EA041610B1 - SELECTIVE ESTROGEN RECEPTOR SUPPRESSORS - Google Patents

Description

State of the art

Selective estrogen receptor suppressors (SSREs) bind to the estrogen receptor (ER) and suppress ER-mediated transcriptional activity. This disruption and downregulation caused by SSREs may be useful in the treatment of cell proliferation disorders such as cancer. Some examples of low molecular weight SSPE are described in the literature (see, for example, WO 2005073204, WO 2014205136 and WO 2016097071). However, the known SSERs are not yet as useful as needed for effective cancer treatment. For example, finding an SSRE with better pharmacokinetic (PK) and pharmacodynamic (PD) properties, higher clinical efficacy, and good oral bioavailability would be very useful in cancer treatment. A pure SSRE antagonist with strong inhibition of RE-mediated transcription would be particularly useful in the treatment of cancer. There is a need for new SSERs for the treatment of cancers such as breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer and lung cancer, as well as mutations due to emerging resistance. In particular, there is a need for new SEERs for the treatment of RE-positive breast cancer, gastric cancer and/or lung cancer.

The essence of the invention

This document proposes a compound of the formula

and its pharmaceutically acceptable salts; and its pharmaceutical compositions.

Also provided are methods of using a compound described herein, pharmaceutically acceptable salts thereof, and pharmaceutical compositions for the treatment of breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer. The methods include administering a therapeutically effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof, to a patient in need.

In addition, the connection described in this document, and its pharmaceutically acceptable salt for use in therapy. The compound described herein and its pharmaceutically acceptable salts can be used in the treatment of breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer.

Also provided is the use of a compound described herein and its pharmaceutically acceptable salts for the manufacture of a medicament for the treatment of breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer or lung cancer.

Description

This document describes a new tetracyclic compound and its pharmaceutical salts, which act as SSRE. These SERMs can be used either as single agents or in combination with other drug classes, including selective estrogen receptor modulators (SERMs), aromatase inhibitors, CDK4 inhibitors, CDK6 inhibitors, PI3K inhibitors, and mTOR inhibitors for the treatment of receptor-positive breast cancer. hormones.

The novel compound described herein is a compound of the formula

Pharmaceutically acceptable salts of the compound are also described. According to the IUPAC nomenclature, the compound can be named (4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl){3-[2-fluoro4-(trifluoromethyl)phenyl]-7-hydroxyquinoline-4 -yl} methanone.

This document also describes a pharmaceutical composition comprising a compound described herein, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable excipient, carrier, or diluent.

The pharmaceutical compositions described herein can be prepared using pharmaceutically acceptable additives. As used herein, the term pharmaceutically acceptable additive(s) refers to one or more carriers, diluents, and excipients that are compatible with other composition or formulation additives and are not harmful to the patient. The compound or its pharmaceutically acceptable salts described herein can be formulated into pharmaceutical compositions administered by various routes, such as orally or intravenously. Bioavailability is often a factor in cancer treatment and it may be useful to be able to tailor administration routes and pharmaceutical compositions to control or optimize

- 1 041610 bioavailability of the active ingredient. An oral bioavailable SSRE formulation would be particularly useful. The compound or pharmaceutically acceptable salts thereof described herein are believed to have oral bioavailability. Examples of pharmaceutical compositions and methods for their preparation can be found in Remington: The Science and Practice of Pharmacy, LV Allen Jr, Editor, ^22nd Ed., Mack Publishing Co., 2012. Non-limiting examples of pharmaceutically acceptable carriers, diluents and excipients include the following: physiological solution, water, starch, sugars, mannitol and silicone derivatives; binding agents such as carboxymethylcellulose and other cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; kaolin and bentonite; polyethyl glycols.

Hereinafter, methods of treating cancer are described. The methods described herein include administering to a patient in need of such treatment an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof. For example, the method of administering an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof, may be oral administration. The cancer may be estrogen sensitive cancer. In addition, the cancer may be breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer. For example, the cancer may be RE-positive breast cancer, RE-positive gastric cancer, or RE-positive lung cancer.

This document also describes the compound described in this document, or its pharmaceutically acceptable salt for use in therapy. The compound described herein, or its pharmaceutically acceptable salts described herein, can be used in the treatment of breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer. For example, the cancer may be RE-positive breast cancer, RE-positive gastric cancer, or RE-positive lung cancer. For example, the compound or a pharmaceutically acceptable salt thereof may be administered orally.

In addition, the compound described herein, or pharmaceutically acceptable salts thereof, can be used in the manufacture of a medicament for the treatment of cancer. For example, the drug can be administered orally. The types of cancer for which the drugs described herein may be used include breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer. In particular, the cancer may be ER-positive breast cancer, ER-positive gastric cancer, or ER-positive lung cancer.

The compound described herein and its pharmaceutically acceptable salts may have clinical use as a single agent or in combination with other anticancer agents for the treatment of cancer such as breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer. and lung cancer. When used in combination with other anticancer agents, the compound described herein, or pharmaceutically acceptable salts thereof, can be used simultaneously, sequentially or separately with other anticancer agents. An example of another anticancer agent that can be combined with a compound described herein is 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H[ 1 ] benzopyrano[4,3-c]quinolin-2-ol.

As used herein, the term "effective amount" refers to an amount or dose of a compound described herein, or pharmaceutically acceptable salts thereof, which, when administered single or multiple times to a patient, provides the desired effect in the patient being diagnosed or treated. Preferably, the desired effect is inhibition of tumor cell proliferation, tumor cell death, or both. The compound described herein, or the pharmaceutically acceptable salts described herein, are generally effective over a wide dosage range. For example, daily dosages are typically in the daily range of about 100 to about 2000 mg.

As used herein, treating, treating, or treating refers to limiting, slowing, stopping, or reversing the progression or severity of an existing symptom or disorder.

As used herein, the term patient refers to a person who is afflicted with a specific disease, disorder, or pathological condition.

The compound described herein, or its pharmaceutically acceptable salts described herein, can be obtained by various methods known in the art, some of which are illustrated below in the syntheses and in the examples. Specific synthetic steps for each of the described methods can be combined in various ways or combined with steps from other methods to obtain the compounds described in this document, or its pharmaceutically acceptable salts. The products of the synthetic steps can be isolated by standard methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. These reagents and starting materials are readily available to those skilled in the art.

In an optional step, a pharmaceutically acceptable salt of a compound described herein can be prepared by reacting the appropriate free base

- 2 041610 of the invention with a suitable pharmaceutically acceptable acid in a suitable solvent under standard conditions. In addition, these salts can be obtained simultaneously with the removal of nitrogen protecting groups. The possible formation of pharmaceutically acceptable salts is well known. See, for example, Gould, P.L., Salt selection for basic drugs, International Journal of Pharmaceutics, 33: 201-217 (1986); Bastin, R.J., et al., Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities, Organic Process Research and Development, 4: 427-435 (2000); and Berge, S. M., et al., Pharmaceutical Salts, Journal of Pharmaceutical Sciences, 66: 1-19, (1977). Those skilled in the art will appreciate that the compound described herein can be easily converted and isolated as a pharmaceutically acceptable salt.

Unless otherwise noted, the abbreviations used in this document are defined in accordance with Aldrichimica Acta, Vol. 17, no. 1, 1984, or generally accepted values for those skilled in the art. Other abbreviations are defined as follows: AUC refers to the area under the curve; BSA refers to bovine serum albumin; DCM refers to dichloromethane or methylene chloride; DMA refers to dimethylamine; DMEM refers to Dulbecco's Modified Eagle's Medium; DMF refers to N,N-dimethylformamide; DMSO refers to dimethyl sulfoxide; DNA refers to deoxyribonucleic acid; cDNA refers to complementary DNA; DNase refers to deoxyribonuclease; DTT refers to dithiothreitol; EC ₅₀ refers to the concentration of an agent that elicits a 50% response of the target activity compared to a predetermined positive control compound (absolute EC ₅₀ ); EDTA refers to ethylenediaminetetraacetic acid; CEA refers to the estrogen receptor alpha; EtOAc refers to ethyl acetate; EtOH refers to ethanol; FBS refers to fetal bovine serum; HBSS refers to Hank's balanced salt solution; HEPES refers to 4-(2-hydroxyethyl)-1 pipazineethanesulfonic acid; IC ₅₀ refers to the concentration of an agent that causes 50% of the maximum inhibitory response possible for that agent (relative IC ₅₀ ) or the concentration of an agent that causes 50% inhibition of target enzyme activity compared to a placebo control (absolute IC ₅₀ ); iPrOH refers to isopropanol or isopropyl alcohol; IV refers to intravenous administration; Ki refers to the inhibition constant; MeOH refers to methyl alcohol or methanol; MTBE refers to methyl tert-butyl ether; PBS refers to phosphate buffered saline; The software refers to oral administration; RPa refers to the progesterone receptor alpha; QD refers to once daily dosage; RNA refers to ribonucleic acid; RNase refers to ribonuclease; RT-PCR refers to reverse transcription polymerase chain reaction; RT-qPCR refers to quantitative reverse transcription polymerase chain reaction; THF refers to tetrahydrofuran; and XPhos Pd G2 refers to chloro-(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium( P).

The following syntheses and examples further illustrate the present invention.

Syntheses and examples

Synthesis 1. 2-[3-(Fluoromethyl)azetidin-1-yl]ethan-1-ol ^f Vn^ _0H

Add sodium triacetoxyborohydride (405 g, 1.91 mol) portionwise over 15 min to a stirred solution of 3-(fluoromethyl)azetidine hydrochloride (160 g, 1.28 mol) in DCM (2.4 L) under N ₂ at 0 °C and stirred at 0°C for 10 min. Add 1,4-dioxane-2,5-diol (99 g, 0.83 mol) at 0°C in 6 portions over 1 hour, then stir at 0-5°C for 15 minutes. Allow the reaction mixture to warm to room temperature and stir for 2 h under N ₂ . Cool the reaction mixture to 10-15°C for 20 min. Add water (800 ml) over 25-30 min at 10-15°C, allow the mixture to warm to room temperature over 5-10 min and then separate the layers. The aqueous layer was washed with DCM (800 ml), the layers were separated, then the combined aqueous layers were cooled to 10-15° C. and the pH was adjusted to 13-14 using 50% sodium hydroxide solution (~540 ml). Allow the aqueous layer to warm to room temperature, extract with DCM (4x800 ml), dry with sodium sulfate (80 g), filter and concentrate to dryness to give the title compound. This synthesis gave 139 g (82%) of the title compound as a thick yellow oil with ES/MS (m/z): 134.1 (M+H).

Synthesis of 2. 2-[3-(Fluoromethyl)azetidin-1-yl]ethan-1-ol hydrochloride

HCI

Dissolve 2-[3-(fluoromethyl)azetidin-1-yl]ethan-1-ol (529 g, 4 mol) in MTBE (2.6 L) and cool to 0°C. An HCl/EtOH solution (492 ml, 30 wt%) was added dropwise over 30 min, then stirred at 0° C. for 30 min. Filter the solids and wash the cake on an MTBE filter (2x200

- 3 041610 ml). Dry under N2 for 8 hours to give the title compound. This synthesis gave 580 g (86%) of the title compound as a white solid with

ES/MS (m/z): 134.0 (M+H).

Synthesis 3. (3-Chloro-7-methoxyquinolin-4-yl)-(4-fluorophenyl)methanone

Cool a mixture of 4-bromo-3-chloro-7-methoxyquinoline (70 g, 254 mmol) and THF (1 L) to -40° C. under N2, which causes the material to precipitate. Add isopropyl magnesium chloride (2M in THF, 254 ml, 509 mmol) over 20 min and stir the mixture for 1 h. Add a solution of 4fluorobenzoyl chloride (66 ml, 559 mmol) in THF (140 ml) dropwise, then allow the mixture to warm up to room temperature. Quench the reaction with saturated ammonium chloride solution (300 ml) and water (200 ml) and separate the layers. The organic layer was washed with saturated ammonium chloride solution (300 ml), dried over MgSO ₄ , filtered and concentrated to give an oily residue. The crude brown oil was filtered through silica gel, eluted with MTBE/hexane (1:1) to give the crude product as a yellow solid (84 g). Treat the solid with 10% methyl acetate/heptane (800 ml) and stir at room temperature overnight. Filtered to collect solids and stock. Concentrate the filtrate and purify on silica gel, elute with 10-40% EtOAc/hexanes, then treat the product with 10% methyl acetate/heptane (200 ml) and stir at room temperature for 3 hours. The resulting solids are filtered, combined with solids from previous filtration and dried in vacuo overnight to give the title compound. This synthesis gave 31 g (38%) of the title compound as a yellow solid with ES/MS (m/z): 316.0 (M+H).

Synthesis 4. (3-Chloro-7-hydroxyquinolin-4-yl)-(4-fluorophenyl)methanone

Add boron tribromide (1M in DCM, 295 ml, 295 mmol) to a mixture of (3-chloro-7-methoxyquinolin-4yl)-(4-fluorophenyl)methanone (31 g, 98 mmol) in DCM (217 ml) and stir the mixture at room temperature for 3 days. Pour the mixture slowly into a solution of dibasic potassium phosphate (2M in water, 700 ml) and water (200 ml) at 0°C. Allow the mixture to warm to room temperature and stir for 1 h. Concentrate the solution in vacuo to remove organic solvents, filter, collect the filtrate and dry the filtrate in vacuo at 45° C. overnight. Treat the solids with DCM/heptane (1:1, 450 mL) and stir overnight. Collect the solids and dry in vacuo overnight to give the title compound. This synthesis gave 32 g (quantitative) of the title compound as a light brown solid with ES/MS (m/z): 302.0 (M+H).

Synthesis 5

Add 2-[3-(fluoromethyl)azetidin-1-yl]ethan-1-ol hydrochloride (3.90 g, 23.0 mmol) to a stirred solution of (3-chloro-7-hydroxyquinolin-4-yl)-( 4-fluorophenyl)methanone (5.00 g, 15.3 mmol) in DMF (75 ml) followed by sodium hydride (60% in mineral oil, 3.07 g, 76.6 mmol). Stir under N2 atmosphere and heat to 40°C for 45 min. Quench the solution with water and concentrate. Partition the residue between 20% iPrOH/CHCl ₃ and saturated aqueous sodium bicarbonate and separate, extract the aqueous solution with 2x20% iPrOH/CHC^, combine the organic extracts, dry the combined organic layers over magnesium sulfate, filter and concentrate the filtrate to give the crude product as dark red oil. The crude product was purified by silica gel column chromatography, eluting with a gradient of 5-10% 7N NH3 in

- 4 041610

MeOH/DCM to give the title compound. This synthesis gave 5.31 g (84%) of the title compound as a yellow solid with ES/MS (m/z): 415.0 (M+H).

Synthesis 6. (4-Fluorophenyl)-[3-[2-fluoro-4-(trifluoromethyl)phenyl]-7-hydroxy-4-quinolyl]methanone

Degas/purge 5x N ₂ a mixture of (3-chloro-7-hydroxy-4-quinolyl)-(4-fluorophenyl)methanone (140 g, 440.8 mmol), 2-fluoro-4-(trifluoromethyl)phenylboronic acid (183 .3 g, 881.7 mmol), potassium carbonate (184.6 g, 1.3 mol), 2-methyl-2-butanol (1.7 L) and water (0.56 L). XPhos Pd G2 (7.1 g, 8.82 mmol) is added and heated at 80° C. for 2 hours. The mixture is cooled to room temperature and the organic solvent is evaporated. Add EtOAc (1 L) and water (0.2 L). The organic layer is separated and dried over magnesium sulfate. Filter this material through silica gel and concentrate to dryness. Triturate the crude material with a mixture of hexanes (1.25 L) and MTBE (0.25 L) to give a solid. Filter the solid and dry under vacuum. Dissolve the solid in THF (1.5 L) and add the absorbent SiliaMetS® Thiol (150 g). Stir the mixture at room temperature. Filter off the absorbent and evaporate the filtrate to dryness to give the title compound. This synthesis gave 185.5 g (96%) of the title compound as a white solid with ES/MS (m/z): 430.0 (M+H).

Example 1 (4-{2-[3-(Fluoromethyl)azetidin-1-yl]ethoxy}phenyl){3-[2-fluoro-4-(trifluoromethyl)phenyl]-7hydroxyquinolin-4-yl} methanone

To a vessel equipped with an N2 inlet, add THF (2.8 L), potassium tert-butoxide (274.5 g, 2.45 mol) and 2-(3-(fluoromethyl)azetidin-1-yl)ethan- 1-ol (168 g, 1.22 mol). Stir the mixture for 10 min. A solution of (4-fluorophenyl)-[3-[2-fluoro-4-(trifluoromethyl)phenyl]-7hydroxy-4-quinolyl]methanone (350 g, 0.81 mol) in THF (0.7 L) was added dropwise . Stirred at room temperature for one hour. Quench the reaction with 1N HCl to pH 8 and dilute with EtOAc (4 L). Separate the organic layer and wash with brine (2 L). Dry the solution over magnesium sulfate, filter the solution and concentrate to dryness to give the title compound. This synthesis gave 415 g (93.8%) of the title compound as a pale brown solid with S/MS (m/z): 543.2 (M+H).

Alternative example 1.

Degas/purge N ₂ x5 mixture of (3-chloro-7-hydroxyquinolin-4-yl)-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)methanone (200 mg, 0. 48 mmol), 2-fluoro-4-(trifluoromethyl)phenylboronic acid (158 mg, 0.72 mmol), potassium carbonate (202 mg, 1.45 mmol), 2-methyl-2-butanol (3 ml) and water (1 ml) in a microwave bottle. Add XPhos Pd G2 (12 mg, 0.015 mmol), seal the mixture and place in a microwave oven at 80° C. for 2 hours. Partition the residue between MTBE and saturated ammonium chloride solution. Separate the layers and extract the aqueous phase with MTBE. The organic extracts are combined, dried over magnesium sulfate, filtered and the filtrate is concentrated to give an orange residue. The crude material was purified by silica gel column chromatography, eluting with 5% MeOH/DCM to give the title compound. This synthesis gave 205 mg (78%) of the title compound as a yellow solid with ES/MS (m/z): 543.2 (M+H).

Example 2 Racemic 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)5H-[1]benzopyrano[4,3-c]quinoline-2 -ol

Cool the solution of (4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl){3-[2-fluoro-4-(trifluoromethyl)phenyl]-7-hydroxyquinolin-4-yl}methanone ( 5.27 g, 9.71 mmol) in 1,4-dioxane (100 ml) to 5°C.

- 5 041610

Lithium triethylborohydride (1M in THF, 30.0 ml, 30.0 mmol) is added. Remove the cooling bath and stir for 1.5 h at room temperature. The mixture is quenched with water. Add a saturated solution of NH ₄ Cl and EtOAc. Separate the layers and extract the aqueous layer with EtOAc. The organic extracts are combined, dried over anhydrous MgSO ₄ , filtered and the filtrate is concentrated. Dissolve the crude residue in THF (100 ml). Sodium hydride (60% in mineral oil, 1.94 g, 48.5 mmol) is added. Reflux the solution for 1.5 hours. Add more sodium hydride (60% in mineral oil, 1.94 g, 48.5 mmol), then reflux for an additional 30 minutes. Cool the solution to room temperature and quench with water. Add EtOAc and saturated NH4Cl solution. Separate the layers and extract the aqueous layer with EtOAc. Combine the organic extract, dry over anhydrous MgSO4, filter and concentrate the filtrate. Purify the residue by silica gel column chromatography, elute with a gradient of 5-7% MeOH in DCM to give the title compound (3.70 g, 72%) as a light yellow foam. ES/MS (m/z): 525.2 (M+H).

Biological analyzes

The relationship between estrogen receptor expression and some cancers has been described in the literature (for breast cancer, see e.g. Puhalla S., Bhattacharya S., Davidson N., Hormonal therapy in breast cancer: A model disease for the personalization of cancer care, Molecular Oncology, 2012, 6:222-236 Kennecke H., Yerushalmi R., Woods R., Cheang MCU, Voduc D., Speers C.H., Nielsen T.O., Gelmon K., Metastatic behavior of breast cancer subtypes, J. Clin Oncol ., 2010, 28(20):3271-3277, for ovarian cancer see, e.g., O'Donnell A.J., Macleod K.G., Burns D.J., Smyth J.F., Langdon S.P., Estrogen receptor-alpha mediates gene expression changes and growth response in ovarian cancer cells exposed to estrogen, Endocr Relat Cancer, 2005; 12(4):851-66; Walker G., MacLeod K., Williams A.R., Cameron D.A., Smyth J.F., Langdon S.P., Estrogen regulated gene expression predicts response to endocrine therapy in patients with ovarian cancer, Gynecol.Oncol., 2007, 106(3):461-8; Smyth J.F., Gourley C., Walker G ., MacKean M.J., Stevenson A., Williams A.R., et al., Antiestrogen therapy is active in selected ovarian cancer cases: The use of letrozole in estrogen receptor-positive patients, Clin Cancer Res, 2007, 13(12):3617- 22; for prostate cancer, see, for example, Bonkohoff H, Fixemer T, Hunsicker I and Remberger K, Estrogen receptor expression in prostate cancer and premalignant prostate lesions, Am J. Pathol., 1999, 155:641-647; for endometrial and uterine cancer, see, for example, Krasner C., Aromatase inhibitors in gynecologic cancer, J. Steroid Biochem Mol. Biol., 2007, Aug-Sep; 106(l-5):76-80; Boisen M.M., Andersen C.L., Sreekumar S., et al., Treating gynecologic malignancies with selective estrogen receptor downregulators (SERDs): Promise and challenges, Mol. cell. Endocrinol., 2015, 418:322-3330; for lung cancer see e.g. Baik C.S., Eaton K.D. et al., Estrogen signaling in lung cancer: An opportunity for novel therapy, Cancer, 2012, 4:969-988; Marquez-Garban D.C., Chen H-W., Goodglick L., Fishbein M.C. and Pietras R.J., Targeting aromatase and estrogen signaling in human non-small cell lung cancer. Steroid enzymes and cancer, Ann. N. Y. Acad Sci, 2009, 1155:194-205; Hamilton D.H., Griner L.M., Keller J.M., Hu X., Southall N., Marugan J., David J.M., Ferrer M. and Palena C., Targeting estrogen receptor signaling with fulvestrant enhances immune and chemotherapy mediated cytotoxicity of human lung cancer, Clin Cancer Res., 2016, 22(24): 6204-16; Rodriguez-Lara V., Hernandez-Martinez J.M., Arrieta O., Influence of estrogen in non-small cell lung cancer and its clinical implications, J. Thoracic Disease, 2018, 10(1):482-497; for gastric cancer see, for example, Tang W., Liu R., Yan Y., Pan X., Wang M., Han X., Ren H., and Zhang Z., Expression of estrogen receptors and androgen receptor and their clinical significance in gastric cancer, Oncotarget, 2017, 8(25) 40765-777).

The following assays demonstrate that the exemplary compounds are potent disruptors of wild-type REE and mutant proteins. The results of the assays also demonstrate that the exemplary compounds are potent antagonists of wild-type and mutant ERs and inhibit ER-mediated transcriptional activity. In addition, the assays demonstrate that the compound of Example 1 inhibits the proliferation of EC+ breast cancer cell lines and CE signaling and tumor growth in an EC positive breast cancer xenograft model.

Analysis of competitive binding of CEA (wild type) and CEA (Y537S mutant).

The purpose of the following ER competitive binding assays is to determine the binding affinity of a test compound for CEA (wild type) and CEA (Y537S mutant) see Fanning et al., Estrogen receptor alpha somatic mutations Y537S and D538G confer breast cancer endocrine resistance by stabilizing the activating function- 2 binding conformation, eLife 2016; 5:e12792.

Perform a competition binding assay in buffer containing 50 mM HEPES, pH 7.5, 1.5 mM EDTA, 150 mM NaCl, 10% glycerol, 1 mg/ml ovalbumin, and 5 mM DTT using 0.025 µ Ci per well ³ H- estradiol (118 Ci/mmol, 1 mCi/ml), 7.2 ng/well CEA (wild type), or 7.2 ng/well CEA (Y537S mutant). Test compound is added at 10 different concentrations ranging from 10,000 to 0.5 nM and non-specific binding is determined in the presence of 1 μM 17-β estradiol. Incubate the binding reaction (140 µl) for 4 hours at room temperature and then add cold dextran-charcoal buffer (70 µl) (containing 0.75 g charcoal and 0.25 g dextran per 50 ml assay buffer) to each reaction. Mix the plates for 8 minutes on an orbital shaker at 4°C and then centrifuge at 3000 rpm at 4°C for 10 minutes. Transferred

- 6 041610 Aliquot (120 µl) of the mixture into another 96-well white flat bottom plate (Costar) and add Perkin Elmer Optiphase Supermix scintillation fluid (175 µl) to each well. Seal the plates and mix vigorously on an orbital shaker. After incubation for 2.5 hours, the plates are read on a Wallac Microbeta counter. Calculate IC ₅₀ using a 4 parameter logistic curve and calculate % inhibition at 10 μM. Convert the IC ₅₀ values for the compound to Ki using the Cheng-Prusoff equation. The results of this assay show that Example 1 binds to recombinant wild-type CEA with Ki (nM) 3.78±0.74 (n=3) and binds to mutant CEA (Y537S) with Ki (nM) 21.24±2 ,12 (n=3).

The results of this assay demonstrate the binding affinity and efficacy of the exemplary compound against wild-type REa and mutant proteins (ESR1 Y537S).

CEA destruction analysis in MCF7 cells.

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

MCF7 cells (purchased from ATCC HTV-22) are cultured in DMEM supplemented with 10% FBS, 0.01 mg/ml human insulin 1 and 1% penicillin/streptomycin antibiotics and plated in 384-well flat bottom plates at a density of 4000 cells per well in phenol red free DMEM (20 µl) containing 10% charcoal-purified FBS. The cells are incubated overnight in a cell culture incubator (5% CO ₂ , 95% relative humidity and 37° C.) and the cells are allowed to adhere to the plate. The next day, test compound is injected into the cells. Use an Echo 555 acoustic pipette to obtain serial dilutions of the test compound (1:3) in the range of 6 to 0.0003 μM. Dose the cells by adding 5 µl from the serial dilution plate to the cell plate to give a final concentration of DMSO of 0.2% with a final test compound dose range of 2 to 0.0001 µM. For the maximum point, medium containing 0.2% DMSO is used, and for the minimum point, fulvestrant diluted to final concentrations of 2 μM in growth medium containing 0.2% DMSO is used. After test compound dosing, incubate cells with cells at 37° C. and 5% CO2 for 24 hours. Fix 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 with PBS containing 0.05% TWEEN® 20 (2x) and blocked 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) (REA (Clone SP1) rabbit monoclonal antibody No. RM-9101-S, Thermo Scientific) at a dilution of 1% BSA in PBS containing 0.05% TWEEN® 20 per well, seal the plates and incubated overnight at 4°C. The next day, cells are washed with PBS containing 0.05% TWEEN® 20 (2x) and incubated with secondary antibody (20 µl/well) (1:1000 dilution, goat anti-rabbit IgM ALEXA FLUOR™ 488) in PBS 1% BSA in for 105 min at room temperature. After washing the plates with PBS (2x20 μl), RNase (Sigma) (20 μl, 50 μg/ml) and propidium iodide diluted 1:1000 per well (20 μl) in PBS are added. Seal the cups and incubate for 1 hour at room temperature on a table (protected from light). The plates are scanned with ACUMEN EXPLORER™ [Laser Scanning Fluorescent Cytometer for Plates, manufactured by TTP LABTECH LTD] to measure CEA. Image analysis relies on cellular fluorescent signals to identify positive cells. Estrogen receptor positive cells are determined by average intensity. Use a total intensity of 575-640 nm from propidium iodide/DNA to identify individual cells. The result of the analysis is the % of cells positive for the estrogen receptor. IC ₅₀ is determined by curve fitting a logistic equation with four parameters for each output using GENE DATA™.

The results of this analysis demonstrate that the compound of formula (I) is an SSRE with a strong destructive activity of CEA in cells. In particular, the results show a strong destruction of CEA by the compound of example 1 in breast cancer MCF7 cells. Using this assay, the relative IC ₅₀ value (μM) for the compound of Example 1 is 2.16±0.96 nM (n=15).

Induction analysis of RPa in MCF7 cells.

The purpose of the following RPa induction assay is to determine if a test compound has agonistic activity against the CEA receptor (the agonist is expected to activate the receptor).

Cultivate MCF7 (purchased from ATCC HTV-22) in DMEM supplemented with 10% FBS, 0.01 mg/mL human insulin 1, and 1% penicillin/streptomycin antibiotics and plate cells (until 70% confluent) in 384-well plates flat bottom at 4000 cells/well in 20 µl volume in phenol red free DMEM containing 10% FBS (purified charcoal). The cells are incubated overnight in a cell culture incubator (5% CO 2 , 95% relative humidity and 37° C.) and the cells are allowed to adhere to the plate. The next day, enter

- 7 041610 cells test compound. An Echo 555 acoustic pipette is used to obtain serial dilutions of the compound (1:3) ranging from 6 to 0.0003 μM. Dose cells with test compound (5 µl) from serial dilution plate to cell plate to give a final concentration of 0.2% DMSO with a final concentration ranging from 2 to 0.0001 µM of test compound. For the maximum point, medium containing 0.2% DMSO is used, and for the minimum point, fulvestrant diluted to final concentrations of 2 μM in growth medium containing 0.2% DMSO is used. After test compound dosing, cells are incubated with cells at 37° C. and 5% CO ₂ for 24 hours. Cells are fixed 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 twice with PBS (20 µl) containing 0 .05% TWEEN® 20 and blocked 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) (anti-human mouse progesterone receptor monoclonal antibody, clone PgR 636 Dako, M3569) at a 1% BSA/PBS dilution with 0.05 TWEEN® 20 per well, seal the plates and incubate overnight at 4°C.

The next day, cells are washed with PBS 0.05% TWEEN® 20 (2x20 µl) and incubated with secondary antibody (20 µl/well) (1:1000 dilution, goat anti-rabbit IgM ALEXA FLUOR™ 488) in PBS 1% BSA in for 105 min at room temperature. After washing with PBS (2x20 μl), RNase (20 μl, 50 μg/ml) (Sigma) and propidium iodide diluted 1:1000 per well in PBS are added. The plates are sealed and incubated for 1 hour at room temperature on a table (protected from light). The plates are scanned with ACUMEN EXPLORER™ [Laser Scanning Fluorescent Cytometer for Plates, manufactured by TTP LABTECH LTD] to measure progesterone areceptors. Image analysis relies on cellular fluorescent signals to identify positive cells. Cells positive for the progesterone receptor are determined by average intensity. Use a total intensity of 575-640 nm from propidium iodide/DNA to identify individual cells. The result of the analysis is % of cells positive for the progesterone receptor. IC ₅₀ is determined by curve fitting a logistic equation with four parameters for each output using GENE DATA™.

Using this assay, the relative IC ₅₀ (μM) of Example 1 is >2 μM. The results of this assay do not demonstrate significant agonist activity of Example 1 against breast cancer MCF7 cells. These results also demonstrate that Example 1 is a pure CEA antagonist in MCF7 breast cancer cells.

Analysis of RPa inhibition (functional CEA antagonism) in MCF7-ESR1 Y537N 682 CRISPR cells.

The purpose of the following RPa inhibition assay (functional CEA antagonism) in cells is to determine the antagonistic activity of a test compound against the Y537N CEA mutant receptor. The antagonist in this assay is expected to block REA receptor function. RPa (PGR) is a downstream target of CEA transcription and therefore a CEA antagonist is expected to inhibit RPa expression.

MCF7-ESR1 Y537N-682 (generated by editing the CRISPR/Cas9 gene of the ESR1 gene in MCF7 cells, clone no. 682) is cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin antibiotics and placed the cells (until 70% confluence is reached) in Flat bottom 384-well plates at 4,000 cells/well in phenol red-free DMEM, 10% FBS (20 µl volume) (charcoal-purified). The cells are incubated overnight in a cell culture incubator (5% CO ₂ , 95% relative humidity and 37° C.) and the cells are allowed to adhere to the plate. The next day, test compound is injected into the cells. An Echo 555 acoustic pipette is used to obtain serial dilutions of the compound (1:3) ranging from 6 to 0.0003 μM. Dose the cells by adding 5 µl from the serial dilution plate to the cell plate to give a final concentration of DMSO of 0.2% with a final test compound dose range of 2 to 0.0001 µM. For the maximum point, medium containing 0.2% DMSO is used, and for the minimum point, fulvestrant diluted to final concentrations of 2 μM in growth medium containing 0.2% DMSO is used. After test compound dosing, cells are incubated with cells at 37° C. and 5% CO ₂ for 72 hours. Cells are fixed by adding 14% paraformaldehyde (10 μl) for 30 minutes at room temperature. Wash cells with PBS (1x20 µl) and incubate with PBS (20 µl) containing 0.5% (v/v) TWEEN® 20 for 1 hour. Wash cells with PBS (2x20 µl), 0.05% TWEEN ® 20, and blocked with 3% BSA in PBS, 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) (murine anti-human progesterone receptor monoclonal antibody, clone PgR 636 Dako, M3569) at a dilution of 1% BSA/PBS 0.05 TWEEN® 20 per well, seal the plates and incubate overnight at 4°C.

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The next day, cells are washed with PBS 0.05% TWEEN® 20 (2x20 µl) and incubated with secondary antibody (20 µl/well) (1:1000 dilution, goat anti-rabbit IgM ALEXA FLUOR™ 488) in PBS 1% BSA for 105 min at room temperature. After washing with PBS (2x20 μl), RNase (20 μl, 50 μg/ml) (Sigma) and propidium iodide diluted 1:1000 per well in PBS are added. Seal the cups and incubate for 1 hour at room temperature on a table (protected from light). The plates are scanned with ACUMEN EXPLORER™ [Laser Scanning Fluorescent Cytometer for Plates, manufactured by TTP LABTECH LTD] to measure progesterone receptor α. Image analysis relies on cellular fluorescent signals to identify positive cells. Cells positive for the progesterone receptor are determined by average intensity. Use a total intensity of 575-640 nm from propidium iodide/DNA to identify individual cells. The result of the analysis is % of cells positive for the progesterone receptor. IC ₅₀ is determined by curve fitting a logistic equation with four parameters for each output using GENE DATA™.

Using this assay, the relative IC ₅₀ value (µM) for the compound of Example 1 is 7.602±4.804 nM (n=14). These results demonstrate strong RPa inhibition and functional antagonism by Example 1 in MCF7 (ESR1 Y537N, heterozygous mutant) breast cancer cells. Thus, the compound of Example 1 is a strong antagonist of the CEA mutant (Y537N) and a strong inhibitor of CEA-mediated transcription. RPa (PGR) is also a target for CEa transcription, and the results of this assay demonstrate strong inhibition of CE-mediated transcription of RPa.

Analysis of RPa inhibition (functional CEA antagonism) in MCF7 cells.

The purpose of the following RPa inhibition assay (functional CEA antagonism) in cells is to determine the antagonistic activity of a test compound against CEA. The antagonist in this assay is expected to block CEA function. RPa is a downstream transcriptional target of CEA and therefore a CEA antagonist is expected to inhibit RPa expression.

Perform assay conditions as detailed in the Acumen CEA disruption assay above using the MCF7 cell line, except that media is removed from the plate prior to distribution of test compound and all wells except the negative control wells (column 24 of the assay plate) are pretreated. medium containing 0.47 nM estradiol for 30 minutes. In this assay, immunostaining is performed to detect PRα, and plates are scanned with ACUMEN EXPLORER™ [Laser Scanning Microplate Fluorescent Cytometer manufactured by TTP LABTECH LTD] to measure PRα. Image analysis relies on cellular fluorescent signals to identify positive cells. Cells positive for RPa are determined by average intensity. Use a total intensity of 575-640 nm from propidium iodide/DNA to identify individual cells. The result of the analysis is % of cells positive for RP. IC ₅₀ is determined by curve fitting a logistic equation with four parameters for each output using GENE DATA™.

Using this assay, the relative IC ₅₀ value (μM) for the compound of Example 1 is 15.75±9.037 nM (n=15). The results of this assay demonstrate strong RP inhibition and functional antagonism of Example 1 in breast cancer MCF7 cells. Thus, the compound of Example 1 is a strong antagonist of the wild type CEA protein and a strong inhibitor of CEA mediated transcription. RPa (PGR) is also a target for CEa transcription, and the results of this assay demonstrate strong inhibition of CE-mediated transcription of RPa.

Cell proliferation assay in MCF7 and MCF7-ESR1 Y537N-682.

The purpose of the following cell proliferation assays is generally to determine if a test compound has an effect on cell proliferation, cell viability, and cytotoxicity in response to treatment in cell culture experiments. Cell proliferation is monitored by tracking the number of cells over time, and the propidium iodide assay used allows continuous measurement of cell viability over time.

MCF7 cells (purchased from ATCC NTV-22) were plated at 2000 cells per well in phenol red free DMEM, 10% FBS (volume 20 μl) (purified with charcoal) in a 384 well clear bottom cell culture plate. Place MCF7-ESRY537N-682 (created by editing the CRISPR/Cas9 gene of the ESR1 gene in MCF7 cells, clone #682) in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin antibiotics at a density of 1000 cells per well. Incubate plates at 37°C and 5% CO2. The next day, test compound is injected into the cells. Use an Echo 555 acoustic pipette to obtain serial dilutions of the test compound (1:3) in the range of 60 to 0.003 μM. Dose the cells by adding 5 μl from the serial dilution plate to the cell plate to give a final concentration of 0.2% DMSO with a final test compound dose range of 20 to 0.001 μM. For maximum

0.2% DMSO medium is used for the minimum point and fulvestrant diluted to final concentrations of 2 μM in growth medium containing 0.2% DMSO is used for the minimum point. After dosing the test compound, the cells are incubated at 37° C. and 5% CO ₂ . Seven days after the addition of the test compound, remove the plates from the incubator and add 96% cold ethanol (65 µl) to each well. After 30 min, remove the medium and add RNase (20 μl, 50 μg/ml) (Sigma) and a solution of propidium iodide 1:1000 in PBS per well. Seal the cups and incubate for 1 hour at room temperature on a table (protected from light). The plates are scanned using ACUMEN EXPLORER™ [Laser Scanning Fluorescence Cytometer for Plates, manufactured by TTP LABTECH LTD]. The MCF-7 cell line grows by forming aggregates, the number of cells as the number of objects cannot be used for reading; thus, the number of cells can be estimated using the estimated number of cells (calculated using the area parameter (the ratio of the total area to the total cell population (specified FL-1 peak intensity range (PI) and the average area of individual cell populations (determined by the perimeter)) The IC ₅₀ is determined by curve fitting the logistic equation with four parameters for each output using GENE DATA™.

Using this assay, the relative IC ₅₀ (nM) of Example 1 in wild-type MCF7 ESR1 is 9.243±1.741 nM (n=2) and in MCF7-ESR1 Y537N mutant cells is 7.960±3.691 nM (n=6). These results demonstrate the potent antiproliferative activity and cell growth inhibition of Example 1 in breast cancer MCF7 (wild-type ESR1) and MCF7 (ESR1 Y537N mutant) cells.

In Vivo Targeted Inhibition (CI IV) Assay (PGR RT-qPCR assay) in MCF7 tumors.

The purpose of this CI IV assay is to measure the ability of a test compound (SSER) to inhibit the expression (transcription) of the PGR (α-progesterone receptor) gene below CEA in xenograft tumors implanted in mice.

Female mice NOD SCID (22-25 g) from Envigo RMS, Inc., Madison, Wisconsin are implanted subcutaneously 5x10e ⁶ RE-positive breast cancer MCF7 cells (ATCC, No. HTB-22) in the region of the right flank in 1:1 solution of HBSS + MATRIGEL™ (200 µl). 17-β estradiol beads (0.18 mg/bead, 90 day release, from Innovative Research) are implanted subcutaneously 1 day prior to tumor cell implantation. Measure tumor growth and body weight twice a week, starting from the seventh day after implantation. When the tumor size reaches 250-350 mm ³ , animals are randomized and grouped into groups of five animals. Administer multiple doses of test compound to animals in a test compound specific vehicle (1% hydroxyethyl cellulose/0.25% TWEEN® 80/0.05% defoamer in purified water) or vehicle alone orally for 3 days and collect tumors and blood at desired intervals time since the last dose. Animals are sacrificed using isoflurane anesthesia and cervical dislocation. Tumors are flash frozen and stored at -80° C. until processed for RNA isolation and RT-qPCR analysis. Collect blood in tubes with EDTA, slow rotation to collect plasma and freeze at -80°C in a 96-well plate. Determine the effect of the test compound using mass spectrometry.

Tumors were ground in liquid nitrogen and lysed in lxRNA lysis buffer (from RNA isolation kits) using Matrix D beads (MP Biomedical, no. 6913-500) in a FASTPREP-24™ cell disruptor (MP Biomedical). Transfer tumor lysates to fresh tubes after rotation at 14,000 rpm for 20 min at 4°C. RNA is isolated from tumor lysates using the PURELINK® RNA Mini Kit (Invitrogen No. 12183018A) or RNeasy Mini Kit (Qiagen No. 74104 and No. 74106). Remove DNA contamination using the PURELINK® DNase kit (Invitrogen no. 12185010) or the RNase-Free DNase kit (Qiagen no. 79254). Measure the concentration of the isolated RNA by diluting the samples with RNase-free water and measure the absorbance at 260 nm on a plate reader (SpectraMax190). Subtract the average absorbance measurement at 260 nm of the blank (RNase-free water only) from the 260 nm measurements of all other RNA samples. Dilute RNA samples to equal concentrations in RNase-free water. Synthesize cDNA from diluted RNA using a first strand synthesis system for RT-PCR (Invitrogen, no. 18080-051). To perform RT-qPCR, first dilute the cDNA in RNase-free water. Combine 2x Absolute Blue qPCR ROX Mix (Thermo, # AB-4139/A), PGR primer (Thermo, Hs01556702_m1) and diluted cDNA for each reaction in a PCR plate (Applied Biosystems, # 4309849). Amplify the cDNA by incubating the samples for 2 min at 50°C and then 15 min at 95°C in a thermal cycler (ABI Prism 7900HT seventator). Continue incubation at 95°C for 15 seconds and then at 50°C for 60 seconds, for a total of 40 cycles. Cycles are normalized to the home gene and are used to calculate % PGR inhibition compared to vehicle alone. Analyze each sample in duplicate and use averages for calculations. Calculate target percent inhibition (PGR) using Excel and XL Fit.

The results of this assay demonstrate that the compound of Example 1 inhibits the expression of Ppa (PGR) in a xenograft tumor model. In addition, the compound of example 1 inhibits RPa (PGR) expression by 57% in a xenograft tumor model for 24 hours at a dose of 30 mg/kg.

- 10 041610 when administered orally. These results demonstrate a significant and sustained inhibition of CEA antagonistic activity and CEA-mediated transcriptional activity in vivo in a xenograft tumor model.

An in vivo tumor growth inhibition study in MCF7 tumor xenograft implanted in mice.

The purpose of the following xenograft tumor growth inhibition assay is to measure tumor volume reduction in response to test compound administration.

Human breast cancer MCF7 cells (ATCC No. HTB-22) are expanded in culture, harvested and injected 5x10e ⁶ cells in HBSS+MATRIGEL™ 1:1 solution (200 µl) subcutaneously into the posterior right flank of female NOD SCID mice (22-25 g, Envigo RMS, Inc. 24 hours prior to implantation of MCF7 cells, estrogen beads (0.18 mg/bead, 17β estradiol, 90 day release, Innovative Research) were implanted subcutaneously. Measure tumor growth and body weight twice a week, starting from the seventh day after implantation. When the tumor size reaches 250-350 mm ³ , animals are randomized and grouped into groups of five animals. The test compound is prepared in a suitable solvent (1% hydroxyethyl cellulose/0.25% TWEEN® 80/0.05% defoamer in purified water) and administered via gavage for 42 days. Determine the response of the tumor by measuring the volume of the tumor twice a week during the course of treatment. When measuring tumor volume, body weight is taken as a general measure of toxicity.

When used in this assay, the compound of Example 1 was found to have delta T/C% values as shown in the table below. These results indicate that the compound of Example 1 exhibits good oral bioavailability in mice and significant antitumor activity or tumor regression in the MCF7 human breast cancer xenograft model.

An in vivo tumor growth inhibition study in MCF7 tumor xenograft implanted in mice.

Tumor Model Dose (mg/kg) Schedule Delta T/C% or Regression % p-value MCF7 (Breast Cancer Xenograft) thirty QD -36 <0.001*

The tumor volume analysis is based on the Log 10 covariance structure and SpatialPower.

*: Significant (p<0.05) compared to solvent control.

Delta T/C% is calculated when the final tumor volume in the treated group is at or above the initial tumor volume. The formula is 100x(T-T ₀ )/(C-C ₀ ), where T and C are the average final tumor volumes in the treated or control group, respectively. T ₀ and C ₀ - the average initial volume of tumors in these groups.

% Regression is calculated when the final volume is lower than the initial one. The formula is 100x(T-T ₀ )/T ₀ in which T ₀ is the average initial tumor volume for the treated group.

The total mean of all groups from baseline (randomization) at day 32 is used to calculate the % change in T/C.

Analysis of oral bioavailability in rats.

The purpose of the following assay is to demonstrate whether a test compound is orally bioavailable.

The test compound is administered to Sprague-Dawley rats intravenously (IV) at 1 mg/kg (using solvents 20% CAPTISOL® in 25 mM sodium phosphate buffer, sufficient for pH 2; or 25% DMA, 15% EtOH, 10 % propylene glycol, 25% 2-pyrrolidone and 25% purified water) and orally (PO) at 10 mg/kg (using 1% hydroxyethyl cellulose, 0.25% polysorbate 80, 0.05% antifoam 1510-US and sufficient amount of treated water). Serial blood samples were taken at 0.08, 0.25, 0.5, 1, 2, 4, 8, and 12 hours after the IV bolus dose and at 0.25, 0.5, 1, 2, 4, 8, and 12 hours after oral dosing. After treatment with EDTA coagulant, plasma was obtained by centrifugation and stored at -70°C until analysis by LC-MS/MS. The plasma concentration of the test compound was determined and loaded into the Watson LIMSTM system, where non-compartmental analysis was used to calculate the area under the curve (AUC) for the VV and PO branches. Calculate oral bioavailability (% F) using the following equation:

% F \u003d (PPKpoxDozavv) / (PPKinvxDose πo)x 100.

Using this analysis, the compound of example 1 shows a % F value of 27%. This assay demonstrates that Example 1 has good oral bioavailability.

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Claims (18)

CLAIM 1. Compound formula or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, characterized in that the compound is 3. A pharmaceutical composition containing a compound or a pharmaceutically acceptable salt thereof according to claim 1 or a compound according to claim 2 in combination with one or more pharmaceutically acceptable excipients, carriers or diluents. 4. Pharmaceutical composition according to claim 3, additionally containing one or more additional therapeutic agents. 5. A method of treating breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric or lung cancer, comprising administering a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof according to claim 1 or a compound according to claim 2 to a patient in need of such treatment. 6. The method of claim 5, wherein the compound or a pharmaceutically acceptable salt thereof is administered orally. 7. The method of claim 5 wherein the breast cancer is estrogen receptor positive (ER positive) breast cancer. 8. The method according to claim 5, wherein the gastric cancer is an ER-positive gastric cancer. 9. The method according to claim 5, wherein the lung cancer is EC-positive lung cancer. 10. The use of a compound or a pharmaceutically acceptable salt thereof according to claim 1 or a compound according to claim 2 for the treatment of breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer or lung cancer. 11. Use according to claim 10, characterized in that the compound is administered orally. 12. Use according to claim 10, characterized in that the breast cancer is an ER-positive breast cancer. 13. The use according to claim 10, characterized in that the gastric cancer is an ER-positive gastric cancer. 14. Use according to claim 10, characterized in that the lung cancer is an EC-positive lung cancer. 15. The use of a compound or a pharmaceutically acceptable salt thereof according to claim 1 or a compound according to claim 2 for the manufacture of a medicament for the treatment of breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer or lung cancer. 16. Use according to claim 15, characterized in that the drug is intended for oral administration. 17. Method for the preparation of 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H[1]benzopyrano[4,3-c]quinoline-2 -ol, including cooling the solution of (4-{2-[3-(fluoromethyl)azetidin-1yl]ethoxy}phenyl){3-[2-fluoro-4-(trifluoromethyl)phenyl]-7-hydroxyquinolin-4-yl} methanone in 1,4-dioxane to about 5°C, followed by the addition of lithium triethylborohydride to obtain alcohol, dissolving the alcohol in tetrahydrofuran, adding sodium hydride and heating the resulting mixture under reflux to obtain the specified connection. 18. Pharmaceutical composition according to claim 4, characterized in that the additional therapeutic agent is 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H [ 1 ]benzopyrano[4,3-c]quinolin-2-ol. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2