JP2000072765A - A-1211 substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a novel compound having an excellent Cds25B protein tyrosine phosphatase inhibiting activity, and also antifungal and antitumor activities simultaneously by culturing Actinomycetes belonging to the genus Nocardia. SOLUTION: This compound is represented by the formula (R is H or methyl), having following physicochemical properties, e.g. shape: reddish brown powder; melting point: 115 to 120 deg.C; molecular formula: C13H10O4; molecular weight: 230; specific rotation: [α]D26=-85.4 deg. (in chloroform, concentration c: 0.25 wt.%); solubility: soluble in methanol, acetone, ethyl acetate, chloroform and dimethyl sulfoxide, sparingly soluble in water, and insoluble in ether; Rf: 0.42 (chloroform/ ethyl acetate ratio: 10/1). It is isolated from the system in which a bacterial strain (FERM BP-6372), belonging to the genus Norcadia and capable of producing the compound, is cultured in a medium at around neutral pH level and 25 to 40 deg.C for around 3 to 7 days.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to Cdc25B protein tyrosine phosphatase.
The present invention relates to a novel naphthofuranone compound and a salt thereof, which have an inhibitory activity on tase) and are useful as pharmaceuticals.

[0002]

2. Description of the Related Art Regarding the signal transmission system of cell proliferation,
Although research on the role of protein kinases has been progressing conventionally, it has become clear in recent years that protein phosphatase also plays an important role. Cyclin dependent kinase [Cyclin dependent kinase]
kinase (Cdk)] is known as a key enzyme of cell cycle progression, and binds to Cdk such as p16, p21, etc., which have cyclin D and other oncogenic effects, overexpression in cancer cells, and tumor suppressor gene effects Findings such as decreased expression of intracellular inhibitory protein (CDKI) in cancer cells suggest that Cdk activity is involved in cancer cell growth. Many cancer cells have abnormal cell cycle control mechanisms, and Cdk is the most important factor in cell cycle control.
Regulators of k are thought to be a new type of antitumor agent [Meijer, L .: Trends in cell Biology,
6, 393-397, 1996]. Furthermore, Cdk is activated by binding to Cyclin to phosphorylate other molecules, while
k itself undergoes phosphorylation and dephosphorylation, and its activity is regulated. Phosphorylated Cdk becomes an inactive enzyme, which is further dephosphorylated and Cdc25 is reactivated.
Phosphatase. Therefore, inhibitors of phosphorylase and phosphatase are also expected to be a new type of antitumor agent whose mechanism of action is cell cycle inhibition (Nagata: Cancer and Chemotherapy, 24, 1541-1546). , 1997).

[0003] At present, in diseases involving protein kinases, a specific inhibitor for this enzyme is required, but at present, sufficient substances have not been obtained. Especially in human cancer tissues such as breast cancer, head and neck cancer, gastric cancer,
Inhibitors that have an oncogenic effect due to overexpression of Cdc25A or B phosphatase and specifically inhibit Cdc25B protein tyrosine phosphatase, which removes the tyrosine phosphate group using phosphorylated Cdk as a substrate, Not obtained.

[0004]

DISCLOSURE OF THE INVENTION The present invention has been developed in view of the above circumstances, and has been developed for Cdc25B protein tyrosine phosphatas.
It is intended to provide a substance having excellent inhibitory activity against e). The present invention also relates to a method for producing the substance using a microorganism, and an object thereof is to provide a microorganism useful for such production.

[0005]

The present inventors have SUMMARY OF THE INVENTION separates a number of microorganisms newly from soil, where they were to continue the search for new useful substance as a medicine for the metabolites produced, Nocardia (Nocardia) It was found that a novel compound having excellent Cdc25B protein tyrosine phosphatase inhibitory activity and having both antifungal activity and antitumor activity was produced in a culture of the Streptomyces TP-A0248 strain belonging to the genus Genus. The present invention has been accomplished by isolating the active substance and determining its physicochemical properties and structure.

That is, the present invention provides the following general formula (1)

[0007]

Embedded image

(Wherein, R represents a hydrogen atom or a methyl group). Naphthofuranone
It relates to a compound or a salt thereof.

Conventionally, as a microorganism metabolite having a structure similar to the naphthofuranone compound of the present invention, an MS-444 substance [The Journal of Antibiotics (The Journal of Antibiotic)
journal of antibiotics), 48, 94 8−953, 1995] is a myosin light chain kinase.
inase) reported as an inhibitor.

Also, as a compound having a structure similar to the compound of the present invention, Streptocpus duny
apus dunnii ) Dunion isolated from leaves
e) and its analogs, and β-Lapachone isolated from Lapacho trees. These compounds can be used as anticancer agents [International Patent Application Publication No. WO 97/31936, Cancer Research
arch), 57, 620-627, 1997] and Dunnion.
(International Patent Application Publication No. WO97 / 16970) has been reported to be used as an antifungal agent.

[0011] European Patent Publication No. EP0413224 includes:
Naphthoquinone derivatives having the same basic skeleton as the compounds of the present invention have been described as antifungal agents. However,
The publication does not specifically disclose the compound of the present invention, and thus the naphthofuranone compound of the present invention is a novel compound which has hitherto been unknown.

[0012] The present invention is also characterized in that a strain belonging to the genus Nocardia and having the ability to produce the above naphthofuranone compound or a salt thereof is cultured in a medium, and the compound and the like are separated and recovered from the culture. The present invention relates to a method for producing a naphthofuranone compound or a salt thereof.

The present invention also relates to a medicament containing the naphthofuranone compound or a salt thereof as an active ingredient, more specifically, a therapeutic agent for fungal infection or a therapeutic agent for tumor.

Furthermore, the present invention relates to a pharmaceutical composition containing the above naphthofuranone compound or a salt thereof and a pharmaceutically acceptable carrier.

Furthermore, the present invention relates to a strain having an ability to produce the above naphthofuranone compound or a salt thereof.

In the present invention, the naphthofuranone compound or a salt thereof is hereinafter collectively referred to as "A-1211 substance" for convenience, and the bacterium producing the substance is referred to as "A-1211 substance producing bacterium".

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The A-1211 substance of the present invention has the following general formula (1)

[0018]

Embedded image

(Wherein, R represents a hydrogen atom or a methyl group). Here, when R in the formula is a hydrogen atom, embodiments of a salt are also included. The salt is not particularly limited, but is preferably a base salt reacted with a pharmaceutically acceptable basic compound. Examples of the basic compound include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; and alkali metal carbonates such as sodium carbonate and potassium carbonate.

The A-1211 substance has optical isomers, and the present invention includes all such isomers and mixtures thereof. Further, the A-1211 substance may be in an anhydrous state or hydrated at an appropriate ratio. It may be crystalline or amorphous.

In the general formula (1), the physicochemical properties of the A-1211 substance of the present invention wherein R is a hydrogen atom (hereinafter referred to as A-1211-a substance) are shown below.

1) Shape: reddish brown powder 2) Melting point: 115-120 ° C. 3) Molecular formula: C ₁₃ H ₁₀ O ₄ 4) Molecular weight: 230 (C ₁₃ H by high resolution electron impact mass spectrometry (HREI-MS) method) ₁₀ O ₄
Experimental value 230.570, calculated value 230.05 as [M ⁺ ]
79 is shown. 5) Specific rotation: measured at a concentration of C = 0.25% in chloroform. [α] _D ²⁶ = -85.4 ° 6) Ultraviolet absorption spectrum: measured in methanol, methanol containing 0.001N-HCl, and methanol containing 0.001N-NaOH at a concentration of 10 μg / ml, respectively: λ _max ^MeOH (nm) (ε): 203 (16,400), 237 (15,100), 259
(18,000), 291 (sh, 5,700), 416 (br, 4,900); λ _max ^{MeOH / HCl} (nm) (ε): 202 (8,500) 237 (sh), 259 (1
8,300), 293 (sh, 5,800), 415 (br, 5,000); λ _max ^{MeOH / NaOH} (nm) (ε): 205 (15,500), 238 (sh, 16,
300), 259 (14,400), 296 (sh, 5,100), 474 (br, 6,100) 7) Infrared absorption spectrum: KBr tablet method IRνmax (KBr) cm ^-1 : 1640, 1620, 1580, 1450, 1410,
1320, 1270, 1030 (FIG. 1) 8) Nuclear magnetic resonance spectrum: 400 MHz ¹ H-NMR spectrum measured in deuterated chloroform at 30 ° C. (FIG. 2) and 1
Table 1 shows the chemical shifts of the 00 MHz ¹³ C-NMR spectrum.

[0023]

[Table 1]

9) Solubility: Soluble in methanol, acetone, ethyl acetate, chloroform, dimethyl sulfoxide,
Poorly soluble in water, insoluble in ether 10) Rf value on thin layer chromatography: Kieselgel plate 60F254 (Merck, Art. 5175)
When developing with chloroform as the developing solvent, the Rf value is 0.
25, Rf value 0.42 when developed with chloroform / ethyl acetate (10: 1).

11) High performance liquid chromatography (HPL)
Retention time (Rt) in C): mobile phase acetonitrile / 0.05% trifluoroacetic acid (60:40) using a reverse-phase ODS silica gel column (Inertsil ODS-2, 250 × 4.6 mm, Id, GL Science) ), At a flow rate of 0.8 ml / min and detection at 230 nm, a peak is detected at Rt. 5.9 min.

Next, in the general formula (1), the A-1211 substance of the present invention wherein R is a methyl group (hereinafter referred to as A-1211-b)
It is called substance. ) Are shown below.

1) Shape: reddish brown powder 2) Melting point: 79-81 ° C. 3) Molecular formula: C ₁₄ H ₁₂ O ₄ 4) Molecular weight: 244 (electron impact mass spectrum (EI-MS) method) 5) Nuclear magnetic resonance Spectra: 400 MHz ¹ H-NMR spectrum measured in deuterated chloroform at 30 ° C. (FIG. 3) and 1
Table 2 shows the chemical shifts of the 00 MHz ¹³ C-NMR spectrum.

[0028]

[Table 2]

6) High performance liquid chromatography (HPLC)
Retention time (Rt): using a reverse-phase ODS silica gel column (Inertsil ODS-2, 250 × 4.6 mm, Id, GL Sciences), mobile phase acetonitrile / 0.05% trifluoroacetic acid (60:40), When analyzed at a flow rate of 0.8 ml / min and detection at 230 nm, a peak is detected at Rt. 6.3 minutes.

The A-1211 substance of the present invention can be produced, for example, by culturing a strain having the ability to produce the substance (A-1211 substance producing bacterium) under appropriate conditions as described below. . The present invention relates to this A-121.
It also includes one substance producing bacterium.

Examples of the bacteria producing A-1211 substance include strains belonging to the genus Nocardia . The present invention includes an A-1211 substance that can be obtained by culturing the strain belonging to the genus Nocardia in a medium and separating and recovering the resulting culture.

As an example of the strain belonging to the genus Nocardia, Nocardia sp. TP-A0248 ( Nocardia
sp. TP-A0248) strain. This strain is a strain belonging to the genus Nocardia newly isolated from the soil of Kosugi Town, Toyama Prefecture by the present inventors, and is located at 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan.
No. 3 is displayed on May 25, 1998 at the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry of the Ministry of International Trade and Industry.
dia sp. TP-A0248 ", (accession number)" F
ERM BP-6372 ".

TP-A0248 ( Nocardia sp. TP-A024)
8) About the strain, the International Streptomyces Project
t; Sealing and Gottlieb methods recommended by the ISP [International Journal of Sistmaticatic Bacteriology (Shirling, EB & D.
Gottlieb: International Journal of Systematic Bact
eriology), 16 (3), 313-340, 1966], and as a medium, Waxman [Waksman, SA (Ed.): The Actinomycetes), 2,
328-334, 1961, The Williams & Wilkins Co., published]
And Arai [Arai, T. (Ed.): Culture media for Ac
tinomycetes), 1975, The Society for Actinomycetes
TP examined using the one described by [Issue]
-The mycological properties of the A0248 strain are as follows.

(A) Morphology 1) Branching method of spore formation; simple branching 2) Morphology of spore formation; cylindrical spores are linearly linked 3) Number of spores; 4 to 6 4) Surface structure of spores Fine wrinkles are observed 5) size of spores; 0.4 to 0.6 × 1.0 to 1.3 μm 6) presence of flagellar spores; not observed 7) location of spore stalk formation; aerial hyphae 8) bacteria Nucleating ability: not recognized 9) Disruption of base mycelium; not recognized.

(B) Growth on various media: Table 3 shows the growth on seven media.

[0036]

[Table 3]

The color tones relating to the culture properties described in the table are shown based on a new color name dictionary (published by Nippon Color Research Co., Ltd., 1987), and color code numbers are added in parentheses.

This strain grows well on various media, and the underlying mycelium exhibits a bright orange to reddish brown color, and the mature powdery aerial mycelium exhibits a white to pale white color. No characteristic soluble dye formation is observed.

(C) Physiological properties 1) Growth temperature range; (yeast extract / starch agar medium); grows well in the range of 17-45 ° C. 2) Liquefaction of gelatin (glucose / peptone / gelatin medium, 27 ° C.) Negative) 3) Liquefaction of gelatin (simple gelatin medium, 20 ° C.);
Negative 4) Coagulation of milk (37 ° C); Negative 5) Peptonization of milk (37 ° C); Negative 6) Production of melanin-like pigment; Tyrosine agar (ISP-
7) Positive on medium, negative on tryptone / yeast extract / broth (ISP-1) medium 7) Hydrolysis of starch (starch / inorganic salt agar, ISP-
Negative 8) Reduction of nitrate (1% potassium nitrate broth, I
9) Cellulose degradation; Negative 10) Salt tolerance; (Yeast extract / starch agar medium); 4
Grow to the presence of% sodium chloride.

(D) Utilization of carbon source (Preded ham / Gottlieb agar, ISP-9 medium): D-glucose, D-glucose
It grows well using fructose and glycerol, but raffinose, D-xylose, D-mannitol,
L-arabinose, sucrose, inositol, and L-rhamnose are not available.

(E) Chemical taxonomic properties of bacterial cell components: Identification of actinomycetes [edited by Japanese Society of Actinomycetes, 62-70, 198]
5], and Rechevallier et al. [The Actinomycetes (Le
chevalier, HA & MP Lechevalier; The Actitino
mycetes (H. Prauser, ed.), 393-405, 1970, Jena, G
ustav Fischer Verlag] and Stanek et al. [Upride microbiology (Stanech, Jl & GD Ro
berts: Applied Microbiology), 28, 226-231, 1974]
As a result of analysis of the acid hydrolyzate in all the cells by the thin-layer chromatography method described in the above, the presence of meso- type diaminopimelic acid, galactose and arabinose was confirmed. From this, the cell wall of this strain is IV
-Classified as type A.

Lechevalier et al. [Biochemical systematic ecology (Lechevalier, MP, C.D.)
ebievre & HA Lechevalier: Biochemical systemati
c Ecology), 5, 249-260, 1977], phosphatidylinositol, phosphatidylethanolamine and diphosphatidylglycerol were detected from phospholipid analysis performed according to the method described by J. From this, it was classified into P-II type.

Collins et al. [Journal of Applied Microbiology (Collins, MD; HNS)
hah, & DE Minnikin: Journal of Appilied Microbi
ology), 48, 277-282, the main menaquinone of this strain was performed according to the method described by _{1980] MK-8 (H 4} ) 8
6.6% and MK-8 (H ₂ ) were 13.1%.

Suzuki et al. [International Journal of Sistmatic Bacteriology (Suzuk
i, K. & K. Komagata: International Journal of Syst
ematic Bacteriology), 33, 188-200, 1983].
noate 23%, 14-methylhexadecanoate 16%, heptade
canoate 15%, tuberculostearate 14%, pentadecan
oate 10% and 13-methyltetradecanoate 10%.

Further, Uchida et al. [Journal of General Applied Microbiology (Uchid,
K. & K. Aida: Journal of General Applied Microbiol
ogy), 44, 193-203, 1994], and a glycolate test performed according to the method described by Yano et al. [by Ikuya Yano and Ikuko Tomiyasu] Mycolic acid; 104-130, 1985], all tested positive for mycolic acid.

Taxonomic properties such as morphological observation, physiological properties, growth properties on various media, and chemical taxonomic properties of bacterial cell components of the TP-A0248 strain described above, especially, To form aerial hyphae having a chain of spores, that the cell wall composition is IV-A type, that the phospholipid is P-II type, and that the main menaquinone is MK-8 (H ₄ )
, The mycolic acid test and the glycolate test are both positive, and the fact that they do not form flagellar spores or sporangia revealed that the strain belongs to the genus Nocardia. Therefore, this strain is nocardia
Nocardia sp. TP-A0248.

The A-1211 substance of the present invention is obtained by culturing various A-1211 substance producing bacteria belonging to the genus Nocardia, such as the above-mentioned TP-A0248 strain or a mutant thereof, in an appropriate medium, and then obtaining a culture solution From the crude extract containing the substance of the present invention, and further isolate and purify the A-1211 substance from the crude extract.

The cultivation of the above-mentioned microorganisms is carried out in principle according to the cultivation of general microorganisms, but is preferably carried out under aerobic conditions such as a shaking culture method by liquid culture and an aeration-agitation culture method.

The medium used for the cultivation includes: A-1
Any medium containing a nutrient source that can be used by the 211 substance-producing bacteria may be used, and any of various synthetic media and natural media can be used. Glucose,
Sucrose, fructose, glycerin, dextrin, starch, molasses, corn steep liquor, organic acids, etc., alone or in combination of two or more; as a nitrogen source, pharma media, peptone, meat extract, yeast extract, soybean powder , Casein, amino acids, organic nitrogen sources such as urea, and inorganic nitrogen sources such as sodium nitrate and ammonium sulfate are used alone or in combination of two or more.
Further, a sodium salt, a potassium salt, a magnesium salt, a phosphate, and other heavy metal salts are added to the medium as needed.

When effervescence during the culturing is remarkable, for example, vegetable oils such as soybean oil and linseed oil, higher alcohols such as octadecanol, tetradecanol and heptadecanol;
An antifoaming agent such as various silicon compounds can be appropriately added to the medium.

The pH of the medium is around neutral, preferably pH 6
It is desirable to set to ８8. The cultivation temperature is preferably maintained at a temperature at which the A-1211 substance-producing bacterium grows favorably, usually at 25 to 40 ° C, particularly around 27 to 35 ° C. The culturing time is preferably about 3 to 7 days in both the liquid shaking culture and the aeration stirring culture.

The various culture conditions described above can be appropriately changed according to the type and characteristics of the microorganism used, external conditions, and the like, and the optimum conditions can be appropriately selected and adjusted from the above range according to each.

The crude extract containing the A-1211 substance can be separated from the culture broth according to a general method for collecting a fermentation product, for example, a conventional method such as solvent extraction, chromatography, crystallization and the like. Can be used alone or in combination of two or more in any order. More specifically, the following method can be used.

That is, A- produced by the above culture
Since the substance 1211 is mainly present in the culture filtrate and cells, the culture filtrate and the cell solids are separated by filtration, centrifugation, etc., according to a conventional method to obtain the obtained A-1211.
The solid substance containing the substance is eluted with a substance A-1211 using a solvent such as methanol or acetone. Then, the solvent is distilled off under reduced pressure to obtain a crude concentrated liquid containing the substance A-1211. An organic solvent that does not mix with water, such as ethyl acetate, chloroform, or butanol, is added to the crude concentrated solution to transfer the substance A-1211 into the organic solvent layer, and Glauber's salt is added to the obtained solvent layer, followed by dehydration. Is distilled off under reduced pressure to obtain a crude extract containing the substance A-1211. Furthermore, a crude extract can be obtained by performing the same operation as described above in which the culture filtrate is dissolved in the organic solvent layer. Further, if necessary, the pH can be adjusted with sodium hydroxide or hydrochloric acid, or by adding industrial salt, extraction efficiency can be increased, and a method of preventing emulsion formation can be taken.

Further, in order to isolate and purify the substance A-1211 from the crude extract, usual means for isolating and purifying a fat-soluble low-molecular substance, for example, activated carbon, silica gel, alumina,
Various kinds of adsorption chromatography using an adsorbent such as a macroporous nonionic adsorption resin or reverse phase chromatography using ODS-bonded silica gel or the like can be used. Among them, silica gel chromatography using a mixed solvent system such as chloroform, chloroform / ethyl acetate, chloroform / methanol, chloroform / acetone, benzene / acetone as an elution solvent, and acetonitrile or methanol / 0.05% trifluoroacetic acid or 10%
Reverse phase chromatography using a mixed solvent system such as mM monopotassium phosphate for elution is particularly preferred. When further purification is required, the above-mentioned chromatography is repeatedly carried out, or appropriately combined with column chromatography using Sephadex LH-20 (manufactured by Pharmacia) using chloroform, methanol or the like as an elution solvent. Thereby, a high-purity A-1211 substance can be obtained.

The confirmation of the substance A-1211 during the purification step was carried out according to the antifungal activity against Candida albicans or Cdc2.
A method of measuring a physiological activity or a pharmacological activity such as an inhibitory activity against 5B protein tyrosine phosphatase and a detection method based on the physical properties of the substance using thin layer chromatography or high performance liquid chromatography or the like are used in combination. Good.

The specific methods for measuring the antifungal activity against Candida albicans and the inhibitory activity against Cdc25B protein tyrosine phosphatase are described in Examples and Test Examples. As shown in the test examples, the substance A-1211 of the present invention has inhibitory activity, antifungal activity and antitumor activity on Cdc25B protein tyrosine phosphatase. Therefore, the substance of the present invention is useful as an inhibitor of the above-mentioned enzyme and also as an active ingredient of a prophylactic / therapeutic agent for a disease associated with the enzyme and a prophylactic / therapeutic agent for fungal infections and tumors.

From this viewpoint, the present invention also provides a pharmaceutical composition containing the A-1211 substance produced and purified as described above as an active ingredient.

The A-1211 substance of the present invention is particularly useful for Aspergillus ( Aspergillus ) as shown in the test examples described later.
s) the genus Trichophyton (Trichophyton) filamentous fungi belonging to the like; Candida (Candida) spp, Cree Kluyveromyces (K
a bacterium belonging to the genus luyveromyces ; Cryptococcus ( Cryp)
It has antibacterial activity against fungi such as bacteria belonging to the genus tococcus . Therefore, the therapeutic agent for fungal infection of the present invention can be widely applied to diseases caused by such fungi. For example, applicable diseases include respiratory aspergillosis, systemic aspergillosis, aspergillosis including aspergillosis of skin and nails; trichophyton tinea, oral candidiasis, broncho-pulmonary candidiasis, vaginitis, urinary tract Candidiasis, including infections, meningitis, sepsis, etc .;
Examples include cryptococcosis including meningitis, pulmonary mycosis, skin cryptococcosis and the like.

Further, the substance A-1211 of the present invention was tested on U937 (human histiocytic lymphoma) as shown in the test examples described later.
P388 (mouse leukemia cell), HeLa (human cervical squamous cell carcinoma cell), KB (human oral epidermal squamous cell carcinoma cell), SBC-5
(Human-derived non-small cell lung cancer cells) also exhibit antitumor activity against cancer cells. Therefore, the antitumor agent of the present invention is mainly applied to epithelial malignant tumors such as lung cancer, breast cancer, stomach cancer, uterine cancer, and colon cancer; non-epithelial malignant tumors such as sarcoma; malignant lymphoma; can do.

As the dosage form when the substance A-1211 is used as a pharmaceutical composition, various pharmaceutical dosage forms can be widely used depending on the purpose. Specific examples of the dosage form include tablets, capsules, powders, and granules. Preparations, fine granules, solutions, pills, emulsions, suspensions, etc .; oral preparations such as injections, suppositories, ointments, plasters, patches, aerosols, eye drops and the like. You. Each of these administration forms can be produced by a commonly used production method known to those skilled in the art.

The oral solid preparation is the active ingredient A- of the present invention.
Excipients, binders, disintegrants as needed,
It can be manufactured as tablets, capsules, powders, granules, fine granules and the like according to a conventional method by adding a lubricant, a coloring agent, a flavoring agent, a flavoring agent and the like. As an excipient, for example, lactose,
Sucrose, starch, talc, magnesium stearate, crystalline cellulose, methylcellulose, carboxymethylcellulose, glycerin, sodium alginate, gum arabic, etc., as binders polyvinyl alcohol, polyvinyl ether, ethyl cellulose, gum arabic,
Shellac and sucrose, etc., as disintegrants are dried starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate and lactose, etc., as lubricants magnesium stearate and talc etc. As a flavoring agent, sucrose, orange peel, citric acid, tartaric acid and the like can be used. In addition, commonly known coloring agents and odor correctors can be used. The tablets may be coated by a known method.

The oral liquid preparation is the active ingredient A- of the present invention.
A flavoring agent, a buffering agent, a stabilizing agent, a flavoring agent, and the like can be added to the 1211 substance to produce an oral solution, a syrup, an elixir and the like according to a conventional method. In this case, those mentioned above may be used as the flavoring agent, and sodium citrate or the like may be used as a buffer, and tragacanth, gum arabic, gelatin or the like may be used as a stabilizer. An injection is prepared by adding a pH adjuster, a buffer, a stabilizer, a tonicity agent, a local anesthetic and the like to the active ingredient A-1211 substance of the present invention, and preparing it according to a conventional method. Injections for internal, subcutaneous, intradermal and intraperitoneal injections can be produced. Sodium citrate, sodium acetate, sodium phosphate and the like can be used as pH adjusters and buffers, and sodium pyrosulfite, ethylenediaminetetraacetic acid, thioglycolic acid, thiolactic acid and the like can be used as stabilizers. Sodium chloride and glucose can be used as tonicity agents, and procaine hydrochloride and lidocaine hydrochloride can be used as local anesthetics.

A suppository can be produced by adding a base and, if necessary, a surfactant to the active ingredient A-1211 substance of the present invention, and then preparing the suppository according to a conventional method. As the base, for example, oily bases such as macrogol, lanolin, cocoa oil, fatty acid triglyceride, and Witepsol (manufactured by Dynamite Nobels) can be used.

The ointment is the active ingredient A-1211 of the present invention.
A base, a stabilizer, a wetting agent, a preservative, and the like, which are usually used for the substance, are blended as necessary, and mixed by a conventional method to form a formulation. As a base, liquid paraffin, white petrolatum, beeswax, octyldodecyl alcohol, paraffin, etc., as a preservative, methyl paraoxybenzoate,
Ethyl paraoxybenzoate and propyl paraoxybenzoate can be used.

A patch can be produced by applying an ointment, cream, gel, paste or the like containing the active ingredient of the present invention to a usual support in a conventional manner. As the support, a woven or nonwoven fabric made of cotton, cloth, or chemical fiber, a film of soft vinyl chloride, polyethylene, polyurethane, or the like, or a foam sheet can be used.

The amount of the active ingredient A-1211 of the present invention to be incorporated in each of the above-mentioned dosage unit forms is not constant depending on the symptoms, age and weight of the patient to which this substance is to be applied, or on the dosage form thereof. However, generally, about 1 to 1000 mg for an oral preparation and about 0.1 to 5 mg for an injection per dosage unit form.
Desirably, the dose should be about 00 mg and about 5 to 1000 mg for suppositories. The daily dose of the drug having the above dosage form is appropriately selected depending on the condition, weight, age, sex, and other conditions of the patient. 1000 mg / kg, preferably about 1-100
mg / kg, which can be administered once a day or divided into 2 to 4 times.

[0068]

EXAMPLES The present invention will be described more specifically with reference to examples and test examples, but the present invention is not limited to these examples.

Embodiment 1 Production of A-1221-a substance (a) Cultivation step Soluble starch 1.0%, glucose 0.5%, Enzet case (NZ-case) 0.3%, yeast extract 0.2%, tryptone 0.5%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate・ Heptahydrate 0.05%, precipitated calcium carbonate
500 ml of a medium (pH 7.0) consisting of 0.3%
Into a K-shaped flask, sterilized (121 ° C., 20 minutes), and then treated with Nocardia SP. TP-A0248 strain (Accession No. FERM BP-6372) was inoculated with a monobacterial ear,
The cells were shake-cultured at 30 ° C. for 4 days to obtain inoculum (200 in
rotation). Next, glucose 0.5%, glycerol 2.0
%, Soluble starch 2.0%, Pharmamedia 1.
5%, yeast extract 0.2%, Diaion HP-20
(Diaion HP-20, manufactured by Mitsubishi Kasei) 1.0% of a medium (pH 7.0) was dispensed into a 500-ml K-type flask, dispensed 100 ml at a time, and sterilized (121 ° C, 20 minutes). Add the above inoculum at a ratio of 5% (v / v) and add 30%
C. for 6 days at 200.degree. C. (200 revolutions per minute).

(B) Separation Step Immediately after the completion of the culture, the culture was centrifuged (8000 rpm, 15 minutes).
After the separation, the cells were separated into the cells and the filtrate. The obtained culture filtrate (4.5 liter, pH 6.5) is diluted with diluted hydrochloric acid to pH.
The mixture was adjusted to 2-3, and extracted with stirring twice with ethyl acetate (2.2 liter). The cell portion was extracted with stirring three times with methanol (2.5 liters). After filtration, the methanol extract was concentrated under reduced pressure to remove methanol, and the mixture was stirred and extracted twice with ethyl acetate (0.5 liter). The two ethyl acetate extracts were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a brown oily crude extract (4.83 g).

(C) Isolation and Purification Step The above crude extract was dissolved in chloroform (5 ml), and silica gel (manufactured by Merck, Kieselgel, 270 × 45 mm, i.p.
d.) The mixture was adsorbed on a column, and fractionated in 40 ml portions using chloroform as an elution solvent. Thin layer chromatography of the eluted fractions and detection by high performance liquid chromatography, Can
By examining the antifungal activity against dida albicans, an active fraction containing the A-1211-a substance was confirmed.
The active fractions were collected, evaporated to dryness after evaporation of the solvent to give a brown oil (160 mg).

The measurement of the antifungal activity against Candida albicans for detecting the active fraction containing the substance A-1211 was carried out by a paper disk method as described below.

Specifically, a medium (yeast nitrogen base, manufactured by Difco) consisting of 0.65%, glucose 0.5%, dipotassium hydrogen phosphate 0.36%, disodium hydrogen phosphate 0.57% and agar 1.3% (pH 7. In step 0), a lower agar plate is prepared and solidified. After that, an agar plate of the same medium containing a bacterial solution of Candida albicans (0.1%) in the upper layer is prepared and solidified.

The fraction containing the substance A-1211 was impregnated on a commercially available paper disk, and placed on an agar plate.
It was detected by measuring the diameter of the inhibition circle against Candida albicans that appeared after incubation for hours.

The obtained brown oily substance was dissolved in methanol (5 m
l), and a part thereof is reversed-phase silica gel column (G
L-Science, Inertsil ODS-2,25
0x4.6 mm, i. d. ), Mobile phase; acetonitrile / 10
mM sodium dihydrogen phosphate (60:40), flow rate;
Under the condition of 4.0 ml / min, detection; 230 nm, a peak showing a retention time (Rt) of 7.6 minutes was fractionated and purified. The eluted fractions were collected, the organic solvent was distilled off under reduced pressure, and the aqueous solution was stirred and extracted twice with ethyl acetate (100 ml). After evaporating the organic solvent under reduced pressure, A-1221-a substance purified product (8.0 mg)
I got

The physicochemical properties of the obtained substance A-1221-a are shown below.

1) Shape: reddish brown powder 2) Melting point: 115-120 ° C. 3) Molecular formula: C ₁₃ H ₁₀ O ₄ 4) Molecular weight: 230 (C ₁₃ H by the high resolution electron impact mass spectrometry (HREI-MS) method) ₁₀ O ₄
Experimental value 230.570, calculated value 230.05 as [M ⁺ ]
79 is shown. 5) Specific rotation: measured at a concentration of C = 0.25% in chloroform. [α] _D ²⁶ = -85.4 ° 6) Ultraviolet absorption spectrum: measured in methanol, methanol containing 0.001N-HCl, and methanol containing 0.001N-NaOH at a concentration of 10 μg / ml, respectively: λ _max ^MeOH (nm) (ε): 203 (16,400), 237 (15,100), 259
(18,000), 291 (sh, 5,700), 416 (br, 4,900); λ _max ^{MeOH / HCl} (nm) (ε): 202 (8,500) 237 (sh), 259 (1
8,300), 293 (sh, 5,800), 415 (br, 5,000); λ _max ^{MeOH / NaOH} (nm) (ε): 205 (15,500), 238 (sh, 16,
300), 259 (14,400), 296 (sh, 5,100), 474 (br, 6,100) 7) Infrared absorption spectrum: KBr tablet method: IRνmax (KBr) cm ^-1 : 1640, 1620,1580, 1450, 1410, 1
320, 1270, 1030 (FIG. 1) 8) Nuclear magnetic resonance spectrum: 400 MHz ¹ H-NMR spectrum (FIG. 2) and 1 measured in deuterated chloroform at 30 ° C.
Table 4 shows the chemical shifts of the 00 MHz ¹³ C-NMR spectrum.

[0078]

[Table 4]

The above positions are shown in the following structural formula.

[0080]

Embedded image

9) Solubility: Soluble in methanol, acetone, ethyl acetate, chloroform, dimethyl sulfoxide,
Poorly soluble in water, insoluble in ether 10) Rf value on thin layer chromatography: Kieselgel plate 60F254 (Merck, Art. 5175)
When developing with chloroform as the developing solvent, use an Rf value of 0.2
5, Rf value 0.42 when developed with chloroform / ethyl acetate (10: 1) 11) Retention time (Rt) in high performance liquid chromatography (HPLC): reverse phase ODS silica gel column (Inertsil
Using ODS-2, 250 x 4.6 mm, Id, manufactured by GL Sciences), the mobile phase was analyzed using acetonitrile / 0.05% trifluoroacetic acid (60:40) at a flow rate of 0.8 ml / min and detection at 230 nm. It is detected as a peak at 5.9 minutes.

Embodiment 2 FIG . A-1221-a substance and A-1
Production of 221-b substance (a) Culture step Under the same conditions as in Example 1, a rotary shaking culture at 30 ° C. for 6 days (200 rotations per minute) was performed.

(B) Separation Step Immediately after the completion of the culture, the culture was centrifuged (8000 rpm, 15 minutes).
After the separation, the cells were separated into the cells and the filtrate. The obtained culture filtrate (10.7 liters, pH 6.5) is diluted with diluted hydrochloric acid.
The mixture was adjusted to pH 2-3, and extracted with stirring twice with ethyl acetate (3.0 liter). The cell portion was extracted three times with methanol (3.0 liters) while stirring. After filtration, the methanol extract was concentrated under reduced pressure to remove methanol, and ethyl acetate (1 liter) was added.
Liter) for 2 times. The two ethyl acetate extracts were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a brown oily crude extract (8.38 g).

(C) Isolation and Purification Step The above crude extract was dissolved in chloroform (15 ml), and silica gel (Merck, Kieselgel, 370 × 65 mm, i.p.
d.) Adsorb to the column and use chloroform as the elution solvent.
Each 60 ml was fractionated. Thin layer chromatography of the eluted fractions and detection by high performance liquid chromatography, Can
By examining the antifungal activity against dida albicans, active fractions containing the A-1211-a substance and the A-1211-b substance were respectively confirmed. The active fractions were collected, and the solvent was distilled off. The residue was evaporated to dryness to obtain a brown oily crude substance (A-1211-a substance fraction (700 mg) and A-1211).
A -b substance fraction (60 mg) was obtained.

The A-1211-a substance fraction was dissolved in 6 ml of chloroform / methanol (1: 1), and 1/3 volume thereof was separated by Sephadex LH-20 (Pharmacia, 950).
x23mm, i. d. ), And eluted and fractionated using an elution solvent chloroform / methanol (1: 1). The active fraction containing the A-1211-a substance was detected by checking the above-mentioned HPLC method and the antibacterial activity against Candida albicans , the active fraction was collected, and the organic solvent was distilled off under reduced pressure to obtain a brown substance (78.6 mg). I got

The obtained brown substance was dissolved in methanol (2 ml), and the reverse-phase silica gel column (GL Science,
Inertsil ODS-2, 250x4.6 m
m, i. d. ), Mobile phase; acetonitrile / 0.05% trifluoroacetic acid (60:40), flow rate: 4.0 ml / min,
Detection: Purification was performed by repeatedly collecting a peak showing a retention time (Rt) of 7.6 minutes under the condition of 230 nm. The eluted fractions were collected, the organic solvent was distilled off under reduced pressure, and the aqueous solution was freeze-dried to obtain a purified A-1221-a substance (24.5 mg).
I got

The physicochemical properties of the obtained A-1221-a substance were as described in Example 1. Was consistent with the data for the A-1221-a substance obtained in the above.

Next, the fraction of the substance A-1211-b was dissolved in 6 ml of chloroform / methanol (1: 1).
/ 3 volume was adsorbed on Sephadex LH-20 (Pharmacia, 950 × 23 mm, id), and eluted and fractionated using an elution solvent chloroform / methanol (1: 1). HPLC method described above and Candida
A-12 by examining the antibacterial activity against Albicans
An active fraction containing the 11-b substance was detected, the active fractions were collected, and the organic solvent was distilled off under reduced pressure to obtain a brown substance (12.6 mg).

The obtained brown substance was dissolved in methanol (2 ml), and the mixture was dissolved in a reversed-phase silica gel column (manufactured by GL Science;
Inertsil ODS-2, 250x4.6 m
m, i. d. ), Mobile phase; acetonitrile / 0.05% trifluoroacetic acid (60:40), flow rate: 4.0 ml / min,
Detection: Purification was performed by repeatedly collecting a peak showing a retention time (Rt) of 8.5 minutes under the condition of 230 nm. The eluted fractions were collected, the organic solvent was distilled off under reduced pressure, and the aqueous solution was freeze-dried to obtain a purified A-1221-b substance (1.2 mg).

The physicochemical properties of the obtained A-1221-b substance are shown below.

1) Shape: reddish brown powder 2) Melting point: 79-81 ° C. 3) Molecular formula: C ₁₄ H ₁₂ O ₄ 4) Molecular weight: 244 (electron impact mass spectrum (EI-MS) method) 5) Nuclear magnetic resonance Spectra: 400 MHz ¹ H-NMR spectrum measured in deuterated chloroform at 30 ° C. (FIG. 3) and 1
Table 5 shows the chemical shifts of the 00 MHz ¹³ C-NMR spectrum.

[0092]

[Table 5]

The above positions are shown in the following structural formula.

[0094]

Embedded image

6) High performance liquid chromatography (HPLC)
Retention time (Rt): using a reverse-phase ODS silica gel column (Inertsil ODS-2, 250 × 4.6 mm, Id, GL Sciences), mobile phase acetonitrile / 0.05% trifluoroacetic acid (60:40), When analyzed at a flow rate of 0.8 ml / min and detection at 230 nm, a peak is detected at Rt. 6.3 minutes.

[0096]

[Test Example] Test Example 1. Assay of enzyme inhibitory activity Cdc25B protein tyrosine phosphatase of substance A-1211-a of the present invention
The measurement of the inhibitory activity against se) was performed as follows.

That is, 50 mM containing a sample diluted to an appropriate concentration with dimethylsulfoxide.
10 mM HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethansulfoni) consisting of sodium chloride (Sodium chlolide) and 5 mM dithiothreitol
c acid, pH 8.0), 50 μl; 200 mM disodium para-nitrophenyl phosphate hexahydrate (Disodium p-nitroph)
enylphosphate hexahydrate), 25 µl; Cdc25B protein tyrosine phosphatase (protein tyrosine p)
hosphatase, 200 mU / ml)
For 60 minutes. After the reaction is completed, use a microplate reader for OD41.
The absorbance at 5 nm was measured. As a control, an enzyme reaction system to which no inhibitor was added was prepared, and the enzyme inhibitory activity was calculated by the following equation.

Enzyme inhibitory activity (%) = [1- (BD) /
A−C)] × 100, where A is the absorbance in the reaction system containing no inhibitor, B is the absorbance in the reaction system containing various concentrations of the inhibitor, and C and D do not add the enzymes of A and B respectively. The absorbance in the reaction system is shown.

The inhibition rates obtained with various concentrations of the inhibitor were inserted into a graph with the probit on the vertical axis and the logarithm of the concentration on the horizontal axis, and if a straight line connecting the points was obtained, 50% from this straight line was obtained. The inhibitory concentration was determined.

Cdc25B of A-1211-a substance of the present invention
Protein tyrosi phosphatase
As a result of measuring the inhibitory activity against ne phosphatase, its 50% inhibitory concentration (IC ₅₀ ) was 17 μM.

Test Example 2 Measurement of antifungal activity The antifungal activity of the substance of the present invention against fungi such as yeasts and filamentous fungi was measured by a serial agar plate dilution method as follows. After dissolving the substance A-1211-a to 10 mg / ml with dimethylsulfoxide, a two-step dilution series was prepared to obtain an appropriate concentration.
A Sabouraud agar plate medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing the 1-a substance was prepared. The antifungal activity against various test bacteria (approximately 10 ⁶ cells / ml) was measured at 30 ° C. at a minimum growth inhibitory concentration (MIC, μg after culturing for 18 hours for yeast and 36 hours for filamentous fungi). / ml). Table 6 shows the results.

[0102]

[Table 6]

Test Example 3 The 50% growth inhibitory concentration (IC ₅₀ ) against antitumor active U937 cells (established cell line derived from pleural effusion of histiocytic lymphoma patient) was measured as follows. U937 cells were cultured at 37 ° C. in a 5% CO 2 incubator using an RPMI 1640 medium (RPMI 1640 medium, manufactured by Nissui Pharmaceutical Co.) supplemented with 10% calf serum to prepare 5 × 10 ⁴ cells / ml. did. Then, after dissolving the substance A-1211-a in dimethyl sulfoxide, add 96 μl of the medium to 96 μl to obtain an appropriate concentration.
A two-step dilution series was made per well of the well assay plate, and cultured for 2 days with 50 μl of the cell suspension. After adding 25 μl of MTT solution to each well and reacting for 4 hours, culture supernatant 1
Discard 00 μl and add acid propanol
l) 100 μl was added, and the absorbance at 590 nm was measured with a microplate reader, and the IC ₅₀ value was determined by comparison with the untreated group. As a result, A-12
The 50% growth inhibitory concentration (IC ₅₀ value) of the 11-a substance is 0.22
μg / ml was indicated.

[0104]

[Formulation Example] Formulation Example 1. Capsule A-1211-a substance 10 mg Lactose 50 mg Maize starch 47 mg Crystalline cellulose 50 mg Talc 2 mg Magnesium stearate 1 mg 160 mg per capsule A capsule was prepared according to a conventional method at the above mixing ratio.

Formulation Example 2 Injection A-1211-a substance 5 mg distilled water for injection 5 ml in an appropriate amount of 1 ampule An injection was prepared according to a conventional method at the above mixing ratio.

Formulation Example 3 Suppository A-1211-a substance 20 mg Witepsol W-35 1380 mg (registered trademark, manufactured by Dynamite Nobel) 1400 mg per one A suppository was prepared in the above-mentioned mixing ratio according to a conventional method.

[0107]

The substance A-1211 of the present invention has excellent Cd
It has c25B protein tyrosine phosphatase inhibitory activity and is useful as a preventive or therapeutic agent for fungal infections or tumors.

Further, since the substance A-1211 of the present invention has both antitumor activity and antifungal activity, it is accompanied by a decrease in immunity in anticancer treatment (including use in combination with other anticancer agents). It is also useful as a preventive for fungal infections.

[Brief description of the drawings]

FIG. 1 shows A-1211-a of the present invention obtained in Example 2.
It is an infrared absorption spectrum figure of a substance.

FIG. 2 shows A-1211-a of the present invention obtained in Example 2.
FIG. ^{1 is} a ¹ H-NMR spectrum of a substance.

FIG. 3 shows A-1211-b of the present invention obtained in Example 2.
FIG. ^{1 is} a ¹ H-NMR spectrum of a substance.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12N 1/20 C12R 1: 365) (C12P 17/04 C12R 1: 365) F term (Reference) 4B064 AE45 BA03 BC01 BC02 BE09 BE12 BE14 BG04 BH01 BH02 BH04 BH06 CA03 CE08 CE09 CE10 DA02 DA05 4B065 AA38X AC14 AC15 BA22 BB40 BD15 BD16 BD30 CA18 CA44 4C037 TA10 4C086 AA01 AA02 AA05 MA05 MA05

Claims (12)

[Claims] (1) The following general formula (1): (Wherein, R represents a hydrogen atom or a methyl group.) Or a salt thereof. 2. A bacterium belonging to the genus Nocardia which has the ability to produce the naphthofuranone compound or its salt according to claim 1, is cultured in a medium, and the compound or its salt is separated from the culture. The method for producing a naphthofuranone compound or a salt thereof according to claim 1, wherein 3. The method according to claim 1, wherein the strain is Nocardia sp. TP-A0
The method for producing a naphthofuranone compound or a salt thereof according to claim 2, which is a 248 (Nocardia sp. TP-A0248) strain or a mutant thereof. 4. A medicament comprising the naphthofuranone compound according to claim 1 or a salt thereof as an active ingredient. 5. The medicament according to claim 4, which is a therapeutic agent for a fungal infection. 6. The medicament according to claim 4, which is a therapeutic agent for a tumor. 7. A pharmaceutical composition comprising an effective amount of the naphthofuranone compound according to claim 1 or a salt thereof and a pharmaceutically acceptable carrier. 8. The pharmaceutical composition according to claim 7, which is used for treating a fungal infection. 9. The pharmaceutical composition according to claim 7, which is used for treating a tumor. 10. A strain having an ability to produce the naphthofuranone compound according to claim 1 or a salt thereof. 11. The strain according to claim 10, which belongs to the genus Nocardia . (12) Nocardia sp. TP-A024
The strain according to claim 10 or 11, which is No. 8 ( Nocardia sp. TP-A0248) or a mutant thereof.