JP2012077030A - NEW COMPOUND AND β-SECRETASE INHIBITOR - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new medicinal composition by isolating a useful new compound contained in Hericium erinaceum, and utilizing the compound.SOLUTION: The new compound is a compound represented by general formula (1) or (2), or a pharmaceutically acceptable salt thereof. The β-secretase inhibitor contains the compound as an active ingredient.

Description

  The present invention relates to a compound obtained from Hericium erinaceum and a β-secretase inhibitor containing the compound as an active ingredient.

  It is common practice to search for useful substances or components having specific pharmacological action from natural products and use them for drug discovery. Such techniques are frequently used because isolation of such useful substances or components from natural products often leads to elucidation of the onset mechanism, application to new therapeutic agents, and the like. Mushrooms are one of the natural products that are attracting attention.

  For example, a large number of useful substances such as helicenone that induce nerve growth factor have been isolated from Yamabushitake, a mushroom of the agaric family (see, for example, Patent Documents 1 and 2 and Non-Patent Document 1).

JP-A-8-73486 Japanese Unexamined Patent Publication No. 7-69961

Kawagishi, H. et al., Drugs of the Future 2008, 33 (2): 149-155

  The object of the present invention is to isolate a useful novel compound contained in Hericium erinaceum and to provide a new pharmaceutical composition using the compound.

  The present inventors have found that a novel compound is contained in an extract of Yamabushitake (Hericium erinaceum), and that this novel compound has an excellent inhibitory action on β-secretase activity, thereby completing the present invention. It came to.

  That is, the present invention provides the following compounds and β-secretase inhibitors.

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

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

  Item 3. A preventive or therapeutic agent for senile dementia, comprising the β-secretase inhibitor according to Item 2.

  Item 4. A preventive or therapeutic agent for Alzheimer's dementia, comprising the β-secretase inhibitor according to Item 2.

  Item 5. Cosmetics containing the β-secretase inhibitor according to Item 2.

  Item 6. A carrier oil for aromatherapy comprising the β-secretase inhibitor according to item 2.

  Item 7. A food additive comprising the β-secretase inhibitor according to Item 2.

  The compounds represented by the general formulas (1) and (2) of the present invention are novel and have high β-secretase inhibitory activity. Therefore, it is useful as a β-secretase inhibitor containing this as an active ingredient, and can be used for pharmaceuticals, cosmetics, food compositions and the like. Since the β-secretase inhibitor of the present invention uses a component extracted from Yamabushitake as an active ingredient, it can treat senile dementia such as Alzheimer-type dementia without adversely affecting the human body.

  The β-secretase inhibitor of the present invention can be used as cosmetics, aromatherapy carrier oils, food additives and the like.

It is process drawing which shows isolation operation of the active ingredient from Yamabushitake (Hericium erinaceum). It is a figure which shows the chemical structure and physical-property value of the compounds 1 and 2. It is a figure which shows the spectrum data of ¹ H-NMR and ¹³ C-NMR of compounds 1 and 2. It is a figure which shows the beta-secretase activity inhibitory effect of the compound 1 (isoheririne), the compound 2 (2-N-deethylphenyl isohelerin), and impalatrin.

  Hereinafter, the present invention will be described in detail.

  The present invention relates to a compound represented by the general formula (1) or (2) or a pharmaceutically acceptable salt thereof.

  The compound represented by the general formula (1) (hereinafter also referred to as “compound 1”) and the compound represented by the general formula (2) (hereinafter also referred to as “compound 2”) are both novel substances, They were named isohericerin and 2-N-deethylphenyl isohericerin.

  Examples of the salt of “pharmaceutically acceptable salt” in the present invention include alkali metal salts such as sodium salt and potassium salt. The compound represented by the general formula (1) or (2) or a pharmaceutically acceptable salt thereof may be a solvate such as water.

  Compounds 1 and 2 can be isolated by extracting ericinum (Hericium erinaceum) with a solvent.

  Yamabushitake (Hericium erinaceum) used in the present invention is a mushroom belonging to the family Hyderaceae (Hydnaceae) and the genus Coralus (Hericium). If it is Yamabushitake, the production area will not be specifically limited.

  Yamabushitake can be subjected to extraction as it is, but may be subjected to extraction after being pulverized more finely. Moreover, after making into powder, it can further dry and use for extraction, or it can grind | pulverize in water and can be made into a slurry form and can use for extraction. It is also possible to use a commercially available dry powder.

  Examples of the extraction solvent include alcohols such as methanol, ethanol, propanol and butanol; esters such as ethyl acetate and butyl acetate; ethers such as ethyl ether, propyl ether, isopropyl ether, tetrahydrofuran and dioxane; dichloromethane, chloroform and the like Halogenated hydrocarbons of; ketones such as acetone, methyl ethyl ketone, diethyl ketone, and cyclohexanone; nonpolar organic solvents such as hexane, cyclohexane, and petroleum ether can be used. Of these, ethyl acetate, acetone, methanol, ethyl ether and the like are preferably used. These solvents can be used alone or in admixture of two or more. 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 extraction method is not particularly limited. After adding a solvent (for example, ethyl acetate) to the fruit body of Yamabushitake, heating and heating to such an extent that the activity of the active ingredient contained in the extract is not inactivated. An extraction method, a supercritical extraction method, or the like can be applied as appropriate. Further, various known extraction methods such as an immersion extraction method in which Yamabushitake is immersed in a certain amount of solvent and batch processing, and a continuous extraction method in which the solvent is continuously fed can be applied.

  As an example of a specific extraction method, for example, for Yamabushitake, about 5 to 10 times the dry weight, preferably about 3 to 5 times the extraction solvent is added and immersed and heated, The active ingredient can be extracted by refluxing the solvent for about 1 to 3 hours. Alternatively, an active ingredient is added to Yamabushitake by adding an extraction solvent of about 5 to 10 times by weight, preferably about 3 to 5 times by weight of the dry weight, and leaving it at room temperature for about 1 to 14 days. It is also possible to extract. Of course, the type of solvent, the amount of solvent, the heating temperature, the heating time, etc. may be appropriately adjusted so that the active ingredient can be extracted efficiently.

  After obtaining an extract from Yamabushitake by the method described above, by solvent fractionation operation using one or more organic solvents such as methanol, ethanol, propanol, butanol, dichloromethane, ethyl acetate, toluene, hexane, acetone, The active fraction (compounds 1 and 2) can be separated from the obtained extract. Furthermore, if necessary, one or more suitable separation and purification means such as alumina column chromatography, silica gel chromatography, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and high performance liquid chromatography may be combined. It can also be purified.

  In addition, isolation and identification of compounds 1 and 2 from Yamabushitake can be performed specifically as described in Example 1.

  Thus, the compounds 1 and 2 of the present invention can be isolated and purified from natural products, but the production method of the compound is not limited thereto. The compound may be synthesized based on a known chemical synthesis method, or may be produced by subjecting a substance obtained from a natural product to a raw material as a raw material.

Since the compounds 1 and 2 of the present invention have a strong β-secretase inhibitory activity as described in detail in the following examples, they can be used for pharmaceuticals. For example, it is useful as a β-secretase inhibitor. Moreover, the β-secretase inhibitor of the present invention can be used for various applications.

  For example, when the β-secretase inhibitor of the present invention is used as a pharmaceutical, it is used as a preventive or therapeutic agent for senile dementia in mammals (especially humans), particularly as a preventive or therapeutic agent for Alzheimer-type dementia. .

  The β-secretase inhibitor of the present invention is a tablet, powder, fine granule, granule, coated tablet, capsule, syrup, troche, inhalant, suppository, injection, ointment by a conventional method. , Eye ointments, eye drops, nasal drops, ear drops, poultices, lotions and the like.

  Excipients, binders, lubricants, coloring agents, flavoring agents, and if necessary stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives, Antioxidants and the like can be used. In general, ingredients and blending amounts that are generally used as raw materials for pharmaceutical preparations are appropriately selected to prepare a preparation by a conventional method.

  When administering the pharmaceutical formulation of this invention, the form is not specifically limited, What is necessary is just the method used normally, and oral administration or parenteral administration may be sufficient. The pharmaceutical dosage according to the present invention can be appropriately selected from pharmaceutically effective doses according to the degree of symptoms, age, sex, body weight, dosage form / salt type, specific type of disease, etc. . As an example of the dosage, in the case of oral administration, the amount of active ingredient is usually about 0.001 to 1000 mg / kg for an adult, and if this is administered once to several times a day, Good.

  Moreover, since the β-secretase inhibitor of the present invention has a strong β-secretase inhibitory activity, it is used in addition to products such as bathing agents, soaps, cosmetics, aromatherapy carrier oils, perfumes, hairdressing agents, and the like. Can do.

  The β-secretase inhibitor of the present invention may be usually contained in an amount of about 0.001 to 100% by weight (preferably about 0.1 to 5% by weight) with respect to the entire product.

  When the product containing the β-secretase inhibitor of the present invention is used, a relaxation effect and a refreshing effect due to the scent of essential oil are exhibited, and a physical and mental healing effect is exhibited in a stressful modern society. By using the above products in an environment where elderly people, sick people, etc. live, the effects of preventing and improving senile dementia are also exhibited. Of course, the effect which the essential oil or essential oil component to use, for example, antiseptic | preservation effect, an antimicrobial effect, etc. are exhibited.

  In addition, the β-secretase inhibitor of the present invention includes various foods and beverages such as soft drinks, dairy products (processed milk, yogurt), and confectionery (jelly, chocolate, biscuit, gum, tablet confectionery) as food additives. It can also be blended. The β-secretase inhibitor of the present invention can be blended not only as a food additive in such human foods and drinks but also as a food additive in pet foods such as dogs and cats.

  When used as a food additive, the amount added is not particularly limited, and may be appropriately determined according to the type of food. As an example, what is necessary is just to add so that content may become in the range of about 0.0005 weight%-50 weight% as dry weight of an above-described extract.

  Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1
(1) Extraction and isolation of compounds from Yamabushitake The extraction and isolation of compounds from Yamabushitake were performed according to FIG. Note that a rotary evaporator was used for concentration under reduced pressure. Yamabushitake (manufactured by Marimo Pharmaceutical Co., Ltd.) (600 g) was divided into three 200 g portions and extracted using a Soxhlet continuous extractor. Yamabushitake (200 g) was continuously extracted with refluxing ethyl acetate (1.2 liters) for 3 hours. Fractions eluted three times were combined and concentrated under reduced pressure to obtain an ethyl acetate extract (14 g). By subjecting the ethyl acetate extract to silica gel column chromatography (eluent: hexane-ethyl acetate and methanol), four fractions (hereinafter referred to as Fr.) 1 to Fr. Fractionated into 4. Fr. 3 (0.9 g) was subjected to silica gel column chromatography (eluent: hexane-ethyl acetate and methanol) to fractionate it into four fractions (Fr. 3.1-3.4). Compound 1 (120 mg) was isolated by separating and purifying 3.3 by medium pressure silica gel chromatography (dichloromethane: ethyl acetate = 4: 1). Furthermore, this was crystallized from diethyl ether-dichloromethane to obtain an analytically pure sample (86 mg).

  In addition, Fr. 4 (4.7 g) was subjected to silica gel column chromatography (eluent: hexane-acetone and methanol) to fractionate into three fractions (Fr. 4.1-4.3). 4.3 was subjected to silica gel column chromatography (eluent: dichloromethane-acetone and methanol) to be fractionated into four fractions (Fr. 4.3.1 to 4.3.4). Furthermore, Fr. Compound 2 (74 mg) was isolated by separating and purifying 4.3.2 by medium pressure silica gel chromatography (dichloromethane: acetone = 3: 2). Further, this was crystallized from hexane-acetone to obtain an analytically pure sample (36 mg).

(2) Structure determination of compounds 1 and 2 In order to determine the structures of compounds 1 and 2, HR-EI-MS, IR, ¹ H-NMR and ¹³ C-NMR were measured.

  JEOL the Tandem MS station JMS-700 manufactured by JEOL Ltd. was used for the measurement of HR-EI-MS.

  JASCO FT / IR-470 plus Fourier transform infrared spectrometer manufactured by JASCO Corporation was used for the measurement of IR (infrared absorption) spelltle.

For measurement of NMR spectrum, JEOL ECA-500 (500 MHz, ¹ H; 125 MHz, ¹³ C) spectrometer was used. Compound 1 was measured in CDCl ₃ and compound 2 was measured in DMSO-d ₆ .

  The chemical structures and physical properties of compounds 1 and 2 are shown in FIG.

Compound 1 showed molecular formula _C 27 _H 33 NO ₃ from the measurement of HR-EI-MS. Further, for analysis of ¹ H-NMR and ¹³ C-NMR spectra, two-dimensional NMR was measured to examine the correlation between protons and carbon. As a result, all signal assignments became clear ( ¹ The spectral data of H-NMR and ¹³ C-NMR are shown in FIG. The NMR data of Compound 1 is similar to that of the known compound Helisine (Kimura Y., et ai., Agric. Biol. Chem. 55 p.2673-2674 (1991)), suggesting that it is an isomer. It was done. When the correlation between remote ¹ H- ¹³ C heteronuclei (HMBC) in two-dimensional NMR was examined in detail, the relationship between H3 and C1, C3a, C4, C7a, and H7 and C1, C3a, C5, C6 From the fact that a correlation was observed with C7a, it was found that helicerine is a positional isomer of carbonyl of the isoindolinone skeleton.

  From these results, Compound 1 was obtained from (E) -5- (3,7-dimethylocta-2,6-dien-1-yl) -4-hydroxy-6-methoxy-2-phenylethylisoindoline-1 -On ((E) -5- (3,7-dimethylocta-2,6-dien-1-yl) -4-hydroxy-6-methoxy-2-phenylethylisoindolin-1-one) (Isohelicerin; named isohericerin) ).

Compound 2 exhibited a molecular formula _C 19 _H 25 NO ₃ from the measurement of HR-EI-MS. The spectrum data of ¹ H-NMR and ¹³ C-NMR obtained from the two-dimensional NMR measurement are shown in FIG. Compound 2 was found to be a compound lacking a phenylethyl group from Compound 1, as is apparent from the NMR data in FIG. Further, when the HMBC correlation was examined in order to confirm the position of the carbonyl of the indolinone skeleton, the same correlation between proton and carbon as in Compound 1 was observed. -(3,7-dimethylocta-2,6-dien-1-yl) -4-hydroxy-6-methoxyisoindoline-1-one ((E) -5- (3,7-dimethylocta-2,6 -dien-1-yl) -4-hydroxy-6-methoxyisoindolin-1-one) (2-N-deethylphenyl isohericerin; named 2-N-deethylphenyl isohericerin).

Example 2 (β-secretase inhibitory activity test)
β-secretase inhibitory activity was evaluated using a BACE1 (recombinant human BACE1) assay kit purchased from PanVera. The evaluation test was performed according to a method (Jeon SY, et al., Bioorg. Med. Chem. Lett. 13: 3905-3908 (2003)) in which the description of the instruction manual of the kit produced by the manufacturer was modified. A brief description is given below.

  Test by mixing 10 μL BACE1 (1.0 U / ml), 10 μL substrate (750 nM Rh-EVNLDAEFK-Quencher in 50 mM ammonium bicarbonate), and 10 μL sample solution (sample dissolved in 30% DMSO) A sample was used. Moreover, what added 10 microliters assay buffer (30% DMSO containing, 50 mM sodium acetate, pH4.5) instead of the said sample solution was set as control. These were incubated at room temperature for 60 minutes in the dark and then excited by irradiation with light having a wavelength of 550 nm, and the emission intensity at a wavelength of 590 nm was measured.

The percent inhibition of β-secretase activity is expressed as:
Secretase activity (%) = [1-{(S−S ₀ ) / (C−C ₀ )}] × 100
(Where C represents the luminescence intensity of the control (enzyme, buffer, and substrate) after 60 minutes of incubation, C ₀ represents the luminescence intensity of the control at 0 o'clock, and S represents the test sample (enzyme, buffer). The luminescence intensity of the sample solution and the substrate) is shown, and S ₀ is the luminescence intensity of the sample at 0 o'clock). All data is the average of 3 test results. Imperatorin was used as a positive control (Marumoto S. and Miyazawa M. Phytother. Res. 24: 510-513 (2010)). The results are shown in FIG.

From the results of FIG. 4, it is found that the IC ₅₀ values which are 50% inhibitory concentrations of Compound 1 (Isohelicerin) and Compound 2 (2-N-deethylphenylisohelicerin) are 79.1 μM and 61.7 μM, respectively. It was.

Claims (7)

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

  A β-secretase inhibitor comprising as an active ingredient the compound represented by the general formula (1) or (2) according to claim 1 or a pharmaceutically acceptable salt thereof.   A preventive or therapeutic agent for senile dementia, comprising the β-secretase inhibitor according to claim 2.   A preventive or therapeutic agent for Alzheimer-type dementia, comprising the β-secretase inhibitor according to claim 2.   Cosmetics containing the β-secretase inhibitor according to claim 2.   A carrier oil for aromatherapy containing the β-secretase inhibitor according to claim 2.   A food additive comprising the β-secretase inhibitor according to claim 2.

Images