JP3067904B2 - A new penicillium mold - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a new bacterium which produces a novel isochroman carboxylic acid derivative having antibacterial activity, FERM BP-3959.

[0002]

2. Description of the Related Art In recent years, bacterial topoisomerase has been developed.
II (gyrase) inhibitors have attracted attention as targets for antimicrobial agents. This is based on the fact that the mechanism of action of the currently widely used synthetic quinolone antibacterial agents is mainly attributed to inhibition of gyrase activity. Quinolone-based synthetic antibacterial agents show a wide range of antibacterial activities, but like other antibacterial agents, the emergence of resistant bacteria gradually becomes a problem. Novobiocin, kumamycin A1, and the like are known as gyrase inhibitors produced by microorganisms, but their antibacterial spectrum is narrow, and they do not extend to quinolone-based synthetic antibacterial agents as antibiotics.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a bacterium which produces a novel isochroman carboxylic acid derivative having an excellent antibacterial activity in which resistant bacteria hardly appear.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have developed a novel isozyme having anti-gyrase activity, a mold N990-12 isolated from a soil sample collected in a forest community near Flagstaff, Arizona, USA. It has been found to produce a roman carboxylic acid derivative.

That is, the present invention provides a novel isochroman carboxylic acid derivative-producing bacterium represented by the following formula.

[0006] The mycological properties of N990-12 are as follows.

[0007] N990-12 was inoculated from potato-glucose agar slant culture at one and three places on an identification agar plate medium and cultured at 25 ° C for 1 to 5 weeks. The color is Robert R
idgway's Color Standards and Color Nomenclature (19
It was determined by comparing with the color chip of 12). The media used for strain identification are as follows.

1. Corn meal agar (CM): Carmichae
l, JW, Mycologia 49: 820-830, 1957. 2. Tzapek sucrose agar (CZ): Raper, KB and
Fennell, DI The Genus Aspergillus, p. 36, 196
Five. 3. Malt extract agar (MEA): ibid, p. 4. Tzapek Yeast Agar (CYA): Pitt, JI, Pen
icillium and Its Teleomorphic States Eupenicillium
and Talaromyces, p. 18, 1979. 5. 25% glycerin / nitrate agar (G25N): Same as above. 6. Oatmeal agar (OA): ISP medium No. 3, Difco. 7. Potato and glucose agar (PDA): ATCC medium N
o. 336, ATCC Media Handbook, p. 17, 1984. 8. V-8 juice agar: ATCC medium No. 343, ibid. 9. Phytone Yeast Extract Agar (Phytone YEA): BBL
. 10. Yeast extract / soluble starch agar (YPSS): Emerso
n, R., Mycologia 50, 589-621, 1958.

[0009] 1. Growth state in each medium reaches 4.2 cm in diameter in CYA (25 ° C, 7 days) ; white to maize yellow (IV), and in 11 days, pale orange to pale ochraceous. salmon to
It becomes pale ochraceous buff, XV, and the periphery is light celandine green to celandine g
reen, XLVII); radial wrinkles, moderate elevation; pale yellow orange to pale yellow orange to
orange buff, III) secretion, which is 11
In the day it becomes orange (orange, III), red-orange or dark red-orange (bittersweet orange to mars orange, II);
Reverse side is gray-blue (dusky orient blue, XXXIV); no soluble pigment is produced.

[0010] CZ (25 ° C., 7 days) reaches 3.3 cm in diameter; russian green (XLII), velvety, flat; colorless on the reverse side; ocharaceous orange, XV) or orange (apri
cot orange, XIV).

In MEA (25 ° C., 7 days), the diameter reaches 2.7 cm; light green (celandine green, XLVII), velvety, flat; the back side is dark gray green to dark green gray (dusky blue)
-greento dusky bluish-green, XXXIII); no soluble pigment is produced.

G25N (25 ° C., 7 days) reaches 2.1 cm in diameter; light yellow (baryta yellow, IV), light green to green (gnaphalium green to pea green, X) on day 11
LVII); a circular dent is formed at the periphery and center.
Wrinkles radiate in one day; moderate ridges, felt-like; reverse green to verdigris gr
een, XIX) but the periphery is light green yellow (light chalcedony
yellow, XVII). On the 11th, turquoise, dark turquoise, dark gray green (porcelain green, dark porcelaingree
n, dusky blue-green, XXXIII); no soluble pigment is produced.

[0013] CYA (5 ° C., 7 days) reaches 0.8 cm in diameter; white, grows thinly, but moderately bulges on 11 days, flat, felt-like; reverse side white; does not produce soluble pigment.

CYA (37 ° C., 7 days) reaches 2.2 cm in diameter; white to pale olive buff,
XL), markedly irregularly wrinkled, highly raised; reverse side orange-yellow, deep orange-yellow to brown (warm-buff, antimony
yellow, ochraceous orange tocinnamon-brown, XV
), 11 days dark orange yellow to dark brown (raw sienna to
antique brown, III); no soluble pigment is produced.

At CM (25 ° C., 7 days) it reaches 3.7 cm in diameter; thin, flat, velvety; colorless to pale green to yellow (V); no soluble pigment is produced.

YPSS (25 ° C., 7 days) reaches 2.9 cm in diameter; pale green to green grey (celandinegreen to artemisi
a green, XLVII) or deep bluish gra
y-green, XLII), flat, velvety; colorless on the reverse side, buff yellow (IV), light yellow pink to deep orange yellow (ochraceous buff to ochraceous-orange, XV); no soluble pigment is produced.

2. Morphological characteristics Morphological characteristics were observed after 7 and 11 days of culture on CYA and CZ agar. Conidiophores usually arise from aerial mycelium and produce a smooth or sparsely rough surface, 50-200 × 2-4 μm, either phialide or metre; metre is 2 to 4
12-20 x 2-4 μm rings, smooth or rough wall; 6 to 10 phialides per metre, smooth, ampoule-shaped, sharply tapered, 7-9 x 2.0-2.5 μm; conidia spherical or subspherical, often elliptical, 2.5-3.
According to observation with a scanning electron microscope at 0 μm or 3.0-3.5 × 2.5-3.0 μm, the surface was smooth or finely rough.

3. Growth temperature Good growth at 15, 20, 28 ° C; moderate to good growth at 37 ° C; no growth at 45 ° C and 50 ° C.

Summarizing the above mycological properties, N990-12 has a monocyclic or bicyclic penicillium, a smooth or coarsely rough conidia, and a smooth or finely rough wall. Characterized by spherical or subspherical conidia. The color of the conidium mass is generally green. On CYA, MEA and G25N media, the color of the back of the colonies was blue to turquoise. Therefore, this strain belongs to the genus Penicillium.

The strain N990-12 was obtained from Raper and Thom (A Manual
of the Penicillia, The Williamsand Wilkins Compan
y, Baltimore, 1949), Pen
icillium raistrickii series and Penicillium brevi-co
It is closely related to the strains of the mpactum series, but differs from them as follows.

N990-12 is in the form of metre and phialide,
P. raistrickii s sparsely rough conidia and conidia
Similar to eries but lacking sclerotium, color on the back of the colony, shorter conidiophores, thinner phialides, and a smoother or finer conidia surface than a smooth surface Differs in being rough.

N990-12 and Penicillium brevi-compactum
The strains of the series have the same growth inhibition on most of the media tested, common conidia head color, metre and phialide morphology, and sparsely rough conidia pattern. Three members of this series (P. brevi-compactum, P. stoloniferum
And P. paxilli), P. brevi-compactum
P. stoloniferum refers to the lack of a lami, the shorter color of the colonies, the shorter color of the conidia, the smaller conidia, and the smaller conidia. P.paxilli means that the color of the back side of the colony is longer, and that the majority of the conidia are spherical or subspherical rather than elliptical or subspherical. Each is different.

From the above properties, N990-12 did not match any of the species described in the genus Penicillium and was named Penicillium sp. This is Penicillium Sp.
(Penicillium sp. N990-12)
Deposit No. 9 (FERM BP-3959) on July 31, 1992 under the terms of the Budapest Treaty.

[Culture of Bacteria] Culture of the strains of the present invention is preferably carried out as follows. For small-scale culture,
First, an appropriate amount of nutrient medium is placed in a flask, sterilized by a known method, and inoculated with N990-12 spores or vegetatively growing cells in the flask, and then inoculated on a rotary shaker at a constant room temperature of about 26 ° C. Culture for 2 to 10 days. For large scale cultivation, it is desirable to carry out in a suitably sized tank equipped with a stirrer, aeration device and sterilization device. A nutrient medium is prepared in a tank, sterilized, and inoculated with N990-12 spores or vegetatively growing cells. The pH of the medium is between 5 and 9, preferably
6 to 8 is preferred. The culturing is carried out at a temperature in the range of about 20 to 40 ° C. for about 2 to 10 days with stirring and / or aeration of the nutrient medium. The degree of aeration needs to be varied depending on several factors (eg, incubator size, agitation speed, etc.). Generally, stirring is carried out at 0 to 2,000 rpm, preferably 100 to 360 rpm, and aeration is preferably carried out by blowing air at 0 to 500%, preferably 80 to 120% of the medium volume per minute.

[Recovery of Product] The recovery of the novel isochromancarboxylic acid derivative from the whole culture can be separated by solvent extraction and various chromatographic techniques. The substance is only slightly soluble in water but soluble in organic solvents. By utilizing this property, it is possible to easily recover a novel isochromancarboxylic acid derivative. For example, the whole culture solution is extracted with a water-immiscible solvent such as chloroform, ethyl acetate, and methyl isobutyl ketone. These extracts are dried and concentrated using a known method, and then subjected to various types of chromatography. Column chromatography using a medium such as silica gel, reversed-phase silica gel, alumina oxide, or dextran gel, and eluting these columns with various solvents and / or changing the ratio of two or more solvents, and eluting them is an example. . Liquid chromatography and thin layer chromatography are convenient for the detection of isochroman carboxylic acid derivatives. Its presence can be determined by measuring the antibacterial and anti-gyrase activities of the various chromatographic fractions as described below.

[Measurement of activity] The gyrase inhibitory activity of the novel isochroman carboxylic acid derivative was measured by a known method using gyrase derived from Escherichia coli (Mizuuchi, K.,
O'dea, MH, andGellert, M., Proc. Natl. Acad. Sc
i. USA, 75, 5960-5963, 1978). The inhibitory activity on eukaryotic cell-derived topoisomerase II was measured using a commercially available Drosophila-derived topoisomerase II according to the method specified by the manufacturer (US Biochemical Corporation). The inhibitory activities (IC ₅₀ values) of the novel isochroman carboxylic acid derivative against gyrase and topoisomerase II were 3 μg / ml and 1 μg / ml, respectively.
ml. These biological activities suggest that isochroman carboxylic acid derivatives have utility not only as antibiotics but also as antitumor agents. This is based on the mechanism of action of some currently widely used antitumor agents by inhibiting topoisomerase II activity (see Znang, HD, D'Arpa, P. and Liu,
LF, Cancer Cells, 2, 23-26, 1990).

The antibacterial activity of the novel isochromancarboxylic acid derivative was measured by a standard method (Ericsson, HM and Scherr).
is, JC, Acta Pathol. et Microbiol. Suppl. 217B,
64-68, 1971) according to the agar plate dilution method using BHI medium (pH 7.4). Table 1 shows the minimum inhibitory concentration of the novel isochroman carboxylic acid derivative for each test bacterium.
Shown in

[0028]

[Table 1] Test bacteria Minimum growth inhibitory concentration (μg / ml) Staphylococcus aureus 01A005 25 (Staphylococcus aureus) Staphylococcus aureus 01A063 50 (Staphylococcus aureus) # Staphylococcus epidermides 01B087 50 (Staphylococcus epidermides) 100 (Enterococcus faecalis) Streptococcus pyogenes 02C054 50 (Streptococcus pyogenes) Escherichia coli 51A266> 100 (Escherichia coli) Pseudomonas aeruginosa 52A104> 100 (Pseudomonas aeruginosa) Maltida 59A001 100 (Pasturella multocida) Serratia marcescens 63A017> 100 (Serratia marcescens) Enterobacter aerogenes 67A040> 100 (Enterobacter aerogenes) Enterobacter aerogenes 67B00 9> 100 (Enterobacter cloacae) Providencia stuarti 77A013> 100 (Providencia stuartii) Providencia rettgeri 77C025> 100 (Provedencia rettgeri) Morganella morgani 97A001> 100 (Morganella morganii) # Ciprofloxacin resistant bacteria

[0029]

The novel isochroman carboxylic acid derivative produced by the fungus of the present invention is useful for preventing or treating human and other animal diseases caused by a wide range of pathogenic bacteria including the fungi shown in Table 1, and is useful for oral or oral administration. Administration can be by either parenteral or topical routes. Generally, the most desirable doses of these compounds will be between about 300 mg to about 3000 mg per day, but will, of course, vary with the weight and condition of the patient and the particular route of administration. However, about 1 day per kilogram of body weight
A dose of 5 mg to about 50 mg is most desirable. When administered to an animal, the dosage varies depending on the type of animal to be treated, the difference in sensitivity of the animal to the drug, the method of prescribing the drug, the administration period and the administration interval. In some cases, a dose lower than the lower limit of the above range may be appropriate, and even if the dose is larger than the above range, it may cause harmful side effects if it is divided into several times a day and administered in small amounts. Not always.

The novel isochroman carboxylic acid derivative can be administered alone or together with a pharmaceutically acceptable carrier or diluent by any of the above three administration routes, and the administration can be carried out once or several times. Can be done separately. More specifically, it can be administered in a variety of dosage forms, such as tablets, capsules, lozenges, lozenges, troches, hard candy, powder in combination with various pharmaceutically acceptable inert carriers. Agent, spray,
It can be in the form of creams, salves, suppositories, jellies, gels, pastes, lotions, ointments, aqueous suspensions, injections, elixirs, syrups and the like. These carriers include solid diluents or excipients, sterile aqueous media, various non-toxic organic solvents, and the like. In the case of a drug for oral administration, sweetening and / or flavoring may be appropriately performed. Generally, the therapeutically effective compounds of the present invention will be administered in a concentration as described above in a concentration ranging from about 5% to 70% by weight.

For oral administration, various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium phosphate, glycine may be added to starch, preferably corn, potato or tapioca starch, and alginic acid. It can be used with various disintegrants, such as certain silicic acid double salts, and granule-forming binders such as polyvinylpyrrolidone, sucrose, gelatin, gum arabic. Lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are also often very effective for tablet formation. The same kind of solid composition can be used by filling it into a gelatin capsule. Suitable substances in this connection include, in addition to lactose or milk sugar, high molecular weight polyethylene glycols. In the case of aqueous suspensions and / or elixirs for oral administration the active ingredient may be combined with various sweetening or flavoring agents, coloring agents or dyes and, if necessary, emulsifiers and / or suspending agents. And can be used with water, ethanol, propylene glycol, glycerin, and the like, and diluents thereof in combination.

For parenteral administration, a solution in which the novel isochroman carboxylic acid derivative is dissolved in either sesame oil or peanut oil, or in an aqueous propylene glycol solution can be used. The aqueous solutions should be suitably buffered (preferably pH 8 or more) if necessary and the liquid diluent first rendered isotonic. Such aqueous solutions are suitable for intravenous injection, and oily solutions are suitable for intra-articular, intramuscular and subcutaneous injection. The production of all these solutions under sterile conditions can be readily achieved by standard pharmaceutical techniques well known to those skilled in the art. Furthermore, the novel isochroman carboxylic acid derivative can be administered topically, such as on the skin. In this case, it is desirable to apply topically in the form of creams, jellies, pastes and ointments according to standard pharmaceutical practice.

[0033]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to the details specified in these examples. The melting point used in the examples is a Yanaco micro melting point analyzer (uncorrected), the optical rotation is a DIP-370 digital polarimeter manufactured by JASCO Corporation, and the ultraviolet absorption spectrum is a spectrophotometer manufactured by JASCO Corporation in a methanol solution ( Ubest-30), the infrared absorption spectrum was measured by Shimadzu infrared spectrophotometer (IR-470). Unless otherwise indicated, ¹ H and ¹³ C nuclear magnetic resonance spectra (NMR) were measured with a solution of deuterated chloroform (CDCl ₃ ) using a JEOL nuclear magnetic resonance apparatus (JNM-GX270, 270 MHz). It is a thing. The peak position is expressed in parts per million (ppm) from tetramethylsilane to downfield. The peak shape is represented as follows.
s: singlet, d: doublet, t: triplet, m: multiplet, br: broad. The EI mass spectrum was measured with a Hitachi mass spectrometer (model M-80), and the HRFAB mass spectrum was measured with a NaI matrix using a Kratos mass spectrometer (model 1S).

Example 1 Medium 1 (glucose 2%, polypeptone 3%, meat extract
0.3%, yeast extract 0.5%, blood meal 0.3%, Ca
Inoculate Penicillium sp. N990-12 into a 500 ml Erlenmeyer flask containing 100 ml of CO ₃ 0.4%, pH 7.0).
This flask was cultured at 26 ° C. for 4 days on a rotary shaker having a diameter of 7 cm and rotating in a circle (220 rpm) to obtain a first seed culture solution.

7.5 ml of the first seed culture is inoculated into five 500 ml Erlenmeyer flasks each containing 150 ml of Medium 1. This flask was cultured at 26 ° C. for 4 days on a rotary shaker having a circular rotation (220 rpm) having a diameter of 7 cm to obtain a second seed culture solution.

Medium 2 (glucose 1%, corn starch
2%, Enuzetto amine 0.5%, 0.5% wheat germ, 0.5% yeast _{extract, CoCl 2 · 6H 2 O 0.00001} %, CaCO 3 0.4
%, PH 7.2) is added to 5 6 L glass fermenters containing 150 L of the second seed culture solution. The fermenter was ventilated at 3 L / min, rotating at 1700 rpm,
Stir at 26 ° C. for 72 hours.

After completion of the culture, 15 L of the whole culture was freeze-dried,
The resulting dried broth is extracted with 5 L of 70% aqueous acetone.
After removing acetone under reduced pressure, the ethyl acetate extract obtained by extracting twice with 2 L of ethyl acetate is dried over anhydrous sodium sulfate and concentrated to dryness. An oily residue containing 3.0 g of the new isochromancarboxylic acid derivative is obtained. This oily substance
Chromatography is performed on a column packed with 200 g of silica gel. The column is eluted sequentially with 1000 ml each of chloroform, chloroform-methanol (9: 1) and methanol. The fractions eluted with methanol are collected, concentrated, and subjected to 1.2 L Sephadex LH-20 column chromatography packed with methanol. The active fraction eluted with methanol is collected, concentrated to dryness, and 209 mg of crude powder is obtained. Place 75 mg of this on 5 thin-layer chromatograph plates (Kieselgel 60 F-254, 20 cm X 20 cm, 0.25 mm) and develop with chloroform-methanol (3: 1). The band exhibiting UV absorption is collected, and 62 mg of the novel isochromancarboxylic acid derivative is obtained as a white powder. The product was 3-n-heptyl-1,6,8-trihydroxyisochroman-7-carboxylic acid represented by the above formula.

Property: white powder Molecular formula: C ₁₇ H ₂₄ O ₆ HRFAB-mass spectrum: [m / z, (M + H) ⁺ -H ₂ O] 307.15
44 (calculated value 307.1545) Melting point:> 145 ° C (decomposition) Specific rotation: [α] D (24 ° C) +32.36 (c = 0.13, MeOH) UV absorption spectrum (nm) λ (ε): 214.5 (29700 ), 2
15.5 (sh), 252.0 (9700), 307.0 (4600) TLC (Rf): 0.28 (CHCl ₃ / CH ₃ OH, 3: 1, Merck Kieselgel 60 F254, Art, 5719); 0.47 (MeOH / H ₂ O, 3: 1, Merck HPTLC RP-18 F254S, Art, 13724) ¹ H NMR (CD ₃ OD): 6.02 (1H, s), 5.49 (1H, s), 4.04
(1H, m), 2.56 (1H, dd, J = 4.0, 16.9 Hz), 2.46 (1H,
dd, J = 10.6, 16.9 Hz), 1.58 (2H, br), 1.50 (1H,
m), 1.3 -1.4 (9H, m), 0.90 (3H, t, J = 6.6 Hz) ¹³ C NMR (CD ₃ OD): 178.8 (s), 163.6 (s), 161.8 (s),
141.8 (s), 113.5 (s), 106.5 (d), 104.0 (s), 98.0
(d), 68.4 (d), 37.5 (t), 36.2 (t), 33.8 (t), 31.5
(t), 31.2 (t), 27.6 (t), 24.5 (t), 15.2 (q).

Example 2 The first seed culture of Penicillium sp. N990-12 obtained in Example 1 was added to Medium 2 (100 ml) of Example 1.
Inoculate 5 ml in a 00 ml Erlenmeyer flask. The flask is cultured at 28 ° C. for 3 days on a rotary shaker having a circular rotation (220 rpm) of 7 cm in diameter. The resulting 100 ml culture contains the novel isochroman carboxylic acid derivative and has anti-gyrase activity. The substance can be isolated in the same manner as in Example 1.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ identification symbol FI (C12P 17/06 C12R 1:80) (58) Investigated field (Int.Cl. ⁷ , DB name) WPI (DIALOG) BIOSIS ( DIALOG) CAS ONLINE (STN)

Claims (1)

(57) [Claims] 1. A penicillium sp. Deposited as Microfabrication Kenji No. 3959 (FERMBP-3959) for producing isochroman carboxylic acid derivatives.
ium sp.) N990-12.