JP3086114B2 - Separating agents for chromatography - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chromatographic separating agent comprising an amine derivative of cyclodextrin as an active ingredient, and has excellent separation characteristics, and is particularly effective when used for separating optical isomers. Of a separating agent.

[0002]

BACKGROUND OF THE INVENTION As is known in general biochemistry, the optical isomers of compounds that are chemically the same usually have different effects on living organisms. Therefore, in the field of medicine and agricultural chemicals, preparation of an optically pure compound has become a very important task for the purpose of improving the medicinal effect per unit and preventing side effects and phytotoxicity.

In order to separate, that is, optically resolve, a mixture of optical isomers, a preferential crystallization method and a diastereomer method have been conventionally used. However, these methods are limited in the types of compounds to be optically resolved, and often require a long test time and a great deal of labor. For this reason, it is strongly desired to develop a technique for easily performing the optical splitting operation by a chromatography method or the like.

[0004]

Studies on optical resolution by chromatography have been carried out for a long time, and many studies have already been made on the use of cyclodextrin. When a cyclodextrin solution is used as a mobile phase in the use of cyclodextrin, a serious problem arises due to inadequate solubility in a solvent. For this reason, attempts have been made to covalently attach cyclodextrin to a solid matrix.

[0005] As a method for bonding a cyclodextrin and a support, a bond containing nitrogen (Fujimura, K. et al, An
al. Chem. 55, 446 (1983) or Kawaguchi, Y. et al, An.
al. Chem. 55, 1852 (1983)), a nitrogen-free ether bond (see U.S. Pat. No. 4,539,399), a bond via a carbamic acid group (see JP-A-4-230852), and a compound having one carbon atom. There is a bond via a bonding group or a bonding not via the bonding group (see JP-A-2-228558). However, the separating agents obtained by these covalent bonds have various problems such as poor separation efficiency, poor stability, and a low amount of cyclodextrin carried, and a poor recovery rate of the optically active compound. Remains.

[0006] The present invention has been proposed to improve the above-mentioned problems relating to the conventional separation agent for chromatography, and has a chemical structure different from that of the existing separation agent even when cyclodextrin is used. It is an object of the present invention to provide a separation agent for chromatography having separation characteristics superior to those of existing separation agents. Another object of the present invention is to provide a separating agent for chromatography having a high optical isomer discriminating ability.

[0007]

Means for Solving the Problems To achieve the above object, a separating agent for chromatography according to the present invention is obtained by converting a part or all of the hydroxyl groups of cyclodextrin into the following compound.
The cyclodextrin derivative substituted with an amino group represented by the formula is used as the active ingredient.

Embedded image (Wherein, R ₁ represents a hydrogen atom, an aliphatic group containing a methyl group or a substituent thereof, an alicyclic group, an aromatic group containing a phenyl group, a heteroaromatic group or a substituent thereof, and R ₂ represents methyl Represents an aliphatic group containing a group or a substituent thereof, an alicyclic group, an aromatic group containing a phenyl group, a heteroaromatic group or a substituent thereof)

[0008] The cyclodextrin used in the present invention is
There is no limit on the number of glucose units, but α-,
β- or γ-cyclodextrin is preferred in terms of ease of handling.

The amino group forming the amine derivative of cyclodextrin as an active ingredient has 1 to 3 carbon atoms.
6 is preferably 3 to 18. The substituent on the nitrogen atom of the amino group is, for example, an aliphatic group containing a methyl group or a substituent thereof, an alicyclic group, an aromatic group containing a phenyl group, a heteroaromatic group or a substituent thereof.

In the amine derivative of cyclodextrin as an active ingredient, 1 to 100%, preferably 20 to 40%, of all the hydroxyl groups of the corresponding cyclodextrin is substituted with an amino group. The hydroxyl group not substituted with an amino group may be a free hydroxyl group, but may be further esterified, etherified, or carbamated as long as the isomer separation ability of the amine derivative is not impaired. .

As a specific example of the amine derivative of cyclodextrin as an active ingredient, heptakis (6-dodecylamino-6-deoxy) -β-cyclodextrin, heptaquis {6-[(2S) -2-carboxypyrrolidino]- 6-deoxy} -β-cyclodextrin, heptakis {6-[(2R) -2-carboxypyrrolidino]
-6-deoxy}-[beta] -cyclodextrin, heptakis {6-[(1R) -1- (1-naphthyl) ethylamino] -6-deoxy}-[beta] -cyclodextrin, heptakis {6-[(1S)- 1- (1-naphthyl) ethylamino] -6-deoxy} -β-cyclodextrin,
Heptakis (6-octadecylamino-6-deoxy}
-Β-cyclodextrin.

The separation of the mixture of optical isomers with the separating agent of the present invention may be performed by a chromatography method such as gas chromatography, liquid chromatography, thin layer chromatography, etc., and in some cases, membrane separation may be performed. In order to apply the separating agent of the present invention to a chromatography method, a method of retaining an amine derivative of cyclodextrin on a suitable support, a method of coating the amine derivative on a capillary column, or the like may be used.

In order to hold the amine derivative of cyclodextrin on the support, the amine derivative may be dissolved in a soluble solvent, and this solution may be passed through a column filled with the support. Alternatively, the solution of the amine derivative described above may be mixed well with the support, and the solvent may be distilled off under reduced pressure or heated airflow.

When the amine derivative of cyclodextrin is held on the support, the amount of the amine derivative held is 0.1 to 100% by weight, preferably 1 to 5% by weight, based on the support.
0% by weight. The size of the support varies depending on the size of the column or plate to be used or the purpose of use, but is generally 1 μm to 10 mm in diameter, preferably 1 to 300 mm in diameter.
μm.

The support is preferably a porous organic or inorganic support having an average pore size of 10 Å to 10 μm, preferably 50 to 50,000 Å. As the porous organic support, a polymer material such as polystyrene, polyacrylamide, and polyacrylate is suitable. As the porous inorganic support, a synthetic or natural substance such as silica gel, alumina, magnesia, titanium oxide, glass, silicate, kaolin and the like is suitable.

The above-mentioned inorganic support may be subjected to a chemical surface treatment to improve the affinity with the amine derivative of cyclodextrin. As this chemical surface treatment method, there is a silanization treatment method using an organic silane compound.
Examples of the chemically decorated support obtained by this method include octadecylsilylated silica gel, octyl silylated silica gel, butyl silylated silica gel, aminopropyl silylated silica gel, and 3- (2,3-dihydroxypropoxy) propyl silylated silica gel. Especially effective.

There is no particular limitation on the developing solvent used when performing liquid chromatography or thin-layer chromatography, except for the liquid that dissolves or reacts with the separating agent of the present invention. If insolubilized, only the reactive liquid needs to be removed. In the separating agent of the present invention, the separation characteristics of the compound or the optical isomer are increased or decreased depending on the type of the developing solvent. Therefore, it is necessary to sufficiently examine a suitable developing solvent to be used.

[0018]

The separating agent according to the present invention is prepared, for example, by mixing a solution of heptakis (6-dodecylamino-6-deoxy) -β-cyclodextrin with octadecylsilylated silica gel and distilling off the solvent. When this separating agent is packed in a stainless steel column and dansyl-D, L-phenylalanine, dansyl-D, L-leucine or hexobarbital is separated, excellent results are obtained as shown in the chromatograms of FIGS. Racemates can be separated with the same separation characteristics.

[0019]

Next, the present invention will be described based on examples, but the present invention is not limited to the following examples. Example 1 Heptakis (6-o-methylstyrenesulfonyl) -β-cy
Preparation of Clodextrin 5 g (4.4 mmol) of β-cyclodextrin was dried at 100 ° C. under reduced pressure for 4 hours.
Dissolve in 0 ml. To this solution was added 13.6 g (62 mmol) of styrene sulfonyl chloride in 40 m of pyridine.
The solution dissolved in 1 is added dropwise over 1 hour under water cooling.

The obtained solution is further reacted at room temperature for 24 hours. The reaction solution was poured into 1 liter of ice water, and the resulting solid was filtered off with suction.
Wash in liter order and dry. 9.5 g of this crude product was subjected to column chromatography (SIL, 70/2).
After purification by 30 mesh, 60 angstroms), recrystallization using methanol gives 5.5 g of heptakis (6-o-methyesulfonyl) -β-cyclodextrin.

Heptakis (6-dodecylamino-6)
Preparation of deoxy) -β-cyclodextrin 5 g (2 mmol) of the above compound was added to 100 m of methanol.
and then add 24.8 g (134 mmol) of dodecylamine and stir in a pressure reactor at 100 ° C. for 4 days. The reaction solution is precipitated by adding methanol, and the precipitate is washed with methanol and further with hot methanol to obtain 1.6 g of heptakis (6-dodecylamino-6-deoxy) -β-cyclodextrin.

The physical constant data of this compound is as follows. Melting point: 236-240 ° C. (decomposition) ¹ H NMR (CDCl ₃ , ppm), 0.80-0.85
(Multiline, 21H) 0.90 to 1.25 (Multiline, 140H), 1.95 to 3.
15 (multiple line, 28H) 3.10 to 3.70 (multiple line, 14H), 3.70 to 4.2
5 (multiple line, 14H) 4.80-4.95 (multiple line, 14H). IR (Nujol method, cm ^-1 ), 3630 (= CH-O
H) 3350 (= N-H ), 2960 (-CH 3), 292
0 (-CH ₂ -)

Elemental analysis values

Preparation of separating agent for chromatography Heptakis (6-dodecylamino-6-deoxy) obtained
-Β-cyclodextrin 600 mg in chloroform 4
Dissolve in a mixture of 0 ml and 20 ml of methanol and mix with 3 g of octadecylsilylated silica gel. A separating agent is prepared by removing the solvent.

Test Example 25 cm × 0.46 (id) c of the separating agent obtained in Example 1
m stainless steel column by the slurry filling method. This column has a theoretical plate number of 7,000 to 8,000 for a compound having a low adsorptive power, and performs high-performance liquid chromatography at a flow rate of 1 ml / min at room temperature. The results of the optical resolution of the three racemates are shown in Table 1 below.

[0026]

[Table 1]

[0027]

Volume ratio Note) separation factor of the adsorbed optical isomer strongly than [Formula 1] (α) = ─────────────────── optical isomer adsorbed more rather weak Body volume ratio

Dansyl with the separating agent obtained in Example 1
The chromatogram of D, L-phenylalanine is shown in FIG. 1, where peaks 1 and 2 are the optical resolution of the target. FIG. 2 shows a chromatogram of dansyl-D, L-leucine by this separating agent. Peaks 1 and 2 are optical resolutions of the target substance. In addition, FIG. 3 shows a chromatogram of hexobarbital by this separating agent. Peaks 1 and 2 are optical resolutions of the target substance.

[0029]

The separating agent for chromatography according to the present invention hardly limits the target compound when separating a compound, especially a mixture of optical isomers, as compared with the preferential crystallization method or the diastereomer method. Applicable to Moreover, the separation time of the separation agent of the present invention is relatively short, and the separation is completed with little effort, so that the optical resolution test can be easily performed.

The separating agent of the present invention has better stability than cyclodextrin covalently bonded to a support, and has a larger loading amount of cyclodextrin. In addition, the separating agent of the present invention has generally superior separating properties than existing separating agents,
A mixture of optical isomers can be separated with particularly good recovery.

[Brief description of the drawings]

FIG. 1 is a chromatogram of dansyl-D, L-phenylalanine obtained by using the separating agent of the present invention containing heptakis (6-dodecylamino-6-deoxy) -β-cyclodextrin as an active ingredient.

FIG. 2 is a chromatogram of dansyl-D, L-leucine by the separating agent of the present invention.

FIG. 3 is a chromatogram of hexobarbital with the separating agent of the present invention.

Continuation of the front page (56) References JP-A-3-275635 (JP, A) JP-A-59-228936 (JP, A) JP-A-61-237057 (JP, A) JP-A-4-500229 (JP) , A) (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 30/48

Claims (2)

(57) [Claims] (1) When separating a mixture of optical isomers by liquid chromatography, heptakis (6) as an active ingredient is separated.
-A separating agent having dodecylamino-6-deoxy) -β-cyclodextrin retained on a support. 2. The support is a chemically decorated inorganic support such as octadecyl-silylated silica gel, octyl silylated silica gel, butyl silylated silica gel, aminopropyl silylated silica gel or 3- (2,3-dihydroxypropoxy) propyl silyl. Use activated silica gel
The separating agent according to claim 1 .