The present invention relates generally to compositions and methods for treating cancer and neoplastic disease. Provided herein are substituted pyrido[3,4-d]pyrimidin-4-one derivative compounds and pharmaceutical compositions comprising said compounds. The subject compounds and compositions are useful for inhibition of histone demethylase. Furthermore, the subject compounds and compositions are useful for the treatment of cancer, such as prostate cancer, breast cancer, bladder cancer, lung cancer and/or melanoma and the like.

BACKGROUND

A need exists in the art for an effective treatment of cancer and neoplastic disease.

BRIEF SUMMARY OF THE INVENTION

Provided herein are substituted pyrido[3,4-d]pyrimidin-4-one derivative compounds and pharmaceutical compositions comprising said compounds. The subject compounds and compositions are useful for inhibition histone demethylase. Furthermore, the subject compounds and compositions are useful for the treatment of cancer, such as prostate cancer, breast cancer, bladder cancer, lung cancer and/or melanoma and the like. The substituted pyrido[3,4-d]pyrimidin-4-one derivative compounds described herein are based upon a substituted pyrido[3,4-d]pyrimidin-4-one ring system bearing a hydroxy group at the 4-position, and an oxygen-based substituent at the 2-position. The 8-position substituent, in various embodiments, is selected from a wide variety of groups, such as, but not limited to, hydrogen, alkyl, aryl, carbocyclyl, and the like.

One embodiment provides a compound of Formula (I), or pharmaceutically acceptable salt thereof,

wherein,
X is alkyl, or -L-R1;L is a bond, or C1-C6 alkylene;R1is carbocyclyl, aryl, heterocyclyl, or heteroaryl;
Y is hydrogen or

One embodiment provides a pharmaceutical composition comprising a compound of Formula (I), or pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

One embodiment provides a method for inhibiting a histone demethylase enzyme comprising contacting a histone demethylase enzyme with a compound of Formula (I).

One embodiment provides a method for treating cancer in subject comprising administering to the subject in need thereof a composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

INCORPORATION BY REFERENCE

DETAILED DESCRIPTION OF THE INVENTION

As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an agent” includes a plurality of such agents, and reference to “the cell” includes reference to one or more cells (or to a plurality of cells) and equivalents thereof known to those skilled in the art, and so forth. When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary between 1% and 15% of the stated number or numerical range. The term “comprising” (and related terms such as “comprise” or “comprises” or “having” or “including”) is not intended to exclude that in other certain embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, described herein, may “consist of” or “consist essentially of” the described features.

Definitions

“Amino” refers to the —NH2radical.

“Cyano” refers to the —CN radical.

“Nitro” refers to the —NO2radical.

“Oxa” refers to the —O— radical.

“Oxo” refers to the ═O radical.

“Thioxo” refers to the ═S radical.

“Imino” refers to the ═N—H radical.

“Oximo” refers to the ═N—OH radical.

“Hydrazino” refers to the ═N—NH2radical.

“Alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to fifteen carbon atoms (e.g., C1-C15alkyl). In certain embodiments, an alkyl comprises one to thirteen carbon atoms (e.g., C1-C13alkyl). In certain embodiments, an alkyl comprises one to eight carbon atoms (e.g., C1-C8alkyl). In other embodiments, an alkyl comprises one to five carbon atoms (e.g., C1-C5alkyl). In other embodiments, an alkyl comprises one to four carbon atoms (e.g., C1-C4alkyl). In other embodiments, an alkyl comprises one to three carbon atoms (e.g., C1-C3alkyl). In other embodiments, an alkyl comprises one to two carbon atoms (e.g., C1-C2alkyl). In other embodiments, an alkyl comprises one carbon atom (e.g., C1alkyl). In other embodiments, an alkyl comprises five to fifteen carbon atoms (e.g., C5-C15alkyl). In other embodiments, an alkyl comprises five to eight carbon atoms (e.g., C5-C8alkyl). In other embodiments, an alkyl comprises two to five carbon atoms (e.g., C2-C5alkyl). In other embodiments, an alkyl comprises three to five carbon atoms (e.g., C3-C5alkyl). In other embodiments, the alkyl group is selected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (iso-propyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (iso-butyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl). The alkyl is attached to the rest of the molecule by a single bond. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted by one or more of the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, —ORa, —SRa, —OC(O)—Ra, —N(Ra)2, —C(O)Ra, —C(O)ORa, —C(O)N(Ra)2, —N(Ra)C(O)ORa, —OC(O)—N(Ra)2, —N(Ra)C(O)Ra, —N(Ra)S(O)tRa(where t is 1 or 2), —S(O)tORa(where t is 1 or 2), —S(O)tRa(where t is 1 or 2) and —S(O)tN(Ra)2(where t is 1 or 2) where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl.

“Alkoxy” refers to a radical bonded through an oxygen atom of the formula —O— alkyl, where alkyl is an alkyl chain as defined above.

“Alkenyl” refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one carbon-carbon double bond, and having from two to twelve carbon atoms. In certain embodiments, an alkenyl comprises two to eight carbon atoms. In other embodiments, an alkenyl comprises two to four carbon atoms. The alkenyl is attached to the rest of the molecule by a single bond, for example, ethenyl (i.e., vinyl), prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like. Unless stated otherwise specifically in the specification, an alkenyl group is optionally substituted by one or more of the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, —ORa, —SRa, —OC(O)—Ra, —N(Ra)2, —C(O)Ra, —C(O)ORa, —C(O)N(Ra)2, —N(Ra)C(O)ORa, —OC(O)—N(Ra)2, —N(Ra)C(O)Ra, —N(Ra)S(O)tRa(where t is 1 or 2), —S(O)tORa(where t is 1 or 2), —S(O)tRa(where t is 1 or 2) and —S(O)tN(Ra)2(where t is 1 or 2) where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl.

“Alkynyl” refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, having from two to twelve carbon atoms. In certain embodiments, an alkynyl comprises two to eight carbon atoms. In other embodiments, an alkynyl has two to four carbon atoms. The alkynyl is attached to the rest of the molecule by a single bond, for example, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless stated otherwise specifically in the specification, an alkynyl group is optionally substituted by one or more of the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, —ORa, —SRa, —OC(O)—Ra, —N(Ra)2, —C(O)Ra, —C(O)ORa, —C(O)N(Ra)2, —N(Ra)C(O)ORa, —OC(O)—N(Ra)2, —N(Ra)C(O)Ra, —N(Ra)S(O)tRa(where t is 1 or 2), —S(O)tORa(where t is 1 or 2), —S(O)tRa(where t is 1 or 2) and —S(O)tN(Ra)2(where t is 1 or 2) where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl.

“Alkylene” or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing no unsaturation and having from one to twelve carbon atoms, for example, methylene, ethylene, propylene, n-butylene, and the like. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon in the alkylene chain or through any two carbons within the chain. In certain embodiments, an alkylene comprises one to eight carbon atoms (e.g., C1-C8alkylene). In other embodiments, an alkylene comprises one to five carbon atoms (e.g., C1-C5alkylene). In other embodiments, an alkylene comprises one to four carbon atoms (e.g., C1-C4alkylene). In other embodiments, an alkylene comprises one to three carbon atoms (e.g., C1-C3alkylene). In other embodiments, an alkylene comprises one to two carbon atoms (e.g., C1-C2alkylene). In other embodiments, an alkylene comprises one carbon atom (e.g., C1alkylene). In other embodiments, an alkylene comprises five to eight carbon atoms (e.g., C5-C8alkylene). In other embodiments, an alkylene comprises two to five carbon atoms (e.g., C2-C5alkylene). In other embodiments, an alkylene comprises three to five carbon atoms (e.g., C3-C5alkylene). Unless stated otherwise specifically in the specification, an alkylene chain is optionally substituted by one or more of the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, —ORa, —SRa, —OC(O)—Ra, —N(Ra)2, —C(O)Ra, —C(O)ORa, —C(O)N(Ra)2, —N(Ra)C(O)ORa, —OC(O)—N(Ra)2, —N(Ra)C(O)Ra, —N(Ra)S(O)tRa(where t is 1 or 2), —S(O)tORa(where t is 1 or 2), —S(O)tRa(where t is 1 or 2) and —S(O)tN(Ra)2(where t is 1 or 2) where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl.

“Aryl” refers to a radical derived from an aromatic monocyclic or multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or multicyclic hydrocarbon ring system contains only hydrogen and carbon from five to eighteen carbon atoms, where at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) π-electron system in accordance with the Hückel theory. The ring system from which aryl groups are derived include, but are not limited to, groups such as benzene, fluorene, indane, indene, tetralin and naphthalene. Unless stated otherwise specifically in the specification, the term “aryl” or the prefix “ar-” (such as in “aralkyl”) is meant to include aryl radicals optionally substituted by one or more substituents independently selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, —Rb—ORa, —Rb—OC(O)—Ra, —Rb—OC(O)—ORa, —Rb—OC(O)—N(Ra)2, —Rb—N(Ra)2, —Rb—C(O)Ra, —Rb—C(O)ORa, —Rb—C(O)N(Ra)2, —Rb—O—Rc—C(O)N(Ra)2, —Rb—N(Ra)C(O)ORa, —Rb—N(Ra)C(O)Ra, —Rb—N(Ra)S(O)tRa(where t is 1 or 2), —Rb—S(O)tORa(where t is 1 or 2), —Rb—S(O)tRa(where t is 1 or 2) and —Rb—S(O)tN(Ra)2(where t is 1 or 2), where each Rais independently hydrogen, alkyl, fluoroalkyl, cycloalkyl, cycloalkylalkyl, aryl (optionally substituted with one or more halo groups), aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl, each Rbis independently a direct bond or a straight or branched alkylene or alkenylene chain, and Rcis a straight or branched alkylene or alkenylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated.

“Aralkyl” refers to a radical of the formula —Rc-aryl where Rcis an alkylene chain as defined above, for example, methylene, ethylene, and the like. The alkylene chain part of the aralkyl radical is optionally substituted as described above for an alkylene chain. The aryl part of the aralkyl radical is optionally substituted as described above for an aryl group.

“Aralkenyl” refers to a radical of the formula —Rd-aryl where Rdis an alkenylene chain as defined above. The aryl part of the aralkenyl radical is optionally substituted as described above for an aryl group. The alkenylene chain part of the aralkenyl radical is optionally substituted as defined above for an alkenylene group.

“Aralkynyl” refers to a radical of the formula —Rc-aryl, where Rcis an alkynylene chain as defined above. The aryl part of the aralkynyl radical is optionally substituted as described above for an aryl group. The alkynylene chain part of the aralkynyl radical is optionally substituted as defined above for an alkynylene chain.

“Aralkoxy” refers to a radical bonded through an oxygen atom of the formula —O—Rc-aryl where Rcis an alkylene chain as defined above, for example, methylene, ethylene, and the like. The alkylene chain part of the aralkyl radical is optionally substituted as described above for an alkylene chain. The aryl part of the aralkyl radical is optionally substituted as described above for an aryl group.

“Carbocyclyl” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms. In certain embodiments, a carbocyclyl comprises three to ten carbon atoms. In other embodiments, a carbocyclyl comprises five to seven carbon atoms. The carbocyclyl is attached to the rest of the molecule by a single bond. Carbocyclyl may be saturated, (i.e., containing single C—C bonds only) or unsaturated (i.e., containing one or more double bonds or triple bonds.) A fully saturated carbocyclyl radical is also referred to as “cycloalkyl.” Examples of monocyclic cycloalkyls include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. An unsaturated carbocyclyl is also referred to as “cycloalkenyl.” Examples of monocyclic cycloalkenyls include, e.g., cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. Polycyclic carbocyclyl radicals include, for example, adamantyl, norbornyl (i.e., bicyclo[2.2.1]heptanyl), norbomenyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwise stated specifically in the specification, the term “carbocyclyl” is meant to include carbocyclyl radicals that are optionally substituted by one or more substituents independently selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, —Rb—ORa, —Rb—OC(O)—Ra, —Rb—OC(O)—ORa, —Rb—OC(O)—N(Ra)2, —Rb—N(Ra)2, —Rb—C(O)Ra, —Rb—C(O)ORa, —Rb—C(O)N(Ra)2, —Rb—O—Rc—C(O)N(Ra)2, —Rb—N(Ra)C(O)ORa, —Rb—N(Ra)C(O)Ra, —Rb—N(Ra)S(O)tRa(where t is 1 or 2), —Rb—S(O)tORa(where t is 1 or 2), —Rb—S(O)tRa(where t is 1 or 2) and —Rb—S(O)tN(Ra)2(where t is 1 or 2), where each Rais independently hydrogen, alkyl, fluoroalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl, each Rbis independently a direct bond or a straight or branched alkylene or alkenylene chain, and Rcis a straight or branched alkylene or alkenylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated.

“Carbocyclylalkyl” refers to a radical of the formula —Rc-carbocyclyl where Rcis an alkylene chain as defined above. The alkylene chain and the carbocyclyl radical is optionally substituted as defined above.

“Carbocyclylalkoxy” refers to a radical bonded through an oxygen atom of the formula —O—Rc-carbocyclyl where Rcis an alkylene chain as defined above. The alkylene chain and the carbocyclyl radical is optionally substituted as defined above.

“Heterocyclyl” refers to a stable 3- to 18-membered non-aromatic ring radical that comprises two to twelve carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems. The heteroatoms in the heterocyclyl radical may be optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heterocyclyl radical is partially or fully saturated. The heterocyclyl may be attached to the rest of the molecule through any atom of the ring(s). Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in the specification, the term “heterocyclyl” is meant to include heterocyclyl radicals as defined above that are optionally substituted by one or more substituents selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, —Rb—ORa, —Rb—OC(O)—Ra, —Rb—OC(O)—ORa, —Rb—OC(O)—N(Ra)2, —Rb—N(Ra)2, —Rb—C(O)Ra, —Rb—C(O)ORa, —Rb—C(O)N(Ra)2, —Rb—O—Rc—C(O)N(Ra)2, —Rb—N(Ra)C(O)ORa, —Rb—N(Ra)C(O)Ra, —Rb—N(Ra)S(O)tRa(where t is 1 or 2), —Rb—S(O)tORa(where t is 1 or 2), —Rb—S(O)tRa(where t is 1 or 2) and —Rb—S(O)tN(Ra)2(where t is 1 or 2), where each Rais independently hydrogen, alkyl, fluoroalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl, each Rbis independently a direct bond or a straight or branched alkylene or alkenylene chain, and Rcis a straight or branched alkylene or alkenylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated.

“N-heterocyclyl” or “N-attached heterocyclyl” refers to a heterocyclyl radical as defined above containing at least one nitrogen and where the point of attachment of the heterocyclyl radical to the rest of the molecule is through a nitrogen atom in the heterocyclyl radical. An N-heterocyclyl radical is optionally substituted as described above for heterocyclyl radicals. Examples of such N-heterocyclyl radicals include, but are not limited to, 1-morpholinyl, 1-piperidinyl, 1-piperazinyl, 1-pyrrolidinyl, pyrazolidinyl, imidazolinyl, and imidazolidinyl.

“C-heterocyclyl” or “C-attached heterocyclyl” refers to a heterocyclyl radical as defined above containing at least one heteroatom and where the point of attachment of the heterocyclyl radical to the rest of the molecule is through a carbon atom in the heterocyclyl radical. A C-heterocyclyl radical is optionally substituted as described above for heterocyclyl radicals. Examples of such C-heterocyclyl radicals include, but are not limited to, 2-morpholinyl, 2- or 3- or 4-piperidinyl, 2-piperazinyl, 2- or 3-pyrrolidinyl, and the like.

“Heterocyclylalkyl” refers to a radical of the formula —Rc-heterocyclyl where Rcis an alkylene chain as defined above. If the heterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl is optionally attached to the alkyl radical at the nitrogen atom. The alkylene chain of the heterocyclylalkyl radical is optionally substituted as defined above for an alkylene chain. The heterocyclyl part of the heterocyclylalkyl radical is optionally substituted as defined above for a heterocyclyl group.

“Heterocyclylalkoxy” refers to a radical bonded through an oxygen atom of the formula —O—Rc-heterocyclyl where Rcis an alkylene chain as defined above. If the heterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl is optionally attached to the alkyl radical at the nitrogen atom. The alkylene chain of the heterocyclylalkoxy radical is optionally substituted as defined above for an alkylene chain. The heterocyclyl part of the heterocyclylalkoxy radical is optionally substituted as defined above for a heterocyclyl group.

“N-heteroaryl” refers to a heteroaryl radical as defined above containing at least one nitrogen and where the point of attachment of the heteroaryl radical to the rest of the molecule is through a nitrogen atom in the heteroaryl radical. An N-heteroaryl radical is optionally substituted as described above for heteroaryl radicals.

“C-heteroaryl” refers to a heteroaryl radical as defined above and where the point of attachment of the heteroaryl radical to the rest of the molecule is through a carbon atom in the heteroaryl radical. A C-heteroaryl radical is optionally substituted as described above for heteroaryl radicals.

“Heteroarylalkyl” refers to a radical of the formula —Rc-heteroaryl, where Rcis an alkylene chain as defined above. If the heteroaryl is a nitrogen-containing heteroaryl, the heteroaryl is optionally attached to the alkyl radical at the nitrogen atom. The alkylene chain of the heteroarylalkyl radical is optionally substituted as defined above for an alkylene chain. The heteroaryl part of the heteroarylalkyl radical is optionally substituted as defined above for a heteroaryl group.

“Heteroarylalkoxy” refers to a radical bonded through an oxygen atom of the formula —O—Rc-heteroaryl, where Rcis an alkylene chain as defined above. If the heteroaryl is a nitrogen-containing heteroaryl, the heteroaryl is optionally attached to the alkyl radical at the nitrogen atom. The alkylene chain of the heteroarylalkoxy radical is optionally substituted as defined above for an alkylene chain. The heteroaryl part of the heteroarylalkoxy radical is optionally substituted as defined above for a heteroaryl group.

The compounds disclosed herein may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-. Unless stated otherwise, it is intended that all stereoisomeric forms of the compounds disclosed herein are contemplated by this disclosure. When the compounds described herein contain alkene double bonds, and unless specified otherwise, it is intended that this disclosure includes both E and Z geometric isomers (e.g., cis or trans). Likewise, all possible isomers, as well as their racemic and optically pure forms, and all tautomeric forms are also intended to be included. The term “geometric isomer” refers to E or Z geometric isomers (e.g., cis or trans) of an alkene double bond. The term “positional isomer” refers to structural isomers around a central ring, such as ortho-, meta-, and para-isomers around a benzene ring.

A “tautomer” refers to a molecule wherein a proton shift from one atom of a molecule to another atom of the same molecule is possible. The compounds presented herein may, in certain embodiments, exist as tautomers. In circumstances where tautomerization is possible, a chemical equilibrium of the tautomers will exist. The exact ratio of the tautomers depends on several factors, including physical state, temperature, solvent, and pH. Some examples of tautomeric equilibrium include:

“Pharmaceutically acceptable salt” includes both acid and base addition salts. A pharmaceutically acceptable salt of any one of the substituted pyrido[3,4-d]pyrimidin-4-one derivative compounds described herein is intended to encompass any and all pharmaceutically suitable salt forms. Preferred pharmaceutically acceptable salts of the compounds described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

“Prodrug” is meant to indicate a compound that may be converted under physiological conditions or by solvolysis to a biologically active compound described herein. Thus, the term “prodrug” refers to a precursor of a biologically active compound that is pharmaceutically acceptable. A prodrug may be inactive when administered to a subject, but is converted in vivo to an active compound, for example, by hydrolysis. The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, e.g., Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam).

A discussion of prodrugs is provided in Higuchi, T., et al., “Pro-drugs as Novel Delivery Systems,” A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated in full by reference herein.

The term “prodrug” is also meant to include any covalently bonded carriers, which release the active compound in vivo when such prodrug is administered to a mammalian subject. Prodrugs of an active compound, as described herein, may be prepared by modifying functional groups present in the active compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent active compound. Prodrugs include compounds wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the active compound is administered to a mammalian subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol or amine functional groups in the active compounds and the like.

Substituted pyrido[3,4-d]pyrimidin-4-one derivative compounds are described herein that inhibit a histone demethylase enzyme. These compounds, and compositions comprising these compounds, are useful for the treatment of cancer and neoplastic disease.

The compounds described herein are useful for treating prostate cancer, breast cancer, bladder cancer, lung cancer and/or melanoma and the like.

One embodiment provides a compound of Formula (I), or pharmaceutically acceptable salt thereof,

wherein,
X is alkyl, or -L-R1;L is a bond, or C1-C6 alkylene;R1is carbocyclyl, aryl, heterocyclyl, or heteroaryl;
Y is hydrogen or

Another embodiment provides the compound of Formula (I), wherein Y is hydrogen. Another embodiment provides the compound of Formula (I), wherein Y is

Another embodiment provides the compound of Formula (I), wherein X is alkyl. Another embodiment provides the compound of Formula (I), wherein X is alkyl and Y is hydrogen. Another embodiment provides the compound of Formula (I), wherein X is alkyl and Y is

Another embodiment provides the compound of Formula (I), wherein the alkyl is a C1-C6 alkyl. Another embodiment provides the compound of Formula (I), wherein the alkyl is substituted with at least one fluoro substituent. Another embodiment provides the compound of Formula (I), wherein the alkyl is substituted with at least one group selected from hydroxy, alkoxy, aryloxy, amino, alkylamino, arylamino, or diakylamino. Another embodiment provides the compound of Formula (I), wherein the alkyl is substituted with at least one group selected from —NHCOR3, —NHCO2R3, —NHCONHR3, —N(R4)COR3, —N(R4)CO2R3, —N(R4)CONHR3, —N(R4)CON(R4)R3, —NHSO2R3, or —NR4SO2R3, wherein each R3is independently selected from alkyl, aryl, heteroaryl, carbocyclyl, or heterocyclyl, and each R4is an alkyl. Another embodiment provides the compound of Formula (I), wherein the alkyl is substituted with at least one group selected from —CONH2, —CONHR3, —CON(R3)2, —COR3, —SO2NH2, —SO2NHR3, —SO2N(R3)2, or —SO2R3, wherein each R3is independently selected from alkyl, aryl, heteroaryl, carbocyclyl, or heterocyclyl.

Another embodiment provides the compound of Formula (I), wherein X is -L-R1. Another embodiment provides the compound of Formula (I), wherein X is -L-R1and Y is hydrogen. Another embodiment provides the compound of Formula (I), wherein X is -L-R1and Y is

Another embodiment provides the compound of Formula (I), wherein L is a bond.

Another embodiment provides the compound of Formula (I), wherein L is a bond and R1is carbocyclyl. The compound of claim8or9, wherein L is a bond. Another embodiment provides the compound of Formula (I), wherein R1is heterocyclyl. Another embodiment provides the compound of Formula (I), wherein L is a bond. Another embodiment provides the compound of Formula (I), wherein R1is aryl. Another embodiment provides the compound of Formula (I), wherein the aryl is a phenyl group. Another embodiment provides the compound of Formula (I), wherein the phenyl is substituted with at least one halogen substituent. Another embodiment provides the compound of Formula (I), wherein the phenyl is substituted with at least one alkyl substituent. Another embodiment provides the compound of Formula (I), wherein the phenyl is substituted with at least one group selected from hydroxy, alkoxy, aryloxy, amino, alkylamino, arylamino, or diakylamino. Another embodiment provides the compound of Formula (I), wherein the phenyl is substituted with at least one group selected from —NHCOR3, —NHCO2R3, —NHCONHR3, —N(R4)COR3, —N(R4)CO2R3, —N(R4)CONHR3, —N(R4)CON(R4)R3, —NHSO2R3, or —NR4SO2R3, wherein each R3is independently selected from alkyl, aryl, heteroaryl, carbocyclyl, or heterocyclyl, and each R4is an alkyl. Another embodiment provides the compound of Formula (I), wherein the phenyl is substituted with at least one group selected from —CONH2, —CONHR3, —CON(R3)2, —COR3, —SO2NH2, —SO2NHR3, —SO2N(R3)2, or —SO2R3, wherein each R3is independently selected from alkyl, aryl, heteroaryl, carbocyclyl, or heterocyclyl. Another embodiment provides the compound of Formula (I), wherein the phenyl is substituted with a group selected from aryl, heteroaryl, carbocyclyl, or heterocyclyl.

Another embodiment provides the compound of Formula (I), wherein L is a bond and R1is heteroaryl. Another embodiment provides the compound of Formula (I), wherein the heteroaryl is a group selected from benzimidazolyl, benzofuranyl, furanyl, isothiazolyl, imidazolyl, indazolyl, indolyl, isoxazolyl, oxazolyl, pyrrolyl, pyrazolyl, pyridinyl, prrazinyl, pyrimidinyl, pyridazinyl, thiazolyl or thiophenyl. Another embodiment provides the compound of Formula (I), wherein the heteroaryl group is substituted with at least one halogen substituent. Another embodiment provides the compound of Formula (I), wherein the heteroaryl group is substituted with at least one alkyl substituent. Another embodiment provides the compound of Formula (I), wherein the heteroaryl group is substituted with at least one group selected from hydroxy, alkoxy, aryloxy, amino, alkylamino, arylamino, or diakylamino. Another embodiment provides the compound of Formula (I), wherein the heteroaryl group is substituted with at least one group selected from —NHCOR3, —NHCO2R3, —NHCONHR3, —N(R4)COR3, —N(R4)CO2R3, —N(R4)CONHR3, —N(R4)CON(R4)R3, —NHSO2R3, or —NR4SO2R3, wherein each R3is independently selected from alkyl, aryl, heteroaryl, carbocyclyl, or heterocyclyl, and each R4is an alkyl. Another embodiment provides the compound of Formula (I), wherein the heteroaryl group is substituted with at least one group selected from —CONH2, —CONHR3, —CON(R3)2, —COR3, —SO2NH2, —SO2NHR3, —SO2N(R3)2, or —SO2R3, wherein each R3is independently selected from alkyl, aryl, heteroaryl, carbocyclyl, or heterocyclyl. Another embodiment provides the compound of Formula (I), wherein the heteroaryl group is substituted with a group selected from aryl, heteroaryl, carbocyclyl, or heterocyclyl. Another embodiment provides the compound of Formula (I), wherein the heteroaryl is a pyrazolyl having the structure

wherein R5is a group selected from alkyl, carbocyclyl, heterocyclyl, carbocyclylalkyl, heterocyclylalkyl, aralkyl, or heteroarylalkyl. Another embodiment provides the compound of Formula (I), wherein the R5group is a C1-C6 alkyl, optionally substituted with at least one group selected from hydroxy, C1-C4 alkoxy, amino, C1-C4 alkylamino, C1-C4 diakylamino, piperdinyl, pyrrolidnyl, or morpholinyl. Another embodiment provides the compound of Formula (I), wherein the R5group is a heterocyclyl selected from 4-tetrahydropyranyl, 1-morpholinyl, or 4-piperdinyl having the structure

Another embodiment provides the compound of Formula (I), wherein X is -L-R1. Another embodiment provides the compound of Formula (I), wherein L is a C1-C6 alkylene. Another embodiment provides the compound of Formula (I), wherein L is a C1-C4 alkylene. Another embodiment provides the compound of Formula (I), wherein R1is 3- to 7-membered carbocyclyl. Another embodiment provides the compound of Formula (I), wherein R1is phenyl. Another embodiment provides the compound of Formula (I), wherein R1is a 5- or 6-membered heteroaryl. Another embodiment provides the compound of Formula (I), wherein R1is a 4- to 6-membered oxygen containing heterocyclyl.

Another embodiment provides the compound of Formula (I), wherein R2is alkyl. Another embodiment provides the compound of Formula (I), wherein the alkyl is methyl. Another embodiment provides the compound of Formula (I), wherein the alkyl is C2-C4 alkyl. Another embodiment provides the compound of Formula (I), wherein the alkyl is substituted with at least one fluoro substituent. Another embodiment provides the compound of Formula (I), wherein the alkyl is substituted with at least one group selected from hydroxy, alkoxy, amino, alkylamino, or diakylamino.

Another embodiment provides the compound of Formula (I), wherein R2is heterocyclyl. Another embodiment provides the compound of Formula (I), wherein R2is heterocyclylalkyl.

Another embodiment provides the compound of Formula (I), wherein the heterocyclyl is a 4- to 6-membered oxygen or nitrogen containing heterocyclyl. Another embodiment provides the compound of Formula (I), wherein the heterocyclylalkyl consists of a 4- to 6-membered oxygen or nitrogen containing heterocyclyl, and a C1-C3 alkylene.

Another embodiment provides the compound of Formula (I), wherein R2is carbocyclylalkyl. Another embodiment provides the compound of Formula (I), wherein the carbocyclylalkyl consists of a 3- to 7-membered carbocyclyl, and a C1-C3 alkylene.

One embodiment provides a compound of Formula (Ia), or pharmaceutically acceptable salt thereof,

wherein,
X is -L-R1;L is a bond, or C1-C6 alkylene;R1is heteroaryl substituted with a methylene group bearing at least one aryl group and at least one cycloalkyl group;
Y is hydrogen or

One embodiment provides a compound of Formula (II), or pharmaceutically acceptable salt thereof,

wherein,Y is

Another embodiment provides the compound of Formula (II), wherein R2is methyl. Another embodiment provides the compound of Formula (II), wherein R2is C1-C4 alkyl. Another embodiment provides the compound of Formula (II), wherein the alkyl is substituted with at least one fluoro substituent. Another embodiment provides the compound of Formula (II), wherein the alkyl is substituted with at least one group selected from hydroxy, alkoxy, aryloxy, amino, alkylamino, arylamino, or diakylamino. Another embodiment provides the compound of Formula (II), wherein the alkyl is substituted with at least one group selected from —NHCOR3, —NHCO2R3, —NHCONHR3, —N(R4)COR3, —N(R4)CO2R3, —N(R4)CONHR3, —N(R4)CON(R4)R3, —NHSO2R3, or —NR4SO2R3, wherein each R3is independently selected from alkyl, aryl, heteroaryl, carbocyclyl, or heterocyclyl, and each R4is an alkyl. Another embodiment provides the compound of Formula (II), wherein the alkyl is substituted with at least one group selected from —CONH2, —CONHR3, —CON(R3)2, —COR3, —SO2NH2, —SO2NHR3, —SO2N(R3)2, or —SO2R3, wherein each R3is independently selected from alkyl, aryl, heteroaryl, carbocyclyl, or heterocyclyl. Another embodiment provides the compound of Formula (II), wherein R2is heterocyclylalkyl. Another embodiment provides the compound of Formula (II), wherein R2is heterocyclylalkyl, and the alkylene portion of the heterocyclylalkyl is a C1-C4 alkylene. Another embodiment provides the compound of Formula (II), wherein R2is heterocyclylalkyl and the heterocyclyl portion of the heterocyclylalkyl is a 4- to 7-membered heterocyclyl containing at least one nitrogen atom, or at least one oxygen atom. Another embodiment provides the compound of Formula (II), wherein R2is carbocyclylalkyl. Another embodiment provides the compound of Formula (II), wherein R2is carbocyclylalkyl, and the alkylene portion of the carbocyclylalkyl is a C1-C4 alkylene. Another embodiment provides the compound of Formula (II), wherein R2is carbocyclylalkyl, and the carbocyclyl portion of the carbocyclylalkyl is a 4- to 7-membered carbocyclyl.

In some embodiments, the compound disclosed herein has a structure provided in Table 1.

In some embodiments, the compound disclosed herein has a structure provided in Table 2.

Preparation of the Substituted Pyrido[3,4-d]pyrimidin-4-one Derivative Compounds

Specific and analogous reactants may also be identified through the indices of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases (the American Chemical Society, Washington, D.C., may be contacted for more details). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (e.g., those listed above) provide custom synthesis services. A reference for the preparation and selection of pharmaceutical salts of the substituted pyrido[3,4-d]pyrimidin-4-one derivative compounds described herein is P. H. Stahl & C. G. Wermuth “Handbook of Pharmaceutical Salts”, Verlag Helvetica Chimica Acta, Zurich, 2002.

The substituted pyrido[3,4-d]pyrimidin-4-one derivative compounds are prepared by the general synthetic routes described below in Schemes 1-3.

Referring to Scheme 1, compound A is converted to compound B by condensation with urea. The azaquinazolinedione compound B is converted to compound C using an appropriate chlorinating agent, such as POCl3. Compound C is selectively hydrolyzed to form compound D under a variety of basic conditions, such as hydrolysis in a NaOH solution. Nucleophilic substitution of the chloride in compound D is carried out with an alcohol, such as G-OH, under a variety of basic conditions to form compound F. For example, compound D can be treated with the sodium salt of the alcohol E. Additionally, compound D can be heated with the alcohol or phenol G-OH in the presence of CuI and CsCO3in an appropriate solvent to form compound F.

Referring to Scheme 2, compound H is chlorinated to produce compound J. For example, chlorination can occur through the formation of the pyridine N-oxide in the presence of a chlorine source such as HCl. The biaryl compound L is prepared from aryl halide compound J using aryl coupling conditions, such as Stille conditions with the N-alkyl-imidiazole stannane K. Compound L is converted to compound M by condensation with urea. The azaquinazolinedione compound M is converted to dichloro compound N using an appropriate chlorinating agent, such as POCl3. Compound N is selectively hydrolyzed to form compound P under a variety of basic conditions, such as hydrolysis in a NaOH solution. Nucleophilic substitution of the chloride in compound P is carried out with an alcohol G-OH under a variety of basic conditions to form compound Q. For example, compound P can be treated with the sodium salt of the alcohol E. Additionally, compound P can be heated with the alcohol or phenol G-OH in the presence of CuI and CsCO3in an appropriate solvent to form compound Q.

Referring to Scheme 3, compound R is converted to compound S by condensation with triethyl orthoformate. The compound U is prepared from aryl halide compound S using aryl coupling conditions, such as Stille conditions with the N-alkyl-imidiazole stannane T.

In each of the above reaction procedures or schemes, the various substituents may be selected from among the various substituents otherwise taught herein.

Pharmaceutical Compositions

In certain embodiments, a substituted pyrido[3,4-d]pyrimidin-4-one derivative compound as described by Formula (I) or (II) is administered as a pure chemical. In other embodiments, the substituted pyrido[3,4-d]pyrimidin-4-one derivative compound as described by Formula (I) or (II) is combined with a pharmaceutically suitable or acceptable carrier (also referred to herein as a pharmaceutically suitable (or acceptable) excipient, physiologically suitable (or acceptable) excipient, or physiologically suitable (or acceptable) carrier) selected on the basis of a chosen route of administration and standard pharmaceutical practice as described, for example, inRemington: The Science and Practice of Pharmacy(Gennaro, 21stEd. Mack Pub. Co., Easton, Pa. (2005)), the disclosure of which is hereby incorporated herein by reference, in its entirety.

Accordingly, provided herein is a pharmaceutical composition comprising at least one substituted pyrido[3,4-d]pyrimidin-4-one derivative compound, or a stereoisomer, pharmaceutically acceptable salt, hydrate, solvate, or N-oxide thereof, together with one or more pharmaceutically acceptable carriers. The carrier(s) (or excipient(s)) is acceptable or suitable if the carrier is compatible with the other ingredients of the composition and not deleterious to the recipient (i.e., the subject) of the composition.

One embodiment provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of Formula (I) or a pharmaceutically acceptable salt thereof. One embodiment provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of Formula (II) or a pharmaceutically acceptable salt thereof.

In certain embodiments, the substituted pyrido[3,4-d]pyrimidin-4-one derivative compound as described by Formula (I) or (II) is substantially pure, in that it contains less than about 5%, or less than about 1%, or less than about 0.1%, of other organic small molecules, such as contaminating intermediates or by-products that are created, for example, in one or more of the steps of a synthesis method.

Suitable oral dosage forms include, for example, tablets, pills, sachets, or capsules of hard or soft gelatin, methylcellulose or of another suitable material easily dissolved in the digestive tract. Suitable nontoxic solid carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. (See, e.g.,Remington: The Science and Practice of Pharmacy(Gennaro, 21stEd. Mack Pub. Co., Easton, Pa. (2005)).

The dose of the composition comprising at least one substituted pyrido[3,4-d]pyrimidin-4-one derivative compound as described herein may differ, depending upon the patient's (e.g., human) condition, that is, stage of the disease, general health status, age, and other factors that a person skilled in the medical art will use to determine dose.

Pharmaceutical compositions may be administered in a manner appropriate to the disease to be treated (or prevented) as determined by persons skilled in the medical arts. An appropriate dose and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen provides the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (e.g., an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity. Optimal doses may generally be determined using experimental models and/or clinical trials. The optimal dose may depend upon the body mass, weight, or blood volume of the patient.

Oral doses can typically range from about 1.0 mg to about 1000 mg, one to four times, or more, per day.

Chromatin is the complex of DNA and protein that makes up chromosomes. Histones are the major protein component of chromatin, acting as spools around which DNA winds. Changes in chromatin structure are affected by covalent modifications of histone proteins and by non-histone binding proteins. Several classes of enzymes are known which can covalently modify histones at various sites.

Proteins can be post-translationally modified by methylation on amino groups of lysines and guanidino groups of arginines or carboxymethylated on aspartate, glutamate, or on the C-terminus of the protein. Post-translational protein methylation has been implicated in a variety of cellular processes such as RNA processing, receptor mediated signaling, and cellular differentiation. Post-translational protein methylation is widely known to occur on histones, such reactions known to be catalyzed by histone methyltransferases, which transfer methyl groups from S-adenyosyl methionine (SAM) to histones. Histone methylation is known to participate in a diverse range of biological processes including heterochromatin formation, X-chromosome inactivation, and transcriptional regulation (Lachner et al., (2003) J. Cell Sci. 116:2117-2124; Margueron et al., (2005) Curr. Opin. Genet. Dev. 15:163-176).

Unlike acetylation, which generally correlates with transcriptional activation, whether histone methylation leads to transcription activation or repression depends on the particular site of methylation and the degree of methylation (e.g., whether a particular histone lysine residue is mono-, di-, or tri-methylated). However, generally, methylation on H3K9, H3K27 and H4K20 is linked to gene silencing, while methylation on H3K4, H3K36, and H3K79 is generally associated with active gene expression. In addition, tri- and di-methylation of H3K4 generally marks the transcriptional start sites of actively transcribed genes, whereas mono-methylation of H3K4 is associated with enhancer sequences.

A “demethylase” or “protein demethylase,” as referred to herein, refers to an enzyme that removes at least one methyl group from an amino acid side chain. Some demethylases act on histones, e.g., act as a histone H3 or H4 demethylase. For example, an H3 demethylase may demethylate one or more of H3K4, H3K9, H3K27, H3K36 and/or H3K79. Alternately, an H4 demethylase may demethylate histone H4K20. Demethylases are known which can demethylate either a mono-, di- and/or a tri-methylated substrate. Further, histone demethylases can act on a methylated core histone substrate, a mononucleosome substrate, a dinucleosome substrate and/or an oligonucleosome substrate, peptide substrate and/or chromatin (e.g., in a cell-based assay).

The first lysine demethylase discovered was lysine specific demethylase 1 (LSD1/KDM1), which demethylates both mono- and di-methylated H3K4 or H3K9, using flavin as a cofactor. A second class of Jumonji C (JmjC) domain containing histone demthylases were predicted, and confirmed when a H3K36 demethylase was found using a formaldehyde release assay, which was named JmjC domain containing histone demethylase 1 (JHDM1/KDM2A).

More JmjC domain-containing proteins were subsequently identified and they can be phylogenetically clustered into seven subfamilies: JHDM1, JHDM2, JHDM3, JMJD2, JARID, PHF2/PHF8, UTX/UTY, and JmjC domain only.

The JMJD2 family of proteins are a family of histone-demethylases known to demethylate tri- and di-methylated H3-K9, and were the first identified histone tri-methyl demethylases. In particular, ectopic expression of JMJD2 family members was found to dramatically decrease levels of tri- and di-methylated H3-K9, while increasing levels of mono-methylated H3-K9, which delocalized Heterochromatin Protein 1 (HP1) and reduced overall levels of heterochromatin in vivo. Members of the JMJD2 subfamily ofjumonji proteins include JMJD2C and its homologues JMJD2A, JMJD2B, JMJD2D and JMJD2E. Common structural features found in the JMJD2 subfamily of Jumonji proteins include the JmjN, JmjC, PHD and Tdr sequences.

JMJD2C, also known as GASC1 and KDM4C, is known to demethylate tri-methylated H3K9 and H3K36. Histone demethylation by JMJD2C occurs via a hydroxylation reaction dependent on iron and α-ketoglutarate, wherein oxidative decarboxylation of α-ketoglutarate by JMJD2C produces carbon dioxide, succinate, and ferryl and ferryl subsequently hydroxylates a methyl group of lysine H3K9, releasing formaldehyde. JMJD2C is known to modulate regulation of adipogenesis by the nuclear receptor PPARγ and is known to be involved in regulation of self-renewal in embryonic stem cells.

As used herein, a “JARID protein” includes proteins in the JARID1 subfamily (e.g., JARID1A, JARID B, JARID C and JARID D proteins) and the JARID2 subfamily, as well as homologues thereof. A further description and listing of JARID proteins can be found in Klose et al. (2006) Nature Reviews/Genetics 7:715-727. The JARID1 family contains several conserved domains: JmjN, ARID, JmjC, PHD and a C5HC2 zing finger.

JARID1A, also called KDM5A or RBP2, was initially found as a binding partner of retinoblastoma (Rb) protein. JARID1A was subsequently found to function as a demethylase of tri- and di-methylated H3K4, and has been found to promote cell growth, while inhibiting senescence and differentiation. For instance, abrogation of JARID1A from mouse cells inhibits cell growth, induces senescence and differentiation, and causes loss of pluripotency of embryonic stem cells in vitro. JARID1A has been found to be overexpressed in gastric cancer and the loss of JARID1A has been found to reduce tumorigenesis in a mouse cancer model. Additionally, studies have demonstrated that loss of the retinoblastome binding protein 2 (RBP2) histone demethylase suppresses tumorigenesis in mice lacking Rbl or Menl (Lin et al. Proc. Natl. Acad. Sci. USA, Aug. 16, 2011, 108(33),13379-86; doi: 10.1073/pnas.1110104108) and lead to the conclusion that RBP2-inhibitory drugs would have anti-cancer activity.

JARID1B, also referred to as KDM5B and PLU1, was originally found in experiments to discover genes regulated by the HER2 tyrosine kinase. JARID1B has consistently been found to be expressed in breast cancer cell lines, although restriction of JARID1B has been found in normal adult tissues, with the exception of the testis. In addition, 90% of invasive ductal carcinomas have been found to express JARID1B. In addition, JARID1B has been found to be up-regulated in prostate cancers, while having more limited expression in benign prostate, and has also been found to be up-regulated in bladder cancer and lung cancer (both SCLC and NSCLC). JARID1B has also been found to repress tumor suppressor genes such as BRCA1, CAV1 and 14-3-30, and knockdown of JARID1B was found to increase the levels of tri-methylated H3K4 at these genes.

In an additional embodiment is a method for inhibiting a histone-demethylase enzyme comprising contacting a histone demethylase enzyme with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In an additional embodiment is a method for inhibiting a histone-demethylase enzyme comprising contacting a histone demethylase enzyme with a compound of Formula (II) or a pharmaceutically acceptable salt thereof

In an additional embodiment is the method for inhibiting a histone-demethylase enzyme, wherein the histone-demethylase enzyme comprises a JmjC domain. In an additional embodiment is the method for inhibiting a histone-demethylase enzyme, wherein the histone-demethylase enzyme is selected from JARID1A, JARID1B, JMJD2C, or JMJD2A.

Methods of Treatment

Disclosed herein are methods of modulating demethylation in a cell or in a subject, either generally or with respect to one or more specific target genes. Demethylation can be modulated to control a variety of cellular functions, including without limitation: differentiation; proliferation; apoptosis; tumorigenesis, leukemogenesis or other oncogenic transformation events; hair loss; or sexual differentiation. For example, in particular embodiments, the invention provides a method of treating a disease regulated by histone methylation and/or demethylation in a subject in need thereof by modulating the activity of a demethylase comprising a JmjC domain (e.g., a histone demethylase such as a JHDM protein(s)).

In an additional embodiment is a method for treating cancer in subject comprising administering a composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In an additional embodiment is a method for treating cancer in subject comprising administering a composition comprising a compound of Formula (II) or a pharmaceutically acceptable salt thereof

In a further embodiment is the method for treating cancer in a subject wherein the cancer is selected from prostate cancer, breast cancer, bladder cancer, lung cancer or melanoma.

In an additional embodiment is a method for inhibiting the growth of a tumor comprising administering a composition comprising a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, wherein the tumor is characterized by a loss of retinoblastoma gene (RB1) function.

In an additional embodiment is a method for inhibiting the growth of a tumor comprising administering a composition comprising a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, wherein the tumor is characterized by a loss of multiple endocrine neoplasia type 1 gene (Menl) function.

Other embodiments and uses will be apparent to one skilled in the art in light of the present disclosures. The following examples are provided merely as illustrative of various embodiments and shall not be construed to limit the invention in any way.

EXAMPLES

I. Chemical Synthesis

Unless otherwise noted, reagents and solvents were used as received from commercial suppliers. Anhydrous solvents and oven-dried glassware were used for synthetic transformations sensitive to moisture and/or oxygen. Yields were not optimized.

Reaction times are approximate and were not optimized. Column chromatography and thin layer chromatography (TLC) were performed on silica gel unless otherwise noted. Spectra are given in ppm (δ) and coupling constants, J are reported in Hertz. For proton spectra the solvent peak was used as the reference peak.

To a solution of 2,4-dichloropyrido[3,4-d]pyrimidine (1 g, 5 mmol) in THF (20 mL) was added a solution of NaOH (0.5 g, 12.5 mmol) in water (20 mL). The reaction mixture was stirred at rt for 2 h. The solution was adjusted to pH=2 using 5N HCl and the resulting precipitate was filtered and washed with water and THF, and dried to give 0.8 g (88%) of the title compound.1H NMR (400 MHz, DMSO-d6): δ 13.61 (s, 1H), 8.99 (s, 1H), 8.69 (d, 1H, J=5.2 Hz), 7.94 (d, 1H, J=5.2 Hz).

The title compound was prepared in 16% yield from cyclobutylmethanol and 2-chloropyrido[3,4-d]pyrimidin-4-ol according to the procedure for the preparation of Example 10. [M+H] Calc'd for C12H13N3O2, 232. Found, 232.

A solution of 1-(2-methoxyethyl)-1H-indazol-6-amine (1.0 g, 5.23 mmol) in H2SO4/H2O (1:1, 15 mL) was cooled to 0° C. and a solution of NaNO2(0.36 g, 5.23 mmol) in H2O (1.5 mL) was added dropwise. This dark solution was stirred for 2 h and water (5 mL) was added and then heated at 110° C. for 2 h. The reaction was cooled to rt, carefully neutralized with a saturated solution of NaHCO3and extracted with ethyl acetate. The extracts were washed with brine, dried, and evaporated. The residue was purified by silica gel chromatography (0 to 100% EtOAc:Hexanes) to give 620 mg (62%) the title compound as a white solid. [M+H] Calc'd for C10H12N2O2, 193. Found, 193.

To a solution of 1-ethyl-1H-pyrazole-4-boronic acid, pinacol ester (320 mg, 1.44 mmol) in acetone/H2O (5 mL, 1:1) was added NaIO4(925 mg, 4.32 mmol) and NH4OAc (277 mg, 3.60 mmol). The reaction mixture was stirred at rt for 16 h and concentrated in vacuo. The crude was purified by gel chromatography (5% MeOH:DCM) to give 127 mg (60%) of the title compound as yellow oil. [M+H] Calc'd for C5H9BN2O2, 141. Found, 141.

A mixture of 1-ethylpyrazole-4-boronic acid (127 mg, 0.90 mmol), AcOH (0.35 mL), H2O2(0.32 mL), H2O (0.32 mL) in Et2O (5 mL) was stirred at rt for 1 h and then refluxed for 16 h. The pH was adjusted to 7 using aqueous NaHCO3. The solution was concentrated in vacuo and purified by gel chromatography (5% MeOH:DCM) to give 30 mg of the title compound (30%) as colorless oil. [M+H] Calc'd for C5H8N2O, 113. Found, 113.

To a solution of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.0 g, 5.2 mmol) in DMF (10 mL) was added NaH (0.3 g, 7.5 mmol) at 0° C. and the mixture was stirred at rt for 30 min. Tetrahydro-2H-pyran-4-yl methanesulfonate (1.1 g, 6.1 mmol) was added and the mixture was stirred at 110° C. overnight. The reaction mixture was cooled to rt and filtered. The filtrate was concentrated and the residue was purified by silica gel chromatography (30% EA:PE) to give 550 mg of the title compound (40%). [M+H] Calc'd for C14H23BN2O3, 279. Found, 279.

The title compound was prepared in 67% yield from 1-ethylindazol-6-amine according to the procedure for the preparation of 61A. [M+H] Calc'd for C9H10N2O, 163. Found, 163.

The title compound was prepared in 57% yield from 1,3-dimethylindazol-6-amine according to the procedure for the preparation of 61A. [M+H] Calc'd for C9H10N2O, 163. Found, 163.

The title compound was prepared in 38% yield from 2-fluorobenzyl bromide according to the procedure for the preparation 86A. [M+H] Calc'd for C16H20BFN2O2, 303. Found, 303.

The title compound was prepared in 82% yield from 1-(2-fluorobenzyl)-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-pyrazole according to the procedure for the preparation 66A. [M+H] Calc'd for C10H9FN2O, 193. Found, 193.

The title compound was prepared in 22% yield from 1-bromoethylbenzene according to the procedure for the preparation 86A. [M+H] Calc'd for C17H23BN2O2, 299. Found, 299.

The title compound was prepared in 67% yield from 1-(1-phenylethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole according to the procedure for the preparation 66A. [M+H] Calc'd for C11H12N2O, 189. Found, 189.

To a mixture of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.97 g, 5 mmol), 1-Phenyl-1-propanol (1.36 g, 10 mmol) and triphenylphosphine (2.63 g, 10 mmol) in THF (50 mL) was slowly added a solution of di-tertbutyl azodicarboxylate (2.3 g, 10 mmol) in THF (5 mL). The reaction solution was stirred for 30 min at reflux and concentrated. The residue was purified by silica gel chromatography (0-30%, EA:Hexanes) to give 1.36 g (45%) of the title compound as yellow oil. [M+H] Calc'd for C18H25BN2O2, 313. Found, 313.

1-(1-phenylpropyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole (1.9 g, 6.9 mmol) was dissolved in THF (20 mL) and cooled to 0 OC. NaOH 2.5 M (6 mL, 15.8 mmol) and H2O230 percent solution in water (1.6 mL, 15.8 mmol) were added and the reaction mixture was stirred at room temperature for 45 min. Then the pH was adjusted to 2 by the addition of aqueous 2N HCl and the mixture was extracted with dichloromethane. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give the title compound 1-benzyl-1H-pyrazol-4-ol as an off-white solid (0.56 g, 40%). [M+H] Calc'd for C12H14N2O, 203. Found, 203.

To a mixture of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.97 g, 5 mmol), 1-α-cyclopropylbenzyl alcohol (1.04 g, 7 mmol) and triphenylphosphine (1.45 g, 5.5 mmol) in THF (10 mL) was slowly added a solution of di-tertbutyl azodicarboxylate (1.15 g, 5 mmol) in THF (5 mL). The reaction solution was stirred for 30 min and concentrated. The residue was purified by silica gel chromatography (0-30%, EA:Hexanes) to give 1.12 g (70%) of the title compound as yellow oil. [M+H] Calc'd for C19H25BN2O2, 325. Found, 325.

1-[cyclopropyl(phenyl)methyl]-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole (1.12 g, 3.6 mmol) was dissolved in THF (10 mL) and cooled to 0° C. NaOH 2.5 M (2.9 mL, 7.2 mmol) and H2O230 percent solution in water (0.8 mL, 7.2 mmol) were added and the reaction mixture was stirred at room temperature for 45 min. Then the pH was adjusted to 2 by the addition of aqueous 2N HCl and the mixture was extracted with dichloromethane. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give the title compound 1-benzyl-1H-pyrazol-4-ol as an off-white solid (0.48 g, 62%). [M+H] Calc'd for C13H14N2O, 215. Found, 215.

To a mixture of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (2 g, 10 mmol), 2-chlorobenzyl alcohol (2.9 g, 20 mmol) and triphenylphosphine (5.3 g, 20 mmol) in THF (50 mL) was slowly added a solution of di-tertbutyl azodicarboxylate (4.6 g, 20 mmol) in THF (10 mL). The reaction solution was stirred for 30 min at reflux and concentrated. The residue was purified by silica gel chromatography (0-30%, EA:Hexanes) to give 1.9 g (59%) of the title compound as yellow oil. [M+H] Calc'd for C16H20BClN2O2, 319. Found, 319.

1-[(2-chlorophenyl)methyl]-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole (1.9 g, 6.9 mmol) was dissolved in THF (20 mL) and cooled to 0 OC. NaOH 2.5 M (6 mL, 15.8 mmol) and H2O230 percent solution in water (1.6 mL, 15.8 mmol) were added and the reaction mixture was stirred at room temperature for 45 min. Then the pH was adjusted to 2 by the addition of aqueous 2N HCl and the mixture was extracted with dichloromethane. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give the title compound 1-benzyl-1H-pyrazol-4-ol as an off-white solid (0.48 g, 34%). [M+H] Calc'd for C10H9ClN2O, 209. Found, 209.

To a mixture of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (2 g, 10 mmol), 3-chlorobenzyl alcohol (2.9 g, 20 mmol) and triphenylphosphine (5.3 g, 20 mmol) in THF (50 mL) was slowly added a solution of di-tertbutyl azodicarboxylate (4.6 g, 20 mmol) in THF (10 mL). The reaction solution was stirred for 30 min at reflux and concentrated. The residue was purified by silica gel chromatography (0-30%, EA:Hexanes) to give 2.9 g (91%) of the title compound as yellow oil. [M+H] Calc'd for C16H20BClN2O2, 319. Found, 319.

1-[(3-chlorophenyl)methyl]-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole (2.9 g, 9.1 mmol) was dissolved in THF (20 mL) and cooled to 0 OC. NaOH 2.5 M (7.9 mL, 18.3 mmol) and H2O230 percent solution in water (2.1 mL, 18.3 mmol) were added and the reaction mixture was stirred at room temperature for 45 min. Then the pH was adjusted to 2 by the addition of aqueous 2N HCl and the mixture was extracted with dichloromethane. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give 0.56 g (40%), of the title compound as an off-white solid. [M+H] Calc'd for C10H9ClN2O, 209. Found, 209.

The title compound was prepared in 45% yield from 4-[2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazol-1-yl]ethyl]morpholine according to the procedure for the preparation 115 B. [M+H] Calc'd for C9H15N3O2, 198. Found, 198.

To a solution of 2-[1-((3S)pyrrolidin-3-yl)pyrazol-4-yl]pyridino[3,4-d]pyrimidin-4-ol (150 mg, 0.50 mmol) and DIEA (1 mL) in THF was added chloro-acetyl chloride (68 mg, 0.60 mmol) at 0° C. The reaction mixture was stirred for 2 h at RT, filtered and concentrated. The residue was purified by FC (20:1, DCM:MeOH) to give 188 mg (91%) of the title compound.

The title compound was prepared in prepared in 85% yield from 2-[1-((3S)pyrrolidin-3-yl)pyrazol-4-yloxy]pyridino[3,4-d]pyrimidin-4-ol and acetic acid chlorocarbonylmethyl ester according to the procedure for the preparation of Example 126. [M+H] Calc'd for C188H1N6O5, 399. Found, 399.

II. Biological Evaluation

Example 1: In Vitro Enzyme Inhibition Assay

This assay determines the ability of a test compound to inhibit Jarid1A, Jarid1B, and JMJD2C demethylase activity. Baculovirus expressed Jarid1A (GenBank Accession #NM_001042603, AA1-1090) was purchased from BPS Bioscience (Cat#50110). Baculovirus expressed Jarid1B (GenBank Accession #NM_006618, AA 2-751) was purchased from BPS Bioscience (Cat #50121) or custom made by MolecularThroughput. Baculovirus expressed JMJD2C (GenBank Accession #BC 143571, AA 2-372) was purchased from BPS Bioscience (Cat#50105).

The enzymatic assay of Jarid1A activity is based upon Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) detection. The ability of test compounds to inhibit the activity of Jarid1A was determined in 384-well plate format under the following reaction conditions: 1 nM Jarid1A, 300 nM H3K4me3-biotin labeled peptide (Anaspec cat #64357), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-mono- or di-methylated histone H3 lysine 4 (H3K4me1-2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 25 nM and 1 nM, respectively.

The assay reaction was initiated by the following: 2 μl of the mixture of 900 nM H3K4me3-biotin labeled peptide and 6 μM alpha-ketoglutaric acid with 2 μl of 11-point serial diluted inhibitor in 3% DMSO was added to each well of plate, followed by the addition of 2 μl of 3 nM Jarid1A to initiate the reaction. The reaction mixture was incubated at room temperature for 30 minutes, and terminated by the addition of 6 μl of 5 mM EDTA in LANCE detection buffer containing 50 nM Phycolink Streptavidin-allophycocyanin and 2 nM Europium-anti-H3K4me1-2 antibody. Plates were read by EnVisionMultilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hour incubation at room temperature. A ratio was calculated (665/615) for each well and fitted to determine inhibition constant (IC50).

The ability of test compounds to inhibit the activity of Jarid1B was determined in 384-well plate format under the following reaction conditions: 0.8 nM Jarid1B, 300 nM H3K4me3-biotin labeled peptide (Anaspec cat #64357), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 M sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-mono- or di-methylated histone H3 lysine 4 (H3K4me1-2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 25 nM and 1 nM, respectively.

The assay reaction was initiated by the following: 2 μl of the mixture of 900 nM H3K4me3-biotin labeled peptide and 6 μM alpha-ketoglutaric acid with 2 μl of 11-point serial diluted inhibitor in 3% DMSO was added to each well of the plate, followed by the addition of 2 μl of 2.4 nM Jarid1B to initiate the reaction. The reaction mixture was incubated at room temperature for 30 minutes, and terminated by the addition of 6 μl of 5 mM EDTA in LANCE detection buffer containing 50 nM Phycolink Streptavidin-allophycocyanin and 2 nM Europium-anti-H3K4me1-2 antibody. Plates were read by EnVisionMultilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hour incubation at room temperature. A ratio was calculated (665/615) for each well and fitted to determine inhibition constant (IC50).

The ability of test compounds to inhibit the activity of JMJD2C was determined in 384-well plate format under the following reaction conditions: 0.3 nM JMJD2C, 300 nM H3K9me3-biotin labeled peptide (Anaspec cat #64360), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-di-methylated histone H3 lysine 9 (H3K9me2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 50 nM and 1 nM, respectively.

The assay reaction was initiated by the following: 2 μl of the mixture of 900 nM H3K9me3-biotin labeled peptide and 6 μM alpha-ketoglutaric acid with 2 μl of 11-point serial diluted inhibitor in 3% DMSO were added to each well of the plate, followed by the addition of 2 μl of 0.9 nM JMJD2C to initiate the reaction. The reaction mixture was incubated at room temperature for 30 minutes, and terminated by the addition of 6 μl of 5 mM EDTA in LANCE detection buffer containing 100 nM Phycolink Streptavidin-allophycocyanin and 2 nM Europium-anti-H3K9me2 antibody. Plates were read by EnVisionMultilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hour incubation at room temperature. A ratio was calculated (665/615) for each well and fitted to determine inhibition constant (IC50).

The ability of the compounds disclosed herein to inhibit demethylase activity was quantified and the respective IC50value was determined. Table 3 provides the IC50values of various compounds disclosed herein.

Example 2: In Vitro Cell-Based Assay

An assay to measure the degree of cellular inhibition of KDM5A and 5B was developed. This quantitative immuno-blotting assay measures the amount tri-methylated histone H3 at amino acid Lysine number 4, a specific substrate and product of the direct enzymatic activity of the histone demethylases KDM5A and KDM5B from extracts of the ZR-75-1 breast cancer cell line.

Assay Principle

This assay is a fluorometric immunoassay for the quantification of tri-methyl H3K4 extracted from cells treated with test compound and is used as a measure of the cellular inhibition of KDM5A/B.

Assay Method

ZR-75-1(PTEN null, ER+) breast cancer cells numbering 50,000 (ATCC) were seeded into each well of a 96-well tissue culture treated plate and then exposed to an 11 point dilution of test compound with final concentration ranges of test compound ranging from 1250 μM to 10 nM. Cells were left in the presence of test compound for 72 hours. Extracts were prepared containing all of the cellular histone material using detergent based lysis and sonication methods. These lysates were subsequently normalized for total protein content using a colorimetric bicinchonic acid assay (MicroBCA Pierce/Thermo Scientific). Normalized cell extracts were then subjected to typical immuno-blotting procedures using NuPage reagents (Life Technologies). Electrophoretically separated histones were then transferred and immobilized using polyvinylidene difluoride membrane (Immobilon-FL Millipore). The amount of tri-methylated lysine 4 of histone H3 was detected using an antibody specific to the tri-methylated state (Cell Signaling Technologies) and quantified on an infrared imager using a densitometry software package (Odyssey CLx, Image Studio, Li-Cor). This background subtracted densitometry value was reported as a ration of the GAPDH amount for that sample and then calculated as a percent of the DMSO treated sample. The software package XL-fit (IDBS) was then used to calculate a relative IC50value for the dilution series of a given test compound according to the equation:
fit=(D+((Vmax*(x^n))/((x^n)+(Km^n)))).

Table 4 provides the cellular IC50values of various compounds disclosed herein.

Example 3: In Vivo Xenograph Study

Time release pellets containing 0.72 mg 17-β Estradiol are subcutaneously implanted into nu/nu mice. MCF-7 cells are grown in RPMI containing 10% FBS at 5% CO2, 37° C. Cells are spun down and re-suspended in 50% RPMI (serum free) and 50% Matrigel at 1×107cells/mL. MCF-7 cells are subcutaneously injected (100 μL/animal) on the right flank 2-3 days post pellet implantation and tumor volume (length×width2/2) is monitored bi-weekly. When tumors reach an average volume of ˜200 mm3animals are randomized and treatment is started. Animals are treated with vehicle or compound daily for 4 weeks. Tumor volume and body weight are monitored bi-weekly throughout the study. At the conclusion of the treatment period, plasma and tumor samples are taken for pharmacokinetic and pharmacodynamic analyses, respectively.

III. Preparation of Pharmaceutical Dosage Forms

Example 1: Oral Tablet

A tablet is prepared by mixing 48% by weight of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, 45% by weight of microcrystalline cellulose, 5% by weight of low-substituted hydroxypropyl cellulose, and 2% by weight of magnesium stearate. Tablets are prepared by direct compression. The total weight of the compressed tablets is maintained at 250-500 mg.