METHOD FOR PREPARING 1 alpha -HYDROXYVITAMIN D COMPOUNDS

Abstract: Process for separating 1 alpha -hydroxyvitamin D compounds from their corresponding 1 alpha -hydroxy-5,6-trans isomers in a mixture thereof by treating the mixture with a dienophile and then separating the resulting dienophil-adduct of the 1a-hydroxy-5,6-trans vitamin D isomer from the unreacted 1 alpha -hydroxyvitamin D compound.

Description: Description Method for Preparing lα-Hydroxyvitamin D Compounds This invention was made with Government support under Hϊ Grant No. AM-14881 awarded by the Department of Health and Hunan Services. Ωie Government has certain rights in this invention. Technical field This invention relates to a novel method for the preparation of lα-hydroxyvitamin D compounds. More specifically, the invention relates to a novel procedure for obtaining substantially pure lα-hydroxyvitamin. D compounds frαti mixtures containing such compounds and their corresponding 5,6-trans-vitamin D iscmers. Background lα-Hydro^vitamin D compounds, specifically lα,25- dihydroxyvitamin D- and lot,25-dihydroxyvitamin D_, are known as irrportant regulators of calcium hcmeostasis and of proper bone formation in animals and humans. These cαspounds and certain structural analogs (e.g. lα-hydroxyvitamin. D_, lctr-hydroxyvitamin D_ and related compounds) therefore find, or have been proposed for, many important uses in both human and veterinary medicine. Such uses include the prophylaxis and/or treatment of calcium metabolism disorders, such as renal osteαfystrσphy, rickets, ostecanalacia, osteoporosis, the milk fever condition in animals, etc. As a consequence of the medical utility of lα-hydroxy- vitamin D compounds, a variety of methods for their preparation have been developed. Summaries of these known methods have been presented, for example by Yakhimσvich, uss. Chen. Rev. 49,371 (1980), and DeLuca et al. Topics in Current Cbem. vol. J33, p. 1-65 (1979) , and DeLuca & Schnoes, Ann. Rev. Biochem. 52, 411 (1983). Several of these preparatory methods for lα-hydroxy- vitamin D ∞mpσunds result in mixtures of 5,6-cis and trans iscmers (i.e. lα-hydroxyvitamin D and the corresponding 5,6-trans iscmer) . Although for some therapeutic or other applications such mixtures may be used directly, it is generally the 5,6-cis product that is desired especially for medicinal formulations. Hence all synthetic methods yielding such cis/trans mixtures usually require separation, of the iscmers which is difficult and very laborious, and markedly reduces the yield of pure product. Relevant to the present invention is specifically the lα-hydroxylation method via 3,5-cyclovitamin D intermediates / as described in U.S. Patents 4,195,027 and 4,260,549. In this method the C-3-hydroxy group of a vitamin D compound is tosylated, and the tosylate is subjected to solvolysis to obtain a 3,5-cyclσvitamin intermediate. This intermediate is then oxidized to the lα-hydroxycyclσvitamin ccmpcund and the latter is solvolyzed to obtain a mixture of the 5,6-cis and 5,6-trans-lα-hydroxyvitamin D compounds (usually as the 3-acetate derivatives) . Whenever, for pharmaceutical -use, the 5,6-cis compound is the desired product, the cis/trans mixture resulting from solvolysis must be separated. Also relevant is the method of Saimond, U.S. Patent 4,206,131, for the preparation of lα-hydroxyvitainin D com¬ pounds. In this procedure, lα-hydroxy-5,6-trans-vitamin D compounds are produced as intermediates, which are then isαnerzied to the desired 5,6-cis vitamins. Since known isαnerization methods result in mixtures of cis and. trans vitamin D compounds, separation of the desired cis-product from the mixture is again required. Similarly, the lα-hydrox¬ ylation methods proposed in U.S. Patents 4,202,829, 4,263,215, 4,265,822, and 4,338,250, which involve direct 1-hydroxylation of 5,6-trans-vitarπin D compounds, followed by 5,6-dσuble bond iscmerization, require, if the pure cis product is desired. methods for the separation of the 5,6-cis compound from the cis/trans mixture. Because of the very similar chrc atographic properties of the cis and trans iscmers, such separation, though feasible, on a small scale with efficient columns, is very difficult, laborious and expensive, especially on a preparative scale. This difficulty in separating cis and trans iscmers is thus a major disadvantage of the above methods. Disclosure of Invention A new method for the preparation of 5,6-cis-lα-hydroxy- vitamin D cαrpounds has now been found which allows for the effective and efficient removal of 5,6-trans-lα-hydroxyvitamin D compounds from mixtures of the 5,6-cis- and trans-iscmers so as to obtain the desired 5,6-cis-vitamin D isc er in substantially pure form. Specifically, this novel method comprises treatment of a mixture of 5,6-cis- and trans-lα- hydroxyvitamin D compounds with a dienophile under reaction conditions so chosen as to produce selectively the Diels-Alder adduct of the 5,6-trans-vitamin D compound. The resulting mixture of unreacted 5,6-cis-vitamin D compound and the Diels-Alder adduct of the 5 ,6-trans-vitamin D compound is-now readily separated in a chrcmatographic or extractive step. The purpose of the reaction with the dienophile is thus to convert the two chr natographically very similar constituents of the mixture, the 5,6-cis and trans-vitamin D compounds, into a mixture of two chemically and chrcmato- graphically very different components, the adduct of the trans-ccπcound and the unreacted 5,6-cis-vitamin, from which the adduct is readily removed so as to obtain the desired 5,6-cis-vitamin D compounds in substantially pure form. The 5,6-cis and 5,6-trans vitamin D compounds, the mixture of which may be separated by the above surrenarized procedure, are characterized by the formulae shown below: where X, and X- represent, independently, hydrogen or a hydro:xy-prσtecting group, and where R may be any steroid side chain, e.g. R may be hydrogen or an alkyl radical, or R may be a substituted side chain of the type wherein , , R_ and R~ are each selected from the group consisting of hydrogen, hydroxy, halogen and protected hydroxy, R. is hydrogen, halogen or alkyl, a d ' Rg and & are selected, independently, from hydrogen, hydroxy, and protected hydroxy, or taken together, may form a carbon-carbon double bond. In this specification and in the claims, a hydrσxy- protecting group represents any of the groups well known in the art used for the temporary protection of hydroxy functions, such as an acyl group, an alkylsilyl group, or an ether group. A protected hydroxy is thus a hydroxy function derivatized (i.e. acylated, etherified) with one of these protecting groups. An acyl group is an alkanoyl group of 1 to 6 carbons, in all iscmεric forms, e.g. foπryl, acetyl, propionyl, butyryl, pivalcyl etc., or an arαyl group, such as benzσyl, or an alkyl-, halo- or nitro-substituted benzαyl group. Suitable alkylsilyl hydroxy-protecting groups are for example, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl and analogous alkyl-substituted silyl-radicals. Suitable ether protecting groups are methoxymethyl or ethoxymethyl groups, or tetrahydrofuranyl or tetrahydropyranyl groups, all of which are well-known in the art. The term "alkyl" signifies a hydrocarbon radical of frα 1 to 6 carbons, in all its isαneric forms, e.g. methyl, ethyl, prσpyl, isσpropyl, butyl, etc. The term "aryl" signifies a phenyl group or an alkyl-, halo-, or nitro-substituted phenyl group. The key aspect of the separation of a mixture of cis and trans vitamin D compounds is the treatment of such a mixture with a reactive dienophile. Although both 5,6-cis and trans vitamin D compounds can react with dienophiles, the 5,6-trans cαipounds undergo the reaction more rapidly, so that, under seme conditions treatment of a mixture of cis and trans ccmpσunds with a dienophile, will yield almost exclusively the Diels-Alder adduct of the trans-iscmer, while the cis-cempound remains unaltered. Suitable dienophiles for the above application are olefinic, acetylenic, or diazene compounds, characterized, respectively, by the general structures below Λ = C N Y 2 - C≡C - Y 1 * 2 - N = N - Y l Y 4 Y 3 wherein each of the substituents Y, Y_, Y_, and Y λ is selected 1 2 3 4 from the group consisting of hydrogen, alkyl, and an electron-withdrawing group, and where any two of the Y-substituents taken together may form a carbocyclic or heterocyclic ring, but where, in each case, at least one of the substituents Y is an electron-withdrawing group. An electron-withdrawing group, in the context of this invention, is a group which activates the dienophile towards reaction with a diene. Such activating groups are well-known in the art (see, for example, M. C. Kloetzel, Organic Reactions, vol. jj, p. 2-4; H. L. Holmes", Organic Reactions, vol. 5, p. 65) and include, for example, such groupings as keto, cyano, nitro, O-alkyl, O-acyl, halogen, aryl, carbαxyaldehyde, carbσxylic acid, alkyl carbαxplate, carboxamide, or the imide, or anhydride-form of a dicarboxylic acid. Specific examples of suitable dienophiles aret acrylic acids and alkyl esters thereof, acrylamide and acrylonitrile; acetylenic acids and esters, such as prqpiolic acid and its alkyl esters, or acetylene dicarboxylic acid and its mono- or di-al yl esters; maleic acid and its derivatives, such as maleic anhydride, maleimide, N-substituted maleimides, and maleic acid mono- or di-alkyl esters; quinones such as benzoquinone or naphthoquinone; and diazeπe-diencphiles such " as N(4)-alkyl- or N4-aryl-substituted triazoline-3,5-diσne, diazo-p j -benzoquinone , diazo-p-naphthcxjuinone , or the alkyl esters of azodicarroxylic acid. For work on larger scale, dienophiles such as maleic acid and its derivatives, e.g. maleic anhydride, are particularly useful because of their low cost, and the possibility of removing the resulting adducts from the mixture by hydrolytic/ extractive wαrk-up (as described belcw) . Other preferred dienophiles include acetylene dicarboxylic acids and esters, and N(4)-alkyl or phenyl substituted triazoline-3,5-diones. The diazo-p-benzoquinαne or diazonaphthcxjuiπone dienophiles, although also useful for purposes of the present invention, because of their extremely high reactivity requires conducting the reaction at exceedingly lew temperatures to achieve, discrimination in adduct formation between cis and trans vitamin D compounds, offer less advantage. The reaction of the above dienophiles with the cis/trans vitamin D mixture is conducted by adding the dienophile to the vitamin mixture in an organic solvent. It should be noted that in such a mixture the 5,6-cis and trans vitamin D compounds can be present in any proportion relative to each other. Tb assure complete reaction of the trans-vitamin, the dienophile reagent is added in a 1.5-5-fold excess over the known (or estimated) amount of 5,6-trans compound present. Suitable solvents are hydrocarbons, chlorinated hydrocarbons, . lew-molecular weight ethers, or low molecular weight acid or ester solvents; specific examples include, benzene, hexane, toluene, chlorobenzene, ethyl ether or ethyl acetate. As pointed out above, the dienophile is advantageously added in about 1.5-5-fold excess over the amount of 5,6-trans ccirpound estimated to be present. The reaction can be conducted over a wide temperature range, e.g. from about -50°C to the boiling temperature of the solvent used, and for a time sufficient to react all of the 5,6-trans-cαπpound present. The specific temperature and time chosen depends on the reactivity of the. dienophile. For highly reactive dienophiles, temperatures in the lower part of the range indicated are appropriate, for the less reactive ones the upper temperature range is preferred. The time required for the reaction also depends on the reactivity of the dienophile, and varies from minutes to hours. For example, for the reaction of a cis/trans vitamin D mixture with the reactive dienophile 4-phenyl-triazoline-3,5- dione, suitable reaction conditions are a temperature of 0-10°C, for 1-2 hr, using ca. 2-3-fold excess of reagent. For the reaction of a cis/trans vitamin mixture with maleic anhydride, a somewhat less reactive reagent, appropriate conditions are, a temperature of 30-40°C, for ca. 12-24 hr, using a 4-fold excess of the dienophile. At higher temperatures, e.g. 50-60°C the reaction is complete within 1-2 hrs and at about 80°C, a reaction time of 10-20 min. is appropriate. In general, for any given dienophile, appropriate reaction conditions (e.g. time and temperature) are easily determined by simply treating a small test sample of a cis/trans vitamin D mixture with the dienophile and checking the progress of the reaction (i.e. completeness of reaction with the txans-ccmpαund) by analysis of the reaction mixture (e.g. by thin layer or high performance liquid chrcmatography) . Obviously, unduly prolonged reaction of a cis/trans vitamin D mixture with a dienophile is to be avoided to prevent losses of the desired 5,6-cis-vitamin D ccπpound owing to its reaction (adduct formation) with the dienophile. After treatment with the dienophile, the reaction mixture consists of the Diels-Alder adduct of the 5,6-trans ccπpσund and the unreacted 5,6-cis compound, as represented by the structures below, where R and X. and X ? represent substituents as defined above, where Z represents carbon or nitrogen, which may be joined by a single or double bond, depending on the dienophile used in the reaction, and where Y-, Y 2 , Y_ and Y. each have the meaning as defined above, except that when Z is nitrogen, or double-bonded carbon, Y, and Y. will be absent, as is self-evident to one skilled in the art. For example, the reaction of the 5,6-trans compound with maleic anhydride, dimethyl acetylene dicarbox late and 4-phenyltriazoline-3,5-dione, respectively, yields the adducts haracterized, respectively, by the structures shewn below: chrcmatographically very similar to the 5,6-cis iscmers, the Diels-Alder adducts of the trans ccπpounds, as is readily apparent from inspection of the above structures, are substantially altered and thus behave very differently from .the free 5,6-cis cαrpounds on chromatography. Therefore, such adducts ' are readily separated by chromatography from the free 5,6-cis-compounds, e.g. on silica gel columns, or by preparative high pressure liquid chromatography. Even more advantageous, especially for large-scale preparations, is the separation of the adduct from the free 5,6-cis vitamin compound by simple solvent extraction. This is possible whenever the dienophile used in the above Diels-Alder reaction contains an acidic function (e.g. a phenolic or acid function) , or a function, such as ester or anhydride, that is readily saponifiable to a carbox late group. The 5,6-trans- vitamin-adduct can then be removed from the 5,6-cis-vitamin by extraction of the organic phase with dilute base. For example, treatment of a cis/trans vitamin D mixture with maleic anhydride yields a mixture of the free 5,6-cis-vitamin D compound and the maleic anhydride adduct of the 5,6-trans compound. This reaction product mixture can then be treated with aqueous alkali metal hydroxide (e.g. NaOH or KOH) to saponify the anhydride adduct to the corresponding dicarboxylate. The resulting adduct-dicarbox late derivative, being water-soluble, is then easily separated from the organic-soluble 5,6-cis-lα-hydroxyvitamin D compound by simple partitioning of the basic solution against an iirmiscible organic solvent and separation of the phases. The desired lα-hydroxy-5,6-cis-vitamin product, retained in the organic phase, is then isolated by evaporation of the solvent, and if required, further purified in the customary fashion, i.e. chromatography and/or crystallization. Alternatively, the undesired 5,6-trans-vitamin D adduct carboxylate can be removed from the free lα-hydro^vitamin D compound by treatment of the mixture with (or passage over) an ion exchange resin in the standard manner, whereby the free ' lα-hydroxyvitamin D compound is recovered, and if required, further purified. It will be noted that the above method for preparing lα-hydroxyvitamin D compounds can be usefully applied to any mixture of the 5,6-cis- and trans-iscmers, regardless of the synthetic origin of such mixtures. The new method is particularly useful for those cases where the direct separation (e.g. by chromatography) of 5,6-cis- and trans-iscmer mixtures is especially difficult. Such, is true, for example, whenever the substituents X. and X 2 in the above-shewn structures of 5,6-cis and trans compounds are alike or very similar (e.g. X. and X„ are both hydrogen or both acyl) . In addition, it is evident that the new method can be applied advantageously in all preparative work conducted on a large (e.g. cαtmercial) scale. It is also obvicus that, if required, the above described separation procedure using dienophiles can be repeatedly applied to the same preparation. If for example, the lα-hydroxyvitamin D product obtained by the process of this invention is found to still contain seme residual undesired 5,6-trans-iscmer (as might occur, for example, if reaction time was insufficient), this product may simply be treated again with the same or a different dienophile, according to the above procedure, to remove such undesired residual material. Example 1 A mixture (1 g) of 5,6-cis- and 5,6-trans-lα-hydroxy- vitamin D_ (containing about 20-25% of the trans compound) dissolved in ethyl acetate (25 ml) was treated with recrystallized maleic anhydride (4-fold molar excess over the 5,6-trans-vitamin compound present) and warmed to 35°C under nitrogen for 24 hr. The solvent was then removed under vacuum and 10% aqueous NaCH (25 ml) was added to hydrolyze the anhydride adduct of the 5,6-trans compound. After 15 minutes the mixture was extracted with ether and the ether extracts were washed with 10% NaCH, water and brine and then dried over MgSO.. The solvent was removed in vacuo and the resulting residue was applied to a silica gel column (2 x 30 cm) and product was eluted with ethyl acetate/hexane mixtures (500 ml of 10% ethyl acetate; then 500 ml of 30% and 500 ml of 50% ethylacetate in hexane) . The fractions containing the desired lα-hydroxyvitamin D_ were pooled, solvent was evaporated, and a portion of the resulting oil was crystallized frcm methyl formate. After two recrystallizations frcm the same solvent crystalline lα-hydroxyvitamin D_, mp 135-37° was obtained. Example 2 A mixture (0.75 g) of lα-hydroxyvitamin D_ 3-acetate and lα-hydroxy-5,6-trans-vitamin D_ 3-acetate (containing about 30% of the trans isαner) , was dissolved in 25 ml of ethyl acetate and treated with excess maleic anhydride (ca. 4-fold molar excess over the estimated amount of 5,6-trans isαπer present) . After 24 hr at 35°C, the solvent was removed in vacuo, 10% aqueous sodium hydroxide (25 ml) was added and the mixture was stirred for 10-20 min to saponify the anhydride adduct of the 5,6-trans-compound. (This saponification also removes the acetyl groups.) This mixture, transferred to a separatory funnel, was then extracted repeatedly with ether, and the combined ether extracts were washed with 10% aqueous NaCH, water, and saturated NaCl-solution and then dried (MgSO . The solvent was evaporated to obtain the desired lα-hydroxyvitamin D~ product which was purified by chrcmatography on a silica gel column (eluted with ethyl acetate/hexane mixtures) and then by preparative TEC (50% ethyl acetate/hexane) to yield 290 mg of lα-hydroxyviamin D_. Example 3 A mixture (840 mg) of 5,6-cis and trans lα-hydroxyvitarain D 3 (containing ca. 20-25% trans compound) in 10 ml ethyl acetate was treated with 820 mg of maleic anhydride under N_ at 55°C for 90 min. Solvent was evaporated, the residue was treated with aqueous NaCH (20 min., room temperature) and then the mixture was extracted repeatedly with ether; the organic phase was washed with H-0, and saturated NaCl solution, then dried over MgSO,, filtered and solvent was evaporated. The residue (cαtprising the desired lα-hydroxyvitamin D_ cc poun ) was chrαπatographed on a 3 x 27 Florisil column. After passage of 300 ml of 15% ethyl acetate in hexane, the product (lα-hydroxyvitamiή D. was eluted with 25% ethyl acetate in hexane. Evaporation of solvent gave 440 mg of colorless oil, which was crystallized frcm methyl formate to give crystalline lα-bydroxyvitamin D_ product. Example 4 An ca. 1:1 mixture of lα-hydroxyvitamin D, 3-acetate and lα-hydroxy-5,6-trans-vitamin D_ 3-acetate was dissolved in hexane and treated with a 1.5-fold molar excess (over the txans-ccmpound present) of 4-phenyltriazoline-3,5-dicne (1 mg/ml as solution in ethyl acetate) . The resulting mixture was allowed to react at 0-10°C for 65 min. Excess reagent was consumed by addition of isoprene, and the mixture was then directly chrcmatographed to obtain lα-hydroxyvitamin D_ in pure form.

Claims: Claims 1. A method for preparing lα-hydroxyvitamin D compounds which comprises treating a mixture containing said ccarpounds and their corresponding lα-hydroxy-5,6-trans- vitamin D iscmers, with a dienophile, wherefcy a mixture containing the dienσphile-adduct of the lα-hydroxy-5,6- trans-vitamin D isαmer and the unreacted lα-hydroxy¬ vitamin D compound is obtained, and separating said mixture. 2. The method of Claim 1 wherein the dienophile is selected from the group consisting of maleic acid, maleic acid mono alkyl ester, maleic acid di-alkyl ester and maleic anhydride. 3. The method of Claim 1 wherein the dienophile used is acetylene dicarboxylic acid, or an alkyl-or dialkyl ester thereof. 4. The method of Claim 1 wherein the dienophile is a 4-alkyl- or 4-aryl-triazoline-3,5-dione compound. 5. The method of Claim 1 wherein the desired lα-hydroxy¬ vitamin D ccirpound is separated frαn the adduct of the 5,6-trans iscmer by chromatography. 6. The method of Claim 2 or 3 wherein the desired lα-hydroxyvitamin D compound is separated frcm the adduct of 5,6-trans-vitamin D iscmer by treatment of the mixture with an alkaline metal hydroxide and partioπing between an aqueous and organic solvent medium whereby the lα-hydroxyvitamin D compound can be recovered from the organic solvent. 7. The method of Claim 1 wherein the cis/trans vitamin D mixture contains lα-hydroxyvitamin D_ and lα-hydroxy-5,6- trans-vitamin D 3 . 8. The method of Claim 1 wherein the cis/trans vitamin D mixture contains lα-hydroxyvitamin D„ and lα-hydroxy- 5,6-trans-vitamin D_. 9. The method of Claim 1 wherein the cis/trans vitamin D mixture contains lα,25-dihydroxyvitamin D^ and lα,25- dihydroxy-5,6-trans-vitamin D_. 10. The method of Claim 1 wherein the cis/trans vitamin D mixture contains lα,25-dihydroxyvitamin D 2 and lα,25- dihydroxy-5 ,6-trans-vitamin D 2 .

Inventors: DELUCA HECTOR F, LEE SEOK HO, PHELPS MARY E, SCHNOES HEINRICH K, DELUCA, HECTOR, F., LEE, SEOK, HO, PHELPS, MARY, E., SCHNOES, HEINRICH, K.
Applicants: WISCONSIN ALUMNI RES FOUND, WISCONSIN ALUMNI RESEARCH FOUNDATION
Application year: 1985
Main classification: C07J9
IPC codes: A61K31, A61P3, C07C67, C07C401