WO2023128033A1

WO2023128033A1 - Novel anti-fungal composition for inhibiting biofilm production of infectious fungi

Info

Publication number: WO2023128033A1
Application number: PCT/KR2021/020377
Authority: WO
Inventors: 이종승; 이동기
Original assignee: (주)앰틱스바이오
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2023-07-06

Abstract

The present invention provides: a compound which inhibits biofilm production; and an anti-fungal composition including same. Specifically, the present invention provides: a compound which inhibits the expression of fungal genes HWP1, ALG3, and/or ECE1 related to biofilm production; and an anti-fungal composition including same. Therefore, the composition according to the present invention may be used in the production of an anti-fungal composition for preventing or treating fungal infections.

Description

Novel antifungal composition inhibiting biofilm formation of infectious fungi

The present invention relates to antifungal compositions. Specifically, the present invention relates to an antifungal composition that inhibits fungal biofilm formation.

Fungal infection seriously threatens animal and plant ecosystems and human health, causing great economic losses in agriculture, industry and public health. A quarter of the world's population suffers from superficial or systemic fungal infections, and it is reported that approximately 1.5 to 2 million people die each year from invasive fungal infections. These fungal infections commonly affect patients with HIV/AIDS, cancer, diabetes, respiratory diseases, and patients after surgery and transplantation. Opportunistic fungi such as Cryptococcus , Candida and Aspergillus species are the major fungal pathogens causing the most serious fungal diseases. Among these fungal pathogens, Candida ( Candida speciese ) is the most widely distributed fungal species in humans. Most species of Candida live asymptomatically in the gastrointestinal tract, genital tract, oral cavity and skin of humans. However, if the host's state of health deviates from its normal state, these Candida species can cause infections due to changes in the human immune system or changes in the microbiome. For example, systemic candidiasis is a form of candidiasis in which Candida infection sequentially infects various organs of the body, such as the blood, heart, brain, eyes, and bones. Systemic candidiasis is the most common form of fungal infection in developed countries and is difficult to diagnose by bloodstream because of its invasive nature. Candida albicans ( C. albicans) is It is a representative fungus that causes candidiasis, and its morphological change is one of the main virulence factors. These morphological changes caused by changes in various environmental conditions are very important for pathogen penetration, growth, and virulence. In addition, these morphological changes are involved in the attachment ability of fungi to form hyphae. Furthermore, it changes into a structure that can be entangled with each other to form an extracellular matrix to form a Candida albicans biofilm ( C. albicans) biofilm. Accordingly, Candida albicans ( C. albicans ) exports substances to the outside of the cell and acquires an environment favorable to survival surrounded by an extracellular matrix.

Biofilm formation is known to be a very important characteristic for microbial pathogenicity. In particular, it has been reported that the secreted extracellular matrix is involved in drug resistance. Therefore, inhibition of biofilm generation has become a target for antifungal drug development. Currently, azole and echinocandin are mainly used for the treatment of candidiasis in clinical practice, but it is known that they do not effectively inhibit or remove the formation of biofilm. Accordingly, the inventors of the present invention have completed the present invention as a result of constant efforts to develop a drug that inhibits fungal biofilm production. The compounds of the present invention and compositions containing them inhibit morphological changes in the most important initiation step in the fungal biofilm production process, and inhibit the induction of HWP1 , ALG3 , and/or ECE1 involved in this process.

The present invention is the result of the following R&D project support from the Korean government: (Task identification number: 1465033261, task number: HI20C0326010021. Name of department: Ministry of Health and Welfare, name of task management (specialized) institution: Korea Health Industry Development Institute, research project name: Infectious disease prevention and treatment technology development project (R&D), research project title: development of active amino acid derivatives with fungal cell wall glycoprotein chain inhibitory mechanisms as a treatment for deep-seated and antifungal-resistant pathogenic fungal infections, project executing agency name: Amticsbio Co., Ltd., research Period: 20200423 ~ 20211231)

An object of the present invention is to provide a compound that inhibits biofilm production and an antifungal composition comprising the same.

Another object of the present invention is to provide a compound that inhibits the expression of fungal genes HWP1 , ALG3 , and/or ECE1 associated with biofilm production, and an antifungal composition comprising the same.

Another object of the present invention is to provide an antifungal composition for preventing, treating, or preventing and treating fungal infections, including the antifungal compound.

Another object of the present invention is to provide an antifungal composition for the prevention, treatment, or prevention and treatment of fungal infections in animals other than humans, and a method for treating fungal infections, including the antifungal compound.

Another object of the present invention is to provide an antifungal composition for preventing, treating, or preventing and treating fungal infections in plants, including the antifungal compound.

Another object of the present invention is to provide a cosmetic composition comprising an antifungal compound.

In order to achieve the above object, the present invention provides a compound having the structure of Formula 1 below, an enantiomer or optical isomer thereof, a stereoisomer, a diastereomer or a tautomer thereof, or a pharmaceutical thereof, which inhibits fungal biofilm production. It provides an antifungal composition comprising an acceptable salt, and an excipient.

[Formula 1]

(In Formula 1 above,

R ₁ and R ₂ are each independently H or C _1-6 alkyl;

X is a halogen group (provided that the halogen group excludes -F), m halogenated C _1-6 alkyl groups and m halogenated C _1-6 alkoxy groups that are the same as or different from each other selected from the group consisting of alkoxy groups (m is an integer from 1 to 5). ) is a substituent of).

In one embodiment of the present invention related to this, in Formula 1, R ₁ and R ₂ are each independently H or C _1-6 alkyl, and X is 3,4-Cl, which provides an antifungal composition .

In another embodiment related to this, the present invention provides an antifungal composition that regulates the expression of one or more of genes composed of HWP1 , ALG3 and ECE1 as genes involved in fungal biofilm production.

In addition, the present invention is an antifungal composition for preventing, treating, or preventing and treating fungal infection, comprising the antifungal composition. In a related embodiment, the fungal infection is Cryptococcus neoformans , Candida albicans, Candida auris, Candida glabrata , and Aspergillus It may be caused by one or more fungal species (sepcies) selected from the group consisting of Aspergillus fumigatus , but is not limited thereto. In one embodiment related to this, the fungal infection may be caused by Candida albicans .

The antifungal composition of the present invention can effectively inhibit fungal biofilm production. Therefore, it can be effectively used for the prevention, treatment or prevention and treatment of fungal infections in humans, animals and plants.

1 is a result of microscopic confirmation of inhibition of biofilm generation according to treatment with the compound of the present invention.

Figure 2 is a graph showing changes in biofilm production according to the treatment of the compound of the present invention as OD values.

Figure 3 is a result confirming the inhibitory effect of the compound of the present invention on the mycelial growth of Candida albicans in RPMI-1640 medium.

4 is a result of confirming the inhibitory effect of the compound of the present invention on the mycelial growth of Candida albicans in an agar medium.

Figure 5 is the result of confirming whether the compound of the present invention inhibits the expression of genes involved in yeast-hyphal conversion and mycelial growth of C. albicans .

The present invention relates to a compound having the structure of Formula 1 below, an enantiomer or optical isomer, a stereoisomer, a diastereomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof, which inhibits fungal biofilm production, and An antifungal composition comprising an excipient is provided.

[Formula 1]

(In Formula 1 above,

R ₁ and R ₂ are each independently H or C _1-6 alkyl;

The synthesis of Compound 1 of the present invention is described in detail in Korea Patent Registration No. 10-2282897, a prior patent of the present applicant. Specifically, the reaction scheme for synthesizing the compound of the present invention is as follows.

[Scheme a] - Introduction of butoxycarbonyl protecting group (Boc protecting group)

Norleucine (1.0 equivalent), Boc anhydride (1.5 equivalent), and sodium bicarbonate (1.5 equivalent) were dissolved in a 1:1 mixed solvent of distilled water and methanol, and reacted at room temperature for 36-48 hours. After the mixture was concentrated in vacuo, the pH of the water layer was adjusted to 2 with 1.0 M hydrochloric acid. Thereafter, water in the organic layer obtained by extraction with ethyl acetate was removed with sodium sulfate, and the solvent was evaporated in vacuo to obtain the title compound.

[Scheme b] - Methylation of amine group

The compound obtained from [Scheme a] (1.0 equivalent) and iodomethane (10 equivalent) were dissolved in a tetrahydrofuran solvent, and sodium hydride (10 equivalent) was added dropwise very slowly at 0°C. The reactants were reacted at room temperature for 24 hours. After completion of the reaction, it was diluted with an ether solvent and distilled water was added. The pH of the water layer was adjusted to 2 with a 20% citric acid solution. Thereafter, water in the organic layer obtained by extraction with ethyl acetate was removed with sodium sulfate, and the solvent was evaporated in vacuo. The obtained residue was separated and purified through chromatography using silica gel to obtain the title compound.

[Scheme c] - Introduction of a Boc protecting group on a primary amine group

After dissolving 4-bromophenethylamine (1.0 equivalent) in a dimethylchloride solvent, potassium carbonate (1.5 equivalent) and Boc anhydride (1.05 equivalent) were added, and the reaction proceeded at room temperature for about 12-18 hours. The reaction mixture was diluted in dimethylchloride and washed twice with distilled water. The organic layer was dried over sodium sulfate and concentrated in vacuo. The obtained residue was washed with hexane and then evaporated in vacuo to give the title compound.

[Scheme d] - Synthesis of biphenylamine hydrochloride derivatives

The compound obtained from [Scheme c], tert-butyl (4-bromobenzyl) carbamate or tert-butyl (4-bromophenyl) carbamate (1.0 equivalent) and benzeneboronic acid (1.5 equivalent), sodium carbonate (5.0 equivalent) and tetrakis(triphenylphosphine)palladium (0.04 equivalent) are dissolved in a 2:1 to 2.5:1 mixed solvent of degassed toluene and distilled water for 12-18 hours at a temperature of 140°C. countercurrent reaction. After the reaction, the catalyst was removed by filtration through celite, and the solvent was evaporated from the filtered organic layer in vacuum. The obtained residue was separated and purified through chromatography using silica gel. After dissolving the purified product in ethyl acetate solvent, 4.0 M hydrochloric acid (6.0-10.0 equivalent) was added thereto and stirred at room temperature. The obtained white solid in the form of a salt was washed with ethyl acetate and then completely dried in a vacuum to obtain the title compound.

[Scheme e] - Mixed anhydride bonding (MAC) reaction

The compound synthesized according to [Scheme a] or the compound synthesized according to [Scheme b] (1.0 equivalent) and N-methylmorpholine (NMM, 2.5-2.8 equivalent) were added to the distilled tetrahydrofuran solvent. After stirring for 15 minutes, isobutyl chloroformate (IBCF, 1.3 equivalent) was added, followed by further stirring for 15 minutes, and then the compound obtained from [Scheme d] (1.05 equivalent) was added. The reaction mixture was reacted at room temperature for about 3-5 hours. The mixture was filtered and the solvent evaporated in vacuo. The obtained residue was separated and purified through chromatography using silica gel to obtain the title compound.

[Scheme f] - Removal of Boc protecting group

The compound derivative (1.0 equivalent) obtained from [Scheme e] was dissolved in ethyl acetate solvent, and then stirred at room temperature while adding 4.0 M hydrochloric acid (6.0-10.0 equivalent). The white solid in the form of a salt obtained was washed with ethyl acetate and then completely dried in a vacuum to obtain the title compound.

[Scheme g] - Dimethylation of amine group

The compound obtained from [Scheme f] (1.0 equivalent) was dissolved in methanol, triethylamine (6.0 equivalent) was added, and formaldehyde (37% by weight solution, 1.0-2.5 equivalent) and 10% palladium catalyst ( 0.1-0.5 eq.) were added sequentially. The reaction was allowed to react at room temperature for 18 hours. After the reaction, the catalyst was removed by filtration through celite, and the filtered organic layer was evaporated in a vacuum to obtain a white solid. The obtained product was recrystallized from methanol and diethyl ether to obtain the title compound.

The compounds of the present invention may exist in the form of pharmaceutically acceptable salts. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. The term "pharmaceutically acceptable salt" of the present invention is a concentration that has a relatively non-toxic and harmless effective effect on patients, and any of the compounds represented by Formula 1 do not reduce the beneficial effects of the compound represented by Formula 1 by side effects caused by the salt. means any organic or inorganic addition salt of Pharmaceutically acceptable salts of the compounds of the present invention, unless otherwise indicated, include salts of acidic or basic groups which may be present in the compounds of Formula 1 above. For example, pharmaceutically acceptable salts may include sodium, calcium, and potassium salts of a hydroxy group, and other pharmaceutically acceptable salts of an amino group include hydrobromide, sulfate, hydrogen sulfate, phosphate, and hydrogen phosphate. , dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, etc., preparation of salts known in the art It can be produced through the method. Therefore, the salt of the compound of Formula 1 of the present invention is a pharmaceutically acceptable salt, and any salt of the compound of Formula 1 exhibiting pharmacological activity equivalent to that of the compound of Formula 1, for example, exhibiting antifungal activity, can be used without limitation. do.

Acid addition salts are prepared by conventional methods, for example, by dissolving a compound in an excess of an aqueous acid solution and precipitating the salt using a water-miscible organic solvent, such as methanol, ethanol, acetone or acetonitrile. Equimolar amounts of the compound and an acid or alcohol (eg, glycol monomethyl ether) in water may be heated, and then the mixture may be evaporated to dryness, or the precipitated salt may be suction filtered. At this time, organic acids and inorganic acids can be used as the free acid, and hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. can be used as the inorganic acid, and methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, and maleic acid can be used as the organic acid. (maleic acid), succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid (gluconic acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, etc. can be used. , but not limited to these.

In addition, a pharmaceutically acceptable metal salt may be prepared using a base. The alkali metal salt or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable for preparing a sodium, potassium, or calcium salt, but is not limited thereto. In addition, the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

The present invention provides an antifungal composition comprising the compound of Formula 1, a stereoisomer, diastereomer, enantiomer, optical isomer, tautomer or pharmaceutically acceptable salt thereof as an active ingredient. As another aspect related to this, the present invention provides an antifungal composition for inhibiting fungal biofilm production. In another embodiment related to this, the present invention provides an antifungal composition for inhibiting the expression of genes HWP1 , ALG3 , and/or ECE1 associated with fungal biofilm production. For example, since the composition of the present invention can exhibit antifungal activity against opportunistic fungi, it can be used as an antifungal composition, and further used for preventing or treating fungal infections.

As used herein, the term "prevention" refers to any action that inhibits or delays the occurrence, spread, and recurrence of a target disease by administration of the pharmaceutical composition, and "treatment" means that symptoms of a target disease are reduced by administration of the pharmaceutical composition. It means any action that improves or changes beneficially.

For example, non-limiting examples of fungal infections that can be prevented or treated by the pharmaceutical composition of the present invention include Cryptococcus neoformans , Candida albicans , Candida auris , Candida Glabrata ( Candida glabrata ), Aspergillus fumigatus ( Aspergillus fumigatus ) It may include infectious diseases caused by bacteria. Specifically, the fungal infection may be a fungal infection caused by Candida albicans, but is not limited thereto.

The antifungal composition according to the present invention may contain the compound represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier, diluent or excipient may be added. can be included with For example, it can be formulated and used in various forms such as oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and injections of sterile injection solutions according to conventional methods according to each purpose of use. , oral administration or administration through various routes including intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like. Examples of suitable carriers, excipients or diluents that may be included in such compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil; and the like. In addition, the composition of the present invention may further include fillers, anti-agglomerating agents, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the composition, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc. Formulated by mixing. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients.

Oral liquid preparations may include suspensions, solutions for internal use, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may be included in addition to water and liquid paraffin, which are commonly used simple diluents. can

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. The suppositories are Witepsol, Macrogol, and Tween 61. Cacao fat, laurin fat, glycerogeratin and the like can be used. Meanwhile, conventional additives such as solubilizers, tonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives may be included in the injection.

The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dose level is the patient's health condition, Depending on the type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field can The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art. For example, the dosage may increase or decrease depending on the route of administration, severity of disease, sex, weight, age, etc., so the dosage is not limited to the scope of the present invention in any way.

The term "administration" of the present invention means providing a predetermined substance to a patient by any suitable method, and the administration route of the composition of the present invention may be administered through any general route as long as it can reach the target tissue. there is. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or intrarectal administration may be administered, but is not limited thereto. In addition, the pharmaceutical composition of the present invention may be administered by any device capable of transporting an active substance to a target cell. Preferred administration modes and preparations are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, and the like. Injections are formulated with aqueous solvents such as physiological saline and IV, non-aqueous solvents such as vegetable oil, higher fatty acid esters (e.g., ethyl oleate, etc.), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Stabilizers (e.g., ascorbic acid, sodium hydrogensulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.) to prevent deterioration, emulsifiers, buffers to control pH, A pharmaceutical carrier such as a preservative (eg, phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may be included.

The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art. For example, the dosage may increase or decrease depending on the route of administration, severity of disease, sex, weight, age, etc., so the dosage is not limited to the scope of the present invention in any way. In one embodiment, the compound of the invention is preferably administered topically or orally. When administered topically, a therapeutically effective amount of a compound of the present invention may be, but is not limited to, 0.00001 to about 20% (w/w), preferably 0.001 to 3%. When administered orally, the therapeutically effective amount of the compound of the present invention may be 0.01 to about 1,000 mg/kg, preferably 1 to about 300 mg/kg, but is not limited thereto.

Furthermore, the present invention provides a method for treating fungal infections comprising the step of administering the pharmaceutical composition to a subject in need thereof.

In the present invention, the subject to which the antifungal composition of the present invention is administered is any animal, including monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig, including humans. In other words, by administering the pharmaceutical composition of the present invention to a subject, the disease can be effectively prevented or treated. In addition, since the pharmaceutical composition of the present invention exhibits a therapeutic effect for diseases induced by fungal infection through antifungal activity, synergistic effects can be exhibited by administering in parallel with existing therapeutic agents.

The term "therapeutically effective amount" used in combination with an active ingredient in the present invention refers to a derivative compound obtained by introducing a biphenyl group into an aminoalkanoic acid effective for preventing or treating a target disease, a stereoisomer thereof, or a pharmaceutically acceptable compound thereof. amount of possible salt.

Hereinafter, the present invention will be described in detail based on examples. The following examples are for explaining the present invention, but the scope of the present invention is not limited thereto.

1. 재료 및 방법1. Materials and Methods

1-1. Structure and Synthesis of Compounds of the Invention

The compound of the present invention has a structure represented by Formula 1 below.

In Formula 1,

R ₁ and R ₂ are each independently H or C _1-6 alkyl;

X is a halogen group (provided that the halogen group excludes -F), m halogenated C _1-6 alkyl groups and m halogenated C _1-6 alkoxy groups that are the same as or different from each other selected from the group consisting of alkoxy groups (m is an integer from 1 to 5). ) is a substituent of

In addition, the compound represented by Chemical Formula 1 is a compound with no limitation on the three-dimensional arrangement structure of substituents bonded to chiral carbon, and may include all structurally possible optical isomers or enantiomeric compounds. Specifically, the compound represented by Formula 1 may be provided in the form of its (R) or (S) isomer alone or a mixture thereof, for example, a racemate. In addition, it may be provided as a stereoisomer, diastereomer or tautomer, but is not limited thereto.

The present invention may include within the scope of the present invention not only Compound 1 or a pharmaceutically acceptable salt thereof, but also a solvate or hydrate that can be prepared therefrom and exhibits the same efficacy.

1-2. strain information

Candida albicans strain was cultured in YPD medium (Sigma-Aldrich). Candida albicans SC5314 strain (distributed from Professor Ban Yong-sun, Yonsei University) was used for in vitro assays including biofilm analysis, hyphal analysis and gene expression analysis. ATCC90028 strain was used to infect mice for in vivo drug efficacy testing.

1-3. Biofilm assay

Candida albicans SC5314 cells were cultured overnight at 30 °C in 2 ml of YPD medium. The cells were suspended in RPMI 1640 medium (Sigma-Aldrich) at a final concentration of 10 ⁶ cells/ml, and 200 μL of the suspension was added to a flat-bottom 96-well plate with or without drug treatment. Plates were incubated at 37° C. for 90 minutes and washed 3 times with PBS. Next, further incubation was performed with 200 μl of fresh RPMI 1640 for the cells remaining as biofilms in the wells at 37 °C. After 24 hours, the bottom surface of the well was photographed under a microscope (equipment name, manufacturer) and the OD was measured in a plate reader. Each data point represents an independent experiment.

1-4. Yeast-to-hyphae switching assay

SC5314 cells cultured overnight were seeded onto fresh YPD and further incubated in a 30° C. shaking incubator until an OD600 of 0.8 was reached. Cultured cells were washed three times with PBS and resuspended in 50% RPMI-1640 diluted in PBS. Samples were fixed with 2% formalin at each time point.

1-5. Hyphal growth assay on plate

Candida albicans SC5314 cells were cultured overnight at 30 °C in 2 ml of YPD medium. Cells were counted and 10,000 cells were seeded into 5 ml 10% FBS agar medium in a 60 mm plate (#10060, SPL, Korea). Plates were incubated for an additional 24 hours at 37°C.

1-6. Expression analysis

Candida albicans SC5314 cells were inoculated into fresh YPD and incubated in YPD medium at 30 °C until OD600 reached 0.8. Cells were washed three times with PBS, resuspended in 10% FBS in YPD, further incubated at 37° C., and sampled at each time point. Total RNA was extracted and cDNA for qRT-PCR was used for synthesis. The Ct values of each gene were normalized to the Ct values of the housekeeping genes ( ACT1 , EFB1 and PMA1 ). Relative expression was expressed as fold-change compared to the expression level at t = 0. Statistical significance was analyzed by one-way ANOVA analysis with Turkey hypothesis test. (*<0.05, **<0.005 versus each basal expression)

1-7. In vivo drug efficacy test in candidiasis animal model

Candida albiancs ATCC90028 cells were inoculated (2 x 10 ⁶ cells per mouse) into the tail vein of 7-week-old male ICR mice (Orient Bio, Korea). As a positive control, fluconazole (FCZ) was orally administered. Statistics were calculated by Log-rank (Mantel-Cox) test using Prism 8.0.

2. 결과2. Results

2-1. Inhibition of C. albicans biofilm production according to treatment with the compound of the present invention

MIC (Minimum Inhibitory Concentration) test according to CLSI guidelines was performed in RPMI-1640 based medium. The MIC value of the compound of the present invention against C. albicans was about 4 μg/ml. It was confirmed that the compounds of the present invention inhibit not only the growth of C. albicans but also biofilm formation. Therefore, in order to measure the change in C. albicans biofilm according to the treatment with the compound of the present invention , C. albicans biofilm analysis was performed. As a result of observing the growth of C. albicans in a flat-bottomed petri dish under a microscope (Nikon, eclipse Ti), it was confirmed that the treatment with the compound of the present invention inhibited biofilm production. The results are shown in Figure 1. In addition, biofilm production was quantitatively measured and compared through optical density (see FIG. 2).

2-2. Delay in biofilm production by treatment with the compound of the present invention

Yeast-to-mycelial conversion is one of the major virulence of C. albicans , and this heteromorphism, along with proteins involved in extracellular secretion and adhesion, is a key factor in biofilm formation. Therefore, the present inventors investigated the effect of the compound of the present invention on the yeast-to-mycelial transition and mycelial growth of C. albicans . The yeast-mycelial conversion in liquid medium proceeded very rapidly. Mycelia began to be produced within 30 minutes in RPMI-1640 medium diluted to 50% in PBS, and it was confirmed that mycelial growth was actively performed thereafter (Fig. 3, upper panel). In particular, it was observed that these changes were more brittle when the cells sank due to the elongated cell shape by hyphae, and protruded from the surface without aggregation (Fig. 3, lower panel). On the other hand, conversion from yeast to hyphae occurred relatively slowly in the group treated with the compound of the present invention. In particular, when the compound of the present invention was treated at an MIC concentration (4 μg/ml), the change was slow by about 1 hour compared to the untreated group (FIG. 3). This retardation was observed more clearly than in the solid medium. In the agar medium supplemented with fetal bovine serum, C. albicans grew out of colonies with active mycelial growth and yeast cell proliferation (FIG. 4). On the other hand, mycelial growth was inhibited in the plates treated with the compound of the present invention (FIG. 4). In particular, in the group treated with the compound of the present invention at a high concentration, the colony size was small, and the transition from yeast to hyphae did not occur at all. This phenomenon was confirmed in a concentration-dependent manner and was similarly observed with fluconazole (FIG. 4). In conclusion, the compound of the present invention inhibits the transition from yeast to hyphae of C. albicans, thereby inhibiting biofilm initiation.

2-3. Inhibition of mycelial growth-related gene expression by the compound (22h) of the present invention

Genes involved in yeast-hyphal conversion and hyphal growth of C. albicans have been reported in previous studies. First, the response of C. albicans to drugs was confirmed by induction of CDR1 and CDR2 genes encoding multiple drug transporters (Fig. 5A). Similarly, the expression of the EFG1 gene involved in adhesion was partially increased under serum induction, but was not changed by compound (22h) treatment (Fig. 5B). Gene expression levels of TEC1, BCR1, ALS3, HWP1 and ECE1 were increased by serum induction in a wide range related to the onset of biofilm production (FIG. 5C). Induction of expression of ALS3, HWP1 and ECE1 was suppressed by compound 22h treatment (Fig. 5C). Among the genes whose expression was induced by serum, the expression level of only these three genes decreased. These results support that the compound 22h of the present invention inhibits the initiation of biofilm production by suppressing the gene expression of ALS3, HWP1 and ECE1 in C. albicans serum-induced condition.

2-4. Efficacy of compound (22h) in a mouse model of C. albicans systemic infection

It was confirmed whether the compound (22h) of the present invention was effective against systemic infection of C. albicans . A mouse systemic infection model was induced by injecting large numbers of C. albicans cells into mice via intravenous injection. C. albicans 2 x 10 ⁶ cells were administered to ICR mice (7-week-old female, 7 mice per group), and all mice were killed within 1 week. As a positive control group for the group administered with the compound of the present invention, fluconazole was orally administered, and then the therapeutic efficacy was confirmed. The compound of the present invention (22h) showed a dose-dependent increase in viability. In particular, when compound 22h was orally treated at 20 mpk or 30 mpk, survival of mice induced with systemic candidiasis significantly increased.

Compositions comprising the compounds of the present invention can delay the onset of biofilm production of Candida albicans and inhibit biofilms. Therefore, the composition of the present invention can be used for the treatment of cardiosis caused by Candida albicans.

Claims

A compound having the structure of Formula 1 below, an enantiomer or optical isomer, a stereoisomer, a diastereomer or a tautomer thereof, or a pharmaceutically acceptable salt thereof, and an excipient that inhibits fungal biofilm production Antifungal composition comprising:

[Formula 1]

(In Formula 1 above,

R 1 and R 2 are each independently H or C 1-6 alkyl;

X is a halogen group (provided that the halogen group excludes -F), m halogenated C 1-6 alkyl groups and m halogenated C 1-6 alkoxy groups that are the same as or different from each other selected from the group consisting of alkoxy groups (m is an integer from 1 to 5). ) is a substituent of).
According to claim 1,

Inhibiting the biofilm production is by regulating the expression of one or more fungal genes consisting of HWP1 , ALG3 and ECE1 , antifungal composition.
According to claim 1 or 2,

In Formula 1, R 1 and R 2 are each independently H or C 1-6 alkyl, and X is 3,4-Cl, the antifungal composition.
An antifungal composition for preventing, treating, or preventing and treating fungal infection, comprising the antifungal composition of claim 1 or 2.
According to claim 4,

The fungal infection is Cryptococcus neoformans , Candida albicans, Candida auris, Candida glabrata , and Aspergillus fumigatus By one or more fungal species (sepcies) selected from the group consisting of, antifungal composition.
According to claim 5,

The fungal infection is caused by Candida albicans , an antifungal composition.
An antifungal composition for preventing, treating, or preventing and treating fungal infections in non-human animals, comprising the antifungal composition of claim 1 or 2.
An antifungal composition for preventing, treating, or preventing and treating fungal infections in plants, comprising the antifungal composition of claim 1 or 2.
A cosmetic composition comprising the antifungal composition of claim 1 or 2.