General synthetic procedure for thiosemicarbazones (3a-y)
To a solution of benzaldehyde derivatives (1a–y) (0.01 mol) in warm ethanol (30 mL) was added two drops of acetic acid and thiosemicarbazide (2) (0.01 mol) in warm water (30 mL). The reaction mixture was stirred at room temperature for 4 h and monitored by TLC. The precipitate was filtered off and recrystallized from ethanol to afford the target compounds 3a-y.
Determination of drug similarity properties of compounds using Swiss ADME and Admet SAR programs
Today, computer-based calculation methods are used to reduce or eliminate the harm and undesirable effects of chemicals. Before starting preclinical studies, it is possible to make predictions about the pharmacokinetic properties, bioactivity, and drug similarity of the compounds by conducting theoretical studies. While designing a drug molecule, preliminary information about many properties such as water solubility, water carrying capacity, absorption in the gastrointestinal tract, protein affinity and toxicity can be obtained. As a result of many years of studies on drugs, various drug similarity rules such as Lipinski, MDDR-like, Veber, Ghose filter, BBB, CMC-50-like rules, and Quantification of Drug Similarity (QED) have been established. In this study, the properties of the compounds synthesized using the Swiss ADME and Admet SAR programs will be evaluated by investigating their similarities and superiorities with the standard drug [11–14].
Hexe 8.0.0 Docking program was used in the calculations [12–15]. The molecular formulas of each of the 22 thiosemicarbazone derivatives (3a-y) selected as Ligands in the program were drawn with the MarvinSketch 21.20 program and stored as a pdb (Protein Date Bank/PDB) file. The pdb file of the receptor proteins (carbonic anhydrase I-II isoenzymes and acetylcholinesterase) was obtained from RCSB PDB (http://www.rscb.org/pdb). To compare the data obtained, standards currently used as market drugs were used ligands (thiosemicarbazone derivatives) and targeted proteins (protein-78 (GRP78) for C6 and quinone reductase-2 (4ZVM for MCF 7). Receptor and Ligand files were imported in the Hex 8.0 software. Energy of docking (E value) was calculated using Hex 8.0. [15, 19].
Cell Culture Studies
The C6 glioma (ATCC® CCL-107) cell line and the MCF 7 breast cancer (ATCC® HTB-22) cell lines were obtained from ATCC. Penicillin/streptomycin (10,000U/mL), DMEM, Fetal Bovine Serum (FBS), Trypsin-EDTA solution and various consumables required for cell culture were used.
Cell Culture Study and Consumables
Cells proliferated in DMEM cell culture medium containing 1% L-glutamine, 1% penicillin-streptomycin and 10% fetal bovine serum in 25 cm2 flasks in an oven at 37°C and 5% CO2. Cells were passaged when they reached at 80% density and studies were started after a certain passage.
XTT Cell Viability Assay
The effects of the synthesized compounds on the cell viability of C6 and MCF 7 cells were evaluated by applying the XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) test. This study was carried out by using the method applied by Wolf et al . Synthesized (thiosemicarbazone derivatives) compounds (3a-y) at determined concentrations were treated to each cell line, and the XTT cell viability test was performed for each cell line. Imatinib was considered as a positive control group. XTT method is based on the principle that metabolically active cells convert XTT, a tetrazolium salt, into orange colored formazan crystals. The resulting dye is water-soluble, and the dye density can be read at certain wavelengths (450 nm) using an ELISA reading device. The dye intensity in orange is proportional to the number of metabolically active cells. For cytotoxicity experiments, cells were seeded into a 96-well microplate with 10x103 cells per well in 100 µL of DMEM (containing 10% FBS + 1% antibiotic) medium and incubated overnight for cells to attach. Following day, after removing the medium on the cells and washing the wells with PBS, fresh medium was added to the cells. Samples of compounds (3a-y) at concentrations of 2, 5, 10, 25, 50 µg/ml were treated with cells and incubated for 24 hours. At the end of this period, the medium was removed, and the cells were washed three times with PBS.
Then, 100 µl of colorless DMEM and 50 µl of XTT solution were added to each well and incubated for 4 hours in a CO2 incubator. After the incubation, the optical density value was read at 450 nm in the microplate reader, the cell viability rate of the control group was accepted as 100% and it was calculated using the formula:
% Cell viability = (Concentration O.D. / Control O.D.) X 100.
According to the results obtained, IC50 values of compounds (3a-y) and imatinib were calculated.