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WO2007137196A2 - Tubulin binding anti cancer compounds and prodrugs thereof - Google Patents

Tubulin binding anti cancer compounds and prodrugs thereof Download PDF

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Publication number
WO2007137196A2
WO2007137196A2 PCT/US2007/069297 US2007069297W WO2007137196A2 WO 2007137196 A2 WO2007137196 A2 WO 2007137196A2 US 2007069297 W US2007069297 W US 2007069297W WO 2007137196 A2 WO2007137196 A2 WO 2007137196A2
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WO
WIPO (PCT)
Prior art keywords
compound
cancer
alkylamino
crc
group
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PCT/US2007/069297
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French (fr)
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WO2007137196A3 (en
Inventor
Mark Matteucci
Jian-Xin Duan
Xiaohong Cai
Jiayao Li
Jason Lewis
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Threshold Pharmaceuticals, Inc.
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Publication of WO2007137196A2 publication Critical patent/WO2007137196A2/en
Publication of WO2007137196A3 publication Critical patent/WO2007137196A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole

Definitions

  • the present invention provides compositions and methods for treating cancer and other hyperproliferative disease conditions and generally relates to the fields of chemistry, biology, molecular biology, pharmacology, and medicine.
  • Tubulin and its polymerized form microtubules are useful for diverse cellular functions, including chromosome segregation during cell division, intracellular transport, development and maintenance of cell shape, cell motility, and possibly distribution of molecules on cell membranes (Bacher er a/., Pure Appl. Chem., 73(9): 1459-1464, 2001 , incorporated herin by reference).
  • Tubulin binding can inhibit many important biological functions that depend on tubulin and microtubules. Inhibition of tubulin polymerization or prevention of the disassembly of microtubules causes cell cycle arrest, disrupts formation of vasculature, and leads to cell death.
  • tubulin is considered to be a target in cancer therapy and drugs that interfere with tubulin function can be useful anti-cancer agents.
  • Certain anti-cancer agents targeting tubulin including combretastatins, taxanes (paclitaxel, docetaxel), vinca alkaloids (vincristine, vinblastine, vinorelbine), and epothilones suffer from one or more of the disadvantages as described below (Angerer et a/., Curr. Opin. Drug Discov. Dei/., 2000, 3(5): 575-584, incorporated herein by reference). Cancer cells can remove these anti-cancer agents by over-expressing transmembrane pumps and/or by other mechanisms and cause drug resistance. For example, the colon cancer cell HT-29 is resistant to combretastatin-A4. The aqueous insolubility of some of these drugs makes drug administration difficult. The complex chemical structures of these drugs make their synthesis and/or isolation from natural resources difficult. And, administration of certain anti-cancer agents can lead to toxic side effects in patients.
  • anti-cancer compounds preferably tubulin binding anti-cancer compounds and their prodrugs that are less problematic due to drug resistance and/or have less toxic side effects in patients and/or are easier to administer due to enhanced aqueous solubility compared to those known.
  • the present invention meets these needs.
  • the present invention provides tubulin binding anti- cancer compounds and their prodrugs wherein the tubulin binding anti-cancer compounds are 3-aroylindazoles.
  • the 3-aroylindazoles are substituted with an 1-alkynyl, 1-alkenyl, or an alkyl group at the 7-position of the indazole.
  • the 3-aroylindazoles are substituted with a substituted or unsubstituted amino group at the 7-position of the indazole.
  • the tubulin binding compounds inhibit the polymerization of tubuin to microtubule.
  • the tubulin binding compounds bind to the colchicine binding site of tubulin.
  • the tubulin binding compounds are vascular disrupting agents.
  • the prodrugs of the tubulin binding anti-cancer compounds have enhanced aqueous solubility compared to the corresponding tubulin binding anti-cancer compounds.
  • the prodrugs comprise an aqueous solubility enhancing moiety and the tubulin-binding anti-cancer compound.
  • the aqueous solubility enhancing moiety is covalently bonded to the 1-alkynyl, 1- alkenyl, or the alkyl groups substituting the 7-position of the corresponding tubulin binding compounds.
  • solubility enhancing moiety is covalently bonded to the 1 -position of the indazole of the corresponding tubulin binding compounds, directly or via a methylene linker.
  • aqueous solubility enhancing moieties are useful in the prodrugs of the present invention, including but not limited to, amino acid esters, sugar moieties, phosphates, phosphonates, ammonium groups, and heterocyclic amines.
  • the present invention provides hypoxia activated prodrugs of the tubulin binding anti-cancer compounds and their aqueous- solubility enhanced prodrugs. Therefore, a prodrug compound of the present invention can possess an enhanced aqueous solubility and also an enhanced hypoxia selective cytotoxicity compared to the corresponding tubulin binding anti-cancer compound.
  • the hypoxia activated prodrugs comprise tubulin binding anti-cancer compounds and a hypoxic activator (or Hyp).
  • the Hyp moiety is covalently bonded to the 1 -position of the indazole.
  • the Hyp moiety is covalently bonded to the substituted or unsubstituted amino group at the 7- position of the indazole.
  • the Hyp moiety is covalently bonded via alkylene or heteroalkylene linkers to the 1-alkynyl, 1-alkenyl, or the alkyl groups substituting the 7-position of the indazole.
  • the present invention provides a compound having a formula selected from:
  • Q 7 is hydrogen; amino; C 1 -C 6 alkylamino; di CrCe alkylamino; hydroxyl; CrC 6 alkoxy; nitro; cyano; CrC 6 alkyl; CrC 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; C 3 -Ce cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi 8 ; SO 2 Ri 5 ; PO 3 Ri 5 or a monosaccharide; with the proviso that in formula (II) Q 7 excludes hydrogen;
  • Qs is hydrogen; halo; amino; CrCe alkylamino; di CrC 6 alkylamino; hydroxyl; CrC 6 alkoxy; nitro; cyano; CrC 6 alkyl; CrC 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; C 3 -C 8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi 5 ; SO 2 Ri 5 or PO 3 R 15 ; each Q 9 independently is hydrogen; halo; amino; Ci-C 6 alkylamino; di
  • X is O, -NNHRi 6 , NR 16 , or NORi 6 ;
  • Y is hydrogen, hydroxyl, or halogen;
  • Z is -CH- or -N-;
  • R 15 is hydrogen, CrC 6 alkoxy, amino, CrC 6 alkylamino, di CrC 6 alkylamino, NHOH, NHNH 2 , C r C 6 alkyl, C r C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 - C 6 alkynyl, C 3 -Ca cycloalkyl, heterocyclyl, aryl, or heteroaryl;
  • R 16 is hydrogen, C 1 -C 6 alkyl, aryl, C 1 -C 6 alkylsulphonyl, arylsulfonyl, d-
  • the compounds are tubulin binding compounds.
  • the present invention provides a compound of formula (XXI)-(XXVII):
  • each Qi , Q 2 , and Q 6 independently is hydrogen; halo; amino; Ci-C 6 alkylamino; di Ci-C 6 alkylamino; hydroxyl; C 1 -C 6 alkoxy; nitro; cyano; C 1 -C 6 alkyl; C r C 6 heteroalkyl; C 1 -C 6 alkenyl; C 1 -C 6 alkynyl; C 3 -C 8 cycloalkyl; heterocyclyl; aryl; heteroaryl; COR 18 ; SO 2 R 18 ; or PO 3 R 15 ; each Q 3 -Q 5 C 1 -C 6 alkyl; d-C 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; C 3 -C 8 cycloalkyl; and heterocyclyl;;
  • Q 7 is hydrogen; halo; amino; CrC 6 alkylamino; di C 1 -C 6 alkylamino; hydroxyl; Ci-C 6 alkoxy; nitro; cyano; C 1 -C 6 alkyl; d-C 6 heteroalkyl; C 1 -C 6 alkenyl; C 1 -C 6 alkynyl; C 3 -C 8 cycloalkyl; heterocyclyl; aryl; heteroaryl; COR-is; SO 2 R 18 ; or PO 3 R 18 or a monosaccharide; with the proviso that in formula (II) Q 7 excludes hydrogen;
  • Q 8 is CrC 6 alkyl; CrC 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; C 3 -C 8 cycloalkyl; and heterocyclyl; each Q 9 independently is CrC 6 alkyl; d-C 6 heteroalkyl; C 2 -C 6 alkenyl;
  • V is -NHR 16 ; -NHNHRi 6 ; -NHN(Ri 6 ) 2 ; -NRi 6 NHRi 6 ; or -ORi 7;
  • Y is hydrogen, hydroxyl or halogen;
  • Z is -CH- or -N-;
  • R 15 is hydrogen, d-C 6 alkoxy, amino, Ci-C 6 alkylamino, di Ci-C 6 alkylamino, NHOH, NHNH 2 , C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 1 -C 6 alkenyl, C r C 6 alkynyl, C 1 -C 6 cycloalkyl, CrC 6 heterocyclyl, aryl, or heteroaryl;
  • Rie is hydrogen, C 1 -C 6 alkyl, aryl, Ci-C 6 alkylsulphonyl, arylsulfonyl, Ci- C 6 alkoxycarbonyl, aminocarbonyl, C 1 -C 6 alkylaminocarbonyl, di C 1 -C 6 alkylaminocarbonyl, C 1 -C 6 acyl, aroyl, aminothiocarbonyl, C 1 -C 6 alkylaminothiocarbonyl, di Ci-C 6 alkylaminothiocarbonyl, C 1 -C 6 thioacyl, or thioaroyl; and R' is CrC 6 alkyl or aryl; with the proviso that when V is NRi 6 , Ri 6 excludes hydrogen;
  • Ri 7 is CrC 6 alkyl; aryl; or di C r C 6 alkylamino; Rie is hydrogen, hydroxyl, C r C 6 alkoxy, amino, CrC 6 alkylamino, di
  • Ci-C 6 alkylamino NHOH, NHNH 2 , Ci-C 6 alkyl, Ci-C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
  • these compounds are tubulin binding compounds.
  • the present invention provides prodrug compounds wherein the novel compound of the invention is bonded to a hypoxic activator (Hyp) through a hydroxyl oxygen (-OHyp) or an amine nitrogen (-NHyp) in the tubulin binding compound.
  • Hyp hypoxic activator
  • -OHyp hydroxyl oxygen
  • -NHyp amine nitrogen
  • the present invention provides prodrug compounds of known tubulin binding anti-cancer compounds wherein the tubulin binding compound is bonded to the hypoxic activator (Hyp) through an hydroxyl oxygen (-OHyp) or an amine nitrogen (-NHyp) in the tubulin binding compound.
  • Hyp hypoxic activator
  • -OHyp hydroxyl oxygen
  • -NHyp amine nitrogen
  • the hypoxic activator can be nitrobenzene moieties, nitrobenzoic acid amide moieties, nitroazole moieties, nitroimidazole moieties, nitrothiophene moieties, nitrothiazole moieties, nitrooxazole moieties, nitrofuran moieties, and nitropyrrole moieties.
  • Hyp is selected from:
  • each X 2 is N or CR 32 ;
  • X 3 is NR 3I 1 S, or O;
  • each R 30 is independently hydrogen or alkyl;
  • R 31 is hydrogen, hydroxyl, Ci-C 6 alkyl or heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, CrC 6 alkoxy, CrC 6 alkylamino, CrC 6 dialkylamino, aryl or heteroaryl, Ci-C ⁇ acyl or heteroacyl, aroyl, or heteroaroyl;
  • Hyp is selected from wherein X 2 , R 30 , R 31 , R3 2 and n are as defined above.
  • the hypoxic activator is a substituted or unsubstituted nitroimidazole moiety.
  • Hyp is
  • the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a novel compound or a novel prodrug compound of the invention.
  • the present invention provides a method of treating cancer comprising administering a therapeutically effective amount of a novel compound or a novel prodrug compound of the invention alone or in combination with one or more other anti-cancer agents to a subject in need of such treatment.
  • Figure 1 illustrates the effect of Compound 30 alone and in combination with TaxolTM on tumor volume in xenograft mouse.
  • Figure 2 illustrates the effect of Compound 30 alone and in combination with TaxolTM on the bodyweight of tumor bearing xenograft mouse.
  • Figure 3 illustrates the effect of Compound 30 on tubulin binding of colchicine.
  • Figure 4 illustrates the effect of Compound 37 on tubulin binding of colchicine.
  • Figure 5 illustrates the in vitro vascular disruption caused by Compound 30.
  • Figure 6 illustrates the effect of Compound 30 on vascular permeability
  • Section I provides definitions of terms used herein; Section Il provides the compounds of the present invention including their synthesis, formulation, and functional characterization; Section III provides useful therapies employing the compounds of the present invention; Section IV provides illustrative examples of synthesizing the compounds of the present invention and demonstrating their efficacy in treatment of cancer. 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.
  • CrC 6 alkyl or (CrC 6 ) alkyl refers to substituted or unsusbstituted straight or branched chain alkyl groups having 1-6 carbon atoms such as, for example, methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl and 3-methyl pentyl.
  • a CrC 6 alkyl substituent may be covalently bonded to an atom within a molecule of interest via any chemically suitable portion of the CrC 6 alkyl group.
  • "CrC 6 alkyl" or (CrC 6 ) alkyl may be further substituted with substituents, including for example, hydroxy, amino, mono or di(Cr Ce)a[kyl amino, halogen, C 2 -C 6 alkyl ether, cyano, nitro, ethenyl, ethynyl, C 1 - C 6 alkoxy, C 1 -C 6 alkylthio, -COOH, -CONH 2 , mono- or d J-(C 1 -C 6 )alkyl- carboxamido, -SO 2 NH 2 , -OSO 2 -(d-C 6 )alkyl, mono or di(CrC 6 )alkylsulfon- amido, aryl, and heteroary.
  • Substituted CrC 6 alkyl groups include, for example, -CH 2 -CH 2 -OH, -CH 2 -CH 2 -halogen, -CH 2 -CH 2 -NH 2 , -CH 2 -CH 2 -O- CH 2 -CH 2 -OH, -CH 2 -CH2-CH2-NH-CH 2 -CH 2 -OH and -CH 2 -CH 2 -NH-CH 2 -CH 2 - OH and the like.
  • amino acid refers to naturally occurring ⁇ -amino acids and their stereoisomers, as well as unnatural amino acids such as amino acid analogs, amino acid mimetics, synthetic amino acids, ⁇ -amino acids, p-amino acids, and N-substituted glycines in either the L- or D- configuration that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Naturally occurring amino acids and analogs referred to herein may be described by shorthand designations as follows in Table A.
  • amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • amino acid side chain is bonded to the ⁇ -carbon atom of the carboxyl group such that there are two carbon atoms between the amino and carboxyl groups.
  • amino acid side chain is bonded to the K-carbon atom of the carboxyl group such that there are three carbon atoms between the amino and carboxyl groups.
  • the side-chains or R-groups of ⁇ - amino acids, y amino acids, and N-substituted glycines can be, in either stereo configuration, the same as the side chain groups found on naturally occurring and later modified ⁇ -amino acids, as well as side chains found on amino acid analogs and amino acid mimetics.
  • the term "N-substituted glycine” refers to a glycine amino acid where an amino acid side chain is attached to the glycine nitrogen atom.
  • amino acid side chains or “R groups” include, but are not limited to, side chains present in naturally occurring amino acids and side chains present in unnatural amino acids such as amino acid analogs, amino acid mimetics, synthetic amino acids, / 0-amino acids, and ⁇ -amino acids.
  • N-substituted glycines suitable for use in the present invention include, without limitation, N-(2-aminoethyl)glycine, N- (3-aminopropyl)glycine, N-(2-methoxyethyl)glycine, N-benzylglycine, (S)-N-(I- phenylethyi)glycine, N-cyclohexylmethylglycine, N-(2-phenylethyl)glycine, N- (3-phenylpropyl)glycine, N-(6-aminogalactosyl)glycine, N-(2-(3'- indolylethyl)glycine, N-(2-(p-methoxyphenylethyl))glycine, N-(2-(p- chlorophenylethyl)glycine, and N-[2-(p-hydroxyphenylethyl)]glycine.
  • N- substituted glycines can have an L- or D-configuration.
  • N-substituted glycine oligomers referred to herein as "peptoids” have been shown to be protease resistant (Miller et a/., Drug Dev. Res., 35:20-32 (1995), incorporated herein by reference).
  • a peptoid linker containing at least one ⁇ -amino acid having an L-configu ration is within the scope of the present invention.
  • cycloalkyl refers to a monovalent cyclic hydrocarbon radical of three to seven ring carbons.
  • the cycloalkyl group may have double bonds which may but not necessarily be referred to as "cycloalkene” or "cycloalkenyl".
  • the cycloalkyl ring may be optionally substituted independently with one, two, or three substituents selected from alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkylalkyl, halo, nitro, cyano, hydroxy, alkoxy, amino, mono-alkylamino, di-alkylamino, haloalkyl, haloalkoxy, -COR (where R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl), -(CR'R") n -COOR (n is an integer from 0 to 5, R' and R" are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloal
  • cycloalkyl includes, for example, cyclopropyl, cyclohexyl, cyclohexenyl, phenylcyclohexyl, A- carboxycyclohexyl, 2-carboxamido-cyclohexenyl, 2-dimethylaminocarbonyl- cyclohexyl, and the like.
  • heteroalkyl means an alkyl radical as defined herein with one, two or three substituents independently selected from cyano, -OR W , -NR x R y , and -S(O) P R Z (where p 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.
  • R y is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, aryl, araalkyl, alkoxycarbonyl, aryloxycarbonyl, carboxamido, mono- or di- alkylcarbamoyl or alkylsulfonyl.
  • R z is hydrogen (provided that p is 0), alkyl, cycloalkyl, cycloalkyl-alkyl, aryl, araalkyl, amino, mono-alkylamino, di- alkylamino, or hydroxyalkyl.
  • Representative examples include, for example, 2-hydroxyethyl, 2,3-dihydroxy-propyl, 2-methoxyethyl, benzyloxymethyl, 2- cyanoethyl, and 2-methylsulfonyl-ethyl.
  • R w , R x , R y , and R z can be further substituted by amino, fluorine, alkylamino, dialkylamino, OH or alkoxy.
  • the prefix indicating the number of carbon atoms refers to the total number of carbon atoms in the portion of the heteroalkyl group exclusive of the cyano, -OR W , -NR x R y , or -S(O) p R z portions.
  • heteroalkyl by itself or in combination with another term, also refers to a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • the heteroatom(s) O, N and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule.
  • heteroalkylene by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH 2 -CH 2 - S-CH 2 -CH 2 - and -CH 2 -S-CH 2 -CH 2 -NH-CH 2 -.
  • heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O) 2 R'- represents both - C(O) 2 R'- and -R 1 C(O) 2 -. As used herein, "heterocycle”, “heterocyclyl”, “heterocycloalkyl” or
  • cycloheteroalkyl means a saturated or unsaturated non-aromatic cyclic radical of 3 to 8 ring atoms in which one to four ring atoms are heteroatoms selected from O, NR (where R is independently hydrogen or alkyl) or S(0) p (where p is an integer from O to 2), the remaining ring atoms being C, where one or two C atoms may optionally be replaced by a carbonyl group.
  • the heterocyclyl ring may be optionally substituted independently with one, two, or three substituents selected from alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl, halo, nitro, cyano, hydroxy, alkoxy, amino, mono-alkylamino, di-alkylamino, haloalkyl, haloalkoxy, -COR (where R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl), -(CR'R") n - COOR (n is an integer from O to 5, R' and R" are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl), or -(CR'R") n -C0NR x
  • heterocyclyl includes, but is not limited to, pyridyl, tetrahydropyranyl, N-methylpiperidin-3-yl, N-methylpyrrolidin-3-yl, 2- pyrrolidon-1-yl, furyl, quinolyl, thienyl, benzothienyl, pyrrolidinyl, piperidinyl, morpholinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, 1 ,1-dioxo- hexahydro-1 ⁇ 6 -thiopyran-4-yl, tetrahydroimidazo [4,5-c] pyridinyl, imidazolinyl, piperazinyl, and piperidin-2-onyl.and the derivatives thereof.
  • the prefix indicating the number of carbon atoms refers to the total number of carbon atoms in the portion of the cycloheteroalkyl or heterocyclyl group exclusive of the number of heteroatoms.
  • R x and R y together is heterocyclyl. More specifically the term aryl includes, but is not limited to, phenyl, biphenyl, 1-naphthyl, and 2-naphthyl, and the substituted forms thereof.
  • CrC ⁇ alkoxy means a substituted or unsubstituted alkyl group of 1 to 6 carbon atoms covalently bonded to an oxygen atom.
  • a C-I-C ⁇ alkoxy group has the general structure -O-(Ci-C 6 alkyl) wherein alkyl is as described above.
  • Ci-C 6 alkoxy 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.
  • Ci-C 6 alkoxycarbonyl refers to an alkoxy group covalently bonded to a carbonyl.
  • CrC 6 alkylamino means a substituted or unsubstituted alkyl group of 1 to 6 carbon atoms covalently bonded to -NH-.
  • a C 1 -C 6 alkylamino group has the general structure -NH-(Ci-C 6 )alkyl wherein alkyl is as described above.
  • CrC 6 alkylamino groups include, for example, methylamino, ethylamino, propylamino and butylamino.
  • C 2 -C 6 alkyl ether refers to an ether substituent with 2 to 6 carbon atoms, positioned such that at least one carbon atom is located on either side of the oxygen atom.
  • Ci-C 6 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-C 6 alkyl and aryl groups.
  • aryl refers to substituted or unsusbstituted moieties that include 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.
  • halogen refers to fluorine, chlorine, bromine, and/or iodine.
  • heteroaryl refers to substituted or unsusbstituted monocyclic aromatic groups having 5 or 6 ring atoms, or fused ring bicyclic aromatic groups having 8 to 20 atoms, in which the ring atoms are C, O, S, SO, SO 2 , or N and at least one of the ring atoms is a heteroatom, i.e., O, S, SO, SO 2 , 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, is
  • the arrangement of the hetero atoms within the ring may be any arrangement allowed by the bonding characteristics of the constituent ring atoms.
  • Aryl or heteroaryl groups may be further substituted with substituents, including for example, hydroxy, amino, mono or di(CrC 6 )alkyl amino, halogen, C 2 -C 6 alkyl ether, cyano, nitro, ethenyl, ethynyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, -COOH, -CONH 2 , mono- or di- (C r C 6 )alkyl-carboxamido, -SO 2 NH 2 , -OSO 2 -(Ci-C 6 )alkyl, mono or di(d- C 6 )alkylsulfon-amido, aryl, and heteroaryl.
  • C 1 -C 6 heteroalkylene refers to a C 1 -C 6 alkylene as defined above wherein 1 - 3 carbon atoms in the hydrocarbon radical or a branched saturated divalent hydrocarbon radical is replaced with a heteroatom.
  • C 1 -C 6 heteroalkylene groups include, for example, -CH 2 CH 2 -O- CH 2 CH 2 - and -CH 2 CH 2 -S-CH 2 CH 2 -.
  • hydroxy(C 1 -C 6 )alkyl refers to a substituted or unsubstituted aliphatic group having from 1 to 6 carbon atoms, and further comprising at least one hydroxyl group on the main carbon chain and/or on a side chain.
  • Hydroxy(C- ⁇ -C 6 )alkyl groups include, for example, -CH 2 -CH 2 -OH and -CH 2 -CH 2 -CH 2 -OH.
  • hypoxic activator or “hypoxia activated trigger” refers to a group or moiety that is capable of releasing another compound, such as an antineoplastic agent or analogs thereof upon hypoxic reduction.
  • the hypoxic activator is a group that is capable of releasing the antineoplastic agent or analogs thereof upon reduction of the hypoxic activator under hypoxic conditions but does not release any antineoplastic agent or analog under normoxic conditions.
  • one hypoxic activator is a nitroimidazole that may be substituted with a variety of groups.
  • hypoxic activators include, but are not limited to, groups based on nitrobenzenes, nitrobenzoic acid amides, nitroazoles, nitroimidazoles, nitrothiophenes, nitrothiazoles, nitrooxazoles, nitrofurans, and nitropyrroles, where each of these classes of moieties may be substituted or unsubstituted, such that the redox potential for the group lies within a range where the group can undergo reduction in the hypoxic regions of a tumor.
  • hypoxic activators are described in Matteucci ef a/., PCT Publication Nos. WO 04/087075 and WO 07/002931 , each of which is incorporated herein by reference.
  • nitrothiophene, nitrofuranfuran, and nitrothiazole groups may be substituted with one or more electron donating groups, including but not limited to methyl, methoxy, or amine groups, to provide a hypoxic activator with the desired redox potential.
  • the nitropyrrole moiety can be substituted with an electron withdrawing group, including but not limited to cyano, carboxamide, -CF 3 , and sulfonamide groups, to achieve a group with the desired redox potential.
  • prodrug refers to a compound that, after administration, is metabolized or otherwise converted to an active or more active form 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).
  • substituted refers to a molecular moiety that is covalently bonded to an atom within a molecule of interest.
  • substitution refers to replacing a hydrogen atom in a molecular structure with a substituent such that the valence on the designated atom (for example 4 for carbon) is not exceeded, and a chemically stable compound (a compound that can be isolated, characterized, and/or tested for biological activity) results.
  • a combination of substituents or variables is permissible only if such a combination results in a stable or chemically feasible compound.
  • a stable compound or chemically feasible compound is one in which the chemical structure is not substantially altered when kept at a temperature of 4°C or less, in the absence of moisture or other chemically reactive conditions, for at least a week.
  • isomers refer to compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”. Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers”. When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible.
  • An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S- sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (-)-isomers respectively).
  • a chiral compound can exist as either individual enantiomer or as a mixture thereof.
  • a mixture containing equal proportions of the enantiomers is called a "racemic mixture".
  • the compounds of this invention may exist in stereoisomeric form if they possess one or more asymmetric centers or a double bond with asymmetric substitution and, therefore, can be produced as individual stereoisomers or as mixtures.
  • salts include:
  • (1 ) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethane- disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, A- chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsul
  • a metal ion e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion
  • coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, trimethylamine, N- methylglucamin ⁇ , and the like.
  • protecting group refers to a grouping of atoms that when attached to a reactive group in a molecule masks, reduces or prevents that reactivity. Examples of protecting groups can be found in T.W. Greene and P.G. Wuts, PROTECTIVE GROUPS IN ORGANIC CHEMISTRY, (Wiley, 2nd ed. 1991 ) and Harrison and Harrison et al., COMPENDIUM OF SYNTHETIC ORGANIC METHODS, VOIS. 1-8 (John Wiley and Sons. 1971-1996).
  • Representative amino protecting groups include formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ) 1 tert-butoxycarbonyl (Boc), trimethyl silyl (TMS), 2- trimethylsilyl-ethanesulfonyl (SES), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC), nitro- veratryloxycarbonyl (NVOC) and the like.
  • hydroxy protecting groups include those where the hydroxy group is either acylated or alkylated such as benzyl and trityl ethers as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers.
  • aqueous solubility enhancing group refers to a moiety that can be covalently bonded to a compound to increase the compound's aqueous solubility.
  • aqueous solubility enhancing groups include, for example, an amino acid or its ester, a phosphate moiety, a heterocyclic amine, a sugar moiety, and an amino substituted benzoate moiety.
  • sugar refers to a monosaccharide, a disaccharide, or a polysaccharide.
  • sugars include, glucose, glucuronic acid, mannose, and other aldoheptoses, ketohexoses, aldopentoses, and their dimmers or polymers.
  • patient typically refers to a human but more generally refers to a mammal including but not limited to a human.
  • compositions and methods of the invention can be used to treat cancer or other hyperproliferative diseases in any mammal, including non-human primates, and experimental models of human cancers.
  • treating refers to taking steps to obtain beneficial or desired therapeutic results, including clinical results.
  • beneficial or desired therapeutic results include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer, diminishment of extent of disease, delay or slowing of disease progression, palliation or stabilization of the disease state, and other beneficial results, as described below.
  • reduction of a symptom or symptoms (and grammatical equivalents of this phrase) refers to decreasing of the severity or frequency of the symptom(s) or eliminating the symptom(s).
  • administering or "administration of a drug refers to a subject (and grammatical equivalents of this phrase) refer to direct administration, including self-administration and/or indirect administration, including the act of prescribing a drug.
  • 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.
  • an effective amount or a “therapeutically effective amount” of a drug refers to an amount of a drug that, when administered to a subject with cancer or any other hyperproliferative disease condition, will have (i) the intended therapeutic effect, e.g., alleviation, amelioration, palliation or elimination of one or more manifestations of cancer or other disease in the subject; or (ii) a prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of disease or symptoms or reducing the likelihood of the onset (or reoccurrence) of disease or symptoms.
  • the full therapeutic or prophylactic effect does not necessarily occur by administration of one dose and can occur only after administration of a series of doses.
  • a therapeutically or prophylactically effective amount can be administered in one or more administrations.
  • prophylactically effective amount of a drug refers to an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of disease or symptoms, or reducing the likelihood of the onset (or reoccurrence) of disease or symptoms.
  • the full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses.
  • a prophylactically effective amount may be administered in one or more administrations.
  • pharmaceutically acceptable carrier or excipient refers to a carrier or excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes a carrier or excipient that is acceptable for veterinary use as well as human pharmaceutical use. Examples include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like, used in the preparation of a pharmaceutical composition. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the pharmaceutical compositions or pharmaceutical formulations of the invention is contemplated. Supplementary active ingredients can be incorporated into the compositions of the invention.
  • a "pharmaceutically acceptable carrier or excipient" as used in the specification and claims includes both one and more than one such carrier or excipient.
  • a naturally occurring compound, colchicine, binds to tubulin and interferes with the function of the mitotic spindles, causing depolymerization and disappearance of tubulin polymers known as microtubules.
  • colchicine-like tubulin binder such as Combretastatin A can selectively target the vascular system of tumors.
  • the morphological changes induced in the endothelial cells of the tumor's blood vessels irreversibly shut down the blood flow to cancer cells while leaving the blood supply to healthy cells intact.
  • Heterocyclic indole, benzofuran, and benzothiophene containing tubulin binding di- and tri-aryl compounds constitute a sub-class of these compounds (see Nam et al. supra). These compounds, for example, have an aromatic moiety such as an aryl or an aroyl (-CO-Aryl) moiety or a CH group in the 2 position (as illustrated in structures below).
  • Ri is CO or CHfe
  • R 3 is H, methyl, aryl or aroyl
  • R 2 and R 4 are methyl or OMe. None of these compounds have yet been approved for treatment of cancer.
  • the compounds of the invention can be described in part as compounds which can bind to tubulin and/or arrest mitosis, and/or disrupt vaculature.
  • the compounds of the present invention are prodrugs of known and novel compounds that can bind to tubulin and/or arrest mitosis, and/or disrupt vaculature; such prodrugs comprise a hypoxic activator and/or an aqueous solubility enhancer moiety.
  • the compounds are anti-cancer compounds which can bind to tubulin, and prodrugs thereof comprising a hypoxic activator.
  • the present invention provides compounds of formulas (I)-(VIII):
  • each Qi, Q 2 , and Q 6 independently is hydrogen; halo; amino; Ci-Cs alkylamino; di Ci-Ce alkylamino; hydroxyl; CrC 6 alkoxy; nitro; cyano; C 1 -C 6 alkyl; C 1 -Ce heteroaikyl; C2-C6 alkenyl; C 2 -C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi 5 ; SO 2 R 15 ; or PO 3 Ri 5 ; each Q 3 -Q 5 is hydrogen; halo; amino; C 1 -C 6 alkylamino; di CrC 6 alkylamino; hydroxyl; d-C 6 alkoxy; nitro; cyano; C 1 -C 6 alkyl; Ci-C 6 heteroaikyl; C 2 -C 6 alkenyl
  • Q 7 is hydrogen; amino; Ci-C 6 alkylamino; di C 1 -C 6 alkylamino; hydroxyl; C 1 -C 6 alkoxy; nitro; cyano; C 1 -C 6 alkyl; C 1 -C 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; COR 15 ; SO 2 R-Ie; PO 3 R 18 or a monosaccharide; with the proviso that in formula (II) Q 7 excludes hydrogen;
  • Qs is hydrogen; halo; amino; CrC 6 alkylamino; di C 1 -C 6 alkylamino; hydroxyl; C 1 -C 6 alkoxy; nitro; cyano; Ci-C 6 alkyl; C 1 -C 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; C 3 -C 8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi 8 ; SO 2 RiS or PO 3 Ri 8 ; each Qg independently is hydrogen; halo; amino; Ci-C 6 alkylamino; di CrC 6 alkylamino; hydroxyl; C 1 -C 6 alkoxy; nitro; cyano; CrC 6 alkyl; CrC 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; C 3 -C 8 cycloalkyl; heterocyclyl; aryl; hetero
  • X is O, -NNHR 16 , or NR 16 , or NOR 16 ;
  • Y is hydrogen, hydroxyl, or halogen
  • Z is -CH- or -N-;
  • R 15 is hydrogen, CrC 6 alkoxy, amino, CrC 6 alkylamino, di CrC 6 alkylamino, NHOH, NHNH 2 , C r C 6 alkyl, CrC 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 - C 6 alkynyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroaryl;
  • Ri 6 is hydrogen, CrC 6 alkyl, aryl, CrC 6 alkylsulphonyl, arylsulfonyl, d- C 6 alkoxycarbonyl, aminocarbonyl, CrC 6 alkylaminocarbonyl, di C 1 -C 6 alkylaminocarbonyl, CrC 6 acyl, aroyl, aminothiocarbonyl, C 1 -C 6 alkylaminothiocarbonyl, di C 1 -C 6 alkylaminothiocarbonyl, C 1 -C 6 thioacyl, or thioaroyl; with the proviso that when X is NRi 6 , Ri 6 excludes hydrogen;
  • Ris is hydrogen, hydroxyl, CrC 6 alkoxy, amino, C 1 -C 6 alkylamino, di CrC 6 alkylamino, NHOH, NHNH 2 , C 1 -C 6 alkyl, CrC 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
  • the present invention provides compounds of formulas (I)-(VIII), wherein Q 1 is hydrogen; halo; amino, CO 2 H, cyano; nitro;
  • ⁇ — R 13 ; ⁇ Ri 3 ; C 1 -C 6 alkoxy; halo; amino; or hydroxy; each Q 3 , Q 4 and Q 5 independently is hydrogen,C- ⁇ -C 6 alkoxy, halo, amino, hydroxyl, Q 3 and Q 4 together is methylenedioxy, or Q 4 and Q5 together is methylenedioxy, provided that in any compound only one of the Q 3 , Q 4 and Q 5 is hydrogen;
  • Q 7 is C 1 -C 6 alkyl optionally substituted independently with one or more aryl, heteroaryl, hydroxyl, amino, C 1 -C 6 alkylamino, di Ci-C 6 alkylamino, CO 2 H, or CONH 2 ; CORi 8 ; SO 2 R 18 ; or PO 3 R 18 ; or a monosaccharide; each Q 8 and Q 9 is hydrogen;
  • Ri 3 is hydrogen; C 1 -C 5 alkyl, CrC 6 heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroalkyl each optionally substituted with hydroxyl, C 1 - C 6 alkoxy, amino, C 1 -C 6 alkylamino, di Ci-C 6 alkylamino, NHCORi 5 , or COR 18 ; and
  • R 18 is hydrogen, hydroxyl, C 1 -C 6 alkoxy, amino, CrC 6 alkylamino, di C 1 -C 6 alkylamino, NHOH, NHNH 2 , C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
  • Q 1 is ⁇ 0 — Rs wherein Rs is a monosaccharide or a disaccharide.
  • the present invention provides compounds of formula (I) - (VIII) wherein each Q 3 and Q 5 is 1 ,1 '-difluoroethyl and Q 4 is fluoro.
  • the present invention provides compounds of formulas (VIII)-(XIII) wherein X is O.
  • the present invention provides a compound of formula:
  • n 1 or 2.
  • the present invention provides compounds of formulas (IX)-(XIII):
  • the present invention provides compounds of formula (IX-i)-(XIII-i)
  • R 13 is H, Me, CH 2 OH, CH(Me)OH, CH 2 CH 2 OH, CH 2 NH 2 , CH 2 PO 3 H 2 , PO 3 H 2 , CO 2 H, or CONH 2 and R 14 is H, Me, or B(OH) 2 ; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
  • the present invention provides a compound of formula (XIV):
  • Qi is : -Ri3 or -Ri 3 ;
  • Q 2 is C 1 -C 6 alkoxy; halo; amino; or hydroxy;
  • each Q 3 -Q 5 is C r C 6 alkyl; d-C 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; C 3 -C 8 cycloalkyl;
  • R 13 is hydrogen; CrC 6 alkyl, CrC 6 heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroalkyl each optionally substituted with hydroxyl, Cr C 6 alkoxy, amino, C 1 -C 6 alkylamino, di C r C 6 alkylamino; CORi 8 or NHCOR- I5 ;
  • R 15 is hydrogen, hydroxyl, CrC 6 alkoxy, amino, CrC 6 alkylamino, di CrC 6 alkylamino, NHOH, NHNH 2 , C 1 -C 6 alkyl, CrC 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
  • the present invention provides a compound of formula (XV):
  • each Q 3 , Q 4 , and Q 5 independently is C 1 -C 6 alkyl; C r C 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; and C 3 -C 8 cycloalkyl;
  • Q 7 is hydrogen; amino; CrC 6 alkylamino; di C 1 -C 6 alkylamino; hydroxyl; C 1 -C 6 alkoxy; nitro; cyano; C 1 -C 6 alkyl; C 1 -C 6 heteroalkyl; C 1 -C 6 alkenyl; C 1 -C 6 alkynyl; C 3 -C 8 cycloalkyl; heterocyclyl; aryl; heteroaryl; COR 15 ; SO 2 R 18 ; or PO 3 R 18 or a monosaccharide;
  • R 1 is CH 2 or CO
  • R 3 is hydrogen, halo, C 1 -C 6 alkyl, aryl or heteroaryl; Ri 3 is hydrogen; Ci-C 6 alkyl, Ci-C 6 heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroalkyl each optionally substituted with hydroxyl, Cr C 6 alkoxy, amino, CrC 6 alkylamino, di CrC 6 alkylamino; NHCORi 5 or CORi 8 ;
  • Ri 5 is hydrogen, hydroxyl, d-C 6 alkoxy, amino, Ci-C 6 aikylamino, di C r C 6 alkylamino, NHOH, NHNH 2 , Ci-C 6 alkyl, d-C 6 heteroalkyl, C r C 6 alk ⁇ nyl, CrC 6 alkynyl, C 3 -C 6 cycloalkyl, C 3 -C 6 heterocyclyl, aryl, or heteroaryl;
  • Ri 8 is hydrogen, hydroxyl, Ci-C 6 alkoxy, amino, CrC 6 alkylamino, di CrC 6 alkylamino, NHOH, NHNH 2 , CrC 6 alkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
  • the present invention provides a compound selected from formulas (XVI)-(XX):
  • each each Q 3 -Q 5 is CrC 6 alkyl; CrC 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; C 3 -C 8 cycloalkyl; or heterocyclyl;
  • Q 6 is hydrogen; halo; amino; CrC 6 alkylannino; di C 1 -C 6 alkylamino; hydroxyl; CrC 6 alkoxy; nitro; cyano; C 1 -C 6 alkyl; CrC 6 heteroalkyl; C r C 6 alkenyl; C r C 6 alkynyl; C 3 -C 8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi 8 ; SO 2 Ri 8 or PO 3 Ris;
  • Q 7 is hydrogen; amino; C r C 6 alkylamino; di C r C 6 alkylamino; hydroxyl;
  • R 5 is hydrogen, halo, or C 1 -C 6 alkoxy;
  • R 6 is formyl or a protected form thereof;
  • Ri 3 is hydrogen; C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroalkyl each optionally substituted with hydroxyl, C r C 6 alkoxy, amino, CrC 6 alkylamino, di CrC 6 alkylamino, NHCOR-is or COR 18 ;
  • Ri 5 is hydrogen, hydroxyl, CrC 6 alkoxy, amino, CrC 6 alkylamino, di CrC 6 alkylamino, NHOH, NHNH 2 , C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroaryl;
  • Ris is hydrogen, CrC 6 alkoxy, amino, CrC 6 alkylamino, di CrC 6 alkylamino, NHOH, NHNH 2 , C 1 -C 6 alkyl, C r C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 - C 6 alkynyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
  • the present invention provides a compound of formulas (XXI)-(XXVII):
  • each Q 1 , Q 2 , and Q 6 independently is hydrogen; halo; amino; C 1 -C 6 alkylamino; di C 1 -C 6 alkylamino; hydroxyl; CrC 6 alkoxy; nitro; cyano; C 1 -C 6 alkyl; C 1 -C 6 heteroalkyl; Ci-C 6 alkenyl; CrC 6 alkynyl; C 3 -C 8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi 8 ; SO 2 Ri 8 or PO 3 Ri 8 ; each Q3-Q5 is C 1 -C 6 alkyl; C 1 -C 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; C 3 -C 8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi 8 ; SO 2 R 18 ; or PO 3 R 13 ; Q 3 and Q
  • Q 7 is hydrogen; halo; amino; Ci-C 6 alkylamino; di C 1 -C 6 alkylamino; hydroxyl; CrC 6 alkoxy; nitro; cyano; Ci-C 6 alkyl; Ci-C 6 heteroalkyl; C 1 -C 6 alkenyl; CrC 6 alkynyl; C 3 -C 8 cycloalkyl; heterocyclyl; aryl; heteroaryl; COR 15 ; SO 2 Ri 8 ; or PO 3 Ri 8 or a monosaccharide; with the proviso that in formula (II) Q 7 excludes hydrogen;
  • Qs is hydrogen; halo; amino; C 1 -C 6 alkylamino; di C 1 -C 6 alkylamino; hydroxyl; CrC 6 alkoxy; nitro; cyano; aryl; heteroaryl; CORi 5 ; SO 2 Ri 5 or PO 3 Ri 5 ; each Q 9 independently is hydrogen; halo; amino; C 1 -C 6 alkylamino; di Ci-C 6 alkylamino; hydroxyl; Ci-C 6 alkoxy; nitro; cyano; aryl; heteroaryl; COR 15 ;
  • V is -NHR 16 ; -NHNHR 16 ; -NHN(R 16 ) 2 ; -NR 16 NHR 16 ; or -OR 17; Y is hydrogen, hydroxyl or halogen;
  • Z is -CH- or -N-;
  • R- 15 is hydrogen, CrC 6 alkoxy, amino, C 1 -C 6 alkylamino, di CrC 6 alkylamino, NHOH, NHNH 2 , C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 1 -C 6 alkenyl, C 1 - C 6 alkynyl, CrC 6 cycloalkyl, CrC 6 heterocyclyl, aryl, or heteroaryl; R- 15 is hydrogen, C 1 -C 6 alkyl, aryl, C 1 -C 6 alkylsulphonyl, arylsulfonyl, d-
  • R 17 is CrC 6 alkyl; aryl; or di CrC 6 alkylamino;
  • R 18 is hydrogen, CrC 6 alkoxy, amino, C 1 -C 6 alkylamino, di CrC 6 alkylamino, NHOH, NHNH 2 , C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 - C 6 alkynyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
  • the present invention provides the compounds:
  • the present invention provides the compounds:
  • each X and Y independently is CH or N.
  • the present invention provides the compounds:
  • U 3 is Cl or F;
  • Q 1 is OH, NH 2 , CO 2 H, CN, halo, C 1 -C 6 alkyl,
  • the present invention provides the compounds: wherein Q 4 is H or CH 2 U 3 ; each Ui is N or CU 4 ; U 2 is C 1 -C 6 alkyl or CrC 6 heteroalkyl; U 3 is F, CN, OMe, and OH; and U 4 is nitro, nitrile, halo, CF 3 , or carboxamide.
  • Q 4 is H, in another embodiment, Q 4 is CH 2 U 3 .
  • the present invention provides the compound:
  • each Q 4 independently is H, C 1 -C 6 alkyl, or C 1 -C 6 heteroalkyl
  • U 5 is N or CU 2
  • U 6 is O, S, or NH
  • U 7 is halo or alkylsulfonyloxy, or arylsulfonyloxy
  • Us is H or Hyp
  • U 2 is H, C 1 -C 6 alkyl, or CrC 6 heteroalkyl
  • Hyp is defined as in formula XXI.
  • Us is H. in another embodiment, Us is Hyp.
  • Hyp is 2-nitroimidazoIemethyloxycarbonyl.
  • Hyp is 2-nitroimidazolemethyl.
  • the present invention provides prodrugs of the novel tubulin binding comounds of the present invention and those known wherein the prodrugs are more water soluble than the corresponding novel tubulin binding compounds of the present invention or those known.
  • the prodrugs comprise a solubility enhancing moiety and the tubulin-binding anti-cancer compound.
  • the solubility enhancing moiety is covalently bonded to the 1-alkynyl, 1-alkenyl, or the alkyl groups substituting the 7-position of the corresponding tubulin binding compounds.
  • the solubility enhancing moiety is covalently bonded to the 1 -position of the indazole of the corresponding tubulin binding compounds.
  • Various solubility enhancing moieties are useful in the prodrugs of the present invention, including but not limited to, amino acid esters, sugar moieies, phosphates, phosphonates, ammonium groups, heterocyclic amines.
  • the present invention provides a compound having a structure of formula:
  • the aqueous solubility enhancing group is selected from the group consisting of an amino acid, a phosphate moiety, a sugar moiety, and an amino substituted benzoate moiety.
  • the aminoacid substituted benzoate moiety has structure of formula:
  • L 3 is a bond or a linker selected from alkylene or heteroalkylene and W 3 sn amine or a heterocycle.
  • L 3 is methylene.
  • W 3 is piperazine or alkylpiperazine.
  • Other suitable aqueous solubility enhancing groups are included in Table 1 E under the "Q 7 " column.
  • Q 7 is hydrogen.
  • the solubility enhancing group is selected from the group consisting of:
  • Qi is NH 2 .
  • the present invention provides a compound having structure of formula:
  • Qi Ci-C 6 alkynyl
  • R 2 i is selected from the group consisting of C r C 6
  • R22 is selected from:
  • the C r C 6 alkynyl is 1 -alkynyl.
  • water soluble prodrugs of the present invention include, but are not limited to, Compounds 91 - 94, 104, 1 15, 117, 157, 170, 178, 182, 184, 185, 187 - 190, and 192 - 196.
  • the present invention also provides prodrugs of known and novel tubulin binding compounds of this invention.
  • prodrug aspect of the invention an understanding of tumor biology is helpful. Cancer cells generally divide more frequently than normal cells.
  • Tubulin binding-drug mediated cancer therapies include cytotoxic agents selective for dividing cells.
  • tubulin binding compounds target cancer cells, as opposed to normal cells, generally because cancer cells undergo cell division more frequently than normal cells.
  • drugs targeting dividing cells do not kill all of the cancer cells in the solid tumor.
  • cancer cells can acquire mutations that confer drug resistance.
  • Another is that not all cancer cells divide more frequently than normal cells.
  • These slowly-dividing cancer cells are generally located in the hypoxic region of the tumor and can be as, or even more, insensitive to such inhibitors as normal cells. The formation and consequences of the tumor hypoxic region is described below.
  • the new vasculature that supports tumor growth is often disordered, leaving significant regions of the tumor under-vascularized and even the vascularized regions subject to intermittent blockage. Cells in these regions are unable to generate the energy required for cell division. These under-vascularized and blocked regions of the tumor become hypoxic - they have a lower oxygen concentration than the corresponding normal tissue. Thus, the median oxygen concentration of only ten percent of solid tumors falls in the normal range of 40-60 mm Hg, and fifty percent of solid tumors exhibit median oxygen concentrations of less than 10 mm Hg.
  • hypoxic regions of the tumor can constitute a significant reservoir of cancer cells resistant to therapy.
  • low tumor oxygen levels are associated with a poor response to therapy, increased metastases, and poor survival.
  • cancer cells do not divide significantly faster than normal cells, and can be resistant to therapeutic agents such as tubulin binding compounds that target dividing cells.
  • hypoxic region is conducive to biochemical reduction that can be used to generate reduced derivatives of a variety of chemical groups (see Workman et al., 1993, Cancer and Metast. Rev. 12: 73-82), and prodrugs of cytotoxins can be developed to exploit such hypoxic regions (see, Matteucci et al., PCT Publication No. WO 04/087075).
  • Compounds of the present invention arise in part out of the discovery that, cancer cells in the hypoxic region can be targeted by prodrug compounds comprising a tubulin binding cytotoxin and a hypoxia labile protecting group.
  • the hypoxic cells of the tumor generate the active toxin from the inactive, relatively non-toxic prodrug.
  • the active drug diffuses from the hypoxic cells and kills the cancer cells in adjacent regions, including the more frequently dividing cells.
  • the hypoxic region acts as a drug-factory to produce a cytotoxin within a tumor for killing adjacent normoxic cancer cells leading to a higher concentration of the cytotoxin within the tumor, relative to normal tissues.
  • a prodrug to generate the cytotoxin within the tumor, toxic side-effects arising due to normal cell toxicity can be reduced.
  • a hypoxic region can become normoxic and start dividing.
  • tubulin binding cytotoxins generated from the prodrug compounds of this invention, or by administering compounds of this invention in combination with other cytoxins, including for example, tubulin binding compounds and other anti-cancer cytotoxins.
  • the present invention also provides novel prodrugs of previously known tubulin binding anti-cancer compounds.
  • the tubulin binding compound is bonded to the hypoxic activator (Hyp) through a hydroxyl oxygen (-OHyp) or an amine nitrogen (-NHyp) in the tubulin binding compound to yield a hypoxia actived prodrug.
  • the hypoxic activator can be electron deficient nitrobenzene moieties, electron deficient nitrobenzoic acid amide moieties, nitroazole moieties, nitroimidazole moieties, nitrothiophene moieties, nitrothiazole moieties, nitrooxazole moieties, nitrofuran moieties, and nitropyrrole moieties.
  • the hypoxic activator is a substituted or unsubstituted nitroimidazole moiety.
  • hypoxic activator is selected from:
  • each X 2 is N or CR 32 ;
  • X 3 is NR 3 i, S, or O; each R 30 is independently hydrogen or alkyl;
  • R 31 is hydrogen, hydroxyl, C 1 -C 6 alkyl or heteroalkyl, C 3 -Ce cycloalkyl, heterocyclyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 1 -C 6 dialkylamino, aryl or heteroaryl, C 1 -C 6 acyl or heteroacyl, aroyl, or heteroaroyl;
  • Hyp is selected from
  • tubulin binding compounds can be derivatized to yield prodrugs having the following structures
  • these derivatized compounds in general are less active or inactive compared to the parent compound yielding a hypoxia activated prodrug compound.
  • the prodrug compounds demonstrate a 5-1000 fold loss of anticancer activity upon derivatization with respect to the starting compound.
  • activity data can be obtained from structure activity relationship data as described in this disclosure and by using methods known to one of skill in the art.
  • the present invention provides prodrug compounds as defined above wherein the tubulin binding compound is bonded to the hypoxic activator (Hyp) through an hydroxyl oxygen (-OHyp) or an amine nitrogen (-NHyp) in the tubulin binding compound.
  • the hypoxic activator can be electron deficient nitrobenzene moieties, electron deficient nitrobenzoic acid amide moieties, nitroazole moieties, nitroimidazole moieties, nitrothiophene moieties, nitrothiazole moieties, nitrooxazole moieties, nitrofuran moieties, and nitropyrrole moieties.
  • the hypoxic activator is a substituted or unsubstituted nitroimidazole moiety.
  • the present invention provides compounds of the invention wherein X is -NN(Hyp)R wherein Hyp and R are defined as above.
  • the present invention provides a prodrug of the compound of formula (l-i):
  • the present invention provides a compound of formulas (XXVIIIHXXXII):
  • each Q 1 , Q 2 , and Q 6 independently is hydrogen; halo; amino; Ci-C 6 alkylamino; di Ci-C 6 alkylamino; hydroxyl; CrC 6 alkoxy; nitro; cyano; C 1 -C 6 alkyl; C 1 -C 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; C 3 -C 8 cycloalkyl; heterocyclyl; aryl; heteroaryl; COR 18 ; SO 2 R 18 ; or PO 3 R 18 ; each Q 3 -Q 5 is C 1 -C 6 alkyl; C 1 -C 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; C 3 -C 8 cycloalkyl; heterocyclyl; aryl; or heteroaryl;
  • Q 8 is hydrogen; halo; amino; C 1 -C 6 alkylamino; di Ci-C 6 alkylamino; hydroxyl; C 1 -C 6 alkoxy; nitro; cyano; d-C 6 alkyl; CrC 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; C 3 -C 8 cycloalkyl; heterocyclyl; aryl; heteroaryl; COR 15 ; SO 2 R 15 or PO 3 R 15 ; each Qg independently is hydrogen; halo; amino; C 1 -C 6 alkylamino; di C 1 -C 6 alkylamino; hydroxyl; C 1 -C 6 alkoxy; nitro; cyano; CrC 6 alkyl; CrC 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; C 3 -C 8 cycloalkyl; heterocyclyl; ary
  • V is -NHNHR 16 ; -HNR 16 ; -N(Hyp)NHR 16 ; -NHN(Hyp)R 16 ; or
  • Hyp is a hypoxic activator as defined above;
  • X is O, -NNHR 16 , NR 16 , -NN(Hyp)R 16 , or NOR 16 wherein R 16 is C 1 -C 6 alkyl, aryl, C 1 -C 6 alkylsulphonyl, arylsulfonyl, C 1 -C 6 alkoxycarbony, aminocarbonyl, C 1 -C 6 alkylaminocarbonyl, di C 1 -C 6 alkylaminocarbonyl, C 1 -C 6 acyl, aroyl, aminothiocarbonyl, C 1 -C 6 alkylaminothiocarbonyl, di C 1 -C 6 alkylaminothiocarbonyl, C 1 -C 6 thioacyl, or thioaroyl; with the proviso that when X is NR 16 , R 16 excludes hydrogen;
  • Y is hydrogen, hydroxyl, or halogen
  • Z is -CH- or -N-;
  • Ri 5 is hydrogen, CrC 6 alkoxy, amino, C 1 -C 6 alkylamino, di C 1 -C 6 alkylamino, NHOH, NHNH 2 , Ci-C 6 alkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 - C 6 alkynyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroaryl;
  • R 16 is hydrogen, CrC 6 alkyl, aryl, C 1 -C 6 alkylsulphonyl, arylsulfonyl, C 1 - C 6 alkoxycarbonyl, aminocarbonyl, C 1 -C 6 alkylaminocarbonyl, di C 1 -C 6 alkylaminocarbonyl, C 1 -C 6 acyl, aroyl, aminothiocarbonyl, C 1 -C 6 alkylaminothiocarbonyl, di C 1 -C 6 alkylaminothiocarbonyl, C 1 -C 6 thioacyl, or thioaroyl; with the proviso that when X is NRi 6 , Ri 6 excludes hydrogen;
  • R- 18 is hydrogen, hydroxyl, C 1 -C 6 alkoxy, amino, CrC 6 alkylamino, di C 1 -C 6 alkylamino, NHOH, NHNH 2 , CrC 6 alkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
  • the present invention provides the compound:
  • the present invention provides a compound having structure of formula:
  • L-i is selected from Ci-C 6 alkylene and C 1 -Ce heteroalkylene
  • W 1 is selected from the group consisting of hydrogen, amino, C 1 -C 6 alkylamino, di(C r C 6 ) alkylamino, C 1 -C 6 alkoxy, heterocyclyl, heteroaryl, an aqueous solubility enhancing group
  • NR 26 Hyp and R 26 is selected from C 1 -C 6 alkyl or hydrogen and
  • Q 7 is selected from the group consisting of hydrogen, Hyp, a solubility enhancing group, and a methyl group substituted with a solubility enhancing group or a Hyp-NR 2 o moiety wherein R 2 o is Ci-C 6 alkyl group with the proviso that if Q 7 is a solubility enhancing group or a methyl group substituted with a solubility enhancing group, then Q 1 is N R 26 Hyp or W 1 is NR 26 Hyp; and if W- I is selected from the group consisting of amino, CrC 6 alkylamino, di(Ci- C 6 ) alkylamino, C 1 -C 6 alkoxy, heterocyclyl, heteroaryl, and an aqueous solubility enhancing group, then Q 7 is Hyp or a methyl group substituted with a Hyp-NR 2 o moiety; and if Q 1 is ethynyl, then Q 7 is Hyp or a methyl group substituted with a
  • the present invention provides a compound having structure of formula:
  • R 1 OH, NH 2 , OR, NR 2 Q 1 is C 1 -C 6 alkynyl, Hyp is selected from the group consisting
  • R 23 is OH, NH 2 , C 1 -C 6 alkoxy, and C 1 -C 6 alkylamino, and di(Ci-C ⁇ ) alkylamino; and R 24 is C 1 -C 6 alkyl.
  • the present invention provides a compound having a structure of formula:
  • Q 1 is selected from the group consisting of
  • the present invention provides compounds having stuctures of formulas:
  • the present invention provides compounds having structures of formulas: wherein Qi is selected from the group consisting of: H, NH 2 , and a Ci-C ⁇ alkyne.
  • Qi is selected from the group consisting of: H, NH 2 , and a Ci-C ⁇ alkyne.
  • the C r C 6 alkyne is a 1-alkyne.
  • the1 -alkyne is selected from the group consisting of acetylene, propyne, cyclopropylacetylene, and methoxypropyne.
  • examples of these classes of compounds include, but are not limited to:
  • R 2 5 is C-i-C ⁇ alkyl.
  • the present invention provides a compound of formula (XIV):
  • each Q 3 -Q 5 is Ci-C 6 alkyl; C 1 -C 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 - C 6 alkynyl; C 3 -C 8 cycloalkyl; heterocyclyl; (-OHyp) or (-NHyp) with the proviso that in any one compound, at least one of Q 3 -Q 5 is (-OHyp) or (-NHyp);
  • Q 1 , Q 2 , R 13 , R 15 and Hyp are as defined above; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
  • the present invention provides a compound of formula (XXXIV):
  • Q 2 is C 1 -C 6 alkoxy; halo; amino; or hydroxy; each Q 3 , Q 4 , and Q 5 independently is CrC 6 alkyl; C 1 -C 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; C 3 -C 8 cycloalkyl; or heterocyclyl;
  • R 3 is hydrogen, halo, C 1 -C 6 alkyl, aryl or heteroaryl;
  • Ri 3 is hydrogen; C r C 6 alkyl, C r C 6 heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroalkyl each optionally substituted with hydroxyl, d- C 6 alkoxy, amino, CrC 6 alkylamino, di C 1 -C 6 alkylamino; NHCOR 15 Or COR-is R 15 is hydrogen, hydroxyl, CrC 6 alkoxy, amino, CrC 6 alkylamino, di Ci-C 6 alkylamino, NHOH, NHNH 2 , CrC 6 alkyl, C r C 6 heteroalkyl, C r C 6 alkenyl, C r C 6 alkynyl, C 3 -C 6 cycloalkyl, C 3 -C 6 heterocyclyl, aryl, or heteroaryl;
  • R 18 is hydrogen, hydroxyl, Ci-C 6 alkoxy, amino, C r C 6 alkylamino, di CrC 6 alkylamino, NHOH, NHNH 2 , d-C 6 alkyl, Ci-C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroaryl;
  • Hyp is hypoxic activator; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
  • the present invention provides a compound of formulas (XXXV)-(XXXIX):
  • each Q 3 -Q 5 is C 1 -C 6 alkyl; Ci-C 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; C 3 -C 6 cycloalkyl; or heterocyclyl;
  • Q 6 is hydrogen; halo; amino; CrC 6 alkylamino; di CrC 6 alkylamino; hydroxyl; C r C 6 alkoxy; nitro; cyano; CrC 6 alkyl; CrC 6 heteroalkyl; CrC 6 alkenyl; C 1 -C 6 alkynyl; C 3 -C 8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi 8 ;
  • Q 7 is hydrogen; amino; CrC 6 alkylamino; di CrCs alkylamino; hydroxyl; CrC 6 alkoxy; nitro; cyano; CrC 6 alkyl; Ci-C 6 heteroalkyl; CrC 6 alkenyl; Ci-C 6 alkynyl; C 3 -C 8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi 5 ; SO 2 Ri 8 ; or PO 3 Ri 8 ; or a monosaccharide;
  • R 5 is hydrogen, halo, or Ci-C 6 alkoxy
  • R 6 is formyl or a protected form thereof
  • R 13 is hydrogen; CrC 6 alkyl, d-C 6 heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroalkyl each optionally substituted with hydroxyl, d- C 6 alkoxy, amino, CrC 6 alkylamino, di CrC 6 alkylamino, NHCORi 5 Or C0Ri 5;
  • R 15 is hydrogen, C r C 6 alkoxy, amino, C 1 -C 6 alkylamino, di C 1 -C 6 alkylamino, NHOH, NHNH 2 , CrC 6 alkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 - C 6 alkynyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; Ris is hydrogen, hydroxyl, CrC 6 alkoxy, amino, CrC 6 alkylamino, di
  • CrC 6 alkylamino NHOH, NHNH 2 , Ci-C 6 alkyl, CrC 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroaryl;
  • Hyp is hypoxic activator; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
  • the present invention provides a compound of formulas (XL)-(XLIII)
  • each Q 3 -Q 5 is C 1 -C 6 alkyl; CrC 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; C 3 -Cs cycloalkyl; or heterocyclyl;
  • Ri 5 is hydrogen, hydroxyl, CrC 6 alkoxy, amino, Ci-C 6 alkylamino, di Ci-C 6 alkylamino, NHOH, NHNH 2 , C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, Cs-C 8 cycloalkyl, heterocyclyl, aryl, or heteroaryl;
  • Hyp is hypoxic activator; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
  • the present invention provides a compound of formula (XLIV):
  • each Q 3 -Q 5 is Ci-C 6 alkyl; C 1 -C 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkynyl; C 3 -C 8 cycloalkyl; or heterocyclyl;
  • Rg is CrC 6 alkyl; aryl; or heteroaryl;
  • Ri 5 is hydrogen, hydroxyl, CrC 6 alkoxy, amino, C 1 -C 6 alkylamino, di CrC 6 alkylamino, NHOH, NHNH 2 , C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -Cg cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
  • the present invention provides a compound of formula (XLV):
  • each Q 3 -Qs is CrC 6 alkyl; CrC 6 heteroalkyl; C 2 -C 6 alkenyl; C 2 -
  • R 15 is hydrogen, hydroxyl, Ci-C 6 alkoxy, amino, Ci-C 6 alkylamino, di Ci-C 6 alkylamino, NHOH, NHNH 2 , C 1 -C 6 alkyl, Ci-C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; Hyp is hypoxic activator; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
  • each Q 3 -Q 5 is OMe.
  • the present invention provides a compound of formula (I)-(XLVII), wherein Q 1 is
  • the present invention provides a compound of formula (I)-(XLVII), wherein Q 1 is
  • the present invention further includes all salts thereof, and particularly, pharmaceutically acceptable salts thereof.
  • the invention includes compounds that are single isomers of the above formula (e.g., single enantiomers of compounds having a single chiral center), as well as solvate, hydrate, a prodrug and tautomeric forms thereof.
  • isomers include single geometric isomers such as cis, trans, E and Z forms of compounds with geometric isomers, or single tautomers of compounds having two or more tautomers.
  • examples of compounds of the present invention including tubulin binding anti cancer compounds and their water soluble and hypoxia activated prodrugs include but are not limited to the following compounds: 82 - 124, 126 - 159, and 160 - 196 described in the EXAMPLES section and in Tables 1 E and 1 F, and a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
  • a hydroxyl oxygen or an amine nitrogen is covalently bonded to a hypoxic activator (Hyp) yielding compounds having (-OHyp) or (-NHyp) moieties.
  • the compounds of the present invention can be synthesized following methods known to one of skill in the art and/or upon reading this disclosure. Methods for synthesizing aroylindazole compounds are described in PCT Pat. Pub. No. WO 06/057946 in page 71 , line 30 - page 74, line 14 and page 96, line 10 - page 116, line 9 and can be used for synthesizing the compounds of the present invention upon reading this disclosure (incorporated herein by reference).
  • the present invention provides a method for synthesizing a compound of the present invention comprising the steps of
  • the 1-N position of an aroylindzole compound of the present invention can be substituted with an aqueous solubility enhancing moiety or a Hyp containing moiety by reacting a 1-H indazole compound with an acyloxymethyl halide or acyloxymethyl tosylate wherein the acyl group contains a aqueous solubility enhancing moiety or a Hyp containing moiety.
  • Suitable aqueous solubility enhancing moieties or Hyp containing moieties are included in Table 1 E under the "Q 7 " column.
  • DIAD diisopropyl azodicarboxylate
  • DEAD diethyl azodicarboxylate
  • Scheme 10 provides a method for synthesis of a compound of the invention containing an aziridine moiety as described below:
  • Scheme 11 provides a method for synthesis of a compound of the invention containing an aldehyde moiety as described below:
  • the water soluble prodrugs of the present invention are synthesized by reacting a 1-chloromethyl-7-(1-alkylnyl)-3-aroylindazole or a 1-hydroxymethyl- 7-(1-alkylnyl)-3-aroylindazole with a water soluble carboxylic acid moiety.
  • Suitable water soluble carboxylic acid moieties include benzoic acids where the aryl ring is substituted with amine containing moieties.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a novel compound or a novel prodrug of a novel compound of the invention or a known compound; novel compounds and novel prodrugs of novel compounds of the invention or known compounds are described above.
  • the present invention provides a a novel compound or a novel prodrug of a novel compound of the invention or a known compound in substantially pure form.
  • a compound of the present invention disclosed herein is usually formulated as a pharmaceutical composition comprising the compounds or the prodrugs of this invention and a pharmaceutically-acceptable carrier.
  • pharmaceutically acceptable carrier refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically-acceptable material such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the subject composition and its components and not injurious to the patient.
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • Pharmaceutical preparations for oral use can be obtained through combining active compounds with solid excipient and, optionally, other compounds.
  • Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Further details on techniques for formulation can be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.); GOODMAN AND GILMAN'S: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS 10 TH EDITION 2001 by Louis Sanford Goodman et al., McGraw-Hill Professional; PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS 7 th Edition Howard C.
  • the present invention provides a pharmaceutically acceptable formulation of the compounds of the invention, wherein the pharmaceutically acceptable carrier, dilutent, or excipient is selected from a polyethylene glycol (PEG).
  • the pharmaceutically acceptable formulation comprises a compound of formula (I)-(XLVII).
  • the pharmaceutically acceptable formulation comprises a compound of formula (I)-(VIII).
  • the pharmaceutically acceptable formulation comprises a compound of formula (I).
  • Compound 30, for example, can be formulated with a PEG to yield a pharmaceutically acceptable formulation.
  • the compounds and prodrugs suited for use in the invention are tubulin binding compounds when administered to a human, non- human primate, or other mammal.
  • a compound e.g., a tubulin binding compound
  • Active forms can be identified by routine screening of the compounds of the invention for the activity.
  • assays and tests can be used to assess pharmacological activity of a compound or novel prodrug of the invention, including in vitro assays, such as those described below and elsewhere herein, in vivo assays in humans, non-human primates and other mammals, and/or clinical studies.
  • tubulin binding compound in which a tubulin binding compound is used for treatment or prevention of cancer or its manifestations, a tubulin binding compound with apoptosis-inducing activity similar to that of Combretastatin A-4 phosphate is selected. In some embodiments of the invention in which a tubulin binding compound is used for treatment or prevention of cancer or its manifestations, a tubulin binding compound with vascular disrupting activity similar to that of Combretastatin A-4 phosphate is selected.
  • a compound of the present invention that is cytotoxic to cancer cells such as H460, PC3, CCRF, LNCaP, HT29, HCT-15, ACHN, NCI-H69, H69 AR, HL-60, HL-60/MX2, MESSA and MES-SA/DX5 is administered to treat cancer.
  • cancer cells such as H460, PC3, CCRF, LNCaP, HT29, HCT-15, ACHN, NCI-H69, H69 AR, HL-60, HL-60/MX2, MESSA and MES-SA/DX5 is administered to treat cancer.
  • the present invention provides compounds having a Gl 50 ,Gl 90 , IC 50 , or IC 50 of about 0.001 to about 1000 nM, about 0.01 to about 100 nm, about 0.1 to about 50 nM, and about 1 to about 10 nM in a cancer cell antiproliferation assay.
  • the present invention provides a compound of formula (I) having a GI 50 or IC 50 of about 0.01 to about 100 nm, about 0.1 to about 50 nm, and about 1 to about 10 nm in a cancer cell antiproliferation assay.
  • said antiproliferation assays employ cancer cell including but not limited to gastric, colon, breast, leukemia, renal, small cell lung, and non-small cell lung cancer.
  • the gastric cancer cell used is MESSA or doxorubicin resistant MESSA/DX5 cell;
  • the colon cancer cell is HT29 cell and HCT-15;
  • the breast cacner cell is T47D cell;
  • the leukemic cell line used are HL-60 and HL- 60/MX2,
  • the small cell lung cancer cell is NCI-H69 and the non-small cell lung cancer cell is H460 cell.
  • the present invention provides a compound having a GI 50 or IC 50 of about 1 to about 50 nM in a cancer cell antiproliferation assay, such as, for example, compounds 87, 88, 93, 97, 100, and 118.
  • a tubulin binding compound having an IC 50 of tubulin polymerization of about 0.1 to about 10 ⁇ M as determined in a tubulin polymerization inhibition assay, such as for example, compounds 30 and 39.
  • the present invention provides a compound which when subjected to a liver microsomal stability study, remains about 10 to about 100, about 20 to about 80, about 80 to about 100% unmetabolized. In one embodiment, the liver microsomal study is conducted for between 10-60, 20- 40, or 25-35 minutes. In one embodiment, mouse liver microsome is employed in the study. In one aspect, the present invention provides a compound which when subjected to a plasma stability study, remains about 10-100, 20-80, or 80-100 % unmetabolized. In one embodiment, the plasma stability study is conducted for between 10-60, 20-40, or 25-35 minutes. In one embodiment, the plasma employed is from the same species of mammal the liver of whch is employed in the liver microsomal stability study.
  • the present invention provides a compound which upon administration to a human cancer cell xenograft tumor bearing mice, can reduce the tumor volume to about 5-70% of a control tumor volume.
  • the human cancer cell used is H460 or HT 29 cell.
  • the present invention provides a method of treating cancer and other hyperproliferative diseases comprising administering a therapeutically effective amount of a novel compound or a novel prodrug compound of the invention alone or in combination with one or more other anti-cancer agents to a subject in need of such treatment.
  • the methods of the present invention can in general be used for treatment of any cancer.
  • 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.
  • 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
  • the dose, schedule and duration of administration of the compound and/or prodrug of the invention will depend on a variety of factors.
  • the primary factor is the choice of a specific compound or prodrug of the present invention.
  • Other important factors include the age, weight and health of the subject, the severity of symptoms, if any, the subject's medical history, co-treatments, goal (e.g., prophylaxis or prevention of relapse), preferred mode of administration of the drug, the formulation used, patient response to the drug, and the like.
  • a compound and/or a prodrug of the invention can be administered at a dose in the range of about 0.1 mg to about 500 mg of a compound and/or prodrug of the invention per kg of body weight of the patient to be treated per day, optionally with more than one dosage unit being administered per day, and typically with the daily dose being administered on multiple consecutive days.
  • the compounds of the present invention include novel compounds of the invention, novel prodrug thereof, and novel prodrugs of known compounds.
  • a compound and/or a prodrug of the invention is administered in a daily dose in the range of about 0.5 mg to about 400 mg/Kg; about 1.0 mg to about 300 mg/Kg; about 1.5 mg to about 250 mg/Kg; about 2.0 mg to about 200 mg/Kg; about 2.5 mg to about 150 mg/Kg; about 5 to about 100 mg/Kg; about 10 to about 50 mg/Kg; and about 10 to about 70 mg per kg of body weight of the patient to be treated.
  • Cell culture studies are frequently used in the art to optimize dosages, and the assays disclosed herein can be used in determining such doses.
  • a therapeutically or prophylactically effective dose of a compound and/or a prodrug of the invention can be administered daily or once every other day or once a week to the patient. Controlled and sustained release formulations of the analogs can be used. Generally, multiple administrations of the compound and/or prodrug of the invention are employed. For optimum treatment benefit, the administration of the prophylactically effective dose can be continued for multiple days, such as for at least five consecutive days, and often for at least a week and often for several weeks or more.
  • the compound and/or prodrug of the invention is administered once (qday), twice (bid), three times (tid), or four times (qid) a day or once every other day (qod or q2d) or once a week (qweek or q7d), and treatment is continued for a period ranging from three days to two weeks or longer.
  • the present invention provides a method for treating cancer or other hyperproliferative diseases by administering to a patient in need of therapy thereof a therapeutically effective dose of a compound or prodrug compound of the invention. In one embodiment, the present invention provides a method for treating cancer or other hyperproliferative diseases by administering about 0.1 to about 500 mg/Kg of a compound or a prodrug compound of the invention to a patient in need of therapy thereof.
  • a compound and/or a prodrug of the invention is administered in a daily dose in the range of about 0.5 mg to about 400 mg/Kg; about 1.0 mg to about 300 mg/Kg; about 1.5 mg to about 250 mg/Kg; about 2.0 mg to about 200 mg/Kg; about 2.5 mg to about 150 mg/Kg; about 5 to about 100 mg/Kg; about 10 to about 50 mg/Kg; and about 10 to about 70 mg per kg of body weight of the patient to be treated.
  • the present invention provides a unit dosage form of about 1 to about 200 mg of a compound or prodrug compound of the invention to a patient in need of therapy thereof.
  • Combretastatin A-4 phosphate (CA4P), a tubulin-binding compound, is reported to have a maximum tolerated daily dose of 60 - 68 mg/m 2 , and has, for example, been administered to patients in clinical trials in daily doses of 27 and 36 mg/m 2 , by a 10-minute infusion, once every 21 days (Young et a/., 2004 , Expert Opin. Investigat Drugs, 13(9): 1171 -82 and Bilenker ef a/., 2005, Clin. Cancer Res., 11 (4): 1527-33, each of which is incorporated herein by reference).
  • CA4P Combretastatin A-4 phosphate
  • a compound of the present invention can be administered in similar daily doses for treatment of cancer. Therefore, in one embodiment, a compound of the present invention can be administered in a therapeutically affective daily dose of about 10 to about 100 mg/m 2 , about 20 to about 80 mg/m 2 , about 30 to about 70 mg/m 2 , about 40 to about 60 mg/m 2 , and about 45 to about 55 mg/m 2 to treat cancer.
  • a dose in mg/m 2 can be converted to a mg/kg dose in adult humans by dividing the mg/m 2 dose by a factor of 37; in children the corresponding dividing factor is 25.
  • a compound of the present invention can be administered in a therapeutically affective daily dose of about 0.3 to about 3 mg/kg, about 0.6 to about 2.4 mg/kg, about 0.9 to about 2.1 mg/kg, about 1.2 to about 1.8 mg/kg, and about 1.4 to about 1.6 mg/kg to treat cancer.
  • HED human equivalent dose
  • HED human equivalent dose
  • Cancer therapy often involves administering of a drug "cocktail" in which several anti-cancer drugs are contemporaneously administered to a cancer patient.
  • the novel compounds of the present invention and the prodrug compounds of the invention can be used in such therapies either in addition to or in substitution of one or more of the co-administered drugs.
  • co-administering a prodrug of the invention with one or more other drugs that target normoxic cells can, in one embodiment of the invention, co-administering a prodrug of the invention with one or more other drugs that target normoxic cells.
  • a compound and/or a prodrug compound of the invention can be co-administered in combination with other anti-cancer agents ("anticancer agent").
  • anticancer agent an anti-cancer agent
  • co-administration can in some cases provide one or more of several advantages over known cancer therapies, such as, for example co-administration of a compound and/or a prodrug compound of the invention and the anticancer agent has a synergistic effect on induction of cancer cell death.
  • Co-administration provides a better therapeutic result than administration of the anticancer agent alone, e.g., greater alleviation or amelioration of one or more symptoms of the cancer, diminishment of extent of disease, delay or slowing of disease progression, amelioration, palliation or stabilization of the disease state, partial or complete remission, prolonged survival or other beneficial therapeutic results.
  • the co-administration of a compound and/or a prodrug compound compound of the invention increases the sensitivity of cancer cells to the anticancer agent, allowing lower doses of the anticancer agent to be administered to the patient or allowing an anticancer agent to be used for treatment of cells otherwise resistant to the anticancer agent or otherwise refractory to treatment.
  • a compound and/or a prodrug compound of the invention is "co-administered" with another anticancer agent (also referred to herein as, "Agent") wherein a compound and/or a prodrug compound of the invention and Agent are administered as part of the same course of therapy.
  • Agent another anticancer agent
  • a compound and/or a prodrug compound of the invention is first administered prior to administration of the Agent, (i.e., the initiation of the other cancer therapy), and treatment with the compound and/or prodrug compound of the invention is continued throughout the course of administration of the Agent (i.e., the course of the other therapy).
  • a compound and/or a prodrug compound of the invention is administered after the initiation or completion of the other cancer therapy.
  • a compound and/or a prodrug compound of the invention is first administered contemporaneously with the initiation of the other cancer therapy.
  • a compound and/or a prodrug compound of the invention is first administered prior to administration of the Agent, and treatment with the compound and/or prodrug compound of the invention is continued after the cessation of administration of the Agent.
  • a compound and/or a prodrug compound of the invention is first administered prior to administration of the Agent, and treatment with the compound and/or prodrug compound of the invention is continued during part of the period of administration of the Agent.
  • administration of a compound and/or a prodrug compound of the invention can be initiated and completed prior to the administration of the second drug.
  • the present invention provides a method wherein a compound and/or a prodrug compound of the invention administered in combination with a chemoprotective agent or a chemoprotectant.
  • a chemoprotective agent protect healthy tissue from the toxic effects of anticancer drugs.
  • the chemoprotective agent is a thiol or a disulfide.
  • the chemoprotectant can reduce superoxide.
  • the chemoprotectant can react with the "Michael-receptor" generated from a hypoxia activated prodrug of the invention and prevent "Michael-receptor” from reacting with proteins and nucleic acid.
  • Anticancer drug therapy today typically involves multiple rounds, or
  • cycles of administration of the anti-cancer agent(s).
  • each cycle of administration (as well as a complete set of cycles) can be viewed as administration of a second drug.
  • a compound and/or a prodrug compound of the invention can be administered in any or all of the multiple cycles of treatment with the other Agent; in general, the compound and/or prodrug compound of the invention is administered on a daily basis for at least two or more days during each cycle.
  • a compound and/or a prodrug compound of the invention is co-administered with the Agent according to a schedule repeated at each round.
  • the compound and/or prodrug compound of the invention is administered in combination with an effective amount of one or more chemotherapeutic agents, an effective amount of radiotherapy, an appropriate surgery procedure, or any combination of such additional therapies.
  • the compound and/or prodrug compound of the invention and additional therapy can be administered at the same time or can be administered separately.
  • the two agents can be administered simultaneously or can be administered sequentially with some time between administrations.
  • One of skill in the art will understand methods of administering the agents simultaneously and sequentially and possible time periods between administrations.
  • Chemotherapeutic agents that can be used in combination with the compound of the invention include, but are not limited to, busulfan, improsulfan, piposulfan, benzodepa, carboquone, 2-deoxy-D-glucose, lonidamine and analogs thereof (refrence apps), glufosfamide, meturedepa, uredepa, altretamine, imatinib, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamine, chlorambucil, chlomaphazine, estramustine, ifosfamide, gefitinib, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozoto
  • a compound and/or a prodrug compound of the invention can be used in combination with an angiogenesis inhibitor including but not limited to Avastin and similar therapeutics.
  • an angiogenesis inhibitor including but not limited to Avastin and similar therapeutics.
  • a subject is treated with an angiogenisis inhibitor and subsequently treated with a compound and/or a prodrug compound of the invention.
  • the method is used to treat breast cancer.
  • a compound and/or a prodrug compound of the invention is administered with an anti-cancer agent that acts, either directly or indirectly, to inhibit the epidermal growth factor or EGFR receptor.
  • EGFR inhibitors suitable for coadministration with a compound of the invention include gefitinib and erlotonib.
  • a compound and/or a prodrug compound of the invention is administered with an anti-cancer agent that acts, either directly or indirectly, to inhibit hypoxia-inducible factor 1 alpha (HIFI a) or to inhibit a protein or enzyme, such as a glucose transporter or VEGF, whose expression or activity is increased upon increased HIFI a levels.
  • an anti-cancer agent that acts, either directly or indirectly, to inhibit hypoxia-inducible factor 1 alpha (HIFI a) or to inhibit a protein or enzyme, such as a glucose transporter or VEGF, whose expression or activity is increased upon increased HIFI a levels.
  • HIFIa inhibitors suitable for use in this version of the methods and compositions described herein include P13 kinase inhibitors; LY294002; rapamycin; histone deacetylase inhibitors such as [(E)-(I S,4S,10S,21 R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2- oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22- pentanone (FR901228, depsipeptide); heat shock protein 90 (Hsp90) inhibitors such as geldanamycin, 17-allylamino-geldanamycin (17-AAG), and other geldanamycin analogs, and radicicol and radicicol derivatives such as KF58333; genistein; indanone; staurosporin; protein kinase-1 (
  • a compound and/or a prodrug compound of the invention is administered with an anti-angiogenic agent, including but not limited to anti-angiogenic agents selected from the group consisting of angiostatin, an agent that inhibits or otherwise antagonizes the action of VEGF, batimastat, captopril, cartilage derived inhibitor, genistein, endostatin, interleukin, lavendustin A, medroxypregesterone acetate, recombinant human platelet factor 4, Taxol, tecogalan, thalidomide, thrombospondin, TNP-470, and Avastin.
  • an anti-angiogenic agent selected from the group consisting of angiostatin, an agent that inhibits or otherwise antagonizes the action of VEGF, batimastat, captopril, cartilage derived inhibitor, genistein, endostatin, interleukin, lavendustin A, medroxypregesterone acetate, recomb
  • angiogenesis inhibitors for purposes of the combination therapies provided by the present methods and compositions described herein include Cox-2 inhibitors like celecoxib (Celebrex), diclofenac (Voltaren), etodolac (Lodine), fenoprofen (Nalfon), indomethacin (Indocin), ketoprofen (Orudis, Oruvail), ketoralac (Toradol), oxaprozin (Daypro), nabumetone (Relafen), sulindac (Clinoril), tolmetin (Tolectin), rofecoxib (Vioxx), ibuprofen (Advil), naproxen (Aleve, Naprosyn), aspirin, and acetaminophen (Tylenol).
  • Cox-2 inhibitors like celecoxib (Celebrex), diclofenac (Voltaren), etodolac (Lodine), fenoprofen
  • pyruvate mimics and glycolytic inhibitors like halopyruvates, including bromopyruvate can be used in combination with an anti-angiogenic compound and a compound and/or a prodrug compound of the invention to treat cancer.
  • a compound and/or a prodrug compound of the invention is administered with an anti-angiogenic agent and another anti- cancer agent, including but not limited to a cytotoxic agent selected from the group consisting of alkylators, Cisplatin, Carboplatin, and inhibitors of microtubule assembly, to treat cancer.
  • the present methods and compositions described herein provides a variety of synergistic combinations of the compound and/or prodrug compound of the invention and other anti-cancer drugs.
  • Those of skill in the art can readily determine the anticancer drugs that act "synergistically" with a compound and/or a prodrug compound of the invention as described herein.
  • the reference Vendetti "Relevance of Transplantable Animal-Tumor Systems to the
  • the present invention provides a method of cancer treatment, wherein there is synergy between a compound and/or a prodrug compound of the invention and another anticancer agent.
  • Two drugs can be said to possess therapeutic synergy if a combination dose regimen of the two drugs produces a significantly better tumor cell kill than the sum of the single Agents at optimal or maximum tolerated doses.
  • the "degree of synergy” can be defined as net log of tumor cell kill by the optimum combination regimen minus net log of tumor cell kill by the optimal dose of the most active single Agent. Differences in cell kill of greater than ten-fold (one log) are considered conclusively indicative of therapeutic synergy.
  • the compound and/or prodrug compound of the invention When a compound and/or a prodrug compound of the invention is used with another anti-cancer agent, the compound and/or prodrug compound of the invention will, at least in some versions, be administered prior to the initiation of therapy with the other drug or drugs and administration will typically be continued throughout the course of treatment with the other drug or drugs. In some versions, the drug co-administered with a compound and/or a prodrug compound of the invention will be delivered at a lower dose, and optionally for longer periods, than would be the case in the absence of administering the compound and/or prodrug of the invention.
  • Such “low dose” therapies can involve, for example, administering an anti-cancer drug, including but not limited to paclitaxel, docetaxel, doxorubicin, cisplatin, or carboplatin, at a lower than approved dose and for a longer period of time together with a compound and/or a prodrug compound of the invention administered in accordance with the methods described herein.
  • an anti-cancer drug including but not limited to paclitaxel, docetaxel, doxorubicin, cisplatin, or carboplatin
  • the other anti-cancer agent or agents will be administered at the same dose levels used when a compound and/or a prodrug compound of the invention is not co-administered.
  • the additional anti-cancer agent(s) is dosed using either the standard dosages employed for those Agents when used without the compound and/or prodrug compound of the invention or are less than those standard dosages.
  • a compound and/or a prodrug compound of the invention in accordance with the methods described herein can therefore allow the physician to treat cancer with existing (or later approved) drugs at lower doses (than currently used), thus ameliorating some or all of the toxic side effects of such drugs.
  • the exact dosage for a given patient varies from patient to patient, depending on a number of factors including the drug combination employed, the particular disease being treated, and the condition and prior history of the patient, but can be determined using only the skill of the ordinarily skilled artisan in view of the teachings herein.
  • chemotherapeutic agents or antineoplastic agents i.e., the recommended effective dose
  • physicians are given, for example, in the product descriptions found in the Physician's Desk Reference 2003, (Physicians' Desk Reference, 57th Ed) Medical Economics Company, Inc., Oradell, NJ and/or are available from the Federal Drug Administration.
  • Illustrative dosage regimens for certain anti-cancer drugs are also provided below.
  • Cancer drugs can be classified generally as alkylators, anthracyclines, antibiotics, aromatase inhibitors, bisphosphonates, cyclo- oxygenase inhibitors, estrogen receptor modulators, folate antagonists, inorganic aresenates, microtubule inhibitors, modifiers, nitrosoureas, nucleoside analogs, osteoclast inhibitors, platinum containing compounds, retinoids, topoisomerase 1 inhibitors, topoisomerase 2 inhibitors, and tyrosine kinase inhibitors.
  • a compound and/or a prodrug compound of the invention can be coadministered with any anti-cancer drug from any of these classes or can be administered prior to or after treatment with any such drug or combination of such drugs.
  • a compound and/or a prodrug compound of the invention can be administered in combination with a biologic therapy (e.g., treatment with interferons, interleukins, colony stimulating factors and monoclonal antibodies).
  • a biologic therapy e.g., treatment with interferons, interleukins, colony stimulating factors and monoclonal antibodies.
  • Biologies used for treatment of cancer are known in the art and include, for example, trastuzumab (Herceptin), tositumomab and 131 I Tositumomab (Bexxar), rituximab (Rituxan).
  • Alkylators useful in the practice of the methods described herein include but are not limited to busulfan (Myleran, Busulfex), chlorambucil (Leukeran), ifosfamide (with or without MESNA), cyclophosphamide (Cytoxan, Neosar), glufosfamide, melphalan, L-PAM (Alkeran), dacarbazine (DTIC- Dome), and temozolamide (Temodar).
  • a compound and/or a prodrug compound of the invention is co-administered with an alkylator to treat cancer.
  • the cancer is chronic myelogenous leukemia, multiple myeloma, or anaplastic astrocytoma.
  • the present invention provides a method of treating cancer treatable by administering a compound and/or a prodrug compound of the invention alone or in combination with at least another alkylator or a prodrug thereof.
  • Alkylators such as, for example, cyclophosphamide, ifosfamide, glufosfamide, mechlorethamine, melphalan, chlorambucil, dacarbazine, temozolomide, carmustine, streptozocin, bendamustin, busulfan, thiotepa, cisplatin, carboplatin, and oxaliplatin, and types of cancers treated using any one of such alkylators alone or in combination with other anti cancer or chemoprotective agents are described for example in the reference Hardman et al., (see Hardman et al., The
  • the present invention provides a method of treating cancer by administering a compound and/or a prodrug compound of the invention with a cancer treatment regimen using at least the alkylator Glufosfamide.
  • Glufosfamide is in the clinic for the treatment of pancreatic cancer or Gemzar resistant pancreatic cancer.
  • Glufosfamide can be used for treating breast cancer, Morbus Hodgkin, gastrointestinal tract cancer, or as part of the GCE (Glufosfamide, Carboplatin, and Etoposide) or RGCE (Rituxan and GCE) regimen, for treating lymphomas. (Tidmarsh et al., PCT Pat. Pub. Nos.
  • the present invention provides a method of treating cancer by administering a compound and/or a prodrug compound of the invention with a cancer treatment regimen using at least a platinum coordination complex alkylator.
  • the platinum coordination complex alkylator is Cisplatin.
  • Cisplatin can be used to treat cancer of bladder, head and neck, endometrium, small cell carcinoma of the lung, and some neoplasms of childhood.
  • Cisplatin alone or with cyclophosphamide is used to treat advanced ovarian cancer.
  • Combination chemotherapy of Cisplatin with Bleomycin, Etoposide, and Vinblastine is used to treat advanced testicular cancer; and with one of Paclitaxel, Cyclophosphamide, or Doxorubicin to treat ovarian carcinoma.
  • Anthracyclines useful in the practice of the methods described herein include but are not limited to, doxorubicin (Adriamycin, Doxil, Rubex), mitoxantrone (Novantrone), idarubicin (Idamycin), valrubicin (Valstar), and epirubicin (Ellence).
  • doxorubicin Adriamycin, Doxil, Rubex
  • mitoxantrone Novantrone
  • idarubicin Idamycin
  • valrubicin valrubicin
  • Ellence epirubicin
  • a compound and/or a prodrug compound of the invention is co-administered with an anthracycline to treat cancer.
  • the cancer is acute nonlymphocytic leukemia, Kaposi's sarcoma, prostate cancer, bladder cancer, metastatic carcinoma of the ovary, and breast cancer.
  • Doxorubicin has been used successfully to produce regression in disseminated neoplastic conditions such as acute lymphoblastic leukemia, acute myeloblasts leukemia, Wilm's tumor, neuroblastoma, soft tissue and bone sarcomas, breast carcinoma, ovarian carcinoma, transitional cell bladder carcinoma, thyroid carcinoma, lymphomas of both Hodgkin and non-Hodgkin types, bronchogenic carcinoma, and gastric carcinoma.
  • Doxorubicin is typically administered in a dose in the range of 30- 75 mg/m 2 as a single intravenous injection administered at 21 -day intervals; weekly intravenous injection at doses of 20 mg/m 2 ; or 30 mg/m 2 doses on each of three successive days repeated every four weeks.
  • a compound and/or a prodrug compound of the invention is co-administered starting prior to and continuing after the administration of doxorubicin at such doses (or at lower doses).
  • Cyclic Anthracycline cytotoxin prodrugs useful in the practice of the methods described herein are provided by the reference Matteuci et al., PCT Patent App. Pub. No. WO 07/060534.
  • Antibiotics useful in the practice of the methods described herein include but are not limited to dactinomycin, actinomycin D (Cosmegen), bleomycin (Blenoxane), daunorubicin, and daunomycin (Cerubidine, DanuoXome).
  • a compound and/or a prodrug compound of the invention is co-administered with an antibiotic to treat cancer.
  • the cancer is a cancer selected from the group consisting of acute lymphocytic leukemia, other leukemias, and Kaposi's sarcoma.
  • Aromatase inhibitors useful in the practice of the methods described herein include but are not limited to anastrozole (Arimidex) and letroazole (Femara).
  • a compound and/or a prodrug compound of the invention is co-administered with an aromatase inhibitor to treat cancer.
  • the cancer is breast cancer.
  • Bisphosphonate inhibitors useful in the practice of the methods described herein include but are not limited to zoledronate (Zometa).
  • a compound and/or a prodrug compound of the invention is co-administered with a biphosphonate inhibitor to treat cancer.
  • the cancer is a cancer selected from the group consisting of multiple myeloma, bone metastases from solid tumors, or prostate cancer.
  • Cyclo-oxygenase inhibitors useful in the practice of the methods described herein include but are not limited to celecoxib (Celebrex).
  • a compound and/or a prodrug compound of the invention is co-administered with a cyclo-oxygenase inhibitor to treat cancer.
  • the cancer is colon cancer or a pre-cancerous condition known as familial adenomatous polyposis.
  • Estrogen receptor modulators useful in the practice of the methods described herein include but are not limited to tamoxifen (Nolvadex) and fulvestrant (Faslodex).
  • a compound and/or a prodrug compound of the invention is co-administered with an estrogen receptor modulator to treat cancer.
  • the cancer is breast cancer or the treatment is administered to prevent the occurrence or reoccurrence of breast cancer.
  • Folate antagonists useful in the practice of the methods described herein include but are not limited to methotrexate and tremetrexate.
  • a compound and/or a prodrug compound of the invention is co-administered with a folate antagonist to treat cancer.
  • the cancer is osteosarcoma.
  • the compound N-[4-[[(2,4-diamino-6- pteridinyl)methyl methylamino]benzoyl]-L-glutamic acid is an antifolate drug that has been used in the treatment of gestational choriocarcinoma and in the treatment of patients with chorioadenoma destruens and hydatiform mole. It is also useful in the treatment of advanced stages of malignant lymphoma and in the treatment of advanced cases of mycosis fungoides. Methotrexate is administered as follows.
  • intramuscular injections of doses of 15 to 30 mg are administered daily for a five-day course, such courses repeated as needed with rest period of one or more weeks interposed between courses of therapy.
  • twice weekly intramuscular injections are administered in doses of 30 mg/m 2 .
  • weekly intramuscular injections of doses of 50 mg or, alternatively, of 25 mg are administered twice weekly.
  • a compound and/or a prodrug compound of the invention is co-administered with methotrexate administered at such doses (or at lower doses).
  • 5-Methyl-6- [[(3,4,5-trimethoxyphenyI)-amino]methyl]-2,4-quinazolinediamine (commonly known as trimetrexate) is another antifolate drug that can be co-administered with a compound and/or a prodrug compound of the invention.
  • Inorganic arsenates useful in the practice of the methods described herein include but are not limited to arsenic trioxide (Trisenox).
  • a compound and/or a prodrug compound of the invention is co-administered with an inorganic arsenate to treat cancer.
  • the cancer is refractory acute promyelocytic leukemia (APL).
  • Microtubule inhibitors are any agent that interferes with the assembly or disassembly of microtubules
  • a microtubule inhibitor is any agent that interferes with the assembly or disassembly of microtubules
  • useful in the practice of the methods described herein include but are not limited to vincristine (Oncovin), vinblastine (Velban), paclitaxel (Taxol, Paxene), vinorelbine (Navelbine), docetaxel (Taxotere), epothilone B or D or a derivative of either, and discodermolide or its derivatives.
  • a compound and/or prodrug of the invention is co-administered with a microtubule inhibitor to treat cancer.
  • the cancer is ovarian cancer, breast cancer, non-small cell lung cancer, Kaposi's sarcoma, and metastatic cancer of breast or ovary origin.
  • the compound 22-oxo-vincaleukoblastine also commonly known as vincristine, is an alkaloid obtained from the common periwinkle plant (Vinca rosea, Linn.) and is useful in the treatment of acute leukemia. It has also been shown to be useful in combination with other oncolytic agents in the treatment of Hodgkin's disease, lymphosarcoma, reticulum-cell sarcoma, rhabdomyosarcoma, neuroblastoma, and Wilm's tumor.
  • Vincristine is administered in weekly intravenous doses of 2 mg/m 2 for children and 1.4 mg/m 2 for adults.
  • a compound and/or prodrug compound of the invention is co-administered with vincristine administered at such doses.
  • a compound and/or prodrug compound of the invention is not administered prior to treatment with a microtubule inhibitor, such as a taxane, but rather, administration of a compound and/or prodrug compound of the invention is administered simultaneously with or within a few days to a week after initiation of treatment with a microtubule inhibitor.
  • Modifiers useful in the practice of the methods described herein include but are not limited to Leucovorin (Wellcovorin), which is used with other drugs such as 5-fluorouracil to treat colorectal cancer.
  • a compound and/or prodrug compound of the invention is co-administered with a modifier and another anti-cancer agent to treat cancer.
  • the cancer is colon cancer.
  • the modifier is a compound that increases the ability of a cell to take up glucose, including but not limited to the compound N-hydroxyurea.
  • N- hydroxyurea has been reported to enhance the ability of a cell to take up 2- deoxyglucose (see the reference Smith et al., 1999, Cancer Letters 141: 85, incorporated herein by reference), and administration of N-hydroxyurea at levels reported to increase 2-deoxyglucose uptake or to treat leukemia together with administration of 2-deoxyglucose and a compound of the invention is one version of the therapeutic methods provided herein.
  • a compound and/or prodrug compound of the invention is co-administered with nitric oxide or a nitric oxide precursor, such as an organic nitrite or a spermineNONOate, to treat cancer, as the latter compounds stimulate the uptake of glucose.
  • Nitrosoureas useful in the practice of the methods described herein include but are not limited to procarbazine (Matulane), lomustine, CCNU (CeeBU), carmustine (BCNU, BiCNU, Gliadel Wafer), and estramustine (Emcyt).
  • a compound and/or prodrug compound and/or prodrug compound of the invention is co- administered with a nitrosourea to treat cancer.
  • the cancer is prostate cancer or glioblastoma, including recurrent glioblastoma multiforme.
  • Nucleoside analogs useful in the practice of the methods described herein include but are not limited to mercaptopurine, 6-MP (Purinethol), fluorouracil, 5-FU (Adrucil), thioguanine, 6-TG (Thioguanine), hydroxyurea (Hydrea), cytarabine (Cytosar-U, DepoCyt), floxuridine (FUDR), fludarabine (Fludara), azacytidine (Vidaza), pentostatin (Nipent), cladribine (Leustatin, 2- CdA), gemcitabine (Gemzar), and capecitabine (Xeloda).
  • a compound and/or prodrug compound of the invention is co-administered with a nucleoside analog to treat cancer.
  • the cancer is B-cell lymphocytic leukemia (CLL), hairy cell leukemia, adenocarcinoma of the pancreas, metastatic breast cancer, non-small cell lung cancer, or metastatic colorectal carcinoma.
  • CLL B-cell lymphocytic leukemia
  • hairy cell leukemia adenocarcinoma of the pancreas
  • metastatic breast cancer non-small cell lung cancer
  • non-small cell lung cancer or metastatic colorectal carcinoma.
  • the compound 5-fluoro-2,4(1 H,3H)-pyrimidinedione is an antimetabolite nucleoside analog effective in the palliative management of carcinoma of the colon, rectum, breast, stomach, and pancreas in patients who are considered incurable by surgical or other means.
  • 5-Fluorouracil is administered in initial therapy in doses of 12 mg/m 2 given intravenously once daily for 4 successive days with the daily dose not exceeding 800 mg. If no toxicity is observed at any time during the course of the therapy, 6 mg/kg are given intravenously on the 6th, 8th, 10th, and 12th days. No therapy is given on the 5th, 7th, 9th, or 11th days.
  • a daily dose of 6 mg/kg is administered for three days, with the daily dose not exceeding 400 mg. If no toxicity is observed at any time during the treatment, 3 mg/kg can be given on the 5th, 7th, and 9th days. No therapy is given on the 4th, 6th, or 8th days. A sequence of injections on either schedule constitutes a course of therapy.
  • a compound and/or prodrug compound of the invention is co-administered with 5-FU administered at such doses or with the prodrug form Xeloda with correspondingly adjusted doses.
  • the compound 2- amino-1 ,7-dihydro-6H-purine-6-thione is a nucleoside analog effective in the therapy of acute non- pymphocytic leukemias.
  • 6-Thioguanine is orally administered in doses of about 2 mg/kg of body weight per day. The total daily dose can be given at one time. If after four weeks of dosage at this level there is no improvement, the dosage can be cautiously increased to 3 mg/kg/day.
  • a compound and/or prodrug compound of the invention is co-administered with 6-TG administered at such doses (or at lower doses).
  • Osteoclast inhibitors useful in the practice of the methods described herein include but are not limited to pamidronate (Aredia).
  • a compound and/or prodrug compound of the invention is co-administered with an osteoclast inhibitor to treat cancer.
  • the cancer is osteolytic bone metastases of breast cancer, and one or more additional anti-cancer agents are also co-administered with a compound and/or prodrug compound of the invention.
  • Platinum compounds useful in the practice of the methods described herein include but are not limited to cisplatin (Platinol) and carboplatin (Paraplatin).
  • a compound and/or prodrug compound of the invention is co-administered with a platinum compound to treat cancer.
  • the cancer is metastatic testicular cancer, metastatic ovarian cancer, ovarian carcinoma, and transitional cell bladder cancer.
  • the compound cis-Diaminedichloroplatinum (II) commonly known as cisplatin, is useful in the palliative treatment of metastatic testicular and ovarian tumors, and for the treatment of transitional cell bladder cancer which is not amenable to surgery or radiotherapy.
  • Cisplatin when used for advanced bladder cancer, is administered in intravenous injections of doses of 50-70 mg/m 2 once every three to four weeks.
  • a compound and/or prodrug compound of the invention is co-administered with cisplatin administered at these doses (or at lower doses).
  • One or more additional anticancer agents can be co-administered with the platinum compound and a compound and/or prodrug compound of the invention.
  • Platinol, Blenoxane, and Velbam can be co-administered with a compound and/or a prodrug compound of the invention.
  • Platinol and Adriamycin can be co-administered with a compound and/or a prodrug compound of the invention.
  • Retinoids useful in the practice of the methods described herein include but are not limited to tretinoin, ATRA (Vesanoid), alitretinoin (Panretin), and bexarotene (Targretin).
  • a compound and/or a prodrug compound of the invention is co-administered with a retinoid to treat cancer.
  • the cancer is a cancer selected from the group consisting of APL, Kaposi's sarcoma, and T- cell lymphoma.
  • Topoisomerase 1 inhibitors useful in the practice of the methods described herein include but are not limited to topotecan (Hycamtin) and irinotecan (Camptostar).
  • a compound and/or a prodrug compound of the invention is co-administered with a topoisomerase 1 inhibitor to treat cancer.
  • Topoisomerase inhibitors and prodrugs thereof useful in the practice of the methods of the present invention are provided in the reference Matteucci et al., PCT Pat. App. Pub. No. WO 06/065448, incorporated herein by reference.
  • the cancer is metastatic carcinoma of the ovary, colon, or rectum, or small cell lung cancer.
  • topoisomerase 2 inhibitors useful in the practice of the methods described herein include but are not limited to etoposide, VP-16 (Vepesid), teniposide, VM-26 (Vumon), and etoposide phosphate (Etopophos).
  • a compound and/or prodrug compound of the invention is co-administered with a topoisomerase 2 inhibitor to treat cancer.
  • the cancer is a cancer selected from the group consisting of refractory testicular tumors, refractory acute lymphoblastic leukemia (ALL), and small cell lung cancer.
  • ALL refractory acute lymphoblastic leukemia
  • small cell lung cancer a cancer selected from the group consisting of refractory testicular tumors, refractory acute lymphoblastic leukemia (ALL), and small cell lung cancer.
  • administration of a compound and/or a prodrug of the invention either precedes or follows, or both, administration of a topoisomerase 2 inhibitor but is not administered concurrently therewith.
  • Tyrosine kinase inhibitors useful in the practice of the methods described herein include but are not limited to imatinib (Gleevec).
  • a compound and/or a prodrug compound of the invention is co-administered with a tyrosine kinase inhibitor to treat cancer.
  • the cancer is CML or a metastatic or unresectable malignant gastrointestinal stromal tumor.
  • a compound and/or a prodrug compound of the invention or a pharmaceutically acceptable salt thereof and one or more additional anti-cancer agents are administered to a patient.
  • additional anti-cancer agents include without limitation 5-methyl-6-[[(3,4,5-trimethoxyphenyl)amino]-methyl]- 2,4-quinazolinediamine or a pharmaceutically acceptable salt thereof, (8S,10S)-10-(3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl )oxy]-8- glycoloyl-Z. ⁇ . ⁇ .iO-tetrahydro-e. ⁇ .H-trihydroxy-i-methoxy-S, ⁇ - naphthacenedione or a pharmaceutically acceptable salt thereof; 5-fluoro- 2,4(1 H,3H)-pyrimidinedione or a pharmaceutically acceptable salt thereof; 2- amino-1 ,7-dihydro-6
  • the present invention provides a method of treatment of non-cancer hyperproliferative diseases characterized by cellular hyperproliferation (e.g., an abnormally increased rate or amount of cellular proliferation).
  • the hyperproliferative disease treated according to the present method 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 an
  • the hyperproliferative disease is selected from the group consisting of angiofibroma, atherosclerosis, benign prostatic hyperplasia, corneal graft rejection, gout, graft versus host disease, glaucoma, inflammatory diseases such as inflammatory bowel disease, ischemic heart and peripheral vascular disease, Karposi's sarcoma, keloids, life threatening infantile hemangiomas, macular degeration, myocardial angiogenesis, myocardial infraction, multiple sclerosis, neovascular-based dermatological conditions, Osier-Webber Syndrome, osteoarthritis, psoriasis, psoriatic arthritis, pulmonary fibrosis, psoriasis, rheumatoid arthritis, restenosis, rheumatoid arthritis, scleroderma, telangectasia, and wound granularization.
  • angiofibroma atherosclerosis
  • benign prostatic hyperplasia corneal graf
  • a tubulin binding compound with similar apoptosis- inducing activity similar to that of Combretastatin A-4 phosphate is selected.
  • a tubulin binding compound that induces apoptosis in skin, epithelial or endothelial, nerve, and T cells is administered to treat a hyperproliferative disease, e.g. psoriasis, rheumatoid arthritis, restenosis, benign prostatic hyperplasia, and multiple sclerosis.
  • the hyperpriliferative disease treated is psoriasis, a disease characterized by the cellular hyperproliferation of keratinocytes which builds up on the skin to form elevated, scaly lesions.
  • the hyperproliferative disease treated is multiple sclerosis, a disease characterized by progressive demyelination in the brain.
  • the hyperproliferative diseases treated is rheumatoid arthritis, a multisystem chronic, relapsing, inflammatory disease that can lead to destruction and ankyiosis of joints affected.
  • the compounds of the present invention are administered to prevent a hyperproliferative disease resulting from cellular proliferation on a prosthesis implanted in a subject by coating the prosthesis with a composition containing a compound of the present invention.
  • the hyperproliferative disease treated is benign prostatic hyperplasia, a disease in which prostate epithelial cells grow abnormally and thereby block urine flow.
  • Example 1 provides methods of synthesizing Compounds 82-86
  • Example 2 provides a method of synthesizing Compounds 87-90
  • Example 3 provides a method of synthesizing Compounds 93, 94, and 157
  • Example 4 provides a method of synthesizing Compounds 97-99
  • Example 5 provides a method of synthesizing Compounds 100 - 102
  • Example 6 provides a method of synthesizing Compounds 103-104
  • Example 7 provides a method of synthesizing Compound 30
  • Example 8 provides a method of synthesizing Compound 107
  • Example 9 provides a method of synthesizing Compound 108
  • Example 10 provides a method of synthesizing Compound 109
  • Example 11 provides a method of synthesizing Compound 110
  • Example 12 provides a method of synthesizing Compounds 111 and 112
  • Example 14 provides a method of synthesizing Compound 117.
  • Example 15 provides a method of synthesizing Compounds 118 and 119.
  • Example 16 provides a method of synthesizing Compound 121.
  • Example 17 provides a method of synthesizing Compounds 123 and
  • Example 18 provides a method for synthesizing an aqueous solubility enhancing prodrug, compound 187, of tubulin binding compound 67.
  • the hydrochloride salt of compound 187 was synthesized by reacting the free base form of compound 187 (334 mg) with 0.5 M HCI/Dioxane (1.17 mL) in DCM (1 ml_) at O 0 C. The reaction mixture was diluted with ether and the hydrochloride salt of compound 844 isolated by filtration (309 mg). Certain other prodrugs of the present invention that have enhanced aqueous solubility compared to the corresponding tubulin binding compound can be synthesized employing this method upon appropriate substitution of the aqueous solubility enhancing component and the tubulin binding compound.
  • Example 19 describes the synthesis of a 7-amino aroylindazole compound, 159, of the present invention and its Hyp derivative, 155.
  • the staring compound AC was synthesized according to a method described in PCT Pat. Pub. No. WO 04/043932, incorporated herein by reference.
  • the antiproliferative activity of these compounds was tested in a multi-well Alamar Blue based assay (at 2 h and 3 days). Cell growth in the presence and absence of the test compound as tabulated in Table 1 was compared, as measured by a fluorescence plate reader at excitation 550 nm and emission 590 nm (see Biosource International Inc., Tech Application Notes, Use of Alamar Blue in the measurement of Cell Viability and Toxicity, Determining IC 50 ).
  • H460 cells ATCC HTB-177 (NCI-H40), 4,000 cells/well/200 //l
  • LNCap cells ATCC CRL-1740, 6,000 cells/well/200 ⁇
  • a test compound was added to each plate in the treatment groups (2h and 3 day) at a concentration as tabulated in Table 1 (in 50 ⁇ l of medium).
  • the cells were rinsed to remove the test compound and incubated for 3 days, followed by staining with AlamarBlue.
  • the cells in the 3-day treatment group were incubated for 3 days, followed by staining with AlamarBlue.
  • AlamarBlue was added to the plate at (i) day 0 and (ii) day 3 and measured to establish the control reading.
  • the capacity of the cells to proliferate was measured 6 hours after addition of AlamarBlue by a fluorescence plate reader at excitation 550 nm and emission 590 nm.
  • a sample of cell free tubulin polymerizes and the sample's fluorescence emission increases.
  • Inhibition of tubulin polymerization by a tubulin binding compounds of the present invention was measured by the dose dependence of cell free tubulin fluorescence.
  • concentration of compound that reduced tubulin fluorescence by 50% compared to untreated tubulin are tabulated below in Table 3:
  • Compound 67 was effective in inhibiting tubulin polymerization as demonstrated by the same assay.
  • Example 24 demonstrates the usefulness of compounds of the present invention for treating xenograft tumors in mouse.
  • mice Female CB17/SCID mice (purchased from Taconic, Oxnard, CA), 7- 8 weeks of age, were allowed to acclimatize for at least three days, and handled under pathogen-free conditions.
  • Human non-small cell lung cancer cell line NCI-H60 was obtained from the American Type Culture Collection. The cell lines were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum. Cells were maintained in a 37 0 C incubator with 5% CO 2 . The H460 cells were harvested from culture and inoculated at 1 x 10 6 cells/ animal in the peritoneal subcutaneous space.
  • each group of mice (ten per group) was administered for five days, vehicle alone (the vehicle group), compound 30 alone at a daily dose of 5, 20, and 50 mg/kg (treatment group), and compound 30 alone at a daily dose of 5, and 20 mg/kg in combination with Taxol® at a daily dose of 10 mg/kg (combination group). Taxol was administered approximately 2-3 hours before that of compound 30.
  • Compound 30, administered at doses greater than 5 mg/kg were toxic and caused lethality in both treatment and combination groups perhaps indicating that the maximum tolerated dose of compound 30 was between 5 and 20 mg/kg.
  • the results from the experiment employing a daily dose compound 30 (5 mg/kg) are shown graphically in Figures 1 and 2 below.
  • the body weight of each mouse was recorded twice per week ( Figure 1 ).
  • the treatment group administered a daily dose of 5 mg/kg exhibited a weight pattern similar to that of the vehicle group with a mean weight loss of 8% on day 22 from the start of treatment on day 8.
  • Animals in the combination group displayed a weight loss of 13%.
  • One animal in the treatment group was found dead on day 18, and two were found dead on day 22 in the combination group.
  • Figure 2 graphically illustrates the mean tumor volume for each treatment group. Growth of each xenograft was monitored by externally measuring tumors in two dimensions using a digital caliper twice per week.
  • the xenograft data for compound 30 demonstrates that compared to the known anticancer agent taxol, compound 30 can show in vivo anti tumor activity both as a single agent and in combination with taxol.
  • Other compounds of the present invention compounds 94, 117, 157, and 187, whose structures are shown below, were tested in vivo in xenograft HT-29 tumor bearing nude mice, alone and in combination with other anti-cancer agents.
  • the compounds were formulated as follows: 94 and 117 in hydroxypropyl yff-cyclodextrin (HBSS), 157 in 2% Tween/Hank's buffered salt solution or in 2% Tween in HBSS, and 187 in 2.5% DMSO and 5% Tween/80-D5W.
  • the compounds were administered as follows: Compound 94 was administered at 100 mg/kg, intraperitoneally (ip), q2d x 3/wk x 2 wk, or at 50 mg/kg, ip, 2/wk x 2 wk alone, i.e.
  • Compound 117 was administered at 30 mg/kg, ip, q2d x 3 alone and in combination with CDDP at 6 mg/kg, intravenously (iv), q7d x 2.
  • 157 was administered at 100 mg/kg, ip, q2d x 3 alone; it was also administered at 50 mg/kg, ip, q2d x 3/wk x 2 wk alone and in combination with CPT-11 at 50 mg/kg, ip, q7d x 3 and CDDP at 6 mg/kg, iv, q7d x 2.
  • Compound 187 was administered at 10 mg/kg, iv, q2d x 3/wk x 2 or 5 mg/kg for the first 2 doses followed by 25 mg/kg for the next 4 doses, iv, q2d x 3/wk x 2; Compound 187 being administered alone and in combination with CPT-1 1 50 mg/kg, ip, q7d x 3.
  • Compound 94 when administered for 16 days demonstrated a 34% tumor growth inhibition following monotherapy at 50 mg/kg, and a 75% tumor growth inhibition following combination therapy with CPT-11.
  • Compound 157 demonstrated a tumor growth delay-500, determined by the additional time taken for a treated tumor to reach a 500 mm 3 compared to vehicle administered tumor, of 1day when administered alone and 3 days when administered in combination with CPT-11 ;
  • Compound 157 demonstrated a corresponding tumor growth delay-1000 of 2 days when administered alone and 3 days when administered in combination with CPT-11.
  • Compound 187 demonstrated that it was effective in inhibiting tumor growth alone and in combination with CPT-11.
  • Administration of 187 at 5 mg/kg for the first 2 doses followed by 25 mg/kg for the next 4 doses was well tolerated in mice administration at 25 mg/kg showed toxic effects in certain mice.
  • reaction mixture containing tubulin, Na glutamate, GTP, glucose phosphate, and MgCb (98 ⁇ l) was added compound 30 (2 ⁇ l per reaction), preincubated for 10 min at 37 0 C followed by the addition of [ 3 H] colchicine (1 ⁇ l per reaction, see also the reference Tse et al., J. Neurochem., 1980; 35(4): 767-74, incorporated herein by reference).
  • the reaction mixture was incubated for 30 min at 37 0 C and spotted on a DE81 filter, air dried for 5 min, wash 3 times with 1 :10 diluted general tubulin buffer and the filter added to a well followed by the addition of the scintillant.
  • a MatrigelTM matrix was kept on ice for 24 h (400 ⁇ l Matrigel per well). Gelation was performed at 37 0 C for 30min, the gel overlaid with 300 ⁇ l of medium containing 3 x 10 4 HUVEC cells followed by the addition of 300 ⁇ l of 10ng/ml of FGF-2 and the test compound, and incubated for 6 h (See for example the reference Neoplasia, 2004, 6(5):513-22).
  • the capillary tube formation was inspected under an inverted light microscope and the disruption of vessel formation observed as shown in Figure 5 demonstrating that compound 30 disrupts vessel formation.
  • compound 67 disrupted vessel formation Blood vessel formation is observed in many cancers particularly in solid tumors and such vessel formation is necessary for providing nutrients to the tumor. By disrupting these tumor blood vessels, 30, 67 and such other compounds of the invention can act as anti cancer agents.
  • Proliferating HUVEC-C cells were treated with various concentrations of compound 67 for 24 h.
  • Microtubules and F-actin filaments were visualized using fluorescence microscopy and demonstrated that 1-10 nM of compound 67 disrupted microtutbules and F-actin in a dose dependent manner.

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Abstract

Novel tublin binding compuunds and hypoxia activated prodrugs of novel and known tubulin binding compounds are useful for treating cancer and other hyperproliferative diseases.

Description

TUBULIN BINDING ANTI CANCER COMPOUNDS AND PRODRUGS THEREOF
CROSS-REFERENCES TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No.
60/802,267 filed 19 May 2006, the contents of which is incorporated herein by reference.
BACKGROUND OF THE INVENTION Field of the Invention
The present invention provides compositions and methods for treating cancer and other hyperproliferative disease conditions and generally relates to the fields of chemistry, biology, molecular biology, pharmacology, and medicine.
Description of Related Art
Tubulin and its polymerized form microtubules are useful for diverse cellular functions, including chromosome segregation during cell division, intracellular transport, development and maintenance of cell shape, cell motility, and possibly distribution of molecules on cell membranes (Bacher er a/., Pure Appl. Chem., 73(9): 1459-1464, 2001 , incorporated herin by reference). Tubulin binding can inhibit many important biological functions that depend on tubulin and microtubules. Inhibition of tubulin polymerization or prevention of the disassembly of microtubules causes cell cycle arrest, disrupts formation of vasculature, and leads to cell death. Thus, tubulin is considered to be a target in cancer therapy and drugs that interfere with tubulin function can be useful anti-cancer agents.
Certain anti-cancer agents targeting tubulin including combretastatins, taxanes (paclitaxel, docetaxel), vinca alkaloids (vincristine, vinblastine, vinorelbine), and epothilones suffer from one or more of the disadvantages as described below (Angerer et a/., Curr. Opin. Drug Discov. Dei/., 2000, 3(5): 575-584, incorporated herein by reference). Cancer cells can remove these anti-cancer agents by over-expressing transmembrane pumps and/or by other mechanisms and cause drug resistance. For example, the colon cancer cell HT-29 is resistant to combretastatin-A4. The aqueous insolubility of some of these drugs makes drug administration difficult. The complex chemical structures of these drugs make their synthesis and/or isolation from natural resources difficult. And, administration of certain anti-cancer agents can lead to toxic side effects in patients.
There remains a need for anti-cancer compounds, preferably tubulin binding anti-cancer compounds and their prodrugs that are less problematic due to drug resistance and/or have less toxic side effects in patients and/or are easier to administer due to enhanced aqueous solubility compared to those known. The present invention meets these needs.
SUMMARY OF THE INVENTION In one aspect, the present invention provides tubulin binding anti- cancer compounds and their prodrugs wherein the tubulin binding anti-cancer compounds are 3-aroylindazoles. In one embodiment, the 3-aroylindazoles are substituted with an 1-alkynyl, 1-alkenyl, or an alkyl group at the 7-position of the indazole. In another embodiment, the 3-aroylindazoles are substituted with a substituted or unsubstituted amino group at the 7-position of the indazole. In one embodiment, the tubulin binding compounds inhibit the polymerization of tubuin to microtubule. In another embodiment, the tubulin binding compounds bind to the colchicine binding site of tubulin. In another embodiment, the tubulin binding compounds are vascular disrupting agents. In one embodiment, the prodrugs of the tubulin binding anti-cancer compounds have enhanced aqueous solubility compared to the corresponding tubulin binding anti-cancer compounds. In another embodiment, the prodrugs comprise an aqueous solubility enhancing moiety and the tubulin-binding anti-cancer compound. In another embodiment, the aqueous solubility enhancing moiety is covalently bonded to the 1-alkynyl, 1- alkenyl, or the alkyl groups substituting the 7-position of the corresponding tubulin binding compounds. In another embodiment, the solubility enhancing moiety is covalently bonded to the 1 -position of the indazole of the corresponding tubulin binding compounds, directly or via a methylene linker. Various aqueous solubility enhancing moieties are useful in the prodrugs of the present invention, including but not limited to, amino acid esters, sugar moieties, phosphates, phosphonates, ammonium groups, and heterocyclic amines.
In one embodiment, the present invention provides hypoxia activated prodrugs of the tubulin binding anti-cancer compounds and their aqueous- solubility enhanced prodrugs. Therefore, a prodrug compound of the present invention can possess an enhanced aqueous solubility and also an enhanced hypoxia selective cytotoxicity compared to the corresponding tubulin binding anti-cancer compound. In one embodiment, the hypoxia activated prodrugs comprise tubulin binding anti-cancer compounds and a hypoxic activator (or Hyp). In one embodiment, the Hyp moiety is covalently bonded to the 1 -position of the indazole. In another embodiment, the Hyp moiety is covalently bonded to the substituted or unsubstituted amino group at the 7- position of the indazole. In another embodiment, the Hyp moiety is covalently bonded via alkylene or heteroalkylene linkers to the 1-alkynyl, 1-alkenyl, or the alkyl groups substituting the 7-position of the indazole.
In one aspect, the present invention provides a compound having a formula selected from:
Figure imgf000005_0001
Figure imgf000005_0002
(VII) (VIII)
Figure imgf000006_0001
(S-VII) (S-VIII)
Figure imgf000006_0002
(S-IX)
wherein each rii is 0, 1 , or 2; each Qi , Q2, and QQ independently is hydrogen; halo; B(OH)2 or a boronic acid ester; amino; CrCβ alkylamino; di C-i-Cθ alkylamino; hydroxyl; Ci-Cβ alkoxy; nitro; cyano; C-I-CΘ alkyl; CI-CΘ heteroalkyl; C2-C6 aikenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; COR15; SO2R15; or PO3Ri5; each Q3-Q5 is hydrogen; halo; amino; Ci-C6 alkylamino; di Ci-C6 alkylamino; hydroxyl; Ci-C6 alkoxy; nitro; cyano; CHO, NHCOCH=CH2, <] Cr -CC6 alkyl; Ci-C6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; COR15; SO2R-Is or PO3R15with the proviso that in any one compound, only one of Q3-Q5 is hydrogen; Q3 and Q4 together form heterocycle, an aryl, or a heteroaryl; or Q4 and Q5 together form a heterocycle, an aryl, or a heteroaryl;
Q7 is hydrogen; amino; C1-C6 alkylamino; di CrCe alkylamino; hydroxyl; CrC6 alkoxy; nitro; cyano; CrC6 alkyl; CrC6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-Ce cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi8; SO2Ri5; PO3Ri5 or a monosaccharide; with the proviso that in formula (II) Q7 excludes hydrogen;
Qs is hydrogen; halo; amino; CrCe alkylamino; di CrC6 alkylamino; hydroxyl; CrC6 alkoxy; nitro; cyano; CrC6 alkyl; CrC6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi5; SO2Ri5 or PO3R15; each Q9 independently is hydrogen; halo; amino; Ci-C6 alkylamino; di
Ci-C6alkylamino; hydroxyl; CrC6 alkoxy; nitro; cyano; CrC6 alkyl; CrC6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi5; SO2R15 or PO3R15;
X is O, -NNHRi6, NR16, or NORi6; Y is hydrogen, hydroxyl, or halogen;
Z is -CH- or -N-;
R15 is hydrogen, CrC6 alkoxy, amino, CrC6 alkylamino, di CrC6 alkylamino, NHOH, NHNH2, CrC6 alkyl, CrC6 heteroalkyl, C2-C6 alkenyl, C2- C6 alkynyl, C3-Ca cycloalkyl, heterocyclyl, aryl, or heteroaryl; R16 is hydrogen, C1-C6 alkyl, aryl, C1-C6 alkylsulphonyl, arylsulfonyl, d-
C6 alkoxycarbonyl, aminocarbonyl, CrCe alkylaminocarbonyl, di C1-C6 alkylaminocarbonyl, C1-C6 acyl, aroyl, aminothiocarbonyl, C1-C6 alkylaminothiocarbonyl, di CrC6 alkylaminothiocarbonyl, CrC6thioacyl, or thioaroyl; with the proviso that when X is NR16, R16 excludes hydrogen; R18 is hydrogen, hydroxyl, C1-C6 alkoxy, amino, CrC6 alkylamino, di
CrC6 alkylamino, NHOH, NHNH2, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof. In one embodiment, the compounds are tubulin binding compounds.
In another aspect, the present invention provides a compound of formula (XXI)-(XXVII):
Figure imgf000008_0001
(XXVII) wherein each Qi , Q2, and Q6 independently is hydrogen; halo; amino; Ci-C6 alkylamino; di Ci-C6 alkylamino; hydroxyl; C1-C6 alkoxy; nitro; cyano; C1-C6 alkyl; CrC6 heteroalkyl; C1-C6 alkenyl; C1-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; COR18; SO2R18; or PO3R15; each Q3-Q5 C1-C6 alkyl; d-C6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; and heterocyclyl;;
Q7 is hydrogen; halo; amino; CrC6 alkylamino; di C1-C6 alkylamino; hydroxyl; Ci-C6 alkoxy; nitro; cyano; C1-C6 alkyl; d-C6 heteroalkyl; C1-C6 alkenyl; C1-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; COR-is; SO2R18; or PO3R18 or a monosaccharide; with the proviso that in formula (II) Q7 excludes hydrogen;
Q8 is CrC6 alkyl; CrC6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; and heterocyclyl; each Q9 independently is CrC6 alkyl; d-C6 heteroalkyl; C2-C6 alkenyl;
C2-C6 alkynyl; C3-C8 cycloalkyl; and heterocyclyl;;
V is -NHR16; -NHNHRi6; -NHN(Ri6)2; -NRi6NHRi6; or -ORi7;
Y is hydrogen, hydroxyl or halogen; Z is -CH- or -N-; R15 is hydrogen, d-C6 alkoxy, amino, Ci-C6 alkylamino, di Ci-C6 alkylamino, NHOH, NHNH2, C1-C6 alkyl, C1-C6 heteroalkyl, C1-C6 alkenyl, Cr C6 alkynyl, C1-C6 cycloalkyl, CrC6 heterocyclyl, aryl, or heteroaryl;
Rie is hydrogen, C1-C6 alkyl, aryl, Ci-C6 alkylsulphonyl, arylsulfonyl, Ci- C6 alkoxycarbonyl, aminocarbonyl, C1-C6 alkylaminocarbonyl, di C1-C6 alkylaminocarbonyl, C1-C6 acyl, aroyl, aminothiocarbonyl, C1-C6 alkylaminothiocarbonyl, di Ci-C6 alkylaminothiocarbonyl, C1-C6 thioacyl, or thioaroyl; and R' is CrC6 alkyl or aryl; with the proviso that when V is NRi6, Ri6 excludes hydrogen;
Ri7 is CrC6 alkyl; aryl; or di CrC6 alkylamino; Rie is hydrogen, hydroxyl, CrC6 alkoxy, amino, CrC6 alkylamino, di
Ci-C6 alkylamino, NHOH, NHNH2, Ci-C6 alkyl, Ci-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof. In one embodiment, these compounds are tubulin binding compounds.
In another aspect, the present invention provides prodrug compounds wherein the novel compound of the invention is bonded to a hypoxic activator (Hyp) through a hydroxyl oxygen (-OHyp) or an amine nitrogen (-NHyp) in the tubulin binding compound.
In another aspect, the present invention provides prodrug compounds of known tubulin binding anti-cancer compounds wherein the tubulin binding compound is bonded to the hypoxic activator (Hyp) through an hydroxyl oxygen (-OHyp) or an amine nitrogen (-NHyp) in the tubulin binding compound.
The hypoxic activator can be nitrobenzene moieties, nitrobenzoic acid amide moieties, nitroazole moieties, nitroimidazole moieties, nitrothiophene moieties, nitrothiazole moieties, nitrooxazole moieties, nitrofuran moieties, and nitropyrrole moieties.
In one embodiment, Hyp is selected from:
Figure imgf000010_0001
wherein each X2 is N or CR32; X3 is NR3I 1 S, or O; each R30 is independently hydrogen or alkyl;
R31 is hydrogen, hydroxyl, Ci-C6 alkyl or heteroalkyl, C3-C8 cycloalkyl, heterocyclyl, CrC6 alkoxy, CrC6 alkylamino, CrC6 dialkylamino, aryl or heteroaryl, Ci-Cβ acyl or heteroacyl, aroyl, or heteroaroyl;
R32 is hydrogen, halogen, nitro, cyano, CO2H, CrC6 alkyl or heteroalkyl, CrC6 cycloalkyl, CrC6 alkoxy, Ci-C6 alkylamino, d-C6 dialkylamino, aryl, CON(R7)2 , CrC6 acyl or heteroacyl, or aroyl or heteroaroyl; and n = 0, 1.
In an additional embodiment, Hyp is selected from
Figure imgf000011_0001
wherein X2, R30, R31, R32 and n are as defined above. In one embodiment, the hypoxic activator is a substituted or unsubstituted nitroimidazole moiety. In another embodiment, Hyp is
Figure imgf000011_0002
In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a novel compound or a novel prodrug compound of the invention. In another aspect, the present invention provides a method of treating cancer comprising administering a therapeutically effective amount of a novel compound or a novel prodrug compound of the invention alone or in combination with one or more other anti-cancer agents to a subject in need of such treatment. These and other aspects and embodiments of the present invention are described in greater detail in the following sections.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the effect of Compound 30 alone and in combination with Taxol™ on tumor volume in xenograft mouse.
Figure 2 illustrates the effect of Compound 30 alone and in combination with Taxol™ on the bodyweight of tumor bearing xenograft mouse.
Figure 3 illustrates the effect of Compound 30 on tubulin binding of colchicine.
Figure 4 illustrates the effect of Compound 37 on tubulin binding of colchicine. Figure 5 illustrates the in vitro vascular disruption caused by Compound 30.
Figure 6 illustrates the effect of Compound 30 on vascular permeability
DETAILED DESCRIPTION OF THE INVENTION
This detailed description of the aspects and embodiments of the present invention is organized as follows." Section I provides definitions of terms used herein; Section Il provides the compounds of the present invention including their synthesis, formulation, and functional characterization; Section III provides useful therapies employing the compounds of the present invention; Section IV provides illustrative examples of synthesizing the compounds of the present invention and demonstrating their efficacy in treatment of cancer. 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
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 to represent a substantial difference over the definition of the term as generally understood in the art.
As used herein, "a" or "an" means "at least one" or "one or more." As used herein, "CrC6 alkyl" or (CrC6) alkyl refers to substituted or unsusbstituted straight or branched chain alkyl groups having 1-6 carbon atoms such as, for example, methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl and 3-methyl pentyl. A CrC6 alkyl substituent may be covalently bonded to an atom within a molecule of interest via any chemically suitable portion of the CrC6 alkyl group. "CrC6 alkyl" or (CrC6) alkyl may be further substituted with substituents, including for example, hydroxy, amino, mono or di(Cr Ce)a[kyl amino, halogen, C2-C6 alkyl ether, cyano, nitro, ethenyl, ethynyl, C1- C6 alkoxy, C1-C6 alkylthio, -COOH, -CONH2, mono- or d J-(C1 -C6)alkyl- carboxamido, -SO2NH2, -OSO2-(d-C6)alkyl, mono or di(CrC6)alkylsulfon- amido, aryl, and heteroary. Substituted CrC6 alkyl groups include, for example, -CH2-CH2-OH, -CH2-CH2-halogen, -CH2-CH2-NH2, -CH2-CH2-O- CH2-CH2-OH, -CH2-CH2-CH2-NH-CH2-CH2-OH and -CH2-CH2-NH-CH2-CH2- OH and the like.
As used herein, "amino acid" refers to naturally occurring σ-amino acids and their stereoisomers, as well as unnatural amino acids such as amino acid analogs, amino acid mimetics, synthetic amino acids, β-amino acids, p-amino acids, and N-substituted glycines in either the L- or D- configuration that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, κ-carboxyglutamate, and O-phosphoserine. Naturally occurring amino acids and analogs referred to herein may be described by shorthand designations as follows in Table A.
Table A: Amino Acid Nomenclature
Figure imgf000013_0001
Figure imgf000014_0001
Those of skill in the art will readily recognize that additional unlisted amino acid analogs are applicable to the present invention. "Stereoisomers" of naturally occurring amino acids refers to mirror image isomers of the naturally occurring amino acids, such as L- and D-amino acid stereoisomers. Both D- and L- isomers are included in the present invention. "Amino acid analogs" refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetics" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. In /?-amino acids, the amino acid side chain is bonded to the β-carbon atom of the carboxyl group such that there are two carbon atoms between the amino and carboxyl groups. In κ-arnino acids, the amino acid side chain is bonded to the K-carbon atom of the carboxyl group such that there are three carbon atoms between the amino and carboxyl groups. The side-chains or R-groups of β- amino acids, y amino acids, and N-substituted glycines can be, in either stereo configuration, the same as the side chain groups found on naturally occurring and later modified σ-amino acids, as well as side chains found on amino acid analogs and amino acid mimetics. The term "N-substituted glycine" refers to a glycine amino acid where an amino acid side chain is attached to the glycine nitrogen atom. Suitable "amino acid side chains" or "R groups" include, but are not limited to, side chains present in naturally occurring amino acids and side chains present in unnatural amino acids such as amino acid analogs, amino acid mimetics, synthetic amino acids, /0-amino acids, and κ-amino acids. Examples of N-substituted glycines suitable for use in the present invention include, without limitation, N-(2-aminoethyl)glycine, N- (3-aminopropyl)glycine, N-(2-methoxyethyl)glycine, N-benzylglycine, (S)-N-(I- phenylethyi)glycine, N-cyclohexylmethylglycine, N-(2-phenylethyl)glycine, N- (3-phenylpropyl)glycine, N-(6-aminogalactosyl)glycine, N-(2-(3'- indolylethyl)glycine, N-(2-(p-methoxyphenylethyl))glycine, N-(2-(p- chlorophenylethyl)glycine, and N-[2-(p-hydroxyphenylethyl)]glycine. Such N- substituted glycines can have an L- or D-configuration. N-substituted glycine oligomers, referred to herein as "peptoids," have been shown to be protease resistant (Miller et a/., Drug Dev. Res., 35:20-32 (1995), incorporated herein by reference). As such, a peptoid linker containing at least one σ-amino acid having an L-configu ration is within the scope of the present invention. As used herein, "cycloalkyl" refers to a monovalent cyclic hydrocarbon radical of three to seven ring carbons. The cycloalkyl group may have double bonds which may but not necessarily be referred to as "cycloalkene" or "cycloalkenyl". The cycloalkyl ring may be optionally substituted independently with one, two, or three substituents selected from alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkylalkyl, halo, nitro, cyano, hydroxy, alkoxy, amino, mono-alkylamino, di-alkylamino, haloalkyl, haloalkoxy, -COR (where R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl), -(CR'R")n-COOR (n is an integer from 0 to 5, R' and R" are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl), or -(CR'R")n-CONRxRy (where n is an integer from 0 to 5, R' and R" are independently hydrogen or alkyl, Rx and Ry are, independently of each other, hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl). More specifically, the term cycloalkyl includes, for example, cyclopropyl, cyclohexyl, cyclohexenyl, phenylcyclohexyl, A- carboxycyclohexyl, 2-carboxamido-cyclohexenyl, 2-dimethylaminocarbonyl- cyclohexyl, and the like.
As used herein, "heteroalkyl" means an alkyl radical as defined herein with one, two or three substituents independently selected from cyano, -ORW, -NRxRy, and -S(O)PRZ (where p 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. Rw is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, aryl, araalkyl, alkoxycarbonyl, aryloxycarbonyl, carboxamido, or mono- or di-alkylcarbamoyl. Rx 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. Rz is hydrogen (provided that p is 0), alkyl, cycloalkyl, cycloalkyl-alkyl, aryl, araalkyl, amino, mono-alkylamino, di- alkylamino, or hydroxyalkyl. Representative examples include, for example, 2-hydroxyethyl, 2,3-dihydroxy-propyl, 2-methoxyethyl, benzyloxymethyl, 2- cyanoethyl, and 2-methylsulfonyl-ethyl. For each of the above, Rw, Rx, Ry, and Rz can be further substituted by amino, fluorine, alkylamino, dialkylamino, OH or alkoxy. Additionally, the prefix indicating the number of carbon atoms (e.g., C1-C10) refers to the total number of carbon atoms in the portion of the heteroalkyl group exclusive of the cyano, -ORW, -NRxRy, or -S(O)pRz portions. The term "heteroalkyl," by itself or in combination with another term, also refers to a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2- CH2-NH-CH3, -CH2-CH2-N(CH3KH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, - CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3J3, -CH2-CH=N-OCH3, and - CH=CH-N(CH3)-CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2- S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'- represents both - C(O)2R'- and -R1C(O)2-. As used herein, "heterocycle", "heterocyclyl", "heterocycloalkyl" or
"cycloheteroalkyl" means a saturated or unsaturated non-aromatic cyclic radical of 3 to 8 ring atoms in which one to four ring atoms are heteroatoms selected from O, NR (where R is independently hydrogen or alkyl) or S(0)p (where p is an integer from O to 2), the remaining ring atoms being C, where one or two C atoms may optionally be replaced by a carbonyl group. The heterocyclyl ring may be optionally substituted independently with one, two, or three substituents selected from alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl, halo, nitro, cyano, hydroxy, alkoxy, amino, mono-alkylamino, di-alkylamino, haloalkyl, haloalkoxy, -COR (where R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl), -(CR'R")n- COOR (n is an integer from O to 5, R' and R" are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl), or -(CR'R")n-C0NRxRy (where n is an integer from O to 5, R' and R" are independently hydrogen or alkyl, Rx and Ry are, independently of each other, hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl). More specifically the term heterocyclyl includes, but is not limited to, pyridyl, tetrahydropyranyl, N-methylpiperidin-3-yl, N-methylpyrrolidin-3-yl, 2- pyrrolidon-1-yl, furyl, quinolyl, thienyl, benzothienyl, pyrrolidinyl, piperidinyl, morpholinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, 1 ,1-dioxo- hexahydro-1Δ6-thiopyran-4-yl, tetrahydroimidazo [4,5-c] pyridinyl, imidazolinyl, piperazinyl, and piperidin-2-onyl.and the derivatives thereof. The prefix indicating the number of carbon atoms (e.g., C3-Ci0) refers to the total number of carbon atoms in the portion of the cycloheteroalkyl or heterocyclyl group exclusive of the number of heteroatoms. In one embodiment, Rx and Ry together is heterocyclyl. More specifically the term aryl includes, but is not limited to, phenyl, biphenyl, 1-naphthyl, and 2-naphthyl, and the substituted forms thereof.
As used herein, "CrCβ alkoxy," means a substituted or unsubstituted alkyl group of 1 to 6 carbon atoms covalently bonded to an oxygen atom. A C-I-CΘ alkoxy group has the general structure -O-(Ci-C6 alkyl) wherein alkyl is as described above. Ci-C6 alkoxy 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.
As used herein, "CrC6 alkoxycarbonyl" refers to an alkoxy group covalently bonded to a carbonyl. A Ci-C6 alkoxycarbonyl group has the general structure -C(=O)-O-(CrC6)alkyl wherein alkyl is as described above.
As used herein, "CrC6 alkylamino," means a substituted or unsubstituted alkyl group of 1 to 6 carbon atoms covalently bonded to -NH-. A C1-C6 alkylamino group has the general structure -NH-(Ci-C6)alkyl wherein alkyl is as described above. CrC6 alkylamino groups include, for example, methylamino, ethylamino, propylamino and butylamino.
As used herein, "C2-C6 alkyl ether" refers to an ether substituent with 2 to 6 carbon atoms, positioned such that at least one carbon atom is located on either side of the oxygen atom.
As used herein, "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 and aryl groups.
As used herein, "aryl" refers to substituted or unsusbstituted moieties that include 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.
As used herein, the term "halogen" or "halo" refers to fluorine, chlorine, bromine, and/or iodine.
As used herein, "heteroaryl" refers to substituted or unsusbstituted monocyclic aromatic groups having 5 or 6 ring atoms, or fused ring bicyclic aromatic groups having 8 to 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, octahydroisoquinolinyl, 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 hetero atoms within the ring may be any arrangement allowed by the bonding characteristics of the constituent ring atoms. Aryl or heteroaryl groups may be further substituted with substituents, including for example, hydroxy, amino, mono or di(CrC6)alkyl amino, halogen, C2-C6 alkyl ether, cyano, nitro, ethenyl, ethynyl, C1-C6 alkoxy, C1-C6 alkylthio, -COOH, -CONH2, mono- or di- (CrC6)alkyl-carboxamido, -SO2NH2, -OSO2-(Ci-C6)alkyl, mono or di(d- C6)alkylsulfon-amido, aryl, and heteroaryl.
As used herein, "C1-C6 heteroalkylene" refers to a C1-C6 alkylene as defined above wherein 1 - 3 carbon atoms in the hydrocarbon radical or a branched saturated divalent hydrocarbon radical is replaced with a heteroatom. C1-C6 heteroalkylene groups include, for example, -CH2CH2-O- CH2CH2- and -CH2CH2-S-CH2CH2-.
As used herein, "hydroxy(C1-C6)alkyl" refers to a substituted or unsubstituted aliphatic group having from 1 to 6 carbon atoms, and further comprising at least one hydroxyl group on the main carbon chain and/or on a side chain. Hydroxy(C-ι-C6)alkyl groups include, for example, -CH2-CH2-OH and -CH2-CH2-CH2-OH.
As used herein, "protected forms of formyl" refers to acetals, oximes, and hydrazones. As used herein, "hypoxic activator" or "hypoxia activated trigger" refers to a group or moiety that is capable of releasing another compound, such as an antineoplastic agent or analogs thereof upon hypoxic reduction. In one embodiment, the hypoxic activator is a group that is capable of releasing the antineoplastic agent or analogs thereof upon reduction of the hypoxic activator under hypoxic conditions but does not release any antineoplastic agent or analog under normoxic conditions. For example, and as described in more detail below, one hypoxic activator is a nitroimidazole that may be substituted with a variety of groups. Other examples of hypoxic activators include, but are not limited to, groups based on nitrobenzenes, nitrobenzoic acid amides, nitroazoles, nitroimidazoles, nitrothiophenes, nitrothiazoles, nitrooxazoles, nitrofurans, and nitropyrroles, where each of these classes of moieties may be substituted or unsubstituted, such that the redox potential for the group lies within a range where the group can undergo reduction in the hypoxic regions of a tumor. One of skill in the art will understand, in view of the description herein, how to substitute these and other hypoxia labile protecting groups to provide a redox potential that lies within said range. Additional examples of hypoxic activators are described in Matteucci ef a/., PCT Publication Nos. WO 04/087075 and WO 07/002931 , each of which is incorporated herein by reference.
Generally, one of skill in the art can "tune" the redox potential of a hypoxic activator by substituting that activator with electron withdrawing groups, electron donating groups, or some combination of such groups. For example, nitrothiophene, nitrofuranfuran, and nitrothiazole groups may be substituted with one or more electron donating groups, including but not limited to methyl, methoxy, or amine groups, to provide a hypoxic activator with the desired redox potential. In another example, the nitropyrrole moiety can be substituted with an electron withdrawing group, including but not limited to cyano, carboxamide, -CF3, and sulfonamide groups, to achieve a group with the desired redox potential. For this purpose, strong electron withdrawing groups such as cyano, sulfone, sulfonamide, carboxamide, or - CF3, and milder electron withdrawing groups such as -CH2-halogen, where halogen is -F, -Cl, or -Br, can be used. As used herein, "prodrug" refers to a compound that, after administration, is metabolized or otherwise converted to an active or more active form 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).
As used herein, "substituent" refers to a molecular moiety that is covalently bonded to an atom within a molecule of interest. As used herein, "substitution" refers to replacing a hydrogen atom in a molecular structure with a substituent such that the valence on the designated atom (for example 4 for carbon) is not exceeded, and a chemically stable compound (a compound that can be isolated, characterized, and/or tested for biological activity) results. A combination of substituents or variables is permissible only if such a combination results in a stable or chemically feasible compound. A stable compound or chemically feasible compound is one in which the chemical structure is not substantially altered when kept at a temperature of 4°C or less, in the absence of moisture or other chemically reactive conditions, for at least a week.
As used herein, "isomers" refer to compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers". Stereoisomers that are not mirror images of one another are termed "diastereomers" and those that are non-superimposable mirror images of each other are termed "enantiomers". When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S- sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (-)-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a "racemic mixture". The compounds of this invention may exist in stereoisomeric form if they possess one or more asymmetric centers or a double bond with asymmetric substitution and, therefore, can be produced as individual stereoisomers or as mixtures. Unless otherwise indicated, the description is intended to include individual stereoisomers as well as mixtures. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art (see discussion in Chapter 4 of ADVANCED ORGANIC CHEMISTRY, 4th edition J. March, John Wiley and Sons, New York, 1992).
As used herein, "pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include:
(1 ) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethane- disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, A- chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or
(2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, trimethylamine, N- methylglucaminθ, and the like.
As used herein, "protecting group" refers to a grouping of atoms that when attached to a reactive group in a molecule masks, reduces or prevents that reactivity. Examples of protecting groups can be found in T.W. Greene and P.G. Wuts, PROTECTIVE GROUPS IN ORGANIC CHEMISTRY, (Wiley, 2nd ed. 1991 ) and Harrison and Harrison et al., COMPENDIUM OF SYNTHETIC ORGANIC METHODS, VOIS. 1-8 (John Wiley and Sons. 1971-1996). Representative amino protecting groups include formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ)1 tert-butoxycarbonyl (Boc), trimethyl silyl (TMS), 2- trimethylsilyl-ethanesulfonyl (SES), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC), nitro- veratryloxycarbonyl (NVOC) and the like. Representative hydroxy protecting groups include those where the hydroxy group is either acylated or alkylated such as benzyl and trityl ethers as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers.
As used herein, "aqueous solubility enhancing group" refers to a moiety that can be covalently bonded to a compound to increase the compound's aqueous solubility. Examples of aqueous solubility enhancing groups include, for example, an amino acid or its ester, a phosphate moiety, a heterocyclic amine, a sugar moiety, and an amino substituted benzoate moiety.
As used herein, "sugar" refers to a monosaccharide, a disaccharide, or a polysaccharide. Examples of sugars include, glucose, glucuronic acid, mannose, and other aldoheptoses, ketohexoses, aldopentoses, and their dimmers or polymers.
As used herein, "patient" or "subject" typically refers to a human but more generally refers to a mammal including but not limited to a human. Those of skill in the art will appreciate that the compositions and methods of the invention can be used to treat cancer or other hyperproliferative diseases in any mammal, including non-human primates, and experimental models of human cancers.
As used herein, "treating" a condition or patient refers to taking steps to obtain beneficial or desired therapeutic results, including clinical results. Beneficial or desired therapeutic results include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer, diminishment of extent of disease, delay or slowing of disease progression, palliation or stabilization of the disease state, and other beneficial results, as described below. As used herein, "reduction" of a symptom or symptoms (and grammatical equivalents of this phrase) refers to decreasing of the severity or frequency of the symptom(s) or eliminating the symptom(s).
As used herein, "administering" or "administration of a drug refers to a subject (and grammatical equivalents of this phrase) refer to direct administration, including self-administration and/or indirect administration, including the act of prescribing a drug. For example, as used herein, 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. As used herein, "effective amount" or a "therapeutically effective amount" of a drug refers to an amount of a drug that, when administered to a subject with cancer or any other hyperproliferative disease condition, will have (i) the intended therapeutic effect, e.g., alleviation, amelioration, palliation or elimination of one or more manifestations of cancer or other disease in the subject; or (ii) a prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of disease or symptoms or reducing the likelihood of the onset (or reoccurrence) of disease or symptoms. The full therapeutic or prophylactic effect does not necessarily occur by administration of one dose and can occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or more administrations.
As used herein, "prophylactically effective amount" of a drug refers to an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of disease or symptoms, or reducing the likelihood of the onset (or reoccurrence) of disease or symptoms. The full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations. As used herein, "pharmaceutically acceptable carrier or excipient" refers to a carrier or excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes a carrier or excipient that is acceptable for veterinary use as well as human pharmaceutical use. Examples include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like, used in the preparation of a pharmaceutical composition. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the pharmaceutical compositions or pharmaceutical formulations of the invention is contemplated. Supplementary active ingredients can be incorporated into the compositions of the invention. A "pharmaceutically acceptable carrier or excipient" as used in the specification and claims includes both one and more than one such carrier or excipient.
II. Compounds
A naturally occurring compound, colchicine, binds to tubulin and interferes with the function of the mitotic spindles, causing depolymerization and disappearance of tubulin polymers known as microtubules.
Disappearance of microtubules disrupts spindle formation as a result of which colchicine arrests mitosis in metaphase. Cancer cells with a high rate of cell division are affected by mitotic arrest and high concentrations of colchicines can completely prevent cells from entering mitosis, resulting in cell death. In addition, a colchicine-like tubulin binder such as Combretastatin A can selectively target the vascular system of tumors. The morphological changes induced in the endothelial cells of the tumor's blood vessels irreversibly shut down the blood flow to cancer cells while leaving the blood supply to healthy cells intact. Certain di- and tri-aryl compounds having tubulin binding ability have been synthesized (see, for example, Nam et al., Curr. Med. Chem., 2003, -/0:1697-1722 and Hsieh et al., US Patent Publication No. 2003/0195244, each of which is incorporated herein by reference). Heterocyclic indole, benzofuran, and benzothiophene containing tubulin binding di- and tri-aryl compounds constitute a sub-class of these compounds (see Nam et al. supra). These compounds, for example, have an aromatic moiety such as an aryl or an aroyl (-CO-Aryl) moiety or a CH group in the 2 position (as illustrated in structures below).
Figure imgf000026_0001
Colchicine Combretastatin-A
Figure imgf000026_0002
Ri is CO or CHfe; R3 is H, methyl, aryl or aroyl; and R2 and R4 are methyl or OMe. None of these compounds have yet been approved for treatment of cancer.
The compounds of the invention can be described in part as compounds which can bind to tubulin and/or arrest mitosis, and/or disrupt vaculature. In another part, the compounds of the present invention are prodrugs of known and novel compounds that can bind to tubulin and/or arrest mitosis, and/or disrupt vaculature; such prodrugs comprise a hypoxic activator and/or an aqueous solubility enhancer moiety. In one embodiment, the compounds are anti-cancer compounds which can bind to tubulin, and prodrugs thereof comprising a hypoxic activator.
Tubulin Binding Anti-cancer Compounds
In one aspect, the present invention provides compounds of formulas (I)-(VIII):
Figure imgf000027_0001
Figure imgf000027_0002
(VII) (VIII)
Figure imgf000028_0001
(S-VII) (S-VIII)
Figure imgf000028_0002
(S-IX) wherein each ni is 0, 1 , or 2; each Qi, Q2, and Q6 independently is hydrogen; halo; amino; Ci-Cs alkylamino; di Ci-Ce alkylamino; hydroxyl; CrC6 alkoxy; nitro; cyano; C1-C6 alkyl; C1-Ce heteroaikyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi5; SO2R15; or PO3Ri5; each Q3-Q5 is hydrogen; halo; amino; C1-C6 alkylamino; di CrC6 alkylamino; hydroxyl; d-C6 alkoxy; nitro; cyano; C1-C6 alkyl; Ci-C6 heteroaikyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi5; SO2Ri5 or PO3Ri5 with the proviso that in any one compound, only one of Q3-Q5 is hydrogen; Q3 and Q4 together form heterocycle, an aryl, or a heteroaryl; or Q4 and Q5 together form a heterocycle, an aryl, or a heteroaryl;
Q7 is hydrogen; amino; Ci-C6 alkylamino; di C1-C6 alkylamino; hydroxyl; C1-C6 alkoxy; nitro; cyano; C1-C6 alkyl; C1-C6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; COR15; SO2R-Ie; PO3R18 or a monosaccharide; with the proviso that in formula (II) Q7 excludes hydrogen;
Qs is hydrogen; halo; amino; CrC6 alkylamino; di C1-C6 alkylamino; hydroxyl; C1-C6 alkoxy; nitro; cyano; Ci-C6 alkyl; C1-C6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi8; SO2RiS or PO3Ri8; each Qg independently is hydrogen; halo; amino; Ci-C6 alkylamino; di CrC6 alkylamino; hydroxyl; C1-C6 alkoxy; nitro; cyano; CrC6 alkyl; CrC6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; COR18; SO2Ri8 or PO3R18;
X is O, -NNHR16, or NR16, or NOR16;
Y is hydrogen, hydroxyl, or halogen;
Z is -CH- or -N-; R15 is hydrogen, CrC6 alkoxy, amino, CrC6 alkylamino, di CrC6 alkylamino, NHOH, NHNH2, CrC6 alkyl, CrC6 heteroalkyl, C2-C6 alkenyl, C2- C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl;
Ri6 is hydrogen, CrC6 alkyl, aryl, CrC6 alkylsulphonyl, arylsulfonyl, d- C6 alkoxycarbonyl, aminocarbonyl, CrC6 alkylaminocarbonyl, di C1-C6 alkylaminocarbonyl, CrC6 acyl, aroyl, aminothiocarbonyl, C1-C6 alkylaminothiocarbonyl, di C1-C6 alkylaminothiocarbonyl, C1-C6 thioacyl, or thioaroyl; with the proviso that when X is NRi6, Ri6 excludes hydrogen;
Ris is hydrogen, hydroxyl, CrC6 alkoxy, amino, C1-C6 alkylamino, di CrC6 alkylamino, NHOH, NHNH2, C1-C6 alkyl, CrC6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof. In one embodiment, the present invention provides compounds of formulas (I)-(VIII), wherein Q1 is hydrogen; halo; amino, CO2H, cyano; nitro;
C1-C6 alkyl; CORi8; SO2Ri8; PO3Ri8;
Figure imgf000030_0001
R13
^= — R13; ^Ri3; C1-C6 alkoxy; halo; amino; or hydroxy; each Q3, Q4 and Q5 independently is hydrogen,C-ι-C6 alkoxy, halo, amino, hydroxyl, Q3 and Q4 together is methylenedioxy, or Q4 and Q5 together is methylenedioxy, provided that in any compound only one of the Q3, Q4 and Q5 is hydrogen;
Q7 is C1-C6 alkyl optionally substituted independently with one or more aryl, heteroaryl, hydroxyl, amino, C1-C6 alkylamino, di Ci-C6 alkylamino, CO2H, or CONH2; CORi8; SO2R18; or PO3R18; or a monosaccharide; each Q8 and Q9 is hydrogen;
Ri3 is hydrogen; C1-C5 alkyl, CrC6 heteroalkyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroalkyl each optionally substituted with hydroxyl, C1- C6 alkoxy, amino, C1-C6 alkylamino, di Ci-C6 alkylamino, NHCORi5, or COR18; and
R18 is hydrogen, hydroxyl, C1-C6 alkoxy, amino, CrC6 alkylamino, di C1-C6 alkylamino, NHOH, NHNH2, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
In an additional embodiment, the present invention provides compunds of formulas (I)-(VIII), wherein Qi is hydrogen; halo; cyano; CO2H; CONH2; ^= — R13; or = — R13; and each Q2 - Q6 independently is hydrogen, C1-C6, alkoxy; halo; amino; or hydroxy; with the proviso that in any compound only one of the Q3, Q4, and Q5 is hydrogen; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof. In another embodiment,
Q1 is \ 0 — Rs wherein Rs is a monosaccharide or a disaccharide. In another embodiment, the present invention provides compounds of formula (I) - (VIII) wherein each Q3 and Q5 is 1 ,1 '-difluoroethyl and Q4 is fluoro.
In another embodiment, the present invention provides compounds of formulas (VIII)-(XIII) wherein X is O.
In another embodiment, the present invention provides a compound of formula:
wherein Qi is
Figure imgf000031_0001
and n is 1 or 2.
In one embodiment, the present invention provides compounds of formulas (IX)-(XIII):
Figure imgf000031_0002
Figure imgf000031_0003
(XII) (XIII) wherein R14 is H, Me, or B(OH)2; and QrQ9 are as defined above; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
In another aspect, the present invention provides compounds of formula (IX-i)-(XIII-i)
Figure imgf000032_0001
(XIM) (XIIM)
wherein R13 is H, Me, CH2OH, CH(Me)OH, CH2CH2OH, CH2NH2, CH2PO3H2, PO3H2, CO2H, or CONH2 and R14 is H, Me, or B(OH)2; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
In another embodiment, the present invention provides a compound of formula (XIV):
Figure imgf000032_0002
(XIV) wherein
Ri3
^ R13
Qi is : -Ri3 or -Ri3; Q2 is
Figure imgf000033_0001
C1-C6 alkoxy; halo; amino; or hydroxy; each Q3-Q5 is CrC6 alkyl; d-C6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl;
R13 is hydrogen; CrC6 alkyl, CrC6 heteroalkyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroalkyl each optionally substituted with hydroxyl, Cr C6 alkoxy, amino, C1-C6 alkylamino, di CrC6 alkylamino; CORi8 or NHCOR-I5;
R15 is hydrogen, hydroxyl, CrC6 alkoxy, amino, CrC6 alkylamino, di CrC6 alkylamino, NHOH, NHNH2, C1-C6 alkyl, CrC6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
In another aspect, the present invention provides a compound of formula (XV):
Figure imgf000033_0002
wherein
Figure imgf000033_0003
alkoxy; halo; amino; or hydroxy; each Q3, Q4, and Q5 independently is C1-C6 alkyl; CrC6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; and C3-C8 cycloalkyl;
Q7 is hydrogen; amino; CrC6 alkylamino; di C1-C6 alkylamino; hydroxyl; C1-C6 alkoxy; nitro; cyano; C1-C6 alkyl; C1-C6 heteroalkyl; C1-C6 alkenyl; C1-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; COR15; SO2R18; or PO3R18 or a monosaccharide;
R1 is CH2 or CO;
R3 is hydrogen, halo, C1-C6 alkyl, aryl or heteroaryl; Ri3 is hydrogen; Ci-C6 alkyl, Ci-C6 heteroalkyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroalkyl each optionally substituted with hydroxyl, Cr C6 alkoxy, amino, CrC6 alkylamino, di CrC6 alkylamino; NHCORi5 or CORi8;
Ri5 is hydrogen, hydroxyl, d-C6 alkoxy, amino, Ci-C6 aikylamino, di CrC6 alkylamino, NHOH, NHNH2, Ci-C6 alkyl, d-C6 heteroalkyl, CrC6 alkθnyl, CrC6 alkynyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, aryl, or heteroaryl;
Ri8 is hydrogen, hydroxyl, Ci-C6 alkoxy, amino, CrC6 alkylamino, di CrC6 alkylamino, NHOH, NHNH2, CrC6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
In another aspect, the present invention provides a compound selected from formulas (XVI)-(XX):
Figure imgf000034_0001
or hydroxy; each each Q3-Q5 is CrC6 alkyl; CrC6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; or heterocyclyl; Q6 is hydrogen; halo; amino; CrC6 alkylannino; di C1-C6 alkylamino; hydroxyl; CrC6 alkoxy; nitro; cyano; C1-C6 alkyl; CrC6 heteroalkyl; CrC6 alkenyl; CrC6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi8; SO2Ri8 or PO3Ris; Q7 is hydrogen; amino; CrC6 alkylamino; di CrC6 alkylamino; hydroxyl;
CrC6 alkoxy; nitro; cyano; CrC6 alkyl; CrC6 heteroalkyl; CrC6 alkenyl; CrC6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; COR-I5; SO2Ri8; or PO3Ri8; or a monosaccharide;
R5 is hydrogen, halo, or C1-C6 alkoxy; R6 is formyl or a protected form thereof;
Ri3 is hydrogen; C1-C6 alkyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroalkyl each optionally substituted with hydroxyl, Cr C6 alkoxy, amino, CrC6 alkylamino, di CrC6 alkylamino, NHCOR-is or COR18;
Ri5 is hydrogen, hydroxyl, CrC6 alkoxy, amino, CrC6 alkylamino, di CrC6 alkylamino, NHOH, NHNH2, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl;
Ris is hydrogen, CrC6 alkoxy, amino, CrC6 alkylamino, di CrC6 alkylamino, NHOH, NHNH2, C1-C6 alkyl, CrC6 heteroalkyl, C2-C6 alkenyl, C2- C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
In another aspect, the present invention provides a compound of formulas (XXI)-(XXVII):
Figure imgf000036_0001
(XXVII) wherein each Q1, Q2, and Q6 independently is hydrogen; halo; amino; C1-C6 alkylamino; di C1-C6 alkylamino; hydroxyl; CrC6 alkoxy; nitro; cyano; C1-C6 alkyl; C1-C6 heteroalkyl; Ci-C6 alkenyl; CrC6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi8; SO2Ri8 or PO3Ri8; each Q3-Q5 is C1-C6 alkyl; C1-C6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi8; SO2R18; or PO3R13; Q3 and Q4 together form heterocycle, an aryl, or a heteroaryl;
Q7 is hydrogen; halo; amino; Ci-C6 alkylamino; di C1-C6 alkylamino; hydroxyl; CrC6 alkoxy; nitro; cyano; Ci-C6 alkyl; Ci-C6 heteroalkyl; C1-C6 alkenyl; CrC6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; COR15; SO2Ri8; or PO3Ri8 or a monosaccharide; with the proviso that in formula (II) Q7 excludes hydrogen;
Qs is hydrogen; halo; amino; C1-C6 alkylamino; di C1-C6 alkylamino; hydroxyl; CrC6 alkoxy; nitro; cyano; aryl; heteroaryl; CORi5; SO2Ri5 or PO3Ri5; each Q9 independently is hydrogen; halo; amino; C1-C6 alkylamino; di Ci-C6 alkylamino; hydroxyl; Ci-C6 alkoxy; nitro; cyano; aryl; heteroaryl; COR15;
Figure imgf000037_0001
V is -NHR16; -NHNHR16; -NHN(R16)2; -NR16NHR16; or -OR17; Y is hydrogen, hydroxyl or halogen;
Z is -CH- or -N-;
R-15 is hydrogen, CrC6 alkoxy, amino, C1-C6 alkylamino, di CrC6 alkylamino, NHOH, NHNH2, C1-C6 alkyl, C1-C6 heteroalkyl, C1-C6 alkenyl, C1- C6 alkynyl, CrC6 cycloalkyl, CrC6 heterocyclyl, aryl, or heteroaryl; R-15 is hydrogen, C1-C6 alkyl, aryl, C1-C6 alkylsulphonyl, arylsulfonyl, d-
C6 alkoxycarbonyl, aminocarbonyl, C1-C6 alkylaminocarbonyl, di C1-C6 alkylaminocarbonyl, C1-C6 acyl, aroyl, aminothiocarbonyl, C1-C6 alkylaminothiocarbonyl, di C1-C6 alkylaminothiocarbonyl, C1-C6 thioacyl, or thioaroyl; and R' is C1-C6 alkyl or aryl; with the proviso that when V is NR16, Ri6 excludes hydrogen;
R17 is CrC6 alkyl; aryl; or di CrC6 alkylamino;
R18 is hydrogen, CrC6 alkoxy, amino, C1-C6 alkylamino, di CrC6 alkylamino, NHOH, NHNH2, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2- C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
In another embodiment, the present invention provides the compounds:
Figure imgf000038_0001
wherein each Q2-Q5 and Q7 independently is definded as in formula (I).
In another embodiment, the present invention provides the compounds:
Figure imgf000038_0002
wherein Ri is
Figure imgf000039_0001
R2 is
H, NH2, NO2, I, Br1 CI1OH1CN, "OH
Figure imgf000039_0002
Figure imgf000039_0003
each X and Y independently is CH or N.
In another aspect, the present invention provides the compounds:
Figure imgf000039_0004
Figure imgf000039_0005
wherein Q4 is CHO, C(H)=CH2, ECH1 CH2U3, OCH2U3, NHCOCH=CH2,
or "Xl; U3 is Cl or F; Q1 is OH, NH2, CO2H, CN, halo, C1-C6 alkyl,
R13
-Ri3 or ■ R13.
In another aspect, the present invention provides the compounds:
Figure imgf000040_0001
wherein Q4 is H or CH2U3; each Ui is N or CU4; U2 is C1-C6 alkyl or CrC6 heteroalkyl; U3 is F, CN, OMe, and OH; and U4 is nitro, nitrile, halo, CF3, or carboxamide. In one embodiment, Q4 is H, in another embodiment, Q4 is CH2U3. The compound:
Figure imgf000040_0002
reported in the reference Ohsumi et al., J. Med. Chem., 1998, 41 :3022 is excluded from any of these embodiments.
In another aspect, the present invention provides the compound:
Figure imgf000040_0003
wherein each Q4 independently is H, C1-C6 alkyl, or C1-C6 heteroalkyl; U5 is N or CU2; U6 is O, S, or NH; U7 is halo or alkylsulfonyloxy, or arylsulfonyloxy; and Us is H or Hyp; U2 is H, C1-C6 alkyl, or CrC6 heteroalkyl; Hyp is defined as in formula XXI. In one embodiment, Us is H. in another embodiment, Us is Hyp. In another embodiment, Hyp is 2-nitroimidazoIemethyloxycarbonyl. In another embodiment, Hyp is 2-nitroimidazolemethyl. Other chloroethylureas reported in the references Mounetou et al., J. Med. Chem. 2003, 46: 5055-63 and Legault et al., Cancer Res., 2000, 60,985-92 are excluded from these embodiments.
In one embodiment the present invention provides the following compounds:
Figure imgf000041_0001
Figure imgf000041_0002
Figure imgf000041_0003
wherein R is a group which undergoes a tumor specific release and R1 = NH2, OH, Cl, F, and Br Water Soluble Prodrugs
In one aspect, the present invention provides prodrugs of the novel tubulin binding comounds of the present invention and those known wherein the prodrugs are more water soluble than the corresponding novel tubulin binding compounds of the present invention or those known. In another embodiment, the prodrugs comprise a solubility enhancing moiety and the tubulin-binding anti-cancer compound. In another embodiment, the solubility enhancing moiety is covalently bonded to the 1-alkynyl, 1-alkenyl, or the alkyl groups substituting the 7-position of the corresponding tubulin binding compounds. In another embodiment, the solubility enhancing moiety is covalently bonded to the 1 -position of the indazole of the corresponding tubulin binding compounds. Various solubility enhancing moieties are useful in the prodrugs of the present invention, including but not limited to, amino acid esters, sugar moieies, phosphates, phosphonates, ammonium groups, heterocyclic amines.
In another embodiment, the present invention provides a compound having a structure of formula:
Figure imgf000042_0001
wherein Qi is selected from
Figure imgf000042_0002
L1-W1, ethynyl, NH2,
C1-C6 alkylamino, and di(CrC6) alkylamino, wherein L1 is selected from C1-C6 alkylene and CrC6 heteroalkylene and W-i is selected from the group consisting of hydrogen, amino, C1-C6 alkylamino, di(Ci-Cβ) alkylamino, C1-C6 alkoxy, heterocyclyl, heteroaryl, and an aqueous solubility enhancing group and Q7 is selected from the group consisting of hydrogen, an aqueous solubility enhancing group, and a methyl group substituted with an aqueous solubility enhancing group with the proviso that if Q7 is hydrogen, then Q1 is H2 not V^= — c NH2, and if Q7 is hydrogen, then Wi is not hydrogen. In one embodiment, the aqueous solubility enhancing group is selected from the group consisting of an amino acid, a phosphate moiety, a sugar moiety, and an amino substituted benzoate moiety. In one embodiment, the aminoacid substituted benzoate moiety has structure of formula:
Figure imgf000043_0001
wherein L3 is a bond or a linker selected from alkylene or heteroalkylene and W3 sn amine or a heterocycle. In another embodiment, L3 is methylene. In another embodiment, W3 is piperazine or alkylpiperazine. Other suitable aqueous solubility enhancing groups are included in Table 1 E under the "Q7" column. In another embodiment, Q7 is hydrogen. In another embodiment, Qi is ΛΛΛ/^= CH3. Within this embodiment, the solubility enhancing group is selected from the group consisting of:
Figure imgf000043_0002
In another embodiment, Qi is NH2. In another embodiment, the present invention provides a compound having structure of formula:
Figure imgf000043_0003
wherein Qi is defined as in any embodiment above and W4 is an aqueous solubility enhancing group. In another embodiment, the present invention provides compounds having structures of formulas:
Figure imgf000044_0001
wherein Qi is Ci-C6 alkynyl, R2i is selected from the group consisting of CrC6
Figure imgf000044_0002
alkoxy, V OH , and CH2R22 wherein R22 is selected from:
HCI ΛΛ
\ 1 M* m
/ V / n and \ ' HCK OH
In another embodiment, the CrC6 alkynyl is 1 -alkynyl.
Examples of water soluble prodrugs of the present invention include, but are not limited to, Compounds 91 - 94, 104, 1 15, 117, 157, 170, 178, 182, 184, 185, 187 - 190, and 192 - 196.
Hypoxia Activated Prodrugs
The present invention also provides prodrugs of known and novel tubulin binding compounds of this invention. To understand the prodrug aspect of the invention, an understanding of tumor biology is helpful. Cancer cells generally divide more frequently than normal cells. Tubulin binding-drug mediated cancer therapies include cytotoxic agents selective for dividing cells.
For example, tubulin binding compounds target cancer cells, as opposed to normal cells, generally because cancer cells undergo cell division more frequently than normal cells.
However, drugs targeting dividing cells do not kill all of the cancer cells in the solid tumor. One reason for the lack of this complete killing is that cancer cells can acquire mutations that confer drug resistance. Another is that not all cancer cells divide more frequently than normal cells. These slowly-dividing cancer cells are generally located in the hypoxic region of the tumor and can be as, or even more, insensitive to such inhibitors as normal cells. The formation and consequences of the tumor hypoxic region is described below.
As a tumor grows, it requires a blood supply and, consequently, growth of new vasculature. The new vasculature that supports tumor growth is often disordered, leaving significant regions of the tumor under-vascularized and even the vascularized regions subject to intermittent blockage. Cells in these regions are unable to generate the energy required for cell division. These under-vascularized and blocked regions of the tumor become hypoxic - they have a lower oxygen concentration than the corresponding normal tissue. Thus, the median oxygen concentration of only ten percent of solid tumors falls in the normal range of 40-60 mm Hg, and fifty percent of solid tumors exhibit median oxygen concentrations of less than 10 mm Hg.
The hypoxic regions of the tumor can constitute a significant reservoir of cancer cells resistant to therapy. Generally, low tumor oxygen levels are associated with a poor response to therapy, increased metastases, and poor survival. In the hypoxic region of a tumor, cancer cells do not divide significantly faster than normal cells, and can be resistant to therapeutic agents such as tubulin binding compounds that target dividing cells.
However, the hypoxic region is conducive to biochemical reduction that can be used to generate reduced derivatives of a variety of chemical groups (see Workman et al., 1993, Cancer and Metast. Rev. 12: 73-82), and prodrugs of cytotoxins can be developed to exploit such hypoxic regions (see, Matteucci et al., PCT Publication No. WO 04/087075). Compounds of the present invention arise in part out of the discovery that, cancer cells in the hypoxic region can be targeted by prodrug compounds comprising a tubulin binding cytotoxin and a hypoxia labile protecting group. The hypoxic cells of the tumor generate the active toxin from the inactive, relatively non-toxic prodrug. The active drug diffuses from the hypoxic cells and kills the cancer cells in adjacent regions, including the more frequently dividing cells. The hypoxic region acts as a drug-factory to produce a cytotoxin within a tumor for killing adjacent normoxic cancer cells leading to a higher concentration of the cytotoxin within the tumor, relative to normal tissues. As a result, by employing a prodrug to generate the cytotoxin within the tumor, toxic side-effects arising due to normal cell toxicity can be reduced. After the cancer cells die in the normoxic region of the tumor, a hypoxic region can become normoxic and start dividing. At this point, such cells can be killed by the tubulin binding cytotoxins generated from the prodrug compounds of this invention, or by administering compounds of this invention in combination with other cytoxins, including for example, tubulin binding compounds and other anti-cancer cytotoxins.
The present invention also provides novel prodrugs of previously known tubulin binding anti-cancer compounds. In this aspect, the tubulin binding compound is bonded to the hypoxic activator (Hyp) through a hydroxyl oxygen (-OHyp) or an amine nitrogen (-NHyp) in the tubulin binding compound to yield a hypoxia actived prodrug. The hypoxic activator can be electron deficient nitrobenzene moieties, electron deficient nitrobenzoic acid amide moieties, nitroazole moieties, nitroimidazole moieties, nitrothiophene moieties, nitrothiazole moieties, nitrooxazole moieties, nitrofuran moieties, and nitropyrrole moieties. In one embodiment, the hypoxic activator is a substituted or unsubstituted nitroimidazole moiety.
In one embodiment, the hypoxic activator (Hyp) is selected from:
Figure imgf000046_0001
wherein each X2 is N or CR32;
X3 is NR3i, S, or O; each R30 is independently hydrogen or alkyl;
R31 is hydrogen, hydroxyl, C1-C6 alkyl or heteroalkyl, C3-Ce cycloalkyl, heterocyclyl, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 dialkylamino, aryl or heteroaryl, C1-C6 acyl or heteroacyl, aroyl, or heteroaroyl;
R32 is hydrogen, halogen, nitro, cyano, CO2H, C1-C6 alkyl or heteroalkyl, C1-C6 cycloalkyl, Ci-C6 alkoxy, C1-C6 alkylamino, C1-C6 dialkylamino, aryl, CON(Ry)2 , CrC6 acyl or heteroacyl, or aroyl or heteroaroyl; and n = 0, 1.
In an additional embodiment, Hyp is selected from
Figure imgf000047_0001
wherein X2, R3o, R31, R32 and n are as defined above. In another embodiment, Hyp is
Figure imgf000047_0002
wherein n = O or 1 , provided that in -OHyp n = O. For example, the tubulin binding compounds can be derivatized to yield prodrugs having the following structures
Figure imgf000047_0003
These derivatized compounds in general are less active or inactive compared to the parent compound yielding a hypoxia activated prodrug compound. In certain embodiments, the prodrug compounds demonstrate a 5-1000 fold loss of anticancer activity upon derivatization with respect to the starting compound. In general, such activity data can be obtained from structure activity relationship data as described in this disclosure and by using methods known to one of skill in the art.
In another aspect, the present invention provides prodrug compounds as defined above wherein the tubulin binding compound is bonded to the hypoxic activator (Hyp) through an hydroxyl oxygen (-OHyp) or an amine nitrogen (-NHyp) in the tubulin binding compound. The hypoxic activator can be electron deficient nitrobenzene moieties, electron deficient nitrobenzoic acid amide moieties, nitroazole moieties, nitroimidazole moieties, nitrothiophene moieties, nitrothiazole moieties, nitrooxazole moieties, nitrofuran moieties, and nitropyrrole moieties. In one embodiment, the hypoxic activator is a substituted or unsubstituted nitroimidazole moiety.
In one embodiment, the present invention provides compounds of the invention wherein X is -NN(Hyp)R wherein Hyp and R are defined as above. In another embodiment, the present invention provides a prodrug of the compound of formula (l-i):
Figure imgf000048_0001
(l-i) wherein one or more -NH-, enol form of a C=O, and/or -OH moiety or moieties
therein is converted to
Figure imgf000048_0002
or
Figure imgf000048_0003
In one embodiment, the present invention provides a compound of formulas (XXVIIIHXXXII):
Figure imgf000049_0001
(XXVIII)
Figure imgf000049_0002
Figure imgf000049_0003
wherein each Q1, Q2, and Q6 independently is hydrogen; halo; amino; Ci-C6 alkylamino; di Ci-C6 alkylamino; hydroxyl; CrC6 alkoxy; nitro; cyano; C1-C6 alkyl; C1-C6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; COR18; SO2R18; or PO3R18; each Q3-Q5 is C1-C6 alkyl; C1-C6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; or heteroaryl;
Q8 is hydrogen; halo; amino; C1-C6 alkylamino; di Ci-C6 alkylamino; hydroxyl; C1-C6 alkoxy; nitro; cyano; d-C6 alkyl; CrC6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; COR15; SO2R15 or PO3R15; each Qg independently is hydrogen; halo; amino; C1-C6 alkylamino; di C1-C6 alkylamino; hydroxyl; C1-C6 alkoxy; nitro; cyano; CrC6 alkyl; CrC6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi5; SO2R15 or PO3Ri5;
V is -NHNHR16; -HNR16; -N(Hyp)NHR16; -NHN(Hyp)R16; or
-N(Hyp)N(Hyp)R; wherein Hyp is a hypoxic activator as defined above;
X is O, -NNHR16, NR16, -NN(Hyp)R16, or NOR16wherein R16 is C1-C6 alkyl, aryl, C1-C6 alkylsulphonyl, arylsulfonyl, C1-C6 alkoxycarbony, aminocarbonyl, C1-C6 alkylaminocarbonyl, di C1-C6 alkylaminocarbonyl, C1-C6 acyl, aroyl, aminothiocarbonyl, C1-C6 alkylaminothiocarbonyl, di C1-C6 alkylaminothiocarbonyl, C1-C6 thioacyl, or thioaroyl; with the proviso that when X is NR16, R16 excludes hydrogen;
Y is hydrogen, hydroxyl, or halogen;
Z is -CH- or -N-;
Ri 5 is hydrogen, CrC6 alkoxy, amino, C1-C6 alkylamino, di C1-C6 alkylamino, NHOH, NHNH2, Ci-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2- C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl;
R16 is hydrogen, CrC6 alkyl, aryl, C1-C6 alkylsulphonyl, arylsulfonyl, C1- C6 alkoxycarbonyl, aminocarbonyl, C1-C6 alkylaminocarbonyl, di C1-C6 alkylaminocarbonyl, C1-C6 acyl, aroyl, aminothiocarbonyl, C1-C6 alkylaminothiocarbonyl, di C1-C6 alkylaminothiocarbonyl, C1-C6 thioacyl, or thioaroyl; with the proviso that when X is NRi6, Ri6 excludes hydrogen;
R-18 is hydrogen, hydroxyl, C1-C6 alkoxy, amino, CrC6 alkylamino, di C1-C6 alkylamino, NHOH, NHNH2, CrC6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
In one embodiment, the present invention provides the compound:
Figure imgf000050_0001
wherein Q3-Q5 are defined as is in formula (I)
In another embodiment, the present invention provides a compound having structure of formula:
Figure imgf000050_0002
wherein Qi is selected from
Figure imgf000051_0001
and L1-W1 , ethynyl,
NH2, C1-C6 alkylamino, di(C-ι-C6) alkylamino, and N F^Hyp wherein L-i is selected from Ci-C6 alkylene and C1-Ce heteroalkylene; W1 is selected from the group consisting of hydrogen, amino, C1-C6 alkylamino, di(CrC6) alkylamino, C1-C6 alkoxy, heterocyclyl, heteroaryl, an aqueous solubility enhancing group, and NR26Hyp and R26 is selected from C1-C6 alkyl or hydrogen and
Q7 is selected from the group consisting of hydrogen, Hyp, a solubility enhancing group, and a methyl group substituted with a solubility enhancing group or a Hyp-NR2o moiety wherein R2o is Ci-C6 alkyl group with the proviso that if Q7 is a solubility enhancing group or a methyl group substituted with a solubility enhancing group, then Q1 is N R26Hyp or W1 is NR26Hyp; and if W-I is selected from the group consisting of amino, CrC6 alkylamino, di(Ci- C6) alkylamino, C1-C6 alkoxy, heterocyclyl, heteroaryl, and an aqueous solubility enhancing group, then Q7 is Hyp or a methyl group substituted with a Hyp-NR2o moiety; and if Q1 is ethynyl, then Q7 is Hyp or a methyl group substituted with a Hyp-NR2o moiety. Suitable Hyp moieties useful in these embodiments are are included in Table 1 E under the "Q7" column. In one embodiment, Hyp is selected from the group consisting of
Figure imgf000051_0002
consisting of an amino acid, a phosphate moiety, a sugar moiety, and an amino substituted benzoate moiety. In another embodiment, Q-i is NR26Hyp. In another embodiment, R26 is hydrogen. Within this embodiment, Hyp is
Figure imgf000051_0003
In another embodiment, the present invention provides a compound having structure of formula:
Figure imgf000052_0001
R1: OH, NH2, OR, NR2 Q1 is C1-C6 alkynyl, Hyp is selected from the group consisting
Figure imgf000052_0002
R23 is OH, NH2, C1-C6 alkoxy, and C1-C6 alkylamino, and di(Ci-Cβ) alkylamino; and R24 is C1-C6 alkyl.
In another embodiment, the present invention provides a compound having a structure of formula:
Figure imgf000052_0003
wherein Q1 is selected from the group consisting of
, Br1 Cl1CN ^ ' <$? <øϊ ^ "OH OH
Figure imgf000052_0004
and Q7 is selected from the group consisting of
Figure imgf000053_0001
In another embodiment, the present invention provides compounds having stuctures of formulas:
Figure imgf000053_0002
wherein
Figure imgf000053_0003
and
Q1 = NH2, NO2, I, Br, CI1OH1CN ^ ' "OH OH
O
.OH NH7 "OH NH7
/^N
O7N
Figure imgf000053_0004
X= CH, Y=N X=N, Y=CH X=Y=CH
In another embodiment, the present invention provides compounds having structures of formulas:
Figure imgf000054_0001
wherein Qi is selected from the group consisting of: H, NH2, and a Ci-Cε alkyne. In one embodiment, the CrC6 alkyne is a 1-alkyne. In another embodiment, the1 -alkyne is selected from the group consisting of acetylene, propyne, cyclopropylacetylene, and methoxypropyne. In another embodiment, examples of these classes of compounds include, but are not limited to:
Figure imgf000054_0002
wherein R25 is C-i-Cβ alkyl. In another embodiment, the present invention provides a compound of formula (XIV):
Figure imgf000055_0001
(XIV) wherein;each Q3-Q5 is Ci-C6 alkyl; C1-C6 heteroalkyl; C2-C6 alkenyl; C2- C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; (-OHyp) or (-NHyp) with the proviso that in any one compound, at least one of Q3-Q5 is (-OHyp) or (-NHyp);
Q1, Q2, R13, R15 and Hyp are as defined above; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
In one embodiment, the present invention provides a compound of formula (XXXIV):
Figure imgf000055_0002
(XXXIV)
wherein Q1 is : -Ri3 or
Figure imgf000055_0003
Q2 is
Figure imgf000055_0004
C1-C6 alkoxy; halo; amino; or hydroxy; each Q3, Q4, and Q5 independently is CrC6 alkyl; C1-C6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; or heterocyclyl;
Figure imgf000055_0005
R3 is hydrogen, halo, C1-C6 alkyl, aryl or heteroaryl;
Ri3 is hydrogen; CrC6 alkyl, CrC6 heteroalkyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroalkyl each optionally substituted with hydroxyl, d- C6 alkoxy, amino, CrC6 alkylamino, di C1-C6 alkylamino; NHCOR15 Or COR-is R15 is hydrogen, hydroxyl, CrC6 alkoxy, amino, CrC6 alkylamino, di Ci-C6 alkylamino, NHOH, NHNH2, CrC6 alkyl, CrC6 heteroalkyl, CrC6 alkenyl, CrC6 alkynyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, aryl, or heteroaryl;
R18 is hydrogen, hydroxyl, Ci-C6 alkoxy, amino, CrC6 alkylamino, di CrC6 alkylamino, NHOH, NHNH2, d-C6 alkyl, Ci-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl;
Hyp is hypoxic activator; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
In one embodiment, the present invention provides a compound of formulas (XXXV)-(XXXIX):
Figure imgf000056_0001
(XXXVIII) (XXXIX) wherein
Figure imgf000056_0002
alkoxy; halo; amino; or hydroxy; each Q3-Q5 is C1-C6 alkyl; Ci-C6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C6 cycloalkyl; or heterocyclyl;
Q6 is hydrogen; halo; amino; CrC6 alkylamino; di CrC6 alkylamino; hydroxyl; CrC6 alkoxy; nitro; cyano; CrC6 alkyl; CrC6 heteroalkyl; CrC6 alkenyl; C1-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi8;
Figure imgf000057_0001
Q7 is hydrogen; amino; CrC6 alkylamino; di CrCs alkylamino; hydroxyl; CrC6 alkoxy; nitro; cyano; CrC6 alkyl; Ci-C6 heteroalkyl; CrC6 alkenyl; Ci-C6 alkynyl; C3-C8 cycloalkyl; heterocyclyl; aryl; heteroaryl; CORi5; SO2Ri8; or PO3Ri8; or a monosaccharide;
R5 is hydrogen, halo, or Ci-C6 alkoxy;
R6 is formyl or a protected form thereof;
R13 is hydrogen; CrC6 alkyl, d-C6 heteroalkyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroalkyl each optionally substituted with hydroxyl, d- C6 alkoxy, amino, CrC6 alkylamino, di CrC6 alkylamino, NHCORi5Or C0Ri5;
R15 is hydrogen, CrC6 alkoxy, amino, C1-C6 alkylamino, di C1-C6 alkylamino, NHOH, NHNH2, CrC6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2- C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; Ris is hydrogen, hydroxyl, CrC6 alkoxy, amino, CrC6 alkylamino, di
CrC6 alkylamino, NHOH, NHNH2, Ci-C6 alkyl, CrC6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl;
Hyp is hypoxic activator; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
In one embodiment, the present invention provides a compound of formulas (XL)-(XLIII)
Figure imgf000057_0002
(XL) (XLI) (XLII)
Figure imgf000058_0001
(XLIII) wherein each Q3-Q5 is C1-C6 alkyl; CrC6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-Cs cycloalkyl; or heterocyclyl;
Ri5 is hydrogen, hydroxyl, CrC6 alkoxy, amino, Ci-C6 alkylamino, di Ci-C6 alkylamino, NHOH, NHNH2, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, Cs-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl;
Hyp is hypoxic activator; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
In one embodiment, the present invention provides a compound of formula (XLIV):
Figure imgf000058_0002
(XLIV) each Q3-Q5 is Ci-C6 alkyl; C1-C6 heteroalkyl; C2-C6 alkenyl; C2-C6 alkynyl; C3-C8 cycloalkyl; or heterocyclyl;
Rg is CrC6 alkyl; aryl; or heteroaryl;
Ri5 is hydrogen, hydroxyl, CrC6 alkoxy, amino, C1-C6 alkylamino, di CrC6 alkylamino, NHOH, NHNH2, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-Cg cycloalkyl, heterocyclyl, aryl, or heteroaryl; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof. In one embodiment, the present invention provides a compound of formula (XLV):
Figure imgf000059_0001
(XLV) wherein each Q3-Qs is CrC6 alkyl; CrC6 heteroalkyl; C2-C6 alkenyl; C2-
C6 alkynyl; C3-C8 cycloalkyl; or heterocyclyl;
R15 is hydrogen, hydroxyl, Ci-C6 alkoxy, amino, Ci-C6 alkylamino, di Ci-C6 alkylamino, NHOH, NHNH2, C1-C6 alkyl, Ci-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl; Hyp is hypoxic activator; or a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof. In one embodiment, each Q3-Q5 is OMe.
In one embodiment, the present invention provides a compound of formula (I)-(XLVII), wherein Q1 is
-CH2OH , CH2NH2 , CO2H , CONH2
-PO3H2 -CH2PO3H2 , -CH3, C CH
Figure imgf000059_0002
-CH2OH -(CH2)2OH
N- '
-CH2-CH2-OH, -CH2-CH2-CH2-OH, -CONH2, -CO2H, -CN, or halo.
In one embodiment, the present invention provides a compound of formula (I)-(XLVII), wherein Q1 is
-CH2OH , -CO7H -CH; 3, :CH
-CH(Me)OH , -CH2-CH2OH -CH2OH -(CH2)2OH
In addition to compounds having formulas (I)-(XLVII) above, the present invention further includes all salts thereof, and particularly, pharmaceutically acceptable salts thereof. Still further, the invention includes compounds that are single isomers of the above formula (e.g., single enantiomers of compounds having a single chiral center), as well as solvate, hydrate, a prodrug and tautomeric forms thereof. In other embodiments isomers include single geometric isomers such as cis, trans, E and Z forms of compounds with geometric isomers, or single tautomers of compounds having two or more tautomers.
In one embodiment, examples of compounds of the present invention, including tubulin binding anti cancer compounds and their water soluble and hypoxia activated prodrugs include but are not limited to the following compounds: 82 - 124, 126 - 159, and 160 - 196 described in the EXAMPLES section and in Tables 1 E and 1 F, and a tautomer or an individual isomer or a racemic or non-racemic mixture of isomers, a hydrate, a prodrug or a pharmaceutically acceptable salt or solvate thereof.
For example and not as a limitation, in compounds 82-124, 143, 155, 157, 179, 190, 192, and 196, a hydroxyl oxygen or an amine nitrogen is covalently bonded to a hypoxic activator (Hyp) yielding compounds having (-OHyp) or (-NHyp) moieties.
Methods of Synthesis The compounds of the present invention can be synthesized following methods known to one of skill in the art and/or upon reading this disclosure. Methods for synthesizing aroylindazole compounds are described in PCT Pat. Pub. No. WO 06/057946 in page 71 , line 30 - page 74, line 14 and page 96, line 10 - page 116, line 9 and can be used for synthesizing the compounds of the present invention upon reading this disclosure (incorporated herein by reference). The Fedenok ef a/. Tetrahedron Lett., 2003, 44: 5453-5455, Yokoe et al., Heterocycles 1985, 23 (6): 1395-1398, Hachiken et al., J. Heterocyclic Chem., 1988, 25:327-331 , Makosza et al., Eur. J. Org. Chem., 2000, ?:193- 198, Nefedov et al., Russ. J. Org. Chem., 1994, 30(11): 1724-1728, Hlastav et a/. , 1998, Heterocycles, 48, 5: 1015-1022, Wu et al. , J. Fluorine Chem. , 2003, 122(2): 171-174, and Scholtz et al., Chem. Ber., 1913, 46: 1077 references describe method for the synthesis of various aroyl-heterocycles useful for other purposes. Novel compounds including prodrugs of this invention can be synthesized by adapting these aforementioned procedures and other procedures described in literature and/or known to one of skill in the art, upon reading this disclosure. The aroylindazole compounds of this invention can be synthesized by adapting known method to synthesize aroyl indazoles useful for other purposes according to the methods provided by this invention. Prodrug compounds of this invention can be synthesized using the novel compounds of the invention and known tubulin binding compounds as starting material as described herein. Known tubulin binding compounds and methods of their synthesis are described, for example, in the references, Martino et al., J. Med. Chem., 2004, ASAP articles; Mahboobi et al., J. Med. Chem. 2001 , 44, 4535-53; Gastper et al. J. Med. Chem., 1998, 49, 4965-72; Bacher et a/., Pure Appl. Chem, 2001 , 73(9): 1459-64; Lee et al., WO 98/39332; Combeau et al., WO 02/072575; Nam et al.; and Hsieh et al. (supra, each of which is incorporated herein by reference) and can be used in accordance with the present methods to synthesize prodrug compounds of the present invention.
In another aspect the present invention provides a method for synthesizing a compound of the present invention comprising the steps of
Figure imgf000061_0001
(i) reacting a compound having structure of formula:
Figure imgf000061_0002
wherein Q1 is Cl, Br, or i
with the 1-alkyne o — SiMe2(CMe3) and employing a catalyst selected from the group consisting of Cu(O), a Pd(II), and Pd(O) to yield the product.
The 1-N position of an aroylindzole compound of the present invention can be substituted with an aqueous solubility enhancing moiety or a Hyp containing moiety by reacting a 1-H indazole compound with an acyloxymethyl halide or acyloxymethyl tosylate wherein the acyl group contains a aqueous solubility enhancing moiety or a Hyp containing moiety. Suitable aqueous solubility enhancing moieties or Hyp containing moieties are included in Table 1 E under the "Q7 " column.
Certain other methods for making compound of the present invention are described below in Schemes 1-1 1 :
In another embodiment, a compound of formula:
Figure imgf000062_0001
is made as shown in the scheme 6 below. Scheme 6
Figure imgf000062_0002
wherein DIAD is diisopropyl azodicarboxylate and DEAD is diethyl azodicarboxylate.
In another embodiment, compounds of the present invention are synthesized as described in Schemes 7 and 8 below:
Scheme 7
Figure imgf000063_0001
Scheme 8
Figure imgf000063_0002
wherein Ar is
Figure imgf000063_0003
Scheme 9
Scheme 9 provides methods for synthesis of compounds having formula:
aminoacides amides, ester
Figure imgf000064_0001
employing the following synthetic steps:
Figure imgf000064_0002
OTBDMS
Figure imgf000064_0003
Figure imgf000064_0004
Scheme 10
Scheme 10 provides a method for synthesis of a compound of the invention containing an aziridine moiety as described below:
Figure imgf000065_0001
a. HNO3, H2SO4 b. MeOH, MeONa c. PdCI2(PPh3)2, CO, TEA d. SOCI2 e. 6- methoxyindole EtMgBr, ZnCI2, AICI3, f. H2, Pd/C g. 1 ,2-Dibromoethane, MeONa.
Scheme 11
Scheme 11 provides a method for synthesis of a compound of the invention containing an aldehyde moiety as described below:
Figure imgf000065_0002
Scheme 12
The schemes below describes the method of synthesizing aqueous soluble produgs of the present invention containing a 1-N-methyl group substituted with phosphate aqueous solubility enhancing groups.
Figure imgf000066_0001
1M NaOH
MeOH 45oC
Figure imgf000066_0002
compound 67 K2CO3
Figure imgf000066_0003
Scheme 13
The Scheme below describes the method of synthesizing an aqueous soluble prodrug of the present invention.
Figure imgf000067_0001
solubility enhancing group
Figure imgf000067_0002
Methods for synthesis of other compounds reported in J. Med. Chem., 2006, 49:947-54 and J. Med. Chem., 2004, 47:6120-3; PCT App. Pub. Nos. WO 06/057946 and WO 06/041961 ; and US Pat. No. 6,933,316 can be modified according to the present methods for synthesizing compounds and prodrugs of the present invention, each reference being incorporated herein in its entirety. Various prodrugs of the present invention can be synthesized employing methods well known in literature upon reading this disclosure and/or upon appropriate substitution of staring materials. In one embodiment, the water soluble prodrugs of the present invention are synthesized by reacting a 1-chloromethyl-7-(1-alkylnyl)-3-aroylindazole or a 1-hydroxymethyl- 7-(1-alkylnyl)-3-aroylindazole with a water soluble carboxylic acid moiety. Suitable water soluble carboxylic acid moieties include benzoic acids where the aryl ring is substituted with amine containing moieties. Illustrative methods for making anti-cancer compounds and prodrugs of the present invention in accordance with this disclosure are provided in the EXAMPLES section below.
Pharmaceutically Acceptable Formulation
In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a novel compound or a novel prodrug of a novel compound of the invention or a known compound; novel compounds and novel prodrugs of novel compounds of the invention or known compounds are described above. In one embodiment, the present invention provides a a novel compound or a novel prodrug of a novel compound of the invention or a known compound in substantially pure form. For use as a prophylactic or therapeutic agent, a compound of the present invention disclosed herein (including pharmaceutically acceptable salts, solvates, hydrates, and prodrugs) is usually formulated as a pharmaceutical composition comprising the compounds or the prodrugs of this invention and a pharmaceutically-acceptable carrier. The term "pharmaceutically acceptable carrier" is art-recognized and refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the subject composition and its components and not injurious to the patient.
Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. Pharmaceutical preparations for oral use can be obtained through combining active compounds with solid excipient and, optionally, other compounds. Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Further details on techniques for formulation can be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.); GOODMAN AND GILMAN'S: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS 10TH EDITION 2001 by Louis Sanford Goodman et al., McGraw-Hill Professional; PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS 7th Edition Howard C. Ansel, et a/., 2004, Lippincott Williams & Wilkins Publishers; PHARMACEUTICAL CALCULATIONS 1 1th Edition, 2001 , by Mitchell J. Stoklosa et al., Lippincott Williams & Wilkins;. PHYSICAL PHARMACY: PHYSICAL CHEMICAL PRINCIPLES IN THE PHARMACEUTICAL SCIENCES 4th Edition by Pilar Bustamante, et al., 1993, Lea & Febiger.
In one embodiment, the present invention provides a pharmaceutically acceptable formulation of the compounds of the invention, wherein the pharmaceutically acceptable carrier, dilutent, or excipient is selected from a polyethylene glycol (PEG). In one embodiment, the pharmaceutically acceptable formulation comprises a compound of formula (I)-(XLVII). In one embodiment, the pharmaceutically acceptable formulation comprises a compound of formula (I)-(VIII). In one embodiment, the pharmaceutically acceptable formulation comprises a compound of formula (I). Compound 30, for example, can be formulated with a PEG to yield a pharmaceutically acceptable formulation.
Functional Characterization
In one embodiment, the compounds and prodrugs suited for use in the invention are tubulin binding compounds when administered to a human, non- human primate, or other mammal. As is usual in the pharmaceutical arts, not every structural analog of a compound (e.g., a tubulin binding compound) is pharmacologically active. Active forms can be identified by routine screening of the compounds of the invention for the activity. A variety of assays and tests can be used to assess pharmacological activity of a compound or novel prodrug of the invention, including in vitro assays, such as those described below and elsewhere herein, in vivo assays in humans, non-human primates and other mammals, and/or clinical studies.
In some embodiments of the invention in which a tubulin binding compound is used for treatment or prevention of cancer or its manifestations, a tubulin binding compound with apoptosis-inducing activity similar to that of Combretastatin A-4 phosphate is selected. In some embodiments of the invention in which a tubulin binding compound is used for treatment or prevention of cancer or its manifestations, a tubulin binding compound with vascular disrupting activity similar to that of Combretastatin A-4 phosphate is selected. Thus, in some embodiments of the invention, a compound of the present invention that is cytotoxic to cancer cells such as H460, PC3, CCRF, LNCaP, HT29, HCT-15, ACHN, NCI-H69, H69 AR, HL-60, HL-60/MX2, MESSA and MES-SA/DX5 is administered to treat cancer.
In one aspect, the present invention provides compounds having a Gl50 ,Gl90, IC50, or IC50 of about 0.001 to about 1000 nM, about 0.01 to about 100 nm, about 0.1 to about 50 nM, and about 1 to about 10 nM in a cancer cell antiproliferation assay. In one embodiment, the present invention provides a compound of formula (I) having a GI50 or IC50 of about 0.01 to about 100 nm, about 0.1 to about 50 nm, and about 1 to about 10 nm in a cancer cell antiproliferation assay. In various embodiments, said antiproliferation assays employ cancer cell including but not limited to gastric, colon, breast, leukemia, renal, small cell lung, and non-small cell lung cancer. In various embodiments, the gastric cancer cell used is MESSA or doxorubicin resistant MESSA/DX5 cell; the colon cancer cell is HT29 cell and HCT-15; the breast cacner cell is T47D cell; the leukemic cell line used are HL-60 and HL- 60/MX2, the small cell lung cancer cell is NCI-H69 and the non-small cell lung cancer cell is H460 cell.
In one embodiment, the present invention provides a compound having a GI50 or IC50 of about 1 to about 50 nM in a cancer cell antiproliferation assay, such as, for example, compounds 87, 88, 93, 97, 100, and 118. In one embodiment, the present invention provides a tubulin binding compound having an IC50 of tubulin polymerization of about 0.1 to about 10 μM as determined in a tubulin polymerization inhibition assay, such as for example, compounds 30 and 39.
In one aspect, the present invention provides a compound which when subjected to a liver microsomal stability study, remains about 10 to about 100, about 20 to about 80, about 80 to about 100% unmetabolized. In one embodiment, the liver microsomal study is conducted for between 10-60, 20- 40, or 25-35 minutes. In one embodiment, mouse liver microsome is employed in the study. In one aspect, the present invention provides a compound which when subjected to a plasma stability study, remains about 10-100, 20-80, or 80-100 % unmetabolized. In one embodiment, the plasma stability study is conducted for between 10-60, 20-40, or 25-35 minutes. In one embodiment, the plasma employed is from the same species of mammal the liver of whch is employed in the liver microsomal stability study.
In one aspect, the present invention provides a compound which upon administration to a human cancer cell xenograft tumor bearing mice, can reduce the tumor volume to about 5-70% of a control tumor volume. In one embodiment, the human cancer cell used is H460 or HT 29 cell.
III. Therapies
In one aspect, the present invention provides a method of treating cancer and other hyperproliferative diseases comprising administering a therapeutically effective amount of a novel compound or a novel prodrug compound of the invention alone or in combination with one or more other anti-cancer agents to a subject in need of such treatment. The methods of the present invention can in general be used for treatment of any cancer. In various embodiments, 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 other embodiments, 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, small cell lung cancer, topical skin lesion, veticulum cell sarcoma, and Wilm's tumor. Dosage and Administration
A variety of routes, dosage schedules, and dosage forms are appropriate for administration of pharmaceutical compositions of the invention. Appropriate dosage schedules and modes of administration will be apparent to the ordinarily skilled practitioner upon reading the present disclosure and/or can be determined using routine pharmacological methods and/or methods described herein.
The dose, schedule and duration of administration of the compound and/or prodrug of the invention will depend on a variety of factors. The primary factor, of course, is the choice of a specific compound or prodrug of the present invention. Other important factors include the age, weight and health of the subject, the severity of symptoms, if any, the subject's medical history, co-treatments, goal (e.g., prophylaxis or prevention of relapse), preferred mode of administration of the drug, the formulation used, patient response to the drug, and the like.
For example, a compound and/or a prodrug of the invention can be administered at a dose in the range of about 0.1 mg to about 500 mg of a compound and/or prodrug of the invention per kg of body weight of the patient to be treated per day, optionally with more than one dosage unit being administered per day, and typically with the daily dose being administered on multiple consecutive days. In one embodiment, the compounds of the present invention include novel compounds of the invention, novel prodrug thereof, and novel prodrugs of known compounds. In one embodiment, a compound and/or a prodrug of the invention is administered in a daily dose in the range of about 0.5 mg to about 400 mg/Kg; about 1.0 mg to about 300 mg/Kg; about 1.5 mg to about 250 mg/Kg; about 2.0 mg to about 200 mg/Kg; about 2.5 mg to about 150 mg/Kg; about 5 to about 100 mg/Kg; about 10 to about 50 mg/Kg; and about 10 to about 70 mg per kg of body weight of the patient to be treated. Cell culture studies are frequently used in the art to optimize dosages, and the assays disclosed herein can be used in determining such doses.
For illustration, a therapeutically or prophylactically effective dose of a compound and/or a prodrug of the invention can be administered daily or once every other day or once a week to the patient. Controlled and sustained release formulations of the analogs can be used. Generally, multiple administrations of the compound and/or prodrug of the invention are employed. For optimum treatment benefit, the administration of the prophylactically effective dose can be continued for multiple days, such as for at least five consecutive days, and often for at least a week and often for several weeks or more. In one embodiment, the compound and/or prodrug of the invention is administered once (qday), twice (bid), three times (tid), or four times (qid) a day or once every other day (qod or q2d) or once a week (qweek or q7d), and treatment is continued for a period ranging from three days to two weeks or longer.
In one aspect, the present invention provides a method for treating cancer or other hyperproliferative diseases by administering to a patient in need of therapy thereof a therapeutically effective dose of a compound or prodrug compound of the invention. In one embodiment, the present invention provides a method for treating cancer or other hyperproliferative diseases by administering about 0.1 to about 500 mg/Kg of a compound or a prodrug compound of the invention to a patient in need of therapy thereof. In one embodiment, a compound and/or a prodrug of the invention is administered in a daily dose in the range of about 0.5 mg to about 400 mg/Kg; about 1.0 mg to about 300 mg/Kg; about 1.5 mg to about 250 mg/Kg; about 2.0 mg to about 200 mg/Kg; about 2.5 mg to about 150 mg/Kg; about 5 to about 100 mg/Kg; about 10 to about 50 mg/Kg; and about 10 to about 70 mg per kg of body weight of the patient to be treated. In one embodiment, the present invention provides a unit dosage form of about 1 to about 200 mg of a compound or prodrug compound of the invention to a patient in need of therapy thereof.
Additional guidance concerning administration of the compounds of the present invention may be obtained from such information known for other tubulin binding compounds. For example, Combretastatin A-4 phosphate (CA4P), a tubulin-binding compound, is reported to have a maximum tolerated daily dose of 60 - 68 mg/m2, and has, for example, been administered to patients in clinical trials in daily doses of 27 and 36 mg/m2, by a 10-minute infusion, once every 21 days (Young et a/., 2004 , Expert Opin. Investigat Drugs, 13(9): 1171 -82 and Bilenker ef a/., 2005, Clin. Cancer Res., 11 (4): 1527-33, each of which is incorporated herein by reference). The compounds of the present invention can be administered in similar daily doses for treatment of cancer. Therefore, in one embodiment, a compound of the present invention can be administered in a therapeutically affective daily dose of about 10 to about 100 mg/m2, about 20 to about 80 mg/m2, about 30 to about 70 mg/m2, about 40 to about 60 mg/m2, and about 45 to about 55 mg/m2 to treat cancer. A dose in mg/m2 can be converted to a mg/kg dose in adult humans by dividing the mg/m2 dose by a factor of 37; in children the corresponding dividing factor is 25. In one embodiment, a compound of the present invention can be administered in a therapeutically affective daily dose of about 0.3 to about 3 mg/kg, about 0.6 to about 2.4 mg/kg, about 0.9 to about 2.1 mg/kg, about 1.2 to about 1.8 mg/kg, and about 1.4 to about 1.6 mg/kg to treat cancer.
Guidance concerning administration can also be provided by and obtained from studies in humans and other mammalian animals. A therapeutically effective dose determined for an animal can be converted to the corresponding human equivalent dose (HED) as described in the table below:
Figure imgf000074_0001
a To convert animal dose in mg/kgto HED (assumes a 60 kg human) in mg/kg, divide animal dose by HED convertion factor. For species not listed or for weights outside the standard ranges, human equivalent dose (HED) can be calculated from the formula: HED = animal dose in mg/kg x (animal weight in kg/human weight in kg)0'33. b For example, cynomolgus, rhesus, or stumptail. Combination Therapies
Cancer therapy often involves administering of a drug "cocktail" in which several anti-cancer drugs are contemporaneously administered to a cancer patient. The novel compounds of the present invention and the prodrug compounds of the invention can be used in such therapies either in addition to or in substitution of one or more of the co-administered drugs. Also, because there may be cancer cells in a patient that are normoxic and located adjacent to a hypoxic region of a tumor, one can, in one embodiment of the invention, co-administering a prodrug of the invention with one or more other drugs that target normoxic cells.
In one embodiment, a compound and/or a prodrug compound of the invention can be co-administered in combination with other anti-cancer agents ("anticancer agent"). Without intending to be bound by any particular mechanism or effect, such co-administration can in some cases provide one or more of several advantages over known cancer therapies, such as, for example co-administration of a compound and/or a prodrug compound of the invention and the anticancer agent has a synergistic effect on induction of cancer cell death. Co-administration provides a better therapeutic result than administration of the anticancer agent alone, e.g., greater alleviation or amelioration of one or more symptoms of the cancer, diminishment of extent of disease, delay or slowing of disease progression, amelioration, palliation or stabilization of the disease state, partial or complete remission, prolonged survival or other beneficial therapeutic results. The co-administration of a compound and/or a prodrug compound compound of the invention increases the sensitivity of cancer cells to the anticancer agent, allowing lower doses of the anticancer agent to be administered to the patient or allowing an anticancer agent to be used for treatment of cells otherwise resistant to the anticancer agent or otherwise refractory to treatment. Generally anti-cancer agents target rapidly dividing cells in the normoxic region, the prodrug compounds of the invention target the hypoxic cells in the regions of tumors that are not efficiently killed by the anticancer agent alone. As used herein, a compound and/or a prodrug compound of the invention is "co-administered" with another anticancer agent (also referred to herein as, "Agent") wherein a compound and/or a prodrug compound of the invention and Agent are administered as part of the same course of therapy. In one embodiment, a compound and/or a prodrug compound of the invention is first administered prior to administration of the Agent, (i.e., the initiation of the other cancer therapy), and treatment with the compound and/or prodrug compound of the invention is continued throughout the course of administration of the Agent (i.e., the course of the other therapy). In another embodiment, a compound and/or a prodrug compound of the invention is administered after the initiation or completion of the other cancer therapy. In other embodiments, a compound and/or a prodrug compound of the invention is first administered contemporaneously with the initiation of the other cancer therapy. In one embodiment, a compound and/or a prodrug compound of the invention is first administered prior to administration of the Agent, and treatment with the compound and/or prodrug compound of the invention is continued after the cessation of administration of the Agent. In one embodiment, a compound and/or a prodrug compound of the invention is first administered prior to administration of the Agent, and treatment with the compound and/or prodrug compound of the invention is continued during part of the period of administration of the Agent. For certain drugs, such as certain topoisomerase inhibitors, administration of a compound and/or a prodrug compound of the invention can be initiated and completed prior to the administration of the second drug.
In the presence of oxygen, the radical anion formed upon the reduction of Hyp reacts with oxygen to yield superoxide and Hyp. Superoxide is a cytotoxin and the production of superoxide in normoxic tissues can lead to unwanted side effects. In one embodiment, the present invention provides a method wherein a compound and/or a prodrug compound of the invention administered in combination with a chemoprotective agent or a chemoprotectant. Chemoprotective agents protect healthy tissue from the toxic effects of anticancer drugs. In one embodiment, the chemoprotective agent is a thiol or a disulfide. In one embodiment, the chemoprotectant can reduce superoxide. In another embodiment, the chemoprotectant can react with the "Michael-receptor" generated from a hypoxia activated prodrug of the invention and prevent "Michael-receptor" from reacting with proteins and nucleic acid. Anticancer drug therapy today typically involves multiple rounds, or
"cycles," of administration of the anti-cancer agent(s). In the context of administering a compound and/or a prodrug compound of the invention, each cycle of administration (as well as a complete set of cycles) can be viewed as administration of a second drug. A compound and/or a prodrug compound of the invention can be administered in any or all of the multiple cycles of treatment with the other Agent; in general, the compound and/or prodrug compound of the invention is administered on a daily basis for at least two or more days during each cycle. In one aspect of the invention, a compound and/or a prodrug compound of the invention is co-administered with the Agent according to a schedule repeated at each round.
In one version of the method of treating cancer using the a compound and/or a prodrug compound of the invention, the compound and/or prodrug compound of the invention is administered in combination with an effective amount of one or more chemotherapeutic agents, an effective amount of radiotherapy, an appropriate surgery procedure, or any combination of such additional therapies.
When a compound and/or a prodrug compound of the invention is used in combination with one or more of the additional therapies, the compound and/or prodrug compound of the invention and additional therapy can be administered at the same time or can be administered separately. For example, if a compound and/or a prodrug compound of the invention is administered with an additional chemotherapeutic agent, the two agents can be administered simultaneously or can be administered sequentially with some time between administrations. One of skill in the art will understand methods of administering the agents simultaneously and sequentially and possible time periods between administrations.
The Agents can be administered as the same or different formulations and can be administered via the same or different routes. Chemotherapeutic agents that can be used in combination with the compound of the invention include, but are not limited to, busulfan, improsulfan, piposulfan, benzodepa, carboquone, 2-deoxy-D-glucose, lonidamine and analogs thereof (refrence apps), glufosfamide, meturedepa, uredepa, altretamine, imatinib, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamine, chlorambucil, chlomaphazine, estramustine, ifosfamide, gefitinib, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, nimustine, ranimustine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, aclacinomycins, actinomycin F(1 ), anthramycin, azaserine, bleomycin, cactinomycin, carubicin, carzinophilin, chromomycin, dactinomycin, daunorubicin, daunomycin, 6-diazo-5-oxo-1-norleucine, mycophenolic acid, nogalamycin, olivomycin, peplomycin, plicamycin, porfiromycin, puromycin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5- fluorouracil, tegafur, L-asparaginase, pulmozyme, aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, bestrabucil, bisantrene, carboplatin, defofamide, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucid, flutamide, gallium nitrate, hydroxyurea, interferon-alpha, interferon-beta, interferon-gamma, interleukin-2, lentinan, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, podophyllinic acid, 2-ethylhydrazide, procarbazine, razoxane, sizofiran, spirogermanium, paclitaxel, tamoxifen, erlotonib, teniposide, tenuazonic acid, triaziquone, 2,2',2"-trichlorotriethylamine, urethan, vinblastine, cyclophosphamide, and vincristine. Other chemotherapeutic agents that can be used include platinum derivatives, including but not limited to cis platinum, carboplatin, and oxoplatin.
In one version, a compound and/or a prodrug compound of the invention can be used in combination with an angiogenesis inhibitor including but not limited to Avastin and similar therapeutics. In one version of the combination treatment methods, a subject is treated with an angiogenisis inhibitor and subsequently treated with a compound and/or a prodrug compound of the invention. In one version of these combination methods of treatment using an angiogenesis inhibitor, the method is used to treat breast cancer.
In another embodiment, a compound and/or a prodrug compound of the invention is administered with an anti-cancer agent that acts, either directly or indirectly, to inhibit the epidermal growth factor or EGFR receptor. EGFR inhibitors suitable for coadministration with a compound of the invention include gefitinib and erlotonib.
In another version, a compound and/or a prodrug compound of the invention is administered with an anti-cancer agent that acts, either directly or indirectly, to inhibit hypoxia-inducible factor 1 alpha (HIFI a) or to inhibit a protein or enzyme, such as a glucose transporter or VEGF, whose expression or activity is increased upon increased HIFI a levels. HIFIa inhibitors suitable for use in this version of the methods and compositions described herein include P13 kinase inhibitors; LY294002; rapamycin; histone deacetylase inhibitors such as [(E)-(I S,4S,10S,21 R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2- oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22- pentanone (FR901228, depsipeptide); heat shock protein 90 (Hsp90) inhibitors such as geldanamycin, 17-allylamino-geldanamycin (17-AAG), and other geldanamycin analogs, and radicicol and radicicol derivatives such as KF58333; genistein; indanone; staurosporin; protein kinase-1 (MEK-1 ) inhibitors such as PD98059 (2'-amino-3'-methoxyflavone); PX-12 (1- methylpropyl 2-imidazolyl disulfide); pleurotin PX-478; quinoxaline 1 ,4- dioxides; sodium butyrate (NaB); sodium nitropurruside (SNP) and other NO donors; microtubule inhibitors such as novobiocin, panzem (2- methoxyestradiol or 2-ME2), vincristines, taxanes, epothilones, discodermolide, and derivatives of any of the foregoing; coumarins; barbituric and thiobarbituric acid analogs; camptothecins; and YC-1 , a compound described in Biochem. Pharmacol., 15 Apr 2001 , 67(8):947-954, incorporated herein by reference, and its derivatives.
In another version, a compound and/or a prodrug compound of the invention is administered with an anti-angiogenic agent, including but not limited to anti-angiogenic agents selected from the group consisting of angiostatin, an agent that inhibits or otherwise antagonizes the action of VEGF, batimastat, captopril, cartilage derived inhibitor, genistein, endostatin, interleukin, lavendustin A, medroxypregesterone acetate, recombinant human platelet factor 4, Taxol, tecogalan, thalidomide, thrombospondin, TNP-470, and Avastin. Other useful angiogenesis inhibitors for purposes of the combination therapies provided by the present methods and compositions described herein include Cox-2 inhibitors like celecoxib (Celebrex), diclofenac (Voltaren), etodolac (Lodine), fenoprofen (Nalfon), indomethacin (Indocin), ketoprofen (Orudis, Oruvail), ketoralac (Toradol), oxaprozin (Daypro), nabumetone (Relafen), sulindac (Clinoril), tolmetin (Tolectin), rofecoxib (Vioxx), ibuprofen (Advil), naproxen (Aleve, Naprosyn), aspirin, and acetaminophen (Tylenol).
In addition, because pyruvic acid plays an important role in angiogenesis, pyruvate mimics and glycolytic inhibitors like halopyruvates, including bromopyruvate, can be used in combination with an anti-angiogenic compound and a compound and/or a prodrug compound of the invention to treat cancer. In another version, a compound and/or a prodrug compound of the invention is administered with an anti-angiogenic agent and another anti- cancer agent, including but not limited to a cytotoxic agent selected from the group consisting of alkylators, Cisplatin, Carboplatin, and inhibitors of microtubule assembly, to treat cancer.
In addition to the combination of a compound and/or a prodrug compound of the invention with the Agents described above, the present methods and compositions described herein provides a variety of synergistic combinations of the compound and/or prodrug compound of the invention and other anti-cancer drugs. Those of skill in the art can readily determine the anticancer drugs that act "synergistically" with a compound and/or a prodrug compound of the invention as described herein. For example, the reference Vendetti, "Relevance of Transplantable Animal-Tumor Systems to the
Selection of New Agents for Clinical Trial," Pharmacological Basis of Cancer Chemotherapy, Williams and Wilkins, Baltimore, 1975, and Simpson Herren et al., 1985, "Evaluation of In Vivo Tumor Models for Predicting Clinical Activity for Anticancer Drugs," Proc. Am. Assoc. Cancer Res. 26: 330, each of which is incorporated herein by reference, describe methods to aid in the determination of whether two drugs act synergistically.
While synergy is not required for therapeutic benefit in accordance with the methods of described herein, in one embodiment, the present invention provides a method of cancer treatment, wherein there is synergy between a compound and/or a prodrug compound of the invention and another anticancer agent. Two drugs can be said to possess therapeutic synergy if a combination dose regimen of the two drugs produces a significantly better tumor cell kill than the sum of the single Agents at optimal or maximum tolerated doses. The "degree of synergy" can be defined as net log of tumor cell kill by the optimum combination regimen minus net log of tumor cell kill by the optimal dose of the most active single Agent. Differences in cell kill of greater than ten-fold (one log) are considered conclusively indicative of therapeutic synergy. When a compound and/or a prodrug compound of the invention is used with another anti-cancer agent, the compound and/or prodrug compound of the invention will, at least in some versions, be administered prior to the initiation of therapy with the other drug or drugs and administration will typically be continued throughout the course of treatment with the other drug or drugs. In some versions, the drug co-administered with a compound and/or a prodrug compound of the invention will be delivered at a lower dose, and optionally for longer periods, than would be the case in the absence of administering the compound and/or prodrug of the invention. Such "low dose" therapies can involve, for example, administering an anti-cancer drug, including but not limited to paclitaxel, docetaxel, doxorubicin, cisplatin, or carboplatin, at a lower than approved dose and for a longer period of time together with a compound and/or a prodrug compound of the invention administered in accordance with the methods described herein.
These methods can be used to improve patient outcomes over currently practiced therapies by more effectively killing cancer cells or stopping cancer cell growth as well as diminishing unwanted side effects of the other therapy. In other versions, the other anti-cancer agent or agents will be administered at the same dose levels used when a compound and/or a prodrug compound of the invention is not co-administered. When employed in combination with a compound and/or a prodrug compound of the invention, the additional anti-cancer agent(s) is dosed using either the standard dosages employed for those Agents when used without the compound and/or prodrug compound of the invention or are less than those standard dosages. The administration of a compound and/or a prodrug compound of the invention in accordance with the methods described herein can therefore allow the physician to treat cancer with existing (or later approved) drugs at lower doses (than currently used), thus ameliorating some or all of the toxic side effects of such drugs. The exact dosage for a given patient varies from patient to patient, depending on a number of factors including the drug combination employed, the particular disease being treated, and the condition and prior history of the patient, but can be determined using only the skill of the ordinarily skilled artisan in view of the teachings herein.
Specific dose regimens for known and approved chemotherapeutic agents or antineoplastic agents (i.e., the recommended effective dose) are known to physicians and are given, for example, in the product descriptions found in the Physician's Desk Reference 2003, (Physicians' Desk Reference, 57th Ed) Medical Economics Company, Inc., Oradell, NJ and/or are available from the Federal Drug Administration. Illustrative dosage regimens for certain anti-cancer drugs are also provided below.
Cancer drugs can be classified generally as alkylators, anthracyclines, antibiotics, aromatase inhibitors, bisphosphonates, cyclo- oxygenase inhibitors, estrogen receptor modulators, folate antagonists, inorganic aresenates, microtubule inhibitors, modifiers, nitrosoureas, nucleoside analogs, osteoclast inhibitors, platinum containing compounds, retinoids, topoisomerase 1 inhibitors, topoisomerase 2 inhibitors, and tyrosine kinase inhibitors. In accordance with the methods described herein, a compound and/or a prodrug compound of the invention can be coadministered with any anti-cancer drug from any of these classes or can be administered prior to or after treatment with any such drug or combination of such drugs. In addition, a compound and/or a prodrug compound of the invention can be administered in combination with a biologic therapy (e.g., treatment with interferons, interleukins, colony stimulating factors and monoclonal antibodies). Biologies used for treatment of cancer are known in the art and include, for example, trastuzumab (Herceptin), tositumomab and 131I Tositumomab (Bexxar), rituximab (Rituxan).
Alkylators useful in the practice of the methods described herein include but are not limited to busulfan (Myleran, Busulfex), chlorambucil (Leukeran), ifosfamide (with or without MESNA), cyclophosphamide (Cytoxan, Neosar), glufosfamide, melphalan, L-PAM (Alkeran), dacarbazine (DTIC- Dome), and temozolamide (Temodar). In accordance with the methods described herein a compound and/or a prodrug compound of the invention is co-administered with an alkylator to treat cancer. In one version, the cancer is chronic myelogenous leukemia, multiple myeloma, or anaplastic astrocytoma.
In one embodiment, the present invention provides a method of treating cancer treatable by administering a compound and/or a prodrug compound of the invention alone or in combination with at least another alkylator or a prodrug thereof. Alkylators, such as, for example, cyclophosphamide, ifosfamide, glufosfamide, mechlorethamine, melphalan, chlorambucil, dacarbazine, temozolomide, carmustine, streptozocin, bendamustin, busulfan, thiotepa, cisplatin, carboplatin, and oxaliplatin, and types of cancers treated using any one of such alkylators alone or in combination with other anti cancer or chemoprotective agents are described for example in the reference Hardman et al., (see Hardman et al., The
Pharmacological Basis of Therapeutics, 2001 , 1389-1399, McGraw-Hill, New York, USA).
In one embodiment, the present invention provides a method of treating cancer by administering a compound and/or a prodrug compound of the invention with a cancer treatment regimen using at least the alkylator Glufosfamide. Glufosfamide is in the clinic for the treatment of pancreatic cancer or Gemzar resistant pancreatic cancer. Glufosfamide can be used for treating breast cancer, Morbus Hodgkin, gastrointestinal tract cancer, or as part of the GCE (Glufosfamide, Carboplatin, and Etoposide) or RGCE (Rituxan and GCE) regimen, for treating lymphomas. (Tidmarsh et al., PCT Pat. Pub. Nos. WO 06/071955, WO 06/122227, and WO 07/035961 , each of which is incorporated herein by reference). Additional examples of Agents include Terciva, Iressa, Cytarabine and Erbitux. In one embodiment, the present invention provides a method of treating cancer by administering a compound and/or a prodrug compound of the invention with a cancer treatment regimen using at least a platinum coordination complex alkylator. In one embodiment, the platinum coordination complex alkylator is Cisplatin. Cisplatin can be used to treat cancer of bladder, head and neck, endometrium, small cell carcinoma of the lung, and some neoplasms of childhood. Cisplatin alone or with cyclophosphamide is used to treat advanced ovarian cancer. Combination chemotherapy of Cisplatin with Bleomycin, Etoposide, and Vinblastine is used to treat advanced testicular cancer; and with one of Paclitaxel, Cyclophosphamide, or Doxorubicin to treat ovarian carcinoma.
Anthracyclines useful in the practice of the methods described herein include but are not limited to, doxorubicin (Adriamycin, Doxil, Rubex), mitoxantrone (Novantrone), idarubicin (Idamycin), valrubicin (Valstar), and epirubicin (Ellence). In accordance with the methods described herein a compound and/or a prodrug compound of the invention is co-administered with an anthracycline to treat cancer. In one version, the cancer is acute nonlymphocytic leukemia, Kaposi's sarcoma, prostate cancer, bladder cancer, metastatic carcinoma of the ovary, and breast cancer. As one example the compound (8S,10S)-10-[(3-Amino-2,3,6-trideoxy- alpha.-L-lyxo-hexopyranosyl)oxy]-8-glycoloyl-7,8,9,10-tetrahydro-6,8,1 1- trihydroxy-1-methoxy-5,12-naphthacenedione, more commonly known as doxorubicin, is a cytotoxic anthracycline antibiotic isolated from cultures of Streptomyces peucetius var. caesius. Doxorubicin has been used successfully to produce regression in disseminated neoplastic conditions such as acute lymphoblastic leukemia, acute myeloblasts leukemia, Wilm's tumor, neuroblastoma, soft tissue and bone sarcomas, breast carcinoma, ovarian carcinoma, transitional cell bladder carcinoma, thyroid carcinoma, lymphomas of both Hodgkin and non-Hodgkin types, bronchogenic carcinoma, and gastric carcinoma. Doxorubicin is typically administered in a dose in the range of 30- 75 mg/m2 as a single intravenous injection administered at 21 -day intervals; weekly intravenous injection at doses of 20 mg/m2; or 30 mg/m2 doses on each of three successive days repeated every four weeks. In accordance with the methods of the methods described herein, a compound and/or a prodrug compound of the invention is co-administered starting prior to and continuing after the administration of doxorubicin at such doses (or at lower doses). Cyclic Anthracycline cytotoxin prodrugs useful in the practice of the methods described herein are provided by the reference Matteuci et al., PCT Patent App. Pub. No. WO 07/060534.
Antibiotics useful in the practice of the methods described herein include but are not limited to dactinomycin, actinomycin D (Cosmegen), bleomycin (Blenoxane), daunorubicin, and daunomycin (Cerubidine, DanuoXome). In accordance with the methods described herein a compound and/or a prodrug compound of the invention is co-administered with an antibiotic to treat cancer. In one version, the cancer is a cancer selected from the group consisting of acute lymphocytic leukemia, other leukemias, and Kaposi's sarcoma.
Aromatase inhibitors useful in the practice of the methods described herein include but are not limited to anastrozole (Arimidex) and letroazole (Femara). In accordance with the methods described herein a compound and/or a prodrug compound of the invention is co-administered with an aromatase inhibitor to treat cancer. In one version, the cancer is breast cancer. Bisphosphonate inhibitors useful in the practice of the methods described herein include but are not limited to zoledronate (Zometa). In accordance with the methods described herein a compound and/or a prodrug compound of the invention is co-administered with a biphosphonate inhibitor to treat cancer. In one version, the cancer is a cancer selected from the group consisting of multiple myeloma, bone metastases from solid tumors, or prostate cancer.
Cyclo-oxygenase inhibitors useful in the practice of the methods described herein include but are not limited to celecoxib (Celebrex). In accordance with the methods described herein a compound and/or a prodrug compound of the invention is co-administered with a cyclo-oxygenase inhibitor to treat cancer. In one version, the cancer is colon cancer or a pre-cancerous condition known as familial adenomatous polyposis.
Estrogen receptor modulators useful in the practice of the methods described herein include but are not limited to tamoxifen (Nolvadex) and fulvestrant (Faslodex). In accordance with the methods described herein a compound and/or a prodrug compound of the invention is co-administered with an estrogen receptor modulator to treat cancer. In one version, the cancer is breast cancer or the treatment is administered to prevent the occurrence or reoccurrence of breast cancer.
Folate antagonists useful in the practice of the methods described herein include but are not limited to methotrexate and tremetrexate. In accordance with the methods described herein a compound and/or a prodrug compound of the invention is co-administered with a folate antagonist to treat cancer. In one version, the cancer is osteosarcoma.
As one example, the compound N-[4-[[(2,4-diamino-6- pteridinyl)methyl methylamino]benzoyl]-L-glutamic acid, commonly known as methotrexate, is an antifolate drug that has been used in the treatment of gestational choriocarcinoma and in the treatment of patients with chorioadenoma destruens and hydatiform mole. It is also useful in the treatment of advanced stages of malignant lymphoma and in the treatment of advanced cases of mycosis fungoides. Methotrexate is administered as follows. For choriocarcinoma, intramuscular injections of doses of 15 to 30 mg are administered daily for a five-day course, such courses repeated as needed with rest period of one or more weeks interposed between courses of therapy. For leukemias, twice weekly intramuscular injections are administered in doses of 30 mg/m2. For mycosis fungoides, weekly intramuscular injections of doses of 50 mg or, alternatively, of 25 mg are administered twice weekly. In accordance with the methods described herein, a compound and/or a prodrug compound of the invention is co-administered with methotrexate administered at such doses (or at lower doses). 5-Methyl-6- [[(3,4,5-trimethoxyphenyI)-amino]methyl]-2,4-quinazolinediamine (commonly known as trimetrexate) is another antifolate drug that can be co-administered with a compound and/or a prodrug compound of the invention. Inorganic arsenates useful in the practice of the methods described herein include but are not limited to arsenic trioxide (Trisenox). In accordance with the methods described herein a compound and/or a prodrug compound of the invention is co-administered with an inorganic arsenate to treat cancer. In one version, the cancer is refractory acute promyelocytic leukemia (APL). Microtubule inhibitors (as used herein, a "microtubule inhibitor" is any agent that interferes with the assembly or disassembly of microtubules) useful in the practice of the methods described herein include but are not limited to vincristine (Oncovin), vinblastine (Velban), paclitaxel (Taxol, Paxene), vinorelbine (Navelbine), docetaxel (Taxotere), epothilone B or D or a derivative of either, and discodermolide or its derivatives. In accordance with the methods described herein a compound and/or prodrug of the invention is co-administered with a microtubule inhibitor to treat cancer. In one version, the cancer is ovarian cancer, breast cancer, non-small cell lung cancer, Kaposi's sarcoma, and metastatic cancer of breast or ovary origin. As one example, the compound 22-oxo-vincaleukoblastine, also commonly known as vincristine, is an alkaloid obtained from the common periwinkle plant (Vinca rosea, Linn.) and is useful in the treatment of acute leukemia. It has also been shown to be useful in combination with other oncolytic agents in the treatment of Hodgkin's disease, lymphosarcoma, reticulum-cell sarcoma, rhabdomyosarcoma, neuroblastoma, and Wilm's tumor. Vincristine is administered in weekly intravenous doses of 2 mg/m2 for children and 1.4 mg/m2 for adults. In accordance with the methods described herein, a compound and/or prodrug compound of the invention is co-administered with vincristine administered at such doses. In one version, a compound and/or prodrug compound of the invention is not administered prior to treatment with a microtubule inhibitor, such as a taxane, but rather, administration of a compound and/or prodrug compound of the invention is administered simultaneously with or within a few days to a week after initiation of treatment with a microtubule inhibitor.
Modifiers useful in the practice of the methods described herein include but are not limited to Leucovorin (Wellcovorin), which is used with other drugs such as 5-fluorouracil to treat colorectal cancer. In accordance with the methods described herein a compound and/or prodrug compound of the invention is co-administered with a modifier and another anti-cancer agent to treat cancer. In one version, the cancer is colon cancer. In one version, the modifier is a compound that increases the ability of a cell to take up glucose, including but not limited to the compound N-hydroxyurea. N- hydroxyurea has been reported to enhance the ability of a cell to take up 2- deoxyglucose (see the reference Smith et al., 1999, Cancer Letters 141: 85, incorporated herein by reference), and administration of N-hydroxyurea at levels reported to increase 2-deoxyglucose uptake or to treat leukemia together with administration of 2-deoxyglucose and a compound of the invention is one version of the therapeutic methods provided herein. In another such version, a compound and/or prodrug compound of the invention is co-administered with nitric oxide or a nitric oxide precursor, such as an organic nitrite or a spermineNONOate, to treat cancer, as the latter compounds stimulate the uptake of glucose. Nitrosoureas useful in the practice of the methods described herein include but are not limited to procarbazine (Matulane), lomustine, CCNU (CeeBU), carmustine (BCNU, BiCNU, Gliadel Wafer), and estramustine (Emcyt). In accordance with the methods described herein a compound and/or prodrug compound and/or prodrug compound of the invention is co- administered with a nitrosourea to treat cancer. In one version, the cancer is prostate cancer or glioblastoma, including recurrent glioblastoma multiforme. Nucleoside analogs useful in the practice of the methods described herein include but are not limited to mercaptopurine, 6-MP (Purinethol), fluorouracil, 5-FU (Adrucil), thioguanine, 6-TG (Thioguanine), hydroxyurea (Hydrea), cytarabine (Cytosar-U, DepoCyt), floxuridine (FUDR), fludarabine (Fludara), azacytidine (Vidaza), pentostatin (Nipent), cladribine (Leustatin, 2- CdA), gemcitabine (Gemzar), and capecitabine (Xeloda). In accordance with the methods described herein a compound and/or prodrug compound of the invention is co-administered with a nucleoside analog to treat cancer. In one version, the cancer is B-cell lymphocytic leukemia (CLL), hairy cell leukemia, adenocarcinoma of the pancreas, metastatic breast cancer, non-small cell lung cancer, or metastatic colorectal carcinoma. As one example, the compound 5-fluoro-2,4(1 H,3H)-pyrimidinedione, also commonly known as 5- fluorouracil, is an antimetabolite nucleoside analog effective in the palliative management of carcinoma of the colon, rectum, breast, stomach, and pancreas in patients who are considered incurable by surgical or other means. 5-Fluorouracil is administered in initial therapy in doses of 12 mg/m2 given intravenously once daily for 4 successive days with the daily dose not exceeding 800 mg. If no toxicity is observed at any time during the course of the therapy, 6 mg/kg are given intravenously on the 6th, 8th, 10th, and 12th days. No therapy is given on the 5th, 7th, 9th, or 11th days. In poor risk patients or those who are not in an adequate nutritional state, a daily dose of 6 mg/kg is administered for three days, with the daily dose not exceeding 400 mg. If no toxicity is observed at any time during the treatment, 3 mg/kg can be given on the 5th, 7th, and 9th days. No therapy is given on the 4th, 6th, or 8th days. A sequence of injections on either schedule constitutes a course of therapy. In accordance with the methods described herein, a compound and/or prodrug compound of the invention is co-administered with 5-FU administered at such doses or with the prodrug form Xeloda with correspondingly adjusted doses. As another example, the compound 2- amino-1 ,7-dihydro-6H-purine-6-thione, also commonly known as 6- thioguanine, is a nucleoside analog effective in the therapy of acute non- pymphocytic leukemias. 6-Thioguanine is orally administered in doses of about 2 mg/kg of body weight per day. The total daily dose can be given at one time. If after four weeks of dosage at this level there is no improvement, the dosage can be cautiously increased to 3 mg/kg/day. In accordance with the methods described herein, a compound and/or prodrug compound of the invention is co-administered with 6-TG administered at such doses (or at lower doses).
Osteoclast inhibitors useful in the practice of the methods described herein include but are not limited to pamidronate (Aredia). In accordance with the methods described herein a compound and/or prodrug compound of the invention is co-administered with an osteoclast inhibitor to treat cancer. In one version, the cancer is osteolytic bone metastases of breast cancer, and one or more additional anti-cancer agents are also co-administered with a compound and/or prodrug compound of the invention.
Platinum compounds useful in the practice of the methods described herein include but are not limited to cisplatin (Platinol) and carboplatin (Paraplatin). In accordance with the methods described herein a compound and/or prodrug compound of the invention is co-administered with a platinum compound to treat cancer. In one version, the cancer is metastatic testicular cancer, metastatic ovarian cancer, ovarian carcinoma, and transitional cell bladder cancer. As one example, the compound cis-Diaminedichloroplatinum (II), commonly known as cisplatin, is useful in the palliative treatment of metastatic testicular and ovarian tumors, and for the treatment of transitional cell bladder cancer which is not amenable to surgery or radiotherapy. Cisplatin, when used for advanced bladder cancer, is administered in intravenous injections of doses of 50-70 mg/m2 once every three to four weeks. In accordance with the methods described herein, a compound and/or prodrug compound of the invention is co-administered with cisplatin administered at these doses (or at lower doses). One or more additional anticancer agents can be co-administered with the platinum compound and a compound and/or prodrug compound of the invention. As one example, Platinol, Blenoxane, and Velbam can be co-administered with a compound and/or a prodrug compound of the invention. As another example, Platinol and Adriamycin can be co-administered with a compound and/or a prodrug compound of the invention. Retinoids useful in the practice of the methods described herein include but are not limited to tretinoin, ATRA (Vesanoid), alitretinoin (Panretin), and bexarotene (Targretin). In accordance with the methods described herein a compound and/or a prodrug compound of the invention is co-administered with a retinoid to treat cancer. In one version, the cancer is a cancer selected from the group consisting of APL, Kaposi's sarcoma, and T- cell lymphoma.
Topoisomerase 1 inhibitors useful in the practice of the methods described herein include but are not limited to topotecan (Hycamtin) and irinotecan (Camptostar). In accordance with the methods described herein a compound and/or a prodrug compound of the invention is co-administered with a topoisomerase 1 inhibitor to treat cancer. Topoisomerase inhibitors and prodrugs thereof useful in the practice of the methods of the present invention are provided in the reference Matteucci et al., PCT Pat. App. Pub. No. WO 06/065448, incorporated herein by reference. In one version, the cancer is metastatic carcinoma of the ovary, colon, or rectum, or small cell lung cancer. As noted above, however, in one version of the methods described herein, administration of a compound and/or a prodrug compound of the invention either precedes or follows, or both, administration of a topoisomerase 1 inhibitor but is not administered concurrently therewith. Topoisomerase 2 inhibitors useful in the practice of the methods described herein include but are not limited to etoposide, VP-16 (Vepesid), teniposide, VM-26 (Vumon), and etoposide phosphate (Etopophos). In accordance with the methods described herein a compound and/or prodrug compound of the invention is co-administered with a topoisomerase 2 inhibitor to treat cancer. In one version, the cancer is a cancer selected from the group consisting of refractory testicular tumors, refractory acute lymphoblastic leukemia (ALL), and small cell lung cancer. As noted above, however, in one version of the methods described herein, administration of a compound and/or a prodrug of the invention either precedes or follows, or both, administration of a topoisomerase 2 inhibitor but is not administered concurrently therewith.
Tyrosine kinase inhibitors useful in the practice of the methods described herein include but are not limited to imatinib (Gleevec). In accordance with the methods described herein a compound and/or a prodrug compound of the invention is co-administered with a tyrosine kinase inhibitor to treat cancer. In one version, the cancer is CML or a metastatic or unresectable malignant gastrointestinal stromal tumor.
Lonidamine analogs useful in the practice of the present invention are provides in the reference PCT Pat. Appl. Nos. PCT/US2005/026929 and PCT/US2005/027092 and PCT/US2005/024434, each of which is incorporated herein by reference.
Thus, described herein are methods of treating cancer in which a compound and/or a prodrug compound of the invention or a pharmaceutically acceptable salt thereof and one or more additional anti-cancer agents are administered to a patient. Specific versions of such other anti-cancer agents include without limitation 5-methyl-6-[[(3,4,5-trimethoxyphenyl)amino]-methyl]- 2,4-quinazolinediamine or a pharmaceutically acceptable salt thereof, (8S,10S)-10-(3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl )oxy]-8- glycoloyl-Z.δ.θ.iO-tetrahydro-e.δ.H-trihydroxy-i-methoxy-S,^- naphthacenedione or a pharmaceutically acceptable salt thereof; 5-fluoro- 2,4(1 H,3H)-pyrimidinedione or a pharmaceutically acceptable salt thereof; 2- amino-1 ,7-dihydro-6H-purine-6-thione or a pharmaceutically acceptable salt thereof; 22-oxo-vincaleukoblastine or a pharmaceutically acceptable salt thereof; 2-bis[(2-chloroethyl)amino]tetrahydro-2H-1 ,3,2-oxazaphosphorine, 2- oxide, or a pharmaceutically acceptable salt thereof; N-[4-[[(2,4-diamino-6- pteridinyl)methyl]-methylamino]benzoyl]-L-glutamic acid, or a pharmaceutically acceptable salt thereof; or cisdiamminedichloro-platinum (II).
Treatment of Non-Cancer Hyperproliferative Diseases
In another aspect, the present invention provides a method of treatment of non-cancer hyperproliferative diseases characterized by cellular hyperproliferation (e.g., an abnormally increased rate or amount of cellular proliferation). In one embodiment, the hyperproliferative disease treated according to the present method 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. In one embodiment, the hyperproliferative disease is selected from the group consisting of angiofibroma, atherosclerosis, benign prostatic hyperplasia, corneal graft rejection, gout, graft versus host disease, glaucoma, inflammatory diseases such as inflammatory bowel disease, ischemic heart and peripheral vascular disease, Karposi's sarcoma, keloids, life threatening infantile hemangiomas, macular degeration, myocardial angiogenesis, myocardial infraction, multiple sclerosis, neovascular-based dermatological conditions, Osier-Webber Syndrome, osteoarthritis, psoriasis, psoriatic arthritis, pulmonary fibrosis, psoriasis, rheumatoid arthritis, restenosis, rheumatoid arthritis, scleroderma, telangectasia, and wound granularization.
In some embodiments of the invention in which a tubulin binding compound is used for treatment or prevention of a hyperproliferative disease or its manifestations, a tubulin binding compound with similar apoptosis- inducing activity similar to that of Combretastatin A-4 phosphate is selected. Thus, in some embodiments of the invention, a tubulin binding compound that induces apoptosis in skin, epithelial or endothelial, nerve, and T cells, is administered to treat a hyperproliferative disease, e.g. psoriasis, rheumatoid arthritis, restenosis, benign prostatic hyperplasia, and multiple sclerosis. In one embodiment, the hyperpriliferative 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 diseases treated is rheumatoid arthritis, a multisystem chronic, relapsing, inflammatory disease that can lead to destruction and ankyiosis of joints affected. In another embodiment, the compounds of the present invention are administered to prevent a hyperproliferative disease resulting from cellular proliferation on a prosthesis implanted in a subject by coating the prosthesis with a composition containing a 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. The invention, having been described in summary and in detail, is illustrated but not limited by the Example below, which describe the synthesis of compounds of the present invention and demonstrates the usefulness of the compounds of the present invention in treating cancer.
IV. EXAMPLES Compounds 9, 22, 30, 35, 68, 67, and 72, used as starting material in the following synthetic methods were synthesized as decribed in page 96, line 10 - page 116, line 9 of PCT Pat. Pub. No. WO 06/057946, incorporated herein by reference.
Example 1 Example 1 provides methods of synthesizing Compounds 82-86
Figure imgf000094_0001
86 R1=H
Compound 82: To a solution containing compound J (69 mg, 1.3 eq.
1.2-1.4 eq.) and compound 30 (120 mg, 1 eq.) in 10 ml_ anhydrous acetone K2CO3 (30 mg) was added. The mixture was refluxed for 6 hours and filtered.
The filtrate was concentrated under reduced pressure. The residue was separated employing flash chromatography on silica gel (Hex: AcOEt =100:10
~ 0:100(v/v)) to yield compound 82. Compounds 83-86 were synthesized following this procedure as specified below. Compound 83: Compound J (60 mg, 1.4 eq.), compound 35 (100 mg, 1 eq.), and K2CO3 (30 mg) were reacted and the crude product was separated by flash column chromatography (Hex: AcOEt =100:20 ~100:95(v/v)) to yield
60 mg of compound 83.
Compound 84: Compound J (16 mg, 1.3 eq.), compound 68 (27 mg, 1 eq.), and K2CO3 (10 mg) were reacted and the crude product was separated by flash column chromatography (Hex: AcOEt =100: 0 ~100:60(v/v)) to yield
12 mg of compound 84. Compounds 85 and 86: Compound J (13 mg, 1.2 eq.), compound 72 (20 mg, 1 eq.), and K2CO3 (10 mg) were reacted and the crude product was separated by flash column chromatography (Hex: AcOEt =1 :1 (v/v)) to yield a mixture of compounds 85 and 86.
Example 2 Example 2 provides a method of synthesizing Compounds 87-90
Figure imgf000095_0001
89 n = 1
90 n = 2
Compound 87: DBU (2 eq.) was added to a solution containing compound K (2 eq.) and compounds 30 (1 eq.) in anhydrous THF (3 ml_) under a nitrogen atmosphere at rt followed by the addition of DMAP (3 mg). The mixture was stirred overnight at rt. The solvent was removed under reduced pressure. The residue was separated by flash chromatography on silica gel (Hex: AcOEt =1 :1 (v/v)) to yield compounds 87. Using compound 35 as the starting material, compound 88 was synthesized according to this procedure.
Compound 89: To a suspension of compound 30 (1 eq.) and compound K (1.5 eq.) in anhydrous THF (20 ml_) at rt under N2 a solution of tetrazole (0.45 M, 4.5 eq.) in CH3CN was added. The mixture was stirred at rt overnight and then cooled to -780C. To this cooled reaction mixture solid MCPBA (2.5 eq.) was added, and the resulting mixture was stirred at -780C for 1 hour and at rt for another hour. The solvent was removed under reduced pressure and the residue was separated by flash chromatography on silica gel (Hex: AcOEt =1 :1 (v/v)) to yield compounds 89. Using compound 35 as the starting material, compound 90 was synthesized according to this procedure.
Example 3 Example 3 provides a method of synthesizing Compounds 93, 94, and 157
Figure imgf000096_0001
93 R2 = H
Figure imgf000096_0002
Compound 93: To a solution of compounds 30 (200 mg, 1 eq.) and compound M (136 mg, 1.7 eq.) in anhydrous THF (10 ml_) at O0C, under N2, TEA (240 μl_, 3.4 eq.) and DMAP (3 mg) were added. The mixture was stirred and the temperature was raised from O0C to rt in 6 h. After removal of the solvent under vacuum, the residue was separated by flash chromatography on silica gel (DCM: MeOH:TEA =100:10:1 (v/v)) to yield compound 91. To a suspension of compounds 91 (1 eq.) in anhydrous DCM (10 ml_) at O0C a solution of HCI (4 M, 3 eq.) in dioxane was added. The mixture was stirred from O0C to rt for 30 min. After removal of solvent under vacuum, the residue was washed with anhydrous ether twice (10 mLx2) to yield 160 mg of compound 93. Using compound 82 as the starting material compounds 92 and 94 were synthesized using this procedure.
Compound 157: To a solution of compound 83 (1.6 g) in THF (120 ml_) and DCM (80 ml_) was added compound M (1.4 g) at O0C, followed by the dropwise addition of triethylamine (2.5 ml_), and DMAP (5 mg). The reaction mixture was stirred for 16 h and the temperature raised from O0C to room temperature. Volatiles were removed in vacuo and the residue was separated employing flash chromatography on silica gel (DCM to DCM: MeOH: TEA = 100:10:2 (v/v)) to yield a solid that was washed twice with ether to yield the free base corresponding to compound 157 (1.65 g). To a solution of the free base corresponding to compound 157 (900 mg) in DCM (10 ml_) was added dropwise a solution of HCI in dioxane (2 ml_) at O0C, the temperature raised to room temperature, and the reaction mixture stirred for 1 h. Afterwards, volatiles were removed and the residue washed with ether twice to yield compound 157 (750 mg).
Example 4 Example 4 provides a method of synthesizing Compounds 97-99
Figure imgf000098_0001
PdCI2(PPh3)2/Cul/TEA
Figure imgf000098_0002
Compound 97: A mixture of compound 22 (1.87 g, 1eq.), PdCI2(PPh3)2 (140 mg 0.05 eq.) and CuI (38 mg 0.05 eq.) in Et3N (200 ml_) was thrice degassed and exchanged with Ar followed by addition of TBDMS protected propargyl alcohol (2.4 ml_ 3 eq.) at room temperature and stirred at 550C overnight and filtered. The filtrate was concentrated under reduced pressure and the residue was separated employing flash chromatography on silica gel using as eluent 100:25 Hexanes/EtOAc to yield 1.4 g of compound 95. DBU (360 μl_ 3 eq.) was added to a solution containing compound K (500 mg 3 eq.) and compounds 95 (408 mg 1 eq.) in dry THF (15 ml_) under N2 at -780C followed by the addition of 5 mg of DMAP. The mixture was stirred from - 780C to rt for 5 hours. The solvent was removed under reduced pressure. The residue was separated by flash chromatography on silica gel (Hex: AcOEt =100:40(v/v)) to yield compound 96. To a solution of compound 96 (100 mg) in CH3CN (3 ml_) at O0C 0.1 mL of 48% HF was added. After the mixture was stirred from O0C to rt for 5 hours, the reaction mixture was diluted with water (10 ml_) and extracted with DCM (10 ml_ x 3). The organic phase was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. Chromatography of the residue on silica gel (Hex:AcOEt = 100:70(V/V)) afforded 40 mg of compound 97.
Compound 99: Under N2, DIAD (39 //L 2 eq.) was added to a solution containing compound 95 (51 mg 1 eq.), compound N (32 mg 2 eq.) and PPh3 (52 mg 2 eq.) in anhydrous THF (5 ml_) at rt. The mixture was heated at 5O0C overnight. After removal of solvent under vacuum, the residue was separated by flash chromatography on silica gel (Hex:AcOEt=100:35(V/V)) to yield compound 98. Deprotection of the TBDMS group following the method described for compound 23 starting from compound 98 (25 mg) and 48% HF (50 μl_) afforded compound 99.
Example 5 Example 5 provides a method of synthesizing Compounds 100 - 102
Figure imgf000100_0001
Compound 100: To a solution containing compound O (17 mg, 1.1 eq.) and compouds 30 (30 mg, 1 eq.) in dry acetone (8 ml_) K2CO3 (20 mg) was added. The mixture was refluxed (4 hrs) and filtered. The filtrate was concentrated under reduced pressure. The residue was separated employing flash chromatography on silica gel (Hex:AcOEt =100:60(V/V)) to yield compound 100.
Compound 101 : DIAD (39 μL, 2 eq.) was added under N2 to a solution containing compound 30 (40 mg, 1 eq.), 2-nitroimdazole (23 mg, 2 eq.) and PPh3 (52 mg, 2 eq.) in anhydrous THF (5 ml_) at rt. The mixture was stirred at rt for 6 hours. After removal of solvent under vacuum, the residue was separated by flash chromatography on silica gel (Hex:AcOEt =100:50(V/V)) to yield compound 101.
Compound 102: A solution of compound 30 (10 mg) in 95% formic acid (5 ml_ ) was heated under reflux overnight. After removal of solvent under vacuum, the residue was separated by flash chromatography on silica gel (Hex:AcOEt =100:50(V/V)) to yield compound 102.
Example 6 Example 6 provides a method of synthesizing Compounds 103-104
Figure imgf000101_0001
A mixture of compound 22 (468 mg 1eq.), PdCI2(PPh3)2 (35 mg 0.05 eq.) and CuI (10 mg 0.05eq.) in Et3N (40 ml_) was thrice degassed and exchanged with Ar followed by addition of compound P (282 mg 2 eq.) at room temperature (rt) and stirred at 540C overnight and filtered. The filtrate was concentrated under reduced pressure and the residue was separated employing flash chromatography on silica gel using as eluent 100:45 Hexanes/EtOAc to yield 60 mg of compound 103. To a solution containing compound 103 (55 mg) in MeOH (5 mL) was added 25% NaOMe (50 //L) in MeOH at rt. The mixture was stirred at rt for 1 hour. After removal of solvent under vacuum, the residue was separated by flash chromatography on silica gel (Hex:AcOEt =100:60(V/V)) to yield 37 mg of compound 104.
Example 7 Example 7 provides a method of synthesizing Compound 30
Figure imgf000102_0001
To a solution containing compound 95 (1.4 g ) in 95% THF (50 mL) of 1 M tetrabutylammonium fluoride solution (8ml_) was added at O0C. The mixture was stirred at room temperature overnight. After the solvent was removed under reduced pressure, DCM (20 mL) was added. The organic phase was washed with water and dried over Na2SU4 and concentrated under reduced pressure. The residue was washed with MeOH to give compound 30 (0.7 g) without further purification in 44% overall yield from compound 22.
Example 8 Example 8 provides a method of synthesizing Compound 107
Figure imgf000103_0001
Following the method employed to synthesize compound 103,
Compound 22 (117 mg 1 eq.) and compound Q (93 mg 2 eq.) were reacted to yield 100 mg of compound 106. N2H4-H2O (0.2 ml_) was added to a solution of compound 106 (100 mg) in 13 ml_ mixture solvents (10 ml_ MeOH, 3 mL THF) at rt. The mixture was refluxed for 3 hours. After removal of solvent under vacuum, the residue was separated by flash chromatography on silica gel (DCM:MeOH:TEA =100:10:1(VΛ/)) to yield compound 53. To a suspension of compounds 53 (17 mg 1 eq.) in 2 ml_ of anhydrous DCM at O0C was added a 4 M solution of HCI (3 eq.) in dioxane. The mixture was stirred from O0C to rt for 30 min. After removal of solvent under vacuum, the residue was washed with anhydrous ether twice (2 ml_x2) to yield compound 107. Example 9 Example 9 provides a method of synthesizing Compound 108
Figure imgf000104_0001
Under N2, a solution of POCI3 (73 μl_ 2.eq.) in 2 ml_ of DMF was added dropwise to a solution of compound 9 (137 mg, 1 eq.) in 5 ml_ of DMF at rt. The mixture was stirred at rt for 4 hours. After removal of solvent under vacuum, the residue was separated by flash chromatography on silica gel (Hex:AcOEt =100:25(V/V)) to yield compound 108.
Example 10
Example 10 provides a method of synthesizing Compound 109
Figure imgf000104_0002
To a solution containing compound O (80 mg 1.3 eq.) and compound 95 (153 mg 1 eq.) in 6 ml_ of dry acetone was added K2CO3 (150 mg). The mixture was refluxed for 1 hr and filtered. The filtrate was concentrated under reduced pressure. The residue was separated employing flash chromatography on silica gel (Hex:AcOEt =100:50(V/V)) to yield 80 mg of compound 109. Example 11 Example 11 provides a method of synthesizing Compound 110
Figure imgf000105_0001
Under N2, DIAD (39 μL 2 eq.) was added to a solution containing compound 35 (41 mg 1 eq.), 2-nitroimdazole (23 mg 2 eq.) and PPh3 (52 mg 2 eq.) in 5 ml_ anhydrous THF at rt. The mixture was stirred at rt for 3 hours. After removal of solvent under vacuum, the residue was separated by flash chromatography on silica gel (Toluene:Acetone =100:60(V/V)) to yield compound 110.
Example 12 Example 12 provides a method of synthesizing Compounds 111 and 112
Figure imgf000105_0002
Acetic anhydride (9 μl_ 1.7 eq.) was added under N2,to a solution containing compound 107 (10 mg 1 eq.) in 2 ml_ anhydrous THF at rt following addition of pyridine (50//L). The mixture was stirred at rt overnight. After removal of solvent under vacuum, the residue was separated by flash chromatography on silica gel (DCM:MeOH =100:20(V/V)) to yield compound 111 and 112. Example 13 Example 13 provides a method of synthesizing Compound 115:
Figure imgf000106_0001
To a solution of compound 95 (prepared with same method as described in this patent, 100 mg, 0,196 mmol) in DMF (3 ml) NaH (12 mg, 0.294 mmol, 60% in oil) was added at O 0C, compound R, 121 mg, 0.294 mmol) and stirred for 5 min. The reaction was warmed up to rt slowly (10 minute) and stirred for 10 minutes, until a TLC indicated the completion of the reaction. The reaction mixture was poured into brine and extracted with ethyl acetate. The organic layer was washed with brine, dried and concentrated, and the residue was purified by a silica gel column (EA in Hex 0-70%) to give product 113. To a solution of compound 1 13 (120 mg, 0.143 mmol) in anhydrous MeOH (5 ml) was added at rt NaOMe (0.008 ml, 0.0143 mmol, 3.7 M in MeOH). After 10 minutes, the reaction mixture was poured into brine and extracted with dichloromethaπe. The organic layer was dried, concentrated and co-evaperated with toluene to yield compound 114 as a residue which was dissolved in THF (5 ml) followed by the addition of TBAF (0.142 ml). After 10 minutes, solvent was removed and the resulting residue was purified with flash silica gel chromatography (MeOH in DCM 0 to 15%) to give final compound 1 15. Example 14 Example 14 provides a method of synthesizing Compound 117.
Figure imgf000107_0001
116 117
Compound S was synthesized by glycosilation of compound R with propargyl alcohol. To a solution of compound 22 (145 mg, 0.31 mmol) in DMF (1 ml) and TEA (4 ml) was added compound S (120 mg, 0.31 mmol). The solution was degased three times, and PdCI2(PPh3)2 (11 mg, 0.016 mmol) and CuI (0.003 mg, 0.016mmol) were added at rt under argon. The reaction mixture was heated at 50 0C for two hours, diluted with ethyl acetate and washed with brine. The organic layer was separated, dried with MgSO4 and concentrated to yield a residue which was purified with silica gel column with ethyl acetate in hexane 0 to 65% to give final product 116. To a solution of compound 116 (58 mg) in anhydrous MeOH (3 ml) NaOMe (0.01 ml, 25% in MeOH) was added at rt. After 10 minutes, the reaction was concentrated and the resulting residue was purified by flash silica gel chromatography (MeOH in DCM, 0 to 30%) to give final compound 117. Example 15
Example 15 provides a method of synthesizing Compounds 118 and 119.
Figure imgf000108_0001
Compound 30 (50 mg) was dissolved in anhydrous pyridine (1 ml) and acetic anhydride (0.5 ml) at rt. The resulting solution was stirred overnight and concentrated. The residue was purified by silica gel flash chromatography with solvents ethyl acetate in hexane from 0 to 80% to yield product 118. To a solution of compound 30 (70 mg, 0.177 mmol) in DMF NaH (21 mg, 0.531 mmol, 60% in oil) was added at rt, followed by addition of succinic anhydride (53 mg, 0.531 mmol). After two hours, the reaction was poured into brine and extracted with ethyl acetate twice. The combined organic layers were dried and concentrated. The residue was purified by silica gel flash chromatography with solvents acetone in toluene from 0 to 70% to yield 119.
Example 16 Example 16 provides a method of synthesizing Compound 121.
Figure imgf000109_0001
Figure imgf000109_0002
120 121
To a solution of CA 4 (250 mg, 0.79 mmol) in DCM (0.5 ml) and pyridine (1 ml) a solution Of Tf2O (0.15 ml, 0.87 mmol) in DMC (0.5 ml) was added at rt. After two hours, TLC indicated the completion of the reaction. The reaction mixture was diluted with ethyl acetate and washed with 5% of HCI and brine. The organic layer was dried and concentrated. The residue was purified by silica gel flash chromatography with solvents ethyl acetate in hexane from 0 to 80% to give compound T. To a solution of compound T (140 mg, 0.31 mmol) in DMF (2 ml) and TEA (1 ml) was added trimethylsilylacetylene (61 mg, 0.62 mmol). The mixture was degased three times, and PdCI2(PPh3)2 (11 mg, 0.016 mmol) and CuI (0.003 mg, 0.016mmol) were added at rt under argon. The reaction solution was heated at 90 0C for two hours. The reaction mixture was diluted with ethyl acetate and washed with brine. The organic layer was dried with MgSO4 and concentrated. The residue was purified by silica gel column with ethyl acetate in hexane 0 to 80% to yiled compound 120. Compoung 120 was dissolved in THF (5 ml) and TBAF (0.05 ml) was added. After 10 minutes, solvent was removed and the resulting residue was purified by flash silica gel chromatography with ethyl acetate in hexane from 0 to 80% to yield 121. Example 17 Example 17 provides a method of synthesizing Compounds 123 and
124
Figure imgf000110_0001
123 124 To a solution of reagent U (5 g, 27.5 mmol) in DCM and Py (30 ml, 1 :2) was added Tf2θ slowly ay O 0C. After stirring at rt for one hour, the reaction was diluted with EA and washed with 10% of HCI and then with Brine. Organic layer was dried and concentrated. The residue was passed through a silica gel column with EA in Hex from O to 65% to give product V. A solution of compound V (800 mg, 2.55 mmol) and W (677 mg, 3.82 mmol) in DMF (8 ml) and TEA (1.5 ml) was degassed for three times and PdCI2(PPh3)2 (90 mg, 0.128 mmol) was added at rt under argon and the reaction solution was heated at 90 0C for two hours. Diluted with EA and washed with brine. Organic layer was dried and concentrated. The residue was purified with silica gel column with EA in Hex 0-60% to produce X. To a solution of compound X (140 mg, 0.41 mmol) in THF (10 ml) was added LAH (0.34 ml, 0.81 mmol, 2.4 M in THF) at - 78 0C. The reaction was done in one hour. Solvent was removed and the resulting residue was purified with a silica gel column with EA in Hex from 0 to 100% to give product Y. A mixture of compound Y (60 mg) and Zn (50 mg) in AcOH (10 ml) was stirred at rt for two hours. The reaction was concentrated and the residue was passed through a silica gel column with EA in Hex from 10 to 100% to give product Z. To a suspension of compound Z (37 mg, 0.108 mmol) in acetone/water (15 ml, 2:1 ) was added HCI (0.5 ml, 10%) at - 10 0C with vigorously stirring. After 10 min, a solution of NaNO2 (12 mg, 0.162 mmol) in water (0.25 ml) was added and the reaction was stirred at - 10 0C for one hour. Diluted with 200 ml of water and warmed up to rt. Extracted with EA 4x50 ml. Combined organic layers was washed with satd. NaHCC>3, dried, and concentrated. The residue was purified with flash silica gel chromatography with EA in hex 20 to 100% to give 123. To a solution of compound 123 (16 mg, 0.047 mmol) in THF (5 ml) was added SOCI2 (0.007 ml, 0.094 mmol) at rt. The reaction solution was heated at 60 0C for 1 h, cooled to rt and quenched with MeOH. Solvent was removed and the resulting residue was purified with a silica gel column with EA in Hex from 20 to 100% to give product 124.
Example 18
Example 18 provides a method for synthesizing an aqueous solubility enhancing prodrug, compound 187, of tubulin binding compound 67.
Figure imgf000112_0001
Compound AA
Compound AA ClCH2OSO2Cl
Figure imgf000112_0002
Compound AB
67 Compound AB-
Figure imgf000112_0003
187
To a solution of 3-chloromethylbenzoic acid (2.3 g) in THF (2 ml_) was added TEA (3.74 ml_) and N-methylpiperazine (3 ml_) at room temperature and stirred for 72 h. The reaction mixture was filtered, the residue washed with THF, and the filtrate was concentrated to yield a residue that was washed with EtOAc to yield compound AA (1.9 g). To a mixture of compound AA (1 g), NaHCO3 (1.42 g), tetrabutylammoniumbisulfate (145 mg), water (16 ml_), and DCM (16 ml_) was added a solution of CICH2OSO2CI (0.71 g) in DCM (4 ml_) at O0C. The reaction mixture was stirred at that temperature for 1 h, extracted with DCM, the DCM portion dried, adsorbed into silica gel and separated on flash column choromatography employing (EtOAc/Hex = 40- 80%(v/v)) to yield an aqueous solubility enhancing component, compound AB (530 mg). A mixture of tubulin binding compound 67 (271 mg), compound AB (242 mg), and K2CO3 (296 mg) in DMF (4 ml_) was stirred at room temperature for 16 h. The reaction mixture was diluted with EtOAc, washed with waster, and brine, and the organic portion separated. The organic portion was dried and concentrated to yield a residue that was separated employing flash chromatography on silica gel (DCM to 10% DCM: MeOH (Wv)) to yield the free base of compound 187 (335 mg). The hydrochloride salt of compound 187 was synthesized by reacting the free base form of compound 187 (334 mg) with 0.5 M HCI/Dioxane (1.17 mL) in DCM (1 ml_) at O0C. The reaction mixture was diluted with ether and the hydrochloride salt of compound 844 isolated by filtration (309 mg). Certain other prodrugs of the present invention that have enhanced aqueous solubility compared to the corresponding tubulin binding compound can be synthesized employing this method upon appropriate substitution of the aqueous solubility enhancing component and the tubulin binding compound. Example 19
Example 19 describes the synthesis of a 7-amino aroylindazole compound, 159, of the present invention and its Hyp derivative, 155. The staring compound AC was synthesized according to a method described in PCT Pat. Pub. No. WO 04/043932, incorporated herein by reference.
Figure imgf000114_0001
Figure imgf000114_0002
Figure imgf000114_0003
156 159
To a suspension of compound AC (3.8 g) in acetic anhydride (22 ml_) and H2SO4 (19.2 ml_) stirred at room temperature (rt) was added dropwise acetyl nitrate (prepared from 1.0 ml_ 90% HN03 and 3.2 ml_ acetic anhydride). The reaction mixture was stirred at rt for 16 h, water (60 ml_) added to it and stirred for another 1 h. The solid was filtered and separated employing flash chromatography on silica gel (20% - 80% EtOAc: Hexane (v/v)) to yield compound AD as yellow needles. To a solution of compound AD (2.25 g) in ethanol (285 ml_) and water
(45 ml_) at 750C was added iron (6.0 g) followed by the dropwise addition of concentrated HCI (0.94). The mixture was stirred at 850C for 15 min, filtered, and the filtrate concentrated to yield compound AD as a dark brown solid (2.23 g). To a solution of compound AE (1.9 g, 10.3 mmol) in dioxane (28 ml_) and water (14 ml_) was added TEA (4.31 ml_) and BoC2O ( 4.5 g). A precipitate formed immediately and the reaction mixture was stirred for 3 h. The precipitate was filtered, washed with ethyl acetate to yield AF as a yellow solid (2.4 g). To a suspension of compound AF in anhydrous THF (8 ml_) at O0C was added a solution of Grignard reagent AG (1 M, 32 ml_). The mixture was stirred for 1 h at O0C1 quenched with 30% acetic acid (40 ml_) and stirred for 2 h more. Volatiles were removed in vacuo and the residue extracted with EtOAc. The EtOAc portion was washed with water, NaHCO3, and brine, dried, and concentrated to yield a residue that was separated employing flash chromatography on silica gel (Hexane - 30% EtOAc: Hexane (v/v)) to yield compound AE (907 mg) containing an impurity as identified by TLC; compound AH was used for the next step without further purification. Compound AH (907 mg) was added to a solution of trifluoroacetic acid
(28 ml_) and water (1.5 ml_). The mixture was stirred at O0C for 45 min, concentrated, and the residue separated employing flash chromatography on silica gel (EtOAc: Hexane (v/v)) to yield amine compound 159 (480 mg).
To a solution of 20% phosgene/toluene (9 ml_) was added dropwise at 450C a mixture of compound 159 (290 mg) in THF (3 ml_). The mixture was warmed to 6O0C, stirred at 60oC for 20 min, concentrated to yield a residue that was co-evaporated with toluene to yield compound AJ which was used without further purification.
To a solution of compound AJ was and 1-N-methyl-2-nitroimidazole-5- methanol (191 mg) in THF (6 ml_) was added N-methylimidazole (184 μl_) and a catalytic amount of DMAP. The mixture was stirred at rt for 2 h, concentrated, and the residue separated employing flash chromatography on silica gel (DCM - 5 % MeOH/DCM (v/v)) to yield compound 155 as a yellow solid (290 mg).
Example 20
Demonstration of In Vitro Antiproliferation Effect of the Compounds of the present Invention
To determine the effect of the compounds of the present invention on cell proliferation, the antiproliferative activity of these compounds was tested in a multi-well Alamar Blue based assay (at 2 h and 3 days). Cell growth in the presence and absence of the test compound as tabulated in Table 1 was compared, as measured by a fluorescence plate reader at excitation 550 nm and emission 590 nm (see Biosource International Inc., Tech Application Notes, Use of Alamar Blue in the measurement of Cell Viability and Toxicity, Determining IC50). H460 cells (ATCC HTB-177 (NCI-H40), 4,000 cells/well/200 //l) and LNCap cells (ATCC CRL-1740, 6,000 cells/well/200 μ\) were seeded in a 96 well plate in RPMI medium (Invitrogen Corporation, Carlsbad, CA). After 24 hours, these plates were divided into 3 groups - Control group, 2h treatment group and 3 day treatment group.
A test compound was added to each plate in the treatment groups (2h and 3 day) at a concentration as tabulated in Table 1 (in 50 μl of medium). In the 2h treatment group, after 2h the cells were rinsed to remove the test compound and incubated for 3 days, followed by staining with AlamarBlue. The cells in the 3-day treatment group were incubated for 3 days, followed by staining with AlamarBlue. In the Control group, AlamarBlue was added to the plate at (i) day 0 and (ii) day 3 and measured to establish the control reading. In all the groups, the capacity of the cells to proliferate was measured 6 hours after addition of AlamarBlue by a fluorescence plate reader at excitation 550 nm and emission 590 nm. Methods for testing compounds in an antiproliferation assay under hypoxic conditions, in nitrogen, are described in US Pat. App. Nos. PCT App. Pub. Nos. WO 04/087075, WO 05/086952, and WO 07/002931 and can be used, with suitable modifications where necessary, for testing HAP compounds of the present invention (each of which incorporated herein by reference).The results of the assay are tabulated in Tables 1 A - 1 F.
Table 1 B
Figure imgf000116_0001
1 - MES-SA Adryamycin resistant - Combretastatin resistant - Adryamycin resistant NCI-H69 - Mitoxantrone resistant HL-60
Table 1 C
Figure imgf000117_0001
Table 1 D
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
Table 1 E
Figure imgf000122_0001
Figure imgf000122_0002
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0003
Figure imgf000126_0001
131 135 144
Figure imgf000126_0002
161 167
Figure imgf000127_0001
168 176
Example 21
A sample of cell free tubulin polymerizes and the sample's fluorescence emission increases. Inhibition of tubulin polymerization by a tubulin binding compounds of the present invention was measured by the dose dependence of cell free tubulin fluorescence. The concentration of compound that reduced tubulin fluorescence by 50% compared to untreated tubulin (IC50) are tabulated below in Table 3:
Table 3
Figure imgf000127_0002
Compound 67 was effective in inhibiting tubulin polymerization as demonstrated by the same assay.
Example 22
An in vitro assessment of metabolic stability of compounds was performed using commercially available mouse liver microsomes (MLM) containing cytochrome P450 enzymes (Cedra Corp, Austin, TX). Solutions (5 μM) of a compound and microsomes (1 mg/mL protein) was prepared. P450 enzymatic reactions were initiated by adding an NADPH solution. Enzymatic reactions were carried out in a thermostated shaking water bath kept at 370C. Fifty μl of the reaction mixture was withdrawn immediately and 30 minutes after the addition of the NADPH solution and the proteins were precipitated with acetonitrile. The clear supernatant was analyzed by reversed phase LC- MS/MS (Applied Biosystems API-3000 with Hypersil-BDS C18 column and gradient elution), with internal standard area ratio quantification for the amount the compound remaining as shown below.
Example 23
Demonstration of pharmacological stability and pharmacokinetics of the compounds of the present invention.
The pharmacokinetics of the compounds of the present invention was demonstrated by injecting these compounds in mice intraperitoneally (ip) and intravenously (iv), drawing blood at various intervals, and determining the relavant pharmacokinetic parameters as shown below in Tables 5 and 6.
Table 5
Figure imgf000128_0001
Table 6
Figure imgf000128_0002
Example 24
Example 24 demonstrates the usefulness of compounds of the present invention for treating xenograft tumors in mouse.
(i). Female CB17/SCID mice (purchased from Taconic, Oxnard, CA), 7- 8 weeks of age, were allowed to acclimatize for at least three days, and handled under pathogen-free conditions. Human non-small cell lung cancer cell line NCI-H60 was obtained from the American Type Culture Collection. The cell lines were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum. Cells were maintained in a 370C incubator with 5% CO2. The H460 cells were harvested from culture and inoculated at 1 x 106 cells/ animal in the peritoneal subcutaneous space. When the tumors grew to an average volume of 100 mm3 (day 8), each group of mice (ten per group) was administered for five days, vehicle alone (the vehicle group), compound 30 alone at a daily dose of 5, 20, and 50 mg/kg (treatment group), and compound 30 alone at a daily dose of 5, and 20 mg/kg in combination with Taxol® at a daily dose of 10 mg/kg (combination group). Taxol was administered approximately 2-3 hours before that of compound 30.
Compound 30, administered at doses greater than 5 mg/kg were toxic and caused lethality in both treatment and combination groups perhaps indicating that the maximum tolerated dose of compound 30 was between 5 and 20 mg/kg. The results from the experiment employing a daily dose compound 30 (5 mg/kg) are shown graphically in Figures 1 and 2 below.
The body weight of each mouse was recorded twice per week (Figure 1 ). The treatment group administered a daily dose of 5 mg/kg exhibited a weight pattern similar to that of the vehicle group with a mean weight loss of 8% on day 22 from the start of treatment on day 8. Animals in the combination group displayed a weight loss of 13%. One animal in the treatment group was found dead on day 18, and two were found dead on day 22 in the combination group. Figure 2 graphically illustrates the mean tumor volume for each treatment group. Growth of each xenograft was monitored by externally measuring tumors in two dimensions using a digital caliper twice per week. Tumor volume (V) was determined by the following equation: V = (L x W2) /2, where L is the length and W is the width of a xenograft. Tumor volumes were measured twice weekly. On day 11 , the Treated/Control (T/C) ratio was 65% and 52% in the treatment and combination groups, respectively. At the final measurement (day 22), the T/C ratio for the treatment and combination groups respectively were 43% and 19%. Employing the same mouse model, when taxol was administered alone in the same dose and schedule as used for the combination group above, and when treatment began at 150 mm3 volume of xenograft tumors, the T/C for day 21 was 56%. The xenograft data for compound 30 demonstrates that compared to the known anticancer agent taxol, compound 30 can show in vivo anti tumor activity both as a single agent and in combination with taxol. (ii). Other compounds of the present invention, compounds 94, 117, 157, and 187, whose structures are shown below, were tested in vivo in xenograft HT-29 tumor bearing nude mice, alone and in combination with other anti-cancer agents. The compounds were formulated as follows: 94 and 117 in hydroxypropyl yff-cyclodextrin (HBSS), 157 in 2% Tween/Hank's buffered salt solution or in 2% Tween in HBSS, and 187 in 2.5% DMSO and 5% Tween/80-D5W. The compounds were administered as follows: Compound 94 was administered at 100 mg/kg, intraperitoneally (ip), q2d x 3/wk x 2 wk, or at 50 mg/kg, ip, 2/wk x 2 wk alone, i.e. as a monotherapy, and in combination with CPT-1 1 at 50 mg/kg, ip, 2/wk x 2 wks. Compound 117 was administered at 30 mg/kg, ip, q2d x 3 alone and in combination with CDDP at 6 mg/kg, intravenously (iv), q7d x 2. 157 was administered at 100 mg/kg, ip, q2d x 3 alone; it was also administered at 50 mg/kg, ip, q2d x 3/wk x 2 wk alone and in combination with CPT-11 at 50 mg/kg, ip, q7d x 3 and CDDP at 6 mg/kg, iv, q7d x 2. Compound 187 was administered at 10 mg/kg, iv, q2d x 3/wk x 2 or 5 mg/kg for the first 2 doses followed by 25 mg/kg for the next 4 doses, iv, q2d x 3/wk x 2; Compound 187 being administered alone and in combination with CPT-1 1 50 mg/kg, ip, q7d x 3.
Compound 94 when administered for 16 days demonstrated a 34% tumor growth inhibition following monotherapy at 50 mg/kg, and a 75% tumor growth inhibition following combination therapy with CPT-11. Compound 157 demonstrated a tumor growth delay-500, determined by the additional time taken for a treated tumor to reach a 500 mm3 compared to vehicle administered tumor, of 1day when administered alone and 3 days when administered in combination with CPT-11 ; Compound 157 demonstrated a corresponding tumor growth delay-1000 of 2 days when administered alone and 3 days when administered in combination with CPT-11. Compound 187 demonstrated that it was effective in inhibiting tumor growth alone and in combination with CPT-11. Administration of 187 at 5 mg/kg for the first 2 doses followed by 25 mg/kg for the next 4 doses was well tolerated in mice administration at 25 mg/kg showed toxic effects in certain mice.
Figure imgf000131_0001
94 117 157
Figure imgf000131_0002
187
Example 25
Demonstration of the effect of the compounds of the present invention on colchicine binding of tubulin
To a reaction mixture containing tubulin, Na glutamate, GTP, glucose phosphate, and MgCb (98 μl) was added compound 30 (2 μl per reaction), preincubated for 10 min at 370C followed by the addition of [3H] colchicine (1μl per reaction, see also the reference Tse et al., J. Neurochem., 1980; 35(4): 767-74, incorporated herein by reference). The reaction mixture was incubated for 30 min at 370C and spotted on a DE81 filter, air dried for 5 min, wash 3 times with 1 :10 diluted general tubulin buffer and the filter added to a well followed by the addition of the scintillant. Each well was counted using a scintillation counter. 3H colchicine binding to tubulin reduces as the tubulin is incubated with increasing concentrations of compounds 30, 37 whose structure is shown below, and 67, as shown for example in Figures 3 and 4, and demonstrate that these compounds bind to the colchicine binding site of tubulin.
Figure imgf000132_0001
37
Example 26 Disruption of In vitro vessel formation by compound 30
A Matrigel™ matrix was kept on ice for 24 h (400 μl Matrigel per well). Gelation was performed at 370C for 30min, the gel overlaid with 300μl of medium containing 3 x 104 HUVEC cells followed by the addition of 300 μl of 10ng/ml of FGF-2 and the test compound, and incubated for 6 h (See for example the reference Neoplasia, 2004, 6(5):513-22). The capillary tube formation was inspected under an inverted light microscope and the disruption of vessel formation observed as shown in Figure 5 demonstrating that compound 30 disrupts vessel formation. Similarly compound 67 disrupted vessel formation. Blood vessel formation is observed in many cancers particularly in solid tumors and such vessel formation is necessary for providing nutrients to the tumor. By disrupting these tumor blood vessels, 30, 67 and such other compounds of the invention can act as anti cancer agents.
Example 27
Demonstration of the effect of compound 30 on in vitro vascular permeability HUVEC cells were seeded in a 24 well plate at 0.083 x 106 cells/500 μl /well and 0.25 x 106 cells/500 μl /well (also see, for example, the reference Nicholson et a/., Anticancer Drugs, 2006, 17(1 ):25-31 ). The cells were allowed to attach for 3 days (90% confluence). Then, the growth media was removed, the cells were washed once with PBS and 500 μl media containing the test compound (2 μM) was added and incubated for 15 min at 370C. 1 mg/ml FITC- dextran was added to the upper chamber, incubate for 30min, and fluorescence intensity measured at the lower chamber at 485 excitation and 530 emission. Increased in fluorescene with time was observed in the lower chamber indicating increased vascular permeability upon incubation with compound 30 (Figure 6).
Example 28
Demonstration of concentration-dependent Disruption of Microtubules and Actin Filaments by compound 67
Proliferating HUVEC-C cells were treated with various concentrations of compound 67 for 24 h. Microtubules and F-actin filaments were visualized using fluorescence microscopy and demonstrated that 1-10 nM of compound 67 disrupted microtutbules and F-actin in a dose dependent manner.
***
Although the present invention has been described in detail with reference to specific embodiments, those of skill in the art will recognize that modifications and improvements are within the scope and spirit of the invention, as set forth in the claims which follow. All publications and patent documents (patents, published patent applications, and unpublished patent applications) 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 admission that any such document is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been described by way of written description and example, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples are for purposes of illustration and not limitation of the following claims.

Claims

CLAIMS We claim: 1. A compound having a structure of formula:
Figure imgf000134_0001
wherein Qi is selected from
Figure imgf000134_0002
LrW1, ethynyl, NH2,
CrC6 alkylamino, and di(Ci-C6) alkylamino, wherein Li is selected from CrC6 alkylene and CrC6 heteroalkylene and W1 is selected from the group consisting of hydrogen, amino, C1-C6 alkylamino, di(Ci-C6) alkylamino, C1-C6 alkoxy, heterocyclyl, heteroaryl, and an aqueous solubility enhancing group and
Q7 is selected from the group consisting of hydrogen, an aqueous solubility enhancing group, and a methyl group substituted with an aqueous solubility enhancing group with the proviso that if Q7 is hydrogen, then Q1 is not
H2 -C- -NH2 OrW1 is not hydrogen.
2. The compound of claim 1 wherein the aqueous solubility enhancing group is selected from the group consisting of an amino acid, a phosphate moiety, a sugar moiety, and an amino substituted benzoate moiety.
3. The compound of claim 2 wherein Q7 is hydrogen.
4. The compound of claim 2 wherein Q1 is ΛΛΛΛ- -CH3.
5. The compound of claim 4 wherein the solubility enhancing group is selected from the group consisting of:
Figure imgf000135_0001
6. The compound of claim 2 wherein Qi is NH2.
7. A compound having structure of formula:
Figure imgf000135_0002
wherein Qi is selected from
Figure imgf000135_0003
L1-W1, ethynyl, NH2,
CrC6 alkylamino, di(CrCs) alkylamino, and NR26Hyp wherein L1 is selected from C1-C6 alkylene and C1-C6 heteroalkylene; W1 is selected from the group consisting of hydrogen, amino, C1-C6 alkylamino, di(CrC6) alkylamino, C1-C6 alkoxy, heterocyclyl, heteroaryl, an aqueous solubility enhancing group, and
NR26HyP; and R26 is selected from C1-C6 alkyl and hydrogen and
Q7 is selected from the group consisting of hydrogen, Hyp, a solubility enhancing group, and a methyl group substituted with a solubility enhancing group or a Hyp-NR20 moiety wherein R20 is C1-C6 alkyl group; with the proviso that if Q7 is hydrogen, a solubility enhancing group, or a methyl group substituted with a solubility enhancing group, then Q1 is N
R26Hyp Or W1 is NR26Hyp; Jf W1 is selected from the group consisting of amino, C1-C6 alkylamino, di(Ci-C6) alkylamino, CrC6 alkoxy, heterocyclyl, heteroaryl, and an aqueous solubility enhancing group, then Q7 is Hyp or a methyl group substituted with a HyP-NR20 moiety; and if Qi is ethynyl, then Q7 is Hyp or a methyl group substituted with a
HyP-NR20 moiety.
8. The compound of claim 7 wherein the solubility enhancing group is selected from a group consisting of an amino acid, a phosphate moiety, a sugar moiety, and an amino substituted benzoate moiety.
9. The compound of claim 7 wherein Hyp is selected from the group consisting of
Figure imgf000136_0001
10. The compound of claim 7 wherein Q1 is NR26Hyp.
11. The compound of claim 9 wherein R26 is hydrogen.
12. The compound of claim 11 wherein Hyp is
Figure imgf000136_0002
13. A pharmaceutical composition comprising a compound of claims 1- 12 and a pharmaceutically acceptable carrier, excipient, and/or diluent.
14. A method of treating cancer comprising administering a therapeutically effective amount of a compound of Claims 1-12 to a patient in need of such treatment.
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