WO2008151253A1

WO2008151253A1 - Hypoxia activated prodrugs of antineoplastic agents

Info

Publication number: WO2008151253A1
Application number: PCT/US2008/065812
Authority: WO
Inventors: Hailong Jiao; Jason Lewis; Mark Matteucci; Difei Sun
Original assignee: Threshold Pharmaceuticals Inc
Current assignee: Molecular Templates Inc
Priority date: 2007-06-04
Filing date: 2008-06-04
Publication date: 2008-12-11
Anticipated expiration: 2009-12-04

Abstract

Hypoxia activated prodrugs of antineoplastic agents are useful in treatment of cancer and other hyperproliferative diseases.

Description

HYPOXIAACTIVATED PRODRUGS OF ANTINEOPLASTIC AGENTS

CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application claims priority to U.S. provisional application Serial No. 60/941 ,753, filed 4 June 2007 and U.S. provisional application Serial No. 60/985,795 filed 6 November 2007, each of which is incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] NOT APPLICABLE

REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER

PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK. [0003] NOT APPLICABLE

FIELD OF THE INVENTION

[0004] The present invention provides compositions and methods for the treatment of cancer, and generally relates to the fields of medicinal chemistry, medicine, pharmacology, molecular biology, and biology.

BACKGROUND OF THE INVENTION

[0005] Hypoxia activated (or bioreducible) prodrugs of antineoplastic agents, ("HAPs"), are useful for treating cancer. See, for example, PCT Pat. Pub. No. WO 00/064864, WO 04/087075, WO 06/057946, WO 07/002931, WO 07/137196 and PCT Pat. Appl. No. US07/88645, each of which is incorporated herein by reference. HAPs contain a bioreductive group, a linker, and an antineoplastic agent and are less cytotoxic than the corresponding antineoplastic agents. Under hypoxic conditions or hypoxia, the bioreductive group present in the HAP is reduced, and a cytotoxic antineoplastic agent is generated and/or released. Under normoxic conditions or normoxia such as those existing in a normal cell, a HAP can be non toxic or less toxic. Under hypoxic conditions such as those existing in tumor cells within hypoxic zones of solid tumors, the cytotoxin generated and/or released from a HAP can selectively kill cancer cells in and around the hypoxic tumor zone.

[0006] A certain class of antineoplastic agents, known as vascular disrupting agents, tubulin binding agents or anti-tubulin agents, can be cytotoxic to tumor cells by collapsing or otherwise disrupting the tumor vasculature. See, for example PCT Pat. Pub. No. WO 06/057946 and WO 07/137196, each of which is incorporated herein by reference. The collapse of the tumor vasculature reduces blood and oxygen flow into the tumor and can increase tumor hypoxia. Hypoxia activated prodrugs of vascular disrupting agents are reported in PCT Pat. Pub. No. WO 06/057946 and WO 07/137196.

[0007] There remains a need for additional HAPs for the treatment of cancer, including HAPs containing vascular disrupting agents. The present invention meets such needs.

BRIEF SUMMARY OF THE INVENTION

[0008] In one aspect, the present invention provides HAPs having structures of the formula Hyp-L-M wherein the Hyp moiety is a bioreductive group, L is a releasable linker comprising an ammonium moiety, and M is an antineoplastic agent. In another embodiment, L is a releasable linker comprising a methylene-ammonium-methylene moiety. In another embodiment, L has a structure of the formula -L₂-Lr, or in other words the corresponding HAP has a structure of the formula Hyp-L₂-Li-M, wherein -L_\- has a structure of the formula

-CRiR₂-N(R₃R₄)^R₅R₆-

and -L₂- is selected from a bond and a moiety having a structure of the formula:

wherein each Of Ri-R₈ is selected from the group consisting Of Ci-C₆ alkyl, Cj-C₆ heteroalkyl, C₃-C₈ cycloalkyl, heterocyclyl, aryl, and heteroaryl, or together Ri and R₂, R₅ and R₆, and R₇ and R₈ form a C₃-C₈ cycloalkyl or a heterocyclyl group, or together R₃ and R₄ form a heterocyclyl group and each of Ra-Rd is selected independently from the group consisting of hydrogen, halo, nitro, CO₂H, Ci-C₆ alkyl, and Ci-C₆ alkoxy. In one embodiment, each Ri, R₂, R₅, and R₆ is hydrogen. In another embodiment, each R₃ and R₄ is alkyl. In another embodiment, each R₃ and R₄ is methyl. In one embodiment, L₂ is a bond. In one embodiment, Li has a structure of the formula -CH₂-NMe₂CH₂-. In another embodiment, L has a structure of the formula -CH₂-NMe₂CH₂-.

[0009] In one embodiment, the bioreductive group, Hyp, is selected from the group consisting of a nitroaryl, a nitroheteroaryl, an indoloquinonyl, a naphthoquinonyl, an amine oxide, and a disulfide moiety. In another embodiment, the HAPs of the present invention contain novel 4-substituted-2-nitroimidazole bioreductive groups. In another embodiment, Hyp has a structure of the formula:

wherein Ri₅ is selected from the group consisting of hydrogen, Ci-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl, heteroaryl, and halo. In another embodiment, Rj₅ is a terminal C₂-C₆ alkenyl moiety. In another embodiment, the terminal C₂-C₆ alkenyl moiety has a structure of the formula C(Ri_8a)=C(Ri_8b)Ri_8c wherein each of Ri_8a-c independently is selected from hydrogen, C_rC₆ alkyl, and CpC₄ alkyl provided that however, at least one of Ri_8a-_C is Ci-C₄ alkyl. In another embodiment, Ri ₅ is a terminal C₂-C₆ alkynyl moiety. In another embodiment, the terminal C₂-C₆ alkynyl moiety has a structure of the formula

wherein R₁₉ is selected from the group consisting Of C)-C₆ alkyl, aryl, and heteroaryl.

[0010] Various antineoplastic agents can be employed as M in the HAPs of the present invention. In another embodiment, the antineoplastic agent is an aroylindazole compound that can bind tubulin, inhibit microtubule formation, and/or disrupt tumor vasculature (see

PCT Pat. Pub. Nos. WO 06/057946 and WO 07/137196, each of which is incorporated herein by reference).

[0011] In another embodiment, the present invention provides HAPs wherein M is an aroylindazole moiety having a structure of the formula:

wherein R⁹ is selected from the group consisting of hydrogen, halo, hydroxy, nitro, cyano, amino, CrQalkylamino, di(C]-C₆)alkylamino, Ci-C₆alkoxy, CrQalkyl, Ci-C₆heteroalkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₈ cycloalkyl, heterocyclyl, aryl, and heteroaryl. In another embodiment, the present invention provides a HAP containing as an antineoplastic agent an aroylindazole compound that can bind tubulin and wherein the HAP has a structure of the formula:

wherein R₃, R₄, and Hyp are defined as in any embodiment above. In another embodiment, the HAP compound is compound 36 as disclosed herein.

[0012] In another embodiment, the antineoplastic agent is selected from camptothecin and analogs and Taxol® (paclitaxel, Bristol Meyers Squibs) and analogs.

[0013] In another embodiment, the present invention provides Hyp containing compounds having structures of the formula selected from Hyp-CH₂-OH and Hyp-CH₂-Br wherein Hyp has a structure of the formula:

wherein R₁₅ substituents include but are not limited to phenyl, pyridyl, isopropenyl, propenyl, and vinyl. The novel as well as known Hyp-CH₂-OH and HyP-CH₂-Br compounds are intermediates useful in the synthesis of the HAP compounds of the present invention.

[0014] In another aspect, the present invention provides methods of synthesizing HAPs and various intermediates useful in the synthesis of the HAP compounds of the present invention.

[0015] In another aspect, the present invention provides methods of synthesizing a HAP of the present invention.

[0016] In another embodiment, the present invention provides HAPs in substantially pure forms. In another aspect, the present invention provides a pharmaceutically acceptable formulation comprising a compound of the present invention, including but not limited to the HAPs of the present invention and pharmaceutically acceptable carriers, diluents, and/or excipients.

[0017] In another aspect, the present invention provides a method of treating cancer and other hyperproliferative diseases comprising administering a therapeutically effective amount of a HAP of the present invention to a patient in need of such treatment.

[0018] In another aspect, the present invention provides methods of disrupting vasculature associated with cancer or other hyperproliferative diseases by contacting said vasculature with a compound of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1. Demonstration of growth reduction of HT29 xenograft tumor in mice upon administration of Compound 15 alone and in combination with CPT-I l.

[0020] Figure 2. Demonstration of the safe administration of Compound 15 alone and in combination with CPT-11 by plotting the percent change in animal body weight compared to the bodyweight at the beginning of the treatment.

[0021] Figure 3. Demonstration of growth reduction of HT29 xenograft tumors in mice upon administration of 23 and 36 as single agents.

[0022] Figure 4. Demonstration of growth reduction of HT29 xenograft tumors in mice upon administration of 23 and 36 in combination with CPT-I l.

DETAILED DESCRIPTION OF THE INVENTION

[0023] This detailed description of the different aspects and embodiments of the present invention is organized as follows: Section I provides useful definitions; Section II describes the compounds of the present invention, including HAPs and prodrugs that are activated by a reaction other than a reduction reaction, and the methods of their synthesis; Section III describes therapies provided by the present invention; and Section IV provides illustrative examples for synthesizing compounds of the present invention, including HAPs and prodrugs that are activated by a reaction other than a reduction reaction, and demonstrates in vitro and in vivo efficacy of compounds of the present invention, including HAPs and prodrugs that are activated by a reaction other than a reduction reaction. This detailed description is organized into sections only for the convenience of the reader, and disclosure found in any section is applicable to disclosure elsewhere in the specification.

I. DEFINITIONS

[0024] The following definitions are provided to assist the reader. Unless otherwise defined, all terms of art, notations, and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the chemical and medical arts. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference over the definition of the term as generally understood in the art.

[0025] ">ΛΛ^" refers to a position on a moiety which is covalently bonded to the rest of the molecule via a single bond.

[0026] "C₂-C₆alkenyl" by itself or as part of another substituent refers to a straight or branched chain, which may be mono or polyunsaturated, having the number of carbon atoms designated. For example, "C₂-C₆alkenyl" means an alkenyl radical having from 2, 3, 4, 5 or 6 atoms that is derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane. Examples include, but are not limited to vinyl, 2 propenyl i.e. - CH=C(H)(CH₃), -CH=C(CH₃)₂, -C(CH₃)=C(H)₂, -C(CH₃)=C(H)(CH₃), C(CH₂CH₃)=CH₂, butadienyl e.g. 2 (butadienyl), pentadienyl e.g. 2,4 pentadienyl and 3 (1,4 pentadienyl), and hexadienyl, among others, and higher homologs and stereoisomers thereof. The phrase "substituted alkenyl" has the same meaning with respect to alkenyl groups that substituted alkyl groups had with respect to unsubstituted alkyl groups. A substituted alkenyl group includes alkenyl groups in which a non-carbon or non-hydrogen atom is bonded to a carbon double bonded to another carbon and those in which one of the non-carbon or non-hydrogen atoms is bonded to a carbon not involved in a double bond to another carbon. Each site of unsaturation may be either cis or trans configuration about the double bond(s).

[0027] "C]-C₆alkoxy" refers to a substituted or unsubstituted alkyl group of 1-6 carbon atoms covalently bonded to an oxygen atom. In other words, a Ci-C₆ alkoxy group has the general structure -O-(Ci-C₆)alkyl. Q-C_δalkoxy groups include, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, 2-pentoxy, 3- pentoxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy. [0028] "Q-C₆alkoxycarbonyl" refers to an Ci-C₆ alkoxy group covalently bonded to a carbonyl. In other words, a CpC₆ alkoxycarbonyl group has the general structure -C(=O)-O- (Ci-QOalkyl.

[0029] "C_I-C_O alkyl" refers to a substituted or unsusbstituted straight or branched chain alkyl groups having 1-6 carbon atoms. Ci-C₆ alkyl groups include, for example, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl and 3-methylpentyl. A C]-C₆ alkyl substituent may be covalently bonded to an atom within a molecule of interest via any chemically suitable portion of the Q- C₆ alkyl group.

[0030] "Amino" refers to a monovalent radical -NR^aR^b or divalent radical -NR^a-. The term "alkylamino" refers to the group -NR^aR^b where R^a is alkyl and R^b is H or alkyl. The term "arylamino" refers to the group -NR^aR^b where R^a is aryl and R^b is hydrogen, alkyl, aryl, or heterocyclyl. The term "(alkyl)(aryl)amino" refers to the group -NR^aR^b where R^a is alkyl and R^b is aryl. Additionally, for dialkylamino groups, the alkyl portions can be the same or different and can also be combined to form a 3-7 membered ring with the nitrogen atom to which each is attached. Accordingly, a group represented as -NR^aR^b is meant to include piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl and the like.

[0031] "Ci-C₆ alkylamino," refers to a substituted or unsubstituted alkyl group of 1-6 carbon atoms covalently bonded to -NH- . In other words, a Ci-C₆ alkylamino group has the general structure -NH- (Ci -C₆) alkyl. Similarly a di(Ci-C₆)alkylamino group has the general structure -N-[(Q-C₆)alkyl]2. Ci-C₆ alkylamino groups include, for example, methylamino, ethylamino, propylamino and butylamino.

[0032] "Ci-C₆ alkylene" refers to a linear saturated divalent substituted or unsubstituted hydrocarbon radical or a branched saturated divalent hydrocarbon radical having 1 - 6 carbon atoms. Alkylene groups include, for example, methylene, ethylene, propylene, butylene, 2- methylpropylene, pentylene. A substituted alkylene can be substituted, among other groups, with Ci-C₆ alkyl groups.

[0033] "C₂-C₆ alkyl ether" refers to a substituent with an oxygen atom and 2 - 6 carbon atoms positioned such that at least one carbon atom is located on either side of the oxygen atom. [0034] "C₂-C₆ alkynyl" by itself or as part of another substituent, means a straight or branched chain hydrocarbon radical, which may be mono- or polyunsaturated, having the number of carbon atoms designated. For example, "C₂-C₆ alkynyl" means an alkynyl radial having from 2 to 6 carbon atoms that is derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane. "Unsubstituted alkynyl" refers to straight and branched chain groups such as those described with respect to unsubstituted alkyl groups as defined above, except that at least one triple bond exists between two carbon atoms. Examples include, but are not limited to ethynyl e.g. -C≡C(H), 1- propynyl e.g. -C=C(CH₃), -C=C(CH₂CH₃), -C(H₂)CsC(H), -C(H)₂C=C(CH₃), and -C(H)₂C=C(CH₂CH₃) among others, and higher homologs and isomers thereof. The phrase "substituted alkynyl" has the same meaning with respect to alkynyl groups that substituted alkyl groups had with respect to unsubstituted alkyl groups. A substituted alkynyl group includes alkynyl groups in which a non-carbon or non-hydrogen atom is bonded to a carbon triple bonded to another carbon and those in which a non-carbon or non-hydrogen atom is bonded to a carbon not involved in a triple bond to another carbon.

[0035] "Aroylindazole compound" refers to a compound wherein a substituted or unsubstituted benzoyl moiety is covalently bonded to the 3-position of a substituted or unsubstituted indazole moiety. Aroylindazole compounds are described for example in PCT Pat. Pub. WO 06/057946 and WO 07/137196 (each of which is incorporated herein by reference).

[0036] "Aryl" refers to a substituted or unsubstituted cyclic moiety that includes one or more monocyclic or fused ring aromatic systems. Such moieties include any moiety that has one or more monocyclic or bicyclic fused ring aromatic systems, including but not limited to phenyl and naphthyl.

[0037] "Bioreductive group", in one aspect, refers to a substituted or unsubstituted nitroaryl, nitroheteroaryl, indoloquinonyl or a naphtoquinonyl moiety that can undergo reduction. In another aspect, "bioreductive group" refers to a substituted or unsubstituted , amine oxide or a disulfide moiety that can undergo reduction. Bioreductive groups are described for example in the US Pat. Nos. 5,750,782; 5,780,585; 5,872,129; 6,251,933; 5,306,727; 5,403,932; 5,190,929; and 6,656,926; PCT Pat. Appl. Pub. Nos. WO 00/64864; WO 04/85361; WO 04/85421; WO 04/87075; 06/57946; and WO 07/002931; US Pat. Appl. Pub. Nos. 2003/0008850; 2004/254103; and 2005/043244, and the references deGroot et al, 2001, Current Med. Chem. 8: 1093-22; Borch et al., J. Med. Chem. 2000, 43: 2258-65; Borch et al., J. Med. Chem. 2001, 44: 69-73; Borch et al., J. Med. Chem. 2001, 44: 74-7; Hernick et al. J. Med. Chem. 2002, 45: 3540-48; Hernick et al., J. Med. Chem. 2003, 46: 148-54; Papot et al., Curr. Med. Chem., 2002, 2, 155-85; Tercel et al., J. Med. Chem. 1996, 39: 1084-94; and Tercel et al., J. Med. Chem. 2001, 44: 3511-22 (each of which is incorporated herein by reference).

[0038] In each of the above embodiments designating a number of atoms e.g. "C_1-6" is meant to include all possible embodiments that have one fewer atom. Non-limiting examples include Ci_-6, C_2-6, C_2-6, C₃_₆, C_3-6 and the like.

[0039] Each of the terms herein (e.g., "alkyl," "heteroalkyl," "aryl" and "heteroaryl") is meant to include both "unsubstituted" and optionally "substituted" forms of the indicated radical, unless otherwise indicated. Typically each radical is substituted with 0, 1, 2 3 4 or 5 substituents, unless otherwise indicated. Examples of substituents for each type of radical are provided below.

[0040] "Substituted" refers to a group as defined herein in which one or more bonds to a carbon(s) or hydrogen(s) are replaced by a bond to non-hydrogen and non-carbon atom "substituents" 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, aryloxy, and acyloxy groups; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as amino, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, alkoxyamino, hydroxyamino, acylamino, sulfonylamino, N-oxides, imides, and enamines; and other heteroatoms in various other groups. 'Substituents" also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom is replaced by a higher-order bond (e.g., a double- or triple- bond) to a heteroatom such as oxygen in oxo, acyl, amido, alkoxycarbonyl, aminocarbonyl, carboxyl, and ester groups; nitrogen in groups such as imines, oximes, hydrazones, and nitriles. "Substituents" further include groups in which one or more bonds to a carbon(s) or hydrogen(s) atoms is replaced by a bond to a cycloalkyl, heterocyclyl, aryl, and heteroaryl groups. Representative "substituents" include, among others, groups in which one or more bonds to a carbon or hydrogen atom is/are replaced by one or more bonds to fluoro, chloro, or bromo group. Another representative "substituent" is the trifluoromethyl group and other groups that contain the trifluoromethyl group. Other representative "substituents" include those in which one or more bonds to a carbon or hydrogen atom is replaced by a bond to an oxygen atom such that the substituted alkyl group contains a hydroxyl, alkoxy, or aryloxy group. Other representative "substituents" include alkyl groups that have an amine, or a substituted or unsubstituted alkylamine, dialkylamine, arylamine, (alkyl)(aryl)amine, diarylamine, heterocyclylamine, diheterocyclylamine, (alkyl)(heterocyclyl)amine, or (aryl)(heterocyclyl)amine group. Still other representative "substituents" include those in which one or more bonds to a carbon(s) or hydrogen(s) atoms is replaced by a bond to an alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group.

[0041] The herein-defined groups may include prefixes and/or suffixes that are commonly used in the art to create additional well-recognized substituent groups. As examples, "alkylamino" refers to a group of the formula -NR^aR^b. Unless stated otherwise, for the following groups containing R^a, R^b, R^c, R^d and R^e: R^a, and R^b are each independently selected from H, alkyl, alkoxy, thioalkoxy, cycloalkyl, aryl, heteroaryl, or heterocyclyl or are optionally joined together with the atom(s) to which they are attached to form a cyclic group. When R^a and R^b are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6- or 7-membered ring. For example, -NR^aR^b is meant to include 1-pyrrolidinyl and 4-morpholinyl.

[0042] R^c, R^d, R^e and R^f , unless otherwise indicated, are each independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl or alkylenearyl as defined herein.

[0043] Typically, a particular radical will have 0, 1, 2 or 3 substituents, with those groups having two or fewer substituents being preferred in the present invention. More preferably, a radical will be unsubstituted or monosubstituted. Most preferably, a radical will be unsubstituted.

[0044] In some embodidments, "substituents" refers to an atom or group, including, for example, amino, Ci-C₆alkylamino or di(Ci-C₆)alkylamino, C]-C₆alkoxy, Ci-C₆alkylthio, aryl, -COOH, -CONH₂, cyano, ethenyl, ethynyl, halo, heteroaryl, hydroxy, mono- or di(Cr C₆)alkylcarboxamido, mono or di(Ci-C₆)alkylsulfonamido, nitro, -OSO₂-Ry, and -SO₂NH₂.

[0045] "Substituents" for the alkyl and heteroalkyl radicals (as well as those groups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocyclyl) can be a variety of groups selected from: -OR^a, =0, =NR^a, =N-0R'\ -NR^aR^b, -SR^a, halogen, -SiR^aR^bR^c, -OC(O)R^a, -C(O)R³, -CO₂R³, -CONR^aR^b, -OC(O)NR^aR^b, -NR^bC(O)R^a, -NR³- C(O)NR^bR^c, -NR^a-SO₂NR^bR^c, -NR^bCO₂R^a, -NH-C(NH₂)=NH, -NR^aQNH₂)=NH, -NH-C(NH₂)=NR^a, -S(O) R^a, -S0₂R^a, -SO₂NR^aR^b, -NR^bSO₂R, -CN and -NO₂, in a number ranging from zero to three, with those groups having zero, one or two substituents being particularly preferred.

[0046] In some embodiments, "substituents" for the alkyl and heteroalkyl radicals are selected from: -OR³, =0, - NR^aR^b, -SR³, halogen, -SiR³R^bR^c, -OC(O)R³, -C(O)R^a, -CO₂R^a,

-C0NR^aR^b, -0C(0)NR^aR^b, -NR^bC(O)R^a, -NR^bCO₂R^a, -NR^a-SO₂NR^bR^c, -S(O)R³, -SO₂R^a, -SO₂NR^aR^b, -NR⁰SO₂R, -CN and -NO₂, where R^a and R^b are as defined above. In some embodiments, substituents are selected from: -0R^a, =0, - NR^aR^b, halogen, -OC(O) R^a,

-CO₂R³, -C0NR^aR^b, -OC(O)NR^aR^b, -NR^bC(O)R^a, -NR⁵CO₂R³, -NR^a-SO₂NR^bR^c, -SO₂R³,

-SO₂NR³R⁶, -NR¹¹SO₂R, -CN and -NO₂.

[0047] Examples of substituted alkyl are: -(CH₂)₃NH₂, -(CH₂)₃NH(CH₃), -(CH₂)₃NH(CH₃)₂, -CH₂Q=CH₂)CH₂NH₂, -CH₂Q=O)CH₂NH₂, -CH₂S(=O)₂CH₃, - CH₂OCH₂NH₂, -CO₂H_. Examples of substituents of substituted alkyl are: CH₂OH, -OH, - OCH₃, -OC₂H₅, -OCF₃, -OQ=O)CH₃, -OQ=O)NH_2, -OC(=O)N(CH₃)_2, -CN, -NO₂, - Q=O)CH₃, -CO₂H, -CO₂CH₃, -CONH₂, -NH₂,-N(CH₃)₂, -NHSO₂CH₃, -NHCOCH₃, -NHQ=O)OCH₃, -NHSO₂CH₃, -SO₂CH₃, -SO₂NH₂, and halo.

[0048] Similarly, "substituents" for the aryl and heteroaryl groups are varied and are selected from: -halogen, -OR³, -OC(O) R^a, -NR^aR^b, -SR³, -R^a, -CN, -NO₂, -CO₂R³,

-C0NR³R^b, -C(O) R³, -0C(0)NR^aR^b, -NR⁶C(O) R³, -NR^bC(O)₂R^a, -NR^a-C(0)NR^bR^c, -NH-QNH₂)=NH, -NR^aC(NH₂)=NH, -NH-C(NH₂)=NR^a, -S(O) R^a, -S(O) ₂ R³, -S(O) ₂NR'R^b, -N3, -CH(Ph)₂, perfluoroCj.galkoxy, and perfluoroCi.galkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R^a, R^b and R° are independently selected from hydrogen, C;μgalkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-Cj_galkyl, and (unsubstituted ary^oxy-C^galkyl.

[0049] Two of the "substituents "on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(O)-(CH₂)q-U-, wherein T and

U are independently -NH-, -O-, -CH₂. or a single bond, and q is O, 1 or 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)_r_B-, wherein A and B are independently -CH2-, -O-, -NH-, -S-, -S(O)-, -S(O)₂., -S(O) ₂NR^a- or a single bond, and r is 1, 2 or 3. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CH₂)_S-X-(CH₂)I- -, where s and t are independently integers of from O to 3, and X is -0-, -NR^a-, -S- , -S(O)-, -S(O)₂-, or -S(O) ₂NR^a-. The substituent R^a in -NR^a- and -S(O)₂NR³- is selected from hydrogen or unsubstituted Ci .βalkyl. Otherwise, R' is as defined above.

[0050] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-O-C(O)-.

[0051] "C₃-C₈ Cycloalkyl" or "carbocycle", by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl", "alkenyl" and "alkynyl" in which all ring atoms are carbon. "Cycloalkyl" or "carbocycle" refers to a mono- or polycyclic group. When used in connection with cycloalkyl substituents, the term "polycyclic" refers herein to fused and non-fused alkyl cyclic structures. "Cycloalkyl" or "carbocycle" may form a bridged ring or a spiro ring. The cycloalkyl group may have one or more double or triple bond(s). The term "cycloalkenyl" refers to a cycloalkyl group that has at least one site of alkenyl unsaturation between the ring vertices. The term "cycloalkynyl" refers to a cycloalkyl group that has at least one site of alkynyl unsaturation between the ring vertices. When "cycloalkyl" is used in combination with "alkyl", as in C₃_₈cycloalkylC₃-₈alkylene-, the cycloalkyl portion is meant to have the stated number of carbon atoms (e.g., from three to eight carbon atoms), while the alkylene portion has from one to eight carbon atoms. Typical cycloalkyl substituents have from 3 to 8 ring atoms. Examples of cycloalkyl include cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.

[0052] "Eschenmoser salt" refers to an ammonium salt having a structure of the formula H₂C=NMe₂I; similar salts include H₂C=N(R^X)₂ wherein each R^x is independently selected from the group consisting of C₁-C₆ alkyl, Ci-C₆ heteroalkyl, C₃-C₈ cycloalkyl, heteroalkyl, aryl, heteroaryl, or together two R^x groups form a CpC₆ heteroalkyl moiety. [0053] "Acylamino-" refers to the group -NR^aC(=O)R^c where R^c is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl or heterocyclyl.

[0054] "Acyloxy" refers to -OC(=O)-R^C where R^c is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl or heterocyclyl.

[0055] "Halo" or "halogen" by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl", are meant to include alkyl in which one or more hydrogen is substituted with halogen atoms which can be the same or different, in a number ranging from one up to the maximum number of halogens permitted e.g. for alkyl, (2m'+l), where m' is the total number of carbon atoms in the alkyl group. For example, the term "haloC^.galkyl" is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. The term "perhaloalkyl" means, unless otherwise stated, alkyl substituted with (2m'+l) halogen atoms, where m¹ is the total number of carbon atoms in the alkyl group. For example, the term "perhaloCi.galkyl", is meant to include trifluoromethyl, pentachloroethyl, l,l,l-trifluoro-2-bromo-2-chloroethyl, and the like. Additionally, term "haloalkoxy" refers to an alkoxy radical substituted with one or more halogen atoms. "Halide" refers to the acid or anionic form of a halo group.

[0056] "Heteroaryl" refers to a substituted or unsubstituted monocyclic aromatic system having 5 or 6 ring atoms, or a fused ring bicyclic aromatic system having 8 - 20 atoms, in which the ring atoms are C, O, S, SO, SO₂, or N and at least one of the ring atoms is a heteroatom, i.e., O, S, SO, SO₂, or N. Heteroaryl groups include, for example, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothio-furanyl, benzothiophenyl, benzoxazolyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, NH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, dithiazinyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, naphthyridinyl, octahydroisoqiiinolinyl, oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazolyl, pyridoimidazolyl, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, quinuclidinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, thiadiazinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl and xanthenyl. Unless indicated otherwise, the arrangement of the heteroatoms within the ring may be any arrangement allowed by the bonding characteristics of the constituent ring atoms.

[0057] "Heterocyclyl" refers to a monocyclic or fused ring multicyclic cycloalkyl group at least a portion of which is not aromatic and in which one or more of the carbon atoms in the ring system is replaced by a heteroatom selected from O, S, SO, SO₂, P, or N. Examples of heterocyclyl groups include but are not limited to imidazolinyl, morpholinyl, piperidinyl, piperidin-2-only, piperazinyl, pyrrolidinyl, pyrrolidine-2-onyl, tetrahydrofuranyl, and tetrahydroimidazo [4,5-c] pyridinyl.

[0058] "Heteroalkyl" means an alkyl radical as defined herein with one, two or three substituents independently selected from cyano, -OR^W, -NR^XRY, and -S(O)_nR² (where n is an integer from 0 to 2 ), with the understanding that the point of attachment of the heteroalkyl radical is through a carbon atom of the heteroalkyl radical. R^w is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, aryl, araalkyl, alkoxycarbonyl, aryloxycarbonyl, carboxamido, or mono- or di-alkylcarbamoyl. R^x is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, aryl or araalkyl. Ry is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, aryl, araalkyl, alkoxycarbonyl, aryloxycarbonyl, carboxamido, mono- or di-alkylcarbamoyl or alkylsulfonyl. R^z is hydrogen (provided that n is 0), alkyl, cycloalkyl, cycloalkyl-alkyl, aryl, araalkyl, amino, mono- alkylamino, di-alkylamino, or hydroxyalkyl. Representative examples include, for example, 2-hydroxyethyl, 2,3-dihydroxypropyl, 2-methoxyethyl, benzyloxymethyl, 2-cyanoethyl, and

2-methylsulfonyl-ethyl. For each of the above, R^w, R^x ,RY, and R^z can be further substituted by amino, fluorine, alkylamino, di-alkylamino, OH or alkoxy. Additionally, the prefix indicating the number of carbon atoms (e.g., C ^ -C ^Q) refers to the total number of carbon atoms in the portion of the heteroalkyl group exclusive of the cyano, ~OR^W, -NR^XRY, or - S(O)_nR² portions.

[0059] "Leaving group" refers to a moiety that can be replaced by a nucleophile. Examples of leaving groups include but are not limited to halo and sulfonate.

[0060] "Secondary amine" refers to an amine having structure of the formula R^X2NH wherein each R^x is independently selected from the group consisting of Ci-C₆ alkyl, CpC₆ heteroalkyl, C₃-C₈ cycloalkyl, heteroalkyl, aryl, heteroaryl, or together two R^x groups form a C]-C₆ heteroalkyl moiety.

[0061] The term "pharmaceutically acceptable salts" is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of salts derived from pharmaceutically-acceptable inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc and the like. Salts derived from pharmaceutically-acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally- occurring amines and the like, such as arginine, betaine, caffeine, choline, N,N'- dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like {see, e.g., Berge, S.M. et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science, 66:1-19, 1977). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. [0062] The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

[0063] "Substituent" refers to an atom or group, including, for example, amino, C]-C₆ alkylamino or di(C_rC₆)alkylamino, Cj-C₆ alkoxy, C]-C₆ alkylthio, aryl, COOH, CONH₂, cyano, ethenyl, ethynyl, halo, heteroaryl, hydroxy, mono- or di(Ci-C₆)alkylcarboxamido, mono or di(Ci-C₆)alkylsulfonamido, nitro, -OSO₂-R^y, and -SO₂NH₂.

[0064] "Carboxy" or "carboxyl" refers to the group -CO₂H. "Carboxylate" refers to the acid or anionic form of the group R⁰CO₂H wherein R^c is Ci-C₆alkyl, C₂-C₆alkenyl, alkynyl, C₃-C₈cycloalkyl, C₃-C₈cycloalkenyl, aryl, C_rC₆heteroalkyl, heteroaryl or heterocyclyl.

[0065] "Phosphanyl" refers to the group -PO(R^f)₃ where R^f is hydroxyl or alkoxy. "Phosphate" refers to the acid or anionic form of a phosphanyl group.

[0066] "Nitro" refers to -NO₂. "Nitrate" refers to the acid or anionic form of a nitro group.

[0067] "Sulfonyloxy" refers to the group -OSO₂-R⁰ where R° is C_rC₆alkyl, C₂-C₆alkenyl, alkynyl, C₃-Cscycloalkyl, C₃-C₈cycloalkenyl, aryl, Ci-Qheteroalkyl, heteroaryl or heterocyclyl. "Sulfonate" refers to the acid or anionic form of a sulfonyloxy group.

[0068] "Sulfanyl" refers to the group -SO₂R^f ₂ where R^f is hydroxyl or alkoxy. "Sulfate" refers to the acid or anionic form of a sulfanyl group.

[0069] "Administering" or "administration of a drug to a patient (and grammatical equivalents of this phrase) refers to direct administration, which may be administration to a patient by a medical professional or may be self-administration, and/or indirect administration, which may be the act of prescribing a drug. For example, a physician who instructs a patient to self-administer a drug and/or provides a patient with a prescription for a drug is administering the drug to the patient.

[0070] "Pharmaceutically acceptable carrier, excipient, or diluent" refers to a carrier, excipient, or diluent that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes a carrier, excipient, or diluent that is acceptable for veterinary use as well as human pharmaceutical use. A "pharmaceutically acceptable carrier, excipient, or diluent" includes both one and more than one such carrier, excipient, or diluent.

[0071] "Prodrug" refers to a compound that, after administration, is metabolized or otherwise converted to a biologically active or more active compound (or drug) with respect to at least one property. A prodrug, relative to the drug, is modified chemically in a manner that renders it, relative to the drug, less active or inactive, but the chemical modification is such that the corresponding drug is generated by metabolic or other biological processes after the prodrug is administered. A prodrug may have, relative to the active drug, altered metabolic stability or transport characteristics, fewer side effects or lower toxicity, or improved flavor (for example, see the reference Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392, incorporated herein by reference). A prodrug may be synthesized using reactants other than the corresponding drug.

[0072] QnD or qnd refers to drug administration once every n days. For example QD (or qd) refers to once every day or once daily dosing, Q2D (or q2d) refers to a dosing once every two days, Q7D refers to a dosing once every 7 days or once a week, Q5D refers to dosing once every 5 days.

[0073] "Reduction" of a symptom or symptoms (and grammatical equivalents of this phrase) refers to decreasing the severity or frequency of the symptom(s), or elimination of the symptom(s).

[0074] "Therapeutically effective amount" of a drug refers to an amount of a drug that, when administered to a patient with cancer or another hyperproliferative disease, will have the intended therapeutic effect, e.g., alleviation, amelioration, palliation or elimination of one or more manifestations of cancer or another hyperproliferative disease in the patient. A therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations.

[0075] "Treating" or "treatment of" a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer or another hyperproliferative disease; diminishment of extent of disease; delay or slowing of disease progression; amelioration, palliation, or stabilization of the disease state; or other beneficial results.

II. HYPOXIA ACTIVATED PRODRUGS OF ANTINEOPLASTIC AGENT "HYP-L-M"

[0076] In one aspect, the present invention provides HAPs having structures of the formula Hyp-L-M wherein the Hyp moiety is a bioreductive group, L is a releasable linker comprising an ammonium moiety, and M is an antineoplastic agent. Without being bound by mechanism, it is believed that when the bioreductive group of a HAP compound of the present invention is reduced, the releasable linker undergoes a fragmentation and eliminates a tertiary amine molecule, thereby releasing the antineoplastic agent. A variety of bioreductive groups, releasable linkers, and antineoplastic agents can be combined in the HAPs of the present invention. A variety of combinations of Hyp, L, and M, and the resulting structure of the HAPs of the present invention will be apparent to one of skill in the art upon reading this disclosure.

[0077] In one embodiment, the present invention provides HAPs of tubulin binding aroylindazole compounds, wherein the aroylindazole moiety is covalently bonded to the bioreductive group via a releasable ammonium linker. The bioreductive group and releasable ammonium linkers of the HAPs of the present invention are described in the following subsections.

Bioreductive Group "Hyp "

[0078] In one embodiment, the present invention provides HAPs wherein the Hyp moiety is selected from the group consisting of a nitroaryl, a nitroheteroaryl, an indoloquinonyl, a naphthoquinonyl, an amine oxide, and a disulfide moiety.

[0079] In another embodiment, the Hyp moiety is selected from the group consisting of:

wherein Ri₅ is selected from hydrogen, Ci-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, aryl, heteroaryl, and halo and Rn is Ci-C₆ alkyl Ci-C₆ heteroalkyl, C₃-Cg cycloalkyl, heterocyclyl, aryl and heteroaryl. One of skill in the art will appreciate upon reading this disclosure that each of the aryl and heteroaryl moieties shown above as part of the Hyp moieties can be further substituted in accordance with the present invention. Suitable substituents include, but are not limited to, groups that as part of the compounds of the present invention enhance aqueous solubility of these compounds. Other suitable substituents include electron withdrawing groups such as nitro, halo, cyano, haloalkyls, and the like. Certain other suitable substituents include electron donating substituents such as alkoxy, amino, alkylamino, dialkylamino, and the like.

[0080] In another embodiment, Hyp has a structure of the formula:

[0081] In another embodiment, Ri₅ is a terminal C₂-C₆ alkenyl moiety. In another embodiment, the terminal C₂-C₆ alkenyl moiety has a structure of the formula

each of Ri8a-c independently is selected from hydrogen, Ci-C₆ alkyl, and Cj-C₄ alkyl provided that however, at least one of Ri_8a-_c is Cj-C₄ alkyl. In another embodiment, R₁₅ is a terminal C₂-C₆ alkynyl moiety. In another embodiment, the terminal C₂-C₆ alkynyl moiety has a structure of the formula:

wherein R₁₉ is selected from the group consisting of Q-C_όalkyl, aryl, and heteroaryl. In another embodiment, Rj ₉ is selected from a phenyl and a furanyl moiety wherein the phenyl and furanyl moieties can be suitably substituted. Suitable substituents include, but are not limited to, CpC₆ alkyl and halo. In another embodiment, R₁₅ is selected from the group consisting of ethyl, isopropyl, -CH=CH₂, -CH=CHMe, and -CH(Me)=CH₂, hydrogen, phenyl, and bromo. In another embodiment, R₁₅ is hydrogen, phenyl, and bromo.

[0082] In another embodiment, Hyp has a structure of the formula:

[0083] Without being bound by mechanism, it is believed that as the bioreductive group is reduced, electron pairs flow from the reduced bioreductive group to the releasable linker, fragmenting the releasable linker and release of the antineoplastic agent, for example, as shown below for a nitroimidazole moiety:

[0084] In one embodiment, the Hyp moiety is reduced under hypoxia. In another embodiment, the Hyp moiety is reduced by cytochrome P450 reductase, cytochrome P450, DT diaphorase, nitric oxide synthase, and/or a thiol.

Releasable Linker "L"

[0085] In one embodiment, the present invention provides HAPs having structures of the formula Hyp-L-N wherein L has a structure of the formula -L₂-Li- wherein -Li- has a structure of the formula

-CRiR₂-N(R₃R₄)-CR₅R₆-

and -L₂- is selected from a bond and a moiety having a structure of the formula: 'WV

wherein each of RpR₈ is selected from the group consisting of CpC₆ alkyl, Ci-C₆ heteroalkyl, C₃-C₈ cycloalkyl, heterocyclyl, aryl, and heteroaryl, or together Ri and R₂, R₅ and R₆, and R₇ and R₈ form a C₃-C₈ cycloalkyl or a heterocyclyl group, or together R₃ and R₄ form a heterocyclyl group and each of R_a-R_d is selected independently from the group consisting of hydrogen, halo, nitro, CO₂H, Ci-C₆ alkyl, and C_rC₆ alkoxy. In one embodiment, each R₁, R₂, R₅, and R₆ is hydrogen. In another embodiment, each R₃ and R₄ is alkyl. In another embodiment, each R₃ and R₄ is methyl. In one embodiment, Li has a structure of the formula -CH₂-NMe₂CH₂-. In one embodiment, the present invention provides HAPs having structure of the formula (I) wherein L has the structure of the formula:

Rd Rc

WCH₂ O-P % CH₂-N(R₃R₄)-CH>/w

Ra Rb

[0086] In another embodiment, L has a structure of the formula -CH₂-NMe₂CH₂-.

[0087] The Lj moiety having a structure of the formula -CR]R₂-N(R₃R₄)-CR₅R₆-, contains an ammonium group, is positively charged, and is associated with an anion. Anions suitable for the HAPs of the present invention include any pharmaceutically acceptable anion including but not limited to carboxylates, halides, phosphates, nitrate, sulphonates, and sulphates. In one embodiment, the HAPs of the present invention are more water soluble than the corresponding antineoplastic agents due in part to the presence of the Li moiety containing an ammonium group. Because a number of antineoplastic agents including, but not limited to, taxanes and analogs and epothilone and analogs, are lipophilic, sparingly soluble in water, and difficult to formulate for human administration, the enhanced water solutbility of the HAPs of the present invention including these antineoplastic agents provide an advantage in administering these HAPs compared to the corresponding antineoplastic agents.

[0088] In one embodiment, the present invention provides HAPs having structure of the formula Hyp-L-N wherein the releasable linker, L, comprises a heteroaryl ammonium moiety. In another embodiment, the heteroaryl ammonium moiety has a structure of the formula:

V₁=Y₂

wherein each Yj - Y₃ is independently selected from N and CR^X wherein each R^x is independently selected from the group consisting of Ci-C₆alkyl, Ci-C₆heteroalkyl, C₃-

Qcycloalkyl, heteroalkyl, aryl, and heteroaryl, with the proviso that at least one of Yi - Y₃ is CR\

[0089] Without being bound by mechanism, as the releasable linker fragments, the antineoplastic agent M is released. M moieties of the HAPs of the present invention are described in the following subsection.

Antineoplastic Agent "M"

[0090] A variety of antineoplastic agent M moieties are useful in the HAPs of the present invention. In one embodiment of the present invention, the M moiety is covalently bonded to the releasable linker L via a nitrogen, oxygen, sulfur, and/or a carbon atom of the M moiety. Antineoplastic agents useful in the HAPs of the present invention in accordance with the present teachings, are described, for example, in Physicians' Desk Reference, 2003, 57th Ed., Medical Economics Company, Inc., Oradell, N.J; Goodman & Gilman's The Pharmacological Basis of Therapeutics" 2001, 10th Edition, McGraw-Hill, New York. In one embodiment, the present invention provides HAPs wherein the antineoplastic agent M is selected from antineoplastic agents containing one or more NH, amino, C)-C₆ alkylamino, di(Ci-C₆)alkylamino, mercapto, hydroxyl, and phenoxy moieties. In another embodiment, M is selected from campthothecin, Taxol® (paclitaxel, Bristol Meyer Squibs) and other taxanes, epothilone and its analogs, and etoposide and its analogs. In another embodiment, the analogs are clinically useful analogs. In another embodiment, M is selected from antiandrogens and their analogs. Suitable antiandrogens include flutamide, nilutamide, and bicalutamide. Other suitable antineoplastic agents useful in HAPs of the present invention are described in U.S. Pat. Pub. No. 2006/0258656 (incorporated herein by reference). In another embodiment, the present invention provides HAPs of antineoplastic agents that are tubulin binding, and/or microtubule inhibiting agents, and/or vascular disrupting agents, as described below. [0091] In one embodiment, the antineoplastic agent M is a tubulin binding aroylindazole compound. In another embodiment, the tubulin binding aroylindazole compound has a structure of the formula:

Formula (I)

wherein R₉ is selected from the group consisting of hydrogen; halo, hydroxy, nitro, cyano, amino, C_rC₆ alkylamino, di Ci-C₆ alkylamino, Ci-C₆ alkoxy, Ci-C₆ alkyl, Ci-C₆ heteroalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0092] In another embodiment, R₉ is selected from the group consisting of: H, halo, amino, CO₂H, cyano, nitro, C-C₆ alkyl,

and

wherein each Rio is independently selected from the group consisting of hydrogen, Cj-C₆ alkyl, Ci-C₆ heteroalkyl, C₃-Cg cycloalkyl, heterocyclyl, aryl, and heteroaryl. In another embodiment, Rio has a structure of the formula -(CH₂)_n-Rn wherein n is 1-4 and Rn is selected from the group consisting of hydroxy, acyloxy, amino, acylamino, and OMe. In another embodiment, R₉ is selected from the group consisting of:

-CH₂OH , ~™ CO₂H , ""^-= CH₃, ^C=CH ,

^"CH(Me)OH , ~^w^ CH₂-CH₂OH CH₂OH X= (CH₂)₂OH and Λ ^Λ (CHa)₃OH .

[0093] In another embodiment, R₉ is propynyl. In another embodiment, the antineoplastic agent M is a tubulin binding aroylindazole compound described in claim 6, pages 129-30, PCT Pat. Pub. No. WO 06/057946. [0094] In another embodiment, the antineoplastic agent is a camptothecin analog having a structure of the formula:

Formula (II)

wherein each of Ri_2a-_d is selected from the group consisting of hydrogen, fluoro, hydroxy, nitro, amino, Q-Qalkoxy, Ci-C₆alkylamino, and di (Ci-C₆)alkylamino, or together any two of R_12a-d that are adjacent to each other form a methylenedioxy moiety, and Rj₃ is selected from an oximyl (-CH=N-OCi -C₆alkyl), a methyl and an ethyl group, each of which is optionally substituted with an amino, Ci-C₆ alkylamino, a di(Ci-C₆) alkylamino, or a heterocyclic group. In another embodiment, the heterocyclic group is selected from piperazine and (Ci-C₆) alkylpiperazine. In another embodiment, each of Ri_2c and Ri_2d is hydrogen. In another embodiment, R_]2b is hydroxyl. In another embodiment, Ri_2a is di (C₁- C₆) alkylamino. In another embodiment, R] _2a is dimethylaminomethyl. In another embodiment, the camptothecin analog has a structure of the formula selected from:

[0095] In another embodiment, the camptothecin analog is irinotecan.

[0096] In another embodiment, the antineplastic agent is a taxane or analog having a structure of the formula selected from:

Formula (IIIA) and Formula (IIIB)

[0097] In another embodiment, the antineplastic agent is a combretastatin analog. In another embodiment, the combretastatin analog is combretastatin A-4 or one of its analogs. In another embodiment, the combretastatin analog has a structure of the formula:

[0098] In certain other embodiments, the antineoplastic agents are selected from epothilone analogs. Epothilone analogs are described for example in Nicolaou et al., Angew Chem. Int Ed., 1998, 37: 2014-45; Waltman et al., Curr. Pharm. Design, 2005, 11: 1595-613; and Watkins et al., Curr. Pharm. Design, 2005, 1 1: 1615-53. A suitable epothilone analog useful in the HAPs of the present invention include, but is not limited to, epothilone-B. In another embodiment, the HAPs of epothilone analogs are compounds wherein the Hyp-L- moiety is attached to a nitrogen atom in a heterocycle moiety present in the epothilone analogs. Suitable heterocycle moieties include, but are not limited to, pyridines, benzthiazoles, benzoxazoles, and quinalines {see Watkins et al. supra). In another embodiment, a HAP of epothilone-B has a Hyp-CRiR₂- moiety covalently bonded to a thiazole ring in epothilone-B.

[0099] In another embodiment, the present invention provides a HAP having a structure of the formula:

Formula (IA)

wherein Rio is selected from the group consisting of hydrogen, CpC₆ alkyl, CpC₆ heteroalkyl, C^-Cs cycloalkyl, heterocyclyl, aryl, and heteroaryl; each of R₃ and R₄ is selected from alkyl; and Hyp is selected from the group consisting of:

wherein R₁₅ is selected from the group consisting of hydrogen, CrC₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl, heteroaryl, and halo. In another embodiment, R1₅ is selected from the group consisting of hydrogen, phenyl and bromo. In another embodiment, Ri₀ is methyl. In another embodiment, each of R₃ and R₄ is methyl. In another embodiment, Hyp has a structure of the formula:

wherein Ri₅ is defined as in any embodiment above.

[0100] In certain other embodiments, the present invention provides HAPs having a structure of the formulas:

wherein R_12a, Ri₂b, and R_i3 and each of R₃, R₄, and Hyp is defined as in any embodiment above or are defined as in formula (IA).

[0101] Certain examples of HAPs of the present invention including a tubulin binding antineoplastic agent are shown below:

10 11 12

13 14 15

16 17 18

19 20 21

22 23 24

28 29 30

36 37

[0102] Certain other examples of HAPs of the present invention including a camptothecin antineoplastic agent are shown below:

31 32

33 34 35

[0103] Because the L moiety, having a structure of the formula -CH₂-N(Me₂)-CH₂-, in the examples of the HAPs shown above, is positively charged, these HAPs exemplified above, and similar compounds described elsewhere in this disclosure, are associated with an anion. In other words, the compounds of the present invention are salts. Other anions, which may not be shown which are suitable for the HAPs of the present invention include any pharmaceutically acceptable anion including, but not limited to, carboxylates, halides, phosphates, nitrate, sulfonates, and sulfates. For example compound 23 contains a bromide anion and compound 36, a compound having the same Hyp, L, and M moieties as in compound 23, contains a carboxylate anion, e.g. lactate.

[0104] In certain embodiments, the present invention provides HAPs that under normoxic conditions are 5-10,000, 10-1,000, 20-500, and 50-100 times less cytotoxic than the corresponding antineoplastic agents. Without being bound by mechanism, when a bioreductive group of a HAP compound of the present invention is reduced under hypoxia, the releasable linker fragments and the cytotoxic antineoplastic agent is released. Because tumors contain regions of hypoxia, a HAP compound of the present invention is more cytotoxic to hypoxic tumors compared to normal tissue residing in normoxic conditions, and demonstrate tumor selective cytotoxicity. In certain embodiments of the present invention, the HAPs of the present invention are 5-10,000, 10-1,000, 10-50 times 20-500, and 50-100 times more cyctoxic under hypoxia than under normoxia. Examples of HAPS of the present invention that are 10-50 fold or more cyctoxic under hypoxia than under normoxia include but are not limited to compounds 1, 3, 13, 15, 23, 25, 26, 28, 30 and 36.

[0105] The releasable linker, L, useful in the HAPs of the present invention can also be employed in other prodrugs of antineoplastic agents as described below.

Other Antineoplastic Agent Prodrugs

[0106] In another aspect, the present invention provides prodrugs having structure of the formula Q-L-M wherein M is an antineoplastic agent, L is a releasable linker, and Q is an activating group that upon activation, by a reaction other than a reduction reaction, leads to a fragmentation of the releasable linker and release of the antineoplastic agent. In one embodiment, the Q moiety is an aliphatic or and aromatic ester, or a phenolic carbamate and the activation occurs upon hydrolysis of the ester. The hydrolysis can be catalyzed by an esterase or a carbamase. In another embodiment, the Q moiety is a carbohydrate moiety and the activation occurs upon hydrolysis. The hydrolysis can be catalyzed by a glycosidase. Within this embodiment, the carbohydrate moiety is a monosaccharide, including, but not limited to, glucose and glucuronic acid.

Methods of Synthesis

[0107] The present invention also provides methods of synthesizing the HAPs and the other compounds of the present invention as described below:

Method 1

[0108] In one aspect, the present invention provides a method of synthesizing a HAP compound of the present invention having structure of the formula Hyp-L-M wherein the Hyp moiety is a bioreductive group; L has a structure of the formula

-L₂-L]- wherein -L]- has a structure of the formula -CRiR₂-N(R₃R-O-CR₅R₆- and -L₂- is selected from a bond and a moiety having a structure of the formula:

wherein each of Ri-R₈ and R_a-R_d is defined as in any embodiment above and M is an antineoplastic agent, said method comprising the steps of:

[0109] (i) reacting the antineoplastic agent, M, with a compound having the formula:

Zf ⁺N(R₃R₄)=CR₅R₆; and a base, wherein Z₁ ^" is an anion selected from the group consisting of halide and sulfonate to obtain a compound having a structure of the formula M-CR₅R_O- N(R₃R₄) and

(ii) reacting the compound obtained in step (i) with a compound having the formula:

Z₂-CR₁R₂-HyP; or

wherein Z₂ is a leaving group, to make the compound having the structure of the formula Hyp-L-M. One of skill in the art will appreciate upon reading this disclosure in its entirety that other anions suitable as Zi include a variety of pharmaceutically acceptable anions.

[0110] In one embodiment, the antineoplastic agent is an aroylindazole compound and Zf ⁺N(R₃R₄)=CR₅R₆ is I^{" +}N(Me)₂=CH₂. In another embodiment, the reacting in step (ii) is performed using Z₂-CR) R₂-Hyp. In another embodiment, Z₂ is selected from the group consisting of chloro, bromo, and iodo. In another embodiment, -CRiR₂- is -CH₂-. In another embodiment, Z₂ is a substitutued or unsusbstituted 2-nitroimidazole moiety. In another embodiment Z₂-CR[R₂-Hyp has a structure of the formula:

wherein Z₂ is selected from the group consisting of bromo and chloro.

Method 2

[0111] In another aspect, the present invention provides a method of synthesizing a HAP compound of the present invention having structure of the formula Hyp-L-M comprising the steps of:

[0112] (i) reacting an antineoplastic agent, a secondary amine, and formaldehyde to yield a compound of step (i) as described above, and

[0113] (ii) reacting the compound obtained in step (i) and a compound having the formula:

Z₂-CR₁R₂-HyP; or

to make a compound having the formula Hyp-L-M. In one embodiment, the antineoplastic agent is an aroylindazole compound; and the secondary amine is selected from the group consisting of dimethylamine, diethylamine, piperidine, and pyrrolidine.

Method 3

[0114] In another aspect, the present invention provides a method of making a compound having a structure of the formula: Hyp-L-M; comprising the steps of reacting an antineoplastic agent derivative having a structure of the formula:

M-CR₅R₆-Z₂;

wherein Z₂ is a leaving group; with a compound having a structure of the formula selected from the group consisting of Hyp-CR]R₂-N(R₃R₄) and

to make a compound having the formula Hyp-L-M. In one embodiment, Z₂ is a halo group. In one embodiment, -CR₅R₆-Z₂ is -CH₂Cl or -CH₂Br. For example, 20-chloromethyl campthothecin and analogs, 1-chloromethyl aroylindazole antineoplastic agents, and various other chloromethylated antineoplastic agents can be employed according to the present methods in the compounds of the present invention. Various chloromethyl ethers of hydroxyl groups can be conveniently synthesized via a methylthiomethyl ether by reacting the methylthiomethyl ether with a halogen molecule. A suitable, nonlimiting, halogen molecule is bromine. The methylthiomethyl ethers can be synthesized following a reaction of an antineoplastic agent containing the hydroxyl group, dimethyl sulfoxide, and a carboxylic acid anhydride. Suitable carboxylic acid anhydrides include, but are not limited to, acetic anhydride and trifluoromethyl acetic anhydride.

Method 4

[0115] In another aspect, the present invention provides a method of synthesizing a compound having a structure of the formula:

wherein R₁₅ has a structure of the formula -CH(Ri_8a)Me wherein Ris_a is selected from hydrogen and C₂-C₅ alkyl, and R_]7 is selected from Ci-C₆ alkyl, Cj-C₆ heteroalkyl, C₃-C₈ cycloalkyl, heterocyclyl, aryl, and heteroaryl, said method comprising the steps of:

(i) reacting a solution of a compound having the structure of the formula:

in dimethyl formamide with bromine to yield a compound having a structure of the formula:

(ii) reacting the compound yielded in step (i) with a compound having a structure of the formula:

and Pd(DPPF)Cl₂ to yield the compound having structure of the formula:

(iii) reacting the compound yielded in step (ii) with potassium diazodicarboxylate and an acid to yield the compound having the structure of the formula:

[0116] Methods for synthesizing halomethyl and methyl thiomethyl ethers of various antineoplastic agents are described in the references Wong et al., Bioorg. Med. Chem. Lett., 1996, 6(15): 1837-42 and Zhao et al., J. Org. Chem., 2000, 65: 4601-4606 and can be adapted in accordance with the present methods by one of skill in the art upon reading this disclosure. Methods for synthesizing epothilone analogs are described for example in Waltman et al., Bioorg. Med. Chem. Lett., 2000, 10: 2765 -68; Nicolou et al., J. Am. Chem. Soc, 2001, 123: 9313-23; Biswas et al., J. Am. Chem. Soc, 2002, 124: 9825-32; Nicolou et al., Angew. Chem. Int. Ed., 2003, 42: 3515 -20; Waltman et al., Curr. Pharm. Design, 2005, 11: 1595- 613; and Watkins et al., Curr. Pharm. Design, 2005, 11: 1615-53; and can be adapted in accordance with the present methods for synthesizing HAPs of epothilone analogs by one of skill in the art upon reading this disclosure.

[0117] In certain embodiments of Methods 1-3, the bioreductive group, Hyp, has a structure of the formula selected from the group consisting of:

wherein Ri₅ and Ri₇ is defined as in any one of the embodiments above. In certain other embodiment, Hyp, has structure of the formula selected from the group consisting of:

wherein R_1S is defined as in any embodiment above. Suitable R₁₅ moieties useful in the present methods include, but are not limited to, those described in the Examples section below.

[0118] The synthesis of a HAP compound of the present invention containing an aroyl indazole antineoplastic agent is schematically shown below:

Bu₃SnR₁₅ or

(i) yl hydrogenation

[0119] The scheme involves the synthesis of Hyp-CH₂0H intermediates, converting the HyP-CH₂OH intermediates to Hyp-CH₂Br, and reacting the Hyp-CH₂Br and an aroylindazole antineoplastic agent following the Method 1 described above. Hyp-CH₂0H compounds such as Compounds i-vii can also be converted to the corresponding Hyp-CH₂Br or Hyp-CH₂C1 compounds by a variety of routine synthetic procedures known to one of skill in the art. For example and not as a limitation, the Hyp-CH₂OH compounds can be reacted with PPh₃ and Br₂ or CBr₄ in a suitable solvent to yield Hyp-CH₂Br compounds. Other bromomethylated and chloromethylated bioreductive groups are commercially available or were synthesized according to the procedures described in PCT Pat. Pub. Nos. WO 04/087075; WO 05/086951 ; and WO 07/002931 upon appropriate substitution of starting materials (each of which is incorporated herein by reference). Other examples of Hyp or bioreductive groups that are halomethylated and employed to synthesize various HAPs of the present invention include, but are not limited to:

[0120] The synthesis of various Hyρ-CH₂OH and other intermediates useful in the present methods are described in Example IA-F below. In certain embodiments, the present invention provides novel Hyp-CH₂OH intermediates.

[0121] The synthesis of a HAP compound of the present invention containing a camptothecin antineoplastic agent is schematically shown below:

Methylthiomethyl intermediate

A ¹¹HyP-CR₁R₂-NR₃R₄" moiety

A bromomethyl ether or "MCH₂Br" intermediate

[0122] The prodrugs of the present invention, having structure of the formula Q-L-M, that are activated by reactions other than reduction reactions, can be synthesized employing the methods useful for synthesizing HAPs of the present invention and suitably substituting the bioreductive group, Hyp, with a suitably substituted Q moiety.

[0123] Other prodrug compounds of the present invention including other HAPs can be synthesized following synthetic methods described in literature and/or upon reading this disclosure and upon suitable substitution of reactants. Syntheses of HAPs of the present invention employing the methods of the present invention are described in the EXAMPLES Section below.

III. THERAPIES

[0124] In other aspects, the present invention provides methods of treating cancer and other hyperproliferative diseases comprising administering a therapeutically effective amount of a prodrug compound of the present invention to a patient in need of such treatment. In one embodiment, the prodrug compound is a HAP of the present invention. In one embodiment, the HAP has a structure of the formula Hyp-L-N, wherein N is an antineoplastic agent having a structure of the formula (I), (IA), (II), (IIIA), (HIB) as shown above, and Hyp and L are defined as in any embodiments above.

[0125] In another embodiment, the therapeutically effective amount is administered as a pharmaceutically acceptable formulation comprising a HAP compound of the present invention and one or more pharmaceutically acceptable diluents or excipients.

[0126] In another embodiment, the therapeutically effective amount is administered in a daily dose. The therapeutically effective daily dose can be administered by employing suitable unit dose forms of the HAPs of the present invention. In another embodiment, the daily dose is administered from once every day, once every two weeks, up to, once every month. In another embodiment, the daily dose is administered parenterally or orally.

[0127] Examples of administering compounds of the formula (I) and (IA) in accordance with the present methods for treating lung and colon cancer is described, for example, in Examples 6A-D below. Treatment methods including administration of compounds of the formula (I) and (IA) in accordance with the present methods for treating diseases that result from, and/or cause, angiogenesis and/or neovasculature is described, for example, in Example 6E below.

[0128] A compound having a structure of the formula (II), 33, was administered to HT29 tumor bearing mice alone and in combination with 5-FU. The monotherapy and the combination therapy demonstrated tumor regression in comparison with vehicle administration. Dosing once daily was toxic under the conditions tested. Less frequent dosing, twice weekly, was better tolerated.

Treatment of Cancers [0129] Various cancers can be treated according to the methods of the present invention by administering the HAPs the present invention. In certain embodiment, the cancer treated is selected from the group consisting of cancer of the adrenal gland, bone, brain, breast, bronchi, colon and/or rectum, gallbladder, head and neck, kidneys, larynx, liver, lung, neural tissue, pancreas, prostate, parathyroid, skin, stomach, and thyroid. In another embodiment, the cancer treated is selected from the group consisting of acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and in situ carcinoma, Ewing's sarcoma, epidermoid carcinomas, giant cell tumor, glioblastoma multiforma, hairy-cell tumor, intestinal ganglioneuroma, hyperplastic corneal nerve tumor, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemias, lymphomas, malignant carcinoid, malignant melanomas, malignant hypercalcemia, marfanoid habitus tumor, medullary carcinoma, metastatic skin carcinoma, mucosal neuroma, myeloma, mycosis fungoides, neuroblastoma, osteo sarcoma, osteogenic and other sarcoma, ovarian tumor, pheochromocytoma, polycythemia vera, primary brain tumor, small-cell lung tumor, squamous cell carcinoma of both ulcerating and papillary type, hyperplasia, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumor, topical skin lesion, veticulum cell sarcoma, and Wilm's tumor.

[0130] In one embodiment, the HAP compound of the present invention is administered for the treatment of cancer in combination with other anticancer agents or other anticancer therapies. Suitable anticancer therapies useful in accordance with the present methods include radiation therapy and surgery. Methods for treating cancer employing other hypoxia activated prodrugs are described, for example, in PCT Pat. Appl. No. US06/025881; PCT Pat. Pub. Nos. WO 06/57946 and WO 04/87075; WO 07/002931; and WO 07/137196, incorporated herein by reference, and can be used for the treatment of cancer according to the present methods upon appropriate substitution of those other hypoxia activated produgs with the HAPs of the present invention.

Treatment of Hyperproliferative Diseases

[0131] In certain embodiments, the present invention provides methods of treating non- cancer hyperproliferative diseases characterized by cellular hyperproliferation (e.g., an abnormally increased rate or amount of cellular proliferation) in accordance with the present methods. In certain embodiments, the hyperproliferative disease is selected from the group consisting of allergic angiitis and granulomatosis (Churg-Strauss disease), asbestosis, asthma, atrophic gastritis, benign prostatic hyperplasia, bullous pemphigoid, coeliac disease, chronic bronchitis and chronic obstructive airway disease, chronic sinusitis, Crohn's disease, demyelinating neuropathies, dermatomyositis, eczema including atopic dermatitis, eustachean tube diseases, giant cell arteritis, graft rejection, hypersensitivity pneumonitis, hypersensitivity vasculitis (Henoch-Schonlein purpura), irritant dermatitis, inflammatory hemolytic anemia, inflammatory neutropenia, inflammatory bowel disease, Kawasaki's disease, multiple sclerosis, myocarditis, myositis, nasal polyps, nasolacrimal duct diseases, neoplastic vasculitis, pancreatitis, pemphigus vulgaris, primary glomerulonephritis, psoriasis, periodontal disease, polycystic kidney disease, polyarteritis nodosa, polyangitis overlap syndrome, primary sclerosing cholangitis, rheumatoid arthritis, serum sickness, surgical adhesions, stenosis or restenosis, scleritis, scleroderma, strictures of bile ducts, strictures (of duodenum, small bowel, and colon), silicosis and other forms of pneumoconiosis, type I diabetes, ulcerative colitis, ulcerative proctitis, vasculitis associated with connective tissue disorders, vasculitis associated with congenital deficiencies of the complement system, vasculitis of the central nervous system, and Wegener's granulomatosis.

[0132] In one embodiment, the hyperproliferative disease treated is psoriasis, a disease characterized by the cellular hyperproliferation of keratinocytes which builds up on the skin to form elevated, scaly lesions. In another embodiment, the hyperproliferative disease treated is multiple sclerosis, a disease characterized by progressive demyelination in the brain. In another embodiment, the hyperproliferative disease treated is rheumatoid arthritis, a multisystem chronic, relapsing, inflammatory disease that can lead to destruction and ankylosis of joints affected. In another embodiment, a HAP compound of the present invention is administered to prevent a hyperproliferative disease resulting from cellular proliferation on a prosthesis implanted in a patient by coating the prosthesis with a composition containing a HAP compound of the present invention. In another embodiment, the hyperproliferative disease treated is benign prostatic hyperplasia, a disease in which prostate epithelial cells grow abnormally and thereby block urine flow.

[0133] In another embodiment, the present invention provides methods of treating a variety of cancer and other hyperproliferative diseases by disrupting existing vasculature and/or neovasculature. Without being bound by mechanism, existing vasculature and/or neovasculature are useful for transporting nutrients and oxygen to the diseased tissue. Compounds of the present invention can disrupt and/or shut down such vasculature (see, for example Example 6E), and disrupt the flow of nutrients to the diseased tissue and inhibit the growth of, and/or kill, such tissue. By disrupting such vasculature, the compounds of the present invention also disrupt oxygen transport into such diseased tissue and make the tissue increasingly hypoxic and increasing susceptible to certain HAP compounds of the present invention. For example, and without limitation, inflammatory diseases and diseases that cause and/or result from angiogenesis are treatable by administering the compounds of the present invention in accordance with the present methods.

[0134] The invention, having been described in summary and in detail, is illustrated but not limited by the Examples below, which describe methods for synthesizing HAPs of the present invention, and demonstrate the efficacy and administration of HAPs of the present invention for treatment of cancer and other hyperproliferative diseases.

IV. EXAMPLES

[0135] Examples IA - F, below, describe the synthesis of the intermediate compounds, Hyp-CH₂OH and Hyp-CH₂Br, wherein Hyp has a structure of the formula:

R₁₅ is selected from the group consisting of bromo, Ci-C₆ alkyl, CpC₆ alkenyl, substituted or unsubstituted phenyl; that are useful in the synthesis of compounds of the present invention.

Example 1: Synthesis of Hyp-CH₂OH and Hyp-CH₂Br Intermediates

[0136] The following examples A-E describe the synthesis of Hyp-CH₂OH having the structure of the formula:

containing various R]₅ substituents.

Example IA

[0137] This example describes the synthesis of an intermediate wherein Ri ₅ is Br, starting from Compound i that can be synthesized as described in PCT Pat. App. Pub. No. WO 07/002931 and PCT Pat. App. No. US07/88645.

L R₁₅ = H ii. Ri₅ = Br

[0138] To a solution of Compound i (1 g) in DMF (15 mL) was added N- bromosuccinimide (NBS, 1.25 g) and the reaction mixture stirred at 60⁰C for 3h. Then the reaction mixture was diluted with brine, extracted with EtOAc, dried and concentrated to yield a residue that was separated by column chromatography using 0 - 80% EtOAc/Hexanes to yield Compound ii (1.3 g).

Example IB

[0139] This example describes the synthesis of an intermediate wherein R_1S is Ph as shown below, starting from Compound ii synthesized as described above.

11. R₁₅ = Br iii. R₁₅ = Ph

[0140] To a solution of Compound ii (1 g) in DMF (4 mL) purged with argon was added Pd(PPh₃)₄ (490 mg), the reaction mixture was purged with argon, PhSnBu₃ (4.7 g) was added, and the reaction mixture was again purged with argon. The reaction mixture was stirred at HO⁰C for 16 h, diluted with EtOAc, washed with 1 : 1 brine water and the aqueous portion reextracted with EtOAc. The combined EtOAc portions were dried, concentrated to yield a residue, and purified by column chromatography using 0 - 80% EtOAc/Hexanes to yield Compound iii (690 mg). Other Hyp-CH₂OH compounds having a structure of the formula:

wherein Ri ₅ is an aryl or a heteroaryl moiety can be synthesized according to the method described in Example IB. Hyp-CH₂OH compounds wherein R1₅ is selected from the group consisting of 4-fluorophenyl, 2-pyridyl, and 2-fluoro-5-methoxyphenyl were synthesized starting from compound ii and following this method. Example 1C

[0141] This example describes the synthesis of a compound wherein Ri₅ is vinyl.

[0142] A solution of l-methyl-4-bromo-2-nitroimidazolemethanol (compound (ii), 3 g) in dimethylformamide (DMF, 100 mL) was degassed by evacuation and purged three times with argon. Tetrakistriphenylphosphine palladium (1.47 g) was added to the reaction mixture and the ensuing solution purged with argon followed by the addition of vinyl tributyltin (11.1 mL). The solution was again purged with argon and the reaction mixture was heated to 110 ⁰C and stirred under argon for 16 h. The solution was cooled and diluted with water (100 mL), filtered through CELITE, and the CELITE pad washed twice with hexanes. The aqueous portion of the filtrate was diluted with saturated aqueous NaCl (100 mL) and extracted with EtOAc (250 mL) four times. The combined organic portions were washed twice with brine, dried over MgSO₄, and separated by column chromatography on silica gel using EtOAc/Hexane (0-100%) as eluent to yield compound (iv) (479 mg) as yellow crystals that was used in the next step. Other Hyp-CH₂OH compounds containing a terminal alkenyl R₁₅ moiety were synthesized following this method with the appropriately substituted starting material.

[0143] An intermediate wherein Ri ₅ is 1-propenyl (CH=CHMe) was synthesized using 1- propenyl tributyltin according to the method described in Example 1C. Other Hyp-CH₂OH compounds wherein Rj₅ is a terminal alkenyl moiety can be synthesized according to the method described according to the foregoing method.

Example ID

[0144] This example describes the synthesis of an intermediate wherein R₁₅ is ethyl.

(iv)

(V)

[0145] A mixture of compound iv (161 mg) and potassium diazodicarboxylate (10 equivalents) in methanol (MeOH, 36 mL) was stirred, and a solution of acetic acid (HOAc, 0.68 mL) in MeOH (1.8 mL) was added to it over 10 min. The resulting mixture was stirred overnight, filtered, concentrated, and the residue was separated by column chromatography on silica gel using EtOAc/Hexanes (0-100%) to yield compound v (162 mg) as a yellow syrup that was used in the next step.

[0146] Other compounds containing a Hyp moiety substituted with an alkenyl group can similarly be reduced to convert the alkenyl group to an alkyl group. 100 mg of a Hyp- CH₂OH compound having a structure of the formula:

wherein Ri ₅ is isopropenyl was reduced under similar conditions to yield 74 mg of the corresponding isopropyl substituted Hyp-CH₂OH compound.

Example IE

[0147] This example describes the synthesis of an intermediate wherein Ri ₅ is isopropenyl.

(ϋ) (vϋ)

[0148] A solution of 1 -methyl -4-bromo-2-nitroimidazolemethanol (compound (ii), 500 mg) in dimethyl formamide (DMF, 20 mL) was degassed by evacuation and purged three times with argon. [l,l '-bis(diphenylρhosphino) ferrocene] dichloro palladium(II) (Pd(DPPF)Cl₂, 155 mg) and K₃PO₄ (900 mg) were added to the reaction mixture and the ensuing solution purged with argon then stirred at room temperature for 10 minutes. Next, isopropenylboronic acid pinacol ester was added to the solution, the solution was purged with argon, and the reaction mixture was stirred at 60⁰C overnight. The reaction mixture was diluted with a wateπbrine mixture (1: 1) and extracted with EtOAc three times. The combined organic portions were dried, concentrated, and separated by column chromatography on silica gel using EtOAc/Hexane (0- 100%) as eluent to yield compound vii (350 mg) as a yellow solid.

[0149] Other Hyp-CH₂OH intermediates wherein R₁₅ is a terminal alkenyl moiety can be synthesized according to the foregoing method. Compound iv was also synthesized according to this method and employing:

Example IF

[0150] The following example F describes the synthesis of Hyp-CH₂Br having the structure of the formula:

containing various R₁₅ substituents.

[0151] A Hyp-CH₂OH compound was converted to the corresponding Hyp-CH₂Br compound by reacting with thionyl bromide. Compound v ( 162 mg) was added to thionyl bromide (SOBr₂, 1 mL) and stirred at room temperature (15 min). Then, the mixture was quenched with ice and extracted with ethyl acetate (EtOAc). Next, the organic layer was washed with water and brine, dried, concentrated, and the residue separated by column chromatography on silica gel using EtOAc/Hexanes (0-100%) to yield the compound vi (67 mg), as a yellow syrup.

Example 2 [0152] This Example describes in parts A-C below the synthesis of compounds of the present invention in accordance with the methods described in Section II above.

A: Synthesis of Compound 1

[0153] Example 2A describes the synthesis of Compound 1 according to Method 1 described in Section II above.

Aroylindazole A Compound 1

[0154] To a mixture of an antineoplastic agent, aroylindazole A, (400 mg) and Eschenmoser salt (214 mg) in dichloromethane (DCM, 20 mL) was added K₂CO₃ (218 mg) and the reaction mixture was stirred for 8 h. The reaction mixture was filtered and the filtrate evaporated to yield a residue that was co-evaporated with dry toluene (10 mL); MeCN (15 mL) and 5-bromomethyl-2-nitrofuran (239 mg) was added to the residue and the resultant mixture was heated at 45°C for 3 h. Volatiles were removed in vacuo to yield a residue that was triturated with diethyl ether and filtered to yield a solid residue. The solid residue was separated by column chromatography using 0-20% methanol (MeOH)/DCM to yield Compound 1 (316 mg).

[0155] Compounds of the present invention, including but not limited to Compounds 2-9, 13, 15, 23, 25, 27, and 30 were synthesized following the method described in Example 2 upon appropriate substitution of 5-bromomethyl-2-nitrofuran with other halomethylated bioreductive groups, such as, bromomethylated and chloromethylated 2-nitroimidazole groups. For example, the bromomethylated compound vi was reacted with antineoplastic compound A (100 mg) according to the method described in Example 2A to yield, Compound 23 (62 mg) after separation by column chromatography using MeOH/DCM (0- 10%).

10 11

[0156] Compound 10 and 11 were synthesized by reacting 5-bromomethyl-2-nitrofuran and either

B: Synthesis of Compound 12

12

[0157] This Example describes the synthesis of Compound 12 using a secondary amine and aqueous formaldehyde, instead of using an Eschenmoser salt like reagent according to Method 2 described in Section II above. To a mixture of the antineoplastic agent A (140 mg), K₂CO₃ (112 mg), and DCM (5 mL) was added sequentially diethylamine (12 μL) and aqueous formaldehyde (37%, 33 μL) and the reaction mixture stirred for 14 h. Analysis of the reaction mixture by ¹H-NMR demonstrated the formation of a diethylaminomethyl intermediate as described above, of the antineoplastic agent A. The reaction mixture was filtered, the residue washed with dry toluene (10 mL) and the filtrate evaporated in vacuo to yield a residue to which was added MeCN (4 mL) and 5-bromomethyl-2-nitrofuran (86 mg) and heated at 5O⁰C for 2h. The reaction mixture was cooled down to room temperature and the residue collected by filtration to yield compound 12.

C. Synthesis of Compound 36

[0158] This example describes the synthesis of compound 36 by reacting compound 23,that contains a bromide anion, with silver lactate. After the reaction, silver bromide precipitates out and compound 36 containing a lactate anion is isolated in the filtrate after filtration. To a solution of compound 23 (40 mg) in MeOH/MeCN (1: 1, 2 mL) was added a solution of silver lactate (11.5 mg) in water (1 mL) and stirred vigorously for 3 days. The reaction mixture was filtered through a celite pad and the filtrate concentrated to yield compound 36 (35 mg) that was characterized by ¹H-NMR. Compound 23 was transformed into its citrate salt by using silver citrate and following this method.

D. ¹H-NMR and Other Analytical Data for Certain Compounds of the Present Invention

[0159] The ¹H-NMR data for certain compounds of the present invention are provided below. The data corresponding to the compound of the present invention is provided after that compound's structure.

Compound 1

[0160] ¹H NMR (DMSO) δ: 8.32 (d, IH, J = 9.0 Hz), 7.77 (d, IH, J = 3.9 Hz) , 7.55 (s, 2H), 7.43 (d, IH, J = 9.2 Hz), 7.23 (d, IH, J = 3.7 Hz), 6.43 (s, 2H), 5.07 (s, 2H), 3.99 (s, 3H), 3.81 (s, 6H), 3.78 (s, 3H), 3.24 (s, 6H), 2.31 (s, 3H).

Compound 37

[0161] ¹H NMR (DMSO) 5: 8.32 (d, IH, J = 8.8 Hz), 7.61 (s, IH), 7.60 (s, 2H), 7.43 (d, IH, J = 9.2 Hz), 6.45 (s, 2H), 5.06 (s, 2H), 4.04 (s, 3H), 3.99 (s, 3H), 3.80 (s, 6H), 3.78 (s, 3H), 3.16 (s, 6H), 2.26 (s, 3H).

Compound 15

[0162] ¹H NMR (DMSO) δ: 8.27 (d, IH, J = 9.2 Hz), 7.64-7.61 (m, 2H), 7.55 (s, 2H), 7.51- 7.48 (m, 3 H), 7.38 (d, IH, 7 = 9.2 Hz), 6.39 (s, 2H), 5.29 (s, 2H), 4.12 (s, 3H), 3.95 (s, 3H), 3.79 (s, 9H), 2.86 (bs, 6H), 2.26 (s, 3H); HPLC: 8 min linear gradient at 0.2 mL/min; 0-100% MeOH 0.1% HCO₂H in H₂O 0.1% HCO₂H; Waters YMC™ Ci₈ S-3 (120A , 2.0 x 50mm): (t_R = 4.05).

Compound 19

[0163] ¹H NMR (DMSO) δ: 8.27 (d, IH, 7= 8.8 Hz), 7.71-7.66 (m, 2H), 7.55 (s, 2H), 7.40- 7.33 (m, 3H), 6.38 (s, 2H), 5.26 (s, 2H), 4.12 (s, 3H), 3.96 (s, 3H), 3.79 (s, 9H), 2.89 (bs, 6H), 2.25 (s, 3H); HPLC: (t_R = 4.05).

Compound 23,

[0164] ¹H NMR (DMSO) δ: 8.31 (d, IH, J = 9.2 Hz), 7.61 (s, 2H), 7.42 (d, IH, 7 = 9.2 Hz), 6.45 (s, 2H), 5.05 (s, 2H), 4.00 (s, 3H), 3.98 (s, 3H), 3.83 (s, 6H), 3.79 (s, 3H), 3.17 (bs, 6H), 2.77-2.70 (m, 2H), 2.19 (s, 3H), 1.22 (t, 3H, 7 = 7.4 Hz); HPLC: (t_R = 3.98).

Compound 25

[0165] (Primarily trans isomer) ¹H NMR (CDCl₃) δ: 8.31 (d, IH, H3), 7.63 (s, 2H, Hl), 7.14, (d, IH, H2), 7. 01 (bd, IH, H9), 6.75 (b, 2H, H14), 5.95 (b, IH, HlO), 5.20 (b, 2H, H13), 4.2-3.8 (4s, 15H, Me4, 5, 6, 8), 3.45 (b, 6H, Me7), 2.22(s, 3H, H12), 2.18(b, 3H, HI l).

Compound 36

[0166] ¹H NMR (CD₃OD) δ: 8.38 (d, IH, H3), 7.66 (s, 2H, Hl), 7.36, (d, IH, H2), 6.61 (s, 2H, H14), 5.05 (s, 2H, H13), 4.1-3.8 (4s, 15H, Me 4, 5, 6, 8), 3.4 (m, IH, HlO), 3.19 (s, 6H, Me7), 2.75 (q, 2H, H15), 2.25 (s, 3H, H12), 1.4-1.2 (m, 6H, Me9, 16).

Example 3: Hypoxia Selective Cytotoxicities of HAPs of the Present Invention

[0167] This example demonstrates the cytotoxicities of HAPs of the present invention employing an AlamarBlue fluorescence intensity based detection of cell survival. H460 (10,000 - 15,000 cells/well/500 μL, ATCC HTB-177) and HT 29 cells (20,000 - 30,000 cells/well/500 μL, ATCC HTB-38) were seeded in glass inserts on 24- well plates in RPMI1640 medium supplemented with 10% FBS and 1% Penicillin/Streptomycin (Invitrogen Corporation, Carlsbad, CA). The cells were incubated overnight at 37°C in 5% CO₂, 95% air and 100% relative humidity (these incubation conditions were used throughout the experiment unless otherwise mentioned) and divided into 2 groups: a "control group" (no test compound), and "treatment groups" (in which the cells were kept in contact with the test compound at various concentrations for 2 h). The control fluorescence intensity, or FO, proportional to the cell population of the control group at the beginning of the experiment, was determined following an AlamarBlue assay (λ_ex = 550 nm and λ_em = 590 nm). See also, Biosource International Inc., Tech Application Notes, Use of Alamar Blue in the measurement of Cell Viability and Toxicity, Determining IC_5O- The cells in the treatment groups were incubated for 2 hours with 6 different concentrations of a test compound, under hypoxia (5% CO₂, 5% H₂, 90% N₂) or normoxia (5% CO₂, 95% air), media containing the test compound removed, fresh media added, and the cells incubated for 3 days. The fluorescence intensities of the various treatment group cells incubated with different concentrations of the test compound and having different cell populations, and the control group cells at the end of the experiment (F_t) having the highest cell population among all the groups, was determined following an AlamarBlue assay. The fluorescence intensities determined were background corrected by subtracting FO, and normalized by dividing with F₁-F₀. The background corrected and normalized fluorescence intensities of the control group after 3 days of incubation, and the various treatment groups after 3 days of incubation, were plotted against the corresponding concentrations of the test compound. The IC₅₀ value for the test compound, i.e., the concentration of the test compound that killed, or made unviable, 50% of the cells, was calculated based on a best-fit plot using an F test (GraphPad Prism4 software, San Diego, CA).

[0168] The cytotoxicity of the HAPs of the present invention were also demonstrated in other cell lines following the methods described for H460 cells. The results, tabulated below, demonstrate that HAPs of the present invention are more cytotoxic under hypoxia than under normoxia. Certain compounds, under the conditions tested and in an H460 cell line, may not show enhanced cytotoxicity under hypoxia than under normoxia, but can be more cytotoxic under hypoxia than under normoxia when different test conditions and/or cell lines are used. Such different test conditions and cell lines useful for these purposes are known to one of skill in the art and described elsewhere in this disclosure and, for example, in PCT Pat. App. Pub. No. WO 07/002931.

Table 1

Example 4: Cytotoxicity of HAP Compounds in Drug Resistance Cell Lines

[0169] The cytotoxicity of HAP compounds of the present invention, Compounds 1, 3, 5, 13, and 15, were demonstrated in a parental, sensitive cell lines, H69, MESSA, and HEK293/pcDNA and the corresponding resistant cell lines, H69AR that over expresses multi-drug resistance-associated protein (MRP), MDRi that over expresses MDR-I efflux pump, and HEK293/pcDNA/BCRP that over expresses the BCRP efflux pump. HAPs of the present invention were equally cytotoxic to the parental, sensitive cell lines and the resistant cell lines, demonstrating that these compounds were not susceptible to the various resistance mechanisms that protect cancer cells and were useful in cancer therapy.

Table 2

* Mitoxantrone, instead of daunorubicin was used in this cell line as the control compound

Example 5: Pharmacokinetic Properties of HAP Compounds

[0170] The pharmacological stability of HAP compounds of the present invention was demonstrated by incubating these compounds with mouse plasma and in mouse liver microsome (MLM). The pharmacokinetics of the compounds of the present invention were demonstrated by injecting these compounds in mice intraperitoneally (i.p.) and intravenously (Lv.). The results are tabulated below.

Table 3

Example 6: Demonstration of In Vivo Efficacy of HAPs

[0171] This Example describes in parts A-F below the formulation, administration, and efficacy of compounds of the present invention in the treatment of cancer and other hyperproliferative diseases in accordance with the present invention.

Example 6A

[0172] This example describes a formulation of Compound 1 and the in vivo administration of that formulation for treating cancer. Compound 1 was formulated in the vehicle employed in this experiment, 5% DMSO, 5% Tween/80-D5W. The formulation of compound 1 was administered parenterally, by intraperitoneal (i.p.) administration, alone and in combination with CPT-11, to colorectal, HT 29, xenograft tumor bearing nude mice. Compound 1 was administered, for 2 weeks, at doses of 7.5mg/Kg,daily for 8 days, followed by 10 and 15 mg/kg, daily for 2 days each. CPT-11 was administered i.p., once a week, for 2 weeks, at a dose of 50 mg/kg, and was given 24 hours earlier if combination therapy is scheduled. The growth inhibition of the xenograft mice tumor upon treatment with Compound 1, alone and in combination, demonstrated the usefulness of Compound 1, a compound of the present invention, in the treatment of cancer.

Example 6B

[0173] This example describes a formulation of Compound 3 and the in vivo administration of that formulation for treating cancer. Compound 3 was formulated in the vehicle employed in this experiment, 5%DMSO5%Tw80 in D5W, and administered to H460 xenograft tumor bearing mice at 10 mg/kg, i.p., Q2D x 6, alone and in combination with Gemcitabine at 60 mg/kg, i.p., Q3Dx4. Administration of Compound 3 alone reduced tumor growth in tumor bearing mice compared to administration of the vehicle.

Example 6C

[0174] This example demonstrated the efficacy of Compound 15, a HAP of the present invention, to treat xenografted HT29 colon carcinoma in nude mice, by administering Compound 15 as a monotherapy, i.e. as a single agent, and in combination with irinotecan (CPT-11). Compound 15 was formulated (1.5 mg/mL and 1.0 mg/mL) in 5% DMSO/5%Tween80 in WFI (vehicle). CPT-11 was formulated in 2%Tween80-saline. The drug formulations were diluted and filtered though a 0.2 μm filter and administered to animals within about an hour's preparation. [0175] HT29 cells (5 x 10⁶) were implanted subcutaneously on the right flank of the mice. Tumor lumps were measurable on the seventh day post-implantation and were about 150 mm³ on the fifteenth day. Mice with similar tumor size were randomized for treatment and treated as follows: Compound 15 was administered intraperitoneally (i.p.), every other day for total 6 doses (q2Dx6), at dosages of 15 and 10 mg/kg, alone and in combination with

CPT-11 (i.p., 50 mg/kg, q7Dx3); totally 4 groups of 10 mice each were thus treated. A group of 10 tumor bearing mice were used as no-treatment control.

[0176] The tumor growth delays (TGDs), measured by extra days for the tumor to reach 500 mm³ in treatment group as compared to the vehicle group, were between 8 and 9 days for mice undergoing monotherapy with Compound 15 at the two regimens administered (Figure 1). For the combination therapy groups, the TGDs were 14 days for the "10 mg/kg" group and 19 days for the "15 mg/kg group." Tumor growth was about 56% and 59% inhibited by administering Compound 15 alone at 15 mg/kg and 10 mg/kg respectively, and about 86% and 74% inhibited by administering Compound 15 at 15 mg/kg and 10 mg/kg respectively in combination with CPT-11. The body weight changes of Compound 15 treated groups are presented in (Figure 2). The treatment groups lost body weight loss but the lost body weights were recovered when the treatment was stopped demonstrating the safety of the administration of Compound 15.

Example 6D

[0177] This example demonstrated the efficacy of 23 and 36, compounds of the present invention, to treat xenografted HT29 colon carcinoma in nude mice, by administering them as monotherapy, i.e. as a single agents, and in combination with irinotecan (CPT-11). Compound 36 is a HAP having the same structure as that of 23 and having lactate as a counter anion. Compounds 23 and 36 were administered to HT29 xenograft tumor bearing mouse, alone and in combination with CPT-11, in substantial accordance with the method described in Example 7B as modified below. The HAPs were formulated in 5% DMSO/ 2%Tween80-D5W, and CPT-11, in 2%Tween80-saline. The HAPs were administered at 15 mg/kg 6 times over a period of 2 weeks at a frequency of q2d, 3 times a week, in an administration cycle wherein every sixth and seventh days in a cycle were drug-free holidays. In another mode of administration for Compound 23, the 3rd-6th administrations employed 20 mg/kg, instead of 15 mg/kg of the drug. CPT-11 was administered once every week for 3 weeks. Various administration parameters were as tabulated below. The effect of drug administration on tumor growth was demonstrated by comparison with vehicle administration.

[0178] The results are described in Figures 3 and 4. The results demonstrated that, 23 and 36, HAPs of the present invention, were effective in treating cancer alone and in combination with another anticancer agent. During the initial period of treatment with 23 or 36, certain animals lost about 5% of their weight. The animals gained back, completely or substantially, the lost weight during the remaining period of treatment.

[0179] Mouse doses can be converted to human equivalent doses by dividing the mouse dose by -12. Using this conversion factor, and according to Example 6C, Compound 15 can be administered to humans for the treatment of cancer at a therapeutically effective amount of about (15/12 = ) 1.3 mg/kg and about (10/12 = ) 0.7 mg/kg alone and in combination with another anticancer agent and/or anticancer therapy. According to Example 6D, 23 and 36 can be administered to humans for the treatment of cancer at a therapeutically effective amount of about (20/12 = ) 1.7 mg/kg and about (15/12 = ) 1.3 mg/kg alone and in combination with another anticancer agent and/or anticancer therapy.

[0180] Thus, in certain embodiments of the present invention, a compound of the present invention having a structure of the formula (IA) can be administered in a therapeutically effective amount in a range of about 0.05 mg/kg - about 40 mg/kg for treating cancer in a human patient. In another embodiment, the cancer treated is a solid tumor. In another embodiment, the cancer treated is selected from lung cancer and colon cancer.

Example 6E

[0181] This example demonstrates in vivo treatment of cancer and tumor vessel disruption upon administration of compound 36. Vascular disrupting agents (VDA) can shut down the neo- and existing vasculature in tumors, extend the hypoxic zone and lead to increased necrosis of tumor tissue. In one embodiment, as used herein, disrupting vasculature, refers to shrinking the diameter of vasculature and/or impeding the flow of blood through the vasculature. A disruption of the function of tumor vasculature leads to lowered delivery of oxygen and nutrients to cancer and other hyperproliferative diseases. Since hyperproliferative diseases including cancer are often associated with rapid cell division and large consumption of nutrients and oxygen, disrupting vasculature associated with cancer and other hyperpriliferative diseases impedes cell division and/or kills cancer or other hyperpriliferative disease cells. Compound 36 is a HAP of the antineoplastic agent, Aroylindazole A (see, example 2A), which is a VDA and microtubule inhibitor. Once the antineoplastic VDA is generated under hypoxia, the vascular disrupting action of the VDA initially generated can enhance tumor hypoxia and release more of the anti tumor antineoplastic agent and more tumor cells are killed or their growth diminished.

[0182] HT29 cells (3x10⁶) were subcutaneously implanted into the mice. In the efficacy studies, mice were treated with Vehicle, CPT-11 alone (50mg/Kg, Q7D x3, ip), compound 36 alone (Mono, 15 mg/Kg, Q2Dx3/wk x 2wks), and combination of the two compounds (Combo). Daily dosing of compound 36 for 5 days/wk (QDx5/week), total 2 wks, ip was also tested. In vessel perfusion studies, time course and dose response experiments were performed. Combretastatin A-4 phosphate (CA4P), a VDA, served as a positive control.

[0183] Every other day treatment with compound 36 inhibited and delayed the tumor growth. 23 days after the treatment, tumor growth inhibitions (TGIs) were 51.3%, 42.7% and 81.4% in Mono, CPT-11 alone, and Combo groups respectively (Figure 3 and Figure 4). There was no body weight loss in any group during the study period demonstrating that it was safe to administer compound 36 at such doses. Daily dosing of 36 resulted in TGIs of 13.5% and 54.9% in Mono and Combo groups respectively. Only 1 animal death (out of 10 animals) was observed in the daily (QDx5/week) dosing, demonstrating that it was safe to administer compound 36 at such doses.

[0184] Tumor vessel perfusion status was demonstrated by Hoechst 33342 staining. Untreated tumor samples were well perfused throughout the tumor area in both time course and dose response studies. 1 hour after treatment with compound 36, vessels were shut down to much greater extent compared to the partial of vessel closure observed after 24 hours. In dose response study, 5mg/Kg and 1.5mg/Kg of compound 36 induced partial shut down or collapse of the vessels.

[0185] These results demonstrate that compound 36 alone, and in combination with the anti cancer agent CPT- 11 , is effective in treating cancer.

Example 6F

[0186] This example demonstrates compound 36' s efficacy to kill hypoxic tumor cells. The HT29 cells are prepared in 50% matrigel in PREVI medium. 3 x 10⁶ cells are implanted in the subcutaneous space of the right flank in 100 mice (0.2ml/mouse). Mice bearing similar tumor sizes, preferably 300-500 mm³, are selected. Compound 36 is formulated appropriately as disclosed in Example 6 herein and a single dose is administrated intraperitoneally at 1.5 -15mg/Kg.

[0187] Animals are placed in carbogen (95% O2), air or 10% O2 chamber for 1.5 h before the dosing and returned to the chamber for 1 hour immediately after dosing. After total 2.5 h breathing, animals receive intravenous injection of Heochest33342 at 10 mg/kg. Tumors are removed 1 min after Heochest33342 injection and quickly frozen in OCT. 8 μm sections are made and Heochest33342 stained cells, showing vessel status including vessel disruption, are viewed under fluorescent microscopy. CA-4P is used as a control in a a similar way.

***

[0188] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes can be made and equivalents can be substituted without departing from the scope of the invention. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, process, process step or steps, to achieve the benefits provided by the present invention without departing from the scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

[0189] AU publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an indication that any such document is pertinent prior art, nor does it constitute any admission as to the contents or date of the same.

Claims

WHAT IS CLAIMED IS:

1. A compound having a structure of the formula Hyp-L-M wherein

M is an antineoplastic agent; the Hyp moiety is selected from the group consisting of:

Ri₅ is selected from the group consisting of hydrogen, C]-C₆alkyl, Cj-C₆ heteroalkyl, C₃-Cg cycloalkyl, heterocyclyl, C2-C₆alkenyl, C₂-C₆alkynyl, aryl, heteroaryl, and halo; Ri₇ is selected from the group consisting of Ci-C₆ alkyl, Ci-C₆heteroalkyl, C₃- C₈cycloalkyl, heterocyclyl, aryl and heteroaryl; L has a structure of the formula -L₂-Li-; Li has a structure of the formula: CRiR₂-N(R₃R₄)-CR₅R₆; L₂ is selected from the group consisting of a bond and a moiety having a structure of the formula:

-VW- denotes the point of attachment to the rest of the molecule; each of Ri-R₈ is independently selected from the group consisting of Ci-C₆ alkyl, Ci-C₆ heteroalkyl, C₃-C₈ cycloalkyl, heterocyclyl, aryl, and heteroaryl, or together Ri and R₂, R₅ and R₆, or R₇ and R₈ independently form a C₃-C₈ cycloalkyl or a heterocyclyl group; or together R₃ and R₄ independently form a heterocyclyl group; and each of R_a-R_d is independently selected from the group consisting of hydrogen, halo, nitro, CO₂H, Q-Qalkyl, and C)-C₆ alkoxy; and pharmaceutically acceptable salts thereof.

2. The compound of claim 1 wherein the antineoplastic agent M is selected from the group consisting of aroyl indazole tubulin binding compounds, taxanes and analogs, camptothecin and analogs, combretastatin and analogs, epothilone and analogs, and etoposide and analogs.

3. The compound of claim 2 wherein the aroyl indazole tubulin binding compound has a structure of the formula:

Formula (I) wherein R₉ is selected from the group consisting of hydrogen, halo, hydroxy, nitro, cyano, amino, CrC₆alkylamino, diC]-C₆ alkylamino, Ci-C₆alkoxy, Q-Qalkyl, Q-Qheteroalkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₈cycloalkyl, heterocyclyl, aryl, and heteroaryl.

4. The compound of claim 2 wherein the camptothecin analog has a structure of the formula:

Formula (II) wherein each of Ri_2a-_d is independently selected from the group consisting of hydrogen, fluoro, hydroxy, nitro, amino, Ci-C_δalkoxy, Ci-C_δalkylamino and di(Ci-C₆)alkylamino; or together Ri_2b and Ri_2c form a methylenedioxy moiety, and R]₃ is selected from the group consisting of methyl, ethyl and oximyl having the formula:

alkyl; each of which is optionally substituted with at least one group selected from the group consisting of amino, Ci-C_δalkylamino, di(Ci-C₆) alkylamino, and heterocyclyl.

5. The compound of claim 2 wherein the taxane analog has a structure of the formula selected from:

Formula (UIA) and Formula (IIIB).

6. The compound of claim 2 wherein the combretastatin analog has a structure of the formula:

7. The compound of claim 3 wherein R₉ has a structure of the formula: w∞ — _{R i 0} wherein R_1O is selected from the group consisting of hydrogen, Ci-C₆ alkyl, Cj-C₆ heteroalkyl, C₃-C₈ cycloalkyl, heterocyclyl, aryl, and heteroaryl.

8. The compound of claim 3 wherein Hyp has a structure of the formula selected from the group consisting of:

9. The compound of claim 7 wherein Rio is methyl.

10. The compound of claim 9 wherein Li has a structure of the formula CH₂-N(R₃R4)-CH₂ and L₂ is a bond.

11. The compound of claim 10 wherein Hyp has a structure of the formula selected from the group consisting of:

wherein Ri₅ is selected from the group consisting of hydrogen, ethyl, phenyl and bromo.

12. The compound of claim 10 wherein each of R₃ and R₄ is methyl and Hyp has a structure of the formula:

wherein R₎₅ is selected from a group consisting of hydrogen, CpC₆ alkyl, C₂-C₆ alkenyl, C₂- C₆ alkynyl, aryl, heteroaryl, and halo.

13. The compound of claim 12 wherein Ri ₅ has a structure of the formula ~C(Ri8a)=C(Risb)R₁₈c wherein each of R₁Sa-C is independently selected from the group consisting of hydrogen and Ci-C₆ alkyl.

14. The compound of claim 12 wherein Ri₅ is selected from the group consisting of ethyl, isopropyl, -CH=CH₂, -CH=CHMe, -CH(Me)=CH₂, hydrogen, phenyl, and bromo.

15. The pharmaceutically acceptable salt of a compound of claim 14.

16. The salt of claim 15 wherein the anion of said salt is selected from the group consisting of a carboxylate, halide, phosphate, nitrate, sulfonate and sulfate.

17. The compound of claim 16 having a structure of the formula selected from the group consisting of:

18. A pharmaceutically acceptable formulation comprising a compound of any one of claims 1-17 and a pharmaceutically acceptable carrier, excipient, or diluent.

19. A method of treating cancer comprising administering a therapeutically effective amount of a compound of one of claims 1-17 to a patient in need of such treatment.

20. The method of claim 19 further comprising administering at least another anti cancer agent or anticancer therapy.

21. A method of disrupting vasculature associated with cancer or other hyperproliferative diseases by contacting said vasculature with a compound of any one of claims 1-17.

22. The method of claim 21 wherein the compound has the structure of the formula selected from the group consisting of:

23. A method of making a compound having a structure of the formula:

wherein R1₅ has a structure of the formula -CH(Rj _8a)Me;

R i_8a is selected from the group consisting of hydrogen and Ci-C₅ alkyl, said method comprising the steps of:

(i) reacting a compound having the structure of the formula:

in dimethyl formamide with bromine to obtain a compound having a structure of the formula:

(ii) reacting the compound obtained in step (i) with a compound having a structure of the formula:

and Pd(DPPF)Cl₂ to obtain a compound having structure of the formula:

(iii) reacting the compound obtained in step (ii) with potassium diazodicarboxylate and an acid to synthesize the compound having the structure of the formula:

24. A method of making a compound having a structure of the formula Hyp-L-M; wherein M is an antineoplastic agent, Hyp is selected from the group consisting of:

Ri₅ is selected from the group consisting of hydrogen, Ci-C₆ alkyl, Ci-C₆ heteroalkyl, C₃-C₈ cycloalkyl, heterocyclyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl aryl, heteroaryl, and halo; Ri₇ is selected from the group consisting of C₁-C₆ alkyl, Ci-C₆ heteroalkyl, C₃- C₈ cycloalkyl, heterocyclyl, aryl, and heteroaryl; L has a structure of the formula: -CR₁R₂-N(R₃R^-CR₅R₆- each R₁, R₂, R₅, and R₆ is independently selected from the group consisting of hydrogen, Ci-C₆ alkyl, C₁-C₆ heteroalkyl, C₃-C₈ cycloalkyl, heterocyclyl, aryl, and heteroaryl, or together Ri, R₂, R₅, and R₆ form a C₃-C₈ cycloalkyl or a heterocyclyl group, and each R₃ and R₄ is independently selected from the group consisting of C₁-C₆ heteroalkyl, C₃-C₈ cycloalkyl, heterocyclyl, aryl, and heteroaryl, or together R₃ and R₃ form a heterocyclyl group; and said method comprising the steps of: (i) reacting the antineoplastic agent, M, with a compound having the formula: Zf ⁺N(R₃R₄O=CR₅R₆; and a base, wherein Z₁ ^" is an anion selected from the group consisting of halide and sulfonate to obtain a compound having a structure of the formula M-CR₅R₆- N(R₃R₄) and (ii) reacting the compound obtained in step (i) with a compound having the formula: Z₂-CR₁R₂-HyP; wherein Z₂ is a leaving group to make the compound having the structure of the formula Hyp-L-M.

25. The method of claim 24 wherein M has a structure of the formula:

Formula (I) wherein Zf ⁺N(R₃R₄)=CR₅R₆ is I^{" +}N(Me)₂=CH₂; and Z₂-CRiR₂-Hyp has a structure of the formula:

wherein Z₂ is selected from the group consisting of bromo and chloro.