JPH04154795A - Separation of steroid compound - Google Patents

Abstract

PURPOSE:To simplify separation of isomers, etc., having constitution extremely similar to that of the title compound by separating a compound having a steroid skeleton using a separating agent containing a polysaccharide derivative as an active ingredient. CONSTITUTION:A compound having a steroidal skeleton (a basial skeleton by condensing 6-membered rings A, B and C to 5-membered ring D) expressed by formula I is separated, using a separating agent containing a polysaccharide derivative [preferably obtained by replacing >=70% of OH radicals and single H on amino which the polysaccharide has, and where one or two kinds of above two saccharide residues are intramolecularly regularly bonded in the saccharide derivative, with an atomic group expressed by formula II, formula III (R<1> is 1-3C aliphatic group, 4-20C aromatic group, etc., which may have substituent groups)] as an active ingredient of the separating agent to carry out the objective separation by chromatography.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method of separating structurally similar compounds having a steroid skeleton, particularly a mixture of isomers, to obtain each isomer as a pure product. be.

[Conventional technology]

Compounds having a steroid skeleton are ubiquitous in living organisms, and many of them exhibit strong physiological activity as adrenal cortical hormones, sex hormones, and the like.

When synthesizing this as a medicine, it is often the case that a naturally abundant and inexpensive steroid compound is used as a precursor and the target product is led through chemical conversion. However, this chemical conversion often produces by-product isomers that are difficult to separate. Furthermore, it may be difficult to even accurately determine the mixing ratio of these isomers.

Chromatography is a powerful method for separating and analyzing substances with similar structures. In the chromatography method, a separation agent such as silica gel or silica gel bonded with a hydrocarbon group (so-called 005) is used, and separation is performed by taking advantage of the fact that the adsorption or distribution ratio to these agents differs depending on the substance.

[Problems to be Solved by the Invention] However, even when such a method is used, it is often extremely difficult to separate isomers with severely deformed structures. Structurally similar compounds with steroid skeletons,
In particular, isomers are no exception.

Therefore, an object of the present invention is to provide a method for effective separation and analysis of compounds having steroid skeletons, especially isomers. is expected to be used.

[Means to solve the problem]

In view of the importance of separating and analyzing the aforementioned compounds having steroid skeletons, the present inventors have conducted extensive studies in order to find a separating agent that can more effectively separate them.

As a result, the present inventors discovered that a separation method using an adsorbent containing a polysaccharide derivative as an active ingredient is suitable for separating isomers of compounds having a steroid skeleton, and completed the present invention.

That is, the present invention relates to a method for separating steroid compounds, which is characterized in that a separation agent containing a polysaccharide derivative as an active ingredient is used in the method for separating compounds having a steroid skeleton.

The compound having a steroid skeleton according to the present invention is shown in the following formula and has a basic skeleton in which six-membered rings A, B, and C and a five-membered ring D are condensed.

Naturally obtained steroids may have an alkyl group at the 17-β-position, a methyl group at the 10-β- and 13-β-positions, a double bond at the 5-position, and an oxo or oxy group at the 3-position. There are many, but they also take on a variety of other structures. The compounds to be separated by the present invention are the presence or absence and position of substituents (including their stereochemistry) on the basic skeleton, the presence or absence and position of unsaturated bonds, or rings, regardless of whether they are natural or synthetic. It refers to compounds with structural differences caused by differences in condensation stereochemistry (cis or trans), etc. Especially when these are isomers, and even stereoisomers,
There are many cases where separation is difficult, and therefore the present invention is of great importance.

The separating agent used in the present invention contains a polysaccharide derivative as an active ingredient. The term polysaccharide here refers to synthetic polysaccharides, natural polysaccharides, or modified natural polysaccharides, in which multiple tetrahydrofuran or tetrahydropyran rings having one or more hydroxyl or amino groups on the ring are linked via acetal or ketal bonds. It is a combination of individual parts.

Examples of naturally occurring polysaccharides include cellulose, amylose, cyclodextrin, β-1,4-chitosan,
Chitin, β-1,4-mannan, β-1,4-xylan, inulin, α-1,3-glucan, β-1,3-glucan (so-called curdlan, schizophyllan, etc.), β-1,2
-Glucan, agarose, glucomannan, β-1,2
-Glucan, pullulan, etc. In recent years, polysaccharides that fall into this category have been obtained by methods such as ring-opening polymerization of two-ring acecur. Among these polysaccharides, polysaccharides in which one or two types of sugar residues are regularly bonded are preferred, and the number of sugar residues in the molecule is preferably two or more on average.

A polysaccharide derivative is one in which part or all, preferably 70% or more, of one hydrogen atom on the hydroxyl group or amino group of the polysaccharide has been replaced with another atomic group.

Examples of the atomic group here include a group represented by the following, where R1 is an aliphatic group having 1 to 3 carbon atoms, a cycloaliphatic group having 3 to 8 carbon atoms, and a cycloaliphatic group having 4 to 20 carbon atoms. It is an aromatic group, an araliphatic group (aralkyl group), or a heteroaromatic group, and any of them may have a substituent. Among the above groups, the main ones include acetyl group, propionyl group, phenylacetyl group, phenoxyacetyl group, optically active or racemic α-phenylpropionyl group, benzoyl group, naphthoyl group, and aromatic ring groups thereof. having one or more substituents such as methyl, chlorphenyl, etc.
Methylcarbamoyl group, phenylcarbamoyl group, naphthylcarbamoyl group, hensylcarbamoyl group, naphthylmethylcarbamoyl group, optically active or racemic α-phenylethyl group, α-naphthylethyl group, and methyl, chlor, or phenyl on their aromatic rings. Those having one or more other tags, benzyl groups, naphthylmethyl groups, and those having one or more substituents such as methyl, chloro, phenyl, etc. on their aromatic rings;
Examples include methyl group, ethyl group, propyl group, 2-hydroxyethyl group, 2-hydroxy-2-methylpropyl group, and optically active or racemic 2-hydroxypropyl group.

These atomic groups may be one type or multiple types, and in addition to these atomic groups, the polysaccharide derivative has plasticity and 1. In order to impart non-volatility, the molecule N100 to 1
000 high molecular weight atomic groups may be combined.

These polysaccharide derivatives can be easily obtained using various known chemical reactions. These polysaccharides and their derivatives are particularly suitable for industrial chromatographic separation because of their ease of raw material availability and stability.

In the separation method of the present invention, an appropriate one may be selected from among these multi-1i derivatives depending on the compound to be separated and the separation method.

Separating agents containing polysaccharide derivatives as active ingredients, even if their secondary structures are the same, have different molecular weights, types of solvents used for molding, heat treatment, swelling by solvents, liquid crystal formation, etc. The separability changes depending on the Furthermore, in order to increase the degree of separation in chromatography, it is necessary to reduce the so-called theoretical plate height of the separating agent for chromatography, and various embodiments for this purpose are possible. For example, in liquid chromatography or thin layer chromatography separation, the column theoretical plate height is
It varies significantly depending on the shape, size, fine structure, etc. of the separating agent particles. For example, the particle size is smaller than larger, the particle size distribution is smaller than larger, the particle shape is more spherical than amorphous, porous particles are more porous than dense particles, or there is an active layer for adsorption of multi-tR derivatives only on the surface. Preferably. In addition, in order to improve the theoretical plate height, mechanical strength, operability, and solvent resistance of the separation agent, the polysaccharide derivative may be supported physically or by chemical bonding on a carrier made of an inorganic or organic material or both. be. In addition, other additives may be added to the polysaccharide derivative for the purpose of improving the thermal stability of the polysaccharide derivative, or imparting plasticity to increase the theoretical plate height.

Typical chromatographic separation techniques include liquid chromatography, thin layer chromatography, and gas chromatography. The shape of the separating agent may be selected appropriately for each method.

The ability of a separating agent to discriminate between different substances (indicated by the magnitude of the separation coefficient α) itself is determined by the chemical properties of the adsorbing active component of the separating agent. The most important aspect of the present invention is that separation agents made of polysaccharide derivatives often exhibit higher α values than general separation agents such as silica gel and ODS in separating compounds with steroid skeletons. be. However, the advantages of using multi-IPi derivatives are not limited to this, for example, the elution order between compounds, sample loading, and in the case of liquid and thin layer chromatography, the type of mobile phase that provides suitable retention strength. In terms of organic properties, they often exhibit characteristics that are different from those of other packings, and these can also be an advantage of the separation method of the present invention. In the separation of compounds having steroid skeletons, the above-mentioned characteristics exhibited by separation agents containing polysaccharide derivatives as active ingredients are based on the chemical interaction between the polysaccharide derivatives and the substance to be separated. What is common in any variation of the embodiments can be easily deduced by analogy. Therefore, the present invention includes any embodiment thereof as long as a separation agent containing a polysaccharide derivative as an active ingredient is used.

[For production]

The reason why the multi-Pi derivative used in the present invention is effective in separating compounds having a steroid skeleton is not completely clear. However, according to the results of various studies conducted to date,
It has been found that a substituent that modifies a polysaccharide in a polysaccharide derivative associates with the steroid compound to be separated through mechanisms such as dipolar interaction, π-π overlap interaction, and hydrogen bonding. The skeleton of sugar residues is characterized by restricted conformation due to its cyclic structure and the presence of numerous substituents, and the arrangement of the residues is generally regular. The substituents bonded to such a polysaccharide skeleton form a regular arrangement and form an adsorption field with a specific three-dimensional shape. This fixed shape of the adsorption field means that
This is essentially different from amorphous substances such as silica gel and ODS. This is considered to be a factor that provides high discrimination ability for the chemical environment, especially the stereochemical environment, around the adsorption group of the compound to be separated.

〔Effect of the invention〕

The present invention has established an effective method for separating mixtures of compounds having steroid skeletons. Separation by chromatography is convenient as a means of analysis if the column or thin layer used is small, but it can also be used for purification purposes by simply increasing the size of the column or thin layer and increasing the amount of sample. I can do it.

Thus, the separation method of the present invention will provide a means for analyzing, separating and purifying various steroid compounds, and will ultimately contribute to the progress of physiological research and the development of medicines.

〔Example〕

The present invention will be specifically explained below using examples and comparative examples using silica gel and ODS, but it is clear that the present invention is not limited to these examples for the reasons already stated.

In the following examples, a separation column (Daicel Chemical Industry ■, C
IIIRAL to EL O or CHIRAL, r'AK
A) was used. In addition, as a comparative example, separation power °lum using silica gel (Daicel Chemical Industry ■, DC-PAK
The separation of the same mixture on a separation column (Gascro Kogyo ■, Inertsil 0DS) using a so-called ODS stationary phase in which octadecyl groups are bonded to S (Si 5-100)) and silica gel is illustrated.

Below, the parameter α used in Examples and Comparative Examples
is defined as below.

Example 1 5α-antrostane-3,17-dione (5α-an
drostan-3+17-dione) (1) and 5β-antrostane-3,17-dione (5β-an
drostan-3,17-dione) (2)
(Both SIGMA CHEMICAL Co.

Separation was performed using a separation column Cl1rRALCEL OJ made of cellulose tris (4-methylhenshade) (manufactured by Co., Ltd.). The chromatography conditions, retention times, volume ratios, separation coefficients, and elution orders of both isomers are shown in Table 1.

Comparative Example 1 The same mixture as in Example 1 was separated using a column using silica gel and ODS. Ria madography conditions, both isomers 1. The retention time, volume ratio, separation coefficient, and elution order of (2)) are shown in Table 1.

Table 1 Example 2 5α-antrostane-3,17-dione (5α-an
drostan-3+17-dione) (1) and 5α-antrostan-1-ene-3,17-dione (
5α-androstan-1-en-3,17-d
ione) (3) (Both SIGMA CHEMI
(manufactured by CAL CO, Ltd.) using a separation column CI made of amylose-tris (3,5-dimethylphenyl carbamate).
Separated using IIRALPAK AD.

The chromatography conditions, retention times, volume ratios, separation coefficients, and elution orders of both compounds are shown in Table 2.

Comparative Example 2 The same mixture as in Example 2 was separated on a column using silica gel and ODS. Chromatography conditions, both compounds 1. (3)) The retention time, capacity ratio, and separation coefficient are shown in Table 2.

Table 2 Example 3 Human tll lutisone (hydrocortisone)
(4) (manufactured by SIGMACII EMICAL Co.) and prednisolone (pred-nisolone)
(5) (manufactured by Tokyo Kasei Co., Ltd.) was separated using a separation column (JIIRALCEL AD) made of amylose-tris (3,5-dimethylphenylcarbamate). Chromatography conditions, retention time of both compounds, volume ratio, separation The coefficients and elution ranks are shown in Table 3.

Comparative Example 3 The same mixture as in Example 3 was separated on a column using silica gel and OOS. Table 3 shows the chromal lubrication conditions, retention time, volume ratio, separation coefficient, and elution order of both compounds ((4), (5)).

Table 3 Example 4 5α-Anthrostan-3-one (5α-andros
tan-3-one) (6) and 5α-androstane-17-one (5a-androstane-
17-one) (7) (Both EMIC to SIGM 8CI
(manufactured by AL Co.) was separated into a separation column CIII made of cellulose tris (3,5-dimethylphenyl carbamate).
Separation was performed using RALC [L OD and the separation column CHIRALr'AK AD used in Examples 2 and 3. Chromatography conditions, retention time of both compounds, volume ratio,
The separation coefficient and elution order are shown in Table 4.

Comparative Example 4 The same mixture as in Example 4 was separated on a column using silica gel and ODS. Table 4 shows the chromatography conditions, retention time, volume ratio, separation coefficient, and elution order of both compounds.

Table 4

Claims (1)

[Claims] 1. A method for separating steroid compounds, the method comprising using a separating agent containing a polysaccharide derivative as an active ingredient.