KR830002568B1 - Method for preparing triazene compound - Google Patents

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Description

Method for preparing triazene compound

The present invention relates to a method for preparing a novel triazene compound exhibiting smooth muscle relaxation and blood pressure lowering action. According to the fermentation process of the present invention, the following two triazene compounds, FR-900184 and FR-900190, are prepared:

FR-900184 Compound:

[1-hydroxy-3- (trans, trans, trans-2,4,6-undecatri-enylidene) triazene]

FR-900190 Compound:

[1-hydroxy-3- (trans, trans, trans-2,4,7-undectrienyl-idene) triazene]

More particularly, FR-900184 and FR-900190 compounds ferment in strains strains belonging to the genus Streptomyces, such as Streptomyces aureofaciens, which produce FR-900184 and / or FR-900190 compounds. It can manufacture.

Specific microorganisms used to prepare FR-900184 and / or FR-900190 compounds and methods for their preparation are described in detail below.

microbe

Microorganisms that can be used for the production of FR-900184 and / or FR-900190 are strains belonging to the genus Streptomyces, among which belong to Streptomyces and suitable for the production of FR-900184 and / or FR-900190 compound strains. Ens strains were newly isolated from soil samples taken from Japan, Metropley, and Tokyo.

The culture of freshly isolated live bacteria was deposited on October 12, 1978, in the American Type Culture Collection, Accession No. ATCC No. 3314, consisting of permanent storage cultures. The culture was also deposited on October 9, 1978, with the accession number FERM-P4671 to the Fermentation Research Institute, Agency of Industrial Science and Technology (Japan).

It is to be understood that the specific microorganisms described herein are not limited to the use of the novel FR-900184 and / or FR-900190 of the present invention, which are provided for illustrative purposes only. The present invention is also directed from known microorganisms by conventional methods such as X-rays, ultraviolet irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminopurine and nitrogen mustard oils as well as natural mutants. It includes artificial mutants that can be produced, and also includes the use of mutants capable of producing FR-900184 and / or FR-900190 compounds.

Streptomyces oreofaciens ATCC 31442 has the following morphological, culture, biological and physiological properties.

1. Morphological characteristics

Microscopy is performed on cultures incubated at 30 ° C. for 10 to 14 days in sucrose-nitrate agar, glycerin-asparagine agar, yeast-malt extract agar, oatmeal agar and inorganic salt-starch agar.

2. Culture Characteristics

For several cultures, cultures for 10 days at 30 ° C. were observed as follows:

3. Biological and Physiological Properties

(1) Temperature requirement (Bennett's sloped agar phase): Growth at 13 ° C to 37 ° C (optimum temperature: 26 ° C)

(2) gelatin liquefaction (glucose-peptone gelatin puncture phase): very weak

(3) Starch hydrolysis (starch-inorganic salt agar phase): negative

(4) Action on milk: weak peptonation without causing aggregation

(5) Production of melanin pigment (tyrosine agar, peptone-yeast iron agar and tryptone-yeast gravy): negative

(6) Use of various carbon compounds (Freedam-Gotrib based agar medium)

(+; Well used, ±; unsure,-; not used)

[I.e., S.A. Waksman's "Bergey's Manual of Determinative Bacteriology" eighth edition (1975), "The Actinomycetes", Vol. II (1961) and E.B. Shirling and D. Gottlieb, "The International Strepto-myces Project Report" (see International Journal of Systematic Bacteriology Vol. 18, pages 69 and 279 (1968), Vol. 19, page 391 (1969) and Vol. 22, page 265 (1972)], and the strains exhibiting the characteristics as mentioned above can be found in Streptomyces olivaceus, Streptomyces oreofaciens, Streptomyces xantholiticus xantholiticus) and Streptomyces viridifaciens were confirmed to exhibit characteristics similar to those of strain ATCC 31442. However, strain ATCC 31442 differs from these similar strains in the following points.

Streptomyces olivasus:

Streptomyces olivaseus exhibits a non-specific color in its substrate mycelium and uses relatively all carbon sources without producing any soluble pigment in the medium.

Streptomyces Oreofaciens:

Streptomyces oreofaciens does not produce any soluble pigment in the medium.

Streptomyces xantholiticus:

The growth of aerial mycelium of Streptomyces xantholiticus is not abundant. The aerial mycelium of Streptomyces xantholiticus differs from the strain of ATCC 31442. The fungal mycelium of Streptomyces xantholiticus is green.

Streptomyces vidiofasion

Streptomyces viridoxidase exhibits a non-specific color in its substrate mycelia. The physiological characteristics of Streptomyces viridoxidase differ from those of the ATCC 31442 strain.

According to the above observations, it can be determined that the ATCC 31442 strain belongs to Streptomyces oreofaciens species because the streptomyces oreofaciens shows a microbiological characteristic almost similar to the ATCC 31442. However, while none of the known strains of Streptomyces oreofaciens produces FR-900184 and / or FR-900190 compounds, ATCC 31442 produces new FR-900184 and / or FR-900190 compounds and therefore is novel. Strain.

FR-900184 and / or FR-900190 compounds are produced by culturing strains in the nutrient medium to produce FR-900184 and / or FR-900190 compounds, which are included in Streptomyces, such as Streptomyces oreofaciens ATCC 31442, At this time, if Streptomyces oreofaciens ATCC 31442 is used, it is simultaneously produced in the medium of the two compounds.

In general, FR-900184 and / or FR-900190 can be used to produce FR-900184 and / or FR-900190 compound-producing strains, preferably under aerobic conditions (eg methods of shaking culture, immersion culture, etc.), carbon and nitrogen. It is prepared by culturing in a nutrient medium containing an assimilation source.

Preferred carbon sources in nutrient media are carbohydrates such as glucose, fructose, glycerin and starch. Other sources that may be used include lactose, adabinose, xylose, textrin, molasses and the like.

Preferred sources of nitrogen include yeast extract, peptone, gluten wheat, cottonseed meal, soy flour, corn steep liquor, dry yeast, as well as ammonium salts (e.g. ammonium, ammonium sulfate, ammonium phosphate, etc.), urea Inorganic and organic nitrogen compounds such as amino acids, and the like.

As a carbon and nitrogen source, compounds in pure form are advantageously used in combination but need not be used in pure form because they are suitable for use with less pure substances containing trace growth factors and significant amounts of inorganic nutrients. Because. If desired, they can be added to the medium with inorganic salts such as calcium carbonate, sodium phosphate or potassium, sodium chloride or potassium, magnesium salts, copper salts and the like. In some cases, defoaming agents such as liquid paradine, higher alcohols, vegetable oils, mineral oils and silicones can be added if the medium foams significantly.

As conditions for mass production, immersion aerobic culture conditions are preferred for the preparation of FR-900184 and / or FR-900190 materials. When producing small amounts, a shake or surface culture method in a flask or bottle is used. In addition, in the case of growing in a large tank, it is preferable to use a growth type of microorganisms for inoculation in a production tank in order to prevent growth delay during the manufacturing process of the FR-900184 and / or FR-900190 compounds. Thus, firstly, a relatively small amount of medium is inoculated with spores or mycelium of the microorganism and the inoculated medium is cultured to produce a proliferative inoculum of the microorganism, followed by aseptic transfer of the cultured proliferative inoculum from a large tank It is preferable. As the medium in which the proliferative inoculum is produced, a medium almost identical to or slightly different from that used in the main production step of the FR-900184 and / or FR-900190 compounds may be used.

Stirring and aeration of the culture mixture can be carried out in several ways. Stirring is accomplished by using a propeller or similar mechanical stirring device, rotating or shaking the fermentation product, using various pumping devices, or by passing sterile air through the medium. Aeration can be accomplished by passing sterile air through the fermentation mixture.

Fermentation is generally performed at a temperature of about 20 ° C. to 40 ° C., preferably about 30 ° C. for 50 acid to 100 hours, which may vary depending on fermentation conditions and scale.

The resulting FR-900184 and / or FR-900190 compounds can be recovered from the medium by conventional methods commonly used for the recovery of other fermentation products, such as antibiotics.

In particular, when streptomyces oreofaciens ATCC 31442 is used, FR-900184 and FR-900190 compounds are simultaneously produced in culture broth. Thus, the two compounds may be separated separately by conventional methods, or may be separated into a mixture thereof.

In general, most of the resulting FR-900184 and FR-900190 compounds are present in the culture filtrate, so that the FR-900184 and FR-900190 compounds are concentrated and lyophilized under reduced pressure from the filtrate obtained by filtering or centrifuging the juice. Conventional solvent extraction, pH adjustment, treatment with conventional resins (e.g. anionic or cation exchange resins, nonionic adsorption resins), treatment with conventional adsorbents (e.g. activated carbon, silicic acid, silica gel, cellulose, alumina), Separation can be carried out by conventional methods such as crystallization or recrystallization.

In addition, FR-900184 and FR-900190 compounds can also be separated by conventional methods such as chromatographic (eg, high pressure liquid chromatography) methods.

According to the present invention, the FR-900184 and FR-900190 compounds are produced in the medium, so that the FR-900184 and FR-900190 compounds produced in the culture broth can be separated in the free form, in the process of extraction, separation or purification. Treatment of the solution or concentrate with an alkali metal compound (e.g., sodium or potassium hydroxide) may separate it in the form of an alkali metal salt of the compound.

The FR-900184 and FR-900190 compounds obtained in the free form may be prepared according to conventional methods, either inorganic bases (eg sodium hydroxide, potassium hydroxide, potassium or sodium alkoxides) or organic bases (eg ethanolamine, trimethylamine, dicyclo Hexylamine, etc.), and may be converted into their salts.

Salts of FR-900184 and FR-900190 can be easily converted to the free form by treatment with an acid such as inorganic acid (eg hydrochloric acid) according to conventional methods.

The FR-900184 and FR-900190 compounds obtained according to the aforementioned method exhibit the following physical and chemical properties.

FR-900190 Compound:

(A) Physicochemical properties of crystalline FR-900190 compound obtained by the method described in Preparation Example 1

(1) Elemental Analysis (%): C 63.58; H 8.28; N 20.01

(2) Molecular Weight: [Mass Spectrometry]

M + = 207

(3) Molecular formula: C ₁₁ H ₁₇ N ₃ O

(4) Melting point: 125 to 128 ° C (decomposition)

(C=1.0, 메탄올)(5) Specific light intensity:

(C = 1.0, methanol)

(6) UV absorption spectrum:

(7) infrared absorption spectrum:

=3300-2400(브로드), 1630, 1610, 1595, 1570(쇼울더), 1540(쇼울더), 1460, 1410, 1380, 1370(쇼울더), 1260, 1200(쇼울더), 1170, 1150, 1080, 1030, 1010, 970, 900, 875 860, 830, 730cm^-1

= 3300-2400 (Broad), 1630, 1610, 1595, 1570 (Shoulder), 1540 (Shoulder), 1460, 1410, 1380, 1370 (Shoulder), 1260, 1200 (Shoulder), 1170, 1150, 1080, 1030, 1010, 970, 900, 875 860, 830, 730 cm ^-1

(8) nuclear magnetic resonance spectrum:

S (ppm) (CDCl ₃ ); 0.90 (3H, t, J = 6 Hz), about 1.17-1.67 (2H, m), about 1.83-2.42 (2H, m), 2.88 (2H, m), 5.47 (2H, m), about 6.0-7.0 ( 4H, m), 8.36 (1H, dJ = 9 Hz), 9.00 (1H, broad, this signal is lost in D ₂ O).

(9) Color reaction

Positive: Positive in each reaction with potassium permanganate, iodine vapor and sulfuric acid.

(10) Color of Crystal: Light Yellow

(11) Solubility:

Soluble; Chloroform, ethyl acetate, methanol and acetone

Somewhat soluble; Benzene, hexane and ether

Insoluble; water

(12) Physical properties of compounds: Acidic substances

(13) Thin layer chromatography:

Carrier; Silica gel plate

Development solvent Rf value

Benzene-Ethyl Acetate (3: 1) 0.45

Hexane-acetone (2: 1) 0.3

(8) Physicochemical Properties of Crystalline FR-900190 Compound Obtained by the Method Described in Preparation Example 2

(1) Elemental Analysis (%): C 63,88; H 8,28; N 20.09

(2) Molecular Weight:

M + = 207 [Mass Spectrometry]

(3) Molecular formula: C ₁₁ H ₁₇ N ₃ O

(4) Melting point: 100 to 120 ℃

(5) Specific light intensity:

(c=1.0, 메탄올)

(c = 1.0, methanol)

(6) UV absorption spectrum:

(7) infrared absorption spectrum:

=3170, 2950, 2920, 2860, 2710, 2700-2100(브로드), 2150, 1612, 1580, 1565, 1540, 1490(브로드), 1458, 1410, 1378, 1365, 1310, 1277, 1255, 1185, 1165, 1080, 1025, 1005, 975, 966, 910, 890, 872, 820, 760(쇼울더), 715, 623cm^-1

= 3170, 2950, 2920, 2860, 2710, 2700-2100 (broad), 2150, 1612, 1580, 1565, 1540, 1490 (broad), 1458, 1410, 1378, 1365, 1310, 1277, 1255, 1185, 1165 , 1080, 1025, 1005, 975, 966, 910, 890, 872, 820, 760 (Shoulder), 715, 623 cm ^-1

(8) nuclear magnetic resonance spectrum:

S (ppm) (CDCl ₃ ); 0.90 (3H, t, J = 6.5 Hz), about 1.18-1.56 (2H, m), about 1.80-2.12 (2H, m), 2.88 (2H, m), 5.45 (2H, m), about 5.9-7 (4H, m), 8.36 (1H, dJ = 9 Hz), about 10.8-11.6 (1H, broad; this signal is lost in D ₂ O.)

(9) Color reaction

Positive: Positive in each reaction with potassium permanganate, iodine vapor and sulfuric acid.

(10) Color of Crystal: Light Yellow

(11) Solubility:

Soluble; Chloroform, ethyl acetate, methanol and acetone

Somewhat soluble; Benzene, hexane and ether

Insoluble; water

(12) Physical Properties of Compounds: Acidic Substances

Analysis of the crystals mentioned in paragraph (A) by high pressure liquid chromatography under almost the same conditions as described in Preparation Example 2 showed that the crystals contained 20% of the FR-900184 compound in addition to FR-900190. do. On the other hand, the crystal referred to in the above (B) is a crystal of pure FR-900190 which does not contain FR-900184.

FR-900184 Compound:

(A ') Physicochemical Properties of Crystalline FR-900184 Compound Obtained by the Method Described in Preparation Example 1

(1) Molecular Weight:

M ⁺ = 207 [Mass Spectrometry]

(2) Specific light intensity:

(C=1.0, 메탄올)

(C = 1.0, methanol)

(3) UV absorption spectrum:

(4) Color reaction

positivity ; Positive in each reaction with potassium permanganate, iodine vapor and sulfuric acid.

(5) Crystal color: light yellow

(6) Solubility:

Soluble; Chloroform, ethyl acetate, methanol and acetone

Somewhat soluble; Benzene, hexane and ether

Insoluble; water

(7) nuclear magnetic resonance spectrum:

S (ppm) (CMOS-d ₆ ); 0.88 (3H, t, J = 7.0 Hz), about 1.1-1.6 (4H, m), about 2.13 (2H, m), 5.9-7.5 (6H, m), 8.41 (1H, dJ = 9.5 Hz), 13.93 (1H, broad s)

(B ') Physicochemical Properties of Crystalline FR-900184 Compound Obtained by the Method Described in Preparation Example 2

(1) Elemental Analysis (%): C 63,46; H 8,23; N 20.51

(2) Molecular Weight:

M + = 207 [Mass Spectrometry]

(3) Molecular formula: C ₁₁ H ₁₇ N ₃ O

(4) Melting point: 135-138 degreeC

(5) Specific light intensity:

(C=1.0, 메탄올)

(C = 1.0, methanol)

(6) UV absorption spectrum:

(7) infrared absorption spectrum:

=3150, 2950, 2920, 2860, 2760, 2700-2050(브로드), 1593, 1575(쇼울도), 1538, ,1520, 1458, 1405, 1378, 1363, 1335, 1300, 1280(쇼울더), 1236, 1200, 1177, 1162, 1142, 1078, 1022, 1010, 933, 920, 893, 850, 715, 650(쇼울더), 630cm^-1.

= 3150, 2950, 2920, 2860, 2760, 2700-2050 (Broad), 1593, 1575 (Shouldo), 1538,, 1520, 1458, 1405, 1378, 1363, 1335, 1300, 1280 (Shoulder), 1236, 1200, 1177, 1162, 1142, 1078, 1022, 1010, 933, 920, 893, 850, 715, 650 (Shoulder), 630 cm ^-1 .

(8) nuclear magnetic resonance spectrum:

(ppm)(CDCl₃-CD₃OD) ; 0.90(3H,t,J=6.5Hz), 약 1.1-1.6(4H,m), 약 2.0-2.3(2H,m), 약5.9-7.0(6H,m), 8.31(1H,d.J=9.7Hz)

(ppm) (CDCl ₃ -CD ₃ OD); 0.90 (3H, t, J = 6.5 Hz), about 1.1-1.6 (4H, m), about 2.0-2.3 (2H, m), about 5.9-7.0 (6H, m), 8.31 (1H, dJ = 9.7 Hz )

(9) Color reaction

Positive: Positive in reaction with potassium permanganate, iodine vapor and sulfuric acid.

(10) Color of Crystal: Light Yellow

(11) Solubility:

Soluble; Chloroform, ethyl acetate, methanol and acetone

Somewhat soluble; Benzene, hexane and ether

Insoluble; water

(12) Physical Properties of Compounds: Acidic Substances

Purity of the crystals mentioned in item (A ') was measured by high-pressure liquid chromatography under conditions substantially the same as that of Preparation Example 2. As a result, the crystals contained 10% of FR-900190 in addition to FR-900184. It was found to contain. In addition, the crystal | crystallization of said (B ') is pure crystal of FR-900184 which does not contain FR-900190.

Depending on the physicochemical properties and the relationship between the ultraviolet absorption spectrum and the nuclear magnetic resonance absorption spectrum of the FR-900190 and FR-900184 compounds, the structure of each of the FR-900190 and FR-900184 compounds is determined as follows:

FR-900190:

FR-900184:

In this structure, the terminal triaza group [= N- (N ₂ OH)] is 1-hydroxytriazene [= NN = N-OH] or its tautomer 1-oxotriazane [= N-NH-N = O], and it is confirmed by the synthesis process of these compounds.

The FR-900184 and FR-900190 compounds of the present invention have smooth muscle relaxation (eg, relaxation cardiovascular expansion on bronchial muscle) and blood pressure lowering.

Therefore, the target compound of the present invention is useful as a bronchodilator for the treatment of bronchial asthma, chronic bronchitis, asthmatic bronchitis and emphysema, and also useful as an vasodilator for the treatment of coronary insufficiency, angina pectoris and myocardial infarction, and also high blood pressure. It is also useful as a blood pressure lowering agent used in therapy.

As examples showing the biological and pharmacological effects of the above-mentioned FR-900184 and FR-900190 compounds, several pharmacological test data have been given.

Trial 1: Relaxation of Rat Aorta

After killing 8 week old Sprague-Dawley rats, the thoracic aorta is isolated. Helical strips (2 mm x 50 mm) are removed from the aorta and suspended in an organ bath containing Tyrode's solution, ventilated at 37 ° C with a mixture of 95% oxygen and 5% carbon dioxide.

The maximum relaxation of the aorta induced by the test compound and the duration of relaxation are measured by the conventional Superfusion method under the following conditions.

Initial tension: 1 g

Dropping acceleration: (a) Ti-rod solution (10ml / min)

(b) Noradrenaline-saline solution (0.6 μ / ml) (0.5 ml / min)

(c) 300 mmol KCl-tirod solution (1 ml / min)

The results of the test are shown in Table 1 below.

TABLE 1

Test 2: Blood pressure lowering effect in experimental animals

Eight week old Sprague-Dawley rats are anesthetized with urethane (1.7 g / kg intraperitoneally). Blood pressure is measured from the femoral artery using a transducer connected to a Biophy siograph 180 system (manufactured by Sanei Sokuki Co., Ltd.). Intubate the femoral vein for intravenous injection of the test compound. Test compound is dissolved in saline and injected into a 0.2 ml dose. The test results are shown in Table 2 below.

TABLE 2

Note) Initial blood pressure: 80mmHg

Trial 3: Coronary Relaxation of Dogs

Thick and thin coronary arteries with external diameters of 2.0 mm and 0.5 mm, respectively, were isolated from dogs anesthetized with pentobarbital. Helical strips of about 15 mm and 5 mm in length are removed from the coarse and fine coronary arteries, respectively, and suspended at 37 ° C. in an organ bath containing a thiroded solution, which is ventilated with a gas mixture of 95% oxygen and 5% carbon dioxide. The tension of the strip is recorded using a force-displacement transducer and a polygraph. The initial resting period was adjusted to 1.0 g for the thick artery and 100 mg for the thin artery, then 35 mmol of potassium chloride was added to the trachea to 1.4-1.6 g for the thick artery strip, and to the thin artery strip. Increase from 120 to 140 mg. After that, the measured concentration of the test compound is added, and finally, papaverine (10 ^-4 moles) is added to determine the maximum relaxation. The tension concentration required to reduce _50% (ED 50 value) is the average storage capacity - will be improved by interpolation (interpolation) from the response curves (effect = 100% by papaverine ^10-4 mol). The test results are shown in Table 3 below.

TABLE 3

Trial 4: Relaxation of the guinea pig on the trachea

Male guinea pigs weighing 610-800 g are struck and stunned. Immediately thereafter, the trachea is excised from the animal and cut into rings about 2 mm wide. The chains are made by connecting six chains with thin threads. This chain is suspended in 25 ml of an engine bath containing a thiroded solution, vented with a gas mixture of 95% oxygen and 5% carbon dioxide. The temperature of the bath is maintained at 37 ° C. The engine chain is connected to a force-shift transducer with an initial tension of 0.5 to 0.6 g and the tension is recorded as isotonic on the polygraph. Isoproterenol (1.0 × 1.0 ^-8 g / ml) is added to the tracheal bath to obtain maximum relaxation and the test compound is added to the bath. The concentration required to reduce the strain 50% (ED ₅₀ ) is calculated by interpolation from the average dose-response curve (effect of isoproterenol 1.0 × 1.0 ⁻⁸ / ml = 100%).

TABLE 4

Test 5: Acute Toxicity in Mice

Male DDY species mice (30 g body weight) are administered intraperitoneally with a 1% aqueous methylcellulose solution containing FR-900190 or FR-900184 compound. One week after the administration of the test compound, the LD ₅₀ value is obtained from the number of surviving mice. The results are shown in Table 5 below.

TABLE 5

The FR-900184 and FR-900190 compounds of the present invention are mixtures with pharmaceutically acceptable carriers, including humans in the form of pharmaceutical compositions such as capsules, tablets, granules, powders, oral tablets, sublingual tablets and solutions. It can be administered to a mammal.

Pharmaceutically acceptable carriers can include various organic or inorganic carrier materials commonly used for pharmaceutical purposes, including, for example, excipients (eg, sucrose, starch, mannite, sorbet, lactose, glucose, Cellulose, talc, calcium phosphate, calcium carbonate, etc., binders (e.g. cellulose, methyl cellulose, hydroxypropyl cellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch, etc.), disintegrants ( Examples include starch, carboxymethyl cellulose, calcium salts of carboxymethyl cellulose, hydroxypropyl starch, sodium glycol-starch, sodium bicarbonate, calcium phosphate, calcium citrate, etc., lubricants (e.g. magnesium stearate, aerosil, talc, Sodium laurylsulfate, etc.), flavoring agents (e.g. citric acid, menthol, ammonium salts of glycyrrisin, glycine, orange powder, etc.), preservatives (sodium benzoate, sodium bisulphate) Yitrate, methylparaben, propylparaben, etc.), stabilizers (citric acid, sodium citrate, acetic acid, etc.), suspending agents (e.g. methyl cellulose, polyvinylpyrrolidone, aluminum stearate, etc.), dispersants [e.g., polysorbate 80 , Emalen 408 (surfactant), emasol (surfactant) and the like], an aqueous diluent (eg water), a wax base (eg cacao butter, polyethylene glycol, stomach depsol, white petrolatum).

The dose of the target compound of the present invention varies depending on the type of disease, the weight and / or age of the patient, and the method of administration. For example, when used as a cardiovascular and bronchodilator of the compound of the present invention, the preferred injection dose may be selected in the range of about 10 μg to 1 mg / kg / day and the oral dose may be in the range of 100 μg to 10 mg. You can choose from, but are not limited to these.

The following examples illustrate the invention in detail.

Example 1

Medium (100 ml, pH 7.0) containing 1% potato starch, 1% gluten mill, 0.5% corn steep liquor and 0.5% dry yeast is poured into each of five 500 ml Erlenmeyer flasks and sterilized at 120 ° C. for 20 minutes. Each medium was inoculated with a platinum loopfull of Streptomyces Oreofaciens ATCC 31442 slope culture and incubated at 30 ° C. for 72 hours. The resulting culture was sterilized at 120 ° C. for 20 minutes in advance of 3% water soluble starch in a 30-l jar-fermener, 0.5% gluten mill, 0.5% peanut powder, 0.5% dry yeast and 0.06% sodium carbonate. It is inoculated in the containing medium (20l) and incubated for 72 hours at 30 ℃.

After filtration of the culture broth obtained in this way, the filtrate (18l) was adjusted to pH 6.8 with 6N hydrochloric acid, and then Daiion Hp-20 (trade name, Mitsubishi Chemical Industries Ltd.), a macroporous nonionic adsorption resin, was used. Product) (1.5 l). The column is washed with water (3 l) and then eluted with methanol (3 l). The eluate is dried under reduced pressure and the resulting residue is chromatographed on a silica gel column. The column is washed with benzene and then eluted with a mixture of benzene and ethyl acetate (4: 1) (500 ml). The eluate is concentrated to a volume of about 20 ml under reduced pressure to obtain crystalline precipitate which is collected by filtration (yield: 50 mg).

Crystalline precipitation is subjected to high pressure liquid chromatography under the following conditions.

Equipment: M-6000A type liquid chromatography equipment (products from WATERS ASSOCIATES, INT.)

Stationary phase: Merck RP-18 (10㎛)

Column length: 500mm

Inner diameter of column: 8mm

Fluidized bed: 75% methanol

Column Temperature: Ambient Temperature

Column pressure: 2000psi

Flow rate: 5ml / min

Detector: UV absorption at 254mm

Liquid chromatography fraction A (retention time: 10.10 minutes) and fraction B (retention time: 12.40 minutes) are obtained. Fraction (A) contains crystals of FR-900190 (30 mg) and fraction (B) contains crystals of FR-900184 (3 mg).

Example 2

Medium (100 ml, pH 7.3) containing 0.4% glucose, 0.4% yeast extract and 1.0% malt extract is poured into two 500 ml Erlenmeyer flasks and sterilized at 120 ° C. for 30 minutes. Platinum is inoculated into the medium with a tetraculture of Streptomyces Oreofaciens ATCC 31442 and incubated at 30 ° C. for 72 hours. 100 ml of each culture broth produced, medium containing 20% of starch, 2% of gluten wheat, 0.5% of corn steep liquor and 0.5% of dry yeast in two 30-l bottle-fermenters, previously sterilized at 120 ° C. for 30 minutes. , pH 7.0), and incubate at 30 ° C. for 48 hours. The resulting culture broth was previously sterilized at 120 ° C. for 30 minutes in a medium containing a starch 3%, gluten wheat 0.5%, dry yeast 0.5%, peanut powder 0.5% and Na ₂ CO ₃ 0.06% in a 2000 l fermentation tank ( 1.760 l) and incubated for 72 hours at 30 ℃. The resulting broth was filtered to obtain filtrate (1,500 l), which was concentrated to 500 l under reduced pressure and extracted with ethyl acetate (620 l). The extract (370 l) is concentrated under reduced pressure to a volume of 500 ml. The concentrate is mixed with silica gel (700 g) and column chromatographed on silica gel. The column is washed with a mixture of hexane and acetone (9: 1, 2l) and (8: 2, 2l) and then eluted with a mixture of hexane and acetone (7: 3, 3l). The eluate was concentrated under reduced pressure to give a mixed crystal (7: 3) (20 g) of FR-900190 and FR-900184.

250 mg of mixed crystals are dissolved in acetonitrile (5 ml) and subjected to high pressure liquid chromatography under the following conditions.

Device: System 500 for liquid chromatography (system 500, WATERS ASSOCIATES, manufactured by INT.)

Stationary phase: Prep Park-500 C-18 (Prep PAK-500 C-18, WATERS ASSOCIATES, INT.)

Column length: 30mm

Inner diameter of column: 57mm

Fluid bed: mixture of acetonitrile, water and acetic acid (50: 50: 0.1)

Column pressure: 30kg / ㎠

Column Temperature: Ambient Temperature

Flow rate: 200ml / min

Detector: UV absorption at 254mm

This chromatography is repeated three times. Liquid chromatography gives fraction A (retention time: about 28 minutes) and wheat fraction B (retention time: about 34 minutes). Each fraction is neutralized with sodium bicarbonate, concentrated under reduced pressure and extracted with ethyl acetate, respectively.

The extract obtained from fraction (A) was washed with water, dried over magnesium sulfate and concentrated to give FR-900190 (40 mg) crystals free of ER-900184.

In addition, the extract obtained from fraction (B) was washed with water, dried over magnesium sulfate and concentrated under reduced pressure to give crystals (5 mg) of FR-900184 that did not contain FR-900190.

Example 3

To a suspension of FR-900184 (1.05 g) in methanol (20 ml) is added with stirring 28% sodium methoxide methanol solution (1 ml) at ambient temperature. The reaction mixture is stirred at the same temperature for 10 minutes and then filtered to remove insoluble matters from the mixture. The solution was concentrated under reduced pressure to obtain crystals, which were collected by filtration to dry the crystals to obtain sodium crystals (500 mg) of FR-900184. The same crystal (200 mg) is also recovered from the mother liquor.

Melting point: about 210 ℃ (decomposition)

Example 4

To a suspension of FR-900184 (1.05 g) in methanol (20 ml) is added potassium tert-butoxide (600 mg) at ambient temperature. After the mixture is stirred for 20 minutes, it is filtered to remove insoluble matters from the mixture. The filtrate was concentrated under reduced pressure to obtain crystals which were collected by filtration and dried to give crystals of potassium salt (450 mg) of FR-900184. The same crystal (230 mg) is recovered from the mother liquor.

Claims (1)

After culturing FR-900184 and / or FR-900190 producing strains belonging to the genus Streptomyces in aqueous nutrient medium under aerobic conditions, recovering FR-900184 and / or FR-900190 compounds from the resulting culture broth. Characterized in that the FR-900184 compound of formula (I) and / or FR-900190 compound of formula (II).