JPH0329053B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Technical Field The present invention relates to novel phenalen-1-one derivatives.

Structure of the Invention The phenalen-1-one derivative of the present invention is a novel compound that has not been described in any literature, and is represented by the following structural formula (1).

The present inventors conducted a series of studies on culture products obtained by culturing microorganisms, and as a result, microorganisms belonging to the genus Aspergillus isolated from the soil of Kochi City were found to contain cyclic adenosine monophosphate (hereinafter referred to as "C-AMP"
discovered the fact that a substance having specific phosphodiesterase (hereinafter referred to as "PDE") inhibitory activity is produced, and the fact that this substance is a novel compound represented by the above structural formula (1),
The present invention has now been completed.

The compound of the present invention having the above-mentioned PDE inhibitory activity is
It is produced by culturing a microorganism belonging to the genus Aspergillus and capable of producing the compound in a commonly used nutrient medium containing glucose, starch, etc. as the main carbon source, and then isolating and purifying the culture.

A specific example of the above-mentioned microorganism most suitable for producing the compound of the present invention is a strain belonging to the genus Aspergillus that the present inventors isolated from soil. Its mycological properties are as follows.

(A) Growth status on each medium 1 Malt extract agar medium Growth is rapid, with colonies having a diameter of 65 mm after 8 days of culture.
Reach ~70mm. The colony is circular and flat, and white to grayish-white mycelium grows thinly and sparsely. From around the 3rd day of culture, black-brown conidial heads were observed to grow, and on the 8th day, a dense growth of conidial heads was observed in concentric circles in the center of the village. No coloring is seen on the back side of the colony or in the agar.

2. Tuapetsk agar medium Growth was good, and after 8 days of culture, the colony had a diameter of 45 ~
Reaches 50mm. The colony is circular and flat, with white to grayish-white hyphae extending sparsely and long, and on the fifth day, conidial heads begin to grow, with blackish brown in the center and white in the periphery. After that, the center becomes round and swollen, and the length of the conidia in this part reaches nearly 5 mm, and the entire village is covered with blackish brown conidial heads. No coloring is seen on the back side of the colony or in the agar.

(B) Physiological properties The pH and temperature range in which this strain can grow, and the optimal growth pH and temperature are as shown below.

PH Temperature Growth range 3.0-11.4 12-42℃ Optimum growth conditions 3.6-8.9 32-37℃ For pH, use Sabouraud liquid medium at 27℃,
Sabouraud agar (PH
6.2) for 7 days.

(C) Morphological properties Lower fertile hyphae have septa and form white or grayish-white, circular, flat colonies. As conidia settle, the surface of the colony changes to blackish brown. The conidial head is spherical with a diameter of 60 to 200 μm and is blackish brown.
The conidiophores extend in a straight line, and in rare cases have septa. The wall is rather thick, the surface is smooth, and the diameter is
It is 8.3 to 17 μm. The apical capsule is approximately spherical with a diameter of 50-60 μm and is colorless and transparent. The sycamores are double epiphytes, the primary sycamore is 17-20μm, and the secondary sycamore is 8.3-15μm.
It is m. The conidia are approximately spherical black particles with a diameter of 3.1 to 4.2 μm that are connected in a long chain, and the surface has a curved ridge structure. Sclerotia are not observed.

Based on the above mycological properties, Gilman (J.
“A Manual of Soil Fungi,” by C. Gilman.
2nd, The Iowa State University Press,
Ames, Iowa, USA 1957) and KBRaper and DIFennell, The Geuus Aspergillus,
The Williams & Wilkins, Co.Baltimore,
1965), this strain was found to be Aspergillus niger.
However, since there is no clear basis for determining the species, it was named Aspergillus sp. No. 2970 and transferred to the Microtech Institute. Bacteria number 7390
It was entrusted as the No.

The compound of the present invention can be produced using the compound-producing bacteria of the genus Aspergillus, including the Aspergillus sp. No. 2970, according to a conventional method. The culture medium for culturing microorganisms may be the same as that commonly used, and glucose, soluble starch, etc. can be used as the carbon source. The nitrogen source is not particularly limited, but natural organic nitrogen sources such as peptone obtained from soybean flour hydrolyzate are preferred.Inorganic salts include sodium chloride, calcium carbonate, copper sulfate, manganese chloride, zinc sulfate, etc. can be used as appropriate. In addition, micronutrients contained in yeast extract and the like can be used.

Culture conditions vary depending on the medium composition, but are usually PH
4 to 9. It is desirable to keep the temperature in the range of 25 to 37 degrees Celsius. Although shaking culture blades and aerated agitation culture are also possible, stationary liquid culture is preferable, and by this method, a significant amount of the desired target compound can be obtained. Production is seen. In the stationary liquid culture, culture for about 5 to 20 days is appropriate.

After completion of the culture, the target substance produced in the culture solution is isolated and purified. The isolation and purification method is not particularly limited;
Any of various known methods utilizing the physical and chemical properties of the produced substance can be employed. As a specific example, the following method can be exemplified. That is, the culture solution is preliminarily separated into bacterial cells and liquid by filtration or centrifugation according to a conventional method. Next, methanol etc. are added to the bacterial cells to extract the culture, and the extract is further mixed with methanol, ethyl acetate, chloroform, ether, n-
Extraction is carried out using an appropriate combination of solvents such as hexane and benzene, and after distilling off the solvent, the residue is gel-filtered with silica gel or Sephadex LH-20 (Pharmacia), etc., and each of the obtained fractions is further extracted as needed. Solvent extraction, gel filtration, neutralization, concentration, as appropriate
Isolation and purification can be achieved by repeating operations such as crystallization alone or in appropriate combinations.

The physicochemical properties of the compound of the present invention thus obtained are as follows.

Elemental analysis values C: 62.70%, H: 4.01% Molecular weight SIMS spectrum (Figure 1) shows molecular weight 288 Melting point 280℃ (decomposition point) Ultraviolet absorption spectrum (UV; nm) The UV analysis diagram is as shown in Figure 2. , 412n
It has maximum absorption at m.

Infrared absorption spectrum (IR; cm- ¹ ) The IR spectrum of the KBr tablet is shown in Figure 3, and the main absorption peaks are from 3400 to
3200, 1600, 1160 etc. are accepted.

Nuclear magnetic resonance spectrum ( ^1H -NMR; ppm) ^1H -NMR measured using DMSO- _d6 as a solvent
The spectrum is as shown in Figure 4, and the main peaks are 18.3 (brs, 1H), 18.0 (brs, 1H),
6.25 (d-like, 0.9Hz, 1H) 5.96 (s, 1H),
3.71 (s, 3H) and 2.80 (brs, 3H) are accepted.

Nuclear magnetic resonance spectrum ( ^13C -NMR; ppm)
^13C -NMR measured using DMSO- _d6 as a solvent
The spectrum is as shown in FIG. 5, and the following main peaks are observed.

172.2 (s) 170.1 (s) 170.0 (s) 170.0 (s) 115.0 (d) 144.1 (s) 130.6 (s) 99.3 (d) 167.7 (s) 107.0 (s) 110.0 (s) 101.1 (s) 128.0 ( s) 58.8 (q) 25.2 (q) Thin layer chromatography (TLC) As a result of TLC analysis using silica gel, the Rf value was as follows.

Rf value = 0.40 (chloroform: methanol: water =
(13:6:1) Color reaction Positive for ferric chloride reaction Solubility in solvents Insoluble in chloroform, ethyl acetate, ether, and cyclohexane, almost insoluble in water,
Dissolve in methanol, dimethylformamide, and 5% aqueous sodium hydroxide solution.

Distinction between basic, acidic, and neutral Acid Color of substance Light yellowish brown From the analysis of the various physical and chemical properties mentioned above, the compound of the present invention has a structure represented by the above structural formula (1),
It is presumed to be a compound called 2,3,4,9-tetrahydrokyl-7-methoxy-6-methyl-1H-phenalen-1-one.

Compound of the present invention (phenalen-1-one derivative
(1)) has C-AMP-specific PDE inhibitory activity, and is associated with various diseases caused by C-AMP metabolic abnormalities, such as arteriosclerosis, hypertension, bronchial asthma, diabetes, and cancer. It is useful as a prophylactic or therapeutic agent, and can be particularly effectively used as a therapeutic agent for hypertension.

In general, C-AMP is widely distributed in animal tissues,
It is a substance that plays an important physiological and biochemical role as a secondary transmitter of various hormonal actions. It is also known to be involved in cell proliferation and differentiation, hemodynamics, effects on the central nervous system, and the secretion of insulin and histamine. C-AMP is biosynthesized from adenosine triphosphate (ATP) by the action of adenyl cyclase, and C-AMP is
It is degraded by AMP/phosphodiesterase (PDE), and the intracellular concentration is regulated by the action of both enzymes. Therefore, generally, by administering a PDE inhibitor to a living body, PDE, which is a C-AMP degrading enzyme, is inhibited and the intracellular C-
Since the AMP concentration increases, pharmacological effects such as inhibiting platelet aggregation, lowering blood pressure, anti-asthma, increasing insulin secretion, and anti-cancer can be expected.

Examples Next, in order to further clarify the present invention, production examples of the compounds of the present invention will be given as examples, and then test examples will be given.

Example 1 Aspergillus sp. No. 2970 (Feikoken Bacteria No. 7390) was inoculated into a Monod tube containing 10 ml of a medium with the following composition, and incubated at 27°C and pH
= 7.2 for 3 days in a Monod type shaking culture apparatus.

Glucose 2% Starch 2% Phytone Peptone 2% (manufactured by BBL) Yeast extract 0.5% Sodium chloride 0.25% Calcium carbonate 0.32% Copper sulfate (pentahydrate) 0.0005% Manganese chloride (tetrahydrate) 0.0005% Zinc sulfate (7%) Water salt) 0.005% Each 500 ml Erlenmeyer flask containing 100 ml of the above medium was inoculated with one seed culture obtained above, and statically cultured at 27°C for 11 days.

The obtained culture solution was heat-treated (at 85° C. or higher for 6 minutes) and then separated into the solution and the bacterial cells by vacuum filtration. Methanol was added to this bacterial cell (culture solution 16.2 minutes).
16.2 was added, and the mixture was stirred under NaCl saturated and acidic (about 3) conditions. After filtration under reduced pressure, the methanol extract was concentrated to dryness under reduced pressure. Water was added to the dried product to dissolve it, extracted three times with ethyl acetate, and the extracts were combined and concentrated. Then add 5% to this ethyl acetate extract concentrate.
An aqueous solution of NaHCO ₃ was added, extraction was performed three times, the extracts were collected, acidified with hydrochloric acid, and then extracted three times with ethyl acetate. The extracts were combined and concentrated, and washed with water and dehydrated during the process to obtain 6.9 g of an oily concentrate. This oily concentrate was applied to a silica gel dry column and sequentially eluted with a solvent system of chloroform and methanol.
I got g. Further, the mixture was applied to a Sephadex LH-20 column and eluted with methanol to obtain a fraction eluted with the target product. After concentrating this under reduced pressure, it was fractionated using preparative thin layer chromatography (developing solvent: chloroform:methanol:water = 13:6:1), applied to a Sephadex LH-20 column again, eluted with methanol, and concentrated. The mixture was dried to obtain 155 mg of the compound of the present invention.

The physical and chemical properties of this product were as described above.

Test Example 1 <Inhibitory activity against C-AMP/PDE> (1) Measurement method As enzyme reaction solution, 50μ of 20mM magnesium sulfate solution, 150μ of 6mM MC-AMP (manufactured by Boehringer Mannheim, Yamanouchi) solution, 7U/ml alkaline phosphatase ( Boehringer Mannheim, Yamanouchi), calf intestine) solution 50 μ, 0.05 U/ml Sample solution containing 200 μ of phosphodiesterase (Boehringer Mannheim, Yamanouchi, derived from bovine heart) solution and the compound of the present invention or theophylline as a control compound, respectively. A mixed solution consisting of 100μ and a total volume of 550μ was prepared. All except the sample solution are 50mM.
It was used after being dissolved in Tris-HCl buffer (PH7.5).
After reacting the above reaction solution at 37℃ for 45 minutes,
% trichloroacetic acid was added to stop the reaction, centrifugation was performed (3000 rpm, 10 minutes), and inorganic phosphorus in the supernatant was detected using Phosphor B-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). It was quantified using

C-AMP/PDE inhibitory activity is calculated by subtracting the amount of inorganic phosphorus produced when adding the sample solution from the amount of inorganic phosphorus produced when distilled water is added to the reaction solution instead of the sample as a control, and then subtracting this amount. It was expressed as a percentage of the value divided by the amount of inorganic phosphorus produced in the control.

(2) Results The inhibitory activity of the compound of the present invention against C-AMP/PDE was compared with that of theophylline, a positive control, and the results are shown in FIG. In Fig. 6, the horizontal axis indicates the final concentration of the compound of the present invention (indicated as (1) in the figure) or theophylline (indicated as (2) in the figure) in the antiliquid on a logarithmic scale. It is
The vertical axis shows inhibition rate %.

From the figure, the 50% inhibitory concentration (IC ₅₀ ) of the compound of the present invention
is 6.5×10 ₋₆ M, which clearly shows about 350 times stronger inhibitory activity than theophylline, which was used as a control and had a concentration of 2.3×10 ₋₃ M.

[Brief explanation of the drawing]

Figures 1 to 5 are mass spectrum analysis diagrams, ultraviolet absorption spectrum analysis diagrams, and
These are an infrared absorption spectrum analysis diagram, ^{a 1} H-NMR analysis diagram, and ^{a 13} C-NMR analysis diagram, and FIG. 6 is a graph showing the C-AMP/PDE inhibitory activity of the compound of the present invention.

Claims (1)

[Claims] 1. Structural formula A phenalen-1-one derivative represented by