JPS5929652A - Purification of spergualin or its derivative - Google Patents

Abstract

PURPOSE:To prepare the titled compound useful as a carcinostatic agent, in high purity and yield, by purifying a crude product of an acid salt of spergualin or its derivative with a specific packing material and an eluting solution. CONSTITUTION:An acid salt of spergualin of formula (R is H, hydroxyl or 1- 10C aliphatic acyloxy; n is 4-6) or its derivative is subjected to column chromatography using an insoluble carrier having carboxyl groups as packing material (e.g. CM-Sephadex, carboxymethyl cellulose, etc.). A water-containing hydrophilic organic solvent (e.g. methanol, ethanol, acetone, etc.) containing an inorganic salt is used as the elution solution, and the purification is carried out at <=room temperature, preferably <=10 deg.C. EFFECT:The impurities can be seprated economically and the concentration can be carried out at low temperature in a short time by the use of the water-containing hydrophilic organic solvent.

Description

Detailed Description of the Invention The present invention is represented by the general formula (wherein R represents a hydrogen atom, a hydroxyl group, or an aliphatic acyloxy group having 1 to 10 carbon atoms, and n represents an integer of 4 or 6). Sperguajin”
! Regarding the purification method of the acid salts of spagarin or its derivatives, more specifically, 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, an inorganic salt is used as an eluent. The present invention relates to a method for purifying spagarin or its derivative acid salt represented by the general formula (I), which is characterized by using a water-containing hydrophilic solvent containing the following.

In the compound of general formula (I), R is a hydroxyl group, n
A substance with 4 is called spagarin, and the configuration is (11
J, 15S) is a compound isolated as a hydrochloride from microbial culture F solution by Hira et al., and is expected to be a non-toxic anticancer agent (J, AntibioLics).
34: I 619-1627).

15
8) -, <It is a new substance that has the same anticancer effect as pagarin (see Tokko El" No. 56-69340 and Japanese Patent Application No. 159503/1982).

Conventionally, the acid salt of spagarin of the general formula (I) has been purified by, for example, column chromatography of the crude product using sodium salt of CM-cefazo-nox as a packing material and water containing sodium chloride as an eluent. The fraction containing the acid salt of spagarine was eluted and 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 removing residual sodium chloride by elution with methanol, 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-CefAdenox.

Unfortunately, even if the acid salts of the compounds of general formula (I) 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 a highly pure acid salt of spagarin or its derivatives, the present inventors first conducted various studies on the reasons why it was not possible to obtain a highly pure product using the above purification method. . As a result, the cause is
The acid salt of spagarin or its derivatives with the general formula (Il is
Gradually decomposes in water by an intramolecular reversible reaction shown by the following formula to form H2NCNH(CH2)nCHCH2CONHCHCO
NH(CH2)4NH(CH2)3NH2111 NHR0H fi+ (2) (In the formula, R is the same as above.) General formula (marked ω-guanidino fatty acid amide acid salt and general formula (m) N(:4 -(3-aminopropyl)aminobutyl] -2,2-dihydroxyethanamide, so it is 1, (11←l, 15S)-spargarate is separated from bacterial culture P solution, General formula (2)
It was found that this is because it contains many impurities in addition to the acid salt of the compound of formula (m) and the acid salt of the compound of general formula (m).

Based on the above knowledge, the present inventors have conducted intensive studies on purification methods for the acid salts of spagarin or its derivatives, and have found that in purifying them using column chromatography 5-E using an insoluble carrier having a carboxyl group as a packing material. The 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 an eluent, and have completed the present invention.

To explain the present invention in more detail, in the acid salt of spagarin or a derivative thereof having the general formula (Il) 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 - An aliphatic acyloxy group having 1 to 10 carbon atoms, such as a butanoyloxy group, an isobutanoyloxy group, and an octanoyloxy group, where 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. of lower fatty acids, succinic acid, fumaric acid,
Examples include polybasic acids such as maleic acid, tartaric acid, and citric acid, and sulfonic acids such as benzene-6-nesulfonic acid, toluenesulfonic acid, mechnesulfonic acid, and ethanesulfonic acid.

Typical specific examples include spagarin, 15-0-pormylspargarin, 15-O-acetyl spagarin, 15
-0-Fropionylshakarin, 15-0-butanoylspargarin, 15-0-octanoylspargarin, 15
-deoxyspargarin and hydrochloride salts such as N-(4-(3-aminopropyl)aminobutyl]-2-(9-guanidinononanamide)-2-hydroxyethanamide;
Examples include sulfates.

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

There are no particular restrictions on the crude acid salt of the compound of general formula (I), but in the case of spagarin, it can be obtained by subjecting the culture solution to IT (C-50 column chromatography) by a known method. Alternatively, those isolated by conventional CM-Sephadex column chromatography using only water as a solvent are used, and in the case of spagarin derivatives, those obtained by concentrating the reaction mixture under reduced pressure are used. It will be done.

The purity of the crude product is not particularly limited, but is preferably 30-95%.

The insoluble carrier having a carboxyl group is not particularly limited, but CM-Sephadex, carboxymethyl cellulose, etc. are preferred.

There are no particular restrictions on the inorganic salts contained in the eluent, but
Examples include chlorides, bromides, sulfates, nitrates, and phosphates of alkali metals such as sodium and potassium, with sodium chloride and potassium chloride being preferred.

The hydrophilic organic solvent used as the eluent has a boiling point of 1
There is no particular restriction as long as it is an organic solvent that mixes with water at a temperature of 20C or less, preferably 90C or less, but lower alcohols such as methanol, n-propatool, and isopropatool; ketones such as acetone and 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%, 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 run to elute most of the impurities, and then an eluent with a high concentration of inorganic salt is used. There are two methods: a stepwise method in which the target substance is eluted by flowing a liquid, and a method 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 that dissolves 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 it is preferable to vary it from 0 to 1 molar concentration. It is preferable to first run an eluent with a 0.2 to 0.5 molar concentration, and then run an eluent with a 0.7 to 1.5 molar concentration.

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

The acid salt of spagarin or its derivative represented by general formula (I) is isolated by a known method. for example,
After performing the column chromatography of the present invention, the fraction containing the target product is concentrated to dryness at 40C or lower, 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 in a furnace. Concentrate under reduced pressure. Further, the residue = 10- can be isolated by dissolving the residue 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 freeze-drying the fraction containing the target product after column chromatography, it is more important to concentrate when using a water-containing hydrophilic organic solvent than when using conventional water alone as a solvent. Since this 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 the general formula (I) of spagarin or its derivatives, which is unstable in an aqueous solution. Therefore, according to the present invention, a target product of higher purity can be obtained in a higher yield than in the conventional method.

The crude product spagarin, which is the raw material of the present invention, is disclosed in Japanese Patent Application Laid-open No. 57
-48957. Furthermore, the compound represented by the general formula (I) which is a raw material of the present invention excluding spagarin is disclosed in Japanese Patent Application No. 56-69340 and Japanese Patent Application No. 56-1
Synthesized according to No. 59503. That is, it was produced by reacting an acid salt of an ω-guanidino fatty acid amide derivative of the general formula % with an acid salt of the general formula % in the presence of an acid 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 (m) are known.

Next, the present invention will be specifically explained with reference to Examples. In addition,
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-Sephadex: CM-Sephadex ■ Su C-25 (sodium form) 1 (PLC: High Performance Liquid Chromatography Example 1゜■ CM-Sephadex was pre-hydrated with 60% sodium chloride to make it 0.3M) Prepare by swelling with a solution dissolved in methanol (V/V) and filling 200ml of it into a column.Sunokugarin hydrochloride 2.01 with a purity of 921%, separated and purified from the culture P solution by the usual method. Dissolve in 20 ml of 60% aqueous methanol (v/v) and inject this solution into the column prepared above. Then add a solution of sodium chloride in aqueous methanol with the same composition as described above.2.
14 to elute impurities, and then flow 600 ml of a solution of 1.0 M sodium chloride in 60% aqueous methanol (v/v) to separate the eluted portion containing spagarin.

This eluate is concentrated under reduced pressure to remove methanol, and the residual liquid is lyophilized. Next, methanol is added to the freeze-dried product, and after removing insoluble matter in a furnace, the P solution is concentrated under reduced pressure at room temperature.

13- ■ Seadex LH-20 further swollen with methanol
, 200rnlJ is prepared.

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 lyophilized to obtain purified spagarine hydrochloride with a purity of 981.
7 g (yield 83.5%) was obtained.

The HPLC chromatogram of spagarin purified by this method is shown in FIG. 1, and the HPLC chromatogram of spagarin purified again by the conventional method from the raw material used in this example is shown in FIG. 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.

=14- Example 2 ■ CM-Sephadex was swollen with a solution of sodium chloride dissolved in 50% aqueous ethanol (v/v) to a concentration of 0.3M. , e3 are packed into a column and prepared.

Spagarin hydrochloride 148ψ with a purity of 91.8% was added to a sodium chloride-containing aqueous ethanol solution with the same composition as described above.
Dissolve in 50 ml and inject into the column prepared above.

Next, an aqueous sodium chloride-containing ethanol solution 5.6.8 having the same composition as mentioned above was poured to elute the impurities that were to be dissolved, and then sodium chloride was added to
Solution 1.7 in 0% aqueous ethanol (V/V)
Drain the swamp and collect the eluted portion containing the target substance.

Thereafter, by processing in the same manner as in Example 1, the purity was 98.
0% purified spagarin hydrochloride 10.9p (yield 73.6
%) was obtained.

Example 3 Prepare by swelling with 100 ml of this (V/V) into a column.

1.01 ml of spagarine hydrochloride with a purity of 949% is dissolved in 10 ml of the same aqueous methanol as described above and injected into the column. 60% aqueous methanol (Cv/v) 500 ml
Continuously chlorinate using 500 ml of a solution of sodium chloride dissolved in 60% aqueous methanol (v/v) to a concentration of 0.8 M) Elute using a gradient method that increases the concentration of IJium. , separate the eluted portion containing the target product.

Thereafter, by processing in the same manner as in Example 1, the purity was 98.
0% purified spagarin hydrochloride 0.70PC yield 700%
) was obtained.

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

Obtained by condensing 0.31% of 3-acetoxy-7-guanidinohebutanamide hydrochloride and 0.32% of N-(4-(3-aminopropyl)aminobutyl]-2,2-dihydroxyethanamide dihydrochloride. The reaction mixture containing 15-0-acetyl spagarin hydrochloride 0.75y-(
Target content: 0.21. P) is chlorinated with the same composition as mentioned above) 2 ml of IJum-containing aqueous methanol solution
and inject it into the column prepared above. 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, by processing in the same manner as in Example 1, the purity was 98.
7% purified 15-0-acetyl spagarin hydrochloride 0.1
59-(yield 75.0%) was obtained.

] 5-0-Propionyl spagarin crude and product purification can be carried out in the same manner.

Example 5 7-Guanidinohebutanoic acid amide hydrochloride 2.201 and N
-r:4-(3-aminopropyl]aminobutyl]-2
, 2-dihydroxyethanamide dihydrochloride 2.905
A reaction mixture containing 15-deoxyspargarin hydrochloride obtained by condensing 1- (6.10 PC target product content: 2.75 fi') was carried out using a column consisting of CM-cefazo 17- ■Tuxuf 00 ml. Example 1
By performing column chromatography and post-treatment in the same manner as above, purified 15-deoxyspargarin hydrochloride 1.80P with a purity of 98.0% (recovery rate 65.5%) was obtained.

Example 6 Condensation of 3-hydroxy-7-guanidinohebutanamide hydrochloride 0.24.7 and N-[4-(3-aminopropyl)aminobutyl]-2,2-dihydroxyethanamide dihydrochloride 01291 0.60 P of the reaction mixture containing 11(to)-spargarin hydrochloride (target product content: 0.215') was treated in the same manner as in Example 1 using a column consisting of 100 ml of CM-70 Fadex. Purification 11 (up to 985% purity) by column chromatography and post-treatment according to method
- Spagarin 0.14? (recovery rate of 67%) was obtained.

Reference example 115-deoxyspargarin (N-(4-(3-
Synthesis of 4-guanidibucunamide hydrochloride 36 om9 (2 mmol), N-(4-( 3-Aminopropyl)aminobutyl, 1-2,2-dihydroxyethanamide hydrochloride 70
1 mg (2,4 mmol), glutaric acid 2647+1&
(2 mmol) and 0.36 rnl of water, 60r
The mixture was reacted for one day to obtain a reaction mixture containing 15-deoxyspargarin.

Reference Example 2.15-Synthesis of 0-acetyl spagarin (S)
-7-guanidino-3-hydroxyheptanamide hydrochloride 813 m9 to 7 ml pyridine and 7 m acetic anhydride
1 was added, and the mixture was stirred at room temperature overnight. 100m of ice water after reaction
1 was added, concentrated under reduced pressure, and the concentrated solution was transferred to a CM-Sephadex C-25 (Na type) column (450 ml, diameter 2.5 Cr
n), and the eluate with a salt concentration of 0.16 to 0.2 mol was collected and concentrated to dryness under reduced pressure. The obtained residue was extracted with methanol, and this extract was mixed with methanol-swollen Cef.
Cus LH-20(r) tower (1,5,, e.

diameter 5.6 cm), developed with methanol, and desalted. The effective fractions were collected and dried under reduced pressure to form a white powder (S
753 mg (yield 788%) of hydrochloride of -7-guanidino-3-acetoxyhebutanamide was obtained.

Proton NMR of this product measured in heavy methanol (
With TMS as the standard, 1.4 to 1.9 (CH2X) at 60 MHz (hereinafter simply referred to as NMR)
3), 2.00 (C0CH2), 2.49 (C
J-72), 3.1.8 (NCH2) 5.1.9 (C
H) showed a characteristic signal.

This (81-7-guanidino-3-acetoxyhepcunamide hydrochloride 354 m9, N-[4,-(3-aminopropyl)aminobutyl]-2,2-dihydroxyethanamide hydrochloride 429 m9 and glutaric acid 333m
g was added, dissolved in 0.5 ml of water, and heated at 60C for 1
The reaction was carried out overnight to obtain a reaction mixture containing 15-0-acetyl spagarin.

[Brief explanation of the drawing]

FIG. 1 is an HPLC chromatogram of spagarin hydrochloride purified by the method of the present invention. Figure 2 shows spagari (■)) purified by the conventional method;
(2) is spagarin. Patent applicant Nippon Kayaku Co., Ltd.=21=

Claims (1)

[Claims] (1) Spagarin or The general formula ( A method for purifying the acid salt of spagarin or its derivative represented by I).