JPH0543591A - Method for separating 24-position epimer of 24-hydroxycholesterol derivative - Google Patents

Abstract

PURPOSE:To separate the title epimer selectively and industrially from an epimer mixture of a hydroxycholesterol derivative by using liquid chromatography filled with a specific packing material. CONSTITUTION:A 24 epimer mixture of a 24-hydroxycholesterol derivative is treated by liquid chromatography filled with a silica gel (preferably spherical one) prepared by reacting a silicon compound shown by the formula (R1 is 1-22C aliphatic group and/or 6-10C aromatic group; R2 to R4 are halogen, lower alkoxy or lower alkyl with the proviso that at least one of R2 to R4 is halogen or lower alkoxy) as a packing material to separate the 24-position epimer of the derivative. 1alpha,24-Hydroxycholesterol or #1a,24-dihydroxycholester-5,7-diene may be preferable as the 24-hydroxycholesterol derivative.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for separating the 24-position epimer of a 24-hydroxycholesterol derivative.

24-hydroxycholesterol derivative,
For example, 1α, 24-dihydroxycholesterol is a vitamin D ₃ active metabolite, 25-hydroxycholecalciferol [Biochemistry 7 , 3317 (1968)], 1,
25-dihydroxycholecalciferol [Biochemist
ry, 10 , 2,799 (1971)], 24,25-dihydroxycholecalciferol [Biochemistry, 11 , 4251 (1972)],
It is a very useful compound as a synthetic intermediate for 1α, 24-dihydroxycholecalciferol, which is an analog such as 1α, 24,25-trihydroxycholecalciferol [J.Biol.Chem.248,6691 (1973)]. ..

It is known that, in this 1α, 24-dihydroxycholecalciferol, either one of the epimers at the 24-position exhibits physiological activity, or that the epimers differ in qualitative or quantitative physiological activity. ing. Therefore, in the synthesis of such 1α, 24-dihydroxycholecalciferol, it is necessary to separately synthesize the 24-position epimer.

By the way, 1α, 24-dihydroxycholecalciferol is synthesized by a known reaction via a 1α, 24-dihydroxycholesterol derivative.

Therefore, an efficient method for separating the epimer at the 24-position of the 1α, 24-dihydroxycholesterol derivative, which is a synthetic intermediate of 1α, 24-dihydroxycholecalciferol, should be used in the selective and industrial synthesis of the epimer. You can

[0006]

2. Description of the Related Art Conventionally, the separation of the 24-position epimer of a 24-hydroxycholesterol derivative has
By conducting fractional crystallization [J.Am.Chem.Soc., 75,3284 (1953)] or thin layer chromatography [J.Chromatogr., 49,555 (1970)] by conducting benzoyloxycholesteryl benzoate, 24- A 24-benzoyloxycholesterol derivative obtained by benzoylating the 24-position of a hydroxycholesterol derivative is used by silica gel column chromatography [Japanese Patent Publication No. 53-27268], or 24-hydroxycholesterol-3
-There is a report that it can be achieved by using silica gel column chromatography [JP-A-57-116096] with acetate or the like.

Further, 1α, 24-dihydroxycholesterol having a hydroxyl group introduced at the 1α position in addition to the 24th position is present in seaweed in a large amount and is easily obtained as an industrial raw material by a simple operation such as extraction. As a starting material [Chem.Pharm.Bull. 21 , 2568-2570 (1973)],
First, 24-oxocholesterol, then 1α, 24
-Dihydroxycholesterol is synthesized, and the hydroxyl group of the obtained 1α, 24-dihydroxycholesterol is protected to separate the epimer at the 24-position.

However, a method for directly separating the 24-position epimer of 1α, 24-dihydroxycholesterol having no protective group is not known.

[0009]

As described above, in the prior art, there was no method for efficiently separating the epimer at the 24-position without protecting the hydroxy group of the 24-hydroxycholesterol derivative.

Therefore, in the synthesis of active vitamin D ₃ , the total number of reaction steps is increased due to the protection and deprotection process of the hydroxy group in the separation of the epimeric intermediate at the 24-position, and the yield is more industrial. Effective 2 suitable for production
A method for separating the 4-position epimer has been desired.

The present inventors have arrived at the present invention as a result of extensive studies in view of the problems of the prior art.

[0012]

That is, the present invention provides a 24-
When separating the 24-position epimer of the hydroxycholesterol derivative from the mixture of the 24-position epimer, the following formula [I]

[0013]

[Chemical 2]

[In the formula, R ₁ represents a C _{1 to} C ₂₂ aliphatic group and / or a C _{6 to} C ₁₀ aromatic group, and R ₂ , R ₃ and R ₄
Is a halogen atom, a lower alkoxy group or a lower alkyl group, and at least one of R _{2 to} R ₄ represents a halogen atom or a lower alkoxy group. ] The method for separating the 24-position epimer of the derivative, which comprises using a liquid chromatography packed with a filler made of silica gel obtained by reacting a silicon compound represented by the formula:

The 24-hydroxycholesterol derivative of the present invention means cholesterol having a hydroxy group at least at the 24-position and derivatives thereof, such as 24-hydroxycholesterol; 1α, 24-dihydroxycholesterol; 24,25-dihydroxycholesterol; 1
α, 24,25-trihydroxycholesterol; 1
α, 24-dihydroxycholesta-5,7-diene; 2
4-hydroxycholesta-5,7-diene and the like. Among these, 1α, 24-hydroxycholesterol, 1α, 24-dihydroxycholesta-5,7-diene and the like are preferable as the 24-hydroxycholesterol derivative of the present invention.

In the present invention, the 24-position epimer of the derivative is a stereoisomer (R) based on an asymmetric carbon atom at the 24-position.
Body, S body).

In the above formula [I], R ₁ is C _{1 to} C _22.
Of the aliphatic groups and / or C _{6 to} C ₁₀ aromatic groups. As such an aliphatic group and / or aromatic group, as long as it has a suitable hydrophobic group, it may be linear or branched, and may contain an unsaturated group and other substituents, but it is preferable. The linear one is preferable. Among them, R ₁ is C
Preferable are _4- C ₁₈ aliphatic groups and C ₆ aromatic groups. More preferably, a C _{4 to} C ₁₈ saturated aliphatic group, for example, an n-butyl group (C ₄ ), an n-octyl group (C ₈ ), an octadecyl group (C ₁₈ ), or a phenyl group (C ₆ ) is mentioned. Be done.

In the above formula [I], R ₂ , R ₃ ,
R ₄ is a halogen atom, a lower alkoxy group or a lower alkyl group, and at least one of R _{2 to} R ₄ is a halogen atom or a lower alkoxy group.

The halogen atom is fluorine,
Examples thereof include chlorine atom and bromine atom. Among them, chlorine atom is preferable. Examples of the lower alkoxy group include C _{1 to} C ₆ linear or branched alkoxy groups such as methoxy group, ethoxy group, propoxy group and butoxy group. Of these, a methoxy group and an ethoxy group are preferable. Examples of the lower alkyl group include C ₁ -C ₆ linear and branched alkyl groups such as methyl group, ethyl group, propyl group and butyl group. Of these, a methyl group is preferable.
Among them, R ₂ , R ₃ and R ₄ are R ₂ and R 4.
₃ , R ₄ is a halogen atom, a C ₁ -C ₄ lower alkoxy group or a C ₁ -C ₄ lower alkyl group, and R ₂ -R
_At least one of ₄ is a halogen atom or C _{1 to} C
_It is preferably ₄ lower alkoxy groups. More preferably, one or two of R _{2 to} R ₄ are chlorine atoms,
The other is a methyl group.

The filler of the present invention is a filler made of silica gel obtained by reacting the silicon compound represented by the above formula [I] with silica gel. The reaction between the silicon compound and silica gel is a conventionally known reaction. Can be done by Further, such a filler is an object of the present invention,
Other than the above, silica gel or the like may be included as a filler within the range where the effect is achieved.

The filler of the present invention comprises silica gel obtained by reacting with a silicon compound, and the degree of reaction can be expressed as the carbon content of the filler. In such a case, the carbon content of the filler is preferably in the range of 1 to 25 wt%. Among them, the range of 5 to 25 wt% is preferable, and the range of 10 to 25 wt% is more preferable.

As the silica gel reacted with the silicon compound, that is, the base gel, known silica gel can be mentioned. As the silica gel, spherical and crushed silica gel is mainly used, but spherical silica gel is preferably used.

The particle size of the filler of the present invention is 3 to 30.
Those having a diameter of 0 μm are used, preferably 5 to
Those having a diameter of 100 μm are used. The particle size is too small and the resolution tends to be low. 50 as pore size
˜500 angstroms, preferably 60 to 300 angstroms, and more preferably 80 to 200 angstroms are used.

Examples of such a filler include C8-120-S5 (column name: YMC-A-202) and ODS-A1 manufactured by YMC.
20-S5 (column name: YMC-A-302), C4-120-S5 (column name: YMC-A-802), YMC-ODS-20AM (or YMC-ODS-AM)
-120-S20), 50AQ (or YMC-ODS-)
AQ120-S50) etc .; Develosi of Nomura Chemical Co.
l ODS-120-S5 (column name: Develosil-ODS), Develosi
l ODS-A-120-S5 (column name: Develosil-A), Develosi
l ODS-T-120-S5 (column name: Develosil-T); Inertsil (registered trademark) ODS-2 (column name: ODS- of Gas Chloro Industry Co., Ltd.)
2); Shiseido ODS-SG-120 (column name: Capcell Pack
SG-120) and the like.

In the present invention, the following formula [II] is used in the eluent system having a composition of acetonitrile / water = 30/70 as the packing material.

[0026]

[Equation 2] Fillers having a Kp value of -1.0 to 0.2 are preferred. Above all, the Kp value is preferably -1.0 to-.
One is 0.1.

Conditions for obtaining the Kp value of the present invention include, for example, high performance liquid chromatography (HPLC); for example, JASCO PU-880, 875UV, column; various columns, eluent; acetonitrile / water = 30/70, empty cylinder Flow rate: 0.1 cm / sec, detection wavelength: 207 nm, column temperature: 30 ± 1 ° C. The Kp value generally varies depending on the degree of treatment of silica gel with a silicon compound containing a substituent R ₁ and further treatment with a low molecular weight silicon compound such as trimethylchlorosilane. The higher the processing rate, the larger the absolute value of X at Kp = -X.

The separation method of the present invention is carried out by using liquid chromatography with a 24-hydroxycholesterol derivative as a sample without protecting its hydroxyl group. Specifically, glass, stainless steel,
Alternatively, a column made of titanium or the like is tightly packed with a packing material, and a liquid feed pump and a sample injector are installed at the inlet side of the column
Also, connect an ultraviolet detector or a differential refraction detector, etc. on the outlet side, and a fraction collector if necessary,
The 24-hydroxycholesterol derivative injected by feeding the eluent with a liquid feeding pump is developed in a column and separated.

Preferred eluent systems include, for example, acetonitrile / water, methanol / water, ethanol / water, n-
Propyl alcohol / water, isopropyl alcohol /
Examples include water, acetone / water, tetrahydrofuran / water, dioxane / water, dimethylformamide / water, dimethylacetamide / water, dimethyl sulfoxide / water, N-methylpyrrolidone / water, and ethylene glycol monomethyl ether / water system. Of these, acetonitrile / water, methanol / water, ethanol / water, acetone / water are particularly preferable, and the respective solvents may be mixed in any proportion, but if the amount of water increases, the elution time will be delayed and It takes a long time and is not economical. Further, when the amount of the organic solvent is too large, the separation of the 24-position epimer becomes poor.

Therefore, the optimum composition differs depending on each solvent. For example, in the acetonitrile / water system, 40 / 60-
85/15 is preferred, 60/4 in methanol / water system
0 to 90/10 is preferable. Further, two or more kinds of organic solvents may be mixed.

The pH of the eluent is preferably kept in the range of 4.0 to 8.0. More preferably, pH 5.0 to 7.5 is preferable from the column life.

As a typical solvent for dissolving the sample, dimethylformamide, dimethylacetamide, dimethylsulfoxide, N-methylpyrrolidone, dioxane, tetrahydrofuran, methanol, ethanol, isopropyl alcohol, acetone, acetonitrile which are miscible with water. Etc. are preferred. Moreover, the thing which added water to these solvents may be sufficient.

The sample concentration is 0.1 to 35 w / w%, preferably 2 to 30 w / w%. If the concentration is too low, the separation will be poor especially in a solvent that dissolves the sample well. On the other hand, if the concentration is too high, there is a risk of precipitation when coming into contact with the eluent. Therefore, an optimum concentration exists depending on the eluent used and the sample dissolving solvent.

The temperature of the column and the eluent depends on the packing material used, the eluent, the sample concentration, and the flow rate, but is 5
~ 60 ° C is good. It is preferably 15 to 50 ° C. If the temperature is too low, the viscosity of the eluent will increase, the solubility of the sample will decrease, and the separation performance will decrease. On the other hand, if the temperature is too high, the separation performance is deteriorated and the composition change due to the volatilization of the eluent is not preferable.

The flow rate of the eluent differs depending on the size of the column, the amount of load, the composition of the eluent, and the temperature of the eluent. On the other hand, if it is too fast, the column pressure will increase and the separation performance will decrease.

On the other hand, if the loading amount is too large, the separation performance will deteriorate and the desired purity cannot be obtained. If the load is too small, the number of separations will increase, which is not economical.

Thus, according to the separation method of the present invention, 24
-Without protecting the hydroxy group of the hydroxycholesterol derivative, for example, in the case of 24-hydroxycholesterol, any of the 3-position and 24-position hydroxy groups, or in the case of 1α, 24-dihydroxycholesterol, the 1α-position It is possible to selectively and industrially separate the 24-position epimer while leaving both 3- and 24-hydroxy groups free.

Hereinafter, the present invention will be described in more detail with reference to examples.

[0039]

Examples 1 to 4, Comparative Examples 1 and 2 Relationship between R ₁ and Degree of Separation Columns filled with various packing materials manufactured by YMC (YMC) shown in Table 1 (column size; 4.6 × 250 Lmm /
The particle size of the packing material; 5 μm) was used to separate the 24R and 24S forms of 1α, 24-dihydroxycholesterol.

For the separation, samples 1α, 24-
Using dihydroxycholesterol 20% THF solution,
Injection volume 1.0 μl, eluent acetonitrile / water =
6/4, flow rate 1.0 ml / min.

Table 1 shows the results of the S form regarding the degree of separation and the retention time. From Table 1, R ₁ is nC ₄ H ₉ , nC ₈ H ₇ ,
It can be seen that the resolution is excellent when the nC ₁₈ H ₃₇ group is used.

In Examples 1 to ₄ , at least one of R _{2 to} R ₄ is a methyl group or a chlorine atom.

[0043]

[Table 1]

[0044]

[Examples 5 to 11] Relationship between R ₁ and Kp value, etc. Columns packed with the packing materials of the respective commercial companies listed in Table 2 (column size; 4.6 × 250 Lmm / particle size of packing materials; 5 μm)
2 of 1α, 24-dihydroxycholesterol
The 4R form and the 24S form were separated.

The separation was performed under the same conditions as in Example 1 for the sample, the eluent and the flow rate, and the degree of separation was determined in the same manner.

5 samples each of pyridine and phenol
Injection amount of 3 mg / 50 ml was 1.5 μl, and Kp value was eluent acetonitrile / water = 3/7, flow rate 1.0 ml / min.
Under the conditions of, the retention time of each sample was calculated by the above formula [II].

Table 2 shows these Kp values and the degree of separation.

[0048]

[Table 2] From Table 2, it can be seen that excellent separation is obtained when the Kp value is -1.0 to 0.1.

[0049]

Example 12 1250 mg of a crude product of 1α, 24-dihydroxycholesterol was added to 5 ml of tetrahydrofuran.
0.47 ml of the sample solution prepared by adjusting the volume to a commercially available filler (YMC, ODS-20AM, particle size 20 μm, pore size 120)
_{Å, Kp = -0.42, R 1 =} C
₁₈ H ₃₇ , at least one of R _{2 to} R ₄ is injected into a column [YMC-SH-343-20AM, column size: φ20 × 250 Lmm] packed with a chlorine atom or a methyl group, and acetonitrile / water = 75 / 25, flow rate 20 ml / min (0.10
(cm / sec) eluent was developed in 20 minutes. The elution peaks of the R and S forms were divided into 8 parts, and the purity of each fraction was confirmed by the following analysis method. The corresponding fractions were combined and the solvent was distilled off to give 1α, 24R-dihydroxycholesterol 54 mg (yield 95% , Purity 99.1%) was obtained.

The column performance for obtaining the target product 1α, 24R-dihydroxycholesterol was 3.4 × 10 ^-2 mg / min.
-It was cm ³ (column volume). <Analysis conditions> Column: YMC-AM-303 (φ4.6 × 250 Lmm ODS-AM, particle size 5 μm, pore size 120 Å), eluent: acetonitrile / water = 60/40, flow rate: 1.5 ml / min, detection Wavelength: 207 nm, Column temperature: 30 ° C, HPLC: JASCO PU-880, UV-875 <Column performance> Target product, 1α, 24R-dihydroxy per unit time (min), per unit column empty volume (cc) Expressed as the weight (mg) that separates cholesterol.

[0051]

Example 13 2000 mg of 1α, 24-dihydroxycholesterol crude product was dissolved in tetrahydrofuran to give 10
1.30 ml of the sample solution prepared by adjusting the volume to ml was used as a commercially available filler (YMC, ODS-50AQ, particle size 50 μm, pore size 12
0 _{angstrom, Kp = -0.88, R 1 =} C 18 H
₃₇ , and at least one of R _{2 to} R ₄ was injected into a column [YMC-SH-343-50AQ, column size: φ20 × 250 Lmm] packed with a chlorine atom or a methyl group). Methanol / water = 75/25 as eluent, flow rate 30 ml / min, 3
Elution was performed at 7 ° C for 44 minutes. The peak was divided into eight. Purity of each fraction was confirmed by the same analytical method as in Example 12, and the corresponding fractions were combined and the solvent was distilled off to give 99 mg of 1α, 24R-dihydroxycholesterol (yield 80%, purity 98.0%). Got

The target product 1α, 24R-dihydroxycholesterol was obtained. Column performance is 2.9 × 10 ^-2 mg / mi
It was n · cm ³ (column volume).

[0053]

Example 14 1α, 24-dihydroxycholesta
600 μl of a sample solution prepared by adjusting 500 ml of a crude product of 5,7-diene to 5 ml with tetrahydrofuran.
Is a commercially available filler (YMC, ODS-50AQ, particle size 50 μm,
Pore size 120 angstrom, Kp = -0.88, R
₁ = C ₁₈ H ₃₇ , at least one of R _{2 to} R ₄ is a column packed with a chlorine atom or a methyl group (column size:
φ20 × 250 Lmm). Acetonitrile / water = 57/43 as eluent, flow rate 28 ml / min, 25 ° C
Elution and development at about 20 minutes, and the peak was divided into eight. The purity of each fraction was confirmed by the same analytical method as in Example 12, and the corresponding fractions were combined and the solvent was distilled off to give 1α, 24R-dihydroxycholesta-5.
23 mg of 7-diene (yield 80%, purity 98.0%) was obtained.

[0054]

Comparative Example 3 2000 mg of a crude product of 1α, 24-dihydroxycholesterol was added to 40 ml of isopropyl alcohol.
Apply the solution applied to the commercially available filler [Baso Gel IR-1
Column packed with 20, silica gel, particle size 10 to 25 μm, pore size 120 Å [φ50 × 1000
L-mm], n-hexane / isopropyl alcohol = 95/5, flow rate 114 ml / min (0.10 cm / se
The eluent of c) was eluted and developed at 25 ° C. It took 280 minutes to complete the elution. The peak was divided into 30 parts. Each fraction is analyzed in the analytical column under the same preparative conditions as above,
The purity was confirmed, and the corresponding fractions were combined and the solvent was distilled off to obtain 824 mg of 1α, 24R-dihydroxycholesterol (yield 84%, purity 99.7%).

The column performance for obtaining the target product 1α, 24R-dihydroxycholesterol was 1.5 × 10 ⁻³ mg / min.
-It was cm ³ (column volume).

Claims (4)

[Claims] 1. When separating the 24-position epimer of a 24-hydroxycholesterol derivative from a mixture of 24-position epimers, the following formula [I]: [In the formula, R ₁ is a C _{1 to} C ₂₂ aliphatic group and / or C ₆ to
C ₁₀ aromatic group, wherein R ₂ , R ₃ and R ₄ are a halogen atom, a lower alkoxy group or a lower alkyl group, and R
_At least one of _{2 to} R ₄ represents a halogen atom or a lower alkoxy group. ] The method for separating the 24-position epimer of the derivative, which comprises using a liquid chromatography packed with a silica gel filler obtained by reacting a silicon compound represented by the formula: 2. In the above formula [I], R ₁ is a C ₄ -C ₁₈ aliphatic group or a C ₆ phenyl group, and R ₂ , R ₃ and R
₄ is a halogen atom, a C ₁ -C ₄ lower alkoxy group, or a C ₁ -C ₄ lower alkyl group, and at least one of R ₂ -R ₄ is a halogen atom or a C ₁ -C ₄ lower alkoxy group. The separation method according to claim 1, which is a group. 3. The filler is acetonitrile / water = 30/7.
In an eluent system having a composition of 0, the following formula [II] The Kp value represented by is 1.0 to 0.2.
Separation method described. 4. The derivative is 1α, 24-dihydroxycholesterol, or 1α, 24-dihydroxycholester
The separation method according to claim 1, which is 5,7-diene.