JPS6219156B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a new anti-cancer antibiotic, NCS-C. When Streptomyces carcinostaticus variant F41 Kuroya is cultured, the polymeric anticarcinogenic substance neocarcinostatin (Special Publication No. 42-21752),
It is already known that N-1 fraction, N-2 fraction (Special Publication No. 47-680) and Ma fraction (Special Publication No. 13516-1982) can be obtained. The present inventors further investigated the culture and found that
We have discovered the existence of a novel anticancer antibiotic, NCS-C, which has different physical properties from neocarzinostatin, N-1 fraction, N-2 fraction, and Ma fraction. Therefore, the present invention belongs to the genus Streptomyces.
The present invention provides a method for producing the anticancer antibiotic NCS-C, which is characterized by culturing NCS-C substance-producing bacteria and extracting the culture solution under acidic conditions with a polar organic solvent that is immiscible with water. According to the present invention, the NCS-C material is produced as follows. The bacterial species used in the production of the NCS-C substance of the present invention are neocarcinostatin-producing bacteria belonging to the genus Streptomyces, including Streptomyces carcinostaticus variant F41 Kuroya described in the above patent specification. The mutant bacteria and NCS-C
All bacterial species belonging to the genus Streptomyces that produce substances are used. Streptomyces carcinostaticus variant F41 Kuroya has already been deposited in the National Institute of Microbial Technology, Agency of Industrial Science and Technology as accession number 2257, and also in the American Type Culture Collection.
It has been deposited as ATCC.No.15945. Streptomyces carcinostaticus variant when producing the NCS-C substance of the present invention
As for the cultivation method of F41 Kuroya, the method described in Japanese Patent Publication No. 42-21752 and Japanese Patent Publication No. 54-13516 is applied. The NCS-C substance can be collected from the above culture solution by the following method. First, when a culture solution is separated into bacterial cells and a culture solution by a known method such as filtration or centrifugation, the NCS-C substance is mainly contained in the culture solution. If the pH of this culture solution is adjusted to 1 to 4 with a mineral acid such as hydrochloric acid or sulfuric acid, or an organic acid such as acetic acid or formic acid, and then extracted with an organic solvent that is immiscible with water, such as butanol or chloroform, most of the NCS
-C substances migrate to the organic layer. This organic layer is then concentrated and subjected to adsorption chromatography using silica gel, _alumina , Amberlite
-20, NCS-C can be obtained by purification using gel permeation chromatography such as TSK gel.
Substances are obtained. In this process, the NCS-C substance is obtained as an acid salt, and the hydrochloride exhibits the following physical properties. (1) Physicochemical properties (a) Properties Pale yellow to brown powder (b) Decomposition point 125℃ (c) Specific rotation [α] ²⁰ _D = -171° (c = 1.4 × 10 ^-3 , methanol) . (d) Molecular weight 686.5-688.5 (vapor pressure method) 695 (mass spectrometry) Figure 3 (e) Nuclear magnetic resonance spectrum ¹ H-NMR spectrum Figure 4 ¹³ C-NMR spectrum Figure 5 (f) Ultraviolet absorption spectrum ( Figure 1) 240, 265, 274, 290, 305, 330nm shoulder (G) Infrared absorption spectrum (Figure 2) 3400, 1780, 1610, 1400, 1190,
It has absorption at 1080 and 1010 cm ^-1 . (H) Color reaction Positive for permanganic acid reaction and diazo coupling reaction, negative for xanthoprotein reaction, Ehrlich reaction, ninhydrin reaction, orcinol reaction, and Moritsch reaction (li) Solubility Easily reacted with methanol, ethanol, propanol, butanol Soluble in acetone, slightly soluble in chloroform and ethyl acetate, slightly soluble in water, ether and benzene. (n) Stability

【table】

【table】

[Table] (2) Acute toxicity Mouse intravenous LD ₅₀ = 1.8 mg/Kg Mouse peritoneal LD ₅₀ = 10 mg/Kg or more (3) Biological properties (i) Antitumor effect

[Table] Hela S3 cells The NCS-C substance does not show antitumor effects at a concentration of 1.0 mg/ml, but the mixture of preneocarzinostatin and NCS-C substance at a ratio of 10:1 is 0.1 mg/ml. It showed antitumor effect at a concentration of ml. (ii) Antimicrobial action

[Table] As described above, the NCS-C substance of the present invention has anticancer and antimicrobial effects by itself, but its effects are significantly increased when used in combination with preneocarzinostatin. As mentioned above, the physicochemical and biological properties of the NCS-C substance of the present invention are neocarzinostatin, N-1 fraction, N-2 produced from Streptomyces carcinostaticus variant F41 Croya.
It is completely different from the carcinostatin complex (Special Publication No. 5400 of 1983) produced from the fraction, Ma fraction, and the closely related strain Streptomyces carcinostaticus, as well as other anticancer antibiotics. Therefore, the NCS-C substance of the present invention is recognized as a new substance. Next, an example will be given and explained. Example 1 Starch 2.0%, soybean flour 2.0%, dry yeast 0.5%, sodium chloride 0.25%, calcium carbonate 0.35%, manganese chloride 0.005%, copper sulfate 0.005%, zinc sulfate 0.005
Medium with a composition of 200% (adjusted to PH6.6)
The culture tank was adjusted to 400 °C and heat sterilized at 120°C for 30 minutes. Add seed mother liquor 3 of Streptomyces carcinostaticus variant F41 Kuroya, which had been cultured for 24 hours in a medium with the same composition,
Aerated agitation culture at 27℃ (aeration rate 300/min, number of agitation
180 rotations/min) for 50 hours to obtain 180 culture fluids. This culture solution was filtered through a filter press, and after adjusting the pH to 2.0 with concentrated hydrochloric acid, 100% n-butanol was added, stirred at 180 rpm for 2 hours, and then left for 24 hours. Next, the butanol layer that was sufficiently separated from the aqueous layer was separated and concentrated using a flash evaporator at 50°C to obtain 1200 ml of a concentrated solution of the NCS-C substance. Example 2 The concentrated solution obtained in Example 1 was adsorbed on a column filled with silica gel 5 equilibrated with chloroform in advance, and eluted with methanol:chloroform (1:1) to obtain 400 ml of active fraction. This was concentrated to dryness under reduced pressure to obtain 2.1 g of a crude powder of NCS-C substance. Example 3 2.1 g of the coarse powder obtained in Example 2 was added to 50 ml of methanol.
Dissolved in 1.0N hydrochloric acid: methanol (1:
The mixture was passed through a column packed with Sephadex LH-20 1 equilibrated with 9), and chromatography was performed using 1.0N hydrochloric acid:methanol (1:9) as an eluent to obtain 90 ml of an active fraction. This was concentrated to dryness to obtain 126 mg of NCS-C substance powder.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of the anticancer antibiotic NCS-C of the present invention, Figure 2 shows the infrared absorption spectrum of the same substance, Figure 3 shows the FAB mass spectrum of the same substance, and Figure 4 shows the same substance. Figure 5 shows the ¹ H-NMR spectrum of the substance, and Figure 5 shows ^{the 13} C-NMR spectrum of the same substance.

Claims (1)

[Claims] 1. A method for producing the anticancer antibiotic NCS-C, which comprises culturing NCS-C substance-producing bacteria belonging to the genus Streptomyces and extracting the culture solution under acidic conditions with a polar organic solvent that is immiscible with water.