2.1. Materials and methods
Sigma-Aldrich and Alfa-Aesar provided all of the reagents and solvents utilized in this research. Micro heating table HMK 67/1825 Kuestner (Büchi Apparatus) has been used to determine the melting point of the synthesized derivatives. A Nicolet 205 has been used to acquire FT-IR spectra. TMS was employed as an internal standard in the NMR analysis, and the spectra were recorded on a Bruker Avance spectrometry (300 II, 2007). Advion expression S electrospray ionization mass spectrometer (ESI–MS) (Shimadzu Corporation, Kyoto, Japan) with a TLC interface was utilized to invistgate the Mass spectra.
2.2 Preparation of derivatives 6a-f
Aldehydes (0.005 mol) were dissolved in dry DMF (20 ml) and p-TsOH (0.2 g, 0.001 mole), then (1.59 g, 0.005 mole) of compound (5) was added to the mixture. The mixture was refluxed (14-15 hours) then left it to cool. Into the mixture, crushed ice was added and kept under stirring overnight. Finally, the mixture was filtrated, the precipitate was washed with water and a solution of ethanol/dioxane was used for recrcystalization to acquire the targets (6a-f).
6-(4-Bromophenyl)-3-{[(6-methoxy-2-methylquinolin-4-yl)oxy]methyl}-5,6-dihydro-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole(6e).
Yield: 75%; yellow crystals, M.p. 208-210oC. IR (KBr) [υ, cm−1]: 3340 (NH), 2951 (CH), 1630 (C=N). 1H NMR (300 MHz, CDCl3): δ=2.17 (s, 3H, CH3),3.62 (s, 3H, OCH3),4.20 (s, 1H, CH), 4.52 (s, 2H, OCH2),5.61 (s, 1H, NH), 6.72 (s, 1H, ArH), 7.11 (d, 2H, J=8.4Hz, ArH), 7.21 (d, 1H, J=2.9Hz, ArH), 7.26 (dd, 1H, J=2.7, 9.5Hz, ArH), 7.30-7.38 (m, 1H, ArH), 8.11 (d, 1H, J=9.1Hz, ArH), 8.25 (d, 1H, J=7.8Hz, ArH). 13C NMR (62.9 MHz, CDCl3): δ=32.1 (CH3), 55.2 (OCH3), 60.3 (OCH2), 112.1, 112.9, 120.9, 122.1, 123.2, 124.1 (CH), 125.2, 126.6, 129.4, 130.1, 130.2, 130.9 (C), 135.5 (CH), 137.5, 158.1, 159.0 (C). HRMS (ESI): [M+H]+ found, 483.0429; calcd for C21H19BrN5O2S, 483.0425.
6-(3,4-Dimethoxyphenyl)-3-{[(6-methoxy-2-methylquinolin-4-yl)oxy]methyl}-5,6-dihydro-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole(6f).
Yield: 76%; yellow crystals, M.p. 220-222oC. IR (KBr) [υ, cm−1]: 3361 (NH), 2919 (CH), 1635 (C=N). 1H-NMR (300 MHz, CDCl3): δ=2.17 (s, 3H, CH3),3.42, 3.63, 3.76 (s, 9H, 3OCH3),4.27 (s, 1H, CH), 4.54 (s, 2H, OCH2),5.67 (s, 1H, NH), 6.70 (s, 1H, ArH), 7.10 (d, 1H, J=8.0Hz, ArH), 7.20-7.23 (m, 1H, ArH), 7.24-7.27 (m, 1H, ArH), 7.32-7.35 (m, 1H, ArH), 8.10 (d, 1H, J=8.7Hz, ArH), 8.25 (d, 1H, J=7.8Hz, ArH). 13C NMR (75.5 MHz, CDCl3): δ=32.0 (CH3), 55.0, 59.9, 60.3 (3OCH3), 62.1 (OCH2), 103.7, 111.2, 116.4, 120.1, 122.3, 124.8, 124.9 (CH), 127.0, 127.3, 127.9, 129.3 139.2, 140.8, 144.1 (C), 146.6 (CH), 148.8, 153.1, 157.9 (C). HRMS (ESI): [M+H]+ found, 465.1548; calcd for C23H24N5O4S, 465.1544.
2.3. Preparation of derivatives 7a-g
Compound 5 (1.59 g, 0.005 mol) were mixed with different substituted isothiocyanates (0.005 mol) dissolved in dry DMF (10ml) and stirred under reflux for 18-20 hours. Crushed ice was added to the reaction mixture and stirred overnight. Filtration and washing were carried out followed by recrystallization from ethanol/ether to obtain the targets 7a-g.
N-(4-chlorophenyl)-3-{[(6-methoxy-2-methylquinolin-4-yl)oxy]methyl}-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-amine7f .
Yield: 74%; M.p. 199-201oC. IR (KBr) [υ, cm−1]: 3350 (NH), 3065 (C=C aryl), 1627 (C=N). 1H NMR (300 MHz, CDCl3): δ=2.28 (s, 3H, CH3), 3.76 (s, 3H, OCH3),4.54 (s, 2H, OCH2), 6.70 (s, 1H, ArH), 7.11 (d, 1H, J=7.8Hz, ArH), 7.20-7.25 (m, 1H, ArH), 7.27-7.31 (m, 1H, ArH), 7.35 (dd, 1H, J=8.0, 3.5Hz, ArH), 7.52 (d, 1H, J= 8.1 Hz, ArH), 8.12 (d, 1H, J=7.9Hz, ArH), 8.28 (d, 1H, J=7.8Hz, ArH), 9.28 (s, 1H, NH). 13C NMR (62.9 MHz, CDCl3): δ=30.1 (CH3), 51.4 (OCH3), 60.4 (OCH2), 102.1, 117.6, 121.8, 122.8, 122.9 (CH), 123.1, 126.1, 127.4, 127.6, 128.0, 128.3 (C), 128.7 (CH), 129.6, 132.7, 136.0, 155.3 (C). HRMS (ESI): [M+H]+ found, 452.0872; calcd for C21H18ClN6O2S, 452.0867.
3-{[(6-Methoxy-2-methylquinolin-4-yl)oxy]methyl}-N-(p-tolyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-amine(7g).
Yield: 79%; M.p. 203-205oC. IR (KBr) [υ, cm−1]: 3379 (NH), 3058 (C=C aryl), 2982, 2953, 2863 (CH3 and CH2), 1637 (C=N). 1H NMR (300 MHz, CDCl3): δ=2.16, 2.41 (s, 6H, 2CH3), 3.85 (s, 3H, OCH3),4.61 (s, 2H, OCH2), 6.51 (s, 1H, ArH), 6.91-7.06 (m, 1H, ArH), 7.11 (dd, 1H, J=1.8, 8.5Hz, ArH), 7.32 (d, 1H, J=8.0Hz, ArH), 7.40 (d, 1H, J=8.0Hz, ArH), 7.61 (s, 1H, ArH), 8.30 (d, 1H, J=7.9Hz, ArH), 9.38 (s, 1H, NH). 13C NMR (75.5 MHz, CDCl3): δ=25.4, 28.7 (2CH3), 55.3 (OCH3), 60.3 (OCH2), 102.1, 116.6, 120.7, 120.9 (CH), 127.0, 127.6 (C), 128.4, 128.7 (CH), 129.2, 129.4, 129.8, 132.7, 133.6, 136.0, 141.2, 155.5 (C). HRMS (ESI): [M+H]+ found, 432.1492; calcd for C22H21N6O2S, 432.1487.
2.4. Preparation of derivatives 8a-e
In 50ml of ethanol, compound 5 (1.59 g, 0.005 mole) was added to substituted 2-bromoacetophenones (0.01 mol) and refluxed for 8-10 hours before cooling. The mixture was poured over ice and stirred overnight. To obtain 8a-e targets, the solid was washed with water and dried, then recrystallized from an ethanol/dioxane solution.
3-{[(6-Methoxy-2-methylquinolin-4-yl)oxy]methyl}-6-(p-tolyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine(8e).
Yield: 76%; M.p. 213-215oC. IR (KBr) [υ, cm−1]: 3064 (C=C aryl), 2972, 2861 (CH2), 1610 (C=N). 1H NMR (300 MHz, CDCl3): δ=2.27, 2.47 (s, 6H, 2CH3), 3.65 (s, 3H, OCH3),4.24 (s, 2H, CH2),4.60 (s, 2H, OCH2), 6.72 (s, 1H, ArH), 7.12 (d, 1H, J=8.0Hz, ArH), 7.31-7.39 (m, 1H, ArH), 7.41-7.48 (m, 1H, ArH), 7.54 (d, 1H, J= 9.7Hz, ArH), 7.81 (s, 1H, ArH), 8.13 (d, 1H, J=8.1Hz, ArH), 8.32 (d, 1H, J=7.8Hz, ArH). 13C NMR (75.5 MHz, CDCl3): δ=31.4, 32.7 (2CH3), 46.8 (CH2), 55.4 (OCH3), 62.3 (OCH2), 113.2, 116.1, 117.2, 123.3 (CH), 127.6, 128.0, 128.9 (C), 129.1, 129.3 (CH), 130.0, 133.6, 134.2, 134.8, 147.8, 152.6, 158.7 (C). HRMS (ESI): [M+H]+ found, 431.1460; calcd for C23H22N5O2S, 431.1456.
2.5. Docking Study
Toshiba Portege Z30C series Ultrabook with an Intel™ Core [email protected] GHz and Windows 10 Pro 64-bit operating system was used. ChemDraw Professional (20.0) was utilized for molecular modeling, Chem3D Ultra (20.0) for the minimization of energy, for ligand and conversion of receptor format was used OpenBabel (3.1.1), for the configuration of docking protocol was used AutoDockTools 1.5.7rc1, for the process of docking was used Autodock Vina (1.1.2), for the validation of the docking protocol was used PyMOL (2.4.1), for the preparation of docking results UCSF Chimera 1.15rc and for visualization and observation of docking results was used Discovery Studio Visualizer (20.1) [29–32]. The obtained data of three-dimensional receptor structures was obtained at www.rscb.org
Ligandʼs Preparation
The ligands test was consisted of 14 quinoline-based heterocyclic derivatives as test compounds, while the reference ligand was ampicillin. The two-dimensional structure was sketched, and energy minimization was carried out with the Merck Molecular Force Field (MMFF94) [33]. The format of optimized structure was converted from *.hin to *.pdb, using AutoDockTools 1.5.7rc1, the charge and torque of the ligands were given by default.
Receptors Preparation
Penicillin-binding proteins (PBPs) were used as receptors, which represent two types of Gram-positive and negative bacteria, consisting of S. aureus (PDB ID 3HUN) and E. coli (PDB ID 2EX6). The two receptors used to form a complex with ampicillin, which is used as the reference ligand, are consistent with the in vitro test carried out. Both receptors have a resolution of not more than 2 Å, with Ramachandran outliers not more than 0.2% [34, 35].
Docking Protocol Validation
Docking protocol validation was carried out based on the method reported by Pratama et al. [36]. PyMOL 2.4.1 was used for investigation of the root-mean-square deviation (RMSD), If an RMSD value of no more than 2 Å is attained, then the docking protocol is valid.
Molecular Docking
The docking process is carried out with a previously validated protocol. Three times replication for the docking process and ΔG is used and the limit values of the standard deviation to be 0.12 kcal/mol maximum. *.pdb format using Chimera 1.15rc was utilized for storing the ligand pose that has lowest ΔG. Discovery Studio Visualizer (19.1.0). was used in order to investigate two-dimensional analyses. Discovery Studio Visualizer (19.1.0) was used to perform two-dimensional analyses of docking results. There are two parameters observed including the difference ΔG and the similarity of amino acid interactions, both of which are compared with reference ligands. The method of determining both is carried out as reported by Pratama et al. [37].