Two New Compounds Containing Pyridinone or Triazine Heterocycles Have Antifungal Properties against Candida albicans

Candidiasis, caused by the opportunistic yeast Candida albicans, is the most common fungal infection today. Resistance of C. albicans to current antifungal drugs has emerged over the past decade leading to the need for novel antifungal agents. Our aim was to select new antifungal compounds by library-screening methods and to assess their antifungal effects against C. albicans. After screening 90 potential antifungal compounds from JUNIA, a chemical library, two compounds, 1-(4-chlorophenyl)-4-((4-chlorophenyl)amino)-3,6-dimethylpyridin-2(1H)-one (PYR) and (Z)-N-(2-(4,6-dimethoxy-1,3,5-triazin-2-yl)vinyl)-4-methoxyaniline (TRI), were identified as having potential antifungal activity. Treatment with PYR and TRI resulted in a significant reduction of C. albicans bioluminescence as well as the number of fungal colonies, indicating rapid fungicidal activity. These two compounds were also effective against clinically isolated fluconazole- or caspofungin-resistant C. albicans strains. PYR and TRI had an inhibitory effect on Candida biofilm formation and reduced the thickness of the mannan cell wall. In a Caenorhabditis elegans infection model, PYR and TRI decreased the mortality of nematodes infected with C. albicans and enhanced the expression of antimicrobial genes that promote C. albicans elimination. Overall, PYR and TRI showed antifungal properties against C. albicans by exerting fungicidal activities and enhancing the antimicrobial gene expression of Caenorhabditis elegans.


Introduction
Opportunistic fungi have become an increasingly important cause of nosocomial bloodstream infections, with high rates of morbidity and mortality in intensive care units [1,2]. These fungal infections are particularly problematic for immunocompromised patients, as well as patients with solid-organ malignancies or those recovering from abdominal surgery [1,2]. Candida albicans is an opportunistic yeast that colonizes the oropharyngeal, esophageal, and gastrointestinal mucosa in most healthy humans. Overgrowth of C. albicans in these niches can result in mucosal infections and life-threatening systemic disease, making C. albicans an important opportunistic pathogen. C. albicans is the most commonly Introducing diethylamine in compound TRI9 instead of p-anisidine of TRI slightly decreased the activity, which could be related to the importance of the aromatic ring or the H-bonding capacity of the -NH-link (Table 1, entry 10). To confirm the role of the -NHlink, compound C, in which the p-methoxyphenyl group was conserved but the aminovinyl linker was modified, was also screened (Figure 1). Compound C was 6-fold less active than TRI against C. albicans. This highlights the importance of a -NH-linker for activity. Derivatives of PYR compounds were also evaluated, with chemical modifications on the aromatic moieties (Table 1, series B). In this experiment, it is notable that the parachloroaniline moiety had the highest activity against C. albicans. Introducing inductive or mesomeric electro-donating substituents such as methyl-or methoxy-on the aromatic rings led to a decrease in biological activity (Table 1, entries [12][13][14][15][16]. Moreover, replacing one aromatic ring by a piperazine part as in compounds PYR7 and PYR8 did not improve the activity (Table 1, entries [17][18]. While in series A, the p-methoxyaniline was the most effective ring for antifungal activity, it appeared that in series B, p-chloroaniline was the best choice. Gathering these data, compounds TRI and PYR were selected for further biological studies. Some derivatives of TRI and PYR were also screened against C. albicans to determine the importance of each moiety in antifungal activity. In a first experiment, modification of the amine moiety was performed, while keeping the 4,6-dimethoxy-1,3,5-triazine and ethylene linkers constant (Table 1). Suppression of the para-methoxy substituent on the phenyl ring (TRI1) led to a decrease in activity, measured at 32 µg/mL and confirmed at 0.8 mmol/L (Table 1, entry 2). Polymethoxylated substitution or addition of a dioxolane ring did not improve the activity against C. albicans, whatever the position of the substituent on the aromatic ring (Table 1, entries [3][4][5][6][7][8]. Introducing a 3-hydroxy-4-methoxyphenyl unit, similar to DHMB, an antifungal previously described by our group, did not result in any improvement in activity (Table 1, entry 6) [12]. Moreover, replacing the methoxy groups with chloro substituents did not increase the antifungal potency (Table 1, entry 9). Introducing diethylamine in compound TRI9 instead of p-anisidine of TRI slightly decreased the activity, which could be related to the importance of the aromatic ring or the H-bonding capacity of the -NH-link (Table 1, entry 10). To confirm the role of the -NH-link, compound C, in which the p-methoxyphenyl group was conserved but the amino-vinyl linker was modified, was also screened (Figure 1). Compound C was 6-fold less active than TRI against C. albicans. This highlights the importance of a -NH-linker for activity.
Derivatives of PYR compounds were also evaluated, with chemical modifications on the aromatic moieties (Table 1, series B). In this experiment, it is notable that the parachloroaniline moiety had the highest activity against C. albicans. Introducing inductive or mesomeric electro-donating substituents such as methyl-or methoxy-on the aromatic rings led to a decrease in biological activity (Table 1, entries 12-16). Moreover, replacing one aromatic ring by a piperazine part as in compounds PYR7 and PYR8 did not improve the activity (Table 1, entries [17][18]. While in series A, the p-methoxyaniline was the most effective ring for antifungal activity, it appeared that in series B, p-chloroaniline was the best choice. Gathering these data, compounds TRI and PYR were selected for further biological studies.

Effect of PYR and TRI on C. albicans Viability
To determine the antifungal effect of PYR and TRI on C. albicans viability, C. albicans cells were challenged with PYR or TRI and viability was assessed by culture-plate assay ( Figure 2). PYR and TRI at their 1× MIC (12.5 µg/mL and 50 µg/mL, respectively) reduced C. albicans viability and this antifungal activity was more pronounced than with caspofungin at 2× MIC ( Figure 2). Additionally, the viability of C. albicans cells was monitored in real time using a bioluminescent C. albicans strain challenged with either caspofungin,

Effect of PYR and TRI on C. albicans Viability
To determine the antifungal effect of PYR and TRI on C. albicans viability, C. albicans cells were challenged with PYR or TRI and viability was assessed by culture-plate assay ( Figure 2). PYR and TRI at their 1× MIC (12.5 µg/mL and 50 µg/mL, respectively) reduced C. albicans viability and this antifungal activity was more pronounced than with caspofungin at 2× MIC ( Figure 2). Additionally, the viability of C. albicans cells was monitored in real time using a bioluminescent C. albicans strain challenged with either caspofungin,

Effect of PYR and TRI on C. albicans Viability
To determine the antifungal effect of PYR and TRI on C. albicans viability, C. albicans cells were challenged with PYR or TRI and viability was assessed by culture-plate assay ( Figure 2). PYR and TRI at their 1× MIC (12.5 µg/mL and 50 µg/mL, respectively) reduced C. albicans viability and this antifungal activity was more pronounced than with caspofungin at 2× MIC ( Figure 2). Additionally, the viability of C. albicans cells was monitored in real time using a bioluminescent C. albicans strain challenged with either caspofungin, Biological activity was evaluated at a concentration of a 32 µg/mL or b 0.8 mmol/L corresponding to 230, 206, 255, 190, 287, and 255 µg/mL for TRI, TRI1, TR2, TRI9, PYR, and PYR1, respectively, on C. albicans strain ATCC90028 grown for 3 days on yeast extract-peptone dextrose (YPD) agar at 30 °C. A yeast suspension of 1 × 10 6 -5 × 10 6 cfu/mL (determined by OD550) was prepared from five colonies. The suspension was diluted and added to each well of the compound-containing plates to give a final fungal cell concentration of 2.5 × 10 3 cfu/mL and a total volume of 50 µL. All plates were covered and incubated at 35 °C for 24 h without shaking. Inhibition of C. albicans growth was determined by measuring the absorbance at 530 nm (OD530). Fluconazole was used as a positive control. CPD: compound. ND: not determined

Effect of PYR and TRI on C. albicans Viability
To determine the antifungal effect of PYR and TRI on C. albicans viability, C. albicans cells were challenged with PYR or TRI and viability was assessed by culture-plate assay ( Figure 2). PYR and TRI at their 1× MIC (12.5 µg/mL and 50 µg/mL, respectively) reduced C. albicans viability and this antifungal activity was more pronounced than with caspofungin at 2× MIC ( Figure 2). Additionally, the viability of C. albicans cells was monitored in real time using a bioluminescent C. albicans strain challenged with either caspofungin, Biological activity was evaluated at a concentration of a 32 µg/mL or b 0.8 mmol/L corresponding to 230, 206, 255, 190, 287, and 255 µg/mL for TRI, TRI1, TR2, TRI9, PYR, and PYR1, respectively, on C. albicans strain ATCC90028 grown for 3 days on yeast extract-peptone dextrose (YPD) agar at 30 °C. A yeast suspension of 1 × 10 6 -5 × 10 6 cfu/mL (determined by OD550) was prepared from five colonies. The suspension was diluted and added to each well of the compound-containing plates to give a final fungal cell concentration of 2.5 × 10 3 cfu/mL and a total volume of 50 µL. All plates were covered and incubated at 35 °C for 24 h without shaking. Inhibition of C. albicans growth was determined by measuring the absorbance at 530 nm (OD530). Fluconazole was used as a positive control. CPD: compound. ND: not determined

Effect of PYR and TRI on C. albicans Viability
To determine the antifungal effect of PYR and TRI on C. albicans viability, C. albicans cells were challenged with PYR or TRI and viability was assessed by culture-plate assay ( Figure 2). PYR and TRI at their 1× MIC (12.5 µg/mL and 50 µg/mL, respectively) reduced C. albicans viability and this antifungal activity was more pronounced than with caspofungin at 2× MIC ( Figure 2). Additionally, the viability of C. albicans cells was monitored in real time using a bioluminescent C. albicans strain challenged with either caspofungin, Biological activity was evaluated at a concentration of a 32 µg/mL or b 0.8 mmol/L corresponding to 230, 206, 255, 190, 287, and 255 µg/mL for TRI, TRI1, TR2, TRI9, PYR, and PYR1, respectively, on C. albicans strain ATCC90028 grown for 3 days on yeast extract-peptone dextrose (YPD) agar at 30 °C. A yeast suspension of 1 × 10 6 -5 × 10 6 cfu/mL (determined by OD550) was prepared from five colonies. The suspension was diluted and added to each well of the compound-containing plates to give a final fungal cell concentration of 2.5 × 10 3 cfu/mL and a total volume of 50 µL. All plates were covered and incubated at 35 °C for 24 h without shaking. Inhibition of C. albicans growth was determined by measuring the absorbance at 530 nm (OD530). Fluconazole was used as a positive control. CPD: compound. ND: not determined

Effect of PYR and TRI on C. albicans Viability
To determine the antifungal effect of PYR and TRI on C. albicans viability, C. albicans cells were challenged with PYR or TRI and viability was assessed by culture-plate assay ( Figure 2). PYR and TRI at their 1× MIC (12.5 µg/mL and 50 µg/mL, respectively) reduced C. albicans viability and this antifungal activity was more pronounced than with caspofungin at 2× MIC ( Figure 2). Additionally, the viability of C. albicans cells was monitored in real time using a bioluminescent C. albicans strain challenged with either caspofungin,

ND
Biological activity was evaluated at a concentration of a 32 µg/mL or b 0.8 mmol/L corresponding to 230, 206, 255, 190, 287, and 255 µg/mL for TRI, TRI1, TR2, TRI9, PYR, and PYR1, respectively, on C. albicans strain ATCC90028 grown for 3 days on yeast extract-peptone dextrose (YPD) agar at 30 • C. A yeast suspension of 1 × 10 6 -5 × 10 6 cfu/mL (determined by OD 550 ) was prepared from five colonies. The suspension was diluted and added to each well of the compound-containing plates to give a final fungal cell concentration of 2.5 × 10 3 cfu/mL and a total volume of 50 µL. All plates were covered and incubated at 35 • C for 24 h without shaking. Inhibition of C. albicans growth was determined by measuring the absorbance at 530 nm (OD 530 ). Fluconazole was used as a positive control. CPD: compound. ND: not determined.

Effect of PYR and TRI on C. albicans Viability
To determine the antifungal effect of PYR and TRI on C. albicans viability, C. albicans cells were challenged with PYR or TRI and viability was assessed by culture-plate assay ( Figure 2). PYR and TRI at their 1× MIC (12.5 µg/mL and 50 µg/mL, respectively) reduced C. albicans viability and this antifungal activity was more pronounced than with caspofungin at 2× MIC ( Figure 2). Additionally, the viability of C. albicans cells was monitored in real time using a bioluminescent C. albicans strain challenged with either caspofungin, fluconazole, PYR, or TRI (at 0, 30, 60, and 120 min). PYR and TRI at their MICs (12.5 µg/mL and 50 µg/mL, respectively) led to a significant reduction in bioluminescence of C. albicans when compared to that of C. albicans unchallenged with antifungal compounds (Figure 3). This reduction was observed rapidly after C. albicans treatment with PYR and TRI indicating the fungicidal effect of these compounds against C. albicans. We then analyzed whether derivatives of PYR (PYR1 and PYR2) and TRI (TRI1) had antifungal activity against C. albicans ( Figure 3). In contrast to TRI1, the derivatives PYR1 and PYR2 had antifungal activity against C. albicans. Of note, the derivatives PYR1 and PYR2 had less antifungal activity against C. albicans when compared to their original compound, PYR ( Figure 3). These data confirmed that the substitution of the aromatic moiety is highly important in the activity. For PYR derivatives, p-chloro substituent is superior to the p-methoxy one whereas the p-methoxy group is needed for activity in the TRI series of compounds.
Antibiotics 2021, 10, x FOR PEER REVIEW 5 of 16 fluconazole, PYR, or TRI (at 0, 30, 60, and 120 min). PYR and TRI at their MICs (12.5 µg/mL and 50 µg/mL, respectively) led to a significant reduction in bioluminescence of C. albicans when compared to that of C. albicans unchallenged with antifungal compounds (Figure 3). This reduction was observed rapidly after C. albicans treatment with PYR and TRI indicating the fungicidal effect of these compounds against C. albicans. We then analyzed whether derivatives of PYR (PYR1 and PYR2) and TRI (TRI1) had antifungal activity against C. albicans (Figure 3). In contrast to TRI1, the derivatives PYR1 and PYR2 had antifungal activity against C. albicans. Of note, the derivatives PYR1 and PYR2 had less antifungal activity against C. albicans when compared to their original compound, PYR (Figure 3). These data confirmed that the substitution of the aromatic moiety is highly important in the activity. For PYR derivatives, p-chloro substituent is superior to the p-methoxy one whereas the p-methoxy group is needed for activity in the TRI series of compounds.  To evaluate whether PYR and TRI had antifungal activity against drug-resistant C. albicans clinical isolates, we selected five C. albicans clinical isolates resistant to either fluconazole or caspofungin (Tables 2 and 3). PYR and TRI significantly decreased the viability of drug-resistant C. albicans clinical isolates at 1× MIC and 2× MIC in terms of viable colony counts using fungal culture media (Table 3). We next assessed the effect of PYR and TRI on the C. albicans cell wall (Figure 4). Quantification of the fluorescence intensity of C. albicans cells labeled with concanavalin A from 30 different images extracted from confocal microscopy using ImageJ showed that PYR and TIR reduced the thickness of the mannan part of the C. albicans cell wall while the labeling of C. albicans with wheat germ agglutinin (WGA) did not show any changes in the chitin part of the cell wall ( Figure 4). Antibiotics 2021, 10, x FOR PEER REVIEW 6 of 16 To evaluate whether PYR and TRI had antifungal activity against drug-resistant C. albicans clinical isolates, we selected five C. albicans clinical isolates resistant to either fluconazole or caspofungin (Tables 2 and 3). PYR and TRI significantly decreased the viability of drug-resistant C. albicans clinical isolates at 1× MIC and 2× MIC in terms of viable colony counts using fungal culture media (Table 3). We next assessed the effect of PYR and TRI on the C. albicans cell wall (Figure 4). Quantification of the fluorescence intensity of C. albicans cells labeled with concanavalin A from 30 different images extracted from confocal microscopy using ImageJ showed that PYR and TIR reduced the thickness of the mannan part of the C. albicans cell wall while the labeling of C. albicans with wheat germ agglutinin (WGA) did not show any changes in the chitin part of the cell wall ( Figure 4).
To determine whether PYR and TRI had an impact on biofilm formation, which is involved in resistance to antifungal agents by acting as a shield that delays or prevents drug diffusion to host cells, C. albicans biofilms were challenged with the two new antifungal compounds ( Figure 5). The cell density of C. albicans challenged with caspofungin, fluconazole, PYR, or TRI at their MICs (0.03 µg/mL, 0.5 µg/mL, 12.5 µg/mL, and 50 µg/mL, respectively) was clearly lower when compared to that of untreated C. albicans. In this assay, the measurement of C. albicans cell-wall thickness was difficult from microscope images since the biofilm matrix was a dense and highly compact structure in all conditions (C. albicans alone or C. albicans challenged with antifungal compounds).      To determine whether PYR and TRI had an impact on biofilm formation, which is involved in resistance to antifungal agents by acting as a shield that delays or prevents drug diffusion to host cells, C. albicans biofilms were challenged with the two new antifungal compounds ( Figure 5). The cell density of C. albicans challenged with caspofungin, fluconazole, PYR, or TRI at their MICs (0.03 µg/mL, 0.5 µg/mL, 12.5 µg/mL, and 50 µg/mL, respectively) was clearly lower when compared to that of untreated C. albicans. In this assay, the measurement of C. albicans cell-wall thickness was difficult from microscope images since the biofilm matrix was a dense and highly compact structure in all conditions (C. albicans alone or C. albicans challenged with antifungal compounds).

PYR and TRI Reduced the Virulence of C. albicans in the C. elegans Infection Model
The effects of PYR and TRI on C. albicans virulence in vivo were investigated using a C. elegans nematode infection model. Nematodes infected with C. albicans were treated with either PYR or TRI ( Figure 6). The survival of nematodes was followed daily by microscopic observation. C. albicans infection was observed to cause 85% mortality of C. elegans at day 4. PYR treatment of C. elegans infected with C. albicans at 12.5 µg/mL increased nematode survival to about 53% at day 4 while TRI treatment at 50 µg/mL protected the nematodes to about 30% survival indicating that PYR and TRI are active agents in prolonging nematode survival against C. albicans infection ( Figure 6).

PYR and TRI Reduced the Virulence of C. albicans in the C. elegans Infection Model
The effects of PYR and TRI on C. albicans virulence in vivo were investigated using a C. elegans nematode infection model. Nematodes infected with C. albicans were treated with either PYR or TRI ( Figure 6). The survival of nematodes was followed daily by microscopic observation. C. albicans infection was observed to cause 85% mortality of C. elegans at day 4. PYR treatment of C. elegans infected with C. albicans at 12.5 µg/mL increased nematode survival to about 53% at day 4 while TRI treatment at 50 µg/mL protected the nematodes to about 30% survival indicating that PYR and TRI are active agents in prolonging nematode survival against C. albicans infection ( Figure 6).
To explore the effect of these two compounds on the immune response of C. elegans, we studied the expression of lys-1, lys-7, and cnc-4 involved in the antimicrobial response and pmk-1 (p38 MAPK signaling pathway), which concerns the immune reaction. In comparison with Escherichia coli OP50 control conditions, C. albicans increased the expression of lys-1, lys-7, cnc-4, and pmk-1 (Figure 7). PYR treatment of C. elegans infected with C. albicans enhanced the expression of lys-7 and cnc-4 while no significant change in pmk-1 expression was observed. TRI treatment of C. elegans infected with C. albicans induced the up-regulation of lys-1, lys-7, and cnc-4 while a reduction of pmk-1 expression was noted indicating that these antimicrobial genes, including the p38 MAPK pathway, have a significant role in C. elegans during C. albicans infection. Thus, PYR or TRI treatment enhanced the expression of antimicrobial genes that promote the elimination of C. albicans (Figure 7). Nematodes infected with C. albicans SC5314 strain were examined for survival daily for 4 days and the percentage nematode survival was determined on day 4. Nematodes were considered dead when they did not respond to being touched with a platinum-wire pick. Ca + PYR represents C. elegans infected with C. albicans and treated with PYR. Ca + TRI represents C. elegans infected with C. albicans and treated with TRI. Data are presented as mean ± SD of four independent measurements.
To explore the effect of these two compounds on the immune response of C. elegans, we studied the expression of lys-1, lys-7, and cnc-4 involved in the antimicrobial response and pmk-1 (p38 MAPK signaling pathway), which concerns the immune reaction. In comparison with Escherichia coli OP50 control conditions, C. albicans increased the expression of lys-1, lys-7, cnc-4, and pmk-1 (Figure 7). PYR treatment of C. elegans infected with C. albicans enhanced the expression of lys-7 and cnc-4 while no significant change in pmk-1 expression was observed. TRI treatment of C. elegans infected with C. albicans induced the up-regulation of lys-1, lys-7, and cnc-4 while a reduction of pmk-1 expression was noted indicating that these antimicrobial genes, including the p38 MAPK pathway, have a significant role in C. elegans during C. albicans infection. Thus, PYR or TRI treatment enhanced the expression of antimicrobial genes that promote the elimination of C. albicans ( Figure  7). Figure 6. Effects of PYR and TRI on C. albicans virulence in C. elegans. Nematodes infected with C. albicans SC5314 strain were examined for survival daily for 4 days and the percentage nematode survival was determined on day 4. Nematodes were considered dead when they did not respond to being touched with a platinum-wire pick. Ca + PYR represents C. elegans infected with C. albicans and treated with PYR. Ca + TRI represents C. elegans infected with C. albicans and treated with TRI. Data are presented as mean ± SD of four independent measurements.
Antibiotics 2021, 10, x FOR PEER REVIEW Figure 7. Relative expression of antimicrobial peptides (lys-1, lys-7, and cnc-4) and the imm gene (pmk-1) in C. elegans infected with C. albicans. L4 nematodes were infected with C. albi SC5314 lawns for 6 h and then treated with PYR or TRI at their 1x MIC for 12 h. E. coli repre C. elegans fed with E. coli. Ca represents C. elegans infected with C. albicans. Ca + PYR corres to C. elegans infected with C. albicans and treated with PYR. Ca + TRI represents C. elegans in with C. albicans and treated with PYR. Data are presented as mean ± SD of four independen urements.

Discussion
The development of new antifungal drugs remains a major challenge to over the spread of drug-resistant clinically-relevant fungal pathogens, as strains are i ingly becoming less sensitive to conventional antifungal compounds [14,15].
In the present study, we selected two compounds, TRI and PYR, belonging triazine and pyridine chemical families, and showing antifungal activity against cans and improved efficacy against fluconazole-or caspofungin-resistant clinical C Figure 7. Relative expression of antimicrobial peptides (lys-1, lys-7, and cnc-4) and the immune gene (pmk-1) in C. elegans infected with C. albicans. L4 nematodes were infected with C. albicans SC5314 lawns for 6 h and then treated with PYR or TRI at their 1x MIC for 12 h. E. coli represents C. elegans fed with E. coli. Ca represents C. elegans infected with C. albicans. Ca + PYR corresponds to C. elegans infected with C. albicans and treated with PYR. Ca + TRI represents C. elegans infected with C. albicans and treated with PYR. Data are presented as mean ± SD of four independent measurements.

Discussion
The development of new antifungal drugs remains a major challenge to overcoming the spread of drug-resistant clinically-relevant fungal pathogens, as strains are increasingly becoming less sensitive to conventional antifungal compounds [14,15].
In the present study, we selected two compounds, TRI and PYR, belonging to the triazine and pyridine chemical families, and showing antifungal activity against C. albicans and improved efficacy against fluconazole-or caspofungin-resistant clinical Candida strains.
Triazine is an important heterocyclic nucleus, which is a major component in some natural products such as reurhycin, toxoflavin, and fervenulin [16]. The chemistry of 1,2,4triazines and their derivatives has attracted considerable attention owing to their broad spectrum of biological activity, including antifungal, anti-cancer, antiviral, cyclin-dependent kinase inhibitory, and anti-inflammatory activities [17][18][19][20][21]. Some 1,3,5-triazine derivatives, such as altretamine or almitrine have been approved by the FDA as antineoplastic agents or as respiratory stimulants, but none as antimicrobial agents. Moreover, very few reports have been published on the antimicrobial activity of such derivatives. In 1989, Reich et al. reported pyrido [3,4-e]-1,2,4-triazines and related heterocycles as having antifungal activity, with moderate activity against C. albicans, with MIC values ranging from 8 to 16 µg/mL for the lead compounds [22]. As shown in Figure 1, the reported compounds are bis-heterocycles and differ chemically from TRI.
Pyridin-2(1H)-compounds are naturally occurring products and display a variety of biological properties, including antimicrobial activity. Oxysporidinone was isolated after the fermentation of Fusarium oxysporum and has demonstrated antifungal activity against Aspergillus niger, Botrytis cinerea, Alternaria alternata, and Venturia inequalis, with MIC values of 10, 1, 50, and 10 µg/mL, respectively [23]. Its parent compound, funicolosin, is also a natural broad-spectrum antibiotic [24]. The related compound, ciclopirox, is a synthetic approved antifungal agent used as a topical treatment for superficial mycoses, particularly against Tinea versicolor [25]. As observed for TRI series, the compounds from the PYR series are chemically different from the reported oxysporidone, funicolosin, or ciclopirox parent molecules. Although these three reported antibiotics bear cyclic aliphatic moieties, PYR derivatives are composed of a pyridinone moiety decorated with two distinct aromatic rings.
Treatment with PYR and TRI resulted in a rapid reduction of C. albicans viability indicating that these compounds have fungicidal activity against C. albicans. Additionally, they reduced the viability of clinically isolated fluconazole-or caspofungin-resistant Candida strains. C. albicans cells in biofilms exhibit phenotypic traits, such as increased resistance to antifungal drugs and protection from host defenses that are dramatically different from their planktonic counterparts [5]. In the present study, we showed that challenge of C. albicans biofilms with either TRI or PYR contributed to a significant reduction in biofilm formation. Derivatives of PYR had low antifungal activity against C. albicans when compared to the original compound, while the derivative TRI1 did not have any antifungal activity at all.
The cell wall of C. albicans is the first point of contact between the fungus and the innate immune system [2]. The cell wall is composed of an outer layer enriched in mannan and mannosylated glycoproteins and an inner layer enriched in β-glucan and chitin [2,26]. We observed that PYR or TRI reduced the thickness of the mannan part of the C. albicans cell wall indicating that these two compounds can modulate the fungal cell wall and affect C. albicans virulence. However, no changes in chitin levels were observed in the fungal cells. These observations are consistent with our previous experimental studies, which showed that mannans were involved in Candida virulence and a deficiency in cell wall mannan contributed to a reduction in mouse mortality and intestinal inflammation induced by DSS [27].
In the C. elegans model, both TRI and PYR reduced the mortality of a high percentage of nematodes infected with C. albicans supporting the antifungal properties of these compounds against C. albicans infection [28]. C. elegans relies on its innate immune system to defend itself against fungal and bacterial pathogens by producing different antimicrobial proteins [29]. Caenacins and lysozyme, which are expressed in C. elegans, play a direct role against pathogens that infect worms via the intestinal lumen or cuticle [9,30,31]. Mallo et al. reported that overexpression of the lysozyme gene, lys-1, augmented the protection of C. elegans against Serratia marcescens infection [9]. In the current study, we showed that treatment with the two compounds enhanced the expression of antimicrobial genes (lys-1, lys-7, and cnc-4) in nematodes infected with C. albicans, indicating that PYR and TIR treatments not only had beneficial antifungal effects during infection but were also capable of improving the innate immune response of nematodes against C. albicans infection.

Chemical Synthesis of PYR and TRI
Compound PYR was synthesized as described previously by Boisse et al. [35]. The reaction was performed between an amine (4-chloroaniline for Cpd PYR) and an allenic precursor ( Figure 8). This methodology led to new 1-aryl-3,6-dimethyl-4-aminoaryl-2pyridones 1 in good yields. These syntheses can be performed in two distinct steps, allowing for the possibility of introducing different substituents in the positions 1 and 4. Using this strategy, all compounds from the PYR series (1) were obtained ( Figure 8). All the procedures and chemical characterizations of PYR compounds are detailed in the publication described by Boisse et al. [35].

Chemical Synthesis of PYR and TRI
Compound PYR was synthesized as described previously by Boisse et al. [35]. The reaction was performed between an amine (4-chloroaniline for Cpd PYR) and an allenic precursor ( Figure 8). This methodology led to new 1-aryl-3,6-dimethyl-4-aminoaryl-2pyridones 1 in good yields. These syntheses can be performed in two distinct steps, allowing for the possibility of introducing different substituents in the positions 1 and 4. Using this strategy, all compounds from the PYR series (1) were obtained ( Figure 8). All the procedures and chemical characterizations of PYR compounds are detailed in the publication described by Boisse et al. [35]. The synthesis of TRI compounds has not yet been described in the literature. The full synthetic study will be reported in due course in a global article of organic chemistry. Briefly, TRI compounds were obtained by reaction of arylamine with 2-ethinyl-4,6dimethoxy-1,3,5-triazine in dichloromethane under magnetic stirring at room The synthesis of TRI compounds has not yet been described in the literature. The full synthetic study will be reported in due course in a global article of organic chemistry. Briefly, TRI compounds were obtained by reaction of arylamine with 2-ethinyl-4,6-dimethoxy-1,3,5-triazine in dichloromethane under magnetic stirring at room temperature for 2 days (Figure 9).

Antifungal Compounds
PYR and TRI were designed, synthesized, and provided by JUNIA (a graduate school of general engineering-Hautes Etudes d'Ingénieur, Lille, France). Several small aliquots were prepared for each compound and stored in the freezer at −20°C. These fresh aliquots were adjusted to the appropriate dilutions in PBS for each experiment. PYR and TRI were used at their minimum inhibitory concentrations (MICs; 12.5 µg/mL and 50 µg/mL, respectively), diluted in PBS during the various in vitro and in vivo experiments. Commercially available caspofungin (Merck, Semoy, France) and fluconazole (Fresenius, Sèvres, France) were used as positive controls. The negative control was PBS. respectively). Serial dilutions ranging from 10 −1 to 10 −4 were performed on the samples suspended in PBS and 100 µL of each dilution was spread onto Sabouraud agar at 37 • C.

Effect of PYR and TRI on C. albicans Biofilm Formation
C. albicans at a concentration 10 7 colony-forming units (cfu) in 200 µL of RPMI was added to each well of a 96-well polystyrene plate (Greiner Bio-one) and incubated for 24 h. PYR and TRI were then added to the plate at their 1× MIC (12.5 µg/mL and 50 µg/mL, respectively) for 24 h. Caspofungin or fluconazole were also used at their MICs as positive controls. After three washes with PBS to remove non-adherent cells, 110 µL of crystal violet (0.4%; Fluka) was added to each well. After three washes with PBS, crystal violet staining was decolorized by adding 200 µL of ethanol to the plate. The absorbance of the decolorization solution that reflected the numbers of viable cells was measured at 550 nm using a spectrophotometer (FLUOstar; BMG Labtech, Champigny sur Marne, France). The results are presented as the mean of six replicates from two independent experiments. 4.6. C. elegans Survival Assay and RT-PCR Quantification of Antimicrobial Genes of C. elegans C. albicans SC5314 strain was inoculated in 2 mL of Sabouraud broth and incubated at 36 • C for 24 h. Candida lawns were prepared by spreading 10 µL of the C. albicans culture onto plates containing solid BHI medium with amikacin (45 µg/mL). These plates were then incubated at 36 • C for 24 h. N2 wild-type C. elegans was grown on nematode growth medium seeded with E. coli OP50 as a food source at room temperature for 72 h, until they reached the L4 stage. Approximately 100 worms were selected and washed with M9 buffer containing 90 µg/mL amikacin to eliminate E. coli. These worms were then picked onto the Candida lawn and allowed to feed for 6 h. The nematodes were washed three times with M9 buffer to remove C. albicans cells from their cuticles. Approximately 70-80 nematodes were then picked to wells in a 6-well microtiter dish that contained 2 mL of liquid 80% M9 buffer, 20% BHI, 10 µg/mL cholesterol in ethanol, and 90 µg/mL amikacin. TRI and PYR at their MICs (12.5 µg/mL and 50 µg/mL, respectively) were added to each well. The plates were incubated at room temperature for 12 h. The worms were examined for survival daily for 4 days. Nematodes were considered dead when they did not respond to being touched by a platinum-wire pick.
For the RT-PCR assay, total RNA was isolated from N2 after 12 h of antifungal treatment using a NucleoSpin RNA ® kit (Macherey-Nagel, Hoerdt, France). Nematode RNA was quantified by spectrophotometry (Nanodrop; Nyxor Biotech, Paris, France). cDNA synthesis was carried out using a High Capacity DNA Reverse Transcription (RT) kit, with Master Mix (Applied Biosystems, CA, USA). To amplify the cDNA, Fast SYBR green (Applied Biosystems) was employed in a one-step system (Applied Biosystems) [39]. SYBR green dye intensity was assessed using one-step software. All results were normalized to the reference gene, act-2.

Statistical Analysis
The Mann-Whitney U test was used to analyze the differences between the groups and the results were considered to be statistically significant when the p value was as follows: p < 0.05; p < 0.01; p < 0.001. All statistical analyses were carried out using GraphPad Prism version 6 (GraphPad, La Jolla, CA, USA).

Conclusions
In conclusion, two series of triazine (TRI) and pyridinone (PYR) derivatives were selected for their antifungal potential. These series share some similarities such as a 6membered N-heterocycle, a free -NH-link and a para-substituted aromatic moiety. Whereas in series TRI, the p-methoxyaniline was the most effective ring for antifungal activity, it appeared that in series PYR, p-chloroaniline was the best choice. TRI and PYR were found to have fungicidal activity against C. albicans which was more pronounced than their analogues, and both reduced the viability of clinically isolated fluconazole-or caspofungin-