CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/523,608 entitled "INHIBITORS OF ANDROGEN RECEPTOR ACTIVATION

FUNCTION-2 (AF2) AS THERAPEUTICS AND METHODS FOR THEIR USE" filed on 15 August 2011.

TECHNICAL FIELD

This invention relates to therapeutics, their uses and methods for the treatment of various indications, including various cancers. In particular the invention relates to therapies and methods of treatment for cancers such as prostate cancer.

BACKGROUND

Androgens are known to mediate their effects through the androgen receptor (AR). Androgens play a role in a wide range of developmental and physiological responses, for example, male sexual differentiation, maintenance of spermatogenesis, and male gonadotropin regulation (R. K. Ross, G. A. Coetzee, C. L. Pearce, J. K. Reichardt, P.

Bretsky, L. N. Kolonel, B. E. Henderson, E. Lander, D. Altshuler & G. Daley, Eur Urol 35, 355-361 (1999); A. A. Thomson, Reproduction 121 , 187-195 (2001); N. Tanji, K. Aoki & M. Yokoyama, Arch Androl 47, 1-7 (2001)). Also, androgens are associated with the development of prostate carcinogenesis. Induction of prostatic carcinogenesis in rodent models has been associated with androgens (R. L. Noble, Cancer Res 37, 1929-1933 (1977); R. L. Noble, Oncology 34, 138-141 (1977)) and men receiving androgens in the form of anabolic steroids are reported to have a higher incidence of prostate cancer (J. T. Roberts & D. M. Essenhigh, Lancet 2, 742 (1986); J. A. Jackson, J. Waxman & A. M. Spiekerman, Arch Intern Med 149, 2365-2366 (1989); P. D. Guinan, W. Sadoughi, H. Alsheik, R. J. Ablin, D. Alrenga & I. M. Bush, Am J Surg 131 , 599-600 (1976)).

Furthermore, prostate cancer does not develop if humans or dogs are castrated before puberty (J. D. Wilson & C. Roehrborn, J Clin Endocrinol Metab 84, 4324-4331 (1999); G. Wilding, Cancer Surv 14, 113-130 (1992)). Castration of adult males causes involution of the prostate and apoptosis of prostatic epithelium (E. M. Bruckheimer & N. Kyprianou, Cell Tissue Res 301 , 153-162 (2000); J. T. Isaacs, Prostate 5, 545-557 (1984)). This dependency on androgens provides the underlying rationale for treating prostate cancer with chemical or surgical castration (i.e. androgen ablation).

Prostate cancer is the second leading cause of male cancer-related death in Western countries (Damber, J. E. and Aus, G. Lancet (2008) 371 : 1710-1721). Numerous studies have shown that the androgen receptor (AR) is central not only to the development of prostate cancer, but also the progression of the disease to the castration resistance state (Taplin, M. E. et al. J. Clin. Oncol. (2003) 21 :2673-8; and Tilley, W. D. et al. Cancer Res. (1994) 54:4096-4102). Thus, effective inhibition of human AR remains one of the most effective therapeutic approaches to the treatment of advanced, metastatic prostate cancer.

The AR possesses a modular organization characteristic of all nuclear receptors. It is comprised of an N-terminal domain, a central DNA binding domain, a short hinge region, and C-terminal domain that contains a hormone ligand binding pocket and the Activation Function-2 (AF2) site (Gao, W. Q. et al. Chem. Rev. (2005) 105:3352-3370). The latter represents a hydrophobic groove on the AR surface which is flanked with regions of positive and negative charges -"charge clamps" that are significant for binding AR activation factors (Zhou, X. E. et al. J. Biol. Chem. (2010) 285:9161-9171).

The activation of AR follows a well characterized pathway: in the cytoplasm, the receptor is associated with chaperone proteins that maintain agonist binding conformation of the AR (Georget, V. et al. Biochemistry (2002) 41 : 11824-11831). Upon binding of an androgen, the AR undergoes a series of conformational changes, disassociation from chaperones, dimerization and translocation into the nucleus (Fang, Y. F. et al. J. Biol. Chem. (1996) 271 :28697-28702; and Wong, C. I. et al. J. Biol. Chem. (1993) 268: 19004- 19012) where it further interacts with co-activator proteins at the AF2 site (Zhou, X. E. et al. J. Biol. Chem. (2010) 285:9161-9171). This event triggers the recruitment of RNA polymerase II and other factors to form a functional transcriptional complex with the AR. The association of co-activators at the AF2 site is significant for the transcriptional activities of the receptor.

Notably, the current anti-androgens such as bicalutamide, flutamide, nilutamide and MDV3100, all target this particular process. However, instead of affecting the AR-cofactor interaction directly, these anti-androgens act indirectly, by binding to the AR ligand binding site. Thus, by preventing androgens from binding they also prevent conformational changes of the receptor that are necessary for co-activator interactions. While treatment with these AR inhibitors can initially suppress the prostate cancer growth, long term hormone therapy becomes progressively less effective (Taplin, M. E. et al. J. Clin. Oncol. (2003) 21 :2673-8; and Tilley, W. D. et al. Cancer Res. (1994) 54:4096-4102). Factors that make the AR less sensitive to conventional anti-androgens include resistance mutations at the ligand binding site that can even lead AR antagonists to act as agonists further contributing to cancer progression (Chen, Y. et al. Lancet Oncol. (2009) 10:981-991).

Recently, Gunther et al evaluated a library of peptide-mimetic compounds and some of these peptides were capable of inhibiting the AR/SRC association at low micro-molar levels (Gunther, J. R. et al. ACS Chem. Biol. (2009) 4:435-440). In a similar study, a screen of -10,000 chemicals identified several structurally diverse micro-molar hits that could prevent association of the AR with its co-activator gelsoline (Joseph, J. D. et al. Proc. Nat. Acad. Sci. USA (2009) 106: 12178-12183). Furthermore, a large-scale crystallographic screen demonstrated that several approved drugs, such as hormone analogues 3,3' ,5-triiodo thyroacetic acid, 1-tertbutyl-, thriiodothyronine, 3-((l-tert-butyl-4-amino-lHpyrazolo[3,4- D]pyrimidin-3-yl)methyl)phenol and a kinase inhibitor 3-(2,5-dimethyl-benzyl)-lH- pyrazolo[3,4-D]pyrimidin-4-ylamine (later noted as K10), can non-selectively bind to the AF2 and moderately (IC5o~50uM) interfere with the AR transcriptional activity (Estebanez- Perpina, E. et al. Proc. Nat. Acad. Sci. USA(2007) 104: 16074 - 16079).

Androgens also play a role in female cancers. One example is ovarian cancer where elevated levels of androgens are associated with an increased risk of developing ovarian cancer (K. J. Helzlsouer, A. J. Alberg, G. B. Gordon, C. Longcope, T. L. Bush, S. C. Hoffman & G. W. Comstock, JAMA 274, 1926-1930 (1995); R. J. Edmondson, J. M. Monaghan & B. R. Davies, Br J Cancer 86, 879-885 (2002)). The AR has been detected in a majority of ovarian cancers (H. A. Risch, J Natl Cancer Inst 90, 1774-1786 (1998); B. R. Rao & B. J. Slotman, Endocr Rev 12, 14-26 (1991); G. M. Clinton & W. Hua, Crit Rev Oncol Hematol 25, 1-9 (1997)), whereas estrogen receptor- alpha (ERa) and the progesterone receptor are detected in less than 50% of ovarian tumors.

SUMMARY

This invention is based in part on the fortuitous discovery that compounds described herein modulate androgen receptor (AR) activity. Specifically, compounds identified herein, show inhibition of Androgen Receptor Activation Function-2 (AF2), which may make these compounds suitable for use in the treatment of at least one indication selected from the group consisting of: prostate cancer, breast cancer, ovarian cancer, and endometrial cancer, hair loss, acne, hirsutism, ovarian cysts, polycystic ovary disease, precocious puberty, and age-related macular degeneration, breast cancer, ovarian cancer, and endometrial cancer.

In accordance with a first embodiment, there is provided a use of a compound or pharmaceutically acceptable salt thereof having a structure of Formula I

wherein, D may be H, OH, F, Br, or CI; E may be H, OH, Br, CI, F, C≡N, CH ₂ OH, or COOH; G may be H, OH, F, Br, or CI; D ₂ may be H, OH, F, Br, or CI; E ₂ may be H, OH, CI, Br, F, C≡N, CH ₂ OH, Me, or COOH; G ₂ may be H, OH, F, Br, or CI; or D and E or E

and G or D ₂ and E ₂ or E ₂ and G ₂ may form a ring having the structure ; or A and

D or J and G or A ₂ and D ₂ or J ₂ and G ₂ may form a ring having the structure ; or one of E and D may be OMe or OEt or NHEt and the other may be OH; or one of E and G may be OMe or OEt or NHEt and the other may be OH; or one of E ₂ and D ₂ may be OMe or OEt or NHEt and the other may be OH; or one of E ₂ and G ₂ may be OMe or OEt or NHEt and the other may be OH; or one of D or G or D ₂ or G ₂ may be -N ⁺ 00 ^" ; or one of D, E, and G may be OMe or OEt or NHEt, one may be F, Br, or CI and one may be OH; or one of D ₂ , E ₂ , and G ₂ may be OMe or OEt or NHEt, one may be F, Br, or CI and one may be OH; or one of A or J may be F , CI or Br and one of D or G may be CI, Br, or F; or one of A ₂ or J ₂ may be F , CI or Br and one of D ₂ or G ₂ may be CI, Br, or F; or one of A or J may be OH and one of D or G may be OMe or OEt; or one of A ₂ or J ₂ may be OH and one of D ₂ or G ₂ may be OMe or OEt; or one of A or J may be OH, F, CI or Br and E may be OH, CI, NH(Et) ₂ ; or one of A ₂ or J ₂ may be OH, F, CI or Br and E ₂ may be OH, CI, NH(Et) ₂ ; or one of A or J or A ₂ or J ₂ may be CF ₃ , CBr ₃ , CCI ₃ , OH, F, CI or Br; for modulating androgen receptor (AR) activity.

In accordance with another embodiment, there is provided a use of a compound or pharmaceuticall acceptable salt thereof having a structure of Formula II

wherein, R may be O, S, or NH; T may be H, OH, Me, Et, OMe, OEt, NHEt, NHMe, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; X may be H, OH, Me, Et, OMe, OEt, NHEt, NHMe, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Z may be H, OH, Me, Et, OMe, OEt, NHEt, NHMe, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; for modulating androgen receptor (AR) activity.

In accordance with another embodiment, there is provided a use of a compound or pharmaceuticall acceptable salt thereof having a structure of Formula III

wherein, Li may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt,

NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; L ₂ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; L ₃ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; L ₄ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; L5 may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; M may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Qi may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Q ₂ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Q ₃ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Q ₄ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Q ₅ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; for modulating androgen receptor (AR) activity.

In accordance with another embodiment, there is provided a pharmaceutical composition comprising a compound or pharmaceutically acceptable salt thereof having a structure of Formula I

wherein, D may be H, OH, F, Br, or CI; E may be H, OH, Br, CI, F, C≡N, CH ₂ OH, or COOH; G may be H, OH, F, Br, or CI; D ₂ may be H, OH, F, Br, or CI; E ₂ may be H, OH, CI, Br, F, C≡N, CH ₂ OH, Me, or COOH; G ₂ may be H, OH, F, Br, or CI; or D and E or E

and G or D ₂ and E ₂ or E ₂ and G ₂ may form a ring having the structure ; or A and

D or J and G or A ₂ and D ₂ or J ₂ and G ₂ may form a ring having the structure ; or one of E and D may be OMe or OEt or NHEt and the other may be OH; or one of E and G may be OMe or OEt or NHEt and the other may be OH; or one of E ₂ and D ₂ may be OMe or OEt or NHEt and the other may be OH; or one of and G ₂ may be OMe or OEt or NHEt and the other may be OH; or one of D or G or D ₂ or G ₂ may be -N ⁺ 00 ^" ; or one of D, E, and G may be OMe or OEt or NHEt, one may be F, Br, or CI and one may be OH; or one of D ₂ , E ₂ , and G ₂ may be OMe or OEt or NHEt, one may be F, Br, or CI and one may be OH; or one of A or J may be F , CI or Br and one of D or G may be CI, Br, or F; or one of A ₂ or J ₂ may be F , CI or Br and one of D ₂ or G ₂ may be CI, Br, or F; or one of A or J may be OH and one of D or G may be OMe or OEt; or one of A ₂ or J ₂ may be OH and one of D ₂ or G ₂ may be OMe or OEt; or one of A or J may be OH, F, CI or Br and E may be OH, CI, NH(Et) ₂ ; or one of A ₂ or J ₂ may be OH, F, CI or Br and E ₂ may be OH, CI, NH(Et) ₂ ; or one of A or J or A ₂ or J ₂ may be CF ₃ , CBr ₃ , CCI ₃ , OH, F, CI or Br; and a pharmaceutically acceptable excipient. In accordance with another embodiment, there is provided a pharmaceutical composition comprising a compound or pharmaceutically acceptable salt thereof having a structure of Formula II

wherein, R may be O, S, or NH; T may be H, OH, Me, Et, OMe, OEt, NHEt, NHMe, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; X may be H, OH, Me, Et, OMe, OEt, NHEt, NHMe, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Z may be H, OH, Me, Et, OMe, OEt, NHEt, NHMe, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; and a pharmaceutically acceptable excipient.

In accordance with another embodiment, there is provided a pharmaceutical composition comprisin mpound or pharmaceutically acceptable salt thereof having a

structure of Formula III wherein, Li may be H, OH, Me, Et, OMe, NH ₂ ,

NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; L ₂ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; L ₃ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; L ₄ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; L ₅ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; M may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Qi may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Q ₂ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Q ₃ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Q ₄ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Q ₅ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; and a pharmaceutically acceptable excipient.

In accordance with another embodiment, there is provided a method for modulating AR activity, the method comprising administering to a mammalian cell a compound or pharmaceutically acceptable salt thereof as described herein. The mammalian cell may be a human cell.

In accordance with another embodiment, there is provided a compound or pharmaceutically acceptable salt thereof having a structure of Formula I

wherein, D may be H, OH, F, Br, or CI; E may be H, OH, Br, CI, F, C≡N, CH ₂ OH, or COOH; G may be H, OH, F, Br, or CI; D ₂ may be H, OH, F, Br, or CI; E ₂ may be H, OH, CI, Br, F, C≡N, CH ₂ OH, Me, or COOH; G ₂ may be H, OH, F, Br, or CI; or D and E or E

and G or D ₂ and E ₂ or E ₂ and G ₂ may form a ring having the structure ; or A and

D or J and G or A ₂ and D ₂ or J ₂ and G ₂ may form a ring having the structure ; or one of E and D may be OMe or OEt or NHEt and the other may be OH; or one of E and G may be OMe or OEt or NHEt and the other may be OH; or one of E ₂ and D ₂ may be OMe or OEt or NHEt and the other may be OH; or one of E ₂ and G ₂ may be OMe or OEt or NHEt and the other may be OH; or one of D or G or D ₂ or G ₂ may be -N ⁺ 00 ^" ; or one of D, E, and G may be OMe or OEt or NHEt, one may be F, Br, or CI and one may be OH; or one of D ₂ , E ₂ , and G ₂ may be OMe or OEt or NHEt, one may be F, Br, or CI and one may be OH; or one of A or J may be F , CI or Br and one of D or G may be CI, Br, or F; or one of A ₂ or J ₂ may be F , CI or Br and one of D ₂ or G ₂ may be CI, Br, or F; or one of A or J may be OH and one of D or G may be OMe or OEt; or one of A ₂ or J ₂ may be OH and one of D ₂ or G ₂ may be OMe or OEt; or one of A or J may be OH, F, CI or Br and E may be OH, CI, NH(Et) ₂ ; or one of A ₂ or J ₂ may be OH, F, CI or Br and E2 may be OH, CI, NH(Et) ₂ ; or one of A or J or A ₂ or J ₂ may be CF3, CBr3, CCI3, OH, F, CI or Br; excluding those compounds set out in TABLES 1 and 2.

In accordance with another embodiment, there is provided a compound or pharmaceutically acceptable salt thereof having a structure of Formula II

wherein, R may be Ο, S, or NH; T may be H, OH, Me, Et, OMe, OEt, NHEt, NHMe, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; X may be H, OH, Me, Et, OMe, OEt, NHEt, NHMe, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Z may be H, OH, Me, Et, OMe, OEt, NHEt, NHMe, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; excluding those compounds set out in TABLES 1 and 2.

In accordance with another embodiment, there is provided a compound or pharmaceuticall acceptable salt thereof having a structure of Formula III

wherein, Li may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt,

NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; L ₂ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; L ₃ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; L ₄ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Ls may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; M may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Qi may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Q ₂ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Q ₃ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Q ₄ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; Q ₅ may be H, OH, Me, Et, OMe, NH ₂ , NHMe, OEt, NHEt, NEt ₂ , NMe ₂ , F, Br, CI, or COOH; excluding those compounds set out in TABLES 1 and 2.

In accordance with another embodiment, there is provided a commercial package, including a compound or pharmaceutically acceptable salt thereof as described herein and instructions for use in modulating androgen receptor (AR) activity. The compounds or pharmaceutically acceptable salt thereof having a structure of Formula IA, IB, IIA, IIB, or III as described herein may also be used in the manufacture of a medicament for modulating androgen receptor (AR) activity. Alternatively, the pharmaceutical composition described herein may be used for modulating androgen receptor (AR) activity. The modulating of androgen receptor (AR) activity may be used for the treatment of at least one indication selected from the group consisting of: prostate cancer, breast cancer, ovarian cancer, and endometrial cancer, hair loss, acne, hirsutism, ovarian cysts, polycystic ovary disease, precocious puberty, and age-related macular degeneration. The compounds or pharmaceutically acceptable salt thereof or

pharmaceutical composition described herein may be used for the treatment of one or more 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. The compounds or pharmaceutically acceptable salt thereof or pharmaceutical composition described herein may be used for the treatment of prostate cancer. The compounds or pharmaceutically acceptable salt thereof or pharmaceutical composition described herein may be used for the treatment of breast cancer. The compounds or pharmaceutically acceptable salt thereof or pharmaceutical composition described herein may be used for the treatment of ovarian cancer. The compounds or pharmaceutically acceptable salt thereof or pharmaceutical composition described herein may be used for the treatment of endometrial cancer. The compounds or pharmaceutically acceptable salt thereof or pharmaceutical composition described herein may be used for the treatment of hair loss. The compounds or pharmaceutically acceptable salt thereof or pharmaceutical composition described herein may be used for the treatment of acne. The compounds or pharmaceutically acceptable salt thereof or pharmaceutical composition described herein may be used for the treatment of hirsutism. The compounds or pharmaceutically acceptable salt thereof or pharmaceutical composition described herein may be used for the treatment of ovarian cysts. The compounds or pharmaceutically acceptable salt thereof or pharmaceutical composition described herein may be used for the treatment of polycystic ovary disease. The compounds or pharmaceutically acceptable salt thereof or pharmaceutical composition described herein may be used for the treatment of precocious puberty. The compounds or pharmaceutically acceptable salt thereof or pharmaceutical composition described herein may be used for the treatment of age-related macular degeneration. D may be H, OH, F, Br, or CI; D ₂ may be H, OH, F, Br, or CI; G may be H, OH, F, Br, or CI; G ₂ may be H, OH, F, Br, or CI; E may be H, OH, CI, F, or C≡N; E ₂ may be H, OH, CI, C≡N, or Me; or one of E and D may be OMe or OEt and the other may be OH; or one of E and G may be OMe or OEt and the other may be OH; or one of E ₂ and D ₂ may be OMe or OEt and the other may be OH; or one of E ₂ and G ₂ may be OMe or OEt and the other may be OH; or one of D, E, and G may be OMe or OEt, one may be F, Br, or CI and one may be OH; or one of D ₂ , E^, and G ₂ may be OMe or OEt, one may be F, Br, or CI and one may be OH.

D may be H, OH, F, Br, or CI; D ₂ may be H, OH, F, Br, or CI; G may be H, OH, F, Br, or CI; G ₂ may be H, OH, F, Br, or CI; E may be H, OH, CI, or F; E ₂ may be H, OH, F, or CI; or one of E and D may be OMe or OEt and the other may be OH; or one of E and G may be OMe or OEt and the other may be OH; or one of E ₂ and D ₂ may be OMe or OEt and the other may be OH; or one of E ₂ and G ₂ may be OMe or OEt and the other may be OH; or one of D, E, and G may be OMe or OEt, one may be F, Br, or CI and one may be OH; or one of D ₂ , E ₂ , and G ₂ may be OMe or OEt, one may be F, Br, or CI and one may be OH.

D may be H, OH, F, Br, or CI; D ₂ may be H, OH, F, Br, or CI; G may be H, OH, F, Br, or CI; G ₂ may be H, OH, F, Br, or CI; E may be H, OH, CI, or F; E ₂ may be H, OH, or CI; or one of E and D or E ₂ and D ₂ may be OMe or OEt and the other may be OH; or one of E and G or E ₂ and G ₂ may be OMe or OEt and the other may be OH; or one of D, E, and G may be OMe or OEt, one may be F, Br, or CI and one may be OH; or one of D ₂ , E ₂ , and G ₂ may be OMe or OEt, one may be F, Br, or CI and one may be OH.

Alternatively, one of A or J may be F, CI or Br and one of E or D or G may be CI, Br, or F. One of A ₂ or J ₂ may be F , CI or Br and one of E ₂ or D ₂ or G ₂ may be CI, Br, or F. One of A or J may be OH and one of D or G may be OMe or OEt. One of A ₂ or J ₂ may be OH and one of D ₂ or G ₂ may be OMe or OEt. One of A or J may be OH, F, CI or Br and E may be OH, CI, NH(Et) ₂ . One of A ₂ or J ₂ may be OH, F, CI or Br and E ₂ may be OH, CI, NH(Et) ₂ . One of A or J or A ₂ or J ₂ may be CF ₃ , CBr ₃ , CC1 ₃ , OH, F, CI or Br. A and D may be CI. A and E may be CI. A may be F and G may be Br. A and E may be OH. D and E may be OH. A may be Br. A may be CI. A may be F. A ₂ and D ₂ may be CI. A ₂ and may be CI. A ₂ may be F and G ₂ may be Br. A ₂ and E ₂ may be OH. D ₂ and E ₂ may be OH. A ₂ may be Br. A ₂ may be CI. A ₂ may be F. E or E ₂ may be Me. E or E ₂ may be -C≡N. A or A ₂ may be CF3. A or A ₂ may be OH and E or may be -N ⁺ H(Et) ₂ . D or D ₂ may be - N (=0)-0 . A or A ₂ may be OH. E or E ₂ may be -CI. E or E ₂ may be F. A may be OH and D may be OMe. A ₂ may be OH and D ₂ may be OMe. E may be OH and D may be OH. E ₂ may be OH and D ₂ may be OH. E may be COOH. E ₂ may be COOH. D may be OH and E may be OMe. D ₂ may be OH and E ₂ may be OMe. D may be OEt and E may be OH. D ₂ may be OEt and E ₂ may be OH. D or D ₂ may be CI. E or E ₂ may be CI. E or E ₂ may be OH. D or D ₂ may be Br. D or D ₂ may be F. D may be OMe and E may be OH. D ₂ may be OMe and E ₂ may be OH. D and E may be CI. D ₂ and E ₂ may be CI. A, D, E, G, and J may be H. A ₂ , D ₂ , E ₂ , G ₂ , and J ₂ may be H. D may be OMe, E may be OH, and G may be Br. D ₂ may be OMe, E ₂ may be OH, and G ₂ may be Br.

R may be O, S, or NH; T may be H, Me, Et, OMe, OEt, F, Br, CI, or COOH; X may be H, Me, Et, OMe, OEt, F, Br, CI, or COOH; and Z may be H, Me, Et, OMe, OEt, F, Br, CI, or COOH. R may be O, S, or NH; T may be H or Me; X may be H or Me; and Z may be H or Me.

R may be O or NH; T may be H, Me, Et, OMe, OEt, F, Br, CI, or COOH; X may be H, Me, Et, OMe, OEt, F, Br, CI, or COOH; and Z may be H, Me, Et, OMe, OEt, F, Br, CI, or COOH. R may be O or S; T may be H, Me, Et, OMe, OEt, F, Br, CI, or COOH; X may be H, Me, Et, OMe, OEt, F, Br, CI, or COOH; and Z may be H, Me, Et, OMe, OEt, F, Br, CI, or COOH. R may be S or NH; T may be H, Me, Et, OMe, OEt, F, Br, CI, or COOH; X may be H, Me, Et, OMe, OEt, F, Br, CI, or COOH; and Z may be H, Me, Et, OMe, OEt, F, Br, CI, or COOH. R may be O or S; T may be H or Me; X may be H or Me; and Z may be H or Me. R may be O or NH; T may be H or Me; X may be H or Me; and Z may be H or Me. R may be S or NH; T may be H or Me; X may be H or Me; and Z may be H or Me.

Li_5 may be H, OH, Me, Et, OMe, F, Br, CI, or COOH; M may be H, OH, Me, Et, F, Br, CI, or COOH; Qi_ ₅ may be H, OH, Me, Et, OMe, OEt, F, Br, CI, or COOH. Li_ ₅ may be H; M may be H, OH, Me, Et; Qi_ ₅ may be H, OH, Me, Et, or COOH.

The compound may be selected from one or more of the compounds set out in

TABLE 2. The com ound may be . The compound may be . The compound may be . The compound may be . The compound may be . The compound may be The compound may be . The compound may be The compound may be

. The compound may be The compound may be . The compound may be The compound may be . The compound may be . The compound may be . Th m ound may be

. The compound may be . The compound may

be . The compound may be . The compound may be ie compound may be The compound may be . The compound may be . The compound may be . The compound may be . The compound may . The compound may be . The compound may be

. The compound may

be . The compound may be . The compound may be . The compound may be . The compound may be

 BRIEF DESCRIPTION OF THE DRAWINGS

shows a graphic representation of the AF2 site on the surface of the androgen receptor, with hydrophobic residues (M734, V730, L712, 1737, and V716), charged residues are shown as E897, E893, 1898, M894, and K720 and the

SRC2-3 peptide is shown as a coil overlayed on the AF2 site.

shows inhibition of AF2 co-activator binding and AR transcriptional activity by the identified compounds VPC-4107, VPC-0100 and VPC-0061 as follows:

1) FP dose response curves (0-200μΜ) for displacement of SRC2-3 peptide from the AF2 site by the compounds;

2) The AR- transcription inhibition dose response curves (0-ΙΟΟμΜ) illustrating inhibiting effect of the compounds on the AR transcriptional activity in cells; and

3) Biolayer interferometrydose response curves (0-50 μΜ) reflecting direct binding of the compounds to the AR protein.

shows a graphic representation of the AF2 site on the surface of the androgen receptor. Hydrophobic residues are V730, M734, 1737, V716, and L712, whereas charged residues are K720, M894, E893, 1898, and E897. The experimentally resolved structure of a bound compound VPC-0061 is shown, with the docking pose overlayed.

shows a superimposition of the Androgen Receptor - VPC-0061 complex with Androgen Receptor from 2PIP PDB structure. Dotted line illustrates anchoring cation-arene interaction.

shows docking poses of selected compoundsVPC-4107 (A), VPC-4040 (B), VPC-4023 (C) and VPC-0100 (D) inside the AF2 site of the AR. Hydrogen bonds are shown with broken lines, charged residues are E893, E897, K720, and Q733 whereas hydrophobic residues are indicated are the remaining residues shown.

shows a plot from the FP assay of % of inhibition (mP) against log concentration for compounds 7001, 7002, 7009, and 7022 (where

P=Polarization value and mP is P/1000) vs the control). Figure 5B shows a plot from the FP assay of % of inhibition (mP) against log concentration for compounds 7004 and 7007.

Figure 6A shows a plot from the FP assay of % of inhibition (mP) against log

concentration for compounds 7050, 7051 , and 7056.

Figure 6B shows a plot from the FP assay of % of inhibition (mP) against log

concentration for compounds 7065 and 7071.

DETAILED DESCRIPTION

In silico virtual screening for potentialAF2 binders.

The Activation Function 2 (AF2) site represents a hydrophobic groove on the AR surface flanked with charged amino acids that are significant for the binding of receptor co- activators. Being a protein-protein interaction site, the AF2 proved to be a challenging target. However, the AF2 site is also an attractive target for direct inhibition of the AR co- activation. In silico computational drug discovery methods were used to conduct a virtual screen of ~4 million purchasable lead-like compounds from the ZINC database (Irwin, J. et al. Abstracts of Papers Am. Chem. Soc. (2005) 230:U1009) to identify potential AF2 binders. The in silico methods included large-scale docking, in-site rescoring and consensus voting procedures.

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.

TABLE 1 Shows all of the tested compounds by structure and the associated identifiers, for the ZINC database and the identifiers as used in the description. If more than one identifier is used they are in "()".

TABLE 1 Compounds Tested for AF2 binding

ZINCO

ZINCOO

06253 7007 7047

135767

33

ZINCO

ZINCOO

02322 7008 7048

028678

45

ZINCO

ZINCOO

02322 7009 7049

135808

58

7050

ZINCO

ZINCOO (4040) 21789 7010

228765 (VPC- 38

3)

ZINCO

ZINCOO

20830 7011 7051

135780

66



TABLE 2 shows compounds by structure and the associated identifiers for those compounds identified herein as modulators of androgen receptor (AR) activity. In particular, those compounds identified as AF2 binding compounds or cellular transcription inhibitors. The compounds in TABLE 2 represent embodiments of Formula IA/IB and Formula IIA/IIB or Formula III above.

TABLE 2

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.

In some embodiments, compounds of Formula I or Formula II or Formula III above may be 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, breast cancer, acne, hirsutism, ovarian cysts, polycystic ovary disease, precocious puberty and age-related macular degeneration. In some embodiments compounds of Formula I or Formula II or Formula III 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.

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 (Berge S. M. et al., /. Pharm. Set (1977) 66(1): 1-19). 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 may be 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 may be 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, Ν,Ν-dibenzylphenethylamine, 1 -ephenamine, Ν,Ν'-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.

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.

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.

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.

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.

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, 20 ^th 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 may be oily solutions for administration in the form of nasal drops, or as a gel.

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.

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.

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 judgement 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 may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be 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.

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, hair loss, breast cancer, ovarian cysts, polycystic ovary disease, precocious puberty and age-related macular degeneration. For example, compounds and all their different forms as described herein may be used as neoadjuvant (prior), adjunctive (during), and/or adjuvant (after) therapy with surgery, radiation (brachytherapy or external beam), or other therapies (eg. HIFU).

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. Systemic therapy that targets the AR will not likely cause major problems to other tissues since antiandrogens and androgen insensitivity syndrome are not fatal. Compounds as described herein may be administered to a subject. As used herein, a "subject" may be 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, hair loss, breast cancer, 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.

Definitions used include ligand-dependent activation of the androgen receptor (AR) by androgens such as dihydrotestosterone (DHT) or the synthetic androgen (R1881) used for research purposes. Ligand-independent activation of the AR refers to transactivation of the AR in the absence of androgen (ligand) by, for example, stimulation of the

cAMP-dependent protein kinase (PKA) pathway with forskolin (FSK).

Some compounds and compositions as described herein may interfere with a mechanism specific to ligand-dependent activation (e.g., accessibility of the ligand binding domain (LBD) to androgen) or to ligand-independent activation of the AR.

Various alternative embodiments and examples of the invention are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.

METHODS

Heterologous Expression of Androgen Receptor

The AR ligand binding domain was expressed and purified as previously described (Estebanez-Perpina, E. et al. Proc. Nat. Acad. Sci. USA(2007) 104:16074 - 16079). eGFP Cellular AR Transcription Assay (eGFP transcription assay IC50 (μΜ)) AR transcriptional activity was assayed as previously described (Tavassoli, P. et al. Prostate (2007) 67:416-426). Briefly, stably transfected eGFP-expressing LNCaP human prostate cancer cells (LN-ARR2PB-eGFP) containing an androgen responsive probasin- derived promoter (ARR2PB) were grown in phenol red free RPMI 1640 supplemented with 5% CSS. After 5 days, the cells were plated into a 96-well plate (35,000 cells/well) with O.lnM R1881 and increasing concentrations (0-100 μΜ) of compound. The cells were incubated for three days and the fluorescence was then measured (excitation 485 nm, emission 535nm). The viability of these cells has been assayed by MTS cell proliferation assay (CellTiter 961™ Aqueous One Solution Reagent, Promega™) according to the manufacturer's instructions.

MTS assay ( Cell Viability at 50 μΜ (MTS))

Cell viability was determined using the MTS cell proliferation assay following incubation with the compound (0-100 μΜ) over 72hr (CellTiter 961™ AQueous One Solution Reagent, Promega™). In brief, 20 μΐ of the reagent was added to cells in each well of the 96-well plate containing 100 μΐ of media and incubated for 2hr at 37.8°C in 5% C0 ₂ . The production of formazan was measured at 490 nm.

Biolayer interferometry

The direct interaction between small molecules and the AR was quantified by biolayer interferometry using an OctetRED™ (ForteBio™). Biotinylated AR-LBD (bAR- LBD) was produced in situ with AviTag technology™ (Tirat, A. et al. Int. J. Biol. Macromol. (2006) 39:66-76). The AviTag™ sequence (GLNDIFEAQKIEWHE) followed by a six residue glycine serine linker (GSGSGS) was incorporated at the N-terminus of the AR-LBD (669-919). E. coli BL21 containing both biotin ligase and AR-LBD vectors were induced with 0.5 mM IPTG in the presence of DHT and biotin at 16°C overnight. The bacteria were then lysed by sonication and the resulting lysate was purified by IMAC with Ni-NTA resin and cation exchange chromatography (HiTrap SP™).

Purified bAR-LBD (50 μg/ml) was bound to the super-streptavidin sensors over 50 min. at room temperature. The sensor was kept in assay buffer (20 mM HEPES, 150mM NaCl, 500μΜ TCEP, 500nM DHT, 1 % DMSO). In all experiments, a known AF2 interacting peptide was used as a control to confirm functionality of the bAR-LBD. AF2 Fluorescence Polarization Assay (FP peptide displacement assay)

Purified AR-LBD (2 μΜ) and FITC labeled SRC2-3 peptide (5 nM) were incubated with increasing concentrations of compound (0-200 μΜ) in 25 mM HEPES pH 7.5, 50 mM NaCl, 0.01 % NP40 and 2 μΜ DHT. The samples were incubated for 2 hours and then the FP was measured (excitation = 485 nm, emission = 530 nm). The experiments were conducted in triplicate and the means ± standard deviations are shown. The data was analyzed by nonlinear regression with the software GraphPad (Kenakin, T. P. Pharmacologic Analysis of Drug-Receptor Interaction. Lippincott Williams & Wilkins 1997) and fit using the following equation.

Top— Bottom

B ottom +

1 + ioVOSiC _{3 c} -X)-HiiiSiape

Kj values were calculated with the Kenakin Equation (Tirat, A. et al. Int. J. Biol. Macromol. (2006) 39:66-76). i(LL)[(/q = _c .XK _d )

Where, ¾ = Equilibrium dissociation constant; L _b = Bound Tracer Concentration; Lo = Total Tracer Concentration; Ro = Total Receptor Concentration.

Androgen Displacement Assay (IC50 (μΜ) ligand displacement)

Androgen displacement was assessed with the PolarScreen™ Androgen Receptor Competitor Green Assay Kit as per the manufacturer's instructions (Invitrogen™ Catalog # P3018).

Protein expression, purification, crystallization and data collection

The LBD of human AR containing amino acid residues 663-919 was expressed as a glutathione S-transferase (GST) fusion protein in E. coli BL21 (DE3) cells, which were grown in 2-YT medium at 18°C. Testosterone (200 μΜ) was added into cell culture medium before induction with 100 mM isopropyl-b-D-thiogalactoside (IPTG). The fusion protein was purified by glutathione-sepharose affinity chromatography and subsequently cleaved with thrombin. The protein was further purified by cation exchange chromatography. In order to stabilize the AR-LBD, all solutions used for purification contained 50 μΜ testosterone.

The binary complex of AR-LBD and testosterone was crystallized using the sitting drop vapour-diffusion method at 294 K. The protein sample contained 3 mg/ml AR-LBD, 50 μΜ testosterone, 50 mM NaCl, 70 mM Li ₂ S0 ₄ , 0.1 % n-octyl-beta-glucoside, 40 mM Tris-HCl, pH 7.5. The well solution contained 0.35 M Na ₂ HP0 ₄ /K ₂ HP0 ₄ , 0.1 M (NH ₄ ) ₂ HP0 ₄ , 7.0 % PEG 400, 50 mM Tris-HCl, pH 7.5. Crystals were selected and then soaked in 8.0 mM of compound VPC-0061.

Single crystals were flash frozen in liquid nitrogen after soaking with the compound for 16 hours. X-ray diffraction data sets were collected using beam line 5.0.3 at the Lawrence Berkeley National Laboratory Advanced Light Source. Data sets were processed with iMosflm™. The best data set collected had 98% completeness at 2.2 A resolution. Crystal space group is P212121 with unit cell parameters of a=55.9, b=66.2, c=72.9.

Structure Solution and Refinement

The ternary complex structure was solved by molecular replacement using Phaser (McCoy, A. J. et al. J. Appl. Cryst. (2007) 40:658-674).

The coordinate of the AR-LBD-testosterone complex (Protein Data Bank™ entry, 2AM9) was used as the search model; however, with testosterone removed. The structure was refined to 2.2 A resolution using Refmac™ (Murshudov, G. N. et al. Acta cryst. Sec. D, Biological crystallography (1997) 53:240-255). The extra density of testosterone was clearly observed at the initial refinement step. A characteristic electron density of the compound was observed at the AF2 binding site.

The compound VPC-0061 was fitted according to the electron density map using the COOT program (Emsley, P. et al. Acta Crystallographica Sec D (2010) D66:486-501). As the compound binding is quite flexible, its occupancy was set as 0.5 during the refinement. The free R factor and R factors of the final mode of the ternary complex of are 25.9% and 21.1%, respectively, with good stereochemistry (TABLE 7).

All crystallographic experiments have been carried out as contract research by the Structure-Based Design (Structure Based Design, Inc.™ San Diego, CA ).

Cell Proliferation Assay LNCaP and PC3 cells were seeded into 96-well black plates (NalgeNunc

International™, Rochester, NY) using 5% CSS, phenol red-free RPMI medium (Gibco™, Life Technologies, Inc.™, Gaithersburg, MD) at cell density of 5,000 cells per well. After 24 hours of growth, cells were treated with a combination of R1881 (0.1 nM) and various concentrations of small molecule inhibitors (0-12 μΜ) for 6 days. Media and compounds were replaced with fresh components after 3 days. Cell numbers were determined at the termination of each experiment (Day 6) using the MTS cell proliferation assay (CellTiter 961™ AQueous One Solution Reagent, Promega™). 20 ml of the reagent was added each well of the 96-well plate containing 200 μΐ media and incubated for 2 hr at 37.8 °C in 5% C0 ₂ . Light absorbance of formazan was measured at 490 nm on a 96-well plate reader equipped with KC4™ software (Biotek Instruments, Inc.™, Winooski, VT).

After a series of in silico virtual screening steps for AF2 binders, numerous compounds were identified as candidates and were purchased and tested experimentally as described below. Of the compounds identified in the in silico virtual screen, 18 compounds were found to have over 36% inhibition in a Fluorescence Polararization (FP) assay for peptide displacement, wherein the compounds demonstrated an ability to displace the co- activator peptide (SRC2-3) from the target AF2 site.

EXAMPLES

EXAMPLE 1 - eGFP Cellular AR Transcription Assay (IC ₅₀ (μΜ))

Following in silico virtual screening, compounds were selected for further evaluation, for their ability to inhibit AR cellular transcriptional activity in vitro using a non-destructive cell-based eGFP AR transcriptional assay as described above. In this experimental model, these compounds were able to effectively inhibit cellular AR transcription with IC5 ₀ values in the low micro-molar range (TABLE 3).

TABLE 3 eGFP Cellular AR Transcription Assay (eGFP transcription assay IC ₅₀ (μΜ))

7006 0.19 7031 1.4 7056 (4021) 11.1

7007 51.48 7032 N/A 7057 1.2

7008 1.5 7033 0.5 7058 N/A

7009 1.5 7034 N/A 7059 N/A

7010 0.1 7035 N/A 7060 8.4

7011 1.3 7036 N/A 7061 N/A

7012 4.3 7037 N/A 7062 N/A

7013 7 7038 6 7063 4.7

7014 N/A 7039 N/A 7064 N/A

7015 N/A 7040 N/A 7065 26.7

7016 N/A 7041 N/A 7066 N/A

7017 (4023) 17.95 7042 62 7067 N/A

7018 N/A 7043 N/A 7068 N/A

7019 N/A 7044 N/A 7069 N/A

7020 N/A 7045 N/A 7070 1.4

7021 N/A 7046 N/A 7071 200

7022 6.7 7047 1 7072 2.6

7023 2 7048 N/A 7073 N/A

7024 N/A 7049 N/A

EXAMPLE 2 - Fluorescence Polararization (FP) assay for peptide displacement

Compounds were also tested to determine their ability to prevent co-activator (SRC2-3 peptide) interactions with the androgen receptor in vitro. TABLE 4 below shows 17 compounds that have an inhibition of > 36% (i.e. 0061, 0100, 4107, 7004, 7007, 7009, 7017, 7022, 7038, 7041, 7042, 7050, 7051 , 7053, 7056, 7065, and 7071). Of these 14 compounds show inhibition > 50% (i.e. 0061 , 0100, 4107, 7004, 7007, 7009, 7017, 7038, 7042, 7050, 7051, 7056, 7065, and 7071). Figures 5A, 5B, 6A and 6B also show percent inhibition as compared to a control for compounds 7001 , 7002, 7009, 7022, 7004, 7007, 7050, 7051 , 7056, 7065, and 7071.

TABLE 4 - Percent inhibition (%) by compound in FP assay (grey highlighting is

4107 72 7028 0.252525 7053 40.15152

7004 57.61124 7029 1.767677 7054 13.63636

7005 9.133489 7030 3.787879 7055 3.535354

7006 8.665105 7031 3.535354 7056 (4021) 70.75

7007 85.2459 7032 16.91919 7057 4.292929

7008 29.74239 7033 5.050505 7058 35.10101

7009 50.81967 7034 11.11111 7059 25.75758

7010 19.43794 7035 8.838384 7060 6

7011 19.20375 7036 15.65657 7061 3.118503

7012 10.30445 7037 28.0303 7062 24.11642

7013 12.64637 7038 72.9798 7063 9.355509

7014 4.449649 7039 11.36364 7064 12.26611

7015 11.7096 7040 0.757576 7065 89.39709

7016 5.30303 7041 36.86869 7066 3.534304

7017 (4023) 70 7042 62.12121 7067 17.46362

7018 7.323232 7043 3.282828 7068 14.34511

7019 5.808081 7044 23.73737 7069 23.07692

7020 19.94949 7045 24.24242 7070 16.21622

7021 14.39394 7046 20.45455 7071 64.86486

7022 44.94949 7047 3.282828 7072 19.33472

7023 30.30303 7048 7.575758 7073 24.74012

7024 19.69697 7049 13.88889

7025 7.828283 7050 (4040) 72

Similarly, compounds were initially tested at 50 and 100 μΜ concentrations and 13 compounds were effective at interfering with DHT binding in the 8-35 μΜ range (TABLE 5).

TABLE 5 - Pe tide Dis lacement Assa (IC ₅₀ ( Μ) e tide dis lacement)

7007 14.7 7033 N/A 7072 N/A

7008 N/A 7034 N/A 7061 N/A

7009 >200 7035 N/A 7073 N/A

7010 N/A 7036 N/A 0100 26

7011 N/A 7037 N/A 0061 30.5

7012 N/A 7038 27.62 4023 30.7

7013 N/A 7039 N/A 4040 32.9

7014 N/A 7040 N/A 4021 31.5

7015 N/A 7041 N/A 4107 8.2

7016 N/A 7042 62 7056 33.47

7017 30.7 7043 N/A 7057 N/A

7018 N/A 7044 N/A 7058 N/A

7019 N/A 7045 N/A 7059 N/A

7020 N/A 7046 N/A 7060 N/A

7021 N/A 7047 N/A 7061 N/A

7022 167 7048 N/A 7062 N/A

7023 N/A 7049 N/A 7063 N/A

7024 N/A 7050 89.42 7064 N/A

7025 N/A 7051 28.68 7065 N/A

7026 N/A 7052 N/A 7066 N/A

7027 N/A 7053 N/A 7067 N/A

7028 N/A 7054 N/A 7068 N/A

7029 N/A 7055 N/A

EXAMPLE 3 - Androgen Displacement Assay (IC ₅₀ (μΜ) ligand displacement)

Compounds were tested for their ability to displace hormone DHT from the androgen receptor (AR) in vitro to demonstrate that compounds did not interfere with the hormone binding site of the AR, an androgen displacement assay was used. The compounds did not exhibit any detectible hormone displacement when tested at 10 μΜ, suggesting that the mechanism of action is not due to conventional androgen displacement (data not shown). EXAMPLE 4 - MTS assay (% Cell Viability at 50 μΜ (MTS))

To rule out any non-specific inhibition of the AR transcriptional activity general cytotoxicity was tested using an MTS assay for cell viability as set out above. Many of the compounds demonstrated no detectible effect on cell viability when administered at a concentration of 50μΜ for over 72 hour. Compounds 7004, 7007, 7009, 7017, 7022, 7038, 7041, 7042, 7051, 7071, 0100 decreased cell viability, MTS assay as shown in TABLE 6.

TABLE 6 Shows a summary of the results for the compounds listed in TABLE 2 and some additional com ounds with reduced activit for com arison ur oses.

7051 59.34343 28.68 105.435 2.018 2.5 60.979 58.8

65

7052 15.90909 N/A 110.629 2.162 98.836 46.6

43

7053 40.15152 >200 110.177 3.549 97.135 57.5

88

7054 N/A 98.460 3.454 94.397 57.4

13.63636 68

7055 N/A 113.840 2.489 6.9 91.157 55.4

3.535354 02

7056 70.75 33.47 108.111 3.298 11.1 137.789 30.2

07

7057 4.292929 N/A 106.470 1.257 1.2 88.942 68.1

01

7065 89.39709 17.15 93.938 4.077 26.7 146.674 33.0

23

7067 17.46362 N/A 110.123 3.162 99.946 42.1

23

7071 64.86486 >200 111.316 2.533 87.799 17.9

51

7072 N/A 103.488 1.392 84.308 31.9

19.33472 75

7073 N/A 114.480 1.748 65.430 29.5

24.74012 34

100 59 26 88.0 5 33.4 50.9 14.2

61 75 30.5 100.3 4 23.2 120.8 2.2

4023 82 30.7 92.8 1 5.9 106.1 5.0

4040 88 32.9 72.6 0 4.6 129.0 3.3

4021 88 31.5 97.9 2 11.1 121.7 8.0

4107 72 8.2 70.9 1 14.42 144.9 6.2

EXAMPLE 5 - Biolayer Interferometry (BLI)

To further confirm that there was a direct interaction between the compounds and the AR, Biolayer Interferometry (BLI) was conducted 6 of the compounds. Figure 2 (column 3) shows BLI data obtained for VPC-4107, VPC-0100, and VPC-0061, demonstrating a direct interaction between the AR and these small molecules. It should be noted that none of the BLI tested compounds could be fit with a simple 1:1 model at higher concentrations, suggesting that there may be multiple sites of binding at higher concentrations. EXAMPLE 6 - Crystallographic structure of AR in complex with AF2 inhibitor.

To confirm that the identified compounds bind directly to the AR AF2 site, crystallographic soaking experiments with compound VPC-0061 was conducted. Following optimization of the soaking protocol, the structure of the AR in complex with VPC-0061 was determined to a 2.3A resolution (TABLE 7). In the resolved structure, VPC-0061 was found to bind directly to the AF2 with a good structural fit in the target cavity. Importantly, the experimentally determined configuration of the AF2-bound molecule turned out to be very similar to the predicted docking pose (RMSD=1.1 A; Figure 3 A) giving confidence to the in silico protocol used to identify potential compounds.

The structure of the AR/VPC-0061 complex revealed that structural changes occurred in the AF2 site as compared to its previously published structure containing K10 ligand (PDB code 2PIP conducted (Estebanez-Perpina, E. et al. Proc. Nat. Acad. Sci. USA (2007) 104: 16074 - 16079)). As Figure 3 illustrates, there is a substantial repositioning of Lys720 side chain towards the benzene ring of VPC-0061, which results in arene-cation conjugation (see Figure 3B). In addition, Met734 side chain in the resolved complex is pushed away from the site (compared to other AF2 structures), which is likely caused by repulsion with VPC-0061.

A number of the tested compounds are effective AR binders (as confirmed by the BLI analysis) which can inhibit AR transcriptional activity (demonstrated by the cell-based assay) without interfering with its hormone binding site like the conventional anti- androgens (confirmed by the androgen displacement assay).

For example, inhibiting the AR by targeting co-activator interactions may offer several advantages over conventional anti-androgens for the treatment of advanced prostate cancer. First, many of the mechanisms of drug resistance that arise in castration resistant prostate cancer such as point mutations at the ligand binding sites would not affect the efficacy of an AF2 inhibitor. In addition, these mutations would not convert an AF2 inhibitor from antagonist to agonist and inadvertently increase the growth of the cancer as has been observed with anti-androgens (Culig, Z. et al. British J Cancer (1999) 81(2):242- 51). Furthermore, as all anti-androgens are structurally very closely related, they cannot be used in combination. By targeting a different site on the AR, an AF2 inhibitor could be taken concurrently with anti-androgens. This could potentially be useful in decreasing the time to cancer remission in prostate cancer patients. Similar to highly active anti-retroviral therapy, by taking several therapeutics concurrently, some with the same target, the rate of drug resistance may be reduced, which could in turn lead to an improvement in the survival rate of patients with advanced cancer.

The compounds described herein are potent inhibitors of both AF2 co-activator binding and cellular AR transcriptional activation (Figure 2). Importantly, the mechanism of action of these small molecule inhibitors has been demonstrated through BLI (Figure 2) and X-ray crystallography (Figure 3).

The identified structures offer two novel classes of molecular scaffolds capable of inhibiting protein-protein interaction, which is seen as a notoriously difficult drug design target (Arkin, M. R. and Wells, J. A. Nat. Rev. Drug Disc. (2004) 3:301-317). The identified AF2 binders belong to two distinctive types: derivatives of 2, 3-dihydro-lH- perimidine (compounds 4021 , 4023, 4040, 4107 and 0061 in TABLES 1 and 2), and a substituted lH-pyrazol-5-(4H)-one (compound VPC-0100). According to the crystal structure, VPC-0061 binds to the AF2 site in an orientation previously predicted by docking (Figure 3A) and maintains close hydrophobic contacts with the floor of the site constituted of Val730, Met734 and Val737 residues. One of the hydroxyl groups attached to a benzene ring of the VPC-0100 forms a weak hydrogen bond with Val730 backbone carbonyl group, while the ring itself engages into arene-cation conjugation with a charged nitrogen of Lys720 significantly stabilizing the protein-ligand complex.

The analysis of the docking poses of other compounds inside the AF2 site illustrates possible formation of hydrogen bonds with the residues of "charge-clamp" regions. Previous studies have demonstrated that this "charge-clamp" may be significant for co- activator binding (Zhou, X. E. et al. J. Biol. Chem. (2010) 285:9161-9171). From our docking models, 4107 is likely to be anchored by two hydrogen bonds with the Lys720 side chain (shown on Figure 4A), while a closely related molecule VPC-4040 is slightly turned away from Lys720 and forms only one hydrogen bond with Glu733 (Figure 4B). This may account for its lowered peptide displacement potential (IC5 ₀ = 32.9 μΜ versus IC5 ₀ = 8.2 μΜ for VPC-4107). Compounds 4021 and 4023, similarly to 4040, both carry a para- hydroxyl substituent and form similar single hydrogen bond with the Glu733 residue (VPC- 4023shown on Figure 4C). Notably, a structurally different compound,VPC-0100 adopts a different predicted binding mode inside the AF2, whereby it directly anchors to theGlu893 and Glu897 residues through short 2.2A hydrogen bonds (Figure 4D). The above considerations indicate that the identified chemicals are likely to interact with the key residues Lys720, Glu893 and Glu897 that belong to a "charged clamp" region of the AF2 site responsible for direct anchoring of AR co-activators. This distinguishes compounds VPC-4021, VPC-4023, VPC-4040, VPC-4107 and VPC-0061 from other crystallographically confirmed AF2 binders including 3,3' ,5-triiodo thyroacetic acid, 1- tertbutyl-, thriiodothyronine, 3-((l-tert-butyl-4-amino-lHpyrazolo[3,4-D]pyrimidin-3- yl)methyl)phenol and 3 -(2,5 -dimethyl-benzyl)- 1 H-pyrazolo [3 ,4-D]pyrimidin-4-ylamine (Estebanez-Perpina, E. et al. Proc. Nat. Acad. Sci. USA (2007) 104: 16074 - 16079), which do not make these important contacts within the target site. This may explain why other crystallographically confirmed AF2 binders are unable to efficiently prevent SRC co- activator peptide binding to the AR AF2 (IC5 ₀ >50μΜ (Estebanez-Perpina, E. et al. Proc. Nat. Acad. Sci. USA (2007) 104: 16074 - 16079)).

It should be noted that the details presented in this study of the protein-ligand interactions derived from the experimentally resolved and predicted binding poses of the AF2 inhibitors are overall in good agreement with the measured AF2 binding potentials. It should also be emphasized that our peptide displacement assay directly quantifies interactions between the chemicals and the AR AF2 site and, thus, provides first hand evidence for binding. Hence, the described AF2-directed inhibitors can be viewed as promising new drug candidates capable of the direct targeting of co-activation of the AR, rather than disrupting its co-activating interactions through indirect induced conformational changes eluted by androgen replacement at the ligand binding site.

TABLE 7. Data collection and refinement statistics.

Data collection

Space group P2 ₁ 2 ₁ 2 ₁

Cell dimensions

a, b, c (A) 57.53 66.275 73.68

α, β, γ (°) 90.0, 90.0, 90.0

Resolution (A) 2.2

^sym Or ?merge 0.089

Completeness (%) 98.8

Redundancy 5.2

Refinement

Resolution (A) 2.2

No. reflections 12805

^work / ^free 21.1/25.9

No. atoms

Protein 2071

Lig and/ion 42/1 Water 23

R.m.s. deviations

Bond lengths (A) 0.023

Bond angles (°) 1.8

Ramachandran statistics (%)

Core region 93.0

Additional allowed region 6.6

Disallowed 0.4

¾ _work and ¾ _ee =∑ _h ll F ₀ (h) - F _c (h) ll/∑ _h llfor the working set and test set (5%) of reflections, where the F ₀ (h) and F _c (h) are the observed and calculated structure factor amplitudes for reflection

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. Any priority document(s) and all publications, including but not limited to patents and patent applications, cited in this specification are incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.