DE19817521C1 - Preparation of 4,4-dimethyl-5-alpha-cholesta-8,14,24-trien-3-beta-ol useful as meiosis regulator for the promotion of fertility - Google Patents

Abstract

Multi-stage preparation of 4,4-dimethyl-5 alpha -cholesta-8,14,24-trien-3 beta -ol is carried out starting with a 3 beta -(alkoxy)pregna-5,7-diene-20-methanol derivative. Preparation of 4,4-dimethyl-5 alpha -cholesta-8,14,24-trien-3 beta -ol of formula (I) comprises: (a) conversion of the primary OH group of a 3 beta -(alkoxy)pregna-5,7-diene-20-methanol of formula (XVII) to a leaving group followed by reaction with a copper-containing Grignard reagent and cleavage of the 3-OH protecting group to give cholesta-5,7,25-trien-3 beta -ol of formula (XX); (b) oxidizing (XX) to give cholesta-5,7,25-trien-3-one of formula (XXI); (e) dimethylating (XXI) to give 4,4-dimethyl-cholesta-5,7,25-trien-3-one of formula (XXII); (f) reducing (XXII) to give 4,4-dimethyl-cholesta-5,7,25-trien-3 beta -ol of formula (XXIII); and (g) isomerising (XXIII) to give (I). R<2> = H, CH3, alkanoyl, alkoxymethyl or trialkylsilyl. Independent claims are also included for compounds of formula (XXI)-(XXIII).

Description

The invention relates to a process for the preparation of 4,4-dimethyl-5α-cholesta 8,14,24-trien-3β-ol (1) and intermediates in the process.

Studies by Byskov et al. (Nature 1995, 374, 559) show that 4,4-dimethyl-5α-cholesta-8,14,24-trien-3β-ol, formula 1, hereinafter referred to as FF-MAS, isolated from human follicular fluid, an endogenous substance that regulates meiosis which is attributed to interesting hormonal effects. So this substance is for pharmaceutical applications, for example to promote fertility, of importance.

A synthesis of this natural product, which is used in the biosynthesis of cholesterol from lanosterol is followed by Dolle et al. (J. Am. Chem. Soc. 1989, 111, 278) described. Starting from ergosterol, FF-MAS is complex in an 18-step process Obtained synthesis sequence. Large parts of the synthesis are the chemical partial degradation of the Ergosterol side chain, the subsequent assembly of the FF-MAS side chain and the Achievement of this goal dedicated to protecting group chemistry.

Another synthesis of FF-MAS was by Schroepfer et al. starting from Dehydrocholesterol in a 13-step synthesis described (Bioorg. Med. Chem. Lett. 1997, 8, 233). In this synthesis, too, complex protection is required for building the side chain of the diene system. Four steps alone (epoxidation and rearrangement for protection; Reduction and elimination for regeneration of the diene system) are the Protection group strategy owed.

The object of this invention is a new method for the synthesis of FF-MAS. Likewise This invention relates to the new, previously unknown compounds, which in Be run through the syntheses and per se or derivatized as Starting materials can be used for the synthesis of other target molecules for example for the synthesis of FF-MAS analogs (see WO 96/00235).  

Method according to the invention:

According to Scheme 1, FF-MAS is started from a compound of the general formula 17 [in which R ² = hydrogen, a methyl, an alkanoyl (e.g. formyl, acetyl, propanoyl), a trialkylsilyl ether or an alkoxymethyl -, (e.g. methoxymethyl group) is] prepared in a seven-step sequence. The compounds mentioned here as starting material can be obtained in various ways from commercially available steroids. The starting material here is preferably obtained in a microbiological process from ergosterol, or a derivative suitable for microbiological side chain degradation. A method which uses the methoxymethyl ether of ergosterol for microbiological side chain degradation is described in WO 92/03465 A1. By using the microbiological process, complex protection of the Δ5,7-diene system can be dispensed with.

Scheme 1

The 22-hydroxy function of a compound of general formula 17 is converted into an easily substituted leaving group. The conversion into a suitable sulfonic acid ester is preferred here. For example, reaction with p-toluenesulfonic acid chloride in pyridine to give a compound of the formula 18 in which R ³ then stands for p-toluenesulfonyl (for example JJ Org. Chem. 1944, 9, 235). If the methanesulfonic acid ester is desired, a compound of formula 17 is used in which R ^{2 is} not hydrogen in order to avoid selectivity problems. However, a suitable reactive derivative of 2,4,6-trimethylbenzenesulfonic acid can also be used to convert a compound of the formula 17 into a compound of the formula 18. A compound of the general formula 18 with R ² in the meaning of methoxymethyl and R ³ in the meaning of p-toluenesulfonyl has already been described (JP 08333385 A2 961217).

This is followed by a copper-catalyzed reaction of the Grignard compound of 3-methyl-3-butenyl bromide. This nucleophilic substitution of a sulfonic acid ester in position 22 of a compound of formula 18 is carried out in the presence of copper salts as a catalyst (e.g. Li ₂ CuCl ₄ , Li ₂ CuCl ₃ , CuJ, CuBr) (e.g. Chem. Pharm. Bull. 1980, 28, 606). In the case of ester protecting groups in position 3, an excess of the Grignard reagent used cleaves this protecting group and compound 20 is obtained directly. On the other hand, when using acetal or silyl ether protecting groups for R ^2, the protecting group in position 3 is retained and compound 19 is obtained.

In this case, connection 19 at position 3 is deprotected in the next step without the Isomerize 5,7-diene, which in the case of acetal protecting groups, for example, by Sales of pyridinium para-toluenesulfonate in tert-butanol can be achieved (Synth. Commun. 1983, 13, 1021). Other reagents such as trimethysilyl bromide (Tetrahedron Lett. 1984, 25, 2515), dimethylborbromide (Tetrahedron Lett. 30, 1989, 3201) or aqueous hydrochloric acid (J. Am. Chem. Soc. 1982, 104, 1767) can be used for deprotection be used. In the case of silyl ether protecting groups, deprotection can also be done by fluoride-containing reagents, such as tetrabutylammonium fluoride (e.g. Tetrahedron 1986, 42, 4035) or hydrofluoric acid in various solvents (J. Am. Chem. Soc. 1989, 111, 2967) can be achieved. In this way, the alcohol 20 is obtained, for which one already other, complex synthesis is described (J. Chem. Soc. Perk. I 1976, 731).

The 3-hydroxy group is then migrated with a double bond oxidized from Δ5 to Δ4 to the keto function. This is done, for example, with aluminum tris (isoproylate) and cyclohexanone in the sense of Oppenauer oxidation (e.g. Helv. Chim. Acta 1939, 22, 1178). The compound 19 obtained in this way can according to Standard methods can be dimethylated and reduced.  

The reaction of a compound of formula 21 into a compound of formula 23 takes place according to processes known per se (e.g. Helv. Chim. Acta 1980, 63, 1554; J. Am. Chem. Soc. 1954, 76, 2852). For example, a compound of formula 21 in the presence of bases such as the alkali salts of lower alcohols, but preferably potassium tert-butylate, with a Alkylating agents such as dimethyl sulfate, dimethyl carbonate or methyl iodide in one Solvent or solvent mixture implemented. Lower solvents are preferred, preferred tertiary alcohols and ethers, for example methyl tertiary butyl ether or tetrahydrofuran and whose mixtures are used. The use of tertiary butanol or a mixture of tertiary butanol and tetrahydrofuran. The reaction is in one Temperature range from 0 ° C to 65 ° C carried out, but preferably in the temperature range from 15 to 50 ° C.

The reaction of a ketone of formula 23 in the corresponding 3-alcohol of formula 23 can be carried out with a variety of reducing agents. Examples include: BH ₃ complexes (e.g. with tertiary-butylamine or trimethylamine), Selectride, sodium and lithium borohydride, braked lithium aluminum hydrides (e.g. LiAl (O ^t Bu) ₃ H); are also microorganisms such. B. baker's yeast or enzymes, for example 3β-hydroxysteroid dehydrogenase, can be used.

It is known to the person skilled in the art that, depending on the reagent used, different solvents or Solvent mixtures and reaction temperatures are used. Are preferred here however, borohydrides such as sodium borohydride in suitable solvents such as lower ones Alcohols or mixtures of alcohols with aprotic solvents, for example Dichloromethane or tetrahydrofuran. The implementations are in a temperature range from -20 to 40 ° C, but preferably in the range from 0 to 30 ° C.

In addition to the reagents specified there for the reduction of the 3-ketone, the Carry out the reduction with lithium aluminum hydride under standard conditions.

Compound 23, an isomer of FF-MAS, can then be used under acidic conditions be transferred to FF-MAS 1. This can be done, for example, by heating with sulfuric acid happen in dioxane. As already described in method A, others can also Reagents or solvents are used for isomerization. This acid-catalyzed rearrangement causes the rearrangement of the Δ5,7-diene system to the Δ8,14 diene system and on the other hand the rearrangement of the isolated double bond in the Side chain from Δ25 to Δ24. An analogous side chain isomerization under catalysis of iodine has already been described (J. Org. Chem. 1987, 52, 2586).  

Examples

• a) (20S) -3ß- (methoxymethoxy) pregna-5,7-diene-20-methanol-4-methylbenzenesulfonate:
  5.0 g of 3β- (methoxymethoxy) pregna-5,7-diene-20-methanol are dissolved in 100 ml of pyridine and 4.12 g of para-toluenesulfonic acid chloride are added in portions at room temperature. Then allowed to stir for eight hours at room temperature. For working up, dilute with ethyl acetate, wash twice with 2 normal hydrochloric acid and once with saturated sodium chloride solution. The organic phase is dried over sodium sulfate and then concentrated on a rotary evaporator. After drying under high vacuum, 6.9 g of 3β- (methoxymethoxy) pregna-5,7-diene-20-methanol-4-methylbenzenesulfonate are obtained as a yellowish powder.
  ¹ H NMR (CDCl ₃ ): δ = 0.58 ppm (s, 3H, H-18); 0.93 (s, 3H, H-19); 1.04 (d, J = 7 Hz, 3H, H-21); 3.38 (s, 3H, 3-OCH ₂ OCH _3); 3.55 (m, 1H, H -3); 3.82 (m, 1H, H-22); 4.00 (m, 1H, H-22); 4.72 (s, 2H, 3-O-CH ₂ -O); 5.38 (m, 1H, H-7); 5.57 (m, 1H, H-6); 7.46 (m, 2H, 22-OSO ₂ -R); 7.80 (m, 2H, 22-OSO ₂ R)
• b) 3β- (methoxymethoxy) cholesta-5,7,25-triene:
  From 34.93 g of 3-methyl-butenyl bromide and 23.43 g of magnesium chips, a Grignard solution is prepared in 350 ml of tetrahydrofuran, which is boiled under reflux for one hour to complete the formation of the Grignard reagent. The supernatant warm solution is then transferred to a three-neck flask flushed with argon. The mixture is cooled to -30 ° C. and a dilithium trichlorocuprate (I) solution (48 ml, 0.1 M, THF) freshly prepared from two parts of lithium chloride and one part of copper (I) chloride is added. At this temperature, the mixture is stirred for 30 minutes and then a solution of 6.21 g of the compound described in step a) in 150 ml of tetrahydrofuran is added dropwise. The mixture is stirred for a further three hours at -30 ° C and then allowed to warm to room temperature overnight. For working up, the mixture is poured onto saturated ammonium chloride solution and extracted three times with ethyl acetate. The combined organic extracts are washed twice with saturated sodium chloride solution, dried over sodium sulfate and concentrated on a rotary evaporator. The residue is chromatographed on silica gel with a mixture of hexane and diethyl ether. 3.55 g of 3β- (methoxymethoxy) -cholesta-5,7,25-triene are obtained as a white solid.
  ¹ H NMR (CDCl ₃ ): δ = 0.63 ppm (s, 3H, H-18); 0.93 (s, 3H, H-19); 0.94 (d, J = 7 Hz, 3H, H-21); 1.72 (s, 3H, H-27); 3.38 (s, 3H, 3-OCH ₂ OCH _3); 3.55 (m, 1H, H -3); 4.67 (m, 2H, H-26); 4.72 (s, 2H, 3-O-CH ₂ -O); 5.38 (m, 1H, H-7); 5.57 (m, 1H, H-6)
• c) Cholesta-5,7,25-trien-3β-ol:
  2.81 g of the compound described in step b) and 16.5 g of pyridinium para toluenesulfonate are dissolved in 70 ml of tert-butanol and the reaction mixture is heated to 95 ° C. under reflux. After two hours, the mixture is allowed to cool, diluted with water and extracted with diethyl ether. The combined organic extracts are washed once with saturated sodium chloride solution, dried over sodium sulfate and then concentrated on a rotary evaporator. The residue is chromatographed on silica gel with a mixture of hexane and ethyl acetate. 1.82 g of cholesta-5,7,25-trien-3β-ol are obtained as a white solid.
  ¹ H NMR (CDCl ₃ ): δ = 0.63 ppm (s, 3H, H-18); 0.93 (s, 3H, H-19); 0.94 (d, J = 7 Hz, 3H, H-21); 1.72 (s, 3H, H-27); 3.65 (m, 1H, H-3); 4.67 (m, 2H, H-26); 5.38 (m, 1H, H-7); 5.57 (m, 1H, H-6)
• d) Cholesta-4,7,25-trien-3-one:
  1.83 g of the compound described in step c) are dissolved in 18 ml of toluene and 3.6 ml of cyclohexanone, and the mixture is first heated to a bath temperature of 140 ° C. for 10 minutes. A solution of 0.48 g of aluminum tris (isopropylate) in 3.6 ml of toluene and 1.1 ml of cyclohexanone is then added and the mixture is heated to a bath temperature of 140 ° C. for a further 30 minutes. Allow to cool, diluted with ethyl acetate, washed with 2 normal sulfuric acid, sodium hydrogen carbonate solution and with sodium chloride solution. After drying with sodium sulfate, the mixture is concentrated on a rotary evaporator. The residue is chromatographed on silica gel with a mixture of hexane and ethyl acetate. 1.33 g of Cholesta-4,7,25-trien-3-one are obtained as a light yellow solid.
  ¹ H NMR (CDCl ₃ ): δ = 0.59 ppm (s, 3H, H-18); 0.96 (d, J = 7 Hz, 3H, H-21); 1.18 (s, 3H, H-19); 1.72 (s, 3H, H-27); 4.67 (m, 2H, H-26); 5.18 (m, 1H, H-7); 5.70 (s, 1H, H-6)
• e) Preparation of 4,4-dimethylcholesta-5,7,25-trien-3-one:
  1.33 g of the compound described in step d), dissolved in 20 ml of tetrahydrofuran, are added to 186.6 g of potassium tert-butylate in one liter of tert-butanol at 45 ° C. After 10 minutes, 1.83 ml of methyl iodide are added dropwise. After a further 50 minutes, it is poured onto 1.0 liter of ice water, acidified with 4 normal hydrochloric acid and extracted with ethyl acetate. After washing the organic phase with water, sodium bicarbonate solution and saturated sodium chloride solution, it is dried over sodium sulfate, filtered, concentrated and the evaporation residue is chromatographed on silica gel with a mixture of n-hexane and ethyl acetate. 0.97 g of 4,4-dimethyl-cholesta-5,7,25-trien-3-one is obtained as a white solid.
  ¹ H NMR (CDCl ₃ ): δ = 0.62 ppm (s, 3H, H-18); 0.94 (s, 3H, H-19); 0.97 (d, J = 7 Hz, 3H, H-21); 1.30 (s, 3H, 4-CH _3); 1.32 (s, 3H, 4-CH _3); 1.73 (s, 3H, H-27); 4.67 (m, 2H, H-26); 5.53 (m, 1H, H-7); 5.82 (d, J = 6 Hz, 1H, H-6)
• f) Preparation of 4,4-dimethylcholesta-5,7,25-trien-3β-ol:
  0.97 g of the compound described in step e) is dissolved in 22 ml of tetrahydrofuran and a suspension of 56 mg of lithium aluminum hydride in 6 ml of tetrahydrofuran is added dropwise at room temperature. The reaction mixture is stirred for 30 minutes at room temperature. It is then carefully hydrolyzed with water and extracted three times with ethyl acetate. The combined organic phases are washed once with saturated sodium chloride solution, dried over sodium sulfate and concentrated on a rotary evaporator. The raw material is chromatographed on silica gel with a mixture of hexane and ethyl acetate. 0.87 g of 4,4-dimethyl-cholesta-5,7,25-trien-3β-ol is obtained as a white solid.
  ¹ H NMR (CDCl ₃ ): δ = 0.60 ppm (s, 3H, H-18); 0.96 (d, J = 7 Hz, 3H, H-21); 0.98 (s, 3H, H-19); 1.14 (s, 3H, 3-CH _3); 1.22 (s, 3H, 4-CH _3); 1.73 (s, 3H, H-27); 3.39 (m, 1H, H -3); 4.67 (m, 2H, H-26); 5.53 (m, 1H, H-7); 5.93 (d, J = 6 Hz, 1H, H-6)
• g) 4,4-Dimethyl-5α-cholesta-8,14,24-trien-3β-ol (FF-MAS):
  60 mg of the compound described in step f) are boiled in 15 ml of 1,4-dioxane with 0.5 ml of 6 normal sulfuric acid for 170 hours. After the solvent has been largely removed, the evaporation residue is distributed between sodium hydrogen carbonate solution and ethyl acetate. After washing the organic phase with sodium bicarbonate solution and saturated sodium chloride solution, drying over sodium sulfate, filtration and concentration, the evaporation residue is chromatographed on silica gel with a mixture of hexane and ethyl acetate. After concentrating the eluate and crystallizing the evaporation residue from ethanol, 30 mg of 4,4-dimethyl-5α-cholesta-8,14,24-trien-3β-ol with 90% purity are obtained. The NMR data agree with those of the literature (J. Am. Chem. Soc. 111, 1989, 278)

Claims (4)

1. Process for the preparation of 4,4-dimethyl-5α-cholesta-8,14,24-trien-3β-ol of formula 1
from 3β- (alkyloxy) pregna-5,7-diene-20-methanol of the general formula 17
wherein
R ^{2 is} hydrogen or a methyl, alkanoyl, alkoxymethyl or trialkylsilyl group,

• a) by converting the primary hydroxyl group into an easily replaceable leaving group, reaction with a copper-containing Grignard reagent and cleavage of the 3-hydroxy protecting group to give cholesta-5,7,25-trien-3β-ol of formula 20
  
• b) by oxidation to cholesta-4,7,25-trien-3-one of formula 21
  
• c) by dimethylation to give 4,4-dimethylcholesta-5,7,25-trien-3-one of formula 22
  
• d) by reduction of the 3-keto group to 4,4-dimethylcholesta-5,7,25-trien-3β-ol of the compound of formula 23
  and by subsequent isomerization to give 4,4-dimethyl-5α-cholesta-8,14,24-trien-3β-ol (1)

2. Cholesta-4,7,25-trien-3-one (compound 21)
3. 4,4-dimethylcholesta-5,7,25-trien-3-one (compound 22)
4. 4,4-dimethylcholesta-5,7,25-trien-3β-ol (compound 23)