JPH04224559A - Arterialization-inhibiting substance fr-901448 and fr-901449 - Google Patents

Abstract

PURPOSE:To obtain an arterialization-inhibiting agent such as an antiulcer agent containing FR-901448 substance and FR-901449 (new) substance obtained by culturing a microorganism belonging to the genus Chaetasbolisia as an active ingredient. CONSTITUTION:An arterialization-inhibiting agent containing a compound expressed by the formula (X is H or Cl) as an active ingredient is produced. The compound expressed by the formula is obtained by culturing FR-901448 substance and FR-901449 substance-producing mold belonging to the genus Chaetasbolisia and the FR-901449 substance therein is a new substance.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to the FR-901448 substance and the FR-901449 substance (WF16775, respectively).
-A1 or A2) as an active ingredient.

The present invention also relates to a new method for producing these materials. Among these substances, FR-9
Substance 01449 is a new substance that has not been published in any literature.

[0003] These substances have an excellent angiogenesis inhibiting effect and are useful as various medicines including anticancer agents.

0004

BACKGROUND OF THE INVENTION Suppressing or inhibiting the formation of new blood vessels in a living body has very important significance from a physiological standpoint. For example, if we inhibit the formation of blood vessels in cancer cells or their surrounding cells, oxygen and various nutrients will no longer be delivered to these (those) cells, so they will eventually die, and indirectly, The cancer cells will be subdued. Therefore, excellent angiogenesis inhibitors can be highly expected as excellent anticancer agents.

The substance according to the present invention was discovered in the process of screening for angiogenesis inhibitors using inhibition of lumen formation in vascular endothelial cells as an indicator. It has not been previously known that pyridine derivatives have an angiogenesis inhibitory effect, and this is novel.

As described above, the present invention is new from the viewpoint of action, but the present invention is also novel from the viewpoint of substance, and the FR-901449 substance is a new compound. Furthermore, the FR-901448 substance is a known compound (T
he Journal of Antibiotics
, VOL. XLIII, No. 9, 1064-1068
(1990)), although its antifungal effect has been clarified, nothing has been disclosed about its angiogenesis inhibitory effect.

[0007] The present invention also provides these FR-90144
The present invention also relates to a method for producing the substances 8 and FR-901449 by culturing Ketoasborisia bacteria, but such a method is also completely unknown and new. That is, since the FR-901449 substance is a new substance, the method of the present invention for producing it is naturally new, and the FR-901448 substance is a Peni
cilium sp. It is known that it is an antifungal antibiotic produced by FO-125 (cited above)
, it is completely unknown that it is produced by Ketoasborisia as in the present invention.

[0008]

OBJECTS OF THE INVENTION The present invention was made for the purpose of developing a highly safe and extremely effective angiogenesis inhibitor or antitumor agent.

[0009]

[Means for Solving the Problems] The present invention has been made to achieve the above-mentioned objects.

[0010] Therefore, the present inventors focused on natural products from the viewpoint of safety, and came to focus on fermentation products of microorganisms.
As a result of searching for various microorganisms, we found filamentous fungus No. 1 belonging to the suborder Phialopycnidia, which was newly isolated from soil collected in the mountains of Hakusan, Ishikawa Prefecture. It was discovered that the 16775 strain accumulates the target substance within its bacterial body. Furthermore, as a result of detailed research into the physical and chemical properties of this substance, they discovered that it consists of two types of substances, and these were combined into WF16775-
They named them A1 and WF16775-A2, and conducted further studies and completed the present invention.

The WF16775-A2 substance according to the present invention has the physical and chemical properties shown in Table 1 below.

0012

[Table 1]

[0013] A substance having such physical and chemical properties has not been previously known, and it has been confirmed that this substance is a new substance. As a result of trying to determine the chemical structure of this new substance, WF16775-A2, we succeeded, and the structural formula of this WF16775-A2 substance was shown below:
was determined as in formula II.

[0014]

[C4]

[0015] Furthermore, in the method of the present invention, the above-mentioned WF
Along with the 16775-A2 substance, the WF16775-A1 substance was also produced, and as a result of determining its structure, it was found to be a compound represented by the following formula III.

[0016]

[C5]

W having the structural formula represented by formula III
As mentioned above, the F16775-A1 substance is a substance already known as a fermentation product of Penicillium genus bacteria.

[0018] In this way, along with the structure determination of the WF16775-A1 and WF16775-A2 substances, these substances were reclassified as FR-901448 and FR-901449, respectively.
This name may also be used hereafter.

The WF16775 substance (general name of WF16775-A1 and A2) used in the present invention has a structure shown by the formula I shown in the following chemical formula 6, and is obtained from soil collected by the present inventors in Hakusan, Ishikawa Prefecture. Filamentous fungus No. newly isolated from Produced by 16775.

[0020]

[C6]

[0021] Filamentous fungus No. Strain 16775 was recognized as belonging to the genus Chaetoasvolisia, and was identified as Chaetoasborisia erysihoides No. 16775 (Chaetasbol
isia erysiphoides No. 16
775).

[0022] A freeze-dried sample of this strain was obtained from the Institute of Microbial Technology, Agency of Industrial Science and Technology (1 Higashi, Tsukuba City, Ibaraki Prefecture, 305 Japan).
-1-3) under the number FERM P-11873. (Date of deposit = November 29, 1990)

002
3. It should be understood that the production of WF16775 material is not limited to the use of the particular microorganisms described herein, which are mentioned for illustrative purposes only. This invention provides X-ray irradiation, ultraviolet irradiation,
N-methyl-N'-nitro N-nitrosoguanidine,
This includes the use of all mutant strains that can produce the WF16775 substance, including artificial mutant strains and natural mutant strains that can be obtained by mutation treatment with 2-aminopurine, etc.

[0024] This strain (strain No. 16775) spreads in a somewhat suppressed manner on various media and forms dark brown colonies. No. Strain 16775 formed conidial anamorphs on various media. The conidiation mode was phialo type. Also, no teleomorphs were formed. From these morphological characteristics, No. Strain 16775 belongs to the suborder Phia.
lopycnidiineae Sutton 1
980).

[0025] The mycological properties of this bacterium are shown below.

[0026] The culture properties on various media are shown in Table 2 below. When inoculated at the center of a malt extract agar medium and cultured at 25°C for 14 days, growth was somewhat suppressed, with a diameter of 3.0-3.
It expanded to 5cm. The surface of the settlement was felt-like, with radial grooves, and was reddish-brown to black in color. It also produced a red soluble pigment. The back side of the village was dark brown. It produced abundant conidia. When a similar culture was performed on potato dextrose agar medium, growth was suppressed (2.5-3.0 cm in diameter). The surface of the colony was flat to cotton-like, with radial grooves, and reddish-brown to black in color. It also produced a red soluble dye. The back side of the colony was grayish red to black. Abundant conidia were seen.

Observation of morphological characteristics was carried out based on growth on autoclaved leaves. Conidia superficial, dark brown;
Spherical, porous, with setae, cells 1-2 of polyhedral tissue
It consisted of thin walls of layers, up to 60 μm in diameter. The bristles are
There were no septa, dark brown, warty, up to 25 μm long and 3.5-4.5 μm wide. No conidiophores were observed;
Phialides were arranged in conidia. The phialide is round-shaped, 4.0-9.0 μm long, and the base is 3.0 μm wide.
-4.0μm, the tapered tip is 0.5-2.0μm wide
Met. Colorless conidia were formed from the phialides. Conidia are 1 cell, smooth, subglobose, 3.0-3.5×
It was 2.5-3.0 μm. Vegetative hyphae have septa;
Brown, rough, branched. Hyphal cells are cylindrical or barrel-shaped and 2.0-4.0 μm wide.

[0028]No. The 16775 strain can grow at 7-32°C, and the optimal growth temperature is 22-25°C. (potato·
(measured on dextrose agar).

From the above characteristics, No. Strain 16775 is Ketoasborisia erysifoides spegaccini (Ch
aetasbolisia erysiphoide
s Spegazzini 1918) (1). Therefore, this producing strain was designated as Ketoasborisia erysihoides No. 16775 (Chaeta
sbolisia erysiphoides N
o. 16775).

[0030]No. The culture characteristics of strain 16775 are summarized in Table 1 of Table 2 below.

[0031]

[Table 2]

The above characteristics were observed after culturing at 25° C. for 14 days. For descriptions of color tones, please refer to the Methuen Handbook of Color.
ofColour) (2).

[0033] The documents referred to above are shown in Table 3 below.
It is as follows.

[0034]

[Table 3]

The WF16775 substance according to the present invention is produced by a substance-producing bacterium belonging to the genus Ketoasvolisia (for example, Cha
etasbolisia erysiphoides
No. 16775) into a nutrient medium containing assimilable carbon and nitrogen sources and cultured under aerobic conditions (for example, shaking culture, aerated stirring culture, etc.).

As carbon sources, glucose, sucrose, starch, fructose, glycerin and other carbohydrates are preferably used.

Nitrogen sources include oatmeal, yeast extract, peptone, gluten meal, cottonseed flour,
soybean meal, corn steep liquor, dried yeast,
Wheat germ, peanut flour, chicken bone meal, etc. are preferably used, but inorganic and organic nitrogen compounds such as ammonium salts (e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea, amino acids, etc. may also be used advantageously. I can do it.

Although it is advantageous to use these carbon sources and nitrogen sources in combination, it is not necessary to use pure sources. This is because impure substances contain growth factors and trace elements.

If necessary, the following inorganic salts may be added to the medium: sodium carbonate, potassium carbonate, sodium phosphate, potassium phosphate, sodium chloride, potassium chloride, sodium iodide, iodine. potassium salts, magnesium salts, copper salts, cobalt salts, etc.

In particular, if the medium foams strongly, liquid paraffin, animal oil, vegetable oil, mineral oil, silicone, etc. may be added as necessary.

[0041] In order to industrially produce a target substance in large quantities, it is preferable to carry out aerated agitation culture as in the case of other fermentation products. For small-scale production, shaking culture using a flask is suitable.

[0042] Furthermore, when culturing is carried out in a large tank, W
In order to prevent growth delay of bacteria in the production process of F16775 substance, it is preferable to first seed culture the production bacteria in a relatively small amount of medium, and then transfer the culture to a large production tank and culture there for production. In this case, the compositions of the medium used for preculture or seed culture and the medium used for production culture or main culture may be the same, or may be changed if necessary.

[0043] Cultivation is preferably carried out under aeration and agitation conditions,
For example, known methods such as stirring using a propeller or other mechanical device, rotating or shaking a fermenter, pumping, and blowing air may be used as appropriate. Use sterile air for ventilation.

[0044] The culture temperature can be changed as appropriate within the range that allows the present WF16775 substance-producing bacteria to produce this substance, but it is usually 1 to 40°C, preferably 7 to 32°C, particularly preferably 2°C.
It is preferable to culture at 2 to 25°C. The culture time varies depending on culture conditions and culture amount, but is usually about 1 day to 1 week.

[0045] After completion of fermentation, the desired WF1 is extracted from the culture.
6775 substances were recovered. That is, the bacterial cells can be extracted directly with water and/or an organic solvent, or destroyed mechanically or using known means such as ultrasonic waves, extracted with water and/or an organic solvent, and then extracted using a conventional method. Therefore, it is collected and purified. In the case of a culture solution, it may be directly collected and purified according to a conventional method.

Recovery and purification methods include, for example, solvent extraction with water, organic solvents, and mixed solvents thereof; chromatography; and recrystallization from single solvents or mixed solvents; conventional methods may be used alone or in combination as appropriate. can.

Recovery and purification of the WF16775 substance are carried out using appropriate known methods as described above, but the following method may also be used, for example. First, an acetone extract of a culture, for example, mycelium, is extracted with ethyl acetate, hexane, or a mixed solvent thereof, and the extract is concentrated by evaporation or distillation, if necessary. For the concentrate and/or extract, WF1677
Since both 5-A1 and A2 are contained, if they are to be used without being separated, the concentrate and/or extract may be used as is or after being purified according to a conventional method.

In addition, when separating and purifying WF16775-A1 and A2, the extract and/or concentrate obtained above is further purified, and fractions containing these A1 and A2 components are separated, for example, by chromatography. It is sufficient to separate the substance, and in order to obtain a substance with even higher purity, purification treatment such as chromatography treatment, extraction treatment, recrystallization treatment, etc. may be performed according to conventional methods, and if necessary, further lyophilization etc. may be performed. good. In this way, WF16775-A1 and A2 can be obtained in pure form.

The WF16775 substance thus obtained (
A1, A2 and mixtures thereof) may be used in free form or in the form of pharmaceutically acceptable salts by conventional methods such as treatment with the following bases; are also included within the scope of the present invention.

Examples of suitable bases are: alkali metals (eg sodium, potassium etc.)
, alkaline earth metals (eg, magnesium, calcium, etc.), their hydroxides or carbonates, alkali metal alkoxides (eg, sodium methoxide, sodium ethoxide, potassium t-butoxide, etc.), and others.

[0051] The pharmaceutical composition according to the present invention comprises WF1677
5 substances (A1 and/or A2) and/or their salts as active ingredients and a commonly used inorganic or organic carrier, in the form of solid, semi-solid or liquid, for oral administration as well as for external use. Formulated into parenteral drugs such as drugs.

Preparations for oral administration include tablets, pills, granules, soft/hard capsules, powders, fine granules, powders,
Examples include emulsions, suspensions, syrups, elixirs, and the like. Preparations for parenteral administration include injections, drops, infusions, ointments, lotions, tonics, sprays, suspensions, oils, emulsions, suppositories, and the like. The active ingredients of the present invention may be formulated according to conventional methods, including surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonic agents, etc. Use drugs and other commonly used adjuvants as appropriate.

The dosage of the pharmaceutical composition according to the present invention varies depending on the type thereof, the type of disease to be treated or prevented, the method of administration, the age of the patient, the symptoms of the patient, the treatment time, etc.
For intravenous administration, the active ingredient (WF
16775 substances and/or their salts) from 0.1 to 100
mg/kg, and in the case of intramuscular administration, the same dose is 0.1 to 1.
00mg/kg, and in the case of oral administration, 1 to 1
000 mg/kg.

[0054] The present invention will now be explained in more detail with reference to examples.

[0055]

[Example 1]

[0056]

[(1) Fermentation production] Seed culture medium 7.71 shown in Table 4 below in 48 500ml Erlenmeyer flasks
was divided into equal portions and sterilized at 120°C for 30 minutes. In each of these media, Chaetasbolisia ery
Siphoides No. 16775 (FERM
P-11873) strain culture was inoculated one platinum loop at a time, and placed in a rotary shaker (220 rpm) at 25°C for 3 days.
m, approximately 5.1 cm stroke).

[0057]

[Table 4]

Next, the main culture medium shown in Table 5 below was used for 20
After injecting 150 liters into three 0 liter fermenters and sterilizing them at 120°C for 30 minutes, divide the entire volume of the preculture obtained above into three equal parts, inoculate each of these, and inoculate them at 25°C.
The cells were cultured for 7 days. Stirring is 200 rpm, aeration rate is 15
Culture was carried out under pressure of 0 L/min and 1.0 kg/cm2 or more.

[0059]

[Table 5]

The amount of bacterial cells was determined after centrifugation at 3000 rpm for 10 minutes. Whole broth extract (extracted for 1 hour at room temperature after addition of an equal volume of acetone) was used as the assay sample, and its activity was monitored by a tube formation test using endothelial cells.

[0061]

[(2) Separation and Purification] The fermentation broth obtained above was filtered using 10 kg of diatomaceous earth as a filter aid. The obtained bacterial cells (wet weight 54 kg) were extracted twice using 50 liters of acetone. The acetone was evaporated under reduced pressure and the pH was adjusted to 4 by adding 6N HCl to the resulting aqueous solution. Extraction was carried out three times with 20 l of ethyl acetate and then concentrated under reduced pressure, which was dried over Na2SO4, resulting in an oil.

The obtained oily substance was mixed with 21 silica gel (7
0-230 mesh (Merck & Co.) and the resulting powder was applied to a 1 liter column packed with the same silica gel as above in n-hexane. Next, this column was washed with a mixed solution of n-hexane and ethyl acetate (1:0; 8
:1; 4:1; 3:1; 2:1; 1:1) were eluted stepwise using three column volumes (91) in each step. Elution was performed with 8:1 and 3:1 mixtures of n-hexane and ethyl acetate. Since activity was observed in the fractions, these fractions were combined and concentrated under reduced pressure to obtain 56.3 g of an oily substance.

This oil was re-dried with 150 ml of silica gel and then reapplied to a 1 liter column filled with the same silica gel as above with n-hexane. As a result of stepwise development using the same mixed solvent as described above, the active compound was eluted in a 3:1 and 2:1 mixture of n-hexane and ethyl acetate. This was concentrated under reduced pressure to obtain 13.6 g of a yellowish brown oil.

After diluting this oil with 340 ml of ethanol, 510 ml of water was added. A yellow precipitate was formed, so 1N NaOH was added to it to adjust the pH to 7.5.
This was redissolved by adjusting the temperature, resulting in a red solution. On the other hand, YMC gel (ODS-AM
120-S50, manufactured by Yamamura Chemical Research Institute Co., Ltd.), 10 m
A 1 liter column was packed in 40% methanol water containing M potassium phosphate buffer (pH 7), and the above red solution was applied to this YMC gel column. 6 liters of the above methanol water
After washing the column with
It was eluted with 50% methanol water containing potassium phosphate buffer (pH 7). At the same time, HPLC (column: YMC-
AM 303 S-5 120A ODS (manufactured by Yamamura Kagaku Kenkyusho Co., Ltd.), solvent: 65% methanol water containing 10 mM potassium phosphate buffer (pH 7), flow rate: 1
．． The above eluate was monitored using 0 ml/min, detection: 210 nm). As a result, active compounds were detected at retention times of 10.1 minutes and 14.6 minutes, respectively. Among these two active compounds, the former is WF1677
5-A1 (=FR-901448) and the latter as WF1
They were named 6775-A2 (=FR-901449).

[0065] WF16775-A1 is 700ml to 37
00 ml fraction (fraction I), while WF16775-A2 was eluted in 4000 ml to 830 ml fractions.
It was eluted in a 0 ml fraction (fraction II).

Fraction I (containing 1270 mg of WF16775-A1) was combined, water was added to give a final concentration of 35% methanol and water, and chromatography was performed again as follows. i.e. 10m
The above sample was loaded onto a 1 liter YMC gel column packed with 40% methanol water containing M potassium phosphate buffer (pH 7). After washing the column with 6 liters of the above aqueous methanol solvent, the compound was eluted with 45% methanol water. This active fraction (WF1
6775-A1 (containing 1176 mg), its pH was adjusted to 4 using 1N HCl.

The obtained suspension was mixed with a mixture of n-hexane and ethyl acetate (1:2) five times (equal amounts of each mixture were used).
Extracted and dried. The resulting red oily substance (1547m
g) was redissolved in a hot mixture of n-hexane:ethyl acetate (6:1) and applied to a 150 ml silica gel column packed using the same solvent as above.

[0068] This column was treated with WF16 using the above solvent.
The reaction mixture was developed sufficiently until the elution of 775-A1 was completed. The solvent was removed under reduced pressure and the pale yellow oily residue was heated with a small amount of heat.
Redissolved in hexane. When this solution was left at room temperature overnight, 822 mg of white needle-shaped crystals were obtained.

On the other hand, fraction II exhibiting red color
(containing 328 mg of WF16775-A2) was diluted with water to a final concentration of 40% methanol and water, and chromatography was performed again on a 1 liter YMC gel column. 10mM potassium phosphate buffer (p
After washing this column with 5 liters of 40% methanol water containing H7), WF16775-A2 was eluted using 50% methanol water containing 10 mM of the above buffer.

Fractions with a purity of 95% or higher were combined and concentrated under reduced pressure to remove methanol. This active substance was extracted with n-hexane at pH 7 to obtain a pale yellow extract. Concentrate this and make WF16775-A
273 mg of colorless needle-like crystals of No. 2 were obtained.

[0071]

[Example 2] WF16775-A1 manufactured in Example 1
and the same A2 substance (sometimes simply referred to as A1 and A2, respectively), the inhibition of tube formation on vascular endothelial cells was measured, and in addition to the angiogenesis inhibitory activity, antitumor activity was also confirmed, and the safety was also confirmed. confirmed.

[0072]

[(1) Tube formation inhibitory activity] Endothelial cells were derived from bovine carotid artery, cultured in Eagle's MEM (minimum essential) medium containing 10% FBS (fetal bovine serum), treated with trypsin, and collected. Ta. Neutralized type I collagen was added to each well of a 96-well microtiter plate to form a gel, and 1.5 x 104 endothelial cells suspended in MEM medium supplemented with 10% FBS were seeded onto the gel. After culturing in a CO2 incubator for 3 days, the medium was replaced with serum-free MEM medium, and the sample and bFGF (20 ng
/ml) and continued culturing. After 3 days, the degree of development of the lumen-like structure was observed using a microscope.

As a result, the MIC values for tube formation were as shown in Table 6 below.

[0074]

[Table 6]

[0075] At this time, A1 and A2 are applied to the endothelial cells.
Both showed no cytotoxicity at a concentration of 10 μg/ml.

[0076]

[(2) Cytotoxicity] Bovine carotid artery endothelial cells are 10%
The cells cultured in Eagle's MEM medium containing FBS were treated with trypsin and collected. Mouse lymphoma EL-4 cells were cultured in Dulbecco's medium containing 10% FBS. In addition, mouse fibrosarcoma Meth A cells are Balb/
c Mouse (female, 8 weeks old) passaged intraperitoneally,
The cells were suspended in Dulbecco's medium containing % FBS. Add 1 x 104 cells suspended in the above medium to each well of a 96-well microtiter plate, add the sample thereto, and incubate endothelial cells or Met in a CO2 incubator.
h After culturing for 72 hours in the case of A cells and 48 hours in the case of EL-4 cells, cell growth was observed using a microscope.

As a result, the MIC values for cell proliferation were as shown in Table 7 below.

[0078]

[Table 7]

[0079] These effects are caused by endothelial cells, Meth
It was cytostatic to A cells, but cytotoxic to EL-4 cells.

[0080]

[(3) Antibacterial activity] Antibacterial activity is determined by diluting the sample using bouillon medium when using bacteria as the test bacteria, and using Sabouraud medium when using yeast and mold.
MIC values were determined by vapor disk-agar diffusion method. The results are shown in Table 8 below.

[0081]

[Table 8]

[0082]

[(4) Acute toxicity] Balb/c mouse (female, 8 weeks old,
The LD100 value obtained by intraperitoneal administration using (n=2) was A1.
was 5 mg/kg, and A2 was 10 mg/kg. In addition, A
2 was administered intraperitoneally for 5 consecutive days, and at a concentration of 3 mg/kg or less, a weight increase comparable to that of controls was observed.

[0083]

[(5) Angiogenesis inhibitory effect] The angiogenesis inhibitory effect in vivo was investigated using the chicken embryo chorioallantoic membrane (CAM) method. The assay is N. G. The method of Tanaka et al. (Ex
p. Pathol. 30, 143, 1986) with some improvements. That is, after extracting about 3 ml of albumen from a 3-day-old fertilized egg, a small window of about 1 cm square was made in the egg shell, and the egg shell membrane was removed to expose the CAM. Then,
After incubating for 2 days with the small window closed, a silicon ring (inner diameter 3 mm, outer diameter 5 mm, thickness 1 mm: Ikeda Rika) was placed on the 5-day old CAM, and R. Langer
et al.'s method (Nature 263, 797, 1979
) was placed in a sustained release pellet containing 0.1 to 10 μg of the present compound. After incubating for another 2 days with the small window closed, an appropriate amount of 10% fat emulsion (Intralipos, Midori Juji) was injected into the chorioallantoic cavity to make it easier to see the blood vessels on the CAM, and the sample pellet was examined under a stereomicroscope. An anti-angiogenic zone was observed around the tube.

Angiogenesis inhibitory activity (percentage) was calculated as the number of specimens forming an angiogenesis inhibition zone per total specimens tested. Note that 8 to 10 fertilized eggs were used in each group.

As a result, as shown in Table 2 of Table 9 below, all of the present compounds inhibited angiogenesis in the CAM in a concentration-dependent manner.

[0086]

[Table 9]

[0087]

[(6) Antitumor activity] Mouse fibrosarcoma Meth A cells were subcultured intraperitoneally in Balb/c mice (female, 8 weeks old), and 8 days later, the cells were intradermally transferred to 1 x 10 cells of Balb/c mice. Ported. This compound is used for days 0 to 3, day 6
-10 and day 13, each concentration was intraperitoneally administered, and tumor size was measured over time. Note that the relative tumor weight is N
Calculated according to CI (USA) manual. The results are shown in Table 3 of Table 10 below.

[0088]

[Table 10]

[0089] From the above results, it was revealed that the substance WF16775 has excellent angiogenesis inhibitory and antitumor effects. That is, in the chicken egg chorioallantoic membrane assay system, which is an in vivo angiogenesis evaluation system, A1(0
．． 5-5 μg/egg) and A2 (0.2-2 μg/egg)
It was confirmed that the inhibitory effect was strong at the concentration of egg).

[0090] In addition, mouse solid tumor Meth A (i
d→ip administration), a tendency to suppress tumor growth was observed at 1 to 3 mg/kg.

[0091] Therefore, the WF16775 substance was found to be useful, for example, in inhibiting angiogenesis and in preventing and treating cancer.

[0092]

Example 3 Tablets were manufactured using the raw materials shown in Table 11 below.

[0093]

[Table 11]

That is, (1), (2) and (3) (17 g) were mixed together and granulated together with the paste prepared from (3) (7 g). Add (3) to the obtained granules (
However, 5g) and (4) are added and mixed well, and this mixture is compressed using a compression tablet machine to obtain the active ingredient (1) per tablet.
1000 tablets containing 50 mg of

[0095]

Example 4 An injection was manufactured using the raw materials shown in Table 12 below.

[0096]

[Table 12]

That is, all the components (1) to (4) were dissolved in 1000 ml of distilled water, and then 1 ml each was dispensed into ampoules to produce 1000 injections.

[0098]

Effect of the invention The present invention provides WF16775-A1 and A2
These substances (FR-901448 and FR-901449) exhibit excellent angiogenesis inhibitory effects and are useful in medicines, such as cancer and tumor prevention and/or treatment.
It is also very useful as a therapeutic agent. Also, FR-9
Since the 01449 substance is also a new substance, it is highly expected that new inventions will be developed in terms of compounds as well.

Furthermore, according to the present invention, an industrial method for producing the above substance using microorganisms has also been established.

Claims (4)

[Claims] Claim 1: A compound represented by the formula I shown below, FR-
901448 substance and/or FR-901449 substance,
An angiogenesis inhibitor characterized by having as an active ingredient. [Chemical formula 1] [Claim 2] FR-9 represented by the following formula II:
01149 substance. [Case 2] [Claim 3] Chaetasb.
FR-901 was obtained by culturing FR-901448 and FR-901449 substance-producing bacteria belonging to the genus Olisia.
448 substance and FR-901449 substance,
FR-9014 characterized by collecting these (these)
48 substance and/or method for producing FR-901449 substance. [Claim 4] A compound represented by the formula I of the following chemical formula 3, F
An antitumor agent characterized by containing R-901448 substance and/or FR-901449 substance as an active ingredient. [Chemical formula 3]