GB1564810A - Cholesterol derivatives - Google Patents

Description

PATENT SPECIFICATION ( 11) 1564810

C' ( 21) Application No 23142/77 ( 22) Filed 8 Oct 1976 -I ( 62) Divided out of No 1 564 806 ( 19) ( 31) Convention Application No 621 319 ( 32) Filed 10 Oct 1975 k ( 31) Convention Application No 623 859 ( 32) Filed 20 Oct 1975 in ( 33) United States of America (US) ( 44) Complete Specification published 16 April 1980 ( 51) INT CL 3 C 07 J 9/00 ( 52) Index at acceptance C 2 U 4 A 1 B 4 C 4 X 6 B ( 54) CHOLESTEROL DERIVATIVES ( 71) We, F HOFFMANN-LA ROCHE & CO, AKTIENGESELLSCHAFT, a Swiss Company of 124-184 Grenzacherstrasse, Basle, Switzerland, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: 5

The present invention relates to cholesterol derivatives More particularly, the invention is concerned with cholesterol derivatives and a process for the preparation thereof.

The isolation and characterisation of 24,25-dihydroxycholecalciferol ( 24, 25dihydroxyvitamin D 3) lM F Holick et al, Biochemistry, 11, 4251 l 197211, and the 10 subsequent finding that this second most abundant metabolite of vitamin D 3 lJ L.

Omdahl and H F De Luca, Physiological Reviews, 53, 327 l 19731 l preferentially stimulates intestinal calcium transport without, at comparable dosage levels, mobilising bone calcium and is biologically synthesised in the kidney at the expense of the production of la,25-dihydroxycholecalciferol, the potent, rapidacting, 15 natural metabolite of vitamin D 3 (J L Omdahl and H F De Luca, supra), prompted fairly extensive investigation of the physiological role played by this metabolite lsee, for example, H K Schnoes and H F De Luca, Vitamins and Hormones, 32, 395 l 197411 These investigations have been hampered by the minute amounts of the metabolite available from natural sources, the lack of information 20 concerning the stereochemistry of the metabolic hydroxyl group at C-24 and the effect of the configuration of this group on the biological activity exhibited by 24,25-dihydroxycholecalciferol.

Recently, M Seki et al lChem Pharm Bull lJapanl, 21, 2783 l 19731 l described the non-stereoselective conversion of desmosterol acetate to 24, ,25 25 dihydroxycholesterol by either epoxidation with m-chloroperbenzoic acid followed by hydrolysis or hydroxylation with osmium tetroxide and subsequent reductive hydrolysis The diol of undefined stereochemical composition at C-24, as well as the epoxide, were subsequently used for the preparation of 24,25dihydroxycholecalciferol Shortly thereafter, H -Y Lam et al lBiochemistry, 12, 30 4851 1973 ll reported a non-stereosetective synthesis of 24 t,25dihydroxycholecalciferol starting from 3 p 3-acetoxy-27-nor-5-cholesten-25-one and proceeding via 24,25-dihydroxycholesterol J Redel et al lCompt rend Acad Sos lParisl, 278, 529 l 1974 ll disclosed a non-stereo-selective process for the preparation of the vitamin D 3 metabolite The latter process started with desmosterol acetate, 35 proceeded through an undetermined mixture of 24 R,25 and 245,25dihydroxycholesterols and gave an extremely poor (about 1 %) yield of an undefined mixture of 24 R,25 and 24 S,25-dihydroxycholecalciferol Thus, the stereospecific synthesis of 24 R and 24 S,25-dihydroxycholecalciferol using 24,25dihydroxycholesterol derivatives of known stereochemistry at C-24 to overcome 40 the deficiencies of the prior art processes and to make this important metabolite of vitamin D 3 readily available for biological, clinical and therapeutic use would represent a major contribution to the advancement of the state of the art in the vitamin D field.

The present invention provides a compound of the general formula 45 1,564,810 CM 3H h H Ia-Il, hu M H H a-1 M 413 o 1 wherein R 90 represents a lower alkanoyloxy group and wherein the absolute configuration at C-24 is R, said compound being substantially free of the 24-S epimer 5 The invention further provides a compound of the general formula 3 I Ib-I, wherein Ro represents a lower alkanoyloxy group and wherein the absolute configuration at C-24 is S, said compound being substantially free of the 24 R 10 epimer.

As used in this description and in the claims appended hereto, the term "lower alkyl" refers to a straight-chain or branched-chain saturated monovalent substituent consisting solely of carbon and hydrogen and containing from 1 to 8 carbon atoms Examples of lower alkyl groups are methyl, ethyl, n-propyl, 15 isopropyl, tert butyl, hexyl and octyl The term "lower alkoxy" refers to a monovalent substituent which consists of a lower alkyl group linked through an ether oxygen atom having its free valence bond from the ether oxygen atom.

Examples of lower alkoxy groups are methoxy, ethoxy, isopropoxy and tert butoxy.

The term "phenyl-(lower alkoxy)" refers to a lower alkoxy group which is 20 substituted by a phenyl group Examples of phenyl-(lower alkoxy) groups are benzyloxy, 2-phenylethoxy and 4-phenylbutoxy The term "lower alkanoyloxy" refers to the residue of a C,-C 8 alkanoic acid formed by removal of the hydrogen atom from the hydroxyl moiety of the carboxyl group Example of lower alkanoyloxy groups are formyloxy, acetoxy, butyryloxy and hexanoyloxy 25 In the formulae given in this description and in the accompanying claims, the various substituents and hydrogen atoms are shown as being joined to the steroid nucleus by one of these notations: namely, a solid line (-) indicating a substituent or hydrogen atom which has the p-configuration (i e above the plane of the molecule), a broken line ( 1111111) indicating a substituent or hydrogen atom which has 30 the a-configuration (i e below the plane of the molecule) or a wavy line (') indicating a substituent or hydrogen atom which may have the a or 3configuration The formulae all show the compounds in their absolute stereochemical configurations Since the starting materials are derived from naturally occurring stigmasterol, the products exist in the single absolute 35 configuration shown herein.

The Greek letter xi () in the name of a vitamin D 3 intermediate or metabolite indicates that the stereochemistry of the substituent to which it refers is undefined.

The nomenclature adopted to define the absolute configuration of a substituent bound to carbon atom 24 of the steroid nucleus is described in The 40 Journal of Organic Chemistry, 35, 2849 ( 1970) under the title "IUPAC Tentative Rules for the Nomenclature of Organic Chemistry Section E Fundamental Stereochemistry".

The present invention also provides a synthesis of 24 R,25 and 245,25dihydroxycholesterol and of alkanoyl derivatives thereof which involves the retro-i 45 rearrangement of compounds of the general formula 1,564,810 c 1 3 (I), wherein R 1 represents a hydroxy, lower alkoxy, phenyl-(lower alkoxy), lower alkanoyloxy or benzoyloxy group, to cholesterols of the general formulae OH NCH, H e Ia 33 H CH 3 -_ -1 (Iha), wherein R 9 represents a hydroxy or lower alkanoyloxy group, configuration at C-24 is R, H and the absolute (I Ib), wherein R 9 has the significance given earlier and the absolute configuration at C-24 is H H CH 3 H (II Ia), wherein R and Ro each represent the same lower alkanoyloxy group and the absolute configuration at C-24 is R, (II Ib), Cl O" 4 1,564,810 4 wherein R 9 o and R 10 each represent the same lower alkanoyloxy group and the absolute configuration at C-24 is S.

These conversions can be carried out by treating an i-cholesteryl diol of formula I with an acid in a suitable solvolytic medium For example, 24 R and 5 24 S,25-dihydroxycholesterol (i e a compound of formula I Ia or I Ib in which R 9 represents a hydroxy group) can be prepared by solvolysing a compound of formula I with a strong acid in an aqueous medium containing a miscible cosolvent.

Suitable stong acids for this purpose include mineral acids such as hydrochloric acid, hydrobromic acid and sulphuric acid and organic sulphonic acids such 10 as benzenesulphonic acid and p-toluenesulphonic acid Sulphuric acid is preferred As suitable miscible co-solvents there may be mentioned ethereal solvents such as tetrahydrofuran and dioxane and ketones such as acetone and methyl ethyl ketone Ethereal solvents are preferred Dioxane is most preferred.

While the retro-i-steroid rearrangement proceeds readily over a wide 15 temperature range, it is preferable to carry out the rearrangement within the temperature range of from 25 C to the boiling point of the medium For most solvent systems, a temperature of about 80 C is most preferred.

It it is desired to prepare a 3-(lower alkanoyloxy) derivative (i e a compound of formula I Ia or I Ib in which R 9 represents a lower alkanoyloxy group) , the retro-i 20 rearrangement is carried out in a solvent system containing the alkanoic acid corresponding to the alkanoyloxy group desired at the 3-position For example, 24 R,25 or 24 S,25-dihydroxycholesteryl 3-acetate is prepared using glacial acetic acid as the solvent medium In this case, a strong acid is not required since the alkanoic acid solvent is sufficiently strong to serve as the acidic source In order to 25 promote the solvolysis, sodium acetate is preferably added to the medium, the solvolysis may be carried out at an elevated temperature between 40 C and the boiling point of the medium A temperature of about 60 C is most preferred.

If it is desired to prepare a 3,24-di(lower alkanoyloxy) derivative (i e a compound of formula II Ia or II Ib), the solvolysis is carried out in a solvent medium 30 comprising the lower alkanoic acid and lower alkanoic acid anhydride corresponding to the alkanoyloxy group desired at the 3 and 24-positions For example, 24 R,25 or 24 S,25-dihydroxycholesterol 3,24-diacetate is prepared using glacial acetic acid and acetic anhydride as the solvent medium and sodium acetate to promote the solvolysis Elevated temperatures within the range of from 40 C to 35 the boiling point of the solvent system may also be used to promote the retro-irearrangement A solvolysis temperature of about 60 C is most preferred.

24 R,25 and 24 S,25-dihydroxycholesterol and the alkanoyl derivatives thereof are useful intermediates in the preparation of the C-24 stereoisomers of the biologically important metabolite of vitamin D 3, 24 R,25 40 dihydroxycholecalciferol and the unnatural 24 S-stereoisomer by wellknown routes This transformation is carried out by the introduction of the A 7double bond (generally by a halogenation/dehydrohalogenation procedure) followed by photolysis of the diene, thermal isomerisation of the pre-vitamin and hydrolysis of the alkanoyl groups, if necessary (see, for example, J Redel, et al, supra, and 45 H.-Y Lam et al, supra).

The invention also provides compounds of general formula II Ia, substantially free of the 24 S epimer; compounds of general formula II Ib, substantially free of the 24 R epimer; 24 S,25-dihydroxycholesterol substantially free of the 24 R epimer; and 24 R,25-dihydroxycholesterol substantially free of the 24 S epimer 50 The following Examples illustrate the present invention:

Example 1.

24 S,25-Dihydroxycholesterol A solution of 0 108 g ( 0 00025 mol) of 24 S,25-dihydroxy-6 p-methoxy-3 a, 5cyclo-5 a-cholestane, I ml of O 1-N aqueous sulphuric acid and 4 ml of dioxane 55 was stirred at 80 C for 4 hours 50 ml of water were added to the mixture and the solution was extracted with three 50 ml portions of methylene chloride The combined organic extracts were washed with 50 ml of saturated aqueous sodium bicarbonate solution, dried over anhydrous magnesium sulphate and filtered.

Recrystallisation of the residue (obtained by evaporation of the filtrate) from 60 methanol gave 0 085 g ( 81 %) of 24 S,25-dihydroxycholesterol of melting point 196 -198 C: lai D 5 = -46 O (c = 0 99 in methanol).

Example 2.

24 S,25-Dihydroxycholesteryl 3-acetate A mixture of 0 200 g ( 0 00046 mol) of 24 S,25-dihydroxy-6/3-methoxy-3 o C,5cyclo-5 a-cholestane and 3 ml of 1-M sodium acetate in acetic acid was heated for 32 hours at 60 C 50 ml of methylene chloride were added to the mixture and the 5 solution was washed with 25 ml of water and then with 25 ml of saturated aqueous sodium bicarbonate solution and dried over anhydrous magnesium sulphate.

Evaporation of the solvent (after removal of the drying agent by filtration) gave 0.210 g ( 99 %) of 24 S,25-dihydroxycholesteryl 3-acetate Recrystallisation from ethyl acetate gave a pure sample of melting point 157 -158 C; llD 5 = 58 3 (c = 10 1.03 in chloroform).

Example 3.

24 S,25-Dihydroxycholesterol 3,24-diacetate A mixture of 0 020 g ( 0 000046 mol) of 24 S,25-dihydroxy-6/3-methoxy-3 a, 5cyclo-5 a-cholestane, 0 024 g ( 0 000235 mol) of acetic anhydride and 1 ml of I-M 15 sodium acetate in acetic acid was stirred at 60 C for 24 hours 25 ml of methylene chloride were added to the mixture and the solution was washed with 25 ml of water and 25 ml of saturated aqueous sodium bicarbonate solution and dried over anhydrous magnesium sulphate Evaporation of the solvent (after removal of the drying agent by filtration) and recrystallisation of the residue from gave 0 018 g 20 ( 78 %) of 24 S,25-dihydroxycholesterol 3,24-diacetate of melting point 173 -174 C; la 25 = -40 90 (c = 0:9 in chloroform).

Example 4.

24 R,25-Dihydroxycholesterol A solution of 0 194 g ( 0 00045 mol) of 24 R,25-dihydroxy-6/3-methoxy-3 a, 5 25 cyclo-5 a-cholestane, 2 ml of O 1-N aqueous sulphuric acid and 6 ml of dioxane was stirred at 80 C for 4 hours 50 ml of water were added to the mixture and the solution was extracted with three 50 ml portions of methylene chloride The combined organic extracts were washed with 50 ml of saturated aqueous sodium bicarbonate solution, dried over anhydrous magnesium sulphate and filtered 30 Recrystallisation of the residue obtained by evaporation of the solvent gave 0 150 g ( 79 %) of 24 R,25-dihydroxycholesterol of melting point 200 -202 C; la 125 D = 11 6 (c = 0 93 in methanol).

Example 5.

24 R,25-Dihydroxycholesteryl 3-acetate 35 A solution of 0 200 g ( 0 00046 mol) of 24 R,25-dihydroxy-6/-methoxy-3 a, 5cyclo-5 a-cholestane and 3 ml of 1-M sodium acetate in 3 ml of acetic acid was heated at 60 C for 24 hours 25 ml of methylene chloride were added to the mixture and the solution was washed with 25 ml of water and then with 25 ml of saturated aqueous sodium bicarbonate solution and dried over anhydrous magnesium 40 sulphate The drying agent was collected on a filter and the filtrate was evaporated.

Recrystallisation of the residue from gave 0 187 g ( 88 %) of 24 R,25dihydroxycholesteryl 3-acetate of melting point 164 -165 C; lal 5 = -31 2 (c = 0 95 in chloroform).

Example 6 45

24 R,25-Dihydroxycholesterol 3,24-diacetate A mixture of 0 030 g ( 0 00007 mol) of 24 R,25-dihydroxy-6/3-methoxy-3 a, 5cyclo-5 a-cholestane, 0 020 g ( 0 00020 mol) of acetic anhydride and 1 ml of 1-M sodium acetate in acetic acid was stirred at 60 C for 24 hours 25 ml of water were added to the mixture and the solution was extracted with three 25 ml portions of 50 methylene chloride The combined organic layers were washed with two 25 ml portions of saturated aqueous sodium bicarbonate solution, dried over anhydrous magnesium sulphate and filtered Evaporation of the filtrate followed by recrystallisation of the residue yielded 0 030 g ( 85 %/) of 24 R,25dihydroxycholesterol 3,24-diacetate of melting point 122 -123 C; lal 25 = -36 8 55 (c = 1 10 in chloroform).

The compounds of formula I herein are described and claimed in our copending Application No 41938/76 (Serial No 1,564,806).

1,564,810 S 1,564,810

Claims (1)

1. WHAT WE CLAIM IS:-

   1 A compound of the general formula I Ia-I, H 1 wherein R 9 o represents a lower alkanoyloxy group and wherein the absolute configuration at C-24 is R, said compound being substantially free of the 245 epimer.

   2 24 R,25-Dihydroxycholesteryl 3-acetate.

   3 A compound of the general formula I Ib-1, wherein R 9,0 represents a lower alkanoyloxy group and wherein the absolute configuration at C-24 is S, said compound being substantially free of the 24 R epimer.

   4 24 S,25-Dihydroxycholesteryl 3-acetate.

   A process for the preparation of a compound of the general formula OH (II), wherein R 9 represents a hydroxy or lower alkanoyloxy group, and wherein the absolute configuration at C-24 is R or S, which process comprises treating a compound of the general formula CH 3 OH (I), wherein R 1 represents a hydroxy, lower alkoxy, phenyl-(lower alkoxy), lower alkanoyloxy or benzoyloxy group, with an acid in a solvent medium comprising Rg H (R 9 having the significance given earlier in this claim) at a temperature between 25 C and the boiling point of the medium.

   6 A process according to claim 5, wherein R 1 represents a lower alkoxy group.

   7 A process according to claim 6, wherein R 1 represents a methoxy group.

   8 A process according to any one of claims 5 to 7 inclusive, wherein the 5 absolute configuration of the C-24 hydroxyl group is R.

   9 A process according to any one of claims 5 to 7 inclusive, wherein the absolute configuration of the C-24 hydroxyl group is S.

   A process according to any one of claims 5 to 9 inclusive wherein'the acid is a strong acid 10 11 A process according to claim 10, wherein the strong acid is sulphuric acid or an organic sulphonic acid.

   12 A process according to claim 11, wherein the strong acid is sulphuric acid.

   13 A process according to any one of claims 5 to 12 inclusive, wherein the solvent medium comprises water 15 14 A pr'ocess according to any one of claims 5 to 12 inclusive, wherein the solvent medium comprises acetic acid.

   A process according to any one of claims 5 to 14 inclusive, wherein the temperature is 80 C.

   16 A compound of the general formula 20 3 H s R 1 O Ct 32,, /H 3 11 C 3 (II 1 b), wherein Rgo and Ro 10 each represent the same lower alkanoyloxy,group and the absolute configuration at C-24 is S, said compound being substantially free of the 24 R epimer 25 17 A compound of the general formula CHIP 33 (I Ila), R g o wherein Ro and Ro each represent the same lower alkanoyloxy group and the absolute configuration at C-24 is R, said compound being substantially free of the 245 30 epimer.

   18 24 R,25-Dihydroxycholesterol 3,24-diacetate.

   19 24 S,25-Dihydroxycholesterol 3,24-diacetate.

   A process for the preparation of a compound of the general formula Cl 131 3 row "HH 35 3 (III), 1,564,810 8 1,564,810 8 wherein R 0, and R 10 each represent the same lower alkan 6 yloxy group, and wherein the absolute configuration at C-24 is R or S, which process comprises contacting a compound of the general formula CH' H 3 CR 13 c 13 wherein R 1 represents a hydroxy, lower alkoxy, phenyl-(lower alkoxy), lower alkanoyloxy or benzoyloxy group, with an acid comprising R 9 o H (R 90 representing a lower alkanoyl group) in a solvent medium comprising R 90 H and (Rs)20 (R 5 representing a lower alkanoyl group) at a temperature between 40 C and the 10 boiling point of the medium.

   21 A process according to claim 20, wherein R 1 represents a lower alkoxy group.

   22 A process according to claim 21, wherein R 1 represents a methoxy group.

   23 A process according to any one of claims 20 to 22 inclusive, wherein the 15 absolute configuration of the C-24 hydroxyl group is R.

   24 A process according to any one of claims 20 to 22 inclusive, wherein the absolute configuration of the C-24 hydroxyl group is S.

   A process according to any one of claims 20 to 24 inclusive, whereini the acid comprises acetic acid 20 26 A process according to any one of claims 20 to 25 inclusive, wherein the solvent medium comprises acetic acid and acetic anhydride.

   27 A process according to any of claims 20 to 26, wherein the temperature is about 60 C.

   28 24 S,25-Dihydroxycholesterol substantially free of the 24 R epimer 25 29 24 R,25-Dihydroxycholesterol substantially free of the 24 S epimer.

   A process for the preparation of cholesterol derivatives, substantially as hereinbefore described with reference to any one of the foregoing Examples.

   For the Applicants, CARPMAELS & RANSFORD, Chartered Patent Agents, 43 Bloomsbury Square, London, WCIA 2 RA.

   Reference has been directed in pursuance of section 9, subsection ( 1) of the Patents Act 1949, to patent No 1,455,789.

   Printed for Her Majesty's Stationery Office by the Courier Press, Leamington Spa, 1980.

   Published by the Patent Office, 25 Southampton Buildings, London, WC 2 A l AY, from which copies may be obtained.