IE57951B1 - 1alpha-hydroxyvitamin d2 analogs and process for preparing same - Google Patents

Abstract

Novel vitamin D derivatives, analogs of vitamin D2 compounds which lack the 24-methyl substituent and are identified as 1 alpha -hydroxy-28-norvitamin D2 and 1 alpha ,25-dihydroxy-28-norvitamin D2. The compounds of the invention are characterized by unexpectedly high vitamin D-like activity as well as a novel activity pattern. Because of such activity they would find ready application as substitutes for vitamin D or various of the known vitamin D metabolites in their various application for the treatment of calcium disorders.

Description

WISCONSIN ALUMNI RESEARCH FOUNDATION, a Corporation organised and existing under the laws of the State of Wisconsin, United States of America, of 61//, North Walnut Street, Madison, Wisconsin 53705, United States of America.

The invention relates to biologically active 1 Because of the well-known and clearly established activity of 1«-hydroxyvitamin D compounds in regulating calcium and pnosphate homeostasis in the animal or numan, there nas been interest in the preparation of the natural metabolites and in the discovery of analogs with useful biological properties. This nas led to the preparation of a variety of compounds exhibiting biological activity. Interest in sucn compounds is continuing especially now that it has been recognized that in addition to their classical function as regulators of calcium homeostasis, certain vitamin D derivatives, specifically 1O<, 25-dihydroxyvitamin and 10i-hydroxyvitamin , also affect cellular differentiation processes and are capable of inhibiting the growth and proliferation of certain leukemic cells [see U.S. Patent 4,391,802; Proc. Natl. Acad. USA 80 , 201 (1 983); Nature, 306, 492-494 ( 1 983 ) ] .

Most of the known vitamin D metabolites and analogs are derivatives of the vitamin series, i.e. they possess saturated steroid side chains. Side cnain unsaturated vitamin D compounds are, however, also known, namely certain hydroxy-derivatives of vitamin such as 25-hydroxy-,1«,25-dihydroxy-, 24-hydroxyand 24,25-dihydroxy- metabolites and Ic^-hydroxy-vitamin D2, as well as certain related 22-trans-dehydro compounds lacking the 24-methyl substituent.

The present invention provides the vitamin D analogs which are characterized by the structure: wherein R is hydrogen or a hydroxy group. The compound of this invention wherein R is hydrogen can be obtained as an intermediate in the preparation of the compound where R is hydroxy. Structurally the compounds of this invention are analogs of hydroxyvitamin compounds which lack the 24-methyl substituent, i.e. the compounds are iQi-hydroxy28-norvitamin and 1α<, 25-dihydroxy-28-norvitamin , respectively.

Both the product and the intermediate exhibit high biological activity and are characterized by an unexpected and novel pattern of activity.

An embodiment of preparing the compounds of this invention is depicted in Process Scheme I. In the following description, compound designations by Arabic Numerals (e.g. (/), (2), (/),... (/0), (//), (/2)) refer to the structure so numbered in the Process Scheme.

The starting material is the diene-protected aldehyde of structure (/), which can be prepared from ergosterol acetate according to the method of Barton et al. (J. Chem. Soc. (C) 1968) (1971)). Reaction of aldehyde (/) with 3-methyl-1-butylphenylsulfone having the structure shown below: (CH3) 2CHCH2CH2-SO2Ph typically in an organic solvent and in tne presence of a strong 15 organic base, gives the hydroxy-sulfone intermediate (/).

Reduction of intermediate (/)eg. with sodium amalgam in in buffered alcohol solution, provides the 22,23-trans olefin (2zE-olefin) (/).

Reaction of the 22E-olefin (/) with a strong 20 nydride reducing reagent (e.g. LiAlH^) in an ether solvent gives the 5,7-diene intermediate (/). This intermediate can then be irradiated with ultraviolet light in an organic solvent to obtain the corresponding previtamin D derivative, which can be isolated and heated in an organic solvent at a temperature from room temperature to reflux to isomerize the previtamin D ciiromophore to the vitamin D chromophore, thus affording the 22E-dehydrovitamin D3 compound of structure (5) Conpound (5} is a known vitamin D analog, having been prepared previously by a less convenient method(J. Gen. Chem. USSR 48 (4), 828 (197«) ) .

Intermediate (^) can then be hyaroxylated at carbon Ί, according to the general procedure of DeLuca et al. (U.S. Patents 4,195,027 and 4,260,549). These reaction steps typically involve the reaction of conpound (5) with toluenesulfonyl cnloride in the conventional manner to obtain the corresponding 3B-tosylate derivative which is directly solvolyzed in buffered methanol to obtain the 3,5-cyclovitamin D intermediate (6); reaction with a different lcwer alkanol, eg. of 1 to 4 carbon atoms will yield the corresponding lower alkoxy conpound. By treatment of the latter with selenium dioxide and tert, -butyl hydroperoxide, there is obtained after chranatographic purification the corresponding 1hydroxylated product comprising chiefly the li-hydroxycyclovitamin D compound (_7) - A small amount of the corresponding Ιβ-hydroxy epimer may also be present in the product, but separation of the epimers, though possible by chromatography, is not necessary at this stage. Heating of this 1-hydroxycyclovitamin D intermediate in, say, glacial acetic acid at 40-60°C then provides a mixture of the 3-acetylated solvolysis products frcm wnich can be isolated by chromatography the 5,6-cis and 5,6-trans compounds (8) and (9), respectively. If the 1-hydroxycyclovitamin D product subjected to solvolysis contained sane 1β-hydroxy-epimer, then tne solvolysis mixture will contain also the 1ii-hydroxy epimers corresponding to canpounds (8j or (9} and, if desired, these can also be isolated by chromatography.

Conventional base hydrolysis of the acetate function in compound (8) yields the diol product of structure (10).

This diol (10) can then be subjected to in vitro enzymatic hydroxylation at carbon 25, using a liver homogenate prepared frcm vitamin D-deficient rats, (as described in United States Patent No. 4,307,025) to obtain, after chranatographic separation of the product mixture, the desired 1 In the following detailed description of the synthetic process the physicochemical data was obtained using the below referenced methods arid apparatus. High pressure liquid chromatography (HPLC) was performed on a Waters Associates Model ALC/GPC 204 using a Zorbax-Sil (Dupont) column (6.2 nm x 25 an column, flew rate 4 ml/min, I 1500 psi; 105 kg/αη ). Column chranatcyrapny was performed on Silica Gel 60, 70-230 mesh AS7M (212-63 nm) (Merck). Preparative thin-layer chromatography (TLC) was carried out on Silica 60 PF-254 (20 x 20 cm plates, 1 nm silica gel). Irradiations were carried out using a Hanovia 608A36 mercury arc lamp fitted with a Vycor filter. All reactions are preferably performed under an inert atmosphere (e.g. argon). 3-Methy 1-1-butylphenylsulfone (I sopentylphenylsulfone).

PhSOjNa (1.97 g, 12 nmol) was added to a stirred solution of 3-methyl-l-brranobutane (1.51 g, 1.2 ml, 10 nmol) in DMF (20 ml) at 75°C. Ihe mixture was heated at 75*C for 5 h, then cooled, poured into water and extracted with benzene. The organic layer was washed with 5% HCl, 5% NaHCO^ and water, dried over Na^SO^ and evaporated. The oily sulfone product (1.69 g, 80%) was substantially pure and was used without any purification; NMR δ 0.88 (6H, d, J=6.5 Hz, ΣχΟΗ^), 1.61 (3H, m, -CH2-CH), 3.08 (2H, m, SO2~CH2-), 7.50-7.95 (5H, m, Ar-H); IR; 1300 (br), 1147,1088,745,692,564,539 cm \ mass spectrum, m/e 212 (M+, 3), 143 (92), 77 (57), 71 (73), 70 (73), 55 (42), 43 (100), 41 (47). (22E) -5α, 8a- (4-pheny 1-1,2-urazolo) -cholesta-6,22-diene3g-ol (3). n-Butyllithium (1.7 M solution in hexane, 4.12 ml, mol) was added to a stirred solution of di-isopropylamine (707 mg, 1 ml, 7 nmol) in dry THF (14 ml) and the mixture was stirred for 15 min at roam temperature. The sulfone as prepared above (1.50 g, 7.07 mmol) in dry THF (11 ml) was added dropwise in 10 min. The solution was stirred at roan tenperature for an additional 15 min, then cooled to 0°C and aldehyde (1) (545 mg, 1 nmol) in dry THF (7 ml) was added.

The stirring was continued for 2 h at 0°C and solution was slcwly warmed to rocm tenperature (30 min). The mixture (containing hydroxy-sulfone product (2.)) was poured into saturated solution of Na2HPO4 in methanol (200 ml), sodium amalgam (5.65%, 10 g) was added and the reaction mixture was 35 stirred at 4 °C for 17 h. Precipitated mercury was filtered off and after concentration of the reaction mixture to 5 ml, it was diluted with water and extracted with methylene chloride. Organic extract was washed with water, dried (Na2SO4), concentrated in vacuo and the oily residue was chranatographed on silica gel column. Excess of sulfone reagent was eluted with benzene-ether (7:3) mixture. Elution with benzene ether (6:4) afforded pure adduct (3) (375 mg, 67%) as a foam: NMR 6 0.81 (3H, s, 18-¾), 0.86 (6H, d, J=6.7 Hz, 26-¾ and 27-¾) , 0.97 (3H, s, 19-¾), 1.03 (3H, d, J=6.8 Hz, 21-¾), 3.16 (IH, dd, ^=4.4 Hz, J2=14Hz, 9-H), 4.44 (IH, m, 3-H), 5.25 (2H, br m, 22-H and 23-H), 6.22 and 6.39 (2H, AB q, J=8.5 Hz, 6-H and 7-H), 7.40 (5H, br m, Ar-H); IR: 3444, 1754,1701,1599,1402,969,757 cm mass spectrum, m/e 557 (M+, 1%), 382 (70), 349 (51), 253 (28), 251 (45), 119 (PhNOO, 83), 55 (100). (22E) -Cholesta-5,7,22-trien-3fi-ol (4) . The adduct (3) (330 mg, 0.6 mmol) and lithium aluminum hydride (700 mg) in dry THF (40 ml) was heated under reflux for 18 h. The excess of reagent was decomposed with a few drops of water.

Anhydrous Na2SO4 was added and the organic phase was decanted and evaporated to give crystalline residue which was purified on a column of silica gel. Elution with benzene ether (94:6) mixture gave pure diene (4) (180 mg, 80%) which was crystallized from methanol: mp 119.5-122.5°C; [a]^= -118° (c=1.2, CHC13); NMR δ 0.63 (3H, s, 18-¾), 0.87 (6H, d, J=6.7 Hz, 26-¾ and 27-¾), 0.95 (3H, s, 19-¾), 1.03 (3H, d, J=6.8 Hz, 21-¾), 3.64 (IH, m, 3-H), 5.25 (2H, br m, 22-H and 23-H), .38 and 5.57 (2H, AB q, J=6 Hz, 7-H and 6-H); UV λ 281 nm; , max IR: 3436,1461,1382,1366,1062,1036,968 cm ; mass spectrum, m/e 382 (M+, 100), 349 (M+-H20-Me, 71), 323 (34), 271 (M+-side chain, 16), 253 (M+-side chain-H20, 32). (5Z,7E,22E)-9,10-Secocholesta-5,7,10(19),22-tetraen-3p-ol (5). A solution of compound (4) (100 mg, 0.26 mmol) in ether (120 ml)-benzene (30 ml) mixture was degassed with argon for 40 min. The solution was irradiated at 0°C for 13 min in a quartz immersion well equipped with UV lamp and filter. The solvent was removed under reduced pressure and the residue separated by HPLC, using 1% 2-propanol in hexane as eluent to obtain the pure previtamin D derivative (40.4 mg, 40%) which was collected at 24 ml; NMR 6 0.72 (3H, s, 18-H^), 0.87 (6H, d, J=6.7 Hz, 26-H3 and 27-H3), 1.04 (3H, d, J=6.8 Hz, 21-H3), 1.65 (3H, s, I9-H3), 3.91 (1H, m, 3-H), 5.28 (2H, br m, 22-H and 23-H), 5.50 (IH, m, 11-H), 5.69 and 5.96 (2H, AB q, J=12.5 Hz, 7-H and 6-H); UV λ 260.5 nm; λ . 234 nm. The max mm previtamin (40 mg, 0.1 nmol) in ethanol (100 ml) was heated under reflux for 3 h. After removal of solvent, the product mixture was separated by HPLC (elution with 1% 2-propanol in hexane). The yield of the vitamin (5) (collected at 34 ml) was 30.8 mg (77%); mp (hexane) 99-101°C; NMR 6 0.56 (3H, s, 18-H3), 0.88 (6H, d, J=6.7 Hz, 26-H3 and 27-H3), 1.02 (3H, d, J=6,6 Hz, 21-H3), 3.96 (IH, m, 3-H), 4.82 and 5.05 (2H, each narr. m, 19-^), 5.27 (2H, br m, 22-H and 23-H,, 6.03 and 6.24 (2H, ABq, J=11.4 Hz, 7-H and 6-H); UV λ 265 nm, λ . 228 nm; IR: 3420,1458,1441,1378,1366,1050,970,943,891,862 on ; mass spectrum, m/e (M+, 22), 349 (M+-H20-Me, 4), 271 (M+-side chain, 8), 253 (M+-side chain-H20, 13), 136 (100), 118 (80). (5Z,7E,22E)-3g-Acetoxy-9,10-secocholesta-5,7,10(19),22tetraen-la-ol (8). A freshly recrystallized p-toluenesulfonyl chloride (50 mg, 0.26 mmol) was added to a solution of vitamin (5) (30 mg, 0.08 mmol) in dry pyridine (300 μΐ). After 30 h at 4°C, the reaction mixture was poured into ice/saturated NaHCOg with stirring. The mixture was stirred for 15 min and extracted with benzene. The organic extract was washed with saturated NaHC03, saturated copper sulfate and water, dried (Na2SO4) and concentrated in vacuo to obtain the oily 36-tosyl derivative. The crude tosylate was treated with NaHC03 (150 mg) in anhydrous methanol (10 ml) and the mixture was stirred for 8.5 h at 55°C. After cooling and concentration to 2 ml the mixture was diluted with benzene (80 ml), washed with water, dried (Na2S04) and evaporated under reduced pressure.

The resulting oily 3,5-cyclovitamin D analog (6) was sufficiently pure to be used for the following oxidation step without any purification. To a vigorously stirred suspension of SeO2 (4 mg, 0.036 rnnol) in dry CH2C12 (5 ml), tert, -butylhydroperoxide (13.2 μΐ, 0.094 mmol) was added. After 30 min dry pyridine (40 μΐ) was added and the mixture was stirred for additional 25 min at room temperature, diluted with CH2C12 (3 ml) and cooled to 0°C. Ihe crude 3,5-cyclovitamin product (6) in CH2C12 (4.5 ml) was then added and the reaction permitted to proceed at O°C for 15 min. The mixture was then allowed to warm slcwly (30 min) to rocm temperature. The mixture was transferred to a separatory funnel and shaken with 30 ml of 10% NaOH. Ether (150 ml) was added and the separate organic phase was washed with 10% NaOH, water and dried over Na2S0^. Concentration to dryness in vacuo gave a yellow oily residue which was purified on silica gel TLC plates developed in 7; 3 hexane-ethyl acetate (R^ 0.35) giving 1-hydroxycyclo- vitamin product (14.4 mg, 45%): NMR 6 0.55 (3H, s, 18-H3), 0.64 (1H, m, 3-H), 0.88 (6H, d, J=6.9 Hz, 26-H3 and 27-H.*), 1.03 (3H, d, J=6.9 Hz, 21-H3), 3.26 (3H, s, -OCH3), 4.2 (2H, m, 1-H and 6-H), 4.95 (1H, d, J=9.3 Hz, 7-H), 5.1-5.4 (4H, br m, 19-H2, 22-H and 23-H); mass spectrum, m/e 412 (M+, 27), 380 (M+-MeOH, 46), 339 (22), 269 (M+-side chain-ifeOH, 29), 245 (18), 135 (100). Ihe above product comprised chiefly the la-hydroxycyclovitamin D analog of structure C7) and a small amount of the corresponding Ιβ-epimer. A solution of this 1-hydroxyclovitamin D product (12 mg) in glacial acetic acid (0.5 ml) was heated at 55°C for 15 min. Ihe mixture was carefully poured into ioe/saturated NaHC03 and extracted with benzene and ether. Ihe combined extracts were washed with water, dried (Na2S0^) and evaporated. The resulting product mixture was subjected to HPLC (1.5% 2-propanol in hexane as eluant) to obtain 4.9 mg of conpound (8) (eluting at 42 ml) and 3.1 mg of compound (9) (eluting at 50 ml).

Compound (8): NMR 6 0.56 (3H, s, 18¾), 0.88 (6H, d, J=7.0 Hz, 26¾ and 27¾) , 1.02 (3H, d, J=6.8 Hz, 21¾) , 2.04 (3H, s, -000¾), 4.41 (IH, m, 1-H), 5.02 (IH, narr. m, 19-H), .1-5.4 (4H, br m, 3-, 19-, 22- and 23-H), 6.03 and 6.35 (2H, AB q, J=11.4 Hz, 7-H and 6-H); UV λ 264 nm, λ . 227.5 nm; max min mass spectrum m/e 440 (Μ , 15), 380 (M -HOAc, 84), 362 (M+-HQAc-H2O, 9), 269 (M+-side chain-HQAc, 40), 251 (15), 135 (100), 134 (94).

Ccxipound (9): NMR 6 0.57 (3H, s, 18¾), 0.89 (6H, d, J=7.0 Hz, 26¾ and 27¾), 1.03 (3H, d, J=6.8 Hz, 21¾), 2.04 (3H, s, -000¾), 4.49 (IH, m, 1-H), 5.00 and 5.14 (2H, each narr. m, 19¾), 5.25 (3H, br m, 3-, 22- and 23-H), 5.81 and 6.58 (2H, AB q, J=12.0 Hz, 7-H and 6-H); UV λ 269.5 nm; λ . 228 max min nm; mass spectrum, m/e 440 (Μ , 6), 380 (47), 269 (15), 135 (100), 134 (62).

Also obtained frcm the solvolysis product mixture was a small amount (0.87 mg of the Ιβ-hydroxy-epimer corresponding to ccxipound (8), characterized by the following data: NMR 6 0.55 (3H, s, 18¾), 0.87 (6H, d, J=6.9 Hz, 26¾ and 27¾), 1.01 (3H, d, J=6.9 Hz, 21¾), 2.06 (3H, s, -000¾), 4.17 (IH, m, 1-H), 4.99 (2H, m, 3-H and 19-H), 5.1-5.4 (3H, br m, 19- , 22- and 23-H), 6.00 and 6.38 (2H, AB q, J=11.3 Hz, 7-H and 6-H); UV λ 262.5 nm, λ . 227 nm; mass spectrum, m/e max min 44$ (Μ , 27), 380 (78), 362 (12), 269 (28), 251 (20), 135 (100), 134 (78).

Hydrolysis of 3fi-acetoxy group in compounds (8) and (9).

A solution of the 33-acetoxyvitamin derivative (8) (0.7-6 mg) in ethanol (0.1 ml) was treated with 10% KOH in methanol (0.8 ml) and the mixture was heated for 1 h at 50°C. After usual work-up and final HPLC purification (8% 2-propanol in hexane as eluent), there was obtained the la,3p-diol of structure (10) in 84% yield: NMR 6 0.56 (3H, s, 18¾), 0.87 (6H, d, J=7.0 Hz, 26¾ and 27¾), 1.02 (3H, d, J=6.8 Hz, 21¾), 4.22 (IH, m, 3-H), 4.42 (IH, m, 1-H), 5.00 (IH, narr. m, 19-H), 5.1-5.4 (3H, br m, 19-, 22- and 23-H), 6.01 and 6.38 (2H, AB q, J=11.4 Hz, 7-H and 6-H); UV λ 264.5 nm, λ max min 227.5 nm; mass spectrum, m/e 398 (Μ , 21), 380 (M -^0, 9), 2.87 (M+-side chain, 6), 269 (M+-side chain-HjO, 8), 251 (5), 152 (38), 134 (100). Compound (10) elutes at 40 ml in the above HPLC system.

Analogous treatment of the acetoxy derivative (/), gave after HPLC purification the 5,6-trans-la,3 β-diol of structure (11) in 72% yield: NMR 6 0.58 (3H, s, 18-H3), 0.87 (6H, d, J=7.0 Hz, 26-H3 and 27-H.j), 1.03 (3H, d, J=6.8 Hz, 21-H3), 4.24 (IH, m, 3-H), 4.49 (IH, m, 1-H), 4.97 and 5.13 (2H, each narr. m, 19-^), 5.25 (2H, br m, 22-H and 23-H), 5.88 and 6.58 (2H, AB q, J=11.5 Hz, 7-H and 6-H); UV λ 273 nm, λ . 229.5 max min nm; mass spectrum, m/e 398 (Μ , 21), 380 (5), 287 (6), 269 (5), 251 (4), 152 (33), 134 (100). Coirpound (11) elutes at 38 ml.

-Hydroxylation of conpound (10). The 5,6-cis-la,3βdiol of structure (10) as obtained above was then 25-hydroxylated by the following procedure: Male weanling rats were fed a low calcium vitamin D-deficient diet as described by Suda et al. J. Nutr. 100, 1049 (1970) for 2 weeks. They were killed by decapitation and their livers were removed. A 20% (w/v) homogenate was prepared in ice-cold 0.25 M sucrose. Incubation was carried out in 10 ml incubation medium in a 125 ml Erlenmayer flask containing an aliquot of liver homogenate representing 1 g of tissue, 0.125 M sucrose, 50 mM phosphate buffer (pH 7.4), 22.4 mM glucose-6-phosphate, 20 m ATP, 160 mM nicotinamide, 25 mM succinate, 0.4 mM NADP, 5 mM MgC^, °·1 M KCI and 0.5 units glucose-e-phosphatedehydrogenase. The reaction was initiated by addition of 400 pg of compound (10) dissolved in 100 pi 95% ethanol. The mixture was incubated at 37°C with shaking at 80 oscillations/ min for 3 h. The reaction was stopped by the addition of 20 ml methanol and 10 ml dichloromethane. After further addition of 10 ml dichloromethane, the organic phase was collected 35v;hile aqueous phase was re-extracted with 10 ml dichloro1 2 methane. The organic p’.ases frcm total of three extractions were combined and evaporated with a rotary evaporator. The residue containing the desired product was dissolved in 1 ml of CHCl^:Hexane (65:35) mixture and applied to a Sephadex LH-2O column (0.7 cm x 14 cm) packed, equilibrated and eluted with the same solvent. The first 10 ml was discarded while the next 40 ml was collected and evaporated. The residue was then dissolved in 8% 2-propanol in hexane and subjected to high performance liquid chromatography (Model LC/GPC 204 HPLC, Waters Associates, Medford, MA) using a Zorbax-Sil column (4.6 nm x 25 cm, Dupont, Inc., Wilmington, DE) operating under 2 pressure of 1000 psi (70 kg/cm ) with a flew rate of 2 ml/min.

The desired 25-hydroxylated product was eluted at 42 ml. The product was further purified by high performance liquid chromatography using a reverse phase column (Richrosorb Rp-18, 4.6 nm x 25 cm, E. Merck, Darmstadt, West Germany) operated 2 under pressure of 1200 psi (84 kg/αη ) and a flew rate of 2 ml/min The column was eluted with 22 % H^O in methanol and the product was eluted at 50 ml. The product was further purified by HPLC using Zorbax-Sil and conditions as described above. The resulting product was characterized by the following data: UV absorption (95% ethanol) λ 265 nm, λ . 228 nm; mass r max min ' spectrum, m/z 414 (mol. ion M ), 396 (M-^0), 378 (M -2x^0), 287 (M+-side chain), 269 (M+-side chain-H20), 251 (M+-side chain-2xH2O), 152 (ring A, C 7/8 bond cleavage), 134 (152-H2O) and 59 ((CH^)2C=0H)+ resulting frcm C24/25-bond cleavage).

The above data, especially the characteristic ultraviolet absorption and the prominent and diagnostic peaks at m/z 152, 134 and 59 in the mass spectrum, establish the product to be the desired 25-hydroxylated compound represented by structure (12) If desired, the canpounds of this invention can be readily obtained in crystalline form by crystallization frcm suitable solvents, e.g. hexane, ethers, alcohols, or mixtures thereof, as will be apparent to those skilled in the art. 3 The biological potency of the dehydrovitamin D of this invention was estaf)! i shed by in vivo assays in the rat and comparison with lot-hydroxyvitamin D compounds of known potency. Both product (12) and the key intermediate, compound (10) were tested and found to be highly active.

Biological Activity of compound (10). The assay was conducted as follows; Male weanling rats were purchased frcm Holtzman Co., Madison, WI and fed ad libtum water and a lew calcium-adequate phosphorus, vitamin D-deficient diet as described by Suda et al. (J. Nutr. 100, 1049 (1970)) for three weeks. Rats were then divided into groups of 6 rats each and were given 650 pmol of either compound (10) or la-hydroxyvitamin D^ (la-OH-D^) dissolved in 0.05 ml 95% ethanol intra jugular ly 18 h prior to sacrifice. The rats in the control group were given ethanol vehicle in the same manner.

The rats were killed by decapitation and the blood was collected. Serum obtained by centrifugation of the blood was diluted with 0.1% lanthanum chloride solution (1:20) and serum calcium concentration was measured with an atomic absorption spectrophotometer. Results are shown in the Table I below: Table 1 Increase in serum calcium concentration in response to a single dose of 650 pmol of either compound (10) or or Ια-ΟΗ-Dy given 18 h prior to sacrifice Compound Given Serum Calcium Concentration (mg/100 ml) + Standard Deviation ethanol compound (10) la-0H-D3 cl) is significantly different from p <0.001. 3.2 + 0.1 a 4.5 + 0.4 b) 4.7 + 0.5 b) b) Biological Activity of compound (12). The assay was performed as follows: Male weanling rats were fed a low calcium vitamin D-deficient diet for 3 weeks as described above. They were then divided into groups of 5 rats each. Rats in a control group received 0.05 ml 95% ethanol intra jugular ly while rats in the test groups were given 325 pmol of either compound (12) or of la, 25-dihydroxyvitamin D^ (la, 25- (OH) dissolved in 0.05 ml 95% ethanol. Eighteen hours later they were killed by decapitation and blood was collected. Serum calcium concentration was determined with an atomic absorption spectrophotometer as described above. Results are shown in Table II belcw: Table II Increase in serum calcium concentration in response to single dose of 325 pool of either conpound (12) or la, 25- (OH) 2D3 given 18 h prior to sacrifice Ccmpound Given Serum Calcium Concentration (mg/100 ml) + Standard Deviation ethanol 4.2 + 0.1 ccmpound (12) 5.0 + 0.4 b) la, 25- (OH) 2D3 5.4 + 0.4 is significantly different frcm a^ p<0.001.

The above results show that both conpound (10) and the final product (12) of this invention are highly active in promoting a rise in serum calcium levels of vitamin D-deficient rats. They are, in fact, equivalent in biological potency to the corresponding known side chain-saturated ccmpounds, Ια-ΟΗ-D^ and la,25-(OH) 2D^, the high potency and pharmaceutical utility of which is well documented by many reports in the general and patent literature (e.g. U.S.

Patent 4,225,596.

Particularly noteworthy and unexpected is the superior activity of compound (10) in mineralizing the bones of an animal (chick) that discriminates against the physiological utilization of the vitamin D2 compounds. This is amply illustrated by the following data.

Method Day-old white Leghorn male chicks were obtained frcm Northern Hatcheries (Beaver Dam, WI). They were fed the vitamin D-deficient soy protein diet described in Cmdahl et al (Biochemistry 10, 2935-2940, 1971) for 3 weeks at which time they were vitamin D deficient. They were then divided into groups of 6 chicks each. One group received Wesson oil vehicle by mouth; the other groups received the indicated compounds (see Table III) dissolved in the same amount of Wesson oil each day for 7 days. Twenty-four hours after the last dose, all chicks were killed by cervical dislocation. Their tibiae were removed and freed of adherent soft and connective tissue and extracted for 24 hours in alcohol followed by 24 hours in diethyl ether using a Soxhlet extractor. The bones were then dried to constant weight at 100°F and weighed. They were then ashed at 650°C for 24 hours in a muffle furnace. The ash was weighed and the percent ash in each of the tibiae was calculated. Results are shown in Table III below. i16 Table III Mineralization of Rachitic Chick Bone Compound Amount Number of Animals % Ash (pmole/d/7 days) -D 0 6 32.0 + 1.0 1,25-(QH)2D3 130 6 41.0 + 3.4 * la-OH-D. 130 6 42.0 + 3.2 * 3 la-0H-D_ 1300 6 38.0 + 4.0 * 2 — Carpound (10) 130 6 38.0 + 3.9 * ♦These values are not significantly different Ihe results demonstrate that 130 pmoles of either 1,25(OH)2D3 or la—OH—or the novel conpound (10) of this invention, were equally effective in increasing the mineral content of the rachitic tibia. Hcwever, to achieve the same degree of effectiveness, 1300 pmoles of la-OH15 TJhis agrees with previous results which shew that a ΙΟ-fold greater dose of la-0H-D2 than Ια-ΟΗ-D^ is required to produce the same mineralization of the tibia of rachitic chickens. These results demonstrate, surprisingly, that although compound (10) is an analog of la-0H-D2, it offers unexpected and superior activity in mineralizing the bones of an animal that is known to discriminate against the vitamin D2 compounds in physiological utilization. This is an unexpected characteristic of and utility for this new analog. Since the unexpected in vivo activity of conpound (10) is undoubtedly manifested after hydroxylation to the corresponding la, 25-dihydroxy compound (compound (12)) in vivo, that compound, i.e. compound (12), which is the 24-desmethyl analog of the known la, 25-dihydroxyvitamin D^, should function to provide bone mineralization in chicks equivalent to that shown by la, 25-dihydroxyvitamin D^; thus this novel vitamin D2 analog would be fully active in the chick. This high potency of compounds (10) and (12) in an animal that discriminates against vitamin D2-type derivatives, is a novel and distinctive characteristic of these substances. ; Because of this marked biological activity, the compounds of this invention will find ready application as substitutes ‘ for various of the known vitamin D metabolites in the therapy or prophylaxis of calcium metabolism disorders such as rickets, hypoparathyroidism, osteodystrophy, osteomalacia or osteoporosis in the human, or related calcium deficiency diseases (e.g. milk fever) in animals. Likewise, these ccmpounds may be used for the treatment of nertain malignancies, such as human leukemia. In view of the marked and unexpected activity of the compounds of this invention in promoting the mineralization of bone in birds (see Table III), these ccmpounds would have particular utility in the prevention or treatment of calcium imbalance-induced conditions (e.g. egg shell thinness, poultry leg weakness) in poultry where all the known vitamin D2 derivatives exhibit very poor activity.

For therapeutic purposes, the ccmpounds may be administered by any conventional route of administration and in any form suitable for the method of administration selected. The ccmpounds may be formulated with any acceptable and innocuous pharmaceutical carrier, in the form of, for exanple, pills, tablets, gelatin capsules, or suppositories, or as solutions, emulsions, dispersions or suspensions in innocuous solvents or oils; such formulations may contain also other , therapeutically active and beneficial ingredients as may be appropriate for the specific applications. For human 1 applications, the compounds are advantageously administered in amounts from 0.5 to 10 pg per day, the specific dosage being adjusted in accordance with the specific compound administered, the disease treated and the condition and response of the subject, as is well understood by those skilled in the art.

Claims (11)

1. A compound having the formula: HO wherein R is hydrogen or hydroxy.
2. 2. The compound according to claim 1 wherein R is hydrogen.
3. 3. The compound according to claim 2 in crystalline form.
4. 4. The compound according to claim 1 wherein R is hydroxy.
5. 5. The compound according to claim 4 in crystalline form.
6. 6. A process for preparing a compound as claimed in any one of claims 1 to 5 which coinprises hydroxylating the loc-position of the compound of the formula: HO in known manner and, optionally, hydroxylating the 25 position of the resulting 1d-hydroxy compound in known manner.
7. 7. A process according to claim 6 substantially as hereinbefore described.
8. 8. A compound as defined in claim 1 whenever prepared by a process as claimed in claim 6 or 7. 5
9. 9. A pharmaceutical composition which comprises a compound as claimed in any one of claims 1 to 5 and a pharmaceutically acceptable excipient.
10. 10. A composition according to claim 9 which comprises the compound of claim 2. 10
11. 11. A composition according to claim 9 which comprises the compound of claim 4.