Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C401/00—Irradiation products of cholesterol or its derivatives; Vitamin D derivatives, 9,10-seco cyclopenta[a]phenanthrene or analogues obtained by chemical preparation without irradiation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/02—Nutrients, e.g. vitamins, minerals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
  • A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C35/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring
  • C07C35/48—Halogenated derivatives
  • C07C35/52—Alcohols with a condensed ring system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C2601/00—Systems containing only non-condensed rings
  • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
  • C07C2601/14—The ring being saturated
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C2602/00—Systems containing two condensed rings
  • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
  • C07C2602/14—All rings being cycloaliphatic
  • C07C2602/24—All rings being cycloaliphatic the ring system containing nine carbon atoms, e.g. perhydroindane

Definitions

• This invention relates to a compound which is characterized by vitamin D-like activity.
• this invention relates to a derivative of vitamin D 3 .
• Vitamin D 3 is a well-known agent for the control of calcium and phosphorous homeostasis. In the normal animal or human this compound is known to stimulate intestinal calcium transport and bone-calcium mobilization and is effective in preventing rickets.
• vitamin D 3 must be converted in vivo to its hydroxylated forms.
• the vitamin is first hydroxylated in the liver to form 25-hydroxyvitamin D 3 and is further hydroxylated in the kidney to produce 1 ⁇ ,25-dihydroxy vitamin D 3 or 24, 25-dihydroxy vitamin D 3 .
• the 1 ⁇ - hydroxylated form of the vitamin is generally considered to be the physiologically active or hormonal form of the vitamin and to be responsible for what are termed the vitamin D-like activities, such as increasing intestinal absorption of calcium and phosphate, mobilizing bone mineral, and retaining calcium in the kidneys.
• vitamin D-like compounds have been synthesized. See, for example, U. S. Patents Nos.
• vitamin D A new derivative of vitamin D has now been prepared, which is at least as potent as 1 ⁇ , 25-dihydroxyvitamin D 3 , as measured by its ability to stimulate calcium transport in intestine or its ability to mobilize calcium from bone.
• This derivative has been identified as 24,24-difluoro-1 ⁇ , 25-dihydroxycholecalciferol (24, 24-difluoro-1 ⁇ , 25-dihydroxyvitamin D 3 or 24-F 2 -1, 25(OH) 2 D 3 ).
• This new derivative may represent a preferred agent for man therapeutic applications, because it is blocked to further metabolism at the carbon 24. It is well known that 1 ⁇ , 25-dihydroxyvitamin D 3, can undergo further metabolism in vivo to yield 1 ⁇ , 24R, 25-trihydroxyvitamin D 3 .
• This 24-hydroxylated form is however less active than 1 ⁇ , 25-dihydroxyvitamin D 3 itself, and 24-hydroxylation may indeed represent the first step towards degradation and elimination of this compound from the animal system.
• the presence of two fluorine atoms at carbon-24 will, of course, prevent hydroxylation of this carbon atom and the compound is not, therefore, subject to the activity attenuating metabolism affecting 1, 25-dihydroxycholecalciferol.
• 24, 24 -difluoro- 1 ⁇ , 25-dihydroxyvitamin D 3 is readily prepared from 24, 24-difluoro-25-hydroxyvitamin D 3 by in vitro enzymatic hydroxylation of the latter compound at carbon 1 as illustrated in the following schematic:
• Hydroxylation at carbon 1 is accomplished by incubating the precursor 24, 24-difluoro-25-hydroxyvitamin D 3 with a homogenate prepared from kidney tissue of vitamin D-deficient chickens.
• a homogenate prepared from kidney tissue of vitamin D-deficient chickens One day-old leghorn chickens are fed a vitamin D-deficient diet contain ⁇ ing 1% calcium for one month (Omdahl et al, Biochemistry, 10, 2935-2940 (1971). They 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 (trihydroxymethylaminoethane acetate) (pH 7. 4) and 1. 9 mM magnesium acetate. (Omdahl, J.
• a typical small-scale incubation involves the addition of 3 ⁇ g of 24, 24-difluoro-25-hydroxyvitamin D 3 ( in 25 ,ul of 95% ethanol) to an aliquot of the kidney homogenate (prepared as described avove 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 mM Tris-acetate, 1. 9 mM magnesium acetate and 25 mM succinate.
• the product is further purified by high pressure liquid chromatography using a model ALC/GPC 204 high pressure liquid chromatograph (Waters Associates, Medford, Mass. ) 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 ⁇ 25 cm, manufactured by Dupont, Inc. ) operating under a pressure of 1000 psi which produces a flow rate of 2 ml/min.
• 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 ⁇ 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), recycled once and then collected.
• the collected fractions are evaporated and the residue is rechromatographed on the straight-phase silica gel column (Zorbax-SIL, 0. 46 ⁇ 25 cm) using conditions exactly as described above. After recycling twice, the sample is collected, and after evaporation of the solvent, pure product is obtained.
• the identity of the product as 24, 24 -difluoro- 1 ⁇ , 25-dihydroxyvitamin D 3 can be confirmed by its spectroscopic properties.
• the product shows the typical vitamin D-like ultraviolet absorption with a maximum at 264 nm;
• the mass spectrum of the product contains a molecular ion at m/e 452 as required for a 24, 24-difluoroderivative of 1 ⁇ , 25-dihydroxyvitamin D 3 .
• Fragment ions at m/e 434 and 416 represent elimination of one and two molecules of H 2 O. Loss of the entire steroid side chain results in the fragment of m/e 287 which, by elimination of one and two molecules of H 2 O, gives rise to the peaks at m/e 269 and 251.
• the spectrum shows prominent peaks at m./e 152 and m/e 134 (152-H 2 O) which represent ring A fragments and are diagnostic for 1 ⁇ 3 ⁇ -dihydroxyvitamin D 3 compounds.
• the starting material, 24, 24-difluoro-25-hydroxyvitamin D 3 , required for preparation of the 1 ⁇ hydroxy analog of this invention can be prepared according to the following process and schemttic.
• Cholenic acid 1 is treated with dihydropyran in a suitable organic solvent (CH 2 Cl 2 ) at 0° in the presence of p-toulene sulfonic acid (PtsOH) and then with IN Na OH in ethanol at 20° to form the cholenic acid tetrahydropyranyl ether (protection of the hydroxyl group in the A ring).
• PtsOH p-toulene sulfonic acid
• the methylketone 2 when refluxed for seven hours in acetic anhydride in the presence of p -TsOH (enolacetylation) yields the diacetate 3 (mp 109-110°). (m/e 396 (M-60)).
• the diacetate is then converted to the difluorocyclopropane 4 by heating with sodium chlorodifluoro-acetate in digiyme at 170° for 0. 5 hours (yield, 34%; mp 112-115°).
• the difluoroketone 5 is then reacted with an excess of CH 3 Mgl in ethyl ether at 0° C for 15 minutes and is subsequently acetylated (AC 2 0-pyridine-CH 2 Cl 2 , 20°, 20 hr. ) to furnish the 25-carbinol, 6, in 85% yield (mp 163-164. 5°, ⁇ 1. 28 (6H, s, C-26, 27), m/e 420 (M-60)).
• the carbinol, 6, is allylically brominated by reacting it with N-bromo-succinimide in refluxing CC1 4 for 25 minutes.
• the brominated compound is then directly dehydrobrominated by treatment with s-collidine in refluxing xylene for 15 minutes to give a mixture of the 4, 6-diene and the 5, 7-diene, 7.
• the 5, 7-diene ( ⁇ max 263, 272, 282 and 292 nm) is isolated after treatment of the reaction mixture with p- TsOH in acetone at 20° for 15 hours followed by preparative thin-layer chromatography (benzene-ethyl acetate (15: 1), 3 times).
• the recovered 5, 7-diene after saponification by treatment with 5% KOH-methanol at 20° C for 15 hours, is irradiated (Hanovia high pressure quartz mercury vapor lamp, model 654A36;200 W) in a mixture of ethanol and benzene for 2. 5 minutes at 0° C to give the previtamin 8 in solution.
• the irradiated solution is refluxed for one hour and then fractionated by thin-layer chromatography (silica gel, benzene-ethyl acetate, (5:1), 3 times) and high pressure liquid chromatography (Zorbax SIL, 25 cm ⁇ 2. 1 mm i. d. , available through the Dupont Co.
• 24, 24-difluoro-1 ⁇ , 25-dihydroxyvitamin D 3 can, of course, also be accomplished by purely chemical methods.
• 24,24-difluoro-25-hydroxyvitamin D 3 as starting material a particularly convenient process involves the direct C- 1-hydroxylation via 3, 5-cyclovitamin D- intermediates according to the general procedures described by Paaren et al. (Proc. Nat. Acad. Sci. , U. S. A. 75, 2080-2081 (1978)). The synthesis is shown in the process schematic below.
• the cyclovitamin D product can be purified by silica gel thin-layer chromatography using hexane /ethyl acetate (8:2) as solvent system, but it can also be used directly for the next step, which involves treatment of the cyclovitamin intermediate, dissolved in a halo-carbon solvent (e. g. CH 2 Cl 2 ), with SeO 2 (0. 5 equiv. ) and t-butyl hydroperoxide (2 equiv. ) according to the conditions described by Paaren et al (supra). After a 15 minute reaction time at room temperature, a 10% NaOH- solution is added, and the product is extracted with ether.
• a halo-carbon solvent e. g. CH 2 Cl 2
• the 5, 6-trans -24, 24-difluoro-1 ⁇ , 25-dihydroxyvitamin D 3 compound can of course be converted to 24, 24-difluoro- 1 ⁇ , 25-dihydroxyvitamin D 3 by the well-known photochemical isomerization of the 5, 6 -double bond, using the method described, for example by Inhoffen et al (Chem. Ber. 90 2544 (1957).
• the 5, 6-trans -3-acetate intermediate (13) obtained from the solvoysis of the cyclovitamin compound as described above can be isomerized by irradiation with ultraviolet light to the 5, 6-cis-3-acetate (12) a the conversion of the material, to the desired 24, 24-difluoro-1 ⁇ , 25-dihydroxyvitamin D 3 analog then is accomplished by saponification in mild base as already described.
• mice Male weanling rats are fed the low-calcium vitamin D-supplemented diet of Suda et al (J. Nutr. 100 , 1049-1052 (1970)) for 3. 5 weeks. They are then divided into 3 groups of 5-6 animals each. The animals of one group (the control group) receive 0. 05 ml of ethanol by intrajugular injection. The seco nd and third groups receive a known amount of 24, 24-difluoro- 1 ⁇ , 25-dihydroxyvitamin D 3 (24, 24-F 2 -1, 25-(OH) 2 D 3 ) and 1 ⁇ , 25-dihydroxyvitamin D 3 (1 ⁇ ,25-(OH) 2 D 3 ), respectively, as solutions in 0. 05 ml ethanol by intrajugular injection.
• test compounds are determined by standard assay procedures (Martin and DeLuca, Am. J. Phys. 216, 1351-1359 (1969), and Tanaka et al, Biochemist 14, 3293-3296 (1975))with the following results:
• the 24, 24-difluoro-1 ⁇ ,25-dihydroxycholecalciferol and 24,24-difluoro-1 ⁇ ,25-dihydroxy-5, 6-trans-cholecalciferol compounds of this invention may be readily administered as sterile parenteral solutions by injection or intravenously or by alimentary canal in the form of oral dosages, or by suppository. Doses of from about 0.1 ⁇ g to about 2. 5 ug per day are effective in obtaining the physiological calcium balance responses described and which are characteristic of vitamin D-like activity, with maintenance doses of about 0.25 ⁇ g being suitable.
• Dosage form of the compounds can be prepared by combining them with a non-toxic pharmaceutically acceptable carrier as is well known in the art.
• a non-toxic pharmaceutically acceptable carrier as is well known in the art.
• Such carriers may be either solid or liquid such as, for example, corn starch, lactose, sucrose, peanut oil, olive oil, sesame oil and water. If a solid carrier is used the dosage forms of the compounds of the invention may be tablets, capsules, powders, troches or lozenges. If a liquid carrier is used, soft gelatin capsules, or syrup or liquid suspensions, emulsions or solutions may be the dosage form.
• the dosage forms may also cotain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, etc. They may also contain other therapeutically valuable substances.

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• 1979
  • 1979-03-28 US US06/024,848 patent/US4201881A/en not_active Expired - Lifetime
  • 1979-10-15 CH CH8817/80A patent/CH650240A5/de not_active IP Right Cessation
  • 1979-10-15 NL NL7920190A patent/NL7920190A/nl unknown
  • 1979-10-15 WO PCT/US1979/000899 patent/WO1980002028A1/en not_active Ceased
  • 1979-10-15 JP JP55500243A patent/JPS6342618B2/ja not_active Expired
  • 1979-10-22 IL IL58526A patent/IL58526A/xx unknown
• 1980
  • 1980-02-08 NZ NZ191900A patent/NZ191900A/xx unknown
  • 1980-02-22 AU AU55810/80A patent/AU530015B2/en not_active Ceased
  • 1980-03-07 BE BE0/199728A patent/BE882143A/fr not_active IP Right Cessation
  • 1980-03-11 IT IT20508/80A patent/IT1140776B/it active
  • 1980-03-13 GB GB8035585A patent/GB2067567B/en not_active Expired
  • 1980-03-13 GB GB8008488A patent/GB2045765B/en not_active Expired
  • 1980-03-17 FR FR8005935A patent/FR2452481A1/fr active Granted
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• 1987
  • 1987-09-16 JP JP62229955A patent/JPS63179855A/ja active Granted

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