JPS58180487A - Antibiotic dc-81 and its preparation - Google Patents

Abstract

NEW MATERIAL:An antibiotic DC-81 shown by the formula. USE:An antibacterial agent, and disinfectant. Having antibacterial activity and antitumor activity. PROCESS:A bacterium such as DC-81 strain (FERM-P 6502) belonging to the genus Streptomyces, capable of producing DC-81, is cultivated in a medium, DC- 81 is accumulated in the culture, and DC-81 shown by the formula is collected from the culture. Properly, the culture temperature is 25-40 deg.C, and the pH of the medium is 4-10. Having the following physical and chemical properties. Melting point: 98-105 deg.C, molecular weight: 246 (mass spectrum method), molecular formula: C13H14O3N2; specific rotatory power; [alpha]<22>D=+135 deg. (c 0.2, methanol); solubility: easily soluble in DMSO, methanol, etc., soluble in ethyl acetate, and water, slightly soluble in ethyl ether, and n-hexane.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic and a method for producing the same, and particularly to a novel antibiotic named Do-81 by the present inventor and a method for producing the same.

The present invention is based on the finding that certain microorganisms belonging to the Streptomyces genus produce the novel antibiotic T) O-81.

An object of the present invention is to provide new and useful substances.

The novel substance D C-81 according to the present invention is characterized by being a novel compound specified by the following planar structural formula.

As will be described later, since DO-81 exhibits antibacterial activity against certain types of bacteria, it is expected to have a therapeutic effect on infectious diseases caused by these types of bacteria. In addition, DC-81 was found to exhibit antitumor effects.

This substance belongs to the so-called 1,4-benzodiazepine derivatives, and may also be used as an analgesic, sedative, and antispasmodic agent.

The physicochemical and biological properties of the DO-81 substance according to the present invention are as follows.

(2) Physical and chemical properties (melting point = 98-105°C (2) min-1it: 246 (mass spectrometry) (3) Molecular formula: "s 8H14" 3N2 (4) Ultraviolet absorption spectrum (in methanol): 224. 236
, 260 (ah), 316 (nm) (5) Infrared absorption spectrum (KBr tablet method): Shown in FIG.

(5) PMR, spectrum (TM8 group in deuterium-substituted chloroform) (ppm): 1.8-2.33 (4H), 3.3-3.8 (3H)
, 3.84 (3H), 6.89 (IH).

7.48 (IH), 7.63 (IH) (7) OMR spectrum (in deuterated chloroform,
TMS Standard): 24.2.29.5.46.7.53.7. F16.
0°111.4, 113.1, 119.4, 140.8
゜146.2, 149.2, 162.5, 164.9 (
8) Specific rotation; [α:]'j=+135° (00
, 2°methanol) (9) Soluble in dimethyl sulfoxide, methanol, chloroform, and acetone. Soluble in ethyl acetate and water, almost insoluble in ethyl ether and n-hexane.

(10) Rf value: Thin layer chromatography [Silica gel (trade name: Kleselgel 60 Art.

5721, E, Merck, West Germany) at room temperature.
The Rf values in [time evolution] are shown in Table 1.

Table 1 Developing agent R, f Chloroform/acetone (33:67v/V) 0.3
8rioo form metayr (9: Jv/v)
0.290.05N N) (40H saturated ethyl acetate
0.12 Toluene/Ethanol/Aqueous ammonia 0,27 (40:10:0.1 v/v) (1) to (9) From the chemical properties of the shell shell, the compound of the present invention has the following planar structural formula. It was determined that the

■, biological properties (1) Antibacterial activity The antibacterial activity (agar dilution method, pH 7.0) is shown in Table 2.

As shown in the table below, substance D C', -81 has antibacterial activity and can be expected to be used as an antibacterial agent or disinfectant.

Table 2 Test bacteria MIO (μIIAnl Staphylococcus aureus 50ATO
O6538P Bacillus fistula 50107
07 Escherichia coli 200TO
O26 Salmonella Φ Taihosa 50ATO
O9992 Shigella zonei 50A
TOO9290 (2) Acute antecedent toxicity (LD5o) is 42Tn9/9 when administered intraperitoneally to mice.

(3) Antitumor activity Effect on Lymphocyts lykemia P-388 tumors Groups of 5 0DF1 male mice weighing approximately 229 were given Lymphocyts lykemia P-388 tumor.
eukemia) P-388 tumor cells IX1
06 cells were implanted intraperitoneally.

DO-81 substance in saline solution 24 hours after implantation
, 2 ml was administered intraperitoneally once.

As a comparative example, a group was established in which 0.2 ml of mitomycin 0 in physiological saline was administered intraveinally 24 hours after tumor cell transplantation.
The average survival days of the test examples (O: average survival days of the controls) are shown in Table 3.

Table 3 The method for producing the antibiotic DC-81 according to the present invention involves culturing in a medium a microorganism capable of producing genus Streptomyces [2, DC-8], and culturing DC-81 in the culture. DO-81 is collected from this culture.

The microorganism used in the present invention belongs to the genus Streptomyces, and any microorganism can be used as long as it has the ability to produce DC-81. Bacterial strain 1) isolated from soil in the city is strain O-81 (Micronige [Bacteria No. 6502]). The mycological properties of this strain are as follows.

■ Morphological properties This strain shows good or normal growth on various natural and synthetic media, and the color of its aquatic hyphae is generally pale yellow to brown, but it is especially suitable for glycerol-asparagine agar, egg, and alda barium. When used on agar medium or glucose/yeast extract agar medium, it becomes reddish. This dye (7) is not a pH indicator. Aerial hyphae colonization is good on starch/agar media, but generally shows normal colonization and is white to gray in color.

Spores are 1 spore on a sporophyte that is simply branched from an elongated aerial hyphae.
Zero or more spirals (fil(spiralg)
It grows as an epiphyte. The shape of the spore is elliptical or oval, and the size is 1.0-1.1μ x 0.4-0.6μ.The spore surface is smooth or warty according to electron microscopy, and the flagellum is unacceptable.

Also, no spores were found.

(2) Growth status on various media The characteristics of growth and color when cultured on various media at 28° C. for 2 weeks are shown below. Color display is 0olor Har
mony Manual (0ontaln@rOor
According to the color classification according to the United Nations Corporation of America). No soluble dye is detected in any of the media used.

(1) Growth on sucrose/nitrate agar medium: Good, flat (8) Color of front and back surfaces of aquatic mycelia: Fresh pink (4c)
a) or fleck φ pink (5ca) Annaka mycelium: normal, white (a) (2) Growth on glucose-asparagine agar medium:
Color of the surface and back of poor, raised aquatic hyphae Nirite Ipoly (2c
a) Or fleck pink (5ca) Aerial mycelium: None 3) Growth on glycerol-asparagine agar medium:
Normally, the color of the front and back sides of flat aquatic mycelium: chestnut brown (
4nl) Aerial hyphae: Poor, white (&) (4) Growth on starch/inorganic salt agar medium: Good, color of the surface and back of raised aquatic hyphae: Marble (41θ) or light brown (4ng) Aerial hyphae : Abundant, White (a) No 1-Fresh 9 Pink (4ca) (5) Growth on egg/albumin agar medium: Poor, flat base Color of surface and back of hyphae: Dark lacquer-red (6pe) Also medium hyphae : Poor, white (a) (6) Nutrient agar medium growth: W-through, flat base medium hyphae color nirite yellow (13/
2c a) Aerial hyphae: Normal, pink tint (7ba) (7) Yeast extract/malt extract Agar medium raw *: Normal, color of the surface and back of the raised basal hyphae Nirite wheat (2.a)
) Aerial hyphae: Normal, Burl shell tint (3ba) (8) Autobar terrarium growth: Good, color of surface and underside of raised base hyphae: Bamboo (2ge) Aerial hyphae: Normal , white (a) or ipoly tent (2e
b) (9) Growth on glucose/yeast extract agar medium:
Good color of the surface and back of the 2 granular hyphae Nilite/Ipoly (2c
a) or deep red brown (6/2 pl) Aerial hyphae: Normal, white (a) or gray (5fe) (10) Growth on Bennett's agar medium: Normal, color of the surface and back of the hyphae in the raised base : Bamboo (2gc) Aerial mycelium: Normal, Sand (3ab) (11) Emerson's agar medium Growth: Normal 2 granular base Medium color of surface and back side of mycelium: Burl pink (2gc)
) Aerial hyphae: Ordinary, Orchid Chin) (10ba) (12) Hitzky-Tressner's agar medium (11) Growth: Good, color of the surface and back of the raised basal hyphae Nirite ipoly- (2c)
m) Aerial mycelium: normal, pearl fist shell tint (3ba) (13) Peptone/yeast extract/iron agar medium growth:
Normally, the color of the front and back sides of the hyphae in the raised base is bivar pink (3ca) Aerial hyphae: Normal, white (&) (14) Growth on tyrosine agar medium: Normal, the color of the front and back sides of the hyphae in the raised base Color: Fresh pink (5c
m) or burgundy (7 pl) Aerial mycelium: normal, fresh pink (4 cm) <15
) Growth on glycerol/calcium malate agar medium: normal, flat base, surface of medium hyphae, surface color: old wa (]2) in (7/!ng) aerial hyphae poor, white (a) ■, physiological Properties (1) Assimilation of carbon sources (on Pridham-Godelive agar medium): D-glucose, L-7 rabinose, D-xylose, l-inositol, D-mannitol, D-fructose, L-rhamnose, Assimilates claus and D-raffinose.

(2) Liquefaction effect of gelatin: None.

(3) Effect on milk: Neither coagulate nor liquefy.

(4) Starch hydrolysis effect: Yes.

(5) Growth temperature range: 20-40'C (6)
Production of melanin-like pigment: None.

However, (2) the liquefaction effect of gelatin occurs after 3 weeks at 20℃, (3) the effect on bulk gelatin after 3 weeks at 28℃, (5) the growth temperature range after 5 days, and the other conditions
These are the observation results after 2 weeks at 8°C.

(2) Cell wall composition As a result of analyzing diaminopimelic acid, which is one of the amino acids constituting the cell wall, LL-2,6-diaminopimelic acid was detected.

In terms of the above-mentioned mycological properties, this strain forms aerial hyphae, has simple branches, forms long spore chains at the tips, and contains L-diaminopimelic acid in its cell wall, so this strain belongs to the order Actinobacteria. It is classified as a member of the genus Streptomyces.

■Identification of one species In this strain, the spore chain has a spiral shape (Bpl
ral), and the spore surface is smooth (amoo).
th) or a rough surface (warty). The color of aerial mycelia on various agar media is generally white, but may also be pale yellow or pinkish gray. However, it does not exhibit green or blue colors. The color of the basal hyphae ranges from cream to orange or brown, and is particularly characteristically red on glycerol/calcium malate agar and egg/albumin agar (]5). In either case the dye is not a pH indicator.

Production of sweet, soluble pigments and melanin-like pigments is absent. As a carbon source, it has a wide range of sugar assimilation abilities such as L-arabinose, D-xylose, l-inositol, D-mannitol, L-rhamnose, and D-raffinose.

Similar strains of this strain were listed on the Approved List of Bacterial Scientific Names (Int, J
, System, Bacterol, 30 volumes.

Int.

J, System, Bacterol, vol. 18, p. 69.

279 pages, 1968, vol. 19, p. 391.

1969, Volume 22, Page 265, 1972, the following six bacterial species are listed as closely related species.

Streptomyces roseis fleuroticus (Str.
eptomyces roselsclerotlcu
m) Streptomyces soufflelothiaras (8,
aclerotlalug), Streptomyces livanii (S, 1lbanl), Streptomyces (
16) Ises Fist Okaseiscleoteikas (S.

ochraceiacleoticus)X Streptomyces flocculus (S, flocculus)
and Streptomyces binaceus sutrabus (S
, vinaoeus-drappus) n Among these strains, Streptomyces roseis scleroteicus, Streptomyces soufflerochiaras, and Streptomyces ocaseis scleroteicus are all types that form sclerotia. However, the formation of sclerotia is not observed in this strain. However, it is known that even in bacterial species that form sclerotia, sclerotia do not become M if they are relatively well attached to aerial mycelia. Therefore, the presence or absence of sclerotia was excluded from consideration when identifying this strain, which forms abundant aerial hyphae.

When these six strains were compared with the present strain in more detail in the literature, differences were found in the color tone of aerial hyphae and basal hyphae.

For +i r+IWi yarn, Streptomyces
Both Roseis fleuroticus and Streptomyces soufflerotiaras are similar to Honmakura, but the other four strains have a darker brown tone compared to Honmakura.

In terms of basal hyphae, all six strains exhibited a yellow or brown color, but only Strephtomyces roseis fleuroticus had a red color, which can be considered a characteristic of this pillow.

Therefore, it was determined that Streptomyces roseis fleuroticus was relatively similar to this strain, except for the dark color of the basal hyphae on the oatmeal agar medium.

Therefore, this strain was transformed into Streptomyces roseis fleuroticus DO-81 (Strepto-myces r.
oseisclerotlcus Do-81), and was deposited with the Institute of Microbiology, Agency of Industrial Science and Technology as Microbiological Laboratory Deposit No. 6502.

Next, we will discuss the culture method. The culture method of the present invention is similar to the culture of total actinomycetes. That is, as a charcoal source for the medium, for example, grape viscosity, starch, dextrin, mannose, fructose, sucrose, lactose, and molasses are used alone or in combination. Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, organic acids, etc. may also be used. Crab sources include nitrogen compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, and urea, as well as peptone, meat extract, yeast extract, and dried
Chichrine-containing natural products such as milk, corn steep liquor, soybean flour, and casamino acids are used singly or in combination. If necessary, add inorganic salts such as common salt, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferric chloride, calcium chloride, manganese sulfate, zinc sulfate, copper sulfate, etc. Good too.

Furthermore, vitabane Bl, biotin, etc., which promote the growth of the microorganism used and the production of O-81, may be appropriately added.

As a culture method, a liquid culture method, particularly deep agitation 10, especially 6 to 8, is suitable, and the pH is adjusted with aqueous ammonia, aqueous ammonia carbonate, or the like. In the case of liquid culture, usually 1 day f, c
After 7 days of culture, a significant amount of the target substance no-si was produced and accumulated in the culture solution. When the amount accumulated in the culture reaches the maximum, the culture is stopped and the bacterial cells are separated from P.

i) Isolation and purification of the O-81 substance from the culture solution can be performed using conventional separation and purification methods used to separate microbial metabolic products from the culture solution. For example, after passing a culture solution (for example, p 16.0) through activated carbon (Wako Tsumugi Co., Ltd.) to adsorb active ingredients,
Methanol/pyridine/ammonia/water (86:3:1
:10v/v) etc. to elute the adsorbed substance from the activated carbon. The solution is concentrated to dryness, dissolved in a suitable buffer solution having a pH of 7.0, and extracted with a solvent such as n-butanol. The extract was concentrated to dryness and dissolved in ethyl acetate saturated with aqueous ammonia. This solution is suspended in the same solvent in advance, and then chromatography is performed using silica gel packed in a column.

Elute with aqueous ammonia and saturated ethyl acetate, concentrate the active fraction to dryness, and dissolve in a small amount of methanol.

This methanol solution was suspended in methanol in advance and then packed into a column using Sephadex L H-20 (Phar
macla Fine Chemicals Inc.
.

(Sweden) column to obtain both portions of DO-81. This was crystallized from ethyl acetate or a mixed solvent of chloroform, ethyl ether, and petroleum ether.
-81 can be obtained.

Example 1 Streptomyces roseis fleuroticus Do-81 was used as a seed strain.

In a 21-volume Erlenmeyer flask, a strain of the following was added to the seed medium [dextrin 20 &/A' r glucose 109/l, peptone 1011/11. Corn φ Steep Liquor 51
1/l, yeast extract 1g/), K) (2PO40-
5F/l, N1gSO4・7H2C) 0.5
g/ l t Oaoo a[7/j! (pH7,2
)] Inoculated into 3 ood and shaken at 30℃ for 48 hours (22
0r, p, m,) was cultured. The obtained culture solution was added to a fermentation medium 15 with the following composition in a 301 volume jar fermenter.
1 to 5 cm (volume), and stirred with aeration at 30°C (number of revolutions: 25 Or, p, m6, aeration i'15) 7
Culture was carried out using (m1n).

Fermentation medium composition: dex) IJn 509/l, soybean meal 2
0 E/l, KH2PO40,5! //1. Mg8
04.7H200,5&/A', 0a00s 5
y/it, p) (7,2 (before sterilization) adjusted with NaOH.

During the culture, the pH of the medium was not controlled, and the culture was continued for 72 hours. The bacterial cells and precipitate were separated from the P from the culture solution to obtain a slurry 131. Pi, IA' activated carbon (Wako Tsumugi Pharmaceutical Co., Ltd.) is passed through the tower to adsorb the active substance, and the water is approximately 3A! After washing with water, the sample is further washed with methanol to remove impurities. Next, methanol, pyridine, ammonia e water (86:
The adsorbed substances are eluted from the activated carbon using 3:1:10 v/v) 51. The eluate is concentrated to dryness and then dissolved in a small amount of ethyl acetate saturated with 0.05 N NH,OH. This solution is suspended in the same solvent in advance, and then chromatography is performed using silica gel (manufactured by Merck & Co., Ltd.) packed in a column. The active fraction was chromatographed again in the same manner and after concentration, a small amount of toluene-ethanol solution NH40H (45:5:0.1 v/v
), and after suspension in the same solvent, chromatography was performed using silica gel packed in a column. After collecting and concentrating the active fractions, ethyl acetate was added to obtain a powder of DO-81. This powder was dried at 40°C under reduced pressure to obtain DO-
I was able to get 81 shaves (approximately 8 2001n9).

The physicochemical properties, antibacterial activity, and antitumor activity of DO-81 thus obtained were as described above.

This substance belongs to the so-called (1.4) benzodiazepine compounds, and as it is widely accepted that compounds of this type have water or alcohol (methanol, etc.) added to the 0-11 positions (7. are easily obtained. Their structures can be shown as follows.

However, these substances are easily converted to Do-81 by drying under reduced pressure as described above.

Example 2 The same procedure as in Example 1 was carried out except that the fermentation medium composition was replaced with the following and bamboo was used.
I got 0■.

Fermentation medium composition: soluble starch 40 y/1. Soybean meal powder 3
0I/ll Corn Steve Liquor 51//l,
K2HPO40,51/ 11. MgSO4'7H
200.5g/11. Kolo, 3g/l, 0
a0033.011/l, pH was adjusted to 7.2 (before sterilization) with NaOH.

[Brief explanation of drawings]

FIG. 1 shows the infrared absorption spectrum of Do-81. Continuation of page 1 (7■ Originator Yukizo Asano 3-9-10 Nakamachi, Machida City (C author: Makoto Morimoto) 13-9 Miyukicho, Numazu City (7■ Presenter: Ryoji Imai 1014-9 Tokukura, Mishima City (Old founder Kazuhisa Fujimoto 1188 Shimotsukari, Nagaizumi-cho, Sunto-gun, Shizuoka Prefecture

Claims (3)

[Claims] (1) New compound D specified by the following planar structural formula
C-81゜ (2) A microorganism that belongs to the genus Streptomyces and has the ability to produce Do-81 is cultured in a medium, and DC-8
A method for producing compound DC-81 according to claim 1, which comprises accumulating DC-81 in a culture and collecting DC-81 from the culture. (3) The production method according to claim 2, wherein the microorganism is Streptomyces roseis phleroticus DO-81 (Feikokenbokuyori No. 6502).