DK147525B - 24,24-Difluoro-25-hydroxy-6-alkoxy-3,5-cyclovitamin D3 compounds - Google Patents

Description

147525 i

The invention relates to novel 24,24-difluoro-25-hydroxy-6-alkoxy-3,5-cyclovitamin D compound of the formula

RO J

^ OH

wherein R represents a hydrocarbon group of 1-4 carbon atoms which are intermediates in the preparation of the novel 24,24-difluoro-ΐα, 25-dihydroxy vitamin Dj.

Vitamin Dj is a well-known means of controlling calcium and phosphorus homeostasis. In normal animals or humans, this compound is also known to stimulate intestinal calcium transport and bone calcium immobilization, and it is able to prevent rickets.

It is also well known that to be effective, vitamin Dj must be converted in vivo to its hydroxylated forms. Eg. the vitamin is first hydroxylated in the liver to form 25-hydroxy-vitamin D 2 and further hydroxylated in the kidneys, thereby forming 1α, 25-dihydroxy-vitamin D eller or 24, 25-dihydroxy-vitamin D ^ · The Ια-hydroxylated the form of the vitamin is usually believed to be the physiologically active or hormonal form of the vitamin and to be responsible for what is termed the vitamin D-like effect, such as increased intestinal absorption of calcium and phosphate, mobilization of bone mineral and retention of calcium in the kidneys.

Since the discovery of biologically active metabolites (metabolism products) of vitamin D, there has been much interest in the preparation of structural analogues of these metabolites because such compounds may constitute valuable therapeutic agents for the treatment of diseases caused by calcium metabolism irregularities.

A variety of vitamin D-like compounds have been synthesized. Cf.. U.S. Patent 3,741,996, which relates to 1-hydroxycholecalciferol; 3,907,843, which discloses Ια-hydroxyergocalciferol; No. 3,786,062 to 22-dehydro-25-hydroxycholecalciferol; 3 906 014, which discloses 3-deoxy-1a-hydroxycholecalciferol and 4,069,321, 20 which disclose the preparation of various side chain fluorinated vitamin D 1 derivatives and side chain fluorinated dihydrotachysterol analogs. Indeed, many of these compounds have been shown to possess potent vitamin D-like effects, and some of these offer other practical advantages, such as e.g. that they are relatively easy to prepare or that they have partially selective mode of action, but none have yet been shown to be as active in vivo as 1α, 25-dihydroxycholecalciferol (1α, 25-dihydroxy vitamin D D), which is now usually believed to be the target cell-30 active hormonal form of vitamin D.

A new derivative of vitamin D has now been prepared which is at least as potent as 1α, 25-dihydroxy vitamin D D when measuring its ability to stimulate intestinal calcium transport or its ability to mobilize calcium from 3 bones This derivative has been identified as 24, - 24-difluoro-1α, 25-dihydroxycholecalciferol (24,24-difluoro-1α, 25-dihydroxy vitamin Dj or 24-F2 ~ 1,25 (OH) 2 ·

The subject derivative may constitute a preferred agent 5 for many therapeutic purposes because it is blocked for further metabolism by the carbon atom 24. It is well known that la, 25-dihydroxy vitamin Dj can undergo further degradation in vivo, thereby producing la, 24R, - 25- Trihydroxy Vitamin Dj. However, this 24-hydroxylated form is less active than the 1α, 25-dihydroxy vitamin itself

Dj, and the 24-hydroxylation may actually represent a first step towards degradation and removal of this compound from the organism. In the derivative in question, the presence of 2 fluorine atoms in the carbon-24 naturally prevents the hydroxylation of this carbon atom, and therefore the bond is not sensitive to the activity-reducing degradation affecting 1,25-dihydroxychole-calciferol. Prevention of this side-chain metabolism means that it is possible to maintain higher cell levels of the analog over a longer period of time, a factor which has obvious benefits for many therapeutic purposes.

The identity of the compound as 24,24-difluoro-ΐα, 25-dihydroxy vitamin Dj can be confirmed from the spectroscopic properties. The compound exhibits the typical vitamin D-like ultraviolet absorption with a maximum at 264 nm. The mass spectrum of the compound contains a molecular ion at m / e 452, as required for a 24,25-difluoro derivative of 1α, 25-dihydroxy vitamin Dj. Frag-30 mentions m / e 434 and 416 represent the removal of 1 and 2 molecules ^ 0. The loss of the entire steroid side chain causes fragmentation of m / e 287, which upon removal of 1 and 2 molecules 1 ^ 0 causes two peaks at m / e 269 and 251. In addition, the spectrum shows pronounced peaks at m / e 152 and m / e 4 14752 -6 134 (152- O) representing ring A fragments and which are typical of 1α, 3 [3-dihydroxy-vitamin D D compounds.

Preparation of 24,24-difluoro-ΐα, 25-dihydroxy vitamin 5 can be carried out by direct C1-hydroxylation via the 3,5-cyclo-vitamin D intermediates of the invention by general methods described by Paaren et al., (Proc Nat.

Acad. Sci., U.S.A. 75, 2080-2081 (1978)). The synthesis is shown on the schematic process diagram below V f f 'χΧχ'® »-Q' h in" ^ "F F P P P p vX ^ .T r ^ H ^ L pv oh

iT AcO HO HO

ΗΟ · * 'νν> ^ ΟΗ 1 12 - 13 15 5 147525

Treatment of a pyridine solution of 24,24-difluoro-25-hydroxy vitamin (9) with 1-1.5 equivalents of p-toluene sulfonyl chloride for 24 hours at 3 ° C, after addition, gives a saturated solution of NaHCO with. ether, and subsequent evaporation of the ether, the corresponding 3-monotosylated derivative. This material is dissolved in anhydrous methanol and then treated with 5-10 equivalents of NaOAc. After heating to 55 ° C for 20 hours, the mixture is diluted with HgO and extracted with ether.

The desired compound 24,24-difluoro-25-hydroxy-6-methoxy-3,5-cyclovitamin D 2 (10) (R = methyl) is obtained after evaporation of the ether in a 50% total yield. The use of other alcoholic solvents, such as ethanol and propanol, in the above reaction gives analogous cyclo-vitamin vitamin D compounds of the general formula (10) where R = ethyl, propyl. These analogs are equally applicable to the subsequent synthetic steps described below. If desired, the cyclovitamin D compound can be purified by silica gel thin layer chromatography using hexane / ethyl acetate (8: 2) as the solvent system, but it can also be used directly in the next step involving treatment of The cyclovitamine intermediate, dissolved in a halohydrocarbon solvent (e.g., Ct ClCl ^) with SeC ^ (0.5 equivalents and 25 t-butyl hydroperoxide (2 equivalents), was sold as described by Paaren et al. ( After 15 minutes of reaction at room temperature a 10% NaOH solution is added and the product is extracted with ether, the ether phase is washed with aqueous alkali and then with water which is evaporated and after thin layer chromatography of the residue (using silica gel and hexane / ethyl acetate (6: 4) as the solvent) gives the pure 24,24-difluoro-1α, 25-dihydroxy-6-methoxy-3,5-cyclovitamine (11) (R = methyl) (yield 55%). Treatment of this compound with ice-55 acetic acid at 60 ° C for 15 minutes followed by neutralization Acidification of the acetic acid with aqueous alkali and extraction with ether, after evaporation of the solvent, give a mixture of 24,24-difluoro-1α, 25-dihydroxy vitamin Dj-3-acetate (12) and the corresponding transisomers (13) (ratio ca. 3: 1). These compounds can be conveniently separated either by column chromatography or thin layer chromatography (e.g., silica gel, hexane: ethyl acetate solvent) or high pressure liquid chromatography, and the thus obtained pure 24,24-difluoro-1α, 25-dihydroxy vitamin Dj-3 Acetate is hydrolyzed by treatment with base (eg 5% 10 NaOH / MeOH, 2 hours at room temperature) and then by extraction with ether and evaporation of the ether the desired analog of 24,24-difluoro-1α, 25-dihydroxy is obtained. Vitamin Dj (14) in pure form, exhibiting the same spectral properties as a similar compound prepared enzymatically by hydroxylation of 24,24-difluoro-25-hydroxy vitamin Dj.

5.6- The trans-24,24-difluoro-1α, 25-dihydroxy-vitamin Dβ-3-acetate (13) isomer, obtained in pure form following the chromatography described above, is hydrolyzed in dilute base 20 (5¾ NaOH). MeOH, 2 hours at room temperature) to give, after ether extraction and evaporation of the ether, the pure 5,6-trans-24,24-difluoro-1α, 25-dihydroxy vitamin Dj (15). This compound shows a mass spectrum very similar to the corresponding 5,6-cis isomer, but it exhibits an ultraviolet spectrum with a maximum at 270 nm, which is characteristic of the 5,6-trans vitamin D chromophore .

5.6- The trans-24,24-difluoro-1α, 25-dihydroxy-vitamin D-compound can, of course, be converted to 24,24-difluoro-1α, 25-dihydroxy-vitamin D ved by the well-known photochemical isomerization of 5.6- using the method, e.g. described by Inhoffen et al., (Chem. Cal. 9, 2544 (1957)). Alternatively, the 5,6-trans-3-acetate intermediate (13) obtained by the solvolysis of the cyclovitamine compound, as described above, can be isomerized by irradiation with ultraviolet light to give the 5,6-cis-3 acetate (12). ), and the conversion of this material to the desired 24,24-difluoro-1α, 25-5 dihydroxy-vitamin D 2 analogs can then be achieved by mild base hydrolysis, as already described.

Biological activity

The biological efficacy of the subject analog is confirmed by in vivo experiments against rats.

10 Weaned male rats are fed a low-calcium, vitamin D-free diet, described by Suda et al. (J. Nutr.100, 1049-1052 (1970)) for 3.5 weeks. They are divided into three groups with 5-6 animals in each. The animals from this group (control group) received 0.05 ml of ethanol intrajugularly. The second and third groups 15 received a known amount of 24,24-difluoro-1α, 25-dihydroxy-viatmin D D (24,24-F₂-1,25- (Oh -hydroxy vitamin D 1 (1a, 25- (OH) 3) as solutions in 0.05 ml ethanol intrajugularly. (Martin and DeLuca, Am. 0. Phys. 216, 1351 - 1359 (1969) and Tanaka et al., Biochemistry 14, 3293 - 3296 (1975) with the following results: 8 1Λ7525

Biological activity of 24,24-F2 ~ 1,25- (OH) 2D3 T arm Ca

Hours Transport Serum

Dose administered after .45r (Calcium compound (pmol) dose (4503 mucos) m9 / 100 ml

EtOh 1.9 | 0.4® 3.2ίθ, 1® 24.24-F 65 6 4.6 ± 0.8 ° 4.3 ± 0.2b 1.25- (00) D 24 4.7 ± 0.9 ° 4, l ± 0.3b 1.25- (0H) ^ d £ 65 6 4.8 ± 0.7d 4.2 ± 0.3b 2 3 24 5; 7io, 7e 4; ι ± 0; 4 °

Significance b, c, subtract a b from a of difference P <0.001; P <0.001; c from e c from a do not signi- P <0.005; fikant

The above data shows that 24,24-F2-1,25- (OH) 2D3 is effective both in the intestine and in the bones, and that the compound is at least as effective as 1.25- (OH) 2D3, the most active , known vitamin D derivative.