JPH0363281A - Novel antibiotics endynamicin and its production - Google Patents

Abstract

NEW MATERIAL:A compound expressed by the formula (R represents H or OH). USE:An antibiotic against Gram-positive and Gram-negative bacteria. PREPARATION:An endynamicin-producing bacterium belonging to the genus Micromonospora [example; MG331-hF6 strain (FERM-P-10651) or Micromonospora globosa)] is cultured and a compound expressed by the formula is collected from the culture.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel antibiotic endinamycin and a method for producing the same.

[Conventional technology]

Many antibiotics produced by microorganisms have been discovered so far and are widely used to treat infectious diseases. Furthermore, among antibiotics, those having anticancer effects are used as anticancer agents.

[Problem to be solved by the invention]

However, in the treatment of infectious diseases, the emergence of resistant bacteria is unavoidable, and there is a constant need for antibiotics with new skeletons that are effective against resistant bacteria. Moreover, existing anticancer drugs cannot be said to have sufficient therapeutic effects, and new regulatory cancer antibiotics are also expected.

[Means to solve the problem]

To summarize the present invention, the first invention of the present invention relates to a new antibiotic endynamicin ([indynamicin]), which has the following formula I: (Endynamycin A is R=OH, endynamicin B is a compound represented by R=H. Further, the second invention of the present invention relates to a method for producing the antibiotic endinamycin, and the invention relates to a method for producing the antibiotic endinamycin, which
The method is characterized in that an endinamycin-producing bacterium belonging to the genus romonospora is cultured, and endinamycin represented by the above general formula I is collected from the culture.

An example of an endinamycin-producing bacterium belonging to the genus Micromonospora is MG3, which was isolated from a soil sample collected by the present inventors in May 1980 at Minobuyama, Yamanashi Prefecture.
31. - There is an actinomycete with the strain number hF6. The mycological properties of this strain are as follows.

1. Morphology Under an electron microscope, the MG331-hF6 strain shows that the basal hyphae are long and elongate in a straight line or with loose spacing, and there is a single dark olive (almost black) on the short, axle-branched sporophyte. ) spores are punished. Spores are spherical and approximately 0.8-0.9
The size is μm, and the surface is smooth. Discipline of aerial mycelia is not permitted.

2. Regarding the description of growth status colors in various media
taine'r Corporation of
The Colorharmony manual (America) was used.

(1) Cisucrose/nitrate agar medium (cultivated at 30°C) Relatively good growth was observed, and the initial growth rate [3g
c, Lt Tan]~Bright yellowish Daidai [4gc,
Apricot], but it gradually becomes darker from around the 10th day of culture. No aerial mycelia are attached, and no soluble pigments are observed.

(2) Glucose-asparagine agar medium (30
℃ culture) The growth was good, with wrinkled and raised growth, and 1
Knot yellow daikon [31c, Amher] ~ dark brown [3
pn, Dk Brown], and from around the 14th day after culture, olive ash C1'Aig, 01ive6ray
]~Dark Olive C1'Apn, Dk, 01ive
], and a slightly dark olive soluble pigment is observed. Aerial mycelium does not grow.

(3) Glycerin-asparagine agar medium (SP
-Medium 5.30℃ culture) No aerial mycelium attached, growth was slow [3ea, L
Melon Yellow) ~Bright yellowish [4ia, Apricot] ~Dark olive [4ia, Apricot]
IWpn, Dk 0live], and no soluble dye was observed.

(4) Starch/Inorganic Salt Agar Medium (ISP-Medium 4)
．． (Cultivated at 30℃) Growth is bright yellow [3ia, Drite Me
Jon Yellow) ~Bright yellow Daidai C41
a.

Apricot], and after culturing, it gradually turns black from around the 14th pewter and becomes a dark olive [1y2ρn, DkOl
ive ]. No aerial mycelia are attached, and no soluble pigments are observed.

(5) Tyrosine agar medium (ISP-medium 7.30℃ culture) Growth is bright yellow [3nc, Amher] ~
Light brown [: 31e, Cinnamon] ~ dark olive C1'Apn, Dk 01ive,], no aerial mycelium is attached, and no soluble pigment is observed.

(6) Nutrient agar medium (cultured at 30°C) Growth is pale C, 3aa, It Melon
Ye-11ow~3ga, Melon Yellow
w] ~ Bright fIi Daidai C3ia, Br1te
Melon yellow], no aerial mycelia were attached, and no soluble pigment was observed.

(7) Yeast/malt agar medium (ISP-medium 2.
(Cultivated at 30℃) No aerial mycelia were attached, good growth with raised wrinkles was observed, bright) 1 C3nc, hemb
er] ~ yellow tea C3ne, 'ropaze] ~ light brown C4pg, Dk Luggage Tan],
Gradually darker olive [1'Apn, Dk 01ivc
〕become. No soluble dyes are observed.

(8) Oatmeal agar medium (ISP-medium 3.3
(Culture at 0℃) The growth is medium-sized, colorless to pale [
3gc, Lt Tan]~Dark Olive CI Hpn
, Dk 01ivel, no aerial mycelia were attached, and no soluble pigment was observed.

(9) Glycerin/nitrate agar medium (30℃ culture) Growth was slight and olive ash [1%ig.

0live Gray) ~Dark Olive [1'Apn
, DKOlive), and neither aerial mycelia nor soluble pigments were observed.

αO starch agar medium (cultured at 30°C) Growth is colorless to pale C3ea, Lt Me-1
ion Yellow ~3 gc, Lt Tan]
Slightly raised and good growth is observed, and no aerial mycelia or soluble pigments are observed.

01) Malate lime agar medium (30℃ culture) Growth is slow and poor, pale to dark olive [1%pn
, Dk 01ive, :l, and neither aerial mycelia nor soluble pigments were observed.

(Support) Cellulose (synthetic solution added with filter paper, cultured at 30°C) Grows thinly on the surface of the filter paper and the wall of the test tube, exhibiting a pale to gray color. No aerial mycelia or soluble pigments are observed.

Q31 Gelatin puncture culture In 15% simple gelatin medium (cultured at 20°C), the growth is colorless, no aerial mycelium is attached, and no soluble pigment is observed. In the case of glucose-peptone-gelatin medium, no growth was observed at 30°C, so 20°C, 24°C, 27°C, 30°C
Although the culture was repeated at ℃, no growth was observed at any temperature.

α Skim milk powder (cultured at 30°C) Growth is colorless, no aerial mycelia are attached, and no soluble pigments are observed.

3. Physiological properties (1) Growth temperature range Pennet agar medium [Yeast extract (Difco?)]
0.1%, beef extract (manufactured by Difco) 0.1%, N-
Z-amine type A 0.2%, glucose, 1.0%
, string agar 3.0%, pH 7.3] at 20°C, 2
As a result of tests at each temperature of 4℃, 27℃, 30℃, 37℃, and 50℃, it grew at all temperatures except 50℃,
The optimum growth temperature seems to be around 30°C to 37°C.

(2) Liquefaction of gelatin (15% simple gelatin, cultured at 20°C; glucose peptone gelatin medium, 20°C, 2
4°C, 27°C, 30°C culture> In simple gelatin medium, liquefaction is tolerated for a month from around the 7th day after culture, and thereafter it progresses slowly and its effect is moderate to strong. On the other hand, no growth was observed in the glucose-peptone-gelatin medium at any temperature.

(3) Hydrolysis of starch (starch/1 ltu
K'U salt agar medium and starch agar medium, both 30
(°C culture) Starch/inorganic salt agar media are observed to have ice-melting properties from around 5 days after cultivation, while starch agar media are found to have ice-melting properties from around 7 days after cultivation, and the effect is stronger in both cases.

(4) Coagulation and peptonization of skimmed milk (skimmed milk, 37℃
(Culture) Coagulation started around the 8th day after culturing, completed immediately, and reached 100
Peptoneization begins around the same day.

Peptonization progresses extremely slowly and is not completed even after 3 weeks after culture.

(5) Production of melanin-like pigment (tryptone yeast broth, l5P-medium 1.27°C; peptone yeast soft agar medium, l5P-medium 6.30°C; tyrosine agar medium, l5P-medium 7.30°C #) Tryptone yeast broth, peptone yeast broth
Negative on iron agar and tyrosine agar.

(6) Utilization of carbon sources (Pridham-God IJ-Bu agar medium, 15P-medium 9.30°C culture) Utilizing D-glucose, L-arabinose, D-arabinose, D-xylose, sucrose, and raffinose. It grows well.

Fructose, rhamnose, and lactose are probably used, but inositol and D-mannitol are not used.

(7) Dissolution of malate lime (malate lime agar medium, 3
(cultured at 0°C) negative.

(8) Nitrate reduction reaction (peptone water containing 0.1% potassium nitrate, l5P-medium 8.30°C culture) Positive.

(9) Decomposition of cellulose (synthetic solution with filter paper pieces added, 30℃
Culture) Although it is extremely weak, it seems to be slightly decomposed. (Observed until 130 days after culture) To summarize the above properties, M.
The G331-hFG strain does not grow aerial hyphae, and a single dark olive (almost black) spore is formed on the sporophyte, which is short and uniaxially branched from the basal hyphae, which are linear or gently curved and elongated. be done.

The spores are spherical and have a smooth surface. Relatively good growth is observed on various media, especially on natural agar media, where the growth is wrinkled and thick and raised. At the early stage of cultivation, the growth exhibits a pale to bright yellowish color, but it becomes a dark olive color from around 14 days after cultivation. Almost no soluble pigments are observed. The production of melanin-like pigments is caused by tryptone, yeast broth, peptone,
It was negative on both yeast/iron agar medium and tyrosine agar medium. Proteolytic power is moderate to strong,
Starch also has strong ice-melting properties. Furthermore, 2 and 6 contained in the cell wall
- Diaminopimelic acid is mainly in the meso form,
LL type is also slightly recognized.

Based on these properties, the M0331-hFO strain is considered to belong to Micromonospora. Furthermore, when searching for closely related known bacterial species, Micromonospora globosa [Micr
omonosporaglobosa, The Journal of Antibiotics
l of Antibiotics) Volume 35, No. 14
30 pages, (1982); Journal of Bacteriology
146, page 527, (1981)]
, and Micromonospora 1 velchyllosa [Micro
monospora verruculosa, the.
Journal of Antibiotics Vol. 35, No. 1430, (1982); Special Publication No. 51-33195
]. Therefore, we first conducted a comparative experiment on the MG331-hFG strain and Micromonospora globosa stored at our institute. Next, we obtained Micromonospora verculosa and compared it with the MG331-hFe strain based on the literature, focusing on the differences in its properties. The results and comparisons in literature are summarized in Table 1.

As is clear from Table 1, the MG331-bF6 strain exhibited properties similar to Micromonospora globosa and Micromonospora verculosa. Above all, it is extremely closely related to Micromonospora globosa.
The MG331-h F6 strain differs from Micromonospora globosa in its use of rhamnose as a carbon source and in its cell
This is the strength of decomposition of 7-su (observed for 130 days).

In the process of searching for similar products in history, the anti-sedative substance 'I''-
Micromonospora bavrosus producing 42318 (
Micromonospora papuloSus,
I learned that Tokuko No. 59-5279) was similar. Although comparison experiments with Micromonospora babrosus could not be carried out due to manual labor, it is considered to be a species more closely related to Micromonospora verculosa than the MG331-hF6 strain in the following respects. That is, Micromonospora pavlosas has a warty or blunt short linear spore surface, and its cell wall composition does not contain LL-diaminopimelic acid.

Taking all the above points into account, the MG331-bF6 strain is considered to be the species most closely related to Micromonospora globosa. Therefore, the MG331-hFG strain was identified as Micromonospora globosa MG331-hF6. Furthermore, MG33
1. We filed an application for depositing the hFG strain with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and on April 10, 1989, we submitted the application to the Institute of Microbiology, No. 1065.
No. (FER, MP-10651).

Bacterial strains that can be used in the present invention include the above-mentioned strains, their mutant strains, and all endinamycin-producing bacteria belonging to the genus Micromonospora.

The antibiotic endinamycin of the present invention is produced by inoculating the above-mentioned strain into a medium suitable for producing endinamycin and culturing it. The medium may be a nutrient-containing medium commonly used for culturing actinomycetes, or may not contain a nutrient source.

Examples of nutritional sources include commercially available peptone, meat extract, corn staple liquor, cottonseed powder, peanut powder,
Soy flour, yeast extract, NZ amine, casein melt,
Nitrogen sources such as sodium nitrate, ammonium nitrate, ammonium sulfate, and carbon sources such as commercially available carbohydrates such as glycerin, sucrose, starch, glucose, galactose, mannose, molasses, or fats, and salt, phosphates, carbonates. Inorganic salts such as magnesium sulfate and magnesium sulfate can be used. In addition, trace amounts of metal salts, animal/vegetable/mineral oils as antifoaming agents, etc. can also be added as necessary. These materials can be used as long as they are useful for the production of endinamycin and can be used by production bacteria, and all known culture materials for actinomycetes can be used. Furthermore, by adding an inorganic or organic iodine salt or iodine compound to the above-mentioned nutrient source-containing medium, the production amount of endinamycin can be increased.

Examples of the medium containing no nutrient source include aqueous solutions of inorganic or organic iodine salts and iodine compounds.

By adding reverse-phase silica gel powder to these media, endinamycin can be easily extracted from the culture solution, and the production amount can also be slightly increased. Liquid culture is preferable for mass production of endinamycin, and the culture temperature can be applied within a range that can produce endinamycin.

Cultivation can be carried out under the above-mentioned conditions, appropriately selected according to the properties of the endinamycin-producing bacteria used.

Endinamycin is present in both the culture filtrate and the bacterial cells. The culture filtrate can be extracted with a water-immiscible organic solvent such as butyl acetate, chloroform, butanol, etc. at pH 10 or less.

After extracting the bacterial cells with an organic solvent such as methanol or acetone, the extract can be concentrated under reduced pressure and further extracted in the same manner as the culture filtrate. When reverse-phase silica gel is added to the culture medium, it can be collected together with bacterial cells and extracted using the same method as for extraction from bacterial cells. In addition to the above-mentioned extraction methods, there are known methods used to collect fat-soluble substances, such as adsorption chromatography, gel filtration chromatography, scraping from thin layer chromatography, centrifugal countercurrent partition chromatography, and high performance liquid chromatography. By appropriately combining or repeating the above, pure collection can be achieved.

The physicochemical properties of antibiotics endinamycin A and B are as follows.

Table 2 shows the shapes, field desorption mass spectrometry (FDMS), molecular formulas, ultraviolet and visible absorption maxima, solubility, acidity, neutrality, and basicity of Endinamycin A and B. The chemical shifts of the proton nuclear magnetic resonance spectra of mycin and B are shown in Table 3.

Furthermore, FIG. 1 shows the infrared absorption spectrum of endinamycin by potassium bromide tablets.

The horizontal axis in Figure 1 is the wave number (cm-'), and the vertical axis is the transmittance (%).
) represents.

Endinamycin B was determined to have a structure of R=H in the above general formula I by X-ray crystal analysis. Furthermore, by comparing various physical and chemical properties and spectral data with endinamycin B, it was determined that endinamycin A has the structure of R=OH in the general formula I.

The biological properties of the antibiotics endinamycin A and B are as follows.

Table 4 shows the inhibitory concentrations of endinamycin A and B against various bacteria on a nutrient agar medium (according to the agar plate dilution method). As is clear from Table 4, endinamycins A and B had strong antibacterial activity against Durum-positive and -negative bacteria, and particularly inhibited the growth of Durum-positive bacteria at extremely low concentrations. Also Escherichia Co IJK
-12 It also inhibited the growth of resistant bacteria such as ML1629.

LD when endinamycin A was administered intraperitoneally to female ICR mice. was 3°1 to 6.3 mg/kg.

Table 5 shows the results of experiments on treating tumor-bearing mice with endinamycin. Leukemia L1210 or Ehrlich ascites carcinoma was transplanted, respectively, and endinamycin was injected into the peritoneal angle for IO days immediately after transplantation. From Table 5, endinamycin A was effective against both leukemia L1210 and Ehrlich ascites carcinoma.

00 50 25 62.5 31.3 15.6 20th 52 30 74 〉399 30 Table 70 〉337 〉2G1 〉294 39 As mentioned above, the physicochemical properties and biological properties of endinamycin A and B are detailed. As mentioned above, M-92VA-2 (
Special equipment 11i'35133195) and T-42318 (
Special equipment lpj 5 Q-5279). Although there is no description regarding the structure and only physical and chemical properties are shown, M-92VA-2 has an infrared absorption spectrum of 1720 to 1730 cm-', which is seen in endinamycin A, when compared with endinamycin A. There is no absorption (seen even when measured with Nujol), and the absorption in the visible region is also shifted by 10 to 20 nm. Also M-92VA
When comparing Endinamycin-2 with Endinamycin B, the absorption in the ultraviolet and visible regions is clearly different. T-42318 has elemental analysis values for carbon and oxygen that are different from the calculated values for endinamycin A and B, and the intensity of the visible absorption maximum is about twice that of endinamycin A, making it an enzyme. Absorption in the visible region is clearly different from that of dynamycin B. Furthermore, M
Both -92VA-2 and T-42318 are described as acidic substances, whereas Endinamycin A and B are amphoteric substances. Therefore, M-92VA-2 and T-
Although both of 42318 are considered to be in the same group as endinamycin, they were found to be different substances, and endinamycin A and B were determined to be new substances.

〔Example〕

Next, the present invention will be explained by examples, but the present invention is not limited thereto.

Example 1 Micromonospora globosa M cultured on agar slant medium
G331-hF6 strain [Feikoken Bibori No. 10651 (FE
RM P-10651)], glucose 1%,
Liquid medium (pH 7,
One platinum loopful was inoculated into two Erlenmeyer flasks with Watsufuru tubes containing i o Ord of 0), and cultured with shaking at 30° C. for 6 hours. This was used as the primary seed culture solution, and the same medium was dispensed in 100rnl portions into Erlenmeyer flasks 48 with Watsufuru.
The seeds were inoculated into 4 days each and cultured with shaking at 30°C for 4 days to obtain a secondary seed culture.

Potassium iodide 1 mM, silanized silica gel (
Art, 7719) manufactured by Merck & Co., Ltd. 1%, antifoaming silicone oil (Silicone KM70 manufactured by Shin-Etsu Chemical Co., Ltd.)
A jar containing 2OA of liquid medium consisting of 0.025%.
8 bottles of the secondary seed culture solution were inoculated into 6 fermentors each, and aerated and stirred for 3 days at 37°C, aeration rate of 41/min, and agitation rate of 100 rpm.

The bacterial cells and silica gel powder were collected by filtration, and extracted with 71 methanol. After concentrating the extract under reduced pressure, 41
of water was added and extracted with 4f of chloroform at pH 7.0. Add an equal volume of 25mM sodium borate buffer (pllio, o) to the chloroform layer and transfer to the aqueous layer, then adjust water 1i''4 to pH 8, () and extract again with an equal volume of chloroform. The mixture was concentrated under reduced pressure to obtain 125 mg of a blue candy-like substance. A small amount of methanol was added to it and centrifuged to obtain 34.0 mg of a purple powder. It was dissolved in dimethylformamide (DMF) manufactured by Shoisha. After centrifuging and discarding the precipitate, it was concentrated and cooled.It was centrifuged to collect the precipitate, and the supernatant was concentrated and cooled by adding a small amount of methanol.It was then centrifuged to collect the precipitate, and the supernatant was reused in the same manner. The operation was repeated to obtain a precipitate three times. By collecting the three precipitates, 6.5 mg of reddish-purple powder was obtained.

This powder was dissolved in DMF and swollen with DMF.
0mj? ) and eluted with DMF.
, by thin layer chromatography using a 5715 silica gel plate, Rf*I0.3-0.4 [Developing solution: chloroform-methanol (1(11)]) showed a single reddish-purple band. Both fractions were collected and concentrated under reduced pressure. 5.1 mg of endinamycin was obtained.
Dissolve mg in DMF (1mg/mf), 0.2mj
! Zutsu High Performance Liquid Chromatography [■ Senshu Kagaku, Senshu Back Nucleosil (Nucleos)
iJ) 5 Cto, 4,6 x 250 mm] and DMF-0.1% ammonium carbonate aqueous solution (1:
Using 1) as a mobile phase, peaks at 20 minutes and 25 minutes were separated and concentrated under reduced pressure at a flow rate of 0.5nf1 minute while detecting absorption at 590 nm. As a result, 2.4 mg of endinamycin was obtained from the 20 minute peak, and 0.5 mg of endinamycin B was obtained from the 25 minute peak, both as reddish-purple powder.

Each of these gave a single peak in high performance liquid chromatography under the above conditions.

Furthermore, endinamycin B was dissolved in a small amount of DMF, concentrated, and then stored at 5°C to obtain 0.2 mg of reddish-purple crystals.

〔Effect of the invention〕

As explained in detail below, the present invention provides a newly regulated cancer antibiotic and a method for producing the same. The newly regulated cancer antibiotic according to the present invention is effective against murine leukemia and Ehrlich ascites cancer, and has remarkable effects in that it exhibits strong antibacterial activity.

[Brief explanation of drawings]

FIG. 1 is an infrared absorption spectrum diagram of endinamycin A.

Claims (1)

[Claims] 1. A compound represented by the following general formula I: ▲There are mathematical formulas, chemical formulas, tables, etc.▼... [I] (In the formula, R represents H or OH). and the antibiotic endinamycin. 2. A method for producing the antibiotic endinamycin, which comprises culturing an endinamycin-producing bacterium belonging to the genus Micromonospora, and collecting the endinamycin according to claim 1 from the culture.