Geranylnerol glycoside or its geometric isomer

Abstract

To provide a novel geranyl nerol glycoside or geometric isomer thereof useful as an antibacterial agent, a food additive, or an emulsifier.SOLUTION: A compound of the present invention is represented by the following general formula (1) [where, the wavy line is a single bond; the configuration of the double bond to which it is bonded represents E configuration or Z configuration, each independently; the group R represents a monosaccharide moiety excluding the 1-position hydroxyl group; and the bond of the monosaccharide moiety is an α-bond or a β-bond].SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to geranyl nerol glycoside or a geometric isomer thereof. [Background technology]

[0002] Human mycoses are broadly divided into superficial mycoses of the skin and deep mycoses of organs and the whole body, the latter of which are more lethal. Most deep mycoses are opportunistic infections, such as aspergillosis, candidiasis, and Schizophyllum commune infection.

[0003] In a search for candidate pathogenic compounds of Schizophyllum commune, the causative fungus of Schizophyllum commune infection, the inventors cultivated Schizophyllum commune at both 27°C and 37°C, and searched for compounds that were significantly produced at 37°C, the internal environment, using HPLC analysis and bioactivity such as antibacterial activity as indicators. As a result, they discovered a new acyclic diterpene glycoside (glycoside), geranylnerol β-D-mannoside (2Z,6E,10E), which exhibits antibacterial activity. Furthermore, we synthesized two geometric isomers of the above-mentioned novel natural compound, geranylgeraniol β-D-mannoside (2E,6E,10E) and geranylgeraniol α-D-mannoside (2E,6E,10E), and confirmed that these newly synthesized compounds exhibited higher antibacterial activity.

[0004] In relation to the novel natural and synthetic compounds that the present inventors have obtained for the first time, four plant components are known to have a disaccharide glycoside structure of geranylgeraniol: vernanolide (Non-Patent Document 1), cupasinoside with anthelmintic activity

[12] , 6'-de-O-acetylcupasinoside with anthelmintic activity (Non-Patent Document 2), and 2",3",4",6'-de-O-acetylcupasinolide with antiproliferative activity (Non-Patent Document 3). However, the monosaccharide glycoside of geranylgeraniol or its geometric isomer glycoside (for example, the monosaccharide glycoside of geranylnerol) has not been known as either a natural compound or a synthetic compound.

[0005] Meanwhile, the monosaccharide glycosides of the acyclic sesquiterpene farnesol, namely farnesol β-D-glucoside, farnesol β-D-mannoside, and farnesol β-D-galactoside, have already been synthesized (Non-Patent Document 4), and farnesol β-D-glucoside has been reported to have inhibitory activity against aldose reductase (Non-Patent Document 5).

[0006] Furthermore, with regard to geranylgeraniol and geranylnerol, which are the aglycones in the novel compounds of the present invention, the former, geranylgeraniol, has been reported to have various biological activities, such as antibacterial, anti-inflammatory, antitumorogenic, neuroprotective, and testosterone production regulation (Non-Patent Document 6), whereas research on the latter, geranylnerol, has not progressed much, and its activity as a heat shock protein inducer is known (Patent Document 1). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2001-172171 [Non-patent literature]

[0008] [Non-Patent Document 1] Cavalvanti SBT et al. New tetra-acetylated oligosaccharide diterpene from Cupania vernalis. J Braz Chem Soc. 2001; 12:413-16. [Non-patent document 2] Gachet MS et al. Antiparasitic compounds from Cupania cinerea with activities against Plasmodium falciparum and Trypanpsoma brucei Rhodesian. J Nat prod. 2011;74:559–66.

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[0009] As described above, the present inventors have discovered the natural compound geranylnerol β-D-mannoside (2Z,6E,10E) for the first time from Schizophyllum commune. The compound was unexpectedly found to have antibacterial activity during a search for candidate compounds for the pathogenic factor of Schizophyllum commune, the causative agent of Schizophyllum commune infection. Furthermore, as is clear from its structural formula, the compound exhibits surface activity, making it suitable for use as a food additive or emulsifier.

[0010] Geranylgeraniol and geranylnerol, which are the aglycones in the novel compounds of the present invention, and compounds having a disaccharide glycoside structure of geranylgeraniol, were already publicly known at the time of filing of this application. However, the monosaccharide glycoside of geranylgeraniol or its geometric isomeric glycoside (e.g., the monosaccharide glycoside of geranylnerol) was not publicly known, and its existence as a natural compound in Schizophyllum commune was not predicted from the prior art.

[0011] In particular, the novel natural compound of the present invention was isolated and purified from Schizophyllum commune by cultivating Schizophyllum commune at both 27°C and 37°C, and focusing on the compound that was produced significantly at 37°C, the internal environment of the mushroom. Without using this method, the compound would not be easily obtained in terms of production volume by cultivating Schizophyllum commune only at 27°C, the mushroom's natural growth environment, and the novel compound of the present invention is therefore a significant inventive step.

[0012] Under these circumstances, an object of the present invention is to provide a novel geranyl nerol glycoside or a geometric isomer thereof that is useful as an antibacterial agent, a food additive, or an emulsifier. [Means for solving the problem]

[0013] The above-mentioned problems can be solved by the present invention as follows: [1] General formula (1): [ka] (In the formula, the wavy lines represent single bonds, and the configurations of the double bonds to which they are attached are independently E or Z configurations. The group R represents a monosaccharide moiety excluding the hydroxyl group at the first position, and the bond of the monosaccharide moiety is an α-bond or a β-bond.) or a mixture thereof in any proportion. [2] A pharmaceutical composition containing the compound or mixture of [1] above as an active ingredient. [3] An antibacterial agent containing the compound or mixture of [1] above as an active ingredient. [4] A food additive containing the compound or mixture described in [1] above. [5] An emulsifier comprising the compound or mixture of [1] above. [Effects of the Invention]

[0014] According to the present invention, it is possible to provide a novel geranyl nerol glycoside or a geometric isomer thereof which is useful as an antibacterial agent, a food additive, or an emulsifier. [Brief explanation of the drawings]

[0015] [Figure 1] 1H-1H COSY spectrum (CDCl3) of compound 1. [Figure 2] 1H NMR spectrum (500 MHz, CDCl3) of Compound 1. [Figure 3] 13C NMR spectrum (125 MHz, CDCl3) of Compound 1. [Figure 4] 1 is an HMQC spectrum (CDCl3) of compound 1. [Figure 5] 1 is an HMBC spectrum (CDCl3) of compound 1. [Figure 6] This is a summary of the two-dimensional NMR spectrum data used in the structural analysis of compound 1. [Figure 7]1H NMR spectrum (500 MHz, CDCl3) of compound 2. [Figure 8] 13C NMR spectrum (125 MHz, CDCl3) of compound 2. [Figure 9] 1H NMR spectrum (500 MHz, CDCl3) of compound 3. [Figure 10] 13C NMR spectrum (125 MHz, CDCl3) of compound 3. DETAILED DESCRIPTION OF THE INVENTION

[0016] (Compounds of the present invention) General formula (1): [ka] (In the formula, the wavy lines represent single bonds, and the configurations of the double bonds to which they are attached are independently E or Z configurations. The group R represents a monosaccharide moiety excluding the hydroxyl group at the first position, and the bond of the monosaccharide moiety is an α-bond or a β-bond.) The compound of the present invention, represented by the formula (I), is an acyclic diterpene glycoside. Geranylnerol β-D-mannoside, whose aglycone (general formula (1) has a hydrogen atom attached to the non-monosaccharide portion excluding the group R) is geranylnerol (2Z,6E,10E), is a novel compound discovered by the present inventors for the first time from Schizophyllum commune, and all of its geometric isomers are also novel compounds.

[0017] In the compound represented by general formula (1), the three double bonds (positions 2, 6, and 10, in order from the side closest to the glucoside bond) connected to the single bond represented by the wavy line can each independently be in the E or Z configuration. Therefore, there are eight geometric isomers of the aglycone moiety with respect to positions 2, 6, and 10: (2Z,6E,10E), (2E,6E,10E), (2Z,6Z,10E), (2E,6E,10Z), (2E,6E,10Z), (2Z,6Z,10Z), and (2E,6Z,10Z). The known aglycone geranylnerol is (2Z,6E,10E), and the known aglycone geranylgeraniol is (2E,6E,10E).

[0018] The group R in general formula (1) is not particularly limited as long as it is a monosaccharide moiety obtained by removing the first-position hydroxyl group from a monosaccharide. For example, it can be a monosaccharide moiety obtained by removing the first-position hydroxyl group from a monosaccharide selected from the group consisting of monosaccharides having 3 to 7 carbon atoms constituting the sugar skeleton, typically hexasaccharide aldoses and ketoses such as allose, altrose, glucose, mannose, gulose, idose, galactose, talose, psicose, fructose, sorbose, and tagatose, pentasaccharide aldoses and ketoses such as ribose, arabinose, xylose, lyxose, ribulose, and xylylose, as well as derivatives such as deoxysugars and aminosugars of these unsubstituted hexasaccharides and pentasaccharides, and derivatives of all of these monosaccharides in which some or all of the hydroxyl groups or amino groups have been modified as esters, ethers, amides, or the like. The glycosidic bond between the aglycone moiety and the monosaccharide moiety in general formula (1) can be an α-bond or a β-bond.

[0019] The present invention includes not only individual compounds represented by general formula (1), but also mixtures of two or more of these compounds in any ratio. In the case of a mixture of three or more compounds, the ratios of any two or more of the compounds may be the same, or the ratios may all be different.

[0020] Among the compounds of the present invention represented by general formula (1), geranylnerol β-D-mannoside (2Z,6E,10E) can be isolated and purified from a culture of Schizophyllum commune obtained by culturing it on a solid medium at 37°C, as shown in Example 1 below. For example, a neutral fraction can be obtained by extracting a culture (including the bacterial cells and medium) grown on solid medium at 37°C for 30 to 40 days with methanol, and then extracting the resulting methanol extract with ethyl acetate. The resulting neutral fraction can be purified by two-step HPLC according to the conditions described in Example 1 to obtain geranylnerol β-D-mannoside (2Z, 6E, 10E).

[0021] The eight geometric isomers of the aglycone moiety in the compound represented by general formula (1) were separated from geranylgeraniol by a known method of high-performance liquid chromatography after ultraviolet irradiation (Yasuyuki Tanaka, Hisaya Sato, Akira Kagefu, and Toshihiko Tomita, Separation of geranilgeraniol isomers by high-performance liquid chromatography and identification by 13 C nuclear magnetic resonance spectroscopy, Journal of Chromatography, 347, 275-283, 1985.) Each of the prepared geranylgeraniol geometric isomers is glycosylated by reaction with a monosaccharide donor in which an activating group such as trichloroacetimidate has been introduced at the 1-position and the other hydroxyl groups have been protected with acetyl groups or the like, followed by deprotection of the hydroxyl groups, thereby preparing monoglycosides in which various monosaccharides are bound to eight geometric isomers of geranylgeraniol, including geranylneol.

[0022] (Pharmaceutical composition of the present invention) The compound of the present invention represented by general formula (1), or a mixture thereof in any ratio, can be used as an active ingredient of the pharmaceutical composition of the present invention. The pharmaceutical composition of the present invention can be administered to a subject (e.g., an animal, preferably a mammal, particularly a human) in an effective amount as a medicament, either alone or preferably together with a pharmaceutically acceptable carrier or diluent, or can be provided in the form of a food or drink.

[0023] When the pharmaceutical composition of the present invention is provided as a medicine, it can be administered as an oral preparation, an injection, an external preparation, etc., and is preferably an oral preparation. Examples of the oral preparation include liquid preparations such as suspensions, emulsions, syrups, and extracts, and solid preparations such as tablets, capsules, powders, fine granules, and granules. Examples of the external preparation include ointments, eye drops, ear drops, nasal drops, and inhalants.

[0024] Examples of pharmaceutically acceptable carriers or diluents include excipients, disintegrants, binders, flavoring agents, effervescent agents, sweeteners, flavoring agents, lubricants, buffers, antioxidants, surfactants, and fluidizing agents.

[0025] The dosage of the pharmaceutical composition of the present invention can be appropriately selected depending on, for example, the target disease, the patient's condition, body weight, age, sex, route of administration, etc., but generally the lower limit of the dosage can be selected from the range of 0.001 μg to 0.1 μg per day for an adult, and the upper limit of the dosage can be selected from the range of 100 μg to 10,000 μg per day for an adult, and the composition can be administered in 1 to 3 divided doses per day.

[0026] The pharmaceutical composition of the present invention can be used as an antibacterial agent.

[0027] (Food additive or emulsifier of the present invention) The compound of the present invention represented by general formula (1) exhibits surfactant activity because it is composed of a hydrophobic aglycone moiety and a hydrophilic monosaccharide moiety. Therefore, the compound of the present invention represented by general formula (1) or a mixture thereof in any ratio can be used as the food additive of the present invention or the emulsifier of the present invention. However, the emulsifier of the present invention is not limited to use in food. [Example]

[0028] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0029] Example 1: Isolation and identification of geranylnerol β-D-mannoside (2Z,6E,10E) (1) Small-scale cultivation of Schizophyllum commune and HPLC analysis A stock mycelium suspension of Schizophyllum commune (Schizophyllum commune LSEM 2741 strain; Ogawa H et al. The influence of Schizophyllum commune on asthma severity. Lung. 2011;189:485-492) was inoculated onto potato dextrose agar solid medium (9 cm diameter Petri dish, 25 mL) and cultured at 37°C and 27°C for 30 days. Each culture (mycelium and medium) was extracted with methanol, and the resulting methanol extract was extracted with ethyl acetate to obtain a neutral fraction. HPLC analysis of the resulting neutral fraction (detected at 210 nm) revealed multiple peaks that were detected only in the 37°C culture.

[0030] (2) Mass cultivation of Schizophyllum commune and fractionation of the culture To isolate the peak detected only in the 37°C culture, Schizophyllum commune LSEM2741 strain was inoculated into 35 petri dishes (9 cm diameter, 25 mL of potato dextrose agar solid medium per dish, total volume 875 mL) and cultured at 37°C. The entire culture was collected, extracted twice with methanol (1000 mL), and concentrated to 60 mL. Water was added to make 200 mL, the pH was adjusted to 2, and the extract was three times with ethyl acetate (100 mL). After washing three times with saturated aqueous NaHCO3, the ethyl acetate solution was dried and evaporated to obtain a residue (59.2 mg).

[0031] The resulting residue was dissolved in 90% CH3CN (6.0 mL) and purified by two-stage HPLC. The first-stage column was a 250 mm x 4.6 mm i.d. Capsule Pack C18 (Osaka Soda), and the second-stage column was a 250 mm x 4.6 mm i.d. Senshu Pack C22 (Senshu Scientific). The mobile phase flow rate was 1 mL / min (first and second stages: 100% HO for 2 min isocratic elution, 0-100% CH3CN in HO for 15 min gradient elution, 100% CH3CN for 23 min isocratic elution). Detection was at 210 nm. The retention times were 20.7 min for the first stage and 22.4 min for the second stage. 1.4 mg of compound 1 was obtained as a colorless oil. ESIMS m / z 475.3030 (M+Na) + (calcd for C 26 H 44 NaO6, 475.3036); [α] 23 D -31 (c = 0.36, MeOH); UV end absorption (CH3CN); IR νmax (film) cm -1 3387, 1666, 1447, 1377, 1076, 1026.

[0032] (3) Identification of isolated compounds High-resolution ESIMS analysis showed that compound 1 is C 26 H 44 The compound 1 was found to have the molecular formula O6.1 H- 1 The H COSY spectrum (Figure 1) shows that compound 1 has six monosaccharides bound as pyranose residues. 1 Coupling constants (J H-1’,H-2’ =<1Hz,J H-2’,H-3’ =2.5Hz,J H-3’,H-4’ =9Hz,J H-4’,H-5’ =9 Hz), it was found that the linked sugar was mannose. The binding mode of the mannose residue was 1 J C,H From the value (154Hz), it was clear that it was β. 13 Analysis of the C NMR, HMQC, and HMBC spectra (Figures 3, 4, and 5) revealed that the aglycone moiety of compound 1 was geranylnerol. Long-range coupling was observed in the HMBC spectrum between the anomeric protons of the mannose residue and the oxymethylene of geranylnerol, confirming that mannose was glycosidicly linked to geranylnerol in compound 1. The mannopyranosylgeranylnerol structure contains all atoms present in the molecular formula of compound 1, confirming that the structure of compound 1 is mannopyranosylgeranylnerol. The absolute configuration of the mannose moiety in compound 1 was determined to be D-isomer by preparing 1,2,3,4,6-pentabenzoyl-α-mannose from compound 1 and comparing the CD spectrum with that of an authentic sample.

[0033] From the above, the structure of compound 1 was determined to be geranylnerol β-D-mannopyranoside. Figure 6 shows the two-dimensional NMR spectrum data used in the structural analysis. Table 1 summarizes the assignments in the NMR spectrum.

[0034] [Table 1]

[0035] Example 2: Geranylgeraniol β-D-mannoside (2E, 6E, 10E) and Synthesis of Geranylgeraniol α-D-Mannoside (2E, 6E, 10E) Under a nitrogen gas flow, trichloroacetonitrile (1.43 mL, 14.3 mmol, Fujifilm Wako Pure Chemical Industries) and 1,8-diazabicylo[5.4.0]undec-7-ene (0.18 mL, 1.2 mmol, Sigma-Aldrich) were added dropwise to a solution of 2,3,4,6-tetra-O-acetyl-D-mannopyranose (0.83 g, 2.4 mmol, Thermo Fisher Scientific) in anhydrous dichloromethane (15 mL, Kanto Chemical) at -10 °C. The reaction mixture was gradually warmed to room temperature and stirred overnight at room temperature. The reaction mixture was concentrated and purified by silica gel column chromatography (150 g, silica gel 60, Merck) using hexane-ethyl acetate (4:1, v / v) as the eluent to yield 0.91 g (1.9 mmol, 79%) of 2,3,4,6-tetra-O-acetyl-D-mannopyranosyl trichloroacetimidate.

[0036] A solution of 2,3,4,6-tetra-O-acetyl-D-mannopyranosyl trichloroacetimidate (0.26 g, 0.54 mmol) and geranylgeraniol (0.1 g, 0.34 mmol, Sigma-Aldrich) in 8 mL of anhydrous dichloromethane was added with a small amount of molecular sieves 4A (Sigma-Aldrich) and stirred at room temperature for 40 min under a nitrogen gas stream. The reaction mixture was cooled to -10 °C, and boron trifluoride diethyl etherate (0.07 mL, Sigma-Aldrich) was added. The mixture was gradually warmed to room temperature with stirring and stirred overnight. 10 mL of dichloromethane was added to the reaction mixture, and the molecular sieves 4A were filtered off. The dichloromethane solution was washed with 10% sodium thiosulfate and water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield 0.25 g of residue.

[0037] The residue was purified by silica gel column chromatography (20 g, silica gel 60, eluent: hexane-ethyl acetate (1:1, V / V)) to obtain 125 mg of crude 2,3,4,6-tetra-O-acetyl-D-mannopyranosyl geranylgeraniol fraction. The obtained crude fraction was dissolved in 5 mL of dehydrated methanol (Kanto Chemical), and a small amount of sodium methoxide (Tokyo Chemical Industry) was added. After confirming that the solution became basic with pH test paper, the fraction was immediately transferred to a cation exchange resin (H + A small amount of Dowex 50Wx8 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to neutralize the solution.

[0038] The reaction mixture was filtered off and purified by silica gel column chromatography (20 g, silica gel 60, eluent: ethyl acetate) to obtain 25 mg of crude geranylgeraniol D-mannoside. The crude fraction was purified by HPLC (column: Senshu Pak C22, 4.6 x 250 mm, Senshu Scientific; elution: 0-2 min 100% water, 2-17 min 0-100% acetonitrile in water gradient elution, 17-40 min 100% acetonitrile; flow rate: 1 mL / min; detection: UV 210 nm) to obtain 0.7 mg and 3.4 mg of geranylgeraniol β-D-mannoside (hereinafter, compound 2) and geranylgeraniol α-D-mannoside (hereinafter, compound 3), respectively (retention times: 22.0 min and 22.5 min).

[0039] Geranylgeraniol β-D-mannoside: ESIMS m / z 475.3005 (M+Na) + (calcd for C 26 H 44 NaO6, 475.3036); 1 H NMR spectrum (Figure 7); 13 C NMR spectrum (Figure 8); assignment of hydrogen and carbon signals in the NMR spectrum (Table 2) Geranylgeraniol α-D-mannoside: ESIMS m / z 475.3004 (M+Na) + (calcd for C 26 H 44NaO6, 475.3036); 1 H NMR spectrum (Figure 9); 13 C NMR spectrum (Figure 10); assignment of hydrogen and carbon signals in the NMR spectrum (Table 2)

[0040] [Table 2]

[0041] Example 3: Evaluation of the antibacterial activity of geranylnerol β-D-mannoside (2Z, 6E, 10E) and its analogues The antibacterial activities of geranylnerol β-D-mannoside (2Z, 6E, 10E; Compound 1) isolated and purified in Example 1, geranylgeraniol β-D-mannoside (2E, 6E, 10E; Compound 2) synthesized in Example 2, and geranylgeraniol α-D-mannoside (2E, 6E, 10E; Compound 3) were evaluated by a standard disk diffusion assay. The Gram-positive bacteria used for evaluation were Kocuria rhizophila (ATCC9341) and Bacillus subtilis (KB211 (ATCC6633)).

[0042] Each strain was cultured in 10 mL of Mueller-Hinton broth (BBL TM After pre-cultivation for 18 hours at 37°C and 230 rpm in test tubes containing Mueller Hinton Broth (Becton, Dickinson and Company), the pre-cultivation broth was added to nutrient agar medium to a concentration of 3%, which was used to prepare assay plates (10 mL per 9 cm diameter Petri dish). A two-fold dilution series of the test sample was prepared, and 30 μL of methanol solution was dropped onto each paper disk (Advantec). After drying, the disk was placed on an assay plate. After standing overnight at 37°C, the diameter of the growth inhibition zone on the plate was measured.

[0043] Table 3 shows the diameters of the inhibition zones of Compounds 1 to 3 at 30 μg per disk.

[0044] [Table 3] [Industrial Applicability]

[0045] The compounds of the present invention can be used as active ingredients in pharmaceutical compositions, particularly antibacterial agents, or as food additives or emulsifiers.

Claims

1. General formula (1): 【Chemistry 1】 (In the formula, the wavy lines represent single bonds, and the configurations about the double bonds to which they are attached are independently E or Z configurations. The group R represents D-mannose from which the hydroxyl group at the first position has been removed, and the bond of the D-mannose from which the hydroxyl group at the first position has been removed is an α-bond or a β-bond.) or a mixture thereof in any proportion.

2. A pharmaceutical composition comprising the compound or mixture according to claim 1 as an active ingredient.

3. An antibacterial agent comprising the compound or mixture according to claim 1 as an active ingredient.

4. A food additive comprising the compound or mixture of claim 1.

5. An emulsifier comprising a compound or mixture according to claim 1.

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