Method of obtaining novel 1 alpha-hydroxyvitamin d4 and novel transition and analog compounds

The present invention relates to a process for the preparation of new 5,6-cis-1a-hydroxyvitamin D 4.

It is known that vitamin D plays an important role in regulating calcium metabolism in animals and humans, see Harrison's Principals of Internal Medicine: Part 11, Disorders of Bone and Mineral Metabolism, ch. 335, E. Braunwald et al. (Ed.). McGraw-Hill, New York 1987, pp. 1860-1865. Vitamin D 3 and vitamin D2 are two of the best-known useful forms of vitamin D. Vitamin D3 is synthesized endogenously in animal and human skin, while vitamin D 2 is a form of vitamin D supplied by plants. Vitamin D 2 differs from vitamin D 3 in that it contains a double bond between C22 and C23 and a methyl group at C24. In humans and rats, vitamin D 3 and vitamin D2 have equivalent biopotence.

Vitamin D4, also known as irradiated 22.23-dihydroergosterol or 22.23-dihydro-vitamin D2 or 22.23-dihydroergocalciferol, differs from vitamin D 3 in that it contains a methyl group at C24. Vitamin D4 was first described in 1936. See, W. Grab. Z. Physiol. Chem. 243: 63 (1936); FG McDonald, J. Biol. Chem. 14: IVX (1936). See also, A. Windaus and G. Trautmann, Z. Physiol. Chem. 247: 185-188 (1937). In these publications, there were some disagreements regarding the level of biological activity of the vitamin, suggesting that in the rat vitamin D 4 activity is 1/3 or 3/4 of vitamin D4 activity, and in the chick 1/10 or 1/5 of vitamin D 3 activity.

More specific studies of the biological activity of vitamin D4 were conducted by DeLuca et al. In 1968 (DeLuca et al., Arch. Biochem. Biophys., 124: 122-128 (1968). The authors confirmed that vitamin D4 is less active than vitamin D 3. DeLuca et al. Report that, according to their study, vitamin D4 activity is 2/3 of vitamin D 3 or vitamin D2 activity in a rat and 1/5 of vitamin D 3 activity in a chick.

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DeLuca et al. Write that since Vitamin D 4 was first described by Windhaus and Trautmann, its synthesis is apparently little used and they comment that this is perhaps due to the fact that vitamin D4 is only of academic interest .

According to the Applicant, vitamin D4 remains only in the sphere of academic interest, because the Applicant is not aware of any further research on vitamin D4 since reports by DeLuca et al. In fact, The Merck Index found about vitamin D4 that its biological activity seems questionable. Merck Index. S Budawari (ed.), Ed. 11th, Merck a. Co., Rahway, NJ (1989) p. 1579 # 9930.

Since DeLuca et al., They have discovered the active form of vitamin D 3, 1,25-dihydroxyvitamin D 3 (US Patent No. 3,697,559) and its synthetic precursor, la-hydroxyvitamin D 3 (US Patent No. 3,741,996) the greatest interest focused on the therapeutic use of these active metabolites of vitamin D 3. Unfortunately, although vitamin D3 metabolites were very promising as therapeutic agents, they were never fully utilized because of their extremely high toxicity. For example, toxicity limits the effectiveness of vitamin D 3, its active forms and analogues, in preventing bone defects or renewing bone defects.

Many studies indicate that at the doses required to ensure the effectiveness of these agents in the prevention of bone defects or in the restoration of defects, in hypercalcaemia and excessive excretion of calcium in the urine, there are problems.

1 α-hydroxyvitamin D 3 at a daily dose of 2 ^ g / day (which has been shown to be effective in preventing bone loss) has been reported to cause toxicity in approximately 67% of patients. There is therefore a need for a biologically potent metabolite of vitamin D with low toxicity, so that the drug is practically a therapeutic agent.

The new compound produced according to the invention, 5,6-cis-1a-hydroxyvitamin D4 and the biological products of its metabolism are bioactive forms of vitamin D4. The inventors of the present invention have found that these active forms of vitamin D 4 have a much higher biopotence than could have been foreseen on the basis of previously described biological tests with vitamin D4. They also found that bioactive new compounds are less toxic than could have been predicted based on their biopotency. This combination of high activity and low toxicity makes these compounds useful as therapeutic agents in the treatment of calcium metabolism disorders.

The novel compound of the present invention is used as an active compound in pharmaceutical compositions intended for the treatment of diseases caused by abnormal calcium metabolism.

Research on a new compound required work on the processes for its production. During the synthesis of α-hydroxyvitamin D4, new intermediates were also produced. 1,25-dihydroxyvitamin D4 and 1,24-dihydroxyvitamin D4 have been isolated as biological products of 1a-hydroxyvitamin D4 metabolism.

Other advantages and a more complete assessment of specific adaptations, compositional variations, physical and chemical features of the present invention will be apparent after analyzing the following detailed description of the invention including the accompanying drawings.

Figure 1 illustrates the preparative steps for the synthesis of vitamin D4, and Figure 2 illustrates the preparative steps for the synthesis of 1a-hydroxyvitamin D4, in which vitamin D 4 is used as the starting compound.

The terms bilogical or biologically active activity as used herein should be understood to refer to the biochemical properties of compounds, such as exerting influence on metabolism, e.g. effect on serum calcium concentration or binding to the appropriate receptor protein, e.g. binding to vitamin D receptor protein .

170 447

The method of the invention produces a compound of formula (I)

<img file="PL170447B1_D0001.tif" />

wherein Rx and R2 are H.

The method of the invention comprises the following steps:

a) tosylation of vitamin D4 to form vitamin D4 tosylate;

b) solvolysis of vitamin D4 tosylate to 3,5-cyclovitamin D4;

c) oxidation of 3,5-cyclo-vitamin D4 to 1a-hydroxy-3,5-cyclo-vitamin D4 and

d) successively solvolysis and Diels-Alder reaction of 1? -hydroxy-3,5-cyclovitamin D4 to form 1a-hydroxyvithin D4 and isolation of 5,6-cis-1 α-hydroxyvitamin D4.

The scheme in Figure 1 shows the synthesis of vitamin D4, which is used as the starting compound in the process of the invention.

Ergosterol is used as the starting compound in the synthesis of Figure 1. Ergosterol is saturated in the side chain in a 6-step process to obtain 22.23-dihydroergosterol (VIII) using a procedure similar to that of Barton et al., JCS Perkin I, 1976, 821-826.

Then 22.33-dihydroergosterol is irradiated as described by Windaus et al., Z. Psysiol. Chem., 1937, 147: 185 and vitamin D4 [22.23-dihydrocalciferol] (IX) is obtained. As can be seen in Fig. 2, vitamin D4 is then hydroxylated in a 4-step process to obtain 1α-hydroxyvitamin D4 using a similar procedure to that described by Paaren et al., J. Org. Chem. 1980, 45: 3253.

More specifically, ergosterol is acetylated to 3P-acetate. Ergosterol acetate is subjected to hydroxychlorination with a 5.6 double bond to give a 6? -Chloro-5? 4-hydroxyl derivative. This chlorohydrin is reduced and again acetylated to a 5-hydroxyl derivative (i.e. to 5α-ol). 5a-ol is hydrogenated to saturate the side chain. The 3e-acetoxyergost-7-en-5a-ol produced is reduced to 22.23-dehydroergosterol acetate. 22.23-dehydroergosterol is then irradiated to produce vitamin D4. Vitamin D 4 is tosylated and 3P-tosylovitamin D 4 is obtained. Then tosylate is solvolyzed to give 6-methoxy-3,5-cyclovitamin D 4. Cyclovitamin D4 is subjected to allyl oxidation and a 1α-hydroxyl cyclo-vitamin derivative is produced. The hydroxy derivative is subsequently subjected to solvolysis and a Diels-Alder reaction which removes the 5-methoxy group and separates 5,6-cis-1 α-hydroxy vitamin D 4 from 5,6-trans-1a-hydroxyvitamin D4.

170 447

Step a) in the process according to the invention is preferably carried out by reacting vitamin D 4 with toluenesulfonyl chloride in the presence of dry pyridine.

In step b), preferably vitamin D4 tosylate is subjected to buffered solvolysis.

In step c), preferably 3,5-cyclo-vitamin D4 is subjected to allylic oxidation of selenium dioxide.

In contrast, step d) preferably comprises solvolysis of 1α-hydroxycyclovitamin D4 with a mixture of dimethyl sulfoxide and organic acid to form a mixture of 5,6-cisil-hydroxy- and 5,6-trans-α-hydroxyvitamin D 4 and the Diels Alder reaction of the resulting mixture which produces the 5,6-trans ia-hydroxyvitamin D4 adduct and the separately prepared 5,6-cis-1α-hydroxyvitamin D 4.

1,24-dihydroxyvitamin D4 and 1,25-dihydroxyvitamin D4 - metabolites of α-hydroxyvitamin D4, are synthesized by incubating the α-hydroxy derivatives with human liver cells, culturing the cells and recovering 1,24-dihydroxy- or 1,25-dihydroxyvitamin D4. Using vitamin D binding receptor proteins, these metabolites were found to be biologically active.

The compound of formula (I) has been found to have valuable pharmacological activity, namely as a calcium metabolic regulating agent, especially serum calcium. Namely, the compound of formula (I) increases the serum calcium concentration in rats with vitamin D deficiency. It has also been found that the compound of formula (I) has low toxicity, which improves its pharmaceutical properties. The toxicity of the compound of formula (I) measured in the LD 50 test is similar to the toxicity of the corresponding vitamin D2 compounds and lower than the toxicity of the corresponding vitamin D3 compounds. Thus, the compound of the present invention is suitable for use in various clinical and veterinary cases and is particularly useful in the treatment of abnormal calcium and phosphorus metabolism.

The invention enables the treatment of abnormal calcium metabolism caused e.g. by hepatic insufficiency, renal failure, gastrointestinal failure, etc. The compound of formula (I) can be used prophylactically or therapeutically in vitamin D deficiency diseases and related diseases, e.g. in renal osteodystrophy, fat diarrhea, anticonvulsant bone softening, vitamin D-opom phosphate rickets, osteoporosis, including postmenopausal osteoporosis, senile osteoporosis, steroid-induced osteoporosis, and other disease conditions characterized by bone loss, in which bone loss is defective from vitamin D), rickets associated with nutrition and rickets associated with poor absorption of food, in secondary bone softening and osteopenia associated with hypoparathyroidism, in postoperative hypoparathyroidism, in spontaneous hypoparathyroidism, supposed hypoparathyroidism and in alcoholism.

The compound of formula (I), and preferably its metabolite, such as 1,24-dihydroxyvitamin D4, is valuable in the treatment of skin diseases associated with excessive proliferation, such as psoriasis.

The compound of formula (I) is useful as an active compound in pharmaceutical compositions that exhibit reduced side effects and low toxicity compared to known analogs of active vitamin D3 forms, for use, e.g., in diseases caused by abnormal calcium metabolism.

The pharmacologically active compound of the present invention can be converted into conventional methods of pharmacy into therapeutic agents intended for administration to patients, e.g., mammals including humans. For example, the compounds of formula (I) can be used in mixtures with conventional excipients, e.g. with pharmaceutically acceptable carriers suitable for enteral use (e.g. oral), parenteral or topical, which do not react with active compounds in a harmful manner.

Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, acacia, vegetable oils (e.g., corn oil, cottonseed oil, peanut oil, olive oil, coconut oil), fish oil, oily esters , such as Polysorbate 80, polyethylene glycols, gelatin, hydrocarbons (e.g. lactose, amylose or starch), magnesium stearate, talc, silicic acid, liquid paraffin, monoglycerides and diglycerides

170 447 fatty acids, fatty acid esters of pentaerythritol, hydroxymethyl cellulose, polyvinylpyrrolidone, etc.

Pharmaceutical preparations can be sterilized and, if necessary, can be mixed with auxiliaries, e.g. with lubricants, preservatives, stabilizers, wetting agents, emulsifiers, with salts that affect the osmotic pressure, with buffers, coloring, flavoring and / or with one or more other active compounds, e.g. with vitamin D 3 or D2 and their 1α-hydroxyl metabolites, with conjugated estrogens and their equivalents, with antiestrogens, calcitonin, bisphosphonates, with calcium supplements, cobalomin, pertussis toxin and boron.

Sterile injectable solutions, preferably an oily or aqueous solution, as well as suspensions, emulsions or implants, and suppositories are particularly suitable for parenteral administration. Ampoules are a convenient form of use.

For enteral administration, tablets, dragees, liquids, drops, suppositories, lozenges, powders or capsules are particularly suitable. If a carrier with a sweet taste is indicated, a syrup, elixir or similar form may be used.

Sustained or direct release compositions may also be prepared, e.g. with liposomes or in which the active compound is protected by differentially degrading coatings, e.g. by microencapsulation, multiple coating, etc.

Topically, suitable non-sprayable, viscous, semi-solid or solid forms can be used, which include a carrier suitable for topical use, with a dynamic viscosity preferably greater than that of water. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, dusting powders, liniments, lotions, sprays, transdermal patches, etc., which, if desired, are sterilized or mixed with auxiliaries, e.g. with preservatives, stabilizers, demulsifiers, wetting agents etc.

For rectal administration, the compound is formulated into a pharmaceutical composition containing a suppository base, such as cocoa oil or other triglycerides. To extend the shelf life, the composition preferably contains an antioxidant such as ascorbic acid, butylated hydroxyanisole or hydroquinone.

Preferably, the pharmaceutical compositions are administered orally. Generally, the compound of the present invention is prepared in a dosage form containing about 0.5 pg to about 25 pg of the compound in a pharmaceutically acceptable carrier per unit dose. The dose of compound is usually about 0.01 to about 0.5 pg / kg / day, preferably about 0.04 to about 0.03 pg / kg / day.

Of course, the actual preferred amounts of active compound in a particular case will vary depending on the effectiveness of the particular compound employed, the particular form of the composition, the method of use, the particular site being treated and the organism. Eg. the specific dose for a given patient depends on the age, body weight, general health, sex, diet, time and method of administration, the rate of excretion and the drugs used in combination, as well as the severity of the disorder being treated. Dosages for a given patient can be determined in a conventional manner, e.g., by simply comparing the differential activity of the given compounds and the known agent, e.g., using the appropriate conventional pharmacological protocol.

The compound of the present invention may also be advantageously used in veterinary compositions, e.g., compositions for feeding pet animals to treat or prevent hypocalcaemia. Usually, the compound of the present invention is introduced into animal feed in such an amount that normal consumption of such feed provides the animal with about 0.01 to 0.5 (tg of compound / kg / day.

The following examples are given for the purpose of illustration only and do not limit the invention. In the examples, all temperatures are given in degrees Celsius, unless otherwise indicated, all parts and percentages are by weight. Proton nuclear magnetic resonance (Ή NMR) spectra were obtained on an IBM Sy-200 (200 MHz) and Bruker Am-400 (400 MHz) with a 3000 computer in CDCb solution with CHCl3 as the internal standard. Infrared spectra were recorded with Fourier transform (FTIR) using samples in form

170 447 tablets with potassium bromide (KBr) or in the form of liquids. Mass spectra were recorded on a Finnigan MAT-90 mass spectrometer at 20 eV / CI. Melting points were determined on Hoover-Thomas (capillary) Uni-Melt and Fisher-Johns (cover-slip) devices.

Example 1: Synthesis of 1a-hydroxy vitamin D 4

Ergosterol (II) was converted into ergostrerol (III) acetate by dissolving 100 g (0.25 mol) ergosterol in 600 ml anhydrous pyridine and 68 ml (0.7 mol) acetic anhydride. The solution was stirred overnight at room temperature, then cooled by adding 1.2 L of ice, which caused a precipitate to form. The precipitate was washed 5 times with 400 ml portions of water, then once with 400 ml CH 3 CN. The resulting product was air-dried to obtain 79 g (71%) of ergosterol acetate as a white crystalline solid with the following characteristics: Melting point (mp): 169-171 ° C;

1 H NMR: (400 MHz, CDCls), δ ppm 2.05 (3H, s, 3p-CH3CO), 4.65-4.75 (1H, m, 3a-H), 5.15-5.25 (2H, m, 22-H and 23-H), 5.4 (1H, d, 6-H), 5.6 (1H, d, 7-H); FTIR [KBr]: 1734 cm '¹ (C = 0 stretching) 968 cm⁴ (CH twisting).

Ergosterol (III) acetate (26 g, 0.062 M) was dissolved in 2.5 L of freshly distilled deoxygenated toluene. To this solution, 9 ml (0.111 mol) of chromyl chloride dissolved in 240 ml of dry CH2Cl2 was added under nitrogen at -78 ° C over 30 minutes. The reaction system was stirred at 78 ° C for a further 15 minutes, after which 62 ml of a saturated solution of sodium borohydride in ethanol were added once. After stirring at -78 ° C for another 15 minutes, the reaction solution was poured into a two-phase system of 3N hydrochloric acid (3 L) and benzene (3 L). The organic layer was separated, then washed with water (2 1), twice with brine solution (2 x 1 1) and dried with anhydrous MgSO 4. The dried solution was filtered and concentrated in vacuo. The crude crystalline product was then treated with CH3 CN (280 mL) and filtration of the resulting suspension gave 12.5 g (41%) of white crystalline 33-acetoxy-6-chloroergosta-7,22-dien-5a-ol (IV) with the following characteristics: mp 190 - 192C; 'HNMR (400 MHz, CDCls), δ ppm 2.05 (3H, s, 3P-OAc), 4.65 (1H, d, 6 β-Η), 5.1 (1H, s, 7-H) , 5.1-5.3 (2H, m, 22-H and 23-H); FTIR [KBr}; 1732 c '¹ (C = O stretching), 968 cm-¹ (CH twisting), 3437 cm-1 (OH stretching).

3P-acetoxy-6a-chloroergosta-7,22-dien-5a-ol (IV) (21.4 g, 0.0444 mol) in dry THF (900 ml) was slowly added to a stirred suspension of lithium aluminum hydride (2, 66 g, 0.07 mol) in dry THF (750 ml) at room temperature under nitrogen. The mixture was heated to reflux for 3 hours and cooled to 0 ° C. Excess hydride decomposed with a saturated Na2SO4 solution. Filtration through anhydrous Na2SO4 and evaporation of the filtrate gave a solid which was immediately treated with acetic anhydride (110 ml) and dry pyridine (220 ml) at 0 ° C. Removal of the solvent under reduced pressure gave acetate (12.75 g, 61%) 3 (^ - ac ^^ tt4 ^ k ^; ^ y ^ ir ^ (^^, stinn-7,; ^ '2-dien-5a -ol (V) with the following characteristics: mp: 229 - 232 ° C; FTIR [KDr] 1736 cm^at (C = O stretching), 3460 cm⁴(OH stretching), 972 cm- (CH torsion).

3P-acetoxyergosta-7,22-diene-5a-ol (V) (2.5 g, 0.0055 mol) was shaken for 16 hours with freshly prepared PtO2 (0.5 g) in ethyl acetate (820 ml) under H2 gas ( 15 psi). The catalyst was filtered off and the filtrate evaporated to give crude acetate, which was dissolved in CH 2 Cl 2 and chromatographed on silica gel. Elution with CH2O2 gave essentially pure 3P-acetoxyergost-7-en5a-ol (VI) (2.15 g, 85%) as a white crystalline substance with the following characteristics: mp: 228-232 ° C;

'H NMR (400 MHz, CDCls), 5 ppm 2.05 (3H, s, 3 β-OAc), 5.05-5.20 (2H, m, 3a-H and 7-H; FTIR [KBr] : 1736 cm⁴ (C = O stretching), 3462 cm⁴ (OH stretching).

To 3e-acetoxyergost-7-en-5a-ol (VI) (12.0 g, 0.0262 mol) in dry pyridine (800 ml) double-distilled thionyl chloride (9.7 ml) was added at 0 ° C under nitrogen in dry pyridine (170 ml). After 2.5 hours the solution was diluted with ice-cold water (1.5 L) and extracted with two portions of ether (2.5 L + 1.51). The combined ether extracts were washed with a NaHCO3 solution (1.01 x 2), then with IN HCl (1.51 x 2), then with water (11). The ether solution was dried with MgS04, and after filtration and evaporation under reduced pressure, the crude product was obtained, which was suspended in CH3CN (100 ml). The product was collected by

170 Filtration and recrystallization from CH 3CN gave 4.5 g (39%) of white crystalline 22.23-dihydroergosteryl acetate (VII) with the following characteristics: mp: 144-147 ° C;

Η NMR (400 MHz, CDCb), 5 ppm 2.05 (3H, s, 3P-OAc), 4.65-4.75 (1H, m, 3a-H), 5.4 (1H, d, 6-H), 5.6 (1H, d, 7-H); FTIR [KBr]: 1734 cm '¹ (C = O stretching).

22.23-dihydroergosteryl acetate (VII) (4.8 g, 0.011 mol) was added immediately to a stirred suspension of lithium aluminum hydride (2.5 g, 0.066 mol) in dry ether (1.11) at room temperature. The mixture was stirred for 2 hours at room temperature. 5N NaOH was added to destroy the excess lithium aluminum hydride, followed by the addition of water (500 mL). The aqueous solution was then extracted with four 250 ml portions of ether. The combined ether extracts and the combined organic layer were washed with brine solution (1 L) and then dried with Na2SO4. Evaporation of the ether under reduced pressure gave 22.23-dihydroergosterol, (VIII) (4.1 g, 91%) as a white crystalline substance with the following characteristics: mp: Χ4Ί - 150 ° C;

^lH NMR (400 MHz, CDCb), δ ppm 3.6-3.7 (1H, m, 3a-H), 5.4 (1H, d, 6H), 5.6 (1H, d, 7-H ); FTIR [KBr]: 3400 cm⁴ (OH stretching).

22.23-dihydroergosterol (VIII) (2.0 g, 5.0 mmol) was dissolved in a solution of ethyl ether and benzene (4: 1, 600 ml) and irradiated (Hannovia diving lamp, 450 W), stirring under nitrogen in the vessel quartz cooled with water for 3 hours. The solution was concentrated in vacuo to give a viscous substance, which was dissolved in 100 ml of ethanol and heated to reflux under argon for 8 hours. The solution was then concentrated in vacuo and the residue adsorbed on a silica gel column and eluted with 30% ethyl acetate in hexane. Vitamin D4 (22.23-dihydroergocalciferol) (IX) was obtained in a yield of 1.2 g (60%) with the following characteristics:

-H NMR (400 MHz, CDCb), δ ppm 0.55 (3H, s, 18-H3) 0.78 (6H, dd, 26-H3 and 27-H₃), 0.87 (3H, d, 21-H3) 0.93 (3H, d, 28-H3), 3.94 (1H, m, 3-H), 4.82 (1H, m, acute, 19-H), 5.04 (1H, m (acute), 19-H), 6.04 (1H, d, 7-H), 6.24 (1H, d, 6-H).

To a mixed solution of vitamin D4 (IX) (3.0 g, 7.5 mmol) in 10 mL dry pyridine was added freshly recrystallized toluenesulfonyl chloride (3.6 g, 19 mmol) at 0 ° C. The reaction mixture was stirred at 5 ° C for 24 hours before being quickly quenched by pouring the mixture on ice and saturated NaHCO 3 (100 mL) and the mixture.

The aqueous suspension was extracted with CH 2 Cl (3 x 300 mL). The combined organic extracts were washed with 10% HCl (3 x 200 mL), saturated NaHCO3 (3 x 200 mL) and saturated NaCl (2 x 200 mL), dried over MgSO4 and concentrated in vacuo to give 3.5 g (84%) of the new compound transient, vitamin D4 (X) tosylate with the following characteristics:

* H NMR (400 MHz, CDCb), 8 ppm 0.54 (3H, s, 18-H3), 0.78 (6H, dd, 26-H3 and 27-H3) 0.87 (3H, d, 21 -H3), 0.96 (3H, d, 28-H3), 2.45 (3H, s, CH3 (tosylate)) 4.68 (3H, m, 3-H), 4.82 (1H, m (acute), 19-H) 5.04 (1H, m, (acute), 19-H), 5.95 (1H, d, 7-H), 6.09 (1H, d, 6-H) , 7.34 and 7.79 (4H, d, aromatic).

To a mixed suspension of NaHCO3 (17.0 g, 202 mmol) in methanol (200 mL), a solution of vitamin D4 (X) tosylate (3.5 g, 6.3 mmol) in dry CH2Ck (10 mL) was added dropwise. The reaction mixture was refluxed under argon overnight, then cooled to room temperature and concentrated in vacuo to about 50 mL. The concentrate was diluted with ether (600 mL), washed with water (3 x 300 mL), dried over MgSO 4 and concentrated in vacuo. The residue was passed through a silica gel column and eluted with 10% ethyl acetate in hexane to give a new intermediate, 3,5-cyclovitamin D4 (XI) (heavy oil) in the amount of 1.5 g (58%) with the following characteristics:

'H NMR (400 MHz, CDCls), δ ppm 0.56 (3H, s, 18-H3) 0.78 (6H, dd, 26-H3 and 27-H₃), 0.87 (3H, d, 21-H3) 0.94 (3H, d, 28-H3), 3.28 (3H, s, OCH3), 4.2 (1H, d, 6-H) , 4.91 (1H, m (acute), 19-H), 4.98 (1H, d, 7-H), 5.08 (1H, m (acute), 19-H).

To a suspension of selenium dioxide (0.22 g, 2 mmol) in dry CH2Cb (150 mL) stirred in a three-necked flask was added anhydrous t-butyl hydroperoxide in toulen (3M) (2.6 mL, 7.8 mmol). The mixture was stirred for 3 hours under argon. Pyridine (0.3 mL, 3.7 mmol) was then added, followed by the addition of cyclo-vitamin D 4 (XI) (1.5 g, 3.6 mmol) as a solution in CH 2 O 2 (50 mL). After stirring for 30 minutes, a 10% aqueous NaOH solution was added

170 447 (200 ml). The reaction mixture was then diluted with ether (500 mL) and the phases separated. The organic phase was washed with 10% NaOH (3 x 200 mL), water (2 x 200 mL) and saturated NaCl solution (2 x 200 mL), dried over MgSO 4 and concentrated in vacuo. The residue was absorbed on a silica gel column and eluted with 30% ethyl acetate in hexane to give 0.45 g (29%) of a new intermediate, 1-hydroxy-3,5-cyclovitamin D4 (XII) (oil) with the following characteristics:

H NMR (400 MHz, CDCb), δ ppm 0.54 (3H, s, 18-H3) 0.78 (6H, dd, 26-H3 and 27-¾) 0.86 (3H, d, 21-H3 ), 0.95 (3H, d, 28-H3), 3.26 (3H, s, OCH3) 4.2 (1H, d, 6-H), 4.22 (1H, m, 1-H) , 4.95 (1H, d, 7-H), 5.18 (1H, dd, 19-H), 5.25 (1H, d, 19-H).

A solution of 1a-hydroxy-3,5-cyclo-vitamin D4 (XII) (0.45 g, 1.05 mmol) in a solution of dimethyl sulfoxide (4.5 mL) and glacial acetic acid (3.6 mL) was heated to 50 ° C under argon within 1 hour. The reaction mixture was then poured through ice and saturated NaHCO3 solution (100 mL) and extracted with ether (3 x 200 mL). The combined ether extracts were washed with saturated NaCl solution (3 x 200 mL), dried over MgSO 4, concentrated in vacuo to give a mixture containing 5,6-cis and 5,6-trans 1a-hydroxyvitamin D 4 (about 4: 1, by NMR method) ) with a yield of 0.4 g (92%). A mixture of 5,6-cis and 5,6-trans 1α-hydroxyvitamin D4 (0.4 g, 0.97 mmol) was dissolved in ethyl acetate (25 mL) and freshly recrystallized maleic anhydride (0.08 g, 0.8 mmol). The reaction mixture was heated to 35 ° C under argon for 24 hours. After evaporation of the solvent in vacuo, the crude mixture was chromatographed on a silica gel column using ethyl acetate and hexane (1: 1) as the eluent to give the new active form of vitamin D4, 5,6-cis 1 α-hydroxy-vitamin D 4 (XIII) in yield 90 mg (23%) with the following characteristics: mp 128 - 60 ° C; IR v_max (clean): 3400 cm '¹ (OH stretching);

Ή NMR (400 MHz, CDCb), 5 ppm 0.55 (3H, s, 18-H), 0.79 (6H, dd, 26-¾ and 27-¾) 0.87 (3H, d, 21- H3) 0.94 (3H, d, 28-H3), 4.24 (1H, m, 3-H), 4.44 (1H, m, 1-H), 5.02 (1H, n, ( acute), 19-H), 5.34 (1H, m (acute), 18-H), 6.02 (1H, d, 7-H), 6.4 (1H, d, 6-H); Mass spectrum [Ci] m / e (relative intensity): 415 (M + 1.41%) 397 (M + 1-OH 100%), 379 (27%), 135 (22%).

Example 2: Bioassay for 1a-hydroxyvitamin D4

Young freshly weaned male rats (Holtzman strain, Holtzman Company, Madison, Wisconsin) were fed a vitamin D deficient diet containing calcium (0.47%) and phosphorus (0.3%), respectively. Within 3-4 weeks, this diet caused exceptional vitamin D deficiency characterized by low serum calcium levels and poor growth. After 4 weeks on such a diet, the serum calcium content fell below 7 mg / dl. The rats were then divided into 4 groups and bi-hydroxyvitamin D4 was orally administered in a carrier such as coconut oil or carrier alone (control) daily for 14 days. 24 hours after the last dose, rats were killed and blood calcium levels were measured by standard laboratory technique. The results of these determinations are shown in Table 1.

Table 1

Increase in serum calcium

<td>Relationship</td><td>Dose (Pg / kg / day)</td><td>number rats</td><td>Serum calcium concentration (mg / dl) ± Standard deviation</td>

<td>Control test</td><td> -</td><td> 10</td><td> 6,1± 0,48</td>

<td>1 -OH-D4</td><td> 0,042</td><td> 8</td><td> 7,1±0,80</td>

<td>1 -OH-D4</td><td> 0,250</td><td> 7</td><td> 11,60 ±0,45</td>

<td>1-OH-D4</td><td> 1,500</td><td> 9</td><td> 12,7 ±0,37</td>

The data in Table 1 indicate that 1α-hydroxyvitamin D4 is effective in increasing serum calcium levels in vitamin D deficient rats and that the response is dose-dependent. Surprisingly, the level of response is favorable compared to the response reported by Wientroub et al. For 1,25-dihydroxyvitamin D 3 administered to rat

170 447 rom with vitamin D deficiency under experimental conditions similar to those described above. See, S. Wientroub, PA Price, AH Raddi, The Dichotomy in the Effects of 1,25 dihydroxy vitamin D3 and 24.25 dihydroxy vitamin D 3 on Bone Gamma-Carboxyglutamic Acid-Containing Protein in Serum Bone in vitamin D-DeficientRats, Calcif. Tissue Int. (1987) 40: 166-172.

Example 3: Toxicity tests

The acute oral toxicity of ia-OH-Dp in rats was assessed by determining the mean lethal dose (LD50) using a well-known method. Rats were fed a standard laboratory diet for 8-10 weeks. Five animals of each sex were given one oral dose of 1a-OH-D4. The animals were observed for 14 days and the number of dead animals recorded. The determined LD 50 value was approximately 1.0 mg / kg for males and 3.0 mg / kg for females.

By comparison, the LD50 value for? -Hydroxyvitamin D2 under the same conditions found by the inventors is 1.7 and 1.8 mg / kg for male and female rats, respectively. Toxicity 1 (χ-hydroxyvitamin D 2 has been previously reported to be lower than 1 α-hydroxyvitamin D3. G. Sjoden, C. Smith, U. Lindgren 1 HF DeLuca, Proc. Soc. Experimental Biol. Med. 178-432- 436 (1985).

Example 4: Preparation and isolation of 1,25-dihydroxy vitamin D4

-α-hydroxyvitamin D4 produced by the method of the present invention is incubated with human liver cells in culture that metabolize the compound to several products, including the metabolite -1,25-dihydroxyvitamin D4. The 1.25 metabolite is isolated and purified by high performance liquid chromatography and identified by gas chromatography and mass spectrometry. Binding studies show that 1,25-dihydroxyvitamin D 4 has good affinity for binding to the vitamin D receptor protein in mammals, indicating its biological activity. The procedures used are similar to those described by Strugnell et al., Biochem. Pharm. vol. 40: 333-341 (1990).

Example 5: Preparation and isolation of 1.24-dihydroxyvitamin D4

Preparation and isolation of 1,24-dihydroxyvitamin D 4 is carried out as described above in Example 4. The 1a-hydroxyvitamin D4 prepared by the method of the present invention is incubated with human liver cells in culture that metabolize the compound to several products including the metabolite 1,24-dihydroxyvitamin D4. The 1.24 metabolite is isolated and purified by high performance liquid chromatography and identified by gas chromatography and mass spectrometry. Binding studies with the new metabolite show that the metabolite has good affinity for binding to the vitamin D receptor protein in mammals, indicating that the drug is biologically active.

Example 6: Study of hypercalcemia

Female rats were fed a commercial diet containing calcium (0.8%) and phosphorus (0.6%). The rats were divided into 4 groups and each group was orally administered daily for 13 weeks with 1α-OH D4 in a carrier such as coconut oil or carrier alone (control). 24 hours after the last dose, rats were killed and serum calcium was determined by a standard method.

This procedure shows no or only slight increase in serum calcium at 1a-OH-D4 doses up to 2.5 pg / kg / day.

Example 7: Further biological studies

Young male rats weaned are fed a diet with a vitamin D deficiency and low calcium content (0.02%). After 4 weeks, the rats were divided into 4 groups and given 1a-OH D4 intravenously in a vehicle such as ethanol or vehicle alone (control). 16 hours after administration, the rats were killed and intestinal calcium transport was measured using inverted duodenal pouches, according to the method of Martin and DeLuca Am. J. Phvsiol. 216: 1352-1359.

Following the procedure, stimulation of intestinal calcium transport is demonstrated in a dose-dependent manner.

Example 8: A clinical trial is conducted with patients with postmenopausal osteoporosis, aged 55-75. The study includes up to 120 patients randomly divided into three treatment groups and are conducted for 12-24 months. Two of the treatment groups receive fixed doses of 1-vitamin D4 (uid; two different dose levels above 3.0 pg / day) and the remainder

170 Group 447 receives the appropriate placebo. All patients receive a normal dose of calcium in the diet (50θ to 800 mg / day) and do not take calcium supplements. Efficacy is assessed by comparing patient groups before and after treatment in terms of (a) bone density throughout the body, radius, femur and vertebral bone as determined by X-ray absorptiometry (DRXA), (b) iliac crest bone biopsy and (c) osteocalcin level in serum. Safety is assessed by comparing urinary excretion of hydroxyproline, serum and urine calcium levels, creatinine clearance, blood urea nitrogen and other routine determinations.

This study shows that patients treated with 1 α-vitamin D 4 have significantly higher bone density throughout the body, radial, femur and / or vertebral bones compared to patients treated with placebo. The treated patients also show significantly increased serum osteocalcin. Bone biopsy in treated patients shows that 1 ^ vitamin D4 stimulates normal bone formation. The monitored safety parameters confirm the low incidence of hypercalcaemia or excessive calcium excretion in the urine, or other metabolic disorders associated with 1 «-vitamin D4 therapy.

Example 9: Clinical trials are conducted with healthy postmenopausal women aged 55-60. The study covers up to 80 patients randomly divided into two treatment groups and is carried out for 12-24 months. One treatment group receives a fixed dose of Ια-vitamin D 4 (uid; dose level above 3.0 pg / day) and the other receives the appropriate placebo. The test is carried out as indicated above in Example 2.

This study shows that patients treated with 1a-vitamin D4 have reduced bone density throughout the body, radial, femur and / or vertebral bone compared to baseline. In contrast, placebo-treated patients show significant losses in these parameters relative to standard values. The monitored safety parameters confirm the safety of long-term administration of 1a-vitamin D4 at this dose level.

Example 10: A double-blind, placebo-controlled clinical trial was performed with 30 men and / or women with kidney disease who undergo chronic hemodialysis. All patients undergo an 8-week follow-up period during which they receive a maintenance dose of vitamin D3 (400 IU / day). After this control period, patients are randomly divided into two treatment groups: one group receives a fixed dose of 1a-vitamin D 4 (uid; dose above 3.0 pg / day) and the other group receives the corresponding placebo. Both treatment groups receive a maintenance dose of vitamin D 3, maintain a normal dose of calcium in their food, and do not take calcium supplements. Efficacy was assessed by comparing two groups of patients before and after treatment for a) direct measurements of intestinal calcium absorption, b) bone density mineralization throughout the body of radial, femoral and / or vertebral bones and c) determination of serum calcium and osteoclacin. Safety is assessed by regularly reading serum calcium levels.

Analysis of clinical data shows that 1a-vitamin D 4 significantly increases serum osteocalcin and intestinal calcium absorption as determined by single- or double-isotope technique measurements. Patients treated with this compound show normalized levels of serum calcium, with unchanged bone density values ​​throughout the body, radial, femur and / or vertebral bones relative to reference values. In contrast, placebo-treated patients often exhibit hypocalcaemia, a significant reduction in bone density throughout the body, radial, femur and / or vertebral bones. In the treated group, slight hypercalcaemia is observed.

Although the present invention has been described and illustrated by specific examples, it is obvious to those skilled in the art that various modifications, including variations, additions and omissions, can be made within the scope described.

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