Manufacturing Process
1. Preparation of 1,4-cyclized adduct of cholesta-1,5,7-trien-β-ol and 4-
phenyl-1,2,4-triazoline-3,5-dione: a solution of 400 mg of cholesta-1,5-7-
trien-3β-ol in 30 ml of tetrahydrofuran is cooled with ice, and 190 mg of 4-
phenyl-1,2,4-triazoline-3,5-dione is added little by little to the solution under
agitation. The mixture is agitated at room temperature for 1 hour and the
solvent is distilled under reduced pressure. The residue is purified by
chromatography using a column packed with silica gel. Fractions eluted with
ether-hexane (7:3 v/v) are collected and recrystallization from ether gives
550 mg of a 1,4-cyclized adduct of cholesta-1,5,7-trien-3β-ol and 4-phenyl-
1,2,4-triazoline-3,5-dione having a melting point of 178°C to 182°C.
2. Preparation of 1,4-cyclized adduct of cholesta-5,7-dien-3β-ol-1α-epoxide
and 4-phenyl-1,2,4-triazoline-3,5-dione: 1.25 g of the 1,4-cyclized adduct of
cholesta-1,5,7-trien-3β-ol and 4-phenyl-1,2,4-triazoline-3,5-dione is dissolved
in 50 ml of chloroform, and 560 mg of m-chloroperbenzoic acid is added to
the solution. The mixture is agitated for 20 hours at room temperature, and
200 mg of m-chloroperbenzoic acid is further added and the mixture is
agitated again for 20 hours. The reaction mixture liquid is diluted with
chloroform, washed with a 10% aqueous solution of potassium carbonate and
dried with magnesium sulfate. Then, the solvent is distilled under reduced
pressure. The residue is purified by silica gel chromatography, and first
effluent fractions eluted with ether are collected, and recrystallization from
methanol gives 680 g of a crystal melting at 172°C to 173°C. The second
ether effluent fractions are collected, and recrystallization from methanol gives
400 mg of a 1,4-cyclized adduct of cholesta-5,7-dien-3β-ol-1α,2α-epoxide and
4-phenyl-1,2,4-triazoline-3,5-dione having a melting point of 152°C to 154°C.
3. Preparation of cholesta-5,7-diene-1α,3β-diol: a solution of 500 mg of the
1,4-cyclized adduct of cholesta-5,7-dien-3β-ol-1α,2α-epoxide and 4-phenyl-
1,2,4-triazoline-3,5-dione in 40 ml of tetrahydrofuran is added dropwise under
agitation to a solution of 600 mg of lithium aluminum hydride in 30 ml of THF.
Then, the reaction mixture liquid is gently refluxed and boiled for 1 hour and
cooled, and a saturated aqueous solution of sodium sulfate is added to the
reaction mixture to decompose excessive lithium aluminum hydride. The
organic solvent layer is separated and dried, and the solvent is distilled. The residue is purified by chromatography using a column packed with silica gel.
Fractions eluted with ether-hexane (7:3 v/v) are collected, and
recrystallization from the methanol gives 400 mg of cholesta-5,7-diene-1α,3β-
diol.
4. Preparation of 1α,3β-dihydroxyprovitamin D3: a solution of 25 mg of
cholesta-5,7-diene-1α,3β-diol in 650 ml of ether is subjected to radiation of
ultraviolet rays for 14 minutes in an argon gas atmosphere by passing it
through a Vycor filter using a 200-W high pressure mercury lamp (Model
654A-36 manufactured by Hanobia). The solvent is distilled at room
temperature under reduced pressure. This operation is repeated twice, and 50
mg of the so obtained crude product is fractionated by chromatography using
a column packed with 20 g of Sephadex LH-20. The first effluent fractions
eluted with chloroform-hexane (65:35 v/v) give 13.5 mg of oily 1α,3β-
dihydroxyprovitamin D3. The composition exhibits a maximum ultraviolet
absorption at 260 nm in an ether solution.
5. Preparation of 1α-hydroxycholecalciferol: a solution of 13.5 mg of 1α,3β-
dihydroxyprovitamin D3 in 200 mi of ether is allowed to stand still in the dark
at room temperature in an argon gas atmosphere for 2 weeks. During this
period, the position of the maximum ultraviolet absorption is shifted from 260
nm to 264 nm, and the absorption intensity becomes 1.6 times as high as the
original intensity. The solvent is distilled at room temperature under reduced
pressure, and the residue is purified by chromatography using a column
packed with 10 g of Sephadex LH-20. The fractions eluted with chloroformhexane
(65:35 v/v) give 6.5 mg of oily 1α-hydroxycholecalciferol.
