JP2013014546A - Dna synthase inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a compound having DNA synthase inhibitory activity, and to provide a pharmaceutical composition (a DNA synthase inhibitor and an anticancer drug) containing the compound as an active ingredient.SOLUTION: The compound is expressed by general formula (1). In the formula, R1 and R2 are different from each other, representing a hydrogen atom or a benzoyl group in which the second position is substituted with a methyl group, the fourth and the sixth positions are substituted with a specified substituent such as a hydroxy group and an alkoxy group; and R3 represents an alkenyl group optionally having a hydroxy group. The pharmaceutical composition containing the above compound as an active ingredient is also provided.

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.

  Eukaryotic DNA synthases (DNA polymerases) are α, β, γ, δ, ε, ζ, η, θ, ι, κ, λ, μ, σ and TdT (terminal deoxynucleotidyl transferase). ), 15 molecular species of Rev1 are 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 both ζ-κ type and Rev1 type have different functions depending on the type of repair, such as being responsible for repair.

  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).

Japanese Patent Laid-Open No. 11-106395 JP 2000-143516 A

Annual Review of Biochemistry, 2002, 71, 133-163

  An object of this invention is to provide the compound which has DNA synthetase inhibitory activity, and the pharmaceutical composition (DNA synthetase inhibitor and anticancer agent) which contains this compound as an active ingredient.

  As a result of intensive studies to solve the above problems, the present inventors have found that the compound represented by the general formula (1) has an excellent DNA synthase inhibitory activity. Furthermore, it has also been found that this compound specifically suppresses the growth of cancer cells. As a result of further research based on this knowledge, the present invention has been completed.

  That is, the present invention has the following configuration.

  Item 1. General formula (1):

[Wherein R ¹ and R ² are different from each other, hydrogen, or general formula (2):

(In the formula, R ⁴ and R ⁵ are the same or different and each represents hydrogen, a lower alkyl group, or an alkanoyl group.)
R ³ represents an alkenyl group which may have a hydroxyl group. ]
A DNA synthase inhibitor comprising a compound represented by the formula:

Item 2. R ³ is an alkenyl group C3~5 which may have a hydroxyl group, DNA synthesis inhibitor according to claim 1 ^{R 4} and ^{R 5} are hydrogen.

  Item 3. An anticancer agent comprising the compound represented by the general formula (1) according to Item 1 as an active ingredient.

  Item 4. General formula (3):

[Wherein R ⁶ and R ⁷ are different from each other, hydrogen or general formula (2):

(In the formula, R ⁴ and R ⁵ are the same or different and each represents hydrogen, a lower alkyl group, or an alkanoyl group.)
When R ⁶ is a group represented by the general formula (2) and R ⁷ is hydrogen, R ⁸ represents an alkenyl group, R ⁶ is hydrogen and R ⁷ Is a group represented by the general formula (2), R ⁸ represents an alkenyl group having a hydroxyl group. ]
A compound represented by

  Item 5. Item 5. A pharmaceutical composition comprising the compound represented by the general formula (3) according to Item 4 as an active ingredient.

  Item 6. A food composition comprising the compound represented by the general formula (3) according to Item 4.

  Item 7. A method for producing a compound represented by the general formula (1), comprising a step of purifying a culture of penicillium pinofhilum Hedgcock.

  Item 8. General formula (4):

(In the formula, R ⁴ and R ⁵ are the same as above.)
A process for producing a compound represented by the general formula (5):

A compound represented by formula (6):

(In the formula, R ⁹ and R ¹⁰ are the same or different and each represents a protecting group.)
The manufacturing method characterized by making the compound represented by these react.

  ADVANTAGE OF THE INVENTION According to this invention, the pharmaceutical composition (DNA synthetase inhibitor and anticancer agent) which contains a compound which has DNA synthetase inhibitory activity, and this compound as an active ingredient can be provided.

A structural formula showing the structures and carbon numbers of compounds 1a and 1b and a structural formula showing the structure of compound 1c are shown. (A) shows the HMBC correlation of Compound 1a and HMQC correlation of Compound 1b, and (B) shows the ¹ H- ¹ H NOESY correlation of Compound 1a and Compound 1b. ¹ H-NMR and ¹³ C-NMR data of compound 1a and 1b measured in CDCl ₃ are shown. 1 shows a synthetic scheme for compound 1c. The evaluation result of the inhibitory activity with respect to a DNA synthetase of compound 1a, 1b, or 1c is shown. The determination result of the inhibition mode of a DNA synthetase of compound 1a is shown. The evaluation result of the influence of the compound 1a, 1b, or 1c on the proliferation of human cancer cells and human normal cells is shown.

  Hereinafter, the present invention will be described in detail.

1. Compound The compound of the present invention has the general formula (1):

[Wherein R ¹ and R ² are different from each other, hydrogen, or general formula (2):

(In the formula, R ⁴ and R ⁵ are the same or different and each represents hydrogen, a lower alkyl group, or an alkanoyl group.)
R ³ represents an alkenyl group which may have a hydroxyl group. ]
It is a compound represented by these.

R ¹ and R ² in the general formula (1) are different and represent hydrogen or a group represented by the general formula (2). That is, when R ¹ is a group represented by the general formula (2), R ² is hydrogen, and when R ¹ is hydrogen, R ² is a group represented by the general formula (2). is there. R ¹ is preferably a group represented by the general formula (2), and R ² is hydrogen.

R ³ in the general formula (1) represents an “alkenyl group optionally having a hydroxyl group”.

  Examples of the “alkenyl group” in the “alkenyl group optionally having a hydroxyl group” include 2 to 8 carbon atoms (hereinafter referred to as “C”), preferably C3 to 5, more preferably C3 to C4. An alkenyl group is mentioned. The “alkenyl group” may be either linear or branched, but is preferably a linear alkenyl group. Specific examples include vinyl, 1-propenyl, 2-propenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl and 2-pentenyl. , 3-pentenyl, 4-pentenyl, 1,3-butadienyl, 1,3-pentadienyl, 2-pentene-4-ynyl, 2-hexenyl, 1-hexenyl, 5-hexenyl, 3-hexenyl, 4-hexenyl 3,3-dimethyl-1-propenyl, 2-ethyl-1-propenyl, 1,3,5-hexatrienyl, 1,3-hexadienyl, 1,4-hexadienyl and the like.

  Examples of the “alkenyl group having a hydroxyl group” include a group having a hydroxyl group of 1 to 4, preferably 1 to 3, more preferably 1 to 2, on the carbon of the “alkenyl group”. Specific examples include 3-hydroxy-1-propenyl, 4-hydroxy-1-butenyl, 4-hydroxy-2-butenyl and the like.

R ³ in the general formula (1) is preferably an alkenyl group.

R ⁴ and R ⁵ in the general formula (2) are the same or different and each represents hydrogen, a lower alkyl group, or an alkanoyl group.

  Examples of the lower alkyl group include C1-6, preferably C1-4, more preferably C1-2 alkyl groups. The lower alkyl group may be either linear or branched, and preferably a linear lower alkyl group. Specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and the like.

  Examples of the alkanoyl group include C1-6, preferably C1-4, more preferably C1-2 alkanoyl groups. The alkanoyl group may be either linear or branched, but preferably a linear alkanoyl group. Specific examples include formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, tert-butylcarbonyl, hexanoyl and the like.

R ⁴ and R ⁵ in the general formula (2) are preferably the same or different and include hydrogen or a lower alkyl group, more preferably hydrogen.

  The compound represented by the general formula (1) includes stereoisomers and optical isomers, and these are not particularly limited. Among the compounds represented by the general formula (1), as a compound having a preferable steric structure, the following general formula (1 ′):

[Wherein, R ¹ and R ² are different and represent hydrogen or a group represented by the general formula (2), and R ³ represents an alkenyl group which may have a hydroxyl group. The formula indicates relative placement. ]
The compound represented by these is mentioned.

As a particularly preferred specific example of the compound represented by the general formula (1),
Formula (1a):

A compound represented by
Formula (1b):

Or a compound represented by the formula (1c):

The compound represented by these is mentioned. Formulas (1a), (1b), and (1c) indicate relative arrangement. Among these, from the viewpoint of higher DNA inhibitory activity and a stronger action of suppressing the growth of cancer cells, the compound represented by formula (1a) or formula (1c) is preferred, more preferably formula The compound represented by (1a) is mentioned.

  As another preferred embodiment of the compound represented by the general formula (1), the general formula (3):

[Wherein R ⁶ and R ⁷ are different and each represents hydrogen or a group represented by the general formula (2), R ⁶ represents a group represented by the general formula (2), and R ⁷ represents hydrogen. In some cases, R ⁸ represents an alkenyl group, and when R ⁶ is hydrogen and R ⁷ is a group represented by the general formula (2), R ⁸ represents an alkenyl group having a hydroxyl group. ]
The compound represented by these is mentioned.

R ⁶ in the general formula (3) represents hydrogen or a group represented by the general formula (2).

R ⁷ in the general formula (3) represents hydrogen or a group represented by the general formula (2).

However, when R ⁶ is a group represented by the general formula (2), R ⁷ represents hydrogen, and when R ⁶ is hydrogen, R ⁷ represents a group represented by the general formula (2). Show.

R ⁸ in the general formula (3) represents an alkenyl group or an alkenyl group having a hydroxyl group.

However, when R ⁶ is a group represented by the general formula (2) and R ⁷ is hydrogen, R ⁸ represents an alkenyl group, R ⁶ is hydrogen and R ⁷ is the general formula (2). R ⁸ represents an alkenyl group having a hydroxyl group.

The alkenyl group in R ⁸ is the same as the alkenyl group in R ³ , and the alkenyl group having a hydroxyl group in R ⁸ is the same as the alkenyl group having a hydroxyl group in R ³ .

  Particularly preferred specific examples of the compound represented by the general formula (3) include compounds represented by the formula (1a) or the formula (1b). Among these, the compound represented by the formula (1a) is preferable from the viewpoint of higher DNA inhibitory activity and a stronger action of suppressing cancer cell growth.

2. Compound production method-1
The production method-1 of the present invention is a method for producing a compound represented by the general formula (1), and includes a step of purifying a culture of penicillium pinofhilum Hedgcock.

  Among the compounds represented by the general formula (1), the compounds represented by the formula (1a), the formula (1b) and the formula (1c) can be obtained by purifying a culture of Penicillium pinofhilum Hedgcock. . After purification, if necessary, formula (2a) in formula (1a), (1b), or (1c):

The compound represented by General formula (1) can be manufactured by passing through the process which protects the hydroxyl group in group represented by a lower alkyl group or an alkanoyl group.

Penicillium pinofhilumHedgcock is a kind of known blue mold and can be obtained from various microorganism deposit institutions. For example, it can be obtained from NBRC (NITE Biological Resource Center) as mold having IFO number 33285 ^T , 100533 ^T , 6345 or 106907.

  As a method for obtaining a culture, Penicillium pinofhilum Hedgcock is allowed to stand still in a known medium such as PDB medium (potato dextrose liquid medium), a mixed medium of corn meal and rose bengal, a malt extract medium, or a meat extract medium. A method of culturing and extracting a filtrate obtained by removing cells from the culture solution with a solvent may be mentioned. 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 Ethers such as ether, isopropyl ether, tetrahydrofuran and dioxane, polar organic solvents such as halogenated hydrocarbons such as methylene chloride and chloroform; non-polar organic solvents such as hexane, cyclohexane and petroleum ether, and the like. Can 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, alcohols such as methanol and ethanol, 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.

  The method for purifying the culture is not particularly limited, but preferably includes a method for purification using DNA synthase inhibitory activity as an indicator. The purification method is not particularly limited, and a known purification method can be adopted, and preferably, chromatographic purification is used.

  Examples of the chromatography include alumina column chromatography, silica gel chromatography, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, high performance liquid chromatography and the like, and silica gel chromatography is preferably exemplified.

  Examples of the mobile phase for chromatography include a combination of one or more organic solvents such as methanol, ethanol, propanol, butanol, chloroform, ethyl acetate, toluene, hexane, and benzene.

  The purified product obtained in the step of purifying the culture may be further purified. The purification conditions in this case may be the same conditions as in the step of purifying the culture, or may be the conditions in which the type of carrier or mobile phase is changed.

The group for protecting the hydroxyl group in the group represented by the formula (2a) is a lower alkyl group or an alkanoyl group. The lower alkyl group is the same as the lower alkyl group represented by R ⁴ and R ⁵ in the general formula (2). The alkanoyl group is the same as the alkanoyl group of R ⁴ and R ⁵ in the general formula (2). The method for protecting is not particularly limited, and a known method can be employed.

  By the production method-1 of the present invention, the compound included in the compound represented by the general formula (1), for example, the compound represented by the formula (1a), the compound represented by the formula (1b), the formula ( A compound represented by 1c), a compound represented by the general formula (3), or a compound represented by the following general formula (4) can also be produced.

  In addition, the production and identification of the compound from the culture can be performed specifically as described in Example 1.

3. Compound production method-2
The production method-2 of the present invention has the general formula (4):

(In the formula, R ⁴ and R ⁵ are the same as above.)
A process for producing a compound represented by the general formula (5):

A compound represented by formula (6):

(Wherein R ⁹ and R ¹⁰ are the same or different and each represents a protecting group, and R ¹¹ represents chlorine, iodine, bromine, or fluorine)
A production method characterized by reacting a compound represented by the formula:

  The compound represented by the general formula (5) can be obtained, for example, by a method for synthesizing the precursor 13 in Example 2 described later.

R ⁹ and R ¹⁰ in the general formula (6) are the same or different and represent a protecting group. The protective group is not particularly limited, and is a known protective group such as an acyl group (acetyl, propanoyl, benzoyl group, etc.), a silyl group (trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl group, etc.), Alkyl groups (such as methyl, ethyl, propyl, and butyl groups), alkenyl groups (such as allyl and crotyl groups), aralkyl groups (such as benzyl and phenethyl groups), alkoxymethyl groups (such as methoxymethyl groups), sulfonyl groups (such as methanesulfonyl, Benzenesulfonyl group and the like). R ⁹ is preferably a benzyl group, and R ¹⁰ is preferably an alkyl group, more preferably a methyl group.

R ¹¹ in the general formula (6) represents chlorine, iodine, bromine, or fluorine. R ¹¹ is preferably chlorine, bromine, or iodine, more preferably chlorine or bromine, and particularly preferably chlorine.

  The reaction of the compound represented by the general formula (5) and the compound represented by the general formula (6) is not particularly limited, and a known method can be adopted. For example, there is a method in which the compound represented by the general formula (5) and the compound represented by the general formula (6) are reacted in the presence of a base, preferably in the presence of an organic base, more preferably in the presence of a tertiary amine. Can be mentioned.

  Examples of the base include trimethylamine, triethylamine, tri-n-propylamine, triisopropylamine, tributylamine, N-methyl-diethylamine, N-ethyl-dimethylamine, N-ethyl-diamilamine, N, N-diisopropylethylamine, Aliphatic amines such as N, N-dimethyl-cyclohexylamine and N, N-diethyl-cyclohexylamine; aromatic amines such as N, N-dimethylaniline and N, N-diethylaniline; pyridine, picoline, N, N- Heterocyclic amines such as dimethylaminopyridine and 4-pyrrolidinopyridine; 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5-diazabicyclo [4.3.0] non-5-ene And alicyclic amines such as quinuclidine and 1,4-diazabicyclo [2.2.2] octane Preferably, N, N-diisopropylethylamine or N, N-dimethylaminopyridine is used. These may be used alone or in combination of two or more.

  Although it does not specifically limit as a solvent, For example, an organic solvent can be used. Examples of the organic solvent include heptane, hexane, octane, dichloromethane, chloroform, ethylene dichloride, acetone, acetonitrile, cyclohexanone, cyclopentanone, 2-heptanone, γ-butyrolactone, methyl ethyl ketone, methyl isobutyl ketone, 2-methoxyethyl acetate, Ethylene glycol monoethyl ether acetate, propylene glycol monomethyl ether acetate, benzene, toluene, xylene, ethyl acetate, butyl acetate, methyl lactate, ethyl lactate, methyl methoxypropionate, ethyl ethoxypropionate, methyl pyruvate, ethyl pyruvate, pyruvin Propyl acid, N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, diethyl ether Di t- butyl ether, cyclopentyl methyl ether, tetrahydrofuran, 1,4-dioxane and the like, with preference given to dichloromethane. These may be used alone or in combination of two or more.

Known conditions can be adopted for both the reaction temperature and the reaction time. Moreover, a well-known method (separation, distillation, chromatography, recrystallization etc.) can also be employ | adopted for the means to isolate and refine | purify the compound represented by General formula (4) from the reaction liquid after completion | finish of reaction.

As described above, after reacting the compound represented by the general formula (5) and the compound represented by the general formula (6), the protecting groups represented by R ⁹ and R ¹⁰ are publicly known as necessary. You may deprotect by the method.

Furthermore, after the deprotection of the protecting group represented by R ⁹ and R ¹⁰ , the hydroxyl group generated after the deprotection may be protected with a lower alkyl group or an alkanoyl group as necessary. The lower alkyl group is the same as the lower alkyl group represented by R ⁴ and R ⁵ in the general formula (2). The alkanoyl group is the same as the alkanoyl group of R ⁴ and R ⁵ in the general formula (2). The method for protecting is not particularly limited, and a known method can be employed.

  By the production method-2 of the present invention, a compound included in the compound represented by the general formula (4), for example, a compound represented by the formula (1c) can also be produced.

4). Use of the compound The compound represented by the general formula (1) of the present invention has a selective inhibitory action on DNA synthase, so that it can be used for pharmaceuticals, specifically, a DNA synthase inhibitor and the like. It is. In addition, this compound has the property of selectively inhibiting mammalian DNA synthase, and is non-competitive inhibition against template DNA, and is competitive inhibition against deoxyribonucleotide triphosphate (dNTP). Therefore, it can also be used as a DNA synthase inhibitor for applications specialized in these properties. Furthermore, the compound represented by the general formula (1) does not affect the proliferation of human normal cells (for example, normal human dermal fibroblasts, normal human umbilical vein endothelial cells, etc.), and human cancer cells (human lung cancer) Cells, human leukemic B cells, human colon cancer cells, human cervical cancer cells, human gastric cancer cells and the like). Therefore, a compound represented by the general formula (1) or a DNA synthase inhibitor containing the compound represented by the general formula (1) as an active ingredient is a preventive or therapeutic agent for cancer, tumor, etc., specifically Can also be used as an anticancer agent. The same structure-activity relationship is observed between the DNA synthase inhibitory activity and the cancer cell growth inhibitory activity.

  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).

  A pharmaceutical 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, tablets, capsules, granules, fine granules, powders and other oral preparations, injections, and suppositories Parenteral preparations such as agents, and the like, and preferably 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 a 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 be used as an edible composition (for example, health food) having an anticancer effect or an anticarcinogenic effect by being added to and blended with food and drink.

  That is, the compound of the present invention is used as it is in a liquid, gel or solid food such as 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.

  Furthermore, the compound of the present invention can also be used as a biochemical reagent, specifically, a DNA synthase inhibitor, or a cancer 7 cell-specific growth inhibitor. That is, the compound of the present invention can be used as a biochemical reagent alone or together with known components such as buffers, salts and the like to be contained in a biochemical reagent.

  Further, the compound of the present invention can also be used as a lead compound in the above-mentioned drug development process. By examining the inhibitory activity against DNA synthase using the compound of the present invention as a lead, efficient screening of candidate compounds for anticancer agents can be expected. 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 examples, and a suitable method may be selected from known test methods.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Example 1. Purification of compounds
(1) Isolation of strains Seaweeds living on the coast of Sado Island were treated with 5% acetic acid and then suspended in sterilized water. The suspension was added dropwise to a PDA medium plate (potato dextrose agar plate), and the plate was cultured at 27 ° C. After culturing, the strain was isolated from the plate by a known method. The isolated strain was identified as Penicillium pinofhilum Hedgcock (IFO No. 33285 ^T , 100533 ^T , 6345, or 106907) as a result of strain identification (Techno Suruga Lab Co., Ltd.).

(2) Purification of the compound The isolated strain was statically cultured in two 3 L Erlenmeyer flasks containing 2 L × 2 PDB medium (potato dextrose liquid medium) in the dark for 14 days. From the culture solution in which this strain was cultured, the cells were removed using gauze. The obtained filtrate was extracted with methylene chloride (CH ₂ Cl ₂ ), and the solvent was distilled off using a rotary evaporator to obtain a crude extract (160 mg). This crude extract was fractionated into four fractions (Fr. 1 to Fr. 4) by column chromatography using silica gel as a carrier and chloroform-methanol (100: 0 → 0: 100) as a developing solvent.

  Fr. 2 eluted with chloroform-methanol (80: 1 → 60: 1) was obtained by column chromatography using silica gel as a carrier and toluene-ethyl acetate (12: 1 to 10: 1) as a developing solvent. Fractions were fractionated into two fractions (Fr.2-1 to Fr.2-3). Then, from Fr. 2-1 eluted with toluene-ethyl acetate (12: 1), compound 1a as a yellow powder, and from Fr. 2-2 eluted with toluene ethyl acetate (10: 1), yellow powder As a result, Compound 1c was obtained.

  Fr. 4 eluted with chloroform-methanol (40: 1 → 20: 1) was obtained by column chromatography using silica gel as a carrier and toluene-ethyl acetate (4: 1 to 3: 1) as a developing solvent. Fractions were fractionated into two fractions (Fr.4-1 to Fr.4-3). Compound 1b was obtained as a yellow solid from Fr. 4-1, which was eluted with toluene-ethyl acetate (4: 1 to 3: 1).

(3) Structure determination of purified compound The compounds 1a, 1b, and 1c were determined by HR-ESIMS, IR, ¹ H-NMR, ¹³ C-NMR, ¹ H- ¹ H NOESY, HMBC, and HMQC. FIG. 1 shows the structures of compounds 1a and 1b, the structural formulas indicating the carbon numbers, and the structural formula of compound 1c. FIG. 2A shows the HMBC correlation of compound 1a (“1a” in FIG. 2A) and the HMQC correlation of compound 1b (“1b” in FIG. 2A). Shows the ¹ H- ¹ H NOESY correlation of compound 1a ("1a" in FIG. 2B) and the ¹ H- ¹ H NOESY correlation of compound 1b ("1b" in FIG. 2B). . FIG. 3 shows ¹ H-NMR and ¹³ C-NMR data of Compound 1a measured in CDCl ₃ and Compound 1b measured in MeOD.

Various properties and spectrum data (excluding NMR data) of Compound 1a are as follows. Yellow solid; mp 213-215 ° C (decomp.); [Α] ²³ _D -114.5 (c 0.065, MeOH); IR (neat) ν _max 3373, 2919, 2828, 1728, 1648, 1585 cm ^-1 ; HR ESIMS m / z 387.1430 (M + H ⁺ ) (calcd for 387.1438).

Various properties and spectrum data (excluding NMR data) of Compound 1b are as follows. Yellow solid; mp 212-214 ° C (decomp.); [Α] ²³ _D +92.4 (c 0.62, MeOH); IR (neat) ν _max 3376, 2923, 2853, 1725, 1651, 1585 cm ^-1 ; HR ESIMS m / z 403.1390 (M + H ⁺ ) (calcd for 403.1387).

  As a result of analysis, Compound 1c was found to be Sch 725680, which is a known compound. “1c” in FIG. 1 shows the structural formula.

Compound 1a was shown by HR-ESIMS to have the molecular formula C ₂₂ H ₂₂ O ₇ . The IR spectrum suggested the presence of a hydroxyl group (3373 cm ⁻¹ ), a conjugated ester group (1728 cm ⁻¹ ), and a conjugated ketone group (1648 cm ⁻¹ ). From the ¹ H and ¹³ C-NMR spectra shown in FIG. 3, it was suggested that compound 1a has an azaphyrone skeleton and 2,4-dihydroxy-6-methylbenzoic acid as partial structures. The azaphyllon skeleton was determined mainly by the ¹ H- ¹³ C long range correlation measured in the HMBC experiment. From the HBC correlation from H-3 'to C-1' and C-2 ', the methyl group showing a double double line in ¹ H-NMR is adjacent to the double bond consisting of two methine carbons. Determined to be. Based on the HMBC correlation from H-4 to C-1 ′ and C-5, and the HMBC correlation from H-5 to C-4, this double bond is further conjugated to two double bonds and C -6 'leads to ketones. Judging from the HMBC correlation from H-5 to C-7 (δ18.4), next to the ketone (C-6) was a quaternary carbon atom (C-7) with an oxygen functional group. Based on the HMBC correlation from 7-CH ₃ to C-6, and C-7, C-8, this is a methine group (C-8) with a methyl group (7-CH ₃ ) and an oxygen functional group. It was replaced. Aliphatic methine (C-8a) showing long range correlation from H-1, H-5, and H-8 was determined to be adjacent to C-1, and C-4a, C-8 It was. From the above, the cyclohexenone ring was determined. The HMBC correlation from proton signals (H-1α and H-1β) of methylene with oxygen functional group to olefin carbon (C-3) suggested the presence of dihydropyran moiety. From the above, the 6,7,8,8a-tetrahydro-1H-isochromene ring skeleton was determined. HMBC correlation from H-4 "to C-2" and C-3 ", C-5" and C-6 ", from H-6" to C-2 "and C-4" and 7 "-Me The remaining 7 carbons were determined to be 2,4-dihydro-6-methylbenzoyl groups from the HMBC correlation and the HMBC correlation from 7 ″ -Me to C-2 ″ and C-6 ″, C-7 ″. Based on the correlation between H-8 and 8-OH, the position of the benzoyl group was determined at the 7-O position, and the relative configuration of compound 1a was determined from the ¹ H- ¹ H binding constant and the NOESY correlation. The relative configuration of -8 and H-8a was determined to be anti from the binding constant (J _8-8a = 10.0 Hz). The relative stereochemistry of H-8a and 7-O-benzoyl group was 7 "- NOE correlation between CH ₃ and H-8a, NOE correlation between 7-CH ₃ and 8-H, and NOE correlation between 7-CH ₃ and 8-OH, determined to be syn did. The absolute stereochemistry of compound 1a was determined using CD exciton chirality method. Since the CD spectrum of Compound 1a has a negative Cotton effect of 325 nm (Δε = −28.2) and 297 nm (Δε = + 9.2), the absolute stereochemistry of C-7 was determined to be S. From the above, compound 1a was converted to (7S, 8S, 8aS) -8-hydroxy-7-methyl-6-oxo-3 [(1E) -prop-1-en-1-yl] -6,7,8,8a -tetrahydro-1H-isochromen-7-yl 2,4-dihydroxy-6-methylbenzoate. “1a” in FIG. 1 shows the structural formula.

Compound 1b was shown by HR-ESIMS to have the molecular formula C ₂₂ H ₂₂ O ₈ . From the ¹ H and ¹³ C-NMR spectra shown in FIG. 3, it was suggested that Compound 1b has a similar structure to Compound 1a and Compound 1c, and is different only in the side chain C-3 ′. . In the HMQC spectrum, in compound 1b, the ¹ H signal of H-3 ′ (δ1.84) of compound 1c becomes H-3 ′ (δ4.22) that correlates with C-3 ′ (δ61.1). It was replaced. This is consistent with the results of HR-ESIMS and suggests the presence of C-3 ′ alcohol. The relative configuration of the compound 1b was determined from ¹ H- ¹ H coupling constants and NOESY correlation. The relative stereochemistry of H-8 and H-8a was determined to be anti from the binding constant (J _8-8a = 10.0 Hz). The relative stereochemistry of 7-OH and H-8 was determined to be anti from the NOE correlation between 7 ″ -CH ₃ and H-8. From the above, compound 1b was determined to be (7R *, 8R *, 8aR *)-7-hydroxy-3-[(1E) -3-hydroxyprop-1-en-1-yl] -7-methyl-6-oxo-6,7,8,8a-tetrahydro-1H-isochromen- It was determined to be 8-yl 2,4-dihydroxy-6-methylbenzoate, and the structural formula is shown in FIG.

  The absolute stereochemistry of compound 1c was determined using the CD exciton chirality method. Since the CD spectrum of Compound 1c has a positive cotton effect of 362 nm (Δε = 6.7) and 307 nm (Δε = −5.3), the absolute stereochemistry of C-8 was determined to be S.

Example 2 Compound 1c Organic Synthesis Compound 1c was synthesized from compound 1, which is a known compound, according to the synthesis plan shown in FIG. The structural formula shown in FIG. 4 shows a relative arrangement. Details of the synthesis method are as follows.

  Synthesis of Precursor 2: A tetrahydrofuran solution of 5.0 g of a known imide (Compound 1) was added to a THF solution of lithium diisopropylamide (LDA, 1.6 equivalents) and stirred at −78 ° C. for 30 minutes. A hexane solution (1 M, 56.4 mL) of chlorotriisopropyl orthotitanate was added, and the mixture was stirred at −40 ° C. for 3 hours. After cooling again to −78 ° C., a tetrahydrofuran solution of 4.1 g of a known aldehyde (Precursor 1) was added and stirred overnight at −45 ° C. A saturated aqueous ammonium chloride solution was added to the reaction solution at room temperature, and the organic layer was separated. The aqueous layer was extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain 5.71 g.

Various properties and spectral data of the precursor 2 are as follows. Clear oil; [α] _D ²² +54.8 (c 0.085, CHCl ₃ ); ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.29-7.39 (m, 8H), 7.18 (d, J = 8.7 Hz, 2H), 5.21 (s, 1H), 4.67 (dd, J = 8.9, 4.6 Hz, 1H), 4.39 (s, 2H), 3.98 (ddd, J = 11.9, 4.6, 0.8 Hz, 1H), 3.94 (dd, J = 11.7, 9.1 Hz, 1H), 3.92 (dd, J-= 11.9, 9.1 Hz, 1H), 3.79 (ddd, J = 11.7, 4.6, 0.8 Hz, 1H), 2.62 (d, J = 8.9 Hz, 1H, OH), 1.92 (ddddd, J = 9.1, 9.1, 4.6, 4.6, 4.6 Hz, 1H), 1.82 (s, 3H), 1.59 (s, 3H), 1.41 (s, 3H), 1.36 (s, 3H) , 0.99 (s, 3H); ¹³ H NMR (150 MHz, CDCl ₃ ) δ173.3, 151.9, 137.9, 136.2, 128.9, 128.8, 128.4, 127.7, 127.6, 97.7, 86.4, 82.4, 72.4, 69.1, 67.0, 62.9, 60.9, 37.4, 28.8, 26.7, 23.9, 21.4, 16.7; IR (ATR) ν _max 3510, 2990, 1781, 1697, 1456, 1369, 831 HR ESIMS m / z 520.2326 (M + Na ⁺ ) (calcd for 520.2305).

  Synthesis of Precursor 3: Zinc nitrate hexahydrate (13.7 g) was added to an acetonitrile solution (100 ml) of Precursor 2 (5.71 g), and the mixture was stirred at 60 ° C. for 30 minutes. After cooling to 0 ° C., saturated aqueous sodium hydrogen carbonate was added to the reaction solution, and the organic layer was separated. The aqueous layer was extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain 2.55 g.

  Synthesis of Precursors 4 and 5: Dimethoxypropane (610 μl) and camphorsulfonic acid (29 mg) were sequentially added to a dichloromethane solution of Precursor 3 (309 mg) and stirred overnight. Saturated sodium hydrogen carbonate solution was added to the reaction liquid, and the organic layer was fractionated. The aqueous layer was extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain Precursor 4 (266.3 mg) and Precursor 5 (68.1 mg).

Various properties and spectral data of the precursor 4 are as follows. Clear oil; [α] _D ²³ -6.14 (c 0.12, CHCl ₃ ); ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.30-7.35 (m, 5H), 4.70 (d, J = 11.7 Hz, 1H), 4.50 (d, J = 11.7 Hz, 1H), 4.30 (dd, J = 11.5, 5.5 Hz, 1H), 3.89 (dd, J = 11.5, 5.5 Hz, 1H), 3.88 (dd, J = 11.5, 11.5 Hz , 1H), 3.77 (d, J = 11.5 Hz, 1H), 3.65 (dd, J = 11.5, 11.5 Hz, 1H), 2.99 (ddddd, J = 11.5, 11.5, 11.5, 5.5, 5.5 Hz, 1H), 1.58 (s, 3H), 1.49 (s, 3H), 1.49 (s, 3H); ¹³ H NMR (150 MHz, CDCl ₃ ) δ 169.1, 138.5, 128.2, 127.4, 127.2, 99.3, 77.1, 74.4, 67.9, 66.4, 60.8, 29.9, 29.5, 18.9, 16.1; IR (ATR) ν _max 3483, 2992, 2947, 1740, 1455, 1382, 1143, 876; HR ESIMS m / z 329.1347 (M + Na ⁺ ) (calcd for 329.1359 ).

Various properties and spectral data of the precursor 5 are as follows. Clear oil; [α] _D ²⁵ +34.9 (c 0.24, CHCl ₃ ); ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.40 (d, J = 7.6 Hz, 1H), 7.32 (dd, J = 7.6, 7.6 Hz, 1H), 7.25 (d, J = 7.6 Hz), 4.87 (d, J = 11.0, 11.0 Hz, 1H), 4.83 (d, J = 11.7 Hz, 1H), 4.80 (d, J = 11.7 Hz, 1H), 4.36 (dd, J = 11.0, 7.0 Hz, 1H), 4.35 (d, J = 2.4 Hz, 1H), 4.10 (dd, J = 12.3, 2.4 Hz, 1H), 3.62 (dd, J = 12.3 , 2.4 Hz, 1H), 2.27 (ddddd, J = 11.0, 7.0, 2.4, 2.4, 2.4 Hz, 1H), 1.56 (s, 3H), 1.40 (s, 3H), 1.39 (s, 3H); ¹³ C NMR (150 MHz, CDCl ₃ ) 139.5, 171.7, 128.2, 127.3, 127.2, 99.6, 76.9, 74.9, 74.2, 69.5, 67.2, 60.1, 32.1, 29.2, 24.1, 18.7; IR (ATR) ν _max 3503, 2991, 2941, 1746, 1455, 1382, 1142, 851; HR ESIMS m / z 329.1351 (M + Na ⁺ ) (calcd for 329.1359).

  Synthesis of Precursor 6 (From Precursor 4): A tetrahydrofuran solution (10 ml) of Precursor 4 (343 mg) was cooled to 0 ° C., and N, O-dimethylhydroxyamine hydrochloride (328 mg) and isopropylmagnesium bromide in tetrahydrofuran Solution (0.75 M, 9 ml) was added in order. After stirring at 0 ° C. for 30 minutes, a saturated aqueous ammonium chloride solution was added to the reaction solution, and the organic layer was separated. The aqueous layer was extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried and concentrated under reduced pressure. The obtained alcohol was directly used in the next reaction. Molecular sieves 4A (750 mg) and pyridinium chlorochromate (362 mg) were sequentially added to the obtained alcohol solution in dichloromethane at room temperature, and the mixture was stirred overnight. The reaction solution was filtered through a Florisil layer, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography to obtain Precursor 6 (266 mg).

Various properties and spectral data of the precursor 6 are as follows. Clear oil; [α] _D ²⁴ +22.2 (c 2.9, CHCl ₃ ); ¹ H NMR (600 MHz, CDCl ₃ ) δ 9.7 (d, J = 3.4 Hz, 1H), 7.22-7.35 (m, 5H), 5.02 (d, J = 9.5 Hz, 1H), 4.69 (d, J = 11.1 Hz, 1H), 4.31 (d, J = 11.1 Hz, 1H), 4.08 (dd, J = 11.7, 9.5 Hz, 1H), 3.88 (dd, J = 11.7, 5.4 Hz, 1H), 3.55 (s, 3H), 3.25 (s, 3H), 2.99 (dddd, J = 9.5, 9.5, 5.4, 3.4 Hz, 1H), 1.66 (s, 3H), 1.44 (s, 3H), 1.36 (s, 3H); ¹³ H NMR (150 MHz, CDCl ₃ ) δ 200.1, 171.3, 137.4, 128.4, 127.5, 127.2, 99.2, 83.4, 71.9, 66.4, 60.8, 58.9, 49.5, 34.9, 29.3, 19.9, 16.6; IR (ATR) ν _max 2993, 2940, 2877, 2738, 1719, 1657, 1455, 1381, 1103, 858; HR ESIMS m / z 388.1715 (M + Na ⁺ ) (calcd for 329.1730).

  Synthesis of Precursor 7: A tetrahydrofuran solution (20 ml) of Precursor 5 (614 mg) was cooled to 0 ° C., and a tetrahydrofuran solution (0.75 M, 16 ml) of N, O-dimethylhydroxyamine hydrochloride (590 mg) and isopropylmagnesium bromide. ) In order. After stirring at 0 ° C. for 30 minutes, a saturated aqueous ammonium chloride solution was added to the reaction solution, and the organic layer was separated. The aqueous layer was extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried and concentrated under reduced pressure. The obtained alcohol was directly used in the next reaction. Molecular sieves 4A (1.5 g) and pyridinium chlorochromate (862 mg) were successively added at room temperature to the obtained alcohol solution in dichloromethane (40 ml), followed by stirring for 2 hours. The reaction solution was filtered through a Florisil layer, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography to obtain Precursor 7 (498 mg).

Various properties and spectral data of the precursor 7 are as follows. Clear oil; [α] _D ²⁴ -75.4 (c 0.85, CHCl ₃ ); ¹ H NMR (600 MHz, CDCl ₃ ) δ 10.9 (d, J = 4.9 Hz, 1H), 7.25-7.36 (m, 5H), 4.88 (d, J = 11.0 Hz, 1H), 4.68 (d, J = 2.6 Hz, 1H), 4.61 (d, J = 11.0 Hz, 1H), 4.20 (dd, J = 12.1, 2.6 Hz, 1H), 4.04 (dd, J = 12.1, 2.6 Hz, 1H), 3.50 (s, 3H), 3.20 (s, 3H), 2.54 (dddd, J = 4.9, 2.6, 2.6, 2.6 Hz, 1H), 1.70 (s, 3H), 1.53 (s, 3H), 1.50 (s, 3H); ¹³ H NMR (150 MHz, CDCl ₃ ) δ 204.1, 172.2, 137.9, 128.4, 127.6, 127.5, 100.37, 82.5, 77.1, 67.3, 62.4, 61.1, 48.3, 34.9, 29.2, 19.0, 17.4; IR (ATR) ν _max 2991, 2940, 2873, 1713, 1650, 1454, 1381, 1195, 1097, 987, 865; HR ESIMS m / z 388.1741 (M + Na ⁺ ) (calcd for 329.1730).

  Synthesis of precursor 6 (from precursor 7): 1,8-diazabicyclo [5.4.0] -7-undecene (50 μl) was added to a tetrahydrofuran solution (500 μl) of precursor 7 (10.3 mg) at room temperature and stirred for 2 hours. did. The reaction solution was concentrated and then purified by silica gel column chromatography to obtain precursor 6 (9.8 mg).

  Synthesis of Precursor 8: A tetrahydrofuran solution of precursor alkynyllithium prepared from (3E) -1,1-dibromo-1,3-pentadiene after cooling a tetrahydrofuran solution of precursor 6 (81.1 mg) to −78 ° C. (About 0.2M, 1.65ml) was added at -78 ° C and stirred for 5 minutes. The temperature was raised to 0 ° C. and stirred for 15 minutes, and then cooled again to −78 ° C. After adding methyllithium (1.07M, 1.0 ml), the temperature was raised again to 0 ° C. and stirred for 2 hours. A saturated aqueous ammonium chloride solution was added to the reaction solution, and the organic layer was separated. The aqueous layer was extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried and concentrated under reduced pressure. The obtained alcohol was directly used in the next reaction. Molecular sieves 4A (380 mg) and pyridinium chlorochromate (190 mg) were sequentially added to the obtained alcohol solution in dichloromethane at room temperature, and the mixture was stirred overnight. The reaction solution was filtered through a Florisil layer, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography to obtain Precursor 8 (77 mg).

Various properties and spectral data of the precursor 8 are as follows. Clear oil; ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.23-7.35 (m, 5H), 6.41 (dq, J = 15.9, 6.8 Hz, 1H), 5.60 (dq, J = 15.9, 1.7 Hz, 1H) , 4.65 (d, J = 9.9 Hz, 1H), 4.52 (d, J = 11.5 Hz, 1H), 4.14 (dd, J = 11.6, 9.9 Hz, 1H), 4.11 (d, J = 11.5 Hz, 1H) , 3.94 (dd, J = 11.6, 5.2 Hz, 1H), 3.20 (ddd, J = 9.9, 9.9, 5.2 Hz, 1H), 2.17 (s, 3H), 1.87 (dd, J = 6.8, 1.7 Hz, 3H ), 1.49 (s, 3H), 1.47 (s, 3H), 1.34 (s, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 208.8, 185.0, 147.9, 137.5, 128.2, 127.4, 127.1, 108.7, 98.8, 91.8, 87.2, 84.8, 73.3, 66.1, 61.1, 50.8, 28.7, 24.1, 19.3, 19.2, 13.2; IR (ATR) ν _max 3419, 2992, 2924, 1719, 1383, 1092; HR ESIMS m / z 407.1817 (M + Na ⁺ ) (calcd for 407.1828).

  Synthesis of Precursors 9 and 10: A tetrahydrofuran solution of Precursor 8 (209 mg) was cooled to 0 ° C., then a tetrahydrofuran solution of tetrabutylammonium fluoride (1M, 160 μl) was added, and the mixture was stirred at 0 ° C. for 50 minutes. Further, a tetrahydrofuran solution (1M, 380 μl) of tetrabutylammonium fluoride was added and further stirred for 1 hour. A saturated aqueous ammonium chloride solution was added to the reaction solution, and the organic layer was separated. The aqueous layer was extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain Precursor 9 (144 mg) and Precursor 10 (22.2 mg).

Various properties and spectral data of the precursor 9 are as follows. Data of E body which occupies mainly. Clear oil; ¹ H NMR (600 MHz, CDCl ₃ ) δ7.33-7.35 (m, 4H), 7.26-7.30 (m, 1H), 6.15 (dq, J = 15.9, 6.8 Hz, 1H), 5.46 (dq , J = 15.9, 1.9 Hz, 1H), 4.75 (d, J = 11.7 Hz, 1H), 4.30 (d, J = 11.7 Hz, 1H), 4.19 (dd, J = 11.1, 4.9 Hz, 1H), 4.10 (dd, J = 11.1, 11.1 Hz, 1H), 3.92 (d, J = 11.1 Hz, 1H), 3.34 (d, J = 13.6 Hz, 1H), 2.78 (ddd, J = 11.1, 11.1, 5.3 Hz, 1H), 2.56 (d, J = 13.6 Hz, 1H), 1.92 (s, 1H, OH), 1.79 (dd, J = 6.8, 1.9 Hz, 3H), 1.48 (s, 3H), 1.45 (s, 3H ), 1.42 (s, 3H); ¹³ C NMR (150 MHz, CDCl ₃ ) δ 204.3, 141.5, 138.7, 128.3, 127.4, 127.1, 109.4, 99.1, 86.4, 84.6, 82.2, 73.9, 68.3, 66.6, 60.3, 52.2, 41.0, 29.5, 19.1, 18.6, 13.3; IR (ATR) ν _max 3419, 2924, 2854, 1723, 1675, 1455, 1382, 1198, 1089, 876; HR ESIMS m / z 407.1846 (M + Na ⁺ ) (calcd for 407.1828).

Various properties and spectral data of the precursor 10 are as follows. Data of E body which occupies mainly. Clear oil; ¹ H NMR (600 MHz, CDCl ₃ ) δ7.22-7.38 (m, 5H), 6.32 (dq, J = 15.5, 6.8 Hz, 1H), 6.10 (d, J = 2.6 Hz, 1H), 5.69 (dq, J = 15.5, 1.9 Hz, 1H), 4.63 (d, J = 11.7 Hz, 1H), 4.34 (d, J = 11.7 Hz, 1H), 4.24 (dd, J = 11.7, 5.3 Hz, 1H ), 3.80 (d, J = 9.1 Hz, 1H), 3.79 (dd, J = 11.7, 11.7 Hz, 1H), 3.24 (dddd, J = 11.7, 9.1, 5.3, 2.6 Hz, 1H), 1.87 (dd, J = 6.8, 1.9 Hz, 3H), 1.49 (s, 3H), 1.46 (s, 3H), 1.44 (s, 3H); ¹³ C NMR (150 MHz, CDCl ₃ ) δ 194.8, 144.0, 140.1, 138.8, 130.5, 128.2, 127.2, 127.2, 110.1, 102.1, 99.4, 83.8, 78.3, 77.3, 66.1, 62.2, 36.2, 29.6, 19.0, 18.8, 13.3; IR (ATR) ν _max 3421, 2992, 2924, 2854, 1724, 1381, 1197, 1091, 754; HR ESIMS m / z 389.1712 (M + Na ⁺ ) (calcd for 389.1723).

Synthesis of Precursor 10 (From Precursor 9): Methyl-N- (triethylammoniumsulfonyl carbamate (37 mg) carbamate was added to a toluene solution of precursor 9 (60 mg) at room temperature, followed by stirring at 100 ° C. for 1 hour. After cooling to room temperature, a saturated aqueous ammonium chloride solution was added to the reaction mixture, and the organic layer was separated, the aqueous layer was extracted twice with ethyl acetate, washed with saturated brine together with the organic layer, dried and concentrated under reduced pressure. Purification by silica gel column chromatography gave precursor 10 (55 mg) Synthesis of precursor 11: Camphorsulfonic acid (3.0 mg) was added to a methanol solution of precursor 10 (53 mg) and stirred overnight at room temperature. Saturated aqueous sodium bicarbonate was added to the solution, and the organic layer was separated, and the aqueous layer was extracted twice with ethyl acetate, combined with the organic layer, washed with saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography. Precursor 11 (36.1m g) was obtained.

Various properties and spectral data of the precursor 11 are as follows. ¹ H NMR (600 MHz, CDCl ₃ ) Z: δ7.20-7.34 (m, 5H), 6.31 (d, J = 3.0 Hz, 1H), 6.23 (dq, J = 11.0, 6.8 Hz, 1H), 5.73 (dq, J = 11.0, 1.9 Hz, 1H), 4.49 (d, J = 11.3 Hz, 1H), 4.28 (d, J = 11.3 Hz, 1H), 4.20-4.25 (m, 2H), 3.92 (dd , J = 10.6, 8.9 Hz, 1H), 2.87 (dddd, J = 8.9, 4.4, 3.0, 3.0 Hz, 1H), 2.49 (d, J = 10.6 Hz, 1H), 2.08-2.13 (br s, 1H, OH), 1.95 (dd, J = 6.8, 1.9 Hz, 3H), 1.55 (s, 3H); E form; δ7.20-7.34 (m, 5H), 6.37 (dq, J = 15.9, 6.8 Hz, 1H ), 6.27 (d, J = 3.0 Hz, 1H), 5.73 (dq, J = 15.9, 1.9 Hz, 1H), 4.48 (d, J = 11.3 Hz, 1H), 4.27 (d, J = 11.3 Hz, 1H ), 4.14-4.24 (m, 1H), 3.89 (dd, J = 10.6, 8.9 Hz, 1H), 2.84 (dddd, J = 8.9, 4.4, 3.0, 3.0 Hz, 1H), 2.48 (d, J = 10.6 Hz, 1H), 2.08-2.13 (m, 1H), 1.88 (dd, J = 6.8, 1.9 Hz, 3H), 1.55 (s, 3H); ¹³ C NMR (150 MHz, CDCl ₃ ) Z form: δ 195.1 , 142.7, 138.3, 138.0, 131.7, 128.4, 127.7, 127.6, 109.4, 101.7, 91.1, 79.2, 75.4, 66.1, 61.6, 45.4, 16.5, 14.7; E form; δ 193.9, 144.2, 138.3, 138.2, 131.5, 128.4 , 127.7, 127.6, 110.1, 101.5, 85.0, 79.2, 75.4, 66.1, 61.6, 45.5, 19.0, 14.7; IR (ATR) ν _max 2924, 2953, 1464, 1121; HR ESIMS m / z 349.1420 (M + Na ⁺ ) (calcd for 349.1410).

  Synthesis of Precursor 12: To a dichloromethane solution of Precursor 11, silver trifluoromethanesulfonate (12.3 mg) was added at room temperature, and then heated to reflux in the dark for 8 hours. A saturated aqueous ammonium chloride solution was added to the reaction solution, and the organic layer was separated. The aqueous layer was extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain Precursor 12 (25.1 mg).

Various properties and spectral data of the precursor 12 are as follows. Clear oil; [α] _D ²¹ -81 (c 0.09, CHCl ₃ ); ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.20-7.32 (m, 5H), 6.45 (dq, J = 15.5, 7.0 Hz, 1H ), 5.90 (dq, J = 15.5, 1.5 Hz, 1H), 5.73 (d, J = 1.9 Hz, 1H), 5.53 (s, 1H), 4.78 (dd, J = 11.0, 5.3 Hz, 1H), 4.50 (d, J = 11.0 Hz, 1H), 4.35 (d, J = 11.0 Hz, 1H), 3.76 (dd, J = 14.0, 11.0 Hz, 1H), 3.47 (dd, J = 11.7, 9.8 Hz, 1H) , 3.13 (dddd, J = 14.0, 9.8, 5.3, 1.9 Hz, 1H), 2.37 (d, J = 11.7 Hz, 1H, OH), 1.87 (dd, J = 7.0, 1.5 Hz, 1H), 1.55 (s , 1H); ¹³ C NMR (150 MHz, CDCl ₃ ) δ 194.4, 160.7, 151.8, 138.4, 133.7, 128.3, 127.6, 127.0, 125.5, 116.2, 102.8, 79.1, 76.1, 68.9, 65.9, 36.9, 183, 15.1 IR (ATR) ν _max 3390, 2924, 2854, 1726, 1644, 1584, 1455, 1386, 1276, 1120; HR ESIMS m / z 349.1401 (M + Na ⁺ ) (calcd for 349.1410).

  Synthesis of Precursor 13: After cooling a dichloromethane solution of precursor 12 (20 mg) to 0 ° C., a hexane solution of boron trichloride (1 M, 180 μl) was added at 0 ° C. and stirred for 2 hours. Saturated sodium hydrogen carbonate solution was added to the reaction liquid, and the organic layer was fractionated. The aqueous layer was extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain precursor 13 (9.8 mg).

Various properties and spectral data of the precursor 13 are as follows. Amorphous solid; [α] _D ²¹ -75 (c 0.02, CHCl ₃ ); ¹ H NMR (600 MHz, CDCl ₃ ) δ 6.46 (dq, J = 15.5, 7.0 Hz, 1H), 6.90 (dq, 15.5 , 1.5 Hz, 1H), 5.70 (d, J = 1.9 Hz, 1H), 5.52 (s, 1H), 4.78 (dd, J = 10.2, 5.5 Hz, 1H), 3.76 (dd, J = 13.4, 10.2 Hz , 1H), 3.30 (dd, J = 11.7, 9.1 Hz, 1H), 2.89 (dddd, J = 13.4, 9.1, 5.5, 1.9 Hz, 1H), 2.30 (d, J = 11.7 Hz, 1H, OH), 1.87 (dddd, J = 7.0, 1.5 Hz, 3H), 1.55 (s, 3H); ¹³ C NMR (150 MHz, CDCl ₃ ) δ 196.3, 160.9, 152.1, 134.0, 125.5, 115.6, 102.9, 74.4, 74.3, 66.6, 36.7, 20.6, 16.4; IR (ATR) ν _max 3415, 2929, 1584, 1276; HR ESIMS m / z 259.0929 (M + Na ⁺ ) (calcd for 259.0940).

  Synthesis of precursor 14: After cooling a solution of precursor 13 (5.4 mg), diisopropylethylamine (12 μl) and N, N-dimethylaminopyridine (2.4 mg) to 0 ° C., the known 4-benzyloxy- Acid chloride (about 0.25 M, 140 μl) prepared from 2-methoxy-6-methylbenzoic acid (68 mg) by a known method was added at 0 ° C. and stirred at room temperature for 1 hour. Further, acid chloride (about 0.25 M, 140 μl) was added and stirred for 2 hours, and then 1 N hydrochloric acid was added to the reaction solution to separate the organic layer. The aqueous layer was extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain precursor 14 (6.0 mg).

  Synthesis of Compound 1c (Sch 725680): A dichloromethane solution of precursor 14 (4.0 mg) was cooled to 0 ° C., then a hexane solution of boron trichloride (1M, 10 μl) was added at 0 ° C., and the mixture was stirred for 30 minutes. 1N hydrochloric acid was added to the reaction solution, and the organic layer was separated. The aqueous layer was extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain compound 1c (2.0 mg).

Various properties and spectrum data of Compound 1c (synthetic product) are as follows. Yellow solid; [α] _D ²² +103 (c 0.1, MeOH); ¹ H NMR (600 MHz, CDCl ₃ ) δ 6.46 (dq, J = 15.5, 7.0 Hz, 1H), 6.26 (d, J = 2.5 Hz , 1H), 6.20 (d, J = 2.5 Hz, 1H), 6.01 (dd, J = 15.5, 1.7 Hz, 1H), 5.76 (d, J = 1.9 Hz, 1H), 5.72 (s, 1H), 5.29 (d, J = 9.8 Hz, 1H), 4.46 (dd, J = 11.0, 4.9 Hz, 1H), 3.84 (dd, J = 13.6, 11.0 Hz, 1H), 3.42 (dddd, J = 13.6, 9.8, 4.9 , 1.9 Hz, 1H), 2.58 (s, 3H), 1.85 (dd, J = 7.0, 1.7 Hz, 3H), 1.31 (s, 3H); ¹³ C NMR (150 MHz, CDCl ₃ ) δ 197.3, 172.0, 166.1, 164.3, 162.1, 153.6, 145.0, 134.9, 126.6, 116.8, 112.8, 105.5, 103.8, 101.9, 76.6, 75.0, 69.3, 36.3, 24.7, 19.7, 18.4; UV λ _max ^-MeOH nm (log ε): 345 (0.19); CD (c 1.0 × 10-4 M, MeOH) Δε (nm): +8.7 (378), -3.2 (308); IR (ATR) ν _max 3370, 2926, 2855, 1720, 1649, 1579 , 1445, 1255; HR ESIMS m / z 409.1251 (M + Na ⁺ ) (calcd for 409.1257).

Test Example 1 Evaluation of inhibitory activity against DNA synthase (in vitro)
The DNA synthase is only used when the template DNA is present, and only four precursors, that is, four deoxynucleoside triphosphates (deoxyadenosine triphosphate (hereinafter abbreviated as dATP), deoxyguanosine triphosphate (hereinafter referred to as “deoxyguanosine triphosphate”). DNA is synthesized using deoxycytidine triphosphate (hereinafter abbreviated as dCTP) and deoxythymidine triphosphate (hereinafter abbreviated as dTTP)] as substrates. This fact by utilizing the DNA polymerase activity of the synthetic DNA (poly (dA) / oligo (dT) 18) as a template primer, when the dTTP in the substrate ³ H previously labeled by a polymerization reaction of the polymerase ³ H- dTTP is incorporated as a thymine residue in the DNA strand. This DNA strand is adsorbed on DEAE-cellulose (diethylaminoethyl-cellulose) filter paper. The specific activity of the adsorbed DNA strand becomes the DNA synthase activity. At this time, the presence or absence of inhibition is judged from the fact that when the test substance inhibits the polymerization reaction of the DNA synthase, the incorporation of the substrate into the DNA strand is reduced or eliminated, and the radioactivity decreases.

  Utilizing this fact, the activities of the compounds 1a, 1b and 1c obtained in Example 1 against various DNA synthase groups were measured by the following method.

(1) Preparation of material Compound 1a, 1b, or 1c was used as a test compound. From the results of NMR analysis, the degree of purification of compounds 1a, 1b, and 1c was 98% or more.

  As the DNA synthase, mammalian DNA synthase {A-Family (Human polγ), B-Family (Calf polα, Human polδ, Human polε), X-Family (Rat polβ, Human polλ, Calf TdT), Y-Family (Human pol η, Mouse pol ι, Human pol κ)}, plant DNA synthase {Cauliflower pol α}, and prokaryotic DNA synthase {E. coli pol I, Taq pol, T4 pol} were used. As other DNA metabolic enzymes, Calf primase of polα, HIV-1 reverse transcriptase, T7 RNA polymerase, T4 polynucleotide kinase, and Bovine deoxyribonuclease I were used.

Calf polα was purified from bovine thymus by immunoaffinity column chromatography according to the method described in known literature (tamai et al., Biochim. Biophys. Acta, 1988, 950, 263). Rat polβ was purified from E. coli JMpβ5 as a recombinant protein according to the method described in known literature (Date et al., Biochemistry 1988, 27, 2983). Human polγ is expressed using BAC-TO-BAC HT Baculovirus Expression System (Life Technologies, MD, USA) as a recombinant protein with a coding region incorporated into pFastBac (Invitrogen Japan KK, Tokyo Japan) and a histidine tag linked. And purified using ProBoundresin (Invitrogen Japan KK, Tokyo Japan) according to the method described in known literature (Umeda et al., Eur. J. Biochem. 2000, 267, 200). Human pol δ and Human pol ε were obtained by analyzing the nuclear fraction of peripheral blood cancer cells (Molt-4) by affinity column chromatography of the second subunit of pol δ or pol ε (Ohige et al., Protein Expr. Purif. 2004, 35, 248). Human polη is expressed in E. coli cells as a recombinant protein (amino acid numbers 1 to 511) with a His ₆ tag linked to the C-terminus and described in known literature (Kusumoto et al., Genes Cells 2004, 9, 1139). Purified according to the method of Mouse polι is a recombinant protein in which a His ₆ tag is linked to the C terminus, by Ni-NTA column chromatography according to the method described in known literature (McDonald et al., J. Exp. Med. 2003, 198, 635). Purified. Human pol kappa is expressed in E. coli cells as a recombinant protein (amino acid number 1 to 560) with a His ₆ tag linked to the C terminus, and is described in known literature (Ohashi et al., Genes Cells 2004, 9, 523). Purified according to the method described. Human polλ was overexpressed and purified as a recombinant protein linked with a histidine tag according to the method described in the known literature (Shimazaki et al., Genes Cells 2000, 7, 639). Cauliflower polα was purified according to the method described in known literature (Sakaguchi et al., 1980, 5, 323). Calf primase of polα and Bovine deoxyribonuclease I were obtained from Stratagene Cloning Systems (La Jolla, CA, USA). E. coli pol I and HIV-1 reverse transcriptase were purchased from Worthington Biochemical Corp. (Freehold, NJ, USA). Taq pol, T4 pol, T7 RNA polymerase, and T4 polynucleotide kinase were purchased from Takara Bio.

(2) Measurement of enzyme inhibitory activity
(2-1) Measurement of DNA synthase inhibitory activity
Preparation of reaction solution
Calf pol α, Rat pol β, Human pol η, Mouse pol ι, Human pol κ, Cauliflower pol α, E. coli pol I, Taq pol, and T4 pol are known (Mizushina et al., Biochim. Biophys. Acta 1996, 1308, 256, and Mizushina et al., Biochim. Biophys. Acta 1997, 1336, 509). For human polγ, human polδ, human polε, and human polλ, publicly known literature (Umeda et al., Eur. J. Biochem. 2000, 267, 200, and Ogawa et al. Jpn. J. Cancer Res. 1998, 89 , 1154) to prepare a reaction solution. For HIV-1 reverse transcriptase, a reaction solution was prepared according to the description in the known literature. The reaction solution for Calf polα is shown below as an example.
<Reaction solution for Calf polα>
Tris-HCl buffer (pH 7.5): 50 mM
Dithiothreitol (DTT): 1mM
Magnesium chloride: 5mM
Glycerin: 15% (v / v)
poly (dA) /: 10μg / ml
oligo ₁₈ (dT): 5μg / ml
DTTP containing tritium-labeled deoxythymidine triphosphate ( ³ H-dTTP): 10 μM (100 cpm / pmol)
Measurement of inhibitory activity
8 μl of a mixture of 16 μl of enzyme-containing solution (enzyme amount: 0.05 unit) and 4 μl of a test compound (compound 1a, 1b, or 1c) dissolved in DMSO (the final concentration of the test compound is 0 to 200 μM) The reaction solution was added to 16 μl of the reaction solution described in “Preparation of reaction solution” above. The mixture was incubated at 37 ° C. for 60 minutes (74 ° C. for 60 minutes for Taq pol). After incubation, 18 μl of each test solution was adsorbed on DEAE-cellulose filter paper, and the filter paper was washed with 5% (w / v) Na ₂ HPO ₄ , water, and ethanol. Under this washing condition, polymers of 10 nucleotides or more are adsorbed on the filter paper. Subsequently, it was air-dried and submerged in a vial containing a toluene scintillator, and the radiation dose was measured with a liquid scintillation counter. From the measurement results, the inhibition rate of the enzyme reaction was determined by the following formula.

Inhibition rate (%) = (1−specific activity of test solution (cpm) / specific activity of control solution (cpm)) × 100
Based on the inhibition rates at various concentrations of the test compound, the 50% inhibitory concentration (IC ₅₀ ) (μM) was determined.

(2-2) Measurement of Other DNA Metabolizing Enzyme Inhibitory Activities The activity of other DNA metabolizing enzymes (Calf primase of polα, T7 RNA polymerase, T4 polynucleotide kinase, and Bovine deoxyribonuclease I) can be measured using known literature (Tamiya et al. Biochem. Mol. Biol. Int. 1997, 41, 1179, Nakayama et al., J. Biochem. (Tokyo) 1985, 97, 1385, Mizushina et al., Biochimie 2007, 89, 581, Soltis et al. , J. Biol. Chem. 1982, 257, 11332, and Lu et al., J. Biol. Chem. 1991, 266, 21060), and the test compound (compound 1a, 1b, or 1c) Inhibition rates for these enzymes were measured to determine 50% inhibitory concentration (IC ₅₀ ) (μM).

(2-3) Results The results are shown in FIG. From FIG. 5, mammalian DNA synthase {A-Family pol (Human pol γ), B-Family pol (Calf pol α, Human pol δ, Human pol ε), and Y-Family pol (Human pol) polη, Mouse polι, Human polκ)} was about 45-100 μM. The strength of the inhibitory activity of compounds 1a, 1b, and 1c was compound 1a> compound 1c> compound 1b in the order of increasing inhibitory activity. On the other hand, the compounds 1a, 1b, and 1c include mammalian DNA synthase {X-Family (Rat pol β, Human pol λ, Calf TdT)}, plant DNA synthase {Cauliflower pol α}, prokaryotic DNA synthase {E Inhibitory activity against .coli pol I, Taq pol, T4 pol} and other DNA metabolizing enzymes {Calf primase of polα, HIV-1 reverse transcriptase, T7 RNA polymerase, T4 polynucleotide kinase, Bovine deoxyribonuclease I} There wasn't.

Test Example 2 Determination of inhibition pattern of DNA synthase
Regarding the mode of inhibition of Compound 1a against Calf polα and Human polκ, changes in pol activity by adding 0-30 μM of Compound 1a were analyzed by a reciprocal plot. Among the two substrates, the template DNA (DNA template-primer) inhibition mode was calculated by measuring the pol inhibitory activity by changing the template DNA concentration to 0 to 40 μM. The inhibition mode of nucleotide (dNTP substrate) was examined by measuring the pol inhibitory activity by changing the nucleotide concentration to 0 to 10 μM. The inhibition constant (Ki) was calculated by analyzing the obtained data by the Dixon plot method.

  The results are shown in FIG. Compound 1a had non-competitive inhibition against the template DNA and antagonistic inhibition against the nucleotide for both polα and polκ. Moreover, since the Ki value in nucleotide was smaller than the Ki value in template DNA, it is considered that compound 1a has affinity for nucleotide rather than template DNA.

Test Example 3 Evaluation of the effects on proliferation of human cancer cells and normal human cells (in vitro)
The cancer cell growth inhibitory activity of compound 1a, 1b, or 1c was evaluated using the following method.
In this experiment, A549 (lung cancer cells), BALL-1 (leukemia B cells), HCT116 (colon cancer cells), Hela (cervical cancer cells), and NUGC-3 (gastric cancer cells) are used as human cancer cells. Used as human normal cells were HDF (skin fibroblasts) and HUVEC (umbilical vein endothelial cells). These human cells were obtained from ATCC (American Type Culture Collection). Human cancer cells were cultured in McCoy's 5A medium supplemented with fetal bovine serum (final concentration 10%), penicillin (final concentration 100 units / mL), and streptomycin (final concentration 100 mg / mL). Normal human cells consist of glucose (final concentration 4.5 g / L), fetal bovine serum (final concentration 10%), L-glutamine (final concentration 5 mM), penicillin (final concentration 50 units / mL), and streptomycin (final concentration). The cells were cultured in Eagle's Minimum Essential Medium (MEM) medium supplemented with 50 mg / mL. The culture was performed in a humid environment of 37 ° C., 5% carbon dioxide / 95% air.
The culture of this test was performed in a 96-well microplate. 1.0 × 10 ⁴ cells were seeded with each test compound (Compound 1a, 1b, or 1c) in each well. The test compound was added so that the final concentration in the medium was 0 to 200 μM. The test compound was dissolved in DMSO at various concentrations before addition to the medium, and added so that the DMSO concentration in the medium was 1%. As a positive control, a medium containing 1% DMSO (no test substance) was used. After addition of the test compound, the cells were cultured at 37 ° C. for 24 hours in a 5% CO ₂ incubator. Then, the cell viability of each test group was determined by the WST-1 method. That is, after 24 hours, the tetrazolium salt WST-1 was added and further cultured for 4 hours. The amount of formazan produced through reduction by viable cells was considered to be proportional to the number of viable cells, and quantified with an optical density (OD) of 450 nm. Cell viability was calculated by the following formula.

  Cell viability (%) = (OD of test group [450 nm] −OD of medium-only well [450 nm]) / (OD of control group [450 nm] −OD of well of medium only] [450 nm]) .

Based on the cell viability at various concentrations of the test compound, the 50% growth inhibitory concentration (LD ₅₀ ) (μM) was determined. The results are shown in FIG.

  From FIG. 7, the 50% growth inhibitory concentration of compound 1a, 1b, or 1c on human cancer cells was about 50-100 μM. The intensity of the inhibitory activity of compound 1a, 1b, or 1c was compound 1a> compound 1c> compound 1b in the order of the inhibitory activity. On the other hand, compounds 1a, 1b, and 1c did not show growth inhibitory activity against human normal cells.

Claims (8)

General formula (1):

[Wherein R ¹ and R ² are different from each other, hydrogen, or general formula (2):

(In the formula, R ⁴ and R ⁵ are the same or different and each represents hydrogen, a lower alkyl group, or an alkanoyl group.)
R ³ represents an alkenyl group which may have a hydroxyl group. ]
A DNA synthase inhibitor comprising a compound represented by the formula: The DNA synthase inhibitor according to claim 1, wherein R ³ is a C3-5 alkenyl group which may have a hydroxyl group, and R ⁴ and R ⁵ are hydrogen.   The anticancer agent which contains the compound represented by General formula (1) of Claim 1 as an active ingredient. General formula (3):

[Wherein R ⁶ and R ⁷ are different from each other, hydrogen or general formula (2):

(In the formula, R ⁴ and R ⁵ are the same or different and each represents hydrogen, a lower alkyl group, or an alkanoyl group.)
When R ⁶ is a group represented by the general formula (2) and R ⁷ is hydrogen, R ⁸ represents an alkenyl group, R ⁶ is hydrogen and R ⁷ Is a group represented by the general formula (2), R ⁸ represents an alkenyl group having a hydroxyl group. ]
A compound represented by   The pharmaceutical composition which contains the compound represented by General formula (3) of Claim 4 as an active ingredient.   The food composition formed by mix | blending the compound represented with General formula (3) of Claim 4 with foodstuffs.   A method for producing a compound represented by the general formula (1), comprising a step of purifying a culture of penicillium pinofhilum Hedgcock. General formula (4):

(In the formula, R ⁴ and R ⁵ are the same as above.)
A process for producing a compound represented by the general formula (5):

A compound represented by formula (6):

(In the formula, R ⁹ and R ¹⁰ are the same or different and each represents a protecting group.)
The manufacturing method characterized by making the compound represented by these react.

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