*2.3. Effect on the Cell Wall Integrity of B. cinerea*

To analyze the effect of the compounds on the cell wall integrity, the dye calcofluor white (CFW) was used. This dye binds to β-1,3 and β-1,4 polysaccharides, for example chitin, which is a primary component of the cell wall in fungi, and fluorescence of the hyphae can be detected [39]. Figure 2 shows the effect of compound **3a** on the cell wall of *B. cinerea*. Treatment with this compound showed lower fluorescence intensity than the negative control (acetone), indicating that this compound can damage the cell wall of this fungus. The same assay was performed using compounds **3b–j**. The ten synthesized compounds caused a decrease of the fluorescence intensity compared to the control; relative fluorescence intensity is observed in Figure 3. This result could be attributed to the toxicity of quinones, which could be connected to the production of reactive oxygen species (ROS) which cause oxidation of cell molecules [40]. Quinone derivative *N*-acetyl-*p*-benzoquinone imine (NAPQI) can react with nucleophiles such as thiol groups of proteins or glutathione [41,42]; this last molecule is an important antioxidant molecule in fungi [43]. On the other hand, some aromatic antifungal compounds have shown effects on cell wall synthesis [6] by inhibiting chitin and glucan synthases [44], enzymes that catalyze the synthesis of the main polymers of the cell wall in fungi.

**Figure 2.** Effect of compound **3a** on the cell wall of *B. cinerea*. Hyphae of *B. cinerea* incubated with liquid medium along with (**A**) lysing enzymes (positive control), (**B**) acetone 5% (*v*/*v*) (negative control), and (**C**) compound **3a** at 0.16 mM. *B. cinerea* hyphae were treated with calcofluor white (CFW) stain. Assays were carried out in triplicate.

**Figure 3.** Effect of the compounds on the cell wall integrity of *B. cinerea*. The effect was measured as relative fluorescence intensity compared to maximum fluorescence (negative control).
