DK154290B - VITAMIN-D DERIVATIVES AND PHARMACEUTICAL PREPARATIONS CONTAINING THESE - Google Patents

Description

DK 154290 B

The present invention relates to novel vitamin D derivatives and pharmaceutical compositions containing them.

5 It is well known that vitamin D regulates calcium and phosphorus metabolism in animals and humans, and it is now clearly established that the biological effect of vitamin D depends on the metabolic conversion of the vitamin in vivo to hydroxylated derivatives. Thus, in vivo, vit-10 min-D3 is hydroxylated to 25-hydroxy vitamin-D3 in the liver, which in turn is converted to 1α, 25-dihydroxyvitamin-D3 in the kidney. It is the latter compound that is recognized as being the circulating hormonal form of vitamin D.

Due to the biological ability of the compounds to promote calcium and phosphorus transport in the intestine and to mobilize and mineralize bones, these forms of vitamin D are important pharmaceutical compounds which are particularly suitable for use in the treatment of various bone disorders.

Vitamin-D derivatives and their preparation and their use are discussed in a variety of patents and in 25 other literature. For example, OS discloses patent specification no.

3,556,924 25-dihydroxy cholecalciferol; U.S. Patent No. 3,697,559 discloses 1,25-dihydroxycholecalciferol; U.S. Patent No. 3,471,996 discloses 1-hydroxycholecalciferol; U.S. Patent No. 3,786,062 discloses 22-30 dehydro-25-hydroxycholecalciferol; U.S. Pat.

3,880,894 discloses 1,25-dihydroxyergocalciferol; U.S. Patent No. 4,201,881 discloses 24,24-difluoro-α, 25-dihydroxycholecalciferol; U.S. Patent No. 4,196,133 discloses 24,24-diflubr-1α, 25-dihydroxycholecalciferol.

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2

The vitamin-D derivatives of the invention have been found to exert excellent vitamin-D-like action, and for that reason they can be readily used in place of vitamin-D3 or derivatives thereof for known purposes such as e.g. for the treatment of various morbid conditions which appear in calcium and phosphorous imbalances such as hypoparathyroidism, osteodystrophy, osteomalacia and osteoporosis.

In addition, these derivatives exhibit an unexpectedly high antineoplastic effect relative to 1α-10 hydroxyvitamin D3 and 1α, 25-dihydroxyvitamin D3, and this effect is unexpectedly and surprisingly exhibited with no increased calcemic effect.

These properties make the compounds particularly suitable as substitutes for Ια-hydroxyvitamin D3 and 1α, 25-dihydroxyvitamin D3 suggested for the treatment of leu-chemoid disease.

These derivatives are 24-homovitamins and especially 1α, 25-dihydroxy-22E (or Z) -dehydro-24-homovitamin-D3 and 20α, 25-dihydroxy-24-homovitamin-D3.

The derivatives of the invention are characterized by the following formula I: Yrs, Rs 25 ~ 30

Rt0 λ wherein R 1, R 2 and R 3 are each selected from hydrogen, a. acyl group having from 1-4 carbon atoms and benzoyl, and R4 and R5 each represent hydrogen atoms or form 3

DK 154290 B

together a carbon-carbon double bond.

The pharmaceutical compositions of the invention are characterized by the characterizing part of claim 7.

5

The present compounds can be prepared as set forth in the following reaction scheme and as described below. In the schematic and detailed description of the process, the same numbers identify the same compounds.

10 15 20 25 30 35 4

DK 154290 E

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OH

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X Λ R · * ^ Y | γ = ν ^; κ ^ χόγ ^ 0Χυγ .νοΧΧγ '* ^ ι »^ Ν / r« ^ tw

Hc - * - CCcK ΗΟ '-' ^ ζοΗ ΙΛ »♦ 5

DK 154290B

The process for preparing the derivatives of the invention is further described below:

Bisnorcholenoic acid acetate (a) was reduced with lithium aluminum hydride and then oxidized with dichlorodicyanobenzoquinone to 1,4,6-trien-3-one (b) in 47% yield.

The 22-THP ether of (b) was treated with alkaline hydrogen peroxide to give the 1α, 2α epoxide (1) in a 41% yield. Reduction of (1) with lithium and ammonium chloride 10 in liquid ammonia tetrahydrofuran at -78 ° C and subsequent treatment with chloromethyl methyl ether gave the dimethoxymethyl ether (2) in a 38% yield. Removal of the THP group followed by Swern oxidation gave the aldehyde (4) in a yield of 81%. This was reacted with vinyl magnesium bromide to give the allyl alcohol (5) in a yield of 94%. The alcohol was heated at reflux in xylene with triethyl orthoacetate and a catalytic amount of propionic acid to give the ester (6) in a 93% yield. Subsequently, the ester (6) was reacted with methylmagnesium bromide to give the alcohol (7) in a yield of 93%. Removal of the MOM group followed by acetylation gave (22E) -1α, 38-diacetoxy-25-hydroxy-24-homo-cholesterol-5,22-diene (9) in 73% yield.

Allyl bromination of (9) with N-bromosuccinimide followed by treatment with tetra-n-butylammonium bromide and then with tetra-n-butylammonium fluoride afforded 5,7,22-triene (10) as a major product in a 24% yield. The 5,7,22-triene (10) was irradiated with an intermediate-pressure mercury lamp in benzene-ethanol for 5 minutes, then heated at reflux and hydrolyzed to give (22E) -la, 25-dihydroxy-22-dehydro-benzene. 24-homovitamin-D3 (11) in a yield of '22%.

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6 The 5,22-diene (9) was selectively hydrogenated to give the 5-ene (12) in a 92% yield. This compound was converted to 1α, 25-dihydroxy-24-homovitamin-D3 (14) via the 5,7-diene (13) as described above in a total yield of 12%.

In the following detailed description of the method, the melting points were determined by hot-stage microscope and indicated uncorrected. The HNMR spectra were recorded with a Hitachi R-24A (60 MHz) NMR spectrometer in CDCl3 with Me4Si as the internal standard, unless otherwise indicated. Mass spectra were recorded with a Shimadzu QP-1000 mass spectrometer at 70 eV. DV spectra were obtained in ethanolic solution with a Shimadzu UV-200 dual-beam spectrophotometer. Column chromatography was performed using silica gel (E. Merck, silica gel 60, 70-230 mesh). Preparative thin layer chromatography was performed on pre-coated sheets of silica gel (E. Merck, silica gel 60 F254, 0.25 mm thickness). The usual preparation is by dilution with water, extraction with an organic solvent indicated in brackets, washing of the extract for neutral reaction, drying over anhydrous magnesium sulfate, filtration and removal of the solvent under reduced pressure. The following abbreviations are used: THP - tetrahydropyranyl; THF - tetrahydrofuran; ether - diethyl ether, MeOH - methanol, MOM - methoxymethyl. Temperatures are given in ° C.

22-Hydroxy-23,24-dinorchola-1,4,6-trien-3-one (b)

To a solution of 3B-acetoxydinorcholenic acid (a) (7.0 g, 18.04 mmol) in THF (20 ml) was added lithium aluminum hydride (3.0 g, 78.95 mmol). The mixture was stirred at 60 ° C for 14 hours. To the reaction mixture was carefully added 35

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7 water and ethyl acetate. Filtration and removal of the solvent gave a residue (5.2 g). This was treated in dioxane (140 ml) with dichlorodicyanobenzoquinone (11.7 g, 51.54 mmol) under reflux for 14 hours. After cooling to room temperature, the reaction mixture was filtered and the filtrate was evaporated to a residue applied to a column of alumina (200 g). Elution with dichloromethane gave the trienone (b) (2.8 g 47%) m.p. 156-157 ° C (ether) UVX EtOH / max nm (epsilon): 299 (13000), 252 (9200), 224 (12000), 1 H NMR (CDCl 3) δ: 0.80 (3H, S, 18 - H3), 1.04 (3H, d, J = 6 Hz, 21-H3), 1.21 (3H, s, 19-H3), 3.10-3.80 (3H, m, 22-H2 and OH), 5.90-6.40 (4H, m, 2-H, 4-H, 6-H and 7-H), 7.05 (1H, d, J = 10 Hz, 1-H) , MS m / z: 326 (M +), 311, 308, 293, 267, 112.

1α, 2α-epoxy ~ 22-tetrahydropyranyloxy-23,24-dinorchola-4,6-dien-3-one (1)

The alcohol (b) (2.7 g, 8.28 mmol) in dichloromethane (50 ml) was treated with dihydropyran (1.5 ml, 16.42 mmol) and p-toluenesulfonic acid (50 mg) at room temperature for 1 hour.

Ordinary work-up (ethyl acetate for the extraction) gave a crude compound. To a solution of this compound in MeOH (70 ml) was added 30% H 2 O 2 (4.8 ml) and 10% NaOH / MeOH (0.74 ml) and the mixture was stirred for 14 hours at room temperature. Usual work-up (ethyl acetate for the extraction) gave a crude compound which was applied to a column of silica gel (50 g). Elution with benzene ethyl acetate (100: 1) gave the epoxide (1) (1.45 g, 41%): 30 m.p. 113-115 ° C (hexane) UVX Et 1 H / max nm (epsilon): 290 (22000), 1 H-NMR (CDCl 3) δ: 0.80 (3H, s, 18-H3), 1.04 (3H , d, J = 6 Hz, 2I-H3), 1.21 (3H, s, 19-H3), 3.38 (1H, dd, J = 4 and 1.5 Hz, 1-H), 3, 55 (1H, d, J = 4 Hz, 2-H), 3.30-4.10 (4H, m, 22-H2 and THP), 4.50 (1H, m, THP), 5.58

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8 (1H, d, J = 1.5 Hz, 4-H), 6.02 (2H, s, 6-H and 7-H), MS m / z: 342 (M + -DHP), 324 (M + - THPOH), 309, 283, 85.

1α, 38-dimethoxy-23,24-dinorchol-5-ene-22-tetrahydropyranyl ether (2)

Lithium (5.00 g) was added in small portions to liquid ammonia (200 ml) at -78 ° C in an argon atmosphere over 30 minutes. After stirring for 1 hour at -78 ° C to -10, 1α, 2α-epoxy-22-tetrapyranyloxy-23,24-dinorcho-1α-4,6-dien-3-one (1) (2.00 g, 4.69 mmol) in dry THF (50 ml) at -78 ° C over 30 minutes and the mixture was stirred for 1 hour at -78 ° C. To the reaction mixture was added anhydrous NH 4 Cl (60 g) in small portions over 1 hour at -78 ° C. After 1.5 hours, the cooling bath was removed and most of the ammonia was removed by bubbling with argon. Usual work-up (ether was used as solvent) gave an impure compound.

This was treated with chloromethyl methyl ether (2.0 mL, 20.34 mmol) and N, N-diethylcyclohexylamine (4.6 mL, 24.93 mmol) in dioxane (20 mL) at 45 ° C for 24 hours. Usual work-up (ethyl acetate) gave an impure compound which was applied to a column of silica gel (40 g). Elution with hexane-ethyl acetate (5: 1) gave the dimethoxymethyl ether (2) (922 mg, 38%) as an oil. 1 H-NMR δ: 0.70 (3H, s, 18-H3), .1.02 (3H, s, 19-H3), 1.04 (3H, d, J = 6 Hz, 21-H3), 3.34 (3H, s, -0-CH 3), 3.37 (3H, s, -0-CH 3), 4.63 (2H, ABq, J = 7 Hz, δ AB = 11 Hz, 1 CX- O-CH 2 -O-), 4.64 (2H, S, 3β-O-CH 2 -O-> and 5.50 (1H, m, 6-H).

30 35 9

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1α, 38-dimethoxy-23,24-dinorchol-5-en-22-ol (3) THP-ether (2) (922 mg, 177 mmol) in THF (8 ml) and MeOH (8 ml) were treated with 2 M HCl (1 ml) for 2 hours at 5 room temperature. Usual work-up (ethyl acetate) gave an impure compound which was applied to a column of silica gel (40 g). Elution with hexane-ethyl acetate (2: 1) gave the alcohol (3) (678 mg, 88%) as an amorphous solid. 1 H-NMR δ: 0.70 (3H, s, 18-H3), 1.02 (3H, s, 19-H3), 1.04 (3H, d, J = 6 Hz, 2I-H3) , 3.34 (3H, s, -O-CH 3), 3.38 (3H, s, -O-CH 3), 4.65 (2H, ABq, J = 7 Hz, δ AB = 11 Hz, 1a - O-CH 2 -O-), 4.66 (2H, s, 38-O-CH 2 -O-), 5.53 (1H, m, 6-H).

1α, 3g-dimethoxy-23,24-dinorchol-5-en-22-al (4) 15

To a solution of oxalyl chloride (0.27 mL, 3.09 mmol) in dichloromethane (8 mL) was added at -78 ° C under argon dimethyl sulfoxide (0.44 mL, 6.21 mmol). The mixture was stirred for 10 minutes at -78 ° C. To the solution was added the alcohol 20 (3) (660 mg, 1.51 mmol) in dichloromethane (5 ml) at -78 ° C. After stirring for 15 minutes, triethylamine (1.89 mL, 13.6 mmol) was added. The mixture was stirred at -78 ° C under argon for 5 minutes and warmed to room temperature.

Usual work-up (ether) gave an impure compound which was applied to a column of silica gel (30 g). Elution with hexane-ethyl acetate (4: 1) gave the aldehyde (4) (607 mg, 92%) as a crystalline mass, m.p. 71-72 ° C (hexane), 1 H NMR 6: 0.74 (3H, s, 1.8-H3), 1.04 (3H, s, 19-H3), 1.12 (3H, d, J = 6 Hz, 21-H3), 3.35 (3H, s, -O-CH 3), 3.39 (3H, s, -O-CH 3), 3.7 (1H, m, 1β-Η), 4.65 (2H, ABq, J = 7 Hz, δ AB = 11 Hz, Ia-0 -CH2-O-), 4.66 (2H, s, 3 & -O-CH-O-), 5.52 (1H, m, 6-H) and 9.61 (1H, d, J = 3 Hz , -CHO), analysis calculated for -G2gH4205: C, 71, -85; H 9, 74. Found: C, 71.71; H, 9.68.

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1α, 3g-dimethoxychola-5,23 dien-22-ol (5)

To magnesium (70 mg, 2.92 mmol) in THF (3 ml) was added a 50% solution of vinyl bromide in THF (0.42 ml, 2.98 mmol).

The mixture was stirred under argon at room temperature for 30 minutes. To the prepared Grignard reagent, the aldehyde (4) (5.95 mg, 1.37 mmol) in THF (6 ml) was added at room temperature. The mixture was stirred for 1 hour at room temperature. Usual work-up (ether) gave an impure 10 compound applied to a column of silica gel (30 g).

Elution with hexane-ethyl acetate (3: 1) gave the allyl alcohol (5) (595 mg, 94%) as an amorphous solid. 1 H-NMR δ: 0.70 (3H, s, 18-H3), 1.02 (3H, s, m 19-H3), 3.35 (3H, s, -O-CH 3), 3.38 (3H, s, -O-CH 3), 3.69 (1H, m, IgH), 4.20 (1H, m, 22-H), 4.64 (2H, ABq, J = 7 Hz, δ AB = 11 Hz, Ia-O-CH2-0-), 4.65 (2H, s, 38-0-CH2-0-), 5.52 (1H, m, 6-H), 4.90- 6.0 (3H, m, 23-H and 24-H2).

(22E) -la, 3g-dimethoxymethoxy-27-norcholesta-5,22-diene-26-oanoic acid ethyl ester (6)

A solution of the allyl alcohol (5) (590 mg, 1.28 mmol), triethyl orthoacetate (1.0 mL, 5.64 mmol), propionic acid (4 drops) and xylene (8 mL) was heated under argon for 2 hours at reflux . Removal of the solvent under reduced pressure gave a residue applied to a column of silica gel (30 g). Elution with hexane / ethyl acetate (4: 1) afforded the ester (6) (630 mg, 93%) as an oil. 1 H-NMR δ: 0.68 (3H, s, 18-H3), 0.97 (3H, d, J = 6 Hz, 2I-H3), 1.03 (3H, s, 19-H3) , 1.24 (3H, t, J = 7 Hz, -CO 2 CH 2 CH 3), 3.35 (3H, s, -O-CH 3), 3.39 (3H, s, -O-CH 3), 3.70 ( IH, m, ΙβΗ), 4.11 (2H, q, J = 7 Hz, -CO2CH2CH3), 4.64 (2H, ABq, J = 7 Hz, δ AB = 11 Hz, Ia-O-CH2-0 -), 4.65 (2H, s, 3β-O-CH 2 -O-), 5.29 (2H, m, 22-H and 23-H), 5.52 (1H, m, 6-H ).

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11

Optionally, the 22E stereoisomeric compound (6) can be readily converted to the 22Z stereoisomer by iodine treatment.

Thus, treatment of the compound (6) in ether with a catalytic amount of iodine (2%) relative to the compound (6) causes, under diffuse daylight, for 1 hour a trans to cis isomerization which after HPLC purification (Zorbax-Sil column, 4.6 x 25 cm, 6% 2-propanol / hexane) gives the 22Z stereo isomer.

10 (22E) -lot, 3g-dimethoxymethoxy-24-homo-cholesta-5,22-diene 25-01 (7)

To a solution of the ester (6) (605 mg, 1.14 mmol) in THF (6 ml) was added a 1 M solution of methylmagnesium bromide in THF (4.5 ml, 4.5 mmol) at room temperature.

The mixture was stirred for 1 hour at room temperature. Usual work-up (ether) gave an impure compound applied to a column of silica gel (30 g). Elution with hexane / ethyl acetate (3: 1) gave the alcohol (7) (548 mg, 93%) as an oil. 1 H NMR δ: 0.68 (3H, s, 18 H3), 0.97 (3H, d, J = 6 Hz, 21-H3), 1.01 (3H, s, 19-H3), 1 , 21 (6H, s, 26-H3 and 27-H3) 3.33 (3H, s, -0-CH3), 3.38 (3H, s, -O-CH3), 3.70 (1H, m , Ig-H), 4.64 (2H, ABq, J = 7 Hz, δ AB = 11 Hz, Ia-O-CH2-0-), 4.65 (2H, s, 3B-O-CH2-0 -), 5.29 (2H, m, 22-H and 23-H) and 5.50 (1H, m, 6-H).

(22E) -24-homocholesta-5,22-diene-1α, 3g, 25-triol (8)

A solution of the dimethoxymethyl ether (7) (540 mg, 1.04 30 mmol) in THF (15 ml) was treated with 6 M HCl (3 ml) at 50 ° C for 2.5 hours. Usual work-up (ethyl acetate) gave an impure compound which was applied to a column of silica gel (20 g). Elution with hexane / ethyl acetate (1: 1) gave the triol (8) (428 mg, 95%) as a crystalline product.

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12

Mp. 164-166 ° C (hexane / ethyl acetate) 1 H-NMR δ: 0.68 (3H, s, 18-H3), 0.95 (3H, s, J = 6 Hz, 2I-H3), 1.00 (3H, s, 19-H3), 1.20 (6H, s, 26-H3 and 27-H3), 3.80 (1H, m, 18H), 3.92 (1H, m, 3a-H) , 5.30 (2H, 22-H and 23-H) and 5.53 (1H, 5 mr 6-H) - (22E) -la, 38-diacetoxy-25-hydroxy-24-homocholesta-5.22 -diene (9) 10 A solution of the triol (8) (395 rag, 0.919 mmol) in pyridine (2 ml) was treated with acetic anhydride (1 ml) at room temperature for 16 hours. Usual work-up (ethyl acetate) gave an impure compound applied to a column of silica gel (20 g). Elution with hexane / ethyl acetate (2: 1) gave the diacetate (9) (361 mg, 77%) as an oil. 1 H NMR δ: 0.67 (3H, s, 18-H3), 0.97 (3H, d, J = 6 Hz, 21-H3), 1.07 (3H, S, 19-H3), 1 , 21 (6H, s, 26-H3 and 27-H3), 2.01 (3H, s, acetyl), 2.04 (3H, s, acetyl), 4.98 (1H, m, 3a-H) , 5.05 (1H, m, 18-H), 5.31 (2H, m, 22-H and 23-H) and 5.52 (1H, m, 6-H).

(22E) -la, 3B-diacetoxy-25-hydroxy-24-homocholesta-5,7,22-triene (10) A solution of 5-ene (9) (51 mg, 0.0992 mmol) and N bromosuccinimide (21 mg, 0.118 mmol) was refluxed under argon for 20 minutes in carbon tetrachloride (3 ml). After the mixture was cooled to 0 ° C, the precipitated precipitate was filtered off. The filtrate was concentrated below 40 ° C to give a residue. This was treated in THF (5 ml) with a catalytic amount of tetra-n-butylammonium bromide for 50 minutes at room temperature.

Then the mixture was treated with a solution of tetra-n-butylammonium fluoride in THF (3.5 ml, 3.5 mmol) in 35

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13 minutes at room temperature. Usual work-up (ethyl acetate) gave a crude compound which was subjected to thin-layer preparative chromatography (hexane / ethyl acetate, 4: 1, 5 times). The band with the Rf value 0.48 was removed and eluted with ethyl acetate. Removal of the solvent gave the 5,7,22 triene (10) (12.5 mg, 24%), UVX EtOH / max nm: 293, 282 and 271.

lot, 25-dihydroxy-22E-dehydro-24-homovitamin-D7 (11) 10

A solution of the 5,7,22-triene (10) (7.3 mg, 0.0143 mmol) in benzene (90 ml) and ethanol (40 ml) was irradiated with a medium-pressure mercury lamp through a Vycor filter at 0 ° C. 5 minutes under argon. The reaction mixture was heated under argon for 1 hour at reflux.

Removal of the solvent under reduced pressure gave a crude compound which was subjected to preparative thin layer chromatography (hexane / ethyl acetate, 4: 1, which was developed 5 times). The band with Rf value 0.38 was removed and eluted with ethyl acetate. Removal of the solvent gave the vitamin D3 diacetate (1.8 mg, 25%). The band with Rf value 0.43 was removed and eluted with ethyl acetate. Removal of the solvent recovered the 5,7,22-triene (10) (2.1 mg, 29%).

25

Vitamin D3 diacetate (1.8 mg, 2.15 mmol) in THF (4 mL) was treated with 5% KOH / MeOH (1 mL) at room temperature for 20 minutes. Usual work-up (ethyl acetate) gave an crude compound which was subjected to thin-layer preparative chromatography (hexane / ethyl acetate, 1: 2, induced 3 times). The band with the Rf value 0.43 was removed and eluted with ethyl acetate. Removal of the solvent gave the vitamin D3 analog (11) (1.4 mg, 90%). The purity of the compound (11) was determined to be 100% at high pressure liquid chromosome 14

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matography (Shimadzu LC-3A? column Zorbax ZIL normal phase, 4.6 mm internal diameter x 15 cm; solvent MeOH-CH 2 Cl 2, 1:49, flow rate 3 ml / minute; retention time 11.5 minutes). The vitamin D3 analog (11) 5 had the following spectral data: DVX EtOH / max: 265 nm, UVX EtOH / min: 228 nm, MS m / z: 428 (M +), 410, 392 (base peak), 374, 287, 269, 251, 152, 134, 123, 59 1 H-NMR (360 MHz) δ: 0.55 (3H, s, 18-H3), 1/02 (3H, d / J = 6.6 Hz, 21- H3), 1.22 (6H, s, 26-H3 and 27-H3), 2.32 (1H, dd, J = 13.2 and 6.7 Hz), 2.60 (1H, dd, J = 13.0 and 3.0 Hz), 2.83 (1H, dd, J = 12.0 and 3.0 Hz), 4.23 (1H, m, Wjy2 = 18 »4 Hz, 3a-H) , 4.43 (1H, m, W1 / 2 = 16.9 Hz, 1β-Η), 5.00 (1H, bs, W1 / 2 = 3.2 Hz, 19-H), 5.30 (1H , dd, J = 15.0 and 7.1 Hz, 22-H or 23-H), 5.33 (1H, bs, Wi / 2 = 3.2 Hz, 19-H), 5.37 (1H , 15 dd, J = 15.0 and 5.8 Hz, 22-H or 23-H), 6.01 (1H, d, J = 11.0 Hz, 7-H), 6.32 (1H, d, J = 11.0 Hz, 6-H).

1α, 38-diacetoxy-24-homocholesta-5-en-25-ol (12) 20 A mixture of 5,22-diene (9) (40 mg, 0.0778 mmol) and 10% Pd-C (4 mg ) in ethyl acetate (2 ml) was stirred under hydrogen for 3 hours at room temperature. The Pd catalyst was filtered off and the filtrate was concentrated to give a residue applied to a column of silica gel (5 25 g). Elution with hexane / ethyl acetate (4: 1) afforded 5-ene (12) (37 mg, 92%) as an oil. 1 H-NMR δ: 0.66 (3H, s, 18-H3), 1.08 (3H, s, 19-H3), 1.20 (6H, s, 26-H3 and 27-H3), 2 , 02 (3H, s, acetyl), 2.05 (3H, s, acetyl), 4.97 (1H, m, 3a-H), 5.07 (1H, m, 18-H), 5.53 (1H, m, 6-H).

30 Ia, 38-diacetoxy-24-homocholesta-5,7-diene-25-ol (13) The 5-ene (12) (19 mg, 0.037 mmol) was converted as described for compound (9) to 5,7-diene (13) (5.8 mg, 31%).

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UVX EtOH / max: 293, 282, 271 nm.

1α, 25-dihydroxy-24-homovlaniin-D 2 (14) 5 14) (890 µg, 19%). The retention time of (Γ4) under the HPLC conditions described above was 11, D minutes DVX EtOH / max. 265 am, UVX E ^ H / min: 228 nm,. ; MS 10 m / z: 430 (M +), 412, 394 ((base top), 376, 287, 269, TESI, 152, 134, 59).

The compound of the invention may be readily obtained in crystalline form by recrystallization with suitable solvents such as hexane, ether, alcohol, or mixtures thereof, as will be apparent to those skilled in the art.

Biological Activity Bone Calcium Mobilization Effect of Ia, 25- (OH)? 2,4-Ho-Mo-D 2 Compounds

The bone calcium mobilizing effect was investigated by measuring the increase in serum calcium levels in response to the compound administered. Male-weaned rats (Holtzman Co., Madison, Wl) were fed a low-calcium-vitamin-D-poor diet (Suda et al., J. Nutr. 100, 1049-1059, 1970) and water. ad libitum for 3 weeks. Subsequently, the rats were divided into 3 groups of 5-6 rats in each group and intrajugularly administered either 1, 2'5 (OH) 2D3 or the test compound dissolved in 0.05 ml of 95% ethanol. The rats in the control group fi: k 0.05 ml ethanol vehicle on. same way. 18 hours after administration, the rats were sacrificed and their blood collected and centrifuged for 35 minutes.

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reaching serum. Serum calcium concentrations were determined with a Model 403 atomic absorption spectrometer (Per-kin-Elmer Co., Norwalk, Conn.) In the presence of 0.1% lanthanum chloride.

5

The results are given in the following table: 10 15 20 25 30 35

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17 "Table 1

Increase in serum calcium concentration in response to a administered compound 5

Administered Administered Serum calcium compound amount concentration (pmol / rat) (mg / 100 out) 10 Ex. In Ethanol - 3.6 ± 0.3a / 1a, 25-650 4.9 ± 0.2bl) (OH) 2-D3a, 25- (650 4.4 ± 0.2b ') (OH) ) 2-24-15 homo-D3

Ex. II Ethanol - 4.2 ± 0.1, 1a, 25- (OH) 2D3 325 5.0 + 0.5d) 1a, 25- 650 5.0 ± 0.5d) (OH) 2-22E- dehydro-24-homo-D3 25 * Standard deviation from mean significantly different from b) from a) and p <0.001 d) from c).

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18

From the above results, it can be concluded that in the vitamin D-dependent systems of vitamin D-deficient animals, the compounds of the invention exhibit the same effect as 1α, 25-dihydroxyvitamin-D3, the circulating hormonal form of the vitamin, although in the case of about the 22-dehydro derivative, the dose was significantly higher.

If the subject compounds are administered to human promyelocytic leukemia cells (HL-60 cells) grown in tissue culture, they induce differentiation of these cells into macrophages (monocytes). In several standard methods for measuring the differentiation activity, these compounds were found to be more effective than 1α, 25- (OH) 2D3, the most active hitherto known vitamin D-15 derivative. These experiments were performed as follows:

Investigation of the Differential Effect of Homovitamin D Compounds 20 The human promyelocytic leukemia cell line (HL-60) was maintained in suspension culture in RPM1 1640 substrate (Gibco,

Grand Island, NY) added 10 volume / volume% heat-inactivated fetal calf serum, 100 µg / ml penicillin, 100 µg / ml streptomycin and 0.25 µg / ml fungizone. Cells 25 were grown in a humidified atmosphere with 5% CO 2. Cell viability was investigated by standard methods e.g. by trypan blue exclusion. The morphological assessment was done on Wright stained glass slides.

The cells were seeded at concentrations of 1.5-2 x 10 6 cells / ml in 10 ml substrate in tissue culture dishes. After 20 hours, the dishes were treated in pairs with Ia, 25- (OH) 2D3 - and one of the compounds Ί "or II at various concentrations as indicated below. (Compounds I and II 35

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19 is defined below). The test compounds were added as solutions in 100% ethanol so that the total ethanol concentration in each culture dish did not exceed 0.2%. Control cultures were treated with the same concentration of ethanol. After 4 days (96 hours) of incubation with the test compounds, the cells from these culture dishes were harvested and cell counts and viability determined. The degree of differentiation induced by the studied vitamin D derivatives was expressed as the percentage of cell lines that exhibited funchonal and enzymatic markers characteristic of monoeytes. The two markers examined were a) the ability of the cells to phagocytize dead yeast, and b) the ability of the cells to produce superoxide (reduced nitrotetrazole azimuth) when stimulated with phorbol esters.

a) Phagocytosis study for differentiation activity; The harvested cells were suspended in RPMI substrate containing 20% AB serum and 20% fetal calf serum to obtain a composition containing 2 x 10 6 cells / mL.

To 0.5 ml (10® cells) of the above suspension was then added 0.5 ml of a suspension (in phosphate-buffered saline) of heat-killed Sacctoaromyces cerevisiae cells (1x104 cells) which had been stained with tryphanb'låt.

After incubating this mixture for 1 hour at 37 ° C, the number of phagocylic cells was counted (determined by trypan blue-stained yeast that appeared intracellularly) and expressed as a percentage of the total number of living cells present. This "% phagocytic cells" gives the differentiation percentage induced by the test compounds. The results are summarized in Table 2 below.

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TABLE 2

Percentage of phagocytic (differentiated) cells produced in HL-60 cell cultures treated with vitamin D compounds at various concentrations

Admini- Concentration (mol / liter) Strained Compound 0 (a'b) 3x1O-10 5x10 ~ 10 lx10_9 (bJ lx10 "8 (b) lx10_7 (b) lx10 -7 la, 25- (OH) 2D3 1Q ± 1 , 5 17 23 28 ± 4 47 ± 1 67 ± 6 69 15

Compound I * 10 ± 1.5 29 38 47 ± 6 69 ± 3 77 + 1 78

Compound II * 10 ± 1.5 31 48 51 ± 4 59 + 0.5 77 ± 1 78 a control level; cell cultures were treated only with the solvent ethanol 25 h ° The results reported in these columns represent mean ± standard deviation of three experiments, each performed twice.

Compound I - 1a, 25-dihydroxy-24-homovitamin D3

Compound II-1α, 25-dihydroxy-22E-dehydro-24-homo-vitamin D3

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The results in Table 2 show that the 24-homo compounds had very similar differentiation activity and are significantly more active than 25- (OH) 2D3. At all concentrations, the homo compounds cause a greater degree of differentiation of the leukemia cells than Ia, 25- (OH) 2D3, the most active known compound to date. Eg. the homo compounds at a concentration of 10 “8 mol / L cause a 70% differentiation, while 1a, 25- (OH) 2D3 at the same concentration gives only approx. 47% differentiated cells.

10 To achieve an approximate 50% differentiation, a concentration of 1 x 10-9 M of the homo compounds is required, but approx. 1 x 10-8 M of Ia, 25- (OH) 2D3 ie. the homo compounds have a differentiation activity that is approx. 10 ganpe higher.

(B) NBT reduction experiments for differentiation:

This assay utilizes the ability of monocyte-like leukemia cells to reduce the nitro-blue tetrazolium 20 (NBT) reagent to a blue-black precipitate (formazan) when stimulated with phorbol esters. The study was carried out as described by Yen et al. (J. Cellular Physiol. 11U, 277 (1984). The cells are harvested as described above and suspended in RPMI substrate; to 0.2 ml of this suspension (containing 1.4 x 108 cells / ml) was added 0.2 ml of nitro blue tetrazolium (NBT) reagent (NBT reagent was prepared by mixing a solution containing 50 mg of nitro blue tetrazolium in 50 ml of phosphate-buffered saline with 10 μΐ of acetone / water (1: 1) solution containing 30 mg of 0.5 mg / ml of 48-phorbol-1,2-myristate-13-acetate) After standing for 30 minutes in a water bath, the differentiated cells (i.e., cells showing formazan blue deposits as one indication - on NBT) were counted. reduction) by a haemocytometer and expressed as a percentage of the total 35

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22 number of living cells present. The results of this study are given in Table 3.

TABLE 3 5

Percent of cells in HL-60 cell cultures exhibiting nitro blue tetrazolium (NBT) reducing ability after treatment with vitamin D compounds at various concentrations 10 -

Admini- Concentration (mol / liter) Strained Compound o (a'b> 3x10 - 10 5x10 "10 lxl0_9 (b) lxl0" 8 (b> lxl0-7 <b) lxl0-7 15 -I-10.25 - (OH) 2D3 10 ± 1.5 15 27 31 + 4 45 ± 4 69 ± 7 65

Compound I1 10 ± 1.5 33 39 46 ± 4 72 ± 1 79 ± 5 77

Compound II1 10 ± 1.5 33 45 52 + 5 71 ± 1 78 ± 5 79 25 a control level; cells treated only with the solvent ethanol k the results given in these columns represent 2q mean ± standard deviation of three separate experiments # each made twice.

Compound I - Io, 25-dihydroxy-24-homovitamin D3 Compound II - Ia # 25-dihydroxy-22E-dehydro-24-homo-vitamin D3

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The results reported in Table 3 show again that the 24-homo compounds examined are more active than 1α, 25- (OH) 2D3 to induce differentiation of human renal cell leukemia cells into normal cells, in vitro, in all 5 in the previous experiment (Table 2) are equally active. To achieve 60% differentiation of leu germ cells measured by this NBT reduction method, a concentration of 2 x 10 10 M of the homo compounds is required; to achieve the same degree of differentiation with Ia, 25- (OH ') 3> I> 3 10, a concentration of 3.5 x 10 6 M is required, a 17 fold difference in activity.

Thus, both studies confirm the high activity of the subject 24-homovitamin D compounds to induce differentiation of leukemia cells. In addition, the above results * indicate that with respect to this differentiation activity, the subject homovitaminin compounds are 10-20 times more active than Ia, 25- (®H;) jiD3.

The compounds of this invention can be readily administered as sterile parenteral solutions by injection or intravenously or per os in oral doses or as suppositories or transcutaneously. Doses on from about ..

0.1 µg to approx. 2.5 µg per day is capable of providing physiologic calcium balance response characteristic of vitamin D-like action, with a maintenance dose of about 0.1 pg to approx. 0.5 µg is appropriate.

Dosage forms of the subject compounds can be prepared by combining these with a non-toxic pharmaceutically acceptable carrier well known in the art. Such carriers may be either solid or liquid such as, for example, corn starch, lactose, sucrose, peanut oil, olive oil, sesame oil and water.

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24

If a solid carrier is used, the dosage forms of the compounds of the invention may be tablets, capsules, powders, dragons or lozenges. If a liquid carrier is used, soft gelatin capsules or syrups or liquid suspensions, emulsions or solutions may be the dosage form. The dosage forms may also contain adjuvants, e.g. preservatives, stabilizers, wetting agents or emulsifiers, solvents, etc. They may further contain other therapeutically active compounds.

The dose administered to a patient depends on the specific disease case, on the outcome desired in that case, on the patient's physical size 15, as well as on other factors well known to those skilled in the art.

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Claims (6)

Vitamin D derivatives, characterized in that they have the general formula I -10 - 10 RJ 15 wherein Rx, R2 and R3 are each selected from hydrogen, an acyl group having from 1 to 4 carbon atoms and benzoyl, and R4 and R5 each represent hydrogen atoms or together form a carbon-carbon double bond. Derivative according to claim 1, characterized in that R 1, R 2 and R 3 represent hydrogen and R 4 and R 5 represent hydrogen atoms. Derivative according to claim 1, characterized in that R 1, R 2 and R 3 represent hydrogen and R 4 and R 5 together form a carbon-carbon double bond. 4. A derivative according to claim 2 or 3, characterized in that it is in crystalline form. A derivative according to claim 3, characterized in that the Δ-22 bond is in the E configuration. DK 154290 B Pharmaceutical composition, characterized in that it contains a derivative according to any one of the preceding claims and a pharmaceutically acceptable excipient. 5 10 15 20 25 30 35