DE3429590A1 - DIASTEREOISOMERS OF GLYCYRRHETINSAE DERIVATIVES - Google Patents

Description

ΐ /

Patent attorneys Dipl.-Ing-. H. Weicr-mann, -TDipl.-Phys. Dr. K. Fincke

Dipl.-Ing. F. A. Weickmann, Dipl.-Chem. B. Huber Dr.-Ing. H. Liska, Dipl.-Phys. Dr. J. Prechtel

342959Q

8000 MUNICH 86 POST BOX 860820 ._

MöHLSTRASSE «1 0.

TELEPHONE (089) 98 03 51 TELEX 5 ₂ 2621

TELEGRAM PATENTWEICKMANN MÖNCHEN

H / By / Va

Biorex Laboratories Limited,
Biorex House, Canonbury Villas,
London. N. 1.2HB., England

Diastereoxsomers of glycyrrhetinic acid derivatives

description

The present invention relates to new and pharmaceutically useful diastereoisomers of certain derivatives of glycyrrhetinic acid.

GB-PS 1,387,499 describes and claims glycyrrhetinic acid derivatives of the general formula

COOR

COOR

V- /

(D

R is a hydrogen atom or an alkyl radical and R ^{1 is} also a hydrogen atom or an alkyl radical and the salts of these compounds in which R and / or R ^{1 is} a hydrogen atom. This application also describes that the compounds in question have valuable pharmaceutical properties and, in particular, have a good anti-inflammatory effect.

It was then discovered that various derivatives of glycyrrhetinic acid, including those with the general formula (I) are very suitable for the treatment of viral infections and diseases, arising from virus infections.

The compounds of the general formula (I) which are derived from cyclohexane-1,1-dicarboxylic acid exist from a mixture of diastereoisomers because of the chirality of the ring carbon atoms Cl and C2 of Cyclohexane-1,2-dicarboxylic acid, which are bonded in the C3 position of glycyrrethinic acid.

As a result of further research, it was found that the antiviral activity of one diastereoisomer is far greater than that of the others. Therefore it was found by way of examples in a series of experiments that a diastereoisomer of the disodium salt of mono- (18ß-glycyrreth-3-yl) - cis cyclohexane-1,2-dicarboxylic acid, namely 3ß- ( cis -2S- carboxycyclohexane-IS-carbonyloxy) -ll-oxo-iess-olean-12-en-30-acid disodium salt, has antiviral activity against HSV-2 strain HG52 that is about 50 times greater than the antiviral activity of the other diastereoisomer, namely 3ß- ( cis -2R-carboxycyclohexane-IR-carboxylic acid) -11-oxo-18ß-12-en-30-carboxylic acid disodium salt.

Therefore, according to the present invention, diastereoisomers of the general formula:

COOR

COOR ₁

(II)

made available, wherein

-COOR and -COOR ₂ , which can mean the same or something different, can be free carboxylic acid groups or carboxylic acid groups in salt or ester form.

If -COOR ₁ and -COOR _{2 are} esterified carboxylic acid groups, R ₁ and R _{0 are} preferably aliphatic hydrocarbon radicals with up to 6 carbon atoms, which can be straight-chain or branched, saturated or unsaturated and substituted or unsubstituted.

For the preparation of the new compounds according to the present invention of the general formula (II), the 18- or 18β-glycyrrhetinic acid or an ester thereof can be used, which reacts either with the commercially available cis -cyclohexane-1,2-dicarboxylic acid anhydride to give a mixture of the diastereoisomers which are separated in known manner, e.g. B. by fractional crystallization or with a suitable isomer of cyclohexane-1,2-dicarboxylic anhydride.

If the diastereoisomers obtained contain free carboxylic acid groups, they can be brought into salt form are or are esterified in a known manner. The esterification is preferably carried out by reaction with a suitable diazo compound, e.g. B. diazomethane, if it is desired to obtain the methyl ester or a suitable alcohol and sulfuric acid.

The present invention also provides pharmaceutical compositions containing at least one of the new compounds contained in a mixture with a solid or liquid pharmaceutical diluent or Porters available.

The following examples are presented to the present invention further explain:

example 1

3ß- (cis- ^ S-carboxycyclohexane-IS-carbonyloxy) -11-oxo-18ß-olean-12-en-30-acid.

a) 100 g of the sodium salt of mono- (glycyrrhet-3-yl) - cis -cyclohexane-1,2-dicarboxylic acid (obtained in the usual way by reacting 18ß-glycyrrhetinic acid with cis -cyclohexane-1,2-dicarboxylic anhydride, followed by neutralization with sodium hydroxide) were dissolved with heating in 600 ml acetone / water (1/1 v / v) and the solution was filtered. Acetone was added to the hot filter until a faint cloudiness persisted. 200 ml of acetone were required for this. The solution was allowed to cool to ambient temperature and the resulting solid was filtered off, washed with acetone / water (85/15 v / v) and then washed with acetone and dried in vacuo at 6O ⁰ C. The yield was 40 g.

A high pressure liquid chromatogram made on a 25 cm χ 4 mm Spherisorb ODS 10 μ column using the solvent system methanol / 0.1 M phosphate buffer (pH 7) (75/25 v / v) showed the solid contained 95% of the slower running isomer.

The solid was dissolved with heating in 300ml acetone / water (1/1 v / v) and acetone became to added to the hot solution until a weak one

Turbidity persisted, 90 ml of acetone were required for this. The solution was allowed to cool to ambient temperature and the solid was filtered off, washed with acetone / water (85/15 v / v), then washed with acetone and dried to constant weight in vacuo at 60 ⁰ C. The yield was 22.6 g.

A high pressure liquid chromatogram made as shown above showed less than 1% of the faster moving isomer. A thin layer chromatogram using n-Butanol / 0.88 ammonia (5/1 v / v) showed no impurities.

An infrared absorption spectrum (Nujöl) showed larger bands at V _mv = 1710 (ester), 1650

Iu el X *

(Enone), 1560 (broad, CO ₃ Na) cm ". The material had a melting point of about 36O ⁰ C and an optical rotation of / ° {J '= + 118 ° + 2 ° (1% in methanol / 0, 2% sodium carbonate solution).

The corresponding free acid had a melting point of 295 to 297 ^° C. (with decomposition). The infrared absorption spectrum (Nujol) had larger bands at V _max = 1740 to 1700 (acids and esters), 1660 (enone) cm ".

b) 3g of the free diastereoisomeric diacid were mixed with 30 ml of methanol and enough dichloromethane was added to make a clear solution to which an excess of ethereal diazomethane was added. After a few minutes

a little acetic acid was added to destroy the excess diazomethane, a clear solution being obtained. This was then concentrated on a cooker until crystallization began and then allowed to cool to ambient temperature. The crystalline solid was filtered off, washed with methanol and dried ^{to constant weight in a vacuum oven at 80 ° C.} The yield was 3.16 g of the dimethyl ester of the diastereoisomeric diacid *

The product had a melting point of 197 to 198 ^° C. and the infrared absorption spectrum (Nujol) had larger bands at f _v = 1740 (C-3 and side chain ester), 1710 (C-30 ester) and 1655 (enone) cm ". The NMR spectrum (CDCl ₃ ) supported the structure.

Examination of the product by thin layer chromatography using petroleum ether (boiling point 60 to 80 ° C) / dichloromethane / acetone (6/3/1 v / v / v) showed that the product had a degree of purity of had more than 99%.

c) Ig of the free diastereoisomeric diacid was suspended in 20 ml of dry toluene and 0.6 ml Oxalyl chloride was added. The resulting mixture was magnetically stirred under anhydrous Conditions at ambient temperature for one hour. After this time the reaction mixture was still a suspension. Another 0.6 ml Oxalyl chloride was added and the reaction mixture was allowed to stir overnight. the

clear solution was rotary evaporated and the residue dried to constant in a vacuum oven Weight. 1.07 g of the diacid chloride were obtained.

1.07 g of the diacid chloride was added to a mixture of 2 ml of anhydrous pyridine and 0.8 ml of hexan-1-ol. The resulting mixture was refluxed in an oil bath at 14O ⁰ C under anhydrous conditions for 2 hours. A thin-layer chromatogram (petroleum ether 60-80 / dichloromethane / acetone; 6: 3: 1 v / v / v) showed that the reaction was 95% complete. The reaction mixture was refluxed for an additional hour and then allowed to cool. The reaction mixture was poured into ice / concentrated hydrochloric acid and extracted twice with ethyl acetate. The ethyl acetate phase was then washed with distilled water until the wash waters were neutral, dried over anhydrous sodium sulfate, filtered and rotary evaporated to dryness. 1.35 g of the di-n-hexyl ester of the diastereoisomeric diacid were obtained.

A thin layer chromatogram, prepared as above, showed the product to have a purity of 95% had.

25 g of silica gel (Merck 7734) was packed into a column. The column was eluted with a Solvent mixture of petroleum ether 60 to 80 / dichloromethane / acetone (6: 3: 1 v / v / v) and then became

a solution of 1.35 g of the dihexyl ester in the same Solvent mixture added to the column, followed by the same solvent mixture on the column was abandoned. A 25ml fraction was made first collected and discarded. 10 ml fractions were then collected and transferred to thin layer chromatography plates dabbed on. The pure fractions were combined and rotary evaporated to dryness. It 0.88 g of the purified dihexyl ester was obtained.

A thin layer chromatogram (petroleum ether 60 to 80 / dichloromethane / acetone; 6: 3: 1 v / v / v) showed that the product had a purity of 99%.

0.88 g of the dihexyl ester was dissolved in 8 ml of petroleum ether 60 to 80 and one week in one Freezer for left. The crystalline obtained Product was filtered through a cold sintered glass funnel with a small amount Washed methanol and dried to constant weight in a vacuum dryer. Yield 0.56g.

A thin layer chromatogram, prepared as above, showed the product to be greater than 99% pure. The material had a melting point of 118 to 112 ° C and the infrared spectrum had larger bands at: y = 1710

^ J ΙΠαΧ

to 1730 (ester), 1660 (enone) cm ".

Al

example

3ckr (cis-2S-carboxycyclohexane-IS-carbonyloxy) -11-oxo-18β-olean-12-en-30-acid.

a) 23 g laöt-glycyrrhetinic acid, 17.6 g of cis -1,2-cyclohexane-dicarboxylic acid anhydride and 28.2 ml of anhydrous pyridine was heated under reflux for four hours at an oil bath temperature of 140 to 160 ⁰ C. After this time, thin layer chromatography showed the reaction to be nearly 90% complete. To the cooled reaction mixture was added 352.5 ml of acetone and while stirring the solution magnetically, a solution of 42.3 ml of concentrated hydrochloric acid in 112 ml of distilled water was added at ambient temperature.

The resulting precipitate was filtered off, washed with 70% aqueous acetone and then with distilled water until the washings were neutral and dried in a vacuum oven at 100 ⁰ C. The yield was 28.28 g.

28.28 g of the above diacid was slurried with 84.8 ml of acetone and a solution of 3.64 g Sodium hydroxide in 84.8 ml of distilled water was slowly added with stirring until the pH was 9 was. The reaction mixture was still a suspension so it was warmed in a hot water bath. This made no change, so an additional 24 ml of distilled water was added and the mixture was added was reheated. The slightly cloudy solution was filtered hot and under continuous Acetone was added to heating until the solution became cloudy. This was then left to cool down 4 ° C.

The precipitated solid was filtered off, with acetone / water (85:15 v / v) and then with acetone washed and dried to constant weight in a vacuum oven. The yield of the disodium salt the diacid was 17.60 g.

The above disodium salt was dissolved in 1056 ml of hot aqueous acetone (1: 1 v / v) (6 χ volume), filtered and with continuous heating acetone was added until the mixture became cloudy after which it was cooled to 4 ° G. The crystalline solid obtained was filtered off, washed with about 50 ml acetone / water (85:15 v / v) and then with acetone. The product was in one Vacuum oven dried to constant weight, the yield was 12.05 g. HPLC indicated that the Was diastereoisomer of 97.5% purity.

Two more recrystallization of the disodium salt using the above conditions gave 6.95 g of white crystals.

HPLC using a 75:25 v / v methanol / 0.1M phosphate buffer (pH 7) solvent mixture showed that the material contained greater than 99% of the desired diastereoisomer.

The material had a melting point of more than 360 ^° C. and the infrared absorption spectrum had larger bands at V * "= 1710 (ester), 1660 (enone), 1570 (CO ₂ Na) cm". The pH of a 10% aqueous Solution of the disodium salt was 8.6.

0.5 g of the disodium salt was converted into the corresponding acid by treating an aqueous solution thereof with dilute hydrochloric acid. The resulting precipitate was filtered off, washed with water until the washings were neutral and dried to constant weight in a vacuum oven at 100 ⁰ C. The yield was 0.29 g.

The diacid had a melting point of 303-305 ⁰ C and the infrared spectrum had (d, - pyridine) larger bands at 1705-1725 ψ = (CO + H ₀ ester), 1665 (enone) cm "The NMR spectrum. supported the expected structure.

b) 40 ml of dry methanol and 1 ml of concentrated sulfuric acid were added to 2 g of the disodium salt and the resulting mixture was refluxed on a hot water bath for 7 hours cooked. After this time, a thin layer chromatogram (petroleum ether 60-80 / dichloromethane / Acetone; 6: 3: 1; v / v / v) that the reaction was incomplete.

0.4 ml of concentrated sulfuric acid was added to the reaction mixture and the solution became refluxed for a further 4 hours, after which a thin layer chromatogram showed that the Reaction was 95% complete.

The cooled reaction mixture was poured into ice / water and extracted with ethyl acetate. This The procedure was repeated and the ethyl acetate extracts were combined, washed with water

until the wash waters were neutral and above anhydrous Dried sodium sulfate. The dried ethyl acetate solution was then filtered and rotary evaporated to dryness. The yield was 1.66 g.

The above product was dissolved in methanol / dichloromethane (2: 1 v / v) and concentrated on a cooker until crystallization began. The crystalline solid was filtered off, washed with methanol and dried in a vacuum oven at 8O ⁰ C, was until constant weight attained. The yield was 1.36 g. A thin layer chromatogram showed that the product was more than 99% pure.

The infrared spectrum had larger bands at V = 1740 (ester), 1710 (ester) and 1655

IUciX «

(Enone) cm ".

The material had a melting point of 235 to 237 ^° C. Gas chromatography (11% OV-17 column at 305 ^° C.) showed that the product was approximately 99% pure and contained less than 1% of the corresponding 18β compound. A nuclear magnetic resonance spectrum (CDCl ₃ ) supported the expected structure.

c) The diacid chloride was prepared in a manner analogous to Example Ic), but the last work-up was different.

The clear solution was rotary evaporated to about half the original volume. Dry Toluene was added and the same

Process repeated. This was done until no oxalyl chloride remained. The final solution was concentrated to 10 ml and placed in a freezer for 15 minutes. The received crystalline solids were filtered off and washed with a small amount of dry toluene. The product was used for the next step without drying.

The di-n-hexyl ester was prepared in a manner analogous to that described in Example 1 c), the The yield was 0.83 g.

A thin layer chromatogram (petroleum ether 60-80 / dichloromethane / acetone; 6: 3: 1 v / v / v) showed that the Product had a purity greater than 99%.

0.63 g of the dihexyl ester was dissolved in 7 ml of petroleum ether and left in a freezer for 3 weeks. The crystalline product obtained was filtered off, washed with methanol and ^{dried in a vacuum oven at 40 °} C. to constant weight. The yield was 416 mg.

The material had a melting point of 99 to 101 ⁰ C and the infrared spectrum had larger bands at ψ _v - 1700-1720 (ester), 1660 (enone)

Claims (5)

Patent claims 1. / Compounds of the general formula: COOR, .H
O Il wherein
-COOR COOR, and -COOR ,,. which either the same or can be something different, free carboxylic acid groups or carboxylic acid groups in salt or Are ester form. 2. 3β- (Ci £ -2S-carboxycyclohexane-1S-carbonyloxy) -11-oxo-18β-olean-12-en-30-acid and the disodium salt, dimethyl ester or di-n-hexy ester thereof. 3. 3β- (c_is_-2S-carboxycyclohexane-1S-carbonyloxy) -11-oxo-18iX -olean-12-en-30-acid and its disodium salt, dimethyl ester and di-n-hexyl ester. 4. A process for the preparation of the compounds according to claim 1, characterized in that 18ö (- or 18ß-glycyrrhetinic acid or one of its esters either reacts with cis -cyclohexane-1,2-dicarboxylic acid anhydride to give a mixture of the diastereoisomers, which then in a known manner be separated or reacted with a suitable isomer of cyclohexane-l ^ -dicarboxylic acid anhydride, after which, if desired, free carboxylic acid groups in the product obtained in the salt or ester form be transferred in a known manner. 5. Pharmaceutical compositions containing at least one compound according to claim 1 in a mixture with a solid or liquid pharmaceutical diluent or carrier.