Synthesis of the compound 2
All commercially available chemicals were obtained from Sigma-Aldrich and used without further purification. To a 100 mL one-necked round-bottomed flask equipped with a condenser were added benzidine (1) (0.5 g, 2.71 mmol) and ethanol (50 mL). To the above solution was added formaldehyde solution (54.28 mmol, 1.63 g, 20 equiv), and the reaction mixture was refluxed for 12 h and monitored by TLC. After complete consumption of benzidine (1), the reaction mixture was concentrated under reduced pressure. The mixture was then cooled to room temperature. Upon cooling the resulting reaction solution to ambient temperature, white solids precipitated from solution, the solid product was filtered and washed with cold methanol and then dried in vacuo to give the compound (2) (yield, 98%). N4,N4,N4',N4'-Tetrakis(ethoxymethyl)-[1,1'-biphenyl]-4,4'-diamine (2): Mp: 137–139°C IR (cm− 1): 3091, 3019, 1979, 2932, 2879, 2877, 1610, 1505, 1422, 1388, 1367, 1330, 1265, 1203, 1161, 1100, 1058, 1034, 997, 966, 897, 836, 817, 764, 751, 677, 631. 1H NMR (400 MHz, CDCl3): δ 7.45 (d, J = 8.7 Hz, 4H), 7.11 (d, J = 8.7 Hz, 4H), 4.88 (s, 8H), 3.52 (q, J = 7.0 Hz, 8H), 1.24 (t, J = 7.0 Hz, 12H). 13C NMR (100 MHz, CDCl3): δ 145.60, 132.60, 127.09, 114.98, 82.76, 62.36, 15.16. HRMS (Q-TOF): m/z [(M-C10H24O4) + H]+ calcd. for C14H13N2: 209.10787, found: 209.10787.
General procedure for synthesis of the compounds 4a-b
Benzidine (1) (1.000 mmol) and aromatic aldehydes 3a or 3b (2.100 mmol) were dissolved in EtOH, and the resulting reaction mixture was heated at the refluxed temperature for 12 h and monitored by TLC. After complete consumption of aromatic aldehyde, the reaction mixture was concentrated under reduced pressure. The mixture was then cooled to room temperature. Upon cooling the resulting reaction solution to ambient temperature, crystals precipitated from solution, the solid product was filtered and washed with cold methanol and then dried in vacuo to give the compound 4a-b [34, 44].
(1E,1'E)-N,N'-([1,1'-biphenyl]-4,4'-diyl)bis(1-phenylmethanimine) (4a): Mp: 231–233°C. 1H NMR (400 MHz, CDCl3): δ 8.53 (s, 2H, CH = N), 7.98–7.89 (m, 4H), 7.71–7.63 (m, 4H), 7.53–7.46 (m, 6H), 7.36–7.29 (m, 4H).
(1E,1'E)-N,N'-([1,1'-biphenyl]-4,4'-diyl)bis(1-(4-nitrophenyl)methanimine) (4b): Mp: 218–220°C. 1H NMR (400 MHz, CDCl3): δ 8.62 (s, 2H), 8.33 (d, J = 8.8 Hz, 6H), 8.09 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.6 Hz, 4H), 7.33 (d, J = 8.6 Hz, 4H).
Anticancer activity of the compounds 2, 4a-b
MDA-MB-231 breast cancerand DLD1 colorectal cancer cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin. Cancer cell lines were maintained at 37°C and 5% CO2 in an incubator.
Measurement of cell survival
Cancer cells were seeded in 96-well plates at a density of 0.5 x 104 in 200 µl culture medium and incubated at 37°C and 5% CO2 until the cells were grown to reach about 70% confluency before chemical treatments. Then, the cells were treated with different concentrations of the compound 2 for 24 hrs. Cell survival was determined using the Eco-Tech CVDK-8 Cell Viability kit (EcoTech Biotechnology, Turkey) according to the manufacturer’s recommendations. The assay is based on the reduction of WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt). As a result of WST-8 bio-reduction, the amount of yellow-colored formazan is produced in the culture medium and this is directly proportional to the number of living cells. The absorbance of the medium in each well was measured at 450 nm via a spectrophotometer.
The data set was created by transferring the absorbance values and folding time calculation results of the wells to IBM SPSS 20.0 software. The variance homogeneity test was applied according to the results obtained by applying the Shapiro-Wilk normality test to the created data set and p < 0.05 was considered significant. One-Way ANOVA test was applied to all the data and the appropriate Post-Hoc test was selected according to the variance homogeneity test results and p < 0.05 was considered significant.
1H NMR spectra were recorded on a Bruker Ultrashield Plus Biospin spectrometer at 400 MHz. NMR chemical shifts were determined relative to internal standard TMS at δ 0.0 ppm. The chemical shifts (δ) are reported in ppm, and coupling constants (J) are in Hertz (Hz). Melting Points (M.p) were recorded on a Stuart melting point SMP30 device and are uncorrected. FTIR spectra were recorded using a Perkin Elmer Frontier FT-IR spectrophotometer. Mass spectra were recorded on an Agilent Technologies 6530 Accurate-Mass Q-TOF-LC/MS. The absorbance of the cell culture medium was measured via an ELx800 BioTek spectrophotometer.
Molecular docking calculations
Molecular docking calculations are the common method used to compare the activities of molecules. In this method, the interactions of molecules with proteins are examined. These calculations are made using the Maestro Molecular modeling platform (version 12.2) by Schrödinger . For these calculations, it is necessary to prepare both proteins and molecules, which consists of many stages. It is the preparation process of the proteins studied in the first stage. At this stage, active sites of proteins are determined. All proteins in this active region were given freedom of movement, because the molecules were easier to interact with the proteins. At this stage, the protein preparation module  is used. The next stage is the preparation of molecules for calculations. In this process, all conformers of the molecules are prepared. For the interaction of proteins and molecules, each structure is tested one by one, in an attempt to find the most stable interaction pose. the LigPrep module  is used in the calculations of this stage. In the next step, the interactions of molecules with proteins are made. At this stage, calculations are made with The Glide ligand docking module  for calculations. In these calculations, the preparation of molecules and proteins is calculated by the OPLS3e method. In the next stage, the properties of molecules to be drugs are examined. This analysis is performed to predict the actions of drug molecules in human metabolism. These calculations are calculated by The Qik-prop module  of the Schrödinger software.