Antifungal compositions containing carbazole compounds as active ingredients

Abstract

This invention relates to an antifungal composition containing a carbazole compound as an active ingredient. According to the invention, the carbazole compound not only inhibits the growth of Candida species fungi but also inhibits the growth of molds such as yeast, and inhibits the morphological transformation of Candida species fungi and neutralizes pathogenicity, thus being very useful as an antifungal composition capable of treating Candida infections.

Description

Technical Field

[0001] This invention relates to an antifungal composition containing a carbazole compound as an active ingredient. Background Technology

[0002] Candida is one of the most common pathogens and causes a wide range of infections, from mucosal infections in healthy individuals to systemic infections, particularly serious infections in immunocompromised and immunocompromised patients. Candida lives in the mouth, skin, vagina, and intestines of healthy humans and is controlled by a normal immune system, but can cause pathogenic symptoms due to accumulated fatigue, the secretion of toxins from improper diet, and weakened immunity caused by conditions such as diabetes and malnutrition. Additionally, symptoms caused by Candida may occur during long-term treatment with antibiotics, corticosteroids, or immunosuppressants. In particular, this pathogen is known to be an endogenous factor involved in hospital-acquired infections. It is a life-threatening factor not only in immunocompromised patients due to organ transplantation but also in patients undergoing chemotherapy and AIDS patients. In the United States, Candida infections accounted for 6% of all hospital-acquired infections from 1980 to 10.4% in 1990 and have recently been recognized as the most common type of hospital-acquired infection. Furthermore, according to recent results reported by the National Nosocomial Infections Surveillance System (NNIS) from 1992 to 1997, Candida infections rank fourth among bloodstream infections and second among infections of the urinary tract, eyes, ears, nose, and throat. To date, damage caused by Candida in hospitals worldwide is at a serious level and is becoming a priority to be addressed. Attempts have been made to eradicate these Candida bacteria with antibiotics, but treating Candida has been challenging because treatments vary from patient to patient, and even the same antibiotic can differ in its scope of action or dosage. In South Korea, Candida infections account for approximately 18% of hospital-acquired infections, and the mortality rate for systemic Candida infections is 50% or higher.

[0003] Candida albicans is the most representative species of the Candida genus because it has been scientifically and academically studied since the 1950s, resulting in a wealth of data available for it. Candida albicans is a dimorphic fungus, existing most of the time as a single oval yeast cell and growing by binary fission. However, under suitable conditions (i.e., body temperature, pH, and serum), Candida albicans forms filamentous branching hyphae. While the relationship between this morphological change and the conversion to pathogenic forms is unclear, most Candida species isolated from patients with pathogenic Candida species in vivo have developed hyphae. Furthermore, in experiments using mice with mutant Candida species that exhibit defects in hyphal formation, Candida species did not develop candidiasis. Considering these results, a series of correlations are considered to exist between morphological transformation and the acquisition of pathogenicity. Therefore, many scientists have conducted extensive functional analyses of genes associated with morphological changes in Candida species, revealing numerous findings.

[0004] For the treatment of candidiasis, many antifungal drugs, such as amphotericin B and fluconazole, have been developed and used, and their efficacy has been proven. However, strong resistance to fluconazole has become a new problem (Infect Drug Resist. 2017; 10:237–245). Therefore, there is an urgent need to develop a new drug that can avoid resistance to fluconazole.

[0005] Therefore, the inventors have made considerable efforts to develop an antifungal treatment agent that is safer and more effective than conventional agents used to treat candidiasis, and have found that compounds containing carbazole exhibit excellent antifungal activity not only against Candida spp. but also against other fungi such as yeast and Aspergillus spp., thus completing this invention. Summary of the Invention

[0006] The purpose of this invention is to provide the antifungal use of carbazole compounds that have growth-inhibiting and pathogenicity-neutralizing effects not only against Candida spp. but also against fungi such as yeast.

[0007] To achieve the above objectives, the present invention provides an antifungal composition containing a carbazole compound as an active ingredient, the carbazole compound being selected from Formula 1 to Formula 8.

[0008] [Formula 1]

[0009]

[0010] R1 and R2 are either H or Br.

[0011] [Equation 2]

[0012]

[0013] Where R is H or NO2.

[0014] [Formula 3]

[0015]

[0016] [Formula 4]

[0017]

[0018] [Formula 5]

[0019]

[0020] [Formula 6]

[0021]

[0022] [Formula 7]

[0023]

[0024] [Formula 8]

[0025]

[0026] The present invention also provides the antifungal use of carbazole compounds selected from Formulas 1 to 8.

[0027] In this invention, the antifungal use can be a use for inhibiting fungal infections, a use for treating fungal infections, or a use for killing fungal infections.

[0028] The present invention also provides the use of carbazole compounds selected from Formula 1 to Formula 8 for the manufacture of antifungal drugs.

[0029] The present invention also provides a method for killing fungi, the method comprising the steps of: treating a subject requiring fungal killing with a carbazole compound selected from Formula 1 to Formula 8.

[0030] The present invention also provides a method for inhibiting fungal infection, the method comprising the steps of treating a subject who may have a fungal infection with a carbazole compound selected from Formula 1 to Formula 8.

[0031] The present invention also provides a method for treating fungal infections, the method comprising the steps of administering a carbazole compound selected from Formulas 1 to 8 to a subject with a fungal infection. Attached Figure Description

[0032] Figure 1 shows the results of absorbance measurements of Candida species treated with carbazole compounds. Figure 1a The results of testing molecules A, B, C, D, and E for their MICs against Candida albicans are shown. Figure 1b The results of testing for the MICs of molecules F, G, H, I, and J against Candida albicans are shown.

[0033] Figure 2 The results of measuring the MICs of carbazole compounds B and C against Saccharomyces cerevisiae are shown.

[0034] Figure 3 The results of measuring the MICs of carbazole compounds B and C against Aspergillus fumigatus are shown.

[0035] Figure 4 The results of an evaluation of the cytotoxicity of carbazole compounds B and C on mammalian cells (HeLa cells) are shown.

[0036] Figure 5 The results show the survival rate of mouse models of candidiasis treated with carbazole compounds B and C via tail vein injection.

[0037] Figure 6 The results show the survival rate of mouse models of candidiasis treated with carbazole compounds B and C diluted in drinking water.

[0038] Figure 7 The results show the assessment of the degree of Candida infection in the kidneys of a mouse model of candidiasis treated with carbazole compounds B and C via tail vein injection. Detailed Implementation

[0039] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Generally, the nomenclature used in this specification is well-known and commonly used in the art.

[0040] In this invention, carbazole compounds with various structures that can act on Candida to inhibit the growth of Candida and neutralize the pathogenicity of Candida have been demonstrated to inhibit the growth of Candida and various fungi, and to exhibit therapeutic effects in mice infected with Candida. Furthermore, the carbazole compounds of this invention exhibit excellent antifungal activity in mammalian cells while remaining safe.

[0041] Therefore, in one aspect, the present invention relates to an antifungal composition containing a carbazole compound as an active ingredient, said carbazole compound being selected from the following formulas 1 to 8:

[0042] [Formula 1]

[0043]

[0044] R1 and R2 are either H or Br.

[0045] [Equation 2]

[0046]

[0047] Where R is H or NO2.

[0048] [Formula 3]

[0049]

[0050] [Formula 4]

[0051]

[0052] [Formula 5]

[0053]

[0054] [Formula 6]

[0055]

[0056] [Formula 7]

[0057]

[0058] [Formula 8]

[0059]

[0060] Unless otherwise stated, the carbazole compounds of the present invention comprise both the salt of the compound and the optical isomer of the compound.

[0061] When carbazole compounds are used in the form of salts, it is preferable to select from the possible salts of the carbazole compound as the active ingredient that are suitable or acceptable for each use. Preferably, the salts that can be used in this invention are acid addition salts formed from pharmaceutically acceptable free acids.

[0062] Examples of free acids include organic or inorganic acids. Examples of inorganic acids include hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid, etc., and examples of organic acids include citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, pyruvic acid, glutamic acid, citric acid, aspartic acid, etc., among which methanesulfonic acid or hydrochloric acid are preferred.

[0063] In examples of the present invention, a compound in Formula 1 where R1 is Br and R2 is H can be represented as molecule B. Additionally, in examples of the present invention, a compound in Formula 1 where R1 is H and R2 is Br can be represented as molecule J.

[0064] In examples of the present invention, compounds in which R1 in Formula 2 is H can be represented as molecule C. Additionally, in examples of the present invention, compounds in which R1 in Formula 2 is NO2 can be represented as molecule G.

[0065] In addition, in the examples of the present invention, Formula 3 can be represented as molecule A, Formula 4 can be represented as molecule D, Formula 5 can be represented as molecule E, Formula 6 can be represented as molecule F, Formula 7 can be represented as molecule H, and Formula 8 can be represented as molecule I.

[0066] Compounds of Formulas 1 to 8 were produced according to the method described in Bioorganic & Medicinal Chemistry Letters 18:4670-4674, 2008.

[0067] The carbazole compounds of this invention exhibit excellent antifungal activity against Candida and various fungi. The therapeutic effect against Candida infections is due to the carbazole's ability to inhibit the hyphae of Candida, which are a pathogenic factor in morphological transformation.

[0068] In the examples of this invention, molecules B and C exhibited MICs of 8 μg / ml and 16 μg / ml against Candida spp., respectively, indicating that they possess excellent antifungal activity.

[0069] In another example of the invention, molecules B and C exhibited MICs of 4 μg / ml and 4 μg / ml against budding yeast (Saccharomyces cerevisiae) and Aspergillus fumigatus, respectively, indicating their excellent antifungal activity. Furthermore, both molecules B and C exhibited an MIC of 64 μg / ml against Aspergillus fumigatus.

[0070] Preferably, the fungi in this invention are selected from any of the following: Candida sp., Saccharomyces sp., Kazakhstania sp., Aspergillus sp., Cladosporium sp., Penicillium sp., and combinations thereof, but are not limited thereto.

[0071] As described above, the carbazole compounds according to the present invention exhibit excellent antifungal activity against various pathogenic microorganisms, are non-toxic, and show excellent effects even when used in trace amounts. Therefore, carbazole compounds can be used as additives in various antifungal agents and pharmaceutical compositions, food preservation additives, cosmetic additives, additives for antifungal purposes in building materials, additives for pest control agents, and additives for household products.

[0072] The antifungal composition of the present invention contains a carbazole compound with antifungal activity as an active ingredient, and can therefore be used as an antifungal agent or pharmaceutical composition for inhibiting the activity of various pathogenic microorganisms or inhibiting hyphal formation.

[0073] In addition to carbazole compounds, the antifungal composition may further contain at least one active ingredient that exhibits the same or similar function as the carbazole compound.

[0074] In addition to the active ingredients described above, the compositions of the present invention can also be prepared to contain one or more pharmaceutically acceptable carriers for administration. As pharmaceutically acceptable carriers, one or more selected from saline, sterile water, Ringer's solution, buffered saline, dextran solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof can be used, and other conventional additives such as antioxidants, buffers, and antibacterial agents can be added if necessary. Furthermore, diluents, dispersants, surfactants, binders, and lubricants can be added to prepare injectable formulations (such as aqueous solutions, suspensions, emulsions, etc.), pills, capsules, granules, or tablets. Moreover, the compositions of the present invention can preferably be formulated according to each disease or component using suitable methods known in the art or by methods disclosed in Remington's Pharmaceutical Science (latest version), Mack Publishing Company, Easton, Pennsylvania.

[0075] The compositions according to the invention can be administered orally or parenterally during clinical application and can be used in the form of general pharmaceutical formulations. For formulations, diluents or excipients may be used, such as commonly used antifungal agents, fillers, expanders, binders, wetting agents, disintegrants, surfactants, etc. As an antifungal agent, at least one of the following can be used: ketoconazole, itraconazole, fluconazole, miconazole, clotrimazole, fenticonazole, econazole, bifonazole, oxiconazole, cloconazole, tolciclate, amphotericin B, flucytosine, griseofulvin, terbinafine, nystatin, tolnaftate, naftifine, haloprogin, ciclopirox, triclosan, norfloxacin, ciprofloxacin, and their salts.

[0076] Solid formulations for oral administration can be prepared by mixing one or more carbazole compounds according to the invention with at least one excipient (e.g., starch, calcium carbonate, sucrose, lactose, or gelatin). In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral administration include suspensions, solutions, emulsions, or syrups, and for liquid formulations, in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be used.

[0077] Examples of formulations intended for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories.

[0078] As a non-aqueous solvent or suspending agent, propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), injectable esters (such as ethyl oleate), etc. can be used, and as a suppository base, Witepsol, Macrogol, Tween 61, cocoa butter, lauryl ester, glycerin, gelatin, etc. can be used.

[0079] The compositions of the present invention can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or locally) or orally, depending on the desired method, and the dosage of the composition can vary according to the patient's weight, age, sex, health condition and diet, time of administration, method of administration, excretion rate, and severity of disease. More preferably, the composition is administered once or several times a day.

[0080] In addition, the carbazole compounds of the present invention can be used as food preservation additives to extend the shelf life of food. Examples of food include processed food products, fish products, tofu, gelatinous foods, health supplements, seasonings, baked goods and confectionery, dairy products, pickled foods, fermented foods, or beverages, and feed applications are limited (including livestock feed), and can be solid or liquid.

[0081] In addition, carbazole compounds with antifungal activity can be used as cosmetic additives to prepare cosmetic compositions for keeping hands or feet clean. Examples of cosmetic compositions include soaps (solid soaps, liquid soaps, foaming soaps, bath soaps, hand soaps, etc.), cleansing foams, shampoos (hair shampoos, dry shampoos, etc.). Soaps are preferred, and in particular, carbazole compounds can be used in formulations such as liquid soaps and bath soaps.

[0082] Furthermore, the carbazole compounds of the present invention can be used in a variety of applications (including as additives in antifungal building materials, pesticides, household products, etc.) and can be used in combination with antifungal agents known in the art. For example, pesticide-related antifungal agents are described in the Pesticide Dictionary (Pesticide Manual, 12th edition, British Crop Protection Council, 2000).

[0083] For the above-described uses, the carbazole compound of the present invention may be contained in an amount of 0.001 wt% to 99.9 wt%, preferably 0.1 wt% to 99 wt%, more preferably 1 wt% to 50 wt% based on the total weight of the composition, in order to exhibit its effect in each use.

[0084] In another aspect, the present invention relates to the antifungal use of carbazole compounds selected from the following formulas 1 to 8:

[0085] [Formula 1]

[0086]

[0087] R1 and R2 are either H or Br.

[0088] [Equation 2]

[0089]

[0090] Where R is H or NO2.

[0091] [Formula 3]

[0092]

[0093] [Formula 4]

[0094]

[0095] [Formula 5]

[0096]

[0097] [Formula 6]

[0098]

[0099] [Formula 7]

[0100]

[0101] [Formula 8]

[0102]

[0103] In this invention, the antifungal use can be a use for inhibiting fungal infections, a use for treating fungal infections, or a use for killing fungal infections.

[0104] In another aspect, the present invention relates to the use of carbazole compounds for manufacturing antifungal drugs selected from Formulas 1 to 8.

[0105] In another aspect, the present invention relates to a method for killing fungi, the method comprising the steps of treating a subject requiring fungal killing with a carbazole compound selected from Formula 1 to Formula 8.

[0106] In another aspect, the present invention relates to a method for inhibiting fungal infection, the method comprising the steps of treating a subject who may have a fungal infection with a carbazole compound selected from Formula 1 to Formula 8.

[0107] In another aspect, the present invention relates to a method for treating fungal infections, the method comprising the steps of administering a carbazole compound selected from formulas 1 to 8 to a subject with a fungal infection.

[0108] [Example]

[0109] The invention will now be described in more detail with reference to embodiments. These embodiments are for illustrative purposes only, and it will be apparent to those skilled in the art that the scope of the invention should not be construed as limited to these embodiments.

[0110] Example 1: Inhibition of fungal growth

[0111] 1-1: Inhibition of Candida growth

[0112] Inhibition of Candida growth was assessed using the CLSI M27 method, a known standard method for antifungal susceptibility testing. The assessment was performed using the broth microdilution method according to the CLSI M27-A2 guidelines. As the culture medium, 10.4 g of RPMI-MOPS (RPMI-1640 medium supplemented with L-glutamine (Gibco, Gaithersburg, MD, USA)) was dissolved in 900 mL of distilled water, and then 34.53 g of 0.165 M 3-N-morpholinopropanesulfonic acid (MOPS) buffer was added to bring the final volume to 1,000 mL, followed by pH adjustment to 7.0. The solution was then filtered through a 0.2 μm filter to prepare the culture medium.

[0113] Dissolve the carbazole compound in triple-distilled water or dimethyl sulfoxide (DMSO) (Sigma) to a concentration of 20 mg / mL. Serially dilute this antifungal agent from a concentration of 64 μg / mL to a final concentration between 0.125 μg / mL.

[0114] Candida strains were incubated in yeast extract peptone dextrose (YPED) at 30°C for 16 hours, and the turbidity was adjusted to 1.0 (measured by spectrophotometer at 600 nm) to achieve a fungal concentration of approximately 2.0 x 10⁻⁶. 7 CFU / mL. The fungal solution was washed with triple-distilled water and then diluted again with RPMI-MOPS medium. 100 μL of the fungal solution was aliquoted into each well of a 96-well microplate from well 1 to well 10 (final cell concentration: 2.0 x 10⁻⁶). 4 (CFU / mL). Additionally, 100 μL of fungal solution was aliquoted into well 11 (growth control well), and only 100 μL of RPMI-MOPS medium was aliquoted into well 12 (medium control well). After fungal inoculation, the microplates were incubated at 37°C for 24 hours.

[0115] To prevent errors caused by inspectors, absorbance data were obtained by measuring absorbance using a microplate spectrophotometer for evaluation of the results. Table 1 shows the absorbance of Candida strains treated with compounds containing carbazole.

[0116] [Table 1]

[0117]

[0118] In the data, the concentration at which growth was inhibited based on the culture medium control wells was determined as the MIC (minimum inhibitory concentration), and the results are shown in Table 2 and Figure 1 below.

[0119] [Table 2]

[0120]

[0121] As a result, the MICs of molecules B and C against Candida spp. were 8 μg / ml and 16 μg / ml, respectively (Figure 1). This indicates that these molecules have an inhibitory effect on the growth of Candida spp.

[0122] 1-2: Growth inhibition of Saccharomyces cerevisiae and Aspergillus fumigatus

[0123] MIC tests were performed on budding yeast (Saccharomyces cerevisiae) and Aspergillus fumigatus in the same manner as in Examples 1-1.

[0124] As a result, the MICs for both molecules B and C against yeast were 4 μg / ml, which was lower than the MIC against Candida spp. Figure 2 Furthermore, in the measurement of the MIC of Aspergillus fumigatus, which causes aspergillosis, it can be seen that molecules B and C inhibited the growth of Aspergillus fumigatus at the same concentration of 64 μg / ml. Figure 3 ).

[0125] Therefore, it can be confirmed that the two compounds are effective not only against Candida spp., but also against other fungi.

[0126] Example 2: Cytotoxicity Assessment

[0127] The cytotoxicity of carbazole compounds to mammalian cells was evaluated using the human cervical cancer cell line HeLa.

[0128] Cells were spaced at 1.0 × 10⁶ cells per well. 4Cells were seeded at a density of 100 cells per well in 96-well microplates and cultured in DMEM containing 10% FBS at 37°C for 24 hours. The culture medium was then removed, each well was washed once with PBS, and then treated with a synthetic compound serially diluted in DMEM from 64 μg / ml to 1 μg / ml. Cytotoxicity was measured using MTS 16 hours after compound treatment.

[0129] The results confirmed that molecule C did not exhibit toxicity to mammalian cells until a concentration of 32 μg / ml, and molecule B did not exhibit toxicity to mammalian cells until a concentration of 16 μg / ml. Figure 4 Furthermore, it can be seen that this concentration has an inhibitory effect on the growth of Candida species and does not show toxicity to mammalian cells. Therefore, it is confirmed that the compound of the present invention is harmless to mammalian cells and can inhibit the growth of Candida species.

[0130] Example 3: Measurement of survival rate in a mouse model of candidiasis

[0131] For Candida infections, SC5314 (wild-type strain) was used in 6-week-old female Balb / C mice. Mice were divided into two groups of 5 mice each, and survival rates in each group were monitored.

[0132] 3-1: Infecting mice with Candida spp. via tail vein injection

[0133] 5.0x10 5 Candida cells were diluted in 200 μl of PBS and mice were infected via tail vein injection. Starting from day 1 post-infection, the control group was inoculated with 200 μl of PBS via tail vein injection, while the experimental group was weighed and treated with either molecule B (diluted in 100 μl of PBS) at a dose of 8 mg / kg via tail vein injection or molecule C (diluted in 100 μl of PBS) at a dose of 16 mg / kg via tail vein injection.

[0134] As a result, the group that did not receive the carbazole compound showed a 0% survival rate on day 9, while the other mice that received both compounds survived for more than 30 days. Figure 5 ).

[0135] 3-2: Infecting mice with Candida spp. via drinking water

[0136] Following Candida infection via tail vein injection as described in Example 3-1, normal drinking water was provided daily to the experimental control group (which used drinking water), while the experimental group was provided daily with either drinking water treated with molecule B at a concentration of 16 μg / ml or drinking water treated with molecule C at a concentration of 32 μg / ml. Mice survival was monitored daily for two weeks.

[0137] As a result, Candida-infected mice that drank normal drinking water showed a 0% survival rate within 9 days, while mice that drank drinking water treated with molecule B or molecule C showed survival rates of 100% and 80%, respectively. Figure 6 In other words, it can be seen that the compounds of the present invention have a therapeutic effect on Candida infections.

[0138] 3-3: Assessment of the degree of kidney infection in mice infected with Candida.

[0139] Following Candida infection via tail vein injection as described in Example 3-1, mice exhibiting the following conditions based on humane euthanasia criteria were euthanized with CO2, and kidneys were collected from their bodies for comparison of the degree of kidney infection: inability to eat or drink; abnormal breathing; dull and stiff fur; reduced movement; or hunched posture or trembling.

[0140] The results of examining the degree of infection after collecting kidneys from control mice and mice in the two experimental groups showed that the mice in the groups treated with molecules B and C exhibited very low degrees of kidney infection compared to the control mice. Figure 7 In other words, it can be seen that the carbazole compound of the present invention exhibits a significant therapeutic effect on Candida infections.

[0141] Although the invention has been described in detail with reference to specific features, it will be apparent to those skilled in the art that this description is merely a description of preferred embodiments of the invention and does not limit the scope of the invention. Therefore, the essential scope of the invention will be defined by the appended claims and their equivalents.

[0142] Industrial applicability

[0143] The carbazole compound according to the present invention not only inhibits the growth of Candida spp. but also inhibits the growth of fungi such as yeast, and neutralizes the pathogenicity of Candida spp. by inhibiting the morphological transformation of Candida spp. Therefore, the carbazole compound is very useful as an antifungal composition capable of treating Candida spp. infections.

Claims

1. Use of the carbazole compound of formula 1 or a salt thereof in the preparation of a medicament for inhibiting fungal infections: [Formula 1] Where R1 is Br and R2 is H; and The fungus mentioned is Saccharomyces cerevisiae.

2. Use of the carbazole compound of Formula 1 or a salt thereof in the preparation of a medicament for treating fungal infections: [Formula 1] Where R1 is Br and R2 is H; and The fungus mentioned is Saccharomyces cerevisiae.

3. Use of carbazole compounds of formula 1 or salts thereof in the preparation of medicaments for killing fungi: [Formula 1] Where R1 is Br and R2 is H; and The fungus mentioned is Saccharomyces cerevisiae.

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