lα _y 25-D-U-ydrcxy^22Z-dehydrovitaιt-^ D Cαnpound

This invention was made with Government support under. -CH Grant No. AM 14881 awarded by the Departπeπt of Health and Human Services. The Government: has certain rights in this invention. Technical Field

This invention relates to a biologically active vitamin D cciηpound. Specificially, the i-πventicn relates to a novel lα,25-d-i-hydrαxy-lated vitamin D cαrpound with a 22,23-cis- dσuble bond in the side chain, and to a method far its preparation. Background

Calcium and phosphate hαneostasis in animals and humans is regulated by vitamin D metabolites, and the cααpound lα,25-d-lhydrox-yvit----r-in D. is generally considered as the most active and most important vitamin D-derived regulator of normal calcium and phosphate balance. This natural metabolite and cαπpσunds structurally related to it are therefore of great ph--_rmaceutical interest as effective agents for the prevention and treatment of bone diseases and related calcium metabolism disorders. In addition to the natural D_ metabolites, a number of ccnrpσunds have been prepared in recent years which, because of their high potency, find use or show very considerable promise as therapeutic agents, among them lα-h-yd-rQxyvitarr-in D _g , lcHydroxyvitamin D_, lα,25- dihydroxyvita-c-vin D and certain fluorinated analogs (U.S. Patents 3,741,996; 3,907,843; 3,880,894; 4,226,788; 4,358,406) . Most of the known active analogs are characterized by the type of sterol side chain as it occurs in vitamin D_ (i.e. saturated side chain). Known analogs with 22,23-unsaturated side -ain are represented by cαipounds of

the vitamin D_ series (i.e. 22,23-trans-unsaturated with a C-24-methyl substituent) , and include, in addition to the cαtpounds named above, 25-hydroxyvitamin D ₂ (U.S. Patent 3,585,221) and the 24- and derivatives (Jones et al. Acch. Biochem. Biophys. 202, 450 (1980)) and three compounds lacking the 24-methyl sυbstituent (U.S. Patent 3,786,062; Bogoslovskϋ et al. J. Gen. Chem. USSR, 48(4) 828 (1978); Chan. Abstr. 89, 163848j, 209016s). Disclosure of Invention

A novel vitamin D analog has now been found which may be represented by structure I sshheewwnn bbeellooww:

This novel compound is characterized by a 22,23-double b d in the side chain having the cis (or Z) geometry. Because of the presence of this 22Z^double bond, which results in a quite different side chain geometry from that pertaining to compounds having the normal saturated side chain (e.g. as in lα,25-d---hydroxyvitamin D_) or a 22,23-trans (22E)-unsat- urated side chain (e.g. as in lα,25-dihydraxyvitamin D_) , it was assumed that this cis-unsaturated product would exhibit lew biological activity, if any. Surprisingly, this material, in spite of its altered side chain st-zπicture, shows high activity, being as active as lα,25-d-ihyd-coxyvitamin D in its ability to raise serum calcium levels in test animals. Preparation of the Cαπpound

The novel product of this invention, cαrpound I, above, was prepared from a 22Z^ehydrovitamin D precursor having the structure II shown below, by in vitro enzymatic hydroxylation at carbon 25 using a liver hαnogenate preparation frαn vitamin D-teficient rats.

The following procedure was used: Male weanling rats were fed a low calcium and vitamin D-deficient diet as described by Suda et al. [J. utr. ICO, 1049 (1970)] for 2 weeks. They were killed by decapitation and their livers were removed. A 20% (w/v) hαmogenate was prepared in ice-cold 0.25 M sucrose. Incubation was carried out in 10 ml incubation medium in a 125 ml Erleπmayer flask containing an aliquot of liver hαmogenate representing 1 g of tissue, 0.125 M sucrose, 50 mM phosphate buffer (pH 7.4) , 22.4 m glucose-6-phosphate, 20 m ATP, 160 M nicotinamide, 25 mM succinate, 0.4 M NADP, 5 mM MgCl_, 0.1 M KC1 and 0.5 units glucose-6-phosphate- dehydrogenase. The reaction was initiated by addition of 400 μg of the substrate, cαipound II above, dissolved in 100 j-tl 95% ethanol. The incubation mixture was incubated at 37°C with shaking at 80 oscillations/min for 3 h. The reaction was stopped by addition of 20 ml ethanol and 10 ml dichloromethane. After further addition of 10 ml dichlorσmethane, the organic phase was collected while the aqueous phase was re-extracted with 10 ml dichloromethane. The organic phases from total of three extractions were combined and evaporated with a rotary evaporator. The residue containing the desired product was dissolved in 1 ml of CHCl-,: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 next 40 ml was collected and evaporated. The residue was then dissolved in 8% 2-propanol in hexane and subjected to high performance liquid chro atography (Model C/GPC 204 HPLC,

ϊfeters Associates, ϊfedford, MA) using a Zorbax-SIL column (4.6 mm x 25 cm, Dupont, Wilmington, Delaware) operating under pressure of 1000 psi with a flow rate of 2 ml/min. The desired 25-hydrαxylated product was eluted at 44 ml. This product was further purified by high performance liquid chrαmatography using a reversed phase column (Richrosorb I -18, 4.6 irm x 25 cm, E. Merck, Darmstadt, West Germany> operated under pressure of 1200 psi and a flow rate of 2 ml/min. The column was eluted with 22% H.0 in methanol, and the cαipσund was eluted at 50 ml.. The product was further purified by HPLC using the Zorbax-SIL column and ∞nditions as described above. The resulting product was then subjected to physical characterization. Characterization of the Product

Ihe W absorption of the product in 95% ethanol exhibited a nm and a n indicating - the p ^r resence of the

5,6-cis-triene chrcmcphore.

The mass spectrum of the substance contains a molecular ion at m/e 414 as required for a 25-hydroxylated product. Elimination of one and two molecules of H_0 gives fragment ions at m/e 396 and 378. Loss of the entire steroid side chain (cleavage of ,_/C_ ₀ bond) results in the fragment of m/e 287, which by elimination of one and two molecules of H_0, gives rise to the peaks at m/e 269 and 251. The spectrum shews prα-iinent peaks at m/e 152 and 134 (152-H -)) which represent ring A fragments and are diagnostic for lα,3B-dihydroxyvita_-τ-in D compounds. In addition, the spectrum shews a very prominent fragment peak at m/e 59 which results from cleavage of the ₂₄ /C _2c bond. The presence of this ion confirmed the presence of 25-hydroxy group in the product. Thus, these data established the structure of the product obtained as the lα,25-dihydroxylated compound, as represented by structure I, above.

Biological Activity

The biological activities of the novel analog was demonstrated by in vivo assay in the rat. Male weanling rats were fed the low calcium vitamin D-deficient diet of Suda et al. (supra) for 3 weeks. They were then divided into groups of 5 rats each _. . Rats in a control group received 0.05 ml 95% ethanol intrajugularly while rats in the other groups were given 325 pmole of either cαrpound I or lα,25-dihydroxyvit_amin

Dm. dissolved in 0.05 ml 95% ethanol. Eighteen hours _, later, 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 ^' atonic absorption spectrophotcmeter. Results are shown in the following Table:

Increase in serum calcium concentration in response to a single dose of 325 pmole of either compound I or lα,25- dihydroxyvitamin D_ given 18 h prior to sacrifice

Compound Given Serum Calcium Concentration

(mg/100 ml) +_ standard deviation

ethanol 4.2 + 0.1 ^a) coπpound I 5.2 + 0.2 lα,25-d-ihydroxyvitamin D ₃ 5.4 + 0.4

is significantly different from p<0.001

• The above results show the new analog to be highly potent and to exhibit biological activity essentially equivalent to that of l ,25-diJhydrox-yvitamin D_.

Because of this high potency, the compound of this invention will find application as a therapeutic agent in the therapy or prophylaxis of disorders such as the various types of rickets, hypoparat±yroidism, osteodystrσphy, osteomalacia or osteoporosis in the human, or for the treatment of related calcium deficiency diseases (e.g. milk fever, leg weakness,

egg shell thinness) in animals. Likewise the compound may be used for the treatment of certain malignancies, such as human leukemia.

For therapeutic purposes, the compound may be administered by any conventional route of adrtiinistration and in any form suitable for the method of adm-uiistration selected. The compound may be formulated with any acceptable and innocuous pharmaceutical carrier, in the form of pills, tablets, gelatin capsules, or suppositories, or as solutions, emulsions, dispersions or suspensions ^" in innocuous solvents or oils, and such fo-απulations may contain also other therapeutically active and beneficial ingredients as may be appropriate for the specific applications. For human applications, the cαrpound is advantageously administered in amounts from 0.25 to 10 μg per day, the specific dosage being adjusted in accordance with the disease to be treated and the- medical history, condition and response of the subject, as is well understood by those skilled in the art.

The 22^-dehydro precursor substrate, ccirpound II above, required for the preparation of the novel product of this invention is itself prepared by the process depicted in Process Scheme I, appended, and described below. In the description, compound designation by Arabic numerals (e.g. (1) _r (2) , (3) , etc.) refer to the structures so numbered in the Process Scheme. The desired substrate (compound II) for the above described 25-hydroxylation reaction is identified by Arabic numeral (11) in Process .Scheme I and in the following description.

(22Z)-3β-(Methoxymethoxy)-5α,8 -(4-phenyl-l,2-urazolo)cholest- a-6,22-dien _) . Isopentyl phosphonium bromide [(CH ₃ ) ₂ CHCH ₂ CH ₂ Ph ₃ Br] (1.67 g, 4.04 mmol) in dry tetrahydrofuran (73 ml) was treated with n-butyllithium (1.7 M solution in hexane, 2.42 ml, 4.11 mmol) at 3-5°C with stirring. After stirring for 1 h at room temperature, the orange-red solution was cooled to 3°C and aldehyde (jL) (1.84

g, 3.36 mmol) in dry THF (24 ml) was added. The colorless reaction mixture was stirred overnight at room temperature and then poured into water and extracted with benzene. The organic extract was washed with 5% HC1, saturated sodium bicarbonate and water, dried (Na-SO.) and concentrated in a vacuo 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 δ 0.83 (3H, s, 18-H ₃ ) , 0.89 and 0.91 (6H, each d, J=6.8 Hz, 26-H ₃ and 27-H ₃ ) , 0.97 (3H, d, J=6.8 Hz, 21-H ₃ ), 0.98 (3H, s, 19-H ₃ ) , 3.30 (IH, dd, J _χ =4.4 Hz, J ₂ =14Hz, 9-H), 3.38 (3H, s, O y , 4.33 (IH, m, 3-H) , 4.70 and 4.81 (2H, ABq, J=6.8 Hz, CCH ₂ 0), 5.21 (2H, br m, 22-H and 23-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 spectrum, m/z.601 (M ⁺ , 1%) , 426 (4) , 364 (61) , 349 (16) , 253 (18), 251 (18), 119 (PhNCO, 100).

(22Z)-5o.,8 -(4-phenyl-l,2-urazolo)cholesta-6,22-dien-3β-ol (3) . A solution of adduct (2) (601 mg, 1 irmol) and p-toluenesulfonic acid (523 mg, 2.75 i ol) in methanol (20 ml)-THF (12 ml) mixture was stirred for 2 days at room teitperature. The reaction mixture was poured into saturated scxuum bicarbonate and extracted several times with benzene. Extracts were washed with water, dried (Na^SO.) and evaporated under reduced pressure. Purification of the crude product by column chromatography (benzene ether 70:30 as eluant) gave the hydrcxy adduct (3) (550 mg, 99%) as a foam: NMR δ 0.83 (3H, s, 18-H ₃ ), 0.89 and 0.91 (6H, each d, J=6.8 Hz, 26-H ₃ and 27-H ₃ ) , 0.95 (3H, s, 19-H ) , 0.98 (3H d, J=6.8 Hz, 21-H ) , 3.16 (IH, dd, J =4.4 Hz, J ₂ =14 Hz, 9-H)., 4.44 (IH, 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 , Ar-H); IR: 3447,1754,1700,1600,1397

-1 + cm ; mass spectrum, τ_/τ_ (557 (M , 1%) , 382 (35) , 349 (33) ,

253 (20), 251 (33), 119 (100), 55 (82).

(22Z)-Cholesta-5.,7,22-trien-3β-ol (4) . The adduct (3_) (530 mg, 0.95 irmol) was converted to the diene (4) by reduction

with lithium aluminum hydride (1 g) , in tetrahydrofuran (60 ml) at reflux for 18 h. After conventional work-up, the product was purified by chromatograpby over silica

(benzene-ether 94:6 as eluant) to afford pure diene (4_) (290 mg, 76%) after crystallization from ethanol:mp 148-151 ^β C;

[α]^ ⁴ = -132° (c=0.9, CHC1 ₃ ); NMR δ 0.66 (3H, s, 18-H ₃ ) , 0.90 and 0.91 (6H, each d, J=6.8 Hz, 26-H ₃ and 27-^), 0.96 (3H, s, 19-H ₃ ), 0.98 (3H, d, J=6.9 Hz, 21-^), 3.64 (IH, m, 3-H) , 5.20

(2H, brm, 22-H and 23-H) , 5.39 and 5.57 (2H, ABq, J=6 Hz, 7-H and 6-H); UV λ 281 nm; IR: 3346,1463,1375,1364,1067,1040, , max

831 cm ; mass spectrum, m/z 382 (M , 100) , 349 (65); 323 (32), 271 (15), 253 (30).

(5Z,7E,22Z)-9,lO-Secocholesta-5,7,10(19) ,22-tetraen-3β-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 min) was performed at 0°C for 13 min using a UV-lamp and Vycor filter. HPLC (1% of 2-propanol in hexane) of the resulting mixture afforded the previtamin (56.9 mg, 38%) as a colorless oil: NMR δ 0.75 (3H, s, 18-CH ₃ ) , 0.90 and 0.91 (6H, each d, J=6.7 Hz, 26-H ₃ and 27-H ₃ ) , 0.99 (3H, d, J=6.8 Hz, 21-H ₃ ) , 1.64 (3H, s, 19-H ₃ ), 3.90 (IH, m, 3-H) , 5.20 (2H, brm, 22-H and 23-H) , 5.69 and 5.95 (2H, ABq, J=12 Hz, 7-H and 6-H) ; UV λmax 261 nm, ^r λmi.n 234 nm.

Thermal isomerization of this previtamin intermediate (56 mg, 0.15 mmol) in refluxing ethanol (3 h) gave the oily vitamin analog (5) (43 g,.77%) after separation by HPLC. NMR δ 0.60 (3H, s, 18-H ₃ ), 0.89 and 0.90 (6H, each d, J=6.7 Hz,

26-H ₃ and 27-H ₃ , 0.97 (3H, d, J=6.6 Hz, 21-H ₃ ) , 3.96 (IH, s,

3-H), 4.82 and 5.05 (2H, each narr. , 19-H ₂ ) , 5.20 (2H, brm,

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 on ^"" - ¹ ; mass spectrum, _lτ_ 382 (M , 21), 349 (5), 271 (8) , 253 (14) , 136 (100) , _. 118 (82) . Vitamin analog (5_) is a known compound (Bogoslovskii et al., supra) .

1-Hydroxylation of compound (5_) . Freshly recrystallized p-toluenesulfσnyl chloride (50 mg, 0.26 mmol) was added to a solution of vitamin (5) (50 mg, 0.13 mmol) in dry pyridine (300 μl) . After 30 h at 4°C, the reaction mixture was poured into ice/saturated NaHC0 ₃ with stirring. The mixture was stirred for 15 min and extracted with benzene. The organic extract was washed with saturated NaHCO-, saturated copper sulfate and water, dried (Na ₂ SO.) and concentrated in vacuo to obtain the oily tosylate (6) . The crude tosylate (6) was treated with NaECO- (150 mg) in anhydrous methanol (10 ml) and the mixture was stirred for 8.5 h at 55°C. After cooling and concentration to 2 ml the mixture was diluted with benzene (80 ml) , washed with water, dried (Na ₂ S0.) and evaporated under reduced pressure. The oily 3,5-cyclσvit-_-ctιin D compound ( ) thus obtained was sufficiently pure to be used for the following oxidation step without any purification. To a vigorously stirred suspension of Se0 ₂ (5.1 mg, 0.046 mmol) in dry CH ₂ C1 ₂ (5 ml), tert-butylhydroperoxide (16.5 μl, 0.118 iitnol) was added. After 30 min dry pyridine (50 μl) was added and the mixture was stirred for additional 25 min at room temperature, diluted with CH ₂ C1 ₂ (3 ml) and cooled to 0°C. The crude 3,5-cyclovitamin product (7) in CH Ξl- (4.5 ml) was then added. The reaction proceeded at 0°C for 15 min and then it was allowed to warm slowly (30 min) to room tarperature. The mixture was transferred to a separatory funnel and shaken with 30 ml of 10% NaOH. Ether (150 ml) was added and the separate organic phase was washed with 10% NaOH, water and dried over Na ₂ S0.. Concentration to dryness in vacuo gave a yellow oily residue which was purified on silica gel TLC plate developed in 7:3 hexane-ethyl acetate giving 1-hydroxycyclo- vitamin product (20 mg, 37%): NMR δ 0.59 (3H, s, 18-H ) , 0.63 (IH, m, 3-H), 0.89 and 0.90 (6H, each d, J=6.9 Hz, 26-H ₃ and 27-H ₃ ), 0.96 (3H, d, J=6.9 Hz, 21-H ₃ ) , 3.25 (3K, s, -CCH ₃ ) , 4.17 (2H, m, 1-H and 6-H) , 4.96 (IH, d, J=9.3 Hz, 7-H) , 5.1-5.4 (4H, br m, 9~H ₂ , 22-H and 23-H) ; mass spectrum, m/z

412 (M ⁺ , 26), 380 (48), 339 (22), 269 (28), 245 (20), 135

(100) . This product is cx-mposed chiefly of the lα-hydroxycyclovit--ιmin D compound of st-racture (8) , as well as small amount of the corresponding lβ-h-ydro->y-ep---mer. These components may be separated at this stage, if desired, but such separation is not required.

The l-hydro^cyclovitamin product (18 mg) as obtained above was heated (55°C/15 min) in glacial acetic acid (0.8 ml) , the mixture was neutralized (ice/saturated NaHCO and extracted with benzene and ether, to yield after HPLC (1.5% of

2-propanol in hexane as eluent) separation pure lα-hydroxy-

3β-acetC3φvitamins (9) (6.60 mg, 34%, eluting at 42 ml) and

(10) (4.2D mg, 22%, eluting at 50 ml) .

Cαrpound (9) : NMR δ 0.60 (3H, s, 18-H ₃ ) , 0.90 and 0.92 (6H, each d, J=7.0 Hz, 26-H ₃ and 27-H ₃ ) , 0.97 (3H, d, J=6.8 Hz,

21-H ₃ ), 2.04 (3H, s, -OCCOy , 4.41 (B, m, 1-H) , 5.02 (IH, narr. m, 19-H) , 5.1-5.4 (4H, brm, 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, ¹ max λ . 227.5 nm; mass spectrum, m/z 440 (M , 10), 380 (72), 362

(7), 269 (31), 251 (12), 135 (100), 134 (99).

Cαrpound (10) : NMR δ 0.60 (3H, s, 18-H ) , 0.90 and 0.91 (6H, each d, J=7.0 Hz, 26-H and 27-H , 0.97 (3H, d, J=6.9 Hz,

21-H ₃ ) , 2.05 (3H, s, -CCC y , 4.49 (IH, m, 1-H) , 5.00 and

5.14 (2H, each narr. m, 19-H , 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 λmax 270 nm; λmi.n 228 nm; mass spectrum, m/z 440 (M , 4)-, 380

(30) , 269 (10) , 135 (100) , 134 (52) .

Hydrolysis of 3S-acetαxy group in compounds (9) and (10) . Each of the 3β-acetoxy-derivatives (9) or (10) was separately hydrolyzed, using the same procedure. A solution of 3β-acetoxyvitami-n (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 (8% of 2-propanol in hexane as eluent) the corresponding 1-hydroxyvitamins were obtained, namely:

Compound (11): NMR δ 0.59 (3H, s, 18-H _j ) , 0.89 and~0.90 (6H, each d, J=7.0 Hz, 26-H ₃ and 27-H ₃ ) , 0.96 (3H, d, J=6.8 Hz,

21-H ), 4.23 (IH, m, 3-H) , 4.43 (IH, m, 1-H) , 5.00 (IH, narr. m, 19-H), 5.1-5.4 (3H, brm, 19-, 22-, and 23-H) , 6.02 and

6.39 (2H, ABq, J=11.4 Hz, 7-H and

227.5 nm; mass spectrum, mAz 398 269 (7) , 251 (5) , 152 (36) , 134 (100) . (Elution volume 39 ml) . Cαpound (12): NMR δ 0.61 (3H, s, 18-H ₃ ) , 0.89 and 0.91 (6H, each d, J=7.0 Hz, 26-H ₃ and 27-H ₃ ) , 0.97 (3H, d, J=6.9 Hz, 21-H ₃ ), 4.25 (IH, m, 3-H) , 4.51 (IH, m, 1-H) , 4.98 and 5.13 (2H, each narr. m., 19-H ₂ ) , 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 273 nm, λ . 229.5 nm; mass spectrum, m/z_ 398 (M , 17) , 380 (4) , 287 (5), 269 (5), 251 (4), 152 (29), 134 (100). (Elution volume 38 ml).

In the above described process, high pressure liquid chrcrnatography (HPLC) was performed on a Waters Associates Model ALC/GPC 204 using a Zorbax-Sil (DuPont) (6.2 irm x 25 cm column, flow rate 4 ml/min, 1500 psi) . Column chromatography was performed on Silica Gel 60, 70-230 mesh ASTM (Merck) . Preparative thin-layer chromatography (TLC) was carried out on Silica 60 PF-254 (20 x 20 cm plates, 1 mm silica gel) . Irradiations were carried out using a Hanσvia 608A36 mercury arc lamp fitted with a Vycor filter. All reactions are preferably performed under an inert atmosphere (e.g. argon) .

The cαrpound of this invention can, if desired, be readily obtained.in crystalline form by crystallization from suitable solvents such as hexane, ethers and alcohols (absolute or aqueous) , and mixtures thereof as will be evident and well known to those skilled in the art.

Process Scheme X

(1)R = CH ₂ 0CH ₃ {_,) R*CH ₂ 0CH-. (1) R=H

(5) R*H

(6) R*Ts

,R= = H .) X, = Acetyl X,=Ace.yl

(8) R- = OH (M) X,= H (]Z) X- =