Selective estrogen receptor degraders

Abstract

Provided herein are novel selective estrogen receptor degraders (SERDs) according to the following formula, and pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof, wherein R 1 or one of R 2 is independently selected from Cl, F, -CF3, or -CH3, and the other is hydrogen, and methods of use thereof.

Description

[0001] This application is a divisional application. The original application has the application number 201980046849.3, the application date is July 11, 2019, the priority date is July 12, 2018, and the invention title is "Selective Estrogen Receptor Degrader". background

[0002] Selective estrogen receptor degraders (SERDs) bind to the estrogen receptor (ER) and downregulate ER-mediated transcriptional activity. This degradation and downregulation induced by SERDs can be used to treat cell proliferation disorders, such as cancer. Several small-molecule examples of SERDs have been disclosed in the literature (see, for example, WO2005073204, WO2014205136, and WO2016097071). However, known SERDs are not yet as useful as needed for effective cancer treatment. For example, discovering SERDs with better pharmacokinetic (PK) and pharmacodynamic (PD) properties, higher clinical efficacy, and good oral bioavailability would be very helpful in cancer treatment. Pure antagonist SERDs that effectively inhibit ER-mediated transcription would be particularly beneficial in cancer treatment. New SERDs are needed to treat cancers such as breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, and lung cancer, as well as mutations resulting from resistance. In particular, new SERDs are needed to treat ER-positive breast cancer, gastric cancer, and / or lung cancer. Invention Overview

[0003] This article provides compounds with the following formulas:

[0004]

[0005] And its pharmaceutically acceptable salts, and pharmaceutical compositions thereof. In this formula, R 1 Or R 2 One of them is independently selected from Cl, F, -CF3 or -CH3, and the other is hydrogen.

[0006] Methods for treating breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer using compounds as described herein, pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof are also provided. The methods include administering a therapeutically effective amount of a compound as described herein or a pharmaceutically acceptable salt thereof to a patient in need.

[0007] Also provided are compounds as described herein and their pharmaceutically acceptable salts for therapeutic purposes. The compounds described herein and their pharmaceutically acceptable salts may be used to treat breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer.

[0008] The use of the compounds described herein and their pharmaceutically acceptable salts in the preparation of medicaments for the treatment of breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer is also provided. Invention Details

[0009] This article discloses novel tetracyclic compounds used as SERDs and their pharmaceutically acceptable salts. The novel SERDs described herein provide ER-mediated transcriptional inhibition, which could be used to treat cancers such as breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, and lung cancer, as well as mutations resulting from resistance. These SERDs can be used as a single active agent or in combination with other classes of drugs, including selective estrogen receptor modulators (SERMs), aromatase inhibitors, CDK4 inhibitors, CDK6 inhibitors, PI3K inhibitors, and mTOR inhibitors, to treat hormone receptor-positive cancers such as breast cancer, gastric cancer, and / or lung cancer.

[0010] The new compound described in this article is represented by formula I:

[0011]

[0012] and its pharmaceutically acceptable salts, of which R 1 Or R 2 One of the chiral centers is independently selected from Cl, F, -CF3, or -CH3, and the other is hydrogen. Those skilled in the art will understand that the compounds of Formula I or their pharmaceutically acceptable salts contain a chiral center, the position of which is indicated by the asterisk (*). Those skilled in the art will also understand that the Cahn-Ingold-Prelog(R) or (S) nomenclature for the chiral center will vary depending on the substitution pattern surrounding the chiral center. The chiral center in the compound of Formula I provides the R-enantiomer form shown in Formula II.

[0013]

[0014] The S-enantiomer form shown in Formula III:

[0015]

[0016] All individual stereoisomers, enantiomers and diastereomers of compounds of formulas I, II and III, as well as mixtures of enantiomers and diastereomers (including racemates), are included within the scope of the compounds described herein. Pharmaceutical compounds containing chiral centers are typically isolated as single enantiomers or diastereomers, and compounds of formulas I, II and III isolated in this way are included within the scope of the compounds disclosed herein. Those skilled in the art will also understand that compounds of formulas I, II and III described herein, and their pharmaceutically acceptable salts, may be deuterated (where hydrogen may be replaced by deuterium), and such molecules are considered to be included within the scope of the compounds disclosed herein.

[0017] Specific examples of compounds of formula I (including IUPAC nomenclature) are shown here:

[0018]

[0019] 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol;

[0020]

[0021] 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-7-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol;

[0022]

[0023] 8-Chloro-5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol;

[0024]

[0025] 7-Chloro-5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol;

[0026]

[0027] 8-Fluoro-5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol;

[0028]

[0029] 7-Fluoro-5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol;

[0030]

[0031] 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-8-methyl-5H-[1]benzopyrano[4,3-c]quinoline-2-ol; and

[0032]

[0033] 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-7-methyl-5H-[1]benzopyrano[4,3-c]quinoline-2-ol.

[0034] Due to the aforementioned chiral centers, these specific examples of the compounds of Formula I each have the R- and S-enantiomers shown in Table 1 (i.e., the R-enantiomers of Formula II and the S-enantiomers of Formula III).

[0035] Table 1: Enantiomers of Compounds of Formula I

[0036]

[0037]

[0038] This document also describes pharmaceutical compositions comprising compounds of formulas I, II, and III as described herein, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable excipients, carriers, or diluents. The pharmaceutical compositions described herein can be prepared using pharmaceutically acceptable additives. As used herein, the term "pharmaceutically acceptable additive" refers to one or more carriers, diluents, and excipients that are compatible with other additives in the composition or formulation and are harmless to the patient. The compounds of formulas I, II, and III as described herein, or pharmaceutically acceptable salts thereof, can be formulated as pharmaceutical compositions for administration via multiple routes, such as oral or IV. Bioavailability is often a factor in cancer treatment, and the ability to select administration methods and pharmaceutical compositions to control or optimize the bioavailability of the active ingredient is useful. For example, orally bioavailable SERD compositions would be particularly useful. Compounds of formulas I, II, and III as described herein, or pharmaceutically acceptable salts thereof, are considered to have oral bioavailability. Examples of pharmaceutical compositions and methods for their preparation can be found in *Remington: The Science and Practice of Pharmacy*, edited by L.V. Allen Jr., 22nd edition, Mack Publishing Co., 2012. Non-limiting examples of pharmaceutically acceptable carriers, diluents, and excipients include: saline, water, starch, sugar, mannitol, and silica derivatives; binders such as carboxymethyl cellulose and other cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; kaolin and bentonite; and polyethylene glycol.

[0039] This article also describes methods for treating cancer. The methods described herein involve administering to a patient requiring said treatment an effective amount of a compound of formulas I, II, and III as described herein, or a pharmaceutically acceptable salt thereof. For example, the method of administering an effective amount of a compound of formulas I, II, and III as described herein, or a pharmaceutically acceptable salt thereof, may be oral administration. The cancer may be an estrogen-responsive cancer. Additionally, 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 ER-positive breast cancer, ER-positive gastric cancer, or ER-positive lung cancer.

[0040] This document also describes compounds of formulas I, II, and III as described herein, or pharmaceutically acceptable salts thereof, for therapeutic purposes. This document also provides compounds of formulas I, II, and III as described herein, or pharmaceutically acceptable salts thereof, for the treatment of breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer. In particular, breast cancer can be ER-positive breast cancer, ER-positive gastric cancer, or ER-positive lung cancer. For example, compounds of formulas I, II, and III, or pharmaceutically acceptable salts thereof, can be administered orally.

[0041] Furthermore, the compounds of formulas I, II, and III described herein, or their pharmaceutically acceptable salts, can be used to prepare medicaments for treating cancer. For example, these medicaments can be administered orally. The types of cancers that can be treated with the medicaments described herein include breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer. In particular, the cancers can be ER-positive breast cancer, ER-positive gastric cancer, or ER-positive lung cancer.

[0042] Compounds of Formulas I, II, and III, and their pharmaceutically acceptable salts, as described herein, have clinical efficacy as a single active agent or in combination with one or more other therapeutic agents (e.g., anticancer agents) for the treatment of cancers such as breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer. When used in combination with other therapeutic agents (e.g., anticancer agents), compounds of Formulas I, II, and III, or their pharmaceutically acceptable salts, as described herein, can be used simultaneously, sequentially, or separately with other therapeutic agents. Examples of drug classes in which compounds of Formulas I, II, and III, or their pharmaceutically acceptable salts, as described herein, can be combined include SERMs, aromatase inhibitors, CDK4 inhibitors, CDK6 inhibitors, PI3K inhibitors, and mTOR inhibitors for the treatment of hormone receptor-positive breast cancer. More specific examples of drugs in which compounds of Formulas I, II and III, or their pharmaceutically acceptable salts, as described herein, can be combined include abemaciclib (CDK4 / 6 inhibitor), everolimus (mTOR inhibitor), alpelisib (PIK3CA inhibitor), and 8-[5-(1-hydroxy-1-methylethyl)pyridin-3-yl]-1-[(2S)-2-methoxypropyl]-3-methyl-1,3-dihydro-2H-imidazo[4,5-c]quinoline-2-one (PI3K / mTOR inhibitor).

[0043] As used herein, the term "effective amount" refers to the amount or dose of a compound of formulas I, II, and III, or a pharmaceutically acceptable salt thereof, as described herein, which provides the desired effect in a diagnosed or treated patient after administration of a single or multiple doses. Preferably, the desired effect is inhibition of tumor cell proliferation, tumor cell death, or both. Compounds of formulas I, II, and III, or pharmaceutically acceptable salts thereof, as described herein, are generally effective over a wide dose range. For example, the daily dose is typically in the range of about 100 mg to about 2000 mg per day.

[0044] As used in this article, "treatment" or "management" refers to suppressing, slowing, stopping, or reversing the progression or severity of existing symptoms or disorders.

[0045] As used in this article, the term "patient" refers to a person who suffers from a particular disease, disorder, or condition.

[0046] Compounds of Formulas I, II, and III, or pharmaceutically acceptable salts thereof, as described herein, can be prepared by a variety of methods known in the art, some of which are illustrated in the preparation and examples below. Specific synthetic steps for each of these pathways may be combined in different ways, or steps from different methods may be combined, to prepare compounds of Formulas I, II, and III, or pharmaceutically acceptable salts thereof, as described herein. Products can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, grinding, and crystallization. Reagents and starting materials are readily available to those skilled in the art.

[0047] Intermediates and methods used to synthesize compounds of Formulas I, II, and III as described herein are intended to be included in this specification. Additionally, some intermediates described herein may contain one or more protecting groups. Depending on the specific reaction conditions and the specific transformation to be performed, the variable protecting group may be the same or different each time it appears. Protecting and deprotecting conditions are well known to those skilled in the art and are described in the literature (see, for example, "Greene's Protective Groups in Organic Synthesis", 4th edition, Peter GMWuts and Theodora W. Greene, John Wiley, and Sons, Inc. 2007).

[0048] Using methods such as selective crystallization or chiral chromatography, those skilled in the art can separate or resolve the individual isomers, enantiomers, and diastereomers at any convenient point in the synthesis of compounds of Formulas I, II, and III as described herein (see, for example, J. Jacques et al., “Enantiomers, Racemates, and Resolutions”, John Wiley and Sons, Inc., 1981, and E.L. Leeel and S.H. Wilen, “Stereochemistry of Organic Compounds”, Wiley-Interscience, 1994). While the individual isomers, enantiomers, and diastereomers can be separated or resolved as described, the Cahn-Ingold-Prelog(R) or (S) nomenclature of their chiral centers may not yet be determined. In cases where the Cahn-Ingold-Prelog(R) or (S) nomenclature is unavailable, the identifiers “Isomer 1” and “Isomer 2” are used, and these are combined with IUPAC names that do not have a Cahn-Ingold-Prelog stereochemical nomenclature. They are separated as defined in the specific experimental description below. Whether isomer is "1" or "2" refers to the order in which compounds of formulas I, II, and III elute from the chiral column under the listed conditions; that is, "isomer 1" elutes first from the column under the stated conditions. If chiral chromatography begins early in the synthesis, the same nomenclature is applied to subsequent intermediates and compounds of formulas I, II, and III.

[0049] Unless otherwise specified, the abbreviations used herein are based on the definitions in Aldrichimica Acta, Vol. 17, No. 1, 1984. Other abbreviations are defined as follows: “ACN” refers to acetonitrile; “BSA” refers to bovine serum albumin; "A Pd G3" refers to [(di(1-adamantyl)-butylphosphine)-2-(2′-amino-1,1′-biphenyl)]palladium(II) methanesulfonate; "DCM" refers to dichloromethane or methylene dichloromethane; "DMA" refers to dimethylacetamide; "DMEA" refers to dimethylethylamine; "DMEM" refers to Dalberg modified Eagle 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" refers to... 50 "This refers to the comparison with the predetermined positive control compound (absolute EC50). 50The concentration of the active agent that produces a 50% target activity response; "EDTA" refers to ethylenediaminetetraacetic acid; "ee" refers to enantiomer excess; "ERα" refers to estrogen receptor α; "ERβ" refers to estrogen receptor β; "EtOAc" refers to ethyl acetate; "EtOH" refers to ethanol or ethanol; "FBS" refers to fetal bovine serum; "HBSS" refers to Hank's balanced salt solution; "HEC" refers to hydroxyethyl cellulose; "HEPES" refers to 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; "HPLC" refers to high performance liquid chromatography; "IC 50 "" refers to the concentration of the surfactant at which it may produce a 50% maximum inhibitory response (relative to IC50) 50 Or the concentration of the active agent that produces 50% inhibition of target enzyme activity compared to the placebo control (absolute IC50). 50 "IPA" refers to isopropylamine; "iPrOH" refers to isopropanol or isopropyl alcohol; "IV" refers to intravenous administration; "K" refers to... i "MeOH" refers to methanol or methanol; "MTBE" refers to methyl tert-butyl ether; "PBS" refers to phosphate-buffered saline; "PO" refers to oral administration; "PRα" refers to progesterone receptor α; "QD" refers to once-daily administration; "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; "SFC" refers to supercritical fluid chromatography; "TED" refers to... 50 "THF" refers to the effective dose that achieves 50% inhibition of the target tumor; "THF" refers to tetrahydrofuran; "t" refers to the effective dose that achieves 50% inhibition of the target tumor. (R) "" refers to retention time; "XantPhosPd G2" refers to chloro[(4,5-bis(diphenylphosphino)-9,9-dimethylxanthanyl)-2-(2′-amino-1,1′-biphenyl)]palladium(II); and "XPhos Pd G2" refers to chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II).

[0050] The following preparation examples and embodiments further illustrate the present invention.

[0051] Preparation examples and embodiments

[0052] Process 1

[0053]

[0054] Procedure 1 describes the synthesis of compound I.

[0055] In step A, a Grignard reaction is completed. Grignard reactions are well known in the art as reactions for forming carbon-carbon bonds. This reaction involves an organometallic reaction in which an aryl magnesium halide (a Grignard reagent) adds to the acyl chloride of a carbonyl group, such as compound 2, to give the compound of step A. For example, a 4-chloro-substituted quinolone (compound 1) is treated with a Grignard reagent, such as isopropyl magnesium chloride, to form a Grignard intermediate, followed by the addition of an acyl chloride (4-fluorobenzoyl chloride) (compound 2) to a solvent, such as THF. Upon completion of the reaction, the reaction can be quenched with water to give compound 3.

[0056] In step B, the aryl methyl ether of compound 3 can be demethylated under a variety of conditions recognizable to a person skilled in the art, such as treatment with boron tribromide. For example, compound 3 is slowly treated with boron tribromide in a solvent such as DCM at a temperature of about 0°C. The mixture is stirred at room temperature and quenched with dipotassium hydrogen phosphate to give compound 4.

[0057] In step C, the azacyclic butane ether 6 can be formed as follows: in a suitable polar aprotic solvent such as DMF or THF, the corresponding p-fluorophenyl ketone 4 and the azacyclic butane alkoxide 5 or the corresponding free base are treated with a suitable base, such as sodium hydride, sodium tert-butoxide or potassium tert-butoxide, to obtain the ether compound 6.

[0058] Compound 6 is then alkylated with a suitable substituted arylboronic acid (compound 7) in a Suzuki cross-coupling reaction to give compound 8 in step D. Those skilled in the art will recognize that various conditions exist that can be used to promote this cross-coupling reaction. Suitable palladium reagents may include XantPhos Pd G2, A Pd G3, bis(triphenylphosphine)palladium(II) chloride, tris(dibenzylacetone)palladium(O) and tricyclohexylphosphine, (1,1'-bis(diphenylphosphine)ferrocene)palladium(II) chloride, tetra(triphenylphosphine)palladium or palladium acetate(II). Suitable bases may include potassium fluoride, cesium carbonate, sodium carbonate, potassium carbonate, lithium tert-butoxide or tripotassium phosphate monohydrate. For example, compound 6 can be reacted with a suitable boric acid compound 7 (e.g., 2-fluoro-4-(trifluoromethyl)phenylboronic acid) in a solvent (e.g., 2-methyl-2-butanol) with a base (e.g., potassium carbonate) and a catalyst (e.g., XPhos Pd G2), and heated under microwave conditions to about 80°C to give compound 8.

[0059] Those skilled in the art will recognize that step D, the Suzuki cross-coupling reaction, can be completed before the formation of the aza-butane ether in step C.

[0060] In step E, those skilled in the art will recognize that compound 8 can be cyclized by initial reduction of a ketone. This can be accomplished using a reducing agent (such as lithium triethylborohydride) in a solvent (such as 1,4-dioxane and THF) and at a temperature of about 0°C to room temperature to give the corresponding secondary alcohol. This intermediate alcohol can be supported on the crude product and deprotonated with a suitable base such as cesium carbonate, sodium hydride, sodium tert-butoxide, or potassium tert-butoxide in a solvent such as THF, DMSO, or DMF. The resulting alkoxide can be cyclized to an aryl fluoride by heating to reflux at room temperature or at a temperature of about 60°C. The substituted cyclic ether formed upon fluoride substitution can then be obtained to give the compound of formula I.

[0061] Alternatively, ketone 8 can be reduced to an alcohol and chiral purified in step F to obtain chiral alcohol 9, which is then cyclized in step G as described above for step E to obtain the compound of formula I.

[0062] In another alternative reaction, the ketone can be reduced directly by a chiral reagent such as (R)-(+)-α,α-diphenyl-2-pyrrolidinemethanol with trimethyl borate and borane-dimethyl sulfide to give the desired chiral alcohol compound 9. Then, compound 9 can be cyclized in step G as described above for step E to give the compound of formula I.

[0063] In an optional step, pharmaceutically acceptable salts of the compounds of formulas I, II, and III described herein can be formed by reacting a suitable free base of the compounds of formulas I, II, and III described herein with a suitable pharmaceutically acceptable acid in a suitable solvent under standard conditions. Additionally, the formation of such salts can occur simultaneously with the deprotection of the nitrogen protecting group. The possible formation of pharmaceutically acceptable salts is well known. See, for example, Gould, PL, “Salt selection for basic drugs,” International Journal of Pharmaceutics. 33 :201-217(1986); Bastin, RJ et al. "Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities," Organic Process Research and Development, 4 :427-435 (2000); and Berge, SM et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, 66:1-19, (1977). Those skilled in the art will understand that the compounds of formulas I, II, and III described herein are readily convertible into pharmaceutically acceptable salts and can be isolated into pharmaceutically acceptable salts. Examples of useful salts include, but are not limited to, benzenesulfonates and 4-methylbenzenesulfonates. 4-Methylbenzenesulfonates are also known as toluenesulfonates.

[0064] Preparation Example 1

[0065] 2-[3-(fluoromethyl)azacyclobutan-1-yl]ethane-1-ol

[0066]

[0067] Sodium triacetoxyborohydride (405 g, 1.91 mol) was added in portions to a stirred 0°C solution of 3-(fluoromethyl)azacyclobutane hydrochloride (160 g, 1.28 mol) in 2.4 L of DCM over N2 for 15 minutes, and stirred at 0°C for 10 minutes. 1,4-Dioxane-2,5-diol (99 g, 0.83 mol) was added in six portions over 1 hour at 0°C, and then stirred at 0–5°C for 15 minutes. The reaction was allowed to cool to room temperature and stirred under N2 for 2 hours. The reaction was cooled to 10–15°C over 20 minutes, then warmed to 25–30°C and maintained at that temperature for 2 hours. Water (800 mL) was added at 10–15°C over 25–30 minutes, and the mixture was allowed to cool to room temperature for 5–10 minutes, after which the layers were separated. The aqueous layer was washed with DCM (800 mL), the layers were separated, and the combined aqueous layer was then cooled to 10–15 °C and the pH was adjusted to 13–14 using a 50% sodium hydroxide solution (~540 mL). The aqueous layer was allowed to cool to room temperature, extracted with DCM (4 x 800 mL), dried over sodium sulfate (80 g), filtered, and concentrated to dryness to give the title compound (139 g, 82%) as a viscous yellow oil. ES / MS (m / z): 134.1 (M+H).

[0068] Preparation Example 2

[0069] 2-[3-(fluoromethyl)azacyclobutane-1-yl]ethanol-1-ol hydrochloride

[0070]

[0071] 529 g (4 mol) of 2-[3-(fluoromethyl)azacyclobutan-1-yl]ethanol-1-ol was dissolved in 2.6 L of MTBE and cooled to 0 °C. HCl / EtOH solution (492 mL, 30 wt%) was added dropwise over 30 minutes, followed by stirring at 0 °C for another 30 minutes. The solid was filtered, and the filter cake was washed with MTBE (2 x 200 mL). The mixture was dried under N2 for 8 hours to give the title compound (580 g, 86%) as a white solid. ES / MS (m / z): 134.0 (M+H).

[0072] Preparation Example 3

[0073] (3-Chloro-7-methoxyquinoline-4-yl)-(4-fluorophenyl)methyl ketone

[0074]

[0075] A mixture of 4-bromo-3-chloro-7-methoxyquinoline (70 g, 254 mmol) and THF (1 L) was cooled to -40 °C under N2, resulting in a precipitate. Isopropyl magnesium chloride (2 M in THF, 254 mL, 509 mmol) was added over 20 minutes, and the mixture was stirred for 1 hour. A solution of 4-fluorobenzoyl chloride (66 mL, 559 mmol) in THF (140 mL) was added dropwise, and the mixture was allowed to warm to room temperature. The reaction was quenched with saturated NH4Cl solution (300 mL) and water (200 mL), and the layers were separated. The organic layer was washed with saturated NH4Cl solution (300 mL), dried over MgSO4, filtered, and concentrated to obtain an oily residue. The crude brown oily residue was eluted with silica gel using a 1:1 mixture of MTBE and hexane to obtain a crude product, a yellow solid (84 g). The solid was treated with 10% methyl acetate / heptane (800 mL) and stirred overnight at room temperature. The solid was collected by filtration and stored. The filtrate was concentrated and purified by elution with silica gel using 10–40% EtOAc / hexane. The product was then treated with 10% methyl acetate / heptane (200 mL) and stirred at room temperature for 3 hours. The filtered solid was combined with the previously filtered solid and dried under vacuum overnight to give the title compound (31 g, 38%) as a yellow solid. ES / MS (m / z): 316.0 (M+H).

[0076] Preparation Example 4

[0077] (3-Chloro-7-hydroxyquinoline-4-yl)-(4-fluorophenyl)methyl ketone

[0078]

[0079] Boron tribromide (1 M, in DCM, 295 mL, 295 mmol) was added to a mixture of (3-chloro-7-methoxyquinoline-4-yl)-(4-fluorophenyl) methyl ketone (31 g, 98 mmol) in DCM (217 mL), and the mixture was stirred at room temperature for 3 days. This mixture was then slowly poured into a 0°C solution of dipotassium hydrogen phosphate (2 M, in water, 700 mL) and water (200 mL). The mixture was allowed to warm to room temperature and stirred for 1 hour. The solution was concentrated under vacuum to remove the organic solvent, filtered, and the filtrate was collected and dried under vacuum at 45°C overnight. The solid was treated with DCM / heptane (1:1, 450 mL) and stirred overnight. The solid was collected and dried under vacuum overnight to give the title compound (32 g, quantitative yield) as a light brown solid. ES / MS (m / z): 302.0 (M+H).

[0080] Preparation Example 5

[0081] (3-Chloro-7-hydroxyquinoline-4-yl)-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)methyl ketone

[0082]

[0083] 2-[3-(fluoromethyl)azacyclobutan-1-yl]ethanol-1-ol hydrochloride (3.90 g, 23.0 mmol) was added to a stirred solution of (3-chloro-7-hydroxyquinoline-4-yl)-(4-fluorophenyl) methyl ketone (5.00 g, 15.3 mmol) in DMF (75 ml), followed by the addition of sodium hydride (60% in mineral oil, 3.02 g, 76.8 mmol). The mixture was stirred under N2 and heated to 40 °C for 45 minutes. The solution was quenched with water and concentrated. The residue was partitioned between 20% iPrOH / CHCl3 and saturated aqueous sodium bicarbonate solution and separated. The aqueous phase was extracted with 2 x 20% iPrOH / CHCl3. The organic extracts were combined, the combined organic layers were dried over magnesium sulfate, filtered, and the filtrate was concentrated to give a crude product as a dark red oil. The crude substance was purified by silica gel column chromatography and eluted with a gradient of 5-10% 7N in MeOH / DCM with NH3 to give the title compound (5.31 g, 84%) as a yellow solid. ES / MS (m / z): 415.0 (M+H).

[0084] Preparation Example 6

[0085] (4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl){3-[2-fluoro-4-(trifluoromethyl)phenyl]-7-hydroxyquinoline-4-yl}methyl ketone

[0086]

[0087] A mixture of (3-chloro-7-hydroxyquinoline-4-yl)-(4-{2-[3-(fluoromethyl)azacyclobutan-1-yl]ethoxy}phenyl) methyl ketone (200 mg, 0.48 mmol), 2-fluoro-4-(trifluoromethyl)phenylboronic acid (158 mg, 0.72 mmol), boric acid (202 mg, 1.45 mmol), 2-methyl-2-butanol (3 ml), and water (1 ml) was degassed with N2 (5x) in a microwave-safe vial. XPhos Pd G2 (12 mg, 0.015 mmol) was added, the vial was sealed, and microwaved at 80 °C for 2 hours. The residue was partitioned between MTBE and a saturated NH4Cl solution. The layers were separated, and the aqueous layer was extracted with MTBE. The organic extracts were combined, dried over magnesium sulfate, filtered, and the filtrate was concentrated to give an orange residue. The crude substance was purified by silica gel column chromatography with elution of 5% MeOH / DCM to give the title compound (205 mg, 78%) as a yellow solid. ES / MS (m / z): 543.2 (M+H).

[0088] The following compounds were prepared in a manner substantially similar to that of Preparation Example 6, with the following modifications: heating for 1-2 hours, extraction with MTBE or EtOAc, and drying of the organic layer with magnesium sulfate or sodium sulfate. Purification was performed by silica gel column chromatography using a maximum of 10% (MeOH or 7M ammonia-containing MeOH) DCM solution (Preparation Example 10: DCM solution with a gradient of 3-8% 7M ammonia-containing MeOH; Preparation Examples 9 and 11: DCM solution with a gradient of 4-10% 7M ammonia-containing MeOH), and / or by high-pH reversed-phase chromatography as described above.

[0089] Table 2: Compounds prepared according to Preparation Example 6

[0090]

[0091]

[0092] a High pH reversed-phase rapid chromatography (RediSep Rf) Purification was performed using a high-efficiency C18 column and eluted with a 10 mM ammonium bicarbonate aqueous solution containing 35-45% ACN (containing 5% MeOH).

[0093] b After purification by elution of silica with 4-10% 7M ammonia-containing MeOH in DCM, it was further purified by high pH reversed-phase rapid chromatography (RediSep Rf). Purification was performed using a high-efficiency C18 column and eluted with a 10 mM ammonium bicarbonate aqueous solution containing 30-44% ACN (containing 5% MeOH).

[0094] Preparation Example 14

[0095] Racemic 4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)(hydroxy)methyl]-3-[2-fluoro-4-(trifluoromethyl)phenyl]quinoline-7-ol

[0096]

[0097] Add (305 g, 562.2 mmol) and THF (1.5 L) together under N2 atmosphere and cool the solution to 0–5 °C. Add triethyllithium borohydride (1 M in THF, 1.5 L, 1.5 mol) dropwise. Stir the mixture at 0–5 °C for 1 hour. Add water (300 mL) and saturated NH4Cl (1 L) dropwise. Warm the mixture to room temperature. Add EtOAc (2 L) and collect the organic layer. Wash the organic layer with brine (500 mL), dry with MgSO4, filter, and concentrate to dryness. The residue was dissolved in a 95:5 mixture of acetone and 2M ammonia in MeOH, and filtered through silica gel to give the title compound (264 g, 86.2%) as an orange solid. ES / MS (m / z): 545.2 (M+H).

[0098] Preparation Example 15

[0099] 4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)(hydroxy)methyl]-3-[2-fluoro-4-(trifluoromethyl)phenyl]quinoline-7-ol, isomer 1

[0100]

[0101] Racemic 4-{2-[3-(fluoromethyl)azacyclobutan-1-yl]ethoxy}phenyl)(hydroxy)methyl]-3-[2-fluoro-4-(trifluoromethyl)phenyl]quinoline-7-ol (354 g, 0.62 mol) was purified by chiral chromatography under the following conditions: column Chiralpak AD-H, 150 x 50 mm, flow rate 300 g / min, UV 350 nm, mobile phase 35% iPrOH containing 0.5% DMEA / CO2, column temperature 40 °C, yielding the title compound (171.4 g, 48%) of the first eluted isomer. Enantiomer enrichment of isomer 1 was confirmed by chiral analysis using SFC, >98% ee, t (R)=0.79 min, column: 4.6×150 mm Chiralpak AD-H, eluted with 35% iPrOH (containing 0.5% DMEA) mobile phase in CO2, flow rate: 0.6 mL / min, UV detection: 350 nm.

[0102] Alternative preparation example 15

[0103] Trimethyl borate (65 mg, 0.62 mmol) was added to a solution of (R)-(+)-α,α-diphenyl-2-pyrrolidinemethanol (132 mg, 0.52 mmol) in THF (20 mL). The mixture was stirred at room temperature under N2 for 1 hour. Borane-dimethyl sulfide (2.0 M in THF, 2.6 mL, 5.2 mmol) was added, followed by (4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl){3-[2-fluoro-4-(trifluoromethyl)phenyl]-7-hydroxyquinoline-4-yl} methyl ketone (1.0 g, 1.73 mmol). The reaction was heated at 45 °C overnight. Another borane-dimethyl sulfide (2.0 M in THF, 2.6 mL, 5.2 mmol) was added, and the mixture was stirred at 45 °C for 5 hours. Slowly add saturated NH4Cl solution (25 mL) to separate the organic phase. Re-extract the water extract with 20% iPrOH / CHCl3. Combine the organic extracts, dry over Na2SO4, filter, and evaporate to give a borane complex intermediate (1.2 g). Dissolve one-third of the borane complex intermediate (0.4 g, 0.6 mmol) in 1,4-dioxane (4 mL) and ethanolamine (0.3 mL, 5 mmol), and heat the reaction to 70 °C for 3 hours. Quench the reaction with saturated NH4Cl solution (25 mL) and separate the organic phase. Re-extract the water extract with 20% iPrOH / CHCl3 (4 x 25 mL). Combine the organic extracts, dry over Na2SO4, filter, and concentrate to dryness to give the title compound as an orange solid (0.33 g, 0.57 mmol, 100% yield). LC / MS (m / z): [M+H] + 545. Enantiomer enrichment of isomer 1 was confirmed by chiral analysis (SFC), 96% ee,t (R) =0.79 min, column: 4.6×150 mm Chiralpak AD-H, eluted with a mobile phase containing 0.5% DMEA in CO2 and 35% iPrOH, flow rate: 0.6 mL / min, UV detection: 350 nm.

[0104] Example 1

[0105] Racemic 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol

[0106]

[0107] A solution of (4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl){3-[2-fluoro-4-(trifluoromethyl)phenyl]-7-hydroxyquinoline-4-yl} methyl ketone (5.27 g, 9.71 mmol) in 1,4-dioxane (100 mL) was cooled to 5 °C. Triethyllithium borohydride (1 M in THF, 30.0 mL, 30.0 mmol) was added. The cooling bath was removed, and the mixture was stirred at room temperature for 1.5 h. The mixture was quenched with water. A saturated NH4Cl solution and EtOAc were added. The layers were separated, and the aqueous layer was extracted with EtOAc. The organic extracts were combined, dried over anhydrous MgSO4, filtered, and the filtrate was concentrated. The crude residue was dissolved in THF (100 mL). Sodium hydride (60% in mineral oil, 1.94 g, 48.5 mmol) was added. The solution was refluxed for 1.5 h. Add additional sodium hydride (60% in mineral oil, 1.94 g, 48.5 mmol), and reflux for another 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 extracts, dry over anhydrous MgSO4, filter, and concentrate the filtrate. Purify the residue by silica gel column chromatography with a 5–7% MeOH gradient in DCM to give the title compound (3.70 g, 72%) as a pale yellow foam. ES / MS (m / z): 525.2 (M+H).

[0108] The following compounds were prepared in a manner substantially similar to that of Example 1, with the following operational changes. For reduction, 3 to 5 equivalents of lithium triethylborohydride were used, the reaction time was 30 minutes to 1 hour, and the organic layer was dried over magnesium sulfate or sodium sulfate. Prior to cyclization, purification was performed directly using the crude residue or by silica gel column chromatography with a 0–5–7.5–10% MeOH gradient in DCM. Cyclolysis was completed by refluxing in THF for up to 16 hours, or by refluxing in DMF at room temperature for 2 hours (for Example 2) and at 85°C for 2 hours (for Example 8). Extraction was performed with DCM or EtOAc, and the organic layer was dried over magnesium sulfate or sodium sulfate. Purification was performed by silica gel column chromatography using a maximum of 10% (MeOH or 7M ammonia-containing MeOH) DCM solution (Example 2: gradient 0-10% MeOH DCM solution; Example 5: gradient 4-10% 7M ammonia-containing MeOH DCM solution; Example 8: gradient 5-7.5% 7M ammonia-containing MeOH DCM solution), or by high pH reversed-phase HPLC as described above.

[0109] Table 3: Compounds prepared according to Example 1

[0110]

[0111] a High pH reversed-phase HPLC Purification was performed using a C18, 5μm, 30×250mM column, eluted with a 10mM ammonium bicarbonate aqueous solution containing 35-50% ACN (containing 5% MeOH).

[0112] b High pH reversed-phase HPLC Purification was performed using a C18, 5μm, 30×250mm column, eluted with a 10mM ammonium bicarbonate aqueous solution containing 35-43% ACN (containing 5% MeOH).

[0113] c After purification by elution with silica gel using 4-10% 7M ammonia-containing MeOH in DCM, the sample was further purified by high-pH reversed-phase HPLC. Purification was performed using a C18, 5μm, 30×250mm column, eluted with a 10mM ammonium bicarbonate aqueous solution containing 30-44% ACN (containing 5% MeOH).

[0114] d High pH reversed-phase HPLC Purification was performed using a C18 5μm OBD, 30×75mm column, eluted with 10mM ammonium bicarbonate aqueous solution containing 10-75% ACN (containing 5% MeOH).

[0115] e High pH reversed-phase HPLC Purification was performed using a C18 5μm OBD, 30×75mm column, eluted with 10mM ammonium bicarbonate aqueous solution containing 10-60% ACN (containing 5% MeOH).

[0116] Example 1A

[0117] 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 1

[0118] and

[0119] Example 1B

[0120] 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 2

[0121]

[0122] Two enantiomers of 5-(4-{2-[3-(fluoromethyl)azacyclobutan-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol were separated by chiral SFC under the following conditions: Column: Cellulose-1, 5×25 cm; eluted with a mobile phase of 30% iPrOH (containing 0.5% DMEA) in CO2; column temperature: 40 °C; flow rate: 300 g / min; UV detection wavelength: 270 nm, yielding Example 1A, the first eluted enantiomer (isomer 1). ES / MS (m / z): 525.2 (M+H). Enantiomer enrichment of isomer 1 was confirmed by chiral analysis using SFC, >99% ee,t. (R) :1.30 minutes; Column: OD-H, 4.6 × 150 mm; eluted with a mobile phase of 30% MeOH (0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 5 mL / min; UV detection wavelength: 225 nm. The title compound of Example 1B was separated to give a second eluted enantiomer (isomer 2). ES / MS (m / z): 525.2 (M+H). Enantiomer enrichment of isomer 2 was confirmed by chiral analysis using SFC, 98% ee,t. (R) 2.03 minutes; Column: OD-H, 4.6 × 150 mm; eluted with a mobile phase of 30% MeOH (0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 5 mL / min; UV detection wavelength: 225 nm.

[0123] Alternative preparation example 1B

[0124] Crystalline 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 2

[0125] 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, 4-methylbenzenesulfonic acid, isomer 2 (23.8 g, 0.034 mol) was stirred at 1000 rpm in water (250 mL). NaOH (76 μL) was added and the solution was stirred for 2 hours. DCM (600 mL) was added. The mixture was separated, the DCM extract was dried with magnesium sulfate, the material was filtered through a syringe filter (0.45 μm) and concentrated to dryness. The material was placed under a flow of N2 for the weekend. EtOH / water (80 mL) was added and the mixture was stirred under sonication. The brown solid was collected by filtration through a nylon membrane to give the title compound (10.47 g, 0.02 mol, 59%).

[0126] X-ray powder diffraction (XRD)

[0127] XRPD patterns of the crystalline solid were obtained on a Bruker D4 Endeav X-ray powder diffractometer equipped with a CuKα source and a Vantec detector, operating at 35 kV and 50 mA. The sample was scanned between 4 and 40 2θ° with a step size of 0.008 2θ° at a scan rate of 0.5 s / step, using a divergence of 1.0 mm, a fixed antiscattering of 6.6 mm, and a detector slit of 11.3 mm. The dried powder was mounted on a quartz sample holder, and a smooth surface was obtained using a glass slide. Crystalline diffraction patterns were collected at ambient temperature and relative humidity. In an MDI-Jade, the crystal peak positions were determined after full pattern movement according to internal NIST 675 standards, with peak values ​​at 8.853 and 26.774 2θ°. It is well known in crystallography that for any given crystal form, the relative intensity of diffraction peaks can vary due to preferred orientations caused by factors such as crystal morphology and habit. When the influence of preferred orientation is present, the peak intensity changes, but the characteristic peak positions of the polymorph remain unchanged. See, for example, United States Pharmacopeia #23, National Formulary #18, pp. 1843-1844, 1995. Furthermore, it is known in crystallography that the angular peak positions can vary slightly for any given crystal form. For example, peak positions may shift due to temperature variations in the analyzed sample, sample substitution, or the presence or absence of an internal standard. In this case, a peak position variation of ±0.2 2θ° is considered to account for these potential variations without impeding the definitive identification of the indicated crystal form. The crystal form can be confirmed based on any unique combination of distinguishing peaks.

[0128] The prepared crystalline sample of 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol (isomer 2) was characterized by CuKa radiation XRD pattern, which had the diffraction peaks (2θ values) described in Table 4 below, particularly the combination of a peak at 19.8 with one or more peaks selected from 6.8, 16.0 and 22.1; the deviation of the diffraction angle was 0.2 degrees.

[0129] Table 4: X-ray powder diffraction peaks of crystallization example 1B

[0130] 1 6.8 29.40 2 15.3 8.30 3 16.0 20.10 4 17.4 7.60 5 18.1 16.00 6 19.8 100.00 7 21.1 14.60 8 22.1 28.90 9 24.9 16.40 10 25.4 21.90

[0131] Alternative preparation example 1B

[0132] 4-{2-[3-(fluoromethyl)azacyclobutan-1-yl]ethoxy}phenyl)(hydroxy)methyl]-3-[2-fluoro-4-(trifluoromethyl)phenyl]quinoline-7-ol, isomer 1 (63.05 g, 104.7 mmol), was dissolved in DMSO (1.3 L) at room temperature under N2. Cesium carbonate (108 g, 331 mmol) was added in portions over 5 minutes. The mixture was heated to 60 °C for 15 hours. The mixture was cooled to room temperature and diluted with water (2.1 L) and EtOAc (1.3 L). The mixture was stirred for 5 minutes and then separated. The aqueous residue was re-extracted with EtOAc (1.3 L) and stirred for 5 minutes. The organic extracts were separated and combined, and washed with brine, water, and EtOAc. The organic extract was dried over MgSO4, concentrated, and dried overnight at room temperature under high vacuum to give the title compound as a brown solid (52.69 g, 95.9%). Enantiomer enrichment in Example 1B was confirmed by chiral analysis (SFC), 98.1% ee,t. (R) 2.03 minutes; Column: OD-H, 4.6 × 150 mm; eluted with a mobile phase of 30% MeOH (0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 5 mL / min; UV detection wavelength: 225 nm.

[0133] Example 2A

[0134] 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-7-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 1

[0135] and

[0136] Example 2B

[0137] 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-7-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 2

[0138]

[0139] Two enantiomers of 5-(4-{2-[3-(fluoromethyl)azacyclobutan-1-yl]ethoxy}phenyl)-7-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol were separated by chiral SFC under the following conditions: Column: IC, 21×250cm; eluted with a mobile phase of 30% iPrOH (containing 0.2% IPA) in CO2; column temperature: 40℃; flow rate: 70 g / min; UV detection wavelength: 225 nm, yielding Example 2A, the first eluted enantiomer (isomer 1). ES / MS (m / z): 525.1 (M+H). Chiral analysis by SFC confirmed enantiomer enrichment of isomer 1, >99% ee,t. (R) 1.56 minutes; Column: IC, 4.6 × 150 mm; eluted with a mobile phase of 30% iPrOH (containing 0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 5 mL / min; UV detection wavelength: 225 nm. The title compound of Example 2B was separated to give a second eluted enantiomer (isomer 2). ES / MS (m / z): 525.2 (M+H). Enantiomer enrichment of isomer 2 was confirmed by chiral analysis using SFC, 98% ee,t. (R) 2.33 minutes; Column: IC, 4.6 × 150 mm; eluted with a mobile phase of 30% iPrOH (containing 0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 5 mL / min; UV detection wavelength: 225 nm.

[0140] Example 3A

[0141] 8-Chloro-5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 1

[0142] and

[0143] Example 3B

[0144] 8-Chloro-5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 2

[0145]

[0146] Two enantiomers of 8-chloro-5-(4-{2-[3-(fluoromethyl)azacyclobutan-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol were separated by chiral SFC under the following conditions: Column: OD-H, 21 × 250 cm; eluted with a mobile phase of 35% MeOH (containing 0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 80 g / min; UV detection wavelength: 225 nm, yielding Example 3A, the first eluted enantiomer (isomer 1). ES / MS (m / z): 491.0 (M+H). Chiral analysis by SFC confirmed enantiomer enrichment of isomer 1, >99% ee,t. (R) 1.55 minutes; Column: OD-H, 4.6 × 150 mm; eluted with a mobile phase of 35% MeOH (0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 5 mL / min; UV detection wavelength: 225 nm. The title compound of Example 3B was separated to give a second eluted enantiomer (isomer 2). ES / MS (m / z): 491.0 (M+H). Enantiomer enrichment of isomer 2 was confirmed by chiral analysis using SFC, >99% ee,t. (R) 2.26 minutes; Column: OD-H, 4.6 × 150 mm; eluted with a mobile phase of 35% MeOH (0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 5 mL / min; UV detection wavelength: 225 nm.

[0147] Example 4A

[0148] 7-Chloro-5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 1

[0149] and

[0150] Example 4B

[0151] 7-Chloro-5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 2

[0152]

[0153] Two enantiomers of 7-chloro-5-(4-{2-[3-(fluoromethyl)azacyclobutan-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol were separated by chiral SFC under the following conditions: Column: OD-H, 21 × 250 cm; eluted with a mobile phase of 35% MeOH (containing 0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 80 g / min; UV detection wavelength: 225 nm, yielding Example 4A, the first eluted enantiomer (isomer 1). ES / MS (m / z): 491.0 (M+H). Enantiomer enrichment of isomer 1 was confirmed by chiral analysis using SFC, >99% ee,t. (R) 1.71 minutes; Column: OD-H, 4.6 × 150 mm; eluted with a mobile phase of 35% MeOH (0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 5 mL / min; UV detection wavelength: 225 nm. The title compound of Example 4B was separated to give a second eluted enantiomer (isomer 2). ES / MS (m / z): 491.0 (M+H). Enantiomer enrichment of isomer 2 was confirmed by chiral analysis using SFC, >99% ee,t. (R) 2.38 minutes; Column: OD-H, 4.6 × 150 mm; eluted with a mobile phase of 35% MeOH (0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 5 mL / min; UV detection wavelength: 225 nm.

[0154] Example 5A

[0155] 8-Fluoro-5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 1

[0156] and

[0157] Example 5B

[0158] 8-Fluoro-5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 2

[0159]

[0160] Two enantiomers of 8-fluoro-5-(4-{2-[3-(fluoromethyl)azacyclobutan-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol were separated by chiral SFC under the following conditions: Column: OD-H, 21 × 250 cm; eluted with a mobile phase of 30% MeOH (containing 0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 80 g / min; UV detection wavelength: 225 nm, yielding Example 5A, the first eluted enantiomer (isomer 1). ES / MS (m / z): 475.0 (M+H). Chiral analysis by SFC confirmed enantiomer enrichment of isomer 1, >99% ee,t. (R) 1.56 minutes; Column: OD-H, 4.6 × 150 mm; eluted with a mobile phase of 30% MeOH (0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 5 mL / min; UV detection wavelength: 225 nm. The title compound of Example 5B was separated to give a second eluted enantiomer (isomer 2). ES / MS (m / z): 475.0 (M+H). Enantiomer enrichment of isomer 2 was confirmed by chiral analysis using SFC, >99% ee,t. (R) 2.29 minutes; Column: OD-H, 4.6 × 150 mm; eluted with a mobile phase of 30% MeOH (0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 5 mL / min; UV detection wavelength: 225 nm.

[0161] Example 6A

[0162] 7-Fluoro-5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 1

[0163] and

[0164] Example 6B

[0165] 7-Fluoro-5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 2

[0166]

[0167] Two enantiomers of 7-fluoro-5-(4-{2-[3-(fluoromethyl)azacyclobutan-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol were separated by chiral SFC under the following conditions: column: OD-H, 21 × 250 cm; eluted with a mobile phase of 35% MeOH (containing 0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 80 g / min; UV detection wavelength: 225 nm, yielding Example 6A, the first eluted enantiomer (isomer 1). ES / MS (m / z): 475.0 (M+H). Enantiomer enrichment of isomer 1 was confirmed by chiral analysis using SFC, >99% ee,t. (R) 1.32 minutes; Column: OD-H, 4.6 × 150 mm; eluted with a mobile phase of 35% MeOH (0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 5 mL / min; UV detection wavelength: 225 nm. The title compound of Example 6B was separated to give a second eluted enantiomer (isomer 2). ES / MS (m / z): 475.0 (M+H). Enantiomer enrichment of isomer 2 was confirmed by chiral analysis using SFC, >99% ee,t. (R) 1.95 minutes; Column: OD-H, 4.6 × 150 mm; eluted with a mobile phase of 35% MeOH (0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 5 mL / min; UV detection wavelength: 225 nm.

[0168] Example 8A

[0169] 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-7-methyl-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 1

[0170] and

[0171] Example 8B

[0172] 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-7-methyl-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 2

[0173]

[0174] Two enantiomers of 5-(4-{2-[3-(fluoromethyl)azacyclobutan-1-yl]ethoxy}phenyl)-7-methyl-5H-[1]benzopyrano[4,3-c]quinoline-2-ol were separated by chiral SFC under the following conditions: column: OD-H, 21 × 250 cm; eluted with a mobile phase of 30% iPrOH (containing 0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 80 g / min; UV detection wavelength: 265 nm, yielding Example 8A, the first eluted enantiomer (isomer 1). ES / MS (m / z): 471.2 (M+H). Enantiomer enrichment of isomer 1 was confirmed by chiral analysis using SFC, >99% ee,t. (R) 1.47 minutes; Column: OD-H, 4.6 × 150 mm; eluted with a mobile phase of 30% iPrOH (containing 0.2% IPA) in CO2; column temperature: 40 °C; flow rate: 5 mL / min; UV detection wavelength: 225 nm. The title compound of Example 8B was separated to give a second eluted enantiomer (isomer 2). ES / MS (m / z): 471.2 (M+H). Enantiomer enrichment of isomer 2 was confirmed by chiral analysis using SFC, >99% ee,t. (R) 2.05 minutes; Column: OD-H, 4.6 × 150 mm; eluted with a mobile phase of 30% iPrOH (containing 0.2% IPA) in CO2; column temperature: 40℃; flow rate: 5 mL / min; UV detection wavelength: 225 nm.

[0175] Example 9

[0176] 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 2, benzenesulfonic acid

[0177]

[0178] A slurry of 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 2 (Example 1B) (100 mg, 0.19 mmol) in ACN (3 mL) was heated at 50 °C. A solution of benzenesulfonic acid monohydrate (40 mg, 0.23 mmol) in ACN (1 mL) was added. The clear yellow solution was heated at 50 °C for 10 minutes. Heating was stopped, the reaction mixture was cooled to room temperature, and the mixture was stirred overnight. Toluene (2 mL) was added, and the reaction mixture was stirred for 2 hours. The solution was filtered, the resulting solid was collected, and the solid was washed with ACN (1 mL). The solid was dried under vacuum to give the title compound (74 mg, 55%).

[0179] Alternative preparation example 9

[0180] A slurry of 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 2 (Example 1B) (124.1 mg, 0.24 mmol) in MEK (4 mL) was heated at 50 °C. A solution of benzenesulfonic acid monohydrate (50 mg, 0.28 mmol) dissolved in MEK (1 mL) was added. Heating was stopped, the reaction mixture was cooled to room temperature, and the mixture was stirred over the weekend. The mixture was concentrated under a stream of N2. MEK (1 mL) and the slurry were added to give a yellow crystalline solid. The solid was collected, washed with MEK, and dried under vacuum at room temperature to give the title compound (78.8 mg, 48%).

[0181] XRD, Example 9

[0182] XRD was performed as described in Example 1B. The prepared sample of -(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 2, benzenesulfonic acid was characterized by CuKa radiation XRD pattern, which had diffraction peaks (2θ values) as described in Table 5 below, particularly a peak at 20.5 and a combination of one or more peaks selected from 12.3, 22.2 and 23.1; the deviation of the diffraction angle was 0.2 degrees.

[0183] Table 5: X-ray powder diffraction peaks of crystallization example 9

[0184]

[0185]

[0186] Example 10

[0187] Crystalline 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, 4-methylbenzenesulfonic acid, isomer 2

[0188]

[0189] 5-(4-{2-[3-(fluoromethyl)azacyclobutan-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, isomer 2 (Example 1B) (204.2 g, 389 mmol) and EtOAc (5 L) were added together and stirred at 60 °C. MeOH (200 mL) was then added at 60 °C to give a clear brown solution. The title product (11.48 g) was added to inoculate the solution, followed by a premixed solution of 4-methylbenzenesulfonic acid; hydrate (81.4 g, 428 mmol) in EtOAc (800 mL) to give a yellow suspension. The suspension was stirred at 50 °C for 30 min. The suspension was concentrated to half volume. The solution was cooled to room temperature for 1 h, filtered, the solid was collected, and washed with EtOAc. The solid was dried under vacuum at 30°C over the weekend to give the title compound (239 g, 343 mmol). To further purify this substance, the title compound (229 g, 328.7 mmol) and 2-propanol (4.6 L) were added together, and the mixture was heated to 60°C for 2 hours. The mixture was cooled to room temperature for 30 minutes. The solid was filtered and washed with iPrOH (100 mL). The solid was dried under N2 flow overnight to give the title compound (174.4 g, 76.2%). The batches of title compounds prepared in essentially the same manner were combined, and heptane (2 L) was added. The suspension was stirred for 30 minutes, the solid was filtered, and washed with heptane (300 mL). The collected solid was dried under N2 flow overnight to give the title compound (199.7 g, 99.5%).

[0190] XRD, Example 10

[0191] XRD was performed as described in Example 1B. The preparation of the sample of 5-(4-{2-[3-(fluoromethyl)azacyclobutane-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinoline-2-ol, 4-methylbenzenesulfonic acid, isomer 2 (Example 10) is characterized by an XRD pattern obtained using CuKa radiation, which has diffraction peaks (2-θ values) as described in Table 6 below, particularly a peak at 20.1 combined with one or more peaks selected from 12.8, 19.5 and 22.8; the deviation of the diffraction angle is 0.2 degrees.

[0192] Table 6: X-ray powder diffraction peaks of crystallization example 10

[0193] 1 7.6 25.70 2 12.4 27.90 3 12.8 36.80 4 18.9 26.50 5 19.5 56.90 6 20.1 100.00 7 20.9 41.50 8 21.8 40.90 9 22.8 39.40 10 25.4 29.70

[0194] Biological assay

[0195] Evidence of a relationship between ER expression and certain cancers is well-known in the field.

[0196] The results of the following measurements demonstrate that the compounds of formulas I, II and III in the examples are active SERDs and are considered to be useful for the treatment of cancer.

[0197] Competitive binding assays of ER (wild-type), ERα (Y537S mutant) and ERβ

[0198] The purpose of the following ER competitive binding assay is to determine the affinity of the test compound for ERα (wild type), ERα (Y537S mutant), and ERβ.

[0199] Competitive binding assays were performed using a 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 μCi / well. 3 H-estradiol (118 Ci / mmol, 1 mCi / mL), 7.2 ng / well ERα (wild type), 7.2 ng / well ERα (Y537S mutant), or 7.7 ng / well ERβ receptor. Ten different concentrations of the test compound were added at concentrations ranging from 10,000 nM to 0.5 nM, and nonspecific binding was measured in the presence of 1 μM 17β-estradiol. The binding reaction mixture (140 μL) was incubated at room temperature for 4 h, and then cold dextran-char buffer (70 μL) was added to each reaction mixture (each 50 mL of assay buffer contained 0.75 g char and 0.25 g dextran). Plates were mixed on a fixed-track shaker at 4 °C for 8 min, and then centrifuged at 3000 rpm for 10 min at 4 °C. Aliquots (120 μL) of the mixture were transferred to another 96-well white substrate (Costar), and 175 μL of Perkin Elmer Optiphase Supermix scintillation solution was added to each well. The substrate was sealed and vibrated vigorously on a track vibrator. After incubation for 2.5 hours, the substrate was read using a Wallac Microbeta counter. The IC was calculated using 4-parameter logic curve fitting. 50 The % inhibition at 10 μM was calculated. The IC50 of the compound was determined using the Cheng-Prusoff equation. 50 Value converted to K i The results of this assay demonstrate that Examples 1, 1A, and 1B (and other examples) combined recombinant ERα wild-type and ERα mutant (Y537S), as shown in Table 7 below, and also measured Example 1B with a K0.11 + 0.07. i (nM)ERβ competes with ERβ, n=3.

[0200] Table 7: Results of competitive binding of ERα (wild type), ERα (Y537S mutant) and ERβ

[0201] 1 0.87 5.80 1A 12.45±9.32,n＝3 57.18±39.13,n＝3 1B 0.31±0.38,n＝5 2.79±3.00,n＝5 2 2.17 6.78 2A 0.65 7.92 2B 60.4 293.6 3 2.36 6.69 3A 8.11 27.23 3B 0.59 2.79 4 0.64 12.11 4A 16.78 54.97 4B 0.34 2.34 5 2.82 19.47 5A 12.54 81.15 5B 1.30 6.56 6 4.14 15.77 6A 8.53 45.99 6B 1.13 5.71 7 1.55 8.55 8 3.20 11.4 8A 9.33 66.94 8B 0.94 5.44

[0202] In the exemplary compounds tested, the Ki range for the wild-type ERα was about 0.300 nM to about 65 nM. The Ki range for the ERαY537S mutant was about 2 nM to 300 nM. The results of this assay demonstrate the binding affinity and potency of the example compounds to the wild-type ERα, the mutant (ESR1 Y537NS), and the ERβ protein.

[0203] ERα degradation assay in MCF7 cells

[0204] The purpose of the following ERα degradation assay is to determine the degradation of ERα by the test compound in ERα-positive breast cancer cell lines such as MCF7.

[0205] MCF7 cells (purchased from ATCC HTB-22) were cultured in DMEM medium supplemented with 10% FBS, 0.01 mg / mL human insulin, and 1% penicillin / streptomycin antibiotics, and seeded at a density of 4,000 cells / well in 20 μL of phenol red-free DMEM medium containing 10% activated charcoal-treated FBS in 384-well flat-bottom plates. Cells were incubated overnight in a cell culture incubator (5% CO2, 95% relative humidity, and 37°C) to allow cell attachment. The test compound was administered to the cells the following day. Serial dilutions (1:3) of the test compound were prepared using an Echo 555 acoustic dispenser, ranging from 6 μM to 0.0003 μM. 5 μL of the serial dilution was added to the cell plate to administer the compound, resulting in a final DMSO concentration of 0.2%, with the final test compound concentration ranging from 2 to 0.0001 μM. For the maximum point, medium containing 0.2% DMSO was used; for the minimum point, fulvestrant was diluted to a final concentration of 2 μM in growth medium containing 0.2% DMSO. After administration of the test compound, the cell plates were incubated at 37°C and 5% CO2 for 24 hours. Cells were fixed by adding 14% paraformaldehyde (10 μL) for 30 minutes at room temperature. Cells were washed once with PBS (20 μL) and treated with 0.5% (v / v) fulvestrant. Incubate with 20 μL of PBS for 1 hour. Use a solution containing 0.05%... Wash cells with 20 μL PBS (2×) and use a solution containing 0.05% PBS. 20 and 0.1% TRITON TMX-100 was blocked in PBS with 3% BSA (20 μL / well) for 1 hour at room temperature. 20 μL of primary antibody (ERα (clone SP1) monoclonal rabbit antibody #RM-9101-S, Thermo Scientific) was added to each well at a 1:500 dilution in PBS containing 0.05% BSA. In step 20), the sealed plate was incubated overnight at 4°C. The next day, it was incubated with a solution containing 0.05%... Cells were washed with 20 (2×) PBS and then mixed with secondary antibody (20 μL / well) in PBS containing 1% BSA (1:1000 dilution, goat anti-rabbit IgM ALEXA FLUOR). TM 488) Incubate at room temperature for 105 minutes. After washing the plate with PBS (2 × 20 μL), add RNase (Sigma) (20 μL, 50 μg / mL) and a 1:1000 dilution of propidium iodide in PBS per well (20 μL). Seal the plate and incubate at room temperature for 1 hour (protected from light). Use ACUMENEXPLORER. TM [TTP LABTECH LTD Laser Scanning Fluorescence Microplate Cytometer] Scans the plate to measure ERα. Image analysis is based on cell fluorescence signals for identifying positive cells. ER-positive cells are identified by average intensity. Individual cells are identified by total intensity of propidium iodide / DNA at 575-640 nm. The experimental output value is %ER-positive cells. This is achieved using GENE DATA. TM For each output value, curve fitting is performed to a four-parameter logic curve to determine the IC. 50 The results of this experiment demonstrate the potent degradation of ERα induced by compounds of formulas I, II, and III described herein in MCF7 breast cancer cells. Relative IC50 values ​​for Examples 1, 1A, and 1B are also provided. 50 The values ​​are shown in Table 8.

[0206] Table 8: ERα degradation assay in MCF7 cells

[0207]

[0208]

[0209] Specifically, the results in Table 8 show that the compounds of Example 1 potently degrade ERα in MCF7 breast cancer cells. Among the exemplary compounds tested, relative to IC50... 50 The range was 0.003 to >2 μM, indicating that all compounds except those in Example 2B showed activity at the tested concentrations. This analysis demonstrates that the compound of formula (I) is a SERD with potent ERα-degrading activity in cells.

[0210] Assay for PRα induction in MCF7 cells

[0211] The purpose of the following PRα induction assay is to determine whether the test compound has ERα receptor agonist activity (the agonist is expected to activate the receptor).

[0212] MCF7 cells (purchased from ATCC HTB-22) were cultured in DMEM medium supplemented with 10% FBS, 0.01 mg / mL human insulin, and 1% penicillin / streptomycin antibiotics. Cells (before reaching 70% confluence) were seeded in 384-well plates at a density of 4,000 cells per well in 20 μL of phenol red-free DMEM medium (treated with activated charcoal) containing 10% FBS. Cells were incubated overnight in a cell culture incubator (5% CO2, 95% relative humidity, 37°C) to allow cell attachment. The next day, the test compound was administered to the cells. Serial dilutions (1:3) of the compound were prepared in the range of 6 μM to 0.0003 μM using an Echo 555 acoustic dispenser. 5 μL of the test compound from the serial dilution plate was added to the cell plate to administer the compound to the cells, resulting in a final DMSO concentration of 0.2%. The final concentration dose of the test compound ranged from 2 to 0.0001 μM. For the maximum point, medium containing 0.2% DMSO was used; for the minimum point, fulvestrant was diluted to a final concentration of 2 μM in growth medium containing 0.2% DMSO. After administration of the test compound, the cell plates were incubated at 37°C and 5% CO2 for 24 hours. Cells were fixed by adding 14% paraformaldehyde (10 μL) for 30 minutes at room temperature. Cells were washed once with PBS (20 μL) and treated with 0.5% (v / v) fulvestrant. Incubate with 20 μL of PBS for 1 hour. Then, at room temperature, use a solution containing 0.05%... Wash cells twice with 20 μL of PBS and use a solution containing 0.05% PBS. 20 and 0.1% TRITON TM X-100 cells were blocked for 1 hour with 3% BSA (20 μL / well) in PBS. 1:500 primary antibody (20 μL) (PR monoclonal mouse anti-human antibody, clone PgR 636Dako, M3569) was added to each well in 1% BSA / PBS (containing 0.05... The diluted solution in 20) was sealed in a plate and incubated overnight at 4°C.

[0213] The next day, use 0.05% PBS. Wash cells with 20 μL (2 × 20 μL) of secondary antibody in PBS and 1% BSA (20 μL / well) (1:1000 dilution, goat anti-rabbit IgM ALEXA FLUOR). TM488) Incubate at room temperature for 105 minutes. After washing with PBS (2 × 20 μL), add RNase (20 μL, 50 μg / mL) (Sigma) and a 1:1000 dilution of propidium iodide in PBS to each well. Seal the plate and incubate at room temperature for 1 hour (protected from light). Analyze using ACUMEN EXPLORER. TM [TTP LABTECH LTD Laser Scanning Fluorescence Microplate Cytometer] Scans the plate to measure PRα. Image analysis is based on cell fluorescence signals for identifying positive cells. PR-positive cells are identified by average intensity. Individual cells are identified by total intensity of propidium iodide / DNA at 575-640 nm. The experimental output value is %PR-positive cells. This is achieved using GENE DATA. TM For each output value, curve fitting is performed to a four-parameter logic curve to determine the IC. 50 The results of this experiment showed that Examples 1, 1A, and 1B did not exhibit significant agonistic activity in MCF7 breast cancer cells. For the compounds tested, the relative IC50 values ​​in this experiment were... 50 >2 μM. The results of this experiment indicate that the example compounds tested in MCF7 breast cancer cells did not exhibit significant agonistic activity. These results also demonstrate that the exemplary compounds tested are ERα antagonists in MCF7 breast cancer cells (i.e., they possess SERD activity).

[0214] PRα inhibition (ERα antagonism) cell assay in MCF7-ESR1 Y537N 682CRISPR cells

[0215] The purpose of the following PRα inhibition (ERα function antagonism) cellular assay is to determine the antagonistic activity of the test compound against the Y537N mutant ERα receptor. In this assay, the antagonist is expected to block the function of the ERα receptor. PRα is a downstream transcriptional target of ERα, therefore, ERα antagonists are expected to inhibit PRα expression.

[0216] MCF7-ESR1Y537N-682 cells (generated by CRISPR / Cas9 gene editing of the ESR1 gene in MCF7 cells, clone #682) were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin antibiotics. Cells (before reaching 70% confluence) were seeded in 384-well plates in phenol red-free DMEM medium with 10% FBS (20 μL volume) (treated with activated charcoal) at a density of 4,000 cells per well. Cells were incubated overnight in a cell culture incubator (5% CO2, 95% relative humidity, and 37°C) to allow cell attachment to the plate. The test compound was administered to the cells the following day. Serial dilutions (1:3) of the compound were prepared in the range of 6 μM to 0.0003 μM using an Echo 555 acoustic dispenser. Add 5 μL of the serial dilution medium from the cell plate to administer the compound to the cells, producing a final DMSO concentration of 0.2%. The final test compound concentration ranged from 2 to 0.0001 μM. For the maximum point, medium containing 0.2% DMSO was used, and for the minimum point, fulvestrant was diluted to a final concentration of 2 μM in growth medium containing 0.2% DMSO. After administration of the test compound, the cell plates were incubated at 37°C and 5% CO2 for 72 hours. Cells were fixed by adding 10 μL of 14% paraformaldehyde at room temperature for 30 minutes. Cells were washed with PBS (1 × 20 μL) and treated with 0.5% (v / v) DMSO. Incubate with 20 μL of PBS for 1 hour. Then incubate with 2 × 20 μL of PBS and 0.05% PBS. Wash cells with 20 ml of 3% BSA / PBS and 0.05% PBS. 20, 0.1% TRITON TM X-100 (20 μL / well) was used for blocking at room temperature for 1 hour. 1:500 primary antibody (20 μL) (PR monoclonal mouse anti-human antibody, clone PgR 636Dako, M3569) was added to each well in 1% BSA / PBS (containing 0.05%...) The diluted solution in 20) was sealed in a plate and incubated overnight at 4°C.

[0217] The next day, with PBS Wash cells (2 × 20 μL) and add secondary antibody (20 μL / well) in PBS and 1% BSA (1:1000 dilution, goat anti-rabbit IgM ALEXA FLUOR) to the cells. TM488) Incubate at room temperature for 105 minutes. After washing with PBS (2 × 20 μL), add RNase (20 μL, 50 μg / mL) (Sigma) and a 1:1000 dilution of propidium iodide in PBS per well. Seal the plate and incubate at room temperature for 1 hour (protected from light). Analyze with ACUMEN EXPLORER. TM [TTP LABTECHLTD Laser Scanning Fluorescent Microculture Plate Hemocytometer] The plate is scanned to measure PRα. Image analysis is based on cell fluorescence signals for identifying positive cells. PR-positive cells are identified by average intensity. Individual cells are identified by total intensity of propidium iodide / DNA at 575-640 nm. The experimental output value is %PR-positive cells. This is achieved using GENE DATA. TM For each output value, curve fitting is performed to a four-parameter logic curve to determine the IC. 50 .

[0218] The results of this experiment indicate that Examples 1, 1A, and 1B potently inhibit PRα and antagonize its function in MCF7 (ESR1 Y537N, heterozygous mutant) breast cancer cells. In this experiment, the relative IC50 values ​​of Examples 1, 1A, and 1B (and other examples) were significantly higher. 50 The relative IC50 values ​​of exemplary compounds tested are shown in Table 9 below. 50 The range of approximately 0.0118 to >1.6 μM indicates that the exemplary compound is a potent antagonist of ERα mutants (Y537N) and a potent inhibitor of ERα-mediated transcription, except for Example 2B (1.6 μM). PRα (PGR) is also a transcriptional target of ERα, and the results of this assay demonstrate potent inhibition of ERα-mediated PRα transcription.

[0219] Table 9: PRα inhibition (ERα antagonism) cell assay in MCF7Y537N 682 CRISPR cells

[0220] 1 0.01679±0.00003,n＝2 1A 1.20±0.29,n＝2 1B 0.0130±0.0059,n＝14 2 0.01451±0.002619,n＝2 2A 0.02494±0.007386,n＝3 2B 1.639±0.2228,n＝3 3 0.07717±0.01154,n＝2 3A 0.6117 3B 0.016 4 0.03854+0.003865,n＝2 4A 0.5052 4B 0.01181 5 0.06614±0.01551,n＝2 5A 0.3945 5B 0.01822+0.009815,n＝2 6 0.06319±0.01609,n＝2 6A 0.2364 6B 0.0136 7 0.1271 8 0.04124+0.006572,n＝2 8A 0.4335+0.1946,n＝3 8B 0.008926±0.003828,n＝3 10 0.007936±0.003163,n＝3

[0221] PRα inhibition (ERα antagonism) cell assay in MCF7 cells

[0222] The purpose of the following PRα inhibition (ERα function antagonism) cell assay is to determine the antagonistic activity of the test compound against the ERα receptor. In this assay, the antagonist is expected to block the function of the ERα receptor. PRα is a downstream transcriptional target of ERα, therefore, ERα antagonists are expected to inhibit PRα expression.

[0223] The assay conditions detailed in the above-described ERα-degrading cell-based Acumen assay were performed using the MCF7 cell line, with the difference that the culture medium was removed from the cell plate before dispensing the test compounds, and all wells except the negative control wells (column 24 of the plate) were pretreated for 30 minutes with assay medium containing 0.47 nM estradiol. In this assay, immunostaining was performed to detect PRα, and the assay was performed using ACUMEN EXPLORER. TM [Laser Scanning Fluorescence Microplate Cytometer manufactured by TTP LABTECH LTD] Scans the plate to measure PRα. Image analysis is based on cell fluorescence signals for identifying positive cells. PRα-positive cells are identified by average intensity. Individual cells are identified using a total intensity of 575-640 from propidium iodide / DNA. The experimental output value is %PRα-positive cells. This is achieved using GENE DATA. TM For each output value, curve fitting is performed to a four-parameter logic curve to determine the IC. 50 The results of this experiment indicate that Examples 1, 1A, and 1B potently inhibit PRα and antagonize its function in MCF7 breast cancer cells. In this experiment, the relative IC50 values ​​of Examples 1, 1A, and 1B were... 50 The relative IC50 values ​​of exemplary compounds are shown in Table 10 below. 50 The range of approximately 0.029 to >2 μM indicates that all exemplary compounds tested, except for 1A and 2B, are potent antagonists of ERα wild-type protein and potent inhibitors of ERα-mediated transcription. PRα (PGR) is also a transcriptional target of ERα, and the results of this assay demonstrate potent inhibition of ERα-mediated PRα transcription at the tested concentrations.

[0224] Table 10: PRα inhibition (ERα antagonism) cell assay in MCF7 cells

[0225] 1 0.1283±0.0226,n＝3 1A >2.000 1B 0.04129±0.03370,n＝16 2 0.1634 2A 0.1215±0.05368,n＝2 2B >2.000 3 0.07666±0.02101,n＝3 3A 0.9274 3B 0.03435 4 0.07626±0.1676,n＝3 4A 0.8465 4B 0.02866 5 0.1180±0.01230,n＝2 5A 0.6002 5B 0.03203±0.005306,n＝2 6 0.08258+0.005682,n＝3 6A 0.2528 6B 0.02835 7 0.1134±0.02087,n＝2 8 0.06835±0.02273,n＝2 8A 0.2058 8B 0.04848±0.02944,n＝2 10 0.02633+0.004459,n＝3

[0226] Cell proliferation assays in MCF7 and MCF7-ESR1 Y537N-682

[0227] The purpose of the following cell proliferation assays can be summarized as detecting whether the test compound has an effect on cell proliferation.

[0228] MCF7 cells (purchased from ATCC HTB-22) at a density of 2,000 cells / well were seeded in phenol red-free DMEM medium with 10% FBS (20 μL volume) (treated with activated charcoal) in 384-well cell culture plates with clear bottoms. MCF7-ESRY537N-682 cells (generated by CRISPR / Cas9 gene editing of the ESR1 gene in MCF7 cells, clone #682) were seeded in DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin antibiotics at a density of 1,000 cells / well. The plates were incubated at 37°C and 5% CO2. The test compound was administered to the cells the following day. Serial dilutions (1:3) of the test compound were prepared in the range of 60 μM to 0.003 μM using an Echo 555 acoustic dispenser. Add 5 μL of the serial dilution plate to the cell plate to administer the compound to the cells, producing a final DMSO concentration of 0.2%. The final test compound concentration ranged from 20 to 0.001 μM. For the maximum point, medium containing 0.2% DMSO was used; for the minimum point, fulvestrant diluted to a final concentration of 2 μM in growth medium containing 0.2% DMSO was used. After administration of the test compound, the cell plate was incubated at 37°C and 5% CO2. Seven days after the addition of the test compound, the plate was removed from the incubator, and 65 μL of cold EtOH 96% was added to each well. After 30 minutes, the medium was removed, and RNase (20 μL, 50 μg / mL) (Sigma) and a 1:1000 dilution of propidium iodide in PBS were added to each well. The plate was sealed and incubated at room temperature for 1 hour on the bench (protected from light). The cells were then analyzed using ACUMEN EXPLORER. TM [TTPLABTECH LTD Laser Scanning Fluorescent Microculture Plate Cytometer] Scanning plate. MCF-7 cell lines grow to form aggregates, and the number of cells as a physical quantity may not be usable as a reading; therefore, the cell number can be assessed by estimation (calculated using area parameters (the ratio of the total area of ​​the total cell population to the specified range of peak intensity of FL-1(PI) and the average area of ​​a single cell population (defined by perimeter))). This can be achieved using GENE DATA. TM For each output value, curve fitting is performed to a four-parameter logic curve to determine the IC. 50 Relative IC50 values ​​of Examples 1, 1A, and 1B (and other examples) in MCF7 ESR1 wild-type and MCF7-ESR1 Y537N mutant cells. 50 The results are shown in Table 11 below. These results demonstrate the potent antiproliferative activity and cell growth inhibition of Examples 1, 1A, and 1B (and other examples) in MCF7 (ESR1 wild-type) and MCF7 (ESR1 Y537N mutant) breast cancer cells. Relative IC50 values ​​of the exemplary compounds are also shown. 50The concentrations were approximately 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 the exemplary compounds tested exhibited potent antiproliferative activity and cell growth inhibition in both MCF7 (ESR1 wild-type) and MCF7 (ESR1 Y537N mutant) breast cancer cells.

[0229] Table 11: Cell proliferation assays in MCF7 and MCF7-ESR1 Y537N-682 cells

[0230]

[0231]

[0232] In vivo target inhibition (IVTI) assay in MCF7 tumors (PGR RT-qPCR assay)

[0233] The purpose of this IVTI trial was to determine the ability of the test compound (SERD) to inhibit the expression (transcription) of the PRα gene downstream of ERα in xenograft tumors implanted in mice.

[0234] Female NOD SCID mice (22-25g) from Envigo RMS, Inc., Madison, Wisconsin were subcutaneously implanted with 5×10e cells in the right flank region. 6 MCF7 ER-positive breast cancer cells (ATCC, #HTB-22), which were detected in a 1:1 HBSS+MATRIGEL assay. TM Solution (200 μL). One day prior to tumor cell implantation, 17-β estradiol pills (0.18 mg / pill, 90-day release, from Innovative Research) were subcutaneously implanted. Tumor growth and body weight were measured twice weekly starting on day 7 post-implantation. When the tumor size reached 250-350 mm... 3 Animals were randomly selected and divided into groups of five. The animals were orally administered a solution specific to the test compound (1% hydroxyethyl cellulose / 0.25%). Multiple doses of the test compound or the solvent alone were administered orally over 3 days in 80 / 0.05% antifoaming agent (in purified water), with tumors and blood collected at desired intervals after the last administration. Animals were euthanized using isoflurane anesthesia and cervical dislocation. Tumors were rapidly frozen and stored at -80°C until processing for RNA isolation and RT-qPCR assays. Blood was collected from EDTA tubes, centrifuged to obtain plasma, and frozen in 96-well plates at -80°C. Test compound exposure was determined using mass spectrometry.

[0235] The tumor was crushed in liquid nitrogen and then processed in FASTPREP-24. TM Cell lysate was performed using Matrix D beads (MP Biomedical, #6913-500) in 1× RNA lysis buffer (from an RNA isolation kit) using a cell disruptor (MP Biomedical). After rotating at 14,000 rpm at 4°C for 20 minutes, the tumor lysate was transferred to fresh tubes. RNA was isolated from tumor lysates using the RNAMini Kit (Invitrogen #12183018A) or the RNeasy Mini Kit (Qiagen #74104 and #74106). DNA contaminants were removed using a DNase Set (Invitrogen #12185010) or an RNase-Free DNase Set (Qiagen #79254). The concentration of isolated RNA was measured by diluting the sample in RNase-free water and measuring the absorbance at 260 nm on a plate reader (SpectraMax 190). The average 260 nm absorbance measurement of the blank sample (only RNase-free water) was subtracted from the 260 nm measurements of all other RNA samples. The RNA sample was diluted to the same concentration in RNase-free water. cDNA was synthesized from the diluted RNA using an RT-PCR first-strand synthesis system (Invitrogen, #18080-051). For RT-qPCR, the cDNA was first diluted in RNase-free water. 2×AbsoluteBlue qPCR ROX Mix (Thermo, #AB-4139 / A), PGR primers (Thermo, Hs01556702_m1), and diluted cDNA for each reaction were pooled in a PCR plate (Applied Biosystems, #4309849). cDNA was amplified by incubating samples at 50°C for 2 minutes and then at 95°C for 15 minutes in a thermal cycler (ABIPrism 7900HT sequence detection system). This was followed by 40 cycles of incubation at 95°C for 15 seconds and then at 50°C for 60 seconds. Cycles were normalized to housekeeping genes and used to calculate %PGR inhibition compared to media alone. Each sample was analyzed in duplicate, and the mean was used for calculation. The percentage of target (PGR) inhibition was calculated using Excel and XL Fit.

[0236] The results of this study demonstrate that Example 1B inhibited PRα (PGR) expression in a tumor xenograft model. When administered orally at a dose of 30 mg / kg, Example 1B showed a ~78% inhibition of PRα (PGR) expression in the tumor xenograft model over 24 hours. These results demonstrate a significant and sustained inhibition of ERα antagonistic activity and ERα-mediated transcriptional activity in vivo in a tumor xenograft model.

[0237] In vivo tumor growth inhibition study in a mouse xenograft model of ER-positive (ESR1 wild-type) breast cancer.

[0238] The purpose of the following xenograft tumor suppression assays is to measure the reduction in tumor volume in response to the administration of the test compound.

[0239] Human breast cancer cells MCF7 (ATCC#HTB-22) and HCC1428 (ATCC#CRL-2327) were expanded in cultures, harvested, and subcutaneously injected with 1:1 HBSS+MATRIGEL into the right posterior flank of female NOD SCID mice (22-25g, Envigo RMS, Inc.). TM 5×10e in solution (200μL) 6 1,000 cells. Twenty-four hours prior to cell implantation, estrogen pills (0.18 mg / pill, 17β-estradiol, released after 90 days, Innovative Research) were subcutaneously implanted. Human breast cancer cells T47D (ATCC#HTB-22) were expanded in the culture, harvested, and subcutaneously injected (1:1 HBSS + MATRIGEL) into the right posterior flank of female NOD SCID mice (22-25 g, Envigo RMS, Inc.). TM 5×10e in solution (200μL) 6 1,000 cells. Twenty-four hours prior to cell implantation, estrogen pills (0.38 mg / pill, 17β-estradiol, released after 90 days, Innovative Research) were subcutaneously implanted. Human breast cancer cells ZR-75-1 (ATCC#CRL-1500) were expanded in the culture, harvested, and subcutaneously injected with 1:1 HBSS + MATRIGEL into the right posterior flank of female NOD SCID mice (22-25 g, Envigo RMS, Inc.). TM 5×10e in solution (200μL) 6 100 cells. Twenty-four hours before cell implantation, animals were administered 50 μl of estradiol valerate (10 mg / mL) intravenously, followed by administration every 14 days during the study period. Tumor growth and body weight were measured twice weekly starting on day seven post-implantation. Tumors were classified as 250-350 mm in size. 3Animals were randomly selected and grouped into groups of 5. They were then placed in a suitable medium (1% hydroxyethyl cellulose / 0.25%). The test compound of Example 1B was prepared in purified water (80 / 0.05% defoamer) and administered orally via tube feeding for 28 days, QD. Tumor response was determined by tumor volume measurement twice weekly during the treatment process. Body weight was used as a general measure of toxicity whenever tumor volume was measured.

[0240] The compounds of Example 1B were found to have the ΔT / C% values ​​provided in Table 12 below. These results indicate that the compounds of Example 1B exhibit good oral bioavailability in mice and show significant antitumor activity or tumor regression in an ER-positive (ESR1 wild-type) human breast cancer xenograft model.

[0241] Table 12: In vivo tumor growth inhibition studies in ER-positive breast cancer xenograft tumor models implanted in mice

[0242]

[0243]

[0244] Tumor volume analysis was based on Log 10 and SpatialPower covariance structures.

[0245] *: Significantly different from the control (p<0.05).

[0246] When the endpoint tumor volume in the treatment group is equal to or greater than the baseline tumor volume, ΔT / C% is calculated. The formula is 100*(T-T0) / (C-C0), where T and C are the mean endpoint tumor volumes in the treatment or control groups, respectively. T0 and C0 are the mean baseline tumor volumes in those groups.

[0247] When the endpoint volume is lower than the baseline, the regression percentage is calculated. The formula is 100*(T-T0) / T0, where T0 is the mean baseline tumor volume in the treatment group.

[0248] On day 32, the percentage change in T / C was calculated using the total mean of all groups from baseline (randomized).

[0249] In vivo tumor growth inhibition study in a mouse PDX tumor model of ESR1-mutated (Y537S) breast cancer (ST941 / HI)

[0250] The purpose of the following xenograft tumor suppression assay is to measure tumor volume reduction in response to the application of the test compound in a patient-derived xenograft (PDX) model of ESR1-mutant and hormone-independent (HI) breast cancer.

[0251] The ST941 / HIPDX model was derived from and operated at the South Texas Accelerated Research Therapy Center (San Antonio, TX). Tumor fragments were harvested from host animals and implanted into immunodeficient mice (Jackson Lab), at approximately 125-250 mm. 3 The study began with the average tumor volume. In a suitable medium (1% hydroxyethyl cellulose / 0.25%)... The test compound was prepared in 80 / 0.05% defoamer (in purified water), Example 1B, and administered orally via tube feeding for 28 days. Tumor response was determined by measuring tumor volume twice weekly during the treatment process. Body weight was used as a general measure of toxicity whenever tumor volume was measured.

[0252] The compounds of Example 1B were found to have the ΔT / C% values ​​provided in Table 13 below. These results indicate that the compounds of Example 1B exhibit good oral bioavailability in mice and significant antitumor activity or tumor regression in an ESR1-mutant (Y537S) human breast cancer PDX model.

[0253] Table 13: In vivo tumor growth inhibition studies in a mouse model of ESR1-mutant breast cancer PDX tumor implanted in mice

[0254]

[0255] Tumor volume analysis was based on Log 10 and SpatialPower covariance structures.

[0256] *: Significantly different from the control (p<0.05).

[0257] When the endpoint tumor volume in the treatment group is equal to or greater than the baseline tumor volume, ΔT / C% is calculated. The formula is 100*(T-T0) / (C-C0), where T and C are the mean endpoint tumor volumes in the treatment or control groups, respectively. T0 and C0 are the mean baseline tumor volumes in those groups.

[0258] When the endpoint volume is lower than the baseline, the regression percentage is calculated. The formula is 100*(T-T0) / (C-C0), where T0 is the mean baseline tumor volume in the treatment group.

[0259] On day 32, the percentage change in T / C was calculated using the total mean of all groups from baseline (randomized).

[0260] Combinatorial research

[0261] Due to tumor heterogeneity and acquired resistance to endocrine therapy, combination therapy has become necessary in the treatment of ER-positive and advanced / metastatic breast cancer, either for efficacy or to overcome acquired resistance. We have tested in vitro the combination of Example 1B with the CDK4 / 6 inhibitor abemiclib, the mTOR inhibitor everolimus, the PIK3CA inhibitor alpelisib, and the PI3K / mTOR inhibitor 8-[5-(1-hydroxy-1-methylethyl)pyridin-3-yl]-1-[(2S)-2-methoxypropyl]-3-methyl-1,3-dihydro-2H-imidazo[4,5-c]quinoline-2-one (“Compound A”) in five ER-positive breast cancer cell lines.

[0262] Cell viability assays for combined studies

[0263] Cells at the densities shown in Table 14 were seeded into 20 μL volumes of the culture medium described in the table into 384-well clear-bottom cell culture plates.

[0264] Table 14: Cell line information for cell viability assay

[0265]

[0266] The plates were incubated at 37°C and 5% CO2. The next day, the compound—Example 1B—was applied to the cells.

[0267] The compounds were prepared as 10 mM DMSO stock solutions and used for dose-response studies, with the highest concentration starting at 10 or 1 μM. The two compounds were tested together at a fixed ratio, followed by the preparation of 1:3 serial dilutions and solutions for IC50 analysis. 50 The individual compounds were tested, starting at a concentration of 20 μM. 5 μL of the serial dilution plate was added to the cell plate to administer the compound to the cells, producing a final DMSO concentration of 0.2%. The final test compound concentration dose ranged from 20 to 0.001 μM for a single treatment or a lower range for combinations. For the maximum point, medium containing 0.2% DMSO was used; for the minimum point, astrosporin diluted to a final concentration of 2 μM in growth medium containing 0.2% DMSO was used. After administration of the test compound, the cell plates were incubated at 37°C and 5% CO2. After two doubling times of incubation with the compound, the plates were removed from the incubator, and 65 μL of cold EtOH 96% was added to each well. After 30 minutes, the medium was removed, and each well was added with RNase (20 μL, 50 μg / mL) (Sigma) and a 1:1000 dilution of propidium iodide in PBS. The plates were sealed and incubated at room temperature for 1 hour (protected from light). The cells were then analyzed using ACUMEN EXPLORER. TM[Laser Scanning Fluorescent Microplate Cytometer manufactured by TTP LABTECH LTD] Scanning plate. As some cell lines grow and form aggregates, the number of cells as a physical quantity may not be usable as a readout value; therefore, the total area population (a specified range of peak intensity of FL-1 (PI)) or the total intensity of PI is used to assess the cell number.

[0268] In vitro combination data showed that the combination of Example 1B with abecilibi or everolimus had a synergistic effect in all five of the five ER-positive breast cancer cell lines (as defined below), as shown in Table 15A. The combination of Example 1B with compound A had a synergistic effect in all four of the four ER-positive breast cancer cell lines tested. The combination of Example 1B with abecilibi was additive in two of the four ER-positive breast cancer cell lines and synergistic in two of the four ER-positive breast cancer cell lines.

[0269] Table 15A: In vitro combination of Example 1B with other targeted active agents in ER-positive breast cancer cell lines

[0270]

[0271] Data analysis and interpretation of combined effects

[0272] In vitro combination effects were calculated using methods published in the literature (L. Zhao et al., Front Biosci, 2010, 2:241-249 and L. Zhao et al., Clin Cancer Res, 2004, 10(23):7994-8004). To identify synergistic or antagonistic interactions between the two drugs, curve shift analysis was performed using a custom XL template with XLFit 5 add-ins. Curves for single active agents were adjusted using 4-parameter logistic regression. The criteria and limits used for fitting were (i) bottom < (-20) fixed to 0 and (ii) top > 120 fixed to 100. If all observations were below the user-set threshold, a constant fit with hill = 0 was performed, and IC50 was considered acceptable. 50 Above the maximum containment concentration. Once the absolute IC50 for each individual active agent is obtained... 50 We then calculate the equivalent concentrations of the single and combined components at 50% activity. Using these equivalent concentrations and the measured activity, we recalculate the absolute IC50. 50 At an eq concentration value of 1, the curve for a single active agent will reach 50% activity, while synergistic combinations will reach 50% at lower values, causing a leftward shift, and antagonistic combinations will show a rightward shift. CI was also calculated using equivalence concentration. 50 (Combination index at 50% activity), where CI50 equal to the absolute IC of the combination curve 50 With CI 50 Together, other CIs (combination indices) for different activity percentages can be calculated (CI10, CI20, CI30, CI40, CI60, CI70, CI80, CI90). To calculate CInn, the equivalent concentrations at different activity percentages are calculated. For each activity percentage, we calculate an error limit within a 95% confidence interval, and using this confidence interval, we calculate the upper limit as CI plus the error limit, and the lower limit as CI minus the error limit. Upper limit = CI + 95% confidence interval and lower limit = CI - 95% confidence interval. These limits are then used to interpret the results.

[0273] The statistical interpretation of each percentage of activity is as follows:

[0274] Upper limit < 1 Collaboration Lower limit > 1 Antagonism

[0275] The biological interpretation of each percentage of activity is as follows:

[0276] CInn>0.5 and CInn<2 Addition CInn>2 Antagonism

[0277] In vivo combination studies

[0278] Due to tumor heterogeneity and acquired resistance to endocrine therapy, combination therapy has become necessary in the treatment of ER-positive and advanced / metastatic breast cancer, either for its effectiveness or to overcome acquired resistance. It is hypothesized that combinations of targeted therapies have the potential to be more effective in slowing or even halting ER-positive breast cancer. The combination of a CDK4 / 6 inhibitor and fulvestrant has been approved for the treatment of ER-positive metastatic breast cancer, but a high percentage of patients develop resistance due to acquired mutations in ESR1 or PIK3CA. Oral SERDs, such as those in Example 1B, as potent degraders and antagonists of ERα, have the potential to more effectively slow or halt ESR1-mutant or PIK3CA-mutant breast cancer, either as a single active agent or in combination with CDK4 / 6 inhibitors such as abexicillin or PI3K / mTOR inhibitors such as compound A. In this context, the tumor growth inhibition of the compound of Example 1B in combination with abexicillin (patent reference) or compound A (patent reference) is tested. More specifically, in ESR1 wild-type and PIK3Ca-mutant MCF7 breast cancer xenograft models, the compounds of Example 1B were tested in combination with abexilide or compound A.

[0279] Human breast cancer cells MCF7 (ATCC#HTB-22) were expanded in culture, harvested, and subcutaneously injected with 1:1 HBSS + MATRIGEL into the right posterior flank of female NOD SCID mice (22-25g, Enviou RMS, Inc.).TM 5×10e in solution (200μL) 6 Cells. Twenty-four hours prior to cell implantation, estrogen pills (0.18 mg / pill, 17β-estradiol, 90-day release, Innovative Research) were subcutaneously implanted. Tumor growth and body weight were measured twice weekly starting on day seven post-implantation. When the tumor size reached 250-350 mm... 3 Animals were randomly selected and grouped into groups of 5. They were then placed in a suitable medium (1% hydroxyethyl cellulose / 0.25%). The test compound (Example 1B) was prepared in purified water (80 / 0.05% defoamer) and administered orally via tube feeding for 42 days. The CDK4 / 6 inhibitor (abecile) was prepared in 25 mM sodium phosphate buffer (1% HEC), pH 2.0. In 1% hydroxyethyl cellulose / 0.25%... The PI3K / mTOR inhibitor (compound A) was formulated in purified water containing 80% defoamer and 0.05% antifoaming agent. Tumor response was determined by measuring tumor volume twice weekly during treatment. Body weight was used as a general measure of toxicity whenever tumor volume was measured. Tumor volume was estimated using the formula v = l x w² x 0.535, where l = the longer of the measured diameter and W = the shorter of the vertical diameter.

[0280] Statistical analysis

[0281] Statistical analysis of tumor volume data began with data conversion to a logarithmic scale to balance differences between time and treatment groups. Logarithmic volume data were analyzed using the MIXED procedure in SAS software (version 9.3) via two-way repeated measures analysis to assess changes over time and treatment. The correlation model used for repeated measures was spatial power. Treatment groups were compared to control groups at each time point. The MIXED procedure was also used individually for each treatment group to calculate the adjusted mean and standard error at each time point. Both analyses accounted for autocorrelation within each animal and data loss that occurred when animals with large tumors were removed from the early stages of the study. The adjusted mean and standard error for each treatment group were plotted against time. Tumor volume analysis was based on logarithmic... 10 And spatial power covariance structure. The p-value is based on a comparison between two specific groups.

[0282] Combinatorial analysis method (Bliss independence for the IVEF study)

[0283] First, a typical repeated measures model is fitted to the relationship between logarithmic volume and group and time. Then, contrast statements are used to test the interaction effects of the combination of two specific treatments at each time point. This is equivalent to the Bliss independence method and assumes that tumor volume can theoretically reach zero, i.e., complete regression. The expected additive response (EAR) of the combination is calculated on the tumor volume scale as: EAR volume = V1 * V2 / V0, where V0, V1, and V2 are the estimated mean tumor volumes of the medium control, treatment 1 alone, and treatment 2 alone, respectively. If the interaction test is significant, the combination effect is declared statistically greater than or less than additive, depending on whether the observed combination mean volume is less than or greater than the EAR volume. Otherwise, the statistical conclusion is additive. Furthermore, the biologically relevant range of additiveness can be defined as X% above and below the EAR volume. Typically, X is 25-40%. If the observed combination mean volume is below, within, or above the additive range, a biological conclusion of greater than, additive, or less than additive can be drawn for that combination.

[0284] There may be situations where stagnation is the optimal expected response. In these cases, the Bliss method can be directly applied to the %ΔT / C value to obtain the EAR percentage response: EAR%ΔT / C = Y1*Y2 / 100, where Y1 and Y2 are the percentage ΔT / C values ​​of single-drug treatments. Currently, there are no statistical tests to compare %ΔT / C with EAR observed in the combination group, but the aforementioned biological criteria can be applied.

[0285] As shown in Tables 15B and 16, treatment with either Example 1B alone or abexicillin as a single active agent resulted in tumor regression of 32% (% dT / C = -32) and 52% (% dT / C = -52), respectively, both of which were statistically significant compared to the mediator control (p < 0.001). The combined effect of Example 1B and abexicillin was "less than additive," but the combined effect of Example 1B and abexicillin was significantly better than that of Example 1B alone (p < 0.001). However, the efficacy of abexicillin as a single active agent was not statistically significant in the combination (p = 0.055). The combination was well tolerated in animals without significant weight loss.

[0286] Table 15B: Efficacy of the combination of Example 1B and abecilibry in an MCF7 ER-positive breast cancer model

[0287]

[0288] b Difference = Treatment 1 – Treatment 2; *p-value: significant (p<0.05)

[0289] SE – Standard Error

[0290] Table 16: Efficacy of the combination of Example 1B and abecilibry in an MCF7 ER-positive breast cancer model

[0291]

[0292] Tumor volume analysis is based on Log. 10 And spatial power covariance structure.

[0293] *p-value: significant (p<0.05); NA: not applicable

[0294] ΔT / C% is calculated when the endpoint tumor volume in the treatment group is equal to or higher than the baseline tumor volume; and regression percentage is calculated for tumor volumes below baseline. The formula is 100*(T-T0) / (C-C0), where T and C are the mean endpoint tumor volumes in the treatment or control groups, respectively. T0 and C0 are the mean baseline tumor volumes in those groups.

[0295] As shown in Tables 17 and 18, treatment with Example 1B or Compound A as a single active agent resulted in tumor regression of 32% (%dT / C = -32) and 36% (%dT / C = -36), respectively, both of which were statistically significant compared to the median control (p < 0.001). The combined potency of Example 1B and Compound A was "less than additive," but the combination of Example 1B and Compound A was significantly better than Example 1B alone (p < 0.001) or Compound A alone (p = 0.002*). The combination was well tolerated in animals without significant weight loss.

[0296] Table 17: Combination efficacy of Example 1B and compound A in an MCF7 ER-positive breast cancer model

[0297]

[0298]

[0299] b Difference = Treatment 1 – Treatment 2; *p-value: significant (p<0.05)

[0300] SE – Standard Error

[0301] Table 18: Efficacy of the combination of Example 1B and abecilibry in an MCF7 ER-positive breast cancer model

[0302]

[0303] Tumor volume analysis is based on Log. 10 And spatial power covariance structure.

[0304] *p-value: significant (p<0.05); NA: not applicable

[0305] ΔT / C% is calculated when the endpoint tumor volume in the treatment group is equal to or higher than the baseline tumor volume; and regression percentage is calculated for tumor volumes below baseline. The formula is 100*(T-T0) / (C-C0), where T and C are the mean endpoint tumor volumes in the treatment or control groups, respectively. T0 and C0 are the mean baseline tumor volumes in those groups.

[0306] As shown in Tables 19 and 20, treatment with either Example 10 alone or abexicillin as a single active agent resulted in tumor regression of 51% (%dT / C = -51) and 70% (%dT / C = -70), respectively, both of which were statistically significant compared to the mediator control (p < 0.001). The efficacy of the combination of Example 10 and abexicillin was “less than additive,” but the efficacy of Example 10 plus abexicillin was significantly superior to that of Example 10 alone (p = 0.039). However, the efficacy of the combination of Example 10 plus abexicillin was not significantly different from that of abexicillin alone (p = 0.905). The combination was well tolerated in animals without significant weight loss.

[0307] Table 19: Efficacy of the combination of Example 10 and abexilide in an MCF7 ER-positive breast cancer model

[0308]

[0309] b Difference = Treatment 1 – Treatment 2; *p-value: significant (p<0.05)

[0310] SE – Standard Error

[0311] Table 20: Efficacy of the combination of Example 10 and abecilibin in an MCF7 ER-positive breast cancer model

[0312]

[0313]

[0314] Tumor volume analysis is based on Log. 10 And spatial power covariance structure.

[0315] *p-value: significant (p<0.05); NA: not applicable

[0316] ΔT / C% is calculated when the endpoint tumor volume in the treatment group is equal to or higher than the baseline tumor volume; and regression percentage is calculated for tumor volumes below baseline. The formula is 100*(T-T0) / (C-C0), where T and C are the mean endpoint tumor volumes in the treatment or control groups, respectively. T0 and C0 are the mean baseline tumor volumes in those groups.

[0317] As shown in Tables 21 and 22, treatment with Example 10 or apelipis alone as a single active agent resulted in 51% (% dT / C = -51) and 21% (% dT / C = -21) tumor regression, respectively, both of which were statistically significant compared to the mediator control (p < 0.001 and p = 0.013). The combined efficacy of Example 10 and apelipis was "additive," and the combined efficacy of Example 10 plus apelipis was significantly superior to Example 10 alone (p = 0.009). The combined efficacy of Example 10 plus apelipis was also significantly superior to apelipis alone (p < 0.001). This combination was well tolerated in animals without significant weight loss.

[0318] Table 21: Efficacy of the combination of Example 10 and apegliflozin in an MCF7 ER-positive breast cancer model

[0319]

[0320] b Difference = Treatment 1 – Treatment 2; *p-value: significant (p<0.05)

[0321] SE – Standard Error

[0322] Table 22: Efficacy of the combination of Example 10 and apegliflozin in an MCF7 ER-positive breast cancer model

[0323]

[0324]

[0325] Tumor volume analysis is based on Log. 10 And spatial power covariance structure.

[0326] *p-value: significant (p<0.05); NA: not applicable

[0327] ΔT / C% is calculated when the endpoint tumor volume in the treatment group is equal to or higher than the baseline tumor volume; and regression percentage is calculated for tumor volumes below baseline. The formula is 100*(T-T0) / (C-C0), where T and C are the mean endpoint tumor volumes in the treatment or control groups, respectively. T0 and C0 are the mean baseline tumor volumes in those groups.

[0328] As shown in Tables 23 and 24, treatment with Example 10 or everolimus as a single active agent resulted in 51% (%dT / C = -51) and 50% (%dT / C = -50) tumor regression, respectively, both of which were statistically significant compared to the mediator control (p < 0.001 and p < 0.001). The combination efficacy of Example 10 and everolimus was "additive," and the combination efficacy of Example 10 plus everolimus was significantly superior to Example 10 alone (p = 0.004). The combination efficacy of Example 10 plus apegliflozin was also significantly superior to everolimus alone (p = 0.04). This combination was well tolerated in animals without significant weight loss.

[0329] Table 23: Efficacy of the combination of Example 10 and everolimus in an MCF7 ER-positive breast cancer model

[0330]

[0331] b Difference = Treatment 1 – Treatment 2; *p-value: significant (p<0.05)

[0332] SE – Standard Error

[0333] Table 24: Efficacy of the combination of Example 10 and everolimus in an MCF7 ER-positive breast cancer model

[0334]

[0335]

[0336] Tumor volume analysis is based on Log. 10 And spatial power covariance structure.

[0337] *p-value: significant (p<0.05); NA: not applicable

[0338] ΔT / C% is calculated when the endpoint tumor volume in the treatment group is equal to or higher than the baseline tumor volume; and regression percentage is calculated for tumor volumes below baseline. The formula is 100*(T-T0) / (C-C0), where T and C are the mean endpoint tumor volumes in the treatment or control groups, respectively. T0 and C0 are the mean baseline tumor volumes in those groups.

[0339] Oral bioavailability assay in rats

[0340] The purpose of the following assays is to demonstrate whether the test compound is orally bioavailable.

[0341] The test compound was administered at 1 mg / kg (using medium: 20%). Sprague-Dawley rats were administered intravenously in 25 mM sodium phosphate buffer, pH 2, or 25% DMA, 15% EtOH, 10% propylene glycol, 25% 2-pyrrolidone, and 25% purified water, followed by PO administration at 10 mg / kg (medium: 1% hydroxyethyl cellulose, 0.25% polysorbate 80, 0.05% antifoaming agent 1510-US, and purified water). Serial blood samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 8, and 12 hours after intravenous bolus administration and at 0.25, 0.5, 1, 2, 4, 8, and 12 hours after oral administration. Plasma was obtained by centrifugation after treatment with EDTA anticoagulant and stored at -70°C until LC-MS / MS analysis. The concentrations of test compounds in the plasma were determined and uploaded to Watson LIMS. TM In the system, non-compartmental analysis was used to calculate the area under the curve (AUC) for both the IV and PO arms. Oral bioavailability (%F) was calculated using the following equation.

[0342] %F = (AUC) PO X dose IV ) / (AUC IV X dose PO )X 100.

[0343] The compound of Example 1B showed a %F value of ~50% in the above tests. This test demonstrates that Example 1B has excellent oral bioavailability.

Claims

1. The following compound: Where R 1 or R 2 One of them is independently selected from Cl, F, or -CF3, and the other is hydrogen. Or its pharmaceutically acceptable salt.

2. The compound according to claim 1, wherein the compound is Or its pharmaceutically acceptable salt.

3. The compound according to claim 1, wherein the compound is Or its pharmaceutically acceptable salt.

4. The compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is a benzenesulfonate or 4-methylbenzenesulfonate.

5. The compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is 4-methylbenzenesulfonate.

6. A pharmaceutically acceptable salt of the compound according to any one of claims 1-3, wherein the pharmaceutically acceptable salt is a 4-methylbenzenesulfonate.

7. A compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof, for use in combination with a second therapeutic agent, wherein the second therapeutic agent is abexicillin, everolimus, or apelixix.

8. Use of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 in the preparation of a medicament for treating ER-mediated cancer, said cancer being breast cancer, ovarian cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer.

9. The use according to claim 8, wherein the breast cancer is ER-positive breast cancer.

10. The use according to claim 8, wherein the gastric cancer is ER-positive gastric cancer.

11. The use according to claim 8, wherein the lung cancer is ER-positive lung cancer.

12. Use of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 in the preparation of a medicament for the treatment of ER-mediated endometrial cancer.

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