JPS6192576A - Fr-900456 substance, its production and medical composition containing same - Google Patents

Abstract

PURPOSE:Techniques in microbial technology are utilized to produce a novel compound, FR-900456, which is used as a remedy for human hypertension, be cause it inhibits enzymes transforming angiotencin. CONSTITUTION:A microorganism in Doratomyces, capable of producing FR-900456, such as Doratomyces putredenis F-10129 (FERM 7302) is used to effect its cultivation in an enriched medium under aerobic conditions at about 20-40 deg.C for 50-150hr. The culture broth is filtered or centrifuged and the resultant filtrate is subjected to concentration under reduced pressure, freeze-dry ing, ionic adsorption resin treatment, recrystallization and so on according to usual method to give FR-900456.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel substance useful for inhibiting angiotensin converting enzyme, hereinafter referred to as FR-900456 substance, its pharmaceutically acceptable salts, its production method, and pharmaceutical compositions containing the same. .

That is, one object of the present invention is to provide a novel compound useful for the treatment of human hypertension by inhibiting asigiotensin converting enzyme.
FR-900456 substance and pharmaceutically acceptable salts thereof are provided.

Another object of this invention is that Doratomyces (Doratomyces)
atomyces) FR-900456 belonging to %
FR-90045 by fermentation in nutrient medium of substance producing strain
The purpose of the present invention is to provide a method for producing six substances.

Yet another object of this invention is to provide a pharmaceutical composition containing FR-900456 substance or a pharmaceutically acceptable salt thereof as an active ingredient.

The FR-900456 substance of this invention is Doratomyces putretinis F-10129 (Dorato-
It can be produced by fermentation of the FR-900456 substance-producing strain belonging to the genus Doratomyces.

The characteristics of the bacteria used in the production of the FR-900456 substance are described below.

The bacterial strain F-10129 was first isolated from soil samples collected in Ayabe City, Kyoto Prefecture, Japan. Based on its morphological characteristics, this strain is characterized by the filamentous fungus Doratomyces corda (Doratomyces corda).
ratomyces corda). The morphological, cultural, and physiological characteristics of this strain are as follows.

Perfect conditions did not occur on the various culture media. On the other hand, two types of variants, Sphubularicoplaliopsis, consist of compound branches with male hands that produce many conidia at the apical annelophore. Another Doratomyces state is the condensed hyphal form of Scobraliopsis, whose annerophores and conidia are similar in shape.

The smooth, hyaline conidiophores of the F-10129 strain are gigantic filamentous, septated, square, 70-150 mm long.
p 1 The thickness is 4 to 5 μ. These occur on the surface of the medium as branches at right angles to the aerial hyphae, and constitute abundant, dense, and whorled, manly-shaped branches. One to five annelophores are formed at the tip of each branch. Annerophores are hyaline, smooth, flask-shaped or sieve-shaped, with a length of 7 to 15/1 and a width of 3 to 4 μm.

Smooth, hyaline conidia are produced in long underwater chains, forming pale yellow cylinders with a length of 250 μm.60 Conidia are single-celled, oval or oval in shape with no appendages, and have an apex. It is rounded, with a truncated base and a length of 3.5 mm.
-8μ, width 2-4μ.

Concentrated hyphae are abundantly produced during long-term culture in natural media. They consist of a sterile stalk and a fertile head over the upper quarter. The fungal stalk consists of closely spaced parallel pairs of mycelia, 150-400p
The size is 10-30μ. Dry conidial casts are formed by fertile heads, 600-800 voices x 70-1
Grows to a size of 20μ.

The vegetative hyphae are septate, hyaline, smooth, and branched. Hyphal cells are cylindrical, length 7~2211, thickness 3~
It is 71. Chlamydospores are formed at the apex of basal hyphae. They are approximately spherical to ovoid, septated, thick-walled, and measure 7-10/1 x 5-7/1.

Colonies on potato dextrose agar were grown at 25°C.
, each grows to reach a diameter of 2.0 am after two weeks. The surface of the colony is flat, thin and powdery, white or pale yellow to pale yellowish orange.

Conidial structures are abundant and sometimes form condensed hyphae. The back side of the village is pale yellow. Cultures on YpSs agar medium reach a diameter of 5 cm under the same conditions, are flat, powdery, and pale yellowish-orange in color. Conidia are abundantly formed.

The back side is pale yellow.

Strain F-10129 can grow in a temperature range of 4-35°C, with an optimal growth temperature of 24-28°C.

These temperature data were measured on a potato dextrose agar medium using a temperature gradient incubator (manufactured by Toyo Kagaku Sangyo Co., Ltd.). This strain can grow at pH 5-11 and has an optimum growth pH of 8-9 on malt extract yeast medium.

According to the taxonomic criteria of the genus Doratomyces, F-10
Strain 129 was developed by Dratomyces butredinis (Corda) Molton and Gee Smith [Doratomyce
s putredinis (Corda) Mort
onet G, Sm1th). In addition, although there are some exceptions to the above characteristics, the description by Udagawa and Horie [Udagawa and Horie, "Kuxonomiru Noteon Mycogenus Fanjai I" Journal of General and Applied Microbiology Vol. 17, 141] ~159 pages, 1971 ("Tax
Onomical notes on mycoge-
nous fungi L Journal of
General and Applied Micro
biology 17. 141-159 (1971)
] matched. In conclusion, strain F-10129 was identified as a strain of Dratomyces butredinis, and Dratomyces butredinis F-10129 (D
oratomyces putredinis F −
10129>.

The culture of Doratomyces butredinis F-10129 has been deposited with the Moe Research Institute of Microbial Technology as Microtechnology Research Institute No. 7302.

Production of FR-900456 substance The FR-900456 substance of the present invention uses an FR-900456 substance-producing strain belonging to the genus Doratomyces, such as Doratomyces butredinis F-10129 F-10129, as an assimilable carbon source and nitrogen source. It is produced when grown under aerobic conditions, such as shaking culture or deep culture, in a nutrient medium containing .

Preferred carbon sources in the nutrient medium are carbohydrates such as glucose, fructose, glycerin, scooch, and the like.

Carbon sources that may be included in the pond include galactose,
These include maltose and textrin.

Preferred nitrogen sources are yeast extract, peptone, gluten-free cottonseed flour, soybean flour, corn staple liquor, dried yeast, wheat germ, etc., as well as ammonium salts such as ammonium nitrate, ammonium sulfate, ammonium phosphate, urea, amino acids, etc. Inorganic and organic nitrogen compounds such as

Although it is advantageous to use carbon and nitrogen sources in combination, they must be used in their pure form, as less pure substances containing traces of growth factors and significant amounts of inorganic nutrients are also suitable for use. There isn't.

If desired, inorganic salts such as calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, magnesium salts, copper salts, etc. may be added to the medium. If necessary, antifoaming agents such as liquid paraffin, fatty oils, vegetable oils, mineral oils or silicones may be added, especially if the culture medium foams heavily.

Deep aerobic culture conditions are used in FR-90, as is the preferred method used in the case of large-scale production of other biologically active substances.
Preferred for mass production of 0456 substances. In the case of small scale production, cultivation in flasks, bottles, shaking or on the surface is carried out. Furthermore, when growing in a large tank, it is preferable to use the bacterium in a growing form to inoculate the production tank in order to avoid growth delays in the production process of the FR-900456 substance. That is, a fungal culture broth is first produced by inoculating a relatively small amount of culture medium with empty spores or mycelium;
The culture broth is then transferred under aseptic conditions to a large tank. The medium in which the viable inoculum was produced was FR-90045.
The medium used for the production of the 6 substances may be slightly different from the one used in the production of the six substances.

Agitation and aeration of the culture mixture may be accomplished in a variety of ways. For stirring, use a propeller or similar stirring device.
This may be carried out by rotating or shaking the fermentation device itself, by using various pump devices, or by passing sterile air through the medium. Agitation is effectively accomplished by passing sterile air through the fermentation mixture.

Fermentation is usually about 20-40°C, preferably 25-35°C.
The process is carried out at a temperature range of about 50 to 150 hours.

The FR-900456 substance produced in this way is
Recovery of other known biologically active substances; They can be recovered from the culture medium by conventional methods commonly used.

Generally, the majority of the FR-900456 material produced is found in the culture broth and therefore the FR-900456 material produced is
6 substances are obtained from the p liquid obtained by filtration of culture broth or centrifugation with molecules 4f, &, concentrated under reduced pressure, lyophilized, pH adjusted, and treated with resin, such as anion exchange resin, cation exchange resin, or nonionic adsorption resin. treatments, conventional adsorbent treatments such as activated carbon, silicic acid, silica gel, cellulose,
It can be separated by conventional methods such as treatment with alumina, gel filtration, crystallization, etc.

FR-900 thus produced in culture broth
456 material can be isolated in free form, i.e., as FR-900456 material itself, and FR-90
0456 substance-containing solution or its concentrate as a base,
That is, for example, alkali metal compounds such as sodium hydroxide, potassium hydroxide, alkaline earth metal compounds such as calcium hydroxide, magnesium hydroxide, inorganic bases such as ammonia, etc., such as ethanolamine, triethanolamine, dicyclohexylamine, etc. organic base,
or with an acid, i.e. an inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, or an acid such as formic acid, acetic acid, p-toluenesulfonic acid, citric acid, tartaric acid, etc., during the operation of each step.
For example, when processed during extraction, separation, or purification steps, the FR-900456 substance is converted to its corresponding salt form and separated. In particular, the salts of the FR-900456 material thus prepared can be easily converted into the free form, ie, the FR-900456 material itself, by conventional methods.

Furthermore, the FR-900456 substance obtained in free form may be converted into the corresponding salt thereof using the base or acid in a conventional manner.

Therefore, the FR-900456 substance as well as its salts as described above are included within the scope of this invention.

FR-90045 produced by t(1) according to the above manufacturing method
(The three substances have the following physicochemical properties.

(1) Form and color tone: Colorless powder (2) Color reaction: Positive for ninhydrin reaction, Morisch reaction, cerium sulfate reaction, and iodine reaction Negative for Dragendorff reaction (3) Solubility: Soluble in water and methanol Insoluble in hexane, benzene, chloroform 1゛4) Melting point: 90-95℃ (5) Specific light intensity: [α] 23= +12.9° (C=0.38.H2O
) (6) Ultraviolet absorption spectrum: 1% λH20 = 275 nm (E = 25) max
1 cm 1% -280 nm (E = 22) cIn (7) Infrared absorption spectrum: v KBr = 3350. 2950. 300
0-2300 (broad).

max 1720, 1680. 1620. 1540. 1
520°1445, 1380. 1350. 126
0. 1230°1110, 910. 880. 8
30cm” (9) Molecular weight: FABQ MS: rn/z 447 (M++23
) 10113C nuclear magnetic resonance absorption spectrum (D20):
δ (ppm): 211.0. 174.7. 171.
2. 169.4°155.1. 131.2. 12
9.1. 116-OL67.5. 58.2. 56
．． 7. 54.7. 42.2°38.5. 36.9
．． 32゜1. 22.3. 16.7(II)'
H nuclear magnetic resonance absorption spectrum (D20): δ (ppm)
: 7.17 (2H, d, J=8Hz), 6.
83(2T(.

d, J = 8Hz), 4.75 (IH, m),
4.08 (IH, q.

J=7Hz), 3.79 (IH, m), 3.3
2 (IH, dd.

J-15 and 4Hz), 3.17 (IH, dd,
J = 20 and 6.5Hz), 2.97 (2H,
m), 2.78 (IH, dd, J=15
0Hz), 2.55 (2H, m), 1.67 (
2H, m).

1.52 (3H, d, J=7Hz), 1.16 (3
H, d, J = 6.5 Hz) FR-900456 substance and its still pharmaceutically acceptable salt fA are useful in inhibiting asigiotensin converting enzyme.

Some pharmacological test results are shown below as examples showing such angiotensin converting enzyme inhibitory activity.

Test l Inhibition of angiotensin-converting enzyme The activity of angiotensin-converting enzyme was determined by
Aku No. 93@, pp. 215-220, 1979 IC]
1nica Chimica Acta 9:L 2
15-220 (1979)] with some modifications.

Very weak fluorescent substrate O-ami/benzoylglycyl-p
-Nitro-7enylalanyl-L-proline was used as the substrate and guinea pig serum was used as the angiotensin converting enzyme source.

Buffer (1M chloride) in IJ cum pH 8,200,2
0.58 M O-aminobency) L/reIJ'i mil-p-nitro-monophenylalanyl-L-proline (20 (I7!') in buffer solution (I
0.2 M in sodium chloride, pH 8.2)
FR-900456 substance and Hartley male guinea pig serum (40/Ie) (total 250/ll) in hydrochloric acid) were incubated for 20 minutes at 37°C. The substrate O-aminobenzoylglycyl-p-nitro-phenylalanyl-proline is hydrolyzed to form a strong fluorescent substance O-
Amino/benzoylglycine is produced and this fluorescence of 0-aminobenzoylglycine is determined by fluorescence spectrophotometry.
It was 1°. Fluorescence measurements were performed using a Hitachi fluorescence spectrophotometer (650-1).
The test was carried out using a model 0 (newly manufactured by Hitachi). The excitation and emission wavelengths were 360 nm and 410 nm, respectively. Fluorescence intensity is 2.5 x 10 in 0.1 M sulfuric acid.
Normalization was performed using a standard solution of M chinosulfate.

The IC5° value of FR-900456 substance is 2.2X10'
Met.

Trial @ 2 Suppression of angiotensin I blood pressure increase response. 7-week-old Sprague-Dawley rats were
The mice were anesthetized by intraperitoneal injection of 700 mq/kg. Blood pressure was measured from the femoral artery using the Bioquisidiograph 180 system (
Recording was performed using a transducer combined with a transducer (manufactured by NEC/Sanei). FR-900456 material was injected via a polyethylene catheter inserted into the femoral vein. FR-900456 substance (300,tlf/
The inhibitory activity of garlic (intravenous injection) was evaluated in groups of four rats. The results are shown below.

In parentheses: standard error The pharmaceutical compositions of this invention are suitable for e.g. It can be administered in the form of a solid, semi-solid or liquid pharmaceutical preparation containing an acceptable salt thereof as an active ingredient.

The active ingredient can be mixed with conventional non-toxic, pharmaceutically acceptable carriers, for example, in the form of tablets, pellets, capsules, halves, solutions, emulsions, suspensions and any other form suitable for use. good.

Carriers that can be used are water, glucose, ratatose, gum arabic, gelatin, mannylatol, starch paste, magnesium trisilicate, curcum, and corn.
Starch, keratin, colloidal silica, potato starch, urea and other solid, semi-solid or liquid carriers suitable for use in the manufacture of formulations, as well as yJ agents, stabilizers, thickeners, colorants. Agents as well as fragrances may be used. The desired active compound is included in the pharmaceutical composition in an amount sufficient to exert the desired effect according to the disease context or condition.

When this composition is applied to humans, it is preferably administered orally. The dosage or therapeutically effective amount of the FR-900456 substance will vary depending on the age and condition of each individual patient to be treated, but for the treatment of disease the active ingredient will generally be administered in kilograms of one's body weight per day. about 0 for
．． Administered in doses of 1-100 mg, preferably 0.5-5 mfI, usually with an average single dose of about 5'qs
10 mg, 50~. 100 mg, 250 fnq
and 500 tng are administered.

The present invention will be explained below according to examples.

Example fermented corn starch (3'X), gluten meal 1%)
Seed medium (80me) containing , cottonseed flour (1%), dry yeast (1 cup), corn staple liquor (1%) (
pH 6,5) and lucium carbonate (0,2%), 2
Pour each into 20 50 me Erlenmeyer flasks and sterilize at 120° C. for 30 minutes. Dratomyces butredinis F-10129 Microtechnical Research Institute No. 73
A loopful of No. 02 slant culture was inoculated into each flask.
At 25°C, on a rotary shaker, 3 inch stroke, 22 O
Incubate for 4 days at rpm. The resulting culture (400mf
'), 301! Corn starch (4%), gluten meal (3%), dried yeast (0.5%), magnesium sulfate heptahydrate (
0,051), lucium sulfate dihydrate (0,2ti)
Fermentation medium (pH 7,0) containing
Sterilize for 30 minutes at 20°C. Initial pH of inoculation 11f1
Adjust to 5.4 with sterile aqueous sodium hydroxide solution. The culture is incubated at 30° C. with aeration of 20 e/min and agitation of 300 rpm.

Glucose (400y) was added to each fermenter on the 36th and 5th day of culture, and culture was continued for 7 days.

Separation The culture broth thus obtained was mixed with diatomaceous earth (5 kq
). Concentrate the P solution ('40ff) under reduced pressure to a volume of 4e. Me! Leave the f#i solution in the oven for 72 hours, then add water to make the volume 40/. p liquid (401)
is passed through a column of Gukuenox 1 (OH-type, 71 (manufactured by Dow Chemical Company). 0.3N hydroxide) IJ
After washing with um (147?), remove the active ingredient by 0. IN hydrochloric acid (
30j7). Wash the eluate with 50 W
(Apply to H+ type, 1.51 columns. Wash the column with 0.05 M aqueous sodium chloride solution (8I!') and elute with 0.1 M aqueous sodium chloride solution (81). 11, the active ingredient is absorbed by 80%
Elute with % aqueous acetone (2I). Desalt eluate with DE
AE/Sephadex A-25 (pH 6.0, buffer solution,
1. Pack into a 21-liter column (manufactured by Pharmacia Fine Chemicals). The column was washed with 0.01 M aqueous sodium chloride (2e), then 0.03 M
Elute with an aqueous sodium chloride solution (61). Combine the effective fractions and adjust the pH to 2.0 with 6N hydrochloric acid.
e) and absorb the active ingredient in 70% aqueous methanol (2
Elute with 1). The desalted eluate was concentrated under reduced pressure to a volume of 50
Let me be. The concentrated solution was subjected to column chromatography using water using CM-Sephatex C-25 (H type, 1.877) (Pharmacia Fine Chemicals Co., Ltd.). The effective fractions are collected and reduced to a volume of 10 me. Sephatex G 15 concentrate
The sample was subjected to column chromatography using (720me), developed with a 1% acetic acid solution, and collected as both Sme aliquots.

The effective fractions are combined and further purified by preparative HPLC. HPLC is performed using a Hitachi Model 638-30 pump with a Rheogaine Mochilua 125 Injector. Chromatography with UV detector, Hitachi 638-4
1 at 254 nm. Cosmodyl Cl8 (
A steel column (manufactured by Hanui Chemical Co., Ltd.) packed with a steel column (manufactured by Dokei 10
mm, length 250 h) at a flow rate of 5 m to 7 min. 0.05% as mobile phase) Lifluoroacetic acid 98%
Use acetonitrile solution. Sample injection of active ingredient 2
Elutes after 0 minutes. The effective eluate is freeze-dried and processed to obtain a white powder. This powder is dissolved in toluacetic acid (2me>) and the solution is lyophilized to obtain FR-900456 substance 17.1 as a white powder.

Claims (1)

[Claims] 1) (1) Form and color: Colorless powder (2) Color reactions: Positive for ninhydrin reaction, Molisch reaction, cerium sulfate reaction and iodine reaction (3) Negative for Dragendorff reaction Properties: Soluble in water, methanol Insoluble in hexane, benzene, chloroform (4) Melting point: 90-95℃ (5) Specific light intensity: [α]^2^3_D=+12.9° (c=0.38, H
_2O) (6) Ultraviolet absorption spectrum: λ^(H_2)^O_m_a_x=275nm(E^1
^%_1_c_m=25)=280nm(E^1^%_
1_c_m=22)λ^(H_2)^O^+^H^C^
l_m_a_x=275, 280nmλ^(H_2)^
O^+^N^a^O^H_m_a_x=277, 283
, 292 nm (7) Infrared absorption spectrum: ν^K^B^r_m_a_x=3350, 2950, 3
000-2300 (broad), 1720, 1680,
1620, 1540, 1520, 1445, 1380,
1350, 1260, 1230, 1110, 910, 8
80, 830cm^-^1 (8) Thin layer chromatography: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (9) Molecular weight: FABQ MS: m/Z 447 (M^++23) (1
0)^1^3C nuclear magnetic resonance absorption spectrum (D_2O)
:δ (ppm): 211.0, 174.7, 171.2
, 169.4, 155.1, 131.2, 129.1,
116.0, 67.5, 58.2, 56.7, 54.7
, 42.2, 38.5, 36.9, 32.1, 22.3
, 16.7(11)^1H nuclear magnetic resonance absorption spectrum (
D_2O): δ (ppm): 7.17 (2H, d, J=
8Hz), 6.83 (2H, d, J=8Hz), 4.7
5 (1H, m), 4.08 (1H, q, J = 7Hz),
3.79 (1H, m), 3.32 (1H, dd, J=1
5 and 4Hz), 3.17 (1H, dd, J=20 and 6.5Hz), 2.97 (2H, m), 2.78 (
1H, dd, J = 15 and 10Hz), 2.55 (2
H, m), 1.67 (2H, m), 1.52 (3H, d
, J=7Hz), 1.16 (3H, d, J=6.5Hz
) FR-900456 substance and pharmaceutically acceptable salts thereof having the following physicochemical properties. 2) FR-900456 substance-producing strain belonging to the genus Doratomyces in a medium,
FR-9004 from culture broth cultured under aerobic conditions
FR characterized by recovering 56 substances or their salts
-900456 Method for producing substance. 3) The strain producing the FR-900456 substance is Doratomyces putredinis (￥Doratomyces￥￥p
The manufacturing method according to claim 2, which is a type of utre-dinis ¥). 4) The strain producing the FR-900456 substance is Doratomyces putredinis F-10129 (Feikoken Bacteria Collection No. 730).
2), the manufacturing method according to claim 2. 5) A pharmaceutical composition comprising a compound according to claim 1 or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable substantially non-toxic carrier or excipient.