The hemibastadin analogues evaluated in this study were obtained using previously described methods [13][16]. In practical terms, these compounds were prepared in excellent yield in two steps (Scheme 1). 1-(2-Chloroethyl)-4-methoxybenzene was subjected to N-bromosuccinimide to afford 2-bromo-4-(2-chloroethyl)-1-methoxybenzene. Treatment of this halogeno compound by sodium azide in dimethylformamide afforded the 4-(2-azidoethyl)-2-bromo-1-methoxybenzene. Synthesis of the 1-(3-bromo-4-methoxyphenethyl)-1H-1,2,3-triazole-4-carboxylic acid was then achieved by performing the copper(I)-catalyzed 1,3-dipolar cycloaddition of the organic azides with propargylic acid resulting in the formation of 1,2,3-triazoles. In practice, propargylic acid was added at room temperature to a solution of azide, CuSO₄/sodium ascorbate in water/ethanol mixture (50/50) and the reaction time was optimized at 12 hours at room temperature. Access to the different hemibastadin analogues was then allowed by a peptide coupling step using EDC/HOBt methodology (22). All amides were obtained in good yields.

Scheme 1. Synthesis of hemibastadin analogues.

Fungal strains

Strain selection was performed using three isolates provided by the Institut Pasteur de Côte d’Ivoire (IPCI). These were clinical strains collected from vaginal samples during a 2017 survey among female sex workers. The patients were HIV-seronegative and presented with clinical signs of vaginal candidiasis. Strains from this anatomical site were selected because they are frequently implicated in chronic and/or recurrent infections associated with biofilm formation. Additionally, these strains were chosen for their susceptibility to commonly used antifungal agents, particularly azole antifungals.

Biofilm formation

The methods employed in this study were inspired by those of Pierce and Ramage [17]-[19]. Starting from a colony of a C. albicans strain cultured on Yeast Peptone Dextrose (YPD; Sigma-Aldrich, St Louis, USA) agar, 10 ml of liquid YPD were inoculated under sterile conditions and incubated overnight at 30˚C (12 - 14 hours). The resulting suspension was centrifuged at 3000 rpm for 5 minutes. The supernatant was discarded, and the pellet was resuspended in phosphate-buffered saline (PBS) and centrifuged again to remove any residual YPD. After washing, a cell suspension at a density of 10⁶ cells/ml in Roswell Park Memorial Institute 1640 medium (RPMI 1640; Gibco, GB) was prepared and distributed into flat-bottomed, transparent 96-well microplates. Following 48 hours of incubation at 37˚C without agitation, the wells were rinsed three times with PBS. Biofilm detection was performed using crystal violet at a concentration of 1%. The dye was added to the wells, and the plate was incubated for 30 minutes. The plates were then washed again, and the dye fixed by the fungus was resolubilized by adding acetic acid at 30 volumes for 15 minutes. Optical density was measured at 590 nm using an ELISA plate reader, allowing determination of the optical density (OD).

Antibiofilm Activity

Biofilm inhibition tests were conducted by exposing the selected strain to a solution of the compound under evaluation. Compound concentrations ranged from 5 to 200 µM. Results were expressed as the percentage of adhesion at each selected concentration, calculated using the following formula. Fluconazole was used as the reference substance. For data analysis, the percentage of adhesion for each well was calculated as follows:

% adhesion = (ODT − ODPdt)/(ODTB − ODBlk) × 100

where:

ODT = Mean optical density (OD) of triplicates for each tested concentration (compound + yeast).ODPdt = OD of the compound alone in the presence of the fluorochrome, without yeast.ODTB = Mean OD of six replicates for the bacterial adhesion control (yeast).ODBlk = Mean OD of blanks (RPMI1640 + fluorochrome).

Based on the adhesion percentages, the EC₅₀ values for each compound were calculated using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, Calif.).

Visualisationof Biofilms by Confocal Microscopy

Biofilm visualisation was performed using confocal microscopy, following staining with SYTO® RED 9, both in the presence and absence of the most active compound. The culture was carried out under the same conditions as previously described. However, for SYTO RED 9 staining, 24-well microplates were used, into which coverslips previously sterilised by ultraviolet light were immersed. Yeast suspensions were prepared by incubating them with 200 µM of the compound, compared to a suspension without the molecule. After washing with PBS, the coverslips were mounted on microscope slides using ProLong Diamond Antifade. The preparations were then examined under a confocal microscope at 528 nm.

Data Extraction

Biofilm biovolume and average thickness data were extracted using the COMSTAT plugin in ImageJ [20][21].

Evaluation of Antifungal Activity

To determine whether the observed antibiofilm activity was specific and not due to an antifungal effect, a sensitivity test was performed with the most active compound. The method used was the standard CLSI protocol, specifically the microdilution method in liquid medium [22]. This approach allows for the determination of the minimum inhibitory concentration (MIC) of the molecules. The procedure involved culturing yeast for 24 hours on a YPD agar plate. Serial dilutions of both the test molecules and the yeast were prepared to carry out the assay. The molecules to be tested, as well as fluconazole as a reference, were weighed to prepare stock solutions according to the following formula:

Mass (mg) = volume (ml) × concentration (μg/ml)/potency (μg/mg)

The specific activity of fluconazole, according to the technical data sheet, is 99.64%, while that of the test molecule was considered to be 100%. From a stock solution of the molecule, the concentrations tested ranged from 0.125 to 64 µg/mL, diluted in RPMI 1640. The yeast stock solution was prepared at 10⁶ cells/ml. This inoculum was diluted 1:1000 in RPMI. Then, 100 µl were distributed into each well of the plate except for column 11 (sterility control). The plates were incubated at 37˚C for 72 hours.

The characterization of the chemical structures of the various synthesized derivatives has been described in previous articles [13][16].

1-(3-bromo-4-methoxyphenethyl)-N-(4-hydroxyphenyl)-1H-1,2,3-triazole-4-carboxamide (1).

1H NMR (400 MHz, DMSO) δ 10.17 (s, 1H), 9.26 (s, 1H), 8.62 (s, 1H), 7.57 (d, J = 8.9 Hz, 1H), 7.48 (s, 1H), 7.14 (dd, J = 8.4, 2.0 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H), 6.72 (d, J = 8.9 Hz, 2H), 4.68 (t, J = 7.1 Hz, 2H), 3.81 (s, 3H), 3.16 (t, J = 7.1 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 158.3, 154.5, 154.2, 143.4, 133.5, 131.5, 130.5, 129.7 (2C), 127.4, 122.6, 115.4 (2C), 112.9, 110.9, 56.6, 51.2, 34.1.

1-(3-bromo-4-methoxyphenethyl)-N-(4-hydroxybenzyl)-1H-1.2,3-triazole-4-carboxamide(2).

1H NMR (400 MHz, DMSO): δ 9.27 (s, 1H, OH), 8.91 (t, J = 6.3 Hz, 1H, NHCO), 8.50 (s, 1H), 7.45 (d, J = 2.1 Hz, 1H), 7.13 (d, J = 2.1 Hz, 1H), 7.10 (d, J = 8.3 Hz, 2H), 7.00 (d, J = 8.5, 1H), 6.68 (d, J = 8.4 Hz, 2H), 4.64 (t, J = 7.1 Hz, 2H), 4.30 (d, J = 6.2 Hz, 2H), 3.79 (s, 3H), 3.13 (t, J = 7.1 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 159.9, 156.6, 154.5, 143.1, 133.5, 131.7, 130.3, 129.7, 129.2 (2C), 126.8, 115.4 (2C), 112.9, 110.9, 56.6, 51.1, 41.8, 34.5; (ESI, m/z) 383.20 [M + H]⁺, 431.03.

1-(3-bromo-4-methoxyphenethyl)-N-(3.5-dibromo-4-hydroxybenzyl)-1H-1.2,3-triazole-4-carboxamide (3).

1H NMR (400 MHz, DMSO): δ 9.87 (s, 1H, OH), 9.11 (t, J = 6.2 Hz, 1H, NHCO), 8.50 (s, 1H), 7.47 (s, 1H), 7.41 (s, 1H), 7.13 (d, J = 8.4 Hz, 2H), 6.99 (d, J = 8.5 Hz, 1H), 4.64 (t, J = 7.1 Hz, 2H), 4.30 (d, J = 6.2 Hz, 2H), 3.79 (s, 3H), 3.13 (t, J = 7.1 Hz, 4H); 13C NMR (101 MHz, DMSO) δ 160.3, 154.6, 150.3, 142.9, 134.2, 133.5, 131.8, 131.6, 129.8(2C), 127.1, 113.0 (2C), 112.3, 110.9, 56.6, 51.3, 40.5, 34.6; (ESI, m/z) 383.20 [M + H]⁺, 385.17.

1-(3-bromo-4-methoxyphenethyl)-N-(4-hydroxyphenethyl)-1H-1.2,3-triazole-4-carboxamide (4).

1H NMR (400 MHz, MeOD) δ 8.09 (s, 1H), 7.30 (d, J = 2.0 Hz, 1H), 7.05 (m, 2H), 7.02 (dd, J = 8.4, 2.0 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 6.71 (m, 2H), 4.63 (t, J = 7.0 Hz, 2H), 3.81 (s, 3H), 3.55 (t, J = 7.3 Hz, 2H), 3.14 (t, J = 7.0 Hz, 2H), 2.80 (t, J = 7.3 Hz, 2H).13C NMR (101 MHz, MeOD) δ 162.4, 157.0, 156.6, 143.9, 134.5, 132.2, 131.2, 130.7 (2C), 130.1, 127.3, 116.4 (2C), 113.7, 112.8, 56.9, 52.7, 42.0, 36.0, 35.8.

In order to select a strain to serve as a model for antibiofilm assays, the three strains of Candida albicans were subjected to established biofilm formation protocols and compared using optical density (O.D.) as the primary criterion. The results of this comparative analysis are presented in Figure 2.

Figure 2. Optical densities of biofilms formed by the three candida strains used to validate the method.

Differences in the thickness of the biofilms formed were observed. It has previously been demonstrated that biofilm thickness varies significantly according to parameters such as HIV serological status or the anatomical origin of the strain (vaginal or oral) [23].

When observed under phase-contrast optical microscopy as well as confocal microscopy (Figure 3), the biofilm formed by this strain exhibits a high cell density and the presence of cells in the form of hyphae, pseudohyphae, and yeast, which are characteristic features of a Candida albicans biofilm.

Under the selected experimental conditions, this strain forms a biofilm with an average biovolume of 0.14 ± 0.04 µm³/µm² and a biomass exhibiting a mean thickness of 7.15 ± 0.84 µm. These properties enable its use for the evaluation of antibiofilm activity.

The selected compounds have been evaluated against the biofilm of Candida albicans 17649. The results are reported in Table 1.

Figure 3. Strain 17649 observed under phase-contrast optical microscopy (left) and confocal microscopy (right).

Table 1. Chemical structure and biological activity of hemibastadin analogues against the biofilm of Candida albicans, IPCI 17649 strain. Results are expressed as effective concentration to inhibit 50% of biofilm formation (EC50) in micromoles/L (µM). Data represent means ± standard deviations values from three independent experiments.

These results show that three of the four evaluated molecules exhibit antibiofilm activity; only compound 4 lacks this activity. These findings are consistent with those previously obtained for the same molecules against marine bacteria [13][16]. Indeed, compound 3 did not inhibit biofilm formation by marine bacteria. The SAR trends parallel those observed in marine bacteria: maximal activity is achieved with a non-brominated single-carbon eastern region. Hyperbromination, typically beneficial, becomes detrimental on this scaffold and abolishes activity, likely due to steric or electronic disruption. In marine bacteria, replacing the western-region methoxyl group with a phenolic hydroxyl restores the positive effect of hyperbromination [24], a modification that should also be evaluated against C. albicans. Given this reversal and the persistence of a single-carbon, non-brominated east region as the optimal motif, QSAR modelling and molecular docking studies will be essential to elucidate the mechanistic basis of this unfavorable hyperbromination effect.The inhibitory effect of this compound is shown by confocal microscopy (Figure 4).

Figure 4. Confocal laser scanning microscopy images for the Candida albicans 17649 strain with compound 2 (above). Effect on the compound on the biovolume (down left) and the average thickness (down right).

Determination of the minimum inhibitory concentration (MIC) according to CLSI standards showed a value greater than 64 µg·mL⁻¹, compared with fluconazole, which has an MIC of 4.06 µg·mL⁻¹. Therefore, this compound does not possess intrinsic antifungal activity. Thus, as observed with marine bacteria, the effect on C. albicans biofilm appears to be a strictly antibiofilm activity, without antifungal properties. In C. albicans biofilms, the hemibastadin analogues may also modulate the early adhesion phase. Although their precise mode of action in fungal systems remains to be fully elucidated, this hypothesis is consistent with observations reported for marine bacteria, where these molecules interfere with initial surface attachment and the establishment of nascent biofilms. Further investigations will be required to determine whether similar molecular targets or signaling pathways are involved in C. albicans, and to clarify how these compounds influence adhesion dynamics, morphogenesis, and early matrix deposition.