Method for Preparing 1-Hyd-a-yvitaπdn D (-απpounds

This invention was made with Goveriitrent support under NIH Grant No. AM-14881 awarded by the Department of Health and Human Services. The Gσvernπent has certain rights to this iπventicn. Technical Field

This j_πvention relates to hydrαxylated vit-airάn D co-pounds. More specifically, the invention relates to a method for preparing l-hydrαx/vitcϊmin D cαrpounds frαα 3,5-ycl vάtamin D -Lntermediates. Background Art

It is known that the physiological actions of vit-3_ιτ__n D, namely the maintenance of calcium and phosphate hαneostasis and the proper mineralization of bone, is dependent on the in vivo metabolism of the vi-t-Ξaiiin to hydroxlyated derivatives. Particularly irnportant are 1-hydroxylated viteinin D metabolites, and one of these, lα,25-d__hyd__o_ς^itaιπ__n D.., is indeed generally regarded as the physiologically active hormonal form of vitamin D_. This cciripσund and certain of its l-hydroxylated structural analogues, e.g. lα-bydrαxyvitairiin D ₃ , lα-hydro_<yvitam__n D ₂ and lα,25-dihyd_x>_^vi-tømiπ D ₂ , and related ccπpounds are therefore of great interest as therapeutic agents, being useful for the treatment and prophylaxis of various human and animal diseases related to calcium -tibalance. As a result, there has been much effort directed tcwiards the synthesis of such l-hydi-oxyvitarain D ccmpounds, and a variety of useful procedures are documented in the patent and other literature.

Of relevance to the present application is the synthetic method described by Paaren et al. in J. Qrg. Chem. _45, 3253 (1980) and DeLuca et al. in U.S. Patents 4,195,027 and

4,260,549 which disclosures relate to the preparation of lα-hydj-OxyTit-amin D derivatives from vitamin D compounds by hydro-Qrlation at carbon 1. Briefly, this method involves the tosylation of a vitamin D compound at the C-3-hydroxy group, followed by tosyl displacenent with formation of a 3,5-cyclo- vitamin D derivative, and subsequent oxidation of that -_nt_e_πrediate to a lα-hyd_.o_^-3,5-cyclovi1^min D which is then converted to the C-1-acyloxy derivative and subsequently solvolyzed under acid catalysis to obtain a mixture of the 5,6-cis- and 5,6-t--ans-lα-hyd-X-<yvita-r-in D 1-O-acylates. Alternatively, the free lα-hydιra-ψ-3,5-γclcfvita-n_-n D __nt-_-cιtιediate can be directly solvolyzed in an acid medium (e.g. a low- irolecular-*weight organic acid, such as formic or acetic acid) to obt n a mixture of the 5,6-cis-- and 5,6-trans--lo&-hyd__Oxy- vitamin D 3-0-acylates, where the acyl group originates, in this case, frαn the said medium used.

It will be noted, that the irethods taught by the prior art produce the 1- or 3-0-acyl derivatives of the lα-hydrcxy- vitamin D cctπpounds, and since the free (unprotected) lα- hyd_-o_Q^it_aπins are generally the desired products, these acyl groups must be removed by a subsequent hydrolysis or reduction step. Disclosure of Invention

A new process has now been developed which yields directly the desired free l-hydroxyvii _rιin D cciπpounds thus el__πτ__røting the need for the additional acyl-removal step of the known process described above. This process comprises the acid-catalyzed solvolysis of a l-hyά^oxy-3,5-cyclσvi ^* -_a_r__n D compound to obta n directly a mixture of 1-hydroxyvitamin D (5,6-cis ccrpound) and 5,6- ^* t_rans-l-hydroxyvitamin D, i.e. the free, non-acylated, hyd__oxyvitamin D compounds.

More specifically, the .l-hyd_-o-^-3,5-qyclσvit_amin D compound is dissolved in a mixture of di ethylsulfoxide and a

lew-molecular weight organic acid, e.g. glacial acetic acid, halo-substituted acetic acids or formic acid. An equiinolar ratio of diirethylstilfoxide and acid, or a slight excess of acid is preferred. Ωis reaction mixture is then wa med to a temperature ranging from ca. 30° to ca. 100°C, in an inert atmosphere for a time sufficient to achieve cαnplete reaction. In general, reaction teπiperatures of about 50°C and reaction times of 0.5 to 1 hr are appropriate. Under such conditions, the l-hydroxy-cyclσvitamin derivative is solvolyzed to yield the free hydroxyvitarrdn D product, namely the mixture of l-j_ydroxyvita_r__n D and its 5,6-trans-isαner, in a ratio of about 4 to 1. *

Preferred 3,5-cyclσvitaιnin D cαrpounds to be used in the above described process are the lα-hyd_.cκy-3,5-cyclo-vit_a-n__n D ccrrpounds, characterized by the general structure shewn belcw

wherein Z is an alkyl group, and R is a steroid side chain of

wherein each of R., FL and R ₃ is selected frαπ the group consisting of hydrogen, hydroxy, protected hydroxy, and halogen, and where R. is hydrogen, halogen or alkyl, and R,.

and R, represent, independently, hydrogen, hydroxy, protected hydroxy and halogen, or taken together, form a carbon-carbon bond. Particularly preferred are cyclovitamin αapounds in which the side chain R has the structure of the side chain of vitamin D. or of vitamin D ₂ . Also preferred are the compounds in which R is a side chain as it occurs in 25-hydroxyvit-_min D_, 25-hy-ta3xyvit-_min D_, 25-hydroxy-24- epivitamin D_, 24,25-dihydroxy- vitamin D, and 25,26- d__hyd__oxyvitamin D^.

Wherever used in this specification and the claims, the term "alkyl" denotes a hydrocarbon radical of 1 to 6 carbons, in all iscmeric forms, e.g. methyl, ethyl, propyl, isqpropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, etc., the term "protected hydroxy" denotes a hydroxy function protected by acyl, alkylsilyl or ether groups. Acyl groups suitable for such function are, for example, alkanoyl groups of 1 to 6 carbons, e.g. foπnyl, acetyl, butyryl, hexanoyl, or aroyl groups such as benzoyl, or methyl-, halo- or nitro-substituted benzoyl, while examples of suitable ether protecting groups are rtet-hoxyrrethyl, ethoxymethyl, tetrahydrofuranyl or tetrahydrc-pyranyl.

The lα-_ d__oxy-3,5-cyclσvitamin D can be prepared by the general procedures given in U.S. Patent 4,195,027. This preparative procedure involves the 1-hydroxylation of the corresponding 3,5-cyclcvitamin D compound by treatment of the latter with Se0_ and a hydroperoxide (typically t-butyl hydroperoxide) in an organic solvent such as chloroform or methylene chloride. It has been found that the yield of the desired lα-hydro_y-3,5-cyclo-vi'{-_3Jιin D product is increased (and the amount of undesired side products, e.g. the l-oxo-x3ipσunds is substantially decreased) when the reaction is conducted with substantially anhydrous hydroperoxide (e.g. anhydrous t-butylhydroperoxide in toluene, prepared according to the method of Sharpless et al., J. Org. Chem. 4j3, 3607 (1983) ) and in the presence of a small amount of a nitrogenous

base, e.g. pyridine. For example, a reaction mixture containing 0.5 equivalent of SeO-, 2 equivalents of t-buty__hydroperc»_ide (as a ca. 3M solution in toluene) , 1 equivalent of pyridine and 1 equivalent of the 3,5-cyclo- vltamin D ccmpound to be oxidized, all dissolved in an organic solvent such as rπethylene chloride, gives a substantially inproved yield of the desired lα-lψd_-oxy-3,5-cyclovitamin D cenpound.

Solvolysis of such lα-hyd_ro_5y-3,5-cyclσvi ^* laιπins in d____et-_ψl--u_-fc^de/o_-ganic acid as specified above leads to a product mixture comprising lα-hyd_ >2yvitamin D and lα- hydrOxy-5,6-trc-ns-vitamin D, characterized, respectively, by the structures shown below wherein R is a side chain as defined above.

Such product inixture, which consists predcminantly of the 5,6-cis product can be used as such for therapeutic purposes, or it may be separated, e.g. by c-hrαra ^* tog__aphic methods, such as high performance liquid chrcmatography, to obtain either of the cαppounds in pure form. However, as mentioned above, direct d-imratographic separation of such mixtures is exceedingly tedious and difficult especially on a preparative scale. A preferred method for the separation of such mixtures consists of treatment of the mixture as obtained by the above-described solvolysis method, with a dienσphile in an

organic solvent, so as to obtain a mixture cαtprising the dienqphile adduct of the 5,6-trans compound and the unreacted 5,6-cis vitamin compound. The adduct and the free 5,6-cis compound are then easily separated by standard ch__αrcatography so as to obtain the 1-hydroxy- vit-curiin D product in pure form. Examples of suitable dienσphiles are acrylic acid and alkyl esters thereof, acetylenic acids and esters, acetylene dicarbojQ ic acid and mono- or di-alkyl esters thereof, maleic acid and its derviatives, such as maleic anhydride, maleimide, N-substituted maleimides, and maleic acid esters, as well as ι_it__ogen-dienophiles such as N4-alkyl or N4-phenyl- substituted triazol__ne-3,5-dione, or the alkyl esters of azo-dicarbojylic acid.

VJhen a mixture of ^" 5,6-cis- and 5,6-trans-lα-hydroxy- vitamin D compounds is treated with such a di-_nqphile, the trans compound in the mixture reacts preferentially to form the Diels-Alder adduct between the dienqphile and the 5,6-trans-cαrpound. This adduct is conveniently separated from the unreacted 5,6-cis cαrpound by c-_-rc.ratography, so as to recover the latter in pure form. Alternatively, whenever the dienσphile contains an acid, or hydrolyzable ester, or anhydride group, the mixture of adduct and free 5,6-cis compound, may be treated with a base, which converts the acid to the carboxylate and saponifies the anhydride or ester g oups to carboxylate groups, thus rendering the dienσphile- adduct of the 5,6-txans-vi ^* t-arnin D cc pound water soluble, so that the trans-product can be removed by simple partitioning between an aqueous and organic solvent with the desired 5,6-cis-l-hyσro ^* xyvitamin D compound being recovered in the organic phase.

The reaction of cis/ ^* brans-l-hyd_n3xyvit--min D mixtures with dienophile can be conducted in a wide range of organic solvents (e.g. aliphatic or aromatic hydrocarbon solvents, halo-carbon solvents, ethers, or lcw-*molecular weight ester solvents) , the temperature and time of reaction

being adjusted so as to give complete reaction with the 5,6--fa:ans-ccιr ^* pound. To assure complete reaction, the diencphile is preferably added in sane excess over the amount of 5,6-trans-ccιπpound estimated to be present in the mixture (e.g. 1.5-5-fold molar excess) . Reaction terrperatures of from below 0°C to the boiLLng temperature of the solvent are appropriate, the temperature and time being selected in accordance with the inherent reactivity of the dienqphile chosen, as is well- understood in the art. For example, for the reaction of a cis/t_^-_s-lα--iydrθ2^vit--min D mixture with maleic anhydride a reaction temperature of 30-40°C and reaction time of from 12-24 hr is suitable. At higher temperatures, e.g. 5O-60°C, a reaction time of 1-2 hr is adequate, at about 80°C, -reaction time is reduced to about 16-30 min.

The above described solvolysis procedure can also be applie to lβ-_ψd_-o_y-3,5-^clσvitarnin D ccrrpαunds, i.e. compound o£ the general

wherein Z and R represent substituents as defined above. Solvolysis of these lβ-hyd_-ox ^, /-ep__π_ers in dimethylsulfoxide/ organic acid under the conditions specified above leads to a mixture of lβ-hydroxyvitamin D and lβ-hydroxy-5,6-trans- vitamin D which may be separated, if desired, by the procedures previously described. Furtherirore, mixtures of lα- hydroxy- and lβ-hyd_.oxy-3,5-cyclcvitam__n D α_mpσunds can be solvolyzed whereby a product mixture ccnprising lα-hydroxy- vitamin D and the corresponding 5,6-trans-iscmer, and lβ- hydroxyvitamin D and its 5 ,6-trans-iscmer is obtained. If individual ccmpounds are desired, the lα-hydroxy-epimers can be separated frαn the Iβ-hydroxy compounds by direct chrαmato-

graphy (e.g. high pressure liquid chrαmatography) or via boronate ester formation and chromatography (as described in U.S. Patent 4,338,250) and the 5,6-cis/trans pairs can then be separated by additional chromatography on high performance columns or by the dienophile-adduct method as described above.

The D compounds are obtained as minor products in the 1-hydrαxylation process using selenium dioxide and t-bui_y-_hydrσperσxide as described above, or may be obtained by hydride reduction, under standard conditions, of the known l-oxo-3,5-cyclσvitamin D compounds. Example 1 Solvolysis of lα-hyd_^3xy-3,5-cyclovi ^* --_(i_in D-

A solution of lα-i d_-oxy-3,5-_yclovitam__n D_ (1 g) in a mixture of dimethylsulfoxide (10.64 ml, 0.15 mole) and glacial acetic acid (8.59 ml, 0.15 mole) was heated to 50°C under ₂ for 1 hr. Ηie mixture was then poured over ice, and extracted with ether. (3 x' 75 ml) . (Alternatively, the mixture can be neutralized by addition of NaHCO- solution, prior to ether extraction.) The combined extracts were washed with saturated aqueous NaH00 ₃ , water, and saturated NaCl solution, then dried over MgSO., filtered and concentrated in vacuo to give a mixture containing in ca. 4:1 ratio lα-hyd_ >xyvit-_min D^ (5,6-cis-compound) and 5,6-trans-lα- hyd_-oxyviτ_amin D_. Example 2

Separation of 5,6-cis and t_rans-lα-h' ₎ ^* -droxy-vitamin D compounds by reaction with a dienophile

A mixture of 5,6-cis- and 5,6-^ans-lα-hyd__oxyvitamin D_ ccmpounds, as obtained in Example 1 above, was dissolved in ethyl acetate (25 ml) and treated with freshly recrystallized maleic anhydride (4-fold molar excess of the estimated amount of 5,6-trans-ccπpound present) . This reaction mixture was heated to 3_5°C under ₂ for 24 hr. After evaporation of the solvent in vacuo, the crude oil was chrcmatographed over a silica gel column (2 x 30 cm) using ethyl acetate/hexane mixtures as eluent. The fractions conta__ning the desired lα-hydroxyvitamin D_ were collected and pooled, and this

material was repeatedly crystallized from methylfoππate to give the pure product (mp 135-137°C) .

Example 3

Separation of a mixture of 5,6-cis and trans lα-hydroxy- vitamin D_. by dienophile-reaction and saponification

A mixture of 5,6-cis- and 5, -trans-lα-hyd_xxxyvitamin D_, dissolved in ethyl acetate was reacted with maleic anhydride as in Example 2 above. After completion of reaction (24 hr, at 35°C) , the solvent was removed in vacuo and the resulting residue was treated with an aqueous solution of sodium hydroxide (25 ml) for 10-20 min at room teπperature (to saponify the maleic anhydride adduct of the 5,6-trans- cx__ftpound) . Ether was then added and the phases were separated in a separating funnel. After further ether extraction of the aqueous phase, the pooled ether phases were washed with 10% aqueous NaCH, water, and saturated NaCl solution, and then dried qver 5S0.. Evaporation of the ether solvent gave crude lα-hyd-_ox7v±ta_r-_n D_ product, which was further purified by c±trcamatography over silica gel (ethyl acetate/hexane mixtures as eluent) and then crystallized from methyl-formate to obtain the desired lα-hydroxyvitamin D . Example 4 -rproved lαr-hydroxylation of 3 ,5-cyclovitamin D-

Anhydrous tert-butyl hydroperoxide (26 rrmole) in toluene (9.0 ml) was added to a stirred suspension of selenium dioxide (0.722 g, 6.5 itirDle) in dry methylene chloride (150 ml) in a three-necked flask. The mixture was stirred for 3 hr under a slight positive pressure of nitrogen. Pyridine (1.05 ml, 13 mmole) was then added, and then 3,5-cyclcviτ-amin D_ (13 mmole) was introduced as a solution in methylene chloride (50 ml) . After 30 min, 10% aqueous NaOH solution (70 ml) was added, and then the reaction mixture was diluted with ether (500 ml) and the phases were separated. The organic phase was washed with 10% NaOH (3 x 70 ml) , then with water and saturated NaCl solution, and dried over MgSO.. After filtration and

evaporation of the solvent, the crude lα-hydroxy-3,5- cγclcvi_-aτ_in D- product was purified by chromatography over Florisil (6 x 35 cm column) . Elution with ethyl acetate/ hexane mixtures, and pooling of appropriate fractions gave 3.65 g of the desired lα-hyd_.ox^-3,- γclovita_t n D- product (ca. 85-90% pure).