Patent Description:
The decreased production of estrogen in ovariectomized (OVX) or postmenopausal or anti-estrogen therapy women leads to estrogen-deficiency symptoms that may adversely affect their quality of life for decades. Estrogen replacement therapy (ERT) has been utilized to treat these symptoms since the <NUM>.

Estrogen binds to its receptors to regulate RNA transcription, stimulate cell proliferation and modulate metabolic signaling in many tissues during mammalian reproduction and development. Three genes for estrogenic binding proteins have been identified, encoding estrogen receptor (ER) α and β, and G-protein coupled estrogen receptor <NUM> (GPER1). ER-α and β have similar structural and functional domains, containing activation function domain <NUM> (AF-<NUM>), a DNA binding domain (DBD), a dimerization domain and activation function domain <NUM> (AF-<NUM>), which is the ligand binding domain (LBD). They both belong to the nuclear super family of ligand-dependent transcription factors and have highly conserved DBD and LBD regions. They regulate RNA transcription upon ligand binding, which results in ligand-receptor complexes that can dimerize and translocate into the nucleus, where they bind to estrogen response elements (EREs) found in the promoters of estrogen-responsive genes. This type of modulation is typically referred to as the classical estrogen pathway. ER-α and β also regulate diverse biological functions through membrane-initiated estrogen signaling (MIES), associating with plasma membrane by interaction with their ligand binding domain. The detailed molecular mechanisms of signaling by membrane-associated ERs are still unclear. The modulatory effects of estrogen mediated by membrane-associated receptors on cell proliferation, matrix/migration, metabolism and glucose homeostasis have been reviewed (<NUM>, <NUM>). Furthermore, studies on ER knockout mice indicate that ER-α is the dominant functional estrogen receptor, as compared to ER-β. Three transcription variants of ER-α, <NUM>, <NUM> and <NUM>, have been found. ER-α36 lacks the AF-<NUM> domain and contains a partial ligand binding domain. It has been found localized to the cell membrane and cytosol. Since ER-α-<NUM> is restricted to modulating MIES and was found to be uniquely expressed in tamoxifen-resisted cancer cells, such as MDA-MB-<NUM> and Hec1A, MIES modulated by membrane-associated ER is thought to be responsible for the resistance to anti-estrogen therapy found by some researchers (<NUM>, <NUM>).

However, studies showing an increased risk of breast and uterine cancer, as well as thromboembolism morbidity, associated with ERT have led to a decline in its usage. Combined administration of estrogen and progesterone prevents the risk of breast and uterine cancers, but it causes progesteron side effects such as dizziness, nausea, vomiting, fatigue, anxiety, depression and headache etc. The postmenopausal symptoms remain a problem for many older women. Therefore, there remains a continuing need for new development of replacing treatment of estrogen-deficiency symptoms.

<NPL>), <CIT> and <CIT> all disclose processes for the preparation of the known pharmaceutical agent anordrin as well as esters thereof.

The present application provides a method of synthesizing a diastereomeric compound of formula (I) (such as α-anordrin) or salt thereof. In accordance with various embodiments described herein, a diastereomeric compound of formula (I) (such as α-anordrin) or salt thereof is substantially pure.

These and other aspects and advantages of the present invention will become apparent from the subsequent detailed description and the appended claims. It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention.

The present application provides methods of synthesizing a diastereomeric compound of formula (I) (such as α-anordrin) or salt thereof.

As a person of ordinary skill in the art would appreciate, in stereochemistry, each of two or more compounds differing only in the spatial arrangement of their atoms is regarded as a diastereomer.

A compound or a salt thereof prepared by a method according to the present application may in one aspect be substantially pure diastereomeric.

As used in this application, "alkyl" refers to a linear or branched saturated hydrocarbon. In some embodiments, alkyl groups are those having <NUM> to <NUM> carbon atoms (a "C<NUM>-C<NUM> alkyl"). In some embodiments, an alkyl group has <NUM> to <NUM> carbon atoms (i.e., (C<NUM>-C<NUM>alkyl)), or <NUM> to <NUM> carbon atoms (i.e., (C<NUM>-C<NUM>alkyl)), or <NUM> to <NUM> carbon atoms (i.e., (C<NUM>-C<NUM>alkyl)), or <NUM> to <NUM> carbon atoms (i.e., (C<NUM>-C<NUM>alkyl)), or <NUM> to <NUM> carbon atoms (i.e., (C<NUM>-C<NUM>alkyl)). Examples of suitable alkyl groups include, but are not limited to, methyl (Me, -CH<NUM>), ethyl (Et, -CH<NUM>CH<NUM>), <NUM>-propyl (n-Pr, n-propyl, -CH<NUM>CH<NUM>CH<NUM>), <NUM>-propyl (i-Pr, i-propyl, -CH(CH<NUM>)<NUM>), <NUM>-butyl (n-Bu, n-butyl, -CH<NUM>CH<NUM>CH<NUM>CH<NUM>), <NUM>-methyl-<NUM>-propyl (i-Bu, i-butyl, -CH<NUM>CH(CH<NUM>)<NUM>), <NUM>-butyl (s-Bu, s-butyl, -CH(CH<NUM>)CH<NUM>CH<NUM>), <NUM>-methyl-<NUM>-propyl (t-Bu, t-butyl, -C(CH<NUM>)<NUM>), <NUM>-pentyl (n-pentyl, -CH<NUM>CH<NUM>CH<NUM>CH<NUM>CH<NUM>), <NUM>-pentyl (-CH(CH<NUM>)CH<NUM>CH<NUM>CH<NUM>), <NUM>-pentyl (-CH(CH<NUM>CH<NUM>)<NUM>), <NUM>-methyl-<NUM>-butyl (-C(CH<NUM>)<NUM>CH<NUM>CH<NUM>), <NUM>-methyl-<NUM>-butyl (-CH(CH<NUM>)CH(CH<NUM>)<NUM>), <NUM>-methyl-<NUM>-butyl (-CH<NUM>CH<NUM>CH(CH<NUM>)<NUM>), <NUM>-methyl-<NUM>-butyl (-CH<NUM>CH(CH<NUM>)CH<NUM>CH<NUM>), <NUM>-hexyl (-CH<NUM>CH<NUM>CH<NUM>CH<NUM>CH<NUM>CH<NUM>), <NUM>-hexyl (-CH(CH<NUM>)CH<NUM>CH<NUM>CH<NUM>CH<NUM>), <NUM>-hexyl (-CH(CH<NUM>CH<NUM>)(CH<NUM>CH<NUM>CH<NUM>)), <NUM>-methyl-<NUM>-pentyl (-C(CH<NUM>)<NUM>CH<NUM>CH<NUM>CH<NUM>), <NUM>-methyl-<NUM>-pentyl (-CH(CH<NUM>)CH(CH<NUM>)CH<NUM>CH<NUM>), <NUM>-methyl-<NUM>-pentyl (-CH(CH<NUM>)CH<NUM>CH(CH<NUM>)<NUM>), <NUM>-methyl-<NUM>-pentyl (-C(CH<NUM>)(CH<NUM>CH<NUM>)<NUM>), <NUM>-methyl-<NUM>-pentyl (-CH(CH<NUM>CH<NUM>)CH(CH<NUM>)<NUM>), <NUM>,<NUM>-dimethyl-<NUM>-butyl (-C(CH<NUM>)<NUM>CH(CH<NUM>)<NUM>), <NUM>,<NUM>-dimethyl-<NUM>-butyl (-CH(CH<NUM>)C(CH<NUM>)<NUM>, and octyl (-(CH<NUM>)<NUM>CH<NUM>).

"-Alkyl-" refers to a bivalent radical derived from alkyl as described above. In some embodiments, -alkyl- as used herein has at least <NUM> carbon atom, at least <NUM> carbon atoms, at least <NUM> carbon atoms, at least <NUM> carbon atoms, at least <NUM> carbon atoms, at least <NUM> carbon atoms, at least <NUM> carbon atoms; at least <NUM> carbon atoms; at least <NUM> carbon atoms; or at least <NUM> carbon atoms; or <NUM> to <NUM> carbon atoms, <NUM> to <NUM> carbon atoms, <NUM> to <NUM> carbon atoms, <NUM> to <NUM> carbon atoms, <NUM> to <NUM> carbon atoms, <NUM> to <NUM> carbon atoms, or <NUM> to <NUM> carbon atoms. Examples of -alkyl- include, but are not limited to, groups such as methylene (-CH<NUM>-), ethylene (-CH<NUM>CH<NUM>-), propylene (-CH<NUM>CH<NUM>CH<NUM>-), butylene (-CH<NUM>CH<NUM>CH<NUM>CH<NUM>-), and the like.

"Alkenyl" as used herein refers to a linear or branched hydrocarbon with at least one carbon-carbon double bond. In some embodiments, an alkenyl group has <NUM> to <NUM> carbon atoms (i.e., C<NUM>-C<NUM>alkenyl), or <NUM> to <NUM> carbon atoms (i.e., C<NUM>-C<NUM>alkenyl), or <NUM> to <NUM> carbon atoms (i.e., C<NUM>-C<NUM>alkenyl), or <NUM> to <NUM> carbon atoms (i.e., C<NUM>-C<NUM>alkenyl), or <NUM> to <NUM> carbon atoms (i.e., C<NUM>-C<NUM>alkenyl). Examples of suitable alkenyl groups include, but are not limited to, ethylene or vinyl (-CH=CH<NUM>), allyl (-CH<NUM>CH=CH<NUM>) and <NUM>-hexenyl (-CH<NUM>CH<NUM>CH<NUM>CH<NUM>CH=CH<NUM>).

"-Alkenyl -" refers to a bivalent radical derived from alkenyl as described above. In some embodiments, -alkenyl- as used herein has at least <NUM> carbon atoms, at least <NUM> carbon atoms, at least <NUM> carbon atoms, at least <NUM> carbon atoms, at least <NUM> carbon atoms, at least <NUM> carbon atoms; at least <NUM> carbon atoms; at least <NUM> carbon atoms; or at least <NUM> carbon atoms; or <NUM> to <NUM> carbon atoms, <NUM> to <NUM> carbon atoms, <NUM> to <NUM> carbon atoms, <NUM> to <NUM> carbon atoms, <NUM> to <NUM> carbon atoms, <NUM> to <NUM> carbon atoms, or <NUM> to <NUM> carbon atoms. To give an example, -alkenyl- may be - CH=CH-.

"Alkynyl" as used herein refers to an unsaturated linear or branched univalent hydrocarbon chain or combination thereof, having at least one site of acetylenic unsaturation (i.e., having at least one moiety of the formula C≡C). Alkynyl groups can have the number of carbon atoms designated (i.e., C<NUM>-C<NUM> means <NUM> to <NUM> carbon atoms). In some embodiments, alkynyl groups are those having <NUM> to <NUM> carbon atoms (i.e., "C<NUM>-C<NUM> alkynyl"), having <NUM> to <NUM> carbon atoms (i.e., "C<NUM>-C<NUM> alkynyl"), having <NUM> to <NUM> carbon atoms (i.e., "C<NUM>-C<NUM> alkynyl"), having <NUM> to <NUM> carbon atoms (i.e., "C<NUM>-C<NUM> alkynyl"), or having <NUM> to <NUM> carbon atoms (i.e., "C<NUM>-C<NUM> alkynyl"). Examples of alkynyl include, but are not limited to, groups such as ethynyl (or acetylenyl), prop-<NUM>-ynyl, prop-<NUM>-ynyl (or propargyl), but-<NUM>-ynyl, but-<NUM>-ynyl, but-<NUM>-ynyl, homologs and isomers thereof, and the like.

The term "aryl" refers to and includes polyunsaturated aromatic hydrocarbon groups. Aryl may contain additional fused rings (e.g., from <NUM> to <NUM> rings), including additionally fused aryl, heteroaryl, cycloalkyl, and/or heterocyclyl rings. In one variation, the aryl group contains from <NUM> to <NUM> annular carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, biphenyl, and the like.

The term "cycloalkyl" refers to and includes cyclic univalent hydrocarbon structures, which may be fully saturated, mono- or polyunsaturated, but which are non-aromatic, having the number of carbon atoms designated (e.g., C<NUM>-C<NUM> means one to ten carbons). Cycloalkyl can consist of one ring, such as cyclohexyl, or multiple rings, such as adamantly, but excludes aryl groups. A cycloalkyl comprising more than one ring may be fused, spiro or bridged, or combinations thereof. A preferred cycloalkyl is a cyclic hydrocarbon having from <NUM> to <NUM> annular carbon atoms. A more preferred cycloalkyl is a cyclic hydrocarbon having from <NUM> to <NUM> annular carbon atoms (a "C<NUM>-C<NUM> cycloalkyl"). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, <NUM>-cyclohexenyl, <NUM>-cyclohexenyl, cycloheptyl, norbornyl, and the like.

"Halo" or "halogen" refers to elements of the Group <NUM> series having atomic number <NUM> to <NUM>. Preferred halo groups include fluoro, chloro, bromo and iodo. Where a residue is substituted with more than one halogen, it may be referred to by using a prefix corresponding to the number of halogen moieties attached, e.g., dihaloaryl, dihaloalkyl, trihaloaryl etc. refer to aryl and alkyl substituted with two ("di") or three ("tri") halo groups, which may be but are not necessarily the same halo; thus <NUM>-chloro-<NUM>-fluorophenyl is within the scope of dihaloaryl. An alkyl group in which each hydrogen is replaced with a halo group is referred to as a "perhaloalkyl. " A preferred perhaloalkyl group is trifluoroalkyl (-CF<NUM>). Similarly, "perhaloalkoxy" refers to an alkoxy group in which a halogen takes the place of each H in the hydrocarbon making up the alkyl moiety of the alkoxy group. An example of a perhaloalkoxy group is trifluoromethoxy (-OCF<NUM>).

The term "heteroaryl" refers to and includes unsaturated aromatic cyclic groups having from <NUM> to <NUM> annular carbon atoms and at least one annular heteroatom, including but not limited to heteroatoms such as nitrogen, oxygen and sulfur, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule at an annular carbon or at an annular heteroatom. Heteroaryl may contain additional fused rings (e.g., from <NUM> to <NUM> rings), including additionally fused aryl, heteroaryl, cycloalkyl, and/or heterocyclyl rings. Examples of heteroaryl groups include, but are not limited to, pyridyl, pyrimidyl, thiophenyl, furanyl, thiazolyl, and the like.

The term "heterocycle" or "heterocyclyl" refers to a saturated or an unsaturated non-aromatic group having from <NUM> to <NUM> annular carbon atoms and from <NUM> to <NUM> annular heteroatoms, such as nitrogen, sulfur or oxygen, and the like, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heterocyclyl group may have a single ring or multiple condensed rings, but excludes heteroaryl groups. A heterocycle comprising more than one ring may be fused, spiro or bridged, or any combination thereof. In fused ring systems, one or more of the fused rings can be aryl or heteroaryl. Examples of heterocyclyl groups include, but are not limited to, tetrahydropyranyl, dihydropyranyl, piperidinyl, piperazinyl, pyrrolidinyl, thiazolinyl, thiazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, <NUM>,<NUM>-dihydrobenzo[b]thiophen-<NUM>-yl, <NUM>-amino-<NUM>-oxopyrimidin-<NUM>(<NUM>)-yl, and the like.

"Optionally substituted" unless otherwise specified means that a group may be unsubstituted or substituted by one or more (e.g., <NUM>, <NUM>, <NUM>, <NUM> or <NUM>) of the substituents listed for that group in which the substituents may be the same of different. In one embodiment, an optionally substituted group has one substituent. In another embodiment, an optionally substituted group has two substituents. In another embodiment, an optionally substituted group has three substituents. In another embodiment, an optionally substituted group has four substituents. In some embodiments, an optionally substituted group has <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM> or <NUM> to <NUM> substituents.

It is understood that aspect and embodiments of the invention described herein include "consisting" and/or "consisting essentially of" aspects and embodiments.

Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".

As used herein and in the appended claims, the singular forms "a", "or" and "the" include plural referents unless the context clearly dictates otherwise.

The present disclosure includes a specific stereochemical form of compounds described. In stereochemistry, each of two or more compounds differing only in the spatial arrangement of their atoms is regarded as a diastereomer. A diastereomeric compound as detailed herein may be substantially pure.

Generally speaking, chromatography, recrystallization and other conventional separation procedures may be used with intermediates or final products to obtain a particular isomer of a compound. In some embodiments, a diastereomeric compound as detailed herein can be prepared by a resolution where the diasteromeric compound is separated from a mixture of diasteromers. In other embodiments, a diastereomeric compound is prepared via asymmetric synthesis where a substantially pure diastereomeric compound or salt thereof is obtained.

The present disclosure describes asymmetic synthesis that results in a substantially pure diastereomeric compound or salt thereof. Provided herein are methods of preparing a substantially pure diastereomer form of a compound or a salt thereof.

According to the present invention, compounds are steroid-like compounds as detailed herein. For example, anordrin is a steroid-like estrogen. In some embodiments, a diastereomeric compound is α-anordrin, (<NUM>α,<NUM>α)-diethynyl-(<NUM>β, <NUM>β)-diol-dipropionate-A-nor-<NUM>α-androstane).

The a diastereomeric compound prepared by the method of the present invention has the structure of Formula (I):
<CHM>
or a salt thereof, wherein.

In some embodiments, R<NUM> is hydroxyl.

In some embodiments, R<NUM> is -OC(O)R1a, wherein R1a is C<NUM>-C<NUM>alkyl. In some embodiments, R1a is C<NUM>-C<NUM>alkyl, for example, methyl, ethyl, <NUM>-propyl (n-Pr, n-propyl, - CH<NUM>CH<NUM>CH<NUM>), <NUM>-propyl (i-Pr, i-propyl, -CH(CH<NUM>)<NUM>), <NUM>-butyl. In some embodiments, R1a is C<NUM>-C<NUM>alkyl or C<NUM>-C<NUM>alkyl. In some embodiments, R1a is ethyl.

In some embodiments, R<NUM> is -OC(O)R1bCOOH, wherein R1b is -C<NUM>-C<NUM>alkyl-. In some embodiments, R<NUM> is -OC(O)R1bCOOH, wherein R1b is -C<NUM>-C<NUM>alkenyl-. In some embodiments, R<NUM> is -OC(O)R1bCOOH , wherein R1b is methylene (i.e., -CH<NUM>-) and R<NUM> is -OC(O)CH<NUM>COOH. To give some examples of a salt form, in certain embodiments, R<NUM> is -OC(O)CH<NUM>COOLi, - OC(O)CH<NUM>COONa or -OC(O)CH<NUM>COOK. In some embodiments, R<NUM> is -OC(O)R1bCOOH , wherein R1b is ethylene (i.e., -CH<NUM>CH<NUM>-) and R<NUM> is -OC(O)CH<NUM>CH<NUM>COOH. In certain embodiments, R<NUM> is -OC(O)CH<NUM>CH<NUM>COOLi, -OC(O)CH<NUM>CH<NUM>COONa or -OC(O)CH<NUM>CH<NUM>COOK. In some embodiments, R<NUM> is -OC(O)R1bCOOH, wherein R1b is -CH=CH- and R<NUM> is - OC(O)CH=CHCOOH. In certain embodiments, R<NUM> is -OC(O) CH=CHCOOLi, -OC(O) CH=CHCOONa or -OC(O) CH=CHCOOK.

In some embodiments, R<NUM> is -OC(O)R4a, wherein R4a is C<NUM>-C<NUM>alkyl. In some embodiments, R4a is C<NUM>-C<NUM>alkyl, for example, methyl, ethyl, <NUM>-propyl (n-Pr, n-propyl, - CH<NUM>CH<NUM>CH<NUM>), <NUM>-propyl (i-Pr, i-propyl, -CH(CH<NUM>)<NUM>), <NUM>-butyl. In some embodiments, R4a is C<NUM>-C<NUM>alkyl or C<NUM>-C<NUM>alkyl. In some embodiments, R4a is ethyl.

In some embodiments, R<NUM> is -OC(O)R4bCOOH , wherein R4b is -C<NUM>-C<NUM>alkyl-. In some embodiments, R<NUM> is -OC(O)R4bCOOH, wherein R4b is -C<NUM>-C<NUM>alkenyl-. In some embodiments, R<NUM> is -OC(O)R4bCOOH , wherein R4b is methylene (i.e., -CH<NUM>-) and R<NUM> is -OC(O)CH<NUM>COOH. To give some examples of a salt form, in certain embodiments, R<NUM> is -OC(O)CH<NUM>COOLi, - OC(O)CH<NUM>COONa or -OC(O)CH<NUM>COOK. In some embodiments, R<NUM> is -OC(O)R4bCOOH , wherein R4b is ethylene (i.e., -CH<NUM>CH<NUM>-) and R<NUM> is -OC(O)CH<NUM>CH<NUM>COOH. In certain embodiments, R<NUM> is -OC(O)CH<NUM>CH<NUM>COOLi, -OC(O)CH<NUM>CH<NUM>COONa or -OC(O)CH<NUM>CH<NUM>COOK. In some embodiments, R<NUM> is -OC(O)R4bCOOH, wherein R4b is -CH=CH- and R<NUM> is - OC(O)CH=CHCOOH. In certain embodiments, R<NUM> is -OC(O) CH=CHCOOLi, -OC(O) CH=CHCOONa or -OC(O) CH=CHCOOK.

In some embodiments, R<NUM> is C<NUM>-C<NUM>alkyl. In some embodiments, R<NUM> is C<NUM>-C<NUM>alkenyl. In some embodiments, R<NUM> is C<NUM>-C<NUM>alkyl. In some embodiments, R<NUM> is C<NUM>-C<NUM>alkenyl. R<NUM> and R<NUM> may or may not be the same.

In some embodiments, R<NUM> is hydrogen, -OH, -NH<NUM>, -NO<NUM>, halogen, C<NUM>-C<NUM>alkyl, or C<NUM>-C<NUM>alkenyl. In some embodiments, R<NUM> is hydrogen. In some embodiments, R<NUM> is C<NUM>-C<NUM>alkyl. In some embodiments, R<NUM> is hydrogen, -OH, -NH<NUM>, -NO<NUM>, halogen, C<NUM>-C<NUM>alkyl, or C<NUM>-C<NUM>alkenyl. In some embodiments, R<NUM> is hydrogen. In some embodiments, R<NUM> is C<NUM>-C<NUM>alkyl. R<NUM> and R<NUM> may or may not be the same.

According to the method of the present invention a substantially pure diastereomeric compound described herein or a salt thereof is prepared using a method comprising reacting a di-ketone compound of formula (II)
<CHM>
with silylacetylene of formula (III)
<CHM>
in the presence of an organometallic reagent R<NUM>-M.

R<NUM>, R<NUM>, R<NUM> and R<NUM> of a di-ketone compound of formula (II) are as defined above in various embodiments of a compound has the structure of Formula (I).

With reference to the reaction step III-<NUM> in Method II as illustrated in EXAMPLES below, an exemplary process to prepare α-anordrin comprising reacting a di-ketone product (c) with a silylacetylene, trimethylsilylacetylene (TMS). It was recognized in the present application, a silylacetylene can be used for asymmetric alkynylation of a di-ketone compound described herein. Without being bound to any particular theory, it is believed that two terminal alkynes add to two carbonyl groups of the de-ketone compound one by one.

Ra, Rb and Rc of silylacetylene of formula (III) can be the same or different. In some embodiments, Ra, Rb and Rc are independently selected from the group consisting of hydrogen, - OH, C<NUM>-C<NUM>alkyl optionally substituted by -OH, halogen or C<NUM>-C<NUM>alkyl, C<NUM>-C<NUM>alkoxy optionally substituted by -OH, halogen or C<NUM>-C<NUM>alkyl, C<NUM>-C<NUM> cycloalkyl, C<NUM>-C<NUM> aryl, <NUM>-<NUM>-membered heteroaryl or <NUM>-<NUM> membered heterocyclyl. In some embodiments, Ra, Rb and Rc are independently C<NUM>-C<NUM>alkyl optionally substituted by -OH, halogen or C<NUM>-C<NUM>alkyl. In some embodiments, Ra, Rb and Rc are independently C<NUM>-C<NUM>alkyl, for example, methyl, ethyl, <NUM>-propyl (n-Pr, n-propyl, -CH<NUM>CH<NUM>CH<NUM>), <NUM>-propyl (i-Pr, i-propyl, -CH(CH<NUM>)<NUM>), <NUM>-butyl. In some embodiments, Ra, Rb and Rc are methyl and thus the silylacetylene is trimethylsilylacetylene (TMS). Without being bound to any particular theory, it is believed that one or more of Ra, Rb and Rc has a large size to creat steric hindrance that leads to forming one diastereomer in preference to another.

An organometallic reagent used in accordance with the present methods is defined as R<NUM>-M, wherein M is a metal. To give an example, R<NUM>-M is n-Butyllithium.

According to the invention, M is Li, Na or K. In some somebodiments, M is Li.

According to the invention, R<NUM> is C<NUM>-C<NUM>alkyl, optionally substituted by -OH, halogen, or C<NUM>-C<NUM>alkyl. In some embodiments, R<NUM> is C<NUM>-C<NUM>alkyl. In some embodiments, R<NUM> is n-butyl.

In some embodiments, tetramethylehtylenediamine (TEMED) is added with the organometallic reagent.

Methods provided herein, in various embodiments, are carried out in the presence of an organic solvent. A non-limiting list of suitable organic solvents includes, without limitation, dichloromethane, chloroform, acetoniltrile, dichloroethane, tetrahydrofuran (THF), dimethylsulfoxide, and toluene.

Methods provided herein are carried out under relatively low reaction temperature. A reaction temperature can range from a temperature of from about <NUM>° to about -<NUM>° C. In some embodiments, a reaction temperature is between about -<NUM>° to about -<NUM>° C. In some embodiments, a reaction temperature is between about -<NUM>° to about -<NUM>° C. In some embodiments, a reaction temperature is between about -<NUM>° to about -<NUM>° C.

In accordance with some embodiments of the present invention, a method for stereospecifically preparing a substantially pure diastereomeric compound or a salt thereof further involves removing a silyl group of formula (IV):
<CHM>.

In some embodiments, the removing step is carried out by contacting with a deprotective agent. A non-limiting list of suitable deprotective agents includes, without limitation, tetrabutyl ammonium fluoride (TBAF), hydrofluoric acid and potassium fluoride.

The invention will now be described in greater detail by reference to the following non-limiting examples.

Of the following examples, example <NUM> compares a method for the preparation of alpha-anordrin (see scheme I of example <NUM>) which is not in accordance with the present invention, for the purposes of comparison with another method for the preparation of alpha-anordrin (see scheme II of example <NUM>) which is in accordance with the present invention. Examples <NUM> and <NUM> simply demonstrate the biological effects of alpha-anordrin and its beta-isomer, and as such, do not form part of the present invention but exist only to illustrate the application of the product of the claimed method.

Chemical synthesis of α-anordrin can be prepared as described in using steps detailed below method I (as shown in Scheme I), incorporated herein, or method II (scheme II) by reference <CIT>, and the publication of <NPL>. <CHM>
<CHM>.

Reaction step I: Preparation of Jones reagent: <NUM> CrO<NUM> was soluted in <NUM> H<NUM>O, and then slowly added <NUM> H<NUM>SO<NUM>. The yielding Jones reagent was then colded to <NUM>. <NUM> of 5α-androstane and <NUM> of acetic acid were added into a <NUM> three-necked flask. The mixture was warmed to <NUM>-<NUM>, stirred and dissolved under nitrogen atmosphere. Jones reagent was added dropwise within <NUM> hour. Reaction temperature was increased to <NUM> and reacted for <NUM> hour, and acetic acid was distilled the reduced pressure. The residue was filtrated, washed with H<NUM>O and dryed to give <NUM> of di-acidic product (b).

Reaction step I: Preparation of Jones reagent: <NUM> CrO<NUM> was soluted in <NUM> H<NUM>O, and then slowly added <NUM> H<NUM>SO<NUM>. The yielding Jones reagent was then colded to <NUM>. <NUM> of 5α-androstane and <NUM> of acetic acid were added into a <NUM> three-necked flask. The mixture was warmed to <NUM>-<NUM>, stirred and dissolved under nitrogen atmosphere. Jones reagent was added dropwise within 1hour. Reaction temperature was increased to <NUM> and reacted for <NUM> hour, and acetic acid was distilled the reduced pressure. The residue was filtrated, washed with H<NUM>O and dryed to give <NUM> of di-acidic product (b).

Reaction step <NUM>: <NUM> of di-acid product (b) and <NUM> of acetic anhydride were dissolved in <NUM> three-necked flask and added in <NUM> sodium acetate, stirred and refluxed for <NUM> hours. The residue was filtrated and dried and give <NUM> of di-ketone product (c).

Reaction step III-<NUM> and IV-<NUM>: Preparation of potassium acetylene reagent: <NUM> of KOH solution (<NUM>% w/w) was colded to <NUM> <NUM>C in <NUM> three-necked flask and injected with acetylene to stable mass. <NUM> of di-ketone product (c) was dissolved in <NUM> of THF and then added in three-necked flask thereto <NUM> <NUM>C for <NUM> hours. The product was diluted with H<NUM>O and acidified with HCl. The residue was filtrated, washed with H<NUM>O and dried to give <NUM> of the <NUM>,17α-diethynyl-A-nor-5a-androstane-<NUM>,17β-diol product (e).

Reaction step V: <NUM> of <NUM>,17α-diethynyl-A-nor-5a-androstane-<NUM>,17β-diol product (e), <NUM> of propionic acid and <NUM> of propionic anhydride were mixed in <NUM> of flask, and then stirred for <NUM> hours. <NUM> of H<NUM>O and <NUM> of ethyl acetate were added in flask, stirred and separated water and organic phase. Organic phase was washed by water for <NUM> times and added <NUM> n-hexane. The residue was filtrated and dry to give <NUM> of mixture of product α-anordrin and β-anordrin (fα and fβ), respectively.

In accordance with some embodiments of the present invention, asymmetic synthesis of α-anordrin can be prepared as described in using steps detailed below method II (as shown in Scheme II). <CHM>
<CHM>.

Silimar to Method I, di-ketone product (c) was prepared using reaction steps I and II as described above.

Reaction step III-<NUM>: <NUM> of dried THF, <NUM> of trimethylsilylacetylane (TMS) were added in a dried <NUM> three-necked flask, and then cooled to -<NUM> - -<NUM> <NUM>C, <NUM> of n-buLi was added in solution and stirred for <NUM>, <NUM> of tetramethylehtylenediamine (TEMED). <NUM> of di-ketone product (c) from reaction step II of method I was dissolved in <NUM> of THF, and then cooled to -<NUM> - -<NUM><NUM>C. Mix two solutions together and react for overnight. The reaction was stopped by NH<NUM>Cl solution. <NUM> ethyl acetate was added in flask and stirred for <NUM>. Organic phase was dried to give <NUM> of product (d-<NUM>).

Reaction step IV-<NUM>: <NUM> product (d-<NUM>) was dissolved in the mixture of <NUM> THF and <NUM> tetrabutyl ammonium fluoride (TBAF) solution to react for <NUM> hours. THF was distillated. The <NUM>α,17α-diethynyl-A-nor-5a-androstane-<NUM>β,17β-diol product α-anordiol (e-<NUM>) was dissolved in ethyl acetate. The organic solution was washed by distilled water for <NUM> times. After removing ethyl acetate, <NUM> of <NUM>α,17α-diethynyl-A-nor-5a-androstane-<NUM>β,17β-diol product, α-anordiol (e-<NUM>), was given.

Reaction step V: <NUM> of the <NUM>α,17α-diethynyl-A-nor-5a-androstane-<NUM>β,17β-diol product (e-<NUM>), <NUM> of propionic acid and <NUM> of propionic anhydride were mixed in <NUM> of flask, and then stirred for 3hours. <NUM> of H<NUM>O and <NUM> of ethyl acetate were added into flask, stirred and separated water and organic phase. Organic phase was washed by distilled water for <NUM> times and <NUM> of n-hexane was added. The residue was filtrated and dry to give <NUM> of mixture of product, α-anordrin (fα).

The silica gel chromatography analysis showed that only one diastereomeric compound was synthesized using method II (<FIG>). In contrast, using method I, the mixture of diastereomeric compounds was synthesized (<FIG>).

NMR analysis: Collecting fraction <NUM> (F1) and fraction <NUM> (F2) and dissolving in CCl<NUM> (<NUM>/ mL) for NMR analysis. <FIG> and <FIG> showed α-anordrin from fraction <NUM> (F1) and β-anordrin from fraction <NUM> (F2), respectively.

In this Example, studies have been shown the estrogenic effects of α- and β-anordrins on estrogen-modulated metabolic signaling and tissue atrophy. The ovaries of <NUM> week old mice were surgically excised. One week post surgery, the mice were given isoflavone, α-anordrin or β-anordrin by food. Body mass, blood glucose and food intake were measured monthly. After three months. Mice were sacrificed. The leg and spine bone, liver, uterus and vagina were harvested.

The uterus and vagina wet mass and H&E staining showed that α-anordrin but not β-anordrin prevented vulvovagina atrophy (<FIG>). The α-anordrin-treated OVX mice showed less atrophy symptom in uterus compared with those mice treated by β-anordrin and blank groups (<FIG>). The α-anordrin showed the less blood gluose, TG and body mass compared with β-anordrin (<FIG> and <FIG>), respectively, but initiation body mass and food uptake was not significantly different (<FIG>), respectively. These data indicated that the differences were due to changes in energy expenditure. The liver H&E section and statistical analysis showed that α-anordrin but not β-anordrin decreased the amount of liver TG in OVX mice (<FIG>). The micro-CT result shows that α-anordrin but not β-anordrin prevented osteoporosis in OVX mice (<FIG>).

Tamoxifen was the first FDA-approved drug for breast cancer patients with positively expressed ER. However, tamoxifen also induces side effects, such as uterine endometrial cancer and NASH. Importantly, for women with ER-positive (ER+) cancer, continuing tamoxifen treatment for up to <NUM> years, rather than stopping at <NUM> years, produces further reductions in recurrence and mortality, particularly after year <NUM>. We found that anordrin can eliminate tamoxifen-induced endometrial epithelial cell (EEC) mitosis. We further tested whether α-anordrin or β-anordrin eliminates tamoxifen-induced EEC mitosis. The <NUM> week old mice were given by food containing isoflavone (Blank), α-anordrin (a-ANO), β-anordrin (b-ANO), α-anordrin+tamoxifen (TAM+a-ANO), β-anordrin+tamoxifen (TAM+b-ANO) or tamoxifen (TAM) for <NUM> months. Mice were then sacrificed to harvest uterus and liver. Uterine and liver tissues were fixed.

H&E staining sections showed that EECs in TAM+b-ANO and TAM groups are rough and mitotic compared with smooth and single layer EEC in TAM+a-ANO and sham groups (<FIG>), and the high fatty deposition in liver cells of TAM+b-ANO and TAM groups compared with those liver cells in TAM+a-ANO and sham groups (<FIG>). The statistical analysis of H&E staining sections showed that α-anordrin but not β-anordrin inhibited tamoxifen-induced EEC mitosis (<FIG>) and NASH (<FIG>).

Aromatase inhibitors were administrated to ER positive breast in post-menopausal women. It also induced side effects such as tissue atrophy and osteoporosis et al. Anastrozole is an aromatase inhibitor. It was administrated to ER positive breast cancer in post menopausal women. Anastrozole (ANA) also induced vagina atrophy and osteoporosis. Vulvovagina atrophy usually results in infection of vagina and urinary tract. As an example, we tested whether α-anordrin or β-anordrin eliminates anastrozole-induced uterus and vulvovagina atrophy, and osteoporosis. The <NUM> week old mice were given by food containing isoflavone (Blank), α-anordrin (a-ANO), β-anordrin (b-ANO), α-anordrin+anastrozole (ANA+a-ANO), β-anordrin+anastrozole (ANA+b-ANO) or anastrozole (ANA) for <NUM> months. Mice were then sacrificed to harvest vagina, leg and spine bone. The tissues were fixed in paraformaldehyde. Micro-CT reults showed that α-anordrin but not β-anordrin inhibited anastrozole-induced osteoporosis (<FIG>). The statistical analysis of vagina wet mass and H&E staining sections of vulvovagina showed that that α-anordrin but not β-anordrin inhibited anastrozole-induced vagina atrophy (<FIG>).

Tamoxifen and anastrozole (MPG USP GRADE) were offered by Okahata (Shanghai) Trading Co. Epiandrostenestone was purchase from Jiangsu Jiaerke Pharmaceuticals Group, LTD. All other compounds were purchased from Sigma or Aladdin. Mice food was made by Trophic Animal Feed High-tech Co. , Ltd, Nantong, China. Mice were purchased from BK animal Inc. The animal experiment was performed in the Southern Center of Pesticide Research, Shanghai.

Glucose concentration assay: glucose concentration in medium or total blood from mouse tail was measured using glucose assay kits following manufacturer's instructions (Yicheng, Beijing).

Construction of ovariectomized (OVX) mice model and administration of drugs: The ovaries of <NUM> week old mice were excised by surgery. <NUM> days post surgery drugs were administered by gastric tract injection every day or mixed with food.

Preparation of paraffin sections and HE staining: The tissue of mice was excised by surgery and fixed using <NUM>% paraformaldehyde in 1X DPBS (Beijing Solar Bioscience&Technology co. Paraffin section preparation and HE staining was performed by GLP laboratory of BK animal model, Inc.

Measurement of TG in mice liver: <NUM>-<NUM> of mouse liver was excised by surgery and homogenized in <NUM> Cholroform:Methanol (<NUM>: <NUM>) mixture and extracted using <NUM> ddH<NUM>O. The organic phase was transferred to new tubes and air dried. The amount of TG was measured using kits following manufacturer's instructions. The error was corrected by using internal standard controls.

Measurement of the EEC height, uterine diameter and circular muscle thickness: EEC height was measured from <NUM>-200x magnification EEC images of Paraffin-embedded H&E mice uterus sections.

Measurement of the thickness of vulvovaginal wall: Vagina was cut at the middle site. Both uterine site and vulvovaginal site were embedded in paraffin at the same direction for H&E section preparation. The thickness of vulvovaginal wall was measured from 40x magnification images of Paraffin-embedded H&E mice vagina sections.

Bone density assay using micro-CT: Thighbone was fixed in 1X DPBS containning <NUM>% formaldehyde for two weeks. The fixation solution was exchanged after one week. The density of thighbone was measured by Siemens Inveon Micro-CT. Inveon Research Workplace (IRW) was used to analyze the HU2000 value at the following measurement conditions: 80KVP, 500mA, <NUM> exposure time; CCD Readout installation: 2048axial, <NUM> binning; FOV Transaxial: <NUM>, axial: <NUM>, pixels size: <NUM>.

Claim 1:
A method for stereospecifically preparing a substantially pure diastereomeric compound of formula (I):
<CHM>
or a salt thereof, wherein
R<NUM> is -OH, -OC(O)-R1a or, -OC(O)R1bCOOH and R<NUM> is -OH, -OC(O)-R4a or, - OC(O)R4bCOOH, wherein R1a and R4a are independently hydrogen, C<NUM>-C<NUM>alkyl, C<NUM>-C<NUM>alkenyl, C<NUM>-C<NUM> alkynyl or C<NUM>-C<NUM> cycloalkyl, and R1b and R4b are independently -C<NUM>-C<NUM>alkyl- or -C<NUM>-C<NUM>alkenyl-;
R<NUM> and R<NUM> are -C=CH;
R<NUM> and R<NUM> are independently C<NUM>-C<NUM>alkyl, C<NUM>-C<NUM>alkenyl, C<NUM>-C<NUM> alkynyl or C<NUM>-C<NUM> cycloalkyl;
R<NUM> and R<NUM> are independently hydrogen, -OH, -NH<NUM>, -NO<NUM>, halogen, C<NUM>-C<NUM>alkyl, C<NUM>-C<NUM>alkenyl, C<NUM>-C<NUM> alkynyl, C<NUM>-C<NUM> cycloalkyl, C<NUM>-C<NUM> aryl, <NUM>-<NUM>-membered heteroaryl or <NUM>-<NUM> membered heterocyclyl,
comprising:
(a) reacting a di-ketone compound of formula (II)
<CHM>
wherein R<NUM>, R<NUM>, R<NUM> and R<NUM> are defined as for formula (I),
with a silylacetylene of formula (III)
<CHM>
wherein Ra, Rb and Rc are independently selected from the group consisting of hydrogen, -OH, C<NUM>-C<NUM>alkyl optionally substituted by -OH, halogen or C<NUM>-C<NUM>alkyl, C<NUM>-C<NUM>alkoxy optionally substituted by -OH, halogen or C<NUM>-C<NUM>alkyl, C<NUM>-C<NUM> cycloalkyl, C<NUM>-C<NUM> aryl, <NUM>-<NUM>-membered heteroaryl or <NUM>-<NUM> membered heterocyclyl,
in the presence of an organometallic reagent R<NUM>-M, wherein M is Li, Na or K, and R<NUM> is C<NUM>-C<NUM>alkyl, optionally substituted by -OH, halogen or C<NUM>-C<NUM>alkyl.