IE850520L - 1ó-hydroxy vitamin d2 analogues - Google Patents

Abstract

Novel vitamin D derivatives, analogs of vitamin D2? compounds which lack the 24-methyl substituent and are identified as 1$g(a)-hydroxy-28-norvitamin D2? and 1$g(a),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. [WO8503939A1]

Description

E> i n .> i PATENTS ACT, 1964 COMPLETE SPECIFICATION let-HYDROXYVITAMIN D2 ANALOGS AND PROCESS FOR PREPARING SAME ""PLICATION No 520 h'f 1 f io,\> r.^{.eo 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. ( ;j L 2 The invention relates to biologically active 1o(-hydroxyvitamin analogs which exhibit unexpected biological properties.

Because of the well-known and clearly established 5 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 zo the preparation of a variety 10 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 , 25-dihydroxyvitamin and 15 lQt-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 8Jj, 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 3 unsaturated vitamin D compounds are, however, also known, namely certain hydroxy-aerivatives of vitamin such as 25-hydroxy-, lot, 25-dihydroxy-, 24-hydroxy-and 24,25-dihydroxy- metabolites and M-hydroxy-vitamin 5 D^, as well as certain related 22-trans-dehydro compounas 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 15 are analogs of hydroxyvitamin compounds which lack the 24-methyl substituent, i.e. the compounds are 1<*-hydroxy-28-norvitamin and 1 o#,25-dihydroxy-28-norvitamin , respectively.

Both the product and the intermediate exhibit 20 high biological activity and are characterized by an unexpected and novel pattern of activity. 4 An embodiment of preparing the compounds of this invention is depicted in Process Scheme I. In the following description, compound designations by Araoic Numerals (e.g. (1), (2), (3),... (10), (J_J_) , (12)) refer 5 to the structure so numbered in the Process Scheme.

The starting material is the diene-protected aldehyde of structure Q), which can be prepared from ergosterol acetate according to the method of Barton et al. (J. Chem. Soc. (C) 1968) (1971)). Reaction of 10 aldehyde (1) with 3-methyl-1-butylphenylsulfone having the structure shown below: (CH3) 2CHCH2CH2-S02Ph typically in an organic solvent and in tne presence of a strong 15 organic base, gives the hydroxy-sulfone intermediate (2). Reduction of intermediate {2)eg. with sodium amalgam in in buffered alcohol solution, provides the 22,23-trans olefin (2zE-olefin) (_3) .

Reaction of the 22E-olefin (3) with a strong 20 hydride reducing reagent (e.g. LiAlH^) in an ether solvent gives the 5,7-diene intermediate M). 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 25 temperature from room temperature to reflux to isomerize the previtamin D cnromophore to the vitamin D chromophore, thus affording the 22E-dehydrovitamin compound of structure (5).

Compound (j>) is a known vitamin D analog, having been prepared previously by a less convenient method(J. Gen. Chem. USSR 48 (4), 828 (197b) ) .

Intermediate (50 can then be hyaroxylated at carbon i, according to the general procedure of DeLuca et al. (U.S. Patents 4,195,027 and 5 4,260,549). These reaction steps typically involve the reaction of compound (Jb) with toluenesulfonyl cnloride in the conventional manner to obtain the corresponding 36-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 10 carbon atoms will yield the corresponding lower alkoxy compound. By treatment of the latter with selenium dioxide and tert. -butyl hydroperoxide, there is obtained after chromatographic purification the corresponding 1-hydroxylated product catprising chiefly the Id-hydraxycyclovitamin D compound (2)- A small amount of the corresponding 113-hydroxy epijmer may also be pre-sent 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 20 compounds (8) and (9), respectively. If the 1-hydroxycyclovitamin D product subjected to solvolysis contained sctne 16-hydroxy-epimer, then tne solvolysis mixture will contain also the 1 (3-hydroxy epimers corresponding to compounds (£}) or (9J and, if desired, these can also be isolated by chrcmatography.

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

This diol (J_0) can then be subjected to ijn vitro enzymatic hydroxylation at carbon 25, using a liver hcmogenate prepared frcm vitamin D-deficient rats, (as described in United States Patent 30 No. 4,307,025) to obtain, after chromatographic separation of the product mixture, the desired 1 cc ,25-dihydroxylated product (J_2), in pure form.

In the following detailed description of the synthetic process the physicochemical data was obtained using the below referenced methods and 35 apparatus. High pressure liquid chrcmatography (HPLC) was performed on a Waters Associates Model ALC/GPC 204 using a Zorhax-Sil (Dupont) column (6.2 mm x 25 cm column, flew rate 4 ml/'min, s 2 1500 psi; 105 kg/cm ). Column chromatography was performed on Silica Gel 60, 70-230 mesh ASTM (212-63 nm) (Merck). Preparative thin-layer chrcmatography (TLC) was carried out on Silica 60 PF-254 (20 x 20 cm plates, 1 nm silica gel). Irradiations were carried out 5 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-Methyl-l-butylphenylsulfone (I sopentylphenylsulfone).

PhSO^Na (1.97 g, 12 imol) was added to a stirred solution of 10 3-rethyl-l-bromobutane (1.51 g, 1.2 ml, 10 iiiidI) in IMF (20 ml) at 75°C. The 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% HC1, 5% NaHCO^ and water, dried over Na^SO^ and evaporated. The oily sulfane product 15 (1.69 g, 80%) was substantially pure and was used without any purification; NMR 6 0.88 (6H, d, J=6.5 Hz, 2x01^), 1.61 (3H, m, -CH2-CH), 3.08 (2H, m, S02~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), 20 43 (100), 41 (47). (22E) -5a, 8a- (4-phenyl-1,2-urazolo) -cholesta-6,22-diene-3g-ol (3) • n-Butyllithium (1.7 M solution in hexane, 4.12 ml, 7 rrmol) was added to a stirred solution of di-isppropylamine (707 mg, 1 ml, 7 rmol) in dry THF (14 ml) and the mixture was ^5 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 drqpwise in 10 min. The solution was stirred at roan temperature for an additional 15 min, then cooled to 0°C and aldehyde (1) (545 mg, 1 rrmol) in dry THF (7 ml) was added. 30 The stirring was continued for 2 h at 0°C and solution was slowly warmed to roan temperature (30 min). The mixture (containing hydroxy-sulfone product (2_)) was poured into saturated solution of Na2HP04 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 7 off and after concentration of the reaction mixture to f 5 ml, it was diluted with water and extracted with methylene chloride. Organic extract was washed with water, dried (Na2SOg), concentrated in vacuo and the oily residue was 5 chranatographed on silica gel column. Excess of sulfane 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-H^), 0.86 (6H, d, J=6.7 Hz, 26-H3 and 27-H3), 0.97 (3H, s, 19-H3), 1.03 (3H, d, J=6.8 10 Hz, 21-H3), 3.16 (1H, dd, ^=4.4 Hz, J2=14Hz, 9-H), 4.44 (1H, 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), 15 55 (100). (22E) -Cholesta-5,7,22-trien-3ft-ol (4). The adduct (3) (330 mg, 0.6 rtmol) 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 Na2S0^ 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 frcm methanol: mp 119.5-122.5°C; [a]-118° (c=1.2, CHC13); NMR 6 0.63 (3H, s, 18-H3), 0.87 (6H, d, J=6.7 Hz, 26-H3 and 27-H.j) , 0.95 (3H, s, 19-H3), 1.03 (3H, d, J=6.8 Hz, 21-H3), 3.64 (1H, 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 A 281 nm; max IR: 3436,1461,1382,1366,1062,1036,968 cm ; mass spectrum, 30 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-3 p-ol (_5). A solution of compound H) (100 mg, 0.26 ntnol) in ether (120 ml)-benzene (30 ml) mixture was degassed with argon for 35 40 min. The solution was irradiated at 0°C for 13 min in a 8 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, 19-H3), 3.91 (1H, m, 3-H), 5.28 (2H, br m, 22-H and 23-H), 5.50 (1H, m, 11-H), 5.69 and 5.96 (2H, AB q, J=12.5 Hz, 7-H and 6-H); UV X 260.5 nm; X . 234 nm. The max mm previtamin (40 mg, 0.1 itmol) 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%); irp (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 (1H, 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=ll.4 Hz, 7-H and 6-H); UV X 265 nm, X . 228 max min , nm; IR: 3420,1458,1441,1378,1366,1050,970,943,891,862 cm; 20 mass spectrum, m/e (M+, 22), 349 (M+-H20-Me, 4), 271 (M+-side chain, 8), 253 (M+-side chain-^O, 13), 136 (100), 118 (80). (52,7E,22E)-3 B-Acetoxy-9,10-secocholesta-5,7,10(19),22-tetraen-la-ol (8). A freshly recrystallized p-toluenesulfonyl chloride (50 mg, 0.26 mmol) was added to a solution of vitamin 25 (5) (30 mg, 0.08 rrmol) in dry pyridine (300 yil). After 30 h at 4°C, the reaction mixture was poured into ice/saturated NaHG03 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 30 (Na^SO^) and concentrated in vacuo to obtain the oily 38-tosyl derivative. The crude tosylate was treated with NaH003 (150 mg) in anhydrous methanol (10 ml) and the mixture was stirred for 8.5 h at 55°C. After cooling and concentration to -v 2 ml the mixture was diluted with benzene (80 ml), washed with 35 water, dried (Na2S0^) and evaporated under reduced pressure. 9 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 Se02 (4 mg, 0.036 nmol) in dry CH2C12 (5 ml), tert. -butylhy-5 droperoxide (13.2 )il, 0.094 rrmol) was added. After 30 min dry pyridine (40 pi) was added and the mixture was stirred for additional 25 min at room temperature, diluted with CH^Cl^ (3 ml) and cooled to 0°C. Hie crude 3,5-cyclovitamin product (j6) in CH2C12 (4.5 ml) was then added and the reaction permitted 1 o to proceed at 0°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^. 1 5 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-H3), 1.03 (3H, d, 20 J=6.9 Hz, 21-H3), 3.26 (3H, s, -0CH3), 4.2 (2H, m, 1-H and 6-H), 4.95 (IH, 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-flfeOH, 29), 245 (18), 135 (100). The above product comprised chiefly the la-hydroxy-25 cyclovitamin D analog of structure (J7) and a small amount of the corresponding 18-epiroer. A solution of this 1-hydroxy-clovitamin D product (12 mg) in glacial acetic acid (0.5 ml) was heated at 55°C for 15 min. The mixture was carefully poured into ioe/saturated NaHC03 and extracted with benzene 30 and ether. The 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 compound (8) (eluting at 42 ml) and 3.1 mg of compound (9) (eluting at 50 ml). 1 0 Compound (£) : NMR 6 0.56 (3H, s, 18-H.j) , 0.88 (6H, d, 3=1.0 Hz, 26-H3 and 27-H3), 1.02 (3H, d, J=6.8 Hz, 21-H3), 2.04 (3H, s, -ocoqy, 4.41 (1H, m, 1-H), 5.02 (1H, 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 X 264 nm, X . 227.5 ran; ^ max min nass spectrum m/e 440 (M , 15), 380 (M -HQAc, 84), 362 (M+-HQAc-^O, 9), 269 (M+-side chain-HQAc, 40), 251 (15), 135 (100), 134 (94).

Canpound (9): NMR 6 0.57 (3H, s, 18-H3), 0.89 (6H, d, J=7.0 Hz, 26-H3 and 27-H3), 1.03 (3H, d, J=6.8 Hz, 21-H3), 2.04 (3H, s, -OCOqy, 4.49 (1H, m, 1-H), 5.00 and 5.14 (2H, each narr. m, 19-^), 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 X 269.5 nm; X . 228 ^ max min nm; mass spectrum, m/e 440 (M , 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 1p-hydroxy-epimer corresponding to compound (8), characterized by the following data: NMR 6 0.55 (3H, s, 18-H3), 0.87 (6H, d, J=6.9 Hz, 26-H3 and 27-H3), l.oi (3H, d, J=6.9 Hz, 21-H.j), 2.06 (3H, s, -00X3^), 4.17 (1H, m, 1-H), 4.99 (2H, m, 3-H and 19-H), 5.1-5.4 (3H, br mf 19- , 22- and 23-H), 6.00 and 6.38 (2H, AB q, J=11.3 Hz, 7-H and 6-H); UV X 262.5 nm, X . 227 nm; mass spectrum, m/e max min 44$ (M , 27), 380 (78), 362 (12), 269 (28), 251 (20), 135 25 (100), 134 (78).

Hydrolysis of 3ft-acetoxy group in compounds (8) and (9). A solution of the 3p-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 30 work-up and final HPLC purification (8% 2-propanol in hexane as eluent), there was obtained the la,3(5-diol of structure (10) in 84% yield: NMR 6 0.56 (3H, s, 18-H3), 0.87 (6H, d, J=7.0 Hz, 26-H3 and 27-H3), 1.02 (3H, d, J=6.8 Hz, 21-H3), 4.22 (1H, m, 3-H), 4.42 (1H, m, 1-H), 5.00 (1H, narr. m, 35 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 X 264.5 nm, X ^ max min 227.5 nm; mass spectrum, m/e 398 (M , 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 5 above HPLC system.

Analogous treatment of the acetoxy derivative (9), gave after HPLC purification the 5,6-trans-la,3 B-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-H3), 1.03 (3H, d, J=6.8 Hz, 21-H3), 4.24 (1H, m, 3-H), 4.49 (1H, 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 X 273 nm, X . 229.5 max min nm; mass spectrum, m/e 398 (M , 21), 380 (5), 287 (6), 269 (5), 251 (4), 152 (33), 134 (100). Compound (11) elutes at 38 15 ml- -Hydroxylation of cornpound (10). The 5,6-cis-la,3fl-diol 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 20 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) hcmogenate 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 25 liver hcmogenate 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^, 0.1 M KC1 and 0.5 units glucose-6-phosphate-dehydrogenase. The reaction was initiated by addition of 400 30 jig 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 dichlorornethane. After further addition of 10 ml dichlorornethane, the organic phase was collected 35 while aqueous phase was re-extracted with 10 ml dichloro- methane. The organic p'.-ases frcn total of three extractions were combined and evaporated with a rotary evaporator. The residue containing the desired product was dissolved in 1 ml of CHCLjtHexane (65:35) mixture and applied to a Sephadex LH-20 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 chrcmatography (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/an ) 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 chrcmatography using a reverse phase column (Richrosorb Rp-18, 4.6 ran x 25 an, E. Merck, Darmstadt, West Germany) operated under pressure of 1200 psi (84 kg/an ) 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) X 265 nm, X . 228 ran; mass max min ' spectrum, m/z 414 (mol. ion M ), 396 (M -H^0), 378 (M -2x^0), 287 (M+-side chain), 269 (M+-side chain-H20), 251 (M+-side chain-2x^0), 152 (ring A, C 7/8 bond cleavage), 134 (152-^0) 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 rn/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 compounds 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. 1 3 The biological potency of the dehydrovi'camin D of this invention was established 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 5 (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 lib turn water and a low calcium-adequate phosphorus, vitamin D-deficient diet as 10 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 pnol of either compound (10) or la-hydroxy-vitamin D^ (la-OH-D^) dissolved in 0.05 ml 95% ethanol intra jugular ly 18 h prior to sacrifice. The rats in the 15 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 20 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 lot-OH-Dy given 18 h prior to sacrifice Compound Given Serum Calcium Concentration (mg/100 ml) + Standard Deviation ethanol 3.2 + 0.1 a) compound (10) la-0H-D3 4.5 + 0.4 b) 4.7 + 0.5 b) ^ is significantly different from p <0.001. 14 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. Fats in a control 5 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) ^3^) dissolved in 0.05 ml 95% ethanol. Eighteen hours later they were killed by decapitation and blood was collected. Serum calcium 10 concentration was determined with an atonic absorption spectrophotometer as described above. Results are shown in Table II below: Table II Increase in serum calcium concentration in response to single dose of 325 pncl of either cotpound (12) or 15 la, 25- (OH) ^>2 given 18 h prior to sacrifice Compound Given Serum Calcium Concentration (mg/100 ml) + Standard Deviation ethanol 4.2 + 0.1 a) compound (12) .0 + 0.4 b) la,25-(OH)2D3 .4 + 0.4 b) ^ is significantly different frcm p<0.001.

The above results show that both compound (10) and the final product (12) of this invention are highly active in promoting a rise in serum calcium levels of vitamin 25 D-deficient rats. They are, in fact, equivalent in biological potency to the corresponding known side chain-saturated compounds, lct-OH-D^ and la,25- (OH) the high potency and pharmaceutical utility of which is well documented by many reports in the general and patent literature (e.g. U.S. 30 Patent 4,225,596. 1 5 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 compounds. This is amply 5 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 Gndahl et al (Biochemistry 10, 2935-2940, 1971) for 3 weeks at which time 10 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 15 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 20 100°F and weighed. They were then ashed at 650°C for 24 hours in a muffle furnace. Hie ash was weighed and the percent ash in each of the tibiae was calculated. Results are shown in Table III below. 16 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-(OH) 2D3 130 6 41.0 + 3.4 * 1q-oh-d3 130 6 42.0 + 3.2 * la-0H-D2 1300 6 38.0 + 4.0 * Ccrnpound (10) 130 6 38.0 + 3.9 * ♦These values are not significantly different The results demonstrate that 130 proles of either 1,25- (0H) 2D3 or la-OH-D^ or the novel compound (10) of this invention, were equally effective in increasing the mineral content of the rachitic tibia. However, to achieve the same degree of effectiveness, 1300 pmoles of la-OH-15 TJhis agrees with previous results which shew that a lO-fold greater dose of la-OH-D^ than la-OH-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-OH-D^, it offers 20 unejqpected and superior activity in mineralizing the bones of an animal that is knewn to discriminate against the vitamin compounds in physiological utilization. This is an unexpected characteristic of and utility for this new analog. Since the unexpected in vivo activity of compound (10) is undoubtedly 25 iranifested 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 30 by la, 25-dihydroxyvitamin D^; thus this novel vitamin analog would be fully active in the chick. This high potency 1? of ocnpounds (10) and (12) in an animal that discriminates against vitamin D^-type derivatives, is a novel and distinctive characteristic of these substances. ; Because of this marked biological activity, the oonpounds 5 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 hunan, or related calcium deficiency 10 diseases (e.g. milk fever) in animals. Likewise, these ocrrpounds may be used for the treatment of certain malignancies, such as human leukemia. In view of the narked and unexpected activity of the compounds of this invention in promoting the mineralization of 1 5 bone in birds (see Table III), these compounds 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 derivatives ejdiibit very poor activity.

For therapeutic purposes, the compounds may be administered by any conventional route of administration and in any form suitable for the method of administration selected. The compounds may be formulated with any acceptable and innocuous pharmaceutical carrier, in the form of, for example, pills, tablets, gelatin 25 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 ccrrpounds are advantageously administered in amounts from 0.5 to 10 yg 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 35 skilled in the art. 1 8 CMO <4> ACO HO vr >n >C J>—Nf>h 12) <4> (fi)y-h 1z> y-oh (B) X»Ac (15) x-H (fi) X • Ac (HI X « H 1 9

Claims (11)

1. A compound having the formula: 10 wherein R is hydrogen or hydroxy.

2. The compound according to claim 1 wherein R is hydrogen.

3. The compound according to claim 2 in crystalline form.

4. The compound according to claim 1 wherein R is hydroxy.

5. The compound according to claim 4 in crystalline form.

6. A process for preparing a compound as claimed in any one of claims 1 to 5 which comprises hyaroxylating the 1o<-position of the compound of the formula: 15 in known manner and, optionally, hydroxylating the 25-position of the resulting lot -hydroxy corpound in known manner. 20

7. A process according to claim 6 substantially as hereinbefore described.

8. A compound as defined in claim 1 whenever prepared by a process as claimed in claim 6 or 7.

9. A pharmaceutical composition which comprises a compound as claimed in any one of claims 1 to 5 and a pharmaceutically acceptable excipient.

10. A composition according to claim 9 which comprises the compound of claim 2.

11. A composition according to claim 9 which comprises the compound of claim 4. Dated this the 1st day of March, 1985 ■ —-v. EXECUTIVE 2 7 Clyde Roapf, Ballsbridge, Dublin 4 AGENTS FOR .THE APPLICANTS.