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Title:
SMALL MOLECULE INHIBITORS OF APEX, ALONE AND IN COMBINATION WITH OTHER CANCER DRUGS TO INHIBIT GROWTH OF CANCER CELLS
Document Type and Number:
WIPO Patent Application WO/2022/093789
Kind Code:
A1
Abstract:
Disclosed are compounds and their pharmaceutically acceptable salts that possess inhibitory activity against APEX, and methods of using the compounds and salts to treat cancer and precancerous conditions.

Inventors:
MUNSHI NIKHIL C (US)
SHAMMAS MASOOD A (US)
Application Number:
PCT/US2021/056605
Publication Date:
May 05, 2022
Filing Date:
October 26, 2021
Export Citation:
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Assignee:
DANA FARBER CANCER INST INC (US)
MUNSHI NIKHIL C (US)
SHAMMAS MASOOD A (US)
International Classes:
A61K31/538; C07D265/34; C07D265/38
Foreign References:
US20180243299A12018-08-30
Other References:
DATABASE Pubchem [online] 25 May 2018 (2018-05-25), ANONYMOUS: "SID 371395985", XP055939178, retrieved from NCBI Database accession no. 371395985
TAKAGI, K. MIZUNO, A. IWAMOTO, H. OOTA, M. SHIRAI, K. MATSUOKA, M.: "A colour sensor system based on the photo- or thermal-isomerisation of anthraisoxazoles to phenoxazinequinones", DYES AND PIGMENTS, vol. 45, no. 3, 1 June 2000 (2000-06-01), GB , pages 201 - 208, XP004222175, ISSN: 0143-7208, DOI: 10.1016/S0143-7208(00)00004-8
Attorney, Agent or Firm:
CLARKE, J.D., Daniel W. et al. (US)
Download PDF:
Claims:
What is claimed is:

1. A method of treating cancer or a pre-cancerous condition with apurinic/apyrimidinic (AP) deoxyribonuclease (APEX) expression and/or activity, comprising administering to a subject in need thereof a therapeutically effective amount of any one or more of compounds 1, 2, 3, 4, 5, 6, 7, 8, and 9: or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

2. A method of treating cancer or a precancerous condition with APEX expression and/or activity, which comprises co-administering to a subject in need thereof a therapeutically effective amount of any one or more of compounds 1, 2, 3, 4, 5, 6, 7, 8, and 9:

or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an additional anti-cancer agent to a subject in need thereof, wherein the co-administering results in a greater anti-cancer effect than the effect of the additional anti-cancer agent when administered alone as a sole active agent, without any of compounds 1-9.

3. The method of claim 2, wherein the anti-cancer agent is selected from the group consisting of melphalan, vincristine, cyclophosphamide, etoposide, doxorubicin, bendamustine, bevacizumab, irinotecan hydrochloride, capecitabine, cetuximab, ramucirumab, oxaliplatin, cetuximab, fluorouracil, ipilimumab, pembrolizumab, nivolumab, panitumumab, regorafenib, abemaciclib, paclitaxel, everolimus, anastrozole, atezolizumab, docetaxel, epirubicin hydrochloride, exemestane, toremifene, fulvestrant, letrozole, gemcitabine hydrochloride, eribulin mesylate, trastuzumab, palbociclib, ixabepilone, ribociclib, olaparib, methotrexate, pertuzumab, thiotepa, alectinib, brigatinib, dabrafenib, dacomitinib, durvalumab, gefitinib, lorlatinib, sunitinib malate, mitomycin C, osimertinib mesylate, dactinomycin, ifosfamide, vinblastine sulfate, bleomycin sulfate, topotecan hydrochloride, hydroxyurea, megestrol acetate, glasdegib, veneto cl ax, paxopanib, temsirolimus, abiraterone acetate, apalutamide, bicalutamide, cabazitaxel, degarelix, leuprolide acetate, enzalutamide, flutamide, goserelin acetate, nilutamide, sipuleucel-T, radium 223 dichloride, aldesleukin, auelumab, axitinib, cabozantinib-S-malate, lenvatinib mesylate, sorafenib tosylate, bortezomib, carfilzomib, ixazomib, thalidomide, lenalidomide, pomalidomide, iberdomide, CC-885, dexamethasone, cisplatin, venetoclax, a DNA-damaging agent, a topoisomerase inhibitor, a telomerase inhibitor, a poly ADP ribose polymerase (PART) inhibitor, cellular and antibody-drug conjugates, antibody-based therapies, bispecific antibodies and cellular therapies.

4. The method of claim 3, wherein the anti-cancer agent is melphalan.

5. The method of claim 3, wherein the anti-cancer agent is cyclophosphamide.

6. The method of claim 2, wherein the anti-cancer agent is a proteasome inhibitor.

7. The method of claim 6, wherein the proteasome inhibitor is bortezomib, carfilzomib, or ixazomib.

8. The method of claim 7, wherein the proteasome inhibitor is bortezomib.

9. The method of claim 2, wherein the anti-cancer agent is an immunomodulating agent.

10. The method of claim 9, where the immunomodulating agent is thalidomide, lenalidomide, pomalidomide, or CC-885.

11. The method of claim 10, where the immunomodulating agent is lenalidomide.

12. The method of claim 1 or 2, wherein the cancer is thyroid, skin, cervical, esophageal, stomach, or a hematological cancer.

13. The method of claim 12, wherein the cancer is multiple myeloma.

14. The method of claim 12, wherein the cancer is esophageal adenocarcinoma.

15. The method of claim 1 or 2, wherein the precancerous condition is monoclonal gammopathy of undetermined significance (MGUS).

16. The method of claim 1 or 2, wherein the precancerous condition is Barrett’s esophagus.

17. The method of claim 1 or 2, wherein the cancer is a virus-induced cancer.

18. The method of claim 17, wherein the virus-induced cancer is induced by Epstein-Barr virus (EBV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Human immunodeficiency' virus (HIV), Human herpes virus 8 (HHV-8), Human papillomavirus (HPV), or Human T-cell leukemia virus type 1 (HTLV-1).

19. The method of claim 1 or 2, comprising co-administering to the subject compound 1 and cyclophosphamide wherein the subject has multiple myeloma.

20. The method of claim 1 or 2, comprising co-administering to the subject compound 1 and melphalan wherein the subject has multiple myeloma.

21. The method of claim 1 or 2, comprising co-administering to the subject compound 1 and lenalidomide wherein the subject has multiple myeloma.

22. The method of claim 1 or 2, comprising co-administering to the subject compound 1 and bortezomib wherein the subject has multiple myeloma.

23. The method of claim 1 or 2, comprising co-administering to the subject compound 2 and cyclophosphamide wherein the subject has multiple myeloma.

24. The method of claim 1 or 2, comprising co-administering to the subject compound 2 and melphalan wherein the subject has multiple myeloma.

25. The method of claim 1 or 2, comprising co-administering to the subject compound 2 and lenalidomide wherein the subject has multiple myeloma.

26. The method of claim 1 or 2, comprising co-administering to the subject compound 2 and bortezomib wherein the subject has multiple myeloma.

27. The method of claim 1 or 2, comprising co-administering to the subject compound 3 and cyclophosphamide wherein the subject has multiple myeloma.

28. The method of claim 1 or 2, comprising co-administering to the subject compound 3 and melphalan wherein the subject has multiple myeloma.

29. The method of claim 1 or 2, comprising co-administering to the subject compound 3 and lenalidomide wherein the subject has multiple myeloma.

30. The method of claim 1 or 2, comprising co-administering to the subject compound 3 and bortezomib wherein the subject has multiple myeloma.

31. The method of claim 1 or 2, comprising co-administering to the subject compound 4 and cyclophosphamide wherein the subject has multiple myeloma.

32. The method of claim 1 or 2, comprising co-administering to the subject compound 4 and melphalan wherein the subject has multiple myeloma.

33. The method of claim 1 or 2, comprising co-administering to the subject compound 4 and lenalidomide wherein the subject has multiple myeloma.

34. The method of claim 1 or 2, comprising co-administering to the subject compound 4 and bortezomib wherein the subject has multiple myeloma.

35. The method of claim 1 or 2, comprising co-administering to the subject compound 5 and cyclophosphamide wherein the subject has multiple myeloma.

36. The method of claim 1 or 2, comprising co-administering to the subject compound 5 and melphalan wherein the subject has multiple myeloma.

37. The method of claim 1 or 2, comprising co-administering to the subject compound 5 and lenalidomide wherein the subject has multiple myeloma.

38. The method of claim 1 or 2, comprising co-administering to the subject compound 5 and bortezomib wherein the subject has multiple myeloma.

39. The method of claim 1 or 2, comprising co-administering to the subject compound 6 and cy clophosphamide wherein the subject has multiple myeloma.

40. The method of claim 1 or 2, comprising co-administering to the subject compound 6 and melphalan wherein the subject has multiple myeloma.

41. The method of claim 1 or 2, comprising co-administering to the subject compound 6 and lenalidomide wherein the subject has multiple myeloma.

42. The method of claim 1 or 2, comprising co-administering to the subject compound 6 and bortezomib wherein the subject has multiple myeloma.

43. The method of claim 1 or 2, comprising co-administering to the subject compound 7 and cyclophosphamide wherein the subject has multiple myeloma.

44. The method of claim 1 or 2, comprising co-administering to the subject compound 7 and melphalan wherein the subject has multiple myeloma.

45. The method of claim 1 or 2, comprising co-administering to the subject compound 7 and lenalidomide wherein the subject has multiple myeloma.

46. The method of claim 1 or 2, comprising co-administering to the subject compound 7 and bortezomib wherein the subject has multiple myeloma.

47. The method of claim 1 or 2, comprising co-administering to the subject compound 8 and cyclophosphamide wherein the subject has multiple myeloma.

48. The method of claim 1 or 2, comprising co-administering to the subject compound 8 and melphalan wherein the subject has multiple myeloma.

49. The method of claim 1 or 2, comprising co-administering to the subject compound 8 and lenalidomide wherein the subject has multiple myeloma.

50. The method of claim 1 or 2, comprising co-administering to the subject compound 8 and bortezomib wherein the subject has multiple myeloma.

51. The method of claim 1 or 2, comprising co-administering to the subject compound 9 and cyclophosphamide wherein the subject has multiple myeloma.

52. The method of claim 1 or 2, comprising co-administering to the subject compound 9 and melphalan wherein the subject has multiple myeloma.

53. The method of claim 1 or 2, comprising co-administering to the subject compound 9 and lenalidomide wherein the subject has multiple myeloma.

54. The method of claim 1 or 2, comprising co-administering to the subject compound 9 and bortezomib wherein the subject has multiple myeloma.

55. The method of any one of claims 1-22, wherein a therapeutically effective amount of compound 1 or a pharmaceutically acceptable salt thereof is administered to the subject.

56. The method of any one of claims 1-18 or 23-26, wherein a therapeutically effective amount of compound 2 or a pharmaceutically acceptable salt thereof is administered to the subject.

57. Hie method of any one of claims 1-18 or 27-30, wherein a therapeutically effective amount of compound 3 or a pharmaceutically acceptable salt thereof is administered to the subject.

58. The method of any one of claims 1-18 or 31-34, wherein a therapeutically effective amount of compound 4 or a pharmaceutically acceptable salt thereof is administered to the subject.

59. The method of any one of claims 1-18 or 35-38, wherein a therapeutically effective amount of compound 5 or a pharmaceutically acceptable salt thereof is administered to the subject.

60. The method of any one of claims 1-18 or 39-42, wherein a therapeutically effective amount of compound 6 or a pharmaceutically acceptable salt thereof is administered to the subject.

61. The method of any one of claims 1 -18 or 43-46, wherein a therapeutically effective amount of compound 7 or a pharmaceutically acceptable salt thereof is administered to the subject.

62. The method of any one of claims 1-18 or 51-54, wherein a therapeutically effective amount of compound 9 or a pharmaceutically acceptable salt thereof is administered to the subject.

63. A method of reducing ddeelleetteeririoouuss effects ooff cchheemmiiccaall or carcinogen exposure, petrochemical and pesticide exposure, metal exposure or radiation exposure, comprising administering to a subject in need thereof a therapeutically effective amount of compound 1, 2, 3, 4, 5, 6, 7, 8, or 9, or a pharmaceutically acceptable salt thereof.

64. The method of any one of claims 1-63, wherein the subject is human.

Description:
SMALL MOLECULE INHIBITORS OF APEX, ALONE AND IN COMBINATION WITH OTHER CANCER DRUGS TO INHIBIT GROWTH OF CANCER CELLS

RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No: 63/106,175, filed October 27, 2020, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] The APEX gene encodes the major apurinic/apyrimidinic (AP) endonuclease in human cells. It encodes the APEX endonuclease, a DNA repair enzyme with AP activity. Such AP activity sites occur frequently in DNA molecules by spontaneous hydrolysis, by DNA damaging agents or by DNA glycosylases that remove specific abnormal bases. The AP sites are the most frequent pre-mutagenic lesions that can prevent normal DNA replication.

[0003] The majority of cancers display a complex spectrum of diverse genetic alterations at diagnosis, and acquire additional changes with progression of disease, indicating a genomic instability, which seems to arise early at the premalignant stage. Genomic instability enables cells to acquire new genomic characteristics for progression and resist therapy. The number of mutations correlates with overall and event free survival in multiple myeloma (MM), suggesting a role of genomic instability in poor clinical outcome. Dysregulated homologous recombination (HR) is a key mediator of genomic instability and drug resistance in MM, as well as contributes to telomere maintenance and tumor growth in cancer cells.

SUMMARY OF THE INVENTION

10004] The present invention is based, at least in part, on a discovery that APEX nucleases (APEX1 and APEX2), major base excision repair proteins, contribute to dysregulation of homologous recombination (HR) activity and genome stability in cancer cells at multiple levels. Data presented in the working examples show that inhibition of APEX activity in cancer cells reduces DNA breaks, inhibits HR and genomic instability', and induces cell cycle arrest. The working examples also show that inhibition of APEX activity increases sensitivity of MM cells to cancer drugs such as melphalan and PARP inhibitor. Although inhibition of AP activity increases cytotoxicity, it reduces genomic instability and heterogeneity caused by melphalan, a known chemotherapeutic agent. Data herein further show that APEX1 and APEX2 also contribute to regulation of HR and cell cycle (G2/M) checkpoint in cancer cells. The present invention includes methods for using APEX nuclease inhibitors in combination with chemotherapeutics, such as melphalan, to treat cancer and pre-cancerous conditions. Embodiments of the invention might achieve a synergistic effect in terms of cytotoxicity and/or reduced genomic instability in treated subjects.

[0005] A first aspect of the present invention is directed to a method of treating cancer or a pre-cancerous cancer or pre-cancer express apurinic/apyrimidinic (AP) deoxyribonuclease (APEX) (and/or the cancer or pre-cancerous condition has APEX activity), comprising administering a therapeutically effective amount of compound 1, 2, 3, 4, 5, 6, 7, 8, or 9: or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

[0006] A related aspect is directed to a method of treating cancer or a precancerous condition wherein cells of the cancer or pre-cancer express APEX (and/or the cancer or pre-cancerous condition has APEX activity), which comprises co-administering a therapeutically effective amount of compound 1, 2, 3, 4, 5, 6, 7, 8, or 9:

or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an additional anti-cancer (e.g., chemotherapeutic) agent to a subject in need thereof, wherein the additional anti-cancer (e.g., chemotherapeutic) agent exhibits greater cytotoxicity to cancer cells of the subject when administered in combination than when administered alone without any of compounds 1-9.

[0007] Without intending to be bound by theory, the inventive methods may inhibit, reduce or delay genomic evolution, increase tytotoxidty of existing cancer drugs, and/or reduce genetic damage/instability caused by anti-cancer (chemotherapeutic) agents in cancer and pre- cancerous cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1 is a bar graph showing the dose-dependent inhibition of APEX activity by compound 1 and 2 in a high throughput screen.

[0009] FIG. 2A-FIG. 2C are immunoblots depicting the biological activity of compound 1. FIG. 2A is an immunoblot showing the inhibition of yH2AX (a marker of DNA breaks) and p- RPA32 (a marker of DNA end resection which is a distinctive step in the initiation of HR) with various concentrations of compound 1 ; APB, a known inhibitor of APEX1, is used as a positive control. FIG. 2B is an immunoblot showing that compound 1 reduced melphalan-induced DNA damage/breaks (as indicated by yH2AX expression) in MM1S cells; nilotinib (NILO), a clinically approved drug which inhibits HR by targeting ABL kinase, is used as a control. FIG. 2C is an immunoblot showing that compound 1 can prevent/reduce spontaneous as well as melphalan-induced DNA damage in MM cells.

[0010] FIG. 3 is an immunoblot showing the inhibition of yH2AX and p-RPA32 (indicating reduced DNA breaks and HR) with compound 2 at indicated concentrations and 3750 (2-(lH- pyrrol-l-yl)terephthalic acid).

[0011] FIG. 4 is an illustration depicting micronucleus formation. Micronuclei are small DNA containing structures surrounded by one lipid bilayer, which are independent from the main nucleus in a cell, and used as a marker of genomic instability.

[0012] FIG. 5A is a bar graph showing that compounds 1 and 2 (at indicated concentrations) and 3776 (5-(lH-pyrrol-l-yl)benzene-l,3-dicarboxylic acid) stabilized the genome in live cell fraction in MM1 S cells. FIG. 5B is a bar graph showing that compound 1 stabilized the genome in live cell fraction in RPMI cells at indicated concentrations.

[0013] FIG. 6A-FIG. 6B show that treatment with compound 1 reduces the acquisition of genomic changes over time in MM cells. Control and treated cells were cultured for 3 weeks and genomic changes acquired by these cells during this period were identified by single nucleotide polymorphism (SNP) arrays, using the genome of “day 0” cells as a baseline. FIG. 6A is a series of plots of copy number changes (red dots, amplifications; blue dots, deletions) acquired on different chromosomes in control (DMSO-treated) cells and those treated with 8 and 16 pM compound 1. FIG. 6B is a series of bar graphs showing copy number changes over whole genome in control (DMSO-treated) cells and those treated with 8 and 16 pM compound 1.

[0014] FIG. 7A-FIG. 7B show that treatment with compound 2, APB (a known APEX1 inhibitor), 3750 (5-(lH-pyrrol-l-yl)benzene-l,3-dicarboxylic acid), and 7045 (2V,A-diethyl-7V- (5-imino-5H-benzo|a|phenoxazin-9-yl)amine) reduces the acquisition of genomic changes over time in MM cells. Control and treated cells were cultured for 3 weeks and genomic changes acquired by these cells during this period were identified by SNP arrays, using the genome of “day 0” cells as a baseline. FIG. 7 A is a series of plots of copy number changes (red dots, amplifications; blue dots, deletions) acquired on different chromosomes in control (DMSO-treated) cells and those treated with inhibitors. FIG. 7B is a series of bar graphs showing copy number changes over whole genome in control (DMSO-treated) cells and those treated with inhibitors.

[0015] FIG. 8A-FIG. 8B show that treatment with compound 2 at a lower dose also reduces the acquisition of genomic changes over time in MM cells. Control and treated cells were cultured for 3 weeks and genomic changes acquired by these cells during this period were identified by SNP arrays, using tire genome of “day 0” cells as a baseline. FIG. 8 A is a series of plots of copy number changes (red dots, amplifications; blue dots, deletions) acquired on chromosomes 1 and 7 in control (DMSO-treated) cells and those treated with inhibitors. FIG. 8B is a bar graph showing copy number changes over whole genome in control (DMSO- treated) cells and those treated with inhibitor.

[0016] FIG. 9A-FIG. 9F is a series of bar graphs showing that compounds 1 and 2 increase cytotoxicity of existing myeloma drugs. MM cells were treated with compounds 1 and 2, alone and in combination with existing drugs. FIG. 9A is a bar graph depicting cell viability for MM1S cells treated with compound 1 in combination with cyclophosphamide. FIG 9B is a bar graph depicting cell viability for RPMI cells treated with compound 1 in combination with cyclophosphamide. FIG. 9C is a bar graph depicting cell viability for RPMI cells treated with compound 1 in combination with melphalan. FIG. 9D is a bar graph depicting cell viability for MM1 S cells treated w-ith compound 1 in combination with lenalidomide. FIG. 9E is a bar graph depicting cell viability for MM1S cells treated with compound 1 in combination with bortezomib. FIG. 9F is a bar graph depicting cell viability for MM1S cells treated with compound 2 in combination with bortezomib.

[0017] FIG. 10 is a series of bar graphs showing inhibition of APEX activity of compounds 3-7 assessed by fluorescence-based assay. Top bar graph shows relative APEX activities. Bottom bar graph shows IC 50 values of APEX activity.

[0018] FIG 11 A-FIG. 11 D is a series of bar graphs showing the impact of the treatment on cell viability. FIG. 11 A shows the three-day treatment with compound 9. FIG. 1 IB shows the three-day treatment with compound 7. FIG. 11C shows the five-day treatment with compound 9. FIG. 1 ID shows the five-day treatment with compound 7. Normal fibroblasts (HDF) and MM cell lines (RPMI, MM1S and H929) are shown.

[0019] FIG. 12A-FIG. 12D is a series of bar graphs showing the impact of the treatment on cell viability. FIG. 12A shows the three-day treatment with compound 5. FIG. 12B shows the three-day treatment with compound 6. FIG. 12C show's the five-day treatment with compound 5. FIG. 12D shows the five-day treatment with compound 6. Normal fibroblasts (HDF) and MM cell lines (RPMI, MM1S and H929) are shown.

10020] FIG. 13A-FIG. 13D is a series of bar graphs showing the impact of the treatment on cell viability. FIG. 13A shows the three-day treatment with compound 3. FIG. 13B shows the three-day treatment with compound 4. FIG. 13C show's the five-day treatment with compound 3. FIG. 13D shows the five-day treatment with compound 4. Normal peripheral blood mononuclear cell samples (PBMC1, PBMC2) and MM cell lines (RPMI, MM IS and H929) are shown.

[0021] FIG. 14A is a series of bar graphs showing the impact of 3-day treatment with compound 8 on cell viability. FIG. 14B is a series of bar graphs showing the impact of 5-day treatment with compound 8 on cell viability. Normal peripheral blood mononuclear cell samples (PBMC1, PBMC2) and MM cell lines (RPMI, MM1 S and H929) are shown.

[0022] FIG. 15 is a series of Western blots showing the impact on DNA damage. MM cells were treated with APE1 inhibitor III (APB) and compound 4 or 9 for 48 h, live cells purified and evaluated for expression of pRPA32 (RPA; a marker of DNA damage and end resection), y-H2AX (DNA break marker) and GAPDH (internal control).

DETAILED DESCRIPTION

10023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used in tiie specification and the appended claims, unless specified to the contrary', the following terms have the meaning indicated in order to facilitate the understanding of the present invention.

[0024] As used in the description and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an inhibitor” includes mixtures of two or more such inhibitors, and the like.

[0025] Unless stated otherwise, the term “about” means within 10% (e.g., within 5%, 2% or 1%) of the particular value modified by the term “about.”

[0026] The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim The transitional phrase “consisting essentially of’ limits the scope of a claim to tire specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. f 0027] Methods of the present invention utilize the following compounds, as follows:

7-(phenylamino)-8H -naphtho[2,3-a ]phenoxazine-8, 13(14H ) -dione

2-(diethylamino)benzo[b]phenazine-6,11 -dione

N-((5S,8R )-3-(benzo[d ]thiazol-2-yl)-9-ethyl-5,6,7,8-tetrahydro-4H -5,8- epiminocyclohepta[b]thiophen-2-yl)acetamide “0212”)

N-((5S,8R )-3-(benzo[d ]thiazol-2-yl)-5,6,7,8-tetrahydro-4H -5,8- ep iminocy clohepta[b ] thiophen -2-yl)acetamide “0238”) N-((5S,8R )-3-(benzo[d ]thiazol-2-yl)-9-ethyl-5,6,7,8-tetrahydro-4H -5,8- epiminocyclohepta[b]thiophen-2-yl)-1H -pyrazole-4 -carboxamide

(5, 0282”), N-((5S',8R )-3-(benzo[d ]thiazol-2-yl)-9-ethyl-5,6,7,8-tetrahydro-4H -5,8- epiminocyclohepta[6]thiophen-2-yl)- 1 -methyl- 1H -pyrazole-4-carboxamide “0285”) N-((5S',8R )-3-(benzo[d]thiazol-2-yl)-9-etiiyl-5,6,7,8-tetrahydro-4H -5,8- epiminocyclohepta[6]thiophen-2-yl)-l-methyl-lH -imidazole-5-carboxami “e “0288”) N-((5S',8R )-3-(benzo[d ]thiazol-2-yl)-9-propyl-5,6,7,8-tetrahydro-4H -

5,8-epiminocyclohepta[b]thiophen-2-yl)acetamide hydrogen chloride “0214”)

N -(3-(l,3-benzothiazol-2-yl)-6-(propan-2-yl)-4H,5H,6H,7H- thieno[2,3-c]pyridin-2-yl)acetamide "2328”) or a pharmaceutically acceptable salt thereof. The compounds may be synthesized in accordance with known procedures and/or are commercially available.

[0028] The compounds may be in the form of a free acid or free base, or a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable" in the context of a salt refers to a salt of the compound that does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the compound in salt form may be administered to a subject without causing undesirable biological effects (such as dizziness or gastric upset) or interacting in a deleterious manner with any of the other components of the composition in which it is contained. The term "pharmaceutically acceptable salt" refers to a product obtained by reaction of the compound of the present invention with a suitable acid or a base. Examples of pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic bases such as Li, Na, K, Ca, Mg, Fe, Cu, Al, Zn and Mn salts. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, 4-methylbenzenesulfonate or p-toluenesulfonate salts and the like. Certain compounds of tire invention can form pharmaceutically acceptable salts with various organic bases such as lysine, arginine, guanidine, diethanolamine or metformin.

Pharmaceutical Compositions

[0029] Compounds 1-9 may be formulated into a variety of compositions for purposes of treating patients. Thus, practice of the inventive methods may entail formulating compounds 1-9, or a pharmaceutically acceptable salt thereof, with a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier,” as known in the art, refers to a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals. Suitable carriers may include, for example, liquids (both aqueous and non-aqueous alike, and combinations thereof), solids, encapsulating materials, gases, and combinations thereof (e.g., semi-solids), and gases, that function to earn' or transport the compound from one organ, or portion of the body, to another organ, or portion of the body'. A carrier is “acceptable” in the sense of being physiologically inert to and compatible with the other ingredients of the formulation and not injurious to the subject or patient Depending on the type of formulation, the composition may further include one or more pharmaceutically acceptable excipients.

[0030] Broadly, compounds 1-9 and their pharmaceutically acceptable salts may be formulated into a given type of composition in accordance with conventional pharmaceutical practice such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping and compression processes (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. S warbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York). The type of formulation depends on the mode of administration which may include enteral (e.g., oral, buccal, sublingual and rectal), parenteral (e.g., subcutaneous (s.c.\ intravenous (z.v.), intramuscular (i.mf, and intrastemal injection, or infusion techniques, intra-ocular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, interdermal, intravaginal, intraperitoneal, mucosal, nasal, intratracheal instillation, bronchial instillation, and inhalation) and topical (e.g., transdermal). In general, the most appropriate route of administration will depend upon a variety of factors including, for example, the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration). For example, parenteral (e.g., intravenous) administration may also be advantageous in that the compound may be administered relatively quickly such as in the case of a single-dose treatment and/or an acute condition.

[0031] In some embodiments, the compounds are formulated for oral or intravenous administration (e.g., systemic intravenous injection).

[0032] Accordingly, compounds 1-9 may be formulated into solid compositions (e.g., powders, tablets, dispersible granules, capsules, cachets, and suppositories), liquid compositions (e.g., solutions in which the compound is dissolved, suspensions in which solid particles of the compound are dispersed, emulsions, and solutions containing liposomes, micelles, or nanoparticles, syrups and elixirs); semi-solid compositions (e.g., gels, suspensions and creams); and gases (e.g., propellants for aerosol compositions). The compounds may also be formulated for rapid, intermediate or extended release.

10033] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with a carrier such as sodium citrate or dicalcium phosphate and an additional carrier or excipient such as a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, carboxy methylcellulose, sodium carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as crosslinked polymers (e.g., crosslinked polyvinylpyrrolidone (crospovidone), crosslinked sodium carboxymethyl cellulose (croscarmellose sodium), sodium starch glycolate, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also include buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings. They may further contain an opacifying agent.

[0034] In some embodiments, compounds 1-9 may be formulated in a hard or soft gelatin capsule. Representative excipients that may be used include pregelatinized starch, magnesium stearate, mannitol, sodium stearyl fumarate, lactose anhydrous, microcrystalline cellulose and croscarmellose sodium. Gelatin shells may include gelatin, titanium dioxide, iron oxides and colorants.

[0035] Liquid dosage forms for oral administration include solutions, suspensions, emulsions, micro-emulsions, syrups and elixirs. In addition to the compound, the liquid dosage forms may contain an aqueous or non-aqueous carrier (depending upon the solubility of the compounds) commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Oral compositions may also include an excipients such as wetting agents, suspending agents, coloring, sweetening, flavoring, and perfuming agents. [0036] Injectable preparations may include sterile aqueous solutions or oleaginous suspensions. They may be formulated according to standard techniques using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. The effect of the compound may be prolonged by slowing its absorption, which may be accomplished by the use of a liquid suspension or crystalline or amorphous material with poor water solubility. Prolonged absorption of the compound from a parenterally administered formulation may also be accomplished by suspending the compound in an oily vehicle.

[0037] In certain embodiments, compounds 1-9 may be administered in a local rather than systemic manner, for example, via injection of the conjugate directly into an organ, often in a depot preparation or sustained release formulation. In specific embodiments, long acting formulations are administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Injectable depot forms are made by forming microencapsule matrices of the compound in a biodegradable polymer, e.g., polylactidepolyglycolides, poly(orthoesters) and poly(anhydrides). The rate of release of the compound may be controlled by varying the ratio of compound to polymer and the nature of the particular polymer employed. Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues. Furthermore, in other embodiments, the compound is delivered in a targeted drug delivery system, for example, in a liposome coated with organ-specific antibody. In such embodiments, the liposomes are targeted to and taken up selectively by the organ.

10038] Compounds 1-9 may be formulated for buccal or sublingual administration, examples of which include tablets, lozenges and gels.

[0039] Compounds 1-9 may be formulated for administration by inhalation. Various forms suitable for administration by inhalation include aerosols, mists or powders. Pharmaceutical compositions may be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In some embodiments, the dosage unit of a pressurized aerosol may be determined by providing a valve to deliver a metered amount. In some embodiments, capsules and cartridges including gelatin, for example, for use in an inhaler or insufflator, may- be formulated containing a powder mix of tire compound and a suitable powder base such as lactose or starch.

[0040] Compounds 1-9 may be formulated for topical administration which as used herein, refers to administration intradermally by application of the formulation to the epidermis. These types of compositions are typically in the form of ointments, pastes, creams, lotions, gels, solutions and sprays.

[0041] Representative examples of carriers useful in formulating compositions for topical application include solvents (e.g., alcohols, poly alcohols, water), creams, lotions, ointments, oils, plasters, liposomes, powders, emulsions, microemulsions, and buffered solutions (e.g., hypotonic or buffered saline). Creams, for example, may be formulated using saturated or unsaturated fatty adds such as stearic acid, palmitic acid, oleic acid, palmito-oleic add, cetyl, or oleyl alcohols. Creams may also contain anon-ionic surfactant such as polyoxy-40-stearate. [0042] In some embodiments, the topical formulations may also include an excipient, an example of which is a penetration enhancing agent. These agents are capable of transporting a pharmacologically active compound through the stratum comeum and into the epidermis or dermis, preferably, with little or no systemic absorption. A wide variety of compounds have been evaluated as to their effectiveness in enhancing the rate of penetration of drugs through the skin. See, for example, Percutaneous Penetration Enhancers, Maibach H. I. and Smith H. E. (eds.), CRC Press, Inc., Boca Raton, Fla. (1995), which surveys the use and testing of various skin penetration enhancers, and Buyuktimkin et al, Chemical Means of Transdermal Drug Permeation Enhancement in Transdermal and Topical Drug Delivery Systems, Gosh T. K., Pfister W. R., Yum S. I. (Eds.), Interpharm Press Inc., Buffalo Grove, Ill. (1997). Representative examples of penetration enhancing agents include triglycerides (e.g., soybean oil), aloe compositions (e.g., aloe-vera gel), ethyl alcohol, isopropyl alcohol, octolyphenylpolyethylene glycol, oleic acid, polyethylene glycol 400, propylene glycol, N- decylmethylsulfoxide, fatty' acid esters (e.g., isopropyl myristate, methyl laurate, glycerol monooleate, and propylene glycol monooleate), and N-methylpyrrolidone.

[0043j Representative examples of yet other excipients that may be included in topical as well as in other types of formulations (to the extent they are compatible), include preservatives, antioxidants, moisturizers, emollients, buffering agents, solubilizing agents, skin protectants, and surfactants. Suitable preservatives include alcohols, quaternary amines, organic acids, parabens, and phenols. Suitable antioxidants include ascorbic acid and its esters, sodium bisulfite, butylated hydroxytoluene, butylated hydroxyanisole, tocopherols, and chelating agents like EDTA and citric acid. Suitable moisturizers include glycerin, sorbitol, polyethylene glycols, urea, and propylene glycol. Suitable buffering agents include citric, hydrochloric, and lactic acid buffers. Suitable solubilizing agents include quaternary ammonium chlorides, cyclodextrins, benzyl benzoate, lecithin, and polysorbates. Suitable skin protectants include vitamin E oil, allatoin, dimethicone, glycerin, petrolatum, and zinc oxide.

[0044] Transdermal formulations typically employ transdermal delivery devices and transdermal delivery patches wherein the compound is formulated in lipophilic emulsions or buffered, aqueous solutions, dissolved and/or dispersed in a polymer or an adhesive. Patches may be constructed for continuous, pulsatile, or on demand delivery- of pharmaceutical agents. Transdermal delivery of the compounds may be accomplished by means of an iontophoretic patch. Transdermal patches may provide controlled delivery of the compounds wherein the rate of absorption is slowed by using rate-controlling membranes or by trapping the compound within a polymer matrix or gel. Absorption enhancers may be used to increase absorption, examples of which include absorbable pharmaceutically acceptable solvents that assist passage through the skin.

[0045[ Ophthalmic formulations include eye drops.

[00461 Formulations for rectal administration include enemas, rectal gels, rectal foams, rectal aerosols, and retention enemas, which may contain conventional suppository bases such as cocoa butter or other glycerides, as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and the like. Compositions for rectal or vaginal administration may also be formulated as suppositories which can be prepared by mixing the compound with suitable non-irritating carriers and excipients such as cocoa butter, mixtures of fatty acid glycerides, polyethylene glycol, suppository' waxes, and combinations thereof, all of which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the compound.

Dosage Amounts

[0047] As used herein, the term, "therapeutically effective amount" refers to an amount of compound 1, 2, 3, 4, 5, 6, 7, 8, or 9, or a pharmaceutically acceptable salt thereof, effective in producing the desired therapeutic response in a particular patient suffering from cancer or a precancerous condition. The term "therapeutically effective amount" includes the amount of the compound or a pharmaceutically acceptable salt thereof, when administered, may induce a positive modification in the cancer or precancerous condition to be treated (e.g., to inhibit or reduce APEX activity), or is sufficient to prevent progression of the cancer or precancer, or alleviate to some extent, one or more of the symptoms of the cancer or precancer being treated in a subject, or which simply kills or inhibits the growth of diseased cells, or reduces the amount of APEX or other relevant markers in diseased cells. In the context of co-therapy, the amount of compound 1, 2, 3, 4, 5, 6, 7, 8, or 9 may inhibit, reduce or delay genomic evolution, increase cytotoxicity of the additional anti-cancer agent, or reduce genetic damage/instability caused by' the anti-cancer (e.g., chemotherapeutic) agents in cancer or precancerous cells.

[0048] A “prophylactically effective amount” of a compound described herein is an amount sufficient to prevent a condition, or one or more symptoms associated with the condition or prevent its recurrence. A prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition. The term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy' of another prophylactic agent.

[0049] The total daily dosage of the compounds and usage thereof may be decided in accordance with standard medical practice, e.g., by the attending physician using sound medical judgment. The specific therapeutically effective dose for any particular subject may depend upon a variety of factors including the disease or disorder being treated and the severity thereof (e.g. , its present status); the age, body' weight, general health, sex and diet of the subj ect; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see, for example, Goodman and Gilman's, The Pharmacological Basis of Therapeutics, 10th Edition, A Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001).

10050] The amount of compound 1, 2, 3, 4, 5, 6, 7, 8, or 9 that is used for a therapeutic application varies with standard considerations, such as die nature of the condition being treated and die age and condition of the subject, and may be ultimately determined by die attending physician.

[0051] Compounds 1-9 may be effective over a wide dosage range. In some embodiments, the total daily dosage (e.g., for adult humans) may range from about 0.0001 to about 3000 mg, from about 0.0001 to about 2000 mg, from about 0.0001 to about 1000 mg, from about 0.001 to about 1000 mg, from about 0.01 to about 1000 mg, from about 0.1 to about 1000 mg, from about 1 to about 1000 mg, from about 1 to about 100 mg, from about 10 to about 1000 mg, from about 100 to about 1000 mg, and in yet other embodiments from about 0.001 to about 100 mg/kg or from about 1.0 to about 100 pg/kg. Individual dosages may be formulated to contain the desired dosage amount depending upon the number of times the compound is administered per day. By way of example, capsules may be formulated with from about 5 to about 25 mg of compound (e.g., 1, 2, 2.5, 3, 4, 5, 10, 15, 20, 25, 50, 100, 150, and 200 mg).

Methods of Use

10052] Aspects of the present invention are directed to treating cancer or a precancerous condition. In some embodiments, the cancer or precancerous condition may be characterized by expression of APEX. In some embodiments, the cancer or precancerous condition may be characterized by APEX activity. In some embodiments, the cancer or precancerous condition may be characterized by expression of APEX and APEX activity.

[0053] The term “subject” (or “patient”) as used herein includes all members of the animal kingdom prone to or suffering from the indicated disease or disorder. In some embodiments, the subject is a mammal, e.g., a human or a non-human mammal. The methods are also applicable to companion animals such as dogs and cats as well as livestock such as cows, horses, sheep, goats, pigs, and other domesticated and wild animals. A subject “in need of’ the treatment may be suffering from or suspected of suffering from a specific disease or disorder may have been positively diagnosed or otherwise presents with a sufficient number of risk factors or a sufficient number or combination of signs or symptoms such that a medical professional could diagnose or suspect that the subject was suffering from the disease or disorder. Thus, subjects suffering from a specific disease or disorder versus subjects suspected of suffering from a specific disease or disorder are not necessarily two distinct groups. [0054] In some embodiments, compounds 1-9 and their pharmaceutically acceptable salts may be used to treat cancer or a precancerous condition by way of monotherapy. The method entails administering a therapeutically effective amount of compound 1, 2, 3, 4, 5, 6, 7, 8, or 9: or a pharmaceutically acceptable salt thereof, to a subject in need thereof. Thus, in some embodiments, compound 1, 2, 3, 4, 5, 6, 7, 8, or 9 may be administered to a patient as monotherapy as a front/first-line therapy who has not undergone any prior anti-cancer treatment or as follow-on, e.g., “second line” therapy e.g., for a patient who is unresponsive to front line therapy, who has had previous treatments which have been partially successful but are intolerant to die particular treatment, or as an adjuvant treatment, z.e., to prevent reoccurrence of cancer in a patient with no currently detectable disease or after surgical removal of a tumor.

[0055] Compounds 1-9 and their pharmaceutically acceptable salts may also be used to treat cancer or a precancerous condition by way of combination therapy. The method entails coadministering a therapeutically effective amount of compound 1, 2, 3, 4, 5, 6, 7, 8, or 9:

or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an additional anti-cancer (e.g., chemotherapeutic) or active agent (that is not compound 1, 2, 3, 4, 5, 6, 7, 8, or 9) to a subject in need thereof, wherein the additional anti-cancer (e.g., chemotherapeutic) agent exhibits greater cytotoxicity to cancer cells of the subject than when co-administered alone without any of compounds 1-9.

[0056] As used herein, the term “in combination” means that the two active agents are coadministered. Co-administration includes substantially contemporaneous administration, by way of the same or separate dosage forms, and by the same or different modes of administration, or sequentially (either one before the other), e.g., as part of the same treatment regimen, or by way of successive treatment regimens. Therefore, the method is not limited to the administration of the active agents at exactly the same time. If administered sequentially, administration of the second agent is timed such that it is capable of augmenting the anti-cancer effect of the first and previously administered agent. In some embodiments, the coadministration of compound 1 and/or 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and the additional anti-cancer agent achieves a synergistic anti-cancer effect. For example, in some embodiments, the additional anti -cancer (e.g., chemotherapeutic) agent may exhibit greater cytotoxicity to cancer cells of the subject when administered in combination with compound 1 and/or 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 than when administered alone as a single active agent, without any of compounds 1-9. In some embodiments, the synergistic anti-cancer effect may be achieved by reducing genomic instability and heterogeneity caused by known chemotherapeutic agents such as melphalan.

[0057] In view of these effects, in some embodiments, the dosage of the additional anticancer therapeutic may be the same or even lower than known or recommended doses. See, Hardman etal., eds., Goodman & Gilman's The Pharmacological Basis Of Basis Of Therapeutics, 10th ed., McGraw-Hill, New York, 2001; Physician's Desk Reference 60th ed., 2006.

10058] The methods of the present invention may entail administration of compounds 1 and/or 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or the additional anti-cancer agent, or pharmaceutical compositions thereof, to the patient in a single dose or in multiple doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more doses). For example, the frequency of administration may range from once a day up to about once every' eight weeks. In some embodiments, the frequency of administration ranges from about once a day for 1, 2, 3, 4, 5, or 6 weeks, and in other embodiments entails a 28-day cycle which includes daily administration for 3 weeks (21 days). In other embodiments, the compound may be dosed twice a day (BID) over the course of two and a half days (for a total of 5 doses) or once a day (QD) over the course of two days (for a total of 2 doses). In other embodiments, the compound may be dosed once a day (QD) over the course of five days. The length of the treatment period depends on a variety of factors, such as severity of the disease, age of the subject, the concentration and the activity of the compound, or a combination thereof. It will also be appreciated that the effective dosage of the compound used for the treatment may increase or decrease over the course of a particular treatment regime.

[0059] Representative examples of additional anti-cancer agents that may be effective in tiie inventive methods include chemotherapeutic agents such as melphalan, vincristine, cyclophosphamide, etoposide, doxorubicin, bendamustine, bevacizumab, irinotecan hydrochloride, capecitabine, cetuximab, ramucirumab, oxaliplatin, cetuximab, fluorouracil, ipilimumab, pembrolizumab, nivolumab, panitumumab, regorafenib, abemaciclib, paclitaxel, everolimus, anastrozole, atezolizumab, docetaxel, epirubicin hydrochloride, exemestane, toremifene, fulvestrant, letrozole, gemcitabine hydrochloride, eribulin mesylate, trastuzumab, palbociclib, ixabepilone, ribociclib, olaparib, methotrexate, pertuzumab, thiotepa, alectinib, brigatinib, dabrafenib, dacomitinib, durvalumab, gefitinib, lorlatinib, sunitinib malate, mitomycin C, osimertinib mesylate, dactinomycin, ifosfamide, vinblastine sulfate, bleomycin sulfate, topotecan hydrochloride, hydroxyurea, megestrol acetate, glasdegib, venetoclax, paxopanib, temsirolimus, abiraterone acetate, apalutamide, bicalutamide, cabazitaxel, degarelix, leuprolide acetate, enzalutamide, flutamide, goserelin acetate, nilutamide, sipuleucel-T, radium 223 dichloride, aldesleukin, auelumab, axitinib, cabozantimb-S-malate, lenvatinib mesylate, sorafenib tosylate, bortezomib, carfilzomib, ixazomib, thalidomide, lenalidomide, pomalidomide, iberdomide, CC-885, dexamethasone, cisplatin, venetoclax, a DNA-damaging agent, a topoisomerase inhibitor, a telomerase inhibitor, a poly ADP ribose polymerase (PARP) inhibitor, cellular and antibody-drug conjugates, antibody-based therapies (e.g., daratumumab and elotuzumab), bispecific antibodies and cellular therapies.

[0060] In some embodiments, the other anti-cancer agent is a proteasome inhibitor, examples of which include bortezomib, carfilzomib, and ixazomib.

[0061] In some embodiments, the other anti-cancer agent is a topoisomerase inhibitor, examples of which include irinotecan, topotecan, camptothecin, diflomotecan, lamellarin D, etoposide, teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, ellipticines, aurintricarboxylic acid, HU -311, genistein, quercetin, and resveratrol.

[0062] In some embodiments, the other anti-cancer agent is a telomerase inhibitor, examples of which include azidothymidine (AZT), GRN163, GRN163L, tamoxifen, epigallocatechin gallate, purpuromycin, rubromycin, BIBR-1532, U-73122, carbovir, dideoxyguanosine (DDG), carbocyclic oxetanocin G triphosphate (C-OXT-GTP), TMPyP4, BRACO19, telomestatin, fluoroquinophenoxazine QQ58, 12459 triazine, pentacyclic acridine RHPS4, and dibenzo phenanthroline.

[0063] In some embodiments, the other anti-cancer agent is a poly ADP ribose polymerase (PARP) inhibitor, examples of which include talazoparib, veliparib, pamiparib, olaparib, rucaparib, CEP 9722, E7016, iniparib, and 3-aminobenzamide.

[0064] In other embodiments, the other anti-cancer agent is an immunomodulating agent, examples of which include thalidomide, lenalidomide, pomalidomide and CC-885.

[0065] In certain embodiments, the other anti-cancer agent is cyclophosphamide.

[0066] In certain embodiments, the other anti-cancer agent is melphalan.

[0067] In certain embodiments, the other anti-cancer agent is lenalidomide.

[0068] In certain embodiments, the other anti-cancer agent is bortezomib.

[0069] In some embodiments, compound 1 is used in combination with cy clophosphamide. [0070] In some embodiments, compound 1 is used in combination with melphalan.

[0071] In some embodiments, compound 1 is used in combination witti lenalidomide.

]0072] In some embodiments, compound 1 is used in combination with bortezomib.

[0073] In some embodimmts, compound 2 is used in combination with cyclophosphamide.

[0074] In some embodimmts, compound 2 is used in combination with melphalan.

[0075] In some embodimmts, compound 2 is used in combination with lenalidomide.

[0076] In some embodimmts, compound 2 is used in combination with bortezomib.

[0077] In some embodimmts, compound 3 is used in combination with cyclophosphamide.

[0078] In some embodimmts, compound 3 is used in combination witti melphalan.

|0079] In some embodimmts, compound 3 is used in combination with lenalidomide.

|0080] In some embodimmts, compound 3 is used in combination with bortezomib.

[0081] In some embodimmts, compound 4 is used in combination with cy clophosphamide.

[0082] In some embodimmts, compound 4 is used in combination with melphalan.

[0083] In some embodimmts, compound 4 is used in combination with lenalidomide.

[0084] In some embodimmts, compound 4 is used in combination with bortezomib.

[0085] In some embodimmts, compound 5 is used in combination witti cyclophosphamide.

]0086] In some embodimmts, compound 5 is used in combination with melphalan.

10087] In some embodimmts, compound 5 is used in combination with lenalidomide.

[0088] In some embodimmts, compound 5 is used in combination with bortezomib.

[0089] In some embodimmts, compound 6 is used in combination with cyclophosphamide.

[0090] In some embodimmts, compound 6 is used in combination with melphalan.

[0091] In some embodimmts, compound 6 is used in combination with lenalidomide.

[0092] In some embodimmts, compound 6 is used in combination witti bortezomib.

10093] In some embodimmts, compound 7 is used in combination with cyclophosphamide.

10094] In some embodimmts, compound 7 is used in combination with melphalan.

[0095] In some embodimmts, compound 7 is used in combination with lenalidomide.

[0096] In some embodimmts, compound 7 is used in combination with bortezomib.

[0097] In some embodimmts, compound 8 is used in combination with cyclophosphamide.

[0098] In some embodimmts, compound 8 is used in combination with melphalan.

[0099] In some embodimmts, compound 8 is used in combination witti lenalidomide.

[00100] In some embodimmts, compound 8 is used in combination with bortezomib.

[00101] In some embodimmts, compound 9 is used in combination with cyclophosphamide.

[00102] In some embodimmts, compound 9 is used in combination with melphalan. [00103] In some embodiments, compound 9 is used in combination with lenalidomide. [00104] In some embodiments, compound 9 is used in combination with bortezomib. |00105] Cancers which may be amenable to combination therapy according to the present invention include carcinomas (solid tumors including both primary and metastatic tumors), sarcomas, melanomas, and hematological cancers (cancers affecting blood including lymphocytes, bone marrow and/or lymph nodes) such as leukemia, lymphoma and multiple myeloma Adult tumors/cancers and pediatric tumors/cancers are included. The cancers may be vascularized, or not yet substantially vascularized, or non-vascularized tumors.

[00106] Sarcomas that may be treatable with the methods of the present invention include both soft tissue and bone cancers alike, representative examples of which include osteosarcoma or osteogenic sarcoma (bone) (e.g., Ewing’s sarcoma), chondrosarcoma (cartilage), leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle), mesothelial sarcoma or mesothelioma (membranous lining of body cavities), fibrosarcoma (fibrous tissue), and angiosarcoma or hemangioendothelioma (blood vessels).

[00107] In some embodiments, methods of the present invention entail treatment of subjects having a cancer of the thyroid, skin, cervix, esophagus, stomach, or hematological system. |00108] In certain embodiments, the cancer may be an esophageal or hematological cancer. [00109] Representative examples of hematological cancers that may be amenable to treatment with the methods of the present invention include lymphoma, leukemia, myeloid neoplasms, mast cell neoplasms, myelodysplasia, benign monoclonal gammopathy, lymphomatoid papulosis, polycythemia vera, chronic myelocytic leukemia, agnogenic myeloid metaplasia, and essential thrombocythemia. Representative examples of hematologic cancers may thus include multiple myeloma, lymphoma (including T-cell lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma (diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL) and ALK+ anaplastic large cell lymphoma (e.g., B-cell non-Hodgkin’s lymphoma selected from diffuse large B-cell lymphoma (e.g., germinal center B-cell-like diffuse large B-cell lymphoma or activated B-cell-like diffuse large B-cell lymphoma), Burkitt’s lymphoma/leukemia, mantle cell lymphoma, mediastinal (thymic) large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, metastatic pancreatic adenocarcinoma, refractory B-cell non-Hodgkin’s lymphoma, and relapsed B-cell non-Hodgkin’s lymphoma, childhood lymphomas, and lymphomas of lymphocytic and cutaneous origin, e.g., small lymphocytic lymphoma, leukemia, including childhood leukemia, hairy-cell leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloid leukemia (e.g., acute monocytic leukemia), chronic lymphocytic leukemia, small lymphocytic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, and mast cell leukemia, myeloid neoplasms, mast cell neoplasms and virus-induced cancers (e.g, Epstein-Barr virus (EBV)- induced cancers, hepatitis B virus (HBV)-induced cancers, and human immunodeficiency virus (HIV)-induced cancers).

[00110] Representative examples of esophageal cancers that may be amenable to treatment with the methods of the present invention include all forms of cell proliferative disorders affecting esophageal cells such as esophageal cancer (e.g., esophageal adenocarcinoma, esophageal squamous cell carcinoma, esophageal leiomyosarcoma, esophageal rhabdomyosarcoma, esophageal melanoma, and esophageal lymphoma), a precancer or precancerous condition of the esophagus, hyperplasia of the esophagus, dysplasia of the esophagus, benign growths or lesions of the esophagus, and malignant growths or lesions of the esophagus, and metastatic lesions in tissue and organs in the body other than the esophagus. [00111] In certain embodiments, the methods may be useful in tire treatment of multiple myeloma.

[00112] In certain embodiments, the methods may be useful in the treatment of esophageal adenocarcinoma.

[00113] Inventive methods that treat pre-cancerous conditions may deter or delay onset of cancer.

[00114 j In certain embodiments, the methods may be usefill in the treatment of monoclonal gammopathy of undetermined significance (MGUS) and/or to prevent or delay its progression. [00115] In certain embodiments, the methods may be useful in the treatment of Barrett’s esophagus and/or to prevent or delay its progression.

[00116] In some embodiments, the methods may be useful in the treatment of a virus induced cancers. In some embodiments, the virus is Epstein-Barr virus (EBV). EBV infection is known to cause Burkitt lymphoma, Hodgkin’s and non-Hodgkin’s lymphoma, and stomach cancer. In some embodiments, the virus is hepatitis B virus (HBV). Hepatitis B is known to cause liver cancer. In some embodiments, the virus is hepatitis C virus (HCV). Hepatitis C is known to cause liver cancer and non-Hodgkin’s lymphoma. In some embodiments, the virus is human immunodeficiency virus (HIV). It is known that HIV can cause Kaposi sarcoma, non- Hodgkin’s and Hodgkin’s lymphoma, cervical cancer, and cancers of the anus, liver, mouth and throat and lung. In some embodiments, the virus is human herpes virus 8 (HHV-8). HHV- 8 is known to cause Kaposi sarcoma. In some embodiments, the virus is human papillomavirus (HPV). HPV is known to cause anal, cervical, penile, throat, vaginal and vulvar cancer. In some embodiments, the virus is human T-cell leukemia virus type 1 (HTLV-1). HTLV-1 is known to cause to adult T-cell leukemia/lymphoma.

[00117] In some embodiments, compound 1 is used in combination with cyclophosphamide to treat multiple myeloma.

[00118] In some embodiments, compound 1 is used in combination with melphalan to treat multiple myeloma.

[00119] In some embodiments, compound 1 is used in combination with lenalidomide to treat multiple myeloma.

[00120] In some embodiments, compound 1 is used in combination with bortezomib to treat multiple myeloma.

[00121] In some embodiments, compound 2 is used in combination with cyclophosphamide to treat multiple myeloma.

[00122] In some embodiments, compound 2 is used in combination with melphalan to treat multiple myeloma.

[00123] In some embodiments, compound 2 is used in combination with lenalidomide to treat multiple myeloma.

[00124] In some embodiments, compound 2 is used in combination with bortezomib to treat multiple myeloma.

[00125] In some embodiments, compound 3 is used in combination with cyclophosphamide to treat multiple myeloma.

[00126] In some embodiments, compound 3 is used in combination with melphalan to treat multiple myeloma.

[00127] In some embodiments, compound 3 is used in combination with lenalidomide to treat multiple myeloma.

[00128] In some embodiments, compound 3 is used in combination with bortezomib to treat multiple myeloma.

[00129] In some embodiments, compound 4 is used in combination with cyclophosphamide to treat multiple myeloma.

[00130] In some embodiments, compound 4 is used in combination with melphalan to treat multiple myeloma. [00131 ] In some embodiments, compound 4 is used in combination with lenalidomide to treat multiple myeloma.

[00132] In some embodiments, compound 4 is used in combination with bortezomib to treat multiple myeloma.

[00133] In some embodiments, compound 5 is used in combination with cyclophosphamide to treat multiple myeloma.

[00134] In some embodiments, compound 5 is used in combination with melphalan to treat multiple myeloma.

[00135] In some embodiments, compound 5 is used in combination with lenalidomide to treat multiple myeloma.

[00136] In some embodiments, compound 5 is used in combination with bortezomib to treat multiple myeloma.

[00137] In some embodiments, compound 6 is used in combination with cyclophosphamide to treat multiple myeloma.

[00138] In some embodiments, compound 6 is used in combination with melphalan to treat multiple myeloma.

[00139] In some embodiments, compound 6 is used in combination with lenalidomide to treat multiple myeloma.

[00140] In some embodiments, compound 6 is used in combination with bortezomib to treat multiple myeloma.

[00141 ] In some embodiments, compound 7 is used in combination with cyclophosphamide to treat multiple myeloma.

[00142] In some embodiments, compound 7 is used in combination with melphalan to treat multiple myeloma.

[00143] In some embodiments, compound 7 is used in combination with lenalidomide to treat multiple myeloma.

[00144] In some embodiments, compound 7 is used in combination with bortezomib to treat multiple myeloma.

[00145] In some embodiments, compound 8 is used in combination with cyclophosphamide to treat multiple myeloma.

[00146] In some embodiments, compound 8 is used in combination with melphalan to treat multiple myeloma. [00147] In some embodiments, compound 8 is used in combination with lenalidomide to treat multiple myeloma.

(00148] In some embodiments, compound 8 is used in combination with bortezomib to treat multiple myeloma.

(00149] In some embodiments, compound 9 is used in combination with cyclophosphamide to treat multiple myeloma.

[00150] In some embodiments, compound 9 is used in combination with melphalan to treat multiple myeloma.

[00151] In some embodiments, compound 9 is used in combination with lenalidomide to treat multiple myeloma.

[00152] In some embodiments, compound 9 is used in combination with bortezomib to treat multiple myeloma.

[00153] Another aspect of the present invention pertains to a method of reducing deleterious effects of chemical or carcinogen exposure (e.g., asbestos, radon, vinyl chloride, benzidene, and benzene), petrochemical and pesticide exposure (e.g., arsenic, ethylene oxide, lindane, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)), metal exposure (e.g., nickel and cadmium), radiation exposure or preservatives or potential carcinogens in food and food metabolites. Such inventive methods entail administering to aa subject in need thereof a therapeutically/prophylatically effective amount of compound 1, 2, 3, 4, 5, 6, 7, 8, or 9, or a pharmaceutically acceptable salt thereof. Subjects in need thereof have been exposed or are at risk of exposure to carcinogens, petrochemicals, pesticides, metals or radiation known to place subjects at risk of developing cancer. Deleterious effects may include the alteration of cellular metabolism or damage to DNA.

Pharmaceutical Kits

(00154] The compounds and/or compositions containing them may be assembled into kits or pharmaceutical systems. Kits or pharmaceutical systems according to this aspect of the invention include a carrier or package such as a box, carton, tube or the like, having in close confinement therein one or more containers, such as vials, tubes, ampoules, or bottles, which contains compound 1, 2, 3, 4, 5, 6, 7, 8, or 9, or a pharmaceutical composition thereof. The kits or pharmaceutical systems of the invention may also include printed instructions for using the compounds and compositions. [00155] These and other aspects of the present invention will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain particular embodiments of the invention but are not intended to limit its scope, as defined by the claims.

EXAMPLES

[00156] These and other aspects of the present invention will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain particular embodiments of the invention but are not intended to limit its scope, as defined by the claims.

[00157] Example 1: Materials and Methods

Identification of novel APEX inhibitors

[00158] A custom library of 100,000 small molecules to identify novel inhibitors of APEX1 activity in myeloma cells. These inhibitors were evaluated for impact on genome stability and growth, alone as well as in the presence of existing MM drugs, in MM cell lines.

Detection of DNA breaks

[00159] DNA breaks were estimated by evaluating MM cells for levels of y-H2A.X. Expression of y-H2AX was measured by Western blotting using anti-H2A.X (Serl39, antibody # 2577; Cell Signaling Technology®) as previously reported (Pal, et al., J. Gastroenterol. Hepatol. Res. 6(1): 2286-2295 (2017)). DNA double strand break end resection, a decisive and initiating step in HR which requires phosphorylation of RPA32 on S4/8. Levels of phosphorylated RPA32 were detected using anti-phospho-RPA32 (S4/8) or RPA32 antibodies (Bethyl Laboratories).

Apurinic/apyrimidinic (AP) nuclease activity

[00160] Apurinic/apyrimidinic (AP) nuclease activity was assessed using a fluorescence based assay (Dorjsuren et al, PLos One 7(70):e47974 (2012)). The oligonucleotides carrying AP site mimic (Tetrahydrofuran; THF) and a fluorescein label on one strand and a quenching moiety (Dabcyl-Q) on other strand were synthesized commercially (Eurogentec Ltd.). A stretch of G and C base pairs before and after the THF moiety were used to prevent spontaneous dissociation of the short-labeled fragment prior to cleavage by AP nuclease. Complementary single stranded oligonucleotides w-ere annealed and resulting double stranded DNA substrate was incubated with MM cell lysates in assay buffer (50 mM Tris pH 8.0, 1 mM MgCh, 50 mM NaCl, 2 mM dithiothreitol (DTT)) and changes in fluorescence measured at 37°C over time, using a Plate Reader. The APEX endonuclease activity cleaves AP site releasing the short fluorescein-labeled fragment, thus resulting in the increase in fluorescence. This assay was miniaturized to screen the small molecule library in a high throughput fashion. Compounds 1-9 were evaluated for various parameters of genome stability' and growth. Myeloma cells were treated with inhibitors, alone or in combination with existing drugs, and impact on DNA breaks evaluated measuring levels of y-H2AX (marker of DNA breaks) and impact on DNA end resection detected by monitoring levels of p-RPA32 using Western blotting.

Evaluating impact on micronuclei, a marker of genomic instability

[00161 j To evaluate impact on genomic instability, the cells were cultured in the presence or absence of inhibitor of AP nuclease activity (APB),

N S

NHAc

/J

A-(3-(l,3-benzo[d ]thiazol-2-yl)-6-isopropyl-4, 5,6,7- tetrahydrothieno[2,3-c]pyridin-2-yl)acetamide (API3), melphalan or combination of both, and cells evaluated for micronuclei, a marker of unstable genome (Balmus et al., Nat. Protoc. 70<7):205-215 (2015); Terradas et al., Arch. Toxicol. 90(17):2657 -2667 (2016)), using Micronucleus Assay MicroFlow® kit (Litron Laboratories).

Genomewide copy number and loss of heterozygosity analyses

|00162] Genomic instability was monitored through microarray screens by evaluating changes in loss of heterozygosity (LOH) and copy number, using 10K and SNP 6.0 arrays (Affymetrix®). Genomic DNA was isolated from myeloma cells in which activity of APEX1 and/or APEX2 was either transgenically or chemically modulated, and analyzed using SNP arrays according to the manufacturer’s directions. Genome of “Day 0” cells (those harvested at the beginning of experiment) was used as a baseline to detect changes in the treated/modulated cells as described previously (Shammas et al., Blood 773/70/2290-2297 (2009)).

Cell Viability Assays

Cells were treated as indicated for 72 h and viability assessed using CellTiter-Glo® Luminescent Cell Viability Assay (Promega TM ) or Cell Counting Kit-8 (CCK-8) assay (Sigma Aldrich®) according to the manufacturer’s protocol. Significance of Results

[00163] The majority of cancers display a complex spectrum of diverse genetic alterations at diagnosis, and acquire additional changes with progression of disease. Genomic instability, which seems to arise early at the premalignant stage, enables cells to acquire new characteristics for progression and resist therapy. There are a number of mutations that correlate with overall and event free survival in MM, suggesting a role of genomic instability in poor clinical outcome. The data presented herein in MM and esophageal cancers show that elevated homologous recombination (HR) is a key mediator of genomic instability and drug resistance in cancer, and contributes to telomere maintenance and tumor growth. Elevated expression and/or activity of apurinic/apyrimidinic nucleases, APEX1 and/or APEX2, contribute to increased HR activity, DNA breaks (both endogenous as well as DNA damaging agent-induced) and cell cycle progression, and thus drive genomic evolution in MM cells. Inhibition of APEX expression, through transgenic as well as chemical manipulations, reduces DNA breaks and genomic instability, and induces G2/M arrest in MM cells. Consistent with these data, transgenic upregulation of APEX1 and APEX2 in normal human cells, induces DNA breaks and genomic instability, as evident from new copy number and chromosomal changes including aneuploidy, leading to oncogenic transformation enabling these cells to grow as tumors in mice. The overexpression of APEX1 or APEX2 in transgenic animal models led to formation of tumors in these animals, and tumorigenesis was associated with increased DNA breaks and genomic instability, as assessed by Comet assay and whole genome sequencing. These data demonstrate that APEX overexpression can drive genomic evolution leading to tumorigenesis, and its inhibitors have potential to not only inhibit growth but also inhibit or reduce genomic damage and evolution, both endogenous or that induced by chemotherapy, in MM cells (FIG. 2-FIG. 15). APEX nucleases (APEX1 and APEX2) are critical targets and their inhibitors, compounds 1-9, have ability to: (1) inhibit or reduce genomic evolution in multiple myeloma and esophageal cancer, preventing or delaying the progression of cancer to advanced stages including development of drug resistance, (2) prevent or reduce the DNA damage and genomic instability caused by chemotherapeutic agents, and (3) increase cytotoxicity (or efficacy) of existing cancer drugs.

[00164] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

|00165] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.