*2.3. Antibacterial Susceptibility Testing*

Antibacterial properties against *Bacillus subtilis* and *Staphylococcus aureus* as two Gram-positive, and *Escherichia coli* and *Pseudomonas aeruginosa* as Gram-negative bacterial strains were tested for all synthesized compounds. Working cultures were grown overnight in Mueller-Hinton broth (MHB) (Fluka, BioChemica, Germany) under optimal conditions (37 ◦C with 50% humidity). Modified broth microdilution method [17] was used for MIC values determination as described in our previous work [18]. One hundred microliters of bacterial cultures in MHB was added to 100 μL of a serially diluted compound (250 to 0.122 μg mL−1) in sterile TPP 96-well plates (TPP Techno Plastic Products AG Trasadingen, Switzerland). Growth control and background control were included in each plate in amounts corresponding to the highest amount in the test solution and subtracted from the results. The antibacterial standard amikacin sulphate was co-assayed under the same conditions in concentration range of 0.122–250 μg mL−1. After incubation at 37 ◦C for 24 h in an atmospheric incubator with 5% CO2 and 50% humidity, an additional incubation for three hours at 37 ◦C was performed with triphenyl tetrazolium chloride as a reducing agen<sup>t</sup> indicator for microbial growth. The lowest concentrations of compound at which there was no color change or visual turbidity due to

microbial growth was defined as the MIC value, derived from triplicate analyses and expressed as micrograms per milliliter.

### *2.4. Tyrosinase Inhibiting Activity*

All synthesized compounds were tested for monophenolase and diphenolase inhibitory activity of mushroom tyrosinase according to a slightly modified procedure of Molnar et al. [19]. In brief, reaction mixture (1 mL) for determination of monophenolase activity contained 100 U of mushroom tyrosinase, 1 mM L-tyrosine and 100 μM inhibitor, while for diphenolase activity 0.5 mM L-DOPA instead L-tyrosine. In both cases, final phosphate buffer (pH 6.5) concentration in the reaction mixture was 100 mM, and the amount of DMSO was 1%.

IC50 value was determined for kojic acid by nonlinear regression using a dose-response inhibition model using GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla, CA, USA).
