Selective estrogen receptor degrader

Abstract

To provide new SERDs to treat cancers as well as mutations due to emerging resistance.SOLUTION: The present invention provides a novel selective estrogen receptor degrader (SERD) of the following formula (where either R1 or R2 is independently selected from Cl, F, -CF3, or -CH3, and the other is hydrogen), a pharmaceutically acceptable salt thereof, and a pharmaceutical composition thereof, and methods for use thereof.SELECTED DRAWING: None

Description

[Background technology]

[0001] Selective estrogen receptor degraders (SERDs) bind to estrogen receptors (ERs) and downregulate ER-mediated transcriptional activity. This degradation and downregulation caused by SERDs may be useful in treating 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 as useful as needed to effectively treat cancer. For example, finding a SERD with good pharmacokinetic (PK) and pharmacodynamic (PD) properties, high clinical efficacy, and good oral bioavailability would be extremely useful for cancer treatment. Pure antagonist SERDs that potently inhibit ER-mediated transcription would clearly be beneficial for 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 emerging resistance-causing mutations. In particular, new SERDs are needed to treat ER-positive breast, gastric, and / or lung cancer. Summary of the Invention

[0002] formula: [ka] Provided herein are compounds of the formula R 1 or R 2 are independently Cl, F, or -CF 3、 or —CH3, and the other is hydrogen.

[0003] Also provided are methods of using the compounds described herein, pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof for treating breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer, comprising administering a therapeutically effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof, to a patient in need thereof.

[0004] Further provided are compounds described herein, and pharmaceutically acceptable salts thereof, for use in therapy.The compounds described herein, and pharmaceutically acceptable salts thereof, can be used to treat breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer.

[0005] Further provided is the use of a compound described herein, and a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer. DETAILED DESCRIPTION OF THE INVENTION

[0006] Novel tetracyclic compounds and pharmaceutical salts thereof that act as SERDs are disclosed herein. The newly invented SERDs described herein inhibit ER-mediated transcription, which is useful for treating cancers such as breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, and lung cancer, as well as new resistance mutations. These SERDs can be used as single agents or in combination with other classes of drugs, such as 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.

[0007] The novel compounds described herein have formula I: [ka] or a pharmaceutically acceptable salt thereof, wherein R 1 or R 2are independently selected from Cl, F, -CF3, or -CH3, and the other is hydrogen. One of ordinary skill in the art will appreciate that compounds as described by Formula I, or pharmaceutically acceptable salts thereof, contain chiral centers, the positions of which are indicated by * above. One of ordinary skill in the art will also appreciate that the Cahn-Ingold-Prelog (R) or (S) designation of a chiral center varies depending on the substitution pattern around the chiral center. The chiral centers of compounds of Formula I may be in the R-enantiomeric form, as shown by Formula II: [ka] and the S-enantiomeric form represented by formula III: [ka]

[0013] All individual stereoisomers, enantiomers, and diastereomers of the compounds according to Formula I, Formula II, and Formula III, including racemates, and mixtures of enantiomers and diastereomers thereof, are included within the scope of the compounds described herein. Compounds for pharmaceutical use that contain chiral centers are often isolated as single enantiomers or diastereomers, and such isolated compounds of Formula I, Formula II, and Formula III are included within the scope of the compounds disclosed herein. Those skilled in the art will also understand that the compounds of Formula I, Formula II, and Formula III described herein, and their pharmaceutically acceptable salts, can be deuterated (hydrogen can be replaced with deuterium), and such molecules are considered to be included within the scope of the compounds disclosed herein.

[0008] Specific examples of compounds of formula I (including their IUPAC nomenclature names) are shown below. [ka] 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol; [ka] 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-7-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol; [ka] 8-chloro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol; [ka] 7-chloro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol; [ka] 8-Fluoro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol; [ka] 7-Fluoro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol; [ka] 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-methyl-5H-[1]benzopyrano[4,3-c]quinolin-2-ol; [ka] 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-7-methyl-5H-[1]benzopyrano[4,3-c]quinolin-2-ol.

[0009] Due to the chiral centers described above, each of these specific examples of the compound of Formula I described above has R- and S-enantiomeric forms (i.e., R-enantiomeric compounds of Formula II and S-enantiomeric compounds of Formula III), as shown in Table 1. [Table 1-1] [Table 1-2]

[0010] Also described herein are pharmaceutical compositions comprising the compounds of Formula I, Formula II, and Formula III described herein, or pharmaceutically acceptable salts thereof, in combination with a pharmaceutically acceptable excipient, carrier, or diluent. The pharmaceutical compositions described herein can be prepared using pharmaceutically acceptable additives. As used herein, the term "pharmaceutically acceptable additive" refers to one or more carriers, diluents, and excipients that are compatible with the other components of the composition or formulation and are not harmful to the patient. The compounds of Formula I, Formula II, and Formula III described herein, or pharmaceutically acceptable salts thereof, can be formulated as pharmaceutical compositions administered by various routes, such as orally or intravenously. Bioavailability is often a factor in cancer therapy, and the ability to select administration methods and pharmaceutical compositions to control or optimize the bioavailability of active ingredients is useful. For example, orally bioavailable SERD compositions would be particularly useful. The compounds of Formula I, Formula II, and Formula III described herein, or pharmaceutically acceptable salts thereof, are believed to have oral bioavailability. Examples of pharmaceutical compositions and processes for their preparation can be found in "Remington: The Science and Practice of Pharmacy", L.V. Allen Jr., Editor, 22nd Ed., Mack Publishing Co., 2012. Non-limiting examples of pharmaceutically acceptable carriers, diluents, and excipients include: saline, water, starch, sugars, mannitol, and silica derivatives, carboxymethylcellulose and other cellulose derivatives, alginates, gelatin, and binders such as polyvinylpyrrolidone, kaolin and bentonite, and polyethyl glycol.

[0011] Further described herein is a method for treating cancer.The method described herein comprises administering to a patient in need of such treatment an effective amount of a compound of Formula I, Formula II, and Formula III described herein, or a pharmaceutically acceptable salt thereof.For example, the method of administering an effective amount of a compound of Formula I, Formula II, and Formula III described herein, or a pharmaceutically acceptable salt thereof, can be oral administration.The cancer can be an estrogen-responsive cancer.Furthermore, the cancer can be breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer.For example, the cancer can be ER-positive breast cancer, ER-positive gastric cancer, or ER-positive lung cancer.

[0012] Also described herein are compounds of formula I, formula II, and formula III, or their pharmaceutically acceptable salts, for use in treatment.Also provided herein are compounds of formula I, formula II, and formula III, or their pharmaceutically acceptable salts, for use in 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 formula I, formula II, and formula III, or their pharmaceutically acceptable salts, can be orally administered.

[0013] Furthermore, the compounds of formula I, formula II, and formula III described herein, or their pharmaceutically acceptable salts, can be used to manufacture medicaments for treating cancer.For example, the medicament can be administered orally.The types of cancer that the medicament described herein can be used to treat include breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer.In particular, the cancer can be ER-positive breast cancer, ER-positive gastric cancer, or ER-positive lung cancer.

[0014] The compounds of Formula I, Formula II, and Formula III described herein, and their pharmaceutically acceptable salts, may be clinically useful as single agents 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, and lung cancer. When used in combination with other therapeutic agents (e.g., anticancer agents), the compounds of Formula I, Formula II, and Formula III described herein, or their pharmaceutically acceptable salts, can be used simultaneously, sequentially, or separately with the other therapeutic agents. Examples of classes of drugs that can be combined with the compounds of Formula I, Formula II, and Formula III described herein, or their pharmaceutically acceptable salts, 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 with which the compounds of Formula I, Formula II, and Formula III described herein, or pharmaceutically acceptable salts thereof, 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]quinolin-2-one (PI3K / mTOR inhibitor).

[0015] As used herein, the term "effective amount" refers to the amount or dosage of the compounds of formula I, formula II, and formula III described herein, or their pharmaceutically acceptable salts, that, when administered to a patient in a single or multiple doses, provides the desired effect to the patient under diagnosis or treatment.Preferably, the desired effect is the inhibition of tumor cell growth, tumor cell death, or both.The compounds of formula I, formula II, and formula III described herein, or their pharmaceutically acceptable salts, are generally effective over a wide dosage range.For example, the daily dosage is usually within the range of about 100 mg to about 2000 mg per day.

[0016] As used herein, "treat," "treating," or "treatment" refers to inhibiting, slowing, halting, or reversing the progression or severity of an existing condition or disorder.

[0017] As used herein, the term "patient" refers to a human suffering from a particular disease, disorder, or condition.

[0018] The compounds of Formula I, Formula II, and Formula III described herein, or their pharmaceutically acceptable salts, can be prepared by various procedures known in the art, some of which are illustrated in the preparations and examples below. The specific synthetic steps of each described route may be combined with various methods or steps from different procedures to prepare the compounds of Formula I, Formula II, and Formula III described herein, or their pharmaceutically acceptable salts. The products can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. The reagents and starting materials are readily available to those skilled in the art.

[0019] Intermediates and processes useful for synthesizing the compounds of Formula I, Formula II, and Formula III described herein are intended to be included in this description. Furthermore, certain intermediates described herein may contain one or more protecting groups. Variable protecting groups may be the same or different from one occurrence to the next, depending on the specific reaction conditions and the specific transformation being performed. Protection and deprotection 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," Fourth Edition, by Peter G.M.Wuts and Theodora W. Greene, John Wiley and Sons, Inc. 2007).

[0020] Individual isomers, enantiomers, and diastereomers can be separated or resolved by one skilled in the art at any convenient point in the synthesis of the compounds of Formula I, Formula II, and Formula III described herein using methods such as selective crystallization techniques or chiral chromatography (see, e.g., J. Jacques, et al., "Enantiomers, Racemates, and Resolutions," John Wiley and Sons, Inc., 1981, and E.L. Eliel and S.H. Wilen, "Stereochemistry of Organic Compounds," Wiley-Interscience, 1994). While individual isomers, enantiomers, and diastereomers can be separated or resolved as described above, the Cahn-Ingold-Prelog (R) or (S) assignment of the chiral centers may not yet be determined. When a Cahn-Ingold-Prelog (R) or (S) designation is not available, the identifiers "Isomer 1" and "Isomer 2" are used in conjunction with the IUPAC name without the Cahn-Ingold-Prelog stereochemical designation. Compounds of Formula I, Formula II, and Formula III identified herein as "Isomer 1" or "Isomer 2" are isolated as defined in the specific experimental descriptions below. Whether an isomer is "1" or "2" refers to the order in which compounds of Formula I, Formula II, and Formula III elute from a chiral chromatography column under the described conditions, i.e., "Isomer 1" is the first to elute from the column under the above conditions. If chiral chromatography is initiated early in the synthesis, the same designation applies to subsequent intermediates and compounds of Formula I, Formula II, and Formula III.

[0021] Abbreviations used herein are defined according to Aldrichimica Acta, Vol. 17, No. 1, 1984. Other abbreviations are defined as follows: "ACN" refers to acetonitrile. "BSA" refers to bovine serum albumin. "cataCXium® A Pd G3" refers to [(di(1-adamantyl)-butylphosphine)-2-(2'-amino-1,1'-biphenyl)]palladium(II) methanesulfonate. "DCM" refers to dichloromethane or methylene chloride. "DMA" refers to dimethylacetamide. "DMEA" refers to dimethylethylamine. "DMEM" refers to Dulbecco's modified Eagle's medium. "DMF" refers to N,N-dimethylformamide. "DMSO" refers to dimethyl sulfoxide. "DNA" refers to deoxyribonucleic acid. "cDNA" refers to complementary DNA. "DNase" refers to deoxyribonuclease. "DTT" refers to dithiothreitol. "EC 50 ” is the concentration of a predefined positive control compound (absolute EC 50 ) refers to the concentration of a drug that produces a response of 50% of the target activity compared to the target activity. "EDTA" refers to ethylenediaminetetraacetic acid. "ee" refers to enantiomeric excess. "ERα" refers to estrogen receptor alpha. "ERβ" refers to estrogen receptor beta. "EtOAc" refers to ethyl acetate. "EtOH" refers to ethanol or ethyl alcohol. "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-piperazineethanesulfonic acid. "HPLC" refers to high performance liquid chromatography. "IC 50 ” is the concentration of a drug that produces 50% of the maximum inhibitory response possible for that drug (relative IC 50 ), or the concentration of drug that produces 50% inhibition of target enzyme activity compared to a placebo control (absolute IC 50 "IPA" refers to isopropylamine. "iPrOH" refers to isopropanol or isopropyl alcohol. "IV" refers to intravenous administration. "k i" refers to the inhibition constant. "MEK" refers to methyl ethyl ketone. "MeOH" refers to methyl alcohol or methanol. "MTBE" refers to methyl t-butyl ether. "PBS" refers to phosphate buffered saline. "PO" refers to oral administration. "PRα" refers to progesterone receptor alpha. "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 reverse transcription quantitative polymerase chain reaction. "SFC" refers to supercritical fluid chromatography. "TED 50 " refers to the dose effective to achieve 50% inhibition of the target in the tumor. "THF" refers to tetrahydrofuran. "t (R) " refers to retention time. "XantPhos Pd G2" refers to chloro[(4,5-bis(diphenylphosphino)-9,9-dimethylxanthene)-2-(2'-amino-1,1'-biphenyl)]palladium(II). "XPhos Pd G2" refers to chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II).

[0022] The following preparations and examples further illustrate this invention.

[0023] Preparations and Examples [ka]

[0024] In step A, a Grignard reaction is achieved. The Grignard reaction is well known in the art as a reaction for the formation of carbon-carbon bonds. This reaction involves an organometallic reaction in which a Grignard reagent, an aryl magnesium halide, adds to a carbonyl group, such as an acid chloride, of compound 2, resulting in 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 the acid chloride, 4-fluorobenzoyl chloride, and compound 2 in a solvent, such as THF. Upon completion, the reaction can be quenched with water to give compound 3.

[0025] In Step B, the aryl methyl ether of compound 3 can be demethylated under a variety of conditions that will be recognized by those 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 potassium phosphate dibasic to give compound 4.

[0026] In step C, the azetidine ether 6 can be formed by treating the corresponding p-fluorophenyl ketone 4 and the azetidine alcohol salt 5, or the corresponding free base, with a suitable base, such as sodium hydride, sodium t-butoxide, or potassium t-butoxide, in a suitable polar aprotic solvent such as DMF or THF to produce the ether compound 6.

[0027] Compound 6 is then alkylated with an appropriate 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 there are a variety of conditions that may be useful for promoting such cross-coupling reactions. Suitable palladium reagents may include XantPhos Pd G2, cataCXium® APd G3, bis(triphenylphosphine)palladium(II) chloride, tris(dibenzylideneacetone)dipalladium(0) with tricyclohexylphosphine, (1,1'-bis(diphenylphosphino)ferrocene)palladium(II) chloride, palladium tetrakistriphenylphosphine, or palladium(II) acetate. Suitable bases may include potassium fluoride, cesium carbonate, sodium carbonate, potassium carbonate, lithium t-butoxide, or potassium phosphate tribasic monohydrate. For example, compound 6 can be reacted with an appropriate boronic acid, compound 7, e.g., 2-fluoro-4-(trifluoromethyl)phenylboronic acid, in a solvent such as 2-methyl-2-butanol containing a base such as potassium carbonate and a catalyst such as XPhos Pd G2, and heated to about 80°C under microwave conditions to give compound 8.

[0028] Those skilled in the art will recognize that Step D, the Suzuki cross-coupling reaction, can be completed prior to Step C, the azetidine ether formation.

[0029] In Step E, those skilled in the art will recognize that compound 8 can be cyclized by initial reduction of the ketone. This can be accomplished using a reducing agent such as lithium triethylborohydride in a solvent such as 1,4-dioxane and THF at temperatures between about 0°C and room temperature to give the corresponding secondary alcohol. This intermediate alcohol can be carried forward crude and deprotonated with a suitable base such as cesium carbonate, sodium hydride, sodium t-butoxide, or potassium t-butoxide in a solvent such as THF, DMSO, or DMF. The resulting alkoxide can be cyclized to an aryl fluoride at room temperature, by heating to reflux, or at a temperature of about 60°C. The substituted cyclic ether formed upon displacement of the fluoride can then be obtained to give compounds of Formula I.

[0030] Alternatively, ketone 8 can be reduced to the alcohol and chiral purified in step F to give chiral alcohol 9, which can then be cyclized in step G as described above for step E to give compounds of formula I.

[0031] In another alternative reaction, the ketone can be reduced using a chiral reagent such as (R)-(+)-α.α-diphenyl-2-pyrrolidinemethanol with trimethyl borate and borane-dimethyl sulfide to directly provide the desired chiral alcohol, compound 9, which can then be cyclized in step G as described above for step E to provide compounds of formula I.

[0032] In any step, pharmaceutically acceptable salts of the compounds of Formula I, II, and III described herein can be formed by reacting the appropriate free base of the compounds of Formula I, II, and III described herein with a suitable pharmaceutically acceptable acid in a suitable solvent under standard conditions. Moreover, the formation of such salts can occur simultaneously with the deprotection of nitrogen-protecting groups. The possibility of forming pharmaceutically acceptable salts is well known. For example, see 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 formula I, formula II, and formula III described herein can be easily converted into pharmaceutically acceptable salts and separated as pharmaceutically acceptable salts.Examples of useful salts include, but are not limited to, benzenesulfonate and 4-methylbenzenesulfonate.4-methylbenzenesulfonate is also known as tosylate salt.

[0033] Preparation 1 2-[3-(fluoromethyl)azetidin-1-yl]ethan-1-ol [ka] Sodium triacetoxyborohydride (405 g, 1.91 mol) was added portionwise over 15 minutes under N2 to a stirred 0°C solution of 3-(fluoromethyl)azetidine hydrochloride (160 g, 1.28 mol) in DCM (2.4 L) 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 stirred at 0-5°C for 15 minutes. The reaction was allowed to warm 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 this temperature for 2 hours. Water (800 mL) was added over 25-30 minutes at 10-15°C and allowed to warm to room temperature for 5-10 minutes before separating the layers. The aqueous layer is washed with DCM (800 mL) and the layers are separated before the combined aqueous layers are cooled to 10-15 °C and the pH is adjusted to 13-14 using 50% sodium hydroxide solution (approximately 540 mL). The aqueous layer is warmed to room temperature and 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 thick yellow oil. ES / MS (m / z): 134.1 (M+H).

[0034] Preparation 2 2-[3-(Fluoromethyl)azetidin-1-yl]ethan-1-ol hydrochloride [ka] Dissolve 2-[3-(fluoromethyl)azetidin-1-yl]ethan-1-ol (529 g, 4 mol) in MTBE (2.6 L) and cool to 0 °C. Add HCl / EtOH solution (492 mL, 30 wt%) dropwise over 30 minutes and stir at 0 °C for 30 minutes. Filter the solid and wash the filter cake with MTBE (2 × 200 mL). Dry under N for 8 hours to obtain the title compound (580 g, 86%) as a white solid. ES / MS (m / z): 134.0 (M+H).

[0035] Preparation 3 (3-chloro-7-methoxyquinolin-4-yl)-(4-fluorophenyl)methanone [ka] A mixture of 4-bromo-3-chloro-7-methoxyquinoline (70 g, 254 mmol) and THF (1 L) is cooled to -40 °C under N2 to precipitate the material. Isopropylmagnesium chloride (2 M in THF, 254 mL, 509 mmol) is added over 20 min, and the mixture is stirred for 1 h. A solution of 4-fluorobenzoyl chloride (66 mL, 559 mmol) in THF (140 mL) is added dropwise, then allowed to warm to room temperature. The reaction is quenched with saturated NH4Cl solution (300 mL) and water (200 mL), and the layers are separated. Wash the organic layer with saturated NH4Cl solution (300 mL), dry over MgSO4, filter, and concentrate to yield an oily residue. The crude brown oil is filtered through silica gel eluting with a mixture of MTBE / hexane (1:1) to give the crude product as a yellow solid (84 g). Treat the solid with 10% methyl acetate / heptane (800 mL) and stir overnight at room temperature. Filter to collect the solid and save. Concentrate the filtrate and purify on silica gel eluting with 10-40% EtOAc / hexanes. Then treat the product with 10% methyl acetate / heptane (200 mL) and stir at room temperature for 3 hours. Filter the resulting solid, combine it with the solid from the previous filtration, and dry under vacuum overnight to give the title compound (31 g, 38%) as a yellow solid. ES / MS (m / z): 316.0 (M+H).

[0036] Preparation 4 (3-chloro-7-hydroxyquinolin-4-yl)-(4-fluorophenyl)methanone [ka] Boron tribromide (1 M in DCM, 295 mL, 295 mmol) is added to a mixture of (3-chloro-7-methoxyquinolin-4-yl)-(4-fluorophenyl)methanone (31 g, 98 mmol) in DCM (217 mL), and the mixture is stirred at room temperature for 3 days. The mixture is slowly poured into a 0°C solution of dibasic potassium phosphate (2 M in water, 700 mL) and water (200 mL). The mixture is warmed to room temperature and stirred for 1 hour. The solution is concentrated in vacuo to remove the organic solvent, filtered, the filtrate is collected, and the filtrate is dried under vacuum at 45°C overnight. The solid is treated with DCM / heptane (1:1, 450 mL) and stirred overnight. The solid is 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).

[0037] Preparation 5 (3-chloro-7-hydroxyquinolin-4-yl)-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)methanone [ka] To a stirred solution of (3-chloro-7-hydroxyquinolin-4-yl)-(4-fluorophenyl)methanone (5.00 g, 15.3 mmol) in DMF (75 mL) was added 2-[3-(fluoromethyl)azetidin-1-yl]ethan-1-ol hydrochloride (3.90 g, 23.0 mmol), followed by sodium hydride (60% in mineral oil, 3.02 g, 76.8 mmol). Stir under N and warm to 40 °C for 45 min. Quench the solution with water and concentrate. Partition the residue between 20% iPrOH / CHCl and saturated aqueous sodium bicarbonate, separate, extract the aqueous layer with 2 × 20% iPrOH / CHCl, combine the organic extracts, dry the combined organic layer over magnesium sulfate, filter, and concentrate the filtrate to give the crude product as a dark red oil. The crude material is purified by silica gel column chromatography eluting with a gradient of 5-10% 7N NH in MeOH / DCM to give the title compound (5.31 g, 84%) as a yellow solid. ES / MS (m / z): 415.0 (M+H).

[0038] Preparation 6 (4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl){3-[2-fluoro-4-(trifluoromethyl)phenyl]-7-hydroxyquinolin-4-yl}methanone [ka] A mixture of (3-chloro-7-hydroxyquinolin-4-yl)-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)methanone (200 mg, 0.48 mmol), 2-fluoro-4-(trifluoromethyl)phenylboronic acid (158 mg, 0.72 mmol), potassium carbonate (202 mg, 1.45 mmol), 2-methyl-2-butanol (3 ml), and water (1 ml) in a microwave vial is degassed with N (5x). XPhos Pd G (12 mg, 0.015 mmol) is added, the vial is sealed, and the vial is microwaved at 80 °C for 2 h. The residue is partitioned between MTBE and saturated NH Cl solution. The layers are separated, and the aqueous layer is extracted with MTBE. The organic extracts are combined, dried over magnesium sulfate, filtered, and the filtrate is concentrated to give an orange residue. The crude material is purified by silica gel column chromatography eluting with 5% MeOH / DCM to give the title compound (205 mg, 78%) as a yellow solid. ES / MS (m / z): 543.2 (M+H).

[0039] Prepare the following compounds essentially similar to that of Preparation 6, with procedural variations of 1-2 hours of heating time, extraction with MTBE or EtOAc, and drying the organic layer over magnesium sulfate or sodium sulfate. Purify by silica gel column chromatography using up to 10% (MeOH or 7M ammoniated MeOH) in DCM (Preparation 10: gradient 3-8% 7M ammoniated MeOH in DCM; Preparations 9 and 11: gradient 4-10% 7M ammoniated MeOH in DCM) and / or by high pH reverse-phase chromatography as described above. [Table 2-1] [Table 2-2]

[0040] Preparation 14 Racemic 4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)(hydroxy)methyl]-3-[2-fluoro-4-(trifluoromethyl)phenyl]quinolin-7-ol [ka] Under N2, (4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl){3-[2-fluoro-4-(trifluoromethyl)phenyl]-7-hydroxyquinolin-4-yl}methanone (305 g, 562.2 mmol) and THF (1.5 L) are added together and the solution is cooled to 0-5 °C. Lithium triethylborohydride (1 M in THF, 1.5 L, 1.5 mol) is added dropwise. The mixture is stirred at 0-5 °C for 1 h. Water (300 mL) is added dropwise and saturated NH4Cl (1 L) is added. The mixture is cooled to room temperature. EtOAc (2 L) is added and the organic layer is collected. The organic layer is washed with brine (500 mL), dried over MgSO4, filtered, and concentrated to dryness. The residue is 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).

[0041] Preparation 15 4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)(hydroxy)methyl]-3-[2-fluoro-4-(trifluoromethyl)phenyl]quinolin-7-ol, isomer 1 [ka] Purify racemic 4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)(hydroxy)methyl]-3-[2-fluoro-4-(trifluoromethyl)phenyl]quinolin-7-ol (354 g, 0.62 mol) using 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 with 0.5% DMEA / CO2, column temperature 40 °C to obtain the title compound (171.4 g, 48%) as the first-eluting isomer. (R) = 0.79 min, column: 4.6 × 150 mm Chiralpak AD-H, eluted with a mobile phase of 35% iPrOH with 0.5% DMEA in CO2, flow rate of 0.6 mL / min, chiral analytical SFC with UV detection at 350 nm confirms enantiomeric enrichment of isomer 1 of greater than 98% ee.

[0042] Alternative Preparation 15 To a solution of (R)-(+)-α.α-diphenyl-2-pyrrolidinemethanol (132 mg, 0.52 mmol) in THF (20 mL) was added trimethyl borate (65 mg, 0.62 mmol). The mixture was stirred at room temperature under N for 1 h. Borane dimethyl sulfide (2.0 M in THF, 2.6 mL, 5.2 mmol) was added, followed by (4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl){3-[2-fluoro-4-(trifluoromethyl)phenyl]-7-hydroxyquinolin-4-yl}methanone (1.0 g, 1.73 mmol). The reaction was heated at 45 °C overnight. Additional 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 h. Slowly add saturated NH4Cl solution (25 mL) and isolate the organic phase. Re-extract the aqueous extract with 20% iPrOH / CHCl3. Combine the organic extracts, dry over Na2SO4, filter, and evaporate to give the 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 at 70 °C for 3 h. Quench the reaction with saturated NH4Cl solution (25 mL) and isolate the organic phase. Re-extract the aqueous extract with 20% iPrOH / CHCl3 (4 × 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.t (R) = 0.79 min, column: 4.6 × 150 mm Chiralpak AD-H, eluted with a mobile phase of 35% iPrOH with 0.5% DMEA in CO2, flow rate of 0.6 mL / min, chiral analytical SFC with UV detection at 350 nm confirms enantiomeric enrichment of isomer 1 of 96% ee.

[0043] Example 1 Racemic 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol [ka] A solution of (4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl){3-[2-fluoro-4-(trifluoromethyl)phenyl]-7-hydroxyquinolin-4-yl}methanone (5.27 g, 9.71 mmol) in 1,4-dioxane (100 mL) is cooled to 5 °C. Lithium triethylborohydride (1 M in THF, 30.0 mL, 30.0 mmol) is added. The cooling bath is removed and stirred at room temperature for 1.5 h. The mixture is quenched with water. Saturated NH4Cl solution and EtOAc are added. The layers are separated and the aqueous layer is extracted with EtOAc. The organic extracts are combined, dried over anhydrous MgSO4, filtered, and the filtrate is concentrated. The crude residue is dissolved in THF (100 mL). Sodium hydride (60% solution in mineral oil, 1.94 g, 48.5 mmol) is added. The solution is refluxed for 1.5 hours. Additional sodium hydride (60% in mineral oil, 1.94 g, 48.5 mmol) is added, followed by refluxing for an additional 30 minutes. The solution is cooled to room temperature and quenched with water. EtOAc and saturated NH4Cl solution are added. The layers are separated, and the aqueous layer is extracted with EtOAc. The organic extracts are combined, dried over anhydrous MgSO4, filtered, and the filtrate is concentrated. The residue is purified by silica gel column chromatography eluting with a gradient of 5-7% MeOH in DCM to give the title compound (3.70 g, 72%) as a pale yellow foam. ES / MS (m / z): 525.2 (M+H).

[0044] The following compounds are prepared essentially similar to the method of Example 1, with variations on the following procedure. For the reduction, 3-5 equivalents of lithium triethylborohydride are used, the reaction time is 30 minutes to 1 hour, and the organic layer is dried over magnesium sulfate or sodium sulfate. The crude residue is used directly or purified by silica gel column chromatography eluting with a gradient of 0-5-7.5-10% MeOH in DCM prior to cyclization. The cyclization is completed by refluxing in THF for up to 16 hours or in DMF, from 2 hours at room temperature in Example 2 to 2 hours at 85°C in Example 8. Extract with DCM or EtOAc and dry the organic layer over magnesium sulfate or sodium sulfate. Equilibrate by silica gel column chromatography using up to 10% (MeOH or 7M ammoniated MeOH) in DCM (Example 2: Gradient 0-10% MeOH in DCM; Example 5: Gradient 4-10% 7M ammoniated MeOH in DCM; Example 8: Gradient 5-7.5% 7M ammoniated MeOH in DCM) or by high pH reverse phase HPLC as previously described. [Table 3-1] [Table 3-2]

[0045] Example 1A 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 1 and Example 1B 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 2 [ka] The two enantiomers of 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol are separated by chiral SFC under the following conditions: Column: LUX® Cellulose-1, 5 × 25 cm; Elution 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. Example 1A is obtained as the first eluting enantiomer (isomer 1). ES / MS (m / z): 525.2 (M+H). (R) = 1.30 min, Column: CHIRALCEL® OD-H, 4.6 × 150 mm; Elution 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. Enantiomeric enrichment of Isomer 1 is confirmed to be greater than 99% ee by chiral analytical SFC. The title compound of Example 1B is isolated to give the second eluting enantiomer (Isomer 2). ES / MS (m / z): 525.2 (M+H). t (R) = 2.03 min, column: CHIRALCEL® OD-H, 4.6 × 150 mm; elution 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 confirms enantiomeric enrichment of isomer 2 with 98% ee.

[0046] Alternative Preparation Example 1B Crystalline 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 2 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, 4-methylbenzenesulfonic acid, isomer 2 (23.8 g, 0.034 mol) in water (250 mL) was stirred at 1000 rpm. NaOH (76 μL) was added and the solution was stirred for 2 h. DCM (600 mL) was added. The mixture was separated, the DCM extract was dried over magnesium sulfate, and the material was filtered through a syringe filter (0.45 μm) and concentrated to dryness. The material was allowed to stand under a stream of N2 over the weekend. 1:1 EtOH / water (80 mL) was added and the mixture was stirred while sonicating. The tan solid was collected by filtration through a nylon membrane to give the title compound (10.47 g, 0.02 mol, 59%).

[0047] X-ray powder diffraction (XRD) XRPD patterns of crystalline solids were obtained on a Bruker D4 Endeavor X-ray powder diffractometer equipped with a CuKα source and Vantec detector, operating at 35 kV and 50 mA. Samples were scanned from 4 to 40° 2θ using a 1.0 mm divergence slit, a 6.6 mm fixed anti-scatter slit, and an 11.3 mm detection slit, at a step size of 0.008° 2θ and a scan rate of 0.5 seconds per step. Dry powder was loaded into a quartz sample holder, and a glass slide was used to obtain a smooth surface. Diffraction patterns of crystalline forms were collected at ambient temperature and relative humidity. Crystalline peak positions of MDI-Jade were determined after shifting the entire pattern based on an internal NIST 675 standard with peaks at 8.853 and 26.774° 2θ. It is well known in the field of crystallography that for any given crystalline form, the relative intensities of diffraction peaks can vary due to preferred orientation resulting from factors such as crystal morphology and crystal habit. When preferred orientation effects exist, peak intensities change, but the characteristic peak positions of the polymorphs do not. See, for example, The United States Pharmacopeia #23, National Formulary #18, pages 1843-1844, 1995. Furthermore, it is well known in the field of crystallography that for any given crystalline form, angular peak positions may vary slightly. For example, peak positions may vary due to variations in the temperature at which the sample is analyzed, sample displacement, or the presence or absence of an internal standard. In the present invention, a peak position variation of 0.2 2θ° is estimated to account for these potential variations without preventing unambiguous identification of the indicated crystalline form. Confirmation of a crystalline form can be based on any unique combination of prominent peaks.

[0048] The prepared crystalline 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, Isomer 2 sample is characterized by an XRD pattern using Cu Ka radiation with a diffraction peak (2-theta value) of 0.2 degrees, specifically a peak at 19.8 in combination with one or more peaks selected from the group consisting of 6.8, 16.0, and 22.1, as set forth in Table 3 below. [Table 4]

[0049] Alternative Preparation Example 1B 4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)(hydroxy)methyl]-3-[2-fluoro-4-(trifluoromethyl)phenyl]quinolin-7-ol, Isomer 1 (63.05 g, 104.7 mmol) is dissolved in DMSO (1.3 L) at room temperature under N2. Cesium carbonate (108 g, 331 mmol) is added portionwise over 5 minutes. The mixture is heated to 60 °C for 15 hours. The mixture is cooled to room temperature and diluted with water (2.1 L) and EtOAc (1.3 L). The mixture is stirred for 5 minutes and separated. The aqueous material is re-extracted with EtOAc (1.3 L) and stirred for 5 minutes. The separated organic extracts are combined and washed with brine, water, and EtOAc. Dry the organic extract with MgSO4, concentrate, and dry under high vacuum at room temperature overnight to give the title compound (52.69 grams, 95.9 percent) as a brown solid. (R) = 2.03 min, Column: CHIRALCEL® OD-H, 4.6 × 150 mm; elution 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 confirms the enantiomeric enrichment of Example 1B of 98.1% ee.

[0050] Example 2A 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-7-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 1 and Example 2B 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-7-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 2 [ka] The two enantiomers of 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-7-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol are separated by chiral SFC under the following conditions: Column: CHIRALPAK® IC, 21 x 250 cm; elution with a mobile phase of 30% iPrOH (containing 0.2% IPA) in CO2; Column temperature: 40°C; Flow rate: 70 g / min; UV detection wavelength: 225 nm. Example 2A is obtained as the first eluting enantiomer (isomer 1). ES / MS (m / z): 525.1 (M+H). (R) = 1.56 min, Column: CHIRALPAK® IC, 4.6 × 150 mm; Elution with a mobile phase of 30% iPrOH (0.2% IPA) in CO2, Column temperature: 40 °C; Flow rate: 5 mL / min; UV detection wavelength: 225 nm. Chiral analytical SFC confirms enantiomeric enrichment of Isomer 1 of greater than 99% ee. The title compound of Example 2B is isolated to give the second eluting enantiomer (Isomer 2). ES / MS (m / z): 525.2 (M+H). t (R) = 2.33 min, column: CHIRALPAK® IC, 4.6 × 150 mm; elution with a mobile phase of 30% iPrOH (0.2% IPA) in CO2, column temperature: 40 °C; flow rate: 5 mL / min; UV detection wavelength: 225 nm confirms enantiomeric enrichment of isomer 2 with 98% ee.

[0051] Example 3A 8-chloro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 1 and Example 3B 8-chloro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 2 [ka] The two enantiomers of 8-chloro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol are separated by chiral SFC under the following conditions: CHIRALCEL® OD-H, 21 × 250 cm; elution 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. Example 3A is obtained as the first eluting enantiomer (isomer 1). ES / MS (m / z): 491.0 (M+H). (R) = 1.55 min, Column: CHIRALCEL® OD-H, 4.6 × 150 mm; Elution 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. Enantiomeric enrichment of Isomer 1 is confirmed to be greater than 99% ee by chiral analytical SFC. The title compound of Example 3B is isolated to give the second eluting enantiomer (Isomer 2). ES / MS (m / z): 491.0 (M+H). t (R) = 2.26 min, column: CHIRALCEL® OD-H, 4.6 × 150 mm; elution 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 confirms enantiomeric enrichment of isomer 2 to greater than 99% ee.

[0052] Example 4A 7-chloro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 1 and Example 4B 7-chloro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 2 [ka] The two enantiomers of 7-chloro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol are separated by chiral SFC under the following conditions: CHIRALCEL® OD-H, 21 × 250 cm; elution 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. Example 4A is obtained as the first eluting enantiomer (isomer 1). ES / MS (m / z): 491.0 (M+H). (R) = 1.71 min, Column: CHIRALCEL® OD-H, 4.6 × 150 mm; Elution 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. Chiral analytical SFC confirms enantiomeric enrichment of Isomer 1 of greater than 99% ee. The title compound of Example 4B is isolated to give the second eluting enantiomer (Isomer 2). ES / MS (m / z): 491.0 (M+H). t (R) = 2.38 min, column: CHIRALCEL® OD-H, 4.6 × 150 mm; elution 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 confirms enantiomeric enrichment of isomer 2 to greater than 99% ee.

[0053] Example 5A 8-Fluoro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 1 and Example 5B 8-Fluoro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 2 [ka] The two enantiomers of 8-fluoro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol are separated by chiral SFC under the following conditions: CHIRALCEL® OD-H, 21 x 250 cm; elution 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. Example 5A is obtained as the first eluting enantiomer (isomer 1). ES / MS (m / z): 475.0 (M+H). (R) = 1.56 min, Column: CHIRALCEL® OD-H, 4.6 × 150 mm; Elution 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. Chiral analytical SFC confirms enantiomeric enrichment of Isomer 1 of greater than 99% ee. The title compound of Example 5B is isolated to give the second eluting enantiomer (Isomer 2). ES / MS (m / z): 475.0 (M+H). t (R) = 2.29 min, column: CHIRALCEL® OD-H, 4.6 × 150 mm; elution 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 confirms enantiomeric enrichment of isomer 2 to greater than 99% ee.

[0054] Example 6A 7-Fluoro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 1 and Example 6B 7-Fluoro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 2 [ka] The two enantiomers of 7-fluoro-5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol are separated by chiral SFC under the following conditions: CHIRALCEL® OD-H, 21 × 250 cm; elution 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. Example 6A is obtained as the first eluting enantiomer (isomer 1). ES / MS (m / z): 475.0 (M+H). (R) = 1.32 min, Column: CHIRALCEL® OD-H, 4.6 × 150 mm; Elution 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. Chiral analytical SFC confirms enantiomeric enrichment of Isomer 1 of greater than 99% ee. The title compound of Example 6B is isolated to give the second eluting enantiomer (Isomer 2). ES / MS (m / z): 475.0 (M+H). t (R) = 1.95 min, Column: CHIRALCEL® OD-H, 4.6 × 150 mm; Elution 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 confirms enantiomeric enrichment of isomer 2 to greater than 99% ee.

[0055] Example 8A 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-7-methyl-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 1 and Example 8B 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-7-methyl-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 2 [ka] The two enantiomers of 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-7-methyl-5H-[1]benzopyrano[4,3-c]quinolin-2-ol are separated by chiral SFC under the following conditions: Column: CHIRALCEL® OD-H, 21 × 250 cm; elution 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. Example 8A is obtained as the first eluting enantiomer (isomer 1). ES / MS (m / z): 471.2 (M+H). (R) = 1.47 min, Column: CHIRALCEL® OD-H, 4.6 × 150 mm; Elution with a mobile phase of 30% iPrOH (0.2% IPA) in CO2, Column temperature: 40 °C; Flow rate: 5 mL / min; UV detection wavelength: 225 nm. Chiral analytical SFC confirms enantiomeric enrichment of Isomer 1 of greater than 99% ee. The title compound of Example 8B is isolated to give the second eluting enantiomer (Isomer 2). ES / MS (m / z): 471.2 (M+H). t (R) = 2.05 min, column: CHIRALCEL® OD-H, 4.6 × 150 mm; elution with a mobile phase of 30% iPrOH (0.2% IPA) in CO2, column temperature: 40 °C; flow rate: 5 mL / min; UV detection wavelength: 225 nm confirms enantiomeric enrichment of isomer 2 to greater than 99% ee.

[0056] Example 9 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 2, benzenesulfonic acid [ka] A slurry of 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 2 (Example 1B) (100 mg, 0.19 mmol) in ACN (3 mL) is heated at 50° C. A solution of benzenesulfonic acid monohydrate (40 mg, 0.23 mmol) in ACN (1 mL) is added. The clear yellow solution is heated at 50° C. for 10 minutes. Heating is discontinued, the reaction mixture is cooled to room temperature, and the mixture is stirred overnight. Toluene (2 mL) is added, and the reaction mixture is stirred for 2 hours. The solution is filtered, the resulting solid is collected, and the solid is washed with ACN (1 mL). The solid is dried under vacuum to give the title compound (74 mg, 55%).

[0057] Alternative Preparation Example 9 A slurry of 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, Isomer 2 (Example 1B) (124.1 mg, 0.24 mmol) in MEK (4 mL) is heated at 50° C. A solution of benzenesulfonic acid monohydrate (50 mg, 0.28 mmol) dissolved in MEK (1 mL) is added. Heating is discontinued, the reaction mixture is cooled to room temperature, and the mixture is stirred over the weekend. Concentrate under a stream of N2. Add MEK (1 mL) and the slurry to give a yellow crystalline solid. The solid is collected, washed with MEK, and dried under vacuum at room temperature to give the title compound (78.8 mg, 48%).

[0058] XRD, Example 9 XRD is completed as described in Example 1B. The prepared -(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 2, benzenesulfonic acid sample is characterized by an XRD pattern using Cu Ka radiation with a diffraction peak (2-theta value) of 0.2 degrees, specifically a peak at 20.5 in combination with one or more peaks selected from the group consisting of 12.3, 22.2, and 23.1, as set forth in Table 4 below. [Table 5]

[0059] Example 10 Crystalline 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, 4-methylbenzenesulfonic acid, isomer 2 [ka] 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, isomer 2 (Example 1B) (204.2 g, 389 mmol) and EtOAc (5 L) were added together and stirred at 60° C., followed by the addition of MeOH (200 mL) at 60° C. to give a clear brown solution. The solution was seeded with the title product (11.48 g), followed by the addition of 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 minutes. The suspension was concentrated to half volume. The solution was cooled at room temperature for 1 hour, filtered, the solid 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 the material, the title compound (229 g, 328.7 mmol) was added to 2-propanol (4.6 L) and 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 overnight under a stream of N2 to give the title compound (174.4 g, 76.2%). Various lots of the title compound prepared in essentially the same way were combined and heptane (2 L) was added. The suspension was stirred for 30 minutes, and the solid was filtered and washed with heptane (300 mL). The solid was dried overnight under a stream of N2 and collected to give the title compound (199.7 g, 99.5%).

[0060] XRD, Example 10 XRD is completed as described in Example 1B. The prepared crystalline 5-(4-{2-[3-(fluoromethyl)azetidin-1-yl]ethoxy}phenyl)-8-(trifluoromethyl)-5H-[1]benzopyrano[4,3-c]quinolin-2-ol, 4-methylbenzenesulfonic acid, Isomer 2 (Example 10) sample is characterized by an XRD pattern using Cu Ka radiation with a diffraction peak (2-theta value) of 0.2 degrees, specifically a peak at 20.1 in combination with one or more peaks selected from the group consisting of 12.8, 19.5, and 22.8, as set forth in Table 5 below. [Table 6]

[0061] Biological assays Evidence of a relationship between ER expression and certain cancers is well known in the art.

[0062] The results of the following assays indicate that the compounds of Formula I, Formula II, and Formula III of the Examples are active SERDs and are believed to be useful in the treatment of cancer.

[0063] Competitive binding assay of ERα (wild type), ERα (Y537S mutant), and ERβ The purpose of the following ER competitive binding assay is to determine the affinity of test compounds for ERα (wild type), ERα (Y537S mutant), and ERβ.

[0064] 0.025 μCi per well in 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. 3Competitive binding assays are performed using H-estradiol (118 Ci / mmol, 1 mCi / mL), 7.2 ng / well of ERα (wild-type), or 7.2 ng / well of ERα (Y537S mutant), or 7.7 ng / well of ERβ receptor. Test compounds are added at 10 concentrations ranging from 10,000 nM to 0.5 nM, and nonspecific binding is determined in the presence of 1 μM 17-β estradiol. After incubating the binding reactions (140 μL) at room temperature for 4 h, cold dextran-charcoal buffer (70 μL) (containing 0.75 g of charcoal and 0.25 g of dextran per 50 mL of assay buffer) is added to each reaction. The plate is mixed for 8 min on an orbital shaker at 4 °C and then centrifuged at 3000 rpm for 10 min at 4 °C. Aliquots (120 μL) of the mixture are transferred to separate 96-well white flat-bottom plates (Costar) and Perkin Elmer Optiphase Supermix scintillation fluid (175 μL) is added to each well. The plates are sealed and shaken vigorously on an orbital shaker. After 2.5 hours of incubation, the plates are read on a Wallac Microbeta counter. A four-parameter logistic curve fit is used to determine the IC 50 Calculate the IC of the compound and calculate the % inhibition at 10 μM. 50 The value is calculated using the Cheng-Prusoff equation: i The results of this assay, as shown in Table 7 below, demonstrate that Examples 1, 1A, and 1B (and others) bind to recombinant ERα wild-type and ERα mutant (Y537S), with Example 1B also binding with a K of 0.11 ± 0.07. i Binds to ERβ with ERβ competition (nM), n=3. [Table 7]

[0065] Of the exemplary compounds tested, the K for wild-type ERα ranged from about 0.300 nM to about 65 nM. The K for the ERα Y537S mutant ranged from about 2 nM to 300 nM. The results of this assay demonstrate the binding affinity and potency of the exemplary compounds for wild-type, mutant (ESR1 Y537S), and ERβ proteins.

[0066] ERα degradation assay in MCF7 cells The purpose of the following ERα degradation assay is to measure the degradation of ERα by test compounds in an ERα-positive breast cancer cell line such as MCF7.

[0067] Culture MCF7 (purchased from ATCC HTB-22) cells in DMEM medium supplemented with 10% FBS, 0.01 mg / mL human insulin, and 1% penicillin / streptomycin antibiotics. Seed them into a 384-well flat-bottom plate at a density of 4,000 cells per well in phenol red-free DMEM medium (20 μL) containing 10% charcoal-stripped FBS. Incubate the cells overnight in a cell culture incubator (5% CO2, 95% relative humidity, 37 °C) to allow the cells to adhere to the plate. The next day, administer the test compound to the cells. Prepare test compound serial dilutions (1:3) ranging from 6 μM to 0.0003 μM using an Echo555 acoustic dispenser. Add 5 μL from the serial dilution plate to the cell plate, resulting in a final DMSO concentration of 0.2% and a final test compound concentration ranging from 2 to 0.0001 μM. For the maximum point, medium containing 0.2% DMSO was used, and 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 for 24 hours at 37°C and 5% CO2. Cells were fixed by adding 10 μL of 14% paraformaldehyde for 30 minutes at room temperature. Cells were washed once with 20 μL of PBS and incubated for 1 hour with 20 μL of PBS containing 0.5% (v / v) TWEEN® 20. Cells were washed with PBS containing 0.05% TWEEN® 20 (2x) and blocked with 20 μL of 3% BSA in PBS containing 0.05% TWEEN® 20 and 0.1% TRITON™ X-100 at room temperature for 1 hour. Primary antibody (20 μL) diluted 1:500 in 1% BSA in PBS containing 0.05% TWEEN® 20 (ERα (clone SP1) monoclonal rabbit antibody #RM-9101-S, Thermo Scientific) is added per well, the plate is sealed, and the plate is incubated overnight at 4° C. The next day, cells are washed with PBS containing 0.05% TWEEN® 20 (2×) and incubated with secondary antibody (20 μL / well) (1:1000 dilution, goat anti-rabbit IgM ALEXAFLUOR™ 488) in PBS 1% BSA for 105 minutes at room temperature.After washing the plate with PBS (2 x 20 μL), RNase (Sigma) (20 μL of 50 μg / mL) and a 1:1000 dilution of propidium iodide in PBS were added per well (20 μL). The plate was sealed and incubated on the bench at room temperature for 1 hour (protected from light). ERα was measured by scanning the plate with an ACUMEN EXPLORER™ (a laser-scanning fluorescent microplate cytometer manufactured by TTPLABTECH LTD). Image analysis was based on cellular fluorescence signals to identify positive cells. ER-positive cells were identified by the mean intensity. Individual cells were identified using the total intensity at 575-640 nm from the propidium iodide / DNA assay. The assay output was the % ER-positive cells. The IC was calculated by curve fitting a four-parameter logistic regression for each output using GENE DATA™. 50 The results of this assay demonstrate the potent degradation of ERα induced by the compounds of Formula I, Formula II, and Formula III described herein in MCF7 breast cancer cells. Relative IC of Examples 1, 1A, and 1B 50 The values ​​are shown in Table 8. [Table 8]

[0068] Specifically, the results in Table 7 demonstrate potent degradation of ERα by the compound of Example 1 in MCF7 breast cancer cells. Among the exemplary compounds tested, the relative IC 50 ranged from 0.003 to >2 μM, indicating that all but Example 2B exhibited activity at the concentrations tested. The results of this assay indicate that the compounds of formula (I) are SERDs with potent ERα-degrading activity in cells.

[0069] PRα induction assay in MCF7 cells The purpose of the following PRα induction assay is to determine whether a test compound has agonist activity at the ERα receptor (agonists are expected to activate the receptor).

[0070] Culture MCF7 (purchased from ATCC HTB-22) in DMEM medium supplemented with 10% FBS, 0.01 mg / mL human insulin, and 1% penicillin / streptomycin antibiotics. Seed the cells (before they reached 70% confluence) into 384-well flat-bottom plates at a density of 4,000 cells per well in 20 μL of DMEM phenol red-free medium containing 10% FBS (charcoal-treated). Incubate the cells overnight in a cell culture incubator (5% CO2, 95% relative humidity, 37 °C) to allow the cells to adhere to the plate. The next day, administer the test compound to the cells. Prepare serial compound dilutions (1:3) ranging from 6 μM to 0.0003 μM using an Echo555 acoustic dispenser. Test compounds were dosed to cells by adding 5 μL of test compound from the serial dilution plate to the cell plate, resulting in a final DMSO concentration of 0.2%. The final test compound concentrations ranged from 2 to 0.0001 μM. For the maximum dose, medium containing 0.2% DMSO was used, and for the minimum dose, fulvestrant diluted to a final concentration of 2 μM in growth medium containing 0.2% DMSO was used. After dosing with the test compound, the cell plate was incubated for 24 hours at 37°C and 5% CO2. Cells were fixed by adding 10 μL of 14% paraformaldehyde for 30 minutes at room temperature. The cells were washed once with 20 μL of PBS and incubated for 1 hour in 20 μL of PBS containing 0.5% (v / v) TWEEN® 20. Wash cells twice with 20 μL of PBS containing 0.05% TWEEN® 20 and block with 20 μL / well of 3% BSA in PBS containing 0.05% TWEEN® 20 and 0.1% TRITON™ X-100 for 1 hour at room temperature. Add 20 μL of 1:500 primary antibody (PR monoclonal mouse anti-human antibody, clone PgR 636, Dako, M3569) diluted in 1% BSA / PBS containing 0.05% TWEEN® 20 per well, seal the plate, and incubate overnight at 4°C.

[0071] The next day, wash the cells with PBS 0.05% TWEEN® 20 (2 x 20 μL) and incubate with secondary antibody (20 μL / well) (1:1000 dilution, goat anti-rabbit IgM ALEXAFLUOR™ 488) in PBS 1% BSA for 105 minutes at room temperature. After washing with PBS (2 x 20 μL), add RNase A (20 μL of 50 μg / mL) (Sigma) and a 1:1000 dilution of propidium iodide in PBS per well. Seal the plate and incubate it on the bench for 1 hour at room temperature (protected from light). Scan the plate with an ACUMEN EXPLORER™ (a laser-scanning fluorescence microplate cytometer manufactured by TTPLABTECH LTD) to measure PRα. Image analysis is based on the cellular fluorescence signal to identify positive cells. The average intensity identifies PR-positive cells. The total intensity from propidium iodide / DNA at 575-640 nm is used to identify individual cells. The output of the assay is % PR-positive cells. IC values ​​were calculated by four-parameter logistic curve fitting of each output using GENE DATA™. 50 The results of this assay show no significant agonist activity of Examples 1, 1A, and 1B in MCF7 breast cancer cells. The relative IC in this assay for the compounds tested is 50 is >2 μM. The results of this assay do not demonstrate significant agonist activity of the exemplified compounds tested in MCF7 breast cancer cells. These results also demonstrate that the exemplified compounds tested are antagonists of ERα in MCF7 breast cancer cells (i.e., they have SERD activity).

[0072] PRα inhibition (ERα functional antagonism) cellular assay in MCF7-ESR1 Y537N 682 CRISPR cells The purpose of the following PRα inhibition (ERα functional antagonism) cellular assay is to determine the antagonist activity of test compounds against the Y537N mutant ERα receptor. Antagonists in this assay are expected to block ERα receptor function. Because PRα is a downstream transcriptional target of ERα, antagonists of ERα are expected to inhibit PRα expression.

[0073] MCF7-ESR1 Y537N-682 (clone #682, generated by CRISPR / Cas9 gene editing of the ESR1 gene in MCF7 cells) was cultured in DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin antibiotics. Cells (before reaching 70% confluence) were seeded into 384-well flat-bottom plates at a density of 4,000 cells per well in 20 μL of phenol red-free DMEM medium (charcoal-treated) containing 10% FBS. The cells were incubated overnight in a cell culture incubator (5% CO2, 95% relative humidity, 37 °C) to allow cells to adhere to the plate. The next day, the cells were dosed with test compounds. Using an Echo555 acoustic dispenser, serial compound dilutions (1:3) ranging from 6 μM to 0.0003 μM were prepared. Cells were dosed by adding 5 μL from the serial dilution plate to the cell plate, resulting in a final DMSO concentration of 0.2% and a final test compound concentration ranging from 2 to 0.0001 μM. The maximum point was medium containing 0.2% DMSO, while the minimum point was fulvestrant diluted in growth medium containing 0.2% DMSO to a final concentration of 2 μM. After dosing with the test compound, the cell plate was incubated for 72 hours at 37°C and 5% CO2. Cells were fixed by adding 10 μL of 14% paraformaldehyde for 30 minutes at room temperature. Cells were washed once with 20 μL of 1x PBS and incubated for 1 hour in 20 μL of PBS containing 0.5% (v / v) TWEEN® 20. Wash cells with 20 μL of PBS containing 0.05% TWEEN® 20 and block with 20 μL of 3% BSA / PBS 0.05% TWEEN® 20, 0.1% TRITON™ X-100 (20 μL / well) for 1 hour at room temperature. Add 20 μL of 1:500 primary antibody (PR monoclonal mouse anti-human antibody, clone PgR 636, Dako, M3569) diluted in 1% BSA / PBS 0.05% TWEEN® 20 per well, seal the plate, and incubate overnight at 4°C.

[0074] The next day, wash the cells with PBS 0.05% (2 x 20 μL) and incubate with secondary antibody (20 μL / well) (1:1000 dilution, goat anti-rabbit IgM ALEXAFLUOR™ 488) in PBS 1% BSA for 105 minutes at room temperature. After washing with PBS (2 x 20 μL), add RNase A (20 μL of 50 μg / mL) (Sigma) and a 1:1000 dilution of propidium iodide in PBS per well. Seal the plate and incubate it for 1 hour at room temperature (protected from light) on the bench. Scan the plate with an ACUMEN EXPLORER™ (a laser-scanning fluorescence microplate cytometer manufactured by TTPLABTECH LTD) to measure PRα. Image analysis is based on the cellular fluorescence signal to identify positive cells. The average intensity identifies PR-positive cells. The total intensity from propidium iodide / DNA at 575-640 nm is used to identify individual cells. The output of the assay is % PR-positive cells. IC values ​​were calculated by four-parameter logistic curve fitting of each output using GENE DATA™. 50 Determine.

[0075] The results of this assay demonstrate potent inhibition and functional antagonism of PRα by Examples 1, 1A, and 1B in MCF7 (ESR1 Y537N, heterozygous mutant) breast cancer cells. The relative IC values ​​of Examples 1, 1A, and 1B (and others) in this assay are: 50 are shown below in Table 9. Relative IC 50 ranged from about 0.0118 to >1.6 μM, indicating that the exemplified compounds, with the exception of Example 2B at 1.6 μM, were potent antagonists of the ERα mutant (Y537N) 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 transcription of PRα. [Table 9]

[0076] PRα inhibition (ERα functional antagonism) cellular assay in MCF7 cells The purpose of the following PRα inhibition (ERα functional antagonism) cellular assay is to determine the antagonist activity of test compounds at the ERα receptor. Antagonists in this assay are expected to block ERα receptor function. Because PRα is a downstream transcriptional target of ERα, antagonists of ERα are expected to inhibit expression of PRα.

[0077] The assay conditions were performed using the MCF7 cell line as detailed in the cell-based Acumen assay for ERα degradation described above, except that before dispensing test compounds, the medium was removed from the cell plate and all wells except the negative control well (column 24 of the plate) were pretreated with assay medium containing 0.47 nM estradiol for 30 minutes. This assay involves immunostaining to detect PRα, and then scanning the plate with an ACUMEN EXPLORER™ (a laser-scanning fluorescent microplate cytometer manufactured by TTP LABTECH LTD) to measure PRα. Image analysis relies on cytofluorescence signals to identify positive cells. Mean intensity identifies PRα-positive cells. Individual cells are identified using the total intensity from propidium iodide / DNA at 575–640. The assay output is the % PRα-positive cells. Using GENE DATA™, IC values ​​were calculated by fitting a four-parameter logistic regression to each output. 50 The results of this assay demonstrate the potent inhibition and functional antagonism of PRα by Examples 1, 1A, and 1B in MCF7 breast cancer cells. The relative IC values ​​of Examples 1, 1A, and 1B in this assay are 50 are shown below in Table 10. Relative IC for exemplary compounds 50The ERα ranged from about 0.029 to >2 μM, indicating that all exemplary compounds tested, except for 1A and 2B, were potent antagonists of the 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 transcription of PRα at the concentrations tested. [Table 10]

[0078] Cell proliferation assay of MCF7 and MCF7-ESR1 Y537N-682 The purpose of the cell proliferation assays below is generally to detect whether a test compound affects cell proliferation.

[0079] MCF7 (purchased from ATCC HTB-22) cells were seeded at a density of 2,000 cells per well in charcoal-treated DMEM phenol red-free medium 10% FBS (20 μL volume). MCF7-ESRY537N-682 (generated by CRISPR / Cas9 gene editing of the ESr1 gene in MCF7 cells, clone #682) was seeded at a density of 1,000 cells per well in DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin antibiotics. The plate was incubated at 37 °C and 5% CO2. The following day, the cells were dosed with test compounds. Using an Echo555 acoustic dispenser, serial dilutions (1:3) of test compounds ranging from 60 μM to 0.003 μM were prepared. Cells were dosed by adding 5 μL from the serial dilution plate to the cell plate, resulting in a final DMSO concentration of 0.2% and a final test compound concentration ranging from 20 to 0.001 μM. For the maximum dose, use medium containing 0.2% DMSO, and for the minimum dose, use fulvestrant diluted to a final concentration of 2 μM in growth medium containing 0.2% DMSO. After dosing with the test compound, incubate the cell plate at 37°C and 5% CO2. Seven days after adding the test compound, remove the plate from the incubator and add 65 μL of cold 96% EtOH to each well. After 30 minutes, remove the medium and add RNase (20 μL of 50 μg / mL) (Sigma) and a 1:1000 dilution of propidium iodide in PBS per well. Seal the plate and incubate it on the bench at room temperature for 1 hour (protected from light). The plate is scanned with an ACUMEN EXPLORER™ (a laser-scanning fluorescence microplate cytometer manufactured by TTPLABTECH LTD). MCF-7 cell lines grow to form aggregates, and cell count as a target number may not be used as a readout; therefore, cell number can be assessed by the estimated cell number (calculated by the area parameter (the ratio of the total area of ​​the total cell population (defined by the specified range of peak intensity of FL-1(PI) and the average area of ​​the single cell population (defined by perimeter))).IC by curve fitting a four parameter logistic for each output using GENE DATA™. 50 Determine the relative IC of Examples 1, 1A, and 1B (and others) in MCF7 ESR1 wild-type and MCF7-ESR1 Y537N mutant cells. 50 The results of this assay demonstrate potent antiproliferative activity and cell growth inhibition by Examples 1, 1A, and 1B (and others) in MCF7 (ESR1 wild type) and MCF7 (ESR1 Y537N mutant) breast cancer cells. The relative IC of exemplary compounds 50 ranged from 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 exemplary compounds tested demonstrate potent antiproliferative activity and cell growth inhibition in MCF7 (ESR1 wild-type) and MCF7 (ESR1 Y537N mutant) breast cancer cells. [Table 11-1] [Table 11-2]

[0080] In vivo target inhibition (IVTI) assay (PGR RT-qPCR assay) in MCF7 tumors The purpose of this IVTI assay is to measure the ability of a test compound (SERD) to inhibit PRα gene expression (transcription) downstream of ERα in xenograft tumors implanted in mice.

[0081] Female NOD SCID mice (22–25 g) from Envigo RMS, Inc., Madison, Wisconsin were given 5 × 10 e 6MCF7 ER-positive breast cancer cells (ATCC, #HTB-22) are implanted subcutaneously in a 1:1 HBSS+MATRIGEL™ solution (200 μL) in the right flank area. One day prior to tumor cell implantation, 17-β estradiol pellets (0.18 mg / pellet, 90-day release, from Innovative research) are implanted subcutaneously. Tumor growth and body weight are measured twice weekly, starting 7 days after implantation. Tumors between 150 and 300 mm in size are considered to be resistant to steroids. 3 Upon reaching 100 mg / kg, animals are randomized and divided into groups of 5 animals. Animals are orally administered multiple doses of test compound in a test compound-specific vehicle (1% hydroxyethylcellulose / 0.25% TWEEN® 80 / 0.05% antifoam in purified water) or vehicle alone for 3 days, with tumor and blood samples collected at desired time intervals after the last dose. Animals are sacrificed using isoflurane anesthesia and cervical dislocation. Tumors are snap-frozen and stored at -80°C until processed for RNA isolation and RT-qPCR assays. Blood is collected in EDTA tubes, spun down for plasma, and frozen at -80°C in 96-well plates. Mass spectrometry is used to determine test compound exposure.

[0082] Tumors were disrupted with liquid nitrogen using Matrix D beads (MP Biomedical, #6913-500) in a FASTPREP-24™ Cell Disrupter machine (MP Biomedical) and lysed in 1x RNA lysis buffer (from the RNA isolation kit). After spinning at 14,000 rpm for 20 minutes at 4°C, the tumor lysate was transferred to a new tube. RNA was isolated from the tumor lysate using the PURELINK® RNA Mini Kit (Invitrogen #12183018A) or RNeasy Mini Kit (Qiagen #74104 and #74106). DNA contamination was removed using the PURELINK® DNase Set (Invitrogen #12185010) or RNase-free DNase Set (Qiagen #79254). The isolated RNA concentration was measured by diluting the sample with RNase-free water and measuring the absorbance at 260 nm on a plate reader (SpectraMax190). Subtract the average 260 nm absorbance reading of the blank (RNase-free water only) from the 260 nm readings of all other RNA samples. Dilute the RNA samples to equal concentrations with RNase-free water. Synthesize cDNA from the diluted RNA using the First-Strand Synthesis System for RT-PCR (Invitrogen, #18080-051). To perform RT-qPCR, first dilute the cDNA with RNase-free water. Combine 2x Absolute Blue qPCR ROX Mix (Thermo, #AB-4139 / A), pGR primers (Thermo, Hs01556702_m1), and diluted cDNA for each reaction in a PCR plate (Applied Biosystems, #4309849). Amplify the cDNA by incubating the samples at 50°C for 2 minutes, followed by 95°C for 15 minutes, in a thermocycler (ABI Prism 7900HT Sequence Detection System). Incubation continues at 95°C for 15 seconds, followed by 50°C for 60 seconds, for a total of 40 cycles. Cycles are normalized to a housekeeping gene and used to calculate % PGR inhibition compared to vehicle alone. Each sample is analyzed in duplicate, and the average number is used in the calculation.Percent target (PGR) inhibition is calculated using Excel and XL Fit.

[0083] The results of this assay demonstrate that Example 1B inhibits PRα (PGR) expression in tumor xenograft models. When administered orally, Example 1B inhibits PRα (PGR) expression by approximately 78% in tumor xenograft models at a dose of 30 mg / kg for 24 hours. These results demonstrate in vivo ERα antagonistic activity and significant and sustained inhibition of ERα-mediated transcriptional activity in tumor xenograft models.

[0084] In vivo tumor growth inhibition study in ER-positive (ESR1 wild-type) breast cancer xenograft tumor model implanted in mice The purpose of the following xenograft tumor inhibition assay is to measure the reduction in transplanted tumor volume in response to test compound administration.

[0085] Human breast cancer cells MCF7 (ATCC #HTB-22) and HCC1428 (ATCC #CRL-2327) were grown in culture and harvested to 5 × 10 e 6 Cells were injected subcutaneously into the right hind flank of female NOD SCID mice (22-25 g, Envigo RMS, Inc.) in a 1:1 HBSS + MATRIGEL™ solution (200 μL). 24 hours prior to cell implantation, estrogen pellets (0.18 mg / pellet, 17β-estradiol, 90-day release, Innovative Research) were implanted subcutaneously. Human breast cancer cells T47D (ATCC #HTB-22) were grown in culture and harvested to give 5 × 10 e 6 Cells were injected subcutaneously into the right hind flank of female NOD SCID mice (22-25 g, Envigo RMS, Inc.) in a 1:1 HBSS + MATRIGEL™ solution (200 μL). 24 hours prior to cell implantation, estrogen pellets (0.38 mg / pellet, 17β-estradiol, 90-day release, Innovative Research) were implanted subcutaneously. Human breast cancer cells ZR-75-1 (ATCC #CRL-1500) were grown in culture and harvested to give 5 × 10 e 6Cells are injected subcutaneously in a 1:1 HBSS+MATRIGEL™ solution (200 μL) into the right hind flank of female NOD SCID mice (22-25 g, Envigo RMS, Inc.). 24 hours prior to cell implantation, animals receive an intramuscular injection of 50 μL of estradiol valerate (Delestrogen®) (10 mg / mL), followed by administration once every 14 days for the duration of the study. Tumor growth and body weight are measured twice weekly, starting 7 days after implantation. Tumors measuring 250-350 mm in size are then monitored. 3 When the animals reach 100 mg / kg, they are randomized and divided into groups of 5 animals. The test compound, Example 1B, is prepared in an appropriate vehicle (1% hydroxyethylcellulose / 0.25% TWEEN® 80 / 0.05% antifoam in purified water) and administered by oral gavage once daily (QD) for 28 days. Tumor response is determined by tumor volume measurements performed twice weekly during the treatment course. Body weights are obtained whenever tumor volume is measured as a general measure of toxicity.

[0086] The compound of Example 1B was found to have delta T / C % values ​​as provided below in Table 12. These results indicate that the compound of Example 1B exhibits good oral bioavailability in mice and demonstrates significant antitumor activity or tumor regression in an ER-positive (ESR1 wild-type) human breast cancer xenograft model. [Table 12]

[0087] % regression is calculated when the endpoint volume is below baseline using the formula: 100*(T-T0) / T0, where T0 is the mean baseline tumor volume for the treatment group.

[0088] The % change in T / C is computed using the grand mean of all groups from baseline (randomization) on Day 32.

[0089] In vivo tumor growth inhibition study in ESR1 mutant (Y537S) breast cancer PDX tumor model (ST941 / HI) transplanted into mice The goal of the following xenograft tumor inhibition assay is to measure the reduction in transplanted tumor volume in response to test compound administration in ESR1 mutant and hormone-independent (HI) breast cancer patient-derived xenograft (PDX) models.

[0090] The ST941 / HI PDX model was derived and implemented at South Texas Accelerated Research Therapeutics (San Antonio, TX). Tumor fragments were harvested from host animals and implanted into immunodeficient mice (The Jackson Laboratory). The study consisted of approximately 125–250 mm 3 The treatments were initiated with a mean tumor volume of 100 mg / kg / day. The test compound, Example 1B, was prepared in the appropriate vehicle (1% hydroxyethylcellulose / 0.25% TWEEN® 80 / 0.05% antifoam in purified water) and administered by oral gavage for 28 days. Tumor response was determined by tumor volume measurements taken twice weekly during the treatment course. Body weights were taken whenever tumor volumes were measured as a general measure of toxicity.

[0091] The compound of Example 1B was found to have delta T / C% values ​​as provided below in Table 13. These results indicate that the compound of Example 1B exhibits good oral bioavailability in mice and demonstrates significant anti-tumor activity or tumor regression in an ESR1 mutant (Y537S) human breast cancer PDX model. [Table 13]

[0092] % regression is calculated when the endpoint volume is below baseline using the formula: 100*(T-T0) / T0, where T0 is the mean baseline tumor volume for the treatment group.

[0093] The % change in T / C is computed using the grand mean of all groups from baseline (randomization) on Day 32.

[0094] Combination Studies Due to tumor heterogeneity and acquired resistance to endocrine therapy, combination therapy has become essential for the treatment of ER-positive and advanced / metastatic breast cancer for effective treatment or to overcome acquired resistance. The combinatorial effects of Example 1B with the CDK4 / 6 inhibitor abemaciclib, 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]quinolin-2-one ("Compound A") were tested in vitro in five ER-positive breast cancer cell lines.

[0095] Cell viability assays for combination studies Seed cells in clear bottom 384-well cell culture plates at the density indicated in Table 14 below in a 20 µL volume of the medium listed in the table. [Table 14]

[0096] Plates are incubated at 37° C. and 5% CO 2 . The next day, cells are dosed with the test compound, Example 1B.

[0097] Compounds are prepared as 10 mM DMSO stock solutions and used in dose-response studies, starting at a top concentration of 10 or 1 μM. Two compounds are tested together at a fixed ratio, and 1:3 serial dilutions are prepared. IC 50Compounds for determination start at a concentration of 20 μM. Cells are dosed by adding 5 μL from the serial dilution plate to the cell plate, resulting in a final DMSO concentration of 0.2%. Final test compound concentrations range from 20 to 0.001 μM for single treatments and lower ranges for combinations. For the maximum dose, use medium containing 0.2% DMSO, and for the minimum dose, use staurosporine diluted to a final concentration of 2 μM in growth medium containing 0.2% DMSO. After dosing with the test compound, incubate the cell plate at 37°C and 5% CO2. After a two-doubling incubation with the compound, remove the plate from the incubator and add 65 μL of cold 96% ethanol to each well. After 30 min, remove the medium and add RNase (20 μL of 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 (protect from light). Scan the plate with an ACUMEN EXPLORER™ (a laser-scanning fluorescence microplate cytometer manufactured by TTPLABTECH LTD). Some cell lines proliferate to form aggregates, and cell count as a target number may not be usable as a readout. Therefore, use the total area population (specified range of peak intensity of FL-1 (PI)) or total intensity of PI to assess cell number.

[0098] In vitro combination data suggest synergy (defined below) in combination with Example 1B and abemaciclib or everolimus in five of five ER-positive breast cancer cell lines, as shown in Table 15. The combination of Example 1B and Compound A is synergistic in four of four ER-positive breast cancer cell lines tested. The combined effect of Example 1B and alpelisib is additive in two of four ER-positive breast cancer cell lines and synergistic in two of four ER-positive breast cancer cell lines. [Table 15]

[0099] Data analysis and interpretation of combined effects Published methods were used to calculate in vitro combination effects (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 two drugs, curve shift analysis was performed using a customized XL template with the XLFit5 add-in. Single-drug curves were fitted using four-parameter logistic regression. The criteria and constraints used for fitting were: (i) bottom < (-20) fixed to 0, and (ii) top > 120 fixed to 100. If all observations were below the user-specified threshold, a constant fit was performed with hill = 0, and the IC was calculated. 50 is considered to be higher than the maximum concentration included. The absolute IC 50 Once the IC is obtained, the equivalent concentrations at 50% of activity are calculated for the single agents and combinations. These equivalent concentrations, along with the measured activities, are used to calculate the absolute IC 50 The curves for single agents reach 50% activity at an equivalent concentration value equal to 1, whereas synergistic combinations reach 50% at lower values ​​and show a shift to the left, and antagonistic combinations show a shift to the right. The equivalent concentration is also used to calculate the CI 50 is the absolute IC of the combination curve 50に Equal to, CI 50 Used to calculate the combination index (CI) (at 50% of activity). 50 Along with the CI, other CIs (combination indices) at various percentages of activity can be calculated (CI10, CI20, CI30, CI40, CI60, CI70, CI80, CI90). To calculate CInn, the equivalent concentrations at various activity percentages are calculated. For each activity percentage, a 95% confidence interval, or margin of error, is calculated, and this confidence interval is used to calculate the upper limit as the addition of the margin of error to the CI and the lower limit as the subtraction of the margin of error from the CI. Upper limit = CI + CI 95% and lower limit = CI - CI 95%. These limits are used to interpret the results.

[0100] The statistical interpretation of each activity percentage is as follows: [Table 16] The biological interpretation of each percentage activity is as follows: [Table 17]

[0101] In vivo combination studies Due to tumor heterogeneity and acquired resistance to endocrine therapy, combination therapy has become essential for the treatment of ER-positive and advanced / metastatic breast cancer for effective treatment or to overcome acquired resistance. It has been hypothesized that combinations of targeted therapies may be more effective in slowing or halting ER-positive breast cancer. While the combination of a CDK4 / 6 inhibitor and fulvestrant has been approved for the treatment of ER-positive metastatic breast cancer, a high percentage of patients develop resistance due to acquired mutations in ESR1 or PIK3CA. As potent degraders and antagonists of ERα, oral SERDs such as Example 1B may be more effective in slowing or halting ESR1-mutant or PIK3CA-mutant breast cancer as single agents or in combination with CDK4 / 6 inhibitors such as abemaciclib or PI3K / mTOR inhibitors such as Compound A. In this context, the compound of Example 1B will be tested for tumor growth inhibition in combination with abemaciclib (see patent) or Compound A (see patent). More specifically, the compound of Example 1B is tested in combination with abemaciclib or Compound A in ESR1 wild-type and PIK3Ca mutant MCF7 breast cancer xenograft models.

[0102] Human breast cancer cells MCF7 (ATCC #HTB-22) were grown in culture and harvested at 5 × 10 e 6 Cells are injected subcutaneously into the right hind flank of female NOD SCID mice (22-25 g, Envigo RMS, Inc.) in a 1:1 HBSS+MATRIGEL™ solution (200 μL). 24 hours prior to cell implantation, estrogen pellets (0.18 mg / pellet, 17β-estradiol, 90-day release, Innovative Research) are implanted subcutaneously. Tumor growth and body weight are measured twice weekly, starting 7 days after implantation. Tumors measuring 250-350 mm in size are then injected. 3 Upon reaching 100 mg / kg, animals are randomized and divided into groups of 5 animals. The test compound (Example 1B) is prepared in an appropriate vehicle (1% hydroxyethylcellulose / 0.25% TWEEN® 80 / 0.05% antifoam in purified water) and administered by oral gavage for 42 days. The CDK4 / 6 inhibitor (abemaciclib) is formulated in 1% HEC in 25 mM sodium phosphate buffer, pH 2.0. The PI3K / mTOR inhibitor (Compound A) is formulated in 1% hydroxyethylcellulose / 0.25% TWEEN® 80 / 0.05% antifoam in purified water. Tumor response is determined by tumor volume measurements performed twice weekly during the treatment course. Body weights are taken whenever tumor volume is measured as a general measure of toxicity. Tumor volume is estimated using the formula: v = l x w2 x 0.535, where l = larger measured diameter and w = smaller perpendicular diameter.

[0103] statistical analysis Statistical analysis of tumor volume data begins by transforming the data to a logarithmic scale to equalize variance across time and treatment groups. Log volume data are analyzed using a two-way analysis of variance with replicates by time and treatment using the MIXED procedure in SAS software (version 9.3). The correlation model for repeated measures is Spatial Power. Treatment groups are compared to the control group at each time point. Adjusted means and standard errors at each time point are calculated using the MIXED procedure separately for each treatment group. Both analyses account for autocorrelation within each animal and for missing data that occurs when animals with large tumors are removed from the study early. Adjusted means and standard errors (se) are plotted against time for each treatment group. Analysis of tumor volume is performed using log 10 and based on spatial power covariance structure. P values ​​are based on comparisons between two specific groups.

[0104] Combined Analysis Method (Bliss Independence for IVEF Studies) First, a regular repeated measures model is fitted to the log volume for group versus time. A contrast statement is then used to test for interaction effects at each time point using two specific treatments combined. This is equivalent to the Bliss Independence method, which assumes that tumor volume can theoretically reach zero, i.e., complete regression. The expected additive response (EAR) for the combination is calculated based on the tumor volume scale as EAR volume = V1 * V2 / V0 (where V0, V1, and V2 are the estimated mean tumor volumes for vehicle control, treatment 1 alone, and treatment 2 alone, respectively). If the interaction test is significant, the combination effect is declared statistically superior to additive, depending on whether the observed mean volume of the combination is smaller than the EAR volume, or inferior to additive, depending on whether it is greater. Otherwise, the statistical conclusion is additive. Furthermore, a biologically relevant additive range can be defined as X% above and below the EAR volume. Typically, X would be 25-40%. A biological conclusion can then be made as combination above, above, or below the addition if the mean volume of the observed combination is below, in the middle, or above the addition interval.

[0105] There may be situations where plateauing is the most expected response. In these situations, the Bliss method can be applied directly to the % delta T / C values ​​to obtain the EAR % response: EAR % delta T / C = Y * Y / 100, where Y and Y are the % delta T / C values ​​for the single agent treatments. Currently, there are no statistical tests to compare the observed % delta T / C in the combination group with the EAR, but the biological criteria described above can be applied.

[0106] As shown in Tables 15 and 16, treatment with Example 1B or abemaciclib alone as a single agent resulted in tumor regression of 32% (%dT / C=-32) and 52% (%dT / C=-52), respectively, both of which were statistically significant (p<0.001) compared to vehicle control. Although the combined effect of Example 1B and abemaciclib was "less than additive," the combined effect of Example 1B and abemaciclib was significantly superior to Example 1B alone (p<0.001). However, the efficacy of single-agent abemaciclib was not statistically significant from the combination (P=0.055). The combination was tolerated in animals without significant weight loss. [Table 18] [Table 19]

[0107] As shown in Tables 17 and 18, treatment with Example 1B or Compound A alone as a single agent resulted in tumor regression of 32% (%dT / C=-32) and 36% (%dT / C=-36), respectively, both of which were statistically significant (p<0.001) compared to vehicle control. The combined effect of Example 1B and Compound A was "less than additive," but the combined effect of Example 1B and Compound A was significantly superior to Example 1B alone (p<0.001) or Compound A alone (p=0.002*). The combination was tolerated in animals without significant weight loss. [Table 20] [Table 21]

[0108] As shown in Tables 19 and 20, treatment with Example 10 or abemaciclib alone as a single agent resulted in 51% (%dT / C=-51) and 70% (%dT / C=-70) tumor regression, respectively, both of which were statistically significant (p<0.001) compared to vehicle control. Although the combined effect of Example 10 and abemaciclib was "less than additive," the combined effect of Example 10 and abemaciclib was significantly superior to Example 10 alone (p=0.039). However, the combined effect of Example 10 and abemaciclib was not significantly different from abemaciclib alone (p=0.905). The combination was tolerated in animals without significant weight loss. [Table 22] [Table 23]

[0109] As shown in Tables 21 and 22, treatment with Example 10 or alpelisib alone as a single agent resulted in tumor regression of 51% (%dT / C=-51) and 21% (%dT / C=-21), respectively, both of which were statistically significant compared to vehicle controls (p<0.001 and p=0.013). The combined effect of Example 10 and alpelisib was "additive," and the combined effect of Example 10 and alpelisib was significantly superior to Example 10 alone (p=0.009). The combined effect of Example 10 and alpelisib was also significantly superior to alpelisib alone (p=<0.001). The combination was tolerated in animals without significant weight loss. [Table 24] [Table 25]

[0110] As shown in Tables 23 and 24, treatment with Example 10 or everolimus alone as a single agent resulted in tumor regression of 51% (%dT / C=-51) and 50% (%dT / C=-50), respectively, both of which were statistically significant compared to vehicle controls (p<0.001 and p<0.001). The combined effect of Example 10 and everolimus was "additive," and the combined effect of Example 10 and everolimus was significantly superior to Example 10 alone (p=0.004). The combined effect of Example 10 and alpelisib was also significantly superior to everolimus alone (p=0.04). The combination was tolerated in animals without significant weight loss. [Table 26] [Table 27]

[0111] Oral bioavailability in rats The purpose of the following assay is to demonstrate whether a test compound is orally bioavailable.

[0112] Test compounds are administered to Sprague-Dawley rats at 1 mg / kg IV (using either 20% CAPTISOL® in 25 mM sodium phosphate buffer, pH 2 qs., or 25% DMA, 15% EtOH, 10% propylene glycol, 25% 2-pyrrolidone, and 25% purified water as a vehicle), or 3 mg / kg PO (using 1% hydroxyethylcellulose, 0.25% polysorbate 80, 0.05% antifoam 1510-US, and qs. purified water as a vehicle). Serial blood samples are collected 0.08, 0.25, 0.5, 1, 2, 4, 8, and 12 hours after IV bolus administration, and 0.25, 0.5, 1, 2, 4, 8, and 12 hours after oral administration. After treatment with EDTA coagulant, plasma is obtained by centrifugation and stored at -70°C until analysis by LC-MS / MS. Test article concentrations are determined in plasma and uploaded to the Watson LIMS™ system, which uses non-compartmental analysis to calculate the area under the curve (AUC) for both the IV and PO groups. Oral bioavailability (F%) is calculated using the following formula:

number

[0113] The compound of Example 1B exhibits an F% value of about 50% in the above-described assay, which demonstrates that Example 1B has good oral bioavailability.

Claims

1. A compound of the formula 【Chemistry 1】 (In the formula, R １ or R ２ are independently Cl, F, or —CF ３ , or -CH ３ and the other is hydrogen), or a pharmaceutically acceptable salt thereof.

2. The compound is 【Chemistry 2】 2. The compound of claim 1, wherein:

3. The compound is 【Transformation 3】 2. The compound of claim 1, wherein:

4. The compound is 【Chemistry 4】 3. The compound of claim 2, wherein:

5. 5. The compound of claim 4, wherein the pharmaceutically acceptable salt is a benzenesulfonate salt.

6. The compound of claim 4, wherein the pharmaceutically acceptable salt is 4-methylbenzenesulfonate.

7. The compound is 【Transformation 5】 5. The compound of claim 4, wherein:

8. The compound is 【Transformation 6】 4. The compound of claim 3, wherein:

9. 9. The compound of claim 8, wherein the pharmaceutically acceptable salt is a benzenesulfonate salt.

10. The compound of claim 8, wherein the pharmaceutically acceptable salt is 4-methylbenzenesulfonate.

11. The compound is 【Transformation 7】 9. The compound of claim 8, wherein:

12. A pharmaceutical composition comprising a compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable excipient, carrier, or diluent.

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

14. A method for treating breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer, comprising administering to a patient in need of such treatment an effective amount of a compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 12 to 13.

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

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

17. 15. The method of claim 14, wherein the lung cancer is an ER-positive lung cancer.

18. A compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, for use in therapy.

19. 12. The compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, for use in treating breast cancer, ovarian cancer, endometrial cancer, prostate cancer, uterine cancer, gastric cancer, or lung cancer.

20. 20. The compound or salt thereof for use according to claim 19 in the treatment of ER-positive breast cancer.

21. 20. The compound or salt thereof for use according to claim 19 in the treatment of ER-positive gastric cancer.

22. 20. The compound or salt thereof for use according to claim 19 in the treatment of ER-positive lung cancer.

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