The object of the invention is a new method of prepara- tion of cholecalciferol derivatives, particularly la, 24- dihydroxy-cholecalciferol, and new intermediate compounds used in the method.

Cholecalciferol derivatives, that are active metabolites of vitamin D3, exhibit activity that regulates calcium homeo- stasis in the body as well as initiate cell differentiation.

This property, exhibited particularly by la, 24 (R)-dihydroxy- cholecalciferol, make it possible to use the said derivatives in treatment of a serious skin disease, psoriasis, caused by incomplete differentiation and rapid proliferation of epider- mal cells.

The known method of preparation of cholecalciferol de- rivatives, such as for example la, 24 (R)- dihydroxycholecalciferol (known as tacalcitol), and its stereomer of la, 24 (S) configuration, described in the Dutch Patent Application No. 7507268, consists in subjecting natu- ral, hard available fucosterol to multistep linear synthesis.

The main step of the synthesis includes reaction of ozoniza- tion of the double bond in the side chain of fucosterol and reduction of the resulting carbonyl group to the hydroxyl group. After more than ten steps, mixture of diastereomeric alcohols is obtained which has to be separated by chromatog- raphy on silica gel impregnated with AgN03.

Other methods, disclosed for example in Japanese patent publications No. J5 1076253 and J5 2042864, include methods of modification of steroid precursor backbone of cholestadi- ene, containing a carbon side chain substituted with hydroxyl at C-24.

All these methods are time-and labour-consuming because of multistep synthesis and chromatographic separation of the resulting diastereomer mixtures.

A method of stereoselective total synthesis of la, 24 (R)- dihydroxycholecalciferol starting from fragments of A ring and CD ring system of vitamin D molecule and involving inter- mediate dienyl derivative is also described in the literature (Tetrahedron, 53, p. 4703 (1997)). The key step of side chain synthesis is an opening of a chiral epoxy compound with a use of nitrile anion of Inhoffen-Lythgoc diol precursor.

The objective of the present invention was to develop a practical and effective method of synthesis of cholecalcif- erol derivatives starting from readily available raw materi- als, that would enable preparation of optically pure chole- calciferol derivatives directly from chemical synthesis with- out a subsequent step of diastereomer mixture separation.

The essential feature of the present invention is a method of preparation of cholecalciferol derivatives, par- ticularly la, 24-dihydroxycholecalciferol, of general formula 1, wherein R1 and R2 are hydrogen, hydroxyl or hydroxyl pro- tecting group, R1 and R2 being the same or different, that consists in reacting phenylsulfonyl derivative of formula 2, wherein R1 and R2 are hydrogen lub hydroxyl protecting group,

with 1,2-epoxy-3-methylobutane of (S) or (R) configuration, respectively, at carbon atom C-2 prepared in a separate proc- ess or, in situ". The reaction is carried out in the presence of an aprotic solvent and a strong organic base, to yield hy- droxysulfon of (R) or (S) configuration, respectively, of formula 3, wherein Ri and R2 are as defined above, and the configuration at carbon atom C-22 is represented by formula 3a and 3b. In the next step, a phenylsulfonyl group is re- moved from hydroxysulfone of formula 3 by means of reduction, to yield a compound of formula 4, wherein the carbon atom C- 22 is of configuration represented by formula 4a or 4b, from which optionally hydroxyl protecting groups are removed using a known method.

As organometallic compounds, lithium, sodium or potas- sium organic compounds are used, preferably butyl lithium.

As aprotic solvents, alkylated phosphoric acid amides or urea alkyl derivatives, preferably hexamethyltriamide of phosphoric acid (HMPT), are used.

Phenylsulfonyl group may be removed by reduction with metals, preferably with sodium amalgamate.

Hydroxyl protecting group is any protecting group used in vitamin D chemistry to protect hydroxyl groups, such as for example acyl, alkylsilyl or alkoxyalkyl group.

The term"acyl group"includes alkanoyl and carboxyal- kanoyl groups, of 1 do 6 carbon atoms, such as in particular acetate group. Alkylsilyl protecting groups are such groups as trimethylsilyl, triethylsilyl, t-butyldimethylsilyl. Typi- cal alkoxyalkyl groups are: metoxymethyl, etoxymethyl, tetra-

hydrofuranyl and tetrahydropyranyl.

The object of the invention are also new hydroxysulfones represented by the formula 3, prepared as stereomers of dif- ferent configuration at carbon atoms C-22 and C-24. In these formulae substituents R1 and R2 at carbon atoms C-1 and C-3 denote hydrogen or protected hydroxyl group, R1 and R2 being the same or different.

A further aspect of the invention are new compounds of formula 4, of configuration at carbon atom C-24 represented by formulae 4a or 4b, wherein R1 and R2 have the same meaning as given for compounds of formula 3.

All the new compounds are intermediate compounds in the method of the invention.

The starting compounds for cholecalciferol derivatives synthesis: C-22 vitamin sulfones represented by formula 2, are key synthons used in chemistry of vitamins.

Sulfones of formula 2 may be prepared from the respec- tive C-22 alcohols using known methods, analogously to prepa- ration of C-25 sulfones with C-25 vitamin alcohols, as de- scribed in Polish patent application No. P-310454.

The method according to the invention enables to prepare cholecalciferol derivatives, particularly substituted with hydroxyl group at position 24, such as for example la, 24 (R)- dihydroxycholecalciferol of formula la and la, 24 (S)- dihydroxycholecalciferol of formula lb, starting from com- pounds readily commercially available or synthesised in a simple way.

On the Scheme 1 preparation of la, 24 (R)-dihydroxy-

cholecalciferol by the method according to the invention is presented.

On the Scheme 2 preparation of la, 24 (S)-dihydroxy- cholecalciferol by the method according to the invention is presented.

The invention is illustrated by the following examples, which do not limit the scope of the invention.

Example 1.

Preparation of (5Z, 7E)- (lS, 3R, 24R)-1,3-di-tert- butyldimethylsilyl-22-phenylsulfonyl-9,10-secocholesta- 5,7,10 (19)-triene-24-ol (formulae 3a and 3b) Solution of sulfone of formula 2 (110 mg, 158 pmol) in 800 1 THF, containing several crystals of 1,10-phenanthroline, was cooled to-70°C and then solution of n-BuLi in hexane (150 pL, 1.6 M, 240 mole) was added. The solution was stirred at -700C for 20 min and (S)-1,2-epoxy-3-methylbutane { 25D = +4.3° c 2 MeOH, 50 1,580 pmole} diluted with 50 pl THF was added. The solution was stirred for 20 min at-70°C and then 100 ml HMPT was added. The stirring was continued at-30°C for 5 h, and then the solution was allowed to stand at-18°C for 15 h until the substrate disapeared (TLC using 18% ethyl acetate in hexane system). To the mixture 0.5 ml brine was added and the products were extracted with ethyl acetate. The products were dissolved in benzene and subjected to"flash" column chromatography on silica gel (300-400 mesh). The hy- droxysulfones of formulae 3a and 3b (total amount: 75 mg,

60%) were isolated using solvent mixture (hexane-ethyl ace- tate (8-14%) as colourless oil: UV (EtOH) klmax 265.2 nm; B2max 265.0 nm; TLC (18% ethyl acetate in hexane): Rlf = 0.37, Rif = 0.32.

Example 2.

Preparation of (5Z, 7E)- (lS, 3R, 24R)-9,10-secocholesta- 5,7,10 (19)-triene-1,3,24-triol (formula la) To the solution of mixture of sulfones of formulae 3a and 3b (75 mg) in 100 1 THF, 1 ml of methanol saturated with Na2HPO4 and solid Na2HPO4 were added. The resulting suspen- sion was stirred for 15 min at 20°C. Then, the freshly pre- pared sodium amalgamate (5% Na/Hg) was added and the suspen- sion was stirred at 40°C for 1 h until the substrate disap- peared (TLC using 18% ethyl acetate in hexane system). To the suspension 1 ml ethyl acetate was added and filtered through 5 g silica gel (5 g) using 50 ml mixture eluting 12% ethyl acetate in hexane. Alcohol of formula 4a (52 mg, 84%) was ob- tained as colourless oil. The oil was dissolved in 2 ml of metanol-chloroform 1: 1 mixture, then 15 mg of 10- camphorsulfonic acid was added and the resulting mixture was stirred at 20°C for 2.5 h. The solvents were removed under reduced pressure and the product dissolved in chloroform was subjected to"flash"column chromatography on silica gel t (300-400 mesh) (5 g). Chromatographically pure triol of for- mula la (21 mg, 65%) was isolated using a solvent mixture (30-35% ethyl acetate in hexane) as colourless oil. TLC Rf

value was identical as that of 1,25-dihydroxycholecalciferol in straight phases (75% ethyl acetate in hexane) and reverse phases (8% water in methanol); HPLC, Chiralpak AD column, 4,6 x 25 mm, 15% EtOH in hexane, RU = 9.7 ml IR (film) 3357, 2946,1645,1055 cm-1; UV (EtOH) Xmax 265.2 nm, min 228.6 nm, A265/A228 = 1. 70; [a] D23 +33. 8° (c 0.86 CHC13); 1H NMR (CDC13) 8 (ppm): 0.55 (3H, s, 18-CH3), 0.91 and 0.92 (3H and 3H, dd, J=6.8 Hz, 26-and 27-CH3), 0.94 (3H, d, J=5.7 Hz, 21- CH3), 3.32 (1H, m, 24-H), 4.23 (m, 1H, 3-H), 4.43 (1H, m, 1- H), 5.00 (1H, d, J=3 Hz, 19Z-H), 5.33 (1H, d, J=3 Hz, 19E-H), 6.01 (1H, d, J=11.5 Hz, 7-H), 6.38 (1H, d, J=11.5 Hz, 6-H); EIMS m/z (relative intensity) 416 (M+, 18), 398 (27), 380 (13), 365 (5), 251 (20), 152 (42), 134 (100), HRMS, calcu- lated for C27H440 416.3290, found 416.3291.

Example 3.

Preparation of (5Z, 7E)- (lS, 3R, 24S)-1,3-di-tert-butyldimethyl- 10 (19)-triene- 24-ol (formula 3c) Hydroxysulfone of formula 3c was prepared from sulfone of formula 2 (110 mg, 0,158 mmole) and (R)-1,2-epoxy-3- methylbutane ( [a] 25 D-4. 30 c 2.1 MeOH, 40 . 1, 0.46 mmole} according to the method described in Example 1. Sulfone of formula 2 (40 mg) was recovered by column chromatography and hydroxysulfone of formula 3c was obtained (70 mg, Yield 89% recalculated to the reacted substrat); TLC, 18% ethyl acetate in hexane, Rf =0.34; UV (EtOH) Xmax =265 nm.

Example 4.

Preparation of (5Z, 7E)- (lS, 3R, 24S)-9,10-secocholesta- 5,7,10 (19)-triene-1,3,24-triol (Formula lb) Alcohol of formula 4b was prepared from hydroxysulfone of formula 3c (70 mg) according to method described in Example 2. After performing column chromatography, alcohol of formula 4b was obtained (48 mg, Yield 83%) as colourless oil. Triol of formula lb was obtained from alcohol 4b (48 mg) according to the method described in Example 2. After performing column chromatography, triol lb (21 mg, Yield 68%) was obtained as colourless oil. Preparative HPLC chromatography (column 22 x 25 cm, Lichrosorb Si 60,10 mm, 15% propanol-2 in hexane) yielded triol of formula lb as colourless oil (19 mg): HPLC, column 4.4 x 25 cm, Hibar Si 60,14% propanol-2 in hexane, RV 12 ml, Chiralpak AD column, 4.6 x 25 cm, 15% EtOH in hexane, RV = 6.6 ml: IR (film) 3371,2949,1650,1063 cm- ; UV (EtOH) Xmax 265-2 nm, Xmin 228. 6 nm, A26s/A228 = 1-73; [a] D24 +23.50 (c 1.07 CHC13); 1H NMR (CDC13) 8 0.54 (3H, s, 18- CH3), 0.90 and 0.93 (3H and 3H, dd, J=6.5 Hz, 26-and 27- CH3), 0.94 (3H, d, J=5.7 Hz, 21-CH3), 3.32 (1H, m, 24-H), 4.22 (m, 1H, 3-H), 4.43 (1H, m, 1-H), 5.02 (1H, d, J=3 Hz, 19Z-H), 5.32 (1H, d, J=3 Hz, 19E-H), 6.01 (1H, d, J=11.5 Hz, 7-H), 6.39 (1H, d, J=11.5 Hz, 6-H), MS, m/z (relative inten- sity) 416 (M+, 13), 398 (34), 380 (18), 287 (6), 269 (9), 251 (13), 152 (35), 134 (100); HRMS calculated for C27H4403: 416.3290, obtained: 416.3291.