JP5590286B2 - Novel Ca2 + signaling inhibitor - Google Patents

Description

The present invention relates to a novel Ca ²⁺ signaling inhibitor derived from a natural product, which is useful for the prevention, amelioration and treatment of various diseases including hyperimmune reactions.

The immune system is activated by calcineurin activated by Ca ²⁺ (serine / threonine phosphatase 2B), and the transcription factor NF-AT (Nuclear Factor of Activated T cell) is dephosphorylated and translocated into the nucleus. It has been clarified that the transcription of cytokines typified by -2 is activated and immune cells proliferate by the produced cytokines. Therefore, a substance that suppresses the enhanced Ca ²⁺ signaling suppresses the activation of calcineurin and is useful as a prophylactic or therapeutic agent for hyperimmune reactions and allergic symptoms.
One of the functions of regulating Ca ²⁺ entry into cells is a voltage-dependent L-type Ca ²⁺ channel. A substance that binds to this channel and suppresses the influx of Ca ²⁺ into cells antagonizes Ca ^{2+ in} smooth muscle cells such as myocardium and blood vessels, resulting in ischemic heart disease, hypertension, peripheral vascular disorders, and cerebrovascular It is useful as a prophylactic or therapeutic drug for disorders, cardiac arrhythmias, etc.
Furthermore, since the Ca ²⁺ signal transduction system activates Mpk1, a MAP kinase (Mitogen Activated Protein kinase), substances that suppress Ca ²⁺ signal transduction have various abnormalities in their activation regulation. It is useful as a preventive or therapeutic agent for cancer. In addition, since the Ca ²⁺ signaling system is related to Glycogen Synthase Kinase-3 (GSK-3), substances that suppress Ca ²⁺ signaling are diabetic, cognitive It is useful as a prophylactic or therapeutic drug for diseases.
In this way, substances that suppress the Ca ²⁺ signaling system, which is one of the signal transduction systems that form the basis of life, are various diseases caused by the enhancement of Ca ²⁺ signaling (hyperimmune reactions, allergies, cancers). , Dementia, type 2 diabetes, high blood pressure, angina pectoris, etc.) are being attracted attention because they can be prevented, improved or treated by inhibiting their signals. Examples of application of Ca ²⁺ signaling inhibitors to immunosuppressants and antiallergic agents include cyclosporin A and FK506. Examples of application of Ca ²⁺ signaling inhibitors to prophylactic and therapeutic agents for hypertension and angina include nifedipine, valapamil, and diltiazem. However, although these are satisfactory in terms of activity, there is room for improvement in terms of side effects, and the search for natural sources of Ca ²⁺ signaling inhibitors with few or no side effects is a very significant activity.

The present inventors have been energetically conducting research from the above viewpoints, and to date, substances having an inhibitory action on Ca ²⁺ signaling have been added to magnoliaceae plants, celery family plants, araceae plants, pine familys. It is found from plants and the like (for example, Patent Document 1, Patent Document 2, Non-Patent Document 1, and Non-Patent Document 2), and is also found from microorganisms (for example, Patent Document 3).

JP 2006-225361 A JP 2006-225362 A JP 2007-197354 A

Abstracts of Annual Meeting 2004 Agricultural Chemical Society of Japan p123 “2B05p24 Ca2 + signaling inhibitor honokiol, magnolol contained in honoki” Abstracts of the 2006 Agricultural Chemistry Society of Japan Hokkaido / Tohoku Branch Joint Meeting p52 “C9 Isolation and Purification and Biological Activity of Ca2 + Signaling Inhibitors in Pinus densiflora”

However, it is conceivable that naturally there are materials and substances having further novel action of inhibiting Ca ²⁺ signaling.
Accordingly, an object of the present invention is to provide a novel Ca ²⁺ signaling inhibitor.

As a result of intensive studies in view of the above points, the present inventors have found that the extract of cocoon has an inhibitory effect on Ca ²⁺ signaling.

The Ca ²⁺ signaling inhibitor of the present invention made based on the above findings includes a compound represented by the following structural formula (1) as described in claim 1, but includes dehydroabietic acid and pimaric acid. It is characterized by having no soot extract as an active ingredient .
Also, process for the preparation of Ca ²⁺ signaling inhibitors of the present invention, as claimed in claim 2, wherein isolating the compound represented by the above structural formula by extraction with an organic solvent from amber (1) It is characterized by acquiring .
Also, Ca ²⁺ signaling inhibitors of the present invention, as claimed in claim 3, characterized in that a compound or a pharmaceutically active ingredient acceptable salt represented by the structural formula (1) to.
Also, the compounds of the invention, as claimed in claim 4, characterized by being represented by the above structural formula (1).

According to the present invention, a novel Ca ²⁺ signaling inhibitor derived from a natural product, useful as a preventive or therapeutic agent for hyperimmune reactions, allergies, cancer, dementia, type 2 diabetes, hypertension, angina, etc. Can be provided.

It is a principle diagram of a Ca ²⁺ signal transduction inhibition evaluation system using gene disrupted yeast. It is a figure which shows the activity which can be expected with the Ca2 ⁺ signal transduction pathway in gene disruption yeast. 2 is an HPLC analysis chart of an active ingredient of Ca ²⁺ signaling inhibition activity possessed by a Russian cocoon methanol extract in Example 2. FIG. 4 is an HPLC analysis chart of an active ingredient having an inhibitory action on Ca ²⁺ signaling possessed by a methanol extract of Kuji products from Kuji in Example 3. 6 is a chart of UV spectrum of an active ingredient having an inhibitory action on Ca ²⁺ signaling possessed by a methanol extract of Kuji produced in Kuji in Example 3. It is a chart of ¹ H-NMR spectrum. It is a chart of a ¹³ C-NMR spectrum. It is a chart of an EI-MS spectrum. It is the assignment result of ¹ H-NMR spectrum. It is the assignment result of ¹³ C-NMR spectrum.

The Ca ²⁺ signaling inhibitor of the present invention is characterized by using an extract of persimmon as an active ingredient. In the present invention, cocoon means a fossilized plant resin. In addition to Russia, Poland, Dominica, and other localities in Japan, the area around Kuji City in Iwate Prefecture is well known. Regarding the physiologically active action of the koji extract, for example, Japanese Unexamined Patent Application Publication No. 2007-314522 reports a skin turnover promoting action, and Japanese Unexamined Patent Publication No. 2008-266260 reports a hyaluronic acid production promoting action. However, to the best of our knowledge, there has never been a report that ^sputum extract has an inhibitory effect on Ca ²⁺ signaling.

  The extract of koji can be obtained by, for example, adding an organic solvent to powdered koji according to a general extraction method of natural organic components and performing an extraction operation, and then removing the organic solvent from the obtained filtrate. it can. Examples of the organic solvent that can be used include alcohols (such as methanol, ethanol, and isopropanol), benzene, hexane, ethyl acetate, acetonitrile, acetone, chloroform, and dichloromethane. The extraction operation may be performed using a single organic solvent, or may be performed using a mixture of a plurality of organic solvents. In addition, the extraction operation may be performed in multiple stages, for example, extraction with alcohol and then extraction with ethyl acetate to increase the purity of the extract. Furthermore, in addition to separation operations using ion exchange resins, nonionic adsorption resins, gel filtration chromatography, chromatography with adsorbents such as activated carbon, alumina and silica gel and high performance liquid chromatography, crystallization operations, vacuum concentration operations, Various operations such as freeze-drying operations may be appropriately combined.

The entire content of the active ingredient of Ca ²⁺ signaling inhibitory action of the ^cocoon extract is not necessarily clear at present. However, as will be apparent from the examples described below, the present inventors have found that the active ingredient of the Ca ²⁺ signaling inhibitory activity of the extract of Russian cocoon is a diterpenoid compound contained in pine, As a result of our research so far, it has already been known by Non-Patent Document 2 that it has an inhibitory action on ²⁺ signaling, and dehydroabietic acid and pimaric acid (dehydroabietic acid) represented by the following structural formula ( Pimaric acid), but the active ingredient of the inhibitory action of Ca ²⁺ signaling in Kuji extract is a new labdan type represented by the following structural formula (1) It is specified that it is a like compound. Thus, it is very interesting that the active ingredients differ depending on the production areas even if the extracts of camellia from different production areas have the same Ca ²⁺ signal transduction inhibitory effect.

Based on the above findings, the present invention isolates and acquires dehydroabietic acid, pimaric acid, and a compound represented by the following structural formula (1) having an inhibitory action on Ca ²⁺ signaling by an extraction operation from sputum while providing a method for preparing a Ca ²⁺ signaling inhibitors by, Ca ²⁺ signal transduction inhibitors to compounds represented by or a pharmaceutically acceptable salt thereof as an active ingredient by the following structural formula (1) An agent and a compound represented by the following structural formula (1) are provided. Here, pharmaceutically acceptable salts include inorganic salts with sodium, potassium, magnesium, calcium, aluminum, etc., organic salts with lower alkylamine, lower alcoholamine, etc., basic with lysine, arginine, ornithine, etc. In addition to amino acid salts, known ones such as ammonium salts can be mentioned.

The administration method when the Ca ²⁺ signaling inhibitor of the present invention is administered as a pharmaceutical to humans or animals may be an oral administration method or a parenteral administration method. Good. 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 Ca ²⁺ signaling inhibitor of the present invention is formulated and administered in a manner known per se into a form suitable for these administration methods. 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.

^{Examples of} diseases in which the Ca ²⁺ signaling inhibitor of the present invention acts effectively include hyperimmune reaction, allergy, cancer, dementia, type 2 diabetes, hypertension, angina and the like. When the Ca ²⁺ signaling inhibitor of the present invention is administered to a patient, the dose may be appropriately set depending on conditions such as the age and weight of the patient, the degree of symptoms, and the health condition. About 10 mg to about 10 g per day for an adult may be administered 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, the Ca ²⁺ signaling inhibitor of the present invention may be eaten by adding an effective amount sufficient to exert a Ca ²⁺ signaling inhibitory action to various forms of food (including supplements) (body weight). Ingestion of 0.1 mg to 100 mg per kg is standard).

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

In this example, the evaluation of Ca ²⁺ signaling inhibitory activity, gene Zds1 delta disrupted yeast (Saccharomyces cerevisiae) represents Ca ²⁺ hypersensitive, growth in medium containing a high concentration Ca ²⁺ stops G ₂ phases A system with an improved phenotype (Nature, 392, 303-306, 1998) was used.
The usefulness of the basic system has already been demonstrated using a microbial culture solution and a synthetic compound library, and the inhibitory effect of radicicol, a known compound, on Ca ²⁺ signaling has been discovered by this system. Engineering, 77, 406-408, 1999). In a medium containing a high concentration of Ca ²⁺ , this system does not grow yeast because the Ca ²⁺ signal is superactivated, but if the medium contains substances that inhibit the pathway, It is a positive screening that grows (yeast growth circle is generated) (see Fig. 1). Therefore, it is possible to discover a compound having no toxicity and high specificity (Biosci. Biotechnol. Biochem., 64 (9), 1942-1946, 2000). In fact, this system has been used to confirm an inhibitory effect on Ca ²⁺ signaling such as FK506, an immunosuppressant that has been put to practical use as a pharmaceutical, cyclosporin A, and nifedipine, a Ca ²⁺ antagonist. Furthermore, as shown in FIG. 2, according to this system, in addition to Ca ²⁺ antagonists and calcineurin inhibitors, Mpk1 inhibitors and GSK-3 inhibitors can be logically selected. Since the GSK-3β inhibitor produces a growth circle of yeast, the present inventors have confirmed that the GSK-3 inhibitor can be evaluated using this system (J. Antibiotics. , 61 (8), 496-502, 2008).
Improved system used in this embodiment is obtained by using the gene zds1 Δ erg3 Δ pdr1 / 3 Δ disrupted yeast. In order to increase the membrane permeability of the drug of the gene single disruption yeast, this gene quadruple disruption yeast is obtained by further disrupting the membrane components and the gene of the drug efflux pump by a method known per se and having excellent drug sensitivity Therefore, the Ca ²⁺ signaling inhibition effect can be evaluated with higher accuracy than the basic system using the gene-single-disrupted yeast.

Reference Example 1: Evaluation system Details gene quadruple disrupted yeast YPD medium (yeast extract 10 g / l, peptone 20 g / l, dextrose 20g / l, pH6.5) at 28 ° C., and precultured overnight, A ₅₉₀ = 6 ml of 1.0 culture solution, 3 ml of 5M CaCl ₂ and 41 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 were soaked in 40 μl of paper disc (8 mm, thick), placed on a petri dish, cultured at 28 ° C. for 3 days, measured for growth circle size, and inhibition of Ca ²⁺ signaling Activity was evaluated.

Example 1: Inhibition of Ca ²⁺ signal transduction of Russian and Kuji cocoon extracts After pulverizing Russian and Kuji cocoons and adding methanol, extraction was performed. By removing methanol, an oily methanol extract was obtained. After preparation of 10mg / ml solution by adding methanol to the methanol extract, further the solution of various concentrations were prepared by diluting with methanol soaked in a paper disc, growth of the yeast and the Ca ²⁺ signaling inhibitory activity Evaluation was based on the size of the circle. The results are shown in Table 1.

As is clear from Table 1, both the Russian and Kuji methanol extracts of ^moths strongly inhibited Ca ²⁺ signaling, resulting in growth circles in a wide concentration range.

Example 2: Isolation and purification of active ingredient of Ca ²⁺ signaling inhibitory activity possessed by Russian cocoon methanol extract 12.09 g of methanol extract was obtained from 60.80 g of Russian cocoon powder, and the resulting methanol extraction Water and ethyl acetate were added to the product for extraction, and then ethyl acetate was removed from the ethyl acetate layer to obtain 5.95 g of an oily ethyl acetate extract. Methanol was added to the ethyl acetate extract thus obtained to prepare a 100 mg / ml solution, which was developed on silica gel TLC using a developing solvent of hexane: ethyl acetate = 3: 1, and the active band was around Rf = 0.38. Obtained. After scraping the active band and eluting the active ingredient with methanol, methanol was once removed and methanol was added again to prepare a 100 mg / ml solution. A part of the solution was analyzed by HPLC (column: CAPCELL PAK C18 UG120, 5 μm (4.6 φ x 150 mm), solvent: 80% MeOH-0.1% CH ₃ COOH, flow rate: 1 ml / min). An HPLC analysis chart is shown in FIG. In this chart, each of the characteristic peak A eluting at about 15 minutes and peak B eluting at about 24 minutes are isolated and purified (solvent: 80% MeOH-0.1% CH ₃ COOH). The structure was analyzed by UV measurement, ¹ H-NMR measurement, ¹³ C-NMR measurement, and mass spectrometry. As a result, it was identified that the former was dehydroabietic acid and the latter was pimaric acid, and their respective Ca ²⁺ signaling inhibition effects were confirmed.

Example 3: Isolation and purification of an active ingredient having an inhibitory action on Ca ²⁺ signaling possessed by methanol extract of Kuji products from Koji products 7.39 g of methanol extract was obtained from 64.20 g of Kuji products from Koji products, and the resulting methanol extraction Water and ethyl acetate were added to the product for extraction, and then ethyl acetate was removed from the ethyl acetate layer to obtain 5.24 g of an oily ethyl acetate extract. Methanol was added to the ethyl acetate extract thus obtained to prepare a 100 mg / ml solution, which was developed on silica gel TLC using a developing solvent of hexane: ethyl acetate = 3: 1, and the active band was around Rf = 0.38. Obtained. After scraping the active band and eluting the active ingredient with methanol, methanol was once removed and methanol was added again to prepare a 100 mg / ml solution. A part of the solution was analyzed by HPLC (column: CAPCELL PAK C18 UG120, 5 μm (4.6 φ x 150 mm), solvent: 80% MeOH-0.1% CH ₃ COOH, flow rate: 1 ml / min). An HPLC analysis chart is shown in FIG. In this chart, the characteristic peak C eluting at about 12 minutes was isolated and purified (solvent: 75% MeOH-0.1% CH ₃ COOH), and its structure was measured by UV, ¹ H-NMR, ¹³ C -Analyzed by NMR measurement and mass spectrometry. The UV spectrum chart is shown in FIG. 5 (50 μg / ml in MeOH), the ¹ H-NMR spectrum chart in FIG. 6 (CDCl ₃ , 600 MHz), and the ¹³ C-NMR spectrum chart in FIG. 7 (CDCl ₃ , 150 MHz) and EI-MS spectrum charts are shown in FIG. From the results of structural analysis, this peak is derived from a novel labdan-like compound represented by the following structural formula (1) (with molecular weight 260, C ₁₈ H ₂₈ O, HREI-MS, Calcd. 260.2141, Obsrb. 260.2140, assignments of ¹ H-NMR spectrum and ¹³ C-NMR spectrum are shown in FIG. 9 and FIG. 10, respectively, and their Ca ²⁺ signaling inhibition effect was confirmed (the size of the growth circle of yeast) The results of the evaluation are shown in Table 2).

Formulation Example 1: Injection The sodium salt 1.5 g of the compound represented by the following structural formula (1) is dissolved in 100 ml of physiological saline (total 1.5 g / 100 ml), filled in a vial, and then heat sterilized. An injection for intravenous injection was produced.

Formulation Example 2: Tablets Each component was mixed with the following composition and tableted to produce 400 500 mg tablets containing 50 mg of the Russian cocoon methanol extract obtained in Example 1.
Russian cocoon methanol extract ・ ・ ・ 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.
Muji extract of Kuji products 琥珀 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.
Methanol extract of Kuji products
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.
Compound represented by the following structural formula (1): 0.1 g
Sodium chloride ... 0.9g
Benzalkonium chloride ・ ・ ・ Trace amount
1N sodium hydroxide
1N hydrochloric acid: appropriate amount

The present invention provides a novel Ca ²⁺ signaling inhibitor derived from natural products that is useful as a preventive or therapeutic agent for hyperimmune reactions, allergies, cancer, dementia, type 2 diabetes, hypertension, angina, etc. It has industrial applicability in that it can be done.

Claims (4)

A Ca ²⁺ signaling inhibitor characterized by comprising, as an active ingredient, an extract of persimmon that contains a compound represented by the following structural formula (1) but does not contain dehydroabietic acid and pimaric acid .
A method for preparing a Ca ²⁺ signaling inhibitor, comprising isolating and obtaining a compound represented by the following structural formula (1) by an extraction operation using an organic solvent from straw.
A Ca ²⁺ signaling inhibitor comprising a compound represented by the following structural formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
A compound represented by the following structural formula (1).