JPH03201991A - New antibiotic pf1042 substance and production thereof - Google Patents

Abstract

NEW MATERIAL:Antibiotic substance PF1042 substance having the following physical and chemical properties. Appearance: white powder. Molecular formula: C45H62O16. Mass spectrum (SI-MS): m/z 881 (M+Na)<+>. Melting point: 135-137 deg.C (decomposition). Specific rotatory power: [alpha]25=+44.7 deg. (c=1.0, MeOH). Solubil ity: soluble in methanol, slightly soluble in acetone and ethyl acetate and insol uble in water. Weak acidity, etc. USE:An antifungal agent. PREPARATION:A fungus [e.g. imperfect mold fungus PF1042 strain (FERP P-1,1145)] belonging to imperfect mold fungi, capable of producing antibiotic PF1042 substance is cultured preferably by submerged culture at 26 deg.C for 2-10 months.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel antibiotic PF1042 substance and a method for producing the same.

[Prior art 1] Babracandins A, B, C,
D and E (Papulacandins) [Tr
axleret al., J. Antibiotics
, 30.289-296 (1977)], Chaetiacandin [Komor
i etal, J. Antibiotics, 38.
455-459 (1985)], but the antibiotic PF1042 substance has different physical and chemical properties from these substances and can be clearly distinguished from them.

[Problem to be solved by the invention 1 Various antibiotics produced by microorganisms have been known, but not many clinically useful antifungal antibiotics have been found. The emergence of fungal antibiotics is always desired. An object of the present invention is to provide a novel antifungal antibiotic PF1042 substance and a method for producing the same.

[Means for solving the problems] The gist of the first invention is that the novel antibiotic PF
1042 substances and their salts. PF1 according to the invention
The physicochemical and biological properties of the 042 substance are as follows.

(1) Physical and chemical properties of PF1042 substance 1) Appearance:
White powder 2) Molecular formula: C45H62016 % formula %) () ) 6) Ultraviolet absorption spectrum λmax nm (E1%) 1 cm [MeQHl: 205 (607), 226 (501)
).

229 (sh 495), 263 (665).

[0,1N HCL-MeQHl: 205 (533)
, 227 (48k).

230 (sh 471), 264 (673).

[0,IN NaOH-MeQHl: 215(119
1), 230 (sh 620).

261 (675) 7) Infrared absorption spectrum (KBr cm'): 3430.3015.2950
．． 2920.2850°1700.1635.1615
．． 1465.1415°13B0.1350.1310
．． 1270.1210°1180.1150,1095
．． 1075, 1040° 1000, 875.850.7
70.745°15 8)'HNMR spectrum (400MHz, CD30
D) δ (ppm): 0.89 (3H, t), 0.
90 (3H, t).

1.25”1.36 (8H, m), 1.39 (2H,
m).

1.45 (2B, 111), 2.06 (2H, b
r dt).

2.34 (2H, br dt), 2.37 (2H, b
r dd).

3.46 (2H, m), 3.68 (IH, ddd)
.

3.75(IH,br s), 3.78(lH,br
d).

3.95 (IH, dd), 4.00 (2H, m).

4.14 (18, dt), 4.15 (IH, dd).

4.24 (IH, dd), 4.34 (IH, m).

4.36 (IH, d), 4.99 (IH, d).

5.04 (IH, d), 5.42 (IH, dd).

5.56 (IH, dd), 5.67 (LH, dt).

5.90 (LH, d), 5. '14 (IH, br
dt).

5.97 (Ill, d), 6.01 (IH, br
dd).

6.14 (18, dL), 6.17 (1) l, dd).

6.19 (IH, d), 6.21 (IH, d).

6.21 (IH, br dd), 6.28 (lH,
br dd) 7.27 (IH, dd), 7.70 (
IH, ddd) 9) 13CNMR spectrum (100M
H2, CD30D) δ (ppm): 169.0 s,
168.6 s, 161.6 s, 154.
5s.

146, Od, 145.5 s, 143.4
d, 141.5 d.

141.2 d, 136.0 d, 134. O
d, 132.1 d.

+31.9 d, 131.0 d, 127.5
d, 121.7 d.

121.7 d, 116.5 s, 111.9
s, 105.4 d.

103, Od, 100. Od, 77.8 d,
76.4 d.

74.8 d, 74.6 d, 73.9 d,
73.9 S.

72.6 d, 72.5 d, 71.8 d,
70.3 d.

64.7 t, 61.5 t, 42.3 t,
33.6 t.

32.6 t, 32.5 L, 30.2 t,
30.1 t.

29.2 t, 23.6 t, 23.6 t,
14.4 q.

14.4 q 10) Solubility: Soluble in methanol. acetone.

Slightly soluble in ethyl acetate. Insoluble in water.

11) Distinction between basic, acidic, and neutral: Weakly acidic PF10
Salts of the 42 substances include metal salts, particularly alkali metal salts such as sodium salts, alkaline earth metal salts such as calcium salts, and ammonium salts.

(2) Biological activity of PF1042 substance The minimum inhibitory concentrations of the PF1042 substance according to the present invention against various fungi and various bacteria are shown in Tables 1 and 2, respectively.

The gist of the present invention in Table 1 and Table 2 is that soluble: Cultivating bacteria producing the antibiotic PF1042 belonging to methanol,
The present invention relates to a method for producing an antibiotic PF1042 substance, in which the antibiotic PF1042 substance is collected from the culture.

Practical examples of microorganisms used to produce the antibiotic PF1042 substance of the present invention include:

PFI042 newly isolated from soil in Yokohama City, Kanagawa Prefecture
Stocks are one example. The mycological properties of strain PFI042 are as follows.

(1) Mycological properties of strain PF1042 1) Culture characteristics When cultured on potato dextrose agar medium at 25°C for 10 days, the colony size was approximately 3 nm (1 nm), and brown, slimy colonies were formed. Formed.

Colonies are pale orange on malt agar, milky white on Tuape Nku Dox agar, and other characteristics are potato-like.
It was similar to the dextrose agar medium.

When cultured on a potato/carrot agar medium at 25°C for 10 days, the size of the colony was approximately 40 mm, and a flat colony with a pale maroon color was formed.

In any medium, the color tone of the back side was similar to that of the front side, the soluble pigment did not peel off, and it did not grow at 37°C.

2) Morphological characteristics The macroconidia are soialo-shaped conidia, tapered at both ends of the sickle shape. Usually 3-4 cells, size 25-40 x 2-4
It is μm. Chlamydospores are rough, nuchal or interzoan. Usually solitary, they may be chained.

The size is 2.0 to 2.5 x 2.5 to 3.0 μm and oval.

No microconidia were observed.

Based on the above mycological properties, strain PF1042 is considered to belong to the soluble:methanol class Fusarium sp. Therefore, one strain was designated as the soluble methanol PF1042 strain. This strain was submitted to the Institute of Microbiological Technology, Agency of Industrial Science and Technology, as part of the Microbiological Research Institute.
No. 1145 (FERM P-11145). The properties of the PF1042 strain tend to change as seen in other molds. For example, it may be a mutant strain (naturally occurring or induced), a phenozygote, or a genetically recombinant strain derived from the PF1042 strain.

Anything that produces PF1042 material can be used in the present invention.

(2) Cultivation method of PF1042 substance-producing bacteria A PF1042 substance-producing bacteria belonging to Deuteromycetes is cultured in a medium containing nutrients that can be used by ordinary microorganisms. As a source of nutrients,
Known materials conventionally used for culturing mold can be used. for example.

Carbon sources include glucose, sucrose, starch syrup, dextrin, starch, glycerol, and molasses.

Animal/vegetable oils, etc. can be used. Nitrogen sources include soybean flour, wheat germ, corn staple liquor, cottonseed meal, meat extract, peptone, and yeast extract.

Ammonium sulfate, sodium nitrate, urea, etc. can be used. If necessary, it is effective to add inorganic salts capable of containing sodium, potassium, carunium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid, and other ions. It also helps the growth of bacteria and PF
Organic and inorganic substances that promote the production of 1042 substances can be added as appropriate.

The most suitable culture method is a culture method under aerobic conditions, especially a deep culture method. The appropriate temperature for culturing is 15-30℃.
However, in most cases, they are cultured at around 26°C. PF1
Production of 042 substance varies depending on the medium and culture conditions, but the accumulation usually reaches its maximum within 2 to 10 days in both shaking culture and tank culture. When the accumulated amount of the PF1042 substance during culture reaches the maximum, the culture is stopped, and the target substance is isolated and purified from the culture solution.

(3) Purification method for PF1042 substance When collecting the PF1042 substance obtained according to the present invention from a culture, conventional separation methods utilizing its properties 9 such as solvent extraction method, ion exchange resin method, adsorption or distribution column chromatography are used. Law.

Extraction and purification can be carried out by gel filtration, dialysis, precipitation, etc. alone or in appropriate combinations. For example, PF10
The 42 substances are extracted from the cultured bacterial cells using aceto-7-water, methanol-water, ethyl acetate, or the like. In addition, the PF1042 substance accumulated in the culture solution can be removed using organic solvents that are immiscible with water, for example.

When extracted with butanol, ethyl acetate, etc., the PFIO42 substance is extracted into an organic solvent layer.

To further purify the PF1042 substance, use silica gel (Fuco-gel C-200, manufactured by Wako Pure Chemical Industries, Ltd., etc.).

Chromatography using an adsorbent such as alumina, Sephadex LH-20 (manufactured by Pharmacia), Toyopearl HW-40F (manufactured by Tosoh Corporation), etc. may be performed.

The PFIO42 substance thus produced in the culture can be separated in free form, ie as the PF1042 substance itself, and the solution containing the PFI042 substance or its concentrate can be treated with a base, ie for example sodium hydroxide. Alkali metal compounds such as potassium hydroxide, alkaline earth metal compounds such as calcium hydroxide and magnesium hydride, inorganic bases such as ammonium salts, and organic bases such as ethanolamine, triethylamine, and dicyclohexylamine are used in each step. During the operation, for example, during each step of extraction, separation or purification, P
The F1042 substance is converted into its corresponding salt form and separated. In addition, PF1042 manufactured in this way
Salts of the substance can be converted into the free form, the PF1042 substance itself, by conventional methods. Furthermore, the PF1042 substance obtained in free form may be converted into the corresponding salt thereof using the base in a conventional manner. Therefore PF1042
as well as its salts as mentioned above.

shall be included within the scope of this invention.

Examples of the present invention will be shown below, since the properties of the PF1042 substance have been clarified by the present invention.

Various methods for producing PF1042 substances can be devised based on their properties. Therefore, the present invention is not limited to the examples, and the present invention may of course be modified by means of modifying the examples.1 Based on the properties of the PF1042 substance revealed by the present invention, known means may be applied to produce, concentrate, and extract the PF1042 substance. , encompasses all methods of purification.

Example: Detection of antifungal activity in Candida albica
The paper disk method was used using ns M9001 as the test bacteria.

As a seed medium, starch 2.0%, glucose 1.
0%, polypeptone 0.5%, wheat germ 0.6%, yeast extract 083%, soybean meal 0.2%, calcium carbonate 0.
A medium with a composition of 2% was used. In addition, as a production medium, glucose 5.0%, soybean meal 1.0%, meat extract 0
．． 4%, polypeptone 0.4%, yeast extract 0.1%.

Sodium chloride 0.25%, calcium carbonate 0.5%
A medium consisting of the following composition was used. In addition, the pH before sterilization was adjusted to pH 7.0 in all cases.

A 100 mQ Erlenmeyer flask into which the above seed medium (20 m) was dispensed was sterilized at 120°c't'' for 15 minutes, and 1 platinum loop of slanted agar culture of PFIO42 strain (FERM P-11145) was inoculated into it. A seed culture was obtained by culturing with shaking at °C for 2 days.Then, the production medium (100 m
12) 500mQ volume triangular 7 lasco (50 bottles)
was sterilized at 120'o for 15 minutes, and the above seed culture (
2m(+) of each was inoculated and cultured with shaking at 26°C for 4 days.

After the culture was completed, diatomaceous earth was added as a filter aid and the mixture was filtered to obtain a filtrate and bacterial cells.

50% acetone water (40%) was added to the cells, and after stirring for 1 hour, the cells were filtered out to obtain a cell extract.The cell extract was made into a 2Q concentrated solution by distilling off the acetone under reduced pressure. The PF1042 substance was extracted from this concentrated solution with ethyl acetate (20), and the ethyl acetate layer was concentrated to give an oily substance (830 mg).
was gotten. This oily substance was transferred to a silica gel column (80
g) and add chloroform-methanol (10
Chromatography was performed using :1) as a developing solvent, and the eluate was fractionated into 12 g portions. The active fractions of fraction numbers 51 to 58 containing the PF1042 substance were concentrated to dryness to obtain a yellow powder (42.1 mg). Next, this yellow powder was treated with Sephadex L using methanol as a developing solvent.
Purification was performed by H-20 (280ml) column chromatography, and the eluate was fractionated into 5g portions. The active fractions of fraction numbers 14 to 16 were concentrated to dryness to obtain a pale yellow powder (29,6+ag). Further add the pale yellow powder of
After purification by preparative TLC using chloroform-methanol (4:1) as a developing solvent, column chromatography was performed on Toyobar HW-40F (230 ml) using methanol as a developing solvent, and the eluate was fractionated into 4 g portions. When the active fractions of fraction numbers 16 to 17 are concentrated to dryness, PF1
042 material was obtained as a white powder (23.5 mg).

[Effects of the Invention] The PF1042 substance of the present invention has strong antifungal activity as shown in Table 1. Based on this property, PF10 of the present invention
42 substances can be used as antifungal agents or materials for conversion thereto.

[Brief explanation of the drawings] Figure 1: PF1042 substance in methanol (20μg/
ml, solid line), in acidic methanol (20 μg/ml, dashed line) and in basic methanol (20 μg/ml, -dotted chain line). FIG. 2 shows the infrared absorption spectrum of potassium bromide tablets of the substance PF1042. Figure 3: 4 of PF1042 substance in heavy methanol solution
00MHz lHN MR spectrum is shown. Figure 4: I of PF1042 substance in heavy methanol solution
00MHz13CN MR spectrum is shown. Figure 1

Claims (2)

[Claims] (1) New antibiotic PF10 with the following physical and chemical properties
42 substances and their salts 1) Appearance: White powder 2) Molecular formula: C_4_5H_6_2O_1_63) Mass spectrum (SI-MS): ￥m￥/￥z￥881 (M
+Na)^+4) Melting point: 135-137°C (decomposition) 5) Specific optical rotation: [α]^2^5_D=+44.7°(c
1.0, MeOH) 6) Ultraviolet absorption spectrum: Shown in FIG. 7) Infrared absorption spectrum: Shown in Figure 2. 8)^1H NMR spectrum: Shown in Figure 3. 9)^1^3CNMR spectrum: Shown in Figure 4. 10) Solubility: Soluble in methanol. Slightly soluble in acetone and ethyl acetate. Insoluble in water. 11) Distinction between basic, acidic, and neutral: weak acidic (2) Cultivate antibiotic PF1042 substance-producing bacteria belonging to deuteromycetes, and extract antibiotic PF1042 from the culture.
Antibiotic PF1042 characterized by collecting substances
Method of manufacturing a substance.