3β-HSD1 Inhibitors, Compositions, and Their Use

Abstract

Compounds that inhibit the function of 3β-hydroxysteroid dehydrogenase (3βHSD1), pharmaceutical compositions containing such compounds, and methods of using such compounds for the treatment of, for example, prostate cancer, breast cancer, and other diseases that depend on the activity of 3βHSD1 are disclosed herein.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 354,988, filed on June 23, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] Description of Federally Sponsored Research This invention was made with government support under grants CA261995, CA236780, CA172382, and CA249279 awarded by the National Institutes of Health, and W81XWH - 20 - 1 - 0137 awarded by the U.S. Department of Defense. The U.S. government has certain rights in this invention.

[0003] Description of the Sequence Listing The contents of the electronic sequence listing entitled CCF - 40846.601.xml (size: 4,726 bytes, and creation date: June 23, 2023) are incorporated herein by reference in their entirety.

[0004] This disclosure generally relates to compounds that inhibit the function of 3β - hydroxysteroid dehydrogenase (3βHSD1), pharmaceutical compositions containing such compounds, and methods of using such compounds for the treatment of, for example, prostate cancer, breast cancer, endometrial cancer, and other diseases that depend on the activity of 3βHSD1.

Background Art

[0005] The long-term frontline and most effective treatment for metastatic prostate cancer has been androgen deprivation therapy (ADT) with medical or surgical castration. Gonadal testosterone is the major physiological source of androgens in men, and tumor responses occur in 80-90% of patients treated with ADT. In this context, the median response duration varies widely, reflecting tumor heterogeneity. Prostate cancer eventually becomes resistant to ADT, which is often due to mechanisms that allow testosterone and / or dihydrotestosterone (DHT) from extragonadal (mainly adrenal-derived) precursor steroids to occur in the tumor and other mechanisms that allow reactivation of the androgen receptor (AR). Next-generation hormonal therapies have been developed to counter the resistance mechanisms that drive the development of castration-resistant prostate cancer (CRPC). Specifically, abiraterone inhibits the enzyme 17-hydroxylase / 17,20-lyase (CYP17A1), blocking the synthesis of adrenal precursor steroids, and enzalutamide and apalutamide are potent competitive inhibitors of the AR. However, new therapies are needed to treat prostate cancer and related cancers.

SUMMARY OF THE INVENTION

[0006] Herein, a compound of formula (I):

CHEMICAL

[0007] In some embodiments, R 1 is fluoro, chloro, cyano, or trifluoromethyl. In some embodiments, R 1 is fluoro or cyano.

[0008] In some embodiments, R 2 and R 3 are each independently selected from hydrogen, fluoro, and methyl. In some embodiments, R 2 and R 3 are each independently selected from hydrogen and fluoro. In some embodiments, R 2 and R 3 are each hydrogen.

[0009] In some embodiments, Z is CR 4 wherein R 4 is selected from hydrogen, halo, and methyl. In some embodiments, Z is CR 4 wherein R 4 is selected from hydrogen and halo. In some embodiments, Z is N.

[0010] In some embodiments, Q 1 is CH. In some embodiments, Q 1 is N.

[0011] In some embodiments, Q 2 is CR 5 wherein R 5 is selected from hydrogen and C1-C4-alkyl. In some embodiments, Q 2 is CR 5 wherein R 5is hydrogen. In some embodiments, Q 2 is N.

[0012] In some embodiments, --- represents the presence of a bond, X is oxo, and R 6 is absent. In some embodiments, --- represents the absence of a bond, X is -OR a and -NR b R c is selected from, and R 6 is selected independently from hydrogen, C1-C3 alkyl, C3-C4 cycloalkyl, phenyl, N, O, and a monocyclic 5- or 6-membered heteroaryl having one or two heteroatoms selected from N and S, and phenyl-C1-C2-alkyl, and R a is selected from hydrogen, C1-C3 alkyl, heterocyclyl, and heterocyclyl-C1-C4-alkyl, and R b and R c are each hydrogen, and heterocyclyl is a monocyclic 4- to 6-membered heterocyclyl having one heteroatom selected from O and N. In some embodiments, --- represents the absence of a bond, X is selected from -OH and -NH2, and R 6 is selected from hydrogen, C1-C3 alkyl, C3-C4 cycloalkyl, phenyl, pyridyl, oxazolyl, and phenyl-C1-C2-alkyl.

[0013] In some embodiments, Y is selected from -CH2-, -NR d -, -O-, -CH2CH2-, and a bond, and R d is selected from hydrogen, C1-C4 alkyl, and C1-C4-alkoxy-C1-C4-alkyl. In some embodiments, Y is selected from -CH2-, -NH-, -CH2CH2-, and a bond.

[0014] In some embodiments, R 7 and R 8is independently selected from hydrogen, C1-C3 alkyl, hydroxy, cyano, halo, C3-C6 cycloalkyl, a monocyclic 3- to 6-membered heterocyclyl having one heteroatom selected from O and N, aryl, C1-C2-alkoxy-C1-C3-alkyl, hydroxy-C1-C3-alkyl, halo-C1-C3-alkyl, carboxy-C1-C3-alkyl, amino-C1-C3-alkyl, C3-C4-cycloalkyl-C1-C2-alkyl, aryl-C1-C2-alkyl, and heteroaryl-C1-C2-alkyl, wherein the cycloalkyl and heterocyclyl are each independently unsubstituted or substituted with one substituent selected from hydroxy, methyl, halo, and cyano. In some embodiments, R 7 and R 8 are each independently selected from hydrogen, C1-C3 alkyl, hydroxy, cyano, halo, C3-C6 cycloalkyl, aryl, halo-C1-C3-alkyl, carboxy-C1-C3-alkyl, C3-C4-cycloalkyl-C1-C2-alkyl, aryl-C1-C2-alkyl, and heteroaryl-C1-C2-alkyl. In some embodiments, R 7 and R 8 together with the carbon atom to which they are attached form a 3- to 6-membered cycloalkyl or a 3- to 6-membered monocyclic heterocyclyl having one or two oxygen atoms. In some embodiments, R 7 and R 8 together with the carbon atom to which they are attached form a 3- or 4-membered cycloalkyl.

[0015] In some embodiments, R 9 and R 10 are each independently hydrogen or C1-C4 alkyl. In some embodiments, R 9 and R 10 are each independently hydrogen or methyl.

[0016] In some embodiments, the compound is selected from the group consisting of: [Chemistry] [Chemistry] [Chemistry] [Chemistry] [Chemistry] [Chemistry] [Chemistry] and its pharmaceutically acceptable salts.

[0017] In another aspect, a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier is disclosed herein.

[0018] In another aspect, a method of performing it in a subject in need of treatment for cancer, comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, is disclosed herein. In some embodiments, the cancer is prostate cancer, breast cancer, or endometrial cancer. In some embodiments, the cancer is castration-resistant prostate cancer. In some embodiments, the method further comprises treating the subject with one or more additional therapies. In some embodiments, the one or more additional therapies are selected from surgery, chemotherapy, radiation therapy, hormone therapy, immunotherapy, cryotherapy, and hyperthermia, or any combination thereof.

[0019] In another aspect, provided herein is a method of inhibiting cancer cell proliferation, the method comprising contacting a cancer cell with a compound according to any one of claims 1-14, or a pharmaceutically acceptable salt thereof, in an amount effective to inhibit cancer cell proliferation. In some embodiments, the cancer cell is a prostate cancer cell, a breast cancer cell, or an endometrial cancer cell.

[0020] In another aspect, provided herein is a method of inhibiting the activity of 3β-hydroxysteroid dehydrogenase in a sample, the method comprising contacting the sample with a compound according to any one of claims 1-14, or a pharmaceutically acceptable salt thereof, in an amount effective to inhibit the activity of 3β-hydroxysteroid dehydrogenase.

[0021] In another aspect, provided herein is a method of treating a subject in need thereof, the method comprising determining, by assay of a biological sample from the subject, that cytosine nucleotide is present at position 1245 of the HSD3B1 gene or that threonine is present at position 367 of the 3βHSD1 protein, and administering to the subject a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof provided herein. In some embodiments, the cancer is prostate cancer, breast cancer, or endometrial cancer. In some embodiments, the biological sample comprises cancer cells.

[0022] Other aspects and embodiments will be apparent from the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0023]

Figure 1

[0024] This specification discloses compounds that inhibit the function of 3β-hydroxysteroid dehydrogenase (3βHSD1). Also disclosed herein are pharmaceutical compositions containing the compounds, and methods of using the compounds for the treatment of diseases that depend on the activity of 3βHSD1, including cancers such as prostate cancer, breast cancer, and endometrial cancer.

[0025] Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0026] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of the present disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed. (revised) and specific functional groups are generally defined as described therein. In addition, for organic chemistry and the general principles of specific functional moieties and reactivity, see Sorrell, Organic Chemistry, 2 nd edition, University Science Books, Sausalito, 2006, Smith, March’s Advanced Organic Chemistry: Reactions, Mechanism, and Structure, 7 th Edition, John Wiley & Sons, Inc., New York, 2013, Larock, Comprehensive Organic Transformations, 3rd Edition, John Wiley & Sons, Inc., New York, 2018, and Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987, and the entire contents of each of them are hereby incorporated by reference.

[0027] The modifier "about" as used in relation to a quantity includes the recited value and has the meaning indicated by the context (e.g., it includes at least the degree of error associated with the measurement of a particular quantity). The modifier "about" should also be considered to disclose a range defined by the absolute values of two endpoints. For example, the expression "about 2 to about 4" also discloses the range "2 to 4". The term "about" may refer to ±10% of the indicated number. For example, "about 10%" may indicate a range of 9% to 11%, and "about 1" may mean 0.9 to 1.1. Other meanings of "about" may become apparent from the context, such as rounding, for example, "about 1" may also mean 0.5 to 1.4.

[0028] As used herein, the term "alkyl" refers to a radical of a straight-chain or branched saturated hydrocarbon chain. The alkyl chain may have, for example, 1 to 24 carbon atoms (C1-C 24 alkyl), 1 to 16 carbon atoms (C1-C 16 alkyl), 1 to 14 carbon atoms (C1-C 14 alkyl), 1 to 12 carbon atoms (C1-C 12 alkyl), 1 to 10 carbon atoms (C1-C 10(alkyl), which may contain from 1 to 8 carbon atoms (C1-C8 alkyl), from 1 to 6 carbon atoms (C1-C6 alkyl), from 1 to 4 carbon atoms (C1-C4 alkyl), from 1 to 3 carbon atoms (C1-C3 alkyl), or from 1 to 2 carbon atoms (C1-C2 alkyl). Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl.

[0029] As used herein, the term "alkenyl" refers to a radical of a straight-chain or branched hydrocarbon chain that contains at least one carbon-carbon double bond and no triple bonds (see "alkenyl" below). The double bond(s) can be located at any position(s) in the hydrocarbon chain. The alkenyl chain may contain, for example, from 2 to 24 carbon atoms (C2-C 24 alkenyl), from 2 to 16 carbon atoms (C2-C 16 alkenyl), from 2 to 14 carbon atoms (C2-C 14 alkenyl), from 2 to 12 carbon atoms (C2-C 12 alkenyl), from 2 to 10 carbon atoms (C2-C 10 alkenyl), from 2 to 8 carbon atoms (C2-C8 alkenyl), from 2 to 6 carbon atoms (C2-C6 alkenyl), from 2 to 4 carbon atoms (C2-C4 alkenyl), from 2 to 3 carbon atoms (C2-C3 alkenyl), or 2 carbon atoms (C2 alkenyl). Representative examples of alkenyl include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, butadienyl, 2-methyl-2-propenyl, 3-butenyl, pentenyl, pentadienyl, hexenyl, heptenyl, octenyl, octatrieneyl, etc.

[0030] As used herein, the term "alkynyl" means a radical of a straight-chain or branched hydrocarbon chain containing at least one carbon-carbon triple bond. The alkynyl chain may contain, for example, from 2 to 24 carbon atoms (C2-C 24 alkynyl), from 2 to 16 carbon atoms (C2-C 16 alkynyl), from 2 to 14 carbon atoms (C2-C 14 alkynyl), from 2 to 12 carbon atoms (C2-C 12 alkynyl), from 2 to 10 carbon atoms (C2-C 10 alkynyl), from 2 to 8 carbon atoms (C2-C8 alkynyl), from 2 to 6 carbon atoms (C2-C6 alkynyl), from 2 to 4 carbon atoms (C2-C4 alkynyl), from 2 to 3 carbon atoms (C2-C3 alkynyl), or 2 carbon atoms (C2 alkynyl). The triple bond(s) can be located at any position(s) in the hydrocarbon chain. Representative examples of alkynyl include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and the like.

[0031] As used herein, the term "alkoxy" refers to an alkyl group as defined herein attached to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0032] As used herein, the term "amino" refers to the group -NR x R y wherein R x and R y are selected from hydrogen and alkyl (e.g., C1-C4 alkyl).

[0033] As used herein, "aryl" refers to a radical of a monocyclic, bicyclic, or tricyclic 4n+2 aromatic ring system having 6 to 14 ring carbon atoms and 0 heteroatoms (e.g., 6, 10, or 14 π electrons are shared in a cyclic arrangement) ("C6-C 14"Aryl"). In some embodiments, the aryl group has 6 ring carbon atoms ("C6 aryl", i.e., phenyl). In some embodiments, the aryl group has 10 ring carbon atoms ("C 10 aryl", e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms ("C 14 aryl", e.g., anthracenyl and phenanthrenyl).

[0034] As used herein, the term "cycloalkyl" refers to a radical of a saturated carbocyclic ring system containing 3 to 10 carbon atoms and 0 heteroatoms. Cycloalkyl may be monocyclic, bicyclic, bridged, fused, or spirocyclic. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl, and bicyclo[5.2.0]nonanyl.

[0035] As used herein, the term "cyano" refers to the -CN group.

[0036] As used herein, the term "halogen" or "halo" refers to F, Cl, Br, or I.

[0037] As used herein, the term "haloalkyl" refers to an alkyl group as defined herein in which at least one hydrogen atom (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms) is replaced by a halogen. In some embodiments, each hydrogen atom of the alkyl group is replaced by a halogen. Representative examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, and 3,3,3-trifluoropropyl.

[0038] As used herein, the term "haloalkoxy" means a haloalkyl group as defined herein that is attached to the parent molecular moiety through an oxygen atom. Representative examples of haloalkoxy include, but are not limited to, difluoromethoxy, trifluoromethoxy, and 2,2,2-trifluoroethoxy.

[0039] As used herein, "heteroaryl" refers to a radical of a 5- to 10-membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms (e.g., 6 or 10 π electrons are shared in a cyclic arrangement), where each heteroatom independently refers to a radical selected from nitrogen, oxygen, and sulfur ("5- to 10-membered heteroaryl"). In a heteroaryl group containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom as valence permits. A heteroaryl bicyclic ring system can contain one or more heteroatoms in one or both rings. "Heteroaryl" also includes ring systems in which the heteroaryl ring defined above is fused to one or more aryl groups, and the point of attachment is on either the aryl or heteroaryl ring, and in such cases, the number of ring members indicates the number of ring members in the fused (aryl / heteroaryl) ring system. The point of attachment of a bicyclic heteroaryl group in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.) can be on either ring, i.e., on the ring having a heteroatom (e.g., 2-indolyl) or on the ring without a heteroatom (e.g., 5-indolyl). Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl.Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepynyl. Exemplary 5,6-fused bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-fused bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

[0040] As used herein, the term "heterocyclyl" refers to a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (a "3- to 10-membered heterocyclyl"). In a heterocyclyl group containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom as valence permits. A heterocyclyl group can be monocyclic ("monocyclic heterocyclyl"), or can be any of a fused, bridged, or spiro ring system (e.g., a bicyclic system ("bicyclic heterocyclyl")), and can be saturated or partially unsaturated. A heterocyclyl bicyclic ring system can contain one or more heteroatoms in one or both rings. "Heterocyclyl" includes a ring system in which the heterocyclyl ring defined above is fused to one or more cycloalkyl groups and the point of attachment is in either the cycloalkyl ring or the heterocyclyl ring, or a ring system in which the heterocyclyl ring defined above is fused to one or more aryl or heteroaryl groups and the point of attachment is in the heterocyclyl ring. In such cases, the ring member count continues to indicate the ring member count in the heterocyclyl ring system. A heterocyclyl group can be described, for example, as a 3- to 7-membered heterocyclyl, and the term "membered ring" refers to ring atoms other than hydrogen within that moiety, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. Exemplary 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to, aziridinyl, oxiranyl, and thiirenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrol-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one.Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl (e.g., 2,2,6,6-tetramethylpiperidinyl), tetrahydropyranyl, dihydropyridinyl, pyridinonyl (e.g., 1-methylpyridin-2-onyl), and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, pyridazinonyl (2-methylpyridazin-3-onyl), pyrimidinonyl (e.g., 1-methylpyrimidin-2-onyl, 3-methylpyrimidin-4-onyl), dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azocanyl, oxecanyl, and thiocanyl. Exemplary 5-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as a 5,6-bicyclic heterocyclyl ring) include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, etc. Exemplary 5-membered heterocyclyl groups fused to a heterocyclyl ring (also referred to herein as a 5,5-bicyclic heterocyclyl ring) include, but are not limited to, octahydropyrrolopyrrolyl (e.g., octahydropyrrolo[3,4-c]pyrrolyl), etc.

[0041] Exemplary 6-membered heterocyclyl groups fused to a heterocyclic ring (also referred to as 4,6-membered heterocyclyl rings) include, but are not limited to, diazaspiro[3.5]nonanyl (e.g., 2,7-diazaspiro[3.5]nonanyl). Exemplary 6-membered heterocyclyl groups fused to an aryl ring (also referred to herein as 6,6-bicyclic heterocyclyl rings) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like. Exemplary 6-membered heterocyclyl groups fused to a cycloalkyl ring (also referred to herein as 6,7-bicyclic heterocyclyl rings) include, but are not limited to, azabicyclooctanyl (e.g., (1,5)-8-azabicyclo[3.2.1]octanyl). Exemplary 6-membered heterocyclyl groups fused to a cycloalkyl ring (also referred to herein as 6,8-bicyclic heterocyclyl rings) include, but are not limited to, azabicyclononanyl (e.g., 9-azabicyclo[3.3.1]nonanyl).

[0042] As used herein, the term "hydroxy" or "hydroxyl" refers to an -OH group.

[0043] As used herein, the term "nitro" refers to an -NO2 group.

[0044] When a group or moiety may be substituted, the term "substituted" means that one or more (e.g., 1, 2, 3, 4, 5, or 6, in some embodiments 1, 2, or 3, in other embodiments 1 or 2) hydrogens on the group so indicated using the term "substituted" may be replaced with a selected range of enumerated groups, or suitable substituents known to those of skill in the art (e.g., one or more of the groups listed below), provided that the normal valency of the designated atom is not exceeded. Substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amide, amidino, aryl, azide, carbamoyl, carboxyl, carboxyl ester, cyano, cycloalkyl, cycloalkenyl, guanidino, halo, haloalkyl, haloalkoxy, heteroalkyl, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, oxo, nitro, phosphate, phosphonate, sulfonic acid, thiol, thione, or combinations thereof.

[0045] As used herein, in a chemical structure,

Chem.

[0046] As used herein, the term "subject" generally refers to any vertebrate, including but not limited to mammals. Examples of mammals include primates, including monkeys and humans; equine (e.g., horse); canine (e.g., dog); cat; various livestock (e.g., ungulates such as swine, pig, goat, sheep, etc.); and companion animals (e.g., cat, hamster, mouse, and guinea pig). Treatment or diagnosis of humans is of particular interest. In some embodiments, the subject has cancer or is at risk of developing cancer.

[0047] In the compounds described in this specification, the groups and their substituents can be selected according to the acceptable valences of the atoms and substituents, such that, as a result of the selection and substitution, stable compounds are provided that do not spontaneously undergo conversions, for example, by rearrangement, cyclization, elimination, etc.

[0048] Compound In this specification, a compound of formula (I):

Chemical formula

[0049] In some embodiments, R 1 is halo or cyano, Z is selected from CR 4 and N, R 2 , R 3 , and R 4 are independently selected from hydrogen, halo, and C1-C4 alkyl, Q 1 is CH or N, Q 2 is CR 5 or N, R5 is selected from hydrogen, C1-C4 alkyl, halo, and C1-C4 haloalkyl, --- represents the presence or absence of a bond. When --- represents the presence of a bond in the formula, X is oxo, and R 6 is absent. When --- represents the absence of a bond, X is selected from -OH and -NH2, and R 6 is selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, aryl, and aryl-C1-C4-alkyl, Y is selected from -CH2-, -NH-, -O-, -S-, -CH2CH2-, -NHCH2-, -OCH2-, -SCH2-, and a bond, R 7 R 8 R 9 and R 10 are each independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, hydroxy, cyano, halo, C3-C6 cycloalkyl, aryl, heteroaryl, C1-C4-alkoxy-C1-C6-alkyl, hydroxy-C1-C6-alkyl, halo-C1-C6-alkyl, carboxy-C1-C6-alkyl, C3-C6-cycloalkyl-C1-C4-alkyl, aryl-C1-C4-alkyl, and heteroaryl-C1-C4-alkyl. R 7 and R 8 together with the carbon atom to which they are attached optionally form a 3- to 6-membered ring together.

[0050] In some embodiments, R 1 is halo. In some embodiments, R 1 is fluoro. In some embodiments, R 1 is chloro. In some embodiments, R 1 is cyano. In some embodiments, R 1 is C1-C4 haloalkyl. In some embodiments, R 1 is trifluoromethyl. In some embodiments, R1 is fluoro, chloro, cyano, or trifluoromethyl. In some embodiments, R 1 is fluoro or cyano.

[0051] In some embodiments, R 2 and R 3 are each independently selected from hydrogen, halo, and C1-C2 alkyl. In some embodiments, R 2 and R 3 are each independently selected from hydrogen, fluoro, and methyl. In some embodiments, R 2 and R 3 are each independently selected from hydrogen and halo. In some embodiments, R 2 and R 3 are each independently selected from hydrogen and fluoro. In some embodiments, R 2 and R 3 are each hydrogen. In some embodiments R 2 is hydrogen and R 3 is halo. In some embodiments, R 2 is hydrogen and R 3 is fluoro. In some embodiments, R 2 is halo and R 3 is hydrogen. In some embodiments, R 2 is fluoro and R 3 is hydrogen. In some embodiments, R 2 is C1-C4 alkyl and R 3 is hydrogen. In some embodiments, R 2 is methyl and R 3 is hydrogen. In some embodiments, R 2 and R 3 are each halo. In some embodiments, R 2 and R 3 are each fluoro. In some embodiments, R 2 is hydrogen. In some embodiments, R 2is a halo. In some embodiments, R 2 is fluoro. In some embodiments, R 2 is C1-C4 alkyl. In some embodiments, R 2 is methyl. In some embodiments, R 3 is hydrogen. In some embodiments, R 3 is a halo. In some embodiments, R 3 is fluoro.

[0052] In some embodiments, Z is CR 4 In some embodiments, R 4 is selected from hydrogen, fluoro, chloro, and methyl. In some embodiments, R 4 is selected from hydrogen and halo. In some embodiments, R 4 is selected from hydrogen, fluoro, and chloro. In some embodiments, R 4 is selected from hydrogen and fluoro. In some embodiments, R 4 is hydrogen. In some embodiments, R 4 is a halo. In some embodiments, R 4 is fluoro. In some embodiments, R 4 is chloro. In some embodiments, R 4 is C1-C4 alkyl. In some embodiments, R 4 is methyl. In some embodiments, Z is N.

[0053] In some embodiments, the group in formula (I)

Chemical formula

Chemical formula

[0054] In some embodiments, the group in formula (I) [Chemical formula] is selected from the following: [Chemical formula]

[0055] In some embodiments, the group in formula (I) [Chemical formula] is [Chemical formula] .

[0056] In some embodiments, Q 1 is CH or N. In some embodiments, Q 1 is CH. In some embodiments, Q 1 is N. In some embodiments, Q 2 is CR 5 , and R 5 is hydrogen or C1-C4 alkyl. In some embodiments, Q 2 is CR 5 , and R 5 is hydrogen, methyl, ethyl, or iso-propyl. In some embodiments, Q 2 is CR 5 , and R 5 is hydrogen or methyl. In some embodiments, Q 2 is CR 5 , and R 5 is hydrogen. In some embodiments, Q 2 is N. In some embodiments, Q 1 is CH, Q 2 is CR 5 , and R 5 is hydrogen, methyl, ethyl, or iso-propyl. In some embodiments, Q 1 is CH, Q 2 is CR 5and R 5 is hydrogen or methyl. In some embodiments, Q 1 is N, and Q 2 is CR 5 and R 5 is hydrogen. In some embodiments, Q 1 is CH, and Q 2 is N. In some embodiments, Q 1 is CH, and Q 2 is CR 5 and R 5 is hydrogen.

[0057] In some embodiments, --- represents the presence of a bond, X is oxo, and R 6 is absent.

[0058] In some embodiments, --- represents the absence of a bond, X is -OR a and -NR b R c selected from, and R 6 is selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, and aryl-C1-C4-alkyl. In some embodiments, --- represents the absence of a bond, X is -OR a and -NR b R c selected from, and R 6 is selected from hydrogen, C1-C3 alkyl, C3-C4 cycloalkyl, phenyl, monocyclic 5- or 6-membered heteroaryl having one or two heteroatoms independently selected from N, O, and S, and phenyl-C1-C2-alkyl, and R a is selected from hydrogen, C1-C3 alkyl, heterocyclyl, and heterocyclyl-C1-C4-alkyl, and R b and R c are each hydrogen, and heterocyclyl is a monocyclic 4- to 6-membered heterocyclyl having one heteroatom selected from O and N. In some embodiments, --- represents the absence of a bond, X is -ORa and -NR b R c is selected from, and R 6 is selected from hydrogen, methyl, ethyl, isopropyl, cyclopropyl, phenyl, pyridyl, oxazolyl, and benzyl, and R a is selected from hydrogen, methyl, ethyl, oxetanyl, and -CH2-oxetanyl, and R b and R c are each hydrogen.

[0059] In some embodiments, --- represents the absence of a bond, X is selected from -OH and -NH2, and R 6 is selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, aryl, and aryl-C1-C4-alkyl. In some embodiments, --- represents the absence of a bond, X is selected from -OH and -NH2, and R 6 is selected from hydrogen, C1-C3 alkyl, C3-C4 cycloalkyl, phenyl, and phenyl-C1-C2-alkyl. In some embodiments, --- represents the absence of a bond, X is -OH, and R 6 is selected from hydrogen, methyl, cyclopropyl, phenyl, and benzyl. In some embodiments, --- represents the absence of a bond, X is -NH2, and R 6 is hydrogen.

[0060] In some embodiments, Y is selected from -CH2-, -NR d -, -O-, -CH2CH2-, and a bond, and R d is selected from hydrogen, C1-C4 alkyl, and C1-C4-alkoxy-C1-C4-alkyl. In some embodiments, Y is selected from -CH2-, -NR d -, -O-, -CH2CH2-, and a bond, and R d is selected from hydrogen, C1-C2 alkyl, and C1-C2-alkoxy-C1-C2-alkyl. In some embodiments, Y is -CH2-, -NR dSelected from among —, —O—, —CH2CH2—, and a bond, R d is selected from hydrogen, methyl, ethyl, and —CH2CH2OCH3. In some embodiments, Y is selected from —CH2—, —NH—, —CH2CH2—, and a bond. In some embodiments, Y is —CH2—. In some embodiments, Y is —NH—. In some embodiments, Y is —N(CH3)—. In some embodiments, Y is —N(CH2CH3)—. In some embodiments, Y is —N(CH2CH2OCH3)—. In some embodiments, Y is —CH2CH2—. In some embodiments, Y is —O—. In some embodiments, Y is a bond.

[0061] In some embodiments, R 7 and R 8 are each independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, hydroxy, cyano, halo, C3-C6 cycloalkyl, heterocyclyl, aryl, heteroaryl, C1-C4-alkoxy-C1-C6-alkyl, hydroxy-C1-C6-alkyl, halo-C1-C6-alkyl, carboxy-C1-C6-alkyl, amino-C1-C6-alkyl, C3-C6-cycloalkyl-C1-C4-alkyl, aryl-C1-C4-alkyl, and heteroaryl-C1-C4-alkyl. In some embodiments, R 7 and R 8is independently selected from hydrogen, C1-C3 alkyl, hydroxy, cyano, halo, monocyclic 3- to 6-membered heterocyclyl having one heteroatom selected from O and N, aryl, C1-C2-alkoxy-C1-C3-alkyl, hydroxy-C1-C3-alkyl, halo-C1-C3-alkyl, carboxy-C1-C3-alkyl, amino-C1-C3-alkyl, C3-C4-cycloalkyl-C1-C2-alkyl, aryl-C1-C2-alkyl, and heteroaryl-C1-C2-alkyl, wherein cycloalkyl and heterocyclyl are each independently unsubstituted or substituted with one substituent selected from hydroxy, methyl, halo, and cyano. In some embodiments, R 7 and R 8 are independently selected from hydrogen, C1-C3 alkyl, hydroxy, cyano, halo, monocyclic 4- to 5-membered heterocyclyl having one O atom, aryl, methoxy-C1-C2-alkyl, hydroxy-C1-C2-alkyl, halo-C1-C3-alkyl, carboxy-C1-C3-alkyl, amino-C1-C2-alkyl, C3-C4-cycloalkyl-C1-C2-alkyl, aryl-C1-C2-alkyl, and heteroaryl-C1-C2-alkyl, wherein heterocyclyl is unsubstituted or substituted with one hydroxy group. In some embodiments, R 7 and R 8 are independently selected from hydrogen, methyl, ethyl, hydroxy, cyano, fluoro, 3-hydroxyoxetan-3-yl, phenyl, benzyl, -CH2OH, -CF3, -CF2CF3, -CH2CF3, -CH2N(CH3)2, -CH2OCH3, -CH2-cyclopropyl, and -CH2-pyridyl.

[0062] In some embodiments, R 7 and R 8is independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, hydroxy, cyano, halo, C3-C6 cycloalkyl, aryl, heteroaryl, C1-C4-alkoxy-C1-C6-alkyl, hydroxy-C1-C6-alkyl, halo-C1-C6-alkyl, carboxy-C1-C6-alkyl, C3-C6-cycloalkyl-C1-C4-alkyl, aryl-C1-C4-alkyl, and heteroaryl-C1-C4-alkyl. In some embodiments, R 7 and R 8 are independently selected from hydrogen, C1-C3 alkyl, hydroxy, cyano, halo, C3-C6 cycloalkyl, aryl, halo-C1-C3-alkyl, carboxy-C1-C3-alkyl, C3-C4-cycloalkyl-C1-C2-alkyl, aryl-C1-C2-alkyl, and heteroaryl-C1-C2-alkyl. In some embodiments, R 7 and R 8 are independently selected from hydrogen, C1-C3 alkyl, aryl, C3-C4-cycloalkyl-C1-C2-alkyl, and aryl-C1-C2-alkyl. In some embodiments, R 7 and R 8 are independently selected from hydrogen, methyl, ethyl, phenyl, benzyl, and -CH2-cyclopropyl.

[0063] In some embodiments, R 7 and R 8 together with the carbon atom to which they are attached form a 3- to 6-membered cycloalkyl or a 3- to 6-membered monocyclic heterocyclyl having one or two heteroatoms independently selected from O and N. In some embodiments, R 7 and R 8 together with the carbon atom to which they are attached form a 3- to 6-membered cycloalkyl or a 3- to 6-membered monocyclic heterocyclyl having one or two oxygen atoms. In some embodiments, R 7 and R 8Together with the carbon atom to which they are attached, they together form a ring selected from cyclopropyl, cyclobutyl, oxetane and dioxetane. In some embodiments, R 7 and R 8 together with the carbon atom to which they are attached form a 3- to 6-membered cycloalkyl. In some embodiments, R 7 and R 8 together with the carbon atom to which they are attached form a 3- or 4-membered cycloalkyl (e.g., cyclopropyl or cyclobutyl). In some embodiments, R 7 and R 8 together with the carbon atom to which they are attached form a 3- to 6-membered monocyclic heterocyclyl having one or two heteroatoms independently selected from O and N. In some embodiments, R 7 and R 8 together with the carbon atom to which they are attached form a 3- to 6-membered monocyclic heterocyclyl having one or two oxygen atoms independently. In some embodiments, R 7 and R 8 together with the carbon atom to which they are attached form an oxetane or dioxetane ring.

[0064] In some embodiments, R 9 and R 10 are each independently selected from hydrogen and C1-C6 alkyl. In some embodiments, R 9 and R 10 are each independently selected from hydrogen and C1-C4 alkyl. In some embodiments, R 9 and R 10 are each independently selected from hydrogen and methyl. In some embodiments, R 9 and R 10 are each hydrogen. In some embodiments, R 9 and R 10 are each methyl.

[0065] In some embodiments, the group in the compound of formula (I)

Chemical formula

Chemical formula

Chemical formula

[0066] In some embodiments, the group in the compound of formula (I)

Chemical formula

Chemical formula

[0067] In some embodiments, the compound is selected from the group consisting of:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0068] Certain compounds described herein have at least one asymmetric center. Depending on the nature of the various substituents on the molecule, additional asymmetric centers may be present. Compounds having an asymmetric center give rise to enantiomers (optical isomers), diastereomers (stereoisomers), or both, and all possible enantiomers and diastereomers in mixtures and in pure or partially purified compounds are intended to be included within the scope of the present invention. This disclosure is intended to encompass all such isomeric forms of these compounds.

[0069] The independent synthesis of enantiomer- or diastereomer-rich compounds or their chromatographic separation can be achieved as is known in the art by appropriate modification of the methods disclosed herein. The absolute stereochemistry of a compound can be determined, if desired, by X-ray crystallographic analysis of a crystalline product or crystalline intermediate derivatized with a reagent containing an asymmetric center of known absolute configuration.

[0070] If desired, a racemic mixture of a compound can be separated, whereby the individual enantiomers are isolated. This separation can be carried out by methods well known in the art, such as coupling a racemic mixture of the compound with an enantiomerically pure compound to form a mixture of diastereomers and then separating the individual diastereomers by standard methods such as fractional crystallization or chromatography. The coupling reaction is often the formation of a salt using an enantiomerically pure acid or base. The diastereomeric derivative can then be converted to the pure enantiomer by cleavage of the added chiral residue. A racemic mixture of a compound may also be separated directly by chromatography using a chiral stationary phase, a method well known in the art. Alternatively, any enantiomer of a compound may be obtained by stereoselective synthesis using an optically pure starting material or a reagent of known configuration by methods well known in the art.

[0071] The present disclosure also includes isotope-labeled compounds that are identical to those described in formula (I) except that one or more atoms are replaced with atoms having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes suitable for incorporation into the compounds of the present invention include, but are not limited to, 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, and 36 Cl and other hydrogens, carbons, nitrogens, oxygens, phosphors, sulfurs, fluorines, and chlorines. Substitution with heavier isotopes such as deuterium ( 2 H) can provide certain therapeutic advantages such as improved metabolic stability, e.g., an extended in vivo half-life or a reduced required dose of the drug, and may thus be preferred depending on the circumstances. For medical imaging diagnostics and positron emission tomography (PET) studies to determine receptor distribution, a positron-emitting isotope can be incorporated into the compound. Suitable positron-emitting isotopes that can be incorporated into the compounds of formula (I) are 11 C, 13 N, 15 O, and 18 F. The isotope-labeled compounds of formula (I) can generally be prepared by conventional methods known to those skilled in the art or by processes similar to those described in the accompanying examples using appropriate isotope-labeled reagents in place of non-isotope-labeled reagents.

[0072] a. Method of synthesis The compounds disclosed herein can be prepared by various methods. One approach is shown in Scheme 1. Starting from boronic acid 1.1 (e.g., difluorophenolboronic acid when each R is fluoro) and 2-chloropyridinetetralone 1.2, the final example of type 1.3 is obtained by a Suzuki reaction in a heated ether solvent.

[0073] Scheme 1 [Chem.] Compounds and intermediates can be isolated and purified by methods well known to those skilled in the art of organic synthesis technology. Examples of conventional methods for isolating and purifying compounds include, for example, recrystallization at high or low temperatures using pretreatment with any activated carbon, thin-layer chromatography, distillation at various pressures, sublimation under vacuum, and chromatography on solid supports such as silica gel, alumina, or silica derivatized with alkylsilane groups, as described in "Vogel’s Textbook of Practical Organic Chemistry", 5th edition (1989), pub. Longman Scientific & Technical, Essex CM20 2JE, England, by Furniss, Hannaford, Smith, and Tatchell, but are not limited thereto.

[0074] The reaction conditions and reaction times for each individual step may vary depending on the specific reactants used and the substituents present in the reactants used. The reactants can be worked up in a conventional manner (e.g., by removing the solvent from the residue) and, without limitation, can be further purified according to methodologies generally known in the art, such as crystallization, distillation, extraction, trituration, and chromatography. Unless otherwise stated, starting materials and reagents are either commercially available or can be prepared by those skilled in the art from commercially available substances using methods described in the chemical literature.

[0075] Standard experiments, including appropriate manipulation of reaction conditions, reagents, and the order of synthetic routes, protection of any chemical functionality that may not be compatible with the reaction conditions, and deprotection at appropriate points in the reaction sequence of the method, are included within the scope of the present disclosure. Appropriate protecting groups, and methods of protecting and deprotecting various substituents using such appropriate protecting groups, are well known to those skilled in the art, examples of which can be found in the book by Greene, PGM Wuts and TW Greene, titled Protective Groups in Organic Synthesis (4 th ed.), John Wiley & Sons, NY (2006).

[0076] The optically active forms of the disclosed compounds, if necessary, can be obtained by performing one of the procedures described herein using an optically active starting material (e.g., prepared by asymmetric induction in a suitable reaction step), or by resolving a mixture of stereoisomers of the compound or intermediate using standard procedures (e.g., chromatographic separation, recrystallization, or enzymatic resolution).

[0077] Similarly, if a pure geometric isomer of the compound is required, this can be obtained by using the pure geometric isomer as a starting material and performing one of the procedures described herein, or by resolving a mixture of geometric isomers of the compound or intermediate using standard procedures such as chromatographic separation.

[0078] The synthetic schemes and specific examples described are illustrative and should not be read as limiting the scope of the present disclosure or the claims. Alternatives, modifications, and equivalents of the synthetic methods and specific examples are contemplated.

[0079] b. Pharmaceutically acceptable salts The disclosed compounds may exist as pharmaceutically acceptable salts. The term "pharmaceutically acceptable salts" refers to salts or zwitterions of compounds that are water-soluble or oil-soluble or dispersible, suitable for the treatment of disorders without undue toxicity, irritation, and allergic response, commensurate with a reasonable benefit / risk ratio, and effective for their intended use. The salts may be prepared during the final isolation and purification of the compound or separately by reacting the amino groups of the compound with a suitable acid. For example, the compound may be dissolved in a suitable solvent (e.g., but not limited to, methanol and water) and treated with an acid such as at least one equivalent of hydrochloric acid. The resulting salt may precipitate, be isolated by filtration, and dried under reduced pressure. Alternatively, the solvent and excess acid may be removed under reduced pressure to provide the salt. Representative salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzene sulfonate, bisulfate, butyrate, camphorate, camphor sulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylene sulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, glutamate, para-toluene sulfonate, undecanoate, hydrochloride, hydrobromide, sulfate, phosphate, etc. The amino groups of the compound may also be quaternized with alkyl chlorides, alkyl bromides, and alkyl iodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl, etc.

[0080] The base addition salts can be prepared by reaction of the carboxyl groups with a suitable base of a metal cation (e.g., lithium, sodium, potassium, calcium, magnesium, or aluminum) (e.g., hydroxide, carbonate, or bicarbonate) or an organic primary amine, secondary amine, or tertiary amine during the final isolation and purification of the disclosed compounds. Quaternary amine salts can be prepared from, for example, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine and N,N'-dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, etc.

[0081] Pharmaceutical composition The disclosed compounds can be incorporated into pharmaceutical compositions suitable for administration to a subject (e.g., a patient, which may be human or non-human). The pharmaceutical composition may contain an "effective amount for treatment" or "effective amount for prevention" of the agent(s). An "effective amount for treatment" refers to an amount effective at the dosage and for the period required to achieve the desired therapeutic result. The effective amount for treatment of the composition can be determined by one of ordinary skill in the art and may vary depending on factors such as the medical condition of the individual, age, gender, and weight of the individual, as well as the ability of the composition to induce the desired response in the individual. The effective amount for treatment is also an amount in which any toxic or detrimental effects of the compounds of the present invention are exceeded by the therapeutically beneficial effects. An "effective amount for prevention" refers to an amount effective at the dosage and for the period required to achieve the desired preventive result. Usually, since the prophylactic dosage is used in a subject before or at an early stage of a disease or medical condition, the effective amount for prevention will be less than the effective amount for treatment.

[0082] The pharmaceutical composition may contain a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, or any type of formulation adjuvant. Some examples of substances that can function as pharmaceutically acceptable carriers are saccharides (e.g., but not limited to, lactose, glucose, and sucrose); starches (e.g., but not limited to, corn starch and potato starch); cellulose and its derivatives (e.g., but not limited to, sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate); tragacanth powder; malt; gelatin; talc; excipients (e.g., but not limited to, cocoa butter and suppository wax); oils (e.g., but not limited to, peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and soybean oil); glycols (e.g., but not limited to, propylene glycol); esters (e.g., but not limited to, ethyl oleate and ethyl laurate); agar; buffering agents (e.g., but not limited to, magnesium hydroxide and aluminum hydroxide); alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solution, as well as other non-toxic compatible lubricants (e.g., but not limited to, sodium lauryl sulfate and magnesium stearate), as well as coloring agents, release agents, coating agents, sweetening agents, flavoring agents and fragrances, preservatives, and antioxidants may also be present in the composition at the discretion of the formulator.

[0083] Accordingly, the compounds and their pharmaceutically acceptable salts can be formulated to be administered, for example, by solid dosage, eye drops, in topical oily formulations, by injection, inhalation (either via the mouth or nose), by implant, or by oral administration, buccal administration, parenteral administration, or rectal administration. Techniques and formulations can generally be found in "Remington’s Pharmaceutical Sciences" (Meade Publishing Co., Easton, Pa.). Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage.

[0084] Depending on the route of administration of the disclosed compound and the form of the composition, the type of carrier to be used will be determined. The composition may be in various forms suitable for, for example, systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implant, or parenteral) or topical administration (e.g., transdermal, pulmonary, nasal, otic, intraocular, liposomal delivery systems, or iontophoresis).

[0085] Carriers for systemic administration usually include at least one of diluents, lubricants, binders, disintegrants, colorants, flavoring agents, sweetening agents, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, combinations thereof, etc. All carriers are optional in the composition.

[0086] Suitable diluents include sugars (e.g., glucose, lactose, dextrose, and sucrose); diols (e.g., propylene glycol), calcium carbonate, sodium carbonate, sugar alcohols (e.g., glycerin, mannitol, and sorbitol). The amount of diluent(s) in a systemic or topical composition is usually about 50 to about 90% by weight of the composition.

[0087] Suitable lubricants include silica, talc, stearic acid and its magnesium and calcium salts, calcium sulfate, and liquid lubricants (e.g., polyethylene glycol, and vegetable oils (e.g., peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter)). The amount of lubricant(s) in a systemic or topical composition is usually about 5 to about 10% by weight of the composition.

[0088] Suitable binders include polyvinylpyrrolidone, magnesium aluminum silicate, starch (e.g., corn starch and potato starch), gelatin, tragacanth, and cellulose and its derivatives (e.g., sodium carboxymethylcellulose, ethyl cellulose, methyl cellulose, microcrystalline cellulose, and sodium carboxymethylcellulose). The amount of binder(s) in the systemic composition is usually about 5 to about 50% by weight of the composition.

[0089] Suitable disintegrants include agar, alginic acid and its sodium salts, effervescent mixtures, croscarmellose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clay, and ion exchange resins. The amount of disintegrant(s) in the systemic or topical composition is usually about 0.1 to about 10% by weight of the composition.

[0090] Suitable colorants include colorants such as FD&C dyes. When used, the amount of colorant in the systemic or topical composition is usually about 0.005 to about 0.1% by weight of the composition.

[0091] Suitable flavoring agents include menthol, peppermint, and fruit flavors. The amount of flavoring agent(s) used in the systemic or topical composition is usually about 0.1 to about 1.0%.

[0092] Suitable sweeteners include aspartame and saccharin. The amount of sweetener(s) in the systemic or topical composition is usually about 0.001 to about 1% by weight of the composition.

[0093] Suitable antioxidants include butylated hydroxyanisole ("BHA"), butylated hydroxytoluene ("BHT"), and vitamin E. The amount of antioxidant(s) in the systemic or topical composition is usually about 0.1 to about 5% by weight of the composition.

[0094] Suitable preservatives include benzalkonium chloride, methylparaben, and sodium benzoate. The amount of the preservative(s) in the systemic or topical composition is usually about 0.01 to about 5% by weight of the composition.

[0095] Suitable flow promoters include silicon dioxide. The amount of the flow promoter(s) in the systemic or topical composition is usually about 1 to about 5% by weight of the composition.

[0096] Suitable solvents include water, isotonic saline, ethyl oleate, glycerin, hydroxylated castor oil, alcohol (e.g., ethanol), and phosphate buffer solution. The amount of the solvent(s) in the systemic or topical composition is usually about 0 to about 100% by weight of the composition.

[0097] Suitable suspending agents include AVICEL RC-591 (manufactured by FMC Corporation, Philadelphia, Pennsylvania) and sodium alginate. The amount of the suspending agent(s) in the systemic or topical composition is usually about 1 to about 8% by weight of the composition.

[0098] Suitable surfactants include lecithin, polysorbate 80, and sodium lauryl sulfate, and TWEENS (manufactured by Atlas Powder Company, Wilmington, Delaware). Suitable surfactants include those disclosed below: C.T.F.A. Cosmetic Ingredient Handbook, 1992, pp.587-592; Remington’s Pharmaceutical Sciences, 15th Ed. 1975, pp. 335-337, and McCutcheon’s Volume 1, Emulsifiers & Detergents, 1994, North American Edition, pp. 236-239. The amount of the surfactant(s) in the systemic or topical composition is usually about 0.1% to about 5% by weight of the composition.

[0099] The amounts of the components in the systemic composition can vary depending on the type of systemic composition to be prepared. Generally, however, the systemic composition comprises from 0.01% to 50% by weight of an active compound and from 50% to 99.99% by weight of one or more carriers. Compositions for parenteral administration usually contain from 0.1% to 10% by weight of the active ingredient and from 90% to 99.9% by weight of a carrier (such as a diluent and a solvent).

[0100] Compositions for oral administration can have various dosage forms. For example, solids include tablets, capsules, granules, and bulk powders. These oral dosage forms contain a safe and effective amount, usually at least about 5% by weight, more specifically from about 25% to about 50% by weight, of the active ingredient. Oral dosage compositions contain from about 50% to about 95% by weight, more specifically from about 50% to about 75% by weight, of a carrier.

[0101] Tablets can be compressed tablets, powder tablets, enteric-coated tablets, sugar-coated tablets, film-coated tablets, or multi-compressed tablets. Tablets usually contain the active constituent as well as components selected from carriers (such as diluents, lubricants, binders, disintegrants, colorants, flavorings, sweeteners, glidants, and combinations thereof). Specific diluents include calcium carbonate, sodium carbonate, mannitol, lactose, and cellulose. Specific binders include starch, gelatin, and sucrose. Specific disintegrants include alginic acid and croscarmellose. Specific lubricants include magnesium stearate, stearic acid, and talc. Certain colorants can be FD&C dyes added for appearance. Chewable tablets preferably also contain a sweetener (such as aspartame and saccharin) or a flavoring (such as menthol, peppermint, fruit flavor), or a combination thereof.

[0102] Capsules (e.g., implants, sustained release formulations, and extended release formulations) typically contain, within a capsule that usually contains gelatin, an active compound (e.g., a compound of formula (I)) and a carrier comprising one or more of the diluents disclosed above. Granules typically contain the disclosed compound, preferably a flow promoting agent (e.g., silicon dioxide) to improve flow properties. Implants can be of the biodegradable or non - biodegradable type.

[0103] The selection of the components of the carrier for oral compositions is determined by secondary considerations such as taste, cost, and storage stability, which are not important for the purposes of the present disclosure.

[0104] Solid compositions can typically be coated in a conventional manner using a pH - or time - dependent coating, and the disclosed compounds are released within the gastrointestinal tract in the vicinity of the desired application or at various times and over time to extend the desired action. Coatings typically include one or more components selected from the group consisting of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, ethyl cellulose, EUDRAGIT® coatings (available from Evonik Industries, Essen, Germany), waxes, and shellac.

[0105] Compositions for oral administration can be in liquid form. For example, suitable liquid forms include aqueous solutions, emulsions, suspensions, solutions reconstituted from non - effervescent granules, suspensions reconstituted from non - effervescent granules, effervescent preparations reconstituted from effervescent granules, elixirs, tinctures, syrups, and the like. Liquid oral dosage compositions typically contain the disclosed compound as well as a carrier (i.e., a carrier selected from diluents, colorants, flavorants, sweeteners, preservatives, solvents, suspending agents, and surfactants). Oral liquid compositions preferably contain one or more components selected from colorants, flavorants, and sweeteners.

[0106] Other compositions useful for achieving systemic delivery of the subject compound include sublingual, buccal, and nasal dosage forms. Such compositions typically include a soluble filler (e.g., a diluent including sucrose, sorbitol, and mannitol) and one or more of a binder (e.g., acacia, microcrystalline cellulose, carboxymethyl cellulose, and hydroxypropyl methylcellulose). Such compositions may further include a lubricant, a coloring agent, a flavoring agent, a sweetening agent, an antioxidant, and a glidant.

[0107] The disclosed compounds can be administered topically. Topical compositions that can be applied topically to the skin can be in any form, including solids, solutions, oils, creams, ointments, gels, lotions, shampoos, rinse-off hair conditioners, milks, detergents, moisturizers, sprays, skin patches, etc. Topical compositions include the disclosed compound (e.g., a compound of formula (I)), or a pharmaceutically acceptable salt thereof, and a carrier. The carrier of the topical composition preferably aids in the penetration of the compound into the skin. The carrier may further include one or more optional components.

[0108] The amount of carrier used in combination with the disclosed compound is sufficient to provide a practical amount of the composition for administration per unit dose of the compound. Techniques and compositions for manufacturing dosage forms useful in the methods of the present disclosure are described in the following references: Modern Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979), Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981), and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).

[0109] The carrier may comprise a single component or a combination of two or more components. In a topical composition, the carrier comprises a topical carrier. Suitable topical carriers include phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, symmetrical alcohols, aloe vera gel, allantoin, glycerin, vitamins A and E oils, mineral oil, propylene glycol, PPG-2 myristyl propionate, dimethyl isosorbide, castor oil, and one or more components selected from combinations thereof. More specifically, carriers for skin application include propylene glycol, dimethyl isosorbide, and water, and even more specifically, phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, and symmetrical alcohols.

[0110] The carrier of the topical composition may further comprise one or more components selected from skin softeners, propellants, solvents, humectants, thickeners, powders, fragrances, pigments, and preservatives, all of which are optional.

[0111] Suitable skin softeners include the following: stearyl alcohol, glyceryl monolinoleate, glyceryl monostearate, propane-1,2-diol, butane-1,3-diol, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, sesame oil, coconut oil, peanut oil, castor oil, acetylated lanolin alcohol, petroleum, mineral oil, butyl myristate, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate, and combinations thereof. Specific skin softeners for the skin include stearyl alcohol and polydimethylsiloxane. The amount of the skin softener(s) in the topical composition based on the skin is usually about 5% to about 95% by weight of the composition.

[0112] Suitable propellants include propane, butane, isobutane, dimethyl ether, carbon dioxide, nitrous oxide, and combinations thereof. The amount of the propellant(s) in the topical composition is usually about 0% to about 95% by weight of the composition.

[0113] Suitable solvents include water, ethyl alcohol, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran, and combinations thereof. Specific solvents include ethyl alcohol and homotropic alcohol. The amount of the solvent(s) in the topical composition is usually about 0% to about 95% by weight of the composition.

[0114] Suitable humectants include glycerin, sorbitol, sodium 2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate, gelatin, and combinations thereof. A specific humectant is glycerin. The amount of the humectant(s) in the topical composition is usually from 0 wt% to 95 wt% of the composition.

[0115] The amount of the thickener(s) in the topical composition is usually from about 0 wt% to about 95 wt% of the composition.

[0116] Suitable powders include beta-cyclodextrin, hydroxypropyl cyclodextrin, chalk, talc, fuller's earth, kaolin, starch, gum, colloidal silicon dioxide, sodium polyacrylate, tetraalkylammonium smectite, trialkylarylammonium smectite, chemically modified magnesium aluminum silicate, organically modified montmorillonite clay, hydrated aluminum silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethyl cellulose, ethylene glycol monostearate, and combinations thereof. The amount of the powder(s) in the topical composition is usually from 0 wt% to 95 wt% of the composition.

[0117] The amount of the fragrance in the topical composition is usually from about 0 wt% to about 0.5 wt%, particularly from about 0.001 wt% to about 0.1 wt% of the composition.

[0118] Suitable pH adjusting additives include HCl or NaOH in an amount sufficient to adjust the pH of the topical pharmaceutical composition.

[0119] Method of Use As described herein, the disclosed compounds inhibit the function of 3βHSD1. Thus, the present compounds and pharmaceutical compositions containing the compounds can be used for the treatment of disorders that can benefit from the inhibition of 3βHSD1, such as cancer. In particular, the disclosed compounds and pharmaceutical compositions can also be used in methods for prostate cancer, such as castration-resistant prostate cancer (CRPC), breast cancer, and endometrial cancer. The methods further include co-therapies for improving treatment outcomes in connection with cancer.

[0120] In the overview of steroid metabolism in CRPC, a very important role of 3β-HSD1 has been pointed out. There are at least three possible pathways to dihydrotestosterone (DHT) synthesis from non-gonadal precursors. 1) The canonical pathway that requires the conversion of adrenal dehydroepiandrosterone (DHEA) → androstenedione (AD) → testosterone → DHT, 2) the 5α-androstane-dione pathway that bypasses testosterone and converts androstenedione → 5α-androstane-dione → DHT, but still utilizes adrenal precursors, 3) the "backdoor" pathway that can occur using de novo steroidogenesis from cholesterol, bypassing testosterone via 5α-reduction of progesterone or 17OH-progesterone, and then 5α-androstanediol is required as an intermediate metabolite, which is then converted to DHT. Evidence exists for all three pathways, and prior studies have suggested that the 5α-androstane-dione pathway is dominant in models and freshly collected metastatic CRPC tissues where the tumor utilizes adrenal precursors (Chang et al. P. Natl. Acad. Sci. USA 108, 13728-13733 (2011). Nevertheless, it is important to note that all pathways for the synthesis of testosterone and / or DHT require 3β-HSD enzyme activity. 3βHSD performs two reactions necessary to convert the 3β-OH, Δ 5 -structure to biologically active androgens. The first is the oxidation of 3β-OH to 3-keto, and the second is the conversion of Δ 5 →Δ 4is isomerization (Evaul et al. Endocrinology 151, 3514-3520, (2010), Simard et al. Endocr. Rev. 26, 525-582, (2005)). Humans have two isozymes for 3β-HSD, and 3β-HSD1 is the dominant peripheral expression isozyme (Simard 2005, Sharifi, Endocrinology 154, 4010-4017 (2013). Taken together, these observations shed light on the central role of 3β-HSD1 in prostate cancer.

[0121] Further investigation of the HSD3B1 cell metabolic phenotype revealed a gain-of-function missense in 3β-HSD1 that increases the conversion of adrenal-derived DHEA to AD and downstream DHT synthesis in a human prostate cancer cell line model (Chang et al. Cell 154, 1074-1084 (2013)). A single nucleotide change that converts A to C at position 1245 of HSD3B1 results in the exchange of asparagine (N) for threonine (T) at amino acid position 367 of 3β-HSD1. This missense is a frequent germline variant that exists at an allelic frequency of approximately 25-35%, meaning that at least one copy is present in approximately 50% of all men (Chang 2013). The variant HSD3B1(1245C) that encodes the 3β-HSD1(367T) enzyme missense prevents ubiquitination and rapid proteasome-mediated degradation, thereby increasing the steady-state 3β-HSD1 protein level. Thus, the HSD3B1(1245C) allele is an adrenal-tolerant allele that allows the conversion of DHEA to potent downstream androgens (e.g., testosterone and DHT) that stimulate the AR, while the HSD3B1(1245A) allele is an adrenal-restrictive allele that prevents the conversion of DHEA to potent androgens (Sabharwal et al. Endocrinology 160, 2180-2188 (2019)).

[0122] Inheritance of the adrenal-tolerant HSD3B1(1245C) allele speeds up the normal rate-limiting step in the conversion from DHEA to DHT, enabling more rapid regeneration of potent androgens in men treated with ADT for prostate cancer, which may thereby worsen the clinical outcomes associated with CRPC. This was first retrospectively tested in three cohorts of 443 men treated with ADT (two with biochemical recurrence after prostatectomy and one with metastatic CSPC). All three cohorts showed poor outcomes (including worsening progression-free survival and overall survival) in men who inherited the adrenal-tolerant HSD3B1 genotype. These findings are currently being further validated in at least 10 cohorts (Agarwal et al. JAMA Oncol. 3, 856-857 (2017), Garcia Gil et al. In Proceedings of the 4th European Conference of Oncology Pharmacy, 25-27 October 2018 (Nantes, France) Abstract 107, Hearn et al. JAMA Oncol. 4, 558-562 (2018), Hearn et al. JAMA Oncol. 6(4):e196796 (2020)).

[0123] In HSD3B1 genotype analysis, silencing of 3β-HSD1(367T) in LNCaP cells that endogenously express HSD3B1(1245C) encoding 3β-HSD1(367T) 3Metabolic flux from [H]-DHEA (100 nM) to AD and further downstream conversion to 5α-androstenedione and DHT was shown to be silenced. Gene depletion also inhibited AR-regulated gene expression (PSA, TMPRSS2) and blocked the growth of CRPC in LNCaP xenografts (shHSD3B1 #1, p = 0.002 and shHSD3B1 #2, p = 0.003). Collectively, these data demonstrate that the adrenal permissive HSD3B1 (1245C)-encoded enzyme is a pharmacological target that genetically drives androgen synthesis and CRPC in approximately 50% of all men (Chang 2013).

[0124] Multiple reports have documented an association between 3βHSD1 and breast cancer (see, for example, Kruse et al. JCI Insight. 6:29):e150403 (2021), Flanagan et al. Ann. Surg. Oncol. 29(11): 7194-7201 (2022)). Furthermore, trilostane is known to inhibit 3βHSD1 and has been shown to be active against metastatic breast cancer (Chu et al. Breast Cancer Res. Treat. 13(2):117-21 (1989)).

[0125] Accordingly, a method of doing so in a subject in need of cancer treatment, the method comprising administering to the subject a therapeutically effective amount of a compound disclosed herein (e.g., a compound of formula (I)), or a pharmaceutically acceptable salt thereof, is disclosed herein. In some embodiments, the cancer is prostate cancer, breast cancer, or endometrial cancer.

[0126] In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is castration-resistant prostate cancer. In some embodiments, the cancer is prostate cancer and the subject is a human having the HSD3B1(1245C) allele. Identification of the HSD3B1 genotype in a subject can be performed as previously disclosed (Thomas et al. Urology 145, 13-21 (2020)). In some embodiments, the method comprises identifying a human subject having prostate cancer, determining whether the subject has the HSD3B1(1245C) allele, and, if the subject has the HSD3B1(1245C) allele, administering to the subject a therapeutically effective amount of a compound disclosed herein (e.g., a compound of formula (I)) or a pharmaceutically acceptable salt thereof.

[0127] In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is breast cancer and the subject is a human having the HSD3B1(1245C) allele. See, for example, Kruse et al. JCI Insight. 6(20):e150403 (2021). In some embodiments, the method comprises identifying a human subject having breast cancer, determining whether the subject has the HSD3B1(1245C) allele, and, if the subject has the HSD3B1(1245C) allele, administering to the subject a therapeutically effective amount of a compound disclosed herein (e.g., a compound of formula (I)) or a pharmaceutically acceptable salt thereof. In some embodiments, the cancer is breast cancer resistant to standard hormonal therapies (e.g., aromatase inhibitors, selective estrogen receptor modulators (SERMs), estrogen receptor degraders, oophorectomy, combinations thereof, and combinations with non-hormonal therapeutic agents including CDK4 / 6 inhibitors).

[0128] In some embodiments, the cancer is endometrial cancer.

[0129] Companion diagnostic In some embodiments, the compounds provided herein are used in methods of personalized cancer therapy. In some embodiments, the compounds provided herein are used in companion diagnostic methods involving first determining whether a subject is responsive to treatment with the compounds provided herein, and then providing / administering the compound to the subject if a positive response is predicted. Thus, in some embodiments, the methods provided herein include determining whether a subject has cancer that would be responsive to inhibition of 3βHSD1 using the compounds provided herein. A positive response to a compound (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof) indicates that the compound has effective anti-cancer properties in the subject. In contrast, a non-response or negative response to the compound indicates that the compound is likely not effective in treating cancer in the subject.

[0130] In some embodiments, the methods provided herein include determining whether a subject has cancer that would be responsive to inhibition of 3βHSD1 using the compounds provided herein, and administering to the subject a compound (e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof) if a positive response to inhibition of 3βHSD1 is predicted. In some aspects, determining whether a subject has cancer that would be responsive to inhibition of 3βHSD1 (e.g., predicting responsiveness to treatment with the compounds provided herein) includes determining whether a gain-of-stability mutation that results in a gain-of-function in 3β-hydroxysteroid dehydrogenase type 1 (3βHSD1) is present in the subject. In some embodiments, the subject is human, and the gain-of-stability mutation includes a cytosine nucleotide at position 1245 of the human HSD3B1 gene that causes threonine at position 367 of the human 3βHSD1 protein. The HSD3B1 gene (human) refers to NCBI Gene ID 3283, and its sequence is NCBI Reference Sequence: NG_050909.1. In some embodiments, the sequence of the native human 3βHSD1 protein that does not have the gain-of-stability mutation at position 367 is MTGWSCLVTGAGGFLGQRIIRLLVKEKELKEIRVLDKAFGPELREEFSKLQNKTKLTVLEGDILDEPFLKRACQDVSVIIHTACIIDVFGVTHRESIMNVNVKGTQLLLEACVQASVPVFIYTSSIEVAGPNSYKEIIQNGHEEEPLENTWPAPYPHSKKLAEKAVLAANGWNLKNGGTLYTCALRPMYIYGEGSRFLSASINEALNNNGILSSVGKFSTVNPVYVGNVAWAHILALRALQDPKKAPSIRGQFYYISDDTPHQSYDNLNYTLSKEFGLRLDSRWSFPLSLMYWIGFLLEIVSFLLRPIYTYRPPFNRHIVTLSNSVFTFSYKKAQRDLAYKPLYSWEEAKQKTVEWVGSLVDRHKENLKSKTQ (SEQ ID NO: 1). In some embodiments, the sequence of the human 3βHSD1 protein having threonine at position 367 is MTGWSCLVTGAGGFLGQRIIRLLVKEKELKEIRVLDKAFGPELREEFSKLQNKTKLTVLEGDILDEPFLKRACQDVSVIIHTACIIDVFGVTHRESIMNVNVKGTQLLLEACVQASVPVFIYTSSIEVAGPNSYKEIIQNGHEEEPLENTWPAPYPHSKKLAEKAVLAANGWNLKNGGTLYTCALRPMYIYGEGSRFLSASINEALNNNGILSSVGKFSTVNPVYVGNVAWAHILALRALQDPKKAPSIRGQFYYISDDTPHQSYDNLNYTLSKEFGLRLDSRWSFPLSLMYWIGFLLEIVSFLLRPIYTYRPPFNRHIVTLSNSVFTFSYKKAQRDLAYKPLYSWEEAKQKTVEWVGSLVDRHKETLKSKTQ (SEQ ID NO: 2). Thus, in some embodiments, the method comprises determining whether a subject expresses a gain-of-stability mutation by determining whether cells in a sample obtained from the subject express SEQ ID NO: 2 or a gene encoding SEQ ID NO: 2 (indicating the presence of a mutation), or whether the cells express SEQ ID NO: 1 or a gene encoding SEQ ID NO: 1 (indicating the absence of a gain-of-stability mutation).

[0131] Thus, in some embodiments, the method comprises determining whether the HSD3B1(1245C) gene or the 3βHSD1(367T) protein is expressed in a biological sample obtained from a subject, and predicting a positive response to a compound if the HSD3B1(1245C) gene or the 3βHSD1(367T) protein is expressed.

[0132] In some embodiments, the method comprises obtaining a biological sample from a subject. As used herein, "biological sample" means any biological sample from a subject suitable for analysis for the detection of the HSD3B1(1245C) gene or the 3βHSD1(367T) protein. Suitable biological samples include, but are not limited to, body fluids such as blood-related samples (e.g., whole blood, serum, plasma, and other blood-derived samples), urine, saliva, mucus, cerebrospinal fluid, bronchoalveolar lavage fluid, etc. Another example of a biological sample is a tissue sample. In some embodiments, the biological sample is a cancer cell or a tissue containing cancer cells. The HSD3B1(1245C) gene or the 3βHSD1(367T) protein can be evaluated either quantitatively or qualitatively, and the detection can be determined either in vitro or ex vivo.

[0133] The method comprises providing or obtaining a biological sample from a subject, and the biological sample can be obtained by any known means including, for example, needle puncture, needle biopsy, swab, etc. In an exemplary method, the biological sample is a blood sample, which can be obtained, for example, by venipuncture.

[0134] The biological sample may be fresh or stored. The biological sample may be stored or preserved or deposited under appropriate tissue storage conditions. The biological sample can be a tissue sample explicitly obtained for the assays of the present invention or a tissue sample obtained for another purpose that can be subsampled for the assays of the present invention. Preferably, if the biological sample is stored to prevent sample degradation, it is refrigerated or frozen immediately after collection.

[0135] The sample can be pre-treated as needed by diluting in an appropriate buffer solution, heparinizing, concentrating if desired, or fractionating by ultracentrifugation, fast protein liquid chromatography (FPLC) or HPLC, or any number of methods including but not limited to precipitation of apolipoprotein B containing protein using dextran sulfate or other methods. Any of various standard buffered aqueous solutions of physiological pH such as phosphate, Tris, etc. can be used.

[0136] Either a variant form of a gene (HSD3B1(1245C)) or a variant form of a protein (3βHSD1(367T)) can be detected in a sample obtained from a subject and used to determine whether the subject has a positive response to the compounds provided herein. The gene or protein can be detected or measured by an analytical device such as a kit or a conventional laboratory apparatus, which can be either portable or stationary. In some embodiments, the level of the variant gene or protein can be compared to the level of a corresponding internal standard in one or more samples when performing an analysis to quantify the amount of the detected gene or protein.

[0137] The HSD3B1(1245C) gene or 3βHSD1(367T) protein is typically detected in a biological sample obtained from a subject. However, in some embodiments, non-invasive imaging means are used to detect this mutation. For example, administration of 18F-DHEA in combination with PET imaging may be able to detect tumors carrying the mutant enzyme. This is because these tumors are expected to have high fluidity from DHEA to downstream androgen metabolites.

[0138] In some embodiments, the presence of the 3βHSD1(367T) protein is determined. The presence and / or amount of the 3βHSD1(367T) protein in a biological sample can be determined using polyclonal or monoclonal antibodies that are immunoreactive with the 3βHSD1(367T) protein variant. Use of the antibody involves contacting a sample taken from an individual with one or more antibodies and assaying for the formation of a complex between the antibody and a protein or peptide in the sample. To facilitate detection, the antibody can be bound to a substrate such as a column, plastic dish, matrix, or membrane, preferably nitrocellulose. The sample may be untreated or subjected to precipitation, fractionation, separation, or purification prior to combination with the antibody. The interaction between the antibody and the 3βHSD1(367T) protein in the sample is detected by radiometric, colorimetric, or fluorometric means, size separation, or precipitation. Preferably, detection of the antibody-protein or peptide complex is by addition of a secondary antibody conjugated to a detectable tag, such as an enzyme, fluorophore, or chromophore. Formation of the complex indicates the presence of the 3βHSD1(367T) protein in the sample.

[0139] Antibodies specific to 3βHSD1(367T) are produced and labeled using standard procedures and can then be used in immunoassays to detect the presence of 3βHSD1(367T) in a sample. Suitable immunoassays include, by way of example, immunoprecipitation, particle immunoassay, immunonephelometry, radioimmunoassay (RIA), enzyme immunoassay (EIA) including enzyme-linked immunosorbent assay (ELISA), sandwich, direct, indirect, or competitive ELISA assays, enzyme-linked immunospot assay (ELISPOT), fluorescence immunoassay (FIA), chemiluminescent immunoassay, flow cytometry assay, immunohistochemistry, Western blot, and protein chip assays using, for example, antibodies, antibody fragments, receptors, ligands, or other agents that bind to the target analyte. Polyclonal or monoclonal antibodies produced against 3βHSD1(367T) are produced according to established procedures. Generally, in the preparation of polyclonal antibodies, the protein or its peptide fragment is used as the first step to immunize the host animal. General overviews of immunoassays are available in Methods in Cell Biology v. 37: Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York (1993), and Basic and Clinical Immunology 7th Ed., Stites & Ten, eds. (1991).

[0140] In some embodiments, the 3βHSD1(367T) protein is detected using methods other than immunoassays. For example, the 3βHSD1(367T) protein can be detected using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). The 3βHSD1(367T) protein can also be detected by purifying the 3βHSD1 protein and determining its sequence using peptide sequencing methods. Protein purification techniques are well known to those skilled in the art. These techniques involve, at one level, crudely fractionating the cellular environment into a polypeptide fraction and a non-polypeptide fraction. Once the polypeptide has been separated from other proteins, the polypeptide of interest can be further purified and / or quantified using chromatography and electrophoresis techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for the preparation of pure peptides are immunohistochemistry, ion-exchange chromatography, size-exclusion chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. A particularly efficient method for purifying peptides is high-performance liquid chromatography, or HPLC for short. Similarly, various methods of protein sequencing are known to those skilled in the art. For example, the sequence can be identified using mass spectrometry or an Edman degradation reaction.

[0141] The 3βHSD1(367T) protein can also be detected based on its different characteristics compared to the wild-type version of the protein. The inventors have demonstrated that the 3βHSD1(367T) protein is more resistant to ubiquitination and degradation than the 3βHSD1(367N) protein. Thus, the presence of the 3βHSD1(367T) protein can be detected using ubiquitination and / or degradation assays. Similarly, the inventors have shown that the autocrine motility factor receptor (AMFR), which is involved in protein degradation, exhibits a lower affinity for the 3βHSD1(367T) protein than for the 3βHSD1(367N) protein. Thus, the presence of the 3βHSD1(367T) protein can also be determined by assessing the affinity of 3βHSD1 for AMFR. Some tumors may also have a somatic loss of expression of AMFR or other means of stabilizing the wild-type enzyme (3βHSD1(367N)) that can serve the tumor as an alternative mechanism for protein stabilization, which can increase DHT synthesis and treatment resistance.

[0142] In some embodiments, the presence of 3βHSD1(367T) can be indirectly detected by observing the serum steroid profile. Evaluation of the serum steroid profile may correlate with the HSD3B1 genotype. For example, the mutant enzyme may be associated with an increase in the ratio of enzyme product to precursor (i.e., androstenedione / DHEA and progesterone / pregnenolone).

[0143] In some embodiments, the presence of the HSD3B1(1245C) allele is confirmed. The presence and / or level of the HSD3B1(1245C) allele can be determined by any currently known or later developed assay or method for detecting and / or determining expression levels, such as quantitative RT-PCR, Northern blot, real-time PCR, PCR, allele-specific PCR, pyrosequencing, SNP chip technology, sequencing, or restriction fragment length polymorphism (RFLP).

[0144] Many methods for determining nucleotide sequences include PCR. As used herein, the term "polymerase chain reaction" ("PCR") refers to the methods of U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188 for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification, all of which are incorporated by reference. As used herein, the terms "PCR product" and "amplification product" refer to a mixture of compounds obtained after completion of two or more cycles of PCR steps of denaturation, annealing, and extension. These terms include the case where there is amplification of one or more segments of one or more target sequences. Thus, in some embodiments, detecting the presence and / or level of the HSD3B1(1245C) allele includes extending a primer that hybridizes to a sequence adjacent to the polymorphic nucleotide. In some embodiments, determining the presence and / or level of the HSD3B1(1245C) allele includes hybridizing a probe to a region containing the polymorphic nucleotide.

[0145] In some embodiments, hybridization with a complementary sequence can be used to detect the presence of the HSD3B1(1245C) allele based on the different properties of sequences having a perfect or imperfect sequence match. For example, as described herein, an asymmetric PCR assay can be used, where fluorescence melting reveals two different melting temperatures of the probe / target duplex specific for the amplified allele. This assay can be used to detect the HSD3B1(1245C) allele in the germline or somatic cells.

[0146] In some embodiments, the presence of the HSD3B1(1245C) allele is detected by sequencing, including next-generation sequencing (NGS) techniques. The term "sequencing" encompasses various suitable sequencing technologies that can be used to determine the amount and type of nucleic acids present in a sample, indicating whether the HSD3B1(1245C) allele is present within the subject. In some embodiments, sequencing includes isolating RNA from a sample, generating a cDNA library from the RNA, and then sequencing the cDNA. In some embodiments, sequencing includes next-generation sequencing. In some embodiments, sequencing includes directly sequencing nucleic acid molecules, such as by next-generation direct RNA sequencing. Suitable sequencing methods include sequencing by synthesis (SBS), reversible terminator sequencing, sequencing by ligation, sequencing by hybridization and ligation, sequencing by hybridization and synthesis, nanopore sequencing, nanoball sequencing, and the like.

[0147] Once the presence and / or level of any variant form of a gene (HSD3B1(1245C)) or protein variant form (3βHSD1(367T)) is determined, they can be presented in various ways. For example, the level can be presented graphically on a display as a numerical value or a proportional bar (i.e., a bar graph), or any other presentation method known to those skilled in the art. Presentation by a graph can provide a visual representation of the amount of the variant gene or protein in the biological sample being evaluated.

[0148] Further methods of use Also disclosed herein is a method of inhibiting cancer cell proliferation, which comprises contacting a cancer cell with a compound disclosed herein (e.g., a compound of formula (I)) or a pharmaceutically acceptable salt thereof in an amount effective to inhibit cancer cell proliferation. In some embodiments, the cancer cell is a prostate cancer cell. In some embodiments, the cancer cell is a castration-resistant prostate cancer cell. In some embodiments, the cancer cell is a breast cancer cell. In some embodiments, the cancer cell is an endometrial cancer cell. In some embodiments, the cancer cell is predicted to have a positive response to the compound as described above.

[0149] Also disclosed herein is a method of inhibiting the activity of 3β-hydroxysteroid dehydrogenase in a sample, which comprises contacting the sample with a compound disclosed herein (e.g., a compound of formula (I)) or a pharmaceutically acceptable salt thereof in an amount effective to inhibit the activity of 3β-hydroxysteroid dehydrogenase.

[0150] Route of Administration and Combination Therapy In the treatment methods described herein, the compound or pharmaceutical composition can be administered to a subject by any convenient route of administration, including, but not limited to, orally (e.g., by ingestion), topically (e.g., including transdermal, intranasal, intraocular, buccal, and sublingual), pulmonary (e.g., via the mouth or nose, e.g., by inhalation or insufflation therapy using an aerosol), rectal, vaginal, parenterally, e.g., by injection including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subepidermal, intraarticular, subarachnoid, and intrasternal, or including implantation of a subcutaneous or intramuscular depot. In some embodiments, the administration includes oral administration. Additional modes of administration can include supplying the compound as part of the diet of an animal by adding the compound and / or the composition containing the compound to a food or beverage containing water for animals.

[0151] It will be appreciated that the appropriate dosage of the compound and of the composition containing the compound may vary from patient to patient. Determining the optimal dosage generally involves balancing the level of therapeutic effect against any risks or adverse side effects of the treatment of the present disclosure. The dosage level selected depends on a variety of factors including, but not limited to, the activity of the particular agent, the route of administration, the time of administration, the rate of secretion of the compound, the duration of the treatment, other drugs, compounds, and / or materials used in combination, the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of the compound and the route of administration are ultimately at the discretion of the physician, but generally the dosage is such as to achieve a local concentration at the site of action that will achieve the desired effect without substantially causing harmful or adverse side effects.

[0152] Administration in vivo can be achieved in a single dose, continuously, or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. The most effective means of administration and the method of determining the dosage will be well known to those skilled in the art and will vary depending on the formulation used for treatment, the purpose of the treatment, the target cells being treated, and the subject being treated. Single or multiple administrations can be carried out at dosage levels and patterns selected by the physician conducting the treatment. Generally, a suitable dosage of the compound is in the range of about 100 μg to about 250 mg per kilogram of the subject's body weight per day.

[0153] The compound or composition can be administered once, on a continuous basis (e.g., by intravenous infusion), or on a periodic / intermittent basis, which includes once every about 1 hour, once every about 2 hours, once every about 4 hours, once every about 8 hours, once every about 12 hours, once every about 1 day, once every about 2 days, once every about 3 days, twice a week, once a week, and once a month. The composition may be administered until the desired alleviation of symptoms is achieved.

[0154] The compounds described herein may be used in combination with other known therapies. As used herein, "administered in combination" means that two (or more) different therapies are delivered to a subject during the course of the subject's suffering from a disorder, e.g., the two or more therapies are administered after the subject has been diagnosed and before the disorder has healed or disappeared or before the treatment has been discontinued for other reasons. In some embodiments, the delivery of one therapy is still being performed at the start of the delivery of the second therapy, as if there were an overlap with respect to administration. This may be referred to herein as "simultaneous" or "co-delivery". In other embodiments, the delivery of one therapy ends before the start of the delivery of the other therapy. In some embodiments in either case, the therapies are more effective for combination administration. For example, the second therapy is more effective, e.g., an equivalent effect is seen with less of the second therapy compared to the effect when the second therapy is administered without the first therapy, or the second therapy reduces the symptoms more, or a similar situation is seen with the first therapy. In some embodiments, the delivery is made such that the reduction of symptoms or other parameters related to the disorder is greater than that observed with one of the therapies delivered in the absence of the other. The effects of the two therapies may be partly additive, completely additive, or supra-additive. The delivery may be made such that the effect of the first therapy delivered is still detectable when the second therapy is delivered.

[0155] The compounds or compositions described herein and at least one additional therapeutic agent may be administered simultaneously, in the same or separate compositions, or sequentially. For sequential administration, the compound described herein may be administered first, followed by the additional agent, or the order of administration may be reversed.

[0156] In some embodiments, the compounds described herein are administered in combination with other therapeutic modalities including surgery, chemotherapy, radiation therapy, hormone therapy, immunotherapy, cryotherapy, and hyperthermia. Such combination therapies advantageously may avoid the potential for toxicity or complications associated with the various therapies, using lower dosages of the administered drug and / or other chemotherapeutic agents. The phrase "radiation treatment" includes external beam therapy, including three-dimensional conformal radiation therapy designed such that the radiation field matches the size of the tissue being treated, brachytherapy where seeds of a radioactive compound are implanted into tissue using ultrasound guidance, and combinations of external beam therapy and brachytherapy, but is not limited thereto. In some embodiments, the second therapy includes immunotherapy. Immunotherapy includes administration of chimeric antigen receptor (CAR) T cell therapy or T cell transfer therapy, cytokine therapy, immunomodulatory agents, cancer vaccines, or antibodies (e.g., monoclonal antibodies).

[0157] In some embodiments, the compounds described herein are administered in combination with at least one additional therapeutic agent, such as a chemotherapeutic agent. In certain embodiments, the compounds described herein are administered in combination with one or more additional chemotherapeutic agents. The chemotherapeutic agent may be a chemotherapeutic agent identified in the "A to Z List of Cancer Drugs" issued by the National Cancer Institute. In some embodiments, the chemotherapeutic agent is selected from abiraterone, apalutamide, bicalutamide, cabazitaxel, capecitabine, cyclophosphamide, darolutamide, degarelix, docetaxel, dutasteride, enzalutamide, estradiol, estramustine, finasteride, flutamide, goserelin, histrelin, leuprolide, mitoxantrone, nilutamide, olaparib, radium-223, rucaparib, sipuleucel-T, and triptorelin, or any combination thereof. In some embodiments, the chemotherapeutic agent is selected from the following: abemaciclib, ado-trastuzumab emtansine, alpelisib, anastrozole, atezolizumab, capecitabine, carboplatin, cisplatin, cyclophosphamide, docetaxel, doxorubicin, epirubicin, eribulin, everolimus, exemestane, fam-trastuzumab deruxtecan, 5-fluorouracil, fulvestrant, gemcitabine, goserelin, ixabepilone, lapatinib, letrozole, margetuximab-cmkb, megestrol, methotrexate, neratinib, olaparib, paclitaxel, palbociclib, pamidronate, pembrolizumab, pertuzumab, raloxifene, ribociclib, sacituzumab govitecan-hziy, talazoparib, tamoxifen, thiotepa, toremifene, trastuzumab, tucatinib, vinblastine, and vinorelbine, or any combination thereof. In some embodiments, the chemotherapeutic agent is selected from carboplatin, cisplatin, docetaxel, dostarlimab-gxly, doxorubicin, ifosfamide, lenvatinib, megestrol, paclitaxel, and pembrolizumab, trastuzumab, or any combination thereof.

[0158] Kit The present disclosure also provides a kit for indicating a direction for the treatment of cancer in a subject using the compounds described herein. For example, the kit involves evaluating 3βHSD1(367T) or HSD3B1(1245C) in a sample obtained from a subject having cancer or at risk of having cancer, and providing the compounds described herein to the subject if a positive response to the compound is predicted based on the expression of 3βHSD1(367T) or HSD3B1(1245C) in the sample, which can be used in a method for personalized medicine or a companion diagnostic method.

[0159] The kit includes one or more primers or probes capable of detecting 3βHSD1(367T) or HSD3B1(1245C), and a package for holding the primers or probes. The kit generally includes one or more containers for holding reagents, which can be included as one or more separate compositions or, optionally, as a mixture if the compatibility of the reagents permits. The kit can further include an enzyme (e.g., polymerase), buffer, labeling agent, nucleotides, control, and any other materials necessary for detecting 3βHSD1(367T) or HSD3B1(1245C). The kit can also include a tool for obtaining a sample from a subject, such as a punch tool for obtaining a punch biopsy or a needle biopsy.

[0160] In some embodiments, the kit includes a primer capable of detecting HSD3B1(1245C). For example, the kit can include an oligonucleotide primer of an appropriate size that amplifies a region of HSD3B1 including the A→C conversion. For example, in some embodiments, a region from HSD3B1 including the A→C conversion and including from about 10 nucleotides to about 100 nucleotides can be used. Specific appropriate primers are described in the examples herein. The primer may be labeled.

[0161] In another aspect, there is provided a kit for indicating the direction of treatment, comprising an array and / or a microarray, oligonucleotide primers that amplify a portion of HSD3B1 from approximately nucleotide 1200 to approximately nucleotide 1300, and instructions for use. Alternatively or additionally, primers may be provided that amplify approximately nucleotide 1210 to approximately nucleotide 1280 of HSD3B1, approximately nucleotide 1220 to approximately nucleotide 1270 of HSD3B1, approximately nucleotide 1230 to approximately nucleotide 1260 of HSD3B1, approximately nucleotide 1235 to approximately 1250 of HSD3B1, or other portions that a person skilled in the art determines to be necessary or appropriate to amplify HSD3B1(1245C) using PCR or other sequencing techniques known to those skilled in the art and detect its presence.

[0162] In some embodiments, the kit includes a probe capable of detecting 3βHSD1(367T). A preferred type of probe is an antibody capable of specifically binding to 3βHSD1(367T). Examples of antibodies that can be used in the present disclosure include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, recombinant antibodies, single-chain Fv ("scFv"), affinity matured antibodies, single-chain antibodies, single-domain antibodies, F(ab) fragments, F(ab') fragments, disulfide-linked Fv ("sdFv"), and anti-idiotype ("anti-Id") antibodies and functionally active epitope-binding fragments of any of the above. As used herein, the term "specifically binds" refers to the interaction of an antibody with a second chemical species, and this interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species. For example, an antibody recognizes and binds to a specific protein structure rather than a general protein.

[0163] The kit may also include a solid phase to which an antibody that functions as a capture antibody and / or a detection antibody in a sandwich immunoassay format binds. The solid phase can be a material such as magnetic particles, beads, test tubes, microtiter plates, cuvettes, membranes, scaffold molecules, quartz crystals, films, filter papers, disks, or chips. The kit may also include a detectable label that can be an antibody such as an antibody that functions as a detection antibody or can be conjugated thereto. The detectable label can be a direct label such as, for example, an enzyme, an oligonucleotide, a nanoparticle chemiluminescent group, a fluorophore, a fluorescence quencher, a chemiluminescence quencher, or biotin. The test kit can optionally include any additional reagents necessary for the detection of the label.

[0164] The kit may also include instructions for using the kit to carry out a method for indicating the direction of treatment of a steroid-dependent disease in a subject. In some embodiments, the steroid-dependent disease is a steroid-dependent cancer, for example, prostate cancer. The instructions included in the kit can be affixed to the packaging material or enclosed as an attachment to the package. The instructions can typically be provided in writing or in print, but are not limited thereto. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROM), etc. As used herein, the term "instructions" can include the address of an Internet site that provides the instructions.

[0165] The following examples further illustrate aspects of the present disclosure, but of course should not be construed as limiting the scope thereof in any way.

Examples

[0166] In the examples, the following abbreviations are used: "DCM" means dichloromethane, "DMAP" means 4-dimethylaminopyridine, "DMF" means N,N-dimethylformamide, "DMSO" means dimethyl sulfoxide, "DTBAD" means di-tert-butyl azodicarboxylate, "EtOAc" means ethyl acetate, "HPLC" means high performance liquid chromatography, "LCMS" or "LC-MS" means liquid chromatography / mass spectrometry, "LiHMDS" means lithium bis(trimethylsilyl)amide, "KO t Bu" means potassium tert-butoxide, "MeI" means methyl iodide, "MeOH" means methanol, "[M+H] + " means the protonated mass of the free base of the compound, "mp" means melting point, "NMR" means nuclear magnetic resonance, "Pd(amphos)Cl2" means bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(II), "R t " means retention time (minutes), "THF" means tetrahydrofuran, "tR" means retention time, "Xphos palladacycle G2" means chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II).

[0167] Experiment The hydrogenation reaction was carried out using an atmospheric pressure balloon or a Parr hydrogenation shaker apparatus. Analytical thin layer chromatography (TLC) was performed on Keislegel 60 F 250 micron precoated glass plates and eluted using reagent grade solvents. The TLC plates were visualized with UV light and iodine.

[0168] Normal-phase flash silica gel-based column chromatography was performed using cartridges that can be immediately connected to the Teledyne Isco Combiflash® NextGen 300 system. The cartridges that can be immediately connected are Teledyne RediSep® Rf preparative silica columns with an average particle size of 35 - 70 microns.

[0169] High-resolution mass spectra were recorded on an Agilent 1290 Infinity II series 6230B TOF LC / MS. The detection methods were diode array (DAD) at 210, 254 nM, and positive / negative electrospray ionization (ESI), with a mass range of 25 - 20,000 m / z possible. The MS detector was set to a mass range of 100 - 1700 m / z, and nitrogen was used as the nebulizer gas. High-resolution acquisition rate mass spectra (MS mode) were acquired in electrospray mode by scanning at a rate of up to 40 spectra per second. The system maintains a mass accuracy / stability of 2 ppm with a drift of within 2 °C per hour. Data acquisition was performed using MassHunter Walkup software. Unless otherwise stated, in all methods, an Agilent InfinityLab Poroshell 120 EC-C18 column with dimensions 4.6×50 mm, 2.7 μm, Poroshell 120 EC-C18, 2.1 mm, 1.9 μm guard was used. Mobile phase A was 0.1% TFA in H2O, and mobile phase B was 0.1% TFA in CH3CN.

[0170] For the LC-MS property evaluation of the compounds of the present invention, the following method was used.

[0171] Method 1: Reverse-phase HPLC was performed at a flow rate of 0.4 mL / min, at 55 °C, using positive ESI mode. The injection volume was 1 μL. The gradient conditions used were 5% mobile phase B for 0.2 minutes, followed by a gradient of 5 - 95% mobile phase B for 2.0 minutes, then held at 95% mobile phase B for 0.45 minutes, and returned to the initial conditions at 2.65 minutes.

[0172] Method 2: Reverse-phase HPLC was performed at a flow rate of 0.4 mL / min, at 55 °C, using the positive ESI mode. The injection volume was 1 μL. The gradient conditions used were 40% mobile phase B for 0.2 min, followed by a gradient of 40 - 95% mobile phase B for 2.5 min, then held at 95% mobile phase B for 0.5 min, and returned to the initial conditions at 3.2 min.

[0173] Method 3: Reverse-phase HPLC was performed at a flow rate of 0.4 mL / min, at 55 °C, using the negative ESI mode. The injection volume was 1 μL. The gradient conditions used were 5% mobile phase B for 0.2 min, followed by a gradient of 5 - 95% mobile phase B for 2.0 min, then held at 95% mobile phase B for 0.45 min, and returned to the initial conditions at 2.65 min.

[0174] Preparative RP-HPLC purification was carried out using either a Gilson GX-271 fraction collector or a Teledyne ACCQ-Prep purification system. Both instruments used UV-based peak detection and a Phenomenex Kinetex C18 column with an acetonitrile - water (0.1% TFA) custom gradient. The compound obtained initially as the TFA salt after purification was dissolved in EtOAc and washed with saturated aqueous K2CO3 solution, or eluted via a Biotage ISOLUTE® SCX-II cartridge, loaded and washed with MeOH, and then eluted with 2N NH3 in MeOH to obtain the free base.

[0175] Chiral purification of the racemic mixture was readily achieved using a Lab Hybrid 10 - 100 supercritical CO2 chromatography system from PIC Instrument Solutions, followed by a supercritical fluid chromatography (SFC) apparatus. The chiral analytical column and semi-preparative SFC purification column were obtained from Chiral Technologies.

[0176] 11H NMR spectra were recorded on a Bruker AVANCE NEO NanoBay 400 spectrometer using a standard pulse sequence operating at 400 MHz. Acquisition and processing were performed with TopSpin4 software. Chemical shifts (δ) were reported in parts per million (ppm) from the low-field side relative to tetramethylsilane (TMS), which was used as an internal standard. Coupling constants (J values) were reported in Hz. The following abbreviations (or combinations thereof) were used to describe the splitting patterns: s, singlet; d, doublet; t, triplet; q, quartet; pent, pentuplet; m, multiplet; br, broad.

[0177] General synthetic procedure General procedure 1: [Chemical formula] A mixture of arylboronic acid pinacol ester (1 equiv), aryl halide (1 equiv), and 2 M potassium carbonate (3 equiv) in dioxane (0.25 M) was degassed for 10 min, then Xphos palladacycle G2 (5 mol%) was added. The reaction mixture was stirred at 80 °C for 1 - 16 h. After completion, the mixture was diluted with EtOAc, poured into a separatory funnel containing 1 M HCl, the organic layer was separated, dried over Na2SO4, concentrated in vacuo, and purified by ISCO flash chromatography using EtOAc in hexanes to afford the final product.

[0178] General procedure 2: [Chemical formula] To SEM-protected phenol in THF (0.1 M) was added 1 M tetra-n-butylammonium fluoride (5 equiv) in THF, and the reaction mixture was stirred at 65 °C for 1 h. The reaction mixture was quenched with saturated aqueous NH4Cl, extracted with EtOAc, the organic layer was separated, dried over Na2SO4, concentrated in vacuo, and purified by ISCO flash chromatography using EtOAc in hexanes to afford the final product.

[0179] General Procedure 3: [Chemical formula] A mixture of intermediate L, alkylboronic acid pinacol ester (2 - 5 equivalents), and 2M potassium carbonate (3 equivalents) in dioxane (0.1M) was degassed for 10 minutes, and then Pd(PPh3)4 (5 mol%) was added. The reaction mixture was stirred at 120 °C for 30 minutes in a microwave oven. After completion, the mixture was diluted with EtOAc, poured into a separatory funnel containing 1M HCl, the organic layer was separated, dried over Na2SO4, concentrated in vacuo, and purified by ISCO flash chromatography using EtOAc in hexanes to give the final product.

[0180] Preparation of Intermediates Intermediates A and B: [Chemical formula] Diisopropylamine (1.16 mL, 8.26 mmol) and n-BuLi (3.3 mL, 8.26 mmol) in 12 mL of THF were stirred at 0 °C for 40 minutes. To this solution, 2-chloro-7,8-dihydroquinolin-5(6H)-one (1.5 g, 8.26 mmol) dissolved in 12 mL of THF was added at -78 °C. The reaction mixture was stirred at the same temperature for 30 minutes, then MeI (0.51 mL, 8.26 mmol) was added and stirring was continued at room temperature for 1.5 hours. The reaction mixture was quenched with saturated aqueous NH4Cl, extracted with EtOAc, dried over Na2SO4, and concentrated in vacuo. Purification by ISCO flash chromatography (40 g, 10% EtOAc in hexanes) gave 2-chloro-6-methyl-7,8-dihydro-6H-quinolin-5-one A (250 mg, 1.2778 mmol, 15% yield) as a white solid. Purity ≥95% by LCMS (210, 254 nm), tR = 1.07 m / z = 196.05 [M+H] +and 2-chloro-6,6-dimethyl-7,8-dihydroquinolin-5-one B of off-white solid (330 mg, 1.5739 mmol, 19% yield). Purity ≥95% by LCMS (210, 254 nm), tR = 1.32 m / z = 210.06 [M+H] +

[0181] Intermediate C:

Chemical formula

[0182] To 2-(3,5-difluoro-4-hydroxy-phenyl)-6-methyl-7,8-dihydro-6H-quinolin-5-one (212 mg, 0.73 mmol) in DCM (6 mL) was added SEM-Cl (0.17 mL, 0.95 mmol) and diisopropylethylamine (0.26 mL, 1.47 mmol) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. After completion, the reaction mixture was quenched with water, extracted with DCM, the organic layer was separated, dried over Na2SO4, and concentrated in vacuo. Purification by ISCO flash chromatography (12 g, 15% EtOAc in hexanes) gave 2-[3,5-difluoro-4-(2-trimethylsilylethoxymethoxy)phenyl]-6-methyl-7,8-dihydro-6H-quinolin-5-one C (250 mg, 0.5959 mmol, 81% yield) as a viscous oily liquid. Purity ≥95% by LCMS (210, 254 nm), tR = 2.88 m / z = 420.17 [M+H] +

[0183] Intermediates D and E:

Chemical formula

[0184] To 2-chloro-5,6,7,8-tetrahydroquinolin-5-ol (40 mg, 0.22 mmol) in DMSO (1.5 mL), KOH (122.2 mg, 2.18 mmol) was followed by the addition of ethyl iodide (0.16 mL, 1.96 mmol) at room temperature, and the mixture was stirred for 1 hour. Water was added to the reaction mixture, and the mixture was extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, and concentrated. Purification by ISCO flash chromatography (12 g, 50% EtOAc in hexane) gave 2-chloro-5-ethoxy-5,6,7,8-tetrahydroquinoline E (17 mg, 0.0803 mmol, 36% yield) as a viscous colorless liquid. Purity ≥95% by LCMS (210, 254 nm), tR = 1.39 min, m / z = 212.07 [M+H] +

[0185] Intermediate F:

Chemical Structure

[0186] 2-Chloro-6,6-bis(hydroxymethyl)-7,8-dihydroquinolin-5-one (80 mg, 0.33 mmol), zinc bis(dimethyldithiocarbamate) (404.94 mg, 1.32 mmol), and triphenylphosphine (130.24 mg, 0.5 mmol) in THF (4 mL) were added with diisopropyl azodicarboxylate (0.1 mL, 0.5 mmol) dissolved in toluene (0.4 mL) in an ice-water bath. The mixture was stirred overnight at room temperature. The mixture was diluted with toluene (4 mL) and filtered. The filtrate was washed with 1 M aqueous NaOH (3 times), water, and brine, dried over Na2SO4, and concentrated in vacuo. Purification by ISCO flash chromatography (12 g, 40% EtOAc in hexane) gave 2-chlorospiro[7,8-dihydroquinoline-6,3'-oxetane]-5-one F as a white solid (66 mg, 0.2951 mmol, 89% yield). Purity ≥ 95% by LCMS (210, 254 nm), tR = 1.65, m / z = 224.04 [M+H] +

[0187] Intermediate G: [Chemical formula] To 2-chloro-7,8-dihydroquinolin-5(6H)-one (151.03 mg, 0.83 mmol) in tert-butanol (2 mL), sodium iodide (23.74 mg, 0.16 mmol) and sodium hydride (63.36 mg, 1.58 mmol) were slowly added at room temperature, followed by stirring for 20 minutes. 2-Chloroethyl(dimethyl)sulfonium, iodide (200 mg, 0.79 mmol) was added to the reaction mixture, followed by stirring at room temperature for 48 hours. The reaction was quenched with water, extracted with EtOAc, the organic layer was separated, dried over Na2SO4 and concentrated in vacuo. Purification by ISCO flash chromatography (12 g, 15% EtOAc in hexane) gave 2-chlorospiro[7,8-dihydroquinoline-6,1'-cyclopropane]-5-one G as a white solid (68 mg, 0.3275 mmol, 41% yield). Purity ≥95% by LCMS (210, 254 nm), tR = 1.07 m / z = 208.05 [M+H] +

[0188] Intermediate H: [Chemical Structure] To 2-chloro-5H,6H,7H-cyclopenta[b]pyridin-5-one (63 mg, 0.38 mmol) in THF (1.5 mL), sodium hydride (60.15 mg, 1.5 mmol) was added at 0 °C and stirred for 10 minutes. MeI (0.14 mL, 2.26 mmol) was slowly added at 0 °C and the reaction mixture was stirred at room temperature for 1 hour. The reaction was diluted with EtOAc and water, extracted with EtOAc, dried over Na2SO4 and concentrated in vacuo. Purification by ISCO flash chromatography (12 g, 10% EtOAc in hexane) gave 2-chloro-6,6-dimethyl-7H-cyclopenta[b]pyridin-5-one H as a white solid (42 mg, 0.2147 mmol, 57% yield). Purity ≥95% by LCMS (210, 254 nm), tR = 1.04 m / z = 196.05 [M+H] +

[0189] Intermediates I and J: [Chemical formula] Cycloheptane-1,3-dione (3.88 mL, 33.85 mmol) was taken up in N,N-dimethylformamide dimethylacetal (8.96 mL, 67.7 mmol), and the mixture was subsequently stirred at 100 °C for 2 h. The reaction mixture was then concentrated to give 2-(dimethylaminomethylene)cycloheptane-1,3-dione 9 (6.1 g, 33.659 mmol, crude) as a black oil. LCMS (210, 254 nm), tR = 1.18 min, m / z = 182.11 [M+H] +

[0190] Sodium hydride (1.62 g, 67.32 mmol) was slowly added to a solution of 2-cyanoacetamide (5.66 g, 67.32 mmol) in DMF (20 mL) at 0 °C. After stirring at 0 °C for 30 min, a solution of 2-(dimethylaminomethylene)cycloheptane-1,3-dione (6.1 g, 33.66 mmol) in DMF (30 mL) was added, and the mixture was subsequently stirred at room temperature for 16 h. The DMF was removed under reduced pressure. The residue was dissolved in water, washed three times with ethyl acetate, and neutralized to pH 2 - 3 with 1.0 N aqueous HCl. The dark brown precipitate was collected by filtration and dried in vacuo. The filtrate was concentrated to a minimal water volume, ice was added to afford a further precipitate, which was collected by filtration and dried in vacuo. From the combined dark brown solids, 2,5-dioxo-6,7,8,9-tetrahydro-1H-cyclohepta[b]pyridine-3-carbonitrile 10 (4.4 g, 21.76 mmol, 64% yield over 2 steps) was obtained. Purity by LCMS (210, 254 nm) ≥ 95%, tR = 1.32 min, m / z = 203.08 [M+H] +

[0191] 2,5-Dioxo-6,7,8,9-tetrahydro-1H-cyclohepta[b]pyridine-3-carbonitrile (4.4 g, 21.76 mmol) was dissolved in 35 mL of 50% H2SO4 and stirred at 130 °C for 2 h. The reaction mixture was poured into ice and the resulting black-brown precipitate was collected by filtration and dried in vacuo to give 2,5-dioxo-6,7,8,9-tetrahydro-1H-cyclohepta[b]pyridine-3-carboxylic acid 11 (3.6 g, 16.274 mmol, 74% yield). Purity by LCMS (210, 254 nm) ≥ 95%, tR = 1.29 min, m / z = 222.05 [M+H] +

[0192] Copper (1.72 g, 27.12 mmol), 2,5-dioxo-6,7,8,9-tetrahydro-1H-cyclohepta[b]pyridine-3-carboxylic acid (2 g, 9.04 mmol) and quinoline (7.5 mL, 63.29 mmol) were placed in a reaction vial. The vial was then heated at 235 °C for 2 h. The reaction mixture was then diluted with chloroform, washed three times with 1 N HCl (100 mL) to remove most of the quinoline, dried over Na2SO4 and concentrated in vacuo to give a brown solid of 6,7,8,9-tetrahydro-1H-cyclohepta[b]pyridine-2,5-dione 12 (900 mg, 5.079 mmol, crude). LCMS (210, 254 nm), tR = 1.22 min, m / z = 178.04 [M+H] +

[0193] A solution of 6,7,8,9-tetrahydro-1H-cyclohepta[b]pyridine-2,5-dione (900 mg, 5.08 mmol) in acetonitrile (11 mL) was added with POCl3 (1.42 mL, 15.24 mmol) at room temperature. The reaction mixture was stirred at 100 °C for 2 h. The reaction mixture was concentrated, the flask was placed in an ice bath, aqueous ammonium hydroxide was slowly added dropwise to quench the reaction mixture, EtOAc was added, the organic layer was washed with water, dried over Na2SO4, and concentrated in vacuo. Purification by ISCO flash chromatography (4 g, 0 - 100% EtOAc in hexane) gave 2-chloro-6,7,8,9-tetrahydrocyclohepta[b]pyridin-5-one I as a yellow solid (646 mg, 3.3018 mmol, 65% yield in 2 steps). Purity ≥95% by LCMS (210, 254 nm), tR = 1.95 min, m / z = 196.00 [M+H] +

[0194] To 2-chloro-6,7,8,9-tetrahydrocyclohepta[b]pyridin-5-one (308 mg, 1.57 mmol) in THF (8 mL) was added sodium hydride (346.33 mg, 8.66 mmol) at 0 °C and stirred for 15 min. After adding MeI (0.44 mL, 7.08 mmol) at 0 °C, the reaction mixture was stirred at room temperature for 3 h. The reaction was slowly diluted with water, diluted with EtOAc, extracted with EtOAc, dried over Na2SO4, and concentrated in vacuo. Purification by ISCO flash chromatography (12 g, 20% EtOAc in hexane) gave 2-chloro-6,6-dimethyl-8,9-dihydro-7H-cyclohepta[b]pyridin-5-one J as a white solid (219 mg, 0.9790 mmol, 62% yield). Purity ≥95% by LCMS (210, 254 nm), tR = 1.54 m / z = 224.04 [M+H] +

[0195] Intermediate K:

Chemical Structure

[0196] A mixture of 3-chloro-1-oxide-5,6,7,8-tetrahydroquinoxalin-1-ium (840 mg, 4.55 mmol) and trifluoroacetic anhydride (2.53 mL, 18.2 mmol) in DCM (16 mL) was stirred at 45 °C for 48 h. The reaction mixture was concentrated, the residue was redissolved in MeOH, potassium carbonate (628.84 mg, 4.55 mmol) was added and stirred at room temperature for 15 min. The solvent was evaporated, diluted with EtOAc and water, the aqueous layer was extracted with EtOAc, dried over Na2SO4 and concentrated in vacuo. Purification by ISCO flash chromatography (40 g, 60% EtOAc in hexane) gave 2-chloro-5,6,7,8-tetrahydroquinoxalin-5-ol 15 (200 mg, 1.0833 mmol, 23% yield) as a colorless liquid. Purity ≥95% by LCMS (210, 254 nm), tR = 1.44 m / z = 167.03 [M+H-H2O] +

[0197] To 2-chloro-5,6,7,8-tetrahydroquinoxalin-5-ol (183 mg, 0.99 mmol) in DCM (10 mL) was added Dess-Martin periodinane (546.54 mg, 1.29 mmol) at room temperature, and the mixture was stirred for 1.5 h. The reaction mixture was diluted with DCM, washed with NaHCO3, the organic layer was separated, dried over Na2SO4, and concentrated in vacuo. Purification by ISCO flash chromatography (24 g, 50% EtOAc in hexanes) gave 2-chloro-7,8-dihydro-6H-quinoxalin-5-one K as a white solid (145 mg, 0.7940 mmol, 80% yield). Purity ≥95% by LCMS (210, 254 nm), tR = 1.44 min, m / z = 183.03 [M+H] +

[0198] Intermediate L:

Chemical Structure

[0199] Intermediate M:

Chem.

[0200] To a solution of 5-bromo-3,4-difluoro-2-hydroxy-benzaldehyde (1.7 g, 7.17 mmol) in methanol (15 mL) was added NaBH4 (1.82 g, 8.61 mmol) very slowly at 0 °C over 30 min. The reaction mixture was warmed to room temperature and stirred for 2 h. MeOH was removed in part and the reaction mixture was diluted with EtOAc. After acidifying the mixture with 1N HCl, the organic layer was separated, washed with brine, dried over Na2SO4 and concentrated in vacuo. Purification by ISCO flash chromatography (80 g, 60% EtOAc in hexanes) gave 4-bromo-2,3-difluoro-6-(hydroxymethyl)phenol 19 (1.10 g, 4.4768 mmol, 60% yield over 2 steps) as a pink solid. Ionization of the product was not observed by LCMS. 11H NMR (400 MHz, MeOD) δ 7.37 - 7.30 (m, 1H), 4.62 (s, 2H). 19 19F NMR (376 MHz, MeOD) δ -134.58 (d, J = 20.1 Hz), -160.40 (d, J = 20.1 Hz).

[0201] To a solution of 4-bromo-2,3-difluoro-6-(hydroxymethyl)phenol (1.10 g, 4.84 mmol) and triethylsilane (3.87 mL, 24.21 mmol) in DCM (20 mL) was added BF3.OEt2 (2.57 mL, 9.68 mmol) at 0 °C. After stirring at 0 °C for 10 minutes, the reaction mixture was warmed to room temperature and stirred for 20 hours. The reaction mixture was poured into ice water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. Purification by ISCO flash chromatography (40 g, 15% EtOAc in hexane) gave 4-bromo-2,3-difluoro-6-methyl-phenol 20 as a white solid (690 mg, 3.094 mmol, 63% yield). Ionization of the product was not observed by LCMS. 1 1H NMR (400 MHz, MeOD) δ 7.09 (d, J = 7.2 Hz, 1H), 2.17 (s, 3H). 19 19F NMR (376 MHz, MeOD) δ -136.42 (d, J = 20.2 Hz, 1F), -160.51 (d, J = 20.2 Hz, 1F).

[0202] 4-Bromo-2,3-difluoro-6-methyl-phenol (250 mg, 1.12 mmol), bis(pinacolato)diboron (569.35 mg, 2.24 mmol), Pd(dppf)Cl2.DCM (91.32 mg, 0.11 mmol) and KOAc (275.07 mg, 2.8 mmol) were combined in dioxane (8 mL) in a sealed tube, degassed under Ar for 10 minutes and then heated at 85 °C for 16 h. The reaction was diluted with EtOAc, washed with water, the organic layer was dried over Na2SO4 and concentrated in vacuo. Purification by ISCO flash chromatography (24 g, 15% EtOAc in hexanes) gave 2,3-difluoro-6-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol M as a yellow solid (415 mg, 0.7683 mmol, 50% purity). 1 H NMR (400 MHz, MeOD) δ 7.14 (d, J = 6.0 Hz, 1H), 2.16 (s, 3H), 1.33 (s, 12H). 19 F NMR (376 MHz, MeOD) δ -133.76 (dd, J = 20.3, 2.4 Hz, 1F), -166.00 (dd, J = 20.3, 2.4 Hz, 1F).

[0203] Intermediate N:

Chemical Structure

[0204] Intermediate O:

Chem.

[0205] Intermediate P:

Chem.

[0206] Intermediate Q:

Chem.

[0207] Intermediate R:

Chemical Structure

[0208] Intermediates S and T:

Chemical formula

[0209] To intermediate S (85 mg, 0.38 mmol) in DCM (1.2 mL) at 0 °C were added 2,6-di-tert-butylpyridine (0.13 mL, 0.56 mmol) and methyl trifluoromethanesulfonate (0.06 mL, 0.49 mmol). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was quenched with water, the product was extracted with DCM, the organic layer was washed with saturated aqueous NaHCO3, dried over Na2SO4, and concentrated in vacuo. Purification by ISCO flash chromatography (12 g, 30% EtOAc in hexanes) gave 2-chloro-6-(methoxymethyl)-6-methyl-7,8-dihydroquinolin-5-one T (5 mg, 0.0209 mmol, 5% yield) as a viscous colorless liquid. Purity ≥95% by LCMS (210, 254 nm), tR = 2.12 min, m / z = 240.30 [M+H] +

[0210] Intermediates U and V:

Chemical formula

[0211] To a solution of 6-bromo-2-chloro-6-methyl-7,8-dihydroquinolin-5-one (110 mg, 0.4 mmol) in THF (4 mL) and water (4 mL), NaOH (24.04 mg, 0.6 mmol) was added at 0 °C and the mixture was stirred at the same temperature for 10 minutes. The reaction was quenched with saturated aqueous NH4Cl and extracted with EtOAc. The organic layer was dried over Na2SO4 and concentrated in vacuo. Purification by ISCO flash chromatography (12 g, 45% EtOAc in hexanes) gave 2-chloro-6-hydroxy-6-methyl-7,8-dihydroquinolin-5-one U as an off-white solid (79 mg, 0.3733 mmol, 93% yield). Purity ≥ 95% by LCMS (210, 254 nm), tR = 0.65 min, m / z = 212.04 [M+H] +

[0212] To 2-chloro-6-hydroxy-6-methyl-7,8-dihydroquinolin-5-one (74 mg, 0.35 mmol) in DMSO (3.5 mL), potassium hydroxide (98.08 mg, 1.75 mmol) was added followed by methyl iodide (0.09 mL, 1.4 mmol) at room temperature. The reaction was stirred at the same temperature for 1 h. Water was added to the reaction mixture and the mixture was extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. Purification by ISCO flash chromatography (12 g, 20% EtOAc in hexanes) gave 2-chloro-6-methoxy-6-methyl-7,8-dihydroquinolin-5-one V as a viscous colorless liquid (44 mg, 0.1950 mmol, 55% yield). Purity ≥ 85% by LCMS (210, 254 nm), tR = 1.34 min, m / z = 226.24 [M+H] +

[0213] Intermediate W:

Chemical Structure

[0214] Intermediate X:

Chemical Structure

[0215] Intermediate Y:

Chemical Structure

[0216] Intermediate Z:

Chemical Structure

[0217] To 2-[3,5-difluoro-4-(2-trimethylsilylethoxymethoxy)phenyl]-6-(hydroxymethyl)-6-methyl-7,8-dihydroquinolin-5-one (40 mg, 0.09 mmol) in DCM (2 mL), Dess-Martin periodinane (56.61 mg, 0.13 mmol) was added at 0 °C and stirred at room temperature for 1 h. The reaction was quenched with saturated aqueous NaHCO3, extracted with DCM, the organic layer was separated, dried over Na2SO4 and concentrated in vacuo to give aldehyde 26. To the aldehyde (44 mg, 0.1 mmol) in DCE (1.5 mL), dimethylamine hydrochloride (72.15 mg, 0.88 mmol) and acetic acid (0.01 mL, 0.1 mmol) were added at room temperature. After stirring for 5 min, sodium triacetoxyborohydride (58.34 mg, 0.28 mmol) was added and stirring continued for 24 h. Saturated aqueous NaHCO3 was added to the reaction mixture, extracted with DCM, the organic layer was dried over Na2SO4 and concentrated in vacuo to give viscous liquid 2-[3,5-difluoro-4-(2-trimethylsilylethoxymethoxy)phenyl]-6-[(dimethylamino)methyl]-6-methyl-7,8-dihydroquinolin-5-one Z (40 mg, crude, used directly in the next step). LCMS (210, 254 nm); tR = 1.67, m / z = 477.24 [M+H] +

[0218] Intermediate AA: [Chemical Formula] A mixture of diisopropylamine (0.02 mL, 0.16 mmol) and n-BuLi (0.07 mL, 0.16 mmol) in 1 mL of THF was stirred at 0 °C for 40 minutes. To this solution was added 2-[3,5-difluoro-4-(2-trimethylsilylethoxymethoxy)phenyl]-6-methyl-7,8-dihydro-6H-quinolin-5-one (57 mg, 0.14 mmol) dissolved in 1 mL of THF at -78 °C, and the mixture was stirred for 50 minutes. Subsequently, chloro(triethyl)silane (0.03 mL, 0.16 mmol) in 0.2 mL of THF was added at the same temperature. The reaction mixture was stirred at -78 °C for 30 minutes and then at room temperature for 3 hours. The reaction mixture was quenched with saturated aqueous NaHCO3 at 0 °C, extracted with EtOAc, the organic layer was separated, dried over Na2SO4, and concentrated in vacuo. Purification by ISCO flash chromatography (4 g, 0 - 100% EtOAc in hexane containing 0.1% Et3N) gave the viscous liquid 2-[[2,6-difluoro-4-(6-methyl-5-triethylsilyloxy-7,8-dihydroquinolin-2-yl)phenoxy]methoxy]ethyl-trimethyl-silane 27 (50 mg, 0.0937 mmol, 68% yield). 1 H NMR (400 MHz, MeOD) δ 7.72 - 7.65 (m, 4H), 5.25 (s, 2H), 3.91 (t, J = 8.4 Hz, 2H), 2.97 (t, J = 8.0 Hz, 2H), 2.41 (t, J = 8.0 Hz, 2H), 1.88 (s, 3H), 1.01 (t, J = 8.0 Hz, 9H), 0.93 (t, J = 8.4 Hz, 2H), 0.75 (q, J = 8.0 Hz, 6H), 0.02 (s, 9H). 19 F NMR (376 MHz, MeOD) δ -129.46.

[0219] Under nitrogen, to CuSCN (2.79 mg, 0.02 mmol) and 1-trifluoromethyl-1,2-benziodoxol-3-(1H)-one (78.32 mg, 0.25 mmol), 2-[[2,6-difluoro-4-(6-methyl-5-triethylsilyloxy-7,8-dihydroquinolin-2-yl)phenoxy]methoxy]ethyl-trimethyl-silane (49 mg, 0.09 mmol) dissolved in DMF (1.2 mL) was added. The reaction mixture was stirred at 50 °C for 16 h. The reaction mixture was quenched with saturated aqueous NH4Cl, extracted with EtOAc, the organic layer was separated, dried over Na2SO4, and concentrated in vacuo. Purification by ISCO flash chromatography (12 g, 10% EtOAc in hexanes) gave the off-white solid 2-[3,5-difluoro-4-(2-trimethylsilylethoxymethoxy)phenyl]-6-methyl-6-(trifluoromethyl)-7,8-dihydroquinolin-5-one AA (12 mg, 0.0246 mmol, 26% yield). Purity ≥95% by LCMS (210, 254 nm), tR = 3.32 m / z = 488.26 [M+H] +

[0220] Intermediate AB:

Chemical Structure

[0221] Intermediate AC:

Chem.

[0222] (2E)-2-(Dimethylaminomethylene)-4,4-dimethyl-cyclohexane-1,3-dione (2.7 g, 13.83 mmol) in methanol (12 mL) was added with methylcarbamimidate sulfate (4.76 g, 27.66 mmol) and potassium carbonate (3.82 g, 27.66 mmol), and refluxed for 3 h. Water and EtOAc were added, the organic layer was separated, the aqueous layer was extracted twice with EtOAc, the combined organic layers were dried over Na2SO4 and concentrated in vacuo. Purification by ISCO flash chromatography (40 g, 0 - 10% MeOH in DCM) gave 2-methoxy-6,6-dimethyl-7,8-dihydroquinazolin-5(6H)-one 31 as a yellow solid (222 mg, 1.08 mmol, 8% yield). Purity ≥ 95% by LCMS (210, 254 nm), tR = 1.58 m / z = 207.10 [M+H] +

[0223] Potassium trimethylsilanolate (1.06 g, 8.24 mmol) was added to 2-methoxy-6,6-dimethyl-7,8-dihydroquinazolin-5-one (170 mg, 0.82 mmol) in dioxane (4.5 mL) at room temperature, and the mixture was stirred at 140 °C for 30 min. The reaction mixture was concentrated, diluted with saturated aqueous NH4Cl and 10% MeOH / DCM, and the aqueous layer was extracted multiple times with 10% MeOH / DCM. The combined organic layers were dried over Na2SO4 and concentrated in vacuo. Purification by ISCO flash chromatography (12 g, 7% MeOH in DCM) gave 2-hydroxy-6,6-dimethyl-7,8-dihydroquinazolin-5-one 32 as an off-white solid (93 mg, 0.4838 mmol, 58% yield). Purity ≥ 95% by LCMS (210, 254 nm), tR = 1.23, m / z = 193.09 [M+H] +

[0224] A mixture of 6,6-dimethyl-7,8-dihydro-1H-quinazoline-2,5-dione (93 mg, 0.48 mmol) and POCl3 (3 mL) in a vial was stirred at 100 °C for 1 minute. The reaction mixture was concentrated, ice water was added, the aqueous layer was extracted with DCM, the organic layer was separated, dried over Na2SO4, and concentrated in vacuo. Purification by ISCO flash chromatography (12 g, 20% EtOAc in hexane) gave 2-chloro-6,6-dimethyl-7,8-dihydroquinazolin-5-one AC (45 mg, 0.2136 mmol, 44% yield) as a white solid. Purity ≥95% by LCMS (210, 254 nm), tR = 0.91 min, m / z = 211.06 [M+H] +

[0225] Intermediate AD-1-2: [Chemical Structure] To 7-bromo-2,3-dihydro-1,8-naphthyridin-4(1H)-one (163 mg, 0.72 mmol) in THF (2 mL), Boc anhydride (0.38 mL, 3.95 mmol) and DMAP (58.47 mg, 0.29 mmol) were added at room temperature and stirred at 45 °C for 16 hours. The reaction was diluted with EtOAc and water, extracted with EtOAc, dried over Na2SO4, and concentrated in vacuo. Purification by ISCO flash chromatography (12 g, 20% EtOAc in hexane) gave tert-butyl 7-bromo-4-oxo-2,3-dihydro-1,8-naphthyridine-1-carboxylate 34 (62 mg, 0.1895 mmol, 26% yield) as a white solid. Purity ≥95% by LCMS (210, 254 nm), tR = 1.62 m / z = 270.97 [M+H-Boc] +

[0226] A mixture of diisopropylamine (0.05 mL, 0.35 mmol) and n-BuLi (0.14 mL, 0.35 mmol) in 1.5 mL of THF was stirred at 0 °C for 40 minutes. To this solution was added tert-butyl 7-bromo-4-oxo-2,3-dihydro-1,8-naphthyridine-1-carboxylate (58 mg, 0.18 mmol) dissolved in 1 mL of THF at -78 °C, and the mixture was stirred for 30 minutes, followed by the addition of MeI (0.02 mL, 0.35 mmol). The reaction mixture was stirred at -78 °C for 15 minutes and then at room temperature for 3.5 hours. The reaction mixture was quenched with saturated aqueous NH4Cl, extracted with EtOAc, dried over Na2SO4, and concentrated in vacuo. Purification by ISCO flash chromatography (4 g, 10% EtOAc in hexanes) gave tert-butyl 7-bromo-3,3-dimethyl-4-oxo-2H-1,8-naphthyridine-1-carboxylate AD-1 as a white solid (15 mg, 0.0422 mmol, 23% yield). Purity ≥ 95% by LCMS (210, 254 nm), tR = 2.12 m / z = 299.00 [M+H - Boc] +

[0227] tert-Butyl 7-bromo-3,3-dimethyl-4-oxo-2H-1,8-naphthyridine-1-carboxylate (50 mg, 0.140 mmol) was taken up in 1:4 TFA and DCM (1 mL) and stirred at room temperature for 30 minutes. The solvent was evaporated to give a solid, which was subjected to hexane washing. The solid was redissolved in THF (1.5 mL) and cooled to 0 °C. To this mixture, sodium hydride (5 mg, 0.211 mmol) was added and stirred at the same temperature for 15 minutes, followed by the addition of methyl iodide (13 μL, 0.211 mmol). The reaction was stirred at room temperature for 16 hours. Ice water was added, the aqueous layer was extracted with EtOAc, the organic layer was separated, dried over Na2SO4 and concentrated in vacuo. Purification by ISCO flash chromatography (12 g, 15% EtOAc in hexane) gave the white solid 7-bromo-1,3,3-trimethyl-2H-1,8-naphthyridin-4-one AD-2 (21 mg, 0.078 mmol, 55% yield over 2 steps). Purity ≥95% by LCMS (210, 254 nm), tR = 1.73 m / z = 269.03 [M+H] +

[0228] Intermediate AE:

Chemical Structure

[0229] Intermediate AF:

Chemical Structure

[0230] Intermediate AG:

Chemical Structure

[0231] Intermediate AH-1-2:

Chemical Structure

[0232] To a stirred solution of 4-benzyl-4-methylcyclohexane-1,3-dione (1.5 g, 6.944 mmol) in toluene (25 mL), ammonium acetate (2.13 g, 27.77 mmol) was added at room temperature and the mixture was stirred for 5 minutes. The resulting reaction mixture was heated at 140 °C for 16 hours. The reaction mixture was concentrated directly in vacuo. Purification by ISCO flash chromatography (40 g, 70% EtOAc in hexanes) gave 3-amino-6-benzyl-6-methylcyclohex-2-en-1-one 38 as a yellow solid (1.20 g, 5.57 mmol, 81% yield). MS (ESI) m / z [M+H] + : (C 14 H 17 NO calculated for: 215.13); found: 216.21 [M+H] +

[0233] To a stirred solution of 3-amino-6-benzyl-6-methylcyclohex-2-en-1-one (0.800 g, 3.720 mmol) in ethanol (4 mL), ethyl propionate (6.0 mL, 13.07 mmol) was added at room temperature. The reaction mixture was heated at 140 °C and stirred for 16 hours. After completion of the reaction time, the reaction mixture was cooled to room temperature. To the resulting reaction mixture, ethanol (4 mL) was added followed by sodium ethoxide (6 mL, 18.60 mmol) at room temperature. The reaction mixture was heated at 80 °C for 5 hours. The reaction mixture was concentrated directly in vacuo. Purification by ISCO flash chromatography (24 g, 80% EtOAc in hexanes) gave 6-benzyl-6-methyl-7,8-dihydroquinoline-2,5(1H,6H)-dione 39 as a yellow solid (0.250 g, 0.935 mmol, 25% yield). MS (ESI) m / z [M+H] + : (C 17 H 17 NO2 calculated for: 267.33); found: 268.32 [M+H] +

[0234] To 6-benzyl-6-methyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (250 mg, 0.936 mmol), POCl3 (2.5 mL) was added at room temperature. The reaction mixture was heated at 100 °C for 2 hours. The reaction mixture was concentrated in vacuo, and the resulting residue was dissolved in water and quenched with saturated aqueous NaHCO3. The aqueous layer was extracted with EtOAc, and the organic layer was dried over Na2SO4 and concentrated in vacuo. Purification by ISCO flash chromatography (12 g, 70% EtOAc in hexane) gave off-white solid 6-benzyl-2-chloro-6-methyl-7,8-dihydroquinolin-5(6H)-one AH (170 mg, 0.595 mmol, 64% yield) as a racemic mixture. Achiral HPLC: Peak 1: HPLC = 49.63%, Rt = 6.856 min; Peak 2: HPLC = 50.36%, Rt = 7.671 min

[0235] AH (90 mg) of the resulting racemic mixture was subjected to chiral preparative HPLC to give colorless oil AH-1 (Peak 1, 24.12 mg): MS (ESI) m / z [M+H] + : (C 17 H 16 ClNO calculated: 285.1); found: 286.1 [M+H] + , HPLC: 100%, Rt = 6.857 min, and AH-2 (Peak 2, 21.39 mg): MS (ESI) m / z [M+H] + : (C 17 H 16 ClNO calculated: 285.1); found: 286.1 [M+H] + , HPLC: 99.55%, Rt = 7.672 min, were obtained.

[0236] Chiral preparative HPLC method: Column: Chiral Pak-IG (30 mm X 250 mm, 5 μ), Mobile phase A: 0.1% DEA in n-hexane, Mobile phase B: DCM:MeOH (1:1), Mobile phase A: B - 98:02, Flow rate: 36 mL / min, Loading: 16 mg / Inj / 20 min

[0237] Intermediate AI-1-2:

Chem.

[0238] To a stirred solution of 4-ethyl-4-methylcyclohexane-1,3-dione (5.80 g, 37.66 mmol) in toluene (80 mL) was added ammonium acetate (11.5 g, 150.6 mmol) at room temperature and the mixture was stirred for 5 min. The resulting reaction mixture was heated at 140 °C for 16 h. The reaction mixture was concentrated directly in vacuo. Purification by ISCO flash chromatography (80 g, 70% EtOAc in hexanes) gave 3-amino-6-ethyl-6-methylcyclohex-2-en-1-one 42 (4.30 g, 28.06 mmol, 75% yield) as a yellow solid. MS (ESI) m / z [M+H] + : (Calculated for C9H 15 NO: 153.23); Found: 154.21 [M+H] +

[0239] To a stirred solution of 3-amino-6-ethyl-6-methylcyclohex-2-en-1-one (2.0 g, 13.07 mmol) in xylene (10 mL), ethyl propionate (6.60 mL, 65.35 mmol) was added at room temperature. The reaction mixture was heated at 140 °C and stirred for 16 h. After completion of the reaction time, the reaction mixture was cooled to room temperature. To the resulting reaction mixture, xylene (10 mL) was added followed by sodium ethoxide (21.0 mL, 65.35 mmol) at room temperature. The reaction mixture was heated at 100 °C for 5 h. The reaction mixture was concentrated directly in vacuo. Purification by ISCO flash chromatography (40 g, 70% EtOAc in hexane) gave 6-ethyl-6-methyl-7,8-dihydroquinoline-2,5(3H,6H)-dione 43 as a yellow solid (1.10 g, 5.36 mmol, 41% yield). MS (ESI) m / z [M+H] + : (C 12 H 15 Calculated for (C +

[0240] To 6-ethyl-6-methyl-7,8-dihydroquinoline-2,5(3H,6H)-dione (500 mg, 2.439 mmol), POCl3 (5 mL) was added at room temperature. The reaction mixture was heated at 100 °C for 2 h. The reaction mixture was concentrated in vacuo and the resulting residue was dissolved in water and quenched with saturated aqueous NaHCO3. The aqueous layer was extracted with EtOAc, the organic layer was dried over Na2SO4 and concentrated in vacuo. Purification by ISCO flash chromatography (12 g, 50% EtOAc in hexane) gave 2-chloro-6-ethyl-6-methyl-7,8-dihydroquinolin-5(6H)-one AI as a viscous yellow oil (350 mg, 1.56 mmol, 64% yield).

[0241] The resulting racemic mixture of 2-chloro-6-ethyl-6-methyl-7,8-dihydroquinolin-5(6H)-one (350 mg) was subjected to chiral preparative HPLC to give AI-1 as a colorless oil (peak 1, 120.83 mg): MS (ESI) m / z [M+H] + : (C12 H 14 Calculated value for ClNO: 223.70); Measured value: 224.1 [M+H] + , HPLC: 99.74%, Rt = 8.778 minutes, and AI-2 (peak 2, 169.85 mg): MS (ESI) m / z [M+H] + : (C 12 H 14 Calculated value for ClNO: 223.70); Measured value: 224.1 [M+H] + , HPLC: 98.79%, Rt = 8.515 minutes, were obtained.

[0242] Chiral preparative HPLC method: Column: ChiralPak-IG (30mmX250mm, 5μ), Mobile phase A: 0.1% DEA in n-hexane, Mobile phase B: DCM:MeOH (80:20), Mobile phase A:B - 98:02, Flow rate: 36 mL / min, Loading: 12 mg / Inj / 20 min

[0243] Intermediate AJ:

Chemical Structure

[0244] To 2-chloro-5-isopropyl-7,8-dihydro-6H-quinolin-5-ol (91 mg, 0.4032 mmol) in DMSO (2 mL), KOH (210 mg, 3.23 mmol) was added, followed by iodomethane (0.2 mL, 3.23 mmol) at room temperature, and the mixture was stirred for 1 hour. Water was added to the reaction mixture, and it was extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, and concentrated in vacuo. Purification by ISCO flash chromatography (12 g, 30% EtOAc in hexane) gave 2-chloro-5-isopropyl-5-methoxy-7,8-dihydro-6H-quinoline AK as a white solid (80 mg, 0.333 mmol, 82% yield). Purity ≥95% by LCMS (210, 254 nm), tR = 1.74 m / z = 240.04 [M+H] +

[0245] Intermediate AL:

Chemical Structure

[0246] Example 1: Synthesis of the Compound Compound 1: 2-(3,5-Difluoro-4-hydroxyphenyl)-7,8-dihydroquinolin-5(6H)-one

Chem.

[0247] Compound 2 - 2-(3-Fluoro-4-hydroxyphenyl)-7,8-dihydroquinolin-5(6H)-one

Chem.

[0248] Compound 3 - 2-hydroxy-5-(5-oxo-5,6,7,8-tetrahydroquinolin-2-yl)benzonitrile

Chem.

[0249] Compound 4 - 5-(6,6-dimethyl-5-oxo-5,6,7,8-tetrahydroquinolin-2-yl)-2-hydroxybenzonitrile

Chem.

[0250] Compound 5 - 2-(3,5-Difluoro-4-hydroxyphenyl)-6,6-dimethyl-7,8-dihydroquinolin-5(6H)-one

Chem.

[0251] Compound 6 - 2-(3-Fluoro-4-hydroxyphenyl)-6,6-dimethyl-7,8-dihydroquinolin-5(6H)-one

Chem.

[0252] Compound 7 - 2-(3,5-difluoro-4-hydroxyphenyl)-6-methyl-7,8-dihydroquinolin-5(6H)-one

Chemical Structure

[0253] Compound 8 - 2-(3,5-Difluoro-4-hydroxyphenyl)-6-ethyl-7,8-dihydroquinolin-5(6H)-one

Chem.

[0254] Compound 9 - 6-Benzyl-2-(3,5-difluoro-4-hydroxyphenyl)-7,8-dihydroquinolin-5(6H)-one [Chemical formula] The title compound (91 mg, 0.25 mmol, yield 45%) was prepared according to the procedure described in Example 1, using 6-benzyl-2-chloro-7,8-dihydroquinolin-5(6H)-one obtained after the synthesis of Intermediate A with benzyl bromide in the Suzuki reaction step instead. Purity ≥ 95% by LCMS (210, 254 nm), tR = 1.86 m / z = 366.26 [M+H] + 。 1 H NMR (400 MHz, MeOD) δ 8.30 (d, J = 8.3 Hz, 1H), 7.82 (d, J = 8.3 Hz, 1H), 7.77 - 7.67 (m, 2H), 7.33 - 7.28 (m, 4H), 7.22 (dp, J = 8.7, 4.4, 3.8 Hz, 1H), 3.62 (dd, J = 13.6, 5.2 Hz, 1H), 3.46 - 3.42 (m, 1H), 2.94 (dd, J = 13.6, 9.6 Hz, 1H), 2.82 (ddd, J = 17.3, 9.0, 4.5 Hz, 1H), 2.58 (ddd, J = 17.3, 8.3, 4.7 Hz, 1H), 2.18 (ddt, J = 13.8, 9.1, 4.4 Hz, 1H), 1.93 (p, J = 7.7, 7.1 Hz, 1H). 19 F NMR (376 MHz, MeOD) δ -135.23.

[0255] Compound 10 - 2-(3,5-Difluoro-4-hydroxyphenyl)-6-phenyl-7,8-dihydroquinolin-5(6H)-one [Chemical formula] The title compound (23 mg, 0.065 mmol, 26% yield) was prepared according to the procedure described in Example 1, using 2-chloro-6-phenyl-7,8-dihydroquinolin-5(6H)-one obtained after the synthesis of Intermediate AE instead in the Suzuki reaction step. Purity ≥95% by LCMS (210, 254 nm), tR = 1.62 m / z = 352.11 [M+H] +。1 H NMR (400 MHz, MeOD) δ 8.34 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.77 (dd, J = 8.1, 2.0 Hz, 2H), 7.36 - 7.33 (m, 2H), 7.30 - 7.17 (m, 3H), 3.96 (dd, J = 11.5, 4.9 Hz, 1H), 3.39 - 3.24 (m, 2H), 2.48 (dtd, J = 18.1, 9.8, 8.7, 4.6 Hz, 2H). 19 F NMR (376 MHz, MeOD) δ -135.19.

[0256] Additional compounds The compounds shown in Table 1 were synthesized by the same or similar methods as above. The synthetic examples shown in Table 1 refer to the compounds specified above and the corresponding synthetic methods described therein. The required starting materials were commercially available (CA), those described above, those described in the literature, or those easily synthesized by those skilled in organic synthesis. The mass spectrometry data were obtained using the above TOF LC-MS methods 1 - 3. LC-MS [M+H] means the protonated mass of the free base of the compound.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

[0257] Example 2: 3β-HSD1 Inhibition Assay Using a colorimetric NADH detection assay, it was demonstrated that the compound enzymatically inhibits human 3β-HSD1. Enzyme turnover was determined experimentally by NADH production by purified recombinant human 3β-HSD1 (expressed in Sf9 insect cells) in the presence of the steroid substrate trans-dehydroandrosterone (DHEA) placed slightly above the experimentally determined Km and saturating amounts of the cofactor nicotinamide adenine dinucleotide (NAD + ). The sequence of the recombinant human 3β-HSD1 used was MHHHHHHENLYFQGTGWSCLVTGAGGFLGQRIIRLLVKEKELKEIRVLDKAFGPELREEFSKLQNKTKLTVLEGDILDEPFLKRACQDVSVIIHTACIIDVFGVTHRESIMNVNVKGTQLLLEACVQASVPVFIYTSSIEVAGPNSYKEIIQNGHEEEPLENTWPAPYPHSKKLAEKAVLAANGWNLKNGGTLYTCALRPMYIYGEGSRFLSASINEALNNNGILSSVGKFSTVNPVYVGNVAWAHILALRALQDPKKAPSIRGQFYYISDDTPHQSYDNLNYTLSKEFGLRLDSRWSFPLSLMYWIGFLLEIVSFLLRPIYTYRPPFNRHIVTLSNSVFTFSYKKAQRDLAYKPLYSWEEAKQKTVEWVGSLVDRHKETLKSKTQ (SEQ ID NO: 3). DHEA and NAD+ were enzymatically converted to androstenedione and NADH, respectively. The final NADH concentration was spectrophotometrically quantified using a commercially available Amplite™ colorimetric NADH assay kit utilizing a NADH probe at an absorbance wavelength of 460 nm. Enzyme assays were performed in 384-well Greiner Bio-One UV-STAR® assay plates with a final volume of 25 μL in 50 mM Tris pH 8.0 assay buffer. The IC 50Values were experimentally determined in duplicate using a 10-point concentration response curve (CRC) at a maximum concentration of 50 μM and a minimum concentration of 25 pM. A 10 mM compound stock in DMSO was serially diluted 5-fold in DMSO to generate an ECHO® compatible source plate. Then, 125 nL of DMSO, trilostane, or serially diluted compound was acoustically transferred to an empty UV-STAR® assay plate to obtain a final assay concentration of 0.5% v / v DMSO. The stamp volume and dilution scheme can be adjusted to correspond to the potency of the compound. This assay can tolerate a final assay concentration of up to 5% v / v DMSO. 15 μL of 1 μg of purified 3β-HSD1 and 50 μM of DHEA (final assay volume concentration) were dispensed into the stamped plate per well, capped, centrifuged at 1000 RPM for 1 minute, and incubated at room temperature for 15 minutes. The enzyme reaction was initiated upon addition of 10 μL of NAD + diluted in assay buffer to obtain a final assay volume concentration of 2 mM. The assay plate was capped, centrifuged at 1000 RPM for 1 minute, and incubated at room temperature for 1 hour. 25 μL of Amplite™ Colorimetric NADH Assay Kit detection solution (prepared as recommended) was added to all reaction wells, the plate was capped, centrifuged at 1000 RPM for 1 minute, and incubated for 15 minutes. The final absorbance value (λ = 460 nm) was spectrophotometrically measured using a BioTek® Cytation5 or Synergy4 plate reader. The background signal was determined by averaging 32 completely inhibited reactions (trilostane-treated) and subtracting across the plate. The uninhibited enzyme signal (DMSO-treated) was averaged from 32 control reactions from the value minus the background. The percent inhibition value was calculated by Inhibition % = (1 - ((Inhibited reaction minus background) / (Uninhibited average minus background))) × 100. The percent inhibition obtained was then plotted in duplicate against the corresponding concentration values. The CRC obtained was a 4-parameter variable slope logarithm (inhibitor) vs. response equation where Y = minimum 値 +(maximum 値 - minimum 値 ) / (1 + 10^((logIC50 Apply to (-X)*Hillslope) to determine the IC value of the tested compound. 50 value. [Table 2-1] [Table 2-2]

[0258] Example 3. Cell viability of human tumor cell lines C4-2 cells were routinely maintained in RPMI + 2 mM L-glutamine + 1X antibiotic-antimycotic (Anti-Anti) + 10% FBS. C4-2 cells were harvested by trypsinization in 0.25% trypsin-EDTA for 90 seconds, counted using a Vi-Cell Blu cell counter (Beckman Coulter), and diluted to a density of 10,000 cells / mL in complete survival medium (RPMI + 2 mM L-glutamine + 1X antibiotic-antimycotic + 1% FBS + 500 mM DHEA). A 12-point, two-fold dilution series of test compounds (starting from 10 mM) was prepared in 100% DMSO, and then 450 nL of the dilution series was stamped in triplicate into a 384-well white opaque-bottom TC-treated CulturePlate™ (Perkin Elmer 6007680) using an acoustic dispenser (Labcyte Echo 550). Diluted C4-2 cells (500 cells / well) at 50 μL / well were added directly to the plates containing the dispensed compounds to obtain a final [DMSO] of 0.9% and a final top [compound] of 90 μM. The plates were centrifuged at 500 × g for 1 minute and then incubated at 37 °C, 5% CO2 for 6 days. The cell density at the end of the assay was measured using 35 μL / well of CellTiter-Glo® (Promega) according to the manufacturer's recommendations, and the total luminescence was measured with a Cytation 5 plate reader. The raw luminescence values were normalized from 0 (empty survival medium + 0.9% DMSO, no cells) to 100 (cells + 0.9% DMSO, maximum growth), the triplicate values were averaged, a dose-response curve was fitted, and the GI50 values were extracted using variable slope non-linear regression in GraphPad Prism. The Z'-values for all test plates exceeded 0.5, and the positive control compound D4-abiraterone was reproducible (GI 50 = 2.4 ± 0.4 μM, n = 7), and at least two biological replicates were completed for the primary compounds.

Table 3

[0259] Example 4. Steroid Metabolism in Cell Lines Cells were seeded at 150,000 cells / well in a 12-well plate in RPMI-1640 medium containing 10% FBS and incubated at 37°C for about 24 hours. Then, the medium was replaced with a mixture of serum-free RPMI-1640 and radioactive ( 3 H] labeled) and non-radioactive androgen (final concentration, 25 nM, approximately 600,000 cpm / well, PerkinElmer, Waltham, MA), and a specific concentration of inhibitor compound was added. Aliquots of the medium were collected at 24 hours and 48 hours, then extracted with ethyl acetate:isooctane (1:1) and concentrated under nitrogen gas.

[0260] High-performance liquid chromatography (HPLC) analysis was performed on a Waters 1525 HPLC system (Waters Corp., Milford, MA). The dried samples were reconstituted in 50% methanol and injected into the HPLC. The steroids were separated on a Luna 150×4.6 mm, 3.0 μm particle size C18 reverse-phase column (Phenomenex, Torrance, CA) at 50°C using a gradient of methanol / water. The column effluent was mixed with Liquiscint scintillation cocktail (National Diagnostics, Atlanta, GA) and analyzed using a β-RAM model 4 in-line radioactivity detector (LabLogic, Brandon, FL).

Table 4

[0261] Example 5. Activity of Compounds Using the CRPC C4-2 Flank Tumor Model in Mice The mouse studies were conducted under a protocol approved by the Institutional Animal Care and Use Committee of the Cleveland Clinic Lerner Research Institute. All NOD scid gamma (NSG) male mice (6 - 8 weeks old) were purchased from the Jackson Laboratory. 10 7C4-2 cells were subcutaneously injected into mice with Matrigel (total injection volume 100 μL). After the tumors reached 100 mm 3 (length × width × height × 0.52), the mice were surgically orchidectomized and implanted with a 5 mg 90-day sustained-release DHEA pellet (Innovative Research of America) to mimic human adrenal DHEA production in CRPC patients, and then the mice were arbitrarily (but not strictly randomized) assigned to vehicle (n = 6), compound 1 (n = 6), or compound 5 (n = 6). The mice were force-fed vehicle, compound 1 (100 mg / kg in water containing 20% Captisol), or compound 5 (100 mg / kg in water containing 20% Captisol) twice daily. The data are shown in Figure 1, showing a decrease in tumor volume for both test compounds compared to the vehicle control.

[0262] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference in their entirety to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0263] In the context of the present invention (particularly in the context of the following claims), the use of the terms "a", "an", and "the" and the use of the term "at least one" and the like to refer to an object shall, unless otherwise indicated herein or unless clearly contradicted by the context, be construed to cover both the singular and the plural. The use of the term "at least one" followed by a listing of one or more items (e.g., "at least one of A and B") shall, unless otherwise indicated herein or unless clearly contradicted by the context, be construed to mean either one of the items selected from the listed items (A or B) or any combination of two or more of the listed items (A and B). The terms "comprising", "having", "including", and "containing" shall, unless otherwise stated, be construed as open-ended terms (i.e., meaning "including but not limited to"). The recitation of a range of values herein is intended, unless otherwise indicated herein, merely as a shorthand way of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually recited herein. All methods described herein may be performed in any suitable order, unless otherwise indicated herein or unless clearly contradicted by the context. The use of any example or exemplary language (e.g., "such as") presented herein is merely intended to clarify the invention better and is not intended to limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0264] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that such variations will be adopted by those skilled in the art as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is included in the invention unless otherwise indicated herein or clearly contradicted by context.

Claims

1. Compound of formula (I): 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, in the formula, R 1 is halo, cyano, or C 1 -C 4 It is a haloalkyl, Z is CR 4 Selected from N, R 2 、 R 3 and R 4 are each independently selected from hydrogen, halo, and C 1 -C 4 -alkyl, Q 1 is CH or N, Q 2 CR 5 or N, R 5 is hydrogen, C 1 -C 4 Alkyl, halo, and C 1 -C 4 Selected from haloalkyl groups, --- indicates the presence or absence of a bond, and in the expression, if --- indicates the presence of a bond, then X is an oxo and R 6 If it does not exist, and --- represents the absence of a connection, then X is - OR a and -NR b R c Selected from and R 6 is hydrogen, C 1 -C 6 Alkyl, C 2 -C 6 Alkenil, C 2 -C 6 Alkinyl, C 1 -C 6 Haloalkyl, C 3 -C 6 Cycloalkyl, aryl, heteroaryl, and aryl-C 1 -C 4 - Selected from alkyl groups, Y is -CH 2 -, -NR d -, -O-, -S-, -CH 2 CH 2 -, -NHCH 2 -, -OCH 2 -, -SCH 2 - Selected from the combinations, R 7 , R 8 , R 9 , and R 10 These are, independently, hydrogen and C 1 -C 6 Alkyl, C 2 -C 6 Alkenil, C 2 -C 6 Alkinyl, C 1 -C 6 Alkoxy, hydroxy, cyano, halo, C 3 -C 6 Cycloalkyl, monocyclic 3-6 membered heterocyclyl, aryl, heteroaryl, C having one heteroatom selected from O and N 1 -C 4 -Alkoxy-C 1 -C 6 -Alkyl, hydroxy-C 1 -C 6 -Alkyl, Halo-C 1 -C 6 -Alkyl, carboxy-C 1 -C 6 -Alkyl, amino-C 1 -C 6 - Alkyl, C 3 -C 6 -Cycloalkyl-C 1 -C 4 -Alkyl, aryl-C 1 -C 4 -Alkyl and heteroaryl-C 1 -C 4 - Selected from alkyl groups, R 7 and R 8 These, along with the carbon atoms to which they are bonded, can optionally come together to form a 3- to 6-membered ring. R a , R b , R c , and R d These are, independently, hydrogen and C 1 -C 4 Alkyl, C 1 -C 4 -Alkoxy-C 1 -C 4 - Alkyl, heterocyclyl, and heterocyclyl-C 1 -C 4 - Selected from alkyl groups, Each cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently unsubstituted or substituted with 1, 2, or 3 substituents independently selected from C 1 -C 6 alkyl, C 1 -C 6 alkoxy, hydroxy, cyano, halo, and C 3 -C 6 cycloalkyl The compound of formula (I) or a pharmaceutically acceptable salt thereof.

2. R 1 The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein is fluoro, chloro, cyano, or trifluoromethyl.

3. R 1 The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R is fluoro or cyano.

4. R 2 and R 3 Each of these is independently selected from hydrogen, fluoro, and methyl, and is the compound according to claim 1, or a pharmaceutically acceptable salt thereof.

5. R 2 and R 3 Each of these is independently selected from hydrogen and fluoro, the compound according to claim 4, or a pharmaceutically acceptable salt thereof.

6. R 2 and R 3 The compound according to claim 5, or a pharmaceutically acceptable salt thereof, wherein each of the elements is hydrogen.

7. Z is CR 4 And R 4 The compound according to claim 1, or a pharmaceutically acceptable salt thereof, selected from hydrogen, halo, and methyl.

8. Z is CR 4 And R 4 The compound according to claim 7, or a pharmaceutically acceptable salt thereof, selected from hydrogen and a halo.

9. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein Z is N.

10. Q 1 The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein is CH.

11. Q 1 The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein is N.

12. Q 2 CR 5 And R 5 is hydrogen and C 1 -C 4 - A compound according to claim 1, selected from alkyl groups, or a pharmaceutically acceptable salt thereof.

13. Q 2 CR 5 And R 5 The compound according to claim 12, or a pharmaceutically acceptable salt thereof, wherein is hydrogen.

14. Q 2 The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein is N.

15. --- indicates the presence of a bond, X is an oxo, and R 6 The compound described in claim 1, or a pharmaceutically acceptable salt thereof, does not exist.

16. --- indicates the absence of a connection, and X is -OR a and -NR b R c Selected from, R 6 is hydrogen, C 1 -C 3 Alkyl, C 3 -C 4 Monocyclic five-membered or six-membered heteroaryls having one or two heteroatoms independently selected from cycloalkyl, phenyl, N, O, and S, and phenyl-C 1 -C 2 - Selected from alkyl groups, R a is hydrogen, C 1 -C 3 Alkyl, heterocyclyl, and heterocyclyl-C 1 -C 4 - Selected from alkyl groups, R b and R c The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein each of the atoms is hydrogen, and the heterocyclyl is a monocyclic 4- to 6-membered heterocyclyl having one heteroatom selected from O and N.

17. --- indicates the absence of a bond, and X represents -OH and -NH 2 Selected from, R 6 is hydrogen, C 1 -C 3 Alkyl, C 3 -C 4 Cycloalkyl, phenyl, pyridyl, oxazolyl, and phenyl-C 1 -C 2 - A compound according to claim 16, selected from alkyl groups, or a pharmaceutically acceptable salt thereof.

18. Y is -CH 2 -, -NR d -, -O-, -CH 2 CH 2 -, and selected from the combination, R d is hydrogen, C 1 -C 4 Alkyl and C 1 -C 4 -Alkoxy-C 1 -C 4 - A compound according to claim 1, selected from alkyl groups, or a pharmaceutically acceptable salt thereof.

19. Y is -CH 2 -, -NH-, -CH 2 CH 2 -, and a compound selected from the combinations, or a pharmaceutically acceptable salt thereof, according to claim 18.

20. R 7 and R 8 These are, independently, hydrogen and C 1 -C 3 Alkyl, hydroxy, cyano, halo, C 3 -C 6 Cycloalkyl, monocyclic 3-6 membered heterocyclyl, aryl, C having one heteroatom selected from O and N 1 -C 2 -Alkoxy-C 1 -C 3 -Alkyl, hydroxy-C 1 -C 3 -Alkyl, Halo-C 1 -C 3 -Alkyl, carboxy-C 1 -C 3 -Alkyl, amino-C 1 -C 3 - Alkyl, C 3 -C 4 -Cycloalkyl-C 1 -C 2 -Alkyl, aryl-C 1 -C 2 -Alkyl and heteroaryl-C 1 -C 2 - A compound according to claim 1, or a pharmaceutically acceptable salt thereof, selected from alkyl groups, wherein the cycloalkyl and heterocyclyl groups are each independently unsubstituted or substituted with one substituent selected from hydroxy, methyl, halo, and cyano.

21. R 7 and R 8 These are, independently, hydrogen and C 1 -C 3 Alkyl, hydroxy, cyano, halo, C 3 -C 6 Cycloalkyl, aryl, halo-C 1 -C 3 -Alkyl, carboxy-C 1 -C 3 - Alkyl, C 3 -C 4 -Cycloalkyl-C 1 -C 2 -Alkyl, aryl-C 1 -C 2 -Alkyl and heteroaryl-C 1 -C 2 - A compound according to claim 20, selected from alkyl groups, or a pharmaceutically acceptable salt thereof.

22. R 7 and R 8 The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the carbon atoms to which they are bonded together form a 3-6 membered cycloalkyl or a 3-6 membered monocyclic heterocycline having one or two oxygen atoms.

23. R 7 and R 8 The compound according to claim 22, or a pharmaceutically acceptable salt thereof, wherein the carbon atoms to which they are bonded together form a three-membered or four-membered cycloalkyl group.

24. R 9 and R 10 Each of them is independently of hydrogen or C 1 -C 4 The compound according to claim 1, or a pharmaceutically acceptable salt thereof, which is alkyl.

25. R 9 and R 10 The compound according to claim 24, or a pharmaceutically acceptable salt thereof, wherein each is independently hydrogen or methyl.

26. A compound according to claim 1, selected from the group consisting of the following: 【Chemistry 2-1】 【Chemistry 2-2】 [Chemistry 2-3] [Chemistry 2-4] 【Chemistry 2-5】 【Chemistry 2-6】 【Chemistry 2-7】 or a pharmaceutically acceptable salt thereof.

27. A pharmaceutical composition comprising a compound according to any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

28. A pharmaceutical composition for treating cancer, comprising a compound according to any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof.

29. The pharmaceutical composition according to claim 28, wherein the cancer is prostate cancer, breast cancer, or endometrial cancer.

30. The pharmaceutical composition according to claim 28, wherein the cancer is castration-resistant prostate cancer.

31. An in vitro method for inhibiting cancer cell proliferation, comprising contacting cancer cells in vitro with a compound according to any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, in an amount effective for inhibiting cancer cell proliferation.

32. The method according to claim 31, wherein the cancer cells are prostate cancer cells, breast cancer cells, or endometrial cancer cells.

33. An in vitro or ex vivo method for inhibiting the activity of 3β-hydroxysteroid dehydrogenase in a sample, comprising contacting the sample with a compound according to any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, in an amount effective for inhibiting the activity of 3β-hydroxysteroid dehydrogenase.

34. A pharmaceutical composition for treating cancer in a subject determined to have a cytosine nucleotide at position 1245 of the HSD3B1 gene or a threonine at position 367 of the 3βHSD1 protein, comprising a compound according to any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof.

35. The pharmaceutical composition according to claim 34, wherein the cancer is prostate cancer, breast cancer, or endometrial cancer.

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