NL193245C - A 1,25-dihydroxyvitamin D2 related compound and pharmaceutical composition having a calcium metabolism-regulating action containing such a compound. - Google Patents

Description

1 193245

1a, 25-dihydroxyvitamin D2 related compound and pharmaceutical composition having a calcium metabolism regulating action containing such compound

The present invention relates to a compound related to 1a, 25-dihydroxyvitamin D2.

The compound 1a, 25-dihydroxyvitamin D2 is known from US patent 3,880,894. This reference describes the isolation and identification of this vitamin D2 metabolite. In this compound, represented in the present formula 9a, the methyl group at position 24 has the S configuration. This reference also mentions that 1a, 25-dihydroxyvitamin D2 has significantly greater antirachitic activity than vitamin D3.

US Patent 4,225,596 describes the use of 1a, 25-dihydroxyvitamin D3 for improving calcium balance and calcium absorption, and in particular for the treatment of post-menopausal osteoporosis. Some related derivatives of Vitamin D3 or Vitamin D2. including the 1 a, 25-dihydroxyvitamin D2 obtained in U.S. Pat. No. 3,880,894 would give essentially the same result.

New, 1a, 25-dihydroxyvitamin Da related compounds have now been found to have unexpectedly beneficial properties as the calcium metabolism regulators.

The invention thus provides a compound related to 1a, 25-dihydroxyvitamin D2, characterized in that the compound has the formula A, wherein R 1, R 2 and R 3, which may be the same or different, represent a hydrogen atom or an acyl group, X an alkyl group of 1-6 carbon atoms and the methyl group at position 24 has the R configuration.

As used herein, the term "acyl" means an aliphatic acyl group (alkanoyl group) of 1-6 carbon atoms in all possible isomeric forms, for example, formyl, acetyl, butyryl, isobutyryl or valeryl, or an aromatic acyl group (aroyl group) such as benzoyl, or the methyl, halogen or nitro substituted benzoyl groups, or an acyl group derived from a dicarboxylic acid of the general formulas ROOC (CH2) nCO- or R00CCH2-0-CH2C0, where n is an integer from 0 to 4 and wherein R represents hydrogen or an alkyl radical. Representative examples of such dicarboxylic acyl groups are oxalyl, malonyl, succinoyl, glutaryl, adipyl and diglycolyl. The invention "alkyl group of 1-6 carbon atoms" relates to all isomeric forms of such a group, for example methyl, ethyl, propyl, isopropyl, butyl, isobutyl.

The compounds of the invention can be prepared according to the reactions indicated in reaction scheme I. A wavy line to the substituent (methyl) at C-24 means that this substituent can have either the R or S configuration. The final product of the invention of the formula A indicated in reaction scheme I is the compound of the formula 9b (X is methyl).

A suitable starting material for preparing the compounds of the invention is the vitamin 35 D ketal derivative of the formula 1. This compound can be used as a mixture of the 24-R and 24-S epimers, whereby the separation of the individual 24-R and 24-S epimers are accomplished in a later step of the process. Using the pure 24-R epimer of (1), after treatment via the indicated synthetic steps, only the (24-R) -la, 25-dihydroxylated product of the formula 9b will be obtained.

The starting material of formula 1 (the preparation of which is described in British patent application 2,127,023 published April 4, 1984) is converted into the desired 1a-hydroxylated form via cyclovitamin D derivatives, according to the method of DeLuca et al., U.S. Patent 4,260 .549. Thus, the treatment of compound (1) with toluene sulfonyl chloride conventionally results in the corresponding 3-tosylate of the formula 2 which is solvolysed in an alcoholic medium to form the 3,5-cyclovitamin D derivative of the formula 3, where Z is the organic residue from the alcohol of the formula Z-OH. The solvolysis in, for example, methanol gives the cyclovitamine of the structure (3), wherein Z = methyl. The allyloxidation of intermediate (3) with selenium dioxide and a hydroperoxide gives the 1a-hydroxy analog of the formula 4, wherein Z has the meaning just mentioned. A subsequent acetylation of compound (4) provides the corresponding 50 l acetate of the formula 5 (R1 = acetyl). If desired, other 1-O-acylates (structure 5, wherein R 1 = acyl, e.g. the formate, propionate, butyrate, benzoate) are prepared according to analogous conventional acylation reactions. The 1-O-acyl derivative is then subjected to an acid catalyzed solvolysis. When this solvolysis is carried out in a solvent medium containing water, the 5,6-cis-vitamin D intermediate of the formula 6 (Rn = acyl, R2 = H) and the corresponding 55,6-trans- compound in a ratio of about (3-4): 1. These 5,6-cis and 5,6-trans isomers can be separated in this step, for example for HPLC. If desired, the acyl group (R 1) can be removed by hydrolysis with a base to give the compound of formula 6 wherein 193245 represents 2 R and R 2. Alternatively, the 1-O monoacylates can be further acylated at the C- 3-hydroxyl groups under conventional acylation conditions to give the corresponding 1,3-di-O-acylates of the formula 6 wherein R 1 and R 2, which may be the same or different, represent acyl groups. Furthermore, the hydroxycyclovitamine of the formula IV can be subjected to an acid-catalyzed solvolysis in a medium containing a low molecular organic acid to obtain the 5,6-cis compounds of the formula 6 wherein R 1 = H and R2 = acyl, the acyl group being derived from the acid used in the solvolysis reaction, as well as the corresponding 5,6-trans compounds.

The next step of the process involves the removal of the ketal protecting group to form the corresponding 25-ketone of the formula 8. This step is an essential step, because the conversion of the ketal to the ketone must be effected without any concomitant isomerization of the 22 (23) double bond to the conjugated 23 (24) position, which can occur under the acidic conditions required for the ketal hydrolysis. In addition, the conditions should be chosen to avoid elimination of the sensitive C-1 allylic oxygen group. The conversion is successfully accomplished by gentle hydrolysis at moderate temperatures using an organic acid catalysis, according to the method of converting 25,25-ethylenedioxy-27-norcholest-5- described in U.S. Patent 4,264,513. en-1a, 3B-diol in 1a, 3B-dihydroxy-27-norcholest-5-en-25-one. Thus, treatment of the 5,6-cis compound (6) in aqueous alcohol with p-toluenesulfonic acid gives the corresponding ketone (8). In order to prevent an undesired elimination of the C-1 oxygen group during this reaction, it is advantageous that the C-1 hydroxyl group in compound (6) is protected (eg R. = acyl, R2 = hydrogen or acyl) . See in this regard the aforementioned U.S. Patent 4,260,549.

The subsequent reaction of ketone (8) with a methyl-Grignard reagent is analogous to the method described in U.S. Patent 4,264,513 for preparing 1a, 25-dihydroxyvitamin 25 Dg from 1a-hydroxy-25-oxo-27 -norvitamin D3. It provides the desired 1a, 25-dihydroxyvitamin D2 compound of the formula 9b. If the starting material, compound (1), used in the above process, is a mixture of the two C-24 epimers, in addition to the compound of the invention of the formula 9b, the corresponding 24-S epimer of the formula will also be obtained. 9a. The separation of this epimer mixture can be accomplished by chromatographic methods to obtain 30 1 a, 25-dihydroxyvitamin D2 (structure 9a, 24-S stereochemistry) and its 24-R epimer, 1a, 25-dihydroxy- 24- I epivitamin D2, with structure 9b, both in pure form.

The side chain ketones of the formula 8 can also be used for the preparation of the other compounds of the invention according to the invention of the formula 1a, 25-dihydroxyvitamin D2, which are 5,6-cis-1a, 25-dihydroxyvitamin D2 analogues with the general side chain of formula 7, wherein X and the configuration of the methyl group at position 24 have the meanings previously mentioned in connection with the invention. For example, the treatment of ketone (8) with ethylmagnesium bromide gives the corresponding hydroxyvitamin D2 analogous to the side chain structure indicated above, wherein X represents an ethyl group. Similarly, treatment of (8) with isopropylmagnesium bromide gives the side chain analog wherein X represents isopropyl.

Also in analogy to that disclosed in U.S. Pat. No. 4,264,513, the reaction of the keto intermediates (8) with an isotopically labeled Grignard reagent (e.g., C3H3MgBr, 14CH3MgBr, C2H3MgBr) provides a suitable method for preparing 1a , 25-dihydroxy-24-epivitamin D2 in an isotopically labeled form, i.e. as the compounds of the above side chain, wherein X represents C3H3,14CH3, C2H3,13CH3, or any other isotopically labeled 45 alkyl group having 1- 6 carbon atoms.

The compounds of the formula A wherein X represents an alkyl group having from 2 to 6 carbon atoms are useful substitutes for the compounds of formula 9b in situations where a higher degree of lipophilicity is desirable, while the above isotopically labeled compounds find use as reagents in analytical applications.

For therapeutic applications, the compounds of the invention are generally used in the form of the free hydroxy compounds represented by the above formula A, wherein R 1 (R 2 and R 3 represent H). For some such applications, the corresponding acylated compounds represented by the above general formula A, wherein one or more of the groups R 1 (R 2 and R 3 represent an acyl group) are useful, preferably or not.

Such acyl derivatives are suitably prepared from the free hydroxy compounds using conventional acylation procedures, for example, with an acyl halide or acid anhydride in a suitable solvent, such as pyridine or an alkyl pyridine. By a suitable choice of the reaction time, the acylating agent, the temperature and the solvent, as is known in the art, the partially or fully acylated derivatives represented by the above formula A are obtained. For example, the treatment of 1 a, 25-dihydroxy-24-epivitamin D2 (9b) in pyridine as a solvent with acetic anhydride at room temperature gives the corresponding 1,3-diacetate, while the same reaction, performed at elevated temperature, results in the corresponding 1 , 3,25-triacetate. The 1,3-diacetate can be further acylated at C-25 with another acyl group; for example, 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 hydrolysed in a mild base to form the 1,3-dihydroxy-25-O-acyl compound, the free hydroxy groups of which can be reacylated if desired with various (other) acyl groups. Similarly, a 1,3-diacyl derivative can be subjected to partial acyl hydrolysis to yield the 1-O-acyl and 3-O-acyl compounds, which in turn can be reacylated with other acyl groups.

Like the previously known vitamin D2 metabolite, 1a, 25-dihydroxyvitamin D2 (9a), the new compounds according to the invention show a pronounced vitamin D-like activity. The compounds according to the invention, as will be explained hereinafter, have only a very limited effect on the mobilization of bone calcium, but a good effect on stimulating the intestinal calcium transport and the mineralization of new bones. Therefore, in many therapeutic and veterinary applications, they are desirable replacements for the known vitamin D2 or D3 metabolites. In this regard, the compounds of formula 9b and their acylated derivatives are particularly preferred. The new compounds can be used to correct or ameliorate various calcium and phosphate balance disorders resulting from a variety of diseases such as, in particular, osteoporosis.

It is well known that during the menopause female individuals exhibit a significant loss of bone mass, ultimately leading to osteopenia, which in turn gives rise to spontaneous crushing fractures of the vertebrae and long bone fractures. Commonly known as postmenopausal osteoporosis, this disease is a major medical problem both in the United States and in most other countries in which women reach ages of at least 60 years. Generally, the disease, often accompanied by bone pain and decreased physical activity, is diagnosed by one or two vertebral (vertebral) crushing fractures with reduced x-ray evidence of bone mass. This disease is known to be associated with a decreased ability to absorb calcium, decreased levels of the sex hormones, especially estrogen and androgen, and a negative calcium balance.

The methods used to treat the disease to date vary considerably. For example, a calcium supplement alone is not successful in preventing or curing the disease. Injection of sex hormones, especially estrogen, which has been reported to be effective in preventing the rapid loss of bone mass occurring in postmenopausal women is complicated by fears of its possible carcinogenicity. Other treatments, for which again variable results were reported, included a combination of vitamin D in large doses, calcium and fluoride. The main problem with this treatment is that fluoride causes structurally unhealthy bone, referred to as woven bone, and also produces a number of side effects, such as an increased incidence of fractures and a gastrointestinal response to the large amounts of fluoride administered.

Analogous symptoms characterize the senile osteoporosis and the steroid-induced osteoporosis, the latter being a recognized result of long-term cortico-steroid therapy for 45 certain disease states.

Although varying vitamin D3 metabolites enhance calcium absorption and calcium retention within the body of mammals that have a proven or physiological tendency to lose bone mass, they are also characterized by the complementary vitamin D-like property of mobilizing the calcium in the bones in response to physiological needs. It has now been found that the 24-epi compounds of this invention of the formula A, in particular 1 a, 25-dihydroxy-24-epivitamin D2 (24-epi-1,25- (OH) 2D2), are extremely well suited are for the prevention or treatment of physiological disorders in mammals, characterized by loss of bone mass, because although they have some of the recognized vitamin D-like properties, which affect calcium metabolism, such as increasing calcium transport in the 55 intestines and effecting bone mineralization, they do not increase serum calcium levels, even at high doses. This observed property proves that the compounds of the invention do not mobilize the bone material after administration. This fact, together with the 193245 4 ability of the compounds to mineralize the bones after administration, indicates that they are ideal compounds for the prevention or treatment of prevailing calcium disorders, which are evidenced by loss of bone mass, for example, postmenopausal osteoporosis, senile osteoporosis and steroid-induced osteoporosis. It will be clear that the compounds can also be used for the prevention or treatment of other disease states, in which the loss of bone mass is indicative, such as in the treatment of patients undergoing kidney dialysis, in which a loss of bone mass occurs as a result of dialysis.

The following tests A and B illustrate the properties of 24-epi-1,25- (OH) 2D2, which contribute to its extraordinary suitability for the prevention or treatment of 10 disease states, with loss of bone mass. .

Trial A

Newly weaned male rats are placed on the vitamin D deficient diet, described by Suda et al., Journal of Nutrition 100,1049-1052 (1970), modified to contain 0.02% calcium and 0.3% phosphorus . After two weeks on this diet, the animals were administered either 1a, 25-dihydroxyvitamin D2 or 1a, 25-dihydroxy-24-epivitamin D2 daily by subcutaneous injection in 0.1 ml 5% ethanol in propanediol. The animals were sacrificed 12 hours after the last dose and blood calcium and visceral calcium transport were measured. The results of these measurements for the indicated amounts of the administered compounds are shown in Figures 1 and 2. The visceral calcium transport measurements, indicated in Figure 2, were performed according to the method of Martin and DeLuca, American Journal of Physiology 216.1351- 1359 (1969).

Trial B

Newly weaned male rats were placed on a high calcium (1.2% calcium) and low phosphorus (0.1% phosphorus) diet as described by Suda et al. (Supra). The rats were fed this diet for a period of 3 weeks, after which they were separated into two groups. One group was administered 1.25- (OH) 2D2 (Formula 9a) while the other groups were administered 24-epi-1.25- (OH) 2D2 (Formula 9b), both in 0.1 ml 5% s ethanol in propanediol, the administration being effected subcutaneously at dosage amounts of the compounds as indicated by the data points in Figure 3.

These doses were continued daily for a period of 7 days after which time the animals were sacrificed and the serum inorganic phosphorus was determined. The results are shown in Figure 3.

The amount of bone ash was determined by removing the femurs from the rats. The thighs were cut free from adherent connective tissue and then extracted for 24 hours in absolute ethanol and 24 hours in diethyl ether using a Soxhlet extractor. The bones were then ashed at 600 ° F for 24 hours. The axle weight was determined by weighing to constant weight. The results are shown in Figure 4.

The results of the two studies described in Runs A and B above show that the activity of 24-epi-1,25- (OH) zD2 is approximately equal to that of 1a, 25-dihydroxyvitamin D2 (1 , 25- (OH) 2D2) for effecting bone mineralization and stimulating visceral calcium transport. There is no significant difference between the two groups in Figure 2 and Figure 4. On the other hand, the increase in serum inorganic phosphorus resulting from the mobilization of bone material in the case of the low phosphorus diet is very markedly affected 45 by 1.25- (OH) 2D2, but hardly stimulated by 24-epi-1.25- (OH) 2D2. Similarly, in the mobilization of calcium from bone material, as evidenced by the serum calcium levels (Figure 1), even at the extraordinarily high dosage rate of about 750 pmoles / day, the 24-epi compound had no effect, while the mobilization effect is evident in much lower doses of 1a, 25-dihydroxyvitamin D2. Since the increase in serum calcium of rats on a low calcium diet measures the ability to mobilize bone material and the increase in blood phosphorus of animals on a low phosphorus diet also measures bone mobilization, these results show that 24-epi- 1,25- (OH) 2D2 provides a surprising property, namely that it has a minimal effect on the mobilization of bone calcium, while it is fully capable of stimulating the intestinal calcium transport and the mineralization of new bones, which properties this compound highly suitable for the treatment of disease states in which bone loss occurs.

The unique properties of 24-epi-1,25- (OH) 2D2, as noted above, provide the rare opportunity to control the varying vitamin D responsive processes (visceral calcium absorption, bone mineral mobilization and bone mineralization) in a manner and in a degree, which has hitherto not been practicable. This possibility results from the fact that the 24-epi compound of this invention can be administered to the mammal either alone (with suitable and acceptable excipients) or in combination with other vitamin D derivatives, which display the full spectrum of D-like activity. administered.

By such measures it is therefore possible (to any desired degree) to combine the specificity of the action of the 24-epi analog with the generality of the action of other vitamin D metabolites or analogues. The administration of 24-epi-1,25- (OH) 2D2 alone will, as shown above, stimulate visceral 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 more of the known vitamin D derivatives (e.g. 1a, 25- (OH) 2D3, 1a, 25- (OH) 2D2,1a-OH-D3). By adjusting the relative amounts of the administered compounds, a degree of control over the relative magnitudes of the visceral calcium absorption relative to the bone mineral mobilization processes can be exerted in a manner not possible with the hitherto known vitamin D derivatives. The co-administration of the 24-epi compound and other vitamin D compounds with a bone mobilization activity can be particularly advantageous in situations where a certain degree of bone mobilization is desirable. For example, it is believed that in certain circumstances the bone material must first be mobilized before new bone material can be deposited. In such situations, treatment with vitamin D or a vitamin D derivative, which will effect bone mobilization, for example 1a-hydroxyvitamin D3 or -d2, 20 1a, 25-dihydroxyvitamin D3 or -D2, 25-hydroxyvitamin D3 or -D2, 24 .24 * defluoro-25-hydroxyvitamin D3, 24,24-difluoro-1 <x, 25-dihydroxyvitamin D3, 24-fluoro-25-hydroxyvitamin D, 24-fluoro-1a, 25-dihydroxyvitamin D3, 2B-fluoro-1 α-hydroxyvitamin D3, 2B-fluoro-25-hydroxyvitamin D3, 2B-fluoro-1a, 25-dihydroxyvitamin D3, 26,26,26,27,27,27-hexafluoro-1a, 25-dihydroxyvitamin D3, 26,26 , 26,27,27,27-hexafluoro-25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3,1a, 24,25-trihydroxyvitamin D3, 25,26-dihydroxyvitamin D3 or 1a, 25,26-25 trihydroxyvitamin D3, in combination with 24-epi-1,25- (OH) 2D2 according to the invention, by adjusting the amounts of the 24-epi compound and the bone mobilizing vitamin D compound in the treatment regimen, it is possible to control the extent of the bone mineralization to set in order to achieve the desired medical and physiological goals. Suitable and effective mixtures are, for example, the combination of 1a, 25-dihydroxyvitamin D2 and 1a, 25-dihydroxy-24-epivitamin D2 (9a and 9b).

The invention therefore also relates to a pharmaceutical composition having a calcium metabolism-regulating action comprising a compound 1a, 25-dihydroxyvitamin D2 and a pharmaceutically acceptable excipient, characterized in that the compound has the formula A, wherein R R2, R3, X and the configuration of the methyl group at position 24 have the meanings mentioned previously in connection with the invention. Preferably, the pharmaceutical preparation according to the invention additionally contains at least one compound which promotes bone mobilization.

The compounds of this invention or combinations thereof with other vitamin D derivatives or other therapeutic agents can easily be administered as sterile parenteral solutions by injection or intravenously. They can also be administered via the digestive tract in the form of oral doses, or transdermally or via suppositories. The compounds are advantageously administered in dosage amounts of 0.1-100 micrograms per day. In the case of osteoporosis, doses of about 0.5 to about 25 micrograms per day are generally effective. The compounds of the formula A according to the invention can be administered either alone or in combination with other vitamin D derivatives, the amounts of each of the compounds in the combination being dependent on the special disease condition to be treated and on the desired degree of bone mineralization and / or bone mobilization. In the treatment of osteoporosis, wherein the preferred compound of the invention is 24-epi-1a, 25- (OH) aD2, the amount of the 24-epi compound actually used is not limited. In all cases, a sufficient amount of the compound should be used to induce bone mineralization. Amounts greater than about 50 micrograms per day of the 24-epi compound or the combination of this compound with bone mobilization-inducing vitamin D derivatives are generally not necessary to achieve the desired results and may be economically considerations are undesirable. In practice, higher doses of the compounds are used when the therapeutic treatment of a disease condition is the desired goal, while the lower doses are generally used for 55 prophylactic purposes, it being understood that the specific dose administered in any given case will be established in accordance with the specific compounds administered, the disease to be treated, the patient's condition and other relevant medical facts, which may modify the activity of the drug or the patient's response, as is well known to experts 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 can be either solid or liquid, such as, for example, corn starch, lactose, sucrose, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the dosage form of the compounds may be in the form of tablets, capsules, powders, troches or lozenges. When a liquid carrier is used, soft gelatin capsules, or syrup or liquid suspensions, emulsions or solutions may be the dosage form. The dosage forms may also contain additives, such as preservatives, stabilizers, wetting or emulsifying agents, or solution promoting agents. They can also contain other therapeutically valuable substances, such as vitamins, salts, sugars, proteins or hormones.

The method of preparing the compounds of the present invention is more particularly described in the example below. In this example, the designation of the specific products by Arabic numerals (eg compounds 1,2, 3) refers to the structures thus numbered in reaction scheme I.

Example a. 25,25-Ethylenedioxy-1a-hydroxy-3,5-cyclovitamin D2 (4, Z = methyl).

A solution of 50 mg of compound (1) as a mixture of the 24-R and S epimers in dry pyridine (300 µl) is treated with 50 mg of p-toluenesulfonyl chloride at 4 ° C for 30 hours. The mixture is poured onto ice / saturated NaHCO3 with stirring and the product is extracted with benzene. The combined organic phases are washed with aqueous NaHCO 3, H 2 O, aqueous CuSO 4 and water, then dried over MgSO 4 and then evaporated.

The crude 3-tosyl derivative (2) is directly solvolysed in anhydrous methanol (10 ml) and NaHCO 3 (150 mg) by heating at 55 ° C for 8.5 hours with stirring. The reaction mixture is then cooled to room temperature and concentrated to about 2 ml under reduced pressure. Benzene (80 ml) is then added and the 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 CH2CI2 (4.5 ml) is added to an ice-cooled solution of SeOz (5.05 mg) and t-BuOOH (16.5 µl) in CH2CI2 (8 ml) containing anhydrous pyridine (50 μΙ). After stirring at 0 ° C for 15 minutes, the temperature of the reaction mixture is allowed to rise to room temperature. After a further 30 minutes, the mixture is transferred to a separatory funnel and shaken with 10% NaOH (30 ml).

Diethyl ether (150 ml) is added and the separated organic phase is washed with 10% NaOH and water, then dried and then evaporated. The oily residue is purified on silica gel thin layer plates (20 x 20 cm plates; AcOEt / hexane 4: 6) to give 20 mg of 1a-hydroxy derivative (4) [Z = methyl]: mass spectrum, m / e: 470 (M +, 5), 438 (20), 87 (100); NMR (CDCI3), maxima at δ values: 0.53 (3H, s), 0.63 (1H, m), 4.19 (1H, d, J = 9.5 Hz), 4.2 (1H , m), 4.95 (1H, d, J = 9.5 Hz), 5.17 and 5.25 (2H, each m), 5.35 (2H, m).

b. Acetylation of compound (4).

An 18 mg solution of cyclovitamine (4) [A = methyl] in pyridine (1 ml) and acetic anhydride (0.33 ml) is heated at 55 ° C for 2 hours. The mixture is poured into ice-cooled 45 saturated NaHCO 3 and extracted with benzene and diethyl ether. The combined organic extracts are washed with water, saturated CuSO 4 and aqueous NaHCO 3 solutions, then dried and then evaporated to give the 1-acetoxy derivative (5) [Z = methyl, R 1 = acetyl] (19 mg): mass spectrum m / e: 512 (M +, 5), 420 (5), 87 (100); NMR (CDCI3), maxima at δ values: 0.53 (3H, s), 4.18 (1H, d, J = 9.5 Hz), 4.97 (2H, m), 5.24 (2H , m), 5.35 (2H, m).

50 c. Solvolysis of 25,25-ethylenedioxy-1oc-acetoxy-3,5-cyclovitamin (5) [R1 = acetyl, Z = methyl].

A solution of 4.5 mg of cyclovitamine (5) in a 3: 1 mixture of dioxane / H 2 O (1.5 ml) is heated to 55 ° C. p-Toluene sulfonic acid (1 mg in 20 μΙ H 2 O) is then added and heating is continued for 15 minutes. The mixture is poured into saturated NaHCOg / ice and extracted with benzene and diethyl ether. The organic phases are washed with NaHCO 3 and water and dried over MgSO 4. A residue containing compounds is obtained by evaporation of the solvents

(6) (where R 1 = acetyl and R 2 = H) and the corresponding trans isomer, which are separated by HPLC

Claims (3)

7 193245 over a column of 6.2 mm x 25 cm Zorbax-Sil (microparticle silica gel) using 2% 2-propanol in hexane as eluent. If necessary, the compound (6) can be further purified by repeated chromatography. 5 d. Ketal hydrolysis of compound (6) to give ketone (8). To a solution of 1.35 mg of ketal (6) [R 1 = acetyl, R 2 = H] in ethanol (1.5 ml) is added p-toluenesulfonic acid (0.34 mg and 45 µl H 2 O) and the mixture is heated to reflux for 30 minutes. The reaction mixture is poured into dilute NaHCOa and extracted with benzene and diethyl ether. The combined organic extracts are washed with water, dried over MgSO 4 and evaporated. High pressure liquid chromatography of the crude mixture (4% 2-propanol / hexane, 6.2 mm x 25 cm Zorbax-Sil) gives a small amount of unreacted ketal (6) [0.12 mg, collected at 48 ml] and the desired ketone (8) [R 1 = acetyl, R 2 = H] (0.36 mg, collected at 52 ml), which is characterized by the following data: mass spectrum, m / e: 454 (M +, 9), 394 (17), 376 (10), 134 (23), 43 (100); NMR (CDCl3), maxima at 5 values: 0.53 (3H, s), 1.03 (3H, d, J = 6.5 Hz), 1.13 (3H, d, 15 J = 7.0 Hz), 2.03 (3H, s), 2.12 (3H, s), 4.19 (1H, m), 5.03 (1H, m), 5.33 (3H, wide m), 5 .49 (1H, m), 5.93 (1H, d, J = 11Hz), 6.37 (1H, d, J = 11Hz); UV (EtOH) 266 nm, 250 nm, λ ^ η 225 nm. e. Reaction of ketone (8) with methyl magnesium bromide to give products (9a) and (9b). Ketone (8) [R1 = acetyl, R2 = H] in anhydrous diethyl ether is treated with an excess of CH3MgBr 20 (2.85M solution in diethyl ether). The reaction mixture is stirred at room temperature for 30 minutes, then quenched with aqueous NH4CI and then extracted with benzene, diethyl ether and CH2CI2. The organic phases are washed with dilute NaHCO 3, dried over MgSO 4 and evaporated. The mixture of (9a) and (9b) thus obtained is separated by HPLC (6% 2-propanol / hexane, 4.6 mm x 25 cm Zorbax-Sil), to obtain, in the elution order, the US patent 3,880,894 discloses S epimer (9a) and the corresponding R epimer (9b) of the invention. 1a, 25-Dihydroxyvitamin D2 (9a): UV (EtOH) 265.5 nm, λ1, η 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 (CDCI3), maxima at δ values: 0.56 (3H, s), 1.01 (3H, d, J = 6.5 Hz), 1.04 (3H, d, J = 6.5 Hz ), 1.14 and 1.18 (6H, each s), 4.24 (1H, m), 4.43 (1H, m), 5.01 (1H, m), -5.34 (3H, broad m), 6.02 (1H, d, J = 11 Hz), 6.39 (1H, d, J = 11, 30 Hz). 1a, 25-Dihydroxy-24-epivitamin D2 (9b): UV (EtOH) Xmax 265.5 nm, 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 ). 35 Conclusions A compound related to 1a, 25-dihydroxyvitamin D2, characterized in that the compound has the formula A, wherein R 1, R 2 and R 3, which may be the same or different, represent a hydrogen atom or an acyl group, X an alkyl group with 1 -6 is carbon and the methyl group at position 24 has the R configuration 40. 2. A pharmaceutical composition having a calcium metabolism-regulating action, comprising a compound of 1a, 25-dhydroxyvitamin D2 and a pharmaceutically acceptable excipient, characterized in that the compound has the formula A, wherein R 1, R 2, R 3, X and the configuration of the methyl group at position 24 have the meanings set forth in claim 1. Pharmaceutical preparation according to claim 2, characterized in that it additionally contains at least one compound which promotes bone mobilization. Hereby 3 sheets drawing