-Microwave-assisted synthesis, antimicrobial, antiquorum-sensing and cytotoxic activities of a new series of isatin-β-thiocarbohydrazones

Abstract New isatin-β-thiocarbohydrazone hybrids 4-17 were designed relied on isatin scaffold to generate various analogs with expected antimicrobial activity. The new hybrids were estimated for antibacterial effectiveness over S. aureus, B. cereus and E. coli, and antifungal efficacy over C. albicans and A. fumigatus. Compound 14 evinced the highest efficacy over A. fumigatus and C. albicans. In addition, 5 and 12 showed eminent efficacy toward A. fumigatus, where compounds 4, 7, 9, 11, 13, 15 and 16 exhibited moderate activity over the same fungus. Moreover, 6 and 9 displayed moderate activity over C. albicans. The new compounds were also estimated for antiquorum-sensing effectiveness against C. violacium, where compound 17 showed interesting activity. In vitro cytotoxicity assay of the new analogs was done over Hela and COS-7 cancer cell lines. All analogs have IC50 values >50 μM toward both cell lines. Graphical Abstract Compound 14 demonstrated the highest efficacy over A. fumigatus (MIC = 39.06 µg/mL). Also, it displayed acceptable antiquorum-sensing activity, and it was proven to be an excellent candidate for evolution of new more efficient antifungal analogs.


Biological screening
Antimicrobial and antiquorum-sensing testing

Antibacterial testing
All the bacterial strains were propagated in Luria Bertani (LB) broth (1% peptone, 0.5% yeast extract, 0.5% NaCl) and solidified with 1.5% agar. Melted Muller Hinton agar (50 mL) at 50 °C were seeded with 50 µl of 1x10 6 CFU/mL of 18 hrs culture of the tested bacteria. The inoculated agar was mixed and poured into 15-cm-diameter plates to solidify. Wells were made in agar using cork borer. Tested compounds were dissolved in DMSO in eppendorff tubes for final concentration 5 mg/mL. Aliquots each of (100 µl) of each compound were applied into the wells, DMSO was also included as a negative control, and ampicillin in a concentration of 5 mg/mL was used as a reference antibacterial agent. The compounds were allowed to diffuse for 2 hrs at 4 °C and incubated at 37 °C for 24 hrs. [4][5][6] Inhibition zones were measured using Vernier caliper and the activity of the tested compounds was estimated in comparison to ampicillin ( Table 1). The inhibition zone diameter of DMSO was subtracted from the antibacterial activity of the tested compounds.

Determination of diameter of inhibition zone (mm)
Sabouraud medium (50 mL) inoculated with 50 µl of 1x10 6 CFU/mL of 24 hrs culture of Candida albicans. Glucose minimal medium (50 mL) was inoculated with 50 µl of 1x10 3 SFU/mL of Aspergillus fumigatus 293. Wells were made in agar using cork borer. The tested compounds were dissolved in DMSO in eppendorff tubes for final concentration 5 mg/mL and 100 µl of test solution was applied in the wells. The standard antifungal drug (fluconazole) was also added at the same concentration to each plate. In addition, DMSO (control solvent) was added to each plate. Plates were incubated at 37 °C for 48 hrs. [6][7][8][9] Antifungal activity of the tested compounds was determined by measuring the inhibition zone diameter ( Table 1). The inhibition zone diameter of DMSO was subtracted from the antifungal activity of tested compounds.

Determination of minimal inhibitory concentrations (MICs)
Minimal inhibitory concentrations (MICs) were determined by serial dilution technique using 96-multiwell microtiter plates. The investigated compounds 4-17 were dissolved in DMSO 100% to prepare stock solutions of 5000 µg/mL. Two fold serial dilutions of the dissolved compounds were performed in glucose minimal medium providing eight different concentrations (2500, 1250, 625, 312.5, 156.25, 78.125, 39.06 and 19.53 µg/mL). A. fumigatus 293 was diluted to 1x10 3 SFU/mL in glucose minimal medium, and C. albicans diluted to 1x10 6 CFU/mL in Sabouraud's medium. 20 µL of the diluted culture was added to the test solutions (50 µL) in the wells (one well per concentration). The plates were incubated at 37 °C for 48 hrs for C. albicans and at 30 °C for 48 hrs for A. fumigatus 293. [6,8,9] MIC was detected visually as the least concentration inhibiting fungal growth (no turbidity) and the activity of the tested compounds was estimated in comparison to fluconazole ( Table 2).

Antiquorum-sensing testing
The culture was prepared by growing C. violaceum ATCC 12472 in LB broth and incubated for 16-18 hours in an orbital incubator running at 28 °C and 150 rpm. The culture was then adjusted to 0.5 McFarland standard (Ca. 1x10 6 CFU/mL). C. violaceum (50 µL) was inoculated into LB agar (50 mL), poured into plates and solidified. Wells were made in LB agar medium using cork borer. The tested compounds, 4-17 were dissolved in DMSO 100% in eppendorff tubes for final concentration 5 mg/mL, and 50 µL of the test solution was applied into the wells. The positive control (catechin) was also added at the same concentration and volume to each plate. In addition, DMSO (control solvent) was added to each plate. Plates were incubated at 30 °C for 48 hours to check the inhibition of pigment production around the wells. Bacterial growth inhibition would result in a clear halo around the disc, while a positive quorum sensing inhibition is exhibited by a turbid halo harboring pigmentless bacterial cells of C. violaceum ATCC 12472 monitor strain. [6,10] Bacterial growth inhibition by the tested compounds was measured as radius (r 1 ) in mm, while both growth and pigment inhibition was measured as radius (r 2 ) in mm. The pigment inhibition (QS inhibition) was determined by subtracting bacterial growth inhibition (r 1 ) from total radius (r 2 ); thus, QS inhibition = (r 2 -r 1 ) in mm (Table  1).
Cells were placed in a 96-multiwell microtiter plate (10 4 cells/well), for 24 hrs at 37 °C and in an atmosphere of 5% CO 2 before treatment with the compounds to allow attachment of the cells to the wall of the plate. The tested compounds were dissolved in DMSO and diluted with phosphate buffer solution (PBS) to obtain different concentrations. Tested compounds of different concentrations were added to each well and cells were incubated with the compounds for 48 hrs at 37 °C and in an atmosphere of 5 % CO 2 . All tests were performed in triplicates using doxorubicin as a reference antitumor agent. The treated cells were washed with PBS and 100 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (MTT) (5 mg/mL MTT stock in PBS diluted to 1 mg/mL with 10% RPMI-1640 medium) was added. The 96-multiwell plates were read by microarray reader Perkinelmer vector 3V multilabel counter model 1420 (Perkinelmer, Boston, MA) for optical density at 490 nm. [11][12][13] The relative percentage cell viability was calculated from the following equation: The relation between surviving fraction and drug concentration is plotted to get the survival curve for cervical cancer (Hela) and kidney fibroblast cancer (COS-7) cell lines. The concentration required for 50% inhibition of cell viability (IC 50 ) was obtained for each compound from the curve fitting using Sigma plot10.