Identification of Novel Steroidal Androgen Receptor Degrading Agents Inspired by Galeterone 3β-Imidazole Carbamate

Abstract

Degradation of all forms of androgen receptors (ARs) is emerging as an advantageous therapeutic paradigm for the effective treatment of prostate cancer. In continuation of our program to identify and develop improved efficacious novel small-molecule agents designed to disrupt AR signaling through enhanced AR degradation, we have designed, synthesized, and evaluated novel C-3 modified analogues of our phase 3 clinical agent, galeterone (5). Concerns of potential in vivo stability of our recently discovered more efficacious galeterone 3β-imidazole carbamate (6) led to the design and synthesis of new steroidal compounds. Two of the 11 compounds, 3β-pyridyl ether (8) and 3β-imidazole (17) with antiproliferative GI50 values of 3.24 and 2.54 μM against CWR22Rv1 prostate cancer cell, are 2.75- and 3.5-fold superior to 5. In addition, compounds 8 and 17 possess improved (∼4-fold) AR-V7 degrading activities. Importantly, these two compounds are expected to be metabolically stable, making them suitable for further development as new therapeutics against all forms of prostate cancer.

Keywords: Androgen receptor (AR); androgen receptor degrading agents (ARDAs); prostate cancer; splice variant AR (AR-V7).

Figure 1

Chemical structures of compounds 1–5 (clinical anti-PC agents) and selective ARDAs (6–7).

Scheme 1. Synthesis of Androstene Derivatives (8–13, 15–17)

Reagents and conditions: (i) NaH, DMF, R-X, rt or heat, 1–12 h; (ii) 1,1′-thiocarbonyldiimidazole, CH₃CN, DCM, reflux, 5 h; (iii) 1,1′-carbonyldiimidazole, CH₃CN, rt, 2 h; (iv) dry heat at 195 °C, 1 h; (v) mesyl chloride, pyridine, ice (5 h), then rt (5 h); (vi) toluene, imidazole, reflux, overnight.

Scheme 2. Synthesis of Estrogen-3-hydroxy (22) and 3-Carboxyl Derivative (28)

Reagents and conditions: (i) pyridine, acetic anhydride, rt, 12 h, (ii) PBr₃, DMF, CHCl₃, reflux, 5 h; (iii) ethanol, 10% ethanolic-KOH, rt, 12 h; (iv) BzIm, K₂CO₃, DMF, 80 °C, 1–5 h; (v) benzonitrile, 10% Pd/C, 185 °C, 12 h; (vi) triflic anhydride, TEA, DCM, 0 °C, 1 h; (vii) palladium(II) acetate, dppp, TEA, MeOH, DMF, carbon monoxide, 0 °C, 9 h; (viii) MeOH, 10% methanolic-KOH, reflux, 2 h.

Figure 2

(A) Effects of compounds at 10 μM on dihydrotestosterone (DHT)-stimulated transcription of AR. LNCaP cells were transfected with the AAR2 reporter construct + the Renilla luciferase reporting vector pRL-null and treated with novel compounds for 24 h in the presence of 10 nM dihydrotestosterone (DHT). Control, baseline activity without androgen stimulation. Androgen-stimulated luciferase activity (luminescence) was measured in a Victor 1420 plate reader. The results are presented as the fold induction (i.e., the relative luciferase activity of the treated cells divided by that of the control) normalized to that of the Renilla luciferase activity. *, P < 0.01; ^#, P < 0.05 compared with DHT treated cells. (B) Inhibitory effects of ARDAs/MDV3100 on DHT induced AR transcriptional activity in LNCaP cells. EC₅₀ = the concentration of inhibitor (ARDAs/MDV3100) required to inhibit the DHT-induced AR transcriptional activity by 50%. EC₅₀ values of the compounds were determined from dose–response curves. Points, mean of replicates from three independent experiments; bars, SE. Solid line, best-fit sigmoidal dose response (variable slope).

Figure 3

(A–D) Differential effect of compounds on suppressing AR expression in LNCaP and CWR22Rv1 prostate cancer cells: Western blot analysis of f-AR/AR-V7 expression in LNCaP or CWR22Rv1 cells treated with various compounds. Cells were exposed to individual compounds (15 μM) for 24 h, and the protein lysates were subjected to Western blot analysis. (E) EC₅₀ values (for fAR and AR-V7) for compounds were determined from dose–response curves following compound treatments (0 → 7.5 μM) of CWR22Rv1 cells for 72 h followed by Western blot analysis of lysates.