NO162562B - 3-ALFA, -7BETA-DIHYDROXY-12-KETO-5BETA-CHOLANIC ACID, AND THIS TRIS-TRIMETHYL DERIVATIVE. - Google Patents

Description

This invention relates to new compounds, namely 3a,7(3-dihydroxy-12-keto-5|3-cholanic acid and its tris-trimethylsilyl derivative, with the general formula:

where is hydrogen or -SifCH^-j. The compounds are suitable as intermediates in the production of ursodeoxycholic acid (3a, 7|B-dihydroxycholic acid) with high purity, which above all is not contaminated with the chenodeoxycholic acid epimer (3a,7a-dihydroxycholic acid). The production of ursodeoxycholic acid using these intermediates is the subject of Norwegian patent no. (Norwegian patent application no. 821194).

The methods currently used for the production of ursodeoxycholic acid all essentially fall within the same reaction core, which as the main step comprises the oxidation of chenodeoxycholic acid (A) to 3a-hydroxy-7-ketocholic acid (B), which is hydrogenated to ursodeoxycholic acid (C ) :

The disadvantage of the known methods which include the aforementioned reaction sequence is that the reduction of compound B

in all cases leads to a mixture of isomers consisting of approx. 80% of compound C and 20% of compound A or chenodeoxycholic acid, which is difficult to remove.

All the methods that have been proposed so far to separate the mixture of products A and C are expensive when they are to be carried out on an industrial scale, and have a limited degree of efficiency.

With the help of the present invention, new compounds are provided which enable the production of ursodeoxycholic acid directly and with pharmaceutical purity, especially in view of the absence of chenodeoxycholic acid, without the need for subsequent, complicated and expensive purification processes.

In the overall process where the new compounds are used as intermediates, cholic acid is used as starting material, but ursodeoxycholic acid is arrived at via a completely new series of reactions.

Essentially, this total process comprises the following steps: 1. Selective oxidation of cholic acid or 3<x, 7ot, 12cx- trihydroxycholanic acid (I) to 3a, 12a-dihydroxy-7-ketocholanic acid (II) according to known methods (J.A.C.S. 71, 3935 , 1949 - J.A..S. 72, 5530, 1950)

The necessary step for the production of the acid

(II) from cholic acid, which can be easily obtained on the market, is carried out according to known methods as indicated above and therefore forms no part of the invention. 2. Reduction of the acid (II) to 3ot, 73, 12a-trihydroxycholanic acid (III') by treating a solution of this in a C1, -4„ alcohol with alkali metals

In this reaction, the acid (III') is actually formed together with a limited amount of between 10 and 15%

of the corresponding 7-a-isomer or cholic acid (I).

3a, 7&, 12a-trihydroxycholanic acid (III' ) can be obtained in a pure state by crystallization from suitable solvents or through the production of products that are esterified in whole or in part in positions 3, 7, 12 and 24, and purification of the obtained esters by crystallization from suitable solvents. The acid (III') is released from the thus obtained pure ester by alkaline saponification.

For the purposes of the invention, however, it is not necessary to obtain pure acid (III'), and it is used in the next step in the total synthesis in the state in which it is obtained, i.e. contaminated with cholic acid, but esterified in the appropriate manner with acid in the positions 3 and 7 and optionally with a C^_^ alcohol in position 24. These esterification reactions can, if necessary, be carried out one after the other, using the normal esterification methods. The ester compounds that are produced have the formula:

where R is an aliphatic acid group with 2-6 carbon atoms, unsubstituted or substituted benzoic acid, succinic acid or glutaric acid, and R' is hydrogen or an alkyl group containing 1-4 carbon atoms. 3. Oxidation of the esterified 3a, 7|3, 12a-trihydroxy-5-|3-cholanic acid (III) to the esterified 3a, 7|3-dihydroxy-12-keto-5|3-cholanic acid with alkaline hypochlorite in acetic acid according to the equation:

where R and R<1> have the meanings given above.

The excess of hypochlorite is destroyed with bisulphite. Free 3a, 73-dihydroxy-12-keto-5|3-cholanic acid is obtained by saponification with dilute strong alkali followed by acidification.

The thus obtained free acid (IV-a; see below) contains impurities consisting mainly of 3a, 7a-dihydroxy-12-keto-5|3-cholanic acid and unreacted 3a, 7|3-trihydroxycholanic acid.

4. Purification of the 3α, 7β-dihydroxy-12-keto-5β3-cholanic acid in the preparation of its tris-trimethylsilyl derivative by treating the acid, in solution in an organic solvent, with a silanizing agent, such as bis-trimethylsilylurea, hexamethyldisilazane or bis -trimethylsilylacetamide at a temperature between 30° and 100° C.

In connection with this, it has unexpectedly been shown that compound (IV-b) has very little solubility in organic solvents, while the tris-trimethylsilyl derivatives of the impurities, and especially of 3a, 7a-dihydroxy-12-keto-5(3-cholanic acid, are very soluble in the same solvents 5. Possible removal of the trimethylsilyl groups by acid hydrolysis, preferably with HC1 in aqueous solution or in an organic solvent according to the equation: 6. Reduction of 3a, 7P-dihydroxy-12-keto-53 -cholanic acid to ursodeoxycholic acid according to the Wolff-Kishner method, by heating to 200°C with hydrazine hydrate in the presence of an alkaline base and triethylene glycol:

A hydrolysis like that carried out in step 5 can optionally be carried out after a reduction like that carried out in step 6, in the sense that the Wolff-Kishner reduction (step 6) can be carried out on the tris-trimethylsilane derivative and the trimethylsilyl groups are eliminated from the already reduced compound, i.e. .from the tris-trimethylsilyl derivative of the ursodeoxycholic acid of high purity. Step (6) and the aforementioned alternative are the subject of Norwegian patent no. (Norwegian patent application no. 821194).

The new compounds of formula (IV) according to the invention can thus be prepared from readily available starting materials and can be transferred to ursodeoxycholic acid through a limited number of steps using reactions that can be carried out easily and with high selectivity. By using the new compounds as intermediates, an ursodeoxycholic acid of pharmaceutical purity is obtained, without requiring subsequent complicated and expensive purification processes.

The transfer of the new compounds of formula (IV) to ursodeoxycholic acid with/high purity is carried out by reducing a compound of formula (IV) with hydrazine hydrate in the presence of an alkali base and triethylene glycol, and the product obtained, if R^ = -Si(CH3 )3, is subjected to acid hydrolysis.

This preparation is described in more detail in Norwegian patent document no. (Norwegian patent application no. 821194), which is referred to in the introduction of this description.

The following examples illustrate the preparation of the new compounds of formula (IV). First, however, the preparation of a pair of starting materials of formula (III) which are used for the preparation of the compounds of formula (IV) is described.

E??1§£iiiiD2_Ai_3i2l^i§2?£?£§5 _5Y_52fiZ£iI2a^ir^)}Yd^2x^9hoiani_ acid_£III2^

100 g of 3α,12α-dihydroxy-7-ketocholic acid (II) obtained by selective oxidation of cholic acid (I) (according to J.A.C.S. 71, 3935/1949; J.A.C.S. 72, 5530/1950) was dissolved in 2000

ml of secondary butyl alcohol. The solution was heated to boiling and 100 g of metallic sodium was added. Boiling was carried out for 2 hours under reflux conditions, after which

the butyl alcohol was distilled off with the simultaneous addition of an equal amount of water. When all the butyl alcohol had been removed, the mixture was cooled and acidified with 20% HCl. The resulting slurry was filtered off, washed with water and dried, whereby 95 g of a dry, amorphous product was obtained which, by HPLC analysis, was found to consist of 88.9% 3a, 73.12oc-trihydroxycholanic acid and 9.2% cholic acid.

Acidimetric titer = 99.1%.

[a]p° = +71.2° (C = 1% in dioxane).

200 ml of benzene, 50 ml of pyridine and 50 ml of acetic anhydride were added to 50 g of the cholic acid-contaminated 3α,73,12α-trihydroxycholanic acid. The mixture was allowed to react for 48 hours at 20-25°C, after which 50 ml of water was added with stirring and then, a few minutes later, a mixture of 200 ml of water and 60 ml of 37% HCl. The aqueous phase was removed, and the organic phase was evaporated almost to dryness, after which a residue containing the 3,7-diacetate of 3a,73,12a-trihydroxycholanic acid was obtained.

Preparation_B£_3i7-disuccinate_of_3 a.j.7 3^12a-trihydroxycholanic acid_ (^III|.

100 g of metallic potassium was added under boiling to 100 g of 3α,12α-dihydroxy-7-ketocholanic acid (II) dissolved in 2000 ml of n-butyl alcohol, and the mixture was heated under reflux conditions for 2 hours. All butyl alcohol was then distilled off, while water was added at the same time. After the end of the distillation, the mixture was acidified with 20% HCl, and the precipitated solid was filtered off.

When the solid (95 g) was subjected to HPCL analysis, it was found to consist of 90% 3a,7(3,12a-trihydroxycholanic acid and 10% cholic acid. The characteristic properties of the mixture corresponded to those according to Example 1.

100 g of a mixture of 3a,73,12a-trihydroxycholanic acid and the cholic acid obtained in the previous step were dissolved in 150 ml of pyridine. 100 g of succinic anhydride was added, and the mixture was heated at 80°C for 5 hours. The mixture was then cooled to 0°C and 100 ml of water was added. The mixture was allowed to stand under stirring for 2 hours. 1000 was added

ml of ethyl acetate, and the pH was adjusted to 3-3.5 with HCl. The aqueous phase was separated. The remaining organic solution contained the desired 3,7-disuccinate of 3a,7|3,12a-trihydroxycholanic acid.

Example 1

Preparation of 3a,73-dihydroxy-12-ketocholanic acid (IV-a) and its 3a,73-bistrimethoxysilylether-24-trimethylsilyles-

ter (IV-b)

The residue containing the 3,7-diacetate of 3a,7|3,12a-trihydroxycholanic acid, which was obtained in Preparation A above, was taken up in 500 ml of ethyl acetate, to which was added 30 ml of 80% acetic acid and 15% sodium hypochlorite. The mixture was stirred for 1 hour, the excess of hypochlorite was destroyed with sodium metabisulfite, and the ethyl acetate was washed with 500 ml of water. The organic phase was evaporated to dryness and boiled with 200 ml of 10% NaOH for 5 hours. It was acidified with HCl and extracted with ethyl acetate. 45 g of residue was obtained from the organic phase by evaporation and this to dryness. By HPLC analysis, the product was found to consist of:

- 75% 3a, 7|3-dihydroxy-12-ketocholanic acid

8% 3α,7α-dihydroxy-12-ketocholanic acid

- 15% 3a, 7(J, 12a-trihydroxycholanic acid

2% cholic acid.

50 g of impure product obtained in the previous step was dissolved in 500 ml of N,N-dimethylformamide. 50 was added

g of bis-trimethylsilylurea, and the mixture was heated to 100°C and held at this temperature for 1 hour with stirring, after which it was cooled. The 3α,73-bis-trimethylsilyl ether-24-trimethylsilyl ester was filtered off and washed with 50 ml of N,N-dimethylformamide. The product was then dried in an oven at 70°C

under vacuum. 60 g of product with the following characteristics were obtained:

Molecular weight: 62 3.13

Melting point = 155-157°C

[a]^°= +89° (C = 2% in dioxane).

Example 2

Preparation of 3a, 7ft- dihydroxy- 12- ketocholanic acid ( IV- a) and its 3a, 7[ 3- bis- trimethylsilyl ether- 2 4- trimethylsilyles-

ter (IV-b)

50 ml of acetic acid and 50 ml of a 10% aqueous solution of KBr were added to the organic solution obtained during Preparation B, and the temperature was set to 20°C. While maintaining this temperature, 150 ml of a 15% solution of sodium hypochlorite was added. The mixture was allowed to stand under stirring for 30 minutes, after which the product was filtered off, washed with 200 ml of ethyl acetate and dried in an oven at 80°C. The dry product was boiled for 2 hours with 1000 ml of 10% NaOH. The mixture was then cooled, after which 1000 ml of ethyl acetate was added and the mixture was acidified with 20% HCl. The organic phase was separated and evaporated to a volume of approx. 300 ml. Cooling to 0°C was carried out

and filtering. The product was washed with 100 ml of ethyl acetate and dried in an oven at 80°C. 70 g of impure, 3a, 7|3-dihydroxy-12-ketocholanic acid were obtained.

100 ml of acetonitrile, 700 ml of hexamethyldisilazane and 300 ml of trimethylchlorosilane were added to 100 g of impure 3α,73-dihydroxy-12-ketocholanic acid obtained in the previous step. The mixture was heated under reflux conditions for 1 hour, after which it was cooled. The 3a, 7|3-bis-trimethylsilyl ether-2 4-t trimethylsilyl ester which crystallized was filtered off, washed with acetonitrile and dried at 60°C under vacuum.

50 g of the thus obtained tris-trimethylsilyl derivative was dissolved with stirring and during 10 minutes in 500

ml of ethyl acetate and 100 ml of 10% HCl at a temperature of 50°C. The organic phase was separated and washed with 100 ml of 10% HCl at 50°C for 10 minutes. The organic phase was separated and washed with 100 ml of water. Upon concentration, very pure 3α,73-dihydroxy-12-keto-53-cholanic acid crystallized. This was filtered off, washed with ethyl acetate and dried in an oven at 80°C.

Characteristics:

Molar weight = 406.5

Melting point = 190°C

[a]p°= +108° - 3 (C = 1% in dioxane)

Total amount of impurities, determined by chromatography 0.5%.

Example 3

The procedure described in Example 1 was repeated

in exactly the same way, except for the step where the tris-trimethylsilyl derivative was prepared, which step was carried out as follows: 50 ml of bis-trimethylsilylacetamide was added to 50 g of impure 3a, 7 |3-dihydroxy-12-ketocholic acid dissolved in 500 ml of N, N-dimethylformamide. The mixture was heated to 100°C, held at this temperature for 1 hour with stirring and then cooled. The precipitate was filtered off and recrystallized from N,N-dimethylformamide in the presence of 10 ml of bis-trimethylsilylacetamide at 100°C for 1 hour. The product was filtered off, washed and dried at 70°C under vacuum. 61 g of the tris-trimethylsilyl derivative of 3a,7|3-dihydroxy-12-ketocholanic acid were obtained. The product had the characteristics stated in Example 1.

Claims (1)

3a,-7(3-dihydroxy-12-keto-5|3-cholanic acid and its tris-trimethylsilyl derivative, characterized by the general formula: where is hydrogen or -Si(CH3)3.