CH665834A5 - METHOD FOR PRODUCING 1ALPHA, 25-DIHYDROXYLATED VITAMIN D (2) AND RELATED COMPOUNDS. - Google Patents

Description

DESCRIPTION

Technical field

The invention relates to la, 25-dihydroxylated compounds of the vitamin D2 series and their preparation.

In particular, the compound relates to the preparation of Icc, 25-dihydroxyvitamin D2 and its (24R) epimer, the corresponding 5,6-trans isomers and certain C-25-A1-alkyl or aryl analog compounds, as well as the acylderi- vate of these connections.

State of the art

The importance of the hydroxylated forms of vitamin D as regulators of calcium and phosphate metabolism in animals and humans is now recognized due to many publications in the patent literature and the general literature, and as a result, these hydroxyvitamin D derivatives find increasing clinical and veterinary use as medicines for the treatment and healing of diseases of calcium metabolism and related bone diseases. Vitamin D3 is known to be hydroxilated in vivo to 25-hydroxyvitamin D3 and then to la, 25-dihydroxyvitamin D3, the latter generally accepted as the active hormonal form of vitamin D3. Similarly, the very effective vitamin di-metabolite, la, 25-dihydroxyvitamin D2 (la, 25- (OH) 2D2) is formed from vitamin D2 or 25-hydroxyvitamin D2 (25-OH-D2). These two hydroxylated vitamin D2 compounds have been isolated and identified (DeLuca et al, U.S. Patents 3,585,221, 3,880,894); when derived from vitamin D2, these metabolites are characterized by the (S) stereochemistry at carbon atom 24.

Presentation of the invention

A chemical process for the production of la, 25-dihydroxylated compounds of the vitamin D2 series has now been developed. In particular, this method provides a convenient means of making connections of general structures A and B shown below

OR

A

Often

B

wherein R], R2 and R3 are selected from the group of hydrogen and acyl, and wherein X represents an alkyl or aryl radical. In these structures, the asymmetric center on carbon atom 24 can have the (R) or ^ configuration.

Specific examples of the compounds to be obtained by the above method include la, 25-dihydroxyvitamin D2, the corresponding (24R) epimer, la, 25-dihydroxy-24-epivitamin D2, the corresponding 5,6-trans isomers, i.e. 5,6-trans -cc, 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 formulas shown above, wherein X is ethyl, propyl, isopropyl or phenyl.

In the following description, the term "acyl" means an aliphatic acyl radical (alkanoyl group) having 1 to 6 carbon atoms, in all possible isomeric forms, for example formyl, acetyl, butyryl, isobutyryl, valeryl, etc., or an aromatic acyl radical (aroyl group ) such as benzoyl or the benzoyl groups substituted with methyl, halogen or nitro, or an acyl radical which is derived from a dicarboxylic acid of the general formulas ROOC (CH2) nCO- or R00CCH2-0-CH2 CO-, where n is an integer with the values from 0 to 4 inclusive and R represents a hydrogen atom or an alkyl radical. Representative of such dicarboxylic acid acyl radicals are oxalyl, malonyl, succinoyl, glutaryl, adipyl and diglycolyl. The term “alkyl” relates to a hydrocarbon radical having 1 to 6 carbon atoms in all isomeric forms, for example methyl, ethyl, propyl, isopropyl, butyl, isobutyl, etc. The term “aryl” relates to an aromatic radical, such as phenyl, benzyl or the isomeric alkyl-substituted phenyl radicals.

An embodiment of the chemical process for the preparation of the compounds according to the invention is recorded in the attached process scheme I. In the following description of this process, the numbers (for example 1, 2, 3, etc.) denote specific products and relate to the structures designated in process scheme I. A wavy line on the substituent (methyl) at C-24 indicates that this substituent can have both R and S configuration.

A suitable starting material for the process is the vitamin D ketal derivative of structure (1). It is generally convenient (e.g. in the case when both C-24 epimers of la, 25-dihydroxyvitamin D2 compounds are desired) to use Compound (1) as a mixture of 24R and S epimers, the Separation of the individual 24R and S epimers is carried out in a later stage of the process. However, the pure 24S or pure 24R epimer of (1) is equally suitable as a starting material, the first-named compound after the

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

665 834

Processing via the indicated synthesis steps provides the (24S) -la, 25-dihydroxy product, while the latter compound provides the corresponding (24R) -la, 25-dihydroxylated product under analogous treatment.

The starting material (1) is converted into the desired la-hydroxylated form via cyclovitamin D derivatives (DeLuca et al., US Pat. Nos. 4,195,027 and 4,260,549). Treatment of compound (1) with toluenesulfonylchloride in a conventional manner gives the corresponding C-3 tosylate (2), which is solvolysed in an alcoholic medium to give the new 3,5-cyclovitamin D derivative (3) surrender. Solvolysis in methanol gives the cyclovitamin of structure (3) where Z is methyl, while the use of other alcohols, e.g. Ethanol, 2-propanol, butanol, etc., in this reaction gives the analog cyclovitamin D compounds (3), wherein Z represents an alkyl radical derived from the alcohol, e.g. Ethyl, isopropyl, butyl, etc. Allyl oxidation of the intermediate compound (3) with selenium dioxide and a hydroperoxide gives the la-hydroxy analogue compound of structure (4). The subsequent acetylation of compound (4) creates the 1-acetate of the structure (5, R, = acetyl). If desired, other 1-0 acylates (structure 5, where R is = acyl, e.g. the formate, propionate, butyrate, benzoate, etc.) are prepared by analogous conventional acylation reactions. The 1-0 acyl derivative is then subjected to acid catalyzed solvolysis. If this solvolysis is carried out in a solvent medium which contains water, the 5,6-cis-vitamine D intermediate compound of the structure (6, Rj = acyl, R2 = H) and the corresponding 5,6-trans Compound (structure 7, R, = acyl, R2 = H) in a ratio of about 3 to 4: 1. These 5,6-cis and 5,6-trans isomers can be separated at this stage, e.g. through high performance liquid chromatography. If desired,

the C-1-0-acyl group can be removed by base hydrolysis to obtain compounds (6) and (7) where Rt and R2 = H. Furthermore, if desired, these 1-0-monoacylates can be further acylated on the C-3-hydroxyl groups using conventional acylation conditions to obtain the corresponding 1,3-di-O -acylates of structure (6) or (7) wherein R, and R2, which may be the same or different, represent acyl groups. Alternatively, the hydroxycyclovitamin of structure (4) may be subjected to acid catalyzed solvolysis in a medium containing a low molecular weight organic acid to give the 5,6-cis and trans compounds of structures (6) and (7) to obtain where R, = H and R2 = acyl, the acyl group being derived from the acid used in the solvolysis reaction.

The next step in the process involves removal of the ketal protecting group to produce the corresponding 25-ketone. This step is critical since the conversion of the ketal to the ketone without accompanying isomerization of the 22 (23) double bond to the conjugated 23 (24) position that can occur under the acidic conditions required for ketal hydrolysis must be carried out. Furthermore, the conditions must be selected so as to avoid elimination of the sensitive allyl-C-1 oxygen function. The conversion is successfully accomplished by careful hydrolysis at moderate temperatures using organic acid catalysis. Treatment of the 5,6-cis compound (6) in aqueous alcohol with p-toluenesulfonic acid gives the corresponding ketone (8). In order to avoid the undesired elimination of the C-1-oxygen function during this reaction, it is advantageous that the C-1-hydroxyl group in compound (6) is protected (e.g. Rj = acyl, R2 = hydrogen or acyl).

The subsequent reaction of the ketone (8) with a methyl Grignard reagent then gives the desired compound Ia, 25-dihydroxyvitamin D2 of structure (9). When the starting material, compound (1) used in the above process is a mixture of the two C-24 epimers, compound (9) is considered a mixture of the 24S and R epimers (9a and 9a, respectively). 9b) received. This epimer mixture can be separated by chromatographic methods in order to obtain la, 25-dihydroxyvitamin D2 (structure 9a, 24S stereochemistry), and its 24R epimer, la, 25-dihydroxy-24-epivitamin D2 of structure 9b, both in are in pure form. Such separation of epimers is of course not necessary if the compounds are to be used as a mixture.

The 5,6-trans-25-ketal intermediate of structure (7), which is subjected to ketal hydrolysis in an analogous manner, gives the 5,6-trans-ketone compound of structure (10), which is obtained via a Grignard reaction with Me -thylmagnesiumbromid or an analogous reagent the 5,6-trans-la, 25- -dihydroxyvitamin D2 compounds of structure (11) as the 24S or 24R epimer or as a mixture of both epimers depending on the nature of the starting material (1) which is used in the process. If the epimers are obtained as an epimeric mixture, they can be separated by chromatography to give 5,6-trans-la, 25 -dihydroxyvitamin D2 (IIa) and its 24R-epimer, 5,6-trans-la, 25 -dihydroxy -24-epivitamin D2 of structure (1 lb). These reaction steps using 5,6-trans intermediate are carried out in a manner completely analogous to that applied to the 5,6-cis compounds as described above.

The new side chain ketones of structures (8) or (10) are very useful and versatile intermediates, since they can be used to produce a variety of 1 a, 25-dihydroxyvitamin D2 side chain analog compounds. In particular, these keto intermediates for the preparation of 5,6-cis or 5,6-trans-la, 25 -dihydroxyvitamin D2 analog compounds with the general side chain formula according to the following illustration can be located

✓v X

wherein X represents an alkyl or aryl radical can be used.

For example, treatment of ketone (8) with ethyl magnesium bromide gives the corresponding hydroxyvitamin D2 analogue compound having the side chain structure shown above, wherein X is an ethyl group. Similarly, treatment of (8) with isopropyl magnesium bromide or phenyl magnesium bromide gives the side chain analog compounds where X is isopropyl and phenyl, respectively. Analogous treatment of the 5,6-trans-25-ketone intermediate compound of structure (10) with alkyl or aryl Grignard reagents gives the 5,6-trans-vitamin D2 analogue compound with the above side chain, in which X is the alkyl or aryl residue introduced by the Grignard reagent used.

It is also evident that the reaction of the ke-to intermediates (8) or (10) with an isotopic

4th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

5

665 834

labeled Grignard reagent (e.g. C3H3MgBr,

CI4H3MgBr, C2H3 MgBr, etc.) provides a suitable agent for the production of la, 25-dihydroxyvitamin D2 or its transisomer and the corresponding C-24 epimers in isotopically labeled form, i.e. than the compounds with the side chain shown above, wherein X is C3H3, C14H3, C2H3, C13H3 or any other selected alkyl or aryl group labeled with isotopes.

The above alkyl or aryl homologues of 5,6-cis or trans-la, 25-dihydroxy-vitamins D2 are valuable substitutes for the parent compounds in situations where a greater degree of lipophilicity is desired, while those mentioned above , compounds labeled with isotopes can be used as reagents in analytical applications.

Although the free hydroxyl compounds represented by the above structures A and B (in which Rh R2 and R3 = H) are generally used for therapeutic applications, the corresponding hydroxyl-protected derivatives may be expedient or preferred for some such fields of application. Such hydroxy-protected derivatives are, for example, the acylated compounds which are indicated by the general formulas A and B above, in which one or more of the radicals Rb R2 and R3 represent an acyl radical.

Such acyl derivatives are conveniently prepared from the free hydroxyl compounds by conventional acylation processes, i.e. by treating one of the hydroxyvitamin D2 products with an acyl halide or acid anhydride in a suitable solvent such as pyridine or an alkyl pyridine. By appropriately selecting the reaction time, the acylating agent, the temperature and the solvent, as are known to the person skilled in the art, the partially or completely acylated derivatives which are indicated by structures A or B above are obtained. For example, treatment of la, 25-dihydroxy vitamin D2 (9a) in pyridine solvents with acetic anhydride at room temperature gives 1,3-diace-tat, while the same reaction performed at elevated temperature gives the corresponding 1,3,25-triacetate results. The 1,3-diacetate can be further acylated at C-25 with a different acyl group; for example, treatment with benzoyl chloride or succinic anhydride gives the 1,3-diacetyl-25-benzoyl or the 1,3-diacetyl-25-succinoyl derivative. A 1,3,25-triacyl derivative can be selectively hydrolyzed in a mild base to create the 1,3-dihydroxy-25-0-acyl compound, the free hydroxyl groups of which again, if desired, with different acyl groups can be acylated. Similarly, a 1,3-diacyl derivative can be subjected to partial acyl hydrolysis to obtain the 1-0-acyl and 3-0-acyl compounds, which in turn can be reacted with different acyl groups. Similar treatment of any of the other hydroxyvitamin D2 products (e.g., 9b, 1 la / b or their corresponding 25-alkyl or aryl analog compounds) provides the corresponding desired acyl derivatives indicated by structures A or B, wherein any or all of Rb R2 and R3 are acyl residues.

Like the previously known vitamin D2 metabolite, la, 25-dihydroxyvitamin D2 (9a), the new compounds according to the invention have pronounced vitamin D-like activity and thus represent desirable substitutes for the known vitamin D2 or D3 metabolites in many therapeutic agents or veterinary uses. Particularly preferred in this regard are the products of structure 9b and IIa and IIb or their acylated derivatives. The new connections can

Correction or improvement of a variety of balance disorders of calcium and phosphate balance, which result from a variety of diseases, such as those used for vitamin D-resistant rickets, bone softening, underactive parathyroid glands, osteodystrophy, pseudohypoparathyroidism, osteoporosis, Pagets disease and similar bone diseases and diseases originating from the mineral balance, which are known from medical practice. The compounds can also be used to treat disturbed mineral balance in animals, such as milk fever, poultry leg weakness, or to improve poultry eggshell quality. Their use in combating osteoporosis is particularly noteworthy.

It is known that women experience a marked loss of bone mass at the time of menopause, which ultimately gives rise to the appearance of bone diseases (osteopenia), which in turn give rise to spontaneous fractures of the vertebrae (vertebrae) and fractures of the long bones. This condition is commonly known as postmenopausal osteoporosis and is a major medical problem in the United States and most other countries where women have at least 60 and 70 years of life. General . the disease, which is often accompanied by bone pain and reduced physical activity, is diagnosed by one or two vertebral fractures, with x-rays indicating the decreased bone mass. It is known that this disorder is accompanied by a decreased ability to absorb calcium, decreased levels of sex hormones, especially estrogen and androgen, and a negative calcium balance.

The procedures for treating the disease have changed remarkably. For example, the calcium supplement itself has been unsuccessful in preventing or curing the disease and injecting sex hormones, especially estrogen, which has been reported to be effective in preventing rapid loss of bone mass suffered by postmenopausal women the fear of possible carcinogenicity of the hormone complicates. Other treatments, of which various results have been reported, included a combination of large doses of vitamin D, calcium and fluoride. The primary problem with this approach is that fluoride induces structurally unhealthy bones, known as woven bones, which also cause a number of side effects such as increased fracture incidence and gastrointestinal response to large amounts of fluoride administered.

Similar symptoms characterize old-age osteoporosis and steroid-induced osteoporosis, the latter being a recognized result of long-term glucocorti-coid treatment (Coritco-Steroid) for certain disease states.

While various metabolites of vitamin D3 increase calcium absorption and calcium retention within the body of mammals and have a physiological tendency or provide evidence of loss of bone mass, they are also characterized by the complementary vitamin D-like properties of calcium mobilization in bone in Response to physiological needs. It has been found that the epi compounds according to the invention, in particular

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

665 834

6

whose 24-epi-la, 25-dihydroxyvitamin D2 (24-epi-l, 25- (OH) 2-D2) are extraordinarily suitable for the prevention or treatment of physiological diseases in mammals which are characterized by the loss of bone mass, since, although they do have some of the known vitamin D-like traits that influence calcium metabolism, such as increasing intestinal calcium transport and causing bone mineralization, they do not affect serum calcium levels, even at high doses increase. This observed feature indicates that the compounds do not mobilize the bone after administration. This fact, along with the ability of the compounds to mineralize bone after administration, indicates that they are ideal compounds for preventing or treating common calcium disorders indicated by loss of bone mass, such as postmenopausal osteoporosis, Age osteoporosis and steroid-induced osteoporosis. It is evident that the compounds will find rapid application in the prevention or treatment of other diseases in which the loss of bone mass is an indication, such as the treatment of patients undergoing kidney dialysis, where loss of bone mass occurs as a result of dialysis .

The following examples serve to illustrate the characteristics of 24-Epi-l, 25- (OH) 2D2, which contribute to the extraordinary suitability for the prevention or treatment of diseases which cause a loss of bone mass.

example 1

Male weanling rats were put on a low-vitamin D diet as described by Suda et al., Journal of Nutrition 100. Pages 1049-1052 (1970), which was modified to include 0.02% calcium and Contains 0.3% phosphorus. After 2 weeks of this diet, the animals were given either 1,25-dihydroxyvitamin D2 or 24-epi-1,25-di-hydroxyvitamin D2 daily by subcutaneous injection in 0.1 ml of 5% ethanol in propanediol. The animals were sacrificed 12 hours after the last dose and blood calcium and intestinal calcium transport were measured. The results of these measurements for the indicated levels of the administered compounds are shown in Figures 1 and 2. The intestinal calcium transport measurements shown in Figure 2 were performed according to the method of Martin and DeLuca, American Journal of Physiology 216, pages 1351-1359 (1969).

Example 2

Male weanling rats were put on a high calcium (1.2% calcium) and low phosphorus (0.1% phosphorus) diet as described by Suda et al. (above) has been described. The rats were fed this diet for a period of 3 weeks, after which they were separated into two groups. One group was given 1,25 (OH) 2D: while the other groups received 24-Epi-1,25 (OH) 2D2, each in 0.1 ml of 5% ethanol in propanediol subcutaneously with the dosage levels of the compounds that shown by the data points in Figure 3. These doses were continued daily for a period of 7 days, after which the animals were sacrificed and inorganic phosphorus was determined in the serum. The results are shown in Figure 3.

The bone ash was examined by removing the femur from the rats. The attached connective tissue was removed from the thighbones, extracted in absolute ethanol for 24 hours and extracted in diethyl ether for 24 hours using a Soxhlet extractor. The bones were incinerated at 600 ° F for 24 hours. The ash weight 5 was determined by weighing to constant weight. The results are shown in Figure 4.

The results of the two tests described in Examples 1 and 2 above indicate that 24-epi-l, 25- (OH) 2D2 is approximately as effective as la, 25-di-10 hydroxyvitamin D3 (l, 25- (OH) 2D3) in terms of causing mineralization of bones and stimulating intestinal calcium transport. In short, there is no significant difference between the two groups in Figure 2 and Figure 4. On the other hand, the increase in inorganic phosphorus in the serum caused by bone mobilization in the case of the diet with a low phosphorus content is very clear by 1.25 - (OH) 2D2 influenced, but hardly stimulated by 24-Epi-1, 25 (OH) 2D2. Similarly, the mobilization of calci-20 µm from bone, as indicated by the serum calcium levels (Figure 1), even at the extraordinarily high dose level of about 750 pmol / day, has no effect on the 24-epi compound , while the mobilization effect is clear at much lower doses of 1,25-dihydroxyvitamin 25 D2. Since the increase in serum calcium in rats on a low calcium diet measures the ability to mobilize bones, and since the increase in blood phosphorus in animals on a low phosphorus diet also measures bone mobilization, 30 and since the increase in blood phosphorus in animals on a diet With low phosphorus levels also measuring bone mobilization, these results show that 24-Epi-1, 25 (OH) 2D2 provides an unexpected feature, namely that it is of minimal potency to mobilize IC bone calcium while being fully capable is to stimulate intestinal calcium transport and mineralization of new bone, properties that make this compound very suitable for the treatment of diseases that cause bone loss.

The unique features of 24-Epi-1,25- (OH) 2D2, as mentioned above, offer the rare opportunity to test the various vitamin D response processes (intestinal calcium absorption, bone mineral mobilization 45 and bone mineralization) in one way and in one an extent that was previously not feasible. This possibility arises from the fact that the 24-epi compound according to the invention is available to the mammal either alone (with suitable and compatible carriers) or in combination with other vitamin D derivatives which cover the full spectrum of the D- show similar activity. Such measurements make it possible (to whatever extent desired) to combine the specificity of the mode of action of the 24-epi analogue with the generality of the action of other vitamin D metabolites or analogue compounds. Administration of 24-epi-1,25- (OH) 2D2 alone, as shown above, stimulates intestinal calcium transport and bone mineralization with or without minimal bone mineral-60 mobilization, but the latter effect can be induced by co- Administration of one or more of the known vitamin D derivatives (for example l, 25- (OH) 2D3, la, 25- (OH) 2D2, la-OH-D3 and related analog compounds). By regulating the relative amounts of the 65 compounds administered, a measure of control over the relative amounts of intestinal calcium absorption against the bone mineral mobilization procedures can be performed in a manner that with the previously known vitamins

D derivatives were not possible. Co-administration of the 24-epi compound and other vitamin D compounds with bone mobilizing activity may be particularly advantageously able to achieve a certain level of bone mobilization. For example, it is assumed that, under certain circumstances, the bone must first be mobilized before new bone can be deposited. In such situations, treatment with vitamin D or a vitamin D derivative induces bone mobilization, e.g. la-hydroxy vitamin D3 or -D2, la, 25-dihydroxyvitamin D3 or -D2, 25-hydroxyvitamin D3 or -D2, 24,24-difluoro-25-hydroxyvitamin D3,

24.24-difluoro-la, 25-dihydroxyvitamin D3, 24-fluoro-25-hydroxy-vitamin D3, 24-fluoro-la, 25-dihydroxyvitamin D3, 2ß-fluoro-hydroxyvitamin D3, 2ß-fluoro-25-hydroxyvitamin D3 , 2ß-fluoro-la, 25-dihydroxyvitamin D3, 26,26,26,

27,27,27-hexafluoro-la, 25-dihydroxy vitamin D3, 26,26,26,27,27, 27-hexafluoro-25-hydroxyvitamin D3,

24.25-Dihydroxyvitamin D3, la, 24.25-Trihydroxyvitamin D3, 25.26-Dihydroxyvitamin D3, la, 25.26-Trihydroxyvitamin D3 in combination with 24-Epi-1, 25 (OH) 2D2 allowed by adjusting the ratios of the 24th -Epi compound and the bone mobilizing vitamin D compound in the treatment sample regulating the rate of mineralization of the bone in order to obtain the desired medical and physiological results. Suitable and effective mixtures are, for example, the combination of la, 25-dihydroxy vitamin D2 and la, 25-dihydroxy-24-epivitamin D3 (9a and 9b), or mixtures of the corresponding 5,6-trans compounds (IIa and IIb) or any combination of these four products other than the free hydroxyl compounds or their acylated forms.

The compounds of the invention or their combinations with other vitamin D derivatives or other therapeutic agents can easily be administered as sterile parenteral solutions by injection or intravenously or through a nutritional channel in the form of oral dosages or transdermally or by suppositories. The compounds are advantageously administered in dosage amounts of 0.1 to 100 micrograms per day. Doses of about 0.5 to about 25 micrograms per day are generally effective in osteoporosis. The compounds can be administered either alone or in combination with other vitamin D derivatives, the ratios of each of the compounds in the combination depending on the particular condition being addressed and the desired level of bone mineralization and / or bone mobilization. In the treatment of osteoporosis, the preferred compound being 24-epi-la, 25- (OH) 2D2, the actual amount of 24-epi compound used is not critical. In all cases, a sufficient amount of the compound should be used to induce bone mineralization. Amounts above about 25 micrograms per day of the 24 epi compound or the combination of the compound with bone mobilization inducing vitamin D derivatives are generally required to achieve the desired results and may not appear practical from an economic point of view. In practice, higher doses of the compounds are used where 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 particular dosage administered will be adjusted in each given case in accordance with the specific compounds used, the disease to be treated, the patient's condition and the other rele7 665 834

vanten medical facts that may modify the activity of the drug or patient response, as is known to those skilled in the art.

Dosage forms of the compounds can be prepared by combining this compound with non-toxic pharmaceutically acceptable carriers as are known to those skilled in the art. Such carriers can be either solid or liquid, such as corn starch 10 ke, lactose sucrose, peanut oil, olive oil, sesame oil and propylene glycol.

If a solid carrier is used, the dosage form of the compounds can be tablets, capsules, powder, troches or chewable tablets (lozenges). If a liquid carrier is used, the dosage forms can be soft gelatin capsules, or syrup or liquid suspensions, emulsions or solutions. The dosage form can also contain additives such as preservatives, stabilizers, wetting agents or emulsifiers, solution promoters, etc. They can also contain other therapeutically valuable substances such as other vitamins, salts, sugars, proteins, hormones or other medical compounds.

The process according to the invention is described in greater detail in Examples 3 to 9 below. In these examples, the designations of the specific products by Arabic numbers (e.g. compounds 1, 2, 3, etc.) refer to the structures numbered in process scheme I.

30 Example 3

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

A solution of compound (1) (50 mg) (as a mixture of the 24R and S epimers) in anhydrous pyridine (300 yr 1) is treated with 50 mg of p-toluenesulfonyl chloride at 4 ° C for 30 35 hours. The mixture is poured over ice / saturated NaHCO 3 with stirring and the product is extracted with benzene. The combined organic phases are washed with aqueous NaHC03, H20, aqueous CuS04 and water, dried over MgS04 and evaporated.

The crude 3-tosyl derivative (2) is solvolysed directly 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 brought to room temperature cooled and concentrated under reduced pressure to ~ 2 ml. Benzene (80 ml) is then added and the organic layer is washed with water, dried and evaporated. The cyclovitamin (3, Z = methyl) 50 obtained can be used in the subsequent oxidation without further purification.

The crude product (3) in CH2C12 (4.5 ml) is an ice-cooled solution of Se02 (5.05 mg) and tert-BuOOH (16.5 | xl) in CH2CL2 (8 ml), the anhydrous pyridine (50 (il) contains, 55. After 15 minutes stirring at 0 ° C. the reaction mixture is warmed to room temperature, after an additional 30 minutes the mixture is transferred to a separatory funnel and shaken with 10% NaOH (30 ml) ml) was added and the separated organic phase was washed with 10% NaOH and water, dried and evaporated, and the oily residue was purified on thin-layer silica gel plates (20 × 20 cm plates, AcOE + / hexane 4: 6) Obtain 20 g of the la-hydroxy derivative (4, Z = methyl): mass spectrum, m / e: 470 65 (M +, 5), 438 (20), 87 (100); NMR (CDC13) 5 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 (2H, m, 22-H and 23-H).

665 834

Example 4

Acetylation of compound (4).

A solution of cyclovitamin (4, Z = methyl) (18 mg) in pyridine (1 ml) and acetic anhydride (0.33 ml) is heated to 55 ° C for 2 hours. The mixture is poured into ice-cooled saturated NaHCO 3 and extracted with benzene and ether. The combined organic extracts are washed with water, saturated CuS04 solution and aqueous NaHC03 solution, 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 (CDC13) § 0.53 (3H, s, 18-H3), 4.18 (IH, 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 la-acetoxy-3,5-cyclovitamin (5) (R, = acetyl).

A solution of cyclovitamin (5) (4.5 mg) in a 3: 1 mixture of dioxane / H20 (1.5 ml) is heated to 55 ° C. p-Toluenesulfonic acid (1 mg in 20 ml H20) is then added and heating continued for 15 minutes. The mixture is poured into saturated NaHCO 3 / ice and extracted with benzene and ether. The organic phases are washed with NaHC03 and water and dried over MgS04. Evaporation of the solvents gives a residue which is the compounds (6) (wherein R, = acetyl and R2 = H) and (7) (where R) = acetyl and R; = H), which are separated by chromatography on HPLC (6.2 mm x 25 cm Zorbax style), 2%

2-propanol in hexane is used as an eluent. If necessary, the products are further purified by rechromatography.

Example 6

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

To the solution of the ketal (6, Rj = acetyl, R2 = H) (1.35 mg) in ethanol (1.5 ml) is added p-toluenesulfonic acid (0.34 mg in 45 | il H; 0) and the mixture is heated under reflux for a period of 30 minutes. The reaction mixture is poured into dilute NaHC03 and extracted with benzene and ether. The combined organic extracts are washed with water, dried over MgSO4 and evaporated. Applying high pressure liquid chromatography to the crude mixture (4% 2-propanol hexane, 6.2 mm x 25 cm Zorbax-Sil) provides some unreacted ketal (6) (0.12 mg, collected at 48 ml) and the desired ketone (8, R, = acetyl, R2 = 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 (CDC13) 5 0.53 (3H, s, 18-H3), 1.03 (3H, d. J = 6.5 Hz, 21-H3), 1.13 (3H, d, J = 7, 0 Hz, 28-H3), 2.03 (3H. S, CH, COO), 2.12 (3H, s, CH3CO), 4.19 (1H, m,

3-H), 5.03 (1H, m, 19-H), 5.33 (3H, broad m, 19-H, 22-H and 23-H), 5.49 (1H, m, 1- H), 5.93 (IH, d, J = 11 Hz, 7-H), 6.37 (1 H, d. J = 11 Hz, 6-H); UV (EtOH) A. max 266 nm, 250 nm, 225 nm.

Example 7

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

The ketone (8, R, = acetyl, R2 = H) in anhydrous ether is treated with an excess of CH3MgBr (2.85 M solution in ether). The reaction mixture is stirred at room temperature for 30 minutes, then quenched with aqueous NH4C1, extracted with benzene, ether and CH2C12. The organic phases are washed with dilute NaHC03, dried over MgS04 and evaporated. The mixture of (9a) and (9b) thus obtained is separated by high-performance liquid chromatography (6% 2-propanol / hexane, 4.6 mm x 25 cm Zorbax-Sil) in order to obtain the pure epimers (9a ) and (9b), la, 25-dihydroxy vitamin D2 (9a): UV (EtOH) lmax 265.5 nm, ^ .min 227.5 nm; Mass spectrum, m / e: 428 (M +, 6), 410 (4), 352 (4), 287 (6), 269 (10), 251 (10), 152 (42), 134 (100), 59 ( 99); NMR (CDC13) S 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), 4.43 (1H, m , 1-H), 5.01 (1H, m, 19-H), -5.34 (3H, broad m, 19-H, 22-H and 23-H), 6.02 (1H, d, J = 11 Hz, 7-H), 6.39 (1H, d, J = 11 Hz, 6-H).

la, 25-dihydroxy-24-epivitamin D2 (9b): UV (EtOH) A, max 265.5 nm, /.min 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) into 5,6-trans-la, 25-di-hydroxyvitamin D2 compounds (1 la) and (IIb).

Hydrolysis of the ICetal intermediate (7, R, = acetyl, R2 = H) under the conditions described in Example 4 creates the corresponding 5,6-trans-25-ketone of the structure (10, Ri = acetyl, R2 = H) and the subsequent reaction of this ketone with methyl magnesium bromide using conditions analogous to those of Example 5 gives a mixture of epimers (IIa) and (IIb) separated by high performance liquid chromatography (HPLC) to give in pure form la , 25-Dihydroxy-5,6-trans-vitamin D2 (1 la) and la, 25-dihydroxy-5,6-trans-24-epivitamin D2 (IIb). If necessary, the structure determination can be confirmed by isomerization to the corresponding 5,6-cis compounds (9a, 9b) according to known methods.

5,6-trans la, 25-dihydroxy vitamin D2 (IIa): UV (EtOH) A. max 273.5 nm, Ìmin 230nm; Mass spectrum, m / e: 428 (M +, 8), 410 (3), 287 (3), 269 (7), 251 (7), 152 (34), 134 (100), 59 (78).

5,6-trans-la, 25-dihydroxy-24-epivitamin D2 (IIb): UV (EtOH) A.m., x 273.5 nm, 2imin 230 nm; Mass spectrum, m / e: 428 (M +, 10), 410 (4), 352 (4), 287 (5), 269 (9), 251 (8), 152 (37), 134 (100), 59 ( 82).

Example 9

Production of alkyl and aryl analog compounds of la, 25-dihydroxy vitamin D2 compounds.

By reacting the ketone intermediate (8) (Ri = acetyl, R2 = H) with each corresponding

(a) ethyl magnesium bromide

(b) Propyl magnesium bromide

(c) isopropyl magnesium bromide,

(d) butyl magnesium bromide or

(e) Phenylmagnesium bromide under conditions analogous to the conditions described in Example 7, the corresponding hydroxyvitamin D2 products of the formula given below are obtained

■ OH

OH

HC?

8th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

9

665 834

where X each

(a) ethyl,

(b) propyl,

(c) isopropyl,

(d) butyl or

(e) represents phenyl.

By similar treatment of the 5,6-trans-ketone intermediate (10) (Ri = acetyl, R2 = H) with the Grignard reagents listed above, the corresponding 5,6-trans-hydroxyvitamin D2 products are obtained, which have the following formula:

where X corresponds accordingly

(a) ethyl,

(b) propyl,

(c) isopropyl,

5 (d) butyl or

(e) is phenyl.

A suitable starting material for the process according to the invention is the vitamin D-ketal derivative of structure (1), which can be obtained according to the following process schemes II and III, as described in GB Pat. No. 2,127,023 or US Pat 4,448,721. It is generally convenient (e.g. if 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 epimers is performed later. However, the pure 24S or pure 24R epimer of (1) are equally suitable, the former 20 providing the 24S-la, 25-dihydroxy product and the latter providing the corresponding 24R product.

Process Scheme I

HO * - "OH 9o: 24 S b: 24R

b: 24 rows

665 834

Process scheme II

10th

AcO

cHO

•>

1

Sul fo *

AcO

Acû

'WC HO

ÖS6

ocH,

iï)

I OH

CCHi

(6a.)

Process scheme III

> IsPv ^ A ^ -

'A -f! -

II O G O i_j

S ulfott A

-) Ph -s o

I.

o o

C.

1 sheet of drawings

Claims (19)

665 834 2nd PATENT CLAIMS 1. Connection, characterized by the formula 27,27-hexafluoro-la, 25 -dihydroxyvitamin D3, 26,26,26, 27,27,27 -hexafluoro-25 -hydroxyvitamin D3, 2ß-fluoro-la-hydroxyvitamin D3, 2ß-fluoro-25-hydroxyvitamin D, , 24,25-dihydroxyvitamin D3, la, 24,25-trihydroxyvitamin 5 D3, 25,26-dihydroxyvitamin D3 or la, 25,26-trihydroxyvitamin D3. 15. A process for preparing a compound having a formula as defined in claim 1, wherein each of Rb R2 and R3, which may be the same or different, represents a hydrogen atom or an acyl group, and X is an alkyl or aryl group or one represents an isotopically labeled alkyl or aryl radical, characterized in that a ketal of the formula wherein each of the radicals Rb R2 and R3, which may be the same or different, is hydrogen or acyl and X is an alkyl radical, or aryl or isotopically marked alkyl or Aryl. with the proviso that, provided the C-24-methyl substituent has the S configuration and X is methyl, R,. R2 and R3 cannot all be hydrogen. 2. Connection according to claim 1, characterized. that the asymmetric center at C-24 has the ^ configuration. 3. Connection according to claim 1, characterized. that the asymmetric center at C-24 has the ^ configuration. 4. A compound according to claim 1, characterized in that it is la, 25-dihydroxy-24-epivitamin D2. 5. A compound according to claim 1, characterized. that it is la, 25-dihydroxy-5,6-trans-24-epivitamin D2. 6. A compound according to claim 1, characterized in that X is methyl. 7. Pharmaceutical composition, characterized by the content of a compound according to one of claims 1 to 6 and a pharmaceutically acceptable carrier. 8. The composition according to claim 7, characterized by a content of la, 25-dihydroxy -5,6-trans-vitamin D2 and la, 25-dihydroxy-vitamin D2. 9. The composition according to claim 7, characterized by a content of la, 25-dihydroxy -5,6-trans-vitamin D2 and lct, 25-dihydroxy-5,6-trans -24-epivitamin D2. 10. The composition according to claim 7, characterized by a content of la, 25-dihydroxyvitamin D2, la, 25-dihydroxy -24-epivitamin D2, la, 25-dihydroxy-5,6-trans-vitamin D-. and 1 a, 25-dihydroxy-5,6-trans-24-epivitamin D: - 11. The composition according to claim 7, characterized by a content of la, 25-dihydroxyvitamin D2, and la, 25-dihydroxy -24-epivitamin D2. 12. The composition according to claim 7, characterized. with a content of la, 25-dihydroxy -5,6-trans-vitamin D2 and either la, 25-dihydroxy -5,6-trans-25-epivitamin D2 or 1 a.25-dihydroxy-24-epivitamin D2. 13. Composition according to one of claims 7 to 12, characterized in that it additionally contains at least one compound which induces bone mobilization. 14. The composition according to claim 13, characterized. that it contains as the bone mobilizing compound: 25-Hydroxyvitamin D3, 25-Hydroxyvitamin D2, 1 a-Hydroxyvitamin D3, la-Hydroxyvitamin D2, 1 a, 25-Dihydroxyvitamin D3, la, 25-Dihydroxyvit-amine D2, 24 , 24-difluoro-25-hydroxyvitamin D3, 24,24-di-fluoro-1a, 25-dihydroxyvitamin D3, 24-fluoro-25-hydroxyvitamin D3, 24-fluoro-la, 25-dihydroxyvitamin D3, 26, 26,26,27, wherein R] and R2 correspond to the above definition, the hydrolysis at a temperature of 10 to 38 C under 30 acidic conditions are subjected and the ketone obtained is reacted with a corresponding Grignard reagent. 16. Compound, characterized by the formula wherein IC represents an oxygen atom residue, and R | and R2, which may be the same or different, represent a hydrogen atom or an acyl radical, as an intermediate in the process according to claim 15. 17. A compound according to claim 16, characterized in that it is la-hydroxy -25-oxo-27-norvitamin D2 or the acetate thereof. 55 18. la-hydroxy-25-oxo-27-nor -24-epivitamin D2 as a compound according to claim 16. 19. Compound characterized by the formula 60 65 or 3rd 665 834 wherein K represents an ethylenedioxy radical, and Rj and R2, which may be the same or different, represent a hydrogen atom or an acyl radical, as starting material in the process according to claim 15 20. Use of a compound as defined in any one of claims 1 to 6 in a composition for the treatment of a disease which entails the need to regenerate or avoid loss of bone mass, preferably postmenopausal osteoporosis, aging osteoporosis or steroid-induced Osteoporosis.