JPS60228471A - Furanone derivative - Google Patents

Abstract

NEW MATERIAL:A furanone derivative expressed by formula I (R1 is OH, and R2 is 3-methyl-2-butenyl or both together represent a group expressed by formula II; formula III is a group expressed by formula IV or V). EXAMPLE:3-( 7-Hydroxy-2,2-dimethyl-2H-1-benzopyran-6-yl )-4-hydroxy-5-{[ 4-hydroxy-3-(3-methyl-2-butenyl)phenyl]methylene}-(E)-2(5H)-furanone. USE:Useful as a preventing and treating agent for arteriosclerosis, hypertension, bronchial osthma, diabetes, cancer, etc., and having phosphodiesterase inhibitory activity specific for cyclic adenosine monophosphate, particularly as a remedy for hypertension. PREPARATION:For example, a strain, separated from soil, and belonging to the genus Aspergillus is cultivated in a culture medium usually used for cultivating microorganisms at 4-9pH and 25-37 deg.C to give the aimed compound expressed by formula I.

Description

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

1 The furanone derivative of the present invention is a novel compound that has not been described in any literature, and is represented by the following general formula (1).

[In the formula, R1 represents a hydroxyl group and R2 represents a 3-methyl-2-butenyl group, or the compounds of the present invention that are included in the above general formula (1) are specifically named as follows.

(1) 3-(7-hydroxy-2,2-dimethyl-2
H-1-benzobilan-6-yl)-4-hydroxy-
5-((4-hydroxy-3-(3-methyl-2-butenyl)phenyl)methylene)-(E)-2(5H)-furanone (hereinafter referred to as "Compound A") (2) 3-(2, 3-dihydro-6-hydroxy-2
-(1-hydroxy-1-methylethyl)benzofuran-
5-Ilk-4-hydroxy-5-((4-hydroxy-3-(3-methyl-2-butenyl)phenyl)methylene)-(E)-2(5H)-furanone
(3) 3-(2,4-dihydroxy-5-(3-medy-2-butenyl)phenyl-4-hydroxy-5-
((4-Hydroxy-3-(3-methyl-2-butenyl)phenyl)methylene]-(E)-2(5H)-furanone (hereinafter referred to as "Compound 11!IcJ") The present inventors cultured microorganisms. As a result of a series of studies on the cultured products obtained from Aspergillus (A.
The fact that microorganisms belonging to the genus C-AMPJ (hereinafter referred to as "C-AMPJ") produces a substance that has a phosphogesterase (rPDEJ) inhibitory activity specific to cyclic adenosine monophosphate (hereinafter referred to as "C-AMPJ"); The present invention was completed based on the discovery that the compound is a novel compound represented by the above general formula (1).

The compound having PDE inhibitory activity of the present invention is obtained by culturing a microorganism belonging to the genus Aspergillus and having the ability to produce the compound in a commonly used nutrient medium containing glucose, starch, etc. as the main carbon source, and is isolated from the culture. Manufactured by refining.

As an example of the above-mentioned microorganism most suitable for producing the compound of the present invention, a strain belonging to the genus Aspergillus that the present inventors isolated from soil can be exemplified. 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 75 mm after 10 days from the start of culture.
~80++ m. The village is mostly white, circular and flat. The hyphae grow densely in concentric circles, and as many conidia are formed, the entire surface of the colony takes on a grayish-white powdery appearance, which then changes from ocher to yellowish brown. The underside of the colony is ocher-colored, and secretion of a yellow-brown pigment is observed within the agar.

2) Growth on Czapek agar medium was good, and after 14 days of culture, the colonies had a diameter of 50 to 60 cm.
Reach 18. The village is white and almost circular, but the periphery has an irregular wavy shape, and shallow bends can be seen around the entire village. Conidia are formed from the periphery toward the center of the colony, and the surface of the colony turns into powder. Afterwards, colorless and transparent droplets are formed in the center of the village, and the surface changes from pale yellow to yellow. The underside of the colony is yellowish-brown to brownish-brown, and secretion of pale yellow pigment can be seen within the agar.

(B) Physiological properties pH and temperature range in which this strain can grow, and optimal growth 1
1H and temperature are as shown below.

pH/temperature growth range 2.0-11.4 12-42°C Optimal growth conditions 3.6-8.9 32-37°C For pH, use Sabouraud liquid medium at 27°C for 7 days; for temperature, use Sabouraud agar. Each was cultured for 7 days using medium (I]H6,2).

(C) Morphological properties Sterile hyphae have septa and form white or grayish-white, circular, flat colonies. As conidia settle, the surface of the colony turns powdery and ocher-colored. Conidial heads are ocher-colored with spherical and radial formations, and the diameter is 50-150 μm.
It is. Conidiophores exist with or without septate walls, with somewhat thick walls and uneven or knob-like protrusions on the surface. The diameter is 5 to 12.5 μm, and the length is 2
It is 50 μm or more and is colorless and transparent. The top layer is colorless and transparent and approximately spherical with a diameter of 17 to 40 μm. Kyoko is 2
It is heavily epiphyted, with the primary stoma being 12.5 to 17 μm in diameter, and the secondary stoma being 2.5 to 50 μm in diameter. Conidia are 2.5-3 in diameter
．． It has a nearly spherical shape of 3 μm, is connected in a chain, and is colorless and transparent. No sclerotia are observed.

Based on the above mycological properties, Gilman (J.

C, GNHn)'s "A Manual of 5oilFu"
nai, 2nd ed, The Iowa 5ta
tauntverstty Press, A+++e
S,)owa, u, s.

A., 1957) and Labor et al. (K., B.).

Raper and D, 1. Fennell)'s [The Gerus Aspergillus (The Gen.
usAspergillus, The Willa
ms & Wilkins.

Go, 3altiw+ore, 1965), this strain was found to be Aspergillus ochraeus.
ceus) or a bacterial species very closely related to it, but as no clear evidence has been found for determining the species, this is thought to be Aspergillus sp.N.
o, named 2853 and transferred to the Microtech Institute as 7389
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 Aspergillus sp. No. 2853, according to a conventional method. The culture medium for culturing microorganisms may be the same as those commonly used, and the carbon source may normally be glycose, soluble starch, or the like. Although the nitrogen source is not particularly limited, natural organic nitrogen sources such as peptone and soybean flour hydrolyzate are preferred. As the inorganic salts, sodium chloride, calcium carbonate, copper sulfate, manganese chloride, zinc sulfate, etc. can be used as appropriate. Other micronutrients that can be used include yeast extract and malt extract.

Culture conditions vary depending on the medium composition, but are usually pH 4-9.
The temperature is preferably in the range of 25 to 37°C, and although shaking culture and aerated agitation culture are also possible, static liquid culture is better, as this produces a significant amount of the desired target compound. . 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 method for producing single 11ifI is not particularly limited, and any of various known methods that utilize 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 fluid is filtered or centrifuged according to a conventional method to separate the bacterial cells and the fermentation solution in advance. Next, methanol or the like is added to the bacterial cells to extract the culture, and the extract is further extracted with an appropriate combination of solvents such as methanol, ethyl acetate, chloroform, ether, n-hexane, benzene, etc., and the solvent is distilled off. After that, the remaining residue is gel-filtered with silica gel or Sephadex LH-20 (Pharmacia), etc., and each of the obtained fractions is further subjected to operations such as solvent extraction, gel filtration, neutralization, concentration, and crystallization as necessary. Isolation and purification can be achieved by repeating these steps alone or in appropriate combinations.

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

<Compound A> ■ Molecular weight: 446 (I!l/z=572 was obtained in the mass spectrum of 1-lyacetate (C3383209).) ■ Melting point: Does not show a clear melting point.

(2) Ultraviolet absorption spectrum (UV; nm) As shown in FIG.

(2) Infrared absorption spectrum (IR: cm-') The IR spectrum of the KBr tablet is as shown in FIG.

■ Nuclear magnetic resonance spectrum ('H-NMR:llI)1
m) I)(-NM measured using CDa OD as solvent)
The R spectrum is as shown in FIG.

(1) Nuclear Magnetic Resonance Spectrum (1T C-NMR; I) The 13C-NMR spectrum measured using CDa OD as a solvent is as shown in FIG. 4, and the following main peaks are observed.

176.5 (s) 123.6 (d) 174.5 (s) 116.0 (ci) 157.6 (s
) 115.0 (d) 156.0 (s) 114.6<5) 153.3 (s) 106.0 (d) 145.5 (s) 105.1 (s) 132.
8 (s) 94.8 (r, > 132.7 (d) 77.0 (s) 129.8 (d > 29.2 (t) 129.3 (S) 28.2 ((1) 128. 0 (d) 25.8 (Q) 127.0 (s) 25.8 (Q) 126.0 (d) 17.9 (q) 123.8 (d) From the above various physical and chemical properties, the present invention Compound A is estimated to have the following structure.

Compound B> ■ Molecular weight 464 (m/z in the mass spectrum of t-lyacetate
=590 was obtained. ] ■ Melting point: Does not show a clear melting point.

(2) Ultraviolet absorption spectrum (UV: nm) As shown in FIG.

■ Infrared absorption spectrum (TR: cIIl-') KB
The IR spectrum of the r tablet is shown in FIG.

■ Nuclear magnetic resonance spectrum ('l-l-1-N: DI
llI) IH-NM measured using CD300 as a solvent
The R vector is as shown in FIG.

■+1 gas resonance spectrum ("C-NMR:l)I)m) The 13C-NMR spectrum measured using CD30D as a solvent is as shown in Figure 8, and the main peaks are:
The following are accepted:

176.1 (S) 116.0 (d > 174.7 (s) 114.3 (s) 160.3 (s) 104.8 (d) 156.5 (s) 99.5 (d) 156. 0(s) 95.5(s) 145.5(s) 91.0(d) 132.8(s) 72.5(s) 132.6(d) 31.1(t) 129.8< d) 29.2(t) 129.3(s) 25°7(q) 127.0(s
) 25.0(q) 123.9(d> 25.0(Q) 123.7(d) 17.7(Q) 118.8(s) From the above various physical and chemical properties, compound B of the present invention is estimated to have the following structure.

<Compound C> ■ Molecular weight 462 (m/z in mass spectrum of t-lyacetate
=588 was obtained. ] ■Melting point: Does not show a clear melting point.

(2) Ultraviolet absorption spectrum (LJV: nm) As shown in FIG.

(2) Infrared absorption spectrum (IR: c'') The IR spectrum of the KBr tablet is as shown in Figure 10.

■ Nuclear magnetic resonance spectrum ('H-NMR: ppm)
The l)l-NMR spectrum measured using CD30D as a solvent is shown in FIG.

(1) Nuclear magnetic resonance spectrum ("C-NMR:E) 111I) The 130-NMR spectrum measured using CD30D as a solvent is as shown in FIG. 12, and the following main peaks are observed.

176.0 (s) 114.6 (s) 174.3 (S) 109.7 (d) 161.0 (S) 106.0 (d) 157.5 (s) 105.0 (d) 153. 3 (s) 95.0 (s) 145.5 (s) 90.7 (d) 131.5 (s) 77.0 (s) 128.9 (d) 72.3 (s) 128.1 ( S) 31.3(t) 128.0(d) 28. 2(Q) 127° 5(d) 25.2(q) 126.0(d > 25.2(q) 123.5(
d) 25.1(Q) 114.7(s) From the various physicochemical properties mentioned above, it is estimated that the compound C of the present invention has the following structure.

The compound of the present invention has a PDE inhibitory activity specific to C-AMP, and is associated with various diseases caused by a decrease in C-AMP metabolism, such as arteriosclerosis, hypertension, bronchial asthma, etc.
It is useful as a preventive or therapeutic agent for 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, and 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 azonyl cyclase, and C-AMP
- Decomposed by phosphogesterase (PDE),
The action of these two enzymes regulates the level within the i cell. 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-AMP concentration increases. It is expected to have pharmacological effects such as , hexagonal insulin secretion, and anticancer effects.

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. 2853 (Feikoken Bacteria No. 7389) was inoculated into a mono test tube containing 10 mQ of a medium with the following composition, and 27
Culture was carried out at ℃ and pH=7.2 for 3 days in a mono (Monod) type shaking culture apparatus.

Glycose 2% Starch 2% Phyton 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) Salt 0.0005% zinc sulfate (7
One seed culture obtained above was inoculated into each 500 mG Erlenmeyer flask containing 100 mf2 of the above medium, and statically cultured at 27°C for 12 days.

The obtained culture solution was heat-treated (at 85° C. or higher for 6 minutes), and then separated into furnace solution and bacterial cells by vacuum filtration. Add methanol 179 to this bacterial cell (culture solution 17 (2 minutes),
The mixture was stirred under CQ-saturated and acidic (pH approximately 3) conditions, filtered under reduced pressure, and the methanol extract was concentrated to dryness under reduced pressure. Water was added to the dried product to dissolve it, and the mixture was extracted successively three times with n-hexane and ethyl acetate, and the ethyl acetate extracts were combined and concentrated. Then, 5% NaH was added to this ethyl acetate extraction concentrate.
Extraction was carried out by sequentially adding a CO3 aqueous solution and a 5% NaOH aqueous solution, and the remaining ethyl acetate extracts were combined and concentrated, and washed with water and dehydrated midway through to obtain 6.3 g of an oily concentrate. This oily concentrate was subjected to medium pressure liquid chromatography on silica gel and sequentially eluted with a solvent system of chloroform:methanol to obtain fractions (1) and (2) eluting the target product. (1)
and (2) were separated by preparative thin layer chromatography (developing solvent, ethyl acetate:methanol = 6:1), then applied to a column of Sephadex LH-20, eluted with methanol, and concentrated to dryness. , from (1), the present compound A is 3811 (], and from (2), the present invention compound C is 38
*O got it.

These physical and chemical properties were as described above.

Example 2 The culture furnace liquid (17Q) obtained in the same manner as in Example 1 was
a CQ saturated, acidic (p)I about 3), extraction was performed three times with n-butanol, and the extracts were combined and 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, to this ethyl acetate extraction concentrate, 5% Na HCOa aqueous solution, 5% Na 01
-1 aqueous solution was sequentially added and extracted, the remaining ethyl acetate extracts were combined and concentrated, and washed with water and dehydrated in the middle.
2.6 g of oily concentrate was obtained. This oil was subjected to medium pressure liquid chromatography on silica gel, sequentially eluted with a solvent system of chloroform:methanol, concentrated the fraction eluted with the target product, and then subjected to preparative thin layer chromatography (developing solvent, ethyl acetate: methanol 5:1), then applied to a column of Sephadex LH-20 and eluted with methanol. 231 g of the compound of the present invention and 3 g of the compound C of the present invention
01<+ obtained.

In addition, the 5% NaOH extracted aqueous solution was acidified with hydrochloric acid, extracted three times with ethyl acetate, the extracts were combined and concentrated, and washed with water and dehydrated midway through to obtain an oily concentrate with 7.7
I got g. This oily concentrate was applied to a silica gel dry column and sequentially eluted with a solvent system of chloroform/rotanol to obtain a fraction eluted with the target product. Furthermore, Sephadex L
The mixture was applied to a H-20 column, eluted with methanol, concentrated under reduced pressure, and fractionated using preparative thin layer chromatography (developing solvent: chloroform:methanol = 2:1) to obtain 241!1 g of Compound B of the present invention. .

The compounds A, B, and C of the present invention obtained above each had the above-mentioned physical and chemical properties.

Test Example 1 <Inhibitory activity against C-AMP-PDE> (1) Assay method As an enzyme reaction solution, 20 mM magnesium sulfate solution 50
μ9.61 150 μQ of MC-AMP (Boehringer Mannheim, Yamanouchi) solution, 50 μQ of 7U/IIQ alkaline phosphatase (Boehringer Mannheim, Yamanouchi origin) solution, 0.05 LI/mQ phosphogesterase (Boehringer Mannheim, Yamanouchi) 10 sample solutions each containing 200 μQ of the bovine heart-derived) solution and the compound of the present invention or theophylline as a control compound, respectively.
A mixed solution consisting of 0 μQ and a total of 1550 μQ was prepared. All except the sample solution were 50Il1M Tris-HCl buffer (p
It was used after being dissolved in H7,5>. After reacting the above reaction solution at 37°C for 45 minutes, 55% trichloroacetic acid 50%
Add μQ to stop the reaction, centrifuge (3000 rpm)
10 minutes), and the inorganic phosphorus in the supernatant was analyzed using Phosphor-8 Test Wako (Phosphor B
The 0 C-AMP-PDE inhibitory activity was determined using the 0C-AMP-PDE inhibitory activity (C-Test Wako, manufactured by Wako Junkuwa Industries, Ltd.), based on the amount of inorganic phosphorus produced when distilled water was added to the reaction solution instead of the reagent solution as a control. , subtract the amount of inorganic phosphorus produced when adding the reagent solution,
This value was further divided by the amount of inorganic phosphorus produced in the control and expressed as a percentage.

(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 were
Shown in the figure. In FIG. 13, the horizontal axis indicates the maximum n in the anti-liquid of the secondary sample, using the compound of the present invention or theophylline as the sample.
The concentration is shown on a logarithmic scale, and the vertical axis is the inhibition rate (%
) is shown. In addition, (1) in the figure shows the compound A of the present invention, (2
) represents the same compound B, (3) represents the same compound C, and (4) represents theophylline, respectively.

From the figure, the 50% inhibitory concentration (TCso) of the compound of the present invention is 3.4X10-5M (Compound A>, 9.4X10-
5 M (Compound B) and 8.5×10 M (Compound C), compared to 2.3 for theophylline as a control.
It can be seen that the inhibitory activity is about 68 times, about 24 times, and about 27 times stronger than that of X10-3M, respectively.

[Brief explanation of drawings]

Figures 1, 5 and 9 are ultraviolet absorption spectrum analysis diagrams of the compound of the present invention, Figures 2, 6 and 10 are infrared absorption spectrum analysis diagrams of the same compound, respectively.
Figures 7 and 11 are I H-NMR analysis diagrams of the same compound, respectively, and Figures 4, 8, and 12 are +
The 3C-NMR analysis diagram and Figure 13 are the PD of the same compound.
It is a diagram showing the E inhibitory activity. (Continued from page 1) ■Int, C1,' Identification symbol Internal docket number Procedure amendment exporting party September 10, 1980 Commissioner of the Japan Patent Office Manabu Shiga 1 Case indication 1988 Patent Application No. No. 85949 2 Name of the invention Furanone derivative 3 Relationship with the case of the person making the amendment Patent applicant Otsuka Pharmaceutical Factory Co., Ltd. 4 Spontaneity of the agent 6 Contents of the amendment subject to the amendment 1) Page 4, line 5 and 7 of the specification In line 16 of the page, “A
5perallusJ and each r A 5pe
Correct it as ro i l lus J. 2) On page 7, lines 14-15 of the specification, "Geas" is corrected to "Genus". 3) On page 7, lines 18-19 of the specification, [A spergillus ochraceus]
ceus J [Aspergillus ochraceus]
Correct it with J. 4) Specification page 8, line 10 and page 18, line 9 L-r
Correct "glucose" to "glucose". On page 21, line 10 of the specification, "rotanol" is corrected to "methanol J." 6) On page 22, line 2 of the specification, "r6mMC-P" is corrected to r6mM C-AMPJ. On page 22, line 18 of the specification, the text "r8-testJ" is corrected to "FB-Test J." 8) In the first line and second line of page 23 of the specification, the words "reagent liquid" are corrected to read "sample liquid." 9) On page 24, line 8 of the specification, the text rlH-NMRJ is corrected to r'H-NMRJ. (that's all)

Claims (1)

[Claims] ■ A furanone derivative represented by the general formula (wherein R1 is a hydroxyl group and R2 is 3-methyl-2).