JPS62215551A - Novel physiologically active substance tpi and production thereof - Google Patents

Abstract

NEW MATERIAL:The compound of formula (R is beta-D-glucopyranosyl, beta-D- galactopyranosyl, 6'-O-acetyl-beta-D-glucopyranosyl, 6'-O-acetyl-beta-D-galactopyranosyl or H) or its salt. USE:It has an activity to inhibit cyclic adenosine-3',5'-monophosphate phosphodiesterase and is useful as a medicine such as brochodilator, cardiotonic agent, smooth muscle relaxant, hormone secretagogue, etc., or a reagent for research. PREPARATION:The physiologically active substance TPI can be separated from the culture product of a microbial strain capable of producing the physiologically active substance TPI of formula wherein R is not H such as Nodulisporium sp M5220 (FERM P-8133). Another compound of formula wherein R is H can be produced by treating the compound of formula wherein R is other than H with a glycoside bond hydrolase (e.g. beta-glucosidase) in an aqueous medium.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application J The present invention relates to a novel physiologically active substance TPI having cyclic adenosine-3', 5'-monophosphate phosphozoesterase inhibitory activity and a method for producing the same.

[Conventional technology]

Cyclic adenosine-3', 5'-monophosphoric acid (hereinafter referred to as c
-AMP) is said to be involved in the cell membrane machinery btL, cell proliferation and differentiation as a second messenger in vivo. This c -AA'I
P is decomposed to form 5'-adenosine monoly7r1! The enzyme that converts cyclic adenosine-
3',5'-monophosphate phosphozoesterase (hereinafter referred to as P
(abbreviated as DE), and substances that inhibit this increase c-AMP levels in the body, and therefore have various physiological effects, such as bronchodilation, cardiac inotropy, smooth muscle relaxation, and promotion of hormone secretion. It is known that it has the following effects.

Conventionally, PDE inhibitors produced by microorganisms include PDE
-1, PDE-n (Agricultural Biological Chemistry (Agr, Biol.

Chem, ), 42 (7)-1331-1336.
1978), Terferol
(The Sharnal Op Antibiotics (Th
e Journal of Antibiotics)
, Dan (1), 6-9, 1984), CC-1065
[Id., 33(8), 902-903° 1980), Reticulol (Id.).

28 (7), 558-560.1975], but all of these are produced by actinomycetes. In addition, APD is produced by bacteria.
-1%APD -II, APD-■ [Ibid., Royal, 19
4-196.1983) are known.

Further, as chemically synthesized products that inhibit PDE, theophylline, noverine, nicardipine, etc. are known, and these are used as pharmaceuticals such as bronchodilators and vasodilators.

[Problems that the invention attempts to solve]

As mentioned above, the search for physiologically active substances produced by microorganisms is medically useful, and in particular, the search for *J substances that inhibit PDE' is medically useful and c-
This is extremely useful for research on AMP, etc.

[Means for solving problems]

In order to obtain new physiologically active substances, the present inventors isolated many microorganisms from the natural world and investigated the biological properties of the products. It has been found that a substance that strongly inhibits the activity of PDH is produced in microorganisms belonging to the genus Nodulisporium. Then, the PDE inhibitor was collected from the culture and its physicochemical properties were examined, and as a result, it was confirmed that it was a new substance of 4 types having the structural formula described below.
Each of them is TPI-1, TPI-2, TPI-3, T
It was named PI-4.

Furthermore, the present inventors have determined that the above TPI-1゜TPI-2
, learned that a glycosidic bond-eliminating substance having the structural formula described below can be obtained by treating TPI-3 or TPI-4 with a glycosidic bond hydrolase to decompose the glycosidic bond, and this substance t-TPI-5 It was named.

In this specification, TPI-1% TPI-2, TPI-3
, TPI-4 and TPI-5 are collectively referred to as TPI.

That is, the present invention relates to a compound of the formula The present invention provides a novel physiologically active substance TPI represented by -D-galactopyranosyl group or hydrogen atom) or a salt thereof, and a method for producing the same.

In formula (I), a substance in which R is a β-D-glucopyranosyl group is TPI-1, a substance in which R is a /-D-galactopyranosyl group is TPI-2, and R is 6
The substance in which R is 6′-〇-acetyl-β-D-glucopyranosyl group is TPI-3.
-D-galactobyranosyl group is TPI-4
The substance in which R is a hydrogen atom is TPI-5.

The formula isolated by the present inventors (wherein " is β-D-glucopyranosyl, β-D-galactopyranosyl, 6'-0-acetyl-β-D-glucopyranosyl or 6'-〇-acetyl-β -D-galactopyranosyl group)
The strain M5220 that produces I (11) has the following characteristics.

U) Morphological characteristics M5220 strain is grown on Czapek agar medium, wort agar medium,
Cultivate on potato-glucose agar medium, tt! The findings expressed are as follows.

Vegetative hyphae are solitary or hyphal bundles (S)mnemata)
f-shaped, yellowish brown and 2.5 to 3. Stretched to θμm,
Its wall is m-. Conidial size is 50-130X
2.5-3. It is colorless to pale yellow in color and has branches7 and septa, the walls of which are steel surfaces. Uniconidiogenic cells are solitary or Fi2-3 whorled at the tip of the conidiophore, and the size It is 6-20×2.0-2.5 μm and colorless to pale yellow.

Is the mode of conidia formation symposozoid? Sympod
ulosporae). The conidia are colorless, oval, slightly tapered at the base, and 3.0 in size.
~5.0 x 2.0~2.8 tim, one cell with steel surface walls.

Growth status on each culture medium VC Culture was performed at 26° C. for 10 days on Tanimune medium, and the following findings were observed. The colors shown are Stingray, Kone 2 Tuzo and Zoe.

Ecchi, Wansha, Metan Handbook Oshi
Color 3rd edition, Yale Meta-7, 07 Don CA +
Kornerup and J+H, Wanscher
.

Methuen handbook of colou
r # 3 rded, I E) rreMethuen
+ London (1978)).

■ Growth on Zapek agar medium is slow, reaching a diameter of 11 to 13 mm in 10 days at 26℃, and the bacterial flora is flat and velvety to slightly fluffy. The color is dark green (dar
k green 30 F 6 ). The periphery is smooth. Exudates and diffusible dyes are not released.

The back side is dark green (dark green 27 F 5
).

■ Wort agar medium growth is slow and iti diameter is 15-171m1 in 10 days at 26℃
．． The bacterial flora is thick and swollen and slightly fluffy. The color is dark green (da
rk green 27 F 3 )-1 peripheral area is smooth.

Exudates and diffusible dyes are not released. The back side is dark green (d
ark green 27f;'3).

■ Vallejo Grape Shield Agar Medium Growth is slow at 26℃lO days, with a diameter of 15~16u0, the bacterial flora becomes thick and swollen and slightly fluffy.

The color is dark green (dark green 27 F 3)
o Is the peripheral area flat r1? . Exudates and diffusible dyes are not released.

The m side is dark green (dark green 27 F 3
).

(3) Physiological properties ■ Growth pH range: 1 to 9.5 Optimal growth pH: 3 to 7 ■ Raw temperature range: 13 to 40°C Optimal growth degree = 25 to 30C Mycological properties, especially This strain belongs to Deuteromycetes because it forms conidia without sexual formation and the hyphae have septa. In addition, this strain has a synsynthetic mode of conidia formation. It is Schlossubilla type, the conidiophores are well branched, and the conidia-forming cells are often whorled.
It is a strain belonging to the genus Jum.

Therefore, in order to distinguish this strain from known strains, we used M5220 (Nodulisp sp.
orium sp.
Deposited as No. 3 (FEBMP-8133).

The physiologically active substance TPI (n) of the present invention is, for example, a physiologically active substance TPI (n) that deceives the genus Nodouris and IJum.
n) It is produced by culturing the producing bacteria and collecting the physiologically active substance TPI(u)'fr from the culture.

The physiologically active substance TPI (II) producing bacteria used in the present invention include the above-mentioned Nodourisborium S.B., M52
20 are available, including natural mutant strains and artificial mutant strains,
Nodori squirrel? TPI (n), a physiologically active substance belonging to the genus Rium
Any strain capable of producing can be used in the present invention.

In the method of the present invention, culturing can be carried out under conditions generally used for culturing molds.

As the medium, a nutrient medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be digested, and mineral salts and vitamins as necessary is used.

Charcoal: A sources include sugars such as glucose, fructose, maltose, sucrose, glucose, galactose, dextrin, starch, glycerin, and sorbitol, soybean flour such as soybean oil, cottonseed flour, corn stew, etc. Liquor, malt extract, casein, amine ffl, urine g, -/
Ammonium salts, nitrates, etc. may be used alone or in combination. Other inorganic salts such as phosphates, 1-gnesium salts, calcium salts, sodium salts, potassium salts, iron salts, manganese salts, and vitamins such as B vitamins and calcium chloride may be added as appropriate. Ru.

In culturing, the medium may be either liquid or solid as long as it contains the above-mentioned nutrient source, but it is usually preferable to use a liquid medium and perform shaking culture or aerated agitation culture. The pH of the medium is 3
~7. The culture temperature is preferably 25 to 30°C. In the case of liquid culture, the culture time is usually 2 to 10 days, but the 8 weight of the physiologically active substance TPI (11) in the culture reaches its maximum in about 5 days, so the culture for 4M is terminated at this point. is preferable. It goes without saying that culture conditions such as medium composition, medium liquid properties, culture temperature, culture time, etc. should be adjusted and selected as appropriate to obtain favorable results depending on the type of bacterial strain used and external conditions. . If foaming occurs in liquid culture, antifoaming agents such as silicone oil, vegetable oil, and surfactants are used as appropriate.

Physiological activity '#negative TPI (11) thus obtained
is present in the culture medium 11F as a king, so the culture should be filtered with a filter aid such as Celite, Nori-Lite, High Flow Suit Q-cell, etc., or centrifuged to separate the culture P solution and bacterial cells. It is a good idea to separate the TPI (II) from the culture and collect the menstrual period from the cultured P fluid.

In order to separate and purify the physiologically active substance TPI (■) from the culture medium filtrate, extract the F solution with a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate, butanol, etc. Bioactive TP as a composition by
I (II) is destroyed. To further make 4#,
Adsorption chromatography using silica gel, alumina, activated carbon, nonionic adsorption resin, etc., inverse 1 (1) partition chromatography using alkyl group-bonded silica gel, gel filtration using a gel RI carrier, etc., to separate and purify ordinary organic heavy substances. It is preferable to use a suitable combination of means.Specifically, after adsorbing the composition in a column completely filled with an adsorbent such as silica gel or alumina, chloroform-inzolo, q-nol-acetic acid, ethyl acetate-inzolo This is carried out by bathing out using a mixed solvent such as nolinol-acetic acid, ethyl acetate-methanol-acetic acid, etc. Checking the bathing out of the target product is carried out using silica gel thin I-chromatography or 9
Inhibition activity using PDE of heart mountain rice 4! +: Can be performed with a dramatic shout.

By these column chromatography, TPI-1
, TPI-2, TPI-3 and TPI-4 can be separated and isolated respectively. R in formula (1)
TPI-5, where is a hydrogen atom, the moiety has the formula CH! (CH
z ) scHs CHz (CHz ) sCHs (
In the formula, R# represents a hydrogen atom), the physiologically active substance TPI is a physiologically active substance TP represented by the formula (1[)
It can be obtained by enzymatically decomposing glycosidic bonds by treating I or a salt thereof with a glycosidic bond hydrolase or a treated product thereof in an aqueous medium.

As a glycosidic bond hydrolase, TPI (II
) is a TPI-1 or TPI-3 substance, β-glucosidase may be used;
If the substance is PI-24 or TPI-4, β-galactosidase may be used.

The above-mentioned enzyme may be a glycoside bond hydrolase derived from any animal, plant, or microorganism, and commercially available β-glucosidase and I-galactosidase are usually used.

When the enzyme is derived from a microorganism, it exists as an extracellular enzyme or an intracellular enzyme depending on the enzyme-producing microorganism. In the case of an extracellular enzyme, an enzyme preparation is used which has been purified to various stages by a known enzyme separation and purification method from a culture p solution which has been sterilized from a culture of the present enzyme-producing bacterium. When the enzyme is an intracellular enzyme, it can be used in the form of live cells obtained from a culture of the enzyme-producing microorganism. Furthermore, bacterial cells treated by physical or chemical methods, such as dried bacterial cells ground with a hydrophilic organic solvent such as acetone, methanol, ethanol, etc., materials destroyed by ultrasonic treatment, cetyl bildiram chloride, A bacterial cell lysate treated with a surfactant or the like or an enzyme agent separated therefrom by known enzyme separation and purification means can also be used in the present invention.

As mentioned above, the processed product of the enzyme refers to all products that have the present enzyme activity and exist in the form of woodcutter, but are not limited to this, and include immobilized enzymes or immobilized enzymes immobilized on a suitable carrier. It means a bacterial cell producing hydrostatic water.

The above-mentioned immobilized enzyme or immobilized omega form can be prepared by a known method for producing an immobilized enzyme and an immobilized image. For example, enzymes can be prepared using covalent bonding, ionic bonding,
An immobilization method using a carrier binding method such as a physical adsorption method,
A method in which enzymes or bacterial cells are immobilized by a cross-linking method, an entrapment method or a lattice entrapment method in which enzymes or bacterial cells are coated in a microcapsule, hollow system, membrane, etc. with a semipermeable membrane. This can be done by a method of immobilization. As mentioned above, by immobilizing the present enzyme or the microbial cells that produce it, it can be used continuously.

The above enzyme reaction is preferably carried out within the optimum pH range of the enzyme. The reaction temperature is preferably carried out within the optimum temperature range of the enzyme, but is usually carried out at about 25 to 37C. The reaction time was determined using thin J-chromatography (TLC), high performance liquid chromatography (TLC), high performance liquid chromatography (TLC), high performance liquid chromatography (TLC), etc. Togelaf E (
Since TPI-5 can be tracked using K such as HPIc), the reaction can be appropriately terminated after waiting for TPI-5 to accumulate to the maximum. When the above-mentioned enzyme is used in the form of an immobilized enzyme or immobilized bacterial cells, the enzymatic reaction can be carried out continuously.

From the enzyme reaction solution thus obtained, TPI-5t
- To collect, make the pH of the reaction solution acidic, extract with a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate, butanol, and concentrate the extract to obtain a composition. For further purification, bioactive product 'J[TPI
A can be expressed by chromatography, gel filtration, etc. in the same manner as the separation and purification method (n).

To convert the physiologically active substance TPI into a salt, it can be converted into a salt by a conventional method at the composition stage, but it is preferable to convert it into a salt after isolation. Preferred salts include alkali metal salts such as sodium and potassium, alkaline earth salts such as calcium and magnesium, aluminum salts, other gold salts, ammonium salts, salts with known basic amino acids, and known organic amines. Examples include salt.

The physicochemical properties of each of the physiologically active substances TPI obtained as described above are shown below.

Physiologically active substance TPI-1> ■ Color and properties of the substance White powder ■ Distinction between neutral, acidic, and basic Acidic ■ Elemental analysis (Cs4H4sOtz ・3/2H*
CB Actual value 60.43% 7.48 Theoretical value 60.44 7.56% ■ Molecular formula % formula % ■ Molecular weight (according to FAB mass spectrum) ■ Specific optical rotation (a)"5- 20.60 (C=1.0, Mea
l ) ■ Ultraviolet absorption spectrum in MeOH or acidic MeOH λmax (nm): 253 (E ''250 )cI
n 311 Shoulder λmax (nm) in alkaline MeOH: 240M 300 (E""350) crR (Figure 1) (a) Infrared absorption spectrum (KBr method) 3400
, 2930.2850.1720.1590.1470
．． 1430.1370.1340.1250.1170
．． 1140.1060 cm 'K has a characteristic absorption band.

(Figure 6) ■ Proton nuclear magnetic resonance spectrum (in heavy methanol,
100 horses) δ (ppm): 0.8B (t, 3H), 0.90
(t, 3H) 1.31 (m, 20) 1), 1.6
1 (m, 4H) 2.68 (t, 2H), 2.94
(t, 2) 1) 3.40-4.00 (5H), 4
．． 89 (d, IFl) 6.42 (d, IH), 6
．． 61 (d, d, 2M) 6.71 (d, IH) (6) C-13 nuclear magnetic resonance spectrum (in heavy methanol, 25 MHz) 174.1 (41168, 2 (sl 1 64.2 (
aλ 161.8 ← entering 158.4 (s), 156.0 (
Q entry 149.1 (a), 145.2 (string 116.5 (
d), 11 6.1 (s:% 11 3.5 (smu 1
12.1 (d included 109.3(d), 103.4(d),
102.7 (dK78.6 (dK78.4(d), 7
5.3 (d), 71.5 (d included 63.0 (t),
37.2 (t), 35.2 (enter 33.3 (L),
33.3(t,% 33.3(t), 33.0(t
), 31.1(t), 30.9(tl, 30.6(t)
, 30.6 (t), 24.0 (t), 24.0 (t;
%14.8(q), 14.8(q), ■ Solubility Soluble in lower alcohols such as methanol, ethanol, butanol, ethyl acetate, dimethyl sulfoxide, and alkaline water. Insoluble in hexane, benzene and petroleum ether.

■ Color reactions Potassium permanganate reaction, ferric chloride reaction, iodine reaction, Molisch reaction positive. Ninhydrin reaction and iodoform reaction were negative.

0 Thin layer chromatography (manufactured by Tokyo Kasei Co., Ltd., using silica gel f) Development Solvent Rf value Chloroform: Inderonol: 6rfRO, 25
(10:4:0.1) Ethyl acetate: isozoro, e-nol: acetic acid 0.
36 (10:4:0.1) Ethyl acetate:acetone:acetic acid 0.1
7 (6:6:0.1) Rioroform: Methanol: Acetic acid 0.38 (10:2:
0.1) Physiologically active substance TPI-2> ■ Color and properties of the substance White powder ■ Distinction between neutral, acidic, and basic Acidic ■ Elemental analysis (C34&s 012・) as hO) C
H Actual value 61.79% 7.56% Theoretical value 61.26% 7.51% ■ Molecular formula % Formula % ■ Molecule k (according to FAB mass spectrum) ■ Specific optical rotation ('') B-12,60 (C= 0.9-MeOH
) ■ Ultraviolet absorption spectrum λmax (nm) in MeOH or acidic MeOH: 253 (E" 230) Infrared absorption spectrum (KBr method) 3400, 2
930.2850%1720.1600.1470.1
410.1320.1240.1170.1140.1
05Q3″″1 has a characteristic absorption band.

(Figure 7) ■ Proton nuclear magnetic resonance spectrum (in heavy methanol,
100MH2) δ (ppm): 0.87 (t, 3H), 0-89 (t
, 3H) 1.31 (m, 20H), 1.60 (m,
4H) 2.68 (t, 2H), 2.94 (t
, 2) 1) 3.50-4.00 (5H), 4.88
(d, IH) 6.42 (d, IH), 6.64 (d,
d, 2H) 6.74 (d, IH) [Phase] C-13 nuclear magnetic resonance spectrum (in heavy methanol, 25 MHz) 174.1 (s), 168.2 (s), 164.2
(s), 161.8(a), 158.6(s), 156
．． 0 (a), 149.1 (s), 145.2 (+9
), 116, '6(d), 116.1(s), l
l 3.4(s), 112.1(d), 109.4(d
), 104.1 (d), 102.8 (d), 77.4
(d), 75.3 (d included 72.7 (d), 70.5 (d
Enter 62.7 (t), 37.2 (enter 35, 2 (t), 3
3.3 (t), 33.3 (this book 33.3 (t), 33
．． 0(t)%31.1(difficult 31.0(t), 30.6
(t), 30.6(t), 24.0(t), 24.0(
t), +4.8(q), 14.8(q)■ Solubility Soluble in lower alcohols such as methanol, ethanol, butanol, ethyl acetate, dimethyl sulfoxide, and alkaline water. Insoluble in hexane, benzene and petroleum ether.

@ Color reaction Potassium permanganate reaction, ferric chloride reaction, iodine reaction, Molisch reaction positive. Ninhydrin reaction and iodoform reaction were negative.

◎ Thin layer chromatography (manufactured by Tokyo Kasei Co., Ltd., using silica gel f) Development Solvent Rf value Chloroform: Isozoro/Q-Nol: Acetic acid 0.19 (10:4
:0.1) Ethyl acetate: Isoderono Q-nol: Vinegar fi
0.21 (10:4:0.1) Ethyl acetate:acetone:vinegar r110.14 (6:6:0
．． 1) Chloroform: methanol: vinegarffi
0.22 (10:2:0.1) Physiologically active substance TPI-3> ■ Color and properties of the substance White powder ■ Distinction between neutral, acidic, and basic Acidic ■ Elemental analysis CH Actual value 61.93% 7 .36% Theoretical value 62.59% 7.30% ■ Molecular formula % formula % ■ Molecule t (according to FAB mass spectrum) ■ Specific optical rotation Cα] f3-19.6° (C=0.6, Menu
) ■ Ultraviolet absorption spectrum 31O (waste) (Figure 3) ■ Infrared absorption spectrum (KBr method) 3400.29
25.2850.1720.1650.1600.14
60, 1240.1130.1040m with characteristic absorption @.

(#!8 figure) ■ Proton nuclear magnetic resonance spectrum (in heavy methanol,
100M days 2) δ (ppm): 0.88 (t, 3)1), 0
．． 90 (t, 3H) 1.31 (m, 20H),
1.61 (m, 11) 2.08CB, 3H), 2.68
(t, 2H) 2.94 (t, 2)i), 3.20
~4.00 (m.

3H), 4.00-4.60 (m, 2H)4.
35 (d, LH), 6.44 (d, IH) 6.59 (d
, d, 2H), 6.73 (d, I H) [Yes]
C-13 nuclear magnetic resonance spectrum (in heavy methanol, 25
MHz) 174, 2(a), 173.1(a), 167
．． 9 (aL 164.4 (s included 161.7 (a),
158.3 (IH included 156.0 (8 included 1 49.2
(s), 145, 2(s), 116.5 (dk 116
．． 3(a), 113.3(s), 112.1(d),
109.4(d), 103.3(dk 103.0(d
), 78.3(d), 75.8(d), 75.2(d)
%71.8(dk65.0(tj%37, 2(t), 3
5.2(t), 33.3(t), 33.3(t), 33
．． 3(t), 32, 9(t), 3 t-t(tk 30
．． 9 (tK30.6(t), 30.6(t), 24.0
(t), 24.0( enter 21.0(q), 14.8(q
), 14.8 ■ Soluble Soluble in lower alcohols such as methanol, ethanol, butanol, diethyl vinegar, dimethyl sulfoxide, and alkaline water. Insoluble in hexane, benzene and petroleum ether.

■ Color reactions Potassium permanganate reaction, ferric chloride reaction, iodine reaction, Molisch reaction positive. Ninhydrin reaction and iodoform reaction were negative.

◎ Thin layer chromatography (manufactured by Tokyo Kasei Co., Ltd., using Kurikadal f) Development Solvent Rf value dichloroform isoderono Q-nor: Vinegar rRO, 54 (10
:4:0.1) Ethyl acetate: isofrononol:acetic acid 0.
51 (10:4:0.1) ethyl acetate:acetone:vinegar MO,
35 (6:6:0.1) Chloroform:methanol:vinegar fi (
152 (10:2:0.1) Physiologically active substance TPI-4> ■ Color and properties of the substance White powder ■ Distinction between neutral, acidic, and basic Acidic ■ Elemental analysis C) 1 Actual value 62.35% 7 .57% Theoretical value 62.59 arrogant 7.30 ring ■ Molecular formula % formula % ■ Molecule i (according to FAB mass spectrum) ■ Specific optical rotation ('' ) 3-13.3 (C = 0.4, Men
u ) ■ Ultraviolet absorption spectrum λy'B0H (nrn): 252 (E' is 217
2) ax 310/Tl (Figure 4) (a) Infrared absorption spectrum (KBr method) 3420
．． 2930.2860.1730.1660.1610
．． 1470.1240.1140.1040cIn has a characteristic absorption band.

(Figure 9) ■ Proton nuclear magnetic resonance spectrum (7 in methanol, 100 MHz) δ (ppm): 0.87 (t, 3 H), 0.9
0(t, 3) 1) 1.31(m, 201), 1.61(
m, 4H) 2.73 (a, 3B), 2.68 (t, 2H
) 2.95 (t, 2H), 3.20-4.00
(m.

2B), 4.00-4.60 (m, 2H) 4.87 (d
, IH), 6.43 (d, 1) 1) 6.60 (ci,
c+, 2H), 6.73 (d, 1) 1) Q) oC-13
Nuclear magnetic resonance spectrum (in heavy methanol, 25 MHz
) 1 74.2 (s), 173.0 (s tile 168.0
(aj% 164.2(sL161.8(11), 1
58.4(sL 156.0(sL149.1(s),
145.2 (11 in 1 16.5 (dK 1 16.
1(s), 113.5(sL111.9(d)% 1
09.4(d), 103.7(d), 102.9(
d included 75.2 (dK 74.7 (d), 72.5 (d
K 70.4 (d), 64.9 (t), 37.2 (enter 35.2 (t), 33.3 (t), 33゜3 (t
), 33.3(input 33.0(t), 31.1(t,%
31.0 (1), 30.6 (enter 30.6 (t), 24
．． 0(t), 24.0(entered 21.0(q entered 14
．． 8 (Q included) 14.8 (Q) ■ Solubility Soluble in lower alcohols such as methanol, ethanol, butanol, ethyl acetate, dimethyl sulfoxide, and alkaline water. Insoluble in hexane, benzene, and petroleum ether.

@ Color reaction Potassium permanganate reaction, ferric chloride reaction, iodine reaction, Molisch reaction positive. Ninhydrin reaction and iodoform reaction were negative.

0 Thin layer black thornfish (manufactured by Tokyo Kasei Co., Ltd., using silica gel f) Development Solvent Rf value dichloroform isoderono Q-nol: Vinegar rIR0,46
(10:4:0.1) Ethyl acetate: Inderononol: Vinegar #0
．． 43 (10:4:0.1) Ethyl acetate:acetone:vinegar rRO, 29 (6:6:0.
1) Chloroform:methanol:vinegar [0.48 (10:2:
0,1) Physiologically active substance TPI-5> ■ Color and properties of the substance White powder ■ Distinction between neutral, acidic, and basic Acidic ■ Elemental analysis CH Actual value 69.30'% 7.72% Theoretical value 69. 11 whisper 7.87% ■ Molecular formula % Formula % ■ Ultraviolet absorption spectrum 1 ”eoH (nm): 271 (E i2389)
aX 305 (E1% 243) 1 country (Figure 5) ■ Optical external absorption spectrum (KBr method) 3320.29
20.2850.1650.1600.1580.14
50%1300%1240.1200.1140.10
It has a characteristic absorption band of 60 oaK.

(Figure 1θ) ■ Proton nuclear magnetic resonance spectrum (1i chloroform, 100MHz) δ (PPffl): 0.85 (t, 3H), o
, ss (t, 3H) 1.27 (m, 201), 1
．． 60 (m, 4H) 3.02 (t, t, 4F]), 6.
32 (8, 2) 1) 6.62 (d, IH), 6.74 (
d, IH) 11.27 (s, 1fi) (yu C-13 nuclear magnetic resonance spectrum (■ in chloroform, 25 8 pulls 2) 174.7 (sk l 69.3 (s1% 166.3 (
sk 165.3 (toge 1 61.4 (sk l 55)
．． 0(aL 1 50.0(al l 49.5
(aL116.2(d+, 111.5(d), l 0
9.011 entry 108.7(s to 104.2(at,
Enter 101.8(d), 37.3(t), 36.7(32.3(t), 31.9(tl, 31.9(
Enter 31.9 (L enter 29.9 (t), 29.9 (t
), 29.3(t), 29.2(t), 22.8(t)
, 22.8(t), 14.2(q), 14.2(q) ■ Solubility Compatible with lower alcohols such as methanol, ethanol, butanol, ethyl acetate, dimethyl sulfoxide, and alkaline water. Insoluble in hexane, petroleum ether.

Color reaction, potassium reaction, ferric chloride reaction, iodine reaction. Ninhydrin reaction, iodoform reaction, Molisch reaction positive test Thin m chromatography (manufactured by Tokyo Kasei Co., Ltd., using silica gel f) Development Solvent Rf value dichloroform indelonol: Vinegar [0,87 (1
0:4:0.1) Ethyl acetate:isozolo/Qnor:acetic acid 0.
68 (10:4:0.1) Ethyl acetate: Acetone: Vinegar fi 0
．． 78 (6:6:0.1) Chloroform: Methanol: Vinegar [0,77 (10:2:
0.1) From the above physicochemical properties, the physiologically active substance TPI of the present invention
-3, TPI -4 and TPI -5 are presumed to have the structure represented by formula (I) as stated in i>iJ.

The physiologically active substance TPI of the present invention is a new substance that is completely different from conventional PDE inhibitors produced by microorganisms. That is, the aforementioned PDE-I.

PDE-n, CC-1065, APD-1, APD
-II and APD-III are both nitrogen-containing compounds, and lecticlor and til-7erol are CI
IHIGO5s A compound having the molecular formula of C15HtsOs, which is clearly different from the substance of the present invention.

[Effect]

Physiologically active substance of the present invention II! tTPI has potent PDE inhibitory activity. Below are PDEs derived from bovine heart (EC3,
1,4,17) Indicates the effect on activity.

Method Enzyme reaction solution 1.0-40mM)'JS hydrochloric acid buffer (p
H7,5) 2mM magnesium chloride, 0.4mMc-A
Add MP, PDE (30 μg protein amount, Pellinger Mannheim) and the test substance, react at 30°C for 30 minutes, then add 55% trichloroacetic acid to stop the reaction. were measured using high-performance liquid chromatography (Hitachi, 655 system).

The measurement conditions for high performance liquid chromatography are column Nihitachi + 30.
56 4Xl 50311. Mobile layer 10mM KH*
POa (pH 2,0): MeOH (10: l),
Flow rate L 5 ml/ml ns detection 262 mμ. The inhibition rate was calculated using the following formula.

Inhibition rate = (A-B)/AX IOO (%) A: 5'-# amount when test substance is not included B: 5'-AMP amount when test substance is added Test when inhibition rate is 50 substance concentration I
C5et-The obtained results are shown below.

Results Test substance ICgo concentration (μf/111t) TP
I-12,8 TPI-25,4 TPI-32,8 TPI -43,5 TPI -52,3 No♀NoQ Verine 25 Dicalcibin 6.0 Theophylline 470 Furthermore, TPI-1i? 1 o o wry/Kp
B There were no deaths after intracavitary administration.

〔Effect of the invention〕

As mentioned above, the physiologically active substance TPI of the present invention not only strongly inhibits PDE activity but also has high safety.
It is useful as a bronchodilator, cardiotonic agent, smooth muscle relaxant, hormone secretagogue, etc., or as a research reagent.

〔Example〕

Next, the present invention will be explained with reference to Examples.

Example 1 Nodori squirrel? One platinum loop was taken from a slant culture medium of Rium sp. , 1% of Celite 1 (pH 6.5) was dispensed into a 500-mL Erlenmeyer flask, sterilized by autoclaving at 120°C for 20 minutes, inoculated, and cultured with shaking at 26°C for 4 days. death,
I named this ft'PJi mother. This mother is Dulcyth 5%
, Pharmamedia 2%, C3L 0.5%, potassium dibasic phosphate 0.1%, magnesium sulfate 0.1%, calcium carbonate 0.5%, ferrous sulfate 0.0005%, zinc sulfate 0.0002% , cobalt chloride 0.000131
Manganese sulfate 0.0003%, copper sulfate 0.0002% (
A 500-mL Erlenmeyer flask containing 100-mL of a sterilized medium having a composition of pH 7.0) was inoculated with 3-mL, and cultured with shaking at 26° C. for 5 days. The culture solution 10j obtained under this pressure was centrifuged to remove the bacterial cells, and the culture solution was centrifuged. I got it. Add 5j of ethyl acetate to this supernatant, and add 2N.
A 1-tcz aqueous solution was added dropwise to adjust p)I2 to 1, and ethyl acetate extraction was performed to obtain 4.51 ml of ethyl acetate. T.P.
Most of the I material was transferred to ethyl acetate MVC. Water 3j was added to this polyethyl acetate, and while stirring, concentrated aqueous ammonia was added to the mixture to pH 9KL, and most of the TPI substance was dissolved in the water. 2 g of ethyl acetate was added to the obtained aqueous layer 31, the pH was adjusted to 2K with a 2N ice aqueous solution while stirring, and ethyl acetate extraction was performed to obtain about 2 g of an ethyl acetate layer.

After dehydrating this ethyl acetate layer with anhydrous sodium sulfate,
When concentrated under reduced pressure, about 6 tons of brown powder was obtained.

Example 2 6 tons of coarse powder containing TPI m quality obtained in Example 1
Dissolved in 30-h methanol and 4 t of silica dal powder
was added, stirred well, reduced under reduced pressure, dried to remove methanol, and then placed on a silica gel column (400 m) filled in advance with a mixed solvent of chloroform:indelononol:acetic acid (10:2:0.1). and developed with the same mixed solvent. Fractionate 20 tons each, and separate each fraction with chloroform:isoderonol:acetic acid (10:4:0.1).
) Silica dull thin layer chromatography (TLC) was performed using t-developing solvent, and Rf = 0.54, Rf = 0.46, ■ Begging f=0.25
and Rf=0.19 components were separated.

Rf=0.54' for fractions 413-428! i-show T
TPI − showing PI −3 substance and Rf=0.46
Contains a mixture of 4 substances, fraction A 71 = 4
122 contains TPI-1 substance showing Rf=0.25', and fractions 123 to 187 contain Rf=0.25'.
廿 knee 1 showing 25! #T indicating responsibility and Iyaf=0.19
It contained a mixture with PI-2 substance.

All fractions 13 to 428 were collected, concentrated under reduced pressure to 20°C, and the residue was preliminarily divided into ethyl acetate:acetone:vinegar 1! ! ! l! (10
:6:0.1) was charged onto a silica dull column (200 m/), and elution and development was performed using the same mixed solvent. The alcoholic acid was fractionated in 20' increments, and each fraction was subjected to silica gel '1' LC using ethyl acetate: acetone: acetic acid (6:6:0.1) as the developing solvent, and 0.29 components of 7 Nasrula were extracted. Both fractions were separated. Rf=0.
Fractions A35 to A40, containing only material showing 35, were collected and concentrated under reduced pressure to about 3 m/ml. N-hexane was added to the residue to precipitate Tl)I-3 substance. This precipitate was collected on a glass filter, washed with n-hexane, and dried under reduced pressure to obtain 20 g of TPI-3 substance.

Fraction 4 containing substances exhibiting only Rf = 0.29
8-A69 were collected and concentrated under reduced pressure to approximately 3 ml-f. N-hexane was added to the residue to precipitate TPI-4 substance. This precipitate was poured onto a glass filter, washed with n-hexane, and dried under reduced pressure to obtain 15 pieces of TPI-4 substance.

Example 3 Rf=0°25ft obtained in Example 2 TPI -1 including the purchase 11t11A71~Mu122 was disappointed, 20-
After concentrating to
00m was added, and after stirring vigorously, ethyl acetate Jdtk was fractionated and condensed under reduced pressure* to about 10m7! The mixture was closed and n-hexane was added to precipitate TPI-1. The precipitate was collected on a glass filter, washed with n-hexane, and dried under reduced pressure to obtain 1.82 pure TPI-1.

Fractions 4123 to A187t containing a mixture of the TPI-1 substance showing Rf=0.25 and the TPI-2 substance showing Rf=0.191 obtained in Example 2 were collected, concentrated under reduced pressure, and added to methanol at about 201Rt. Dissolve silica gel powder 2
Add t, mix well, and remove methanol under reduced pressure.
Prepare chloroform:methanol:acetic acid (10:2) in advance.
:0.1) silica gel column (
200-), and bath development was carried out using the same mixed solvent. The eluate was fractionated into 20t portions. Each fraction V (chloroform:isoglono9ol:acetic acid (10:4:
Using silica gel T, L, C using 0.1) as a developing solvent, ultraviolet detection by Manaslu lamp irradiation and detection by potassium permanganate decolorization reaction revealed that Rf = 0.19.
Fractional plates 54 to 4130 containing substances showing only ? It was collected and concentrated under reduced pressure in about 20 ml, and 200 ml of ethyl acetate was added.

Add 200 mjyfr of water, stir vigorously, perform ethyl acetate extraction, separate the ethyl acetate layer, and concentrate under reduced pressure to about 10-
Then, n-hexane was added to precipitate the TPI-2 substance. The precipitate was collected on a glass filter, washed with n-hexane, and dried to obtain 600 m9 of TPI-2v/J.

Example 4 50 lIg of TPI-1 substance obtained in Example 3 was reduced to 0.1
Dissolved in MU acid buffer (p86.5) 4-, and added β-glucosidase (manufactured by Jig! Co., Ltd., 4.60/Q) to this solution.
)4 When Q is added and the enzymatic reaction is carried out at 26℃ for 7 days, TP
Approximately 50 microns of I-1 material was converted to TPI-5 material. This reaction solution was adjusted to 3K and extracted with ethyl acetate. Separate ethyl acetate and add to sulfuric anhydride)
After dehydration with IJum, the mixture was concentrated under reduced pressure.

The residue was prepared in advance in chloroform:methanol:acetic acid (20:l).
:0.1) was charged onto a silica dull column (2Bm), and the mixture was developed using the same mixed solvent. Is the eluate 5? Fractions &28 to &31 were collected and concentrated under reduced pressure.

N-hexane was added to the residue to precipitate TPI-5 material. This precipitate was collected on a glass filter, washed with n-hexane, and then dried under reduced pressure to obtain white powder 15Q of TPI-5 substance.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the ultraviolet absorption spectrum of TPI-1. FIG. 2 is a diagram showing the ultraviolet absorption spectrum of TPI-2. FIG. 3 is a diagram showing the ultraviolet absorption spectrum of TPI-3. FIG. 4 is a diagram showing the ultraviolet absorption spectrum of TPI-4. FIG. 5 is a diagram showing the ultraviolet absorption spectrum 7 of TPI-5. FIG. 6 is a diagram showing the optical external absorption spectrum of TPI-1. FIG. 7 is a diagram showing the optical external absorption spectrum 7 of TPI-2. FIG. 8 is a diagram showing the optical external absorption spectrum of TPI-3. FIG. 9 is a diagram showing the optical external absorption spectrum of TPI-4. FIG. 1θ is a diagram showing the optical external absorption spectrum of TPI-5.

Claims (1)

[Claims] 1), Formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, R is β-D-glucopyranosyl, β-D-galactopyranosyl, 6'-O-acetyl-β
-D-glucopyranosyl, 6'-O-acetyl-β-D
- a physiologically active substance TPI or a salt thereof. 2) Formulas belonging to the genus Nodurisporium ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, R' is β-D-glucopyranosyl, β-D-galactopyranosyl, 6'-O-acetyl- Physiologically active substance T represented by β-D-glucopyranosyl or 6'-O-acetyl-β-D-galactopyranosyl group
PI-producing bacteria are cultured, and the physiologically active substance T is obtained from the culture.
A method for producing a physiologically active substance TPI or a salt thereof, which comprises collecting PI. 3) The production method according to claim 2, wherein the physiologically active substance TPI-producing bacterium is Nodurisporium sp. M5220 (Feikoken Bibori No. 8133). 4), Formulas ▲ Numerical formulas, chemical formulas, tables, etc. '-O-acetyl-β-D-galactopyranosyl group)) or its salt is treated with a glycoside bond hydrolase or a treated product thereof in an aqueous medium. There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, R'' represents a hydrogen atom) A method for producing a physiologically active substance or its salt. 5) If the glycoside bond hydrolase is β-glucosidase or β-galactosidase. A manufacturing method according to claim 4.