Table 1.

Initial Biological Evaluation of Indole AR Analogues

ID	Structure	Binding (Ki)/Transactivation (IC50) (μM)		SARD Activity (% degradation)		Metabolic Stability in Mouse Liver Microsomes (MLM)f
		Ki (DHT = 1 nM)a	IC50b	Full Lengthc,d (LNCaP)@1μM	Splice Variantc (22RVl)@10μM	T1/2 (min)	CLint (mL/min/mg)
8 13		0.267	0.085	76	87	12.35	5.614
19a 13		0.547	0.157	32	69	21.77	3.184
19b		0.332	0.045	68	62	9.29	7.46
20		0.757	0.027	57, 97d	N/Ae	14.6	4.70
26		>10	>10	0	0	N/Ae	N/Ae

^aAR binding was determined by competitive ligand binding, as described.^13,21,23,24 The recombinant ligand binding domain (LBD) of wildtype AR was incubated with 1 nM [³H]-mibolerone and the candidate SARD compound (1 pm – 100 μM) or 1 nM DHT; following incubation, the complex was precipitated with hydroxyapatite and washed, and the bound radioactivity was eluted and quantified on a scintillation counter. SigmaPlot software was used to determine the resulting inhibitory constant (K_i) for each compound relative to 1 nM DHT.

^bInhibition of AR transactivation was determined by transfecting human embryonic kidney HEK-293 cells with full-length wildtype AR, GRE-LUC, and CMV-renilla luciferase as a transfection control. Twenty-four h later, the cells were treated for 24 h with 0.1 nM agonist R1881 or varying doses of the indicated antagonist compounds (1 pM to 10 μM in log units). After incubation, the cells were collected, lysed, and analyzed by luciferase assay, as described.^13,21

^cSARD activity was determined as a function of AR protein levels in cultured cells, as described.^13,21,22 Cells used were of the androgen-sensitive human prostate adenocarcinoma cell line LNCaP that expresses full-length (FL) AR or the xenograft-derived human prostate carcinoma epithelial cell line 22RV1 that expresses a c-terminally truncated splice variant (SV) of AR (AR-V7). Cells were treated for 24 h with the indicated doses of the candidate antagonist compounds in the presence of 0.1 nM agonist R1881. Cells were harvested, lysed, and FL or SV AR was detected by immunoblot with AR-N20 antibodies directed against the AR N-terminal domain (NTD). Blots were stripped and reprobed with antibodies specific for the cellular protein actin as an internal control. FL and SV AR species were quantified and normalized to actin signals.

^dSARD activity of compound 20 against FL AR was evaluated in LNCaP cells at 10 μM concentration.

^eSARD activity of compound 20 against SV AR in 22RV1 cells was not available (N/A).

^fThe metabolic stability (half-life, hepatic clearance) of each compound was determined by incubation for varying time periods with mouse liver microsomes (MLM) and cofactors for phase I and phase II metabolism, as described in the Experimental section in Supporting Information. T_1/2 (h) after oral administration in humans as previously reported.¹³ CL (mL/h/kg) after oral administration in humans as previously reported.^23,24