NL8320218A - NEW VITAMIN D ANALOGS. - Google Patents

Description

- 1 - 832 02 1 8

Description

New vitamin D analogues 5 Technical field

The invention relates to compounds which are characterized by vitamin D-like action.

More particularly, the invention relates to fluorinated vitamin Dg analogues.

Technical background

It is now clearly established that the action of vitamin D in the regulation of calcium and phosphate hemeostasis in animals and humans depends on the metabolism of the vitamin in vivo to hydroxylated forms.

In the case of vitamin Dg, this metabolic activation involves an initial hydroxylation of carbon 25 to give 25-hydroxyvitamin Dg, followed by another hydroxylation of carbon 1 to give 1, 25-dihydroxyvitamin Dg. The latter compound is generally considered to be the active form of vitamin Dg and as such it is directly responsible for the stimulation of intestinal calcium transport and bone mineral mobilization in vivo.

However, 1 <V ', 25-dihydroxyvitamin Dg is itself subject to further metabolism in vivo, for example converting it into 10,24,30,30-trihydroxyvitamin Dg by enzymatic hydroxylation on C-24. However, this 24-hydroxylated form is less active than 10 25, 25-dihydroxyvitamin D 2 itself, and 24-hydroxylation may even represent a step towards inactivation and degradation of the active form of the vitamin. Thus, as 24-hydroxylation occurs, it depletes the 35 body stores of the most physiologically active metabolite, 8320218 - 2 - 1, 25-dihydroxyvitamin D 1. 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-dampening 24-hydroxylation reactions. It has even been shown that such compounds have actions that are at least equivalent and generally superior to the corresponding non-fluorinated metabolite (see U.S. Patents 4,196,133 and 4,201,881, Tanaka 10 et al., J. Biol Chem. 254, 7163 (1979) Tanaka et al., Arch.

Biochem. Biophys. 199, 473 (1980)).

Description of the invention The present invention relates to new compounds with the desired properties of strong potency and a blocked C-24 position. An example of such a compound is 24-fluoro-1,25-dihydroxy-vitamin D 1, which can be represented by formula 1 and 20 on corresponding acyl derivatives of these compounds, as well as their 5,6 transisomers.

The above structure is characterized by the presence of the activity-enhancing hydroxyl groups on carbons 1 and 25, as they occur in the natural metabolite and fluorine as a blocking substituent on carbon 24. The blocking substituent interferes with the in vivo metabolism of the 24 -hydroxyl group. Due to the presence of a single fluorine substituent, such compounds have the advantage of previously proposed 24-blocked analogs (US patents cited above, 4,201,881 and 4,196,133) / that they are biologically and chemically more similar. ...

and related to the natural 1,25-dihydroxy metabolite and thus be a more desirable substitute for the natural compound. Also because of this less fluorine substitution, the new analog can be used more advantageously and better in the treatment of bone diseases and related calcium disorders, since it is often desirable in the practical pharmaceutical applications of drugs, the introduction of unnatural avoid foreign elements as much as possible.

24-fluoro-1 (Y, 25-dihydroxyvitamin) can be prepared by the in vitro enzymatic hydroxylation of 24-fluoro-25-hydroxyvitamin (U.S. Patent 4,305,880) to carbon 1, using a homo-10 gene from kidney tissue, which is obtained from vitamin D-deficient chicks The required homogenate is obtained as follows: one-day-old leghorn chicks are fed for one month on a vitamin D-deficient diet, which contains 1% calcium (Omdahl et al., Biochemistry, 10, 2935-2940 15 (1971)) The chicks are then sacrificed, 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 (trihydroxymethylaminoethane acetate) pH 7.4)) and contains 1.9 mM magnesium acetate. (Omdahl, J., et al., Biochemistry, 20 J0, 2935-2940 (1971) and Tanaka, Y., et al., Arch. Biochem.

Biophys. 171, 521-526 (1975)).

To prepare the desired analog, 24-fluoro-1e ^, 25-dihydroxyvitamin D ^, the starting material, 24-fluoro-25-hydroxyvitamin D ^, is then incubated with this homogenate. The following procedure shows small-scale incubation, but if desired, the amounts can of course be increased to an appropriate scale.

A sample (approx. 3 µg) of 24-fluoro-25-hydroxyvitamin D ^ (in 25 µl of 95% ethanol) is added to an amount of renal homogenate (prepared as described above and representing about 600 mg of renal tissue ), which is suspended in 4.5 ml buffer solution (pH 7.4) containing 0.19 M sucrose, 1.5 mM Tris acetate, 1.9 mM magnesium acetate and 25 mM succinate. After shaking the mixture for 2 hours at 37 ° C, the reaction is stopped by adding a 2: 1 8320218-4 solvent mixture of methanolsCHCl 3. The organic phase of the resulting mixture is separated and evaporated to give the residue Containing 24-fluoro-100, 25-dihydroxy-tamine DI, is subjected to chromatography.

Initial purification of the product is easily accomplished by chromatography on small Sepha-dex LH-20 (Pharmacia Corp., Piscataway, NJ) columns, using solvent systems such as CHCl 3 hexane (65:35, v / v) or hexane CHCl 2 MeOH (9: 1: 1, v / v), as many as 10, the product is purified by high pressure liquid chromatography. (A chromatograph suitable for such purpose is a model ALC / GPC 204 high performance liquid chromatograph (Waters Associates, Medford, MA) equipped with an ultraviolet detector operating at 254 nm.) The above mixture 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, producing a flow rate of about 2 mm / min. Using a solvent system containing 9% 2-propanol in hexane, the sample is returned through this column twice (by switching the instrument to its return position) and then collected. The solvent is evaporated and the residue is further purified on an inverted phase column / Zorbax-ODS (octadecylsilan, which is bound 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 H 2 O / MeOH (1: 3), returned once and then collected. The fractions collected are evaporated and the residue is re-chromatographed on the straight phase silica gel column (Zorbax-SIL, 0.46 x 25 cm) using exactly the same conditions as described above. After returning twice, the sample is collected and after evaporation of the solvent, pure product (24-fluoro-1,025,2-dihydroxyvitamin 8320218 - 5 -

Dg) obtained and characterized and quantified from its ultraviolet absorption maximum at 264 nm and its characteristic mass spectrum pattern (molecular weight = 434).

From the 24-fluoro-1 D (, 25-dihydroxyvitamin 5), the corresponding 5,6-trans isomer is readily prepared by iodine-catalyzed isomerization according to the general procedure of Verloop et al., Ree. Trav. Chim. Rays-Bas 78, 1004 (1969) Thus, treatment of 24-fluoro-1 tX ', 25-dihydroxyvitamin Dg in ether solution containing 10 drops of pyridine with an iodine solution in hexane (0.5 mg / ml) for 15 minutes, followed by addition of an aqueous sodium thiosulfate solution, separation of the organic phase and evaporation of the solvent to give a residue from which the desired 24-fluoro-1 D <', 25-dihydroxy-15 5,6-transvitamin Dg is recovered by combination of reverse phase and straight phase high pressure liquid chromatography (HPLC) using the systems described above or by thin layer chromatography (TLC) on silica gel using ethyl acetate / hexane solvent mixtures.

Acylated derivatives of both of the above-mentioned compounds, which may be desirable for certain applications, are prepared in conventional ways, i.e. by treating a solution of the hydroxyl compound with an acylating agent, such as an acyl anhydride or acyl chloride. Various embodiments for such acylations are well known in the art, as are the required acylation reagents. For example, treatment of 24-fluoro-1,25-dihydroxyvitamin Dg 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, while under more powerful conditions, for example 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 analogously 8320218-6 - by reaction of the vitamin alcohol with the corresponding anhydrides or acid chlorides. If desired, acyl groups in such acylated derivatives are removed in a conventional manner, such as soft base hydrolysis (KOH / MeOH or similar conditions). By selective acylation and / or deacylation carried out on 24-fluoro-1,25-dihydroxyvitamin D ^ or its 5,6-trans isomer, one can obtain compounds of the structural formulas 2 or 3, wherein and Rg, which may be the same or different each represent hydrogen or acyl.

In this description and in the claims, the term acyl refers to an aliphatic acyl group of 1 to 6 carbon atoms in all possible isomeric forms, such as acetyl, propionyl, butyryl, etc., or to conventional aromatic aryl groups, such as benzoyl, nitro- benzoyl, halobenzoyl, methylbenzoyl, etc., or on a dicarboxylic acid acyl group with 2-6 atom chain length, as represented by oxalyl, malonyl, succinyl, glutaryl, adipyl, diglykolyl, etc.

The strong vitamin D-like activity of the novel compounds of the invention, similar to that of the natural hormone, 1,25-dihydroxyvitamin Dg, strongly encourages their use as therapeutic agents for conditions involving calcium and phosphate metabolism, with perhaps better maintenance of the compound stock in vivo, due to the disruption of the 24-hydroxy lation.

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83 2 0 2 H

Claims (3)

1. Compounds of formula 2 or 3, wherein R 1 and R 2, which may be the same or different, are each selected from hydrogen or acyl. Compounds according to claim 1, wherein R., R. and R. and R. are each selected from the group consisting of hydrogen, acetyl and benzoyl. 3. 24-fluoro-1 Q (, 25-dihydroxyvitamin D ^. 4 24-fluoro-1 ty-S-dihydroxy-S, 6-transvifamine D ^. 10 8320218