RENNIE PAUL (CA)
BAN FUQIANG (CA)
LEBLANC ERIC (CA)
CARABET LAVINIA (CA)
LALLOUS NADA (CA)
SINGH KRITI (CA)
MORIN HELENE (CA)
TON ANH-TIEN (CA)
| WO2020047668A1 | 2020-03-12 |
DATABASE REGISTRY 28 July 2019 (2019-07-28), ANONYMOUS : "Benzoic acid, 3,4,5-trifluoro-, 2-[4-cyano-2- (trifluoromethyl)phenyl]hydrazide (CA INDEX NAME)", XP093090120, retrieved from STN Database accession no. 2361823-16-5
DATABASE REGISTRY 28 July 2019 (2019-07-28), ANONYMOUS : "Benzoic acid, 3-chloro-2,4-difluoro-, 2-[4-cyano-2-(trifluoromethyl)phenyl]hydrazide (CA INDEX NAME)", XP093090121, retrieved from STN Database accession no. 2361823-00-7
DATABASE REGISTRY 28 July 2019 (2019-07-28), ANONYMOUS : "Benzoic acid, 3-fluoro-4-(trifluoromethyl)-, 2-[4-cyano-2-(trifluoromethyl)phenyl]hydrazide (CA INDEX NAME)", XP093090124, retrieved from STN Database accession no. 2361755-48-6
DATABASE REGISTRY 28 July 2019 (2019-07-28), ANONYMOUS : "Benzoic acid, 2,4,5-trifluoro-, 2-[4-cyano-2- (trifluoromethyl)phenyl]hydrazide (CA INDEX NAME)", XP093090128, retrieved from STN Database accession no. 2361718-33-2
DATABASE REGISTRY 28 July 2019 (2019-07-28), ANONYMOUS : "Benzoic acid, 4-fluoro-3-(trifluoromethyl)-, 2-[4-cyano-2-(trifluoromethyl)phenyl]hydrazide (CA INDEX NAME)", XP093090132, retrieved from STN Database accession no. 2361699-20-7
| What is Claimed is: 1. A compound, the compound having the structure of Formula I: alternatively, R1 and M2 form a 5 or 6 membered ring having a structure selected from one of provided that at least one of Q1 and Q2 is H or OH, or provided that when Q1 and Q2 are both F then one of M1 and M2 is F, or R1 is CF3, OCF3, SCF3, provided that when one of Q1 and Q2 is F, then R1 is selected from I, Br, Cl, F, CF3, C(CH3)3, CHF2, NHC(=O)CH3, C(=O)OC(CH3)3, CH=CH2, N(CH2CH3)CH(CH3)2, C(CH3)2CF3, CH2CH2CF3, OCH2CF3, N(CH3)2, OCH2CH=CH2, CH2CH2CH3, CH2SCH3, OCF3, SCF3, SF5, provided that when one of Q1 and Q2 is Cl, Br or I, then R1 is selected from I, Br, Cl, F, CF3, C(CH3)3, CHF2, NHC(=O)CH3, C(=O)OC(CH3)3, CH=CH2, N(CH2CH3)CH(CH3)2, C(CH3)2CF3, CH2CH2CF3, OCH2CF3, N(CH3)2, OCH2CH=CH2, CH2CH2CH3, CH2SCH3, OCF3, SCF3, SF5, F provided that at least one of M1, M2 and R1 is I, Br, Cl, F, CF3, C(CH3)3, CHF2, OCF3, SCF3, SF5, provided the compound is not wherein the compound is for use in the treatment of cancer. 2. The compound of claim 1, wherein the compound is for use in the treatment of one or more of the following: one or more of the following: prostate cancer; lymphoma; neuroblastoma; breast cancer; colon cancer; cervical cancer; small-cell lung carcinoma; osteosarcoma; glioblastoma; melanoma; and myeloid leukaemia. 3. The compound of claim 1 or 2, wherein E1 is selected from H, CH3, F, Cl, Br and I; Q1 is selected from H, OH, I, Br, Cl, F and CF3; Q2 is selected from H, OH, I, Br, Cl, F and CF3; M1 is selected from H, I, Br, Cl, F, CF3, SCH3 M2 is selected from H, I, Br, Cl, F, CF3, SCH3, R1 is selected from H, I, Br, Cl, F, CF3, C(CH3)3, CHF2, C(=O)OC(CH3)3, CH=CH2, N(CH2CH3)CH(CH3)2, C(CH3)2CF3, CH2CH2CF3, OCH2CF3, N(CH3)2, OCH2CH=CH2, CH2CH2CH3, CH2SCH3, OCH3, SCH3, OCH(CH3)2, OC(CH3)3, CF2CH3, C(=O)OCH3, C(=O)OCH2CH3, OCF3, SCF3, alternatively, R1 is , provided that M1 and M2 are selected from H, I, Br, Cl, F, SCH3, 2 are selected from H and F; = N alternatively, R1 is CH3, provided that M1 or M2 is F, Cl, Br or ' ; alternatively R1 is NHC(=O)CH3, provided that M1 or M2 are H; alternatively, one of M1 or M2 is OH, provided that Q1 and Q2 are H or provided that R1 is selected from I, Br, Cl, F, CF3, C(CH3)3, CHF2, NHC(=O)CH3, C(=O)OC(CH3)3, CH=CH2, N(CH2CH3)CH(CH3)2, C(CH3)2CF3, CH2CH2CF3, OCH2CF3, N(CH3)2, OCH2CH=CH2, CH2CH2CH3, CH2SCH3, OCF3, SCF3, and SF5; alternatively, one of Q1 and Q2 is CH3, provided that R1 is selected from I, Br, Cl, F, CF3, C(CH3)3, CHF2, NHC(=O)CH3, C(=O)OC(CH3)3, CH=CH2, N(CH2CH3)CH(CH3)2, C(CH3)2CF3, provided that at least one of M1, M2 and R1 is I, Br, Cl, F, CF3, C(CH3)3, CHF2, OCF3, SCF3, SF5, 4. The compound of claim 1, 2 or 3, wherein E1 is selected from H, CH3, F, Cl, Br and I; Q1 is selected from H, OH, Br, Cl and F; Q2 is selected from H, OH, Br, Cl and F; o- M1 is selected from H, Br, Cl, F, CF3 and 0 ; O- M2 is selected from H, Br, Cl, F, CF3 and 0 ; R1 is selected from H, I, Br, Cl, F, CF3, C(CH3)3, CHF2, C(=O)OC(CH3)3, CH=CH2, N(CH2CH3)CH(CH3)2, C(CH3)2CF3, CH2CH2CF3, OCH2CF3, N(CH3)2, OCH2CH=CH2, CH2CH2CH3, CH2SCH3, OCH3, SCH3, OCH(CH3)2, OC(CH3)3, CF2CH3, C(=O)OCH3, C(=O)OCH2CH3, OCF3, SCF3, alternatively, R1 is , provided that M1 and M2 are selected from H, I, Br, Cl, F, SCH3, and Q1 and Q2 are selected from H and F; alternatively, R1 is CH3, provided that M1 or M2 is F, Cl, Br or ; alternatively R1 is NHC(=O)CH3, provided that M1 or M2 are H; provided that when one of Q1 and Q2 is F, then R1 is selected from I, Br, Cl, F, CF3, C(CH3)3, CHF2, NHC(=O)CH3, C(=O)OC(CH3)3, CH=CH2, N(CH2CH3)CH(CH3)2, C(CH3)2CF3, CH2CH2CF3, OCH2CF3, N(CH3)2, OCH2CH=CH2, CH2CH2CH3, CH2SCH3, OCF3, SCF3, SF5, provided that when one of Q1 and Q2 is Cl, Br or I, then R1 is selected from I, Br, Cl, F, CF3, C(CH3)3, CHF2, NHC(=O)CH3, C(=O)OC(CH3)3, CH=CH2, N(CH2CH3)CH(CH3)2, C(CH3)2CF3, or then one of M1 and M2 is F, Cl, Br or I. 5. The compound of any one of claims 1-4, wherein E1 is selected from H, CH3, F, Cl and Br; Q1 is selected from H, OH, Br, Cl and F; Q2 is selected from H, OH, Br, Cl and F; o- M1 is selected from H, Br, Cl, F, CF3 and 0 ; o- M2 is selected from H, Br, Cl, F, CF3 and 0 ; R1 is selected from H, I, Br, Cl, F, CF3, C(CH3)3, CHF2, C(=O)OC(CH3)3, CH=CH2, N(CH2CH3)CH(CH3)2, C(CH3)2CF3, CH2CH2CF3, OCH2CF3, N(CH3)2, OCH2CH=CH2, CH2CH2CH3, CH2SCH3, OCH3, SCH3, OCH(CH3)2, OC(CH3)3, CF2CH3, C(=O)OCH3, C(=O)OCH2CH3, OCF3, SCF3, alternatively, R1 is provided that M1 and M2 are selected from H, I, Br, Cl or F, and Q1 and Q2 are selected from H and F; and alternatively R1 is NHC(=O)CH3, provided that M1 or M2 are H. 6. The compound of any one of claims 1-5, wherein E1 is selected from H, CH3, F, Cl and Br; Q1 is selected from H, OH, Br, Cl and F; Q2 is selected from H, OH, Br, Cl and F; M1 is selected from H, Br, Cl, F, CF3 and M2 is selected from H, Br, Cl, F, CF3 and ; and R1 is selected from H, Br, Cl, F, CF3, C(CH3)3, CHF2, C(=O)OC(CH3)3, CH=CH2, N(CH2CH3)CH(CH3)2, C(CH3)2CF3, CH2CH2CF3, OCH2CF3, N(CH3)2, OCH2CH=CH2, CH2CH2CH3, CH2SCH3, OCH3, SCH3, OCH(CH3)2, OC(CH3)3, CF2CH3, C(=O)OCH3, C(=O)OCH2CH3, OCF3, SCF3, 7. The compound of any one of claims 1-6, wherein E1 is selected from H, CH3, F and Cl and Br; Q1 is selected from H, OH, Br, Cl and F; Q2 is selected from H, OH, Br, Cl and F; M1 is selected from H, Br, Cl, F, CF3 and ; M2 is selected from H, Br, Cl, F, CF3 and ; and 8. The compound of claim 1, wherein the compound is selected from TABLE 1, provided that it has at least 60% inhibition or is selected from TABLE 2. 10. The compound of claim 1, wherein the compound is selected from one or more of: 11. A compound of any one of claims 1-10, wherein the compound is for use in the treatment of one or more of the following: prostate cancer; lymphoma; neuroblastoma; breast cancer; colon cancer; cervical cancer; small-cell lung carcinoma; osteosarcoma; and myeloid leukaemia. 12. A compound of any one of claims 1-11, wherein the compound is for use in the treatment of one or more of the following: one or more of the following: prostate cancer; lymphoma; and neuroblastoma. 13. A pharmaceutical composition for treating cancer, comprising compound of any one of claims 1 and 3-10 and a pharmaceutically acceptable carrier. 14. The pharmaceutical composition of claim 13, wherein the cancer is selected from one or more of the following: prostate cancer; lymphoma; neuroblastoma; breast cancer; colon cancer; cervical cancer; small-cell lung carcinoma; osteosarcoma; glioblastoma; melanoma; and myeloid leukaemia. 15. Use of compound of any one of claims 1 and 3-10 for treating cancer. 16. Use of compound of any one of claims 1 and 3-10 in the manufacture of a medicament for treating cancer. 17. The use of claim 15 or 16, wherein the cancer is selected from one or more of the following: prostate cancer; lymphoma; neuroblastoma; breast cancer; colon cancer; cervical cancer; small-cell lung carcinoma; osteosarcoma; glioblastoma; melanoma; and myeloid leukaemia. 18. A commercial package comprising (a) compound of any one of claims 1-10 and a pharmaceutically acceptable carrier; and (b) instructions for the use thereof for treating cancer. 19. A commercial package comprising (a) a pharmaceutical composition comprising compound of any one of claims 1-10 and a pharmaceutically acceptable carrier; and (b) instructions for the use thereof for treating cancer. 20. The commercial package of claim 18 or 19, wherein the cancer is selected from one or more of the following: prostate cancer; lymphoma; neuroblastoma; breast cancer; colon cancer; cervical cancer; small-cell lung carcinoma; osteosarcoma; glioblastoma; melanoma; and myeloid leukaemia. 21. A compound of Formula I, provided that the compound excludes all of the compounds set out in TABLE 1. 22. A compound, the compound having the structure of Formula II: wherein, E2 is selected from H, CH3, F, Cl, Br and I; Q3 is selected from H, OH, I, Br, Cl, F and CF3; Q4 is selected from H, OH, I, Br, Cl, F and CF3; M3 is selected from H, I, Br, Cl, F, CF3, SCH3, M4 is selected from H, I, Br, Cl, F, CF3, SCH3, alternatively, R2 is selected from H, F, Cl, Br, I, CF3, SF5, , , CH(CH3)2, C(CH3)3, CHF2, C(=O)OC(CH3)3, CH=CH2, N(CH2CH3)CH(CH3)2, C(CH3)2CF3, CH2CH2CF3, OCH2CF3, N(CH3)2, OCH2CH=CH2, CH2CH2CH3, CH2SCH3, OCH3, SCH3, OCH(CH3)2, OC(CH3)3, CF2CH3, C(=O)OCH3, C(=O)OCH2CH3, OCF3, SCF3, F , F that E2 is selected from CH3, F, Cl, Br and I; alternatively, R2 is selected from CF3, F, Br, and I, provided that M3, M4 or both M3 and M4 are alternatively, R2 is 0 , provided that at least one of M3 or M4 is F, Cl, or Br; and alternatively, R2 is ' , provided that M3 and M4 are both F, Cl, or Br, or one of M3 or M4 is F 23. The compound of claim 22, wherein, E2 is selected from H, CH3, F, Cl and Br; Q3 is selected from H, OH, Br, Cl, F and CF3; Q4 is selected from H, OH, Br, Cl, F and CF3; M3 is selected from H, Br, Cl, F, CF3, SCH3, and M4 is selected from H, I, Br, Cl, F, CF3, SCH3, and R2 is selected from alternatively, R2 is selected from H, F, Cl, Br, I, CF3, SF5, , CH(CH3)2, C(CH3)3, CHF2, C(=O)OC(CH3)3, CH=CH2, N(CH2CH3)CH(CH3)2, C(CH3)2CF3, CH2CH2CF3, OCH2CF3, N(CH3)2, OCH2CH=CH2, CH2CH2CH3, CH2SCH3, OCH3, SCH3, OCH(CH3)2, OC(CH3)3, CF2CH3, C(=O)OCH3, ; and alternatively, R2 is selected from CF3, F, Br, and I, provided that M3, M4 or both M3 and M4 are CF3. 24. The compound of claim 22 or 23, wherein, E2 is selected from H, CH3, F, and Cl; Q3 is H; Q4 is H; M3 is selected from H, Br, Cl, F, and CF3; M4 is selected from H, Br, Cl, F, and CF3; alternatively, R2 is selected from CF3, F, Br, and I, provided that M3, M4 or both M3 and M4 are CF3. 25. The compound of claim 22, 23 or 24, wherein the compound is selected from the following: |
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/313,915 filed 25 February 2022 entitled “MYC-MAX INHIBITOR COMPOUNDS FOR CANCER TREATMENT, METHODS AND USES ASSOCIATED THEREWITH”.
TECHNICAL FIELD
[0001] The present invention relates to the field of Myc-Max inhibitors. In particular, the invention relates to Myc-Max inhibitor compounds for use in the treatment of cancer.
BACKGROUND
[0002] Myc is a transcription factor that regulates growth in normal cells, but in many cancers overactivity of Myc results in high rates of growth needed for tumor proliferation and progression [1, 2]. Myc drives tumorigenesis by transcriptional programming of a large number of target genes that promote cell growth, proliferation, metabolism and apoptosis, and block differentiation [3-7]. Myc is estimated to contribute to most if not all human cancers, including prostate, breast, colon, cervical cancers, small-cell lung carcinomas, neuroblastoma, osteosarcomas, glioblastomas, melanoma, and myeloid leukaemia, most of which are aggressive and respond poorly to the current therapies [1, 8, 9].
[0003] In prostate cancer (PCa), which is the second leading cause of cancer-related death in men, the Myc family members - L-Myc, c-Myc and N-Myc - are implicated in pathogenesis and progression across the full spectrum of PCa, from localized adenocarcinoma to the most advanced and treatmentresistant subtypes - castration-resistant (CRPC) and its neuroendocrine phenotype (NEPC). Amplifications of Myc family members are the most frequently observed genomic alterations associated with specific clinical stages and subtypes of PCa [10-16]. L-Myc is amplified in -27% of localized PCa, in a mutually exclusive manner to c-Myc [11], whereas c-Myc is commonly amplified in all PCa stages and subtypes [17]. Notably, c-Myc overexpression antagonizes the transcriptional activity of the androgen receptor (AR), which is a driving force in PCa and constitutes the main drug target for advanced cases of disease [18]. Besides influencing clinically relevant AR target genes, c- Myc upregulation also affects critical splicing programs [19] and increases levels of AR-V7 - the constitutively active ligand-independent AR splice variant that promotes CRPC [20, 21] and is also observed in NEPC [14]. Importantly, N-Myc amplifications induce the NEPC phenotype [14, 15, 22]. [0004] To elicit its oncogenic effects, Myc must form a heterodimer with its obligate partner Max, which together bind to the DNA and activate transcription of the target genes [23-26]. Although Myc could qualify as an ideal cancer target, applying conventional structure-based drug design approaches is inherently challenging in drugging Myc. Myc and Max are intrinsically disordered proteins (IDP), which exist as dynamic ensembles, with no effective pockets on their surfaces [27-29]. The disordered basic-helix-loop-helix-leucine zipper (bHLHLZ) domain of the Myc monomer forms DNA-binding functionalities only via association with the homologous bHLHLZ domain of Max [23, 30]. Only upon such heterodimerization does the resulting Myc- Max complex adopt a stable helical configuration which can bind specific DNA recognition sequences 5’-CACGTG-3’, termed E-boxes, at enhancers and promoters of target genes, and thereby trigger the recruitment of chromatin-remodeling complexes and assembly of the transcriptional machinery to drive the transcriptional program [31, 32]. Myc and Max oligomerize through their helix-loop-helix (HLH) and leucine zipper (LZ) regions and bind DNA mainly through highly positively charged basic (b) region and specific residues located in the HLH region [33, 34].
[0005] Although Myc inactivation may have undesired effects on normal cells, experimental mouse models of KRAS-driven lung cancer carrying a conditionally inducible Omomyc construct - a Myc dominant negative, 93 residue bHLHZ protein fragment with 4 single-point mutations in the LZ region - established that periodic inhibition is effective at stopping cancer growth with mild and tolerable side effects, suggesting a viable therapeutic strategy [35, 36].
[0006] Small molecule inhibition of Myc, a therapeutically compelling oncogenic transcription factor, has been a challenge for a long time. Current strategies that directly target Myc in cancer include inhibitors of Myc-Max protein-protein interactions, such as 10058-F4, 10074-G5, and JY-3-094 [37, 38], or protein-DNA interactions, such as Mycro3 [39] and KJ-Pyr-9 [40], and inhibitors of Myc expression with G-quadruplex stabilizers, antisense oligonucleotides, and siRNA [41, 42]. Indirect approaches have been reviewed elsewhere [41, 43, 44].
[0007] Compounds 10058-F4, 10074-A4, and 10074-G5 are among the first identified direct small molecules Myc inhibitors that bind with mid-micromolar range affinity at 3 independent sites on the disordered bHLHLZ domain of the Myc (c- and N-Myc) monomer (as validated by mutagenesis and NMR experiments) [37, 45, 46]. The efforts to identify them relied on functional screening of finite libraries unlikely to contain clinically-optimized structures. Attempts to find more potent and selective analogs have yet to succeed given the inconsistent behaviour of compounds in in vitro assays [47, 48]. Moreover, these compounds lack proper antitumor activity in vivo due to rapid metabolism to inactive metabolites, resulting in low tumoral concentrations insufficient to inhibit Myc-Max dimerization [49, 50]. Thus, further more effective small molecule inhibitors of Myc-Max are needed. SUMMARY
[0008] The present invention is based in part, on the surprising discovery that the compounds described herein modulate Myc-Max activity. Specifically, some compounds identified herein, also show inhibition of Myc-Max in prostate cancer cells.
[0009] In accordance with one embodiment, there is provided a compound, the compound having the structure of Formula I , wherein, E 1 may be selected from H, CH 3 , F, Cl,
Br and I; Q 1 may be selected from H, OH, I, Br, Cl, F and CF 3 ; Q 2 may be selected from H, OH, I, Br, Cl, ; R 1 may be selected from H, I, Br, Cl, F, CF 3 , C(CH 3 ) 3 , CHF 2 , C(=O)OC(CH 3 ) 3 ,
CH=CH 2 , N(CH 2 CH 3 )CH(CH 3 ) 2 , C(CH 3 ) 2 CF 3 , CH 2 CH 2 CF 3 , OCH 2 CF 3 , N(CH 3 ) 2 , 0CH 2 CH=CH 2 ,
CH 2 CH 2 CH 3 , CH 2 SCH 3 , OCH 3 , SCH 3 , OCH(CH 3 ) 2 , OC(CH 3 ) 3 , CF 2 CH 3 , C(=O)OCH 3 , C(=O)OCH 2 CH 3 , , provided that M 1 and M 2 are selected from H, I, Br, Cl, F,
SCH 3 , are selected from H and F; alternatively, R 1 may be CH 3 , provided that M 1 or M 2 may be F, Cl, Br or ; alternatively R 1 may be NHC(=O)CH 3 , provided that M 1 or M 2 are H; alternatively, one of M 1 or M 2 is OH, provided that Q 1 and Q 2 are H or provided that R 1 may be selected from I, Br, Cl, F, CF 3 , C(CH 3 ) 3 , CHF 2 , NHC(=O)CH 3 , [0011] The compound may be for use in the treatment of one or more of the following: one or more of the following: prostate cancer; lymphoma; neuroblastoma; breast cancer; colon cancer; cervical cancer; small-cell lung carcinoma; osteosarcoma; glioblastoma; melanoma; and myeloid leukaemia.
[0012] E 1 may be selected from H, CH 3 , F, Cl, Br and I. Q 1 may be selected from H, OH, I, Br, Cl, F and CF 3 . Q 2 may be selected from H, OH, I, Br, Cl, F and CF 3 . M 1 may be selected from H, I, Br, Cl, F, CF 3 , or may be selected from H and F; alternatively, R 1 may be CH 3 , provided that M 1 or M 2 may be F, Cl, Br or ; alternatively R 1 may be NHC(=O)CH 3 , provided that M 1 or M 2 may be
H. Alternatively, one of M 1 or M 2 may be OH, provided that Q 1 and Q 2 are H or provided that R 1 may be selected from I, Br, Cl, F, CF 3 , C(CH 3 ) 3 , CHF 2 , NHC(=O)CH 3 , C(=O)OC(CH 3 ) 3 , CH=CH 2 ,
N(CH 2 CH 3 )CH(CH 3 ) 2 , C(CH 3 ) 2 CF 3 , CH 2 CH 2 CF 3 , OCH 2 CF 3 , N(CH 3 ) 2 , OCH 2 CH=CH 2 , CH 2 CH 2 CH 3 , CH 2 SCH 3 , OCF 3 , SCF 3 , and SF 5 . Alternatively, one of Q 1 and Q 2 may be CH 3 , provided that R 1 may be selected from I, Br, Cl, F, CF 3 , C(CH 3 ) 3 , CHF 2 , NHC(=O)CH 3 , C(=O)OC(CH 3 ) 3 , CH=CH 2 ,
N(CH 2 CH 3 )CH(CH 3 ) 2 , C(CH 3 ) 2 CF 3 , CH 2 CH 2 CF 3 , OCH 2 CF 3 , N(CH 3 ) 2 , OCH 2 CH=CH 2 , CH 2 CH 2 CH 3 ,
CH 2 SCH 3 , OCF 3 , SCF 3 , SF 5 , Alternatively, one or both of M 1 or M 2 may be CH 3 or OCH 3 , provided that R 1 may be selected from I, Br, Cl, F, CF 3 , C(CH 3 ) 3 , CHF 2 , NHC(=O)CH 3 , C(=O)OC(CH 3 ) 3 , CH=CH 2 , N(CH 2 CH 3 )CH(CH 3 ) 2 , C(CH 3 ) 2 CF 3 , CH 2 CH 2 CF 3 ,
OCH 2 CF 3 , N(CH 3 ) 2 , OCH 2 CH=CH 2 , CH 2 CH 2 CH 3 , CH 2 SCH 3 , OCF 3 , SCF 3 , SF 5 , , or provided that wherein only one of M 1 or M 2 may be CH 3 or OCH 3 and the other maybe selected from I, Br, Cl, F, CF 3 , OCF 3 , SCF 3 , . Provided that at least one of Q 1 and Q 2 may be H or OH, or provided that when Q 1 and Q 2 are both F then one of M 1 and
M 2 may be F, or R 1 may be CF 3 , OCF 3 , SCF 3 , Provided that when one of Q 1 and Q 2 maybe F, then R 1 maybe selected from I, Br, Cl, F, CF 3 , C(CH 3 ) 3 , CHF 2 , NHC(=O)CH 3 , C(=O)OC(CH 3 ) 3 , CH=CH 2 , N(CH 2 CH 3 )CH(CH 3 ) 2 , C(CH 3 ) 2 CF 3 , CH 2 CH 2 CF 3 , OCH 2 CF 3 , N(CH 3 ) 2 ,
OCH 2 CH=CH 2 , CH 2 CH 2 CH 3 , CH 2 SCH 3 , OCF 3 , SCF 3 , SF 5 , , or then one of M 1 and M 2 may be F. Provided that when one of Q 1 and Q 2 may be Cl, Br or I, then R 1 may be selected from I, Br, Cl, F, CF 3 , C(CH 3 ) 3 ,
CHF 2 , NHC(=O)CH 3 , C(=O)OC(CH 3 ) 3 , CH=CH 2 , N(CH 2 CH 3 )CH(CH 3 ) 2 , C(CH 3 ) 2 CF 3 , CH 2 CH 2 CF 3 ,
OCH 2 CF 3 , N(CH 3 ) 2 , OCH 2 CH=CH 2 , CH 2 CH 2 CH 3 , CH 2 SCH 3 , OCF 3 , SCF 3 , SF 5 , , or then one of M 1 and M 2 may be F, Cl, Br or I. Provided that at least one of M 1 , M 2 and R 1 may be I, Br, Cl, F, CF 3 , C(CH 3 ) 3 , CHF 2 ,
OCF 3 , SCF 3 , SF 5
[0013] E 1 may be selected from H, CH 3 , F, Cl, Br and I. E 1 may be selected from CH 3 , F, Cl, Br and I.
E 1 may be selected from H, CH 3 , F, Cl and Br. E 1 may be selected from H, CH 3 , F and Cl. E 1 may be selected from CH 3 , F and Cl. Q 1 may be selected from H, OH, Br, Cl and F. Q 1 may be selected from H, OH, Cl and F. Q 1 may be selected from OH, Br, Cl and F. Q 1 may be selected from H, Br, Cl and F. Q 1 may be selected from H, Cl and F. Q 2 may be selected from H, OH, Br, Cl and F. Q 2 may be selected from H, OH, Cl and F. Q 2 may be selected from OH, Br, Cl and F. Q 2 may be selected from H, Br, Cl and F. Q 2 may be selected from H, Cl and F. M 1 may be selected from H, Br, Cl, F, CF 3 and may be selected from H, Br, Cl, F and M 2 may be selected from H, Br, Cl, F, CF 3 and o- may be selected from H, Br, Cl, F and ° .
[0014] R 1 maybe selected from H, I, Br, Cl, F, CF 3 , C(CH 3 ) 3 , CHF 2 , C(=O)OC(CH 3 ) 3 , CH=CH 2 , N(CH 2 CH 3 )CH(CH 3 ) 2 , C(CH 3 ) 2 CF 3 , CH 2 CH 2 CF 3 , OCH 2 CF 3 , N(CH 3 ) 2 , OCH 2 CH=CH 2 , CH 2 CH 2 CH 3 , CH 2 SCH 3 , OCH 3 , SCH 3 , OCH(CH 3 ) 2 , OC(CH 3 ) 3 , CF 2 CH 3 , C(=O)OCH 3 , C(=O)OCH 2 CH 3 , OCF 3 , SCF 3 ,
[0015] Alternatively, R 1 may be , provided that M 1 and M 2 may be selected from H, I, Br, Cl, F,
SCH 3 , and Q 1 and Q 2 may be selected from H and F. Alternatively, R 1 may be CH 3 , provided that M 1 or M 2 may be F, Cl, Br or . Alternatively, R 1 may be NHC(=O)CH 3 , provided that M 1 or M 2 are H. Provided that when one of Q 1 and Q 2 may be F, then R 1 may be selected from I, Br, Cl, F, CF 3 , C(CH 3 ) 3 , CHF 2 , NHC(=O)CH 3 , C(=O)OC(CH 3 ) 3 , CH=CH 2 , N(CH 2 CH 3 )CH(CH 3 ) 2 , C(CH 3 ) 2 CF 3 , CH 2 CH 2 CF 3 , OCH 2 CF 3 , N(CH 3 ) 2 , OCH 2 CH=CH 2 , CH 2 CH 2 CH 3 , CH 2 SCH 3 , OCF 3 , SCF 3 , SF 5 , then one of M 1 and M 2 may be F. Provided that when one of Q 1 and Q 2 may be Cl, Br or I, then R 1 may be selected from I, Br, Cl, F, CF 3 , C(CH 3 ) 3 , CHF 2 , NHC(=O)CH 3 , C(=O)OC(CH 3 ) 3 , CH=CH 2 , N(CH 2 CH 3 )CH(CH 3 ) 2 , C(CH 3 ) 2 CF 3 , CH 2 CH 2 CF 3 , OCH 2 CF 3 , N(CH 3 ) 2 , OCH 2 CH=CH 2 , CH 2 CH 2 CH 3 ,
[0016] E 1 may be selected from H, CH 3 , F, Cl and Br. Q 1 may be selected from H, OH, Br, Cl and F. Q 2 may be selected from H, OH, Br, Cl and F. M 1 may be selected from H, Br, Cl, F, CF 3 and may be selected from H, Br, Cl, F, CF 3 and . R 1 may be selected from H, I, Br, Cl, F, CF 3 , C(CH 3 ) 3 , CHF 2 , C(=O)OC(CH 3 ) 3 , CH=CH 2 , N(CH 2 CH 3 )CH(CH 3 ) 2 , C(CH 3 ) 2 CF 3 , CH 2 CH 2 CF 3 , OCH 2 CF 3 , N(CH 3 ) 2 , OCH 2 CH=CH 2 , CH 2 CH 2 CH 3 , CH 2 SCH 3 , OCH 3 , SCH 3 , OCH(CH 3 ) 2 , OC(CH 3 ) 3 , CF 2 CH 3 , C(=O)OCH 3 ,
[0017] Alternatively, R 1 may be , provided that M 1 and M 2 are selected from H, I, Br, Cl or F, and Q 1 and Q 2 are selected from H and F; and alternatively R 1 may be NHC(=O)CH 3 , provided that M 1 or M 2 are H.
[0018] E 1 may be selected from H, CH 3 , F, Cl and Br. Q 1 may be selected from H, OH, Br, Cl and F. Q 2 may be selected from H, OH, Br, Cl and F. M 1 may be selected from H, Br, Cl, F, CF 3 and may be selected from H, Br, Cl, F, CF 3 and . R 1 may be selected from H, Br, Cl, F, CF 3 , C(CH 3 ) 3 ,
CHF 2 , C(=O)OC(CH 3 ) 3 , CH=CH 2 , N(CH 2 CH 3 )CH(CH 3 ) 2 , C(CH 3 ) 2 CF 3 , CH 2 CH 2 CF 3 , OCH 2 CF 3 , N(CH 3 ) 2 , 0CH 2 CH=CH 2 , CH 2 CH 2 CH 3 , CH 2 SCH 3 , OCH 3 , SCH 3 , OCH(CH 3 ) 2 , OC(CH 3 ) 3 , CF 2 CH 3 , C(=O)OCH 3 ,
C(=O)OCH 2 CH 3 , OCF 3 , SCF 3 , SF 5 ,
[0019] E 1 may be selected from H, CH 3 , F and Cl and Br. Q 1 may be selected from H, OH, Br, Cl and F.
Q 2 may be selected from H, OH, Br, Cl and F. M 1 may be selected from H, Br, Cl, F, CF 3 an may be selected from H, Br, Cl, F, CF 3 and R 1 may be selected from H, Br, Cl, F, CF 3 , C(CH 3 ) 3 ,
[0020] The compound may be selected from TABLE 1, provided that it has at least 6o% inhibition or selected from TABLE 2.
[0022] The compound may be selected from one or more of:
[0024] The compound may be for use in the treatment of one or more of the following: prostate cancer; lymphoma; neuroblastoma; breast cancer; colon cancer; cervical cancer; small-cell lung carcinoma; osteosarcoma; and myeloid leukaemia. The compound may be for use in the treatment of one or more of the following: one or more of the following: prostate cancer; lymphoma; and neuroblastoma.
[0025] In accordance with another embodiment, there is provided a pharmaceutical composition for treating cancer, comprising compound as described herein and a pharmaceutically acceptable carrier.
[0026] In accordance with another embodiment, there is provided a compound as described herein for use in the treatment of cancer.
[0027] In accordance with another embodiment, there is provided a pharmaceutical composition for treating cancer, comprising compound described herein and a pharmaceutically acceptable carrier.
[0028] The cancer maybe selected from one or more of the following: prostate cancer; lymphoma; neuroblastoma; breast cancer; colon cancer; cervical cancer; small-cell lung carcinoma; osteosarcoma; glioblastoma; melanoma; and myeloid leukaemia.
[0029] In accordance with another embodiment, there is provided a use of compound described herein for treating cancer. [0030] In accordance with another embodiment, there is provided a use of compound described herein in the manufacture of a medicament for treating cancer.
[0031] In accordance with another embodiment, there is provided a commercial package including (a) compound described herein and a pharmaceutically acceptable carrier; and (b) instructions for the use thereof for treating cancer.
[0032] In accordance with another embodiment, there is provided a commercial package including (a) a pharmaceutical composition comprising compound described herein and a pharmaceutically acceptable carrier; and (b) instructions for the use thereof for treating cancer.
[0033] The compound may exclude all of the compounds set out in TABLE 1.
[0034] In accordance with another embodiment, there is provided a compound, the compound having the structure of Formula I wherein, E 2 may be selected from H, CH 3 ,
F, Cl, Br and I; Q 3 may be selected from H, OH, I, Br, Cl, F and CF 3 ; Q 4 may be selected from H, OH, I, be selected from CH 3 , F, Cl, Br and I; and alternatively, R 2 may be selected from F, Br, and I, provided that M3, M4 or both M3 and M 4 are CF 3 . provided that M 3 , M 4 or both M 3 and M 4 are CF 3 . Alternatively, R 2 may , provided that at least one of M 3 or M 4 is F, Cl, or Br. Alternatively, R 2 may be , provided that M 3 and M 4 are both
F, Cl, or Br, or one of M 3 or M 4 is F or Br.
[0036] E 2 may be selected from H, CH 3 , F, Cl and Br. E 2 may be selected from CH 3 , F, Cl and Br. E 2 may be selected from H, CH 3 , F and Cl. E 2 may be selected from CH 3 , F and Cl. Q3 may be selected from H, OH, Br, Cl, F and CF 3 . Q3 may be selected from H, OH, Br, Cl and F. Q3 may be selected from H, Br, Cl, F and CF 3 . Q3 may be selected from OH, Br, Cl, F and CF 3 . Q3 may be selected from H, OH, Cl, F and CF 3 . Q 4 maybe selected from H, OH, Br, Cl, F and CF 3 . Q 4 maybe selected from H, OH, Br, Cl and F. Q 4 may be selected from H, Br, Cl, F and CF 3 . Q 4 may be selected from OH, Br, Cl, F and CF 3 . Q 4 maybe selected from H, OH, Cl, F and CF 3 . M3 maybe selected from H, Br, Cl, F, CF 3 , SCH 3 , and M 3 may be selected from H, Br, Cl, F, CF 3 and M 3 may be selected from H, Br, Cl, F [0037] The compound may be selected from the compounds set out in TABLE 2. The compound may
DETAILED DESCRIPTION
[0038] The invention is not limited to the precise arrangements, examples, and instrumentalities shown.
[0039] Any terms not directly defined herein shall be understood to have the meanings commonly associated with them as understood within the art of the invention.
[0040] The Myc-Max complex is an attractive target for direct inhibition. In silica computational drug discovery methods were used to conduct a virtual screen of more than 6 million purchasable compounds from the ZINC database (53, 54, 70 and 89) to identify potential Myc-Max complex binders. The in silica methods included large-scale docking, in-site rescoring and consensus voting procedures.
[0041] It will be understood by a person of skill that COOH and NR 2 may include the corresponding ions, for example carboxylate ions and ammonium ions, respectively. Alternatively, where the ions are shown, a person of skill in the art will appreciate that the counter ion may also be present.
[0042] Those skilled in the art will appreciate that the point of covalent attachment of the moiety to the compounds as described herein may be, for example, and without limitation, cleaved under specified conditions. Specified conditions may include, for example, and without limitation, in vivo enzymatic or non-enzymatic means. Cleavage of the moiety may occur, for example, and without limitation, spontaneously, or it may be catalyzed, induced by another agent, or a change in a physical parameter or environmental parameter, for example, an enzyme, light, acid, temperature or pH. The moiety may be, for example, and without limitation, a protecting group that acts to mask a functional group, a group that acts as a substrate for one or more active or passive transport mechanisms, or a group that acts to impart or enhance a property of the compound, for example, solubility, bioavailability or localization.
[0043] In some embodiments, compounds of Formula I, as described herein, maybe used for systemic treatment of at least one indication selected from the group consisting of: prostate cancer, breast cancer, ovarian cancer, endometrial cancer, hair loss, acne, hirsutism, ovarian cysts, polycystic ovary disease, precocious puberty and age related macular degeneration. Alternatively, the compounds of Formula I maybe used for systemic treatment of at least one indication selected from the group consisting of: prostate cancer; lymphoma; neuroblastoma; breast cancer; colon cancer; cervical cancer; small-cell lung carcinoma; osteosarcoma; glioblastoma; melanoma; and myeloid leukaemia. In some embodiments compounds of Formula I may be used in the preparation of a medicament or a composition for systemic treatment of an indication described herein. In some embodiments, methods of systemically treating any of the indications described herein are also provided. [0044] Compounds as described herein may be in the free form or in the form of a salt thereof. In some embodiment, compounds as described herein may be in the form of a pharmaceutically acceptable salt, which are known in the art (90). Pharmaceutically acceptable salt as used herein includes, for example, salts that have the desired pharmacological activity of the parent compound (salts which retain the biological effectiveness and/or properties of the parent compound and which are not biologically and/or otherwise undesirable). Compounds as described herein having one or more functional groups capable of forming a salt may be, for example, formed as a pharmaceutically acceptable salt. Compounds containing one or more basic functional groups may be capable of forming a pharmaceutically acceptable salt with, for example, a pharmaceutically acceptable organic or inorganic acid. Pharmaceutically acceptable salts may be derived from, for example, and without limitation, acetic acid, adipic acid, alginic acid, aspartic acid, ascorbic acid, benzoic acid, benzenesulfonic acid, butyric acid, cinnamic acid, citric acid, camphoric acid, camphorsulfonic acid, cyclopentanepropionic acid, diethylacetic acid, digluconic acid, dodecylsulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, glucoheptanoic acid, gluconic acid, glycerophosphoric acid, glycolic acid, hemisulfonic acid, heptanoic acid, hexanoic acid, hydrochloric acid, hydrobromic acid, hydriodic acid, 2-hydroxyethanesulfonic acid, isonicotinic acid, lactic acid, malic acid, maleic acid, malonic acid, mandelic acid, methanesulfonic acid, 2-napthalenesulfonic acid, naphthalenedisulphonic acid, p- toluenesulfonic acid, nicotinic acid, nitric acid, oxalic acid, pamoic acid, pectinic acid, 3- phenylpropionic acid, phosphoric acid, picric acid, pimelic acid, pivalic acid, propionic acid, pyruvic acid, salicylic acid, succinic acid, sulfuric acid, sulfamic acid, tartaric acid, thiocyanic acid or undecanoic acid. Compounds containing one or more acidic functional groups maybe capable of forming pharmaceutically acceptable salts with a pharmaceutically acceptable base, for example, and without limitation, inorganic bases based on alkaline metals or alkaline earth metals or organic bases such as primary amine compounds, secondary amine compounds, tertiary amine compounds, quaternary amine compounds, substituted amines, naturally occurring substituted amines, cyclic amines or basic ion-exchange resins. Pharmaceutically acceptable salts maybe derived from, for example, and without limitation, a hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation such as ammonium, sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese or aluminum, ammonia, benzathine, meglumine, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2- diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, glucamine, methylglucamine, theobromine, purines, piperazine, piperidine, procaine, N-ethylpiperidine, theobromine, tetramethylammonium compounds, tetraethylammonium compounds, pyridine, N,N-dimethylaniline, N-methylpiperidine, morpholine, N- methylmorpholine, N-ethylmorpholine, dicyclohexylamine, dibenzylamine, N,N- dibenzylphenethylamine, 1-ephenamine, N,N'-dibenzylethylenediamine or polyamine resins. In some embodiments, compounds as described herein may contain both acidic and basic groups and may be in the form of inner salts or zwitterions, for example, and without limitation, betaines. Salts as described herein may be prepared by conventional processes known to a person skilled in the art, for example, and without limitation, by reacting the free form with an organic acid or inorganic acid or base, or by anion exchange or cation exchange from other salts. Those skilled in the art will appreciate that preparation of salts may occur in situ during isolation and purification of the compounds or preparation of salts may occur by separately reacting an isolated and purified compound.
[0045] In some embodiments, compounds and all different forms thereof (e.g. free forms, salts, polymorphs, isomeric forms) as described herein may be in the solvent addition form, for example, solvates. Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent in physical association the compound or salt thereof. The solvent may be, for example, and without limitation, a pharmaceutically acceptable solvent. For example, hydrates are formed when the solvent is water or alcoholates are formed when the solvent is an alcohol.
[0046] In some embodiments, compounds and all different forms thereof (e.g. free forms, salts, solvates, isomeric forms) as described herein may include crystalline and amorphous forms, for example, polymorphs, pseudopolymorphs, conformational polymorphs, amorphous forms, or a combination thereof. Polymorphs include different crystal packing arrangements of the same elemental composition of a compound. Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability and/ or solubility. Those skilled in the art will appreciate that various factors including recrystallization solvent, rate of crystallization and storage temperature may cause a single crystal form to dominate.
[0047] In some embodiments, compounds and all different forms thereof (e.g. free forms, salts, solvates, polymorphs) as described herein include isomers such as geometrical isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, individual enantiomers, individual diastereomers, racemates, diastereomeric mixtures and combinations thereof, and are not limited by the description of the formula illustrated for the sake of convenience.
[0048] In some embodiments, pharmaceutical compositions as described herein may comprise a salt of such a compound, preferably a pharmaceutically or physiologically acceptable salt. Pharmaceutical preparations will typically comprise one or more carriers, excipients or diluents acceptable for the mode of administration of the preparation, be it by injection, inhalation, topical administration, lavage, or other modes suitable for the selected treatment. Suitable carriers, excipients or diluents (used interchangeably herein) are those known in the art for use in such modes of administration. [0049] Suitable pharmaceutical compositions may be formulated by means known in the art and their mode of administration and dose determined by the skilled practitioner. For parenteral administration, a compound may be dissolved in sterile water or saline or a pharmaceutically acceptable vehicle used for administration of non-water soluble compounds such as those used for vitamin K. For enteral administration, the compound may be administered in a tablet, capsule or dissolved in liquid form. The tablet or capsule may be enteric coated, or in a formulation for sustained release. Many suitable formulations are known, including, polymeric or protein microparticles encapsulating a compound to be released, ointments, pastes, gels, hydrogels, or solutions which can be used topically or locally to administer a compound. A sustained release patch or implant may be employed to provide release over a prolonged period of time. Many techniques known to one of skill in the art are described in Remington: the Science & Practice of Pharmacy by Alfonso Gennaro, 20th ed., Lippencott Williams & Wilkins, (2000). Formulations for parenteral administration may, for example, contain excipients, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for modulatory compounds include ethylene vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene 9 lauryl ether, glycocholate and deoxycholate, or maybe oily solutions for administration in the form of nasal drops, or as a gel.
[0050] Compounds or pharmaceutical compositions as described herein or for use as described herein may be administered by means of a medical device or appliance such as an implant, graft, prosthesis, stent, etc. Also, implants may be devised which are intended to contain and release such compounds or compositions. An example would be an implant made of a polymeric material adapted to release the compound over a period of time.
[0051] An “effective amount” of a pharmaceutical composition as described herein includes a therapeutically effective amount or a prophylactically effective amount. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduced tumor size, increased life span or increased life expectancy. A therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as smaller tumors, increased life span, increased life expectancy or prevention of the progression of prostate cancer to an androgen independent form. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.
[0052] It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners. The amount of active compound(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the subject. Dosage regimens maybe adjusted to provide the optimum therapeutic response. For example, a single bolus maybe administered, several divided doses maybe administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
[0053] In some embodiments, compounds and all different forms thereof as described herein may be used, for example, and without limitation, in combination with other treatment methods for at least one indication selected from the group consisting of: prostate cancer, breast cancer, ovarian cancer, endometrial cancer, hair loss, acne, hirsutism, ovarian cysts, polycystic ovary disease, precocious puberty and age related macular degeneration. Alternatively, the compounds described herein may be useful for the treatment of one or more of the following: prostate cancer; lymphoma; neuroblastoma; breast cancer; colon cancer; cervical cancer; small-cell lung carcinoma; osteosarcoma; glioblastoma; melanoma; and myeloid leukaemia. Alternatively, the treatment may be for one or more of the following: prostate cancer; lymphoma; and neuroblastoma. For example, compounds and all their different forms as described herein maybe used as neo-adjuvant (prior), adjunctive (during), and/or adjuvant (after) therapy with surgery, radiation (brachytherapy or external beam), or other therapies (for example, HIFU).
[0054] In general, compounds as described herein should be used without causing substantial toxicity. Toxicity of the compounds as described herein can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population). In some circumstances however, such as in severe disease conditions, it may be appropriate to administer substantial excesses of the compositions. Some compounds as described herein may be toxic at some concentrations. Titration studies may be used to determine toxic and non-toxic concentrations. Toxicity may be evaluated by examining a particular compound’s or composition’s specificity across cell lines using PC3 cells as a negative control that do not express AR. Animal studies may be used to provide an indication if the compound has any effects on other tissues. On balance, a systemic therapy that targets the AR is still of interest despite the effects of anti-androgens, including androgen insensitivity in non-prostate tissues.
[0055] Compounds as described herein may be administered to a subject. As used herein, a “subject” maybe a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. The subject may be suspected of having or at risk for having a cancer, such as prostate cancer, breast cancer, ovarian cancer or endometrial cancer, or suspected of having or at risk for having acne, hirsutism, alopecia, benign prostatic hyperplasia, ovarian cysts, polycystic ovary disease, precocious puberty, or age related macular degeneration. Diagnostic methods for various cancers, such as prostate cancer, breast cancer, ovarian cancer or endometrial cancer, and diagnostic methods for acne, hirsutism, alopecia, benign prostatic hyperplasia, ovarian cysts, polycystic ovary disease, precocious puberty, or age related macular degeneration and the clinical delineation of cancer, such as prostate cancer, breast cancer, ovarian cancer or endometrial cancer, diagnoses and the clinical delineation of acne, hirsutism, alopecia, benign prostatic hyperplasia, ovarian cysts, polycystic ovary disease, precocious puberty, or age related macular degeneration are known to those of ordinary skill in the art.
[0056] Various alternative embodiments and examples are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.
MATERIALS AND METHODS
[0057] Virtual screening of potential Myc-Max DBD inhibitors.
[0058] The published 1.9 A crystal structure of c-Myc-Max heterodimer bound to its DNA- recognition sequence (PDB ID:1NKP [34], chains A, B; waters and DNA excluded) was subjected to the Site Finder algorithm implemented in MOE [51]. Site Finder is a geometric method which uses alpha spheres (virtual atoms) to probe a protein surface for suitable small molecule binding pockets. Briefly, Site Finder first identifies regions of tight atomic packing, filters out highly solvent exposed sites, calculates alpha spheres on sites and classifies them as either hydrophobic or hydrophilic depending on whether the virtual atom is in a good hydrogen bonding spot in the receptor, and then produces a collection of sites based on pruning (alpha spheres corresponding to inaccessible regions or exposed to solvent are eliminated) and clustering (by number and chemical type) of alpha spheres. The sites are then ranked according to their Propensity for Ligand Binding (PLB) score. The top PDB-ranked pocket was used for subsequent in silica screening. Virtual screening of the ZINC12 database [53, 54] was performed using structure-based methods including molecular docking algorithms and pharmacophore screening. The Glide™ program [55, 56], part of Maestro 9.3™ suite, Schrodinger LLC™ [57], was used as the starting point to perform rigid docking of 4.7 million drug-like chemicals. Following Maestro’s standard protein preparation protocol [87, 88], applied to the Myc-Max X-ray structure, a docking grid was defined as a 20 A box centered on the residues of predicted Myc-Max DBD binding site for Glide sampling and scoring of screening compounds. Prior to docking, each chemical was washed and energy- minimized under the MMFF94X force field and Born solvation as per ligand preparation protocol implemented in MOE [51]. Docking was conducted using Glide standard precision mode with all other settings set to default. The generated docking poses were ranked by the Glide score, an interaction energy score that includes hydrogen bonding and hydrophobic interactions contributions. Potentially weak binders (Glide score > -5.5 kcal/mol) were discarded. The remaining top-ranked 12503 remaining compounds were further filtered by structure-based pharmacophore screening using MOE’s tools [51]. A pharmacophore model of two essential hydrophobic features (1.5 A diameter each) of the binding site (formed primarily by Leugi , Ile2i8, Phe92i and Phe222) was built and used to search for matching hits in the database of top ranked Glide poses. 1019 pharmacophore-matching hits were then selected for manual inspection using the 3D visual environment in MOE. Compounds having a good balance of Glide docking score and ligand efficiency and making favorable interactions with the surrounding side chains in the pocket were purchased for subsequent experimental testing.
[0059] Cell culture and reagents
[0060] LNCaP and PC3 cells were purchased from the ATCC and grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS). HO15.19 cells were a generous gift from John Sidivy at Brown University and were cultured in Dulbecco’s modified Eagle’s medium DMEM (ATCC 30-2002) supplemented with 10% FBS. 10058-F4 and 10074-G5 were obtained from Sigma™. The UBE2C reporter plasmid was purchased from GeneCopoeia (product ID #HPRM16429). The Biolux Gaussia™ luciferase assay kit was purchased from New England Biolab™ (#E33OOL). PrestoBlue™ cell viability reagent was purchased from Invitrogen™ (#A- 13262).
[0061] Transfection and Reporter Assays
[0062] Cell transfection was performed using TransIT-2020™ transfection reagents according to the manufacturer’s instructions (Minis™). LNCaP cells were plated at 10000 cells per well and treated for 1 day with the indicated concentration of compound. Myc reporter activity was measured using the Cignal Myc Reporter Assay Kit™ from Qiagen™ (#336841) according to the manufacturer’s instructions. For the UBE2C reporter assay, 22rvi cells were plated at 10000 cells per well in 96-well plates in RPMI media supplemented with 5% charcoal-stripped serum (CSS) and treated for 1 day with 5 pM, 10 pM and 25 pM of compound.
[0063] Cell viability assays
[0064] LNCaP were plated at 5000 cells per well in RPMI 1640 containing 5% CSS in a 96-well plate, treated with test compounds (0-25 pM) for 96 hours. Cell density was measured using the PrestoBlue™ assay according to the manufacturer’s protocol. The percentage of cell survival was normalized to the cell density of control wells treated by vehicle. Viability of Myc-negative HO15.19 cells was done similarly but in DMEM supplemented with 5% CSS.
[0065] c-Myc-Max purification
[0066] Histidine tagged Max (residues 23-102) and GST tagged Myc (residues 368-454) were overexpressed in E. coli BL21-DE3 cells. Cells were co-lyzed in lysis buffer (20 mM Tris pH 8, 500 mM NaCl, 5% glycerol, 10 mM imidazole, 8 mM BME, 2.1 mM PMSF). After sonication and centrifugation, the complex was first purified by using a Ni-NTA affinity resin. After overnight dialysis to remove the imidazole, the protein sample was applied to a size exclusion chromatography equilibrated with (20mM Tris pH 8, lsomM NaCl, 5% glycerol, o.2mM TCEP). Fractions containing equal amount of Myc and Max on SDS PAGE were collected and used for the binding assay. The presence of both proteins was validated by Western blot using a specific antibody of each protein (Max (h2) Sc-8011 and c-Myc (9E10) Sc-40, Santa Cruz Biotechnology™).
[0067] Microsomal stability - Sample preparation
[0068] This is a very strictly timed experiment, wherein it is necessary to make sure that everything is ready (tubes identified and aliquots prepared) before starting and that you won't be interrupted once you start.
[0069] Reagents: 100 mM potassium phosphate buffer (pH 7.4) (8oopl of 1M K2HPO4 + 200pl of 1M KH2PO4 + 9ml ddH20); NADPH Regenerating system (Solution A and Solution B, kit from BD); Pooled mouse liver microsome (Xenotech, M1000) = MLM; 50UM DMSO stock of test coumpound (keep on ice); Acetonitrile+0.05% fomic acid with internal standard (i50ng/ml dsT) = stopping solution; 2ml clear glass tubes (Canadian Life Science, VT010M-1232); 250pl PP insert bottom spring, 6x29mm (the plastic ones) (Canadian Life, I025PBS-629); 10-425 black screw cap, red PTFE/white silicone, pre-slit (Canadian Life, C06050-10S); Water bath or bloc heater at 37°C.
[0070] Preparation of compounds
[0071] (1) Prepare IOOUM working stock of test compounds (and controls) in DMSO.
[0072] 2pl of compound in 998pl of DMSO.
[0073] Preparation of microsomal reaction assays & reagents (most important: ratio of compound & MLM): [0074] (2) For each compound, prepare duplicate assays by mixing: 461.25PI of the phosphate buffer (for a total reaction of 5OOul); 4pl of MLM (final concentration of O.i5mg/ml); and 5pl of toopM compound (final concentration of lpM)
[0075] (3) Vortex briefly and incubate at 37°C.
[0076] (4) For each MLM mix prepare a series of 4 tubes (t=o, t=iomin, t=20min, t=45min) with 3oopl stopping buffer.
[0077] (5) Prepare the t=o control by taking a 94pl aliquot from the MLM master mix into 3OOul of stopping solution. Add 6pl of NADPH later (after it has been added to start assays just to be sure not to run out).
[0078] (6) Prepare NAPDH mix using Solution A&B in 5:1 ratio.
[0079] (Addition of NADPH to start the reaction, so don't add to MLM mix until ready to start.)
[0080] Start & stop reaction
[0081] (7) Add 24pl of NADPH mix to the first MLM mix and start timer. Vortex briefly and reincubate.
[0082] (8) At 3Osec, add NADPH to the next MLM mix.
[0083] (9) Continue adding NADPH to MLM mixes at 3Osec intervals.
[0084] (10) At desired time points (10, 20, & 45mm), transfer a tooul aliquot of the master mix into a tube of stopping solution.
[0085] Preparation of samples for LC-MS analysis
[0086] (11) Vortex all samples, then centrifuge at 18,000g 4°C for lomin.
[0087] (12) Transfer the organic layer into a new set of 1.5ml microtubes. (If samples are to be kept for future use.)
[0088] Transfer 200pl of sample into LCMS vials for analysis.
EXAMPLES
[0090] EXAMPLE 1: In silico identification of hit compounds targeting the Myc-Max DBD site.
[0091] A virtual screed of over 9 million drug-like purchasable chemical compounds from the ZINC15 database [70], was further reduced to 4.7 million compounds by filtering by physicochemical properties such as charge, number of rings and rotatable bonds. The resulting set of 4.7 million structures was virtually screened against the identified pocket on the Myc-Max dimer DBD. Glide™ (Maestro 9.3™ suite, Schrodinger LLC™) software [55-57] was employed as the primary structurebased docking technique (with the standard precision mode). The generated docking poses were then filtered by the Glide™ docking score (binding energy score used to rank docking poses and distinguish strong binders in their optimal placement in the respective pocket from compounds that bind weakly) using a -5.5 kcal/mol cutoff. The top ranked 12503 remaining compounds were further filtered by structure-based pharmacophore screening using MOE’s tools. A pharmacophore model of two essential hydrophobic features (1.5 A diameter each) of the binding site (formed primarily by Leugiy, Ile2i8, Phe92i and Phe222) was built and used to search for matching hits in the database of top ranked Glide™ poses. 1019 pharmacophore-matching hits were then selected for visual inspection and 116 compounds having a good balance of Glide™ docking score and ligand efficiency (the ratio of binding affinity over the number of heavy atoms) made additional side-chain or backbone hydrogen bonds with the charged residues in the site. Compounds were selected for purchase, in particular those predicted to form hydrogen bonds with the backbone carbonyl oxygen of Arg2i5. The purchased compounds were then subjected to rapid evaluation using a primary screening transcriptional assay as described below. From the primary cell -based hits were identified showing better than 60% inhibition of Myc-Max transcriptional activity at 10 pM and those have less inhibitory activity.
[0092] EXAMPLE 2: Effects of hit compounds on Myc-Max transcriptional activity.
[0093] Compounds were subjected to experimental evaluation using the commercially available transcriptional assay Cignal c-Myc luciferase reporter assay in LNCaP cells. Compounds 10058-F4 and 10074-G5, known Myc inhibitors from the literature, were used as positive controls. A transiently transfected Myc-driven luciferase reporter allowed the monitoring of Myc- regulated signal in LNCaP upon treatment with the in silico identified compounds. From a larger number of hits, 133 compounds caused more than 60% reduction of the Myc-driven luciferase levels at 10 pM (see TABLE 1). A dose response analysis was performed using LNCaP cells to evaluate the potency of hit compounds.
[0094] The compounds shown in TABLE 1 below represent tested ZINC compounds, having Myc- Max inhibitory activity. [0095] TABLE 1: TESTED ZINC COMPOUNDS
[0096] The compounds shown in TABLE 2 below represent novel tested compounds, falling under Formula I and having Myc-Max inhibitory activity.
[0097] TABLE 2: NOVEL COMPOUNDS TESTED
[0098] The compounds shown in TABLE 3 below represent novel tested compounds, not having Myc-Max inhibitory activity.
[0099] TABLE 3: NOVEL COMPOUNDS TESTED LACKING OR WITHOUT SIGNIFICANT MYC-MAX INHIBITORY ACTIVITY [00100] EXAMPLE 3: Effects of hit compounds on cell viability and half-life.
[00101] Some of the compounds showing inhibition of Myc-Max transcriptional activity were also tested for IC 50 values (half-maximal inhibitory concentration with 95% Confidence Intervals) and half-life in minutes. The effect of hit compounds on Myc-driven cell proliferation was evaluated by measuring the cell viability of LNCaP cells after treatment with increasing concentrations of compounds. Compounds 70596, 70614 and 70812 showed the very good inhibition of LNCaP cell proliferation (i.e. IC 50 = 2 pM; [95%CI: 2.1-2.8 pM]). Similarly, reasonable IC 50 vales were shown for 70551 (4 pM), 70613 (3 pM), 70589 (3 pM), 70599 (3 pM), 70693 (3 pM), 70815 (3 pM), 70632 (5 pM), 70818 (5 pM), 70816 (6 pM), 70582 (8 pM), 70604 (8 pM), 70616 (9 pM), 70811 (10 pM), 70593 (12 pM), 70597 (12 pM), 70595 (12 pM), 70681 (12 pM), 70819 (12 pM), 70615 (15 pM), 70590 (15 pM), 70598 (15 pM) and 70591 (25 pM).
[00102] Some of the compounds were also tested for half-life in minutes as described herein and ranged from 2310 minutes to 13 minutes as follows: 70551 (140), 70589 (141), 70599 (301, 138), 70582 (210), 70604 (68), 70593 (2310), 70597 (62), 70596 (217), 70614 (139), 70595 (13), 70590 (112), 70804 (122), 70815 (78), 70816 (267), 70811 (105), 70812 (98) and 70818 (578).
[00103] In the absence of clinically approved anti-Myc drugs, targeting the Myc-Max complex represents a critical step towards creating new therapeutics for lethal CRPC and NEPC. In this study, we identified a possible druggable site on the DNA-binding domain (DBD) of the structurally ordered Myc-Max complex and employed a computer-aided rational drug discovery approach to identify small molecules that inhibit Myc-Max functionality.
[00104] Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word “comprising” is used herein as an open-ended term, substantially equivalent to the phrase “including, but not limited to”, and the word “comprises” has a corresponding meaning. As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a thing” includes more than one such thing. Citation of references herein is not an admission that such references are prior art to an embodiment of the present invention. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples.
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