Process for the Preparation of g _r 25--Dihydroxylated Vitamin D and Related Coπipounds

Technical Field

This invention relates to the preparation of la,25- dihydrσylated ccπtpounds of the vitamin D ₂ series.

More specifically, this invention relates to the preparation of ltt,25-d__hyd_ ^* Oxyvit- ^* - ^* mn O and its (24R)-epimer, the corresponding 5,6-trans-isσrrers, and to certain C-25-alk l or aryl analogs as well as the acyl derivatives of these compounds. Background

The __πportance of the hydroxylated forms of vitamin D as regulators of calcium and phosphate metabolism in animals and humans is by now well recognized through many disclosures in the patent and general literature, and as a consequence these hyd_roxyvit3m_ιι D derivatives are finding increasing clinical and veterinary use as medicaments for the treatment and cure of disorders of calcium metabolism and associated bone diseases. Vitamin D-, is known to be hydroxylated in vivo to 25-hydroxyvitamin D_ and then to lor, 25-di_hydroxyvitamin D , the latter being generally accepted as the active hormonal form of vitamin D-. Similarly, the very potent vitamin D ₂ metabolite, lff,25-dihydroxγvitamin D_ (lor,25-{0H) ₂ D ₂ ) is formed from vi1_amin D ₂ via 25-_ψd_π2xγvit2min D _? (25-OH-D ₂ ) . Both of these hydroxylated vitamin D_ coiripσunds have been isolated and identified (DeLuca et al, U.S. Patents 3,585,221; 3,880,894); being derived from vitamin D ₂ , these metabolites are characterized by the (S_)-stereochemistry at carbon 24. Disclosure of Invention

A chemical process for preparing lff,25-dihydroxylated ccripounds of the vitamin D ₂ series has now been developed. Specifically, this process provides a convenient means for

preparing compotinds having the general structures A ^' and 15

aryl group. In these structures the asyirrnetriσ center at carbon 24 may have the (R) or (S) configuration.

.Specific examples of corrpounds obtainable by the present process include lor,25-d__hyd__oxyvita_t-in D ₂ , the corresponding (24R)-epimer,- D ₂ , the respective 5,6-trans-isomers, i.e. 5,β-trans-lg,25-~d___yd_x«yv±tamin D ₂ , and 5.6-trans-lor,25--d:ihyd- ^* oxy-24-epivitH ^* nin D ₂ , as well as the C-25-alkyl or aryl hαtologs of these cempounds, i.e. the cciπpounds having the formulae shown above where X is ethyl, propyl, isopropyl or phenyl.

As used herein the term "acyl" signifies an aliphatic acyl group (alkanoyl group) of from 1 to 6 carbons, in all possible isomeric forms, e.g. formyl, acetyl, butyryl, isobutyryl, valeryl, etc., or an aromatic acyl group (aroyl group) such as benzcyl, or the methyl, halo, or nitro-substituted benzcyl groups, or an acyl group derived from a dica__bo_ ^* ylic acid having the general formulae R0CC(CH ₂ ) _n CX--, or where n is an integer having the values of 0 to 4 inclusive, and R is hydrogen or an alkyl radical. Representative of such dicarbox lic acyl groups are oxalyl, malonyl, succinoyl, glutaryl, adipyl and diglycolyl. The term "alkyl" refers to a hydrocarbon group of 1 to 6 carbons in all isomeric forms, e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl, etc. The term "aryl"

refers to an arσnatic radical such as phenyl, benzyl, or the isomeric alkyl-substituted phenyl radicals.

An e ^* - ^* bod ^* _ment of the chemical process of this invention is depicted in appended Process Scheme I. In the following description of this process, numerals (e.g. 1, 2 , 3_, etc) designating specific products refer to i±e structures so numbered in Process Scheme I. A wavy line to the substituent (methyl) at C-24 indicates that this substituent may have either the RorS configuration.

A suitable starting material for the process of this invention 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-d__hydrcκyvitamin t ⁾ „ compounds -are desired) to use cαtpound (1) as a mixture of the 24R and S_ epimers, separation of the individual 24R and S_-eρimers being acα_tπplished at a later stage of the process. However, the pure 24£>, or the pure 24R-epimer of (jL) are equally suitable starting materials, whereby the former compound upon being processed through the indicated synthetic steps will provide the (24£)-lα-,25-d_ιydroxy product, whereas the latter, treated analogously, will yield the corresponding (24R)-1__,25- dihydroxylated product.

Starting material (1) is converted to the desired l ~ hydroxylated form via cyclovitamin D derivatives {DeLuca et al., U.S. patents 4,195,027 and 4,260,549). Thus, treatment of compound (1) with toluenesulfonyl chloride in the conventional manner yields the corresponding C-3-tosylate (2) , which is solvolyzed in an alcoholic medium to produce the novel 3,5-cyclovitamin D derivative (3_) . Solvolysis in methanol yields the cyclovitamin of structure (3_) where Z=methyl, whereas the use of other alcohols, e.g. ethanol, 2-propanol, butanol, etc. , in this reaction provides the analogous cyclcvitamin D ccmpounds (3_) , where Z is an alkyl group derived from the alcohol, e.g. ethyl, isopropyl, butyl, etc. Allylie oxidation of intermediate (3) with selenium

dioxide and a hydroperoxide yields the lα-hyd__oxy-.analog of structure (4) . Subsequent acetylatiαn of compound (4) provides the 1-acetate of structure (5_, R. ^* =acetyl) . If desired, other 1-0-acylates (structure 5_, where R,=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. When this solvolysis is ccra3ucted in a solvent medium containing water, there is obtained the 5,6-cis-vitaroin D intermediate of structure (6, R-=acyl, y= ) and the corresponding 5,6-trans-coι- ound (structure 7_, R=acyl, -^-"-H)) in a -ratio of about 3-4:1. These 5,6-cis and 5,6-trans- iso ers can be separated at this stage, e.g. by high performance liquid chrc ^* ιatography. If desired _e the C-1-O-acyl group may be removed by base hydrolysis to obtain compounds (6) and (7) where R. and ,*-* ⁸ _* Also if desired, these 1-0-monoacylates may be further acylated a the C-3-hydroxy groups, using conventional acylation conditions to obtain the corresponding 1,3-di-O-acylates of st__ucture (6) or (7) where R-. and R-, which may be the same or different, represent acyl groups. Alternatively, the hydrαxy cyclovitamin of structure (4) can be subjected to acid-catalyzed solvolysis ±n a medium conta__r ng a low-molecular weight organic acid to obtain the 5,6-cis and trans compounds of structures (6) and (7_) where R=H and R =acyl, where the acyl group is derived frcm the acid used in the solvolysis reaction.

The next step of the process comprises the removal of the ketal protecting group to produce the corresponding- 25-ketone. This step is a critical one, since the ketal to ketone conversion must be acccmplished without conccmitant iscmerization of the 22(23)-double bond to the conjugated 23(24)-position, which can occur under the acidic conditions required for ketal hydrolysis. Ficthermore, conditions must be chosen so as to avoid elimination of the sensitive allylie C-1-oxygen function. The conversion is accomplished

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successfully by careful hydrolysis at moderate terrperatures using organic acid catalysis. Thus, treatment of the 5,δ-cis-c-c-πtpound (6) in aqueous alcohol with p-toluenesulfonic acid gives the corresponding ketone (8) . To avoid undesired elimination of the C-1-oxygen function during this reaction, it is advantageous that the C-1-hydrσxy group in compound (6) be protected (e.g. R-,=acyl, R ₂ =hydrogen or acyl) .

Subsequent reaction of ketone (8 with a πethyl-Grignard reagent then provides the desired lαr,25--<lihyd__oxyvitamin D ₂ compound of structure (9) . If the starting material, compound (1) , used in the above process, is a mixture of the two C-24-epiιrers, then cxaipound (9) will be obtained as a mixture of the 24S_ and R-epimers (9a and 9b, respectively) . Separation of this epimer mixture can be achieved by ckromatographic methods, to obtain l _ff ,25-d_Jιydroxyvii-aπ n D ₂ (structure >a, 24S-stereochemistry) and its 24R-epimer, ____, 25-d__hydroxy-24-epivitamin D ₂ , of structure 9b, both in pure form. Such separation of epimers is, of course, not necessary if the compounds are intended to be used as a mixture.

The 5,6-trans-25-ketal-intermediate of structure {!) , subjected to ketal hydrolysis in an analogous manner, provides the 5,6-trans ketone intermediate of structure (10) , which via a Grignard reaction with methyl magnesium bromide or analogous reagent gives the 5,6-tra -lαr,25-dihydroxyvitamin D _? compounds of structure (11) , as the 24£5 or 24R-epimer, or as a mixture of both epimers depending on the nature of the starting material (1) used in the process. If obtained as an epi εric mixture, the epimers can be separated by chrαmatography, to obtain 5,6-trans-l<-f,25-d_Jaydrcκyvitamin D ₂ (11a) and its 24R-epimer, 5,6-trans-3__.,25-dihydroxy-24- epivitamin D_, of structure (lib) . These reaction steps utilizing the 5,6-trans-intermediate are conducted in a manner entirely analogous to those applicable to the 5,6-cis- ccmpounds described above.

The novel side chain ketones of structures (8) or (10) are most useful and versatile interrriediates in that they can be used to prepare a variety of __2,25-d__hyd_χιxyv_-tamin D ₂ -side chain analogs. Specifically, these keto-inteπrεdiates can serve for the preparation of 5,6-cis- or 5,6-trans-lα-, 25-d__hyd__OX$ ^* vitamin D ₂ analogs having the general side chain formula shown below,

where X is an alkyl or aryl group.

For example, treatment of ketone (8) with, ethyl magnesium bromide gives the corresponding hyd_-oxyvitamin D ₂ analog having the side chain structure shown above wherein X is ethyl group. Likewise, treatment of (8) with isopropyl magnesium brαnide or phenyl magnesium bromide gives the side chain analogs where X is isopropyl or phenyl, respectively. Analogous treatment of the 5,6-trans-25-ketone intermediate of structure (10) with alkyl or aryl-Grignard reagents gives the 5,6-trans-vitamin D ₂ analog having the side chain above where X is the alkyl or aryl radical -Introduced by the Grignard reagent employed.

It is also evident that the reaction of the keto-inter ediates (8) or (10) with, an isotopically-labeled

3 14 2. Grignard reagent (e.g. C HJtøgBr, O ϊgBr, C H-MgBr, etc.) provides a convenient means for preparing la,25-dihydroxy- vitamin D ₂ or its trans isc er, and the corresponding

C-24-epimers, in isotopically-labeled form, i.e.. as the cor_po ^* _nds having the side chain shown above, wherein X is

3 14 2 13 C .ft,, CH-, C H_, CH ₃ , or any other isotopically-labeled alkyl or aryl group selected.

The above alkyl or aryl homologues of the 5,6-cis or trans-lor,25-d_ιydrOxy-vitamin D ₂ are useful substitutes of the parent compounds in situations where a greater degree of lipophilicity is desired, whereas the isotopically labeled

compounds referred to above, find use as reagent in -analytical applications.

Further, although for therapeutic applications, the free hydroxy compounds represented by structures A and B_ above (where R., K. and R- _j =H) are generally used, for some such applications, the corresponding hydroxy-protected derivatives may be useful or preferred. Such hydroxy-protected derivatives are for example the acylated compounds represented by general formulae A and B above, wherein one or more of R-, R _j , and R. represents an acyl group.

Such acyl derivatives are conveniently prepared from the free hydroxy coπipounds by conventional acylation procedures _F i.e. treatment of any of the hydroxyvitamin D ₂ products with an acyl halide, or acid anhydride in a suitable solvent such as pyridine, or an alkyl-pyridine. By appropriate selection of reaction time, acylating agent, temperature and solvent, as is well-known in the art, the partially or fully acylated derivatives represented by structures A or 13 above are obtained. For example, treatment of l*,25-di ydroxyvita_r_Ln D ₂ (9a) in pyridine solvent with acetic anhydride at room temperature gives the 1,3-diacetate, while the same reaction conducted at elevated terrperature yields the corresponding 1,3,25-triacetate. The 1,3-diacetate can be further acylated at C-25 with a different acyl group; e.g. by treatrrent with benzqyl chloride or succinic anhydride there is obtained the l,3-diacetyl-25-benzoyl-, or l,3-diacetyl-25-succincyl- derivative, respectively. A 1,3,25-triacyl derivative can be selectively hydrolyzed in mild base to provide the l,3-dihydroxy-25-0-acyl compound, the free hydroxy groups of which can be reacylated, if desired, with different acyl groups. Likewise, a 1,3-diacyl derivative can be subjected to partial acyl hydrolysis to obtain the 1-0-acyl and the 3-0-acyl cαrpounds, which in turn can be reacylated with different acyl groups. Like treatment of any of the other hydroxyvii_amin D ₂ products (e.g. 9b, lla/b, or their

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corresponding 25-alkyl or aryl analogs) provides the corresponding desired acyl derivatives represented by structures A or B_, where any or all of , , R,, and R, are acyl.

Like the previously known vitamin D ₂ metabolite, lor, 25-di_ayd_s__xyvitamin D ₂ (9a) , the novel compounds of this invention exhibit pronounced vitaminD-like activity, and thus represent desirable substitutes for the kncwn vitamin D ₂ or D metabolites in many therapeutic,or veterinary applications. Particularly pref rred in this regard are the products of structure 9b and 11a and lb, or their acylated derivatives. The novel compounds may be used for correcting or improving a variety of calcium and phosphate imbalance conditions resulting from a variety of diseases, such as vitamin D-resistant rickets, osteomalacia, hypoparathyroidisn, osteo- dystrophy, pseudohypαparathyroidism, osteoporosis, Paget's disease, and similar bone and ir neral-related disease states known to the medical practice. The cαrpounds can also be used for the treatment of mineral imbalance conditions in animals, for example, the milk fever condition, poultry leg weakness, or for improving egg shell quality of fcwl. Their use in the treatment of osteoporosis is particul-arly noteworthy.

It is well know that females at the time of menopause suffer a marked loss of bone mass giving rise ultimately to osteopenia, which, in turn gives rise to spontaneous crush fractures of the vertebrae and fractures of the long bones. This disease is generally known as postmencpausal osteoporosis and presents a major medical problem, both in the United States and most other coutnries where the life-span of females reaches ages of at least 60 and 70 years. Generally the disease which is often accompanied by bone pain and decreased physical activity, is diagnosed by one or two vertebral crush fractures with X-ray evidence of diminished bone mass. It is known that this disease is accompanied by diminished ability to absorb calcium, decreased levels of sex hoinrones.

especially estrogen and androgen, and a negative calcium balance.

Methods for treating the disease have varied considerably. For example, calcium supplementation by itself has not been successful in preventing or curing 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 experienced in post enopausal women, has been cαrplicated by the fear of its possible carcincgenicity. Other treat- ments, for which vari-able results have again been reported, have included a cα±iination of vitamin D in large doses, calcium and fluoride. The primary problem with this approach is that fluoride induces structurally unsound bone, called woven bone, and in addition, produces a number of side effects such as increased incidence of fractures and gastrointestinal reaction to the large amounts of fluoride administered.

Similar symptoms characterize senile osteoporosis and steroid-induced osteoporosis, the iatter being a recognizied result of long term glucocorticoid (cortico-steroid) therapy for certain disease states.

While various metabolites of vitamin D_ increase calcium absorption and retention within the body of mammals displaying evidence of or having a physiological tendency toward loss of bone mass they are also characterized by the cαrplementary vitamin D-like characteristic of mobilizing the calcium in bone in response to physiological needs. It has now been found that the epi compounds of this invention, especially 24-epi-lα,25-dihydroxyvitamin D- ₂ (24-epi-l,25-(OH) ₂ D ₂ ) , are eminently suitable for the prevention or treatment of physiological disorders in mammals which are characterized by the loss of bone mass because, although they express some of the recognized vitamin D-like characteristics affecting calcium metabolism, such as, increasing intestinal calcium transport, and effecting bone mineralization, they do not

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increase serum calcium levels, even at high dosages. This observed characteristic evinces that the compounds upon administration, do not mobilize bone. This fact, along with the ability of the comounds upon admirάstration to mineralize bone, indicates that they are ideal cαrpounds for the prevention or treatment of prevalent calcium disorders which are evidenced by loss of bone mass, for example postmenopausal osteoporosis, senile osteoporosis and steroid-induced osteoporosis. It will be evident that the coπpounds will find ready application for the prevention or treatment of other disease states in which the loss of bone mass is an indiσationsuch as in the treatment of patients undergoing renal dialysis where loss of bone mass as a consequence of the dialysis is encountered.

The following Examples will serve to illustrate the characteristics of 24-epi-l,25-(OH) -) ₂ which contribute to its eminent suitability for the prevention or treatment of disease states that evince bone mass loss. Example 1

Weanling 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 .02% calcium and .3% phosphorus. After two weeks on this diet, the animals were given either l,25-d__hydroxyvitamin D ₂ , or 24-epi-l,25- di droxy-'itamin D ₂ daily by subcutaneous injection in 0.1 ml of 5% ethanol in propanediol. Twelve hours after the last dose, the animals were killed and the blood calcium and intestinal calcium transport measured. The results of these measurements for the indicated levels of the compounds administered are shown in Figures 1 and 2. The intestinal calcium transport measurements shown in Figure 2 were performed by the method of Martin and DeLuca, American Journal of Physiology 216, 1351-1359 (1969).

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Example 2

Weanling male rats were placed on a high calcium (1.2% calcium) and low phosphorus (.1% phosphorus) diet described by Suda et al (supra) . The rats were fed this diet for a period of three weeks at which time they were separated into two groups. One group was given l,25(OH) ₂ D ₂ while the other groups was given 24~epi-l,25(OH) ₂ D ₂ , both in 0.1 ml of 5% ethanol in propane diol subcutaneously at the dosage levels of the compounds shown by the data points in Figure 3. These doses were continued daily for a period of seven days, at which ^' time the animals were killed and serum inorganic phosphorus determined. Results are shown in Figure 3.

Bone ash was determined by removing the femurs frαn rats. The femurs were dissected free of adhering connective tissue, extracted for 24 hours in absolute ethanol, and 24 hours in diethyl ether, using a Soxhlet extractor. The bones are ashed at 600°F for 24 hours. The ash weight was determined by weighing to constant weight. Results are shown in Figure 4.

The results of the two studies described in Examples 1 and 2, above, illustrate that 24-epi-l,25-(OH) -> ₂ is approximately equal in potency to lc.,25-dihydrαxyvitamin D ₃ (l,25-(OH) ₂ D_) in causing the mineralization of bone and in st mulating 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 elevation of serum inorganic phosphorus which results from mobilization of bone in the case of the low phosphorus diet is very markedly • affected by 1,25-{0H) ₂ D ₂ , but hardly stimulated by 24-epi- l,25(OH) ₂ D ₂ . Similarly, in the mobilization of calcium from bone, as indicated by the serum calcium levels (Figure 1) even at the extremely high dose level of about 750 pmoles/day, the 24-epi compound had no effect, while the mobilization effect is evident at much lower doses of 1,25-dihydroxyvitamin D_. Since the rise in serum calcium of rats on a low calcium diet measures the ability to mobilize bone, and since the elevation

of blood phosphorus of animals on a low phosphorus diet also measures bone mobilization, these results show that 24-epi-l,25-(OH) ₂ D ₂ provides an t_ι_expected property, namely that it is of ιtri_-rimal effectiveness in mobilizing bone calcium, while being fully able to st±πulate .intestinal calcium transport and the mineralization of new bone, properties which make this cαrpound highly suitable for the treatment of disease states that evince bone loss.

The unique characteristics of 24-epi-l,25-(0H) ₂ D ₂ , as set forth above, offer the rare opportunity to control the various vitamin D-responsive processes (intestinal calcium absorption, bone mineral mobilization, and bone mineralization) in a manner and to a degree heretofore not feasible. This possibility arises from the fact that the 24-epi compound of this invention may be administered to the :m__ ^* rmal either alone (with suitable and acceptable exeipients) or in combination with other vitamin D-derivatives which exhibit the full spectrum of D-like activity. By such measures, it is possible therefore to combine (to whatever degree desired) the specificity of action of the 24-epi-analog with the generality of action of other vitamin D metabolites or analogs. _dministration of 24-epi-l,25-(OH) ₂ D- alone will, as shown above, stimulate intestinal calcium transport and bone mineralization with no or minimal bone mineral mobilization, but the latter activity can be induced by <_o-adπdnistration of one or more of the known vitamin D derivatives (e.g., l,25-(OH) ₂ D ₃ , l_.,25-(OH) ₂ D ₂ , lorOH-D.-, and related analogs) . By adjusting the relative amounts of coirpounds administered, a degree of control over the relative magnitudes of the intestinal calcium absorption vs. bone mineral mobilization processes can be exercised in a manner not possible with the heretofore known vitamin D derivatives. Q_^administration of the 24-epi compound and other vitamin D α ^** j- ^* po ^* _nds wit bone mobilizing activity can be particularly advantageous in situation where some degree of bone mobilization is desired.

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For exaπple, it is believed that in certain circumstances, bone must first be mobilized before new bone can be laid down. In such situations treatment with vitamin D or a vitamin D derivative which will induce bone mobilization, e.g. lor- hydroxyvitamin D ₃ or -D ₂ , lα,25-d__hydroxyvitamin D ₃ or - D ₂ , 25-hydroxyvitamin D ₃ or - D ₂ , 24,24-difluoro-25- hydroxyvltamin D ₃ , 24,24~difluoro^3α,25-dihydroxy- vitamin D ₃ , 24-fluoro-25-hydroxy ^** itaιr__n D ₃ , 24-fluoro-lα-,25- d___-ydroxyvitaιr n D ₃ , 2j3-flιιoro-to-hydroxyvitemin D ₃ , 2j3- fluoro-25-hyd_π_«vita_nin D ₃ , 2β-fluoro-lor,25-dihydroxy- vitamin D ₃ , 26,26,26,27,27,27-hexaflι ^* oro-l£-r,25-dihydro_Q-- vitamin D ₃ , 26,26,26,27,27,27-hexafluoro-25-hyά^xyvitamin D~, 24,25-dil ^* ydroxyvitamin D ₃ , lα,24,25-trihydroxyvitamin D_, 25,26-d_U-ydroxyvitamin D ₃ , l_.,25,26-trihydroxyvitamin D ₃ , in . combination with 24-epi-l,25(OH) ₂ D will, by adjustment of the proportions of the 24-epi compound and the bone-i ^** x_bilizing vitamin D compound in the treatment regimen permit the rate of mineralization of bone to be adjusted to achieve the desired medical and physiological ends. Suitable and effective mixtures are for example, the combination of lα-,25-dihydroxy- vitamin D ₂ and lα,25-d__hydroxy-24-epivitamin D_ (9a and 9b) , or mixtures of the corresponding 5,6-ta ^* nj3-co ^* τpounds (11a and lib) , or any other combination of these four products as the free hydroxy compounds or as their acylated forms.

The compounds of this invention or cαnbinations thereof with other vitamin D derivatives or other therapeutic agents, can be readily administered .as sterile parenteral solutions by injection or intravenously, or by alimentary canal in the form of oral dosages, or trans-dermally, or by suppository. Advan¬ tageously the coπpounds are administered in dosage amounts of from 0.1 to 100 micrograms per day. In relation to osteoporo¬ sis, doses from about 0.5 to about 25 micrograms per day are generally effective. The compounds can be administered either alone or in o bination with other vitamin D derivatives, the proportions of each of the coπpounds in the cαrbination being

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dependent upon the particular disease state being addressed and the degree of bone mineralization and/or bone mobilization desired. In the treatment of osteoporosis where the preferred cxπpound is 24-epi-lα,25-(OH) ₂ D ₂ the actual amount of the 24-epi-cαrpound used is not critical. In all cases, sufficient of the c rpound should be used to induce bone mineralization. Amounts in excess of about 25 micrograms per day of the 24-epi-coπpound or the combination of that compound with bone .n-cbiliLzatim-JLnducing vitemin D derivatives, are generally unnecessary to achieve the desired results and may not be economically sound practice. In practice, higher doses of the compounds are used where therapeutic treatment of a disease state is the desired end w_rLle the lower doses are generally used for p ^* -ophylactic purposes, it being understood that the specific dosage administered in any given case will be adjusted in accordance with the specific corrpounds being administered, the disease to be treated, the condition of the subject and the other relevant medical facts that may modify the activity of the drug or the response of the subject, as is well known by those skilled in the art.

Dosage forms of the compounds can be prepared by c ^* c ^* ±>-_ning them with non-toxic pharmaceutically acceptable carriers as is well known in the art. Such carriers may be either solid or liquid such as, for example, 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, troches or lozenges. If a liquid carrier is used, soft gelatin ceipsules, or syrup or liquid suspensions, emulsions or solutions may be the dosage form. The dosage forms may also contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, etc. They may also contain other therapeutically valuable substances such as other vitamins,

salts, sugars, proteins, hormones or other medicinal compounds.

The process of the present invention is more particularly described by Examples 3 through 9 which follow. In these examples the designation of specific products by Arabic numerals (e.g. corrpounds 1_, 2_, 3_, etc.) refer to the structures so numbered in Process Scheme I. Example 3 l<>-hydroxy-3,5-eyclσvitamin D (4, Z=methyl) . A solution of compound (1) (50 mg) (as a mixture of the 24 R and S_ epimers) in dry pryridine (300 ul) is treated with 50 mg of p-toluenesulfαnyl chloride at 4°C for 30 h. The mixture is poured over ice/sat. NaHCO, with stirring and the product is extracted with benzene. The combined organic phases are washed with aqueous NaHCO-, H_0, aqueous CuSO. and -water, dried over MgSO. and evaporated.

The crude 3-tosyl derivative (2) is directly solvolyzed in anhydrous methanol (10 ml) and NaHC0 ₃ (150 mg) by heating at 55°C for 8.5 h with stirring. The reaction mixture is then cooled to room terrperature and concentrated ml under vacuo. Benzene (80 ml) is then added and organic layer is washed with water, dried and evaporated. The resulting cyclovitamin (_3_, Z=methyl) can be used in the subsequent oxidation without further purification.

The crude product (3) in CΑJ21- (4.5 ml) is added to an ice-cooled solution at Se0 ₂ (5.05 irg) and t-BuCOH (16.5 ul) in C3_ ₂ C1 ₂ (8 ml) containing anhydrous pyridine (50 μl) . After being stirred for 15 min at 0°C, the reaction mixture is allowed to warm to room terrperature. After an additional 30 min, the mixture is transferred to a separatory funnel and shaken with 10% NaOH (30 ml) . Ether (150 ml) is added and the separated organic phase is washed with 10% NaOH, water, dried and evaporated. The oily residue is purified on silica gel thin layer plates (20 :< 20 cm plates, AcOEt/hexane 4:6) to

yield 20 mg of lα-hydroxy derivative ( , Z=methyl) : mass spectrum, me: 470 (M ⁺ , 5),, 438 (20) , 87 (100); NMR (CDC1 ₃ ) Sθ.53 (3H, s, 18-H ₃ ), 0.63 (]fi, m, 3-H) , 4.19 (1H, d, J=9.5 Hz, 6-H), 4.2 (1H, , 1-H), 4.95 (1H, d, J=9.5 Hz, 7-H) , 5.17 and 5.25 (2H, each , 19-^) , 5.35 (2H, , 22-H and 23-H) . Example 4

Acetylation of ccπpound (4) .

A -solution of cyc__cιvitamin (4_, Z ^* =methyl) (18 mg) in pyridine (1 ml) and acetic anl ^* ydride (0.33 ml) is heated at 55°C for 2 h„ The mixture is poured into ice-cooled sat. NaHXλ, and extracted with benzene and ether. The combined organic extracts are washed with water, saturated CuSO^ and aqueous NaHG0 ₃ solutions, dried and evaporated to give 1-acetcxy derivative (5_, Z ^** -methyl, acyl=acetyl) (19 mg) : mass spectrum, m/e: 512 (M ⁺ , 5), 420 (5), 87 (100); NMR (CDC1 ₃ ) Sθ.53 (3H, s, 18-H ₃ ), 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 l^r-acetαxy-3,5-cyclovitamin (5_) (R.=acetyl) .

A solution of cyclovitamin (5) (4.5 mg) in 3:1 mixture of dioxane/H ₂ 0 (1.5 ml) is heated at 55°C. p-Toluenesulfonic acid (1 mg in 20 /(l of H ₂ 0) is then added and heating is continued for 15 min. The mixture is poured into saturated NaHCO-/ice, and extracted with benzene and ether. The organic phases are washed with NaHC0 ₃ and water and dried over MgSO.. Evaporation of solvents gives a residue containing cαrpounds (6) (where R.=acetyl and R^H) and (7_) (where R.=acetyl and R^H) which are separated by ch__cmatography on HPLC (6.2 irm x 25 cm Zorbax-Sil) using 2% of 2-propanol in hexane as an eluent.. If necessary, the products are further purified by rechrcmatography. Example 6

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

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To the solution of ketal (6, R=acetyl, P^H) (1.35 mg) in ethanol (1.5 ml) , p-toluenesulfonic acid (0.-34 mg in 45 uL of H-O) is added and the mixture is heated under reflux for 30 min. The reaction mixture is poured into diluted NaKX , and extracted with benzene and ether. The cxπibined organic extracts are washed with water, dried over MgSO. and evaporated. High-pressure liquid chrcmatography of the crude mixture (4% 2-propanol/hexane, 6.2 mm x 25 cm Zorbax-Sil) affords some unreacted ketal {§) (0.12 mg, collected at 48 ml) and desired ketone (£, R.=acetyl, ₂ =H) (0.36 mg, collected at 52 ml) , characterized by the following data: mass spectrum, m/e: 454 (M ⁺ , 9) , 394 (17) , 376 (10) , 134 (23) , 43 (100) ; NMR (CDCI 0.53 (3H, s, 18-H , 1.03 (3H, d, J=6.5 Hz, 21-H , 1.13 (3H, d, J=7.0 Hz, 28-^), 2.03 (3H, s, CH ₃ C00) , 2.12 (3H, s _f .CH _j C0), 4.19 (1H, m, 3-H) , 5.03 (1H, , 19-H) _f 5.33 (3H, broad m, 19-H, 22-H and 23-H) , 5.49 (IH, m, 1-H) , 5.93 (1H, d, J=ll Hz, 7-H), 6.37 (IH, d, J=ll Hz, 6-H); ϋV (EtOH) _m _ _v 266

Example 7

Reaction of ketone (8) with ^* ret_πylmagnesium bromide to obtain products (9a) and (9b) .

Ketone (8,. R-= ^* acetyl, R-^H) in anhydrous ether is treated with the excess of O IgBr (2.85 M solution in ether) . The reaction mixture is stirred at room terrperature for 30 min, then quenched with aq. NH _j Cl, extracted with benzene, ether and CH-C1 ₂ . The organic phases are washed with dilute NaHCO-, dried over MgSO. and evaporated. The mixture of (9a) and (9b) thus obtained is separated by high performance liquid chrcmatography (6% 2-propanol/hexane, 4.6 rm x 25 cm Zorbax-Sil) , to obtain, in order of elution, pure epimers (9a) and (9b "). 3^,25-dihydroxyvitamin D ώ„ (9a -): ϋV (EtO ,H ro3__*c

265.5 rr , - ^; * _j ^ 227.5 nm; mass spectrum, m/e 428 ( , 6) , 410 (4) , 352 (4) , 287 (6) , 269 (10) , 251 (10) , 152 (42) , 134 (100) , 59 (99) ; NMR (CDC^) 50.56 (3H, s, 18-^) , 1.01 (3H, d, J=6.5 Hz, 28-H ₃ ) , 1.04 (3H, d, J=6.5 Hz, 21-^) , 1.14 and

1.18 (6H, each s, 26-H ₃ and 27-H ₃ ) , 4.24 (IH, m, 3-H) , 4.43

( H, m, 1-H) , 5.01 ( H, m, 19-H) , / 5.34 (3H, broad m, 19-H,

22-H and 23-H) , 6.02 ( H, d, J=ll Hz, 7-H) , 6.39 (IH, d, J ^* =ll

Hz, 6-H) . l^,25-dinydro^-24-epivitamin D (9b) : UV (EtOH) r? 265.5 nm, λ . 227.5 nm; mass spectrum, m/e 428 (M , 13) , 410 (9) , min

352 (7) , 287 (11) , 269 (15) , 251 (13) , 152 (52) , 134 (100) , 59

(97) .

Example 8

Conversion of corrpound (7) to 5,6-t ^* rans-lg,25-dihydroxy- vitamin D ₂ cαrpounds (11a) and (lib).

Hydrolysis of ketel-intermediate {7_, R.=acetyl,. ^H using the conditions described in Example 4 provides the corresponding 5,6-trans-25-ketone of structure (10, R. ^* =acetyl,

R----H) , and subsequent reaction of this ketone with methyl magnesiumbromide, using conditions analogous to those of

Example 5, gives amixture of epimers (11a) and (lib) which are separated by high performance liquid cforomatography (HPLC) to obtain in pure form __3f,25-d___ψdroxy- 5,6-trans-vitamin D-

(11a) and la,25-dJhydroxy-5,6-trans-24-epivita- min D ₂ (lib) <.

If required, structure assignment can be confirmed by isomerization to the respective 5,6 cis cαrpounds (9a, 9b) according to known procedures.

5,g-trans-lg,25-d__hyd__cgcyvit_i_rf__n p (11a): UV (EtOH)

- ^• J.

' 7m-a _w x 273.5 nm, _m_i.n 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-lff,25--d-_hydro-W-24-epivitamin D^ (lib): UV

(EtOH) λ 273.5 rm,^ . 230 nm; mass spectrum, m/e 428 (M ⁺ _r 10) , 410*1$), 352 (4) , 28? (5), 269 (9) , 251 (8) , 152 (37) ,

134 (100), 59 (82).

Example 9

Preparation of alkyl and aryl analogs of la,25-dihydroxy- vitamin D ₂ compounds.

By reaction of ketone intermediate (8) (R.=acetyl, R=H) with, respectively.

VIPO

(a) ethyl magnesium b_ ^* omide

(b) propyl magnesium brαrάde

(c) isopropyl magnesium bromide

(d) butyl magnesium h_OTU.de

(e) phenyl magnesium bromide using conditions -analogous to those described in Exairple 7, there are obtained the corresponding hydrc«yvii_amin D ₂

(a) ethyl

(b) propyl

(c) isopropyl

(d) butyl

(e) phenyl

By like treatment of 5,6-trans-ketone __nteπrιediate (10) (R.=acetyl, R^H) with the above listed Grignard reagents, there are obtained the corresponding 5,6-trans-hydroxyvitamin D ₂ products, having the formula shown below

^■ -STΓ ET,

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wherein X is, respectively

(a) ethyl

(b) propyl

(c) isopropyl

(d) butyl

(e) phenyl

A suitable starting material for the process of this invention is the vitamin D-ketal derivative of structure (1) which can be obtained following Process Schemes II and III as described in British Specification No. 2,127,023 or United States Letters Patent No. 4,448,721. It is generally convenient (e.g. when both C-24-ep__mers are desired) to use compound (1) as a mixture of 24R and 24£3 epimers, separation of the individual 24R and 24£3 epimers being accomplished later. However, pure 24&-, or pure 24R-epimer of (1) are equally suitable, the former providing the 24S-l<z,25-dihy- droxy product and the latter the corresponding 24R-product.

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Process Scheme I

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Process Scheme H

ORffl - .

Process Scheme III