Selective estrogen receptor degraders

Abstract

Novel selective estrogen receptor degraders (SERDs) according to the formula: ##STR00001##
and pharmaceutically acceptable salt thereof and pharmaceutical compositions thereof, wherein R is selected from ##STR00002##

Claims

1. A compound of the formula: ##STR00026## wherein R is selected from ##STR00027## or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein the compound is ##STR00028## wherein R is selected from ##STR00029## or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1, wherein the compound is ##STR00030## wherein R is selected from ##STR00031## or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 2, wherein the compound is ##STR00032## or a pharmaceutically acceptable salt thereof.

5. The compound according to claim 2, wherein the compound is ##STR00033## or a pharmaceutically acceptable salt thereof.

6. The compound according to claim 2, wherein the compound is ##STR00034##

7. The compound according to claim 2, wherein the compound is ##STR00035##

8. The compound according to claim 3, wherein the compound is ##STR00036## or a pharmaceutically acceptable salt thereof.

9. The compound according to claim 3, wherein the compound is ##STR00037## or a pharmaceutically acceptable salt thereof.

10. The compound according to claim 3, wherein the compound is ##STR00038##

11. The compound according to claim 3, wherein the compound is ##STR00039##

12. A pharmaceutical composition comprising a compound according to claim 1, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents, or excipients.

13. The pharmaceutical composition according to claim 12, comprising one or more other therapeutic agents.

14. A method of treating cancer, comprising administering to a patient in need of such treatment an effective amount of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the cancer is selected from breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, and lung cancer.

15. The method according to claim 14, wherein the breast cancer is ER-positive breast cancer.

16. The method according to claim 14, wherein the gastric cancer is ER-positive gastric cancer.

17. The method according to claim 14, wherein the lung cancer is ER-positive lung cancer.

18. A method of treating cancer, comprising administering to a patient in need of such treatment an effective amount of a pharmaceutical composition according to claim 12 wherein the cancer is selected from breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, and lung cancer.

19. The method according to claim 18, wherein the breast cancer is ER-positive breast cancer.

20. The method according to claim 18, wherein the gastric cancer is ER-positive gastric cancer.

21. The method according to claim 18, wherein the lung cancer is ER-positive lung cancer.

Description

EXAMPLE 1

[(5R)-5-[4-[2-[3-(Fluoromethyl)azetidin-1-yl]ethoxy]phenyl]-8-(trifluoromethyl)-5H-chromeno[4,3-c]quinolin-2-yl]hydrogen sulfate

(1) ##STR00023##

(2) Sodium methoxide solution in MeOH (0.5 M, 0.6 mL, 0.3 mmol) is added to a solution of (5R)-5-[4-[2-[3-(fluoromethyl)azetidin-1-yl]ethoxy]phenyl]-8-(trifluoromethyl)-5H-chromeno[4,3-c]quinolin-2-ol (54 mg, 0.1 mmol) in anhydrous THF (10 mL). After stirring at room temperature for 0.5 hours, sulfur trioxide trimethylamine complex (57 mg, 0.4 mmol) is added every hour in four equal portions over 4 h. The solvent is then evaporated under a stream of nitrogen gas and the reaction mixture is diluted with water (5 mL). Aqueous NaOH (1 M, 2 drops) is added adjusting the pH to 8. The solution is directly loaded onto an Iterchim automated chromatography system (30 g RediSep® Rf Gold reverse-phase C18 column), eluting with a gradient of 10 to 90% acetonitrile in water to give the title product (46 mg, 74%) as a light-yellow solid. ES/MS (m/z): 605.6 (M+H).

Preparation 10

Methyl (2S,3S,4S,5R,6S)-6-[[(5R)-5-[4-[2-[3-(fluoromethyl)azetidin-1-yl]ethoxy]phenyl]-8-(trifluoromethyl)-5H-chromeno[4,3-c]quinolin-2-yl]oxy]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylate

(3) ##STR00024##

(4) Lithium hydroxide (90.9 mg, 3.80 mmol) is added to a suspension of (5R)-5-[4-[2-[3-(fluoromethyl)azetidin-1-yl]ethoxy]phenyl]-8-(trifluoromethyl)-5H-chromeno[4,3-c]quinolin-2-ol (830.0 mg, 1.58 mmol) in anhydrous MeOH (16 mL) at room temperature. The mixture is stirred until the starting material dissolved (around 20 min). Acetobromo-α-D-glucuronic acid methyl ester (1.19 g, 3.01 mmol) is added. The reaction is stirred at room temperature for 4 hours at which point LC/MS analysis of the reaction mixture indicates 28% conversion to the desired product and 60% unreacted starting material. Additional lithium hydroxide (92.0 mg, 3.84 mmol) is added. After stirring for 10 minutes, additional acetobromo-α-D-glucuronic acid methyl ester (1.19 g, 3.01 mmol) is added. After an additional 3 hours, LC/MS analysis indicates 35% conversion to product and 50% unreacted starting material. The reaction is discontinued and the mixture is used in the subsequent step without purification.

EXAMPLE 2

(2S,3S,4S,5R,6S)-6-[[(5R)-5-[4-[2-[3-(Fluoromethyl)azetidin-1-yl]ethoxy]phenyl]-8-(trifluoromethyl)-5H-chromeno[4,3-c]quinolin-2-yl]oxy]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid

(5) ##STR00025##

(6) The reaction mixture from Preparation 10 is added to a solution of lithium hydroxide (114.5 mg, 4.78 mmol) in water (10 mL). The reaction is stirred for 2 hours at which point LC/MS analysis of the reaction mixture indicates the hydrolysis is not complete. Additional lithium hydroxide (114.5 mg, 4.78 mmol) and MeOH (8 mL) are sequentially added. The reaction is stirred at room temperature for 1.5 hours and LC/MS analysis indicates the hydrolysis reaction is complete. The pH of the mixture is adjusted to pH 7 with concentrated acetic acid and separated into two equal portions. Each portion is loaded onto a reverse phase C18 column (275 g RediSep® Rf Gold reverse-phase C18 column) eluting with a gradient of 0 to 100% acetonitrile in water. The fractions are combined and concentrated under reduced pressure at 25° C. The residue is lyophilized to give the title compound as a light-yellow solid (80 mg, 7% yield over two steps). ES/MS (m/z): 701.6 (M+H).

Biological Assays

(7) The evidence for a relationship between ER expression and certain cancers is well known in the art.

(8) The results of the following assays demonstrate that the compounds of Formula I, Formula II, and Formula III of the examples are active SERDs and are conceived to be useful in treating cancer.

ERα Degradation Assay in MCF7 Cells

(9) The purpose of the following ERα degradation assay is to measure the degradation of ERα by a test compound in an ERα positive breast cancer cell line such as MCF7.

(10) Culture MCF7 (purchased from ATCC HTB-22) cells in DMEM media supplemented with 10% FBS, 0.01 mg/mL human insulin 1 and 1% penicillin/streptomycin antibiotics and plate in 384-well flat-bottom plates at a density of 4,000 cells per well in phenol red free DMEM media (20 μL) containing 10% charcoal stripped FBS. Incubate the cells overnight in a cell culture incubator (5% CO.sub.2, 95% relative humidity and 37° C.) and allow the cells to attach to the plate. The following day dose the cells with the test compound. Use an Echo 555 acoustic dispenser to prepare test compound serial dilutions (1:3) in a range from 6 μM to 0.0003 μM. Dose the cells with the addition of 5 μL from the serial dilution plate to the cell plate producing a final DMSO concentration of 0.2% with a final test compound concentration dose range between 2 and 0.0001 μM. For the maximum point, use media containing 0.2% of DMSO and for the minimum point, use fulvestrant diluted at 2 μM final concentrations in the growth media containing 0.2% DMSO. After dosing with the test compound, incubate the cell plates at 37° C. and 5% CO.sub.2 for 24 hours. Fix the cells by adding 14% paraformaldehyde (10 μL) for 30 minutes at room temperature. Wash the cells once with PBS (20 μL) and incubate with PBS (20 μL) containing 0.5% (v/v) TWEEN® 20 for 1 hour. Wash the cells with PBS containing 0.05% TWEEN® 20 (2×) and block with 3% BSA in PBS containing 0.05% TWEEN® 20 and 0.1% TRITON™ X-100 (20 μL/well) for 1 hour at room temperature. Add 1:500 Primary antibody (20 μL) (ERα (Clone SP1) monoclonal rabbit antibody #RM-9101-S, Thermo Scientific) dilution in 1% BSA in PBS containing 0.05% TWEEN® 20 per well, seal the plates and incubate overnight at 4° C. The following day wash the cells with PBS containing 0.05% TWEEN® 20 (2×) and incubate with secondary antibody (20 μL/well) (1:1000 dilution, Goat anti-rabbit IgM ALEXA FLUOR™ 488) in PBS 1% BSA for 105 minutes at room temperature. After washing plates with PBS (2×20 μL), add RNase (Sigma) (20 μL of 50 μg/mL) and 1:1000 propidium iodide dilution in PBS per well (20 μL). Seal the plates and incubate 1 hour at room temperature on the bench (preserved from light). Scan the plates with ACUMEN EXPLORER™ (Laser-scanning fluorescence microplate cytometer manufactured by TTP LABTECH LTD) to measure ERα. Image analysis is based on cellular fluorescent signals for identifying positive cells. Identify ER positive cells by mean intensity. Use total intensity at 575-640 nm from propidium iodide/DNA to identify individual cells. Assay output is % ER positive cells. Determine the IC.sub.50 by curve fitting to a four parameter logistic for each output using GENE DATA™. The relative IC.sub.50 values for Examples 1 and 2 are shown in Table 2. The results of this assay demonstrate degradation of ERα induced by Examples 1 and 2 as described herein in MCF7 breast cancer cells.

(11) TABLE-US-00002 TABLE 2 ERα degradation assay in MCF7 cells Example # Relative IC.sub.50 (μM) 1 0.115 ± 0.0184, n = 4 2 0.211 ± 0.0692, n = 4

PRα Induction Assay in MCF7 Cells

(12) The purpose of the following PRα induction assay is to determine whether a test compound has agonistic activity against ERα receptor (an agonist would be expected to activate the receptor).

(13) Culture MCF7 (purchased from ATCC HTB-22) in DMEM media supplemented with 10% FBS, 0.01 mg/mL human insulin 1 and 1% penicillin/streptomycin antibiotics and plate the cells (prior to becoming 70% confluent) in 384-well flat-bottom plates at a density of 4,000 cells per well in 20 μL volume in DMEM phenol red free media containing 10% FBS (charcoal stripped). Incubate the cells overnight in a cell culture incubator (5% CO.sub.2, 95% relative humidity at 37° C.) and allow the cells to attach to the plate. The following day dose the cells with test compound. Use an Echo 555 acoustic dispenser to prepare compound serial dilutions (1:3) in a range from 6 μM to 0.0003 μM. Dose the cells with the addition of the test compound (5 μL) from the serial dilution plate to the cell plate producing a final DMSO concentration of 0.2% with a final concentration of the test compound dose range between 2 and 0.0001 μM. For the maximum point use media containing 0.2% of DMSO and for the minimum point, use fulvestrant diluted at 2 μM final concentrations in the growth media containing 0.2% DMSO. After dosing with the test compound, incubate the cell plates at 37° C. and 5% CO.sub.2 for 24 hours. Fix the cells by adding 14% paraformaldehyde (10 μL) for 30 minutes at room temperature. Wash cells once with PBS (20 μL) and incubate with PBS (20 μL) containing 0.5% (v/v) TWEEN® 20 for 1 hour. Wash cells twice with PBS (20 μL) containing 0.05% TWEEN® 20 and block with 3% BSA in PBS containing 0.05% TWEEN® 20 and 0.1% TRITON™ X-100 (20 μL/well) for 1 hour at room temperature. Add 1:500 primary antibody (20 μL) (PR monoclonal mouse anti-human antibody, clone PgR 636 Dako, M3569) dilution in 1% BSA/PBS with 0.05 TWEEN® 20 per well, seal the plates and incubate overnight at 4° C.

(14) The following day wash cells with PBS 0.05% TWEEN® 20 (2×20 μL) and incubate with secondary antibody (20 μL/well) (1:1000 dilution, Goat anti-rabbit IgM ALEXA FLUOR™ 488) in PBS 1% BSA for 105 minutes at room temperature. After washing with PBS (2×20 μL), add RNase (20 μL of 50 μg/mL) (Sigma) and 1:1000 propidium iodide dilution in PBS per well. Seal plates and incubate 1 hour at room temperature on the bench (preserved from light). Scan plates with ACUMEN EXPLORER™ (Laser-scanning fluorescence microplate cytometer manufactured by TTP LABTECH LTD) to measure PRα. Image analysis is based on cellular fluorescent signals for identifying positive cells. Identify PR positive cells by mean intensity. Use total intensity at 575-640 nm from propidium iodide/DNA to identify individual cells. Assay output is % PR positive cells. Determine the IC.sub.50 by curve fitting to a four parameter logistic for each output using GENE DATA™. The results of this assay demonstrate no significant agonistic activity of Examples 1 and 2 in MCF7 breast cancer cells. For the compounds tested, the Relative IC.sub.50s in this assay are >2 μM. The results of this assay demonstrate no significant agonistic activity of the exemplified compounds tested in MCF7 breast cancer cells. These results also demonstrate that the exemplified compounds tested are antagonists of ERα in MCF7 breast cancer cells (i.e., they have SERD activity).

PRα Inhibition (ERα Functional Antagonism) Cell Assay in MCF7-ESR1 Y537N 682 CRISPR Cells

(15) The purpose of the following PRα inhibition (ERα functional antagonism) cell assay is to determine the antagonistic activity of a test compound against the Y537N mutant ERα receptor. An antagonist in this assay is expected to block the function of the ERα receptor. PRα is a downstream transcriptional target of ERα and hence an antagonist of ERα is expected to inhibit the expression of PRα.

(16) Culture MCF7-ESR1 Y537N-682 (generated by CRISPR/Cas9 gene editing of ESR1 gene in MCF7 cells, clone #682) in DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin antibiotics and plate the cells (prior to becoming 70% confluent) in 384-well flat-bottom plates at a density of 4,000 cells per well in DMEM phenol red free media 10% FBS (20 μL volume) (charcoal stripped). Incubate the cells overnight in a cell culture incubator (5% CO.sub.2, 95% relative humidity and 37° C.) and allow the cells to attach to the plate. The following day dose the cells with the test compound. Use an Echo 555 acoustic dispenser to prepare compound serial dilutions (1:3) in a range from 6 μM to 0.0003 μM. Dose the cells with the addition of 5 μL from the serial dilution plate to the cell plate producing a final DMSO concentration of 0.2% with a final test compound concentration dose range between 2 and 0.0001 μM. For the maximum point use media containing 0.2% of DMSO and for the minimum point, use fulvestrant diluted at 2 μM final concentrations in the growth media containing 0.2% DMSO. After dosing with test compound, incubate the cell plates at 37° C. and 5% CO.sub.2 for 72 hours. Fix the cells by adding 14% paraformaldehyde (10 μL) for 30 minutes at room temperature. Wash the cells with PBS (1×20 μL) and incubate with PBS (20 μL) of containing 0.5% (v/v) TWEEN® 20 for 1 hour. Wash the cells with PBS (2×20 μL), 0.05% TWEEN® 20, and block with 3% BSA/PBS 0.05% TWEEN® 20, 0.1% TRITON™ X-100 (20 μL/well) for 1 hour at room temperature. Add 1:500 primary antibody (20 μL) (PR monoclonal mouse anti-human antibody, clone PgR 636 Dako, M3569) dilution in 1% BSA/PBS 0.05 TWEEN® 20 per well, seal the plates and incubate overnight at 4° C.

(17) The following day wash the cells with PBS 0.05%® (2×20 μL) and incubate with secondary antibody (20 μL/well) (1:1000 dilution, Goat anti-rabbit IgM ALEXA FLUOR™ 488) in PBS 1% BSA for 105 minutes at room temperature. After washing with PBS (2×20 μL), add RNase (20 μL of 50 μg/mL) (Sigma) and 1:1000 propidium iodide dilution in PBS per well. Seal the plates and incubate 1 hour at room temperature on the bench (preserved from light). Scan the plates with ACUMEN EXPLORER™ [Laser-scanning fluorescence microplate cytometer manufactured by TTP LABTECH LTD] to measure PRα. Image analysis is based on cellular fluorescent signals for identifying positive cells. Identify PR positive cells by mean intensity. Use total intensity at 575-640 nm from propidium iodide/DNA to identify individual cells. Assay output is % PR positive cells. Determine the IC.sub.50 by curve fitting to a four parameter logistic for each output using GENE DATA™.

(18) The Relative IC.sub.50s of Examples 1 and 2 in this assay are shown in Table 3 below. The results of this assay demonstrate inhibition of PRα and functional antagonism by Examples 1 and 2 in MCF7 (ESR1 Y537N, heterozygous mutant) breast cancer cells. PRα (PGR) is also a transcriptional target of ERα and the results from this assay demonstrate inhibition of ERα-mediated transcription of PRα.

(19) TABLE-US-00003 TABLE 3 PRα inhibition (ERα functional antagonism) cell assay in MCF7 Y537N 682 CRISPR cells Example # Relative IC.sub.50 (μM) 1 0.330 ± 0.116, n = 3 2 0.470 ± 0.058, n = 3

Cell Proliferation Assay in MCF7 and MCF7-ESR1 Y537N-682

(20) The purpose of the following cell proliferation assays generally is to detect whether a test compound has effects on cell proliferation.

(21) Seed MCF7 (purchased from ATCC HTB-22) cells at a density of 2,000 cells per well in DMEM phenol red free media 10% FBS (20 μL volume) (charcoal stripped) into a clear bottom 384-well cell culture plate. Plate MCF7-ESRY537N-682 (generated by CRISPR/Cas9 gene editing of ESr1 gene in MCF7 cells, clone #682) in DMEM media supplemented with 10% FBS, and 1% penicillin/streptomycin antibiotics at a density of 1000 cells per well. Incubate the plates at 37° C. and 5% CO.sub.2. The following day dose the cells with the test compound. Use an Echo 555 acoustic dispenser to prepare test compound serial dilutions (1:3) in a range from 60 μM to 0.003 μM. Dose the cells with the addition of 5 μL from the serial dilution plate to the cell plate, producing a final DMSO concentration of 0.2% with a final test compound concentration dose range between 20 and 0.001 μM. For the maximum point use media containing 0.2% of DMSO and for the minimum point use fulvestrant diluted at 2 μM final concentrations in the growth media containing 0.2% DMSO. After dosing with the test compound, incubate the cell plates at 37° C. and 5% CO.sub.2. Seven days after test compound addition, remove the plates from the incubator and add cold EtOH 96% (65 μL) to each well. After 30 minutes, remove the media and add RNase (20 μL of 50 μg/mL) (Sigma) and 1:1000 propidium iodide dilution in PBS per well. Seal the plates and incubate 1 hour at room temperature on the bench (preserved from light). Scan the plates with ACUMEN EXPLORER™ (Laser-scanning fluorescence microplate cytometer manufactured by TTP LABTECH LTD). The MCF-7 cell line grows forming aggregates, cell number as number of objects may not be able to be used as readout, so the cell number may be evaluated through estimated number of cells (calculated through the area parameter (ratio of total area of the total cells population (a designated range of peak intensity of FL-1 (PI) and the mean area of the single cells population (defined by perimeter)). Determine the IC.sub.50 by curve fitting to a four parameter logistic for each output using GENE DATA™. The Relative IC.sub.50 of Examples 1 and 2 in MCF7 ESR1 wild type and MCF7-ESR1 Y537N mutant cells are shown in Table 4 below. The results of this assay demonstrate anti-proliferative activity and cell growth inhibition by Examples 1 and 2 in MCF7 (ESR1 wild type) and MCF7 (ESR1 Y537N mutant) breast cancer cells. The Relative IC.sub.50 of the exemplified compounds range from about 0.0035 to 1.176 μM in MCF7 ESR1 wild type and 0.014 to 1.86 μM in MCF7 (ESR1 Y537N mutant) breast cancer cells indicating that all exemplified compounds tested demonstrate anti-proliferative activity and cell growth inhibition in MCF7 (ESR1 wild type) and MCF7 (ESR1 Y537N mutant) breast cancer cells.

(22) TABLE-US-00004 TABLE 4 Cell Proliferation Assay in MCF7 and MCF7-ESR1Y537N-682 Relative IC.sub.50 (μM) Relative IC.sub.50 (μM) MCF7 ESR1 Y537N Example # MCF7 ESR1 wild type mutant cells 1 0.518 ± 0.0499, 0.702 ± 0.389, n = 3 n = 4 2 0.550 ± 0.238,   1.50 ± 0.966, n = 6 n = 4