DK171397B1 - 24-Epi-1alpha,25-dihydroxylated vitamin D2 derivatives, and preparations for pharmaceutical use which comprises these derivatives - Google Patents

Description

in DK 171397 B1

The present invention relates to novel 24-epi-1α, 25-dihydroxylated vitamin D 2 derivatives which are characterized in that they have the formula I as set forth in claim 1, pharmaceutical compositions 5 The characterizing part of claim 4, and the use of the derivatives for the preparation of a composition for the treatment of diseases requiring regeneration of bone mass or to prevent loss of bone mass, preferably postmenopausal osteoporosis, senile osteoporosis or steroid-induced osteoporosis.

The importance of the hydroxylated forms of vitamin D as regulators of calcium and phosphorus metabolism in animals and humans is well known and described in several patents 15 and in the literature, and as a result, these hydroxyvitamin D derivatives increasingly find clinical and veterinary use as drugs for the treatment of morbid conditions in calcium metabolism and associated bone disorders. It is known that vitamin D3 is hydroxylated in vivo to 25-hydroxyvitamin D3 and then to 1α, 25-dihydroxyvitamin D3, the latter compound being widely accepted as the active hormonal form of vitamin D3. Also, the extremely potent vitamin D2 metabolite Ia, 25-dihydroxyvitamin D2 (Ia, 25-25 (OH) 2 ^ 2) is formed from vitamin D2 via 25-hydroxyvitamin D2 (25-OH-D2). Both of these hydroxylated vitamin D2 compounds have been isolated and identified (DeLuca et al., U.S. Patent Nos. 3,585,221 and 3,880,894); derived from vitamin D2, these metabolites are characterized by (S) stereochemistry at carbon 24.

Surprisingly, it has been found that the epi-compounds in question exhibit a potent vitamin D-like effect. However, although they very effectively stimulate intestinal calcium-calcium transport and mineralization of new bones, they have minimal effect on the mobilization of bone calcium.

DK 171397 B1 2

These properties therefore open up the possibility of controlling various vitamin D-dependent processes in a way that has not previously been possible with more common vitamin D derivatives.

Particular examples of the subject compounds are 1α, 25-dihydroxy-24-epivitamin D 2, as well as the C-25 alkyl homologues of this compound, i. compounds of the above formula wherein X represents ethyl, propyl, isopropyl, butyl or isobutyl.

Typical R 1, R 2 and R 3 groups include alkylcarbonyl as formyl, acetyl, butyryl, isobutyryl and valeryl or aromatic carbonyl such as e.g. benzoyl, or an acyl group derived from a dicarboxylic acid of the general formula R00C (CH2) nCO or R00CCH2-0-CH2CO- such as e.g. oxalyl, malonyl, succinoyl, glutaryl, adipyl and diglycolyl.

An embodiment of the preparation of the subject compounds is illustrated in attached Scheme I.

In the following depiction of this process, the numbers (1, 2, 3, etc.), denoting the specific ones for bonds, refer to the formulas numbered in this way in Scheme I.

25

A suitable starting material for the process is the vitamin D ketal derivative of formula (1). It may be convenient to use compound (1) as a mixture of the 24R and S epimers, since the R-configuration derivative at 30C-24 can be isolated during a later step of the process.

However, the pure 24R epimer of (1) is also well suited as starting materials to obtain the corresponding (24R) -la, 25-dihydroxylated final compound. 1

The starting material (1) is converted to the desired 1α, hydroxylated form via the cyclovitamin D derivatives (DeLuca DK 171397 B1 3 et al., U.S. Patents 4,195,027 and 4,260,549). Thus, treatment of the compound (1) with toluene sulfonyl chloride in the usual manner gives the corresponding C-3 tosylate (2), which is solvolysed in an alcoholic substrate to obtain the novel 3,5-cyclovitamin D derivative (3). Solvolysis in methanol gives the cyclovitamin of formula (3) where Z = methyl, while the use of other alcohols such as e.g. ethanol, 2-propanol or butanol in this reaction give the analogous cyclovitamin D compounds (3) wherein Z represents an alkyl group derived from the alcohol e.g. ethyl, isopropyl or butyl. Allyl oxidation of the intermediate (3) with selenium dioxide and a hydroperoxide gives the Ια-hydroxy analog of formula (4). Subsequent acetylation of the compound (4) gives the 1-acetate of formula (5, R 1 = acetyl). Optionally, other 1-O acylates (formula 5 wherein R 1 = acetyl, for example, the formate, propionate, butyrate or benzoate) are prepared by analogous ordinary acylations. The 1-O-acyl derivative is then subjected to acid-catalyzed solvolysis. If this solvolysis is carried out in a solvent containing water, the 5,6-cis-vitamin D intermediate of formula (6, R 1 = acyl, R 2 = H) and the corresponding 5,6-trans compound (formula 7, R 1 acyl, R2 = H) in a ratio of 3-4: 1. These 5,6-cis and 5,6-trans isomers can be separated at this stage e.g. using high capacity liquid chromatography. If desired, the C-1-O acyl group can be removed by base hydrolysis to give compounds (6) and (7), wherein R 1 and R 2 = H. Optionally, these 1-O monoacylates can also be further acylated at C-3 hydroxy groups using ordinary acylation conditions to obtain the corresponding 1,3-di-0-acylates of formulas (6) and (7), wherein R 1 and R 2, which may be the same or different, represent acyl groups. Alternatively, the hydroxycyclovitamin red formula (4) may be subjected to acid-catalyzed solvolysis in a substrate containing a low molecular weight organic acid to give 5,6-cis and trans-compounds of formulas (6) and (7). ) wherein R 1 = H and R 2 = acyl, wherein the acyl group is derived from the acid used in the solvolysis.

The next step of the process comprises removing the metal protecting group to prepare the corresponding 25 ketone. This step is critical as the conversion of the ketone to the ketone must be accomplished without simultaneous isomerization of the 22 (23) double bond to the conjugated 23 (24) position which may occur under the acidic conditions required for ketal hydrolysis. Further, the conditions must be selected so as to avoid the removal of the sensitive allyl C-1 oxygen function. The conversion is successfully carried out by gentle hydrolysis at moderate temperatures using organic acid catalysis. Thus, treatment of the 5,6-cis compound (6) in aqueous alcohol with p-toluene-sulfonic acid gives the corresponding ketone (8).

In order to avoid unwanted removal of the C-1 oxygen function during the reaction, it is advantageous to protect the C-1-hydroxy group 20 in the compound (6) (eg R 1 - acyl, R 2 = acyl, R 2 = hydrogen or acyl).

Subsequent reaction of ketone (8) with a methyl Grig-nard reagent then gives the desired 1α, 25-dihydroxy-vitamin D 2 compound of formula (9). If the starting material, compound (1) used in the above process is a mixture of the two C-24 epimers, then the compound (9) will be obtained as a mixture of the 24-S and R epimers (9a and 9b, respectively). ). The separation of this epimeric mixture can be obtained by chromatographic methods to obtain 1α, 25-dihydroxyvitamin D2 (formula 9a, 24S stereochemistry) and its 24R epimer, 1α, 25-dihydroxy-24-epivitamin D2 of formula 9b, both compounds are available in pure form. Of course, such separation of the epimers is not necessary if the compounds are to be used in the form of a mixture. The 5,6-trans-25-ketal intermediate of the product of formula (7) is similarly subjected to ketal hydrolysis to yield the 5,6-transketone intermediate of formula (10) which, via a Grignard reaction with methyl magnesium bromide or a by analogy, 5,6-trans-5α, 25-dihydroxyvitamin Dz gives the compounds of formula (11) as the 24S or 24R epimer, or as a mixture of both epimers depending on the nature of the starting material (1) used in method. If obtained as an epimeric mixture, the epimers can be separated by chromatography to give 5,6-trans-1α, 25-dihydroxyvitamin Oz (Ha) and its 24R epimer 5,6-trans-1α, 25-dihydroxy-24 -epivitamin D2 of formula (11b). These reactions using the 5,6-trans-intermediates are carried out in a manner completely analogous to those used for the 5,6-cis compounds described above.

The novel side chain ketones of formulas (8) or (10) are the most valuable and easily convertible intermediates because they can be used to prepare a full 20 series of 1α, 25-dihydroxyvitamin D2 side chain analogs. Specifically, these keto intermediates may serve to prepare 5,6-cis or 5,6-trans-1α, 25-dihydroxyvitamin D2 analogs of the general side chain formula set forth below.

✓ν »X

wherein X represents an alkyl group.

For example, treatment of the ketone (8) with ethylmagnesium bromide gives the corresponding hydroxyvitamin D2 analog having the above side chain formula, wherein X represents an ethyl group. Similarly, treatment of (8) with propyl, butyl or isopropylmagnesium bromide gives the side chain analogs where X represents propyl, butyl or isopropyl, respectively. Analogous treatment of the 5,6-trans-25-ketone intermediate of formula (10) with alkyl-Grignard reagents yields the 5,6-trans-vitamin D2 analog having the above side chain, wherein X represents alkyl introduced by the Grignard reagent used.

5

The above alkyl homologue of 5,6-cis- or trans-1α, 25-dihydroxyvitamin D2 is valuable substitutes for the parent compounds in situations where greater fat solubility is desired.

10

Although the free hydroxy compound of formula I above (wherein R 1, R 2 and R 3 = H) is further usually used for therapeutic purposes, it may in some cases be advantageous to use the corresponding hydroxy protective derivative. This hydroxy protected derivative is e.g. the acylated compounds of general formula I above, wherein one or more of R 1, R 2 and R 3 represents an acyl group. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

These acyl derivatives are conveniently prepared from the free hydroxy compound by ordinary acylation methods, i.e.

3 treating any of the hydroxyvitamin D 2 final compounds with an acyl halide or acid anhydride in a suitable solvent such as e.g. pyridine or an alkyl pyridine. By appropriate selection of reaction time, acylating agent, temperature and solvent well known in the art, the partially or fully 9 permanently acylated derivatives of the above form are obtained in clay 10, e.g. gives treatment of 1α, 25-dihydroxyvitamin 11 D2 (9a) in pyridine solvent with acetic anhydride 12 at room temperature the 1,3-diacetate, while the same reaction 13 carried out at elevated temperature gives the corresponding 14 1,3,25-triacetate. The 1,3-diacetate can be further acylated at C-25 with another acyl group e.g. by treatment 16 with benzoyl chloride or succinic anhydride to give the 1,3-diacetyl-25-benzoyl or 1,3-diacetyl-25-succinoyl derivative, respectively. The 1,3,25-triacyl derivative can be seen to be selectively hydrolyzed in the mild base to give the 1,3-dihydroxy-25-O-acyl compound, the free hydroxy groups of which can be acylated again optionally with other acyl groups. Similarly, the 1,3-diacyl derivative may be partially subjected to acyl hydrolysis to obtain the 1-O-acyl and the 3-O-acyl compounds, which in turn can be acylated with other acyl groups. Similar treatment of any of the other hydroxyvitamin D2 end products (e.g.

9b, 11a / b or their corresponding 25-alkyl analogs) give the corresponding acyl derivatives of formula I wherein any or all of R 1, R 2 and R 3 represent acyl.

Like the prior art vitamin D2 metabolite Ia, 25-15 dihydroxyvitamin D2 (9a), the compounds of this invention exhibit pronounced vitamin D-like activity and are thus suitable substitutes for the known vitamin D2 or D3 metabolites for a variety of therapeutic or veterinary purpose. Particularly preferred for this is the compounds of formulas 9b and 11a and 11b and their acylated derivatives. The present compounds can be used to repair or improve a whole range of calcium and phosphate imbalances caused by a variety of diseases such as Vitamin D-resistant rakis, osteomalacia, hypoparathyroidism, osteodystrophy, pseudohypoparathyroidism, osteoporosis, Paget's disease and similar bone and mineral dependent medical conditions well known in the medical practice. The compounds may also be used in the treatment of mineral imbalances in animals e.g. calving fever, weakness in the legs of poultry or to improve the eggshell quality of poultry. Their use in the treatment of osteoporosis is particularly noteworthy. 1

It is well known that women around the menopause suffer from a marked loss of bone mass, which can eventually cause osteopenia, which in turn causes spontaneous vertebra fractures and long bone fractures. This disease is commonly referred to as postmenopausal osteoporosis and is a significant medical problem in 5 countries where women's lifespan is around 60-70 years. Usually, this disease, often accompanied by bone pain and decreased physical activity, is diagnosed by one or two vertebral fractures, with an X-ray finding that there is a diminished bone mass. It is well known that this disease is accompanied by diminished ability to absorb calcium, reduced levels of sex hormones, especially estrogen and androgen and a negative calcium balance.

15 Methods for treating this disease have varied considerably. For example. the calcium supplement itself has not been able to prevent or cure the disease, and the injection of sex hormones, especially estrogen, which has been reported to be effective in preventing the rapid loss of bone mass seen in postmenopausal women has been accompanied by the fear of the possible carcinogenic effect of estrogen. Other treatments for which different results have been reported again have included a combination of vitamin D in large doses of calcium and fluoride. The primary problem with this solution is that fluoride induces structurally unhealthy bones, called open structured bones, and in addition produces a variety of effects such as increased cases of fractures and gastrointestinal reactions over 30 excessive amounts of fluoride.

Similar symptoms characterize senile osteoporosis and steroid-induced osteoporosis, the latter being recognized as a result of long-term glucocorticoid (corticosteroid) therapy for certain morbid conditions.

DK 171397 B1 9

While various metabolites of vitamin D3 enhance calcium absorption and retention in mammalian bodies showing signs or physiological tendency for bone loss, they are also characterized by the complementary vitamin D-like property in the form of mobilizing calcium in the bones as answers to physiological needs. It has now been found that the epi compounds, in particular 24-epi-1α, 25-dihydroxyvitamin D2 (24-epi-1.25- (OH) 2D2), are remarkably well suited to prevent or treat physiological morbid conditions in mammals characterized by loss of bone mass because, although they possess some of the recognized vitamin D-like properties that affect calcium metabolism such as increased intestinal calcium transport, and affects bone mineralization, does not increase serum calcium levels even at high doses. This observed property indicates that the compounds do not mobilize bone upon administration. This fact, together with the ability of the compounds to mineralize bone upon administration, indicates that they are ideal compounds for preventing or treating widespread morbid potassium teeth seen in the loss of bone mass, e.g. postmenopausal osteoporosis, senile osteoporosis, and steroid-induced osteoporosis. It is to be understood that the compounds 25 will readily be used in the prophylaxis or treatment of other morbid conditions where bone loss is observed e.g. in the treatment of patients undergoing renal dialysis where loss of bone mass as a consequence of the dialysis may occur.

The following examples illustrate the properties of 24-epi-1.25- (OH) 2D2, which demonstrate the excellent ability of this compound to prevent or treat morbid conditions in which bone loss occurs.

EXAMPLE 1

Weaned male rats were fed the vitamin D deficient diet described by Suda et al., Journal of Nutrition 5 100, 1049-1052 (1970), modified to contain 0.02% calcium and 0.3% phosphorus. After 2 weeks on this diet, the animals received either 1,25-dihydroxyvitamin D2 or 24-epi-1,25-dihydroxyvitamin D2 daily by subcutaneous injection in 0.1 ml of 5% ethanol in propanediol. 12 hours 10 after the last dose, the animals were sacrificed and blood calcium and intestinal calcium transport were measured. The results of these measurements for the indicated levels of the administered compounds are given in FIG. 1 and 2. The intestinal calcium transport measurements shown in FIG. 2 was performed as described 15 by Martin and DeLuca, American Journal of Physiology 216, 1351-1359 (1969).

Example 2 Weaned male rats were fed a high calcium (1.2% calcium) and low phosphorus (0.1% phosphorus) diet described by Suda et al. (See above). The rats were fed this diet for 3 weeks, at which time they were divided into two groups. One group was assigned 1.25 (OH) 2D2, while the other group was assigned 24-epi-1.25 (OH) 2D2, both in 0.1 ml of 5% ethanol in propanediol subcutaneously in the dosage amounts of the compounds which is indicated by the points in FIG. 3. These doses were continued daily for 7 days, at which time the animals were sacrificed and the serum inorganic phosphorus determined. The results are shown in FIG. Third

Bone ash was determined by removing the femur from the rats. The bones were dissected free of adherent connective tissue, extracted 24 hours in absolute ethanol and 24 hours in diethyl ether using a Soxhlet ex-171797 B1 11 tractor. The bones were ashed at ca. 315 ° C for 24 hours. The ash weight was determined by weighing until constant weight. The results are shown in FIG. 4th

The results of the two studies described in Examples 1 and 2 above show that 24-epi-1,25- (OH) 2D2 is about as potent in effect as Ia, 25-dihydroxyvitamin D3 (1,2,25- (OH)). 2D3) to effect bone mineralization and to stimulate intestinal calcium transport. In short, there is no significant difference between the two groups in FIG.

2 and FIG. 4. On the other hand, the increase of the inorganic phosphorus in serum arising from the mobilization of the bone in the case of the low phosphorus diet is very markedly affected by 1,25- (OH) 2D3 / D2, but barely 15 stimulated with 24-epi-1.25 (OH) 2D2. Likewise, at the mobilization of calcium from the bone, the 24-epi compound had been indicated as the serum calcium levels (Fig. 1) even at extremely high doses of ca. 750 pmol / day no effect, while the mobilizing effect is evident at 20 significantly lower doses of 1,25-dihydroxyvitamin D2 · As the increase in serum calcium in rats on a low calcium diet is an expression of the ability to mobilize the bone and then increase the blood phosphorus in animals on a low phosphorus diet is also an indication of bone mobilization, 25 these results show that 24-epi-1,25- (OH) 2D2 exhibits an unexpected property, namely that it has very little ability to mobilize bone calcium, whereas it is completely capable of stimulating intestinal calcium transport and mineral iron none of the new bones, properties that make this compound particularly suitable for treating the morbid conditions that appear in bone loss.

The unique properties of the 24-epi-1,25- (OH) 2D2 listed above offer the rare opportunity to control 35 different vitamin D-dependent processes (intestinal calcium transport, bone mineral mobilization and bone mineralization) in a manner and in extent that so far has not been possible. This possibility is due to the fact that the subject 24-epi compound can be administered to mammals either alone (with appropriate and acceptable adjuvants) or together with other vitamin D derivatives exhibiting the full spectrum of vitamin D-like effect. . Because of this, it is therefore possible to combine (depending on the degree to which you want) the specific effect of the 24-epi analog with the general effect of 10 other vitamin D metabolites or analogs. Administration of 24-epi-1,25- (OH) 2D2 alone will, as indicated above, stimulate intestinal calcium transport and bone mineralization with no or minimal bone mineral mobilization, but the latter effect may be induced by co-administration of one or more of the known vitamin D derivatives (e.g., 1,25- (OH) 2D3, Ia, 25- (OH) 2D2, I01-OH-D3 and related analogues). By adjusting the relative amounts of the administered compounds, one can gain a degree of control over the relative magnitudes of the processes of bone calcium absorption relative to bone mineral mobilization in a manner not possible with the known vitamin D derivatives. Co-administration of the 24-epi compound and other vitamin D compounds with bone mobilizing activity may be particularly advantageous in the situation where some degree of bone mobilization is desired. For example. it is believed that in some cases the bone must first be mobilized before a new bone can be formed. In these situations, treatment with vitamin D or a vitamin D derivative will induce bone mobilization such as e.g. 1α, 25-dihydroxyvitamin D3 or -D2, 25-hydroxyvitamin D3 or -D2, 24,24-difluoro-25-hydroxy vitamin D3, 24,24-difluoro-ΐα, 25-dihydroxyvitamin D3, 24-fluoro 25-hydroxyvitamin D3, 24-fluoro-1α, 25-dihydroxyvitamin D3, 2β-fluoro-1α-hydroxyvitamin D3,2β-isluoro-25-hydroxyvitamin D3, 2β-fluoro-1α, 25-dihydroxyvitamin D3, 26,26, 26,27,27,27-hexafluoro-ΐα, 25-dihydroxyvitamin D3, 26,26,26,27,27, - DK 171397 B1 13 27-hexafluoro-25-hydroxyvitaminin D3, 24,25-dihydroxyvitamin D3, la , 24,25-trihydroxyvitamin D3, 25,26-dihydroxyvitamin D3, Ia, 25,26-trihydroxyvitamin D3 in combination with 24-epi-1,25 (OH) 2D2 by adjusting the ratios of the 24-5 epi compound to the bone mobilizing agent Vitamin D compounds within the treatment area allow the rate of bone mineralization to be adjusted to achieve the desired medical and physiological end results. Suitable and effective mixtures are e.g. the combination of Ia, 25-dihydroxyvitamin D2 and Ia, - 25-dihydroxy-24-epivitamin D2 (9a and 9b) or mixtures of the corresponding 5,6-trans compounds (11a and 11b) or any other combination of these four final compounds as the free hydroxy compounds or in their acylated forms.

The present compounds 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 orally in the form of oral doses or transdermally or by suppositories. Advantageously, the compounds are administered in doses ranging from 0.1 to 100 micrograms per day. day. For osteoporosis, doses of 0.5 to 25 mi-25 hook-grams per day usually effective. The compounds can be administered either alone or with other vitamin D derivatives, the amount of each compound in the combination being dependent on the disease state to be treated and on the desired degree of bone mineralization and / or bone mobilization. In the treatment of osteoporosis, where the preferred compound is 24-epi-1α, 25- (OH) 2D2, the actual amount of the 24-epi compound used is not critical. In all cases, it is sufficient for the compound to be used to induce bone mineralization. Quantities over approx. 25 micrograms per day of the 24-epi compound or the combination of this compound with bone mobilization-inducing vitamin D derivatives are usually unnecessary to achieve the desired results and are not relevant economically. In practice, higher doses of the compounds are used, with therapeutic treatment of a diseased condition being the desired target, while the lower doses are usually used for prophylactic purposes, as it is clear that the dose administered in each case is adjusted according to 10 the particular compound being administered, depending on the disease to be treated, and on the patient's condition and other relevant medical facts that may influence the activity of the drug or the patient's response, as is well known to those skilled in the art.

Dosage forms of the compounds can be prepared by combining these with non-toxic pharmaceutically acceptable carriers well known in the art. Such carriers may be either solid or liquid, e.g. corn starch, lactose, sucrose, peanut oil, olive oil, sesame oil and propylene glycol. If a solid carrier is used, the dosage form of the compounds may be tablets, capsules, powders, dragees or lozenges.

If a liquid carrier is used, the dosage form may be soft gelatin capsules or syrups or liquid suspensions, emulsions or solutions. The dosage forms may also contain adjuvants, e.g. preservatives, stabilizers, wetting agents or emulsifiers, solvents and the like. They may also contain other therapeutically valuable compounds, e.g. other vitamins, salts, sugars, proteins, hormones or other medical compounds. 1

The preparation of the subject compounds is described in more detail in Examples 3-8 below. In these examples, DK 171397 B1 15 designates the individual end products as compounds 1, 2 and 3 which correspond to the numbers given in Scheme I.

EXAMPLE 3 1a-Hydroxy-3,5-cyclovitamin D (4, Z - methyl).

A solution of compound (1) (50 mg) (as a mixture 10 of 24 R and S epimers) in dry pyridine (300 μΐ) is treated with 50 mg of p-toluenesulfonyl chloride for 30 hours at 4 ° C. The mixture is poured over ice / saturated NaHCC> 3 with stirring and the compound is extracted with benzene. The combined organic phases are washed with aqueous NaHOCl3, H2O, aqueous CuSO4, 15 and water, dried over MgS (> 4 and evaporated).

The crude 3-tosyl derivative (2) is directly solvated in anhydrous methanol (10 ml) and NaHOC3 (150 mg) by heating 8.5 hours with stirring at 55 ° C. The reaction mixture is then cooled to room temperature and concentrated to about 2 ml in vacuo. Benzene (80 ml) is then added and the organic layer is washed with water, dried and evaporated. The cyclovitamine (3, Z - methyl) produced can be used in the subsequent oxidation without further purification.

The crude compound (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 μΐ) in CH 2 Cl 2 (8 mL) containing anhydrous pyridine (50 30 μΐ). After stirring at 0 ° C for 15 minutes, allow the reaction mixture to rise to room temperature. After an additional 30 minutes, transfer the mixture to a separatory funnel and shake 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 DK 171397 B1 16 20 cm plates, AcOEt / hexane 4: 6) to give 20 mg of the Ια-hydroxy derivative (4, Z = methyl): mass spectrum, m / e: 470 (M; 5), 438 (20), 87 (100); NMR (CDCl 3) δ 0.53 (3H, s, 18-H3), 0.63 (1H, m, 3-H), 4.19 (1H, d, j = 9.5 Hz, 6-5 H ), 4.2 (1H, m, 1-H), 4.95 (1H, d, J = 9.5 Hz, 7-H), 5.17 and 5.25 (2H each m, 19-H2 ), 5.35 (2H, m, 22-H and 23-H).

EXAMPLE 4 Acetylation of Compound (4).

A solution of 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-cold saturated NaHCO 3 and extracted with benzene and ether. The combined organic extracts are washed with water, saturated CuSO4 and aqueous NaHCO3 solutions, dried and evaporated to give 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 25

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

A solution of cyclovitamine (5) (4.5 mg) in a 3: 1 mixture of dioxane / H 2 O (1.5 ml) is heated at 55 ° C. Then p-toluenesulfonic acid (1 mg in 20 ml of water) is added and heating is continued for 15 minutes. The mixture is poured into saturated NaHCO 3 / ice and extracted with benzene and ether.

The organic phase is washed with NaHCO 3 and water and dried over MgSO 4. Evaporation of the solvents gives a residue containing compounds (6) (wherein R 1 = acetyl, R 2 = H) and (7) (wherein R 1 = acetyl and R 2 = H) which is separated by chromatography on HPLC (6.2 mm x 25 cm "Zor-bax-Sil") using 2% 2-propanol in hexane as eluant. Optionally, the final compounds can be further purified by repeated chromatography.

gyS3MREL, .f

Ketal hydrolysis of compound (6) to give the ketone 10 (8).

To the solution of the ketal (6, R 1 = acetyl, R 2 = H) (1.35 mg) in ethanol (1.5 ml) is added p-toluenesulfonic acid (0.34 mg in 45 μΐ water) and the mixture is heated for 30 minutes with 15 reflux. The reaction mixture is poured into dilute NaHCO 3 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 20 "Zorbax-Sil") gives unreacted ketone (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), characterized by the following results: 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, 21-H3), 1.13 (3H, d, J = 7 2.0 Hz, 28-H3), 2.03 (3H, s, CH3COO), 2.12 (3H, s, CH3CO), 4.19 (1H, m, 3-H), 5.03 (1H, m, 19-H), 5.33 (3H, wide m, 19-H, 22-H and 23-H), 5.49 (1H, m, 1H), 5.93 (1H, d, J = 11 Hz, 7-H), 6.37 (1H, d, J 11 Hz, 6-30 H); UV (EtOH λ ^ χ 266 nm, 250 nm, λ ^ η 225 run.

Example 17

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

5

Ketone (8, R 1 = acetyl, R 2 = H) in anhydrous ether is treated with excess CH 3 MgBr (2.85 M solution in ether). The reaction mixture is stirred for 30 minutes at room temperature, then quenched with aqueous NH 4 Cl, extracted with benzene, ether and CH 2 Cl 2 · The organic phases are washed with dilute NaHCO 3, dried over MgSO 4 and evaporated. The mixture of (9a) and (9b) prepared in this way is separated by high-pressure liquid chromatography (6% 2-propanol / hexane, 4.5 mm x 25 cm "Zorbaz-Sil") to obtain, after elution, the pure epimers ( 9a) and (9b). 1α, 25-dihydroxy vitamin D 2 (9a); UV (EtOH) λ 2 χ 265.5 nm, 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 (CDCl 3) δ 0.56 (3H, s, 18-H3), 1.01 (3H, d, J = 6.5 Hz, 28-H3), 1.03 (3H, d, J = 6 , 5 Hz, 21-H3), 1.14 and 1.18 (6H, each s, 26-H3 and 27-H3), 4.24 (1H, m, 3-H), 4.43 (1H, m, 1-H), 5.01 (1H, m, 19-H), ~ 5.34 (3H, 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).

25a, 25-dihydroxy-25-epivitamin D2 (9b): UV (EtOH) λmax 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) ).

EXAMPLE 8

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

5

Hydrolysis of the ketal intermediate (7, R 1 = acetyl, R 2 = H) under the conditions of Example 4 gives the corresponding 5,6-trans-25 ketone of formula (10, R 1 = acetyl, R 2 = H) and subsequent reaction of this ketone 10 with methyl magnesium bromide using conditions analogous to those set forth in Example 5 yields a mixture of the epimeric compounds (11a) and (11b) which are separated by high pressure liquid chromatography (HPLC) to give of the pure 1α, 25-dihydroxy-5,6-trans-vitamin-D2 (Ha) and 1α, 25-dihydroxy-5,6-trans-24-epi-vitamin D2 (11b). If desired, the formula can be configured by isomerization to the respective 5,6-cis compounds (9a, 9b) by well known methods.

5,6-trans-1α, 25-dihydroxyvitamin D2 (11a); UV (EtOH) λmax 273.5 nm, λ, ηΐη 230 nm, mass spectrum, m / e 428 (M +, 8), 410 (3), 287 (3), 269 (7), 251 (7), 152 ( 34), 134 (100), 59 (78).

5,6-trans-1α, 25-dihydroxy-24-epi-vitamin D2 (11b); UV

(EtOH) Araax 273.5 nm, λ, ηΐη 230 nm, mass spectrum, m / e 428 (M +, 10), 410 (4), 352 (4), 287 (5), 269 (9), 251 (8 ), 152 (37), 134 (100), 59 (82). 1 2 3 4 5 6

A suitable starting material for the compounds of the present invention is the vitamin D ketal derivative of formula (1), which can be obtained by working as described in Reaction Schemes II and 4 III as disclosed in English Patent No. 2 127 023 or US Patent 5 No. 4,448,721. It is usually convenient (e.g., if both C-24 epimers are desired) to use compound (1) as a mixture of 24R and 24S epimers, since the separation of the individual 24R and 24S epimers can be performed later. However, the pure 24S or pure 24R epimer of (1) is suitable, with the former providing the 24S, Ia, 25-dihydroxy final compound and the latter compound the corresponding 24R final compound.

SCHEME OF REACTION I

DK 171397 B1 21 5 / Yes o o .aA o o o o f j i i_i. r j] i_i r j] i_i

JJ so ... Jf so · .. JT

10 ROV ^ 's / ^ v'OR |

I: R = H 3 4: R, = H

5: R f = Acyl

> νΑ o o ^ X O JL »OH

R20 ** "kv ^ 0R, R20 **" ^ Av0R | ΗΟ · '' ^ "ΌΗ

6 '8 9o: 24 p

b · 24R

25 fl "" - jl b> fj r, ov '' ^ or2 r, ov '^ A "or2 hovvVv ^^ oh

7 10 IIo: 24 S

35 h>: 24R

DK 171397 B1 22

SCHEME OF REACTION II

γαΗ ° "γΓ ~ ΗΟ O * O V tt«

</) I * «1 ^ W

1 ° X Satf-οΐΓ ^ Λ J 2 ^ v «o ^

OØO Yp'iT

is> <vi 'V' WO T.

ocri ° Va 3 ^ 2 s 1 * ^ _, r ^ ^ I / lCi Oo.) λΑ 1—1 J ^ oo 30 UA7 _ ^ * rt_ | ^ jj (?) Jl »/ p, (<?) 35 DK 171397 B1 23

SCHEME OF REACTION III

5 -> Ts0 ^ —Pl> ~ S S ^ Y '

o O O

l n-L · ^ * - 10 II o o o o o l — I i t

suffotf A

15 20 25 1 35

Claims (8)

15 I having R configuration at C-24, wherein each of R 1, R 2 and R 3, which are the same or different, represents hydrogen, alkylcarbonyl of 1-6 carbon atoms, arylcarbonyl or an acyl group of the formula: ROOC ( CH2) nCO- or ROOCCH2-0-CH2CO- where n is an integer from 0 to 4 and R represents an alkyl group of 1-6 carbon atoms and X represents alkyl of 1-4 carbon atoms. Derivative according to claim 1, characterized in that it is 1α, 25-dihydroxy-24-epivitamin D2. 30 A composition for pharmaceutical use, characterized in that it comprises at least one derivative according to claim 1 or 2 and a pharmaceutically acceptable excipient. 35 DK 171397 B1 Composition according to claim 3, characterized in that it comprises Ia, 25-dihydroxyvitamin D2, Ia, 25-dihydro-xy-24-epi-vitamin D2, Ia, 25-dihydroxy-5,6-trans-vitamin D2 and 1α, 25-dihydroxy-5,6-trans-24-epi-vitamin D2. 5 Composition according to claim 3, characterized in that it comprises Ia, 25-dihydroxyvitamin D2 and Ia, 25-dihydroxy-24-epi-vitamin D2. Composition according to claim 3, characterized in that it comprises Ia, 25-dihydroxy-5,6-trans vitamin D2 and Ia, 25-dihydroxy-24-epi-vitamin D2 · A composition according to any one of claims 3-6, characterized in that it further comprises at least one bone mobilization-inducing compound, preferably 25-hydroxyvitamin D3, 25-hydroxyvitamin D2, 1α-hydroxyvitamin D3, Ια-hydroxyvitamin D2, Ia, 25-dihydroxyvitamin D3, Ia, 25-dihydroxyvitamin D2, 24,24-difluoro-25-hydroxyvitamin D3, 24,24-difluoro-βα, 25-dihydroxyvitamin D3, 24-fluoro-25-hydroxyvitamin D3, 24-fluoro-1α, 25-dihydroxyvitamin D3, 26,26,26,27,27,27-hexafluoro-1α, 25-dihydroxy vitamin D3, 26,26,26,27,27,27-hexafluoro 25-hydroxyvitamin D3, 2β-isluoro-1α-hydroxyvitamin D3, 2β-ίluoro-25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3, 1α, 24,25-trihydroxy vitamin D3, 25,26-dihydroxyvitamin D3 or Ia, 25,26-tri-hydroxyvitamin D3. Use of a derivative according to claims 1 or 2 to 30 in the preparation of a composition for the treatment of diseases requiring regeneration of bone mass or to prevent loss of bone mass, preferably postmenopausal osteoporosis, senile osteoporosis or steroid-induced osteoporosis. 35