Methods for preparing 17-alkynyl-7-hydroxy steroids and related compounds

[0001] Embodiments of the invention relate to methods for the preparation of 17-ethynyl-10R, 13S-dimethyl-2,3,4,7,8R9S, 10,11,12,13,14S, 15,16, 17-hexadechydro-1H-cyclopenta [a] phenanthreno3R, 7R, 17S-triol and other related pharmaceutically active compounds that are substantially free of steroidal impurities of a reaction displaying unwanted binding activity to the sex steroid receptors.

BACKGROUND OF THE INVENTION [0002] 17-Ethynyl-10R, 13S-dimethyl-2,3,4,7,8 R9S, 10,11,12,13,14S, 15,16, 17-hexadecanoic-1-cyclopent [a] phenanthrene-3R, 7R, 17S-triol (also referred to herein as 17α-ethynylandrost-5-ene-3e, 7e, 17e-triol or Compound 1) is effective in treating conditions attributable to chronic non-productive inflammation. WO 2008/039566 A2 describes the use of compound 1 (17α-ethynylandrostrost-5-ene-3e, 7e, 17e-triol) as a drug. In contrast to other anti-inflammatory steroids, Compound 1 has been shown to be substantially free of binding activity to the sex steroid receptor, which is an activity that can contribute to the unwanted side effects of such compounds. Steroid synthetic intermediates, by-products, or other such impurities, which may affect or modulate the activity of the sex steroid receptor (s) and may be present in the preparations of compound 1, are undesirable because they contribute to side effects. Thus, methods for making Compound 1 and analogs and derivatives that avoid the production of impurities such as synthetic intermediates, steroidal by-products that affect or modulate the activity of the sex steroid receptor (s) are useful.

SUMMARY OF THE INVENTION [0003] It has surprisingly been found that the 17α-alkynyl-5-ene-3e, 7e, 17e-triol production sequences produce a substance containing an undesired steroidal impurity (s) inducing sex steroid receptor activity (s), which in Otherwise, they would not be present in the substance. Such impurities adversely affect the pharmaceutical acceptability of 17α-alkynylandrost-5-ene-3e, 7e, 17e-triol. The presence of these impurities in 17α-alkynylandrost-5-ene-3e, 7e, 17e-triol formulations has not been described and their presence leads to the additional cost of removing or reducing their presence to a level where no activity (s) is present. ) sex steroids. The reaction sequences disclosed herein allow to avoid the production of unwanted steroid (s) contamination (s),

[0004] One embodiment of the invention provides a reaction sequence for introducing an alkynyl group at position 17 and a functional oxygen group at the 7-position to androst-5-ene having an oxygen-linked group at the 3-position and a keto group at the 17-position such that a by-product is excluded devoid of the oxygen substituent at C-7, and thus exhibiting undesirable binding activity with sex steroid receptors.

Another embodiment of the invention provides a reaction sequence for the introduction of the ethynyl group in the 17-position and the hydroxyl group in the 7-position to the dehydroepiandrosterone (also referred to herein as DHEA or 3e-hydroxyandrost-5-ene-17-one), so that the preparation of compound 1 results. , which is essentially free of binding activity to sex steroid receptors.

[0006] In another embodiment, the invention provides a reaction sequence using DHEA as a substrate to form a formulation comprising Compound 1 that is substantially free of the estrogenic compound, 17α-ethynylandandrost-5-ene-3e, 17e-diol, or its potential for binding. precursor, 17α-ethynyl-3-acetoxyandrost-5-ene-17e-ol.

[0007] In one embodiment of the invention, the reaction sequence comprises an earlier oxidation step of a suitably protected androst-5-ene having first and second oxygen-bound group at positions 3 and 17, such that the third oxygen-bound group is introduced at position 7, and a subsequent step the reaction of the intermediate, in which the second oxygen-bound group in position 17 is = O, with an alkyne-derived anion.

[0008] In another embodiment of the invention, the reaction sequence comprises a prior oxidation step of DHEA, properly protected, to introduce a third oxygen-bound group at position 7 and a subsequent reaction step of an intermediate having a substituent = O at position 17 with an acetylene anion, optionally protected.

In another embodiment of the invention, the reaction sequence comprises an androst-5-ene-17-one-3e, 7e-diol reaction, suitably protected, with an alkyne-derived anion. DETAILED DESCRIPTION Definitions As used herein, and unless otherwise specified or implied by the context, terms defined herein have the specified meanings. The descriptions of the forms and the examples described illustrate the invention and are not intended to limit it in any way. If there is no other contraindication or implication, e.g. by including mutually exclusive elements or options, in these descriptions and in this specification, the terms in the singular shall mean one or more, and the term "or" means and / or.

[0011] "Alkyl" as used herein refers to combined normal, secondary, tertiary or cyclic carbon atoms, i.e. linear, branched, cyclic or any combination thereof. The alkyl groups or moieties, as used herein, may be saturated or unsaturated, i.e. the moiety may include one, two, three or more independently selected double bonds or triple bonds. Unsaturated alkyl moieties include moieties as described below for alkenyl, alkynyl, cycloalkyl and aryl moieties. The number of carbon atoms in the alkyl moiety is 1-20, preferably 1 to 8. C1-8 alkyl or C1-8 alkyl means an alkyl moiety containing 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms and C1-6 alkyl or C 1-6 alkyl means an alkyl moiety containing 1, 2, 3, 4, 5 or 6 carbon atoms. When mentioning an alkyl moiety, the species may include methyl, ethyl, 1-propyl (n-propyl), 2-propyl (iso-propyl, -CH (CH3) 2), 1-butyl (n-butyl), 2-methyl -1propyl (iso-butyl, -CH 2 CH (CH 3) 2), 2-butyl (sec-butyl, - CH (CH 3) CH 2 CH 3), 2-methyl-2-propyl (t-butyl, -C (CH 3) 3), 1 -pentyl (n-pentyl), 2-pentyl (-CH (CH 3) CH 2 CH 2 CH 3), 3-pentyl (-CH (CH 2 CH 3) 2) and 2-methyl-2-butyl (-C (CH 3) 2 CH 2 CH 3).

[0012] "Cycloalkyl", as used herein, refers to a monocyclic, bicyclic or tricyclic ring system composed of only carbon atoms. The number of carbon atoms in the cycloalkyl group or moiety may vary and is typically 3 to about 20, e.g. 3-8. C 3-8 alkyl or C 3 -C 8 alkyl means a cycloalkyl moiety containing 3, 4, 5, 6, 7 or 8 carbon atoms, and C 3-6 alkyl or C 3 -C 6 is a cycloalkyl moiety containing 3, 4, 5 or 6 carbon atoms. Preferred cycloalkyl substituents are cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl and adamantyl. Cycloalkyl substituents having a double bond in the cyclic ring system are sometimes referred to as cycloalkenyl substituents.

[0013] "Alkenyl" as used herein means a moiety or group that includes one or more double bonds (-CH = CH-), e.g. 1, 2, 3, 4, 5, 6 or more, typically 1, 2 or 3, and includes related normal, secondary, tertiary or cyclic carbon atoms, i.e. linear, branched, cyclic or any combination thereof, if the alkenyl moiety is not vinyl (-CH = CH 2). The alkenyl moiety with multiple double bonds may have double bonds arranged in a tiled (i.e., 1,3 butadienyl moiety) or non-adjacent one or more interposed between saturated carbon atoms or combinations thereof, provided that the cyclic neighboring double bond system does not form cyclically coupled system

4n + 2 electrons (i.e. aromatic). The number of carbon atoms in the alkenyl moiety may be 2-20, preferably 2-8. C 2-8 alkenyl or C 2-8 alkenyl means an alkenyl moiety of 2, 3, 4, 5, 6, 7 or 8 carbon atoms and C 2-6 alkenyl or C 2-6 alkenyl is an alkenyl moiety of 2, 3, 4, 5 or 6 carbon atoms. When mentioning an alkenyl moiety, the genera include, for example, any alkyl moieties described above that have one or more double bonds, such as methylene (= CH 2), methylmethylene (= CH-CH 3), ethylmethylene (= CH-CH 2 -CH 3 ), propylmethylenes (= CH-CH2-CH2-CH3), vinyl (-CH = CH2), allyl (-CH = CHCH3), 1-methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl or 1- pentenyl.

[0014] "Alkynyl" as used herein refers to linked normal, secondary, tertiary or cyclic carbon atoms where one or more triple bonds (-C = C-), typically 1, 2 or 3, usually 1, exist. optionally containing 1, 2, 3, 4, 5, 6 or more double bonds, wherein the remaining bonds (if any) are single bonds, and include related normal, secondary, tertiary or cyclic carbon atoms, i.e. linear, branched, cyclic or any combination thereof if the alkynyl moiety is not ethynyl. The number of carbon atoms in the alkynyl group or moiety is from 2 to 20, preferably from 2 to 8. C2-8 alkynyl or C2-8 alkynyl is an alkynyl moiety containing 2, 3, 4, 5, 6, 7 or 8 carbon atoms. When an alkynyl substituent is mentioned, preferred types include -C CH, -C CCII ;. -C CH 2 CH 3, -C CC; IC and -C CCH 2 C 3 H-. Ethynyl, propynyl and 1-butynyl are particularly preferred types, and ethynyl is particularly preferred.

[0015] "Aryl" as used herein refers to an aromatic ring system or condensed ring system without ring heteroatoms including 1, 2, 3 or 4 to 6 rings, typically 1 to 3 rings; where the rings are composed only of carbon atoms; and refers to a cyclic conjugated 4n + 2 electron system (Huck's rule), typically 6, 10 or 14 electrons, some of which may additionally participate in exocyclic conjugation. When mentioning an aryl group, the species may include phenyl, biphenyl, naphthyl, phenanthryl and quinone.

[0016] "Ieteroaryl" as used herein means an aryl ring system wherein one or more, typically 1, 2 or 3, but not all carbon atoms including an aryl ring system are replaced with a heteroatom which is a non-carbon atom, including N, O, S, Se, B, Si, P, typically, oxygen (-O-), nitrogen (-NX-) or sulfur (-S-), where X is -I, a protecting group or optionally substituted C1-6 alkyl, wherein the heteroatom participates in a system conjugated by linking pi to an adjacent ring atom or a single electron pair on a heteroatom and may be optionally substituted at one or more carbon atoms or heteroatoms, or a combination of both, including heterocycle a method that maintains a cyclically coupled system.

[0017] A "protecting group" as used herein means a moiety that prevents or limits the participation of an atom or a functional group to which it is associated in unwanted reactions. For example, for -OR^PR, R^PR is a protecting group for the oxygen atom present in the hydroxyl, while for = 0 the protecting group is a ketal or thiocetal group in which the divalent oxygen is replaced, for example, by -X- (Cl2) nY-, in which X and Y independently are S and O, n is 2 to 3, to form a spiro or oxime ring system in which the divalent oxygen is replaced by = N-OR, where R is -I, alkyl or aryl. For -C (O) -OR^PR, R^PR is a carbonyloxy protecting group, for -SR^PR, R^PR is a protecting group for a sulfur atom for example in thiols, and for -NIR^PR or -N (R.^PR) 2 _RPR is a nitrogen protecting group for primary or secondary amines. Protecting groups for sulfur or nitrogen atoms, or monovalent oxygen are usually used to prevent unwanted reactions with electrophilic compounds. Protecting groups for bivalent oxygen atoms (i.e. = O) are usually used to prevent unwanted reactions with nucleophilic compounds.

[0018] "Optionally substituted alkyl", "optionally substituted alkenyl", "optionally substituted alkynyl", "optionally substituted heterocycle", "optionally substituted aryl", "optionally substituted heteroaryl" and the like are alkyl, alkenyl, alkynyl, aryl, heteroaryl or another group or moiety as defined or disclosed herein that has substituent (s) optionally substituted for hydrogen atom (s). Such substituents are as described above. For a phenyl moiety (-Ph), the arrangement of any two substituents present on the aromatic ring may be ortho (o), meta (m), or para (p) relative to each other. Preferred optionally substituted moieties are -CF3, -CH2OH, -CC-Cl and -Ph-F.

[0019] A "group bound by O", a substituent bonded by O, and like terms as used herein, refers to a group or substituent that is attached to the moiety directly via the oxygen atom of the group or substituent. The group bound by O may be monovalent, including such groups as -OH, acetoxy, i.e. -OC (O) -CH3), acyloxy, i.e. -OC (O) -R, wherein R is -H, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl or optionally substituted heterocycle, aryloxy (aryl-O-), phenoxy (Ph-O-), heteroaryloxy (heteroaryl-O-), silyloxy, i.e. R3SiO-, wherein R is independently alkyl or aryl, optionally substituted, or -OR^PRwhere R^PR is a protecting group as defined previously, or it may be bivalent, i.e. = O.

[0020] The term "preparation of compound 1" refers to a substance prepared according to any of the synthetic schemes specifically or generically described, and includes Compound 1, where Compound 1 is found as the major component in weight, and the impurities present in the Compound after its initial isolation. as a solid or after recrystallization and / or purification of a solid.

[0021] The term "binding activity" refers to the ability of a particular compound, a preparation containing Compound 1 or an impurity (or impurity) in a preparation of Compound 1 to bind or bind to a receptor, typically a sex steroid receptor such as an androgen receptor or an estrogen receptor, for causing or modulating biological activity of the receptor. Binding is usually measured in tests as the ability of a compound, usually a contaminant in a formulation containing Compound 1, to displace the physiologically relevant ligand of a receptor (i.e., a reference ligand) that is bound to that receptor in a competition assay. The reference ligand is typically a natural receptor ligand or a receptor agonist that has been labeled with a radioactive or spectroscopic probe,

[0022] A radioactive probe is typically ³H and / or ¹⁴C, where the radioactive (s) atom (s) is replaced with one or more ligand atoms at positions where there will be no loss of radiolabelling to the extent that the test could complicate or entangle the interpretation of the test results. A spectroscopic probe is typically a fluorophore that is bound to a reference ligand at a position to provide a labeled reference ligand that has a Kd in the range of from 0.1-100 nM at positions and does not lose a fluorescent label to a degree under test conditions that could complicate or entangle interpretation of test results. The binding activity of a given compound or preparation of Compound 1 is typically expressed by Ki, where Ki is determined from the concentration of a given compound or preparation displacing a labeled reference ligand on the receptor at 50%, and Kd of a labeled reference ligand.

[0023] A "sex steroid receptor" refers to nuclear receptors normally associated with affecting the growth or function of reproductive organs and the development of secondary sexual characteristics, and includes the androgen receptor, the estrogen receptor α (ERa), the estrogen receptor β (ERe) and the progesterone receptor.

[0024] "Substantially free" as used herein refers to the property or impurity in a preparation of Compound 1 as absent or measurable in an amount that would adversely affect or impair the desired pharmacological activity of Compound 1. For example, the term "substantially free of receptor binding activity sex steroids "refers to the absence of receptor binding activity of Compound 1 preparation with nuclear sex steroid receptors as defined by K> 10 μΜ values ​​for binding to these receptors, as determined by standard receptor binding assay conditions, and is independent of the nature of the impurity. present in the preparation that would induce sex receptor binding activity. Similarly, Estrogen receptors that are essentially free of estrogen receptor binding activity relate to the absence of receptor binding activity of Compound 1 preparation with the EREA and ERe nuclear estrogen receptors as defined by K> 10 μΜ values ​​for binding to these receptors, as determined by standard binding assay conditions the receptor, and regardless of the nature of the impurity present in the preparation, which would induce binding activity to the estrogen receptor. When the term & quot; substantially free & quot; is used to describe the amount of impurity present in the preparation of Compound 1, the term means that the contaminant is absent in amount, which would adversely affect the pharmacological activity of Compound 1 in its intended use by causing side effects usually attributable to the activation of the nuclear estrogen receptor due to the activity of binding the contaminants to these receptors. Impurity (e.g., 17-acetinylandrost-5-ene-3e, 17e-diol) may directly affect the pharmacological activity of Compound 1 by binding or modulating the estrogen receptor or may indirectly affect the pharmacological activity of Compound 1 by its conversion in a subject treated with a composition containing the formulation. Compound 1 by hydrolysis (spontaneously or enzymatically) into a compound that affects or modulates the estrogen receptor (e.g., 17α-ethynyl-3e-acetoxyandrost-5-ene-17e-ol converted to 17-acetylandrost-5-ene-3β-ββ ol).

The terms "impurity" or "process contamination" as used herein refer to a component in a preparation of Compound 1 which is a steroidal by-product, by-product or degradation product formed during the synthesis of compound 1 and represents a minority share of the total weight of the preparation , typically less than about 2%.

[0026] A "formulation" or "pharmaceutically acceptable formulation" as used herein refers to a composition comprising a preparation of Compound 1 and one or more pharmaceutically acceptable excipients.

[0027] The inventions described herein provide methods for preparing 17-alkynylnteroids having oxygen substituents at positions 3, 7 and 17 that are substantially free of contaminants having undesired binding activity to the sex steroid receptors. The 17-alkynyleroids thus produced are substantially free of one or more impurities characterized by the absence of an aerobic substituent at C7, which is responsible for the undesired binding activity.

[0028] The present methods were developed in connection with the unexpected estrogenic effects found for Compound 1. Compound 1 was prepared by a process comprising the following steps, referred to as Process A:

(1) contacting a suitably protected acetylene anion with a suitably protected dehydroepiandrosterone to introduce the ethynyl group at position 17a by adding an acetylene anion to the functional group = O at position 17;

(2) contacting a suitably protected 17α-ethynylandrost-5-o-3e, 17α-diol with an oxidizing agent to introduce a functional group = O at the 7-position; and (3) contacting a suitably protected 17α-ethynylandrost-5-ene-7-it-3e, 17α-diol with a reducing agent to directly convert the functional group = O at the 7-position in β-hydroxyl.

[0029] Process Form A is depicted in Scheme I (herein referred to as Process A, Route 1). The Compound 1 thus produced has surprisingly been shown to have estrogenic effects. The profiling of the impurities of Compound 1 prepared in this way showed the presence of 17α-ethynyl-provrost-5-ene-3e, 17e-diol, which has the same structure as Compound 1 except that there is no β-hydroxy group at the 7-position. that 17α-ethynylandrost-5-ene-3e, 17β-diol showed significant binding activity to the estrogen receptors ERa and ERe, whereas Compound 1 essentially free of this contamination did not exhibit activity at these receptors during testing to 10 μΜ. Based on this unwanted sex steroid activity, a new method was developed, referred to as Process B, which omitted the production of an estrogenic by-product,

[0030] Process B includes the following steps.

(a) Connecting a suitably protected dehydroepiandrosterone with an oxidizing agent to direct introduction of the functional group = O at the 7-position;

(b) contacting properly protected androst-5-ene-7,17-dione-3e-ol with a reducing agent to carry out the functional group = O at the 7-position in β-hydroxyl;

(c) contacting a suitably protected acetylene anion with a suitably protected androst-5-en-17-ite-3e, 7e-diol to introduce the ethynyl group at position 17α by adding the acetylene anion to the functional group = O at position 17.

[0031] The procedures for carrying out step (a) include microbial oxidation as described in Wuts, PGM "A chemobiological synthesis of eplerenone" Synlett (3): 418-422 (2008); oxidation with oxygen-chromium based reagents [e.g. see Koutsourea, et al., "Synthetic approaches to the synthesis of cytostatic steroidal BD bilactam" Steroids 68: 569666 (2003) and Condom, et al., "Preparation of steroid-antigens through positions of the steroid not bearing functional groups" Steroids 23: 483-498 (1974)], peroxide-mediated allylic oxidation [e.g. see Marwah, P., et al. "An economical and green approach to the oxidation of olefins to eneones" Green Chem. 6: 570-577 (2004) and Marwah, P., et al., "Ergosteroids IV: clinical and biological activity of steroid glucuronosides, ethers and alkylcarbonates" Steroids 66: 581-595 (2001)], oxidation with N-hydroxysuccinimide / AIBN [e.g. see Lardy, et al. "Ergosteroids II: Biologically active metabolites and synthesis derivatives of dehydroepiandrosterone" Steroids 63: 158-165 (1998)].

to provide 7e-hydroxy as the dominant isomer and (4) removal under conditions in which the allyl alcohol produced by the functional group reduction = 0 is of sufficient stability. The hydroxyl protecting groups satisfying conditions (1) - (4) include an ester, usually an aryl ester or a C1-6 alkyl ester, when the protecting group for the functional group = 0 at position 17 is a ketal and the reducing agent used is a borohydride-based reducing agent. The use of a stronger hydride as a reducing agent would require a hindered ester or a substituted methyl ether as a hydroxyl protecting group to prevent premature loss of the hydroxy protecting group. Preferred protecting groups = O are a ketal, such as dimethyl ketal, diethyllketal or spiroketal made from ethylene glycol.

[0033] The procedures for performing step (b) include reduction with metal hydride reagents such as borohydride reagents that include Zn (BH4) 2, NaBH4, optionally with a transition metal salt such as CeCl3, NiCl2, CoCl2 or CuCl2. , L-Selectride (lithium tri-sec-butylborohydride) or N-Selectride (sodium tri-sec-butylborohydride). Licloruminum hydride or sodium aluminum hydride reagents may also be used, although the selectivity may deteriorate due to the reducing power of such reagents. This can be alleviated by reagents based on lithium aluminum hydride having alkoxy ligands on the aluminum to reduce reactivity. Such reagents have the general formula LiAl-Hn (OR) 4-n, in which n = 1, 2, 3, R is C1-6 alkyl, while reductions with aluminum hydride reagents require an ether solvent such as THF. The selectivity may be increased, particularly for aluminum hydride reagents, by conducting the reaction at a temperature of from 0 ° C to -78 ° C, with lower temperatures being more suitable for aluminum hydride reagents. while reductions with aluminum hydride reagents require an ether solvent such as THF. The selectivity may be increased, particularly for aluminum hydride reagents, by conducting the reaction at a temperature of from 0 ° C to -78 ° C, with lower temperatures being more suitable for aluminum hydride reagents.

[0034] The procedures for performing step (c) involve the in situ generation of an acetylene anion and subsequent contacting of the acetylenecane so formed with a suitably protected androst-5-en-17-ite-3e, 7e, 17e-triol. Acetylene can be prepared by contacting acetylene with an amide anion (e.g., NaNH2) in a hydrocarbon solvent such as benzene, toluene or xylene, such as in US 2251939, with sodium or potassium as a metal in liquid ammonia, such as in US 2267257, or by contacting a monosilyl-protected acetylene, such as trimethylsilylacetylene, with an organolithium reagent. Suitable organolithium reagents include commercially available n-butyl lithium, sec-butyl lithium, methyl lithium, t-butyl lithium or phenyllithium or may be prepared by reacting an alkyl or aryl bromide with a metallic lithium in an inert solvent such as diethyl ether or tetrahydrofuran. The acetylenet thus formed is then contacted with the appropriately protected androst-5-en-17-it-3β, β-diol.

[0035] For step (c), suitably protected androst-5-en-17-it-3e, 7e-diol will have hydroxyl-protecting groups typically used in carbanion chemistry and can be removed under conditions compatible with the presence of terminal alkyne and alcohol allyl, and include protecting groups that are removed under neutral or weakly acidic conditions, typically at pH 3-7, and which can be introduced under conditions compatible with allyl alcohol. Preferred protecting groups are silyl ethers of the formula (R.¹) 3SiO-, in which R¹ are independently aryl or C 1-6 alkyl, and include trimethylsilyl ether, triethylsilyl, t-butyldimethylsilyl, isopropyl dimethylsilyl, t-butyldiphenylsilyl, methyl diisopropylsilyl, methyl-t-butylsilyl, tribenzylsilyl and triphenylsilyl. Preferred silyl ethers are trimethylsilyl ether and t-butyldimethylsilyl ether. Certain substituted methyl ethers may be used and include 2- (trimethylsilyl) ethoxymethyl ether (SEM ether), tetrahydropyranyl ether (THP ether), tetrahydrothiopyranyl ether, 4-methoxytetrahydropyranyl ether, 4-methoxytetrahydrotiopyranyl ether, tetrahydrofuranyl ether and tetrahydrotiofuranyl ether. Some substituted ethyl ethers that can be used as hydroxyl protecting groups include 1-ethoxyethyl ether and t-butyl ether.

[0036] Other procedures for carrying out step (c) involve contacting suitably protected androst-5-en-17-ite-3e, 7e-diol with sodium acetylene, lithium acetylide (as its complex with ethylenediamine), ethynyl magnesium halide (e.g. chloride or bromide) or ethynyl zinc halide, such as, for example, in US 2243887, in diethyl ether or other ether solvents such as tetrahydrofuran, 1,2-dimethoxyethane, 2-methoxyethyl ether and the like.

[0037] In one embodiment of Process B, 3e-acetoxyandrost-5-en-7-one-17,17-ethylenedioxy is used as properly protected androst-5-ene-7,17-dione-3e-ol (see Scheme II; as Process B, Route 1).

[0038] In another embodiment of Process B, 3e-acetoxyandrost-5-en-7-one-17-oxime is used as properly protected androst-5-ene-7,17-dione-3e-ol (see Scheme III; as Process B, Route 2)).

[0039] The following examples and schemes further illustrate the invention and have no intention

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[0040] Example 1 Synthesis of 3e-trimethylsilyloxyandrost-5-en-17-one (TMS-DHEA):

DIEA combines with 1,1,1,3,3,3-hexamethyldisilazane (IMDS) and saccharin (as catalyst) in acetonitrile. The reaction mixture was heated at reflux for several hours while stirring under nitrogen. The released ammonia was removed under a small vacuum. The volume was then reduced by distillation, and then the mixture was cooled and the precipitated product was collected by filtration. The product filter cake, TMS-DIEA, was washed with cold acetonitrile and dried with warm nitrogen to a loss on drying (LOD) of no more than 0.5% to give the title compound with a yield of approximately 90%.

[0041] Example 2. Synthesis of 17α-ethynylandrost-5-ene-3e, 17e-diol: n-Butyllithium is slowly added to Me3Si-C CI in TIF under a nitrogen atmosphere at approximately 0 ° C to form Me3Si acetylenate -C C-Li. The temperature was raised to about 20 ° C, and TMS-DIEA was added as a solution in TIF and stirred for about 3 hours. The reaction was stopped by raising the temperature to about 40 ° C, and then methanol was slowly added. The released acetylene is removed under a small vacuum. Concentrated KOI was then slowly added to give way to gas evolution and the volume was reduced by approximately 50% by vacuum distillation at approximately 45 ° C. An excess of 6 N IC1 was slowly added, maintaining the temperature at approximately 40 ° C. The reaction mixture was diluted with water and cooled to approximately 5 ° C before collecting the product by filtration and washing the filter cake with cold methanol and 50/50 water. The product was dried with warm nitrogen to no more than 0.5% LOD to give the title compound with a yield of approximately 87%.

[0042] Example 3. Synthesis of 17α-ethynyl-3e-acetoxyandrost-5-en-17e-ol: 17-anynylandrost-5-ene-3e, 17e-diol was mixed with acetic anhydride, triethylamine and catalytic amount of DMAP in TIF at boiling temperature condensate return conditions for at least 4 hours. The progress of the reaction was monitored by IPLC and allowed to remain no more than 1% of the substrate. The mixture was then cooled to 30-50 ° C and water was added, followed by cooling to approximately 0 ° C for 1 hour. The crude product was collected by filtration, washed with cold acetonitrile and dried with warm nitrogen to a LOD of no more than 5%. The crude product was assessed by IPLC and recrystallized from acetonitrile if the title compound was present with a purity of less than 95% of the peak area of ​​the IPLC method. The recrystallized product was collected by filtration and dried,

[0043] Example 4: Synthesis of 17a-ethynyl-3e-acetoxy-17e-ol-androst-5-en-7-one: 17-aetinyl-3e-acetoxyandrost-5-en-17e-ol was combined with tert-butyl hydroperoxide and iodide copper (I) in acetonitrile and heated at reflux for two hours. The reaction was then quenched by cooling to about 50 ° C, and then a large excess of aqueous sodium thiosulfate was added over at least 30 minutes while stirring. Under these conditions, organic and aqueous phases do not mix with each other. Stirring was stopped to allow separation of the phases and the aqueous (lower) phase was discharged. The organic phase was extracted twice with an aqueous sodium sulfite solution and brine by stirring at a temperature of about 45 ° C for at least 30 minutes, followed by separation of the phases and removal of the aqueous phase. The resulting organic phase was concentrated to approximately 25% of the initial volume by vacuum distillation at a temperature lower than 45 ° C, and then cooled to approximately 5 ° C. Raw product,

17α-ethynyl-3e-acetoxylandrost-5-en-7-one-17e-ol, was collected by filtration. The filtered cake was washed with cold acetonitrile / water and dried with warm nitrogen to a LOD of no more than 1.0%. The crude product was recrystallized twice by dissolving N5 methylpyrrolidinone (NMP) at approximately 90 ° C, followed by cooling to 0 ° C for approximately one hour. The title compound was collected by filtration at a total yield of approximately 30%.

[0044] Example 5: Synthesis of 17α-ethynylandrostrost-5-ene-3e, 7e, 17e-triol: 17a-ethynyl-3-aceto-oxyandrost-5-en-7-one-17e-ol was reduced with NaBH4 in THF / methanol in the presence of

CeCl3 at approximately 0 ° C for approximately 2 hours. The progress of the reaction was monitored by HPLC. The reaction was quenched by the slow addition of dilute HCl with the liberated hydrogen removed under slight vacuum. Methyl tert-butyl ether (MTBE) was added and the reaction mixture was washed twice with brine, discarding the aqueous phases. The 3e-acetoxy group from the crude product was removed by adding methanol KOH to the organic phase at approximately 0 ° C for about 2 hours while the reaction was monitored by HPLC. After completion, the reaction mixture was neutralized with acetic acid and approximately half the volume of the reaction mixture was removed by vacuum distillation at a temperature lower than 45 ° C. Approximately two-thirds of the initial volume of the reaction mixture was added as isopropanol (IPA), and the volume is in turn reduced to approximately one quarter of the initial volume by vacuum distillation at a temperature lower than 45 ° C. The remaining mixture was cooled to 0 ° C and the crude 17α-ethynylandandrost-5-ene-3e, 7e, 17e-triol was collected by filtration, washed with cold IPA and dried with warm nitrogen.

[0045] Example 6. Recrystallization of 17α-ethynyllandrost-5-ene-3e, 7e, 17e-triol from the Process

A: The crude 17α-ethynylthrost-5-ene-3e, 7e, 17e-triol of Example 5 was dissolved in boiling methanol / water (~ 10/1). The methanol was removed by vacuum distillation while stirring and replaced with sufficient water to maintain the suspension of the crystallized product. The suspension is cooled to approximately 5 ° C during stirring and the product is recovered by melting. The filtered cake was washed with water and dried under vacuum to less than 0.5% water to give the title compound in yield for isolation.

<img file="PL2300489T3_D0003.tif" />

<img file="PL2300489T3_D0004.tif" />

[0046] Example 7. Synthesis of 3e-acetoxyandrost-5-ene-17,17-ethylendioxy: 36 kg of triethyl orthoformate, 20 kg of 3e-acetoxy-5androstene-17-one, 12.6 kg of glycol were introduced into a 300-L reactor. ethylene and 400 g of p-toluenesulfonic acid. The mixture was heated at reflux under nitrogen until complete reaction (about 2-3 hours). The mixture was then cooled to 60 ° C and 16 kg of anhydrous ethanol and 400 ml of pyridine were added. The resulting solution was transferred to a container and cooled overnight. The resulting solids were filtered and washed with 80 kg of 50% ethanol and dried at 40-50 ° C to yield 18.5-21.0 kg (81.5-92.5%) of the title compound.

[0047] Example 8. Synthesis of 3e-acetoxyandrost-5-en-7-one-17,17-ethylendioxy: 200 kg of ethyl acetate and 25 kg of 3e-acetoxyandrost-5-ene-17,17-ethylenedioxy were introduced into a 500-L reactor. . The mixture was stirred for 30 minutes, after which 55 kg was added

70% t-butyl peroxide and 9 kg sodium bicarbonate. The reaction mixture was then cooled to 0 ° C and 116 kg of 13% sodium perchlorate (aqueous solution) was added over 10 hours in such a way that the reaction temperature was kept below 5 ° C and the pH was from 7.5 to 8.5. After completion of the reaction, the organic layer was separated and the aqueous phase was extracted with ethyl acetate (35 kg x 2). The combined organic phase was combined from a solution of 33 kg of sodium sulfite in 167 kg of water and the resulting mixture was stirred at 40 ° C for 3 hours. The organic phase was washed with 50 kg of brine and concentrated to 55-60 kg, after which 50 kg of methanol was added. After cooling overnight, a white solid was formed, which was filtered and washed with 10 kg of methanol and dried at 40-50 ° C, yielding 7.1-7.8 kg (27.4- 30.1%) of the title compound. .

[0048] Example 9. Synthesis of 3e-acetoxyandrost-5-ene-17.17-ethylenedioxide-7e-ol. To a 500 L reactor, 48 kg of THF, 10 kg of 3e-acetoxyandrost-5-en-7ono-17, 17-ethylendioxy and a solution of 9.6 kg of CeCl 3 7H 2 O in 95 kg of methanol were charged. This mixture was cooled to 0 ° C, and 2.0 kg of NaBH4 was added in portions over 3 hours to maintain the temperature below 5 ° C. After stirring for another 30 minutes, 28 kg of acetone was slowly added to maintain the temperature below 5 ° C, while stirring continued for a further 30 minutes. 240 kg of water was added to the mixture while stirring continued for 1 hour. The organic solvents were removed in vacuo and the residue was extracted with ethyl acetate (100 kg + 50 kg). The combined organic phase was washed with brine. The solvent was then removed to give 8.6-8.9 kg (85.1-88.1%) of the title compound.

[0049] Example 10. Synthesis of 3e-acetoxyandrost-5-en-17on-7e-ol: 315 kg of acetone and 18 kg of 3e-acetoxyandrost-5-ene-17, 17-ethylenedioxine-7e-ol were introduced into a 500-L reactor. The mixture was cooled to 5 ° C and 2.34 kg of p-toluenesulfonic acid was slowly added to keep the temperature below 10 ° C. After stirring the mixture at 8-15 ° C for 36-48 hours, 3.0 kg of sodium bicarbonate was added with stirring continued for 1 hour. Acetone was removed in vacuo and 100 kg of water was added to the residue. The mixture was placed in a refrigerator overnight to give a white solid, which was filtered to give 33 kg (wet) of the title compound.

[0050] Example 11. Synthesis of androst-5-en-17-it-3e, 7e-diol: 230 kg of methanol, 33 kg of (wet) 3e-acetoxy-7e-hydroxy-5androstene-17 were introduced into a 500 l reactor. -one, 108 kg of water and 15 kg of Na2CO3. The mixture was heated at reflux for 3 hours. The methanol was removed in vacuo and 250 kg of water was added to the residue. The mixture was placed in a refrigerator overnight to give a precipitate. The solids were collected by filtration, then washed with water and dried at 40-50 ° C to afford 9.5-10.5 kg (67.9-75.0%) of the title compound as a white solid.

[0051] Example 12. Purification of androst-5-en-17-it-3e, 7e-diol: 20 kg of crude 3e, 7e-dihydroxyandrost-5-en-17-one and 200 kg of methanol were introduced into a 500 l reactor. and heated to dissolve all solids. The solution was filtered hot and after cooling the filtrate a white crystalline solid was formed. The solids were collected by filtration, washed with a small amount of methanol and dried at 40-50 ° C. The solid was then heated at reflux in 50 kg of ethyl acetate for 20 minutes. After cooling, the solid was filtered and dried at 40-50 ° C under vacuum to afford 15.2 kg (76%) of the purified title compound.

[0052] Example 13. Synthesis of 3e, 7e-bis- (trimethylsiloxy) -5-androsten-17-one: A mixture of 14.87 kg androst-5-en-17-it-3e, 7e-diol, 23.8 kg HMDS and 0.7 kg of saccharin as catalyst in 100 L of acetonitrile were heated at reflux for 8 hours while stirring under nitrogen. The released ammonia was removed under a small vacuum. The volume of the reaction mixture was then reduced by distillation by collecting 30 L of distillate (requires about 2 h). The volume of the reaction mixture was further reduced to half the initial volume of the reaction mixture by distillation under reduced pressure (700 mm Hg), which requires about 2 h of heating at 50 ° C. The resulting homogeneous thick suspension is cooled to 5 ° C (requires about 3 hours), with additional acetonitrile added to maintain the minimum mixing volume,

[0053] Example 14. Synthesis of 17α-ethynyl-5-androstene-3e, 7e, 17e-triol: up to 11.02 kg of TMS-acetylene in 56.5 L of tetrahydrofuran (THF) at -27 ° C under nitrogen atmosphere was added 8.51 l 10M n-BuLi. n-Butyllithium was added very slowly to maintain the temperature -7 to -27 ° C (requires about 2 h) and the resulting reaction mixture was stirred for 10 min at approximately 0 ° C to form lithium TMS-acetylide. A solution of 25.41 kg of 3e, 7e-bis- (trimethylsiloxy) -5androsten-17-one in 95.3 1 of THF filtered through a 25 μm filter was added to the solution of lithium TMS-acetyl, allowing the reaction temperature to rise to 20-25 ° C. After the addition was complete, the reaction temperature was raised to 40-45 ° C. To work up the contents of the reactor, 31.8 1 of methanol was added in about 1 h, followed by 3.81 kg of KOH in 18.4 L of water to give a final reactor temperature of 50 ° C. The released acetylene is removed under a small vacuum. The reactor contents were then concentrated by distillation at 80 ° C for 1 h, then under vacuum (175 mm Hg) at a temperature of about 70 ° C (with an initial temperature of 25 ° C to avoid turbulent boiling) to half the initial volume of the vessel. The residue was cooled to about 10 ° C and 35.0 kg of deionized water was added, followed by 16.4 kg of 12 N HCl, keeping the vessel at about 10 ° C to give a final pH of 1. An additional 26.0 kg of deionized water was added and the resulting the mixture was stirred at about 5 ° C for 1 h. The resulting suspension was filtered and washed with a 75/25 methanol / water mixture (16.9 L methanol, 5.6 L water). Collected solids were dried under vacuum (28 inches Hg column) at 45 ° C for 12 h to a loss on drying of no more than 0.5% to obtain 9,

[0054] Example 15. Recrystallization of 17α-ethynyl-5-androstene-3e, 7e, 17e-triol: 9.6 kg of the crude 17α-ethynyl-5-androstene-3e, 7e, 17e-triol prepared in Example 14 was dissolved in a heated boiling at methanol condensate / 50/50 water (4.2 kg methanol and 5.4 kg water). 33.4 kg of methanol was added to the solution followed by 37.6 kg of THF. The mixture was heated at reflux and stirring was continued until all solids dissolved, after which 99.8 kg of deionized water were added, maintaining the reactor temperature at 60-75 ° C. The mixture was cooled to 0-5 ° C over 2 h and kept at this temperature for 1 h with continued stirring. The solids were recovered by filtration, washed with 9.6 kg of cold methanol and 50/50 water and dried under vacuum (28 inches Hg) at 50 ° C for 8 h, as a result of which 8.2 kg of 17α-ethynyl-5-androstenee, 7e, 17e-triol were obtained. This first recrystallisation is used to remove trace colored impurities from the initial product. The second recrystallization was carried out by heating the solid from the first recrystallization in methanol: water ~ 10: 1 (145.8 kg methanol and 18.2 kg water) to 80 ° C until all solids dissolved. The solution at 55-60 ° C was filtered through a 25 μm filter to remove particulate contaminants, after which 2.5 kg of methanol was added at 55-60 ° C (used to flush the reactor). Vacuum distillation was carried out at 125 mmHg at 70 ° C, as much as 0.9 to 1.2 times the volume of methanol, which was added to the reactor, collected as a distillate with water added as needed to allow stirring (about 120-160 kg of added water ). The final volume of the reaction mixture was 200-225 L. The reaction mixture was cooled to 0-5 ° C and kept at this temperature for 1 h. The resulting suspension was filtered and the filter cake was rinsed with 10 kg of deionized water and dried under vacuum (28 inches Hg column). ) at 50 ° C for 12 hours to a residual water content of less than 0,5%. Isolation procedures were used to reduce the THF content in the final product. The yield was 8.0 kg recrystallized title compound (83% yield). Isolation procedures were used to reduce the THF content in the final product. The yield was 8.0 kg recrystallized title compound (83% yield). Isolation procedures were used to reduce the THF content in the final product. The yield was 8.0 kg recrystallized title compound (83% yield).

Scheme II. Process B, Route 2

<img file="PL2300489T3_D0005.tif" />

[0055] Example 16. Synthesis of 3e-acetoxyandrost-5-en-7-one-17-oxime: 3e-Acetoxyandrost-5-ene-7,17-dione (45 g, 130 mmol) was dissolved in 800 ml of methanol, 200 mL of dichloromethane and 14.5 g of Et3N (144 mmol). A solution of 10 g of hydroxylamine hydrochloride dissolved in 200 ml of methanol was added to the solution at room temperature. After stirring overnight, 200 ml of water were added, and volatile organic substances were then removed by evaporation under reduced pressure. Additional 1 L of water was added to the resulting residue to give a white solid that was filtered and washed thoroughly with water. 45 g of crude title oxime with a purity of 95% according to¹H-NMR, which was used in the next step without further purification.

[0056] Example 17. Synthesis of 3e-acetoxyandrost-5-ene-17-oxime-7e-ol: To a solution of 44 g

3 g of acetic acid-5-en-7-one-17-oxime (100 mol%) in 800 ml of methanol and 200 ml of tetrahydrofuran was added 50 g of cerium chloride heptahydrate (110 mol%) in 20 ml of methanol. The resulting mixture was stirred until the solids dissolved completely. To a solution cooled to about -5 ° C was added 7 g of sodium borohydride over 30 min. After stirring for an additional 1.5 h at -5 ° C, the reaction mixture was quenched with acetone (100 mL) and then allowed to warm to room temperature over 30 min. The reaction mixture was concentrated in vacuo to remove volatile organic matters. 800 ml of water was added to the residue, followed by extraction with ethyl acetate (3 x 500 ml). The combined organic extracts were washed with brine, dried over Na 2 SO 4, then concentrated to give 42 g of the title compound as a white foam,

[0057] Example 18. 3e-acetoxyandrost-5-en-17-one-7e-ol: To a solution of 42 g of 3β-acetoxy-8-oxo-5-ene-17-oxime-7e-ol (100 mol%) in 200 ml of ethanol was added 100 ml of water, followed by 80 g (400 mol%) of sodium hydrosulphite. The reaction mixture was heated at 55 ° C and stirred for 16 h. After cooling, the reaction mixture was concentrated under reduced pressure. The residue was diluted with 100 mL of water and the resulting solid was collected by filtration and redissolved in 1 L of dichloromethane. 1 g of active carbon was added to the DCM solution. After stirring overnight, the mixture was filtered and the resulting filtrate was washed with water, dried and concentrated to give 25 g of crude product. Recrystallization from ethyl acetate gave 22 g of the title compound.

[0058] Example 19. Estrogen Receptor Binding Assay: A suitable example system is the estrogen receptor kit manufactured by PanVera for ERe, which contains a recombinant estrogen receptor ligand β, FLUORMONE ™ ES2 (ES2), a fluorescent labeled estrogen ligand and a suitable buffer. The system was used in a competitive fluorescence polarization assay in which the test article, such as Compound 1 preparation or positive control, displaces ES2 from its binding site. After binding to ERe, ES2 breaks down slowly and has a high fluorescence polarization value. Unbound ES2 breaks down quickly and has a low fluorescence polarization value. The change in the polarization value in the presence of the test compound then determines the relative binding affinity of this test compound with respect to ERe expressed by its IC50, which is the concentration of the test compound providing half the maximum polarization change. Based on IC50, a Ki was calculated using the Cheng-Prusoff equation [Biochem. Pharmacol. 22: 30993108, (1973)]: Ki = IC50 / (1 + D / Kd), where D is the concentration of ES2 and Kd is the dissociation constant for binding ES2 to ERe (Kd = 4 ± 2 nM).

[0059] The competition test was carried out according to the manufacturer's instructions (Lit. # L0712, Rev. 10/03). The reagents used in the study were baculovirus-expressed full-length human ERe 4.5 pmol / Lil in 50 mM bis-tris-propane (pH = 9), 400 mM KCl, 2 mM DTT, 1 mM EDTA, 10% glycerol, 400 nM ES2 in methanol and selection buffer E2 consisting of 100 mM potassium phosphate (pH = 7.4), 100 Lig / ml BGG, 0.02% NaN3. The ES2ERe complex was prepared with 20 μl of 20 nM ERe (0.020 pmol ^ 1) and 20 μL of 2 nM ES2 (0.002 pmol-1). The positive control (estrogen) solution was prepared using 20 μl of a 1.0 mM stock solution in DMSO and 80 μl DMSO. During the first dilution, 50 μl of this solution is added to 50 μl DMSO, which is then diluted 2 times to produce a 14 point dilution curve. During the second dilution, to 4 μl of each DMSO solution from the first dilution, 400 μl ES2 selection buffer is added. To 20 μl of the test compound, serially diluted as described immediately above, in a 384-well black microtiter flat bottom plate, 20 μl of ES2ERe complex (0.5% final concentration of DMSO) was added, followed by incubation in the dark at 20-30 ° C for 1 hour. -4 h. Test compound was similarly treated, except that the initial concentration was 10 mM. Fluorescence polarization values ​​are obtained using interference filters at excitation at 485 nm and emission at 530 nm. ERa binding assay was performed as for ERe except that the Baculovirus-expressed full-length 2.8 pmol / Lil ERa was used as a reagent with the ERa-ES2 complex made with 20 μl 30 nM (0.030 pmol / dl) and 20 g 2 nM ES2 (0.002 pmol / dl).

[0060] Example 20: AR, GR and PR receptor binding assays. The AR competition test was carried out in accordance with the manufacturer's instructions (Lit. No. L0844, Rev. 05/02) as described for ERe with the following exceptions. The reagents used were the recombinant binding domain of rat androgen receptor ligand labeled His and GST [AR-LBD (HisGST)] 0.38 pmol / Lil in buffer containing protein and glycerol stabilizing agents (pH = 7.5), 200 nM FLUORMONE^™ AL Green, which is a fluorescently labeled androgen ligand in 20 mM Tris, 90% methanol and an AR selection buffer containing stabilizing agents and glycerol (pH = 7.5) with 2 μl 1 mM DTT added per ml of selection buffer (2 mM selection buffer AR in the added DTT), were used as reactants. The AL Green-AR complex was prepared from 20 μl of 50 nM AR (0.050 pmol ^ 1) and 20 μl 2 nM AL Green (0.002 pmol ^ 1). K, calculated using the dissociation constant for fluorophore binding to the receptor Kd = 20 ± 10 nM. [0061] The PR competition assay was performed according to the manufacturer's instructions (Lit. # L0503, Rev. 06/03) as described for ERe with the following exceptions. The factors used were the recombinant human GST receptor ligand binding domain [PR-LBD (GST)] 3.6 pmoi / Lii in 50 mM Tris (pH = 8.0), 500 mM KCl, 1 M urea, 5 mM DTT. .^™ PL Green, which is a fluorescently labeled progesterone ligand in 20 mM Tris, 90% methanol (pH = 6.8) and a PR selection buffer containing protein and glycerol stabilizing agents (pH = 7.4) with 4 Lil 1 mM DTT added on ml of selection buffer (4 mM PR selective buffer in the added DTT). The PL Green-PR complex was made of 20 g 80 nM PR (0.080 pmol / L) and 20 L of 4 nM PL Green (0.004 pmol / LL). Ki was calculated using the dissociation constant for the fluorophore binding to the receptor Kd = 40 nM.

[0062] The GR competition test was carried out in accordance with the manufacturer's instructions (Lit.

No. L0304, Rev. 12/01) as described for ERe with the following exceptions. The reagents used were a recombinant full-length human glucocorticoid receptor of 0.240 pmol / LL in 10 mM phosphate buffer (pH = 7.4), 200 mM Na2MoO4, 0.1 mM EDTA, 5 mM DTT and 10% glycerol, 200 nM FLUORMONE.^™ GS1, which is a fluorescently labeled glucocorticoid ligand in 75% methanol, and a GR selection buffer containing 100 mM potassium phosphate (pH = 7.4), 200 mM Na2MoO4, 1 mM EDTA, 20% DMSO with 5 Ll 1 mM DTT added on ml of selection buffer (5 mM selection buffer GR in added DTT), 1 mM GR stabilizing peptide, which is a co-activator related peptide [see Chang, CY Mol. Cell Biol. 19: 8226-36 (1999)] in 10 mM phosphate buffer (pH = 7.4) and 1 M

DTT in water, used as reagents. 2.5 ml of GR stabilizing peptide solution and 125 I1 M DTT solution are added to 2.5 ml GR selection buffer to form a GR-glucocorticoid receptor stabilizing peptide complex. The order of addition to the microtiter plate was: 1 L of test compound in 1% DMSO, 10 L of 16 nM GR (0.016 pmol / LL) and finally 10 L of 4 nM GS1, followed by incubation in the dark at 20-30 ° C for 4 h (the whole experiment should not exceed 7 hours). Ki was calculated using the dissociation constant for the binding of the fluorophore to the receptor Kd = 0.3 ± 0.1 nM.

[0063] Example 21. Contamination of the impurities of the 17α-ethynyl-5-androstene3e, 7e, 17e-triol preparations (Compound 1).

[0064] Process A: HPLC conditions for profiling impurities of the preparations of Compound 1 with

Process B is given in Table 1.

Table 1 HPLC conditions for the contamination profiling of preparations of Compound 1 from Process A

<td>Column</td><td colspan="4">Waters XTERA ™ RP18, 3.5 gm, 4.6 mm (ID) χ 150 mm (L)</td>

<td>Mobile phase A</td><td colspan="4">100% deionized water (degassed)</td>

<td>Mobile phase B</td><td colspan="4">100% acetonitrile (degassed)</td>

<td>Column temperature</td><td colspan="4">30 ° C</td>

<td>Detection wavelength</td><td colspan="4">210 nm</td>

<td>Mobile phase (initial)</td><td colspan="4">90% mobile phase A; 10% mobile phase B</td>

<td>Flow rate (initial)</td><td colspan="4">1.0 ml / min</td>

<td rowspan="6">Pump gradient program</td><td>Time (min)</td><td>%AND</td><td>% B</td><td>Flow rate</td>

<td>0.00</td><td>90.0</td><td>10.0</td><td>1.00</td>

<td>40.0</td><td>20.0</td><td>80.0</td><td>1.00</td>

<td>43,00</td><td>20.0</td><td>80.0</td><td>1.00</td>

<td>43,01</td><td>90.0</td><td>10.0</td><td>1.00</td>

<td>50.00 (end)</td><td>90.0</td><td>10.0</td><td>1.00</td>

<td>Injection volume</td><td colspan="4">10 gl</td>

<td>Analysis time</td><td colspan="4">50 minutes</td>

Table 2. Impurities in an exemplary Compound 1 preparation prepared according to Process A, Route 1 before recrystallization

<td>Relationship</td><td>RRT *</td><td>Peak field%</td>

<td>Unknown</td><td>0.63</td><td>0.59</td>

<td>Androst-5-en-17-it-3 β, 7β-diol</td><td>0.87</td><td>0.12</td>

<td>17α-ethynylandrost-5-ene-3e, 7e, 173-triol (Compound 1)</td><td>1.00</td><td>96,58</td>

<td>17α-ethynylandrost-5-ene-3β, 7α, 17β-triol</td><td>1.04</td><td>0.99</td>

<td>17a-etenyloandrost-5-ene-3e, 7e, 173-triol</td><td>1.09</td><td>0.93</td>

<td>17 α-ethynyl oandrost-5-en-7-it-3β, 17β-diol</td><td>1.16</td><td>0.06</td>

<td>Unknown</td><td>1.47</td><td>0.04</td>

<td>Unknown</td><td>1.60</td><td>0.06</td>

<td>17α-etynyloandrost-5-ene-3β, 17β-diol</td><td>1.75</td><td>0.63</td>

<td colspan="3">* RRT-relative retention time (approximate) relative to Compound 1 (ie RRT of Compound 1 assumed to be 1.00)</td>

Table 3. Impurities identified in an exemplary Compound 1 preparation prepared according to Process A, Route 1 after recrystallization

<td>Relationship</td><td>RRT *</td><td>Peak field%</td>

<td>Unknown</td><td>0.55</td><td>0.13</td>

<td>Androst-5-ene-3β, 7β, 17β-triol</td><td>0.87</td><td>0.06</td>

<td>17α-ethynylandrost-5-ene-3β, 7β, 17β-triol (Compound 1)</td><td>1.00</td><td>97.67</td>

<td>17α-ethynylandrost-5-ene-3β, 7α, 17β-triol</td><td>1.05</td><td>0.72</td>

<td>17α-etenyloandrost-5-ene-3β, 7β, 17β-triol</td><td>1.09</td><td>0.65</td>

<td>17 α-ethynyl oandrost-5-en-7-it-3β, 17β-diol</td><td>1.16</td><td>0.05</td>

<td>Unknown</td><td>1.60</td><td>0.05</td>

<td>17α-ethynyl-3 β-acetoxyandrost-5-ene-17β-diol</td><td>1.72</td><td>0.05 **</td>

<td>17α-etynyloandrost-5-ene-3β, 17β-diol</td><td>1.75</td><td>0.61 **</td>

<td colspan="2">* RRT-relative retention time (approximate) relative to Compound 1 (i.e.</td><td>RRT of compound 1</td>

<td>assumed to be 1.00)</td><td></td><td></td>

<td colspan="3">** Impurities with specific binding capacity to estrogen receptors</td>

<td>(directly or from the product obtained after ester hydrolysis)</td><td></td><td></td>

[0065] Process B: HPLC conditions for the contamination profiling of preparations of Compound 1 from Process B are identical to those of Table 1.

Table 4. Impurities identified in the exemplary Compound 1 preparation

<td colspan="2">created in accordance with Process B, Route 1 before the re-name</td><td>.izacją</td>

<td>Relationship</td><td>RRT</td><td>Peak field%</td>

<td>Androst-5-en-17-it-3 β, 7β-diol</td><td>0.94</td><td>0.80</td>

<td>17α-ethynylandrost-5-ene-3β, 7β, 17β-triol (Compound 1)</td><td>1.00</td><td>98.30</td>

<td>17α-ethynylandrost-5-ene-3β, 7α, 17β-triol</td><td>1.02</td><td>0.26</td>

<td>17α-etynyloandrost-5-en-7-one-3β, 17β-diol</td><td>1.06</td><td>0.08</td>

<td>3 β-acetoxyandrost-5-en-17-on-7 β-ol</td><td>1.24</td><td>0.10</td>

<td>DHEA</td><td>1.27</td><td>0.03</td>

<td>3 β-acetoxyandrost-5-en-17-one</td><td>1.46</td><td>0.09</td>

<td>3 β-acetoxy-17-ethylenedioxyandrost-5-ene</td><td>1.50</td><td>0.12</td>

<td>3β, 7β-bis (trimethylsilyloxy) androst-5-en-17-one</td><td>1.68</td><td>0.21</td>

<td colspan="3">* RRT-relative retention time (approximate) relative to Compound 1 (i.e., RRT compound 1 assumed to be 1.00)</td>