JPH0767668A - Wb968 substance group and its production - Google Patents

Abstract

PURPOSE:To obtain a WB968 substance group of new compound useful in the field of medical treatment, having antimicrobial and antitumor activity, by culturing a strain belonging to the genus Chromobacterium, capable of producing a WB968 substance group, in an aqueous nutrient medium under an aerobic condition. CONSTITUTION:A strain [e.g. Chromobacterium violaceum WB968 (FERM-BP-1,968), etc.] belonging to the genus Chromobacterium, capable of producing a WB968 substance group, is cultured in an aqueous nutrient medium under an aerobic condition and the culture product is recovered to give a WB968 substance group including FR900,414 substance, FR901,399 substance and FR901,402 substance. FR900, 414 substance has the following properties. Colorless prism crystal. Neutral substance. Melting point: 167-177 deg.C. Specific rotatory power: [alpha]<23>D=-27 deg. (c=1.0, chloroform:methanol=1:1). Molecular formula C23H34N4O6S2. FR901,399 substance has the following properties. Colorless prism crystal. Neutral substance. Melting point: 225-235 deg.C Specific rotatory power: [alpha]<23>D=--183 deg. (c=0.5). Molecular formula C24H36N4O6S3. FR901,402 substance has the following properties. Colorless powder. Neutral substance. Melting point: 169-179 deg.C. Specific rotatory power: [alpha]<23>D=+3 deg. (c=0.5). Molecular formula C25H38N4O6 S2.

Description

Detailed Description of the Invention

[0001]

This invention relates to novel compounds having biological activity, more specifically FR900414 substance, FR901.
399 substances and FR901402 substances (hereinafter WB96
8 substance group). More specifically, the present invention relates to a novel biologically active WB968 substance group having antibacterial and antitumor activities, a process for producing them, and a pharmaceutical composition containing them.

[0002]

DETAILED DESCRIPTION OF THE INVENTION The WB968 substance group of the present invention is a chromobacterium violaceum ( Chromobact).
erium violaceum ) WB968, etc.,
It can be produced by fermenting a WB968 substance group-producing bacterium belonging to the genus Chromobacterium in a nutrient medium.

This fermentation process will be described in detail below. (1) Microorganism The details of the microorganism used for producing the WB968 substance group will be described below.

Taxonomic study on strain WB968: WB
Strain 968 was isolated from a soil sample collected in Yamagata Prefecture. For this taxonomic study, the methods described in Vergy's Manual of Systematic Bacteriology (Volume 1) were mainly adopted.

(I) Morphological characteristics Morphological observation of the WB968 strain was carried out on cells cultured for 20 hours at 30 ° C. in a nutrient medium and agar under an optical microscope and an electron microscope. Strain WB968 was a Gram-negative, athletic bacterium. The morphology of the cells is bacillus, and the size is about 0.5-0.6 × 1.2-1.8 μm.
Met. The results are shown in Table 1.

[0006]

(Ii) Physiological characteristics Table 2 shows the physiological characteristics of the WB968 strain. The growth temperature range was 15 to 40 ° C. The WB968 strain was oxidase positive and catalase positive, and the OF test showed fermentability. Casein was hydrolyzed and esculin hydrolysis was negative. Glucose, fructose and trehalose were fermented. The indole test was negative. The Foge Supres Couer test was negative. The β-galactosidase test (ONPG test) was negative.

[0008]

(Iii) Identification According to Verzies Manual of Systematic Bacteriology (Vol. 1), the WB968 strain was identified as Chromobacterium violaceum from the above characteristics. A culture of Chromobacterium violaceum WB968 is based on the Budapest Treaty,
As of July 20, 1988, it has been deposited with the Institute of Microbial Technology, Institute of Industrial Technology (1-1-3 Higashi, Tsukuba, Ibaraki) (deposit number FERM BP-1968). WB
When the 968 strain was deposited with the institution, the strain was identified as an Aeromonas bacterium, but now the strain is identified as described above.

(2) Production of WB968 substance group The WB968 substance group of the present invention contains an assimilable carbon source and a nitrogen source of a WB968 substance group-producing bacterium belonging to the genus Chromobacterium (for example, Chromobacterium violaceum WB968). When it is cultivated under aerobic conditions (for example, shaking culture, submerged culture, etc.) in a nutrient medium for production, it is produced.

The preferred carbon sources in the nutrient medium are carbohydrates such as glucose, starch, fructose and glycerin. Preferred nitrogen sources are broth, yeast extract, peptone, gluten powder, cottonseed powder, soybean powder, corn
Steep liquor, dry yeast, wheat malt, and the like, as well as inorganic and organic nitrogen compounds such as ammonium salts (eg, ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea, amino acids and the like. The carbon source and the nitrogen source are advantageously used in combination, but need not be used in pure form. This is because a less pure material containing a trace amount of growth factors and a considerable amount of inorganic nutrients is also suitable for use.

If desired, inorganic salts such as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts and cobalt salts may be added to the medium. If necessary, liquid paraffin, fatty oils,
Defoaming agents such as vegetable oils, mineral oils and silicones may be added. Aerobic submerged culture conditions are preferred for large-scale production of the WB968 substance group, as in the case of the methods preferably used for large-scale production of other bioactive substances. Shake culture in flasks is used for small-scale production.

When the culture is carried out in a large tank, it is preferable to inoculate the production tank with proliferating cells of the bacterium in order to avoid the growth delay in the WB968 substance group production process. Therefore, it is desirable to inoculate the bacterial cells into a relatively small amount of medium, culture the inoculated medium, and transfer the cultured vegetative cells to a large tank. The medium for producing vegetative cells is WB9
It may be substantially the same as or different from the medium used for producing the 68 substance group.

The stirring and aeration of the culture mixture can be carried out in various ways. Agitation may be effected by a mechanical agitation device such as a propeller, by rotation or shaking of the fermenter, by various pump devices, or by passing sterile air through the medium. Aeration can be achieved by passing sterile air through the fermentation mixture. Fermentation is usually at temperatures between about 10 ° C and 40 ° C, preferably 20 ° C to 35 ° C
For about 15 to 20 hours. The fermentation time may be changed depending on the fermentation conditions and scale.

After the fermentation is completed, the culture broth is subjected to various processes conventionally used for recovery and purification of bioactive substances,
For example, solvent extraction with a suitable solvent or a mixture of several solvents, chromatography, recrystallization from a suitable solvent or a mixture of several solvents, WB,
The 968 substance group is collected. According to the invention, in general,
The WB968 substance group is mainly present in bacterial cells. Therefore, the entire culture broth is directly subjected to a WB968 mass isolation process, such as by extraction with a suitable solvent such as hot water, acetone, ethyl acetate, mixtures of these solvents, and the like.

The extract was treated by a conventional method to obtain WB968.
Obtain a substance group. For example, the extract is concentrated to a small amount by evaporation or distillation, and the residue containing the active substance obtained here, namely the WB968 substance group, is subjected to a conventional purification process such as chromatography or a suitable solvent or some kind of solvent. Purify by recrystallization from a mixture of solvents.

Physicochemical Properties of WB968 Substance Group The WB968 substance group obtained according to the above fermentation process has the following physicochemical properties.

(I) FR9004 4 substances: Solubility: Soluble: Chloroform, Ethyl acetate Slightly soluble: Methanol, Acetone Insoluble: Water, n-hexane

Color reaction: Positive: Cerium sulfate reaction, sulfuric acid reaction, iodine vapor reaction Negative: Ninhydrin reaction, ferric chloride reaction, Ehrlich reaction, Maurish reaction Thin layer chromatography: UV absorption spectrum: Terminal absorption (in methanol)

Infrared absorption spectrum: ¹ H nuclear magnetic resonance spectrum: [CDCl ₃ -CD ₃ OH (10: 1), 400 MH
z] δ: 8.34 (1H, wide s, exchangeable), 8.03
(1H, d, J = 8.5Hz, replaceable), 8.01
(1H, s, replaceable), 7.98 (1H, d, J = 5
Hz, replaceable), 5.96 (1H, s), 5.83
(1H, m), 5.73 (1H, m), 5.70 (1
H, s), 5.67 (1H, m), 4.74 (1H, d)
d, J = 8.5 and 3.5 Hz), 4.54 (1H,
m), 3.96 (1H, m), 3.12-2.94 (4
H, m), 2.86-2.78 (2H, m), 2.71.
-2.56 (2H, m), 2.48 (1H, m), 2.
23 (1H, m), 1.11 (3H, d, J = 6H
z), 1.09 (3H, d, J = 6Hz), 1.01
(3H, d, J = 6Hz), 0.98 (3H, d, J =
6Hz) ppm

¹³ C nuclear magnetic resonance spectrum: [CDCl ₃ -CD ₃ OH (10: 1), 100 MH
z] δ: 172.2 (s), 172.0 (s), 169.5
(S), 168.8 (s), 164.5 (s), 13
5.7 (s), 129.4 (d), 128.6 (d),
111.0 (t), 70.3 (d), 62.1 (d),
59.4 (d), 57.3 (d), 37.0 (t), 3
6.9 (t), 35.3 (t), 31.0 (d), 2
9.0 (d), 28.9 (t), 18.8 (q), 1
8.7 (q), 18.5 (q ), 17.3 (q) ppm HRFAB-MS: C 23 H 34 N 4 O 6 S 2 + calculated for H: 52
7.1998 Found: 527.1974.

Amino acid analysis: FR900414 substance (2
mg) in a sealed tube with 6N hydrochloric acid (2 ml) at 110 ° C.
And hydrolyzed for 20 hours. The mixture was analyzed on an automated amino acid analyzer. Amino acid analysis of FR900414 substance revealed the presence of L-valine, D-valine and ammonia (1: 1: 1).

[0023] Solubility: Soluble: Chloroform, Ethyl acetate Slightly soluble: Methanol, Acetone Insoluble: Water, n-hexane

Color reaction: Positive: Cerium sulfate reaction, sulfuric acid reaction, iodine vapor reaction Negative: Ninhydrin reaction, ferric chloride reaction, Ehrlich reaction, Maurish reaction Thin layer chromatography: UV absorption spectrum: Terminal absorption (in methanol)

Infrared absorption spectrum: ¹ H nuclear magnetic resonance spectrum: [CDCl ₃ -CD ₃ OH (5: 1), 400 MHz] δ: 8.37 (1 H, s, exchangeable), 7.59 (1
H, d, J = 4Hz, replaceable), 7.50 (1H,
d, J = 8Hz, replaceable), 7.24 (1H, d, J
= 9Hz, exchangeable), 6.60 (1H, q, J = 7H
z), 5.84-5.74 (2H, m), 5.65 (1
H, m), 5.24 (1H, m), 4.59 (1H, d
d, J = 8 Hz and 4.5 Hz), 4.05-3.9.
7 (2H, m), 3.29 (1H, m), 2.96-
2.85 (2H, m), 2.80-2.67 (3H,
m), 2.56 (1H, m), 2.33 (1H, m),
2.22 (1H, m), 1.72 (3H, d, J = 7H
z), 1.33 (3H, d, J = 6.5Hz), 1.0
9 (3H, d, J = 6.5Hz), 1.00 (3H,
d, J = 6.5 Hz), 0.98 (3H, d, J = 6.
5Hz) ppm

¹³ C nuclear magnetic resonance spectrum [CDCl ₃ -CD ₃ OH (5: 1), 100 MHz] δ: 173.3 (s), 171.9 (s), 170.0
(S), 169.6 (s), 165.6 (s), 13
2.5 (d), 132.0 (d), 130.6 (d),
130.3 (s), 71.6 (d), 63.7 (d),
58.9 (d), 51.1 (d), 48.1 (t), 4
0.7 (t), 40.5 (t), 32.4 (d), 3
0.3 (d), 29.6 (t), 20.3 (q), 1
9.8 (q), 19.3 (q), 18.9 (q), 1
_{3.7 (q) ppm HRFAB-MS} : calculated for _{C 24 H 36 N 4 O 6} S 3 + H: 57
3.1875 actual value: 5731.873

Amino acid analysis: FR901399 substance (1
mg) in a sealed tube with 6N hydrochloric acid (1 ml) at 110 ° C.
And hydrolyzed for 20 hours. The mixture was analyzed on an automated amino acid analyzer. Amino acid analysis of the FR901399 substance revealed the presence of D-valine, L-valine and ammonia (1: 1: 1).

[0028] Solubility: Soluble: Chloroform, Ethyl acetate Slightly soluble: Methanol, Acetone Insoluble: Water, n-hexane

Color reaction: Positive: Cerium sulfate reaction, sulfuric acid reaction, iodine vapor reaction Negative: Ninhydrin reaction, ferric chloride reaction, Ehrlich reaction, Maurish reaction Thin layer chromatography: UV absorption spectrum: Terminal absorption (in methanol)

Infrared absorption spectrum: ¹ H nuclear magnetic resonance spectrum: [CDCl ₃ -CD ₃ OH (10: 1), 400 MHz δ: 8.35 (1 H, s, exchangeable), 7.91 (1
H, wide d, J = 4Hz, replaceable), 7.66 (1
H, d, J = 7Hz, replaceable), 7.36 (1H,
d, J = 8Hz, replaceable), 6.37 (1H, q, J
= 7 Hz), 5.81 (1H, m), 5.74 (1H,
m), 5.73 (1H, wide d, J = 16Hz),
4.74 (1H, m), 4.61 (1H, dd, J = 8)
And 4 Hz), 4.26 (1 H, m), 3.15-
3.10 (3H, m), 2.93 (1H, m), 2.7
9-2.67 (4H, m), 2.38 (1H, m),
1.82-1.64 (3H, m), 1.75 (3H,
d, J = 7 Hz), 1.02 (3H, d, J = 6.5H)
z), 1.01 (3H, d, J = 6.5Hz), 0.9
9 (3H, d, J = 6.5Hz), 0.96 (3H,
d, J = 6.5 Hz) ppm

¹³ C nuclear magnetic resonance spectrum: [CDCl ₃ -CD ₃ OH (10: 1), 100 MH
z] δ: 173.5 (s), 170.3 (s), 169.3.
(S), 168.8 (s), 165.2 (s), 13
1.2 (d), 130.5 (d), 130.1 (s),
129.0 (d), 69.8 (d), 58.0 (d),
56.0 (d), 54.5 (d), 39.1 (t), 3
9.0 (t), 36.9 (t), 34.1 (t), 3
2.1 (d), 30.9 (t), 24.8 (d), 2
2.7 (q), 21.3 (q), 18.5 (q), 1
8.2 (q), 12.8 (q ) ppm HRFAB-MS C 25 H 38 N 4 O 6 S 2 + calculated for H: 55
5.2311 Found: 555.2307.

Amino acid analysis: FR901402 substance (1
mg) was hydrolyzed in a sealed tube with 6N hydrochloric acid (1 ml) at 110 ° C. for 20 hours. The mixture was analyzed on an automated amino acid analyzer. Amino acid analysis of FR901402 substance revealed the presence of L-valine, D-valine and ammonia (1: 1: 1).

[0033]

【The invention's effect】

Biological properties of WB968 substance group As an example showing the biological activity of the WB968 substance group,
Explain some biological data.

Test Example 1 Antibacterial activity of WB968 substance group The antibacterial activity of WB968 substance group was tested by agar dilution method in Sabro-medium. Incubate at 25 ° C. for 48 hours and express the minimum inhibitory concentration (MIC) in μg / ml. The results are shown in Table 3.

[0035]

Test Example 2 Inhibition of human tumor cell growth in vitro by WB968 substance group 10% fetal bovine serum, penicillin (50 units / ml) and streptomycin (50 μg / m) in each well.
Cytotoxicity studies were carried out using microtiter plates containing 3 × 10 ³ tumor cells in 100 μl of Dulbecco's minimal essential medium supplemented with 1). 3 those cells
After culturing at 7 ° C. for 4 days, Mosmann (Journal of Immunological Methods, Vol. 65, 55)
Pp. 63, 1983).
T [3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, Sigma] colorimetric assay was performed. MTT in phosphate buffer (PB
It was dissolved in S) at a concentration of 5 mg / ml and sterile filtered to remove a small amount of insoluble residue. After culturing the human tumor cells, add this MTT solution to all assay wells,
The plate was further incubated at 37 ° C for 4 hours.
Acidic isopropanol (0.04N HCl in all wells
Isopropanol solution (100 μl) was added and mixed well to dissolve dark blue crystals. After all the crystals were dissolved, a 2-wavelength micrometer photometer (MTP-22 type,
Corona Electric Co., Ltd., Katsuta City)
The plate was read at 550 nm, with 0 nm. The target compound of this invention is dissolved in methanol and diluted with Dulbecco's minimum essential medium to give a final concentration of 1 μg / ml.
The cultures were added as follows. The results are shown in Table 4.

[0037] From the test results, it is confirmed that the WB968 substance group has biological activity.

The pharmaceutical composition of the present invention contains the WB968 substance group as an active ingredient as a mixture with an organic or inorganic carrier or excipient suitable for external use, oral administration or parenteral administration, for example, a solid, It can be used in the form of pharmaceutical preparations in semisolid or liquid form. The active ingredient is mixed, for example, with tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and other conventional pharmaceutically acceptable non-toxic carriers for dosage forms suitable for use. be able to. If necessary, additives, stabilizers, thickeners, colorants, and fragrances may be further used. The WB968 substance group may be contained in the pharmaceutical composition in an amount sufficient to exhibit a desired antitumor action or antibacterial action against a disease process or condition.

To apply the composition to humans, it is
It is preferably applied by intravenous, intramuscular or oral administration. The therapeutically effective dose of the WB968 substance group varies depending on the age and condition of each patient to be treated, but in the case of intravenous administration, the daily dose of WB9 per kg of human body weight is WB9.
68 substance group 0.1 to 100 mg, and in the case of intramuscular administration, daily dose 0.1 to 100 mg per 1 kg of human body weight,
In the case of oral administration, the daily dose is 0.1 per kg of human body weight.
It is common to administer -100 mg for the treatment of tumors and infectious diseases.

[0040]

EXAMPLES In order to explain the present invention in more detail, examples are shown below.

Example 1 Fermentation To each of 16 500 ml Erlenmeyer flasks,
Glucose (1%), broth (1%), (NH ₄ ) ₂
SO ₄ (0.1%) and _{MgSO 4 · 7H 2 O (0} .
006%) containing medium (100 ml),
Sterilization was performed at 20 ° C. for 20 minutes. Chromobacterium
Slope culture of Violaceum WB968 1 platinum loop was inoculated into each medium and cultured at 25 ° C. for 24 hours on a rotary shaker. The obtained culture was placed in a 500 l tank fermenter,
Glucose (1%), broth (1%), (NH ₄ ) _{2
SO 4 (0.1%), MgSO} 4 · 7H 2 O (0.00
6%) and Adecanol (antifoam, trademark, manufactured by Asahi Denka Kogyo Co., Ltd.) (0.1%) (300 l) (previously sterilized at 120 ° C for 20 minutes) and inoculated to 300 l
Culturing was carried out at 25 ° C. for 18 hours under aeration of 100 rpm and stirring at 200 rpm. The resulting seed culture in 4000l tank fermentors, glucose (3%), broth _{(2%), (NH 4} ) 2 SO 4 (0.1%), MgSO
_{4 · 7H 2 O (0.006%} ), KH 2 PO 4 (1.1
_%), Medium (3000 containing _{Na 2 HPO 4 · 12H 2 O} (0.72%) and Adekanol (0.1%)
1) (previously sterilized at 120 ° C. for 20 minutes), and cultured at 25 ° C. for 30 hours under aeration of 3000 l / min and stirring at 90 rpm.

Isolation and purification: After completion of the culture, the culture solution (2
800l) was adjusted to pH 2.0 with sulfuric acid, and then at 121 ° C for 3
Sterilized for 0 minutes. This was filtered using diatomaceous earth (60 kg). The pH of the filtrate (3850l) was adjusted to pH with 6N NaOH.
Adjusted to 6.5, activated carbon (150l, Takeda Pharmaceutical Co., Ltd.)
Through the column. 80 after washing with water (300 l)
Elution was performed with% water-containing acetone (1260 l). The eluate was evaporated under reduced pressure to a volume of 180 l and
The pH was adjusted to 3.0 with 1 and extracted with 200 l of ethyl acetate. The extraction was performed twice, and the extracts were combined. The ethyl acetate extract was concentrated under reduced pressure to a volume of 5.5 l. After dehydration over anhydrous sodium sulfate (300 g), the ethyl acetate solution was evaporated under reduced pressure to give an oily residue. This oily residue was dissolved in ethyl acetate (1.21) and packed with n-hexane on silica gel (MS-GEL D-150).
-60A, manufactured by Dokai Kagaku Co., Ltd., 20 kg).

N-hexane-ethyl acetate (1: 1, v /
v, 30 l) and n-hexane-ethyl acetate (1:
2, v / v, 90 l) and then washed with ethyl acetate (10
Elution was carried out with 0 l). Fractions containing the desired compound were combined and concentrated under reduced pressure to give an oily residue. The oily residue was converted to dichloromethane-methanol (25: 1, v /
v) It was dissolved in 3.9 l and the solution was evaporated under reduced pressure to a volume of 0.8 l. Add this concentrate to 5 ° C 1
Hold for 5 hours. Crude FR901228 substance (448
g) was obtained in crystalline form. FR901228 substance,
It is disclosed in European Patent Application No. 891133394.4. These crude crystals were dissolved in hot acetone (5 l) and kept at 28 ° C for 15 hours to give purified FR901228.
The material (329 g) was obtained as colorless crystals. After evaporation of the acetone mother liquor, the powder obtained was dissolved in dichloromethane-methanol (10: 1, v / v, 5 l) and the solution was evaporated under reduced pressure to a volume of 0.2 l. This concentrated solution was kept at 5 ° C. for 3 hours and filtered to obtain a crude crystalline FR.
901228 substance (93 g) (fraction I) and mother liquor (fraction I)
I) and got.

Crude crystalline FR901228 substance (93
g) (fraction I) was dissolved in chloroform (4.5 l),
Silica gel filled with this solution using chloroform (70-230 mesh, manufactured by E. Merck, 4.5 kg)
Column. After washing with chloroform (9 l), chloroform-ethyl acetate (2: 1, v / v, 4
Elution was carried out with 9.5 l). Fractions containing the desired compound were combined and evaporated under reduced pressure to a volume of 150 ml. This concentrate was mixed with 170 g of silica gel (70-230 mesh, manufactured by E. Merck). After evaporation of the solvent, the resulting dry powder was subjected to column chromatography on the same silica gel (1 kg) packed with n-hexane. n-
After washing with hexane (2 l), elution was performed with n-hexane-ethyl acetate (1: 3, v / v, 7.2 l). Fractions containing the target compound were combined and concentrated under reduced pressure to give a white powder.

This white powder was dissolved in 2 ml of chloroform. To this, 30 ml of n-hexane was added to powder 900
mg was obtained. Dissolve this powder in 10 ml of ethanol,
This solution was applied to an ODS column (YMC-pack ODS R3
35, φ50 × 500 mm, manufactured by Yamamura Chemical Laboratory, and developed with a mixture of acetonitrile-methanol-tetrahydrofuran-water (20: 20: 5: 55, v / v) and 0.5% phosphate. Fractions containing the target compound were combined and concentrated under reduced pressure to obtain 400 ml of a solution. To this solution was added 800 ml of ethyl acetate. Extraction was performed, and the ethyl acetate extract was concentrated under reduced pressure to give a white powder. This powder was dissolved in 2 ml of ethanol. This solution is then applied to an ODS column (YMC-pack ODS S-34
3, φ20 × 250 mm, Yamamura Chemical Laboratory), and eluted with a mixture of acetonitrile-methanol-tetrahydrofuran-water (20: 20: 5: 55, v / v) and 0.5% phosphate. Fractions containing the target compound were combined and concentrated under reduced pressure. The concentrate (160 ml) was extracted with 300 ml of ethyl acetate. The ethyl acetate extract was evaporated under reduced pressure to give a white powder. This white powder was dissolved in 2 ml of chloroform. 20 ml of n-hexane in this solution
Was added to obtain purified FR901402 substance (44 mg) in the form of colorless powder.

The mother liquor (fraction II) was concentrated under reduced pressure to give a powder (3.7 g). This powder was dissolved in 5 ml of ethyl acetate, and this solution was applied to a column of silica gel (MS-GEL D-150-60A, 350 g, manufactured by Dokai Kagaku) filled with n-hexane. The column was charged with n-hexane-ethyl acetate (1: 1, v / v, 600 ml and 1: 1).
2, v / v, 1500 ml) and then n-hexane-ethyl acetate (1: 4, v / v, 1500 ml, fraction A) and (1: 6, v / v, 1500 ml and 1:
Elution was performed with 8, v / v, 1000 ml, fraction B). Fraction A was concentrated under reduced pressure to give a powder (400 mg). This powder was dissolved in 2 ml of hot acetone and purified FR
900414 substance (309 mg) was obtained as a colorless prism. Fraction B was concentrated under reduced pressure and the resulting dry powder was dissolved in 10 ml of ethanol. Water in ethanol solution 10
When ml was added and this was kept at 5 ° C. for 15 hours, crystals were formed. After filtering off the crystals, the mother liquor was concentrated to obtain a powder.

This powder was dissolved in 50 ml of chloroform, and this solution was applied to a column of silica gel (70 to 230 mesh, 45 g, manufactured by E. Merck) filled with chloroform. After washing with 100 ml of chloroform,
Chloroform-ethyl acetate (2: 1, v / v, 1300
(ml) was used for elution. Combine the fractions containing the target compound,
It was concentrated under reduced pressure to a volume of 3 ml. This concentrate was mixed with 3 g of silica gel (70-230 mesh, manufactured by E. Merck). After evaporating the solvent, the resulting dry powder was subjected to column chromatography on the same silica gel (85 g) packed with dichloromethane.
Dichloromethane (450 ml) and dichloromethane-
After washing with methanol (75: 1, v / v, 300 ml), dichloromethane-methanol (50: 1, v / v)
(v, 320 ml).

The target compound-containing fraction was evaporated under reduced pressure to give a white powder. This powder was dissolved in 2 ml of chloroform. 20 ml of n-hexane was added to this to obtain a powder. This powder was mixed with dichloromethane-methanol (1
0: 1, v / v, 5 ml). 50 ml of diethyl ether was added to this to obtain purified FR901399 substance (140 mg) as colorless prism crystals.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display area // (C12P 1/04 C12R 1:01)

Claims (4)

[Claims] 1. A FR900414 article, FR90139.
WB96 including 9 substances and FR901402 substance
8 substance groups. Here, the FR900414 substance has the following physicochemical properties: Ultraviolet absorption spectrum: Terminal absorption (in methanol) Infrared absorption spectrum: ¹ H nuclear magnetic resonance spectrum: [CDCl ₃ -CD ₃ OH (10: 1), 400 MH
z] δ: 8.34 (1H, wide s, exchangeable), 8.03
(1H, d, J = 8.5Hz, replaceable), 8.01
(1H, s, replaceable), 7.98 (1H, d, J = 5
Hz, replaceable), 5.96 (1H, s), 5.83
(1H, m), 5.73 (1H, m), 5.70 (1
H, s), 5.67 (1H, m), 4.74 (1H, d)
d, J = 8.5 and 3.5 Hz), 4.54 (1H,
m), 3.96 (1H, m), 3.12-2.94 (4
H, m), 2.86-2.78 (2H, m), 2.71.
-2.56 (2H, m), 2.48 (1H, m), 2.
23 (1H, m), 1.11 (3H, d, J = 6H
z), 1.09 (3H, d, J = 6Hz), 1.01
(3H, d, J = 6Hz), 0.98 (3H, d, J =
6 Hz) ppm ¹³ C nuclear magnetic resonance spectrum: [CDCl ₃ -CD ₃ OH (10: 1), 100 MH
z] δ: 172.2 (s), 172.0 (s), 169.5
(S), 168.8 (s), 164.5 (s), 13
5.7 (s), 129.4 (d), 128.6 (d),
111.0 (t), 70.3 (d), 62.1 (d),
59.4 (d), 57.3 (d), 37.0 (t), 3
6.9 (t), 35.3 (t), 31.0 (d), 2
9.0 (d), 28.9 (t), 18.8 (q), 1
8.7 (q), 18.5 (q ), 17.3 (q) ppm HRFAB-MS: C 23 H 34 N 4 O 6 S 2 + calculated for H: 52
7.1998 Actual value: 527.1974 Amino acid analysis: Results of amino acid analysis, L-valine, D-
Valine and ammonia 1: 1: 1) present; FR9
The 01399 substance has the following physicochemical properties: Appearance: colorless prism crystals Nature: neutral substance Melting point: 225 to 235 ° C (decomposition) Molecular _{weight: C 24 H 36 N 4 O} 6 S 3 as 572.78 FAB-MSm / z573 (M + H) + Solubility: soluble: chloroform, ethyl acetate僅溶: methanol, acetone insoluble: water, n- hexane color Reaction: Positive: Cerium sulfate reaction, sulfuric acid reaction, iodine vapor reaction Negative: Ninhydrin reaction, ferric chloride reaction, Ehrlich reaction, Maurish reaction Thin layer chromatography: Ultraviolet absorption spectrum: Terminal absorption (in methanol) Infrared absorption spectrum: ¹ H nuclear magnetic resonance spectrum: [CDCl ₃ -CD ₃ OH (5: 1), 400 MHz] δ: 8.37 (1 H, s, exchangeable), 7.59 (1
H, d, J = 4Hz, replaceable), 7.50 (1H,
d, J = 8Hz, replaceable), 7.24 (1H, d, J
= 9Hz, exchangeable), 6.60 (1H, q, J = 7H
z), 5.84-5.74 (2H, m), 5.65 (1
H, m), 5.24 (1H, m), 4.59 (1H, d
d, J = 8 Hz and 4.5 Hz), 4.05-3.9.
7 (2H, m), 3.29 (1H, m), 2.96-
2.85 (2H, m), 2.80-2.67 (3H,
m), 2.56 (1H, m), 2.33 (1H, m),
2.22 (1H, m), 1.72 (3H, d, J = 7H
z), 1.13 (3H, d, J = 6.5Hz), 1.0
9 (3H, d, J = 6.5Hz), 1.00 (3H,
d, J = 6.5 Hz), 0.98 (3H, d, J = 6.
5 Hz) ppm ¹³ C nuclear magnetic resonance spectrum: [CDCl ₃ -CD ₃ OH (5: 1), 100 MHz] δ: 173.3 (s), 171.9 (s), 170.0.
(S), 169.6 (s), 165.6 (s), 13
2.5 (d), 132.0 (d), 130.6 (d),
130.3 (s), 71.6 (d), 63.7 (d),
58.9 (d), 51.1 (d), 48.1 (t), 4
0.7 (t), 40.5 (t), 32.4 (d), 3
0.3 (d), 29.6 (t), 20.3 (q), 1
9.8 (q), 19.3 (q), 18.9 (q), 1
_{3.7 (q) ppm HRFAB-MS} : calculated for _{C 24 H 36 N 4 O 6} S 3 + H: 57
3.1875 Actual value: 5731.873 Amino acid analysis: As a result of amino acid analysis, D-valine, L-
The presence of valine and ammonia (1: 1: 1) was found;
And the FR901402 substance has the following physicochemical properties: Solubility: Soluble: Chloroform, Ethyl acetate Slightly soluble: Methanol, Acetone Insoluble: Water, n-hexane Color reaction: Positive: Cerium sulfate reaction, sulfuric acid reaction, iodine vapor reaction Negative: Ninhydrin reaction, ferric chloride reaction, Ehrlich reaction, Maurish reaction Thin layer chromatography: Ultraviolet absorption spectrum: Terminal absorption (in methanol) Infrared absorption spectrum: ¹ H nuclear magnetic resonance spectrum: [CDCl ₃ -CD ₃ OH (10: 1), 400 MH
z] δ: 8.35 (1H, s, replaceable), 7.91 (1
H, wide d, J = 4Hz, replaceable), 7.66 (1
H, d, J = 7Hz, replaceable), 7.36 (1H,
d, J = 8Hz, replaceable), 6.37 (1H, q, J
= 7 Hz), 5.81 (1H, m), 5.74 (1H,
m), 5.73 (1H, wide d, J = 16Hz),
4.74 (1H, m), 4.61 (1H, dd, J = 8)
And 4 Hz), 4.26 (1 H, m), 3.15-
3.10 (3H, m), 2.93 (1H, m), 2.7
9-2.67 (4H, m), 2.38 (1H, m),
1.82-1.64 (3H, m), 1.75 (3H,
d, J = 7 Hz), 1.02 (3H, d, J = 6.5H)
z), 1.01 (3H, d, J = 6.5Hz), 0.9
9 (3H, d, J = 6.5Hz), 0.96 (3H,
d, J = 6.5 Hz) ppm ¹³ C nuclear magnetic resonance spectrum: [CDCl ₃ -CD ₃ OH (10: 1), 100 MH
z] δ: 173.5 (s), 170.3 (s), 169.3.
(S), 168.8 (s), 165.2 (s), 13
1.2 (d), 130.5 (d), 130.1 (s),
129.0 (d), 69.8 (d), 58.0 (d),
56.0 (d), 54.5 (d), 39, 1 (t), 3
9.0 (t), 36.9 (t), 34.1 (t), 3
2.1 (d), 30.9 (t), 24.8 (d), 2
2.7 (q), 21.3 (q), 18.5 (q), 1
8.2 (q), 12.8 (q ) ppm HRFAB-MS: C 25 H 38 N 4 O 6 S 2 + calculated for H: 55
5.2311 Actual value: 555.2307 Amino acid analysis: Results of amino acid analysis, L-valine, D-
The presence of leucine and ammonia (1: 1: 1) was found. 2. WB96 belonging to the genus Chromobacterium
A method for producing a WB968 substance group, which comprises culturing a strain capable of producing the 8 substance group in an aqueous nutrient medium under aerobic conditions to recover the WB968 substance group. 3. A strain of the genus Chromobacterium is Chromobacterium violaceum WB968 (FERM BP
-1968). 4. A pharmaceutical composition containing the WB968 substance group as an active ingredient and a non-toxic pharmaceutically acceptable carrier.