IE841149L - 1, 25-dihydroxylated vitamin d2 compounds and intermediates - Google Patents

Abstract

Compounds of formula <IMAGE> where K is oxygen or ethylene dioxy and R1 and R2 are H or acyl are used as intermediates in production of 1 alpha ,25-dihydroxy Vitamin D2 compounds. [GB2158443A]

Description

58277 PATENTS ACT, 1964 COMPLETE SPECIFICATION la,2 5-DIHYDROXYLATED VITAMIN D2 COMPOUNDS AND INTERMEDIATES IN THE PREPARATION THEREOF "*~U SPECIFICATION application tto.

ION FJLED 'l J WISCONSIN ALUMNI RESEARCH FOUNDATION, a corporation organised and existing under the laws of the State of Wisconsin, United,States of America, of 614, North Walnut Street, Madison, Wisconsin 53705, United States of America. 58277 - lfV- la,25-DIHYDROXYLATED VITAMIN D2 COMPOUNDS AND INTERMEDIATES IN THE PREPARATION THEREOF This invention relates to the preparation of la, 25-dihydroxylated compounds of the vitamin D2 series, especially la,25-dihydroxyvitamin D^ and its (2^R)-epimer, the corresponding 5,6-trans-isomers, and certain C-25-5 alkyl and aryl analogs and acyl derivatives thereof.

The importance of the hydroxylated forms of vitamin D as regulators of calcium and phosphate metabolism in animals and humans is now well recognized and, as a consequence, these hydroxyvitamin D derivatives are finding 10 increasing clinical and veterinary use as medicaments for the treatment and cure of disorders of calciiam metabolism and associated bone diseases. Vitamin D^ is known to be hydroxylated in vivo to 25-hydroxyvitamin D^ and then to la;25-dihydroxyvitamin D^, the latter being generally 15 accepted as the active hormonal form of vitamin D^.

Similarly, the very potent vitamin D2 metabolite, la,25-dihydroxyvitamin D2 (la, 25-(OH)2D2) is formed from vitamin D2 via 25-hydroxyvitamin D2 (25-OH-D2). Both of these hydroxylated vitamin D2 compounds have been isolated and 20 identified; being derived from vitamin D2< these metabolites are characterized by the (S)-stereochemistry at carbon 2k.

A chemical process for preparing la,25-dihydroxylated vitamin D2 compounds having the general 25 structures A and B wherein R^, R2, and R^ are idependently hydrogen or acyl, and X is alkyl or aryl has been found. In these structures the asymmetric center at carbon 2k may have the (R) or (S) configuration.

Specific examples of compounds obtainable by 5 the process include la,25-dihydroxyvitamin D2/ the corresponding (24R)-epimer, la,25-dihydroxy-24-epivitamin D2# the respective 5,6-trans-isomers, i.e. 5,6-trans-la , 25-dihydroxyvitamin D2, and 5,6-trans-la, 25-dihydroxy-24-epivitamin as well as the C-25-alkyl or aryl 10 homologs of these compounds, e.g. where X is ethyl, propyl, isopropyl or phenyl.

As used herein the term "acyl" signifies aliphatic acyl (alkanoyl group) of from 1 to 6 carbons, in all possible isomeric forms, e.g. formyl, acetyl, butyryl, 15 isobutyryl or valeryl, or aromatic acyl (aroyl group) such as benzoyl, or methyl, halo, or nitro-substituted benzoyl, or an acyl group derived from a dicarboxylic acid having the general formulae R00C(CH2)nC0-, or R0CXXH2-0-CH2C0-, where n is 0 or an integer from 1 to k, and R is hydrogen or an 20 alkyl radical, such as oxalyl, malonyl, succinoyl, glutaryl, adipyl or diglycolyl. The term "alkyl" refers to a hydrocarbon group of 1 to, say, 6 carbons in all isomeric forms, e.g. methyl, ethyl, propyl, isopropyl, butyl or isobutyl. The term "aryl" refers to an aromatic 25 radical such as phenyl, benzyl, or the isomeric alkyl-substituted phenyl radicals.

An embodiment of the process of this invention is depicted in Process Scheme I. In the following description, numerals (e.g. 1, 2, 3, etc) refer to the 30 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 35 structure (1) which can be obtained following Process Schemes II and III as described in British Specification No. 2127023 to which reference should be made for further details. It is generally convenient (e.g. when both C-2k- - 3 - epimers are desired) to use compound (1) as a mixture of the 24r and S epimers, separation of the individual 24R and S-epimers being accomplished later. However, pure 24s, or pure 24R-epimer of (1) are equally suitable, the 5 former providing the (24S)-lot,25-dihydroxy product, and the latter the corresponding (24R) product.

Starting material (1) can be converted to the desired loc-hydroxylated form via cyclovitamin D derivatives (see, e.g. U.S. patents 4,195,027 and 4,260,549). Thus, 10 treatment of compound (1^ with toluenesulfonyl chloride in the conventional manner yields the corresponding C-3-tosylate (.2) < which can be solvolyzed in an alcoholic medium to produce the novel 3,5-cyclovitamin D derivative (3). Solvolysis in methanol yields the cyclovitamin (3) 15 where Z=methyl, whereas the use of other alcohols, e.g. ethanol, 2-propanol or butanol, in this reaction provides the analogous compounds (3.), where Z is the alkyl group derived from the alcohol, e.g. ethyl, isopropyl or butyl. Allylic oxidation of intermediate (3.), typically with 20 selenium dioxide and a hydroperoxide, yields the la- hydroxy-analog (4), acetylation of which provides the 1-acetate (f>, R^acetyl). If desired, other 1-0-acylates (structure 5_, where R^=acyl, e.g. the formate, propionate, butyrate or benzoate) can be prepared by analogous 25 conventional acylation reactions. Compound (J5) can then be subjected to acid-catalyzed solvolysis. When this solvolysis is conducted in a solvent medium containing water, there is generally obtained the 5,6-cis-vitamin D intermediate (6, R^=acyl, R2=H) anc* the corresponding 30 5,6-trans-compound (2» Rj^acyl, R2=H) in a ratio of about 3-4:1. These 5,6-cis and 5,6-trans-isomers can be separated at this stage, e.g. by high pressure liquid chromatography. If desired, the C-l-0-acyl group may be removed by base hydrolysis to obtain compounds (6) and (J) 35 where R^ 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-0-acylates of structure (6) or (2) where Rj and R2, which may be the same or different, represent acyl groups. Alternatively, the hydroxy cyclovitamin (4) can be subjected to acid-catalyzed 5 solvolysis in a medium containing a low-molecular weight organic acid to obtain the 5,6-cis and trans compounds (€>) and (2) where R^=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 10 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 accomplished without concomitant isomerization of the 22(23)-double bond to the conjugated 23(24)-position, which can occur under the 15 acidic conditions required for ketal hydrolysis.

Furthermore, conditions must be chosen so as to avoid elimination of the sensitive allylic C-l-oxygen function. The conversion can be accomplished successfully by careful hydrolysis at moderate temperatures using acid catalysis. 20 In general from room temperature to the boiling point of the solvent, especially from 50° to 100°F (10 to 38°C) may be used. Suitable acid catalysts include organic acids such as a haloacetic acid, formic acid or a low molecular weight organic sulfonic acid including p-toluene sulfonic 25 acid which is especially preferred. Thus, treatment of the 5,6-eis-compound (6) in aqueous alcohol with p-toluene-sulfonic acid gives the corresponding ketone (8). To avoid undesired elimination of the C-l-oxygen function during this reaction, it is advantageous that the C-l-hydroxy 30 group in compound (6) be protected (e.g. as R^=acyl, R2=hydrogen or acyl).

Subsequent reaction of ketone (8)with a methyl-Grignard reagent then provides the desired la,25-dihydroxyvitamin D2 compound (9). If the starting material, 35 compound (1), 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 - 5 - epimer mixture can be achieved by chromatographic methods, to obtain la,25-dihydroxyvitamin D2 (9®' 24s-stereochemistry) and its 24R-epimer, la,25-dihydroxy-24-epivitamin D2, of structure 9b, both in pure form. Such 5 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 (2) can be subjected to ketal hydrolysis in an analogous manner, to give the 5,6-trans ketone intermediate 10 (10), which via a Grignard reaction with methyl magnesium bromide or analogous reagent, as with the cis intermediate, gives the 5,6-trans-la,25-dihydroxyvitamin D2 compounds (11), as the 24s or 24R-epimer, or as a mixture of both epimers depending on the nature of the 15 starting material (1.). Again the epimers can be separated by chromatography, to obtain 5,6-trans-la,25-dihydroxyvitamin D2 (11a) and its 24R-epimer, 5,6-trans-la , 25-dihydroxy-24-epi vitamin D2 (lib).

The novel side chain ketones (8) and (10) are 20 most useful and versatile intermediates in that they can be used to prepare a variety of la,25-dihydroxyvitamin D2~ side chain analogs. Specifically, they can serve for the preparation of 5,6-cis- or 5,6-trans-la,25-dihydroxyvitamin D2 analogs having the general side chain formula shown 25 below. where X is an alkyl or aryl group. For example, treatment 30 of ketone (8) with ethyl magnesium bromide gives the corresponding hydroxyvitamin D2 analog having this side chain wherein X is ethyl. Likewise, treatment of (8) with isopropyl magnesium bromide or phenyl magnesium bromide gives side chain analogs where X is isopropyl or phenyl, 35 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- 3 14 5 labeled Grignard reagent (e.g. C H^MgBr, CH^MgBr, C^H^MgBr, etc.) provides a convenient means for preparing la,25-dihydroxyvitamin D2 or its trans isomer, and the corresponding C-24-epimers, in isotopically-labeled form, i.e. as with the side chain shown above, wherein X is 10 c^H3» ^"*CH , or any other isotopically- labeled alkyl or aryl group.

The above alkyl or aryl homologues of the 5,6-cis or trans-la,25-dihydroxy-vitamin D2 are useful substitutes for the parent compounds where a greater 15 degree of lipophilicity is desired, whereas the isotopically labeled compounds find use as reagents in analytical applications.

Further, although for therapeutic applications the free hydroxy compounds of structures A and B above 20 where R-^, R2 and R^=H are generally used, for some applications the corresponding hydroxy-protected derivatives may be useful or preferred. Such hydroxy-protected derivatives include the acylated compounds of general formulae A and B, wherein one or more of R^, R2 and 25 R^ represents an acyl group.

Such acyl derivatives are conveniently prepared from the free hydroxy compounds by conventional acylation procedures, e.g. treatment with an acyl halide, or acid anhydride in a suitable solvent such as pyridine, or an 30 alkyl-pyridine. By appropriate selection of reaction time, acylating agent, temperature and solvent, as is well-known in the art, partially or fully acylated derivatives can be obtained. For example, treatment of la,25-dihydroxyvitamin D2 (9a) in pyridine solvent with 35 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; thus treatment with benzoyl chloride or succinic anhydride gives the 1,3-diacetyl-25-benzoyl-, or 1,3-diacetyl-25-succinoyl- derivative, respectively. A 1,3,25-triacyl derivative can be selectively hydrolyzed 5 in mild base to provide the l,3-dihydroxy-25-0-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 10 3-0-acyl compounds, which in turn can be reacylated with different acyl groups. Like treatment of any of the other hydroxyvitamin D2 products (e.g. 9b, lla/b, or their corresponding 25-alkyl or aryl analogs) provides the corresponding acyl derivatives. 15 Like the known vitamin D2 metabolite, la,25- dihydroxyvitamin D2 (9a), the novel compounds of this invention especially the products of structure 9b-2^R and lla-24S and llb24R, or their acylated derivatives, exhibit pronounced vitamin D-like activity, and thus 20 represent desirable substitutes for known vitamin D2 or metabolites in many therapeutic or veterinary applications. The compounds may be used for correcting or improving a variety of calcium and phosphate imbalance conditions resulting from a variety of diseases, such as 25 vitamin D-resistant rickets, osteomalacia, hypoparathyroidism, osteodystrophy, pseudohypoparathyroidism, osteoporosis, Paget's disease, and similar bone and mineral-related disease states known to the medical practice. The compounds can also be used 30 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 fowl. Their use in the treatment of osteoporosis is particularly noteworthy. 35 It is well known that females at the time of menopause suffer a marked loss of bone mass giving rise to spontaneous crush fractures of the vertebrae and fractures of the long bones. This disease is generally known as postmenopausal osteoporosis and presents a major medical problem. The disease which is often accompanied 5 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 10 estrogen and androgen, and a negative calcium balance.

Methods for treating the disease have varied considerably but to date no really satisfactory treatment is yet known. For example, calcium supplementation by itself has not been successful in preventing or curing the 15 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 treatments, for which 20 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 25 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 30 being a recognizied result of long term steroid (corticosteroid) therapy for certain disease states.

While various metabolites of vitamin increase calcium absorption and retention within the body of mammals displaying evidence of or having a physiological tendency 35 toward loss of bone mass they are also characterized by the complementary vitamin D-like characteristic of mobilizing - 9 - the calcium in bone in response to physiological needs. It has now been found that the epi compounds of this invention, especially 24-epi-loc, 25-dihydroxyvitamin (24-epi-l,25-(OH)2D2), are eminently suitable for the 5 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 calciiam 10 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 moblize bone. This fact, along with the ability of the compounds upon administration to 15 mineralize bone, indicates that they are ideal compounds 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 20 the compounds will find ready application for the prevention or treatment of other disease states in which the loss of bone mass is an indication such as in the treatment of patients undergoing renal dialysis where loss of bone mass as a consequence of the dialysis is 25 encountered.

The unique characteristics of 24-epi-l,25-(OH)2^2 offer the rare opportunity to control mineralization of bone through combination with other of the known vitamin D derivatives which function upon 30 administration to mobilize bone. For example, it is believed that in certain circumstances, such as in the healing of bone fractures, 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 35 will induce bone mobilization, e.g. la-hydroxyvitamin or -D2# 1,25-dihydroxyvitamin D3 or -D2,25-hydroxyvitamin or -D2/ 2h,24-difluoro-25-hydroxyvitamin D^, - 10 - 2k,24-difluoro-la,25-dihydroxyvitamin D^/ 24-fluoro-25-hydroxyvitamin D^24-fluoro-l,25-dihydroxyvitamin , la, 25-dihydroxy-2(3-fluorovitamin D^, 26,26,26,27,27,27-hexafluoro-la,25-dihydroxyvitamin D^, 26,26,26,27,27,27-5 hexafluoro-25-hydroxyvitamin , in combination with 24-epi-(OH)2^2# or other 24-epi compound of this invention, 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 10 of bone to be adjusted to achieve the desired medical and physiological ends.

The compounds of this invention, or combinations thereof with other vitamin D derivatives or other therapeutic agents, specifically la,25-dihydroxy vitamin 15 D2 and la,25-dihydroxy-24-epi-vitamin D2 (9a and 9b) or of the corresponding 5,6-trans-compounds (11a and lib), can be readily administered as sterile parenteral solutions by injection or intravenously or by alimentary canal in the form of oral dosages, or by suppository. 20 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 corn starch, lactose, sucrose, peanut oil, olive oil, sesame oil and 25 propylene glycol. If a solid carrier is used the dosage form of the compounds may be pills, tablets, capsules, powders, troches or lozenges, for example. If a liquid carrier is used, soft gelatin capsules, or syrup or liquid suspensions, emulsions or solutions in innocuous solvents 30 and oils may be the dosage form. The dosage forms may also contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents or solution promoters.

They may also contain other therapeutically valuable substances such as other vitamins, salts, sugars, proteins 35 or hormones.

Advantageously, the compounds of this invention are administered in dosage amounts of 0.1 or 0.5 to 100 ng - 11 - per day, it being understood, of course, that the specific dosage administered in any given case will be adjusted in accordance with the specific compound administered, the disease to be treated, the condition 5 of the subject and 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.

In relation to osteoporosis doses of from one-tenth microgram to one microgram per day of 24-epi-l, 10 25-(OH)2^2 per se are generally effective. Although 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 one microgram per day of the 24-epi-compound or the 15 combination of that compound with bone mobilization- inducing vitamin D derivatives, are generally unnecessary to achieve the desired results and may not be economically sound practice. In practice the higher doses are used where therapeutic treatment of a disease state is the 20 desired end while the lower doses are generally used for prophylactic purposes.

The following Examples further illustrate the present invention.

Example 1 25 la-hydroxy-3,5-cyclovitamin D (4, Z=methyl).

A solution of compound (1) (50 mg) (as a mixture of the 24r and s epimers) in dry pyridine (300 ul) is treated with 50 mg of p-toluenesulfonyl chloride at b°C for 30h. 12 obtained the corresponding 5,6-trans-hydroxyvitamin products, having the formula shown belo-' (b) propyl (c) isopropyl (d) butyl (e) phenyl Example 8 Weanling male rates were placed on the vitamin D deficient diet described by Suda et al., Journal of Nutrition 100, 1049-1052 (1970), modified to contain .02% calcium and .3% phosphorus. After two weeks on this diet, the animals were given the indicated level of vitamin D compound, either 1,25-dihydroxyvitamin D2, or 24-epi-l, HOo wherein X is, respectively (a) ethyl 5 13 25-dihydroxyvitamin D^ daily by subcutaneous injection in 0.1 ml of 5% ethanol in propanediol. Twelve hours after the last dose, the animals were killed and blood calcium taken and intestinal calcium transport measured. The results, shown in Table 1 and Figure 1, represent the blood calcium measurements, and the results in Table 2 and Figure 2 illustrate the intestinal calcium transport measurements performed by the method of Martin and DeLuca, American Journal of Physiology 216, 1351-1359 (1969).

Corrpound Table 1 Dosage Serum Ca (mg/lOOml) 1,25-(OH)2 7p mol/day 20p 70p 325p 650p 3.6 4.2 4.4 5.2 7.9 24-epi-l, 25- (OH) ?P nol/day 3.5 3.6 3.5 3.6 3.7 20p 70p 325p 650p - Ik -Table 2 Compound Dosage Intestinal calcium-transport (ca serosal/ Ca mucosal) l,25-(OH)2D2 6p mol/day 5.0 5.2 5.3 5.9 6.2 13p " 20p " 30p " HOp " 24-epi-l,25-(OH)2D2 6p mol/day 3.0 3.4 3."6 5.0 4.9 13p " 20p " 30p " lOOp " Example 9 Weanling male rats were placed on a high calcium (1.2% calcium) and lew 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 given the indicated doses of the compounds shown in Table 3 in 0.1 ml of 5% ethanol in propanediol subcutaneously. 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 shewn in Table 3 and Figure 3.

Bone ash was determined by removing the femurs from 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 Table 4 and Figure 4.

Compound l,25-(OH)2D2 - 15 -Table 3 Dosage 30p nol/day 70p 140p 325p 650p Serum Inorganic Phos-phorous (mg/100 ml) 3.4 3.3 4.1 5.5 5.3 24-epi-l/25-(OH)2D2 Compound l,25-(OH)2D2 30p mol/day 70p 325p 650p " Table 4 Dosage 65p mol/day 35 Op 750p 2.3 2.2 2.5 2.7 Total bone ash (mg) 45 50 51 24-epi-l, 25- (OH) 2D2 65p nol/day 36 350p " 43 750p " 48 The results of the two studies shewn in Examples 8 and 9, illustrate that 24-epi-l,25-(OH)2D2 is approximately equal in potency to la,25-dihydroxyvitamin D^ (1,25-(OH)2D^ in causing the mineralization of bone and in stimulating intestinal calcium transport. In short, there is no significant difference between the two groups in Table 2 (Figure 2) and Table 4 (Figure 4). On the other hand, the elevation of serum inorganic phosphorus which results frcxn ixbilization of bone in the case of the low phosphorus diet is very markedly affected by 1,25-(OH) 2D2, but hardly stimulated by 24-epi-1,25(OH)2D2. Similarly, in the mobilization of calcium from bone, even at the extremely high dose level of 750 pmoles/day, the 24-epi compound had no effect, while the mobilization - 16 - effect is evident at much lcwer doses of 1,25-dihydroxyvitamin D2> Since the rise in serum calcium of rats on a lew calcium diet measures the ability to mobilize bone, and since the elevation of blood phosphorus of animals on a lew phosphorus 5 diet also measures bone mobilization, these results shew that 24-epi-l,25-(OH)2D2 provides an unexpected property, namely that it is almost 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 corrpound highly suitable for the treatment of disease states that evince bone loss. - 17 - The mixture is poured over ice/sat. NaHCO^ with stirring and the product is extracted with benzene. The combined organic phases are washed with aqueous CuSO^ and water, dried over MgSO^ and evaporated. 5 The crude 3-tosyl derivative (2) is directly solvolyzed in anhydrous methanol (10 ml) and NaHCO^ (150 mg) by heating at 55°C for 8.5 h with stirring. The reaction mixture is then cooled to room terrperature and concentrated to~2 ml under vacuo. Benzene (80 ml) is then added and organic layer is washed with water, dried and 10 evaporated. The resulting cyclovitamin (.3, Z=methyl) can be used in the subsequent oxidation without further purification.

The crude product (3) in CH^Cl^ (4.5 ml) is added to an ice-cooled solution at SeC^ (5.05 mg) and t-BuOOH (16.5 ul) in CH^Cl^ (8 ml) containing anhydrous pyridine (50 jil). After being 15 stirred for 15 min at 0°C, the reaction mixture is allowed to warm to roam terrperature. 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 20 purified on silica gel thin layer plates (20 x 20 cm plates, AcOEt/hexane 4:6) to yield 20 mg of laHhydroxy derivative (£, Z=methyl): mass spectrum, m/e: 470 (M+, 5), 438 (20), 87 (100); NMR (CDC13)<£ 0.53 (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 25 and 5.25 (2H, each m, 19-^), 5.35 (2H, m, 22-H and 23-H).

Example 2 Acetylation of compound (4).

A solution of cyclovitamin (4, Z=methyl) (18 mg) in pyridine (1 ml) and acetic anhydride (0.33 ml) is heated at 55°C for 2 h. The 30 mixture is poured into ice-cooled sat. NaHGO^ and extracted with benzene and ether. The combined organic extracts are washed with water, saturated CuSO^ and aqueous NaHCO^ solutions, dried and evaporated to give 1-acetoxy derivative 05, Z=methy1, acyl=acetyl) (19 mg): mass spectrum, m/e: 512 (M+, 5), 420 (5), 87 (100); NMR - 18 - (CDC13) 6 0.53 (3H, s, 18-H3) r 4.18 (1H, df 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 3 5 Solvolysis of lo-acetoxy-3,5-cyclovitamin (5) (R^=acetyl).

A solution of cyclovitamin (5) (4.5 mg) in 3:1 mixture of dioxane/^O (1.5 ml) is heated at 55°C. p-Toluenesulfonic acid (1 mg in 20^1 of H^O) is then added and heating is continued for 15 min. The mixture is poured into saturated NaHOO^/ice, and extracted 10 with benzene and ether. The organic phases are washed with NaHCO^ and water and dried over MgSO^. Evaporation of solvents gives a residue containing compounds (6) (where Rj=acetyl and and (J) (where R^=acetyl and R^H) which are separated by chromatography on HPLC (6.2 irai x 25 cm Zorbax-Sil) using 2% of 2-prqpanol in hexane as 15 an eluent. If necessary, the products are further purified by rechrorratography.

Example 4 Ketal hydrolysis in compound (6) to obtain ketone (8).

To the solution of ketal (6, Rj=acetyl, R^H) (1.35 mg) in 20 ethanol (1.5 ml), p-toluene sulfonic acid (0.34 mg in 45 pL of HgO) is added and the mixture is heated under reflux for 30 min. The reaction mixture is poured into diluted NaHCO^r and extracted with benzene and ether. The combined organic extracts are washed with water, dried over MgS04 and evaporated. High-pressure liquid 25 chromatography of the crude mixture (4% 2-prqpanol/hexane, 6.2 mm x 25 cm Zorbax-Sil) affords some unreacted ketal (6) (0.12 mg, collected at 48 ml) and desired ketone (j8, R^=acety 1, R^=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); 30 NMR (CDC13) S 0.53 (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, CRjCOO), 2.12 (3H, s, CH3a», 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=ll Hz, 7-H), 6.37 (1H, d, J=ll Hz, 6-H); UV (EtOH) 266 nm, 250 nm, 225 35 11m. - 19 - Example 5 Reaction of ketone (JB) with methylmagnesium bromide to obtain products (9a) and (9b).

Ketone (8, Rj=acetyl, in anhydrous ether is treated with 5 the excess of CH^MgBr (2.85 M solution in ether). The reaction mixture is stirred at room terrperature for 30 min, then quenched with aq. NH^Cl, extracted with benzene, ether and O^CL,. The organic phases are washed with dilute NaHCO^, dried over MgSO^ and evaporated. The mixture of (9a) and (9b) thus obtained is separated 10 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). la,25-dihydroxyvitamin D„ (9a): UV (EtOH) A ■ —■ / max 265.5 227.5 nm; mass spectrum, m/e 428 (M , 6) , 410 (4), 352 (4), 287 (6), 269 (10), 251 (10), 152 (42), 134 (100), 59 (99); 15 NMR (CDC13) S 0.56 (3H, s, 18-Rj) , 1.01 (3H, d, J=6.5 Hz, 28-^), 1.04 (3H, d, J=6.5 Hz, 21-H^), 1.14 and 1.18 (6H, each s, 26-H^ 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=ll Hz, 7-H), 6.39 (1H, d, J=ll Hz, 6-H). 20 la,25-dihydroxy-24-epivitamin (9b): UV (EtOH) Pi 265.5 nm, ^ , max ^min ^27.5 "H1* mass spectrum, m/e 428 (M , 13), 410 (9), 352 (7), 287 (11), 269 (15), 251 (13), 152 (52), 134 (100), 59 (97).

Example 6 Conversion of ccxrpaund (7) to 5,6-trans-la,25-dihydroxyvitamin 25 compounds (11a) and (lib).

Hydrolysis of ketal-intermediate (7, Rj=acety 1, R^^H) using the conditions described in Example 4 provides the corresponding 5,6-trans-2 5 -ketone of structure (10, Rj=acetyl, , and subsequent reaction of this ketone with methyl magnesium bromide, 30 using conditions analogous to those of Example 5, gives a mixture of epimers (11a) and (lib) which are separated by high performance liquid chromatography (HPLC) to obtain in pure form la, 25-dihydroxy-5,6-trans-vitamin (11a) and la, 25-dihydroxy-5,6-trans-24-epiv.ita-min D2 (lib). If required, structure assignment can be confirmed by - 20 - isomerization to the respective 5,6 cis compounds (9a, 9b) according to known procedures. 5,6-trans-la, 25-dihydroxyvitamin D_ (11a): UV (EtOH) /( 2 , max 273.5 230 nm; mass spectrum, m/e 428 (M , 8), 410 (3), 287 5 (3), 269 (7), 251 (7), 152 (34), 134 (100), 59 (78). 5,6-trans-la,25-dihydroxy-24-epivitamin (lib): UV (EtOH) 273.5 nm,/K . 230 nm; mass spectrum, m/e 428 (M+ 10), 410 (4), - max ' nun ^ — — , 352 (4), 287 (5), 269 (9), 251 (8), 152 (37), 134 (100), 59 (82). Example 7 10 Preparation of alkyl and aryl analogs of to, 25-dihydroxyvitamin D2 compounds.

By reaction of ketone intermediate (8) (R^=acetyl, f^H) with, respectively, (a) ethyl magnesium bromide 15 (b) propyl magnesium bromide (c) isopropyl magnesium bromide (d) butyl magnesium bromide (e) phenyl magnesium brcmide using conditions analogous to those described in Example 5, there 20 are obtained the corresponding hydroxyvitamin products having the formula shown below k0H wherein X is, respectively 25 (a) ethyl (b) propyl (c) isopropyl (d) butyl (e) phenyl 30 By like treatment of 5,6-tran s-ketone intermediate (10) (Rj=acetyl, R^^H) with the above listed Grignard reagents, there are - 21 - Process Scheme I b_: 24R - 22 - Process Scheme II - 23 - Process Scheme III { Ph'Sv^/ o>S sulfone A H0> _ o 6 o Ph- - 24 -

Claims (24)

1. A compound having the formula: wherein each of R,, R, and R-,, 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, with the proviso that when the C-24-methyl substituent has the S-configuration and X is methyl, R, and R, are not both hydrogen.

2. A compound according to claim 1, wherein X is methyl.

3. A compound according to claim 1 or 2, wherein the asymmetric center at C-24 has the (R)-configuration.

4. A compound according to claim 1 or 2, wherein the asymmetric center at C-24 has the (S)-configuration.

5. la,25-dihydroxy-24-epi-vitamin D2 .

6. la,25-dihydroxy-5,6-trans-24-epi-vitamin D2 .

7. A pharmaceutical composition which comprises a compound as claimed in any one of claims 1 to 6 and a pharmaceutically acceptable excipient.

8. A composition according to claim 7 which comprises la.25-dihydroxv-5.6-trans-24-epi-vitamin D2 and, optionally, la,25-dihydroxy-5,6-trans-vitamin D2.

9. A composition according to claim 7 or 8, which comprises la.25-dihvdroxv-5.6-trans-vitamin D2 and la,25-dihydroxy-5,6-trans-24-epivitamin D2.

10. A composition according to claim 7 or 8, which comprises la,25-dihydroxyvitamin D2, la,25-dihydroxy-24-epi-vitamin D2, la,25-dihydroxy-5,6-trans-vitamin D2 and la,25-dihydroxy-5,6-trans-24-epi-vitamin D2.

11. A composition according to claim 7, which comprises la,25-dihydroxyvitamin D2 and la,25-dihydroxy-24-epi-vitamin D2.

12. A pharmaceutical composition which comprises la,25-dihydroxy-5,6-trans-vitamin D2 and either la,25-dihvdroxy-5.6-trans-24-epi-vitamin D2 or la,25-dihydroxy-24-epi-vitamin D2.

13. A composition according to claims 7 or 12 which comprises at least one bone mobilisation-inducing compound.

14. A composition according to claim 13 in which the said compound is 25-hydroxyvitamin D3, 25-hydroxyvitamin D2, la-hydroxyvitamin D3, la-hydroxyvitamin D2 , la, 25-dihydroxyvitamin D3, la, 25-dihydroxyvitamin D2, 24,24- difluoro-25-hydroxyvitamin D3, 24,24-difluoro-la,25-dihydroxyvitamin D3, 24-fluoro-25-hydroxyvitamin D3, 24-fluoro-la,25-dihydroxy-2-fluorovitamin D3/ 26,26,26,27,27,27,-hexafluoro-la,25-dihydroxyvitamin D3 or 26,26,26,27,27,27,-hexafluoro-25-hydroxyvitamin D3. - 26 -

15. A composition according to claim 7 or 12 substantially as hereinbefore described.

16. A compound having the formula: wherein K is an oxygen atom or an ethylenedioxy group, and R, and R2, which may be the same or different, are hydrogen or acyl.

17. A compound according to claim 16 wherein K is an oxygen group.

18. A compound according to claim 16 wherein K is an ethylenedioxy group.

19. la-Hydroxy-25-oxo-27-nor vitamin D3 or the acetate thereof.

20. la-Hydroxy-25-oxo-27-nor-24-epivitamin D2 or the acetate thereof.

21. A process for preparing a compound as claimed in claim 1 which comprises subjecting a ketal of the formula: - 27 - wherein R, and R2 are as defined above to hydrolysis at a temperature from 50 to 100°F (10 to 38°C) under acidic conditions and reacting the resulting Ketone with a Grignard reagent.

22. A process according to claim 21 wherein the hydrolysis is carried out using p-toluene-sulfonic acid.

23. A process according to claim 21 substantially as hereinbefore described.

24. A compound as claimed In claim 1 whenever prepared by a process as claimed in any one of claims 21 to 23. F. R. KELLY & CO. AGENTS FOR THE APPLICANTS