Steroid compounds, method for their preparing, pharmaceutical composition, methods for regulation and improving for reproduction, intermediate compounds

FIELD: organic chemistry, steroids, biology.

SUBSTANCE: invention relates to steroid compounds of the general formula (X):

wherein in fragment of the formula XA:

each bond between C6 and C7, between C7 and C8, between C8 and C9, between C8 and C14 and between C14 and C15 is a single or double bond under condition that each atom C6, C7, C8, C9, C14 and C15 is bound with adjacent C-atom by a single bond or one double bond; CR3 means -CHOH; A means methylene or ethylene group; R4 and R4' mean (C1-C4)-alkyl, hydrogen atom (H); R20 means (C1-C4)-alkyl; R23 and R23' mean in common piperidine-1-yl, morpholine-4-yl, pyrrolidine-1-yl, piperazinyl possibly substituted with -OH, benzene, pyridine, pyrimidine, phenyl, alkoxycarbonyl group, or R23 means H and R23' means substituted alkyl. These compounds can be used for stimulation of meiosis in human oocytes. In proposed compounds steroid differs specifically as nitrogen atom of amino-group is bound with C17-atom of steroid skeleton by spacer A.

EFFECT: improved methods of synthesis, valuable biological properties of compounds.

16 cl, 8 dwg, 2 tbl, 30 ex

 

The invention relates to pharmaceutically active steroid compounds, pharmaceutical compositions comprising these compounds, the use of these compounds to obtain a pharmaceutical composition suitable for the regulation of reproduction, especially of meiosis, to apply to obtain a contraceptive or as propertylegal of the drug, the way the regulation of reproduction, such as meiosis, the way of improvement opportunities inherent oocytes ability to develop in a mammal, and methods of obtaining new steroid compounds.

Meiosis is a unique and final event of embryonic cells, which is based on sexual reproduction. Meiosis involves two meiotic division. In the first division are exchanged between the maternal and paternal genes to dividing pairs of chromosomes in the daughter cells. They contain only half the number (1n) chromosomes and DNA double 2s. The second meiotic division takes place without DNA synthesis. Therefore, this division leads to the formation of haploid germ cells only with 1C DNA.

Meiotic events similar in male and female germ cells, but the timing and the processes of differentiation that lead to female embryonic cells and spermatozoa differ fundamentally. All female germ cells entering the in profeso the first meiotic division in the early period of life, often before birth, but they later stop in the form of oocytes in prophase (dictyate state) up until after puberty does not occur ovulation. Thus, since the early period of life, a woman has a stock of oocytes that are used until stock is exhausted. Meiosis in women does not end until, until fertilization, and leads to only one germ cell and two abortive to polotical on the germ cell. In contrast, only some male germ cells enter meiosis after the onset of puberty and leave the population of germ cells throughout life. Once initiated, the meiosis in the male cage passes without significant delay and gives four sperm.

Very little is known about the mechanisms that control the initiation of meiosis in men and women. New studies indicate that in the follicle purines, gipoksantin and adenosine may be responsible for the stop of meiosis in the oocyte [S.M.Downs etc., Dev. Biol., 82, 454-458 (1985); J.J. Epplg. and others, Dev. Biol., 119. 313-321 (1986); S.M.Downs, Mol. Reprod. Dev., 35, 82-94 (1993)]. Presence is able to diffuse substances that regulate meiosis, first described Byskov and others on the culture of embryonic mouse gonads [A.G.Byskov etc., Dev. Biol., 52, 193-200 (1976)]. Activating meiosis substance (MAS) the secret is regulated in the fetal mouse ovary, in which meiosis occurs and prevents meiosis substance (MPS) are distinguished from the morphologically differentiated testis with resting cells, not included in meiosis. It has been suggested that the relative concentrations of MAS and MPS regulate the start, the stop and the resumption of meiosis in male and female germ cells [A.G.Byskov and others: The discrimination of Reproduction (as amended .Knobil and J.D.Neill), Raven Press, New York (1994)]. It is clear that if meiosis can be adjustable, playback can be controlled. In a recent article [A.G.Byskov et al., Nature, 374, 559-562 (1995)] described the isolation of some of sterols, which can activate the meiosis of oocytes from bovine testes and from human follicular fluid [T-MAS (Sterol activating meiosis in the testes) and FF-MAS (Sterol activating meiosis in follicular fluid):4,4-dimethyl-5α-cholesta-8,14,24-triene-3β-ol].

It was also demonstrated that micromolar concentrations of synthetic FF-MAS is able to induce resumption of meiosis dose-dependent manner in rat oocytes, which stopped the phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) [.Hegele-Hartung and others, Biol. Reprod., 64, 418-424 (2001)]. It was shown that this effect can occur when multiple shielded oocytes (CEO) and naked oocytes (DO) were cultured in vitro in the presence of FF-MAS.

Additional substances that reg is irout meiosis, described in WO 96/00235 A1, WO 96/27658 A1, WO 97/00884 A1, WO 98/28323 A1, WO 98/52965 A1, WO 99/58549 A1 and WO 00/68245 A1.

In the application WO 98/52965 A1 describes meiosis activating derivatives 20-aralkyl-5α-pregnane.

In the application WO 00/68245 A1 discloses steroid derivatives, which are able to suppress meiosis, so that these compounds applicable as contraceptives in women and men. These compounds are primarily unsaturated derivatives of cholestane, characterized in that 3β-hydrogen atom is associated with14-carbon atom cholestanol skeleton.

In the application WO 96/00235 A1 describes inducing meiosis sterols, known as intermediate compounds in the biosynthesis of cholesterol, and some structurally related synthetic sterols. It was found that these substances regulate meiosis. Like cholesterol, these sterols contain the side chain at C17skeleton of Sterol and additionally at least one double bond in Δ7that Δ8or Δ8(14).

In the application WO 96/27658 A1 disclosed a method of stimulation of meiotic germ cells, which comprises introducing into the cell in vivo, ex vivo or in vitro an effective amount of a compound that causes an accumulation of endogenous meiosis activating substance to the level at which undergo meiosis. Revealed that these compounds, which cause the accumulation of meiosis activating the substances, are amphotericin b, aminoguanidine, 3α,5α,6β-trihydroxychalcone, melatonin, 6-chloroethane and 5 methoxytryptamine, as well as other derivatives and their agonists. It is also reported that amongst the meiosis activating substance is 5α-cholestan-3β-ol, D-gonochorist-8,14-Dien-3β-ol and 22,25-disforestation, 25-Aza-24,25-digitalnature, 24,25-aminolevulenic, 23 - and 24-assholesteen and derivative 25-zaholustnoe.

In applications WO 97/00884 A1 and WO 98/28323 A1 describes a substance that can be used for stimulation of meiosis in vitro, in vivo or ex vivo. Therefore, the disclosed compounds are agonists of the natural activators of meiosis and therefore can be used in the treatment of infertility caused by insufficient stimulation of meiosis in men and women. This document discloses some compounds that can be antagonists existing in nature activators of meiosis, so that these compounds may be suitable for use as a contraceptive. Disclose, among other compounds include 5α-cholesterol-8-EN-3β-Ola and 5α-cholesterol-8,14-Dien-3β-Ola, which, in particular, can contain an amino group in the side chain, related With17cholesterol skeleton, and the amino group attached to the Sterol skeleton through With4the spacer. With aminogroup the th bound ( 1-C4)alkyl or (C3-C6)cycloalkyl.

Additionally, in the application WO 99/58549 A1 disclosed Sterol derivatives which are effective in the regulation of meiosis. Described that these compounds have the ability to weaken infertility for both males and females, especially man. Derivative of a Sterol, effective as regulating substances, among others, are (20R)-20-methyl-23-dimethylamino-5α-pregna-8,14-Dien-3β-ol, (20R)-20-methyl-23-dimethylamino-5α-pregna-5,7-Dien-3β-ol, 4,4-dimethyl-24-phenyl-amino-5αhole-8,14-Dien-3β-ol, 4,4-dimethyl-24-(N,N-dimethylamino)-24-cyano-5α-cholesta-8,14-Dien-3β-Oli, in addition, many derivatives of sterols with inorganic salts of acids at position 24, which has one double bond or more double bonds in the Sterol skeleton.

Unsaturated derivative of a Sterol having the amino group in the side chain at C17described also J.J.Sheets and L.E.Vickery, "Active Site-directed Inhibitors of Cytochrome P-450scc", J. Biol. Chem., volume 258 (19), 1983, str-11452, in relation to the impact of these sterols to cleave the side chain bullish adenocortical cholesterol cytochrome P-450 (P-450scc). In this document, among other compounds disclosed 22-amino-23,24-binaural-5-ene-3β-ol and 23-amino-24-normal-5-ene-3β-ol.

Additional unsaturated derivatives having an amino group in the side chain at C17described ..Mangla and W.DNes: "Sterol C-methyl Transferase from Prototheca wickerhamii. Mechanism, Sterol Specificity and Inhibition", Bioorg. and Med. Chem. (2000), 8 (5), 925-936. Herein disclosed, in particular, the 32-asasination.

When using previously described regulating meiosis components it was found that the resumption of meiosis occurs in naked oocytes in vitro. However, these compounds had only edge the effectiveness of the stimulation of meiosis in oocytes surrounded by a granular cells of the ovary (SEO multiple shielded oocytes). The disclosure of the above documents are included by citation.

One objective of the present invention is to find substances that are applicable to the regulation of reproduction, particularly of meiosis, in individuals, male and female, mainly in mammals and, more particularly, in humans.

Another objective of the present invention is to provide pharmaceutical compositions that include new substances.

Another preferred aim of the present invention is to provide use of new substances in obtaining pharmaceutical compositions suitable for the regulation of reproduction, particularly of meiosis.

Another preferred aim of the present invention is to provide a new method of regulation play, for example, meiosis.

An additional objective of the present invention is ware the treatment of human infertility.

An additional objective of the present invention is to improve the maturation of human oocytes.

Another objective of the present invention is to improve the synchrony of nuclear maturation, cytoplasmic and/or membrane of oocytes.

Another objective of the present invention is to improve the speed implantation of oocytes during maturation in humans and in vitro fertilization.

Another additional objective of the present invention is to reduce the occurrence of human Precambrian with chromosomal anomalies (aneuploidy).

Another additional objective of the present invention is to increase the rate of cleavage of human Precambrian.

Another additional objective of the present invention is to improve the quality of human Precambrian.

Another objective of the present invention is to provide a method of producing new substances.

In accordance with the present invention steroid compounds of General formula X can be usefully applied in the regulation of reproduction, for example, meiosis in mammals, for example male and female individuals, and, in particular, in humans:

where in the code

each link between5and6that is between 6and7between7and8between8and9between8and14and between14and15means independently single bond or a double bond, and at least one of these bonds is a double bond, and

where each carbon atom With5With6With7With8With9With14and15associated with the adjacent carbon atom by a single bond or, at most one double bond and for all other carbon atoms of the steroid skeleton are a single bond, and

With3R3means

a)3=O or

b)3H-OR3'where R3'selected from the group including hydrogen, unsubstituted or substituted, linear or branched (C1-C10)alkyl and3(O)-R3"attached to part of the SN-O through C(O)-group, where

R3"selected from the group including

i) substituted or unsubstituted, linear or branched (C1-C10)alkyl,

ii) substituted or unsubstituted, linear or branched (C1-C10)foralkyl,

(iii) unsubstituted or substituted (C6-C10)aryl,

(iv) unsubstituted or substituted (C5-C10)heteroaryl,

(v) unsubstituted or substituted, linear or branched (C1-C10)alkyloxy and

vi) unsubstituted or substituted, Lina is hydrated or branched (C 1-C10)alkylamino or

C)3H-SO2-R3"or3=NOR3"where R3"has the same meanings as above, or

g)3H-O-R3"'where R3"'means unsubstituted or substituted, linear or branched (C2-C10)alkylen and forms a cyclic simple ether with a carbon atom of the steroid skeleton, and an oxygen atom, or

d) a cyclic structure with atom With3in which R3'means unsubstituted or substituted, linear or branched (C2-C10)alkylene, or

e)3N-Hal, where Hal denotes F, Cl, Br or I, and

R4, R4'and R20chosen independently from the group comprising hydrogen and unsubstituted or substituted, linear or branched (C1-C4)alkyl, and

R23and R23'chosen independently from the group including:

a) hydrogen,

b) unsubstituted or substituted, linear or branched (C1-C8)alkyl,

C) unsubstituted or substituted, linear or branched (C2-C8)alkenyl,

d) unsubstituted or substituted, linear or branched (C1-C8)alkyl, and at least one of the carbon atoms of the alkyl substituted by any of the atoms of oxygen, nitrogen and sulphur,

d) unsubstituted or substituted, linear or branched (C2-C8)al is Anil, moreover, at least one of the carbon atoms alkenyl substituted by any of the atoms of oxygen, nitrogen and sulfur, and

(e) unsubstituted or substituted, linear or branched (C6-C10)aryl, or

R23and R23'form together

a) unsubstituted or substituted, linear or branched (C2-C7)alkylene, especially (C5-C7)group, or

b) unsubstituted or substituted, linear or branched (C2-C7)alkylene, especially (C5-C7)group, where at least one of alkilinity carbon atoms replaced by oxygen, nitrogen or sulfur, and

A represents methylene or ethylene group, and the group is unsubstituted or substituted methylene or ethylene; in a preferred variant embodiment of the invention a represents methylene or ethylene,

when the condition of the waiver on the following connections: (20R)-20-methyl-23-dimethylamino-5α-pregna-8,14-Dien-3β-ol and (20R)-20-methyl-23-dimethylamino-5α-pregna-5,7-Dien-3β-ol, 23-asasination, 22-amino-23,24-binaural-5-ene-3β-ol, 23-amino-24-normal-5-ene-3β-ol.

Preferably the invention relates to steroid compounds in which at least one double bond is present in the steroid skeleton between carbon atoms With6With7With8With9With14and15accordingly, In one additional variant embodiment of the present invention the double bond can be between 5and6in addition, at least one double bond between C6With7With8With9With14and15respectively. Particularly preferably, when the steroid double bond conjugated with each other, if the steroid skeleton contains more than one double bond.

All indicationsn-alkyl, Cn-foralkyl,n-alkyloxy,n-alkylamino,n-cycloalkyl,n-alkylen,nalkenyl,n-aryl, Cn-heteroaryl and the like are radicals with n carbon atoms in the residue, and the number n of carbon atoms includes all carbon atoms in the side chains, for example, branched radicals. If the context is not described otherwise, alkyl, alkoxy, Allenova or acyl groups contain 1-10 carbon atoms, including carbon atoms of the side chain, if these groups are branched; Alchemilla or Alchemilla group contains 2-10 carbon atoms, including carbon atoms of the side chain, if these groups are branched; in addition, cycloalkyl contains 4-7 carbon atoms; aryl contains 6-10 carbon atoms; and a heterocyclic ring or heteroaryl contains 6-10 ring atoms. In addition, the aryl also means alkylaryl; heteroaryl also means alkylglycerol; and cycloalkyl also means alkylsilanes.

New the e steroid compounds have several chiral centers, so these compounds exist in several isomeric forms. All of these isomeric forms are covered by the scope of the present invention, if the context is not described otherwise.

Steroid compound with the following General formula is preferred:

Especially derivatives Δ5-pregnene, derivative Δ8,14-pregnadien, derivative Δ8-pregnene and derivative Δ5,7-pregnadien applicable as pharmaceutically active steroid compounds for the regulation of reproduction, for example, meiosis, i.e. compounds having the following General formula:

It has been unexpectedly found that the compounds of the present invention have a strong incentive meiosis effect in oocytes, especially in SEO, although these compounds are structurally very different from the Sterol FF-MAS. In this respect, the compounds according to this invention is superior to that previously described substance that regulates meiosis [for example: A.G.Byskov and others, Nature, 374. 559-562 (1995)]. Preferred compounds of General formula X are such compounds that cause the destruction of embryonic vesicles, at least 40%, preferably at least 60%and particularly at least 80% of the study sample with oocytes as described in example 23.

Compounds of the present invention are superior to the previously described connection and on the second aspect: while the FF-MAS is not able to induce maturation in the cultural system of the follicle, the compounds of the present invention have the ability to activate meiosis in these conditions.

For this reason, a new steroid compounds may be used, for example, for use in vivo, and for applying pop in vivo, which in particular includes the use of in vitro. Steroid compounds are particularly suitable for fertilization in vitro and in vivo mammalian, especially humans.

The exceptional properties of new compounds can be attributed to the amino group in the side chain, related to the carbon atom With17in the steroid skeleton through (C2-C3)alkilinity the spacer (including the group With20-R20).

Especially preferred are compounds in which part of the3R3mean SN-HE, in particular, 3β-hydroxyacyl associated with atom With3steroid skeleton. The part can also mean CH-O-C(O)-R3"(=CH-O-R3'where R3'means C(O)-R3"), where R3"has the above values. In particular, R3"can be essential radical monocarboxylic acid, Dakar is about acid or inorganic acid, or any other acid-related atom With3steroid skeleton. In particular, for R3meaning ether radical of the dicarboxylic acid, R3"can be (CH2)n-UNSD, where n is 1, 2, 3, 4, 5 or 6. The ester radical can also be formed from inorganic acids such as phosphoric acid, sulfuric acid and sulfamic acid, furthermore, from a monocarboxylic acid, such as acetic acid, propionic acid, n-butane acid, trimethylhexane acid, benzoic acid, nicotinic acid and isonicotinamide acid. In particular, the ester moiety can be formed from dicarboxylic acids such as succinic acid and glutaric acid.

Additional steroid compounds of the present invention may also include derivatives in which the C-O-R3means a cyclic ether, including the atom With3steroid skeleton.

Deputy R3may also form a cyclic ring structure together with the atom3and R3means unsubstituted or substituted, linear or branched (C2-C10)alkylen. For example, the group With3R3can mean cyclopropylethyl, cyclobutylmethyl, cyclopentyloxy or cyclohexyloxy radical. It can also mean unsaturated cyclic number of the core structure, such as cyclopropene, cyclobutene, cyclopentene and cyclohexene. The ring structure may be substituted with any halogen, hydroxy-group, alkoxygroup, arroceros and the like.

The substances according to the present invention can usefully be compounds in which R3"selected from the group including vermeil, aryl, heteroaryl and (CH2)nCOOH, where n is 1, 2, 3, 4, 5 or 6, particularly compounds in which R3'(=C(O)-R3"means acetyl, propionyl, pivaloyl, butanoyl, benzoyl, nicotinyl, isonicotinic, hemiglutarate, butylcarbamoyl, phenylcarbamoyl, etoxycarbonyl and tert.-butoxycarbonyl. In a particularly preferred steroid connection R3'can be hemisuccinate.

In addition, new steroid compounds R4and R4'means independently preferably hydrogen or linear or branched (C1-C4)alkyl group, i.e. methyl, ethyl, propyl, butyl and especially methyl.

In addition, R4and R4'independently may be (C1-C4)alkyl substituted by halogen, hydroxy, alkoxy or aryloxy.

R20means preferably hydrogen or linear or branched (C1-C4)alkyl, i.e. methyl, ethyl, propyl and butyl. R20means, mainly methyl.

R23and R23' independently can be, in particular, hydrogen or (C1-C8)alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert.-butyl, n-pentyl, isopentyl, tert.-pencil, neopentyl, in addition, hexyl, cyclohexyl and the like. Then R23and R23'independently may be (C2-C6)alkenylphenol group, i.e. unsaturated alkyl group, for example, vinyl, allyl, Isopropenyl and phenyl, then (C6-C10)aryl, such as phenyl and 1-naphthyl, this group, including alkylaryl, bound through the aryl part or through alkyl portion with the nitrogen atom, for example, benzyl and tolyl. R23and R23'may preferably be an alkyl or alkenyl substituted with at least one radical selected from the group comprising linear or branched (C1-C4)alkyl and (C1-C4)alkoxy. Phenyl and 1-nattily radical may also be substituted by halogen, (C1-C4)alkoxy, hydroxy or (C1-C4)alkyl, including derivative feralcode and foralkyl. Then R23and R23'independently can be, for example, 4-hydroxyphenyl, 4-methoxyphenyl, 2,4,6-trimetilfenil, 2,4-dichlorophenyl, 4-florfenicol, 4-cryptomaterial and 2-pentabromodiphenyl.

In addition, R23and R23'nez the performance can also mean alkyl and alkenyl, moreover, at least one of alkyl and alkenyl carbon atoms, respectively, substituted by any of the atoms of oxygen, nitrogen and sulfur, for example, methoxymethyl, Meliksetyan, methoxypropyl, ethoxypropane and the like.

R23and R23'together may also form a heterocyclic ring structure associated with the side chain through the nitrogen atom in the side chain and the nitrogen atom linked to the carbon atom of the steroid skeleton via spacer elements group A. Heterocyclic ring structure formed by the N(R23)(R23'), can be in particular selected from the group comprising piperidine-1-ilen, morpholine-4-ilen, piperazine-1-ilen, pyrrolidin-1-ilen, pyrrol-1-ilen, indol-1-ilen, pyrazole-1-ilen, imidazol-1-ilen, thiazolidin-1-ilen and oxazolidin-3-ilen ring structure and their derivatives. Particularly preferred heterocyclic ring structures are saturated radicals, namely piperidine-1-yl, morpholine-4-yl, piperazine-1-yl and pyrrolidin-1-yl. Heterocyclic ring structure may be substituted by hydroxy, carboxy, amino, alkylamino, dialkylamino, alkyl, alkenyl, quinil, cycloalkyl, cycloalkenyl, alkylthio by alkyl, aryl, alkylaryl, alkoxy, alkylalkoxy, alkyloxyalkyl, alkylacrylate, alkyloxyaryl, Galai the om and acyl, where alkyl, alkenyl, quinil, cycloalkyl, aryl, alkoxy and acyl contain the number of carbon atoms, as described above. Heterocyclic ring structure may be substituted heterocyclic radicals, such as the heterocyclic ring structure with which they can be linked, and in addition to them as a further radicals, for example, pyridinyl, chinoline, ethenolysis, pyridazinyl, pyrimidinyl, pyrazinyl, honokalani, thiazolyl and oxazolyl, including, in addition, all other isomers of data radicals, for example, pyridine - 2-yl, pyridin-3-yl and pyridin-4-yl. Further, if N(R23)(R23'means heterocyclic ring structure, this ring structure may contain in the ring oxoprop.

If N(R23)(R23'means piperazine-1-yl, then this part can be, in particular, substituted pyridine-2-yl, pyridin-3-yl and pyridin-4-yl, preferably to form a corresponding group N(R23)(R23'), in which the balance piperazine-1-yl substituted in the para-position, for example, 4-(pyridin-3-yl)piperazine-1-yl.

The group N(R23)(R23'can be any of the part

related With20in the side chain of the steroid skeleton via a nitrogen atom of this part, where Z denotes O, S, N-R24N-C(O)-R24where R24about the means alkyl, alkenyl, quinil, the aryl, the number of carbon atoms defined above. In addition, R24may be a heterocyclic ring structure, in which the number of ring atoms such as defined above.

The nitrogen atom of the group N(R23)(R23') is not directly related to the atom With20and connected with it through And that means unsubstituted or substituted methylene or ethylene spacer elements group, such as, for example, (unsubstituted) and methylene (unsubstituted) ethylene and, in addition, isopropyl, tert.-butylene and the like. Preferably a represents methylene and ethylene.

Especially preferred are compounds in which R3means hydroxy or hemisuccinate group, where R4, R4'and R20mean each methyl, and where the heterocyclic ring structure N(R23)(R23'), including the nitrogen atom of the amino group is unsubstituted or substituted morpholine-4-yl, piperidine-1-yl, piperazine-1-yl or pyrrolidin-1-yl. The group N(R23)(R23'means, in particular, 3-hydroxypiperidine-1-yl, 4-hydroxypiperidine-1-yl, 3-ketopyperidines-1-yl, 4-keto piperidine-1-yl, 4-dimethylaminopyridine-1-yl, 3,3-dimethylpiperidin-1-yl, 4,4-dimethylpiperidin-1-yl, 3-carboxypeptidase-1-yl, 4-carboxypeptidase, 4-phenylpiperidine-1-yl, 4-benzoylpiperidine-1-yl, 4-(piperidine-1-yl)piperidine-1-yl, 4-metile erasin-1-yl, 4-acetylpiperidine-1-yl, 4-phenylpiperazin-1-yl, 4-benzylpiperazine-1-yl, 4-benzylpiperazine-1-yl, 4-(pyridin-2-yl)piperazine-1-yl, 4-(pyridin-4-yl)piperazine-1-yl, 4-(pyrimidine-2-yl)piperazine-1-yl.

The hydrogen atoms can be joined with all the other carbon atoms of the steroid compounds, i.e. With1With2With6With7With8With9With11With12With14With15and16.

Preferably pharmaceutically acceptable compounds of the present invention are salts of steroid compounds of General formula X. Examples of data salts listed in Journal of Pharmaceutical Science, 66, 2, and placenta. (1977), which are hereby incorporated by citation. Examples of such salts include salts of organic acids such as formic acid, fumaric acid, acetic acid, propionic acid, glycolic acid, lactic acid, grape acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, methanesulfonate acid and the like. Appropriate inorganic acid for the formation of pharmaceutically acceptable salts include hydrochloric acid, Hydrobromic acid, sulfuric acid, phosphoric acid and the like.

The following compounds of the present invention is particularly site is stitely:

1) (20S)-20-[(3,3-dimethylpiperidin-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

2) (20S)-20-[(piperidine-1-yl)methyl]-4,4-dimethyl-5β-pregna-8,14-Dien-3βol

3) (20S)-20-[(4,4-dimethylpiperidin-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

4) (20S)-20-[(4-methylpiperazin-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

5) (20S)-20-[(4-phenylpiperazin-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

6) (20S)-20-[(morpholine-4-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

7) (20S)-20-[(4-(pyrimidine-2-yl)piperazine-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

8) (20S)-20-[(pyrrolidin-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

9) hemisuccinate (20S)-20-[(3,3-dimethylpiperidin-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-Ola

10) (20S)-20-[N-(3-methoxypropyl)aminomethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

11) (20S)-20-aminomethyl-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

12) (20S)-20-[N-di-(2-methoxyethyl)aminomethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

13) (20S)-20-[N-(2,2-dimethylethylene)aminomethyl]-4,4-dimethyl-5β-pregna-8,14-Dien-3βol

14) (20S)-20-[(piperidine-1-yl)methyl]-4,4-dimethyl-5α-pregna-5,7-Dien-3βol

15) (20S)-20-[(4-(pyridin-2-yl)piperazine-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

16) (20S)-20-[(4-phenylpiperazin-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

17) (20S)-20-[(4-methylpiperazin-1-yl)ethyl]-4,-dimethyl-5α -pregna-8,14-Dien-3βol

18) (20S)-20-[(N-dimethylamino)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

19) (20S)-20-[(morpholine-4-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

20) (20S)-20-[(pyrrolidin-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

21) (20S)-20-[(piperidine-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

22) (20S)-20-[(4-phenylpiperazin-1-yl)methyl]-5α-pregna-5-ene-3βol

23) (20S)-20-[(piperidine-1-yl)methyl]-5α-pregna-5-ene-3βol

24) (20S)-20-[(morpholine-4-yl)methyl]-5α-pregna-5-ene-3βol

25) (20S)-20-[(pyrrolidin-1-yl)methyl]-5α-pregna-5-ene-3βol

26) (20S)-20-[(4-carboxyethylpyrrole-1-yl)methyl]-5α-pregna-5-ene-3βol

27) (20S)-20-[(3-hydroxypiperidine-1-yl)methyl]-5α-pregna-5-ene-3βol

28) (20S)-20-[(4-benzoylpiperidine-1-yl)methyl]-5α-pregna-5-ene-3βol

29) (20S)-20-[(4-(piperidine-1-yl)piperidine-1-yl)methyl]-5α-pregna-5-ene-3βol

30) (20S)-20-[(4-thiomorpholine)methyl]-5α-pregna-5-ene-3βol

31) (20S)-20-[(4-dimethylaminopyridine-1-yl)methyl]-5α-pregna-5-ene-3βol

32) (20S)-20-[(4-keto piperidine-1-yl)methyl]-5α-pregna-5-ene-3βol

33) (20S)-20-[(3-ketopyperidines-1-yl)methyl]-5α-pregna-5-ene-3βol

34) (20S)-20-[(4-carboxypeptidase-1-yl)methyl]-5α-pregna-5-ene-3βol

35) (20S)-20-[(3-carboxypeptidase-1-yl)methyl]-5α-pregna-5-ene-3βol

36) (20S)-20-[(4-hydroxypiperidine-1-yl)methyl]-5α-pregna-5-ene-3β-on the Sabbath.

37) (20S)-20-[(3,3-dimethylpiperidin-1-yl)methyl]-5α-pregna-5-ene-3βol

38) (20S)-20-[(4,4-dimethylpiperidin-1-yl)methyl]-5α-pregna-5α-EN-3βol

39) (20S)-20-[(4-piperazine-1-yl)methyl]-5α-pregna-5-ene-3βol

40) (20S)-20-[(4-phenylpiperazin-1-yl)methyl]-5α-pregna-5-ene-3βol

41) (20S)-20-[(4-methylpiperazin-1-yl)methyl]-5α-pregna-5-ene-3βol

42) (20S)-20-[(4-benzylpiperazine-1-yl)methyl]-5α-pregna-5-ene-3βol

43) (20S)-20-[(4-acetylpiperidine-1-yl)methyl]-5α-pregna-5-ene-3βol

44) (20S)-20-[(4-benzylpiperazine-1-yl)methyl]-5α-pregna-5-ene-3βol

45) (20S)-20-[{4-(2-pyridyl)piperazine-1-yl}methyl]-5α-pregna-5-ene-3βol

46) (20S)-20-[{4-(3-pyridyl)piperazine-1-yl}methyl]-5α-pregna-5-ene-3βol

47) (20S)-20-[{4-(4-pyridyl)piperazine-1-yl}methyl]-5α-pregna-5-ene-3βol

48) (20S)-20-[{4-(2-pyrimidyl)piperazine-1-yl}methyl]-5α-pregna-5-ene-3βol

49) (20S)-20-[(piperidine-1-yl)methyl]-4,4-dimethyl-5α-pregna-Δ8(14)1-EN-3βol

Structural formulas of data steroid compounds shown in figa-1K.

An additional objective of the present invention are pharmaceutical compositions comprising at least one steroid compound of General formula X and at least one pharmaceutically acceptable excipient, well known in this field, for example, at least one carrier, diluent, the amplifier pohlad the tion, preservative, a buffer agent to maintain osmotic pressure and rheological properties of the medicinal product, if it is a liquid, the surface-active agent, a solvent, a disintegrator for tablets, microcapsules, filler, moving additive, colorant, flavoring and other ingredients. These substances are commonly used in the art. Steroid compounds according to the invention preferably include pharmaceutical compositions in an effective amount.

Examples of solid carriers are magnesium carbonate, magnesium stearate, dextrin, lactose, sugar, talc, gelatin, pectin, starch, silica gel, tragakant, methylcellulose, carboxymethylcellulose sodium form, low-melting wax, and cocoa butter.

Liquid compositions include sterile solutions, suspensions and emulsions, which can be administered orally, nazalnam by or as ointment. Such liquid compositions may also be applicable for injection or for use in connection with the use of ex vivo or in vivo. For oral administration, the fluid may contain pharmaceutically acceptable oil and/or lipophilic surface-active agent and/or solvent which is miscible with water. In this regard, reference is made to the application WO 97/21440 A1, which is hereby incorporated by citation.

Liquid comp the positions may also contain other ingredients, which is usually used in this field, some of which are mentioned in the list above. In addition, the composition for the transdermal injection of the compounds of the present invention may be provided in the form of a patch. Composition for nasal injection can be provided in the form of a nasal spray in liquid or powder form.

In order to increase the bioavailability of steroid compounds, these compounds may be developed as well as cyclodextrines of clathrates. For this purpose, the compounds are mixed with α-, βor γ-cyclodextrin or its derivatives.

Creams, ointments, lotions and other liquids introduced externally, should be in such a state that steroid compounds of the present invention can be delivered in sufficient quantity to a patient in need of regulation of meiosis. To this end the drug contains additives to regulate its rheological properties, surfactants, preservatives, solvents,

thinners, substances to enhance the ability of penetration through the skin, in addition, flavors and protects the skin substances such as air conditioners and regulators humidity.

The drug may also contain additional active agents to enhance or regulate effective is here steroid compounds or to obtain other desired effects of the drug.

For parenteral administration of steroid compounds may be dissolved or suspended in a pharmaceutically acceptable diluent. Often use oils in combination with solvents, surface-active agents, suspension or emulsion agents, such as olive oil, oil of groundnuts, castor oil and the like. To obtain injectable medicines can be used by any carrier liquid. These fluids often contain agents for the regulation of their viscosity, as well as agents for the regulation of isotonic liquid properties.

Steroid compound of the present invention, moreover, can be introduced in the form of injectable depot forms or in the form of the implant, which may, for example, be introduced subcutaneously, so that it is able to slow the release of steroid compounds. To this end may use a variety of techniques, for example, the introduction of depo-forms, which include the membrane containing the active compound, or slowly dissolving depot-forms. Implants can contain, for example, biologically degradiruete polymers or synthetic silicones as inert material.

The dose used steroid compounds will be determined by the physician and depend inter alia on the specific use of the steroid has been created connection route of administration and purpose. Typically, the compositions of the present invention is obtained by introducing into close contact active compound with a liquid or auxiliary ingredients, and then, if necessary, shaping the product into the desired composition.

Usually no more than 3000 mg, preferably not more than 350 mg, and in some preferred cases, not more than 30 mg of steroid compounds in the day to enter mammals, for example humans.

The present invention relates also to the use of steroid compounds of General formula X to obtain a composition applicable to the regulation of reproduction, for example, meiosis. Preferably the composition is applicable as a medicine.

The present invention relates further to the use of new steroid compounds of General formula X to obtain a contraceptive or propertylegal drug.

The present invention relates further to the use of steroid compounds of General formula X to apply non-in vivo.

The present invention relates also to method of regulation play, for example, meiosis, including introduction to the subject in need of such regulation, an effective amount of at least one steroid compounds of General formula X.

In addition, the present invention relates to a JV is the property of improving the ability of oocytes to develop in a mammal, includes contact of the oocyte, taken from a mammal, with steroid compound of General formula X.

Regulation play, for example, meiosis, is used in the context to show that the compounds of the present invention is particularly suitable for stimulation of the play, for example, meiosis in mammals, especially in humans, oocytes, so that these compounds that are agonistic analogues of the natural activators of meiosis, can be used in the treatment of infertility resulting from insufficient stimulation of meiosis in women and men.

By introducing the compositions containing the compound of the present invention, may be any route, which effectively transports the active steroid compound to the site of its action.

So, when steroid compounds must be administered to a mammal, they are usually provided in the form of a pharmaceutical composition, which comprises at least one steroid compound according to the present invention in combination with a pharmaceutically acceptable carrier. For oral administration, such compositions are preferred in the form of tablets or capsules.

The present invention relates also to a method for producing steroid compounds of General formula X, where R4and R4'means unsubstituted or substituted, linear is whether branched (C 1-C4)alkyl, and not hydrogen.

The above-mentioned steroid compounds can be synthesized similarly to obtain the known compounds. Therefore, the synthesis of steroid compounds of formula X can be followed by conventional synthetic techniques described in the extensive literature on the sterols and steroids. The following literature may be used as the primary source for synthesis: L.F.Fieser and .Fieser: Steroids, Reinhold Publishing Corporation, N.Y., 1959; Rood''s Chemistry of Carbon Compounds (as amended S.Coffrey): Elsevier Publishing Company, 1971; and especially Dictionary of Steroids (as amended R.A.Hill, D.N.Kirk, H.L.J.Makin and G.M.Murphy), Chapman and Hall, thus this literature included by citation. The latter source contains an extensive list of references to original papers, covering the period up to 1990.

In particular, steroid compounds can be synthesized, for example, in accordance with the General method including the following steps:

(a) on the basis of (20S)-20-hydroxymethylene-4-EN-3-one,

(b) the introduction of two alkyl groups at position 4 by alkylation,

(C) recovering ketogroup in the hydroxy-group,

(g) introduction of a double bond Δ7bromirovanii/dehydrobrominated,

(e) isomerization of the diene Δ5,7in Dien Δ8,14by heating in the presence of acid,

(e) oxidation of 17-hydroxy-group in the aldehyde group and

(g) recovering Mininova is their aldehyde group.

The corresponding scheme of the synthesis of this first synthetic method is shown in figure 2. According to the scheme first hydroxyl group in the side chain (20S)-20-hydroxymethylene-4-EN-3-one 1 protects as salelologa ether, for example, in the form of triisopropylsilyl (TIPS) ether, receiving the connection 2. In order to obtain compound 3, two methyl groups introduced at position 4 of the steroid skeleton by alkylation with iodine stands in the presence of such grounds as tert.-butyl potassium. In the next step, 3-ketogroup restore normal regenerating agent such as alumalite lithium or sodium borohydride. The resulting alcohol 4 then protect, turning, for example, acetate (compound 5). Subsequently introduce the second of the double bond using the sequence of reactions of the synthesized-dihydrobromide. Generated Δ5,7-diene system in connection 6 then isomerized in Δ8,14-diene system by heating in the presence of hydrochloric acid to obtain compound 7. At this stage, acid catalyzed, both hydroxyl groups are released and can be obtained diol 7. Soft selective oxidation triacetoxyborohydride on dess-Martin hydroxyl group in the side chain leads to aldehyde 8, which serves as VI is th intermediate compounds for the introduction of various amines in the side chain via reductive amination. This purpose can be applied to various reducing agents like sodium borohydride or Tris(acetoxy)borohydride sodium. The result is a steroid compound 9 of the present invention.

Method for producing steroid compounds of the present invention obtained new compounds, namely, (20S-4,4-dimethyl-20-hydroxymethyl-5α-pregna-8,14-Dien-3β-ol and (20S)-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol-20-carbaldehyde. Therefore, the present invention relates also to these intermediate connections.

Alternative steroid compounds can also be synthesized in the following way:

(a) on the basis of (20S)-20-trimethylpropane-8,14-Dien-3β,21-diol, in particular, from (20S)-4,4,20-trimethylpropane-8,14-Dien-3β,21-diol

(b) nucleophilic substitution at C21mainly with toluensulfonate acid,

(C) the substitution nucleophilic group (tosilata) With21cyanide with the formation of new carbon-carbon links in With21,

(g) recovering the obtained nitrile with the formation of aldehyde,

(d) reductive amination of nitrile with the formation of various amines.

The corresponding scheme of the synthesis of this second synthetic method is shown in figure 3. Tailyour p-toluenesulfonyl chloride (20S)-4,4,20-trimethylpropane-8,14-3β,21-diol 1 on the side chain. Then TOZ the lat 2 replace cyanide as a building block C1. After that, the formed nitrile 3 restore diisobutylaluminium with the formation of aldehyde 4. Aldehyde 4 then reacts with the amine by the reaction of reductive amination in the presence of Tris(acetoxy)borohydride sodium. The resulting final product 5 can be cleaned by standard column chromatography or HPLC.

Examples are given for a more detailed description of the present invention.

Example 1: (20S)-20-[(piperidine-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol (compound No. 2)

(a) (20S)-20-[((Triisopropylsilyl)oxy)methyl]pregna-4-EN-3-one

To a solution of 30 g of (20S)-20-[(hydroxymethyl]pregna-4-EN-3-one and 13.5 g of imidazole in 300 ml of dichloromethane at room temperature was added dropwise 26 ml triisopropylsilane. The reaction mixture was stirred for 20 h at the same temperature and then poured into water. The aqueous layer was extracted with ethyl acetate. The organic layers were combined, washed with saturated saline solution, dried over sodium sulfate, filtered and concentrated under reduced pressure, getting to 45.4 g of the crude (20S)-20-[((triisopropylsilyl)oxy)methyl]pregna-4-EN-3-one as a brown oil which was used without further purification.

MS [chemical ionization with the formation of positive ions (CI+)]: 487 (M+H).

(b) (20S)-4,4-Dimethyl-20-[((triisopropylsilyl)oxy)is etil]pregna-5-ene-3-one

A solution of 45.4 g of the crude (20S)-20-[((triisopropylsilyl)oxy)methyl]pregna-4-EN-3-one in 320 ml of tetrahydrofuran was added to a solution of 42.3 g of tert.-the butyl potassium in 950 ml of tert.-butanol at a temperature of 50°C. the Mixture was stirred for 10 min at the same temperature. Then there was added 50 ml of methyl iodide and continued stirring for 1 h the Reaction mixture was poured into water and was extracted with ethyl acetate. The organic layers were combined, washed with saturated saline solution, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified column chromatography, obtaining of 27.3 g (20S-4,4-dimethyl-20-[((triisopropylsilyl)oxy)-methyl]pregna-5-ene-3-one as pale yellow solid.

MC(CI+): 515(M+H).

(C) (20S)-4,4-Dimethyl-20-[((triisopropylsilyl)oxy)methyl]pregna-5-ene-3βol

To a solution of 27.3 g of (20S)-4,4-dimethyl-20-[((triisopropylsilyl)oxy)methyl]pregna-5-ene-3-one in 500 ml of tetrahydrofuran carefully in small portions was added 1.24 g of lithium aluminum hydride at room temperature. The reaction mixture was stirred for 1 h, then was cooled to 0°C. was Added sequentially 2.5 ml water, 2.5 ml of 0.1 n sodium hydroxide solution and 7.5 ml of water. The mixture was filtered through celite. The filtrate was concentrated under reduced pressure. The residue was purified column chromatography, obtaining of 18.2 g (0S)-4,4-dimethyl-20-[((triisopropylsilyl)oxy)methyl]pregna-5-ene-3β -ol as a pale yellow solid.

MS(CI+):517(M+H).

(g) Acetate (20S)-4,4-dimethyl-20-[((triisopropylsilyl)oxy)methyl)pregna-5-ene-3β-Ola

To a solution of 18.2 g of (20S)-4,4-dimethyl-20-[((triisopropylsilyl)oxy)methyl]-pregna-5-ene-3β-Ola in 175 ml of pyridine was added 6,24 ml of acetic anhydride at room temperature. The reaction mixture was stirred for 20 h and then poured into a mixture of ice/hydrochloric acid. The resulting mixture was extracted with ethyl acetate. The organic layers were combined, washed with saturated saline solution, dried over sodium sulfate and concentrated under reduced pressure, obtaining 16, 2 g acetate (20S)-4,4-dimethyl-20-[((triisopropylsilyl)oxy)-methyl]pregna-5-ene-3β-ol as a white solid, which was used without further purification.

MS (CI+): 559 (M+H).

(e) Acetate (20S)-4,4-dimethyl-20-[((triisopropylsilyl)oxy)methyl]pregna-5,7-Dien-3β-Ola

To a solution of 16.2 g of the acetate (20S)-4,4-dimethyl-20-[((triisopropylsilyl)oxy)-methyl]pregna-5-ene-3β-ol in a mixture of 100 ml of benzene and 100 ml of hexane was added portions is 4.93 g of 1,3-dibromo-5,5-dimethylhydantoin at 70°C. After 30 min the mixture was cooled to 0°and filtered. The filtrate was evaporated in vacuum.

To the mixture was added 160 ml of toluene and 7.8 ml of 2,4,6-trimethylpyridine. The mixture was heated at boiling for 2.5 hours After cooling the reaction, see the camping washed with 1 N. hydrochloric acid, saturated sodium bicarbonate solution and saturated saline solution. The organic layer was dried over sodium sulfate, filtered and evaporated in vacuum. The residue was purified column chromatography, receiving 12.5 g of acetate (20S)-4,4-dimethyl-20-[((triisopropylsilyl)-oxy)methyl]pregna-5,7-Dien-3β-ol as a white solid.

MS (CI+): 557 (M+H).

(e) (20S)-4,4,20-Trimethylpropane-8,14-Dien-3β,21-diol

A mixture of 16.1 g of the acetate (20S)-4,4-dimethyl-20-[((triisopropylsilyl)oxy)methyl]-pregna-5,7-Dien-3β-Ola, 210 ml of ethanol, 28 ml of benzene and 28 ml of concentrated hydrochloric acid was heated at boiling for 6 hours, After cooling the mixture was poured into saturated sodium bicarbonate solution, extracted with ethyl acetate and washed with saturated saline solution. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was recrystallized from dichloromethane and methanol, getting 4,48 g (20S)-4,4,20-trimethylpropane-8,14-Dien-3β,21-diol.

MS [ei with elektrorazpredelenie with the formation of positive ions (EI+)]: 358 (M).

(W) (20S)-3β-hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-al

To a solution of 1 g of (20S)-4,4,20-trimethylpropane-8,14-Dien-3β,21-diol in 10 ml of dichloromethane was added at room temperature of 5.4 ml of 0.5 M solution triacetoxyborohydride socilize on Dess-Martin. The mixture was stirred for 1 h, poured into saturated sodium bicarbonate solution, extracted with ethyl acetate and washed with saturated saline solution. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified column chromatography, getting 230 mg (20S)-3β-hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-Ala in the form of a white solid.

MS (ES+): 356 (M).

(C) (20S)-20-[(piperidine-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

Was added 38 mg of Tris(acetoxy)sodium borohydride to a solution of 42 mg (20S)-3β-hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-Ala and 10 μl of piperidine in 3 ml of tetrahydrofuran at room temperature. The mixture was stirred for 2 h, poured into saturated sodium bicarbonate solution, extracted with ethyl acetate and washed with saturated saline solution. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified column chromatography, receiving 15 mg of (20S)-20-[(piperidine-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol as a white solid.

MS (EI+): 425 (M).

Example 2: (20S)-20-[(3.3-dimethylpiperidin-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

Processed (20S)-3β-hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-al 3,3-dimethylpiperidino and Tris(acetoxy)what orhideja sodium, as described in example 1H. Allocated (20S)-20-[(3,3-dimethylpiperidin-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol as a white solid.

MS (EI+): 453 (M).

Example 3: (20S)-20-[(4-phenylpiperazin-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol (compound No. 5)

Processed (20S)-3R-hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-al N-phenylpiperazine and Tris(acetoxy)borohydride sodium as described in example 1H. Allocated (20S)-20-[(4-phenylpiperazin-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol as a white solid.

MS (EI+): 502 (M).

Example 4: (20S)-20-[(morpholine-4-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol (compound No. 6)

Processed (20S)-3β-hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-al morpholine and Tris(acetoxy)borohydride sodium as described in example 1H. Allocated (20S)-20-[(morpholine-4-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol as a white solid.

MS (EI+): 427 (M).

Example 5: (20S)-20-[(4-(pyrimidine-2-yl)piperazine-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol (compound No. 7)

Processed (20S)-3β-hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-al N-(pyrimidine-2-yl)piperazine and Tris(acetoxy)borohydride sodium as described in example 1H. Allocated (20S)-20-[(4-(pyrimidine-2-yl)piperazine-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol as a white solid.

MS (EI+): 504 (M).

Example 6: (20S)-20-[(pyrrolidin-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol (compound No. 8)

Processed (20S)-3β-hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-al pyrrolidino and Tris(acetoxy)borohydride sodium as described in example 1H. Allocated (20S)-20-[(pyrrolidin-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol as a white solid.

MS (EI+): 411 (M).

Example 7: (20S)-20-N-(3-methoxypropyl)aminomethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol(compound No. 10)

Processed (20S)-3β-hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-al 3-methoxypropylamine and Tris(acetoxy)borohydride sodium as described in example 1H. Allocated (20S)-20-N-(3-methoxypropyl)aminomethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol as a white solid.

MS (EI+): 429 (M).

Example 8: (20S)-20-[(piperidine-1-yl)methyl]-4,4-dimethyl-5α-pregna-5,7-Dien-3β-ol (compound No. 14)

(a) Benzoate (20S)-4,4-dimethyl-20-[((triisopropylsilyl)oxy)methyl]pregna-5-ene-3β-Ola

Added to 34.4 ml of benzoyl chloride to a solution of 70 g of the crude (20S)-4,4-dimethyl-20-[((triisopropylsilyl)oxy)methyl]pregna-5-ene-3β-ol (example 1B) (purified by chromatography) in 670 ml of pyridine at 0-3°C. the Reaction mixture was stirred for 2 h at room temperature and then poured into a mixture of ice and water. The precipitate was separated by filtration, washed with water and recrystallized from Cetona, receiving 42 g of the benzoate (20S)-4,4-dimethyl-20-[((triisopropylsilyl)oxy)-methyl]pregna-5-ene-3β-Ola.

1NAMR (CDCl3that δ): with 8.05 (2H, d); 7,56 (1H, t); 7,45 (2H, t); the ceiling of 5.60 (1H, t); 4,74 (1H, m); 3,68 (1H, dd); to 3.36 (1H, m).

(b) Benzoate (20S)-4,4-dimethyl-20-[((triisopropylsilyl)oxy)methyl]pregna-5,7-Dien-3β-Ola

Was treated with 42 g of the benzoate (20S)-4,4-dimethyl-20-[((triisopropylsilyl)oxy)-methyl]pregna-5,7-Dien-3β-Ola from 13.7 g of 1,3-dibromo-5,5-dimethylhydantoin and 18 ml of 2,4,6-trimethylpyridine as described in example 1D, receiving (without purification column chromatography) of 41.9 g of the benzoate (20S)-4,4-dimethyl-20-[((triisopropylsilyl)oxy)-methyl]pregna-5,7-Dien-3β-Ola.

1H-NMR (CDCl3that δ): with 8.05 (2H, d); EUR 7.57 (1H, t); 7,45 (2H,t); 5,95 (1H, d); to 5.56 (1H, m); is 4.85 (1H, m); 3,7 (1H, dd); 3,39 (1H, m).

(in) 3-Benzoate (20S)-4,4,20-trimethylpropane-5,7-Dien-3β,21-diol

Added 2 g of crushed molecular sieves (4A) and 1.27 g of hydrate tetrabutylammonium to a solution of 2.0 g of (20S)-4,4-dimethyl-20-[((triisopropylsilyl)oxy)methyl]pregna-5,7-Dien-3β-ol in 50 ml of tetrahydrofuran. The mixture was stirred over night at room temperature, concentrated under reduced pressure and was purified column chromatography, obtaining 1,17 g 3-benzoate (20S)-4,4,20-trimethylpropane-5,7-Dien-3β,21-diol.

1H-NMR (CDCl3that δ): of 8.06 (2H, d); 7,56 (1H, t); 7,45 (2H, t); 5,94 (1H, d); to 5.56 (1H, m); is 4.85 (1H, m); 3,66 (1H, m); to 3.41 (1H, m).

(g) Benzoate (20S)-4,4-dimethyl-20-[((toluene-4-sulfonyl)ACS is)methyl]pregna-5,7-Dien-3β -Ola

The solution 0,573 g of toluene-4-sulphonylchloride in 4 ml of pyridine was added dropwise to a solution of 0,926 g 3-benzoate (20S)-4,4,20-trimethylpropane-5,7-Dien-3β,21-diol in 15 ml of pyridine while cooling in a bath of ice. The reaction mixture was stirred for 1 h at 0-3°and over night at room temperature. The reaction mixture was poured into a mixture of ice water and was extracted with ethyl acetate. The organic layers were combined, washed with 1 N. hydrochloric acid, saturated sodium bicarbonate solution and saturated saline solution. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure, obtaining 1.0 g of the benzoate (20S)-4,4-dimethyl-20-[((toluene-4-sulfonyl)oxy)-methyl]pregna-5,7-Dien-3β-Ola, which could further be purified by crystallization from methanol.

1H-NMR (CDCl3that δ): with 8.05 (2H, d); 7,8 (1H, d); 7,56 (1H, t); 7,45 (2H, t); 7,34 (2H, d); 5,94 (1H, d); of 5.55 (1H, m); is 4.85 (1H, m); 4,0 (1H, dd); 3,81 (1H, m); of 2.45 (3H, s).

(d) Benzoate (20S)-20-[(piperidine-1-yl)methyl]-4,4-dimethyl-5α-pregna-5,7-Dien-3β-Ola

Added of 0.625 ml of piperidine to a solution of 0,154 g of the benzoate (205)-4,4-dimethyl-20-[((toluene-4-sulfonyl)oxy)methyl]pregna-5,7-Dien-3β-ol in 3 ml of N,N-dimethylformamide. The reaction mixture was stirred for 48 h at room temperature and then concentrated under reduced pressure. The residue was dissolved in dichloromethane and three times amywali water. The organic layer was dried over sodium sulfate, filtered and evaporated in vacuum.

The residue (125 mg) was led from methanol, receiving 45 mg of the benzoate (20S)-20-[(piperidine-1-yl)methyl]-4,4-dimethyl-5α-pregna-5,7-Dien-3β-Ola.

1H-NMR (CDCl3that δ): with 8.05 (2H, d); EUR 7.57 (1H, t); 7,46 (2H, t); 5,94 (1H, d); to 5.56 (1H, m); 4,88 (1H, t); 2,43 (2H, m).

(e) (20S)-20-[(piperidine-1-yl)methyl]-4,4-dimethyl-5α-pregna-5,7-Dien-3βol

A solution of 45 mg of the benzoate (20S)-20-[(piperidine-1-yl)methyl]-4,4-dimethyl-5α-pregna-5,7-Dien-3β-Ola in 2.5 ml of tetrahydrofuran was added to a suspension of 45 mg alumaguard lithium in 2 ml of tetrahydrofuran. After stirring for 2 h at room temperature was added 3 ml of saturated solution of sodium sulfate and 5 ml of 1 n sodium hydroxide solution. The mixture was extracted three times with 10 ml dichloromethane. The organic layers were combined, washed with 1 N. a solution of sodium hydroxide and water, dried over sodium sulfate, filtered and concentrated under reduced pressure. The remainder (35 mg) was purified column chromatography, obtaining 22 mg (20S)-20-[(piperidine-1-yl)methyl]-4,4-dimethyl-5α-pregna-5,7-Dien-3β-Ola.

1H-NMR (CDCl3that δ): to 5.93 (1H, d); of 5.53 (1H, m); 3,4 (1H, t); to 2.42 (2H, m); 1,22 (3H, s); of 1.13 (3H, s); of 1.02 (3H, d); and 0.98 (3H, s); 0,6 (3H, s).

Example 9: (20S)-20-[(4-(pyridin-2-yl)piperazine-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol (compound No. 15)

(a) (20S)-4,4,20-Trimethyl-21-tosilos pregna-8,14-Dien-3β -ol

Was added 4.7 g of tosylchloramide small portions to a solution of 4.5 g of (20S)-4,4,20-trimethylpropane-8,14-Dien-3β,21-diol in 91 ml of pyridine at room temperature. The mixture was stirred for 3 h at room temperature and then concentrated under reduced pressure. The residue was dissolved in ethyl acetate, washed with water and sodium chloride solution. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified column chromatography, obtaining 3.2 g of (20S)-4,4,20-trimethyl-21-toiletspray-8,14-Dien-3β-ol as a white solid.

MS (EI+): 512 (M).

b) (20S)-3β-Hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-carbonitril

Was added 1.7 g of potassium cyanide to a solution of 4.7 g of (20S)-4,4,20-trimethyl-21-toiletspray-8,14-Dien-3β-ol in 90 ml of DMSO at room temperature. The mixture was stirred for 2 h at 90°C, poured into ice water, extracted with ethyl acetate and washed with water. The organic layer was dried with sodium sulfate, filtered and concentrated under reduced pressure, obtaining 2.8 g of (20S)-3β-hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-carbonitrile in the form of a white solid, which was used without further purification.

MS (EI+): 367 (M).

(C) (20S)-3β-Hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-carbaldehyde

Added 16 ml diisobutylaluminium is a (20% in toluene) dropwise to a solution of 920 mg (20S)-3β -hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-carbonitrile in 60 ml of toluene at a temperature of -72°C. the Mixture was stirred for 2 h at the same temperature, was poured into a solution of 2 N. sulfuric acid, was extracted with ethyl acetate, washed 2 N. sulfuric acid and then washed polysystem solution of sodium chloride. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure, receiving 750 mg (20S)-3β-hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-carbaldehyde as a white solid, which was used without further purification.

MS (EI+): 370 (M).

(g) (20S)-20-[(4-(Pyridin-2-yl)piperazine-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3βol

Added 107 mg of Tris(acetoxy)sodium borohydride to a solution of 125 mg (20S)-3β-hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-carbaldehyde and to 82.6 mg of N-(2-pyridyl)-piperazine in 2 ml of tetrahydrofuran at room temperature. The mixture was stirred for 20 h, poured into water and concentrated under reduced pressure. The residue was purified column chromatography, receiving 84 mg (20S)-20-[(4-(pyridin-2-yl)piperazine-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol as a white solid.

MS(EI+): 517(M).

Example 10: (20S)-20-[(4-phenylpiperazin-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol (compound No. 16)

Processed (20S)-3β-hydroxy-4,4,20-trimethyl who regna-8,14-Dien-21-carbaldehyde N-phenylpiperazine and Tris(acetoxy)borohydride, sodium, as described in example g Allocated (20S)-20-[(4-phenylpiperazin-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol as a white solid.

MS(EI+): 516 (M).

Example 11: (20S)-20-[(4-methylpiperazin-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol (compound No. 17)

Processed (20S)-3β-hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-carbaldehyde 1-methylpiperazine and Tris(acetoxy)boron sodium hydride as described in example g Allocated (20S)-20-[(4-methylpiperazin-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol as a white solid.

MS (EI+): 454 (M).

Example 12: (20S)-20-[(N,N-dimethylamino)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol (compound No. 18)

Processed (20S)-3β-hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-carbaldehyde N,N-dimethylamine and Tris(acetoxy)borohydride sodium as described in example g Allocated (20S)-20-[(N,N-dimethylamino)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol as a white solid.

MS (EI+): 399 (M).

Example 13: (20S)-20-[(morpholine-4-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol (compound No. 19)

Processed (20S)-3β-hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-carbaldehyde morpholine and Tris(acetoxy)borohydride sodium as described in example g Allocated (20S)-20-[(morpholine-4-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol as a white solid.

MS (EI+): 441 (M).

Example 14: (20S-20-[(pyrrolidin-1-yl)ethyl]-4,4-dimethyl-5α -pregna-8,14-Dien-3β-ol (compound No. 20)

Processed (20S)-3β-hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-carbaldehyde by pyrrolidino and Tris(acetoxy)borohydride sodium as described in example g Allocated (20S)-20-[(pyrrolidin-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol as a white solid.

MS (EI+): 425 (M).

Example 15: (20S)-20-[(piperidine-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol (compound No. 21)

Processed (20S)-3β-hydroxy-4,4,20-trimethylpropane-8,14-Dien-21-carbaldehyde with piperidine and Tris(acetoxy) boron sodium hydride as described in example g Allocated (20S)-20-[(piperidine-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol as a white solid.

MS (EI+): 439 (M).

Example 16: (20S)-20-[(4-phenylpiperazin-1-yl)methyl]-5α-pregna-5-ene-3β-ol (compound No. 22)

(a) Methyl ester of 3βtert.-butyldimethylsilyloxy acid

Mixed 3β-hydroxybenzophenone acid (10 g), potassium bicarbonate (12 g), methyl iodide (10 ml) and dimethylformamide for 3 hours at room temperature and poured into water (1.5 ml). A white precipitate was separated by filtration. Was added diethyl ether to a white precipitate, insoluble material was filtered. Concentration of the ether phase in vacuo gave the crude silloway ester, which was dissolved in dimethylformamide (400 ml). Added tre is.-butyldimethylsilyloxy (6 g) and imidazole (6 g), and the mixture was concentrated in vacuo, and purified flash chromatography, getting 5.3g methyl ester 3βtert.-butyldimethylsilyloxy acid.

(b) 3βtert.-Butyldimethylsilyloxy-(20S)-20-methyl-5α-pregna-5-EN-21-ol

Was added 2,2 EQ. diisobutylaluminium (2 M) in toluene to methyl ether 3βtert.-butyldimethylsilyloxy acid, and the reaction mixture was stirred at room temperature. The reaction mixture was added to water, was extracted using diethyl ether, concentrated in vacuo, and purified flash chromatography, obtaining 3,3 g 3βtert.-butyldimethylsilyloxy-(20S)-20-methyl-5α-pregna-5-EN-21-ol.

() 3βtert.-Butyldimethylsilyloxy-(20S)-20-methyl-5α-pregna-5-ene-21-al

The solution dess-Martin (triacetoxyborohydride) (1,9 g in 30 ml dichloromethane) was added to 1.6 g 3βtert.-butyldimethylsilyloxy-(20S)-20-methyl-5α-pregna-5-EN-21-ol in dichloromethane (30 ml)and the reaction mixture was stirred for 24 h, then was poured into diethyl ether and was extracted with 1 N. sodium hydroxide. The organic layer was dried with magnesium sulfate and concentrated in vacuum, obtaining 1.6 g 3βtert.-butyldimethylsilyloxy-(20S)-20-methyl-5α-pregna-5-ene-21-Ala.

(g) 3βtert.-Butyldimethylsilyloxy-(20S)-20-[(4-phenylpiperazin-1-yl)methyl]-5α-pregna-5-ene

Was added 4-phenylpiperidine 0.16 g) to 0.26 g 3β -tert.-butultimately-silyloxy-(20S)-20-methyl-5α-pregna-5-ene-21-Ala in THF (20 ml) followed by addition of Tris(acetoxy)borohydride sodium (0,38 g). After 5 h the reaction mixture was poured into saturated sodium bicarbonate solution, extracted with ethyl acetate, dried and concentrated in vacuum. Flash chromatography gave 76 mg 3β-7-tert.-butyldimethylsilyloxy-(20S)-20-[(4-phenylpiperazin-1-yl)methyl]-5α-pregna-5-ene.

(d) (20S)-20-[(4-Phenylpiperazin-1-yl)methyl]-5α-pregna-5-ene-3βol

Added 6 N. hydrochloric acid (0.2 ml) to a mixture of 100 mg 3βtert.-butyldimethylsilyloxy-(20S)-20-[(4-phenylpiperazin-1-yl)methyl]-5α-pregna-5-ene in 10 ml of ethanol, and the mixture was stirred for 4 days at room temperature. Was added dichloromethane, and the organic phase is washed with sodium bicarbonate solution, dried over magnesium sulfate and concentrated under reduced pressure, obtaining the title compound.

MS (EI+): 478 (M).

Examples 17-22:

Reproduce steps (d) and (e) of example 16 3βtert.-butyldimethylsilyloxy-(20S)-20-methyl-5α-pregna-5-ene-21-ale, receiving the following examples of the compounds.

Example 17: (20S)-20-[(piperidine-1-yl)methyl]-5α-pregna-5-ene-3β-ol (compound No. 23)

Using piperidine in step reductive amination (g) with the subsequent release of stage (d), was obtained the title compound.

MS (EI): 400 (M).

Example 18: (20S)-20-[(morpholine-4-yl)methyl]-5α-pregna-5-ene-3β-ol (with unity No. 24)

Using morpholine in step reductive amination (g) with the subsequent release of stage (d), was obtained the title compound.

MS (EI+): 402 (M).

Example 19: (20S)-20-[(pyrrolidin-1-yl)methyl]-5α-pregna-5-ene-3β-ol (compound No. 25)

Using pyrrolidin recovery amination (g) with the subsequent release of stage (d), was obtained the title compound.

MS (EI+): 386 (M).

Example 20: (20S)-20-[(4-carboxyethylpyrrole-1-yl)methyl]-5α-pregna-5-ene-3β-ol (compound No. 26)

Using 4-carboxyethylpyrrole recovery amination (g) with the subsequent release of stage (d), was obtained the title compound.

MS (EI+): 472 (M).

Example 21: (20S)-20-[(3-hydroxypiperidine-1-yl)methyl]-5α-pregna-5-ene-3β-ol (compound No. 27)

Using 3-hydroxypiperidine recovery amination (g) with the subsequent release of stage (d), was obtained the title compound.

MS (EI+): 416(M).

Example 22: (20S)-20-[(4-benzoylpiperidine-1-yl)methyl]-5α-pregna-5-ene-3β-ol (compound No. 28)

Using 4-benzoylpiperidine recovery amination (g) with the subsequent release of stage (d), was obtained the title compound.

MS (EI+): 504 (M).

Example 23: a study of antivirusines substances in the analysis of mouse oocytes

Animals

Oocytes were obtained from immature female mice (C57BI/6J×DBA/2J F1-hybrids, Bomholtgaard, Denmark) weighing 13-16 g, which were kept under controlled lighting and temperature. Mice received intraperitoneal injection of 0.2 ml of gonadotropins [containing 10 international units (ME) serum gonadotropin foals mares (PMSG), Sigma Cat. No. G-4877)] and after 48 h, animals were scored by displacement of the cervical vertebrae.

The collection and culturing of oocytes

The ovaries were dissected and oocytes were isolated in NC-environment (see below) under a stereo microscope by manual rupture of the follicle, using a pair of needle 27 in size. Spherical multiple shielded oocytes (CEO)with intact germinal vesicle (GV), was placed in minimum essential medium modification α (α-MEM without ribonucleosides, Gibco BRL, Cat. No. 22561) with the addition of 3 mm gipoksantina (NC) (Sigma Cat. No. N-9377), 8 mg/ml human serum albumin (HAS, State Serum Institute, Denmark), 0.23 mm pyruvate (Sigma, Cat. No. S-8636), 2 mm glutamine (Flow Cat. No. 16-801), 100 IU/ml penicillin and 100 µg/ml streptomycin (Flow Cat. No. 16-700) This Wednesday marked the NC environment.

Oocytes were washed three times in NC environment and cultured in 4-cell advance plates (Nunclon, Denmark), in which each cell contained 0.4 ml NC-environment and about 25 oocytes. One control (i.e. approximately 25 oocytes, kultivirovanii is in the NC environment without the addition of the compounds) is always carried out simultaneously with the analyzed crops, which were prepared with different concentrations of the studied compounds. Culture functioned at 37°C and 100% humidity in an atmosphere with 5% carbon dioxide. Time of cultivation was 22 PM

The study of oocytes

Oocytes were stopped in meiosis and characterized with the help of intact nuclei with prominent nucleoli, known as germinal vesicles (GV). If re-initiation of meiosis nucleolus and nuclear envelope disappear, and it is characterized by splitting of GV, which in this case is called the splitting of the embryonic vesicles (GVB). A few hours later, the oocyte completes recovered division comes first, so-called polar body (PB).

At the end of the culture period, the number of oocytes with germinal vesicle (GV) or splitting of the embryonic vesicles (GVB) and oocytes with a polar body (PB) was calculated using stereomicroscope or inverted microscope with differential interference contrast equipment. In the analyzed crops was calculated the percentage of oocytes with GVB from the total number of oocytes and the percentage of oocytes with PB from the total number of oocytes and compared with the data of the control culture.

Table 1:

activation of meiosis in multiple shielded rat e is sanirovanii the oocytes
ConnectionOocytes (n)Activation (%)
GVGVBPBGVB+PB
Control (NC)191421
10 μm FF-MAS136546
0.1 ám compound of example 1147239
1 μm compound of example 1612675
10 μm compound of example 1114795
NC means gipoksantin

GV means germinal vesicle

GVB means splitting germinal vesicle

PB means polar body

n means the number of oocytes

Example 24: a study of meiosis activating substance in the culture system of mouse follicle

Animals

The follicles were obtained from immature 19-21-day-old female mice (C57B1/6J×CBA/J), which were kept under controlled lighting and temperature.

Collection of serum and cultivation of the follicles

Animals were given ether anesthesia and collected blood by removing the eye. After clotting the blood centrifugual is whether for 15 min at 4000g, collected serum was stored at -20° to use. The ovaries were removed and placed in the environment of Leibovitz L-15 (Gibco Cat. No. 41300) supplemented with glutamine, 1 mmol/l, 3 mg BSA/ml human transferrin (iron-free) 5 μg/ml insulin, 5 µg/ml (degree of purity chemicals for crops, Sigma, St. Louis, MO) at 37°C.

Preantral follicles with a diameter of 170-190 μm were isolated mechanically with two needles 27 size attached to the syringe of 1 ml were placed in a 4-cell culture plates (Nunclon, Denmark) and then washed three times minimum essential medium modification α (α-MEM, Gibco Cat. No. 11900) supplemented with glutamine 2 mmol/l, transferrin, 10 μg/ml insulin, 10 μg/ml and 3 mg BSA/ml

Follicles 170-190 microns normal morphological appearance, i.e. the Central spherical oocyte, a high density of granulocytes and a shell layer surrounding the follicle completely, were selected and individually cultured in 96-cell culture the tablet (Nunclon, Denmark) with the introduction of 40 µl of culture medium α-MEM with the addition of immature mouse serum 50 μl/ml, insulin 5 μg/ml, glutamine 2 mmole/l human transferrin, 10 μg/ml and 0.2 international units of follicle-stimulating hormone (FSH) (Gonal F, Serono, Solna, Sweden). Without any oil coating follicles were cultured in humidified thermostat, filled with air with 5% CO 2at 37°C.

The beginning of the cultivation was defined as day 0. Culture medium was changed every day. The follicle diameter was measured every day using a 100-fold increase and a calibrated micrometer. In addition, the survival rate of follicles is controlled by degradation assessment (darkening of the follicle) and disruption (loss of the oocyte). Time of cultivation amounted to 4 days.

In day 2 and 3 of the cultivation of the analyzed connection or bubble volume 1,72 µl was added into the culture medium. The investigated compound was dissolved in ethanol (bubble).

Research follicle

Oocytes were stopped in meiosis and characterized with the help of intact nuclei with prominent nucleoli, known as germinal vesicles (GV). If re-initiation of meiosis nucleolus and nuclear envelope disappear, and it is characterized by splitting of GV, which in this case is called the splitting of the embryonic vesicles (GVB).

In day 4, at the end of the culture period, the follicles controlled on the resumption of meiosis. The number of follicles with oocytes with germinal vesicle (GV) or splitting of the embryonic vesicles (GVB)were counted using a stereo microscope or an inverted microscope with differential interference contrast equipment. In the analyzed crops was calculated the percentage of follicles with GVB from BEGO number of follicles and compared with a control culture of bubbles.

Table 2:

activation of the maturation of oocytes in culture system of mouse follicle
ConnectionFollicles (n)Activation (%)
GVGVBGVB
Control (1,72 ál of ethanol)16211
10 μm FF-MAS900
1 μm compound of example 12778
3 μm of the compound of example 109100
10 μm compound of example 108100
GV means oocytes with germinal vesicles

GVB means oocytes with splitting embryonic bubbles

n means the number of follicles

Example 25: infertility treatment with the use of MAS agonist in vitro

Eggs were extracted using guided by ultrasound transvaginal aspiration of ovarian cancer, or hormone-stimulated or not stimulated by the hormone of the female patient.

Hormonal stimulation could go through the Protocol of in vitro fertilization (IVF) standard length, including origaudio regulation antagonist of gonadotropin, for example, a nasal spray of cinerella, then after 14 days of daily injections of follicle stimulating hormone (FSH) (Gonal-F, subcutaneously) 150 IU/day. For 36 h before collecting eggs, the patient was given human chorionic gonadotropin (hCG) (10000 ME subcutaneously)to trigger the final maturation of the follicle and oocyte.

The connection was added to the culture medium at a concentration of 3 μm and allowed to interact with the reproductive cell prior to fertilization or mediation, or to improve procedures meiotic maturation. Oocytes were fertilized in vitro, cultured in vitro and returned to the uterus of the patient is usually on day 3 after collection of oocytes. The patient gave progesteron (krinon in the form of a vaginal gel, 1 dose per day) and/or estradiol (estradiol valerate, 2 mg/day)to improve implantation and to give the uterus greater susceptibility.

The connection is added to the culture medium significantly improved the quality of the maturation of oocytes, which resulted in higher fertilization, more intensive development prior to implantation, the higher implantation and ultimately to higher metric born healthy human infants.

Example 26: the treatment of female infertility with the use of MAS agonist in vivo

The compound was administered to the patient orally twice daily at a dose of 10 mg/to the since the final maturation of oocytes caused by injection of hCG (10000 ME human chorionic gonadotropin, subcutaneously). Human chorionic gonadotropin can be introduced during a normal cycle. The cycle could be induced by withdrawal of progesterone, administered at least 10 days before canceling to induce bleeding and cyclic activity of a patient with amenorrhea or polycystic ovarian syndrome. Or hCG could be introduced as an integrated part of the hormonal stimulation of the normal duration of the Protocol IVF (using lower regulation antagonist of gonadotropin, for example, a nasal spray of cinerella, then after 14 days by daily injections of follicle stimulating hormone 150-225 IU/day).

The patient received treatment or as an adjunct to conventional therapy IVF with a selection of eggs, IVF and embryo transfer, or an alternative treatment was applied in combination with fertilization of the egg, using insemination or natural sexual intercourse.

The treatment increased the level of MAS agonists in the serum of the patient just before ovulation, thus was obtained improved maturation of oocytes. Quality undergone ovulation, which was enhanced by induction of meiosis daily input connection, which led to higher fertilization, the more intensive development prior to implantation, the higher implantation and ultimately to higher metric born healthy human infants.

Example 27: the treatment of male infertility with the use of MAS agonist in vivo

The compound is administered orally to the patient a male twice daily at a dose of 10 mg/kg consistently for at least 60 (sixty) days.

The treatment increased the level of agonist MAS in the patient's serum, which positively stimulated the progress of meiosis in the testis and, consequently, over time the quality of sperm. Indicators of semen quality (sperm counts, morphology, progressive motility and so on) separately or together improved leading to improved fertility sperm mentioned patient.

It was expressed that it was possible to avoid fertilization by intracytoplasmic sperm injection (ICSI) to achieve fertilization only through IVF or in another example, the fertilization IVF/ICSI were cancelled, and it was possible to achieve fertilization by insemination or natural fertilization of the egg.

Example 28: therapeutic scheme of women's contraceptive use MAS agonist and early maturation of oocytes

The compound is administered orally to the patient twice a day in a dose of 50 mg/kg every day throughout a normal cycle. The patient floor of the Ala therapy or as an Addendum to the usual treatment Protocol IVF with a selection of eggs, IVF and embryo transfer, or alternative therapy was performed in combination with fertilization achieved by insemination or natural sexual intercourse.

Therapy has increased the level of MAS agonists in the serum of the patient long before had ovulated and posredoval maturation of oocytes before ovulation. As a result of ovulation occurring aged oocytes were no longer viable or capable of fertilization. Normal menstrual cycle was not affected, has not changed also normal level and dynamics of steroid hormones.

Example 29: therapeutic scheme female contraception, using the MAS antagonist, blocking the progress of meiosis in the ovary

The compound is administered orally to the patient twice a day in a dose of 50 mg/kg every day throughout a normal cycle.

Therapy was increased in the serum of the patient's level of MAS agonists, which effectively inhibited the passage of the natural maturation of oocytes. The stage of ovulation was normal, and cyclic activity remained unchanged. However, during ovulation meiosis stopped, and so did the output immature and poplocalframe of oocytes. Normal levels and dynamics of steroid hormones remained unaffected, as well as have remained untouched natural cyclic activity of the monthly menstruation.

Example 30: therapeutic scheme of male contraception using the MAS antagonist. blocking the progress of meiosis in the testis

The compound is administered orally to the patient a male twice daily at a dose of 50 mg/kg consistently for at least 60 (sixty) days. The passage of spermatogenesis in men is approximately 60-65 days.

Therapy induced the treated subject level antagonists MAS in serum, which effectively suppressed the natural course of meiosis and differentiation, which led to the formation of fertilizing Mature spermatozoa in the testis of the subject. The progress of spermatogenesis was suppressed, and formed and allocated exclusively for non-fertilizing sperm. However, endocrinology of the testis was not affected, and the normal level and dynamics of steroid hormones remained unchanged.

below is a table that summarizes the physico-chemical properties of some of the compounds obtained.

ExampleConnectionData NMS/MS
11H-NMR: 5.32 s, (1H, H-14); 3.24 dd (1H, H-3 alpha) MS(EI+): 126 (100%), 453 (5%, M+)
121H-NMR: 5.36 s (1H, H-14); 3.19 dd (1H, H-3 alpha) MS (EI+): 98 (100%), 425 (8%, M+)
35 1H-NMR: 7.32 (2H, aryl); 7.05 (3H, aryl); 5.31 s, (1H, H-14); 3.75 m (8H, piperazine), 3.28 dd (1H, H-3 alpha) MS(EI+): 175 (100%), 502 (12%, M+)
461H-NMR: 5.34 s, (1H, H-14); 3.70 m (4H, J morpholine); 3.23 dd (1H, H-3 alpha)
MS (EI+): 100 (100%), 427 (8%, M+)
571H-NMR: 8.30 d (2H, pyrimidine 6.5 m (1H pyrimidine); 5.32 s, (1H, H-14); 3.70 (8H, piperazine) 3.24 dd (1H, H-3 alpha) MS (EI+): 177 (100%), 504 (6%, M+)
681H-NMR: 5.32 s, (1H, H-14); 3.26 dd (1H, H-3 alpha) MS (EI+): 84 (100%), 411 (8%, M+)
7101H-NMR: 5.31 s, (1H, H.14); 3.24 dd (1H, H-3 alpha) MS (EI+): 86 (100%), 370 (8%), 413 (9%, M+)

1. Steroid compound having the General formula X:

where in the code

HA:

each link between6and7between7and8between8and9between8and14and between14and15means independently single bond or double bond, provided that each carbon atom With6With7With8With9With14and15associated with each of adjacent carbon atom by a single bond or, at most one double bond;

CR3mean SN-HE;

A - methylene or ethylene spacer;

4and R4'- C1-C4alkyl, hydrogens;

R20- C1-C4alkyl;

R23and R23'together form a piperidine-1-yl, morpholine-4-yl, pyrrolidin-1-yl, piperazinil, possibly substituted by a hydroxy-group, a benzene, pyridine, pyrimidine, phenyl, alkoxycarbonyl

or R23means hydrogen, and R23'- substituted alkyl.

2. Steroid compound according to claim 1, where the fragment has one of the General formula HA:

3. Steroid compound according to any one of claims 1 and 2, where R4and R4'means independently hydrogen or methyl.

4. Steroid compound according to any one of claims 1 to 3, where R4and R4'means independently (C1-C4)alkyl.

5. Steroid compound according to any one of claims 1 to 4, where R20means methyl.

6. Steroid compound according to any one of claims 1 to 5, where R23and R23'together with the amino nitrogen form a nitrogen-containing heterocyclic ring structure selected from the group including piperidine-1-yl, morpholine-4-yl, piperazine-1-yl, pyrrolidin-1-yl, and their substituted derivatives.

7. Steroid compound according to any one of claims 1 to 6, where a represents methylene.

8. Steroid compound according to any one of claims 1 and 7, selected from the group including

(20S)-20-[(3,3-dimethylpiperidin-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol,

(20S)-20-[(piperidine-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol,

(20S)-20-[(4,4-dimethylpiperidin-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol,

(20S)-20-[(4-methylpiperazin-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol,

(20S)-20-[(4-phenylpiperazin-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol,

(20S)-20-[(morpholine-4-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol,

(20S)-20-[(4-(pyrimidine-2-yl)piperazine-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol,

(20S)-20-[(pyrrolidin-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol,

hemisuccinate(20S)-20-[(3,3-dimethylpiperidin-1-yl)methyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-Ola,

(20S)-20-[(4-(pyridin-2-yl)piperazine-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol,

(20S)-20-[(4-phenylpiperazin-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol,

(20S)-20-[(4-methylpiperazin-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol,

(20S)-20-[(morpholine-4-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol,

(20S)-20-[(pyrrolidin-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol,

(20S)-20-[(piperidine-1-yl)ethyl]-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol.

9. Steroid compound, representing

(20S)-20-[(piperidine-1-yl)methyl]-4,4-dimethyl-pregna-5,7-Dien-3β-ol,

(20S)-20-[(4-phenylpiperazin-1-yl)m is Teal]-pregna-5-ene-3β -ol,

(20S)-20-[(piperidine-1-yl)methyl]pregna-5-ene-3β-ol,

(20S)-20-[(morpholine-4-yl)methyl]pregna-5-ene-3β-ol,

(20S)-20-[(pyrrolidin-1-yl)methyl]pregna-5-ene-3β-ol,

(20S)-20-[(4-carboxyethylpyrrole-1-yl)methyl]pregna-5-ene-3β-ol,

(20S)-20-[(3-hydroxypiperidine-1-yl)methyl]pregna-5-ene-3β-ol,

(20S)-20-[(4-benzoylpiperidine-1-yl)methyl]pregna-5-ene-3β-ol,

(20S)-20-[(4-hydroxypiperidine-1-yl)methyl]pregna-5-ene-3β-ol,

(20S)-20-[(4,4-dimethylpiperidin-1-yl)methyl]pregna-5α-EN-3β-ol,

(20S)-20-[(4-piperazine-1-yl)methyl]pregna-5-ene-3β-ol,

(20S)-20-[(4-phenylpiperazin-1-yl)methyl]pregna-5-ene-3β-ol,

(20S)-20-[{4-(2-pyridyl)piperazine-1-yl}methyl]pregna-5-ene-3β-ol,

(20S)-20-[{4-(3-pyridyl)piperazine-1-yl}methyl]pregna-5-ene-3β-ol,

(20S)-20-[{4-(4-pyridyl)piperazine-1-yl}methyl]pregna-5-ene-3β-ol,

(20S)-20-[{4-(2-pyrimidyl)piperazine-1-yl}methyl]pregna-5-ene-3β-ol.

10. The pharmaceutical composition for regulation of meiosis and migration of awtitow comprising at least one steroid compound of General formula X according to any one of claims 1 to 9 and at least one pharmaceutically acceptable excipient.

11. The pharmaceutical composition of claim 10, where the steroid compound of General formula X is contained in an effective amount.

12. The method of regulation play, especially meiosis is, includes introduction to the subject in need of such regulation, an effective amount of at least one steroid compounds of General formula X according to any one of claims 1 to 9.

13. Way of improving the possibilities of the ability of oocytes to develop in a mammal that includes the contact of the oocyte, taken from a mammal, with the steroid compound according to any one of claims 1 to 9.

14. Method for the production of steroidal compounds of General formula X according to any one of claims 1 to 9, where R4and R4'mean (C1-C4)alkyl, including

(a) on the basis of (20S)-20-methylpregna-8,14-Dien-3β,21-diol

(b) substitution by nucleophilic attack With21,

(C) the substitution nucleophilic group at21cyanide with the formation of new carbon-carbon links in With21,

(g) recovering the obtained nitrile with the formation of aldehyde,

(d) reductive amination of nitrile with the formation of various amines.

15. Method for the production of steroidal compounds of General formula X according to any one of claims 1 to 9, where R4and R4'mean (C1-C4)alkyl, including

(a) on the basis of (20S)-20-hydroxymethylene-4-EN-3-one,

(b) introduction by alkylation of the two alkyl groups at position 4,

(C) recovering ketogroup in the hydroxy-group,

(g) the introduction of dual tie the Δ 7by the synthesized/dihydrobromide,

(e) isomerization of the diene Δ5,7in Dien Δ8,14by heating in the presence of acid,

(e) oxidation of 17-hydroxy-group in the aldehyde group and

(W) reductive amination of aldehyde groups.

16. Intermediate compounds for the production of steroidal compounds according to any one of claims 1 to 9, namely:

(20S)-4,4-dimethyl-20-hydroxymethyl-5α-pregna-8,14-Dien-3β-ol and

(20S)-4,4-dimethyl-5α-pregna-8,14-Dien-3β-ol-20-carbaldehyde.



 

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FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to the substituted 4-benzylaminoquinolines and their heteroanalogs of the general formula (I): P-L-G (I) wherein G means compound of the formula: G(I) wherein K means -OR(7), -NH-CH2-CH2-SO3H, -NH-CH2-CO2H wherein R(7) means hydrogen atom, CH3; R1-R6 mean independently of one another hydrogen atom, -OR(10), -R(10) being one of residues R1-R6 means a bond with L always; R(10) means hydrogen atom, (C1-C4)-alkyl; L means (C1-C15)-alkyl being one or some structural CH2-fragments can be replaced for -C≡C-, -NR(11)-, -CO-, -O- wherein R(11) means hydrogen atom; P means: or wherein A means nitrogen atom (N); B means CH; D means CH; E means CH; R16-R24 mean independently of one another hydrogen atom, F, Cl atoms, (C1-C4)-alkyl being alkyl residues can be mono- or multiple-substituted with fluorine atom, NR(25)R(26), OR(25), COR(25), COOR(25), CONR(25)R(26) being one of residues R16-R(24) means a bond with L always; R25 and R26 mean independently of one another hydrogen atom, (C1-C4)-alkyl or benzyl. Also, invention relates to their pharmaceutically acceptable salts. Also, invention relates to a method for their preparing and to a drug based on thereof for prophylaxis of supersaturation of bile with cholesterol. Invention provides preparing new compounds and a drug based on thereof that can be used for prophylaxis and treatment of patients suffering with gallstones.

EFFECT: improved preparing method, valuable medicinal properties of compounds and drugs.

10 cl, 32 ex

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where

R1- (R2R3N(O)r)-, where r=0, 1, each of R2and R3independently - H, C1-6alkyl; or R1-

where q=0, - 1, Y-(CH2)m-, where m=1, 2, 3, 4, 5; or

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,

where -(CH2)m-, where m=2; R6=H; R7=H, C1-6alkyl, C2-6alkenyl,2-6quinil, perhaps HE or substituted BASED; S=0, 1; each of the8and R9independently - H, HE, C1-6alkyl, R10CO., where R10’- H, C1-6alkyl

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< / BR>
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EFFECT: valuable medicinal properties of steroids.

5 tbl, 7 dwg

FIELD: organic chemistry, steroids, chemical technology.

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EFFECT: improved preparing method.

56 cl, 42 tbl, 30 sch, 5 dwg, 89 ex

FIELD: organic chemistry, steroids, medicine, pharmacy.

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EFFECT: valuable medicinal properties of compounds, improved method for treatment.

38 cl, 1 tbl, 18 ex

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EFFECT: improved method for rehabilitation.

1 ex

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