JPH05230088A - New physiologically active substance and its production - Google Patents

Abstract

PURPOSE:To industrially and advantageously obtain a new physiologically active substance expected to have utility as an anti-cancer medicine, having inhibitory activity against DNA polymerase alpha by culturing a haploid ameba of Physarum polycephalum of true Myxomycetes in a culture. CONSTITUTION:A haploid ameba of Physarum polycephalum MCI-2,526 of true Myxomycetes is inoculated into a liquid medium, cultured at 24 deg.C for one day at a dark place, 2-3 drops of the culture solution is dripped on an agar medium, spread to the whole face, further cultured at 24 deg.C for two days at a dark place, Aerobacter aerogenes MCI-2,517 as a feed of the ameba of Myxomycetes is inoculated to the agar medium and the haploid ameba is cultured at 24 deg.C for four to five days at a dark place. The haploid ameba is collected from the agar medium, subjected to an ultrasonic grinder and centrifuged to collect a supernatant, which is extracted with a chloroform/methanol solvent and the extract is purified by chromatography to give the objective new physiologically active substance of the formula.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel physiologically active substance having a DNA polymerase α inhibitory activity and a cell growth inhibitory activity, and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, various anticancer agents have been put into practical use as pharmaceuticals. However, due to problems such as side effects, new anticancer agents are still required in the medical field.

[0003]

[Means for Solving the Problems] The present inventors have found that the true slime mold Physarum po
Lycephalum ) MCI 2526 was found to be a physiologically active substance having cell growth inhibitory activity from a single-phase amoeba,
The produced substance was isolated and purified.

As a result, it was found that this substance has a DNA polymerase α inhibitory activity, particularly a strong inhibitory activity against cancer cells, and the isolation and purification thereof were performed to obtain a novel physiologically active substance. As a result, the present invention has been completed.

[0005]

That is, the gist of the present invention lies in a novel physiologically active substance represented by the following formula and a method for producing the same.

[0006]

[Chemical 2]

The present invention will be described in detail below. The novel physiologically active substance of the present invention is represented by the above formula. The physiologically active substance is, for example, the strain Mycobacterium lepidoptera
It is produced by a fungus belonging to the genus Mohjikoshi. Examples of such a producing bacterium include, for example, Physalum polycephalum ( P
hysarum polycephalum ) MCI
2526, and such strains include Microorganisms Research Institute No. 115
Deposited as No. 76 (FERM P-11576).

Physalum polycephalum (Physa)
rum polycephalum) MCI 2526
The microbiological properties of (hereinafter, abbreviated as "MCI No. 2526 bacterium") are as follows.

1) Morphological characteristics The multi-phase (2n generation) mutants live on decayed fallen trees and deciduous leaves, and are rotted plants and bacteria that propagate on them. It is a translucent gelatinous organism that has a yellow pigment and spreads flat in a mesh-like shape, ranging in size from several cm to several tens of cm. The thickness is about 0.5 to several mm. This variant is composed of a single cell without a cell wall, and has numerous nuclei with a diameter of about 3 μm inside. As the transformant grows, the nucleus repeats division and other intracellular organelles increase in number, but cytokinesis does not occur. In search of food, it moves to a giant amoeba and continues to grow, but the transformant during growth prefers a moist dark place. When it matures, it moves from under the cracks or fallen leaves of the bark of fallen trees to a dry place where it is exposed to light, and forms fruiting bodies there. The fruiting body is 1 to 1.5 mm high, and several pillow-shaped sporangia are attached to the stalk that is formed by the assembly of protoplasts. The diameter of one sporangium is 0.3-
0.5 mm, the color of the sporangia is dark purple-brown, and it looks whitish when dried due to the adhesion of lime to the sporangial bag (epidermis). The fruiting body is not directly generated on the fallen tree or fallen leaves, but is firmly attached to the fallen tree through a transparent film called a deformed film.

The spores are enclosed in a thick film, have a round shape, a diameter of about 8 μm, and have thorn-like projections on the surface. The color is dark purple. In the sporangia, there are hollow thin tubes called fine capillaries, which maintain the shape of the sporangia and help to disperse the spores. More than 100 fruiting bodies are formed from the deformed body with a diameter of about 10 cm, and each fruiting body produces 1 million spores.

When the spores germinate under conditions of appropriate temperature and humidity, they become single-phase (n-generation) amoeba. This amoeba can live completely independently of the polymorphic variant, undergoes cell division and proliferates. The single-phase amoeba is about 10 μm in diameter and has no dye. It behaves like a general soil amoeba on solids, and cells have no distinct polarity, but in water, they produce two flagella and become distinct polarity. Under conditions unsuitable for survival, a cell wall mainly composed of galactosamine and a protein is formed in the surroundings to form cysts which are dormant cells. Cysts are spherical cells with a diameter of about 5 μm. There is one nucleus with a diameter of about 3 μm inside the single-phase amoeba, but this nucleus is morphologically indistinguishable from that of the deformed body. Mitochondria mimic those of protozoa and have electron-rich nucleoid structures. The size is the same as that of the deformed body. The endoplasmic reticulum has a well-developed rough surface and smooth surface, and the Golgi apparatus exists near the nucleus. (In the deformed body, the endoplasmic reticulum and the Golgi apparatus are not clearly observed.) The centrioles and the basal bodies are structures that can be seen only during the single phase, and the size of the centriole is 180 nm × 470 nm. The single-phase amoeba corresponds to gametes, and when males and females corresponding to each other meet each other, they join to form a multiphase (2n generation). The multiphase zygote repeats fusion and fission, and grows into a deformed body with a yellow pigment. In the present invention, of the life cycles described above, the single-phase amoeba was used as the physiologically active substance-producing bacterium.

2) Physiological Properties A single-phase amoeba will be described.

[Table 1] Optimal growth conditions Optimal pH: 5 to 7 (cultivation on agar medium for 5 days) Optimum temperature: 20 to 25 ° C (cultivation on agar medium for 5 days) Growth range pH range: 4 to 8 (agar medium) Upper, 5 days of culture) Temperature range: Growth at 10 to 30 ° C. (cultured on agar medium for 5 days) At 35 ° C. or higher, cell walls are formed to become dormant cells (cysts).

3) Taxonomical consideration Higher taxonomic position The fungi are categorized mainly by the morphology of fruiting bodies. This microbial cell (1) forms sporangia (endospores), (2) the color of the spores is dark purple-brown, and (3) accumulating granular calcareous crystals in the epidermis of sporangia, ( 4) Since the microcapillaries do not have a protrusion pattern on the surface, Myceto
zoa ; ed. 3. British Museum.
(Nat. Hist.) London (1925), T
he Myxomycetes ; pp. 561, Un
iv. of lowa Press, Iowa (196
9), The Myxomycetes in “Th
e Fungi: An Advanced Treat
is ″ ; vol.4B, pp.39, Acad.Pre
ss, New York (1973), Myxomyc
etes. Flora Neotropica Mon
ogr ; No. 16, pp. 304, New York
Bot. Gard. , New York (1976)
Based on the classification by Myxomycete
It is clear that it belongs to the order Physarales (Physaraceae).

Identification of genus level When the attribution of MCI No. 2526 was examined by the above-mentioned literature, (1) the shape of the sporangia was pillow-like, and (2) the freshly-made sporangia was orange-yellow, As it matures and dries, it becomes whitish-purple brown, and (3) transparent and delicate fine capillaries branch or connect to form a mesh. Further, since it has (4) a pillar axis, it is clear that it belongs to the genus Physarum.

Species Level Identification The Myxomycetes ; pp. 561, Un
iv. of Iowa Press, Iowa (196
9) According to [reference], genus Physar (Physar
um), 84 species have been reported, and at least 10 species have been added thereafter. MCI No. 2526 has multiple sporangia on the stalk of fruiting body,
The seed is Moji dust (Physalum p.
lycephalum).

MCI No. 2526 was identified by Dr. Nobuo Kamiya (N. Kamiya, formerly Professor of Osaka University, Professor of National Institute for Basic Biology) from 1939 to 1942 as Dr. W. It is one of the strains (named PPO strain) obtained from the Seifriz laboratory, and was sold to Ochanomizu University in 1954.
Since 965, Journal of General
Microbiology; 25 , 47, (1961).
Aseptic culturing of the variant and further spore formation and monospore culture according to the method described in (1) above are performed to isolate the single-phase amoeba strains J and F (Botanical Magaz).
ine (Tokyo), 86 , 290, 1973), a single-phase amoeba strain J is used in the present invention.

[0017] The genus Physarum is generally susceptible to changes in its properties, as is the case with other fungi.

[0018] The ATCC includes Physarum Polycephalum.
um ) strains 24112, 24466, 24467, 2
4738, 24739, 26788, 36822, 38
898, 38899, 38900, 38901, 389
02, 42489, 42601, 42602, 4260
4,42605, 42627, 44490, 4449
1,44912,52728 have been deposited, and MCI
Although the 2526 bacterium has a different origin from those of the above, any of those having the ability to produce the physiologically active substance can be used in the method of the present invention. For example, in addition to the above, MCI 2526 bacterium , Or mutants (spontaneous or inducible) derived from this strain, all of which have the ability to produce the physiologically active substance, whether they are zygotes or transgenics. Can be used.

In the present invention, the above-mentioned bacteria are two-membered cultured with bacteria in a medium containing nutrients that can be used by ordinary microorganisms. Glucose, water candy,
Dextrin, sucrose, starch, molasses, animal / vegetable oil, etc. can be used. As a nitrogen source, soybean flour, wheat germ, corn steve liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate,
Urea or the like can be used. In addition, if necessary, it is effective to add sodium, potassium calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other inorganic salts capable of generating ions. In addition, organic and inorganic substances that help the growth of bacteria and promote the production of the physiologically active substance represented by the above formula (hereinafter referred to as the physiologically active substance) can be added appropriately.

The most suitable culture method is a culture method under aerobic conditions, particularly a dark culture on an agar medium. A suitable temperature for culturing is 20 ° C to 25 ° C, but in many cases,
Incubate at around 24 ° C. In addition, before culturing the physiologically active substance-producing bacterium, the microorganism serving as the feed, for example, bacteria of the genus Aerobacterium is inoculated on the agar medium prepared and grown. Specifically, examples of such bacteria belonging to the genus Aerobacterium include Aerobacterium aerogenes ( Aero
vector aerogenes ) MCI 2517
No. bacterium, and such strains are referred to as Micro Inst.
No. (FERM P-11577).

The production of the physiologically active substance of the present invention varies depending on the culture medium, culture conditions and the like, but usually the maximum amount is accumulated in 4 to 5 days. At this point, the culture is stopped and the target substance is isolated and purified from the culture. In the present invention, when collecting the physiologically active substance from the culture, usual separation means utilizing its properties, for example, solvent extraction method, ion exchange resin method, adsorption or partition column chromatography method, gel filtration method, dialysis The extraction method and the precipitation method can be used alone or in combination as appropriate. For example, the physiologically active substance is extracted from the cultured cells with methanol and chloroform / methanol, and concentrated under reduced pressure. The concentrated solution is subjected to ion exchange chromatography, preparative thin layer chromatography,
Purification by combination with gel filtration chromatography, reverse phase high performance liquid chromatography and the like gives the pure physiologically active substance.

[0022]

EXAMPLES The present invention will be more specifically described below with reference to examples.
The present invention will be described below, unless the gist thereof is exceeded.
It is not limited to the examples. Example 1. (1) Culture 5.0 g glucose, 0.5 g yeast extract, bactopep
Tons 5.0 g, potassium dihydrogen phosphate (KH₂PO_Four)
2.3 g, dipotassium hydrogen phosphate (K₂HPO _Four) 1.
5 g, magnesium sulfate 7-hydrate (MgSO 4_Four・ 7H
₂O) 0.5g, agar 30g with 1 liter of distilled water
Prepare, sterilize, and dispense into a petri dish to obtain a 0.7 cm thick agar culture.
The ground was made.

Separately, Aerobacterium aerogenes (Aerobacterium aerobae), which feeds the slime mold Amoeba
Genes) MCI No. 2517 is a liquid medium using a platinum loop from a slant medium (agar removed from the above medium)
The cells were inoculated into the cells and cultured at 24 ° C. in the dark for 1 day. 2 to 3 drops of the seed culture solution were inoculated on the prepared agar medium (a Petri dish with a diameter of 20 cm), spread over one surface, and further cultured at 24 ° C. for 2 days in the dark. On the agar medium on which Aerobactera aerogenes MCI No. 2517 was grown, the growth tip of the single-phase amoeba of the slime mold MCI No. 2526 was scraped off using a platinum loop and inoculated into the petri dish.
It was cultured in the dark at 4 ° C for 4 to 5 days.

A single-phase amoeba grown on the entire surface of a petri dish was collected from the agar medium and aerobic Aerogenes M
In order to remove the CI 2517 bacterium, the suspension was suspended in several tens of volumes of distilled water and centrifuged at 500 to 1000 rpm to remove the supernatant. This operation was repeated several times, and when the supernatant became clear, distilled water was added again to the mixture for 3000 to 35
Centrifugation was performed at 00 rpm to obtain cultured bacterial cells. The cells were suspended in ethanol and stored at -80 ° C.

(2) Purification of culture The cells (50 Petri dishes x 2) obtained in (1) above were
A 20-fold amount of methanol was added in several times, and the mixture was subjected to an ultrasonic disruption device for 30 minutes, and then the supernatant was separated and extracted with a centrifuge. By the same operation, the precipitate was converted into chloroform:
Methanol = 1: 2, chloroform: methanol = 1:
Extraction was carried out in the order of 1, chloroform: methanol = 2: 1, all the obtained extracts were mixed and concentrated to dryness under reduced pressure to obtain an extract.

This was again dissolved in methanol and subjected to an ultrasonic disruption device, and then the supernatant was separated from the precipitate by a centrifuge. By the same operation, chloroform: methanol =
Extraction was carried out in the order of 1: 2, chloroform: methanol = 1: 1, and the obtained extracts were all mixed and concentrated to dryness under reduced pressure to obtain an extract. Then add a little chloroform:
It was dissolved in methanol = 1: 2 and loaded on a DEAE-Sephadex column packed under equilibrium with the same solvent, and ion exchange chromatography was performed with the same solvent. Fractions with DNA polymerase α inhibitory activity were collected and concentrated to dryness under reduced pressure to obtain an extract.

Further, this was dissolved in a small amount of chloroform: methanol = 1: 2 and placed on preparative thin layer chromatography (60 plates) to obtain chloroform: methanol:
The mixture was developed with a mixed solvent of water = 60: 40: 9, and the active fraction (R
f = 0.50 to 0.65) was scraped off, 10 times the amount of methanol was added, the mixture was subjected to an ultrasonic disruption device for 30 minutes, and then the supernatant was separated and extracted with a centrifuge. By the same operation, chloroform: methanol = 1: 2, chloroform: methanol = 1: 1, chloroform: methanol 2: 1 were extracted in this order, and all the obtained extracts were mixed and concentrated to dryness under reduced pressure. And an extract was obtained.

This was again put on preparative thin layer chromatography (14 plates), and chloroform: methanol:
Developing with a mixed solvent of acetic acid: water = 10: 2: 2: 4: 1,
Shaving off the active fraction (Rf = 0.45 to 0.60), 1
After adding 0-fold amount of methanol and subjecting to an ultrasonic homogenizer for 30 minutes, the supernatant was separated and extracted with a centrifuge. By the same operation, chloroform: methanol = 1: 2, chloroform: methanol = 1: 1, chloroform: methanol = 2: 1 were extracted in this order, and all the obtained extracts were mixed and concentrated under reduced pressure to dryness. A solidified extract was obtained.

The obtained extract was dissolved in a small amount of chloroform: methanol = 1: 1, centrifuged to remove the insoluble matter, placed on a Sephadex G-15 column packed with the same solvent, and gel-filtered with the same solvent. I went. Active fractions were collected and concentrated to dryness under reduced pressure to obtain an extract. Further, this was dissolved in methanol: water = 65: 35, centrifuged to remove insoluble matter, and purified by reverse phase high performance liquid chromatography (HPLC Shimadzu 6A series) using the same solvent. The column used was TSK-ODS-80TM, 0.46 × 15.
cm, elution rate was set to 1 ml / min, and temperature was set to 35 ° C. Fraction No. The active substance was eluted in 14 to 17, but the highest activity was No. 15 and about 250 μg active substance was obtained as a result of concentration to dryness under reduced pressure.
The substance is represented by the above formula from the following physical properties.

1) Molecular weight 426 Glycerin was used as a matrix agent and measured by fast atom bombardment ionization mass spectrometry (FAB-MS). At M / z = 427, a protonated ion (M +
H) ⁺ was observed. The measured spectrum is shown in FIG.

2) Molecular formula C ₂₀ H ₃₇ O ₆ PNa

3) Infrared absorption spectrum (FT-IR) The measured spectrum is shown in FIG.

4) Hydrogen nuclear magnetic resonance spectrum A hydrogen nuclear magnetic resonance spectrum of 500.02 MHz measured in a solvent of deuterated chloroform: deuterated methanol = 1: 1 is shown in FIG.
Shown in.

5) J correlation ¹ H- ¹ H two-dimensional nuclear magnetic resonance spectrum (H-H COZY) Using a solvent of deuterated chloroform: deuterated methanol = 3: 1,
The resonance frequency was ¹ H 500.02 MHz, and the phase detection 2 quantum filter method was used for measurement. Observation width is 5 KHz, data points are
For t ₂ axis 512 and t ₁ axis 128, t is used during data processing.
Zero-filing was performed 4 times on only _one axis. The measured spectrum is shown in FIG.

6) Internuclear ( ¹ H- ¹³ C) J-correlation Two-dimensional nuclear magnetic resonance spectrum (HC COZY) HMQC method (by hydrogen nucleus observation) using a solvent of deuterated chloroform: deuterated methanol = 3: 1. High-sensitivity spectrum measurement method)
2J = 3.5 msec (J to 143 Hz), 3.1 mse
The measurement was performed at two points of c (J to 161 Hz). The resonance frequency is ¹ H 500.02 MHz, ¹³ C 125.8 MH
z, observation width is t ₂ ( ¹ H) axis 4 KHz, t ₁ ( ¹³ C) axis 25 KHz, data points are t ₂ axis 512, t ₁ axis 128
At the time of data processing, only the t ₁ axis was zero-folded 4 times. The measured spectra are shown in FIGS.

7) NOE correlation ¹ H- ¹ H two-dimensional nuclear magnetic resonance spectrum (NOESY) Using a solvent of deuterated chloroform: deuterated methanol = 3: 1,
The resonance frequency was measured at ¹ H500.02 MHz.
Data points were taken on the t ₂ axis 512 and the t ₁ axis 128, and at the time of data processing, only the t ₁ axis was zero-filed 4 times.
The measured spectrum is shown in FIG. 7.

8) ³¹ P nuclear magnetic resonance spectrum Using a solvent of deuterated chloroform: deuterated methanol = 3: 1,
The spectrum measured at a resonance frequency of ³¹ P202.5 MHz is shown in FIG. 9) CAD spectrum The physiologically active substance of the present invention was mixed with 5N NaCl at 70 ° C for 5 days.
After hydrolyzing for a period of time, it was extracted with diethyl ether and subjected to negative ion FAB / MS measurement. The parent ion was selected by adjusting the magnetic field intensity from the ions considered to be derived from the carboxylic acid detected at m / z = 267, the electric field voltage was scanned, and the MS / MS analysis was performed. The conditions for FAB / MS measurement and MS / MS analysis are as follows.

[Table 2] FAB / MS measurement conditions Device: VG ZAB ^_ HF mass spectrometer Data processing: VG 11/250 Data processing system Ionization method: FAB Measurement ion: Negative ion Collision atom: Xenon Collision atom acceleration voltage: Approximately 8 kV FAB gun emission current: Approximately 1 mA Matrix: Diethanolamine Ion acceleration voltage: 8.0 kV Scanning speed: 30 sec./decade Scanning interval: 2 sec. Scanning range: m / z = 10 to 1000 MS / MS analysis conditions Scanning part: Electric field scanning Velocity: 10 sec. Scanning range: 8 keV to 500 eV Collision gas: Helium (gas pressure 2 × 1
0 ^-6 mbar) Total number of times: 10 times Other conditions are the same as FAB / MS measurement conditions. The measured spectrum is shown in FIG. Each peak represents CH ₂ , and it was found that the physiologically active substance of the present invention is represented by the above formula.

Test Example 1. [DNA polymerase α inhibitory activity test] As a substrate, activated DNA, deoxyribonucleotide triphosphate (dN
TPs), deoxythymidine triphosphate ( ³ H-dTTP) labeled with tritium, and purified bioactive substance obtained in Example 1 from purified calf thymus-derived DNA polymerase α [Biochemica Biophysica Actor (Bioc
himika et Biophysica Act
a) 950 , 1988 263-273] was measured.

Calf thymus-derived DNA polymerase α5 ng,
Activated DNA 2 μg, dNTPs 20 μM (0.4 μC
i ³ H-dTTP included), 2-mercaptoethanol 2 mM, bovine serum albumin 400 μg / ml, 10% glycerol, magnesium chloride 10 mM, tris hydrochloride 5
0 mM (pH 7.5) was mixed, and the resulting reaction solution was mixed with 50 mM.
μl, and the physiologically active substance is 0, 14, 35, 70,
140, 210, and 280 ng were added, and each was incubated at 37 ° C. for 60 minutes for reaction.

After the reaction, a round filter paper (Whattma
n 3MM), and add 1% with 10% trichloroacetic acid.
After washing for 5 minutes, washing with 5% trichloroacetic acid was repeated 3 times for 15 minutes, further washing with 99% ethanol, and then the filter paper was dried. The radioactivity on the filter paper was analyzed by a liquid scintillation counter (Pac
Kard, Tri-carb3255) was used to quantify the synthesized DNA. The result is shown in Figure 1.
It shows in 0.

Test Example 2 [Various origin DNA polymerase α inhibitory activity test] Tri
s-HCl (pH 7.5) 40 mM, deoxyadenine triphosphate (dATP) 20 μM, deoxycytosine triphosphate (dCTP) 20 μM, deoxyguanine triphosphate (dGTP) 20 μM, deoxythymidine triphosphate (d)
TTP) 10 μM, MgCl ₂ 7 mM, KCl 50 m
M, bovine serum albumin (BAS) 10 μg, 10% glycerose, 2-mercaptoethanol 2 mM, activated DNA 2 μg, DNA polymerase α 0.05 unit, 0.25 μ of the physiologically active substance of the present invention were mixed, and the resulting reaction solution was mixed. 25 μl and below. This reaction solution is 60 at 37 ° C.
The reaction was carried out for a minute (at 25 ° C. for 60 minutes at 25 ° C., containing Physarum Polycephalum- derived DNA polymerase α), and the radioactivity incorporated into the physiologically active substance was measured by the same method as in Test Example 1 above. The results are shown in Table-1.

[Table 3] In Table-1, -PHYLPA shows the case where the physiologically active substance of the present invention was not added, + PHYLPA shows the case where the physiologically active substance of the present invention was added, and "" indicates DE-I and "" indicates DE-II. 3 [Inhibition effect of cancer cell growth in vivo] In MC-D-B-104 (MCDB104) medium, insulin 5 μg / ml, transferrin 5 μg / ml, dexamethasone 10 ng / ml, PDG f (PDG)
F) A serum-free medium supplemented with 125 ng / ml was prepared, and 10 ⁴ cells / cm ^{2 of} human cervical cancer-derived HeLa cells (Hela cells) were inoculated into a petri dish and cultured at 37 ° C. for 4 days in a CO ₂ incubator.

When the physiologically active substance obtained in Example 1 was added to the culture system at a concentration of 0.5-1 μg / ml,
Hela cells were killed almost 100%. On the other hand, under the same conditions, human fetal lung-derived TIII 3 cells (TI
G-3 cells) were inoculated into a petri dish at 10 ⁴ cells / cm ² and 37
The cells were cultured in a CO ₂ incubator at 4 ° C. for 4 days, and the physiologically active substance obtained in Example 1 was added at a concentration of 0.5-1 μg / ml, and the proliferation of TIG-3 cells was suppressed by 100%. In addition, when the physiologically active substance was removed from the culture system, the cell growth ability was restored, and the inhibitory effect was reversible.

[0043]

INDUSTRIAL APPLICABILITY The novel physiologically active substance of the present invention has DNA polymerase α inhibitory activity and is expected to be useful as an anticancer agent.

[Brief description of drawings]

FIG. 1 is a diagram showing a positive ion FAB mass spectrum.

FIG. 2 is a diagram showing an infrared absorption spectrum (FT-IR).

FIG. 3 is a diagram showing the entire region of a hydrogen nuclear magnetic resonance spectrum.

FIG. 4 is a diagram showing a J-correlation hydrogen-hydrogen two-dimensional nuclear magnetic resonance spectrum (H-H COZY).

FIG. 5 shows 1 / 2J = 3.5 using the HMQC method.
between different nuclei measured in msec ( ¹H-¹³C) J correlation secondary
It is a figure which shows a former nuclear magnetic resonance spectrum.

FIG. 6 shows 1 / 2J = 3.1 using the HMQC method.
between different nuclei measured in msec ( ¹H-¹³C) J correlation secondary
It is a figure which shows a nuclear magnetic resonance spectrum.

FIG. 7 is a diagram showing a NOE-correlated hydrogen-hydrogen nuclear magnetic resonance spectrum (NOESY).

FIG. 8 is a diagram showing a phosphorus nuclear magnetic resonance spectrum.

FIG. 9 is a diagram showing a CAD spectrum.

FIG. 10 is a diagram showing the results of a DNA polymerase α inhibitory activity test.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1: 645) 7804-4B

Claims (2)

[Claims] 1. The following formula: A novel physiologically active substance represented by. 2. A single-phase amoeba of the true slime mold Physarum polycephalum is cultured in a medium.
A method for producing the novel physiologically active substance described.