JPH0314588A - Physiologically active compound 3822a and b - Google Patents

Abstract

NEW MATERIAL:A compound expressed by the formula and having the following physicochemical properties: Appearance, white powder; m.p., >=160 deg.C (decomp.); Mass spectrography, m/z 509 (M+H)<+>, cation FABMS spectrum measured as a diacetylated substance; Elementary analysis (%) C 49.71, H 4.72, N 6.32, S 14.69, O 24.56; Molecular weight, 424; Specific rotatory power [alpha] =-480.4 deg. (C=0.05, DMSO solution); Solubility, readily soluble in pyridine and DMSO; sparingly soluble in ethyl acetate, methanol, chloroform and acetone and insoluble in n-hexane, petroleum ether, ethyl ether and water; color reaction, positive to sulfuric acid and iodine, and negative to ninhydrin; neutral, etc. USE:An antitumor agent which is also active against mycosis, Gram-negative and -positive bacteria. PREPARATION:Epicoccum purpurascens F-382 (FERM P-10691) is cultured.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel physiologically active substance that has an antitumor effect and also has a growth-inhibiting effect against fungi and Gram-positive and -negative bacteria.

[Prior Art] The physiologically active substance 3822A of the present invention has a molecular weight of 424 and a molecular formula of CtaHteNtS*Og, and 3822B is a new substance with a molecular weight of 402 and a molecular formula of CxaHxtN*SzO*, and has the same physicochemical properties and The existence of physiologically active substances is not known.

[Problems to be Solved by the Invention] An object of the present invention is to provide a novel physiologically active substance that has an antitumor effect and also has a growth-inhibiting effect against fungi and Gram-positive and -negative bacteria. .

[Means for solving the problem] As a result of repeated exploratory research in order to obtain a new substance with anticancer activity from soil-isolated bacteria, the present inventors discovered that a specific microorganism In completing the present invention, we discovered that we can produce a new physiologically active substance that has a growth-inhibitory effect on cultured cancer cells and also has a growth-inhibitory effect on certain fungi and Duram-positive and -negative bacteria. It's arrived.
The strains that produce physiologically active substances 3822A and B represented by structural formula (I) 0 and structural formula (II) of the present invention are strains newly isolated by the present inventors from soil collected in Wako City, Saitama Prefecture. and the name of the microorganism' Ep
icoccum purpurascens F-38
It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P-10691 (Microorganism Deposit Number 1).
The mycological properties of this strain are shown below.

■ Morphology This strain shows good growth on wort agar, potato/glucose agar, Tsuabetsu Kudox agar, Saiichi Tomyl agar, YpSs agar, etc., but conidia formation is limited to potato/glucose agar. It is slightly observed only when cultured on agar medium or LCA medium for a long period of time.

When colonies grown on potato-glucose agar are observed under a microscope, the hyphae have septa and are highly branched, with a diameter of 1 to 5P, and the hyphae surface is smooth to slightly rough. ．． When cultured at room temperature for 2 to 3 weeks or more, conidia are formed at the periphery of the colony. Conidiophores grow in clusters from hyphae or form on conidiophores, 3 to 8 units x 5 to 1
It is 8μ cylindrical to rod-shaped in appearance and produces 0-2 septa.

The surface is smooth, colorless to light brown, and the areuro-type conidia with dark-colored tips are produced singly. Conidia are blackish brown and have a diameter of 9 to 25 Jf.
It is spherical to subspherical in shape, has irregular septa in the vertical and horizontal directions, and is covered with an outer membrane with mesh-like cracks, giving it a wart-like appearance.

(2) Properties on media The following Table 1 shows the results of macroscopic observation when cultured on various media at 25°C for 14 days.

Table 1 ■ Physiological and ecological properties 1) Optimal growth conditions The optimal growth conditions for this strain are YpSs medium with a pH of 5.
~6, and the temperature is 22-26°C. 2) Growth range This strain grows in the Sabouraud medium at a temperature of 6 to 34°C and has a pH of 3 to 9. 3) Distinction between aerobic and anaerobic; from the morphological characteristics and culture characteristics that are more than aerobic, this strain is
Since it became clear that it belongs to the genus Epicoccum, based on the above-mentioned characteristics, Illustrated Encyclopedia of Fungi by Shunichi Udagawa and Keisuke Tsubaki (197B) and B.B. Written by Ellis'DEMA
rIACEOUS HYPHOMYCEI'ESJ (
Comparative studies were conducted with many known bacterial strains reported in 1971). As a result, this strain was found to be Epicoccutn pu
It has become clear that this species exhibits properties closest to those of C. rpurascens.

This strain is called Epicoccum purpurasc.
It was named ens F-3822J.

Next, the production of physiologically active substances 3822A and 3822B is carried out roughly in the same manner as in the production of general fermentation products. That is, Epieocc is grown in a medium containing various nutritional substances.
ua+ purpurascens F-3822
Cultivate the strain under aerobic conditions. A liquid medium is mainly used as the medium, and glufus, blackstrap molasses, starch, etc. can be used alone or in combination as a carbon source. As a nitrogen source, polypebutone, soybean flour, yeast extract, etc. can be used alone or in combination. In addition, 382 physiologically active substances that help the growth of bacterial strains
Organic substances and inorganic salts that promote the production of 2A and B can be added or mixed as necessary. In addition, physiologically active substances 3822A and B that help the growth of bacterial strains
Organic substances and inorganic salts that promote the production of can be added as necessary. As the antifoaming agent, adecanol, silicone, etc. can be used. Suitable culture methods include aerobic culture such as shaking culture and aerated agitation culture, and the culture is carried out at pH 5-7 and 25-30°C for 3-4 days. Physiologically active substances 3822A and 3822B produced in this culture solution can be isolated according to a general method for collecting fermentation products. That is, after completion of the culture, the culture filtrate separated by centrifugation or filtration is adsorbed onto activated carbon, etc., and eluted with ethyl acetate or the like. The eluted fraction of physiologically active substance 3822A is concentrated to form a syrup. Add this syrup again to ethyl acetate,
After dissolving in an organic solvent such as methanol and filtering the precipitate, it was subjected to column chromatography using silica gel, Sephadex LH-20 (trade name; manufactured by Pharmacia).
Physiologically active substances 3822A and 3822B can be purified and isolated by gel filtration using a bioactive substance 3B22A obtained by the above purification method.
The structural formula of the compound was determined as follows by analysis of its elemental analysis value, molecular weight, ultraviolet absorption spectrum, infrared absorption spectrum, 'H-NMR spectrum, and IC-spectrum.

(Structural formula of Physiologically Active Substance 3g22A) 0 (Physical and chemical properties of Physiologically Active Substance 3822A) (1) Appearance: White powder (2> Melting point; 160°C or higher (decomposition)) (3) Mass spectrometry value: Cation FABMS Spectrum: Measured as diacetylated product.

m/z 509 (M+H)” (4) Elemental analysis value; C+sHt. Calculated value as N.S.O. (%); C 50.94, H 4.47.N
6.60, S L5.09.0 22.64. Actual value:
C 49.71, H 4.72. N 6.32. S
14.69,0 24.56 (5) Molecular weight = 424 (6> [ α co: -480.4 @( c
=0.05. (DMSO solution) (7) UV absorption spectrum: As a result of measurement with a methanol solution, λ. =215 nm (ε-9320) (8) IR absorption spectrum: The spectrum measured in the KBr tablet is shown in 1st ryJ.

(9)'H-NMR spectrum: in heavy dimethyl sulfoxide CDMSO-di), 4
The spectrum measured at 0.00 MHZ is shown in FIG.

(10) "C-NMR spectrum: No. 3150 shows the spectrum measured at toOMHz in deuterated dimethyl sulfoxide.

(11> Solubility: Easily soluble in pyridine and DMSO.

Slightly soluble in acetic acid ethyl ester, methanol, chloroform, and acetone.

Insoluble in n-hexane, petroleum ether, ethyl ether, water.

(12) Color reaction: Positive: sulfuric acid, iodine Negative: ninhydrin (13) Distinction between basic, acidic, and neutral: Neutral In addition, physiologically active substance 3822B has its elemental analysis value, molecular weight, ultraviolet spectrum, and infrared spectrum. ,'H-NMR spectrum and 18C-spectrum analysis, its structural formula was determined as follows. (Structural formula of physiologically active substance 3822B) (Physical and chemical properties of physiologically active substance 3822B) (1> Appearance: Yellow powder (2) Melting point = 65-71°C (3> Mass spectrometry value: Cation FABMS spectrum
m/z 4 0 3 (M10H)” (4) Elemental analysis value; C * m H * t N! S!
Calculated value as Os (%); C 59.68. H
5.51. N 6.96. S 15.93,0
11.92. Actual value. C 58.84. H 5.51
．． N 6.66, S L6.53, 0 12.44 (5
) Molecular weight: 402 (6) [(2 Co: -74.6" (C error 0.
05, chloroform solution) (7) UV absorption spectrum:
As a result of measurement with methanol solution, ^2,"," = 2 1 5 nm (
(ε12700), (8) IR absorption spectrum: The spectrum measured in the KBr tablet is shown in Figure 4. (9)'H-NMR spectrum: The spectrum measured at 400 MHz in heavy dimethyl sulfoxide (DMSO-da) is shown in Figure 5. <10)"C-NMR spectrum: The spectrum measured at 100 MHz in deuterated dimethyl sulfoxide is shown in FIG.

(1l) Solubility: Easily soluble in methanol, chloroform, and DMSO. Poorly soluble in chloroform.

n-hexane, insoluble in water.

(12) Color reaction: Positive: Sulfuric acid, iodine (13) Basic, acidic, neutral distinction: Neutral 2 6 5n
m (E3900) [Effect of the invention] Physiologically active substances 3822A and B of the present invention have a growth-inhibitory effect on cultured cancer cells, and also have a growth-inhibitory effect on certain fungi and Gram-positive and -negative bacteria. It is useful as a medicine because of its action.

[Example] The present invention will be specifically described below with reference to Examples and Test Examples.

Example 1 (Isolation and purification of 3822A) (1) Per 100rd, glucose 2g, yeast extract 0
．． 2g, magnesium sulfate 0.05g, polypebutone 0
．． Strain F3822 was inoculated into a sterile liquid medium containing 5 g of potassium phosphate and 0.1 g of potassium phosphate, and cultured with shaking at 28°C for 96 hours. Next, 0.51 of the seed medium was inoculated into a sterile medium 301 having the same composition as the seed medium using two Jar Fermentors with an internal capacity of 501, and the mixture was stirred and cultured at 30°C for 72 hours. 2) After the completion of the culture, filter the culture solution 552 for the two groups, adsorb the obtained filtrate 5 (l) onto 500 g of activated carbon (manufactured by Wako Pure Chemical Industries, Ltd.), wash with 3 l of methanol, and then and ethyl acetate 101. The eluate was concentrated to 11, the precipitate was filtered, and the filtrate was dehydrated over anhydrous sodium sulfate and further concentrated to obtain 4.5 g of a yellow syrup-like substance.

(3) Add 4.5 g of syrupy substance to 15 m of methanol.
Silica gel (trade name: Wakogel C-200, manufactured by Wako Pure Chemical Industries, Ltd.) prepared with n-hexane and dissolved in
was adsorbed onto a 500 mQ column filled with ethyl acetate, and a mixed solvent of n-hexane and ethyl acetate was eluted while gradually increasing the concentration of ethyl acetate (0 to 100%).
The active fractions were combined and concentrated to dryness to give a yellow syrupy substance 4.
I got 00■.

(4) Dissolve 400 ml of the yellow syrup-like substance from the previous section in 10 mQ of methanol, and use a 200 ml column filled with Sephadex LH-20C (trade name, manufactured by Pharmacia) prepared with methanol. Gel filtration was performed. Collect the active fraction and make 150 μg of coarse powder.
I got it.

(5) 150r+g of the crude powder obtained in the previous section was washed with methanol and further washed with benzene to obtain 90cm of white powder.

Example 2 (isolation and purification of 3922B) (1) and (the details are the same as in Example 1).

<3) 4.5g of syrupy substance and 15ml of methanol
Silica gel dissolved in l and prepared with n-hexane (trade name: Wafugel C-200, manufactured by Wako Pure Chemical Industries, Ltd.)
was adsorbed onto a 500 mit column packed with n-
A mixed solvent of hexane-ethyl acetate was eluted while gradually increasing the concentration of ethyl acetate (0 to 100%), and the active fractions were combined and concentrated to dryness to obtain 190 ml of a yellow syrup-like substance. (4) 190 μ of the yellow syrup-like substance from the previous section was dissolved in 10 mQ of ethyl acetate, and 90 ml was filled with silica gel (trade name: Wako Gel C-200, manufactured by Wako Pure Chemical Industries, Ltd.) prepared with n-hexane. The mixture was adsorbed onto a column of 3, and eluted again with a mixed solvent of n-hexane and ethyl acetate, and the active fractions were collected to obtain 55 cm of a yellow syrup-like substance.

(5) Silica gel (trade name: Chromatorex Fuji Davisio) prepared by mixing 55 cm of the yellow syrup-like substance obtained in the previous section with a mixture of methanol:water and 45:55.
n Chemical Ltd. Co., Ltd.), and eluted with a methanol-water mixture while gradually increasing the methanol concentration (50 to 70%). The active fractions were combined, concentrated, the solvent removed, and an equivalent amount of ethyl acetate was added. Extraction was carried out twice, and the acetic acid ethyl ester layer was dehydrated with anhydrous sodium sulfate and then concentrated to obtain 37.7 cm of a yellow oily substance. This oily substance was dissolved in chloroform and concentrated to dryness to obtain 37.7 cm of yellow powder.

Test Example 1 (Proliferation inhibitory effect on various cultured cells) (Sample) Sample 1: 10 μm of the white powder obtained in Example 1 was dissolved in methanol and diluted with sterile physiological saline to the desired concentration. was used.

Specimen 2: 10 μm of the yellow powder obtained in Example 2 was dissolved in methanol and diluted with sterile physiological saline to the desired concentration.

(Test cells) ■ P388 Mouse leukemia ■ L-1210 Mouse leukemia ■ HL-60 Human leukemia ■ KB Human oral cancer (Culture solution used) ■ RPMI-1640 medium (Test method) Various cancer cells were grown using the above culture solution. , 2X10'~1x1
0s/mfL, 2ml in a 6-hole Petri dish with a diameter of 35mm.
Dispense each liter. Then, the sample soPf1, which had been diluted in advance to the desired concentration, was added at the same time as the start of the culture. The test cells were incubated at 37°C in a 5% carbon dioxide incubator for 3 to 30 minutes.
After continuing the culture for 4 days, live cells were measured and ICs were determined from the sample concentration and inhibition rate. The value (concentration for 50% inhibition) was determined. (Results) The results are shown in Table 2. Table 2 Test Example 2 (Proliferation inhibitory effect on various microorganisms) (Sample) Sample 1: The white powder obtained in Example 1 was dissolved in DMSO to the desired concentration, and the solution was soaked into a paper disk with a diameter of 8 m. I used it.

Specimen 2: The yellow powder obtained in Example 2 was dissolved in DMSO to the desired concentration, and the solution was soaked into a paper disk with a diameter of 100 gll before use. (Medium) (1) Heart infusion agar medium (for bacteria) pH
7. 4 (2) Sabouraud agar medium (for fungi) pH 6.0 (test method) 1% of the bacterial cell suspension prepared in 10" cells/TIII was added to the agar medium, and 10 ml was dispensed into a 9 cm Petri dish. A paper disk impregnated with the specimen was placed on agar, incubated at 30°C for 24 to 48 hours, and the diameter of the inhibition circle formed was measured. (Results) The results are shown in Table 3.

[Brief explanation of drawings]

Figure 1 shows the IR spectrum of 3822A measured in KBr tablets. Figure 2 shows the 'H-NMR spectrum of 3822A measured at 400 MHz in deuterated dimethyl sulfoxide (DMSO 1d.). Figure 3 shows the "C-NM" of 3822A measured at 100 MHz in deuterated dimethyl sulfoxide (DMSO 1d).
The R spectrum is shown. FIG. 4 shows the IR spectrum of 3822B measured in KBr tablets. Figure 5 shows the 'H-NMR spectrum of 3822B measured at 400 MHz in deuterated dimethyl sulfoxide (DMSO-d1). FIG. 6 shows the "C-NMR spectrum of 3822B measured at 100 MHz in deuterated dimethyl sulfoxide (DMSO-d.).

Claims (1)

[Claims] 1) Structural formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ 3822A substance represented by 2) Structural formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ 3822B substance represented by.