The present invention relates to a novel substance DC114-A1 represented by the following formula (I): ##STR1##

This application is filed under 35 USC 371 of PCT/JP94/00956 dated Jun. 14, 
1994. 
TECHNICAL FIELD 
The present invention relates to a novel substance DC114-A1 which has 
antibacterial and anti-tumor activity and is useful as antibacterial and 
anti-tumor agents. 
BACKGROUND ART 
Compound DC114-C which has a skeleton related to the present compound and 
which is represented by the following formula (II): 
##STR2## 
has been known (EP-A-0429209). 
DISCLOSURE OF THE INVENTION 
The present invention provides a novel substance DC114-A1 having 
antibacterial and anti-tumor activity which is represented by the 
following formula (I): 
##STR3## 
This compound can be produced by culturing a microorganism belonging to the 
genus Streptomyces. 
The present invention is described in detail below. 
The physicochemical properties of DC114-A1 are shown below. 
(1) Molecular weight: 548 
(2) Molecular formula: C.sub.29 H.sub.24 O.sub.11 
(3) Mass spectrum: High resolution FAB mass spectrum (matrix: m-nitrobenzyl 
alcohol): m/z amu 
Found: 549.1402 (M+H).sup.+ 
Calculated for C.sub.29 H.sub.25 O.sub.11 : 549.1397 
(4) Specific rotation: [.alpha.].sub.D.sup.26 =-53.8.degree. (c=0.03, 
acetone) 
(5) UV absorption spectrum (measured in methanol) .lambda.max(.epsilon.); 
224.5 (18,000), 275.0 (32,100), 408.0 (7,800) 
(6) IR absorption spectrum (measured by the KBr method): .nu.max cm.sup.-1 
; 3469, 1726, 1697, 1653, 1396, 1294, 1271, 1117, 1093. 
(7) .sup.13 C-NMR spectrum (100 MHz, DMSO-d.sub.6 solution): .delta. ppm 
(multiplicity); 206.0(s), 195.0(s), 189.2(s), 178.1(s), 164.3(s), 
158.4(s), 154.2(s), 138.5(s), 128.2(s), 127.5(s), 125.0(s), 121.6(s), 
120.8(d), 112.9(d), 110.8(d), 110.7(s), 75.3(d), 74.5(d), 64.2(d), 
64.1(d), 63.9(s), 57.8(d), 57.1(s), 48.5(d), 43.7(t), 42.3(t), 22.9(q), 
14.3(q), 12.5(q). 
(8) .sup.1 H-NMR spectrum (400 MHz, DMSO-d.sub.6 solution): .delta. ppm 
[integration, multiplicity, coupling constant (Hz) ]; 12.34(1H, br.s), 
8.18(1H, s), 7.83(1H, s ), 6.46(1H, s), 5.47(1H, m), 4.85 (1H, dd, 11.4, 
3.1), 4.51(1H, dq, 6.7, 6.7), 4.43(1H, br.d, 6.7), 4.31(1H, s), 3.26(1H, 
ddd, 6.6, 4.1, 2.5), 3.19(1H, d, 6.6), 2.95(1H, t, 4.7), 2.88(1H, dd, 5.0, 
2.5), 2.85(1H, dd, 14.0, 11.4), 2.80(3H, d, 0.8), 2.57(1H, dd, 14.0, 3.1), 
1.88(3H, s), 1.15(3H, d, 6.7) . 
(9) Solubility: Soluble in dimethylsulfoxide (DMSO), methanol and acetone; 
sparingly soluble in water, ethyl acetate, chloroform and n-hexane. 
(10) Color reaction: Positive to the iodine test 
(11) Color and property of the substance: Yellow powder 
(12) Thin layer chromatography: silica gel thin layer (HPTLC plate Art. 
15647, produced by Merck & Co., Inc.) 
The Rf value obtained by using toluene:acetone solution (2:1 v/v) as a 
developing solvent was 0.4. 
The Rf value obtained by using chloroform:methanol (20:1 v/v) as a 
developing solvent was 0.6. 
After the development, the spot of DC114-A1 can be detected by bioassay 
using Bacillus subtilis, by using hot sulfuric acid, or by ultraviolet 
absorption. 
The biological activities of DC114-A1 are described below. The compound 
DC114-C described above was used for comparison. 
(A) Antibacterial activity against various bacteria 
The minimum inhibitory concentration (MIC) against the growth of various 
bacteria is shown in Table 1. The antibacterial activity was determined by 
the agar dilution method using a medium (pH 7) which comprises 3 g/l 
Bacto-tryptone (produced by Difco Laboratories), 3 g/l meat extract, 1 g/l 
yeast extract, 1 g/l glucose and 16 g/l agar. 
TABLE 1 
______________________________________ 
MIC (.mu.g/ml) 
Bacteria tested DC114-A1 DC114-C 
______________________________________ 
Staphylococcus aureus ATCC 6538P 
0.04 0.16 
Enterococcus faecium ATCC 10541 
0.16 0.16 
Bacillus subtilis No. 10707 
0.33 0.33 
Klebsiella pneumoniae ATCC 10031 
5.21 5.21 
Escherichia coli ATCC 26 
5.21 20.83 
Pseudomonas aeruginosa Bin H No. 1 
5.21 41.67 
Salmonella typhi ATCC 9992 
5.21 20.83 
Proteus vulgaris ATCC 6897 
2.60 5.21 
Shigella sonnei ATCC 9290 
5.21 20.83 
Candida albicans ATCC 10231 
5.21 &gt;83.33 
______________________________________ 
(B) Growth inhibition against HeLaS.sub.3 cells 
HeLaS.sub.3 cells (ATCC HTB22) were suspended in a medium comprising 10% 
fetal calf serum, 2 mM glutamine and MEM medium (produced by Nissui 
Pharmaceutical Co., Ltd.) (hereinafter referred to as medium A) to a 
concentration of 3.times.10.sup.4 cells/ml. The cell suspension was put 
into wells of a 96-well microtiter plate in an amount of 0.1 ml per well. 
The cells in the plate were cultured at 37.degree. C. for 20 hours in a 
CO.sub.2 -incubator. Subsequently, the test compound appropriately diluted 
with medium A was added to the wells in an amount of 0.1 ml/well. The 
cells were further cultured at 37.degree. C. for one hour in the CO.sub.2 
-incubator, and then the culture supernatant was removed. To the residue 
was added a medium comprising medium A and 0.02% Neutral Red in an amount 
of 0.1 ml per well, followed by culturing at 37.degree. C. for one hour in 
the CO.sub.2 -incubator, whereby the cells were stained. After removal of 
the culture supernatant, the residue was washed once with physiological 
saline. The pigment was extracted with 0.001 N hydrochloric acid/30% 
ethanol, and the absorbance at 550 nm was measured by using a microplate 
reader. The concentration of the test compound at which the growth of the 
cells is inhibited by 50% (IC.sub.50) was calculated by comparing the 
absorbance of untreated cells with those of the cells treated with the 
test compound at known concentrations. The result is shown in Table 2. 
TABLE 2 
______________________________________ 
Test compound IC.sub.50 (nM) 
______________________________________ 
DC114-A1 12 
DC114-C 21 
______________________________________ 
The process for producing DC114-A1 is described below. 
DC114-A1 can be obtained by culturing a microorganism belonging to the 
genus Streptomyces and having the ability to produce DC114-A1 in a medium, 
allowing DC114-A1 to accumulate in the culture, and recovering DC114-A1 
from the culture. 
As the DC114-A1-producing strains of the present invention, any strains 
which belong to the genus Streptomyces and have the ability to produce 
DC114-A1 can be used. In addition, any mutants of such strains which are 
obtained by various artificial mutation methods such as UV irradiation, X 
ray irradiation and treatment with mutagens or by spontaneous mutation may 
also be used in the present invention, insofar as they have the ability to 
produce DC114-A1. A typical example of a suitable strain is DO-114 strain 
(EP-A-0429209). 
The strain has been deposited with the Fermentation Research Institute, 
Agency of Industrial Science and Technology with accession number FERM 
BP-2641 under the Budapest Treaty (date of original deposit: Nov. 8, 
1989). 
The culturing method for the DC114-A1-producing strains is as follows. 
For the culturing of the DC114-A1-producing strains used in the present 
invention, conventional methods for culturing actinomycetes are generally 
employed. 
As the medium, either a synthetic medium or a natural medium may be used 
insofar as it appropriately contains carbon sources, nitrogen sources and 
inorganic substances which can be assimilated by the strains employed and 
the growth- and production-promoting substances required. 
As the carbon sources, glucose, starch, dextrin, mannose, fructose, 
sucrose, lactose, xylose, arabinose, mannitol, molasses, etc. can be used 
alone or in combination. In addition, hydrocarbons, alcohols, organic 
acids, etc. may also be used according to the assimilability of the 
microorganism employed. 
As the nitrogen sources, ammonium chloride, ammonium nitrate, ammonium 
sulfate, sodium nitrate, urea, peptone, meat extract, yeast extract, dry 
yeast, corn steep liquor, soybean powder, casamino acid, etc. can be used 
alone or in combination. 
If necessary, inorganic salts such as sodium chloride, potassium chloride, 
magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, 
ferrous sulfate, calcium chloride, manganese sulfate, zinc sulfate, copper 
sulfate, etc. may be added. In addition, trace ingredients that promote 
the growth of the strain employed and the production of DC114-A1 may also 
be added to the medium. 
As the method of culturing, liquid culture, especially submerged stirring 
culture, is preferably employed. Culturing is carried out at 16.degree. to 
37.degree. C. preferably 25.degree. to 32.degree. C., and at pH 4 to 10, 
preferably 6 to 8. In general, by culturing for 1 to 7 days, the desired 
compound DC114-A1 is produced and accumulated in the culture broth and the 
microbial cells. In order to adjust the pH of the medium, aqueous ammonia, 
ammonium carbonate solution, etc. are used. When the amount of the product 
in the culture reaches the maximum, the culturing is discontinued. 
For the isolation and purification of the desired compound DC114-A1 from 
the culture, an ordinary method for isolating a microbial metabolite from 
the culture can be utilized. For example, the culture is separated into 
culture filtrate and microbial cells by filtration. The microbial cells 
are extracted with chloroform, acetone, or the like. Then, the extract is 
mixed with the culture filtrate, and the resulting mixture is passed 
through a column of polystyrene adsorbent such as Diaion HP20 (produced by 
Mitsubishi Kasei Corporation) to adsorb the active substance, followed by 
elution with ethyl acetate, acetone, or the like. The eluate is 
concentrated, and the concentrate is subjected to silica gel column 
chromatography, high performance liquid chromatography, and the like to 
obtain DC114-A1. During the culture and purification steps, DC114-A1 can 
be traced by bioassay using Bacillus subtilis No. 10707, or by thin layer 
chromatography using the UV absorbance of DC114-A1 as indication.

BEST MODE FOR CARRYING OUT THE INVENTION 
Example 1 
Streptomyces sp. DO-114 strain (FERM BP-2641) was used as the seed strain. 
The strain was inoculated into 300 ml of a seed medium having the 
following composition in a 2-l Erlenmeyer flask, and cultured with shaking 
(rotation: 200 rpm) at 30.degree. C. for 48 hours. 
Composition of the seed medium: 5 g/l Bacto-tryptone (produced by Difco 
Laboratories), 5 g/l yeast extract, 3 g/l meat extract, 10 g/l soluble 
starch, 10 g/l glucose and 5 g/l calcium carbonate (pH 7.2 before 
sterilization) 
The resulting seed culture was transferred into 15 l of a fermentation 
medium having the following composition in a 30-l jar fermentor at the 
rate of 10% (by volume), and culturing was carried out at 28.degree. C. 
with stirring and aeration (rotation: 200 rpm, aeration: 15 l/min.). 
Composition of the fermentation medium: 25 g/l glycerol, 25 g/l glucose, 15 
g/l dry yeast, 0.5 g/l KH.sub.2 PO.sub.4, 0.5 g/l MgSO.sub.4 
.multidot.7H.sub.2 O, 5 g/l calcium carbonate (pH 7.0 before 
sterilization, adjusted with NaOH) 
Culturing was carried out for 80 hours without controlling the pH of the 
medium. 
After the completion of culturing, 15 l of n-propanol was added to the 
culture, followed by stirring. The removal of the cells and precipitates 
by filtration gave 28 l of a filtrate. The filtrate was concentrated, and 
the concentrate was diluted with water. The resulting mixture was passed 
through a column packed with 10 l of a polystyrene adsorption resin, 
Diaion HP20 to adsorb the active substance. After impurities were eluted 
with deionized water and 50% methanol, the active substance was eluted 
with ethyl acetate. The active fraction thus eluted was concentrated and 
water was added to the concentrate, followed by extraction with ethyl 
acetate. The extract was dehydrated over sodium sulfate, and concentrated. 
The residue was applied to a silica gel column (BW300, Fuji Davison 
Chemical Co., Ltd.) and developed with toluene:acetone solution (4:1 v/v). 
The active fraction thus eluted was concentrated, and the concentrate was 
applied to a silica gel column (Lichroprep Si60 Art. 9390; produced by 
Merck & Co., Inc.) and developed with toluene:acetone solution (4:1 v/v). 
The active fraction eluted was concentrated to give 10 mg of DC114-A1 as 
yellow powder. 
Industrial Applicability 
According to the present invention, the novel substance DC114-A1 which has 
antibacterial and anti-tumor activity can be provided.