NL8520009A - 1ALFA.25-DIHYDROXY-22Z-DEHYDROVITAMINE D COMPOUND. - Google Patents

Description

/ 8 5 2 0 0 0 9 la 25-dihydroxy-22Z-dehydrovitamin D compound.

The invention relates to a biologically active vitamin D compound. More particularly, the invention relates to a new 1,25-di-hydroxylated vitamin D compound having a 22.23-cis-double-5 bond in the side chain and a method for its preparation.

The calcium and. phosphate homeostasis in animals and humans is regulated by vitamin D metabolites and the compound la.25-dihydroxyvitamin D1 is generally considered to be the most active and most important vitamin D-derived regulator of the normal calcium and phosphate -balance. This natural metabolite and ver compounds structurally related thereto are therefore of great pharmaceutical importance as effective agents for the prevention and treatment of bone diseases and; related calcium metabolism disorders. In addition to the natural metabolites D, a number of compounds have been prepared in recent years, which, because of their high potency, find application or are very promising as therapeutic agents, including 1-hydroxyvitamin D 1, Ια- hydroxyvitamin, 1,25-dihydroxyvitamin D4 and certain fluorinated analogs (U.S. Pat. Nos. 3,741,996, 3,907,843, 3,880,894, 4,226,788 and 4,358,406).

Most of the known active analogs are characterized by the type of sterol side chain as it exists; in vitamin D ^ (i.e. a saturated side chain).

Known analogues with a 22.23-unsaturated side chain are the compounds of the vitamin D ^ series (i.e., 22.23-trans-unsaturated with a C 1 -methyl substituent), 30 and include, in addition to the above compounds, 25-hydroxyvitamin D 1 U.S. Patent 3,585,221) and the 24- and 24,25-dihydroxy derivatives (Jones et al., Arch.

8520009 - 2 -

Biochem. Biophys. '202, '. 450 (1980)) and three compounds without the 24-methyl substituent (U.S. Patent 3,786,062; Bogoslovskiic., J. Gen. Chem. USSR 48 (4), 828 (1978) Chem. Abstr. 89, 6384.8j and 209016s).

A new vitamin D analog has now been found, which can be represented by the structural formula la indicated on the formula sheet.

This new connection is characterized by a 22.23 double bond in the side chain with the cis (or Z) 10 geometry. Due to the presence of this 22Z double bond, which results in a completely different side chain geometry than that which belongs to big compounds with the normal saturated side chain. (for example, as in la.25-dihydroxyvitamin D ^) or a 22.23-trans (22E) -unsaturated-the side chain (for example, as in la.25-dihydroxyvitamin D ^), this cis-unsaturated product was believed to have a low or would show no biological activity at all. Surprisingly, despite its altered side chain structure, this material exhibits high activity; | 20 te it, wherein it is as active as 1a-25-dihydroxyvita-i'i: | mine for its ability to increase the serum calcium level in laboratory animals.

The novel product of the present invention of the above formula la was prepared from a 22Z-25 dehydrovitamin D precursor of the structural formula 1b by enzymatic hydroxylation in vitro at the 25 carbon atom using a liver homogen. hate preparation from vitamin D deficient rats.

The following procedure was used: Only 30 suckled (weaned) male rats were fed a low calcium and vitamin D deficient diet as a | written by Suda et al. / J. Nutr. 100, 1049 (1970) for 2 weeks. They were killed by decapitation and | their livers were removed. A 20% (w / v) homogenate | 35 was prepared in ice cold 0.25M sucrose. The incubation was performed in 10 ml incubation medium in a 125 ml Erlenmeyer flask 8520009-3 containing a sample of the liver homogenate, which sample included 1 g of tissue, 0.125 M. sucrose, 50 mM phosphate buffer (OH 7, 4), 22.4 mM glucose-6-phosphate, 20 m ATP, 160 mM nicotinamide, 25 mM succinate, 0.4 mM 5 NADP, 5 mM MgCl 3 / 0.1 M KCl and 0.5 units glucose-6- phosphate dehydrogenase. The reaction was started by adding 400 µg of the substrate, the above compound 1b, dissolved in 100 µl of 95% ethanol. The incubation mixture was incubated for 3 hours at 37 ° C with shaking at 80 swings / minute for 3 hours.

The reaction was stopped by adding 20 ml of methanol and 10 ml of dichloromethane. After a further addition of 10 ml of dichloromethane. the organic phase was collected, while the aqueous phase was re-extracted with 10 ml of dichloromethane. The organic phases from a total of three extractions were combined and evaporated on a rotary evaporator. The residue, containing the desired product, was dissolved in 1 ml of a CHCl 2 hexane (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 were collected and evaporated. ; The residue was then dissolved in 8% 2-propanol in hexane and subjected to a liquid chromatography with; 25 High Performance (Model LC / GPC 204 HPLC, Waters; j

Associates, Medford, MA) using a Zorbax-SIL column (4.6 mm x 25 cm, DuPont, Wilmington, Delaware) operating at a pressure of .70 kg / cm 2 at a flow rate of 2 ml / minute. The desired 25-hydroxy-! The leather product was eluted at 44 ml. This product was further purified by high performance liquid chromatography using an inverted phase column (Richrosorb Rp-18, 4.6 mm x 25 cm, E. Merck, Darmstadt, West Germany) operating under a pressure of 35 84 kg / cm2 and a flow rate of 2 ml / minute. The column was eluted with 22% ^ 0 in methanol and the compound - 4 - was eluted at 50 ml. The product was further purified by HPLC using the Zorbax-SIL column and under the conditions described above. The resulting product is then subjected to a physical characterization.

Characterization of the product.

The UV absorption of the product in 95% ethanol showed a λ = 265 nm and a λ = 228 nm, max mm, indicating the presence of the 5.6-cis-triene-10 chromophore.

The mass spectrum of the substance contains a molecular ion at m / e 414 as required for a 25-hydroxylated product. The elimination of 1 and 2 molecules of H 2 O gives fragment ions at m / e 396 and 378. It! Loss of the entire steroid side chain (cleavage of the C17 ^ C20 bond) results in the fragment of m / e 287, which by elimination of one and two molecules of H ^ O gives rise to the peaks at m / e 269 and 251 The spectrum shows prominent peaks at m / e 152 and 134 (152-20 I ^ O), which represent ring A fragments and are diagnostic for 13,3-dihydroxyvitamin D compounds. In addition, the spectrum shows a very prominent fragment peak at m / e 59, resulting from the split

From the C0 / 1 / C '_ bond. The presence of this ion confirmed the presence of the 25-hydroxy group in the product. These data thus confirmed the structure of the resulting product as the 1,25-dihydroxylated compound. bond, as represented by the structural formula la.

j Biological activity I 30 The biological activities of the new i.

analogous were demonstrated by in vivo assays in the rat. Newly suckled male rats were fed the low calcium vitamin D deficient diet of Suda et al. (Literature above) for 3 weeks. They were then divided into groups of 5 rats each. The rats in a control group received 0.05 ml of 95% ethanol @ 520061-5 intrajugular (in the jugular vein), while in the other groups, the rats received 325 pmol of either compound 1a or 1a-25-dihydroxyvitamin D> dissolved in 0.05 ml of 95 Vs ethanol were administered. Eighteen hours later they were killed by decapitation and the blood was collected. The serum obtained by centrifugation of the blood. diluted with 0.1% lanthanum chloride solution (1:20) and the serum calcium concentration was determined with an atomic absorption spectrophotometer. The results are indicated in the table below:

Increase in serum calcium concentration in response to a single dose of 325 pmol of either compound 1a or 1a-25-dihydroxyvitamin D1 administered 18 hours before killing the animals. _ i I Administered Serum Calcium Concentration Compound_ (mg / 10Q ml) ± Standard Deviation | ethanol 4.2 ± 0.1 ^ | compound la 5.2 ± 0.2 ^ | 20 la.25-dihydroxyvitamin D 5.4 ± 0.4 ^

i O

! (b) is significantly different from (a) p <0.001 | The above results show that! the new analog has high efficacy and a! 25 exhibits biological activity that is substantially equivalent to that of 1,25-dihydroxyvitamin D ^

Because of this high potency, the I compound of this invention will find utility as a therapeutic agent in the therapy or prophylaxis of disorders such as the varying types of English disease (rickets), hypoparathyroidism, osteodystrophy, osteomalacia or osteoporosis in humans, or for the treatment of related calcium deficiency diseases (e.g., milk fever, leg (leg) weakness, and egg-shell thinness) in animals. The compound can also be used to treat certain malignant diseases, such as human leukemia.

For therapeutic purposes, the compound can be administered by any conventional route of administration and in any form suitable for the method of administration selected. The compound can be formulated with any acceptable and harmless pharmaceutical carrier, in the form of pills, tablets, gelatin capsules or suppositories, or as solutions, emulsions, dispersions or suspensions in harmless solvents or oils, and the like formulations (pre preparations) may also contain other therapeutically active and beneficial ingredients, such as may be suitable for specific applications. For use in humans, the compound is advantageously administered in amounts of 0.25-10 ^ 9 per day, with the specific | Dosage is adjusted depending on the disease to be treated and the patient's medical history, condition and response, as is apparent to those skilled in the art.

The 22Z-dehydro precursor substrate, the above compound 1b, required For the preparation of the! . .

| new product of this invention is self-prepared according to the method shown in Reaction Scheme A, which will be described below. In the description, the designations of the compounds have by | Arabic numerals (e.g. (1), (2), (3), etc.) refer to the structure thus numbered in the reaction scheme; time formulas. The desired substrate (compound 1b) for the 25-hydroxylation reaction described above is indicated in the reaction scheme A and in the following description by the Arabic numeral (11).

j (22Z) -3β- (methoxymethoxy) -5a.8a- (4-phenyl-1,2-urazolo) cholesta-6.22-diene (2).

isopentylphosphonium bromide / (CH2) 35 (1.67 g, 4.04 mmol) in dry tetrahydrofuran (73 ml) was treated with n-butyl lithium (1.7 M) with stirring at 3-5 ° C.

8520009-7 solution in hexane, 2.42 ml, 4.11 mmol). After stirring for 1 hour at room temperature, the orange-red solution was cooled to 3 ° C and aldehyde (1) (1.84 g, 3.36 mmol) in dry THF (24 ml) was added. The colorless reaction mixture was stirred at room temperature overnight, then poured into water and extracted with benzene. The organic extract was washed with 5% HCl, saturated sodium hydrogen carbonate and water, dried (Na 2 SO 2) and concentrated in vacuo to an oil which was purified on a column of silica gel. The elution with a benzene-ether (94: 6) mixture gave the adduct (2) (1.38 g, 68%) as a foam: NMR: 6 0.83 (3H, s, 18-H3), 0, 89 and 0.91 (6H, each d, J J = 6.8 Hz, 26-H3 and 27-H3), 0.97 (3H, d, J = 6.8 Hz, 21-H3); j 15 0.98 (3H, s, 19-H3), 3.30 (1H, dd, J = 4.4 Hz, J = 14 Hz, | 9-H), 3.38 (3H, s, 0CH3 ), 4.33 (1H, m, 3-H), 4.70 and 4.81 (2H, ABq, J = 6.8 Hz, OCHO), 5.21 (2H, br, in, 22- H and I 23rH) / 6.23 and 6.39 (2H, ABq, J = 8.5 Hz, 6-H and 7-H), 7.41! (5H, br m, Ar-H); IR: 1756, 1703, 1601, 1397, 1046 cm -1; | 20 mass spectrum, m / z 601 (M +, 1%), 426 (4), 364 (61), 349 (16), 253 (18), 251 (18), 119 (PhNCO, 100).

(22Z) -5a.8a- (4-phenyl-1,2-urazolo) cholesta-6,22-dien-38-ol (3).

A solution of adduct (2) (601 mg, l.ramol), 25 and p-toluenesulfonic acid (523 mg, 2.75 mmol) in a methanol (20 ml) -THF (12 ml) mixture was left at room temperature for 2 days stirred. The reaction mixture was poured into saturated sodium hydrogen carbonate and extracted several times with benzene. The extracts were washed with water, dried (Na SO) and evaporated under reduced pressure. The purification of the crude product by column chromatography (benzene ether 70:30 as an eluent) gave the hydroxy adduct (3) (550 mg, 99%) as a foam: NMR 6 0.83 (3H, s, 18-H3) ), 0.89 and 0.91 (6H, each d, 35 J = 6.8 Hz, 26-H3 and 27-H3), 0.95 (3H, s, 19-H3), 0.98 (3H , d, J = 6.8 Hz, 21-H3), 3.16 (1H, dd, 0 ^ = 4.4 Hz, J2 = 14 Hz, 8520009 - 8 - 9-H), 4.44 (1H , m, 3-.H), 5.22 (2H, br, m, 22-H and '23 -H), 6.22 and 6.39 (2H, ABq, J = 8.5 Hz, 6- H and 7-H), 7.40 (5H, br-1 m, Ar-H); IR: 3447, 1754, 1700, 1600, 1397 cm; mass spectrum, m / z (557 (M +, 1%), 382 (35), 349 (33), 253 (20), 5 251. (33), 119 (100), 55 (82).

(22Z) -cholesta-5.7.22-triene-38-ol (4).

The adduct (3) (530 mg, 0.95 mmol) was converted to the diene (4) by reduction with lithium aluminum hydride (1 g) in tetrahydrofuran (60 ml) under reflux for 18 hours. After the usual work-up, the product was purified by chromatography on silica (benzene-ether 94: 6 as an eluent) to give the pure diene (4) (290 mg, 76%) after crystallization from ethanol: melting point = -. 148-151 ° C; -.24 15 L aJn = “132 ° (c = ° '9, chc13)' * n * ® 5 0.66 (3H '? 18-h3), 0.90 and 0.91 (6H, each d , J = 6.8 Hz, 26-H3 and 27-H3), 0.96 (3H, s, 19tH3), 0.98 (3H, d, J = 6.9 Hz, 21-Hj), 3, 64 (1H, m, 3-H), 5.20 (2H, br m, 22-H and 23-H). 5.39 and 5.57 (2h, ABq, J = 6Hz, 7-H and 6-H); UV λ 281 nm; IR: 3346, 1463, max 20 1375, 1364, 1067, 1040, 831 cm; mass spectrum, m / z 382 (M +,] 100), 349 (65), 323 (32), 271 (15), 253 (30).

(5Z, 7E, 22Z) -9.10-secocholesta-5.7.10 (19), 22-; tetraen-3g-ol (5).

Irradiation of 5.7-diene (4) (150 mg, 0.39 mmol), dissolved in ether (120 ml) and benzene (30 ml) (degassed with argon for 40 minutes), was performed at 0 ° C for 13 minutes using a | UV lamp and a Vycor filter. HPLC (1% 2-propanol in hexane) of the resulting mixture gave the previtamin; 30 (56.9 mg, 38%) as a colorless oil: NMR δ 0.75 (3H, s, 18-CH3), 0.90 and 0.91 (6H, each d, j = 6.7 Hz, 26-H3 and 27-H3), 0.99 (3H, d, J = 6.8 Hz, 2I-H3),

1.64 (3H, s, 19-H3), 3.90 (1H, m, 3-H), 5.20 (2H, br m, 22-H

and 23-H), 5.69 and 5.95 (2H, ABq, J = 12 Hz, 7-H and 6-H); UV

35 λ 261 nm, λ. 234 nm.

max min

The thermal isomerization of this pre-8520OOg-9-vitamin intermediate (56 mg, 0.15 mmol) in refluxing ethanol (3 hours) gave the oily vitamin analog (5) (43 mg, 77%) after separation by HPLC.

5 NMR δ 0.60 (3H, s, 18-H3), 0.89 and 0.90 (6H, each d, J = 6.7 Hz, 26-H3 and 21-Έ. ^), 0.97 (3H, d, J = 6.6 Hz, 21-H3), 3.96 (1H, s, 3-H), 4.82 and 5.05 (2H, each narrow m, 19-H2), 5 .20 (2H, br m, 22-H and 23-H), 6.04 and 6.24 (2H, ABq, J = 11.4 Hz, 7-H and 6-H); UV λ 265.5 nm, λ. 228 nm; IR: 3427, max min 10 1458, 1379, 1048, 966, 943, 892 cm-1; mass spectrum, in / z 382 (M +, 21), 349 (5), 271 (8), 253 (14), 136 (100), 118 (82).

Vitamin analog (5) is a known compound (Bogoslovskii et al., Literature above).

1-Hydroxylation of compound (5).

Freshly crystallized p-toluenesulfonyl chloride (50mg, 0.26mmol) was added to a solution of vitamin (5) (50mg, 0.13mmol) in dry pyridine (300µl). After 30 hours at 4 ° C, the reaction mixture was poured into ice / saturated NaHCO 3 with stirring. The mixture was stirred for 15 minutes and extracted with benzene. The organic extract was washed with saturation; the NaHCC> 3, saturated copper sulfate and water, dried (Na 2 SO 4) and concentrated in vacuo to give the oily tosylate (6). The crude tosylate (6) was treated with NaHCO 3 (150mg) in anhydrous methanol (10ml) and the mixture was stirred at 55 ° C for 8.5 hours.

After cooling and concentration to 2 ml, the mixture was diluted 1 with benzene (80 ml), washed with water, dried (Na 2 SO 4) and evaporated under reduced pressure. The oily 3,5-cyclovitamin-D compound (7) thus obtained was sufficiently pure that it could be used for the next oxidation step without any purification. To a vigorously stirred suspension of SeC> 2 (5.1mg, 0.046mmol) in 35 dry CH 2 Cl 2 (5ml) was added tert.-butyl hydroperoxide (16.5 µl, 0.118mmol). After 30 minutes, dry 8520009-10-pyridine (50 µl) was added and the mixture was stirred at room temperature for an additional 25 minutes, diluted with (3 ml) and cooled to 0 ° C. The crude 3.5-cyclo-vitamin product (7) in CH 2 Cl 2 (4.5 ml) was then added. The reaction was allowed to proceed at 0 ° C for 15 minutes and then the temperature of the reaction mixture was slowly allowed to rise to room temperature (30 minutes). The mixture was transferred to a separatory funnel and shaken with 30 ml of 10% NaOH. Ether (150 ml) 10 was added and the separate organic phase was washed with 10% NaOH, water and dried over Na 2 SO 4. Evaporation in vacuo gave a yellow oily residue, which was purified on a silica gel TLC plate developed in 7: 3 hexane-ethyl acetate to produce the 1-hydroxy-15-cyclo-vitamin product (20 mg, 37%). NMR δ 0.59 (3H, s, -187H), 0.63 (1H, m, 3H), 0.89 and 0.90 (6H, each d, J = 6.9 Hz, 26- H3 and 27-H3), 0.96 (3H, d, J = 6.9 Hz, 21-H3), 3.25 (3H, s, -0CH3), 4.17 (2H, m, 1-H and 6-H), 4.96 (1H, d, J = 9.3 Hz, 7-H), 5.1-5.4 (4H, br m, 20 I-H2, 22-H and 23- H); mass spectrum, m / z 412 (M +, 26), 380 (48), 339 (22), 269 (28), 245 (20), 135 (100). This product mainly consisted of the α-hydroxycyclovitamin D compound of the formula (8), as well as a small amount of the corresponding Ιβ-hydroxy epimer. These components can be separated in this step if desired, but such separation is not necessary.

The above-obtained 1-hydrocyclo-! 1 vitamin product (18 mg) was heated (55 ° C / 15 minutes) in 30 glacial acetic acid (0.8 ml), the mixture was neutralized (ice / saturated NaHCC> 3) and extracted with benzene and ether, after a HPLC (1.5% 2-propanol in hexane as eluant) separation of the pure 1a-hydroxy-3β-acetoxy vitamins (9) (6.60 mg, 34%, elution at 42 ml) and (10) 35 (4 .20 mg, 22%, elution at 50 ml) were obtained.

Compound (9): NMR δ 0.60 (3H, s, 18-H3), 8520009 4 - 11 -φ 0.90 and 0.92 (6Η, each d, J = 7.0 Hz, 26-¾) , 0.97 (3H, d, J = 6.8 Hz, 21-¾), 2.04 (3H, s, -OCOCH3), 4.41 (1H, m, 1-H), 5.02 ( 1H, narrow m, 19-H)., 5.1-5.4 (4H, br m, 3-, 19-, 22- and 23-H), 6.03 and 6.35 (2H, ABq, J = 11.4 Hz, 7-H and 5 6-H); UV λ 264.5 nm, λ. 227.5 nm; mass spectrum, max mm m / z 440 (M +, 10), 380 (72), 362 (7), 269 (31), 251 (12), 135 (100), 134 (99).

. Compound 10: NMR δ 0.60 (3H, s, 18-¾), 0.90 and 0.91 (6H, each d, J = 7.0 Hz, 26-H3 and 27-¾), 0.97 (3H, d, 10 J = 6.9 Hz, 21-H3), 2.05 (3H, s, -0000¾), 4.49 (1H, m, 1-H), 5.00 and 5.14 (2H, each narrow m, 19-¾), 5.20 (3H, br m, 3-, 22- and 23-H), 5.82 and 6.59 (2H, ABq, J = 12.0 Hz , 7-H and 6-H); UV λ 280 nm; λ. 228 nm; mass spectrum, m / z 440 max min (M +, 4), 380 (30), 269 (10), 135 (100), 134 (52).

Hydrolysis of the 3β-acetoxy group in compounds (9) and (10).

Any of the 38-acetoxy derivatives (9) or (10)! was hydrolyzed separately using the | same procedure. A solution of 38-acetoxyvitamin 20 (0.7-6mg) in ethanol (0.1ml) was treated with 10% KOH in methanol (0.8ml) and the mixture was stirred at 50 ° for 1 hour C heated. After the usual work-up and final HPLC purification (8% 2-propanol in hexane as the eluent), the corresponding 1-hydroxyvitamins were obtained, namely:

Compound (LI): NMR δ 0.59 (3H, s, 18- ^ ¾), 0.89 and 0.90 (6H, each d, J = 7.0 Hz, 26-H and 27-H) , 0.96 (3H, d, J = 6.8 Hz, 21-H3), 4.23 (1H, m, 3-H), 4.43 (1H, m,; 1-H), 5, 00 (1H, narrow m, 19-H), 5.1-5.4 (3H, br m, 19-, 22-,

30 and 23-H), 6.02 and 6.39 (2H, ABq, J = 11.4 Hz, 7-H and 6-H); UV

λ 264.5 nm, λ, 227.5 nm; mass spectrum, m / z 398 (M +, max mm r '21), 380 (8), 287 (6), 269 (7), 251 (5), 152 (36), 134 (100). (Elution volume 39 ml).

Compound (12): NMR 6 0.61 (3H, s, 18-¾), 0.89 35 and 0.91 (6H, each d, J = 7.0 Hz, 26-H3 and 27-H3) / ), 97 (3H, d, J = 6.9 Hz, 21-H3), 4.25 (1H, m, 3-H =, 4.51 (1H, m, 1-H),

4.98 and 5.13 (2H, each narrow m, 19-¾), 5.21 (2H, br m, 22-H

8520009 Λ - 12 - 0 and 23-Η), 5.89 and 6.59 (2Η, ABq, J = 11.5 Hz, 7-H and 6-H); UV λ 273 nm, λ. 229.5 nm; mass spectrum, m / z 398 max mm (M +, 17), 380 (4), 287 (5), 269 (5), 251 (4), 152 (29), 134 (100). (Elution volume 38 ml).

In the above described method

the high pressure liquid chromatography (HPLC) performed on a Waters Associates Model ALC / GPC 204 using Zorbax-Sil (DuPont) (6.2 mm x 25 cm column, flow rate 4 ml / minute, 105 kg / cm2). The column chromatography was performed on Silica Gel 60, 70-230 mesh ASTM

(Merck). The preparative thin layer chromatography (TLC) was performed on Silica 60 PF-254 (20 x 20 cm plates, 1 mm silica gel). The irradiations were performed using a Hanovia 608A36 mercury arc lamp equipped with a Vycor filter. All reactions are preferably performed in an inert atmosphere (e.g. argon).

! The compound of this invention can if desired, can be easily obtained in crystalline form. Gene by crystallization from suitable solvents, such as hexane, ethers and alcohols (absolute or aqueous) and mixtures thereof, as will be apparent to those skilled in the art.

j! 8520008

Claims (3)

1. The compound of the formula la. The compound of claim 1 in crystalline form. A pharmaceutical composition containing the compound of claim 1 together with a pharmaceutically acceptable excipient. 8320009