NL8520021A - 1-ALPHA-HYDROXYVITAMINE D2 ANALOGA AND METHOD FOR PREPARING THAT. - Google Patents

Description

i> uK '' '8 52 00 2 1 ^ Ία-Hydroxy vitamin D analogues and method for the preparation thereof.

The present invention has been accomplished with the support of the Government under NIH Grant No.

AM 14881 granted by the Department of Health and Human Services. The Government has certain rights in the present invention.

Object of the invention.

The invention relates to biologically active vitamin D compounds and in particular to 1-hydroxyvitamin D 1 analogs with unexpected biological properties.

State of the art.

Because of the well known and conclusively established activity of 1a-hydroxyvitamin D compounds in controlling the calcium and phosphate homeo-15 stasis in humans and animals, there has been an interest in preparing the natural metabolites and finding analogues with useful biological properties .

This has led to the preparation of a variety of compounds with biological activity (see, for example, DeLuca et al., Topics in Current Chem., Vol. 83, p. 1 (1979); Ann. Rev. Biochem. 52, 411 (1983);

Yakhimovich, Russian Chem. Rev. 49, 371 (1980)). Interest in such compounds has been growing, especially now that certain vitamin D 25 derivatives, in particular 1, 25-dihydroxyvitamin D 1, and 1a-hydroxyvitamin D 1, have been found to exist in addition to their known regulators. action on the calcium homeostasis also influence cellular differentiation processes and are able to inhibit the growth and proliferation of certain leukemia cells (Suda et al., U.S. Patent 4,391,802; Suda et al., Proc. Natl. Acad. , USA 80, 201 8520021 J.

- 2 - (1983); Reitsma et al., Nature, 306, 492-494 (1983)).

Most known vitamin D metabolites and analogs are derivatives of compounds from the vitamin series, i.e. they have saturated ste-rolled side chains. However, vitamin D compounds with unsaturated side chain are also known, namely, certain hydroxy derivatives of vitamin such as 25-hydroxy vitamin (U.S. Pat. No. 3,585,221), 1,25-dihydroxyvitamin (U.S. Pat. No. 3,880,894), 24 -hydroxy and 24,25-dihydroxyvitamin D2 metabolites (Jones et al. Arch. Biochem. Biophys. 202, 450 (1980)), Ια-hydroxyvitamin (US Patent 3,907,843), as well as certain related 22-trans-dehydro compounds without 24-methyl substituent as described in U.S. Patent 3,786,062 and by Bogoslovskii et al. (J. Gen. Chem. USSR 48 (4), 828 (1978); Chem. Abstr. 89, 163848j, 89, 209016s).

Description of the invention.

New vitamin D analogs characterized by formula I on the formula sheet have now been prepared. wherein R represents hydrogen or hydroxy. The compound of the invention wherein R is hydrogen can be obtained as an intermediate in preparing the compound wherein R is hydroxy.

Structurally, the compounds of the invention are analogs of hydroxyvitamin D2 compounds having no 24-methyl substituent, ie the compounds are 1a-7hydroxy-28-norvitamin D2 and 1a, 25-dihydroxy-28-norvitamin D2, respectively.

Both the product and the intermediate exhibit strong biological activity and have been characterized by an unexpected and new pattern of activity as described below.

An embodiment of the method that has led to the compounds of the formula I is. shown in Scheme A. In the following description 8520021 - 3 - of this method as well as in the examples and tables, the designations with arabic numerals (for example (IK (2), (3), ... (10), ( 11), (12) refers to the formulas thus numbered in Schedule A.

The starting compound is the diene-protected aldehyde of the formula (JL) (see Scheme A), prepared from ergosterol acetate according to the method of Barton et al.

(J. Chem. Soc. (C) 1968 (1971)). Reaction of the aldehyde (1) with 3-methyl-1-butylphenyl sulfone of the formula 10 (CH3) 2CHCH2CH2-SO2Ph in an organic solvent and in the presence of a strong organic base gives the intermediate hydroxy sulfone of the formula (2) as shown in scheme A. Treatment of the intermediate {2) with sodium amalgam in a buffered alcohol solution then gives the 22,23-trans-olefin (22E-olefin) of the formula (3).

Reaction of the 22E olefin (3) with a Strong hydride reducing agent (e.g. LiAlH 2) in an ether solvent gives the intermediate 5,7-diene 20 (4). This intermediate is then irradiated with ultraviolet light in an organic solvent to obtain the corresponding provitamin D derivative. This is separated and heated in an organic solvent at a temperature between room temperature and reflux temperature to isomerize the provitamin D chromophore to form the vitamin D chromophore to yield the 22E-dehydrovitamin DI compound of formula (5). Compound (5) is a known vitamin D analog previously prepared according to a less suitable one. method (Bogos-30 lovskii et al., supra).

Then, the intermediate (5) of carbon atom 1 is hydroxylated by the general method of DeLuca et al (U.S. Pat. Nos. 4,195,027 and 4,260,549). These reaction steps involve the reaction of compound (5) with toluene sulfonyl chloride on known or conventional free form to obtain the corresponding 8520021-4-33 tosylate derivative which is immediately solvolysed in buffered methanol to produce the 3,5-cyclovitamin D intermediate of the formula (6) available in Schedule A. By treatment of the latter compound 5 with selenium dioxide and tert-butyl hydroperoxide, after chromatographic purification, the corresponding 1-hydroxylated product containing mainly the 1-hydroxycyclovitamin D compound of the formula (T) is obtained. A small amount of the corresponding β-hydroxy-10 epimer may also be present in the product, but separation of the epimers, although possible by chromatography, is not necessary in this step. Heating the 1-hydroxycyclovitamin D intermediate in glacial acetic acid at 40-60 ° C then yields a mixture of the 3-acetylated solvolysis products from which the 5,6-cis and 5,6-trans compounds of the formula (8, respectively) are chromatographed. ) and (_9) are isolated. When the 1-hydroxycyclovitamin D product being solvolysed contains any Ιβ-hydroxypimer, the solvolysis mixture will also contain the Ιβ-hydroxy epimers corresponding to compounds (8) or (-9) which, if desired, also by chromatography can be isolated.

Conventional hydrolysis of the acetate group in the compound (8) with a base gives the diol product of the formula (10).

This diol (10) is then subjected to in vitro enzymatic hydroxylation at carbon using a liver homogenate prepared from vitamin. D-deficient rats (as disclosed in U.S. Pat. No. 4,307,025) to obtain, after chromatographic separation of the product mixture, the desired 1,25-dihydroxylated product of formula (12) in pure form.

In the following detailed description of the above-described method, the physiochemical data are obtained using the following 8520021 -5.-.

indicated methods and equipment. High pressure liquid chromatography (HPLC) was performed with a Waters Associates Model ALC / GPC 204 using a Zorbax-Sil (DuPont) column (6.2 mm x 25 cm column, flow rate 4 ml / min, 10335 kPa ). Column chromatography was performed on Silica gel 60, 0.062-0.210 mm ASTM (Merck).

! Thin layer chromatography (TLC) was performed on Silica 60 PF-254 (20 x 20cm plates, 1mm silica gel). Irradiations were performed with a Hanovia 608A36 mercury arc lamp 10 equipped with a Vycor filter. All reactions were performed under an inert atmosphere (e.g. argon).

3-Methyl-1-butylphenylsulfone (isopentylphenylsulfone).

PhSC ^ Na (1.97 g, 12 mmol) was added to a stirred solution of 3-methyl-1-bromobutane 15 (1.51 g, 1.2 mL, 10 mmol) in DMF (20 mL) at 75 ° C. The mixture was heated at 75 ° C for 5 hours, then cooled, poured into water and extracted with benzene. The organic layer was washed with 5% HCl, 5% NaHCO 2 and water, dried over Na 2 SC> 4 and evaporated. The oily sulfone product (1.69 g, 80%) was practically pure and used without purification. NMR δ 0.88 (6H, d, J = 6.5 Hz, 2 x CH3), 1.61 (3H, m, -CH2-CH), 3.08 (2H, m, SO -CH -), 7.50-7.95 (5H, m, Ar-H); IR: 1300 (br), 12.

1147, 1088, 745, 692, 564, 539 cm; mass spectrum, m / e 25,212 (M +, 3). 143 (92), 77 (57), 71 (73), 70 (73), 55 (42), 43 (100), 41 (47).

(22E) -5a, 8ot- (4-phenyl-1,2-urazolo) -cholesta-6,22-diene-33-Ql (3).

n-Butyl lithium (1.7 M solution in hexane, 4.12 mL, 7 mmol) was added to a stirred solution of diisopropylamine (707 mg, 1 mL, 7 mmol) in dry: THF (14 mL) and the mixture was stirred at room temperature for 15 min. The sulfone as prepared in Example 1 (1.50 g, 7.07 mmol) in dry THF (11 ml) was taken over 10 min.

Dripped. The solution was stirred at room temperature for an additional 15 minutes after which after cooling to 0 ° C aldehyde 8520021-6 U) (545 mg, 1 mmol) in dry THF (7 mL) was added. Stirring was continued for 2 hours at 0 ° C, after which the solution was allowed to slowly come to room temperature (30 min.). The mixture (containing the hydroxysulfone product (2)) was poured into a saturated solution of Na 2 HPO 4 in methanol (200 ml). Sodium amalgam (5.65%, 10 g) was added and the mixture was stirred at 4 ° C for 17 hours. Precipitated mercury was filtered off and the reaction mixture was concentrated to about 5 ml.

: 10 The concentrate was diluted with water and extracted with methylene chloride. The organic extract was washed with water, dried (Na 2 O 4) and concentrated in vacuo. The oily residue was chromatographed on a silica gel column. Excess sulfone was eluted with a benzene ether mixture (7: 3). Elution with benzene ether (6: 4) gave a pure adduct P) (375 mg, 67%) as a foam. NMR δ 0.81 (3H, s, 18-H3), 0.86 (6H, d, J = 6.7 Hz, 26-H3 and 27-H ^), 0.97 (3H, s, 19- H3), 1.03 (3H, d, J = 6.8 Hz, 21-H3), 3.16 (1H, dd, J = 4.4 Hz, J2 = 14 Hz, 20 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 +, 1.1%), 382 (70), 349 (51), 253 (28), 251 (45), 119 (PhNCO, 83), 55 (100).

25 (22E) -Cholesta-5,7,22-trien-33-ol (4).

The adduct P) (330mg, 0.6mmol) and lithium aluminum hydride (700mg) in dry T.HF (40ml) were refluxed for 18 hours. Excess reactant was decomposed with a few drops of water.

Anhydrous Na 2 O 3 was added and the organic phase was decanted and evaporated. The crystalline residue was purified on a silica gel column. Eluting with a benzene ether mixture (94: 6) gave pure diene (4) (180 mg, 80%) which was crystallized from methanol m.p .: 119.5-122-5 ° C, / a_ / D = -118 ° (c = 1.2, CHCl 3); NMR δ 0.63 (3H, s, 18-H3), 0.87 (6H, d, 8520021 - 7 - J = 6.7 Hz, 26-H3 and 27-H3), 0.95 (3H, s , 19-H3'jf, 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), 5.38 and 5.57 (2H, AB q, J = 6 Hz, 7-H and 6-H); UV λ 281 mm; IR: 3436, 1461, 1382, 1366, I 5 1062, 1036, 968 cm-1; mass spectrum, m / e 382 (M +, 100), 349 (M + -H20-Me, 71), 323 (34), 271 (M + side chain, 16), j 253 (M + side chain-H20, 32). J

(5Z, 7E, 22E) -9,10-Secocholesta-5,7,10 (19), 22-tetraen-3β-ol I

(5). I

10 A solution of compound (4_) (100mg, 0.26mmol) in a mixture of ether (120ml) and benzene | (30 ml) was degassed with argon for 40 minutes. The solution ! | was irradiated for 13 min at O ^ C in a quartz immersion before! 1! see a UV lamp and filter. The solvent was removed under reduced pressure and the residue was separated by HPLC eluting with 1% 2-propanol in hexane to obtain the pure provitamin D derivative (40.4 mg, 40%). to be collected at 24 ml.

NMR δ 0.72 (Sn, s, 18 ^ Η), 0.87 (6H, d, J = 6.7 Hz, 26-H3 i 20 and 27-rfl), 104 (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 λ 260.5 nm; λ. 234 nm.

max mm

The provitamin (40mg, 0.1mmol) in ethanol (100ml) was refluxed for 3 hours. After solvent removal, the product mixture was separated by HPLC (eluting 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; j I 30 NMR δ 0.56 (3H, s, 18-H), 0.88 (6H, d, J = 6.7 Hz, 26-H3 j and 27-H-), 1.02 (3H, d, J = 6.6 Hz, 21-tH-), 3.96 (1H, m, 3-H) !, i «5« J '' 'yy 4.82 and 5.05 (2H, each narrow m, 19-fl.), 5.27 (2H, br m, 22-H1; 2 i

1 and 23 ^ H), 6.03 and 6.24 (2H, ABq, J = 11.4 Hz, 7-H and 6-H); UV

ί λ 265 nm, λ. 228 nm; IR: 3420, 1458, 1441, 1378, max mm i 35 1366, 1050, 970, 943, 891, 862 cra-1; mass spectrum, m / e | (M +, 22), 349 (M + -H20-Me, 4), 271 (M + side chain, 8), 253 8520021 - 8 - j (M + side chain-H20, 13), 136 (100), 118 (80 ).

! (5Z, 7E, 22E) -33-Acetoxy-9,10-secocholesta-5,7,10 (19), 22-tetraen-1 a-ol (8), I Freshly recrystallized p-toluenesulfonyl-! | Chloride (50mg, 0.26mmol) was added to a solution release of vitamin (5_) (30 mg, 0.08 mmol) in dry ! pyridine (300 µl). After 30 hours at 4 ° C, the reaction mixture poured into ice / saturated with stirring!

NaHCOg. The mixture was stirred for 15 min and then extracted with benzene. The organic extract was washed with saturated NaHCO 2, saturated copper sulfate and water, dried (Na 2 SO 4) and concentrated in vacuo to give the oily 33 tosyl derivative.

The crude tosylate was treated with NaHCO 2 (150 mg) in absolute methanol (10 ml) and the mixture was stirred at 55 ° C for 8.5 hours. After cooling and concentrating to about 2 ml, the mixture was diluted with benzene (80 ml), washed with water, dried (Na 2 SO 4) and evaporated under reduced pressure.

The resulting oily 3,5-cyclovitamin D analog (6) was sufficiently pure to be without purification i to be used for the next oxidation step. To a vigorously stirred suspension of SeO 2 (4mg, 0.036mmol) in dry Cl 2 Cl (5ml) was added t-butyl hydroperoxide. 25 (13.2 µl, 0.094 mmol) added. After 30 minutes, dry pyridine (40 µl) was added, after which the mixture was added 25 µl stirred at room temperature for minutes. After this, the mixture was diluted with CH2Cl2 (3 ml) and cooled to 0 ° C. Then the crude 3,5-cyclovitamine (6) in 30 CH 2 Cl 2 (4.5 ml) was added and the reaction was allowed to proceed for 15 min; at 0 ° C. Afterwards, the mixing was allowed to proceed come to room temperature slowly (30 min.). The mixture was transferred to a separatory funnel and shaken with 30 ml of 10% NaOH. Ether (150 ml) was added and the separated organic phase was washed with | 10% NaOH, water and dried over Na 2 SO 4. Concentration in vacuo to dryness resulted in a yellow oily 1 µl residue which was purified on silica gel TCL plates underneath! developing with a 7: 3 hexane-ethyl acetate mixture (Rf: 0.35) to yield the 1-hydroxycyclovitamine product: 5 (14.4 mg, 45%). NMR δ 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,. J = 6.9 Hz, 21-H3), 3.26 (3H, s, -0CH3), 4.2 ί (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- ^, 22-H and 23-H); mass spectrum, m / e 412! 10 (M +, 27), 380 (M + -MeOH, 46), 339 (22), 269 (M + -side chain-)

MeOH, 29), 245 (18), 135 (100).

The .prbduct thus obtained mainly contained the 1a-hydroxycyclovitamin D analog of the formula (7) and a small amount of the corresponding Ιβ-epimer. A solution of the 1-hydroxycyclovitamin D product (12 mg) in glacial acetic acid (0.5 ml) was heated at 55 ° C for 15 minutes. Afterwards, the mixture was carefully poured out into ice / saturated NaHCO3 and extracted with benzene and ether. The united extrac | | 20 were washed with water, dried (Na SO) and evaporated. The resulting product mixture was subjected to HPLC (1.5% 2-propanol in hexane as eluent) with 4.9 mg of compound (8) (elution at 42 ml) and 3.1 mg of compound (9) (elution at 50 ml).

Compound (8): NMR δ 0.56 (3H, s, 18-H3), 0.88 (6H, d, J = 7.0 Hz, 26-H3 and 27-H3), 1.02 (3H , d, J = 6.8 Hz, 21-H3), j 2.04 (3H, s, -OCOCH3), 4.41 (1H, m, 1-H), 5.02 (1H, narrow m, 1 19 -H), 5.1-5.4 (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, 30 λ. 227.5 nm; mass spectrum m / e 440 (M +, 15), 380 (M + -; | HOAc, 84), 362 (M + -HOAc-HO, 9), 269 (M + side chain-HOAc, Δ '| | 40),. 251. (15), 135 (100), 134 (94). j

Compound (9): NMR δ 0.57 (3H, s, 18-rH3), 0.89 (6H, d, | i J = 7.0 Hz, 26-H3 and 27-H3), 1.03 ( 3H, d, J = 6.8 Hz, 21-H3); ; 2.04 (3H, s, -OCOCH 3), 4.49 (1H, m, 1-E), 5.00 and 5.14!

(2H, each narrow m, 19- ^), 5.25 (3H, br m, 3-, 22- and 23-H), I

8520021 - 10 - 5.81 and 6.58 (2H, AB q, J = 12.0 Hz, 7-H and 6-H); UV λ j max j 269.5 nm; λ, 228 nm; ' mass spectrum, m / e 440 (M, 6), mm 380 (47), 269 (15), 135 (100), 134 (62). j i ί! Also from the solvolysis product mixture | 5 obtained a small amount (0.87 mg) of the met compound (8) corresponding 1β-hydroxy epimer with the! following characteristics: NMR δ 0.55 (3H, s, Ιδ-Η ^), | 0.87 (6H, d, J = 6.9 Hz, 26-H3 and 27-H3), 1.01 (3H, d, | J = 6.9 Hz, 21-H3), 2.06 (3H , s, -OCOCH3), 4.17 (1H, m, | 10 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-ji); UV λ 262.5 nm, λ. 227 nm; mass spec I max min r trum, m / e 440 (M +, 27), 380 (78), 362 (12), 269 (28), j 251 (20), 135 (100), 134 (78). | Hydrolysis of the 3β-acetoxy group in the compounds j (£) and (9).

A solution of the 38-acetoxyvitamin derivative (8) (0.76: mg) in ethanol (0.1 ml) was treated with 10% KOH in methanol (0.8 ml) and the mixture was heated for 1 hour at 50 ° C. After customary work-up and final HPLC purification (8% 2-propanol in hexane as eleris), the 1,3f5-diol of the formula (10) was obtained in 84% yield. NMR δ 0.56 (3H, s, | 18-H3), 0.87 (6h, d, J = 7.0 Hz, 26-HI and 27-H3), 1.02 y | 25 (3H, d, J = 6.8 Hz, 21-H), 4.22 (1H, m, 3-H), 4.42 (1H, m,! 1-H), 5.00 (1H , narrow 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. 227.5 nm; mass spectrum, m / e 398; max mm (M +, 21), 380 (M + -H20, 9), 2.87 (M + side chain, 6), 269 30 (M + side chain-H20, 8), 251 (5), 152 (38), 134 (100).

Compound (10) elutes at 40 ml in the aforementioned HPLC system. j | ί! By analogous treatment of the acetoxy-! derivative (O) after HPLC purification, the 5,6-trans-1,3β-; [ί

35 diol of formula (11) in 72% yield

got. NMR δ 0.58 (3H, s, 18- ^ H), 0.87 (6h, d, J = 7.0 Hz, 8520021-11-26-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 j ί j | narrow 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, i j max; 5 min min 229.5 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 ml.

| 25-Hydroxylation of compound (10).

The 5,6-cis-1,38-diol of formula (10) j 10 as obtained above was 25-hydroxylated as follows: newly weaned male rats were placed on a low calcium: vitamin D deficient diet as described by Suda et al. in J. Nutr. 100, 1049 (1970) for 2 weeks. After this they were beheaded and their livers were removed! 15 'A 20% w / v homogenate was prepared in ice cold

0.25 M sucrose. Incubation was performed in 10 ml of incubation; medium in a 125 ml Erlenmeyer flask containing an amount of liver homogenate with the following composition: 1g tissue, 0.125 M sucrose, 50 mM phosphate buffer I 20 (pH 7.4), 22.4 mM glucose-6-phosphate, 20 m ATP, 160 mM

nicotinamide, 25 mM succinate, 0.4 mM NADP, 5 mM MgCl 4, 0.1 M KCl, and 0.5 units of glucose-6-phosphate dehydrogenase. The reaction was initiated by adding 400 µg of compound (10) dissolved in 100 µl of 95% ethanol. The mixture was incubated at 37 ° C for 3 hours with shaking at 80 oscillations / min. The reaction was stopped by adding 20 ml of methanol and 10 ml of methylene chloride. After | again adding 10 ml of dichloromethane, the I organic phase was collected and the aqueous phase was re-extracted with 10 ml of dichloromethane. The organic phases from a total of three extractions were combined and evaporated in a rotary evaporator. The residue containing the desired product was dissolved in 1 ml of! a CHCl-hexane mixture (65:35) and applied to a 3 35 Sephadex LH-20 column (0.7 cm x 14 cm) packed, equilibrated and eluted with the same solvent. The first 8520021 - 12 - 10 ml were discarded and the next 40 ml were collected and evaporated. The residue was dissolved in 8% 2-propanol in hexane and subjected to high pressure liquid! chromatography (Model LC / GPC 204 HPLC, Waters J5 Associates, Medford, MA) using a

Zorbax-Sil column (4.6 mm x 25 cm, DuPont, Inc.,} Wilmington, DE) operated under a pressure of 6894 kPa at a flow rate of 2 ml / min. The desired 25-hydroxylated product was eluted at 42 ml. The product was further purified by high pressure liquid chromatography using a reverse phase column (Richrosorb Rp-18, 4.6 mm x 25 cm, E. Merck, Si

Darmstadt, West Germany) operated under a pressure of 8272.8 kPa at a flow rate of 2 ml / min. The column was eluted with 22% <2 <3 in methanol and the product was eluted at 50 ml. The product was further purified by HPLC using Zorbax-Sil under the conditions described above. The product obtained was characterized by the following data: UV absorption (95% ethanol) λ 265 nm ', max!

Snin nm; mas sa spectrum, m / z 414 (mol.ion M +), 396 j (M + -H20), 378 (M + -2xH20), 287 (M + side chain), 269 (M + side chain J-H20), 251 (M + side chain-2xH20), 152 (ring A, C 7/8 breaking bond), 134 (152-HO) and 59 ((CH0) oC = 0H) + 25 due to breaking C24 / 25 bond) . I

The reported data, in particular the characteristic ultraviolet absorption and the prominent and diagnostic; nostic peaks at m / z 152, 134 and 59 in the mass spectrum, support the product to achieve the desired 25% | I is a hydroxylated compound of the formula (12) in Scheme J A).

If desired, the compounds according to: j; i; The invention can be easily crystallized. | obtained by crystallization from suitable solvents,! for example hexane, ethers, alcohols, or mixtures therein ! i! . from.

8520021 - 13 -

Biological activity of side chain dehydro-; | vitamin D compounds.

| The biological activity of the above Written dehydrovitamin D analogs were tested in rat in vivo tests and for comparison with 1a-hydroxy-vitamin D compounds of known strength. Both the I product (12) and the key intermediate, compound (10) were tested and found to be highly active.

| Biological activity of compound (10). I

! 10 The test was carried out as follows: Pas j! weaned male rats from Holtzman Co.,

Madison, WI were on a low calcium adequate i phosphorus, vitamin D deficient diet for 3 weeks as described by! j

Suda et al. (J. Nutr. 100, 1049 (1970)) with water ad libitum 15. The rats, divided into groups of 6, were administered intrajugularly 650 pmol of compound (10) or α-hydroxyvitamin D 1 (1α-OH-D 1) dissolved in 0.05 ml of 95% ethanol 18 hours before killing. The rats in the control group received the ethanol vehicle on the same! 20 administered. The rats were killed by decapitation: and the blood was collected. Serum obtained by the | | centrifuge blood, was diluted with a 0.1% lanthanum chloride solution (1:20) and the serum calcium concentration was measured with an atomic absorption spectrometer. The results are shown in Table A below.

• i j i] l i | ! i 8520021 - 14 -

Table A;

Increase in serum calcium concentration in response to a single dose of 650 pmol compound (JLO) or | 1a-OH-D 4 / administered 18 hours before killing.

5 '\

Serum calcium concentration compound administered (mg / 100 ml) ± standard ethanol deviation 3.2 ± 0.1 a ^ j | compound (10) 4.5 ± 0.4 b ^ | 10 1a-OH-D3 4.7 ± 0.5 1 t)) δ) is significantly different from p <0.001.

Biological activity of compound (12). The experiment was performed as follows: Newly weaned male rats were placed on a low calcium vitamin D deficient diet as described above for 3 weeks. The rats were divided into groups of 5. At the | Rats in a control group were administered 0.05 ml of 95% ethanol 20 intrajugularly and to the rats in the test groups 325 pmol of compound (12) or 1,25-dihydroxy- Vitamin D1 (1a, 25- (OH) 2D3) dissolved in 0.05 ml of 95% strength ethanol. Eighteen hours later they were killed by decapitation! and the blood was collected. The serum calcium concentration was determined with an atomic absorption spectrophotometer as described above. The results are shown in Table B below

Table B

- |

Increase in serum calcium concentration in response to a single dose of 325 pmol of compound (12) or µl, 25- (OH) 2D3 administered 18 hours before killing.

Serum administered calcium concentration compound_ (mg / 100 ml) ± standard deviation ethanol 4.2 ± 0.1 a ^ 35 compound (12) 5.0 ± 0.4 b ^ 1a, 25- (OH) „D„ 5.4 ± 0.4 1! 1 '* “" ϋί "' *" r 1 "r - r 1.

8520021 is significantly different from a) p <0.001.

- 15 - ί The above results show that | both compound (10) and the final product (1 - 2) according to the invention an increase in serum calcium levels at | strongly promote vitamin D deficient rats. In fact 5 they are equivalent in biological strength to the | known side chain saturated compounds, loi-OH-D and i! 3 i I la ^ B-iÖH ^ Dg »whose high strength and pharmaceutical j

j I

Applications have been well described in the general literature and patent literature (see, for example, U.S. Patent 4,225,596.

Particularly noteworthy and unexpected is the excellent activity of compound (10) in the; bone mineralization of animals (chicken), so that this compound lends itself better to physiological application than the vitamin E> 2 compounds. To explain this! serve the following. |

Method j

One-day-old white male Leghorn-j chicks from Northern Hatcheries (Beaver Dam, |! 20 WI). were put on a vitamin D deficient soybean protein diet described by Omdahl et al (Biochemistry J 10, 2935-2940, 1971) for 3 weeks after which they were deficient in | had vitamin D. After this they were divided into groups! of six. One group was administered Wesson oil vehicle through the beak; to the other groups, the compounds listed in Table C below, dissolved in the same amount of Wessen oil, were administered every day for 7 days. 24 Hours after the last administration j! all chicks were killed by cervical dislocation. The i! | 30 tibiae were removed and stripped of adherent soft; and connective tissue and then extracted in alcohol for 24 hours: followed by 24 hours in diethyl ether using | , from a Soxhlet device. The bones: were taken at 100 ° F dried to constant weight and then incinerated for 24 hours:! 35 at 650 ° C in a sleeve bright oven., "The ash was weighed and the percentage of ash in each tibia was calculated. Results 8520021 ·: - 16 -v are listed in Table C below.

Table C

! Mineralization of Rickety chick bone

| Connection Quantity Number% shaft I

(pmol / d / 7 days) _animal_y! -D 0 6 32.0 ± 1.0 | ! 1.25- (OH) 2D3 130 6 41.0 ± 3.4 *! -Οί-ΟΗ-Ό ^ 130 6 42.0 ± 3.2 * | 10 1a-OH-D2 1300 6 38.0 ± 4.0 * |

Compound (10) 130 6 38.0 ± 3.9 *: * These values are not significantly different.

The results show that 130 pmol of 1,25- (OH) 2 D21 Lct-OH-D 1 or the new compound (10) of the invention are equally effective in increasing the mineral content in the rickets tibia. But to achieve the same activity with 1a-OH-D2, 1300 µm pmol is needed. This is in accordance with previous results'

j 4 '* I

demonstrating that a 10-fold dose of 1a-OH-D 1/2 relative to 1a-OH-D 1 is needed to achieve the same tibia-j mineralization in Rickety chickens.

These results surprisingly show that while j compound (10) is an analog of 1a-OH-D 4, compound j 25 (10) has an unexpected and excellent activity over j! spreads to mineralize animal bones; y y y j this in contrast to the vitamin D compounds in; I 1 \ physiological application. This is an unexpected characteristic! and the application of these new analogues. Because! i

30 the unexpected in vivo activity of compound (10) I

undoubtedly manifests itself after hydroxylation to the! corresponding 1a, 25-dihydroxy compound (compound (12)) in vivo, the compound, which is compound (12), which is the 24-desmethyl analog of the known 1a, 25-! Dihydroxyvitamin D ^, are as effective in promoting bone mineralization in chickens or chickens as shown in 8520021-17 for Ια, 25-dihydroxyvitamin. This new vitamin analogue would therefore be fully effective in chickens or chickens. With this high activity of the compounds (10): and (12) in animals they favorably distinguish 1; 5 of vitamin D ^ type derivatives, and this constitutes a new one; and distinctive feature of these compounds.

i! Because of their remarkable biological activities The compounds of the invention will readily facilitate; Find use as substitutes for several known vitamin D metabolites in the therapy or prophylaxis of calcium metabolism disorders such as rickets, hypoparathyroidism, osteodystrophy, osteo-malacia or osteoporosis in humans, or related calcium deficiency diseases ( for example, wake fever) in animals.

Likewise, these compounds can be used to treat certain malignancies such as human leukemia. Due to the remarkable and unexpected activity of the compounds of the invention: (compounds (10) and (12)) in promoting bone mineralization in birds (see Table C), the compounds could be of particular use are to prevent or treat calcium oribalance-induced disorders (eg thin eggshell, weak legs in poultry) in poultry where all known vitamin 25 derivatives show very poor activity.

For therapeutic applications, the compounds can be administered by any conventional route and in any suitable form for the chosen route of administration. The compounds can be formulated with any acceptable and non-toxic pharmaceutical carrier, in the form of pills, tablets, gelatin capsules or suppositories, or as solutions, emulsions, dispersions or suspensions in non-toxic solvents or oils. Such formulations may also contain other therapeutically active and beneficial ingredients as may be appropriate for the specific uses. To man 8520021 ft

V

- 18 -! the compounds are suitably administered in amounts of 0.5-10 µg per day, the specific dosage being is tailored to the compound administered, the

j acting disorder and the patient's condition and response l

I 5 i | ί; j ί ί! f i j | | i i ί ί ί: j: 8520021

Claims (7)

1. R is hydroxy. i; 1. Compounds of the formula I on it:! formula sheet where R represents hydrogen or hydroxy. | 5 A compound according to claim 1 wherein R is hydrogen. A compound according to claim 2 in crystalline form. The compound of claim 1 wherein j A compound according to claim 4 in i; crystalline form. j ί 6. A pharmaceutical composition containing the compound of claim 2 and a pharmaceutically acceptable excipient. Pharmaceutical composition comprising the compound of claim 4 and a pharmaceutically acceptable bar contains excipient. i 20 j ί i | l | i ί I 8520021