CN1671858A - Microbiological method for the production of 7 alpha-substituted 11 alpha-hydroxysteroids - Google Patents
Microbiological method for the production of 7 alpha-substituted 11 alpha-hydroxysteroids Download PDFInfo
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Abstract
Disclosed is a new synthetic pathway for the production of precursors for the production of compounds having general formula (8,10,12). During said synthesis, compounds of general formal (4,B) are produced in a microbiological reaction. The meanings of radicals R<7>, R<10>, R<11>, R<13>, R<17> and R<17'> and group U-V-W-X-Y-Z are defined in the patent claims.
Description
Technical field
The present invention relates to prepare 7 α-substituted 11 Alpha-hydroxy steroides microbial process and can 7 α prepared therefrom, 17 α-substituted 11 beta-halogen steroides, the preparation method of latter's compound and application thereof and the pharmaceutical preparation that comprises these compounds.In addition, the present invention relates to other 7 α-substituted 11 beta-halogen steroides, can be female-1,3 by 7 α that 7 α-substituted 11 Alpha-hydroxy steroides obtain-substituted, 5 (10)-triolefins.
Background technology
Male sex hormone, particularly testosterone are used to treat symptom and be used for that the male genitals grows and male contraception male climacteric.In addition, these hormones also have and can promote for example partial synthesis metabolic activity composition of muscle growth.
The andropausal relevant endogenous male sex hormone of age that is characterized as generates minimizing, therefore it is carried out hormone replacement therapy (HRT: hormone replacement therapy).
Except that spermatogenetic minimizing, take LH-RH and be used for the decline that male contraception also causes discharging LH and testosterone levels and sexual desire, these effects are offset (D.E.Cummings etc. by taking the testosterone medicine, " Prostate-Sparing Effects of the Potent Androgen 7 α-Methyl-19-Nortestosterone:A Potential Alternative to Testosterone forAndrogen Replacement and Male Contraception; " Journal of ClinicalEndocrinology and Metabolism, Vol.83, No.12, pages 4212-4219 (1998)).
Taking male sex hormone and progestin composition carries out combination therapy and can be used for controlling male fertility (for example referring to WO 01/60376 A and institute's citing document thereof).
With regard to the use testosterone is treated, it is found that side effect is owing to the increase of stroma cell and body of gland number develops into particularly prostatomegaly (BPH: benign prostatic hyperplasia).Progesterone be subjected to form dihydroprogesterone (DHT) in the metabolism that 5 regulates, its can cause again for example generation of BPH (Cummings etc., the same; WO 99/13883 A1).Therefore, in clinical practice, treat BPH (finasteride) by suppressing 5.
Male sex hormone steroide testosterone further not only causes deleterious DHT to generate in the intravital rapid metabolism of people, also make must oral higher dosage to reach desirable testosterone curative effect level.Therefore must use other selectable adjustment modes such as intramuscular injection or big paster (largepatch) etc.
For in above-mentioned indication field, substituting testosterone, 7 Alpha-Methyls-19-nortestosterone (MeNT) has been proposed, it is on the one hand because higher thereby have a biology effect higher than testosterone with the bonding force of androgen receptor, on the other hand since 7 Alpha-Methyls sterically hindered and may hinder 5 metabolism (Cummings etc., the same, WO 99/13883 A1, WO 99/13812 A1, US-A-5,342,834).
Between the metabilic stage of testosterone, particularly in brain, liver and fatty tissue, the sub-fraction of this compound also generates estradiol by the aromatization reaction of the A of steroidal system ring.With regard to the general function of testosterone and its meta-bolites, estradiol is the essential reason that sex-specific behavior and gonad-stimulating hormone are regulated.Therefore, its function as testosterone in the function of adult man, be considered to useful (Cummings etc., the same).
Yet, find that the pharmacokinetics of testosterone is also unsatisfactory.Particularly when oral administration, testosterone is immediately drained, and the validity of the feasible medicine of preparation thus and the continuity of effect are unsatisfactory.Therefore the testosterone derivative that has synthesized other.This derivative is recorded in for example US-A-5,952, in 319, be specially 7 α of 19 nortestosterones, 11 beta-dimethyl-derivatives, i.e. 7 α, 11 beta-dimethyl-s-17 beta-hydroxy is female-4-alkene-3-ketone, 7 α, 11 beta-dimethyl-s-17 β-heptan acyloxy is female-4-alkene-3-ketone, 7 α, 11 beta-dimethyl-s-17 β-[[(2-cyclopentyl ethyl)-carbonyl]-oxygen base]-female-4-alkene-3-ketone, 7 α, 11 beta-dimethyl-s-17 β-(phenyl acetoxyl group)-female-4-alkene-3-ketone and 7 α, 11 beta-dimethyl-s-17 β-[[(trans-the 4-[normal-butyl] cyclohexyl)-carbonyl]-the oxygen base]-female-4-alkene-3-ketone.
Above-mentioned 7 α, 11 beta-dimethyl-derivatives such as MeNT have aforementioned advantages, comprise that pharmacokinetic property is improved, and for example the continuity of its validity and effect is compared with testosterone and is improved.Yet these derivatives only can be by expensive synthetic method preparation.
The microbial process of synthetic steroide is recorded in EP 0 900 283B1.This article is pointed out female-4-alkene-3,17-diketone and canrenone can use the microbial transformation that is selected from down group 11 Alpha-hydroxy analogues as correspondence: aspergillus niger (Aspergillus nigricans), unrooted rhizopus (Rhizopus arrhizus) and Pestalotia (Pestelotia) bacterial strain.Yet, in the introduction of its specification sheets also with reference to Shibahara etc., Biochim.Biophys.Acta, 202 (1970), 172-179, it may be unforeseen that microorganism 11 'alpha '-hydroxylations in this article report steroide react.
Therefore, the present invention based on problem be find insensitive to 5, pharmacokinetic property is improved and the testosterone derivative of preparation especially easily.Therefore, very significant aspect of the present invention comprises the wherein easy method of producing of initial product that finds.
The present invention based on problem solve by following aspect: as claim 1,3 and 6 described microbiologies prepare the method and 7 α as claimed in claim 11 of 7 α-substituted steroide, 17 α-substituted 11 beta-halogen steroides, as claim 23,24 and 25 described preparation 7 α, the method of 17 α-substituted 11 beta-halogen steroides, 7 α as claimed in claim 26, the application of 17 α-substituted 11 beta-halogen steroides, these 7 α that contains as claimed in claim 27, the pharmaceutical preparation of 17 α-substituted 11 beta-halogen steroides and 7 α as claimed in claim 21-substituted 11 β-halogen female-1,3,5 (10)-triolefins.Preferred embodiment being recorded in the dependent claims of theme required for protection.
Definition
Following definitions relates to the various piece of this specification sheets and claims and the accompanying drawing I of institute:
All atomic groups, group or other structural units can change in its represented intended scope in all cases independently of one another.
All alkyl, alkylidene group, thiazolinyl, alkenylene, alkynyl and alkynylene group can be straight or branched.For example, propenyl can be represented by one of following chemical structure :-CH=C-CH
3,-CH
2-C=CH
2Or-C (CH
3)=CH
2Therefore, for example methyl, ethyl, n-propyl, sec.-propyl normal-butyl, isobutyl-, the tertiary butyl, n-pentyl, isopentyl, tert-pentyl, neo-pentyl, n-hexyl, 1-methyl n-pentyl, 2-methyl n-pentyl, 3-methyl n-pentyl, 4-methyl n-pentyl, 1-ethyl normal-butyl, 2-ethyl normal-butyl etc. all belong to C
1-C
18Alkyl.
Alicyclic alkyl is cycloalkyl or the cycloalkyl for being replaced by or some alkyl, and it is directly by cycloalkyl ring or by one of the alkyl bonding.
Identical therewith, alicyclic thiazolinyl is cycloalkenyl group or the cycloalkenyl group or the cycloalkyl that replace for one or more thiazolinyl of quilt or one or more alkyl, it is directly by the cyclenes basic ring or by one of thiazolinyl or optional alkyl bonding, and wherein, this alicyclic thiazolinyl comprises a two key at least.
On the other hand, aryl can be phenyl, and 1-naphthyl or 2-naphthyl.In principle, aryl also comprises heteroaryl, particularly 2-, 3-and 4-pyridyl, 2-and 3-furyl, 2-and 3-thienyl, 2-and 3-pyrryl, 2-, 4-and 5-imidazolyl, pyridazinyl, 2-, 4-and 5-pyrimidyl and 3-and 4-pyridazinyl.
Halogen is fluorine, chlorine, bromine or iodine.
Pharmacology consistency additive salt is corresponding compound and salt inorganic or that organic acid forms, and described acid for example is spirit of salt, Hydrogen bromide, hydroiodic acid HI, acetate, citric acid, oxalic acid, tartrate and methylsulfonic acid.Particularly become ester with succsinic acid.
The subscript numeral of mark R, for example R
13Be meant its position on steroidal ring skeleton, the carbon atom on the steroidal ring skeleton is arranged in numerical order according to the IUPAC nomenclature.Subscript on the mark C is C for example
10Be meant the position of each carbon atom in the steroidal ring skeleton.
Summary of the invention
Use the new microbial method to prepare general formula 4,7 α of B-substituted 11 Alpha-hydroxy steroides:
Wherein
R
7Be the P-Q base, and
P is C
1-C
4Alkylidene group, Q are hydrogen, C
1-C
4Alkyl or C
1-C
4Fluoro-alkyl (by fluorinated alkyl partially or completely), and the P-Q base is bonded on the steroid backbone by P,
R
10Represent H, CH
3Or CF
3, and
R
13Be methyl or ethyl.
In the first method embodiment of these materials of preparation, the suitable general formula 3 of hydroxylation and oxidation in a processing step, 7 α of A-substituted steroide:
Wherein, R
7, R
10And R
13Have with general formula 4, identical implication described in the B, the microorganism that is selected from following group is used in described hydroxylation and oxidation: aspergillus (Aspergillus sp.), muscardine (Beauveria sp.), sick mould (Glomerella sp.), sundial shell (Gnomonia sp.), big monospore (Haplosporella sp.) and head mold (Rhizopus sp.).Concrete Aspergillus awamori (Aspergillus awamori) preferably, take Xi Shi aspergillus (Aspergillus fischeri), poisonous aspergillus (Aspergillus malignus), black aspergillus (Aspergillus niger), silkworm muscardine (Beauveria bassiana), apple withered rotten disease mould (Glomerella cingulata), Gnomoniacingulata, Haplosporella hesperedica and Rhizopus stolonifer (Rhizopus stolonifer), wherein that preferred especially use is Aspergillus awamori (CBS), take Xi Shi aspergillus (ATCC1020), poisonous aspergillus (IMI 16061), black aspergillus (ATCC 9142), silkworm muscardine (ATCC 7159), the apple withered rotten disease is mould, and (CBS 15226, CBS 23849, CBS 98069, ATCC 56596, ATCC 64682, and IFO 6425), Gnomonia cingulata (CBS 15226), Haplosporella hesperedica (CBS 20837) and Rhizopus stolonifer (ATCC 15441).
As selection, this microbiology is the preparation method also can implement in two steps, and wherein hydroxylating and oxidizing reaction take place in the successive reactions steps.The process of reaction can be controlled by the reaction times: by interrupting reaction, for example after one period reaction times, can obtain own hydroxylation but still unoxidized type.Therefore, two kinds of processing steps can carry out alone or mixed fermentation.
For this reason, at general formula 3 described in the first microbiology processing step that uses first microorganism, the compound of A can be by 11-position hydroxylation, this first microorganism is selected from following group: aspergillus (Aspergillus sp.), muscardine (Beauveria sp.), red mould (Gibberella sp.), sick mould (Glomerella sp.), sundial shell (Gnomonia sp.), green muscardine fungus (Metarrhizium sp.), black pore fungi (Nigrospora sp.), head mold (Rhizopus sp.) and wheel branch spore (Verticilliumsp.) wherein generate 7 α-substituted steroide that 11 alpha-positions have hydroxyl.This compound has general formula C:
Wherein, R
7, R
10And R
13Has with general formula 4 identical implication described in the B.That specifically use is poisonous aspergillus (Aspergillus malignus), honey aspergillus (Aspergillus melleus), black aspergillus (Aspergillus niger), reddish brown aspergillus (Aspergillus ochraceus), silkworm muscardine (Beauveria bassiana), rice is disliked seedling red mould (Gibberella fujikuroi), Gibberella zeae (Gibberella zeae), apple withered rotten disease mould (Glomerella cingulata), Glomerellafusaroides, Gnomonia cingulata, Metarhizium anisopliae (Metarrhizium anisopliae), nigrospora sphaerica (Nigrospora sphaerica), Rhizopus oryzae (Rhizopus oryzae), Rhizopus stolonifer (Rhizopus stolonifer) and Garden Dahlia wheel branch spore (Verticillium dahliae).In this connection, be particularly preferred for the hydroxylated poisonous aspergillus (IMI 16061) that is, honey aspergillus (CBS), black aspergillus (ATCC 11394), (NRRL 405 for reddish brown aspergillus, CBS 13252, ATCC 46504), (ATCC 7159 for the silkworm muscardine, IFO 5838, ATCC 13144, IFO 4848, CBS 11025, CBS12736), rice is disliked seedling red mould (ATCC 14842), Gibberella zeae (CBS 4474), the apple withered rotten disease is mould, and (ATCC 10534, CBS 23849, CBS 23749, ATCC 16646, ATCC 16052, IFO6459, IFO 6470, CBS 98069, IFO 7478, IFO 5257, ATCC 64682, ATCC15470), Glomerella fusaroides (ATCC 9552), Gnomonia cingulata (CBS15226), Metarhizium anisopliae (IFO 5940), nigrospora sphaerica (ATCC 12772), Rhizopus oryzae (ATCC4858, ATCC 34102, ATCC 34102), Rhizopus stolonifer (ATCC 6227b, ATCC 15441) and Garden Dahlia wheel branch spore (ATCC 11405).
Intermediate product C passes through to use the oxidized generation general formula 4 of second microorganism subsequently in the second microbiology processing step, 7 α of B-substituted 11 Alpha-hydroxy steroides, this second microorganism is selected from following group: genus bacillus (Bacillus sp.), mycobacterium (Mycobacterium sp.), Nocardia bacteria (Nocardia sp.) and pseudomonas (Pseudomonas sp.).Concrete use be bacillus lentimorbus (Bacillus lactimorbus), Bacillus sphaericus (Bacillussphaericus), Mycobacterium neoaurum, M. smegmatics (Mycobacteriumsmegmatis), coral Nocardia bacteria (Nocardia corallina), globule nocardia (Nocardia globerula), minimum Nocardia bacteria (Nocardia minima), limit Nocardia bacteria (Nocardia restrictus), scarlet Nocardia bacteria (Nocardia rubropertincta), salmon look Nocardia bacteria (Nocardia salmonicolor) and testosterone pseudomonas (Pseudomonastestosteroni).That wherein especially preferably uses is bacillus lentimorbus (ATCC 245), Bacillus sphaericus (ATCC 7055), (ATCC 9626 for Mycobacterium neoaurum, NRRLB-3683, NRRL B-3805), M. smegmatics (ATCC 14468), coral Nocardia bacteria (ATCC 31338), globule nocardia (ATCC 9356), minimum Nocardia bacteria (ATCC 19150), limit Nocardia bacteria (NCIB 10027), scarlet Nocardia bacteria (ATCC 14352), salmon look Nocardia bacteria (ATCC 19149) and testosterone pseudomonas (ATCC 11996).
In the second method embodiment, general formula 4, the compound of B can be in microbiology reaction by the 7 α-substituted steroide preparation of general formula D:
Wherein, R
7, R
10And R
13Has with general formula 4 identical implication described in the compound of B.This reaction uses the microorganism that is selected from down group to carry out: aspergillus (Aspergillus sp.), muscardine (Beauveriasp.), curved spore (Curvularia sp.), red mould (Gibberella sp.), sick mould (Glomerella sp.), sundial shell (Gnomonia sp.), big monospore (Haplosporella sp.), convolution branch mould (Helicostylum sp.), black spore (Nigrospora sp.), head mold (Rhizopus sp.) and common mould (Syncephalastrum sp.).Wherein thereby 11 alpha-positions of steroid backbone are generated general formula 4,7 α of B-substituted 11 Alpha-hydroxy steroides by hydroxylation.Be preferably onion aspergillus (Aspergillus alliaceus), Aspergillus awamori (Aspergillus awamori), take Xi Shi aspergillus (Aspergillus fischeri), poisonous aspergillus (Aspergillus malignus), honey aspergillus (Aspergillus melleus), Aspergillus nidulans (Aspergillus nidualans), black aspergillus (Aspergillus niger), reddish brown aspergillus (Aspergillus ochraceus), aspergillus versicolor (Aspergillus variecolor), silkworm muscardine (Beauveria bassiana), new long-radius elbow spore (Curvularia lunata), Gibberella zeae (Gibberella zeae), apple withered rotten disease mould (Glomerella cingulata), Glomerella fusaroides, Gnomonia cingulata, Haplosporella hesperedica, pyriform convolution branch mould (Helicostylum piriformae), nigrospora sphaerica (Nigrospora sphaerica), Rhizopus oryzae (Rhizopus oryzae) and racemose gongtou mould (Syncephalastrum racemosum), wherein especially preferably use onion aspergillus (ATCC10060), Aspergillus awamori (CBS), take Xi Shi aspergillus (ATCC 1020), poisonous aspergillus (IMI 16061), honey aspergillus (CBS), Aspergillus nidulans (ATCC 11267), (ATCC 9142 for black aspergillus, ATCC11394), (NRRL 405 for reddish brown aspergillus, ATCC 13252, ATCC 46504), aspergillus versicolor (ATCC 10067), (IFO 5838 for the silkworm muscardine, ATCC 13144, IFO 4848, CBS 11025, CBS 12736, ATCC 7159), new long-radius elbow spore (IX3), Gibberella zeae (CBS 4474), the apple withered rotten disease is mould, and (ATCC 10534, CBS 23849, CBS 23749, ATCC 16646, IFO 6459, IFO 6425, IFO 6470, ATCC 15093, ATCC 10529, IFO 5257, ATCC 56596, and ATCC 64682), Glomerella fusaroides (ATCC 9552), Gnomonia cingulata (CBS 15226), Haplosporella hesperedica (CBS 20837), pyriform convolution branch mould (ATCC 8992), nigrospora sphaerica (ATCC 12772), Rhizopus oryzae (ATCC 4858) and racemose gongtou mould (IFO 4827).
Suitable especially is to generate general formula 4, the method for 7 α of B-substituted 11 Alpha-hydroxy steroides, wherein R independently of one another
7Represent CH
3And/or R
10Represent H and/or R
13Represent CH
3
Described method is implemented in normal way.For this reason, usually at first for bacterial strain prepares bactericidal nurishing liquid, then this nutritive medium and strain cultured solution are inoculated to cultivate bacterial strain.The pre-culture that will be somebody's turn to do so preparation then adds to can be chosen wantonly in the fermentor tank that is covered by appropriate nutrition liquid.Preferably after the vegetative period of strain culture initial substance being added in the fermentor tank, is general formula 3 thus, the compound of A or the compound of general formula D, thus can carry out reaction of the present invention.Reaction obtains 7 required α-substituted 11 Alpha-hydroxy steroides with compounding substances with the ordinary method purifies and separates after finishing.
With this general formula that obtains 4, the B compound can be by synthetic other the The compounds of this invention of preparation method of the present invention by this.Particularly be general formula 8,10,12 7 α, the additive salt of 17 α-substituted 11 β-halo steroide and their pharmacology consistency, ester, acid amides generate good activeconstituents:
Wherein
U-V-W-X-Y-Z represents one of following ring structure: C
1-C
2-C
3-C
4=C
5-C
10, C
1-C
2-C
3-C
4-C
5=C
10, C
1-C
2-C
3-C
4-C
5-C
10, and in the case, the oxo base (=O) be bonded to W (=C
3) on, perhaps represent ring structure C
1=C
2-C
3=C
4-C
5=C
6, and in the case, OR
3Be bonded to W (=C
3) on,
R
3Represent H, C
1-C
4Alkyl, C
1-C
4Alkyloyl or have OR
3The C of the O atom of base
3-C
7Cyclic ethers,
R
7Be the P-Q base, and P is C
1-C
4Alkylidene group, Q are hydrogen, C
1-C
4Alkyl or C
1-C
4Fluoro-alkyl (by fluorinated alkyl partially or completely), and the P-Q base is bonded on the steroid backbone by P,
R
10Can be in α-or β-position, it represents H, CH
3Or CF
3, and be not C at X-Y-Z only
4-C
5=C
10In time, exist,
R
11Be halogen,
R
13Be methyl or ethyl,
R
17Represent H, C
1-C
18Alkyl, C
1-C
18Alicyclic alkyl, C
1-C
18Thiazolinyl, C
1-C
18Alicyclic thiazolinyl, C
1-C
18Alkynyl, C
1-C
18Alkaryl, C
1-C
8Alkylidene group nitrile or represent the P-Q base, wherein the P-Q base has above-mentioned implication,
R
17Represent H, C
1-C
18Alkyl, C
1-C
18Alicyclic alkyl, C
1-C
18Thiazolinyl, C
1-C
18Alicyclic thiazolinyl, C
1-C
18Alkynyl, C
1-C
18Alkaryl, wherein R
17' also can be bonded to 17 β-oxygen base by ketone group, and R
17' also can be in addition by one or more NR
18R
19Base or one or more SO
xR
20Replace, wherein, x=0,1 or 2, and R
18, R
19And R
20Has R in all cases independently of one another
17Implication.
These compounds can pass through general formula 4, and 7 α of B-substituted 11 Alpha-hydroxy steroides are obtained by other processing step, and it is to have the valuable activeconstituents that strong androgenic effect does not have above-mentioned side effect.These compounds are applicable to preparation medicine, particularly effective contraceptive and are used for the activeconstituents of hormone replacement therapy (HRT).
If R
17 'In addition by NR
18R
19Replace, it can be methylamino, dimethylamino, ethylamino, diethylamino, cyclohexyl amino, dicyclohexyl amino, phenyl amino, diphenyl amino, benzylamino or dibenzyl amino.
Particularly suitable 7 α with general formula 8,10,12,17 α-substituted 11 beta-halogen steroides are represented ring structure C for U-V-W-X-Y-Z wherein
1-C
2-C
3-C
4=C
5-C
10, C
1-C
2-C
3-C
4-C
5=C
10Or C
1=C
2-C
3=C
4-C
5=C
10Compound.
(U-V-W-X-Y-Z represents C to first kind of situation
1-C
2-C
3-C
4=C
5-C
10) be the steroide of general formula 10:
(U-V-W-X-Y-Z represents C to second kind of situation
1-C
2-C
3-C
4-C
5=C
10) be the steroide of general formula 12:
General formula 10 and 12 compound are male hormone compound.
(U-V-W-X-Y-Z represents C to the third situation
1=C
2-C
3=C
4-C
5=C
6) be the steroide of general formula 8:
These compounds are oestrogenic hormon (estrogen receptor binding compounds).
In above-mentioned three kinds of situations, R
3, R
7, R
10, R
11, R
13, R
17And R
17 'Have with general formula 8,10,12 in the corresponding identical implication of group.
Independently of one another, preferably, R
1Represent H and/or R
7Represent CH
3And/or R
11Represent fluorine and/or R
13Represent CH
3And/or R
17Represent H, CH
3, the C of ethynyl particularly
1-C
18Alkynyl, CH
2CN or CF
3And/or R
17 'Represent H.
7 α of general formula 8,10,12, be particularly suitable in 17 α-substituted 11 beta-halogen steroides of the present inventionly be:
17 α-ethynyl-11 β-fluoro-17 beta-hydroxies-7 Alpha-Methyl is female-4-alkene-3-ketone (general formula 10)
Female-5 (10)-alkene of 17 α-ethynyl-11 β-fluoro-17 beta-hydroxies-7 Alpha-Methyl-3-ketone (general formula 12)
17 α-ethynyl-11 β-fluoro-7 Alpha-Methyls are female-1,3,5 (10)-triolefins-3,17-isoallopregnane-3 (general formula 8)
Can adopt following method to prepare these compounds:
U-V-W-X-Y-Z represents ring structure C for preparing wherein
1-C
2-C
3-C
4=C
5-C
107 α with general formula 10,17 α-substituted 11 beta-halogen steroides use by what microbiology preparation method of the present invention obtained to have a general formula 4,7 α of B-substituted 11 Alpha-hydroxy steroides are as initial substance.
In the first step is synthetic, these are converted into corresponding 7 α-substituted 11 beta-halogen steroides 5 with these 7 α that obtain-substituted 11 Alpha-hydroxy steroides by using halo to go hydroxylation reagent to carry out nucleophilic substitution:
All compounds that are generally used for this purpose all can be used as halo and remove hydroxylation reagent, for example hydrofluoric acid, spirit of salt, Hydrogen bromide or hydroiodic acid HI, thionyl chloride or thionyl bromide, phosphorus pentachloride, phosphoryl chloride, N-chlorosuccinimide, triphenylphosphine/tetracol phenixin, HF/ pyridine or diethylamino sulfur trifluoride or be preferably nine fluorine butane sulfonic acid fluoride (nonaflyl fuoride)/1,5-diazabicylo [5.4.0] undecylene.
Then by ring skeleton C
17Selective alkylation by compound 5 preparation compounds 10 (referring to Fig. 1).Can use common alkylating reagent to be used for selective alkylation, for example Grignard compound and organometallic compound, particularly alkyl lithium compounds.For example ethynyl bromination magnesium can be used as alkylating agent by female-4-alkene-3, the 17-diketone prepares corresponding 17 α-ethynyl-17 beta-hydroxy female-4-alkene-3 ketone.
Can use and the compound of isomerization general formula 10 assorted, make Δ
4Double-bond isomerism turns to Δ
5 (10)Two keys, can be used for preparing wherein thus, U-V-W-X-Y-Z represents ring structure C
1-C
2-C
3-C
4-C
5=C
107 α with general formula 12,17 α-substituted 11 beta-halogen steroides.For protection 3-ketone group, generate cyclic ethers at 3 at first for this purpose.Then, with Δ
4Double-bond isomerism turns to Δ
5 (10)Two keys, thus 7 α generated with general formula 12,17 α-substituted 11 beta-halogen steroides, and protecting group ruptures once more.
Represent ring structure C for preparing other wherein U-V-W-X-Y-Z
1=C
2-C
3=C
4-C
5=C
107 α with structure 8,17 α-substituted 11 beta-halogen steroides, its technology is as follows:
At first, as mentioned above, go hydroxylation to use the general formula 4 that is made by microbiology hydroxylation and oxidation by carry out halogen in nucleophilic substitution reaction, 7 α of B-substituted 11 Alpha-hydroxy steroides prepare the corresponding 11 beta-halogen steroides of general formula 5.
It is female-1,3 to generate 7 α of general formula 6-substituted by the former by copper (II) salt oxidation for example, 5 (10)-triolefins:
R wherein
3, R
7, R
11And R
13Implication as mentioned above.If R
3Represent H, then these compounds can directly synthesize.If another group substitutes R
3In H, then corresponding ether or ester must generate 1,3 in oxidation, make by known method after 5 (10)-triolefin rings.
Owing to have 7 α-substituted 11 beta-halogens female-1 of general formula 6; 3; 5 (10)-triolefins with and additive salt, ester and the acid amides of pharmacology consistency be new; therefore; require synthetic 7 α with general formula 8 of conduct in the present invention, the intermediate product of 17 α-substituted 11 beta-halogen steroides is protected.
A kind of 7 α of particularly preferred general formula 6-substituted 11 beta-halogens are female-1,3, and 5 (10)-triolefins are that 11 β-fluoro-3-hydroxyl-7 Alpha-Methyl is female-1,3,5 (10)-triolefins-17-ketone.
7 α of general formula 8 of the present invention, 17 α-substituted 11 beta-halogen steroides can be female-1,3 by 7 α-substituted 11 beta-halogens of general formula 6, the preparation of 5 (10)-triolefins, its method and above-mentioned by selective oxidation ring skeleton C
17The method for preparing general formula 10 is identical.
In addition, 7 α of general formula 9-substituted 11 beta-halogen steroides also can pass through by general formula 3, the microbiology hydroxylation of A or D and the general formula 4 that oxidation makes, the material preparation of B, and described 7 α-substituted 11 beta-halogen steroides also have androgenic effect:
Wherein, R
7, R
11And R
13Implication as mentioned above.A kind of particularly preferred compound be 11 β-fluoro-17 beta-hydroxies-7 Alpha-Methyl female-4-alkene-3-ketone.The compound of general formula 9 with and additive salt, ester and the acid amides of pharmacology consistency also all have androgenic effect.
Be preparation general formula 9 compounds, with female-4-alkene-3,17-diketone 5 usefulness hydroboronss are reduced to 17 beta-hydroxies-female-4-alkene-3-ketone 9.
In addition, general formula 9 compounds can be converted into female-5 (10)-alkene of corresponding 7 α-substituted 11 beta-halogens:
Wherein, R
7, R
10, R
11And R
13Implication identical as described in the general formula 8,10,12.For this purpose, with the Δ of the compound of general formula 9
4Double-bond isomerism turns to Δ
5 (10)Two keys.Be protection 3-ketone group, generate cyclic ethers at first for this purpose in the 3-position.Then, with Δ
4Double-bond isomerism turns to Δ
5 (10)Two keys, thus 7 α-substituted 11 beta-halogen steroides generated, and protecting group ruptures once more.
At last, female-5 (10)-alkene of corresponding 7 α-substituted 11 beta-halogens can pass through Δ
4Double-bond isomerism turns to Δ
5 (10)Two keys are transformed by general formula 5 compounds.
Wherein, R
7, R
10, R
11And R
13Implication identical as described in the general formula 8,10,12.For this purpose, with the Δ of the compound of general formula 5
4Double-bond isomerism turns to Δ
5 (10)Two keys.Be protection 3-ketone group, generate cyclic ethers at first for this purpose in the 3-position.Then, with Δ
4Double-bond isomerism turns to Δ
5 (10)Two keys, thus 7 above-mentioned α-substituted 11 beta-halogen steroides generated, and protecting group ruptures once more.
If 3-or 17 β-positions have corresponding hydroxyl, all above-mentioned compounds can be by further esterification or etherificate.For example, compound 9 can be converted into corresponding 17 β-ether or 17 β-ester.A kind of preferred compound be 11 β-fluoro-17 β-(4-sulfamyl benzoyloxy)-7 Alpha-Methyls female-4-alkene-3-ketone.Basically with R
17 'Described identical group also is suitable as C
17Substituting group on oxygen-Sauerstoffatom.
Especially, 7 α of general formula 8,10,12,17 α-substituted 11 beta-halogen steroides are applicable to the preparation medicine.The present invention therefore relate to above-mentioned general formula 8,10,12 compounds in the preparation medicine application and the pharmaceutical preparation that contains at least a above-mentioned general formula 8,10,12 compounds and at least a pharmacology consistency carrier.
7 α of general formula 10,12 of the present invention, 17 α-substituted 11 beta-halogen steroides have the intensive androgenic effect and do not have above-mentioned side effect for example to stimulate the compound of prostate gland (particularly non-benign hyperplasia of prostate).These compounds are synthetic easily.It is found that, these general formulas 10 of the present invention or 12 compound not only can be used for male sex HRT, if and measure fully with the testosterone that generates in abundant reduction LH, the body and the haemoconcentration of FSH (follicle stimulating hormone), even these compounds also can be used as efficient male contraceptive pill under the situation of not taking other activeconstituentss in addition.This relies on the 11 beta-halogen steroides that the present invention suppresses LH and FSH release.LH stimulates mesenchymal cell, thereby testosterone is secreted.If the haemoconcentration of LH is kept low-level, then the release of endogenous testosterone also descends.Spermatogeny needs testosterone, and FSH stimulates spermatid.Therefore effectively spermatogeny requires enough height of FSH and LH haemoconcentration, and sufficiently high LH haemoconcentration causes the release of the necessary testosterone of spermatogeny.
Since do not use other sterilization activeconstituents only to use 7 α, 17 α-substituted 11 beta-halogen steroides are treated male contraceptive efficiently can be provided, can significantly simplify taking of the medicine that is applicable to this purpose, and the cost of using can reduce significantly thus.
7 α of the present invention, 17 α-substituted 11 beta-halogen steroides also can with the progestogen combined utilization with control male fertility.
And, 7 α of the present invention, 17 α-substituted 11 beta-halogen steroides can effectively suppress 5 and steroidal-11-hydroxylase [CYP11B (P450c11), G.Zhang, W.L.Miller, Journal of Clinical Endocrinology and Metabolism, Vol.81, pages3254-3256 (1996)], thus for example alternative is avoided prostatic stimulation, and these compounds have the pharmacokinetic property through improving.The inhibition of 11-hydroxylase reduces the deactivation of male hormone compound, and causes its secretion from human body to reduce.Therefore, the lasting of the validity of these compounds and effect makes moderate progress for known compound, particularly behind oral administration.
For above-mentioned reasons, these compounds have reduced the taxis of 5 and have obtained the ability of virtueization generation oestrogenic hormon steroide simultaneously and serum lipid and nervus centralis are had beneficial effect, are specially adapted to birth control and androgen replacement therapy.
General formula 10 of the present invention and 12 compounds are carried out the seminal vesicle test above-mentioned side effect do not occur to measure its androgenic effect and to observe it.Carry out the estrogen effect of uterine growth test detection general formula 8 compounds of the present invention.
General formula 10 of the present invention or 12 7 α, the pharmaceutical preparation that 17 α-substituted 11 beta-halogen steroides or the present invention comprise these compounds extremely is suitable for treating non-sterile (non-sterile) male patient, also is suitable for boar basically.The application of male contraception only makes the male patient temporarily sterile.After activeconstituents of the present invention or pharmaceutical preparation application end, get back to original state again, thereby the male patient recovers reproductive performance, and spermatogeny comes back to original degree.Keep temporary transient sterile situation for stable during desirable, should take this activeconstituents or preparation continuously, wherein look form of medication, this administration every day or repeat with shorter or longer gap periods.At this activeconstituents or preparation once or after repeat administration finishes, male patient's non-sterile situation is not randomly recovered at once but is only recovered lentamente, wherein the essential timed interval of this process is depended on various factors, for example parallel administration of dosage, patient's body state and other drug.
If the purpose of administration comprises contraception, 7 α, the dosage of 17 α-substituted 11 beta-halogen steroides must be set at height, thereby the haemoconcentration of LH and FSH is up to 2.5I.E./ml (I.E.: international unit) in all cases, particularly maximum 1.0 I.E./ml, and the haemoconcentration of testosterone is up to 10nmol/l, particularly maximum 3nmol/l.
If 7 α of the present invention, 17 α-substituted 11 beta-halogen steroides are used for HRT and need not to reach contraceptive effect, and it is low that its dosage is set.Thus, attempt to reach can make LH and FSH haemoconcentration separately greater than the haemoconcentration of 2.5 I.E./ml and testosterone effect level greater than 10nmol/l.
General formula 10 of the present invention or 12 7 α, 17 α-substituted 11 beta-halogen steroides are used to regulate LH, FSH and the required dosage of testosterone haemoconcentration depends on many factors, thereby must determine with the administration specific pattern.At first, this dosage depends on the kind of treatment naturally.If this compound is to be used to male contraception, then must provide the dosage that significantly increases under the HRT situation than being used for.This dosage also depends on 7 α, the kind and the bioavailability thereof of 17 α-substituted 11 beta-halogen steroides.Administering mode is also very important for dosage.Whether at last, this dosage also depends on patient's body quality or other factors that will treat, for example walk abreast and take other drug.
But these compound per os and parenteral route be ip (intraperitoneal), iv (intravenously), im (intramuscular) or intradermal administration for example.In their also implantable tissues.If effective dosage, the amount of these compounds of institute's administration can change in wide region.According to the type of the disease that will treat and preparation, the consumption of the drug compound of giving can change in wide region.In human body, every day, dosage was 0.1 to 100mg.Preferably every day, dosage was 0.1 to 10mg in the human body.The administration interval, depend on the purpose that will reach.
Applicable formulation is for example ip, iv, im or intradermal injection etc. of capsule, pill, tablet, coating tablet, creme, ointment, lotion, liquid preparation such as syrup, gel, injectable liquids.Thus, decide on all kinds, various formulations progressively or at short notice discharge the The compounds of this invention of all dosage.
For oral administration, as the pharmaceutical preparation application is capsule, pill, tablet, coated tablet and liquid preparation or other known oral dosage forms.In this case, medicine can be prepared in a certain way, makes itself or release of active ingredients and it is passed in the body at short notice perhaps to make it have the bank effect, thereby is able to for a long time, slowly raises with activeconstituents to health.Remove 7 α, outside 17 α-substituted 11 beta-halogen steroides, this dose unit can contain one or more plants pharmaceutical compatible carrier, for example regulate material, tensio-active agent, solubilizing agent, micro-capsule, particulate, particle, thinner, tackiness agent such as starch, sugar, sorbyl alcohol and the gelatin of medicine rheological property, and weighting agent such as silicic acid and talcum powder, lubricant, dyestuff, spices and other materials.
Especially, 7 α of the present invention, 17 α-substituted 11 beta-halogen steroides also can be configured to the form that is used for oral solution, and it also contains following ingredients except that active 11 beta-halogen steroides: the lipophilic surfactant of drug compatibility oil and/or drug compatibility and/or drug compatibility hydrophilic surfactant active and/or drug compatibility water mutual solubility solvent.Thus, can be with reference to WO-A-97/21440.
For making steroide reach better bioavailability, these compounds also can be mixed with cyclodextrin inclusion compound.When being used for this purpose, with the same α of these compounds-, β-or γ-Huan Hujing or derivatives thereof reaction.
Can local creme, ointment, lotion and the liquid that uses if use, their composition must make compound of the present invention to raise in body with enough amounts.In these formulations, contain that the material of for example regulating the medicine rheological property, tensio-active agent, sanitas, solubilizing agent, thinner increase the percutaneous material that sees through ability of The compounds of this invention, dyestuff, spices and as the auxiliary material of the skin-protecting agent of regulator solution (conditioner) and moisture retention liquid.Other activeconstituentss also can together be contained within this medicine with steroide of the present invention.
When being used for parenteral administration, this activeconstituents solubilized or be suspended in the thinner of physiological compatibility.As thinner, what use usually is the oil that contains or do not contain solubilizing agent, tensio-active agent, suspending agent or emulsifying agent.The example of employed oil is sweet oil, peanut oil, Oleum Gossypii semen, soybean oil, Viscotrol C and sesame oil.For the preparation injectable formulation, can use compound of the present invention solubilized or any liquid carrier of emulsive therein.These liquid contain material, tensio-active agent, sanitas, solubilizing agent, thinner and other additives of regulating viscosity usually, by these additives this solution etc. are oozed.Other activeconstituentss also can with 7 α, 17 α-substituted 11 together administrations of beta-halogen steroide.
11 beta-halogen steroides of the present invention can be for example through the reservoir type injection of skin or the form administration of implantation preparation, and it can be prepared by a definite form, makes activeconstituents slowly to discharge.Thus, can use known technology, the bank that for example can dissolve or together work with film.Implant can contain inert material, and biological example degradable polymer or synthetic siloxanes be silicon rubber for example.11 beta-halogen steroides of the present invention also for example can be contained in and be used for percutaneous dosing in the paster.
Following embodiment is used for explaining in more detail the present invention.
Embodiment
A. microbial process is synthetic
11 Alpha-hydroxies-7 Alpha-Methyl-female-4-alkene-3, and the 17-diketone (compound 4, B)
Embodiment 1
The inclined-plane bacillus culture of Gnomonia cingulata bacterial strain (CBS 15226) is inoculated in to be equipped with contains 3 weight % glucose, 1 weight % corn steep liquor, 0.2 weight %NaNO
3, 0.1 weight %KH
2PO
4, 0.2 weight %K
2HPO
4, 0.05 weight %KCl, 0.05 weight %MgSO
47H
2O and 0.002 weight %FeSO
47H
2The 1000ml of O (pH 6.0) is in autoclave in 2 liters of Erlenmeyer flasks of nutritive mediums of 30 minutes of 121 ℃ of sterilizations, in jolting 72 hours in the gyrate shaker of 165rpm under 28 ℃.After so pre-the cultivation, be inoculated in 20 liters of fermentor tanks coated with 19 liters of aseptic culture mediums identical with the final component of aforesaid pre-culture.In addition, (ethoxylation oxo alcohol is oxoalcoholethoxylate) reduce to bubble to add the synperonic of 1.0ml silicone oil and 1.0ml in addition again.In under the superpressure of 0.7bar, the 28 ℃ of temperature through 12 hours by a definite date vegetative period after, with 20 liters of/minute ventilations, 250rpm stir and add 4.0g 17 beta-hydroxies-7 Alpha-Methyl female-the 40ml DMF solution of 4-alkene-3-ketone.Continue to stir and ventilation.After 135 hours, the results culture broth is with 10 liters of methyl isopropyl Ketone extractions 12 hours, then with 5 liters of methyl isopropyl Ketone extractions 5 hours.Merge organic phase and evaporate to dryness.With hexane eccysis silicone oil.Become the silica gel chromatography purifying of gradient with ethyl acetate with hexane, separate 11 Alpha-hydroxies-7 Alpha-Methyl that obtains 1.64g (39%) female-4-alkene-3, the 17-diketone.
Embodiment 2
The inclined-plane bacillus culture of Glomerella cingulata bacterial strain (IFO 6425) is inoculated in to be equipped with contains 3 weight % glucose, 1 weight % corn steep liquor, 0.2 weight %NaNO
3, 0.1 weight %KH
2PO
4, 0.2 weight %K
2HPO
4, 0.05 weight %KCl, 0.05 weight %MgSO
47H
2O and 0.002 weight %FeSO
47H
2The 1000ml of O (pH 6.0) is in autoclave in 2 liters of Erlenmeyer flasks of nutritive mediums of 30 minutes of 121 ℃ of sterilizations, in jolting 72 hours in the gyrate shaker of 165rpm under 28 ℃.After so pre-the cultivation, be inoculated in 20 liters of fermentor tanks coated with 19 liters of aseptic culture mediums identical with the final component of aforesaid pre-culture.In addition, add the synperonic of 1.0ml silicone oil and 1.0ml in addition again to reduce foaming.In under the superpressure of 0.7bar, the 28 ℃ of temperature through 12 hours by a definite date vegetative period after, with 10 liters of/minute ventilations, 350rpm stir and add 2.0g 17 beta-hydroxies-7 Alpha-Methyl female-the 30ml DMF solution of 4-alkene-3-ketone.Continue to stir and ventilation.After 19 hours, the results culture broth is with 20 liters of methyl isopropyl Ketone extractions 1 hour, then with 20 liters of methyl isopropyl Ketone extractions 23 hours.Merge organic phase and evaporate to dryness.The residue major part is dissolved in the methyl alcohol.Filtering silicone oil.Evaporation concentration becomes the silica gel chromatography purifying of gradient with methylene dichloride with acetone, separate 11 α that obtain 1.55g (73%), and 17 beta-dihydroxyies-7 Alpha-Methyl is female-4-alkene-3-ketone.From acetone/Di Iso Propyl Ether behind the recrystallization, separate that to obtain fusing point be 163 ℃, [α]
D=-16 ° of (CHCl
3, white crystal 827mg (39%) c=0.501).
Four cryospheres (cryosphere) that will be derived from Bacillus sphaericus bacterial strain (ATCC 7055) culture are inoculated in to be equipped with and contain in 0.5 weight % glucose, 0.5 weight % bacterium 2 liters of Erlenmeyer flasks with 500ml nutritive mediums of 30 minutes of 121 ℃ of sterilizations in autoclave of yeast extract, 0.1 weight % peptone and 0.2 weight % corn steep liquor (pH 7.5), in jolting 24 hours in the gyrate shaker of 165rpm under 28 ℃.After so pre-the cultivation, each inoculates this culture broth of 10% in 42 liters of fermentor tanks that contain the 500ml aseptic culture medium identical with the final component of aforesaid pre-culture again.In the gyrate shaker of 165rpm, under 28 ℃ of temperature,, in each flask, adding 11 α of 50mg through after 4 hours by a definite date vegetative period, 17 beta-dihydroxyies-7 Alpha-Methyl is female-the 4-alkene-solution of 3-ketone in 2.5ml DMF.Continue to stir 48 hours.The culture broth that merges extracts 2 times with 2 liters methyl iso-butyl ketone (MIBK).With the organic phase that merges with dried over sodium sulfate and evaporate to dryness.Thus, obtain 630mg oily crystal residue.From acetone/Di Iso Propyl Ether behind the recrystallization, separate that to obtain 103mg (49.2%) fusing point be 189 ℃, [α]
D=+40.4 ° of (CHCl
3, yellow crystals c=0.529) (direct crystallization and without aforementioned chromatogram purification).
Embodiment 3
Half inclined-plane bacillus culture of reddish brown Aspergillus strain (CBS 13252) is inoculated in to be equipped with contains 3 weight % glucose, 1 weight % corn steep liquor, 0.2 weight %NaNO
3, 0.1 weight %KH
2PO
4, 0.2 weight %K
2HPO
4, 0.05 weight %KCl, 0.05 weight %MgSO
47H
2O and 0.002 weight %FeSO
47H
2The 500ml of O (pH 6.0) is in autoclave in 2 liters of Erlenmeyer flasks of nutritive mediums of 30 minutes of 121 ℃ of sterilizations, in jolting 72 hours in the gyrate shaker of 165rpm under 28 ℃.After so pre-the cultivation, be inoculated in 10 liters of fermentor tanks coated with 9.5 liters of aseptic culture mediums identical with the final component of aforesaid pre-culture.In addition, add the synperonic of 0.5ml silicone oil and 0.5ml in addition again to reduce foaming.In under the superpressure of 0.7bar, the 28 ℃ of temperature through 6 hours by a definite date vegetative period after, with 5 liters of/minute ventilations, 350rpm stir and add 1.0g 7 Alpha-Methyls female-4-alkene-3, the 15ml DMF solution of 17-diketone.Continue to stir and ventilation.After 22 hours, the results culture broth is also with 7 liters of methyl isopropyl Ketone extractions 2 times 4 hours.Merge organic phase and evaporate to dryness.The residue major part is dissolved in the methyl alcohol.Filtering silicone oil.Evaporation concentration becomes the silica gel chromatography of gradient to send out purifying with methylene dichloride with acetone, separate 11 Alpha-hydroxies-7 Alpha-Methyl obtain 0.78g (74%) female-4-alkene-3, the 17-diketone.From acetone/Di Iso Propyl Ether behind the recrystallization, separate that to obtain fusing point be 200 ℃, [α]
D=+52 ° of (CHCl
3, white crystal 311mg (29.6%) c=0.5905).
B. chemical preparation process
Embodiment 4:11 β-fluoro-17 beta-hydroxies-7 Alpha-Methyl is female-preparation of 4-alkene-3-ketone
A) 11 β-fluoro-7 Alpha-Methyls-female-4-alkene-3, the 17-diketone
Dropwise 11.5ml perfluorinated butane-1-sulfonic acid fluoride is added 13.08 gram 11 Alpha-hydroxies-7 Alpha-Methyl-female-4-alkene-3 under 0 ℃, 17-diketone (the microorganism synthesis method of [A part] preparation according to the present invention) is at 1 of 250ml toluene and 18.2ml, 8-diazabicylo [5,4,0] in the solution in 11 carbon-7-alkene.After 1 hour, it with the neutralization of 2M hydrochloric acid, is added in the entry, use ethyl acetate extraction 4 times, with the saturated nacl aqueous solution washing, evaporated in vacuo concentrates then.After thick product become the silica gel chromatography purifying of gradient with hexane/ethyl acetate, obtain 8.7 gram 11 β-fluoro-7 Alpha-Methyls-female-4-alkene-3, the 17-diketone.Fusing point: 101.4 ℃, [α]
D=+135.8 ° of (CHCl
3).
B) 11 β-fluoro-17 beta-hydroxies-7 Alpha-Methyl female-4-alkene-3-ketone:
0 ℃ with 8.7 gram 11 β-fluoro-7 Alpha-Methyls-female-4-alkene-3, the solution of 17-diketone in the 148ml tetrahydrofuran (THF) dropwise with 29.5ml 1M tetrahydrofuran (THF) in three tert.-butoxy lithium aluminium hydride mix and to be incorporated in 0 ℃ and to stir 5.5 hours.Then, after 0 ℃ adds dilute sulphuric acid reaction soln is added in the frozen water, with ethyl acetate extraction three times, be washed till neutrality, use dried over sodium sulfate, vacuum-evaporation is concentrated and with hexane/ethyl acetate silica gel chromatography purifying.Obtain 5.8 the gram 11 β-fluoro-17 beta-hydroxies-7 Alpha-Methyls female-4-alkene-3-ketone, fusing point is 143-144 ℃, [α]
D=+89.9 ° of (CHCl
3).
Embodiment 5:11 β-fluoro-17 β-(4-sulfamyl benzoyloxy)-7 Alpha-Methyls are female-preparation of 4-alkene-3-ketone
Under the room temperature with 500mg 11 β-fluoro-17 beta-hydroxies-7 Alpha-Methyl female-N of the 7.5ml pyridine solution of 4-alkene-3-ketone and 750mg 4-sulfamoylbenzoic acid, 800mg, the p-toluenesulphonic acids of N-dicyclohexyl carbodiimide and 125mg mixes also and stirred 8.5 hours.Then it is added in sodium hydrogen carbonate solution, with dichloromethane extraction 4 times, be washed till neutrality, use dried over sodium sulfate, vacuum-evaporation is concentrated and with methylene dichloride/acetone silica gel chromatography purifying then.11 β-fluoro-17 β-(4-sulfamyl benzoyloxy)-7 Alpha-Methyls that obtain 302mg are female-and 4-is rare-3-ketone, its fusing point is 232 ℃.[α]
D＝+100.5°(CHCl
3)。
Embodiment 6:17 α-ethynyl-11 β-fluoro-17 beta-hydroxies-7 Alpha-Methyl is female-preparation of 4-alkene-3-ketone
A) 11 β-fluoro-3-methoxyl group-7 Alpha-Methyl is female-3,5-diene-17-ketone
80 ℃ with 2 gram 11 β-fluoro-7 Alpha-Methyls female-4-alkene-3, the 20ml 2 of 17-diketone, the toluenesulphonic acids pyridine of 2-Propanal dimethyl acetal solution and 200mg stirring 6.5 hours.With the ethyl acetate dilution,, concentrate then with dried over sodium sulfate and vacuum-evaporation with sodium hydrogen carbonate solution and sodium chloride solution washing.It is female-3 to obtain 2 gram 11 β-fluoro-3-methoxyl group-7 Alpha-Methyls, the 5-diene-thick product of 17-ketone.
B) 17 α-ethynyl-11 β-fluoro-17 beta-hydroxies-7 Alpha-Methyl female-4-alkene-3-ketone
Under dropwise being incorporated in 0 ℃ with ethynyl magnesium bromide (tetrahydrofuran (THF) of the 0.5M) solution of 74.2ml is mixed, stirred 1 hour 0 ℃ of 60ml tetrahydrofuran solution with 9.17 gram Cerium II Chlorides (III).It is female-3 dropwise to add 2 gram 11 β-fluoro-3-methoxyl group-7 Alpha-Methyls then, the 40ml tetrahydrofuran solution of 5-diene-thick product of 17-ketone and 0 ℃ of following restir 3.5 hours.Pure system step for add saturated ammonium chloride solution, with its add in the water, with ethyl acetate extraction three times, with half concentrated hydrochloric acid, sodium hydrogen carbonate solution and sodium chloride solution washing, dried over sodium sulfate, vacuum-evaporation is concentrated and with the pure system of hexane/ethyl acetate silica gel chromatography.Obtain 1.15 gram fusing points and be pure 17 α-ethynyl-11 β-fluoro-17 beta-hydroxies-7 Alpha-Methyl of 218-220 ℃ female-4-alkene-3-ketone.[α]
D＝+19.2°(CHCl
3)。
The preparation of female-5 (10)-alkene of embodiment 7:17 α-ethynyl-11 β-fluoro-17 beta-hydroxies-7 Alpha-Methyl-3-ketone
A) 3, female-5 (10)-alkene-17 β-alcohol of 3-ethylenedioxy-17 α-ethynyl-11 β-fluoro-7 Alpha-Methyls
At room temperature with 700mg 17 α-ethynyl-11 β-fluoro-17 beta-hydroxies-7 Alpha-Methyl female-the p-toluenesulphonic acids hydrate of the 7ml methylene dichloride of 4-alkene-3-ketone and the ortho-formiate of 4.7ml ethylene glycol solution and 2.3ml and 30mg stirred 6.5 hours.Then it is added sodium hydrogen carbonate solution, with ethyl acetate extraction three times, be washed till neutrality, use dried over sodium sulfate, vacuum-evaporation is concentrated and with hexane/ethyl acetate silica gel chromatography purifying.Obtain 3 of 205mg, female-5 (10)-alkene-17 β-alcohol of 3-ethylenedioxy-17 α-ethynyl-11 β-fluoro-7 Alpha-Methyls.
B) female-5 (10)-alkene of 17 α-ethynyl-11 β-fluoro-17 beta-hydroxies-7 Alpha-Methyl-3-ketone
At room temperature with 3 of 205mg, female-5 (10)-alkene-17 β-alcohol of 3-ethylidene dioxy-17 α-ethynyl-11 β-fluoro-7 Alpha-Methyls in 27ml methyl alcohol and 3.6ml water solution and the oxalic acid of 361mg together stirred 24 hours.Then it is added sodium hydrogen carbonate solution, with ethyl acetate extraction three times, be washed till neutrality, use dried over sodium sulfate, vacuum-evaporation is concentrated and with hexane/ethyl acetate silica gel chromatography purifying.Obtain the 95mg fusing point and be female-5 (10)-alkene of 17 α-ethynyl-11 β-fluoro-17 beta-hydroxies-7 Alpha-Methyl-3-ketone of 112-114 ℃.
Embodiment 8:17 α-ethynyl-11 β-fluoro-7 Alpha-Methyls are female-1,3,5 (10)-triolefins-3, the preparation of 17-isoallopregnane-3
A) 11 β-fluoro-3-hydroxyl-7 Alpha-Methyl is female-1,3,5 (10)-triolefins-17-ketone
With 500mg 11 β-fluoro-7 Alpha-Methyls female-4-alkene-3, the 16.5ml acetonitrile solution of 17-diketone and 400mg cupric bromide (II) stirred 6.5 hours down at 25 ℃.Then with ethyl acetate dilution, with sodium hydrogen carbonate solution and sodium chloride solution washing, dried over sodium sulfate, vacuum-evaporation concentrates and with hexane/acetone silica gel chromatography purifying.It is female-1,3 to obtain the 280mg fusing point and be pure 11 β-fluoro-3-hydroxyl-7 Alpha-Methyl of 185-186 ℃, 5 (10)-triolefins-17-ketone.
B) 17 α-ethynyl-11 β-fluoro-7 Alpha-Methyls are female-1,3,5 (10)-triolefins-3,17-isoallopregnane-3
Under 0 ℃, the 7.5ml tetrahydrofuran (THF) suspension of 2.03 gram Cerium II Chlorides (III) are dropwise mixed with ethynyl magnesium bromide (tetrahydrofuran (THF) of the 0.5M) solution of 16.5ml and be incorporated in 0 ℃ and stirred 0.5 hour down.It is female-1,3 dropwise to add 280mg 11 β-fluoro-3-hydroxyl-7 Alpha-Methyl then, the 2.8ml tetrahydrofuran solution of 5 (10)-triolefins-17-ketone and 0 ℃ of following restir 3.5 hours.Pure system step for add saturated ammonium chloride solution, with its add in the water, with ethyl acetate extraction four times, be washed till neutrality, with dried over sodium sulfate, vacuum-evaporation is concentrated and with the pure system of hexane/ethyl acetate silica gel chromatography.
It is female-1,3 to obtain the 220mg fusing point and be 17 α-ethynyl-11 β-fluoro-7 Alpha-Methyls of 115-117 ℃, 5 (10)-triolefins-3,17-isoallopregnane-3.
Claims (27)
1, preparation general formula 4, the microbial process of 7 α of B-substituted 11 Alpha-hydroxy steroides:
Wherein, R
7Be the P-Q base, and
P is C
1-C
4Alkylidene group, Q are hydrogen, C
1-C
4Alkyl or C
1-C
4Fluoro-alkyl,
And the P-Q base is bonded on the steroid backbone by P,
R
10Can be α position or β position, and represent H, CH
3Or CF
3, and
R
13Be methyl or ethyl,
Wherein use the microorganism be selected from aspergillus, muscardine, sick mould, sundial shell, big monospore and head mold with general formula 3,7 α of A-substituted steroide hydroxylation and oxidation:
Wherein, R
7, R
10And R
13Has implication same as described above.
2, the method for claim 1 is characterized in that this microorganism is selected from following group: Aspergillus awamori, expense Xi Shi aspergillus, poisonous aspergillus, black aspergillus, silkworm muscardine, apple withered rotten disease are mould, Gnomonia cingulata, Haplosporella hesperedica and Rhizopus stolonifer.
3, preparation general formula 4, the microbial process of 7 α of B-substituted 11 Alpha-hydroxy steroides:
Wherein, R
7Be the P-Q base, and
P is C
1-C
4Alkylidene group, Q are hydrogen, C
1-C
4Alkyl or C
1-C
4Fluoro-alkyl,
And the P-Q base is bonded on the steroid backbone by P,
R
10Can be α position or β position, and represent H, CH
3Or CF
3, and
R
13Be methyl or ethyl,
Wherein, general formula 3,7 α of A-substituted steroide
R wherein
7, R
10And R
13Have implication same as described above,
It uses the first microorganism hydroxylation that is selected from aspergillus, muscardine, red mould, sick mould, sundial shell, green muscardine fungus, black pore fungi, head mold and wheel branch spore at 11 alpha-positions in the first microbial process step, generate 7 α-substituted 11 Alpha-hydroxy steroides of general formula C:
R wherein
7, R
10And R
13Have implication same as described above,
Then, 7 α-substituted 11 Alpha-hydroxy steroides with the general formula C that generated in the second microbial process step use second microbiological oxidation that is selected from genus bacillus, mycobacterium, Nocardia bacteria and pseudomonas, generate general formula 4,7 α of B-substituted steroide.
4, method as claimed in claim 3, wherein said first microorganism is selected from following group: poisonous aspergillus, honey aspergillus, black aspergillus, reddish brown aspergillus, silkworm muscardine, rice dislike that red mould, the Gibberella zeae of seedling, apple withered rotten disease are mould, Glomerella fusaroides, Gnomoniacingulata, Metarhizium anisopliae, nigrospora sphaerica, Rhizopus oryzae, Rhizopus stolonifer and a Garden Dahlia wheel branch spore.
5, as claim 3 or 4 described methods, wherein said second microorganism is selected from following group: bacillus lentimorbus, Bacillus sphaericus, Mycobacterium neoaurum, M. smegmatics, coral Nocardia bacteria, globule nocardia, minimum Nocardia bacteria, qualification Nocardia bacteria, scarlet Nocardia bacteria, salmon look Nocardia bacteria and testosterone pseudomonas.
6, preparation general formula 4, the microbial process of 7 α of B-substituted 11 Alpha-hydroxy steroides:
Wherein, R
7Be the P-Q base, and
P is C
1-C
4Alkylidene group, Q are hydrogen, C
1-C
4Alkyl or C
1-C
4Fluoro-alkyl,
And the P-Q base is bonded on the steroid backbone by P,
R
10Can be α position or β position, and represent H, CH
3Or CF
3, and
R
13Be methyl or ethyl,
Wherein use the 7 αs-substituted steroide hydroxylation of microorganism with general formula D:
Wherein, R
7, R
10And R
13Have implication same as described above, described microorganism is selected from following group: aspergillus, muscardine, curved spore, red mould, sick mould, sundial shell, big monospore, mould, the black spore of convolution branch, head mold and mould altogether.
7, method as claimed in claim 6, wherein said microorganism are selected from following group: onion aspergillus, Aspergillus awamori, expense Xi Shi aspergillus, poisonous aspergillus, honey aspergillus, Aspergillus nidulans, black aspergillus, reddish brown aspergillus, aspergillus versicolor, silkworm muscardine, new long-radius elbow spore, Gibberella zeae, apple withered rotten disease are mould, Glomerella fusaroides, Gnomonia cingulata, Haplosporellahesperedica, pyriform convolution branch are mould, nigrospora sphaerica, Rhizopus oryzae and racemose gongtou mould.
8, as the described microbial process of one of claim 1 to 7, wherein R
7Represent CH
3
9, as the described microbial process of one of claim 1 to 8, wherein R
10Represent H.
10, as the described microbial process of one of claim 1 to 9, wherein R
13Represent CH
3
11, general formula 8,10,12 7 α, and 17 α-substituted 11 beta-halogen steroides and their pharmacology consistency additive salt, ester and acid amides:
Wherein, U-V-W-X-Y-Z represents one of following ring structure: C
1-C
2-C
3-C
4=C
5-C
10, C
1-C
2-C
3-C
4-C
5=C
10, C
1-C
2-C
3-C
4-C
5-C
10, in the case, the oxo base (=O) be bonded to W (=C
3) on, perhaps represent ring structure C
1=C
2-C
3=C
4-C
5=C
6, in the case, OR
3Be bonded to W (=C
3) on.
R
3Represent H, C
1-C
4Alkyl, C
1-C
4Alkyloyl or have OR
3The C of the O atom of base
3-C
7Cyclic ethers,
R
7Be the P-Q base, and P is C
1-C
4Alkylidene group, Q are hydrogen, C
1-C
4Alkyl or C
1-C
4Fluoro-alkyl, and the P-Q base is bonded on the steroid backbone by P,
R
10Can be in α-or β-position, it represents H, CH
3Or CF
3, and be not C at X-Y-Z only
4-C
5=C
10In time, exist,
R
11Be halogen,
R
13Be methyl or ethyl,
R
17Represent H, C
1-C
18Alkyl, C
1-C
18Alicyclic alkyl, C
1-C
18Thiazolinyl, C
1-C
18Alicyclic thiazolinyl, C
1-C
18Alkynyl, C
1-C
18Alkaryl, C
1To C
8The alkylidene group nitrile, or represent the P-Q base, wherein the P-Q base has above-mentioned implication,
R
17' represent H, C
1-C
18Alkyl, C
1-C
18Alicyclic alkyl, C
1-C
18Thiazolinyl, C
1-C
18Alicyclic thiazolinyl, C
1-C
18Alkynyl or C
1-C
18Alkaryl, wherein R
17' also can be bonded to 17 β-oxygen base by ketone group, and R
17' also can add by one or more NR
18R
19Base or one or more SO
xR
20Replace, wherein, x is 0,1 or 2, and R
18, R
19And R
20Has R in all cases independently of one another
17Implication.
12,7 α as claimed in claim 11,17 α-substituted 11 beta-halogen steroides, wherein U-V-W-X-Y-Z represents ring structure C
1-C
2-C
3-C
4=C
5-C
10Or C
1-C
2-C
3-C
4-C
5=C
10Or C
1=C
2-C
3=C
4-C
5=C
10
13, as claim 11 or 12 described 7 α, 17 α-substituted 11 beta-halogen steroide, wherein R
1Represent H.
14, as described 7 α of one of claim 11 to 13,17 α-substituted 11 beta-halogen steroide, wherein R
7Represent CH
3
15, as described 7 α of one of claim 11 to 14,17 α-substituted 11 beta-halogen steroide, wherein R
11Represent fluorine.
16, as described 7 α of one of claim 11 to 15,17 α-substituted 11 beta-halogen steroide, wherein R
13Represent CH
3
17, as described 7 α of one of claim 11 to 16,17 α-substituted 11 beta-halogen steroide, wherein R
17Represent H, CH
3, C
1-C
18Alkynyl, CH
2CN or CF
3
18, as described 7 α of one of claim 11 to 17,17 α-substituted 11 beta-halogen steroide, wherein R
17It is ethynyl.
19, as described 7 α of one of claim 11 to 18,17 α-substituted 11 beta-halogen steroide, wherein R
17' represent H.
20, as described 7 α of one of claim 11 to 19,17 α-substituted 11 beta-halogen steroides, it is:
17 α-ethynyl-11 β-fluoro-17 beta-hydroxies-7 Alpha-Methyl is female-4-alkene-3-ketone,
Female-5 (10)-alkene of 17 α-ethynyl-11 β-fluoro-17 beta-hydroxies-7 Alpha-Methyl-3-ketone or
17 α-ethynyl-11 β-fluoro-7 Alpha-Methyls are female-1,3,5-triolefin-3,17-isoallopregnane-3.
21,7 α of general formula 6-substituted 11 beta-halogens are female-1,3, additive salt, ester and the acid amides of 5 (10)-triolefins and their pharmacology consistency:
R
3Represent H, C
1-C
4Alkyl, C
1-C
4Alkyloyl or have OR
3The C of the O atom of base
3-C
7Cyclic ethers,
R
7Be the P-Q base, and P is C
1-C
4Alkylidene group, Q are hydrogen, C
1-C
4Alkyl or C
1-C
4Fluoro-alkyl, and the P-Q base is bonded on the steroid backbone by P,
R
11Be halogen,
R
13Be methyl or ethyl.
22,7 α as claimed in claim 21-substituted 11 beta-halogens are female-1,3,5 (10)-triolefins, and it is that 11 β-fluoro-3-hydroxyl-7 Alpha-Methyl is female-1,3,5 (10)-triolefins-17-ketone.
23, preparation is as 7 α of the described general formula 10 of one of claim 11 to 20, the method for 17 α-substituted 11 beta-halogen steroides, and wherein U-V-W-X-Y-Z represents ring structure C
1-C
2-C
3-C
4=C
5-C
10, preparation method's step is as follows:
-use halo to remove hydroxylation reagent mutual-through type 4, nucleophilic substitution is carried out in the 11-position of 7 α of B-substituted 11 Alpha-hydroxy steroides;
-7 α-substituted 11 beta-halogen steroides that will so make and alkylating agent are optionally at the C of ring skeleton
17Atom reacts, and generates 7 α of general formula 10,17 α-substituted 11 beta-halogen steroides.
24, preparation is as 7 α of general formula 12 as described in one of claims 11 to 20, the method for 17 α-substituted 11 beta-halogen steroides, and wherein U-V-W-X-Y-Z represents ring structure C
1-C
2-C
3-C
4-C
5=C
10, its method steps is as follows:
-use halo to remove hydroxylation reagent mutual-through type 4, nucleophilic substitution is carried out in the 11-position of 7 α of B-substituted 11 Alpha-hydroxy steroides;
-7 α-substituted 11 beta-halogen steroides that will so make and alkylating agent are optionally at the C of ring skeleton
17Atom reacts, and generates 7 α of general formula 10,17 α-substituted 11 beta-halogen steroides;
7 α of-isomerization general formula 10,17 α-substituted 11 beta-halogen steroides generate the isomer of corresponding general formula 12, and wherein U-V-W-X-Y-Z represents ring structure C
1-C
2-C
3-C
4-C
5=C
10
25, preparation is as 7 α of one of claims 11 to 20 described general formula 8, the method for 17 α-substituted 11 beta-halogen steroides, and wherein U-V-W-X-Y-Z represents ring structure C
1=C
2-C
3=C
4-C
5=C
6, its method steps is as follows:
-use halo to remove hydroxylation reagent mutual-through type 4, nucleophilic substitution is carried out in the 11-position of 7 α of B-substituted 11 Alpha-hydroxy steroides;
-7 α-substituted 11 beta-halogen steroide the oxidations that will so make, it is female-1,3 to generate 7 α as claim 17 or 18 described general formulas 6-substituted, 5 (10)-triolefins;
-7 α-substituted that will so make are female-1,3, and 5 (10)-triolefins and alkylating agent are optionally at the C of ring skeleton
17Atom reacts, and generates 7 α of general formula 8,17 α-substituted 11 beta-halogen steroides.
26, as 7 α of the described general formula 8,10,12 of one of claims 11 to 20, the application of 17 α-substituted 11 beta-halogen steroides in the preparation medicine.
27, a kind of pharmaceutical preparation, its contain at least a as claim 11 to 20 it-7 α of described general formula 8,10,12,17 α-substituted 11 beta-halogen steroides and at least a pharmacology consistency carrier.
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| DE10233723.3 | 2002-07-24 | ||
| DE10233723A DE10233723A1 (en) | 2002-07-24 | 2002-07-24 | Preparation of 7 alpha-substituted 11 alpha-hydroxy-steroids, useful as precursors for new androgenic 7 alpha,17 alpha-substituted 11 beta-halo-steroids, by microbiological conversion of 7 alpha-substituted steroids |
| US40295302P | 2002-08-14 | 2002-08-14 | |
| US60/402,953 | 2002-08-14 |
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| CN101296936B (en) * | 2005-10-27 | 2011-06-22 | 大鹏药品工业株式会社 | Process for production of steroid compound |
| CN103214543A (en) * | 2012-12-25 | 2013-07-24 | 中国人民解放军海军医学研究所 | New maslinic acid derivatives, preparation method, and applications of new maslinic acid derivative in antitumor drug |
| CN104862323A (en) * | 2015-06-02 | 2015-08-26 | 中国农业科学院生物技术研究所 | Hydroxylase gene for modifying benzenediol compound |
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| CN103834712B (en) * | 2012-11-26 | 2016-03-30 | 复旦大学 | The optimization method of desogestrel intermediate nanometer liposome bio-transformation |
| PL442098A1 (en) * | 2022-08-26 | 2024-03-04 | Uniwersytet Przyrodniczy we Wrocławiu | Method of producing 11α-hydroxy-19-nortestosterone |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101296936B (en) * | 2005-10-27 | 2011-06-22 | 大鹏药品工业株式会社 | Process for production of steroid compound |
| CN103214543A (en) * | 2012-12-25 | 2013-07-24 | 中国人民解放军海军医学研究所 | New maslinic acid derivatives, preparation method, and applications of new maslinic acid derivative in antitumor drug |
| CN103214543B (en) * | 2012-12-25 | 2015-09-02 | 中国人民解放军海军医学研究所 | New Crategolic acid derivative, its preparation method and the application in antitumor drug thereof |
| CN104862323A (en) * | 2015-06-02 | 2015-08-26 | 中国农业科学院生物技术研究所 | Hydroxylase gene for modifying benzenediol compound |
| CN104862323B (en) * | 2015-06-02 | 2018-01-16 | 中国农业科学院生物技术研究所 | Modify the '-hydroxylase gene of diphenol compounds |
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| WO2004011663A2 (en) | 2004-02-05 |
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| KR20050026507A (en) | 2005-03-15 |
| EA200500224A1 (en) | 2005-08-25 |
| CN100339486C (en) | 2007-09-26 |
| IL166358A0 (en) | 2006-01-16 |
| EA200601030A1 (en) | 2006-10-27 |
| EA008147B1 (en) | 2007-04-27 |
| MX260952B (en) | 2008-10-01 |
| AU2003281677A1 (en) | 2004-02-16 |
| EA010572B1 (en) | 2008-10-30 |
| NZ549529A (en) | 2008-04-30 |
| RS20050045A (en) | 2007-06-04 |
| JP2006503813A (en) | 2006-02-02 |
| JP4417838B2 (en) | 2010-02-17 |
| PL373808A1 (en) | 2005-09-19 |
| HRP20050172A2 (en) | 2005-04-30 |
| KR101041328B1 (en) | 2011-06-14 |
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