Antifungal compound and composition containing the same

Abstract

To provide a compound having antifungal activity and a composition containing the same.SOLUTION: The invention provides: a compound represented by the formula (I) in the figure; and an antifungal agent and a pharmaceutical composition that comprise the compound. The pharmaceutical composition may be for use in the treatment or prevention of tinea.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present disclosure relates to a compound having antifungal activity and a composition containing the same.

Background Art

[0002] Diseases caused by fungal infections are called mycosis. Mycosis is divided into deep mycosis (which can generally cause severe symptoms) in which fungi infect deep parts of the body such as the lungs and brain, and superficial mycosis in which fungi infect the body surface such as the skin. Among these two types of mycosis, about 99.9% is superficial mycosis, and among them, mycosis called tinea is considered to account for about 87%. Therefore, most of mycosis is tinea.

[0003] Tinea is a mycosis caused by infection with tinea fungi, and is divided into four types: tinea pedis, onychomycosis, tinea corporis, and tinea cruris according to the infection site. In Japan, tinea pedis and onychomycosis are the main types of tinea, and according to the data in 2007, the number of patients is estimated to be about 25 million and about 12 million respectively, which is very large.

[0004] It is predicted that the market size of onychomycosis treatment drugs in Japan will almost double from 2017 to 2025. Currently marketed antifungal drugs include luliconazole, lanoconazole, ketoconazole, neticonazole, and bifonazole, which are imidazole-based drugs, ciclopirox, which is a triazole-based drug, amorolfine, which is a morpholine-based drug, liranaftate, which is a thiocarbamate-based drug, terbinafine, which is an allylamine-based drug, and butenafine, which is a benzylamine-based drug. These antifungal drugs are considered to act by inhibiting the biosynthesis of ergosterol that constitutes the fungal cell membrane, and are classified into the above six groups based on their structural characteristics.

[0005] Thus, at least 10 types of antifungal drugs for tinea have been marketed. However, since these existing drugs are considered to have similar mechanisms of action and / or structures, if resistant bacteria emerge, the effectiveness of any of these drugs may decrease. In addition, despite the availability of treatment drugs for tinea as described above, there are also survey results showing that the proportion of tinea patients among patients visiting dermatology departments is increasing. Against this background, the development of antifungal drugs with mechanisms of action and / or structures different from those of existing drugs is eagerly awaited.

[0006] Several bioactive substances derived from natural products with antifungal activity are known. For example, Patent Document 1 describes that dihydrochalcone compounds contained in polar organic solvent extracts of Eucalyptus plant branches and leaves have antifungal effects, and Patent Document 2 describes that extracts of Primulaceae plants have antifungal effects.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] Various embodiments of the present disclosure aim to provide a compound having antifungal activity and a composition containing the same.

Means for Solving the Problems

[0009] The inventors discovered that an extract of shiitake mushrooms (fungi) contains a substance having antifungal activity. Furthermore, when the active compound contained in the extract was isolated and identified, surprisingly, it was a novel compound with a structure different from any of the compounds that have been marketed so far.

[0010] The present disclosure includes the following embodiments. [1] Compound represented by the following formula (I)

Chemical formula

Chemical formula

Chemical formula

Brief description of the drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0012] Hereinafter, embodiments of the present disclosure will be described in detail, but these are merely examples and the present invention is not limited to these examples.

[0013] Some aspects of the present disclosure provide a novel compound represented by the following structural formula (I) and an antifungal agent containing the compound as an active ingredient. Hereinafter, this compound may also be referred to as compound A in this specification. Compound A is found, for example, in the polar organic solvent extract of Auricularia heimuer or its culture filtrate.

Chemical formula

[0014] The above compound can be obtained by extraction from a mushroom of the family Tremellaceae or its mycelial culture filtrate. Here, the mushroom is preferably a mushroom of the genus Auricularia, and Auricularia heimuer is particularly preferred. As understood by those skilled in the art, the culture filtrate is a liquid obtained by filtering mycelia from the culture solution after culturing the mushroom in a liquid medium. The culture filtrate contains substances derived from the mushroom.

[0015] In one embodiment, the extract is obtained from the filtrate of the cultured mycelium of shiitake mushrooms, but the possible extraction sources are not limited thereto. For example, the mycelium of shiitake mushrooms may be used to obtain the extract. The shiitake mushrooms or their culture filtrate (hereinafter collectively referred to as the shiitake mushroom sample) may be used as they are to obtain the extract, but the treated product of the shiitake mushroom sample, for example, the one subjected to drying, pulverization, sterilization or disinfection, heating, and / or freezing, more specifically, for example, a dry powder may be used to obtain the extract. In the present disclosure, the "extract" refers to a group of substances derived from the extraction source that are separated from the extraction source by being dissolved in an extraction solvent.

[0016] The extract containing compound A can be obtained by extracting shiitake mushrooms or their culture filtrate with a polar organic solvent (for example, a halogenated hydrocarbon, an ether, a lower fatty acid ester, a ketone, a lower alcohol or a mixed solvent thereof), or a mixed solvent of a polar organic solvent and a non-polar organic solvent. The obtained extract may be concentrated under reduced pressure. The extract can also be fractionated by a method known to those skilled in the art such as silica gel chromatography to further purify compound A.

[0017] Extraction methods known to those skilled in the art can be used. For example, a method of immersing the shiitake mushroom sample in the extraction solvent, or a method of performing extraction while heating and stirring at a temperature below the boiling point of the extraction solvent can be used.

[0018] Examples of solvents for extraction include halogenated hydrocarbons such as chloroform, dichloromethane, dichloroethane, and trichloroethane; lower fatty acid esters such as methyl acetate, ethyl acetate, and butyl acetate; lower alcohols such as methanol, ethanol, and propanol; ethers such as methyl ether, ethyl ether, tetrahydrofuran, and dioxane; and ketones such as acetone and methyl ethyl ketone. However, the solvents are not limited to these, and a mixed solvent of any of these polar organic solvents, or a mixed solvent of a polar organic solvent and a non-polar organic solvent may be used. The extraction solvent preferably contains at least one of methanol and ethyl acetate, and more preferably contains ethyl acetate. For example, extraction may be performed with ethyl acetate alone.

[0019] The addition amount of the extraction solvent to the lichen sample is preferably 0.1 to 10 times the amount, for example, based on weight or volume, and may be 0.3 to 8 times the amount or 0.5 to 7 times the amount.

[0020] As an example of the method for producing Compound A, a method for obtaining Compound A by extraction from a lichen culture filtrate will be described in more detail below. However, the possibility that Compound A is produced by other methods, such as chemical synthesis, is not excluded.

[0021] First, the lichen culture filtrate is extracted with a polar organic solvent such as a halogenated hydrocarbon, a lower fatty acid ester, a lower alcohol, an ether, or a ketone, and the extract is concentrated under reduced pressure to obtain an extract. The extract thus obtained is subjected to chromatography known to those skilled in the art (typically column chromatography, but not limited thereto), such as adsorption chromatography using celite, florisil, silica gel, alumina, etc., partition chromatography using ODS, etc., ion exchange chromatography using DEAE, etc., or molecular sieve chromatography using LH-20, etc. to obtain a crude purified product. Further, by combining multiple types of chromatography, for example, by additional purification using thin layer chromatography such as silica gel thin layer chromatography or high performance liquid chromatography, a purer Compound A can be obtained.

[0022] Examples of column chromatography that can be used to obtain a crude product include adsorption chromatography such as celite, florisil, silica gel, etc., and reverse phase chromatography such as ODS. In celite column chromatography, the extract can be adsorbed onto a column filled with celite, and the active compound can be separated and eluted with a mixed solution of hexane-ethyl acetate or the like. In florisil chromatography, the extract can be adsorbed onto a column filled with florisil, and the active compound can be separated and eluted with a mixed solution of hexane-ethyl acetate or the like. In silica gel column chromatography, the extract can be adsorbed onto a column filled with silica gel, and the active compound can be separated and eluted with a mixed solution of hexane-ethyl acetate or the like. As the solvent here, any one of hexane, benzene, toluene, ethyl ether, ethyl acetate, acetone, dichloromethane, chloroform, ethanol, isopropanol, etc. can be used alone or in combination.

[0023] For example, in silica gel chromatography, compound A can be separated and eluted with a two-solvent system of hexane-ethyl acetate. When the volume ratio of ethyl acetate in the solvent is gradually increased, compound A can typically be separated into elution fractions of 30-70% or 40-60% ethyl acetate.

[0024] The separated compound can be identified and confirmed by a fungal growth inhibition assay as exemplified in the Examples section and / or by a structural analysis method such as LC / MS or NMR.

[0025] In one aspect, the present disclosure provides an antifungal agent comprising compound A. As used herein, "antifungal agent" means a composition used for the purpose of suppressing the growth or proliferation of fungi or killing fungi. The fungi referred to herein are particularly molds, i.e., filamentous fungi that do not form macroscopic fruiting bodies such as mushrooms, or yeast-like fungi, more typically skin-infecting fungi, preferably dermatophytosis fungi, i.e., the causative agents of dermatophytosis. It is known to those skilled in the art that there are also examples where one fungus can take both filamentous and yeast forms depending on the environment. The fungus according to this embodiment can be a fungus of the family Arthrodrmataceae including Trichophyton, Microsporum, and Epidermophyton. Trichophyton is the main dermatophyte, and related Microsporum and Epidermophyton belonging to the same family can also contribute to dermatophytosis. The fungi of the genus Trichophyton include Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Trichophyton verrucosum, and Trichophyton mentagrophytes. Trichophyton mentagrophytes is an example of the most preferred fungus. The fungi of the genus Microsporum include Microsporum canis and Microsporum gypseum. The fungus according to this embodiment can be other molds or yeasts, such as fungi of the genus Penicillium like Penicillium citrinum, fungi of the genus Cladosporium like Cladosporium sphaerospermum, etc., but is not limited thereto. In some embodiments of the present disclosure, the antifungal agent is an anti-mold agent.

[0026] The antifungal agent according to this embodiment can be added to or applied to, for example, floor mats in bathrooms, pools, etc., wet tissues, insoles of footwear, and other daily necessities that can come into contact with the human or animal body or clothing, bedding, cosmetics, cleaning supplies, sanitary products, storage supplies, etc. Further, since the active ingredient of the antifungal agent according to this embodiment is a safe one contained in edible shiitake mushrooms, it is also possible to use the antifungal agent as a food additive.

[0027] In one aspect, the present disclosure provides a pharmaceutical composition containing Compound A. The pharmaceutical composition can also be understood as a form of antifungal agent. In certain embodiments, the pharmaceutical composition can be provided as a pharmaceutical composition for use in the treatment or prevention of tinea. Typically, the pharmaceutical composition of the present disclosure is formulated as a topical agent, but embodiments in which it is administered in other forms such as oral medications are not excluded. The pharmaceutical composition or antifungal agent of the present disclosure can be formulated, for example, in the form of a liquid, powder, granule, gel, cream, ointment, etc., and embodiments in which it is applied to the affected area via a support such as a bandage, patch, pad, etc. are also contemplated.

[0028] The antifungal agent and pharmaceutical composition containing Compound A of the present disclosure can also be provided in the form of an extract of, for example, shiitake mushrooms of the family Tricholomataceae such as the genus Auricularia or its culture filtrate. The extract may be partially purified. The Compound A in the extract can preferably be purified to 10% or more, more preferably 30% or more, still more preferably 50% or more, 70% or more, or 90% or more. In the context of the extract, when Compound A is, for example, "purified to 90% or more", it means a state in which substances other than Compound A have been removed so that the proportion of Compound A in the extract is 90% by weight or more.

[0029] The antifungal agent and pharmaceutical composition of the present disclosure can contain, in addition to Compound A, a carrier or excipient known to those skilled in the art (in the case of a pharmaceutical composition, particularly a pharmaceutically acceptable carrier or excipient). In some embodiments, the antifungal agent and pharmaceutical composition can be provided in the form of an aqueous solution or aqueous dispersion.

[0030] The present disclosure is based on the discovery that extracts of Auricularia mushrooms or their culture filtrates have antifungal activity. Thus, in one aspect, the present disclosure provides a method for producing an antifungal agent (particularly an anti-dermatophyte agent) or a pharmaceutical composition (particularly a pharmaceutical composition for the treatment or prevention of dermatophytosis), which includes extracting Auricularia mushrooms or their culture filtrates with a polar organic solvent to obtain an extract containing antifungal activity (e.g., anti-dermatophyte activity). In some embodiments, a method for producing an antifungal agent or a pharmaceutical composition is provided, which includes extracting Auricularia mushrooms or their culture filtrates with a polar organic solvent to obtain an extract containing Compound A (i.e., the compound of formula (I)). The extract can provide an antifungal agent or a pharmaceutical composition as it is or after being partially purified. The Auricularia mushroom is preferably Auricularia heimuer. The polar organic solvent preferably includes ethyl acetate. The method of this embodiment may further include combining the extract with the above-described carrier or excipient.

Examples

[0031] Hereinafter, examples will be shown to explain specific embodiments in more detail, but these are merely examples, and the embodiments of the present disclosure are not limited to these specific examples. In particular, the fungi against which the extract, fraction, or compound exhibits antifungal activity are not limited to those exemplified herein. For example, growth inhibitory activity against various fungi including Penicillium genus such as Penicillium citrinum and Cladosporium genus such as Cladosporium sphaerospermum has been confirmed.

[0032] 1. Establishment of an antifungal activity evaluation method using terbinafine as a positive control A strain of Trichophyton mentagrophytes NBRC16646, which is a type of dermatophyte (hereinafter simply referred to as dermatophyte), was pre-cultured on Sabouraud agar medium. Next, a 5-mm square agar fragment of the above pre-culture containing dermatophyte was inoculated into a test tube containing Sabouraud liquid medium containing terbinafine, a known antifungal agent, at a concentration of 0 to 0.1 μg / ml, and static culture was performed at 25°C for 5 days. The Sabouraud liquid medium is an aqueous solution (pH 5.6 ± 0.1) containing 10 g of peptone and 40 g of glucose per 1000 mL, and the Sabouraud agar medium is solidified by adding 15 g of agar to this composition.

[0033] This assay was designed such that the growth of fungi in the liquid medium is suppressed when the test sample has antifungal activity (in this case, antifungal activity against dermatophytes). In fact, when dermatophytes were cultured in a liquid medium without terbinafine (i.e., terbinafine concentration of 0 μg / ml), white mold grew vigorously, whereas the growth of dermatophytes was completely suppressed in the liquid medium containing terbinafine at a final concentration of 0.1 μg / ml (0.34 μM). In the following experiments, this concentration of terbinafine was used as a positive control.

[0034] 2. Evaluation of Test Extracts Test tubes of Sabouraud liquid medium with each test extract added to a final concentration of 0.5 mg / ml were prepared (this concentration was selected assuming that the active substance is approximately 0.03% of the mass of the crude extract). The pre-culture of dermatophytes was inoculated into these liquid media as described above, and static culture was performed at 25°C for 5 days. Among the more than 160 mushroom sample extracts tested, the growth of dermatophytes was observed to be suppressed to varying degrees in only a few samples containing candidate extracts derived from different types of mushrooms. In the exemplified Figure 1, it can be seen that in most test tubes, white, fuzzy mold has grown near the liquid surface (arrow), whereas the growth of such mold is absent in the first and second test tubes from the right, and the extracts corresponding to these two test tubes were presumed to have antifungal activity.

[0035] To confirm the reproducibility of the fungal growth inhibition observed in the above primary evaluation experiment, the selected candidate extracts were retested in a similar assay for secondary evaluation. As a result, although there were samples with a lower degree of fungal growth inhibition compared to the primary evaluation (the test tubes on the left and right in Figure 2), the sample numbered 4328 consistently showed strong fungal growth inhibition (the test tube in the center of Figure 2). As judged by this assay, this was a strong antifungal activity at least comparable to the positive control terbinafine. Note that the test tube shown at the right end in Figure 1 was the sample numbered 4328. Sample 4328 is an ethyl acetate extract of the culture filtrate of Auricularia heimuer.

[0036] 3. Isolation of Antifungal Active Substances Regarding the extract of Auricularia heimuer culture filtrate, the isolation and identification of the antifungal active substance were carried out. First, 5 L of malt extract medium (800 mL of malt extract per 1 L, 3 g of polypeptone, 30 g of glucose, the rest is distilled water, pH 5.6) was prepared and dispensed into 100-ml Erlenmeyer flasks at 20 ml each. Next, each Erlenmeyer flask was inoculated with Auricularia heimuer (TUFC100803 strain) and statically cultured at 25 °C for 1 month.

[0037] After static culture, the culture solution was filtered, and the filtrate was extracted three times with an equal volume of ethyl acetate. Then, the extract was concentrated under reduced pressure to obtain an extract as a solid. 1477 mg of extract was obtained per 2 L of culture solution.

[0038] The above extract was loaded onto a silica gel column, and fractionation of the extract was performed by eluting with an ethyl acetate / hexane solution with the concentration of ethyl acetate increased stepwise to 25%, 50%, 75%, 100%, and finally methanol. When the solvents were removed from these 25% ethyl acetate fraction, 50% ethyl acetate fraction, 75% ethyl acetate fraction, 100% ethyl acetate fraction, and methanol fraction, solids of 95 mg, 75 mg, 44 mg, 60 mg, and 1145 mg were recovered, respectively.

[0039] The solids of each obtained fraction were redissolved in dimethyl sulfoxide (DMSO). A DMSO solution of each fraction (5 μg) was added to a separate well of a 24-well plate containing a liquid medium inoculated with Trichophyton fungi in the same manner as the above-described assay, and static culture was performed at 25°C for 5 days. Figure 3 shows the 24-well plate after static culture. In Figure 3, a negative control with only the DMSO solvent was added to all the wells in the second row, and to the wells in the third row, from left to right, a negative control, a 25% ethyl acetate fraction, a 50% ethyl acetate fraction, a 75% ethyl acetate fraction, a 100% ethyl acetate fraction, and a methanol fraction were added. It can be seen that the growth of Trichophyton fungi was completely suppressed only in the well corresponding to the 50% ethyl acetate fraction (black arrow).

[0040] Subsequently, the above 50% ethyl acetate fraction was further separated and eluted on silica gel column chromatography using an ethyl acetate / hexane solution (ethyl acetate / hexane = 2 / 3), and finally eluted with methanol to obtain fractions 1 to 5 and a methanol fraction. Solids of 14.8 mg, 22.0 mg, 16.7 mg, 6.4 mg, 5.9 mg, and 9.0 mg were recovered from fractions 1 to 5 and the methanol fraction, respectively.

[0041] Each of the obtained fractions was added to a separate well of a 24-well plate containing a liquid medium inoculated with Trichophyton fungi in the same manner as above, and static culture was performed at 25°C for 5 days. Figure 4a shows the 24-well plate after static culture. In Figure 4, a negative control with only the DMSO solvent was added to all the wells in the second row, and to the wells in the third row, from left to right, fraction 1, fraction 2, fraction 3, fraction 4, fraction 5, and the methanol fraction from the above silica gel column chromatography were added. Antifungal activity was particularly concentrated in fraction 2 (the well indicated by the white arrow) and fraction 3. Figure 4b shows a photograph of a part of the above fractions after further separation on silica gel thin layer chromatography and staining. It was found that fraction 2 (No. 2) contained a single active compound substantially purely with an Rf value of 0.56.

[0042] 4. Determination of Chemical Structure The compound with an Rf value of 0.56 in fraction 2 on silica gel thin-layer chromatography was recovered, and its structure was determined using electrospray ionization mass spectrometry (ESI-MS) and two-dimensional NMR ( 1 1H-NMR and 13 13C-NMR) techniques. As a result, the antifungal active compound contained in fraction 2 was determined to be the novel compound A represented by the chemical formula C8H 10 O2 and described in this specification. The structural formula of the determined compound A is shown below.

Chemical formula

Claims

1. A compound represented by the following formula (I): 【Chemical 1】

2. An antifungal agent comprising a compound represented by the following formula (I):

3. 【Chemical Formula 2】 The antifungal agent according to Claim 2, wherein the fungus is Trichophyton.

4. A pharmaceutical composition comprising a compound represented by the following formula (I):

5. The pharmaceutical composition according to Claim 4, for use in the treatment or prevention of tinea. 【Chemical Formula 3】

6. A method for producing an antifungal agent or a pharmaceutical composition for the treatment or prevention of tinea, comprising extracting an Auricularia mushroom or its culture filtrate with a polar organic solvent to obtain an extract containing anti-Trichophyton activity.

7. The production method according to Claim 6, wherein (a) the mushroom is Auricularia heimuer, (b) the polar organic solvent contains ethyl acetate, or (c) both (a) and (b) are satisfied. ​ ​ ​

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