CN103880909A - Steroidal compound - Google Patents

Abstract

The invention discloses a steroidal compound with enhanced metabolic resisting capability and increased water solubility, and discloses a preparation method of the steroidal compound. The steroidal compound disclosed by the invention is suitable for preparing a drug for curing CNS disorder caused by the steroidal compound or related to the steroidal compound and relieving and preventing or curing the CNS disorder.

Description

Steroide

The application is to be on May 20th, 2008 applying date, application number is 200810127741.0, denomination of invention is the divisional application of the application for a patent for invention of " steroide " (former denomination of invention is " new steroide ", then change " steroide " into).

Invention field

The present invention relates to act on a kind of new steroide of gamma-aminobutyric acid receptor-chlorion carrier (GABAA-R) mixture, described compound can be used for the treatment of central nervous system (CNS) obstacle relevant with GABA-steroide with GABA and/or that steroide causes.

Background of invention

Steroide hormone Vitarrine, the metabolite of progesterone; As pregnant (steroid) alkanol ketone, and testosterone, the known Desoxycortone of metabolite of Androstenedione and dehydroepiandrosterone, cortisone and hydrocortisone, all become the object of various researchs, and illustrated at least partly their effects in mammalian nervous signalling system.Because the compound nomenclature in this field is different from other field, therefore the application's compound all uses IUPAC nomenclature.Cause that the steroide that CNS illness and handicapped steroide metabolite and the application pay close attention to has same constructional feature, they comprise 3 Alpha-hydroxies, 5 α or 5 β pregnane parent nucleus, and one be positioned at ketone group or the hydroxyl of 17,20 or 21.These 3 Alpha-hydroxy-5 α/β-steroides act on gamma-aminobutyric acid receptor-chlorion carrier (GABA _a-R) mixture, thereby be named as GABA-steroide.Table I has provided the example of described steroide:

Table I .3 Alpha-hydroxy-5 α/β-pregnane or-name of androstane compounds

* CAS registration number does not find

All compounds of mentioning in example are not open in the prior art, are therefore new inventions.U.S.5,232,917 (people such as Bolger) and U.S.5,925,630; 5,939,545; 6,143,736; 6,277,838, people such as () Upasani discloses many 3 Alpha-hydroxy steroides and some 3 β-steroides.All compounds of the present invention are formerly not open.The competitiveness that these patents relate to GABA-A acceptor regulates.That is to say, above-mentioned patent lays particular emphasis on 3 Alpha-hydroxies-steroide and their Benzodiazepine sample effect.All steroides as GABA-A receptor modulators all have 3 Alpha-hydroxy structures.

WO99/45931

the antagonistic action of this steroide of 3 β-OH-5 alpha-pregnane-20-ketone is disclosed, but and the mentioned compound of unexposed the application.WO03/059357

disclose several 3 beta-hydroxy steroides and their antagonistic actions to GABA-A acceptor, but also do not disclose the related compound of the application.

The people such as Wang (Wang M.D.,

t. with Landgren S. (2000), allopregnanolone and pregnanolone are to can be by pialloperegnanolone selective exclusion (The inhibitory effects of allopregnanolone and pregnanolone on the population spike by the antagonistic action of the external group's honeybee current potential bringing out of CA1 pyramidal cell at hippocampus, evoked in the rat hippocampal CA1stratum pyramidale in vitro, can be blocked selectively by epiallopregnanolone) acta Physiol Scand169,333-341 and Wang M, He Y, Eisenman LN, Fields C, Zeng CM, Mathews J etc., the pregnant steroide of 3 beta-hydroxy is as Pregnenolone sulfate sample GABA (A) receptor antagonist (3beta-hydroxypregnane steroid are pregnenolone sulfate-like GABA (A) receptor antagonists) J Neurosci2002; 22 (9): 3366-75) antagonistic action of 3 β-OH-5 alpha-pregnane-20-ketone and other 3 β-OH-5 α/β-pregnane steroid-like is disclosed.Some other paper has been recorded the dose-dependently antagonistic action of 3 β-OH-5 α/β-pregnane steroide and sulfuric ester steroide.But above-mentioned paper not mentioned compound of the present invention.

The U.S. Pat 2004/0242549 of Covey and Jiang discloses many steroides, but and not mentioned compound of the present invention.

The present invention relates to pharmaceutical chemistry field, and intend to prepare compound and pharmaceutical composition for regulating the excitability of mammal brain, this regulating effect is by gamma-aminobutyric acid receptor-chlorion carrier (GABA _a-R) mixture and other direct or indirect and GABA _athe neurotransmitter system that-R mixture is relevant carries out.Find that multiple steroide has effective GABA Signal Regulation and excitation, thereby shown various physiological roles.The steroide that comprises 3 Alpha-hydroxy-5 α/β-pregnane-20-ketone/alcohol or androstane-17-ketone/alcohol structure, demonstrates specificity GABA-A{ γ-aminobutyric acid (A) } acceptor enhancement.Table I has been listed 3 Alpha-hydroxy-5 α/β-pregnane steroid-like embodiment.Some compounds in these steroides are very effective, and for example they show the effect of induced anesthesia under high pharmacological dose.Due to these above-mentioned character, these spontaneous pressure steroid hormones and steroid hormone have side effect and cause certain illness.The basis of the side effect of 3 Alpha-hydroxies-pregnane-20-ketone/alcohol and the androstane-17-ketone/alcohol-steroide CNS negative impact that they cause just.

In early days disclose some 3 beta-hydroxy pregnanone compounds and can block these pressure that have side effect or the detrimentally affect of steroid hormone to brain.But the existing problem of the compound of early discovery is that 3 of their keys are easy to be insoluble in water by metabolism and these compounds in vivo.

Due to these 3 Alpha-hydroxies-pregnane-steroides generate in vivo and also be life the metabolite of essential steroide hormone, so their generation is difficult to be prevented from.In the time of the reaction of acute or chronic stress, when the luteal phase of menstrual cycle and gestation, people knows from experience and continues a few days to a few weeks and produce a large amount of above-mentioned steroides.Brain also can generate these compounds.Therefore need specific inhibition agent as treatment.

Due to GABA _athe complicacy of-R composite structure, the direct effect mechanism in this receptor site is not also illustrated completely.GABA receptor family comprises several subunit compositions, and some subunits in them are known relevant with specific function and the illness of CNS.One object of the present invention is exactly to find a kind of new compound to be used for the treatment of the receptor related neurohumoral irregular excitement of GABA acceptor or other and GABA, and these compounds have broad spectrum or specificity to some subunits and function to a certain extent.Endogenous 3 Alpha-hydroxy-5 α steroides or 3 Alpha-hydroxy-5 'beta ' steroids act on the caused illness of GABA-A acceptor and are described and understand by people well.Simultaneously people also recognize, after contact human body, 3 Alpha-hydroxy-5 α/β-steroides can bring out human body to they itself and the resistance of other similar compound, and 3 Alpha-hydroxy-5 α/β-steroides stop the withdrawal symptom after administration.To be further elaborated this below.

In a word, it has been recognized that 3 Alpha-hydroxies-Δ 4-5,5 α/βs-steroide can cause CNS dysfunction by three kinds of possible mechanism as described below: a) direct effect, b) inducible resistance, and c) withdrawal symptom.Compound of the present invention belong to pregnane-, pregnene-, androstane-, androstene-serial steroide, has suitable additional function.These compounds can use separately or combine use as prodrug and/or with preparation and other component, for strengthening or regulating CNS effect.Described in field of the present invention, composition comprises all compositions, thereby as long as the compounds of this invention that contains significant quantity in them accomplishes the end in view.

3α the disease that-hydroxyl-pregnane (pregnene)/androstane-steroide causes

A) direct effect

Now confirm that 3 Alpha-hydroxy-5 α/β-steroides can directly cause the obstacle of CNS function.The obstacle being caused by direct effect by 3 Alpha-hydroxy-5 α/β-steroides and the example of symptom have: premenstrual dysphoric disorder, premenstrual syndrome, dementia, Alzheimer (family name) disease, sedative effect, tired, chronic tired syndrome, dysmnesia, learning disorder, dyskinesia, fracture, clumsiness, appetite and appetite increase, obesity, excessive drinking and the recurrence of Drug abuse symptom, the generation of the negative emotions such as nervous, irritability and depression, the going down of hearing and eyesight, the deterioration of petit mal epilepsy, burnout symptom.

B) resistance

Continue, contact the dysfunction that can cause GABA-A receptor system with 3 Alpha-hydroxy-5 α/β-steroides chronically.Thereby a kind of resistance generation and this resistance are finally to cause pressure sensibility, it is wholwe-hearted to be difficult to, and impulsion control is lost and the initial stage of the lysis of depression.The effect of 3 Alpha-hydroxy-5 α/β-steroides has been found to be a factor that strengthens drug dependence.The emphasis of further studying below:

C) give up

If health continues, contacts with 3 Alpha-hydroxy-5 α/β-steroides in short time, after finishing, this contact can cause withdrawal symptom.That is to say, the process that this phenomenon can be produced 3 Alpha-hydroxy-5 α/β-steroides at menstrual period corpus luteum occurs while being interrupted.This drug withdrawal phenomenon can occur in (postpartum) between breeding time equally, and at this moment placenta generates 3 Alpha-hydroxy-5 α/β-steroides and is blocked.After the pressure in a period finishes, also can observe same phenomenon.As the reaction to pressure, suprarenal gland can synthesize 3 Alpha-hydroxy-5 α/β-steroides.After this generation is blocked, withdrawal symptom also likely can occur.The illness example being affected by this drug withdrawal/give up phenomenon is partial epilepsy, and the patient of this disease has an epileptic focus at pallium, and in the time of menstrual period drug withdrawal, illness can increase the weight of.This phenomenon is called " Menstrual Epilepsy ".

Other example has migraine that menstruation is relevant and the migraine of pressure correlation, postpartum emotional change and headache at weekend.Giving up is a sign of resistance early period of origination.

Can clearly be seen that from foregoing steroide is important drug candidate.But the steroide of Nature creating is easy in vivo, by violent metabolism, not therefore conventionally to be suitable for oral.The metabolism of other route of administration is also very strong, so this compounds is impossible as medicine or treatment, because first the active part of this compounds can be destroyed by metabolism.

The Second Problem of steroide is that they are insoluble in water, thereby is difficult to pass through vivo medicine-feeding.

Above and other problem is resolved in compound of the present invention.

Summary of the invention

Describing before the present invention, need to be appreciated that the term that used is herein only used to specializing of description and does not limit with this, because field of the present invention will only be limited by additional claim and its coordinator.

Especially, must recognize, the singulative " ", " one " and " being somebody's turn to do " that in specification sheets and appended claim, use comprise plural indicator equally, unless context has explicitly pointed out other situation.

In the following description, term " steroide is correlated with " and " steroide causes " in " steroide relative disease " mean that comprising three kinds of steroides acts on the mechanism that neural system is possible, a) direct effect, b) inducible resistance, c) withdrawal symptom.The example of these illnesss provides hereinbefore, but these examples are only used to illustrate each mechanism, and does not mean that the present invention is limited.

Term " blocking-up " is considered to define a kind of effect, and wherein 3 Alpha-hydroxy-5 α/β-steroides are prevented from the effect of GABA-R acceptor.Need to be appreciated that the expressed implication of " blocking-up " and " adjusting " or " inhibition " or similar term is completely different, after these term Suggestions still occur, be only that scope is less or speed is slower.

Term " pharmaceutical composition " uses its widest implication, it comprise all pharmaceutically can with the composition that contains at least one active substance, and selectable carrier, auxiliary material, component etc.Term " pharmaceutical composition " comprises the composition of a kind of derivative that contains described active substance or prodrug forms equally, such as pharmaceutically acceptable salt, and vitriol and ester class form.Within the production of different way of administration pharmaceutical composition all drops into chemicals those skilled in the art's limit of power.

Term " administration " and " administering mode " and " route of administration " are all by they the widest implications.The medication of pharmaceutical composition of the present invention can have many kinds, and whether the administering mode that depends primarily on this part, surface or system is best suited for needing the illness for the treatment of.The example of these different modes of administration has surface (such as through skin), local (comprising by eye and various mucous membrane such as vagina and rectum are sent), and oral or enteron aisle external administration and pulmonary administration, comprise lower respiratory tract.

The preparation of described composition and formulation are known for the personnel of pharmacy and formulation art technology, and can be for the formation of composition of the present invention.

Term " antagonist " represents that a kind of material hinders other material, as agonist, thereby brings out its effect.Term antagonist and blocker in the application use simultaneously.

The present inventors have synthesized the new steroide of a class, and they obtain protection and avoid by metabolism in the 3-position of steroid nucleus.Thereby it is water-soluble that they have greatly improved by change structure feature, such as adding two keys on the steroidal parent nucleus in aforementioned pregnane, pregnene, androstane and androstene series, and replace ketone group with oximido on 20,21 or 17.

In a word, 3 Alpha-hydroxies-Δ 4-5,5 α/βs-steroide is realized now by three kinds of possible mechanism described above and causes CNS dysfunction: a) direct effect, b) inducible resistance, c) withdrawal symptom.Compound of the present invention belong to pregnane-, pregnene-, androstane-, androstene-serial steroide, there is suitable additional function.These compounds can use separately or combine use as prodrug and/or with preparation and other component, for strengthening or regulating CNS effect.Described in field of the present invention, composition comprises all compositions, thereby as long as the compounds of this invention that contains significant quantity in them accomplishes the end in view.

According to the inventor's understanding, this is the steroide of invention antimetabolism enhancing first and water-soluble increase.In addition,, according to the reference of claim and this place combination, these materials can be used for the many specific steroides of preparation treatment now to be caused or the relevant handicapped pharmaceutical preparation of CNS, also can be used for methods for the treatment of simultaneously.

Detailed Description Of The Invention

The present invention relates to have the new compound of antimetabolism and water-soluble increase, and produce causing that the handicapped 3 Alpha-hydroxies-pregnane of CNS (pregnene)-steroide has the method for the compound of antagonism and blocking effect.The present invention starts from discoverable type I or the represented steroide of II and has the effect of GABA receptor signal conduction setter, and they can be used as agonist, antagonist or inverse agonist.

The existence of 3 tertiary alcohol groups shows and can, by preventing internal metabolism oxidation or Decomposition, extend steroide at people's Half-life in vivo.The existence that is bonded to the hydrogen bond receptor/donor groups of steroidal molecule D ring regulates the ability of GABA signal conduction to have a great impact to steroide.

The present invention relates to the represented steroide of formula I or II or its pharmaceutically acceptable salt, prodrug or solvate:

Wherein

R ₁for ethynyl, vinyl, ethyl or other saturated or unsaturated alkyl group; Hydroxyl, described hydroxyl is free form or is combined to form ester or ether or glycosylated compound with carboxylic acid residues, sugar, alkyl group; Fluorine or other halogen; Proton;

R ₂for ethynyl, vinyl, ethyl or other saturated or unsaturated alkyl group; Hydroxyl, described hydroxyl is free form or is combined to form ester or ether or glycosylated compound with carboxylic acid residues, sugar, alkyl group; Fluorine or other halogen; Proton;

R ₃be 5 α-or 5 β-H;

R ₄for nitro, hydroxyl, described hydroxyl be free form or with ester, ether, sugared bonding; And

R ₅for proton.

According to embodiment of the present invention, these compounds are above-mentioned formula I or the represented steroide of II, and its pharmacy can accept derivative, salt, prodrug or solvate, wherein

R ₄, R ₅for the N of O or oxime=NOH form, or carbocyclic ring or heterocycle.

R ₆for methyl, alkyl or-CH ₂oR, wherein R is H, carboxylic acid residues, alkyl group or carbohydrate;-CH ₂x, wherein X is fluorine or other halogen;

R ₇, R ₁₀for being positioned at OH, the CH of 7 ₃or H.

R ₈, R ₉or R ₁₁, R ₁₂represent two Me-groups, or represent respectively Me-and H, or two-H.

According to an embodiment, R ₇, R ₁₀for being positioned at OH or the CH of 7 ₃.

According to embodiment of the present invention, these compounds are the represented steroide of above-mentioned formula I or its pharmaceutically acceptable salt, wherein R ₁for ethynyl; Hydroxyl, described hydroxyl is free form or is combined with carboxylic acid residues; Fluorine; Or proton; R ₂for ethynyl; Hydroxyl, described hydroxyl is free form or is combined with carboxylic acid residues; Fluorine; Or proton; R ₃be 5 α-or 5 β-H; R ₄for hydroxyl, and R ₅for proton, or R ₄, R ₅the N of O or oxime=NOH form together; R ₆for methyl; R ₇for H; And R ₈=R ₉=methyl or H.

According to embodiment of the present invention, these compounds are the represented steroide of above-mentioned formula II or its pharmaceutically acceptable salt, wherein R ₁for ethynyl or hydroxyl; R ₂for ethynyl or hydroxyl; R ₃be 5 α-or 5 β-H; R ₄, R ₅the N of O or oxime=NOH form together; R ₁₀for H; And R ₁₁=R ₁₂=H.

According to one of them embodiment, unsaturated link(age) can be between C4-C5 or C5-C6 or other position of molecule.R ₈, R ₉or R ₁₁, R ₁₂represent two Me-groups, or represent respectively Me-and H, or two-H, if or above-mentioned unsaturated link(age) between C4-C5, R ₈, R ₉or R ₁₁, R ₁₂one of them is respectively Me-or H, and another does not exist.According to this embodiment aspect, these compounds are the represented steroide of above-mentioned formula I, wherein R ₁for ethynyl or hydroxyl; R ₂for ethynyl or hydroxyl; R ₃do not exist; R ₄for hydroxyl; R ₅for proton; Or R ₄, R ₅be together=N of O or oxime=NOH form; R ₆for methyl; R ₇for H; And R ₈, R ₁₁for methyl or H; R ₉, R ₁₂for methyl, H, or, if described unsaturated link(age) is between C4-C5, do not exist.

Can there is optically active isomer in the compound of some formula I and II; The present invention includes and can separate the single isomer obtaining by conventional chromatographic technique and other separation method being well known to those skilled in the art.

The present invention also comprises derivative and the prodrug that all functions are suitable, and wherein ester class and ethers are added on the oh group of steroide.Suitable derivative example includes, but not limited to sulfuric ester, manthanoate, and acetic ester, propionic ester, with the glycosyl compound of carbohydrate or oligose, methylate, ethylate.Those skilled the in art will appreciate that other also can be selected use not included in the functional group listing not included in example above.

Table 2 and 3 discloses according to the example of the structure of a series of compounds of the present invention, thereby they are by adding ethynyl, vinyl or acetic ester structure on the 3-position of this steroide molecule or replacing the position of 3-hydroxyl of hydroxyl protection pregnane, pregnene, androstane or androstene steroid-like not by metabolism with fluorine atom.

table 2: based on the new compound with metabolic defence effect of formula I

table 3: based on the new compound with metabolic defence effect of formula II

Table 4 discloses the structure example of a series of compounds of water-soluble increase compared with 5-saturated compound (US2024), and they are to increase with respect to the oximido of ketone group or simple hydroxyl by adding that it is water miscible, as UC2027, UC2029.

table 4: the new compound example of water-soluble increase

Contriver has been found that pharmaceutically with the 3 β positions that have of the upper suitable dose of practice be that the steroides that ethynyl 3 α positions be hydroxyl and 20 are oximido two dimensional structure for ketone group or hydroxyl or 21 can suppress the interaction in vitro of 3 Alpha-hydroxy-5 α/β-steroides, but the effect of GABA self is only had to very weak antagonistic action.Therefore these compounds have stoped the aggravation of 3 Alpha-hydroxy-5 α/β-steroide negative effects, but the effect of GABA self is only had to slight influence.The mechanism of action of 3 Alpha-hydroxy-5 α/β-steroides in the handicapped aggravation process of CNS and the interaction mechanism of Alpha-hydroxy-5,3 β-ethynyl-3 α/β-pregnane-steroide and Alpha-hydroxy-5,3 β-ethynyl-3 α/β-androstane-steroide and 3 Alpha-hydroxy-5 α/β-steroides all are published out.The example of these compounds all provides in table 2 and table 3.

In addition, contriver has been found that the effect that pharmaceutically can suppress the chlorion flow of GABA-steroide to GABA mediation with the steroide of the upper suitable dose of practice, and these compounds have fluorine and/or on 20 or 21, have ketone group or oh group on 3.Therefore these steroides can be used as therapeutant, for preventing and/or treating disease that GABA self function overstimulation causes as Alzheimer (family name) disease.Table 2 has provided the example of these compounds.

Contriver finds that these steroides are antagonist, effect that can antagonism 3 Alpha-hydroxy-5 α/β-pregnane-steroides, but GABA is only had to very weak impact from the effect in central nervous system (CNS).Surprisingly, the combined treatment of disclosed these steroides of 3 Alpha-hydroxies-pregnane-steroide and table 2/3 and table 4, the chlorion flow that can suppress to be caused by 3 Alpha-hydroxies-pregnane-steroide is through HEK-cell (human embryo kidney (HEK), the mankind GABA-A acceptor recombinant chou of expressing HEK), but the chlorion flow that GABA is induced separately but only has low-down effect.

Have now found that and there is 3 β-ethynyl and 3 alpha-hydroxy steroides are actually antagonist (in table 4).The steroide that now finds that there is equally 3 α-fluorine also has antagonistic action (in table 5) to 3 Alpha-hydroxies-pregnane steroide.

An advantage of the present invention is 3 β-ethynyl-3 Alpha-hydroxy-pregnane-20-ketone or oxime-steroide, particularly 3 β-ethynyl-3 Alpha-hydroxy-Δ 4-pregnene-20-ketone, Alpha-hydroxy-5,3 β-ethynyl-3 α pregnane-20-ketone-oximido, the GABA-A regulation effect of antagonism 3 Alpha-hydroxy-5 α/β-pregnane-steroides effectively of 3 β-ethynyl-3 Alpha-hydroxy-Δ 4-pregnene-20-ketone-oximido, but the GABA-A receptor activation that GABA is caused but only has very weak impact.Special advantage of the present invention is that this blocking effect can reach with pharmacy and the suitable concentration of physiology.

A surprising new discovery of the present invention is, can by give pharmaceutically simultaneously with physiology on can acceptable dose the effect of 3 β-fluoro-5 β-pregnane-20-ketone, 3 β-fluoro-5 β-pregnane-20-(R)-ol, 3 β-fluoro-5 β-pregnane-20-(S)-ol, 3 β-fluoro-5 alpha-pregnanes-20-(S)-one and 3 α-fluoro-5 alpha-pregnanes-20-(S)-one oxime selective exclusion, 3 Alpha-hydroxy-5 α/β-pregnane-steroides, and GABA is only had to slight influence effect.

Another surprising new advantage of the present invention is that some steroides that have androstane skeleton can be by block to administration of selective the effect of 3 Alpha-hydroxy-5 α/β-pregnane-steroides with 3 β-ethynyl-3 Alpha-hydroxy-androstane-17-ketone, 3 β-ethynyl-3 Alpha-hydroxy-androstane-17-ketone-oxime, 3 α-ethynyl-3 beta-hydroxy-androstane-17-ketone-oxime simultaneously.A special advantage is that this blocking effect can reach by pharmacy and the suitable concentration of physiology.

GABA-A acceptor is a kind of chloride channel, and this GABA-A acceptor produces its effect by changing chlorion influx in this passage.As everyone knows, open a large amount of chlorions flow into neuronal cell when GABA-A acceptor, the neuronal activity in brain will reduce.Simultaneously people also recognize between amount that chlorion flows into and the clinical effect of GABA-A receptor active medicine and exist contact.

Benzene diaza

class, some effects of barbiturates and alcohol are the effects that produce them by above-mentioned mechanism.But this is also the reason that these medicines have side effects simultaneously.A problem of GABA-A acceptor is that its effect has impact to the major part of brain.Therefore the complete blocker of GABA-A acceptor is very dangerous and may produce mental diseases symptom and convulsions.When blocking the used time of doing of 3 Alpha-hydroxy-5 α/β-pregnane-steroides, wish to use a kind of can specificity the effect of antagonism 3 Alpha-hydroxy-5 α/β-pregnane-steroides and the material of antagonism GABA self-acting not.

In the present invention, be to stablize recombinant expressed human receptor at HEK-cell for checking the GABA-A acceptor of steroide.This acceptor comprises three kinds of different protein and mRNA, because receptor subunit α 1, β 2, γ 2L are expressed under above-mentioned experiment condition.These three GABA-A receptor subunits are the most common in central nervous system.It is the same in normal position with them that these GABA-A acceptors show in vitro, therefore thought that by those skilled in the art GABA-A receptor active drug effect is in a good in vitro tests system of brain.Chlorion flux is the GABA active function result on GABA-A acceptor by chloride channel, and this effect of GABA can be strengthened as the GABA-A receptor modulators of 3 Alpha-hydroxy-5 α/β-pregnane-steroides and so on.Chlorion flow by acceptor can record by patch clamp technique.The usefulness of a compound can be measured, and can carry out their accurate dosage of comparison by these data of standardizing between compound, and dosage is 10 μ m in the present invention.

Cross just as discussed above, have a lot of symptoms relevant with 3 Alpha-hydroxy-5 α/β-pregnane-steroides with disease, the effect of blocking-up 3 Alpha-hydroxy-5 α/β-pregnane-steroides will be a kind of methods for the treatment of of in question situation.Now, the invention can be used for useful matter and the methods for the treatment of of above-mentioned blocking effect.

Contriver has determined the mechanism of action in the disease being caused by 3 Alpha-hydroxy-5 α/β-pregnane-steroides.Determine that in this technique 3 Alpha-hydroxy-5 α/β-pregnane-steroides cause a) direct effect of CNS dysfunction by three kinds of possible mechanism, b) inducible resistance, and c) withdrawal symptom.

In general, but contriver prepares compounds effective and can block 3 Alpha-hydroxies-pregnane-steroide to the effect of GABA-A acceptor do not block the effect of GABA in the present invention, and partial antagonist to gGABA effect.

Another aspect of the present invention is the pharmaceutical composition that comprises a kind of compound, and this compound can be blocked the effect of 3 Alpha-hydroxies-pregnane-steroide to human body GABA-A acceptor.These compounds can be selected from table 4 and 5, and can exist with the form of suitable and pharmaceutically acceptable salt, as sodium salt.

The composition of said composition can be selected or adjust according to normal pharmacology operation, it is included in pharmaceutically compounds effective of chemical form aspect, the approach that is applicable to choose, and with being for a person skilled in the art applicable auxiliary material and carrier combination conventional and that know.

The conventional auxiliary material of oral administration and carrier for instance, can be weighting agent or suspension agent, as titanium dioxide, lactic acid, silicon-dioxide, colloidal silica, methylcellulose gum, Magnesium Stearate, Microcrystalline Cellulose etc.

Intravenous conventional auxiliary material and carrier for instance, can be water for injection (WFI), sterile buffer (as by shock absorption, solution being adjusted to pH7,4) albumin solution, lipid soln etc.

The conventional auxiliary material of percutaneous dosing and carrier for instance, can be Vaseline, whiteruss, glycerine, water etc.

One embodiment of the invention relate to compound according to the present invention for the preparation of alleviating, the particularly application in the pharmaceutical composition of premenstrual syndrome of prevention or treatment CNS obstacle illness, wherein by give at least one as described herein compound prevent the generation of resistance and the downward of GABA-A acceptor.This methods for the treatment of can maintain the susceptibility of GABA-A system and stop the reduction that susceptibility occurs in luteal phase.In long-term Progesterone Treatment, in rat body, demonstrate the variation of GABA-A acceptor.The present invention uses 3 beta-hydroxy-5 α/β-pregnane-steroides or 3 beta-sulfuric ester-5 α/β-pregnane/pregnene-steroides to treat, its shows can prevent the generation of resistance, therefore gives can stop withdrawal symptom after 3 Alpha-hydroxy-5 α/β-pregnane-steroides when stopping.

The generation of resistance will reduce as 3 Alpha-hydroxy-5 alpha-pregnane-20-ketone or benzene phenodiazines

the susceptibility of this endogenous GABA-A of compounds enhancing substance.When medicine is promptly stopped using, as latter stage luteal phase in the menstrual cycle, after 3 Alpha-hydroxy-5 alpha-pregnane-steroides stop administration, rebound effect will arrive.In intermenstrual period follows hard on body, stop generating and stopping giving steroide, finder's migraine and Epileptic fits can increase.

The example of the illness that the direct effect of 3 Alpha-hydroxy-5 α/β-steroides causes has sedative effect, fatigue, chronic tired syndrome, dysmnesia, learning disorder, dyskinesia, fracture, clumsy, appetite and appetite increase, obesity, the recurrence of excessive drinking and Drug abuse symptom, the generation of nervous, irritability and the negative emotions such as depressed and this are the cardinal symptoms of premenstrual syndrome, and the deterioration of petit mal epilepsy.

The caused illness of resistance occurring after long-term (a couple of days) contact 3 Alpha-hydroxy-5 α/β-steroides has, for example pressure sensibility, it is wholwe-hearted to be difficult to, it is absent minded that pressure or menstrual cycle are correlated with, somnopathy, fatigue, forfeiture and depression that impulsion is controlled, memory and learning disorder.3 Alpha-hydroxy-5 α/β-steroides can strengthen drug dependence equally.According to the present invention, can prevent, alleviate or treat these conditioned disjunction symptoms by giving at least one above-mentioned preferred steroide that is selected from table 4 or table 5.Most preferred above-claimed cpd uses with form suitable and pharmaceutically acceptable salt, most preferably sodium salt.

If health continues, contacts with 3 Alpha-hydroxy-5 α/β-steroides in short time, after finishing, this contact can cause withdrawal symptom.This phenomenon can occur in the time that menstrual period corpus luteum is produced 3 Alpha-hydroxy-5 α/β-steroides and is blocked.This drug withdrawal phenomenon can occur in (postpartum) between breeding time equally, and at this moment placenta generates 3 Alpha-hydroxy-5 α/β-steroides and is blocked.After the pressure in a period finishes, also can notice same phenomenon, now during pressure, 3 Alpha-hydroxy-5 α/β-steroides of suprarenal gland synthesized are blocked.Be partial epilepsy by the illness example that this drug withdrawal/abslinence phenomenon affected, the patient of this disease has an epileptic focus at pallium, and in the time of menstrual period drug withdrawal, illness can increase the weight of.This phenomenon is called " Menstrual Epilepsy ".Other example has migraine, the postpartum emotional change of migraine that menstruation is relevant and pressure correlation.Drug withdrawal phenomenon is a sign of resistance early period of origination.

Be considered to relevant to steroid class or these disease examples that steroid class causes comprise several as follows: epilepsy, menstrual cycle dependency epilepsy, depression, the depression of pressure correlation, migraine, the especially pressure-dependent fatigue of fatigue, premenstrual syndrome, premenstrua anxiety is levied, the anxious state of mind that menstrual cycle is relevant, the memory of pressure correlation changes, and the menstrual cycle, relevant attention was difficult to concentrate, the somnopathy that the menstrual cycle is relevant and fatigue.Have believable evidence to show fat and appetite increase, and the form that some balances such as excessive drinking or drug abuse are broken is also that steroid class causes or steroid class is brought out.

Therefore the invention provides compound and the method that is used for the treatment of, alleviates or prevent these illnesss.

A concrete scheme of the present invention relates to compound as described herein in the purposes for the preparation of alleviating, in prevention or treatment anti-inflammatory steroid side effect and the pharmaceutical composition that carries out treating after menopause in patient.According to the present invention, can prevent, alleviate or treat these situations or symptom by giving at least one above-mentioned preferred steroide that is selected from table 4 or table 5.Most preferred above-claimed cpd uses with form suitable and pharmaceutically acceptable salt, most preferably sodium salt.

One embodiment of the invention relate to as described herein compound for the preparation of alleviating, prevention or treatment oral contraceptive be to the purposes in the pharmaceutical composition of patient's side effect.According to the present invention, can prevent, alleviate or treat these conditioned disjunction symptoms by giving at least one above-mentioned preferred steroide that is selected from table 4 or table 5.Most preferred above-claimed cpd uses with form suitable and pharmaceutically acceptable salt, most preferably sodium salt.

Thereby, a particular embodiments of the present invention is a kind of pharmaceutical composition, it comprises a kind of oral contraceptive and at least one can block the compound of the treatment appropriate dose of 3 Alpha-hydroxies-pregnane-steroide to mankind GABA-A receptor acting, the wherein preferably compound in table 4 or table 5 of above-claimed cpd.Most preferred above-claimed cpd uses with form suitable and pharmaceutically acceptable salt, most preferably sodium salt.

Further, in superincumbent embodiment, preferably the coexist concentration of pressure or intermenstrual period endogenous steroid class of some dosage that can block the compound of 3 Alpha-hydroxies-pregnane-steroide to mankind GABA-A receptor acting is consistent.According to the present invention, can block 3 Alpha-hydroxies-pregnane-steroide can share to alleviate or eliminate with oral contraceptive oral contraceptive side effect to the compound of mankind GABA-A receptor acting, or alleviate or eliminate the periodically variable detrimental action of endogenous steroide.

Substantially, the present invention includes Alpha-hydroxy-5,3 β-ethynyl-3 α/β-pregnane-steroide, Alpha-hydroxy-5,3 β-ethynyl-3 α/β-androstane-steroide or 3 β-fluoro-5 α/βs-pregnane-steroide is the purposes in the pharmaceutical composition of or the disease that causes relevant for the preparation of arbitrary 3 Alpha-hydroxy-5 α/β-pregnane-steroides described in treatment or prevention invention book alone or in combination, especially one or more following diseases: epilepsy, menstrual cycle dependency epilepsy, depressed, the depression of pressure correlation, migraine, the especially pressure-dependent fatigue of fatigue, premenstrual syndrome, premenstrua anxiety is levied, the anxious state of mind that menstrual cycle is relevant, menstrual cycle relevant memory change, the memory of pressure correlation changes, Alzheimer, menstrual cycle, relevant attention was difficult to concentrate, the somnopathy that menstrual cycle is relevant and fatigue, appetite increases and is fat, the recurrence of excessive drinking and drug abuse.Preferred said medicine is to use with the form of suitable and pharmaceutically acceptable salt, most preferably sodium salt.

The invention still further relates to the following New cell line relevant with biological assessment.

As described herein compound synthetic, separate and identify and can use method well-known to those skilled in the art.

Have now found that containing the Grignard reagent of ethynyl and have high selectivity to 3 and do not need the protect/deprotection to the functional group of other ketone group with the reaction of 3,20/17 diketo steroide.Can separate and form α and beta isomer by chromatographic process and recrystallization.

The starting raw material of compound of the present invention is the corresponding steroide that 3-hydroxyl replaces and have on 20 or 17 ketone group.They can be converted into diketone separately by IBX reagent oxidation.This reaction is smooth and transform complete.While needs, also can use other suitable steroide as starting raw material.

These reactions can realize in suitable solvent, the solvent considering appropriate as methyl alcohol, ethanol, water, THF, ether, methylene dichloride or other those skilled in the art.

If possible avoid choice for use or even trace also toxic or in reaction conditions, be difficult to the reagent of removing completely, as Heavy Metal Reagent.

Relate to reagent to air or water sensitive or the reaction of product and carry out in as the inert gas environment such as nitrogen or argon gas, and have the existence of anhydrous solvent.Under the existence condition of benzophenone, ether and tetrahydrofuran (THF) are dried by Na.The syringe of having removed other gas with rare gas element is for transfering reagent and anhydrous solvent.By the chromatographic technique with suitable as TLC or GC/MS monitors the formation of product and thereby the loss of raw material determines preferred time of this reaction and temperature.

Utilize high performance liquid chromatography for preparation (HPLC) the isochromatic spectrum technology of carrying out as sharp separation silica gel chromatography or by HPLC device to carry out product purification.Any that those skilled in the art will recognize that two kinds of purification process all can use, and can be applicable to mass-produced chromatographic column by use and will test chromatographic technique for technical scale.

The confirmation of product is by using suitable analytical technology to carry out, as 1H-NMR, 13C-NMR, mass spectroscopy, IR spectrum and other those skilled in the art think the analytical procedure of the structural identification and the purity testing that are applicable to the compounds of this invention.

Accompanying drawing summary

Accompanying drawing 1 shows the reactivity of a clone of the present invention to GABA, and α 1 β 2 γ 2L, are wherein to have applied GABA;

Accompanying drawing 2 shows the reactivity of a clone of the present invention to THDOC, and α 1 β 2 γ 2 are wherein to apply 30 μ M GABA under 300nM-1 μ M THDOC exists.

The embodiment of steroide in the present invention is provided below.These embodiment are for the present invention is described, but method and composition of the present invention are not limited.Person of skill in the art will appreciate that similar reagent, solvent, condition and parameter can be used in the present invention, this depends on substrate.NMR data are by being used Bruker400MHz spectrograph to carry out record.

Base informula I embodiment:

Embodiment 1UC2016

3 β-fluorine, 5 alpha-pregnanes-20-ketone

3 α-OH5 alpha-pregnane-20-ketone (3mmol), at N ₂under condition, be dissolved in 20mL anhydrous methylene chloride.Slowly drip at ambient temperature DAST (700mg, 4.33mmol), the yellow solution that reaction generates continues to stir at ambient temperature 1h.Carry out subsequently TLC.Slowly add 5%NaHCO ₃solution (60mL) finishes solution reaction.Use methylene dichloride (3 × 20mL) to extract water, collect organic phase, use MgSO ₄be dried, and remove organic solvent under reduced pressure, carry out purifying by silica gel flash chromatography method (pentane: ethyl acetate 9: 1), obtain flaxen oily matter.Product is as follows successively: 2,3 H ₂the elimination (yield 67%) of O; The fluorination of 3-OH is accompanied by 30% configuration reversal (UC2016); Retention of configuration after the fluorination of 3-OH (3%-trace).

¹H?NMR(400MHz，CDCl ₃-d ₆)：δ4.57-4.37(dm，1H)；2.51(t，1H)；2.12(m，1H)；2.11(s，3H)；2.02-1.99(m，2H)；0.83(s，3H)；0.67(m，1H)；0.61(s，3H).

Embodiment 2UC2018

3 α-ethynyl, 5 alpha-pregnane-3 β, 20 (R)-glycol

In a flask that has an air vout, 3 α-ethynyl, 3 beta-hydroxies, are dissolved in the dichloromethane solution of 2mL and the MeOH of 5mL under 5 alpha-pregnanes-20-ketone (0.3mmol) room temperature condition, add a part of NaBH ₄(2.1 equivalent) then continues to stir 3h under this suspension room temperature.Colourless solution is dry under vacuum condition obtains white residue, then uses 20+20mL H ₂o ether extracts residue.Water 30mL methylene dichloride: 1: 1 solution of ether extracts, collects organic phase, uses MgSO ₄be dried, and remove organic solvent under vacuum condition.White solid is carried out to purifying by silica gel flash chromatography method (1: 4 ether: methylene dichloride).Quantitatively total recovery.Measured and determined by NMR, the compound at 20-C with (R)-configuration is main product (90%).

¹H?NMR(400MHz，CDCl ₃-d ₆)：δ3.72(m，1H)；2.41(s，1H)；2.02(m，1H)；1.86(2m，2H)；1.12(d，3H)；0.80(s，3H)；0.74(s，3H).

Embodiment 3UC2019

3 β-ethynyl, 5 alpha-pregnanes-20-ketone, 3 α-acetic ester

3 β-ethynyl, 3 Alpha-hydroxies, 5 alpha-pregnanes-20-ketone (0.25mmol) and pyridine (2 equivalent) are dissolved in anhydrous methylene chloride, drip acetic anhydride (4 equivalent) subsequently under condition of nitrogen gas in room temperature.Mixture is no more than 3 days 40 ℃ of continuously stirring.By adding the HCl of 50m L10%, the reaction of this dark mixture is stopped, and uses subsequently 10%NaHCO ₃the aqueous solution (2 × 30mL) is washed till pH=7.Collect organic phase, use MgSO ₄be dried and concentrate.By silica gel flash chromatography method (1: 4 ether: methylene dichloride), faint yellow residue is carried out to purifying, obtain this ester compound with 87% yield.

¹H?NMR(400MHz，CDCl ₃-d ₆)：δ2.60(s，1H)；2.51(t，1H)；2.45(m，1H)；2.11(s，3H)；2.03(s，3H)；0.82(s，3H)；0.60(s，3H).

Embodiment 4UC2024

3 β-ethynyl, 3 Alpha-hydroxies, Δ 4-pregnene-20-ketone

Progesterone (1mmol) is at N ₂under condition, be dissolved in the anhydrous THF of 25mL in room temperature (rt).Ethynyl magnesium bromide (1.1 equivalent) adds in whipping process in room temperature, and this solution is at N subsequently ₂under protection in stirred overnight at room temperature.Use saturated NH ₄cl _(aq)stop the reaction of yellow solution, and water is extracted with methylene dichloride (3 × 30mL).The organic phase reduction vaporization that collection is obtained, the yellow oil of acquisition is dissolved in methylene dichloride, washes and use MgSO by saturated salt ₄be dried.Solution evaporates minimizing under vacuum condition, and by silica gel flash chromatography method (1: 4 ether: methylene dichloride), residue is carried out to purifying, and general yield is 30%.

¹H?NMR(400MHz，CDCl ₃-d ₆)：δ5.32(s，1H)；2.51(m，2H)；2.14(m，2H)；2.11(s，3H)；1.05(s，3H)；0.64(s，3H).

Embodiment 5UC2026

3 β-ethynyl, 3 Alpha-hydroxies, 5 alpha-pregnanes-20-ketoxime

3 β-ethynyl, 3 Alpha-hydroxies, 5 alpha-pregnanes-20-ketone

3,20-5 alpha-pregnane diketone (1.580g, 5.0mmol) is at N ₂under protection, be dissolved in the anhydrous THF of 50mL in room temperature (rt).Ethynyl magnesium bromide (1.1 equivalent) is added dropwise to this solution in stirring at room temperature process, subsequently at N ₂under protection in stirred overnight at room temperature.

Use saturated NH ₄cl _(aq)stop the reaction of yellow solution, and water is extracted with methylene dichloride (3 × 30mL).The organic phase reduction vaporization that collection is obtained, the yellow oil of acquisition is dissolved in methylene dichloride, washes and use MgSO by saturated salt ₄be dried.Solution evaporates minimizing under vacuum condition, and by silica gel flash chromatography method (1: 4 ether: methylene dichloride), residue is carried out to purifying, and general yield is 72%.Can final a small amount of by product be removed by the further recrystallization in ether.

¹H?NMR(400MHz，CDCl ₃-d ₆)：δ2.51(t，1H)；2.47(s，3H)；2.14(m，1H)；2.11(s，3H)；0.81(s，1H)；0.60(s，3H).

3 α-ethynyl, 3 Alpha-hydroxies, 5 alpha-pregnanes-20-ketone

This compound is the by product obtaining in above-mentioned reaction, is separated and is obtained by silica gel flash chromatography method.General yield is 13%.

¹H?NMR(400MHz，CDCl ₃-d ₆)：δ2.52(t，1H)；2.43(s，1H)；2.11(s，3H)；0.80(s，3H)，0.60(s，3H).

3 β-ethynyl, 3 Alpha-hydroxies, 5 alpha-pregnanes-20-ketoxime

3 β-ethynyl, 3 Alpha-hydroxies, 5 alpha-pregnanes-20-ketone (10mmol) is dissolved in the 250mL round-bottomed flask that 5mL methylene dichloride and 50mL ethanol are housed in room temperature under air conditions.The NH of 4 equivalents ₂the sodium-acetate of OH hydrochloride and 4 equivalents is dissolved in 5mLH ₂o, after added again steroide solution.Add the ethanol of 20mL, mixture refluxes and spends the night.Then by reaction mixture subcooling removal of solvent under reduced pressure.White residue is used 50mL H subsequently ₂o and 50mL methylene dichloride are processed, and water extracts with 3 × 30mL methylene dichloride.Then the organic phase that collection obtains uses MgSO ₄be dried, filter and removal of solvent under reduced pressure.Final residue thing carries out purifying by silica gel flash chromatography method: use methylene dichloride: 4: 1 wash-outs of ether, general yield 95-100%.

¹H?NMR(400MHz，CDCl ₃-d ₆)：δ2.47(s，1H)；2.22(t，1H)；2.05(m，1H)；1.88(s，3H)；1.86(m，1H)；0.81(s，3H)，0.62(s，3H).

Embodiment 6UC2029

3 β-ethynyl, 3 Alpha-hydroxies, Δ 4-pregnene-20-ketoxime

3 β-ethynyl, 3 Alpha-hydroxies, Δ 4-pregnene-20-ketone (10mmol) is dissolved in the 250mL round-bottomed flask that 5mL methylene dichloride and 50mL ethanol are housed in room temperature under air conditions.The NH of 4 equivalents ₂the sodium-acetate of OH hydrochloride and 4 equivalents is dissolved in 5mLH ₂o, after added again steroide solution.Add the ethanol of 20mL, mixture refluxes and spends the night.Then by reaction mixture subcooling removal of solvent under reduced pressure.White residue is used 50mL H subsequently ₂o and 50mL methylene dichloride are processed, and water extracts with 3 × 30mL methylene dichloride.Then the organic phase that collection obtains uses MgSO ₄be dried, filter and removal of solvent under reduced pressure.Final residue thing carries out purifying by silica gel flash chromatography method: use methylene dichloride: 4: 1 wash-outs of ether, general yield 85%.

¹H?NMR(400MHz，CDCl ₃-d ₆)：δ5.32(s，1H)；2.51(s，1H)；2.19(m，2H)；2.06(m，1H)；1.88(s，3H)；2.03(s，3H)；1.05(s，3H)；0.65(s，3H).

Embodiment 7UC2030

3 α-fluorine, 5 alpha-pregnanes-20-ketoxime

3 α-fluorine, 5 alpha-pregnanes-20-ketone

3 α-OH5 alpha-pregnane-20-ketone (3mmol), at N ₂under condition, be dissolved in 20mL anhydrous methylene chloride.In-78 ℃ of slow DAST (700mg, 4.33mmol) that drip, the yellow solution that reaction generates continues to stir at ambient temperature 1h.Carry out subsequently TLC.Add carefully 5%NaHCO ₃solution (60mL) finishes solution reaction.Use methylene dichloride (3 × 20mL) to extract water, collect organic phase, use MgSO ₄be dried, and remove organic solvent under reduced pressure, carry out purifying by silica gel flash chromatography method (pentane: ethyl acetate 9: 1), obtain flaxen oily matter.Product is as follows successively: 2,3 H ₂the elimination (yield 67%) of O; The fluorination of 3-OH is accompanied by 30% configuration reversal; Retention of configuration after 3-OH fluorination (3%-trace).

¹H?NMR(400MHz，CDCl ₃-d ₆)：δ4.87-4.75(d，1H)；2.53(t，1H)；2.11(s，3H)；2.00(m，1H)；0.95(m，1H)；0.80(m，1H)；0.78(s，3H)；0.60(s，3H).

3 α-fluorine, 5 alpha-pregnanes-20-ketoxime

3 α-fluorine, 5 alpha-pregnanes-20-ketone (10mmol) is dissolved in the 250mL round-bottomed flask that 5mL methylene dichloride and 50mL ethanol are housed in room temperature under air conditions.The NH of 4 equivalents ₂the sodium-acetate of OH hydrochloride and 4 equivalents is dissolved in 5mLH ₂o, after added again steroide solution.Add the ethanol of 20mL, mixture refluxes and spends the night.Then by reaction mixture subcooling removal of solvent under reduced pressure.White residue is used 50mL H subsequently ₂o and 50mL methylene dichloride are processed, and water extracts with 3 × 30mL methylene dichloride.Then the organic phase that collection obtains uses MgSO ₄be dried, filter and removal of solvent under reduced pressure.Final residue thing carries out purifying (methylene dichloride: ether 4: 1) by silica gel flash chromatography method.Quantitative yield.

¹H?NMR(400MHz，CDCl ₃-d ₆)：δ4.90-4.78(d，1H)；2.26(t，1H)；2.10(m，1H)；1.90(s，3H)；0.98(m，1H)；0.82(s，3H)；0.65(s，3H).

Embodiment 8UC2034

3 β-fluorine, 5 alpha-pregnanes-20-ketoxime

From corresponding 3 β-fluorine, 5 alpha-pregnanes-20-ketone isomer, as starting raw material, obtains this compound by the method identical with the synthetic UC2030 of embodiment 7.

Embodiment 9UC2032

3-dimethyl, Δ 5-pregnene-3 β, 20 (R)-glycol

3-dimethyl, Δ 5-pregnene-3,20-diketone

525mg Progesterone is dissolved in 10mL dry toluene in room temperature.Stirring and N ₂under atmosphere condition, the sodium tert-butoxide solution that the 1.0M of 3.4mL (2 equivalent) is dissolved in dry toluene is added dropwise to Progesterone solution.Yellow solution continues to stir 20 minutes.The MeI of 2 equivalents is added dropwise to mixture subsequently, and at room temperature and N ₂under condition, stir and spend the night.Add 10mL water and 10mL methylene dichloride to finish mixture reaction, water is extracted with 2 × 30mL methylene dichloride.Collected organic layer, uses MgSO ₄be dried, remove solution and obtain faint yellow residue under vacuum condition, this residue carries out purifying (1: 4 ether: methylene dichloride) by silica gel flash chromatography method.Being further purified by silica gel flash chromatography method (1: 9 ethyl acetate: pentane) of target component completes.Yield: 25%.

¹H?NMR(400MHz，CDCl ₃-d ₆)：δ5.56(m，1H)；2.54(m，3H)；2.13(s，3H)；1.23(s，6H)；0.86(s，3H)；0.64(s，3H).

3-dimethyl, Δ 5-pregnene-3 β, 20 (R)-glycol

In a flask that has an air vout, the 3-dimethyl of 91mg, Δ 5-pregnene-3, are dissolved in 3.0mL methylene dichloride 3.0mL methylene dichloride and 15mLMeOH under 20-diketone room temperature condition.Add a part of NaBH ₄(2.1 equivalent) then continues to stir 3h under this suspension room temperature.Colourless solution is dry under vacuum condition obtains white residue, then uses 20+20mL H ₂o ether extracts residue.Water 30mL methylene dichloride: 1: 1 solution of ether extracts, collects organic phase, uses MgSO ₄be dried, and remove organic solvent under vacuum condition.White solid is carried out to purifying by silica gel flash chromatography method (1: 4 ether: methylene dichloride).95% yield.

¹H?NMR(400MHz，CDCl ₃-d ₆)：δ5.60(m，1H)；3.75(m，1H)；3.26(m，1H)；2.09-2.13(m，2H)；1.18(s，6H)；1.21(s，3H)；1.10(s，3H)；0.80(s，3H).

embodiment based on formula II:

Embodiment 10UC2021

3 β-ethynyl, 3 Alpha-hydroxies, androstane-17-ketone

3 β-ethynyl, 3 Alpha-hydroxies, androstane-17-ketone

3,17 androsterones (5.0mmol) are at N ₂under protection, be dissolved in the anhydrous THF of 50mL in room temperature.Ethynyl magnesium bromide (1.1 equivalent) is added dropwise to this solution in stirring at room temperature process, subsequently at N ₂under protection in stirred overnight at room temperature.

Use saturated NH ₄cl _(aq)stop the reaction of solution, and water is extracted with methylene dichloride (3 × 30mL).The organic phase reduction vaporization that collection is obtained, the yellow oil of acquisition is dissolved in methylene dichloride, washes and use MgSO by saturated salt ₄be dried.Solution evaporates minimizing under vacuum condition, and by silica gel flash chromatography method (1: 4 ether: methylene dichloride), residue is carried out to purifying, and general yield is 65%.Can final a small amount of by product be removed by the further recrystallization in ether.

¹H?NMR(400MHz，CDCl ₃-d ₆)：δ2.47(s，1H)；2.42(m，1H)；2.10-2.04(m，2H)；1.02(m，1H)；0.86(s，3H)；0.83(s，3H).

3 β-ethynyl, 3 Alpha-hydroxies, androstane-17-ketone

This compound is the by product obtaining in above-mentioned reaction, uses HPLC chromatography to separate obtain by preparation.General yield is 8%.

¹H?NMR(400MHz，CDCl ₃-d ₆)：δ2.43(s，1H)；0.86(s，3H)，0.83(s，3H).

Embodiment 11UC2025

3 β-ethynyl, 3 Alpha-hydroxies, androstane-17-ketoxime

3 β-ethynyl, 3 Alpha-hydroxies, androstane-17-ketone (10mmol) is dissolved in the 250mL round-bottomed flask that 5mL methylene dichloride and 50mL ethanol are housed in room temperature under air conditions.The NH of 4 equivalents ₂the sodium-acetate of OH hydrochloride and 4 equivalents is dissolved in 5mLH ₂o, after added again steroide solution.Add the ethanol of 20mL, mixture refluxes and spends the night.Then by reaction mixture subcooling removal of solvent under reduced pressure.White residue is used 50mL H subsequently ₂o and 50mL methylene dichloride are processed, and water extracts with 3 × 30mL methylene dichloride.Then the organic phase that collection obtains uses MgSO ₄be dried, filter and removal of solvent under reduced pressure.Final residue thing carries out purifying by silica gel flash chromatography method: use methylene dichloride: 4: 1 wash-outs of ether, general yield 95-100% (quantitatively).

¹H?NMR(400MHz，CDCl ₃-d ₆)：δ2.56-2.41(m，2H)；2.48(s，1H)；1.87(m，2H)；1.00(m，1H)；0.80(m，1H)；0.90(s，3H)，0.83(s，3H).

Embodiment 12UC2027

3 α-ethynyl, 3 beta-hydroxies, androstane-17-ketoxime

This target compound is with corresponding 3 β-ethynyl, 3 Alpha-hydroxies, androstane-17-ketone obtains by the aforesaid operations step the same with UC2025 as starting raw material, wherein 3 β-ethynyl, 3 Alpha-hydroxies, androstane-17-ketone is the by product obtaining in described synthetic UC2021 reaction.

¹H?NMR(400MHz，CDCl ₃-d ₆)：δ2.51-2.47(m，2H)；2.43(s，1H)；1.00(m，1H)；0.80(m，1H)；0.90(s，3H)，0.83(s，3H).

biological assessment

Stable transfection a kind of expressive function α 1 β 2 γ 2L GABA _athe mankind α 1 β 2 γ 2GABA of acceptor _athe HEK-293 cell of acceptor.

The clone of the present invention of stably having expressed a kind of functional human GABA-A acceptor obtains through the following steps.The Kozac sequence before initiator codon that is positioned at that subunit α 1 (308-1727NM_000806), the β 2 (214-1679NM_000813) of this GABA-A acceptor and γ 2L (290-1785NM_198904) contain introducing, these subunits are cloned on the mammalian expression vector that contains Geneticin, hygromycin B and Zocin resistance separately.In the different subunits of subclone, the production method of the plasmid that uses is at Rahman, M, and Borra, VB, Isaksson, M, Johansson, IM, Ragagnin, G,

t and Wang, MD Clin Exp Pharmacol Physiol2008, PMID18430052, announces in the electronic publication before printing.Expression vector-pcDNA3.1+/Geneticin, pcDNA3.1-/Totomycin, and pcDNA3.1+/Zeocin buys from Invitrogen.The simple description of institute's using method: the coding region of GABA-A receptor subunit α 1, β 2 and γ 2L is accompanied by the recognition sequence EcoRI of restriction enzyme (α 1 and γ 2L) and Not I+BamHI (β 2), it is to be cut off and obtained by early stage generation (Rahman et al2008) purified plasmid.The subunit coding region cutting off is connected in mammalian expression vector pcDNA3.1+/Geneticin (α 1), pcDNA3.1-/Totomycin (β 2), and pcDNA3.1+/Zeocin, and the recognition sequence linearizing of same restriction enzyme is used as coding region.The HEK-293 clone of three GABA-A receptor subunits of stably express is produced by subunit of a transfection.By using Lipofectamine and DNA-Plusreagent (all from Invitrogen) to complete transfection.After transfection, select with suitable microbiotic, and carry out cellular segregation with subunit specific antibody (β 2 and γ 2).The clone of producing is analyzed three GABA-A receptor subunits by immunocytochemistry.Cytospin is used for assembling cell.The ICC experimental design of standard has been used by the secondary antibodies of texas Red and these two kinds of fluorophore institute marks of Alexa flour488.Then cell is analyzed by immunofluorescence technique.Be cytospin for the device of experimental design, and for analyzing the fluorescent microscope of existence of desired GABA-A receptor protein.When a stable clone is established, the experiment of selecting is to show GABA (Fig. 1) and THDOC (Fig. 2) are had normally and the function of the GABA-A acceptor of effecting reaction.

Embodiment, GABA (A) receptor effect of UC-steroide

Object: GABAA receptor stimulant THDOC (THDOC) do not exist or existence condition under, by the Dynaflow in HEK-293 clone ^tMsystem is studied the effect of UC-steroide to GABA (A) function of receptors.In these experiments, the physiological condition in the preferred homo-synapse of experimental design gap is similar.

Cell cultures: stable transfection mankind α 1 β 2 γ 2LGABA _athe HEK-293 cell of receptor subtype is with 3 × 10 ⁴/ 25cm ²density be planted in cellbind culturing bottle.This transfected clone is tested for patch clamp after 3 days in plantation.In the time that this cell is tested for patch clamp, use O ₂bubble EC-solution (seeing below) rinses twice.Add subsequently the EC of about 5mL and cell is retained in incubator to 15 minutes.After 15 minutes, cell becomes loose and aspirates carefully several times with pasteur pipet in the bottom of flask.

Dynadow ^tMsystem: with the Dynadow of DF-16Pro II chip ^tMsystem is for all patch clamp experiments.There is inviscid insert in the hole of DF-16Pro II chip.The high 50 μ m of this channel width 150 μ m.The volume in hole is 280 μ L.Experimental period is take the flow relocity calculation of 26 μ L/min as 180 minutes.Injection device is as follows: the Omniflox 2mL syringe that uses internal diameter 9.65mm.When syringe pump is used for DF-16Pro II chip, flow velocity is 26 μ L/min.

Steroide and GABA: at ambient temperature, GABA, by the ultrasonic EC-solution that is dissolved in, probably needs to reach for 40 minutes the strength of solution of 10mM.All steroides are dissolved in DMSO with the concentration of 6mM.In all whole solution, DMSO concentration is 0.1%, and it comprises washing lotion (EC) and only contains the solution of GABA.Whole solution is the solution joining in chip hole.

Electrophysiology: be raised in the never filamented 1.5mm O.D. of membrane electrode, 0.86mm I.D. borosilicate glass.In the time being full of in cell solution, representational electrode has the resistance of 2-5M Ω.Intracellular solution comprises following component (in mM): 140Cs-sodium stibogluconate, 3.0NaCl, 1.2MgCl ₂, 1.0EGTA, 10HEPES, pH is transferred to 7.2 with CsOH.The solution in the extracellular (EC) using in the time recording contains: (in mM): 137NaCl, 5.0KCl, 1.0CaCl ₂, 1.2MgCl ₂, 10HEPES, 10 glucose.PH is transferred to 7.4 with NaOH.After compensation liquid junction potential, in all experiments, use stable maintenance current potential-17mV.Under physiological condition, HEK-293 has at-40mV the chlorion that has lower concentration in a rest potential and cell.In the time that acceptor is activated, flow in cell by the maintenance current potential of use-17mV with containing solution chloride ion in the cell of low-level chlorinated ion.(21 to 23 ℃) are at room temperature carried out in all experiments.The experimental design of standard is for all experiments.

Experimental design

GABA application: by using Dynaflow instrument, studying transfected HEK-293 under nearly all physiological condition all becomes possibility.Dynaflow system allow solution duration of service from 40ms so short extend in minute.On physiology, in synaptic cleft, approximately the time GABA of 2ms is released the concentration range that reaches mM.In these experiments, we use GABA ± steroide to use the time of 40ms.We find in nearly all cell, a little less than the reaction of the ratio of GABA application first GABA application for the second time.For the second time and three GABA application in reaction there is no what difference.Therefore primary GABA application always repeat twice and secondary reaction for analyzing.

Clean: GABA has good solvability and is easy to clean from acceptor in water.After using GABA separately, scavenging period is set to 1min..And steroide is insoluble in water and is difficult to and cleans from acceptor on the other hand.In our experiment, THDOC is used as gaba agonist by we.With the scavenging period of 2 minutes, the THDOC of 200nM was cleaned completely, and after cleaning completely, showing both not accumulate does not have desensitization yet.

Cultivate: we have been found to be and have understood the effect of steroide and obtain stable result, and before use GABA, steroide must first be cultivated for some time in acceptor.Study the optimal time to obtaining stabilization result and scavenging period be reduced to the shortest to different incubation times.The incubation time of 20s. is shown as the optimum time, and now scavenging period is 2min.

The steroide of conclusion: the experimental design of optimization is as follows: 20s. is cultivated, GABA ± steroide of 40ms., the cleaning of 2min..GABA application first repeats twice and has between applying for the first time and for the second time the scavenging period of a 1min..

Result: we notice if cell under disadvantageous condition, the result between them can be different.Therefore, in intracellular buffering liquid, there are some little variations and steroide done to some and supplemented experiment.

Experiment completes to confirm the effect of GABA (A) acceptor as agonist or antagonist in vitro, and it is capable of blocking has the effect (table 4) of the steroide of pregnane (pregnene) or androstane steroidal parent nucleus and 3 α/βs-ethynyl, 3 beta/alphas-oh group.Complete in vitro further experiment to confirm the effect as agonist or antagonist of GABA (A) acceptor, it can be blocked has pregnane steroidal parent nucleus and 3 α-or the effect (table 5) of 3 β-fluorine steroide.

table 4: with the chlorion variations of flux by GABA-A acceptor judge tested with 3 α/βs-ethynyl, the UC-steroide of 3 beta/alphas-oh group under the concentration conditions of 1 μ M to 200nMGABA-steroid the antagonistic action of compound THDOC (3 Alpha-hydroxy-5 alpha-pregnane-20-ketone-21-alcohol)+30 μ M GABA

table 5: judge the tested UC with 3 α/βs-fluorine with the chlorion variations of flux by GABA-A acceptor -steroide under the concentration conditions of 1 μ M to 200nM GABA-steroide THDOC (3 α- hydroxyl-5 alpha-pregnane-20-ketone-21-alcohol) antagonistic action of+30 μ M GABA

the HPLC retention time of the compounds of this invention

HPLC condition:

HPLC system (water) Column

c ₁₈3.5 μ rn4.6x75mm (water); T:45 ℃; Flow 1.0mL/min; Isocratic elution liquid condition 40: 60v/v H ₂o: MeOH.Inject volume: 100 μ L.

Solvent for elutriant is HPLC grade, and water filters by micropore device; All solvents are filtered and are first used N by the millipore filter of 0.45 μ rn ₂air-flow is removed gas.

Because this analysis is to carry out under anti-phase condition, therefore shorter corresponding wetting ability of retention time is higher.

Table 7

Object of reference:	Retention time [min]
		3 β-5 β-pregnane-20-ketone	24.4
3 β-5 alpha-pregnane-20-ketone	30.0

Table 8

The compounds of this invention:	Retention time [min]
		UC2021	7.8
UC2024	14.0
		UC2025	13.9
UC2026	16.9

The retention time that can find out the compounds of this invention from table 7 above and 8 is shorter compared with object of reference, shows that the former has the wetting ability than the latter Geng Gao.

Claims (3)

1. a compound, described compound is 3 α-ethynyl, 3 beta-hydroxies, androstane-17-ketoxime or its pharmacy acceptable salt.

2. a pharmaceutical composition, the compound of its claim 1 that comprises pharmacy effective dose and pharmaceutical acceptable carrier.

3. the purposes of the compound of claim 1 in the pharmaceutical composition for the preparation of alleviation, prevention or treatment CNS obstacle.

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