GB2089811A - 23,25-Dihydroxyvitamin D3 - Google Patents

Abstract

23,25-Dihydroxyvitamin D3 and its hydroxy-protected derivatives of formula <IMAGE> where R1, R2, & R3 are hydrogen, acyl or alkylsilyl are disclosed. The compound is the 23-hydroxylated analog of biologically potent 23-hydroxy- cholecalciferol and by virtue of such structural similarity should find application as a substitute for such compound or for vitamin D3. It is prepared from <IMAGE>

Description

1 GB 2089811 A 1

SPECIFICATION

23,25-Dihydroxyvitamin D3 This invention relates to a novel derivative of 70 vitamin D3.

Vitamin D3 is a well-known agent for the maintenance of calcium and phosphorus hom eostasis in animals or humans, and is widely used therapeutically for the prevention of rick ets and other bone deseases. It has now been clearly established that the biological effective ness of vitamin D in the animal or human depends upon its being metabolically con verted to 25-hydroxyvitamin D, which inter mediate compound undergoes further side chain hydroxylation in vivo to 24,25-dihy droxyvitamin D3 and 25,26-dihydroxyvitamin D3. The foregoing metabolically produced de rivatives have been isolated and characterised and shown to exhibit pronounced biological activity.

A new side chain-hydroxylated vitamin D compound has now been found. This novel compound is characterized by hydroxy substi tution at carbons 23 and 25 and can be represented by the formula shown below; it can alternatively be referred to as 23,25 dihydroxycholecalciferol,23,25-dihydroxyvi- tamin D. or 23,25-DHCC.

HO OH 0OH H 1 1 tkt r 23,25-Dihydroxy vitamin D3 can be prepared from 25-hydroxyvitamin D, by in vitro enzy- matic hydroxylation, using a kidney homogen- ate system prepared from the kidneys of chickens fed a vitamin Dsupplemented diet. The required kidney homogenate was pre- pared as follows:

Single-comb White Leghorn chicks (North- ern Hatcheries, Beaver Dam, Wisconsin) were fed a normal diet for 10 weeks and given 101 I U vitamin D3 per day for 3 days followed by 1. 5 X 101 1 U for 1 day, 4 days prior to sacrifice. The chickens were killed, their kid- neys removed and a 20% (w/v) homogenate was prepared in ice-cold buffer solution containing 0. 1 M phosphate buffer (pH 7.4) and 0.25 M sucrose.

The kidney homogenate was then used for the preparation of 23,25-dihydroxyvitamin D3 according to the following procedure:

Three ml of the homogenate (containing 600 mg of tissue) was placed in a 125 ml Erlenmeyer flask. To this homogenate was then added 1.5 ml of 0.1 M phosphate buffer (pH 7.4) containing 22.4 mM glucose-6-phosphate, 20 mM ATP (adenosine triphosphate), 160 mM nicotinamide, and 0.4 mM NADP (nicotinamide adenine dinucleotide phosphate), and 1.5 ml of a salt solution contain'12, 25 mM succi- ing 0.1 M KCI, 4 mM Mg' nate, 10 ILg DPPD (N,NI-diphenyi-p-phenylene diamine) and 5 units of glucose-6-phosphate dehydrogenase. The reaction was initiated by addition of 80 ttg 25-hydroxyvitamin D3 dissolved in 20 pl 95% ethanol. The mixtures were incubated at 37'C with shaking at 100 oscillation/min for 2 hours under an atmosphere of air. The reaction was stopped by addition of a methanol: chloroform mixture. The incubation mixture was transferred to a phase separating funnel and the organic phase was collected. The aqueous phase was extracted once more with methanol /chloro- form mixture, and the organic extracts were pooled and solvent evaporated.

The residue was dissolved in CHCI,/hexane (65/35) and applied to a Sephadex LH-20 column (0.7 X 14 cm) packed and eluted in the same solvent. The first 11 ml of elutant was discarded and the next 20 ml of elutant was pooled.

After evaporation of solvent the residue was further purified on high pressure liquid chro- matography (HPLC) as follows: the sample was applied onto a silica gel column (4.6 mm X 25 cm, Zorbax-Sil, a product marketed by Dupont Instruments, Wilmington, Delaware) operated under pressure of 100 psi and a flow rate of 2 ml/min in a model ALC/GPC 204 chromatograph (Waters Associates, Medford, Mass) eqipped with a UV monitor operating at 254 nm. The column was eluted with 6% 2-propanol in hexane. The desired prod- uct was eluted at 24-27 ml. The collected product was then chromatographed on a reverse phase HPLC system using a Lichrosorb RP-1 8 column (0.46 X 25 cm, a product of E Merck, Darmstadt, West Germany) and 25% H,O in MeOH was eluting solvent. The desired product eluting at 40-44 ml was collected and further purified by HPLC using the silica gel column (Zorbax-Sil) and conditions as described above. The resulting puri- fied product was then subjected to physical characterization.

Characterization of the product as 23,25-dihy droxyvitamin D3 UV spectrophotometry of the compound in methanol showed a typical vitamin D, absorption maximum of 265 nm and a minimum at 228 nm indicating the presence of an intact 5,6-cis-triene chromophore in the product.

The mass spectrum of the compound exhib- 2 ited a molecular ion at m/e 416 as expected for a derivative of 25-hydroxyvitamin D3 bear ing an additional hydroxy function. The pres ence of this additional hydroxy function in the side chain was evident from the peaks at m/e 271 and 253 (271-H,O), and the characteris tic ions at m/e 136 and 118 confirmed an unaltered ring A and triene structure. The position of the side chain hydroxy groups was established by the mass spectrum of the tris trimethylsily ether derivative of the com pound. The spectrum showed a molecular ion at m/e 623 as required for a tri-silylated derivative of 23,25-dihydroxyvitamin D3. The spectrum also showed characteristic side 80 chain cleavage peaks at m/e 131 ((CH 3)2C =tSi(CH.W which established the pres ence of a hydroxy group at C-25, and at m/e 487 (M + - 145) vvhich proves the presence of a second hydroxyl at carbon 23.

These data therefore established the struc ture of this new vitamin D, as 23,25-dihy droxyvitamin D3, A convenient process for the chemical syn thesis of this novel vitamin D metabolite and of its hydroxy-protected derivatives, which also form part of the present invention, is described below.

The starting material for this process is a ketosteroid of general structure I shown be low, OR 2 0 R 1 0 1 where each of R, and R, is independently hydrogen, acyl or alkylsilyl, especially hydro- gen, acetyl or benzoyl, and where the substi- 110 tuent at carbon 23 has the stereoconfiguration (R) or (s), desired in the final product.

As used in this specification the word acyl- denotes an aliphatic acyl group of 1 to 5 carbons, i.e. acety], propiony], butyryl, pentoyl and the isomeric forms thereof, or an aromatic acyl group, such as benzoyi, or a halo-or nitro-benzoy]. The word---alkyl- denotes an aikyl group of 1 to 5 carbons i.e.

methyl, ethyl, propyl, butyl, pentyl and the isomeric forms thereof.

The keto steroid ot structure 1, above, where R, and R2 is hydrogen, is available according to the procedure of Wichmann et al, Tetrahedron Letters, 21 p 4667-4670 (1980), and the corresponding hydroxy-protected derivatives (e.g. acyl or alkylsily] derivatives) can readily be prepared from this material. Thus, treatment of the dihydroxy corn- pound with ace-tic anhydride in pyridine gives GB 2 089 81 1A 2 the 3,23-diacetate. Other derivatives such as the dibenzoate or di- trimethylsilyl ether derivative are available by analogous methods well known the art. All of these derivatives are suitable for the subsequent steps of the process; hydroxy-protection is therefore not essential.

Treatment of the 25-keto-starting material with methyl Grignard reagent (e.g. methyl magnesium bromide) or with methyl lithium in a suitable solvent (e.g. diethyl ether or tetrahydrofuran) yields the trihydroxysteroid of general structure Ii shown below 11 R 1 0 1 OR 2 OR 3 During this Grignard or methyl lithium reaction step and subsequent workup, hydroxy protecting groups that may have been present in the starting material (e.g. acyl, alkylsilyl) would normally be removed to yield product 11 above where R, R2 and R, are hydrogen. If desired, the hydroxy groups may be reprotected, e.g. by acylation or alkylsilylation according to standard methods to yield the corresponding partially or completely hydroxy- protected product of structure 11 shown above, where each of R, R2 and R, is independently hydrogen, acyl or alkylsily], especially hydrogen, acetyl or benzoyi. For example, treatment of the trihydroxy product with acetic anhy- dride at room temperature in pyridine yields the 3,23-diacetyl compound, whereas at elevated temperatures (75-1 OWC) the 3,23,25triacetyl derivative is obtained. By analogous methods fully or partially hydroxy- protected derivatives (e.g. derivatives carrying other acyl groups, such as benzoy], e.g. 3,23,25-tribenzoyl or alkylsilyl protecting groups) can be obtained. A partially acylated derivative (e.g. 3,23-diacyl) may also be further acylated (e.g.

at C-25) by a different acyl group, or may be alkylsilylated to obtain derivatives carrying mixed hydroxy-protecting functions, and selective removal of protecting groups is also readily accomplished, e.g. selective hydrolysis (10% KOH/MeOH, room temperature) of a 3,23,25-tri-0-acyl derivative to yield the 3,23-dihydroxy-25-0-acyl compound. Different protecting groups can then be introduced at the free hydroxy-positions, and it is thus obvious that combination of selective protection/deprotection steps will yield derivatives with any desired combination of hydroxy protecting groups in the product. Hydroxy protection is, however, not required for the subse- quent conversions.

k 3 GB 2 089 811 A 3 Ultra-violet irradiation of the above 5,7diene intermediate, 11, in a suitable solvent (e.g. aliphatic or aromatic hydrocarbon, ethanol, methanol or ether) yields the correspond- Ing previtamin derivative of the general structure III shown below R 1 0 i 2 OR 3 75 -5M where each of R, R2 and R3 IS independently hydrogen, acyl or alkylsilyl.

This previtamin D intermediate is allowed to stand in solution at room temperature or slightly elevated temperature for a prolonged time (e.g. several days) or, preferably, is subjected to brief heating, say, 50-80C, in a suitable solvent (e.g. an alcoholic solvent, ether, benzene or an aliphatic hydrocarbon solvent) to yield the desired vitamin D compound of general structure IV below R 1 0 1 1 OR 2 OR 3 A_ where each of IR, R, and R3 is independently hydrogen, acyl, or alkylsilyl. In general a temperature from 25' to 1 OWC is suitable.

If hydroxy protecting groups are present, these may be removed according to standard procedures (e.g. heating in 5% methanolic base for, say, 2 hr, or treatment with a hydride reagent, for removal of acyl groups, or treatment of an alcoholic solution of the compound with very dilute acid at room tem- perature for removal of alkylsilyl groups) to obtain the corresponding free hydroxy-compound, 23,25-dihydroxyvitamin D,, represented by structure IV above, where R, R2 and R3 are hydrogen.

The following Examples further illustrate the 125 present invention.

Example 1

27-nor-23-hydroxy-25-keto-cholesta-5,7-di- ene was prepared as a mixture of the two 130 possible C-23 epimers according to the procedure of Wichmann et al., in Tetrahedron Letters 21, 4667-4670 (1980). These epimers were separated as described in the above reference to yield, in pure form, each of the two epimers, designated here for convenience, isomer A and isomer B, respectively.

Isomer A (5 mg) dissolved in 5 ml of diethylether was treated with a 1 0fold excess of methyl magnesium bromide. The mixture was stirred at room temperature for 15 min, then treated with 25 ml of 1 N HCI and extracted with CH2C'2; the extracts were washed with dilute bicarbonate and saturated NaCksolutions, and after evaporation of Solvents, the product was purified on high-performance liquid chromatography (HPLC), using a ItPorasil column (0.79 X 30 cm; Waters Associates, Milford, MA) and 3.5% isopropa- 8Enol in CH2Cl2 as eluting solvent, to yield 2.4 mg of cholesta-5,7-diene- 3,8-23,25-triolisomer A, eluting at about 39 ml, exhibiting the following mass spectrum: m/e 416, 100% M +; 398, 10%, M -I-H20; 383, 60%, M-1-1-120-Ch3; 357, 21%, M±C3H,O; 342, 23% M±C3H,O-CH,; 271, 31%, M± side chain; 143, 83% C,,H,,+.

A portion of this product (0.5 mg) was irradiated for 15 min in 150 ml of 20% benzene in diethyl ether using a quartz immersion well and a Hanovia608A36 lamp fitted with a Corex filter. After solvent evaporation, the desired previtamin D product (23,25-dihydroxy previtamin D3-'somer A) was purified by HPLC (0.62 cm X 25 cm column of ZorbaxSIL, Dupont, Inc., Wilmington, Delaware) eluted with 3.5% isopropyl alcohol in CH2C'2' The previtamin product eluted at ca. 51 ml in this system.

This previtamin D-product, dissolved in 2 ml of ethanol, was heated to 70'C for 2.5 hours, to effect the conversion of the previtamin to the vitamin carbon skeleton. Purifiaction by HPLC (0.62 X 25 cm, Zorbax-Sil) eluted with 4% isopropyl alcohol in CH2Cl2 gave 0.24 mg of 23,25dihydroxyvitamin D3isomer A, collected at 28 ml, and exhibiting the following spectral properties: UV Xmzn, 265 nm, Amin 228 nm; mass spectrum, mle 416.3319 (calculated, M + = 416.3290) 27%, M +; 398, 2.5%, M + -H20; 383, 13%, M±H20-CH3; 271, 8.5%, M±side chain; 253, 9.7%, 271 -H20; 136, 100%, ring A + C6, C7; 118, 136-H20; NMR (CDCI,), 8 6.23, d, J = 11 Hz, C-6; 6.03, d, J = 11 Hz, C-7; 5.05, m, C-19(E); 4.81, m, C-19(Z); 4.13, m, C-23; 3.94, m, C-3; 1.33, s, C-26; 1.26, s, C-27; 1. 00, d, J 7 Hz, C-2 1; 0. 58, s, C- 18; Example 2

A solution of 5 mg of 27-nor-23-hydroxy25-ketocholesta-5,7-diene, isomer B, obtained as described in Example 1 above, was treated with methyl magnesium bromide using the 4 GB 2089811 A 4 conditions described in Example 1, and after HPLC purification of the resulting product using the conditions described in Example 1, 1.2 mg of cholesta-5,7-diene-3,8-23,25-triol, isomer B, was obtained (eluting at ca 58 mi 70 on HPLC) exhibiting a mass spectrum with mle 416, 83%, M +; 398, 11 %, M + -1-120; 383, 80% M+ HP-Ch,; 357, 33%, M±Ch3H,O; 342, 22%, 357H20; 271, 23%, M 1 -side chain; 143, 100%, Cl 1 H 11.

About 0.5 mg of this product was irradiated using the experimental conditions of Example 1 to give 23,25-dihydroxy-previtamin D, isomer B, which was purified by HPLC using a 0.79 X 30 em gPorasil column, eluted with 7% of 2-propanol in hexane. The desired previtamin was collected at ca 38 mi, and then subjected to thermal isomerization (heating in ethanol solution to 7WC for 2.5 hrs, as described above). Subsequent purification of the resulting product on HPLC, using a 0.62 X 23 em Zorbax-Sil column, and 7% 2propanol in hexane as eluant, gave at ca. 35 mi elution volume, the desired 23,25-dihy- droxyvitamin D3, isomer B, with the following spectral properties: LIV, 265 nm; knin 228 nm; mass spectrum, mle 416.3273 (calculated, 416.3290), 18%, M+; 398, 2%, M-1-120; 383, 10%, M±H20-CH3; 271, 7%, M ±side chain; 253, 8%, 271 -1-120; 136, 100%, A ring + C6 and C7; 118, 97%, 136-1-120; NMR (CDCI,) 8 6.23, d, J = 10.5 Hz, C-6; 6.03, d, J = 10.5 Hz, C7; 5.05, m, C-19ffl; 4.82, m, C-lg(Z); 4.10, m, C-23; 3.95, m, C-3; 1.32, s, 100 C26; 1.28, s, C-27; 0.98, d, J = 6 Hz, C-21; 0.56, s, C-18. This compound is chromatographically and spectrally identical with the natural product, as obtained from the chick kidney homogenates.

From the foregoing it is evident also that ketosteroid of general structure 1 comprising a mixture of both possible C-23-stereoisomers (epimeric mixture) can be subjected to the specified process steps, whereby an epimeric mixture of the vitamin product of general structure IV is obtained, from which, if de sired, the two C-23-epimers can be obtained in pure form by chromatographic separation (e.g. hplc on Zorbax-Sil, using 5% 2-propanol 115 mula in hexane as eluting solvent).

Since 23,25-dihydroxyvitamin D, is the 23 hydroxylated analog of the biologically potent 25-hydroxy vitamin D3, by virtue of such structural similarity the compounds of this invention should find application as a substi tute for 25-hydroxy vitamin D. in various therapeutic applications or as a substitute for vitamin D3.

1 R1 0 OR 2 OR 3 1 1 1 1 wherein each of R, R2 and R3 is independently hydrogen, acyl or alkylsilyl.

2. A compound according to claim 1 wherein each of R, R2 and R. is independently acetyl or benzoy].

3. A compound according to claim 1 or 2 wherein the substituent of carbon 23 has the (R)-configuration.

4. A compound according to claim 1 or 2 wherein the substituent of carbon 23 has the (S)-config u ration.

5. 23,25-dihydroxyvitamin D, 6. A process for preparing a vitamin D, derivative as claimed in any one o claims 1 to 5 which comprises:

treating a compound having the general formula OR 2 0 1 1 p ,,C 1 wherein each of R, and R2 is independently 110 hydrogen, acyl or alkylsilyl with a methylGrignard or methyl lithium reagent, and optionally reprotecting one or more of any free hydroxy groups by acylation or alkylsilylation, whereby a compound having the general for-

Claims (1)

1. CLAIMS 1. A vitamin D, derivative having the formula

   1 i i GB 2089 811 A 5 R 1 0 OR2 OR 3 70 1,1il i where each of R,, R2 and R. is independently hydrogen, acyl or alkylsilyl is obtained, subjecting said compound to actinic radiation 20 whereby a compound having the formula R 1 0 - 90 OR 2 OR 3 is obtained, maintaining said compound in a solvent at a temperature of 25 to 1 OWC, and optionally removing one or more of any hydroxy protecting groups by hydrolysis or hydride reduction, and recovering the desired vitamin D3 derivative.

   7. A process according to claim 6 wherein each of R, and R2 in the keto-steroid subjected to the methyl-Grignard or methyl lithium reagent is independently hydrogen, acetyl or benzoyi.

   8. A process according to claim 7 wherein each of R, and IR,, is hydrogen.

   9. A process according to claim 7 wherein each of R, and R2 is acetyl.

   10. A process according to any one of claims 6 to 9 for producing 23,25dihydroxy- vitamin D3.

   11. A process according to claim 6 substantially as described in Example 1 or 2.

   12. A vitamin D3 derivative as defined in claim 1 whenever prepared by a process as claimed in any one of claims 6 to 11.

   13. A compound of the formula OR 2 OR3 7 5 R10 11.11 -_1 1 wherein each of R, R2 and R3 'S independently hydrogen, acyl or alkylsily].

   14. A compound according to claim 13 wherein each of R,, R, and R3 isindependently acetyl or benzoly.

   15. Cholesta-5,7-diene-3p-23,25-triol.

   16. The compound of claim 15 having the R configuration at carbon 23.

   17. The compound of claim 15 having the S configuration at carbon 23.

   18. A compound of the formula OR 2 OR 3 IR 1 R, 0 wherein each of R, R2 and R. is indepen100 dently hydrogen acyl or alkylsilyl.

   19. A compound according to claim 18 wherein each of R, R2, and R3 is hydrogen.

   20. A compound according to claim 18 wherein each of R, R2 and R3 is indepen105 dently acetyl or benzoyi.

   Printed for Her Majesty's Stationery Office boy Sur ass Et Son (Abingdon) Ltd.-I 982. Publ ad at The Patent Office, 25 Southampton Buildings, London, WC2A 1AY, from which copies may be obtained.