WO1984004527A1

WO1984004527A1 - PROCESS FOR THE PREPARATION OF 1alpha,25-DIHYDROXYLATED VITAMIN D2 AND RELATED COMPOUNDS

Info

Publication number: WO1984004527A1
Application number: PCT/US1984/000714
Authority: WO
Inventors: Hector F Deluca; Heinrich K Schnoes; Rafal R Sicinski; Yoko Tanaka
Original assignee: Wisconsin Alumni Res Found
Priority date: 1983-05-09
Filing date: 1984-05-09
Publication date: 1984-11-22
Also published as: DK183291A; JPH02288854A; JPH0339505B2; JPH0610188B2; DK8685D0; DE3448412C2; DE3448360C2; JPH02288873A; DK172733B1; NL193245B; CH665834A5; DK183291D0; DK184588D0; JPH02288829A; DK184588A; DE3490215T; DK172567B1; NL8420137A; AU568549B2; DK171397B1

Abstract

Novel derivatives of vitamin D2 and more specifically 1alpha,25-dihydroxylated compounds of the vitamin D series. A process for the preparation of such derivatives is also provided as are certain intermediates in such process. The invention provides 1alpha,25-dihydroxyvitamin D2 derivatives and acylates thereof, which find ready application as substitutes for vitamin D3 or D2 or various of the known vitamin metabolites of these vitamins in their various applications to the correction of disorders involving calcium metabolism and associated bone disease.

Description

Process for the Preparation of lα,25-Dihydroxylated Vitamin D₂ and Related Compounds

Technical Field

This invention relates to the preparation of lα,25dihydroylated ccmpounds of the vitamin D₂ series.

More specifically, this invention relates to the preparation of lα,25-dihydroxyvitamin D₂ and its (24R)-epimer, the corresponding 5,6-trans-isorrers, and to certain C-25-alkyl or aryl analogs as well as the acyl derivatives of these compounds. Background

The importance of the hydroxylated forms of vitamin D as regulators of calcium and phosphate metabolism in animals and humans is by now well recognized through many disclosures in the patent and general literature, and as a consequence thesehydroxyvitamin D derivatives are finding increasing clinical and veterinary use as medicaments for the treatment and cure of disorders of calcium metabolism and associated bone diseases. Vitamin D₃ is known to be hydroxylated in vivo to 25-hydroxyvitamin D₃ and then to lα, 25-dihydroxyvitamin D₃ , the latter being generally accepted as the active hormonal form of vitamin D₃. Similarly, the very potent vitamin D₂ metabolite, lα,25-dihydroxyvitamin D₃ (lα,25-(OH)₂D₂) is formed from vitamin D₂ via 25-hydroxyvitamin D₂ (25-OH-D₂). Both of these hydroxylated vitamin D₃ compounds have been isolated and identified (DeLuca et al, U.S. Patents 3,585,221; 3,880,894); being derived from vitamin D₂, these metabolites are characterized by the (S) -stereochemistry at carbon 24. Disclosure of Invention

A chemical process for preparing lα,25-dihydroxylated compounds of the vitamin D₂ series has now been developed. Specifically, this process provides a convenient means for preparing compounds having the general structures A and B shown below.

wherein R₁, R₂ , and R₃ are selected from the group consisting of hydrogen and acyl, and where X is an alkyl or aryl group. In these structures the asymmetric center at carbon 24 may have the (R) or (S) configuration. Specific examples of compounds obtainable by the present process include lα,25-dihydroxyvitamin D₂, the corresponding (24R)-epimer,- lα,25-dihydroxy-24-epivitamin D₂, the respective 5,6-trans-isomers, i.e. 5,6-trans-lα,25-dihydroxyvitamin D₂, and 5,6-trans-lα,25-dihydroxy-24-epivitamin D₂, as well as the C-25-alkyl or aryl homologs of these compounds, i.e. the compounds having the formulae shown above where X is ethyl, propyl, isopropyl or phenyl.

As used herein the term "acyl" signifies an aliphatic acyl group (alkanoyl group) of from 1 to 6 carbons, in all possible isomeric forms, e.g. formyl, acetyl, butyryl, isobutyryl, valeryl, etc., or an aromatic acyl group (aroyl group) such as benzoyl, or the methyl, halo, or nitro-substituted benzcyl groups, or an acyl group derived from a dicarboxylic acid having the general formulae ROOC(CH₂)_nCO- , or ROOCCH₂-0-CH₂CO-, where n is an integer having the values of 0 to 4 inclusive, and R is hydrogen or an alkyl radical. Representative of such dicarboxylic acyl groups are oxalyl, malonyl, succinoyl, glutaryl, adipyl and diglycolyl. The term "alkyl" refers to a hydrocarbon group of 1 to 6 carbons in all isomeric forms, e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl, etc. The term "aryl" refers to an aromatic radical such as phenyl, benzyl, or the isomeric alkyl-substituted phenyl radicals.

An em bodi ment of the chemical process of this invention is depicted in appended Process Scheme I. In the following description of this process, numerals (e.g. 1, 2 , 3, etc) designating specific products refer to the structures so numbered in Process Scheme I. A wavy line to the substituent (methyl) at C-24 indicates that this substituent may have either the R or S configuration.

A suitable starting material for the process of this invention is the vitamin D-ketal derivative of structure (1) . It is generally convenient (e.g. in the case when both C-24-epimers of 1α,25-dihydroxyvitamin D₂ compounds are desired) to use compound (1) as a mixture of the 24R and S epimers, separation of the individual 24R and S-epimers being acc mplished at a later stage of the process. However, the pure 24S, or the pure 24R-epimer of (1) are equally suitable starting materials, whereby the former compound upon being processed through the indicated synthetic steps will provide the (24S)-lα,25-dihydroxy product, whereas the latter, treated analogously, will yield the corresponding (24R)-1α,25dihydroxylated product.

Starting material (1) is converted to the desired lαhydroxylated form via cyclovitamin D derivatives (DeLuca et al., U.S. patents 4,195,027 and 4,260,549). Thus, treatment of compound (1) with toluenesulfonyl chloride in the conventional manner yields the corresponding C-3-tosylate (2) , which is solvolyzed in an alcoholic medium to produce the novel 3,5-cyclovitamin D derivative (3). Solvolysis in methanol yields the cyclovitamin of structure (3) where Z=methyl, whereas the use of other alcohols, e.g. ethanol, 2-propanol, butanol, etc., in this reaction provides the analogous cyclcvitamin D ccmpounds (3) , where Z is an alkyl group derived from the alcohol, e.g. ethyl, isopropyl, butyl, etc. Allylie oxidation of intermediate (3) with selenium dioxide and a hydroperoxide yields the lα-hydroxy -analog of structure (4). Subsequent acetylation of compound (4) provides the 1-acetate of structure (5, R₁=acetyl). If desired, other 1-0-acylates (structure 5, where R₁=acyl, e.g. the formate, propionate, butyrate, benzoate, etc.) are prepared by analogous conventional acylation reactions. The 1-0-acyl derivative is then subjected to acid-catalyzed solvolysis. When this solvolysis is conducted in a solvent medium containing water, there is obtained the 5,6-cis-vitamin D intermediate of structure (6, R₁=acyl, R₂=H) and the corresponding 5,6-trans-compound (structure 7, R₁=acyl, R₂=H)) in a ratio of about 3-4:1. These 5,6-cis and 5,6-transisomers can be separated at this stage, e.g. by high performance liquid chromatography. If desired, the C-1-0-acyl group may be removed by base hydrolysis to obtain compounds (6) and (7) where R₁ and R₂=H. Also if desired, these 1-0-monoacylates may be further acylated at the C-3-hydroxy groups, using conventional acylation conditions to obtain the corresponding 1,3-di-o-acylates of structure (6) or (7) where R₁ and R₂, which may be the same or different, represent acyl groups. Alternatively, the hydroxy cyclovitamin of structure (4) can be subjected to acid-catalyzed solvolysis in a medium containing a low-molecular weight organic acid to obtain the 5,6-cis and trans compounds of structures (6) and (7) where R₁=H and R₂=acyl, where the acyl group is derived from the acid used in the solvolysis reaction.

The next step of the process comprises the removal of the ketal protecting group to produce the corresponding 25-ketone. This step is a critical one, since the ketal to ketone conversion must be acccmplished without concomitant isomerization of the 22 (23) -double bond to the conjugated 23 (24) -position, which can occur under the acidic conditions required for ketal hydrolysis. Furthermore, conditions must be chosen so as to avoid elimination of the sensitive allylie C-1-oxygen function. The conversion is accomplished successfully by careful hydrolysis at moderate temperatures using organic acid catalysis. Thus, treatment of the 5,6-cis-compound (6) in aqueous alcohol with p-toluenesulfonic acid gives the corresponding ketone (8). To avoid undesired elimination of the C-1-oxygen function during this reaction, it is advantageous that the C-1-hydroxy group in compound (6) be protected (e.g. R₁=acyl, R₂=hydrogen or acyl).

Subsequent reaction of ketone (8) with a methyl-Grignard reagent then provides the desired lα,25-dihydroxyvitamin D₂ compound of structure (9). If the starting material, compound (1), used in the above process, is a mixture of the two C-24-epimers, then compound (9) will be obtained as a mixture of the 24S_ and R-epimers (9a and 9b, respectively). Separation of this epimer mixture can be achieved by chromatographic methods, to obtain 1α,25-dihydroxyvitamin D₂ (structure 9a, 24S-stereochemistry) and its 24R-epimer, 1α , 25-dihydroxy-24-epivitamin D₂, of structure 9b, both in pure form. Such separation of epimers is, of course, not necessary if the compounds are intended to be used as a mixture.

The 5,6-trans-25-ketal-intermediate of structure (7), subjected to ketal hydrolysis in an analogous manner, provides the 5,6-trans ketone intermediate of structure (10) , which via a Grignard reaction with methyl magnesium bromide or analogous reagent gives the 5,6-trans-1α,25-dihydroxyvitamin D₂ compounds of structure (11), as the 24S or 24R-epimer, or as a mixture of both epimers depending on the nature of the starting material (1) used in the process. If obtained as an epimeric mixture, the epimers can be separated by chromatography, to obtain 5,6-trans-1α ,25-dihydroxyvitamin D₂ (11a) and its 24R-epimer, 5,6-trans-1α ,25-dihydroxy-24epivitamin D₂, of structure (11b). These reaction steps utilizing the 5,6-trans-intermediate are conducted in a manner entirely analogous to those applicable to the 5,6-ciscompounds described above. The novel side chain ketones of structures (8) or (10) are most useful and versatile intermediates in that they can be used to prepare a variety of 1α,25-dihydroxyvitamin D₂-side chain analogs. Specifically, these keto-intermediates can serve for the preparation of 5,6-cis- or 5,6-trans-1α-, 25-dihydroxyvitamin D₂ analogs having the general side chain formula shown below,

where X is an alkyl or aryl group.

For example, treatment of ketone (8) with, ethyl magnesium bromide gives the corresponding hydroxyvitamin D₂ analog having the side chain structure shown above wherein X is ethyl group. Likewise, treatment of (8) with isopropyl magnesium bromide or phenyl magnesium bromide gives the side chain analogs where X is isopropyl or phenyl, respectively. Analogous treatment of the 5,6-trans-25-ketone intermediate of structure (10) with alkyl or aryl-Grignard reagents gives the 5,6-trans-vitamin D₂ analog having the side chain above where X is the alkyl or aryl radical Introduced by the Grignard reagent employed.

It is also evident that the reaction of the keto-intermediates (8) or (10) with, an isotopically-labeled Grignard reagent (e.g. C³H₃MgBr, ¹⁴CH₃MgBr, C²H₃MgBr, etc.) provides a convenient means for preparing 1α,25-dihydroxyvitamin D₂ or its trans isomer, and the corresponding

C-24-epimers, in isotopically-labeled form, i.e. as the compounds having the side chain shown above, wherein X is C³H₃, ¹⁴CH₃, C²H₃, ¹³CH₃, or any other isotopically-labeled alkyl or aryl group selected.

The above alkyl or aryl homologues of the 5,6-cis or trans-1α,25-dihydroxy-vitamin D₂ are useful substitutes of the parent compounds in situations where a greater degree of lipophilicity is desired, whereas the isotopically labeled compounds referred to above, find use as reagent in analytical applications.

Further, although for therapeutic applications, the free hydroxy compounds represented by structures A and B above (where R₁, R₂ and R₃=H) are generally used, for some such applications, the corresponding hydroxy-protected derivatives may be useful or preferred. Such hydroxy-protected derivatives are for example the acylated compounds represented by general formulae A and B above, wherein one or more of R₁, R₂, and R₃ represents an acyl group.

Such acyl derivatives are conveniently prepared from the free hydroxy compounds by conventional acylation procedures i.e. treatment of any of the hydroxyvitamin D₂ products with an acyl halide, or acid anhydride in a suitable solvent such as pyridine, or an alkyl-pyridine. By appropriate selection of reaction time, acylating agent, temperature and solvent, as is well-known in the art, the partially or fully acylated derivatives represented by structures A or B above are obtained. For example, treatment of 1α,25-dihydroxyvitamin D₂ (9a) in pyridine solvent with acetic anhydride at room temperature gives the 1,3-diacetate, while the same reaction conducted at elevated temperature yields the corresponding 1,3,25-triacetate. The 1,3-diacetate can be further acylated at C-25 with a different acyl group; e.g. by treatrrent with benzqyl chloride or succinic anhydride there is obtained the 1,3-diacetyl-25-benzoyl-, or 1,3-diacetyl-25-succincylderivative, respectively. A 1,3,25-triacyl derivative can be selectively hydrolyzed in mild base to provide the 1,3-dihydroxy-25-O-acyl compound, the free hydroxy groups of which can be reacylated, if desired, with different acyl groups. Likewise, a 1,3-diacyl derivative can be subjected to partial acyl hydrolysis to obtain the 1-0-acyl and the 3-0-acyl cαrpounds, which in turn can be reacylated with different acyl groups. Like treatment of any of the otherhydroxyvitamin D₂ products (e.g. 9b, lla/b, or their corresponding 25-alkyl or aryl analogs) provides the corresponding desired acyl derivatives represented by structures A or B, where any or all of R₁, R₂, and R₃ are acyl.

Like the previously known vitamin D₂ metabolite, 1α, 25-dihydroxyvitamin D₂ (9a), the novel compounds of this invention exhibit pronounced vitamin D-like activity, and thus represent desirable substitutes for the kncwn vitamin D₂ or D₃ metabolites in many therapeutic, or veterinary applications. Particularly preferred in this regard are the products of structure 9b and 11a and 11b, or their acylated derivatives. The novel compounds may be used for correcting or improving a variety of calcium and phosphate imbalance conditions resulting from a variety of diseases, such as vitamin D-resistant rickets, osteomalacia, hypoparathyroidism, osteodystrophy, pseudohypoparathyroidism, osteoporosis, Paget's disease, and similar bone and irdneral-related disease states known to the medical practice. The cαrpounds can also be used for the treatment of mineral imbalance conditions in animals, for example, the milk fever condition, poultry leg weakness, or for improving egg shell quality of fcwl. Their use in the treatment of osteoporosis is particul-arly noteworthy.

It is well know that females at the time of menopause suffer a marked loss of bone mass giving rise ultimately to osteopenia, which, in turn gives rise to spontaneous crush fractures of the vertebrae and fractures of the long bones. This disease is generally known as postmencpausal osteoporosis and presents a major medical problem, both in the United States and most other coutnries where the life-span of females reaches ages of at least 60 and 70 years. Generally the disease which is often accompanied by bone pain and decreased physical activity, is diagnosed by one or two vertebral crush fractures with X-ray evidence of diminished bone mass. It is known that this disease is accompanied by diminished ability to absorb calcium, decreased levels of sex hormones, especially estrogen and androgen, and a negative calcium balance.

Methods for treating the disease have varied considerably. For example, calcium supplementation by itself has not been successful in preventing or curing the disease and the injection of sex hormones, especially estrogen, which has been reported to be effective in preventing the rapid loss of bone mass experienced in postmenopausal women, has been complicated by the fear of its possible carcinogenicity. Other treat-ments, for which variable results have again been reported, have included a combination of vitamin D in large doses, calcium and fluoride. The primary problem with this approach is that fluoride induces structurally unsound bone, called woven bone, and in addition, produces a number of side effects such as increased incidence of fractures and gastrointestinal reaction to the large amounts of fluoride administered.

Similar symptoms characterize senile osteoporosis and steroid-induced osteoporosis, the latter being a recognizied result of long term glucocorticoid (cortico-steroid) therapy for certain disease states.

While various metabolites of vitamin D₃ increase calcium absorption and retention within the body of mammals displaying evidence of or having a physiological tendency toward loss of bone mass they are also characterized by the complementary vitamin D-like characteristic of mobilizing the calcium in bone in response to physiological needs. It has now been found that the epi compounds of this invention, especially 24-epi-1α,25-dihydroxyvitamin D-₂(24-epi-1,25-(OH)₂D₂), are eminently suitable for the prevention or treatment of physiological disorders in mammals which are characterized by the loss of bone mass because, although they express some of the recognized vitamin D-like characteristics affecting calcium metabolism, such as, increasing intestinal calcium transport, and effecting bone mineralization, they do not increase serum calcium levels, even at high dosages. This observed characteristic evinces that the compounds upon administration, do not mobilize bone. This fact, along with the ability of the comounds upon admirάstration to mineralize bone, indicates that they are ideal cαrpounds for the prevention or treatment of prevalent calcium disorders which are evidenced by loss of bone mass, for example postmenopausal osteoporosis, senile osteoporosis and steroid-induced osteoporosis. It will be evident that the compounds will find ready application for the prevention or treatment of other disease states in which the loss of bone mass is an indicationsuch as in the treatment of patients undergoing renal dialysis where loss of bone mass as a consequence of the dialysis is encountered.

The following Examples will serve to illustrate the characteristics of 24-epi-1,25-(OH)₂D₂ which contribute to its eminent suitability for the prevention or treatment of disease states that evince bone mass loss. Example 1

Weanling male rats were placed on the vitamin D deficient diet described by Suda et al., Journal of Nutrition 100, 10491052 (1970), modified to contain .02% calcium and .3% phosphorus. After two weeks on this diet, the animals were given either 1,25-dihydroxyvitamin D₂, or 24-epi-1,25¬dihydroxyvitamin D₂ daily by subcutaneous injection in 0.1 ml of 5% ethanol in propanediol. Twelve hours after the last dose, the animals were killed and the blood calcium and intestinal calcium transport measured. The results of these measurements for the indicated levels of the compounds administered are shown in Figures 1 and 2. The intestinal calcium transport measurements shown in Figure 2 were performed by the method of Martin and DeLuca, American Journal of Physiology 216, 1351-1359 (1969). Example 2

Weanling male rats were placed on a high calcium (1.2% calcium) and low phosphorus (.1% phosphorus) diet described by Suda et al (supra). The rats were fed this diet for a period of three weeks at which time they were separated into two groups. One group was given 1,25(OH)₂D₂ while the other groups was given 24-epi-1,25(OH)₂D₂, both in 0.1 ml of 5% ethanol in propane diol subcutaneously at the dosage levels of the compounds shown by the data points in Figure 3. These doses were continued daily for a period of seven days, at which time the animals were killed and serum inorganic phosphorus determined. Results are shown in Figure 3.

Bone ash was determined by removing the femurs frαn rats. The femurs were dissected free of adhering connective tissue, extracted for 24 hours in absolute ethanol, and 24 hours in diethyl ether, using a Soxhlet extractor. The bones are ashed at 600°F for 24 hours. The ash weight was determined by weighing to constant weight. Results are shown in Figure 4.

The results of the two studies described in Examples 1 and 2, above, illustrate that 24-epi-1,25-(OH)₂D₂ is approximately equal in potency to 1α,25-dihydrαxyvitamin D₃ (1,25-(OH)₂D₃) in causing the mineralization of bone and in stimulating intestinal calcium transport. In short, there is no significant difference between the two groups in Figure 2 and Figure 4. On the other hand, the elevation of serum inorganic phosphorus which results from mobilization of bone in the case of the low phosphorus diet is very markedly affected by 1,25-(OH)₂D₂, but hardly stimulated by 24-epi1,25(OH)₂D₂. Similarly, in the mobilization of calcium from bone, as indicated by the serum calcium levels (Figure 1) even at the extremely high dose level of about 750 pmoles/day, the 24-epi compound had no effect, while the mobilization effect is evident at much lower doses of 1,25-dihydroxyvitamin D₂. Since the rise in serum calcium of rats on a low calcium diet measures the ability to mobilize bone, and since the elevation of blood phosphorus of animals on a low phosphorus diet also measures bone mobilization, these results show that 24-epi-1,25-(OH)₂D₂ provides an unexpected property, namely that it is of minimal effectiveness in mobilizing bone calcium, while being fully able to stimulate intestinal calcium transport and the mineralization of new bone, properties which make this compound highly suitable for the treatment of disease states that evince bone loss.

The unique characteristics of 24-epi-1,25-(OH)₂D₂, as set forth above, offer the rare opportunity to control the various vitamin D-responsive processes (intestinal calcium absorption, bone mineral mobilization, and bone mineralization) in a manner and to a degree heretofore not feasible. This possibility arises from the fact that the 24-epi compound of this invention may be administered to the manmal either alone (with suitable and acceptable exeipients) or in combination with other vitamin D-derivatives which exhibit the full spectrum of D-like activity. By such measures, it is possible therefore to combine (to whatever degree desired) the specificity of action of the 24-epi-analog with the generality of action of other vitamin D metabolites or analogs. Administration of 24-epi-1,25-(OH)₂D₂ alone will, as shown above, stimulate intestinal calcium transport and bone mineralization with no or minimal bone mineral mobilization, but the latter activity can be induced by co-administration of one or more of the known vitamin D derivatives (e.g., 1,25- (OH) ₂D₃, 1α ,25-(OH) ₂D₂, 1α-OH-D₃, and related analogs) . By adjusting the relative amounts of compounds administered, a degree of control over the relative magnitudes of the intestinal calcium absorption vs. bone mineral mobilization processes can be exercised in a manner not possible with the heretofore known vitamin D derivatives. Co-administration of the 24-epi compound and other vitamin D compounds with bone mobilizing activity can be particularly advantageous in situation where some degree of bone mobilization is desired. For example, it is believed that in certain circumstances, bone must first be mobilized before new bone can be laid down. In such situations treatment with vitamin D or a vitamin D derivative which will induce bone mobilization, e.g. lαhydroxyvitamin D₃ or -D₂, lα,25-dihydroxyvitamin D₃ orD₂, 25-hydroxyvitamin D₃ or - D₂, 24,24-difluoro-25hydroxyvltamin D₃, 24,24-difluoro-1α,25-dihydroxyvitamin D₃, 24-fluoro-25-hydroxyvitamin D₃, 24-fluoro-1α-,25dihydroxyvitamin D₃, 2β-fluoro-1α-hydroxyvitemin D₃, 2βfluoro-25-hydroxyvitamin D₃, 2β-fluoro-lor,25-dihydroxyvitamin D₃, 26,26,26,27,27,27-hexafluoro-1α ,25-dihydroxyvitamin D₃, 26,26,26,27,27,27-hexafluoro-25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, 1α,24,25-trihydroxyvitamin D₃, 25,26-dihydroxyvitamin D₃, 1α,25,26-trihydroxyvitamin D₃, in combination with 24-epi-1,25(OH)₂D₂ will, by adjustment of the proportions of the 24-epi compound and the bone-mobilizing vitamin D compound in the treatment regimen permit the rate of mineralization of bone to be adjusted to achieve the desired medical and physiological ends. Suitable and effective mixtures are for example, the combination of lα-,25-dihydroxyvitamin D₂ and 1α,25-dihydroxy-24-epivitamin D₃ (9a and 9b), or mixtures of the corresponding 5,6-trans-compounds (11a and lib), or any other combination of these four products as the free hydroxy compounds or as their acylated forms.

The compounds of this invention or cαnbinations thereof with other vitamin D derivatives or other therapeutic agents, can be readily administered as sterile parenteral solutions by injection or intravenously, or by alimentary canal in the form of oral dosages, or trans-dermally, or by suppository. Advantageously the compounds are administered in dosage amounts of from 0.1 to 100 micrograms per day. In relation to osteoporosis, doses from about 0.5 to about 25 micrograms per day are generally effective. The compounds can be administered either alone or in combination with other vitamin D derivatives, the proportions of each of the compounds in the cαrbination being dependent upon the particular disease state being addressed and the degree of bone mineralization and/or bone mobilization desired. In the treatment of osteoporosis where the preferred compound is 24-epi-1α,25-(OH)₂D₂ the actual amount of the 24-epi-compound used is not critical. In all cases, sufficient of the compound should be used to induce bone mineralization. Amounts in excess of about 25 micrograms per day of the 24-epi-compound or the combination of that compound with bone mobilization-inducing vitemin D derivatives, are generally unnecessary to achieve the desired results and may not be economically sound practice. In practice, higher doses of the compounds are used where therapeutic treatment of a disease state is the desired end while the lower doses are generally used for prophylactic purposes, it being understood that the specific dosage administered in any given case will be adjusted in accordance with the specific compounds being administered, the disease to be treated, the condition of the subject and the other relevant medical facts that may modify the activity of the drug or the response of the subject, as is well known by those skilled in the art.

Dosage forms of the compounds can be prepared by combining them with non-toxic pharmaceutically acceptable carriers as is well known in the art. Such carriers may be either solid or liquid such as, for example, corn starch, lactose, sucrose, peanut oil, olive oil, sesame oil and propylene glycol. If a solid carrier is used the dosage form of the compounds may be tablets, capsules, powders, troches or lozenges. If a liquid carrier is used, soft gelatin ceipsules, or syrup or liquid suspensions, emulsions or solutions may be the dosage form. The dosage forms may also contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, etc. They may also contain other therapeutically valuable substances such as other vitamins, salts, sugars, proteins, hormones or other medicinal compounds.

The process of the present invention is more particularly described by Examples 3 through 9 which follow. In these examples the designation of specific products by Arabic numerals (e.g. compounds 1, 2, 3, etc.) refer to the structures so numbered in Process Scheme I. Example 3

1α -hydroxy-3,5-eyclovitamin D (4, Z=methyl). A solution of compound (1) (50 mg) (as a mixture of the 24 R and S epimers) in dry pryridine (300 μl) is treated with 50 mg of p-toluenesulfonyl chloride at 4°C for 30 h. The mixture is poured over ice/sat. NaHCO₃ with stirring and the product is extracted with benzene. The combined organic phases are washed with aqueous NaHCO₃, H₂O, aqueous CuSO₄ andwater, dried over MgSO4 and evaporated.

The crude 3-tosyl derivative (2) is directly solvolyzed in anhydrous methanol (10 ml) and NaHCO₃ (150 mg) by heating at 55°C for 8.5 h with stirring. The reaction mixture is then cooled to room temperature and concentrated to～ 2 ml under vacuo. Benzene (80 ml) is then added and organic layer is washed with water, dried and evaporated. The resulting cyclovitamin (3, Z=methyl) can be used in the subsequent oxidation without further purification.

The crude product (3) in CΑ₂Cl₂ (4.5 ml) is added to an ice-cooled solution at SeO₂ (5.05 irg) and t-BuOOH (16.5 ul) in CH₂Cl₂ (8 ml) containing anhydrous pyridine (50 μl). After being stirred for 15 min at 0°C, the reaction mixture is allowed to warm to room temperature. After an additional 30 min, the mixture is transferred to a separatory funnel and shaken with 10% NaOH (30 ml). Ether (150 ml) is added and the separated organic phase is washed with 10% NaOH, water, dried and evaporated. The oily residue is purified on silica gel thin layer plates (20 x 20 cm plates, AcOEt/hexane 4:6) to yield 20 mg of 1α-hydroxy derivative (4, Z=methyl) : mass spectrum, m/e: 470 (M⁺, 5), 438 (20), 87 (100); NMR (CDCl₃) δ0.53 (3H, s, 18-H₃), 0.63 (1H, m, 3-H) , 4.19 (1H, d, J=9.5 Hz, 6-H), 4.2 (1H, m, 1-H), 4.95 (1H, d, J=9.5 Hz, 7-H), 5.17 and 5.25 (2H, each m, 19-H₂), 5.35 (2H, m, 22-H and 23-H). Example 4

Acetylation of compound (4).

A solution of cycloivitamin (4, Z=methyl) (18 mg) in pyridine (1 ml) and acetic anlydride (0.33 ml) is heated at 55°C for 2 h. The mixture is poured into ice-cooled sat. NaHCO₃, and extracted with benzene and ether. The combined organic extracts are washed with water, saturated CuSO₄ and aqueous NaHCO₃ solutions, dried and evaporated to give 1-acetoxy derivative (5, Z-methyl, acyl=acetyl) (19 mg) : mass spectrum, m/e: 512 (M⁺, 5), 420 (5), 87 (100); NMR (CDCl₃) δ0.53 (3H, s, 18-H₃), 4.18 (1H, d, J=9.5 Hz, 6-H) , 4.97 (2H, m, 7-H and 19-H), 5.24 (2H, m, 1-H and 19-H), 5.35 (2H, m, 22-H and 23-H). Example 5

Solvolysis of 1α-acetoxy-3,5-cyclovitamin (5) (R.=acetyl).

A solution of cyclovitamin (5) (4.5 mg) in 3:1 mixture of dioxane/H₂O (1.5 ml) is heated at 55°C. p-Toluenesulfonic acid (1 mg in 20 μ1 of H₂O) is then added and heating is continued for 15 min. The mixture is poured into saturated NaHCO₃/ice, and extracted with benzene and ether. The organic phases are washed with NaHCO₃ and water and dried over MgSO₄. Evaporation of solvents gives a residue containing compounds (6) (where R₁=acetyl and R₂=H) and (7) (where R₁=acetyl and R₂=H) which care separated by chromatography on HPLC (6.2 mm x 25 cm Zorbax-Sil) using 2% of 2-propanol in hexane as an eluent.. If necessary, the products are further purified by rechromatography. Example 6

Ketal hydrolysis in compound (6) to obtain ketone (8). To the solution of ketal (6, R₁=acetyl, P₂=H) (1.35 mg) in ethanol (1.5 ml), p-toluenesulfonic acid (0.34 mg in 45 uL of H₂O) is added and the mixture is heated under reflux for 30 min. The reaction mixture is poured into diluted NaHCO₃ and extracted with benzene and ether. The combined organic extracts are washed with water, dried over MgSO₄ and evaporated. High-pressure liquid chromatography of the crude mixture (4% 2-propanol/hexane, 6.2 mm x 25 cm Zorbax-Sil) affords some unreacted ketal (6) (0.12 mg, collected at 48 ml) and desired ketone (8, R₁=acetyl, R₂=H) (0.36 mg, collected at 52 ml) , characterized by the following data: mass spectrum, m/e: 454 (M⁺, 9), 394 (17), 376 (10), 134 (23), 43 (100); NMR (CDCI₃) δ0.53 (3H, s, 18-H₃), 1.03 (3H, d, J=6.5 Hz, 21-H₃) , 1.13 (3H, d, J=7.0 Hz, 28-H₃), 2.03 (3H, s, CH₃COO) , 2.12 (3H, s .CH₃CO), 4.19 (1H, m, 3-H) , 5.03 (1H, m, 19-H), 5.33 (3H, broad m, 19-H, 22-H and 23-H), 5.49 (1H, m, 1-H), 5.93 (1H, d, J=11 Hz, 7-H), 6.37 (1H, d, J=11 Hz, 6-H); UV (EtOH) λ _max 266 nm, 250 nm, λ_min 225 nm.

Example 7

Reaction of ketone (8) with methylmagnesium bromide to obtain products (9a) and (9b).

Ketone (8,. R₁=acetyl, R₂=H) in anhydrous ether is treated with the excess of CH₃MgBr (2.85 M solution in ether). The reaction mixture is stirred at room temperature for 30 min, then quenched with aq. NH₄Cl, extracted with benzene, ether and CH-C1₂. The organic phases are washed with dilute NaHCO₃, dried over MgSO₄ and evaporated. The mixture of (9a) and (9b) thus obtained is separated by high performance liquid chromatography (6% 2-propanol/hexane, 4.6 mm x 25 cm Zorbax-Sil), to obtain, in order of elution, pure epimers (9a) and (9b). 1α,25-dihydroxyvitamin D₂ (9a): UV (EtOH) λ _max

265.5 mm , λ _min 227.5 nm; mass spectrum, m/e 428 (M⁺, 6) , 410 (4) , 352 (4) , 287 (6) , 269 (10) , 251 (10) , 152 (42) , 134 (100) , 59 (99) ; NMR (CDCl₃) 50.56 (3H, s, 18-H₃) , 1.01 (3H, d, J=6.5 Hz, 28-H₃) , 1.04 (3H, d, J=6.5 Hz, 21-H₃) , 1.14 and 1.18 (6H, each s, 26-H₃ and 27-H₃) , 4.24 (1H, m, 3-H) , 4.43

(1H, m, 1-H) , 5.01 (1H, m, 19-H) , ～ 5.34 (3H, broad m, 19-H,

22-H and 23-H) , 6.02 (1H, d, J=11 Hz, 7-H) , 6.39 (1H, d, J=11

Hz, 6-H) .

1α,25-dinydroxy-24-epivitamin D₂ (9b) : UV (EtOH) λ_max 265.5 nm, λ_max. 227.5 nm; mass spectrum, m/e 428 (M⁺, 13) , 410 (9) ,

352 (7) , 287 (11) , 269 (15) , 251 (13) , 152 (52) , 134 (100) , 59

(97) .

Example 8

Conversion of compound (7) to 5,6-trans-1α,25-dihydroxyvitamin D₂ compounds (11a) and (11b).

Hydrolysis of ketel-intermediate (7, R₁=acetyl, R₂=H) using the conditions described in Example 4 provides the corresponding 5,6-trans-25-ketone of structure (10, R₁=acetyl,

R₂=H), and subsequent reaction of this ketone with methyl magnesium bromide, using conditions analogous to those of

Example 5, gives a mixture of epimers (11a) and (11b) which are separated by high performance liquid chromatography (HPLC) to obtain in pure form 1α,25-dihydroxy- 5,6-trans-vitamin D₂

(11a) and 1α,25-dihydroxy-5,6-trans-24-epivita- min D₂ (11b).

If required, structure assignment can be confirmed by isomerization to the respective 5,6 cis compounds (9a, 9b) according to known procedures.

5,6-trans-1α,25-dihydroxyvitamin D₂ (11a) : UV (EtOH) λ_max 273.5 nm,λ_min 230 nm; mass spectrum, m/e 428 (M⁺, 8), 410

(3), 287 (3), 269 (7), 251 (7), 152 (34), 134 (100), 59 (78).

5,6-trans-1α,25-dihydroxy-24-epivitamin D₂ (11b): UV

(EtOH) λ_max 273.5 rm, λ_min 230 nm; mass spectrum, m/e 428 (M⁺ 10), 410 (4), 352 (4), 287 (5), 269 (9), 251 (8), 152 (37),

134 (100), 59 (82).

Example 9

Preparation of alkyl and aryl analogs of 1α,25-dihydroxyvitamin D₂ compounds.

By reaction of ketone intermediate (8) (R₁=acetyl, R₂=H) with, respectively. (a) ethyl magnesium bromide

(b) propyl magnesium bromide

(c) isopropyl magnesium bromide

(d) butyl magnesium bromide

(e) phenyl magnesium bromide using conditions analogous to those described in Example 7, there are obtained the corresponding hydroxyvitamin D₂ products having the formula shown below

wherein X is, respectively

(a) ethyl

(b) propyl

(c) isopropyl

(d) butyl

(e) phenyl

By like treatment of 5,6-trans-ketone intemediate (10) (R₁=acetyl, R₂= H) with the above listed Grignard reagents, there are obtained the corresponding 5 ,6-trans-hydroxyvitamin D₂ products, having the formula shown below

wherein X is, respectively

(a) ethyl

(b) propyl

(c) isopropyl

(d) butyl

(e) phenyl

A suitable starting material for the process of this invention is the vitamin D-ketal derivative of structure (1) which can be obtained following Process Schemes II and III as described in British Specification No. 2,127,023 or United States Letters Patent No. 4,448,721. It is generally convenient (e.g. when both C-24-epimers are desired) to use compound (1) as a mixture of 24R and 24S epimers, separation of the individual 24R and 24£3 epimers being accomplished later. However, pure 24S-, or pure 24R-epimer of (1) are equally suitable, the former providing the 24S-1α ,25-dihydroxy product and the latter the corresponding 24R-product.

Process Scheme I

Process Scheme II

Process Scheme III

Claims

1. A compound selected from the group consisting of

wherein each of R₁, R₂ and R₃, which may be the same or different, is selected from the group consisting of hydrogen and acyl and X is selected from an alkyl or aryl group or an isotopically labeled alkyl or aryl group, with the proviso that when the C-24-methyl substituent in the 5,6-cis compound has the &-configuraticn, and X is methyl, R₁, R₂ and R₃ cannot all be hydrogen.

2. The compounds of claim 1 where X is methyl.

3. The compounds of claim 1 where the asymmetric center at C-24 has the (R)-configuration.

4. The compounds of claim 1 where the asymmetric center at C-24 has the (S)-configuration

5. 1α,25-dihydroxy-24-epivitamin D₂.

6. 1α,25-dihydroxy-5,6-trans-vitamin D₂.

7. 1α,25-dihydroxy-5,6-trans-24-epi-vitamin D₂.

8. A pharmaceutical composition which comprises a compound as claimed in any one of claims 1 to 7 and a pharmaceutically acceptable excipient.

9. A composition according to claim 8, which comprises 1α, 25-dihydroxy-5,6-trans-vitamin D₂ and/or 1α-,25-dihydroxy5,6-trans-24-epi-vitamin D₂.

10. A corrposition according to claim 8 or 9, which comprises 1α,25-dihydroxy-5,6-trans-vitamin D₂ and 1α,25-dihydroxyvitamin D₂.

11. A corrposition according to claim 8 or 9 , which comprises 1α,25-dihydroxy -5,6-trans-vitamin D₂ and 1α,25-dihydroxy5, 6-trans-24-epivitamin D₂.

12. A composition according to claim 8 or 9, which coirprises 1α,25-dihydroxy vitamin D₂ , 1α,25-dihydroxy -24-epi-vitamin D₂, 1α,25-dihydroxy -5, 6-trans-vitamin D₂ and 1α,25dihydroxy-5, 6-trans-24-epi-vitamin D₂

13. A composition according to claim 8, which comprises la, 25-dihydroxyvitamin D₂ and 1α,25-dihydroxy-24-epivitamin D₂.

14. A pharmaceutical composition which co mprises 1α,25dihydroxy-5, 6-trans-vitamin D₂ and either 1α,25dihydroxy-5, 6-trans-25-epi-vitamin D₂ or 1α,25dihydroxy-24-epi-vitamin D₂.

15. A composition according to claims 8 or 14 characterized in that it comprises at least one bone mobilizationinducing compound.

16. A composition according to claim 15 wherein the bone mobilization-inducing compound is a vitamin D derivative selected from the group consisting of 25-hydroxyvitamin D₃ , 25-hydroxyvitamin D₂, 1α-hydroxyvitemin D₃ , 1αhydroxyvitamin D₂, 1α,25-dihydroxyvitamin D₃ , 1α,25dihydroxyvitamin D₂, 24,24-difluoro-25-hydroxyvitamin D₃ , 24,24-difl uoro-1α,25-dihydroxy vitamin D₃ , 24-fluoro25-hydroxyvitamin D₃, 24-fluoro-1α,25-dihydroxy vita min D₃ , 26,26,26,27,27,27-hexafluoro-1α,25-dihydroxy vitamin D₃, 26,26,26,27,27,27-hexafluoro-25-hydroxyvitamin D₃ , 2β-fluoro -1α--hyd roxyvitamin D₃, 2β-fluoro-25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, 1α ,24,25-trihydroxyvitamin D₃, 25,26-dihydroxyvitamin D₃ , 1α ,25, 26-tr ihydroxyvitamin D₃.

17. Compounds having the formula

wherein Y is hydrogen, hydroxy or O-acyl and Z is an alkyl group.

18. The compounds of claim 1 wherein Y is hydrogen.

19. The compounds of claim 1 wherein Y is hydroxy or O-acetyl.

20. The compound of claim 2 where Z is methyl.

21. The compound of claim 3 where Z is methyl.

22. A compound selected from the group consisting of

where K is an oxygen or ethylenedioxy group, and where R₁ andR₂ which may be the same or different, are hydrogen or acyl.

23. The compounds of claim 5 where K is an oxygen group.

24. The compounds of claim 5 where K is an ethylenedioxy group.

25. 1α-hydroxy-25-oxo-27-nor vitamin D₂ and the acetate thereof.

26. A process for preparing a compound having a formula as defined in claim 1 wherein each of R₁, R₂ and R₃, which may be the same or different, is hydrogen or acyl and X is alkyl or aryl or an isotopically labelled alkyl or aryl group which comprises subjecting a ketal of the formula:

wherein R₁ and R₂ are as defined above to hydrolysis at a temperature from 50 to 100°P (10 to 38°C) under acidic conditions and reacting the resulting ketone with a Grignard reagent.

27. A process according to claim 26 wherein the hydrolysis is carried out using p-toluene-sulfonic acid.

28. A method for preventing or treating physiological disorders in mammals, which disorders are characterized by a requirement to regenerate or prevent loss of bone mass, which comprises administering to said mart-rials a therapeutically effective amount of 1α,25-dihydroxy-24epi vitamin D₂, alone or in combination with at least one vitamin D compound characterized by its ability to mobilize bone in vivo.

29. The method of claim 1 wherein the disorder is postmenopausal osteoporosis.

30. The method of claim 1 wherein the disorder is senile osteoporosis.

31. The method of claim 1 wherein the disorder is steroidinduced osteoporosis.

32. The method of claim 2 wherein the compound is administered to women during and subsequent to menopause.

33. The method of claim 2 wherein the cαrpound is administered to women prior to the onset of menopause.

34. The method of claim 1 wherein the compound is administer ed in an amount from about 0.5 microgram to about 25 micrograms per day.

35. The method of claim 1 wherein the compound, in solution in a liquid vehicle ingestible by and nontoxic to said mammals is administered orally in encapsulated form.

36. The method of claim 1 wherein 1α,25-dihydroxy-24-epi vitamin D₂ is the sole compound administered.

37. The method of claim 1 wherein 1α,25-dihydroxy-24-epi vitamin D₂ is administered in combination with at least one vitamin D cxπpound characterized by the ability to mobilize bone in vivo.

38. 1α-dihydrox-25-oxo-27-nor-24-epivitamin D₂.