JPH04321684A - 2-pyridone compound - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to 2-pyridone compounds having antifungal and antitumor effects.

0002

BACKGROUND OF THE INVENTION Hitherto, no compounds have been known that are structurally similar to the compounds of the present invention and have similar physiological activities.

0003

OBJECTS OF THE INVENTION An object of the present invention is to provide a novel compound having excellent antifungal and antitumor effects, and to use the compound as an antifungal and anticancer agent in the treatment of patients.

0004

[Means for Solving the Problems] In order to achieve the above object, the present inventors isolated a large number of bacterial strains from plants and soil,
As a result of various studies on the metabolites of the strain, it was discovered that a certain strain produces a novel physiologically active substance that has a growth-inhibitory effect on fungi and cultured cancer cells, leading to the completion of the present invention. That is, the present invention

00
05]

This is a compound represented by the following formula (hereinafter referred to as AF-110).

The microorganism producing AF-110 of the present invention is a strain newly isolated by the present inventors from a pine cone collected in Yoshino-cho, Omiya City, Saitama Prefecture, and the microorganism is named "Peni".
cilium sp. F-4209” and microorganism deposit number “FERM P-
12163)" and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

[0007] The mycological properties of this strain are shown below. [Morphology] This strain can be used on malt extract agar medium, potato
It grows moderately on glucose agar, oatmeal agar, YpSs agar, etc., and forms spores at a moderate rate. This strain was grown on potato-glucose agar medium at 25°C.
When colonies formed after 14 days of culture were observed under a microscope, the hyphae were white, had septa, and were highly branched. Conidiogenic cells are present at the tips of conidiophores that branch out and stand up from hyphae or loose hyphal bundles.
Penicillium (P
enicilli) is observed. The conidiophore has septa, the surface is smooth, and the diameter is 2.5~
3.8 μm, and the length is 15 to 70 μm. Metoles have 3 to 8 whorls at a relatively narrow angle from the tip of the conidiophore, the surface is smooth, and the size is 10.0 to 16.0.
It is μm×2.0 to 3.0 μm. Phialide has a flask shape with a tapered tip, a smooth surface, and a size of 12.0
~15.0 μm×1.6 to 2.0 μm. Conidia are subglobose to rarely spherical, with a smooth surface and a size of 1
．． It is 8-4.0 micrometers x 1.8-3.0 micrometers. Furthermore, when the culture was extended for more than one month and observation was continued, the formation of sexual reproductive organs such as ascus was not observed, but the formation of conidiophore bundles (Coremia) was observed in some of the colonies. It was done.

[Properties on medium] On various medium, 25
Table 1 shows the results of visual observation when cultured at ℃ for 14 days.
Shown below.

[0009]

[Table 1]

[Physiological properties] 1) Growth pH range and optimum pH This strain grows in a YpSs medium in a pH range of 2 to 7, with an optimum pH of 3 to 6. 2) Growth temperature range and optimum temperature This strain grows in the Sabouraud medium at a temperature range of 10 to 37°C, and the optimum temperature is 25 to 34°C. 3) Distinction between aerobic and anaerobic: aerobic

From the above morphological characteristics and culture properties,
It has become clear that this strain belongs to the genus Penicillium, and based on the above-mentioned properties, it was determined that the present strain belongs to the Deuteromycetes genus Penicillium. B. R
aper, C. “A MANUAL O” by Thom
F THE PENICILLIA” (1949) and J. I. Written by Pitt “A LABORAT”
ORY GUIDETO COMMON Pen
icilium SPECIES” (1985)
A comparison study was made with many known bacterial strains reported in As a result, this strain was found to be more effective in terms of Coremia formation.
icilium vulpinum, and in other respects Penicillium funiculo
It has become clear that the properties are closest to those of sum. However, as a result of checking with descriptions in the literature, Penicill
This strain differs from Penicillium funicul in that it grows at 37°C and in the shape and size of phialides and conidia.
There were some differences from Osum in the size of metres, phialides, conidia, etc., and colony properties. Therefore, it has been suggested that this strain may be one of the above two bacterial species or a variant thereof, but as there is no conclusive evidence, this strain has been classified as Penicillium spp.
p. It was named F-4209.

The production of AF-110 is roughly similar to the production of general antibiotics, in which AF-110 is produced in a medium containing various nutritional substances.
F-110 producing strain Penicillium
sp. This is done by culturing F-4209 under aerobic conditions. The medium is preferably a liquid medium, mainly containing charcoal sources,
It consists of a nitrogen source and inorganic salts, and if necessary, vitamins, precursors, and antifoaming agents can be added, and the pH is adjusted to around 6. As the carbon source, for example, glucose, maltose, dextrin, glycerin, starch, etc. are used alone or in combination. As the nitrogen source, for example, yeast extract, peptone, meat extract, soybean flour, corn tea liquor, urea, ammonium salt, etc. are used alone or in combination. Examples of inorganic salts include potassium phosphate, magnesium sulfate, sodium chloride, and calcium carbonate, which may be used alone or in combination. Adekanol, a silicon compound, etc. can be used as an antifoaming agent.

[0013] As a culture method, aerobic culture such as shaking culture and aerated agitation culture is suitable.
Culture at 4°C, preferably 25-30°C. Since most of the AF-110 produced by this culture is accumulated within the bacterial cells, it can be isolated, for example, by the following method. That is, after completion of the culture, AF-110 is extracted from the bacterial cells separated by centrifugation and filtration with an organic solvent such as a lower alcohol or acetone, and this extract is concentrated under reduced pressure to remove the organic solvent. , transferred to a water-insoluble organic solvent such as chloroform, and concentrated under reduced pressure to form a syrup. This syrup was dissolved again in an organic solvent such as ethyl acetate, benzene, chloroform, acetone, and methanol, and subjected to column chromatography using silica gel, high performance liquid chromatography using an ODS column, and Sephadex LH-20 (trade name, Pharmacia). AF-110 can be isolated as a pale yellow powder by gel filtration using a gel filtration solution (manufactured by Phys. Co., Ltd.).

AF-110, which is the target substance of the present invention obtained through the above purification, has the following elemental analysis values, molecular weight,
Ultraviolet absorption spectrum, 1H-NMR spectrum, 13
The structural formula as shown in Chemical Formula 2 was determined from the analysis results such as C-NMR spectrum. The physical and chemical properties of AF-110 are as follows. (a) Elemental analysis value: Actual value (%) C 67.05, H 8.56,
N 2.99 Theoretical value (%) C 68.21, H 8.63,
N 2.95 (calculated using C27H41NO6) (b) FAB mass spectrum: FAB (+) m/z 476 (M+H) + F
AB(-) m/z 474(MH)-(c) Molecular weight: 475 (d) Melting point: 86-89°C (e) Specific optical rotation: [a] D25 = -184° (c = 0.1 ,methanol)
(f) Ultraviolet absorption spectrum: λmax 290nm (ε=6700) λmax
213 nm (ε=34000) (measured in methanol solution) The measurement results are also shown in FIG. (g) Infrared absorption spectrum: The results measured in potassium bromide tablets are shown in FIG. (h) 1H-NMR spectrum: The results of measurement at 400 MHz in deuterated dimethyl sulfoxide are shown in FIG. (i) 13C-NMR spectrum: The results of measurement at 100 MHz in deuterated dimethyl sulfoxide are shown in FIG. (j) Solubility in solvents: Insoluble in water. Slightly soluble in methanol, acetone, chloroform, and ethyl acetate. Easily soluble in dimethyl sulfoxide. (k) Color reaction; Positive: sulfuric acid, iodine. Negative: ninhydrin, ferrous chloride. (l) Basic, acidic, neutral: Neutral (m) Color of substance: pale yellow powder

[0015]

EFFECTS OF THE INVENTION The compounds of the present invention have a growth-inhibitory effect on bacteria, yeast, filamentous fungi, and cultured cancer cells, and therefore can be useful in the treatment of mycoses and cancer diseases.

[0016]

[Examples] The present invention will be explained in more detail below with reference to Examples and Test Examples. Example 2g of glucose and 0.5g of polypeptone per 100ml
Penicillium sp. F-4209 strain was inoculated and cultured with shaking at 28°C for 72 hours. then 200
Using a L-capacity culture tank, 120 L of a sterile medium with the same composition as the seed culture was inoculated with 1.2 L of the seed culture solution and incubated at 28°C at 96°C.
Cultured with agitation for hours. After the culture was completed, the supernatant and the bacterial cells were separated using a centrifuge. 8.5 kg of the obtained bacterial cells were mixed with 1 part of acetone.
The mixture was extracted twice with 5 L, and the extracts were combined and concentrated under reduced pressure to gradually remove acetone, and then extracted twice with half the amount of ethyl acetate. The ethyl acetate extracted fractions were combined, dehydrated over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain 24 g of brown syrup.

[0017] This brown syrup was mixed with 200 m of chloroform.
500% of silica gel [Wako Gel C-200 (trade name, manufactured by Wako Pure Chemical Industries, Ltd.)] prepared with chloroform and dissolved in
It was adsorbed onto a ml column. After washing with 500 ml of chloroform, this fraction was removed by elution with a mixed solvent of chloroform-methanol (95:5). Next, chloroform-
Elution was performed with a mixed solvent of methanol (90:10), and each fraction (20 g) was fractionated using a fraction collector. After confirming the antibacterial activity of each fraction against Candida albicans by the paper disc method, the fractions with recognized activity were collected and concentrated to dryness under reduced pressure to obtain 1.5 g of crudely purified powder of AF-110. Crude powder of AF-110 obtained by silica gel column purification was dissolved in 20 ml of methanol. This solution was subjected to preparative high performance liquid chromatography using 75% methanol as a mobile phase [equipment used: Senshu Kagaku Co., Ltd. 3110; column: Senshu Pack ODS-
5251-N (20φ x 250 mm)] at a flow rate of 10 ml/min while monitoring with UV absorption at 220 nm.
A peak eluted around 33 minutes under these conditions was fractionated. The fractions obtained by fractionation were combined and concentrated under reduced pressure to remove methanol, and then extracted twice with half the volume of ethyl acetate. The ethyl acetate extracted fractions were combined, dehydrated with anhydrous sodium sulfate, and then concentrated to dryness under reduced pressure. The powder obtained by this operation was dissolved in a small amount of methanol, and Sephadex LH-20 (trade name;
gel filtration with methanol using
The obtained active fractions were collected and concentrated to dryness under reduced pressure to obtain AF-110.
35 mg of pale yellow powder was obtained.

Test Example 1 [Antibacterial Effect Test] The 1000 ug/ml methanol solution of AF-110 obtained in the example was diluted with methanol to the desired concentration, soaked into a paper disk, and then air-dried for about 1 hour. I let it happen. This paper disk was placed on a test bacterium plate prepared by adding sensitive Tisk agar medium as a bacterial medium and Sabouraud agar medium as a fungal medium to give an inoculum size of 106 CFU/ml, and the plate was placed at room temperature for approximately 1. After standing for 18 hours at 37℃ for bacteria and 48 hours at 25℃ for fungi.
The strength of the growth inhibitory effect on various bacteria was determined by culturing for ~72 hours and measuring the inhibition zone after culturing, and the results shown in Table 2 were obtained.

[0019]

[Table 2]

Test Example 2 [Antitumor Effect Test] (Sample) 10 mg of AF-110 obtained in the example was dissolved in methanol, diluted with PBS (-) to the desired concentration, and used. (Test cells) P388 Mouse leukemia K-562 Human bone marrow leukemia HL-60 Human leukemia (Culture solution used) RPM1-1640 medium (Test method) Using the above culture solution, various cancer cells were cultured at 2 x 104 to 1 x 1
05/ml, and put 2ml into a 6-hole petri dish with a diameter of 35mm.
Dispensed in portions. Then, 50 ul of the specimen previously diluted to the desired concentration was added at the same time as the start of the culture. The test cells are
After continuing to culture in a 5% carbon dioxide incubator at 37°C for 3 to 4 days, live cells were measured and IC5 was determined from the sample concentration and inhibition rate.
The 0 value (concentration for 50% inhibition) was determined and the results shown in Table 3 were obtained.

[0021]

[Table 3]

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption spectrum of AF-110 measured in methanol solution.

FIG. 2 shows the infrared absorption spectrum of AF-110 measured in potassium bromide tablets.

[Figure 3] 400MHz in heavy dimethyl sulfoxide
The 1H-NMR spectrum of AF-110 measured in is shown.

[Figure 4] 100MHz in heavy dimethyl sulfoxide
The 13C-NMR spectrum of AF-110 measured in is shown.

Claims (1)

[Claims] [Claim 1] [Chemical formula 1] A compound represented by