Androgen receptor regulation by small molecule enantiomers

Abstract

To provide a new generation anti-androgen drug.SOLUTION: Provided is a compound of Formula I, or a salt thereof [G1 is NHRA, G2 is H or OH, RA is -C(=O)(C1-C6)alkyl, RA is substituted with one or more substituents selected from alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, amino, alkylamino, nitro, trifluoromethoxy, carboxy, carboxyalkyl, and cyano, R1 is H, halo, or -(C1-C6)alkyl, and R3 is CF3 or halo].SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 992,668, filed March 20, 2020, which is incorporated herein by reference. [Background technology]

[0002] Small molecules targeting the androgen receptor (AR) are a mainstay of treatment for lethal castration-resistant prostate cancer (CRPC), yet existing drugs lose their efficacy during ongoing treatment. This evolution of resistance is due to heterogeneous mechanisms, including AR mutations that activate the same drug instead of inhibiting the receptor. Therefore, a detailed molecular understanding of the paradoxical phenomenon in which AR antagonists are converted into agonists by structural mutations in the target receptor is of paramount importance.

[0003] Enzalutamide is the only antiandrogen drug approved by the US Food and Drug Administration (FDA) for metastatic castration-resistant prostate cancer (CRPC), and novel agents are therefore urgently needed. Androgen receptor (AR) activation involves a series of events, including binding to the AR-ligand binding domain (AR-LBD) in the cytoplasm, nuclear translocation, and transactivation "hyperspeckles," and its interaction with androgen response elements (AREs) regulates gene expression. These individual steps can be measured by fluorescence polarization, confocal microscopy, and ARE-luciferase assays.

[0004] Agonist binding induces a conformational change in the AR that allows helix 12 (H12) to close the binding pocket, resulting in activation. Establishing a predictive AR model for antagonists is hindered by the lack of structural information on the AR bound to the antagonist in the open conformation. The AR antagonist BMS-641988 has a chiral center and an endosubstituent at C-5 [(R)-BMS]. Its unknown (S)-enantiomer [(S)-BMS] was hypothesized to also be an antagonist. If a new drug substance contains predominantly one enantiomer, its partner is excluded from the FDA's eligibility and identification thresholds.

[0005] Prostate cancer is the leading cause of cancer death in men worldwide. All stages of prostate cancer have been shown to be androgen receptor dependent. Second-generation antiandrogens, such as enzalutamide, ultimately fail to prevent progression to terminal disease due to drug resistance and are often associated with neurotoxicity. Consequently, there is an urgent clinical need for novel antiandrogens with improved safety profiles that can overcome drug resistance. Summary of the Invention

[0006] In a medicinal chemistry campaign to develop a new generation of antiandrogen drugs, we sought to address these issues using the known AR antagonist, BMS-641988, as a starting point and scaffold. Two potential lead compounds, EITM-1702 and EITM-1707, were identified that inhibited the growth of LNCaP prostate cancer cells expressing the T877A mutant AR. Computational modeling demonstrated that EITM-1702 and EITM-1707 had reduced probability of crossing the blood-brain barrier (logBB<-1). Importantly, BMS-501949, a neurotoxic metabolite of BMS-641988, was not detected in a long-term (8-hour) human liver microsome assay. Therefore, EITM-1702 and EITM-1707 are promising compounds for further preclinical development.

[0007] Thus, the present disclosure provides compounds of formula I: [ka] The salt provide G 1 is NHR A or OH, G 2 is H or OH, R A is —C(═O)heteroaryl, —C(═O)(C1-C6)alkyl, —S(═O)2(C1-C6)alkyl, or —C(═O)heterocycloalkyl, and R A is substituted or unsubstituted; R 1 is H, halo, or -(C1-C6)alkyl; R 3 is CF3 or halo.

[0008] The present disclosure also provides a method of treating cancer in a subject in need thereof by administering to the subject an effective amount of a compound disclosed above, thereby treating the cancer.

[0009] Additionally, the present disclosure provides a pharmaceutical composition comprising the above-disclosed compound and a pharmaceutically acceptable diluent or carrier.

[0010] The present invention provides novel compounds of formula I, IA, or IB, intermediates for synthesizing compounds of the formula, and methods for preparing compounds of the formula. The present invention also provides compounds of the formula that are useful as intermediates for synthesizing other useful compounds. The present invention provides the use of compounds of the formula for the manufacture of a medicament useful for treating cancer in mammals, such as humans.

[0011] The present invention provides the use of the compositions described herein for use in medical therapy.The medical therapy can be treating cancer, for example, breast cancer, lung cancer, pancreatic cancer, prostate cancer, or colon cancer.The present invention also provides the use of the compositions described herein for the manufacture of a medicament for treating mammalian diseases, for example, human cancer.The medicament can include a pharmaceutically acceptable diluent, excipient, or carrier.

[0012] The following drawings form part of the present specification and are included to further demonstrate certain embodiments or various aspects of the present invention. In some cases, embodiments of the present invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may emphasize certain specific examples or aspects of the invention. However, one skilled in the art will understand that some of the examples or aspects may be used in combination with other examples or aspects of the invention. [Brief explanation of the drawings]

[0013] [Figure 1] BMS-641988 is an AR agonist in LNCaP cells expressing AR T878A. A) Luciferase assay in cells treated with 10 µM drug + 1 nM R1881. Signals are relative to 1 nM R1881 and are shown as posterior mean and standard deviation (Bayesian analysis) (n = 2-5 for drug treatments; n = 28 for NTC). B) Individual dose-response curves (n = 2, natural cubic spline of log-log data) used to estimate ED50 values ​​are shown. [Figure 2]Atropisomeric separation of EITM-1712. Enantiopure EITM-1712 separates into two peaks (A) by reverse-phase HPLC (40%-60% acetonitrile in water). Samples from both peaks are equilibrated after overnight incubation in solutions of acetonitrile and water (B, C). Hartree-Fock 3-21G calculations on a Spartan 14 (Wavefunction Inc.) indicate a rotational energy barrier of 58 kJ / mol for (R)-EITM-1707 and 86 kJ / mol for EITM-1712 (D). [Figure 3] LNCaP cell proliferation after 5 days of treatment. Optimal cell proliferation under 60 pM R1181 treatment was normalized to a relative signal of 100%, and the no-treatment control (NTC) was normalized to 0%. Drugs were supplemented at either 1 μM or 10 μM. [Figure 4] Metabolic Prediction. Calculated C-N bond cleavage susceptibility to CYP enzymes: The C-N bond of (R)-EITM-1702 and (R)-EITM-1707 is significantly less susceptible to cleavage by CYP enzymes, as indicated by the score difference (Δ score) between the C-N bond of each molecule and the most sensitive bond. [Figure 5] Neurotoxic potential of lead compounds. A) Calculated logBB of representative compounds. B) Extracted ion chromatograms of metabolites from liver microsome incubations. BMS-641988 is shown as the BMS standard in the third panel, and BMS-501949 is shown as the BMS toxicant in the bottom panel. Intrinsic clearance rates were established by measuring levels of intact compound after increasing incubation times using mass spectrometry. [Figure 6]Enantiomeric agonist / antagonist duality. (A) Confocal microscopy of PC3 GFP-AR cells treated with 10 μM purified enantiomer (180 min) and 1 nM R1881 (90 min). Representative cells are shown. (B) Nuclear speckle quantification of EITM drug pairs. Data (n = 3, encompassing 11,188 cells) are mean ± SD (broad) and SE (narrow), linear model (two-tailed) significance test for difference from control. (C) ARE-luciferase assay in cells treated with 10 μM drug + 1 nM R1881. Data (n ≥ 4) are mean ± SD, linear model (two-tailed), (EITM-drug + R1881) vs. R1881, and (-R1881) vs. NTC. (D) Expression of 82 AR-regulated genes in VCaP cells treated with EITM-1707 enantiomers via qPCR array. Gene expression was projected along the expression change vector between NTC (0%) and DHT (100%). Data (n = 3) are means ± SD, ANOVA with post-hoc correction, versus NTC (left) and DHT (right). (E) Proposed model of AR antagonist / agonist duality. Superposition of (S)-EITM-1703 docked to the AR-LBD in the closed conformation and (R)-EITM-1703 in the open AR homology model. (F) ARE luciferase assay in cells expressing GFP-AR with point mutations relative to the predicted binding site and treated with 10 μM drug + 1 nM R1881. Data (n ≥ 3) are means ± SD (wide) and SE (narrow). Linear model significance test of the difference (relative to NTC-R1881) between WT AR and the corresponding mutations (corrected for multiple comparisons). NTC = untreated control, ENZ = enzalutamide, DHT = dihydrotestosterone, (R)-BMS = BMS-641988 and its (S)-isomer (S)-BMS. *P<0.05, **P<0.01, ***P<0.001, ns not significant. P values ​​were corrected for multiple comparisons with a family-wise error rate of 0.05. [Figure 7]The role of enantiomeric duality in in vitro drug testing. (A and B) Assays in cells treated with 10 μM drug and increasing ratios of (S)-enantiomer + 1 nM R1881. (A) ARE-luciferase was measured after 24 hours, and (B) VCaP cell viability was measured after 6 days of treatment using CellTiter-Glo. Data (n = 3) are means ± SD, one-tailed Wilcoxon test for reduction from R1881 across both drugs. The dashed line represents the relative EC50 of EITM-1702. The 0% data point was not used for curve fitting. (C and D) Pairwise EC50 values ​​calculated from (C) competitive binding curves obtained via fluorescence polarization and (D) ARE-luciferase dose-response curves in antagonist mode with (R)-drug (1 nM R1881) and agonist mode with the respective (S)-isomer (-R1881). Data (n≧3) are mean±SD, two-tailed Welch t-test, **P<0.01, ***P<0.001, ns not significant. NTC = no treatment control, ENZ = enzalutamide. DETAILED DESCRIPTION OF THE INVENTION

[0014] In this study of four BMS-641988 derivatives prepared as two enantiomeric pairs, we discovered that the (R)-enantiomers [(R)-EITM-1702 and (R)-EITM-1707], as well as (R)-BMS itself, are all AR antagonists. Unexpectedly, the corresponding (S)-enantiomers and (S)-BMS proved to be potent AR agonists.

[0015] definition The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the listed terms have the following meanings. All other terms and phrases used herein have the ordinary meanings that would be understood by one of ordinary skill in the art. Such ordinary meanings are defined in Hawley's Condensed Chemical Dictionary 14 by R.J. Lewis, John Wiley & Sons, New York, NY, 2001. th This can be obtained by consulting a technical dictionary such as Edition.

[0016] References herein to "one embodiment," "embodiment," and the like indicate that the described embodiment may include a particular aspect, property, structure, moiety, or characteristic, but not all embodiments necessarily include that aspect, property, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referenced elsewhere in this specification. Furthermore, when a particular aspect, property, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one of ordinary skill in the art to extend or relate such aspect, property, structure, moiety, or characteristic to other embodiments, whether or not explicitly stated.

[0017] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, such as compound X. Furthermore, it should be noted that the claims may be drafted to exclude any element. Accordingly, this statement is intended to serve as a predicate for the use of exclusive terminology, such as "solely," "only," and / or the use of recitations or "negative" limitations of claim elements in connection with any element described herein.

[0018] The term "and / or" refers to any one of the items, any combination of the items, or all of the items with which the term is associated. The phrases "one or more" and "at least one" are readily understood by those skilled in the art, particularly when read in the context of their use. For example, the phrase can mean 1, 2, 3, 4, 5, 6, 10, 100, or any upper limit that is about 10-fold, 100-fold, or 1000-fold higher than the listed lower limit. For example, one or more substituents on a phenyl ring can refer to 1 to 5, or 1 to 4, for example, when the phenyl ring is disubstituted.

[0019] As will be understood by one of ordinary skill in the art, all numbers, including numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and the like, are approximate and are understood in all instances to be optionally modified by the term "about." These values ​​may vary depending upon the desired properties one of ordinary skill in the art seeks to obtain using the teachings set forth herein. It is also understood that such values ​​inherently contain variability necessarily resulting from the standard deviation found in their respective testing measurements. When values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value without the modifier "about" also forms a further aspect.

[0020] The terms "about" and "approximately" are used interchangeably. Both terms may refer to a variation of ±5%, ±10%, ±20%, or ±25% of the specified value. For example, "about 50" percent may, in some embodiments, have a variation of 45 to 55 percent, or may be otherwise defined by the particular claim. For integer ranges, the term "about" can include one or two integers greater than and / or less than the recited integers at both ends of the range. Unless otherwise indicated herein, the terms "about" and "approximately" are intended to include values, e.g., weight percentages, near the recited range that are equivalent in terms of the functionality of the individual component, composition, or embodiment. The terms "about" and "approximately" can also modify the endpoints of the recited ranges, as described above in this paragraph.

[0021] As will be understood by those skilled in the art, for any and all purposes, particularly with respect to providing a written description, all ranges recited herein encompass any and all possible subranges and combinations of subranges, as well as the individual values, particularly integers, that make up the range. Therefore, it is understood that each unit between two specific units is also disclosed. For example, if 10 to 15 is disclosed, 11, 12, 13, and 14 are also individually disclosed as part of the range. A recited range (e.g., weight percent or carbon group) includes each specific value, integer, decimal, or unit within the range. Any recited range can be readily recognized as fully descriptive and allows for division of the same range into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, upper third, etc. As will be understood by those skilled in the art, all terms such as "up to," "at least," "greater than," "less than," "more than," "greater than or equal to," and the like, are inclusive of the recited numbers, and such terms refer to ranges that can subsequently be broken down into subranges as described above. Similarly, all ratios recited herein also include all subratios that fall within the broader ratio range. Thus, specific values ​​recited for radicals, substituents, and ranges are for illustrative purposes only and do not exclude other defined values ​​or other values ​​within defined ranges of radicals and substituents. It will be further understood that each endpoint of a range is significant both in relation to the other endpoint and independently of the other endpoint.

[0022] The present disclosure provides ranges, limits, and deviations for variables such as volume, mass, percentages, and ratios. Ranges such as "number 1" to "number 2" are understood by those skilled in the art to refer to a continuous range of numbers, including integers and fractions. For example, 1 to 10 means 1, 2, 3, 4, 5, ..., 9, 10. This also means 1.0, 1.1, 1.2, 1.3, ..., 9.8, 9.9, 10.0, as well as 1.01, 1.02, 1.03, etc. When a disclosed variable is a number less than "number 10," it refers to a continuous range, as described above, including integers and fractions less than number 10. Similarly, when a disclosed variable is a number greater than "number 10," it refers to a continuous range, including integers and fractions greater than number 10. These ranges may be modified by the term "about," the meaning of which is explained above.

[0023] Those skilled in the art will readily recognize that when members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire recited group as a whole, but also each member of the group individually and all possible subgroups of the main group. Moreover, for all purposes, the invention encompasses not only the main group, but also the main group in which one or more of the group members are absent. Thus, the invention contemplates the explicit exclusion of any one or more of the recited group members. Thus, a qualification can be applied to any of the disclosed categories or embodiments, whereby any one or more of the recited elements, species, or embodiments can be excluded from such category or embodiment, for example, for use in an express negative limitation.

[0024] The term "contacting" refers to the act of touching, making contact, or being in close or proximity, including at the cellular or molecular level, e.g., in solution, in a reaction mixture, in vitro, or in vivo, to bring about, e.g., a physiological reaction, a chemical reaction, or a physical change.

[0025] An "effective amount" refers to an amount effective to treat a disease, disorder, and / or condition, or to produce a recited effect. For example, an effective amount can be an amount effective to reduce the progression or severity of the condition or symptom being treated. Determining a therapeutically effective amount is well within the capabilities of one skilled in the art. The term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, that is effective, for example, to treat or prevent a disease or disorder, or to treat a symptom of a disease or disorder, in a host. Thus, an "effective amount" generally refers to an amount that provides a desired effect.

[0026] Alternatively, the term "effective amount" or "therapeutically effective amount," as used herein, refers to a sufficient amount of an agent or composition or combination of compositions administered that relieves to some extent one or more of the symptoms of the disease or condition being treated. The result can be a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising a compound disclosed herein that is required to provide a clinically significant reduction in a disease symptom. An appropriate "effective" amount in any individual case can be determined using techniques such as a dose escalation study. Doses can be administered in one or more administrations. However, the precise determination of what will be considered an effective dose may be based on factors specific to each patient, including, but not limited to, the patient's age, size, type or extent of disease, stage of disease, route of administration of the composition, type or extent of replacement therapy used, ongoing disease process, and type of treatment desired (e.g., aggressive versus conventional treatment).

[0027] The terms "treating," "treat," and "treatment" include (i) preventing the occurrence of a disease, condition, or medical condition (e.g., prophylaxis), (ii) inhibiting or arresting the development of a disease, condition, or medical condition, (iii) alleviating a disease, condition, or medical condition, and / or (iv) reducing symptoms associated with a disease, condition, or medical condition. Thus, the terms "treat," "treatment," and "treating" can extend to prophylaxis and can include preventing, prevention, preventing, reducing, halting, or reversing the progression or severity of the condition or symptom being treated. Thus, the term "treatment" can include medical, therapeutic, and / or prophylactic administration, as appropriate.

[0028] As used herein, "subject" or "patient" refers to an individual who has or is at risk for symptoms of a disease or other malignancy. A patient may be human or non-human and may include animal strains or species used as "model systems" for research purposes, such as the mouse model described herein. Similarly, a patient may include either an adult or a juvenile (e.g., a child). Furthermore, a patient may refer to any organism, preferably a mammal (e.g., human or non-human), that can benefit from the administration of the compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the mammalian class of laboratory animals, including humans, non-human primates such as chimpanzees, and other apes and monkey species; livestock such as cows, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; and rodents, e.g., rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment of the methods provided herein, the mammal is a human.

[0029] As used herein, the terms "providing," "administering," and "introducing" are used interchangeably herein and refer to the placement of the disclosed compositions into a subject by a method or route that results in at least partial localization of the composition at the desired site. The composition can be administered by any suitable route that results in delivery to the desired location in the subject.

[0030] The compositions described herein can be administered with additional compositions to prolong the stability and activity of the composition, or in combination with other therapeutic agents.

[0031] The terms "inhibit," "inhibiting," and "inhibition" refer to slowing, stopping, or reversing the growth or progression of a disease, infection, condition, or group of cells. Inhibition can be, for example, greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99% compared to growth or progression that occurs in the absence of treatment or contact.

[0032] As used herein, the term "substantially" is a broad term and is used in its ordinary sense, including, but not limited to, a large portion, but not necessarily all, of what is specified. For example, the term may refer to a numerical value that is not 100% of the complete numerical value. The complete numerical value may be about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20% less.

[0033] The present disclosure provides methods for preparing the compounds and compositions of the present invention.Compounds and compositions can be prepared by any of the applicable techniques described herein, optionally in combination with standard techniques of organic synthesis.Many techniques, such as etherification and esterification, are well known in the art. However, many of these techniques are covered in the Compendium of Organic Synthetic Methods (John Wiley & Sons, New York), Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971, Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974, Vol. 3, Louis S. Hegedus and Leroy Wade, 1977, Vol. 4, Leroy G. Wade, Jr., 1980, Vol. 5, Leroy G. Wade, Jr., 1984, and Vol. 6, as well as March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Ed., by M.B. Smith and J. March (John Wiley & Sons, New York, 2001), Comprehensive Organic Synthesis: Selectivity, Strategy & Efficiency in Modern Organic Chemistry. In 9 Volumes, Barry These are described in detail in standard organic reference texts such as M. Trost, Editor-in-Chief (Pergamon Press, New York, 1993 printing), Advanced Organic Chemistry, Part B: Reactions and Synthesis, Second Edition, Cary and Sundberg (1983), and for heterocyclic synthesis see Hermanson, Greg T., Bioconjugate Techniques, Third Edition, Academic Press, 2013.

[0034] Formula and compound described herein can be modified using protecting group.Suitable amino protecting group and carboxy protecting group are known to those skilled in the art (for example, see Protecting Groups in Organic Synthesis, Second Edition, Greene, TW, and Wutz, PGM, John Wiley & Sons, New York and the references cited therein, Philip J. Kocienski; Protecting Groups (Georg Thieme Verlag Stuttgart, New York, 1994) and the references cited therein), and Comprehensive Organic Transformations, Larock, RC, Second Edition, John Wiley & Sons, New York (1999) and the references cited therein).

[0035] As used herein, the term "substituted" or "substituents" is intended to indicate that one or more (e.g., in various embodiments, 1 to 20; in other embodiments, 1 to 10, 1, 2, 3, 4, or 5; in some embodiments, 1, 2, or 3; and in other embodiments, 1 or 2) hydrogens of the group designated in the phrase "substituted" (or "substituents") are replaced with a group selected from the designated group or with a suitable group known to one of ordinary skill in the art, provided that the normal valence of the designated atom is not exceeded and the substitution results in a stable compound. Suitable designated groups include, for example, alkyl, alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, and cyano. Additionally, non-limiting examples of substituents that may be attached to a substituted carbon (or other) atom include F, Cl, Br, I, OR', OC(O)N(R')2, CN, CF3, OCF3, R', O, S, C(O), S(O), methylenedioxy, ethylenedioxy, N(R')2, SR', SOR', SOR', SOR', SOR', SOR', C(O)R', C(O)C(O)R', C(O)CH2C(O)R', C(S)R', C(O)OR', OC(O)R', C(O)N(R')2, OC(O)N(R')2, C(S)N(R')2, (CH2) 0-2and NHC(O)R', N(R')N(R')C(O)R', N(R')N(R')C(O)OR', N(R')N(R')CON(R'), N(R')SOR', N(R')SON(R'), N(R')C(O)OR', N(R')C(O)R', N(R')C(S)R', N(R')C(O)N(R'), N(R')C(S)N(R'), N(COR')COR', N(OR')R', C(=NH)N(R'), C(O)N(OR')R', or C(=NOR')R', where R' may be hydrogen or a carbon-based moiety, which may itself be further substituted.

[0036] The term "halo" or "halide" refers to fluoro, chloro, bromo, or iodo. Similarly, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0037] The term "alkyl" refers to a branched or unbranched hydrocarbon having, for example, 1 to 20 carbon atoms, often 1 to 12, 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms, or in the range of, for example, 1 to 20 carbon atoms, e.g., 2 to 6, 3 to 6, 2 to 8, or 3 to 8 carbon atoms. As used herein, the term "alkyl" also encompasses "cycloalkyl," as defined below.

[0038] The term "cycloalkyl" refers to cyclic alkyl groups, e.g., of 3 to 10 carbon atoms, having a single cyclic ring or multiple condensed rings. Cycloalkyl groups include, by way of example, single-ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple-ring structures such as adamantyl. Cycloalkyls can be unsubstituted or substituted.

[0039] The term "heterocycloalkyl" refers to a saturated or partially saturated monocyclic, bicyclic, or polycyclic ring containing at least one heteroatom selected from nitrogen, sulfur, or oxygen, preferably 1 to 3 heteroatoms, in at least one ring, each ring preferably having 3 to 10 members, more preferably 4 to 7 members.

[0040] The term "aryl" refers to an aromatic hydrocarbon group derived from the removal of at least one hydrogen atom from a single carbon atom of a parent aromatic ring system.

[0041] The term "heteroaryl" refers to a monocyclic, bicyclic, or tricyclic ring system containing one, two, or three aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in the aromatic ring. Heteroaryl can be unsubstituted or substituted with one or more, especially 1 to 3, substituents, e.g., as described in the definition of "substituted."

[0042] The definitions and conventions of stereochemistry used herein are generally as set forth in S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York, and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds," John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric or chiral centers and, therefore, exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, including, but not limited to, diastereomers, enantiomers, and atropisomers, and mixtures thereof, e.g., racemic mixtures, are contemplated and form part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and l or (+) and (-) are used to designate the sign of rotation of plane-polarized light by a compound; (-) or l means the compound is levorotatory. A compound pre-fixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers are also called enantiomers, and mixtures of such isomers are often called enantiomeric mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate (defined below) and can occur when there has been no stereoselection or stereospecificity in a chemical reaction or process.

[0043] The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.

[0044] As used herein, the term "enantiomerically enriched" ("ee") refers to a mixture in which one enantiomer is present in greater abundance than another. Thus, a reaction that provides one enantiomer in greater abundance than another is (or exhibits) "enantioselective."

[0045] Embodiments of the present invention This disclosure provides compounds of formula I: [ka] The salt provide G 1 is NHR A or OH, G 2 is H or OH, R A is —C(═O)heteroaryl, —C(═O)(C1-C6)alkyl, —S(═O)2(C1-C6)alkyl, or —C(═O)heterocycloalkyl, and R A is substituted or unsubstituted; R 1 is H, halo, or -(C1-C6)alkyl; R 3 is CF3 or halo.

[0046] In various embodiments, G 1 is NHR A In other embodiments, R A is pyrrolopyridine, pyrazole or indazole, and R A is unsubstituted. In some embodiments, R A is a pyrazole or triazole, and R A is substituted. In some embodiments, R A are -C(=O)CH3, -S(=O)2CH2CH3, [ka] is.

[0047] In some embodiments, the compound has formula IA: [ka] represented by its enantiomers or salts, R 1 is H, F, methyl or ethyl, R 2 is H, -C(=O)CH3, or -C(OH)CH3.

[0048] In some other embodiments, the compound has formula IB: [ka] represented by its enantiomers and / or salts, X is CH or N; R 1 is H, F, methyl or ethyl, R 4 is H, -C(=O)CH3, or -C(OH)CH3.

[0049] In other embodiments, the compound is EITM-1719 or EITM-1720: [ka]

[0050] In some other embodiments, the compound is [ka] [ka] is.

[0051] In some embodiments, the compound is the (S)-enantiomer. In some embodiments, the compound is the (R)-enantiomer. In other embodiments, the compound is dextrorotatory. In other embodiments, the compound is levorotatory.

[0052] In various embodiments, the compound is an androgen receptor antagonist. In various embodiments, the compound is an androgen receptor agonist. In various other embodiments, the compound is an androgen receptor full agonist. In some embodiments, the agonist is an enantiomer of the antagonist.

[0053] The compound formulas disclosed herein and / or the methods disclosed herein exclude the compound BMS-641988. [ka] For example, G 2 and R 1 For Formula I where is H, R 3 is CF3 and G 1 is NHR A and R A is -S(=O)2(C1-C6)alkyl or -S(=O)2CH2CH3.

[0054] Additionally, the present disclosure provides a compound that is the (S)-enantiomer of BMS-641988, the compound being [ka] [ka] may be.

[0055] Additionally, the present disclosure provides a method for treating cancer in a subject in need thereof by administering to the subject an effective amount of a compound disclosed herein, thereby treating the cancer.

[0056] In some embodiments, the cancer is prostate cancer or breast cancer. In some other embodiments, the cancer is prostate cancer, and the prostate cancer is fatal castration-resistant prostate cancer. In some embodiments, the effective serum concentration of the compound is about 1 nM to about 2000 nM.

[0057] In other embodiments, the effective serum concentration of the compound is about 1 nM, about 10 nM, about 50 nM, about 100 nM, about 250 nM, about 500 nM, about 750 nM, about 1000 nM, about 1500 nM, about 2000 nM, about 2500 nM, about 3000 nM, about 3500 nM, about 4000 nM, about 4500 nM, about 5000 nM, about 7500 nM, about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, or any serum concentration between any two of the listed serum concentrations.

[0058] Additionally, the present disclosure provides a method for treating an endocrine or hormonal disorder in a subject in need thereof by administering to the subject an effective amount of a compound disclosed herein, thereby treating the endocrine disorder.

[0059] The present disclosure also provides for the use of a compound or composition disclosed herein for the treatment of one or more cancer, endocrine, hormonal, or another disorder in a subject in need of treatment by administering an effective amount of a compound disclosed herein to a subject having the disorder, thereby treating the disorder.

[0060] In various embodiments, administration of an effective amount of the compound is by infusion, injection, oral administration, or a combination thereof.

[0061] The present disclosure also provides pharmaceutical compositions comprising the compounds disclosed herein and a pharmaceutically acceptable diluent or carrier.

[0062] Results and Discussion Prostate cancer is the second deadliest cancer in men, resulting in an estimated 29,000 deaths in 2019 in the United States alone. All stages of the disease depend on androgen receptor (AR) pathway signaling. In castration-resistant prostate cancer (CRPC), patients generally initially respond to antiandrogens such as enzalutamide and apalutamide, but progression to end-stage disease is inevitable. Several resistance mechanisms have been identified, including somatic mutations in the AR ligand-binding domain (AR-LBD), which can reduce ligand specificity and even convert AR antagonists into agonists. Enzalutamide is also associated with neurotoxicity, with fatigue and falls frequently reported, and seizures requiring treatment interruption in up to 1% of patients. There is an urgent clinical need for novel antiandrogen drugs with improved safety and reduced susceptibility to resistance.

[0063] AR agonists, including testosterone, bind to the AR-LBD and induce a conformational change that allows helix 12 (H12) to "cap" the binding pocket, a perk of AR activation. In contrast, when AR antagonists bind to the AR-LBD, H12 is prevented from closing. Molecular dynamics simulations suggest that conformational modification of AR antagonists may improve therapeutic efficacy by more completely blocking H12 capping. This also suggests a strategy for alleviating antiandrogen resistance caused by acquired mutations that enlarge the pocket.

[0064] Search for androgen receptor antagonists with an oxabicyclic core. The AR antagonist BMS-641988 possesses a bulky oxabicyclic succinimide core, distinguishing it from current clinical antiandrogens. This core structure confers greater pharmacophore rigidity than bicalutamide, enzalutamide, and darolutamide, which were recently approved for non-metastatic CRPC (ODM-201). BMS-641988 was a promising next-generation anti-AR lead compound that potently antagonized AR in vitro but paradoxically promoted LNCaP proliferation. This drug failed in phase I clinical trials due to poor tumor response and toxicity.

[0065] In an exploratory medicinal chemistry campaign to develop new antiandrogen drugs for CRPC, we designed and synthesized a series of nine novel AR antagonists (EITM-1702-EITM-1712) using the BMS-641988 scaffold as a starting point. Several of these new drugs have: 1) an EC of 1320 nM; 50 These novel compounds were demonstrated to 1) antagonize AR in vitro at median concentrations (ranging from 328 to 3700 nM) with an assay coefficient of variation of approximately 25%, 2) effectively inhibit LNCaP cell proliferation, and 3) not produce a toxic metabolite (BMS-501949) that primarily contributes to the clinical failure of BMS-641988. These novel compounds significantly address the major weaknesses of BMS-641988 and therefore show promise for further preclinical studies.

[0066] Drug Design. BMS-641988 is extensively metabolized in vivo to the active metabolite BMS-501949 via oxidation by CYP 3A4 and subsequent reduction by cytosolic reductases (Scheme 1). BMS-501949 readily crosses the blood-brain barrier (BBB) ​​and promotes GABAergic metabolism. AThis inhibited the receptor, possibly inducing grade 3 seizures that led to clinical failure of the drug. To prevent the production of BMS-501949 and related toxic metabolites in our drug design, we performed in silico metabolic predictions using the Schrodinger P-450 Site of Metabolism (SOM) module. Our calculations suggested that the carboxamide was less susceptible to CYP oxidation than the sulfonamide group of BMS-641988, prompting us to synthesize a series of novel amide analogs of this scaffold.

[0067] Scheme 1. Metabolism of BMS-641988. [ka]

[0068] BMS-641988 is the C-5 (R)-stereoisomer, encompassing a previously uncharacterized (S)-stereoisomer. It is hypothesized that endosubstitution at C-5 or C-6 of the oxabicyclic ring results in direct interaction with H12, creating a classical AR antagonist conformation similar to that predicted for bicalutamide. To optimize the position and size of the substituents in the novel analogs, Schrodinger IFD docking of BMS-641988 and its previously undescribed (S)-enantiomer, (S)-BMS, was performed using the AR-LBD in complex with DHT (PDB: 1T7R). In the preferred conformation, both compounds form hydrogen bonds with R752 and N705 of the AR-LBD, the same residues involved in stable binding of the standard agonists DHT and R1881. However, our docking calculations showed that the orientation of the ethylsulfonamide substituent relative to H11 and H12 is very different for the corresponding stereoisomers. In BMS-641988, the substituents point to H11 and H12, but this is not the case for (S)-BMS. Using ICM-Pro, we calculated the active site surface and ligand-protein contacts of a model constructed using Schrodinger IFD. (S)-BMS fits snugly into the binding site and makes contacts very similar to DHT, including interactions with L704, F764, N705, T877, and W741 (Table 1). On the other hand, BMS-641988 cannot fit inside the pocket and makes stronger interactions with F876, F891, and M895, which are important for stabilizing H12 (Table 1). Compared to its (S)-enantiomer, BMS-641988 induces a significant shift of F876, confirming the destabilization of the closed conformation of H12. These docking results suggested that BMS-641988 is an antagonist, but its (S)-isomer is not. Therefore, it is essential to completely remove the potentially agonistic (S)-enantiomer before functional testing. Finally, the size of the carboxamide C-5 substituent is important for its interaction with H11 and H12 and subsequent destabilization of the closed-pocket conformation.Based on these results, we chose to use pyrazole, alkylpyrazole, 5-(thiophenyl)pyrazole, 5-(furanyl)pyrazole, and indazole as C-5 carboxamide substituents in a novel drug series. To extend the SAR, we also investigated the effect of F or Me substitution at the ortho position of the aniline ring. [Table 1]

[0069] Synthesis. Compounds were synthesized using the general route shown in Scheme 2. Diels-Alder cycloaddition of maleimide 2 to the MEM ester of 2,5-dimethyl-3-furoic acid afforded the racemates (3a and 3b), which were used in the following step without separation. Catalytic hydrogenation led to the formation of esters 4a and 4b. Treatment of 4 with 3N HCl afforded a racemic mixture of acids 5a and 5b. Sequential Curtius rearrangement of the resulting Teoc-carbamates followed by TFA-promoted cleavage afforded racemic amines 7a and 7b. Coupling of amine 7 with carboxylic acid 8 afforded the target amide pharmacophore. In the original synthesis of BMS-641988, 4a and 4b were separated using semi-preparative chiral HPLC. Only the C-5 R stereoisomer (4a) was carried forward to afford the target compound (thus, the corresponding C-5(S)-stereoisomer of BMS-641988 was unavailable). In our synthesis, we did not separate 4, but instead proceeded en route to the final target C-5 (R / S)-mixture, which was then resolved to give both enantiomers of the drug.

[0070] Aniline 1 and carboxylic acid 8 were obtained from commercial sources or synthesized by known procedures (see examples). Of note, for analogs containing a 2'-fluoro or 2'-methyl group, exposure to methanol during purification should be avoided to prevent ring opening of the pyrrolidinedione. The 3aR, 4R, 5R, 7R, 7aS enantiomers of the oxabicyclic scaffold consistently have negative specific rotations, whereas the [α] of BMS-641988 24 DTherefore, after confirming the absolute stereochemistry of BMS-641988 by X-ray crystallography, we calculated its specific rotation as [α] 24 D = -28.1° (c = 0.5, MeOH). The specific rotation of the previously undescribed (S)-stereoisomer was determined to be [α] 24 D = +26.1° (c = 0.5, MeOH), and its stereochemistry was also verified by X-ray crystallography.

[0071] Scheme 2. Synthesis of EITM compounds. [ka]

[0072] Reagents and conditions: (a) maleic anhydride, glacial acetic acid, reflux overnight, 90%; (b) 2-MEM 2,5-dimethylfuran-3-carboxylate, 125°C, 1.5 hours, then room temperature overnight, 25%; (c) H2, Pd / C, EtOAc, 1 atm, overnight, 75%; (d) 3N HCl, THF, room temperature, 16 hours, 99%; (e) 2-trimethylsilylethanol, DPPA, Et3N, 4Å MS, 1,4-dioxane, 75°C, 53%; (f) TFA, CHCl2, room temperature, 2 hours, quantitative; (g) carboxylic acid, DIPEA, HATU, DMF, room temperature, overnight; (h) chiral HPLC separation.

[0073] BMS-641988 is an agonist in LNCaP prostate cancer cells. Previous reports have identified BMS-641988 as an antagonist in LNCaP cells expressing AR T878A, a mutation frequently reported in prostate cancer patients that reduces ligand specificity. However, treatment with the drug paradoxically promoted LNCaP proliferation. We generated an LNCaP cell line stably expressing luciferase regulated by the androgen response element (ARE-luciferase). We performed luciferase experiments after 24 hours of treatment with 10 μM test drug, with and without 1 nM R1881. Unexpectedly, 10 μM BMS-641988 induced ARE luciferase without the addition of R1881 (Figure 1). At increasing doses, the drug was indeed a potent agonist of the T877A mutant AR, demonstrating ED. 50 The agonism was confirmed to be 94 ± 22 nM (mean ± SE, n = 2) compared with 0.1 nM for R1881 (Figure 1B, Table 2). This agonism may explain the limited efficacy of BMS-641988 in clinical studies.

[0074] SAR study of EITM compounds. First, the amide derivatives EITM-1702, -1703, and -1704 were tested. In contrast to BMS-6431988, all three compounds exhibited strong antagonist activity without significant agonism (Table 2). Of the three, EITM-1702 had the highest potency (570 nM) and efficacy (E 最大 8%), supporting the selective ligand design derived from our modeling studies and demonstrating that this design eliminated the partial agonism of BMS-641988 while retaining potent anti-AR properties in prostate cancer cells.

[0075] We used a fragment approach for SAR studies. First, we compared EITM-1705, -1707, and -1712 to investigate the SAR of the 2' (ortho) aniline. Similar to BMS-641988, EITM-1705 exhibited significant AR agonism in LNCaP cells in the ARE-luciferase assay (Table 2). This suggests that the C-5 substituent, rather than the 2'-aniline substituent, plays a key role in defining antagonist activity in this drug cohort. In contrast, EITM-1707 and EITM-1712 are pure antagonists, and experiments performed in agonist mode did not yield measurable EDs for these compounds. 50 The potency (940 nM) and efficacy (E 最大 13%) was comparable to EITM-1702, indicating that the 2'-fluoro substitution does not affect activity. In comparison, the methyl substitution in EITM-1712 resulted in increased potency (2133 nM) and efficacy (E ), likely due to its existence as two atropisomers, only one of which effectively inhibits the AR. 最大 This resulted in a significant loss of 38% of the metastable atropisomers. Evidence supporting the existence of these metastable atropisomers is provided by the observation that, after chiral HPLC separation, EITM-1712, unlike EITM-1707, exhibits two peaks in reversed-phase analytical HPLC (Figure 2A). Both of these species re-equilibrated after standing overnight in a solution of acetonitrile and water (Figure 2B-C).

[0076] Hartree-Fock 3-21G calculations on a Spartan 14 (Wavefunction Inc.) estimated a rotational energy barrier of 58 kJ / mol for EITM-1707 around the N-Ar bond in vacuum (Figure 2D). Because separation of atropisomers requires a rotational energy barrier of at least 93.3 kJ / mol at 300 K (a half-life of at least 1,000 seconds), this calculation confirmed the absence of stable atropisomers of EITM-1707. A similar calculation for EITM-1712 estimated a rotational barrier of 86 kJ / mol around the N-Ar bond, which would not be sufficient for separation of the corresponding atropisomers. 2-Me-N-phenylmaleimide (structurally similar to EITM-1712) has a rotational barrier of 87 kJ / mol. 1 H NMR showed that the imide rotated freely around the N-Ar single bond at room temperature. An additional rotational barrier in the case of EITM-1712 was provided by the interaction of the Me group with solvent molecules, suggesting that it forms a hydrogen bond with the oxygen in the COC bridge. To investigate the role of the oxabicyclic core, EITM-1708 was tested (Scheme 3).

[0077] Scheme 3. Synthesis of N-(2-(4-cyano-3-(trifluoromethyl)phenyl)-1,3-dioxoisoindolin-5-yl)ethanesulfonamide, EITM-1708. [ka]

[0078] Reagents and conditions: (a) acetic acid, 130-140°C, 4.5 hours; (b) H2, Pd / C, EtOAc, overnight, 39% over two steps; (c) EtSO2Cl, TEA, CH2Cl2, room temperature, overnight, 35%.

[0079] Scheme 4. Synthesis of 4-((3aR,4R,5R,7R,7aS)-5-(4-(2-hydroxyethyl)-1H-1,2,3-triazol-1-yl)-4,7-dimethyl-1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-2-(trifluoromethyl)benzonitrile, EITM-1706. [ka]

[0080] Reagents and conditions: (a) 2,5-dimethylfuran, neat, 60 °C, overnight, 75%; (b) BH3 / THF at 0 °C, 30 min; then 0.5 M Na2HPO4 / NaH2PO4 buffer to pH 7.2, 0 °C; then H2O2, 30 min, 71%; (c) Tf2O, pyridine, anhydrous DCM, 0 °C, 1 h, 61%; (d) NaN3, DMF, overnight, 79%; (e) 3-butynol, copper(II) sulfate pentahydrate and sodium ascorbate, 1:1 tBuOH:water, 40 °C, 2 days, 30%; (f) chiral HPLC separation.

[0081] EITM-1708 did not bind to AR in vitro (Table 2), demonstrating that the oxabicyclic core is essential for the binding of this family of compounds. To study the SAR at the C5 position of the oxabicyclic ring, we tested compounds EITM-1706, -1709, -1710, -1711, -1716, and -1717. First, EITM-1706 (Scheme 4) did not bind to AR, demonstrating that the C5 amide or sulfonamide is important for the structure (Table 2). Surprisingly, the AR-LBD appeared to accommodate EITM-1709, -1710, and -1711 despite their size (Table 2). On the other hand, EITM-1716 and -1717 did not bind to AR, suggesting that the flat structure of the aromatic ring is important for binding (Table 2). In summary, several EITM drugs antagonize the AR in vitro and induce EC 50 The median value was 1320 nM (range 328–3700 nM) (Table 2).

[0082] Our SAR studies imply that the dynamics of AR-ligand interaction are not fully explained by the Helix 12 model and that understanding the AR at the molecular level requires more extensive studies. [Table 2-1] [Table 2-2]

[0083] EITM-1702 and EITM-1707 inhibit LNCaP proliferation in vitro. To evaluate EITM-1702 and -1707 as potential preclinical candidates, we treated LNCaP cells with 1 μM and 10 μM of the drugs and 60 pM of R1881 for 5 days and measured cell viability using CellTiter-Glo. As expected, BMS-641988 promoted proliferation. In contrast, we measured a significant decrease in viable cells for the EITM drugs under castration conditions, which EITM-1702 nearly phenocopied (Figure 3). These data and Table 3 support the excellent efficacy of our compounds as promising leads. [Table 3]

[0084] EITM-1702 and EITM-1707 exhibit favorable safety profiles. To estimate the safety profile of our lead compounds, we used the Schrodinger QikProp module to predict BBB penetration compared with known androgen antagonists. Setting logBB < -1 as a practical threshold yielded good correlation with available in vivo data (Figure 5A). For example, flutamide, enzalutamide, and BMS-501949 were predicted to readily penetrate the BBB, whereas bicalutamide, darolutamide, BMS-641988, and our preclinical candidate all had logBB values ​​< -1, suggesting low BBB permeability. Furthermore, we calculated the metabolic susceptibility of our preclinical candidate using SMARTCyp and found that our preclinical candidate was unlikely to be metabolized to BMS-501949 (Figure 4). These calculations were confirmed by an in vitro liver microsome stability assay. Toxic metabolites that accumulated in BMS-641988 samples (BMS-501949 for EITM-1702 and o-fluoro-BMS-501949 for EITM-1707) were not detected in EITM-1702 or EITM-1707, even after 8 h of incubation (Figure 5B, Table 4). Finally, the rates of intrinsic clearance were comparable across EITM-1702 (15.3 μl / min / mg), EITM-1707 (10.3 μl / min / mg), and BMS-641988 (10.1 μl / min / mg). Collectively, these results suggest an improved safety profile for EITM-1702 and EITM-1707 compared with BMS-641988. [Table 4]

[0085] In summary, in a heuristic medicinal chemistry campaign to develop next-generation antiandrogens for lethal CRPC, we synthesized a series of compounds derived from the molecular scaffold of BMS-641988, designed to circumvent the issues of insufficient efficacy and metabolite toxicity of this drug, which entered but failed Phase I clinical trials. We confirmed that BMS-641988 is a potent agonist in LNCaP cells, whereas our designed drugs are pure antagonists of the LNCaP AR. Two of the identified preclinical candidates, EITM-1702 and -1707, have effective in vitro potencies (EDs of 570 nM and 940 nM, respectively). 50 Both computational and microsomal studies suggest that EITM-1702 and -1707 have an improved safety profile compared with existing drugs. EITM-1702 and -1707 are promising lead candidates suitable for further preclinical development as potentially more effective AR antagonists for treating CRPC.

[0086] Paradoxical androgen receptor modulation by small molecule enantiomers Antagonist / agonist duality of chiral molecules. Initial compound testing was performed using PC3 GFP-AR cells and confocal microscopy. First, we tested purified BMS enantiomers (Figure 6A). As expected, (R)-BMS was an antagonist: treatment initiated nuclear translocation but inhibited R1881-induced hyperspeckling. Unexpectedly, (S)-BMS alone induced substantial hyperspeckling comparable to R1881. To examine the significance of this result, which appears to contradict literature speculation (7, 10), we prepared a series of homologous derivatives, "EITM-compounds," (shown below). [ka]

[0087] Notably, their increased molecular size did not overcome the paradoxical AR regulation. Quantification of nuclear spots in over 10,000 cells revealed that all four (R)-enantiomers inhibited R1881-induced hyperspeckles, whereas their (S)-enantiomers induced them by themselves (Fig. 6B). To test the corresponding transcriptional activation, we performed assays in cells expressing ARE-luciferase. The (R)-enantiomers inhibited R1881-induced ARE-luciferase by 24% (18%, 34%) (95% CI) relative to the untreated R1881 control (non-treated control [NTC] + R1881, 100%) (Fig. 6C). In contrast, their (S)-enantiomers activated ARE-luciferase to 110% (87%, 130%) of the untreated control (NTC, 11%) with a 95% CI (Figure 6C). Next, we examined gene expression in VCaP cells, a hormone-responsive but independent model of CRPC that highly expresses AR. A qPCR array assaying 82 AR target genes in response to the natural hormone dihydrotestosterone (DHT) confirmed that (R)-EITM-1707 downregulated AR-dependent gene expression, mimicking androgen starvation. Its (S)-enantiomer rescued the castration phenotype and mimicked DHT (Figure 6D). These experiments demonstrate the enantiomer-dependent antagonist / agonist duality of these C-5 stereoisomers, pointing to a previously unknown mechanism of AR regulation involving a switch from antagonism to agonism.

[0088] Model of AR-enantiomeric duality. To explore the unexpected agonistic properties of the (S)-enantiomer, we performed induced-fit docking with AR-LBD (PDB: 1E3G) (11) and (S)-EITM-1703. In the resulting model (rmsd = 0.28 A), the substituents do not clash with H12 but rather promote a closed conformation, explaining the observed agonism (Figure 6E). Next, we sought to model the antagonistic activity of (R)-EITM-1703. Because no crystal structure of antagonist-bound AR in the open conformation exists, we constructed a three-dimensional (3D) homology model based on the crystal structure of the progesterone receptor (2OVM) (12) (Figure 6E). Here, ring D obstructs H12, preventing closure. Superposition of the two models in Figure 6E shows significantly different compound orientations within the pocket. In both cases, hydrogen bonds are formed with R752 and N705, the same residues that bind DHT (13) and R1881 (11). We confirmed these critical binding sites in our ARE luciferase assays in cells expressing point mutations that significantly reduce the agonist function of both R1881 and the (S)-enantiomer (Figure 6F). Our AR model provides a rationale for the paradoxical agonism of the (S)-enantiomer and emphasizes the importance of a specific, rigid spatial orientation of the ligand within the AR-LBD to ensure that antagonist function does not disrupt agonism. Future studies will systematically examine mutations within H12 that contribute to steric interactions with the ligand.

[0089] The role of enantiomeric duality in drug discovery. This novel AR duality raised the question of whether contaminating agonist enantiomers could interfere with assays commonly used to identify new antiandrogens. To measure the impact of post-resolution impurities, highly purified (R)-drugs were spiked with their (S)-isomers and cells were treated with the mixture at 10 μM. For both EITM-1702 and EITM-1707, the ARE-luciferase signal rapidly increased with increasing contamination levels (Figure 7A). For (S)-EITM-1702, contamination with 2.5% (2.1%, 2.9%) 95% CI halved the drug effect, while contamination with 9% (6.0%, 12.0%) 95% CI completely negated its antagonist effect (Figure 7A). The contamination effect was further detrimental to cell viability as measured by CellTiter-Glo. After 6 days of treatment, pure (R)-EITM-1702 and (R)-EITM-1707 clearly inhibited R1881-induced proliferation, whereas BMS-641988 did not (Figure 7B). Strikingly, only 0.3% of the (S)-enantiomer halved the drug effect of EITM-1702 (0.2%, 0.4%) 95% CI, and 2.2% rescued the proliferation phenotype (0.7%, 3.7%) 95% CI (Figure 7B). Furthermore, the agonist EC values ​​obtained by fluorescence polarization and ARE-luciferase assays were significantly higher than those obtained by BMS-641988. 50 The values ​​were consistently lower than their respective antagonist counterparts, suggesting higher affinity (Figure 7C) and potency (Figure 7D). These experiments reveal the critical importance of enantiopureity in this drug class, even during early discovery when antagonist "hits" may be overlooked or even misidentified as agonists.

[0090] Pharmaceutical preparations The compounds described herein can be used to prepare therapeutic pharmaceutical compositions, for example, by combining the compounds with pharmaceutically acceptable diluents, excipients, or carriers. The compounds can be added to carriers in the form of salts or solvates. For example, if the compounds are sufficiently basic or acidic to form stable, non-toxic acid or base salts, it may be appropriate to administer the compounds as salts. Examples of pharmaceutically acceptable salts include organic acid addition salts formed with acids that form physiologically acceptable anions, such as tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, α-ketoglutarate, and β-glycerophosphate. Suitable inorganic salts can also be formed, including hydrochloride, halide, sulfate, nitrate, bicarbonate, and carbonate.

[0091] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficiently basic compound, such as an amine, with a suitable acid to provide a physiologically acceptable ionic compound. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared by similar methods.

[0092] The compounds of the formulae described herein can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms that can be specifically adapted for a selected route of administration, for example, oral or parenteral administration by intravenous, intramuscular, topical, or subcutaneous routes.

[0093] The compounds described herein may be administered systemically in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. For oral administration, the compounds may be enclosed in hard or soft-shell gelatin capsules, compressed into tablets, or incorporated directly into the food of the patient's diet. The compounds may also be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations typically contain at least 0.1% of the active compound. The percentage of the compositions and preparations may vary and may conveniently be about 0.5% to about 60%, about 1% to about 25%, or about 2% to about 10% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions may be such that an effective dosage level will be obtained.

[0094] Tablets, troches, pills, capsules, etc. may also contain one or more of the following: binders such as gum tragacanth, acacia, cornstarch, or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as cornstarch, potato starch, or alginic acid; and lubricants such as magnesium stearate. Sweeteners such as sucrose, fructose, lactose, or aspartame, or flavorings such as peppermint, wintergreen oil, or cherry flavoring may also be added. When the unit dosage form is a capsule, in addition to the above-mentioned materials, it may contain a liquid carrier such as vegetable oil or polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, sugar, etc. Syrup or elixir may contain active compound, sucrose or fructose as sweetener, methyl and propylparaben as preservative, dye and flavoring, for example, cherry or orange flavor.Any material used to prepare any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amount used.In addition, active compound can be incorporated into sustained-release preparations and devices.

[0095] Active compound can be administered intravenously or intraperitoneally by infusion or injection.The solution of active compound or its salt can be prepared in water, optionally mixed with non-toxic surfactant.Dispersion can be prepared in glycerol, liquid polyethylene glycol, triacetin or their mixture, or in pharmaceutically acceptable oil.Under normal conditions of storage and use, preparation can contain preservative to prevent microbial growth.

[0096] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions, dispersions, or sterile powders containing the active ingredient, optionally encapsulated in liposomes, suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions. The final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. Liquid carriers or vehicles can be solvents or liquid dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and / or antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, such as sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by agents delaying absorption, for example, aluminum monostearate and / or gelatin.

[0097] Sterile injectable solutions can be prepared by incorporating the required amount of active compound into a suitable solvent with various other ingredients as listed above, as needed, and then filter sterilization is required. In the case of sterile powders for preparing sterile injectable solutions, the preparation method can include vacuum drying and freeze-drying techniques to produce powders of the active ingredient plus any additional desired ingredients present in the solution.

[0098] For topical administration, the compounds may be applied in pure form, for example, when they are liquids. However, it will generally be desirable to administer the active agent to the skin as a composition or formulation in combination with a dermatologically acceptable carrier, which may be, for example, a solid, liquid, gel, etc.

[0099] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, etc. Useful liquid carriers include water, dimethyl sulfoxide (DMSO), alcohol, glycol, or water-alcohol / glycol blends, in which the compound can be dissolved or dispersed at an effective level, optionally using a non-toxic surfactant. Adjuvants such as fragrances and additional antibacterial agents can be added to optimize the properties for a given application. The resulting liquid composition can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using a pump-type or aerosol sprayer.

[0100] Thickening agents such as synthetic polymers, fatty acids, salts and esters of fatty acids, fatty alcohols, modified cellulose, or modified mineral materials may also be used with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like for application directly to the user's skin.

[0101] Examples of dermatological compositions for delivering active agents to the skin are known in the art, see, for example, U.S. Patent Nos. 4,992,478 (Geria), 4,820,508 (Wortzman), 4,608,392 (Jacquet et al.), and 4,559,157 (Smith et al.). Such dermatological compositions can be used in combination with the compounds described herein, and components of such compositions can optionally be replaced with the compounds described herein, or compounds described herein can be added to the compositions.

[0102] The useful dosage of the compounds described herein can be determined by comparing their in vitro activity and in vivo activity in animal models.The method for extrapolating the effective dosage in mice and other animals to humans is known in the art, for example, see U.S. Patent No. 4,938,949 (Borch et al.).The amount of compound or its active salt or derivative required for therapeutic use varies not only depending on the specific compound or salt selected, but also on the route of administration, the nature of the condition being treated, and the age and condition of the patient, and is ultimately at the discretion of the attending physician or clinician.

[0103] However, in general, suitable doses will be in the range of about 0.5 to about 100 mg / kg, e.g., about 10 to about 75 mg / kg body weight / day, e.g., 3 to about 50 mg / kg body weight of the recipient / day, preferably in the range of 6 to 90 mg / kg / day, most preferably in the range of 15 to 60 mg / kg / day.

[0104] The compounds are conveniently formulated in unit dosage form, for example containing 5 to 1000 mg, conveniently 10 to 750 mg, and most conveniently 50 to 500 mg of active ingredient per unit dosage form, hi one embodiment, the invention provides a composition comprising a compound of the invention formulated in such a unit dosage form.

[0105] The compounds may conveniently be administered in unit dosage form, for example, 5 to 1000 mg / m per unit dosage form. 2 , advantageously 10 to 750 mg / m 2 , most conveniently 50-500 mg / m 2 The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, e.g., as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into several discrete loosely spaced administrations.

[0106] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, for example, into several discrete loosely spaced administrations, such as by multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

[0107] The compounds described herein are effective anti-cancer agents and may have greater potency and / or reduced toxicity compared to BMS-641988. Preferably, the compounds of the present invention are more potent and less toxic than BMS-641988 and / or avoid potential metabolic sites encountered by BMS-641988, i.e., have a metabolic profile distinct from that of BMS-641988.

[0108] The present invention provides a therapeutic method for treating cancer in a mammal, which comprises administering an effective amount of a compound or composition described herein to a mammal having cancer. Mammals include primates, humans, rodents, dogs, cats, cattle, sheep, horses, pigs, goats, cows, etc. Cancer refers to any of various types of malignant neoplasms, such as colon cancer, breast cancer, prostate cancer, melanoma, and leukemia, and is generally characterized by unwanted cell proliferation, such as uncontrolled proliferation, lack of differentiation, local tissue invasion, and metastasis.

[0109] The ability of the compounds of the invention to treat cancer can be determined by using assays well known in the art, such as those for designing treatment protocols, evaluating toxicity, analyzing data, quantifying tumor cell kill, and the biological significance of using transplantable tumor screening.

[0110] The following examples are intended to illustrate the above invention and should not be construed as narrowing its scope. Those skilled in the art will readily recognize that the examples suggest many other ways in which the invention can be practiced. It should be understood that numerous variations and modifications can be made while remaining within the scope of the invention.

[0111] example Example 1 Materials and General Methods. Unless otherwise stated, all reagents and solvents were commercially available and used as received. The progress of all reactions was monitored on precoated silica gel plates (with fluorescent indicator UV254) using ethyl acetate / hexane or dichloromethane / methanol as the solvent system. Column chromatography was performed using a Teledyne Isco Combiflash with the solvent mixtures specified in the corresponding experiments. NMR spectra were recorded at room temperature on either a Varian 400, 500, or 600. Data are reported as follows: chemical shifts ( 1 H and 13 ppm relative to the C residual solvent peak, δ), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br = broad), coupling constant (Hz), and integral. 19 F NMR spectra were recorded with proton decoupling. Optical rotations were measured using a Jasco P2000 polarimeter. Specific rotation [α] D 20 is deg cm 3 g -1 dm -1 Low-resolution mass spectrometry (LRMS) analysis was performed using an Advion Expression system. Chiral HPLC separations were performed on a Shimadzu Prominence column (Chiralcel OD-H (5 μm, 250 mm × 4.6 mm) or ProntoSIL AX QN (5 μm, 150 mm × 8.0 mm) or ProntoSIL Chiral AX QD-1 (5 μm, 150 mm × 4.0 mm), eluent hexane / isopropanol or acetonitrile) with detection at 254 nm and a column temperature of 20 °C. All final compounds had a purity of >95% as confirmed by HPLC. All final compounds were further purified by reverse-phase HPLC using a Phenomenex Luna C18 column with 50% acetonitrile in water using the same instrument.

[0112] General Synthetic Methods. Aniline 1 was purchased or prepared according to known procedures (Schemes 5, 6). Carboxylic acid 8 was purchased or prepared according to known procedures (Scheme 7). Amine intermediates (7a and 7b) and azide intermediates (15a and 15b) were prepared according to known procedures (Scheme 8).

[0113] Scheme 5. Synthesis of 4-amino-3-methyl-2-(trifluoromethyl)benzonitrile. Reagents and conditions: (a) PivCl, EtN, dry THF, 90%; (b) nBuLi, MeI, dry THF, 10%; (c) CuCN, NMP, 32%; (d) HCl, EtOH, 95%. [ka]

[0114] Scheme 6. Synthesis of 4-amino-3-methyl-2-(trifluoromethyl)benzonitrile. Reagents and conditions: (a) NBS, DMF, 86%; (b) AcO, 96%; (c) CuCN, DMF, 64%; (d) HCl, EtOH, 90%. [ka]

[0115] Scheme 7. Synthesis of 3-acetyl-1H-pyrazole-5-carboxylic acid and 3-(1-hydroxyethyl)-1H-pyrazole-5-carboxylic acid. Reagents and conditions: (a) toluene, overnight, 76%; (b) 5N NaOH, 79%; (c) NaBH4, MeOH, 0°C, 89%. [ka]

[0116] Scheme 8. Synthesis of BMS-641988 and its (S)-enantiomer 9 (S-BMS). Reagents and conditions: (a) 125 °C, 1.5 h, then room temperature overnight, 25%; (b) H2, Pd / C, EtOAc, 1 atm, overnight, 75%; (c) 3 N HCl, THF, room temperature, 16 h, 99%; (d) 2-trimethylsilylethanol, DPPA, Et3N, 4 Å MS, 1,4-dioxane, 75 °C, 53%; (e) TFA, CHCl2, room temperature, 2 h, quantitative; (f) EtSO2Cl, Et3N, CHCl2, room temperature, overnight, 50%; (g) chiral HPLC separation. [ka]

[0117] Scheme 9. Synthesis of (R)-EITM-1702, (R)-EITM-1703, (R)-EITM-1704, and their respective (S)-enantiomers. Reagents and conditions: (a) substituted carboxylic acid, HATU, DIPEA, DMF, room temperature, overnight, 56–72% yield; (b) chiral HPLC separation. [ka]

[0118] Scheme 10. Synthesis of (R)-EITM-1707 and its enantiomer (S)-EITM-1707. Reagents and conditions: (a) 125 °C, 1.5 h, then room temperature overnight, 61%; (b) H2, Pd / C, EtOAc, 1 atm, overnight, 90%; (c) 3N HCl, THF, room temperature, 16 h, 91%; (d) 2-trimethylsilylethanol, DPPA, Et3N, 4 Å MS, 1,4-dioxane, 75 °C, 79%; (e) TFA, CHCl2, room temperature, 2 h, 88%; (f) 5-(1-hydroxyethyl)-1H-pyrazole-3-carboxylic acid, HATU, DIPEA, DMF, room temperature overnight, 66%; (g) chiral HPLC separation. [ka]

[0119] Example 2. Synthesis of compounds. [ka] (2-Methoxyethoxy)methyl (3aR,4R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoindole-5-carboxylate (3a) and (2-methoxyethoxy)methyl (3aS,4S,7S,7aR)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoindole-5-carboxylate (3b). A mixture of 4-(2,5-dioxo-2,5-dihydropyrrol-1-yl)-2-trifluoromethylbenzonitrile (200 mg, 0.75 mmol) and 2,5-dimethylfuran-3-carboxylic acid 2-methoxyethoxymethyl ester (257 mg, 1.13 mmol) was heated at 125 °C for 1.5 h and then stirred at room temperature overnight. The crude product was purified by column chromatography on a silica gel column (0–30% ethyl acetate in hexane) to give compound 3a / b (93 mg, 25%) as a viscous oil and a mixture of two enantiomers, which was used in the following step without further separation. 1 H NMR(400 MHz,CDCl3)δ 7.94(d,J=8.3 Hz,1H),7.85(d,J=1.9 Hz,1H),7.75(dd,J=8.3,1.9 Hz,1H),7.12(s,1H),5.49-5.31(m,2H),3.89-3.75(m,2H),3.63-3.50(m,2H),3.37(s,2H),3.18(d,J=6.6 Hz,1H),3.09(d,J=6.6 Hz,1H),1.89(s,2H),1.79(s,2H).ESI-MS:[M+H] + Calculated value C 23 H 22 F3N2O7, 495.1; measured value 495.2. [ka]

[0120] (2-Methoxyethoxy)methyl (3aR,4R,5R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindole-5-carboxylate (4a) and (2-methoxyethoxy)methyl (3aS,4S,5S,7S,7aR)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindole-5-carboxylate (4b). A solution of 3a / b (93 mg, 0.188 mmol) in ethyl acetate (2 mL) was mixed with 10% Pd / C (10 mg) and stirred overnight at room temperature under a H atmosphere (balloon). The mixture was filtered through a Celite pad, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel flash chromatography (30% EtOAc in hexanes) to give product 4a / b (70 mg, 75%) as a white solid. 1 H NMR(400 MHz,CDCl3)δ 7.94(d,J=8.3 Hz,1H),7.87-7.81(m,1H),7.73(dd,J=8.1,1.8 Hz,1H),5.46(d,J=6.2 Hz,1H),5.34(d,J=6.1 Hz,1H),3.90-3.79(m,2H),3.57(t,J=4.6 Hz,2H),3.38(s,3H),3.33(d,J=7.2 Hz,1H),3.19(d,J=7.3 Hz,1H),3.04(dd,J=11.7,4.9 Hz,1H),2.29(dd,J=12.8,4.9 Hz,1H),2.05(t,J=12.5 Hz,2H),1.78(s,3H),1.64(s,3H).ESI-MS:[M+Na] + Calculated value C 23 H 23 F3N2NaO7,519.1; measured value 519.3. [ka]

[0121] (3aR,4R,5R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindole-5-carboxylic acid (5a) and (3aS,4S,5S,7S,7aR)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindole-5-carboxylic acid (5b). A solution of 4a (0.55 g, 1.11 mmol) in THF (4 mL) was mixed with 3N hydrochloric acid (2.8 mL) and stirred overnight at room temperature. The reaction mixture was diluted with water, and the aqueous layer was extracted with ethyl acetate. The combined organics were dried over Na2SO4, filtered and concentrated to give the product 5a / b (0.45 g, 99%) as a white foam. 1 H NMR(400 MHz,MeOD)δ 8.13(d,J=8.7 Hz,1H),7.93(d,J=2.0 Hz,1H),7.83(dd,J=8.6,2.2 Hz,1H),3.41(d,J=7.2 Hz,1H),3.25(d,J=7.2 Hz,1H),3.02(dd,J=11.7,5.1 Hz,1H),2.26(dd,J=12.7,5.1 Hz,1H),2.02(t,J=12.3 Hz,1H),1.71(s,3H),1.57(s,3H).ESI-MS:[MH] - Calculated value C 19 H 14 F3N2O5, 407.1; measured value 407.1. [ka]

[0122] 2-(Trimethylsilyl)ethyl ((3aR,4R,5R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)carbamate (6a) and 2-(trimethylsilyl)ethyl ((3aS,4S,5S,7S,7aR)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)carbamate (6b). A solution of 5a / b (228 mg, 0.558 mmol), triethylamine (0.1 mL, 0.69 mmol), and powdered 4 Å molecular sieves (228 mg) in dioxane (3 mL) was mixed with diphenylphosphoryl azide (189 mg, 0.148 mmol) and stirred at 50 °C for 1.5 h. The temperature was then increased to 75 °C, and 2-(trimethylsilyl)ethanol (337 mg, 2.85 mmol) was added. After heating at 75 °C for an additional 1.5 h, the reaction mixture was cooled, filtered, and concentrated. The residue was purified by silica gel chromatography (30% ethyl acetate in hexane) to give product 6a / b (154 mg, 53%) as a colorless oil. 1 H NMR(400 MHz,CDCl3)δ 7.93(d,J=8.4 Hz,1H),7.87-7.83(m,1H),7.74(dd,J=8.4,2.1 Hz,1H),4.17(t,J=10.1 Hz,2H),4.07-401(m,1H),3.47(brs,1H),3.13(d,J=7.2 Hz,1H),2.32(t,J=12.4 Hz,1H),1.60(s,6H),1.54(dd,J=13.2,5.0 Hz,1H),0.99(t,J=8.6 Hz),0.04(s,9H).ESI-MS:[M+Na] + Calculated value C 24 H 28 F3N3O5SiNa, 546.2; measured value 546.3. [ka]

[0123] 4-((3aR,4R,5R,7R,7aS)-5-amino-4,7-dimethyl-1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-2-(trifluoromethyl)benzonitrile (7a) and 4-((3aS,4S,5S,7S,7aR)-5-amino-4,7-dimethyl-1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-2-(trifluoromethyl)benzonitrile (7b). A solution of carbamate 6a / b (110 mg, 0.21 mmol) in CHCl (3 mL) was mixed with trifluoroacetic acid (0.55 mL) and stirred at room temperature for 2 h. After this time, the reaction was made basic by the addition of saturated aqueous sodium bicarbonate solution. The organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated to give the product 7a / b (80 mg, quantitative) as a white foam. 1 H NMR (400 MHz, CDCl3): δ 7.93(d,J=8.3 Hz,1H),7.89-7.85(m,1H),7.76(dd,J=8.3,2.0 Hz,1H),3.94(d,J=7.3 Hz,1H),3.40(dd,J=10.7,4.7 Hz,1H),3.07(dd,J=7.4,1.7 Hz,1H),2.19(dd,J=12.6,10.7 Hz,1H),1.60(s,4H),1.53(d,J=0.8 Hz,4H),1.34-1.29(m,1H).ESI-MS:[MH] - Calculated value C 18 H 15 F3N3O3, 378.1; measured value 378.0. [ka]

[0124] N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)ethanesulfonamide (8) and N-((3aS,4S,5S,7S,7aR)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)ethanesulfonamide (9). A solution of 7a / b (80 mg, 0.21 mmol) in anhydrous CHCl (3 mL) was mixed with triethylamine (0.12 mL, 0.84 mmol) and ethanesulfonyl chloride (54 mg, 0.42 mmol) at 0 °C and stirred overnight at room temperature. The reaction mixture was then diluted with CHCl, washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (30–50% ethyl acetate in hexanes) to give the racemic product 8 / 9 (50 mg, 50%) as a white solid. Further separation of the two enantiomers was achieved by chiral HPLC using a Chiralcel OD-H column (250 × 4.6 mm, 5 μm) eluted with 50% isopropanol in hexanes at 1 mL / min and 254 nm detection. Compound 8 had a retention time of 8.4 min, while its enantiomer 9 had a retention time of 12.9 min. 8: [α] 24 D = -28.1° (c = 0.5, MeOH). 1 H NMR(600 MHz,CDCl3)δ 7.93(d,J=8.3 Hz,1H),7.85(s,1H),7.74(d,J=8.4 Hz,1H),5.64(d,J=8.4 Hz,1H),3.75-3.61(m,1H),3.51(d,J=7.3 Hz,1H),3.17(d,J=7.2 Hz,1H),3.15-3.04(m,2H),2.37(t,J=12.2 Hz,1H),1.65(dd,J=13.2,4.8 Hz,1H),1.63(s,3H),1.60(s,3H),1.41(t,J=7.4 Hz,3H). 13C NMR(151 MHz, CDCl3)δ 173.95,173.31,135.79,135.37,133.76(q,J=33.5 Hz),129.57,124.44,121.79(q,J=274.5 Hz),114.78,109.57,87.14,85.59,60.49,53.34,47.84,47.55,44.97,18.28,16.35,8.32. 19 F NMR (564 MHz, CDCl3): δ-62.1.HRMS: [M+H] + Calculation value C 20 H 21 N3O5SF3,472.1154; measured value 472.1158. 9:[α] 24 D =+26.1° (c=0.5, MeOH). 1 H NMR(600 MHz, CDCl3)δ 7.93(d,J=8.3 Hz,1H),7.85(d,J=1.8 Hz,1H),7.74(dd,J=8.3,1.9 Hz,1H),5.62(d,J=8.4 Hz,1H),3.70-3.66(m,1H),3.51(d,J=7.3 Hz,1H),3.16(d,J=7.3 Hz,1H),3.14-3.06(m,2H),2.37(t,J=13.0 Hz,1H),1.65(dd,J=13.1,4.8 Hz,1H),1.63(s,3H),1.60(s,3H),1.41(t,J=7.4 Hz,3H). 13 C NMR(151 MHz, CDCl3)δ 173.95,173.30,135.78,135.37,133.76(q,J=33.5 Hz),129.56,124.44,121.79(q,J=274.3 Hz),114.78,109.58,87.14,85.59,60.50,53.34,47.84,47.56,44.98,18.28,16.36,8.32. 19 F NMR (564 MHz, CDCl3): δ-62.1.HRMS: [M+H] + Calculation value C 20 H 21 N3O5SF3,472.1154; measured value 472.1148.

change

[0125] N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-1H-pyrazole-3-carboxamide (10) and N-((3aS,4S,5S,7S,7aR)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-1H-pyrazole-3-carboxamide (11). A mixture of amine 7 (50 mg, 0.13 mmol), 1H-pyrazole-3-carboxylic acid (24 mg, 0.21 mmol), DIEA (0.073 mL, 0.42 mmol), and HATU (80 mg, 0.21 mmol) in DMF (1 mL) was stirred at room temperature overnight. The volatiles were removed under reduced pressure, and the residue was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was first purified by flash column chromatography (0-5% MeOH in DCM) to afford product 10 / 11 (35 mg, 56%) as a white solid. Further separation of the two enantiomers was achieved by chiral HPLC using a Chiralcel OD-H column (250 × 4.6 mm, 5 μm) eluting with 50% isopropanol in hexane at 1 mL / min and 254 nm detection. Compound 10 had a retention time of 5.0 min, while its enantiomer 11 had a retention time of 6.7 min. 10: [α] 23 D = -28.7° (c = 0.3, MeOH). 1H NMR(600 MHz,MeOD)δ 8.13(d,J=8.3 Hz,1H),7.95(d,J=2.0Hz,1H),7.85(dd,J=8.7,2.0Hz,1H),7.73(s,1H),6.80(s,1H),4.49(dd,J=11.8,5.1). Hz,1H), 3.57(d,J=7.3Hz,1H),3.39(d,J=7.2Hz,1H),2.38-2.25(m,1H),1.87(dd,J=13.0,5.1Hz,1H),1.59(s,3H),1.57(s,3H). 13 C NMR(151 MHz,MeOD)δ 176.40,175.80,165.20,147.44,138.01,137.05,134.10(q,J=32.9 Hz),131.80,131.15,125.94,123.62(q,J=274.5). Hz), 115.94,110.22,106.64,89.32,86.94,57.94,55.10,49.97,43.74,18.64,17.74. 19 F NMR(564 MHz,MeOD):δ-63.6.HRMS:[M+H] + Description C 22 H 19 N5O4F3,474.1389; 11:[α] 23 D =+31.6° (c=0.3, MeOH). 1 H NMR(600 MHz,MeOD)δ 8.13(d,J=8.3 Hz,1H),7.95(d,J=1.9Hz,1H),7.85(dd,J=8.3,1.9Hz,1H),7.73(s,1H),6.80(s,1H),4.49(dd,J=11.8,5.1). Hz,1H), 3.57(d,J=7.2Hz,1H),3.39(d,J=7.3Hz,1H),2.38-2.24(m,1H),1.87(dd,J=13.0,5.1Hz,1H),1.59(s,3H),1.57(s,3H). 13C NMR(151 MHz,MeOD)δ 176.40,175.80,165.23,147.41,138.00,137.05,134.10(q,J=33.1 Hz),131.80,131.15,125.94,123.62(q,J=273.0 Hz),115.94,110.23,106.65,89.32,86.94,57.94,55.09,49.97,43.74,18.64,17.74. 19 F NMR(564 MHz,MeOD):δ-63.6.HRMS:[M+H] + Calculated value C 22 H 19 N5O4F3, 474.1389; measured value 474.1368. [ka]

[0126] 5-acetyl-N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-1H-pyrazole-3-carboxamide (12) and 5-acetyl-N-((3aS,4S,5S,7S,7aR)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-1H-pyrazole-3-carboxamide (13). This compound was prepared as described for 10. It was obtained as a white solid from 7 (50 mg, 0.13 mmol) in 66% yield (45 mg). Separation of the two enantiomers was achieved by chiral HPLC using a ProntoSIL Chiral AX QN-1 column (150 × 8.0 mm, 5 μm) eluted with 50% isopropanol in hexane at 3 mL / min and 254 nm detection. Compound 12 had a retention time of 40.4 min, while its enantiomer 13 had a retention time of 16.3 min. 12: [α] 22 D = -48.7° (c = 0.3, MeOH). 11H NMR (600 MHz, CDCl3) δ 11.39 (s, 1H), 7.94 (d, J = 8.3 Hz, 1H), 7.84 (d, J = 2.1 Hz, 1H), 7.73 (dd, J = 8.3, 2.1 Hz, 1H), 7.35 (s, 1H), 6.99 (d, J = 8.1 Hz, 1H), 4.54 - 4.50 (m, 1H), 3.52 (d, J = 7.2 Hz, 1H), 3.30 (d, J = 7.2 Hz, 1H), 2.58 (s, 3H), 2.42 (dd, J = 13.3, 11.7 Hz, 1H), 1.76 (dd, J = 13.3, 5.0 Hz, 1H), 1.69 (s, 3H), 1.66 (s, 3H). 13 13C NMR (151 MHz, CDCl3) δ 174.23, 173.47, 161.01, 135.75, 135.40, 133.84 (q, J = 33.3 Hz), 124.38, 121.78 (q, J = 274.2 Hz), 114.74, 109.67, 88.31, 85.78, 56.82, 53.67, 48.30, 43.95, 27.03, 18.31, 17.05. 19 19F NMR (564 MHz, CDCl3): δ -62.07. ESI-MS: [M + Na] + Calculated for C 24 H 20 N5O5F3Na, 538.1; Found 538.3. 13: [α] 22 D = +49.0° (c = 0.3, MeOH). 1 1H NMR (600 MHz, CDCl3) δ 7.94 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 2.0 Hz, 1H), 7.73 (dd, J = 8.4, 2.1 Hz, 1H), 7.35 (s, 1H), 6.99 (d, J = 8.0 Hz, 1H), 4.54 - 4.50 (m, 1H), 3.52 (d, J = 7.2 Hz, 1H), 3.29 (d, J = 7.3 Hz, 1H), 2.58 (s, 3H), 2.42 (dd, J = 13.3, 11.7 Hz, 1H), 1.76 (dd, J = 13.3, 5.0 Hz, 1H), 1.69 (s, 3H), 1.66 (s, 3H).<C NMR(151 MHz,CDCl3)δ 174.22,173.46,161.02,135.76,135.40,133.84(q,J=33.5 Hz),124.38,121.79(q,J=274.5 Hz),114.74,109.67,88.31,85.77,56.82,53.68,48.30,43.98,27.02,18.31,17.05. 19 F NMR(564 MHz,CDCl3):δ-62.08.ESI-MS:[2M+Na] + Calculated value C 48 H 40 N 10 O 10 F6, 1053.3; Actual value 1052.9. [ka]

[0127] N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-5-(1-hydroxyethyl)-1H-pyrazole-3-carboxamide (14) and N-((3aS,4S,5S,7S,7aR)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-5-(1-hydroxyethyl)-1H-pyrazole-3-carboxamide (15). This compound was prepared as described for 10. 14 was obtained as a white solid in 64% yield (26 mg) from 7 (30 mg, 0.079 mmol). Separation of the two enantiomers was achieved by chiral HPLC using a ProntoSIL Chiral AX QN-1 column (150 × 8.0 mm, 5 μm) eluted with 50% isopropanol in hexane at 3 mL / min and 254 nm detection. Compound 14 had a retention time of 36.7 min, while its enantiomer 15 had a retention time of 14.9 min. 14: [α] 20 D=-31.0° (c=0.3, MeOH). 1 H NMR(600 MHz,MeOD)δ 8.13(d,J=8.3Hz,1H),7.95(d,J=1.9Hz,1H),7.85(dd,J=8.3,2.0Hz,1H),6.69(s,1H),4.94(q,J=6.2 Hz,1H), 4.47(dd,J=11.7,5.1Hz,1H),3.56(d,J=7.2Hz,1H),3.38(d,J=7.3Hz,1H),2.32-2.25(m,1H),1.85(dd,J=13.0,5.1). Hz,1H), 1.59(s,3H),1.56(s,3H),1.52(d,J=6.8Hz,3H). 13 C NMR(151 MHz,MeOD)δ 176.39,175.79,138.01,137.06,134.10(q,J=33.2 Hz),131.81,125.94,123.62(q,J=273.2). Hz), 110.22,103.35,89.31,86.94,57.94,55.10,49.96,43.75,37.60,23.73,18.64,17.73. 19 F NMR(564 MHz,MeOD):δ-63.61.ESI-MS:[M+Na] + Description C 24 H 22 N5O5F3Na,540.1; 15:[α] 20 D =+34.0° (c=0.3, MeOH). 1 H NMR(600 MHz,MeOD)δ 8.13(d,J=8.3Hz,1H),7.95(d,J=2.0Hz,1H),7.85(dd,J=8.3,2.0Hz,1H),6.66(s,1H),4.94(q,J=6.8). Hz,1H), 4.47(dd,J=11.8,5.1Hz,1H),3.56(d,J=7.2Hz,1H),3.38(d,J=7.2Hz,1H),2.33-2.24(m,1H),1.85(dd,J=13.0,5.1). Hz,1H), 1.59(s,3H),1.56(s,3H),1.52(d,J=6.6Hz,3H). 13C NMR(151 MHz,MeOD)δ 176.39,175.79,138.01,137.06,134.10(q,J=33.0 Hz),131.81,125.94,123.62(q,J=273.2 Hz),115.94,110.23,103.35,89.31,86.94,57.94,55.10,49.96,43.76,37.60,23.73,18.64,17.73. 19 F NMR(564 MHz,MeOD):δ-63.61.ESI-MS:[M+Na] + Calculated value C 24 H 22 N5O5F3Na, 540.1; measured value 540.3.

[0128] N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-2-fluoro-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)ethanesulfonamide (EITM-1705). EITM-1705 was prepared from amine 7 in a manner similar to that described for BMS-641988. The racemic mixture was obtained from 7 (20 mg, 0.050 mmol) as a white solid in 49% yield (12 mg). Separation of the two enantiomers was achieved by chiral HPLC using a ProntoSIL Chiral AX QN-1 column (150 × 8.0 mm, 5 μm) eluted with 50% isopropanol in hexane at 3 mL / min and 254 nm detection. EITM-1705 had a retention time of 8.5 minutes. 20 D = -30.0° (c = 0.2, EtOAc). 1 H NMR(600 MHz,CDCl3)δ 7.75(d,J=8.3 Hz,1H),7.64(s,1H),5.03(s,1H),3.73-3.70(m,1H),3.53(s,1H),3.17(s,1H),3.11(q,J=7.4 Hz,2H),2.40(t,J=12.3 Hz,1H),1.63(s,3H),1.65-1.62(m,1H),1.60(s,3H),1.42(t,J=7.3 Hz,3H). 13C NMR(151 MHz,CDCl3)δ 172.84,172.22,130.75,125.64,120.78(q,J=277.1 Hz),114.11,87.12,85.54,60.49,53.97,48.32,45.17,18.23,16.36,8.37. 19 F NMR(564 MHz,CDCl3):δ-57.77,-112.61.ESI-MS:[M+Na] + Calculated value C 20 H 19 N4O5SF4, 512.1; measured value 512.3.

[0129] 4-((3aR,4R,5R,7R,7aS)-5-(4-(2-hydroxyethyl)-1H-1,2,3-triazol-1-yl)-4,7-dimethyl-1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-2-(trifluoromethyl)benzonitrile (EITM-1706). EITM-1706 was prepared as described in Scheme 4. A mixture of the azide intermediate (36 mg, 0.09 mmol), 3-butynol (9.5 mg, 0.135 mmol), copper(II) sulfate pentahydrate (6.7 mg, 0.027 mmol), and sodium ascorbate (10.7 mg, 0.054 mmol) in 1:1 tert-butanol:water (1 mL) was stirred at 40 °C for 2 days. The volatiles were removed under reduced pressure, and the residue was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by flash column chromatography (0-10% MeOH in DCM) to give the racemic product as a white solid (6.4 mg, 30%). Further separation of the two enantiomers was achieved by chiral HPLC using a Chiralcel OD-H column (250 x 4.6 mm, 5 μm) eluted with 50% isopropanol in hexane at 1 mL / min and 254 nm detection. EITM-1706 had a retention time of 14.5 min. [α] 23 D = -5.3° (c = 0.15, MeOH). 1H NMR(600 MHz, CDCl3)δ 7.95(d,J=8.1 Hz,1H),7.84(s,1H),7.73(d,J=8.3 Hz,1H),7.51(s,1H),4.68(m,1H),4.01(q,J=6.0 Hz,2H),3.53(d,J=7.3 Hz,1H),3.12(d,J=7.3 Hz,1H),3.03-2.99(m,2H),2.97(dd,J=13.4,4.7 Hz,1H),2.49(t,J=12.5 Hz,1H),2.28(t,J=5.8 Hz,1H),1.74(s,3H),1.70(s,3H). 13 C NMR(151 MHz, CDCl3)δ 173.77,173.31,135.69,135.39,134.03,133.70,129.49,124.39(q,J=4.8 Hz),123.13,120.41,114.72,109.71,87.08,86.56,67.66,61.42,53.12,48.61,42.31,28.61,18.29,16.71. 19 F NMR (564 MHz, CDCl3): δ-62.07.ESI-MS: [2M+Na] + Calculate value C 44 H 40 N 10 O8F6Na,973.2832; measured value 973.1.

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[0130] (2-Methoxyethoxy)methyl (3aR,4R,7R,7aS)-2-(4-cyano-2-fluoro-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoindole-5-carboxylate (17a) and (2-methoxyethoxy)methyl (3aS,4S,7S,7aR)-2-(4-cyano-2-fluoro-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoindole-5-carboxylate (17b). This compound was prepared as described for 3. The product was obtained as a viscous oil from compound 16 (100 mg, 0.35 mmol) in a yield of 61% (110 mg). 1 H NMR(600 MHz,CDCl3)δ 7.75(d,J=8.3 Hz,1H),7.66(s,1H),7.12(s,1H),5.47-5.41(m,2H),3.86-3.80(m,2H),3.58- 3.56(m,2H),3.39(s,3H),3.22(s,1H),3.12(s,1H),1.90(s,3H),1.79(s,3H). 13 C NMR(151 MHz,CDCl3)δ 170.93,170.87,161.72,149.68,144.68,135.12,133.19,130.74,125.48,122.05,120.78(q,J=276.5 Hz),114.11,112.26,90.03,88.58,87.66,71.45,69.99,59.12,53.12,52.74,15.37,15.06. 19 F NMR(564 MHz,CDCl3)δ-57.77,-112.58.ESI-MS:[M+Na] + Calculated value C 23 H 20 F4N2NaO7,535.1; measured value 535.1. [ka]

[0131] (2-Methoxyethoxy)methyl (3aR,4R,5R,7R,7aS)-2-(4-cyano-2-fluoro-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindole-5-carboxylate (18a) and (2-methoxyethoxy)methyl (3aS,4S,5S,7S,7aR)-2-(4-cyano-2-fluoro-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindole-5-carboxylate (18b). This compound was prepared as described for 4. It was obtained as a white solid from 17 (0.72 g, 1.41 mmol) in 90% yield (0.65 g). 1 H NMR(600 MHz,CDCl3)δ 7.73(d,J=8.3 Hz,1H),7.60(s,1H),5.45(d,J=6.2 Hz,1H),5.33(d,J=6.2 Hz,1H),3.88-3.79(m,2H),3.56(t,J=4.5 Hz,2H),3.37(s,4H),3.22(s,1H),3.04(dd,J=11.7,4.9 Hz,1H),2.27(dd,J=12.8,5.0 Hz,1H),2.05(t,J=12.4 Hz,1H),1.76(s,3H),1.62(s,3H). 13 C NMR(151 MHz,CDCl3)δ 172.81,172.63,171.26,133.01,130.70,125.75,121.96,120.78(q,J=275.8 Hz),114.12,112.05,90.02,86.87,86.22,59.06,54.20,53.67,50.50,41.34,17.98,17.96. 19 F NMR(564 MHz,CDCl3)δ-57.78,-112.62.ESI-MS:[M+Na] + Calculated value C 23 H 22 F4N2NaO7,537.1; measured value 537.3. [ka]

[0132] (3aR,4R,5R,7R,7aS)-2-(4-cyano-2-fluoro-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindole-5-carboxylic acid (19a) and (3aS,4S,5S,7S,7aR)-2-(4-cyano-2-fluoro-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindole-5-carboxylic acid (19b). This compound was prepared as described for 5. It was obtained as a white foam from 18 (0.65 g, 1.26 mmol) in 91% yield (0.49 g). 1 H NMR(400 MHz,MeOD)δ 7.98-7.95(m,1H),7.86(s,1H),3.44(s,1H),3.28(s,1H),3.02(dd,J=11.8,5.1 Hz,1H),2.25(dd,J=12.7,5.2 Hz,1H),2.04(d,J=12.4 Hz,1H),1.69(s,3H),1.56(s,3H). 19 F NMR(470 MHz,MeOD)δ-59.51,-116.28.ESI-MS:[MH] - Calculated value C 19 H 13 F4N2O5, 425.1; measured value 425.1. [ka]

[0133] 2-(Trimethylsilyl)ethyl ((3aR,4R,5R,7R,7aS)-2-(4-cyano-2-fluoro-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)carbamate (20a) and 2-(trimethylsilyl)ethyl ((3aS,4S,5S,7S,7aR)-2-(4-cyano-2-fluoro-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)carbamate (20b). This compound was prepared as described for 6. Obtained as a white foam from 19 (300 mg, 0.70 mmol) in 79% yield (300 mg). 1 H NMR(400 MHz,CDCl3)δ 7.68(d,J=8.3 Hz,1H),7.54(s,1H),4.15(s,2H),3.94(s,1H),3.37(s,1H),3.06(s,1H),2. 16(s,1H),1.50-1.44(m,7H),0.98-0.95(m,2H),0.00(s,9H).ESI-MS:[M+Na] + Calculated value C 24 H 27 F4N3O5SiNa, 564.2; measured value 564.2. [ka]

[0134] 4-((3aR,4R,5R,7R,7aS)-5-amino-4,7-dimethyl-1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-3-fluoro-2-(trifluoromethyl)benzonitrile (21a) and 4-((3aS,4S,5S,7S,7aR)-5-amino-4,7-dimethyl-1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-3-fluoro-2-(trifluoromethyl)benzonitrile (21b). This compound was prepared as described for 7. Obtained from 20 (218 mg, 0.40 mmol) as a white foam in 88% yield (141 mg). 1H NMR(600 MHz,MeOD)δ 7.86(d,J=8.7 Hz,1H),7.77(s,1H),3.61(s,1H),3.17-3.10(m,2H),2.06(dd,J=12.7,11.1 Hz,1H),1.41(s,3H),1.39(s,3H),1.34(dd,J=12.8,5.0 Hz,1H). 13 C NMR(151 MHz,MeOD)δ 176.29,175.11,157.03,155.26,135.51,132.63,127.66,122.63(q,J=274.5 Hz),115.38,112.68,89.45,86.38,61.10,55.90,49.85,47.23,18.71,16.68. 19 F NMR(564 MHz,MeOD)δ-59.24,-116.13.ESI-MS:[MH] - Calculation value C 18 H 14 F4N3O3,396.1; measured value 396.2.

change

[0135] N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-2-fluoro-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-5-(1-hydroxyethyl)-1H-pyrazole-3-carboxamide (22) and N-((3aS,4S,5S,7S,7aR)-2-(4-cyano-2-fluoro-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-5-(1-hydroxyethyl)-1H-pyrazole-3-carboxamide (23). This compound was prepared as described for 10. 22 was obtained as a white solid in 26% yield (7 mg) from 21 (20 mg, 0.05 mmol). Separation of the two enantiomers was achieved by chiral HPLC using a ProntoSIL Chiral AX QN-1 column (150 × 8.0 mm, 5 μm) and isocratic elution with 50% isopropanol in hexane. Separation of the two enantiomers was achieved by chiral HPLC using a ProntoSIL Chiral AX QN-1 column (150 × 8.0 mm, 5 μm) eluted with 50% isopropanol in hexane at 3 mL / min and 254 nm detection. Compound 22 had a retention time of 20.1 min, while its enantiomer 23 had a retention time of 8.7 min. 22: [α] 20 D = -37.2° (c = 0.5, EtOAc). 1 H NMR(600 MHz,Acetonitrile-d3)δ 11.43(s,1H),7.89(d,J=8.4 Hz,1H),7.78(s,1H),7.39(d,J=7.4 Hz,1H),6.58(s,1H),4.91(q,J=6.6 Hz,1H),4.44-4.39(m,1H),3.64(s,1H),3.45(s,1H),3.39(s,1H),2.26-2.22(m,1H),1.82(dd,J=13.1,5.2 Hz,1H),1.52(s,3H),1.50(s,3H),1.46(d,J=6.6 Hz,3H). 13C NMR(151 MHz, Acetonitrile-d3)δ 175.10,174.60,134.80,132.58,122.35(q,J=273.8 Hz),115.56,112.44,102.77,88.73,86.27,62.84,57.48,55.26,50.15,43.39,23.83,18.62,17.72. 19 F NMR(564 MHz, Acetonitrile-d3)δ-58.62.ESI-MS:[MH] - Calculation value C 24 H 20 N5O5F4,534.1; measured value 534.2. 23:[α] 20 D =+39.0° (c=0.5, EtOAc). 1 H NMR(600 MHz, Acetonitrile-d3)δ 11.37(s,1H),7.89(d,J=8.4 Hz,1H),7.78(s,1H),7.35(d,J=8.2 Hz,1H),6.58(s,1H),4.91(q,J=6.6 Hz,1H),4.44-4.39(m,1H),3.56(s,1H),3.45(s,1H),3.39(s,1H),2.27-2.22(m,1H),1.82(dd,J=13.1,5.2 Hz,1H),1.52(s,3H),1.50(s,3H),1.46(d,J=6.1 Hz,3H). 13 C NMR(151 MHz, Acetonitrile-d3)δ 175.10,174.60,134.95,132.60,122.35(q,J=274.4 Hz),115.57,112.45,102.76,88.78,86.30,63.11,57.55,55.30,50.19,43.47,23.87,18.64,17.74. 19 F NMR(564 MHz, Acetonitrile-d3)δ-58.63.ESI-MS:[MH] - Calculation value C 24 H 20 N5O5F4,534.1; measured value 534.2.

[0136] N-(2-(4-cyano-3-(trifluoromethyl)phenyl)-1,3-dioxoisoindolin-5-yl)ethanesulfonamide (EITM-1708). Step 1: A solution of 5-nitroisobenzofuran-1,3-dione (300 mg, 1.55 mmol) and 4-amino-2-(trifluoromethyl)benzonitrile (289 mg, 1.55 mmol) in 5 mL of acetic acid was heated at 130°C to 140°C for 4.5 hours. After completion of the reaction, the solvent was removed under reduced pressure to give the crude product, 4-(5-nitro-1,3-dioxoisoindolin-2-yl)-2-(trifluoromethyl)benzonitrile (10). To the crude residue, 10% Pd / C (184 mg) and ethyl acetate (15 mL) were added, and the reaction mixture was stirred at room temperature under a H2 atmosphere overnight. The mixture was then filtered through a pad of Celite and concentrated. The residue was purified by column chromatography (50% ethyl acetate in hexane) to give 4-(5-amino-1,3-dioxoisoindolin-2-yl)-2-(trifluoromethyl)benzonitrile 11 (200 mg, 39%) as a yellowish solid. Step 2: To a solution of amine intermediate 11 (20 mg, 0.06 mmol) in anhydrous DCM (1 mL) was added triethylamine (0.034 mL, 0.24 mmol) and ethanesulfonyl chloride (16 mg, 0.12 mmol) at 0 °C. The reaction mixture was stirred at room temperature overnight. The reaction mixture was then diluted with DCM, washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (5% MeOH in DCM) to give EITM-1708 (9 mg, 35%) as a white solid. 1 H NMR(600 MHz,DMSO-d6)δ 10.81(s,1H),8.37(d,J=8.3 Hz,1H),8.17(d,J=2.0 Hz,1H),8.03(dd,J=8.3,2.0 Hz,1H),7.99(d,J=8.2 Hz,1H),7.72(d,J=2.0 Hz,1H),7.65(dd,J=8.3,2.0 Hz,1H),3.34-3.29(m,2H),1.23(t,J=7.3 Hz,3H). 19 F NMR(564 MHz,DMSO-d6)δ-60.95.ESI-MS:[MH]- Calculated value C 18 H 11 N3O4F3S, 422.0; Measured value 422.0.

[0137] N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxo-octahydro-1H-4,7-epoxyisoindol-5-yl)-1H-indazole-3-carboxamide (EITM-1709). EITM-1709 was prepared from amine 7 in a manner similar to that described for EITM-1702. The racemic mixture was obtained from 7 (20 mg, 0.053 mmol) as a white solid in 72% yield (20 mg). Separation of the two enantiomers was achieved by chiral HPLC using a ProntoSIL Chiral AX QN-1 column (150 × 8.0 mm, 5 μm) eluted with 50% isopropanol in hexane at 3 mL / min and 254 nm detection. EITM-1709 had a retention time of 16.8 minutes. 20 D = -5.5° (c = 0.2, MeOH). 1 H NMR(600 MHz,CDCl3)δ 10.26(s,1H),8.38(d,J=8.2 Hz,1H),7.94(d,J=8.4 Hz,1H),7.85(d,J=2.0 Hz,1H),7.74(dd,J=8.3,2.0 Hz,1H),7.54(dt,J=8.5,0.9 Hz,1H),7.49-7.46(m,1H),7.35-7.33(m,1H),7.10(d,J=8.0 Hz,1H),4.59-4.55(m,1H),3.58(d,J=7.2 Hz,1H),3.28(d,J=7.2 Hz,1H),2.46(dd,J=13.4,11.6 Hz,1H),1.79(dd,J=13.3,5.0 Hz,1H),1.74(s,3H),1.68(s,3H). 13C NMR(151 MHz,CDCl3)δ 174.24,173.48,162.69,141.38,138.91,135.80,135.38,133.85(q,J=33.3 Hz),129.49,127.86,124.38,123.41,122.49,121.82,121.80(q,J=274.8 Hz),114.76,109.81,109.64,88.43,85.77,77.21,77.00,76.79,56.72,53.74,48.34,44.27,18.34,17.13. 19 F NMR(564 MHz,CDCl3):δ-62.08.ESI-MS:[M+Na] + Calculated value C 26 H 20 N5O4F3Na, 546.1; Measured value 546.1.

[0138] N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-5-(thiophen-2-yl)-1H-pyrazole-3-carboxamide (EITM-1710). EITM-1710 was prepared from amine 7 in a manner similar to that described for EITM-1702. The racemic mixture was obtained from 7 (25 mg, 0.066 mmol) as a white solid in 68% yield (25 mg). Separation of the two enantiomers was achieved by chiral HPLC using a ProntoSIL Chiral AX QN-1 column (150 × 8.0 mm, 5 μm) eluted with 50% isopropanol in hexane at 3 mL / min and 254 nm detection. EITM-1710 had a retention time of 18.6 minutes. 20 D = -58.0° (c = 0.5, MeOH). 1H NMR(600 MHz,CDCl3)δ 7.92(d,J=8.4 Hz,1H),7.82(d,J=2.0Hz,1H),7.71(dd,J=8.3,2.1Hz,1H),7.37(dd,J=5.1,1.2Hz,1H),7.30(dd,J=3.6,1.2). Hz,1H), 7.10(dd,J=5.1,3.6Hz,1H),7.01(d,J=8.0Hz,1H),6.96(s,1H),4.56-4.52(m,1H),3.54(d,J=7.2Hz,1H),3.28(d,J=7.3). Hz,1H), 2.41(dd,J=13.3,11.6Hz,1H),1.78(dd,J=13.3,5.0Hz,1H),1.70(s,3H),1.65(s,3H). 13 C NMR(151 MHz,CDCl3)δ 174.30,173.50,161.70,135.79,135.37,133.80(q,J=33.7). Hz), 129.50,128.11,126.41,125.28,124.38,121.79(q,J=274.8). Hz), 114.78,109.55,103.78,88.34,85.77,56.81,53.65,48.32,43.96,18.33,17.10. 19 F NMR(564 MHz,CDCl3):δ-62.04.ESI-MS:[M+Na] + Description C 26 H 20 N5O4SF3Na,578.1;

[0139] N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-5-(furan-2-yl)-1H-pyrazole-3-carboxamide (EITM-1711). EITM-1711 was prepared from amine 7 in a manner similar to that described for EITM-1702. The racemic mixture was obtained from 7 (25 mg, 0.066 mmol) as a white solid in 72% yield (20 mg). Separation of the two enantiomers was achieved by chiral HPLC using a ProntoSIL Chiral AX QN-1 column (150 × 8.0 mm, 5 μm) eluted with 50% isopropanol in hexane at 3 mL / min and 254 nm detection. EITM-1711 had a retention time of 16.1 minutes. [α] 20 D = -56.0° (c = 0.3, MeOH). 1 H NMR(600 MHz,CDCl3)δ 7.93(d,J=8.4 Hz,1H),7.83(d,J=2.0 Hz,1H),7.72(dd,J=8.3,2.0 Hz,1H),7.49(dd,J=1.8,0.7 Hz,1H),6.97(brs,2H),6.68(dd,J=3.4,0.7 Hz,1H),6.53(dd,J=3.4,1.8 Hz,1H),4.54-4.50(m,1H),3.54(d,J=7.3 Hz,1H),3.30(d,J=7.3 Hz,1H),2.42(dd,J=13.3,11.6 Hz,1H),1.77(dd,J=13.2,5.0 Hz,1H),1.70(s,3H),1.66(s,3H). 13 C NMR(151 MHz,CDCl3)δ 174.28,173.53,161.69,142.89,135.79,135.37,133.83(q,J=33.3 Hz),129.50,124.38,121.80(q,J=274.5 Hz),114.76,111.98,109.61,108.08,102.22,88.39,85.77,56.81,53.67,48.32,44.04,18.33,17.06. 19F NMR(564 MHz,CDCl3)δ-62.07.ESI-MS:[M+Na] + Calculated value C 26 H 20 N5O5F3Na, 562.1; measured value 562.2.

[0140] N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-2-methyl-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-5-(1-hydroxyethyl)-1H-pyrazole-3-carboxamide (EITM-1712). EITM-1712 was prepared from amine 7 in a manner similar to that described for EITM-1702. The racemic mixture was obtained from 7 (40 mg, 0.10 mmol) as a white solid in 56% yield (30 mg). Separation of the two enantiomers was achieved by chiral HPLC using a ProntoSIL Chiral AX QD-1 (5 μm, 150 × 4.0 mm) column eluted with 100% acetonitrile at 1 mL / min and 254 nm detection. EITM-1712 had a retention time of 4.5 minutes. 23 D = -28.0° (c = 0.3, MeOH). 1 H NMR(600 MHz,CD3CN)δ 7.92(d,J=8.8 Hz,1H),7.61(d,J=8.3 Hz,1H),7.44(d,J=9.1 Hz,1H),6.60(s,1H),4.94(q,J=6.6 Hz,1H),4.49-4.42(m,1H),3.47(d,J=7.1 Hz,1H),3.40(d,J=7.0 Hz,1H),2.29(q,J=2.2 Hz,3H),2.26(d,J=12.3 Hz,1H),1.89-1.84(m,1H),1.55(s,3H),1.52(s,3H),1.49(d,J=6.5 Hz,3H). 13C NMR(151 MHz,CD3CN)δ 176.15,175.68,138.95,134.86,133.69,116.99,102.77,88.79,86.40,57.57,55.23,50.26,43.48,23.80,18.78,17.84,15.39. 19 F NMR(564 MHz,CD3CN)δ-57.6.APCI-MS:[M+H] + Calculated value C 25 H 25 N5O5F3,532.2; measured value 532.1.

[0141] 3-(4-Acetylpiperazin-1-yl)-N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)propenamide (EITM-1716). EITM-1716 was prepared from amine 7 in a similar manner as described for EITM-1702. The racemic mixture was obtained from 7 (19 mg, 0.05 mmol) as a white solid in 14% yield (1.9 mg). [α] 23 D = -10.0° (c = 0.1, MeCN). Separation of the two enantiomers was achieved by chiral HPLC using a Chiralcel OD-H column (250 x 4.6 mm, 5 µm) eluted with 60% to 90% isopropanol in hexane at 1 mL / min and 254 nm detection. EITM-1716 had a retention time of 11.5 min. 1H NMR(600 MHz,CD3CN)δ 8.10(d,J=8.3 Hz,1H),7.88(s,1H),7.81(dd,J=8.3,2.0 Hz,1H),7.68(broad,1H),4.26-4.22(m,1H),3.55-3.50(m,2H),3.48(t,J=6.,2H),3.41(dd,J=7.2,1.7 Hz,1H),3.24(d,J=7.2 Hz,1H),2.78-2.55(m,2H),2.55-2.42(m,3H),2.41-2.27(m,3H),2.22(t,J=13.1 Hz,1H),2.00(s,3H),1.59(dd,J=13.1,5.3 Hz,1H),1.54(s,3H),1.49(s,3H). 13 C NMR (151 MHz, CD3CN) δ 175.11,174.41,172.02,168.57,136.63,136.06,132.64,132.37,130.38,124.63(q,J=5.1 Hz),115.11,108.91,87.60,85.24,56.32,54.00,53.78,52.72,52.23,48.53,45.98,43.25,41.07,32.79,20.53,17.65,16.63. 19 F NMR (564 MHz, CD3CN): δ-62.64.ESI-MS: [M+H] + Calculate value C 27 H 31 N5O5F3,562.2277; measured value 561.4.

[0142] 1-Acetyl-N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)piperidine-4-carboxamide (EITM-1717). EITM-1717 was prepared from amine 7 in a similar manner as described for EITM-1702. The racemic mixture was obtained from 7 (19 mg, 0.05 mmol) as a white solid in 36% yield (4.9 mg). Separation of the two enantiomers was achieved by chiral HPLC using a Chiralcel OD-H column (250 x 4.6 mm, 5 µm) eluted with 40% to 90% isopropanol in hexane at 1 mL / min and 254 nm detection. EITM-1717 had a retention time of 13.0 minutes. [α] 23 D = -8.0° (c = 0.2, MeCN). 1 H NMR(600 MHz,CDCl3)δ 7.95(d,J=8.4 Hz,1H),7.86(s,1H),7.74(d,J=8.0 Hz,1H),5.81(dd,J=22.3,7.2 Hz,1H),4.59(d,J=13.2 Hz,1H),4.31(d,J=6.1 Hz,1H),3.90(d,J=13.8 Hz,1H),3.42-3.22(m,1H),3.15-3.10(m,2H),2.67(t,J=12.7 Hz,1H),2.31-2.37(m,2H),2.11(s,3H),1.97-1.81(m,2H),1.79-1.66(m,2H),1.63(s,3H),1.60(s,3H), 13 C NMR(151 MHz,CDCl3)δ 174.37,174.05,173.33,168.97,135.72,135.38,129.44,124.32(q,J=5.1 Hz),122.69,120.87,114.72,109.66,88.03,85.56,56.99,53.64,48.14,45 .77,43.82,43.23,40.91,29.03,28.91,28.65,28.42,21.45,18.27,16.97. 19F NMR(564 MHz,CDCl3):δ-62.08.ESI-MS:[MH] - Calculated value C 26 H 26 N4O5F3,531.1855; measured value 531.2.

[0143] Example 3. General computational and biological methods. Molecular docking and induced fit docking. Molecular docking and induced fit docking (IFD) were performed with the Schrodinger Suite (Glide, Prime). IFD is intended to circumvent the inflexible binding site requirements of grid-based docking by using a post-docking refinement step. Protein preparation is one of the most important steps in molecular docking and IFD. The three-dimensional atomic coordinates of WT AR-LBD in complex with DHT (PDB:1T7R) were used to prepare the receptor in the protein preparation module. The protein structure was optimized using the OPLS3 force field. This structure was used for IFD of the ligand. The ligand for docking was prepared using LigPrep. IFD included the following steps: a) Each ligand was docked (Glide module) with standard precision (SP) to generate 20 different poses (default settings); b) all side chains within a 5.0 Å radius of each docked ligand pose were searched using the Prime side chain sampling algorithm; c) defined regions of the protein-ligand complex were minimized using OPLS3; d) the top-scoring docked poses (based on GlideScore and Prime energy) were analyzed and compared.

[0144] Agonist docking was performed using WT-AR-LBD (PDB ID: 1E3G) in complex with R1881 using Schrodinger Suite 2018-3 (Glide, Prime). For antagonists, a homology model of the open conformation of WT-AR-LBD was constructed using Schrodinger Prime using the progesterone receptor (PDB ID: 2OVM) as a template.

[0145] Cell culture and treatment. The LNCaP cell line was obtained from ATCC and cultured in RPMI 1640 (Corning) supplemented with 10% heat-inactivated GemCell bovine serum (Gemini Bio-Products) and penicillin-streptomycin (Gemini Bio-Products). Cell lines stably expressing ARE-luciferase (LNCaP-luc) were generated using Cignal Lenti AR Reporter (Qiagen) and continuously cultured with 500 ng / mL puromycin (Gibco) for positive selection. Cells were maintained at 37°C in a humidified incubator containing 5% carbon dioxide. All cell lines were authenticated using NIST-approved short tandem repeat (STR) DNA profiling performed by the University of Arizona Genetics Core and routinely tested negative for mycoplasma.

[0146] PC3 and VCaP cell lines were obtained from ATCC and cultured as recommended. Generation and culture of PC3 GFP-AR cells was previously described (14). ARE-luciferase cells were generated using the Cignal Lenti AR Reporter (Qiagen). Cell lines were authenticated using NIST-approved short tandem repeat DNA profiling and tested negative for mycoplasma. Drug treatment was performed after overnight culture in phenol red-free medium supplemented with charcoal:dextran-stripped FBS.

[0147] Confocal microscopy. PC3 GFP-AR cells were seeded and stained with SiR-DNA (Cytochrome) overnight. Cells were treated with drug for 180 minutes and ligand for 90 minutes and imaged with an Operetta CLS microscope (PerkinElmer).

[0148] Luciferase assay. LNCaP-luc cells were seeded at a density of 12,000 cells / well in clear, flat-bottom, white polystyrene 96-well plates (Corning). The following day, luciferase measurements were performed using a GloMax 96 Microplate Luminometer (Promega) after 90 min of drug and 24 h of competing ligand (1 nM R1881). Dose-response curves were fitted using the delta method and t-distribution (n = 1). Compounds were tested up to 10 μM. An assay coefficient of variation of 25% was estimated from seven experiments using three EITM drugs in both LNCaP-luc and PC3-luc cells.

[0149] LNCaP cell viability assay. Cells were plated on a 96-well plate coated with fibronectin (1 μg / cm 2 Cells were seeded at a density of 5,000 cells / well onto 100-well plates (CellCarrier, PerkinElmer). After 48 hours, the medium was replaced with phenol red-free RPMI (Corning) + 2% charcoal:dextran-stripped FBS (Gemini Bio-Products) supplemented with 60 pM R1881 and EITM drugs, as indicated in the figure legends. Drug-treated cells were lysed using a CellTiter-Glo 3D Cell Viability Assay (Promega) and transferred to white 96-well plates (Corning). Luminescence was measured using a GloMax 96 Microplate Luminometer (Promega). Analysis was performed in the R statistical environment (v3.6.0). The mean fluorescence signal per condition (n = 3) was used to calculate relative viability, scaling R1881 to 100% and starvation (no R1881) to 0%. Visualization was facilitated by the ggplot2 package (v3.2.1), with standard errors (SE) indicated where appropriate.

[0150] Microsomal stability assay. In vitro metabolism was determined as previously described. Specifically, to measure parent compound metabolism, a high-throughput protocol was applied in which 0.5 mg / ml pooled human liver microsomes (Sigma) were mixed with 1 μM drug and phosphate buffer (0.1 M, pH 7.4) in a 96-well plate. The enzymatic reaction was initiated by adding 1 mM NADPH (Sigma-Aldrich) followed by incubation at 37°C. After the indicated incubation time, fractions were quenched by transferring to ice-cold acetonitrile. Samples were analyzed using liquid chromatography-mass spectrometry (LC / MS-MS). The rate of intrinsic clearance was determined from the consumption of parent compound per minute per mg of microsomal protein, as previously described. To analyze accumulating metabolites, the basic protocol was used, and microsomes were incubated with 10 μM drug for 8 h.

[0151] Luminometer assay. For ARE-luciferase, luciferase substrate was added to lysed cells after 24 hours of treatment. For viability, cells were lysed after 6 days of treatment using the CellTiter-Glo 3D Cell Viability Assay (Promega). Measurements were performed in 96-well plates (Corning) using a GloMax 96 Microplate Luminometer (Promega).

[0152] AR binding. Ligand binding was analyzed using the PolarScreen AR Competitor Assay Kit, Green, according to the manufacturer's instructions (Thermo Fisher Scientific). Fluorescence polarization was measured after 4 hours of incubation using an EnVision 2103 multilabel plate reader (PerkinElmer).

[0153] RT-qPCR array. RNA was isolated using the Illustra RNAspin Mini Kit (GE Healthcare). RNA was transcribed into cDNA using the RT2 First Strand Kit (Qiagen). RT-qPCR was performed using a Biorad CFX Connect on the RT2 Profiler PCR Array Human Androgen Receptor Signaling Targets (Qiagen).

[0154] Example 4 Pharmaceutical dosage forms. The following formulations illustrate representative pharmaceutical dosage forms that may be used for the therapeutic or prophylactic administration of compounds of the formulae described herein, compounds specifically disclosed herein, or pharmaceutically acceptable salts or solvates thereof (hereinafter referred to as "Compound X"). (i) 1 tablet mg / tablet Compound X 100.0 Lactose 77.5 Povidone 15.0 Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesium stearate 3.0 300.0 (ii) Tablet 2 mg / tablet Compound X 20.0 Microcrystalline cellulose 410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0 500.0 (iii) Capsules mg / capsule Compound X 10.0 Colloidal silicon dioxide 1.5 Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 600.0 (iv) Injection 1 (1 mg / mL) mg / mL "Compound X" (free acid form) 1.0 Dibasic sodium phosphate 12.0 Sodium phosphate monobasic 0.7 Sodium chloride 4.5 1.0N sodium hydroxide solution (appropriate amount) (pH adjustment to 7.0-7.5) Water for injection: Enough 1mL (v) Injection 2 (10mg / mL) mg / mL "Compound X" (free acid form) 10.0 Sodium phosphate monobasic 0.3 Sodium phosphate dibasic 1.1 Polyethylene glycol 400 200.0 0.1N sodium hydroxide solution (appropriate amount) (pH adjustment to 7.0-7.5) Water for injection: Enough 1mL (vi) Aerosols mg / can compound x 20 Oleic Acid 10 Trichloromonofluoromethane 5,000 Dichlorodifluoromethane 10,000 Dichlorotetrafluoroethane 5,000 (vii) Topical Gel 1 weight% Compound X 5% Carbomer 934 1.25% Triethanolamine (appropriate amount) (pH adjustment to 5-7) Methylparaben 0.2% Purified water (up to 100g) (viii) Topical gel 2 weight% Compound X 5% Methylcellulose 2% Methylparaben 0.2% Propylparaben 0.02% Purified water (up to 100g) (ix) topical ointment weight% Compound X 5% Propylene glycol 1% Anhydrous ointment base 40% Polysorbate 80 2% Methylparaben 0.2% Purified water (up to 100g) (x) Topical cream 1 weight% Compound X 5% White beeswax 10% Liquid paraffin 30% Benzyl alcohol 5% Purified water (up to 100g) (xi) Topical cream 2 weight% Compound X 5% Stearic acid 10% Glyceryl monostearate 3% Polyoxyethylene stearyl ether 3% Sorbitol 5% Isopropyl palmitate 2% Methylparaben 0.2% Purified water (up to 100g)

[0155] These formulations can be prepared by conventional procedures well known in the pharmaceutical arts. It will be understood that the above pharmaceutical compositions can be modified according to well-known pharmaceutical techniques to accommodate different amounts and types of active ingredient "Compound X." Aerosol formulation (vi) can be used with standard metered-dose aerosol dispensers. Furthermore, the specific ingredients and proportions are for illustrative purposes. Ingredients may be substituted for appropriate equivalents, and proportions may be modified according to the desired properties of the intended dosage form.

[0156] While particular embodiments have been described above with reference to disclosed embodiments and examples, such embodiments are illustrative only and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects, which is defined in the following claims.

[0157] All publications, patents, and patent documents are incorporated herein by reference, as if individually incorporated by reference. No limitations inconsistent with the present disclosure should be understood therefrom. The present invention has been described with reference to various specific preferred embodiments and techniques. However, it should be understood that many variations and modifications can be made while remaining within the spirit and scope of the invention.

Claims

1. Compounds of Formula I: 【Chemistry 1】 The salt (G １ is NHR Ａ or OH, G ２ is H or OH, R Ａ is —C(═O)heteroaryl, —C(═O)(C １ ~C ６ ) alkyl, —S(═O) ２ (C １ ~C ６ ) alkyl, or —C(═O)heterocycloalkyl, and R Ａ is substituted or unsubstituted; R １ is H, halo, -(C １ ~C ６ ) alkyl, R ３ is CF ３ or halo).

2. G １ NHR Ａ 2. The compound of claim 1, wherein:

3. R Ａ is pyrrolopyridine, pyrazole or indazole, and R Ａ The compound of claim 2 , wherein is unsubstituted.

4. R Ａ is pyrazole or triazole, and R Ａ The compound of claim 2, wherein:

5. R Ａ が、-C(=O)CH ３ 、-S(=O) ２ CH ２ CH ３ 、 【Chemistry 2】 3. The compound of claim 2, wherein:

6. 10. The compound of claim 1, wherein the compound is dextrorotatory.

7. 8. The compound of claim 6 or 7, wherein the compound is levorotatory.

8. The compound has formula IA: 【Transformation 3】 represented by its enantiomers and / or salts, R １ is H, F, methyl or ethyl, R ２ is H, -C(=O)CH ３ , or —C(OH)CH ３ 2. The compound of claim 1, wherein:

9. The compound has formula IB: 【Chemistry 4】 represented by its enantiomers and / or salts, X is CH or N; R １ is H, F, methyl or ethyl, R ４ is H, -C(=O)CH ３ , or —C(OH)CH ３ 2. The compound of claim 1, wherein:

10. The compound is EITM-1719 or EITM-1720: 【Transformation 5】 2. The compound of claim 1, its enantiomers and / or salts.

11. The compound is 【Chemistry 6-1】 【Chemistry 6-2】 2. The compound of claim 1, wherein:

12. A pharmaceutical composition comprising a compound according to any one of claims 1 to 11 and a pharmaceutically acceptable diluent or carrier.

13. 12. A method for treating cancer in a subject in need thereof by administering to the subject an effective amount of a compound of any one of claims 1 to 11, thereby treating the cancer.

14. 14. The method of claim 13, wherein the cancer is prostate cancer or breast cancer.

15. 15. The method of claim 14, wherein the cancer is prostate cancer, and the prostate cancer is fatal castration-resistant prostate cancer.

16. 14. The method of claim 13, wherein the effective serum concentration of the compound is from about 1 nM to about 2000 nM.

17. 14. The method of claim 13, wherein administering an effective amount of the compound is by infusion, injection, oral administration, or a combination thereof.

18. 14. The method of claim 13, wherein the compound is an androgen receptor antagonist.

19. 12. A method for treating an endocrine disorder in a subject in need thereof by administering to said subject an effective amount of a compound of any one of claims 1 to 11, thereby treating the endocrine disorder.

20. 20. The method of claim 19, wherein the compound is an agonist of the androgen receptor.

21. 20. The method of claim 19, wherein the compound is a full agonist of the androgen receptor.

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