JPH0680659A - New isochromancarboxylic acid derivative - Google Patents

Abstract

PURPOSE:To provide the subject new compound exhibiting a broad spectrum antimicrobial activity owing to a gyrase inhibitory effect and an antitumor effect and useful as an antimicrobial agent, etc. CONSTITUTION:A compound of the formula [R<1> to R<4> are each H, a 1 to 6C alkyl or acyl], e.g. 3-n-heptyl-1,6,8-trihydroxyisochroman-7-carboxylic acid. In addition, the above-mentioned compound exhibits the following physical properties; Appearance: white powder; molecular formula: C17H24O6; HRFAB-mass spectrum: [m/z, (M+H)<+>-H2O]: 307.1544 (Calculated value: 307.1545); melting point: >=145 deg.C (decomposition); specific rotatory power: [alpha] D(24 deg.C): +32.36 (C=0.13, MeOH); ultraviolet absorption spectra (nm) lambda (epsilon): 214.5 (29700), 215.5 (sh), 252.0 (9700), 307.0 (4600). The compound of the formula can be obtained by culturing Penicillium Sp. N 990-12 (FERM-BP-3959) under an aerobic condition.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel isochromancarboxylic acid derivative, a bacterium producing the same, and a method for producing the same and its use. The present invention also relates to a novel isochromancarboxylic acid derivative chemically synthesized from the above isochromancarboxylic acid derivative, a method for producing the same, and a use thereof. In particular, the present invention is directed to Micro Engineering Research Article No. 3959 (FERM BP-39
59), a novel isochromancarboxylic acid derivative produced by culturing Penicillium sp. N990-12 deposited in a nutrient medium and a novel isochromancarboxylic acid synthesized from them by a chemical method. It relates to an acid derivative. The present invention relates to an antibacterial agent or an antitumor preparation containing the novel isochromancarboxylic acid derivative as an active ingredient.

[0002]

2. Description of the Related Art Recently, bacterial topoisomerase
II (gyrace) inhibitors are attracting attention as targets for antibacterial agents. This is based on the fact that the mechanism of action of quinolone synthetic antibacterial agents that are widely used at present is mainly due 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. As gyrase inhibitors produced by microorganisms, novobiocin, coumamycin A1 and the like are known, but these have a narrow antibacterial spectrum and have a problem that their application range as antibiotics does not reach that of quinolone synthetic antibacterial agents. . Therefore, the development of yet another type of gyrase inhibitor is desired.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a group of isochromancarboxylic acid derivatives having excellent antibacterial activity and antitumor activity in which resistant bacteria hardly appear. Furthermore, the present invention provides a mold of the genus Penicillium having the ability to produce an isochroman carboxylic acid derivative, in particular, Micro Works Kenji No.
It is also an object of the present invention to provide a method for recovering a novel isochromancarboxylic acid derivative from a culture of Penicillium sp. N990-12 deposited as 3959), and isolating and purifying it in a pure form. Another object of the present invention is to provide an antibiotic and / or antitumor agent containing these isochromancarboxylic acid derivatives as active ingredients.

[0004]

The present inventors searched a large number of microorganisms for the purpose of discovering a novel gyrase inhibitor, and as a result, Flagstaff (Arizona, USA) (Flagst
We found that mold N990-12 isolated from a soil sample collected in a forest community near aff) produced a novel isochroman carboxylic acid derivative with anti-gyrase activity. It has not been previously known that molds of the genus Penicillium produce an isochromancarboxylic acid derivative, and no isochromancarboxylic acid derivative having a gyrase-inhibiting action has been hitherto known.

According to the present invention, N990-12 cultures produce one or more of the isochroman carboxylic acid derivatives of the general formula below, which have gyrase inhibitory activity. Further, this novel isochroman carboxylic acid derivative may be isolated and used as it is, or can be introduced into another derivative represented by the following general formula by a further chemical method without losing the gyrase inhibiting activity.

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

[0007] [In the formula, R ¹ , R ² , R ³ and R ⁴ each independently represent hydrogen, an alkyl group having 1 to 6 carbon atoms, or an acyl group]. In addition, in the said general formula and this specification, an "alkyl group" means the radical of monovalent | monohydric linear hydrocarbon or branched hydrocarbon, and is methyl, ethyl, n-propyl. , Isopropyl, n-butyl, isobutyl, s-butyl, t-butyl and the like, but are not limited thereto.

The "alkoxy group" is used as a term representing -OR (R is an alkyl group) and includes methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, s-butoxy, t-butoxy and the like. However, the present invention is not limited to these.

The "acyl group" is used as a term representing -COR '(R' is an alkyl group or an aryl group),
It includes, but is not limited to, acetyl, propionyl, optionally substituted benzoyl and the like. Substituents include, but are not limited to, alkyl, alkoxy, halogen, mono and dialkylamino and the like.

The "aryl group" represents an aromatic group which may be substituted, for example, phenyl, naphthyl, pyridyl, quinolyl, thienyl, furyl, oxazolyl, tetrazolyl, thiazolyl, imidazolyl, pyrazolyl and the like, but is not limited thereto. Not something. Substituents on aryl include, but are not limited to, alkyl, alkoxy, phenoxy, halogen, cyano, nitro, monoalkylamino, dialkylamino and the like.

The bacterium (N990-12) that produces the isochroman carboxylic acid derivative of the present invention was discovered by Dr. Martha Christensen, the inventor of the application entitled “Invention title: Novel Penicillium fungi” on the same day as the present application. Were given to the people. The mycological properties of N990-12 are as follows.

N990-12 was inoculated on the agar plate medium for identification from potato-glucose agar slant culture and cultured at 25 ° C. for 1 to 5 weeks. Color by Color Standa by Robert Ridgway
It was judged by comparing with a color chip of rdsand Color Nomenclature (1912). The medium used for identifying the strain is as follows.

1. Cornmeal Agar (CM): Carmichae
l, JW, Mycologia 49: 820-830, 1957. 2. Czapek Sucrose Agar (CZ): Raper, KB and
Fennell, DI The Genus Aspergillus, p. 36, 196
Five. 3. Malt extract agar (MEA): ibid., P. 38. 4. Czapek / 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-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 YEA ： BBL
. 10. Yeast extract and soluble starch agar (YPSS): Emerso
n, R., Mycologia 50, 589-621, 1958.

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

CZ (25 ° C., 7 days) reaches 3.3 cm in diameter; grayish green (russian green, XLII), velvety, flat; backside is colorless; no soluble pigment is produced but deep orange yellow (11 days) ocharaceous orange, XV) or orange (apri
cot orange, XIV).

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

G25 N (25 ° C., 7 days) reaches 2.1 cm in diameter; light yellow (baryta yellow, IV), 11 days light green to green gray (gnaphalium green to pea green, X)
LVII); a circular depression is created at the periphery and in the center, 1
Radially wrinkles in 1 day; medium ridges, felt-like; backside is light green (cobalt 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.

CYA (5 ° C., 7 days) reaches a diameter of 0.8 cm; white, lightly developed, but at 11 days moderately elevated, flat, felt-like; white on the back side; no soluble pigment is produced.

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

In CM (25 ° C., 7 days), the diameter reaches 3.7 cm; thin, flat, velvety; colorless to sulphur yellow (V) on the reverse side; no soluble pigment is produced.

YPSS (25 ° C., 7 days) reaches 2.9 cm in diameter; pale green to green gray (celandinegreen to artemisi)
a green, XLVII) or deep bluish gra
y-green, XLII), flat, velvety; back side is colorless, buff yellow (IV), bright yellow pink to ochraceous buff to ochraceous-orange (XV); soluble pigment is not produced.

2. Morphological characteristics Morphological characteristics were observed after 7 and 11 days of culture on CYA and CZ agar. Conidia peduncles usually arise from aerial hyphae, producing smooth or sparsely roughened surfaces, 50-200 x 2-4 μm, either phialides or methotre; metore 2-4
12-20 x 2-4 μm rim, smooth or rough wall; 6 to 10 phialides per smooth surface, ampoule type with sharp tip, 7-9 x 2.0-2.5 μm; conidia spherical or spheroidal, often oval, diameter 2.5-3.
The surface was 0 μm or 3.0-3.5 x 2.5-3.0 μm, and the surface was smooth or finely rough as observed by a scanning electron microscope.

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

To summarize the mycological characteristics, N990-12 has a monolithic or multi-rotary penicillate, a smooth or sparsely conidial stalk, and a smooth or finely rough wall. Characterized by conidia in the shape of a sphere or a sphere. The conidia mass is generally green in color. On CYA, MEA and G25N medium, the color of the back side of the colonies was blue to turquoise. Therefore, this strain belongs to the genus Penicillium.

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

N990-12 is a form of methotre and phialide,
The conidia sparsely rough surface and the shape of conidia are P. raistrickii s
Similar to the eries species, but lacking sclerotia, colony underside color, shorter conidiophores, thinner phialides, and conidial surfaces smooth or fine rather than smooth. The difference is that it is rough.

N990-12 and Penicillium brevi-compactum
The strains of the series have common growth characteristics on most of the tested media, conidial head color, morphology of methotre and phialide, and sparsely conidial stalks. Three species of this series (P. brevi-compactum, P. stoloniferum
And P. paxilli) compared to P. brevi-compactum
P. stoloniferum is a lack of lami, colony backside color, shorter conidia peduncle, generally smaller penicilli and smaller conidia. Backside color, longer metre, and P.paxilli is the backside color of the colony, longer metre, and the majority of conidia are spherical or spheroidal rather than elliptical or spheroidal. Each is different.

From the above properties, N990-12 was named as Penicillium sp. Because it did not match any of the described species of the genus Penicillium. This is Penicillium SPN990-12
(Penicillium sp. N990-12) is displayed.
No. 9 (FERM BP-3959) was deposited under the Budapest Treaty on July 31, 1992.

[Culture of Bacteria] Cultivation of the strain of the present invention is preferably carried out as follows. For small-scale culture,
First, put an appropriate amount of nutrient medium in a flask and sterilize it by a known method, then inoculate this flask with N990-12 spores or vegetatively growing cells, and incubate on a rotary shaker at a constant room temperature of about 26 ° C. Incubate for 2 to 10 days. For large-scale culture, it is desirable to carry out in an appropriately sized tank equipped with a stirrer, aeration device and sterilization device. The nutrient medium is prepared in a tank, sterilized and inoculated with N990-12 spores or vegetative cells. The pH of the medium is 5 to 9, preferably
6 to 8 is preferable. 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, it is desirable to carry out agitation at 0 to 2,000 rpm, preferably 100 to 360 rpm, and aeration by blowing air at 0 to 500%, preferably 80 to 120% of the medium volume per minute.

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

[Synthetic Modification] In order to modify the compound isolated from the culture to obtain another compound of the above general formula, the following standard methods well known to those skilled in the art can be used. .

For example, 3-n-heptyl-1,6,8-trihydroxyisochroman-7-carboxylic acid isolated as in Example 1 below is added to a suitable solvent at room temperature to reflux temperature. Act as an acylating agent. A suitable base can be added to the reaction if desired. Preferred solvents include, for example, tetrahydrofuran, dioxane, methylene chloride, toluene and the like. Liquid bases such as pyridine, lutidine and triethylamine are also used as the solvent. Preferred bases include potassium hydroxide, sodium hydroxide, potassium carbonate, sodium carbonate and the like, in addition to the above liquid bases. Preferred acylating agents include acid chlorides (eg, acetyl chloride, benzoyl chloride, benzoyl bromide, p-chloride).
-Methoxybenzoyl, p-N, N-dimethylbenzoyl chloride), acid anhydrides (e.g. acetic anhydride, butyric anhydride, benzoic anhydride), active carboxylic acid derivatives (e.g. thiol esters, N-hydroxysuccinide, Active imidazole), mixed acid anhydride (for example, alkyl-CO ₂ SO ₂ CF ₃ , alkyl CO ₂ PO (OH) ₂ ) and the like.

The isochroman-7-carboxylic acid derivative is
It can also be obtained by reacting an alkylating agent in a suitable solvent at room temperature to reflux temperature. A suitable base can be added to the reaction if desired. Alkylating agents include alkyl halides (eg, methyl iodide, benzyl bromide, p-methoxybenzyl chloride), alkylsulfates (eg, dimethylsulfate, diethylsulfate), epoxides (eg, ethylene oxide), diazomethane and the like. Can be mentioned.

The pharmaceutically acceptable salt of the novel isochromancarboxylic acid derivative of the present invention is prepared by contacting the above compound with a stoichiometric amount of a metal hydroxide, alkoxide or amine in an aqueous solution or in a suitable organic solvent. Can be easily obtained. The salts are obtained by subsequent precipitation or by evaporation of the solvent.

All the isochroman carboxylic acid derivatives of the present invention have at least one asymmetric center,
Various optical isomers or configurations may exist. Thus, the compounds of the present invention can exist as (+) and (−) separate optically active forms, and as racemates or (±) mixtures. Further, in the case of a compound having two or more asymmetric centers, diastereomers due to their respective optical isomers may be present. The present invention includes within its scope all these types. For example, diastereomers can be separated by methods well known to those skilled in the art, such as fractional crystallization, and optically active forms can be obtained by well-known organic chemistry techniques for this purpose. be able to.

[Measurement of Activity] The gyrase inhibitory activity of the isochromancarboxylic acid derivative of the present invention is 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 eukaryotic cell-derived topoisomerase II inhibitory activity was measured according to the method instructed by the manufacturer (US Biochemical Corporation) using commercially available Drosophila-derived topoisomerase II. The inhibitory activity (IC ₅₀ value) of gyrase and topoisomerase II of the novel isochroman carboxylic acid derivative obtained in Example 1 described below was 3 μg / m 2, respectively.
1 and less than 1 μg / ml. These biological activities are
This suggests that the isochromancarboxylic acid derivative is useful as an antitumor agent as well as an antibiotic agent. This is based on the mechanism of action of some of the widely used antitumor agents at present by inhibition of topoisomerase II activity (Reference Znang, HD, D'Arpa,
P. and Liu, LF, Cancer Cells, 2, 23-26, 199
0).

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

[0038]

[Table 1] Minimum inhibitory concentration of test bacteria (μg / ml) Staphylococcus aureus 01A005 25 (Staphylococcus aureus) Staphylococcus aureus 01A063 50 (Staphylococcus aureus) # Staphylococcus epidermides 01B087 50 (Staphylococcus epidermides) 100 (Enterococcus faecalis) Streptococcus piogenes 02C054 50 (Streptococcus pyogenes) Escherichia coli Martocida 59A001 100 (Pasturella multocida) Serratia marcescens 63A017> 100 (Serratia marcescens) Enterobacter aerogenes 67A040> 100 (Enterobacter aerogenes) Enterobacter acrogenes 67B00 9> 100 (Enterobacter cloacae) Providencia sturarch 77A013> 100 (Providencia stuartii) Providencia rettgeri 77C025> 100 (Provedencia rettgeri) Morganella morgani 97A001> 100 (Morganella morganii) # Ciprofloxacin-resistant bacteria In order to prevent or treat diseases of humans and other animals caused by a wide range of pathogenic bacteria including the bacteria shown in 1 above, it can be administered orally, parenterally, topically or by other suitable routes. In general,
The most desirable dosage of these compounds for human administration is between about 300 mg and about 3000 mg per adult day, but can vary depending on the weight and condition of the patient and the particular route of administration. However, a dose of about 5 mg to about 50 mg per kg body weight daily is most desirable. When administered to animals, the dose 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 higher than the above range, it may cause harmful side effects if it is administered in small doses divided into multiple doses per day. It may not be.

When the compound of the present invention is used as an antitumor agent, the same administration route and dose as above can be applied.

The isochroman carboxylic acid derivative of the present invention 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 performed once or several times. It can be done in batches. More specifically, the novel therapeutic agent of the present invention can be administered in various types of dosage forms, for example, tablets, capsules, in combination with various pharmaceutically acceptable inert carriers, It can be in the form of a medicated drop, troche, hard candy, powder, spray, cream, ointment, suppository, jelly, gel, paste, lotion, ointment, aqueous suspension, injectable solution, elixir, syrup 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, it may be optionally sweetened and / or flavored. Generally, the therapeutically active compounds of this invention are administered in a concentration range of about 5% to 70% by weight in the form as described above.

For oral administration, various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium phosphate, glycine are added to starch, preferably corn, potato or tapioca starch, and alginic acid or It can be used with various disintegrants such as certain silicic acid double salts, and granulating binders such as polyvinylpyrrolidone, sucrose, gelatin, acacia. Lubricants such as magnesium stearate, sodium lauryl sulfate and talc are also often very effective in tablet formation. The same type of solid composition can be used by filling a gelatin capsule. Suitable substances in this connection include lactose or milk sugar as well as high molecular weight polyethylene glycols. When it is desired to prepare an aqueous suspension and / or elixir for oral administration, the active ingredient is used in combination with various sweetening agents or flavoring agents, coloring agents or dyes, and if necessary, an emulsifying agent and / or suspending agent. However, it can be used with diluents such as water, ethanol, propylene glycol, glycerin, and the like, and combinations thereof.

For parenteral administration, a solution prepared by dissolving the isochroman carboxylic acid derivative of the present invention in either sesame oil or peanut oil or in a propylene glycol aqueous solution can be used. The aqueous solution should be suitably buffered (preferably pH 8 or higher) as needed and the liquid diluent first rendered isotonic. Such aqueous solutions are suitable for intravenous injection, oily solutions for intra-articular, intramuscular and subcutaneous injection. Aseptic preparation of all of these solutions can be readily accomplished by standard pharmaceutical techniques well known to those skilled in the art. Furthermore, the compounds of the present invention can be administered topically, such as the skin. In this case, topical administration in the form of creams, jellies, pastes, ointments is desirable according to standard pharmaceutical practice.

[0043]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to matters specified in detail in these examples. The melting points used in the examples are Yanaco's micro melting point measuring device (uncorrected), the optical rotations are JASCO's DIP-370 digital polarimeters, and the ultraviolet absorption spectra are in methanol solution, JASCO's spectrophotometers ( Ubest-30), the infrared absorption spectrum was measured by Shimadzu infrared spectrophotometer (IR-470). Unless otherwise specified, ¹ H and ¹³ C nuclear magnetic resonance spectra (NMR) were measured by a solution of deuterated chloroform (CDCl ₃ ) by a JEOL nuclear magnetic resonance apparatus (JNM-GX270, 270 MHz). It is a thing. The peak position is expressed from tetramethylsilane to the downfield in units of 1,000,000 (ppm). The peak shape is expressed 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 Kratos mass spectrometer (model 1S) using a NaI matrix.

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

7.5 ml of the first seed culture is inoculated into 5 500 ml Erlenmeyer flasks containing 150 ml of the medium 1. This flask was cultivated on a rotary shaker having a diameter of 7 cm and rotating at 220 rpm at 26 ° C. for 4 days to obtain a second seed culture solution.

Medium 2 (1% glucose, 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) into 5 6 L glass fermenters containing 3 L each of 150 ml of the second seed culture. This fermenter is ventilated at 3 L / min, rotation speed 1700 rpm,
Stir at 26 ° C for 72 hours.

After completion of the culture, freeze-dry 15 L of the whole culture solution,
The dried broth obtained is extracted with 5 L of 70% aqueous acetone.
After removing acetone under reduced pressure, the mixture was extracted twice with 2 L of ethyl acetate, and the obtained ethyl acetate extract was dehydrated with anhydrous sodium sulfate and concentrated to dryness. An oily residue containing 3.0 g of the novel isochromancarboxylic acid derivative is obtained. This oily substance
Chromatograph on a column packed with 200 g of silica gel. Elute the column sequentially with 1000 ml of chloroform, chloroform-methanol (9: 1), and methanol. The methanol elution fractions are collected, concentrated, and subjected to 1.2 L Sephadex LH-20 column chromatography packed with methanol. The active fractions eluted with methanol are collected and concentrated to dryness to obtain 209 mg of crude powder. Of this, 75 mg is placed on 5 thin-layer chromatography plates (Kieselgel 60 F-254, 20 cm X 20 cm, 0.25 mm) and developed with chloroform-methanol (3: 1). The band showing UV absorption is collected to obtain 62 mg of the novel isochromancarboxylic acid derivative as a white powder. This product is 3
It was -n-heptyl-1,6,8-trihydroxyisochroman-7-carboxylic acid.

Properties: 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 5
Inoculate 5 ml into a 00 ml Erlenmeyer flask. This flask is cultivated on a rotary shaker having a diameter of 7 cm and rotating at 220 rpm at 26 ° C. for 3 days. The obtained 100 ml culture broth 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.

Example 3 3-n-heptyl-1,6,8-trimethoxyisochloro
Preparation of methyl man-7-carboxylate 3-n-heptyl-1,6,8-trihydroxyisochroman-7-carboxylic acid (4.9 mg) in methanol solution (2 ml) with stirring at room temperature under an excess amount A solution of diazomethane in diethyl ether was added, and the mixture was stirred overnight. After distilling off the diazomethane and the solvent, reverse phase column chromatography was performed with methanol-water (25: 1) (Kusano, CIG column C-
18 × 22 × 300 mm, 20 μm) to obtain 2.4 mg of methyl 3-n-heptyl-1,6,8-trimethoxyisochroman-7-carboxylate as a white amorphous substance.

HRFAB mass spectrum [m / z, (M) ⁺ ]: 38
0.2197 (calculated 380.2197); EI mass spectrum: 380 (m / z M ⁺ , 3.3% rel.%), 349 (7
1.9), 317 (23.3), 249 (100.0); ¹ H NMR: 6.39 (1H, s), 5.54 (1H, s), 4.11 (1H, m), 3.
89 (3H, s), 3.83 (3H, s), 3.77 (3H, s), 3.50 (3H, s),
2.62 (1H, br, d), 2.59 (1H, d, J = 4.3Hz), 1.25-1.65 (1
2H, m), 0.87 (3H, t, J = 6.5Hz); ¹³ C NMR: 166.7 (s), 157.1 (s), 156.1 (s), 138.7 (s),
121.0 (s), 116.1 (s), 106.3 (d), 95.4 (d), 66.0 (d), 6
2.8 (q), 56.1 (q), 55.0 (q), 52.5 (q), 35.6 (t), 34,3
(t), 31.8 (t), 29.5 (t), 29.3 (t), 25.6 (t), 22.7 (t),
14.1 (q).

[0052]

INDUSTRIAL APPLICABILITY According to the present invention, a novel antibacterial agent having a broad spectrum antibacterial activity and antitumor activity based on the gyrase inhibitory action is provided.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number FI technical display location C12R 1:80) 7804-4B (72) Inventor Nakao Nakajima 3 Aito, Meito-ku, Nagoya, Aichi -153-1-501

Claims (6)

[Claims] 1. The following general formula: [Wherein R ¹ , R ² , R ³ and R ⁴ each independently represent hydrogen, an alkyl group having 1 to 6 carbon atoms, or an acyl group] and a pharmaceutically acceptable compound thereof. Ru salt. 2. The compound according to claim ¹ , wherein R ¹ , R ² , R ³ and R ⁴ are hydrogen. 3. The method according to claim 2, wherein a microorganism of the genus Penicillium having an ability to produce an isochroman carboxylic acid derivative is cultured, and the isochroman carboxylic acid derivative produced in the culture solution is recovered. Process for producing isochromancarboxylic acid derivative. 4. A micromachine research article No. 3959 (FERM BP
No. 3959), Penicillium sp. N990-12 is cultivated, and the novel isochromancarboxylic acid derivative according to claim 2 is recovered from the culture solution. Method for producing derivative. 5. An antibacterial agent comprising a therapeutically effective amount of the compound of claim 1 or 2 and a pharmaceutically acceptable carrier. 6. An antitumor preparation comprising a therapeutically effective amount of the compound according to claim 1 or 2 and a pharmaceutically acceptable carrier.