JPH0434555B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides a general formula that has antibacterial and antitumor activity. The present invention relates to a novel compound represented by the formula (in which R represents a hydrogen atom or a lower alkanoyl group) and a method for producing the same. The present inventors discovered that a newly isolated bacterial strain (hereinafter referred to as Y-32026) from the soil of Okazaki City, Aichi Prefecture, produces a substance with antibacterial and antitumor properties. We succeeded in isolating a new compound (AIL) represented by (see Japanese Patent Application Laid-Open No. 150595/1983). Further examination of this culture revealed that the compound AIL
In addition, a new compound, AIL-B, was discovered. Compounds of formula I in which substituent R is a hydrogen atom
AIL-B has the following physicochemical properties. Molecular formula: ₃₄ H ₄₇ N ₃ O ₉ Molecular weight: 641 Mass spectrum (FDMS): m/e664 (M ⁺ +Na) IR (CHCl ₃ ) absorption: 3200, 1765, 1655, 1507,
1410, 1190, 980cm ^-1 UV absorption: λCH ₃ OH max230, 265 (sh), 275, 285
(sh) nm ¹ HNMR (CDCl ₃ + CD ₃ OD): δ 0.97 (3H, d,
J=5.6Hz), 1.07 (3H, s), 1.20 (1H, m),
1.25 (3H, d, J=7.3Hz), 1.32 (3H, s),
1.73 (1H, m), 1.77 (3H, s), 2.08 (1H, m),
2.48 (1H, q, J = 7.3Hz), 2.86 (3H, s),
3.52 (2H, d, J = 7.3Hz), 3.85-3.95 (3H,
m), 3.93 (2H, s), 4.34 (1H, dd, J=7.0&
6.3Hz), 4.63 (1H, s), 5.63 (1H, dd, J=7.0
&14.9Hz), 5.67 (1H, dt, J=6.3&14.4Hz),
5.77) 1H, dt, J = 7.3 & 15.2Hz), 5.95 (1H,
dd, J=11.9 & 11.9Hz), 6.16(1H, dd, J=
10.6 & 14.4Hz), 6.21 (1H, dd, J = 10.6 & 14.9
Hz), 6.21 (1H, dd, J=11.9Hz), 6.41 (1H,
brd, J=11.9Hz), 6.64(1H, dd, J=11.9&
15.2Hz), 6.80 (1H, s), 7.00 (1H, brt, J=
5.6Hz), 7.84 (1H, s) ¹³ CNMR (CD ₃ OD): δ 11.408 (q), 17.726
(q), 19.656 (q) 21.470 (q), 23.108 (d),
24.278(d), 26.092(q), 29.017(t), 36.096
(t), 42.765 (q), 45.164 (t), 45.339 (s),
58.151(t), 72.192(s), 75.234(d), 76.814(d),
80.792(s), 87.461(d), 122.270(d), 124.025(d),
124.845(d), 128.121(d), 128.530(d), 129.291(d),
129.583(d), 130.870(d), 131.338(d), 134.556(d),
138.534(s), 150.703(d), 151.112(s),
171.764(s), 176.035(s), 178.433(s) Among the compounds of the general formula (I) of the present invention, the compound in which the substituent R is a lower alkanoyl group, that is, the compound of the general formula (-b), has the structural formula It can be produced by reacting the compound (-a) (hereinafter abbreviated as AIL-B) with an acid anhydride in the presence of pyridine to convert it into lower alkanoylation, and is usually produced by reaction with benzene, acetone, ethyl acetate, etc. AIL-B is dissolved in a solvent that does not have an adverse effect on the AIL-B, and excess acid anhydride is reacted in the presence of an excess amine such as pyridine. In this case, an amine such as pyridine can be used as a reaction solvent. The reaction proceeds at room temperature and is completed in about several hours. After completion of the reaction, excess amine and acid anhydride are distilled off, and then the compound of general formula (-b) can be obtained by a known purification and separation method such as silica gel column chromatography. Examples of the lower arganoyl group include an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a pivaloyl group, and an acetyl group is particularly preferred. Examples of the acid anhydride include acid anhydrides such as acetic anhydride, propionic anhydride, butyric anhydride, and isobutyric anhydride. For example, in general formula (I), the substituent R is
A compound having an acetyl group (AIL-B triacetate) is produced by reacting AIL-B with acetic anhydride in the presence of pyridine. AIL-B is oxidized with ozone in methanol at -78°C, the resulting ozonide is reduced with sodium borohydride, and the resulting alcohol is acetylated with acetic anhydride-pyridine to obtain a decomposition product (and). The decomposition products were completely identical to those obtained by subjecting AIL to ozonolysis. The above facts revealed that AIL-B has a structure represented by formula (-a). AIL-B is an AIL belonging to the genus Streptomyces.
-AIL-B can be obtained by culturing B-producing bacteria and collecting AIL-B from the culture solution. In order to produce AIL-B, a strain belonging to the genus Streptomyces that has the ability to produce AIL-B can be used. In particular, it is preferable to use strain Y-3026 isolated by the present inventors or a mutant strain thereof. Strain Y-32026 was submitted to the Institute of Microbiology, Agency of Industrial Science and Technology with Microbiological Research Institute No. 6313 (FERM).
P-6313). The mycological properties were observed at 28°C for 14 days unless otherwise specified, and are as follows. (1) Morphological characteristics Aerial hyphae of 0.6 to 0.7 μm extend from the branched basal hyphae and exhibit a straight or curved shape. When the aerial hyphae of a mature culture are examined under a microscope, 1 oval spore is found.
There are few cases in which 1 to 10 pieces of ×1 μm are connected in a chain shape, and mainly cylindrical shapes with irregular sizes of 0.4 to 1 μm.
Chains of 5 to 30 segmented spores of 1.0 μm x 0.7 to 3 μm were observed on the aerial hyphae. In other words, the number of spores that settle on the sporophyte is 1 to 1.
If there are two, and the consecutive segmental spores are not linear but irregular, it has the characteristics of Sciudonochaldeia, and acrospores and endospores may be seen, and there are also characteristics of Actinomadeula. It was done. No other special organs were observed. (2) Bacterial composition This strain was used as Romano and Solar (Romano
After culturing with shaking at 28°C for 72 hours in a modified medium of J.D. and Sohler), the cells were collected and washed. Journal of Bacteriology Vol. 89 No. 2
An analytical experiment was performed according to the method of Yamaguchi et al., described on pages 44-453 in 1965, and L,L-diaminopimelic acid and glycine were detected. When this result is applied to the family and genus search formula by Lechevalier (The Biology of Diactinomycetes and Related Organisms, Vol. 11, pp. 78-92, 1976), cell wall composition types - It belonged to the genus Streptomyces. (3) Cultural properties Table 1 shows the results of culturing on various media at 28°C for 10 to 14 days.

【table】

[Table] (4) Physiological properties (1) Growth temperature range: 15-37℃ (optimum temperature 25-30℃)
(℃) (2) Liquefaction of gelatin: Positive (3) Hydrolysis of starch: Positive (4) Peptonization of skim milk: Positive Coagulation: Negative (5) Production of melanin-like pigment Tyrosine agar medium: Positive Peptone yeast Iron agar medium: Negative (6) Assimilation of the carbon source (using Pridham-Gottlieb agar) is as shown in Table 2.

[Table] −: Not growing
(5) Other properties The morphology of this strain cultured in a liquid medium through aeration was completely different from that when cultured on slanted agar, and all of the strains exhibited a rod-like shape during the logarithmic growth phase. In addition, Gram staining of the vegetative hyphae of this strain was positive. As mentioned above, strain Y-32026 exhibits bacillary growth in liquid culture, and the electron microscopic image of the spore form shows that it is related to S. This strain was identified as belonging to the genus Streptomyces because of its ability to assimilate carbon sources, have abundant aerial hyphae, produce melanin-like pigments, and have a typical cell wall composition.
of Systematic Bacteriology, Vol. 26, No. 4, 1976; Vol. 21, No. 1, 1971; Applied Microbiology, Vol. 13, No. 2, 1965;
(See Bacterology 8th edition 1974). To produce AIL-B by fermentation, a normal Streptomyces culture method can be used.
It is preferable to culture with aeration in a fermenter. Various carbon sources, nitrogen sources, inorganic substances, growth promoters, growth inhibitors, antifoaming agents, etc. can be used in appropriate combinations in the culture medium. As the carbon source, starch glycerin, glucose, mannose, fructose, sucrose, molasses, etc. are used alone or in combination. Inorganic or organic nitrogen sources include ammonium chloride, ammonium sulfate, ammonium nitrate,
Sodium nitrate, urea, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soybean flour, etc. may be used alone or in combination. In addition, inorganic salts such as common salt, potassium chloride, calcium salts, phosphates, iron salts, copper salts, zinc salts, manganese salts, cobalt salts, magnesium salts, etc. can be added as necessary. The culture temperature is generally 20 to 40°C, particularly preferably 27 to 30°C. The pH of the medium is adjusted to 4 to 10, preferably by adding a commonly used acid or base as necessary.
Adjust the pH to 6-8. When cultured for 1 to 4 days under aeration, AIL-B accumulates within the bacterial cells and in the culture solution. Stop the culture when the maximum production amount is reached,
The culture solution is filtered and separated into wet bacterial cells and culture solution. Conventional methods are used for isolation and purification. The separated bacterial cells are mechanically crushed and extracted in a solvent such as acetone, ethyl acetate, butyl acetate, etc. while stirring.
The liquid is extracted with ethyl acetate or butyl acetate while stirring. Concentrate the extract from the bacterial cells and the extract from the culture solution together or separately, and if necessary,
In particular, when wet bacterial cells are extracted with acetone, the concentrated residue containing water is extracted again with butyl acetate, ethyl acetate, etc., concentrated, and each concentrated residue is adsorbed together or separately on a silica gel column, and then When elution is performed using a solvent such as chloroform-methanol, while gradually increasing the polarity by changing the mixing ratio, AIL-B elutes immediately after AIL elutes. AIL-B can be obtained by collectively concentrating the fractions containing the target product. This is thin layer chromatography (TLC) using a silica gel plate (Kieselgel 60F ₂₅₄ , manufactured by Merck & Co.), chloroform containing 15% methanol as a developing solvent, and sulfuric acid or ultraviolet light as a coloring agent. AIL has an Rf value of 0.60. , AIL-B is the Rf value
0.58, and bioautography using S. lutea also showed antibacterial spots with the same Rf value. The compound AIL-B of the present invention showed a survival effect on Ehrlichi's ascites carcinoma in mice when administered intraperitoneally. Example 1 Soluble starch 15g/, soybean flour 15g/,
K ₂ HPO ₄ 1g/, MgSO ₄・7H ₂ O 0.5g/,
Aqueous medium (PH
7) was inoculated with strain Y-32026 and cultured with shaking in a shaker at 28°C for 30 hours. The obtained seed culture solution was transferred to fermentation medium 140 with the following composition in a tank of 200, and incubated at 28°C for 43 hours.
Culture was carried out under aeration and stirring (130/min, 200 rpm). Fermentation medium composition: glycerin 2.0%, peptone 10%,
Meat extract 0.5%, NaCl 0.1%, _{K2HPO4 0.1} %,
MgSO ₄・7H ₂ O 0.05%, CaCl ₂・2H ₂ O 0.05% The culture solution is filtered, and the liquid is about 125% and the wet bacterial cells are about 5.2Kg.
I got it. The liquid was extracted twice with butyl acetate 60 at pH 7, dried over Na ₂ SO ₄ , and the solvent was distilled off under reduced pressure. Adsorb and elute with 0.5%, 1%, 2%, 3%, 5%, 1.2% each of methanol and chloroform in order while monitoring with TLC. Fractions containing the target product are collected and the solvent is distilled off under reduced pressure. Leave, AIL-
Obtained 20mg of B. Practical example 2 5 mg of AIL-B obtained in Example 1 was dissolved in 0.5 ml of pyridine, 0.5 ml of acetic anhydride was added under cooling, and the mixture was allowed to stand at room temperature overnight. Pyridine monoacetic anhydride was distilled off in vacuo, and further Toluene was added and completely removed azeotropically to obtain 5 mg of AIL-B triacetate. Physicochemical data: In the NMR spectrum, the CH ₂ OH singlet peak observed at δ3.93 in AIL-B undergoes a bottom magnetic field shift due to acetylation, and ABq ( J=12.5
Hz).

[Brief explanation of drawings]

Figure 1 is the infrared absorption spectrum of anti-tumor antibiotic AIL-B, Figure 2 is the anti-tumor antibiotic AIL
-B is an ultraviolet absorption spectrum.

Claims (1)

[Claims] 1. General formula A compound represented by (wherein R represents a hydrogen atom or a lower alkanoyl group). 2. The compound according to claim 1, wherein R is a hydrogen atom. 3. The compound according to claim 1, wherein R is an acetyl group. 4 Structural formula In producing the compound represented by the formula (I-a), a microorganism belonging to the genus Streptomyces and having the ability to produce the compound of the structural formula (I-a) is cultured, and from the culture solution and bacterial cells, the compound represented by the structural formula (I-a) is obtained. A method for producing a compound represented by structural formula (I-a), which comprises collecting a compound represented by formula (I-a). 5 General formula (I-b) characterized by reacting the compound of structural formula (I-a) with an acid anhydride A method for producing a compound represented by (in the formula, R ¹ represents a lower alkanoyl group). 6. The method according to claim 5, wherein the acid anhydride is acetic anhydride and R ¹ is an acetyl group.