NO124125B - - Google Patents

Description

Process for the production of new steroid compounds with anti-inflammatory action.

The present invention relates to a method for the production of new steroid compounds, namely 1-dehydro-6-methyl-cortisone, 1-dehydro-6-methylhydrocortisone and 21-acyl esters of these compounds.

The method according to the present invention and the products obtained from it can be represented by the following form:

In the above general formulas, R denotes the group

while R'

denotes hydrogen or an acyl group with 1-12 carbon atoms.

It has previously been described the introduction of an A1 double bond in known pregnancy steroids by the action of the microorganisms Bacillus sphaericus var. fusiformis or Corynebacterium simplex and hoagii (Australian Patent 211 200 and J. Am. Chem. Soc. Vol. 77, 4184). Also stated in Heiv. Chim. Acta 38, page 835 (1955) use species of microorganisms of the genus Fusarium to introduce an A1 double bond into certain known steroid compounds.

The main characteristic features of the method according to the invention are that a pregnancy steroid with the general formula:

where R and R' have the meanings given above, are dehydrated in the 1,2 position either by treatment with selenium dioxide or by growing a microorganism from the group consisting of of the genera Corynebacterium, Didymella, Calonectria, Alternaria, Colletotrichum, Cylindrocarpon, Ophiobolus, Septomyxa, Fusarium, Listeria and Erysipelothrix, preferably Septomyxa, and recover the corresponding 11-oxygen-6-methyl-17a,21-dihydroxy-1,4-pregnadiene - 3,20-dione and possibly esters in the 21 position.

Particularly advantageous for dehydration in the 1,2-positions of such steroids are microorganisms selected from the genus Septomyxa, and in particular the species Septomyxa affinis ATCC 6737 and Corynebacterium. The species Corynebacterium simplex is particularly preferred for the production of the corresponding 11-oxygen-6-methyl-17a, 21-dihydroxy-1,4-pregnadiene-3,20-dione.

Esterification of the produced 11-oxygen-6-methyl-1 7a,21 -dihydroxy-1,4-pregnadiene-3,20-dione is carried out by usual methods such as by mixing the selected 11-oxygen-6-methyl-17a, 21-dihydroxy-1,4-pregnadiene-3,20-dione with an acylating agent such as a ketene, an isopropenyl acylate, an acid anhydride or an acid halide of an organic carboxylic acid, preferably with from 1 to 12 carbon atoms, at temperatures between 0°C and 100°C, room temperature being preferred (cf. examples 4-6 and 7-9 in the following).

The term 11-oxygen refers here only

to the lip-hydroxy and the 11-keto group.

With the help of the present invention, the 6-methyl-1-dehydro analogues of cortisone and hydrocortisone as well as the 21-esters thereof, in particular 6a-eomers of the same, i.e. l-dehydro-6a-methylhydrocortisone and l-dehydro-6a- methylcortisone.

6a-methylhydrocortisone, which is used as starting material in the method according to the invention, is a compound and can be produced according to the synthesis described below:

6a-methylhydrocortisone is prepared by first treating 11(3, 17a,21-trihydroxy-5-pregnen-3,20-dione-3,20-bis - (ethylene ketal) with perbenzoic acid in chloroform to form 5a,6a-oxido- lla21-trihydroxyallopreg-nan-3,20-dione-3,20-bis-(ethylene ketal). The 5,6-oxido compound thus obtained is then treated with a Grignard reagent such as methylmagnesium bromide dissolved in tetrahydrofuran and ether, thereby obtaining the corresponding 5a,lip,17a,21-tetrahydroxy-6p-methylallopregnan-3,20-dione-3,20-bis-(ethylene ketal). This 6p-methylallopregnan is then treated with an acid such as dilute sulfuric acid in methanol to form 5p , lip,17a,21-tetrahydroxy-6p-methylallopregnan-3,20-dione The 6p-methylallopregnan-3,2p-dione thus obtained is dehydrated with a base such as 0.1 N sodium hydroxy in methanol, preferably under a nitrogen atmosphere, whereby 6a-methylhydrocortisone is obtained. The new compounds, l-dehydro-6-methylcortisone and l-dehydro-6-methylhydrocortisone, especially in the form of 6a-epimers are in high degree active adrenocortical hormones and with greater glucocorticoid activity than hydrocortisone and cortisone or hydrocortisone's and cortisone's 1-dehydroanalogues, as shown in the following table I.

The new compounds, l-dehydro-6a-methylcortisone and l-dehydro-6a-methylhydrocortisone, have the further advantages of low salt retention and strong anti-inflammatory action, which makes them valuable both for systemic and local application. 1-dehydro-6a-methylhydrocortisone's effect against inflammation determined by the "granuloma pouch" test is 6 to 7 times greater than hydrocortisone's anti-inflammatory effect.

The method according to the present invention, which consists in fermentative dehydration of the starting material, can be carried out under conditions as described in detail in US patent no. 2,602,769, using a suitable nutrient medium containing assimilable carbon, nitrogen, sulfur and phosphorus in appropriate amounts, as well as other substances (vitamins, e.g. thiamine, thiamine-thiazole and plant hormones such as auxins) and inorganic substances (e.g. zinc, iron, cobalt, copper, potassium, manganese, etc. in metabolically available state) that is necessary for the life and growth of microorganisms. Air is introduced into the cultivation mixture by regulated aeration by having air present in shaking bottles or in another suitable way. The 6α-methylcortisone, the 6α-methylhydrocortisone or esters of these compounds or their 6(3-epimeric compounds) are added preferably, but not necessarily, in solution or suspension and after giving the microorganism time to grow. It has been found that certain other steroids significantly increase the yield of the cultivation process described here. In order to achieve optimal yield, therefore, steroid compounds are added which are selected from the group consisting of 3-ketobisnor-4-cholen-22-al, 3-ketobisnor-4-cholenic acid, impure 11(3,21-dihydroxy-1,4,17(20)-pregnatrien-3-one or progesterone. Other compounds of a steroidal or non-steroidal nature can also help to increase the yield in the cultivation process. Further details in cultivation the process, in the extraction of the steroids and in the esterification can be found in the examples below.

Example 1.

1-dehydro - 6a - methylhydrocortisone - (6a-methyl-11(3,17a,21-trihydroxy-1,4-pregna-diene-3,20-dione).

In 250 ml Erlenmeyer flasks, 6 portions of 100 ml of a medium containing 1 per cent glucose, 2 per cent grain casting liquid (60 per cent

solids) and tap water adjusted to a pH value of 4.9. The medium was sterilized in

45 minutes at a pressure of 1.05 kg per cm2

and inoculated with a 1-2 day culture of Septomyxa affinis A.T.C.C. 6737. The Erlenmeyer flasks were shaken at room temperature, i.e.

around 24°C, for a period of 3 days. At the end of this period, these portions totaling 600 ml were used as inoculation medium for 10 liters of the same glucose-grain casting liquid medium to which 10 ml of an antifoam agent (a mixture of tallow oil and octadecanol) had been added. The culture medium was placed in a water bath whose temperature was set at 28°C and stirred with a stirrer whose speed was 300 revolutions per minute. min., as well as aerated with 0.5 litres

air per 10 liters of medium. After 17 hours

incubation, good growth had developed and the pH had risen to 6.7. 2 g of 6α-methylhydrocortisone and 1 g of 3-ketobisnor-4-cholen-22-al, dissolved in 115 ml of dimethylformamide, were then added and the incubation (transformation) was carried out at the same temperature and aeration for 24 hours (final pH value 7 ,9). The mycelium (weight in dry state 56 g) was filtered off and the steroid material remained

extracted with methylene chloride. The methylene chloride extracts were evaporated to dryness and the residue obtained was chromatographed in a Florisil column. The column was filled

with 200 g "Florisil" and was washed out with 5

fractions of methylene chloride of 400 ml each,

mixtures of "Skellysolve B" and acetone in the proportions 9:1, 8:2, 7:3, 1:1 as well as

methanol. The fraction that was eluted with a mixture of "Skellysolve B" and acetone in the ratio of 7:3 weighed 1,545 g. On recrystallization from acetone, it gave in 3 portions 928 mg of product with m.p. 210—

235° C. The sample prepared by analysis melted at 245-247° C. Its rotatability [a]n was +83° in dioxane.

The composition of the product was: Calculated for C22<H>a0O5: C, 70.56 per cent., H, 8.08 per cent. Found: c" 70.53 per cent., H, 7.94 per cent.

In Ethanol nAn ,,.„„„

max = 243> b = 14.875.

The sample's absorption in the infrared region of the spectrum gave in Nujol mineral oil suspension: OH 3430 3330 3180cm-i 20-keto 1706

Conjugated 3-keto 1645 AM double bond 1592

Instead of fermentative dehydration, 1-dehydro-6a-methylhydrocortisone or esters of this compound can be obtained by dehydration of 6a-methylhydrocortisone or an ester of the same with selenium dioxyd as shown in the following example

IA.

Example IA. 1 -dehydro-6cc-methylhydrocortisone acetate.

A mixture of 70 mg of 6α-methylhydrocortisone acetate in 4.5 ml of tertiary butyl alcohol, 0.45 ml of acetic acid and 24 mg of selenium dioxide was heated to 75°C and stirred for 24 hours. A further 24 mg portion of selenium dioxide heated to 75° C was then added and the stirring continued. The mixture was then cooled, filtered to remove the selenium dioxide and evaporated. By paper chromatography, it turned out that the residue contained about 55 percent of 1-dehydro-6a-methyl-hydrocortisone acetate, which can be recovered from the residue by recrystallization and chromatography.

Absorption in the infrared region of the spectrum was in Nujol mineral oil suspension:

Example 2.

1-dehydro-6α-methylcortisone.

In the manner indicated in example 1, 6α-methyl-cortisone was subjected to fermentation with Septomyxa affinis in the rich nutrient medium together with impure lip,21-di-hydroxy-1,4,17 (20)-pregnatrien-3-one. One obtained l-dehydro-6a-methylcortisone with m.p. 230-232°C.

Instead of 3-ketobisnor-4-cholen-22-al, progesterone, lip,21-dihydroxy-1,4,17 (20)-pregnatrien-3-one or 3-ketobisnor-4-cholenic acid can be used.

Example 3.

1-dehydro-6β-methylhydrocortisone.

In the manner indicated in Example 1, fermentation of 6β-methylhydrocortisone using impure lip,21-dihydroxy-4,17(20)-pregnadien-3-one as additive gave 1-dehydro-6β-methylhydrocortisone.

Example 4.

l-dehydro-ep-methylcortisone.

In the manner indicated in example 1, 6p-methyl-cortisone was subjected to fermentation with Septomyxa af finis in the same nutrient medium and with impure 11(3,21-dihydroxy-4,17(20)-pregnadien-3-one as additive). One then got l-dehydro-6p-methylcortisone.

Instead of lip,21-dihydroxy-4,17(20)-pregnadien-3-one, progesterone, 3-ketobisnor-4-cholen-22-al or 3-ketobisnor-4-cholenic acid can be used.

Example 5.

1-dehydro-6α-methylhydrocortisone acetate.

A mixture was prepared containing 500 mg of 1-dehydro-6α-methylhydrocortisone in 5 ml of pyridine and 5 ml of acetic anhydride. This mixture was kept at room temperature (22-24° C.) for a period of 6 hours, after which it was poured into 100 ml of ice water. The resulting aqueous mixture was extracted with three 25 mL portions of methylene chloride. The combined methylene chloride extracts were washed, dried over sodium sulfate and evaporated. The residue thus obtained was crystallized three times from a mixture of acetone and "Skellysolve B" hexanes, whereby pure 1-dehydro-6a-methylhydrocortisone-21-acetate (6-methyl-lip,17a-dihydroxy-21-acetoxy- 1,4-pregnadiene-3,20-dione).

Example 6.

1-dehydro-6α-methylcortisone benzoate.

A mixture of 500 mg of 1-dehydro-6-methylcortisone, 5 ml of pyridine and 5 ml of benzoyl chloride was kept at room temperature for 8 hours. The mixture was then poured into an excess of water, the water extract neutralized with sodium bicarbonate and the mixture cooled. It was then filtered and the 1-dehydro-6-methylcortisone benzoate thus obtained was recrystallized from methanol, whereby pure 1-dehydro-6a-methylcortisone benzoate- (6a-methyl- 17a-hydroxy-21-benzoyloxy-1,4- pregnadiene-3,11,20-trione).

Example 7. 1-dehydro-6α-methylhydrocortisone-21-hemisuccinate.

A solution of 2.5 g of succinic anhydride in 25 ml of pyridine was added with stirring to 2.0 g of 1-dehydro-6-methylhydrocortisone. Stirring was continued until 1-dehydro-6α-methylhydrocortisone was completely dissolved. After standing overnight, the reaction mixture was slowly poured into a vigorously stirred mixture of 30 ml of concentrated hydrochloric acid, 102 ml of water and 127 g of ice. Stirring was continued for one hour and the impure crystalline 1-dehydro-6α-methylhydrocortisone-21-hemisuccinate was separated by filtration. This substance was washed on the filtrate with water until the filtrate had a pH value of 4.0, dried and recrystallized from a mixture of 45 ml of methyl ethyl ketone and 36 ml of "Skellysolve B" hexanes, whereby pure l-dehydro-6a-methyl was obtained -hydrocortisone-21-hemisuccinate.

Example 8.

1-dehydro-6α-methylhydrocortisone-21-hemisuccinate sodium salt.

Sodium hydroxide solution (0.1 n) was added slowly to a stirred solution of 2 g of 1-dehydro-6α-methylhydrocortisone-21-hemisuccinate in 50 ml of acetone until the pH rose to 7.4. During the addition of sodium hydroxide solution, 100 ml of water was also added.

The solution was evaporated in vacuo at 25°C to remove the acetone. The resulting aqueous solution of 1-dehydro-6α-methylhydrocortisone-21-hemisuccinate sodium salt was filtered and freeze-dried. The compound was then recrystallized, whereby pure 1-dehydro-6α-methyl-hydrocortisone-21-hemisuccinate sodium salt was obtained.

Example 9.

1-dehydro-6α-methylhydrocortisone-21-((3;(3-di-methylglutarate).

A solution of 260 mg of |3,3-dimethyl-glutaric anhydride in 2 ml of pyridine was added to 200 mg of 1-dehydro-6α-methyl-hydrocortisone. The resulting mixture was stirred until 1-dehydro-6α-methylhydrocortisone was completely dissolved, whereupon nitrogen was passed through the flask. The mixture was allowed to stand for 18 hours, after which it was slowly poured into a stirred cold mixture of 2.4 ml of concentrated hydrochloric acid and 18 ml of water. The resulting mixture was extracted with three 5 ml portions of ethyl acetate. The ethyl acetate layer was washed with dilute hydrochloric acid and water, dried over anhydrous magnesium sulfate and evaporated under reduced pressure to 1.5 ml. 1 ml of "Skellysolve B" hexanes was added and the mixture was cooled to 0° C. After 24 hours allowed to stand, the mixture was filtered, whereby crystals of 1-dehydro-6-methylhydrocortisone-21-((3,(3-dimethylglutarate) were obtained). The spectrum of this compound in the infrared range, measured in Nujol mineral oil, corresponded to the assumed structural formula of the compound .

Claims (3)

1. Process for the production of anti-inflammatory active 1-dehydro-6-methyl-hydrocortisone, 1-dehydro-6-methylcortisone and 21-acyl esters of these compounds, characterized in that a 4-pregnene steroid with the general formula: where R denotes the group and R' denotes hydrogen or an acyl group with 1-12 carbon atoms, is dehydrated in the 1,2 position either by treatment with selenium dioxide or by growing a microorganism from the group consisting of of the genera Corynebacterium, Didymella, Calonectria, Alternaria, Colletotrichum, Cylindrocarpon, Ophiobolus, Septomyxa, Fusarium, Listeria and Erysipelothrix, preferably Septomyxa, and extract the formed 11-oxygen-6-methyl- 17a,21-dihydroxy-1,4 -pregnadiene-3,20-dione and possibly esters in the 21-position. 2. Method according to claim 1, characterized in that Septomyxa affinis A.T.C.C. is used as the microorganism. 6737. 3. Method according to claim 1 or 2, characterized in that the cultivation is carried out in the presence of 3-ketobisnor-4-cholen-22-al; lip 1 -dihydroxy-1 2 -(20)-pregnatrien-3-one; 3-ketobisnor-4-cholenic acid or progesterone.