WO2015112739A9 - Compounds and method for treating parp1-deficient cancers - Google Patents

Abstract

Disclosed are compounds and a method of treating cancer that is PARP1-deficient compared to a wild-type form of the same cancer, the method involving administering an effective amount of a compound selected from and, in which ring A1, ring A2, Q, R1?-R8?, m, n, o, and p are as defined herein.

Description

COMPOUNDS AND METHOD FOR TREATING PARPl -DEFICIENT CANCERS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 61/988,502, filed May 5, 2014, and U.S. Provisional Patent Application No. 61/930,291 , filed January 22, 2014, both of which are incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] Poly (ADP-ribose) polymerase (PARPl), a highly conserved DNA binding protein, is key in maintaining genomic stability, repairing DNA damage, and regulating transcriptional processes. PARPl is recognized as a critical mediator of chromosomal translocations and also plays important roles in multiple DNA damage response pathways. Many cancer therapies utilize DNA-damaging agents to kill tumor cells, which often triggers DNA repair (e.g., by activating PARPl pathways) and renders the cancer cells resistant to the therapies. Therefore, PARPl inhibitors can be applied either as useful sensitizers to increase the efficacy of DNA-damaging agents in general cancer therapy or for selectively targeting certain types of cancer cells with specific DNA repair defects. Although PARPl inhibitors have unprecedented therapeutic potential for cancer treatment, tumor resistance to these drugs can develop due to a defect in PARPl expression, such as PARPl V276A polymorphism, or in some cancers a reversion of the BRCA mutation (see, e.g., Chiarugi, Trends in

Pharmacological Sciences, 33(1): 42-48 (January 2012)).

[0003] Since various PARPl inhibitors are under clinical trials by multiple

pharmaceutical companies, there is a need for compounds that sensitize PARPl deficiency for use in combination with PARPl inhibitors in treating cancer. In addition, there is a need for using such compounds as a single therapeutic agent for treating PARPl deficient tumors.

BRIEF SUMMARY OF THE INVENTION

[0004] The invention provides a method of treating cancer in a subject comprising administering an effective amount of a compound selected from

as described herein, wherein the cancer is PARPl -deficient compared to a wild-type form of the same cancer.

[0005] The invention also provides a method of treating cancer in a subject comprising administering an effective amount of a compound selected from

wherein the cancer is treatable with a PARPl inhibitor.

[0006] Further provided is a compound of formula (I) or (II)

(I)

which are described herein. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0007] Figure 1 is a reaction scheme illustrating the preparation of N-(3,4- difluorophenyl)-2-pyridin-4-ylquinazolin-4-amine (ML367), a compound in accordance with an embodiment of the invention.

[0008] Figure 2 is a series of graphs illustrating the stability of ML367 measured as a percent composition of probe molecule in aqueous solution (contains 20% acetonitrile) at room temperature over 48 hr. Figure 2A includes measurements in (A) DPBS buffer at pH 7.4 and (B) assay buffer. Figure 2B includes measurements in (C) buffer at pH 2 and (D) buffer at pH 10. Figure 2C includes measurement in (E) 5 mM solution of glutathione over

24 hr.

[0009] Figure 3 is a graph illustrating a dose response curve for ML367 against the ATAD5-Luc primary screen (□) and HEK293 cell viability assay (0).

[0010] Figure 4 illustrates ATAD5 protein levels that were visualized by western blotting using an antibody against FLAG (A), and quantified using ImageJ (E).

DETAILED DESCRIPTION OF THE INVENTION

[0011] Enhanced level of genome instability 1 (Elgl), also known as ATAD5, functions to correct DNA sequence errors generated during cell division. ATAD5 is a good biomarker to detect DNA damage in cells. As a result, a series of compounds that trigger a DNA damage response has been discovered using a cell-based assay with ATAD5 as a biomarker. Accordingly, the invention provides a method of treating cancer in a subject comprising administering an effective amount of a compound selected from

wherein

ring Al is substituted aryl, unsubstituted aryl, substituted heteioaryl, or unsubstituted heteroaryl; ring A2 is substituted heterocyclyl or unsubstituted heterocyclyl; Q is NH, O, or S;

R', R⁴, R⁵, and R⁶ are each independently selected from H, alkyl, alkenyl, cycloalkyl, and aryl, wherein any of the foregoing moieties other than H is optionally substituted;

R², R³, R⁷, and R⁸ are each independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkyl, heteroaryl, halo, haloalkyl, haloalkoxy, aryloxy, alkanoyloxy, hydroxy, alkoxy, cycloalkyloxy, heterocylalkyloxy, alkanoyl, aroyl, arylalkyl, alkylaryl, heteroarylalkyl, alkylheteroaryl, amino, alkylamino, arylamino, arylalkylamino, cycloalkylamino, heterocycloalkylamino, alkanoylamino, aroylamino, aralkanoylamino, thiol, alkylthio, arylthio, arylalkylthio, cycloalkylthio, heterocycloalkylthio, alkylthiono, arylthiono, arylalkylthiono, alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, sulfonamido, nitro, cyano, carboxy, alkoxycarbonyl, carbamido, and carbamyl, wherein any of the foregoing moieties is optionally substituted;

m and n are each independently 0 or 1 to 4; and

o and p are each independently 0 or 1 to 3;

or a pharmaceutically acceptable salt thereof, to a subject in need thereof, wherein the cancer is PARP1 -deficient compared to a wild-type form of the same cancer.

[0012] The invention also provides a method of treating cancer in a subject comprising administering an effective amount of a compound selected from

wherein

ring Al is substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

ring A2 is substituted heterocyclyl or unsubstituted heterocyclyl;

Q is NH, O, or S;

R' , R⁴, R⁵, and R⁶ are each independently selected from H, alkyl, alkenyl, cycloalkyl, and aryl, wherein any of the foregoing moieties other than H is optionally substituted ; R², R³, R⁷, and R⁸ are each independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkyl, heteroaryl, halo, haloalkyl, haloalkoxy, aryloxy, alkanoyloxy, hydroxy, alkoxy, cycloalkyloxy, heterocylalkyloxy, alkanoyl, aroyl, arylalkyl, alkylaryl, heteroarylalkyl, alkylheteroaryl, amino, alkylamino, arylamino, arylalkylamino, cycloalkylamino, heterocycloalkylamino, alkanoylamino, aroylamino, aralkanoylamino, thiol, alkylthio, arylthio, arylalkylthio, cycloalkylthio, heterocycloalkylthio, alkylthiono, arylthiono, aryl alkylthiono, alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, sulfonamido, nitro, cyano, carboxy, alkoxycarbonyl, carbamido, and carbamyl, wherein any of the foregoing moieties is optionally substituted;

m and n are each independently 0 or 1 to 4; and

o and p are each independently 0 or 1 to 3;

or a pharmaceutically acceptable salt thereof, to a subject in need thereof, wherein the cancer is treatable with a PARP1 inhibitor.

[0013] In any of the foregoing methods, ring Al is substituted aryl or unsubstituted aryl, such as phenyl. The aryl (e.g., phenyl) can be substituted with a group described herein, such as halo, haloalkyl, alkyl, alkoxy, or amino. Preferably, ring Al is halo-substituted phenyl (e.g., 2-halophenyl, 3-halophenyl, 4-halophenyl, 2,3-dihalophenyl, 2,4-dihalophenyl, 2,5- dihalophenyl, 3,4-dihalophenyl, 3,5-dihalophenyl, 2,3,4-trihalophenyl, and 2,3,5- trihalophenyl). In a preferred embodiment, the halo-substituted phenyl is a fluoro-substituted phenyl (e.g., 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2,3-difluorophenyl, 2,4- di fluorophenyl, 2,5-difluorophenyl, 3,4-difluorophenyl, 3,5-difluorophenyl, 2,3,4- trifluorophenyl, or 2, 3, 5 -tri fluorophenyl). Ring Al can also be substituted heteroaryl or unsubstituted heteroaryl, in which the heteroaryl is as described herein (e.g., pyrazolyl, alkyl- substituted pyrazolyl, aryl-substituted pyrazolyl, oxazolyl, isooxazolyl, thiazolyl,

benzoimidazolyl, alkyl-substituted benzoimidazolyl, benzothiazolyl, or alkyl-substituted benzothiazolyl).

[0014] In any of the foregoing methods, Q is NH.

[0015] In any of the foregoing methods, R¹ is H or alkyl.

[0016] In any of the foregoing methods, each R is halo, haloalkyl, alkyl, alkoxy, or amino.

[0017] In any of the foregoing methods, each R is halo, haloalkyl, alkyl, alkoxy, amino, alkylamino, or substituted or unsubstituted heterocycloalkyl. [0018] In any of the foregoing methods, m is 0, 1 , 2, 3, or 4. Preferably, m is 1 or 2, and most preferably, m is 2.

[0019] In any of the foregoing methods, n is 0, 1 , 2, 3, or 4. Preferably, n is 0.

[0020] In any of the foregoing methods, ring A2 is substituted or unsubstituted heterocyclyl (i.e., heterocycloalkyl or heteroaryl). Suitable moieties for ring A2 include, e.g., imidazolinyl, thiazolinyl, imidazolidinyl, pyrrolinyl, pyranyl, piperidyl, morpholinyl, triazolyl, pyridinyl, pyridazinyl, pyrimidyl, pyrazinyl, benzimidazolyl, triazinyl, imidazolyl, (1,2,3)- and (l,2,4)-triazolyl, pyrazinyl, tetrazolyl, furyl, pyrrolyl, thienyl, isothiazolyl, thiazolyl, isoxazolyl, and oxadiazolyl.

[0021] In any of the foregoing methods, R⁴ and R⁵ are the same or different and each is H or alkyl.

[0022] In any of the foregoing methods, each R⁶ is H or alkyl.

[0023] In any of the foregoing methods, each R⁷ is alkyl, alkoxy, or alkanoyl.

[0024] In any of the foregoing methods, each R⁸ is halo, haloalkyl, alkyl, alkoxy, or amino.

[0025] In any of the foregoing methods, o is 0, 1 , 2, or 3. Preferably, o is 2 or 3, and most preferably, o is 3.

[0026] In any of the foregoing methods, p is 0, 1, 2, or 3. Preferably, p is 0.

[0027] In an aspect, the method further comprises administering an effective amount of a PARP1 inhibitor. For example, the PARP1 inhibitor can be olaparib, veliparib, KU0058958, XAV939, IWRl , IWR2, 3-(4-chlorophenyl)quinoxaline-5-carboxamide, 3-methyl-5-AIQ hydrochloride, 6(5H)-phenanthridinone, 3-aminobenzamide (3AB), 2-nitro-6(5H)- phenanthridinone (2NP), or 4-amino-l ,8-naphthalimide (4AN).

Additional PARP1 inhibitors are set forth in Papeo et al. (Expert Opin. Them. Patents, 23(4): 503-514 (April 2013)), the contents of which are hereby incorporated by reference. In an aspect, the PARP1 inhibitor is olaparib.

[0028] In some embodiments, the combination of a compound described herein and a PARP 1 inhibitor provides synergistic effects. For example, Applicants discovered that a combination of a PARP1 inhibitor and N-(3,4-difluorophenyl)-2-pyridin-4-ylquinazolin-4- amine synergistically killed rapidly dividing cells. Thus, a compound described herein (e.g., N-(3,4-difluorophenyl)-2-pyiidin-4-ylquinazolin-4-amine or 7V-(5-phenyl-7H-pyrazol-3-yl)-2- (pyridin-4-yl)quinazolin-4-amine) would be a desirable combinatorial treatment option with a PARP 1 inhibitor (e.g., olaparib and/or 4-amino-l ,8-naphthalimide (4AN)) for treating rapidly dividing cancer cells.

foregoing methods, embodiments of the compound include

-(3,4-difluorophenyl)-2-pyridin-4-ylquinazolin-4-amine)

-(5-phenyl-iH-pyrazol-3-yl)-2-(pyridin-4-yl)quinazolin-4-amine), or

Z265 (4-acetyl-7V-[5-(diethylsulfamoyl)-2-morpholin-4-ylphenyl]-3,5-dimethyl-7H-pyrrole-2- carboxamide). In preferred treatment methods, the compound is 7V-(3,4-difluorophenyl)-2- pyridin-4-ylquinazolin-4-amine (ML367). [0030] The invention also provides a compound of formula (I) or (II)

wherein

ring Al is substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

ring A2 is substituted heterocyclyl or unsubstituted heterocyclyl;

Q is NH, O, or S;

R¹ , R⁴, R⁵, and R⁶ are each independently selected from H, alkyl, alkenyl, cycloalkyl, and aryl, wherein any of the foregoing moieties other than H is optionally substituted;

R², R³, R⁷, and R⁸ are each independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkyl, heteroaryl, halo, haloalkyl, haloalkoxy, aryloxy, alkanoyloxy, hydroxy, alkoxy, cycloalkyloxy, heterocylalkyloxy, alkanoyl, aroyl, arylalkyl, alkylaryl, heteroarylalkyl, alkylheteroaryl, amino, alkylamino, arylamino, arylalkylamino, cycloalkylamino, heterocycloalkylamino, alkanoylamino, aroylamino, aralkanoyl amino, thiol, alkylthio, arylthio, arylalkylthio, cycloalkylthio, heterocycloalkylthio, alkylthiono, arylthiono, arylalkylthiono, alkylsulfonyl, arylsulfonyl, arylalkylsulfonyi, sulfonamido, nitro, cyano, carboxy, alkoxycarbonyl, carbamido, and carbamyl, wherein any of the foregoing moieties is optionally substituted;

m and n are each independently 0 or 1 to 4; and

o and p are each independently 0 or 1 to 3;

or a pharmaceutically acceptable salt thereof,

wherein

when R¹ is hydrogen, m is 0, and n is 0, then ring Al is not pyrazol-3-yl; 5- alkylpyrazol-3-yl; 1 -naphthyl, 2-naphthyl, 3-hydroxy-2-napthyl, quinolin-8-yl, indazol-5-yl, 7V-acetyl-isoindolin-4-yl, 4-alkylphenyl, 3,5-dialkylphenyl, 1 -halophenyl, 2-halophenyl, 3- halophenyl, 4-halophenyl, 2,3-dihalophenyl, 2,4-dihaloalkyl, 3,5-dihalophenyl, 2- haloalkylphenyl, 3-haloalkylphenyl, 4-haloalkylphenyl, 3-alkoxyphenyl, 4-alkoxyphenyl, 5- alkoxyphenyl, 2,4,5-trialkoxyphenyl, 3,4,5-trialkoxyphenyl, 3-hydroxy-4-alkoxyphenyl, 3- piperidin-l-ylsulfonyl-4-alkoxyphenyl, 2-hydroxyalkylphenyl, TV-phenyl-4-aminophenyl, N- acetyl-4-aminophenyl, N-alkoxycarbonyl-4-aminophenyl, 3-carboxyphenyl, 3- alkoxycarbonylphenyl, 4-alkoxycarbonylphenyl, 3-halo-4-alkoxyphenyl, 4- morpholinylphenyl, 2,4-dialkoxyphenyl, 2,5-dialkoxyphenyl, pyrrolidin-2-on-l-yl, or 2- pyrazinyloxy;

when R¹ is hydrogen, m is 0, n is 1, and R² is halo, then ring Al is not pyrazol-3-yl or 5-alkylpyrazol-3-yl; and

when R⁶ is hydrogen, p is 0, o is 3, R⁷ is 3-methyl, 4-acetyl, and 5-methyl, Q is NH, and ring A2 is unsubstituted morpholinyl, then R⁴ and R⁵ are not alkyl.

[0031] The embodiments of compounds of formula (I) or (II) set forth above are not intended to encompass UT2, Z265, or homologs thereof.

[0032] In any of the foregoing embodiments or other embodiments, the substituents R , and are attached to the core structure at any suitable position on the aromatic ring

(e.g., the 1 -, 2-, 3-, or 4-position).

[0033] In any of the foregoing compounds, ring Al is substituted aryl or unsubstituted aryl, such as phenyl. The aryl (e.g., phenyl) can be substituted with a group described herein, such as halo, haloalkyl, alkyl, alkoxy, or amino. Preferably, ring Al is halo-substituted phenyl (e.g., 2-halophenyl, 3-halophenyl, 4-halophenyl, 2,3-dihalophenyl, 2,4-dihalophenyl, 2,5-dihalophenyl, 3,4-dihalophenyl, 3,5-dihalophenyl, 2,3,4-trihalophenyl, and 2,3,5- trihalophenyl). In a preferred embodiment, the halo-substituted phenyl is a fluoro-substituted phenyl (e.g., 2-fluorophenyl, 3 -fluorophenyl, 4-fluorophenyl, 2,3-difluorophenyl, 2,4- difiuorophenyl, 2,5-difluorophenyl, 3,4-difluorophenyl, 3,5-difluorophenyl, 2,3,4- trifluorophenyl, or 2,3,5-trifluorophenyl). Ring Al can also be substituted heteroaryl or unsubstituted heteroaryl, in which the heteroaryl is as described herein (e.g., pyrazolyl, alkyl- substituted pyrazolyl, aryl-substituted pyrazolyl, oxazolyl, isooxazolyl, thiazolyl,

benzoimidazolyl, alkyl-substituted benzoimidazolyl, benzothiazolyl, or alkyl-substituted benzothiazolyl).

[0034] In any of the foregoing methods, Q is NH.

[0035] In any of the foregoing compounds, R¹ is H or alkyl. [0036] In any of the foregoing compounds, each R is halo, haloalkyl, alkyl, alkoxy, or amino.

[0037] In any of the foregoing compounds, each R is halo, haloalkyl, alkyl, alkoxy, amino, alkylamino, or substituted or unsubstituted heterocycloalkyl.

[0038] In any of the foregoing compounds, m is 0, 1, 2, 3, or 4. Preferably, m is 1 or 2, and most preferably, m is 2.

[0039] In any of the foregoing compounds, n is 0, 1 , 2, 3, or 4. Preferably, n is 0.

[0040] In any of the foregoing methods, ring A2 is substituted or unsubstituted heterocyclyl (i.e., heterocycloalkyl or heteroaryl). Suitable moieties for ring A2 include, e.g., imidazolinyl, thiazolinyl, imidazolidinyl, pyrrolinyl, pyranyl, piperidyl, morpholinyl, triazolyl, pyridinyl, pyridazinyl, pyrimidyl, pyrazinyl, benzimidazolyl, triazinyl, imidazolyl, (1,2,3)- and (l ,2,4)-triazolyl, pyrazinyl, tetrazolyl, furyl, pyrrolyl, thienyl, isothiazolyl, thiazolyl, isoxazolyl, and oxadiazolyl.

[0041] In any of the foregoing compounds, R⁴ and R⁵ are the same or different and each is H or alkyl.

[0042] In any of the foregoing compounds, each R⁶ is H or alkyl.

[0043] In any of the foregoing compounds, each R is alkyl, alkoxy, or alkanoyl.

[0044] In any of the foregoing compounds, each R is halo, haloalkyl, alkyl, alkoxy, or amino.

[0045] In any of the foregoing compounds, o is 0, 1 , 2, or 3. Preferably, o is 2 or 3, and most preferably, o is 3.

[0046] In any of the foregoing compounds, p is 0, 1 , 2, or 3. Preferably, p is 0.

[0047] In an aspect, the compound of formula (I) is a compound of formula (III)

wherein

R¹ is selected from H, alkyl, alkenyl, cycloalkyl, and aryl, wherein any of the foregoing moieties other than H is optionally substituted;

R² and R³ are each independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkyl, heteroaryl, halo, haloalkyl, haloalkoxy, aryloxy, alkanoyloxy, hydroxy, alkoxy, cycloalkyloxy, heterocylalkyloxy, alkanoyl, aroyl, arylalkyl, alkylaryl, heteroarylalkyl, alkylheteroaryl, amino, alkylamino, arylamino, arylalkylamino,

cycloalkylamino, heterocycloalkylamino, alkanoylamino, aroylamino, aralkanoylamino, thiol, alkylthio, arylthio, arylalkylthio, cycloalkylthio, heterocycloalkylthio, alkylthiono, arylthiono, arylalkylthiono, alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, sulfonamido, nitro, cyano, carboxy, alkoxycarbonyl, carbamido, and carbamyl; and

m and n are each independently 0 or 1 to 4;

or a pharmaceutically acceptable salt thereof.

[0048] Preferably, the compound is

7V-(5-phenyl-7H-pyrazol-3-yl)-2-(pyridin-4-yl)quinazolin-4-amine

or a pharmaceutically acceptable salt thereof.

[0049] In any of the embodiments above, the term "alkyl" means a saturated straight chain or branched non-cyclic hydrocarbon having an indicated number of carbon atoms (e.g.,

C₁ C₂₀, C 1 C18, C1 C 16, C1 C14, C1 C12, C 1 C10, C₁ Cg, C₁ C₆, C1 Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, w-pentyl, n-hexyl, n- heptyl, rc-octyl, M-nonyl, w-decyl, n-dodecyl, w-tetradecyl, n-hexadecyl, and 71-octadecyl; while representative saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert- butyl, isopentyl, 2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4- methylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3- dimethylbutyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,3-dimethylhexyl, 2,4- dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylpentyl, 2,2-dimethylhexyl, 3,3- dimtheylpentyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylpentyl, 3-ethylpentyl, 2- ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, 2- methyl-4-ethylpentyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl, 2,2-diethylpentyl, 3,3-diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyl and the like. An alkyl group can be unsubstituted or substituted.

[0050] In any of the embodiments above, the term "alkenyl group" means a straight chain or branched non-cyclic hydrocarbon having an indicated number of carbon atoms (e.g., C₂- C₂₀, C₂-C₁₈, C₂-C₁₆, C₂-C₁₄, C₂-C₁₂, C₂-C₁₀, etc.) and including at least one carbon-carbon double bond. Representative straight chain and branched alkenyls include vinyl, allyl, 1- butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-l -butenyl, 2-methyl-2- butenyl, 2,3 -dimethyl -2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1-heptenyl, 2-heptenyl, 3- heptenyl, 1 -octenyl, 2-octenyl, 3-octenyl, and the like. Any unsaturated group (double bond) of an alkenyl can be unconjugated or conjugated to another unsaturated group. An alkenyl group can be unsubstituted or substituted.

[0051] In any of the embodiments above, the term "alkynyl group" means a straight chain or branched non-cyclic hydrocarbon having an indicated number of carbon atoms (e.g., C₂- C₂₀, C₂-C₁₀, C₂-C₆, etc.), and including at least one carbon-carbon triple bond.

Representative straight chain and branched alkynyls include -acetyl enyl, -propynyl, -1 - butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3 -methyl- 1-butynyl, -4-pentynyl, -1-hexynyl, -2-hexynyl, -5-hexynyl, -1-heptynyl, -2-heptynyl, -6-heptynyl, -1 -octynyl, -2-octynyl, -7- octynyl, -1 -nonynyl, -2-nonynyl, -8-nonynyl, -1 -decynyl, -2-decynyl, -9-decynyl, and the like. Any unsaturated group (triple bond) of an alkynyl can be unconjugated or conjugated to another unsaturated group. An alkynyl group can be unsubstituted or substituted.

[0052] In any of the embodiments above, the term "cycloalkyl," as used herein, means a cyclic alkyl moiety containing from, for example, 3 to 7 carbon atoms, preferably from 5 to 6 carbon atoms. Examples of such moieties include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. A cycloalkyl group can be unsubstituted or substituted.

[0053] In any of the embodiments above, the term "aryl" refers to an unsubstituted or substituted aromatic carbocyclic moiety, as commonly understood in the art, and includes monocyclic and polycyclic aromatics such as, for example, phenyl, biphenyl, naphthyl, anthracenyl, pyrenyl, and the like. An aryl moiety generally contains from, for example, 6 to 30 carbon atoms, preferably from 6 to 18 carbon atoms, more preferably from 6 to 14 carbon atoms and most preferably from 6 to 10 carbon atoms. It is understood that the term aryl includes carbocyclic moieties that are planar and comprise 4n+2 π electrons, according to Huckel's Rule, wherein n = 1, 2, or 3. An aryl group can be unsubstituted or substituted.

[0054] In any of the embodiments above, the term "heterocyclyl" includes both heterocycloalkyls and heteroaryls as described herein.

[0055] The term "heterocycloalkyl" means a stable, saturated, or partially unsaturated monocyclic, bridged monocyclic, bicyclic, and spiro ring system containing 5 to 7 ring members of carbon atoms and other atoms selected from nitrogen, sulfur and/or oxygen. Preferably, a heterocycloalkyl is a 5 or 6-membered monocyclic ring and contains one, two, or three heteroatoms selected from nitrogen, oxygen, and/or sulfur. The heterocycloalkyl may be attached to the parent structure through a carbon atom or through any heteroatom of the heterocycloalkyl that results in a stable structure. Examples of such heterocyclic rings are isoxazolyl, imidazolinyl, thiazolinyl, imidazolidinyl, pyrrolyl, pyrrolinyl, pyranyl, pyrazinyl, piperidyl, morpholinyl, and triazolyl.

[0056] The term "heteroaryl" refers to aromatic 5 or 6 membered monocyclic groups, 9 or 10 membered bicyclic groups, and 1 1 to 14 membered tricyclic groups which have at least one heteroatom (O, S or N) in at least one of the rings. Each ring of the heteroaryl group containing a heteroatom can contain one or two oxygen or sulfur atoms and/or from one to four nitrogen atoms provided that the total number of heteroatoms in each ring is four or less and each ring has at least one carbon atom. The fused rings completing the bicyclic and tricyclic groups may contain only carbon atoms and may be saturated, partially saturated, or unsaturated. The nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atoms may optionally be quaternized. Heteroaryl groups which are bicyclic or tricyclic must include at least one fully aromatic ring but the other fused ring or rings may be aromatic or non-aromatic. The heteroaryl group may be attached at any available nitrogen or carbon atom of any ring. Illustrative examples of heteroaryl groups are pyridinyl, pyridazinyl, pyrimidyl, pyrazinyl, benzimidazolyl, triazinyl, imidazolyl, (1 ,2,3)- and (l ,2,4)-triazolyl, pyrazinyl, tetrazolyl, furyl, pyrrolyl, thienyl, isothiazolyl, thiazolyl, isoxazolyl, and oxadiazolyl. The heteroaryl group can be unsubstituted or substituted.

[0057] In any of the embodiments above, the term "arylalkyl" refers to an alkyl group substituted with an aryl group, both of which are described herein. The arylalkyl connects to the core molecule through the alkyl group. The term "alkylaryl" refers to an aryl group substituted with at least one alkyl group, both of which are described herein. The alkylaryl connects to the core molecule through the aryl group. The terms "heteroarylalkyl" and "alkylheteroaryl" are the same as "arylalkyl" and "alkylaryl," respectively, in which the aryl group is replaced with a heteroaryl moiety.

[0058] In any of the embodiments above, the terms "hydroxy" and "thiol" or "mercapto" refer to the groups -OH and -SH, respectively.

[0059] In any of the embodiments above, the terms "alkoxy" and "thioalkoxy" embrace linear or branched alkyl groups that are attached to a divalent oxygen or sulfur, respectively. The alkyl group is the same as described herein. Examples of alkoxy group include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, n-pentoxy, isopentoxy, n-hexoxy, and the like. The term "aryloxy" refers to substituents that have an aryl group attached to divalent oxygen. The aryl group is the same as described herein.

Examples of such substituents include phenoxy. The remaining oxy groups are the same as an alkoxy but substituted with the appropriate organic group (e.g., haloalkyl, cycloalkyl, heterocycloalkyl, etc.), as described herein.

[0060] In any of the embodiments above, the term "halo" refers to a halogen selected from fluorine, chlorine, bromine, and iodine, preferably fluorine, chlorine, or bromine. The term "haloalkyl" is an alkyl group substituted with a halo group, both of which are described herein. An example of a haloalkyl group is trifluoromethyl (-CF₃).

[0061] In any of the embodiments above, the term "alkylthio" denotes a substituent with an alkyl group directly attached to a divalent sulfur atom. The alkyl group is the same as described herein. Examples of such substituents include methylthio, ethylthio, and the like. Similarly, the term "arylthio" as used herein, denotes a substituent with an aryl group directly attached to a divalent sulfur atom. The aryl group is the same as described herein. The remaining thio groups are the same as an alkylthio but substituted with the appropriate organic group (e.g., arylalkyl, cycloalkyl, heterocycloalkyl, etc.), as described herein.

[0062] In any of the embodiments above, the terms "alkanoyl" and "aroyl" refer to the group -C(0)R, in which R is an alkyl or aryl group, respectively, as described herein. The terms "alkylthiono" and "arylthiono" refer to the groups -C(S)R, in which R is an alkyl or aryl group, respectively, as described herein.

[0063] In any of the embodiments above, the term "carboxy" refers to the group

-C(0)OH. The term "alkoxycarbonyl" refers to the group -C(0)OR, in which R is an alkyl group as described herein.

[0064] In any of the embodiments above, the terms "alkylsulfonyl" and "arylsulfonyl" refer to the group -S0₂R, in which R is an alkyl or aryl group as described herein. [0065] In any of the embodiments above, the term "alkylamino" refers to a secondary amine substituent with one hydrogen and one alkyl group directly attached to a trivalent nitrogen atom. In addition, the term "alkylamino" also refers to a tertiary amine substituent with two of the same or different alkyl groups directly attached to a trivalent nitrogen atom. The alkyl group is the same as described herein. The remaining amino groups are the same as an alkylamino but substituted with the appropriate organic group (e.g., aryl, cycloalkyl, heterocycloalkyl, etc.), as described herein.

[0066] In any of the embodiments above, the term "amido" refers to the group -C(0)NH₂. In any of the embodiments above, the term "alkylamido" refers to substituents of the formula, -C(0)NRR' or -NRC(0)R', in which R and R' are the same or different and each is a hydrogen or alkyl group, as described herein. The term "haloalkylamido" is an alkylamido as described above, in which one or more of the alkyl groups is substituted with a halo moiety, such as, for example, chlorine, bromine, or iodine.

[0067] In any of the embodiments above, the term "sulfonamido" refers to the group -S0₂-amino, which the amino group is -NH₂ or a substituted amino (e.g., alkylamino), as described herein.

[0068] In any of the embodiments above, the term "carbamyl" refers to the group -CONH₂, whereas a "substituted carbamyl" refers to the group -CONH-alkyl, -CONH-aryl, -CONH-arylalkyl, or instances where there are two substituents on the nitrogen selected from alkyl or arylalkyl.

[0069] Any moiety described as substituted preferably comprises at least one substituent (e.g., 1 , 2, 3, 4, 5, 6, etc.) in any suitable position (e.g., 1 -, 2-, 3-, 4-, 5-, or 6-position, etc.). Suitable substituents include, e.g., halo, alkyl, alkenyl, alkynyl, hydroxy, nitro, amino, alkylamino, alkoxy, aryloxy, aralkoxy, carboxyl, carboxyalkyl, carboxyalkyloxy, amido, alkylamido, and haloalkylamido.

[0070] In any of the embodiments above, whenever a range of the number of atoms in a structure is indicated (e.g., a CM2, CI_-8, CI _-6, or C]_-4 alkyl, alkylamino, etc.), it is specifically contemplated that any sub-range or individual number of carbon atoms falling within the indicated range also can be used. Thus, for instance, the recitation of a range of 1-8 carbon atoms (e.g., C]-C₈), 1 -6 carbon atoms (e.g., C₁-C₆), 1 -4 carbon atoms (e.g., C₁-C₄), 1 -3 carbon atoms (e.g., C1 -C3), or 2-8 carbon atoms (e.g., C2-C₈) as used with respect to any chemical group (e.g., alkyl, alkylamino, etc.) referenced herein encompasses and specifically describes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , and/or 12 carbon atoms, as appropriate, as well as any sub-range thereof (e.g., 1 -2 carbon atoms, 1-3 carbon atoms, 1-4 carbon atoms, 1-5 carbon atoms, 1-6 carbon atoms, 1-7 carbon atoms, 1-8 carbon atoms, 1-9 carbon atoms, 1-10 carbon atoms, 1-1 1 carbon atoms, 1-12 carbon atoms, 2-3 carbon atoms, 2-4 carbon atoms, 2-5 carbon atoms, 2-6 carbon atoms, 2-7 carbon atoms, 2-8 carbon atoms, 2-9 carbon atoms, 2-10 carbon atoms, 2-1 1 carbon atoms, 2-12 carbon atoms, 3-4 carbon atoms, 3-5 carbon atoms, 3- 6 carbon atoms, 3-7 carbon atoms, 3-8 carbon atoms, 3-9 carbon atoms, 3-10 carbon atoms, 3-1 1 carbon atoms, 3-12 carbon atoms, 4-5 carbon atoms, 4-6 carbon atoms, 4-7 carbon atoms, 4-8 carbon atoms, 4-9 carbon atoms, 4-10 carbon atoms, 4-1 1 carbon atoms, and/or 4- 12 carbon atoms, etc., as appropriate).

[0071] The compounds of the invention can be prepared by methods known to those skilled in the art. For example, compounds in accordance with an embodiment of the invention can be prepared by following the method disclosed in Figure 1 and Example 2. Variations can be made by those skilled in the art depending on the nature of the substituents or groups desired.

[0072] In any of the embodiments above, the phrase "salt" or "pharmaceutically acceptable salt" is intended to include nontoxic salts synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. For example, an inorganic acid (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, or hydrobromic acid), an organic acid (e.g., oxalic acid, malonic acid, citric acid, fumaric acid, lactic acid, malic acid, succinic acid, tartaric acid, acetic acid,

trifluoroacetic acid, gluconic acid, ascorbic acid, methylsulfonic acid, or benzylsulfonic acid), an inorganic base (e.g., sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or ammonium hydroxide), an organic base(e.g., methylamine, diethylamine, triethylamine, triethanolamine, ethylenediamine,

tris(hydroxymethyl)methylamine, guanidine, choline, or cinchonine), or an amino acid (e.g., lysine, arginine, or alanine) can be used. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington 's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, 1990, p. 1445, and Journal of Pharmaceutical Science, 66, 2-19 (1977). For example, they can be a salt of an alkali metal (e.g., sodium or potassium), alkaline earth metal (e.g., calcium), or ammonium of salt. [0073] The compounds of the invention or a composition thereof can potentially be administered as a pharmaceutically acceptable acid-addition, base neutralized or addition salt, formed by reaction with inorganic acids, such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base, such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases, such as mono-, di-, trialkyl, and aryl amines and substituted ethanolamines. The conversion to a salt is accomplished by treatment of the base compound with at least a stoichiometric amount of an appropriate acid. Typically, the free base is dissolved in an inert organic solvent such as diethyl ether, ethyl acetate, chloroform, ethanol, methanol, and the like, and the acid is added in a similar solvent. The mixture is maintained at a suitable temperature (e.g., between 0 °C and 50 °C). The resulting salt precipitates spontaneously or can be brought out of solution with a less polar solvent.

[0074] The invention provides a pharmaceutical composition comprising a compound or salt as described herein and a pharmaceutically acceptable carrier. In the pharmaceutical compositions described herein, any suitable pharmaceutically acceptable carrier can be used, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the pharmaceutical composition is to be administered and the particular method used to administer the pharmaceutical composition.

[0075] Suitable formulations include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood or other bodily fluid of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In one embodiment, the pharmaceutically acceptable carrier is a liquid that contains a buffer and a salt. The formulation can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use. Extemporaneous solutions and suspensions can be prepared from sterile powders, granules, and tablets. In one embodiment, the pharmaceutically acceptable carrier is a buffered saline solution.

[0076] The pharmaceutical composition can include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like. The pharmaceutical compositions can also include one or more additional active ingredients, such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.

[0077] The pharmaceutical composition comprising a compound as described herein or a pharmaceutically acceptable salt thereof can be formulated for any suitable route of administration, depending on whether local or systemic treatment is desired, and on the area to be treated. The pharmaceutical composition can be formulated for parenteral

administration, such as intravenous, intraperitoneal, intramuscular, or intratumoral injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for suspension in liquid prior to injection, or as emulsions. Additionally, parental administration can involve the preparation of a slow-release or sustained-release system, such that a constant dosage is maintained. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishes (such as those based on Ringer's dextrose), and the like. Preservatives and other additives also can be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0078] Desirably, the pharmaceutical composition also can be administered orally. Oral compositions can be in the form of powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable.

[0079] Suitable carriers and their formulations are further described in A.R. Gennaro, ed., Remington: The Science and Practice of Pharmacy (19th ed.), Mack Publishing Company, Easton, PA (1995).

[0080] The compound or a pharmaceutical composition comprising at least one compound as described herein or a pharmaceutically acceptable salt thereof can be administered in any suitable manner depending on whether local or systemic treatment is desired, and on the area to be treated. Desirably, the pharmaceutical composition is administered orally, but can be administered parenterally, most preferably by intravenous, intraperitoneal, intramuscular, or intratumoral injection. By the term "injecting," it is meant that the pharmaceutical composition is forcefully introduced into the target tissue. Although more than one route can be used to administer the pharmaceutical composition, a particular route can provide a more immediate and more effective reaction than another route. For regional delivery, the pharmaceutical composition can be administered intraarterially or intravenously, e.g., via the hepatic artery for delivery to the liver or the carotid artery for delivery to the brain.

[0081] The compound or a pharmaceutical composition comprising at least one compound as described herein or a pharmaceutically acceptable salt thereof can be administered in or on a device that allows controlled or sustained release of the compound or a pharmaceutically acceptable salt thereof, such as a sponge, biocompatible meshwork, mechanical reservoir, or mechanical implant. Implants (see, e.g., U.S. Patent 5,443,505), devices (see, e.g., U.S. Patent 4,863,457), such as an implantable device, e.g., a mechanical reservoir or an implant or a device comprised of a polymeric composition, are particularly useful for administration of the active agents. The pharmaceutical compositions of the inventive method also can be administered in the form of sustained-release formulations (see, e.g., U.S. Patent 5,378,475) comprising, for example, gel foam, hyaluronic acid, gelatin, chondroitin sulfate, a polyphosphoester, such as bis-2-hydroxyethyl-terephthalate (BHET), and/or a polylactic-glycolic acid. Of course, administration of the compound or

pharmaceutical composition can be accomplished via any route that efficiently delivers the active agents to the target tissue.

[0082] The inventive methods comprise administering an effective amount of a compound as described herein or a pharmaceutically acceptable salt thereof. An "effective amount" means an amount sufficient to show a meaningful benefit in an individual, e.g., promoting at least one aspect of tumor cell cytotoxicity, or treatment, healing, prevention, delay of onset, halting, or amelioration of other relevant medical condition(s) associated with a particular cancer. Preferably, one or more symptoms of the cancer are prevented, reduced, halted, or eliminated subsequent to administration of a compound as described herein or a pharmaceutically acceptable salt thereof, thereby effectively treating the cancer to at least some degree.

[0083] Effective amounts may vary depending upon the biological effect desired in the subject, condition to be treated, and/or the specific characteristics of the compound as described herein or a pharmaceutically acceptable salt thereof, and the individual. In this respect, any suitable dose of a compound as described herein or a pharmaceutically acceptable salt thereof can be administered to the subject (e.g., human), according to the type of cancer to be treated. Various general considerations taken into account in determining the "effective amount" are known to those of skill in the art and are described, e.g., in Gilman et al., eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th Ed., Mack

Publishing Co., Easton, Pa., 1990, each of which is herein incorporated by reference. The dose of the compound or a pharmaceutically acceptable salt thereof desirably comprises about 0.1 mg per kilogram (kg) of the body weight of the mammal (mg/kg) to about 400 mg/kg (e.g., about 0.75 mg/kg, about 5 mg/kg, about 30 mg/kg, about 75 mg/kg, about 100 mg/kg, about 200 mg/kg, or about 300 mg/kg). In another embodiment, the dose of the compound of formula (I) or (II) comprises about 0.5 mg/kg to about 300 mg/kg (e.g., about 0.75 mg/kg, about 5 mg/kg, about 50 mg/kg, about 100 mg/kg, or about 200 mg/kg), about 10 mg/kg to about 200 mg/kg (e.g., about 25 mg/kg, about 75 mg/kg, or about 150 mg/kg), or about 50 mg/kg to about 100 mg/kg (e.g., about 60 mg/kg, about 70 mg/kg, or about 90 mg/kg).

[0084] In any of the embodiments above, the term "treating" means therapeutic therapy. In reference to a particular condition, treating means: (1) to ameliorate the condition or one or more of the biological manifestations of the condition, (2) to interfere with (a) one or more points in the biological cascade that leads to or is responsible for the condition or (b) one or more of the biological manifestations of the condition (3) to alleviate one or more of the symptoms, effects or side effects associated with the condition or one or more of the symptoms, effects or side effects associated with the condition or treatment thereof, or (4) to slow the progression of the condition or one or more of the biological manifestations of the condition. For example, the term "treating cancer" can mean preventing the growth of tumors, preventing metastatic growth, killing of at least one cancer cell, and/or reducing the proliferation of at least one cancer cell.

[0085] It has been found that a compound of formula (I) or (II) (e.g., C418, Z265, ML367) can be used as a sensitizer for chemotherapy and/or radiation therapy. In particular, a DNA damage response by a DNA damaging agent (e.g., a chemotherapeutic agent) or ultraviolet radiation can be attenuated by treatment with at least one compound of fonnula (I) or (II).

[0086] In an example, cancer cells were preincubated with C418 and irradiated with 20 J/m ultraviolet (UV) radiation. DNA damage response proteins ATR, ATM, and phosphorylation (P) of CHKl , RPA32, CHK2 were monitored by western blot analysis, and GAPDH was used as a control. DNA damage response proteins, ATR and ATM, levels were reduced by treatment with C418. It was observed that phosphorylation of CHKl and RPA32, which is used for DNA damage response, was attenuated by C418 treatment upon UV irradiation.

[0087] In another example, in cancer cells treated with a DNA damaging agent (e.g., hydroxyurea), DNA damage responses, such as BRCA1 phosphorylation (p-BRCAl), RPA phosphorylation (RPA-S4S8), and phosphorylation of CHKl and CHK2 (p-CHKl and p- CHK2), were enhanced. Treatment with Z265, however, reduced DNA damage responses in both regular cancer cells and PARP-1 deficient cancer cells. Such inhibition of DNA damage response facilitates more DNA damage in cells. A high level of induction of DNA double strand breaks, which can be detected by phosphorylation of H2AX (g-H2AX), was observed in PARP1 -deficient tumors. It is believed that such observation may help explain why PARP1 -deficient cancer cells are preferentially killed when Z265 is co-administered with a chemotherapeutic agent, such as hydroxyurea.

[0088] In some aspects, any of the methods described herein further comprise

administering an additional chemotherapeutic agent. A chemotherapeutic agent is any compound that causes apoptosis of a cancerous cell. The mechanism by which apoptosis occur is not limited, for example, the chemotherapeutic agent can stall DNA replication, collapse replication forks, and/or produce DNA double-strand breaks (DSBs). The administration of the additional chemotherapeutic agent can occur simultaneously with a compound as described herein, before administration of a compound as described herein, after administration of a compound as described herein, or cyclically with a compound as described herein. Examples of chemotherapeutic agents include platinum compounds (e.g., cisplatin, carboplatin, oxaliplatin), alkylating agents (e.g., methyl methanesulfonate, cyclophosphamide, ifosfamide, chlorambucil, nitrogen mustard, thiotepa, melphalan, busulfan, procarbazine, streptozocin, temozolomide, dacarbazine, bendamustine), antitumor antibiotics (e.g., daunorubicin, doxorubicin, idarubicin, epirubicin, mitoxantrone, bleomycin, mytomycin C, plicamycin, dactinomycin), taxanes (e.g., paclitaxel and docetaxel), antimetabolites (e.g., 5-fluorouracil, 5-fluorouridine, cytarabine, premetrexed, thioguanine, floxuridine, capecitabine, and methotrexate), nucleoside analogues (e.g., fludarabine, clofarabine, cladribine, pentostatin, nelarabine), topoisomerase inhibitors (e.g., topotecan, etoposide, and irinotecan), hypomethylating agents (e.g., azacitidine and decitabine), proteosome inhibitors (e.g., bortezomib), epipodophyllotoxins (e.g., etoposide and teniposide), DNA synthesis inhibitors (e.g., hydroxyurea), vinca alkaloids (e.g., vicristine, vindesine, vinorelbine, and vinblastine), tyrosine kinase inhibitors (e.g., imatinib, dasatinib, nilotinib, sorafenib, sunitinib), monoclonal antibodies (e.g., rituximab, cetuximab, panetumumab, tositumomab, trastuzumab, alemtuzumab, gemtuzumab ozogamicin, bevacizumab), nitrosoureas (e.g., carmustine, fotemustine, and lomustine), enzymes (e.g., L- Asparaginase), biological agents (e.g., interferons and interleukins), hexamethylmelamine, mitotane, angiogenesis inhibitors (e.g., thalidomide, lenalidomide), steroids (e.g., prednisone, dexamethasone, and prednisolone), hormonal agents (e.g., tamoxifen, raloxifene, leuprolide, bicaluatmide, granisetron, flutamide), aromatase inhibitors (e.g., letrozole and anastrozole), arsenic trioxide, tretinoin, nonselective cyclooxygenase inhibitors (e.g., nonsteroidal antiinflammatory agents, salicylates, aspirin, piroxicam, ibuprofen, indomethacin, naprosyn, diclofenac, tolmetin, ketoprofen, nabumetone, oxaprozin), selective cyclooxygenase-2 (COX- 2) inhibitors, or any combination thereof.

[0089] In an embodiment, the chemotherapeutic agent is fluorouracil (5FU), 5- fluorouridine, or methyl methanesulfonate (MMS).

[0090] Ideally, the target tissue is a tumor, e.g., a solid tumor or a tumor associated with soft tissue (i.e., soft tissue sarcoma), in a human. The tumor can be associated with cancers of (i.e., located in) the oral cavity and pharynx, the digestive system, the respiratory system, bones and joints (e.g., bony metastases), soft tissue, the skin (e.g., melanoma), breast, the genital system, the urinary system, the eye and orbit, the brain and nervous system (e.g., glioma), or the endocrine system (e.g., thyroid) and is not necessarily the primary tumor. Tissues associated with the oral cavity include, but are not limited to, the tongue and tissues of the mouth. Cancer can arise in tissues of the digestive system including, for example, the esophagus, stomach, small intestine, colon, rectum, anus, liver, gall bladder, and pancreas. Cancers of the respiratory system can affect the larynx, lung, and bronchus and include, for example, non-small cell lung carcinoma. Tumors can arise in the uterine cervix, uterine corpus, ovary vulva, vagina, prostate, testis, and penis, which make up the male and female genital systems, and the urinary bladder, kidney, renal pelvis, and ureter, which comprise the urinary system. The target tissue also can be associated with lymphoma (e.g., Hodgkin's disease and Non-Hodgkin's lymphoma), multiple myeloma, or leukemia (e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, and the like). [0091] Specific examples of cancers treatable with the present methods include, without limitation, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related lymphoma, AIDS-related malignancies, anal cancer, cerebellar astrocytoma, extrahepatic bile duct cancer, bladder cancer, osteosarcoma/malignant fibrous histiocytoma, brain stem glioma, ependymoma, visual pathway and hypothalamic gliomas, breast cancer, bronchial adenomas/carcinoids, carcinoid tumors, gastrointestinal carcinoid tumors, carcinoma, adrenocortical, islet cell carcinoma, primary central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, clear cell sarcoma of tendon sheaths, colon cancer, colorectal cancer, cutaneous t- cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing's

sarcoma/family of tumors, extracranial germ cell tumors, extragonadal genu cell tumors, extrahepatic bile duct cancer, eye cancers, including intraocular melanoma, and

retinoblastoma, gallbladder cancer, gastrointestinal carcinoid tumor, ovarian germ cell tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, Hodgkin's disease, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, Kaposi's sarcoma, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic, leukemia, chronic myelogenous leukemia, liver cancer, non- small cell lung cancer, small cell lung cancer, Hodgkin's disease, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, intraocular melanoma, merkel cell carcinoma, metastatic squamous neck cancer with occult primary, multiple endocrine neoplasia syndrome, multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplasia syndrome, chronic myelogenous leukemia, myeloid leukemia, multiple myeloma, myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cancer, oral cavity and lip cancer, oropharyngeal cancer, osteosarcoma/malignant fibrous

histiocytoma of bone, ovarian cancer, ovarian low malignant potential tumor, pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, transitional cell cancer (e.g. renal pelvis and ureter), retinoblastoma, rhabdomyosarcoma, salivary gland cancer, malignant fibrous histiocytoma of bone, soft tissue sarcoma, sezary syndrome, skin cancer, small intestine cancer, stomach (gastric) cancer, supratentorial primitive neuroectodermal and pineal tumors, cutaneous T- cell lymphoma, testicular cancer, malignant thymoma, thyroid cancer, gestational trophoblastic tumor, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0092] In certain embodiments, the cancer is cancer that carries a PARPl mutation (e.g., a mutation in V762A), such that the cancer is PARP 1 -deficient (i.e., PARPl defective) compared to a wild-type form of the same cancer. In other words, the wild-type form of the cancer will not have a defect in PARPl expression.

[0093] A cancer that carries a PARPl mutation can be determined by assays known in the art. The PARPl -deficient cancer can be, for example, leukemia, melanoma, liver cancer, lung cancer, colon cancer (e.g., colorectal cancer), brain cancer, endometrial carcinoma, ovarian cancer, breast cancer, prostate cancer, endometrial carcinoma, or renal cancer.

Preferably, the PARPl -deficient cancer is colorectal cancer, renal cancer, or endometrial carcinoma. While not intending to be held to any particular theory or mechanism, it is believed that a compound of formula (I) or (II) can preferentially kill PARPl -deficient cancer cells because the compound is lethal with PARPl -deficient DNA repair pathways. DNA double strand break repair pathways, including homologous recombination (HR), nonhomologous end joining (NHEJ), and single strand annealing (SSA), redundantly repair DNA damage caused by chemotherapeutic agents and radiation with PARPl -deficient pathway. In an example, it was observed that treating PARPl -deficient cancer cells with a compound of formula (I) or (II) (e.g., C418, Z265, or ML367) down-regulated HR, NHEJ, and/or SSA.

[0094] In an effort to identify why HR, NHEJ, and/or SSA are down-regulated by a compound of formula (I) or (II), the expression level of proteins participating in these pathways were studied. In particular, colon cancer cells (HCT1 16) were pre-treated with C418 or DMSO. Half of the cells were treated with 20 J/m² ultraviolet (UV) irradiation. The protein expressions of BRCA1 , PALB2, and RAD51 were monitored by western blot analysis, and GAPDH expression was used as a control. It was observed that the expressions of PALB2 and RAD51 were significantly reduced by C418 and expression of BRCA1 was moderately reduced. Such results demonstrate why C418 treatment reduced the frequency of DNA double strand break repairs.

[0095] Moreover, compounds of formula (I) or (II) are capable of enhancing ATAD5 protein stabilization in response to DNA damage. The major role of ATAD5 in cells is to unload PCNA (proliferating cell nuclear antigen) from chromatin. It was observed that administration of a compound of formula (II) (Z265) facilitates PCNA unloading, since the level of chromatin-bound PCNA was reduced relative to a control (histone H3). [0096] In other embodiments, the cancer is a cancer that is treatable with a PARP 1 inhibitor. A cancer treatable with a PARP1 inhibitor includes a cancer with a BRCA1 and/or BRCA2 mutation. Suitable cancers include those described herein, particularly ovarian cancer, breast cancer, colorectal cancer, prostate cancer, endometrial cancer, a brain tumor, or melanoma.

[0097] The tumor to be treated can be at any stage, and can be subject to other therapies. The inventive method is useful in treating tumors (i.e., destruction of tumor cells or reduction in tumor size) that have been proven to be resistant to other forms of cancer therapy, including chemotherapy-resistant tumors. The tumor also can be of any size. Ideally, in treating the human for cancer, the inventive method results in cancerous (tumor) cell death and/or reduction in tumor size. It will be appreciated that tumor cell death can occur without a substantial decrease in tumor size due to, for instance, the presence of supporting cells, vascularization, fibrous matrices, etc. Accordingly, while reduction in tumor size is preferred, it is not required in the treatment of cancer.

[0098] For purposes of the present invention, the term "subject" preferably is directed to a mammal. Mammals include, but are not limited to, the order Rodentia, such as mice, and the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simioids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

[0099] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLES

[0100] All air or moisture sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Anhydrous solvents such as dichloromethane, N,N- dimethylforamide (DMF), acetonitrile, methanol, and triethylamine were purchased from Sigma-Aldrich (St. Louis, MO). Preparative purification was performed on a Waters semi- preparative HPLC system. The column used was a PHENOMENEX LUNA™ CI 8 (5 micron, 30 x 75 mm) at a flow rate of 45 mL/min. The mobile phase consisted of acetonitrile and water (each containing 0.1% trifluoroacetic acid). A gradient of 10% to 50% acetonitrile over 8 minutes was used during the purification. Fraction collection was triggered by UV detection (220 nm). Analytical analysis was performed on an Agilent LC/MS (Agilent Technologies, Santa Clara, CA). Method 1 : A 7 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with an 8 minute run time at a flow rate of 1 mL/min. A PHENOMENEX LUNA™ CI 8 column (3 micron, 3 x 75 mm) was used at a temperature of 50 °C. Method 2: A 3 minute gradient of 4% to 100%) acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05%> trifluoroacetic acid) was used with a 4.5 minute run time at a flow rate of 1 mL/min. A PHENOMENEX GEMINI™ Phenyl column (3 micron, 3 x 100 mm) was used at a temperature of 50 °C. Purity determination was performed using an Agilent Diode Array Detector for both Method 1 and Method 2. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode. Ή NMR spectra were recorded on Varian 400 MHz spectrometers (Varian, Lincolnshire, IL).

Chemical shifts are reported in ppm with undeuterated solvent (DMSO-d₆ at 2.49 ppm) as internal standard for DMSO-J₆ solutions. All of the analogs tested in the biological assays have purity greater than 95%, based on both analytical methods. High resolution mass spectrometry was recorded on Agilent 6210 Time-of-Flight LC/MS system. Confirmation of molecular formula was accomplished using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02).

[0101] Dimethyl sulfoxide (DMSO) ACS grade was obtained from Fisher Scientific (Hampton, NH), while ferrous ammonium sulfate, Xylenol Orange (XO), sulfuric acid, and Triton X-100 were obtained from Sigma- Aldrich (St. Louis, MO).

EXAMPLE 1

[0102] This example provides a summary of the screening results and identification of ML367 in an embodiment of the invention.

[0103] The performance of the assay was first evaluated by screening the library of pharmacologically active compounds (LOP AC ) in triplicate using the Kalypsys robotic system. The assay was then used to interrogate a library containing 344,385 compounds at six concentrations ranging from 2.9 nM to 46 μΜ, following the qPITS methodology (Inglese et al., Proc Natl Acad Sci USA, 2006, 103(31): 1 1473-8). A total of 1819 plates were screened. The screen performed well overall with an average Z'-Factor of 0.5 ± 0.3 and S/B ratio of 6 ± 1. The plate performance statistics were used to accept or reject plates. In total, 1,523 plates passed the QC criteria and were included in the final dataset.

[0104] Raw plate reads for each titration point were first normalized relative to the positive control compound (5-FUrd: 0%) and DMSO-only wells (-100%), and then corrected by applying a NCGC in-house pattern correction algorithm (Southall et al., Enabling the Large Scale Analysis of Quantitative High Throughput Screening Data, in Handbook of Drug Screening, 2nd Edition, R. Seethala, Zhang, L, Editor 2009, Taylor and Francis: New York, p. 442-463) using compound-free control plates (i.e., DMSO-only plates) at the beginning and end of the compound plate stack. Concentration-response titration points for each compound were fitted to a four-parameter Hill equation yielding concentrations of half- maximal activity (AC₅₀) and maximal response (efficacy) values from the primary screen. Compounds were designated as Class 1-4 according to the type of concentration-response curve observed (Huang et al., Environmental Health Perspectives, 201 1 , 1 19(8): 1142-8). Curve classes are heuristic measures of data confidence, classifying concentration-responses on the basis of efficacy, the number of data points observed above background activity, and the quality of fit.

[0105] A 3D activity plot of the potential antagonists of ATAD5 demonstrates that out of the 344,385 compounds screened, 7,231 were classified as top actives and 41,977 were weak or inconclusive inhibitors. Compounds with concentration response curves classified as curve class 1.1, 1.2, 2.1 or 2.2 with >50% efficacy, AC50 <20 μΜ and >10 fold more potent in the ATAD5 antagonist assay than the luciferase counter screen were declared as top actives. All other compounds that resulted in signal decrease were grouped as weak or inconclusive inhibitors. Total actives shown in the scatter plot represent 14.3% of the entire chemical library screened. Of the remaining compounds, 48,300 gave a signal increase, and the rest were inactive.

[0106] The 7,231 high quality actives were analyzed using a NCGC in-house scaffold detection algorithm resulting in 16,240 structure cores and 1 ,097 singletons. Compounds containing a specific core form a structure series. Each series was further evaluated for selectivity against the luciferase counter screen and promiscuous activity in other NCGC assays, enrichment of ATAD5 actives, and ranges of potency and efficacy. Eighty series were prioritized for cherry pick selection, resulting in 730 compounds selected for confirmation and follow-up studies. Eighty-seven compounds were selected for additional testing that showed activity in both the original 5-FUrd stimulated ATAD5-antagonist assay and the MMS (methyl methanesulfonate) stimulated ATAD5-antagonist assay, not active or less potent (>5 fold in potency) in either the 5-FUrd or MMS stimulated HEK293

cytotoxicity assay, and the biochemical and cell based CMV-luc luciferase counter screens.

[0107] Two SAR-rich chemotypes represented by CID-921541 (ML367) and CID- 658914 were selected based on the frequency of occurrence, potency, efficacy, logP, and the feasibility of medicinal chemistry optimization. Subsequently, CID-921541 (ML367) emerged as the lead series.

EXAMPLE 2

[0108] This example demonstrates the preparation and ADME properties of ML367 in an embodiment of the invention (Figure 1).

[0109] A mixture of 2,4-dichloroquinazoline (2 g, 10 mmol, 1 eq), 3,4-difluoroaniline (1 mL, 10 mmol, 1 eq), and (z^'Pr)₂NEt (5.3 mL, 30 mmol, 3 eq) in 2-propanol (20 mL) was heated at reflux with stirring for 16 hr. The solvent was removed and the crude product was triturated with water and sonicated, which caused the brown oil to become a tan solid. The solid was removed by filtration, and it was washed with water. 2-Chloro-N-(3,4- difluorophenyl)quinazolin-4-amine was isolated as a tan solid (2.9 g, quant.) and used without further purification.

[0110] A mixture of 2-chloro-N-(3,4-difluorophenyl)quinazolin-4-amine (0.25 g, 0.857 mmol, 1 eq), pyridin-4-ylboronic acid (0.137 g, 1.11 mmol, 1.5 eq), 2 molar solution of sodium carbonate (0.860 ml, 1.71 mmol, 2 eq) and Pd(PPh3)4 (0.050 g, 0.043 mmol, 5 mol %) in dimethoxyethane was degassed with argon for 5 min then heated in a microwave for 30 min at 150 °C. The solvent was removed by forced air and the crude product was dissolved in DMSO then stirred with palladium scavenger for 30 min. The solution was passed through a thiol cartridge and finally purified in preparative HPLC to provide N-(3,4-difluorophenyl)- 2-(pyridin-4-yl)quinazolin-4-amine as a TFA salt (ML367).

[0111] The preliminary absorption, distribution, metabolism, excretion (ADME) profile of ML367 supports its use as a valuable probe for ATAD5 destabilization. While both its microsomal stability (in rat and human) and solubility (in PBS buffer) are moderate, the latter was above the IC determined in the cell based assay. As shown in Figure 2, ML367 has good PAMPA permeability, and its overall ADME profile is consistent with its measured Log D of 1.58 (Table 1). Additionally, ML367 showed good stability in mouse plasma as well as a series of aqueous stability assessments including pH 2 and pH 10 buffers and aqueous 5 mM glutathione (Figure 2, panels (C), (D), and (E)). ML367 showed no degradation without NADPH present over a 1 hr period.

EXAMPLE 3

[0112] This example demonstrates the structure activity relationships (SAR) of ML367 in an embodiment of the invention.

[0113] Following confirmation of the ATAD5 destabilization activity of ML367 (1) via resynthesis (Table 2), systematic structural modifications of the 3 main regions of the molecule were studied in an effort to establish tractable SAR. The most challenging region of the molecule to modify was the quinazoline core; the limited number of modifications assessed were generally not tolerated. As shown in Table 2, modifications to the quinazoline core such as truncating the quinazoline to an amino pyrimidine (2) and one of two fused ring system to biaryl linkage modifications (1→ 3 and 1→ 4) were only tolerated in one instance (4). Additionally, one of two quinazoline ring nitrogen atoms is preferred for optimal activity as its removal resulted in a modest drop in potency (5, IC₅₀ = 3.2 μΜ).

[0114] Table 2 recites the IC₅₀ values of compounds 1-7 that represent the average of assays run in triplicate. Activities are marked as "+++" (IC₅₀ < 3 μΜ), "++" (3 μΜ < IC₅₀ < 10 μΜ), or "+" (IC₅₀ > 10 μΜ).

[0115] Given the fact that the core appeared to lack flexibility and synthetic facility favored utilization of the amino quinazoline core, the core of the hit compound was retained for systematic exploration of the remaining two regions of the pharmacophore. Accordingly, the southeastern 4-pyridyl group was modified, in which a larger number of analogs were synthesized given increased synthetic tractability, as shown in Table 3. Both removal of (phenyl, 11) and moving of the pyridine nitrogen around the ring (2-pyridyl, 8; 3-pyridyl, 9) reduced the potency. Several other modifications (only representative compounds are shown here) such as replacement with a quinoline (10) or -anisyl group (12) resulted in complete loss of activity. Attempts to mimic the pyridine with partially saturated or fully saturated heterocycles, as well as, smaller heterocycles (13-18) were also unsuccessful. Given the intolerance to a variety of modifications, substitution of the 4-pyridyl ring was studied in more detail. As depicted in Table 3, the southeastern 4-pyridyl was substituted with both electron-donating and electron- withdrawing groups at both the 2- and 3 -positions (only representative analogs 19-29 are shown in Table 3). None of these modifications provided a boost in potency at the target, however, in a few instances activity was maintained (22, 25 and 27).

[0116] Table 3 recites the IC₅₀ values of compounds 8-29 that represent the average of assays run in triplicate. Activities are marked as "+++" (IC₅₀ < 3 μΜ), "++" (3 μΜ < IC₅₀ < 10 μΜ), or "+" (IC₅₀ > 10 μΜ).

[0117] Having established the desirable motifs specific to two regions of the

pharmacophore, the focus of the SAR effort shifted to the northern, 3,4-difluoroaniline region where a large number of analogs were made (Tables 4-6). The aniline linkage proved optimal for ATAD5 activity as analogs 30-32 resulted in loss in potency (Table 4).

Additionally, saturated northern substituents, such as both secondary (33, 34) and primary amines (35) also resulted in a moderate loss in potency at the target. Continuing onto the aryl segment, a diverse set of heterocyclic replacements (36-43) was tested. Most notably among these are the phenylpyrazole (38) and the benzthiazole (42) which maintain a similar level of potency to that of ML367 (Table 4). Returning to the SAR of the starting aniline ring, a systematic substituent scan was performed to develop a deeper understanding of the optimal electronics and substitution pattern about the phenyl ring (only representative analogs 44-53 are shown in Table 5). These potency trends suggested a preference for electron-withdrawing groups in the 3- and 4-positions (45, 46, 52, 53) though 3-OMe (51) was also tolerated with only a moderate drop in potency. Also, these data indicate a clear preference for fluorine at both of these positions. Continuing in this regard, a variety of disubstituted anilines were tested as well (representative analogs 54-61 are shown in Table 5). A complete scan of difluoro-substituted analogs (1, 54-58) and their corresponding data implies that it is more of an overall electronic effect that is driving potency as now a variety of substitution patterns about the aromatic ring more or less maintain potency at ATAD5 (54, 55, 56, 57). These analogs did suffer from slightly reduced efficacy at the top dose of 46 μΜ compared to 1. A few other simple 3,4-disubstituted analogs were made, and these analogs experienced only a slight loss in potency (59-61). With the developing SAR in this region suggesting a preference for a 3-fluoro substituent as well as indications from early SAR efforts (not depicted) that more substantial substituents at the 4-position may be tolerated, 3-fluoro, 4- substituted anilines in the northern region were studied (Table 6). A variety of changes in this regard were tolerated with only slight drops in potency, including heteroaryl- and cyclic amine substituents (62-69; Table 6).

[0118] Tables 4-6 recite the IC₅₀ values of compounds 30-69 that represent the average of assays run in triplicate. Activities are marked as "+++" (IC₅₀ < 3 μΜ), "++" (3 μΜ < IC₅₀ < 10 μΜ), or "+" (IC₅₀ > 10 μΜ).

Table 6

EXAMPLE 4

[0119] This example describes an assay for determining compounds that destabilize ATAD5 activity and an assay for determining CMV-Luc activity, which is a counter screen to ATAD5-Luc. [0120] ATAD5-luc cells were dispensed at 2,000/4 μL/well into a tissue culture treated 1 ,536-well white/solid bottom assay plates (Greiner Bio-One, Monroe, NC) using a

MULTIDROP™ COMBI dispenser (Thermo Scientific, Hanover Park, IL). After the assay plates were incubated for 3-4 hr at 37 °C for the cell adherence, 23 nL of each compound were transferred via a pin tool (Kalypsys, Los Angeles, CA) to columns 5-48 of the assay plates, resulting in the final concentrations ranging from 1.0 μΜ to 46 μΜ. DMSO was only included in columns 1 to 4. For antagonist screening, the compound transfer was followed by the addition of either 1 μL of culture medium (columns 1 and 3) or 5-fluorouridine (5-FUrd) (10 μΜ final concentration in rest of the columns), a known stabilizer of ATAD5. The assay plates were incubated for 16 hr at 37 °C, followed by the addition of AMPLITE™ Luciferase reagent (AAT Bioquest, Inc., Sunnyvale, CA) at 5 μL/well using a BIORAPTR™ Flying Reagent Dispenser (FRD) (Aurora Discovery). After 30 min incubation at room temperature, the luminescence intensity was quantified using a VIEWLUX™ CCD-based plate reader (Perkin Elmer, Waltham, MA). Raw plate reads for each titration point were first normalized relative to FUrd control (10 μΜ, 100%) and DMSO only wells (basal, 0%) and then corrected by applying a pattern correction algorithm using compound free control plates (DMSO) plates.

[0121] CMV-Luc2-Hygro HEK293 cells were dispensed at 2,000/5 pL/well with assay medium containing 10% FBS into tissue culture treated 1,536-well white/solid bottom assay plates (Greiner Bio-One, Monroe, NC) using a BIORAPTR™ Flying Reagent Dispenser (FRD) (Aurora Discovery). After the assay plates were incubated for 5 hr at 37 °C for the cell adherence, 23 nL of each compound was transferred via a pin tool (Kalypsys, Los Angeles, CA) to rows 1-30 of the assay plates, resulting in the final concentrations ranging from 0.2 nM to 46 μΜ and DMSO only was transferred to row 31. In row 32, final concentrations of N⁶ Phenyl Adenosine at 46 & 23 μΜ were included from 1 -12 and 13-24 wells respectively and DMSO only was included in 25-48 wells. The assay plates were incubated for 16 hr at 37 °C, followed by the addition of ONE-GLO™ Luciferase reagent (Promega, Madison, WI) at 5 μίΛνεΙΙ using FRD. After 30 min incubation at room temperature, the luminescence intensity was quantified using a VIEWLUX™ CCD-based plate reader (Perkin Elmer, Waltham, MA). Raw plate reads for each titration point were first normalized relative to N⁶ Phenyl Adenosine control (46 μΜ, 100%) and DMSO only wells (basal, 0%) and then corrected by applying a pattern correction algorithm using compound free control plates (DMSO) plates.

[0122] The activity of ML367 in these assays is summarized in Table 7.

Table 7

EXAMPLE 5

[0123] This example describes a biochemical luciferase counter screen and a cell viability assay.

[0124] Three μί of 10 μΜ substrate (50 mM tris acetate, 13.3 mM magnesium acetate, 0.01 mM D-Luc, 0.01 mM ATP, 0.01 % TWEEN™, 0.05% BSA and dH₂0) was dispensed into 1 ,536-well white/solid bottom assay plates (Greiner Bio-One, Monroe, NC) using a BIORAPTR™ Flying Reagent Dispenser (FRD) (Aurora Discovery). 23 nL of each compound was transferred via a pin tool (Kalypsys, Los Angeles, CA) to rows 1 - 30 of the assay plates resulting in the final concentrations ranging from 0.2 nM to 46 μΜ and DMSO only was transferred to rows 31 - 32. Then compound addition was followed by adding 1 \iL of buffer (row 32 only) or enzyme; "Pyralis Luciferase" (0.04 μΜ, final concentration) for rest of the plate. After 5 min of incubation at room temperature, the luminescence intensity was quantified using a VIEWLUX™ CCD-based plate reader (Perkin Elmer, Waltham, MA). Raw plate reads for each titration point were first normalized relative to Pyralis luciferase control (0.04 μΜ, 100%) and DMSO only wells (basal, 0%) and then corrected by applying a pattern correction algorithm using compound free control plates (DMSO) plate.

[0125] In a cell viability assay, 1 x 10⁴ HCT1 16 or PARP-1 deficient cells were seeded into each well of a 96-well. After allowing the cells to attach to the bottom of the plate for 24 hours, compounds were added at serial dilutions from starting concentration of 40 μΜ. 48 hours following treatment, cell viability was determined using CELLTITER-GLO™ (Promega, Madison, WI) according to the manufacturer's protocol and quantified on a FLUOROSKAN™ ASCENT™ LUMINOMETER (Thermo Scientific, Hanover Park, IL).

[0126] The results are shown in Figure 3, which demonstrated ML367's dose response inhibition of ATAD5 activity without any significant cytotoxic effect.

EXAMPLE 6

[0127] This example describes cell culture, FLAG-ATAD5 transfection, and western blotting.

[0128] For the cell culture, human embryonic kidney (HEK293T) cells, human colon cancer (HCT1 16) cells, and PARP-1 deficient HCT116 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) + GLUTAMAX™ (Life Technologies, Grand Island, NY) containing 10% fetal bovine serum (FBS; Hyclone, Fisher Scientific, Hampton, NH), 100 U/mL penicillin G, and 100 μg/mL streptomycin (Life Technologies, Grand Island, NY). All cells were maintained at 37 °C under 5 % C0₂ and atmospheric 0₂.

[0129] For the FLAG-ATAD5 transfections, HEK293T cells were transfected with FLAG-tagged ATAD5 using Lipofectamine 2000 (Life Technologies, Grand Island, NY), according to the manufacturer's protocol. 48 hours post-transfection, the cells were treated with the indicated compounds for 16 hours. To obtain total lysate, the cells were resuspended in lysis buffer [50 mM Tris, pH 7.5, 150 mM NaCl, 1 % Nonidet P-40, 5 mM EDTA, protease inhibitors (Roche, Basel, Switzerland)] and lysed on ice for 30 min. Proteins were separated by SDS-PAGE using a 4-15% Tris-glycine gel (Bio-Rad, Des Plaines, IL) and transferred to a polyvinylidene difiuoride membrane. FLAG-ATAD5 protein levels were detected by the ECL Western Blotting Detection System (GE Healthcare, Wauwatosa, WI) using an HRP- conjugated antibody against FLAG (Sigma, St. Louis, MO). Equal protein loading was confirmed using an antibody against tubulin (Abeam, Cambridge, England). The ratio of FLAG/Tubulin was quantified using ImageJ. See Figures 4(A) and 4(B). ML367

demonstrated robust inhibition (~4-fold) of 5-FUrd mediated FLAG-ATAD5 stabilization.

[0130] In a western blot to monitor DNA damage response, cells were grown to confluence on 60 mm plates and then exposed to 60 J/m² UV in the presence or absence of 10 μΜ 7V-(3,4-difluorophenyl)-2-pyridin-4-ylquinazolin-4-amine (ML367). 6 hours after UV treatment, the cells were lysed in whole cell lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 8.0, 0.5 % Triton X-100 and protease inhibitors (Roche, Basel, Switzerland). Western blotting was carried out according to the protocol above using the following antibodies: anti-phospho-RPA32(S4S8) and anti-phospho-RPA32(S33) (Bethyl Laboratories, Inc., Montgomery, TX); anti-RPA (EMD Millipore, Chicago, IL); anti- phospho-CHKl (S345), anti-phospho-Chk2 (T68), and anti-phospho-ATR (Cell Signaling Technology, Danvers, MA); anti-CHKl and anti-CHK2 (Santa Cruz Biotechnology, Inc., Dallas, TX); anti-DNA-PKcs (Neomarkers, Inc., Fremont, CA); and anti-TEL2 (Proteintech, Chicago, IL).

EXAMPLE 7

[0131] This example describes a colony formation assay.

[0132] 1 x 10³ HCT116 or Lig3, Lig4, FancM, FancG and PARP-1 deficient cells were plated in 60 mm dishes. Twenty-four hours after seeding, the cells were treated with DMSO or 10 μΜ ML367 for 14 days. Colonies were counted after fixing with 10% formaldehyde for 5 minutes and staining with 0.05% crystal violet for 30 minutes.

[0133] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0134] The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (for example, "at least one of A and B") is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly

contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the

specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0135] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A compound selected from

wherein ring Al is substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl; ring A2 is substituted heterocyclyl or unsubstituted heterocyclyl; Q is NH, O, or S;

R¹, R⁴, R⁵, and R⁶ are each independently selected from H, alkyl, alkenyl, cycloalkyl, and aryl, wherein any of the foregoing moieties other than H is optionally substituted;

R², R³, R⁷, and R⁸ are each independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkyl, heteroaryl, halo, haloalkyl, haloalkoxy, aryloxy, alkanoyloxy, hydroxy, alkoxy, cycloalkyloxy, heterocylalkyloxy, alkanoyl, aroyl, arylalkyl, alkylaryl, heteroarylalkyi, alkylheteroaryl, amino, alkylamino, arylamino, arylalkylamino, cycloalkylamino, heterocycloalkylamino, alkanoylamino, aroylamino, aralkanoylamino, thiol, alkylthio, arylthio, arylalkylthio, cycloalkylthio, heterocycloalkylthio, alkylthiono, arylthiono, arylalkylthiono, alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, sulfonamido, nitro, cyano, carboxy, alkoxycarbonyl, carbamido, and carbamyl, wherein any of the foregoing moieties is optionally substituted; m and n are each independently 0 or 1 to 4; and o and p are each independently 0 or 1 to 3; or a pharmaceutically acceptable salt thereof; for use in treating cancer, wherein the cancer is PARPl -deficient compared to a wild-type form of the same cancer.

2. The compound for use according to claim 1 , wherein ring Al is substituted aryl or unsubstituted aryl.

3. The compound for use according to claim 1 or claim 2, wherein R¹ is H or alkyl.

4. The compound for use according to any one of claims 1-3, wherein R² is halo, haloalkyl, alkyl, alkoxy, or amino.

5. The compound for use according to any one of claims 1-4, wherein R³ is halo, haloalkyl, alkyl, alkoxy, amino, alkylamino, or substituted or unsubstituted heterocycloalkyl.

6. The compound for use according to any one of claims 1 -5, wherein m is 1 or

2.

7. The compound for use according to any one of claims 1-6, wherein n is 0.

8. The compound for use according to claim 1 , wherein ring A2 is a heterocyclyl selected from imidazolinyl, thiazolinyl, imidazolidinyl, pyrrolinyl, pyranyl, piperidyl, morpholinyl, triazolyl, pyridinyl, pyridazinyl, pyrimidyl, pyrazinyl, benzimidazolyl, triazinyl, imidazolyl, (1 ,2,3)- and (l ,2,4)-triazolyl, pyrazinyl, tetrazolyl, furyl, pyrrolyl, thienyl, isothiazolyl, thiazolyl, isoxazolyl, and oxadiazoly, each of which is optionally substituted.

9. The compound for use according to claim 1 or claim 8, wherein R⁴ and R⁵ are the same or different and each is H or alkyl.

10. The compound for use according to any one of claims 1 , 8, and 9, wherein R⁶ is H or alkyl.

1 1 . The compound for use according to any one of claims 1 and 8-10, wherein R⁷ is alkyl, alkoxy, or alkanoyl.

12. The compound for use according to any one of claims 1 and 8-1 1 , wherein R is halo, haloalkyl, alkyl, alkoxy, or amino.

13. The compound for use according to any one of claims 1 and 8-12, wherein o is

2 or 3.

14. The compound for use according to any one of claims 1 and 8-13, wherein p is

0.

15. The compound for use according to claim 1 , wherein the compound is

16. The compound for use according to any one of claims 1-15, wherein the PARPl -deficient cancer is colorectal cancer, renal cancer, or endometrial carcinoma.

17. The compound for use according to any one of claims 1-16, which is for combined use with an additional chemotherapeutic agent.

18. The compound for use according to claim 17, wherein the additional chemotherapeutic agent is fluorouracil (5FU), 5-fluorouridine, or methyl methanesulfonate (MMS).

19. The compound for use according to any one of claims 1 -18, wherein the treating cancer includes reducing the proliferation of at least one cancer cell.

20. The compound for use according to any one of claims 1-18, wherein the treating cancer includes killing at least one cancer cell.

21. A compound selected from

wherein ring Al is substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl; ring A2 is substituted heterocyclyl or unsubstituted heterocyclyl; Q is NH, O, or S;

R' , R⁴, R⁵, and R⁶ are each independently selected from H, alkyl, alkenyi, cycloalkyl, and aryl, wherein any of the foregoing moieties other than H is optionally substituted;

R², R³, R⁷, and R⁸ are each independently selected from alkyl, alkenyi, alkynyl, cycloalkyl, aryl, heterocycloalkyl, heteroaryl, halo, haloalkyl, haloalkoxy, aryloxy, alkanoyloxy, hydroxy, alkoxy, cycloalkyloxy, heterocylalkyloxy, alkanoyl, aroyl, arylalkyl, alkylaryl, heteroarylalkyl, alkylheteroaryl, amino, alkylamino, arylamino, arylalkylamino, cycloalkylamino, heterocycloalkylamino, alkanoylamino, aroylamino, aralkanoylamino, thiol, alkylthio, arylthio, arylalkylthio, cycloalkylthio, heterocycloalkylthio, alkylthiono, arylthiono, arylalkylthiono, alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, sulfonamido, nitro, cyano, carboxy, alkoxycarbonyl, carbamido, and carbamyl, wherein any of the foregoing moieties is optionally substituted; m and n are each independently 0 or 1 to 4; and o and p are each independently 0 or 1 to 3; or a pharmaceutically acceptable salt thereof, for use in treating cancer, wherein the cancer is treatable with a P ARP 1 inhibitor.

22. The compound for use according to claim 21 , wherein ring Al is substituted aryl or unsubstituted aryl.

23. The compound for use according to claim 21 or claim 22, wherein R¹ is H or alkyl.

24. The compound for use according to any one of claims 21-23, wherein R² is halo, haloalkyl, alkyl, alkoxy, or amino.

25. The compound for use according to any one of claims 21 -24, wherein R³ is halo, haloalkyl, alkyl, alkoxy, amino, alkylamino, or substituted or unsubstituted

heterocycloalkyl.

26. The compound for use according to any one of claims 21 -25, wherein m is 1 or 2.

27. The compound for use according to any one of claims 21-26, wherein n is 0.

28. The compound for use according to claim 21 , wherein ring A2 is a

heterocyclyl selected from imidazolinyl, thiazolinyl, imidazolidinyl, pyrrolinyl, pyranyl, piperidyl, morpholinyl, triazolyl, pyridinyl, pyridazinyl, pyrimidyl, pyrazinyl,

benzimidazolyl, triazinyl, imidazolyl, (1,2,3)- and (1 ,2,4)-triazolyl, pyrazinyl, tetrazolyl, furyl, pyrrolyl, thienyl, isothiazolyl, thiazolyl, isoxazolyl, and oxadiazolyl, each of which is optionally substituted.

29. The compound for use according to claim 21 or claim 28, wherein R⁴ and R⁵ are the same or different and each is H or alkyl.

30. The compound for use according to any one of claims 21, 28, and 29, wherein R⁶ is H or alkyl.

31. The compound for use according to any one of claims 21 and 28-30, wherein R⁷ is alkyl, alkoxy, or alkanoyl.

32. The compound for use according to any one of claims 21 and 28-31, wherein R is halo, haloalkyl, alkyl, alkoxy, or amino.

33. The compound for use according to any one of claims 21 and 28-32, wherein o is 2 or 3.

34. The compound for use according to any one of claims 21 and 28-33, wherein p is 0.

35. The compound for use according to claim 21 , wherein the compound is

36. The compound for use according to any one of claims 21-35, wherein the cancer is ovarian cancer, breast cancer, colorectal cancer, prostate cancer, endometrial cancer, a brain tumor, or melanoma.

37. The compound for use according to any one of claims 21-36, which is for a combined use with another PARP1 inhibitor.

38. The compound for use according to claim 37, wherein the another PARP1 inhibitor is olaparib, veliparib, KU0058958, XAV939, IWRl, IWR2, or 4-amino-l,8- naphthalimide (4 AN).

39. The compound for use according to any one of claims 21-38, wherein the treating cancer includes reducing the proliferation of at least one cancer cell.

40. The compound for use according to any one of claims 21-38, wherein the treating cancer includes killing at least one cancer cell.

41. A com ound of formula (I) or (II)

(I) (Π) wherein ring Al is substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl; ring A2 is substituted heterocyclyl or unsubstituted heterocyclyl;

Q is NH, O, or S; R¹, R⁴, R⁵, and R⁶ are each independently selected from H, alkyl, alkenyl, cycloalkyl, and aryl, wherein any of the foregoing moieties other than H is optionally substituted;

R², R³, R⁷, and R⁸ are each independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkyl, heteroaryl, halo, haloalkyl, haloalkoxy, aryloxy, alkanoyloxy, hydroxy, alkoxy, cycloalkyloxy, heterocylalkyloxy, alkanoyl, aroyl, arylalkyl, alkylaryl, heteroarylalkyl, alkylheteroaryl, amino, alkylamino, arylamino, arylalkylamino, cycloalkylamino, heterocycloalkylamino, alkanoylamino, aroylamino, aralkanoyl amino, thiol, alkylthio, arylthio, arylalkylthio, cycloalkylthio, heterocycloalkylthio, alkylthiono, arylthiono, arylalkylthiono, alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, sulfonamido, nitro, cyano, carboxy, alkoxycarbonyl, carbamido, and carbamyl, wherein any of the foregoing moieties is optionally substituted; m and n are each independently 0 or 1 to 4; and o and p are each independently 0 or 1 to 3; or a pharmaceutically acceptable salt thereof, wherein when R¹ is hydrogen, m is 0, and n is 0, then ring Al is not pyrazol-3-yl; 5- alkylpyrazol-3-yl; 1-naphthyl, 2-naphthyl, 3-hydroxy-2-napthyl, quinolin-8-yl, indazol-5-yl, 7V-acetyl-isoindolin-4-yl, 4-alkylphenyl, 3,5-dialkylphenyl, 1-halophenyl, 2-halophenyl, 3- halophenyl, 4-halophenyl, 2,3-dihalophenyl, 2,4-dihaloalkyl, 3,5-dihalophenyl, 2- haloalkylphenyl, 3-haloalkylphenyl, 4-haloalkylphenyl, 3-alkoxyphenyl, 4-alkoxyphenyl, 5- alkoxyphenyl, 2,4,5-trialkoxyphenyl, 3,4,5-trialkoxyphenyl, 3-hydroxy-4-alkoxyphenyl, 3- piperidin-l -ylsulfonyl-4-alkoxyphenyl, 2-hydroxyalkylphenyl, 7V-phenyl-4-aminophenyl, N- acetyl-4-aminophenyl, N-alkoxycarbonyl-4-aminophenyl, 3-carboxyphenyl, 3- alkoxycarbonylphenyl, 4-alkoxycarbonylphenyl, 3-halo-4-alkoxyphenyl, 4- morpholinylphenyl, 2,4-dialkoxyphenyl, 2,5-dialkoxyphenyl, pyrrolidin-2-on-l -yl, or 2- pyrazinyloxy; when R is hydrogen, m is 0, n is 1 , and R is halo, then ring Al is not pyrazol-3-yl or 5-alkylpyrazol-3-yl; and when R⁶ is hydrogen, p is 0, o is 3, R⁷ is 3 -methyl, 4-acetyl, and 5-methyl, Q is NH, and ring A2 is unsubstituted morpholinyl, then R⁴ and R⁵ are not alkyl.

42. The compound of claim 41 that is a compound of formula (III)

wherein

R¹ is selected from H, alkyl, alkenyl, cycloalkyl, and aryl, wherein any of the foregoing moieties other than H is optionally substituted;

R² and R³ are each independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkyl, heteroaryl, halo, haloalkyl, haloalkoxy, aryloxy, alkanoyloxy, hydroxy, alkoxy, cycloalkyloxy, heterocylalkyloxy, alkanoyl, aroyl, arylalkyl, alkylaryl, heteroarylalkyl, alkylheteroaryl, amino, alkylamino, arylamino, arylalkylamino,

cycloalkylamino, heterocycloalkylamino, alkanoyl amino, aroylamino, aralkanoylamino, thiol, alkylthio, arylthio, arylalkylthio, cycloalkylthio, heterocycloalkylthio, alkylthiono, arylthiono, arylalkylthiono, alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, sulfonamido, nitro, cyano, carboxy, alkoxycarbonyl, carbamido, and carbamyl; wherein any of the foregoing moieties is optionally substituted; and m and n are each independently 0 or 1 to 4; or a pharmaceutically acceptable salt thereof.

43. The compound of claim 42 that is

44. A pharmaceutical composition comprising the compound or salt of any one of claims 41-43 and a pharmaceutically acceptable carrier.