JP5079242B2 - DNA damage checkpoint activator - Google Patents

Description

  The present invention relates to a novel DNA damage checkpoint activator useful as an anticancer agent.

The DNA damage checkpoint, one of the control mechanisms for orderly cell cycle progression, senses DNA damage and replication delay, and pauses the cell cycle until the DNA is successfully repaired. It is known that a decrease or disappearance of the function causes various genetic changes leading to carcinogenesis. Therefore, in recent years, DNA as an anticancer agent having a new mechanism of action.
Substances that activate damage checkpoints have attracted attention, and in this connection, Non-Patent Document 1 utilizes the fact that yeast G1 / S phase checkpoint mutants are highly sensitive to DNA damage. A screening method for substances that activate DNA damage checkpoints in higher eukaryotic cells including humans has been proposed. Although not yet fully elucidated with respect to DNA damage checkpoint, this screening method, DNA damage checkpoint budding yeast is transmitted by signals through MEC1 and RAD53 around the RAD9, transfer of the enzyme DNA repair system Instead of RAD9 , a substance that activates RAD53 related to DNA damage checkpoint signal by bypassing the defective RAD9 by using yeast with disrupted RAD9 gene based on activation and cell cycle arrest In order to find a substance capable of arresting the cell cycle, the same document describes an antibiotic such as Borrelidin as a substance having a DNA damage checkpoint activation action obtained from a culture solution of microorganisms. Yes.
Hideko Tsuchiya, Development of anticancer drugs using yeast, FY 1998-2012 Research on advanced utilization of brewing microbial functions (Hiroshima Industrial Technology Promotion Organization), p53-p60, (2001)

However, natural resources such as plants including foods other than microbial metabolites may contain substances that have a further novel DNA damage checkpoint activation effect.
Accordingly, an object of the present invention is to provide a novel DNA damage checkpoint activator useful as an anticancer agent.

As a result of searching for a substance having a DNA damage checkpoint activating action mainly in plants serving as foodstuffs in view of the above points, the present inventors have found that the compositae plant is a compositae , Cacaliadelphiniifolia : It was found that an endoperoxide compound contained in “Sidke” edible as a wild vegetable has a DNA damage checkpoint activation effect.

The DNA damage checkpoint activator of the present invention made on the basis of the above findings is 3,6-epidioxy- which is an endoperoxide compound represented by the following structural formula (2) as described in claim 1. 1,10-bisabodiadiene (10,6-epidioxy-1,10-bisaboradiene) or a pharmaceutically acceptable salt thereof as an active ingredient (however, a compound represented by the structural formula (2)) Except for the case where the dry powder of rice bran is included as an active ingredient).

Moreover, the preparation method of the DNA damage checkpoint activator of the present invention comprises, as described in claim 2, an endoperoxide represented by the following structural formula (2) by an extraction operation from a asteraceae plant, Momijigasa. 3,6-epidioxy-1,10-bisaboradiene, which is a compound, is isolated and obtained (provided that the compound represented by the structural formula (2) is obtained) Except for the case where the dry powder of rice bran is included as an active ingredient).

Moreover, the anticancer agent of this invention is 3,6-epidioxy-1,10-bisaboradiene (3,6-epidioxy-) which is an end peroxide compound represented by following Structural formula (2) as described in Claim 3. 1,10-bisaboladiene) or a pharmaceutically acceptable salt thereof as an active ingredient (however, an embodiment comprising a dry powder of Momijigasa containing a compound represented by the structural formula (2) as an active ingredient) except).

According to the present invention, a novel DNA damage checkpoint activator useful as an anticancer agent is provided.

  The DNA damage checkpoint activator of the present invention is characterized by comprising an endoperoxide compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Wherein, R ¹ and R ² are the same or different and each represents a hydrogen atom, a lower alkyl group which may have a substituent, or a hydroxyl group. n represents an integer of 1 to 10. ]

Here, the lower alkyl group means a linear or branched alkyl group having 1 to 6 carbon atoms, specifically, methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group. Tert-butyl group and the like. Examples of the substituent that the lower alkyl group may have include a carboxyl group, a hydroxyl group, and a lower alkoxy group. Here, the lower alkoxy group means a linear or branched alkoxy group having 1 to 6 carbon atoms, specifically, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, or an isobutoxy group. And tert-butoxy group.

Among the endoperoxide compounds represented by the general formula (1), as representative compounds,
3,6-epidioxy-1,10-bisaboladiene represented by the following structural formula (2), which is an endoperoxide compound of bisaborane-type sesquiterpene
Is mentioned. 3,6 epidioxy-1,10 Bisaborajien is, for example, a medicinal plant Merikope Borubonika (Melicopeborbonica) already is a known (if necessary when Simonsen et al as a substance having antifungal activity included in the leaf, Phytother. Res., 18, 542-545, 2004), there has been no report that this substance is contained in Sidke that is edible as a wild vegetable, and this substance is contained in Sidke This is a new finding by the present inventors.

When the endoperoxide compound represented by the general formula (1) is separated and purified from natural resources such as Asteraceae, the operation is performed by a general separation method of natural organic compounds, for example, alcohol (methanol, ethanol, etc. ), Extraction using organic solvents such as ethyl acetate, acetone, hexane, chloroform, and dichloromethane, water, ion exchange resin, nonionic adsorption resin, gel filtration chromatography, chromatography with adsorbents such as activated carbon, alumina and silica gel Various operations such as crystallization operation, vacuum concentration operation, lyophilization operation, etc., in addition to separation operations using chromatography and high performance liquid chromatography, may be performed alone or in combination. The endoperoxide compound of the present invention can also be synthesized by a known organic synthetic chemistry method. Moreover, since the endoperoxide compound of the present invention has a plurality of asymmetric carbons, various stereoisomers and optical isomers may exist, and the present invention includes all of them within the scope of the right. .

When the endoperoxide compound represented by the general formula (1) can take the form of a salt, pharmaceutically acceptable salts thereof include inorganic salts with sodium, potassium, magnesium, calcium, aluminum, etc., lower alkyl In addition to organic salts with amines, lower alcohol amines, etc., basic amino acid salts with lysine, arginine, ornithine and the like, and known salts such as ammonium salts.

The endoperoxide compound of the present invention or a pharmaceutically acceptable salt thereof can be used as an active ingredient of a DNA damage checkpoint activator. When administered to humans and animals as a pharmaceutical, the administration method may be an oral administration method or a parenteral administration method. Examples of parenteral administration methods include intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, transdermal administration, pulmonary administration, nasal administration, enteral administration, buccal administration, and transmucosal administration. In this case, the DNA damage checkpoint activator of the present invention is formulated and administered by a method known per se into a form suitable for these administration methods. In this case, the endoperoxide compound as an active ingredient may be formulated in a highly purified form, or may be formulated in the form of a crude extract from an Asteraceae plant or the like. Examples of the dosage form include injections, suppositories, aerosols, transdermal absorption tapes, eye drops, nasal drops and the like. When preparing an injection, a pH adjuster, a buffer, a stabilizer, a solubilizing agent and the like are appropriately added to form an injection. Examples of oral preparations include tablets (including sugar-coated tablets, coated tablets, and buccal tablets), powders, capsules (including soft capsules), granules (including those coated), pills, troches, liquids, and the like. And a pharmaceutically acceptable sustained release preparation. Liquids include suspensions, emulsions, syrups (including dry syrups), elixirs and the like. For example, a tablet can be prepared with a pharmaceutically acceptable carrier, excipient, binder, disintegrant, lubricant, colorant and the like according to known pharmaceutical manufacturing methods. In this case, as the carrier or excipient, for example, lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, licorice powder, gentian powder and the like can be used. As the binder, for example, starch, tragacanth gum, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose and the like can be used. As the disintegrant, for example, starch, agar, gelatin powder, sodium carboxymethyl cellulose, carboxymethyl cellulose calcium, crystalline cellulose, calcium carbonate, sodium hydrogen carbonate, sodium alginate and the like can be used. As the lubricant, for example, magnesium stearate, talc, hydrogenated vegetable oil, macrogol and the like can be used. As the colorant, those allowed to be added to pharmaceuticals can be used. Tablets and granules are sucrose, gelatin, hydroxypropylcellulose, purified shellac, glycerin, sorbitol, ethylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, cellulose phthalate acetate, hydroxypropylmethylcellulose phthalate, methyl methacrylate, methacrylic acid as required It may be coated with a polymer or the like, or may be coated with two or more layers. Further, it may be encapsulated using ethyl cellulose or gelatin. Moreover, when the volatility of the end peroxide compound as an active ingredient is high, the aroma may be used.

The disease in which the DNA damage checkpoint activator of the present invention acts effectively is cancer. When the DNA damage checkpoint activator of the present invention is administered to a patient as an anticancer agent, the dose may be appropriately set according to conditions such as the patient's age, weight, symptom level, health condition, etc. May be administered at a dose of about 10 mg to about 10 g per day for an adult, orally or parenterally, once to several times a day. In the case of eye drops, an eye drop having an active ingredient concentration of 0.003 to 5 (w / v)% may be administered once a day several times.

In addition, since the DNA damage checkpoint activator of the present invention is a substance originally contained in Sidke, which is edible as a wild vegetable, it activates DNA damage checkpoints in various forms of food (including supplements). May be added in an effective amount sufficient to exert the effect (ingestion of 0.1 mg to 100 mg per kg body weight is standard).

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is limited to the following description and is not interpreted at all.

In this example, the DNA damage checkpoint activation effect was evaluated according to the method described in Non-Patent Document 1. The yeast to be used is a yeast in which the RAD9 gene is disrupted as described above, but since this yeast is further temperature sensitive, DNA damage is induced when cultivated at 37 ° C, and the checkpoint control system does not work, so the cell cycle is changed. It has the characteristic of not being able to stop and having the property of rapidly dying and causing cell division. This is considered to be a model of cancer, and when a substance that bypasses RAD9 deficient in culture and activates RAD53 related to the DNA damage checkpoint signal, or a substance that can stop the cell cycle instead of RAD9 , The yeast grows with a lethal effect at 37 ° C. suppressed (see FIG. 1). That is, in the evaluation method described in Non-Patent Document 1, the anticancer activity is opposite to the activity of killing cancer cells so far, and the phenotype (= substance is impregnated) as a growth circle of growing yeast. This is a positive screening system in which yeast grows around the disk). Accordingly, it is possible to find an active substance with low toxicity and high specificity. In fact, since hydroxyurea and mycophenolic acid having anticancer activity show a growth circle, a substance having a DNA damage checkpoint activating action found by this method can be an effective anticancer agent for humans. Is. It is well known that anticancer drugs used in clinical settings today have a need for new chemical structures and mechanisms of action because of problems such as side effects and reduced drug sensitivity. However, according to this method, it is possible to provide a new anticancer agent having an action mechanism different from that of the existing anticancer agent.

Reference example 1: Details of screening system
A temperature-sensitive mutant of yeast with a disrupted RAD9 gene (gene disrupted yeast: Δ RAD9 ) was treated with 28 YPD medium (yeast extract 10 g / l, peptone 20 g / l, dextrose 20 g / 1, pH 6.5).
Precultured overnight at 1 ° C., 1.75 ml of the obtained A ₅₉₀ = 0.8 culture solution and 48.25 ml of YPD agar medium were well suspended, and 12.5 ml each was dispensed into a petri dish. Samples of various concentrations for measurement (concentration adjustment with methanol) were soaked in 40 μl of paper disc (8 mm, thick), placed on a petri dish, induced DNA damage at 37 ° C. for 6 hours, and then at 28 ° C. for 2 days After culturing, the size of the growth circle was measured, and the effect (= anticancer activity) of growing the mutant strain by the DNA damage checkpoint activation action was evaluated. The positive control was mycophenolic acid (average growth circle 18.1 mm, 50 μg / disc), which is an immunosuppressant and known to have anticancer activity.

Example 1:
(1) Isolation and purification of 3,6-epidioxy-1,10-bisaboradiene from Momijigasa 651.7 g of fresh Irikawa-grown Momijigasa (sold as wild vegetable Sidke) is dried at room temperature to 57.28 g A dried product was obtained. This was soaked in methanol and extracted, and the resulting extract was concentrated. The water-soluble residue was extracted with ethyl acetate. The ethyl acetate extract was concentrated, and the resulting concentrate (yield 3.06 g) was subjected to silica gel column chromatography using a mixed solvent system of hexane-ethyl acetate, and a mixed solvent of hexane-ethyl acetate = 10: 1 and 5: 1. The elution fraction (total yield: 1.14 g ) was used and chromatographed with 75% methanol using an ODS column, and DNA damage was measured using the growth circle of the temperature-sensitive mutant of the gene disrupted yeast (Δ RAD9 ) as an index. By separating the peak having the checkpoint activating action, 25.3 mg of 3,6-epidioxy-1,10-bisaboradiene was obtained.
<Physicochemical data of 3,6-epidioxy-1,10-bisaboradiene>
colorless oil, UV (MeOH) λmax (logε) = end absorption, ¹ H and ¹³ C NMR data: Table 1 and Table 2 (major body with larger amount because it is obtained as a mixture of two diastereomers , The lesser one is described as minor body: signal assignment numbering is as follows), HR-ESI-MS m / z 237.1850, [M + H] ⁺ (calcd for C ₁₅ H ₂₅ O ₂ , 237.1855)

(2) DNA damage checkpoint activation of 3,6-epidioxy-1,10-bisaboradiene Impregnated isolated and purified 3,6-epidioxy-1,10-bisaboradiene into paper discs at various concentrations (concentration) Adjustment was performed with methanol), and the DNA damage checkpoint activating effect was evaluated by the size of the growth circle of the temperature-sensitive mutant of the gene-disrupted yeast (Δ RAD9 ). The results are shown in Table 3 together with the evaluation results of mycophenolic acid used as a positive control.

As is clear from Table 3, 3,6-epidioxy-1,10-bisaboradiene was found to exert superior DNA damage checkpoint activation at lower concentrations than mycophenolic acid used as a positive control. .

(3) Anticancer activity of 3,6-epidioxy-1,10-bisaboradiene
96 human chronic myeloid leukemia cells K562 suspended in 10% FBS-RPMI1640 medium at 5 × 10 ⁵ cells / ml
Add 100 μl to the microwell of the well, add various concentrations of 3,6-epidioxy-1,10-bisaboradiene (concentration adjustment with methanol), and incubate for 3 days in the presence of 5% CO _{2 at} 37 ° C. Cytotoxicity was examined by assay to determine 50% inhibitory concentration. In addition, 100 μl of human prostate cancer cell LNCaP suspended in 10% FBS-RPMI1640 medium at 1 × 10 ⁶ cells / ml is seeded in 96-well microwells, and various concentrations of 3,6-epidioxy-1,10- Bisaboradiene (concentration adjustment with methanol) was added, cultured at 37 ° C. in the presence of 5% CO ₂ for 2 days, cytotoxicity was examined by MTT assay, and 50% inhibitory concentration (IC ₅₀ ) was determined. Table 4 shows the relationship between the concentration of 3,6-epidioxy-1,10-bisaboradiene and the 50% inhibitory concentration for each cell. Table 4 also shows α-curcumene (α-Curcumene) represented by the following structural formula (3), which is a bisaborane-type sesquiterpene similar in structure to 3,6-epidioxy-1,10-bisaboradiene as a comparative control. : Obtained from a commercially available ginger essential oil by a known isolation and purification method) and the 50% inhibitory concentration for each cell.

  As is clear from Table 4, 3,6-epidioxy-1,10-bisaboradiene was found to have an excellent anticancer effect compared to α-curcumene.

Formulation Example 1: Injection
Dissolve 1.5 g of 3,6-epidioxy-1,10-bisaboradiene in 100 ml of physiological saline containing ethanol as a solubilizer (total 1.5 g / 100 ml), fill the vial, and then sterilize by heating. An injection for injection was produced.

Formulation Example 2: Tablets Each component was mixed in the following composition and tableted to produce 400 500 mg tablets containing 50 mg of 3,6-epidioxy-1,10-bisaboradiene.
3,6-epidioxy-1,10-bisaboradiene ・ ・ ・ 20g
Potato starch ・ ・ ・ 6g
Talc stearate ・ ・ ・ 4g
6% HPC lactose ... 170g
(Total 200g)

Formulation Example 3: Granules Each component was mixed with the following composition, compression-molded, pulverized, and sized to produce 20-50 mesh 5% granules.
3,6-epidioxy-1,10-bisaboradiene ・ ・ ・ 10g
Lactose ... 187g
Magnesium stearate ・ ・ ・ 3g
(Total 200g)

Formulation Example 4: Capsules Each component was mixed well with the following composition, and the mixture was filled into No. 1 capsules to produce 300 capsules.
3,6-epidioxy-1,10-bisaboradiene ・ ・ ・ 5g
Lactose ・ ・ ・ 40g
Potato starch ... 50g
Hydroxypropyl methylcellulose ... 3.5g
Magnesium stearate ・ ・ ・ 1.5g
(Total 100g)

Formulation Example 5: Eye drops The following components were dissolved in 100 ml of sterilized purified water, and eye drops were produced by a conventional method.
3,6-epidioxy-1,10-bisaboradiene ... 0.1g
Sodium chloride ... 0.9g
Benzalkonium chloride ・ ・ ・ Trace amount
1N sodium hydroxide
1N hydrochloric acid: appropriate amount ethanol: appropriate amount

The present invention has industrial applicability in that it can provide a novel DNA damage checkpoint activator useful as an anticancer agent.

The figure which shows the screening principle of the DNA damage checkpoint activation effect | action using gene disruption yeast.

Claims (3)

3,6-epidioxy-1,10-bisaboradiene or a pharmaceutically acceptable salt thereof, which is an endoperoxide compound represented by the following structural formula (2) A DNA damage checkpoint activator characterized by comprising as an active ingredient (except for a mode in which a dry powder of Momijigasa containing a compound represented by the structural formula (2) is used as an active ingredient).

By an extraction operation from the asteraceae plant, Momijigasa, 3,6-epidioxy-1,10-bisaboradiene (3,6-epidioxy-1,10) which is an endoperoxide compound represented by the following structural formula (2) A method for preparing a DNA damage checkpoint activator characterized by isolating and obtaining (bisaboladiene) (except for a mode in which a dry powder of Momijigasa containing a compound represented by the structural formula (2) is used as an active ingredient) .

3,6-epidioxy-1,10-bisaboradiene or a pharmaceutically acceptable salt thereof, which is an endoperoxide compound represented by the following structural formula (2) As an active ingredient (except for a mode in which a dry powder of Momijigasa containing a compound represented by the structural formula (2) is used as an active ingredient).

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