NL8420137A - A 1,25-dihydroxyvitamin D2 related compound and pharmaceutical composition having a calcium metabolism-regulating action containing such a compound. - Google Patents

Description

- 1 -

Process for the preparation of Ia-25-hydroxydylated vitamin and related compounds.

The invention relates to the preparation of 1,25-dihydroxylated compounds of the vitamin D series.

a

The invention more particularly relates to the preparation of 1a. 2 5-Dihydroxy-vitamin Dg and its (24R) -epimer, the corresponding 5,6-trans isomers, and certain C-25 alkyl or aryl analogues as well as the acyl derivatives of these compounds.

The importance of the hydroxylated forms of vitamin D as regulators of calcium and phosphate metabolism in animals and humans is now widely recognized in many publications in the patent literature and general literature, and consequently these hydroxyvitamin D derivatives are increasingly finding clinical and veterinary use as medicaments for the treatment and cure of calcium metabolism disorders and related bone diseases. Vitamin D ^ is known to be hydroxylated in vivo to 25-hydroxyvitamin D ^ and then to 1,25-dihydroxyvitamin D ^, the latter being generally recognized as the active hormonal form of vitamin D ^ - in an analogous manner. the very powerful vitamin Dg metabolite, la.2 5-dihydroxyvitamin Dg (la.25- (OH) 2 ^ 2 ^ / from vitamin Dg formed via 25-hydroxyvitamin Dg (25-OH-D2). Both of these hydroxylated vitamin 25 Dg compounds have been isolated and identified (Deluca et al., U.S. Pat. Nos. 3,585,221 and 3,880,894) since they are derived from Vitamin D, these metabolites are characterized by the (S) stereochemistry at carbon atom 24.

30 A chemical process for the preparation 8420137 - 2 - ï ')

of la.25-dihydroxylated compounds of the vitamin series has now been developed. More specifically, this method provides a suitable method for the preparation of compounds of the general structures A and B, wherein R 1, R

4 C6 and R6 are selected from hydrogen and acyl and wherein X represents an alkyl or aryl group. In these structures, the asymmetric center at carbon atom 24 may have the (R) or (S) configuration.

Specific examples of compounds obtainable by the present method include 1a-25-dihydroxyvitamin, the corresponding (24R) epimer, 1a-25-dihydroxy-24-epivitamin, and the respective 5.6-trans isomers, i.e., 5.6-trans -la.25-dihydroxy-vitamin D2 and 5.6-trans-la.25-dihydroxy-24-epivitamin D2, as well as the C-25 alkyl or aryl homologues of these compounds, i.e. the compounds with the above formulas, wherein X represents ethyl, propyl, isopropyl or phenyl.

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, valeryl, etc., or an aromatic acyl group (aroyl group), such as benzoyl, or the methyl, halogen or nitro substituted benzoyl-25 groups, or an acyl group derived from a dicarboxylic acid of the general formulas ROOCCC ^ CO- or ROOCC ^ -O-CI ^ CO-, where n is an integer is 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 term "alkyl" refers to a hydrocarbon group of 1-6 carbon atoms in all isomeric forms, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, etc. The term "aryl" refers to an aromatic moiety, such as phenyl , benzyl, or the isomeric alkyl-substituted phenyl radicals.

8420137 'h 4 - 3 -

An embodiment of the chemical process according to this invention is indicated in the accompanying reaction scheme I. In the description of this process below, the numbers (for example 1, 2, 3, etc.), 5 denoting the specific products refer to the thus numbered structures 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.

A suitable starting material for the method according to this invention is the vitamin D-ketal derivative with the structure (1). It is generally convenient (for example, in the case where both C-24 epimers of 1,25-dihydroxyvitamin D0 compounds are desirable) to use compound (1) as a mixture of the 24R and S epimers, wherein the separation of the individual 24R and S epimers is accomplished in a later step of the process. However, the pure 24S or the pure 24R epimer of (1) are equally suitable starting materials, wherein the first compound after the treatment will provide the (24S) -1.25 dihydroxy product via the 20 synthetic steps indicated, while the latter compound after analogous treatment will yield the corresponding (24R) -la25 dihydroxylated product.

Starting material (1) is converted into the desired Ια-hydroxylated form via cyclovitamin D derivatives (DeLuca et al, US Pat. Nos. 4,195,027 and 4,260,549). Thus, treatment of compound (1) with toluene sulfonyl chloride in the usual manner results in the corresponding C-3 tosylate (2), which is solvolysed in an alcoholic medium to form the new 3.5-cyclovitamin D derivative (3). The solvolysis in methanol gives the cyclovitamine of the structure (3), wherein Z = methyl, while the use of other alcohols, for example ethanol, 2-propanol, butanol, etc., in this reaction provides the analogous cyclovitamine D compounds ( 3), wherein Z represents an alkyl group derived from the alcohol, eg, ethyl, isopropyl, butyl, etc. ethyl. The allyl oxidation of intermediate (3) with selenium dioxide and a hydroperoxide gives the Ια-hydroxy analog with the structure (4). A subsequent acetylation of compound (4) provides the 1-acetate with. the structure (5, R 1 - acetyl). If desired, other 1-O-acylates (structure 5, wherein R 3 = acyl, e.g., the formate, propionate, butyrate, benzoate, etc.) 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 having the structure (6, R ^ = acyl, R2 - H) and the corresponding 5.6-trans compound (structure is obtained). 7, R ^ = acyl, R2 = H)) 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, by liquid chromatography performed under drastic conditions. If desired, the C-1-O-acyl group can be removed by hydrolysis with a base to give compounds (6) and (7), wherein R 1 and R 2 represent H. If desired, these 1-O- monoacylates are also further acylated at the C-3 hydroxy groups under conventional acylation conditions to obtain the corresponding 1,3-di-O-acylates of structure (6) or (7), wherein R 1 and R 2, which are the same or can be different, suggest acyl groups. Alternatively, the hydroxycyclovitamine of the structure (4) may be subjected to an acid catalyzed solvolysis in a medium containing a low molecular organic acid to obtain the 5.6-cis and trans compounds of the structures (6) and (7), wherein = H and R2 = acyl, wherein the acyl group is derived from the acid used in the solvolysis reaction.

The next step of the process involves removing the ketal protecting group to form the corresponding 25-ketone. This stage is an essential stage, since conversion of the ketal to the ketone must be accomplished without an associated 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. Furthermore, 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 organic acid catalysis. Thus, treatment of the 5.6-c is compound (6) in aqueous alcohol with p-toluenesulfonic acid gives the corresponding ketone (8). In order to prevent an undesired elimination of the Cl-oxygen group during this reaction, it is advantageous that the C-1-hydroxy group in compound (6) is protected (for example R1 / acyl, R2 = hydrogen or acyl) .

The subsequent reaction of ketone (8) with a methyl-Grignard reagent then provides the desired 1a. 25-dihydroxyvitamin D2 compound with the structure (9). If the starting material, compound (1) used in the above method, is a mixture of the two C-24 epimers, the compound (9) will be obtained as a mixture of the 24S and R epimers (9a and 9b). The separation of this epimer mixture can be accomplished by chromatographic methods to obtain 1,25-dihydroxyvitamin D2 (structure 9a, 24S stereochemistry) and its 24R epimer, 1,25-dihydroxy-24-epi-vitamin D2. , with the structure 9b, both in pure form.

Such separation of the epimers is of course not necessary if the compounds are intended to be used as a mixture.

The 5.6-trans-25-ketal intermediate with the structure (7), analogously subjected to a ketal hydrolysis, provides the 5.β-trans-ketone intermediate of the structure (10), which via a Grignard reaction with methyl magnesium bromide or an analogous reagent provides the 8420137 - 6 - * * 5.6-trans-la.25-dihydroxyvitamin compounds of the structure (11), as the 24S or 24R epimer, or as a mixture of both epimers depending on the nature of the starting material used in the process (1). If obtained as an epimer mixture, the epimers can be separated by chromatography to obtain 5.6-trans-1a-25 dihydroxyvitamin (11a) and its 24R epimer, 5.6-trans-1a-25 dihydroxy- 24-epivita-mine, with the structure (11b). These reaction steps using the 5.6 trans intermediate are completely performed in the same manner as the steps applicable to the 5.6 cis compounds described above.

The new side chain ketones of structures (8) or (10) are very useful and versatile intermediates because they can be used to prepare a variety of 1,25-dihydroxyvitamin D side chain analogs. More specifically, these keto intermediates can serve to prepare 5.6-cis or 5.6-trans-1a-25-dihydroxyvitamin D 1 analogs of the general side chain of formula 12, wherein X is an alkyl or aryl group proposes.

For example, the treatment of ketone (8) with ethylmagnesium bromide gives the corresponding hydroxy-25 vitamin D 1 analog with the above-mentioned side chain structure, wherein X represents an ethyl group. Similarly, treatment of (8) with isopropylmagnesium bromide or phenylmagnesium bromide gives the side chain analogs, wherein X represents isopropyl or phenyl, respectively. An 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 D 1 analog with the above side chain, wherein X represents the alkyl - or aryl residue, which is introduced by the Grignard reagent used.

It is also clear that the reaction of the keto intermediates (8) or (10) with an isotopic 8420137 C Λτ - 7 - 3 14 label Grignard reagent (for example CH_MgBr, CH-MgBr, 2 J 3 CH ^ MgBr, etc.) provides a suitable method for the preparation of la. 25-dihydroxy-vitamin D or the trans isomer thereof, and the corresponding C-24 epimers, in an isotopically labeled form, ie as the compounds 3 of the above side chain, wherein X represents C EL,

14 2 13 J

CH 2 / C CH 2 / or any other selected isotopically labeled alkyl or aryl group.

The above alkyl or aryl homologs of 5,6-cis- or -trans-1,25-dihydroxy vitamin are useful substitutes for the parent compounds in situations where a higher degree of lipophilicity is desirable, while the above isotopically labeled compounds find use as reagent in analytical applications.

Furthermore, while for therapeutic applications, the free hydroxy compounds represented by the above structures A and B (wherein R 1, R 2 and R 1 represent H) may generally be used for some such applications, the corresponding hydroxy- Protected derivatives are useful or preferred.

Such hydroxy-protected derivatives are, for example, the acylated compounds represented by the above general formulas A and B, wherein one or more of the groups R 1 represents an acyl group.

Such acyl derivatives are suitably prepared from the free hydroxy compounds using conventional acylation procedures, ie, by treatment of one of the hydroxy vitamin D products with an acyl halide or acid anhydride in a suitable solvent, such as pyridine or an alkyl pyridine.

By appropriate selection of the reaction time, acylating agent, temperature and solvent, as known in the art, the partially or fully acylated derivatives represented by the above structures 35 A or B are obtained. For example, the treatment of 1a-25-dihydrosyvitamin D2 (9a) in pyridine as a solvent gives 8413,137 * »$ ί - 8 - with acetic anhydride at room temperature the 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 barosthenic anhydride gives the 1,3-diacetyl-25-benzoyl or 1,3-diacetyl-25-succinoyl derivative, respectively. A 1,3.25-tri-acyl derivative can be selectively hydrolyzed 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 different (other) acyl groups. Analogously, a 1,3-diacyl derivative can be subjected to partial acyl hydrolysis to obtain the 1-O-acyl and 3-O-acyl compounds, which in turn can be reacylated with other acyl groups. An analogous treatment of any of the other hydroxyvitamin products (eg, 9b, 11a / b or their corresponding 25 alkyl or aryl analogs) provides the corresponding desired acyl derivatives represented by structures A or B, wherein one or more of the groups R ^, R ^ and R ^ acyl.

Like the previously known vitamin D metabolite, 1a-25-dihydroxyvitamin D2 (9a), the new compounds of this invention exhibit a pronounced vitamin D-like activity and therefore represent desirable substitutes for the known vitamin D2 or metabolites at many therapeutic or veterinary applications. Particularly preferred in this regard are the products having structures 9b and 11a and 11b or their acylated derivatives. The new compounds can be used to correct or improve a variety of calcium and phosphate imbalance states resulting from a variety of diseases, such as vitamin D resistant rickets, osteomalacia, hypoparathyroidism, osteodystrophy, pseudohypoparathyroidis - 8420137 - 9 - me, osteoporosis, Paget's disease, and analogous diseases known in medical practice, in which there is a connection between the bones and the available minerals. The compounds can also be used to treat mineral imbalance conditions in animals, for example, the waking fever disorder, weakness of the legs in birds, or to improve the eggshell quality of the poultry. Its use in the treatment of osteoporosis is particularly noteworthy.

It is well known that during the menopause, female individuals (women) 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 in both the United States and most other countries, in which women reach the ages of 20, 60 and 70 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. It is known that this disease is associated with a decreased ability to absorb calcium, decreased levels of the sex hormones, especially estrogen and androgen, and a negative calcium balance (calcium balance).

The methods used for the treatment of the disease varied considerably. For example, the calcium supplement alone was (in itself) unsuccessful in preventing or curing the disease and injecting sex hormones, especially estrogen, which has been reported to be effective in preventing the rapid loss of bone mass, which occurring in postmenopausal women was complicated by fears 8420137 - 10 - 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 approach is that fluoride causes structurally unhealthy bone (bone), referred to as woven bone, and also produces a number of side effects, such as an increased incidence of fractures and a gastrointestinal reaction to the large amounts administered fluoride.

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

15 Although varying metabolites of vitamin

Dg enhancing the calcium absorption and retention within the mammalian body, which show evidence of or a 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 epi compounds of this invention, especially 24-epi-1,25-dihydroxyvitamin Dg (24-epi-1,25- (OH) gD2), are extremely well suited for prevent or treat 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 levels, transport in the intestines and effecting bone mineralization, they do not increase serum calcium levels, even at high doses.This observed property proves that the compounds do not mobilize bone material after administration. together with the 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 have been found to be from a loss of bone mass, for example, postmenopausal osteoporosis, senile osteoporosis and steroid-induced osteo-5 porosis. It will be understood 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 renal dialysis, in which a 10 groin of bone mass occurs as a result of the dialysis.

The following examples serve to illustrate the properties of 24-epi-1.25- (OH), which contribute to its extraordinary suitability for the prevention or treatment of disease states, including loss of bone mass. occurs.

Example 1

Newly weaned male rats were 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 1.25-dihydroxyvitamin D4 or 24-epi-1,25-dihydroxyvitamin daily by subcutaneous injection in 0.1 ml 5% ethanol in propanediol. The animals were sacrificed 12 hours after the last dose and the blood calcium and visceral scale ium-tran sport was measured. The results of these measurements for the indicated amounts (levels) 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).

Example 2

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. See here-8420137; > - 12 - above). 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) 2 ^ 2 'while the other groups were administered 24-epi-1.25 (OH), both in 0.1 ml 5% ethanol in propanediol, the administration subcutaneously was effected 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 10 animals were sacrificed and the inorganic phosphorus in the serum was determined. The results are shown in Figure 3.

The bone axis was determined by removing the femurs from the rats. The thighs were cut from adherent connective tissue, extracted for 24 hours in absolute ethanol and for 24 hours in diethyl ether using a Soxhlet extractor. The bones were placed at 600 ° for 24 hours. F surprised. The axle weight was determined by weighing to constant weight.

The results are shown in Figure 4.

From the results of the two studies, described in Examples 1 and 2 above, it appears that the activity of 24-epi-1.25- (OH) is approximately equal to that of 1,25-dihydroxyvitamin D ^ (1.25- ( OH) 2) to effect bone mineralization and stimulate 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, becomes very apparent. influenced 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 amount of about 750 pmoles / day, the 24-epi-compound had no effect, while the mobilization effect is 8420137

* V

- 13 - It appears that at much lower doses of 1 · 25-dihydroxy vitamin D ^ · Since the increase in serum calcium of rats on a low calcium diet measures the ability to mobilize bone material and the increase in 5 blood phosphorus from animals on a low phosphorus diet also measures bone mobilization, these results show that 24-epi-1.25- (OE) 2 ^ 2 provides a surprising property, that it exhibits minimal activity in respect of of mobilizing bone calcium, while fully capable of stimulating visceral calcium transport and mineralization of new bones, which properties make this compound highly suitable for the treatment of disease states involving bone loss occurs.

15 The unique properties of 24-epi-1.25- (OH) 2 ° 2 'zoa - * - s indicated above provide the rare opportunity for the varying vitamin D-responsive processes (intestinal calcium absorption, bone mineral mobilization and bone mineralization) to control (control) in a manner and to an extent which has hitherto not been practicable. This possibility results from the fact that the 24-epi compound of this invention 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, can be administered. By such measures (measurements) 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 analogs. The administration of 24-epi-1.25- (OH) 2 ° 2 alone, as shown above, will stimulate visceral calcium transport and bone mineralization with no or minimal bone mineral mobilization, but the latter activity can be induced by simultaneous administration of one or more of the known vitamin D derivatives (for example, 1.25- (OH) 2 ^ 3 '1a 25- (OH) -> 2' 8420137 * - 14 - 1a-OH-D2, and related analogues) . By adjusting the relative amounts of the administered compounds, a degree of control of the relative sizes of the visceral calcium absorption relative to the bone mineral mobilization processes can be exercised in a manner not possible with the heretofore 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 some 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 15 derivative, which will effect bone mobilization, for example, Ια-hydroxyvitamin D ^ or -D ^, 25-dihydroxy-vitamin D ^ or -D ^, 25- hydroxyvitamin D4 or -D2, 24.24-difluoro-. 25-Hydroxyvitamin D ^, 24.24-Difluoro-1a-25-dihydroxyvitamin D ^ / 24-Fluoro-25-hydroxyvitamin D ^, 24-Fluoro-1a-25-dihy-20-hydroxyvitamin D ^, 2 β-fluoro-1a hydroxyvitamin D 2, 23-fluoro-25-hydroxyvitamin D 2, 23-fluoro-1a-25-dihydroxyvitamin D 2, 26.26.26.27.27.2'7-hexafluor-ία. 25-Dihydroxyvitamin D2,26.26.26.27.27.27-Hexafluoro-25-hydroxyvitamin. D ^, 24.25-dihydroxyvitamin D ^, la. 24.25-trihydroxyvitamin D ^, 25.26-di-25 hydroxyvitamin D ^, la.25.26-trihydroxyvitamin D ^ in combination with 24-epi-1.25- (OH) by setting the amounts of the 24-epi compound and the bone mobilizing vitamin D compound in the treatment regimen allow to adjust the extent of the bone mineralization in order to achieve the desired medical and physiological goals. Suitable and effective mixtures are, for example, the combination of 1a-25-dihydroxyvitamin D 2 and 1a-25-dihydroxy-24-epivitamin D 1 (9a and 9b), or mixtures of the corresponding 5.6-trans compounds (11a and 11b), or any combination of these four products as the free hydroxy compounds or as their acylated forms.

8420137 * * - 15 -

The compounds of this invention or combinations thereof with other vitamin D derivatives or other therapeutic agents can be readily administered as sterile parenteral solutions by injection or intravenously or 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, generally doses of from about 0.5 to about 25 micrograms per day are effective. The compounds 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 particular disease condition to be treated and the desired degree of the bone mineralization and / or bone mobilization. In the treatment of osteoporosis, wherein the preferred compound 24-epi-1a25- (0H) 2 ^ 2 ^ s', the amount of the 24-epi compound actually used is not limited.

In all cases, a sufficient amount of the compound must be used to induce (effect) bone mineralization. Amounts greater than about 25 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 for economic reasons are undesirable.

In practice, higher doses of the compounds are used when the therapeutic treatment of a disease state is the desired goal, while the lower doses are generally used for prophylactic purposes, it being understood that the specific dose administered in each given case will be established according to the specific compounds administered, the disease to be treated, the condition of the patient and the other relevant medical facts, which affect the activity of the drug or the response of the patient. patient, as is well known to 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 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, solution-promoting agents, etc. They may also contain other therapeutically valuable substances, such as other vitamins, salts, sugars, proteins, hormones or other medicinal compounds.

The method of the present invention is more particularly described in Examples 3-9 below. In these Examples, the designation of the specific products is by Arabic numerals (e.g., compounds 1, 2., 3, etc.). ) relates to structures thus numbered in reaction scheme I.

Example 3 1a-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 ^ μΐ) is treated with 50 mg of p-toluenesulfonyl chloride at 4 ° C for 30 hours . The mixture is poured onto ice / saturated NaHCO2 with stirring and the product is extracted with benzene. The combined organic phases are washed with aqueous NaHCO2, H2 O, aqueous 8420137

♦ P

- 17 -

CuSO4 and water / dried over MgSO4 and evaporated.

The crude 3-tosyl derivative (2) is directly solvolysed in anhydrous methanol (10 ml) and NaHCO 2 (150 mg) by heating at 55 ° C for 8 hours with stirring for 5 hours. The reaction mixture is then cooled to room temperature and in vacuo concentrated to about 2 ml. Benzene (80 ml) is then added and the organic layer is washed with water, dried and evaporated. The resulting cyclovitamine (3, Z = methyl) can be used in the subsequent oxidation without further purification.

The crude product (3) in CH 2 Cl 2 (4.5 ml) is added to an ice-cooled solution of SeO 2 (5.05 mg) and t-BuOOH (16.5 µl) in CH 2 Cl 2 (8 ml), containing 15 anhydrous pyridine (50 µl). After stirring at 0 ° C for 15 minutes, the temperature of the reaction mixture is allowed to rise to room temperature. After an additional 30 minutes, 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 give 20 mg of 1a-hydroxy derivative (4, Z = methyl): mass spectrum, 25 m / e: 470 (M +, 5), 438 (20), 87 (100); NMR (CDCL1) $ 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 and 5.25 (2H, each m, 19-H2 5.35 (2E, m, 22-H and 23-H).

Example 4 Acetylation of compound (4).

A solution of cyclovitamine (4, Z = methyl) (18 mg) 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 saturated NaHCO2 and extracted with benzene and ether. The combined organic extracts are washed with water, saturated 8420137 - 18% *.

CuSO. and aqueous NaHCO. solutions, dried and evaporated, 4-y to give the 1-acetoxy derivative (5, Z = methyl, acyl = acetyl) (19 mg): mass spectrum, m / e: 512 (M +, 5), 420 (5 ), 87 (100); NMR (CDCl 3) δ 0.53 (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 acetoxy-3,5-cyclovitamin (5) (R 1 = acetyl).

A solution of cyclovitamine (5) (4.5 mg)

10 in a 3: 1 mixture of dioxane-O (1.5 ml) is placed at 55 ° C

heated, p-toluenesulfonic acid (1 mg in 20 µl 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 ether. The organic phases are washed with NaHCO 3 and water and dried over MgSO 4. Evaporation of the solvents gives a residue containing compounds (6) (wherein R 2 = acetyl and R 2 * H) and (7) (where R 2 = acetyl and R 2 = H), which are separated by chromatography on HPLC (6.2 mm x 20 25 cm Zorbax-Sil) using 2% 2-propanol in hexane as an eluent. If necessary, the products can be further purified by repeated chromatography. Example 6

Ketal hydrolysis of compound (6) to give ketone (8).

To the solution of ketal (6, R 2 = acetyl, R 2 = H) (1.35 mg) in ethanol (1.5 ml), p-toluenesulfonic acid (0.34 mg in 45 µl H 2 O) is added and the mixture is heated to reflux for 30 minutes. The reaction mixture is poured into dilute NaHCO2 and extracted with benzene and 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 ^ = acetyl, 8420137 * - 4 - 19 - R = Η) (0.36 mg / collected at 52 ml), which is characterized

a

is expressed by the following data: mass spectrum, m / e: 454 (M +, 9), 394 (17), 376 (10), 134 (23), 43 (100); NMR (CDCl 3> δ 0.53 (3H, s, 18-H3), 1.03 (3H, d, J = 6.5 Hz, 5 21-H3), 1.13 (3H, d, J = 7 , 0 Hz, 28-H3), 2.03 (3H, s, CH3COO), 2.12 (3H, s, CRjCO), 4.19 (1H, m, 3-H), 5.03 (1H, m, 19-H), 5.33 '(3H, wide 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)? V (EtOH) 266 nm, 250 nm, 225 nm.

10 Example 7

Reaction of ketone (8) with methyl magnesium bromide to give products (9a) and (9b).

Ketone (8, R 2 = acetyl, R 2 = H) in anhydrous ether is treated with an excess of CH 3 MgBr (2.85 M solution in ether). The reaction mixture is stirred at room temperature for 30 minutes, then quenched with aqueous NH 4 Cl, extracted with benzene, ether and The organic phases are washed with dilute NaHCC> 3, dried over MgSO 4 and evaporated. The mixture of (9a) and (9b) thus obtained is separated by liquid chromatography (6% 2-propanol / hexane, 4.6 mm x 25 to give Zorbax-Sil) under drastic conditions, to obtain in the elution order, the pure epimers (9a) and (9b). la 25-dihydroxy vitamin D2 (9a): UV (EtOH) 25 λ 265.5 nm, λ. 227.5 nm? mass spectrum, m / e 428 (M +, 6), max min 410 (4), 352 (4), 287 (6), 269 (10), 251 (10), 152 (42), 134 (100), 59 (99); NMR (CDCl-j) δ 0.56 (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), 30 4.43 ( 1H, m, 1-H), 5.01 (1H, m, 19-H), * ^ 5.34 (3H, wide 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), 1a-25-dihydroxy-24-epivitamin D 4 (9b): OV (EtOH ) λ 265.5 nm, λ. 227.5 nm; mass spectrum, m / e 428 (M, 13), mm 35 410 (9), 352 (7), 287 (11), 269 (15), 251 (13), 152 (52), 134 (100), 59 (97).

8420137 - 20 -

Example 8

Conversion of compound (7) into 5.6-trans-1a. 25-dihydroxy-vitamin compounds (11a) and (11b).

Hydrolysis of the ketal intermediate 5 (7, R 1 * acetyl, = H) under the conditions described in Example 4 gives the corresponding 5.6-trans-25 ketone of the structure (10, R 1 = acetyl, R ^ = H) and by a subsequent reaction of this ketone with methyl magnesium bromide under conditions analogous to those of Example 5, a mixture of epimers (11a) and (11b) is obtained, which are separated by draining liquid chromatography (HPLC) under which conditions are obtained in pure form 1a-25-dihydroxy-5.6-trans-vitamin (11a) and 1a-25-dihydroxy-5.6-trans-24-15-epivitamin (11b). If necessary, the structure determination can be confirmed by isomerization to the respective 5.6-cis compounds (9a, 9b) according to known procedures.

5.6-trans-1a-25-dihydroxyvitamin (11a): 20 UV (EtOH) λ 273.5 nm, λ. 230 nm; mass spectrum, m / e max mm 428 (M, 8), 410 (3), 287 (3), 269 (7), 152 (34), 134 (100), 59 (78).

5.6-trans-1a-25-dihydroxy-24-epivitamin D

(11b): üV (EtOH) λ 273.5 nm, λ. 230 nm; mass spectrum, max mm 25 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 analogues of 1,25-dihydroxy vitamin compounds.

By reaction of ketone intermediate (8) (R ^ = acetyl, Rl = H) with (a) ethylmagnesium bromide (b) propylmagnesium bromide (c) isopropylmagnesium bromide 35 (d) butylmagnesium bromide and (e) phenylmagnesium bromide 8420137 - 21 - below Using conditions analogous to those described in Example 7, the corresponding hydroxy vitamin D products of the general formula 13 are obtained, wherein X represents 5 (a) ethyl (b) propyl (c) isopropyl (d) butyl and (e) phenyl.

An analogous treatment of the 5.6-trans-ketone intermediate (10) (R ^ = acetyl, = h) with the Grignard reagents listed above gives the corresponding 5.6-trans-hydroxy vitamin products of the general formula 14, wherein X represents -15, respectively (a) ethyl (b) propyl (c) isopropyl (d) butyl and 20 (e) phenyl.

A suitable starting material for the process of this invention is the vitamin D-ketal derivative of the structure (1), which can be obtained according to reaction schemes II and III, as described in British Patent Specification 2,127,023 or U.S. Patent - script 4,448,721. It is generally convenient (for example, when both C-24 epimers are desired) to use compound (1) as a mixture of 24R and 24S epimers, with the separation of the individual 24R and 24S- epiming is performed later. However, the pure 24S or pure 24R epimer of (1) is also suitable, the former providing for the 24S-1,25 dihydroxy product and the latter for the corresponding 24R product.

35 8420137

Claims (34)

1. Compounds selected from compounds of formulas A and B, in which and / which may be the same or different, are selected from hydrogen and acyl and X is selected from an alkyl or aryl group or an isotopically labeled alkyl or aryl group, wherein, when the C-24-methyl substituent in the 5.6-cis compound has the S configuration and X is methyl, R 1, R 2, and R 1 cannot be all hydrogen. Compounds according to claim 1, characterized in that X represents methyl. Connections according to claim 1, characterized in that the asymmetric center at C-24 has the 15 (R) configuration. Connections according to claim 1, characterized in that the asymmetric center at C-24 has the (S) T configuration. 5. lei.25-dihydroxy-24-epivitamin D2 · 6. la.25-dihydroxy-5.6-trans-vitamin 22 «7. la.25-dihydroxy-5.6-trans-24-epivitamin V 8. A pharmaceutical composition, characterized in that it contains a compound as defined in claims 1-7 and a pharmaceutically acceptable excipient. Preparation according to claim 8, characterized in that it contains 1,25-dihydroxy-5,6-trans-vitamin D2 and / or 1,25-dihydroxy-5,6-trans-24-epivitamin D2. 10. A composition according to claim 8 or 9, 30, characterized in that it contains 1,25-dihydroxy-5,6-trans-vitamin D2 and 1,25-dihydroxyvitamin D2. Product according to claims 8 or 9, characterized in that it contains 1,25-dihydroxy-5,6-trans-vitamin D2 and 1,25-dihydroxy-5,6-trans-24-epivitamin D2. 12. Preparation according to claim 8 or 9, characterized in that the la.25-dihydroxyvitamin D2, la.25- 8420137 - 22- * 5 * dihydroxy-24-epivitamin D ^, .25α.25-dihydroxy-5.6-trans -vi famine and la. 25-dihydroxy-5.6-trans-24-epivitamin. 13. A composition according to claim 8, characterized in that it contains 1,25-dihydroxy-vitamin D2 and 1,25-dihydroxy-24-epivitamin. A pharmaceutical composition, characterized in that it contains 1a.25-dihydroxy-5.6-trans-vitamin and either 1a-25-dihydroxy-5.6-trans-25-epivitamin or 10 1a-25-dihydroxy-24-epivitamin . Preparation according to claims 8-14, characterized in that it contains at least one bone mobilization-inducing compound. 16. A composition according to claim 15, characterized in that the bone mobilization-inducing compound is a vitamin D derivative selected from 25-hydroxy-vitamin, 25-hydroxyvitamin Ό21 la-hydroxyvitamin, 1a-hydroxyvitamin 1a-25-dihydroxyvitamin , la.25-dihydroxyvitaminè 24.24-difluoro-25-hydroxyvitamin, 20 24.24-difluor-la.25-dihydroxyvitamin, 24-fluoro-25- + hydroxyvitamin, 24-fluoro-la.25-dihydroxyvitaminD2, 26.26.26.27.27.27- hexafluoro-la.25-dihydroxyvitamin, 26.26.26.27.27.27-hexafluoro-25-hydroxyvitamin, 2β-fluoro-la-hydroxyvitamin D ^, 28-fluoro-25-hydroxy-25 vitamin D ^, 24.25-dihydroxyvitamin, la.24.25 -tri-hydroxyvitamin, 25.26-dihydroxyvitamin and la.25.26-trihydroxyvitamin 17. Compounds of formula 16, wherein Y represents hydrogen, hydroxy or O-acyl and Z an alkyl group. Compounds according to claim 17, characterized in that Y is hydrogen. Compounds according to claim 17, characterized in that Y represents hydroxy or O-acetyl. A compound according to claim 18, characterized in that Z is methyl. 8420137 1 h. ! · '* - atf- Compound according to claim 19, characterized in that Z is methyl. 22. Compounds selected from the compounds of formulas 17 and 18, wherein K represents an oxygen or ethylenedioxy group and wherein and R 4, which may be the same or different, represent hydrogen or acyl. Compounds according to claim 22, characterized in that K is an oxygen group. 24. Compounds according to claim 22, characterized in that K is an ethylenedioxy group. 25. 1a-hydroxy-25-oxo-27-nor-vitamin D2 and its acetate. A method of preparing a compound of the formula as defined in claim 1, wherein R 1, R 2 and R 2, which may be the same or different, represent hydrogen or acyl and X alkyl or aryl or an isotopically labeled alkyl or aryl group, characterized in that a ketal of the formula 15 or 7 is used, wherein R 1 and R 2 have the meanings indicated above, is subjected to hydrolysis at a temperature of 10-38 ° C under acidic conditions and reacts the resulting ketone with a Grignard reagent. 27. Process according to claim 26, characterized in that the hydrolysis is carried out using p-toluenesulfonic acid. 28. A method for the prevention or treatment of physiological disorders in mammals, which disorders are characterized by the requirement to regenerate or prevent the loss of bone mass, characterized in that a therapeutic target amount of la. 25 is provided to these mammals. dihydroxy-24-epivitamin D2, alone or in combination with at least one vitamin D compound, which is characterized by its ability to mobilize bone material in vivo. 29. A method according to claim 28, characterized in that the disorder is a postmenopausal osteoporosis. 8420137?> A; - «· - A method according to claim 28, characterized in that the disorder is a senile osteoporosis. 31. A method according to claim 28, characterized in that the disorder is a steroid-induced osteoporosis. A method according to claim 29, characterized in that the compound is administered to women during and after menopause. 33. A method according to claim 29, characterized in that the compound is administered to women before the onset of menopause. 34. A method according to claim 28, characterized in that the compound is administered in an amount from about 0.5 micrograms to about 25 micrograms per day. A method according to claim 28, characterized in that the compound is administered orally in capsule form, in solution in a liquid vehicle that is settable by and non-toxic to these mammals. A method according to claim 28, characterized in that 1,25-dihydroxy-24-epivitamin D2 is the only compound administered. 37. A method according to claim 28, characterized in that 1,25-dihydroxy-24-epivitamin D2 is administered in combination with at least one vitamin D compound characterized by the ability to mobilize bone material in vivo. 38. 1a-Hydroxy-25-oxo-27-nor-24-epivitamin D2 · 8420137