Methods for preparation of 1 alpha, 24-dihydroxy vitamin D2

Abstract: 1∝,24-dihydroxy vitamin D 2 which is useful as an active compound of pharmaceutical compositions for the treatment of disorders of calcium metabolism and for various skin disorders. The invention also includes preparation of synthetic 1∝,24-dihydroxy vitamin D 2 starting from ergosterol which is converted in six steps to 24-hydroxyergosterol. 24-hydroxyergosterol is irradiated and thermally converted to 24-hydroxy vitamin D 2 which is converted in five steps to 1∝,24-dihydroxy vitamin D 2 . The syntheses also produce novel intermediates.

Description: This is a divisional of European patent application no 92904947. This invention relates to methods for preparation of hormonally active, natural metabolite 1α,24 dihydroxy vitamin D 2 and to intermediate compounds produced in the course of that preparation. This invention also relates to a method of preparing a pharmaceutical composition which includes a pharmaceutically effective amount of 1α,24-dihydroxy vitamin D 2 . Vitamin D and its active metabolites are known to be important in regulating calcium metabolism in animals and humans. The naturally occurring form of vitamin D in animals and humans is vitamin D 3 . It has been shown that in animals, including humans, vitamin D 3 is activated by being hydroxylated in the C 25 position in the liver, followed by 1α-hydroxylation in the kidney to produce the hormone 1α,25-dihydroxy vitamin D 3 ["1,25-(OH) 2 D 3 "]. See, U.S. Patent No. 3,880,894. The major physiological pathway for catabolism of the vitamin D 3 metabolites, 25-hydroxy vitamin D 3 and 1,25-(OH) 2 D 3 is initiated by C 24 -oxidation. Holick, M.F., Kleiner-Bossallier, A., Schnoes, H.K., Kasten, P.M., Boyle, I.T., and DeLuca, H.F., J. Biol. Chem. , 248, 6691-6696 (1973). Vitamin D 2 is the major, naturally occurring form of vitamin D found in plants. Vitamin D 2 differs structurally from vitamin D 3 in that vitamin D 2 has a methyl group at C 24 and has a double bond between C 22 and C 23 . Shortly after their discovery, it seemed apparent that vitamin D 3 and vitamin D 2 had similar, if not equivalent, biological activity. It has also been commonly believed that the metabolism (i.e., the activation and catabolism) of vitamin D 2 was the same as for vitamin D 3 . See , Harrison's Principles of Internal Medicine: Part Seven, "Disorders of Bone and Mineral Metabolism: Chap. 35," in E. Braunwald, K.J. Isselbacher, R.G. Petersdorf, J.D. Wilson, J.B. Martin and H.S. Fauci (eds.), Calcium, Phosphorus and Bone Metabolism: Calcium Regulating Hormones, McGraw-Hill, New York, pp. 1860-1865. In this regard, the active form of vitamin D 2 is believed to be 1α,25-dihydroxy vitamin D 2 ["1,25-(OH) 2 D 2 "]. Further, 24-hydroxy derivatives of 25-hydroxy vitamin D 2 and 1,25-(OH) 2 D 2 , that is, 24,25-dihydroxy vitamin D 2 and 1α,24,25-trihydroxy vitamin D 2 , are known, suggesting that catabolism of vitamin D 2 , like vitamin D 3 , proceeds through the same C 24 oxidation step. Jones, G., Rosenthal, D., Segev, D., Mazur, Y., Frolow, F., Halfon, Y., Robinavich, D. and Shakked, Z., Biochemistry , 18:1094-1101 (1979). It has recently been found, however, that an active analogue of vitamin D 2 , 1α-hydroxy vitamin D 2 ["1α-(OH)D 2 "] has pharmacological properties distinctly different than those exhibited by its vitamin D 3 counterpart, 1α-hydroxy vitamin D 3 ["1α-(OH)D 3 "]. EP-A-0381750 discloses that 1α-(OH)D 2 will reverse the loss of bone mass in human osteoporotic patients when administered at dosages of 2.0 µg/day or higher. Because of toxicity, dosage levels of 2.0 µg/day or greater are not safely obtained with 1α-(OH)D 3 . Such distinct pharmacological properties may be explained by the present inventors' discovery that pharmacological dosages of 1α-(OH)D 2 administered to humans are metabolized in part to biologically active 1α,24-dihydroxy vitamin D 2 ["1α,24-(OH) 2 D 2 "]. As explained in more detail below, the hydroxylation at the 24 carbon position of the 1-hydroxylated vitamin D 2 molecule, represents an activation pathway peculiar to the vitamin D 2 molecule. While 1α,24-dihydroxy vitamin D 3 ["1α,24-(OH) 2 D 3 "] has been chemically synthesized (U.S. Patent No. 4,022,891) it has not been demonstrated to be a natural compound in biological systems. Furthermore, the present inventors have discovered that 1α,24-(OH) 2 D 2 has distinctly different biological activity from that exhibited by 1α,24-(OH) 2 D 3 . For example, Ishizuka et al., have found that 1α,24-(OH) 2 D 3 binds the 1,25-(OH) 2 D 3 receptor site more tightly than does 1,25-(OH) 2 D 3 itself. Ishizuka, S., Bannai, K., Naruchi, T. and Hashimoto, Y., Steroids , 37:1,33-42 (1981); Ishizuka, S., Bannai, K., Naruchi, T. and Hashimoto, Y., Steroids , 39:1,53-62 (1982). Using a similar assay, the present inventors have discovered that the 1α,24-(OH) 2 D 2 is two-fold less competitive in binding the 1,25-(OH) 2 D 3 receptor site than is 1,25-(OH) 2 D 3 . The present inventors have also found that 1α,24-(OH) 2 D 2 shows a relatively poor binding affinity for the vitamin D serum binding protein of blood which is evidence of a rather short half life indicative of low toxicity. The present inventors have demonstrated the presence of circulating 1α,24-(OH) 2 D 2 in humans administered 1α-(OH)D 2 . This indicates that in animals and man, vitamin D 2 is naturally metabolized to both 1,25-(OH) 2 D 2 and 1α,24-(OH) 2 D 2 . The relative ratios of the two vitamin D 2 hormones appear to vary according to the precursor and the amount of precursor presented to the C 24 pathway. Thus it appears that as dosages of 1α-(OH)D 2 are increased, the ratio of 1α,24-(OH) 2 D 2 to 1,25-(OH) 2 D 2 increases. These results which are presented in more detail below, indicate that 1α,24-(OH) 2 D 2 has the desirable characteristic of high biological activity with low toxicity. The fact that 1α,24-(OH) 2 D 2 is a significant metabolite when pharmacological levels of 1α-(OH)D 2 are administered indicates that 1α,24-(OH) 2 D 2 may be mediating the desirable pharmacological effects of 1α-(OH)D 2 and is a useful therapeutic drug for treating various types of disorders involving calcium metabolism. The invention provides a method for the preparation of synthetic 1α,24-(OH) 2 D 2 which is a bioactive form of vitamin D 2 . The hormone may also be referred to as 1α,24-dihydroxy ergocalciferol and is represented by the structure given hereinafter. The compound has potent biological activity and rapid systemic clearance, indicating low toxicity. In summary, the method of producing 1α,24 dihydroxy vitamin D 2 entails using ergosterol as a starting material, forming the 24-hydroxy vitamin D 2 and then, 1α-hydroxlyating the 24-hydroxy compound. In the course of this synthesis, novel intermediates are also produced. The compound obtainable by the method of the invention is useful in the treatment of various diseases characterized by vitamin D deficiency and various bone depletive disorders, in particular, treatment without the concomitant incidence of hypercalcemia or hypercaluria. The compounds obtainable by the method of the invention are advantageously used as active compounds of pharmaceutical compositions for vitamin D deficiency diseases, for reversing or preventing the loss of bone mass or bone mineral content in persons predisposed to developing such loss, and for stabilizing bone density in persons suffering from renal osteodystrophy. The compound obtainable by the method of the invention is also useful in topical agents for treatment of certain skin disorders. The compound is advantageously used as an active ingredient for topical compositions which may also include other agents capable of ameloriating skin disorders. Other advantages and a fuller appreciation of the specific adaptations, compositional variations, and physical and chemical attributes of the present invention will be gained upon an examination of the following detailed description of the invention, taken in conjunction with the accompanying drawings. The present invention will hereinafter be described in conjunction with the appended drawings, wherein like designations refer to like elements throughout and in which: Figure 1 illustrates preparative steps for the synthesis of 24-hydroxy vitamin D 2 ; and Figure 2 illustrates preparative steps for the synthesis of 1α,24 dihydroxy vitamin D 2 starting with 24-hydroxy vitamin D 2 . The present invention provides a method for the preparation of synthetic 1α,24-dihydroxy vitamin D 2 [1α,24-(OH) 2 -D 2 ]. As used herein, the terms "biological activity", "biologically active", "bioactive", or "biopotent" are meant to refer to biochemical properties of compounds such as affecting metabolism, e.g., affecting serum calcium concentration, or binding to an appropriate receptor protein, e.g., binding to vitamin D receptor protein. The invention involves the preparation of a compound of formula (I). In one aspect the invention provides a method of preparing 1α,24 dihydroxy vitamin D 2 , comprising: (a) acetylating ergosterol to form its 3β-acetate; (b) reacting with a triazoline dione and ozonating to form the 22-oxo-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)23,24-dinor-6-cholene-3β-yl acetate; (c) adding 3-methylbutan-2-one to 22-oxo-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)23,24-dinor-6-cholene-3β-yl acetate to form (22E)5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl) cholesta-6,22-diene-24-one-3β-yl acetate; (d) adding methylmagnesium bromide to (22E)5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2- diyl) cholesta-6,22-diene-24-one-3β-yl acetate to form (22E)-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)-6,22-ergostadiene-3β,24-diol; (e) reducing the (22E)-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)-6,22-ergostadiene-3β,24-diol to form 24-hydroxy ergosterol; (g) irradiating 24-hydroxyergosterol to form 24-hydroxy vitamin D 2 ; (h) tosylating 24-hydroxy vitamin D 2 in the presence of dry pryridine to form 24-hydroxy vitamin D 2 3β-tosylate; (i) solvolyzing 24-hydroxy vitamin D 2 tosylate to form 24-hydroxy-3,5 cyclovitamin D 2 ; (j) allylically oxidizing the 24 hydroxy-3,5 cyclovitamin D 2 with selenium dioxide to form 1α,24 dihydroxy cyclovitamin D 2 ; and (k) hydrolyzing the 1α,24 dihydroxy 3,5 cyclovitamin D 2 with a mixture of dimethylsulfoxide and an organic acid to form an admixture of the 5,6 cis 1α-hydroxy and 5,6 trans 1α,24 dihydroxy vitamin D 2 and forming a Diels-Alder adduct of the 5,6 trans 1α-hydroxy vitamin D 2 to allow purification to yield 1α,24 dihydroxy vitamin D 2 . In a second aspect the invention provides a method of preparing 24 hydroxy vitamin D 2 , comprising: (a) acetylating ergosterol to form its 3β-acetate; (b) reacting with a triazoline dione and ozonating to form the 22-oxo-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)23,24-dinor-6-cholene-3β-yl acetate; (c) adding 3-methylbutan-2-one to 22-oxo-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)23,24-dinor-6-cholene-3β-yl acetate to form (22E)5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2- diyl) cholesta-6,22-diene-24-one-3β-yl acetate; (d) adding methylmagnesium bromide to (22E)5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl) cholesta-6,22-diene-24-one-3β-yl acetate to form (22E)-5α,8α- (4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)-6,22-ergostadiene-3β,24-diol; (e) reducing the (22E)-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)-6,22-ergostadiene-3β,24-diol to form 24-hydroxy ergosterol; and (g) irradiating 24-hydroxyergosterol to form 24-hydroxy vitamin D 2 . In a third aspect, the present invention provides a method of preparing 1α,24 dihydroxy vitamin D 2 , comprising: (a) tosylating 24-hydroxy vitamin D 2 to form 24-hydroxy vitamin D 2 tosylate; (b) methanolyzing the 24-hydroxy vitamin D 2 tosylate to form 24-hydroxy 3,5 cyclovitamin D 2 ; (c) oxidizing the 24-hydroxy 3,5 cyclovitamin D 2 to form 1α,24-dihydroxy-3,5-cyolovitamin D 2 ; and (d) sequentially hydrolyzing and subjecting to a Diels-Alder reaction the 1α,24-dihydroxy-3,5 cyclovitamin D 2 to form 1α,24-dihydroxy vitamin D 2 . In other aspects, the invention provides synthetic intermediates, and methods for their production, as set out in the appended claims. The intermediates are obtainable in the course of carrying out the methods of the first, second, and third aspects. In a further aspect, the invention provides a method for the production of a medicinal agent for treating a vitamin D deficiency induced bone depletive disease in a mammal, comprising preparing 1 α, 24 dihydroxy vitamin D 2 by a method of the first or third aspect, and mixing said 1 α, 24 dihydroxy vitamin D 2 with a pharmaceutically acceptable vehicle. Synthesis of 1α,24-dihydroxy vitamin D 2 is accomplished according to the schema presented in Figures 1 and 2. As seen in Figure 1, the synthesis uses ergosterol as the starting material. Ergosterol is converted to 24-hydroxyergosterol (5,7,22 ergostatriene-3β,24-diol (7)) by a six-step process. The 24-hydroxy ergosterol is then irradiated and thermally converted by methods well known in the art to yield 24-hydroxy vitamin D 2 . As seen in Figure 2, 24-hydroxy vitamin D 2 is then hydroxylated in a six-step process to yield 1α,24 dihydroxy vitamin D 2 , using a procedure similar to that described by Paaren, et al., J. Org. Chem. , vol. 45, p. 3253 (1980). Specifically, ergosterol is acetylated to form the 3β-acetate (2). An adduct (3) is then formed with the B-ring of the ergosterol structure by reaction of the 3β-acetate with a triazoline dione. The adduct (3) is then ozonated to truncate the side chain to form a C-21 aldehyde (4). The side chain is reestablished by reaction of the resulting aldehyde with the appropriate keto-compound to yield the 24-enone (5). The enone is then converted to the 24-methyl, 3β,24-dihydroxy adduct (6). This adduct is then reacted with a lithium aluminum hydride to deprotect the adduct and yield 24-hydroxy ergosterol (7). The 24-hydroxy ergosterol is then irradiated and thermally treated to form 24-hydroxy vitamin D 2 . The 24-hydroxy vitamin D 2 is then tosylated to yield 3β-tosylate of the 24-hydroxy vitamin D 2 . The tosylate is displaced by solvolysis to yield the 6-methoxy-24-hydroxy-3,5-cyclo vitamin D 2 . The cyclovitamin D 2 is subjected to allylic oxidation to form the 1α, 24-dihydroxy cyclovitamin derivative. The 1α,24 dihydroxy cyclovitamin derivative is sequentially solvolyzed and subjected to a Diels-Alder type reaction which removes the 6-methoxy group and separates the 1α,24 dihydroxy vitamin D 2 (5,6 cis) from the 5,6 trans 1α,24 dihydroxy vitamin D 2 . The compound obtainable by the methods of the invention is applicable to various clinical and veterinary fields, and is particularly useful for the treatment of abnormal metabolism of calcium and phosphorus. Specifically, the compound of formula (I) is intended to be used, for example, to stimulate osteoblastic activity, as measured by serum levels of osteocalcin. Osteocalcin is one of the major proteins in the bone matrix. The compound of formula (I) binds to the vitamin D serum binding protein more weakly than does 1,25-(OH) 2 D 3 , indicative of rapid clearance and low toxicity, which enhances its pharmaceutical properties. The compound obtainable by the methods of the invention may be used for controlling calcium metabolism, such as for treating abnormal calcium metabolism caused, e.g., by liver failure, renal failure, gastrointestinal failure, etc. The compounds of formula (I) can be used to treat prophylactically or therapeutically vitamin D deficiency diseases and related diseases, for example, renal osteodystrophy, steatorrhea, anticonvulsant osteomalacia, hypophosphatemic vitamin D-resistant rickets, osteoporosis, including postmenopausal osteoporosis, senile osteoporosis, steroid-induced osteoporosis, and other disease states characteristic of loss of bone mass, pseudodeficiency (vitamin D-dependent) rickets, nutritional and malabsorutive rickets, osteomalacia and osteopenias secondary to hypoparathyroidism, post-surgical hypoparathyroidism, idiopathic hypothyroidism, pseudoparathyroidism, and alcoholism. 1α,24 dihydroxy vitamin D 2 is also of value for the treatment of hyperproliferative skin disorders such as psoriasis, eczema, lack of adequate skin firmness, dermal hydration, and sebum secretion. The compound of formula (I) is useful as an active compound in pharmaceutical compositions having reduced side effects and low toxicity as compared with the known analogs of active forms of vitamin D 3 , when applied, for example, to diseases induced by abnormal metabolism of calcium. 1α,24 dihydroxy vitamin D2 obtainable by the methods of the invention can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, e.g., mammals including humans. For example, the compound of formula (I) can be employed in admixtures with conventional excipients, e.g., pharmaceutically acceptable carrier substances suitable for enteral (e.g., oral), parenteral, or topical application which do not deleteriously react with the active compound. Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils (e.g., almond oil, corn oil, cottonseed oil, peanut oil, olive oil, coconut oil), mineral oil, fish liver oils, oily esters such as Polysorbate 80, polyethylene glycols, gelatine, carbohydrates (e.g., lactose, amylose or starch), magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be sterilized and, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or one or more other active compounds, for example, vitamin D 3 and its 1α-hydroxylated metabolites, conjugated estrogens or their equivalents, anti-estrogens, calcitonin, biphosphonates, calcium supplements, cobalamin, pertussis toxin and boron. For parenteral application, particularly suitable are injectable, sterile solutions, preferably oily or aqueous solution, as well as suspensions, emulsions, or implants, including suppositories. Parenteral administration suitably includes subcutaneous, intramuscular, or intravenous injection, nasopharyngeal or mucosal absorption, or transdermal absorption. Ampoules are convenient unit dosages. For enteral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, lozenges, powders, or capsules. A syrup, elixir, or the like can be used if a sweetened vehicle is desired. For topical application, suitable nonsprayable viscous, semi-solid or solid forms can be employed which include a carrier compatible with topical application and having a dynamic viscosity preferably greater than water, for example, mineral oil, almond oil, self-emulsifying beeswax, vegetable oil, white soft paraffin, and propylene glycol. Suitable formulations include, but are not limited to, creams, ointments, lotions, solutions, suspensions, emulsions, powders, liniments, salves, aerosols, transdermal patches, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, demulsifiers, wetting agents, etc. A cream preparation in accordance with the present invention suitably includes, for example, mixture of water, almond oil, mineral oil and self-emulsifying beeswax; an ointment preparation suitably includes, for example, almond oil and white soft paraffin; and a lotion preparation suitably includes, for example, dry propylene glycol. Topical preparations of the compound useful for the treatment of skin disorders may also include epithelialization-inducing agents such as retinoids (e.g., vitamin A), chromanols such as vitamin E, β-agonists such as isoproterenol or cyclic adenosine monophosphate (cAMP), antiinflammatory agents such as corticosteroids (e.g., hydrocortisone or its acetate, or dexamethasone) and keratoplastic agents such as coal tar or anthralin. Effective amounts of such agents are, for example, vitamin A about 0.003 to about 0.3% by weight of the composition; vitamin E about 0.1 to about 10%; isoproterenol about 0.1 to about 2%; cAMP about 0.1 to about 1%; hydrocortisone about 0.25 to about 5%; coal tar about 0.1 to about 20%; and anthralin about 0.05 to about 2%. For rectal administration, the compound is formed into a pharmaceutical composition containing a suppository base such as cacao oil or other triglycerides. To prolong storage life, the composition advantageously includes an antioxidant such ascorbic acid, butylated hydroxyanisole or hydroquinone. For treatment of calcium metabolic disorders, oral administration of the pharmaceutical compositions is preferred. Generally, the compound of this invention is dispensed by unit dosage form comprising about 0.5 µg to about 25 µg in a pharmaceutically acceptable carrier per unit dosage. The dosage of the compound generally is about 0.01 to about 1.0 µg/kg/day, preferably about 0.04 to about 0.3 µg/kg/day. For topical treatment of skin disorders, the dosage of the compound in a topical composition generally is about 0.01 µg to about 10 µg per gram of composition. It will be appreciated that the actual preferred amounts of active compound in a specific case will vary according to the efficacy of the specific compound employed, the particular compositions formulated, the mode of application, and the particular situs and organism being treated. For example, the specific dose for a particular patient depends on the age, body weight, general state of health and sex, on the diet, on the timing and mode of administration, on the rate of excretion, and on medicaments used in combination and the severity of the particular disorder to which the therapy is applied. Dosages for a given host can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the subject compounds and of a known agent, such as by means of an appropriate conventional pharmacological protocol. The compound can also be advantageously used in veterinary compositions, for example, feed compositions for domestic animals to treat or prevent hypocalcemia. Generally, the compound is dispensed in animal feed such that normal consumption of such feed provides the animal about 0.01 to about 1.0 µg/kg/day. The following examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. In the following examples proton nuclear magnetic ( 1 H NMR) spectra were recorded with a Bruker AM--400(400 MHz) with aspect 3000 Computer in CDCl 3 solutions with CHCl 3 as an internal standard. Chemical shifts are reported in ppm. Ultraviolet spectra were recorded with a Hitachi U-2000 Spectrophotometer and are reported for ethanol solutions. Example 1: Generation, purification and identification of 1α,24-(OH) 2 D 2 in human liver cells incubated with 1α-(OH)D 2 Substantially pure 1α-(OH)D 2 was obtained from Bone Care International, Inc. of Madison, Wisconsin. The 1α-(OH)D 2 was cultured with Hep 3B cells, derived from a human hepatoma using known methods in the art. Lipid extracts were generated by known methods in the art and were subjected to high pressure liquid chromatography (HPLC) on Zorbax-S1L developed with hexane/isopropanol/methanol (91:7:2). The putative 1α,24-(OH) 2 D 2 metabolite eluted between the parent 1α-(OH)D 2 and standard 1,25-(OH) 2 D 2 (also obtained from Bone Care International, Inc. of Madison, Wisconsin). The 1α,24-(OH) 2 D 2 was further purified by this HPLC system before the metabolite's identification was undertaken using mass spectrometry analysis. The purified metabolite was more polar than the starting material, 1α-(OH)D 2 and thus was tentatively concluded to be a dihydroxy vitamin D 2 metabolite. This metabolite also possessed the vitamin D chromophore, indicating retention of the cis-triene system of vitamin D. Since the metabolite was derived from 1α-(OH)D 2 , its structure was thus 1α,X-(OH) 2 D 2 . The trimethylsilyl-derivative of the 1α,X-(OH) 2 D 2 was prepared according to known methods in the art and mass spectrometry was performed on the TMS-derivative and the native compound. The TMS-derivative was analyzed by GC-MS, and the identification was mainly derived from interpretation of the fragmentation pattern of the pyro-metabolite. The molecular ion possessed a m/z of 644 indicating a dihydroxyvitamin D 2 with addition of three TMS groups accounting for 216 units of additional mass. Since 1α-(OH)D 2 has 3β- and 1α- groups and the putative metabolite had one additional hydroxyl, all three hydroxyls were thus derivatized. Distinctive fragments were found at m/z 601, 511, 421, 331 representing loss of a 43 mass unit of fragment alone or in addition to one, two or three TMS groups of 90 units each. This pattern was most likely explained by cleavage of the C-24 to C-25 bond loss of C 3 H 7 accounting for 43 mass units. This represents loss of the C 26 -C 25 -C 27 fragment. Furthermore, the mass spectrum lacked the m/z 131 fragment characteristic of all 25-hydroxylated vitamin D compounds. The mass spectrum showed the m/z 513 fragment indicating loss of 131 mass units due to A-ring cleavage with loss of C 2 -C 3 -C 4 also characteristic of vitamin D compounds. The mass spectrum also contained m/z 143 which was probably derived from C-24 to C-23 cleavage and a loss of a methyl group. The unusual loss of 43 units indicating C 24 -C 25 fragility coupled with the loss of a fragment due to C 23 -C 24 cleavage indicated that the extra hydroxyl in 1α,X-(OH) 2 D 2 was at carbon-24. Thus, the structure was identified as 1α,24-(OH) 2 D 2 . The native metabolite was analyzed by direct probe mass spectrometry. This analysis was consistent with a hydroxyl in the 24 position, and was also consistent with the GC-MS analysis of the TMS-derivative described above. The native metabolite showed the expected molecular ion at m/z 428 and a distinctive fragment at m/z 367, indicating the loss of one water and the C 25 -C 26 -C 27 fragment of 43 mass units. Example 2: Synthesis of 1α,24 dihydroxy vitamin D 2 (22E)-5,7,22-ergostatriene-3β-yl acetate (2) To a solution of 50 gm (0.13 mol) of ergosterol ( 1 ) in 300 ml of anhydrous pyridine was added 33.3 ml (0.35 mol) of acetic anhydride. The mixture was stirred at room temperature overnight and then 600 ml of water was added. The precipitate was filtered and washed three times with 200 ml portions of acetonitrile and then air dried to yield 42.0 g (74%) of ( 2 ). 22-oxo-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)23,24-dinor-6-cholene-3β-yl acetate (4) To a solution of 33.0 g (0.075 mol) of ergosterol acetate ( 2 ) in 1000 ml of chloroform was added 13.2 g (0.075 mol) of 4-phenyl-1,2,4-triazoline-3,5-dione. The solution of the thus formed ( 3 ) was stirred at room temperature for 30 min. and then 5 ml of pyridine was added. The solution was cooled to -78°C and treated at -78°C with an ozone-oxygen mixture for 2 hours and then thoroughly purged with nitrogen. Then 50 ml of dimethylsulfide was added and the mixture was washed with 300 ml of water, then twice with 200 ml of 2 N HCl and finally 300 water. The organic layer was separated, dried over anhydrous MgSO 4 and concentrated to dryness in vacuo . The residue was purified on a silica gel column using 30% ethyl acetate in hexane to yield 16.0 g (39%) of the title compound as a foamy solid. 1 H NMR: (400 MHz; CDCl 3 ): δppm 0.85 (3H, s , 18-CH 3 ), 1.10 (3H, s , 19-CH 3 ), 1.15 (3H, d , 21-CH 3 ), 1.99 (3H, s , 3β-CH 3 CO), 5.45 (1H, m , 3α-H), 6.26 (1H, d . 7-H), 6.40 (1H, d , 6-H), 7.42 (5H, m , Ph), 9.58 (1H, d , H CO). (22E)5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl) cholesta-6,22-diene-24-one-3β-yl acetate (5) Butyllithium (1.6M solution in hexane 8.94 ml, 0.014 mol) was added to a stirred, cooled (0°C) solution of diisopropylamine (1.45 g, 0.014 mol) in dry tetrahydrofuran (20 ml) under nitrogen. 3-Methylbutan-2-one (1.23 g, 0.014 mol) in dry tetrahydrofuran (6 ml) was added dropwise at 0°C over 15 min. The solution was stirred at 0°C for 1 hr. more, then cooled to -70°C and a solution of the aldehyde ( 4 ) (6.0 g, 0.011 mol) in dry tetrahydrofuran (60 ml) was added. The temperature was raised to -20°C and kept at this temperature for 3 hrs. Then glacial acetic acid (20 ml) was added at -20°C and the solution was brought to room temperature. Ether (800 ml) and water (400 ml) were added and the organic layer was separated and washed with 10% hydrochloric acid (2x300 ml), saturated sodium bicarbonate solution (2x300 ml), and water (2x300 ml). Concentration gave the crude product (7.5 g) which was dissolved in tetrahydrofuran (100 ml) containing 1.5 N -hydrochloric acid (12 ml). After refluxing for 1.5 hrs., the mixture was diluted with ether (600 ml), washed with a 5% sodium carbonate solution (2x200 ml) and water (2x200 ml), and dried (anhyd. MgSO 4 ). Concentration under reduced pressure gave the crude product (7.0 g). Chromatography over silica gel (50% ethyl acetate in hexane) gave the enone ( 5 ) 4.0 g (59%). 1 H NMR: (400 MHz): δppm 0.83 (3H, s . 18-CH 3 ), 0.99 (3H, s , 19-CH 3 ), 1.09 (6H, dd , 26 and 27-CH 3 ), 1.12 (3H, d , 21-CH 3 ), 2.0 (3H, s , 3β-CH 3 CO), 2.84 (1H, m , 25-H), 5.45 (1H, m , 3α-H), 6.06 (1H, d , 23-H), 6.24 (1H, d , 7-H), 6.39 (1H, d , 6-H), 6.71 (1H, dd , 22-H), 7.42 (5H, m , Ph). (22E)-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)-6,22-ergostadiene-3β,24-diol (6) The enone ( 5 ) (3.5 g, 5.7 mmol) in dry ether (100 ml) was cooled to 0°C and methylmagnesium bromide (3.0 M solution in ether 6.8 ml, 0.02 mol) was added dropwise. After 1 hr. at 0°C, saturated ammonium chloride (100 ml) was added. The organic layer was separated. The aqueous layer was extracted with ether (2x200 ml). The combined ether phases were dried over anhydrous MgSO 4 and concentrated to dryness in vacuo to yield the crude product 3.0 g (90%) of ( 6 ). (22E)-5,7,22-ergostatriene-3β,24-diol (7) To a solution of 3.0 g (5.1 mmol) of ( 6 ) in dry tetrahydrofuran (250 ml) was added 3.6 g (0.09 mol) of lithium aluminum hydride. The mixture was heated under reflux for 3 hrs., cooled with ice water bath and reaction mixture decomposed by the cautious dropwise addition of ice water (5 ml). The mixture was filtered and the filtrate was concentrated in vacuo to remove most of the tetrahydrafuran. The residue was dissolved in 200 ml of ethyl acetate and washed twice with saturated NaCl solution (2x200 ml), dried over anhyd. MgSO 4 and concentrated in vacuo. The residue was purified on a silica gel column using 30% ethyl acetate in hexane to yield 1.5 g (71%) of ( 7 ). 1 H NMR: (400 MHz, CDCl 3 ): δppm 0.64 (3H, s , 18-H), 0.88 (6H, dd , 26 and 27-CH 3 ), 0.93 (3H, s , 19-CH 3 ), 1.06 (3H, d , 21-CH 3 ), 1.19 (3H, s , 28-CH 3 ), 3.55 (1H, m , 3α-H), 5.36 (1H, d , 7-H), 5.42 (2H, m , 22 and 23-H), 5.52 (1H, d , 6-H). UV (ethanol) λ max : 282 nm. 24-hydroxyvitamin D 2 (8) One gram (2.4 mmol) of ( 7 ) was dissolved in 250 ml of ether and benzene (4:1) and irradiated with stirring under nitrogen in a water-cooled quartz immersion well using a Hanovia medium-pressure UV lamp for 2 hrs. The solution was concentrated in vacuo , redissolved in 100 ml of ethanol and heated under reflux overnight. The solution was concentrated to dryness in vacuo and the residue was purified on a silica gel column using 30% ethyl acetate in hexane to yield 0.55 g (55%) of ( 8 ). 1 H NMR: (400 MHz, CDCl 3 ): βppm 0.57 (3H, s , 18-CH 3 ), 0.92 (6H, dd , 26 and 27-CH 3 ), 1.06 (3H, d , 21-CH 3 ), 1.20 (3H, s , 28-CH 3 ), 3.93 (1H, m , 3-H), 4.79 (1H, m (sharp), 19-H), 5.01 (1H, m , (sharp), 19-H), 5.43 (2H, m , 22 and 23-H), 6.02 (1H, d , 7-H), 6.22 (1H, d , 6-H). UV (ethanol) λ max : 265 nm. 24-hydroxyvitamin D 2 tosylate (9) To a solution of 0.55 g (1.3 mmol) of ( 8 ) dissolved in 5 ml of anhydrous pyridine was added 0.6 g (3.2 mmol) of tosyl chloride. The mixture was stirred under nitrogen at 5°C for 20 hrs. The reaction mixture was poured into 100 ml of cold saturated NaHCO 3 solution and extracted with ether (3x100 ml). The combined organic extracts were washed with 5% HCl solution (2x200 ml) saturated sodium bicarbonate solution (2x200 ml) and saturated NaCl solution (2x200 ml), dried over anhyd. MgSO 4 and concentrated in vacuo to yield 0.62 g (84%) of ( 9 ). 1 H NMR: (400 MHz, CDCl 3 ): δppm 0.57 (3H, s , 18-CH 3 ), 0.92 (6H, dd , 26 and 27-CH 3 ), 1.08 (3H, d , 21-CH 3 ), 1.24 (3H, s , 28-CH 3 ), 2.43 (3H, s , CH 3 (tosylate)), 4.69 (1H, m , 3-H), 4.77 (1H, m , (sharp), 19-H), 5.0 (1H, m , (sharp), 19-H), 5.42 (2H, m , 22 and 23-H), 6.03 (1-H, d , 7-H), 6.25 (1-H, d , 6-H) 7.31 and 7.83 (4H, d , aromatic). 24-hydroxy-3,5-cyclovitamin D 2 (10) To a solution of 0.6 g (1.06 mmol) of ( 9 ) dissolved in 50 ml of anhydrous methanol was added sodium bicarbonate 4.0 (0.047 mol). The mixture was heated at reflux for 6 hrs. The reaction mixture was concentrated in vacuo . Water (100 ml) was added followed by extraction with ether (2x200 ml). The combined ether extracts were dried over anhydrous MgSO 4 and concentrated to dryness in vacuo to yield 450 mg (100%) of ( 10 ) as an oil. 1α,24-dihydroxy-3,5-cyclovitamin D 2 (11) tert-Butyl hydroperoxide (870 µl (2.61 mmol); 3 M in toluene) was added to a suspension of 73 mg (0.66 mmol) of selenium dioxide in 50 ml of anhydrous dichloromethane under nitrogen. The mixture was stirred at room temperature under nitrogen for 3 hrs. Then 0.1 ml of anhydrous pyridine was added followed solution of 450 mg (1.06 mmol) of ( 10 ) dissolved in 15 ml of anhydrous dichloromethane. The mixture was stirred under nitrogen at room temperature for 10 min. then 25 ml of 10% NaOH solution was added and the mixture was extracted with ether (3x100 ml). The combined ether extracts were washed with 10% NaOH solution (2x100 ml), water (2x100 ml), saturated sodium chloride solution (2x100 ml), dried over anhydrous MgSO 4 and concentrated to dryness in vacuo. The residue was purified on a silica gel column using a mixture of 30% ethyl acetate in hexane to yield 110 mg (24%) of ( 11 ). 1 H NMR: (400 MHz, CDCl 3 ) : δppm, 0.55 (3H, s , 18CH 3 ), 0.90 (6H, dd , 26 and 27-CH 3 ), 1.03 (3H, d , 21-CH 3 ), 1.19 (3H, s , 28-CH 3 ), 3.25 (3H, s , -OCH 3 ), 4.19 (1H, d , 6-H), 4.19 (1H, m , 1-H), 4.92 (2H, d , 7-H), 5.15 (1H, m , (sharp), 19-H), 5.2 (1H, m , (sharp), 19-H), 5.42 (2H, m , 22 and 23-H). 5,6-cis and 5,6-trans-1α,24-dihydroxyvitamin D 2 (12, 13) 1α,24-dihydroxy-3,5-cyclovitamin D 2 ( 11 ) 110 mg (0.25 mmol) was dissolved in 2.0 ml of dimethylsulfoxide and 1.5 ml of acetic acid and heated at 50°C under nitrogen for 1 hr. The solution was poured over ice and 50 ml of saturated NaHCO 3 solution. The mixture was extracted with ether (3x100 ml). The combined ether extracts were washed with saturated NaHCO 3 solution (3x100 ml), water (2x100 ml), saturated NaCl solution (2x200 ml), dried over anhyd. MgSO 4 and concentrated in vacuo to yield the crude product 100 mg (93%) of ( 12 ) and ( 13 ). 5,6-cis-1α,24-dihydroxyvitamin D 2 (12) To a solution of ( 12 ) and ( 13 ) in 5 ml of ethyl acetate was added 20 mg (0.2 mmol) of maleic anhydride and the mixture was stirred at 35°C for 24 hrs. under nitrogen. The solution was concentrated to dryness in vacuo . The residue was purified on a silica gel column using 50% ethyl acetate in hexane to yield 20 mg (22%) of ( 12 ). 1 H NMR: (400 MHz, CDCl 3 ): δppm 0.57 (3H, s , 18-CH 3 ), 0.89 (6H, dd , 26 and 27-CH 3 ), 1.04 (3H, d , 21-CH 3 ), 1.21 (3H, s , 28-CH 3 ), 4.23 (1H, m , 3-H), 4.40 (1H, m , 1-H), 5.0 (1H, m , (sharp), 19-H), 5.33 (1H, m , (sharp), 19-H), 5.44 (2H, m , 22 and 23-H), 6.01 (1H, d , 7-H), 6.37 (1H, d , 6-H). UV (ethanol) λ max : 265 nm. While the present invention has now been described and exemplified with some specificity, those skilled in the art will appreciate the various modifications, including variations, additions, and omissions, that may be made in what has been described. Accordingly, it is intended that these modifications also be encompassed by the present invention and that the scope of the present invention be limited solely by the broadest interpretation that lawfully can be accorded the appended claims.

Claims: A method of preparing 1α,24 dihydroxy vitamin D 2 , comprising: (a) acetylating ergosterol to form its 3β-acetate; (b) reacting with a triazoline dione and ozonating to form the 22-oxo-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)23,24-dinor-6-cholene-3β-yl acetate; (c) adding 3-methylbutan-2-one to 22-oxo-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)23,24-dinor-6-cholene-3β-yl acetate to form (22E)5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl) cholesta-6,22-diene-24-one-3β-yl acetate; (d) adding methylmagnesium bromide to (22E)5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2- diyl) cholesta-6,22-diene-24-one-3β-yl acetate to form (22E)-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)-6,22-ergostadiene-3β,24-diol; (e) reducing the (22E)-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)-6,22-ergostadiene-3β,24-diol to form 24-hydroxy ergosterol; (g) irradiating 24-hydroxyergosterol to form 24-hydroxy vitamin D 2 ; (h) tosylating 24-hydroxy vitamin D 2 in the presence of dry pryridine to form 24-hydroxy vitamin D 2 3β-tosylate; (i) solvolyzing 24-hydroxy vitamin D 2 tosylate to form 24-hydroxy-3,5 cyclovitamin D 2 ; (j) allylically oxidizing the 24 hydroxy-3,5 cyclovitamin D 2 with selenium dioxide to form 1α,24 dihydroxy cyclovitamin D 2 ; and (k) hydrolyzing the 1α,24 dihydroxy 3,5 cyclovitamin D 2 with a mixture of dimethylsulfoxide and an organic acid to form an admixture of the 5,6 cis 1α-hydroxy and 5,6 trans 1α,24 dihydroxy vitamin D 2 and forming a Diels-Alder adduct of the 5,6 trans 1α-hydrox vitamin D 2 to allow purification to yield 1α,24 dihydroxy vitamin D 2 . A method of preparing 1α,24 dihydroxy vitamin D 2 , comprising: (a) tosylating 24-hydroxy vitamin D 2 to form 24-hydroxy vitamin D 2 tosylate; (b) methanolyzing the 24-hydroxy vitamin D 2 tosylate to form 24-hydroxy 3,5 cyclovitamin D 2 ; (c) oxidizing the 24-hydroxy 3,5 cyclovitamin D 2 to form 1α,24-dihydroxy-3,5-cyclovitamin D 2 ; and (d) sequentially hydrolyzing and subjecting to a Diels-Alder reaction the 1α,24-dihydroxy-3,5 cyclovitamin D 2 to form 1α,24-dihydroxy vitamin D 2 . A method of producing (22E)-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)cholesta-6,22-diene-24-one-3β-yl acetate comprising reacting 22-oxo-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)23,24-dinor-6-cholene-3β-yl acetate with 3-methylbutan-2-one in the presence of butyllithium and dry tetrahydrofuran. A method of producing (22E)-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)-6,22-ergostadiene-3β,24-diol, comprising reacting 22-oxo-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)23,24-dinor-6-cholene-3β-yl acetate with a Grignard reagent. A method of producing 24-hydroxy ergosterol, comprising reacting (22E)-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)-6,22-ergostadiene-3β,24-diol with lithium aluminum hydride in the presence of dry tetrahydrofuran. A method of producing 24-hydroxy vitamin D 2 tosylate, comprising reaction 24-hydroxy vitamin D 2 with toluenesulfonyl chloride in the presence of dry pyridine. A method of producing 6-methoxy-24-hydroxy-3,5-cyclovitamin D 2 , comprising solvolyzing 24-hydroxy vitamin D 2 tosylate in methanol. A method of producing 6-methoxy-1α,24-dihydroxy-3,5-cyclovitamin D 2 , comprising allylically oxidizing 6-methoxy-24-hydroxy-3,5-cyclovitamin D 2 with selenium dioxide. A method for the production of a medicinal agent for treating a vitamin D deficiency induced bone depletive disease in a mammal, comprising preparing 1 α, 24 dihydroxy vitamin D 2 by a method of claim 1 or claim 2, and mixing said 1 α, 24 dihydroxy vitamin D 2 with a pharmaceutically acceptable vehicle. (22E)5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl) cholesta-6,22-diene-24-one-3β-yl acetate. (22E)-5α,8α-(4-Phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)-6,22-ergostadiene-3β,24-diol. (22E)-5,7,22-Ergostatriene-3β,24-diol. 24-Hydroxy ergosterol. 24-Hydroxy vitamin D 2 tosylate. 6-Methoxy-24-hydroxy-3,5-cyclovitamin D 2 . A method of preparing 24 hydroxy vitamin D 2 , comprising: (a) acetylating ergosterol to form its 3β-acetate; (b) reacting with a triazoline dione and ozonating to form the 22-oxo-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl) 23,24-dinor-6-cholene-3β-yl acetate; (c) adding 3-methylbutan-2-one to 22-oxo-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)23,24-dinor-6-cholene-3β-yl acetate to form (22E)5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidie-1,2- diyl) cholesta-6,22-diene-24-one-3β-yl acetate; (d) adding methylmagnesium bromide to (22E) 5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl) cholesta-6,22-diene-24-one-3β-yl acetate to form (22E)-5α,8α- (4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)-6,22-ergostadiene-3β,24-diol; (e) reducing (22E)-5α,8α-(4-phenyl-3,5-dioxo-1,2,4-triazolidine-1,2-diyl)-6,22-ergostadiene-3β,24-diol to form 24-hydroxy ergosterol; and (g) irradiating 24-hydroxyergosterol to form 24-hydroxy vitamin D 2 . A method according to claim 1 or claim 16 wherein said step of irradiating 24-hydroxyergosterol to form 24-hydroxy vitamin D 2 is carried out in an diethyl ether/benzene solvent.

Inventors: BISHOP CHARLES W, JONES GLENVILLE, KNUTSON JOYCE C, MORIARTY ROBERT M, PENMASTA RAJU, STRUGNELL STEPHEN, BISHOP, CHARLES W., JONES, GLENVILLE, KNUTSON, JOYCE C., MORIARTY, ROBERT M., PENMASTA, RAJU, STRUGNELL, STEPHEN
Applicants: BONE CARE INT INC, BONE CARE INTERNATIONAL, INC.
Application year: 1992
Main classification: A61K31
IPC codes: C07C401, C07J71, C07J9