JPH0149279B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present application relates to a method for separating the 24-position epimer of a 24,25-dihydroxycholesterol derivative.

24(R)-24,25-dihydroxycholesterol derivative is an active form of vitamin _D324
(R)-24,25-Dihydroxycholesterol is an extremely useful compound as a raw material for vitamin _D3 , but 24,25-dihydroxycholesterol, which can be obtained by ordinary synthetic means, contains approximately equal amounts of the 24-position epimer. It is a mixture of As a means for separating and recovering the 24(R)-24,25-dihydroxycholesterol derivative from the 24-position epimer mixture, high performance liquid chromatography (Tetrahedron) is used.
Letters, 15 (1975)), but the throughput is so small that it cannot be a practical separation method.

Attempts to produce 24(R)-24,25-dihydroxycholesterol derivatives by asymmetric synthesis have also been reported (Japanese Unexamined Patent Publication No. 1983-46061).
It is a complicated, multi-step method that uses a special steroid derivative as a starting material, and cannot be said to be a practical or easy method.

The present inventors took the above into consideration and developed a method for quickly and easily separating and recovering 24(R)-24,25-dihydroxycholesterol derivatives from an epimer mixture in an amount sufficient for practical use. As a result of repeated research aimed at this purpose, we have arrived at the method of the present invention.

Formula () used in the method of the invention 24(ξ)-24,25-dihydroxycholesterol represented by the formula () It can be easily produced from hyodeoxycholic acid represented by or its methyl ester using the method described in Japanese Patent Application No. 57-002336 and Japanese Patent Application No. 57-002337.

24(ξ)-24,25-dihydroxycholesterol () is dissolved in a suitable organic solvent such as pyridine, chloroform-triethylamine, methylene chloride-
A carboxylic acid halide, such as benzoyl chloride, p-nitrobenzoyl chloride, α-naphthoyl chloride or β-naphthoyl chloride, dissolved in dimethylaminopyridine or the like, in an amount of 2 to 10 moles, preferably 3 to 5 moles, relative to the substrate. etc., preferably with benzoyl chloride at -30°C to 50°C, preferably 0°C to 30°C, for 0.5 to 10 hours, preferably 1 to 3 hours to obtain the formula (). (In the formula, R ₁ represents a phenyl group, p-nitrophenyl group, α-naphthyl group, or β-naphthyl group.) 24(ξ)-24,25-dihydroxycholesterol-3,24-diacylate represented by . twenty four
(ξ)-24,25-dihydroxycholesterol-
3,24-diacylate () in a suitable organic solvent,
For example, when recrystallized from benzene, ethyl acetate, acetone, methanol, etc., preferably acetone, the 24(S)-epimer is concentrated in the precipitate, and the 24(R)-epimer is concentrated in the liquid phase. By repeatedly subjecting the precipitate to recrystallization, the formula (-
S) (In the formula, R ₁ represents a phenyl group, p-nitrophenyl group, α-naphthyl group, or β-naphthyl group.) Separate 24(S)-24,25-dihydroxycholesterol-3,24-diacylate represented by It can be recovered.

Formula (-R) is present in the recrystallization solution. (In the formula, R ₁ is the same as in formula (-S).) 24(R)-24,25-dihydroxycholesterol-3,24-diacylate represented by 24(S)-
Although it is contained in excess compared to the epimer (-S), the 24(R)-epimer is difficult to precipitate as crystals, so it is difficult to concentrate the liquid and precipitate it for separation and recovery.

The present inventors have demonstrated that 24-dihydroxycholesterol-3,24-diacylate has 24
We have discovered the fact that the crystal precipitation properties of (R)-epimer can be significantly improved, and have thus completed the second half of the present invention.

That is, 24(R) obtained by separating the precipitated crystals
- The epimer-rich liquid is dried, dissolved in a suitable organic solvent such as pyridine, N,N-dimethylformamide, chloroform-triethylamine, methylene chloride-dimethylaminopyridine, etc., and reacted with a trialkylsilylating agent. Here, as the trialkylsilylating agent, a combination of hexamethyldisilazane and trimethylsilane chloride or a combination of tert-butyldimethylsilane chloride and imidazole is used. The reagent is used 1.5 to 10 times, preferably 2 to 5 times by mole, relative to the substrate, and heated at 0°C to 110°C, preferably 20°C to 70°C, for 10 minutes to 5 hours, preferably 30 minutes to 5 hours. Let react for 2 hours. If the reaction product is recrystallized from a suitable organic solvent such as benzene, ethyl acetate, acetone, methanol, etc., preferably acetone, the 24(R)-
The epimer becomes concentrated in the crystal, and by repeating recrystallization, the formula (-R) (In the formula, R ₁ represents a phenyl group, p-nitrophenyl group, α-naphthyl group, or β-naphthyl group, and R ₂ represents a trimethylsilyl group or tert-butyldimethylsilyl group.) -24,25-dihydroxycholesterol-3,24-diacylate-25-trialkylsilyl ether can be separated and recovered.

In exactly the same manner, a mixture of compound (-S) and compound (-R) containing an excess of compound (-S) is trialkylsilylated and recrystallization is repeated to obtain formula (-S). (In the formula, R ₁ and R ₂ are the same as in formula (-R).) 24(S)-24,25-dihydroxycholesterol-3,24-diacylate-25-trialkylsilyl ether represented by can be separated and recovered.

As is clear from the above, the feature of the method of the present invention is that the separation of the 24-position epimer of 24,25-dihydroxycholesterol derivatives, which has been extremely difficult in the past, has been achieved by recrystallization. Since the recrystallization operation is not affected by the amount of treatment, the method of the present invention can easily carry out large-scale treatment that was unimaginable with conventional methods, and can produce highly pure 24(R)-
Epimers and 24(S)-epimers can be produced.

24(R)-24,25-dihydroxycholesterol-3,24-diacylate-25-trialkylsilyl ether (-R) can be prepared by known methods, namely 7-position bromination followed by dehydrobromination, deprotection, 24(R) by ultraviolet light irradiation reaction and thermal isomerization reaction
Since it can be converted to -24,25-dihydroxyvitamin D ₃ , the method of the present invention provides
This is a significant feature that enables the production of 25-dihydroxyvitamin _D3 on a practical scale.

The present invention will be explained below with reference to Examples.

Example 1 24(ξ)-24,25-dihydroxycholesterol
275.3 g (0.658 mol) are dissolved in 2.74 g of dry pyridine and the reaction vessel is placed in a water bath at 15°C. 266 ml (2.31 mol) of benzoyl chloride was added dropwise over about 30 minutes with stirring, and the temperature of the reaction mixture was adjusted so as not to exceed about 20°C.

After stirring for 2.5 hours after the completion of the addition, the reaction mixture in step 7 was added dropwise to the ice-water while stirring vigorously.
The precipitated solid was filtered, washed with water, thoroughly washed with water, dissolved in chloroform 2.6, thoroughly washed with saturated brine, dried over anhydrous sodium sulfate, and then chloroform was distilled off to obtain crude 24 (ξ ) −24, 25
355.3 g (0.567 mol, 86.2%) of -dihydroxycholesterol-3,24-dibenzoate was obtained.

355g of this 24(ξ)-24,25-dihydroxycholesterol-3,24-dibenzoate
(0.567 mol) was dissolved in acetone of 19 at the boiling point, left at room temperature for 16 hours, and the precipitated crystals were separated.
24, 25 whose R/S ratio at the 24-position is 13/87 after drying
157.2 g (0.251 mol) of -dihydroxycholesterol-3,24-dibenzoate () was obtained. This resultant was dissolved in acetone 6.6 at boiling point and left at room temperature for 16 hours to obtain 71.6 g (0.114 mol) of crystals of compound () having an R/S ratio of 7/93 at the 24-position.
Thereafter, when the crystals of compound () were fractionally recrystallized in the same manner, 45.3 g (72.3 mmol) of compound () with an R/S ratio of 2.8/97.2 was obtained in the third time, and R/S in the fourth time. Compound ()27.5 with an S ratio of 0.9/99.1
g (43.9 mmol), the R/S ratio was 0.2/9 in the 5th time.
9.8 24(S)-24,25-dihydroxycholesterol-3,24-dibenzoate 16.9g
(27.0 mmol) was obtained.

Melting point: 195-196°C [α] ²¹ _D : -26° (CHCl ₃ , C,
0.099) IR (KBr, ν〓cm ^-1 ): 3450, 1715, 1275, 710 NMR (100MHz, CDCl ₃ , δppm): 0.63 (3H, s,
18− CH) ₃ , 0.93 (3H, d (6.0Hz), 21−
_CH3 ), 1.06 (3H, s, 19- _CH3 ), 1.28 (6H, s, 26, 27- _CH3 ) 5.07 (2H, m, 3, 24-CH), 5.41 (1H, br, d, 6-
CH), 7.3~7.6 (6H, m,
phenyl proton),
7.9-8.2 (4H, m, phenyl proton) Example 2 24(ξ)-24,25-dihydroxycholesterol
1140 g (2.72 mol) was dissolved in dry pyridine 11, and then benzoyl chloride was prepared in the same manner as in Example 1.
960 ml (8.27 mol) and post-treatment to give 1554 g (2.48 mol,
91%). This was recrystallized from acetone and 24
451 g of a crystal mixture in which the ratio of -position epimers was R/S = 11/89 was obtained. The liquid was dried and recrystallized again from acetone to obtain 229 g of a crystal mixture with R/S=35/65. N-hexane was added to the liquid and the precipitated solid was overdried to obtain 716 g of a mixture with R/S=80/20.

716 g (1.14 mol) of the above mixture was added to dry pyridine.
738ml of hexamethyldisilazane dissolved in 11
(3.50 mol) and 362 ml (2.85 mol) of trimethylsilane chloride
mol), heated to 70°C, reacted for 1 hour, cooled, and stirred vigorously while dropping into 30ml of ice-water. After washing the precipitate separately with water, chloroform 5
After washing with saturated saline and separating the organic layer,
After drying with anhydrous sodium sulfate, removing most of the chloroform with an evaporator and recrystallizing in acetone, the ratio of the 24-position epimer in the first crystal is R/S = 93/724 , 25-dihydroxycholesterol-3,24-dibenzoate-25-trimethylsilyl ether () 410
get g. If this is recrystallized from acetone again,
340 g of a mixture (2) in which the ratio of epimers at the 24-position is R/S = 96/4 was obtained, and this was recrystallized once again from acetone to obtain 24(S)-24,25-dihydroxycholesterol-3,24-dihydroxycholesterol-3,24-dihydroxycholesterol. Contains less than traces of benzoate-25-trimethylsilyl ether
24(R)-24,25-dihydroxycholesterol-
296 g of needle-like crystals of 3,24-dibenzoate-25-trimethylsilyl ether were obtained.

Melting point: 154-155℃ [α] ²⁴ _D : -4゜ (CHCl ₃ , C, 0.11) IR (KBr, cm ^-1 ): 1715, 1275, 840, 710 NMR (100MHz, CDCl ₃ , δppm): 0.11 (9H,s,
25− TMS−CH ₃ ), 0.68 (3H, s, 18−
CH ₃ ) 0.93 (3H, d, 21− CH ₃ ), 1.06 (3H,
s, 19- _CH3 ), 1.26
(3H, s, 26 or 27−
_CH3 ), 1.28 (3H, s, 26 or 27- _CH3 ), 4.80
(1H, br.m, 3-CH),
4.95 (1H, br.m, 24−
CH), 5.38 (1H, br.d, 6
- CH), 7.3~7.6 (6H,
m, phenyl proton),
7.9-8.2 (4H, m, phenyl proton) Example 3 87% of 24(S)-24,25-dihydroxycholesterol-3,24-dibenzoate (-S) obtained in the middle of Example 1 and 24( R)-24,25-dihydroxycholesterol-3,24-dibenzoate (-
157 g (0.251 mol) of a mixture consisting of R) 13% are dissolved in 1.5 ml of dry pyridine, 159 ml (0.753 mol) of hexamethyldisilazane and 80 ml (0.628 mol) of trimethylsilane chloride are added, and after heating and refluxing for 1 hour. Cool and stir vigorously while dropping into ice-water from Step 6. After washing the precipitate separately with water, chloroform
Dissolve in 800 ml of water, wash with saturated saline, separate the organic layer, dry over anhydrous sodium sulfate, remove most of the chloroform in an evaporator, and recrystallize in acetone to obtain the 24-position configuration. 24,25-dihydroxycholesterol-3,24-dibenzoate-25 with an R/S-ratio of 2.6/97.4
-Trimethylsilyl ether () 115g
(0.165mol) is obtained. If this is recrystallized twice from acetone, it will contain only traces of the 24(R)-24
(S)-24,25-dihydroxycholesterol-
78.5 g (0.112 mol) of 3,24-dibenzoate-25-trimethylsilyl ether (-S) is obtained.

Melting point: 148-149℃ [α] ²⁴ _D : -20゜ (CHCl ₃ , C, 0.29) IR (KBr, ν〓cm ^-1 ): 1715, 1275, 840, 710 NMR (100MHz, CDCl ₃ , δppm) :0.11(9H,s,
25−TMS− _CH3 ), 0.63(3H,
s, 18− _CH3 ), 0.93(3H,
d, 21- _CH3 ), 1.06(3H,
s, 19- _CH3 ), 1.26 and 1.28
(2 × 3H, 2 × s, 26
and 27− _CH3 ), 4.80 (1H,
br.m, 3-CH), 4.95 (1H,
br.m, 24−CH), 5.38
(1H, Br. tr, 6-CH), 7.3
~7.6 (6H, m, phenyl proton), 7.9 ~ 8.2 (4H, m,
(phenyl proton) Example 4 24(R)-24,25-dihydroxycholesterol-3,24-dibenzoate-25- obtained in Example 2
Trimethylsilyl ether (-R) 10.0g
(0.014 mol) in 200 ml of tetrahydrofuran and 20 ml of water
ml of the mixture, p-toluenesulfonic acid
1.0g was added and stirred at room temperature for 24 hours. After the reaction was completed, the solvent was removed using an evaporator and chloroform was added.
Dissolve in 150 ml and wash with 5% sodium hydrogen carbonate and saturated saline, separate the organic layer and dry over anhydrous sodium sulfate, remove chloroform with an evaporator and obtain crude 24(R)-24,25-dihydroxycholesterol. -3,24-dibenzoate (
-R) 8.2 g (0.013 mol, 91%) are obtained. If this is recrystallized from acetone, 6.9 g (0.011 mol) of needle crystals of compound (-R) are obtained.

Melting point: 168-169°C [α] ²¹ _D : -6° (chloroform, C, 0.10) IR (KBr, ν〓cm ^-1 ): 3450, 1715, 1275, 710 NMR (100MHz, CDCl ₃ , δppm): 0.68 (3H, s,
18− _CH3 ), 0.93(3H, d(6.0
Hz), 21− _CH3 ), 1.06
(3H, s, 19−CH ₃ ), 1.26 and 1.28 (2×3H, 2×s,
26− and 27− _CH3 ), 5.07
(2H, m, 3, 24−CH), 5.41
(1H, br, d, 6-CH), 7.3-7.6
(6H, m, phenyl proton),
7.9 ~ 8.2 (4H, m, phenyl proton)

Claims (1)

[Claims] 1 Formula () By reacting carboxylic acid chloride with 24(ξ)-24,25-dihydroxycholesterol represented by the formula () (In the formula, R ₁ represents a phenyl group, p-nitrophenyl group, α-naphthyl group or β-naphthyl group.) 24(ξ)-24,25-dihydroxycholesterol-3,24-diacylate represented by By fractionally recrystallizing this from an organic solvent, mainly the 24(S)-epimer is precipitated and separated, and the recrystallization mother liquor rich in 24(R)-epimer is trialkylsilylated to give the formula ( (In the formula, R ₁ represents a phenyl group, p-nitrophenyl group, α-naphthyl group, or β-naphthyl group, and R ₂ represents a trimethylsilyl group or tert-butyldimethylsilyl group.) -24,25-dihydroxycholesterol-3,24-diacylate-25-trialkylsilyl ether, which was fractionally recrystallized from an organic solvent to form the formula (-R) (In the formula, R ₁ and R ₂ are the same as in formula ().) 24(R)-24,25-dihydroxycholesterol-3,24-diacylate-25-trialkylsilyl ether is separated. 24,25-dihydroxycholesterol derivative 24 characterized by
Method for separating -position epimer. 2. The method according to claim 1, wherein the solvent used for fractional recrystallization is acetone. 3. The method according to claim 1 or 2, wherein the acyl group is a benzoyl group and the trialkylsilyl group is a trimethylsilyl group.