Specific stereochemistry of OP-1074 disrupts estrogen receptor alpha helix 12 and confers pure antiestrogenic activity

Complex tissue-specific and cell-specific signaling by the estrogen receptor (ER) frequently leads to the development of resistance to endocrine therapy for breast cancer. Pure ER antagonists, which completely lack tissue-specific agonist activity, hold promise for preventing and treating endocrine resistance, however an absence of structural information hinders the development of novel candidates. Here we synthesize a small panel of benzopyrans with variable side chains to identify pure antiestrogens in a uterotrophic assay. We identify OP-1074 as a pure antiestrogen and a selective ER degrader (PA-SERD) that is efficacious in shrinking tumors in a tamoxifen-resistant xenograft model. Biochemical and crystal structure analyses reveal a structure activity relationship implicating the importance of a stereospecific methyl on the pyrrolidine side chain of OP-1074, particularly on helix 12.


ER-specific luciferase reporter gene (related to Supplementary
45,000 HeLa cells per well were transiently transfected using manufacturer's protocol for Lipofectamine LTX (ThermoFisher). HeLa cells were transfected with 3 ng of plasmids for either ERα or ERβ, and 85 ng ERE-3x-Luc, containing 3 copies of estrogen-response element (ERE) cloned into pGL4.23 luciferase reporter gene containing a minimal TATA promoter (Promega). Transfected cells were treated with OP-1074 in the presence of 100 pM E2 for 22 hours (1.25% total final charcoal dextran stripped FBS). Bright Glo luciferase reagent (Promega) was used to lyse cells and detect firefly luciferase activity, measured in relative light units, at 1 sec intervals using the Varioskan Lux multimodal plate reader. To rule out non-specific nuclear receptor activity, HeLa cells were transfected as above but also with plasmids for full length wild type glucocorticoid receptor (GR), progesterone receptor (PR), or androgen receptor (AR), along with PRE-3x-Luc, containing 3 copies of progesterone response element (

2-yl)oxy)phenyl)chroman-4-ol
To a solution of 90.0 % 2-(4-iodophenyl)-7-((tetrahydro-2H-pyran-2-yl)oxy)-3-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)chroman-4-one (234.0 g, 0.34 mol, 1.0 equiv.) in tetrahydrofuran (0.89 L) at -5 o C, was added methylmagnesium chloride (3.0 M solution in THF, 200 mL, 0.60 mol, 1.8 equiv.) by addition funnel over 30 minutes. The temperature was kept below 0 °C during the addition. After addition of the Grignard reagent was complete, the reaction was removed from the ice bath, allowed to reach room temperature, and stirred at room temperature overnight. TLC (20 % EA/Hex) indicated that the reaction was complete. The reaction was cooled to 0 o C and carefully quenched with saturated ammonium chloride (100 mL), the temperature was kept below 7 o C during the quench. The mixture was stirred at room temperature for 30 min before the solid was filtered through a bed of Celite. The solid was rinsed with DCM twice and the filtrate was dried over anhydrous sodium sulfate, filtered and concentrated. The resulting pale yellow foam was used directly in the next reaction without purification.
The combined aqueous layer was extracted with ethyl acetate (2 x 800 mL). The combined organic layer was washed with brine (2 x 400 mL), dried over anhydrous magnesium sulfate, filtered and the filtrate mixed with the red residue and concentrated to give a dark red residue.
The residue was adsorbed onto silica gel (375 g) and chromatographed on silica gel (7 x

3-(4-hydroxyphenyl)-4-methyl-2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-2H-chromen-7-ol (OP-1040)
To a cooled ( After completion of addition, the reaction mixture was slowly allowed to reach room temperature and stirred for 6 h. Then reaction mixture was cooled to 0 0 C, quenched with ammonium chloride solution (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under vacuum to isolate the crude methyl adduct as yellow oil. The oil was taken in acetic acid (9 mL), water (1 mL) and and pressurized to 97 psi and left to shake for 1.5 days. TLC analysis indicated partial reduction had occurred. Hydrochloric acid (1 M, 5.5 mL) was added to mixture and the flask was flushed with hydrogen and repressurized to 97 psi. The flask was shaken for 16 hours. TLC analysis indicated the reaction was complete. Celite (3 g) was added to the Parr flask and the mixture filtered through a pad of Celite (2 cm). The solid was washed alternating between methanol (3 x 50 mL) and water (3 x 20 mL). The filtrate was concentrated on a rotovap to dryness. The residue was taken up in methanol (10 mL) and 25 % sodium methoxide in methanol (1.17 mL, 5.4 mmol, 0.98 equiv) was added to the methanolic solution to give a white suspension. The mixture was concentrated to dryness and taken up into DCM (10 mL). The suspension was filtered through 0.45 µm PTFE frit and concentrated to a yellow oil. The oil was redissolved into DCM (10 mL), filtered (0.45 µm PTFE frit) and concentrated to furnish a yellow oil (0.5044 g, 59.2 %). This material was used without further purification.
HPLC showed the completion of the reaction. The reaction was concentrated and the residue was neutralized with sat'd aq. NaHCO 3 (50 ml x 2) and extracted with EA (200 ml x 2). The organic layer was washed with brine, dried over anhy. Na 2 SO 4 , filtered and concentrated to give a red residue which was subjected to a silica gel purification (  . The oil was loaded onto silica gel (3 g) washed through with methanol/DCM and concentrated to give a yellow oil (1.1 g, 101.1 %). This material was used in the following reaction.

(S)-1-(2-(benzyloxy)ethyl)-2-methylpyrrolidine
Aluminum trichloride (1.865 g, 14.0 mmol, 3.0 equiv.) was suspended into anhydrous THF (30 mL) and cooled in an ice bath. Lithium aluminum hydride (1.221 g, 32.2 mmol, 6.9 equiv.) was added in small portions to the above suspension and stirred for 10 minutes. The suspension was cooled to -78 °C for 5 minutes and a solution of (S)-2-(Benzyloxy)-1-(2-methylpyrrolidin-1-yl)ethanone (1.088 g, 4.7 mmol, 1.0 equiv.) in anhydrous THF (30 mL) was added dropwise to the cold suspension. The reaction was kept at -78 °C for 1 hour and stirred at room temperature for 2 hours. The reaction was carefully quenched with 6 N HCl solution (5 mL) and stirred for 10 minutes to give grey aqueous layer. A solution 6 N NaOH (7.5 mL) was added to the mixture to give a grey white mixture of solid in the aqueous layer. The mixture was filtered through a pad of Celite (2 cm). The solids were washed with DCM (5 x 50 mL). The filtrate was cooled in a -78 °C bath until the aqueous portion froze to ice. The organic layer was poured into a Erlenmeyer flask. The ice was melted and poured into a separatory funnel (~3 mL aqueous layer recovered) and extracted with DCM (3 x 15 mL). The organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to a faint yellow solid (1.0495 g). This solid was dissolved into DCM (5 mL) and loaded onto silica gel (2.5 g) and chromatographed through silica gel (25 g cartridge) with 50-100 % ethyl acetate in hexanes followed by 10-40 % methanol in dichloromethane to give a white solid (0.5891 g, 57.