JPH04217681A - New physiologically active substance echigunanines a and b and their production - Google Patents

Abstract

PURPOSE:To obtain the subject substances useful as a reagent for elucidating intracellular signal transmission systems through an antitumor agent or inositol phospholipid metabolic turnover due to its manifestation of a powerful specific inhibitory activity against phosphatidylinositol kinase. CONSTITUTION:A compound expressed by formula I (R is formula II or -CH2-NH2) or its salt, e.g. echiguanine A0 that is a compound in which R is formula II having the following properties. Molecular formula; C10H13O2N7. Molecular weight; 263. Melting point; 215-220 deg.C. Property; basic colorless crystalline powdery substance. Solubility; soluble in water, methanol, etc., and insoluble in acetone, chloroform, etc. The echiguanines A and B are preferably obtained by culturing Streptomyces.sp. MI698-50F1 strain (FERM P-11563) preferably by an aerobic submerged culture method, then purifying and separating the objective substances from the prepared culture filtrate of the culture after the culturing according to a well-known method. The culture temperature is preferably 25-30 deg.C.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to ethiguanine A substance or ethiguanine B substance, which are novel physiologically active substances having phosphatidylinositol kinase inhibitory activity and antitumor activity, and a method for producing these substances. Regarding.

[Prior art and problems to be solved by the invention] Regarding antitumor substances produced by microorganisms, many useful compounds have already been put into practical use, but these are not necessarily satisfactory in terms of efficacy and side effects. Therefore, the provision of new, more effective antitumor substances is awaited.

Phosphatidylinositol turnover is an intracellular information transduction mechanism that is triggered and activated by various hormones, cell growth factors, and oncogenes such as ras and src, and this activation is closely involved in cell proliferation and canceration. [Science, Vol. 231, pp. 407-410 (1986)].

Furthermore, by microinjecting antibodies against phosphatidylinositol 4,5-bisphosphate, a phosphatidylinositol metabolite, into cells, DNA synthesis induced by cell growth factors and hormones was inhibited [Science, Vol. 239, 6
40-642 (1988)], and even for cells that have become cancerous due to oncogenes, phosphatidylinositol 4,5-bisphosphate antibodies can restore the cancerous form to its normal form,
The effect of suppressing proliferation was also observed [``Proceedings of the National Academy of Sciences of the USA''.
of The National Academy
f Sciences OF THE USA)” 82
Vol., pp. 9057-9061 (1988)]. Phosphatidylinositol kinase is one of the enzymes involved in phosphatidylinositol turnover, and is considered essential and rate-limiting for the resynthesis of phosphatidylinositol 4,5-bisphosphate [Experimental Medicine, Vol. 7, No. 9 (special issue) 9
2-97 (1989)].

Therefore, inhibitors of this enzyme are expected to exhibit a new type of anticancer activity by regulating the intracellular signal transduction system via inositol phospholipid turnover, and can also be used as a tool to elucidate cell proliferation mechanisms. is also useful.

Although quercetin and orobol are known as conventional phosphatidylinositol kinase inhibitors, their enzyme inhibitory activity is not specific, making it difficult to fully analyze the physiological role of phosphatidylinositol kinase. be.

An object of the present invention is to provide a substance having stronger and more specific phosphatidylinositol kinase inhibitory activity.

[Means to solve the problem]

The present inventors conducted research to achieve the above objective, and as a result, MI69
We cultured the strain numbered 8-50F1 and found that the resulting culture solution produced a substance with the activity of inhibiting phosphatidylinositol kinase. We succeeded in isolating two substances.

First, these two substances were treated with the physiologically active substance MI69.
8-50F1 A and physiologically active substance MI698-50F1
It was also discovered that these substances specifically exhibit phosphatidylinositol kinase inhibitory activity and have antitumor activity (Japanese Patent Application No. 2-250075).

Moreover, it was discovered that these two substances are new substances that do not correspond to conventionally known substances.

The present inventors have recently discovered these MI698-50F1 A
After determining the chemical structure of the substance and substance B, the general formula (I
), and MI
698-50F1 Substance B with ethiguanine A and M
I698-50F1 Substance A was renamed Etiguanine B.

Therefore, according to the first aspect of the present invention, there are provided physiologically active substances ethiguanine A and ethiguanine B represented by the following general formulas, or acid addition salts thereof.

Examples of acid addition salts of ethiguanine A or B include pharmaceutically acceptable inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, or acetic acid, propionic acid, malic acid, methanesulfonic acid, etc.
There are addition salts with pharmaceutically acceptable organic acids such as.

Ethiguanine A and ethiguanine B have the following structural formulas.

Echiguanine A Echiguanine B Echiguanine A has the following physical and chemical properties.

■Molecular formula and molecular weight: C10H13O2N7 Molecular weight = 263 ■Melting point and properties: Basic colorless crystalline powder substance with a melting point of 215-220℃ ■Mass spectrum (FAB): m/z 264 (M+1) ■Infrared absorption spectrum (KBr tablet method): Shown in Figure 1 of the attached drawings. Its main absorption bands are as follows.

νmax, cm-1 3430-3130br, 235
0, 1700sh, 1640, 1600, 1510, 1
420, 1380, 1360, 1340, 1220, 1
160, 1080, 840, 740, 680 ■Ultraviolet absorption spectrum: Shown in Figure 3 of the attached drawings.

[In H2O]: λmax, nm (ε) 296 (880
0), 252sh (9300), 227 (14740)
, 220 (14740) [in 0.1N HCl]: λmax, nm (ε) 28
1 (7160), 250sh (10420), 217 (
15210) [in 0.1N NaOH]: λmax, n
m(ε) 292 (7160), 256 (9630) ■13C-NMR spectrum (ppm, multiplicity, heavy DM
SO): 168.9s, 163.0s, 160.4s
, 153.0s, 152.6s, 122.9d, 114
．． 1s, 95.8s, 35.6t, 32.9t■1H-
NMR spectrum (ppm, in heavy DMSO): 11.5
1 (1H, s br), 10.41 (1H, t), 9.
12 (4H, s br), 7.24 (1H, s), 6.
62 (2H, s), 3.58 (2H, dd), 2.62
(2H, t) ■Solubility: Soluble in water, methanol, dimethyl sulfoxide, etc., but insoluble in organic solvents such as acetone, ethyl acetate, chloroform, etc.

Ethiguanine B has the following physical and chemical properties.

■Molecular formula and molecular weight: C10H14O2N6 Molecular weight = 250 ■Melting point and properties: Basic colorless powder substance with melting point of >270℃ (decomposition) ■Mass spectrum (FAB): m/z 251 (M+1) ■Infrared absorption spectrum (KBr tablet method): Shown in Figure 2 of the attached drawings. Its main absorption bands are as follows.

νmax, cm-1 3375, 3100, 2320,
1680, 1640, 1580, 1500, 1400,
1350, 1140, 820, 740, 680 ■Ultraviolet absorption spectrum: Shown in Figure 4 of the attached drawings.

[In H2O]: λmax, nm (ε) 296 (705
0), 254sh (7400), 228sh (1140
0), 220 (12000) [in 0.1N HCl]: λmax, nm (ε) 28
0 (5900), 250sh (8650), 217 (1
2600) [in 0.1N NaOH]: λmax, nm
(ε) 291 (5750), 255 (7800), 2
28sh (12300) ■13C-NMR spectrum (
ppm, multiplicity, heavy DMSO): 163.0s, 16
0.4s, 152.8s, 152.6s, 122.7s
, 114.3s, 95.8s, 37.1t, 35.4t
, 28.4t 1H-NMR spectrum (ppm, heavy D
(in MSO): 10.22 (1H, t), 8.46 (4H
, s br), 7.23 (1H, s), 6.39 (2H
, s), 3.33 (2H, dd), 2.82 (2H, t
), 1.77 (2H, m) ■ Solubility: Soluble in water, methanol, dimethyl sulfoxide, etc., but insoluble in organic solvents such as acetone, ethyl acetate, chloroform, etc.

Specifically, the physiologically active substances, ethiguanine A and B substances according to the present invention can be obtained in a culture solution by culturing a strain of actinomycetes isolated from soil as described above and assigned a strain number of MI698-50F1. The above bacterial strain was confirmed to be a microorganism belonging to the genus Streptomyces, as described below. The physiologically active substance ethiguanine A according to the present invention can be produced by culturing an ethiguanine A substance producing bacterium belonging to the genus Streptomyces, and the physiologically active substance ethiguanine B can be produced by culturing an ethiguanine B substance producing bacterium belonging to the genus Streptomyces. It is recognized that it can be produced by culturing bacteria.

Therefore, according to the second invention, a bacterium that produces the physiologically active substance ethiguanine A belonging to the genus Streptomyces is cultured,
The physiologically active substance ethiguanine A is collected from the culture, or a physiologically active substance ethiguanine B is collected from the culture by culturing a bacterium that produces the physiologically active substance ethiguanine B belonging to the genus Streptomyces. A method for producing the active substance ethiguanine A or the physiologically active substance ethiguanine B is provided.

One example of a bacterium producing the physiologically active substance ethiguanine A, and one example of a bacterium producing the physiologically active substance ethiguanine B include
MI698-50F, an actinomycete isolated from the soil of Baeksaiji, Aichi District, Shiga Prefecture, was discovered at the Institute of Microbial Chemistry in February 2017.
The above-mentioned strain is numbered 1. However, when this strain having the strain number MI698-50F1 is cultured, the physiologically active substances ethiguanine A and B are produced in parallel in the culture.

Next, the mycological properties of this MI698-50F1 strain will be described.

1. Morphology Under a microscope, strain MI698-50F1 extends spiral-shaped aerial hyphae from branched basal hyphae, and no whorled branches are observed. A mature spore chain is a chain of 50 or more spores, and the size of the spores is 0.5-0.8 x 0.9-
It is about 1.0 micron. The surface of the spore has a thorn-like structure.

2. Regarding the description of growth state colors in various media, the standards shown in brackets are based on the Color Harmony Manual of Container Corporation of America.
color hor of tion of america
mony manual) was used.

(1) Sucrose/nitrate agar medium (cultured at 27°C) Colorless growth with bright brown ash [3dc, Natural] ~
It grows on aerial mycelia of light gray [2fe, Covert Gray].

No soluble dyes are observed.

(2) Glucose-asparagine agar medium (cultured at 27°C) A small amount of white aerial mycelium grows on colorless to pale yellow growth. No soluble dyes are observed.

(3) Glycerin-asparagine agar medium (ISP-medium 5, cultured at 27°C) White to light gray on pale yellow growth [2fe, Cover
Aerial mycelia of tGray to light brown gray [3ge, Beige] are attached, and no soluble pigment is observed.

(4) Starch inorganic salt agar medium (ISP-medium 4, 27
℃ culture) Colorless to pale yellow growth with bright gray [2fe, Cov.
ert Gray~3fe, Silver Gray]
Aerial mycelium is attached, and no soluble pigment is observed.

(5) Tyrosine agar medium (ISP-medium 7, cultured at 27°C) On the pale yellow growth, bright brown ash [3ge, Beige
~3fe, Silver Gray] ~Bright gray [2f
e, Covert Gray], and no soluble pigment is observed.

(6) Nutrient agar medium (cultured at 27°C)
A small amount of white aerial mycelium grows on it, and no soluble pigment is observed.

(7) Yeast. Malt agar medium (ISP-medium 2, 27
℃ culture) Light gray growth on light colored growth [2ih, Dk Cover
tGray~2fe, Covert Gray], and no soluble pigment was observed.

(8) Oatmeal agar medium (ISP-medium 3, 27℃
Culture) Light yellow growth with bright ash [2fe, Cove
rtGray] ~ Bright brown gray [3fe, Silver
Gray to 3ig, Beige Brown] aerial mycelia are attached, and no soluble pigment is observed.

(9) Glycerin/nitrate agar medium (cultured at 27°C) White aerial mycelia are grown on the pale yellow growth, and no soluble pigment is observed.

(10) Starch agar medium (cultured at 27° C.) A slight amount of bright brown gray [3fe, Silver Gray] aerial mycelium was observed on the colorless growth, and no soluble pigment was observed.

(11) Malate lime agar medium (cultured at 27°C) White aerial mycelia are grown on the colorless growth, and no soluble pigment is observed.

(12) Cellulose (27°C medium) The cells were observed for 3 weeks after culturing, but no growth was observed.

(13) Gelatin puncture culture In simple gelatin medium (cultured at 20°C), a small amount of white aerial mycelium grows on colorless to pale yellow growth, and no soluble pigment is observed. Glucose-peptone-gelatin medium (
When cultured at 27°C, white aerial mycelium slightly grows on the growth of pale yellow to pale yellow dailies, and the soluble pigment has a faint orange flavor.

(14) Skimmed milk (cultured at 37°C) Growth of light yellow to pale yellow daisies is observed, but no aerial mycelium is observed and no soluble pigments are observed.

3. Physiological properties (1) Growth temperature range Yeast starch agar medium (soluble starch 1.0%
, East. Extract 0.2%, string agar 3.0%, pH
7.0) at 14℃, 20℃, 24℃, 27℃, 3
As a result of testing at each temperature of 0℃, 37℃, and 50℃, 14℃
, 20°C, 24°C, 27°C, and 30°C, but the growth was extremely poor at 37°C, and it did not grow at 50°C.

The optimum temperature seems to be around 27°C to 30°C.

(2) Liquefaction of gelatin (15% simple gelatin medium, 20%
℃ culture; glucose peptone gelatin medium, 27℃
Culture) In 15% simple gelatin medium, liquefaction occurs from around 10 days after culture, and its effect is moderate; in glucose peptone gelatin medium, liquefaction occurs from around 20 days after culture, and its effect is moderate. is the weaker one.

(3) Hydrolysis of starch (starch/inorganic salt agar medium and starch agar medium, both cultured at 27°C) In both media, water decomposition was observed on the second day after culture, and the effect was stronger. .

(4) Coagulation and peptonization of skimmed milk (skimmed milk, 37℃
Culture) After culturing without coagulation, peptonization begins around the 10th day, and its effect is weak.

(5) Production of melanin-like pigments (tryptone, yeast,
Broth, ISP-medium 1; peptone yeast iron agar medium, ISP-medium 6; tyrosine agar medium, ISP-medium 7; all cultured at 27° C.) All the media are negative.

(6) Utilization of carbon sources (Pridham-Gotlieb agar medium, ISP-medium 9, 27°C culture) Grows using glucose, fructose, inositol, raffinose, D-mannitol, lactose,
D-xylose, sucrose, and rhamnose are not used. It is not clear whether L-arabinose is used or not.

(7) Dissolution of malic acid lime (malic acid stone pond agar, 27℃
Cultivation) After culturing, it begins to grow from around the 5th day and the surrounding malate lime is dissolved. The effect is moderate to strong.

(8) Nitrate reduction reaction (peptone water containing 0.1% potassium nitrate, ISP-medium 8, cultured at 27°C) was negative.

(9) Decomposition of cellulose (synthetic solution added with filter paper pieces, cultured at 27°C) No growth.

To summarize the above properties, the MI698-50F1 strain is:
It has the following characteristics in terms of morphology. That is, no sporangia are observed, the aerial mycelia have a spiral shape, and no whorled branches are observed.

In addition, the surface of the spore is thorn-like. In various media, the growth is colorless to pale yellow or pale yellow in color, and the aerial mycelia are light gray to light brown gray, with no soluble pigments observed. Production of melanin-like pigments was negative in both media.

Starch hydrolyzability is strong, and protein decomposition power is moderate to weak.

The 2,6-diaminopimelic acid contained in the bacterial cells of this strain is of the LL type, and when considered with the above properties, the MI698-50F1 strain is Streptomyces (Streptomyces).
It is clear that it belongs to the genus (Eptomyces).

Based on these properties, a search for known bacterial species closely related to the MI698-50F1 strain resulted in the following three species. That is, Streptomyces toyocaensis (Strepto
myces toyocaensis, Reference 1 “Int.
ernationalJournal of Syst
matic Bacteriology” Volume 18, 1
74 pages, 1968; and document 2: Special Publication No. 32-304
9, 1957), Streptomyces fasciculatus
atus, literature “International Jour
nal of Systematic Bacteri
18, p. 103, 1968), and Streptomyces. natalensis (Streptomyc
es natalensis, literature “Internat
ional Journal of Systematic Bac
22, p. 323, 1972). We are currently conducting a comparative study between these species and the MI698-50F1 strain. For that matter, MI698-50F1
Streptomyces strain. SP (Streptomy)
ces sp. ) MI698-50F1.

An application was made for depositing strain MI698-50F1 to the Institute of Microbial Technology, Agency of Industrial Science and Technology, and the deposit was received on June 28, 1990, as Microtechnology Research Institute No. 11563.

In the second method of the present invention, the physiologically active substance ethiguanine A-producing bacteria or the physiologically active substance ethiguanine B
Cultivation of the producing bacteria can be carried out using a commonly used culture medium and culture method for culturing known bacteria belonging to the genus Streptomyces.

That is, the medium used in the method of the present invention may contain any nutrient source that can be utilized by the bacteria producing the physiologically active substance of the present invention. For example, glycerin, glucose, maltose, sucrose, dextrin, starch, fats and oils, etc. can be used as carbon sources. Nitrogen sources include organic substances such as soybean flour, cottonseed oil, meat extract, peptone, dried yeast, yeast extract, and corn steep liquor, as well as ammonium nitrate, sodium nitrate, calcium carbonate,
Inorganic salts such as ammonium chloride can be used.

Inorganic salts such as common salt, potassium chloride, phosphates, and heavy metal salts can also be added as necessary. In order to suppress foaming during fermentation, a suitable antifoaming agent,
For example, silicone, soybean oil, etc. can also be added dropwise.

The most suitable culture method is an aerobic liquid deep culture method, similar to the commonly used method for producing antibiotics. The appropriate culture temperature is 20 to 30°C, but 25 to 30°C is suitable.
°C is preferred. In this method, the production amount of the physiologically active substance of the present invention reaches its maximum in 4 to 8 days for both osmotic culture and aerated agitation culture.

A culture in which ethiguanine A and B, the two physiologically active substances of the present invention produced in this manner are accumulated, is obtained.

In the culture, the physiologically active substances of the present invention are mainly present in the culture supernatant.

Any suitable method can be used to isolate the physiologically active substance of the present invention from this culture. One method is to adsorb the culture filtrate on a suitable adsorbent, such as porous adsorption resin Diaion HP-20 or a suitable ion exchange resin, and elute it with a suitable solvent. Physiologically active substances can be obtained. Furthermore, these two physiologically active substances can be independently separated using a suitable countercurrent distribution method.

Next, physiologically active substances ethiguanine A and B according to the present invention
Test Example 1 demonstrates that the compound has phosphatidylinositol kinase inhibitory activity, and Test Example 2 demonstrates that it has an inhibitory effect on the growth of tumor cells, and thus has antitumor activity.

Test Example 1 Phosphatidylinositol kinase inhibitory activity of physiologically active substances ethiguanine A and B.

Measurement of phosphatidylinositol kinase inhibitory activity was carried out as follows [``Proceedings of the National Academy of Sciences of the USA''.
National Academy Science
s OF THE USA), vol. 81, 2117-21
21 pages (1984)]. The enzyme phosphatidylinositol kinase was prepared by crudely purifying the membrane fraction of A431 cells, which are human squamous cell carcinoma cells [The Biochemical Journal
Cal Journal)” Volume 168, 187-194
Page (1977)].

Prepared enzyme, substrate phosphatidylinositol,
[γ-32P)ATP and the physiologically active substance at 20 m
The reaction was carried out in M Hepes buffer (pH 7.2) at 20°C for 20 minutes. The reaction was stopped by adding a chloroform-methanol-1N hydrochloric acid mixture, the reaction solution was centrifuged, and the lower chloroform layer was adsorbed onto a silica gel minicolumn (1 ml). Then, the phosphorylated radioactive phosphatidylinositol-phosphoric acid was eluted with a chloroform-methanol-4N ammonia water mixture.
Phosphatidylinositol kinase inhibitory activity was determined by measuring its radioactivity.

As a result, the 50% inhibitory concentration of ethiguanine A and B for phosphatidylinositol kinase was 47 ng each.
/ml and 96ng/ml.

Test Example 2 Cancer cell proliferation inhibitory activity of physiologically active substances ethiguanine A and B.

5 x 104 mouse leukemia L1210 cells/ml
RPMI 1640 containing 10% calf serum to
The cells were suspended in a medium and cultured at 37° C. for 2 days with various concentrations of the physiologically active substance of the present invention, and then the cell proliferation inhibitory activity was measured.

As a result, ethiguanine A inhibited the proliferation of L1210 cells by 60% at a concentration of 100 μg/ml. Etiguanine B inhibited growth by 20% at a concentration of 100 μg/ml, and ethiguanine A was found to have a 50% growth inhibitory concentration of 87.5 μg/ml.

Next, according to Examples, the physiologically active substance Ethiguanine A of the present invention
The production of and B will be explained in more detail.

Example 1 Production of physiologically active substances ethiguanine A and B Glycerol 2.0%, dextrin 2.0%, soy peptone 1.0%, yeast extract 0.3%, (NH4)2S
Dispense 110 ml of a liquid medium containing O4, 0.2%, CaCO2, 0.2%, and 1 drop of silicone oil into Erlenmeyer flasks, sterilize it at 120°C for 20 minutes by a conventional method, and culture it on an agar slant. Streptomyces sp. M
I698-50F1 (Feikoken Kyoiku No. 11563) was inoculated and cultured with rotary shaking (180 revolutions per minute) at 28°C for 96 hours to obtain a seed culture.

Add 2 ml of this seed culture solution to an Erlenmeyer flask and add 2.0% glycerol, 1.5% soybean flour, and 0.1 K2HPO4.
%, CoCl2.6H2O 0.0005%, and 1 drop of silicone oil in 110 ml of a liquid medium (adjusted to pH 6.2 with KH2PO4), and cultured with rotary shaking at 28° C. and 180 rpm for 8 days.

36 liters of this culture solution was filtered to obtain 27 liters of culture supernatant. This culture supernatant was adsorbed onto a 3 liter Diaion HP-20 column, the column was washed with 9 liters of water, and then eluted with 9 liters of 50% methanol water. After concentrating this eluate under reduced pressure and drying it, 1 liter of water was added and silanizate (silanizate) was added.
isiert Art. 7719 manufactured by Merck & Co.) was suspended in water and adsorbed on a packed column (300 ml), washed with water and eluted with 1 liter of 30% methanol water.

After concentrating this elution fraction, CM-Sephadex C-25
Column (200ml) filled with (Na type) suspended in water
), and concentration gradient elution from 0.4M saline to 1.0M saline was performed. Next, for desalination, it was adsorbed on Diaion CHP-20 (40 ml) and 100 ml of water was added.
After washing with 0.005N hydrochloric acid-50% methanol water 1
It eluted at 00ml.

This eluate was neutralized by adding an appropriate amount of Amberlite IR-45 (OH type), the ion exchange resin was removed by filtration, and the two physiologically active substances of the present invention were concentrated to dryness under reduced pressure. A containing fraction (73 mg) was obtained. Furthermore, this fraction was subjected to centrifugal partition chromatography.
Separation and purification were carried out using chromatography (CPC Mikisha Co., Ltd.). Ethyl acetate-1 as a solvent system
- Butanol-water mixed solvent (mixing ratio 4:7:10) The lower layer was used as the stationary phase and the upper layer was used as the mobile phase to isolate and purify 6.7 mg of ethiguanine B substance and 20 mg of ethiguanine A substance.
8.6 mg each was obtained.

〔Effect of the invention〕

The ethiguanine A and ethiguanine B substances of the present invention very strongly inhibit phosphatidylinositol kinase activity derived from human cancer cells, and are excellent new substances expected to be a new type of antitumor substance related to the cell signal transduction system. . Furthermore, it may become a more important reagent in elucidating the intracellular signal transduction system mediated by phosphatidylinositol turnover.

[Brief explanation of the drawing]

Figures 1 and 2 show the infrared absorption spectra of the physiologically active substances ethiguanine A and B of the present invention, respectively, and Figures 3 and 4 show the ultraviolet absorption spectra of the physiologically active substances ethiguanine A and B of the present invention, respectively. The spectrum is shown. In Figure 4 without Figure 3, the solid line indicates the spectrum measured in a neutral aqueous solution, the large broken line indicates the spectrum measured in a basic aqueous solution, and the broken line indicates the spectrum measured in a basic aqueous solution. A spectrum measured in an acidic aqueous solution is shown.

Claims (4)

[Claims] 1. Physiologically active substances ethiguanine A and ethiguanine B represented by the following general formula (I), or acid addition salts thereof. 2. The compound according to claim 1, which is ethiguanine A as a compound. Claim 3: In general formula (I), R is a group -CH2-N
Claim 1: The compound which is H2 is ethiguanine B.
Compounds described in. 4. The physiologically active substance ethiguanine A-producing bacterium according to claim 1 belonging to the genus Streptomyces, or claim 1
Cultivating the bacteria producing the physiologically active substance ethiguanine B,
A method for producing a physiologically active substance ethiguanine A or ethiguanine B, which comprises collecting the physiologically active substance ethiguanine A or the physiologically active substance ethiguanine B from the culture.