IE57936B1 - 1alpha,25-dihydroxy-22z-dehydrovitamin d compound - Google Patents

Abstract

Novel vitamin D derivative, 1 alpha ,25-dihydroxy-22Z-dehydrovitamin D2. The compound is characterized by unexpectedly high ability to raise serum calcium levels. The compound could therefore, find ready application as a substitute for vitamin D or its metabolites in the treatment of metabolic bone diseases or in other of their known applications.

Description

PATENTS ACT, 1964 COMPLETE SPECIFICATION ··· y ;/· α 1 a 5S’Si la,25-DIHYDROXY-22Z-DEHYDROVITAMIN D COMPOUND WISCONSIN ALUMNI RESEARCH FOUNDATION, a corporation organisedand existing under the laws of the State of Wisconsin, UnitedStates of America, of 614 North Walnut Street, Madison, Wisconsin53705, United States of America. 1 2 ,Μ .1 This invention relates to a biologically activevitamin D compound, specifically 10C, 25-dihydroxylatedvitamin D compound with a 22,23-cis-double bond in theside chain, and to a method for its preparation. 5 Calcium and phosphate homeostasis in animals and humans is regulated by vitamin D metabolites; 1oc, 25-dihydroxyvitamin is generally considered as themost active and most important vitamin D-derived regulatorof normal calcium and phosphate balance. This natural 10 metabolite and compounds structurally related to it aretnerefore of great pharmaceutical interest as effectiveagents for tne prevention and treatment of bone diseasesand related calcium metabolism disorders. In additionto the natural metabolites, a number of compounds 15 nave been prepared in recent years which, because oftheir nigh potency, find use or show very considerablepromise as therapeutic agents, e.g. 1 A novel vitamin D analog has now been found,according to this invention, which may be represented bystructure I This novel compound is characterized by a 22,23-doublebond in tne side chain having the cis (or Z) geometry.Because of the presence ol this 222^-double bond, whichresults in a quite different side chain geometry fromthat pertaining to compounds having the normal saturatedside chain (as in 10C, 25-dihyaroxyvitamin D^) or a 22,23-trans (22E)-unsaturated side chain (as in h'x, 25-dihyaroxy-vitamin ), it would be assumed that this cis-unsaturatedproduct would exhibit low biological activity, if any.Surprisingly, this material shows high activity, beingas active as 10c, 25-dihydroxyvitamin in its ability toraise serum calcium levels in test animals.

The product of this invention (compound I) can beprepared from a 22Z-dehydrovitamin D precursor having thestructure II shown below, by in vitro enzymatic hydroxylation at carbon 25 using a liver homogenatepreparation from vitamin D-deficient rats. 4 u ^'^z'oh 21 Ho The following procedure was used: Male weanling ratswere fed a low calcium and vitamin D-deficient diet asdescribed by Suda et al. [J. Nutr. 10O, 1049 (1970)] for 2weeks. They were killed by decapitation and their livers wereremoved. A 20% (w/v) homogenate was prepared in ice-cold 0.25M sucrose. Incubation was carried out in 10 ml incubationmedium in a 125 ml Erlenmayer flask containing an aliquot ofliver homogenate representing 1 g of tissue, 0.125 M sucrose,50 mM phosphate buffer (pH 7.4), 22.4 mM glucose-6-phosphate,20 m ATP, 160 mM nicotinamide, 25 mM succinate, 0.4 mM NADP, 5mM MgC^, 0.1 M KC1 and 0.5 units glucose-e-phosphate-dehydrogenase. The reaction was initiated by addition of 400pg of the substrate, compound II above, dissolved in 100 μΐ95% ethanol. The incubation mixture was incubated at 37°Cwith shaking at 80 oscillations/min for 3 h. The reaction wasstopped by addition of 20 ml methanol and 10 mldichlorcmethane. After further addition of 10 mldichlorcmethane, the organic phase was collected while theaqueous phase was re-extracted with 10 ml dichlorcmethane.

The organic phases from h total of three extractions werecombined and evaporated with a rotary evaporator. The residuecontaining the desired product was dissolved in 1 ml ofCHCl^:hexane (65:35) mixture and applied to a Sephadex LH-20*column (0.7 cm x 14 cm) packed, equilibrated and eluted withthe same solvent. The first 10 ml was discarded while next 40ml was collected and evaporated. The residue was thendissolved in 8% 2-propanol in hexane and subjected to highperformance liquid chromatography (Model IC/GPC 204 HPLC, *Trade Mark 5 Waters Associates, Medford, MA) using a Zorbax-SIL column (4.6mm x 25 an, Dupont, Wilmington, Delaware) operating underpressure of 1000 psi (70 kg/cn/) with a flow rate of 2ml/min. Thedesired 25-hydroxylated product was eluted at 44 ml. This 5 product was further purified by high performance liquidchromatography using a reversed phase column (Richrosorb Rp-18, 4.6 nm x 25 cm, E. Merck, Darmstadt, West Germany) 2 operated under pressure of 1200 psi (84 kg/αη ) and a flow rate of 2ml/min. The column was eluted with 22% Η,,Ο in methanol, and 1 0 the compound was eluted at 50 ml. The product was further purified by HPLC using the Zorbax-SIL column and conditions asdescribed above. The resulting product was then subjected tophysical characterization.

Characterization of the Product 1 5 The UV absorption of the product in 95% ethanol exhibited a λ =265 nm and a λ . =228 nm indicating the presence of themax min 5,6-cis-triene chromophore.

The mass spectrum of the substance contains a molecularion at m/e 414 as required for a 25-hydroxylated product. 20 Elimination of one and two molecules of H^O gives fragmentions at m/e 396 and 378. Loss of the entire steroid sidechain (cleavage of C^7/C2O b°nc^ results in the fragment ofm/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 spectrumshews prominent peaks at m/e 152 and 134 (152-H^O) whichrepresent ring A fragments and are diagnostic forla,3£-dihydroxyvitamin D compounds. In addition, the spectrumshows a very prominent fragment peak at m/e 59 which resultsfrom cleavage of the ^24^25 ^°n^· presence of this ion 30 confirmed the presence of 25-hydroxy group in the product.

Thus, these data established the structure of the productobtained as the la,25-dihydroxylated compound, as representedby structure I, above. 6 Biological Activity The biological activities of the novel analog wasdemonstrated by in vivo assay in the rat. Male weanling ratswere fed the lew calcium vitamin D-deficient diet of Suda et 5 al. (supra) for 3 weeks. They were then divided into groupsof 5 rats each. Rats in a control group received 0.05 ml 95%ethanol intrajugularly while rats in the other groups weregiven 325 pmole of either compound I or la, 25-dihydroxyvitamin dissolved in 0.05 ml 95% ethanol. Eighteen hours later, 1 0 they were killed by decapitation and 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 determined with an atomic absorption spectrophotometer. Results, are shown in the following Table: 1 5 Increase in serum calcium concentration in response to asingle dose of 325 pmole of either compound I or la,25- dihydroxyvitamin given 18 h prior to sacrifice Compound Given Serum Calcium Concentration(mg/100 ml) + standard deviation ethanol 4.2 + 0.1 a) 20 compound I 5.2 + 0.2 la, 25-dihydroxyvitamin D3 5.4 + 0.4 b) is significantly different from a^ p <0.001 The above results show the new analog to be highly potentand to exhibit biological activity essentially equivalent to 2 5 that of la, 25-dihydroxyvitamin D^.

Because of this high potency, the compound of this invention will find application as a therapeutic agent in thetherapy or prophylaxis of disorders such as the various typesof rickets, hypoparathyroidism, osteodystrophy, osteomalacia 30 or osteoporosis in the human, or for the treatment of relatedcalcium deficiency diseases (e.g. milk fever, leg weakness, 7 egg shell thinness) in animals. Likewise the compound maybe used for the treatment of certain malignancies, such ashuman leukemia.

For therapeutic purposes, the compound may be5 administered by any conventional route of administration and in any suitable form. The compound may be formulated r with any acceptable and innocuous pharmaceutical carrier,in the form of,e.g.,pills , tablets, gelatin capsules, orsuppositories, or as solutions, emulsions, dispersions or 10 suspensions in innocuous solvents or oils; such formulations may contain also other therapeutically activeand beneficial ingredients as may be appropriate for thespecific applications. For human applications, thecompound is advantageously administered in amounts, say, 15 0.25 to 10pg per day, tne specific dosage being adjustedin accordance with the disease to be treated and themedical history, condition and response of the subject,as is well understood.

The 22Z-dehydro precursor substrate (compound II), 20 can itself be prepared by the process depicted in ProcessScheme I described below. In the description, compounddesignation by Arabic numerals (e.g. (JJ » (2.) < (3.), etc.)refer to the structures so numbered in the Process Scneme.

The desired substrate (compound II) is identified by Arabic 25 numeral (11) in Process Scheme I. (222)-38-(Methoxymethoxy-5fr,8x-(4-phenyl-1,2-urazolo)cnolest- a-6,22-dien (2). Isopentyl phosphonium bromide[(CH3)2CHCH2CH2PPh3BrJ (1.67 g, 4.04 mmol) in drytetrahydrofuran (73 ml) was treated with n-butyllithium 30 (1.7 M solution in hexane, 2.42 ml, 4.11 mmol) at 3-5°C withstirring. After stirring for 1 h at room temperature, theorange-red solution was cooled to 3°C and aldehyde U) (1.64 8 g, 3.36 nmol) in dry THF (24 ml) was added. The colorlessreaction mixture was stirred overnight at room temperature andthen poured into water and extracted with benzene. Theorganic extract was washed with 5% HC1, saturated sodium 5 bicarbonate and water, dried (Na2SO4) and concentrated in avacuo to an oil, which was purified on a column of silica gel.Elution with benzene-ether (94:6) mixture afforded adduct (2)(1.38 g, 68%) as a foam: NMR 6 0.83 (3H, s, 18-H^), 0.89 and0.91 (6H, each d, J=6.8 Hz, 26-H3 and 27-H3), 0.97 (3H, d, 10 J=6.8 Hz, 21-H3), 0.98 (3H, s, 19-H3), 3.30 (1H, dd, σχ=4.4Hz, J2=14Hz, 9-H), 3.38 (3H, s, OCH^, 4.33 (1H, m, 3-H), 4.70and 4.81 (2H, ABq, J=6.8 Hz, 00^0), 5.21 (2H, br m, 22-H and23-H), 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; mass 1 5 spectrum, m/£ 601 (M+, 1%), 426 (4), 364 (61), 349 (16), 253 (18), 251 (18), 119 (PhNCO, 100). (22Z) -5α, 8a-(4-phenyl-l, 2-urazolo) cholesta-6,22-dien-3ft-ol (3) · A solution of adduct (2) (601 mg, 1 nmol) andp-toluenesulfonic acid (523 mg, 2.75 mmol) in methanol (20 2 0 ml)-THF (12 ml) mixture was stirred for 2 days at roan temperature. The reaction mixture was poured into saturatedsodium bicarbonate and extracted several times with benzene.Extracts were washed with water, dried (Na^O^) and evaporatedunder reduced pressure. Purification of the crude product by 25 column chromatography (benzene ether 70:30 as eluant) gave thehydroxy adduct (3) (550 mg, 99%) as a foam: NMR 5 0.83 (3H, s,18-K3), 0.89 and 0.91 (6H, each d, J=6.8 Hz, 26-H3 and 27-H.j),0.95 (3H, s, 19-H3) , 0.98 (3H, d, J=6.8 Hz, 21-11 ), 3.16 (1H,dd, Jx=4.4 Hz, J2=14 Hz, 9-H), 4.44 (1H, m, 3-H), 5.22 (2H, br 30 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 m, Ar-H); IR: 3447,1754,1700,1600,1397cm Α; rass spectrum, m/z_ (557 (M+, 1%), 382 (35), 349 (33), 253 (20), 251 (33), 119 (100), 55 (82). (22Z)-Cholesta-5,7,22-trien-36-ol (£). The adduct (3) (53035 mg, 0.95 mmol) was converted to the diene (4) by reduction with lithium aluminum hydride (1 g), in tetrahydro furan (60ml) at reflux for 18 h. After conventional work-up, theproduct was purified by chromatography over silica(benzene-ether 94:6 as eluant) to afford pure diene (4) (290mg, 76%) after crystallization frcm ethanol:mp 148-151 °C; [a]p4=-132° (c=0.9, CHCl.^); NMR δ 0.66 (3H, s, 18-iy , 0.90and 0.91 (6H, each d, J=6.8 Hz, 26-H3 and 27-Η^), 0.96 (3H, s,19-H3), 0.98 (3H, d, J=6.9 Hz, 21-H3), 3.64 (1H, m, 3-H), 5.20(2H, br m, 22-H and 23-H), 5.39 and 5.57 (2H, ABq, J=6 Hz, 7-Hand 6-H); UV λ 281 nm; IR: 3346,1463,1375,1364,1067,1040, _1 4- 831 cm ; mass spectrum, m/£ 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 nmol) dissolved in ether (120 ml) and benzene (30 ml) (degassed with argon for 40 min) was performed at 0°C for 13 min using a UV-lamp and Vycor* filter. HPUC (1% of 2-propanol in hexane) of the resulting mixture afforded the previtamin (56.9 mg, 38%) as a colorless oil: NMR 6 0.75 (3H, s, 18-CH.j), 0.90 and 0.91 (6H, each d, J=6.7 Hz, 26-H3 and 27-H.j), 0.99 (3H, d, J=6.8 Hz, 21-H3), 1.64 (3H, s, 19-K3), 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 λ 261 nm, λ . 234 nm. max min Thermal isomerization of this previtamin intermediate (56 mg, 0.15 nmol) in refluxing ethanol (3 h) gave the oily vitamin analog (j>) (43 mg, 77%) after separation by HPLC. NMR δ 0.60 (3H, s, 18-H3), 0.89 and 0.90 (6H, each d, J=6.7 Hz, 26-H3 and 27-H.p , 0.97 (3H, d, J=6.6 Hz, 21-1^), 3.96 (1H, s, 3-H), 4.82 and 5.05 (2H, each narr. m, 19-^), 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: 3427,1458,1379,1048, max , min 966,943,892 cm ; mass spectrum, m/z^ 382 (M , 21), 349 (5), 271 (8), 253 (14), 136 (100), 118 (82). Vitamin analog (5) isa known compound (Bogoslovskii et al., J. Gen. Chem. USSR48 ( 4), 828 ( 1 978). *Trade Mark 1-Hydroxylation of compound (5). Freshly recrystallizedp-toluenesulfonyl chloride (50 mg, 0.26 mmol) was added to asolution of vitamin (5) (50 mg, 0.13 mmol) in dry pyridine(300 μΐ). After 30 h at 4°C, the reaction mixture was pouredinto ioe/saturated NaHCO^ with stirring. The mixture wasstirred for 15 min and extracted with benzene. The organicextract was washed with saturated NaHCO^, saturated coppersulfate and water, dried (Na_SO.) and concentrated in vacuo toobtain the oily tosylate (6) . The crude tosylate (6) wastreated with NaHCO^ (150 mg) in anhydrous methanol (10 ml) andthe mixture was stirred for 8.5 h at 55°C. After cooling andconcentration to 2 ml the mixture was diluted with benzene(80 ml), washed with water, dried (Na^SO^) and evaporatedunder reduced pressure. The oily 3,5-cyclovitamin D compound (7) thus obtained was sufficiently pure to be used for thefollowing oxidation step without any purification. To avigorously stirred suspension of SeO2 (5.1 mg, 0.046 nmol) indry CHjClj (5 ml), tert-butylhydroperoxide (16.5 μΐ, 0.118nmol) was added. After 30 min dry pyridine (50 μΐ) was addedand the mixture was stirred for additional 25 min at roomtemperature, diluted with G^C^ (3 ml) and cooled to 0°C.

The crude 3,5-cyclovitamin product (2) in G^C^ (4.5 ml) wasthen added. The reaction proceeded at 0°C for 15 min and thenit was allowed to warm slowly (30 min) to roan temperature.

The mixture was transferred to a separatory funnel and shakenwith 30 ml of 10% NaOH. Ether (150 ml) was added and theseparate organic phase was washed with 10% NaOH, water anddried over Na^SO^. Concentration to dryness in vacuo gave ayellcw oily residue which was purified on silica gel TLC platedeveloped in 7:3 hexane-ethyl acetate giving the 1-hydroxycyclovitamin product (20 mg, 37%): NMR 6 0.59 (3Π, s, 18-H^) , 0.63(111, m, 3-11), 0.89 and 0.90 (611, each d, J=6.9 Hz, 26-H3 and27-H3), 0.96 (311, d, J=6.9 Hz, 21-H3) , 3.25 (3H, s, -OCI13) , 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, 19-^, 22-H and 23-H); mass spectrum, m/z 1 1 412 (M+, 26), 380 (48), 339 (22), 269 (28), 245 (20), 135 (100). This product is composed chiefly of the Ια-hydroxycyclovitamin D compound (8J, as well as a small amount of the corresponding Ιβ-hydroxy-epimer. These components may be separated at this stage, if desired, but t· such separation is not required.

The 1-hydroxycyclovitamin product (18 mg) as obtainedabove was heated (55°C/15 min) in glacial acetic acid (0.8ml), the mixture was neutralized (ice/saturated NaHCO^) andextracted with benzene and ether, to yield after HPLC (1.5% of2-propanol in hexane as eluent) separation pure la-hydroxy-38-acetoxyvitamins (9) (6.60 mg, 34%, eluting at 42 ml) and(10) (4.20 mg, 22%, eluting at 50 ml).

Compound (9): NMR 6 0.60 (3H, s, 18-H3), 0.90 and 0.92 (6H, each d, J=7.0 Hz, 26-H^ and 27-H^), 0.97 (3H, d, J=6.8 Hz, 21-1I3), 2.04 (3H, s, -OCOCiy, 4.41 (1H, m, 1-H), 5.02 (1H, narr. 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 6-H); UV λ 264.5 nm, \iin 227.5 nm; mass spectrum, m/z 440 (M , 10), 380 (72), 362 (7), 269 (31), 251 (12), 135 (100), 134 (99).

Conpound (10): NMR 6 0.60 (3H, s, 18-H3), 0.90 and 0.91 (6H, each d, J=7.0 Hz, 26-H3 and 27-H3), 0.97 (3H, d, J=6.9 Hz, 21-H3,, 2.05 (3H, s, -OCeCHj), 4.49 (1H, m, 1-H), 5.00 and 5.14 (2H, each narr. m, 19-^), 5.20 (3K, br m, 3--,22- and 23-H), 5.82 and 6.59 (2H, ABq, J=12.0 Hz, 7-H and 6-H); UV Amax η™' λπΰη 1710· ^ass spectrum, m/z 440 (M+, 4), 300 (30), 269 (10), 135 (100), 134 (52).

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

Each of the 3p-acetoxy-derivatives (9) or (10) was separately j hydrolyzed, using the same procedure. A solution of 38-acetoxyvitamin (0.7-6 mg) in ethanol (0.1 ml) was treated with 10% KOH in methanol (0.8 ml) and the mixture was heated for 1 h at 50°C. After usual work-up and final HPLC purification (81 of 2-propanol in hexane as eluent) the corresponding 1-hydroxyvitamins were obtained, namely: 1 2 Corpound (11): NMR δ 0.59 (3H, s, 18-H.j,, 0.89 and 0.90 (6H, each d, J=7.0 Hz, 26-H3 and 27-H3), 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, narr. m, 19-H), 5.1-5.4 (3H, br m, 19-, 22-, 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 . . ϊπειχ nun 227.5 nm; mass spectrum, m/z 398 (M , 21), 380 (8), 287 (6), 269 (7), 251 (5), 152 (36), 134 (100). (Elution volunte 39 ml).

Ccrpound (12); NMR δ 0.61 (3H, s, 18-H3), 0.89 and 0.91 (6H,each d, J=7.0 Hz, 26-H3 and 27-H3), 0.97 (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 narr. m., 19-^), 5.21 (2H, br m, 22-H and 23-H), 5.89 and 6.59 (2H, ABq, J=11.5 Hz, 7-H and 6-H); UV λ 273nm, 229.5 nm; mass spectrum, m/z 398 (M , 17), 380 (4), 287 (5), 269 (5), 251 (4), 152 (29), 134 (100). (Elutionvolume 38 ml).

In the above described process, high pressure liquidchrcmatography (HPLC) was performed on a Waters AssociatesModel ALC/GPC 204 using a Zorbax-Sil (DuPont) (6.2 nm x 25 cmcolumn, flow rate 4 ml/min, 1500 psi [105 kg/cm^ ). Column chrcmatographywas performed on Silica Gel 60, 70-230 mesh ASTM (Merck).

Preparative thin-layer chromatography (TLC) was carried out onSilica 60 PF-254 (20 x 20 cm plates, 1 nm silica gel).

Irradiations were carried out using a Hanovia 6O8A36 mercuryarc lamp fitted with a Vycor filter. All reactions arepreferably performed under an inert atmosphere (e.g. argon).

The compound of this invention can, if desired, bereadily obtained in crystalline form by crystallization fromsuitable solvents such as hexane, ethers and alcohols(absolute or aqueous), and mixtures thereof as will be evidentand well known to those skilled in the art. 1 3 (4) (8) R = OH (10) X, =Acetyl(12) X, = H

Claims (8)

1. The compound having the structure < img-format="tif" img-content="drawing" /> 1 4

2. The compound of Claim 1 in crystalline form.

3. A pharmaceutical composition comprising the 5 compound of Claim 1 or 2 together with a pharmaceuticallyacceptable excipient.

4. A process for preparing a compound as claimed inclaim 1 or 2 which comprises hydroxylating a compoundhaving the structure: < img-format="tif" img-content="drawing" /> in known manner.

5. A process according to claim 4 substantially asHereinbefore described.

6. A compound as claimed in claim 1 or 2 whenever 1 5 prepared by a process as claimed in claim 4 or 5 .

7. A composition according to claim 3 wnerein the compound is one claimed in claim 6.

8. A composition according to claim 3 substantially as hereinbefore described. Dated this the 29th day of January,1985 F. R. KELLY & CO. BY: _EXECUTIVE 2 7 Clyde Road'T^Ballsbridge, Dublin 4AGENTS FOR THE APPLICANTS.