JPH0437826B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention relates to the general formula (In the formula, R is a hydrogen atom, a hydroxyl group, and a carbon number of 1
to 10 aliphatic acyloxy groups, and n represents an integer of 4 or 6. ) or,
Regarding the purification method for the acid salt of its derivative, in more detail, when purifying the acid salt of spagarin or its derivative by column chromatography using an insoluble carrier having a carboxyl group as a packing material, an inorganic salt is used as an eluent. The present invention relates to a method for purifying an acid salt of spagarin or a derivative thereof represented by the general formula (), which is characterized by using a water-containing hydrophilic solvent.

A compound of general formula () in which R is a hydroxyl group and n is 4 is called spagarin, and those with a steric configuration of (11(-), 15S) were found to be hydrochloride from a microorganism culture solution by Umezawa et al. It is a compound isolated as a drug and is expected to be a non-toxic anticancer agent (J. Antibiotics 34: 1619-1627). Also, (11(−)
,
The acid salts of the compounds of the general formula () other than (11(-), 15S)-spargarin are also new substances that have the same anticancer effect as (11(-), 15S)-spargarin (Patent application 1982-
(See No. 69340 and Japanese Patent Application No. 159503/1983).

Conventionally, the acid salt of spagarin of the general formula () has been purified by eluating the crude product using water containing sodium chloride as the eluent in column chromatography using sodium salt of CM-Sephadex as a packing material. The fraction containing the acid salt of spagarine was concentrated to dryness under reduced pressure, and the dried product was then dissolved in a small amount of methanol to remove insoluble sodium chloride.
This was carried out by applying LH-20, eluting with methanol to remove residual sodium chloride, and drying the fraction containing the acid salt of spagarin under reduced pressure (see JP-A-57-48957).

However, it has not been possible to obtain a highly pure salt of spagarin using the conventional purification method, ie, the method of using water containing sodium chloride as an eluent for spagarin adsorbed on CM-Sephadex.

Further, even if the acid salts of the compounds of the general formula () other than spagarin were purified by the above-mentioned method, it was not possible to obtain compounds with high purity similar to spagarin.

Even though they generally have excellent pharmacological effects, compounds with poor purity cannot be used as pharmaceuticals. In order to develop a drug as a drug, it is necessary to purify a target substance with low purity to a high purity, which is extremely important.

Therefore, in order to develop a purification method for obtaining highly purified acid salts of spagarin or its derivatives, the present inventors first conducted various studies on the reasons why it was not possible to obtain highly pure products using the above purification method. . As a result, the cause is that the acid salt of spagarin or its derivatives of the general formula () gradually decomposes in water by an intramolecular reversible reaction shown by the following formula, (In the formula, R is the same as above.) An acid salt of ω-guanidino fatty acid amide of the general formula () and N[4-(3-aminopropyl)aminobutyl]-2,2-dihydroxy of the general formula () Since it becomes an acid salt of ethanamide, (11(−),
15S) - Since spagarate is isolated from the bacterial culture solution, it is believed that this is because it contains many impurities in addition to the acid salt of the compound of general formula () and the acid salt of the compound of general formula (). found.

Based on the above findings, the present inventors conducted extensive studies on purification methods for the acid salts of spagarin or its derivatives. As a result, the present inventors found that when purifying the acid salts of spagarin or its derivatives by column chromatography using an insoluble carrier having a carboxyl group as a packing material, the eluent The present inventors have discovered that a highly purified acid salt of spagarin or its derivatives can be obtained by using a water-containing hydrophilic organic solvent containing an inorganic salt as a liquid, leading to the completion of the present invention.

To explain the present invention in more detail, in the acid salt of spagarin or its derivative of general formula () which is a raw material of the present invention, R is a hydrogen atom, a hydroxyl group, a formyloxy group, an acetyloxy group, a propionyloxy group, n -butanoyloxy group,
It is an aliphatic acyloxy group having 1 to 10 carbon atoms such as isobutanoyloxy group and octanoyloxy group, and n is an integer of 4 or 6.

The acid in the acid salt may be either an inorganic acid or an organic acid. Examples of the inorganic acid include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and boric acid, and examples of the organic acid include acetic acid and propionic acid. Examples include lower fatty acids such as succinic acid, fumaric acid, maleic acid, tartaric acid, polybasic acids such as citric acid, and sulfonic acids such as benzenesulfonic acid, toluenesulfonic acid, methanesulfonic acid, and ethanesulfonic acid. It will be done.

Typical examples include spagarin, 15-O
-Formyl spagarin, 15-O-acetyl spagarin, 15-O-propionyl spagarin, 15-O
-butanoyl spagarin, 15-O-octanoyl spagarin, 15-deoxyspargarin and N-
[4-(3-aminopropyl)aminobutyl]-2
Examples include hydrochloride and sulfate such as -(9-guanidinononanamide)-2-hydroxyethanamide.

In the purification of the present invention, the crude product of the acid salt of spagarin or its derivatives of the general formula () is applied to a column packed with an insoluble carrier having a carboxyl group, and eluted with a water-containing hydrophilic organic solvent containing an inorganic salt as an eluent. , by collecting fractions containing the object.

There are no particular restrictions on the crude acid salt of the compound of general formula (), but in the case of spagarin, it can be obtained by performing IRC-50 column chromatography on a culture solution using a known method, or by conventional methods. In the case of spagarin derivatives, those obtained by concentrating the reaction mixture under reduced pressure are used.

There are no particular restrictions on the purity of the crude product, but it is 30-95.
% is preferred.

The insoluble carrier having a carboxyl group is not particularly limited, but CM-Sephadex, carboxymethylcellulose, and the like are preferred.

The inorganic salt contained in the eluent is not particularly limited, but includes chlorides, bromides, sulfates, nitrates, and phosphates of alkali metals such as sodium and potassium, with sodium chloride and potassium chloride being preferred. .

Hydrophilic organic solvents used as eluents include:
There is no particular restriction as long as the organic solvent has a boiling point of 120°C or lower, preferably 90°C or lower and is miscible with water, but lower alcohols such as methanol, ethanol, n-propanol, and isopropanol, acetone,
Examples include ketones such as methyl ethyl ketone, and cyclic ethers such as tetrahydrofuran and dioxane, with methanol and ethanol being preferred.

The water content of the hydrophilic organic solvent varies depending on the type and amount of the inorganic salt contained, but is 10 to 90%.
and particularly preferably 30 to 60%.

The elution method is a gradient method in which the concentration of inorganic salt in the eluent is continuously changed.First, an eluent with a low concentration of inorganic salt is passed to elute most of the impurities, and then an eluent with a high concentration of inorganic salt is passed. There are two methods: the stepwise method, in which the target substance is eluted by flowing water, and the method, which is carried out using an eluent containing a fixed concentration of inorganic salts. The stepwise method is preferred because it is easy to obtain.

The concentration of the inorganic salt in the eluent is not particularly limited as long as it is within the range of solubility in the solvent, but in the gradient method it is preferable to vary it from 0 to about 1 molar concentration, and in the stepwise method, the
Run eluent at 0.2 to 0.5 molar concentration, then 0.7
It is preferred to run an eluent having a molar concentration of 1 to 1.5 molar.

The temperature during purification is preferably at room temperature or lower, particularly preferably at 10° C. or lower, since the acid salt of spagarin or its derivative of general formula () is unstable to heat.

The acid salt of spagarin or its derivative represented by the general formula () is isolated by a known method. For example, after performing the column chromatography of the present invention, the fraction containing the target product is heated at 40°C.
Below, it is concentrated to dryness, or the organic solvent in the eluate is concentrated under reduced pressure, and the remaining aqueous solution is freeze-dried. Next, methanol was added to the obtained mixture of the target product and inorganic salt to separate the salt, and the liquid was further applied to a column packed with Sephadex LH-20.
Elute with methanol and concentrate the fraction containing the target product under reduced pressure. Furthermore, the residue can be isolated by dissolving it in a small amount of water and freeze-drying it.

As described above, by using a water-containing hydrophilic organic solvent as an eluent, the separation state of impurities can be significantly improved compared to the conventional method using water as a solvent.

In addition, when removing the solvent by vacuum concentration or lyophilization of the fraction containing the target product after column chromatography, when using a hydrophilic organic solvent, compared to the conventional case where only water is used as a solvent, Since concentration can be carried out at a much lower temperature and in a shorter time, it is possible to suppress the decomposition of the acid salt of spagarin or its derivatives of the general formula (), which is unstable in an aqueous solution. Therefore, the present invention has made it possible to obtain a target product of higher purity in a higher yield than with conventional methods.

The crude product spagarin, which is the raw material of the present invention, was produced in accordance with JP-A-57-48957. Furthermore, the compounds represented by the general formula (), which are raw materials of the present invention excluding spagarin, were synthesized according to Japanese Patent Application No. 56-69340 and Japanese Patent Application No. 56-159503. That is, an acid salt of an ω-guanidino fatty acid amide derivative of general formula () and an acid salt of N-[4-(3-aminopropyl)aminobutyl]-2,2-dihydroxyethanamide of general formula () It was produced by reacting in the presence of a catalyst.

Among the ω-guanidino fatty acid amides of the general formula (), compounds in which R is hydrogen or a hydroxyl group are known. A compound in which R is an aliphatic acyloxy group can be obtained by acylating a corresponding compound in which R is a hydroxyl group.

Compounds of formula () are known.

Next, the present invention will be specifically explained with reference to Examples. Note that the purity of raw materials, purified products, etc. was calculated from the area ratio of peaks in reversed phase high performance liquid chromatography. Further, in this embodiment, the following abbreviations are used.

CM-CEPHADEX: CM-CEPHADEX C-25 (sodium form) HPLC: High performance liquid chromatography Example 1 CM-CEPHADEX was preliminarily mixed with 60% aqueous methanol (v/v) and sodium chloride to 0.3M.
Prepare by swelling with a solution dissolved in v) and filling 200 ml of the solution into a column. 20 g of spagarin hydrochloride with a purity of 92.1%, which was separated and purified from the culture solution by a conventional method, is dissolved in 20 ml of 60% aqueous methanol (v/v), and this solution is injected into the column prepared above. Next, aqueous methanol solution 2.1 of sodium chloride with the same composition as mentioned above was passed through to elute impurities, and sodium chloride was added to a concentration of 1.0M.
Solution in 60% aqueous methanol (v/v)
Flow 600ml and collect the eluate containing spagarin.

This eluate is concentrated under reduced pressure to remove methanol, and the residual liquid is lyophilized. Then, methanol is added to the lyophilized product, insoluble materials are separated, and the liquid is concentrated under reduced pressure at room temperature.

Seadex swollen with methanol
Prepare a column consisting of 200 ml of LH-20. A solution of the above concentrated residue dissolved in a small amount of methanol is injected into this column. Then, elute with methanol, separate the eluate containing the target product, and concentrate under reduced pressure to distill off the methanol. The residual solid was dissolved in a small amount of water and freeze-dried to obtain 1.67 g of purified spagarin hydrochloride with a purity of 98.1% (yield: 83.5%).

Figure 1 shows the HPLC chromatogram of spagarin purified by this method, and the HPLC chromatogram of spagarin purified by the conventional method using the raw materials used in this example.
The HPLC chromatogram is shown in Figure 2. As is clear from this figure, in the case of the conventional method, the presence of a considerable amount of impurities is recognized, whereas in the case of the method of the present invention, the impurities disappear and are greatly reduced. Therefore, according to the method of the present invention, spagarine with almost no impurities can be obtained.

Example 2 CM-Sephadex was prepared by adding sodium chloride to 50% aqueous methanol (v/v) to a concentration of 0.3M.
Prepare by swelling with a solution dissolved in v) and filling 1 into a column.

14.8 g of spagarine hydrochloride with a purity of 91.8% is dissolved in 150 ml of an aqueous sodium chloride-containing ethanol solution having the same composition as described above, and the solution is injected into the column prepared above. Next, a sodium chloride-containing ethanol solution 5.6 with the same composition as mentioned above was passed to elute the impurities contained, and then sodium chloride was added to 50% aqueous ethanol (v/v) to a concentration of 1.0M.
Flow the solution 1.7 dissolved in water and collect the eluted portion containing the target product.

The following treatment was carried out in the same manner as in Example 1, and 10.9 g of purified spagarin hydrochloride with a purity of 98.0% (yield:
73.6%).

Example 3 CM-Sephadex is swollen with 60% aqueous methanol (v/v) and 100 ml of this is packed into a column to prepare.

1.0 g of spagarin hydrochloride with a purity of 94.9% is dissolved in 10 ml of the same aqueous methanol as described above and injected into the column. 60% aqueous methanol (v/v) 500
Using 500 ml of a solution of sodium chloride dissolved in 60% aqueous methanol (v/v) to a concentration of 0.8 M and 0.8 M, elution was performed using a gradient method in which the concentration of sodium chloride was continuously increased. Separate the portion.

The following treatment was carried out in the same manner as in Example 1 to obtain 0.70 g of purified spagarin hydrochloride with a purity of 98.0% (yield:
70.0%).

Example 4 CM-Sephadex is swollen with a solution of sodium chloride dissolved in 60% aqueous methanol (v/v) to a concentration of 0.3M, and 20 ml of this is packed into a column.

15 obtained by condensing 0.31 g of 3-acetoxy-7-guanidinoheptanamide hydrochloride and 0.32 g of N-[4-(3-aminopropyl)aminobutyl]-2,2-dihydroxyethanamide dihydrochloride
0.75 g of the reaction mixture containing -O-acetyl spagarin hydrochloride (target product content: 0.21 g) was dissolved in 2 ml of an aqueous sodium chloride-containing methanol solution having the same composition as described above, and this was added to the column prepared above. Inject into. Next, elution is performed with a sodium chloride-containing methanol solution having the same composition as described above, and the eluted portion containing the target product is fractionated.

Thereafter, the same treatment as in Example 1 was carried out to obtain 0.15 g of purified 15-O-acetyl spagarin hydrochloride with a purity of 98.7% (yield 75.0%).

Purification of the crude 15-O-propionyl spagarin product can be carried out in a similar manner.

Example 5 7-guanidinoheptanoic acid amide hydrochloride 2.20 g
6.10 g of a reaction mixture containing 15-deoxyspargarin hydrochloride obtained by condensing 2.90 g of N-[4-(3-aminopropyl)aminobutyl]-2,2-dihydroxyethanamide dihydrochloride ( Target content: 2.75g)
By performing column chromatography and post-treatment in the same manner as in Example 1 using a 700 ml column, 1.80 g of purified 15-deoxyspargarin hydrochloride with a purity of 98.0% (recovery rate 65.5%) was obtained. .

Example 6 Obtained by condensing 0.24 g of 3-hydroxy-7-guanidinoheptanamide hydrochloride and 0.29 g of N-[4-(3-aminopropyl)aminobutyl]-2,2-dihydroxyethanamide dihydrochloride 11
(±)-Reaction mixture containing spagarin hydrochloride
Purification 11 with a purity of 98.5% was obtained by performing column chromatography and post-treatment on 0.60 g (target substance content: 0.21 g) using a column consisting of 100 ml of CM-Sephadex in the same manner as in Example 1.
0.14 g (67% recovery) of (±)-spargarin was obtained.

Reference Example 1 Synthesis of 15-deoxyspargarin (N-[4-(3-aminopropyl)aminobutyl]-2-(4-guanidinobutanamide)-2-hydroxyethanamide) 4-guanidinoptanamide hydrochloride 360 mg (2
mmol), N-[4-(3-aminopropyl)aminobutyl]-2,2-dihydroxyethanamide dihydrochloride 701 mg (2.4 mmol), glutaric acid 264
mg (2 mmol) and 0.36 ml of water and heated to 60°C for 1 hour.
The reaction mixture was reacted for 1 day to obtain a reaction mixture containing 15-deoxyspargarin.

Reference Example 2 Synthesis of 15-O-acetyl spagarin 7 ml of pyridine and 7 ml of acetic anhydride were added to 813 mg of (s)-7-guanidino-3-hydroxyheptanamide hydrochloride, and the mixture was stirred overnight at room temperature. After the reaction, add 100ml of ice water, concentrate under reduced pressure, and transfer the concentrated liquid to CM-
Sephadex C-25 (Na type) tower (450ml, diameter
2.5 cm), and the eluate with a salt concentration of 0.16 to 0.2 mol was collected and concentrated to dryness under reduced pressure. The resulting residue was extracted with methanol, and the extract was swollen with methanol in a Sephadex LH-20 tower (1.5, diameter 5.6 cm).
The mixture was poured over water, developed with methanol, and desalted. The effective fractions were collected and dried under reduced pressure to obtain (s)-7 as a white powder.
-Guanidino-3-acetoxyheptanamide hydrochloride 753 mg (yield 78.8%) was obtained. Proton NMR (TMS) of this product measured in heavy methanol
(60MHz) (hereinafter simply referred to as NMR)
That's what it means. ) 1.4 to 1.9 (CH ₂ × 3), 2.00 (COCH ₂ ),
Characteristic signals were shown at 2.49 (CH ₂ ), 3.18 (NCH ₂ ), and 5.19 (CH).

354 mg of this (s)-7-guanidino-3-acetoxyheptanamide hydrochloride, 429 mg of N-[4-(3-aminopropyl)aminobutyl]-2,2-dihydroxyethanamide hydrochloride and 333 mg of glutaric acid were added, This was dissolved in 0.5 ml of water and reacted overnight at 60°C to obtain a reaction mixture containing 15-O-acetyl spagarin.

[Brief explanation of drawings]

FIG. 1 is an HPLC chromatogram of spagarin hydrochloride purified by the method of the present invention.
FIG. 2 is an HPLC chromatogram of spagarin hydrochloride purified by a conventional method. In the figure, 1, 3, 4, and 5 are impurities (1 is a decomposition product (), ()), and 2 is spagarin.

Claims (1)

[Claims] 1. General formula (In the formula, R is a hydrogen atom, a hydroxyl group, and a carbon number of 1
to 10 aliphatic acyloxy groups, and n represents an integer of 4 or 6. ) When purifying the acid salt of spagarin or its derivatives by column chromatography using an insoluble carrier having a carboxyl group as a packing material, it is recommended to use a water-containing hydrophilic organic solvent containing an inorganic salt as an eluent. A method for purifying an acid salt of spagarin or a derivative thereof represented by the general formula ().