A screening campaign for fungicidal compounds resulted in the identification of a fungicidal 2,6-disubstituted quinoline derivative, namely 4-tert-butyl-N-(4-methyl-2-(4-methylpiperazin-1-yl)quinolin-6-yl)benzamide (9, Table 1a). This screening campaign, which previously led to the identification of antifungal carbazoles [15], consisted of a two-step procedure. In a first step, approximately 5,000 compounds representing diverse chemical classes were screened for antifungal activity against C. albicans, in an antifungal spot assay on C. albicans-inoculated agar using a single compound dose (100 µg/mL). Next, fungicidal activity of antifungal hits was assessed using two-fold dilution series of the compounds, allowing determination of their minimal fungicidal concentration (MFC) against C. albicans in phosphate buffered saline (PBS). The MFC was defined as the minimal compound concentration that results in 99% cell death of the inoculum as compared to the DMSO control treatment [15]. Compound 9, together with the antifungal carbazoles [15], was among the most potent fungicidal compounds identified in this screening campaign. This compound was characterized by fungicidal activity against C. albicans with a MFC value of 50 µM (Table 2).

To analyze the structural determinants for fungicidal activity against C. albicans of this new class of compounds, we assessed the MFC of 18 commercially available derivatives divided into two subseries of 2,6-disubstituted quinolines, consisting of 6-amide derivatives (Table 1a) and 6-urea derivatives (Table 1b), and of the reference antifungals amphotericin B (AmB) and miconazole (Mico) (Table 2). Next, we assessed their fungicidal activity against C. glabrata, as well as their potential antibiofilm activity in terms of the biofilm eradicating concentration 50 (BEC₅₀), which is the minimal concentration of compound resulting in 50% killing of the C. albicans biofilm cells.

Compounds 1, 2, 5, 6 and 17 were the most potent fungicidal derivatives against C. albicans (MFC = 6.25–25 µM). Together with compound 10, these compounds (except for 17) were able to eradicate C. albicans biofilms with a BEC₅₀ value identical to the one obtained with miconazole (BEC₅₀ = 25 µM), whereas compounds 12, 13 and 15 showed moderate antibiofilm activity (BEC₅₀ = 50 µM). Only compounds 1 and 5 showed fungicidal activity against the emerging pathogen C. glabrata (MFC = 25 µM). The early SAR study showed that the antifungal activity against C. albicans was partially driven by the presence of a piperazine moiety on the 2 position of the quinoline scaffold, since compound 9 showed a modest MFC value (50 µM) whereas compound 4 (piperidine moiety) did not exhibit any fungicidal activity at a concentration higher than 50 µM. In addition, the introduction of an aromatic (pyrimidyl) substituent on the piperazine moiety (compound 3) instead of a small methyl group (compound 1) led to a complete loss of both antifungal (C. albicans and C. glabrata) and antibiofilm activities. Inversely, antifungal (C. albicans) as well as antibiofilm activities were improved by changing the phenyl ring (compound 9) into a cyclohexyl ring (compound 6). The urea derivatives (Table 1b) were in general not active or less potent than the corresponding amide derivatives (Table 1a). Only compounds 10 and 17 showed some antibiofilm (BEC₅₀ = 25 µM) or antifungal activity (C. albicans MFC = 25 µM), respectively. Note that the above SAR was only based on biological data. We did not use QSAR or another computational algorithm.

Antifungal compounds that are active against C. albicans biofilms, like miconazole and amphotericin B, were previously shown to induce an increased accumulation of ROS in biofilm cells [9,10] and planktonic cells [16,17,18]. To assess whether the fungicidal 2,6-disubstituted quinoline derivatives were also able to induce ROS, we measured the endogenous ROS levels via the fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), after induction by the most potent derivatives on C. albicans planktonic and biofilm cells. The most active fungicidal derivatives (compounds 1, 2, 5 and 6) against planktonic C. albicans cells, i.e., with MFC values ranging between 6.25–25 µM did not induce ROS accumulation in planktonic cells upon 1, 3 or 5 h of treatment (data not shown). In contrast, C. albicans biofilm cells treated with the most active antibiofilm compounds (compounds 1, 5, 6, 10, 13 and 15), i.e., compounds characterized with BEC₅₀ values ≤ 50 µM, induced endogenous ROS accumulation on biofilm cells after 24 h incubation (Figure 1). Compounds 10, 13 and 15 were characterized by a high capacity to induce ROS accumulation in biofilm cells as indicated by corrected fluorescence values (CFV) > 3,000. Compounds 10 and 15 can induce ROS to the same extent as AmB (p > 0.05). CFV are the fluorescence values of the biofilm cells treated with the antifungal compounds subtracted with the value of the DMSO control. Compounds 1, 5 and 6 were characterized by an intermediate ROS-induction capacity in biofilm cells (1,000 < CFV < 3,000).

All compounds: 4-tert-butyl-N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)benzamide (1); 4-butyl-N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)benzamide (2); 4-tert-butyl-N-(4-methyl-2-(4-(pyrimidin-2-yl)piperazin-1-yl)quinolin-6-yl)benzamide (3); 4-tert-butyl-N-(4-methyl-2-(4-methyl- piperidin-1-yl)quinolin-6-yl)benzamide (4); 4-butyl-N-(4-methyl-2-(4-methylpiperazin-1-yl)quinolin-6-yl)benzamide (5); 4-tert-butyl-N-(4-methyl-2-(4-methylpiperazin-1-yl)quinolin-6-yl)cyclohexane-carboxamide (6); 5-chloro-2-methoxy-N-(4-methyl-2-(4-methylpiperazin-1-yl)quinolin-6-yl)benz-amide (7); 2,4-dimethoxy-N-(4-methyl-2-(4-methylpiperazin-1-yl)quinolin-6-yl)benzamide (8); 4-tert-butyl-N-(4-methyl-2-(4-methylpiperazin-1-yl)quinolin-6-yl)benzamide (9); 1-(3-chlorophenyl)-3-(4-methyl-2-(4-methylpiperazin-1-yl)quinolin-6-yl)urea (10); 1-(4-methyl-2-(4-methylpiperazin-1-yl)-quinolin-6-yl)-3-phenylurea (11); 1-(4-chlorophenyl)-3-(4-methyl-2-(4-methyl-piperazin-1-yl)quinol-in-6-yl)urea (12); 1-(3,5-dimethylphenyl)-3-(4-methyl-2-(4-methylpiperazin-1-yl)quinolin-6-yl)urea (13); 1-(3,4-dimethylphenyl)-3-(4-methyl-2-(4-methylpiperazin-1-yl)quinolin-6-yl)urea (14); 1-(4-ethylphenyl)-3-(4-methyl-2-(4-methylpiperazin-1-yl)quinolin-6-yl)urea (15); 1-(4-methyl-2-(4-methyl-piperazin-1-yl)quinolin-6-yl)-3-o-tolylurea (16); 1-(2-chlorophenyl)-3-(4-methyl-2-(4-methylpiperazin-1-yl)quinolin-6-yl)urea (17); 1-(4-bromophenyl)-3-(4-methyl-2-(4-methylpiperazin-1-yl)quinolin-6-yl)-urea (18) were purchased from ChemDiv (San Diego, CA, USA). Synthesis of the amide derivatives has been described previously [14].

The yeast strains used in this study were Candida albicans strain SC5314 [19] and Candida glabrata strain BG2 [20]. Overnight cultures were grown in YPD (1% yeast extract, 2% peptone and 2% glucose). Biofilms were grown in SC medium (0.8 g/L CSM, complete amino acid supplement mixture, Bio 101 Systems; 6.5 g/L YNB, yeast nitrogen base; 20 g/L glucose). PBS consists of 8 g/L NaCl, 0.2 g/L KCL, 1.44 g/L Na₂HPO₄ and 0.24 g/L KH₂PO₄ (pH 7.4). Reference antimycotics, amphotericin B and miconazole were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The fungicidal activity of antifungal compounds against C. albicans and C. glabrata was determined in PBS and the MFC for each compound was calculated according to the definition of Thevissen and coworkers [15]. To this end, overnight cultures of C. albicans or C. glabrata in YPD were 1/200 and 1/400 diluted in PBS, respectively, and treated with the compounds or DMSO (2.5% as solvent control) for 24 h at 37 °C. After 24 h, the MFC was calculated by counting the number of colony forming units (CFUs) as described previously [21].

The activity of the compounds against 16 h-old C. albicans SC5314 biofilms was determined in PBS and the BEC₅₀ for each compound was calculated. C. albicans biofilms were grown in sterile 96-well microtiter plates (TPP, Trasadingen, Switzerland). Overnight cultures of C. albicans in YPD were washed in milliQ water and 1/20 diluted in fresh SC medium. 100 µL of this culture was added to each well of a round-bottomed polystyrene 96-well microtiter plate. Following 1 h of adhesion at 37 °C, the supernatant was removed, the wells were rinsed using milliQ water and incubated with 100 µL of fresh SC medium for a growth phase of 16 h. After 16 h of biofilm formation at 37 °C, wells were rinced with PBS and incubated with 100 µL of the antifungal compounds or DMSO (2.5%) in PBS at 37 °C. After 24 h of incubation, biofilms were washed and resuspended in PBS by vigorous vortexing. The BEC₅₀ was determined by counting the number of colony forming units (CFU) as described previously [21].

Endogenous amounts of ROS, after treatment of planktonic and biofilm cells with the compounds (2.5% DMSO) were measured by a fluorometric assay with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes, Inc., Eugene, OR, USA) [18,22]. Fluorescence was measured using a fluorescence spectrometer as described [18] and fluorescence values of the samples were corrected by subtracting the fluorescence values of the antifungal compounds with the value of the DMSO control.