JPH0149351B2 - - Google Patents

Description

[Detailed description of the invention] Technical field The present invention relates to novel xanthone derivatives.

Structure of the Invention The xanthone derivative of the present invention is a new compound that has not been described in any literature, and is represented by the following structural formula (1), and is 3,3′-
It is named diacetonyl-1,1',6,6',8,8'-hexahydroxy-2,2'-bixanthone.

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

Compound (1) of the present invention having the above PDE inhibitory activity
As a representative example of a microorganism capable of producing , a microorganism newly isolated and obtained by the present inventors and having the following mycological properties can be exemplified.

(A) Growth status on each medium This strain grows well on various media used for fungal culture, and forms spherical or oval spores on other media except malt extract agar. . The growth status of this strain in typical media is shown below (Culture conditions: 27℃, 30℃
days).

1. Malt extract agar medium Growth is extremely rapid, with colonies covering the entire surface of the agar plate (85φmm) after 3 to 4 days of culture.
The colony grows in a circular shape, with a circular or wavy edge and a flat surface. Fine, long hyphae extend radially, but are not densely grown.

The hyphae are white or grayish white, but take on a horizontal color from around the 5th day. The medium becomes yellowish from around the 13th day, and brown from around the 20th day. 30
No spore formation was observed even after 1 day of culture.

2. Tuapetsk agar medium Growth is relatively slow, and colonies cover the entire surface of the agar plate on the 16th to 19th day. Initially, the colony grows in a nearly circular shape, but from around the 13th day onwards, it becomes irregular in shape. The hyphae are short and grow sparsely, but from around the third day small powdery spots containing many spores appear.

The colony is initially grayish white, and as small spots appear, ocher and green areas become scattered locally, and after the 19th day, the entire area becomes gray. No coloring was observed in the agar even after 30 days of culture.

3. Sabouraud agar medium Growth is good, and colonies cover the entire surface of the agar plate in 7 to 10 days. The village grows in a circular shape,
The periphery is corrugated and the surface is flat. 5
From the first day, dense areas of concentric hyphae appear and contain many spores, giving it a powdery appearance.
From around the 16th day, the hyphae become felt-like, with circular bulges visible in some areas, and wrinkles running vertically and horizontally in these areas. Also, from around the 16th day, many colorless and transparent minute droplets appear in concentric circles.

The colony is almost white, but it becomes yellowish from around the 16th day, and becomes grayish as it is further cultivated. The medium begins to take on a yellowish color from around the 10th day, and then gradually turns brown.

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

PH Temperature Growth range 2.0 to 9.6 2℃ to 32℃ Optimum growth conditions 6.9 to 7.5 22℃ to 27℃ For pH, use malt extract liquid medium at 27℃
The cells were cultured for 7 days using a Sabouraud agar medium at different temperatures.

(C) Morphological properties The sterile hyphae have septa and grow in a radial pattern to form a flat, grass-like structure, forming circular white colonies.

Conidiophores branch oppositely at the tip and form short barrel-shaped twigs. One to several conidia are produced at the tips of the twigs, and the conidia are spherical or oval in shape, approximately 2 microns in size, and have a smooth, hyaline surface.

From the above mycological properties, especially morphological characteristics, it was determined that this strain belongs to the genus Trichoderma, which is a Deuteromycota species. Therefore, the present inventors identified this strain as Trichoderma sp. NO.2810 (Trichoderma) sp.
No. 2810) and deposited it with the Institute of Fine Technology.
Its accession number is FERM No. 7388 (FERM
P-7388).

The xanthone derivative of the present invention is derived from the above-mentioned Trichoderma sp. It is induced by culturing microorganisms including No. 2810 and other microorganisms belonging to the genus Trichoderma that are capable of producing the derivatives in an appropriate nutrient medium.
In culturing the above-mentioned microorganisms, glucose, soluble starch, etc., which are commonly used as culture medium components, can be used as carbon sources. The nitrogen source is not particularly limited, but natural nitrogen sources such as peptone, soybean flour, and hydrolysates thereof are preferred. In addition, inorganic salts such as sodium chloride, calcium carbonate, copper sulfate, manganese chloride, and zinc sulfate, as well as micronutrients contained in yeast extract, malt extract, etc., can also be added.

Although culture can be carried out by shaking culture or aerated agitation culture, static culture is most preferable.
Culture conditions vary depending on the medium composition, but usually pH is approximately 6.
-8C and a temperature of about 20-27C. In the case of static culture, a culture period of approximately 10 to 20 days is generally 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 that utilize the physical and chemical properties of the produced substance can be employed. For example, a culture solution is filtered or centrifuged in a conventional manner to separate the cells from the liquid, then methanol or the like is added to the cells, or butanol or the like is added to the liquid to extract the culture, and then the extract is extracted. Further methanol, ethyl acetate, chloroform, ether, n-
After extraction with a suitable combination of solvents such as hexane and benzene, etc., and distilling off the solvent, the residue is gel-filtered with Sephadex LH-20 etc., and each fraction obtained is further subjected to solvent extraction, as necessary. Examples include methods in which operations such as gel filtration, neutralization, concentration, and crystallization are repeatedly performed alone or in appropriate combinations.

In this way, the compound of the present invention represented by the above structural formula (1) can be obtained. The physicochemical properties of the obtained compound of the present invention are as follows, and its structure can be identified from these properties.

(1) Molecular weight Molecular weight determined by FD mass spectrum (Figure 1)
598 (2) Melting point No clear melting point.

(3) Nuclear magnetic resonance spectrum ( ¹ H-NHR; ppm)
The ¹ H-NMR spectrum measured using DMSO- _d6 as a solvent (internal standard TMS) is shown in Figure 2.

(4) Nuclear magnetic resonance spectrum ( ^13C -NMR;
ppm) DMSO- _{d6- CD3OD} as solvent (internal standard
^{The 13} C-NMR spectrum analysis chart measured as TMS) is shown in Figure 3, and the main peaks were as follows.

201.7(s) 108.0(d) 183.0(s) 106.3(s) 165.6(s) 106.0(s) 162.7(s) 101.9(s) 159.2(s) 100.8(d) 158.1(s) 98.1(d) 151.9( s) 48.0(t) 139.8(s) 29.8(q) The compound of the present invention has a specific PDE inhibitory activity of C-AMP, and is useful for various diseases caused by a decrease in C-AMP metabolism, such as It is useful as a preventive or therapeutic agent for arteriosclerosis, hypertension, bronchial asthma, diabetes, cancer, etc., 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, blood flow dynamics, effects on the central nervous system, and 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-
The AMP concentration increases, which can be expected to have pharmacological effects such as inhibiting platelet aggregation, lowering blood pressure, anti-asthma, increasing insulin secretion, and anti-cancer.

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 Trichoderma sp. No. 2810 (Feikoken Bacteria No. 7388) was inoculated into a Monod tube containing 10 ml of a medium with the following composition, and the temperature was 27℃, PH=
7.2, culture was carried out for 3 days in a Monod type shaking culture apparatus.

Soluble starch 2% by weight Glucose 2% by weight Phyton Peptone (manufactured by BBL) 2% by weight Yeast extract 0.5% by weight Sodium chloride 0.25% by weight Copper sulfate (pentahydrate) 0.0005% by weight Zinc sulfate (7hydrate) 0.005% by weight Manganese chloride (tetrahydrate) 0.0005% by weight Calcium carbonate 0.32% by weight Each 500ml Erlenmeyer flask containing 100ml of the medium with the above composition was inoculated with one seed culture obtained above, and cultured at a culture temperature of 27°C. Culture was carried out for 18 days.

The obtained culture solution was heat-treated (at 85° C. or higher for 6 minutes) and then separated into a solution and bacterial cells by vacuum filtration. Add 21 ml of methanol to this bacterial cell (culture solution 21 min).
The mixture was stirred well under NaCl-saturated and acidic (PH3) conditions, and 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.
Extraction was performed by adding NaHCO ₃ aqueous solution.

This aqueous solution was acidified with hydrochloric acid, then dissolved in ethyl acetate, washed with water, and dehydrated to obtain 1.6 g of a concentrated dry product. Dissolve this in a small amount of methanol and use Sephadex LH-20 (manufactured by Pharmacia).
The mixture was applied to a column and eluted with methanol to obtain 196 mg of the compound of the present invention. The compound has the above-mentioned physical properties, and has the 3,3′-
It was identified as diacetonyl-1,1',6,6',8,8'-hexahydroxy-2,2'-bixanthone.

Test example 1 <Inhibitory activity against C-AMP/PDE> (1) Measurement method As enzyme reaction solution, 50μ of 20mM magnesium sulfate solution, 150μ of 6mMC-AMP (manufactured by Boehringer Mannheim, Yamanouchi) solution, 7U/ml alkaline phosphatase ( Made by the same company (derived from calf intestine)
A mixed solution consisting of 550 μl of a sample solution containing 50 μl of solution, 200 μl of 0.05 U/ml phosphodiesterase (manufactured by the same company, from Gyushinzo), and 100 μl of a sample solution each separately containing the compound of the present invention or theophylline as a control compound was prepared. All components except the sample solution were dissolved in 50mM Tris-HCl buffer (PH7.5) before use. After reacting the above reaction solution at 37°C for 45 minutes, 50μ of 55% trichloroacetic acid was added to stop the reaction, and centrifugation (3000 rpm, 10
), and inorganic phosphorus in the supernatant was detected using Phosphor B-Test Wako (Phosphor B-Test Wako).
Quantification was performed using Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.).

The 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 the sample solution is replaced with residual water and added to the reaction solution as a control. Furthermore, 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 compounds of the present invention against C-AMP/PDE was compared with that of theophylline as a positive control, and the results are shown in Table 4.

In FIG. 4, the horizontal axis shows the final concentration of the compound of the present invention or theophylline (sample) in the reaction solution on a logarithmic scale, and the vertical axis shows the inhibition rate. Further, in the figure, 1 represents the compound of the present invention, and 2 represents theophylline. From the figure, 50% of the compound of the present invention
The % inhibitory concentration was 4.5×10 ⁻⁶ M, which indicates that the inhibitory activity was about 500 times stronger than that of theophylline, which was 2.3×10 ⁻³ M.

Test Example 2 <Contraction inhibitory effect on angiotensin in isolated guinea pig ileum> (1) Experimental method A male Hartley guinea pig was killed by blight, and the ileum was removed. After washing and removing intestinal contents, the ileum was cut into small pieces of approximately 3 cm and placed in a Magnus apparatus (bath volume 10 ml, bath temperature 31°C, O ₂ 95% - CO ₂ 5%).
Suspended in Tyrode's solution (mixed gas ventilation).
Since the compound of the present invention is insoluble in water, 1% Tween 80
(Tween80, manufactured by Wako Pure Chemical Industries, Ltd.) 50mM
Theophylline, which served as a positive control, was emulsified in Tris buffer (PH7.5) and emulsified in 50mM Tris buffer (PH7.5).
7.5) and used them as sample solutions. The sample solution was added to the bath, and after 5 minutes, 3 × 10 ^-8 M (final concentration in the bath) of angiotensin was added.
The contraction inhibition rate (%) was calculated by comparing the amount of contraction at this time with the case where only angiotensin was added.

(2) Results Figure 5 shows the results of examining the contraction-inhibiting effect of the compounds of the present invention on angiotensin using isolated guinea pig ileum. In FIG. 5, the horizontal axis represents the logarithm of the concentration of the specimen ((1) represents the compound of the present invention, and (2) represents theophylline) in the bath, and the vertical axis represents the contraction inhibition rate against angiotensin.

The concentration of theophylline used as a positive control and the compound of the present invention showing 30% contraction inhibition were each 1×
10 ⁻⁴ M and 1×10 ⁻³ M, and it was observed that the contraction inhibitory effect of the compound of the present invention was stronger than that of theophylline.

[Brief explanation of drawings]

Figure 1 is a mass spectrum analysis diagram of the compound of the present invention, Figures 2 and 3 are nuclear magnetic resonance spectrum analysis diagrams of the compound of the present invention, and Figures 4 and 5 are C-AMP/PDE of the compound of the present invention, respectively. 2 is a graph showing the inhibitory activity and contraction suppressing effect on.

Claims (1)

[Claims] 1. Structural formula 3,3'-diacetonyl-1,1',
6,6',8,8'-hexahydroxy-2,2'-bixanthone.