CZ20001888A3 - Novel neuroactive steroids, process of their preparation and use - Google Patents

Abstract

In the present invention, there are disclosed novel neuroactive steroids i.e. û 3{alpha}-hydroxy-5{alpha}-pregnane-12,20-dione of the general formula VI and 3{alpha}-fluoro-5{alpha}-pregnane-12,20-dione of the general formula VII. These derivatives can be prepared from 3{beta}-hydroxy-5{alpha}-pregnane-12,20-dione of the general formula VIII by converting thereof to active intermediates that decompose with configuration inversion either to the said 3{alpha}-hydroxy derivative of the general formula VI or to 3{alpha}-fluoro derivative of the general formula VII and that are capable of modulating {gamma}-aminobutyric acid.

Description

New neuroactive stéroids, their production and use

Technical field

The present invention relates to novel steroid derivatives which modulate the activity of the neurotransmitter γ-aminobutyric acid. These agents are useful in a variety of medical applications ranging from anesthetics through hypnotics to antiepileptics. Their effect is explained by their binding to a specific receptor site allosterically enhancing the effect of the neurotransmitter on the conductivity of the chloride channel. The problem is still alive, many pharmaceutical companies are developing new preparations to replace existing ones. The main problem that individual companies solve in different ways is the low stability of the natural neurosteroid - 3α-hydroxy-5α-pregnan-20-one of formula I (r).

whose half-life of inactivation in the body is 16 minutes. Compound I is inactivated by oxidation to the diketone Π and subsequent reduction to the inactive 3β-hydroxy-5α-pregnan-20-one of formula Π.

For example, some manufacturers increase the stability of a compound of Formula I by introducing a methyl group at the 3-position to form a compound of Formula IV:

• · • 4 4 • • • • •

Object of the invention

The best known neuroactive substance close to 3α-hydroxy-5α-pregnan-20-one is its 11-oxo derivative of formula V

ΓΠΓΗ

H which under the name alphaxolone (alfaxolone) was used as an anesthetic. This substance also suffers from the aforementioned deficiency - rapid metabolism in the body. Moreover, its availability is made difficult by the use of economically demanding microbial hydroxylation. In an attempt to improve, we prepared two compounds of formulas VI and VII, which differ from the alpha group by the position of the oxo group: it was moved to position 12. One of these new compounds (VII) is characterized by a much stronger 3-substituent bond. -hydroxyl group replaced by a fluorine atom.

The introduction of the 12-oxo group into the steroid molecule is usually effected by a suitable choice of the starting material - hecogenin acetate of formula Vin:

as well as subsequent hydrogenation and hydrolysis to 3β-hydroxy-5α-pregnane-12,20-dione of formula X.

9

The 3-position hydroxyl group configuration is generally accomplished in various ways, such as by solvolysis of the appropriate alkyl or arylsulfonate, or by the modern Mitsunobu reaction, where the corresponding alcohol is exposed to formic acid in the presence of diethyl azodicarboxylate and triphenylphosphine. position 3 is hydrolyzed to the corresponding alcohol. We have thus prepared the hitherto unwritten 3α-hydroxy-5α-pregnane 12,20-dione of formula VI.

Solvolysis of the 3β-alkyl or arylsulfonate can also be carried out by other nucleophiles, such as chloride or bromide ions. In this case, 3α-chloro- or 3α-bromo derivatives are formed, but these are worthless in terms of neuroactive activity. Using our earlier knowledge (Kasal A., Slavikova B., Chodounska H., Kohout L., Krištofíková Z., Uhlířová L. and Kršiak M .: New Neuroactive Steroids, Methods of their Preparation and Use. PV4214 / 1999) appropriate solvolysis by treatment with fluoride reagents to give 3oc-fluoro-5α-pregnane-12,20-dione of formula VU

(VII),

H, which surprisingly exhibited γ-aminobutyric acid modulating activity.

Biological activity of the compounds according to the invention

Synaptic membranes from the forebrain of a 3-month old male Wistar rat were incubated with ( ³ H) muscimol (5 nM) or ( ³⁵ S) TBPS (2 nM). Non-specific binding was determined using 1 mM γ-aminobutyric acid (40% of total ( ³ H) muscimol binding) or 200 μΜ. · 9 9

9

9 9 9

99 picrotoxin (33% of total ( ³⁵ S) TBPS binding). The steroids were dissolved in concentrated dimethylsulfoxide, diluted with redistilled water and sonicated (final solvent concentration was 3%).

Results of stimulation of muscimol binding (EC (50)):

JO nM for compound VI, 114 nM for compound VII.

Results of TBPS binding inhibition (binding at 100 nM steroid concentration):

53.1% for compound VI, 64.3% for compound VII.

Examples

Example 1: Preparation of γ-aminobutyric acid receptors.

The forebrain was homogenized in 10 volumes of ice-cold 0.32 M sucrose. The homogenate was centrifuged at 10,000g for 10 min and the resulting supernatant was centrifuged again at 20,000g (20 min). The pellets were resuspended in 10 volumes of ice-redistilled water and centrifuged at 8,000g (20 min). The resulting supernatant was centrifuged at 24,000g (20 min). To completely remove γ-aminobutyric acid, the pellets were frozen for 40 min, after thawing the synaptic membranes were resuspended in 10 volumes of buffer (pH = 7.4; 20 mM KH ₂ PO ₄ + 200 mM KCl) and centrifuged again at 24,000 g (20 min). This operation was repeated three times.

Example 2: 3α-Hydroxy-5α-pregnane-12,20-dione (VI)

From a solution of 3β-hydroxy-5α-pregnane-12,20-dione (120 mg, 0.38 mmol, ref. Callow J; J. Chem. Soc. 1956, 4744-4746), triphenylphosphine (300 mg, 1.14). mmol) in a mixture of toluene (10 mL) and benzene (20 mL) was rapidly distilled off an azeotropic mixture (10 mL). The mixture was stirred with a magnetic stirrer in an ice bath, and after 3 minutes diethyl azodicarboxylate (10 drops, 1.43 mmol) was added with stirring. After an additional 8 min, anhydrous formic acid (3 drops, 1.43 mmol) was added dropwise to the yellow solution. After 3 h, the mixture was shaken with potassium bicarbonate solution (7%, 5 mL) and water, dried over anhydrous sodium sulfate and evaporated to dryness in vacuo. The residue was applied to a thin layer of silica gel (3 plates, 200 x 200 x 1 mm) which was developed with a solution of benzene in ether (50%). Detection of the product was performed by inspection in UV light (234 nm) after _% morine spray (0.02% in methanol). Elution of the lipophilic band with ether gave 106 mg (78%) of the 3-formate. Mp 195-197 ° C (acetone); [α] D + 178 ° (chloroform, c = 0.95). 1 H NMR Spectrum: 0.89 s, 3H (H-19); 0.95 with 3H (H-18); 2.26 s, 3H (H-21); 3.321.1H (t = 9.0 Hz, H11); 5.18 m, 1H (W = 16Hz, H-3); 8.05 s, 1H (HCOO). For C22H32O4 (360.5) calculated: 73.30% C, 8.95% H; Found: C, 73.23; H, 9.0.

The formate (96 mg, 0.26 mmol) and potassium bicarbonate (50 mg, 0.36 mmol) were heated to boiling in a solution of methanol (9 mL) and water (1 mL). After 10 min, the mixture was concentrated in vacuo to a quarter volume and the product was precipitated with water. Crystallization of the precipitate from a mixture of acetone and heptane gave compound VI (71 mg, 80%), mp 199-201 ° C, [α] _D + 170 ° (c 0.9). 1 H NMR Spectrum: 0.87 s, 3H (H-19); 0.95 with 3H (H-18); 2.27 s, 3H (H-21); 2,461.1H (J = 8Hz, H-17); 3.311, 1H (J = 9.0 Hz, H11); 4.07 m, 1 H (W = 16 Hz, H-3). For C 21 H 32 O 3 (332.5) calculated. % C, 75.86;% H, 9.70; Found:% C, 75.86;% H, 9.65.

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Example 3. 3α-Fluoro-5α-pregnane-12,20-dione (VH)

Diethylaminosulfur trifluoride (96.7 mg, 0.60 mmol) was added to a solution of 3B-hydroxy-5α-pregnane-12,20-dione (100 mg, 0.3 mmol) in dichloromethane (1 mL) at 20 ° C . After two hours, the reaction mixture was poured into a 5% potassium bicarbonate solution, the product was extracted with dichloromethane, washed with water, and dried over anhydrous sodium sulfate. The solvent evaporation residue was chromatographed on two preparative silica gel plates, eluting with petroleum ether-ether (7: 3) to give 3α-fluoro-5α-pregnane-12,20-dione (36 mg, 36%). Mp 146-149 ° C (acetone-heptane), [α] _D + 166 ° (c 1.08). @ 1 H-Spectrum: - 12702 (OO), - 992 (CF) @ 4 H NMR: 0, -8-7s, 3H (HH9); 0.95 s, 3H (H-18); 2.27 s, 3H (H-21), 2.48 t, 1H (J = 8 Hz, H-17); 3.321, 1H (J = 9Hz, H-11); 4.82 dm, 1H (J (F, H-3) = 49Hz, H-3). For C ₂₁ H ₃₁ FO ₂ (334.5) calculated: 75.41% C, 9.34% H, 5.68% F; Found:% C, 75.29;% H, 9.36;% F, 5.49.

Example 4. 3α-Fluoro-5α-pregnane-12,20-dione (VIT)

To a solution of 3B-hydroxy-5α-pregnane-12,20-dione (100 mg, 0.3 mmol) in pyridine (1 mL) was added 4-toluenesulfonyl chloride (100 mg, 0.52 mmol) at 20 ° C. After 24 hours, the reaction mixture was poured into water, the product was extracted with chloroform, washed with water, dilute hydrochloric acid (5%), water, potassium bicarbonate solution (5%) and water. After drying over anhydrous sodium sulfate, the solvent was distilled off. A solution of tetrabutylammonium fluoride in tetrahydrofuran (1M, 1.3 mL) was added to the residue and the reaction mixture was left in the thermostat at 50 ° C for 4 days. The reaction mixture was poured into water, the product was extracted with ether, the organic layer was washed with water, dried over anhydrous sodium sulfate, and the solvent was evaporated. The residue was chromatographed on 4 preparative silica gel plates (petroleum ether: ether, 9: 1) to give 28 mg (28%) of 3α-fluoro-5α-pregnane-12,20-dione. M.p. 147-149 ° C (acetone-heptane). The sample was identical to the product prepared in the previous example.

Industrial applicability

Said process is industrially applicable and provides a product useful in medicine. The use of the indirect pathway - solvolysy mesylate according to Example 4 - is preferable in the preparation of larger amounts of the fluoro derivative. The purity of the products for medical use should be monitored by thin layer chromatography and nuclear magnetic resonance, where the samples no longer contain aromatically coupled proton signals.

Claims (7)

PATENT REQUIREMENTS wherein R represents hydroxyl or fluorine, a process for their preparation and their use as neuroactive substances modulating the action of γ-aminobutyric acid. 2. A process according to claim 1, wherein the compound of formula (X) is treated with an alkyl or arylsulfonyl chloride and the fused alkyl or arylsulfonate is solvolysed in a solution of aqueous dimethylformamide or dimethylsulfoxide at temperatures between 75 and 150 ° C to form 3ahydroxy-5a. -pregnan-12,20-dione of formula VI ' (VI) A process according to claim 2 wherein the solvolysis is carried out in the presence of sodium or potassium nitrite. 4. The process of claim 2 wherein the compound of formula X is inverted by treatment with a formic acid solution in the presence of dialkyl azodicarboxylate and triphenylphosphine, and the resulting 3α-formyloxy-5α-pregnane-12,20-dione is alkalinized to 3ahydroxy- 5α-pregnan-12,20-dione of formula VI above. AND: • · 5. A process according to claim 1, wherein the compound of formula X is treated with an alkyl or arylsulfonyl chloride and the resulting alkyl or arylsulfonate is solvolysed with an anhydrous solution of an inorganic or organic fluoride in a suitable solvent at temperatures between 25 and 100 ° C. 3α-Fluoro-5α-pregnane-12,20-dione of formula VII 6. A process according to claim 1, wherein the compound of formula X is treated with a fluorinating agent to replace the 33-hydroxyl group with a fluorine atom to form the 3α-fluoro-5α-pregnane-12,20-dione of the above formula. Vil. The method of claim 6, wherein the fluorinating agent is diethylaminosulfur trifluoride (DAST).