CH665835A5 - 1ALPHA, 25-DIHYDROXY-22Z-DEHYDROVITAMIN D-CONNECTION. - Google Patents

Description

DESCRIPTION TECHNICAL FIELD The invention relates to a biologically active vitamin D compound, namely a new lcc, 25-dihydroxylated vitamin D compound with a 22.23-cis double bond in the side chain.

PRIOR ART Calcium and phosphate homeostasis in animals and humans is regulated by vitamin D metabolites, and the compound la, 25-dihydroxyvitamin D3 is generally considered to be the most active and important vitamin D-derived regulator of normal calcium and phosphate. Viewed equilibrium. This natural metabolite and compounds that are structurally related to it are thus of great pharmaceutical interest as effective agents for the prevention and treatment of bone diseases and related irregularities in calcium metabolism. In addition to the natural D3 metabolites, a number of compounds have been produced in recent years which are used because of their high potency or are very promising as therapeutic agents, including la-hydroxyvitamin D3, la-hydroxy-xyvitamin D; la, 25-Dihydroxyvitamin Di and certain fluorinated analog compounds include (U.S. Patents 3,741,996, 3,907,843, 3,880,894, 4,226,788, 4,358,406). Most of the known active analog compounds are characterized by the type of sterol side chain as it appears in vitamin D3 (i.e. saturated side chain). Known analogue compounds with a 22.23-unsaturated side chain are represented by the compounds of the vitamin D2 series (i.e. 22.23-trans-unsaturated with a C-24-methyl substituent). and include, in addition to the compounds noted above, 25-hydroxyvitamin D2 (U.S. Patent 3,585,221) and the 24- and 24,25-dihydroxy derivatives (Jones et al., Arch. Biochem. Biophys. 202, 450 (1980)) and three compounds without the 24-methyl substituent (U.S. Patent 3,786,062; Bogoslovskii et al., J. Gen. Chem. USSR, 48_ (4) 828 (1978); Chem. Abstr. 89, 163848j, 209016s).

DESCRIPTION OF THE INVENTION A new analogue compound of vitamin D has now been found, which can be represented by structure I below;

Oh

OH

This new compound is characterized by a 22.23 double bond in the side chain with the eis (or Z) geometry. Due to the presence of this 22Z double bond, which leads to a side chain geometry which is quite different from that of the compounds with the normal saturated side chain (for example in the case of la, 25-dihydroxyvitamin D3) or a 22,23-trans- (22E) unsaturated side chain (for example in la, 25-dihydroxyvitamin D2), it was assumed that this cis-unsaturated product would have a low biological activity if there was any biological activity at all. Surprisingly, in contrast to its modified side chain structure, this material shows a high activity and is as active as la, 25-dihydroxyvitamin D3 in its ability to increase the serum calcium levels in test animals.

The new product according to the invention, the above compound I, was prepared from a 22Z-dehydrovitamin D precursor with the structure II below by enzymatic hydroxylation in vitro at carbon atom 25 using a life homogenate preparation of rats with a vitamin D Defect.

OH

Ho

The following procedure was used; Male weanling rats were fed a low calcium and vitamin D deficiency diet as described by Suda et al. [J. Nutr. 100, 1049 (1970)] was fed for two weeks. They were killed by beheading and their livers were removed. A 20% (w / v) homogenate was made in ice cold 0.25 M sucrose. The incubation was carried out in 10 ml of incubation medium in a 125 ml Erlenmayer flask which contained an aliquot of the liver homogenate, consisting of 1 g of tissue, 0.125 M sucrose, 50 mM phosphate buffer (pH 7.4), 22.4 mM glucose- 6-phosphate, 20 mM ATP, 160 mM nicotinamide, 25 mM sucinate, 0.4 mM NADP, 5 mM MgCl2, 0.1 M KCl and 0.5 units glucose-6-phosphate dehydrogenase. The reaction was initiated by adding 400 µg of the substrate, Compound I above, dissolved in 100 µl of 95% ethanol. The incubation mixture was incubated at 37 ° C with shaking at 80 vibrations / minute for 3 hours. The reaction was stopped by adding 20 ml of methanol and 10 ml of dichloromethane, and after further addition of 10 ml of dichloromethane, the organic phase was total5

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665 835

melt while the aqueous phase was extracted again with 10 ml dichloromethane. The organic phases from the entire three extractions were combined and evaporated using a rotary evaporator. The residue, which contained the desired product, was dissolved in 1 ml of a mixture of CHCl3: hexane (65:35) and applied to a column (0.7 cm x 14 cm) packed with Sephadex LH-20, equilibrated and eluted with the same solvent. The first 10 ml was discarded while the following 40 ml was collected and evaporated. The residue was then dissolved in 8% 2-propanol in hexane and subjected to high performance liquid chromatography (model LC / GPC 204 HPLC, from Waters Associates, Medford, MA) using a Zorbax-SIL column (4.6 mm x 25 cm, DuPont, Wilmington, Delaware), which was operated under a pressure of 1000 psi at a flow rate of 2 ml / min. The desired 25-hydroxylated product was eluted at 44 ml. This product was further purified by high performance liquid chromatography, using a reverse phase column (Richrosorb Rp-18, 4.6 mm x 25 cm, E. Merck, Darmstadt, West Germany), under a pressure of 1200 psi and a flow rate of 2 ml / min was operated. The column was eluted with 22% H20 in methanol and the compound was eluted at 50 ml. This product was further purified by HPLC using the Zorbax-SIL column and the conditions described above. The product obtained was then subjected to physical characterization.

The UV absorption of the product in 95% ethanol showed an A. max = 265 nm and an Xmi "= 228 nm, which indicated the presence of the 5,6-cis-triene chromophore.

The mass spectrum of the substance contains a molecular ion at m / e 414, as is required for a 25-hydroxylated product. Elimination of one and two molecules of H20 results in fragment ions at m / e 396 and 378. Loss of the entire steroid side chain (cleavage of the C17 / C20 bond) leads to fragment m / e 287, which is eliminated by eliminating one or two Molecules H20 leads to the peaks at m / e 269 and 251. The spectrum shows clear peaks at m / e 152 and 134 (152-H20), which represent Ring A fragments and are indications for la, 3β-dihydroxyvitamin D compounds. In addition, the spectrum shows a very clear fragment peak at m / e 59, which results from the cleavage of the C24 / C25 bond. The presence of this ion confirmed the presence of the 25-hydroxy group in the product. Thus, these data confirmed the structure of the product obtained as the la, 25-dihydroxylated compound represented by structure I above.

The biological activities of the new analogue compounds were demonstrated by in vivo experiments in rats. Male weanling rats were fed a diet with a low calcium and vitamin D deficiency according to Suda et al. (above literature reference) fed 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 intrajugularly, while the rats in the other groups received 325 pmol of compound I or of la, 25-dihydroxyvitamin D3 dissolved in 0.05 ml of 95% ethanol. 18 hours later, they were beheaded and the blood was collected. The serum obtained by centrifuging the blood was diluted with 0.1% lanthanum chloride solution (1:20) and the serum calcium concentration was determined using an atomic absorption spectrophotometer. The results are shown in the table below:

Increase in serum calcium concentration in response to a single dose of 325 pmol of compound I or of la, 25-dihydroxy vitamin D3, which are administered 18 hours before the killing 5 Serum calcium concentration administered

Compound (mg / 100 ml) ± standard deviation

Ethanol 4.2 ± 0.1a)

io connection I 5.2 ± 0.2 b)

1 a, 25-dihydroxy vitamin D3 5.4 ± 0.4b)

b) is significantly different from a) p <0.001

The above results show that the new analogue compound is highly effective and has a biological activity that is essentially equivalent to that of la, 25-dihydroxy vitamin D3.

Because of this high potency, this compound according to the invention will be used as a therapeutic agent in the therapy or prophylaxis of diseases of such different types as rickets, hy-poparathyroidism, bone dystrophy, bone softening or osteoporosis in humans or for the treatment of related calcium Deficiency diseases (e.g. 25 milk fever, weakness in legs, low eggshell strength) in animals. In the same way, the compound can be used in the treatment of certain malignancies, such as leukemia in humans.

For therapeutic purposes, the compound can be administered by any conventional route and in any suitable form for the route selected. The compound can be formulated with any compatible and harmless pharmaceutical carrier in the form of pills, tablets, gelatin capsules or suppositories or in the form of solutions, emulsions, dispersions or suspensions in harmless solvents or oils, and such formulations can also be other therapeutically effective and beneficial Contain components as they may be suitable 40 for the specific applications. For use in humans, the compound is advantageously administered in amounts of 0.25 to 10 µg per day, the specific dosage being selected in accordance with the disease to be treated and with the medical development, condition and response of the patient becomes, as is familiar to the expert.

The 22Z-dehydro precursor substrate, the above compound II, which is required for the production of the new product according to the invention, is itself produced according to process scheme I according to the appendix and according to the description below. In the description, the connection names according to Arabic numerals (e.g. (1), (2), (3), etc.) refer to the structures numbered in this way in the process diagram. The desired substrate 55 (Compound II) for the 25-hydroxylation reaction described above is identified by the Arabic numeral (11) in Process Scheme I and in the following description.

(22Z) -3ß- (methoxymethoxy) - 5a, 8a- (4-phenyl-1,2-urazole) -60 cholesta -6,22-diene (2)

Isopentylphosphonium bromide [(CH3) 2CHCH2CH2PPh3Br] (1.67 g, 4.04 mmol) in anhydrous tetrahydrofuran (73 ml) was added with n-butyllithium (1.7 M solution in hexane, 2.42 ml, 4.11 mmol) 3-5 ° C 65 treated with stirring. After stirring at room temperature for 1 hour, the orange-red solution was cooled to 3 ° C. and the aldehyde (1) (1.84 g, 3.36 mmol in anhydrous tetrahydrofuran (24 ml) was added. The colorless

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The reaction mixture was stirred overnight at room temperature (1H, m, 3-H), 5.20 (2H, br m, 22-H and 23-H), 5.69 and then poured into water and with benzene 5.95 (2H, ABq, J = 12 Hz, 7-H and 6-H); UVX, max 261 nm,

extracted. The organic extract was extracted with 5% HCl, mm ^ 234 nm.

saturated sodium bicarbonate and water, the thermal isomerization of this provitamin intermediate

dried (Na2S04) and concentrated under reduced pressure to a 5 compound (56 mg, 0.15 mmol) in refluxing oil, which was purified over a column of silica gel with ethanol (3 hours) to give the oily vitamin ana-

has been. Elution with a mixture of benzene ether log compound (5) (43 mg, 77%) after separation by means of

(94: 6) provided the adduct (2) (1.38 g, 68%) as an HPLC. NMR 5 0.60 (3H, s, 18-H3), 0.89 and 0.90 (6H, each

Foam: NMR 5 0.83 (3H, s, 18-H3), 0.89 and 0.91 (6H, each d, J = 6.7 Hz, 26-H3 and 27-H3), 0, 97 (3H, d, J = 6.6

because d, J = 6.8 Hz, 26-H3 and 27-H3), 0.97 (3H, d, J = 6.8 io Hz, 21-H3), 3.96 (1H, s, 3- H), 4.82 and 5.05 (2H, each

Hz, 2I-H3). 0.98 (3H, s, I9-H3), 3.30 (IH, dd, J, = 4.4 Hz, narrow multiplet, 19-H2), 5.20 (2H, br m, 22-H and -

J, = 14 Hz, 9-H), 3.38 (3H, s, OC H3), 4.33 (1H, m, 3-H), 23-H), 6.04 and 6.24 (2H , ABq, J = 11.4 Hz, 7-H and 6- H);

4.70 and 4.81 (2H, ABq, J = 6.8 Hz, 0CH20), 5.21 (2H, br UV ^ max 265.5 nm, Xmin 228 nm; IR: 3427, 1458, 1379, 1048 ,

m, 22-H and 23-H), 6.23 and 6.39 (2H, ABq, J = 8.5 Hz, 966, 943, 892 cm "1; mass spectrum m / z 382 (M +, 21), 349

6-H and 7-H), 7.41 (5H, br m, Ar-H); IR: 1756, 1703, 1601, «(5), 271 (8), 253 (14), 136 (100), 118 (82). The vitamin ana-

1397, 1046 cm "1; mass spectrum m / z 601 (M +, 1%), 426 log connection (5) is a known connection (Bogoslovs-

(4), 364 (61), 349 (16), 253 (18), 251 (18), 119 (PhNCO, 100). kii et al., literature reference above).

(22Z) -5a, 8a- (4-phenyl-l, 2-urazoljcholesta -6,22-dien- 5ß-o /

(3).

A solution of the adduct (2) (601 mg, 1 mmol) and p-toluenesulfonic acid (523 mg, 2.75 mmol) in a mixture of methanol (20 ml) -THF (12 ml) was stirred at room temperature for 2 days. The reaction mixture was poured into saturated sodium bicarbonate and extracted several times with benzene. The extracts were washed with water, dried (Na2S04) and evaporated under reduced pressure. Purification of the crude product by column chromatography (benzene ether 70:30 as eluent) gave the hydroxy adduct (3) (550 mg, 99%) as a foam: NMR 5 0.83 (3 s, I8-H3), 0.89 and 0.91 (6H, each d, J = 6.8 Hz, 26-H3 and 27-H3), 0.95 (3H, s, 19-Hj), 0.98 (3H, d, J = 6 , 8 Hz, 21-H3), 3.16 (lH, dd, J, = 4.4 Hz, J2 = 14 Hz, 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 m, Ar-H; IR: 3447, 1754, 1700, 1600, 1397 cm-1; mass spectrum m / z 557 (m +, 1%), 382 (35), 349 (33), 253 (20), 251 (33), 119 (100), 55 (82).

(22Z) -Cholesta-5,7,22-triene-3 $ -ol (4)

The adduct (3) (530 mg, 0.95 mmol) was added to the diene

(4) Transferred by reduction with lithium aluminum hydride (1 g) in tetrahydrofuran (60 ml) under reflux for 18 hours. After the conventional work-up, the product was purified by chromatography on silica (benzene ether 94: 6 as eluent) to give the pure diene (4) (290 mg, 76%) after recrystallization from ethanol

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give melting point 148-151 "C; [a] ^ = -132"

(c = 0.9, CHC13); NMR § 0.66 (3H, s, 18-H3), 0.90 and 0.91 (6H, each d, J = 6.8 Hz, 26-H3 and 27-H3), 0.96 (3H, s, 19-Hj), 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-H and 6-H); UVmax 281 nm; IR: 3346, 1463, 1375, 1364, 1067, 1040, 831 cm-1; 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-3f> -

ol (5)

Irradiation of 5,7-diene (4) (150 mg, 0.39 mmol), which was dissolved in Ehter (120 ml) and benzene (30 ml) (degassed with argon for 40 minutes), was at 0 C for 13 minutes using a UV lamp and a Vycor filter. HPLC (1% of 2-propanol in hexane) of the resulting mixture provided the provitamin (56.9 mg, 38%) as a colorless oil: NMR 5 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, 21-H3), 1.64 (3H, s, 19- H3), 3.90

1-hydroxylation of compound (5)

20 Freshly recrystallized p-toluenesulfonyl chloride (50 mg, 0.26 mmol) was added to a solution of the vitamin (5) (50 mg, 0.13 mmol) in anhydrous pyridine (300 | il). 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 saturated NaHCO 3, saturated copper sulfate and water, dried (Na 2 SO 4) and concentrated under reduced pressure to give the oily tosylate (6). The crude tosylate (6) was treated with 30 NaHCO 3 (150 mg) in anhydrous methanol (10 ml) and the mixture was stirred at 55 C for 8.5 hours. After cooling and concentrating to 2 ml, the mixture was diluted with benzene (80 ml), washed with water, dried (Na2S04) and concentrated under reduced pressure 35. The oily 3,5-cyclovitamin D compound (7), which is so was of sufficient purity to be used for the subsequent oxidation step without any purification. To a vigorously stirred suspension of Se02 (5.1 mg, 0.046 mmol) in anhydrous CH2C12 (5 ml) 40 was added tert-butyl hydroperoxide (16.5 (il, 0.118 mmol). After 30 minutes, anhydrous pyridine (50 | il) was added and the mixture was stirred for a further 25 minutes at room temperature, diluted with CH2C12 (3 ml) and cooled to 0 C. The crude 3,5-cyclvitamin product (7) in 45 CH2C12 (4.5 ml) was then The reaction proceeded at 0 C for 15 minutes and the reaction mixture was then allowed to warm slowly (room temperature) to room temperature (30 minutes), transferred to a separatory funnel and shaken with 30 ml of 10% NaOH ge-50 ml) was added and the separated organic phase was washed with 10% NaOH, water and dried over Na2SO4 The concentration to dryness under reduced pressure gave a yellow oily residue which was purified on a silica gel 55 TLC plate under development with 7: 3 hexane-ethyl acetate and the 1st -Hydroxycyclovitamin product (20 mg, 37%) gave: NMR S 0.59 (3H, s, 18-H3), 0.63 (IH, m, 3-H), 0.89 and 0.90 (6H , each d, J = 6.9 Hz, I6-H3 and 27-H3), 0.96 (3H, d, J = 6.9 Hz, 21-H3), 3.25 (3H, s, -OCH3 ), 4.17 (2H, 60 m, 1-H and 6-H), 4.96 (1H, d, J = 9.3 Hz, 7-H), 5.1-5.4 (4H, br m, 19-H->, 22-H and 23-H); Mass spectrum m / z 412 (M +, 26), 380 (48), 339 (22), 269 (28), 245 (20), 135 (100). This product was composed mainly of the la-hydroxycyclovitamin D compound of structure 65 (8), as well as a small amount of the corresponding lß-hydroxy epimer. These components can be separated at this stage if desired, but such separation is not required.

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The 1-H-hydroxycyclovitamin product (18 mg) obtained by the method described above was heated (55 ° C./15 min) in glacial acetic acid (0.8 ml), the mixture was neutralized (ice / saturated NaHCO 3) and with benzene and ether extracted to give the pure la-hydroxy-3β-acetoxyvitamine (9) (6.60 mg, 34%, elution at 42 ml) after HPLC (1.5% of 2-propanol in hexane as eluent) separation. and (10) (4.20 mg, 22%, elution at 50 ml).

Compound (9): NMR 5 0.60 (3H, s, 18-H3), 0.90 and 0.92 (6H, each d, J = 7.0Hz, 26-H3 and 27-H3), 0, 97 (3H, d, J = 6.8 Hz, 21-H3), 2.04 (3H, s, -OCOC H3), 4.41 (1H, m, 1 - H), 5.02 (1H, narrow Multiplet, 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); UVX.max 264.5 nm, X min 227.5 nm; mass spectrum m / z 440 (M +, 10), 380 (72), 362 (7), 269 (31) , 251 (12), 135 (100), 134 (99).

Compound (10): NMR §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, -OCOCH3), 4.49 (1H, m, 1-H), 5.00 and 5, 14 (2H, each narrow multiplet, 19-H2), 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); UVXmax 270 nm, /, min 228 nm; Mass spectrum m / z 440 (M +, 4), 380 (30), 269 (10), 135 (100), 134 (52).

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

Each of the 3β-acetoxy derivatives (9) and (10) was hydrolyzed separately using the same procedure. A solution of 3β-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 at 50 ° C for 1 hour. After the usual work-up and the final HPLC purification (8% of 2-propanol in hexane as eluent), the corresponding 1-hydroxy vitamins were obtained, namely:

Compound (11): NMR Ô 0.59 (3H, s, 18-H3), 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 , narrow multiplet, 19-H), 5.1-5.4 (3H, br m, 19-, 5 22- and 23-H), 6.02 and 6.39 (2H, ABq, J = 11, 4 Hz, 7-H and 6-H); UV /, niilx 264.5 nm, À, min 227.5 nm; Mass spectrum m / z 398 (M +, 21), 380 (8), 287 (6), 269 (7), 251 (5), 152 (36), 134 (100). (Elution volume 39 ml).

Compound (12): NMR 5 0.61 (3H, s, 18-H3), 0.89 and 10 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 narrow multiplet, 19-H2), 5.21 (2H, br, 22-H and 23-H), 5.89 and 6.59 (2H, ABq, J = 11.5 Hz , 7-H and 6-H); VA, max 273 nm, min 229.5 nm; Mass-15 spectrum m / z 398 (M +, 17), 380 (4), 287 (5), 269 (5), 251 (4), 152 (29), 134 (100). (Elution volume 38 ml).

In the process described above, high pressure liquid chromatography (HPLC) was performed using a Waters associates Model 20 ALC / GPC 204 device using a Zorbax-Sil (DuPont) column (6.2 mm 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 over silica 60 PF-254 (plates with 20 x 20 cm, 1 mm silica gel). Irradiation was performed using a Hanovia 608A36 mercury arc lamp equipped with a Vycor filter. All reactions are preferably carried out under an inert gas atmosphere (e.g. argon).

If desired, the compound according to the invention can be obtained in crystalline form by recrystallization from suitable solvents, such as hexane, ethers and alcohols (absolute or aqueous) and mixtures thereof er-3; as will be known to those skilled in the art.

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Process Scheme I

um'CHjOCH,

(2) R »CH, OCH, '<11 R * H

l £> R-H 16) R «T«

(â) X, * Actl »l m> X,« H

(£ 1 X, * Ac «trl (12) X |« H

c

Claims (3)

665 835 2. Compound according to claim 1 in crystalline form. 2nd PATENT CLAIMS 1. Connection of the structure oM Oh HO 3. A pharmaceutical formulation containing the compound of claim 1 together with a pharmaceutically acceptable excipient.