Antimycotic drug

Abstract

To provide a metal chelate compound having high binding capability to zinc as an antimycotic drug.SOLUTION: An antimycotic drug comprises, as an active ingredient, a polyamine compound represented by formula (1) or a salt thereof. (In the formula, R1, R2, R3, R4, R5, and R6 are each independently hydrogen, halogen, C1-C5 alkyl, C1-C5 haloalkyl, or C1-C5 haloalkoxy).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an antifungal agent containing a polyamine compound effective against antifungal drug-resistant bacteria.

Background Art

[0002] In recent years, invasive fungal diseases have been on the rise, and particularly among immunocompromised patients, high morbidity and mortality rates have become a problem. The social and economic impact of invasive fungal diseases is also significant, reaching a burden of over $7.2 billion annually in the United States in 2017, and countermeasures are a global issue.

[0003] Antifungal drugs for treating invasive fungal diseases are limited to only three groups: polyenes, azoles, and echinocandins. As for their mechanisms of action, first, polyenes specifically bind to ergosterol, an essential lipid component of the fungal cell membrane, causing cell membrane dysfunction and acting bactericidally. Subsequently, azoles inhibit lanosterol demethylase ERG11, suppress ergosterol synthesis, and inhibit growth. On the other hand, echinocandins inhibit the function of β-D-glucan synthase encoded by the FKS gene and suppress the production of the fungal cell wall. Currently, resistant bacteria to these antifungal drugs have emerged, becoming a social problem. In particular, the emergence of Candida auris is a threat. C. auris was first reported in Japan in 2009, and since then, infection cases have been reported in various countries around the world. Bloodstream infections caused by C. auris have broken out in various regions such as the United States, Europe, South America, South Africa, and India, and its fatality rate is said to be 30-60%. The problem in the treatment of C. auris lies in its high drug resistance rate. Compared with other Candida species, the drug resistance rate of C. auris to antifungal drugs is very high. In a study investigating the drug susceptibility of isolates from multiple countries, approximately 40% were resistant to two or more antifungal drugs. Also, strains showing resistance to all three antifungal drugs have been successively reported. In response to this, the World Health Organization designated C. auris as a fungal priority pathogen list in 2022, emphasizing the urgency of infection control and countermeasures. Fortunately, there have been no reports of C. auris outbreaks in Japan. However, in response to case reports from overseas and cases that cannot be treated with existing antifungal drugs, the development of antifungal drugs with new mechanisms of action is currently an urgent task.

[0004] In recent years, "nutritional immunity" has attracted attention as a new mechanism of action for antifungal activity. "Nutritional immunity" refers to the host's infection defense mechanism in which host molecules capture trace metals such as iron and zinc required by pathogenic microorganisms and prevent them from being given to the microbial side. Among the trace metals targeted by "nutritional immunity", the important role of zinc in particular has been emphasized in recent research. Zinc is an essential trace metal for intracellular processes in eukaryotes. Specifically, about 9% of proteins are zinc-containing proteins, and zinc provides the structural stability of proteins and functions as an enzyme cofactor. Furthermore, zinc-dependent transcription factors also exist in gene regulation. Therefore, the acquisition of zinc is essential for the growth of pathogenic microorganisms. Candida albicans, a representative fungal species causing invasive fungal diseases, is known to be very susceptible to the influence of zinc, such as a decrease in growth and biofilm formation due to zinc depletion (see, for example, Non-Patent Document 1). From this, if there is a compound that captures and prevents the use of zinc required by fungi by mimicking the mechanism of "nutritional immunity", there is an expectation of a new type of antifungal drug here. In fact, the antifungal activities of zinc and iron capture compounds have been reported in a small number, suggesting that they may be effective against pathogens resistant to existing antifungal drugs (see, for example, Non-Patent Document 2).

[0005] Although polyamine compounds have been disclosed for use as antiparasitic agents, particularly for the treatment of leishmaniasis and Chagas disease (for example, Patent Document 1), the use of these polyamine compounds as antifungal drugs has not been studied.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

[0008] An object of the present invention is to provide an antifungal agent containing a cyclic polyamine compound which has high zinc-binding ability and can form a stable zinc complex. [Means for solving the problem]

[0009] For these reasons, the present inventors have conducted extensive research to solve the above problems. Specifically, they have evaluated the antifungal activity of various polyamine compounds (sometimes abbreviated as PA) that can deprive fungi of zinc and form zinc complexes, which have not been evaluated as antifungal drugs until now. As a result, they have found a cyclic polyamine compound that is a zinc chelating compound that shows the formation of a stable zinc complex, and have completed the present invention. That is, the present invention has the following configuration.

[0010] [1] An antifungal agent comprising, as an active ingredient, a polyamine compound represented by formula (1) or a salt thereof. JPEG2025103098000002.jpg58124 (In formula (1), R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently hydrogen, halogen, alkyl having 1 to 5 carbon atoms, haloalkyl having 1 to 5 carbon atoms, or haloalkoxy having 1 to 5 carbon atoms. [2] In formula (1), R 1 , R 2 , R 3 , R 4 , R 5 , and R 6The antifungal agent according to [1] above, which contains, independently of each other, a polyamine compound represented by formula (1) or a salt thereof, which is hydrogen, fluorine, chlorine, bromine, CH3, or CF3, as an active ingredient. [3] The antifungal agent according to [1] above, which contains an alkyl polyamine compound represented by formula (2) or a salt thereof as an active ingredient. JPEG2025103098000003.jpg59123 [4] A method for preventing or treating a fungal infection, which comprises administering the antifungal agent according to any one of [1] to [3] above. [5] Use of the antifungal agent according to any one of [1] to [3] above for preventing or treating a fungal infection. [6] The method for preventing or treating a fungal infection according to [4] above, wherein the fungal infection is candidiasis. [7] Use of the antifungal agent according to [5] above, wherein the fungal infection is candidiasis. [8] The antifungal agent according to any one of [1] to [3] above, wherein the fungus is Candida. [9] The antifungal agent according to any one of [1] to [3] above, wherein the fungus is C. auris.

[10] The method according to [4] above, wherein the fungal infection is a C. auris infection.

[11] Use according to [5] above, wherein the fungal infection is a C. auris infection. [Effect of the Invention]

[0011] The antifungal agent of the present invention exerts antifungal activity by depriving fungi such as Candida of zinc necessary for their growth and forming a stable zinc complex. Further, the antifungal agent of the present invention shows low cytotoxicity against human cell lines, suggesting high specificity of antifungal activity. [Brief Description of the Drawings]

[0012]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Mode for Carrying Out the Invention

[0013] In the present invention, zinc means zinc ions (Zn 2+ ). Chelate refers to the binding (coordination) of a metal ion by a ligand having a plurality of coordination sites (polydentate ligand), and in the present invention, it refers to the binding of zinc by a polyamine compound. An antifungal agent is a pharmaceutical that inhibits the growth of fungi and is used for the treatment of mycosis and as an agricultural chemical.

[0014] <Antifungal agent> The antifungal agent of the present invention contains at least one polyamine compound represented by the formula (1) or a salt thereof as an active ingredient. The antifungal agent may be composed of only one kind of the polyamine compound or a salt thereof, or may be composed of two or more kinds of the polyamine compounds. Further, other polyamine compounds other than the polyamine compound represented by the formula (1) may be contained.

[0015] JPEG2025103098000004.jpg58124

[0016] In formula (1), R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently hydrogen, halogen, alkyl having 1 to 5 carbon atoms, haloalkyl having 1 to 5 carbon atoms, or haloalkoxy having 1 to 5 carbon atoms. Preferably, R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently hydrogen, fluorine, chlorine, bromine, CH3, or CF3. More preferably, R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are all hydrogen, and it is 1,4,7-tris(2-pyridylmethyl)-1,4,7-triazacyclononane represented by formula (2).

[0017] In formula (1), as the alkyl having 1 to 5 carbon atoms, a linear or branched alkyl having 1 to 5 carbon atoms can be used. For example, methyl, ethyl, n-propyl, i-propyl, n-butyl, tert-butyl, sec-butyl, or n-pentyl can be used. Preferably, methyl, ethyl, n-propyl, or i-propyl is used, and more preferably, methyl is used.

[0018] In formula (1), as the haloalkyl having 1 to 5 carbon atoms, for example, the alkyl part of the haloalkyl having 1 to 5 carbon atoms is a group obtained by removing 1 to 3 hydrogens from the groups exemplified for the alkyl having 1 to 5 carbon atoms above. As the halogen, for example, fluorine, chlorine, and bromine can be mentioned. Specifically, trifluoromethyl or 2,2,2-trifluoroethyl can be mentioned.

[0019] In formula (1), examples of the haloalkoxy having 1 to 5 carbon atoms include groups in which 1 to 3 hydrogens are removed from the groups exemplified for the alkyl having 1 to 5 carbon atoms in the alkyl moiety of the haloalkoxy having 1 to 5 carbon atoms, and examples of the halogen include fluorine, chlorine, and bromine. Specifically, examples of the haloalkoxy having 1 to 5 carbon atoms include trifluoromethoxy.

[0020] Examples of the salt of the polyamine compound represented by formula (1) include salts of inorganic or organic acids such as hydrochloride, sulfate, and acetate, and metal salts (excluding zinc salts) such as sodium, potassium, and iron salts.

[0021] The polyamine compound represented by formula (1) chelates zinc, which is important for the growth of fungi, takes zinc away from the fungi, coordinates and fixes zinc with the nitrogen in the molecule, and incorporates it into the molecule. As the polyamine compound represented by formula (1) used in the present invention, the polyamine compound represented by formula (2) (1,4,7-tris(2-pyridylmethyl)-1,4,7-triazacyclononane) can be preferably used. The polyamine compound represented by formula (2) forms a stable zinc complex (Figure 1) by coordinating and fixing zinc with six nitrogens in the molecule. It should be noted that the zinc in the cell is scarcely detected as free zinc, and most of it forms a strong bond with proteins.

[0022] JPEG2025103098000005.jpg59123

[0023] <Other polyamine compounds> Examples of other polyamine compounds include polyalkylpolyamines such as diethylenetriamine, triethylenetriamine, and tetraethylenepentamine, and cyclic polyamines such as 1,4,7,10,13,16-hexaazacyclooctadecane, 1,4,7,10-tetraazacyclodecane, 1,4,8,12-tetraazacyclopentadecane, and 1,4,8,11-tetraazacyclotetradecane. Other polyamine compounds preferably do not inhibit the binding between the polyamine compound represented by formula (1) and zinc.

[0024] The antifungal agents of the present invention may further contain polyene antifungal agents such as amphotericin B deoxycholate, azole antifungal agents such as fluconazole (FLCZ), itraconazole (ITCZ), voriconazole (VRCZ), and echinocandin antifungal agents such as micafungin (MCFG) and caspofungin (CPFG). In particular, echinocandin antifungal agents are characterized by extremely high safety and are mainly used for severe candidiasis. By using these in combination with the polyamine compound, it is expected to enhance the effect of treating infectious diseases. In particular, voriconazole (VRCZ), a new-generation azole, showed efficacy superior to that of AMPH-B for the first time in a randomized clinical trial against invasive aspergillosis, which is refractory and has a poor prognosis. Subsequently, liposomal AMPH-B also showed similar results, and the initial treatment for invasive aspergillosis has been standardized. Echinocandin antifungals have been confirmed to be effective against chronic aspergillosis.

[0025] <Fungus> Fungi are eukaryotes having a nuclear membrane, different from bacteria. They are classified into filamentous fungi and yeasts according to their morphology. Representative filamentous fungi are Aspergillus fungi, and representative yeasts are Candida fungi.

[0026] <Aspergillus fungi> Aspergillus fungi are the causative agents of aspergillosis (mainly pulmonary aspergillosis), and among them, Aspergillus fumigatus is the main causative agent. Mucormycosis (also called zygomycosis) is important as a differential disease of aspergillosis.

[0027] <Candida fungi> Fungi of the genus Candida are the causative agents of candidiasis. Among them, Candida albicans is the most frequent causative agent, followed by Candida glabrata, Candida parapsilosis, Candida tropicalis, etc. These four species account for more than 90% of the causative agents of candidiasis. In addition, candidiasis caused by other species such as Candida krusei, Candida guilliermondii, Candida kefyr, Candida lusitaniae, etc. has been increasing in recent years. Many strains of C.glabrata and C.krusei show low sensitivity to azole drugs and require attention during treatment. In recent years, nosocomial outbreaks caused by Candida auris have been reported one after another, and it has characteristics different from other Candida species, such as easy transmission and a high resistance incidence rate.

[0028] It has been shown as the result of the Japan Nosocomial Infection Surveillance (JANIS) that fungi of the genus Candida are the main pathogens of bacteremia in our country. In addition to the high frequency, combined with the high fatality rate of candidemia of about 30 - 50%, candidemia is an extremely important bloodstream infectious disease similar to bacteremia caused by Staphylococcus aureus, etc. C.albicans accounts for 2% of all blood isolates, but considering that non-albicans species are isolated at a similar or higher rate, it is estimated that the genus Candida is detected in 4 - 5% of blood culture positive cases.

[0029] <Symptoms caused by fungi of the genus Candida> Infections caused by fungi of the genus Candida are broadly classified into superficial candidiasis and deep candidiasis. Representative diseases of superficial candidiasis include oropharyngeal candidiasis (thrush), vulvovaginal candidiasis, candidal dermatitis, etc. In oropharyngeal candidiasis, white coating can be observed on the mucosa, and oral discomfort, abnormal taste, pain, etc. are felt subjectively. In vulvovaginal candidiasis, symptoms such as itching, odorless leukorrhea, and dysuria appear. Candidal dermatitis is caused by a hypersensitivity reaction to Candida cell components caused by chronic maceration of the skin, and presents erythema with local itching and a burning pain.

[0030] Deep candidiasis occurs when Candida invades deep organs and tissues, forming lesions in multiple organs as systemic disseminated lesions. In Japan, it is generally recognized and treated as candidemia, so disseminated lesions rarely become apparent. However, in any case, it presents a serious condition. It disseminates to the liver, spleen, kidney, heart (endocardium), eye, bone, central nervous system, etc. When candidemia develops, it is said that up to 80% is complicated by Candida endophthalmitis.

[0031] In addition, active ingredients for controlling superficial and systemic infections of humans and animals caused by fungi are actually needed. The antifungal agent of the present invention can be used for the prevention or treatment of fungal infections to be administered to the target human or animal, and is particularly effective in the prevention or treatment of candidiasis. It is also effective in the prevention or treatment of infections caused by C. auris.

[0032] Therefore, the main object of the present invention is to provide a compound having high safety and exhibiting antifungal activity against a wide range of fungi. The antifungal agent of the present invention containing the polyamine compound of formula (1) or a salt thereof as an active ingredient can be used for both therapeutic treatment and prophylactic treatment. In addition, the antifungal agent of the present invention can be used not only for human fungal infections but also for veterinary medicine.

[0033] Another preferred object of the present invention is to be used for controlling fungi not only in humans but also in animals such as livestock, food animals, and pets. As the administration method, methods of administering an effective dose of the antifungal agent of the present invention to host humans and animals by oral, parenteral (transdermal, implant, etc.) and the like can be exemplified. Specifically, as the administration forms of the antifungal agent of the present invention for topical use in humans and in the veterinary field, ointments, creams, sprays, infusions, painting, and spot painting can be used. The concentration of the polyamine compound represented by formula (1) or a salt thereof contained in the antifungal agent of the present invention is appropriately adjusted depending on the compound used, administration method, administration site, treatment, administration form, etc. For example, in the case of topical administration, a range of 0.01 to 99% by weight can be used, a range of 0.05 to 5% by weight is preferred, and a concentration range of 0.1 to 1% by weight is more preferred.

[0034] When the administration method of the antifungal agent of the present invention is oral administration, dosage forms as its administration form can be, for example, powders, tablets, granules, water-soluble capsule agents, emulsions, foams, or microcapsules. However, it is not always necessary to directly administer the preparation to humans or animals, and it may be added to beverages, or in the case of humans, it may be mixed into food and in the case of animals, it may be mixed into feed (dog food, cat food, etc.). Additives such as fragrances and flavoring agents may be added if necessary to make it easier to ingest. Oral administration is a preferred administration method because it can be easily administered.

[0035] In addition, when the administration method is parenteral, for example, intravenous injection, subcutaneous injection, topical application, long-term implantation (depot), or microcapsule injection (microsphere) can be used. Examples of transdermal administration forms include subcutaneous, dermal, intramuscular, and intravenous administration of injection dosage forms. In addition to ordinary syringes with needles, needleless systems may also be appropriate in some cases. Implants can also be used, and examples include devices that can be inserted into the animal body to deliver substances. The formulations used in the method of administering the effective dose of the antifungal agent of the present invention, such as ointments, creams, sprays, infusions, paints, spot paints, powders, tablets, granules, water-soluble capsules, emulsions, foams, microcapsules, and formulations such as those used for intravenous injection, subcutaneous injection, topical application, long-term implantation (depot), or microcapsule injection (microsphere), contain an effective amount of the polyamine compound of formula (1) or a salt thereof, and are not particularly limited as long as zinc is not present in the formulation, and can be produced by known methods according to the common general knowledge in the field of pharmaceutical formulations.

[0036] By selecting an appropriate dosage form, it is possible to promote the permeation of the antifungal agent into the living tissue of an animal and maintain its utilization. For example, when using one or more poorly soluble antifungal agents, since the body fluid of an animal can dissolve only a small amount of the active ingredient at a time and thus it is necessary to increase the solubility, this point is important.

[0037] The antifungal agent of the present invention can be used as a matrix formulation. When the matrix formulation is injected into the body, it remains in the body as a depot and continuously releases the active ingredient of the antifungal agent. By using a matrix formulation, the decomposition of the antifungal agent can be physically prevented, and at the same time, the active ingredient can be sustainedly released and maintained. As the matrix formulation, generally, wax-like substances such as high molecular weight polyethylene glycol, degradable polyester copolymer, and semi-solid excipients of vegetable wax can be used.

[0038] The dosage of the antifungal agent of the present invention varies depending on the active ingredient of the antifungal agent used, the administration method, the administration form, and the desired treatment. Generally, satisfactory results can be obtained with a daily dose of 1 to 100 mg / kg of the animal body weight. It may be administered once a day, divided into 2 to 4 times a day, or in a sustained-release form. Preferred dosages are 1 to 50 mg / kg of the animal body weight for systemic administration and 0.1 to 10% for topical administration. In the case of large mammals, the corresponding daily dose ranges from 70 to 2000 mg, and for dosage forms suitable for oral administration, it is 17.5 to 1000 mg. Since the total dosage varies particularly depending on the body weight and constitution of the animal, even with the same active ingredient, it may differ between animal species and within an animal species. The present invention also relates to a method for treating a disease or infection caused by fungi using the compound of formula (I) or a salt thereof.

[0039] To formulate a composition for administration to humans, livestock, food animals, and companion animals, adjuvants known in medical and veterinary medicine can be used in forms such as oral, parenteral, and implants. Some examples are given below, but are not limited thereto.

[0040] As carriers that can be used for oral administration agents, such as tablets, etc., fillers, binders, disintegrants, excipients, etc. are used. Examples of fillers include saccharides (such as lactose, sucrose, mannitol, or sorbitol, etc.), cellulose preparations, and / or calcium phosphate (such as tricalcium phosphate or calcium hydrogen phosphate, etc.). Examples of binders in a broad sense include starch paste using corn, wheat, rice, or potato starch, gelatin, tragacanth gum, or methylcellulose. If desired, examples of disintegrants include the above starches, carboxymethyl starch in a broad sense, cross-linked polyvinylpyrrolidone, agar, alginic acid or its salts (such as sodium alginate, etc.). Excipients are particularly flow regulators and lubricants, and examples include silicic acid, talc, stearic acid or its salts (such as magnesium stearate or calcium stearate, etc.), and / or polyethylene glycol. Furthermore, an appropriate (appropriately enteric-coated) coating may be applied to the tablet core. In particular, a thick sugar solution that may be added with gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol, and / or titanium dioxide, etc., or a coating solution dissolved in an appropriate organic solvent or solvent mixture is used. When manufacturing an enteric coating, an appropriate cellulose preparation solution such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate is used. For example, dyes, flavoring agents, or pigments may be added to the tablets or tablet coatings for the purpose of identification or to indicate different dosages of the active ingredient. It is preferable to use components that do not generate zinc in any case.

[0041] Examples of other pharmaceutical compositions for oral administration include hard capsule preparations composed of gelatin, and soft seal capsule preparations composed of gelatin and a plasticizer (such as glycerol or sorbitol). Hard capsule preparations can be filled by mixing the active ingredient in granular form with, for example, a filler (such as lactose), a binder (such as starch), and / or a lubricant (such as talc or magnesium stearate), and, as appropriate, a stabilizer. In the case of soft capsule preparations, it is preferable to dissolve or suspend the active ingredient in a suitable liquid (such as fatty oil, paraffin oil, or liquid polyethylene glycol), and a stabilizer may also be added as appropriate. As another form, capsule preparations that are easy to chew or swallow whole are preferred.

[0042] Dosage forms suitable for parenteral administration are, in particular, aqueous solutions of the active ingredient in water-soluble form (such as water-soluble salts), but broadly speaking, suspensions of the active ingredient may also be used. For example, suitable oily injection suspensions or aqueous injection suspensions with a thickening agent (such as sodium carboxymethyl cellulose, sorbitol, and / or dextran) and, as appropriate, a stabilizer, using a suitable lipophilic solvent or vehicle (such as oils like sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides).

[0043] The antifungal agent (drug) of the present invention can be produced by a known method by conventional mixing, granulation, coating, dissolution, or freeze-drying methods, taking care, for example, not to incorporate zinc. The antifungal agent for oral administration can be obtained, for example, by adding a solid carrier to the active ingredient, appropriately granulating the resulting mixture, treating the mixture or granules as desired or necessary, and forming tablets or tablet cores after adding a suitable excipient.

[0044] Therefore, the antifungal agent of the present invention can be used as a preparation such as an emulsified concentrate, a suspension concentrate, a directly sprayable and dilutable solution, an applicable paste, a diluted emulsion, a spray powder, a soluble powder, a dispersible powder, a wettable powder, a dusting powder, a granule, or a capsule-encapsulated polymer containing at least one compound represented by formula (1) or a salt thereof.

[0045] In these compositions, the active ingredient may be used in pure form, for example, as a solid active ingredient with a specific particle size. However, it is preferably used in combination with at least one adjuvant commonly used in the formulation field, such as an extender (such as a solvent or a solid carrier) or a surface-active compound (such as a surfactant). Only physiologically acceptable adjuvants are used for the control of fungi in humans, livestock, food animals, and pets.

[0046] Compositions (drugs) for humans or animals are also included in the present invention.

Examples

[0047] Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples.

[0048] <Materials> Dimethyl sulfoxide (DMSO), zinc(II) sulfate heptahydrate, N,N,N',N'',N''-pentamethyldiethylenetriamine (FUJIFILM Wako Pure Chemical Industries), trientine hydrochloride (Merck), 1,1,4,7,10,10-hexamethyltriethylenetetramine, 1,4,7-trimethyl-1,4,7-triazacyclononane (Sigma-Aldrich), 1,4,7-triazacyclononane, 3HCl (CheMatech), and other reagents obtained from commercial sources were used unless otherwise specified.

[0049] <Synthesis of 1,4,7-tris(2-pyridylmethyl)-1,4,7-triazacyclononane (PA6)> 1,4,7-Triazacyclononane (0.77 g, 5.96 mmol) was dissolved in 5N aqueous NaOH solution (40 mL) under N2. 2-(Chloromethyl)pyridine hydrochloride (3.32 g, 19.7 mmol) was added at 0 °C and the mixture was stirred at ambient temperature for 20 h. The mixture was diluted with saturated aqueous NaCl2 solution (40 mL) and the organic layer was extracted three times with CH2Cl2 (20 mL per extraction). The combined organic layers were dried over Na2SO4 and concentrated in vacuo to give the crude product as a brown liquid. Purification of the product by medium pressure column chromatography gave PA6 (2.40 g) as a black liquid. The structure and purity of PA6 were determined by ESI mass spectrometry at MS (ESI, m / z) 403.26 [PA6 + H + + and ESI mass spectrometry at MS (ESI, m / z) 403.26 [PA6 + H

[0050] <Method for Measuring Minimum Inhibitory Concentration (MIC) of Compounds against Fungi> The MIC was measured according to the method described in Clinical and Laboratory Standards Institute (CLSI) M27-Ed4. The fungal growth medium used was Roswell Park Memorial Institute (RPMI) 1640 medium buffered with morpholinepropanesulfonic acid (RPMI-MOPS), and PA6 was added to the medium at concentrations of 0.03125 - 2 μg / mL in a two-fold serial dilution. Growth was evaluated after culturing at 35 °C for 24 h, and the MIC was defined as the minimum concentration that resulted in at least 50% growth inhibition. The effects of adding metals and reducing agents to the medium were also evaluated. In the presence of PA6, FeSO4, FeCl3, MnCl2, ZnSO4, CuCl2, MgCl2, CaCl2, glutathione reduced form (FUJIFILM Wako Pure Chemical Industries), N-acetyl-L-cysteine (Tokyo Chemical Industry), or α-thioglycerol (Tokyo Chemical Industry) were added at concentrations of 1.25 μmol / L - 2000 μmol / L, respectively, and the effects on the MIC were evaluated.

[0051] <RNA Isolation, cDNA Library Construction, and Sequencing (RNA-Seq)> Candida albicans SC5314 (Figure 2) was used at 1×10​7 Inoculate into RPMI-MOPS medium to a concentration of cells / mL, and further add PA6 to concentrations of 0.25 and 1 μg / mL, then culture at 30 °C for 6 hours. Subsequently, use beads and Isogen (Nippon Gene), RNeasy Mini Kit (Qiagen), and RNase-free DNase I (Qiagen) to extract RNA. Furthermore, use 5′ template switching PCR and SMART-Seq v4 Ultra Low Input RNA Kit (Takara Bio Inc.) to purify and lyse the RNA, fragment the amplified cDNA, and add dual-index barcodes using the Illumina Nextera XT DNA Library Preparation Kit (Illumina). The library was verified on an Agilent 4200 TapeStation automated electrophoresis platform (Agilent Technologies), pooled, and sequenced on an Illumina NovaSeq 6000 platform (Illumina). The read sequences were mapped to the reference genome sequence of C. albicans SC5314 from the Candida genome database (version A22-s07-m01-r153). The transcript abundance was estimated as transcripts per million (TPM).

[0052] <Reverse transcription-quantitative real-time PCR (RT-qPCR)> Use beads and Isogen (Nippon Gene), RNeasy Mini Kit (Qiagen), and RNase-free DNase I (Qiagen) to extract and purify RNA. Use this RNA to perform reverse transcription with a ReverTra Ace qPCR RT Master Mix Kit (Toyobo). RT-qPCR was performed using SYBR Premix ExTaq (Takara Bio) in an Mx3000P real-time PCR system (Agilent). The relative gene expression levels were normalized to the ACT-1 gene (C. albicans) or the B9J08_000486 gene (C. auris).

[0053] <ESI-MS spectrometry> The sample was prepared by diluting the corresponding solution with MeOH. PA6 and Zn were synthesized by mixing PA6 and ZnSO4 in DMSO. 2+ The structure and purity of the complex were determined using an ESI-mass spectrometer (Q Exactive, Thermo Fisher Scientific).

[0054] <Measurement of intracellular free zinc concentration using Zinbo-5> Zinbo-5 (Santa Cruz Biotechnology) is a fluorescent probe that specifically binds to Zn 2+ and is used for measuring the intracellular Zn 2+ content. Candida auris AR 0389 cells were grown overnight in yeast extract-peptone-dextrose (YPD) broth [1% Bacto yeast extract (Difco, MI, USA), 2% Bacto peptone (Difco), and 2% glucose; pH 6.8], pelleted, washed three times with PBS, and then resuspended in PBS to adjust to a bacterial suspension with an OD600 of 2.0. Next, this bacterial suspension was inoculated into a 96-well plate, various concentrations of PA6 or TPEN (Tokyo Chemical Industry) were added, and the plate was incubated at 30 °C for 6 h. Then, it was incubated with 5 μmol / L Zinbo-5 for 30 min. Finally, fluorescence was measured using a microplate reader (excitation at 355 nm, emission at 485 nm).

[0055] <Growth inhibition assay> To 5 mL of RPMI-MOPS medium containing Candida auris AR 0389 cells (4 × 10 4 colony-forming units (CFU) / mL), various concentrations of voriconazole, PA6, or both were added, and the culture was carried out at 35 °C for 96 h with gentle stirring. At each time point (0, 4, 6, 8, 24, 48, and 96 h), 10 μL of the bacterial suspension was taken, appropriately diluted, inoculated onto a YPD agar plate, and the plate was cultured at 30 °C for 24 h to count the colonies, thereby evaluating the viable cell count at each time point in the culture solution.

[0056] <Checkerboard assay> To verify the synergistic effect of PA6 and voriconazole against C. auris AR0389, it was carried out. The medium used was RPMI-MOPS, and it was performed according to the microdilution method specified by CLSI. The final concentration of each drug was in the range of 0.25 - 16 μg / ml for voriconazole and 0.03125 - 2 μg / mL for PA6. The evaluation was carried out by culturing at 35°C for 24 hours and visually observing the growth. The minimum inhibitory concentration (MIC) was defined and determined as the minimum concentration that brought about at least 50% growth inhibition. The fractional inhibitory concentration (FIC) index for evaluating the synergistic effect was determined for each drug by dividing the MIC when the drugs were combined by the MIC of the drug when used alone, and it was defined by the sum. An FIC index of 0.5 or less was considered a synergistic effect, 0.5 or more and less than 1 was additive, 1 or more and less than 2 was non-correlated, and 2 or more was an antagonistic effect.

[0057] <Galleria mellonella Survival Assay> A total of 72 G. mellonella larvae were randomly assigned to four groups (untreated, PA6, VRCZ, PA6 and VRCZ). To the right foreleg of each larva, 10 μL of C. auris AR 0389 bacterial solution (2×10 5 CFU per larva) was injected with only the solvent or with the addition of the drug. The amount of each drug was 1.5 μg per larva for PA6, 2 μg per larva for VRCZ, and PBS was used as the solvent. The number of surviving larvae in each group was tracked over 7 days.

[0058] <Crystal Violet Assay> C. albicans SC5314 and C. auris AR0382 were grown on YPD agar at 30°C for 24 hours, collected with an inoculating loop, washed with sterile PBS, and adjusted to 1.0×10 7It was resuspended at cells / mL. 100 μL of it was added to each well of a 96-well flat-bottom plate and cultured at 37 °C for 4 hours to adhere to the plastic on the plate surface. Then, the supernatant in the well was discarded to remove non-adherent free cells. Next, 200 μL of fresh RPMI-MOPS medium was added to each well, and the plate was incubated at 37 °C for 24 hours to form a biofilm. After that, the supernatant was discarded, and RPMI-MOPS medium containing 0.25 μg / mL PA6 and various concentrations of amphotericin B was added. After 24 hours of culture, the supernatant was carefully removed from each well, and the biofilm was fixed with 200 μL of methanol. Then, each well was washed 3 times with ultrapure water, stained with 0.1% crystal violet (FUJIFILM Wako Pure Chemical Industries) for 30 minutes, and washed 3 times with ultrapure water. Finally, the crystal violet bound to the biofilm was dissolved by culturing with 200 μL of methanol at room temperature for 30 minutes. The OD at a wavelength of 595 nm was measured with a microtiter plate reader to evaluate the biofilm amount.

[0059] <Cytotoxicity Test of PA6 Using Human Hepatocytes (HepG2)> The cytotoxicity of PA6 and TPEN against HepG2 was evaluated by an MTT assay using an MTT cell counting kit (Nacalai Tesque). In each well of a 96-well tissue culture plate (Corning), 5000 human hepatocytes (HepG2) (Sigma) were added to 200 μL of growth medium (Eagle's minimum essential medium; Sigma) supplemented with 2 mM GlutaMAX (Gibco), 1% non-essential amino acids, and 10% fetal bovine serum (Life Technologies). The plate was incubated overnight at 37 °C under 5% CO2. The next day, the growth medium was replaced with growth medium containing various concentrations of PA6 or TPEN, and the plate was further incubated at 37 °C under 5% CO2 for an additional 24 h. Then, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution was added to each well, and the plate was incubated at 37 °C under 5% CO2 for 4 h. Next, 100 μL of solubilization solution was added to each well, and the absorbance at 570 nm was measured using a Synergy LX multimode microplate reader (BioTek). The cell viability was measured by expressing it as (%) according to the following formula. Cell viability (%) = 100 × (mean OD of treated cells - mean OD of blank) / (mean OD of untreated cells - mean OD of blank)

[0060] <Statistical analysis> Statistical analysis was performed using GraphPad Prism, version 8 (GraphPad Software, La Jolla). In Figure 6, a one-way analysis of variance (ANOVA) and Dunnett's multiple comparison post hoc test were used to test for significant differences in the mean biofilm amounts among three or more groups. In the Kaplan–Meier survival curves in Figure 7, significance was evaluated by a log-rank test. A p-value of less than 0.05 was considered statistically significant.

[0061] <Evaluation of the antifungal activity of polyamine compounds> The antifungal activities of polyamine compounds PA1 - PA6 (compounds in Table 1) that are expected to chelate transition metal ions important for fungal growth were evaluated. Note that PA6 was obtained by modifying the skeleton of PA4 with three pyridylmethyl groups. As an evaluation of antifungal activity, the minimum inhibitory concentration (MIC) of each of PA1 - PA6 against C. albicans SC5314 was measured.

[0062] <Evaluation results of antifungal activity> As a result of measuring the minimum inhibitory concentration (MIC), PA1 - PA5 did not inhibit the growth of C. albicans, but PA6 inhibited the growth of C. albicans (MIC in Table 1). This is presumably because by introducing three pyridylmethyl groups into the rigid cyclic skeleton of PA4, the binding ability to zinc increased compared to PA4, and antifungal activity was exerted by preferentially capturing zinc.

[0063]

Table 1

[0064] <Measurement of the concentration of metal ions that inhibit antifungal activity> After preparing metal ions (Fe 2+ , Fe 3+ , Mn 2+ , Zn 2+ , Cu 2+ , Mg 2+ , or Ca 2+ ) by serial dilution 10 - fold and adding them to the medium, the change in the MIC of PA6 in C. albicans SC5314 strain and C. auris AR 0389 strain was followed, and the metal ion concentration (unit: μmol / L) at which the MIC increased from 0.5 μg / mL to 2 μg / mL or more was determined (Table 2). As a result, Zn 2+ and Cu 2+ increased the MIC of PA6 at the lowest concentration (1.25 - 12.5 μmol / L). Subsequently, Mn 2+ increased the MIC value at a low concentration (12.5 - 125 μmol / L), but Zn 2+ and Cu2+ Compared to it, the concentration was about 10 times higher. From these data, it was found that PA6 was inactivated in antifungal activity by Zn 2+ and Cu 2+ . Furthermore, PA6 and zinc form a stable metal complex. Moreover, when PA6 acts on C. albicans, there is no change in the transporter-related genes involved in copper uptake, but the transporter-related genes involved in zinc uptake are significantly upregulated, leading to a state of zinc starvation. Therefore, it was suggested that PA6 preferentially captures Zn 2+ rather than Cu 2+ and exerts antifungal activity.

[0065]

Table 2

[0066] [Example 1] <Evaluation method for the binding of PA6 and zinc ions> To evaluate the binding of PA6 and zinc ions in vitro, PA6 and ZnSO4 were mixed and the solution was analyzed using electrospray ionization (ESI) mass spectrometry. A cation peak cluster was detected at m / z 233.09, and the calculated isotope pattern (B in Figure 3) was consistent with that of the 1:1 Zn 2+ :PA6 complex. The ESI mass spectrum showed the formation of a stable zinc complex formulated as [Zn(PA6)] 2+ (Figure 1).

[0067] PA6 showed the lowest MIC (0.5 μg / mL; 1.25 μmol / L). The antifungal activity of PA6 was evaluated against 19 strains of C. auris and one strain each of Candida glabrata, Candida krusei, and Candida parapsilosis (Table 3).

[0068]

Table 3

[0069] The MIC of PA6 was found to be effective against a wide range of Candida species, at 0.25 μg / mL for C. glabrata and 0.5 μg / mL for other Candida species. It was also similarly effective against C. auris AR 0389, a strain known to have high drug resistance due to ERG11 mutation (Y132F) and overexpression of CDR1.

[0070] <Inhibitory effect of PA6 alone and in combination with voriconazole on Candida growth> Since PA6 has a completely different mechanism of action from conventional antifungal agents, it is expected to be effective against fungi currently considered drug-resistant. Among various strains of C. auris, AR 0389 shows a remarkable drug resistance tendency (Table 4). Therefore, the effects of PA6 alone or in combination with the conventional antifungal drug voriconazole on AR 0389 were examined. The growth dynamics of the AR 0389 strain are shown in Figure 4. PA6 at 0.25 μg / mL suppressed growth compared to the control. The combination of 2 μg / mL of voriconazole at 0.5 MIC and 0.25 μg / mL of PA6 suppressed growth more strongly than PA6 or voriconazole alone. An additive effect of PA6 was also observed with 2 MIC of voriconazole. To evaluate the synergistic effect of PA6 and voriconazole, the fractional inhibitory concentration (FIC) index was calculated by checkerboard assay. As a result, the FIC index was 1.0, and no synergistic effect was evident in the checkerboard assay.

[0071]

Table 4

[0072] <Therapeutic effect of PA6 on invasive candidiasis of C. auris in Galleria mellonella larvae> To evaluate the efficacy of antifungal drugs against pathogenic fungi in vivo, the G. mellonella larva infection model is often used. In this study, G. mellonella infected with C. auris was used to examine the therapeutic effects of PA6, voriconazole, or their combination. G. mellonella larvae were infected with C. auris AR 0389 (2×10 5 CFU / larva), and simultaneously administered PA6, voriconazole, or both, and the survival rate was tracked. The survival rate on the 7th day after infection was only 11% in the control group, but 44% in the group administered PA6 (1.5 μg per larva). The therapeutic effect of the group administered voriconazole (2 μg per larva) was similar. When PA6 and voriconazole were combined, the survival rate increased to 67% (Figure 5). PA6 has a therapeutic effect against C. auris infection alone, and it was shown to exhibit an even higher effect when combined with voriconazole.

[0073] G. mellonella larvae (18 per group) were infected with C. auris AR 0389 (2×10 5 CFU / larva), and simultaneously administered PBS (control), PA6 (1.5 μg / larva), VRCZ (2 μg / larva), or a combination of PA6 and VRCZ (1.5 μg / larva and 2 μg / larva respectively), and the survival of G. mellonella was tracked for 7 days. The Kaplan-Meier survival curve of G. mellonella was created, and the significance of the therapeutic effect of each drug was evaluated by the log-rank test. *P<0.05;***P<0.001 vs control

[0074] <Destruction of C. albicans and C. auris biofilms by PA6> There are reports that the high pathogenicity of C.auris strain AR 0382 is due to its remarkable biofilm-producing ability. For the purpose of observing the effect of PA6 on the biofilm of PA6, PA6 was administered to the biofilms of C.albicans SC5314 and C.auris AR 0382, and the amount of biofilm was evaluated by the crystal violet method. Significant biofilm disruption was observed at 0.25 μg / mL PA6 (Figure 6).

[0075] <Effect of PA6 on C.auris AR 0382 and SC5314 biofilms> Cells were cultured in a 96-well plate in RPMI 1640 at 37 °C for 24 hours. The medium was discarded, and medium containing PA6 or amphotericin B (AMPH-B) was added. After 24 hours, the wells were washed three times with water, and the amount of biofilm was quantified by the crystal violet assay. ** P<0.01;***P<0.001;****P<0.0001

[0076] <Cytotoxicity against human cell lines> To evaluate the cytotoxicity of PA6 against the HepG2 human hepatocellular carcinoma cell line, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay) was used. The values of the 50% cytotoxic concentration (CC50) were 9.67 μg / mL for PA6 and 0.99 μg / mL for TPEN (Figure 7), and the selectivity index (defined as the ratio of CC50 to the MIC against C.auris AR 0389) was 44.12 for PA6 and 4.19 for TPEN. These data indicate that PA6 is less toxic to human cells and safer than TPEN, suggesting a high specificity of the antifungal activity of PA6.

[0077] It was revealed that PA6 exerts antifungal activity by chelating zinc and depriving Candida cells of zinc as an essential metal. In addition, PA6 showed lower cytotoxicity against human cell lines compared to TPEN, suggesting a high specificity as an antifungal activity.

[0078]

Industrial Applicability

[0079] In addition to being usable, for example, in the treatment and / or prevention of fungal infections such as candidiasis, the antifungal agent of the present invention can, for example, deprive fungi of zinc necessary for their growth and form a stable zinc complex, and thus is expected to exhibit good antifungal activity against fungi other than Candida. Further, since the antifungal agent of the present invention has low cytotoxicity against human cell lines, it is expected to be used for pharmaceuticals against lethal resistant bacteria such as various drug transplant patients, coating of in-vivo use devices such as catheters, and spraying on biofilms.

Claims

**Claim 1** An antifungal agent containing, as an active ingredient, a polyamine compound represented by formula (1) or a salt thereof. (wherein, R １ , R ２ , R ３ , R ４ , R ５ , and R ６ are each independently hydrogen, halogen, alkyl having 1 to 5 carbon atoms, haloalkyl having 1 to 5 carbon atoms, or haloalkoxy having 1 to 5 carbon atoms.) **Claim 2** In formula (1), R １ , R ２ , R ３ , R ４ , R ５ , and R ６ are each independently hydrogen, fluorine, chlorine, bromine, CH ３ , or CF ３ , and the antifungal agent according to claim 1, which contains, as an active ingredient, a polyamine compound represented by formula (1) or a salt thereof. **Claim 3** The antifungal agent according to claim 1, containing, as an active ingredient, a polyamine compound represented by formula (2) or a salt thereof. **Claim 4** The antifungal agent according to any one of claims 1 to 3, wherein the fungus is Candida. **Claim 5** The antifungal agent according to any one of claims 1 to 3, wherein the fungus is C. auris.

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