BR112015023415B1 - CYTOTOXIC AND ANTI-MITOTIC COMPOUNDS, COMPOSITION, PHARMACEUTICAL COMPOSITION AND USE THEREOF - Google Patents

Abstract

CYTOTOXIC AND ANTI-MITOTIC COMPOUNDS AND METHODS OF USING THE SAME Compounds that have cytotoxic and/or anti-mitotic activity are disclosed. Methods associated with the preparation and use of such compounds, as well as pharmaceutical compositions comprising such compounds, are also disclosed. Also disclosed are compositions having the structure: (T)-(L)-(D), wherein (T) is a targeting moiety, (L) is an optional linker, and (D) is a compound having cytotoxic and/or anti-mitotic activity.

Description

REFERENCE TO PRIOR REQUEST

[0001] This application claims the benefit of U.S. Provisional Patent No. 61/792,020, filed March 15, 2013, and U.S. Provisional Application No. 61/792,066, filed March 15, 2013, the disclosure of each of which is incorporated by reference herein in its entirety.

FUNDAMENTALS Field

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

Description of Related Technique

[0003] Talpir, R. et al. (1994) Tetrahedron Lett. 35:4453-6, describe the naturally occurring compound hemiasterlin, a stable tripeptide obtained from marine sponges that causes microtubule depolymerization and mitotic arrest in cells. Hemiasterlina consists of unusual and highly congested amino acids, features that appear to contribute to its activity. A number of groups have modified certain structural elements of hemiasterlin to evaluate structure-activity relationships and evaluate the activity of hemiasterlin analogs. See, for example, Zask et al., Bioorganic & Medicinal Chemistry Letters, 14:43534358, 2004; Zask et al., J Med Chem, 47:4774-4786, 2004; Yamashita et al., Bioorganic & Medicinal Chemistry Letters, 14:5317-5322, 2004; PCT/GB96/00942; WO 2004/026293; WO96/33211; and U.S. 7,579,323.

[0004] Hemiasterlin analogues, with modifications to the "A-segment", or the amino terminal segment, have been described (see, for example, Zask et al., J Med Chem, 47:4774-4786, 2004; Yamashita et al. al., Bioorganic & Medicinal Chemistry Letters, 14:5317-5322, 2004; U.S. 7,579,323) .. U.S. 7,579,323 discloses a hemiasterlin analog, known as HTI-286, in which the indole moiety is replaced by a phenyl group . HTI-286 exhibits potent anti-mitotic activity and has been evaluated in clinical trials for the treatment of cancer (Ratain et al., Proc Am Soc Clin Oncol, 22:129, 2003).

[0005] Hemiasterlin analogs, with modifications to the "D-segment", or the carboxy terminal segment, have also been reported (See, for example, WO 2004/026293; Zask et al., Bioorganic & Medicinal Chemistry Letters, 14:4353 -4358, 2004; Zask et al., J Med Chem, 47:47744786, 2004). Most modifications at the carboxy terminus result in compounds with substantially decreased potency compared to parent carboxylic acids. See, for example, WO 2004/026293, particularly table 12. Zask et al, (J Med Chem, 47:4774-4786, 2004) also report that amide analogues prepared with simple cyclic and acyclic amines exhibit significantly reduced potency (reduction of one to three orders of magnitude). Among the few tolerated modifications, Zask et al, (Bioorganic & Medicinal Chemistry Letters, 14:4353-4358, 2004) report that the addition of carboxy-terminal esterified cyclic amino acids produces tetrapeptide analogues with prolyl-like ester-containing termini, some of which have comparable potency to the parent compound in a tested cancer cell line.

[0006] Potent cytotoxic and anti-mitotic compositions are highly desired for the treatment of a number of devastating disorders, including cancer. Although a wide variety of hemiasterlin analogs have been generated, many, including a wide variety of compounds with carboxy terminal modifications, exhibit reduced potency that limits usefulness in medical treatment methods.

[0007] For the reasons set out above, although progress has been made in this field, there is a need for additional potent anti-mitotic and cytotoxic compounds having preferred characteristics that make them suitable for the treatment of a variety of diseases, including cancer. This disclosure meets these needs and offers even more related benefits.

BRIEF SUMMARY

[0008] In brief, the present disclosure is directed to biologically active compounds, compositions including the same and methods of using such compounds and compositions.

[0009] In one embodiment, compounds with the following structure (I) are provided:

[0010] where:

[0011] R1 and R2 are each independently selected from the group consisting of: H and a saturated or unsaturated moiety having a non-aromatic linear, branched or cyclic skeleton, containing from 1 to 10 carbon atoms and the carbon atoms are optionally , replaced by: -OH, -I, -Br, -Cl, -F, -CN, -CO2H, -CHO, -COSH, or -NO2; or R2 and R5 are fused and form a ring;

[0012] R3 and R4 are each independently selected from the group consisting of: H, R, ArR-, or R3 and R4 are joined to form a ring;

[0013] R5 is selected from the group consisting of: H, R, ArR-, and Ar;

[0014] or R5 and R2 are fused and form a ring;

[0015] R6 is selected from the group consisting of: H, R, and ArR-;

[0016] R7 and R8 are each independently selected from the group consisting of: H, R, and ArR-; It is

[0017] R9 is:

[0018] in which,

[0019] R is defined as a saturated or unsaturated fraction having a linear, branched or cyclic non-aromatic skeleton, containing from 1 to 10 carbon atoms, zero to four nitrogen atoms, zero to four oxygen atoms and zero to four atoms sulfur, and the carbon atoms are optionally replaced by: =O, =S, OH, -OR10, -O2CR10, -SH, -SR10, -SOCR10, -NH2, -NHR10, -N(R10)2 , -NHCOR10, -NR10COR10, -I, - Br, -Cl, -F, -CN, -CO2H, -CO2R10, -CHO, -COR10, -CONH2, -CONHR10, - CON(R10)2, -COSH, -COSR10, -NO2, -SO3H, -SOR10, -SO2R10, where R10 is a saturated or unsaturated, branched or cyclic linear alkyl group of one to ten carbons;

[0020] the ring formed by joining R3 and R4 is a cyclic skeleton of three to seven non-aromatic members within the definition of R,

[0021] Y is defined as a fraction selected from the group consisting of: a linear alkyl group, saturated or unsaturated, with one to six carbons, optionally substituted with R, ArR—, or X; It is,

[0022] 2, —NHCOR, —NRCOR, —I, —Br, —Cl, —F, —CN, —CO2H, —CO2R, —CHO, —COR, —CONH2, —CONHR, —CON(R)2, —COSH , —COSR, —NO2, —SO3H, —SOR, and —SO2R;

[0023] R14 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl, COR24, -CSR24, -OR24, and -NHR24, wherein each R24 is independently alkyl optionally substituted with halogen, -OH or -SH;

[0024] or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

[0025] In one embodiment, Ar is an aromatic ring selected from the group consisting of: phenyl, naphthyl, anthracyl, pyrrolyl.

[0026] In one embodiment, compounds with the following structure (Ia) are provided:

[0027] where:

[0028] R14 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl, -COR24, -CSR24, -OR24, and -NHR24, wherein each R24 is independently alkyl optionally substituted with halogen, -OH or -SH;

[0029] R15 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl;

[0030] R16 is selected from the group consisting of H and C1-6 alkyl;

[0031] R17 is selected from the group consisting of H and C1-6 alkyl;

[0032] R18 and R30 are independently selected from the group consisting of H, C1-6 alkyl and -SH, with the exception that R18 and R30 cannot both be H;

[0033] R19, R20, R21 and R22 are independently H and C1-6 alkyl, at least one of R19 and R20 is H; or R20 and R21 form a double bond, R19 is H, and R22 is H or C1-6 alkyl; It is

[0034] R23 is selected from the group consisting of H and C1-6 alkyl;

[0035] or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

[0036] In a further embodiment, each optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl is independently optionally substituted with =O, =S, -OH, - OR24OR24, -O2CR24, -SH, -SR24, -SOCR24, -NH2, -N3, -NHR24, -N(R24)2, - NHCOR24, -NR24COR24, -I, -Br, -Cl, -F, -CN , -CO2H, -CO2R24, -CHO, -COR24, -CONH2, -CONHR24, -CON(R24)2, -COSH, -COSR24, -NO2, -SO3H, -SOR24 or -SO2R24 where each R24 is, independently, alkyl optionally substituted with halogen, -OH or -SH.

[0037] In another additional embodiment, each optionally substituted aryl and optionally substituted heteroaryl is independently selected from the group consisting of optionally substituted phenyl, optionally substituted naphthyl, optionally substituted anthracyl, optionally substituted phenantryl, optionally substituted furyl, optionally substituted pyrrolyl, optionally substituted thiophenyl, optionally substituted benzofuryl, optionally substituted benzothiophenyl, optionally substituted quinolinyl, optionally substituted isoquinolinyl, optionally substituted imidazolyl, optionally substituted thiazolyl, optionally substituted oxazolyl, and optionally substituted pyridinyl.

[0038] In another additional embodiment, R15 is selected from one of the following structures (II), (III), (IV), (V):

[0039] where:

[0040] Q is CR25 or N;

[0041] Z is C(R25)2, NR25, S, or O;

[0042] each R25 is independently selected from the group consisting of H, -OH, -R24, -OR24OR24, -O2CR24, -SH, -SR24, -SOCR24, - NH2, -N3, -NHR24, -N( R24)2, -NHCOR24, -NR24COR24, -R24NH2, -I, -Br, -Cl, -F, -CN, -CO2H, -CO2R24, -CHO, -COR24, -CONH2, -CONHR24, -CON(R24 )2, -COSH, -COSR24, -NO2, -SO3H, -SOR24 or -SO2R24, wherein each R24 is independently an alkyl optionally substituted with halogen, -OH or -SH.

[0043] In another additional embodiment, R15 is selected from the group consisting of:

[0044] wherein each R25 is independently selected from the group consisting of H, -OH, -R24, -OR24OR24, -O2CR24, -SH, -SR24, -SOCR24, -NH2, -N3, -NHR24, - N(R24)2, -NHCOR24, -NR24COR24, -R24NH2, -I, - Br, -Cl, -F, -CN, -CO2H, -CO2R24, -CHO, -COR24, -CONH2, -CONHR24, - CON (R24)2, -COSH, -COSR24, -NO2, -SO3H, -SOR24 or -SO2R24, wherein each R24 is independently an alkyl optionally substituted with halogen, -OH or -SH.

[0045] In another additional embodiment, R15 is selected from the group consisting of:

[0046] In another additional embodiment, R15 is:

[0047] In another additional embodiment, R16, R17, R18, and R30 are each methyl.

[0048] In another additional embodiment, R16 is H, R17 is methyl, R18 is methyl, and R30 is methyl.

[0049] It is understood that any embodiment of the compounds of structure (Ia), as set out above, and any specific substituent established for a group R14, R15, R16, R17, R18, R19, R20 and R30 in the compounds of structure (Ia) , as defined above, and may be independently combined with other modalities and/or substituents of compounds of structure (I) to form modalities of the present disclosure not specifically defined above. Furthermore, in the case where a list of substituents is listed for any particular R14, R15, R16, R17, R18, R19, R20, and R30 in a particular embodiment and/or claim, it is understood that each individual substituent may be deleted from the specific embodiment and/or claim and the remaining list of substituents will be considered within the scope of the present disclosure.

[0050] In one embodiment, compounds with the following structure (Ib) are provided:

[0051] where:

[0052] R26 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl;

[0053] R27 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl;

[0054] R16 is selected from the group consisting of H and C1-6 alkyl;

[0055] R17 is selected from the group consisting of H and C1-6 alkyl; It is

[0056] R18 is selected from the group consisting of C 1-6 alkyl and -SH,

[0057] or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

[0058] In a further embodiment, each optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl is independently optionally substituted with =O, =S, -OH, - OR28, - O2CR28, -SH, -SR28, -SOCR28, -NH2, -N3, -NHR28, -N(R28)2, -NHCOR28, - NR28COR28, -I, -Br, -Cl, -F, -CN , -CO2H, -CO2R28, -CHO, -COR28, -CONH2, - CONHR28, -CON(R28)2, -COSH, -COSR28, -NO2, -SO3H, -SOR28 or -SO2R28, where each R28 is , independently, alkyl optionally substituted with halogen, -OH or -SH.

[0059] In another additional embodiment, each optionally substituted aryl and optionally substituted heteroaryl is independently selected from the group consisting of optionally substituted phenyl, optionally substituted naphthyl, optionally substituted anthracyl, optionally substituted phenantryl, optionally substituted furyl, optionally substituted pyrrolyl, optionally substituted thiophenyl, optionally substituted benzofuryl, optionally substituted benzothiophenyl, optionally substituted quinolinyl, optionally substituted isoquinolinyl, optionally substituted imidazolyl, optionally substituted thiazolyl, optionally substituted oxazolyl, and optionally substituted pyridinyl.

[0060] In another additional embodiment, R27 is selected from one of the following structures (II), (III), (IV), (V):

[0061] where:

[0062] Q is CR29 or N;

[0063] Z is C(R29)2, NR29, S, or O;

[0064] each R29 is independently selected from the group consisting of H, -OH, -OR28, -O2CR28, -SH, -SR28, -SOCR28, -NH2, -N3, -NHR28, -N(R28)2 , -NHCOR28, -NR28COR28, -I, -Br, -Cl, -F, -CN, -CO2H, -CO2R28, -CHO, -COR28, -CONH2, -CONHR28, -CON(R28)2, -COSH, - COSR28, -NO2, -SO3H, -SOR28 or -SO2R28, wherein each R28 is independently alkyl optionally substituted with halogen, -OH or -SH.

[0065] In another additional embodiment, R27 is selected from the group consisting of:

[0066] In another additional embodiment, R27 is:

[0067] In another additional embodiment, R16, R17 and R18 are each methyl.

[0068] In another additional embodiment, R16 is H, R17 is methyl, and R18 is methyl.

[0069] It is understood that any embodiment of the compounds of structure (Ib), as established above, and any specific substituent established for a group R25, R26, R16, R17, R18, R18 and R20 in the compounds of structure (Ib), as defined above, and may be independently combined with other modalities and/or substituents of compounds of structure (I) to form modalities of the present disclosure not specifically defined above. Furthermore, in the case where a list of substituents is listed for any particular R25, R26, R16, R17, R18, R18 and R20 in a particular embodiment and/or claim, it is understood that each individual substituent may be deleted from the embodiment. specific and/or claim and the remaining list of substituents will be considered to be within the scope of the present disclosure.

[0070] In one embodiment, the invention provides a method of making a compound having structure (I), (Ia) or (Ib).

[0071] In another embodiment, a pharmaceutical composition is provided, comprising a compound having structure (I), (Ia) or (Ib), or a stereoisomer, pharmaceutically acceptable salt or prodrug and a pharmaceutically acceptable carrier, diluent or excipient .

[0072] In another embodiment, a method of using a compound with structure (I), (Ia) or (Ib), or a stereoisomer, pharmaceutically acceptable salt or prodrug, in therapy, is provided. In particular, the present disclosure provides a method of treating cancer in a mammal comprising administering to a mammal in respective need of an effective amount of a compound having structure (I), (Ia) or (Ib), or a stereoisomer , pharmaceutically acceptable salt or prodrug thereof or a pharmaceutical composition comprising a compound having structure (I), (Ia) or (Ib), or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof and a pharmaceutically acceptable carrier diluent or excipient.

[0073] In another embodiment, the present disclosure provides a method of inhibiting tumor growth in a mammal comprising administering to a mammal in respective need of an effective amount of a compound having structure (I), (Ia) or ( Ib), or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof or a pharmaceutical composition comprising a compound having structure (I), (Ia) or (Ib), or a stereoisomer, pharmaceutically acceptable salt or prodrug and a pharmaceutically acceptable carrier, diluent or excipient.

[0074] In another embodiment, the present disclosure provides a method of killing cancer cells in vitro using a compound having structure (I), (Ia) or (Ib), or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof. In another embodiment, the present disclosure provides a method of killing cancer cells in vivo in a mammal comprising administering to a mammal in respective need of an effective amount of a compound having structure (Y), (Ia) or (Ib). , or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof or a pharmaceutical composition comprising a compound having structure (I), (Ia) or (Ib), or a stereoisomer, pharmaceutically acceptable salt or prodrug and a pharmaceutical carrier acceptable diluent or excipient.

[0075] In another embodiment, the present disclosure provides a method of increasing the survival time of a mammal with cancer, comprising administering to that mammal an effective amount of a compound having structure (I), (Ia) or (Ib ), or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof or a pharmaceutical composition comprising a compound having structure (I), (Ia) or (Ib), or a stereoisomer, pharmaceutically acceptable salt or prodrug and a carrier pharmaceutically acceptable diluent or excipient.

[0076] In one embodiment, compositions comprising biologically active compounds, having the structure or (I), (Ia) or (Ib), or a stereoisomer, pharmaceutically acceptable salt or prodrug, linked directly or indirectly to a fraction of guidance are provided.

[0077] In one embodiment, the invention provides compositions, having the following structure: (T)-(L)-(D) (VI)

[0078] wherein (T) is a target moiety, (L) is an optional linker and (D) is a compound having structure (I), (Ia) or (Ib), or a stereoisomer, pharmaceutically acceptable salt or respective prodrugs. (D) is covalently linked to (L), if (L) is present, or (T), if (L) is not present.

[0079] In a certain embodiment, (D) is a compound having the structure (Ib).

[0080] In one embodiment, the target moiety is an antibody. In this regard, in one embodiment, antibody-drug conjugates (ADCs) consisting of compounds having structure (I), (Ia) or (Ib), or a stereoisomer, pharmaceutically acceptable salt or prodrug, are provided.

[0081] In one embodiment, the invention provides a method of making a composition having structure (VI).

[0082] In another embodiment, a pharmaceutical composition is provided, comprising a composition having structure (VI), or a stereoisomer, pharmaceutically acceptable salt or prodrug and a pharmaceutically acceptable carrier, diluent or excipient.

[0083] In another embodiment, a method of using a composition with structure (VI) in therapy is provided. In particular, the present disclosure provides a method of treating cancer in a mammal comprising administering to a mammal in respective need of an effective amount of a composition having the structure (VI) or a pharmaceutical composition comprising a composition having the structure (VI) VI) and a pharmaceutically acceptable carrier, diluent or excipient.

[0084] In another embodiment, the present disclosure provides a method of inhibiting tumor growth in a mammal comprising administering to a mammal in respective need of an effective amount of a composition having structure (VI) or a pharmaceutical composition comprising a composition having structure (VI) and a pharmaceutically acceptable carrier, diluent or excipient.

[0085] In another embodiment, the present disclosure provides a method of killing cancer cells in vitro using a composition having structure (VI). In another embodiment, the present disclosure provides a method of killing cancer cells in vivo in a mammal, comprising administering to a mammal in respective need of an effective amount of a composition having the structure (VI) or a pharmaceutical composition comprising a having structure (VI) and a pharmaceutically acceptable carrier, diluent or excipient.

[0086] In another embodiment, the present disclosure provides a method of increasing the survival time of a mammal with cancer, comprising administering to a mammal in respective need of an effective amount of a composition having structure (VI) or a composition pharmaceutical comprising a composition, having structure (VI) and a pharmaceutically acceptable carrier, diluent or excipient.

[0087] These and other aspects of the disclosure will be apparent with reference to the following detailed description.

BRIEF DESCRIPTION OF FIGURES

[0088] Figure 1 shows summary cytotoxicity data (EC50) for each of Compounds A-E for two cell lines (HCC1954 and Jurkat).

[0089] Figure 2 shows a graphical representation of cytotoxicity data for compound A in two cell lines (HCC1954 and Jurkat).

[0090] Figure 3 shows a graphical representation of cytotoxicity data for compound B in two cell lines (HCC1954 and Jurkat).

[0091] Figure 4 shows a graphical representation of cytotoxicity data for compound C in two cell lines (HCC1954 and Jurkat).

[0092] Figure 5 shows a graphical representation of cytotoxicity data for compound D in two cell lines (HCC1954 and Jurkat).

[0093] Figure 6 shows a graphical representation of cytotoxicity data for compound E in two cell lines (HCC1954 and Jurkat).

[0094] Figure 7 shows a cell death curve in HCC 1954 cells in vitro with the antibody-drug conjugates: T-LC-SPDP-A (trastuzumab, LC-SPDP linker, compound A) and T-SMCC-A ( trastuzumab, SMCC linker, compound A). EC50 values are shown in the figure.

[0095] Figure 8 shows a cell death curve in HCC 1954 cells in vitro with the antibody-drug conjugates: T - SPDP-B (trastuzumab, LC-SPDP linker, compound B) and T-SMCC-A (trastuzumab, SMCC connector, compound B). EC50 values are shown in the figure.

[0096] Figure 9 shows a cell death curve in HCC 1954 cells in vitro with the antibody-drug conjugate: T-LC-SPDP-C (trastuzumab, LC-SPDP linker, compound C). EC50 value is shown in the figure.

[0097] Figure 10 shows a cell death curve in HCC 1954 cells in vitro with the antibody-drug conjugates: T-MCvcPABC-85 (trastuzumab, MCvc PABC linker, compound 85), T-MCvcPABC-77 (trastuzumab, linker MCvc PABC, compound 77) and T-MCvcPABC-80 (trastuzumab, MCvc PABC linker, compound 80). EC50 values are shown in the figure.

[0098] Figure 11 shows a cell death curve in BxPC-3 cells in vitro with the drug-antibody conjugate C-MCvcPABC-77, (Cetuximab, MCvc PABC linker, compound 77) and a cell death curve in HPAF cells -II in vitro with the drug-antibody conjugate C- MCvcPABC-77, (Cetuximab, MCvc PABC linker, compound 77). EC50 values are shown in the figure.

[0099] Figure 12 shows a cell death curve in HCC 1954 cells in vitro with the antibody-drug conjugates: T-MCvcPABC-77, (trastuzumab, MCvc PABC linker, compound 77), T-MCvcPABC-85, (trastuzumab , MCvc PABC linker, compound 85), T-MCvcPABC-58, (trastuzumab, MCvc PABC linker, compound 58) and T-MCvcPABC-63, (trastuzumab, MCvc PABC linker, compound 63). Heats of EC50 are shown in the figure.

[0100] Figure 13 shows a cell death curve in NCI-N87 cells in vitro with the antibody-drug conjugates: T-MCvcPABC-77, (trastuzumab, MCvc PABC linker, compound 77), T-MCvcPABC-63, ( trastuzumab, MCvc PABC linker, compound 63), T-MCvcPABC-85, (trastuzumab, MCvc PABC linker, compound 85), T-MCvcPABC-77, (trastuzumab, MCvc PABC linker, compound 77) and T-MCvcPABC-80, (trastuzumab, MCvc PABC linker, compound 80). EC50 values are shown in the figure.

[0101] Figure 14 shows the in vivo results of administering compound F, compound 14 or compound 23 to the tumor volume in athymic nude female mice with established tumors.

[0102] Figure 15 shows the in vivo results of administration of the drug-antibody conjugate T-MCC-DM1 (trastuzumab, MCC linker, maytansinoid DM1) in varying dosages as indicated, or T-MCvcPABC-77 in varying dosages as indicated, in the tumor volume in female NOD/SCID Gamma mice with established tumors.

[0103] Figure 16 shows the in vivo results of administering the antibody-drug conjugate T-MCvcPABC-63 at 3mg/kg, or T-MCvcPABC-77 at 3mg/kg, into the tumor volume in NOD/SCID Gamma mice females with established tumors.

[0104] Figure 17 shows a cell death curve in HCC 1954 cells in vitro with the antibody-drug conjugates: T-SPDP-140 (trastuzumab, LC-SPDP linker, compound 140) and T-SMCC-140 (trastuzumab, SMCC connector, compound 140). Compound 140 is linked through the side chain of its N-terminal amino acid... EC50 values are shown in the figure.

[0105] Figure 18 shows a cell death curve in HCC 1954 cells in vitro with the antibody-drug conjugates: T-SPDP-142 (trastuzumab, LC-SPDP linker, compound 142) and T-SMCC-142 (trastuzumab, SMCC connector, compound 142). Compound 142 is linked through the side chain of its N-terminal amino acid... EC50 values are shown in the figure.

[0106] Figure 19 shows a cell death curve in HCC1954 cells in vitro with the antibody-drug conjugates: T-MCvcPABC-58, (trastuzumab, MCvc PABC linker, compound 58) and T-MCvcPABC-41, (trastuzumab, MCvc PABC linker, compound 41) and shows a cell death curve in NCI-N87 cells in vitro with the antibody-drug conjugates: T-MCvcPABC-58, (trastuzumab, MCvc PABC linker, compound 58) and T-MCvcPABC-41 , (trastuzumab, MCvc PABC linker, compound 41). Compound 41 is linked through the side chain of its N-terminal amino acid. Compound 58 is linked through the side chain of its N-terminal amino acid. EC50 values are shown in the figure. EC50 values are shown in the figure.

DETAILED DESCRIPTION

[0107] In the following description, certain specific details are defined to provide a complete understanding of the various embodiments of the disclosure. However, one skilled in the art will understand that disclosure can be practiced without these details.

[0108] Unless otherwise indicated by the context, throughout the present specification and claims below, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be considered in an open, inclusive, or that is, "including, but not limited to."

[0109] Reference throughout this specification to "an embodiment" or "to embodiments" means that a specific feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrases "in an embodiment" or "in the embodiment" in various places throughout this specification are not necessarily referring to the same embodiment. Furthermore, specific features, structures, or characteristics may be combined appropriately in one or more embodiments. Unless otherwise indicated, the following terms and phrases in this document are intended to have the following meanings. When trade names are used in this document, applicants independently intend to include the formulation of the trade name product, the generic drug product, and active pharmaceutical ingredient(s) of the trade name product.

[0110] "Amino" refers to the -NH2 substituent.

[0111] "Cyan" refers to the -CN substituent.

[0112] "Hydroxy" or "hydroxyl" refers to the -OH substituent.

[0113] "Imino" refers to the substituent of =NH.

[0114] "Nitro" refers to the -NO2 substituent.

[0115] "Oxo" refers to the =O substituent.

[0116] "Thiol" refers to the -SH substituent.

[0117] "thioxo" refers to the substituent of =S.

[0118] "Alkyl" refers to a linear or branched hydrocarbon chain substituent consisting only of carbon and hydrogen atoms, which is saturated or unsaturated (i.e., contains one or more double or triple bonds), with de one to twelve carbon atoms (C1-C12 alkyl), preferably one to eight carbon atoms (C1-C8 alkyl) or one to six carbon atoms (C1-C6 alkyl), and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexinyl and the like. Unless otherwise specifically indicated in the specification, an alkyl group may be optionally substituted.

[0119] "Alkylene" or "alkylene chain" refers to a linear or branched divalent hydrocarbon chain linking the rest of the molecule to a substituent group, consisting only of carbon and hydrogen, which is saturated or unsaturated (or i.e., contains one or more double or triple bonds) and has from one to twelve carbon atoms, e.g., methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propylene, n-butynylene and related. The alkylene chain is linked to the rest of the molecule through a single or double bond and to the substituent group through a double bond or a single bond. Attachment points of the alkylene chain to the rest of the molecule and to the substituent group can be through one carbon or any two carbons within the chain. Unless otherwise specifically indicated in the specification, an alkylene group may be optionally substituted.

[0120] "Alkoxy" refers to a substituent of the formula -ORa where Ra is an alkyl substituent as defined above containing from one to twelve carbon atoms. Unless otherwise specifically indicated in the specification, an alkoxy group may be optionally substituted.

[0121] "Alkylamino" refers to a substituent of the formula -NHRa or -NRaRa where each Ra is, independently, an alkyl substituent as defined above containing from one to twelve carbon atoms. Unless otherwise specifically indicated in the specification, an alkylamino group may be optionally substituted.

[0122] "Thioalkyl" refers to a substituent of the formula -SRa where Ra is an alkyl substituent as defined above containing from one to twelve carbon atoms. Unless otherwise specifically indicated in the specification, a thioalkyl group may be optionally substituted.

[0123] "Aryl" refers to a hydrocarbon ring system substituent formed by hydrogen, 6 to 18 carbon atoms and at least one aromatic ring. For purposes of disclosure, the aryl substituent may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems. Aryl substituents include, but are not limited to, aryl substituents, derivatives of aceanthrylene, cenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene , pleiadene, pyrene and triphenylene. Unless otherwise specifically indicated in the specification, the term "aryl" or the prefix "ar-" (as in "aralkyl"), shall include aryl substituents that are optionally substituted.

[0124] "Aralkyl" refers to a substituent of the formula -Rb-Rc where Rb is an alkylene chain as defined above and Rc is one or more aryl substituents as defined above, for example, benzyl, diphenylmethyl and the like. Unless otherwise specifically indicated in the specification, an aralkyl group may be optionally substituted.

[0125] "Cycloalkyl" or "carbocyclic ring" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon substituent consisting only of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms, preferably having three to ten carbon atoms, and which is saturated or unsaturated and linked to the rest of the molecule by a single bond. Monocyclic substituents include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. Polycyclic substituents include, for example, adamantyl, norbornyl, decalinyl, 7, 7-dimethyl-bicyclo[2.2.1]heptanyl and the like. Unless otherwise specifically indicated in the specification, a cycloalkyl group may be optionally substituted.

[0126] "Cycloalkylalkyl" refers to a substituent of the formula -RbRd where Rd is an alkylene chain as defined above and Rg is a cycloalkyl substituent as defined above. Unless otherwise specifically indicated in the specification, a cycloalkylalkyl group may be optionally substituted.

[0127] "Fused" refers to any ring structure described herein that is fused to an existing ring structure in the compounds of the disclosure. When the fused ring is a heterocyclyl or a heteroaryl ring, any carbon atom in the existing ring structure that becomes part of the fused heterocyclyl ring or the fused heteroaryl ring may be replaced by a nitrogen atom.

[0128] "Halo" or "halogen" refers to fluorine, chlorine, bromine or iodine.

[0129] "Haloalkyl" refers to an alkyl substituent, as defined above, that is substituted by one or more halo substituents, as defined above, for example, trifluoromethyl, difluoromethyl, trichloromethyl, 2,2, 2-trifluoroethyl ,1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl and the like. Unless otherwise specifically indicated in the specification, a haloalkyl group may be optionally substituted.

[0130] "Heterocyclyl" or "heterocyclic ring" refers to a stable 3- to 18-membered non-aromatic ring substituent consisting of two to twelve carbon atoms and one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. Unless otherwise specifically indicated in the specification, the heterocyclyl substituent may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl substituent may be optionally oxidized; the nitrogen atom may optionally be quaternized; and the heterocyclyl substituent may be partially or fully saturated. Examples of such heterocyclyl substituents include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, oxopyrrolidinyl, 2-oxopiperazinyl, 2- oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, tritianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl and 1,1-dioxo- thiomorpholinil. Unless otherwise specifically indicated in the specification, a heterocyclyl group may be optionally substituted.

[0131] “N-heterocyclyl” refers to a heterocyclyl substituent, as defined above, containing at least one nitrogen and where the point of attachment of the heterocyclyl substituent to the rest of the molecule is through a nitrogen atom on the heterocyclyl substituent. heterocyclyl Unless otherwise specifically indicated in the specification, an N-heterocyclyl group may be optionally substituted.

[0132] "Heterocyclylalkyl" refers to a substituent of the formula -RbRe where Rb is an alkylene chain as defined above and Re is a heterocyclyl substituent as defined above, and if the heterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl can be associated with the alkyl substituent on the nitrogen atom. Unless otherwise specifically indicated in the specification, a heterocycloalkyl group may be optionally substituted.

[0133] "Heteroaryl" refers to a 5- to 14-membered ring system substituent composed of hydrogen atoms, one to thirteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur and the minus one aromatic ring. For purposes of this disclosure, the heteroaryl substituent may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems; the nitrogen, carbon or sulfur atoms in the heteroaryl substituent may be optionally oxidized; and the nitrogen atom may optionally be quaternized; Examples include, but are not limited to, zepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl , benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinolinyl, dibenzofuranil, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranil, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phen il- 1H-pyrrolyl, phenazinyl, phenothiazinyl, fenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetra zolil, triazinil, and thiophenyl (i.e., thienyl). Unless otherwise specifically indicated in the specification, a heteroaryl group may be optionally substituted.

[0134] “N-heteroaryl” refers to a heteroaryl substituent, as defined above, containing at least one nitrogen and where the point of attachment of the heteroaryl substituent to the rest of the molecule is through a nitrogen atom on the heteroaryl substituent Unless otherwise specifically indicated in the specification, an N-heteroaryl group may be optionally substituted.

[0135] "Heteroarylalkyl" refers to a substituent of the formula -RbRf where Rb is an Alkylene chain as defined above and Rf is a heteroaryl substituent as defined above. Unless otherwise specifically indicated in the specification, a heteroaryl group may be optionally substituted.

[0136] The term "substituted" used herein means any of the above groups (i.e., alkyl, alkylene, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl , N-heteroaryl or heteroarylalkyl) in which at least one atom is replaced by a bond to non-hydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br and I; an oxygen atom in groups such as hydroxyl groups, alkoxy groups and ester groups; a sulfur atom in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups and sulfoxide groups; a nitrogen atom in groups such as azides, amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides and enamines; a silicon atom in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups and triarylsilyl groups; and other heteroatoms in several other groups. "Substituted" also means any of the above groups in which one or more hydrogen atoms are replaced by a higher-order bond (e.g., a double- or triple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl and ester; and nitrogen in groups such as imines, hydrazones, oximes and nitriles. For example, "substituted" includes any of the above groups in which one or more hydrogen atoms are replaced with -NRgRh, -NRgC(=O)Rh, -NRgC(=O)NRgRh, -NRgC(=O)orh, -NRgC(=NRg) NRgRh, -NRgSO2Rh, -OC(=O)NRgRh, -ORg, -SRg, -SORg, -SO2Rg, -OSO2Rg, - SO2oug, =NSO2Rg, and -SO2NRgRh. "Substituted also means any of the above groups in which one or more hydrogen atoms are replaced by -C(=O)Rg, -C(=O)oug, -C(=O)NRgRh, -CH2SO2Rg, -CH2SO2NRgRh. In the foregoing, Rg and Rh are the same or different and independently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl or heteroarylalkyl. Furthermore "substituted" means any of the above groups in which one or more hydrogen atoms are replaced by a bond to an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkoxy, alkylamino, thioalkyl, aryl group In addition, each of the above substituents may also be optionally substituted with one or more of the above substituents.

[0137] 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 undesirable reactions during procedures synthetics. Hydroxyl and amino groups that are protected with a protecting group are referred to herein as "hydroxyl protected groups" and "amino protected groups", respectively. 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. Protective groups as known in the art are generally described in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd ed., 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 on a compound of the disclosure as an azido group that can be chemically converted to the amino group at a desired point in the synthesis. Generally, the groups are protected or present as a precursor that will be inert to reactions that modify other areas of the parent molecule for conversion to their end groups at the right time. More representative protective groups or precursors 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, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2 ,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenyl-methyl, p-nitrobenzyl, triphenylmethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-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), t-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 cyclic imine and imide protecting groups such as phthalimido and dithiasuccinoyl.

[0138] "Prodrug" is intended to indicate a compound that can be converted under physiological conditions or by solvolysis to a biologically active compound of the disclosure. Thus, the term "prodrug" refers to a metabolic precursor of a compound of the disclosure that is pharmaceutically acceptable. A prodrug may be inactive when administered to a subject in need thereof, but is converted in vivo to an active compound of the disclosure. In one embodiment, a prodrug is rapidly transformed in vivo to produce the compound of the disclosure, for example, by hydrolysis in blood. In one embodiment, a prodrug may be stable in plasma or blood. In one embodiment, a prodrug may be a targeted form of a compound of the invention. The prodrug compound often offers solubility, tissue compatibility, or delayed release advantages in a mammalian organism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier ,Amsterdam)). A discussion of examples is provided in Higuchi, T., et al., A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0139] The term "prodrug" should include covalently linked carriers, which release the active compound from disclosure in vivo when such prodrug is administered to a mammalian subject. Conjugates, including ADCs, as disclosed herein, are such examples of compositions having structure (I), (Ia) or (Ib). Prodrugs of a compound of the disclosure can be prepared by modifying the functional groups present in the compound of the disclosure in such a way that the modifications are cleaved, both in routine manipulation and in vivo, in the main compound of the disclosure. Prodrugs include compounds of the disclosure in which a hydroxy, amino or mercapto group is attached to any group that, when the prodrug of the compound of the disclosure is administered to a mammalian subject, cleaves to form a free hydroxy group, mercapto-free or amino-free, respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol groups or amide derivatives of functional amine groups in the compounds of the disclosure and the like.

[0140] The present disclosure is also intended to encompass all pharmaceutically acceptable compounds of structure (I), (Ia) or (Ib), being isotopically-labeled by having one or more atoms replaced by an atom having a different atomic mass or number. of mass. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine and iodine, such as, but not limited to, 2H, 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 31P, 32P, 35S, 18F, 36Cl, 123I, and 125I, respectively. These radiolabeled compounds can be useful in helping to determine or measure the effectiveness of compounds, by characterizing, for example, the site or mode of action, or binding affinity to the pharmacologically important site of action. Certain isotopically labeled compounds of structure (I), (Ia) or (Ib), for example, those that incorporate a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes, tritium, i.e., 3H, and carbon-14, i.e., 14C, are particularly useful for this purpose in view of their ease of modality and ready means of detection.

[0141] Replacement with heavier isotopes, such as deuterium, i.e., 2H, can generate certain therapeutic advantages resulting from greater metabolic stability, for example, greater in vivo half-life or reduced dosage regimens, and, consequently, can be preferred in some circumstances.

[0142] Replacement with positron emitting isotopes, such as 11C, 18F, 15O and 13N, may be useful in Positron Emission Topography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of structure (I), (Ia) or (Ib) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the Examples and Preparations, as shown below, using an appropriate isotopically labeled reagent. in place of the unlabeled reagent previously used.

[0143] The present disclosure is also intended to encompass the in vivo metabolic products of the disclosed compounds. Such products may result from, for example, oxidation, reduction, hydrolysis, amidation, esterification and the like of the administered compound, mainly due to enzymatic processes. Therefore, the present disclosure includes compounds produced by a process consisting of administering a disclosure compound to a mammal for a period of time sufficient to produce a respective metabolic product. Such products are typically identified by administering a radiolabeled compound of the disclosure in a detectable dose to an animal, such as rat, mouse, guinea pig, monkey, or human, leaving sufficient time for metabolism to occur and isolating its conversion products from urine, blood or other biological samples.

[0144] "Stable compound" and "stable structure" are intended to indicate a compound that is robust enough to survive isolation to a useful degree of purity from a reaction mixture and formulation into an effective therapeutic agent.

[0145] The term "antibody" herein is used in the broadest sense and specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments , as long as they exhibit the desired biological activity. The term "antibodies" refers to a full-length immunoglobulin molecule or a functionally active part of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that immunospecifically binds to an antigen of a target of interest or part thereof. The immunoglobulin disclosed herein may be of any type (e.g., IgG, IgE, IgM, IgD and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of the immunoglobulin molecule. Immunoglobulins can be derived from any species. In one aspect the immunoglobulin is of human, murine or rabbit origin. In another aspect, antibodies are polyclonal, monoclonal, multispecific (e.g., bispecific), human, humanized or chimeric, linear antibodies, single chain antibodies, diabodies, maxibodies, minibodies, Fv fragments, Fab, F( ab'), F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDRs, and epitope-binding fragments of any of the above that immunospecifically bind to a target antigen.

[0146] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations, which may be present in smaller quantities. Monoclonal antibodies include "chimeric" antibodies, in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibody, while the remainder of the chain is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibody, as well as fragments of such antibodies (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al. ., 1984, Proc. Natl. Acad. Sci. USA 81:6851-6855). Monoclonal antibodies also include humanized antibodies that may contain a CDRs from a non-human source and a completely human constant region.

[0147] An "intact" antibody is one that comprises an antigen-ligand variable region, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or respective amino acid sequence variant.

[0148] "Antibody fragments" comprise a portion of an intact antibody, preferably comprising the respective antigen-binding or variable region. Examples of antibody fragments are Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single chain antibody molecules; maxibodies; minibodies; and multispecific antibodies formed from antibody fragment(s).

[0149] An "isolated" antibody is an antibody that has been identified and separated and/or recovered from a component of its natural environment. Contaminating components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other protein or non-protein solutes. In some embodiments, the antibody will be purified (1) to greater than 95% by weight antibody as determined by the Lowry method and more preferably greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 N-terminal residues or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining. Isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Typically, however, isolated antibody will be prepared by at least one purification step.

[0150] An antibody "binding" an antigen of interest is one capable of binding to that antigen with sufficient affinity such that the antibody is useful in targeting a cell expressing the antigen.

[0151] A "native sequence" polypeptide is one that has the same amino acid sequence as a naturally derived polypeptide. Such native sequence polypeptides may be isolated from nature or may be produced by recombinant or synthetic means. Thus, a native sequence polypeptide may have the amino acid sequence of the naturally occurring human polypeptide, murine polypeptide, or polypeptide from any other mammalian species.

[0152] The term "intracellular metabolite" refers to a compound resulting from a metabolic process or reaction within a cell in a composition of the invention (for example, 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 drugs that have been separated intracellularly, i.e., following entry, diffusion, absorption, or transport into a cell (e.g., by cleavage). enzymatic analysis of an ADC by an intracellular enzyme).

[0153] In the context of conjugates, including ADCs, the terms "intracellularly cleaved" and "intracellular cleavage" refer to metabolic processes or reactions within a cell in a composition of the invention whereby covalent bonding, e.g. the linker (L) between the drug moiety (D) and the target moiety (T) (e.g., an antibody) is broken, resulting in dissociated free drug (T) within the cell. In one embodiment, the cleaved fractions of the compositions in question are thus intracellular metabolites (e.g., T, T-L fragment, D-L fragment, D). Accordingly, in one embodiment, the invention provides compositions that are cleavage products of a composition having structure (VI), which cleavage products include compositions comprising structure (Y), (Ia) or (Ib), or respective stereoisomers. Similarly, the linker (L) between the microtubule bursting peptide (PT) toxin and the target moiety (T) (e.g., an antibody) can be cleaved intracellularly, resulting in the dissociated PT (T) within the cell. . The cleaved fractions of the compositions in question are thus intracellular metabolites (e.g., T, T-L fragment, PT-L fragment, PT). In this sense, in one embodiment, the invention provides compositions that are cleavage products of a composition having structure (VII), which cleavage products include compositions comprising structure (I), (Ia) or (Ib), or respective stereoisomers .

[0154] The term "extracellular cleavage" refers to a metabolic process or reaction outside a cell in a composition of the invention whereby the covalent bond, for example, the linker (L), between the drug moiety (D) and the target moiety (T) (e.g., an antibody) is cleaved, resulting in dissociated free drug (T) outside the cell. In one embodiment, the cleaved fractions of the compositions in question are therefore initially extracellular metabolites (e.g., T, T-L fragment, D-L fragment, D), which can move intracellularly by diffusion and cell permeability or transport. In this sense, in one embodiment, the invention provides compositions that are cleavage products of a composition having structure (VI), which cleavage products include compositions comprising structure (I), (Ia) or (Ib), or respective stereoisomers . Similarly, the linker (L) between the microtubule bursting peptide (PT) toxin and the target moiety (T) (e.g., an antibody) can be cleaved extracellularly, resulting in dissociated PT (T) outside the cell. . The cleaved fractions of the compositions in question are therefore initially extracellular metabolites (e.g., T, T-L fragment, PT-L fragment, PT). In this sense, in one embodiment, the invention provides compositions that are cleavage products of a composition having structure (VII), which cleavage products include compositions comprising structure (I), (Ia) or (Ib), or respective stereoisomers .

[0155] "Mammal" includes humans and domestic animals such as laboratory animals and pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits) and non-domestic animals such as wild animals and others.

[0156] "Optional" or "optionally" means that the event or circumstances described below may or may not occur and that the description includes cases where said event or circumstance occurs and cases where it does not occur. 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.

[0157] "Pharmaceutically acceptable excipient, diluent or carrier" includes, without limitation, any adjuvant, carrier, excipient, glidant, sweetening agents, diluent, preservative, dye/color, flavor enhancer, surfactant, wetting agent, dispersing agent, suspension, stabilizer, isotonic agent, solvent, or emulsifier that, for example, has been approved by the United States Food and Drug Administration (or similar regulatory agency in another jurisdiction) as being acceptable for use in humans or domestic animals.

[0158] "Pharmaceutically acceptable salts" include both acid and base addition salts.

[0159] "Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological efficacy and properties of free bases, which are non-biologically or otherwise undesirable, and which are formed with inorganic acids, such as, but 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, dodecysulfuric acid, acid ethane-1,2-disulfonic acid, ethanesulfonic 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, 1-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.

[0160] "Pharmaceutically acceptable base addition salt" refers to those salts that maintain the biological efficacy and properties of free acids, which are not biologically or otherwise undesirable. These salts are prepared by adding an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, salts of: sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum and the like. Preferred inorganic salts are ammonia, sodium, potassium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines including naturally substituted amines, cyclic amines and basic ion exchange resins such as ammonia, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benetamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, amine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0161] Crystallizations often produce a solvate of the compound of disclosure. As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of a compound of the disclosure with one or more solvent molecules. The solvent may be water, in which case the solvent may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present disclosure can exist as a hydrate, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like, as well as corresponding solvated forms. The compound of the disclosure may be true solvate, while in other cases, the compound of the disclosure may simply retain accidental water or be a mixture of water with some adventitious solvent.

[0162] A "pharmaceutical composition" refers to a formulation of a compound of the disclosure and a generally accepted means in the art for delivering the biologically active compound to mammals, e.g., humans. Such a medium includes all pharmaceutically acceptable carriers, diluents or excipients.

[0163] Non-limiting examples of disorders to be addressed herein include benign and malignant tumors; leukemia and lymphoid malignancies, in particular of breast, ovary, stomach, salivary gland, endometrium, lung, kidney, colon, thyroid, pancreas, prostate or bladder cancer; neuronal, glial, astrocytic, hypothalamic and other glandular, macrophagal, epithelial, stromal and bastocoelic disorders, autoimmune disease, inflammatory disease, fibrosis and infectious diseases. Taking into account the characteristics and particularly the potency of the compositions in question, it will be apparent to those skilled in the art that the compounds of the invention may be indicated for use in the treatment of any disease where the exertion of a cytotoxic or cytotoxic effect on a target cell it is desirable.

[0164] In one embodiment, compositions of the invention are used to treat autoimmune disease. Immunospecific antibodies to an antigen of a cell that is responsible for the production of autoimmune antibodies can be obtained from any organization (e.g., a university scientist or a company such as Genentech) or produced by any method known to those skilled in the art. such as, for example, recombinant expression techniques or chemical synthesis. In another embodiment, useful ligand antibodies that are immunospecific for treating autoimmune diseases include, but are not limited to, antinuclear antibody; DNA Anti ds; Anti-ss-DNA, IgM Anti Cardiolipin Antibody, IgG; Anti Phospholipid Antibody IgM, IgG; Anti-SM Antibody; Antimitochondrial antibody;

[0165] Thyroid antibodies; Microsomal antibodies; Thyroglobulin antibodies; Anti-SCL-70; Anti-Jo; Anti-U1RNP; Anti-La/SSB; Anti SSA; Anti-SSB; Anti Perital Cell Antibody; Anti histones; Anti RNP; C- HIP; P-HIP; Anti Centromere; Anti-Fibrillarin and Anti-GBM antibody. In certain preferred embodiments, antibodies useful in the present methods can bind to a receptor or receptor complex expressed on an activated lymphocyte.

[0166] The receptor or receptor complex may include a member of the immunoglobulin gene superfamily, a member of the TNF receptor superfamily, an integrin, a cytokine receptor, a chemokine receptor, a histocompatibility protein, a lectin or a complement control protein. Non-limiting examples of suitable immunoglobulin superfamily members are CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152/CTLA-4, PD-1, and ICOS.

[0167] Non-limiting examples of appropriate TNF receptor superfamily members are CD27, CD40, Fas/CD95, CD134/OX40, CD137/4-1BB, TNF-Rl, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-Rl, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3. Non-limiting examples of suitable integrins are CD1la, CD1lb, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103, and CD104. Non-limiting examples of suitable lectins are type C, type S and type I lectins.

[0168] In one embodiment, the ligand is an antibody that binds to an activated lymphocyte that is associated with an autoimmune disease.

[0169] Immunological diseases that are characterized by inadequate activation of immune system cells and that can be treated or prevented with the methods described in this document can be classified, for example, by the types of hypersensitivity reactions that underlie the disorder. Typically, these reactions are 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).

[0170] Specific examples of such immunological diseases include the following: rheumatoid arthritis, demyelinative autoimmune diseases (e.g., multiple sclerosis, allergic encephalomyelitis), endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Grave's disease, glomerulonephritis, hepatologic autoimmune disorder, inflammatory bowel disease (e.g., Crohn's disease), anaphylaxis, allergic reaction, Sjogren's syndrome, type Y diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure , Schmidt 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, Dressler syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, Pemphigus vulgaris, Pemphigus, dermatitis herpetiformis, Alopecia arcata, pemphigoid, Scleroderma, progressive systemic sclerosis, CREST syndrome (Calcinosis, Rainaud's phenomenon, esophageal dysmotility, sclerodactyl and telangiectasia ), male and female autoimmune infertility, ankylosing sponditis, ulcerative colitis, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture syndrome, Chagas disease, sarcoidosis, rheumatic fever, asthma, recurrent miscarriage, anti-phospholipid syndrome, farmer's lung, erythema multiforme, cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird breeders' lung, toxic epidermal necrolysis, Alport syndrome, Alveolitis, Allergic alveolitis, Alveolitis fibrosing, 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, Behçet's disease, Caplan syndrome, Kawasaki disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, Erythema elevated et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman syndrome, Felti syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft-versus-host disease, transplant rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom macroglobulemia, Evan syndrome, and autoimmune gonadal insufficiency . In this sense, the methods described here encompass treatment of B lymphocyte disorders (e.g., systemic lupus erythematosus, Goodpasture syndrome, rheumatoid arthritis, and type I diabetes), T1 lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjogren's disease, Hashimoto's thyroiditis, Grave's disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or acute graft-versus-host disease), or T2 lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, rhinitis allergic disease, Omenn syndrome, systemic sclerosis or chronic graft-versus-host disease). Generally, disorders involving dendritic cells involve t1 or t2 lymphocyte disorders.

[0171] In certain embodiments, the immunological disorder is mediated by T cells, which may include activated T cells. ADCs or derivatives of ADCs can be administered to deplete such activated T cells.

[0172] In one embodiment, compositions of the invention can be used to treat fibrosis. Fibrosis can occur in various tissues within the body, typically as a result of inflammation or damage, and examples include but are not limited to; Lungs, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis; Liver, cirrhosis; Heart, endomyocardial fibrosis, old myocardial infarction, atrial fibrosis; Others, mediastinal fibrosis (soft tissues of the mediastinum), myelofibrosis (bone marrow), Retroperitoneal fibrosis (soft tissues of the retroperitoneum), progressive massive fibrosis (lungs); a complication of coal workers' pneumoconiosis, nephrogenic systemic fibrosis (skin), Crohn's disease (intestine), keloid (skin), systemic sclerosis/Scleroderma (skin, lungs), Arthrofibrosis (knee, shoulder, other joints), Peyronie's (penis) , Dupuytren's contracture (hands, fingers) and some forms of adhesive capsulitis (shoulder).

[0173] With regard to infectious diseases, compositions of the invention can be used directly on certain infectious agents or pathogens, or can be used to exert a cytostatic or cytotoxic effect on a host cell that harbors or otherwise provides for the agent. infectious or pathogenic agent.

[0174] "Effective" or "therapeutically effective amount" refers to the amount of a compound of the disclosure that, when administered to a mammal, preferably a human, is sufficient to effect treatment, as defined below, of the particular indication. (e.g. cancer or cell tumor in the mammal, preferably a human). The amount of a compound of the disclosure that constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the form of administration, and the age of the mammal being treated, but can be determined routinely by those skilled in the art. taking into account their own knowledge and dissemination.

[0175] "Treat" or "treatment" as used herein covers the treatment of the disease or condition of interest in a mammal, preferably a human, having the disease or condition of interest and includes, for example: (I) prevention of disease or condition occurring in a mammal, in particular when such mammal is predisposed to the condition but has not yet been diagnosed as having it; (II) inhibiting the disease or condition, that is, stopping its development; (III) alleviating the disease or condition, that is, causing regression of the disease or condition; or (IV) relieving symptoms arising from the disease or condition, i.e., relieving pain without treating the underlying disease or condition.

[0176] A therapeutically effective amount of compounds in the field of cancer treatment can reduce the number of cancer cells; reduce the size of the tumor; inhibit (i.e., slow down to a certain extent and ideally stop) infiltration of cancer cells into peripheral organs; inhibit (i.e., slow down to a certain extent and ideally stop) tumor metastases; inhibit, to a certain extent, tumor growth; increase survival time; and/or alleviate, to some extent, one or more of the symptoms associated with cancer. To the extent that the drug can prevent the growth of and/or eliminate existing cancer cells, it may be cytostatic and/or cytotoxic. Compounds of the present invention are preferably cytotoxic. For cancer therapy, efficacy can, for example, be measured by assessing time to disease progression (TTP) and/or determining response rate (RR).

[0177] An "effective" amount in relation to a given result to be achieved is an amount sufficient to achieve the desired result. For example, an "effective amount" of drug, when referring to cancer cell killing, refers to an amount of drug sufficient to produce the effect.

[0178] Solid tumors contemplated for treatment using the currently disclosed compounds include but are not limited to: sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing tumor , leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer (e.g., gastrointestinal cancer), cancer oral, nasal cancer, throat cancer, squamous cell carcinoma (e.g. of the lung), basal cell carcinoma, adenocarcinoma (e.g. of the lung), sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, 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, astrocytoma multiforme, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma and retinoblastoma . Blood-borne cancers contemplated for treatment using the currently disclosed compounds include but are not limited to: acute lymphoblastic leukemia "ALL", B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute myeloblastic leukemia "AML", acute promyelocytic leukemia "APL", acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute non-lymphocytic leukemia, acute undifferentiated leukemia, chronic myeloid leukemia "CML", chronic lymphocytic leukemia "CLL", hairy cell leukemia and multiple myeloma . Acute and chronic leukemias contemplated for treatment using the currently disclosed compounds include but are not limited to: lymphoid, myeloid, lymphoid and myeloid leukemias. Lymphomas anticipated for treatment using the currently disclosed compounds include but are not limited to: Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease and polycythemia vera. Other cancers contemplated for treatment using the currently disclosed compounds include but are not limited to: peritoneal cancer, hepatocellular cancer, hepatoma, salivary cancer, vulvar cancer, thyroid cancer, penile cancer, anal cancer, head and neck cancer, renal cell carcinoma, acute anaplastic large cell carcinoma and cutaneous anaplastic large cell carcinoma.

[0179] Cancers, including but not limited to, a tumor, metastasis, or other disease or disorder characterized by uncontrolled or unwanted cell growth can be treated or prevented through the administration of the presently disclosed compounds.

[0180] In other embodiments, methods for treating or preventing cancer are provided, including administering to a patient in need thereof an effective amount of a compound disclosed herein in combination with an additional method of treatment. In one embodiment, the additional method of treatment includes treatment with a chemotherapeutic agent. In one embodiment the chemotherapeutic agent is one with which the cancer treatment has been found to be non-refractory. In another embodiment, the chemotherapeutic agent is one with which the cancer treatment has been found to be refractory. The compound of the invention can be administered before, after or at the same time as the chemotherapeutic agent.

[0181] In one embodiment, the additional method of treatment is radiation therapy. The compound of the invention can be administered before, after or at the same time as radiation.

[0182] Compounds of the invention can also be administered to a patient who has undergone or will undergo surgery as a treatment for cancer.

[0183] In a specific embodiment, the compound of the invention is administered simultaneously with the chemotherapeutic agent or radiotherapy. In another specific embodiment, chemotherapy agent or radiation therapy is administered prior to or subsequent to the administration of compounds of the invention, in one aspect at least one hour, five hours, 12 hours, one day, one week, one month, in more aspects several months (e.g., up to three months), prior to or subsequent to administration of a compound of the invention.

[0184] A chemotherapeutic agent can be administered over a series of sessions. Any one or a combination of the chemotherapeutic agents listed herein or also known in the art can be administered. Regarding radiation, any radiation therapy protocol can be used depending on the type of cancer being treated. For example, but not by way of limitation, x-ray radiation may be administered; In particular, high-energy megavoltage (radiation of greater than 1 MeV energy) can be used for deep-seated tumors, and orthovoltage electron beam x-ray radiation can be used for skin cancers. Gamma-ray-emitting radioisotopes, such as radioactive isotopes of radium, cobalt, and other elements, may also be administered.

[0185] Furthermore, methods of treating cancer with a compound of the invention are provided as an alternative to chemotherapy or radiotherapy therapy where chemotherapy or radiotherapy has proven or may prove too toxic, for example, resulting in side effects. unacceptable or unbearable for the subject being treated. Furthermore, methods of treating cancer with a compound of the invention are provided as an alternative to surgery where surgery has proven or may prove unacceptable or unbearable for the subject being treated.

[0186] The compound of the invention can also be used in vitro or ex vivo, such as for the treatment of certain types of cancer, including, but not limited to, leukemias and lymphomas, such treatment involving autologous stem cell transplants . This may involve a multi-step process in which the animal's autologous hematopoietic stem cells are harvested and purged of all cancer cells, the animal's remaining bone marrow cell population is then eradicated through administration of a high dose of a compound of the invention with or without accompanying high-dose radiation therapy and the stem cell graft is infused back into the animal. Supportive care is provided while bone marrow function is restored and the animal recovers.

[0187] Methods for treating cancer further include administering to a patient in need thereof an effective amount of a compound of the invention and another therapeutic agent that is an anti-cancer agent. Suitable anticancer agents include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards, cytoxane , etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, actinomycin D, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vindesine, vinorelbine, paclitaxel and docetaxel.

[0188] Other chemotherapeutic agents are examples of agents such as thiotepa and cyclophosphamide CYTOXAN®; alkyl sulfonates such as busulfan, treosulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylamelamines, including Altretamine, Triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; TLK 286 (TELCYTA™); acetogenins (especially bulatacin and bulatacinone); Delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); Beta- lapachone; lapachol; Colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin and aminocamptothecin-9); bryostatin; kallistatin; CC-1065 (including its synthetic analogues, adozelesin, carzelesin and bizelesin); Podophyllotoxin; podophinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobine; pancratistatin; a sarcodictiin; spongistatin; nitrogen mustards such as chlorambucil, chlornafazine, cholofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichinine, phenesterine, prednimustine, trofosfamide and uracil mustard; triazines, such as decarbazine; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; epipodophilins, such as etoposide, Teniposide, Topotecan, 9-aminocamptothecin, camptothecin or chrysnatol; bisphosphonates, such as clodronate; antibiotics such as enediine antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omegal1 (See, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)) and anthracyclines such as anamycin, AD 32, alcarubicin, daunorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100, idarubicin, KRN5500, menogaril, dynemycin, including dynemycin A, esperamycin, neocarzinostatin chromophore and related chromoprotein enediine antibiotic chromophores, aclacinomysins, actinomycin , authramycin, azaserine, bleomycins (e.g., A2 and B2), cactinomycin, carabicin, caminomycin, carzinophylline, chromomycinis, dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMICIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin and deoxydoxorubicin), esorubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, chelamycin, rhodorubicin, streptonigrin, streptozocin, tub ercidine , ubenimex, zinostatin and zorubicin; photodynamic therapies such as vertoporfin (BPD-MA), phthalocyanine, 5ALA Pc4 and demethoxyhypocrelin A (2BA-2-DMHA); folic acid analogues such as denopterin, pteropterin and trimetrexate; dpurine analogues such as 6-mercaptopurine fludarabine, thiamiprine and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, azauridine-6, carmofur, cytarabine, cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine and floxuridine; androgens, such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane and testolactone; anti-adrenal drugs such as aminoglutethimide, mitotane and trilostane; folic acid booster, folinic acid (leucovorin); aceglatone; anti-neoplastic anti-folic acid agents such as ALIMTA®, LY231514 pemetrexed, dihydrofolate reductase inhibitors such as methotrexate and trimetrexate; anti-metabolites such as 5-fluorouracil (5-FU) and its examples such as UFT, S-1 and capecitabine, floxuridine, doxifluridine and ratitrexed; and glycinamide ribonucleotide formyltransferase inhibitors and thymidylate synthase inhibitors and such as raltitrexed (TOMUDEX®, TDX); dihydropyrimidine dehydrogenase inhibitors such as enyluracil; aldophosphamide glycoside; aminolevulinic acid; Amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; an epothilone; ethoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; fenamet; pyrarubicin; losoxantrone; 2-ethylhydrazide; Procarbazine; PSK® complex polysaccharide (JHS Natural Products, Eugene, Oreg.); razoxane; didehydrorhizoxin; sizophyran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); Uretan; vindesine (ELDISINE®, FILDESIN®); Dacarbazine; Manomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids and taxanes, e.g., paclitaxel TAXOL® (Bristol-Miers Squibb Oncologi, Princeton, N.J.), Cremofor-free ABRAXANE™, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.) and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antoni, France); chlorambucil; 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; Novantrona; edatrexate; daunomycin; mtoxantrone; aminopterin; Xeloda; Ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); vitamin D3 analogues such as EB 1089, CB 1093 and KH 1060; retinoids, such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen of oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.

[0189] Antihormonal agents that act to regulate or inhibit hormone action on tumors such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen NOLVADEX®), raloxifene, megastrol , droloxifene, 4-hydroxytamoxifen, trioxyfen, keoxifen, LY117018, onapristone and toremifene FARESTON®; Aromatase inhibitors that inhibit the aromatase enzyme that regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, vorozole RIVISOR®, letrozole FEMARA® and anostrozole ARIMIDEX®; and anti-androgens such as flutamide, bicalutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as troxacitabine (a cytosine nucleoside analogue of 1,3-dioxolane); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways implicated in the proliferation of aberrant cells, 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®, LEUVECTIN® vaccine and VAXID®; rIL-2 PROLEUKIN®; Topoisomerase inhibitor 1 LURTOTECAN®; rmRH ABARELIX®; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0190] The compounds of the disclosure, or their pharmaceutically acceptable salts may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereoisomers and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, like (D)- or (L)- for amino acids. The present disclosure is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active isomers (+) and (-), (R)- and (S)-, or (D)- and (L)- can be prepared using chiral tune or chiral reagents, or resolved using conventional techniques, e.g. chromatography and fractional crystallization. Conventional techniques for the preparation/isolation of the individual enantiomers include chiral synthesis of an appropriate optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative), for example, using chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless otherwise specified, the compounds are intended to include both E and Z geometric isomers. Likewise, all tautomeric forms also are intended to be included.

[0191] A "stereoisomer" refers to the compound made of the same atoms linked by the same bonds, but having different three-dimensional structures, which are not interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof and includes "enantiomers", which refer to two stereoisomers whose molecules are non-superimposable mirror images of each other.

[0192] A "tautomer" refers to a change of protons from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any named compounds.

New Compounds

[0193] In one embodiment, compounds with the following structure (I) are provided:

[0194] where:

[0195] R1 and R2 are each independently selected from the group consisting of: H and a saturated or unsaturated moiety having a non-aromatic linear, branched or cyclic skeleton, containing from 1 to 10 carbon atoms and the carbon atoms are optionally , replaced by: -OH, -I, -Br, -Cl, -F, -CN, -CO2H, -CHO, -COSH, or -NO2; or R2 and R5 are fused and form a ring;

[0196] R3 and R4 are each independently selected from the group consisting of: H, R, ArR-, or R3 and R4 are joined to form a ring;

[0197] R5 is selected from the group consisting of: H, R, ArR- and Ar;

[0198] or R5 and R2 are fused and form a ring;

[0199] R6 is selected from the group consisting of: H, R, and ArR-;

[0200] R7 and R8 are each independently selected from the group consisting of: H, R, and ArR-; It is

[0201] R9 is:

[0202] where,

[0203] R is defined as a saturated or unsaturated fraction having a linear, branched or cyclic non-aromatic skeleton, containing from 1 to 10 carbon atoms, zero to four nitrogen atoms, zero to four oxygen atoms and zero to four atoms sulfur, and the carbon atoms are optionally replaced by: =O, =S, OH, -OR10, -O2CR10, -SH, -SR10, -SOCR10, -NH2, -NHR10, -N(R10)2 , -NHCOR10, - NR10COR10, -I, -Br, -Cl, -F, -CN, -CO2H, -CO2R10, -CHO, -COR10, -CONH2, - CONHR10, -CON(R10)2, -COSH, -COSR10, -NO2, -SO3H, -SOR10, -SO2R10, where R10 is a saturated or unsaturated, branched or cyclic linear alkyl group of one to ten carbons;

[0204] The ring formed by joining R3 and R4 is a cyclic skeleton of three to seven non-aromatic members within the definition of R,

[0205] Y is defined as a fraction selected from the group consisting of: a linear alkyl group, saturated or unsaturated, with one to six carbons, optionally substituted with R, ArR—, or X; It is,

[0206] 2, —NHCOR, —NRCOR, —I, —Br, —Cl, —F, —CN, —CO2H, —CO2R, —CHO, —COR, —CONH2, —CONHR, —CON(R)2, —COSH , —COSR, —NO2, —SO3H, —SOR, and —SO2R;

[0207] R14 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl, COR24, -CSR24, -OR24, and -NHR24, wherein each R24 is independently alkyl optionally substituted with halogen, -OH or -SH;

[0208] or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

[0209] In one embodiment, Ar is an aromatic ring selected from the group consisting of: phenyl, naphthyl, anthracyl, pyrrolyl.

[0210] In one embodiment, compounds with the following structure (Ia) are provided:

[0211] where:

[0212] R14 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl, -COR24, -CSR24, -OR24, and -NHR24, wherein each R24 is independently alkyl optionally substituted with halogen, -OH or -SH;

[0213] R15 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl;

[0214] R16 is selected from the group consisting of H and C1-6 alkyl;

[0215] R17 is selected from the group consisting of H and C1-6 alkyl;

[0216] R18 and R30 are independently selected from the group consisting of H, C1-6 alkyl and -SH, with the exception that R18 and R30 cannot both be H;

[0217] R19, R20, R21 and R22 are independently H and C1-6 alkyl, at least one of R19 and R20 is H; or R20 and R21 form a double bond, R19 is H, and R22 is H or C1-6 alkyl; It is

[0218] R23 is selected from the group consisting of H and C1-6 alkyl;

[0219] or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

[0220] In a further embodiment, each optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl is independently optionally substituted with =O, =S, -OH, - OR24, - O2CR24, -SH, -SR24, -SOCR24, -NH2, -N3, -NHR24, -N(R24)2, -NHCOR24, - NR24COR24, -I, -Br, -Cl, -F, -CN , -CO2H, -CO2R24, -CHO, -COR24, -CONH2, -CONHR24, -CON(R24)2, -COSH, -COSR24, -NO2, -SO3H, -SOR24 or -SO2R24 where each R24 is, independently, alkyl optionally substituted with halogen, -OH or -SH.

[0221] In another additional embodiment, each optionally substituted aryl and optionally substituted heteroaryl is independently selected from the group consisting of optionally substituted phenyl, optionally substituted naphthyl, optionally substituted anthracyl, optionally substituted phenantryl, optionally substituted furyl, optionally substituted pyrrolyl, optionally substituted thiophenyl, optionally substituted benzofuryl, optionally substituted benzothiophenyl, optionally substituted quinolinyl, optionally substituted isoquinolinyl, optionally substituted imidazolyl, optionally substituted thiazolyl, optionally substituted oxazolyl, and optionally substituted pyridinyl.

[0222] In another additional embodiment, R15 is selected from one of the following structures (II), (III), (IV), (V):

[0223] where:

[0224] Q is CR25 or N;

[0225] Z is C(R25)2, NR25, S, or O;

[0226] each R25 is independently selected from the group consisting of H, -OH, -R24, -OR24, -O2CR24, -SH, -SR24, -SOCR24, -NH2, - N3, -NHR24, -N( R24)2, -NHCOR24, -NR24COR24, -R24NH2, -I, -Br, -Cl, -F, -CN, -CO2H, -CO2R24, -CHO, -COR24, -CONH2, -CONHR24, -CON(R24 )2, -COSH, -COSR24, -NO2, -SO3H, -SOR24 or -SO2R24, wherein each R24 is independently alkyl optionally substituted with halogen, -OH or -SH.

[0227] In another additional embodiment, R15 is selected from the group consisting of:

[0228] wherein each R25 is independently selected from the group consisting of H, -OH, -R24, -OR24, -O2CR24, -SH, -SR24, -SOCR24, - NH2, -N3, -NHR24, - N(R24)2, -NHCOR24, -NR24COR24, -R24NH2, -I, -Br, -Cl, -F, -CN, -CO2H, -CO2R24, -CHO, -COR24, -CONH2, -CONHR24, -CON (R24)2, -COSH, -COSR24, -NO2, -SO3H, -SOR24 or -SO2R24, wherein each R24 is independently an alkyl optionally substituted with halogen, -OH or -SH.

[0229] In another additional embodiment, R15 is selected from the group consisting of:

[0230] In another additional embodiment, R15 is:

[0231] In another additional embodiment, R16, R17, R18, and R30 are each methyl.

[0232] In another additional embodiment, R16 is H, R17 is methyl, R18 is methyl, and R30 is methyl.

[0233] It is understood that any embodiment of the compounds of structure (Ia), as set out above, and any specific substituent established for a group R14, R15, R16, R17, R18, R19, R20 and R30 in the compounds of structure (Ia) , as defined above, and may be independently combined with other modalities and/or substituents of compounds of structure (I) to form modalities of the present disclosure not specifically defined above. Furthermore, in the case where a list of substituents is listed for any particular R14, R15, R16, R17, R18, R19, R20, and R30 in a particular embodiment and/or claim, it is understood that each individual substituent may be deleted from the specific embodiment and/or claim and the remaining list of substituents will be considered within the scope of the present disclosure.

[0234] In one embodiment, compounds with the following structure (Ib) are provided:

[0235] where:

[0236] R26 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl;

[0237] R27 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl;

[0238] R16 is selected from the group consisting of H and C1-6 alkyl;

[0239] R17 is selected from the group consisting of H and C1-6 alkyl; It is

[0240] R18 is selected from the group consisting of C 1-6 alkyl and -SH,

[0241] or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

[0242] In a further embodiment, each optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl is independently optionally substituted with =O, =S, -OH, - OR28OR28, -O2CR28, -SH, -SR28, -SOCR28, -NH2, -N3, -NHR28, -N(R28)2, - NHCOR28, -NR28COR28, -I, -Br, -Cl, -F, -CN , -CO2H, -CO2R28, -CHO, - COR28, -CONH2, -CONHR28, -CON(R28)2, -COSH, -COSR28, -NO2, -SO3H, - SOR28, or -SO2R28, where each R28 is , independently, alkyl optionally substituted with halogen, -OH or -SH.

[0243] In another additional embodiment, each optionally substituted aryl and optionally substituted heteroaryl is independently selected from the group consisting of optionally substituted phenyl, optionally substituted naphthyl, optionally substituted anthracyl, optionally substituted phenantryl, optionally substituted furyl, optionally substituted pyrrolyl, optionally substituted thiophenyl, optionally substituted benzofuryl, optionally substituted benzothiophenyl, optionally substituted quinolinyl, optionally substituted isoquinolinyl, optionally substituted imidazolyl, optionally substituted thiazolyl, optionally substituted oxazolyl, and optionally substituted pyridinyl.

[0244] In another additional embodiment, R27 is selected from one of the following structures (II), (III), (IV), (V):

[0245] where:

[0246] Q is CR29 or N;

[0247] Z is C(R29)2, NR29, S, or O;

[0248] each R29 is independently selected from the group consisting of H, -OH, -OR28OR28, -O2CR28, -SH, -SR28, -SOCR28, -NH2, - N3, -NHR28, -N(R28)2 , -NHCOR28, -NR28COR28, -I, -Br, -Cl, -F, -CN, -CO2H, -CO2R28, -CHO, -COR28, -CONH2, -CONHR28, -CON(R28)2, -COSH, - COSR28, -NO2, -SO3H, -SOR28 or -SO2R28, wherein each R28 is independently alkyl optionally substituted with halogen, -OH or -SH.

[0249] In another additional embodiment, R27 is selected from the group consisting of:

[0250] In another additional embodiment, R27 is:

[0251] In another additional embodiment, R16, R17 and R18 are each methyl.

[0252] In another additional embodiment, R16 is H, R17 is methyl, and R18 is methyl.

[0253] It is understood that any embodiment of the compounds of structure (Ib), as set out above, and any specific substituent established for a group R25, R26, R16, R17, R18, R18 and R20 in the compounds of structure (Ib), as defined above, and may be independently combined with other modalities and/or substituents of compounds of structure (I) to form modalities of the present disclosure not specifically defined above. Furthermore, in the case where a list of substituents is listed for any particular R25, R26, R16, R17, R18, R18 and R20 in a particular embodiment and/or claim, it is understood that each individual substituent may be deleted from the embodiment. specific and/or claim and the remaining list of substituents will be considered to be within the scope of the present disclosure.

[0254] In one embodiment, the invention provides a method of making a compound having structure (I), (Ia) or (Ib).

[0255] Conjugates Understanding New Compounds

[0256] Compounds with structure (I), (Ia) or (Ib) can be used to form conjugates, for example, antibody-drug conjugates (ADCs). Accordingly, in one embodiment of the present disclosure, conjugate compositions are provided, having the following structure:

[0257] (T)-(L)-(D)

[0258] (VI)

[0259] wherein (T) is a target moiety, (L) is an optional linker and (D) is a compound having structure (I), (Ia) or (Ib), below. In one embodiment, (T) is an antibody. In this sense, in one embodiment, antibody-drug conjugates (ADCs) consisting of compounds (D) with structure (I), (Ia) or (Ib), are provided.

[0260] As will be appreciated by those reasonably skilled in the art, a wide variety of means are available for covalently linking (T)-(L)-(D). Any known method can be used to connect the conjugate components. Any known linker technology can be used to link (T) to (D). Furthermore, (T), (L), and (D) may be modified appropriately, as recognized by those reasonably skilled in the art, in order to facilitate conjugate formation.

[0261] Targeting Fraction (T)

[0262] The Targeting Motion (T) of the compositions in question 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 cell population. determined target. (T) is a molecule that binds to, complexes with, or reacts with an active part of a cell population to be targeted. In one aspect, (T) acts to deliver the drug (D) to the specific targeted cell population with which it reacts (T). Such (T)s include, but are not limited to, large molecular weight proteins such as, for example, complete antibodies, antibody fragments, smaller molecular weight proteins, polypeptides or peptides, lectins, glycoproteins, non-peptides, vitamins, nutrient-transport molecules (such as, but not limited to, transferrin), or any other cell-binding molecule or substance.

[0263] (T) can form a bond to a linker (L) or drug (D) moiety. (T) can form a bond to a unit of (L) through a heteroatom of (T). Heteroatoms that may be present in (T) include sulfur (in one embodiment, from a sulfhydryl group of a (T)), oxygen (in one embodiment, from a carbonyl, carboxyl, or hydroxyl group of a (T)), and nitrogen ( in one embodiment, from a primary or secondary amino group of a (T)). Such heteroatoms may be present in (T) in the natural state of (T), for example a naturally occurring antibody, or may be introduced into (T) through chemical modification.

[0264] In one embodiment, a (T) has a sulfhydryl group and the bonds from (T) to (L) through the sulfur atom of the sulfhydryl group. In another embodiment, (T) has one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. (T) bonds to the (L) unit through the sulfhydryl group. Reagents that can be used to modify lysines include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-Iminothiolane hydrochloride (Traut's reagent).

[0265] In another embodiment, (L) may have one or more carbohydrate groups that may be chemically modified to have one or more sulfhydryl groups. (T) bonds to (L) through the sulfur atom of the sulfhydryl group. In another embodiment, (T) may have one or more carbohydrate groups that may be oxidized to provide an aldehyde group (--CHO) (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 in a portion of one (L). Reactive sites that can react with a carbonyl group of (T) include, but are not limited to, hydrazine and hydroxylamine. Other protocols for modifying proteins for binding or association of (D) are described in Coligan et al, Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002), incorporated herein by reference.

[0266] (T) may include, for example, a peptide, polypeptide or protein, but is not limited to transferrin, epidermal growth factors ("EGF"), bombesin, gastrin, gastrin-releasing peptide, growth factor derived from platelets, IL-2, IL-6, transforming growth factor ("TGF") such as TGF-α or TGF-β, vaccinia growth factor ("VGF"), insulin and insulin-like growth factors insulin I and II, lectins and low-density lipoprotein apoprotein.

[0267] (T) may also include an antibody, such as polyclonal antibodies or monoclonal antibodies. The antibody may be directed to a particular antigenic determinant, including, 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 can be prepared using any technique 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 Todai 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 Lymphocite Antibody Method (SLAM) (Babcook, J.S., et al., A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. Proc Natl Acad Sci U S A, 1996. 93 (15): p. 7843-8.) and (McLean GR, Olsen OA, Watt IN, Ratanaswami P, Leslie KB, Babcook JS, Scrader JW. Recognition of human cytomegalovirus bi human primari immunoglobulins identifies an innate finding in an adaptive immune response. J Immunol. 2005 Apr 15;174(8):4768-78. Such antibodies may be of any class of immunoglobulin including IgG, IgM, IgE, IgA and IgD and any subclass thereof. Hybridomas producing the mAbs of use in this invention may be cultured in vitro or in alive.

[0268] The monoclonal antibody may be, for example, a human monoclonal antibody, a humanized monoclonal antibody, an antibody fragment or a chimeric antibody (e.g., a human-mouse antibody). Human monoclonal antibodies can be made by any of a number of techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; and Olsson et al., 1982, Met. Enzimol. 92:3-16). see also, Huse et al., 1989, Science 246:1275-1281 and McLean et al. J Immunol. 2005 Apr 15;174(8):4768-78.

[0269] The antibody can also be a bispecific antibody. Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin light chain-heavy chain pairs, where the two chains have different specificities (see, for example, Milstein et al., 1983, Nature 305: 537-539; International Publication No. WO 93/08829, Traunecker et al., 1991, EMBO J. 10:36553659.

[0270] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused into the immunoglobulin constant domain sequences. The fusion is preferably with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy chain constant region (CH1) containing the site necessary for light chain binding present in at least one of the fusions. Nucleic acids with sequences encoding immunoglobulin heavy chain fusions and, if desired, immunoglobulin light chain, are inserted into separate expression vectors and are co-transfected into a suitable host organism. This provides flexibility in adjusting the mutual ratios of three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide optimal yield. However, it is possible to insert coding sequences for two or all three polypeptide chains into an expression vector when expressing at least two polypeptide chains in equal ratios results in high yields or when the ratios are not of any particular significance.

[0271] For example, bispecific antibodies may have a hybrid immunoglobulin heavy chain with a first binding specificity on one arm and a hybrid immunoglobulin light chain-heavy chain pair (providing a second binding specificity) on the other. arm. This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides a form of separation (International Publication No. Wo 94 /04690) which is incorporated herein by reference in its entirety.

[0272] For more details on the generation of bi-specific antibodies see, for example, Suresh et al., 1986, Methods in Enzimologi 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. Using these techniques, bispecific antibodies can be prepared for use in treating or preventing diseases as defined herein.

[0273] Bifunctional antibodies are also described in European Patent Publication No. EPA 0 105 360. As disclosed in this reference, hybrid or bifunctional antibodies can be derived biologically, i.e., cell fusion techniques, or chemically, especially with cross-linking agents or reagents. forming a disulfide bond and may include entire antibodies or fragments thereof. Methods for obtaining such hybrid antibodies are disclosed, for example, in International Publication WO 83/03679 and European Patent Publication No. EPA 0 217 577, both of which are incorporated into the addendum by reference.

[0274] The antibody may also be a functionally active fragment, derivative or analog of an antibody that immunospecifically binds to a target antigen (e.g., 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 analogue is capable of recognizing the same antigen as the antibody from which the recognized fragment, derivative or analogue is derived. Specifically, in an exemplary embodiment the antigenicity of the idiotype of the immunoglobulin molecule can be enhanced by deleting structure and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind to the antigen, synthetic peptides containing the CDR sequences can be used in antigen binding assays by any binding assay method known in the art (e.g., the BIA core assay) ( see, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., National Institute of Healt, Bethesda, Md.; Kabat et al., 1980, J. Immunology125(3):961-969 ).

[0275] Other useful antibodies include antibody fragments, such as, but not limited to, F(ab')2 fragments, Fab fragments, Fab', Fv fragments and antibody heavy chain and light chain dimers, or any minimal fragment such as Fvs or single-chain antibodies (SCAs) (e.g., as described in U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Acad Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54).

[0276] Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising human and non-human portions can be made using standard recombinant DNA techniques. (See, for example, U.S. Pat. No. 4,816,567; and U.S. Pat. No. 4,816,397.) Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) of non-human species and a framework region of a human immunoglobulin molecule. (See, for example, U.S. Pat. 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 Publication No. WO 02671/87; European Patent Publication No. 0 184 187; European Patent Publication No. 0 171 496; European Patent Publication No. 0 173 494; International publication No. WO 01533/86; PAT. U.S. No. 4,816,567; European Patent Publication No. 012 023; Berter et al, 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Academic. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al.,1987, Proc. Natl. Academic. Sci USA 84:214-218; Nishimura et al., 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; Hi et al., 1986, BioTechniques 4:214; U.S. Pat. No. 5,225,539; 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.

[0277] Fully human antibodies can be used. Human antibodies can be prepared, for example, using transgenic mice, which are unable to express endogenous immunoglobulin heavy and light chain genes, but which can express human light and heavy chain genes. Transgenic mice are immunized in the normal way with a selected antigen, for example, all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. Human immunoglobulin transgenes harbored by transgenic mice rearrange during B cell differentiation and subsequently undergo somatic mutation and class switching. Thus, using such a technique, it is possible to produce therapeutically useful IgG and IgA, IgM, IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for the production of human antibodies and human monoclonal antibodies and protocols for the production of such antibodies see, for example, U.S. Pat. U.S. Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806.

[0278] Human antibodies that recognize a selected epitope can also 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 fully human antibody recognizing the same epitope. (See, for example, Jespers et al., 1994, Biotechnology 12:899-903.) Human antibodies can also be produced using various techniques known in the art, including 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-IgE and Allergic Disease, Jardieu, P. M. and Fick Jr., R. B, eds., Marcel Dekker, New York, N.Y., Chapter 20, pp. 427-469).

[0279] In other embodiments, the antibody is a fusion protein of an antibody, or a functionally active fragment thereof. For example, an antibody may be fused via a covalent bond (e.g., a peptide bond) at the N-terminus or C-terminus to an amino acid sequence of another protein (or part thereof, such as at least a portion of 10, 20 or 50 amino acids of the protein) that is not the antibody.

[0280] Antibodies also include analogues and derivatives that are also modified, that is, by the covalent attachment of any type of molecule, although such covalent attachment allows the antibody to retain its antigen-binding immunospecificity. For example, but not by way of limitation, derivatives and analogues of antibodies include those that have been further modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by protecting groups/ blocking, proteolytic cleavage, binding to a cellular unit of antibody or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin etc. Additionally, the analogue or derivative may contain one or more unnatural amino acids.

[0281] Antibodies may have modifications (e.g., substitutions, deletions or additions) in amino acid residues that interact with Fc receptors. In particular, antibodies include antibodies having modifications to amino acid residues identified as involved in the interaction between the anti-Fc domain and the FcRn receptor (see, for example, international publication no. Wo 97/34631, which is incorporated herein document by reference, in its entirety). Immunospecific antibodies to a target antigen may be obtained commercially or from another source or produced by any method known to those skilled in the art, such as, for example, recombinant expression techniques or chemical synthesis. The nucleotide sequence encoding immunospecific antibodies to a cancer cell antigen can be obtained, for example, from the GenBank database or a similar database, literature publications, or by routine cloning and sequencing.

[0282] Examples of antibodies available for the treatment of cancer include, but are not limited to, humanized anti HER2 monoclonal antibody, HERCEPTIN® (trastuzumab; Genentech); RITUXAN® (rituximab; Genentech) which is a monoclonal chimeric anti-CD20 antibody for the treatment of patients with non-Hodgkin's lymphoma; OvaRex (AltaRex Corporation, MA), which is a murine antibody for the treatment of ovarian cancer; Panorex (Glaxo Wellcome, NC), which is a murine IgG2a antibody for the treatment of colorectal cancer; Cetuximab Erbitux (Imclone Systems Inc., N.Y.) which is an anti-EGFR IgG chimeric antibody for the treatment of epidermal growth factor positive cancers such as head and neck cancer; Vitaxin (MedImmune, Inc., MD), which is a humanized antibody for the treatment of sarcoma; Campat Y/H (Leukositis, MA), which is a humanized IgG1 antibody for the treatment of chronic lymphocytic leukemia (CLL); Smart MI95 (Protein Design Labs, Inc., CA) which is a humanized anti-CD33 IgG antibody for the treatment of acute myeloid leukemia (AML); LymphoCide (Immunomedics, Inc., NJ), which is a humanized anti-CD22 IgG antibody for the treatment of non-Hodgkin's lymphoma; Smart ID10 (Protein Design Labs, Inc., CA) which is a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma; Oncolim (Techniclone, Inc., CA), which is a murine anti-HLA-Dr10 radiolabeled antibody for the treatment of non-Hodgkin's lymphoma; Allomune (BioTransplant, CA), which is a humanized anti-CD2 mAb for the treatment of Hodgkin's disease or non-Hodgkin's lymphoma; Avastin (Genentech, Inc., CA), which is a humanized anti-VEGF antibody for the treatment of lung and colorectal cancer; Epratuzamab (Immunomedics, Inc., NJ and Amgen, CA), which is an anti-CD22 antibody for the treatment of non-Hodgkin lymphoma; and CEAcide (Immunomedics, NJ), which is a humanized anti-CEA antibody for the treatment of colorectal cancer.

[0283] Other antibodies useful in treating cancer include, but are not limited to, antibodies against the following antigens (exemplary cancers are indicated in parentheses): 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), gp100 (melanoma), MART1 (melanoma), prostate-specific antigen (PSA) (prostate), IL-2 Receptor (leukemia T-cell and lymphoma), 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). Some useful specific antibodies include, but are not limited to, mAb BR96 (Trail et al., 1993, Science 261:212-215), BR64 (Trail et al., 1997, Cancer Research 57:100105), mAbs against CD40 antigen, such as mAb S2C6 (Francisco et al., 2000, Cancer Res. 60:3225-3231) and chimeric and humanized variants thereof, mabs against the cD33 antigen; mAbs against the EfA2 antigen; mAbs against the CD70 antigen, such as mAb 1F6 and mAb 2F2 and chimeric and humanized variants, 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 humanized and chimeric 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 Bioter. Radiofarm. 15:459 76; Murray, 2000, Semin. Oncol. 27: 64 70; Breitling et al., Recombinant Antibodies, John Wiley, and Sons, New York, 1998).

[0284] The antibody can also be an antibody that binds to an antigen that is present on a target cell or target cell population. For example, transmembrane polypeptides and other markers can be specifically expressed on the surface of one or more of certain target cell types (e.g., a cancer cell) compared to one or more normal cells (e.g., a non-cancerous cell). Often, such markers are more abundantly expressed on the surface of target cells, or exhibit greater immunogenicity compared to those on the surface of normal cells. The identification of such cell surface antigen polypeptides has given rise to the ability to specifically target target cells for destruction through antibody-based therapies. Thus, in some embodiments, the antibodies include, but are not limited to, antibodies against tumor-associated antigens (TAA). These tumor-associated antigens are known in the art and prepared for use in generating antibodies using methods and information that are well known in the art.

[0285] See also EP2552957, WO/2012/116453, WO/2012/032080. See also Zybody™, http://www.zyngenia.com/technology.html. See also heavy chain-only human antibody technology, http://www.crescendobiologics.com/. See also WO2010001251, Human yeast-based antibody yeast-based platform http://www.adimab.com/science-e-technology/technology-overview/, mAbLogix™ platform http://www.dna.com/technology , monoclonal discovery platform http://www.igenica.com/technology/, WO2009/157771, EP2560993, WO2013004842, WO2012166560.

[0286] Linker Fraction (L)

[0287] The compositions in question further optionally include a linker moiety (L). (L) is a bifunctional compound that can be used to link a (D) and a (T) to form a conjugated composition, T-L-D. Such conjugates allow selective delivery of drugs to target cells (e.g., tumor cells). (L)s include a divalent substituent such as an alkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as: -- (CR2)nO(CR2)n--, alkyloxy repeating units (e.g., polyethyleneoxy, PEG, polymethylene-oxy ) and alkylamino (e.g. polyethyleneamino, Jeffamine™); and diacid ester and amides including succinate, succinamide, diglycolate, malonate, and caproamide.

[0288] The compositions in question can be prepared using a unit of (L), having a reactive site for binding with (D) and (T). In some embodiments, (L) has a reactive site, which has an electrophilic group that is reactive to a nucleophilic group present in (T). Useful nucleophilic groups in (T) include but are not limited to groups. sulfhydryl, hydroxyl and amino acids The heteroatom of the nucleophilic group of (T) is reactive towards an electrophilic group (L) and forms a covalent bond to (L). Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups. The nucleophilic group on (T) provides a convenient site for attachment to (L).

[0289] In another embodiment, (L) has a reactive site, which has an electrophilic group that is reactive to a nucleophilic group present in (T). Useful electrophilic groups in (T) include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of the nucleophilic group of (L) can react with an electrophilic group of (T) and form a covalent bond to (T). Useful nucleophilic groups in a (L) include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. The electrophilic group at (T) provides a convenient location for attachment to (L).

[0290] Carboxylic acid functional groups and chloroformate functional groups are also useful reactive sites for (L) because they can react with amino acid groups of (D) to form an amide bond. Also useful as a reactive site is a carbonate functional group on (L), such as but not limited to p-nitrophenyl carbonate, which reacts with an amino group on (D) to form a carbamate bond.

[0291] It will be appreciated that any connector parts taught in the prior art, and particularly those that have taught use in the context of drug administration, can be used in the current invention. Without limiting the scope of the previous statement, in one embodiment, (L) comprises a linker moiety disclosed in 2012 WO/113847. In another embodiment, (L) comprises a linker moiety disclosed in US 8,288,352. In another embodiment, (L) comprises a linker moiety disclosed in US 5,028,697. In another embodiment, (L) comprises a linker moiety disclosed in US 5,006,652. In another embodiment, (L) comprises a linker moiety disclosed in US 5,094,849. In another embodiment, (L) comprises a linker moiety disclosed in US 5,053,394. In another embodiment, (L) comprises a linker moiety disclosed in US 5,122,368. In another embodiment, (L) comprises a disclosed linker moiety disclosed in US 5,387,578. In another embodiment, (L) comprises a linker moiety disclosed in US 5,547,667. In another embodiment, (L) comprises a disclosed linker moiety disclosed at 5,622,929. In another embodiment, (L) comprises a linker moiety disclosed in US 5,708,146. In another embodiment, (L) comprises a disclosed linker moiety disclosed at 6,468,522. In another embodiment, (L) comprises a linker moiety disclosed in US 6,103,236. In another embodiment, (L) comprises a disclosed linker moiety disclosed in 6,638,509. In another embodiment, (L) comprises a linker moiety disclosed in US 6,214,345. In another embodiment, (L) comprises a disclosed linker moiety disclosed at 6,759,509. In another embodiment, (L) comprises a linker moiety disclosed in WO 2007/103288. In another embodiment, (L) comprises a linker moiety disclosed in WO 2008/083312. In another embodiment, (L) comprises a linker moiety disclosed in WO 2003/068144. In another embodiment, (L) comprises a linker moiety disclosed in WO 2004/016801. In another embodiment, (L) comprises a linker moiety disclosed in WO 2009/134976. In another embodiment, (L) comprises a linker moiety disclosed in WO 2009/134952. In another embodiment, (L) comprises a linker moiety disclosed in WO 2009/134977. In another embodiment, (L) comprises a linker moiety disclosed in WO 2002/08180. In another embodiment, (L) comprises a linker moiety disclosed in WO 2004/043493. In another embodiment, (L) comprises a linker moiety disclosed in WO 2007/018431. In another embodiment, (L) comprises a linker moiety disclosed in WO 2003/026577. In another embodiment, (L) comprises a linker moiety disclosed in WO 2005/077090. In another embodiment, (L) comprises a linker moiety disclosed in WO 2005/082023. In another embodiment, (L) comprises a linker moiety disclosed in WO 2007/011968. In another embodiment, (L) comprises a linker moiety disclosed in WO 2007/038658. In another embodiment, (L) comprises a linker moiety disclosed in WO 2007/059404. In another embodiment, (L) comprises a linker moiety disclosed in WO 2006/110476. In another embodiment, (L) comprises a linker moiety disclosed in WO 2005/112919. In another embodiment, (L) comprises a linker moiety disclosed in WO 2008/103693. In another embodiment, (L) comprises a disclosed linker moiety disclosed at 6,756,037. In another embodiment, (L) comprises a linker moiety disclosed in US 7,087,229. In another embodiment, (L) comprises a linker moiety disclosed in US 7,122,189. In another embodiment, (L) comprises a linker moiety disclosed in US 7,332,164. In another embodiment, (L) comprises a disclosed linker moiety disclosed at 5,556,623. In another embodiment, (L) comprises a disclosed linker moiety disclosed in 5,643,573. In another embodiment, (L) comprises a disclosed linker moiety disclosed at 5,665,358.

[0292] Ligands (L) comprising self-immolative component can also be used. See, for example Pat. U.S. No. 6,214,345. An example of a self-immolative component is p-aminobenzylcarbamoyl (PABC).

[0293] Commercially available binders can be used in the invention. For example, the commercially available cleavable linker sulfosuccinimidyl 6-[3'(2-pyridyldithio)-propionamido]hexanoate (sulfo-LC-SPDP: term Pierce Cat# 21650) and non-cleavable linker succinimidyl 4-[N-maleimidomethyl]cyclohexane- 1-carboxylate (SMCC: Pierce term Cat# 22360) can be used, as demonstrated in this document.

[0294] See also, WO2012171020, WO2010138719, the range of commercially available binders, for example, from Concortis http://www.concortis.com/home. See also Kim et al., BIOCONJUGATE CHEmistry, 21 (8): 1513-1519 AUGUST 2010. See also EP2326349. See also copper-free click chemistry ligands, Angew.Chem. int. Ed., 2010, 49, p. 9422-9425, ChemBioChem, 2011, 12, p. 1309- 1312,-http://www.sinaffix.com/technology/.

Drug Fraction (D)

[0295] (D) is a compound with structure (I), (Ia) or (Ib), as described in this document. It will be recognized by one reasonably skilled in the art that compounds of structure (I), (Ia) or (Ib), can be modified in the appropriate manner to facilitate a conjugation reaction with (L), or if (L) is not present, with (T) and the formation of a conjugate (T)-(L)-(D) or (T)-(D). Any attachment point in (D) can be used. In one embodiment, the C-terminus of (D) constitutes the attachment point in a (T)-(L)-(D) conjugate. In another embodiment, N-terminus of (D) constitutes the attachment point in a (T)-(L)-(D) conjugate. In another embodiment, a side chain of (D) constitutes the attachment point in a (T)-(L)-(D) conjugate.

Novel Conjugates Comprising Microtubule-Disrupting Peptide Toxins

[0296] In one embodiment of the present disclosure, conjugates comprising microtubule-disrupting peptide toxins covalently linked in the conjugate through the N-terminal amino acid side chain are provided. In one embodiment, the microtubule-disrupting peptide toxin is hemiasterlin or an analogue thereof and the toxin is covalently linked in the conjugate through the indole moiety within the N-terminal amino acid side chain of the peptide toxin. In another embodiment, the microtubule-disrupting peptide toxin is HTI-286 or an analogue thereof and the toxin is covalently linked in the conjugate through the phenyl group within the N-terminal amino acid side chain of the peptide toxin. In one embodiment, the microtubule-disrupting peptide toxin is a compound having structure (I), (Ia) or (Ib), as disclosed herein.

[0297] The compositions in question have anti-mitotic activity and the following structure: (T)-(L)-(PT) (VII)

[0298] wherein (T) is a targeting moiety as described herein, (L) is an optional linker as described herein, and (PT) is a microtubule-disrupting peptide toxin covalently linked to (L) across the chain side chain of the N-terminal amino acid of (PT), or if there is no (L), (PT) is covalently linked to (T) through the side chain of the N-terminal amino acid of (PT).

[0299] In one embodiment, (T) is an antibody. In this regard, in one embodiment, antibody-drug conjugates (ADCs) comprising microtubule-disrupting peptide toxins that are linked to the conjugate through the N-terminal amino acid side chain are provided.

[0300] In one embodiment, (T)-(L)-(PT) has the following structure:

[0301] where,

[0302] R1 and R2 are each independently selected from the group consisting of: H and a saturated or unsaturated moiety having a non-aromatic linear, branched or cyclic skeleton, containing from 1 to 10 carbon atoms and the carbon atoms are optionally , replaced by: -OH, -I, -Br, -Cl, -F, -CN, -CO2H, -CHO, -COSH, or -NO2;

[0303] R3 and R4 are each independently selected from the group consisting of: H, R, ArR-, or R3 and R4 are joined to form a ring;

[0304] R31 is selected from the group consisting of: H, R', ArR-, Ar-R-Ar, R-Ar-Ar, Ar-Ar-R-, and Ar, wherein each R and each Ar can be substituted, and from zero to ten heteroatoms can replace carbon atoms in the chain, for example O or S or N can be incorporated into the carbon chain; in a modality in which R' is where m is an integer from one to fifteen;

[0305] R6 is selected from the group consisting of: H, R, and ArR-;

[0306] R7 and R8 are each independently selected from the group consisting of: H, R, and ArR-; It is

[0307] R32 is selected from:

[0308] wherein,

[0309] Z is defined as a fraction selected from the group consisting of: -OH, -OR; -SH; -SR; -NH2; -NRCH(R11)COOH; and - NHCH(R11)COOH, where R11 is a fraction with the formula: R, or - (CH2)nNR12R13, where n=1-4 and R12 and R13 are independently selected from the group consisting of: H; R; and -C(NH)(NH2),

[0310] Y is defined as a fraction selected from the group consisting of: a linear alkyl group, saturated or unsaturated, with one to six carbons, optionally substituted with R, ArR—, or X; It is,

[0311] 2, —NHCOR, —NRCOR, —I, —Br, —Cl, —F, —CN, —CO2H, —CO2R, —CHO, —COR, —CONH2, —CONHR, —CON(R)2, —COSH , —COSR, —NO2, —SO3H, —SOR, and —SO2R;

[0312] R14 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl, COR24, -CSR24, -OR24, and -NHR24, wherein each R24 is independently alkyl optionally substituted with halogen, -OH or -SH;

[0313] R is defined as a saturated or unsaturated fraction having a non-aromatic linear, branched or cyclic skeleton, containing from 1 to 10 carbon atoms, zero to four nitrogen atoms, zero to four oxygen atoms and zero to four oxygen atoms. sulfur, and the carbon atoms are optionally replaced by: =O, =S, OH, -OR10, -O2CR10, -SH, -SR10, -SOCR10, -NH2, -NHR10, -N(R10)2 , -NHCOR10, - NR10COR10, -I, -Br, -Cl, -F, -CN, -CO2H, -CO2R10, -CHO, -COR10, -CONH2, - CONHR10, -CON(R10)2, -COSH, -COSR10, -NO2, -SO3H, -SOR10, -SO2R10, where R10 is a saturated or unsaturated, branched or cyclic linear alkyl group of one to ten carbons;

[0314] the ring formed by joining R3 and R4 is a cyclic skeleton of three to seven non-aromatic members within the definition of R,

[0315] Y is defined as a fraction selected from the group consisting of: a linear alkyl group, saturated or unsaturated, with one to six carbons, optionally substituted with R, ArR—, or X; It is,

[0316] 2, —NHCOR, —NRCOR, —I, —Br, —Cl, —F, —CN, —CO2H, —CO2R, —CHO, —COR, —CONH2, —CONHR, —CON(R)2, —COSH , —COSR, —NO2, —SO3H, —SOR, and —SO2R;

[0317] or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

[0318] In one embodiment, Ar is an aromatic ring selected from the group consisting of: phenyl, naphthyl, anthracyl, pyrrolyl.

[0319] In one embodiment, R32 is:

[0320] where Z and Y are as defined above.

[0321] In one embodiment, R32 is:

[0322] where Z and R14 are as defined above.

[0323] In one embodiment, R32 is:

[0324] where Y and R14 are as defined above.

[0325] In another embodiment, (T)-(L)-(PT) has the following structure:

[0326] in which,

[0327] R15 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl;

[0328] R16 is selected from the group consisting of H and C1-6 alkyl;

[0329] R17 is selected from the group consisting of H and C1-6 alkyl;

[0330] R18 and R30 are independently selected from the group consisting of H, C1-6 alkyl and -SH, with the exception that both R18 and R30 substituents cannot be H;

[0331] R32 is selected from:

[0332] in which,

[0333] Z is defined as a fraction selected from the group consisting of: -OH, -OR; -SH; -SR; -NH2; -NRCH(R11)COOH; and -NHCH(R11)COOH, where R11 is a fraction with the formula: R, or -(CH2)nNR12R13, where n=1-4 and R12 and R13 are independently selected from the group consisting of: H; R; and -C(NH)(NH2),

[0334] R is defined as a saturated or unsaturated fraction having a non-aromatic linear, branched or cyclic skeleton, containing from 1 to 10 carbon atoms, zero to four nitrogen atoms, zero to four oxygen atoms and zero to four oxygen atoms. sulfur, and the carbon atoms are optionally replaced by: =O, =S, OH, -OR10, -O2CR10, -SH, -SR10, -SOCR10, -NH2, -NHR10, -N(R10)2 , -NHCOR10, - NR10COR10, -I, -Br, -Cl, -F, -CN, -CO2H, -CO2R10, -CHO, -COR10, -CONH2, - CONHR10, -CON(R10)2, -COSH, -COSR10, -NO2, -SO3H, -SOR10, -SO2R10, where R10 is a saturated or unsaturated, branched or cyclic linear alkyl group of one to ten carbons;

[0335] the ring formed by joining R3 and R4 is a cyclic skeleton of three to seven non-aromatic members within the definition of R,

[0336] Y is defined as a fraction selected from the group consisting of: a linear, saturated or unsaturated alkyl group, with one to six carbons, optionally substituted with R, ArR—, or X; It is,

[0337] 2, —NHCOR, —NRCOR, —I, —Br, —Cl, —F, —CN, —CO2H, —CO2R, —CHO, —COR, —CONH2, —CONHR, —CON(R)2, —COSH , —COSR, —NO2, —SO3H, —SOR, and —SO2R;

[0338] or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

[0339] In another embodiment, (T)-(L)-(PT) has the following structure:

[0340] in which,

[0341] R15 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl;

[0342] R16 is selected from the group consisting of H and C1-6 alkyl;

[0343] R17 is selected from the group consisting of H and C1-6 alkyl;

[0344] R18 and R30 are independently selected from the group consisting of H, C1-6 alkyl and -SH, with the exception that both R18 and R30 substituents cannot be H;

[0345] R33 is:

[0346] in which,

[0347] Z is as defined above,

[0348] R is defined as a saturated or unsaturated fraction having a non-aromatic linear, branched or cyclic skeleton, containing from 1 to 10 carbon atoms, zero to four nitrogen atoms, zero to four oxygen atoms and zero to four oxygen atoms. sulfur, and the carbon atoms are optionally replaced by: =O, =S, OH, -OR10, -O2CR10, -SH, -SR10, -SOCR10, -NH2, -NHR10, -N(R10)2 , -NHCOR10, -NR10COR10, -I, - Br, -Cl, -F, -CN, -CO2H, -CO2R10, -CHO, -COR10, -CONH2, -CONHR10, - CON(R10)2, -COSH, -COSR10, -NO2, -SO3H, -SOR10, -SO2R10, where R10 is a saturated or unsaturated, branched or cyclic linear alkyl group of one to ten carbons;

[0349] The ring formed by joining R3 and R4 is a cyclic skeleton of three to seven non-aromatic members within the definition of R,

[0350] Y is defined as a fraction selected from the group consisting of: a linear alkyl group, saturated or unsaturated, with one to six carbons, optionally substituted with R, ArR—, or X; It is,

[0351] 2, —NHCOR, —NRCOR, —I, —Br, —Cl, —F, —CN, —CO2H, —CO2R, —CHO, —COR, —CONH2, —CONHR, —CON(R)2, —COSH , —COSR, —NO2, —SO3H, —SOR, and —SO2R;

[0352] or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

[0353] In another embodiment, (T)-(L)-(PT) has the following structure:

[0354] in which,

[0355] R14 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl, -COR24, -CSR24, -OR24, -SR24, and -NHR24, wherein each R24 is independently alkyl optionally substituted with halogen, -OH or -SH;

[0356] R15 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl;

[0357] R16 is selected from the group consisting of H and C1-6 alkyl;

[0358] R17 is selected from the group consisting of H and C1-6 alkyl;

[0359] R18 and R30 are independently selected from the group consisting of H, C1-6 alkyl and -SH, with the exception that both R18 and R30 substituents cannot be H;

[0360] or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

[0361] In another embodiment, (T)-(L)-(PT) has the following structure:

[0362] in which,

[0363] R14 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl, -COR24, -CSR24, -OR24, -SR24, and -NHR24, wherein each R24 is independently alkyl optionally substituted with halogen, -OH or -SH;

[0364] R15 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl;

[0365] R16 is selected from the group consisting of H and C1-6 alkyl;

[0366] R17 is selected from the group consisting of H and C1-6 alkyl;

[0367] R18 and R30 are independently selected from the group consisting of H, C1-6 alkyl and -SH, with the exception that both R18 and R30 substituents cannot be H;

[0368] or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

[0369] In another embodiment, (T)-(L)-(PT) has the following structure:

[0370] in which,

[0371] R15 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl;

[0372] or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

[0373] In another embodiment, (T)-(L)-(PT) has the following structure:

[0374] in which,

[0375] R is defined as a saturated or unsaturated fraction having a non-aromatic linear, branched or cyclic skeleton, containing from 1 to 10 carbon atoms, zero to four nitrogen atoms, zero to four oxygen atoms and zero to four oxygen atoms. sulfur, and the carbon atoms are optionally replaced by: =O, =S, OH, -OR10, -O2CR10, -SH, -SR10, -SOCR10, -NH2, -NHR10, -N(R10)2 , -NHCOR10, -NR10COR10, -I, - Br, -Cl, -F, -CN, -CO2H, -CO2R10, -CHO, -COR10, -CONH2, -CONHR10, - CON(R10)2, -COSH, -COSR10, -NO2, -SO3H, -SOR10, -SO2R10, where R10 is a saturated or unsaturated, branched or cyclic linear alkyl group of one to ten carbons;

[0376] or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

[0377] In another embodiment, (T)-(L)-(PT) has the following structure:

[0378] in which,

[0379] R is defined as a saturated or unsaturated fraction having a non-aromatic linear, branched or cyclic skeleton, containing from 1 to 10 carbon atoms, zero to four nitrogen atoms, zero to four oxygen atoms and zero to four oxygen atoms. sulfur, and the carbon atoms are optionally replaced by: =O, =S, OH, -OR10, -O2CR10, -SH, -SR10, -SOCR10, -NH2, -NHR10, -N(R10)2 , -NHCOR10, - NR10COR10, -I, -Br, -Cl, -F, -CN, -CO2H, -CO2R10, -CHO, -COR10, -CONH2, - CONHR10, -CON(R10)2, -COSH, -COSR10, -NO2, -SO3H, -SOR10, -SO2R10, where R10 is a saturated or unsaturated, branched or cyclic linear alkyl group of one to ten carbons;

[0380] or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

[0381] In another embodiment of the invention, (PT) is a hemisterlin analog, such as those disclosed in U.S. 7,579,323, which is hereby incorporated by reference, in its entirety for all purposes.

[0382] In synthesizing conjugates, including ADCs, comprising microtubule-disrupting peptide toxins, peptide bonding through the N-terminal amino acid side chain has several advantages. As demonstrated herein, the side chains of such peptide toxins are amenable to chemical modifications and manipulations that facilitate the formation of covalently linked conjugates without compromising potency. As demonstrated herein, such conjugates are potent cytotoxic compositions capable of delivering a peptide toxin payload.

Administration

[0383] For administration purposes, the compounds of the present disclosure can be administered as a raw chemical or can be formulated as pharmaceutical compositions. Pharmaceutical compositions of the present disclosure comprise a compound of structure (I), (Ia) or (Ib) and a pharmaceutically acceptable carrier, diluent or excipient. The compound of structure (I), (Ia) or (Ib) is present in the composition in an amount that is effective to treat a particular disease or condition of interest - for example, in an amount sufficient to treat cancer or tumor cell growth and preferably with acceptable toxicity for the patient. The activity of compounds of structure (I), (Ia) or (Ib) can be determined by one skilled in the art, for example, as described in the examples below. Suitable dosages and concentrations can be readily determined by those skilled in the art.

[0384] Administration of the compounds of the disclosure, or pharmaceutically acceptable salts thereof, in pure form or in an appropriate pharmaceutical composition, can be carried out through any of the accepted modes of administering agents to serve similar utilities. The pharmaceutical compositions of the disclosure may be prepared by combining a compound of the disclosure with an appropriate pharmaceutically acceptable carrier, diluent or excipient and may be formulated into preparations 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 administration of 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, intrasternal, intramuscular, intravenous infusion or injection techniques. Pharmaceutical compositions of the disclosure 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 subject or patient take the form of one or more dosage units, where, for example, a tablet may be a single dosage unit, and a container of a compound of the disclosure 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 the art; for example, see Remington: The Science and Practice of Pharmacy, 20th Ed. (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered must, in any event, contain a therapeutically effective amount of a compound of disclosure, or a pharmaceutically acceptable salt thereof, for treating a disease or condition of interest in accordance with the teachings of this disclosure.

[0385] The pharmaceutical composition of the disclosure may be in the form of a solid or liquid. In one aspect, the carrier(s) are particulate, such that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid(s), with the compositions being, for example, an oral syrup, an injectable liquid or an aerosol, which is useful, for example, in administration by inhalation.

[0386] When intended for oral administration, the pharmaceutical compositions of the present disclosure may be in either solid or liquid form, where semi-solid suspension, semi-liquid, and gel forms are included within the forms considered herein as either solid or liquids.

[0387] As a solid composition for oral administration, the pharmaceutical composition can be formulated in the form of a powder, granule, tablet, pill, capsule, chewing gum, lozenge or similar forms. 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 carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, gum 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 flavor; and a coloring agent.

[0388] 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 a polyethylene glycol or oil.

[0389] The pharmaceutical compositions of the disclosure may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid can be for oral administration or for delivery by injection, as two examples. When intended for oral administration, pharmaceutical compositions of the disclosure may contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/color 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.

[0390] The liquid pharmaceutical compositions of the disclosure, whether solutions, suspensions or other similar forms, may include one or more of the following adjuvants: diluents sterilized with water for injection, saline solution, preferably saline, Ringer's solution, sodium chloride isotonic, fixed oils such as mono- or diglycerides that can serve as the solvent or suspension medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; stabilizers such as amino acids; surfactants such as polysorbates; 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; or agents for adjusting tonicity such as sodium chloride or dextrose. Parenteral preparations can be placed in ampoules, disposable syringes or multi-dose vials made of glass or plastic. Saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

[0391] A liquid pharmaceutical composition of the disclosure intended for parenteral or oral administration must contain an amount of a compound of the disclosure such that a suitable dosage will be obtained.

[0392] The pharmaceutical composition of the disclosure may be intended for topical administration, in which case the carrier may suitably comprise a base solution, emulsion, ointment or gel. The base, for example, may comprise one or more of the following: petroleum jelly, 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.

[0393] The pharmaceutical composition of the disclosure may be intended 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 non-irritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.

[0394] Pharmaceutical compositions of the disclosure 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 capsule coating around the active ingredients. The materials forming the coating capsule are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients can be encased in a gelatin capsule.

[0395] Pharmaceutical compositions of the disclosure can 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 a colloidal nature to systems consisting of pressurized packages. Delivery can be by a compressed or liquefied gas or by a suitable pump system that dispenses the active ingredients. Aerosols of compounds of the disclosure may be delivered in single-phase, two-phase, or three-phase 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. One skilled in the art, without undue experimentation, can determine the preferred aerosols.

[0396] The pharmaceutical compositions of the disclosure can be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection may be prepared by combining a compound of the disclosure with sterile distilled water to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that interact noncovalently with the compound of the disclosure so as to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system.

[0397] The compositions of this disclosure, or their pharmaceutically acceptable salts or solvates, are administered in a therapeutically effective amount that will vary depending on a variety of factors, including the activity of the specific compound employed, the metabolic stability and duration of action of the compound, the patient's age, body weight, general health, gender, diet; the mode and time of administration, rate of excretion, combination of drugs, the severity of the particular condition or disorder and the patient undergoing therapy.

[0398] Compounds of the disclosure, or pharmaceutically acceptable derivatives thereof, may be administered simultaneously, prior to, or subsequent to the administration of one or more therapeutic agents. Such combinatorial therapy includes administration of a single pharmaceutical dose formulation which contains a compound of the present disclosure and one or more additional active agents, as well as administration of the compound of the disclosure and each active agent in its separate pharmaceutical dose formulation. For example, a compound of the disclosure and the other active agent may be administered to the patient together in a single oral dosage composition, such as a tablet or capsule, or each agent may be administered in separate oral pharmaceutical formulations. Where distinct dosage formulations are used, the compounds of the disclosure and one or more additional active agents may be administered essentially simultaneously, that is, simultaneously or at separately staggered times, that is, sequentially; Combination therapy is understood to include all of these regimens.

[0399] It is understood that, in the present description, combinations of substituents and/or variables of the depicted formulas are permitted only if such contributions result in stable compounds.

[0400] It will be appreciated by those skilled in the art that in the synthetic processes described herein the functional groups of Intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto and carboxylic acid. As described above, suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl and the like, and suitable protecting groups for amino, amidino and guanidino include t-butoxycarbonyl , benzyloxycarbonyl and the like. Suitable protecting groups for mercapto include -C(O)R'' (where R'' is alkyl, aryl or arylalkyl), p-methoxybenzyl, trityl and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups may be added or removed in accordance with conventional techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Greene, T. W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one skilled in the art would appreciate, the protecting group may also be a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl chloride resin.

[0401] It will also be appreciated by those skilled in the art, although such protected derivatives of compounds of this disclosure may not possess pharmacological activity as such, they can be administered to a mammal and subsequently metabolized in the organism to form compounds of the disclosure that are pharmacologically active. Such derivatives, therefore, can be described as "prodrugs". All examples of disclosure compounds are included within the scope of the present disclosure.

[0402] Furthermore, compounds of the disclosure that exist in free base or acid form can be converted into their pharmaceutically acceptable salts by treatment with the appropriate inorganic or organic acid or base by methods known to those skilled in the art. Salts of the compounds of the disclosure can be converted to their free acid or base form by standard techniques.

[0403] The following examples illustrate various methods of making disclosure compounds, i.e., composed of structures (I), (Ia), (Ib), (VI) and (VII). It is understood that one of skill in the art may be able to make these compounds by similar methods or by combining other methods known to those of skill 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 structure (I), (Ia), (Ib), (VI) or (VII) not specifically illustrated below using the appropriate raw material components and modifying synthesis parameters as necessary. In general, raw material components can be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maibridge, 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.

[0404] The following examples are provided for purposes of illustration, not limitation.

Examples GENERAL SYNTHETIC SCHEMES

[0405] General Scheme General Procedure 1 - Trifluoroacetamide Installation

[0406] To a stirred suspension of amine in 1,4-dioxane, trifluoroacetic anhydride (1.1 equivalents) was added. The reaction mixture transitioned from a suspension to a solution and back to a suspension again. Reaction progress was monitored by TLC and/or HPLC-MS for completion. Once the raw material was fully consumed, the reaction was diluted with hexanes or diethyl ether, filtered on a Buchner funnel and the resulting solids were dried under reduced pressure to give pure trifluoroacetamide. General procedure 2 - formation of sulfonamide N-acyl DCC/DMAP-mediated

[0407] 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 then N,N-dimethylaminopyridine (1.2 equivalents). Course of the reaction was monitored by HPLC-MS (typically 16 h) and excess byproducts could be precipitated by addition of diethyl ether. Solids were removed by filtration and washed 1:1. of diethyl ether/dichloromethane. The combined organic layers were concentrated, and the residue was purified by silicon gel chromatography or, optionally, prep-HPLC to give the desired N-acyl sulfonamide. General Procedure 3 - General Saponification

[0408] To a solution of trifluoroacetamide or construct-containing ester in 1,4-dioxane or methanol, lithium hydroxide (10 equivalents) and water (10% v/v) were added. The reaction was allowed to stir at room temperature or, optionally, heated to 50o C. The course of the reaction was monitored by HPLC-MS. Upon completion, volatile compounds were removed under reduced pressure, the aqueous layer was adjusted if necessary and washed successively with dichloromethane or ethyl acetate. The organic phases were pooled, dried over MgSO4, filtered and concentrated. The reaction product was used "as is" or purified by silicone gel chromatography if necessary. General Procedure 4 - HATU-Mediated Peptide Bond Formation

[0409] To a stirred solution of the carboxylic acid in a minimum amount of dichloromethane or N,N-dimethylformamide or respective mixture, at 0oC HATU (equivalents) and N,N-diisopropylethylamine (4 equivalents) were added. 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, volatile compounds were removed under reduced pressure and the residual material was purified by silica gel chromatography or reversed-phase HPLC to provide amide in suitable purity. General procedure 7 - Boc group removal

[0410] To a Boc-protected construct solution in dichloromethane, 10% v/v trifluoroacetic acid was added. Course of the reaction was monitored by HPLC-MS. Upon completion, all volatile compounds were removed under reduced pressure. Residual material was purified by reverse HPLC, silica gel chromatography or precipitation from a cold methanol/dichloromethane/diethyl ether mixture. General Procedure 8 - Pd-catalyzed Suzuki Cross Coupling

[0411] A suspension of aryl bromide, aryl (or alkenyl) boronic acid (1.5 eq), Pd(OAc)2 (10 mol %), 2-(di-tert-butylphosphine)biphenyl (20 mol % ), and K34PO (3 eq) in tF was stirred under N2 at room temperature for 16 h (or 50 °C for 2 h). The brown mixture resulting from the reaction was diluted with ether and washed with 1M NaOH (3x). The aqueous washes were combined and extracted with ether (2x). The organic products were combined, dried over MgSO4, filtered, concentrated in vacuo and purified by silica gel column chromatography (eluted with MeOH/CH2Cl2 mixtures) to yield the cross-coupling product. General Procedure 9 - Ullman Cross-Coupling Cu- catalyzed (methoxy installation)

[0412] A mixture of aryl bromide, CuBr (20 mol%), NaOMe (20 eq, 4.9M in MeOH) and EtOAc (1.5 eq) was stirred under N2 at 95 ° C for 16 h. The resulting mixture was diluted with H2O and poured into the cold (0 °C), stirring 1M citric acid. After stirring for 10 min, the mixture was extracted with EtOAc (4x). The combined organics were washed with H2O (100 ml) and brine (100 ml), dried over MgSO4, filtered and concentrated in vacuo. The product was used in the next step without further purification. General procedure 10 - Synthesis of vinyl analogous amino ester

[0413] The procedure for Weinreb Amide synthesis, reduction and subsequent respective olefination, as described by Nieman J. A. et al. J. Nat. Prod. 2003, 66, 183-199 was employed for the desired commercially available amino acids without modifications. General procedure 11 - Establishment of Boct-t-leucine-(Me)-vinyl analog amino acid

[0414] The vinyl analog amino ester was deprotected and coupled to Boc-t-leucine according to the procedures described by Nieman J. A. et al. J. Nat. Prod. 2003, 66, 183-199 without modifications. General procedure 12 - formation of alkyl halide sulfonamide

[0415] To a suspension of the desired alkyl halide in 2:1 H2O/EtOH was added sodium sulfite (1.2 equiv). The resulting mixture was heated to reflux for 6-24h. The reaction was then cooled to room temperature, the solvents were removed under reduced pressure to remove the ethanol, and the product was precipitated. The sodium alkylsulfonates were filtered, collected and dried in a vacuum. These solids were then suspended in dichloromethane and phosphorus pentachloride (2 equiv) was added with stirring. The resulting suspension was heated to reflux for 2h and allowed to cool to room temperature. The reactions were then cooled to 0oC and water was added dropwise to consume the excess phosphorus pentachloride. The mixture was transferred to a separatory funnel and the organic phase was washed with brine, dried over MgSO4, filtered and concentrated to give the desired sulfonyl chloride. The chloride thus derived was subsequently dissolved in tF and added dropwise to a stirred aqueous solution of ammonium hydroxide concentrate at 0oC. After completion of the addition, the reaction was concentrated under reduced pressure and diluted with water and ethyl acetate. The organic phase was washed with brine, dried over MgSO4, filtered and concentrated to give the desired sulfonamide in sufficient purity for further use. General Procedure 13 - Formation of sulfonamide from aryl substituted compounds

[0416] To a stirred mixture of the aryl substituted desired compound in chloroform was added chlorosulfonic acid (4 equiv). The reaction was heated to 70oC for 1h and allowed to cool to room temperature. Thionyl chloride (2 equiv) was added, and the reaction was heated again at 70oC for 1h. The contents of the reaction vessel were concentrated under reduced pressure to give an oil which was subsequently twice dissolved in toluene and concentrated under reduced pressure to remove residual acid. The remaining material was dissolved in tF and added dropwise to a concentrated ammonium hydroxide solution at 0oC. stirred Once the addition was complete, the reaction was concentrated under reduced pressure and the residue was partitioned between water and ethyl acetate. The organic phase was washed with brine, dried over MgSO4, filtered and concentrated to give the desired phenisulfonamide in sufficient purity for further use. General Procedure 14 - Formation of Sulfamamide

[0417] The procedures used to generate the desired sulfamamides were adapted from Winum, J.-Y. et al., Org Lett, 2001, 3(14), 2241-2243 General procedure 15 — Preparation of MC-VC-PABC-toxins

[0418] The appropriate intermediate amine or aniline was taken up in DMF (~ 90 mg/mL) and to this was added 1-hydroxybenzotriazole hydrate (0.3 eq), and then commercially obtained MC-VC-PABC-PNP(4 -(((R)-2-(((R)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5 -ureidopentanamido)benzyl 4-nitrophenyl carbonate) (1.3 eq) as described in Firestone, et al. US6214345 was added followed by pyridine (25 eq). The reaction was covered to protect from light and stirred at room temperature for 24 to 48 h. The reaction mixture could be purified by concentrating the mixture and performing flash chromatography directly on the crude oil, or alternatively, it could be diluted with DMSO to a suitable volume and injected directly into a preparatory HPLC to give the pure MC-VC-PABC-R construct.

[0419] All sulfonamides and sulfamamides or precursors for the materials used in the procedures below were purchased commercially and manipulated, if necessary, such that they were appropriate for use. Specifically, general procedures 1, 12, 13 and 14 were employed to handle commercially available raw materials, except as noted below. Sulfamamide analogues of the N-acyl sulfonamide containing compounds disclosed herein can be synthesized by those skilled in the art based on knowledge in the art and teachings herein and included are within the scope of the invention. REPRESENTATIVE COMPOUNDS Example 1 3-bromopropane-1-sulfonamide

[0420] To a stirred slurry of potassium bromide (1.904 g) in water (2.8 mL) 1,3-propanesultone was added. The reaction was heated to 60oC, stirring for 1h and allowed to cool to room temperature. Ethanol (~45 mL) was added with stirring and formed a precipitate. The suspension was filtered on a Buchner funnel and the solids were collected and dried under high vacuum overnight to give potassium 3-propyl-1-sulfonate (2.90 g, 12.0 mmol) as a white solid.

[0421] the above solid was added to a round bottom flask equipped with a stir bar. Phosphorus pentachloride (3.22 g, 1.3 equiv) was added in a single charge and the flask was gently shaken to mix the solids. A gas was observed to form and the solids became slightly melted. A single drop of water was added to the mixture and vigorous gas evolution was observed, with more significant melting of the reaction mixture. The flask was submerged in an oil bath at 70oC and the melted mixture was manipulated to try to make it as uniform as possible. After 10 minutes of heating, the flask was allowed to cool to room temperature and was charged with ice (~60 mL) and diethyl ether (~80 mL) and shaken vigorously. The two-phase mixture was transferred to a separatory funnel, the organic layer washed with brine, then dried over MgSO4, filtered and concentrated to a total volume of ~25mL. The ethereal layer was added to a 100 mL round bottom flask, a stir bar was added, and the flask was cooled to 0°C in an ice bath. Ammonia (NH4OH, 28% aq, 5ml) was added with vigorous stirring and an emulsion formed. After the emulsion had softened, brine (~20 mL) and diethyl ether (~20 mL) were added and the mixture transferred to a separatory funnel. The organic phase was separated, dried over MgSO4 and concentrated to give the title compound as a hard slurry which stood solid (0.782 g).

[0422] 1H NMR (400MHz, DMSO-d6) δ (ppm) = 2.24 (p, 2H, J = 6.5 Hz), 3.12 (t, 2H, J = 6.5 Hz), 3 .66 (t, 2H, J = 6.5 Hz), 6.91 (s, 2H).Example 2 3-(tritylthio)propane-1-sulfonamide

[0423] To a stirred solution of triphenylmethanethiol (0.276 g) in N,N-dimethylformamide at 0.oC was added sodium hydride (0.04 g, 1 equiv). After effervescence ceased, 3-bromopropane-1-sulfonamide (0.100 g, 0.5 equiv) was added as a solid in a single portion and the reaction was allowed to warm to room temperature. Reaction progress was monitored by HPLC-MS and TLC (40% EtOAc in hexanes). After 2h, the reaction was quenched with water (~0.5 mL) and concentrated on a rotovap in high vacuum. The resulting oil was partitioned between ethyl acetate and brine, transferred to a separatory funnel and the organic phase was washed with brine, dried over MgSO4, concentrated and purified by flash chromatography (5-50% EtOAc in hexanes) to give the title compound (0.135 g) as a white crystalline solid.

[0424] 1H NMR (400MHz, CD3OD) δ (ppm) = 1.77-1.85 (m, 2H), 2.35 (t, 2H, J = 6.5 Hz), 2.95-2, 99 (t, 2H, J = 6.5 Hz), 7.22-7.33 (m, 9H), 7.407.45 (m, 6H) Example 3 (6S,9S,12S,E)-9-tert-butyl-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-6-(2-phenylpropan-2-) acid il)-3-oxa-5,8,11-triazapentadec-13-en-15- oic

[0425] synthesized according to Nieman JA et al. J. Nat. Prod. 2003, 66, 183-199.Example 4 (S,E)-N-(3-mercaptopropisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2- ((S)-3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide (compound A)

[0426] Example 4 was synthesized from examples 2 and 3 according to general procedures 2 and 7, with the inclusion of tri-isopropylsilane (2equiv) for procedure 9.

[0427] 1H NMR (400MHz, CD3OD) δ (ppm) = 0.88 (3H, d, J = 6.2 Hz), 0.94 (3H, d, J = 6.2 Hz), 1.08 (s, 9H), 1.40 (s, 3H), 1.48 (s, 3H), 1.94 (d, 3H, J = 1.29 Hz), 2.03-2.16 (m, 3H), 2.41 (s, 3H), 2.67 (t, 2H, J = 9.76 Hz), 3.16 (s, 3H), 3.46-3.50 (m, 2H), 4.08 (br s, 1H), 4.94 (s, 1H), 5.07 (t, 1H, J = 10.0 Hz), 6.59 (d, 1H, J = 9.5 Hz) , 7.32-7.37 (m, 1H), 7.41-7.48 (m, 2H), 7.507.57 (m, 2H).

[0428] Methods described above were used to generate the following analogous compounds. Example 5 2,2'-disulfanediyldiethanesulfonamide

[0429] Synthesized as described by Lemaire, H. and Rieger, M in J. Org. Chem., 1961, 1330-1331. Example 6 (S,E)-N-(3-mercaptopropisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2- ((S)-3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide (compound B)

[0430] For a solution of acid (6S,9S,12S,E)-9-tert-butyl-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-6- (2-phenylpropan-2-yl)-3-oxa-5,8,11-triazapentadec-13-en-15-oic acid (0.138 g, 2.4 equiv) in dichloromethane (4 mL) was added 2,2' -disulfanediyldiethanesulfonamide (0.028g), diisopropylcarbodiimide (0.044 mL, 2.4 equiv.) and N,N-dimethylpyridine (0.034 g, 2.8 equiv.). Stirring was continued for 16h at which point TLC analysis (5% MeOH (with 5% AcOH) in 70/30 CH2Cl2/Hexanes) indicated total consumption of disulfanedisulfonamide. The reaction was diluted with hexanes (~5 mL), filtered to remove solids, concentrated, and the resulting oil purified by flash chromatography.

[0431] The chromatographically purified materials were then dissolved in dichloromethane (3 mL), a stir bar was added, and then trifluoroacetic acid (0.60 mL) and triisopropylsilane (0.20 mL). The mixture immediately turned yellow, with the color fading over 5 minutes and conversion of the material to the desired product was monitored by HPLC-MS. After complete conversion, the reaction was concentrated to dryness and the residue purified by flash chromatography (0-15% MeOH (containing 5% AcOH) in 80/20 CH2Cl2/hexanes). HPLC-MS showed this isolate as a free mixture of thiol and disulfide.

[0432] 1H NMR (400MHz, CD3OD) δ (ppm) = 0.88 (3H, d, J = 6.2 Hz), 0.93 (3H, d, J = 6.2 Hz), 1.07 (s, 9H), 1.40 (s, 3H), 1.47 (s, 3H), 1.91-2.05 (m, 5H), 2.32 (s, 3H), 2.67 ( t, 2H, J = 9.76 Hz), 3.07-3.18 (m, 5H), 3.52-3.59 (m, 2H), 3.85 (s, 1H),HH 4, 08 (br s, 1H), 4.93 (s, 1H), 5.09 (t, 1H, J = 10.0 Hz), 6.76 (d, 1H, J = 9.5 Hz), 7 .29-7.35 (m, 1H), 7.39-7.46 (m, 2H), 7.49-7.5s (m, 2H), C29H48N4O5S2 calcd. [M+H]+ = 598.15 amu; found m/z = 598.16. Example 7 4-(tritylthiomethyl)methylbenzenesulfonamide

[0433] To a stirred solution of triphenylmethanethiol (0.276 g, 2 equiv), in N,N-dimethylformamide (3 mL) at 0oC, sodium hydride was added (dispersion of 60% w/w in mineral oil, 0.04 g , 2 equiv). When the effervescence ceased, 4-(bromomethyl)methylbenzenesulfonamide (0.125 g, 1 equiv) was added in a single portion and the reaction was allowed to warm to room temperature. HPLC-MS at 20 minutes indicated that the conversion was complete. The reaction was quenched with acetic acid (~0.2 mL), concentrated in vacuo to dryness and the subsequent residue partitioned between ethyl acetate and brine. The organic layer was separated, dried over MgSO4, filtered, concentrated and purified by flash chromatography (0-50% ethyl acetate in hexanes). Fractions containing the desired material were concentrated to dryness to provide the desired compound as a colorless solid (0.200 g).

[0434] 1H NMR (400MHz, DMSO-d6) δ (ppm) = 3.38 (s, 2H), 7.247.35 (m, 7H), 7.36-7.44 (m, 12H), 7, 67-7.73 (m, 2H) Example 8 (S,E)-N-(4-(mercaptomethyl)phenisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2 -(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide (Compound C).

[0435] Title compound prepared from examples 3 and 7 according to general procedures 2 and 7

[0436] 1H NMR (400MHz, CD3OD) δ (ppm) = 0.88 (d, 3H, J = 6.2 Hz), 0.91 (d, 3H, J = 6.2 Hz), 1.06 (s, 9H), 1.38 (s, 3H), 1.47 (s, 3H), 1.86 (s, 3H), 1.99-2.05 (m, 1H), 2.41 ( s, 3H), 2.67 (t, 2H, J = 9.76 Hz), 3.14 (s, 3H), 3.80 (s, 2H), HH 4.10 (br s, 1H), 4.93 (s, 1H), 5.00 (t, 1H, J = 10.0 Hz), 6.54 (d, 1H, J = 9.5 Hz), 7.30-7.51 (m , 5H), 7.52-7.58 (m, 2H), 7.90-7.97 (m, 2H), C34H50N4O5S2 calcd. [M+H]+ = 659.25 amu; found m/z = 659.37.Example 9 (S,E)-2,5-dimethyl-N-tosyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2- (methylamino)-3- phenylbutanamido)butanamido)hex-2-enamide (Compound D)

[0437] Title compound was prepared from example 3 and tosisulfonamide using general procedures 2 and 7.

[0438] 1H NMR (400MHz, CD3OD) δ (ppm) = 0.88-0.94 (m, 6H), 1.06 (s, 9H), 1.35 (s, 3H), 1.45 ( s, 3H), 1.86 (s, 3H), 2.02-2.11 (m, 1H), 2.44 (s, 3H), 2.51 (s, 3H), 3.17 (s , 3H), HH 4.35 (s, 1H), 4.89-4.99 (m, 2H), 6.48 (d, 1H, J = 9.5 Hz), 7.30-7.43 (m, 4H), 7.43-7.50 (m, 2H), 7.51-7.57 (m, 2H), C34H50N4O5S calcd. [M+H]+ = 627.15 amu; found m/z = 627.31.Example 10 (S,E)-2,5-dimethyl-N-(methylsulfonyl)-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)- 3-phenylbutanamido)butanamido)hex-2-enamide (Compound E)

[0439] Title compound was prepared from example 3 and methanesulfonamide using general procedures 2 and 7.

[0440] 1H NMR (400MHz, CD3OD) δ (ppm) = 0.87-0.98 (3H(m, 6H), 1.09 (s, 9H), 1.40 (s, 3H), 1, 49 (s, 3H), 1.97 (s, 3H), 2.03-2.13 (m, 1H), 2.52 (s, 3H), 2.67 (t, 2H, J = 9, 76 Hz), 3.18 (s, 3H), 3.31 (s, 3H), 4.38 (s, 1H), 4.94 (d, 1H, J = 8.2 Hz), 5.07 (t, 1H, J = 10.0 Hz), 6.54 (d, 1H, J = 9.5 Hz), 7.30-7.40 (m, 1H), 7.40-7.51 ( m, 2H), 7.51-7.59 (m, 2H), C28H46N4O5Scalcd. [M+H]+ = 551.30 amu; found m/z = 551.34.Example 11 (S,E)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido acid )hex-2-enoic (Compound F)

[0441] The title compound was synthesized using methods as described by Nieman et al, in J. Nat. Prod. 2003, 66, 183-199.Example 12 (S,E)-N-(meshisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl- 2-(methylamino)- 3-phenylbutanamido)butanamido)hex-2-enamide

[0442] Title compound was prepared from example 3 and mesitisulphonamide using general procedures 2 and 7.

[0443] 1H NMR (400 MHz, Methanol-d4) δ 7.60 - 7.55 (m, 2H), 7.47 (m, 2H), 7.37 (m, 1H), 7.03 (s , 2H), 6.50 (d, J = 6 Hz, 1H), 5.06 - 4.91 (m, 3H), 4.34 (s, 1H), 3.17 (s, 3H), 2 .68 (s, 6H), 2.51 (s, 3H), 2.31 (s, 3H), 2.07 (m, 6.6 Hz, 2H), 1.87 (s, 3H), 1 .48 (s, 3H), 1.36 (s, 3H), 1.09-1.04 (m, J = 16.8 Hz, 10H), 0.92 (t, J = 6.3 Hz, 6H).C36H54N4O5S calcd m/z = 654.38 found [M+H]+ = 655.03 Example 13 (S,E)-2,5-dimethyl-N-(4-(trifluoromethoxy)phensulfonyl)-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2 -(methylamino)-3-phenylbutanamido)butanamido)hex-2- enamide

[0444] Title compound was prepared from example 3 and 4-trifluoromethoxyphenisulfonamide using general procedures 2 and 7.

[0445] 1H NMR (400 MHz, Methanol-d4) δ 8.16 (dd, J = 8.7, 1.4 Hz, 1H), 7.69 - 7.28 (m, 4H), 6.52 (d, J = 9.2 Hz, 1H), 5.02 - 4.95 (m, 1H), 4.92 (s, 0H), 4.35 (s, 1H), 3.17 (s, 1H), 2.51 (s, 1H), 2.05 (ddd, J = 15.9, 10.9, 3.7 Hz, 1H), 1.87 (s, 1H), 1.47 (s , 1H), 1.36 (s, 1H), 1.07 (s, 4H), 0.91 (t, J = 6.1 Hz, 3H).C34H47F3N4O6S calcd m/z = 696.32 found [M +H]+ = 697.26 Example 14 (S,E)-N-(benzisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl- 2-(methylamino)- 3-phenylbutanamido)butanamido)hex-2-enamide

[0446] Title compound was prepared from example 3 and benzisulfonamide using general procedures 2 and 7.

[0447] 1H NMR (400 MHz, Methanol-d4) δ 7.56 (d, J = 7.9 Hz, 2H), 7.47 (t, J = 7.3 Hz, 2H), 7.38 ( brs, 6H), 6.39 (d, J = 9.4 Hz, 1H), 5.06 (t, J = 10.0 Hz, 1H), 4.93 (s, 1H), 4.75 ( s, 2H), 4.36 (s, 1H), 3.13 (s, 3H), 2.51 (s, 3H), 2.06-1.95 (m, 4H), 1.48 (s , 3H), 1.39 (s, 3H), 1.09 (s, 9H), 0.90 (t, J = 6.2 Hz, 6H). C34H47F3N4O6S calcd m/z = 626.35 found [M+H]+ = 626.99 Example 15 (S,E)-2,5-dimethyl-N-(2,4,6-triisopropylphenisulfonyl)-4-((S)-N,3,3-trimethyl- 2-((S)-3-methyl- 2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0448] Title compound was prepared from example 3 and 2.4.6-triisopropylphenisulfonamide using general procedures 2 and 7.

[0449] 1H NMR (400 MHz, Methanol-d4) δ 7.61 - 7.53 (m, 2H), 7.47 (t, J = 7.8 Hz, 2H), 7.41 - 7.33 (m, 1H), 7.27 (s, 2H), 6.50 (dd, J = 9.6, 1.8 Hz, 1H), 5.05 (t, J = 10.0 Hz, 1H) , 4.92 (s, 1H), 4.43 - 4.26 (m, 3H), 3.16 (s, 3H), 2.94 (dd, J = 14.3, 7.4 Hz, 1H ), 2.51 (s, 3H), 2.07 - 1.99 (m, 2H), 1.90 (d, J = 1.4 Hz, 3H), 1.48 (s, 4H), 1 .39 (s, 3H), 1.33 - 1.22 (m, 18H), 1.11 (s, 2H), 1.06 (s, 9H), 0.91 (t, J = 6.0 Hz, 7H).C42H66N4O5S calcd m/z = 738.48 found [M+H]+ = 738.10 Example 16 (S,E)-N-(4-tert-butylphenisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2- (methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0450] Title compound was prepared from example 3 and 4- tertbutylphenisulfonamide using general procedures 2 and 7.

[0451] 1H NMR (400 MHz, Methanol-d4) δ 7.98 (d, J = 8.6 Hz, 2H), 7.64 (d, J = 8.6 Hz, 2H), 7.55 ( d, J = 7.9 Hz, 2H), 7.47 (t, J = 7.7 Hz, 3H), 7.37 (t, J = 7.1 Hz, 1H), 6.48 (dd, J = 9.6, 1.8 Hz, 1H), 4.99 (t, J = 10.0 Hz, 1H), 4.92 (s, 1H), 4.35 (s, 1H), 3, 16 (s, 3H), 2.51 (s, 3H), 1.87 (d, J = 1.4 Hz, 3H), 1.47 (s, 3H), 1.38 (s, 10H), 1.06 (s, 9H), 0.91 (t, J = 6.2 Hz, 7H).C42H66N4O5S calcd m/z = 668.40 found [M+H]+ = 669.28 Example 17 (S,E)-N-(4-chlorophenisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0452] Title compound was prepared from example 3 and 4-chlorophenisulfonamide using general procedures 2 and 7.

[0453] 1H NMR (400 MHz, Methanol-d4) δ 8.03 (d, J = 8.7 Hz, 2H), 7.60 (d, J = 8.7 Hz, 2H), 7.57 - 7.51 (m, 2H), 7.47 (dd, J = 8.6, 6.9 Hz, 2H), 7.42 - 7.32 (m, 1H), 6.50 (dd, J = 9.2, 1.7 Hz, 1H), 4.96 (dd, J = 10.9, 9.1 Hz, 2H), 4.92 (s, 1H), 4.35 (s, 1H), 3.17 (s, 3H), 2.51 (s, 3H), 2.14 - 2.03 (m, 1H), 2.01 (s, 1H), 1.87 (d, J = 1, 4 Hz, 3H), 1.46 (s, 3H), 1.36 (s, 3H), 1.07 (s, 9H), 0.91 (dd, J = 6.5, 4.6 Hz, 7H).C33H47ClN4O5S calcd m/z = 646.30 found [M+H]+ = 647.20 Example 18 (S,E)-N-(3-cyanophenisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0454] Title compound was prepared from example 3 and 3-cyanophenisulfonamide using general procedures 2 and 7.

[0455] 1H NMR (400 MHz, Methanol-d4) δ 8.38 (s, 1H), 8.31 (dt, J = 8.0, 1.5 Hz, 1H), 8.02 - 7.92 (m, 1H), 7.75 (t, J = 7.9 Hz, 1H), 7.53 (d, J = 1.2 Hz, 1H), 7.48 (dd, J = 8.6, 6.9 Hz, 2H), 7.43 - 7.33 (m, 1H), 6.55 (dd, J = 9.3, 1.7 Hz, 1H), 4.93 (d, J = 5 .4 Hz, 2H), 4.35 (s, 1H), 3.18 (s, 3H), 2.51 (s, 3H), 2.15 - 1.98 (m, 2H), 1.87 (d, J = 1.4 Hz, 3H), 1.45 (s, 3H), 1.32 (s, 3H), 1.07 (s, 9H), 0.92 (dd, J = 6, 6, 3.9 Hz, 7H).C34H47N5O5S calcd m/z = 637.33 found [M+H]+ = 638.00 Example 19 (S,E)-2,5-dimethyl-N-(2-nitrophenisulfonyl)-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0456] Title compound was prepared from example 3 and 2-nitrophenisulfonamide using general procedures 2 and 7.

[0457] 1H NMR (400 MHz, Methanol-d4) δ 8.36 - 8.27 (m, 1H), 7.82 (dd, J = 5.9, 3.8 Hz, 3H), 7.61 - 7.51 (m, 2H), 7.47 (dd, J = 8.6, 6.9 Hz, 2H), 7.42 - 7.31 (m, 1H), 6.63 (dd, J = 9.5, 1.7 Hz, 1H), 5.03 (t, J = 10.0 Hz, 1H), 4.93 (s, 1H), 4.36 (s, 1H), 3.18 (s, 3H), 2.51 (s, 3H), 2.12 - 2.01 (m, 1H), 1.88 (d, J = 1.4 Hz, 3H), 1.48 (s, 3H), 1.37 (s, 3H), 1.06 (s, 9H), 0.97 - 0.86 (m, 6H).

[0458] C34H47N5O5S calcd m/z = 657.32 found [M+H]+ =658.21 Example 20 (S,E)-N-(4-methoxy-2-nitrophenisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl- 2-((S)-3-methyl- 2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0459] Title compound was prepared from example 3 and 2-nitro-4-methanesulfonamide using general procedures 2 and 7.

[0460] 1H NMR (400 MHz, Methanol-d4) δ 8.24 (d, J = 8.9 Hz, 1H), 7.59 - 7.51 (m, 2H), 7.47 (t, J = 7.6 Hz, 2H), 7.44 - 7.25 (m, 4H), 6.60 (dd, J = 9.2, 1.7 Hz, 1H), 5.03 (t, J = 10.0 Hz, 1H), 4.93 (s, 1H), 4.36 (s, 1H), 3.97 (s, 3H), 3.18 (s, 3H), 2.51 (s, 3H), 2.13 - 2.02 (m, 1H), 1.89 (d, J = 1.4 Hz, 3H), 1.48 (s, 3H), 1.38 (s, 3H), 1.11 (s, 2H), 1.06 (s, 9H), 0.99 - 0.88 (m, 6H).

[0461] C34H49N5O8S calcd m/z = 687.33 found [M+H]+ =689.23 Example 21 4-(N-((S,E)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2- (methylamino)- 3-phenylbutanamido)butanamido)hex-2-enoyl)sulfamoyl)-3-nitrobenzamide

[0462] Title compound was prepared from example 3 and 3-nitro-4-sulfamoylbenzamide using general procedures 2 and 7.

[0463] 1H NMR (400 MHz, Methanol-d4) δ 8.35 (d, J = 8.0 Hz, 1H), 8.22 (d, J = 8.0 Hz, 2H), 7.59 - 7.51 (m, 2H), 7.47 (t, J = 7.6 Hz, 2H), 7.37 (t, J = 7.3 Hz, 1H), 6.70 - 6.57 (m , 1H), 5.04 (t, J = 10.0 Hz, 1H), 4.94 (s, 1H), 4.37 (s, 1H), 3.17 (s, 3H), 2.52 (s, 3H), 2.05 (ddd, J = 10.3, 7.4, 5.5 Hz, 1H), 1.87 (d, J = 1.4 Hz, 3H), 1.48 ( s, 3H), 1.38 (s, 3H), 1.06 (s, 9H), 0.92 (dd, J = 14.7, 6.8 Hz, 6H).

[0464] C34H48N6O8S calcd m/z = 700.33 found [M+H]+ =701.28 Example 22 (S,E)-N-(4-methoxyphenisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0465] Title compound was prepared from example 3 and 4-methoxyphenisulfonamide using general procedures 2 and 7.

[0466] 1H NMR (400 MHz, Methanol-d4) δ 7.97 (d, J = 9.0 Hz, 2H), 7.54 (d, J = 7.5 Hz, 2H), 7.46 ( t, J = 7.6 Hz, 2H), 7.36 (t, J = 7.2 Hz, 1H), 7.06 (d, J = 9.0 Hz, 2H), 6.48 (dd, J = 9.3, 1.9 Hz, 1H), 4.97 (t, J = 9.9 Hz, 1H), 4.92 (s, 1H), 4.22 (s, 1H), 3, 89 (s, 3H), 3.15 (s, 3H), 2.46 (s, 3H), 2.10 - 1.99 (m, 2H), 1.86 (d, J = 1.4 Hz , 3H), 1.46 (s, 3H), 1.36 (s, 3H), 1.06 (s, 9H), 0.94 - 0.84 (m, 6H).calcd m/z = 642 .35 found [M+H]+ = 643.31

[0467] C34H50N4O6S calcd m/z = 642.35 found [M+H]+ = 643.31 Example 23 (S,E)-2,5-dimethyl-N-(4-(2,2,2-trifluoroacetamido)phensulfonyl)-4-((S)- N,3,3-trimethyl-2-((S) -3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0468] Title compound was prepared from example 3 and 2,2,2-trifluoro-N-(4-sulfamoylphenyl)acetamide using general procedures 2 and 7.

[0469] 1H NMR (400 MHz, Methanol-d4) δ 8.06 (d, J = 8.9 Hz, 2H), 7.88 (d, J = 8.9 Hz, 2H), 7.52 ( d, J = 7.1 Hz, 2H), 7.49 - 7.40 (m, 3H), 7.35 (dd, J = 8.1, 6.1 Hz, 1H), 6.47 (dd , J = 9.2, 1.8 Hz, 1H), 4.33 (s, 1H), 3.15 (s, 3H), 2.48 (s, 3H), 2.13 - 1.96 ( m, 2H), 1.85 (d, J = 1.4 Hz, 3H), 1.43 (s, 3H), 1.33 (s, 3H), 1.04 (s, 9H), 0, 89 (dd, J = 6.8, 4.7 Hz, 6H).C35H48F3N5O6S calcd m/z = 723.33 found [M+H]+ = 724.08 Example 24 (S,E)-N-(4-aminophenisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0470] Title compound was prepared from example 3 and 2,2,2-trifluoro-N-(4-sulfamoylphenyl)acetamide using general procedures 2, 3 and 7.

[0471] 1H NMR (400 MHz, Methanol-d4) δ 7.71 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 7.6 Hz, 2H), 7.47 ( d, J = 6.9 Hz, 2H), 7.37 (t, J = 6.8 Hz, 1H), 6.67 (d, J = 8.8 Hz, 2H), 6.44 (dd, J = 9.2, 1.6 Hz, 1H), 4.97 (t, J = 9.7 Hz, 1H), 4.92 (s, 1H), 4.36 (s, 1H), 3, 16 (s, 3H), 2.51 (s, 3H), 2.16 - 2.00 (m, 1H), 1.87 (d, J = 1.4 Hz, 3H), 1.46 (s , 3H), 1.37 (s, 3H), 1.07 (s, 9H), 0.92 (d, J = 6.4 Hz, 3H), 0.91 (d, J = 6.3 Hz , 3H).

[0472] C33H49N5O5S calcd m/z = 627.35 found [M+H]+ =628.35 Example 25 (S,E)-2,5-dimethyl-N-(phenisulfonyl)-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)- 3-phenylbutanamido)butanamido)hex-2-enamide

[0473] Title compound was prepared from example 3 and phenisulfonamide using general procedures 2 and 7.

[0474] 1H NMR (400 MHz, Methanol-d4) δ 8.06 - 7.95 (m, 2H), 7.63 - 7.40 (m, 8H), 7.40 - 7.30 (m, 1H), 6.53 (dd, J = 9.3, 1.6 Hz, 1H), 5.05 - 4.95 (m, 1H), 4.22 (s, 1H), 3.14 (s , 3H), 2.45 (s, 3H), 2.09 - 1.95 (m, 1H), 1.85 (d, J = 1.4 Hz, 3H), 1.46 (s, 3H) , 1.36 (s, 3H), 1.06 (s, 9H), 0.89 (dd, J = 11.9, 6.5 Hz, 7H).

[0475] C33H48N4O5S calcd m/z = 612.33 found [M+H]+ = 613.06 Example 26 (S,E)-N-(N-(2-fluorobenzyl)sulfamoyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl -2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0476] 2-fluorobenzisulfamamide was prepared from 2-fluorobenzylamine according to general procedure 14; The title compound was prepared from Example 3 and 2-fluorobenzisulfamamide using general procedures 2 and 7.

[0477] 1H NMR (400 MHz, Methanol-d4) δ 7.63 - 7.41 (m, 6H), 7.41 - 7.26 (m, 3H), 7.14 (td, J = 7, 5, 1.2 Hz, 1H), 7.07 (ddd, J = 9.5, 8.2, 1.1 Hz, 1H), 6.37 (dd, J = 9.4, 1.7 Hz , 1H), 5.07 - 4.97 (m, 1H), 4.37 (s, 1H), 4.33 (s, 2H), 3.15 (s, 3H), 2.51 (s, 3H), 2.10 - 1.97 (m, 1H), 1.83 (d, J = 1.4 Hz, 3H), 1.49 (s, 3H), 1.39 (s, 3H), 1.09 (s, 9H), 0.97 - 0.84 (m, 6H).

[0478] C34H50FN5O5S calcd m/z = 659.35 found [M+H]+ = 660.28 Example 27 (S,E)-2,5-dimethyl-N-(piperidin-1-isulfonyl)-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2- (methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0479] Sulfonamide-piperidine-1 was synthesized from piperidine according to general procedure 14; The title compound was prepared from Example 3 and piperidine-1-sulfonamide using general procedures 2 and 7.

[0480] 1H NMR (400 MHz, Methanol-d4) δ 7.55 (d, J = 1.2 Hz, 1H), 7.47 (t, J = 7.6 Hz, 3H), 7.42 - 7.29 (m, 1H), 6.48 (dd, J = 9.7, 1.8 Hz, 1H), 5.05 (t, J = 10.0 Hz, 1H), 4.39 (s , 1H), 3.18 (s, 3H), 2.52 (s, 3H), 2.07 (d, J = 10.5 Hz, 1H), 1.96 (d, J = 1.4 Hz , 3H), 1.61 (ddd, J = 20.0, 10.3, 5.4 Hz, 9H), 1.49 (s, 4H), 1.39 (s, 3H), 1.09 ( s, 9H), 0.99 - 0.84 (m, 9H).

[0481] C32H53N5O5S calcd m/z = 619.38 found [M+H]+ = 620.38 Example 28 (S,E)-2,5-dimethyl-N-(o-tolysulfonyl)-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl- 2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0482] Title compound was prepared from example 3 and 2-toluenesulfonamide using general procedures 2 and 7.

[0483] 1H NMR (400 MHz, Methanol-d4) δ 8.10 (dd, J = 8.0, 1.4 Hz, 1H), 7.60 - 7.33 (m, 11H), 6.52 (dd, J = 9.6, 1.7 Hz, 1H), 5.04 - 4.90 (m, 2H), 4.35 (s, 1H), 3.18 (s, 3H), 2, 67 (s, 3H), 2.51 (s, 3H), 2.15 - 2.03 (m, 2H), 2.01 (s, 1H), 1.87 (d, J = 1.4 Hz , 3H), 1.46 (s, 3H), 1.35 (s, 3H), 1.07 (s, 9H), 0.92 (t, J = 6.3 Hz, 6H).

[0484] C34H50N4O5S calcd m/z = 626.35 found [M+H]+ = 627.05 Example 29 (S,E)-N-(4-bromophenisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0485] Title compound was prepared from example 3 and 4-bromophenisulfonamide using general procedures 2 and 7.

[0486] 1H NMR (400 MHz, Methanol-d4) δ 7.95 (d, J = 8.3 Hz, 2H), 7.76 (d, J = 8.0 Hz, 2H), 7.55 ( d, J = 7.5 Hz, 2H), 7.47 (dd, J = 8.6, 6.9 Hz, 2H), 7.41 - 7.29 (m, 1H), 6.51 (d , J = 9.0 Hz, 1H), 4.35 (s, 1H), 3.16 (s, 3H), 2.50 (s, 3H), 2.06 (dt, J = 10.7, 6.3 Hz, 1H), 1.87 (s, 3H), 1.46 (s, 3H), 1.36 (s, 3H), 1.07 (s, 9H), 0.91 (dd, J = 6.9, 4.9 Hz, 8H).

[0487] C33H47BrN4O5S calcd m/z = 690.25 found [M+H]+ = 691.17, 693.18 Example 30 (S,E)-2,5-dimethyl-N-(naphthalen-2-isulfonyl)-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2- (methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0488] Title compound was prepared from example 3 and 2-naphthysulfonamide using general procedures 2 and 7.

[0489] 1H NMR (400 MHz, Methanol-d4) δ 8.69 - 8.62 (m, 1H), 8.47 (d, J = 8.2 Hz, 1H), 8.14 - 7.95 (m, 5H), 7.71 (dddd, J = 18.4, 8.2, 6.9, 1.4 Hz, 2H), 7.57 - 7.50 (m, 2H), 7.46 (dd, J = 8.6, 6.9 Hz, 2H), 7.42 - 7.33 (m, 1H), 6.50 (dd, J = 9.3, 1.5 Hz, 1H), 4.92 - 4.87 (m, 1H), 4.34 (s, 1H), 3.16 (s, 3H), 2.50 (s, 3H), 2.13 - 1.99 (m, 1H), 1.85 (d, J = 1.4 Hz, 3H), 1.44 (s, 3H), 1.34 (s, 3H), 1.04 (s, 9H), 0.90 ( dd, J = 6.6, 4.0 Hz, 6H).

[0490] C37H50N4O5S calcd m/z = 662.35 found [M+H]+ =663.32 Example 31 methyl 4-(N-((S,E)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino) -3-phenylbutanamido)butanamido)hex-2-enoyl)sulfamoyl)benzoate

[0491] Title compound was prepared from example 3 and 4-carboxymethylphenisulfonamide using general procedures 2 and 7.

[0492] 1H NMR (400 MHz, Methanol-d4) δ 8.24 - 8.10 (m, 4H), 7.58 - 7.50 (m, 2H), 7.47 (dd, J = 8, 6, 6.9 Hz, 2H), 7.41 - 7.33 (m, 1H), 6.52 (dd, J = 9.2, 1.6 Hz, 1H), 4.35 (s, 1H ), 3.97 (s, 3H), 3.18 (s, 3H), 2.50 (s, 3H), 2.15 - 2.00 (m, 1H), 1.86 (d, J = 1.4 Hz, 3H), 1.45 (s, 3H), 1.35 (s, 3H), 1.07 (s, 9H), 0.91 (dd, J = 6.7, 3.8 Hz, 6H).

[0493] C35H50N4O7S calcd m/z = 670.34 found [M+H]+ =671.10 Example 32 (S,E)-2,5-dimethyl-N-(N-(2-(trifluoromethyl)benzyl)sulfamoyl)-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2- enamide

[0494] Title compound was prepared from example 3 and 2-trifluoromethylbenzisulfonamide using general procedures 2 and 7.

[0495] 1H NMR (400 MHz, Methanol-d4) δ 7.78 (d, J = 7.9 Hz, 1H), 7.74 - 7.67 (m, 1H), 7.64 (dd, J = 8.1, 6.7 Hz, 1H), 7.60 - 7.52 (m, 2H), 7.48 (dd, J = 8.5, 6.8 Hz, 4H), 7.42 - 7.33 (m, 1H), 6.48 - 6.40 (m, 1H), 5.11 - 5.02 (m, 1H), 4.45 (s, 2H), 4.37 (s, 1H), 3.17 (s, 3H), 2.52 (s, 3H), 2.11 - 1.99 (m, 2H), 1.92 (d, J = 1.4 Hz, 3H), 1.49 (s, 3H), 1.40 (s, 3H), 1.09 (s, 9H), 0.92 (dd, J = 9.3, 6.7 Hz, 6H).

[0496] C35H50F3N5O5S calcd m/z = 709.35 found [M+H]+ = 710.02 Example 33 (4S,E)-N-(hexan-2-isulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2- (methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0497] Title compound was prepared from example 3 and hexane-2-sulfonamide using general procedures 2 and 7.

[0498] 1H NMR (400 MHz, Methanol-d4) δ 7.56 - 7.48 (m, 2H), 7.42 (t, J = 7.8 Hz, 2H), 7.31 (t, J = 7.3 Hz, 1H), 6.58 - 6.50 (m, 1H), 5.05 (t, J = 10.0 Hz, 1H), 4.92 (s, 1H), 3.84 (s, 1H), 3.65 (dt, J = 10.8, 4.3 Hz, 1H), 3.14 (s, 3H), 2.32 (s, 3H), 2.09 - 1, 96 (m, 2H), 1.93 (d, J = 1.4 Hz, 3H), 1.61 - 1.27 (m, 3H), 1.06 (s, 9H), 0.98 - 0 .90 (m, 6H), 0.87 (d, J = 6.5 Hz, 3H).

[0499] C33H56N4O5S calcd m/z = 620.40 found [M+H]+ =621.55 Example 34 (S,E)-N-(2-methoxyethylsulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0500] Title compound was prepared from example 3 and 2-methoxyethanesulfonamide using general procedures 2 and 7.

[0501] 1H NMR (400 MHz, Methanol-d4) δ 7.56 (d, J = 7.8 Hz, 2H), 7.47 (t, J = 7.6 Hz, 2H), 7.37 ( t, J = 7.3 Hz, 1H), 6.51 (d, J = 9.4 Hz, 1H), 5.07 (t, J = 10.0 Hz, 1H), 4.95 (s, 1H), 4.33 (s, 1H), 3.82 (t, J = 5.8 Hz, 2H), 3.70 (q, J = 5.2 Hz, 2H), 3.18 (s, 3H), 2.50 (s, 3H), 2.18 - 2.00 (m, 1H), 1.95 (d, J = 1.4 Hz, 3H), 1.49 (s, 3H), 1.39 (s, 3H), 1.09 (s, 9H), 0.93 (dd, J = 14.8, 6.6 Hz, 6H).

[0502] C30H50N4O6S calcd m/z = 594.35 found [M+H]+ =595.44 Example 35 (S,E)-N-(cyclopentylmethylsulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2- ((S)-3-methyl-2-(methylamino)- 3-phenylbutanamido)butanamido)hex-2-enamide

[0503] Title compound was prepared from example 3 and cyclopentylmethanesulfonamide using general procedures 2 and 7.

[0504] 1H NMR (400 MHz, Methanol-d4) δ 7.61 - 7.52 (m, 2H), 7.48 (dd, J = 8.6, 6.9 Hz, 2H), 7.38 (t, J = 7.4 Hz, 1H), 6.54 (dd, J = 9.4, 1.7 Hz, 1H), 5.06 (t, J = 10.0 Hz, 1H), 4 .94 (s, 1H), 4.37 (s, 1H), 3.52 (dd, J = 7.0, 5.4 Hz, 3H), 3.18 (s, 3H), 2.52 ( s, 3H), 2.35 (p, J = 8.1 Hz, 1H), 2.16 - 1.89 (m, 6H), 1.77 - 1.53 (m, 4H), 1.49 (s, 3H), 1.45 - 1.26 (m, 5H), 1.09 (s, 9H), 0.93 (dd, J = 11.3, 6.7 Hz, 6H).

[0505] C33H54N4O5S calcd m/z = 618.38 found [M+H]+ =619.54 Example 36 (S)-methyl 2-(tert-butoxycarbonyl(methyl)amino)-3-(4-cyanophenyl)-3-methylbutanoate

[0506] For a mixture of the methyl ester of example 38 (0.06 g, 0.15mmol), tris(dibenzylideneaceton)dipalladium(0) (0.014 g, 0.015mmol), 1,1'-Bis(diphenylphosphine)ferrocene ( 0.02 g, 0.25 equiv), magnesium acetate (0.013 g, 0.06mmol), zinc powder (0.004 g, 0.06mmol) and zinc cyanide (0.0264 g, 0.225mmol) under a bath of nitrogen were added N,N-dimethylformamide/water (0.8/0.08mL). The reaction was left stirring overnight and cooled to room temperature. HPLC-MS analyzes indicated good conversion to the desired product. The reaction was concentrated under reduced pressure, suspended in CH2Cl2 and the resulting suspension purified by silica gel chromatography (15-25% EtOAc in hexanes) to give the final compound as a colorless oil (0.036 g, 69%).

[0507] 1H NMR (400 MHz, Chloroform-d) δ 7.69 - 7.35 (m, 4H), 5.24 (s, 1H), 3.54 (s, 3H), 2.74 (s , 3H), 1.51 (s, 3H), 1.45 - 1.25 (m, 12H).Example 37 (S)-methyl 2-(tert-butoxycarbonyl(methyl)amino)-3-(4-((tert-butoxycarbonylamino)methyl)phenyl)-3-methylbutanoate

[0508] To a solution of benzonitrile (0.300 g, 0.87 mmol) in methanol/acetic acid (10:1, 9 mL) in a stirred container was added palladium black. The flask was charged with hydrogen gas at 60 psi and the shaker was turned on for 24 hours. At that time, the container was purged of H2 under reduced pressure. The reaction was diluted in methanol and the suspension filtered through a celite cap. The filtrate was concentrated to a slightly yellow oil and re-dissolved in dichloromethane (5 mL). T-butyl dicarbonate (0.524 g, 2.0 equiv) and triethylamine (0.846 mL, 5 equiv) were added to the solution at 0oC with stirring. The reaction was left stirring for 3h, at which time HPLC-MS indicated complete consumption of the amine. The reaction was concentrated under reduced pressure and purified by silicon gel chromatography (diethyl ether in hexanes, 15-30%) to yield the title compound as a colorless oil (0.232 g, 60%).

[0509] 1H NMR (400 MHz, Chloroform-d) δ 7.38 (dd, J = 16.6, 8.0 Hz, 2H), 7.23 (d, J = 7.7 Hz, 2H), 5.27 (s, 1H), 4.31 (s, 2H), 3.61 (s, 3H), 2.78 (s, 3H), 1.50-1.61 (m,6H), 1 .47 (d, J = 15.2 Hz, 18H).Example 38 (S)-3-(4-bromophenyl)-2-(tert-butoxycarbonyl(methyl)amino)-3-methylbutanoic acid

[0510] To a stirred solution of (S)-methyl 3-(4-bromophenyl)-2-(tert-butoxycarbonyl(methyl)amino)-3-methylbutanoate (0.710 g, 1.77 mmol) in 1,4 dioxane ( 4 mL) water (1 mL) (2 mL) and lithium hydroxide monohydrate (0.367 g, 8.9 mmol) were added. The reaction was heated to 50oC and monitored by HPLC for completion. The reaction was cooled to room temperature, acidified to f 3 with 1M citric acid and concentrated to near dryness under reduced pressure. The residue was taken up in ~20mL of ethyl acetate, washed with brine, dried over MgSO4, filtered and concentrated to give analytically pure material which was used without further manipulation.

[0511] 1H NMR (400 MHz, Chloroform-d) δ 7.44 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.3 Hz, 2H), 5.18 ( s, 1H), 2.71 (s, 3H), 1.60 - 1.42 (m, 15H).Example 39 (S)-3-(4-azidophenyl)-2-(tert-butoxycarbonyl(methyl)amino)-3-methylbutanoic acid

[0512] To an open pressure tube containing a magnetic stir bar was added example 38 (0.690 g, 1.8 mmol), copper(I) iodide (0.034 g, 0.18 mmol), sodium azide (0.350 g, 5.4mmol), N1,N2-dimethylethane-1,2-diamine (0.029 mL, 0.27mmol), sodium ascorbate (0.036 g, 0.18mmol), sodium hydroxide (0.072 g, 1.8mmol), ethanol (6 mL) and water (1 mL). The suspension was sprayed with nitrogen gas, the container was sealed and immersed in an oil bath at 105oC with vigorous stirring. The course of the reaction was monitored by HPLC-MS over 24h, at which time little raw material remained. The reaction was diluted with ethyl acetate (~20mL) and washed with brine. The aqueous layer was extracted 2x with ~20 mL of ethyl acetate. The organic layers were combined, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by silicon gel chromatography (2065% EtOAc (containing 2%v/v AcOH) in hexanes) to give the title compound as a colorless oil (0.475 g, 75%).

[0513] 1H NMR (400 MHz, Chloroform-d) δ 7.44 (d, J = 8.6 Hz, 2H), 6.99 (dd, J = 9.0, 3.4 Hz, 2H), 5.24 (s, 1H), 2.71 (s, 3H), 1.63 - 1.38 (m, 18H).Example 40 (S,E)-N-(benzisulfonyl)-4-((S)-2-((S)-3-(4-cyanophenyl)-3-methyl-2-(methylamino)butanamido)-N,3, 3-trimethylbutanamido)-2,5-dimethylhex-2- enamide

[0514] Title compound was prepared from example 36 and (S,E)-4-((S)-2-amino-N,3,3-trimethylbutanamido)-N-(benzisulfonyl)-2,5- dimethylhex-2-enamide using general procedures, 3, 4 and 7.

[0515] 1H NMR (400 MHz, Methanol-d4) δ 7.83 (d, J = 8.2 Hz, 2H), 7.73 (d, J = 8.4 Hz, 2H), 7.38 ( d, J = 2.6 Hz, 5H), 6.39 (dd, J = 9.2, 1.8 Hz, 1H), 5.04 (t, J = 10.1 Hz, 1H), 4, 91 (s, 1H), 4.75 (s, 2H), 4.34 (s, 1H), 3.12 (s, 3H), 2.54 (s, 3H), 2.05 - 1.97 (m, 2H), 1.95 (d, J = 1.5 Hz, 3H), 1.52 (s, 3H), 1.41 (s, 3H), 1.09 (s, 9H), 0 .91 (dd, J = 11.2, 4.8 Hz, 6H).C35H49N5O5S calcd m/z = 651.35 found [M+H]+ = 652.4 Example 41 (S,E)-4-((S)-2-((S)-3-(4-(aminomethyl)phenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido )-N-(benzisulfonyl)-2,5-dimethylhex-2-enamide

[0516] Title compound was prepared from example 37 and (S,E)-4-((S)-2-amino-N,3,3-trimethylbutanamido)-N-(benzisulfonyl)-2,5- dimethylhex-2-enamide using general procedures, 3, 4 and 7.

[0517] 1H NMR (400 MHz, Methanol-d4) δ 7.63 (t, J = 8.8 Hz, 2H), 7.54 (d, J = 8.3 Hz, 2H), 7.49 - 7.43 (m, 3H), 7.39 (m, 2H), 6.39 (d, J = 9.4 Hz, 1H), 5.05 - 4.97 (m, 1H), 4.75 (s, 2H), 4.35 (s, 3H), 4.16 (s, 2H), 3.14 (s, 3H), 2.54 (s, 3H), 2.03 (m, 1H) , 1.95 (s, 3H), 1.51 (s, 3H), 1.39 (s, 3H), 1.31 (s, 3H), 1.09 (s, 9H), 0.98 - 0.81 (m, 6H).Example 42 (S,E)-4-((S)-2-((S)-3-(4-azidophenyl)-3-methyl-2-(methylamino)butanamido)- N,3,3-trimethylbutanamido)-N -(benzisulfonyl)-2,5-dimethylhex-2-enamide

[0518] Title compound was prepared from example 39 and (S,E)-4-((S)-2-amino-N,3,3-trimethylbutanamido)-N-(benzisulfonyl)-2,5- dimethylhex-2-enamide using general procedures 4 and 7.

[0519] C34H49N7O5S calcd m/z = 667.35 amu; found [M+H]+ = 668.4 Example 43 (S,E)-4-((S)-2-((S)-3-(4-aminophenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-N -(benzisulfonyl)-2,5-dimethylhex-2-enamide

[0520] To a stirred solution of protected Boc example 42 (0.035 g, 0.046 mmol) in ethanol (1.6 mL) and water (0.5 mL) was added zinc powder (0.015 g, 0.23 mmol ) and ammonium chloride (0.025 g, 0.46 mmol). After 1h HPLC-MS indicated complete consumption of raw materials. The reaction was quenched with ammonium hydroxide (~0.1mL) and diluted with ethyl acetate (5mL). The reaction was filtered, the solids washed with ethyl acetate (5mL) and the two-phase filtrate transferred to a separatory funnel. The aqueous phase was washed twice with ethyl acetate (5mL) and the organic phases were combined, washed with brine, dried over MgSO4, filtered and concentrated. The reaction product was purified by silicon gel chromatography (5-15% MeOH in CH2Cl2) to yield the Boc-protected intermediate as a colorless glass (0.027 g, 66%). The intermediate was deprotected according to general procedure 7 to give the title compound.

[0521] C34H51N5O5S calcd m/z = 641.36 amu; found [M+H]+ = 642.4 Example 44 (S,E)-N-(cyclohexsulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)- 3-phenylbutanamido)butanamido)hex-2-enamide

[0522] Title compound was prepared from example 3 and cyclohexysulfonamide using general procedures 2 and 7.

[0523] 1H NMR (400 MHz, Methanol-d4) δ 7.61 - 7.52 (m, 2H), 7.47 (dd, J = 8.6, 6.9 Hz, 2H), 7.36 (t, J = 7.5 Hz, 1H), 6.61 - 6.50 (m, 1H), 5.11 - 4.99 (m, 1H), 4.94 (s, 1H), 4, 28 (s, 1H), 3.59 - 3.51 (m, 1H), 3.18 (s, 3H), 2.48 (s, 3H), 2.20 - 2.00 (m, 4H) , 1.97 - 1.87 (m, 6H), 1.78 - 1.69 (m, 1H), 1.60 (td, J = 14.2, 10.9 Hz, 2H), 1.48 (s, 3H), 1.44 - 1.23 (m, 6H), 1.09 (s, 9H), 0.93 (dd, J = 13.7, 6.6 Hz, 7H).

[0524] C33H54N4O5S calcd m/z = 618.38 found [M+H]+ =619.47 Example 45 (S,E)-2,5-dimethyl-N-(pyridin-3-ylmethylsulfonyl)-4-((S)-N,3,3-trimethyl-2- ((S)-3-methyl-2- (methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0525] Title compound was prepared from example 3 and pyridin-3-ylmethanesulfonamide using general procedures 2 and 7.

[0526] 1H NMR (400 MHz, Methanol-d4) δ 8.55 (d, J = 1.7 Hz, 1H), 8.48 (dd, J = 5.0, 1.6 Hz, 1H), 7.89 (d, J = 8.0 Hz, 0H), 7.55 (d, J = 7.6 Hz, 2H), 7.50 - 7.39 (m, 2H), 7.35 (s , 1H), 6.52 (dd, J = 9.6, 2.0 Hz, 1H), 5.05 (s, 0H), 4.94 (s, 1H), 4.64 (s, 2H) , 4.19 (s, 1H), 3.11 (s, 3H), 2.45 (s, 3H), 1.91 (d, J = 1.5 Hz, 3H), 1.48 (s, 3H), 1.39 (s, 3H), 1.07 (s, 8H), 0.89 (dd, J = 15.1, 6.5 Hz, 6H).

[0527] C33H54N4O5S calcd m/z = 627.35 found [M+H]+ = 628.35 Example 46 4-(N-((S,E)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino) acid -3-phenylbutanamido)butanamido)hex-2-enoyl)sulfamoyl)benzoic acid

[0528] Title compound was prepared from example 3 and methyl 4-sulfamoylbenzoate using general procedures, 2, 3 and 7.

[0529] 1H NMR (400 MHz, Methanol-d4) δ 8.25 - 8.07 (m, 4H), 7.54 (d, J = 7.8 Hz, 2H), 7.47 (t, J = 7.6 Hz, 2H), 7.37 (t, J = 7.3 Hz, 1H), 6.55 (d, J = 9.3 Hz, 1H), 4.98 (t, J = 9 .9 Hz, 1H), 4.92 (s, 1H), 4.36 (s, 1H), 3.16 (s, 3H), 2.51 (s, 3H), 2.06 (q, J = 9.0, 7.7 Hz, 1H), 1.88 (s, 3H), 1.46 (s, 3H), 1.36 (s, 3H), 1.06 (s, 9H), 0 .91 (t, J = 6.0 Hz, 6H) Example 47 (S,E)-2,5-dimethyl-N-(3-(2,2,2-trifluoroacetamido)phensulfonyl)-4-((S)- N,3,3-trimethyl-2-((S) -3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0530] Title compound was prepared from example 3 and 2,2,2-trifluoro-N-(3-sulfamoylphenyl)acetamide using general procedures 2 and 7.

[0531] 1H NMR (400 MHz, Methanol-d4) δ 8.49 (p, J = 2.2 Hz, 1H), 7.90 (dtd, J = 6.0, 4.8, 2.9 Hz , 2H), 7.64 - 7.56 (m, 1H), 7.53 (tt, J = 5.4, 4.3, 1.8 Hz, 2H), 7.51 - 7.42 (m , 2H), 7.41 - 7.28 (m, 1H), 6.56 - 6.38 (m, 1H), 4.97 (s, 1H), 4.90 (d, J = 3.3 Hz, 1H), 4.35 (s, 1H), 3.16 (d, J = 15.5 Hz, 3H), 2.49 (d, J = 14.2 Hz, 3H), 2.14 - 2.01 (m, 1H), 1.89 - 1.83 (m, 3H), 1.57 - 1.28 (m, 6H), 1.14 - 0.94 (m, 9H), 0, 95 - 0.85 (m, 6H).

[0532] 13C NMR (101 MHz, Methanol-d4) δ 172.26, 168.81, 167.10,167.00, 144.95, 141.82, 138.82, 138.47, 135.31, 130, 71, 130.38, 128.91, 127.36, 126.65, 126.32, 121.39, 71.20, 66.92, 57.87, 57.78, 42.05, 35.83, 34.15, 32.66, 30.84, 29.79, 26.95, 21.39, 19.84, 19.82, 15.45, 14.03,

[0533] 19F NMR (377 MHz, Methanol-d4) δ -76.96, -77.07.

[0534] C35H48F3N5O6S calcd m/z = 723.33 amu; found [M+H]+ = 724.30, [M+Na]+ = 746.30Example 48 (S,E)-N-(3-aminophenisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0535] Title compound was prepared from example 3 and 2,2,2-trifluoro-N-(3-sulfamoylphenyl)acetamide using general procedures 2, 3 and 7.

[0536] 1H NMR (400 MHz, Methanol-d4) δ 7.55 (d, J = 7.5 Hz, 2H), 7.51 - 7.45 (m, 2H), 7.43 - 7.20 (m, 4H), 6.97 (d, J = 8.1 Hz, 1H), 6.48 (d, J = 9.4 Hz, 1H), 5.02 - 4.89 (m, 2H) , 4.36 (s, 1H), 3.17 (s, 3H), 2.50 (s, 3H), 2.14 - 2.00 (m, 1H), 1.88 (d, J = 1 .4 Hz, 3H), 1.46 (s, 3H), 1.35 (s, 3H), 1.07 (s, 9H), 0.92 (d, J = 6.3 Hz, 3H), 0.90 (s, 3H).

[0537] C33H49N5O5S calcd. m/z = 627.35 found [M+H]+ = 628.36 Example 49 (S,E)-2,5-dimethyl-N-(pyridin-3-isulfonyl)-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2- (methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0538] Title compound was prepared from example 3 and pyridine-3-sulfonamide using general procedures 2 and 7.

[0539] 1H NMR (400 MHz, Methanol-d4) δ 9.18 (s, 1H), 8.80 (s, 1H), 8.46 (dt, J = 8.2, 1.8 Hz, 1H ), 7.65 (dd, J = 8.1, 4.9 Hz, 1H), 7.54 (d, J = 7.3 Hz, 2H), 7.47 (t, J = 7.8 Hz , 2H), 7.37 (t, J = 7.3 Hz, 1H), 6.54 (d, J = 9.3 Hz, 1H), 5.01 - 4.88 (m, 2H), 4 .36 (s, 1H), 3.18 (s, 3H), 2.51 (s, 3H), 2.15 - 2.01 (m, 1H), 1.86 (d, J = 1.4 Hz, 3H), 1.46 (s, 3H), 1.33 (s, 3H), 1.07 (s, 9H), 0.92 (d, J = 3.3 Hz, 3H), 0, 91 (d, J = 3.5 Hz, 3H).

[0540] C32H47N5O5S calcd. m/z =613.33 found [M+H]+ = 614.23 Example 50 (S,E)-2,5-dimethyl-N-(thiophen-2-isulfonyl)-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2- (methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0541] Title compound was prepared from example 3 and thiophene-2-sulfonamide using general procedures 2 and 7.

[0542] 1H NMR (400 MHz, Methanol-d4) δ 7.93 - 7.82 (m, 2H), 7.55 (d, J = 8.3 Hz, 1H), 7.48 (t, J = 7.8 Hz, 2H), 7.37 (t, J = 7.2 Hz, 1H), 7.15 (dd, J = 5.0, 3.8 Hz, 1H), 6.51 (d , J = 9.1 Hz, 1H), 5.02 - 4.93 (m, 2H), 4.36 (s, 1H), 3.18 (s, 3H), 2.51 (s, 3H) , 2.15 - 2.01 (m, 1H), 1.89 (d, J = 1.4 Hz, 3H), 1.46 (s, 3H), 1.34 (s, 3H), 1, 08 (s, 9H), 0.93 (d, J = 4.8 Hz, 3H), 0.91 (d, J = 4.7 Hz, 3H).

[0543] C31H46N4O5S2 calcd. m/z = 618.29 found [M+H]+ = 619.24 Example 51 (S,E)-N-(4-hydroxyphenisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0544] Title compound was prepared from example 3 and 4-(tert-butyldimethylsilyloxy)benzenesulfonamide using general procedures 2 and 7.

[0545] 1H NMR (400 MHz, Methanol-d4) δ 7.89 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 7.0 Hz, 1H), 7.47 ( t, J = 7.6 Hz, 2H), 7.37 (t, J = 7.3 Hz, 1H), 6.91 (d, J = 8.9 Hz, 2H), 6.46 (d, J = 9.2 Hz, 1H), 4.97 (d, J = 10.2 Hz, 1H), 4.92 (s, 1H), 4.33 (s, 1H), 3.16 (s, 3H), 2.50 (s, 3H), 2.11 - 2.00 (m, 1H), 1.87 (d, J = 1.4 Hz, 3H), 1.46 (s, 3H), 1.36 (s, 3H), 1.07 (s, 9H), 0.92 (d, J = 6.5 Hz, 4H), 0.89 (d, J = 6.7 Hz, 3H).

[0546] C33H48N4O6S calcd. m/z = 628.33 found [M+H]+ = 629.38 Example 52 4-(tritylthiomethyl)benzonitrile

[0547] Tritylmercaptan (1.48 g, 5.36 mmol, eq 1.05) in tF (5ml) was added dropwise to a stirred suspension of sodium hydride (60% dispersion in mineral oil, 1.05 eq, 214 mg, 5.36 mmol) in tF (5 mL) under N2 at 0°C. After 15 min, 4-(bromomethyl)benzonitrile (1.00 g, 1.0 eq 5.10 mmol) in tF (5 ml) was added and the reaction was allowed to come to rt (room temperature). After 1 h, TLC indicated complete conversion of raw material. The reaction was quenched by adding saturated ammonium chloride, and then some dH2O. The mixture was extracted three times with ether, washed with saturated brine, dried over sodium sulfate and concentrated to a yellow viscous oil. Purification by flash chromatography gave the title compound (1.76 g, 88%) as a light white powder.

[0548] 1H NMR (400 MHz, Chloroform-d) δ 7.52 (d, J = 8.2 Hz, 2H), 7.47 (d, J = 7.1 Hz, 6H), 7.33 ( t, J = 7.5 Hz, 6H), 7.26 (t, J = 7.2 Hz, 3H), 7.19 (d, J = 8.2 Hz, 2H), 3.40 (s, 2H), m/z calcd. for C27H21NS = 391.14, Found [M+Na]+ = 414.13, Rf = 0.32 (10% EtOAc/Hex).Example 53 1-(4-(tritylthiomethyl)phenyl)cyclopropanamine

[0549] 4-(Tritylthiomethyl)benzonitrile (1.47 g, 3.75 mmol, 1.0 eq) was taken up in 40 mL of tF, under an N2 atmosphere, then cooled to -78°C. To this solution was added Ti(O-iPr)4 (1.21 mL, 4.13 mmol, 1.1 eq) and then ethylmagnesium bromide (3M, 2.75 mL, 2.2 eq, 8.26 mmol) was added dropwise for a further 5 min. The dry ice bath was removed, allowing the solution to reach rt. After 45 min at rt, BF3^Et2O (0.93 mL, 7.51 mmol, 2.0 eq) was added to the now very dark reaction mixture. After stirring for an additional 2.5 h, the reaction was quenched with 5 mL of 2 M HCl, followed by adjusting the f to a strong base with about 15 mL of 2 M NaOH. Some water was added to the mixture, and then it was extracted three times with 75 mL of EtOAc, washed once with dH2O, once with saturated brine, dried with sodium sulfate, and concentrated to a clear oil. The material was purified by flash chromatography to yield the title compound (680 mg, 36%) as a clear oil.

[0550] 1H NMR (400 MHz, Chloroform-d) δ 7.49 (d, J = 7.8 Hz, 6H), 7.33 (t, J = 7.7 Hz, 6H), 7.26 ( t, J = 7.2 Hz, 3H), 7.20 (d, J = 8.2 Hz, 2H), 7.11 (d, J = 8.2 Hz, 2H), 3.32 (s, 2H), 1.06 (dd, J = 7.9, 5.0 Hz, 2H), 0.95 (dd, J = 7.9, 4.7 Hz, 2H), m/z calcd. for C29H27NS = 421.19, Found [M+H]+ = 422.19, Rf = 0.21 (50% EtOAc/Hex).Example 54 2,2,2-trifluoro-N-(1-(4-(tritylthiomethyl)phenyl)cyclopropyl)acetamide

[0551] To a stirred solution of 1-(4-(tritylthiomethyl) phenyl)cyclopropanamine (680 mg, 1.0 eq 1.61 mmol) in CH2Cl2 was added trifluoroacetic anhydride (mL 0.448, 3.22 mmol, eq 2, 0) and triethylamine (0.45 mL, 3.22 mmol, eq 2.0). After two hours, TLC and HPLC indicated complete conversion of raw material. The reaction was quenched by adding 3 mL of NaHCO3, then some dH2O was added, and the mixture was extracted three times with CH2Cl2. The combined organics were washed with saturated brine, dried with sodium sulfate and concentrated to a yellow foam, giving the title compound (715 mg, 86%) in sufficient purity to move to the next step.

[0552] 1H NMR (400 MHz, Chloroform-d) δ 7.48 (d, J = 7.7 Hz, 6H), 7.32 (t, J = 7.6 Hz, 6H), 7.25 ( t, J = 7.2 Hz, 3H), 7.19 (d, J = 8.2 Hz, 2H), 7.10 (d, J = 8.3 Hz, 2H), 6.83 (s, 1H), 3.31 (s, 2H), 1.40 - 1.24 (m, 4H), m/z calcd. for C31H26F3NOS = 517.17, Found [M+Na]+ = 540.25, Rf = 0.71 (50% EtOAc/Hex).Example 55 2,2,2-trifluoro-N-(1-(4-(mercaptomethyl)phenyl)cyclopropyl)acetamide

[0553] 2,2,2-trifluoro-N-(1-(4-(tritylthiomethyl)phenyl)cyclopropyl)acetamide (715 mg, 1.38 mmol, 1.0 eq) in 5 mL CH2Cl2 was treated with 2, 5 mL of TFA. After 1 min, TIPSH (0.42 mL, eq 1.5 2.1 mmol) was added, causing the yellow color to fade. After 30 min, TLC indicated that the reaction was complete. The mixture was concentrated and then co-evaporated once with CH2Cl2 and twice with toluene. The residue was purified by flash chromatography to yield the title compound (261 mg, 69%) as a white solid. 1H NMR (400 MHz, Chloroform-d) δ 7.35 - 7.23 (m, 4H), 6.87 (s, 1H), 3.74 (d, J = 7.6 Hz, 2H), 1 .77 (t, J = 7.6 Hz, 1H), 1.36 (s, 4H), Rf = 0.47 (20% EtOAc/Hex).Example 56 2,2,2-trifluoro-N-(1-(4-(sulfamoylmethyl)phenyl)cyclopropyl)acetamide.

[0554] To a stirred solution of 2,2,2-trifluoro-N-(1-(4-(mercaptomethyl)phenyl)cyclopropyl)acetamide (220 mg, 0.799 mmol, 1.0 eq) in acetonitrile was added dH2O ( 0.029 mL, 1.6 mmol, 2.0 eq), tetrabutylammonium chloride (110 mg, 0.40 mmol, 0.5 eq), and then N-chlorosuccinimide (320 mg, 2.40 mmol, 3.0 eq). ). After 20 minutes, no raw material was visible by TLC. After 90 min, concentrated NH4OH (0.18 mL, 4.0 eq, 3.2 mmol) was added. After 10 minutes, 1 mL of NH4Cl was added and the mixture was extracted three times with EtOAc. The combined organics were washed twice with dH2O, once with saturated brine, dried with sodium sulfate, and concentrated to a clear oil. The residue was purified by flash chromatography to yield the title compound (192 mg, 74%) as a white solid.

[0555] 1H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 7.31 (d, J = 8.2 Hz, 2H), 7.16 (d, J = 8.3 Hz, 2H), 6.85 (s, 2H), 4.23 (s, 2H), 1.27 (dt, J = 6.1, 2.3 Hz, 4H), Rf = 0.26 (50 % EtOAc/Hex).Example 57 (S,E)-2,5-dimethyl-N-(4-(1-(2,2,2-trifluoroacetamido)cyclopropyl)benzisulfonyl)-4-((S)-N,3,3-trimethyl-2 -((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0556] Title compound was prepared from example 3 and example 56 using general procedures 2 and 7.

[0557] 1H NMR (400 MHz, Methanol-d4) δ 7.56 (d, J = 8.4 Hz, 2H), 7.48 (t, J = 7.7 Hz, 2H), 7.37 ( t, J = 7.4 Hz, 1H), 7.32 (d, J = 8.5 Hz, 2H),7.28 (d, J = 8.5 Hz, 2H), 6.37 (d, J = 9.6 Hz, 1H), 5.07 (t, J = 10.0 Hz, 1H), 4.94 (s, 1H), 4.72 (s, 2H), 4.37 (s, 1H), 3.13 (s, 3H), 2.52 (s, 3H), 2.08 -1.96 (m, 1H), 1.96 (d, J = 1.5 Hz, 3H), 1.49 (s, 3H), 1.40 (s, 3H), 1.35 - 1.27(m, 4H), 1.10 (s, 9H), 0.92 (d, J = 7, 1 Hz, 3H), 0.89 (d, J = 6.8 Hz, 3H).

[0558] 13C NMR (101 MHz, MeOD) δ 170.93, 168.81, 165.64, 143.58, 142.24, 136.87, 134.19, 130.64, 129.00, 127, 63, 127.53, 125.95, 125.61, 69.90, 57.10, 57.02, 56.39, 40.73, 34.55, 34.25, 32.80, 30.60, 29.33, 28.39, 25.57, 20.11, 18.38, 18.34, 16.21, 16.15, 14.04, 12.85.

[0559] C39H54F3N5O6S calcd. m/z = 777.37 found [M+H]+ =778.55 Example 58 (S,E)-N-(4-(1-aminocyclopropyl)benzisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl -2-(methylamino)-3-phenylbutanamido)butanamido) hex-2- enamide

[0560] Title compound was prepared from example 3 and example 56 using general procedures 2, 3 and 7.

[0561] 1H NMR (400 MHz, Methanol-d4) δ 7.56 (d, J = 8.7 Hz, 2H), 7.51 (s, 4H), 7.47 (t, J = 7.6 Hz, 2H), 7.37 (t, J = 7.3 Hz, 1H), 6.49 (d, J = 9.5 Hz, 1H), 5.07 (t, J = 10.0 Hz, 1H), 4.94 (s, 1H), 4.81 (d, J = 14.0 Hz, 1H), 4.77 (d, J = 13.8 Hz, 1H), 4.39 (s, 1H), 3.16 (s, 3H), 2.52 (s, 3H), 2.11 - 1.99 (m, 1H), 1.97 (d, J = 1.5 Hz, 3H), 1.49 (s, 8H), 1.45 - 1.41 (m, 2H), 1.40 (s, 3H), 1.34 - 1.26 (m, 2H), 1.10 (s, 9H), 0.93 (d, J = 6.2 Hz, 3H), 0.90 (d, J = 6.3 Hz, 3H).

[0562] 13C NMR (101 MHz, MeOD) δ 170.94, 169.00, 165.69, 143.57, 137.54, 137.12, 134.38, 131.43, 129.66, 128, 98, 127.51, 125.98, 69.85,65.51, 57.68, 57.15, 56.39, 40.72, 36.16, 34.51, 32.80, 30.68, 29.42, 28.40, 25.61, 20.14, 18.42, 18.39, 14.05, 12.86, 11.80.

[0563] C37H55N5O5S calcd. m/z = 681.39 found [M+H]+ = 682.49 Example 59 1-phenylcyclopropanamine

[0564] The title compound was prepared as described in Bertus, P., Szimoniak, J. J. Org. Chem., 2003, 68, 7133-7136 of benzonitrile (1.0 mL, 9.7 mmol) to give 270 mg ( 21%).

[0565] 1H NMR (400 MHz, Chloroform-d) δ 7.44 - 7.28 (m, 4H), 7.27 - 7.15 (m, 1H), 1.18 - 1.06 (m, 2H), 1.07 - 0.95 (m, 2H), Rf = 0.28 (5% (5% NH4OH/MeOH)/CH2Cl2).Example 60 2,2,2-trifluoro-N-(1-phenylcyclopropyl)acetamide

[0566] To a stirred solution of 1-phenylcyclopropanamine (270 mg, 2.03 mmol, 1.0 eq) in dioxane (5 mL), trifluoroacetic anhydride (0.310 mL, 2.23 mmol, 1.1 eq) was added. ). After 5 min, TLC indicated complete conversion of material to begin. The mixture was concentrated and then co-evaporated once with CH2Cl2 and once with toluene to yield the title compound (453 mg, 97%) as a white flaky powder.

[0567] 1H NMR (400 MHz, Chloroform-d) δ 7.47 - 7.15 (m, 5H), 6.88 (s, 1H), 1.65 (s, 4H). m/z calcd. for C11H10F3NO = 229.07. Found [M+H]+ = 230.14. Rf = 0.82 (5% (5% NH4OH/MeOH)/CH2Cl2)Example 61 2,2,2-trifluoro-N-(1-(4-sulfamoylphenyl)cyclopropyl)acetamide

[0568] To stirred chlorosulfonic acid (0.78 mL, 11.8 mmol, 6.0 eq) at 0°C, solid 2, 2,2-trifluoro-N-(1-phenylcyclopropyl)acetamide (450) was added. mg, 1.96 mmol, 1.0 eq) in portions, keeping the temperature low. After complete addition, the mixture was heated to 50°C. After 10 minutes, gas evolution ceased, and the reaction was allowed to cool. The mixture was slowly added to a glass of ice, being careful not to splash. The solid left on the ice was filtered out. This solid was dried in vacuo and then taken up in tF (4 mL). NH4OH concentrate (0.44 mL, 4.0 eq, 7.85 mmol) was added, turning the solution green-black. After 2 min, TLC indicated complete consumption of the intermediate sulfonylchloride. 2M HCl was added until the color faded, then the mixture was extracted three times with EtOAc, washed once with saturated NaHCO 3, once with saturated brine, dried with sodium sulfate and concentrated to a flaky solid. . The crude material was purified by flash chromatography to yield the title compound (235 mg, 39%) as a white solid.

[0569] 1H NMR (400 MHz, DMSO-d6) δ 10.28 (s, 1H), 7.76 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.1 Hz, 2H), 7.31 (s, 2H), 1.42 - 1.35 (m, 2H), 1.35 - 1.27 (m, 2H), m/z calcd. for C11H11F3N2O3S = 308.04. Found [M+H]+ = 309.07, Rf = 0.27 (50% EtOAc/Hex).Example 62 (S,E)-2,5-dimethyl-N-(4-(1-(2,2,2-trifluoroacetamido)cyclopropyl)phenisulfonyl)-4-((S)-N,3,3-trimethyl-2 -((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0570] Title compound was prepared from example 3 and example 61 using general procedures 2 and 7.

[0571] 1H NMR (400 MHz, Methanol-d4) δ 8.00 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 7.6 Hz, 2H), 7.48 ( t, J = 7.7 Hz, 2H), 7.48 - 7.33 (m, 4H), 6.47(dd, J = 9.4, 1.6 Hz, 1H), 5.00 (t , J = 10.0 Hz, 1H), 4.92 (s, 1H), 4.35 (s, 1H), 3.15 (s, 3H), 2.51 (s, 3H), 2.11 - 2.00 (m, 1H), 1.86 (d, J = 1.4 Hz, 3H), 1.47 (d, J = 6.2 Hz, 3H), 1.45 (s, 2H) , 1.43 (s, 2H), 1.38 (s, 3H), 1.06 (s, 9H), 0.91 (d, J = 6.1 Hz, 3H), 0.89 (d, J = 6.2 Hz, 3H).

[0572] C37H50F3N5O6S calcd. m/z = 763.36 found [M+H]+ = 764.45 Example 63 (S,E)-N-(4-(1-aminocyclopropyl)phenisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl -2-(methylamino)-3-phenylbutanamido) butanamido) hex-2- enamide

[0573] Title compound was prepared from example 3 and 2,2,2-trifluoro-N-(1-(4-sulfamoylphenyl)cyclopropyl)acetamide using general procedures 2, 3 and 7.

[0574] 1H NMR (400 MHz, Methanol-d4) δ 8.13 (d, J = 8.5 Hz, 2H), 7.66 (d, J = 8.6 Hz, 2H), 7.55 ( d, J = 7.2 Hz, 2H), 7.47 (t, J = 7.6 Hz, 2H), 7.37 (t, J = 7.2 Hz, 1H), 6.50 (dd, J = 9.4, 1.7 Hz, 1H), 5.02 (t, J = 10.0 Hz, 1H), 4.93 (d, J = 4.9 Hz, 1H), 4.38 ( s, 1H), 3.16 (s, 3H), 2.51 (s, 3H), 2.12 - 1.99 (m, 1H), 1.84 (d, J = 1.4 Hz, 3H ), 1.51 - 1.46 (m, 5H), 1.46 - 1.42 (m,2H), 1.38 (s, 3H), 1.07 (s, 9H), 0.91 ( dd, J = 6.7, 1.7 Hz, 6H).

[0575] C36H53N5O5S calcd. m/z = 667.38 found [M+H]+ = 668.40 Example 64 (S,E)-2,5-dimethyl-N-(2-methylbenzisulfonyl)-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0576] Title compound was prepared from example 3 and 2-methylbenzisulfonamide using general procedures 2 and 7.

[0577] 1H NMR (400 MHz, Methanol-d4) δ 7.61 - 7.52 (m, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.37 (t, J = 7.3 Hz, 1H), 7.30 - 7.23 (m, 3H), 7.22 - 7.14 (m, 1H), 6.48 (dd, J = 9.3, 1.7 Hz, 1H), 5.08 (t, J = 10.0 Hz, 1H), 4.94 (s, 1H), 4.81 (s, 2H), 4.34 (s, 1H), 3, 15 (s, 3H), 2.51 (s, 3H), 2.48 (s, 3H), 2.08 - 2.00 (m, 1H), 1.98 (d, J = 1.1 Hz , 3H), 1.49 (s, 3H), 1.40 (s, 3H), 1.10 (s, 9H), 0.93 (d, J = 6.6 Hz, 3H), 0.91 (d, J = 6.6 Hz, 3H).

[0578] C35H52N4O5S calcd. m/z = 640.37 found [M+H]+ = 641.41 Example 65 (S,E)-2,5-dimethyl-N-(4-nitrobenzisulfonyl)-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0579] Title compound was prepared from example 3 and 4-nitrobenzisulfonamide using general procedures 2 and 7.

[0580] 1H NMR (400 MHz, Methanol-d4) δ 8.18 (d, J = 8.7 Hz, 2H), 7.64 (d, J = 8.7 Hz, 2H), 7.52 ( d, J = 7.5 Hz, 2H), 7.42 (t, J = 7.7 Hz, 2H), 7.31 (t, J = 7.3 Hz, 1H), 6.55 (d, J = 9.4 Hz, 1H), 5.04 (t, J = 10.0 Hz, 1H), 4.92 (s, 1H), 4.63 (s, 2H), 3.08 (s, 3H), 2.32 (s, 3H), 1.95 (dt, J = 11.4, 6.6 Hz, 4H), 1.89 (d, J = 1.4 Hz, 3H), 1, 46 (s, 3H), 1.38 (s, 3H), 1.05 (s, 9H), 0.89 (d, J = 6.5 Hz, 3H), 0.85 (d, J = 6 .5Hz, 3H).

[0581] C34H49N5O7S calcd. m/z = 671.34 found [M+H]+ = 672.36

[0582] Example 66 (S,E)-N-(4-chlorobenzisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0583] Title compound was prepared from example 3 and 4-chlorobenzisulfonamide using general procedures 2 and 7.

[0584] 1H NMR (400 MHz, Methanol-d4) δ 7.56 (d, J = 7.9 Hz, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.44 - 7.34 (m, 5H), 6.39 (d, J = 9.5 Hz, 1H), 5.06 (t, J = 10.0 Hz, 1H), 4.94 (s, 1H), 4.75 (s, 2H), 4.35 (s, 1H), 3.13 (s, 3H), 2.51 (s, 3H), 2.06 - 1.95 (m, 1H), 1 .95 (d, J = 1.4 Hz, 3H), 1.49 (s, 3H), 1.39 (s, 3H), 1.09 (s, 9H), 0.91 (d, J = 6.1 Hz, 3H), 0.89 (d, J = 5.9 Hz, 3H).

[0585] C34H49ClN4O5S calcd. m/z = 660.31 found [M+H]+ =661.32 Example 67 (S,E)-2,5-dimethyl-N-(phenethisulfonyl)-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl- 2-(methylamino)- 3-phenylbutanamido)butanamido)hex-2-enamide

[0586] Title compound was prepared from example 3 and homobenzisulfonamide using general procedures 2 and 7.

[0587] 1H NMR (400 MHz, Methanol-d4) δ 7.56 (d, J = 7.6 Hz, 2H), 7.48 (t, J = 7.5 Hz, 2H), 7.38 ( t, J = 7.4 Hz, 1H), 7.34 - 7.28 (m, 2H), 7.28 - 7.20 (m, 3H), 6.47 (dd, J = 9.2, 1.7 Hz, 1H), 5.03 (t, J = 10.0 Hz, 1H), 4.94 (s, 1H), 4.36 (d, J = 2.3 Hz, 2H), 3 .78 (td, J = 7.5, 4.1 Hz, 2H), 3.17 (s, 3H), 3.12 (t, J = 7.8 Hz, 2H), 2.51 (s, 3H), 2.14 - 2.01 (m, 1H), 1.89 (d, J = 1.4 Hz, 3H), 1.49 (s, 3H), 1.39 (s, 3H), 1.09 (s, 9H), 0.94 (d, J = 6.6 Hz, 3H), 0.91 (d, J = 6.6 Hz, 3H).

[0588] C35H52N4O5S calcd. m/z = 640.37 found [M+H]+ = 641.36 Example 68 (S,E)-N-(4-bromobenzisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2- ((S)-3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0589] Title compound was prepared from example 3 and 4-bromobenzisulfonamide using general procedures 2 and 7.

[0590] 1H NMR (400 MHz, Methanol-d4) δ 7.60 - 7.51 (m, 4H), 7.48 (t, J = 7.7 Hz, 2H), 7.39 (s, 1H ), 7.31 (d, J = 8.3 Hz, 2H), 6.38 (d, J = 9.3 Hz, 1H), 5.06 (t, J = 10.0 Hz, 1H), 4.93 (s, 1H), 4.74 (s, 2H), 4.36 (s, 1H), 3.13 (s, 3H), 2.52 (s, 3H), 2.03 - 1 .98 (m, 1H), 1.95 (d, J = 1.4 Hz, 3H), 1.49 (s, 3H), 1.39 (s, 3H), 1.09 (s, 9H) , 0.91 (d, J = 6.1 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H)

[0591] C34H49BrN4O5S calcd. m/z = 704.26 found [M+H]+ =705.23 Example 69 (S,E)-N-(4-cyanobenzisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0592] Title compound was prepared from example 3 and 4-cyanobenzisulfonamide using general procedures 2 and 7.

[0593] 1H NMR (400 MHz, Methanol-d4) δ 7.77 (d, J = 8.3 Hz, 2H), 7.64 - 7.53 (m, 4H), 7.48 (t, J = 7.7 Hz, 2H), 7.38 (t, J = 7.3 Hz, 1H), 6.41 (dd, J = 9.3, 1.7 Hz, 1H), 5.05 (t , J = 10.0 Hz, 1H), 4.94 (s, 1H), 4.87 (s, 2H), 4.36 (s, 1H), 3.14 (s, 3H), 2.52 (s, 3H), 2.06 - 1.98 (m, 1H), 1.95 (d, J = 1.4 Hz, 3H), 1.49 (s, 3H), 1.39 (s, 3H), 1.09 (s, 9H), 0.91 (d, J = 4.0 Hz, 3H), 0.90 (d, J = 4.0 Hz, 3H).

[0594] C35H49N5O5S calcd. m/z = 651.35 found [M+H]+ = 652.38 Example 70 (S,E)-2,5-dimethyl-N-(3-nitrobenzisulfonyl)-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0595] Title compound was prepared from example 3 and 3-nitrobenzisulfonamide using general procedures 2 and 7.

[0596] 1H NMR (400 MHz, Methanol-d4) δ 8.29 (d, J = 8.0 Hz, 1H), 8.26 (s, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 7.56 (d, J = 7.2 Hz, 2H), 7.48 (t, J = 7.7 Hz, 2H), 7.38 (t, J = 7.3 Hz, 1H), 6.43 (dd, J = 9.4, 1.7 Hz, 1H), 5.05 (t, J = 10.0 Hz, 1H), 4.93 (s, 2H), 4.93 (s, 1H), 4.36 (s, 1H), 3.13 (s, 3H), 2.52 (s, 3H), 2.08 - 1.98 (m, 1H), 1.96 (d, J = 1.4 Hz, 3H), 1.48 (s, 3H), 1.39 (s, 3H), 1.07 (s, 9H), 0.89 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H).

[0597] C34H49N5O7S calcd. m/z = 671.34 found [M+H]+ = 672.39 Example 71 (S,E)-N-(4-tert-butylbenzisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2- ((S)-3-methyl-2- (methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0598] Title compound was prepared from example 3 and 4-t-butylbenzisulfonamide using general procedures 2 and 7.

[0599] 1H NMR (400 MHz, Methanol-d4) δ 7.56 (d, J = 7.6 Hz, 2H), 7.48 (t, J = 7.7 Hz, 2H), 7.43 ( d, J = 8.2 Hz, 2H), 7.38 (t, J = 7.3 Hz, 1H), 7.30 (d, J = 8.2 Hz, 2H), 6.39 (dd, J = 9.4, 1.6 Hz, 1H), 5.07 (t, J = 10.0 Hz, 1H), 4.93 (s, 1H), 4.72 (s, 2H), 4, 37 (s, 1H), 3.13 (s, 3H), 2.52 (s, 3H), 2.06 - 1.98 (m, 1H), 1.96 (d, J = 1.4 Hz , 3H), 1.49 (s, 3H), 1.39 (s, 3H), 1.33 (s, 9H), 1.10 (s, 9H), 0.92 (d, J = 6, 6 Hz, 3H), 0.89 (d, J = 6.5 Hz, 3H).

[0600] C38H58N4O5S calcd. m/z = 682.41 found [M+H]+ = 683.47 Example 72 (S,E)-2,5-dimethyl-N-(2-nitrobenzisulfonyl)-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0601] Title compound was prepared from example 3 and 2-nitrobenzisulfonamide using general procedures 2 and 7.

[0602] 1H NMR (400 MHz, Methanol-d4) δ 8.03 (dd, J = 8.0, 1.4 Hz, 1H), 7.72 (td, J = 7.5, 1.5 Hz , 1H), 7.65 (td, J = 7.7, 1.6 Hz, 1H), 7.60 (dd, J = 7.6, 1.6 Hz, 1H), 7.56 (d, J = 7.2 Hz, 2H), 7.48 (t, J = 7.7 Hz, 2H), 7.38 (t, J = 7.3 Hz, 1H), 6.43 (dd, J = 9.4, 1.6 Hz, 1H), 5.31 (d, J = 14.2 Hz, 1H), 5.26 (d, J = 15.3 Hz, 1H), 5.06 (t, J = 10.0 Hz, 1H), 4.94 (s, 1H), 4.37 (s, 1H), 3.15 (s, 3H), 2.52 (s, 3H), 2.08 - 1.98 (m, 1H), 1.96 (d, J = 1.4 Hz, 3H), 1.49 (s, 3H), 1.39 (s, 3H), 1.10 (s, 9H ), 0.92 (d, J = 6.6 Hz, 3H), 0.90 (d, J = 6.6 Hz, 3H).

[0603] C34H49N5O7S calcd. m/z = 671.34 found [M+H]+ = 672.39 Example 73 (S,E)-2,5-dimethyl-N-(4-nitrophenethysulfonyl)-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0604] Title compound was prepared from example 3 and 4-nitro-homobenzisulfonamide using general procedures 2 and 7.

[0605] 1H NMR (400 MHz, Methanol-d4) δ 8.19 (d, J = 8.7 Hz, 2H), 7.58 - 7.51 (m, 4H), 7.47 (t, J = 7.6 Hz, 2H), 7.37 (t, J = 7.3 Hz, 1H), 6.47 (dd, J = 9.5, 1.7 Hz, 1H), 5.00 (t , J = 10.0 Hz, 1H), 4.93 (s, 1H), 4.36 (s, 1H), 3.91 (dd, J = 14.9, 8.5 Hz, 1H), 3 .84 (dd, J = 12.9, 8.5 Hz, 1H), 3.28 (t, J = 7.5 Hz, 2H), 3.16 (s, 3H), 2.51 (s, 3H), 2.12 - 1.98 (m, 1H), 1.87 (d, J = 1.4 Hz, 3H), 1.48 (s, 3H), 1.39 (s, 3H), 1.08 (s, 9H), 0.91 (d, J = 6.6 Hz, 3H),0.91 (d, J = 6.6 Hz, 3H).

[0606] C35H51N5O7S calcd. m/z = 685.35 found [M+H]+ = 686.38 Example 74 methyl 4-chloro-3-(N-((S,E)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl- 2 -(methylamino)-3-phenylbutanamido)butanamido)hex-2-enoyl)sulfamoyl)benzoate

[0607] Title compound was prepared from example 3 and methyl 4-chloro-3-sulfamoylbenzoate using general procedures 2 and 7.

[0608] 1H NMR (400 MHz, Methanol-d4) δ 8.80 (d, J = 2.1 Hz, 1H), 8.20 (dd, J = 8.3, 2.1 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.59 - 7.52 (m, 2H), 7.47 (t, J = 7.7 Hz, 2H), 7.40 - 7 .32 (m, 1H), 6.63 - 6.56 (m, 1H), 5.02 (t, J = 10.0 Hz, 1H), 4.37 (s, 1H), 3.98 ( s, 3H), 3.18 (s, 3H), 2.51 (s, 3H), 2.13 - 2.00 (m, 1H), 1.86 (d, J = 1.4 Hz, 3H ), 1.47 (s, 3H), 1.37 (s, 3H), 1.06 (s, 9H), 0.96 - 0.87 (m, 6H).

[0609] 13C NMR (101 MHz, Methanol-d4) δ 170.87, 165.65, 164.87,143.61, 137.01, 136.04, 134.29, 133.23, 131.81, 129, 16, 128.98, 128.88, 127.50, 125.98, 69.81, 65.53, 57.39, 56.35, 56.15, 55.37, 51.86, 40.70, 34.51, 32.77, 30.80, 29.39, 28.44, 26.18, 25.56, 20.06, 18.40, 14.06, 12.74.

[0610] C35H49ClN4O7S calcd m/z = 704.30 amu; found [M+H]+ = 705.25, [M+Na]+ = 727.25 Example 75 2,2,2-trifluoro-N-(4-(sulfamoylmethyl)benzyl)acetamide

[0611] The title compound was synthesized from commercially available (4-(aminomethyl)phenyl)methanesulfonamide and TFAA using general procedure 1.

[0612] 1H NMR (400 MHz, Acetone-d6) δ 9.05 (s, 1H), 7.48 - 7.40 (m, 2H), 7.40 - 7.32 (m, 2H), 6 .17 (s, 1H), 4.56 (d, J = 6.1 Hz, 2H), 4.35 (s, 2H) Example 76 (S,E)-2,5-dimethyl-N-(4-((2,2,2-trifluoroacetamido)methyl) benzisulfonyl)- 4-((S)-N,3,3-trimethyl-2-( (S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0613] Title compound was prepared from example 3 and example 75 using general procedures 2 and 7.

[0614] 1H NMR (400 MHz, Methanol-d4) δ 7.57 - 7.49 (m, 2H), 7.45 (t, J = 7.5 Hz, 2H), 7.33 (p, J = 8.8, 7.9 Hz, 5H), 6.37 (d, J = 9.7 Hz, 1H), 5.09 - 5.00 (m, 1H), 4.69 (s, 2H) , 4.44 (s, 2H), 4.30 (s, 1H), 3.10 (s, 3H), 2.45 (d, J = 17.5 Hz, 3H), 2.02 - 1, 87 (m, 4H), 1.46 (s, 3H), 1.37 (s, 3H), 1.07 (s, 9H), 0.95 - 0.81 (m, 6H).

[0615] 19F NMR (377 MHz, Methanol-d4) δ -76.94, -77.24.

[0616] C37H52F3N5O6S calcd m/z = 751.36 amu; found [M+H]+ = 752.46, [M+Na]+ = 774.38 Example 77 (S,E)-N-(4-(aminomethyl)benzisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl- 2-((S)-3-methyl-2 -(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0617] Prepared from example 3 and example 75 using general procedures 2, 3 and 7

[0618] 1H NMR (400 MHz, Methanol-d4) δ 7.60 - 7.54 (m, 2H), 7.54 - 7.50 (m, 4H), 7.47 (d, J = 8, 1 Hz, 2H), 7.37 (t, J = 7.4 Hz, 1H), 6.49 (dd, J = 9.5, 1.5 Hz, 1H), 5.07 (t, J = 10.0 Hz, 1H), 4.94 (s, 1H), 4.83 (d, J = 14.3 Hz, 1H), 4.79 (d, J = 13.9 Hz, 1H), 4 .38 (s, 1H), 4.16 (s, 2H), 3.16 (s, 3H), 2.52 (s, 3H), 2.10 - 2.00 (m, 1H), 1, 97 (d, J = 1.4 Hz, 3H), 1.49 (s, 3H), 1.40 (s, 3H), 1.10 (s, 9H), 0.93 (d, J = 6 .9 Hz, 3H), 0.91 (d, J = 7.0 Hz, 3H).

[0619] C35H53N5O5S calcd. m/z = 655.4; found [M+H]+ = 656.3, [M+2H]2+ = 328.8. Example 78 2,2,2-trifluoro-N-(4-(sulfamoylmethyl)phenyl)acetamide

[0620] The title compound was synthesized from commercially available (4-aminophenyl) methanesulfonamide and TFAA using general procedure 1.

[0621] 1H NMR (400 MHz, DMSO-d6) δ 11.31 (s, 1H), 7.79 - 7.51 (m, 2H), 7.51 - 7.23 (m, 2H), 6 .85 (s, 2H), 4.27 (s, 2H). Example 79 (S,E)-2,5-dimethyl-N-(4-(2,2,2-trifluoroacetamido)benzisulfonyl)-4-((S)- N,3,3-trimethyl-2-((S) -3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0622] Title compound was prepared from example 3 and example 78 using general procedures 2 and 7.

[0623] 1H NMR (400 MHz, Methanol-d4) δ 7.68 (d, J = 8.6 Hz, 2H), 7.54 (d, J = 7.1 Hz, 2H), 7.45 ( t, J = 7.6 Hz, 2H), 7.37 (dd, J = 10.6, 5.0 Hz, 3H), 6.34 (d, J = 9.4 Hz, 1H), 5, 04 (t, J = 10.1 Hz, 2H), 4.74 (s, 2H), 4.35 (s, 1H), 3.10 (s, 3H), 2.49 (s, 3H), 2.02 - 1.94 (m, 1H), 1.93 (d, J = 1.4 Hz, 3H),1.46 (s, 3H), 1.37 (s, 3H), 1.06 (s, 9H), 0.88 (d, J = 6.3 Hz, 3H), 0.86 (s, 3H).

[0624] 19F NMR (377 MHz, Methanol-d4) δ -76.97, -77.05.

[0625] C36H50F3N5O6S calcd m/z = 737.34 amu; found [M+H]+ = 738.38, [M+Na]+ = 760.35 Example 80 (S,E)-N-(4-aminobenzisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0626] Title compound was prepared from example 3 and example 78 using general procedures 2, 3 and 7

[0627] 1H NMR (400 MHz, Methanol-d4) δ 7.56 (d, J = 7.6 Hz, 2H), 7.48 (t, J = 7.7 Hz, 2H), 7.37 ( t, J = 7.3 Hz, 1H), 7.20 (d, J = 8.5 Hz, 2H), 6.87 (d, J = 8.5 Hz, 2H), 6.39 (d, J = 9.4 Hz, 1H), 5.07 (t, J = 10.0 Hz, 1H), 4.95 (s, 1H), 4.64 (s, 2H), 4.38 (s, 1H), 3.14 (s, 3H), 2.52 (s, 3H), 2.07 - 1.98 (m, 1H), 1.96 (d, J = 1.4 Hz, 3H), 1.49 (s, 3H), 1.39 (s, 3H), 1.10 (s, 9H), 0.92 (d, J = 6.7 Hz, 3H), 0.90 (d, J = 6.4 Hz, 3H). C34H51N5O5S calcd. m/z = 641.4; found [M+H]+ = 642.3. Example 81 4-(azidomethyl)benzenesulfonamide

[0628] To a stirred solution of 4-(bromomethyl)methylbenzenesulfonamide (0.50 g) in N,N-dimethylformamide (1mL) was added sodium azide (0.20 g). The suspension was heated at 50oC for 3 hours at which point the solvent was removed under reduced pressure. The residue was partitioned between water and ethyl acetate. The organic phase was washed with brine, dried over magnesium sulfate, filtered and concentrated to dryness to give the title compound as a syrup which stood solid.

[0629] 1H NMR (400 MHz, Chloroform-d) δ 8.06 - 7.91 (m, 2H), 7.58 - 7.44 (m, 2H), 4.96 (s, 2H), 4 .48 (s, 2H).Example 82 4-(aminomethyl)benzenesulfonamide

[0630] To a solution of 4-(azidomethyl)methylbenzenesulfonamide (0.354 g) in methanol (10 mL) in a round-bottomed flask equipped with a magnetic stirrer was added 10% Pd/C (~0.05g). The flask was evacuated of gases at reduced pressure and charged with hydrogen. This evacuation and charging was repeated three times at which point the suspension was left to stir overnight. After 16h, TLC analyzes indicated complete consumption of raw materials. The reaction was diluted in methanol (40 mL), celite was added and the mixture was filtered through a fritted glass funnel. The resulting solution was concentrated to dryness. 1H NMR suggested that the material was sufficiently clean at this stage for further use without purification.

[0631] 1H NMR (400 MHz, DMSO-d6) δ 7.77 (m, 2H), 7.53 (m, 2H), 5.76 (s, 2H), 3.76 (d, J = 11 .9Hz, 2H). Example 83 2,2,2-trifluoro-N-(4-sulfamoylbenzyl)acetamide

[0632] The title compound was synthesized by the reaction of 4-(aminomethyl)methylbenzenesulfonamide with TFAA according to general procedure 1, with a 1H NMR spectrum that was complicated by rotamers.

[0633] 1H NMR (400 MHz, DMSO-d6) δ 7.91 - 7.75 (m, 2H), 7.55 - 7.31 (m, 4H), 4.72 (m, 2H), 4 .47 (d, J = 6.0 Hz, 1H), 3.18 (s, 2H).Example 84 (S,E)-2,5-dimethyl-N-(4-((2,2,2-trifluoroacetamido)methyl)phenisulfonyl)-4-((S)-N,3,3-trimethyl-2-( (S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0634] Title compound was prepared from example 3 and example 83 using general procedures 2 and 7.

[0635] 1H NMR (400 MHz, Methanol-d4) δ 8.02 (d, J = 8.5 Hz, 2H), 7.58 - 7.42 (m, 7H), 7.35 (t, J = 7.3 Hz, 1H), 6.46 (d, J = 8.5 Hz, 1H), 4.97 (d, J = 10.4 Hz, 1H), 4.54 (s, 2H), 4.33 (s, 1H), 3.14 (s, 3H), 2.48 (s, 3H), 2.11 - 1.97 (m, 1H), 1.83 (d, J = 1, 4 Hz, 3H), 1.53 (s, 1H), 1.44 (s, 3H), 1.34 (s, 3H), 1.04 (s, 9H), 0.89 (d, J = 3.9 Hz, 3H), 0.88 (d, J = 4.1 Hz, 3H).

[0636] 19F NMR (377 MHz, Methanol-d4) δ -76.94, -77.26.

[0637] C36H50F3N5O6S calcd m/z = 737.34 amu; found [M+H]+ = 738.39, [M+Na]+ = 760.41 Example 85 (S,E)-N-(4-(aminomethyl)phenisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2- ((S)-3-methyl-2 -(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0638] Prepared from example 3 and example 83 using general procedures 2, 3 and 7

[0639] 1H NMR (400 MHz, Methanol-d4) δ 8.13 (d, J = 8.3 Hz, 2H), 7.68 (d, J = 8.3 Hz, 2H), 7.55 ( d, J = 7.6 Hz, 2H), 7.47 (t, J = 7.7 Hz, 2H), 7.37 (t, J = 7.3 Hz, 1H), 6.51 (dd, J = 9.2, 1.8 Hz, 1H), 5.01 (t, J = 10.0 Hz, 1H), 4.37 (s, 1H), 4.24 (s, 2H), 3, 17 (s, 3H), 2.51 (s, 3H), 2.13 - 1.97 (m, 1H), 1.84 (d, J = 1.4 Hz, 3H), 1.47 (s , 3H), 1.37 (s, 3H), 1.07 (s, 9H), 0.91 (dd, J = 6.7, 2.0 Hz, 7H).

[0640] C34H51N5OS calcd m/z = 641.36 amu; found [M+H]+ = 642.4 Example 86 (S,E)-N-(benzisulfonyl)-4-((S)-2-((S)-3-(4-bromophenyl)-3-methyl-2-(methylamino)butanamido)-N,3, 3-trimethylbutanamido)-2,5-dimethylhex-2- enamide

[0641] Title compound was prepared from example 38 and (S,E)-4-((S)-2-amino-N,3,3-trimethylbutanamido)-N-(benzisulfonyl)-2,5- dimethylhex-2-enamide using general procedures 4 and 7.

[0642] 1H NMR (400 MHz, Methanol-d4) δ 7.62 (t, J = 9.2 Hz, 2H), 7.50 - 7.43 (m, 2H), 7.38 (d, J = 2.2 Hz, 5H), 6.38 (dd, J = 9.5, 1.8 Hz, 1H), 5.05 (t, J = 10.0 Hz, 1H), 4.92 (s , 1H), 4.75 (d, J = 2.2 Hz, 2H), 4.30 (s, 1H), 3.12 (s, 3H), 2.53 (s, 3H), 2.06 - 1.97 (m, 1H), 1.95 (d, J = 1.5 Hz, 3H), 1.47 (s, 3H), 1.39 (s, 3H), 1.09 (s, 9H), 0.94 - 0.86 (m, 6H).

[0643] C34H49BrN4O5S calcd m/z = 704.26 amu; found [M+H]+ = 705.29, [M+Na]+ = 727.36 Example 87 (S,E)-4-((S)-2-((S)-3-(4'-acetylbiphenyl-4-yl)-3-methyl-2-(methylamino)butanamido)-N,3,3 -trimethylbutanamido)-N-(benzisulfonyl)-2,5-dimethylhex-2-enamide

[0644] Title compound was prepared according to general procedure 8 and Example 86 Boc-protected and 4-acetylphenylboronic acid.

[0645] 1H NMR (400 MHz, Methanol-d4) δ 8.15 - 8.08 (m, 2H), 7.86 - 7.76 (m, 4H), 7.66 (dd, J = 14, 7, 8.4 Hz, 2H), 7.38 (d, J = 4.9 Hz, 5H), 6.39 (d, J = 9.3 Hz, 1H), 5.05 (t, J = 10.1 Hz, 1H), 4.94 (s, 1H), 4.75 (d, J = 4.1 Hz, 2H), 4.37 (d, J = 16.1 Hz, 1H), 3 .13 (d, J = 3.4 Hz, 3H), 2.67 (s, 3H), 2.53 (d, J = 11.6 Hz, 3H), 2.01 (s, 1H), 1 .96 (d, J = 1.5 Hz, 3H), 1.54 (d, J = 3.7 Hz, 3H), 1.44 (s, 3H), 1.09 (d, J = 2, 7Hz, 9H), 0.96 - 0.83 (m, 6H).

[0646] C42H56N4O6S calcd m/z = 744.39 amu; found [M+H]+ = 745.42, [M+Na]+ = 767.36 Example 88 (S,E)-N-(benzisulfonyl)-4-((S)-2-((S)-3-(4'-methoxybiphenyl-4-yl)-3-methyl-2-(methylamino)butanamido) -N,3,3-trimethylbutanamido)-2,5-dimethylhex-2- enamide

[0647] Title compound was prepared according to general procedure 8 and Example 86 Boc-protected and 4-methoxyphenylboronic acid.

[0648] 1H NMR (400 MHz, Methanol-d4) δ 7.74 - 7.53 (m, 6H), 7.38 (d,J = 4.7 Hz, 5H), 7.08 - 6.99 (m, 2H), 6.43 - 6.35 (m, 1H), 5.06 (s, 1H), 4.94 (s, 1H), 4.75 (d, J = 4.1 Hz, 2H), 4.38 (s, 1H), 3.86 (s, 3H), 3.13 (s, 3H), 2.54 (s, 3H), 1.99 (d, J = 11.0 Hz, 1H), 1.96 (d, J = 1.5 Hz, 3H), 1.51 (s, 3H), 1.43 (s, 3H), 1.09 (s, 9H), 0, 96 - 0.85 (m, J = 6.0, 5.1 Hz, 6H).

[0649] C41H56N4O6S calcd m/z = 732.39 amu; found [M+H]+ = 733.41, [M+Na]+ = 755.40 Example 89 (S,E)-N-(benzisulfonyl)-4-((S)-2-((S)-3-(biphenyl-4-yl)-3-methyl-2-(methylamino)butanamido)-N, 3,3-trimethylbutanamido)-2,5-dimethylhex-2- enamide

[0650] Title compound was prepared according to general procedure 8 and Example 86 Boc-protected and phenylboronic acid.

[0651] 1H NMR (400 MHz, Methanol-d4) δ 7.86 - 7.51 (m, 6H), 7.48 (t, J = 7.6 Hz, 2H), 7.43 - 7.33 (m, 6H), 6.39 (d, J = 9.5 Hz, 1H), 5.06 (t, J = 10.1 Hz, 1H), 4.94 (s, 1H), 4.75 (d, J = 3.3 Hz, 2H), 4.37 (d, J = 14.4 Hz, 1H), 3.13 (d, J = 3.7 Hz, 3H), 2.55 (d , J = 4.5 Hz, 3H), 2.06 - 1.97 (m, 1H), 1.96 (d, J = 1.5 Hz, 3H), 1.52 (s, 3H), 1 .44 (d, J = 4.5 Hz, 3H), 1.09 (d, J = 5.6 Hz, 9H), 0.96 - 0.83 (m, 6H).

[0652] C40H54N4O5S calcd m/z = 702.38 amu; found [M+H]+ = 703.40, [M+Na]+ = 725.45 Example 90 (S,E)-N-(benzisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)- 3-(4-(4-methylstyryl)phenyl)butanamido)butanamido)hex-2- enamide

[0653] Title compound was prepared according to general procedure 8 and Example 86 Boc-protected and 4-methylstyrylboronic acid.

[0654] 1H NMR (400 MHz, Methanol-d4) δ 7.65 (d, J = 8.2 Hz, 2H), 7.54 (d, J = 8.2 Hz, 2H), 7.47 ( d, J = 7.8 Hz, 2H), 7.38 (s, 5H), 7.26 - 7.11 (m, 4H), 6.39 (d, J = 9.3 Hz, 1H), 5.06 (t, J = 10.0 Hz, 1H), 4.97 - 4.91 (m, 1H), 4.76 (s, 2H), 4.36 (s, 1H), 3.12 (d, J = 8.9 Hz, 3H), 2.54 (s, 3H), 2.37 (s, 3H), 2.05 - 1.97 (m, 1H), 1.97 - 1, 93 (m, 3H), 1.49 (s, 3H), 1.41 (s, 3H), 1.09 (d, J = 3.5 Hz, 9H), 0.91 (tq, J = 10 .8, 4.9 Hz, 6H).

[0655] C43H58N4O5S calcd m/z = 742.41 amu; found [M+H]+ = 743.44, [M+Na]+ = 765.41 Example 91 (S,E)-N-(benzisulfonyl)-4-((S)-2-((S)-3-(4-methoxyphenyl)-3-methyl-2-(methylamino)butanamido)-N,3, 3-trimethylbutanamido)-2,5-dimethylhex-2- enamide

[0656] Title compound was prepared according to general procedure 9 and Example 86 Boc-protected.

[0657] Major diastereoisomer:

[0658] 1H NMR (400 MHz, Methanol-d4) δ 7.44 (dd, J = 12.9, 8.6 Hz, 2H), 7.40 - 7.34 (m, 5H), 7.00 (t, J = 8.4 Hz, 2H), 6.38 (d, J = 9.2 Hz, 1H), 5.05 (t, J = 9.9 Hz, 1H), 4.93 (s , 1H), 4.75 (d, J = 1.8 Hz, 2H), 4.29 (s, 1H), 3.84 (s, 3H), 3.12 (s, 3H), 2.51 (s, 3H), 2.04 - 1.98 (m, 1H), 1.95 (d, J = 1.4 Hz, 3H), 1.45 (s, 3H), 1.37 (s, 3H), 1.09 (s, 9H), 0.92 - 0.86 (m, 6H).

[0659] Minor diastereoisomer:

[0660] 1H NMR (400 MHz, Methanol-d4) δ 7.44 (dd, J = 12.9, 8.6 Hz, 2H), 7.40 - 7.34 (m, 5H), 7.00 (t, J = 8.4 Hz, 2H), 6.38 (d, J = 9.2 Hz, 1H), 4.99 (t, J = 10.1 Hz, 1H), 4.93 (s , 1H), 4.75 (d, J = 1.8 Hz, 2H), 4.26 (s, 1H), 3.82 (s, 3H), 3.11 (s, 3H), 2.47 (s, 3H), 2.04 - 1.98 (m, 1H), 1.92 (d, J = 1.4 Hz, 3H), 1.53 (s, 3H), 1.48 (s, 3H), 0.94 (s, 9H), 0.92 - 0.86 (m, 6H).

[0661] C35H52N4O6S calcd m/z = 656.36 amu; found [M+H]+ = 657.35, [M+Na]+ = 679.25Example 92 (S,E)-N-(benzisulfonyl)-4-((S)-2-((R)-3-(3-methoxyphenyl)-3-methyl-2-(methylamino)butanamido)-N,3, 3-trimethylbutanamido)-2,5-dimethylhex-2- enamide

[0662] Title compound was prepared according to general procedure 9 of (S,E)-N-(benzisulfonyl)-4-((S)-2-((S)-3-(3-bromophenyl) Boc-protected -3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enamide. The two diastereomeric products resulted from diastereomerically impure raw materials and were separated by prep-scale HPLC.

[0663] Major diastereoisomer:

[0664] 1H NMR (400 MHz, methanol-d4) 7.51 - 7.32 (m, 6H), 7.14 - 7.07 (m, 1H), 7.06 (t, J = 2.2 Hz, 1H), 6.98 - 6.90 (m, 1H), 6.38 (dd, J = 9.6, 1.7 Hz, 1H), 4.99 (t, J = 10.3 Hz, 1 H), 4.93 (s, 1 H), 4.75 (d, J = 1.8 Hz 2 H), 4.32 (s, 1 H), 3.85 (s, 3 H) , 3.11 (s, 3 H) 2.47 (3h, s) 2.04 - 1.96 (m, 1 H), 1.93 (d, J = 1.4 Hz, 3 H), 1 .54 (s, 3H) 1.47 (s, 3H), 0.96 (s, 9H), 0.89 (dd, J = 6.6, 3.4 Hz, 6H).

[0665] Minor Diastereoisomer: See example 93 (immediately after) for 1H NMR spectral data

[0666] C35H52N4O6S calcd m/z = 656.36 amu; found [M+H]+ = 657.36, [M+Na]+ = 679.29 Example 93 (S,E)-N-(benzisulfonyl)-4-((S)-2-((S)-3-(3-methoxyphenyl)-3-methyl-2-(methylamino)butanamido)-N,3, 3-trimethylbutanamido)-2,5-dimethylhex-2- enamide

[0667] Title compound was prepared according to example 92. The two diastereomeric products resulted from diastereomerically impure raw materials and were separated by pre-scale HPLC.

[0668] 1H NMR (400 MHz, Methanol-d4) δ 7.39 (d, J = 5.5 Hz, 6H), 7.11 (dd, J = 4.9, 2.8 Hz, 3H), 6.38 (d, J = 9.4 Hz, 1H), 5.06 (d, J = 9.5 Hz, 1H), 4.93 (s, 1H), 4.76 (s, 2H), 4.35 (s, 1H), 3.86 (s, 3H), 3.13 (s, 3H), 2.52 (s, 3H), 2.05 - 1.97 (m, 1H), 1 .95 (d, J = 1.6 Hz, 3H), 1.46 (s, 3H), 1.38 (s, 3H), 1.09 (s, 9H), 0.90 (t, J = 6.6Hz, 6H).

[0669] C35H52N4O6S calcd m/z = 656.36 amu; found [M+H]+ = 657.36, [M+Na]+ = 679.32Example 94 (S,E)-N-(benzisulfonyl)-4-((S)-2-((S)-3-(4-(2-hydroxyethoxy)phenyl)-3-methyl-2-(methylamino)butanamido) -N,3,3-trimethylbutanamido)-2,5-dimethylhex-2- enamide

[0670] Title compound was prepared as follows: example 86 Boc-protected, CuI (10 mol%), 3,4,7,8-tetramethyl-1,10-phenanthroline (20 mol%), Cs2CO3 (2 .5 eq), and ethylene glycol (90 eq) was stirred under N2 at 130°C for 20 h. The resulting mixture was diluted with H2O, carefully acidified with 1M citric acid and extracted with CH2Cl2 (5x). The organic products were combined, washed with brine (1x), dried over MgSO4, filtered, concentrated in vacuo and purified by silica gel column chromatography (eluted with AcOH/EtOAc/hexanes mixtures) to yield the cross-coupling product that was subsequently deprotected and purified according to general procedure 7.

[0671] 1H NMR (400 MHz, Methanol-d4) δ 7.46 (d, J = 8.8 Hz, 2H), 7.38 (d, J = 2.5 Hz, 5H), 7.05 ( d, J = 8.4 Hz, 2H), 6.38 (d, J = 9.5 Hz, 1H), 5.05 (t, J = 10.1 Hz, 1H), 4.93 (s, 1H), 4.76 (s, 2H), 4.28 (d, J = 11.0 Hz,1H), 4.13 - 4.04 (m, 2H), 3.90 (t, J = 4 .6 Hz, 2H), 3.12 (d, J = 6.2 Hz, 3H), 2.50 (d, J = 16.9 Hz, 3H), 2.05 - 1.97 (m, 1H ), 1.94 (d, J = 11.0 Hz, 3H),1.56 - 1.34 (m, 6H), 1.09 (s, 9H), 0.90 (t, J = 6, 4Hz, 6H).

[0672] C36H54N4O7S calcd m/z = 686.37 amu; found [M+H]+ = 687.42, [M+Na]+ = 709.37Example 95 S-2-(4-((S)-4-((S)-1-(((S,E)-2,5-dimethyl-6-oxo-6-(benzisulfonamido)hex-4-en- 3-yl)(methyl)amino)-3,3-dimethyl-1-oxobutan-2-ylamino)-2-methyl-3-(methylamino)-4-oxobutan-2-yl)phenoxy)ethyl ethanethioate

[0673] Title compound was prepared as follows: Tributylphosphine (6 eq) was added to a cold (0 °C) stirring solution of di-tert-butyl azodicarboxylate (6 eq) at tF. After 0.5 h, a solution of Example 94 Boc-protected (1 eq) in tF was added, followed by a solution of AcSH (4.5 eq) in tF. The pale yellow mixture was stirred at 0°C for 1 h and then at room temperature for 23 h. The resulting mixture was concentrated in vacuo, dissolved in EtOAc and successively washed with 1M HCl (2x), quenched with NH4Cl (1x) and brine (1x). The organic products were dried over MgSO4, filtered, concentrated in vacuo and purified by silica gel column chromatography (eluted with AcOH/EtOAc/hexanes mixtures) to yield the Boc-protected thioacetate product (HPLC/MS - [M+ Na]+ = 867.47).

[0674] Thioacetate was dissolved in CH2Cl2 and treated with TFA. After stirring for 1 h, the reaction mixture was concentrated in vacuo. The yellow/brown residue was dissolved in a minimum amount of CH2Cl2, cooled to 0 °C and treated with ether to precipitate the desired aminothioacetate as an off-white solid in 10% yield in two synthetic steps.

[0675] 1H NMR (400 MHz, Methanol-d4) δ 7.46 (d, J = 8.7 Hz, 2H), 7.38 (d, J = 2.4 Hz, 5H), 7.03 ( d, J = 8.6 Hz, 2H), 6.38 (d, J = 9.5 Hz, 1H), 5.05 (t, J = 10.0 Hz, 1H), 4.93 (s, 1H), 4.75 (s, 2H), 4.27 (d, J = 11.4 Hz, 1H), 4.14 (t, J = 6.6 Hz, 2H), 3.28 (t, J = 6.6 Hz, 2H), 3.11 (d, J = 6.6 Hz, 3H), 2.49 (d, J = 15.5 Hz, 3H), 2.38 (s, 3H) , 2.05 - 1.97 (m, 1H), 1.95 (s, 3H), 1.45 (s, 3H), 1.37 (s, 3H), 1.08 (s, 9H), 0.96 - 0.85 (m, 6H).

[0676] C38H56N4O7S2 calcd m/z = 744.36 amu; found [M+H]+ = 745.39, [M+Na]+ = 777.32 Example 96 (S,E)-4-((S)-2-((S)-3-(4-(2-aminoethoxy)phenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3 -trimethylbutanamido)-N-(benzisulfonyl)-2,5-dimethylhex-2-enamide

[0677] Title compound was prepared as follows: Et3N (4 eq) was added to a cold (0 °C) stirring solution of MsCl (3.7 eq) in CH2Cl2. After 2 min, a protected Boc solution of example 94 in CH2Cl2 was added. The pale yellow mixture was stirred cold for 5 min and then at room temperature for 72 h. The resulting mixture was diluted with EtOAc and successively washed with 1M citric acid (1x), 1m NaHCO3 (1x) and brine (1x). The organic products were dried over MgSO 4 , filtered and concentrated in vacuo to yield mesylated alcohol (HPLC/MS - [M+Na]+ = 887.42) which was used in the next step without further purification.

[0678] The mesylate was dissolved in DMF and treated with NaN3 (7 eq). The resulting suspension was stirred at room temperature for 18 h and then at 60 °C for 5 h. The reaction mixture was diluted with H2O, acidified with 1M HCl and extracted with CH2Cl2 (4x). The combined organic products were dried over MgSO4, filtered and concentrated in vacuo to yield the azide product (HPLC/MS - [M+Na]+ = 834.44) which was used in the next step without further purification.

[0679] The azide was dissolved in tF/H2O (10:1) and treated with tributylphosphine (3.5 eq). The mixture was stirred at room temperature for 21 h and then concentrated in vacuo. The residue was dissolved in EtOAc and successively washed with 1M HCl (3x), 1M NaHCO3 (3x), H2O (2x) and brine (2x). The organic products were dried over MgSO4, filtered, concentrated in vacuo and purified by silica gel column chromatography (eluted with MeOH/CH2Cl2 mixtures) to yield the primary amine as a white solid (HPLC/MS - [M + H ]+ = 786.45).

[0680] The amine was dissolved in CH2Cl2 and treated with TFA. After stirring for 1 h, the reaction mixture was concentrated in vacuo. The off-white solid residue was dissolved in a minimal amount of MeOH, cooled to 0 °C and treated with ether to precipitate the desired diamine product as an off-white solid in 6% yield over four synthetic steps.

[0681] 1H NMR (400 MHz, Methanol-d4) δ 7.50 (d, J = 8.6 Hz, 2H), 7.37 (s, 5H), 7.09 (d, J = 8.6 Hz, 2H), 6.41 (d, J = 9.4 Hz, 1H), 5.02 (t, J = 10.0 Hz, 1H), 4.91 (s, 1H), 4.70 ( s, 2H), 4.27 (t, J = 5.0 Hz, 2H), 3.40 (t, J = 5.0 Hz, 2H), 3.37 (s, 1H), 3.12 ( s, 3H), 2.47 (s, 3H), 2.06 - 1.95 (m, 1H), 1.94 (d, J = 1.4 Hz, 3H), 1.45 (s, 3H ), 1.37 (s, 3H), 1.08 (s, 9H), 0.89 (dd, J = 9.7, 6.6 Hz, 6H).

[0682] C38H55N5O6S calcd m/z = 685.39 amu; found [M+H]+ = 686.32, [M+Na]+ = 708.27, [(M+2H)/2]2+ = 343.77 Example 97 (S,E)-2,5-dimethyl-N-(2-(2,2,2-trifluoroacetamido)phensulfonyl)-4-((S)- N,3,3-trimethyl-2-((S) -3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0683] Title compound was prepared from example 3 and a 2,2,2-trifluoro-N-(2-sulfamoylphenyl)acetamide according to general procedures 2 and 7.

[0684] 1H NMR (400 MHz, Methanol-d4) δ 8.27 (d, J = 8.4 Hz, 1H), 8.05 (d, J = 7.8 Hz, 1H), 7.67 ( t, J = 7.9 Hz, 1H), 7.54 (d, J = 8.1 Hz, 2H), 7.48 (t, J = 7.7 Hz, 2H), 7.40 (dt, J = 13.3, 7.4 Hz, 2H), 6.57 (d, J = 9.2 Hz, 1H), 4.92 (s, 2H), 4.34 (s, 1H), 3, 17 (s, 3H), 2.50 (s, 3H), 2.06 (m, 1H), 1.87 (d, J = 1.3 Hz, 3H), 1.45 (s, 3H), 1.33 (s, 3H), 1.07 (s, 9H), 0.91 (dd, J = 6.6, 3.5 Hz, 6H).

[0685] 19F NMR (377 MHz, Methanol-d4) δ -76.96, -77.73.

[0686] C35H48F3N5O6S calcd m/z = 723.33 amu; found [M+H]+ = 723.34, [M+Na]+ = 746.23Example 98 (S,E)-N-(2-aminophenisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0687] Title compound was prepared from example 3 and a 2,2,2-trifluoro-N-(2-sulfamoylphenyl)acetamide according to general procedures 2, 3 and 7.

[0688] 1H NMR (400 MHz, Methanol-d4) δ 7.75 (dd, J = 8.2, 1.5 Hz, 1H), 7.55 (d, J = 7.8 Hz, 2H), 7.48 (t, J = 7.7 Hz, 2H), 7.38 (t, J = 7.4 Hz, 1H), 7.33 - 7.27 (m, 1H), 6.81 (d , J = 8.2 Hz, 1H), 6.69 (t, J = 7.5 Hz, 1H), 6.49 (dd, J = 9.1, 1.5 Hz, 1H), 4.97 (t, J = 10.1 Hz, 1H), 4.92 (s, 1H), 4.35 (s, 1H), 3.17 (s, 3H), 2.51 (s, 3H), 2 .07 (m, 1H), 1.88 (d, J = 1.4 Hz, 3H), 1.46 (s, 3H), 1.36 (s, 3H), 1.06 (s, 9H) , 0.92 (t, J = 6.8 Hz, 6H).

[0689] C33H49N5O5S calcd m/z = 627.35 amu; found [M+H]+ = 628.36, [M+Na]+ = 650.37, [(M+2H)/2]2+ = 314.76 Example 99 (S,E)-N-(biphenyl-4-isulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2- (methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0690] Title compound was prepared using example 56 Boc- protected with phenylboronic acid according to general procedures 8 and 7.

[0691] 1H NMR (400 MHz, Methanol-d4) δ 8.12 (d, J = 8.3 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 7.71 ( d, J = 7.7 Hz, 2H), 7.52 (dd, J = 11.6, 7.6 Hz, 4H), 7.45 (t, J = 7.3 Hz, 3H), 7, 36 (t, J = 7.2 Hz, 1H), 6.52 (d, J = 9.4 Hz, 1H), 4.96 (t, J = 9.5 Hz, 1H), 4.92 ( s, 1H), 4.33 (s, 1H), 3.18 (s, 3H), 2.50 (s, 3H), 2.14 - 2.03 (m, 1H), 1.88 (s , 3H), 1.45 (s, 3H), 1.35 (s, 3H), 1.07 (s, 9H), 0.92 (t, J = 6.9 Hz, 6H).

[0692] C39H52N4O5S calcd m/z = 688.37 amu; found [M+H]+ = 689.10, [M+Na]+ = 711.32Example 100 (S,E)-N-(4'-aminobiphenyl-4-isulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl -2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0693] Title compound was prepared from example 68 Boc- protected with 4-(tert-butoxycarbonylamino)phenylboronic acid according to general procedures 8 and 7

[0694] 1H NMR (400 MHz, Methanol-d4) δ 8.05 (d, J = 8.6 Hz, 2H), 7.75 (d, J = 8.6 Hz, 2H), 7.59 - 7.51 (m, 4H), 7.45 (t, J = 7.7 Hz, 2H), 7.36 (t, J = 7.3 Hz, 1H), 6.91 (d, J = 8 .3 Hz, 2H), 6.50 (d, J = 9.1 Hz, 1H), 4.98 - 4.92 (m, 1H), 4.91 (s, 1H), 4.34 (s , 1H), 3.18 (s, 3H), 2.50 (s, 3H), 2.13 - 2.03 (m, 1H), 1.88 (d, J = 1.4 Hz, 3H) , 1.45 (s, 3H), 1.35 (s, 3H), 1.06 (s, 9H), 0.92 (t, J = 6.2 Hz, 6H).

[0695] C39H53N5O5S calcd m/z = 703.38 amu; found [M+H]+ = 704.26, [M+Na]+ = 726.41, [(M+2H)/2]2+ = 352.77 Example 101 (S,E)-N-(4-fluorobenzisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0696] Title compound was prepared from example 3 and 4-fluorobenzisulfonamide according to general procedures 2 and 7.

[0697] 1H NMR (400 MHz, Methanol-d4) δ 7.60 - 7.52 (m, 2H), 7.48 (t, J = 7.7 Hz, 2H), 7.44 - 7.34 (m, 3H), 7.18 - 7.05 (m, 2H), 6.41 (dd, J = 9.5, 1.7 Hz, 1H), 5.06 (t, J = 10.0 Hz, 1H), 4.94 (s, 1H), 4.74 (s, 2H), 4.35 (s, 1H), 3.13 (s, 3H), 2.51 (s, 3H), 2.07 - 1.97 (m, 1H), 1.95 (d, J = 1.4 Hz, 3H), 1.48 (s, 3H), 1.39 (s, 3H), 1.09 (s, 9H), 0.90 (t, J = 6.3 Hz, 6H).

[0698] C34H49FN4O5S calcd m/z= 644.34 found [M+H]+ = 645.32 Example 102 (S,E)-2,5-dimethyl-N-(3-(trifluoromethyl)benzisulfonyl)-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2 -(methylamino)-3-phenylbutanamido)butanamido)hex-2- enamide

[0699] Title compound was prepared from example 3 and 3-trifluorobenzisulfonamide according to general procedures 2 and 7.

[0700] 1H NMR (400 MHz, Methanol-d4) δ 7.74 - 7.64 (m, 3H), 7.61 (d, J = 7.7 Hz, 1H), 7.60 - 7.54 (m, 2H), 7.48 (t, J = 7.7 Hz, 2H), 7.38 (t, J = 7.3 Hz, 1H), 6.42 (dd, J = 9.4, 1.7 Hz, 1H), 5.06 (t, J = 10.0 Hz, 1H), 4.93 (s, 1H), 4.36 (s, 1H), 3.13 (s, 3H) , 2.51 (s, 3H), 2.07 - 1.97 (m, 1H), 1.95 (d, J = 1.4 Hz, 3H), 1.48 (s, 3H), 1, 39 (s, 3H), 1.08 (s, 9H), 0.89 (d, J = 6.5 Hz, 6H).

[0701] C35H49F3N4O5S calcd m/z = 694.34 found [M+H]+ =695.38 Example 103 (S,E)-2,5-dimethyl-N-(3-(trifluoromethoxy)benzisulfonyl)-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2 -(methylamino)-3-phenylbutanamido)butanamido)hex-2- enamide

[0702] Title compound was prepared from example 3 and 3-trifluoromethoxybenzisulfonamide according to general procedures 2 and 7.

[0703] 1H NMR (400 MHz, Methanol-d4) δ 7.56 (d, J = 7.8 Hz, 2H), 7.48 (t, J = 7.9 Hz, 3H), 7.43 - 7.36 (m, 2H), 7.32 (d, J = 9.3 Hz, 2H), 6.43 (dd, J = 9.4, 1.7 Hz, 1H), 5.06 (t , J = 10.0 Hz, 1H), 4.93 (s, 1H), 4.82 (s, 2H), 4.35 (s, 1H), 3.13 (s, 3H), 2.51 (s, 3H), 2.07 - 1.97 (m, 1H), 1.95 (d, J = 1.4 Hz, 3H), 1.48 (s, 3H), 1.39 (s, 3H), 1.08 (s, 9H), 0.90 (dd, J = 6.6, 4.3 Hz, 6H).

[0704] C35H49F3N4O6S calcd m/z = 710.33 found [M+H]+ =711.38 Example 104 (S,E)-N-(3,4-dichlorobenzisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2- ((S)-3-methyl-2- (methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0705] Title compound was prepared from example 3 and 3,4-dichlorobenzisulfonamide according to general procedures 2 and 7.

[0706] 1H NMR (400 MHz, Methanol-d4) δ 7.56 (td, J = 5.2, 4.5, 1.9 Hz, 4H), 7.48 (t, J = 7.7 Hz , 2H), 7.38 (t, J = 7.3 Hz, 1H), 7.33 (dd, J = 8.4, 2.1 Hz, 1H), 6.41 (dd, J = 9, 5, 1.8 Hz, 1H), 5.06 (t, J = 10.0 Hz, 1H), 4.93 (s, 1H), 4.77 (s, 2H), 4.36 (s, 1H), 3.14 (s, 3H), 2.52 (s, 3H), 2.07 - 1.97 (m, 1H), 1.95 (d, J = 1.4 Hz, 3H), 1.49 (s, 3H), 1.39 (s, 3H), 1.08 (s, 9H), 0.90 (dd, J = 6.6, 4.9 Hz, 6H).

[0707] C34H48Cl2N4O5S calcd m/z = 694.27 found [M+H]+ = 695.32 Example 105 (S,E)-N-(2-cyanobenzisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0708] Title compound was prepared from example 3 and 2-cyanobenzisulfonamide according to general procedures 2 and 7.

[0709] 1H NMR (400 MHz, Methanol-d4) δ 7.81 (dd, J = 7.7, 1.3 Hz, 1H), 7.72 (td, J = 7.7, 1.3 Hz , 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.62 - 7.59 (m, 1H), 7.58 - 7.53 (m, 2H), 7.48 ( t, J = 7.7 Hz, 2H), 7.38 (t, J = 7.3 Hz, 1H), 6.50 (d, J = 9.4 Hz, 1H), 5.08 (dd, J = 10.6, 9.3 Hz, 1H), 4.99 (s, 2H), 4.95 (s, 1H), 4.36 (s, 1H), 3.16 (s, 3H), 2.52 (s, 3H), 2.09 - 1.99 (m, 1H), 1.98 (d, J = 1.4 Hz, 3H), 1.49 (s, 3H), 1.39 (s, 3H), 1.10 (s, 9H), 0.94 (d, J = 6.6 Hz, 3H), 0.91 (d, J = 6.6 Hz, 3H).

[0710] C35H49N5O5S calcd m/z = 651.35 found [M+H]+ = 652.38 Example 106 (S,E)-N-(3-chlorobenzisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)- 3-methyl-2-(methylamino )-3-phenylbutanamido)butanamido)hex-2-enamide

[0711] Title compound was prepared from example 3 and 3-chlorobenzisulfonamide according to general procedures 2 and 7.

[0712] 1H NMR (400 MHz, Methanol-d4) δ 7.58 - 7.53 (m, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.43 - 7.34 (m, 4H), 7.32 (d, J = 7.5 Hz, 1H), 6.42 (d, J = 9.5 Hz, 1H), 5.06 (t, J = 10.0 Hz , 1H), 4.94 (s, 1H), 4.74 (s, 2H), 4.33 (s, 1H), 3.13 (s, 3H), 2.50 (s, 3H), 2 .07 - 1.97 (m, 1H), 1.95 (d, J = 1.4 Hz, 3H), 1.48 (s, 3H), 1.39 (s, 3H), 1.08 ( s, 9H), 0.90 (t, J = 7.2 Hz, 6H).

[0713] C34H49ClN4O5S calcd m/z = 660.31 found [M+H]+ = 661.32 Example 107 (S,E)-N-(4-amino-2-ethylphenisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2- ((S)-3-methyl- 2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0714] Title compound was prepared from example 3 and 2-ethylbenzisulfonamide according to general procedures 2 and 7.

[0715] 1H NMR (400 MHz, Methanol-d4) δ 7.79 (d, J = 8.7 Hz, 1H), 7.55 (d, J = 7.9 Hz, 2H), 7.48 ( t, J = 7.6 Hz, 2H), 7.37 (t, J = 7.4 Hz, 1H),6.57 (d, J = 2.3 Hz, 1H), 6.54 (dd, J = 8.8, 2.4 Hz, 1H), 6.46 (d, J = 9.4 Hz, 1H), 5.01 (t, J = 10.0 Hz, 1H), 4.92 ( s, 1H), 4.34 (s, 1H), 3.16 (s, 3H), 2.99 -2.90 (m, 2H), 2.50 (s, 3H), 2.11 - 2 .00 (m, 1H), 1.87 (d, J = 1.4 Hz, 3H), 1.47 (s, 3H), 1.38 (s, 3H), 1.22 (t, J = 7.5 Hz, 3H), 1.06 (s, 9H), 0.91 (dd, J = 6.6 Hz, 6H).

[0716] C35H53N5O5S calcd m/z = 655.38 found [M+H]+ = 656.4 Example 108 (S,E)-N-(4-amino-3-(trifluoromethoxy)phensulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3 -methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0717] Title compound was prepared from example 3 and 2,2,2-trifluoro-N-(4-sulfamoyl-2-(trifluoromethoxy)phenyl)acetamide according to general procedures 2, 3 and 7.

[0718] 1H NMR (400 MHz, Methanol-d4) δ 7.81 - 7.75 (m, 1H), 7.71 (dd, J = 8.7, 2.1 Hz, 1H), 7.55 (d, J = 7.9 Hz, 2H), 7.47 (t, J = 7.6 Hz, 2H), 7.37 (t, J = 7.1 Hz, 1H), 6.89 (d , J = 8.7 Hz, 1H), 6.51 - 6.42 (m, 1H), 4.98 (t, J = 10.0 Hz, 1H), 4.92 (t, J = 4, 1 Hz, 1H), 4.37 (s, 1H), 3.16 (s, 3H), 2.51 (s, 3H), 2.12 - 2.01 (m, 1H), 1.88 ( d, J = 1.4 Hz, 3H), 1.47 (s, 3H), 1.37 (s, 3H), 1.07 (s, 9H), 0.92 (dd, J = 6.6 Hz, 6H).

[0719] C34H48F3N5O6S calcd m/z = 711.33 found [M+H]+ = 712.4 Example 109 (S,E)-N-(4-amino-2,3-dimethylphenisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3- methyl-2-(methylamino)-3-phenylbutanamido)butanamido) hex-2-enamide

[0720] Title compound was prepared from example 3 and 2,2,2-trifluoro-N-(4-sulfamoyl-2,3-dimethylphenyl)acetamide according to general procedures 2, 3 and 7.

[0721] 1H NMR (400 MHz, Methanol-d4) δ 7.75 (d, J = 8.8 Hz, 1H), 7.55 (d, J = 7.9 Hz, 2H), 7.47 ( t, J = 7.7 Hz, 2H), 7.37 (t, J = 6.9 Hz, 1H), 6.63 (d, J = 8.8 Hz, 1H), 6.46 (d, J = 9.7 Hz, 1H), 5.00 (t, J = 10.0 Hz, 1H), 4.93 (s, 1H), 4.32 (s, 1H), 3.17 (s, 3H), 2.54 (s, 3H), 2.49 (s, 3H), 2.09 (s, 3H), 2.08 - 2.02 (m, 1H), 1.87 (d, J = 1.4 Hz, 3H), 1.47 (s, 3H), 1.37 (s, 3H), 1.07 (s, 9H), 0.92 (dd, J = 6.8, 6, 5Hz, 6H).

[0722] C35H53N5O5S calcd m/z = 655.38 found [M+H]+ = 656.4 Example 110 (S,E)-N-(4-amino-5,6,7,8-tetrahydronaphthalen-1-isulfonyl)-2,5-dimethyl-4- ((S)-N,3,3-trimethyl-2 -((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0723] Title compound was prepared from example 3 and 2,2,2-trifluoro-N-(4-sulfamoyl-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide according to general procedures 2 , 3 and 7.

[0724] 1H NMR (400 MHz, Methanol-d4) δ 7.74 (d, J = 8.7 Hz, 1H), 7.55 (d, J = 7.9 Hz, 2H), 7.48 ( t, J = 7.6 Hz, 2H), 7.38 (t, J = 7.2 Hz, 1H), 6.60 (d, J = 8.7 Hz, 1H), 6.46 (d, J = 9.2 Hz, 1H), 5.00 (t, J = 10.0 Hz, 1H), 4.95 - 4.91 (m, 1H), 4.36 (s, 1H), 3, 17 (s, 3H), 3.10 - 3.05 (m, 2H), 2.51 (s, 3H), 2.46 (t, J = 6.5 Hz, 2H), 2.10 - 2 .02 (m, 1H), 1.88 (s, 3H), 1.87 - 1.75 (m, 4H), 1.47 (s, 3H), 1.38 (s, 3H), 1, 07 (s, 9H), 0.92 (dd, J = 7.1 Hz, 6H).

[0725] C37H55N5O5S calcd m/z = 681.39 found [M+H]+ = 682.4 Example 111 (S,E)-N-(4-amino-3-methylphenisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl- 2-((S)-3-methyl- 2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0726] Title compound was prepared from example 3 and 2,2,2-trifluoro-N-(2-methyl-4-sulfamoylphenyl)acetamide according to general procedures 2, 3 and 7.

[0727] 1H NMR (400 MHz, Methanol-d4) δ 7.64 (s, 1H), 7.61 (dd, J = 8.5, 2.3 Hz, 1H), 7.57 - 7.51 (m, 2H), 7.48 (t, J = 7.7 Hz, 2H), 7.41 - 7.35 (m, 1H), 6.71 (d, J = 8.5 Hz, 1H) , 6.43 (dd, J = 9.3, 1.6 Hz, 1H), 4.96 (t, J = 10.0 Hz, 1H), 4.92 (s, 1H), 4.35 ( s, 1H), 3.16 (s, 3H), 2.51 (s, 3H), 2.17 (s, 3H), 2.10 - 2.01 (m, 1H), 1.87 (d , J = 1.4 Hz, 3H), 1.46 (s, 3H), 1.36 (s, 3H), 1.07 (s, 9H), 0.91 (dd, J = 6.3 Hz , 6H).

[0728] C34H51N5O5S calcd m/z = 641.36 found [M+H]+ = 642.4 Example 112 (S,E)-N-(4-amino-3-fluorophenisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl- 2-((S)-3-methyl- 2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0729] Title compound was prepared from example 3 and 2,2,2-trifluoro-N-(2-fluoro-4-sulfamoylphenyl)acetamide according to general procedures 2, 3 and 7.

[0730] 1H NMR (400 MHz, Methanol-d4) δ 7.62 - 7.55 (m, 3H), 7.54 (s, 1H), 7.48 (t, J = 7.7 Hz, 2H ), 7.37 (t, J = 7.3 Hz, 1H), 6.85 (t, J = 8.6 Hz, 1H), 6.45 (d, J = 9.3 Hz, 1H), 4.98 (t, J = 9.9 Hz, 1H), 4.92 (s, 1H), 4.34 (s, 1H), 3.16 (s, 3H), 2.50 (s, 3H ), 2.12 - 2.00 (m, 1H), 1.88 (d, J = 1.4 Hz, 3H),1.46 (s, 3H), 1.37 (s, 3H), 1 .07 (s, 9H), 0.91 (dd, J = 6.8 Hz, 6H).

[0731] C33H48FN5O5S calcd m/z = 645.34 found [M+H]+ = 646.4 Example 113 (S,E)-N-(4-amino-3-ethylphenisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2- ((S)-3-methyl- 2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0732] Title compound was prepared from example 3 and 2,2,2-trifluoro-N-(2-ethyl-4-sulfamoylphenyl)acetamide according to general procedures 2, 3 and 7.

[0733] 1H NMR (400 MHz, Methanol-d4) δ 7.66 (d, J = 2.3 Hz, 1H), 7.61 (dd, J = 8.6, 2.3 Hz, 1H), 7.55 (d, J = 7.6 Hz, 2H), 7.48 (t, J = 7.7 Hz, 2H), 7.37 (t, J = 7.3 Hz, 1H), 6, 71 (d, J = 8.5 Hz, 1H), 6.43 (dd, J = 9.3, 1.7 Hz, 1H), 4.96 (t, J = 9.9 Hz, 1H), 4.92 (s, 1H), 4.35 (s, 1H), 3.16 (s, 3H), 2.54 (dd, J = 7.4, 2.2 Hz, 2H), 2.51 (s, 3H), 2.12 - 1.99 (m, 1H), 1.87 (d, J = 1.4 Hz, 3H), 1.46 (s, 3H), 1.36 (s, 3H), 1.27 (t, J = 7.5 Hz, 3H), 1.07 (s, 9H), 0.91 (dd, J = 6.4 Hz, 6H)

[0734] C35H53N5O5S calcd m/z = 655.38 found [M+H]+ = 656.5 Example 114 (S,E)-N-(4-amino-3-(trifluoromethyl)phensulfonyl)-2,5-dimethyl-4-((S)- N,3,3-trimethyl-2-((S)-3 -methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

[0735] Title compound was prepared from example 3 and 2,2,2-trifluoro-N-(2-trifluoromethyl-4-sulfamoylphenyl)acetamide according to general procedures 2, 3 and 7.

[0736] 1H NMR (400 MHz, Methanol-d4) δ 8.04 (s, 1H), 7.87 (d, J = 8.8 Hz, 1H), 7.55 (d, J = 7.6 Hz, 2H), 7.48 (t, J = 7.3 Hz, 2H), 7.36 (dd, J = 14.5, 7.4 Hz, 1H), 6.89 (d, J = 8 .9 Hz, 1H), 6.47 (d, J = 9.3 Hz, 1H), 4.99 (t, J = 10.2 Hz, 1H), 4.92 (s, 1H), 4, 33 (s, 1H), 3.16 (s, 3H), 2.50 (s, 3H), 2.11 - 2.00 (m, 1H), 1.88 (s, 3H), 1.47 (s, 3H), 1.37 (s, 3H), 1.07 (s, 9H), 0.91 (dd, J = 7.0 Hz, 6H).

[0737] C34H48F3N5O5S calcd m/z = 695.33 found [M+H]+ = 696.4 Example 115 (S)-1-isopropyl-N-((S)-1-(((S,E)-6-(3-mercaptopropisulfonamido)-2,5-dimethyl-6-oxohex-4-en-3-yl )(methyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)piperidine-2-carboxamide

[0738] For a solution of (S,E)-ethyl 4-((S)-2-(tert-butoxycarbonylamino)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoate (0.373 g , 0.905mmol) in CH2Cl2 (5 mL) trifluoroacetic acid (2 mL) was added. The reaction was monitored by HPLC and upon complete conversion of the concentrated raw material under reduced pressure. N-isopropyl-pipecolic acid (0.200 g, 1.3 equiv) was dissolved in CH2Cl2 (5 mL) and stirred at 0oC, to which was added HBTU (0.450 g, 1.3 equiv) and N,N-diisopropylethylamine (0.400 uL, 2.5 equiv). After 10 minutes, the above deprotected dipeptide was added as a solution in CH2Cl2 (~1mL). The reaction was monitored by HPLC for complete consumption of the dipeptide, at which time the entire reaction was concentrated under reduced pressure. The crude reaction mixture was dissolved in CH2Cl2 and purified by silicon gel chromatography (1-20% MeOH (5% NH4OH) in CH2Cl2).

[0739] The resulting ester was saponified with LiOH in 1,4-dioxane. The resulting carboxylic acid (0.128 g, 0.29 mmol) was dissolved in CH2Cl2 (5 mL) and to the stirred solution was added dicyclohexylcarbodiimide (0.084 g, 1.4 equiv), N,N-dimethylaminopyridine (0.05 g, 1 .4 equiv) and 3-(tritylthio)propane-1-sulfonamide (0.174 g, 1.5 equiv). The resulting mixture was stirred overnight and monitored for reaction progress by HPLC-MS. When the reaction was complete, the mixture was concentrated under reduced pressure and the residue was purified by silicon gel chromatography (5-30% MeOH in CH2Cl2) to give S-trityl derivative of the parent compound as a colorless oil ( 0.056 g).

[0740] 1H NMR (400 MHz, Methanol-d4) δ 7.44 - 7.35 (m, 6H), 7.36 - 7.15 (m, 9H), 6.56 (dd, J = 9, 1, 1.7 Hz, 1H), 5.03 (dd, J = 10.6, 9.3 Hz, 1H), 4.73 (s, 1H), 4.05 (dd, J = 11.5 , 3.3 Hz, 1H), 3.51 - 3.37 (m, 2H), 3.25 - 3.15 (m, 2H), 3.09 (s, 3H), 2.92 (td, J = 12.5, 2.9 Hz, 1H), 2.31 (t, J = 7.2 Hz, 2H), 2.18 - 1.70 (m, 15H), 1.61 (ddt, J = 12.8, 8.4, 4.9 Hz, 1H), 1.28 (dd, J = 30.1, 6.7 Hz, 7H), 1.04 (s, 9H), 0.88 ( dd, J = 37.3, 6.5 Hz, 6H).

[0741] Finally, the trityl-protected thiol was dissolved in CH2Cl2 (3ml) and trifluoroacetic acid was added (0.6ml) with triisopropyl silane (0.1ml). The reaction was monitored by HPLC-MS and upon completion concentrated to dryness under reduced pressure. The residue was taken up in CH2Cl2 (~0.8mL) with a few drops of ethyl alcohol and cooled to 0oC in an ice bath. Cold diethyl ether (~3 mL) was added with vigorous stirring to generate a white precipitate, which was collected by filtration in a Buchner funnel and dried under high vacuum to yield the parent compound as a white amorphous solid.

[0742] 1H NMR (400 MHz, Methanol-d4) δ 6.52 (d, J = 9.0 Hz, 1H), 5.06 (dd, J = 10.7, 8.8 Hz, 1H), 4.73 (s, 1H), 4.16 - 4.04 (m, 1H), 3.69 - 3.56 (m, 2H), 3.48 (dd, J = 13.3, 7.2 Hz, 2H), 3.15 (s, 3H), 3.03 - 2.94 (m, 1H), 2.68 (t, J = 6.9 Hz, 1H), 2.24 - 1.77 (m, 11H), 1.61 (s, 1H), 1.31 (dd, J = 27.2, 6.7 Hz, 6H), 1.06 (s, 9H), 0.91 (dd, J = 34.1, 6.6 Hz, 6H). Example 116 S)-N-((S)-1-((S)-2-((E)-3-(3-mercaptopropisulfonamido)-2-methyl-3-oxoprop-1-enyl)pyrrolidin-1-yl) -3,3-dimethyl-1-oxobutan-2-yl)-3-methyl-2-(methylamino)-3-phenylbutanamide

[0743] The title compound was synthesized from Boc-proline and example 2 according to general procedures 10, 11, 2, 3, 7 and others of Nieman J. A. et al. J. Nat. Prod. 2003, 66, 183-199. The compound was isolated as two diastereoisomers in a ratio of approximately 1:1.

[0744] 1H NMR (400 MHz, Methanol-d4) δ 7.57 - 7.12 (m, 5H), 6.39 (dd, J = 9.4, 1.6 Hz, 0.5H), 6 .31 (dd, J = 8.2, 1.5 Hz, 0.5H), 4.72 (q, J = 7.5 Hz, 0.5H), 4.66 - 4.56 (m, 0 .5H), 4.40 (s, 0.5H), 4.28 (d, J = 11.9 Hz, 1H), 3.81 (m, 0.5H), 3.76 - 3.56 ( m, 3H), 2.77 - 2.64 (m, 2H), 2.59 (m, 3H), 2.39 - 2.22 (m, 1H), 2.18 - 1.72 (m, 7H), 1.61 - 1.33 (m, 6H), 1.15 - 0.85 (m, 11H).

[0745] C29H46N4O5S2 calcd m/z = 594.35 found [M+H]+ = 595.3 Example 117 (S)-N-((S)-1-(2-(3-(3-mercaptopropisulfonamido)-2-methyl-3-oxoprop- 1-enyl)piperidin-1-yl)-3,3-dimethyl- 1-oxobutan-2-yl)-3-methyl-2-(methylamino)-3-phenylbutanamide

[0746] The title compound was synthesized from Boc-homoproline and example 2 according to general procedures 10, 11, 2, 3, 7 and others of Nieman J. A. et al. J. Nat. Prod. 2003, 66, 183-199. The compound was isolated as two diastereoisomers in a ratio of approximately 2:3.

[0747] 1H NMR (600 MHz, Methanol-d4) δ 7.55 (d, J = 7.8 Hz, 1H), 7.46 (m, 3H), 7.38 (m, 1H), 6, 81 (d, J = 8.3 Hz, 0.6H), 6.79 (d, J = 7.8 Hz, 0.4H), 5.66 (m, 0.6H), 5.12 (m , 0.4H), 5.05 (s, 0.6H), 4.86 (s, 0.4H), 4.42 (d, J = 14.9 Hz, 0.4H), 4.35 ( s, 0.6H), 4.26 (s, 0.4H), 4.12 (d, J = 13.8 Hz, 0.6H), 3.64 (d, J = 7.6 Hz, 1H ), 3.63 (d, J = 7.4 Hz, 1H), 3.39 (m, 0.6H), 2.94 (td, J = 13.8, 2.6 Hz, 0.4H) , 2.68 (t, J = 6.7 Hz, 2H), 2.56 (m, 3H), 2.10 (m, 3.5H), 1.97 (s, 1.5H), 1, 90-1.70 (m, 7H), 1.65-1.29 (m, 6H), 1.07 (s, 3.5H), 1.04 (s, 4.5H) ppm.

[0748] C30H47N4O5S2 calcd. m/z = 608.31; found [M+H]+ =609.32 Example 118 (S)-N-((S)-1-(2-(3-(4-(mercaptomethyl)phenisulfonamido)-2-methyl-3-oxoprop-1-enyl)piperidin-1-yl)-3,3 -dimethyl-1-oxobutan-2-yl)-3-methyl-2-(methylamino)-3-phenylbutanamide

[0749] The title compound was synthesized from Boc-homoproline and example 7 according to general procedures 10, 11, 2, 3, 7 and others of Nieman J. A. et al. J. Nat. Prod. 2003, 66, 183-199. The compound was isolated as two diastereoisomers in a ratio of approximately 2:3.

[0750] 1H NMR (600 MHz, Methanol-d4) δ 8.02 (d, J = 8.4 Hz, 0.8H), 8.00 (d, J = 8.5 Hz, 1.2H), 7.58 (d, J = 8.5 Hz, 1H), 7.54 (d, J = 8.5 Hz, 2H), 7.45 (t, J = 8.2 Hz, 2H), 7, 40 (d, J = 7.2 Hz, 0.6H), 7.36 (m, 1H), 7.31 (t, J = 7.1 Hz, 0.4H), 6.74 (d, J = 8.2 Hz, 1H), 5.59 (m, 0.6H), 5.06 (m, 0.4H), 5.02 (s, 0.6H), 4.84 (s, 0, 4H), 4.39 (d, J = 12.5 Hz, 0.4H), 4.34 (s, 0.6H), 4.20 (s, 0.4H), 4.08 (d, J = 12.0 Hz, 0.6H), 3.83 (s, 1.2H), 3.73 (s, 0.8H), 3.35 (m, 0.6H), 2.93 (td, J = 13.6, 3.0 Hz, 0.4H), 2.55 (m, 3H), 2.00 (s, 1H), 1,901.51 (m, 7H), 1.51-1.30 (m, 4H), 1.30 (s, 1H), 1.15 (s, 1H), 1.04 (s, 3.5H), 1.01 (s, 4.5H) ppm.

[0751] C34H47N4O5S2 calcd. m/z = 656.31; found [M+H]+ =657.30 Example 119 MC-VC-PABC-77 The title compound was prepared by applying general procedures 15 and 7 of example 77 Boc-protected.

[0752] 1H NMR (400 MHz, Methanol-d4) δ 7.58 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 7.5 Hz, 2H), 7.38 ( t, J = 7.7 Hz, 2H), 7.36 - 7.24 (m, 6H), 7.22 (d, J = 7.8 Hz, 2H), 6.81 (s, 2H), 6.57 (d, J = 9.1 Hz, 1H), 5.08 (s, 2H), 5.04 (t, J = 10.0 Hz, 1H), 4.91 (s, 1H), 4.53 (dd, J = 9.0, 5.1 Hz, 1H), 4.40 (s, 2H), 4.28 (s, 2H), 4.19 (d, J = 7.4 Hz , 1H), 3.49 (t, J = 7.1 Hz, 2H), 3.26 - 3.11 (m, 2H), 3.07 - 2.93 (m, 3H), 2.30 ( t, J = 7.4 Hz, 2H), 2.18 (s, 3H), 2.15 - 2.05 (m, 1H), 1.99 - 1.91 (m, 1H), 1.89 (s, 3H), 1.83 - 1.72 (m, 1H), 1.72 - 1.53 (m, 7H), 1.44 (s, 3H), 1.37 (s, 3H), 1.35 - 1.27 (m, 2H), 1.03 (s, 9H), 1.00 (d, J = 6.8 Hz, 3H), 0.99 (d, J = 6.7 Hz , 3H), 0.88 (d, J = 6.5 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H).

[0753] C64H91N11O13S calcd. m/z = 1253.7; found [M+H]+ =1254.8, Example 120 4-((R)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5- ureidopentanamido)benzyl 4-(N-((S,E)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2-( methylamino)-3-phenylbutanamido)butanamido)hex-2-enoyl)sulfamoyl)benzylcarbamate,MC-VC-PABC-85

[0754] The title compound was prepared by applying general procedures 15 and 7 to example 85 Boc-protected.

[0755] C63H89N11O13S calcd. m/z = 1239.6; found [M+H]+ = 1240.9.Example 121 MC-VC-PABC-80

[0756] The title compound was prepared by applying general procedures 15 and 7 to Boc-protected example 80.

[0757] C63H89N11O13S calcd. m/z = 1239.6; found [M+H]+ = 1240.9. Example 122 MC-VC-PABC-41

[0758] The title compound was prepared by applying general procedure 15 to example 41.

[0759] C64H91N11O13S calcd. m/z = 1253.65; found [M+H]+ = 1254.75, [M+2H]2+ = 628.20. Example 123 (R)-N-(benzisulfonyl)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3- phenylbutanamido)butanamido)hexanamide

[0760] The suspension of example 14 and 10% palladium on carbon (25 mol% Pd) in glacial acetic acid was stirred under an atmosphere of H2 (1 atm) at room temperature. After 142 h, the reaction suspension was passed through a celite bed, washed with MeOH (5x) and concentrated in vacuo. The crude brown residual film was dissolved and purified on preparative HPLC (30-70% MeCN/H2O with 0.1% TFA) and lyophilized to yield a diastereoisomer of the reduced product as a pale yellow solid in 15% yield.

[0761] 1H NMR (400 MHz, Methanol-d4) δ 7.55 (d, J = 7.2 Hz, 2H), 7.46 (t, J = 7.8 Hz, 2H), 7.43 - 7.31 (m, 6H), 5.01 (s, 1H), 4.79 (d, J = 14.1 Hz, 1H), 4.65 (d, J = 14.1 Hz, 1H), 4.35 (s, 1H), 4.24 (s, 1H), 3.07 (s, 3H), 2.52 (s, 3H), 2.27 (m, J = 10.3, 7, 0, 3.2 Hz, 1H), 2.14 (ddd, J = 13.5, 10.6, 2.7 Hz, 2H), 1.78 (d, J = 8.6 Hz, 1H), 1.47 (s, 3H), 1.34 (s, 3H), 1.15 (d, J = 6.9 Hz, 3H), 1.14 (s, 9H), 1.04 (d, J = 6.6 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H).

[0762] C34H52N4O5S calcd m/z = 628.37 amu; found [M+H]+ = 629.6, [M+Na]+ = 651.6 GENERAL SYNTHETIC SCHEMES FOR (T)-(L)-(D) USING SMCC AND LC-SPDP LIGANDS mAb-SPDP-S-R2'-peptide-NHSO2R1

[0763] Composition produced using the SPDP binding method described below. Note that R2' is distinct from R2, since R2 includes R2'-S.

[0764] Composition produced using the SMCC bonding method described below. Note that R2' is distinct from R2, since R2 includes R2'-S.

[0765] Composition produced using the SPDP binding method described below. Note R1' is distinct from R1, since R1 includes R1'-S.

[0766] Composition produced using the SMCC bonding method described below. Note R1' is distinct from R1, since R1 includes R1'-S. Example 124 (Compound A - SPDP - mAb) produced using the compound A synthesis method, above, and the SPDP ligation method described below. Example 125 (Compound B - SPDP - mAb) produced using the compound B synthesis method, above and the SPDP ligation method described below. Example 126 (Compound C - SPDP - mAb) produced using the compound C synthesis method, above and the SPDP ligation method described below. Example 127 (Compound B - SMCC-mAb) produced using the compound B synthesis method, above and the SMCC ligation method described below. Example 128 (Compound A - SMCC - mAb) produced using the compound A synthesis method, above, and the SMCC ligation method described below.Example 129: (Compound C - SMCC- mAb) produced using the compound C synthesis method, above and the SMCC ligation method described below. Example 130 3-methyl-3-(4-bromophenyl)-butanoic acid

[0767] to a vigorously stirred solution of bromobenzene (4.70 g, 30.0 mmol) and 3,3-dimethylacrylic acid (1.00 g, 10.0 mmol) in 20 mL of CH2Cl2 cooled to -10°C in NH4Cl(aq)/ice bath, solid AICI3 was added in portions, keeping the internal temperature below -5 °C. The solution turned yellow and then brown after addition. After one hour, LC and TLC analysis indicated total consumption of the limiting reagent. The reaction was then quenched by the addition of 1M citric acid, causing the brown color to fade into yellow. The resulting aqueous suspension was extracted four times with 20ml of Et2O, the combined organics washed with NaCl(sat), dried over Na2SO4(s) and concentrated in vacuo with heating to 45°C to remove solvent and residual bromobenzene. The resulting oil slowly solidified. Recrystallization of the crude solid from hexanes yielded the title compound (1.29 g, 50%) as clusters of white prisms.

[0768] 1H NMR (400 MHz, Chloroform-d) δ (ppm) 7.42 (d, J = 8.6 Hz, 2H), 7.23 (d, J = 8.6 Hz, 2H), 2 .63 (s, 2H), 1.43 (s, 6H). C11H13BrO2 calcd. [M+H]+ = 257.02 amu; found m/z = 257.03. Rf = 0.21 (20% (2% AcOH/EtOAc)/Hex).Example 131 3-methyl-3-(3-bromophenyl)-butanoic acid

[0769] The title compound was prepared in the same way as 3-methyl-3-phenylbutanoic acid in Nieman J. a., et al. J. Nat. Prod. 2003, 66, 183-199, using bromobenzene in place of benzene as solvent and replacing the acid-base examination with a simple extraction of the 1 M citric acid reaction mixture and three successive recrystallizations of hexanes. From a crude product enriched in the desired metalomer as a 2:1 mixture, the title compound can be obtained as thick white needles in greater than 95% purity.

[0770] 1H NMR (400 MHz, Chloroform-d) δ (ppm) 7.49 (t, J = 1.9 Hz, 1H), 7.34 (ddd, J = 7.9, 1.9, 1 .0 Hz, 1H), 7.29 (ddd, J = 7.9, 1.9, 1.0 Hz, 1H), 7.18 (t, J = 7.9 Hz, 1H), 2.64 (s, 2H), 1.44 (s, 6H), C11H13BrO2 calcd. [M+H]+ = 257.02 amu; found m/z = 257.01, Rf = 0.21 (20% (2% AcOH/EtOAc)/Hex).Example 132 (S)-methyl 3-(4-bromophenyl)-2-(tert-butoxycarbonyl(methyl)amino)-3-methylbutanoate

[0771] The title compound was synthesized from example 130 according to the sequence of procedures described by Nieman et al for the synthesis of (S)-methyl 2-(tert-butoxycarbonyl(methyl)amino)-3-methyl -3- phenylbutanoate.Example 133 (S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-(4-((14-hydroxy-3,6,9,12-tetraoxatetradecyl)oxy)phenyl)-3-methylbutanoic acid

[0772] To a stirred solution of example 68 (157 mg, 0.405 mmol) in pentaethylene glycol (1.5 mL) were added CsCO3 (330 mg, 1.01 mmol), 3,4,7,8-tetramethyl-1 ,10-phenanthroline (57 mg, 0.24 mmol) and CuI (23 mg, 0.12 mmol). Nitrogen was blown into the flask, then sealed and heated to 130°C, the solution quickly turning from red to brown to black. After 40 h, the reaction appeared to be nearly complete by HPLC analysis. Thus, the mixture was allowed to cool to room temperature, diluted with H2O and transferred to a larger Erlenmeier with a stir bar. This mixture was carefully acidified to f ~ 3 with 1M citric acid, paying attention not to allow the foamy mixture to boil over. The mixture was then extracted five times with CH2Cl2, the combined organic extracts washed with NaCl(sat), dried over Na2SO4(s) and concentrated in vacuo to yield about 300 mg of crude oil. Purification by flash chromatography (1 - 10% MeOH/(2% AcOH/EtOAc)) yielded the title compound (66 mg, 30%) as a clear film that existed as an assembly of N-Boc rotamers in a approximate ratio of 2:1.

[0773] 1H NMR (400 MHz, Chloroform-d) δ (ppm) 7.35 (d, J = 7.8 Hz, 1.3H), 7.30 (d, J = 7.6 Hz, 0, 7H), 6.87 (d, J = 7.1 Hz, 2H), 5.07 (s, 0.7H), 4.93 (s, 0.3H), 4.14 (m, 2H), 3.86 (m, 2H), 3.70 (m, 16H), 2.83 (s, 1H), 2.72 (s, 2H), 1.54 (s, 3H), 1.49 (s , 3H), 1.45 (s, 9H), C27H45NO10 calcd. [M+H]+ = 544.31 amu; found m/z = 544.36, Rf = 0.36 (5% MeOH/(2% AcOH/EtOAc)).Example 134 (S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-(4-(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethoxy)phenyl)-3-methylbutanoic acid

[0774] The title compound was prepared according to the above method of example 68 (132 mg, 0.341 mmol), CsCO3 (278 mg, 0.853 mmol), 3,4,7,8-tetramethyl-1,10 -phenanthroline (24 mg, 0.10 mmol) and CuI (10 mg, 0.051 mmol). Flash chromatography (1 - 10% MeOH/(2% AcOH/EtOAc)) gave the title compound (66 mg, 38%) as a clear oil in an approximate 2:1 ratio of N-Boc rotamers.

[0775] 1H NMR (400 MHz, Chloroform-d) δ (ppm) 7.34 (d, J = 8.4 Hz, 1.3H), 7.29 (d, J = 8.1 Hz, 0, 7H), 6.85 (d, J = 8.4 Hz, 2H), 5.05 (s, 0.7H), 4.91 (s, 0.3H), 4.13 (t, J = 4 .6 Hz, 2H), 3.87 - 3.79 (m, 2H), 3.76 - 3.60 (m, 10H), 3.59 (t, J = 4.1 Hz, 2H), 2 .80 (s, 1H), 2.69 (s, 2H), 1.53 (s, 3H), 1.48 (s, 3H), 1.44 (s, 9H), C25H41NO9 calcd. [M+H]+ = 500.29 amu; found m/z = 500.36, Rf = 0.46 (5% MeOH/(2% AcOH/EtOAc)).Example 135 (S)-3-(3-((14-hydroxy-3,6,9,12-tetraoxatetradecyl)oxy)phenyl)-3-methyl-2-(methylamino)butanoic acid

[0776] The precursor to the title compound, (S)-3-(3-bromophenyl)- 2-(((tert-butoxycarbonyl)(methyl)amino)-3-methylbutanoic acid, was prepared from example 131 following the procedures in Neiman et al.

[0777] Thus, following the procedures above, from (S)-3-(3-bromophenyl)-2-(((tert-butoxycarbonyl)(methyl)amino)-3-methylbutanoic acid (166 mg, 0. 43 mmol), CsCO3 (330 mg, 1.01 mmol), 3,4,7,8-tetramethyl-1,10-phenanthroline (31 mg, 0.13 mmol) and CuI (12.3, 0.060 mmol) in 1.5 mL pentaethylene glycol heated at 130°C for two days, the title compound (73 mg 31%) was obtained as a clear oil after flash chromatography (1 - 10% MeOH/(2% AcOH/EtOAc )) in an approximate 2:1 ratio of N-Boc rotamers.

[0778] 1H NMR (400 MHz, Chloroform-d) δ (ppm) 7.17 (t, J = 7.8 Hz,1H), 7.14 - 7.07 (m, 1H), 7.07 - 6.93 (m, 2H), 6.74 (d, J = 8.0 Hz, 1H), 5.11 (s, 0.7H), 4.93 (s, 0.3H), 4.25 - 4.03 (m, 2H), 3.91 - 3.77 (m, 2H), 3.78 - 3.66 (m, 2H), 3.69 - 3.43 (s, 14H), 2 .72 (s, 1H), 2.65 (s, 1H), 1.51 (s, 3H), 1.49 (s, 3H), 1.45 (s, 9H), C27H45NO10 calcd. [M+H]+ = 544.31 amu; found m/z = 544.34.Example 136 (6S,9S,12S,E)-ethyl 9-(tert-butyl)-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-6-(2-(4 -((16-oxo-3,6,9,12-tetraoxa-15-thiaheptadecyl)oxy)phenyl)propan-2-yl)-3-oxa-5,8,11-triazapentadec-13-en-15- the act

[0779] Acid (S)-2-(((tert-butoxycarbonyl)(methyl)amino)-3-(4-((14-hydroxy-3,6,9,12-tetraoxatetradecyl)oxy)phenyl)-3 -methylbutanoic acid (65 mg, 0.120 mmol) was coupled to (S,E)-ethyl 4-(((S)-2-amino-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoate with HATU and DIPEA, following the same stoichiometry and procedure as described in the general coupling procedures in Nieman et al to yield an alcohol-free intermediate after purification by flash chromatography (1-10% MeOH / (2% AcOH/EtOAc)). Then, triphenylphosphine (40 mg, 0.15 mmol) in 0.75 mL of tF under N2 at 0°C, di-tert-butyl azodicarboxylate (35 mg, 0.15 mmol) was added in one portion. After 35 minutes, a white precipitate came out and the reaction became difficult to stir. To this suspension, a solution of the intermediate alcohol (42 mg, 0.050 mmol) in tF to 0.75 mL was added diluting the precipitate sufficiently to restore stirring. Five minutes later, thioacetic acid (5.7 mg, 0.075 mmol) at tF of 0.05 mL was added causing all the yellow color to fade from the mixture. After 30 min, the reaction was allowed to warm to room temperature. The precipitate disappeared after another 15 minutes, and analysis by TLC and LCMS showed almost complete conversion. A further 40 minutes later, the reaction mixture was concentrated in vacuo and then subjected directly to flash chromatography (40-100% EtOAc/Hex then 10% MeOH/EtOAc) to yield the title compound (26 mg, 57% ) as a transparent film.

[0780] 1H NMR (400 MHz, Chloroform-d) δ (ppm) 7.43 (d, J = 8.4 Hz, 1.3H), 7.31 (d, J = 8.3 Hz, 0, 7H), 6.97 - 6.72 (m, 2H), 6.62 (dd, J = 9.3, 1.6 Hz, 1H), 6.14 (d, J = 9.6 Hz, 1H ), 5.22 (s, 0.7H), 5.12 - 4.99 (m, 1H), 4.84 (s, 0.3H), 4.69 (d, J = 9.3 Hz, 0.3H), 4.60 (d, J = 8.9 Hz, 0.7H), 4.19 (q, J = 7.2 Hz, 2H), 4.09 (td, J = 4.6 , 2.3 Hz, 2H), 3.84 (t, J = 4.9 Hz, 2H), 3.77 - 3.70 (m, 2H), 3.70 - 3.61 (m, 10H) , 3.59 (t, J = 6.4 Hz, 2H), 3.07 (t, J = 6.4 Hz, 2H), 2.97 - 2.91 (m, 3H), 2.84 ( s, 3H), 2.32 (s, 3H), 1.87 (s, 3H), 1.49 (s, 3H), 1.43 (s, 9H), 1.35 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H), 0.80 (d, J = 16.6 Hz, 3H), 0, 77 (s, 9H), C46H77N3O12S calcd. [M+H]+ = 896.53 amu; found m/z = 896.77, Rf = 0.56 (80% EtOAc/Hex).Example 137: (6S,9S,12S,E)-ethyl 9-(tert-butyl)-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-6-(2-(4 -((13-oxo-3,6,9-trioxa-12-thiatetradecyl)oxy)phenyl)propan-2-yl)-3-oxa-5,8,11-triazapentadec-13-en-15-oate

[0781] The title compound was prepared from (S)-2-(((tert-butoxycarbonyl)(methyl)amino)-3-(4-(2-(2-(2-(2-hydroxyethoxy) )ethoxy)ethoxy)ethoxy)phenyl)-3-methylbutanoic acid (66 mg, 0.065 mmol) following the same procedure described above to give 32 mg (57%) as a clear film after flash chromatography (20 - 100% EtOAc/Hex) .

[0782] 1H NMR (400 MHz, Chloroform-d) δ (ppm) 7.44 (d, J = 8.5 Hz, 1.3H), 7.32 (d, J = 8.5 Hz, 0, 7H), 6.95 - 6.77 (m, 2H), 6.62 (dd, J = 9.2, 1.7 Hz, 1H), 6.09 (d, J = 9.1 Hz, 1H ), 5.24 (s, 0.7H), 5.13 - 4.95 (m, 1H), 4.84 (s, 0.3H), 4.69 (d, J = 9.6 Hz, 0.3H), 4.60 (d, J = 9.0 Hz, 0.7H), 4.19 (q, J = 7.1 Hz, 2H), 4.09 (td, J = 4.7 , 2.4 Hz, 2H), 3.84 (t, J = 4.9 Hz, 2H), 3.72 (dd, J = 5.7, 3.2 Hz, 2H), 3.70 - 3 .65 (m, 2H), 3.66 - 3.62 (m, 4H), 3.60 (t, J = 6.5 Hz, 2H), 3.09 (t, J = 6.5 Hz, 2H), 2.96 - 2.88 (m, 3H), 2.84 (s, 3H), 2.33 (s, 3H), 1.88 (d, J = 3.5 Hz, 3H), 1.49 (s, 2H), 1.43 (d, J = 5.5 Hz, 11H), 1.35 (s, 2H), 1.30 (t, J = 7.1 Hz, 2H), 0.87 (d, J = 6.6 Hz, 3H), 0.80 (d, J = 15.9 Hz, 3H), 0.76 (s, 9H), C44H73N3O11S calcd. [M+H]+ = 852.51 amu; found m/z = 852.79, Rf = 0.60 (60% EtOAc/Hex).Example 138 (6S,9S,12S,E)-ethyl 9-(tert-butyl)-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-6-(2-(3 -((16-oxo-3,6,9-trioxa-12-thiatetradecyl)oxy)phenyl)propan-2-yl)-3-oxa-5,8,11-triazapentadec-13-en-15-oate.

[0783] The title compound was prepared from (S)-3-(3-((14-hydroxy-3,6,9,12-tetraoxatetradecyl)oxy)phenyl)-3-methyl-2-( methylamino)butanoic acid (73 mg, 0.080 mmol) after the same procedure described above to give 66 mg (47%) as a clear film after flash chromatography (20-100% EtOAc/Hex).

[0784] 1 H NMR (400 MHz, chloroform-d) δ (ppm) 7.25 - 6.92 (m,3H), 6.78-6.70 (m, 1H), 6.62 (d, J = 8.9 Hz, 1 H), 6.12 (d, J = 8.9 Hz, 1 H),5.26 (s, 0.7 H), 5.12 - 4.99 (m, 1 H), 4.89 (s, 0.3 H), 4.74 - 4.56 (m, 1 H),4.19 (q, J = 7.2 Hz, 1 H), 4.16 - 4.03 (m 2 H), 3.84 (td, J = 5.0, 3.2 Hz, 2 H),3.77 - 3.61 (m, 14 H), 3.60 (t , J = 6.4 Hz, 2 H), 3.09 (t, J = 6.5 Hz, 2 H), 2.97 - 2.75 (m, 6 H), 2.33 (s, 3 H), 1.91 - 1.83 (m, 3 H), 1.52 - 1.35 (m, 16 H),1.26 (t, J = 7.1 Hz, 3 H), 0, 87 (d, J = 6.0 Hz, 3 H), 0.81 (d, J = 12.9 Hz, 3H), 0.77 (s, 9H), C46H77N3O12S calcd. [M+H]+ = 896.53 amu; found m/z = 896.68, Rf = 0.61 (75% EtOAc/Hex). Acid disulfide (S,E)-4-((S)-2-((S)-3-(4-((14-mercapto-3,6,9,12- tetraoxatetradecyl)oxy)phenyl)-3 -methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoic acid.

[0785] The title compound was prepared by saponification and then TFAo-promoted Boc removal according to the exact methods described in Nieman et al of (6S,9S,12S,E)-ethyl-9-( tert-butyl)-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-6-(2-(4-((16-oxo-3,6,9,12 - tetraoxa-15-thiaheptadecyl)oxy)phenyl)propan-2-yl)-3-oxa-5,8,11-triazapentadec-13-en-15-oate (26 mg, 0.029 mmol) to yield the title compound (16 mg, 90%) as a clear glass after complete removal of excess TFA.

[0786] 1H NMR (400 MHz, Methanol-d4) δ (ppm) 8.43 (d, J = 8.1 Hz, 1H), 7.47 (d, J = 8.5 Hz, 2H), 7 .08 - 6.94 (m, 2H), 6.80 (dq, J = 9.9, 1.5 Hz, 1H), 5.08 (t, J = 10.1 Hz, 1H), 4, 94 (d, J = 8.1 Hz, 1H), 4.32 (s, 1H), 4.21 - 4.12 (m, 2H), 3.93 - 3.81 (m, 3H), 3 .76 (t, J = 6.4 Hz, 2H), 3.76 - 3.72 (m, 2H), 3.72 - 3.62 (m, 10H), 3.17 (s, 3H), 2.92 (t, J = 6.4 Hz, 2H), 2.61 - 2.47 (m, 3H), 2.14 - 2.00 (m, 1H), 1.94 (d, J = 1.5 Hz, 3H), 1.46 (s, 3H), 1.40 (d, J = 7.7 Hz, 3H), 1.09 (s, 9H), 0.94 (d, J = 5.0 Hz, 3H), 0.92 (d, J = 4.8 Hz, 3H), C74H124N6O18S2 calcd. [M+H]+ = 1449.85 amu; found m/z = 1450.49.Example 140 Example compound 139 is reduced according to the methods below to produce the compound in question. Example 141 Acid disulfide (S,E)-4-((S)-2-((S)-3-(4-(2-(2-(2-(2-mercaptoethoxy)ethoxy)ethoxy)ethoxy)phenyl) -3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoic acid

[0787] The title compound was prepared by saponification and then TFAo-promoted Boc removal according to the exact methods described in Nieman et al of (6S,9S,12S,E)-ethyl 9-(tert -butyl)-12- isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-6-(2-(4-((13-oxo-3,6,9-trioxa- 12-thiatetradecyl)oxy)phenyl)propan-2-yl)-3-oxa-5,8,11-triazapentadec-13-en-15-oate (32 mg, 0.037 mmol to yield the title compound (29 mg, 86%) as a clear glass after complete removal of excess TFA.

[0788] 1H NMR (400 MHz, Methanol-d4) δ (ppm) 8.39 (d, J = 8.2 Hz, 1H), 7.44 (d, J = 8.9 Hz, 2H), 7 .01 (d, J = 8.5 Hz, 2H), 6.77 (d, J = 7.9 Hz, 1H), 5.05 (t, J = 10.1 Hz, 1H), 4.92 (d, J = 8.3 Hz, 1H), 4.28 (s, 1H), 4.15 (dd, J = 5.8, 3.4 Hz, 2H), 3.89 - 3.80 ( m, 2H), 3.73 (t, J = 6.4 Hz, 2H), 3.72 - 3.69 (m, 2H), 3.69 - 3.60 (m, 6H), 3.14 (s, 3H), 2.89 (t, J = 6.4 Hz, 2H), 2.50 (s, 3H), 2.11 - 1.97 (m, 1H), 1.91 (d, J = 1.4 Hz, 3H), 1.43 (s, 3H), 1.36 (s, 3H), 1.06 (s, 9H), 0.92 - 0.87 (m, 6H), C70H118N6O16S2 calcd. [M+H]+ =1361.80 amu; found m/z = 1362.26.Example 142 Example compound 141 is reduced according to the methods below to produce the compound in question. Example 143 Acid disulfide (S,E)-4-((S)-2-((S)-3-(4-((14-mercapto-3,6,9,12- tetraoxatetradecyl)oxy)phenyl)-3 -methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoic acid

[0789] The title compound was prepared by saponification and then TFA-promoted Boc removal according to the exact methods described in Nieman et al of (6S,9S,12S,E)-ethyl 9-(tert -butyl)-12- isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-6-(2-(3-((16-oxo-3,6,9,12- tetraoxa-15-thiaheptadecyl)oxy)phenyl)propan-2-yl)-3-oxa-5,8,11-triazapentadec-13-en-15-oate (56 mg, 0.029 mmol) to yield the title compound ( 43 mg, 82%) as an off-white foam after complete removal of excess TFA.

[0790] 1H NMR (400 MHz, Methanol-d4) δ (ppm) 8.48 (d, J = 8.3 Hz, 1H), 7.47 - 7.29 (m, 1H), 7.21 - 7.04 (m, 1H), 6.95 (t, J = 9.4 Hz, 1H), 6.80 (d, J = 9.7 Hz, 1H), 5.08 (t, J = 10 .1 Hz, 1H), 4.97 - 4.94 (m, 1H), 4.38 (s, 1H), 4.24 - 4.13 (m, 2H), 3.95 - 3.82 ( m, 2H), 3.80 - 3.58 (m, 14H), 3.17 (s, 3H), 2.92 (t, J = 6.4 Hz, 2H), 2.53 (s, 3H ), 2.11 - 2.03 (m, 1H), 1.94 (d, J = 1.4 Hz, 3H), 1.47 (s, 3H), 1.40 (s, 3H), 1 .09 (s, 9H), 0.93 (dt, J = 11.2, 3.4 Hz, 15H), C74H124N6O18S2 calcd. [M+H]+ = 1449.85 amu; found m/z = 1450.06. Example 144 Example compound 143 is reduced according to the methods below to produce the compound in question. Example 145 (Compound 142 - SPDP - mAb) produced using the compound 142 synthesis method, above and the SPDP ligation method described below. Example 146 (mAb - SPDP - Compound 140) produced using the Compound 140 synthesis method, above, and the SPDP ligation method described below.Example 147 (mAb - SPDP - Compound 144) produced using the Compound 144 synthesis method, above, and the SPDP binding method described below. Example 148 (mAb - SMCC - Compound 140) produced using the Compound 140 synthesis method, above, and the SMCC ligation method described below. Example 149 (mAb - SMCC - Compound 142) produced using the Compound 142 synthesis method, above, and the SMCC ligation method described below. Example 150 (mAb - SMCC - Compound 144) produced using the Compound 144 synthesis method, above, and the SMCC ligation method described below. OTHER Examples Example 151 (S,E)-N-(benzisulfonyl)-4-((S)-2-((S)-3-cyclohexyl-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido) -2,5-dimethylhex-2- enamide.

[0791] The title compound was synthesized from (S)-2-(tert-butoxycarbonyl(methyl)amino)-3-cyclohexyl-3-methylbutanoic acid as prepared by Zask et al., J. Med. Chem. 2004, 47, (19), 4774-4786 and (S,E)- 4-((S)-2-amino-N,3,3-trimethylbutanamido)-N-(benzisulfonyl)-2,5-dimethylhex- 2-enamide, prepared using general procedures 10, 11, 3 and 2 by application of general procedures 4 and 7.

[0792] 1H NMR (400 MHz, Methanol-d4) δ 7.38 (s, 5H), 6.37 (dd, J = 9.4, 1.7 Hz, 1H), 5.01 (t, J = 10.0 Hz, 1H), 4.91 (s, 1H), 4.75 (s, 2H), 4.01 (s, 1H), 3.10 (s, 3H), 2.66 (s , 3H), 2.05 - 1.91 (m, 4H), 1.91 - 1.67 (m, 6H), 1.45 - 1.28 (m, 3H), 1.29 - 1.01 (m, 17H), 0.95 - 0.75 (m, 9H).

[0793] C34H56N4O5S calcd m/z = 632.40 found [M+H]+ =633.35 Example 152 (mAb - MCvcPABC - Compound 85) produced using Example Compound 120, above, and the general MCvcPABC conjugation method described below. Example 153 (mAb - MCvcPABC - Compound 77) produced using Example Compound 119, above, and the general MCvcPABC conjugation method described below. Example 154 (mAb - MCvcPABC - Compound 80) produced using Example Compound 121, above, and the general MCvcPABC conjugation method described below. Example 155 (mAb - MCvcPABC - Compound 58) produced using example compound 158 (MCvcPABC58), below, and the MCvcPABC conjugation method described below. Example 156 (mAb - MCvcPABC - Compound 41) produced using Example Compound 122, above, and the general MCvcPABC conjugation method described below. Example 157 (mAb - MCvcPABC - Compound 63) produced using example compound 159 (MCvcPABC30), below, and the MCvcPABC conjugation method described below. Example 158

[0794] The title compound was prepared by applying general procedures 15 and 7 to example 58 Boc-protected.

[0795] 1H NMR (400 MHz, Methanol-d4) δ 7.60 (d, J = 8.1 Hz, 2H), 7.56 (d, J = 7.8 Hz, 2H), 7.47 ( t, J = 7.6 Hz, 2H), 7.37 (t, J = 7.3 Hz, 1H),7.33 (d, J = 8.2 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 7.9 Hz, 2H), 6.81 (s, 2H), 6.37 (d, J = 9.3 Hz, 1H) , 5.13 - 5.01 (m, 3H), 4.96 (s, 1H), 4.70 (s, 2H), 4.56 - 4.51 (m, 1H), 4.38 (s , 1H), 4.23 - 4.16 (m, 1H), 3.50 (t, J =7.1 Hz, 2H), 3.27 - 3.19 (m, 1H), 3.18 - 3.04 (m, 4H), 2.52 (s, 3H), 2.30 (t, J = 7.4 Hz, 2H), 2.15 - 2.05 (m, 1H), 1.96 (s, 3H), 1.98 - 1.88 (m, 1H), 1.83 - 1.73 (m, 1H), 1.64 (dq, J = 23.1, 7.3 Hz, 7H ), 1.48 (s, 3H), 1.39 (s, 3H), 1.37 - 1.30 (m, 2H), 1.27 (s, 2H), 1.21 (s, 2H) , 1.08 (s, 9H), 1.00 (d, J = 6.7 Hz, 3H), 0.99 (d, J = 6.8 Hz, 3H), 0.91 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.5 Hz, 3H).C66H93N11O13S calcd. m/z = 1279.7 found [M+H]+ = 1281.0.Example 159

[0796] The title compound was prepared by applying general procedures 15 and 7 to example 63 Boc-protected.

[0797] C65H91N11O13S calcd. m/z = 1265.7 found [M+H]+ = 1266.7

[0798] It is understood by those skilled in the art that it may be possible to carry out the chemical conversions shown in the schemes above with modifications of one or more parameters. As examples, alternative non-nucleophilic solvents may be suitable for chemistry, such as tF, DMF, toluene, etc. Reaction temperatures can be varied. Alternative reagents may be suitable to act as dehydrating or acid activating agents that are normally used in amide formation reactions, such as pentafluorophenyl esters, NHS esters, EDAC, HBTU, HOBT etc. Other Representative Compounds

[0799] The following representative compounds can be prepared according to the above procedures. As recognized by those skilled in the art, the following compounds are synthetically accessible using disclosure WO 2004/026293 to obtain the precursor reagent and apply general procedures with the appropriate sulfonamide. EXAMPLE 1: BIOLOGICAL TESTS

[0800] Tables 1-8 summarize the cytotoxic activity of the compounds in question in cell lines. Figure 1 summarizes the data for compounds A, B, C, D, and E when tested using the human mammary carcinoma cell line HCC1954 or the human T-cell leukemia cell line Jurkat. Figures 2-6 show graphical representations of cytotoxicity data for individual compounds A-E. Tables 2-6 summarize the results of additional cytotoxicity assays.

[0801] Cell lines used: Jurkat human T-cell leukemia cell line (ATCC: TIB-152); HCC1954 (ATCC: CRL.2338); human pancreatic cell lines: AsPC-1 (ATCC: CRL- 1682), BxPC-3 (ATCC: CRL.1687), HPAF-II (ATCC: CRL.1997), MiaPaCa2 (ATCC: CRL.1420), PANC-1 (ATCC: CRL.1469), Capan-1 (ATCC: HTB-79), Capan-2 (ATCC: HTB-80) and the human gastric carcinoma cell line NCI-N87 (ATCC: CRL. 5822); AML-193 (ATCC: CRL. 9589), CCRF-CEM (ATCC: CCL-119), DU145 (ATCC: HTB-81), PC-3 (ATCC:CRL.1435), A-431 (ATCC: CRL. 1555), HT-29 (ATCC: HTB-38), A-172(ATCC: CRL.1620), NCI-H358 (ATCC: CRL.5807), A549 (ATCC: CCL-185), Colo-205 (ATCC : CCL-222), MDA-MB-231 (ATCC: HTB-26), OVCAR-3 (ATCC: HTB-161), OV-90 (ATCC: CRL. 11732), OE19 (Sigma: 96071721), RT112/ 84 (Sigma: 85061106).

[0802] The day before compound addition, HCC1954 AsPC-1, BxPC-3, HPAF-II, MiaPaCa2, PANC-1, Capan-1, Capan-2 and NCI-N87 cells were added to microtiter plates treated with opaque-walled 96-well tissue culture using complete growth medium at a density of 2500 cells/100 microliters (uL) of medium. These cells from adherent cell lines were incubated overnight at 370C/5% CO2 to allow the cells to attach to the surface of the microtiter plate. On the day compounds were added, Jurkat cells are added to separate 96-well microtiter plates at 2500 cells/100uL using the same growth medium as HCC1954. Compounds were first serially diluted using dimethylsulfoxide, and then the prepared dilutions were added to complete culture medium at five times the final concentration - compounds were then titrated 1:3, eight steps. A no compound control (growth medium alone) was included in each microtiter plate in sextuplicate. Titrations of prepared compounds were added (25 uL/well) in triplicate. Cells and compound titrations were incubated at 370C/5% CO2 for three nights. After incubation, cell viability is measured using the CellTiter-Glo® reagent by adding thirty uL of CellTiter-Glo® preparation to each assay well. The assay is incubated for at least twenty minutes in the dark before measuring the emitted luminescence using a microplate luminometer (500ms integration time). The collected relative luminescence units (RLU) are converted to % cytotoxicity using the control growth medium alone mentioned above (% cytotoxicity = 1 - [well RLU/average control medium alone RLU]).

[0803] GrafPad Prism was used for generating EC50 values using three-parameter non-linear regression curve fitting. Table 1: Cytotoxicity of compounds Table 2: Cytotoxicity of compounds Table 3: Cytotoxicity of compounds in Jurkat Cells Table 4: Cytotoxicity of compounds in HCC-1954 cells Table 5: Cytotoxicity (EC50) of compounds on various tumor cell lines (nM) Table 6: Compound Cytotoxicity in Jurkat Table 7: Cytotoxicity in Jurkat Table 8: Cytotoxicity in various cell lines Example 2: Exemplary Antibody-Drug Conjugates Antibody-Drug Conjugates - Exemplary Ligands

[0804] As recognized by those skilled in the art, the particular linker used for conjugate formation will depend on the reactive group of the reactant compound being used for link formation. As an example and within the scope of the present invention, compounds with a thiol group can be used to form a conjugate. In some of the present examples, the commercially available cleavable linker sulfosuccinimidyl 6-[3'(2-pyridyldithio)-propionamido]hexanoate (sulfo-LC-SPDP: term Pierce Cat# 21650) and non-cleavable linker succinimidyl 4-[N-maleimidomethyl ]cyclohexane-1-carboxylate (SMCC: Pierce term Cat# 22360) were used for antibody-drug conjugation reactions. The coupling procedure is carried out in two main steps: 1) modulating the linkers to the antibody via reaction with antibody primary amine groups (lysine residues)) and the N-Hydroxysuccinimide (NHS) ester moiety of the linkers and 2) the reaction of incorporated maleimide group (SMCC) or 2-pyridyldithio group (LC-SPDP) with thiol-containing compounds.

[0805] Activation of antibodies with Cleavable (LC-SPDP) or Non-cleavable (SMCC) Ligands

[0806] Antibody (Herceptinaa) was diluted in potassium phosphate at f 8 (SPDP-sulfo-LC) or D-PBS (Invitrogen) at f 7.4 (SMCC) at 5mg/mL. To the diluted antibody, freshly dissolved linker was added - using ultra-pure water for sulfo-LC-SPDP or anhydrous N,N-dimethylacetamide (DMA) for SMCC. 10-14 times molar excesses of SMCC:antibody or sulfo-LC-SPDP:antibody results in 5-7 ligands/antibody modality. The antibody-linker "activation" reaction was incubated at 28°C for 2 hours. After incubation, unreacted linker was removed from each antibody sample using Zeba 40kda size-exclusion chromatography/desalting columns (Pierce term Cat# 87771 or 87772 depending on scale). During the same chromatographic analysis the buffer was changed in preparation for the next reaction; f 6.5 phosphate buffer/EDTA (LC-SPDP) or f 5 EDTA/citrate buffer (SMCC). The purified preparations were then analyzed for total protein content against an antibody standard curve using the adapted microplate BCA assay (Pierce term Cat# 23225). To estimate the extent of the linker modality a small-scale reaction with excess (~10-fold compared to the protein concentration) of cysteine was performed. After a 10 minute incubation unreacted cysteine was detected using 5,5-Dithio-bis-(2-nitrobenzoic acid) (Ellman Reagent, Pierce Term Cat# 22582). By interpolating the concentration from a cysteine standard curve, the linker concentration was determined by subtracting the determined value from the known concentration of cysteine used.Reaction of thiol-containing compounds to the linker-activated antibody

[0807] In the second step of the coupling reaction, the activated antibody was used by first diluting the preparation to 2mg/mL with phosphate buffer/EDTA at f 6.5 (LC-SPDP) or EDTA/citrate buffer f 5 (SMCC ). Prior to use, thiol-containing n-acyl sulfonamide compounds or maytansinoid DM1 were reduced using TCEP-agarose beads to ensure that the thiol group was available to react with the incorporated ligands. Briefly, compounds were diluted to 5mM using phosphate/EDTA buffer at f 6.5. In cases where water solubility was a problem, a small volume of 37% HCl was added (1:300) and this was sufficient to solubilize the compounds to 5 mM. TCEP-agarose beads (Pierce term Cat# 77712), were equilibrated with phosphate buffer/EDTA/10% DMA before use. Compound dilutions were rotated with TCEP-agarose beads for at least 0.5 hours, or up to 3 hours. The reduced compounds were collected by centrifugation on a filter that excludes TCEP-agarose. The extent of reduction and thiol concentration was measured using Ellman's reagent (compared to a cysteine standard curve). The reduced thiol-containing compounds were then added to the activated antibody samples in a ∼2-fold molar excess compared to previously determined linker concentrations. In order to monitor coupling reaction efficiency an "overnight" conjugation control was prepared by diluting each compound in phosphate/EDTA buffer of f 6.5 or citrate/EDTA buffer of f 5 for the same dilution factor. which was used in the conjugation reaction. The remaining stocks of compounds were frozen at -80°C. Overnight reactions and controls were incubated at room temperature overnight. The next morning the frozen compound stocks were thawed and another control was prepared for each compound exactly like the "overnight" control - this is the "fresh" control. A small volume of each conjugation reaction was compared to the overnight and fresh control compounds using Ellman's reagent. Unreacted compound was purified away from the ADCs using Zeba 40kda size-exclusion/desalting columns; during the same step buffer was exchanged for D-PBS at f7.4 (Invitrogen).

[0808] Purified ADCs were then analyzed for: total protein content (BCA assay, Pierce microBCA protocol), relative affinity to binding antigen (equilibrium native binding), and selective cytotoxic killing of HER2-positive cells (HCC1954) compared to HER2-negative cells (Jurkat). Cytotoxicity assay

[0809] Tables 9 and 10 summarize the cytotoxic activity of ADCs comprising compounds A, B or C, when tested using the HCC1954 human mammary carcinoma cell line or Jurkat human T-cell leukemia cell line. Figures 7-9 show graphical representations of cytotoxicity data for individual compositions as indicated.

[0810] The day before adding test articles, HCC1954 cells were added to opaque-walled 96-well tissue culture-treated microtiter plates using complete growth medium at a density of 2500 cells/100 microliters (uL). the middle one. These HCC1954 cells were incubated overnight at 370C/5% CO2 to allow the cells to attach to the surface of the microtiter plate. On the day test articles were added, Jurkat cells are added to separate 96-well microtiter plates at 2500 cells/100uL using the same growth medium as HCC1954. To compare the killing of ADC with that obtained from the free compounds, n-acyl sulfonamide compounds were first serially diluted using dimethylsulfoxide or DMA and then the prepared dilutions are added to the complete culture medium at five times the final concentration - compounds were then titrated 1:3, eight steps. To test the ADCs, they were diluted directly into the growth medium at five times the final concentration - ADCs were then titrated 1:3, eight steps. A control with no test article present (growth medium alone) was included in each microtiter plate in sextuplicate. ADC/prepared compound titrations were added (25 uL/well) in triplicate with both HCC1954 cells and Jurkat cells. Cells and titrations were incubated at 370C/5% CO2 for three nights. After incubation, cell viability was measured using the CellTiter-Glo® reagent by adding thirty uL of CellTiter-Glo® preparation to each assay well. The assay was incubated for at least twenty minutes in the dark before measuring the emitted luminescence using a microplate luminometer (500 ms integration time). The collected relative luminescence units (RLU) were converted to % cytotoxicity using the control growth medium alone mentioned above (% cytotoxicity = 1 - [well RLU/average control medium alone RLU]).

[0811] The data indicate that the compounds in question are active cytotoxins in both cell lines used. LC-SPDP-linked compound conjugates demonstrated potent killing of HER2-positive HCC1954 cells. Jurkat cell death was observed with high-dose ADC due to the presence of β-Mercaptoethanol in the cell culture medium, which resulted in the release of free compound (data not shown). Table 9: Cytotoxicity - Coupling #1 Table 10: Cytotoxicity - Coupling #2 Antibody Drug Conjugate (ADC) Analysis by EsiToF Mass Spectrometry.

[0812] Electrospray ionization time-of-flight mass spectrometry instrument -QStar XL Hybrid quadrupole-TOF LC/MSMS- (AB Sciex) was used to determine the molecular weight of ADCs and to evaluate the drug-to-drug ratio antibody (DAR). The EsiToF MS instrument was equipped with electrospray ionization turbo spray source. Data acquisition was performed in positive ion mode, and sample total ion current was acquired over the mass range 2000 m/z to 4000 m/z using Analist QS 1.1 software. The ion source was operated with an ion spray needle voltage of 5.2 KV and a nebulization (gas 1) at 25 (arbitrary units), gas curtain of 30 (arbitrary units), declustering potential of 150 V and the temperature of 150° C. The ADC test sample solutions were introduced at 5uL/min into the ion source by direct infusion through a capillary through fused silica with the help of syringe and syringe pump. ADC for ESI-ToF MS analysis

[0813] All ADC samples were deglycosylated using EndoS(IgGZERO)TM endoglycosidase and buffer exchanged with water prior to EsiToF-MS analysis. Briefly, the original ADC sample was run through a MWCO Amicon 100K concentrator for buffer exchange into sodium phosphate buffer. The buffer-exchanged sample was then treated with IgGZERO (1 unit/1ug of antibody) in sodium phosphate cleavage buffer, which contains 150mM NaCl, and incubated for 30 minutes at 37°C. The resulting deglycosylated ADC was again exchanged. buffer with water using a MWCO Amicon 100K 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.

[0814] Analyzes indicated that antibodies were loaded with a DAR range between 4-7 (data not shown). Example 3: Exemplary Antibody-Drug Conjugates

[0815] Preparation of MCvcPABC-toxin antibody-drug conjugates, general methods:

[0816] To a solution of antibody (1-10 mg/mL) in 25mM sodium borate, 25mM sodium chloride, 1mM DTPA (f 8.0) TCEP was added 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 two hours before cooling on ice. In some cases, the reduced antibody solution was further diluted with ice-cold phosphate-buffered saline containing 1 mM DTPA (2.0 mg/mL final protein concentration) or 25 m m ice-cold sodium borate, chloride 25 mM sodium, 1 mM DTPA (f 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 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 through Zeba Spin desalting columns (40 KDa MWCO; Peirce) previously equilibrated with phosphate buffer saline or 10mM sodium citrate, 150mM sodium chloride, f 5.5. The eluate was pooled, filter-sterilized (Steriflip, Millipore) and stored at 4 °C.

[0817] Purified ADCs were analyzed for total protein content (bicinchonic acid assay, microBCA Pierce protocol, catalog #23225). The ADC product was characterized by reduction and non-reduction of PAGE, HPLC-HIC, SEC, and RP-UPLC-MS. Average DAR and drug distribution were derived from interpretation of HIC and LC-MS data with reference to unreduced PAGE. Average DAR estimates were typically in the range of 3.5-4.5. Relative affinity of ADCs for antigen binding (equilibrium native binding) was performed as described (above/below). The selective cytotoxicity of drug-antibody conjugates was evaluated by testing for mortality of antigen-positive and negative cell lines. Selective in vitro cytotoxicity assay of antigen-positive cells by antibody-drug conjugates:

[0818] Selective killing of an antigen-positive cell line (including HCC1954, NCI-N87, HPAF-II, and BxPC-3 cell lines) over antigen-negative Jurkat cells was demonstrated for each prepared conjugate. Cytotoxicity of example ADCs in various antigen-positive cell lines is summarized in the identified figures and tables 9-13. Furthermore, the conjugates indicated by (*) in Table 11 were tested and showed potent cell killing activity against a human breast cancer cell line (data not shown). Briefly, cells were obtained from ATCC and cultured as described in the provided product sheet. Cells were seeded at 25000 cells/mL (2500 cells/well) in Costar 3904 black-walled, flat-bottom 96-well plates. Cells from adherent cell lines were incubated overnight at 370C in a 5% CO2 atmosphere to allow the cells to attach to the surface of the microtiter plate, while suspension cells (Jurkat) were plated immediately before use. ADCs were diluted directly into the appropriate cell growth medium at five times the desired final concentration. These ADCs were then titrated, typically 1:3, in eight steps. A control with no test article present (growth medium alone) was included in each microtiter plate in sextuplicate. Prepared ADC titrations were added (25 uL/well) in triplicate to each cell line analyzed. Cells and titrations were incubated at 370C/5% CO2 for three nights (Jurkat) and five nights (all other cell lines). After incubation, cell viability was measured using the CellTiter-Glo® reagent by adding thirty uL of CellTiter-Glo® preparation to each assay well. The mixtures were incubated for at least twenty minutes in the dark before measuring the emitted luminescence using a microplate luminometer (500 ms integration time). The collected relative luminescence units (RLU) were converted to % cytotoxicity using the control growth medium alone mentioned above (% cytotoxicity = 1 - [well RLU/average control medium alone RLU]). Data (% cytotoxicity vs. ADC concentration (log10(nM)) were plotted and were analyzed by non-linear regression methods using GrafPad Prism v. 5.02 software to obtain EC50 estimates. Drug and antibody ratio estimation ( TO GIVE):

[0819] The average degree of toxin-ligand conjugation to antibody was assessed by hydrophobic interaction chromatography and high-performance liquid chromatography/mass spectrometry. These techniques are described in Antibodi Drug Conjugates, Methods in Molecular Biologi vol. 1045, 2013. PP 275-284. L. Ducri, Ed., and Asish B. Chakraborti, Scott J. Berger, and John C. Gebler, Characterization of an IgG1 Monoclonal Antibodi and related Sub-structures in LC/ESI-TOF/MS: Application note, Waters Corporation. March 2007. 720002107EN.Method 1 Hydrophobic Interaction Chromatography

[0820] Antibody-drug conjugates were subjected to hydrophobic interaction chromatography (HIC) on a TSKgel butyl-NPR column (Tosoh Bioscience; 4.6 x 35 mm; 2.5 µm particle size) linked to an Agilent HPLC 1100 series. Samples were injected (5 uL) at or above 4 mg/mL. Where necessary, ADCs were 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 minutes (mobile phase A: 1. 5M ammonium sulfate + 25mM sodium phosphate at f 6.95 and mobile phase B: 25% isopropanol, 75% 25mM sodium phosphate at f 6.95). Injection of unmodified antibodies provided a means of identifying the peak with DAR = 0. Antibodies were detected based on absorbance at 280 nm. Method 2. Ultra Performance Liquid Chromatography-Mass Spectrometry for estimation of DAR

[0821] Reverse-phase ultra-performance liquid chromatography spectrometry coupled with ESI-QToF mass spectrometry (UPLC-ESI-MS QToF) was used to characterize drug-antibody conjugates by the extent of drug conjugation after reduction with dithiothreitol. UPLC analysis of the reduced ADC sample was performed at 70° C, with a PolymerX 5u PR-1 100A, 50 x 2.0 mm column (phenomenex, Inc.) and with a mobile phase composed of solvent a: Acetonitrile/water/ formic acid/trifluoroacetic acid (10/90/0.1/0.1, v/v%) and solvent b: Formic acid/acetonitrile (100/0.1, %v/v). Components of the reduced ADC sample were eluted with a linear gradient, starting with Solvent A/Solvent B (80/20 v/v and a flow rate of 0.3 ml/min for Solvent A/Solvent B (40/60, v/v%) for 25 min and then to Solvent A/Solvent B (10/90,v/v%) over 2 minutes before equilibration back to initial conditions. Total run time was 30 minutes. Total ion current (TIC) data from ESI-Tof MS were acquired over the 500-4500m/z range using Masslynx data acquisition software (Waters Corporation). Mass data of the example component was acquired in V-positive ion mode, and the ESI source was operated at source temperature: 150° C, dissolvation temperature: 350° C, dissolvation gas: 800L/h, sample cone voltage: 60 V, capillary voltage: 3.0 kV, dissolving gases: nitrogen and collision gas: argon. The summed TIC mass spectra for each peak were uncomplicated by the MaxEnt1 algorithm to generate the neutral mass data of the peak component. Preparation of the reduced ADC samples for UPLC/ESI- ToF MS analysis

[0822] Reduction of disulfide bonds in the ADC antibody (~ 1 μg/μl solution) generating light and heavy chains was carried out using 20 mM DTT at 60°C for 20 minutes. An injection volume of 5–10 μl of the reduced ADC sample was employed for UPLC/ESI-ToF-MS analysis. Exemplary ADC (PABC) for illustration purposes:

[0823] Note that T = Trastuzumab, which is used interchangeably with "Herceptinaa" in this document; VC = valine-citrulline; C = Cetuximab (Erbitux)Table 11: ADC cytotoxicity (EC50, nM) Table 12: ADC Cytotoxicity (EC50, nM) Table 13: ADC Cytotoxicity (EC50, nM) Example 4: Efficacy study of toxins in mice with PC-3 tumor

[0824] Test articles were administered IV. Dosage was as indicated in Figure 14, each dosed near the maximum tolerated dose. One injection of test article was delivered every seven days for four replicates/injections (compound D) or one injection every seven days for three replicates/injections (23 compound). Vehicle: 6.3% trehalose, 0.05% Tween20, 20mM citrate buffer, f 5.0, 4° C. Procedure overview

[0825] Thirty-six (66) female athymic nude mice, purchased from Harlan Laboratories at 7-8 weeks of age, were vaccinated subcutaneously in the back with 5 x 106 PC-3 tumor cells on experimental day 0. Tumors were measured every Monday, Wednesday and Friday. Once tumors reached 150-200 mm3 in size (experimental day 27 to 34), animals were assigned to one of 4 treatment groups counterbalancing mean tumor size across groups. Animals were treated with their respective compound as indicated, and tumor measurements continued every Monday, Wednesday, and Friday. Data show animal results up to experimental day 54 or until tumors reach 800 mm3 in size. PC-3 Cells Cellular Preparation-Tissue Culture: The PC-3 human prostate adenocarcinoma cell line was obtained from ATCC (Cat# CRL-1435 ) in 2002.

[0826] Cells started from a frozen vial of laboratory stock that were frozen beneath the original ATCC vial, tested for mycoplasma negative, and maintained in laboratory liquid nitrogen tanks. Cell cultures with passage #3 and #10 and a confluency of 80-90% were harvested for in vivo studies. Cells were cultured in Ham's F12 medium supplemented with 2 mM L-glutamine and 10% FBS at 37°C in a 5% CO2 environment. Cells were subcultured once a week with a split ratio of 1:3 to 1:6 and expanded. The medium was renewed once a week. Cell preparation - harvesting for implantation

[0827] Cells were washed briefly once with 2 mL of fresh trypsin/EDTA solution (0.25% trypsin with 4Na EDTA) and then extra trypsin/EDTA was aspirated. Then 1.5 mL of EDTA/Tisine was added, the flask was placed horizontally to ensure that the cells were covered by trypsin/EDTA. The cells were then incubated at 37°C for a few minutes. The cells were observed under an inverted microscope to ensure that the cell layer was dispersed, then fresh medium was added and 50 μl of cell suspension was sampled and mixed with trypan blue (1:1) and the cells were counted and cell viability assessed using the Cellometer Auto T4. The cells were centrifuged at 1,000 rpm for 7 minutes and the supernatant was aspirated. The cells were then resuspended in growth medium to the appropriate concentration for inoculation. Injection volume was 100 μl per animal. Tumor cell implantation - SC Back

[0828] On day 0, 5.0 x 106 tumor cells were implanted subcutaneously into the back of mice in a volume of 100 μL using a 27/28 gauge needle under isoflurane anesthesia. Animal housing

[0829] Animals were housed in ventilated cages, 2 to 5 animals per cage, on a 12-hour light/dark cycle. Animals received sterile food and water ad libitum and animal use and housing was carried out in accordance with Canadian Council on Animal Care guidelines. Animals were treated under sterile conditions and cages changed once every 10-14 days. Data collection (tumor size)

[0830] Mice were monitored each Monday, Wednesday and Friday for tumor development. Established tumor dimensions were measured with calipers. Tumor volumes were calculated according to the equation L x W2 2 with length (mm) being the long axis of the tumor. The animals were also weighed at the time of tumor measurement. Tumors were allowed to grow to a maximum of 800 mm3. Institutional Animal Care Committee

[0831] The methodology used was reviewed and approved by the Universiti of British Columbia Animal Care Committee (ACC) prior to conducting studies to ensure that studies were designed in accordance with Canadian Council on Animal Care guidelines. During the study, the care, housing and use of animals were carried out in accordance with Canadian Council on Animal Care guidelines. Analysis methods Experimental day growth curves X Tumor volume

[0832] Tumor volumes of each group over the days of treatment were graphed. Growth curves were cut for each group at the time point when the first animal reached the endpoint experimental tumor size (800 mm), or on the last day of the study. Any animal that was removed from the study before group growth curve cutoff was removed from the study entirely. Animal Exclusions

[0833] Any animal with ulcerated tumors, requiring animal euthanasia, with a tumor volume of 700 mm3 or less were removed from the study and did not contribute to data analysis (except days of recurrence if the final tumor volume was > 2 .0 times higher than the day of treatment).Example 5: Finding Effective Dose Range of Antibody-Drug Conjugates in the NCI-N87 Tumor Model Using NOD SCID Gamma Mice

[0834] Test articles were administered IV, only one treatment. "T" refers to Trastuzumab. Dosage was as indicated in Figure 15. Vehicle: 20mM, sodium citrate, 6.3% trehalose, 0.02% Tween-20, f 5, 4° C. Procedure overview

[0835] Seventy-six (76) female NOD/SCID Gamma mice (NSG), purchased from The Jackson Laboratori (JAX® Mice) at 7-8 weeks of age were vaccinated subcutaneously in the lumbar region with 5 x 106 NCI cells -N87 tumor on matrigel on experimental day 0. Tumors were measured every Monday, Wednesday and Friday. Once tumors reached 150-200 mm3 in size (experimental day 27), animals were assigned to one of 10 treatment groups counterbalancing mean tumor size across groups. Animals were treated with their respective compound as indicated, and tumor measurements continued every Monday, Wednesday, and Friday. Data show animal results up to experimental day 50 or until tumors reach 800 mm3 in size. Cellular Preparation-Tissue Culture: NCI-N87 Cells NCI-N87 human gastric carcinoma cells were derived from a liver metastasis of a well-differentiated carcinoma of the stomach taken prior to cytotoxic therapy. The tumor was passaged as a heterologous graft into athymic nude mice for three passages before the cell line was established. NCI-N87 cells were obtained under MTA from ATCC (Cat# CRL-5822) in 2013 and tested negative in RADIL for mycoplasma and mouse pathogens. (RADIL certificate #: 10556-2013) Cells started from a frozen laboratory stock vial that were frozen beneath the original ATCC vial and maintained in laboratory liquid nitrogen tanks. Cell cultures with passage #3 and #10 and a confluency of 80-90% were harvested for in vivo studies. NCI-N87 cells were cultured in RPMI 1640 medium with 1.0 mM L-glutamine and 10% FBS at 37oC in a 5% CO2 environment. Cells were subcultured once or twice a week with a split ratio of 1:3 or 1:4 and expanded. The medium was renewed once a week. Cells were frozen with 5% DMSO. Cell preparation - harvesting for implantation

[0836] The cells were washed briefly once with Hanks Balanced Salt Solution without Ca, Mg. Fresh trypsin/EDTA solution (0.25% trypsin with 4Na EDTA) was added, and the flask was placed horizontally to ensure that the cells were covered by trypsin/EDTA and then extra trypsin/EDTA was aspirated. The cells were then incubated at 37°C for a few minutes. Cells were observed under an inverted microscope until the cell layer was dispersed, and fresh medium was added. Then, 50 μl of cell suspension was collected and mixed with trypan blue (1:1) and the cells counted and assessed for viability on a hemocytometer. Viability should be > 90%. The cells were centrifuged at 125 RCF (1000 rpm) for 7 minutes and the supernatant aspirated away. Cells were resuspended in cold growth medium to 2 times the desired final concentration (100 x 106/mL). The suspension was mixed (on ice) with matrigel (1:1). The resulting cell suspensions (50x106 cells/mL) were used to deliver 5x106 cells in an injection volume of 100 μL per animal. All equipment that comes into contact with matrigel (needles, syringes, pipette tips) was refrigerated before injection. Tumor Cell Implantation - subcutaneous (NCI-N87)

[0837] Before inoculation, an area of approximately 2 x 2 cm was shaved on the lower back of each mouse and cleaned with alcohol. On day 0, 5.0 x 106 tumor cells were implanted subcutaneously into the back of mice in a volume of 100 μL using a 27/28 gauge needle under isoflurane anesthesia. Animal housing

[0838] Animals were housed in ventilated cages, 2 to 5 animals per cage, on a 12-hour light/dark cycle. Animals received sterile food and water ad libitum and animal use and housing was carried out in accordance with Canadian Council on Animal Care guidelines. Animals were treated under sterile conditions and cages changed once every 10-14 days. Data collection (tumor size)

[0839] Mice were monitored each Monday, Wednesday and Friday for tumor development. Established tumor dimensions were measured with calipers. Tumor volumes were calculated according to the equation L x W2/2 with length (mm) being the long axis of the tumor. The animals were also weighed at the time of tumor measurement. Tumors were allowed to grow to a maximum of 800 mm3. Institutional Animal Care Committee

[0840] The methodology used was reviewed and approved by the Universiti of British Columbia Animal Care Committee (ACC) prior to conducting studies to ensure that studies were designed in accordance with Canadian Council on Animal Care guidelines. During the study, care, housing and use of animals were carried out in accordance with the Canadian Council on Animal Care guidelines.Methods of analysis Growth curves of Experimental day X Tumor volume

[0841] Tumor volumes of each group over the days of treatment were represented graphically. Growth curves were cut for each group at the time point when the first animal reached the endpoint experimental tumor size (800 mm), or on the last day of the study. Any animal that was removed from the study before group growth curve cutoff was removed from the study entirely. Animal Exclusions

[0842] Any animal with ulcerated tumors, requiring animal euthanasia, with a tumor volume of 700 mm3 or less were removed from the study and did not contribute to data analysis (except days of recurrence if the final tumor volume was > 2 .0 times the day of treatment).Example 6: Comparison of Efficacy of Antibody-Drug Conjugates in the NCI-N87 Tumor Model Using NOD SCID Gamma Mice

[0843] Test articles were administered IV, with one administration. Dosages were as indicated in Figure 16. "T" refers to Trastuzumab. Vehicle: 20mM, sodium citrate, 6.3% trehalose, 0.02% Tween-20, f 5, 4° C. Procedure overview

[0844] Twenty-four (24) female NOD/SCID Gamma (NSG) mice, purchased from The Jackson Laboratori (JAX® Mice) at 7-8 weeks of age were vaccinated subcutaneously in the lumbar region with 5 x 106 NCI cells -N87 tumor on matrigel on experimental day 0. Tumors were measured every Monday, Wednesday and Friday. Once tumors reached 150-200 mm in size (experimental day 27), animals were assigned to one of 3 treatment groups counterbalancing mean tumor size across groups. Animals were treated with their respective compound as indicated, and tumor measurements continued every Monday, Wednesday, and Friday. Data show animal results up to experimental day 88 or until tumors reach 800 mm3 in size. Cellular Preparation-Tissue Culture: NCI-N87 Cells

[0845] NCI-N87 human gastric carcinoma cells were derived from a liver metastasis of a well-differentiated carcinoma of the stomach taken before cytotoxic therapy. The tumor was passaged as a heterologous graft into athymic nude mice for three passages before the cell line was established. NCI-N87 cells were obtained under MTA from ATCC (Cat# CRL-5822) in 2013 and tested negative in RADIL for mycoplasma and mouse pathogens. (RADIL certificate #: 10556-2013)

[0846] Cells started from a frozen laboratory stock vial that were frozen beneath the original ATCC vial and maintained in laboratory liquid nitrogen tanks. Cell cultures with passage #3 and #10 and a confluency of 80-90% were harvested for in vivo studies. NCI-N87 cells were cultured in RPMI 1640 medium with 1.0 mM L-glutamine and 10% FBS at 37oC in a 5% CO2 environment. Cells were subcultured once or twice a week with a split ratio of 1:3 or 1:4 and expanded. The medium was renewed once a week. Cells were frozen with 5% DMSO. Cell preparation-harvest for implantation

[0847] Cells were briefly washed once with Hanks Balanced Salt Solution without Ca, Mg. Fresh trypsin/EDTA solution (0.25% trypsin with 4Na EDTA) was added, and the flask was placed horizontally to ensure that the cells were covered by trypsin/EDTA and then extra trypsin/EDTA was aspirated. The cells were then incubated at 37°C for a few minutes. Cells were observed under an inverted microscope until the cell layer was dispersed, and fresh medium was added. Then, 50 μl of cell suspension was collected and mixed with trypan blue (1:1) and the cells were counted and assessed for viability on a hemocytometer. Viability must be > 90%. The cells were centrifuged at 125 RCF (1000 rpm) for 7 minutes and the supernatant aspirated away. Cells were resuspended in cold growth medium to 2 times the desired final concentration (100 x 106/mL). The suspension was mixed (on ice) with matrigel (1:1). The resulting cell suspensions (50x106 cells/mL) were used to deliver 5x106 cells in an injection volume of 100 μL per animal. All equipment that comes into contact with matrigel (needles, syringes, pipette tips) was refrigerated before injection. Tumor Cell Implantation - subcutaneous (NCI-N87)

[0848] Before inoculation, an area of approximately 2 x 2 cm was shaved on the lower back of each mouse and cleaned with alcohol. On day 0, 5.0 x 106 tumor cells were implanted subcutaneously into the back of mice in a volume of 100 μL using a 27/28 gauge needle under isoflurane anesthesia. Animal housing

[0849] Animals were housed in ventilated cages, 2 to 5 animals per cage, on a 12-hour light/dark cycle. Animals received sterile food and water ad libitum and animal use and housing was carried out in accordance with Canadian Council on Animal Care guidelines. Animals were treated under sterile conditions and cages changed once every 10-14 days. Data collection (tumor size)

[0850] Mice were monitored each Monday, Wednesday and Friday for tumor development. Established tumor dimensions were measured with calipers. Tumor volumes were calculated according to the equation L x W2 2 with length (mm) being the long axis of the tumor. The animals were also weighed at the time of tumor measurement. Tumors were allowed to grow to a maximum of 800 mm3. Institutional Animal Care Committee

[0851] The methodology used was reviewed and approved by the Universiti of British Columbia Animal Care Committee (ACC) prior to conducting studies to ensure that studies were designed in accordance with Canadian Council on Animal Care guidelines. During the study, the care, housing and use of animals were carried out in accordance with Canadian Council on Animal Care guidelines. Analysis methods Experimental day growth curves X Tumor volume

[0852] Tumor volumes of each group over the days of treatment were graphed. Growth curves were cut for each group at the time point when the first animal reached the endpoint experimental tumor size (800 mm), or on the last day of the study. Any animal that was removed from the study before group growth curve cutoff was removed from the study entirely. Animal Exclusions

[0853] Any animal with ulcerated tumors, requiring animal euthanasia, with a tumor volume of 700 mm3 or less were removed from the study and did not contribute to data analysis (except days of recurrence if the final tumor volume was > 2 .0 times greater than the day of treatment).

[0854] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and patent publications not referred to in this specification are incorporated herein by reference, in their entirety, to the extent that not incompatible with this description.

[0855] From the foregoing it will be appreciated that although specific embodiments of the disclosure have been described herein for purposes of illustration, but that various modifications may be made without departing from the principle and scope of the disclosure. In this sense, the disclosure is not limited except by the appended claims.

Claims (21)

1. Compound characterized by having the following structure (I): wherein: R1 and R2 are independently selected from the group consisting of: H and C1-C6 alkyl; or R2 and R5 are fused and form a ring; R3 and R4 are independently selected from the group consisting of: H and R; or R3 and R4 are joined to form a ring, wherein the ring formed by joining R3 and R4 is a non-aromatic three to seven membered cyclic skeleton within the definition of R; R5 is selected from the group consisting of: optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl; or R5 and R2 are fused and form a ring; R6 is H; R7 and R8 are independently selected from the group consisting of: H and R; It is where, R is a saturated or unsaturated fraction, having a non-aromatic, branched or linear cyclic skeleton that contains from one to ten carbon atoms, from zero to four nitrogen atoms, from zero to four oxygen atoms, from zero to four sulfur atoms, and the carbon atoms are optionally independently substituted by: =O, =S, OH, -OR10, -O2CR10, -SH, -SR10, -SOCR10, -NH2, -NHR10, -N(R10 )2, -NHCOR10, - NR10COR10, -I, -Br, -Cl, -F, -CN, -CO2H, -CO2R10, -CHO, -COR10, -CONH2, - CONHR10, -CON(R10)2, - COSH, -COSR10, -NO2, -SO3H, -SOR10, or -SO2R10, wherein R10 is a saturated or unsaturated alkyl group of one to ten carbons, cyclic, branched or linear; Y is a linear, one to six carbon alkyl group, saturated or unsaturated, optionally substituted by R; and 1. 4 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl; or a stereoisomer or pharmaceutically acceptable salt thereof. 2. Compound, according to claim 1, characterized by having one of the following structures: (a) wherein: R14 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl; R15 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl; R16 is selected from the group consisting of H and C1-6 alkyl; R17 is selected from the group consisting of H and C1-6 alkyl; R18 and R30 are independently selected from the group consisting of H, C1-6 alkyl and -SH, with the proviso that both R18 and R30 cannot be H; R19, R20, R21 and R22 are each independently H or C1-6 alkyl, wherein at least one of R19 and R20 is H; or R20 and R21 form a double bond, R19 is H and R22 is H or C1-6 alkyl; and R23 is selected from the group consisting of H and C1-6 alkyl; or a stereoisomer or pharmaceutically acceptable salt thereof; or (b) wherein: R26 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl; R27 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl; R16 is selected from the group consisting of H and C1-6 alkyl; R17 is selected from the group consisting of H and C1-6 alkyl; and R18 is selected from the group consisting of C1-6 alkyl and -SH, or a stereoisomer or pharmaceutically acceptable salt thereof. 3. Compound according to claim 2, characterized in that R15 in (Ia) is optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl or optionally substituted heteroaryl. 4. Compound according to claim 2, characterized by the fact that R15 in (Ia) is selected from the group consisting of: or the group consisting of: wherein each R25 is independently selected from the group consisting of H, -OH, -R24, -OR24, -O2CR24, -SH, -SR24, -SOCR24, -NH2, -N3, -NHR24, -N( R24)2, -NHCOR24, -NR24COR24, -R24NH2, -I, -Br, -Cl, -F, -CN, -CO2H, -CO2R24, -CHO, -COR24, -CONH2, -CONHR24, -CON(R24 )2, -COSH, -COSR24, -NO2, -SO3H, -SOR24 and -SO2R24, wherein each of R24 is independently an alkyl optionally substituted with halogen, -OH or -SH. 5. Compound according to claim 2, characterized by the fact that R27 in (Ib) is selected from the group consisting of: 6. Compound according to claim 2, characterized by the fact that R15 in (Ia) and R27 in (Ib) are each independently optionally substituted phenyl. 7. Compound according to any one of claims 2 to 6, characterized by the fact that: (a) R16, R17 and R18 are each methyl, or (b) R16 is H, R17 is methyl and R18 is methyl. 8. Compound according to any one of claims 2 to 7, characterized by the fact that R14 in (Ia), and R26 in (Ib) are each independently optionally substituted alkyl or optionally substituted aryl. 9. Compound according to any one of claims 2 to 7, characterized by the fact that R14 in (Ia), and R26 in (Ib) are each independently optionally substituted aralkyl or optionally substituted phenyl. 10. Compound according to any one of claims 1 to 9, characterized in that: (a) each optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl is , independently, optionally substituted by = O, = S, - OH, -OR24, -O2CR24, -SH, -SR24, -SOCR -24, -NH2, -N3, -NHR24, -N(R24)2, - NHCOR24 , -NR24COR24, -I, -Br, -Cl, -F, - CN, -CO2H, -CO2R24, - CHO, -COR24, -CONH2, - CONHR24, -CON (R24)2, -COSH, -COSR24, - NO2, -SO3H, -SOR24 or -SO2R24, wherein each R24 is independently an alkyl optionally substituted by halogen, -OH or -SH, and/or (b) each optionally substituted aryl and optionally substituted heteroaryl is independently selected from the group consisting of optionally substituted phenyl, optionally substituted naphthyl, optionally substituted anthracyl, optionally substituted phenantryl, optionally substituted furyl, optionally substituted pyrrolyl, optionally substituted thiophenyl, optionally substituted benzofuryl, optionally substituted benzothiophenyl, optionally substituted quinolinyl, optionally substituted isoquinolinyl substituted, optionally substituted imidazolyl, optionally substituted thiazolyl, optionally substituted oxazolyl and optionally substituted pyridinyl. 11. Compound according to claim 2, characterized by the fact that the compound has one of the following structures: or a stereoisomer or pharmaceutically acceptable salt thereof. 12. Compound, according to claim 1, characterized by having one of the following structures: or a stereoisomer or pharmaceutically acceptable salt thereof. 13. Composition characterized by having the following structure (VI): (T)-(L)-(D) (VI) where (T) is a targeting moiety, (L) is a linker, and (D) is the compound as defined in any one of claims 1 to 12. 14. Composition characterized by having the following structure: (T)-(L)-(PT) (VII) where (T) is a targeting moiety, (L) is a ligand and (PT) is a peptide toxin of microtubule disruption, where (T)-(L)-(PT) has one of the following structures: (a) wherein: R15 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl; R16 and R17 are independently selected from the group consisting of H and C1-6 alkyl; R18 and R30 are independently selected from the group consisting of H, C1-6 alkyl and -SH, with the proviso that both R18 and R30 cannot both be H; R is a saturated or unsaturated fraction, having a non-aromatic, branched or linear cyclic skeleton that contains from one to ten carbon atoms, from zero to four nitrogen atoms, from zero to four oxygen atoms, from zero to four of sulfur, and the carbon atoms are optionally independently substituted by: =O, =S, OH, -OR10, -O2CR10, -SH, -SR10, -SOCR10, -NH2, -NHR10, -N(R10)2, -NHCOR10, -NR10COR10, -I, -Br, -Cl, -F, -CN, -CO2H, -CO2R10, -CHO, -COR10, -CONH2, - CONHR10, -CON(R10)2, -COSH, - COSR10, -NO2, -SO3H, -SOR10, or -SO2R10, wherein R10 is a saturated or unsaturated alkyl group of one to ten carbons, cyclic, branched or linear; Y is a linear, one to six carbon alkyl group, saturated or unsaturated, optionally substituted by R; and R14 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and optionally substituted heteroaryl; or (b) where: R is a saturated or unsaturated fraction, having a non-aromatic, branched or linear cyclic skeleton that contains from one to ten carbon atoms, from zero to four nitrogen atoms, from zero to four oxygen atoms, from zero to four sulfur atoms, and the carbon atoms are optionally independently substituted by: =O, =S, OH, -OR10, -O2CR10, -SH, -SR10, -SOCR10, -NH2, -NHR10, -N(R10 )2, -NHCOR10, - NR10COR10, -I, -Br, -Cl, -F, -CN, -CO2H, -CO2R10, -CHO, -COR10, -CONH2, - CONHR10, -CON(R10)2, - COSH, -COSR10, -NO2, -SO3H, -SOR10, or -SO2R10, wherein R10 is a saturated or unsaturated alkyl group of one to ten carbons, cyclic, branched or linear. 15. Composition according to claim 13 or 14, characterized by the fact that (L) is a cleavable linker optionally comprising a self-immolative component. 16. Composition, according to claim 13 or 14, characterized by the fact that (L) comprises sulfosuccinimidyl 6-[3'(2-pyridyldithio)-propionamido]hexanoate (sulfo-LC-SPDP), succinimidyl 4-[Nmaleimidomethyl ]cyclohexane-1-carboxylate (SMCC), p-aminobenzylcarbamoyl (PABC) or maleimidocaproyl-valine-citrulline-PABC (MC-VC-PABC). 17. Composition according to claim 13, characterized by the fact that (T)-(L)-(D) has one of the following structures: where (T) is a monoclonal antibody. 18. Composition according to any one of claims 13 to 16, characterized by the fact that (T) is an antibody or antibody fragment. 19. Composition according to claim 18, characterized in that the antibody or antibody fragment is a monoclonal antibody or antibody fragment, a bispecific antibody or antibody fragment, or a multispecific antibody or antibody fragment. 20. Pharmaceutical composition characterized by comprising the compound, as defined in any one of claims 1 to 11, or the composition, as defined in any one of claims 13 to 19, and a pharmaceutically acceptable carrier, diluent or excipient. 21. Use of a compound, as defined in any one of claims 1 to 11, or of the composition, as defined in any one of claims 13 to 19, characterized in that it is in the preparation of a medicine for treating cancer.