Antifungal Activity of 1,4-Dialkoxynaphthalen-2-Acyl Imidazolium Salts by Inducing Apoptosis of Pathogenic Candida spp.

Even though Candida spp. are staying commonly on human skin, it is also an opportunistic pathogenic fungus that can cause candidiasis. The emergence of resistant Candida strains and the toxicity of antifungal agents have encouraged the development of new classes of potent antifungal agents. Novel naphthalen-2-acyl imidazolium salts (NAIMSs), especially 1,4-dialkoxy-NAIMS from 1,4-dihydroxynaphthalene, were prepared and evaluated for antifungal activity. Those derivatives showed prominent anti-Candida activity with a minimum inhibitory concentration (MIC) of 3.125 to 6.26 μg/mL in 24 h based on microdilution antifungal susceptibility test. Among the tested compounds, NAIMS 7c showed strongest antifungal activity with 3.125 μg/mL MIC value compared with miconazole which showed 12.5 μg/mL MIC value against Candida spp., and more importantly >100 μg/mL MIC value against C. auris. The production of reactive oxygen species (ROS) was increased and JC-1 staining showed the loss of mitochondrial membrane potential in C. albicans by treatment with NAIMS 7c. The increased release of ultraviolet (UV) absorbing materials suggested that NAIMS 7c could cause cell busting. The expression of apoptosis-related genes was induced in C. albicans by NAIMS 7c treatment. Taken together, the synthetic NAIMSs are of high interest as novel antifungal agents given further in vivo examination.


Introduction
Infections by invasive fungal pathogens result from immunosuppression, long-term broad-spectrum antimicrobials, endocrinopathies, organ transplantation and use of indwelling catheters [1,2]. Candida spp. is a critical invasive fungal pathogen causing disease in humans, normally responsible for 90% of mucosal infections and 60% of candidiasis episodes [3]. Although various compounds are currently used to control Candida infectious diseases, including well-known azoles such as fluconazole, miconazole and others, the mortality of patients with Candida infection is above 15% [4,5]. Antibiotic-resistant Candida spp. have also arisen. The drugs currently used against fungal pathogens have limitations because of their toxicity [6,7]. For instance, Acetaminophen (APAP), amphotericin B deoxycholate (DAMB) and the triazoles may cause hepatic toxicity [8,9]. Therefore, there is an urgent need for a new drug to treat Candida infection.
Imidazolium salts (IMS) have been reported to exhibit fungicidal activity [10]. Due to its ionicity, IMS provides properties that are unusual and highly interesting for pharmaceutical formulation including potential efficacy against some bacteria and fungi [11].
In addition, tuning the toxicity of IMSs as antitumor agents has attracted much attention [12]. Considering these and our previous results of antifungal compound study, we decided to explore in further detail the potential activity of a new hybrid compound formed by attaching an imidazole moiety to 1,4-dialkoxynaphthalen-2-acyl compound to enhance antifungal activity.
Alagebrium, known as an advanced glycation end-products (AGEs) breaker that reverse one of the main mechanisms of ageing, has a structure of phenacyl thiazolium salt [13]. The phenacyl moiety is known to play an important role in biological activity [14,15]. Therefore, we were interested in the napththalenacyl moiety, similar to the phenacyl moiety, as a pharmacophore of antifungal agents. The 1,4-dialkoxy naphthalenacyl compound, derived from 1,4-naphthoquinone, became of particular interest (Figure 1). antifungal activity.
Alagebrium, known as an advanced glycation end-products (AGEs) breaker that reverse one of the main mechanisms of ageing, has a structure of phenacyl thiazolium salt [13]. The phenacyl moiety is known to play an important role in biological activity [14,15]. Therefore, we were interested in the napththalenacyl moiety, similar to the phenacyl moiety, as a pharmacophore of antifungal agents. The 1,4-dialkoxy naphthalenacyl compound, derived from 1,4-naphthoquinone, became of particular interest ( Figure 1).
Induced endogenous fungal apoptotic responses could provide a basis for antifungal therapies. Environmental stress (acetic acid and hydrogen peroxide) and an antifungal agent (amphotericin B, hibicuslide C and coumarin) have been known to induce apoptosis in C. albicans [16][17][18]. Apoptosis is a kind of programmed cell death. Multicellular organisms and even single-celled organisms, such as yeast, can exhibit many features of apoptosis, including DNA fragmentation, reactive oxygen species (ROS) production and the loss of mitochondrial membrane potential [19][20][21].
With the above considerations, a series of novel IMSs linked to a 2-acetyl-1,4-dialkoxynaphthalene moiety were efficiently synthesized through pharmacophore-hybridization strategy (Figure 1) to find promising drugs for dealing with Candida spp. infection. Through microdilution antifungal susceptibility, NAIMS 7c showed the highest antifungal activity among them by inducing Candida apoptosis and cell bursting. Schematic design for synthesis of acylnaphthalene-imidazolium hybrid using pharmacophore-hybridization approach.

General Remarks
The reactions were monitored by thin-layer chromatography (TLC) on Merck Silica gel 60F254. Column chromatography was performed on Merck silica gel 200-300 mesh. Melting points were determined on the melting point apparatus electrothermal A9100X1 and were uncorrected. We recorded 1 H NMR (300 MHz) and 13 C NMR (75 MHz) spectra on a JEOL FT-NMR spectrometer (Tokyo, Japan), respectively. Spectra are referenced relative to the chemical shift of tetramethylsilane (TMS). High-resolution mass spectra were obtained with a JEOL JMS-700 mass spectrometer. All solvents and reagents were commercially available from Acros Organics (Brookline, MA, USA), Aldrich (St. Louis, MO, USA) and TCI (Tokyo, Japan) and were used as received. The chemicals 1-Methylimidaz- Induced endogenous fungal apoptotic responses could provide a basis for antifungal therapies. Environmental stress (acetic acid and hydrogen peroxide) and an antifungal agent (amphotericin B, hibicuslide C and coumarin) have been known to induce apoptosis in C. albicans [16][17][18]. Apoptosis is a kind of programmed cell death. Multicellular organisms and even single-celled organisms, such as yeast, can exhibit many features of apoptosis, including DNA fragmentation, reactive oxygen species (ROS) production and the loss of mitochondrial membrane potential [19][20][21].
With the above considerations, a series of novel IMSs linked to a 2-acetyl-1,4-dialkoxyn aphthalene moiety were efficiently synthesized through pharmacophore-hybridization strategy ( Figure 1) to find promising drugs for dealing with Candida spp. infection. Through microdilution antifungal susceptibility, NAIMS 7c showed the highest antifungal activity among them by inducing Candida apoptosis and cell bursting.

Antifungal Susceptibility Microdilution Assay
Microdilution assay was performed based on the description of CLSI document M27-A [26][27][28]. Compounds were prepared in DMSO (Dimethyl sulfoxide; Junsei, Tokyo, Japan) at a concentration of 10 mg/mL as a stock solution. A colony of each strain was inoculated in 3 mL of YPD broth at 30 • C overnight, and the medium was changed to new fresh medium. The strains were adjusted at 3.0 × 10 4 CFU/mL with the final compound concentrations ranging from 1.5625 to 100 µg/mL. Growth control without treatment and sterilized medium control were included in each experiment and miconazole was used as the positive control [26,29].

Cell Viability Assay
Cell viability was estimated according to previously reported method with slightly modification [30]. A normal cell line, HaCaT (ATCC, VA, USA), was added to a 96well plate at 1.0 × 10 4 cells per well and incubated for 24 h. Various concentrations of NAIMS 7c (3.125-100 µg/mL) were added and further incubated for 24 h. MTT (3-(4,5dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma, St. Louis, MO, USA) in PBS was added into each well, followed by incubation for 3 h at 37 • C. The medium was then removed, and cells were suspended in 100 µL DMSO for 10 m. Viable cells were calculated from optical density (OD 540 ) values measured using a microplate reader (BioTek Instruments, Winooski, VT, USA).

Fungal Cell Growth Test
Inoculation and culture conditions in this study were same as those in the abovementioned antifungal susceptibility microdilution assay. The compound concentrations then ranged from 1.56 to 25 µg/mL. We cultured 96-well plates (SPL, Gyeonggi-do, Korea) at 30 • C and OD 600 were measured using a microplate reader (BioTek Instruments) at indicated time [31].

ROS Detection
ROS detection cell-based assay kit (Cayman Chemical, Ann Arbor, MI, USA) was used according to manufacturer's instructions. Cells were incubated with/without antimycin (positive control) or indicated concentrations of NAIMS 7c for 2 h. Cells were rinsed with ice cold cell-based assay buffer and then incubated with ROS straining buffer. Dihydroethidium (DHE) fluorescence was measured using an excitation wavelength 480 nm and an emission wavelength 580 nm according to manufacturer's instructions by VICTOR2 (Perkin Elmer, Waltham, MA, USA). Antimycin A was used as positive control [32].

Detection for Release of UV Absorbing Materials at 260 and 280 nm
Overnight cultured C. albicans was washed with PBS to stop further proliferation. Cells were treated with indicated concentration of NAIMS 7c for 1 h at 30 • C. Lysate released was measured with the Ultrospec 3000 at 260 and 280 nm. The values were adjusted by subtracting the optical density measured for the corresponding negative control, which was obtained by same compound concentrations in PBS without cells at the same wavelength [33,34].

Quantitative Reverse Transcriptase PCR (qRT-PCR) analysis
qRT-PCR was performed following the method with slight modification [35]. Total RNA was extracted with TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA). Real-time PCR was performed in 96-well PCR plates (Bio-rad, Hercules, CA, USA) using 2×RT Pre-MIX kit (Biofact, Daejeon-si, Korea) and CFX Connect Real-Time PCR Detection System (Bio-rad). Primer sequences used in this study are listed in Table 1.

Statistical Analysis
All data are expressed as the mean ± S.D. Significant differences among the groups were determined using the Student's t-test or one-way ANOVA, and p < 0.05 was considered significant.

Chemistry
First, we tried to synthesize 2-acetyl-1,4-dialkoxynaphthalnes 4, a key intermediate to target NAIMS 7 by direct alkylation of 1,4-dihydroxynaphthalene followed by Friedel-Crafts acylation. However, Friedel-Crafts acylation was found to be inefficient due to its low yield. Therefore, another synthetic route to produce 4 was suggested as shown in Scheme 1. The compounds 2 were synthesized in high yield from 1,4-diacetoxynaphthalene (1) by Fries rearrangement. Then, alkylation of compound 1 with the corresponding alkyl halide afforded compound 3. The removal of the acetyl group with KOH followed by O-alkylation produced key intermediate 4 in two steps with good yield. Various αbromination reactions of compound 4 were tested to find a condition optimized for the synthesis of bromoacyl intermediate 5. Finally, we found that compound 5 was obtained in the best yield when reacted with TBA-Br 3 . N-Arylimidazoles such as 6a and 6d, which are not commercially available, were synthesized from imidazole and 4-substituted benzyl halide in acetonitrile. The coupling reaction of the 2-bromoacyl intermediates 5 with corresponding imidazoles 6 gave nine new 1,4-dialkoynaphthalen-2-acyl imidazolium salts 7a-i. To investigate a simple relationship between structure and activity of NAIMSs, we prepared compounds 8, 9, 9′, NAIMS 10 and NAIMS 11, as shown in Figure 2. Compounds 8, 9 and 9′ have no imidazolium moiety, and NAIMS 10 and NAIMS 11 have no dialkoxy Reagents and conditions. (i) BF 3 2CH 3 COOH (7 eq), reflux, 1h; (ii) Cs 2 CO 3 (1.5 eq), To investigate a simple relationship between structure and activity of NAIMSs, we prepared compounds 8, 9, 9 , NAIMS 10 and NAIMS 11, as shown in Figure 2. Compounds 8, 9 and 9 have no imidazolium moiety, and NAIMS 10 and NAIMS 11 have no dialkoxy substituents in a naphthalene ring. The same coupling reaction of readily available 2-bromoacetylnaphthalene or 2-bromoacetyl-1-methoxynaphthalene (5e) with imidazole 6b gave, respectively, NAIMS 10 and NAIMS 11. Scheme 1. Preparation of 1,4-dialkoxy-NAIMSs 7a-7i.

Antifungal Activity of NAIMS 7c Against Candida spp.
The growth inhibitory activity of 7a-i, 10 and 11 against Candida spp. was evaluated using broth microdilution assays. The MIC values of miconazole were used as a positive control and confirmed over 12.5 μg/mL to Candida spp. after 24 h incubation. Among the tested compounds, NAIMS 7b, NAIMS 7c, NAIMS 7d and NAIMS 7e showed stronger antifungal activity against C. albicans than miconazole. NAIMS 7c showed strongest antifungal activity compared with other derivatives; MIC values ranged from 3.125 to 6.25 μg/mL against all Candida spp. used in this study. In particular, NAIMS 7c showed 3.125 μg/mL MIC value in 24 h assay against C. auris (KCTC17810) which possessed native resistance to miconazole (Table 2)

Antifungal Activity of NAIMS 7c Against Candida spp.
The growth inhibitory activity of 7a-i, 10 and 11 against Candida spp. was evaluated using broth microdilution assays. The MIC values of miconazole were used as a positive control and confirmed over 12.5 µg/mL to Candida spp. after 24 h incubation. Among the tested compounds, NAIMS 7b, NAIMS 7c, NAIMS 7d and NAIMS 7e showed stronger antifungal activity against C. albicans than miconazole. NAIMS 7c showed strongest antifungal activity compared with other derivatives; MIC values ranged from 3.125 to 6.25 µg/mL against all Candida spp. used in this study. In particular, NAIMS 7c showed 3.125 µg/mL MIC value in 24 h assay against C. auris (KCTC17810) which possessed native resistance to miconazole (  Table 3). NAIMS 7c showed stable antifungal activity compared with other derivatives. The yield of NAIMS 7c was fortunately higher than NAIMS 7b and NAIMS 7d, and this suggested that NAIMS 7c is more cost-beneficial than other derivatives (Scheme 1). In vitro cell viability of NAIMS 7c against HaCaT was performed via MTT assays. NAIMS 7c had IC 50 of 21.39 µg/mL against HaCaT. Based on results, cell growth test was also evaluated for the serial diluted concentration of NAIMS 7c in C. albicans (KCTC7965, KCTC7270 and KACC30071) for 48 h (Figure 3). Regardless of the time, NAIMS 7c at 6.25 µg/mL strongly inhibited cell growth compared with miconazole.   The growth of C. albicans was inhibited by NAIMS 7c. Microdilution assay was performed at 3.125 µg/mL for 48 h. Data represent mean ± SD from three independent experiments. ** p < 0.05 and *** p < 0.01.

Inducing ROS Level and the Loss of Mitochondria Membrane Potential by NAIMS 7c.
To identify antifungal activity of NAIMS 7c against C. albicans, ROS production in C. albicans was detected after NAIMS 7c treatment. NAIMS 7c induced higher ROS production than did antimycin. The treatment of 0.78 µg/mL of NAIMS 7c increased the DHE fluorescence with the maximum at 1.56 µg/mL (Figure 4). For further study about the mechanism of NAIMS 7c, C. albicans was stained with JC-1 dye to detect the mitochondria membrane potential. NAIMS 7c decreased a red fluorescence at 1.56 µg/mL in C. albicans. This suggested that NAIMS 7c causes damage to mitochondria and alters the cellular state of C. albicans ( Figure 5). Based on these results, NAIMS 7c induces loss of mitochondria membrane potential and increases the release of ROS in C. albicans. To identify antifungal activity of NAIMS 7c against C. albicans, ROS production in C. albicans was detected after NAIMS 7c treatment. NAIMS 7c induced higher ROS production than did antimycin. The treatment of 0.78 μg/mL of NAIMS 7c increased the DHE fluorescence with the maximum at 1.56 μg/mL (Figure 4). For further study about the mechanism of NAIMS 7c, C. albicans was stained with JC-1 dye to detect the mitochondria membrane potential. NAIMS 7c decreased a red fluorescence at 1.56 μg/mL in C. albicans. This suggested that NAIMS 7c causes damage to mitochondria and alters the cellular state of C. albicans ( Figure 5). Based on these results, NAIMS 7c induces loss of mitochondria membrane potential and increases the release of ROS in C. albicans.    To identify antifungal activity of NAIMS 7c against C. albicans, ROS production in C. albicans was detected after NAIMS 7c treatment. NAIMS 7c induced higher ROS production than did antimycin. The treatment of 0.78 μg/mL of NAIMS 7c increased the DHE fluorescence with the maximum at 1.56 μg/mL (Figure 4). For further study about the mechanism of NAIMS 7c, C. albicans was stained with JC-1 dye to detect the mitochondria membrane potential. NAIMS 7c decreased a red fluorescence at 1.56 μg/mL in C. albicans. This suggested that NAIMS 7c causes damage to mitochondria and alters the cellular state of C. albicans ( Figure 5). Based on these results, NAIMS 7c induces loss of mitochondria membrane potential and increases the release of ROS in C. albicans.

The Cell Lysis and the Apoptosis of C. albicans by NAIMS 7c
To determine the induction of the loss of mitochondria membrane potential by NAIMS 7c treatment, changes in UV absorbing materials were detected by spectrophotometer with NAIMS 7c in the C. albicans culture medium supernatant. The use of 12.5 µg/mL of NAIMS 7c treatment significantly increased the absorbance at 260 and 280 nm after 1 and 2 h ( Figure 6). These results show that treatment of NAIMS 7c at over the MIC value can lead to lysis of Candida spp. and might induce the apoptosis of Candida spp. Three different Candida-apoptosis related genes were used to detect the effect of NAIMS 7c in C. albicans. YPK1 is a serine/threonine protein kinase that affects diverse cellular activities, including sphingolipid homeostasis. HAC1 is a transcription factor and plays a major role in stressrelated transcriptional response. MCA1 is a cysteine protease involved in apoptosis in response to stresses. The expression of these three genes was dramatically increased by NAIMS 7c treatment (Figure 7). To determine the induction of the loss of mitochondria membrane potential by NAIMS 7c treatment, changes in UV absorbing materials were detected by spectrophotometer with NAIMS 7c in the C. albicans culture medium supernatant. The use of 12.5 μg/mL of NAIMS 7c treatment significantly increased the absorbance at 260 and 280 nm after 1 and 2 h ( Figure 6). These results show that treatment of NAIMS 7c at over the MIC value can lead to lysis of Candida spp. and might induce the apoptosis of Candida spp Three different Candida-apoptosis related genes were used to detect the effect of NAIMS 7c in C. albicans. YPK1 is a serine/threonine protein kinase that affects diverse cellular activities, including sphingolipid homeostasis. HAC1 is a transcription factor and plays a major role in stress-related transcriptional response. MCA1 is a cysteine protease involved in apoptosis in response to stresses. The expression of these three genes was dramatically increased by NAIMS 7c treatment (Figure 7).

Discussion
Candida spp. are the most common organism recovered from the skin and blood of hospitalized patients. Although the need to treat them is increasing, the range of antifungal agents available is limited because of their toxicity. In addition, resistant strains and new species that show innate resistance to antifungal agents have been reported [39,40]. Therefore, it is necessary to introduce new antifungal agents to limit the spread of pathogenic fungi.
For the synthesis of 1,4-dialkoy-NAIMS, we developed a highly efficient synthesis method including the synthesis of 1,4-dialkoynaphthalen-2-acyl intermediate 4, the α-bromination reaction of compound 4, and the SN2 reaction of compound 5 and imidazole 6, such as a quarternization of imidazole. We found that compound 4 was produced in better yield by a novel synthetic method including Fries-rearrangement of compound 1 followed by alkylation than the direct alkylation of 1,4-dihydroxynaphthalene and subsequent Friedel-Crafts acylation. In addition, it was confirmed that the α-bromination reaction of compound 4 was best under the reaction conditions of TBA-Br3.
Among the 14 synthesized compounds (7a-i, 8, 9, 9′, 10, 11), NAIMS 7b, 7c, and 7d showed superior activity compared to other synthetic NAIMSs or miconazole. In particular, NAIMS 7c is best when considering its antifungal activity, stability and synthesis efficiency. As shown in Figure 8, the energy calculation (Gaussian B3LYP, 6-311 [d, p]) predicts NAIMS 7c presenting a slightly curved structure similar to phospholipids. Based on these results, we might propose a first-pass reasoning on the relationship between structure and activity as follows. For antifungal activity, the imidazolium ring must be essential. The naphthalene ring requires a 1,4-dialkoxy group, in which the optimal alkyl group is an isoamyl group. Furthermore, it seems better to have no methoxy groups in the naphthalene A ring and no electron withdrawing groups such as NO2 in the benzene ring in order for NAIMS to be activated.
Miconazole was used as an antifungal agent which has a fungicidal activity against planktonic Candida spp., and the cytotoxic concentration of NAIMS 7c can be significantly lower than miconazole [41,42]. NAIMS 7c had IC50 of 21.39 and 7.56 μg/mL against HaCaT and C. albicans (KACC30071), respectively. Miconazole had IC50 of 13.10 and 17.25 μg/mL

Discussion
Candida spp. are the most common organism recovered from the skin and blood of hospitalized patients. Although the need to treat them is increasing, the range of antifungal agents available is limited because of their toxicity. In addition, resistant strains and new species that show innate resistance to antifungal agents have been reported [39,40]. Therefore, it is necessary to introduce new antifungal agents to limit the spread of pathogenic fungi.
For the synthesis of 1,4-dialkoy-NAIMS, we developed a highly efficient synthesis method including the synthesis of 1,4-dialkoynaphthalen-2-acyl intermediate 4, the αbromination reaction of compound 4, and the S N 2 reaction of compound 5 and imidazole 6, such as a quarternization of imidazole. We found that compound 4 was produced in better yield by a novel synthetic method including Fries-rearrangement of compound 1 followed by alkylation than the direct alkylation of 1,4-dihydroxynaphthalene and subsequent Friedel-Crafts acylation. In addition, it was confirmed that the α-bromination reaction of compound 4 was best under the reaction conditions of TBA-Br 3 .
Among the 14 synthesized compounds (7a-i, 8, 9, 9 , 10, 11), NAIMS 7b, 7c, and 7d showed superior activity compared to other synthetic NAIMSs or miconazole. In particular, NAIMS 7c is best when considering its antifungal activity, stability and synthesis efficiency. As shown in Figure 8, the energy calculation (Gaussian B3LYP, 6-311 [d, p]) predicts NAIMS 7c presenting a slightly curved structure similar to phospholipids. Based on these results, we might propose a first-pass reasoning on the relationship between structure and activity as follows. For antifungal activity, the imidazolium ring must be essential. The naphthalene ring requires a 1,4-dialkoxy group, in which the optimal alkyl group is an isoamyl group. Furthermore, it seems better to have no methoxy groups in the naphthalene A ring and no electron withdrawing groups such as NO 2 in the benzene ring in order for NAIMS to be activated.
applications in the future [43]. Thus, NAIMS 7c could serve as a promising lead compound for further research. The 1,4-dialkoxy naphthalene-2-acyl compound 9 without the imidazolium moiety and the naphthalene-2-acyl imidazolium 10 without the dialkoxy substituent on the naphthalene ring showed no antifungal activity. However, its hybrid NAIMS 7c found an excellent antifungal agent. Therefore, as mentioned in the literature, the pharmacophore-hybridization approach is thought to be a useful tool for new drug design and development [44]. NAIMS 7c led to induced ROS production, the loss of mitochondria membrane potential, the release of UV absorbing materials and up-regulated apoptotic gene expression. ROS and mitochondria play an essential part in apoptosis. These results suggest that NAIMS 7c induced apoptosis in C. albicans.
In summary, a series of novel imidazolium salts containing 2-acetylnaphthalene moiety were designed, prepared and evaluated for antifungal activity. The results presented in this study conclusively demonstrate that NAIMS 7c has a long-term enhanced antifungal activity against Candida spp., including especially C. albicans and C. auris, which possess native resistance to miconazole, as compared with other compounds including miconazole. Further studies of structure-activity relationships and a wide range of biological activities are ongoing and will be reported in the future.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest. Miconazole was used as an antifungal agent which has a fungicidal activity against planktonic Candida spp., and the cytotoxic concentration of NAIMS 7c can be significantly lower than miconazole [41,42]. NAIMS 7c had IC 50 of 21.39 and 7.56 µg/mL against HaCaT and C. albicans (KACC30071), respectively. Miconazole had IC 50 of 13.10 and 17.25 µg/mL against HaCaT and C. albicans, respectively. Accordingly, the selective index (IC 50 HaCaT/IC 50 C. albicans) of NAIMS 7c was higher than miconazole. This result shows that NAIMS 7c can be more effective to treat Candida spp. and safe. Additional in vivo research should be provided to determine the property of any potential candidate for therapeutic applications in the future [43]. Thus, NAIMS 7c could serve as a promising lead compound for further research. The 1,4-dialkoxy naphthalene-2-acyl compound 9 without the imidazolium moiety and the naphthalene-2-acyl imidazolium 10 without the dialkoxy substituent on the naphthalene ring showed no antifungal activity. However, its hybrid NAIMS 7c found an excellent antifungal agent. Therefore, as mentioned in the literature, the pharmacophore-hybridization approach is thought to be a useful tool for new drug design and development [44].
NAIMS 7c led to induced ROS production, the loss of mitochondria membrane potential, the release of UV absorbing materials and up-regulated apoptotic gene expression. ROS and mitochondria play an essential part in apoptosis. These results suggest that NAIMS 7c induced apoptosis in C. albicans.
In summary, a series of novel imidazolium salts containing 2-acetylnaphthalene moiety were designed, prepared and evaluated for antifungal activity. The results presented in this study conclusively demonstrate that NAIMS 7c has a long-term enhanced antifungal activity against Candida spp., including especially C. albicans and C. auris, which possess native resistance to miconazole, as compared with other compounds including miconazole. Further studies of structure-activity relationships and a wide range of biological activities are ongoing and will be reported in the future.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.