Compound having pyranobenzopyran as fundamental skeleton

Abstract

To provide a novel compound derived from an extract of Agrimonia pilosa.SOLUTION: The invention provides a compound represented by Chemical Formula 3.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to novel compounds having a pyranobenzopyran skeleton. [Background technology]

[0002] Agrimonia pilosa (scientific name: Agrimonia pilosa var. japonica) is a perennial plant of the genus Agrimonia in the family Rosaceae. Agrimonia has long been used as a traditional Chinese medicine and is known to have various pharmacological activities. Its safety is also well known. A variety of medicinal uses have been proposed for extracts of agrimony, including as an allergen inactivator (Patent Document 1), an anti-androgenic agent and papilla cell proliferation promoter (Patent Document 2), a skin whitening and anti-aging agent (Patent Document 3), a dopa oxidase activity inhibitor (Patent Document 4), a β-glucuronidase inhibitor (Patent Document 5), and a neutral endopeptidase inhibitor (Patent Document 6). However, none of the above-mentioned prior arts specify what the active substance is.

[0003] The inventors of the present application are conducting research into the pharmacological effects of extracts from agrimony. In the course of this research, they discovered that aqueous or ethanolic extracts from agrimony have neuroactivating effects, and have filed a patent application for a neuroactivator (Patent Document 7). It has been confirmed that agrimony extract increases phosphorylated CaMKII (pCaMKII) and phosphorylated ERK1 / 2 (pERK1 / 2) in neurons involved in neurotransmission, enhances the expression of IEGs (immediate early genes) such as c-fos and Arc, and activates neurons involved in memory, thereby improving memory (see Patent Document 7). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2008-174529 [Patent Document 2] Japanese Patent Application Laid-Open No. 2010-65008 [Patent Document 3] Japanese Patent Application Laid-Open No. 2010-65009 [Patent Document 4] Japanese Patent Application Laid-Open No. 2010-195731 [Patent Document 5] Japanese Patent Application Laid-Open No. 2010-220641 [Patent Document 6] Japanese Patent Application Laid-Open No. 2011-190214 [Patent Document 7] Japanese Patent Application Publication No. 2018-8888 Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have conducted research to identify neuroactivators and have discovered novel compounds. An object of the present invention is to provide a novel compound. [Means for solving the problem]

[0006] The main configuration of the present invention is as follows. (1) A compound represented by chemical formula 1.

[0007] [ka] (chemical formula 1)

[0008] (2) A compound represented by chemical formula 2.

[0009] [ka] (Chemical formula 2)

[0010] (3) A compound represented by chemical formula 3.

[0011] [ka] (Chemical formula 3)

[0012] (4) A compound represented by chemical formula 4.

[0013] [ka] (Chemical formula 4) [Effects of the Invention]

[0014] The present invention provides novel compounds having a pyranobenzopyran skeleton. The compounds of the present invention enhance the expression of IEGs (immediate early genes) such as c-fos, thereby activating neurons involved in memory and improving memory. Therefore, the compounds of the present invention are useful for activating the nervous system involved in memory, and can be used as reagents for research into nervous system activity. They can also be used as starting materials for chemically synthesized pharmaceuticals to obtain memory or neuroactivators, or as research reagents. Furthermore, the compounds of the present invention can be used as foods, beverages, or pharmaceuticals. [Brief explanation of the drawings]

[0015] [Figure 1] HPLC analysis results using KM1-1 chiral column [Figure 2] HPLC analysis results using KM1-2 chiral column [Figure 3] HPLC analysis results using KM2-1 chiral column [Figure 4] HPLC analysis results using KM2-2 chiral column [Figure 5] HPLC analysis results using KM3-1 chiral column [Figure 6] HPLC analysis results using KM3-2 chiral column [Figure 7] HPLC analysis results using KM4-1 chiral column [Figure 8]HPLC analysis results using KM4-2 chiral column [Figure 9] Asymmetric unit structure of the crystalline sponge incorporating KM1-1 (the area within the dotted line is the incorporated KM1-1, solvent omitted) [Figure 10] Relative 3D structure of KM1-1 [Figure 11] Asymmetric unit structure of the crystalline sponge incorporating KM1-2 (the area within the dotted line is the incorporated KM1-2, solvent omitted) [Figure 12] Relative 3D structure of KM1-2 [Figure 13] Asymmetric unit structure of the crystalline sponge incorporating KM2-1 (the area within the dotted line is the incorporated KM2-1, solvent omitted) [Figure 14] Relative 3D structure of KM2-1 [Figure 15] Asymmetric unit structure of the crystalline sponge incorporating KM2-2 (the area within the dotted line is the incorporated KM2-2, solvent omitted) [Figure 16] Relative 3D structure of KM2-2 [Figure 17] Asymmetric unit structure of the crystalline sponge incorporating KM3-1 (the area within the dotted line is the incorporated KM3-1, solvent is omitted) [Figure 18] Relative 3D structure of KM3-1 [Figure 19] Asymmetric unit structure of the crystalline sponge incorporating KM3-2 (the area within the dotted line is the incorporated KM3-2, solvent omitted) [Figure 20] Relative 3D structure of KM3-2 [Figure 21] Asymmetric unit structure of the crystalline sponge incorporating KM4-1 (the area within the dotted line is the incorporated KM4-1, solvent is omitted) [Figure 22] Relative 3D structure of KM4-1 [Figure 23] Asymmetric unit structure of the crystalline sponge incorporating KM4-2 (the area within the dotted line is the incorporated KM4-2, solvent omitted) [Figure 24] Relative 3D structure of KM4-2 [Figure 25] Asymmetric unit structure of crystalline sponge incorporating R13 (the dotted line shows the incorporated R13 solvent, omitted) [Figure 26] Absolute stereostructure of R13 [Figure 27] Asymmetric unit structure of a crystalline sponge incorporating R14 (the dotted line shows the incorporated R14 solvent, omitted) [Figure 28] Absolute stereostructure of R14 [Figure 29] Absolute stereostructure of R1~R16 [Figure 30] Enhancement of immediate early gene expression by KM1-1 to KM4-1 [Figure 31] Extraction, purification, and isolation from Agrimony. Overall separation and purification flow chart. DETAILED DESCRIPTION OF THE INVENTION

[0016] Each novel compound of the present invention can be obtained by extraction and purification processes using Agrimonia pilosa (scientific name: Agrimonia pilosa var. japonica) as a raw material. It can also be obtained by chemical synthesis. Furthermore, the compounds of the present invention can be isolated and purified using extracts or crude products from plants other than Agrimonia that have been confirmed to contain the compounds of the present invention, or dried plant products or plant pastes.

[0017] When extracting and purifying the compounds of the present invention from agrimony, any of the extraction and purification processes commonly used industrially can be used in combination as appropriate. The raw material, plant leaves, stems, roots, flowers, etc., are harvested at an appropriate time and then used as is or after a drying process such as forced air drying to obtain the extraction raw material. When extracting from the dried plant matter, known extraction methods can be used.

[0018] That is, after the raw material is crushed or chopped, extraction is carried out using a solvent. The extraction solvent may be water; alcohols such as ethanol, methanol, isopropyl alcohol, etc.; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; or lipophilic solvents such as hexane and chloroform, either alone or as a mixture. A mixed solvent of ethanol and water is preferred. The extraction temperature is usually 0 to 100°C, preferably 5 to 50°C. The extraction time is about 1 hour to 10 days, and the amount of solvent is usually 1 to 30 times, preferably 5 to 10 times, the weight of the dried raw material. The extraction procedure may be by stirring or by soaking and leaving the raw material. The extraction procedure may be repeated two or three times as necessary. Alternatively, commercially available extracts of agrimony may be used as raw materials for isolating and purifying the compounds. The crude extract obtained by the above procedure, from which insoluble residues have been removed by filtration or centrifugation, or the extract obtained from the plant juice may be purified by any known method for separating and purifying herbal medicines. Usually, two-phase solvent partitioning, countercurrent partitioning, column chromatography, preparative high performance liquid chromatography, etc., may be used alone or in combination. For example, two-phase solvent partitioning includes partitioning the extract between water and a solvent such as n-hexane, chloroform, methyl ethyl ketone, ethyl acetate, or methyl acetate, thereby recovering the target compound in the solvent phase. Column chromatography includes ion exchange column chromatography, methods using normal-phase or reverse-phase silica gel as a carrier, adsorption column chromatography using Diaion HP-20 or the like, and gel filtration using modified dextran gel such as Sephadex LH-20 as a carrier. These methods may be performed alone or in combination, or repeatedly. Preparative high-performance liquid chromatography includes methods using reverse-phase columns such as octadecyl silica, and methods using normal-phase columns such as silica gel.

[0019] Examples of food and drink containing the compounds of the present invention include tea made from dried agrimony, pure products of each compound, partially purified products of the novel compounds, and foods containing crude extracts from agrimony.

[0020] The tea may be used alone or in combination with other tea ingredients, such as green tea, oolong tea, pu-erh tea, black tea, roasted green tea, brown rice tea, du zhong tea, persimmon leaf tea, mulberry leaf tea, or any other tea that is normally consumed as tea.

[0021] The food or drink containing the compound of the present invention can be in any form that can be provided as a normal food, such as tea, drinks, jellies, biscuits, tablets, pills, soft capsules, hard capsules, powders, fine granules, granules, etc. Additives such as excipients, binders, lubricants, dispersants, suspending agents, emulsifiers, diluents, buffers, antioxidants, and bacteria inhibitors can also be used as secondary ingredients.

[0022] The effective intake of each novel compound from a food containing the compound of the present invention varies depending on the intake form, the health condition of the subject, the age of the subject, etc., but is generally 0.001 to 100 mg, preferably 0.01 to 10 mg, and more preferably 0.1 to 1 mg per day for an adult.

[0023] The route of administration of pharmaceuticals containing the compounds of the present invention is not particularly limited. Examples include enteral administration such as oral administration or rectal administration, mucosal administration such as nasal administration, and injection administration such as intravenous administration or subcutaneous administration. The dosage form of the pharmaceuticals of the present invention can be a formulation appropriate for the administration method. Examples include solid formulations such as tablets, powders, fine granules, granules, capsules, powders, pills, and lozenges, liquid formulations such as solutions, suspensions, emulsions, syrups, and injections, and gel formulations. Pure, purified, or crudely purified products of each compound may be administered as is, or may be administered together with a pharmacologically acceptable excipient. Furthermore, the compounds of the present invention may be contained alone as active ingredients related to neuronal activation related to memory, etc. Furthermore, other active ingredients related to nervous system activation may be used in combination. Any excipient that can be generally used in pharmaceutical formulations can be used, such as monosaccharides, disaccharides, polysaccharides, inorganic salts, fats and oils, distilled water, etc. When formulating, additives such as binders, lubricants, dispersants, suspending agents, emulsifiers, diluents, buffers, antioxidants, etc. can also be used.

[0024] The dosage of a pharmaceutical containing each compound of the present invention varies depending on the administration route, dosage form, symptoms of the disease, age of the subject, etc. Generally, it is 0.1 to 1000 mg, preferably 0.5 to 300 mg, and more preferably 1 to 100 mg per day for an adult. [Example]

[0025] The following examples show the specific compounds isolated from Agrimony, extracted and purified by the method of the present invention. Furthermore, the present invention will be explained in more detail by showing examples of neuronal activation tests using the purified compounds.

[0026] Extraction / separation / purification Five kg of ground dried agrimony (Agrimonia pilosa var. japonica) was refluxed and extracted with 10 times the volume of 99.5% ethanol for 2 hours. The resulting extract was concentrated under reduced pressure to yield 750 g of ethanol-extracted solids. The ethanol-extracted solids were applied to an open column (35 cm x 11 cm ID) packed with DIANION HP-20 (Mitsubishi Chemical) and passed through with water, 80% methanol, methanol, and acetone, in that order. The methanol and acetone eluates were combined and concentrated under reduced pressure to yield 62 g of methanol+acetone-eluted solids. To remove chlorophyll, the methanol+acetone-eluted solids were redissolved in 1 L of 95% ethanol, 30 g of activated carbon was added, and the mixture was stirred at 45°C for 30 minutes. The filtrate was then concentrated under reduced pressure to yield 24 g of activated carbon-treated product. The active fraction was applied to an open column (50 cm × 5 cm ID) packed with Chromatorex ODS (Fuji Silysia) and a methanol-water mixture (methanol:water = 60:40 → 100:0) was passed through, yielding 4.4 g of an 80% methanol elution fraction. The 80% methanol elution fraction was applied to an open column (10 cm × 5 cm ID) packed with Silica gel 60 (Merck Millipore) and a hexane-ethyl acetate mixture (hexane:ethyl acetate = 80:20 → 0:100) was passed through, yielding 2.4 g of an 80:20 hexane:ethyl acetate elution fraction. The 80:20 hexane:ethyl acetate elution fraction was subjected to preparative HPLC to obtain KM 1-4. The overall separation and purification flow chart is shown in Figure 31. The preparative HPLC conditions were as follows:

[0027] [Preparative HPLC conditions]] TIFF2025172102000006.tif57148 The obtained KM1-4 was subjected to recycle preparative HPLC to obtain KM1-1 to KM4-2, respectively. The conditions for recycle preparative HPLC are as follows.

[0028] [Recycling preparative HPLC conditions for KM 1 and KM 2] TIFF2025172102000007.tif52151

[0029] [Recycling preparative HPLC conditions for KM3 and KM4] TIFF2025172102000008.tif52145 The optical purity of the resulting KM1-1 to KM4-2 was further investigated using a chiral column. The results revealed that KM1-1 was a racemate consisting of R1 and R2, KM1-2 was a racemate consisting of R3 and R4, KM2-1 was a racemate consisting of R5 and R6, KM2-2 was a racemate consisting of R7 and R8, KM3-1 was a racemate consisting of R9 and R10, KM3-2 was a racemate consisting of R11 and R12, KM4-1 was a racemate consisting of R13 and R14, and KM4-2 was a racemate consisting of R15 and R16 (Figure 1-8). The HPLC conditions were as follows:

[0030] [HPLC analysis conditions using chiral columns KM 1-1 to KM 4-2] TIFF2025172102000009.tif62159

[0031] Planar structure determination The planar structures of KM-1-1 to KM-4-2 were determined by mass spectrometry (electrospray ion-time of flight mass spectrometer (ESI-TOFMS), ACQUITY UPLC / Xevo G2 Tof, Waters) and nuclear magnetic resonance spectroscopy (NMR, JNM-ECS 400, JEOL). 1D NMR of each separated and purified compound ( 1 H-NMR, 13 The planar structural formula determined from C-NMR, two-dimensional NMR (COSY, HSQC, HMBC, NOESY), and high-resolution ESI-TOFMS (positive) data is shown below.

[0032] [ka] KM 1-1 Molecular formula: C 25 H 30 O9 HR positive-ion ESI-MS: calculated for C 25 H 31 O9: m / z 475.1968 [M+H] + , found: 475.1953

[0033]

Chem.

[0034]

Chem.

[0035]

Chem.

[0036]

Chem.

[0037]

Chem.

[0038]

Chem.

[0039]

Chem.

[0040] Relative 3D structure determination The relative conformations of the racemates KM1-1 to KM4-2 were determined using the crystal sponge method. Crystal sponges were prepared from 2,4,6-tri(4-pyridyl)-1,3,5-triazine and zinc chloride according to the method described in Chem. Eur. J. 2017, 23, 15035-15040. The incorporation (soaking) of each compound into the crystal sponge was performed according to the method described in IUCrJ 2016, 3, 139-151. Specifically, one crystal sponge grain was placed in a 1.2 mL V-bottom microvial along with 45 μL of n-hexane, and 5 μL of a dimethoxyethane solution containing each compound was added. The vial was capped and incubated at 50°C. The solvent was then slowly evaporated over 1 to 4 days using a needle. The soaking conditions for each compound are shown in Table 1.

[0041] Table 1: Soaking conditions for each compound during crystalline sponge analysis. After soaking, the crystalline sponge was mounted on a single-crystal X-ray diffractometer (Synergy-R, Rigaku) ​​and diffraction measurements were performed at 100 K. Data analysis followed the method described in IUCrJ 2016, 3, 139-151. All compounds were incorporated into the crystalline sponge, allowing their structures to be observed. The crystal data obtained from the analysis of the crystalline sponges incorporating each compound are summarized in Tables 2 and 3.

[0042] Table 2 Crystal data of KM1-1, KM1-2, KM2-1, and KM2-2 analyzed by the crystalline sponge method TIFF2025172102000018.tif223153

[0043] Table 3 Crystal data of KM3-1, KM3-2, KM4-1, and KM4-2 analyzed by the crystalline sponge method TIFF2025172102000019.tif223153

[0044] The asymmetric unit obtained when analyzing the crystalline sponge incorporating KM1-1 is shown in FIG. 9, and the relative three-dimensional structure of the incorporated KM1-1 is shown in FIG. The asymmetric unit obtained by analyzing the crystalline sponge incorporating KM1-2 is shown in FIG. 11, and the relative three-dimensional structure of the incorporated KM1-2 is shown in FIG. The asymmetric unit obtained by analyzing the crystalline sponge incorporating KM2-1 is shown in FIG. 13, and the relative three-dimensional structure of the incorporated KM2-1 is shown in FIG. The asymmetric unit obtained by analyzing the crystalline sponge incorporating KM2-2 is shown in FIG. 15, and the relative three-dimensional structure of the incorporated KM2-2 is shown in FIG. The asymmetric unit obtained by analyzing the crystalline sponge incorporating KM3-1 is shown in FIG. 17, and the relative three-dimensional structure of the incorporated KM3-1 is shown in FIG. The asymmetric unit obtained when analyzing the crystalline sponge incorporating KM3-2 is shown in FIG. 19, and the relative three-dimensional structure of the incorporated KM3-2 is shown in FIG. The asymmetric unit obtained by analyzing the crystalline sponge incorporating KM4-1 is shown in FIG. 21, and the relative three-dimensional structure of the incorporated KM4-1 is shown in FIG. The asymmetric unit obtained by analyzing the crystalline sponge incorporating KM4-2 is shown in FIG. 23, and the relative three-dimensional structure of the incorporated KM4-2 is shown in FIG.

[0045] Absolute 3D structure determination Racemic KM4-1 was resolved into its enantiomers, R13 and R14, as shown in Figure 7, and the enantiopure R13 and R14 were analyzed using the crystalline sponge method. The incorporation of R13 or R14 changed the space group of the crystalline sponge from achiral C2 / c to chiral C2. The crystallographic data obtained from the analysis of the crystalline sponges incorporating each compound are summarized in Table 4.

[0046] TIFF2025172102000020.tif234163

[0047] The asymmetric unit obtained by analyzing the crystalline sponge incorporating R13 is shown in FIG. 25, and the absolute three-dimensional structure of the incorporated R13 is shown in FIG. The asymmetric unit obtained by analyzing the crystalline sponge incorporating R14 is shown in FIG. 27, and the absolute three-dimensional structure of the incorporated R14 is shown in FIG. From the above results, it was found that under the analytical conditions using the chiral column described in Figures 1 to 8, the enantiomer with the 4aR,10aS configuration of the pyranobenzopyran ring elutes early, and the enantiomer with the 4aS,10aR configuration elutes late. The absolute stereostructures of R1 to R16 are summarized in Figure 29.

[0048] Verification of immediate early gene expression enhancement Fetuses were removed from 17-day-pregnant SD rats (Japan SLC), and the cerebral cortex and hippocampus were isolated. Primary neurons were then prepared using a neuronal cell suspension kit (Sumitomo Bakelite) according to the manufacturer's instructions. The prepared rat primary neurons were cultured at a concentration of 4 x 10 in Neurobasal medium (Gibco) containing 2% B27 (Gibco), 0.5 mM L-glutamine (Gibco), and 1% penicillin-streptomycin (Sigma). 5 The cells were suspended to a concentration of 100 cells / ml, and 350 μl of each was seeded onto a poly-L-lysine-coated 48-well plate (Sumitomo Bakelite), followed by incubation at 37°C under 5% CO 2 for 7 days. KM1-1 (3 μM), KM1-2 (30 μM), KM2-1 (10 μM), KM2-2 (1 μM), KM3-1 (3 μM), KM3-2 (3 μM), KM4-1 (3 μM), and KM4-2 (3 μM) were added to the cells and incubated for 1 or 2 hours (KM1-1, 1-2). RNA was then extracted using the RNeasy Mini kit (QIAGEN) according to the manufacturer's instructions. Approximately 50 ng of the extracted RNA was used to prepare cDNA using the PrimeScript RT reagent kit (Takara) according to the manufacturer's instructions. The expression level of c-fos was quantified using Applied Biosystems TaqMan Gene Expression Assay and a rat GAPDH TaqMan probe as an endogenous control. 1 μL of cDNA, rat GAPDH TaqMan probe, rat c-fos TaqMan probe, and Premix Ex Taq were mixed in the specified ratios to make a total volume of 10 μL. The reaction mixture was analyzed using Qunti Studio 5 (Applied Biosystems) under the following reaction conditions: (95°C, 20 seconds) → (95°C, 1 second) → (60°C, 20 seconds) × 45 cycles. The relative gene expression level of each sample was calculated from the Ct values ​​obtained by the analysis using GAPDH as an internal standard by the ΔΔCt method.

[0049] The measurement results are shown in Figure 30. From the above tests using rat primary nervous system culture cells, it was found that each compound activates nerves by increasing the expression of the immediate early gene c-fos.

Claims

1. A compound represented by chemical formula 3. 【Chemistry 1】 (Chemical formula 3)

2. A compound represented by chemical formula 4. 【Chemistry 2】 (Chemical formula 4)

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