Supporting Information

S1

Supporting Information

A New Generation of Selective Androgen Receptor Degraders: Our Initial

Design, Synthesis, and Biological Evaluation of New Compounds with

Enzalutamide-Resistant Prostate Cancer Activity

Dong-Jin Hwang,† _{Yali He,}† _{Suriyan Ponnusamy,}‡ _{Michael L. Mohler,}† _{Thirumagal Thiyagarajan,}‡ Iain J. McEwan, _{Ramesh Narayanan,}‡ _{Duane D. Miller}*,†

†_{Department of Pharmaceutical Sciences, University of Tennessee Health Science Center,} Memphis, TN 38163. ‡_{Department of Medicine, University of Tennessee Health Science Center,} Memphis, TN 38103. # _{GTx, Inc., Memphis, TN 38103.} & _{School of Medicine, Medical Sciences} and Nutrition, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK.

Additional information on information on compound characterization; additional biological experiments and figures (PDF)

Molecular formula strings and some associated data (CSV)

Table of Contents

1. Analytical data for Indolyl CF3 A-ring Series S2

2. Analytical data for Indolyl Cl A-ring Series S49 3. Analytical data for Indolinyl CF3 A-ring Series S73

4. Analytical data for Indolinyl Cl A-ring Series S96 5. Figure S1. Weak GR antagonism of transactivation S121

6. Figure S2. SARDs bind to AF-NTD and AF-1 regions of AR S123 7. Figure S3. Selectivity of degradation: No ER degradation S124

S2

Indolyl Series II-1 (18a ~ 18q)

(S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-3-(1H-indol-1-yl)-2-methylpropanamide (18a) Compound 18a: HRMS Analysis ([M + H] +_{: 388.1270)}

S3 Compound 18a: 1H NMR (400 MHz) Analysis

S4 Compound 18a: UV Analysis

S5 (S)-N-(4-cyano-3-(trifluoromethyl)phenyl)-3-(4-fluoro-1H-indol-1-yl)-2-hydroxy-2-methylpropanamide (18b)

S6 Compound 18b: HRMS Analysis ([M + H] +_{: 406.1171)}

S7 Compound 18b: LC Analysis (Purity: 96.01%)

S8 (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(6-fluoro-1H-indol-1-yl)-2-hydroxy-2-methylpropanamide (18c)

S9 Compound 18c: HRMS Analysis ([M + H] +_{: 406.1179)}

S10 Compound 18c: LC Analysis (Purity: 95.49%)

S11 (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methyl-3-(4-nitro-1H-indol-1-yl)propanamide (18d)

S12 Compound 18d: LC Analysis (Purity: 95.92%)

S13 Compound 18d: 1H NMR (400 MHz) Analysis

S14 (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methyl-3-(5-nitro-1H-indol-1-yl)propanamide (18e)

S15 Compound 18e: LC Analysis (Purity: 98.42%)

S16 Compound 18e: 1H NMR (400 MHz) Analysis

S17 (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methyl-3-(6-nitro-1H-indol-1-yl)propanamide (18f)

S18 Compound 18f: 1H NMR (400 MHz) Analysis

S19 Compound 18f: 2D NMR (1_H-1_{H, COSY) Analysis}

S20 (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(5-fluoro-3-methyl-1H-indol-1-yl)-2-hydroxy-2-methylpropanamide (18g)

S21 Compound 18g: 1H NMR (400 MHz) Analysis

S22 Compound 18g: LC Analysis (Purity: 97.20%)

S23 (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(5-fluoro-6-phenyl-1H-indol-1-yl)-2-hydroxy-2-methylpropanamide (18i)

S24 Compound 18i: UV Analysis

S25 (S)-3-(5-Bromo-1H-indol-1-yl)-N-(4-cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide (18j)

S26 Compound 18j: 1H NMR (400 MHz) Analysis

S27 Compound 18j: 2D NMR (1_H-1_{H, COSY) Analysis}

S28 (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-3-(5-iodo-1H-indol-1-yl)-2-methylpropanamide (18k)

S29 Compound 18k: 1H NMR (400 MHz) Analysis

S30 Compound 18k: 2D NMR (1_H-1_{H, COSY) Analysis}

S31 (S)-1-(3-((4-Cyano-3-(trifluoromethyl)phenyl)amino)-2-hydroxy-2-methyl-3-oxopropyl)-1H-indole-3-carboxylic acid (18l)

S32 Compound 18l: LC Analysis (Purity: 96.12%)

S33 Compound 18l: 1H NMR (400 MHz) Analysis

S34 (S)-Ethyl 1-(3-((4-cyano-3-(trifluoromethyl)phenyl)amino)-2-hydroxy-2-methyl-3-oxopropyl)-1H-indole-3-carboxylate (18m)

S35 Compound 18m: HRMS Analysis ([M + H] +_{: 460.1484)}

S36 Compound 18m: 2D NMR (1_H-1_{H, COSY) Analysis}

S37 (S)-3-(5-Cyano-1H-pyrrolo[3,2-b]pyridin-1-yl)-N-(4-cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide (18n)

S38 Compound 18n: 1H NMR (400 MHz) Analysis

S39 Compound 18n: LC Analysis (Purity: 95.58%)

S40 (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methyl-3-(4-phenyl-1H-indol-1-yl)propanamide (18o)

S41 Compound 18o: 1H NMR (400 MHz) Analysis

S42 Compound 18o: LC Analysis (Purity: 95.58%)

S43 (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(4-fluoro-5-phenyl-1H-indol-1-yl)-2-hydroxy-2-methylpropanamide (18p)

S44 Compound 18p: LC Analysis (Purity: 98.81%)

S45 Compound 18p: UV Analysis

S46 (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(4-fluoro-6-(4-fluorophenyl)-1H-indol-1-yl)-2-hydroxy-2-methylpropanamide (18q) Compound 18q: HRMS Analysis ([M + H] +_{: 500.1397)}

S47 Compound 18q: 1H NMR (400 MHz) Analysis

S48 Compound 18q: LC Analysis (Purity: 98.81%)

S49

Indolyl Series II-2 (19a ~ 19g)

(S)-N-(3-Chloro-4-cyanophenyl)-3-(3-fluoro-1H-indol-1-yl)-2-hydroxy-2-methylpropanamide (19a) Compound 19a: UV Analysis

S50 Compound 19a: 1H NMR (400 MHz) Analysis

S51 Compound 19a: 19F NMR (decoupled) Analysis

S52 (S)-N-(3-Chloro-4-cyanophenyl)-3-(4-fluoro-1H-indol-1-yl)-2-hydroxy-2-methylpropanamide (19b)

S53 Compound 19b: 1H NMR (400 MHz) Analysis

S54 Compound 19b: 2D NMR (1_H-1_{H, COSY) Analysis}

S55 Compound 19b: 19F NMR (decoupled) Analysis

S56 (S)-N-(3-Chloro-4-cyanophenyl)-3-(5-fluoro-1H-indol-1-yl)-2-hydroxy-2-methylpropanamide (19c)

S57 Compound 19c: 1H NMR (400 MHz) Analysis

S58 Compound 19c: 2D NMR (1_H-1_{H, COSY) Analysis}

S59 Compound 19c: 19F NMR (decoupled) Analysis

S60 (S)-N-(3-Chloro-4-cyanophenyl)-3-(6-fluoro-1H-indol-1-yl)-2-hydroxy-2-methylpropanamide (19d)

S61 Compound 19d: 1H NMR (400 MHz) Analysis

S62 Compound 19d: 19F NMR (decoupled) Analysis

S63 Compound 19d: 2D NMR (1_H-1_{H, COSY) Analysis}

S64 (S)-N-(3-Chloro-4-cyanophenyl)-3-(7-fluoro-1H-indol-1-yl)-2-hydroxy-2-methylpropanamide (19e)

S65 Compound 19e: 1H NMR (400 MHz) Analysis

S66 Compound 19e: 19F NMR (decoupled) Analysis

S67 Compound 19e: 2D NMR (1_H-1_{H, COSY) Analysis}

S68 (S)-N-(3-Chloro-4-cyanophenyl)-3-(5-fluoro-6-phenyl-1H-indol-1-yl)-2-hydroxy-2-methylpropanamide (19f)

S69 Compound 19f: 1H NMR (400 MHz) Analysis

S70 (S)-N-(3-Chloro-4-cyanophenyl)-3-(5-fluoro-3-phenyl-1H-indol-1-yl)-2-hydroxy-2-methylpropanamide (19g)

S71 Compound 19g: 1H NMR (400 MHz) Analysis

S72 Compound 19g: UV Analysis

S73

Indolyl Series III-1 (20a ~ 20g)

(S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-3-(indolin-1-yl)-2-methylpropanamide (20a) Compound 20a: HRMS Analysis ([M + H] +_{: 390.1429)}

S74 Compound 20a: 1H NMR (400 MHz) Analysis

S75 Compound 20a: LC Analysis (Purity: 99.23%)

S76 (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(4-fluoroindolin-1-yl)-2-hydroxy-2-methylpropanamide (20b)

S77 Compound 20b: 1H NMR (400 MHz) Analysis

S78 Compound 20b: 2D NMR (1_H-1_{H, COSY) Analysis}

S79 (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(5-fluoroindolin-1-yl)-2-hydroxy-2-methylpropanamide (20c)

S80 Compound 20c: 2D NMR (1_H-1_{H, COSY) Spectrum}

S81 (S)-3-(5-Bromoindolin-1-yl)-N-(4-cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide (20d)

S82 Compound 20d: 1H NMR (400 MHz) Spectrum

S83 Compound 20d: UV Spectrum

S84 (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(6-fluoroindolin-1-yl)-2-hydroxy-2-methylpropanamide (20e)

S85 Compound 20e: 1H NMR (400 MHz) Spectrum

S86 Compound 20e: 2D NMR (1_H-1_{H, COSY) Spectrum}

S87 Compound 20e: LC Analysis (Purity: 97.03%)

S88 (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(5,6-difluoroindolin-1-yl)-2-hydroxy-2-methylpropanamide (20f)

S89 Compound 20f: 1H NMR (400 MHz) Spectrum

S90 Compound 20f: 19F NMR (400 MHz) Spectrum

S91 Compound 20f: UV Spectrum

S92 Compound 20f: 2D NMR (1_H-1_{H, COSY) Spectrum}

S93 (S)-3-(5-Chloro-6-fluoroindolin-1-yl)-N-(4-cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide (20g)

S94 Compound 20g: 1H NMR (400 MHz) Spectrum

S95 Compound 20g: UV Spectrum

S96

Indolinyl Series III-2 (21a ~ 21f)

(S)-N-(3-Chloro-4-cyanophenyl)-3-(4-fluoroindolin-1-yl)-2-hydroxy-2-methylpropanamide (21a) Compound 21a: HRMS Analysis ([M + H] +_{: 374.1072)}

S97 Compound 21a: 1H NMR (400 MHz) Spectrum

S98 Compound 21a: LC Analysis (Purity: 96.28%)

S99 Compound 21a: UV Spectrum

S100 Compound 21a: 2D NMR (1_H-1_{H, COSY) Spectrum}

S101 Compound 21a: 19F NMR (400 MHz) Spectrum

S102 (S)-N-(3-Chloro-4-cyanophenyl)-3-(5-fluoroindolin-1-yl)-2-hydroxy-2-methylpropanamide (21b)

S103 Compound 21b: 1H NMR (400 MHz) Spectrum

S104 Compound 21b: 2D NMR (1_H-1_{H, COSY) Spectrum}

S105 Compound 21b: 19F NMR (400 MHz) Spectrum

S106

(S)-3-(5-Bromoindolin-1-yl)-N-(3-chloro-4-cyanophenyl)-2-hydroxy-2-methylpropanamide (21c) Compound 21c: HRMS Analysis ([M + H] +_{: 434.0271)}

S107 Compound 21c: 1H NMR (400 MHz) Spectrum

S108 Compound 21c: 2D NMR (1_H-1_{H, COSY) Spectrum}

S109 Compound 21c: UV Spectrum

S110 (S)-N-(3-Chloro-4-cyanophenyl)-3-(6-fluoroindolin-1-yl)-2-hydroxy-2-methylpropanamide (21d)

S111 Compound 21d: 1H NMR (400 MHz) Spectrum

S112 Compound 21d: LC Analysis (Purity: 98.22%)

S113 Compound 21d: 2D NMR (1_H-1_{H, COSY) Spectrum}

S114 (S)-N-(3-Chloro-4-cyanophenyl)-3-(5,6-difluoroindolin-1-yl)-2-hydroxy-2-methylpropanamide (21e)

S115 Compound 21e: 1H NMR (400 MHz) Spectrum

S116 Compound 21e: LC Analysis (Purity: 98.73%)

S117 Compound 21e: 2D NMR (1_H-1_{H, COSY) Spectrum}

S118 (S)-N-(3-Chloro-4-cyanophenyl)-3-(5-fluoro-6-phenylindolin-1-yl)-2-hydroxy-2-methylpropanamide (21f)

S119 Compound 21f: 1H NMR (400 MHz) Spectrum

S120 Compound 21e: LC Analysis (Purity: 96.38%)

S121

Figure S1. Weak GR and ER antagonism of transactivation.

A

COS cells were transfected with 0.25 µg GRE-LUC, 0.005 µg CMV-renilla LUC, and 25 ng pCR3.1 rat GR using lipofectamine. Twenty four hours after transfection, the cells were treated as indicated in the figure and luciferase assay was performed 24 hours after treatment. Firefly luciferase values were normalized with renilla luciferase numbers.

S122

B

19f and 19b do not inhibit GR and ER transactivation: HEK-293 cells were transfected with 25 ng pCR3.1 GR or ER, GRE-LUC or ERE-GRE-LUC, respectively, and CMV-renilla GRE-LUC. Cells were treated 24 hours after transfection and luciferase assay performed 48 hours after transfection.

S123

Figure S2. SARDs bind to AF-NTD and AF-1 regions of AR.

19b demonstrates a dose-dependent shift in the fluorescence of aromatic amino acids upon addition of the SARD compound, indicating that 19b binds to NTD and AF1 regions of the AR, which are regions of the N-terminal domain of the AR. This supports the generalizability of the binding affinity of II and III to a domain in the N-terminus of AR. Fluorescence polarization studies to determine the binding of the SARDs to AF-1 domain of the AR was conducted as published earlier in Cancer Res 77(22): 6282 (November 15, 2017).

S124

Figure S3. Selectivity of degradation: No ER degradation.

MCF-7 breast cancer cells were maintained in charcoal-stripped serum-containing medium for 2 days. The cells were treated with the indicated compounds and were harvested 24 hours after treatment. Western blot for ER and Actin was performed.

S125

Figure S4. SARDs function by destabilizing already expressed protein.

LNCaP cells maintained in growth medium were treated with cycloheximide (50 µM), 19f (10 µM), or combination. Cells were harvested at the indicated time-points and Western blot for AR and GAPDH was performed. The numbers under the blots are densitometric quantification of the blots and represented as fold from respective 0 hour time-point.

To validate that SARDs function by reducing the stability of protein (as opposed to decreasing rate of protein synthesis, e.g., lowering mRNA levels, etc.), we conducted an experiment with 19f in the presence of the general protein synthesis inhibitor, cycloheximide (Supplemental Figure S4). Treatment of LNCaP cells with 50 µM cycloheximide or 10 µM 19f did not significantly reduce the protein levels at the evaluated time-points. However, when the cells were treated with the two molecules together, significant down-regulation of the AR was observed. These results suggest that the SARDs function by destabilizing the AR protein that has already been

synthesized as opposing inhibiting protein synthesis. This agrees well with an earlier validation of 3 and 7 using very similar methodology.