JP2001220388A - Compound having antitumor, antibacterial and antiviral activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new compound having topoisomerase I inhibiting action, antibacterial activity against Gram-positive bacteria, especially MRSA and anti-herpes activity and provide an antitumor agent, an antibacterial agent and an antiviral agent useful for the effective treatment and prevention of tumor diseases. SOLUTION: The objective compound is a compound having the structure expressed by formula (1) or (2) or its salt.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel compound having an excellent topoisomerase I inhibitory activity, an antibacterial activity against Gram-positive bacteria, especially against MRSA, and an antiherpes activity. The present invention also relates to the use of the compound, a producing bacterium, and a production method.

[0002]

BACKGROUND OF THE INVENTION Camptothecin, its derivatives, indolocarbazole compounds and the like are known as compounds having topoisomerase I inhibitory activity, and many antitumor agents have been developed and put to practical use. For more effective treatment and prevention of tumor diseases, development of new useful substances is awaited.

[0003] Conventionally, the frequency of infections caused by Gram-positive bacteria such as staphylococci and streptococci is extremely high, and hosts with low resistance often suffer from severe disease states such as bronchitis, pneumonia, and sepsis. In recent years, various antibiotics are frequently used, and resistant bacteria such as so-called methicillin-resistant cocci (MRSA) have become a clinical problem. Recent trends in MRSA include not only resistance to penicillin drugs, but also aminoglycoside,
Strains that are also resistant to quinolone drugs, which are macrolides or DNA gyrase (topoisomerase II) inhibitors, are increasing. Recently, vancomycin-resistant MRSA and enterococci have become problems. For such multidrug-resistant bacteria, drugs having a novel mechanism of action have been desired.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems of the prior art. That is, the present invention provides a novel compound having an activity of inhibiting topoisomerase I, an antibacterial activity against Gram-positive bacteria, particularly MRSA, and an antiherpes activity, and an antitumor agent useful for more effective treatment and prevention of tumor diseases, An object of the present invention is to provide an antibacterial agent and an antiviral agent.

[0005]

Means for Solving the Problems In order to solve the above problems, the present inventors have searched for substances having topoisomerase I inhibitory activity in microbial metabolites. As a result, strains belonging to the genus Penicillium (Penicillium sp. BAUA42
06) produce compounds having topoisomerase I inhibitory activity. Furthermore, as a result of intensive studies on useful physiological activities of the compound, Gram-positive bacteria,
In particular, they have been found to have an antibacterial action against MRSA and an anti-herpes action.

That is, the present invention provides a compound represented by the formula:

Embedded image And a salt having the structure represented by the formula: The present invention also provides an antitumor agent, an antibacterial agent, and an antiviral agent, which comprise these compounds or salts thereof as an active ingredient. Further, the present invention provides a strain of the genus Penicillium (especially, strain number: FERM P-17643) that produces the compound of the formula (1) and the compound of the formula (2), and a method comprising the step of culturing the strain. ) And a process for producing the compound of formula (2).

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The compound of the present invention has the above formula (1)
Alternatively, it has a structure represented by the above formula (2). The compound of the present invention may be produced using a microorganism, or may be produced by artificially reacting a chemical substance using synthetic means. Compounds having the structures represented by formulas (1) and (2) and salts thereof, which are produced by any means, are included in the scope of the present invention.

In the case of production using microorganisms, microorganisms and mutants producing the compound of the present invention can be used without limitation. For example, a compound of formula (1) or a compound of formula (2)
A strain of the genus Penicillium which produces the compound of formula (I) can be preferably used. Specifically, Penicilliu sp. Isolated from coastal soil in Shimajiri-gun, Okinawa
msp.) BAUA4206 strain. This strain has been deposited with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (deposit number: FERM P-17643), and its bacteriological properties are as follows.

(Morphology) The hypha is a septum, colorless, and the conidia are unicellular, spherical, and form a smooth, colorless conidium of the phyarotype. The conidiophore is smooth and produces 3-4 phialides on the branches. The conidia are chained to the tip, and the whole consolidates to form penicilli.

(Properties on Various Media) The growth conditions when cultured on various agar media at 25 ° C. for 14 days are as follows. The color display is A MYCOLOGICAL COLOR CHART
(Prepared by RWRayner, COMMONWEALTH MYCOLOGICAL
INSTITUTE KEW, SURREY & BRITISH MYCOLOGICAL SOCIET
Y, 1970). When cultured on a malt extract agar medium (MEA), the size of the colonies is 30 to 32 mm in diameter. The flora is wool-like and has a moss green (Greenish Grey: 110) surface.

Potato dextrose agar (PDA)
When cultivated on the above, the size of the settlement is 30-35m in diameter
m. The flora surface is entirely covered with long white hyphae,
Moss green (Greenish Grey: 110). When cultured on cornmeal agar medium (CMA), the size of the colonies is 35-42 mm. The flora is powdery, the center is dense, the periphery is sparsely concentric, and is grayish green (Sm
oke Gray: 105).

(Physiological properties) When cultivated in a potato dextrose liquid medium for 14 days, the range of pH that can grow is 3 to 12, and the optimum growth pH is 6.5 to 7.5. On a potato dextrose agar medium, the growth temperature range is 15-37 ° C, and the optimal growth temperature is 25-3.
5 ° C. The compound of the present invention can be obtained by culturing a strain producing the compound of the present invention. As a culture medium, a carbon source, a nitrogen source,
Furthermore, a medium containing an inorganic salt or the like as required may be used, and a synthetic medium, a semi-synthetic medium, or a natural medium is used.

The culturing method may be the same as the method for producing a general microbial metabolite, and may be liquid culture or solid culture. A suitable temperature for culturing is 15-37 ° C,
Preferably it is 25-35 degreeC. The culturing time varies depending on the medium and the culturing conditions, but is usually 1 to 14 days. When the production of the compound of the present invention reaches the maximum, the culture can be stopped, and the compound can be isolated and purified.

In order to collect the desired compound of the present invention from the culture, a separation means usually used for collecting metabolites produced by microorganisms from the culture is appropriately used.
For example, means such as extraction filtration, centrifugation, dialysis, concentration, drying, freezing, adsorption, desorption, precipitation utilizing differences in solubility in various solvents, crystallization, recrystallization, phase transfer, countercurrent distribution, and chromatography. Is mentioned.

BAUA420 having the above mycological properties
By culturing the six strains, a mixture containing at least the compound of the formula (1) and the compound of the formula (2) can be obtained. The mixture may be used as it is, but is usually used after purification. Further, the compound of the formula (1) can be converted to the compound of the formula (2). The conversion method is not particularly limited,
It is preferable to use a method of treating the compound of the formula (1) with an alkali. Although the kind, amount and concentration of the alkali are not particularly limited, it is preferable that the solution has a high concentration, and it is preferable that the solution is set to a concentration that is easy to handle in operation. Specific examples of the alkali include sodium hydroxide.

The present invention also includes a compound of the formula (1) and a salt of the compound of the formula (2). Examples of salts include sodium,
Examples thereof include alkali metal or alkaline earth metal salts such as calcium. These salts can be produced from the compounds represented by the formulas (1) and (2) by a known method.

When the compound of the present invention is used as a medicament, particularly as an antitumor agent or antibacterial agent, it is mixed with pharmaceutically acceptable carriers, excipients and diluents according to a conventional method to give tablets, granules,
Powders, capsules, syrups, ointments, creams,
It can be made into injections and the like. When preparing a medicament,
One compound may be selected from the compound of the formula (1), the compound of the formula (2), and salts thereof to be used as an active ingredient, or a plurality of compounds may be selected and combined as an active ingredient. May be used. Further, it can be used in combination with an active ingredient other than the present invention.

The preferred dose of the active ingredient of the present invention varies depending on the form and composition of the medicament containing the active ingredient of the present invention, the frequency of application, the specific site to be treated, and the host. For example,
The daily dose of the active ingredient per adult is usually 10 to 1000 mg for oral administration, and usually 10 to 100 m for parenteral administration (preferably intravenous injection).
g. The number of administrations varies depending on the administration method and symptoms, but is usually 1 to 5 times.

[0019]

The present invention will be described more specifically with reference to the following examples. The materials, amounts, proportions,
The operation and the like can be appropriately changed without departing from the spirit of the present invention. Therefore, the scope of the present invention is not limited to the following specific examples. In particular, the compound of the present invention produced, concentrated, extracted, and purified using known means based on the properties of the compound of the present invention revealed by the present specification as well as the production means of the present invention, and the compound The antitumor, antibacterial and antiviral agents that are included are all encompassed by the present invention.

Example 1 Preparation of Compound of Formula (1) In a test tube having a diameter of 24 mm, soluble starch 2.0%, glucose 1.0%, soybean powder 1.5%, malt extract 0.5%,
Magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.5%
05%, V8 vegetable juice centrifugal supernatant 10%, potato dextrose 10% artificial seawater for academic research Jamarin S
10 ml of medium containing (Jamarin Laboratory)
Dispensed at a time and steam sterilized at 121 ° C for 20 minutes.
BAUA grown on potato dextrose agar
The hyphae of the 4206 strain were scraped, inoculated, and cultured with shaking at 25 ° C. for 3 days to obtain a seed culture. Next, the above medium was added to 12
00 ml was prepared and dispensed into K1 flasks in 30 ml increments.
The seed culture solution was transplanted at a rate of 1.6% to those steam-sterilized at 121 ° C. for 20 minutes, and shake-cultured at 25 ° C. for 4 days.

After completion of the cultivation, 44
0 ml of butanol was added for extraction. After performing this extraction operation three times, the obtained butanol extract was concentrated under reduced pressure. The concentrated butanol extract was suspended in 300 ml of water adjusted to pH 3.0, and extracted three times with an equal volume of ethyl acetate. After concentrating the obtained ethyl acetate extract to dryness,
The dried product was dissolved in 20 ml of dimethyl sulfoxide and purified under the following conditions. Column: Senshu Pak. PEGASIL ODS 20φ × 250mm (manufactured by Senshu Science) Separation solution: 45% acetonitrile (50 mM) at the start of purification
55% after 65 minutes from the start of purification from acetic acid pH 3.1)
Gradient elution into acetonitrile Flow rate: 8 ml / min Measurement wavelength: 280 nm

The active fraction having a retention time of 28.8 minutes was concentrated under reduced pressure to obtain 10.3 mg of an orange powdery compound of the formula (1). The results of instrumental analysis and solubility test of the compound were as follows. Ultraviolet absorption spectrum (methanol): HR-FABMS as shown in Figure 1: Measured: 337.0322 (M-H) ^- Calculated: 337.0348 (M-H) ^- Solubility: DMSO, pyridine, ethyl acetate Soluble, hardly soluble in water.

Example 2 Preparation of acetyl derivative of compound of formula (1) To 22.9 mg of compound of formula (1), 2 ml of pyridine and 20 ml of acetic anhydride were added, and an acetylation reaction was carried out at room temperature overnight. After adding 200 ml of water to stop the reaction, the reaction solution was extracted with ethyl acetate. The obtained ethyl acetate solution was concentrated, and the acetyl derivative of the compound of the formula (1) was purified using an ODS column. Formula (1) of 15.0 mg
Acetyl derivative of the compound of formula
and purified under the following conditions. Column: Senshu Pak. PEGASIL ODS 20φ × 250mm (manufactured by Senshu Science) Separation solution: 45% acetonitrile (50 mM acetic acid pH 3.
Isocratic elution of 1) Flow rate: 10 ml / min Measurement wavelength: 280 nm The peak at a retention time of 26.1 minutes was concentrated under reduced pressure to obtain 8.9 mg of an acetyl derivative of the compound of the formula (1). The results of instrumental analysis of the compound were as follows. ¹ H-NMR: as shown in FIG. 2 ¹³ C-NMR: as shown in FIG.

(Example 3) Production method 1 of compound of formula (2) An ethyl acetate extract was obtained in the same manner as in Example 1. The obtained extract was dissolved in 100 ml of a 1% (W / W) sodium hydroxide / methanol solution, followed by stirring at room temperature for 7 days. After the reaction solution was concentrated under reduced pressure, the dried product was redissolved in 50 ml of distilled water, and the pH of the aqueous solution was adjusted to 3.0 using 1N HCl. The precipitate generated by centrifugation at 3500 rpm for 10 minutes was collected and dried. 20 dry matter
It was dissolved in ml of dimethyl sulfoxide and purified under the following conditions. Column: Senshu Pak. PEGASIL ODS 20φ × 250mm (manufactured by Senshu Kagaku) Separation solution: 37% acetonitrile (50 mM acetic acid pH 3.
Isocratic elution of 1) Flow rate: 10 ml / min Measurement wavelength: 280 nm

The active fraction having a retention time of 28.0 minutes was concentrated under reduced pressure to obtain 16.1 mg of an orange powdery compound of the formula (2). The results of instrumental analysis and solubility test of the compound were as follows. Ultraviolet absorption spectrum (methanol): Figure 4 as indicated HR-FABMS: measured value: 337.0353 (M-H) ^- Calculated: 337.0348 (M-H) ^- Solubility: DMSO, pyridine, ethyl acetate Soluble, poorly soluble in water.

Example 4 Method 2 for preparing compound of formula (2) 20.6 mg of the compound of formula (1) was dissolved in 20 ml of a 1% by weight sodium hydroxide / methanol solution and stirred at room temperature for 4 days. did. After the reaction solution was concentrated under reduced pressure, the dried product was redissolved in 10 ml of distilled water, and the pH of the aqueous solution was adjusted to 3.0 using 1N HCl. 3500 rpm, 1
The precipitate generated by centrifugation for 0 minutes was collected and dried. The dried product was dissolved in 20 ml of dimethyl sulfoxide and purified under the following conditions. Column: Senshu Pak. PEGASIL ODS 20φ × 250mm (manufactured by Senshu Science) Separation solution: 35% acetonitrile (50 mM) at the start of purification
Gradient elution from 75% acetonitrile 65 minutes after the start of purification from acetic acid (pH 3.1) Flow rate: 10 ml / min Measurement wavelength: 280 nm The peak at a retention time of 23.4 minutes was concentrated under reduced pressure to give the compound of the formula (2) 7 .2 mg were obtained.

Example 5 Preparation of acetyl derivative of compound of formula (2) To 7.2 mg of compound of formula (2), 2 ml of pyridine and 20 ml of acetic anhydride were added, and acetylation was carried out at room temperature overnight. The reaction was stopped by adding 200 ml of water. The resulting precipitate was collected by centrifugation to obtain 10.0 mg of an acetyl derivative of the compound of the formula (2). The results of instrumental analysis of the compound were as follows. ¹ H-NMR: as shown in FIG. 5 ¹³ C-NMR: as shown in FIG.

Next, the physiological activity of the compound of the present invention will be described with reference to test examples. (Test Example 1) Topoisomerase I inhibitory activity C. The method was performed according to the method of Wang et al. (Cancer Research, 49, 6319-6322, 1989). That is, a vector expressing the human topoisomerase I gene under the control of PGAL1 was constructed, and the yeast Saccharomyces cerevi
siae JN2-134. The vector used and the JN2-134 strain were provided by Wang. The resulting recombinant yeast is placed on a galactose-containing YNB medium (DIFCO LABORATORY) when human topoisomerase I is induced, and on a YN medium containing glucose when human topoisomerase I is not induced.
A final bacterial concentration of 1 × 10 ⁵ cells / ml was suspended in the B medium, and 0.1 ml of the suspension was dispensed into a 96-well flat bottom plate. Various dilutions of the compound of the formula (1) or the compound of the formula (2) were added, and the mixture was cultured in an incubator at 30 ° C. for 2 days, and turbidity was measured at a wavelength of 620 nm. Using the turbidity when no drug was added as a control, half the turbidity (50% growth inhibition)
Drug addition concentration when indicating the expressed topoisomerase inhibitory activity as an IC ₅₀ value. Table 1 shows the results.

[0029]

[Table 1]

Test Example 2 Topoisomerase I Inhibitory Activity A Topoisomerase I Drug Screening Kit (TopoGEN) was used. The test protocol followed the manual attached to the kit. That is, 1 unit of topoisomerase I (derived from human) and a compound of the formula (1) and a compound of the formula (2) dissolved in dimethyl sulfoxide are added to the mixture.
After reacting at 30 ° C. for 30 minutes, agarose gel electrophoresis was performed on 1% agarose. From the electrophoresis pattern, the compound of formula (1) was 33.3 μM, and the compound of formula (2) was 3.3.3 μM.
Human topoisomerase I inhibitory activity was observed at a concentration of 70 μM.

(Test Example 3) In vitro anticancer activity HeLa cells (human cervical cancer), Colon 26 (mouse colon cancer), 3LL (mouse Lewis lung cancer), B16 (mouse melanoma cells), P388 cells (mouse leukemia) And L1210 (mouse leukemia) in cell culture (HeLa, Colon 26, 3L, respectively)
L, B16; Eagle MEM medium, P388, L1210; RPMI1
The cells were suspended at 5 × 10 ⁴ cells / ml in 640 medium, each containing 10% fetal bovine serum and 2 mM glutamine, and 0.1 ml of the cell suspension was dispensed into a 96-well microtiter plate. After culturing the plate in a 5% carbon dioxide saturated steam incubator at 37 ° C. for 24 hours, a test substance solution prepared by appropriately diluting with the same medium is added to each well.
Culture was performed at 7 ° C. for 48 hours. Next, 0.01 ml of a living cell measurement reagent SF (manufactured by Nacalai Tesque) was added to the culture solution, and the mixture was cultured at 37 ° C. for 3 hours in the same incubator to cause a color reaction. The absorbance was measured at a wavelength of 450 nm using a microplate reader (manufactured by Nippon Flow Laboratories). 100% absorbance of control cells without test substance
The concentration at which the test substance inhibits cell growth by 50% (I
_C50 value) was calculated. Table 2 shows the results.

[0032]

[Table 2]

(Test Example 4) Antibacterial activity The antibacterial activity was measured by the agar plate dilution method according to the standard method of the Japanese Society of Chemotherapy. That is, the inoculated bacterial solution was obtained by culturing the bacterial solution overnight in a sensitivity measurement broth for 2-3 × 10 ⁶ cels.
It was prepared by diluting to the ls / ml bacterial concentration with the same medium. DMS for the compound of formula (1) and the compound of formula (2)
O, a series of dilutions with dimethyl sulfoxide for enoxacin as a control drug and distilled water for ceftazim were made, and the mixture was diluted with agar medium for a sensitive disk so that the concentration of the solvent would be up to 0.5%. Allowed to solidify.
5 μl of the inoculum was inoculated on a drug-added agar medium with a microplanter and cultured at 37 ° C. for 20 hours. MIC (minimum growth inhibitory concentration)
And Table 3 shows the results.

[0034]

[Table 3]

Test Example 5 Antiviral Activity The compounds of formula (1) and the compound of formula (2) tended to protect against infection in a herpes simplex virus type 1, type 2 and varicella-zoster virus infection protection test. Was seen.

[0036]

As is clear from these results, the compounds of the formula (1) and the compound of the formula (2), which are novel physiologically active substances, are used as antitumor agents characterized by a topoisomerase I inhibitory action. It is expected. Further, the compound of the formula (1) and the compound of the formula (2) are gram-positive bacteria,
In particular, it has antibacterial activity against MRSA, and is therefore expected to be used for treatment and prevention of various disease states caused by the pathogenic bacteria.

[Brief description of the drawings]

FIG. 1 is an ultraviolet absorption spectrum of a compound of the formula (1).

FIG. 2: ¹ H- of an acetyl derivative of the compound of formula (1)
It is an NMR spectrum.

FIG. 3. ¹³ C- of the acetyl derivative of the compound of formula (1)
It is an NMR spectrum.

FIG. 4 is an ultraviolet absorption spectrum of the compound of the formula (2).

FIG. 5 shows ¹ H- of an acetyl derivative of the compound of the formula (2).
It is an NMR spectrum.

FIG. 6 shows ¹³ C- of an acetyl derivative of the compound of formula (2).
It is an NMR spectrum.

Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) A61P 43/00 111 A61P 43/00 111 C12N 1/14 C12N 1/14 A C12P 17/06 C12P 17/06 // ( C12N 1/14 (C12N 1/14 C12R 1:80) C12R 1:80) (C12P 17/06 (C12P 17/06 C12R 1:80) C12R 1:80) (72) Inventor Daisuke Ishiyama Fujieda-shi, Shizuoka 301 Gensuke, Kaken Pharmaceutical Co., Ltd.Development Research Laboratory (72) Inventor Wakao Iwatani 14, Shinanomiya, Minamikawaramachi, Yamashina-ku, Kyoto, Kyoto Prefecture, Japan Kaken Pharmaceutical Co., Ltd.Development Research Laboratory (72) Inventor, Naoto Senda, Kyoto, Kyoto, Japan 14 Minamikawara-cho, Kaken Pharmaceutical Co., Ltd. (72) Inventor Hiroko Takahashi 241 Kariwano, Nishi-Senhoku-cho, Senboku-gun, Akita Prefecture Inside Fungal Function Development Laboratory Co., Ltd. (72) Inventor Hiroshi Konno Nishi-Senhoku, Senboku-gun, Akita Prefecture 241 Karikano, town character Inside the Fungal Function Development Laboratory Co., Ltd. (72) Inventor Susumu Kanazawa 241 Kariwano, Nishi-Senhoku-cho, Senboku-gun, Akita F-term in the Fungal Function Development Laboratory Co., Ltd. MA04 NA14 ZB26 ZB33 ZB35 ZC20

Claims (8)

[Claims] [Claim 1] The formula: Or a salt thereof having the structure represented by: 2. An antitumor agent comprising the compound of claim 1 or a salt thereof as an active ingredient. 3. An antibacterial agent against Gram-positive bacteria, comprising the compound of claim 1 or a salt thereof as an active ingredient. 4. An antibacterial agent against MRSA, comprising the compound of claim 1 or a salt thereof as an active ingredient. 5. An antiviral agent comprising the compound of claim 1 or a salt thereof as an active ingredient. 6. A strain which produces the compound of claim 1 which belongs to the genus Penicillium. 7. A strain having a deposit number of FERM P-17643. 8. A method for producing the compound according to claim 1, comprising a step of culturing the strain according to claim 6 or 7.