JP2795489B2 - New substance DC115A compound - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel substance DC115A compound.

The DC115A compound has antibacterial and antitumor effects, and is useful as an antitumor agent.

2. Description of the Related Art Conventionally, many compounds such as anthracycline compounds, anthraquinone compounds, and mitomycin compounds have been reported as antitumor antibiotics [CRC Handbook of Antibiotic Compounds, CRC Press (CRC Handbook of An)
tibiotic Compounds, CRC Press) USA 1981].

However, a compound having a skeleton related to the present invention is not known.

SUMMARY OF THE INVENTION An object of the present invention is to provide excellent antibiotics and antitumor compounds.

Means for Solving the Problems The present inventors have collected microorganisms belonging to the genus Streptomyces from soil in Kanazawa City, Ishikawa Prefecture. The present inventors have found that a compound obtained by culturing the strain is a novel compound having excellent antibacterial and antitumor activities, and completed the present invention.

According to the present invention, the following general formula [In the formula, R represents an ethyl group, a propyl group, or a 1-propenyl group. And a novel substance DC115A compound represented by the formula:

This compound is produced by culturing a microorganism of the genus Streptomyces.

 Hereinafter, the present invention will be described in detail.

Among the DC115A compounds of the present invention, DC115A1 represents a compound in which R is represented by a propyl group, and D115A1 represents a compound in which R is represented by an ethyl group.
C115A2, a compound in which R is represented by a 1-propenyl group was converted to DC11
Called 5A3.

 The physicochemical properties of DC115A1 are shown below.

Molecular weight: 494 Molecular formula: C ₂₇ H ₂₆ O ₉ Mass spectrometry: SIMS: 495 (M + 1) ⁺ high resolution EIMS: Observed value 450.1659 (M ⁺ −44) Calculated value C ₂₆ H ₂₆ O ₇ : 450.1642 Specific light intensity: ▲ [ α] ²⁵ _D ▼ = −217 ° (c = 0.1, methanol) UV absorption spectrum: (measured in methanol) λ _max (nm): 216,262,375,390 Infrared absorption spectrum: (measured in KBr) cm ⁻¹ : 3440,2960 , 1740, 1650, 1618, 1425, 1365, 1230 PMR spectrum: (measured in CD ₃ OD, 400 MHz, internal standard TM
S) δ (ppm) 7.51 (s, 1H), 7.33 (s, 1H), 6.32 (s, 1
H), 6.15 (dd, J = 7.0, 3.6 Hz, 1H), 4.26 (d, J = 16.7 Hz, 1
H), 4.17 (d, J = 16.7Hz, 1H), 3.24 (t, J = 6.4Hz, 1H),
3.02 (m, 1H), 2.82 (m, 1H), 2.39 (m, 1H), 2.32 (m, 1
H), 2.17 (s, 3H), 1.84 (s, 3H), 1.50 (m, 4H), 0.86
(T, J = 7.0Hz, 3H) CMR spectrum: (measured in CD ₃ OD, 100MHz, internal standard T)
MS) δ (ppm) 205.6 (s), 180.4 (s), 175.6 (s), 1
71.9 (s), 166.8 (s), 165.5 (s), 158.8 (s), 14
1.9 (s), 141.9 (s), 138.4 (s), 129.6 (d), 121.
0 (s), 118.1 (d), 114.6 (s), 112.9 (s), 111.9
(D), 70.5 (d), 67.6 (d), 60.9 (s), 42.8
(T), 35.3 (t), 30.7 (t), 28.7 (t), 21.1
(Q), 20.3 (t), 20.2 (q), 14.0 (q) Solubility: Soluble in chloroform, dimethyl sulfoxide, methanol, ethyl acetate and acetone, water and n
-Poorly soluble in hexane.

Color reaction: Positive iodine color Substance color / property: Yellow acidic substance Thin layer chromatography: Silica gel thin layer (HPTLC pl
ate Art.15647, Merck) Rf in developing solvent of toluene: acetone solution (1: 1v / v)
The value is 0.20.

Chloroform: methanol: acetic acid solution (20: 1: 0.1v / v /
The Rf value in the developing solvent of v) is 0.48.

 The physicochemical properties of DC115A2 are shown below.

Molecular weight: 480 Molecular formula: C ₂₆ H ₂₄ O ₃ Mass spectrometry: SIMS: 481 (M + 1) ⁺ Specific light intensity: ▲ [α] ²⁵ _D ▼ = -237 ゜ (c = 0.1, methanol) Ultraviolet absorption spectrum: (in methanol Λ _max (nm): 216,262,375,392 Infrared absorption spectrum: (measured in KBr) cm ^-1 : 3420,2970,1720,1650,1615,1420,1360,1230 PMR spectrum: (measured in CD ₃ OD, 400MHz, internal standard TM
S) δ (ppm) 7.54 (s, 1H), 7.36 (s, 1H), 6.34 (s, 1
H), 6.18 (dd, J = 6.8, 3.6 Hz, 1H), 4.27 (d, J = 16.6 Hz, 1
H), 4.19 (d, J = 16.6 Hz, 1H), 3.23 (t, J = 6.4 Hz, 1H),
3.04 (m, 1H), 2.84 (m, 1H), 2.40 (m, 1H), 2.33 (m, 1
H), 2.17 (s, 3H), 1.84 (s, 3H), 1.56 (m, 1H), 1.45
(M, 1H), 0.98 (t, J = 7.5Hz, 3H) CMR spectrum: (measured in CD ₃ OD, 100MHz, internal standard TM
S) δ (ppm) 205.7 (s), 180.5 (s), 175.8 (s), 1
72.1 (s), 166.9 (s), 165.6 (s), 158.9 (s), 14
1.9 (s), 141.9 (s), 138.5 (s), 129.7 (d), 121.
0 (s), 118.2 (d), 114.7 (s), 113.0 (s), 111.9
(D), 70.6 (d), 68.7 (d), 61.1 (s), 42.9
(T), 35.3 (t), 28.7 (t), 21.1 (q), 22.3
(T), 20.2 (q), 10.3 (q) Solubility: Soluble in chloroform, dimethyl sulfoxide, methanol, ethyl acetate and acetone, water and n
-Poorly soluble in hexane.

Color reaction: Positive iodine color Substance color / property: Yellow acidic substance Thin layer chromatography: Silica gel thin layer (HPTLC pl
ate Art.15647, Merck) Rf in developing solvent of toluene: acetone solution (1: 1v / v)
The value is 0.16.

Chloroform: methanol: acetic acid solution (20: 1: 0.1v / v /
The Rf value in the developing solvent of v) is 0.48.

 The physicochemical properties of DC115A3 are shown below.

Molecular weight: 492 Molecular formula: C ₂₇ H ₂₄ O ₉ Mass spectrometry: SIMS: 493 (M + 1) ⁺ Specific light intensity: ▲ [α] ²⁵ _D ▼ = -349 ゜ (c = 0.1, methanol) Ultraviolet absorption spectrum: (in methanol Λ _max (nm): 216,262,375,390 Infrared absorption spectrum: (measured in KBr) cm ^-1 : 3460,2950,1742,1650,1620,1426,1370,1230 PMR spectrum: (measured in CD ₃ OD, 400MHz, internal standard TM
S) δ (ppm) 7.55 (s, 1H), 7.37 (s, 1H), 6.38 (s, 1H)
H), 6.19 (dd, J = 6.8,3.5Hz, 1H), 5.83 (m, 1H), 5.15
(M, 1H), 4.26 (d, J = 16.7Hz, 1H), 4.21 (d, J = 16.7Hz,
1H), 4.01 (d, J = 7.9Hz, 1H), 3.05 (m, 1H), 2.86 (m, 1
H), 2.43 (m, 1H), 2.35 (m, 1H), 2.15 (s, 3H), 1.91
(S, 3H), 1.81 (dd, J = 7.1, 1.8 Hz, 3H) CMR spectrum: measured in CD ₃ OD, 100 MHz, internal standard TMS) δ (ppm) 205.7 (s), 181.0 (s), 175.5 (S), 1
72.0 (s), 166.7 (s), 165.5 (s), 158.9 (s), 14
1.9 (s), 141.9 (s), 138.2 (s), 134.8 (d), 129.
7 (d), 124.1 (d), 120.9 (s), 118.1 (d), 114.7
(S), 113.0 (s), 111.5 (d), 70.7 (d), 62.9
(D), 62.2 (d), 42.7 (t), 35.3 (t), 28.7
(T), 21.1 (q), 19.8 (q), 13.7 (q) Solubility: Soluble in chloroform, dimethyl sulfoxide, methanol, ethyl acetate and acetone, water and n
-Poorly soluble in hexane.

Color reaction: Positive iodine color Substance color / property: Yellow acidic substance Thin layer chromatography: Silica gel thin layer (HPTLC pl
ate Art.15647, Merck) Rf in developing solvent of toluene: acetone solution (1: 1v / v)
The value is 0.10.

Chloroform: methanol: acetic acid solution (20: 1: 0.1v / v /
The Rf value in the developing solvent of v) is 0.48.

Next, the biological activities of DC115A1, DC115A2 and DC115A3 will be described.

(A) Antibacterial activity The minimum inhibitory concentration (MIC) for various bacteria was measured by agar dilution method (pH 7.0). Table 1 shows the results.

(B) Growth inhibition on T24 cells F10 medium (GIBCO), 0.1 g / ml fetal bovine serum, 100 Unit
A T24 cell suspension (2 × 10 ⁴ cells / ml) obtained by suspending T24 cells in a medium (hereinafter, referred to as a medium A) having a composition of 100 μg / ml penicillin and 100 μg / ml streptomycin was added in 96 ml amounts.
Dispensed into each well of a well microtiter plate. After the plate was cultured in a carbon dioxide bacteria incubator at 37 ° C. for 20 hours, 0.05 ml of a sample appropriately diluted with the medium A was added to each well, and the plate was cultured in a carbon dioxide bacteria incubator at 37 ° C. for 72 hours. .
After removing the culture supernatant, 0.1 ml of a medium containing neutral red so as to be 0.02% in medium A was added to each well, and 3 ml of the medium was added.
The cells were cultured in a carbon dioxide bacteria incubator at 7 ° C for 1 hour, and the cells were stained. After removing the culture supernatant, the residue was washed once with physiological saline.

Next, the dye was extracted with 0.001N hydrochloric acid / 30% ethanol, and the absorbance at 550 nm was measured using a microplate reader. By comparing the absorbance of the untreated cells with the absorbance of the cells treated with a known concentration of the sample, the concentration of the sample that inhibits cell proliferation by 50% (IC ₅₀ ) was calculated. Table 2 shows the results.

(C) Helas ₃ Growth Inhibition MEM medium to cells (Nissui Pharmaceutical) and Helas ₃ cell suspensions were suspended Helas ₃ cells into the medium from the composition of 2mM glutamine (3
× except for using 10 ⁴ / ml) IC ₅₀ was calculated as in the case of T24 cells. Table 2 shows the results.

From the results of (B) and (C) above, DC115A1, DC115A2, and DC115A3 were found to inhibit the growth of cancer cells, and are expected to be effective antitumor agents.

 Next, a method for producing the DC115A compound will be described.

The DC115A compound belongs to the genus Streptomyces and DC115A
Culturing a microorganism capable of producing the compound in a medium,
Produce and accumulate DC115A compound in the culture, and
It can be obtained by collecting the DC115A compound.

The DC115A compound producing strain belongs to the genus Streptomyces, and any strain can be used as long as it is a DC115A compound producing strain. In addition, these strains may be capable of artificially producing DC115A compounds, such as mutant strains mutated by a mutation method such as ultraviolet irradiation, X-ray irradiation, treatment with a mutagenic agent, or naturally mutated strains. Any strain can be used. A representative strain is DO-115 strain.

 Next, the bacteriological properties of the DO-115 strain will be described.

The determination of the mycological properties is determined by the international Streptomyces
methods recommended by the reptomyces project (ISP) for characterization of Streptomyces species [EBShirling and D. Gottlieb]
International Journal of Systematic Bacteriology from Int.J.Syst.Bacteriol. 16
313-340 (1966)]. The method for analyzing isomers of diaminopimelic acid in the hydrolyzate of whole cells is described by the method of B. Becker et al. [Applied Microbiology (Appl. Microbiol.) 12 421-423 (19)
64)]. For the morphological examination, an optical microscope was used, and particularly for the morphology of the spore surface, a scanning microscope was used. Color names are assigned in the Color Harmony Manual [Container Corporation of America].
oration of America) 4th edition 1958].

(1) Form Aerial hyphae: branched.

Underlying hypha: Branched but not broken.

Spores: settle on aerial hyphae as long chains of segmented spores (10 to 30 or more). The form of the chain is bent or looped.

Spore surface: Sping Spore motility: none Spore shape / size: elliptical (0.5 × 0.7 μm) No sclerotia or spore sac are observed.

(2) Color tone Aerial mycelium: Gray Base mycelium: Light brown to yellow brown Soluble pigment: Light yellow Melanin pigment: Available (3) Chemical organization of cell wall Stereotype of diaminopimelic acid: LL type (4) Physiological properties Assimilation: Assimilate: Glycol Not assimilate: Xylose, inositol, mannitol, arabinose, rhamnose, raffinose, fructose, sucrose Gelatin liquefaction: negative Starch hydrolysis: positive Coagulation of skim milk: negative Peptoneization of skim milk: positive Decomposition of fibrin: positive Growth temperature range: 16-37 ° C (optimal 28-32 ° C) In addition, the effect on gelatin, skim milk and fibrin is 28 ° C, after one month, the growth temperature range is 2
The results after 28 days and the other properties were the results after 28 weeks at 28 ° C.

(5) Growth state on various agar media Table 3 shows the results of culturing DO-115 strain on various agar media at 28 ° C for 28 days.

The abbreviations are as follows: G: degree of growth, AM: degree and color of aerial hyphae, SM: color of basic mycelium, P: color of soluble pigment.

Since DO-115 detects LL-type diaminopimelic acid, MP Lechevalier and H.A. Lechevalier were used.
Classification of Actinomycetes [International Journal of Systematic Bacteriology (Int.J.Syst.B)
acteriol.) 20 435-443 (1970)]. Further combining the morphological characteristics of the strain, it is appropriate to assign this strain to the genus Streptomyces.

In the identification of the species of the genus, the strain has a gray aerial mycelium, a bent or looped spore chain, a stingy spore surface and a utilization pattern of the carbon source as described above,
Based on its ability to produce melanin-like pigments and the ability to produce pale yellow soluble pigments, a list of approved bacterial names [VBDSkerman et al., I.
nt.J.Syst.Bacteriol. 30 225-420 (1980)], a species having similar taxonomic characteristics to the strain is described in ISP [Int.J.Syst.Bacteriol. 18 69-189 (19
68), ibid. 18 279-392 (1968), ibid. 19 391-512 (19
69), the same magazine 22 265-394 (1972) and R.E.
Buchanan (REBuchanan) and NE Gibbons (N.
E. Gibbons, edited by Bergey's Manual of Bergie's Manual of Deterministic Bacteriology (Bergey's Manual)
of Determinative Bacteriology), 8th edition].

As a result of the search, it is difficult to specify a species that matches the properties of the DO-115 strain. The strain has been deposited with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology under the Budapest Treaty as Streptomyces sp. DO-115, FERM BP-2408. .
(Original deposit date: May 1, 1989).

 Next, the culture method will be described.

In the culture of the present invention, a usual method of culturing actinomycetes is generally used. As the medium, either a synthetic medium or a natural medium can be used as long as the medium contains assimilable carbon sources, nitrogen sources, inorganic substances, and substances necessary for promoting growth or production.

As a carbon source, glucose, starch, dextrin, mannose, fructose, sucrose, lactose,
Xylose, arabinose, mannitol, molasses and the like are used alone or in combination. Further, carbon hydrogen, alcohols, organic acids and the like may be used depending on the assimilation ability of the bacteria.

As the nitrogen source, ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dried yeast, corn steep liquor, soybean powder, casamino acid and the like are used alone or in combination.

In addition, if necessary, salt, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate,
Dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride,
Inorganic salts such as manganese sulfate, zinc sulfate and copper sulfate can be added.

Furthermore, a trace component that promotes the growth of the bacterium used and the production of the DC115A compound can be appropriately added.

As a culture method, a liquid culture method, particularly a deep stirring culture method, is most suitable. Culture temperature is 16-37 ℃, especially 25
The temperature of the medium during culturing is preferably maintained at 4 to 10, particularly 6 to 8, by adding aqueous ammonia or an ammonium carbonate solution.

When the culture is performed for 1 to 7 days in liquid culture,
The DC115A compound is produced and accumulated in the culture solution and the cells.

The culture is stopped when the production in the culture reaches a maximum.

The isolation and purification of DC115A1, DC115A2 and CD115A3 from the culture are performed according to the methods commonly used for isolating and purifying microbial metabolites from the culture. For example, the culture is separated into a culture solution and cells by filtration, and the cells are extracted with chloroform, acetone, or the like. Next, the extract and the culture solution are combined and passed through a polystyrene-based adsorbent such as Diaion HP20 (manufactured by Mitsubishi Kasei) to adsorb the active ingredient, and then eluted with ethyl acetate, acetone or the like. The eluate is concentrated and subjected to silica gel column chromatography, high performance liquid chromatography, etc.
DC115A1, DC115A2 and DC115A3 are obtained as pale yellow powder.

In addition, DC115A1, DC115A2 and DC115
The trend of 115A3 can be tracked using a bioassay using Bacillus subtilis No. 10707 or ultraviolet absorption by thin layer chromatography as a guide.

 Hereinafter, examples of the present invention will be described.

Example 1. Streptomyces sp. DO-115 was used as an inoculum. The strain was inoculated into 300 ml of a seed medium having the following composition in a two-volume Erlenmeyer flask and cultured at 30 ° C. for 48 hours with shaking (at 200 rpm).

Seed medium composition: Peptone (Kyokuto Pharmaceutical) 10g /, Yeast extract 1g /, Corn steep liquor 5g /, Soluble starch 20g /, Glucose 10g /, Calcium carbonate 5g /
(Before sterilization, pH 7.2) The obtained seed culture solution was transferred to a fermentation medium 15 having the following composition in a 30-volume culture tank at a rate of 10% (volume) at 28 ° C., and aerated and agitated at 28 ° C. (at 200 rpm, 90 with ventilation rate 15 / min)
Culture was performed for hours.

 During the culture, the pH of the medium was not particularly controlled.

Fermentation medium composition: soluble starch 50g /, dried yeast 14g /
_{, KH 2 PO 4 0.5g /,} MgSO 4 · 7H 2 O0.5g /, calcium carbonate _{5g /, CuSO 4 1.0mg /,} NiSO 4 · 6H 2 O0.5mg /, CrK
(SO ₄ ) ₂・ 12H ₂ O1.0mg / (pH before sterilization, adjusted with NaOH) Separate the culture solution, add acetone 15 to the cell fraction, extract with stirring, separate the precipitate, Extract 14 was obtained. The obtained extract is concentrated to 3 and combined with the already separated culture solution 10 and combined with the polystyrene adsorbent Diaion HP.
The active substance was adsorbed by passing through a column of 20 (2).

After removing impurities with deionized water and 30% methanol, the active substance was eluted with methanol. The obtained active fraction was concentrated and extracted with ethyl acetate. The extract was dehydrated with sodium sulfate and concentrated. This is a silica gel column BW300
(Fuji Devison Chemical Co., Ltd.). It was developed with a toluene: acetone solution (5: 1 v / v). The eluted fraction was fractionated into test tubes in 20 ml increments, and a test tube number was assigned in the order of fractionation.

As a result of examining the activity in each test tube using a thin layer of silica gel (Art. 15647, manufactured by Merck), DC was changed from test tube number 50 to No. 65.
The active fraction containing 115A1 (hereinafter referred to as fraction 1) was 70
No. 80 shows that an active fraction containing DC115A2 (hereinafter, referred to as fraction 2) exists, and from No. 85 to 93, an active fraction containing DC115A3 (hereinafter, referred to as fraction 3) exists. Was.

Concentration of fraction 1 gave a yellow-green concentrate. The obtained concentrate is applied to a silica gel column (Lichroprep Si60, Merc
k company) and adsorbed. Then, while applying a pressure of about 10 kg / cm ³ , a chloroform: methanol: acetic acid solution (100: 2:
1v / v / v). The eluted active fraction was concentrated and passed again through a silica gel column to give chloroform: methanol:
Elution was performed with an acetic acid solution (100: 2: 1 v / v / v).

The obtained active fraction was concentrated and then dissolved in methanol,
The mixture was applied to a Sephadex LH20 column to elute a methanol active fraction. The active fraction was concentrated to a yellow powder DC151A1.
Was obtained.

The same treatment was applied to fractions 2 and 3, and from each fraction, 8 mg of DC115A2 and 2 mg of DC115A3 as yellow powder were obtained.
8 mg were obtained.

Effects of the Invention According to the present invention, a novel fermentation product DC115A compound having antibacterial and antitumor effects can be provided.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 1/20 C12R 1: 465) (C12P 17/16 C12R 1: 465) (58) Fields surveyed (Int.Cl. ⁶ , DB name) C12P 17/00-17/18 C07D 407/04 REGISTRY (STN) CA (STN) WPI (DIALOG) BIOSIS (DIALOG)

Claims (1)

(57) [Claims] (1) General formula [In the formula, R represents an ethyl group, a propyl group, or a 1-propenyl group. ] The new substance DC115A compound represented by these.