Patent Application
20210340155
The field of the invention relates to compounds that function as ligands for estrogen receptors (ERs). In particular, the field of the invention relates to substituted (4′-hydroxyphenyl)cycloalkane compounds and (4′-hydroxyphenyl)cycloalkene compounds that are specific agonists for the estrogen receptor beta (ERβ) and the use of such compounds in pharmaceutical compositions for treating diseases and disorders associated with ER activity in enhancing memory consolidation.
Estrogens are important regulators of many physiological processes that include reproduction, cognition, cardiovascular health, and bone metabolism.66 Based on their widespread role in a number of physiological processes, estrogens have been implicated in a number of diseases and disorders which include cell proliferative diseases and disorders (e.g., breast cancer, ovarian cancer, endometrial cancer, colorectal cancer, and prostate cancer), neurodegenerative diseases and disorders, cardiovascular disease, and osteoporosis to name a few.66 In many of these diseases and disorders, estrogen mediates its effects through the estrogen receptors (ERs).
The ERs exist in 2 main forms, ERα and ERβ, which have different tissue expression patterns.67 ERα and ERβ are encoded by separate genes, ESR1 and ESR2, respectively, found at different chromosomal locations, and numerous mRNA splice variants exist for both ERα and ERβ.68 Because of their role in estrogen-related diseases, ERα and ERβ have been targeted for development of specific ligands that modulate their activities. The ligand specificity of ERα and ERβ differ, and a ligand that binds and functions as an agonist or antagonist for ERα may or may not bind and function as an agonist or antagonist for ERβ.
ERα and ERβ agonists have a wide range of biological effects that implicate disease such as cancer and disorders of the central nervous system (CNS). 17β-estradiol (E2) is a critical modulator of hippocampal synaptic plasticity and hippocampal-dependent memory formation in male and female rodents.6 E2 levels decrease in both sexes as people age, but drop much more precipitously in women during the menopausal transition. ERβ is the predominant ER isoform in the hippocampus and plays an important role in mediating estradiol's effects on neural plasticity and neuroprotection, which could be pivotal during aging and in Alzheimer's disease (AD). For example, overexpression of ERβ in a rat model of AD significantly reduced hippocampal AD pathology and improved learning and memory.7 Moreover, specific alleles of the gene for ERβ (Esr2), but not ERα, are associated with decreased AD risk in men and women,8 supporting ERβ as a putative drug target for AD.
ERβ agonists in particular have a number of promising clinical applications1. Current ERβ agonist drug lead molecules possess a phenolic ring, with varying substituted aromatic ring systems on the other half of the molecule, typically comprising another phenolic or indole-like ring systems (
APOE4 is the most well established genetic risk factor for Alzheimer's disease (AD). Women with the APOE4 genotype are 2-4 times more likely to develop AD than women without APOE4 or than men of any other APOE genotype.9-11 APOE4 carriers are also much more likely to show symptoms of anxiety and depression.12 A major contributor to these risks in women is menopausal estrogen loss, as estrogens are neuroprotective for brain regions like the hippocampus and cortex that mediate cognitive function and deteriorate in AD.13 As such, drugs that facilitate estrogen-mediated effects on cognition, like the selective ERβ agonists (SERBAs) being developed herein, may reverse memory loss and alleviate anxiety and depression in aging females. But, estrogen-based hormone replacement therapy is associated with increased risk of various diseases (thought to be associated with ERα agonist activity), including breast cancer (esp. lobular) as well as stroke, gallbladder disease and venous thromboembolism14-18. Accordingly, any ERβ agonist therapeutic should be selective for ERβ over ERα agonist activity.
Thus, new ligands for estrogen receptors are desirable. In particular, new ligands that exhibit selective agonist or antagonist activity for ERβ versus ERα are desirable. These new ligands should be suitable for treating diseases and disorders associated with ER activity, such as cell proliferative diseases and disorders or psychiatric diseases and disorders. Recently, we reported a novel ERβ agonist that was more selective for ERβ versus ERα activation than previously reported clinical candidates19. This ERβ agonist was in a unique structural class, comprised of a phenol ring tethered to a 4-hydroxymethyl-cycloheptane ring system. However, the presence of the 4-substituted cycloheptane ring presents synthetic and stereochemistry challenges, making it less desirable as a drug lead.
Herein is reported the optimization and characterization of a related class of molecules, comprised of a 4-hydroxymethyl-cyclohexane ring tethered to a phenol ring, making it an A-C estrogen that closely resembles the naturally occurring estrogen molecule, but lacks the B and D rings (
Disclosed are substituted (4′-hydroxylphenyl)cycloalkane compounds and (4′-hydroxylphenyl)cycloalkene compounds and their use as selective agonists of the estrogen receptor beta (ERβ). The disclosed compounds may be formulated as pharmaceutical compositions and administered to treat diseases associated with ER activity.
In some embodiments, the disclosed compounds have a Formula I or a hydroxy-protected form thereof:
wherein:
The disclosed compounds may include 4-substituted-(4′-hydroxyphenyl)cyclohexane compounds. For example, the disclosed compounds may have a Formula Ia:
where X, Y, and Z are as defined for Formula I.
The disclosed compounds include the compound 4-hydroxymethyl-(4′hydroxyphenyl)-cyclohexane and in particular the enantiomer:
otherwise referred to herein as “ISP358-2”.
The disclosed compounds may include 4-substituted-(4′-hydroxyphenyl)cyclohexene compounds. For example, the disclosed compounds may have a Formula Ia(i):
where X, Y, and Z are as defined for Formula I.
The disclosed compounds may be used to prepare and formulate pharmaceutical compositions. As such, also disclosed herein are pharmaceutical compositions comprising an effective amount of any of the compounds disclosed herein, or pharmaceutically acceptable salts of any of the compounds disclosed herein, together with a pharmaceutically acceptable excipient, carrier, or diluent.
The disclosed compounds may be used for preparing a medicament for treating a disease or disorder associated with estrogen receptor 13 (ERβ) activity, and in particular, a disease or disorder that may be treated with an agonist of ERβ. As such, the disclosed compounds may exhibit ERβ agonist activity, and preferable the compounds exhibit specificity as ERβ agonists versus activity as ERβ antagonists and/or versus activity as estrogen receptor α (ERα) agonists or activity as ERα antagonists. The disclosed compounds may be formulated for use in treating psychiatric or neurological diseases or disorders. In particular, the disclosed compounds may be formulated for use in treating a subject in need of enhanced memory consolidation, for example, enhanced memory consolidation under low estrogen conditions observed in post-menopausal women.
The present invention is described herein using several definitions, as set forth below and throughout the application.
Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a substitution” should be interpreted to mean “one or more substitutions.” Similarly, “a substituent group” should be interpreted to mean “one or more substituent groups.”
As used herein, “about,” “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of these terms which are not clear to persons of ordinary skill in the art given the context in which they are used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.
As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
As used herein, a “subject in need thereof” may include a human or a non-human animal. The term “subject” may be used interchangeably with the terms “individual” or “patient.”
As used herein, a “subject in need thereof” may include a subject in need of treatment with an agonist of the estrogen receptor beta isoform (ERβ). A subject in need of treatment with an agonist of ERβ may include a subject having subject having a disease or disorder associated with ERβ activity. Diseases and disorders associated with ERβ activity may include, but are not limited to, cell proliferative diseases and disorders (e.g., cancers such as breast cancer, ovarian cancer, and endometrial cancer), psychiatric diseases and disorders (e.g., depression, anxiety, and schizophrenia), neurodegenerative diseases or disorders (e.g., Alzheimer” s disease including APOE4 associated Alzheimer's disease), memory decline (e.g., memory decline observed under low estrogen conditions as those observed in post-menopausal women), bone metabolic diseases or disorders (e.g. osteoporosis), metabolic diseases or disorders (e.g., obesity or insulin resistance), and cardiovascular diseases or disorders.
A subject in need thereof may include a subject exhibiting low estrogen (i.e., estradiol) serum levels. A subject exhibiting low estrogen serum levels may be exhibiting low estrogen serum levels associated with menopause (e.g., as observed in post-menopausal women). A subject exhibiting low estrogen serum levels may include a subject exhibiting estrogen serum levels of less than about 60 pg/ml, 55 pg/ml, 50 pg/ml, 45 pg/ml, 40 pg/ml, 35 pg/ml, 30 pg/ml, 25 pg/ml, 20 pg/ml, 15 pg/ml, 10 pg/ml, 5 pg/ml, or less, or exhibiting estrogen serum levels within a range bounded by any of these values (e.g., within a range of 15-60 pg/ml).
A subject in need thereof may include a subject exhibiting low estrogen serum levels associated with the subject having been administered a therapy and/or treatment which reduces estrogen serum levels and/or estrogen activity in the subject. A subject in need thereof may include a subject undergoing therapy for cancer treatment or therapy after cancer treatment (e.g., therapy for breast cancer treatment and/or therapy after breast cancer treatment). A subject in need thereof may include a subject undergoing hormone therapy (e.g., hormone therapy for cancer such as breast cancer) and/or a subject undergoing hormone replacement therapy (e.g., hormone replacement therapy after treatment for cancer such as breast cancer). A subject in need thereof may include a subject undergoing treatment with drugs that may include, but are not limited to, tamoxifen, toremifene (Fareston), fulvestrant (Faslodex), aromatase inhibitors (e.g., letrozole (Femara), anastrozole (Arimidex), and exemestane (Aromasin)), lutenizing hormone-releasing hormone (LHRH) analogs (e.g., goserelin (Zoladex) and Leuprolide (Lupron)). A subject in need thereof may include a subject having undergone an oophorectomy and/or a hysterectomy.
Disclosed are substituted (4′-hydroxylphenyl)cycloalkane compounds and (4′-hydroxylphenyl)cycloalkene compounds and there use as selective agonists of the estrogen receptor beta isoform (ERβ). Several compounds of this class of compounds have been previously described in U.S. Patent Pub. No. 2016/0340279 to Donaldson et al., the contents of which are incorporated herein by reference in its entirety. The disclosed compounds may alternatively be referred to as substituted 4-cycloalkylphenol compounds or p-cycloalkyl substituted phenol compounds that include one or more substitutions on the cycloalkyl substituent, which cycloalkyl substituent preferably is a cyclohexyl substituent.
In some embodiments, the disclosed compounds include one or more substitutions on the 4-carbon of the cycloalkyl substituent and have a Formula I:
where:
The alkyl moiety of the X or Y substituents may be a C(1-6)alkyl. In certain embodiments, the alkyl moiety may be a C(1-3)alkyl. The hydroxyalkyl moiety of the X or Y substituents may be a hydroxyl-C(1-6)alkyl. In certain embodiments, the hydroxyalkyl moiety may be a hydroxyl-C(1-3)alkyl. The aminoalkyl moiety of the X or Y substituents may be a amino-C(1-6)alkyl. In certain embodiments, the aminoalkyl moiety may be a amino-C(1-3)alkyl.
The alkyl portion of the carboxyalkylidenyl, esteralkylidenyl, hydroxyalkylidenyl, or aminoalkylidenyl moieties may be a C(1-6)alkyl. In certain embodiments, alkyl portion of the carboxyalkylidenyl, esteralkylidenyl, hydroxyalkylidenyl, or aminoalkylidenyl may be a C(1-3)alkyl. For example, the carboxyalkylidenyl may be a carboxy-C(1-6)alkylidenyl or carboxy-C(1-3)alkylidenyl; the esteralkylidenyl may be a C(1-6)alkyl-ester-C(1-6)alkylidenyl or C(1-3)alkyl-ester-C(1-3)alkylidenyl; the hydroxyalkylidenyl may be a hydroxy-C(1-6)alkylidenyl or hydroxy-C(1-3)alkylidenyl; or the aminoalkylidenyl may be a amino-C(1-6)alkylidenyl or amino-C(1-3)alkylidenyl.
The disclosed compounds may include 4-substituted-(4′-hydroxyphenyl)cyclohexane compounds. For example, in the disclosed compounds having Formula I, A-B may be —CH2CH2—, A′-B′ may be —CH2CH2—, and the compound may have a Formula Ia
where X and Y are as defined for Formula I. In some embodiments of compounds having Formula Ia, substituent X is selected from hydrogen, hydroxyl, and hydroxyalkyl and Y is selected from hydrogen, hydroxyl, alkyl, and —OCH2 and Y and Z form a bridge.
The disclosed compounds having Formula Ia may exhibit specific stereochemistry. For example, where X and Y are as defined for Formula I, the compounds may comprise cis and trans isomers of each other. For example, the compounds may comprise cis and trans isomers having the following formula.
In particular embodiments, the compound may be the isomer having the formula
In some embodiments of compounds having Formula Ia, X may be hydroxyalkyl and Y may be hydrogen. An exemplary compound may have the formula:
In some embodiments of compounds having Formula Ia, X may be hydroxy and Y may be hydrogen. An exemplary compound may have the formula:
In some embodiments of compounds having Formula Ia, X may be hydroxyalkyl and Y may be hydroxyl. An exemplary compound may have the formula:
In some embodiments of compounds having Formula Ia, X may be hydrogen and Y may be —OCH3— and form a bridge with Z. An exemplary compound may have the formula:
The disclosed compounds may include 4-substituted-(4′-hydroxyphenyl)cyclohexene compounds. For example, in the disclosed compounds having Formula I, A-B may be —CH2CH2—, A′-B′ may be ═CHCH2—, and the compound may have a Formula Ia(i)
where X and Y are as defined for Formula I. In some embodiments of compounds having Formula Ia(i), substituent X is selected from hydrogen, hydroxyl, and hydroxyalkyl and Y is selected from hydrogen, hydroxyl, alkyl.
In some embodiments of compounds having Formula Ia(i), X may be hydroxyalkyl and Y may be hydrogen. An exemplary compound may have the formula:
The compounds disclosed herein (e.g., compounds having any of Formula I, Ia, and Ia(i) may have several chiral centers, and stereoisomers, epimers, and enantiomers of the disclosed compounds are contemplated. The compounds may be optically pure with respect to one or more chiral centers (e.g., some or all of the chiral centers may be completely in the S configuration; and/or some or all of the chiral centers may be completely in the R configuration; etc.). Additionally or alternatively, one or more of the chiral centers may be present as a mixture of configurations (e.g., a racemic or another mixture of the R configuration and the S configuration). Compositions comprising substantially purified stereoisomers, epimers, or enantiomers of compound having any of Formula I, Ia, and Ia(i) are contemplated herein (e.g., a composition comprising at least about 90%, 95%, or 99% pure stereoisomer, epimer, or enantiomer).
Compositions contemplated herein may include compositions comprising the compound:
wherein the isomer
of the compound represents the majority of isomers of the compound in the composition (e.g. at least about 90%, 95%, or 99% of the isomers of the compound in the composition).
A compound which is a substantially pure stereoisomer, epimer, or enantiomer is contemplated herein, for example a compound which is at least about 90%, 95%, or 99% pure stereoisomer, epimer, or enantiomer. Contemplated herein is a compound which is a substantially pure (e.g., at least about 90%, 95%, or 99%) stereoisomer, epimer, or enantiomer of the compound:
Also disclosed herein are hydroxy-protected derivatives of the compounds disclosed herein. For example, the compounds disclosed herein (e.g., compounds having any of Formula I, Ia, and Ia(i), may include a hydroxy-protected group at any hydroxy group. As contemplated herein, a “protected-hydroxy” group is a hydroxy group derivatized or protected by any of the groups commonly used for the temporary or permanent protection of hydroxy functions (e.g., alkoxycarbonyl, acyl, silyl, or alkoxyalkyl groups). A “hydroxy-protecting group” signifies any group commonly used for the temporary protection of hydroxy functions, such as for example, alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups (hereinafter referred to simply as “silyl” groups), and alkoxyalkyl groups. Alkoxycarbonyl protecting groups are alkyl-O—CO— groupings such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl or allyloxycarbonyl. As contemplated herein, the word “alkyl” as used in the description or the claims, denotes a straight-chain or branched alkyl radical of 1 to 6 carbons, in all its isomeric forms. “Alkoxy” refers to any alkyl radical which is attached by oxygen (i.e., a group represented by “alkyl-O—”). Alkoxyalkyl protecting groups are groupings such as methoxymethyl, ethoxymethyl, methoxyethoxymethyl, or tetrahydrofuranyl and tetrahydropyranyl. Preferred silyl-protecting groups are trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, dibutylmethylsilyl, diphenylmethylsilyl, phenyldimethylsilyl, diphenyl-t-butylsilyl and analogous alkylated silyl radicals. The term “aryl” specifies a phenyl-, or an alkyl-, nitro- or halo-substituted phenyl group. The terms “hydroxyalkyl”, “deuteroalkyl” and “fluoroalkyl” refer to an alkyl radical substituted by one or more hydroxy, deuterium, or fluoro groups respectively. An “alkylidene” refers to a radical having the general formula —CkH2k— where K is an integer (e.g., 1-6). The term “acyl” signifies an alkanoyl group of 1 to 6 carbons, in all of its isomeric forms, or a carboxyalkanoyl group of 1 to 6 carbons, such as an oxalyl, malonyl, succinyl, glutaryl group, or an aromatic acyl group such as benzoyl, or a halo, nitro or alkyl substituted benzoyl group.
The compounds disclosed herein may exhibit binding and agonist and/or antagonist activity for estrogen receptors. As used herein, “ERα” refers to estrogen receptor-alpha, and in particular, human estrogen receptor-alpha. As used herein, “ERβ” refers to estrogen receptor-beta, and in particular human estrogen receptor-beta. Agonists and antagonists for ERα and ERβ are known in the art as are assays for determining the binding affinity of a compound for ERα and ERβ and determining whether a bound compound is an agonist or antagonist for ERα and ERβ. (See e.g., McCullough et al., “Probing the human estrogen receptor-α binding requirements for phenolic mono- and di-hydroxyl compounds: a combined synthesis, binding and docking study,” Biorg. & Med. Chem. (2014) Jan. 1; 22(1):303-10. doi: 10.1016/j.bmc.2013.11.024. Epub (2013) Nov. 21, and the corresponding Supplementary Information, the contents of which are incorporated herein by reference in their entireties). Suitable assays for determining the binding affinity of a compound for ERα and ERβ and determining whether a bound compound is an agonist or antagonist for ERα and ERβ may include fluorescence polarization displacement assays and cell-based ERα and ERβ luminescence activity assays.
As used herein, the term “selective agonist” may be used to refer to compounds that selectively bind and agonize an estrogen receptor, and in particular ERβ, relative to another estrogen receptor, and in particular ERα. For example, a compound that is a selective agonist for ERβ may have an IC50 (nM) in an assay for ERβ receptor agonist activity that is less than 100 nM, preferably less than 10 nM, even more preferably less than 1 nM; and a compound that is that is a selective agonist for ERβ may have an IC50 (nM) in an assay for ERα receptor agonist activity that is greater than 100 nM, preferably greater than 500 nM, even more preferably greater than 1000 nM.
As used herein, the term “selective agonist” may be used to refer to compounds that selectively bind to an estrogen receptor, and in particular, ERβ, relative to another estrogen receptor, and in particular ERα. For example, a compound that is a selective agonist for ERβ may have a binding affinity for ERβ receptor (e.g., as measured by Kd (nM)) that is at least 3-fold greater (or at least 5-fold greater, at least 10-fold greater, at least 20-fold greater, at least 50-fold greater, at least 100-fold greater, at least 500-fold greater, or at least 1000-fold greater) than a binding affinity for ERα. Preferably, a selective agonist for ERβ has a Kd (nM) for ERβ that is less than 100 nM, more preferably less than 10 nM, or even more preferably less than 1 nM; and preferably, a selective agonist for ERβ has a Kd (nM) for ERα that is greater than 500 nM, more preferably greater than 1000 nM, or even more preferably greater than 2000 nM.
As used herein, the term “selective agonist” may be used to refer to compounds that selectively bind and agonize an estrogen receptor, and in particular ERβ, instead of antagonizing an estrogen receptor, and in particular ERβ. For example, a compound that is a selective agonist for ERβ may have an IC50 (nM) in an assay for ERβ receptor agonist activity that is less than 100 nM, preferably less than 10 nM, even more preferably less than 1 nM; and a compound that is that is a selective agonist for ERβ may have an IC50 (nM) in an assay for ERβ receptor antagonist activity that is greater than 100 nM, preferably greater than 500 nM, even more preferably greater than 1000 nM.
Pharmaceutically acceptable salts of the disclosed compounds also are contemplated herein and may be utilized in the disclosed treatment methods. For example, a substituent group of the disclosed compounds may be protonated or deprotonated and may be present together with an anion or cation, respectively, as a pharmaceutically acceptable salt of the compound. The term “pharmaceutically acceptable salt” as used herein, refers to salts of the compounds which are substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds as disclosed herein with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts. It will be appreciated by the skilled reader that most or all of the compounds as disclosed herein are capable of forming salts and that the salt forms of pharmaceuticals are commonly used, often because they are more readily crystallized and purified than are the free acids or bases.
Acids commonly employed to form acid addition salts may include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of suitable pharmaceutically acceptable salts may include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleat-, butyne-.1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, alpha-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.
Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Bases useful in preparing such salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.
It should be recognized that the particular counter-ion forming a part of any salt of a compound disclosed herein is usually not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole. Undesired qualities may include undesirably solubility or toxicity.
It will be further appreciated that the disclosed compounds can be in equilibrium with various inner salts. For example, inner salts include salts wherein the compound includes a deprotonated substituent group and a protonated substituent group.
The disclosed compounds may be used to prepare and formulate pharmaceutical compositions. As such, also disclosed herein are pharmaceutical compositions comprising an effective amount of any of the compounds disclosed herein, or pharmaceutically acceptable salts of any of the compounds disclosed herein, together with a pharmaceutical excipient. In some embodiments, the disclosed compounds may be used for preparing a medicament for treating a disease or disorder associated with estrogen receptor 13 (ERβ) activity, and in particular, a disease or disorder that may be treated with a specific agonist of ERβ. As such, the disclosed compounds may exhibit ERβ agonist activity, and preferable the compounds exhibit specificity as an ERβ agonist versus an ERβ antagonist, an ERα agonist, and/or an ERα antagonist.
The disclosed compounds may be used to prepare and formulate pharmaceutical compositions for treating diseases that are associated with estrogen ERβ activity. Diseases and disorders associated with ERβ activity may include, but are not limited to, cell proliferative diseases and disorders (e.g., cancers such as breast cancer, ovarian cancer, and endometrial cancer), psychiatric diseases and disorders (e.g., depression, anxiety and/or schizophrenia), neurodegenerative diseases or disorders (e.g., Alzheimer's diseases including APOE4 associated Alzheimer's disease), memory decline (e.g., memory decline observed under low estrogen conditions as those observed in post-menopausal women), bone metabolic diseases or disorders (e.g. osteoporosis), metabolic diseases or disorders (e.g., obesity or insulin resistance), and cardiovascular diseases or disorders. The disclosed pharmaceutical compositions may be administered to subjects in need thereof in methods for treating diseases and disorders associated with ERβ activity.
The compounds and pharmaceutical compositions disclosed herein may be administered to a subject in need thereof to treat a disease or disorder. In some embodiments, the compounds disclosed herein may be administered at an effective concentration such that the compound functions as an agonist for ERβ in order to treat a disease or disorder associated with ERβ activity. In some embodiments, the amount of the disclosed compounds that is effective for the compound to function as an agonist of ERβ is about 0.05-50 μM (or about 0.05-10 μM, or about 0.05-1 μM).
As used herein, a “subject” may be interchangeable with “patient” or “individual” and means an animal, which may be a human or non-human animal, in need of treatment. Suitable subject s for the disclosed methods may include, for example mammals, such as humans, monkeys, dogs, cats, horses, rats, and mice. Suitable human subjects include, for example, those who have a disease or disorder associated with ERβ activity or those who have been determined to be at risk for developing a disease or disorder associated with ERβ activity. A subject in need of treatment may include a post-menopausal woman (e.g., a post-menopausal woman exhibiting low estrogen).
As used herein, a “subject in need of treatment” may include a subject having a disease, disorder, or condition that is responsive to therapy with an ERβ agonist. For example, a “subject in need of treatment” may include a subject having a cell proliferative disease, disorder, or condition such as cancer (e.g., cancers such as breast cancer). In addition, a “subject in need of treatment” may include a subject having a neurological disease or disorder including psychiatric diseases and disorders (e.g., depression, anxiety, and/or schizophrenia). A “subject in need thereof” may include a subject having a neurodegenerative disease or disorder (e.g., Alzheimer's disease including APOE4 associated Alzheimer's disease). In particular, a subject in need thereof may include a subject exhibiting memory loss or the need for enhanced memory consolidation (e.g., a subject having a disease or disorder characterized by a need for enhanced memory consolidation under low-estrogen conditions). A subject in need thereof may include a post-menopausal woman in need of enhanced memory consolidation (e.g., a post-menopausal woman in need of enhanced memory consolidation under low-estrogen conditions).
As used herein, the terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and/or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disorder. As such, the methods disclosed herein encompass both therapeutic and prophylactic administration.
As used herein the term “effective amount” refers to the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment. The disclosed methods may include administering an effective amount of the disclosed compounds (e.g., as present in a pharmaceutical composition) for treating a disease or disorder associated with ERβ activity in a subject, whereby the effective amount induces, promotes, or causes ERβ agonist activity in the subject.
An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.
In some embodiments, a daily dose of the disclosed compounds may contain from about 0.01 mg/kg to about 100 mg/kg (such as from about 0.05 mg/kg to about 50 mg/kg and/or from about 0.1 mg/kg to about 25 mg/kg) of each compound used in the present method of treatment. The dose may be administered under any suitable regimen (e.g., weekly, daily, twice daily).
The pharmaceutical compositions for use according to the methods as disclosed herein may include be a single compound as an active ingredient or a combination of compounds as active ingredients. For example, the methods disclosed herein may be practiced using a composition containing a single compound that is an ERβ agonist. Alternatively, the disclosed methods may be practiced using a composition containing two or more compounds that are ERβ agonists, or a compound that is an ERβ agonist together with a compound that is an ERα antagonist.
Instead of administering a pharmaceutical composition comprising a compound that is an ERβ agonist together with a compound that is an ERα antagonist, the disclosed methods may be practiced by administering a first pharmaceutical composition (e.g., a pharmaceutical composition comprising an ERβ agonist) and administering a second pharmaceutical composition (e.g., a pharmaceutical composition comprising an ERα antagonist), where the first composition may be administered before, concurrently with, or after the second composition. As such, the first pharmaceutical composition and the second pharmaceutical composition may be administered concurrently or in any order, irrespective of their names.
As one skilled in the art will also appreciate, the disclosed pharmaceutical compositions can be prepared with materials (e.g., actives excipients, carriers, and diluents etc.) having properties (e.g., purity) that render the formulation suitable for administration to humans. Alternatively, the formulation can be prepared with materials having purity and/or other properties that render the formulation suitable for administration to non-human subjects, but not suitable for administration to humans.
The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition in solid dosage form, although any pharmaceutically acceptable dosage form can be utilized. Exemplary solid dosage forms include, but are not limited to, tablets, capsules, sachets, lozenges, powders, pills, or granules, and the solid dosage form can be, for example, a fast melt dosage form, controlled release dosage form, lyophilized dosage form, delayed release dosage form, extended release dosage form, pulsatile release dosage form, mixed immediate release and controlled release dosage form, or a combination thereof. Alternatively, the compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition in liquid form (e.g., an injectable liquid or gel)
The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes an excipient, carrier, or diluent. For example, the excipient, carrier, or diluent may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.
The compounds utilized in the methods disclosed herein also may be formulated as a pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Filling agents may include lactose monohydrate, lactose anhydrous, and various starches; examples of binding agents are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™). Suitable lubricants, including agents that act on the flowability of the powder to be compressed, may include colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame. Examples of flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.
Suitable diluents for the pharmaceutical compositions may include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose® DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch; sorbitol; sucrose; and glucose.
The disclosed pharmaceutical compositions also may include disintegrants. Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof.
The disclosed pharmaceutical compositions also may include effervescent agents. Examples of effervescent agents are effervescent couples such as an organic acid and a carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.
Pharmaceutical compositions comprising the compounds may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).
Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.
Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis.
Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams.
For applications to the eye or other external tissues, for example the mouth and skin, the pharmaceutical compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the compound may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the compound may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops where the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.
Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.
Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas.
Pharmaceutical compositions adapted for nasal administration where the carrier is a solid include a coarse powder having a particle size (e.g., in the range 20 to 500 microns) which is administered in the manner in which snuff is taken (i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose). Suitable formulations where the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.
Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators.
Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.
Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
The following embodiments are illustrative and should not be interpreted to limit the scope of the claimed subject matter.
Embodiment 1. A compound having a formula and stereochemistry as follows:
Embodiment 2. The compound of embodiment 1 in substantially pure form.
Embodiment 3. A pharmaceutical composition comprising an effective amount of the compound of embodiment 1 preferably in substantially pure form (e.g., where the stereoisomer represents at least about 90%, 95%, or 99% of the compound in the composition), or a pharmaceutically acceptable salt thereof, together with a pharmaceutical excipient, carrier, or diluent.
Embodiment 4. A method for treating a disease or disorder associated with estrogen receptor β (ERβ) activity in a subject in need thereof, the method comprising administering to the subject the compound of embodiment 1 or 2 or the pharmaceutical composition of embodiment 3.
Embodiment 5. The method of embodiment 4, wherein the disease or disorder is selected from neurological, psychiatric, and cell proliferative diseases and disorders.
Embodiment 6. The method of embodiment 4, wherein the disease or disorder is associated with memory loss or memory dysfunction.
Embodiment 7. A method for enhancing memory consolidation in a subject in need thereof, the method comprising administering to the subject the compound of embodiment 1 or 2 or the pharmaceutical composition of embodiment 3.
Embodiment 8. A method for treating a subject exhibiting low estrogen levels, the method comprising administering to the subject the compound of embodiment 1 or 2 or the pharmaceutical composition of embodiment 3.
Embodiment 9. The method of embodiment 7 or embodiment 8, wherein the subject is a post-menopausal woman.
Embodiment 10. A compound having a formula selected from
Embodiment 11. A pharmaceutical composition comprising an effective amount of the compound of embodiment 10, or a pharmaceutically acceptable salt thereof, together with a pharmaceutical excipient, carrier, or diluent.
Embodiment 12. A method for treating a disease or disorder associated with estrogen receptor β (ERβ) activity in a subject in need thereof, the method comprising administering to the subject the compound of embodiment 10 or the pharmaceutical composition of embodiment 11.
Embodiment 13. The method of embodiment 12, wherein the disease or disorder is selected from neurological, psychiatric, and cell proliferative diseases and disorders (e.g., cancer such as breast cancer, ovarian cancer, endometrial cancer, and the like).
Embodiment 14. The method of embodiment 12, wherein the disease or disorder is associated with memory loss or memory dysfunction.
Embodiment 15. A method for enhancing memory consolidation in a subject in need thereof, the method comprising administering to the subject the compound of embodiment 10 or the pharmaceutical composition of embodiment 11.
Embodiment 16. A method for treating a subject exhibiting low estrogen levels, the method comprising administering to the subject the compound of embodiment 10 or the pharmaceutical composition of embodiment 11.
Embodiment 17. The method of embodiment 15 or 16, wherein the subject is a post-menopausal woman.
Embodiment 18. A method for enhancing memory consolidation in a subject in need thereof, the method comprising administering to the subject a compound or a pharmaceutical composition comprising the compound having a formula:
where:
(a) Z is a carbon atom;
(b) X is selected from the group consisting of hydrogen, hydroxyl, alkyl, hydroxyalkyl, amino, and aminoalkyl; and
(c) Y is selected from the group consisting of hydrogen, hydroxyl, alkyl, and hydroxyalkyl; or Y is —CH2CH2— or —OCH2— and Y and Z form a bridge; or X and Y together form alkylidenyl, carboxyalkylidenyl, esteralkylidenyl, hydroxyalkylidenyl, hydroxyalkylalkylidenyl, aminoalkylidenyl, oxo, or oxime.
Embodiment 19. The method of embodiment 18, wherein the compound has a Formula Ia:
Embodiment 20. The method of embodiment 18 or 19, wherein in the compound X is selected from hydrogen, hydroxyl, alklyl, and hydroxyalkyl; and Y is selected from hydrogen, hydroxyl, alkyl, and hydroxyalkyl; or Y is —OCH2— and Y and Z form a bridge.
Embodiment 21. The method of embodiment 18, wherein the compound has a Formula Ia(i):
Embodiment 22. The method of embodiment 21, wherein in the compound X is selected from hydrogen, hydroxyl, alkyl, hydroxylalkyl and Y is hydrogen.
Embodiment 23. The method of embodiment 18, wherein in the compound X is hydrogen or methyl, and Y is hydroxymethyl (—CH2OH) or hydroxyethyl (—CH2CH2OH).
Embodiment 24. The method of embodiment 18, wherein in the compound X is methyl, and Y is Y is hydroxymethyl (—CH2OH).
The following examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.
Estrogen receptor-beta (ERβ) is a drug target for memory consolidation in post-menopausal women. Herein is reported a series of potent and selective ERβ agonists (SERBAs) with in vivo efficacy that are A-C estrogens, lacking the B and D estrogen rings. The most potent and selective A-C estrogen is selective for activating ER relative to seven other nuclear hormone receptors, with a surprising 750-fold selectivity for the beta over alpha isoform, and with IC50's of 20-30 nM in cell-based and direct binding assays. Comparison of potency in different assays suggests that the ER isoform selectivity is related to the compound's ability to drive the productive conformational change needed to activate transcription. The compound disclosed herein also shows in vivo efficacy after microinfusion into the dorsal hippocampus, and after intraperitoneal injection (0.5 mg/kg) or oral gavage delivery (5 mg/kg). This simple yet novel A-C estrogen is selective, brain penetrant, and facilitates memory consolidation.
Results
Compound synthesis. Commercially available 4-(4-hydroxyphenyl)cyclohexanone 1 was transformed into 2° or 3° alcohols 2 or 3 by reaction with NaBH4 or excess methyl lithium respectively, or into oxime 4 by condensation with hydroxylamine (Scheme 1).
The stereochemistries of 2 and 3 were assigned based on their NMR spectral data. In particular, for 2° alcohol 2, the alcohol methane proton appears as a triplet of triplets at δ 2.38 (J=11.8, 3.4 Hz); the large couplings are consistent with an axial-axial disposition of this proton, and thus the hydroxyl group is equatorial. Signals at δ 69.5 and 31.1 ppm in the 13C NMR spectrum of 3, assigned to the 3° alcohol and methyl carbons, are in good agreement with cis-1,4-alcohols of this type24. The t-butyldimethylsilyl ether 5 underwent olefination with the ylide generated from methyltriphenylphosphonium bromide to give 6. Cleavage of the silyl ether using TBAF gave 7; catalytic hydrogenation of 7 gave 8 as a mixture of stereoisomers. Reaction of 7 with excess paraformaldehyde, MgCl2 and NEt3 gave the substituted salicaldehyde 9, which upon reaction with hydroxylamine afforded the oxime 10. Dihydroxylation of 6, followed by cleavage of the silyl ether gave 12, as a single stereoisomer after chromatographic purification. The stereochemistry of 12 was assigned as indicated, based on the known stereochemistry of osmium-catalyzed dihydroxylation of 4-t-butylmethylenecyclohexane.25 Hydroboration-oxidation of 6 using BH3-THF, produced an inseparable mixture of stereoisomeric primary alcohols cis-13 and trans-14, in a 2:1 ratio as determined by integration of the 1H NMR signals for the hydroxymethylene protons for each (δ 3.60 and 3.39 ppm respectively). The stereochemistry of the isomers was tentatively assigned on the basis of the relative chemical shift of these two signals; the signal for an axial hydroxymethylene (i.e. cis-isomer) appears downfield compared to that for an equatorial hydroxymethylene.24 Alternatively, hydroboration-oxidation using 9-BBN afforded a mixture in which trans-14 was in greater proportion compared to cis-13 (2:3, cis:trans). The use of these two borane reagents to tune the cis:trans outcome for 4-substituted methylenecyclohexanes has previously been reported.26,27 Cleavage of the silyl ether using TBAF gave a mixture of stereoisomeric 4-(4-hydroxymethylcyclohexyl)phenols cis-15/trans-16. Treatment of a mixture of the stereoisomers 15/16 (2:3, cis: trans) with DDQ (0.5 equiv.) led to a separable mixture of a bicyclic ether 17 and trans-16. The tentative structural assignment for trans-16 was corroborated by single crystal X-ray diffraction analysis (
The structure of 19 was assigned on the basis of its NMR spectral data; in particular, the signal for the olefinic proton appears as a narrow multiplet at ca. δ 5.95 ppm. This signal is characteristic of other 1-(4-hydroxyphenyl)cyclohexenes.29
Silyl ether 20 (prepared from 1) underwent Horner-Emmons olefination with triethyl phosphonoacetate to afford the unsaturated ester (±)-21; desilylation with TBAF gave the phenol (±)-22 (Scheme 3).
Reduction of 21 with DIBAL, followed by deprotection of the silyl ether yielded the allylic alcohol (±)-24. Catalytic hydrogenation of 24 gave a separable mixture of alcohol 25 and the over reduced ethyl cyclohexane derivative 26.
TR-FRET and Cell-based Transcriptional Assays. Initial screening of compounds was performed in a TR-FRET displacement assay, which detects binding to the ERβ LBD (see
The most potent compounds were 16 (hereafter referred to as ISP358-2) (hydroxymethyl substitution), 25 (hydroxyethyl substitution), and 8 (methyl substitution) which all had IC50s<30 nM for ERβ. ISP358-2 is the pure trans isomer, and was found to bind with higher affinity to ERβ than the mixture of cis- and trans-stereoisomers (15/16). While having a methylene (ISP358-2) or ethylene (25) linker to the hydroxyl group leads to potency, the direct substitution of the hydroxyl on the cyclohexane ring yields a significant decrease in affinity (IC50 of 7,250 nM for 2). While introduction of unsaturation into the alkyl linker (24) also led to a decrease in affinity (676 nM), introduction of unsaturation into the cyclohexane ring (18) only decreased affinity modestly (49 nM). ISP358-2 was also tested for binding to ERα, and bound with only 12-fold higher affinity to ERβ (IC50 of 24 nM;
ISP358-2 was further screened in a nuclear hormone receptor functional assay (
When the coactivator form of the TR-FRET LBD binding assay was performed (
Finally, a cell-based transcriptional activation assay, which employs a full length and native ER (comprised of an ER LBD and an ER DBD), was performed. Unlike the previous assays, this assay is cell-based, so best mimics the in vivo situation. The most potent and selective compound tested in this assay is ISP358-2, which has an ERβ agonist potency of 31±7 nM (
In Vitro Druggability—CYP450 Binding, hERG and Nephelometry. ISP358-2 shows no inhibition of CYP1A2 and CYP2D6, and only weak inhibition of CYP2C9 (IC50=34+4.7 μM) and CYP3A4 (IC50=89±18 μM) (
Docking Studies. ISP358-2 (
Assessment of Memory Consolidation. Dorsal hippocampal infusion. We first investigated the effects of direct intrahippocampal infusion of ISP358-2 on object recognition and spatial memory consolidation in ovariectomized mice (
Results for object recognition (
Intraperitoneal injection. We next used a new set of mice to investigate whether systemic administration of ISP358-2 also provide similar memory enhancing effects as intrahippocampal infusion (
For object placement (
Similar results were observed for object recognition (
Oral gavage. Given the mnemonic effectiveness of IP injection, we next assessed whether oral administration of ISP358-2 could enhance memory consolidation (
Similar results were observed for oral administration as were observed for IP injection ISP358-2. For object placement (
Finally, we collected preliminary data to assess the effectiveness of orally-gavaged ISP358-2 on spatial memory consolidation in mice who experienced long-term estrogen deprivation. Mice that received i.p. injections of vehicle, DPN, or ISP358-2 above remained in our colony for 4 months after ovariectomy. They were then trained in the object placement task (using new objects) and then immediately given vehicle, DPN, or ISP358-2 via oral gavage in the same doses described above (n=9-12/group). Unlike mice gavaged within 1 month of ovariectomy (
Assessment of Peripheral Pathology or Cell Proliferation Due to ISP358-2 Treatment. In general, all the tissues from the twenty different specimens appeared similar. Heart: the cardiac tissues were all unremarkable. The ventricular walls were intact and normal thickness. The atrial walls were intact with normal thickness. There was no evidence of congenital defects such as myofiber disarray or ischemic heart disease or ischemic injury. There was no evidence of inflammation or myocarditis. Kidney: The kidneys were all unremarkable. The glomeruli were intact. The tubules appeared normal. There was no evidence of inflammation involving any of the structures of the kidneys. Liver: The general architecture of the liver was intact and normal appearing with large portal-types veins running together with hepatic ducts and hepatic arteries. The central veins were present and normal appearing. A generalized appearance of low grade/mild ischemic injury was present in all samples. This seemed non-specific, was appreciated in all specimens, and could be secondary to early ischemic damage or autolysis that occurred post mortem. In several animals, small foci of cellular necrosis were observed likely secondary to ischemia. Two animals showed areas with mild, low grade inflammation that was small and focal. One animal (R15-IP-24 V) had multifocal areas of an organized inflammatory infiltrate composed primarily of mononuclear lymphocytes. Overall there was no evidence of acute inflammation composed of neutrophils or damage to structures in the liver, such as hepatic ducts. Bloodwork chemistry and hematology data (
ERβ has previously been pursued as a drug target for a wide range of conditions, including anxiety, depression, schizophrenia, and Alzheimer's disease, with representative ERβ drug lead agonist compounds shown in
Structure Activity Relationship for the A-C Estrogens. The binding affinity of 4-(4-substituted cyclohexyl)phenols were assessed in a TR-FRET ERβ binding assay (Table 1 and
The potency differences observed in the assays may be due to the nature of what the assays measure. The TR-FRET assay in
All compounds tested showed no significant ERβ or ERα antagonist activity (EC50>10,000 nM), thus also demonstrating their selectivity as agonist vs. antagonist activity (Table 1 and
Assay Differences Suggest Mechanism for Isoform Selectivity. As mentioned above, the cell-based assay for ISP358-2 indicates that it is ≅750 fold selective for ERβ agonist activity over ERα agonist activity (
Consistent with the above hypothesis that ISP358-2 potency and selectivity is related to its ability to drive a productive conformational change, docking studies show that ISP358-2 docks into the ERβ active site in a conformation that differs significantly from that for the ERα binding site. Key differences occur where the estrogen C ring is normally located (
Druggability and Preliminary Safety Toxicity. While ISP358-2 binds to ER and activates transcription, it shows no significant off-target activity with seven other nuclear hormone receptors (
To assess the potential of ISP358-2 to stimulate breast cancer cell growth, MTT assays with MCF-7 human breast cancer cells were performed (
To assess potential peripheral pathology due to ISP358-2 treatment, a histological analysis of tissue slices of treated animals was performed (
In vivo Efficacy. In vivo behavioral assays, measuring object placement or object recognition (
Finally, while it was observed that ISP358-2 is highly selective for ERβ over ERα in the more biologically relevant cell-based assay, it is not known if it has this same selectivity for ERβ in vivo. However, our preliminary studies have shown a correlation between behavioral results and levels of ERβ in the brain, consistent with the effect being related to ERβ agonist activity (
The results of the current study demonstrate that our lead compound, ISP358-2, is selective for ERβ, and shows no obvious signs of peripheral toxicity. Importantly, ISP358-2 also enhances multiple types of memory dependent on the hippocampus, a brain region involved in numerous disorders including AD, depression, and schizophrenia.43, 46 ISP358-2 is distinct from previously reported ERβ agonists in that it has higher selectivity for ERβ over ERα, and in that it more closely resembles that naturally occurring 17β-estradiol molecule (
Experimental Section
Compound Synthesis. All the chemicals were purchased from Sigma-Aldrich, Matrix Scientific, or Alfa Aesar and used as received. Reactions with moisture- or air-sensitive reagents were conducted under an inert atmosphere of nitrogen in oven-dried glassware with anhydrous solvents. Reactions were followed by TLC on precoated silica plates (60 Å, F254, EMD Chemicals Inc) and were visualized by UV lamp (UVGL-25, 254/365 nm). Flash column chromatography was performed by using flash silica gel (32-63μ). NMR spectra were recorded on Varian UnityInova 400 MHz instrument. CDCl3, d6-acetone, and CD3OD were purchased from Cambridge Isotope Laboratories. 1H NMR spectra were calibrated to δ=7.26 ppm for residual CHCl3, δ=2.05 ppm for d5-acetone and δ=3.30 ppm for residual d3-CD3OD. 13C NMR spectra were calibrated from the central peak at δ=77.23 ppm for CDCl3, δ=29.92 ppm for d6-acetone and δ=49.00 ppm for CD3OD. Purity of all compounds was >95%, determined with chromatography and NMR.
trans-4-(4-Hydroxycyclohexyl)phenol (2). To a solution of 1 (0.200 g, 5.30 mmol) in anhydrous methanol (15 mL) at room temperature was added solid NaBH4 (0.400 g, 10.6 mmol). The mixture was stirred for 3 h and then extracted with several times with ethyl acetate. The combined extracts were concentrated to give 2 (0.181 g, 90%) as a colorless solid. mp 196-208° C. 1H NMR (400 MHz, CD3OD) δ 7.00 and 6.67 (AA′ XX′, JAX=8.7 Hz, 4H), 3.61-3.53 (m, 1H), 2.38 (tt, J=11.8, 3.4 Hz, 1H), 2.05-1.98 (m, 2H), 1.87-1.78 (m, 2H), 1.56-1.30 (m, 4H) ppm. 13C NMR (100 MHz, CD3OD) 156.6, 139.2, 128.7, 116.1, 71.4, 49.3, 44.3, 36.9, 34.2 ppm. HRMS m/z 191.1077 [calcd for C12H15O2− (M-H+) 191.1077].
4-(4-Hydroxy-4-methylcyclohexyl)phenol (3). To a solution of 1 (0.100 g, 0.526 mmol) in dry ether (20 mL) at −78° C. under N2, was slowly added a solution of methyllithium-lithium bromide complex (1.5 M in ether, 0.78 mL, 1.2 mmol). The mixture was stirred at −78° C. for 30 min, warmed to room temperature and stirred for another 1 h. The mixture was cooled to 0° C. and quenched with water. The mixture was extracted several times with ether, and the combined extracts dried (Na2SO4) and concentrated. The residue was purified by column chromatography (SiO2, hexanes-ethyl acetate=4:1) to give 3 (0.040 g, 37%) as a colorless solid. mp 126-131° C.; 1H NMR (400 MHz, CD3OD) δ 7.03 and 6.67 (AA′XX′, JAX=8.3 Hz, 4H), 2.35 (tt, J=12.4, 3.6 Hz, 1H), 1.87-1.69 (m, 4H), 1.61-1.44 (m, 4H) 1.21 (s, 3H); 13C NMR (100 MHz, CD3OD) δ 156.5, 140.0, 128.8, 116.1, 69.5, 44.5, 40.0, 31.9, 31.1 ppm. HRMS m/z 205.1234 [calcd for C13H17O2− (M-H+) 205.1234]. Anal. calcd. for C13H18O2: C, 75.69; H 8.79. Found: C, 75.33; H, 8.83.
4-(4-Hydroxyphenyl)cyclohexanone oxime (4). To a solution of 1 (0.050 g, 0.26 mmol) in ethanol (10 mL), were added Amberlyst (0.060 g) and hydroxylamine hydrochloride (0.039 g, 0.560 mmol). The mixture was stirred at room temperature for 2 h and then filtered. The filtrate was concentrated and extracted several times with ethyl acetate. The combined organic extracts were washed with water, dried (MgSO4), and concentrated to give 4 as a colorless solid (0.037 g, 70%). mp 171-174° C.; 1H NMR (400 MHz, CD3OD) δ 7.00 and 6.69 (AA′XX′, JAX=8.2 Hz, 4H), 3.39 (broad d, J=13.5 Hz, 1H), 2.67 (t, J=12.8 Hz, 1H), 2.41 (broad d, J=14.0 Hz, 1H), 2.20 (td, J=14.6, 5.4 Hz, 1H), 1.93 (broad t, J=15.8 Hz, 2H), 1.81 (td, J=14.0, 5.2 Hz, 1H), 1.61-1.42 (m, 2H) ppm. 13C NMR (100 MHz, CD3OD) δ 160.8, 156.7, 138.3, 128.6, 116.2, 44.1, 35.8, 34.6, 32.8, 25.1 ppm. Anal. calcd. for C12H15NO2: C, 70.22; H 7.36; N, 6.83. Found: C, 69.93; H, 7.36; N, 6.63.
4-(4-t-Butyldimethylsilyloxyphenyl)cyclohexan-1-one (5). To a solution of 1 (0.500 g, 2.62 mmol) in anhydrous CH2Cl2 (30 mL) at 0° C. under N2, was added imidazole (0.357 g, 5.24 mmol). After 30 min t-butyldimethylsilyl chloride (0.594 g, 3.94 mmol) was added and the mixture was gradually warmed to room temperature overnight. The resulting mixture was diluted with brine (25 mL) and extracted several times with CH2Cl2. The combined organic extracts were dried (Na2SO4) and concentrated. The residue was purified by column chromatography (SiO2, hexanes-ethyl acetate=9:1) to give 5 (0.664, 83%) as a colorless solid. mp 39-42° C. 1H NMR (400 MHz, CDCl3) δ 7.08 and 6.78 (AA′XX′, JAX=8.4 Hz, 4H), 2.96 (t, J=12.3 Hz, 1H), 2.56-2.40 (m, 4H), 2.25-2.14, (m, 2H), 1.97-1.82 (m, 2H), 0.98 (s, 9H), 0.19 (s, 6H) ppm. 13C NMR (100 MHz, CDCl3) δ 211.6, 154.3, 137.7, 127.7, 120.1, 42.2, 41.6, 34.6, 25.9, 18.4, −4.2 ppm.
t-Butyldimethyl(4-(4-methylenecyclohexyl)phenoxy)silane (6). To a solution of methyltriphenylphosphonium bromide (0.836 g, 2.34 mmol) in dry THF (20 mL) at −10° C. under N2, was slowly added a solution of n-butyllithium (1.6 M in hexane, 1.50 mL, 2.4 mmol). After 30 min, a solution of 5 (0.502 g, 1.17 mmol) in dry THF (8 mL) was added dropwise. The reaction mixture was slowly warmed to room temperature and stirred overnight. After this time, the mixture was diluted with water (20 mL), extracted several times with ethyl acetate, and the combined extracts were dried (Na2SO4) and concentrated. Purification of the crude residue by column chromatography (SiO2, hexanes-ethyl acetate=9:1) gave 6 (1.678 g, 84%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.06 and 6.77 (AA′XX′, JAX=8.3 Hz, 4H), 4.68 (s, 2H), 2.62 (tt, J=12.1, 3.4 Hz, 1H), 2.42 (broad d, J=13.5 Hz, 2H), 2.18 (broad t, J=13.2, 2H), 2.00-1.93 (m, 2H), 1.57-1.45 (m, 2H), 0.99 (s, 9H), 0.20 (s, 6H) ppm. 13C NMR (100 MHz, CDCl3) δ 153.9, 149.2, 139.8, 127.8, 119.9, 107.4, 43.5, 35.9, 35.4, 25.9, 18.4, −4.2 ppm. Anal. calcd. for C19H30O2Si: C, 75.43; H, 9.99. Found: C, 75.71; H, 10.02.
4-(4-Hydroxyphenyl)methylenecyclohexane (7). To a solution of 6 (0.739 g, 0.244 mmol) in anhydrous THF (20 mL) was added a solution of TBAF (1 M in THF, 9.8 mL, 9.8 mmol). The mixture was heated at reflux for 5 h. After cooling, the solution was partitioned between ethyl acetate and water, and the aqueous layer was extracted several times with ethyl acetate. The combined organic layers were washed with brine, dried (Na2SO4) and concentrated. Purification of the residue by column chromatography (SiO2, hexanes-ethyl acetate=4:1) gave 7 (0.379 g, 83%) as a colorless solid. mp 82-84° C.; 1H NMR (400 MHz, CD3OD) δ 6.99 and 6.67 (AA′XX′, JAB=8.5 Hz, 4H), 4.63 (t, J=1.7 Hz, 2H), 2.57 (tt, J=12.3, 4.3 Hz, 1H), 2.41-2.33 (m, 2H), 2.22-2.11 (m, 2H), 1.94-1.85 (m, 2H), 1.45 (qd, J=12.3, 4.3 Hz, 2H); 13C NMR (100 MHz, CD3OD) δ 156.6, 150.3, 139.3, 128.8, 116.2, 107.8, 44.8, 37.3, 36.4 ppm. HRMS m/z 187.1128 [calcd for C13H15O− (M-H+) 187.1128]. Anal. calcd. for C13H16O2: C, 82.93; H 8.57. Found: C, 82.71; H, 8.58.
4-(4-Methylcyclohexyl)phenol (8). To a solution of 7 (0.150 g, 0.797 mmol) in methanol (10 mL) was added 10% Pd/C (85 mg, 10 mol %). The mixtures was stirred under a balloon filled with H2, at room temperature, for 12 h. The reaction mixture was filtered through a pad of celite, dried (Na2SO4) and concentrated. The residue was purified by column chromatography (SiO2, hexanes-ethyl acetate=4:1) to give 8 (0.121 g, 80%) as a colorless solid. This was determined to be a mixture of cis- and trans-stereoisomers by 1H NMR spectroscopy. mp 93-99° C.; 1H NMR (400 MHz, CD3OD) 7.05-6.96 (m, 2H), 6.70-6.64 (m, 2H), 2.48-2.28 (m, 1H), 1.83-1.34 (m, 8H), 1.13-1.04 (m, 1H), 1.03 (d, J=7.2 Hz, 1H), 0.92 (d, J=6.6 Hz, 2H); 13C NMR (100 MHz, CD3OD) δ 156.3, 140.0, 128.6, 116.0, 44.7, 36.9, 35.9, 33.7, 33.1, 30.1, 23.1 ppm.
2-Hydroxy-5-(4-methylenecyclohexyl)benzaldehyde (9). To a solution of 7 (0.100 g, 0.531 mmol) in dry CH3CN (20 mL) were sequentially added MgCl2 (0.076 g, 0.797), triethylamine (0.28 mL, 2.0 mmol), followed by paraformaldehyde (0.108 g, 3.59 mmol). The mixture was heated at reflux for 6 h. The mixture was cooled to room temperature and quenched with 10% HCl (10 mL) and extracted several times with ethyl acetate. The combined extracts were washed with brine, dried (Na2SO4) and concentrated. Purification of the residue by column chromatography (SiO2, hexanes-diethyl ether=4:1) gave 9 (0.046 g, 40%) as a colorless oil. 1H NMR (400 MHz, CD3OD) δ 9.96 (s, 1H), 7.50 (s, 1H), 7.39 (d, J=8.5 Hz, 1H), 6.85 (d, J=8.5 Hz, 1H), 4.65 (t, J=1.7 Hz, 2H), 2.68 (tt, J=12.0, 3.4 Hz, 1H), 2.44-2.34 (m, 2H), 2.24-2.13 (m, 2H), 1.97-1.89 (m, 2H), 1.49 (qd, J=13.0, 4.0 Hz, 2H); 13C NMR (100 MHz, CD3OD) δ 197.5, 161.0, 149.8, 139.9, 137.0, 131.6, 122.5, 118.2, 108.2, 44.3, 36.9, 36.2 ppm. HRMS m/z 231.1027 [calcd for C14H15O3 (M-H+) 231.1027].
2-Hydroxy-5-(4-methylenecyclohexyl)benzaldehyde oxime (10). To a solution of 9 (0.050 g, 0.232 mmol) in pure ethanol (10 mL), were added sodium bicarbonate (0.024 g, 0.278 mmol) and hydroxylamine hydrochloride (0.025 g, 0.348 mmol). The reaction was heated at 80° C. for 5 h and the mixture was extracted several times with ethyl acetate. The combined organic extracts were dried (MgSO4) and concentrated. Purification of the residue by column chromatography (SiO2, hexanes-ethyl acetate=13:7) gave 10 (0.037 g, 69%) as a colorless solid. mp 120-125° C.; 1H NMR (400 MHz, CD3OD) δ 8.20 (s, 1H), 7.09-7.06 (m, 1H), 7.05 (d, J=2.4 Hz, 1.8H), 6.99 (d, J=7.9 Hz, 0.2H), 6.78 (d, J=8.1 Hz, 0.8H), 6.68 (d, J=8.6 Hz, 0.2H), 4.63 (t, J=1.6 Hz, 2H), 2.60 (tt, J=12.2, 3.3 Hz, 1H), 2.42-2.33 (m, 2H), 2.22-2.10 (m, 2H), 1.94-1.85 (m, 2H), 1.46 (qd, J=12.5, 4.0 Hz, 2H); 13C NMR (100 MHz, CD3OD) δ 156.6, 152.4, 150.1, 139.4, 130.2, 129.2, 128.7, 118.5, 117.2, 116.2, 108.0, 107.7, 44.5, 37.3, 37.1, 36.4, 36.3 ppm. HRMS m/z 230.1187 [calcd for C14H16NO3 (M-H+) 230.1186].
4-(4-((t-Butyldimethylsilyl)oxy)phenyl)-1-(hydroxymethyl)cyclohexan-1-01 (11). To a solution of 6 (0.280 g, 0.926 mmol) and N-methylmorpholine-N-oxide (0.13 mL, 1.3 mmol) in acetone (6 mL) and distilled water (0.3 mL) was added a solution of OsO4 in tert-butanol (2.5%, 90 μL). The mixture was stirred overnight and saturated aqueous NaHSO3 (10 mL) was added to quench the reaction. The mixture was diluted with ether and washed several times with water. The organic layer was dried (MgSO4), concentrated and the residue purified by column chromatography (SiO2, hexanes-ethyl acetate=1:4) to give 11 (0.267 g, 86%) as a colorless solid. mp 80-86° C.; 1H NMR (400 MHz, CDCl3) δ 7.04 and 6.75 (AA′XX′, JAX=8.5 Hz, 4H), 3.69 (s, 2H), 2.52 (tt, J=11.4, 3.6 Hz, 1H), 2.04-1.72 (m, 4H, solvent peak overlap), 1.61-1.37 (m, 4H), 0.97 (s, 9H), 0.18 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 154.0, 138.9, 127.7, 120.0, 72.4, 66.2, 42.8, 35.4, 31.3, 25.9, 18.4, −4.2 ppm.
4-(4-Hydroxy-4-(hydroxymethyl)cyclohexyl)phenol (12). To a solution of 11 (0.230 g, 0.683 mmol) in anhydrous THF (10 mL) was added a solution of TBAF (1M in THF, 2.8 mL, 2.8 mmol). The mixture was heated at reflux for 6 h and cooled to room temperature. The solution was partitioned between ethyl acetate and water. The combined organic layers were washed with brine, dried (Na2SO4) and concentrated. Purification of the residue by column chromatography (SiO2, ethyl acetate-methanol=9:1) gave 12 (0.118 g, 78%) as a colorless solid. mp 182-188° C.; 1H NMR (400 MHz, CD3OD) δ 7.02 and 6.68 (AA′XX′, JAX=8.5 Hz, 4H), 3.62 (s, 2H), 2.53-2.42 (m, 1H), 1.99-1.89 (m, 2H), 1.85-1.68 (m, 2H), 1.58-1.43 (m, 4H); 13C NMR (100 MHz, CD3OD) δ 156.6, 138.9, 128.8, 116.2, 73.1, 66.6, 44.3, 36.0, 32.6 ppm. Anal. calcd. for C13H18O3: C, 70.24; H, 8.16. Found: C, 70.18; H, 7.78.
4-(4-t-Butyldimethylsilyloxyphenyl)cyclohexyl)methanol (13/14). To a solution of 6 (0.821 g, 2.71 mmol) in THF (24 mL) at 0° C. under N2, was added a solution of borane-THF complex (1 M in THF, 5.4 mL, 5.4 mmol). The reaction mixture was slowly warmed to room temperature and stirred for 20 h. The mixture was then cooled to 0° C., followed by sequential addition of ethanol (50 mL), hydrogen peroxide solution (30% in water, 4.0 mL) and 1N NaOH solution (20 mL). The mixture was warmed to room temperature and stirred for 90 min. The reaction mixture was quenched with saturated sodium bicarbonate solution (10 mL), diluted with water (20 mL) and extracted with several times with ethyl acetate. The combined organic extracts were washed with brine, dried (Na2SO4,) and concentrated. Purification of the residue by column chromatography (SiO2, hexanes-ethyl acetate=7:3) gave a colorless oil (0.572 g, 66%). This was determined to be a 2:1 mixture of cis-13 and trans-14 by 1H NMR integration of the signals for the CH2OH groups at δ 3.69 and 3.50 ppm respectively. 1H NMR (400 MHz, CDCl3) δ 7.06 and 6.76 (AA′XX′, JAX=8.5 Hz, 4H), 3.69 (d, J=7.4 Hz, 1.3H), 3.50 (d, J=6.5 Hz, 0.7H), 2.59-2.51 (m, 0.5H), 2.42 (tt, J=12.1, 3.8 Hz, 0.5H), 1.96-1.84 (m, 2H), 1.80-1.37 (m, 7H), 0.98 (s, 9H), 0.19 (s, 6H) ppm. 13C NMR (100 MHz, CDCl3) δ 153.7, 140.4, 140.0, 127.8, 119.9, 68.9, 64.6, 43.8, 42.6, 40.3, 36.2, 34.1, 30.0, 29.4, 27.0, 25.9, 18.4, −4.2 ppm. Use of 9-BBN instead of BH3-THF gave a 2:3 mixture of cis-13:trans-14 (74%).
4-(4-(Hydroxymethyl)cyclohexyl)phenol (15/16). To a solution of 13/14 (0.594 g, 1.85 mmol, 2:1 mixture c:t) in dry THF (10 mL) was added a solution of TBAF (1 M in THF, 7.5 mL, 7.5 mmol). The reaction mixture was heated to reflux at 70° C. overnight and cooled to room temperature. The solution was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried (Na2SO4) and concentrated. The residue was purified by column chromatography (SiO2, hexanes-ethyl acetate=3:2) to give a colorless solid (0.280 g, 73%). This was determined to be a 2:1 mixture of cis-13 and trans-14 stereoisomers by 1H NMR integration of the signals for the CH2OH groups at δ 3.60 and 3.39 ppm respectively. mp 118-122° C. 1H NMR (400 MHz, CD3OD) δ 7.04-6.98 (m, 2H), 6.70-6.65 (m, 2H), 3.60 (d, J=7.6 Hz, 1.5H), 3.39 (d, J=6.6 Hz, 0.5H), 2.54-2.44 (m, 1H), 2.37 (tt, J=12.1, 3.4 Hz, 1H), 1.93-1.70 (m, 3H), 1.61 (d, J=6.3 Hz, 4H), 1.46-1.37 (m, 1H), 1.14-1.02 (m, 1H) ppm. 13C NMR (100 MHz, CD3OD) δ 156.2, 139.6, 128.7, 116.0, 68.0, 64.4, 45.2, 44.0, 41.4, 37.0, 35.4, 31.2, 30.5, 28.0 ppm.
1-(4-Hydroxyphenyl)-2-oxabicyclo[2.2.2]octane (17) and trans-(hydroxymethyl)cyclohexyl) phenol (16). To a solution of 15/16 (0.080 g, 0.388 mmol, 2:3 mixture of cis-15:trans-16) in anhydrous CH2Cl2 (20 mL) at −10° C., was slowly added, over a period of 30 min, a suspension of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (0.044 g, 0.194 mmol) in CH2Cl2 (4 mL). The green solution was stirred at 0° C. for 2 h and gradually warm to room temperature and stirred for another 3 h. The mixture was quenched by slow addition of saturated sodium bicarbonate solution at 0° C. After a 10 min the layers were separated and aqueous layer was extracted several times with CH2Cl2. The combined organic extracts were washed with brine, dried (Na2SO4), and concentrated. The residue was purified by column chromatography (SiO2, hexanes-ethyl acetate=3:2) to give 17 (0.029 g, 37%) followed by 16 (0.038 g, 47%) both as colorless solids. Purity of 16 was established by 1H NMR (
17: mp 120-124° C.; 1H NMR (400 MHz, CD3OD) δ 7.18 and 6.64 (AA′XX′, JAX=7.9 Hz, 4H), 4.04 (s, 2H), 2.01 (t, J=7.8 Hz, 4H), 1.94-1.73 (m, 5H); 13C NMR (100 MHz, CD3OD) δ 157.3, 139.0, 127.2, 115.8, 73.2, 71.5, 34.7, 27.5, 26.1 ppm. Anal. calcd. for C13H16O2: C, 76.44; H, 7.89. Found: C, 76.39; H, 7.97.
16: mp 115-120° C.; 1H NMR (400 MHz, CD3OD) δ 7.00 and 6.68 (AA′XX′, JAX=8.7 Hz, 4H), 3.39 (d, J=6.7 Hz, 2H), 2.36 (tt, J=12.1, 3.0 Hz, 1H), 1.87 (broad t, J=15.4, 4H), 1.55-1.36 (m, 3H), 1.14-1.02 (m, 2H); 13C NMR (100 MHz, CD3OD) δ 156.5, 140.0, 128.7, 116.1, 68.9, 45.3, 41.5, 35.5, 31.3 ppm. Anal. calcd. for C13H18O2: C, 75.69; H, 8.79. Found: C, 75.66; H, 9.09.
4′-(Hydroxymethyl)-2′,3′,4′,5′-tetrahydro-[1,1′-biphenyl]-4-ol (±)-19. To a solution of 17 (0.103 g, 0.504 mmol) in dry CH3CN (25 mL) was added MgCl2 (0.072 g, 0.756 mmol) followed by triethylamine (0.26 mL, 1.89 mmol). The mixture was heated at reflux for 8 h, then cooled and quenched with 10% HCl (15 mL). The mixture was extracted several times with ethyl acetate, and the combined extracts washed with brine, dried (Na2SO4) and concentrated. Purification of the residue by column chromatography (SiO2, hexanes-ethyl acetate=13:7) gave 19 (0.080 g, 78%) as a colorless solid. mp 177-184° C.; 1H NMR (400 MHz, CD3OD) δ 7.20 and 6.69 (AA′XX′, JAX=8.6 Hz, 4H), 5.97-5.92 (m, 1H), 3.48 (dd, J=6.4, 2.6 Hz, 2H), 2.49-2.23 (m, 3H), 2.01-1.71 (m, 4H), 1.43-1.31 (m, 1H); 13C NMR (100 MHz, CD3OD) δ 157.5, 137.7, 135.3, 127.2, 122.1, 116.0, 68.0, 37.5, 30.1, 28.2, 27.2 ppm. HRMS m/z 203.1078 [calcd for C13H15O2− (M-H+) 203.1077].
4-(4-((t-Butyldiphenylsilyl)oxy)phenyl)cyclohexan-1-one (21). To a solution of 1 (0.815 g, 4.28 mmol) in dry CH2Cl2 (30 mL) at 0° C., was added imidazole (0.583 g 8.57 mmol) followed by dropwise addition of a solution of t-butyldiphenylsilyl chloride (1.60 mL, 5.57 mmol) in CH2Cl2 (9 mL). The reaction mixture was slowly warmed to room temperature and stirred for 12 h. The mixture was diluted with water and extracted several times with CH2Cl2. The combined extracts were washed with brine, dried (Na2SO4), and concentrated. The residue was purified by column chromatography (SiO2, hexanes-ethyl acetate=4:1) to give 21 (1.70 g, 93%) as a colorless solid. mp 83-84° C.; 1H NMR (400 MHz, CDCl3) δ 7.74-7.70 (m, 4H), 7.45-7.34 (m, 6H), 6.96 and 6.71 (AA′BB′, JAB=8.6 Hz, 4H), 2.90 (tt, J=12.1, 3.3 Hz, 1H), 2.49-2.42 (m, 4H), 2.19-2.10 (m, 2H), 1.91-1.77 (m, 2H), 1.09 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 211.6, 154.3, 137.3, 135.7, 133.2, 130.1, 127.9, 127.5, 119.8, 42.1, 41.6, 34.3, 26.7, 19.7 ppm.
Methyl 2-(4-(4-t-butyldiphenylsilyloxyphenyl)cyclohexylidene)acetate (±)-22. To a solution of trimethyl phosphonoacetate (0.160 mL, 0.980 mmol) in dry THF (5 mL) at 0° C., was added NaH (40 mg, 55% in mineral oil, 0.980 mmol). After stirring for 45 min, a solution of 21 (0.350 g, 0.816 mmol) in dry THF (5 mL) was added and the mixture was warmed to room temperature and stirred for 8 h. The mixture was diluted with water and extracted several times with ether. The combined extracts were dried (MgSO4) and concentrated. The residue was purified by column chromatography (SiO2, hexanes-ethyl acetate=9:1) to give 22 (0.376 g, 95%) as colorless gum. 1H NMR (400 MHz, CDCl3) δ 7.74-7.68 (m, 4H), 7.44-7.32 (m, 6H), 6.91 and 6.69 (AA′XX′, JAX=8.6 Hz, 4H), 5.65 (s, 1H), 3.96-3.88 (m, 1H), 3.69 (s, 3H), 2.66 (tt, J=12.1, 3.4 Hz, 1H), 2.38-2.24 (m, 2H), 2.04-1.93 (m, 3H), 1.59-1.46 (m, 2H), 1.08 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 167.4, 162.7, 154.0, 138.6, 135.7, 133.3, 130.0, 127.9, 127.5, 119.6, 113.3, 51.10, 43.3, 37.9, 35.9, 35.1, 29.7, 26.7, 19.7 ppm.
4-[(4-Hydroxyphenyl)cyclohexylidene]acetic acid ethyl ester (±)-23. To a stirring solution of 22 (60 mg, 0.12 mmol) in dry THF (1 mL) was added a solution of tetrabutylammonium fluoride (0.247 mL, 1.0 M in THF, 0.247 mmol). The solution was stirred at room temperature after 1 h, and then the mixture was diluted with water and extracted with ethyl acetate. The combined extracts were washed with brine, dried and concentrated. The residue was purified by preparative TLC (SiO2, hexanes-ethyl acetate=9:1) to give 23 (20 mg, 64%) as a colorless solid. mp 92-94° C.; 1H NMR (CDCl3, 300 MHz) δ 7.08 and 6.77 (AA′XX′, JAB=8.4 Hz, 4H), 5.68 (s, 1H), 4.58 (s, 1H), 4.17 (q, J=7.1 Hz, 2H), 4.00-3.90 (m, 1H), 2.80-2.68 (m, 1H), 2.45-1.97 (m, 6H), 1.30 (t, J=7.3 Hz, 3H); 13C NMR (CDCl3, 75 MHz) δ 167.0, 162.2, 154.0, 138.6, 128.1, 115.4, 113.9, 59.8, 43.4, 37.9, 36.0, 35.2, 29.7, 14.5 ppm. HRMS m/z 259.1339 [calcd for C16H19O3− (M-H+) 259.1340].
4-(4′-Hydroxyphenyl)(2-hydroxyethylidene)cyclohexane (±)-25. To a solution of 22 (275 mg, 0.551 mmol) in dry CH2Cl2 (2 mL) under N2 at −40° C. was added a solution of diisobutylaluminum hydride (1.0 M in CH2Cl2, 1.41 mL, 1.41 mmol). After 90 min, saturated aqueous potassium sodium tartrate was added and reaction mixture warmed to room temperature. After 2 h the layers were separated and the aqueous layer was extracted several times with CH2Cl2. The combined organic layers were dried, filtered through a pad of celite and concentrated to give 4-(4′-t-butyldiphenylsilyloxyphenyl)(2-hydroxyethylidene)cyclohexane (254 mg, quantitative) as a colorless gum. This compound was used without further purification. To a solution of the crude allylic alcohol (235 mg, 0.514 mmol) in dry THF (1 mL) under nitrogen was added a solution of tetrabutylammonium fluoride in (1.0 M in THF, 1.03 mL, 1.03 mmol). The solution was stirred for 3 h and then diluted with water and the resultant mixture extracted several times with ethyl acetate. The combined extracts were washed with brine, dried and concentrated. The residue was purified by column chromatography (SiO2, hexanes-ethyl acetate=4:1) to give 25 (90 mg, 80%) as a colorless solid. mp 165-166° C.; 1H NMR (d6-acetone, 300 MHz) δ 8.10 (s, 1H), 7.04 and 6.74 (AA′XX′, JAX=8.4 Hz, 4H), 5.36 (t, J=6.6 Hz, 1H), 4.17-4.02 (m, 2H), 2.78-2.70 (m, 1H), 2.64 (tt, J=3.3, 12.0 Hz, 1H), 2.35-2.10 (m, 2H), 1.98-1.80 (m, 4H), 1.54-1.37 (m, 2H); 13C NMR (d6-acetone, 75 MHz) δ 156.5, 141.1, 138.6, 128.5, 123.6, 116.0, 58.5, 44.6, 37.5, 37.0, 36.2, 29.2. Anal. calcd. for C14H18O2: C, 77.03; H 8.31. Found: C, 77.20; H, 8.28.
4-[4-(2-Hydroxyethyl)cyclohexyl]phenol (26) and 4-(4-ethylcyclohexyl)phenol (27). A solution of 25 (50 mg, 0.23 mmol) in methanol (15 mL) with small pinch of 20% Pd/C was stirred under H2 (30 psi) for 12 h. The reaction mixture was filtered through a pad of celite, concentrated and the residue was purified by preparative TLC (SiO2, hexanes-ethyl acetate=13:7) to give 27 (28 mg, 60%), followed by 26 (7 mg, 14%) both as colorless solids.
27: mp 120-125° C.; 1H NMR (d6-acetone, 300 MHz) δ 8.02 (s, 1H), 7.08-7.01 (m, 2H), 6.77-6.71 (m, 2H), 3.65-3.56 and 3.43-3.37 (m, 3H total), 2.52-2.33 (m, 1H), 1.91-1.00 (m, 11H); 13C NMR (d6-acetone, 75 MHz) δ 156.4, 139.5, 128.6, 115.9, 61.1, 60.4, 44.6, 41.4, 35.5, 34.8, 34.5, 31.1, 30.3 ppm. HRMS m/z 219.139 [calcd for O14H19O2− (M-H+) 219.1390].
26: mp 80-81° C.; 1H NMR (CDCl3, 300 MHz) δ 7.08 and 6.76 (AA′XX′, JAX=8.1 Hz, 4H), 4.55 (s, 1H), 2.54-2.35 (m, 1H), 1.92-1.82 (m, 2H), 1.70-1.50 (m, 3H), 1.45-1.00 (m, 6H), 0.91 (t, J=7.2 Hz, 3H) ppm.
TR-FRET Assay. LanthaScreen® TR-FRET ER Alpha and Beta Competitive Binding Assay kits from Thermo Fisher Scientific were used to perform the TR-FRET assays. These included a terbium-labeled anti-GST antibody, a fluorescent small molecule ER Alpha or Beta ligand as a “tracer”, and a human ER Alpha or Beta ligand-binding domain (LBD) that is tagged with glutathione-S-transferase (GST) in a homogenous mix-and-read assay format.
The TR-FRET assay employs a Tb-anti-GST antibody that binds to a GST tag, and a fluorescently labeled estrogen (tracer) binds in the active site pocket. The TR-FRET signal obtained decreases when competitor compounds displace the fluorescently labeled tracer. Assays were performed according to kit instructions. Briefly, 1:5 dilution series of compounds were made with DMSO, then diluted in assay buffer such that the highest concentration tested in the assay was 50 μM for ERβ and 50 μM for ERα and DMSO was 1%. Assays were set up in 384-well white, small volume plates (Corning® 4512). The assay was incubation for 1 hour in the dark at room temperature, after which plates were spun at 1000 rpm in a tabletop centrifuge equipped with a swing-out rotor (Eppendorf 5810, rotor A-4-64). TR-FRET signal was read on a SpectraMax M5 (Molecular Devices) set-up according to Thermo Fisher Scientific machine settings (excitation of 332 nm, emissions 518 nm and 488 nm with a 420 nm cutoff, 50 μs integration delay, 400 μs integration time, and 100 flashes per read). The TR-FRET ratio was calculated using the SoftmaxPro software by dividing the emission at 518 nm (fluorescein) by the emission at 488 nm (Terbium). Data were normalized to E2, which had an IC50 of 0.25±0.06 nM in this assay. Data analysis was done using Prism (GraphPad Software, Inc., La Jolla, Calif.), with fits typically constrained to go to zero at high concentrations of competing ligand. Standard deviations are for the nonlinear least squares fit of the data. When replicate assays and fits were done, curves are shown (
Nuclear Hormone Receptor Specificity Assay. Selectivity measurements were performed using the SelectScreen™ cell-based nuclear receptor profiling service from ThermoFisher (
Repeat assays for ERα and ERβ agonist assays in a 10-point curve were also completed (
Coactivator Assay. The LanthaScreen® TR-FRET assay from ThermoFisher was used (
Cell-based Assays. ERα and ERβ cell-based assays for both agonist and antagonist activity measurements were performed using kits provided by Indigo Biosciences (
As described for the TR-FRET assay fitting, IC50 values and standard deviations are from the nonlinear least squares fit of the data; and, when replicate assays and fits were done, median values were reported in Table 1.
In Vitro Druggability Assays—CYP450 Binding, hERG & Nephelometry. The P450-Glo™ Screening System from Promega Corporation (Madison, Wis.) was used to measure CYP450 (cytochrome P450) inhibition, as described in the kit instructions. Assays were run in 96-well white plates (Corning® 3912), and luminescence was measured on a SpectraMax M5 instrument (
Nephelometry was performed to determine the relative propensity of compounds to aggregate in solution (
hERG assays were performed using the SelectScreen service from ThermoFisher (
MTT Assays. Human breast cancer cells (MCF-7) were provided by Dr. Manish Patankar (Department of Obstetrics and Gynecology, University of Wisconsin-Madison). Cells were cultured in Eagle's Minimum Essential Media (EMEM) supplemented with 10% fetal bovine serum and 0.01 mg/mL human recombinant insulin in 5% CO2 at 37° C. A seeding density of 7,000 cells per well was chosen and applied to a 96 well plate. After 24 hours, treatments of ISP358-2, DPN or estradiol in media containing 0.1% Dimethyl sulfoxide (DMSO), were applied to the cells at varying concentrations (10, 1, 0.1, 0.01, and 0.001 μM). Negative, positive and untreated control cells received 100% DMSO, 0.01 μM estradiol in EMEM or EMEM with 0.1% DMSO content, respectively. Treated cells were incubated for 24 hours after which an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was performed by adding 20% MTT in EMEM solution to each well and incubating for 4 hours. Formazan crystal metabolites were dissolved using 100% DMSO and absorbance was read at OD 570 nm as well as a reference of 650 nm using a VMax kinetic microplate reader (Molecular Devices, CA) running Softmax Pro version 6.1. Absorbances were converted to cell number using a standard growth curve. Two-sample equal variance t-tests were conducted using Microsoft Excel to determine if cell proliferation was significantly different from untreated controls or cells treated with 0.1 μM E2.
Docking. Three dimensional (3D) conformations were prepared for all ligands, before docking (
Assessment of Memory Consolidation. Subjects. C57BL/6 female mice (8-10 weeks of age) were purchased from Taconic Biosciences. Mice were singly housed in a 12 h light/dark cycle room, with food and water ad libitum. All procedures with live mice were performed between 9:00 am and 6:00 pm in a room with a light intensity of dimmer than 100 lux. All procedures were approved by the University of Wisconsin-Milwaukee Institutional Animal Care and Use Committee and observed policies of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
General experimental design. A series of three experiments were conducted in mice that were bilaterally ovariectomized to remove the primary source of circulating estrogens. In each experiment, a negative control (dimethylsulfoxide, DMSO), positive control (2,3-bis(4-hydroxyphenyl)-propionitrile, DPN), and multiple doses of ISP358-2 were administered to separate groups of mice via one of three routes of administration: direct bilateral dorsal hippocampal infusion, intraperitoneal injection, or oral gavage. All drugs were administered acutely immediately after training in object recognition and object locations tasks designed to test hippocampal-dependent object recognition and spatial memory consolidation, respectively, as described below (
Surgery. Four days after arrival in the laboratory, mice were bilaterally ovariectomized as described previously.34, 58, 59 Mice slated to receive dorsal hippocampal infusion of ISP358-2 were also implanted with guide cannulae into the dorsal hippocampus (DH) as described previously.30-32 Mice were anesthetized with isoflurane gas (2% isoflurane in 100% oxygen) and placed in a stereotaxic apparatus (Kopf Instruments) Immediately after ovariectomy, mice were implanted with two guide cannulae (22 gauge; C232G, Plastics One) aimed at the dorsal hippocampus (−1.7 mm AP, ±1.5 mm ML, −2.3 mm DV). Dummy cannulae (C232DC, Plastics One) were placed inside the guide cannulae to conserve patency of the guide cannulae. Dental cement (Darby Dental) was applied to anchor the guide cannulae to the skull and also served to close the wound. Mice were allowed to recover for six days before behavioral testing.
Drugs and infusions. Dorsal hippocampal (DH) infusions or intraperitoneal (IP) injections were conducted immediately post-training as described previously.34, 58, 59 During infusions, mice were gently restrained and drugs delivered using an infusion cannula (C3131, 28-gauge, extending 0.8 mm beyond the 1.5 mm guide). The infusion cannula was connected to a 10 μl Hamilton syringe using PE20 polyethylene tubing. The infusion was controlled by a microinfusion pump (KDS Legato 180, KD Scientific) at a rate of 0.5 μl/minute. Each infusion was followed by a one-minute waiting period to prevent diffusion back up the cannula track and allow the drug to diffuse through the tissue. The negative control (“vehicle”) was 1% DMSO in 0.9% saline. As a positive control, the ERβ agonist DPN (2,3-bis(4-hydroxyphenyl)-propionitrile, Tocris Bioscience) was dissolved in 1% DMSO in saline and infused at a dose of 10 pg/hemisphere.30 DPN has a 70-fold higher affinity for ERβ than ERα,60 and bilateral infusion of 10 pg/hemisphere into the dorsal hippocampal previously enhanced memory consolidation in the object recognition and object placement tasks in young adult ovariectomized mice.34 ISP358-2 was dissolved in 1% DMSO to a concentration of 2 ng/μl and then diluted to administer doses of 1 ng/hemisphere, 100 pg/hemisphere, and 10 pg/hemisphere.
For intraperitoneal injections, ISP358-2 was dissolved in 10% DMSO in physiological saline and injected at doses of 0.5 or 5 mg/kg in a volume of 10 ml/kg. DPN was dissolved in 10% DMSO in saline and injected at a dose of 0.05 mg/kg in volume of 10 ml/kg. This dose previously enhanced object recognition memory consolidation in young adult ovariectomized mice.31 Vehicle controls received 10 ml/kg of 10% DMSO in saline. For oral gavage, all drugs were administered in a volume of 10 ml/kg at the same doses as intraperitoneal injections; 0.5 or 5 mg/kg ISP358-2 and 0.05 mg/kg DPN. Vehicle controls received 10% DMSO in saline. In the procedure, a bulb tipped gastric gavage needle (24 GA, 25 mm) was used to deliver the drugs directly to the stomach.
Memory testing. Object recognition and object placement were performed as described previously.34, 58, 59 Object recognition and object placement evaluated object recognition memory and spatial memory, respectively, and require intact dorsal hippocampal function.39, 61-63 Mice were first handled (30 sec/d) for 3 days to acclimate them to the experimenters. On the second day of handling, a small Lego was placed in the home cage to habituate the mice to objects. This Lego was removed from the cage just before training. After 3 days of handling, mice were habituated to an empty white arena (width, 60 cm; length, 60 cm; height, 47 cm) by allowing them to explore freely for 5 min each day for two days. On the training day, mice were habituated for 2 min in the arena, and then removed to their home cage. Two identical objects were then placed near the northwest and northeast corners of the arena. Mice were returned to the arena allowed to explore until they accumulated a total of 30 s exploring the objects (or until a total of 20 min had elapsed) Immediately after this training, mice were removed from the arena, infused, and then returned to their home cage. Object placement memory was tested 24 h after training by moving one of the training objects to the southeast or southwest corner of the box. Because mice inherently prefer novelty, mice that remember the location of the training objects spend more time with moved object than the unmoved object. Mice performing at chance (15 s) spend an equal amount of time with each object and demonstrate no memory consolidation. Thus, consolidation of memory for the training objects is demonstrated if mice spend significantly more time than chance with the moved object. Object recognition training was conducted two weeks after object placement. The object recognition task used the same apparatus and general procedure as object placement, but instead of changing the object location, one familiar object was replaced with a new object during testing. Object recognition testing occurred 48 h after training. As with object placement, mice accumulated 30 s exploring the novel and familiar objects. Because mice are inherently drawn to novelty, more time than chance spent exploring the novel object indicated memory for the familiar training object. To maintain novelty, different objects were used in the object placement and object recognition tasks. Because vehicle-infused female mice do not remember the location of the training objects 24 h after training,34 a 24-h delay was used to test the memory-enhancing effects of drugs in object placement. Similarly, because vehicle-infused female mice do not remember the familiar object 48 h after training,34 a 48-h delay was used to test the memory-enhancing effects of drugs in object recognition. For both tasks, the time spent exploring each object and elapsed time to accumulate 30 s of exploration were recorded using ANYmaze tracking software (Stoelting).
Behavioral data analysis. One-sample t-tests and one-way analyses of variance (ANOVAs) were conducted using GraphPad Prism 6 (La Jolla, Calif.). One-sample t-tests were used to determine whether mice spent significantly more time than chance (15 s) investigating the novel or moved object, indicating whether each group of mice successfully formed a memory of the identity and location of the training objects. To determine the extent to which DPN or ISP358-2 treatment influenced memory consolidation relative to vehicle, between-group comparisons were conducted for each behavioral task using one-way ANOVAs, followed by Fisher's LSD post hoc tests. Significance was determined at p>0.05.
Assessment of Potential Peripheral Pathology. To assess possible toxicity of ISP358-2 treatment to peripheral organs, ovariectomized mice received a single intraperitoneal injection of vehicle or ISP358-2, and liver, kidney, and heart tissues were collected 24 hours later. Similar to behavioral testing, ISP358-2 was injected at doses of 0.5 or 5 mg/kg in a volume of 10 ml/kg and DPN was injected at a dose of 0.05 mg/kg in a volume of 10 ml/kg. Vehicle controls received 10 ml/kg of 10% DMSO in saline (
The slides were then examined by a pathologist (ACM) who is board certified in anatomical pathology by the American Board of Pathology. All specimens contained three tissue samples corresponding to liver, kidney, and heart. In some instances, portions of adjacent tissues were also present. For example, several specimens had gall bladder. One specimen had a portion of spleen. Each organ was examined for specific pathological changes. Three major categories of change were examined: (1) structural changes to the organs. For liver, the central vein, portal triads, and hepatocytes were examined. For kidneys, the glomeruli and tubules were examined. For heart, the myocytes and coronary vessels were examined, (2) evidence of inflammation was evaluated including hepatitis, glomerulonephritis, interstitial nephritis, and myocarditis, and (3) evidence of ischemic changes was examined See the attached table for a summary of the findings.
In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
Citations to a number of patent and non-patent references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.
The present application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/572,932, filed on Oct. 16, 2017 and U.S. Provisional Application No. 62/478,758, filed on Mar. 30, 2017, the contents of which are incorporated herein by reference in their entireties.
This invention was made with government support under R15GM118304 awarded by the National Institute of General Medical Sciences and R01DA038042 awarded by the National Institute on Drug Abuse. The Government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/025342 | 3/30/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62478758 | Mar 2017 | US | |
| 62572932 | Oct 2017 | US |
