JP2010120882A - New compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To discover a compound having a DNA synthetase-inhibiting activity, and to provide a new medicinal composition (DNA synthetase inhibitor and anticancer agent) utilizing the compound. <P>SOLUTION: The compound represented by general formula (1) or (2), or a pharmaceutically acceptable salt thereof are provided. The medicinal composition (the DNA synthetase inhibitor, the anticancer agent or the like) contains them as active ingredients. (In the formulas, R<SP>1</SP>, R<SP>2</SP>and R<SP>3</SP>are the same or different, and each H or a protective group). <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a compound having a DNA synthase inhibitory action and use thereof. This compound can be used as a DNA synthase inhibitor, for example, as a biochemical reagent, or as an anticancer agent or a lead compound thereof.

  Eukaryotic DNA synthase (DNA polymerase) has so far been classified into 15 molecular species: α, β, γ, δ, ε, ζ, η, θ, ι, κ, λ, μ, σ, TdT, and Rev1. It has been known. These DNA synthase groups are involved in cell growth, division, differentiation, etc., but α type is DNA replication, β type, λ type and TdT are repair and recombination, and δ type and ε type are replication. It is known that the ζ to κ types have different functions depending on the type, such as both repair and ζ to κ.

  Since DNA synthase is involved in cell growth and the like in this way, a DNA synthase inhibitor that inhibits the enzyme activity exhibits, for example, cancer cell growth inhibitory action against cancer and HIV against AIDS. It is considered that it exhibits an inhibitory effect on the derived reverse transcriptase and an immunosuppressive action that suppresses specific antibody production against an antigen against an immune disease. For this reason, development of pharmaceuticals effective for prevention and treatment of cancer diseases, viral diseases such as AIDS, and immune diseases using a DNA synthase inhibitor is expected.

For example, it has been reported that glycolipids having DNA synthase inhibitory activity are useful as anticancer agents, HIV-derived reverse transcriptase inhibitors, and immunosuppressants (see Patent Document 1 below). Currently, dideoxy TTP (ddTTP), N-methylmaleimide, butylphenyl-dGTP, and the like are known as DNA synthetase inhibitors (see Non-Patent Document 1 below). In addition, sulfosynovosyl acylglycerides, which are plant-derived glycolipids, have been found to inhibit DNA synthase (see Patent Document 2 below).
JP 11-106395 A JP 2000-143516 A Annual Review of Biochemistry, 2002, 71, 133-163

  An object of the present invention is to find a novel compound having a DNA synthase inhibitory action and to provide a new pharmaceutical composition (a DNA synthase inhibitor and an anticancer agent) using the compound.

  As a result of intensive studies to achieve the above-mentioned problems, the present inventors have found that Compound 1 (a novel substance) is obtained from a culture solution obtained by culturing Penicillium daleae KM Zalessky in a PDB medium (potato dextrose liquid medium). “Penicilliol A” and Compound 2 (named “Penicilliol B”) were isolated (see Example 1).

  The present inventors investigated and examined the relationship between these compounds and the DNA synthase inhibitory action, and found that these compounds selectively inhibit DNA synthase among DNA metabolic enzymes. confirmed. As a result of further research based on this knowledge, the present invention has been completed.

  That is, the present invention provides the following compound, a DNA synthase inhibitor and an anticancer agent comprising the compound as an active ingredient.

  Item 1 A compound represented by the general formula (1) or (2) or a pharmaceutically acceptable salt thereof.

(In the formula, R ¹ , R ² and R ³ are the same or different and represent H or a protecting group.)
Item 2 A pharmaceutical composition comprising the compound represented by the general formula (1) or (2) according to Item 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

  Item 3. A DNA synthase inhibitor comprising, as an active ingredient, the compound represented by the general formula (1) or (2) according to Item 1 or a pharmaceutically acceptable salt thereof.

  Item 4 An anticancer agent comprising the compound represented by the general formula (1) or (2) according to Item 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

  Item 5 An edible composition comprising the compound represented by the general formula (1) or (2) according to Item 1 or a pharmaceutically acceptable salt thereof in a food.

  The novel compound 1 (Penicilliol A), compound 2 (Penicilliol B), and derivatives thereof of the present invention have a DNA synthase inhibitory action. In particular, Compound 2 selectively inhibits DNA synthases η, ι and κ belonging to the Y family among mammalian DNA synthases.

  Therefore, these compounds can be used as pharmaceutical compositions such as DNA synthase inhibitors and anticancer agents.

  These compounds can also be used as biochemical reagents by utilizing the above-mentioned activity. Furthermore, these compounds can be blended into foods and used as edible compositions.

Hereinafter, the present invention will be described in detail.
1. TECHNICAL FIELD The present invention relates to a compound represented by formula (1) or (2) or a pharmaceutically acceptable salt thereof.

(In the formula, R ¹ , R ² and R ³ are the same or different and represent H or a protecting group.)
The protecting group represented by R ¹ , R ² and R ³ is not particularly limited as long as it is a protecting group for a hydroxyl group. For example, an acyl group (acetyl, propanoyl, benzoyl group, etc.), a silyl group (trimethylsilyl, triethylsilyl, Triisopropylsilyl, tert-butyldimethylsilyl group, etc.), alkyl group (methyl, ethyl, propyl, butyl group, etc.), alkenyl group (allyl, crotyl group, etc.), aralkyl group (benzyl, phenethyl group, etc.), alkoxymethyl group (Methoxymethyl group etc.), sulfonyl group (methanesulfonyl, benzenesulfonyl group etc.) etc. are mentioned.

The compounds represented by the general formulas (1) and (2) have a plurality of asymmetric carbons (sp ³ carbons), and the compound has a stereostructure of a plurality of asymmetric carbons of either R or S. Are also included. For example, any of enantiomers, diastereomers, and mixtures thereof are included.

  Among the compounds represented by the general formula (1), compound 1 (Penicilliol A) in which R is H is preferable. Of the compounds represented by formula (2), compound 2 (Penicilliol B) in which R is H is preferable.

  Examples of the salt of “pharmaceutically acceptable salt thereof” in the present invention include alkali metal salts such as sodium salt and potassium salt; ammonium salt; primary, secondary or tertiary amine salt; quaternary ammonium salt; amino acid Examples include salt. The compound represented by the general formulas (1) and (2) or a pharmaceutically acceptable salt thereof may be a solvate such as water.

  Among the compounds represented by the general formulas (1) and (2), the compound represented by R = H can be isolated and purified from a culture obtained by culturing the strain Penicillium daleae K.M. Zalessky. Specifically, Penicillium daleae K.M. Zalessky is statically cultured in a PDB medium (potato dextrose liquid medium), and the filtrate obtained by removing the cells from the culture is extracted with a solvent. Examples of the extraction solvent include alcohols such as methanol, ethanol, propanol and butanol, glycols such as 1,3-butylene glycol, glycerin and propylene glycol, esters such as ethyl acetate and butyl acetate, ethyl ether and propyl ether. Polar organic solvents such as ethers such as isopropyl ether, tetrahydrofuran and dioxane, halogenated hydrocarbons such as methylene chloride and chloroform; non-polar organic solvents such as hexane, cyclohexane and petroleum ether, etc. It can also be used alone or as a mixed solvent of two or more.

  Among these, it is preferable to use at least one extraction solvent selected from the group consisting of halogenated hydrocarbons such as methylene chloride and esters such as ethyl acetate. When mixing and using a solvent, what is necessary is just to adjust the mixing ratio of each solvent suitably according to the kind of solvent.

  After obtaining the extract by the above-mentioned method, organic solvent such as methanol, ethanol, propanol, butanol, chloroform, ethyl acetate, toluene, hexane, benzene; by solvent fractionation operation using one or more kinds of water, The active fraction (compounds 1 and 2) can be separated from the obtained extract. Furthermore, it can also be purified by combining one or more suitable separation and purification means such as alumina column chromatography, silica gel chromatography, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and high performance liquid chromatography. . In addition, isolation and identification of the active compound from the culture solution can be performed specifically as described in Example 1.

Furthermore, by introducing various protective groups into the isolated compound 1 or 2 using a known method, among the compounds represented by the general formula (1), a compound represented by R ¹ = protective group, and Among the compounds represented by the general formula (2), compounds represented by R ² and R ³ = the same or different protecting groups can be obtained. Alternatively, a protective group (R ² ) is introduced into the isolated compound 1 using a known method, and OR ³ is introduced into a double bond at the C7 position using a known method such as a Michael addition reaction. By doing this, among the compounds represented by the general formula (2), compounds represented by R ² and R ³ = the same or different protecting groups can be obtained.

2. Use of the Compound of the Present Invention The compound represented by the general formula (1) or (2) of the present invention has a DNA synthase selective inhibitory action, and thus can be used for pharmaceuticals. For example, it is useful as a pharmaceutical composition such as a DNA synthase selective inhibitor and an anticancer agent. The compound of the present invention can also be used as a lead compound in the pharmaceutical development process.

  When the compound of the present invention is administered into the body, parenteral administration is preferred over oral administration, and administration in a carrier such as a liposome is preferred. At this time, if a carrier that specifically recognizes cancer cells is used, the compound of the present invention can be efficiently transported to the target site (lesion site).

  Next, a pharmaceutical composition comprising the compound of the present invention will be described. The anticancer agent comprising the compound of the present invention as an active ingredient can be administered to animals and humans as it is or as a pharmaceutical composition together with a conventional pharmaceutical preparation carrier. The dosage form of the pharmaceutical composition is not particularly limited and may be appropriately selected as necessary.For example, oral preparations such as tablets, capsules, granules, fine granules, powders, injections, Non-oral preparations such as suppositories are mentioned, and preferred examples include parenteral preparations.

  In the present invention, oral preparations such as tablets, capsules, granules, fine granules, and powders are produced according to a conventional method using, for example, starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts, and the like. . The compounding amount of the compound of the present invention in these preparations is not particularly limited and can be appropriately designed. In addition to the compound of the present invention, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a corrigent, a colorant, a fragrance and the like can be appropriately used for this type of preparation.

  Examples of the binder include starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch, sodium methylcellulose, hydroxypropylcellulose, crystalline cellulose, ethylcellulose, polyvinylpyrrolidone, macrogol and the like. Examples of the disintegrant include starch, hydroxypropyl starch, carboxymethylcellulose sodium, carboxymethylcellulose calcium, carboxymethylcellulose, and low-substituted hydroxypropylcellulose. Examples of the surfactant include sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polyoxyethylene sorbitan fatty acid ester and the like. Examples of lubricants include talc, waxes, hydrogenated vegetable oils, sucrose fatty acid esters, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol and the like. Examples of the fluidity promoter include light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate and the like. The compounds of the present invention can also be administered as suspensions, emulsions, syrups, and elixirs, and these various dosage forms may contain flavoring agents and colorants.

  In order to express the desired effect of the present invention as a parenteral agent, it varies depending on the age, body weight, and degree of disease of the patient, but usually, 1 to 60 mg intravenously per day as the weight of the compound of the present invention in an adult, Intravenous infusion, subcutaneous injection, and intramuscular injection are suitable. This parenteral preparation is produced according to a conventional method, and generally used as diluent is distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, etc. it can. Furthermore, you may add a disinfectant, antiseptic | preservative, and a stabilizer as needed. In addition, from the viewpoint of stability, this parenteral preparation can be frozen after filling into a vial or the like, the water can be removed by ordinary freeze-drying treatment, and the liquid preparation can be re-prepared from the freeze-dried product immediately before use. Furthermore, you may add an isotonic agent, a stabilizer, an antiseptic | preservative, and a soothing agent as needed. The compounding quantity of the compound of this invention in these formulations is not specifically limited, It can set arbitrarily. Examples of other parenteral agents include liquid preparations for external use, coating agents such as ointments, suppositories for rectal administration, etc., and these are also produced according to conventional methods.

  Moreover, the compound of this invention can be utilized for food other than the utilization to a pharmaceutical. For example, it can also be used as an edible composition (for example, health food) having an anticancer effect by being added to and blended with food and drink.

  That is, the compound of the present invention is used as it is as a liquid, gel or solid food, for example, juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, infant formula, cake. Add to mixes, powdered or liquid dairy products, bread, cookies, etc., and if necessary, process into pellets, tablets, granules etc. with excipients such as dextrin, lactose, starch, flavorings, pigments, etc. Further, it can be coated with gelatin or the like and molded into a capsule to be used as a health food or nutritional supplement.

  Since the DNA synthase structure of humans and other mammals is almost the same, the DNA synthase inhibitor of the present invention can be used as a DNA synthase inhibitor derived from mammals other than humans.

  Therefore, efficient screening of candidate compounds for anticancer agents can be expected by examining the inhibitory activity against DNA synthase using the compound of the present invention as a lead. The present invention includes such a screening method. In this screening method, the method for examining the inhibitory activity against DNA synthase is not limited to the method described in the above-mentioned Examples, and a suitable method may be selected from known test methods.

Next, the present invention will be specifically described, but the present invention is not limited thereto.
Example 1
(1) Isolation and culture of strains After treating moss collected on the beach in Nichinan City, Miyazaki Prefecture with 60% acetic acid, on CMA + rose bengal medium (medium with cornmeal agar medium plus rose bengal) After culturing, this strain was isolated. As a result of strain identification, this strain was identified as Penicillium daleae KM Zalessky. This strain was statically cultured in the dark for 18 days in three 3 L Erlenmeyer flasks (total 3 L) containing 1 L of PDB medium (potato dextrose medium).

(2) Extraction and purification The culture solution obtained by culturing this strain was used to remove the cells using gauze. The obtained filtrate was extracted with methylene chloride, and the solvent was distilled off using a rotary evaporator to obtain a crude extract (22.9 mg). As shown in FIG. 1, this crude extract was subjected to column chromatography using silica gel as a carrier and chloroform-methanol (99: 1 → 0: 100) as a developing solvent, so that six fractions (Fr. A to Fr. G) were obtained. It was fractionated.

  Fr. B (5.7 mg) eluted with chloroform-methanol (98: 2) was separated into three components by column chromatography using silica gel as a carrier and developing solvent as hexane-ethyl acetate (4: 1 → 2: 1). Fractions were fractionated (Fr. BA to Fr. BC). Compound 1 (1.8 mg) was obtained from Fr.B-A eluted with hexane-ethyl acetate (2: 1).

  Fr. F (7.1 mg) eluted with chloroform-methanol (98: 2) was obtained by column chromatography using silica gel as a carrier and developing solvent as hexane-ethyl acetate (2: 1 → 1: 1). Fractionation into three fractions (Fr.FA to Fr.FC). And compound 2 (4.5 mg) was obtained from Fr.F-B eluted with hexane-ethyl acetate (2: 1).

(3) Structure determination Compound 1 and Compound 2 are determined by MS, IR, ¹ H-NMR, ¹³ C-NMR, DEPT, ¹ H- ¹ H COZY, ¹ H- ¹³ C HMQC, ¹ H- ¹³ C HMBC did. 2A shows the carbon number of Compound 1, and FIG. 2B shows the ¹ H- ¹ H COZY and ¹ H- ¹³ C HMBC correlation of Compound 1. FIG. 3 shows ¹ H-NMR and ¹³ C-NMR data of Compound 1 measured in CDCl ₃ . 4A shows the carbon number of Compound 2, and FIG. 4B shows the ¹ H- ¹ H COZY and ¹ H- ¹³ C HMBC correlation of Compound 2. FIG. 5 shows ¹ H-NMR and ¹³ C-NMR data of Compound 2 measured in CDCl ₃ .

Various properties and spectral data (excluding NMR data) of Compound 1 are as follows. Colorless oil, [α] _D ²⁶ +60.7 (c 0.09, CHCl ₃ ); IR ν _max (film): 3450, 2973, 1693, 1643, 1587, 1441, 1390, 1201, 1128, 1043, 991 cm ^-1 ( NaCl); HRESI-MS m / z found 293.13859 [M + H] ⁺
Various properties and spectrum data (excluding NMR data) of Compound 2 are as follows. Colorless oil, [α] _D ²⁶ +62.8 (c 0.23, CHCl ₃ ); IR ν _max (film): 3421, 2974, 1708, 1643, 1581, 1442, 1394, 1205, 1126, 1043, 993 cm ^-1 ( NaCl); HRESI-MS m / z found 311.14924 [M + H] ⁺

The molecular formula of Compound 1 was determined to be C ₁₆ H ₂₀ O ₅ from MS, ¹ H-NMR, ¹³ C-NMR and DEPT. ^{The 1} H- ¹ H COZY correlation suggested the existence of the partial structure shown by the thick line in FIG. The partial structure from C-11 to C-16 was determined from the HMBC correlation of H-16 → C-15 and C-14 and the chemical shift of C-15. In addition, HHM correlations of H-10 → C-2, C-3, and C-11 suggested that a quaternary C-2 had a methyl group, a ketone group, and a partial structure as described above. Here, the partial structure from C-9 to C-6 is determined from the HHMBC correlation of H-9 → C-8, C-7 and H-7 → C-6, and the COZY correlation of H-7 and H-8. did. The HMBC correlation of Me-17 → C-5 and the chemical shift of C-5 were greatly shifted to a low magnetic field, suggesting that the ring shown in the figure was formed.

  From the above, Compound 1 is 4-[(2E) -but-2-enoyl] -2-[(1E, 3E) -5-hydroxyhexa-1,3-dien-1-yl] -5-methoxy-2. -Methylfuran-3 (2H) -one [4-[(2E) -but-2-enoyl] -2-[(1E, 3E) -5-hydroxyhexa-1,3-dien-1-yl] -5 -methoxy-2-methylfuran-3 (2H) -one] (Penicilol A; named Penicilliol A).

The molecular formula of Compound 2 was determined as C ₁₆ H ₂₂ O ₆ from MS, ¹ H-NMR, ¹³ C-NMR and DEPT. Compound 2 has a molecular weight higher than that of Compound 1 by H ₂ O, and the NMR shift is very similar to that of Compound 1, suggesting that Compound 2 is hydroxylated after the double bond of Compound 1 is reduced. It was. Here, H-7 and H-8 were shifted in a higher magnetic field than Compound 1, and the chemical shift of C-8 suggested that a hydroxyl group was bonded to C-8.

  From the above, compound 2 was obtained from 4- (3-hydroxybutanoyl) -2-[(1E, 3E) -5-hydroxyhexa-1,3-dien-1-yl] -5-methoxy-2-methylfuran- 3 (2H) -one [4- (3-hydroxybutanoyl) -2-[(1E, 3E) -5-hydroxyhexa-1,3-dien-1-yl] -5-metoxy-2-methylfuran-3 (2H ) -One] (Penicilol B; named Penicilliol B).

Example 2 (Verification of DNA synthase inhibitory activity)
The activity of Compound 1 (Penicilliol A) and Compound 2 (Penicilliol B) obtained in the above Examples against DNA synthase group was measured by the following method.

  As DNA synthase, mammalian-derived DNA synthase α, β, γ, δ, ε, η, ι, κ, λ and terminal deoxynucleotidyl transferase (TdT), fish-derived DNA synthase δ, insect-derived DNA synthases α, δ and ε, plant-derived DNA synthases α and λ, and prokaryotic DNA synthases were tested. As the DNA synthase α, a sample extracted and purified from calf thymus by a conventional method was used. DNA synthase β is a gene derived from a rat, DNA synthase γ, η, κ, λ is a gene derived from a human, and DNA synthase ι is a gene derived from a mouse. A standard product incorporated into E. coli by the method and produced was used. For DNA synthetases δ and ε, preparations purified from human cancer cell extracts using an antibody column were used. TdT used a calf-derived gene incorporated into Escherichia coli by an ordinary genetic recombination method (Product Code: 2230A) purchased from Takara Bio Inc. and used. For fish DNA synthase δ and plant DNA synthase α, preparations extracted and purified from inflorescence tissues of cherry salmon testis and cauliflower, respectively, were used. Insect DNA synthases α, δ, and ε were prepared by using an antibody column purified from an extract of Drosophila early embryo. For plant DNA synthase λ, a standard product produced by incorporating a rice-derived gene into Escherichia coli by an ordinary genetic recombination method was used. Prokaryotic DNA synthase groups were purchased from Takara Bio Inc. (product code of E. coli DNA synthase I: 2130A, product code of T4 DNA synthase: 2040A, product code of Taq DNA synthase: RR001A).

For the measurement of the inhibitory action of compounds 1 and 2 on these DNA synthases, a general DNA synthase reaction system (edited by the Japanese Biochemical Society, Shinsei Chemistry Laboratory 2, Nucleic Acid IV, Tokyo Kagaku Dojin, pages 63-66) Was used. That is, a DNA synthesis reaction is carried out in a system containing [ ³ H] -TTP labeled with a radioisotope, and the radioactivity is used as an indicator of the amount of product (synthetic DNA chain).

The inhibition rate is (a) the amount of synthetic DNA in the control, and (b) the amount of synthetic DNA in the presence of the test substance.
b / a x 100 = DNA synthase residual activity (%)
As evaluated. The results of the DNA synthase residual activity of compounds 1 and 2 at 100 μM are shown in FIGS. 6 (A) to (C). The data shown in FIG. 6 is an average of three measurement values. For each DNA synthase, the upper graph (black) is the result of Compound 1, and the lower graph (gray) is the result of Compound 2. .

FIG. 6 (A) is a graph showing the inhibitory activity of compounds 1 and 2 on DNA-synthesizing enzymes derived from mammals. As shown in FIG. 6 (A), eukaryotic DNA synthases are classified into four families of A, B, X, and Y based on the homology of their gene sequences and the difference in activity function. Compound 1 and Compound 2 selectively inhibited DNA synthases η, ι and κ called Y family among mammalian DNA synthases. Among the DNA synthetases investigated, DNA synthetase ι was most strongly inhibited, so FIG. 7 shows the activity inhibition curves of compounds 1 and 2 against human DNA synthetase ι. As a result, the 50% inhibitory concentration (IC ₅₀ value) of Compound 1 against DNA synthetase ι was 19.8 μM, and the 50% inhibitory concentration of Compound 2 was 32.6 μM. From the results of FIG. 7, it was found that the inhibitory activity of Compound 1 was stronger than that of Compound 2.

  On the other hand, compounds 1 and 2 do not inhibit DNA synthase γ belonging to the A family, DNA synthase α, δ, ε belonging to the B family, and DNA synthases β, λ, TdT belonging to the X family. It was.

  FIG. 6 (B) is a graph showing the inhibitory activity of compounds 1 and 2 on DNA synthases derived from various organisms. From the results in FIG. 6 (B), it was found that compounds 1 and 2 do not inhibit any of the investigated DNA synthases.

  FIG. 6 (C) is a graph showing the inhibitory activity of compounds 1 and 2 on DNA metabolic enzymes other than DNA synthase. From the results of FIG. 6C, compounds 1 and 2 are calf primase, HIV-1 reverse transcriptase, human telomerase, T7 RNA synthase, mouse IMP dehydrogenase, human DNA topoisomerase I and II, T4. Neither polynucleotide kinase nor bovine DNAase I (DNase I) was found to be inhibited.

  From these results, it was found that compounds 1 and 2 selectively inhibit Y family DNA synthases η, ι and κ.

  DNA synthase has a high enzyme activity for cells and tissues with high cell division such as cancer cells. Therefore, compounds 1 and 2 are expected to have an action as an anticancer agent.

  In particular, since the Y family DNA synthase is responsible for DNA repair, cancer cells can be killed by administering these compounds to cancer tissues while causing damage to the DNA by radiation or chemical substances. . That is, it can be expected that the compound of the present invention will be used in combination with radiotherapy or chemotherapy for cancer.

Example 3 (Verification of cell growth inhibitory activity)
The cancer cell proliferation inhibitory effect of compound 1 (Penicilliol A) and compound 2 (Penicilliol B) was evaluated using the following method.

The cells used in this experiment are human colon cancer-derived HCT116 cells. As the medium, RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) supplemented with 10% (v / v) fetal calf serum was used. The culture was performed at 37 ° C. in a 5% CO ₂ incubator.

Various concentrations of compounds 1 and 2 were dissolved in the media indicated above. However, since these compounds are hardly soluble in water, they were once dissolved in DMSO (dimethyl sulfoxide) and dissolved in the above medium. In addition, the final concentration of DMSO in the medium in the medium is 1% or less in all test groups, and the possibility that DMSO is involved in the suppression of the growth of HCT116 cells used in this measurement example can be denied. State. The culture for this test was performed in a 96-well microplate. Each well was inoculated with 3.0 × 10 ⁵ cells, giving 5 wells for each test concentration. As a positive control, a medium containing 1% DMSO was used.

After the compound was added, the cells were cultured for 24 hours at 37 ° C. in a 5% CO ₂ incubator, and the cell viability of each test group was determined. The determination of survival rate is described in the literature ["Rapid Colorimetric Assay for Cellular Growth and Surviva1: Application to Proliferation and Cytotoxicity Assays", Tim Mosmann, J. Immunol. Methods, 65, 55 (1983)]. The assay method was used. That is, the tetrazolium salt MTT was added after 24 hours, and further cultured for 4 hours. The amount of formazan produced through reduction by viable cells was considered proportional to the viable cells and was quantified with an optical density (OD) of 570 nm.

Cell viability was calculated by the following formula.
Cell viability (%) = OD of test group [570 nm] / OD of control group [570 nm]
The obtained result is shown in FIG. The data shown in FIG. 8 is an average of 5 wells.

From the inhibition curve of FIG. 8, compounds 1 and 2 inhibited the proliferation of human colon cancer cell HCT116 in a concentration-dependent manner. The 50% growth inhibitory concentration (LD ₅₀ value) was 38.1 μM and 54.3 μM, respectively. From this result, it was found that Compound 1 has stronger human cancer cell growth inhibitory activity than Compound 2. Thus, it was shown that Compounds 1 and 2 that selectively inhibit the Y family DNA synthase can actually exert a cell growth inhibitory effect on human cancer cell lines and can be used as anticancer agents.

The process of isolation and purification of compounds 1 and 2 from a culture solution of Penicillium daleae K.M. Zalessky by silica gel column chromatography is shown. 2 is a chemical structure of Compound 1. ¹ H-NMR and ¹³ C-NMR data of Compound 1. 2 is a chemical structure of Compound 2. ¹ H-NMR and ¹³ C-NMR data of Compound 2. It is a graph which shows the residual activity with respect to the DNA synthetase and DNA metabolic system enzyme in each 100 micromol of the compound 1 and 2, Comprising: (A) is a graph regarding DNA synthase derived from a mammal. FIG. 2 is a graph showing the residual activity of compounds 1 and 2 with respect to DNA synthase and DNA metabolizing enzyme at 100 μM, (B) is a graph regarding DNA synthases derived from various organisms, and (C) is a graph showing various DNAs. It is a graph regarding a metabolic enzyme. 2 is a graph showing activity inhibition curves of compounds 1 and 2 against mouse DNA synthetase ι. It is a graph which shows the growth inhibition curve with respect to the human colon cancer cell (HCT116 cells) of the compounds 1 and 2.

Claims (5)

A compound represented by the general formula (1) or (2) or a pharmaceutically acceptable salt thereof.

(In the formula, R ¹ , R ² and R ³ are the same or different and represent H or a protecting group.)   A pharmaceutical composition comprising the compound represented by the general formula (1) or (2) according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.   A DNA synthase inhibitor comprising the compound represented by the general formula (1) or (2) according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.   An anticancer agent comprising the compound represented by the general formula (1) or (2) according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.   An edible composition comprising the compound represented by the general formula (1) or (2) according to claim 1 or a pharmaceutically acceptable salt thereof in a food.

Images