Novel Vitamin D Analogs

Technical Field

This invention relates to compounds characterized by vitamin D-like activity.

More specifically, this invention relates to fluorinated analogs of vitamin D.,. Background Art

It is by now well established that the action of vitairdn D in regulating calcium and phosphate hemeostasis in animals and humans is dependent on metabolism of the vitamin in vivo to hydroxylated forms. In the case of vitamin D ₃ , this metabolic activation involves an initial hydroxylation at carbon 25 to yield 25-hydroxyvitamin D_, followed by another hydroxylation at carbon 1 to produce lc.>25-dihydroxyvi1_amin D_. This latter cccrpound is generally considered the active form of vitamin D , and as such is the agent directly responsible for stimulation of intestinal calcium transport and bone mineral mobilization in vivo. D ₃ is, hcwever, itself subject to further metabolism in vivo, being converted for example to la, 24,25-trihydroxyvitamin D_ by enzymatic hydroxylation at C-24. This 24-hydroxylated form is, however, less active than lc.,25- dihydroxyvitamin D-. itself, and 24-hydroxylation may represent indeed a step towards inactivation and degradation of the active form of the vitamin. Hence, to the extent that 24-hydroxylation occurs, it depletes the body stores of the physiologically most active metabolite, lα,25-ctihydroxyvitamin D_. For this reason, 24,24-difluoro compounds have recently been proposed as pharmacologically desirable analogs and substitutes, because the presence of the 24-difluoro substituents blocks the activity-attenuating 24-hydroxylation

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reactions. Indeed, such compounds have been shown to possess activites at least equivalent, and generally superior, to the corresponding non-fluorinated metabolite (see U.S. Letters Patents Nos. 4,196,133 and 4,201,881; Tanaka et al, J. Biol. Chem. 254, 7163 (1979) ; Tanaka et al, Arch. Biochem. Biophys. 199, 473 (1980)). Disclosure of Invention

The present invention relates to novel compounds possessing the desirable properties of high potency and a blocked C-24-position. An example of such a compound is 24-fluo-.o-l,25-d_i_hydroxγvita_τu_n D_, which may be represented by the following structure:

and to corresponding acyl derivatives of these compounds as well as their 5,6-trans isomers.

The structure shown above is characterized by the presence of the activity-enhancing hydroxy groups at carbons 1 and 25, as they occur in the natural metabolite, and fluorine as a blocking substituent at carbon 24. The blocking substitutent interferes with the introduction, by in vivo metabolism, of the 24-hydro_Q ^r group. Eecause of the presence of a single fluoro substitutent, such compounds have the advantage over previously proposed 24-blocked analogs (U.S. Patents 4,201,881 and 4,196,133 supra) of being biologically and chemically more similar and closely related to the natural

1,25-dihydrαxy metabolite, and hence of being a more desirable substitute for the natural coirpound. Also, by virtue of this lesser fluorine substitution, the novel analog may be used advantageously and more broadly in treatments of bone disease and related calcium disorders, since in the practical pharmaceutical applications of drugs it is often desirable to avoid as far as possible, the introduction of unnatural foreign elements.

24-fluoro-lα,25-dihydro_^^it__-rrιin D can be prepared by the in vitro enzymatic hydroxylation of 24-fluoro-25-hydroxy- vitamin D_ (U.S. Letters Patents No. 4,305,880) at carbon 1, using a hαtogenate of kidney tissue obtained from vitamin D-deficient chickens. The required homogenate is obtained as follows: One day-old leghorn chickens are fed a vitamin D-deficient diet containing 1% calcium for one month (Omdahl et al, Biochemistry, 10_, 2935-2940 (1971)). The chickens are then killed, their kidneys are removed, and a 20% (w/v) homogenate is prepared in ice-cold 0.19 M sucrose solution containing 15 mM Tris-acetate (trihydroxyirethylaitu oethane acetate) pH 7.4) and 1.9 irM magnesium acetate. (Omdahl, J. , et al, Biochemistry , _10_, 2935-2940 (1971) and Tanaka, Y. , et al, Arch. Biochem. Biophys. 171, 521-526 (1975)).

To produce the desired analog, 24-fluoro- l ,25-dihydroxy- vitamin D_, the precursor compound, 24-fluoro-25-hydroxy- vitamin D, is then incubated with this homogenate. The following procedure illustrates a small-scale incubation, but it should be understood that amounts can be scaled-up appropriately if desired.

A sample (ca. 3 ^αg) of 24-fluoro-25-hydroxyvitaιrd_n D-. (in 25 _^ μl of 95% ethanol is added to an aliquote of the kidney hαtogenate (prepared as described above and representing about 600 mg of kidney tissue) suspended in 4.5 ml of buffer solution (pH 7.4) which contains 0.19 M sucrose, 1.5 irM Tris-acetate, 1.9 mM magnesium acetate and 25 M succinate. After shaking the mixture at 37°C for 2 hours, the reaction is

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stopped by adding a 2:1 mixture of methanol:CHCl-. solvent. The organic phase of the resulting mixture is separated and evaporated and the residue containing the desired 24-fluoro- __2,25-d__hydrαxyvitaιrιin D, is subjected to i__omatography.

Initial purification of the product is readily accomplished by chromatography on small Sephadex IH-20 (Pharmacia Corp. , Piscataway, N.J.) columns, using solvent systems such as CHCU:hexane (65:35, v/v) or hexane:CHCl.,:MaOH (9:1:1, v/v) , but most advantageously the product is purified by high pressure liquid chromatography. (A cteomatograph suitable for such purpose is a model ALC/GPC 204 high pressure liquid chromatograph (Waters Associates, Medford, MA) equipped with an ultraviolet detector operating at 254 nm.) The sample obtained as above is injected onto a silica gel column (Zorbax-SIL, 0.46 x 25 cm, manufactured by Dupont, Inc.) operating under a pressure of 1000 psi which produces a flow rate of about 2 ml/min. Using a solvent system containing 9% of 2-propanol in hexane, the sample is recycled twice through this column (by switching the instrument to its recycle mode) and then collected. Solvent is evaporated and the residue is further purified on a reversed-phase column [Zorbax-ODS (octadecylsilane bonded to a fine grained silica gel) 0.45 x 25 cm, a product of Dupont and Co.] using the same high pressure liquid chromatograph operating at a pressure of 3000 psi. The product is eluted with a solvent mixture of E-O/MeOH (1:3) , recycled once and then collected. The collected fractions are evaporated and the residue is rechrcmatographed on the straight-phase silica gel column (Zorbax-SIL, 0.46 x 25 cm) using conditions exactly as described above. After recycling twice, the sample is collected, and after evaporation of the solvent, pure product (24-fluoro-l<__,25-di- hydroxyvitamin D_) is obtained, and characterized and quanti- tated by its ultraviolet absorption maximum at 264 nm, and its characteristic mass spectral pattern (-*S=434) .

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From the 24-fluorcι-lQf,25-dihydroxyvit__-min D, the corresponding 5,6-trans-isomer is readily prepared by iodine-catalyzed isomerization according to the general procedure of Verloop et al, Rec. Trav. Chim. Rays-Bas 2 .' 1®®4 (1969). Thus treatment of 24-fluoro-]_3;,25-dihydroxyvi1_aιrι_Ln D_ in ether solution containing a drop of pyridine, with a solution of iodine in hexane (ca. 0.5 mg/ml) over 15 minutes, followed by addition of an aqueous solution of sodium thiosulfate, separation of the organic phase, and evaporation of solvent yields a residue, from which the desired 24-fluoro- la,25-dihydrαxy-5,6-transvitamin D.. is isolated by a combination of reversed-phase and straight-phase high performance liquid chromatography (HPLC) using the systems described above, or by thin layer chromatography (TLC) on silica gel using ethylacetate/hexane solvent mixtures.

Acylated derivatives of both the above compounds which may be desired for certain applications are prepared by conventional methods, i.e. treatment of a solution of hydroxy coπpound with an acylating agent, such as an acyl anhydride or acyl chloride. Various procedures for such acylations are well known in the art, as are the required acylating reagents. Thus, for example, treatment of 24-fluoro-l,25-dihydrojy- vitamin D ₃ with acetic anhydride in pyridine, or mixtures of pyridine and an inert co-solvent, at temperatures below 50°C yields the corresponding 1,3-diacetate, whereas under more forcing conditions, e.g. 80-90°C, the 1,3,25-triacetate is obtained. Other acylates, such as propionates, butyrates, benzoates, nitrobenzoates, hemisuccinates, hemiglutamates, hemiadipates, hemidiglycolates, etc. are prepared in an analogous fashion by reaction of the vitamin alcohol with the corresponding anhydrides or acyl chlorides. If desired, acyl groups in such acylated derivatives, are removed by conventional means, such as hydrolysis with mild base (KOH/MeOH or similar conditions) . By means of selective acylation and/or deacylation, performed on 24-fluoro-lo_,

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25-dihydroxyvitamin D_ or its 5,6-trans-isoιπer, one can obtain compounds characterized by the structures below:

wherein each of R,, R_, and R~, and which may.be the same or different, is hydrogen or acyl.

In this description, and in the claims, the term acyl refers to an aliphatic acyl group of 1 to 6 carbons in all possible isomeric forms, such as acetyl, propionyl, butyryl, etc., or to conventional aromatic acyl groups, such as benzoyl, nitrdbenzoyl, haldbenzoyl, methylbenzoyl, etc. , or to a dicarboxylic acyl group of 2-6 atoms chain length, such as represented by oxalyl, malonyl, succinyl, glutaryl, adipyl, diglycolyl, etc.

The high vitamin D-like activity of the novel compounds of this invention, similar to that of the natural hormone, l,25-dihydroxyvitam_Ln D_, strongly suggests their application as therapeutic agents for disorders involving calcium and phosphate metabolism with perhaps, better maintenance of the compound pool in vivo because of the interference with 24-hydroxylation.

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