JPH0223541B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention is based on the following formula () The novel antibiotic BMG162-aF2, represented by the following formula (), is hydrolyzed with dilute acid or dilute alkali in an aqueous solution, and becomes an important intermediate for the synthesis of a new anticancer substance. N-[4-(3-aminopropyl)aminobutyl]-2,2-dihydroxyethanamide represented by and the following formula () The present invention relates to a method for producing N-[4-(3-aminopropyl)aminobutyl]-2,2-dihydroxyethanamide represented by:

Novel antibiotic BMG162 represented by formula ()
-aF2 [Japanese Unexamined Patent Publication No. 57-48957 (see Japanese Patent Application No. 55-123585)] is heated in a pure state, crude state, or in a culture solution with a dilute organic acid, mineral acid, or dilute alkali or at room temperature. By treating with and hydrolyzing, N-[4
-(aminopropyl)aminobutyl]-2,2-dihydroxyethanamide (hereinafter abbreviated as substance A) and (S)-7-guanidino-3-hydroxyheptanamide represented by formula () (hereinafter abbreviated as substance B) The present invention was completed based on the discovery that it is possible to collect the following in high yield.

Substance A and Substance B produced by the present invention have already been synthesized by the present inventors
No. 192347 (Japanese Patent Application No. 1983-73510 and (Patent Application No. 1983)
-188562 (see Japanese Patent Application No. 56-73511)], and furthermore, it is possible to synthesize various new anticancer substances by condensing substance A with substance B or various acid amides derived from substance B. It has also been discovered that it is possible to
No. 57-192347 (Japanese Patent Application No. 56-73510) and (Japanese Unexamined Patent Publication No. 57-188562) (see Japanese Patent Application No. 56-73511)].

That is, the following formula () Substance A or its salt represented by and general formula (') When substance B represented by (wherein X represents a hydroxyl group or acyloxy group) or a derivative thereof or a salt thereof is thermally condensed in the presence of an inorganic or organic acid, the following general formula (') is obtained. The anticancer substance represented by the formula (wherein X represents a hydroxyl group or an acyloxy group) can be obtained very easily. Therefore, Substance A and Substance B are compounds that serve as important raw materials for synthesizing various new and useful anticancer substances.

The novel antibiotic BMG162-aF2, which is the raw material for substance A and substance B in the present invention, is
It is collected by the method described in detail in No. 48957 (Japanese Patent Application No. 123585-1987). An overview of this method is to inoculate a strain that produces the new antibiotic BMG162-aF2 (Feikoken Bibori No. 5230) into a nutrient-containing medium and grow it aerobically to create a culture containing BMG162-aF2. is obtained. As the nutrient source, those that can be normally used as a nutrient source for microorganisms can be used.
For example, commercially available nitrogen sources such as peptone, meat extract, corn steep liquor, cottonseed flour, peanut flour, soybean flour, yeast extract, NZ-amine, casein hydrolyzate, ammonium nitrate, sodium nitrate, ammonium sulfate, etc. Carbon sources such as carbohydrates or fats such as glycerin, sucrose, glucose, maltose, and molasses, and inorganic salts such as common salt, phosphate, calcium carbonate, and magnesium sulfate can be used. In addition, trace amounts of metal salts and the like may be added as necessary. These nutritional sources are used by producing bacteria,
Any material useful for producing BMG162-aF2 may be used, and any known culture material for microorganisms can be used. Liquid culture is preferred for mass production of BMG-162-aF2, and the culture temperature is set to allow the production bacteria to grow.
Applicable within the range of producing BMG162−aF2, and usually
The temperature is 15-40°C, preferably 20-40°C, particularly preferably 20-35°C. The pH of the medium is usually 5.0 to 8.2, preferably 6.0 to 7.8. Culture is usually BMG162−
This can be continued until sufficient aF2 has been accumulated. For example, glycerin 2.0%, dextrin 2.0%, peptone 1.0
%, yeast extract 0.3%, ammonium sulfate 0.2%,
Liquid medium consisting of 0.2% calcium carbonate (PH6.0 ~
7.4) BMG162−aF2 cultured on agar slant medium
When the strain was inoculated and cultured aerobically with rotational shaking at 27°C, accumulation of the desired antibiotic was observed from the first day of culture. The quantitative determination of BMG162-aF2 is carried out by a conventional cylindrical plate method using Bacillus subtilis POI219 strain as the test bacterium. BMG162
-When culturing aF2-producing strains, in addition to the above-mentioned shaking culture, a jar incubator generally used for aerated agitation culture of microorganisms, or a large stainless steel
Steel tank culture vessels are also used for mass production. In the case of mass culture, it is preferable to use a culture solution obtained by shaking culture for 20 to 40 hours in the above liquid medium as a seed culture solution, and inoculate it at 0.5 to 2.0%.
BMG162-aF2 is usually collected from a culture solution of BMG162-aF2-producing bacteria by column chromatography using a weak cation exchanger having carboxylic acid as an active group. Weak cations include Amberlite IRC-50, OG-50 (registered trademark: manufactured by Rohm and Haas), Rewacht CNP (registered trademark: manufactured by Bayer), CM-Sephadex (registered trademark: manufactured by Pharmacia) ), H-type, Na
type, NH ₄ type, etc. and mixed types thereof are used. Elution of the adsorbed BMG162-aF2 is carried out by developing with water containing an acid such as hydrochloric acid or a salt such as common salt. In addition to the above-mentioned extraction, separation, or purification methods, a gel filtration method or an ultrafiltration method can be appropriately combined or repeated to obtain a pure product.

A method for collecting substance A and substance B by hydrolyzing the novel antibiotic BMG162-aF2 of the present invention will be described in detail below.

obtained as a crude product or pure
BMG162-aF2 can be decomposed by treating it in an acidic or alkaline aqueous solution.

As the acid, organic acids such as formic acid, acetic acid propionic acid, succinic acid, glutaric acid, adipic acid, fumaric acid, malic acid, and citric acid, or mineral acids such as hydrochloric acid, sulfuric acid, and phosphoric acid are used.

As the alkali, strong bases such as sodium hydroxide and potassium hydroxide, or weak bases such as sodium carbonate, sodium bicarbonate, sodium acetate, and ammonia are used.

Furthermore, a buffer solution with a pH of 3 or lower or a buffer solution with a pH of 5 or higher can also be used.

Although the reaction temperature and reaction time vary depending on the type of acid or alkali used, for example, in a 1N acetic acid aqueous solution, decomposition can be achieved in good yield by heating at 100° C. for 3 hours. Also, use sodium hydroxide to
In an aqueous solution with a concentration of 8.5, it can be decomposed in good yield by stirring at room temperature for one day. After neutralizing or concentrating the obtained hydrolyzate, Amberlite IRC-
50, CN-Sephadex, Diaion HP-
20 (manufactured by Mitsubishi Kasei) and other adsorbent towers,
Purify A substance and B substance by developing with water containing acid such as hydrochloric acid or salt such as common salt. For example, purified BMG162-aF2 is heated under reflux at 100°C for 1.5 hours in dilute acetic acid, and then heated under reduced pressure. Concentrate to remove acetic acid. Add water to the obtained residue and CM-
Substance B is eluted first, followed by substance A. The fraction containing substance B is concentrated to dryness under reduced pressure, and the resulting residue is extracted with methanol. Sephadex this extract.
After desalting with LH-20, crystallize from acetone,
Obtain crystals of the hydrochloride of the substance. The fraction containing Substance A is also treated in the same manner to obtain the dihydrochloride of Substance A in the form of candy.

(1) N-[4-(3-aminopropyl)aminobutyl]-2,2-dihydroxyethanamide (A
Physical and chemical properties of dihydrochloride (substance) This substance is in the form of a hygroscopic candy or powder.

Elemental analysis value (as C ₉ H ₂₁ N ₃ O ₃・2HC1) Experimental value (%): C37.08, H7.94, N13.78,
C121.96 Theoretical value (%): C36.99, H7.93, N14.38,
C124.27 Color reaction is positive for ninhydrin reaction, Lydon-Smith reaction, and 2,4-dinitrophenylhydrazine reaction. Infrared absorption spectrum (potassium bromide tablets): 3400, 2950, 1660, 1540, 1455, 1100
and 1040 cm _-1 . Proton nuclear magnetic resonance spectrum (measured in heavy methanol with tetramethylsilane as internal standard, 60MHz): 1.4-2.3 (CH ₂ × 3),
2.8-3.4 ( _CH2 ×4), 4.90ppm (CH).

(2) Physical and chemical properties of (S)-7-guanidino-3-hydroxyheptanamide (Substance B) hydrochloride This substance is usually obtained as a colorless crystalline powder, and its melting point is 101-103°C. Elemental analysis value (as C ₈ H ₁₈ N ₄ O ₂・HC1) Experimental value (%): C40.67, H7.55, N23.90,
C129.03 Theoretical value (%): C40.25, H7.60, N23.47,
C129.71 Specific optical rotation [α] ²⁴ _D −1° (C1, water), Lydon-Smith reaction and Sakaguchi reaction are positive for color reaction. Infrared absorption spectrum (potassium bromide tablets): 3330,
3175, 2930, 2850, 1655, 1430, 1400, 1175,
1095 and 1030 cm _-1 . Proton nuclear magnetic resonance spectrum (measured in heavy methanol with tetramethylsilane as internal standard, 60MHz): 1.4-1.7 ( _CH2
3), 2.39 (CH ₂ ), 3.20 (CH ₂ ) and 3.95ppm
(CH).

Next, the present invention will be explained by reference examples and examples, but the present invention is not limited thereto.

Reference example Glycerin 2.0%, Dextrin 2.0%, Soy peptone (Bactosoitone manufactured by Difco) 1.0%,
A liquid medium (PH7.4) 5 consisting of 0.3% yeast extract (powdered yeast extract manufactured by Daigo Nutritional Chemical Co., Ltd.), 0.2% ammonium sulfate, and 0.2% calcium carbonate was added to 125
Dispense ml of seed culture solution into Sakaguchi flasks [Bacillus cultured on agar slant medium].
1.0% of the BMG162-aF2 strain (Fiber Science and Technology Research Institute No. 5230) cultured in the same medium with shaking for 2 days] was inoculated and cultured at 28°C for 5 days. Culture solution obtained by filtration4.9
The mixture is passed through a column (500 ml, diameter 5.2 cm) containing Amberlite IRC-50 Na type and H type mixed in a ratio of 7:3 to adsorb the active ingredients. After washing the column with water, the active ingredient was eluted with 1.0N hydrochloric acid 2, and the active fraction was adjusted to pH 6 with 10N sodium hydroxide. This solution was diluted 4 times with water, and CM-
The active ingredients were adsorbed in a Cephadex C-25 column (400 ml, diameter 4.3 cm). The active ingredient was eluted with 0.3 molar saline. The active fraction was concentrated to dryness under reduced pressure, the resulting residue was extracted with 5 ml of methanol, the salt was removed by filtration, and a Sephadex LH-20 column (diameter
2.6 cm, 445 ml), developed with methanol, and dried the active fraction under reduced pressure to obtain 640 mg of pure BMG162-
The trihydrochloride of aF2 was obtained as a white powder.

Example 1 15.3g of BMG162-aF2 trihydrochloride was dissolved in 1N acetic acid.
Dissolve in 300ml and place in an oil bath at 110℃ under a nitrogen stream.
It took up a lot of time. The reaction solution was concentrated to dryness, the resulting residue was dissolved in 100 ml of water, and CM-Sephadex-25
(Na type) Pour into a tower (inner diameter 55 mm) filled with 1200 ml,
A gradient elution of 6 in water and 6 in 1 molar saline was performed. Fractions with a salt concentration of 0.29 to 0.37 mol were combined and evaporated to dryness under reduced pressure, and the resulting residue was extracted three times with 15 ml of methanol. This extract was transferred to a Cephadex LH-20 tower (inner diameter 30 mm, 450
ml) and developed with methanol (10 ml/fraction). Fractions 23-30 were combined and evaporated to dryness under reduced pressure. The resulting residue was crystallized from acetone to give 6.16 g of the hydrochloride of Substance B as colorless crystals. (yield 86.5%).
Further, a fraction with a salt concentration of 0.51 to 0.62 mol was treated in the same manner as described above to obtain 6.79 g of dihydrochloride of substance A as a colorless candy-like substance (yield 78%). Further, a fraction with a salt concentration of 0.72 to 0.76 mol was treated in the same manner, and 1.2 g of trihydrochloride of BMG162-aF2, which was a raw material, was recovered (recovery rate: 8%). The infrared absorption spectrum (potassic bromide) of the obtained substance A dihydrochloride is 3400,
2950, 1660, 1540, 1455, 1100 and 1040cm _-1 ,
Proton nuclear magnetic resonance spectrum (measured in heavy methanol with tetramethylsilane as internal standard,
60MHz) is 1.4 to 2.3 (CH ₂ × 3), 2.8 to 3.4 (CH ₂ ×
4), 4.90ppm (CH).

In addition, the infrared absorption spectrum of the obtained substance B hydrochloride (potassium bromide tablet) is 3330, 3175, 2930, 2850,
1655, 1430, 1400, 1175, 1095 and 1030cm _-1 ,
Proton nuclear magnetic resonance spectrum (measured in heavy methanol with tetramethylsilane as internal standard,
60MHz) is 1.4 to 1.7 (CH ₂ × 3), 2.39 (CH ₂ ), 3.20
(CH ₂ ) and 3.95 ppm (CH).

Example 2 After adjusting the culture solution (PH6.2) of BMG162-aF2 producing bacteria obtained in Reference Example to PH4 with 1N hydrochloric acid,
70 ml of acetic acid was added to this, and the mixture was heated under reflux at 100°C for 1.5 hours. Adjust the pH of this reaction solution with 10N caustic soda.
After adjusting to 6.0, Amberlite IRC-50 (Na/H=
7/3, 50ml) to adsorb substances A and B, and these were eluted with 1N hydrochloric acid. The eluate was dried under reduced pressure and extracted with 50 ml of methanol. After drying this extract under reduced pressure, it was dissolved in 50 ml of water and poured into a column of CM-Sephadex (Na type, 100 ml).
Elution was carried out using 500 ml and 500 ml of 1 molar saline, increasing the salt concentration linearly. A substance or B
The fractions containing the substance were separately evaporated to dryness under reduced pressure and extracted with 20 ml of methanol. The obtained extract was concentrated under reduced pressure, and the supernatant (3 ml) was transferred to a sepadex.
The mixture was poured into a column of LH-20 (200 ml) for desalting and purification. By the above operations, 10.5mg of dihydrochloride of Substance A
And 22.0 mg of the hydrochloride of Substance B was obtained. Obtained A
The infrared absorption spectrum of the substance dihydrochloride (potassium bromide tablet) is 3400, 2950, 1660, 1540, 1455, 1100 and 1040 cm _-1 , the proton nuclear magnetic resonance spectrum (measured in heavy methanol with tetramethylsilane as an internal standard, 60MHz) is 1.4~2.3 (CH ₂ × 3),
It was 2.8 to 3.4 (CH ₂ ×4) and 4.90 ppm (CH).

In addition, the infrared absorption spectrum of the obtained substance B hydrochloride (potassium bromide tablet) is 3330, 3175, 2930, 2850,
1655, 1430, 1400, 1175, 1095 and 1030cm _-1 ,
Proton nuclear magnetic resonance spectrum (measured in heavy methanol with tetramethylsilane as internal standard,
60MHz) is 1.4 to 1.7 (CH ₂ × 3), 2.39 (CH ₂ ), 3.20
(CH ₂ ) and 3.95 ppm (CH).

Example 3 The culture solution 1 obtained in the reference example was mixed with Amberlite.
Pour into a column of IRC-50 (Na/H=7/3, 50ml),
BMG162-aF2 was adsorbed, washed with water, and eluted with 1N hydrochloric acid. The active fraction was adjusted to pH 6.0, concentrated under reduced pressure to dryness, extracted with 50 ml of methanol, and this extract was dried under reduced pressure. Add 5 ml of 1N acetic acid to this,
Heated at 100°C for 1.5 hours. This heated reaction solution was concentrated under reduced pressure to dryness, then dissolved in 30 ml of water, and the pH was adjusted to 6.0. Add this solution to CM-Sephadex (Na type,
50 ml) column, add 300 ml of water and 1 molar saline solution.
Using 300 ml, substance A and substance B were separated by elution with increasing salt concentration linearly. Thereafter, the product was desalted and purified using Sephadex LH-20 in the same manner as in Example 2 to obtain 18 mg of the dihydrochloride of Substance A and 35 mg of the hydrochloride of Substance B. The infrared absorption spectrum of the obtained substance A dihydrochloride (potassium bromide tablet) is 3400, 2950,
1660, 1540, 1455, 1100 and 1040 cm _-1 , proton nuclear magnetic resonance spectra (measured in heavy methanol with tetramethylsilane as internal standard, 60MHz)
is 1.4 to 2.3 (CH ₂ × 3), 2.8 to 3.4 (CH ₂ × 4),
It was 4.90ppm (CH).

In addition, the infrared absorption spectrum of the obtained substance B hydrochloride (potassium bromide tablet) is 3330, 3175, 2930, 2850,
1655, 1430, 1400, 1175, 1095 and 1030cm _-1 ,
Proton nuclear magnetic resonance spectrum (measured in heavy methanol with tetramethylsilane as internal standard,
60MHz) is 1.4 to 1.7 (CH ₂ × 3), 2.39 (CH ₂ ), 3.20
(CH ₂ ) and 3.95 ppm (CH).

Example 4 200 mg of trihydrochloride of BMG162-aF2 obtained in Reference Example
Add 5 ml of 0.5 mol glutaric acid to the mixture and heat to 100℃.
After heating reaction for 1.5 hours, the pH of the reaction solution was adjusted to 6.0, and water was added to make the total volume to 30 ml. Thereafter, chromatography using CM-Sephadex and then Cephadex LH-20 was carried out in the same manner as in Example 3 to obtain 31 mg of the dihydrochloride of Substance A and 62 mg of the hydrochloride of Substance B. The infrared absorption spectrum of the obtained substance A dihydrochloride (potassium bromide tablet) is 3400, 2950, 1600, 1540,
1455, 1100 and 1040 cm _-1 , proton nuclear magnetic resonance spectra (measured in heavy methanol with tetramethylsilane as internal reference, 60 MHz) are 1.4-2.3
(CH ₂ × 3), 2.8 to 3.4 (CH ₂ × 4), 4.90ppm (CH)
It was hot.

In addition, the infrared absorption spectrum of the obtained substance B hydrochloride (potassium bromide tablet) is 3300, 3175, 2930, 2850,
1655, 1430, 1400, 1175, 1095 and 1030cm _-1 ,
Proton nuclear magnetic resonance spectrum (measured in heavy methanol with tetramethylsilane as internal standard,
60MHz) is 1.4 to 1.7 (CH ₂ × 3), 2.39 (CH ₂ ), 3.20
(CH ₂ ) and 3.95 ppm (CH).

Example 5 200 mg of trihydrochloride of BMG162-aF2 obtained in Reference Example
2 ml of 1N ammonia water was added to the mixture, and the mixture was allowed to stand at room temperature for 5 minutes, and then concentrated under reduced pressure to dryness. The obtained residue was dissolved in water, substance A and substance B were separated by CM-Sephadex column chromatography in the same manner as in Example 3, and chromatography was performed on Sephadex LH-20 to separate the two substances of substance A. hydrochloride
15 mg and 59 mg of the hydrochloride of Substance B were obtained. The infrared absorption spectra (potassium bromide tablets) of the obtained dihydrochloride of Substance A are 3400, 2950, 1660, 1540, 1455, 1100 and 1040 cm _-1 , and the proton nuclear magnetic resonance spectra (in heavy methanol with tetramethylsilane internally). Measured as a reference, 60MHz) is 1.4 ~ 2,3 (CH ₂ ×
3), 2.8 to 3.4 (CH ₂ ×4), and 4.90 ppm (CH).

In addition, the infrared absorption spectrum of the obtained substance B hydrochloride (potassium bromide tablet) is 3330, 3175, 2930, 2850,
1655, 1430, 1400, 1175, 1095 and 1030cm _-1 ,
Proton nuclear magnetic resonance spectrum (measured in heavy methanol with tetramethylsilane as internal standard,
60MPH) is 1.4-1.7 (CH ₂ × 3), 2.39 (CH ₂ ), 3.20
(CH ₂ ) and 3.95 ppm (CH).

Example 6 Dissolve 1.87 g of trihydrochloride of BMG162-aF2 in 10 ml of water, add saturated aqueous sodium bicarbonate solution, and adjust the pH.
8.5 and stirred at room temperature for 1 day. reaction solution
After adjusting the pH to 6.0 with 0.2N-HC1, it was concentrated to dryness, the residue was dissolved in 5 ml of water, and the same procedure as in Example 2 was carried out.
CM-Sephadex plus Sephadex LH
-20 chromatography was carried out, and 803 mg (yield 75.5%) of dihydrochloride of substance A and 659 mg of hydrochloride of substance B
mg (yield 75.8%). The infrared absorption spectrum of the obtained substance A dihydrochloride (potassium bromide tablet) is
3400, 2950, 1660, 1540, 1455, 1100 and 1040
cm _-1 , proton nuclear magnetic resonance spectrum (measured in heavy methanol with tetramethylsilane as internal standard, 60 MHz) is 1.4-2.3 (CH ₂ ×3), 2.8-3.4
(CH ₂ ×4), 4.90 ppm (CH).

In addition, the infrared absorption spectrum of the obtained substance B hydrochloride (potassium bromide tablet) is 3330, 3175, 2930, 2850,
1655, 1430, 1400, 1175, 1095 and 1030cm _-1 ,
Proton nuclear magnetic resonance spectrum (measured in heavy methanol with tetramethylsilane as internal standard,
60MHz) is 1.4 to 1.7 (CH ₂ × 3), 2.39 (CH ₂ ), 3.20
(CH ₂ ) and 3.95 ppm (CH).

Claims (1)

[Claims] Linear formula () The following formula () is characterized by hydrolyzing the new antibiotic BMG162-aF2 expressed by dilute acid or dilute alkali in an aqueous solution. N-[4-(3-aminopropyl)aminobutyl]-2,2-dihydroxyethanamide represented by and the following formula () A method for producing (S)-7-guanidino-3-hydroxyheptanamide represented by: