Materials and methods
All the reagents were purchased from Sigma Aldrich and Alfa Aesar and used without further purification. Melting points of the synthesized compounds were recorded using Gallenkamp melting point apparatus. Characterization of the synthesized compounds was done by FTIR and 1HNMR and elemental analysis data. FTIR spectra were recorded on Thermoscientific NICOLET IS10 spectrophotometer, and 1HNMR spectra were taken on Bruker AM300 MHz spectrophotometer, in which DMSO was used as solvent. The progress of reaction was monitored by TLC with pre-coated silica gel 60 F254 plates using ethyl acetate and petroleum ether as mobile phase.
General procedure for the synthesis of Rhodanine-3-acetic acid (1)
Glycine (2.4 g, 0.031 mol) was dissolved in 33% NH4OH (20 mL), carbon disulfide (2.36 g, 0.031mol) was added to the solution and stirred vigorously for 1 hour while color of the solution turned orange. Then aqueous solution of sodium chloroacetate (3.61 g, 0.031 mol) was added and refluxed for 3 hours, after completion, reaction mixture was acidified with dilute HCl to bring the pH to 1.0 and further refluxed for 1 h. Saturated NaHCO3 solution was added to the reaction mixture to neutralize it and the resultant solution was acidified again with dilute HCl. The solid separated was filtered and recrystallized with water to obtain rhodanine-3-acetic acid. Yield: 86.0%. M.p.: 145–148°C [27].
General procedure for the synthesis of 5-benzylidenerhodanine-3-acetic acid 2(a-b)
Equimolar amounts of rhodanine-3-acetic acid, anhydrous sodium acetate and respective aldehyde were dissolved in glacial acetic acid (30mL) and solution was put to reflux for 3–4 hours. After completion the reaction mixture was cooled and the solid separated was filtered, washed with water and recrystallized from ethanol [28].
Z-5-(4-hydroxy-3-methoxybenzylidene) rhodanine-3-acetic acid (2a)
Yield: 75.0%. M.p.: 144°C [28].
(Z)-5-(4-methoxybenzylidene) rhodanine-3-acetic acid (2b)
Yield: 66.0%. M.p.: 249–250°C
General procedure for the synthesis of 5-benzylidenerhodanine-3-acetamide derivatives 3(a-g)
The synthesized 5-benzylidenerhodanine-3-acetic acid 2(a-b) was stirred with excess of thionyl chloride in dichloromethane (20ml) for 2 hours. After reaction completion solvent was evaporated and residue treated with equimolar amount of respective amine in the presence of triethylamine and dichloromethane as solvent. Progress of reaction was monitored by TLC, after completion product was isolated by evaporation and purified by column chromatography [29].
(Z)-5-(4-hydroxy-3-methoxybenzylidene)-3-(2-anilino-2-oxoethyl)-2-thioxothiazolidin-4-one (3a)
Yield 53.5%, Orange solid, m.p 201-202 ℃, Rf 0.42 (ethyl acetate: Pet-ether 2:1), IR (KBR) cm-1, 1635 (C=O), 1201 (C=S), 3440 (NH), 1020 (C-N), 3150 (OH), 1489 (-CH3), 1H-NMR (DMSO, 400MHz δ ppm) 9.98 (s, 1H, OH), 7.51 (s, 1H, vinylic H) 7.15-7.46 (m, 7H, ArH), 7.01 (d, 1H, J= 8.7 Hz, ArH), 6.87 (d, 2H, J= 8.7 Hz, Ar-H) 5.52 (s, 2H, CH2-CO) and 3.65 (s, 3H, OCH3), 13CNMR (DMSO-d6, 100MHz, δ ppm): 149.7, 146.2, 134.3, 126.8, 116.7, 109.2, 138.4, 129.7, 129.7, 125.1, 119.9, 119.9 (Ar-C), 194.3, 167.2, 130.2 (thiazolidine-C), 162.6 (CONH), 133.7 (CH), 57.1 (OCH3), 48.8 (CH2), Anal. Calcd. For C19H16N2O4S2: C, 56.93; H, 4.02; N, 6.99; S, 15.98; Found: C, 56.84; H, 3.99; N, 7.00; S, 15.99.
(Z)-5-(4-hydroxy-3-methoxybenzylidene)-3-(2-morpholino-2-oxoethyl)-2-thioxothiazolidin-4-one (3b)
Yield 61.5%, yellow solid, m.p 211-213 ℃, Rf 0.41 (ethyl acetate: Pet-ether 2:1), IR (KBR) cm-1, 1615 (C=O), 1300 (C=S), 1104 (C-N), 1192 (C-O), 3384 (OH), 1450 (-CH3), 1H-NMR (DMSO, 400MHz δ ppm) 9.76 (s, 1H, OH), 7.80 (s, 1H, vinylic H), 7.37-7.43 (m, 1H, ArH), 7.14-7.22 (m, 1H, ArH), 7.02 (d,d 1H, J= 8.4Hz, J= 2.7Hz ArH), 4.72 (s, 2H, CH2-CO), 3.84 (s, 3H, OCH3), 3.05 (t, 4H, J= 7.2Hz Morpholine H), 1.19 (t, 4H, J= 7.2Hz Morpholine H), 13CNMR (DMSO-d6, 100MHz, δ ppm): 150.1, 148.7, 136.8, 129.3, 117.1, 110.3 (Ar-C), 192.3, 166.1, 128.8 (thiazolidine-C), 70.1, 70.1, 45.3, 45.3 (Morpholine-C), 163.9 (CO), 132.1 (CH), 57.2 (OCH3), 49.2 (CH2), Anal. Calcd. For C17H18N2O5S2: C, 51.71; H, 4.59; N, 7.09; S, 16.22; Found: C, 51.69; H, 4.61; N, 7.10; S,16.20.
2-Hydroxy-4-({[(Z)-5-(4-hydroxy-3-methoxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]acetyl}amino)benzoic acid (3c)
Yield 59.2%, yellow solid, m.p 199-201℃, Rf 0.48 (ethyl acetate: Pet-ether 2:1), IR (KBR) cm-1, 1610 (C=O), 1300 (C=S),1272 (C-N), 3130 (OH), 1370 (-CH3), 1720 (COOH) 1H-NMR (DMSO, 400MHz δ ppm) 12.0 (s, 1H, COOH), 9.75 (s, 1H, OH), 9.35 (d, 1H, OH),7.75 (s, 1H, vinylic H), 7.41 (m, 1H, ArH), 7.19 (m, 1H, ArH), 7.05 (d,d 1H, J= 8.4Hz, J= 2.7Hz), 7.29-7.48 (m, 4H, ArH), 5.52 (s, 2H, CH2-CO), 3.82 (s, 3H, OCH3), 13CNMR (DMSO-d6, 100MHz, δ ppm): 150.2, 149.7, 136.2, 131.3, 118.2, 109.5, 167.1, 141.4, 133.7, 119.7, 101.2, 100.1 (Ar-C), 197.2, 171.6, 130.3 (thiazolidine-C), 174.6 (COOH) 166.2 (CONH), 133.1 (CH), 57.0 (OCH3), 48.3 (CH2), Anal. Calcd. For C20H16N2O7S2: C, 52.12; H, 3.50; N, 6.08; S, 13.98; Found: C, 52.00; H, 3.55; N, 6.11; S, 14.01.
4-({[(Z)-5-(4-hydroxy-3-methoxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]acetyl}amino)benzoic acid (3d)
Yield 84.5%, orange solid, m.p 217-219 ℃, Rf 0.45(ethyl acetate: Pet-ether 2:1), IR (KBR) cm-1, 1622 (C=O), 1299 (C=S), 1050 (C-N), 3330 (OH), 1368 (-CH3), 1704 (COOH) 1H-NMR (DMSO, 400MHz δ ppm), 12.0 (s, 1H, CO-OH), 9.75 (s, 1H, OH) 7.71 (s, 1H, vinylic H), 7.41 (m, 1H, ArH), 7.19 (m, 1H, ArH), 7.05 (d,d 1H, J= 8.4Hz, J= 2.7Hz, ArH), 7.29-7.48 (m, 5H, ArH), 4.75 (s, 2H, CH2-CO), 3.83 (s, 3H, OCH3), 13CNMR (DMSO-d6, 100MHz, δ ppm): 148.5, 147.3, 133.2, 128.6, 115.3, 108.8, 137.9, 130.4, 130.4, 123.2, 119.7, 119.7 (Ar-C), 193.3, 166.5, 129.4 (thiazolidine-C), 168.8 (COOH) 163.5 (CONH), 132.7 (CH), 56.4 (OCH3), 49.6 (CH2), Anal. Calcd. For C20H16N2O6S2: C, 53.99; H, 3.62; N, 6.29; S, 14.39; Found: C, 54.02; H, 3.60; N, 6.31; S, 14.33.
(Z) 5-(4-methoxybenzylidene)-3-(2-anilino-2-oxoethyl)-2-thioxothiazolidin-4-one (3e)
Yield 60%, yellow solid m.p 190-192℃ Rf 0.44 (ethyl acetate: Pet-ether 2:1), IR (KBR) cm-1, 1620 (C=O), 1216 (C=S), 3368 (NH), 1022 (C-N), 1375 (-CH3), 1H-NMR (DMSO, 400MHz δ ppm) 7.56 (s, 1H, vinylic H) 7.29-7.48 (m, 5H, ArH), 7.10 (d, 2H, J= 8.7 Hz, ArH), 6.85 (d, 2H, J= 8.7 Hz, ArH), 5.63 (s, 2H, CH2-CO) and 3.71 (s, 3H, OCH3), 13CNMR (DMSO-d6, 100MHz, δ ppm): 160.0, 132.3, 132.3, 125.8, 114.5, 114.5, 137.6, 129.2, 129.2, 124.6, 120.9, 120.9 (Ar-C), 194.2, 167.7, 129.8 (thiazolidine-C), 164.2 (CONH), 133.3 (CH), 55.7 (OCH3), 50.0 (CH2), Anal. Calcd. For C19H16N2O3S2: C, 59.30; H, 4.19; N, 7.28; S, 16.64; Found: C, 59.28; H, 4.20; N, 7.29; S, 16.61.
(Z) 5-(4-methoxybenzylidene)-3-(2-morpholino-2-oxoethyl)-2-thioxothiazolidin-4-one (3f)
Yield 49.3%, yellow solid m.p 203-205 ℃ Rf 0.48 (ethyl acetate: Pet-ether 2:1), IR (KBR) cm-1, 1650 (C=O), 1225 (C=S), 1101 (C-N), 1173 (C-O), 1375 (-CH3), 1H-NMR (DMSO, 400MHz δ ppm) 7.85 (s, 1H, vinylic H), 7.64 (d, 2H, J= 8.7Hz ArH), 7.13 (d, 2H, J= 8.7Hz ArH), 4.73 (s, 2H, CH2-CO), 3.84 (s, 3H, OCH3), 3.79 (t, 4H, J= 7.5Hz Morpholine H), 1.19 (t, 4H, J= 7.5Hz Morpholine H), 13CNMR (DMSO-d6, 100MHz, δ ppm): 161.7, 133.3, 133.3, 127.2, 117.4, 117.4 (Ar-C), 190.8, 168.0, 128.3 (thiazolidine-C), 66.7, 66.7, 44.3 44.3 (Morpholine-C) 166.3 (CO), 134.9 (CH), 57.6 (OCH3), 45.2 (CH2), Anal. Calcd. For C17H18N2O4S2: C, 53.90; H, 4.79; N, 7.39; S, 16.91; Found: C, 53.87; H, 4.81; N, 7.37; S, 16.88.
(Z) 5-(4-hydroxy-3-methoxybenzylidene)-3-(2-pyrrolidino-2-oxoethyl)-2-thioxothiazolidin-4-one (3g)
Yield 39.2%, yellow solid, m.p 187-189 ℃, Rf 0.31 (ethyl acetate: Pet-ether 2:1), IR (KBR) cm-1, 1640 (C=O), 1215 (C=S),1260 (C-N), 3201 (OH), 1451 (-CH3), 1H-NMR (DMSO, 400MHz δ ppm) 9.76 (s, 1H, OH), 7.79 (s, 1H, vinylic H), 7.40 (m, 1H, ArH), 7.17 (m, 1H, ArH), 7.00 (d,d 1H, J= 8.4Hz, J= 2.7Hz, ArH), 4.72 (s, 2H, CH2-CO), 3.83 (d, 3H, OCH3), 3.04 (m, 4H, Pyrrolidine H),1.19 (t, 4H, J= 7.5Hz, Pyrrolidine H), 13CNMR (DMSO-d6, 100MHz, δ ppm): 159.8, 133.7, 133.7, 124.2, 115.1, 115.1 (Ar-C), 192.7, 165.3, 127.5 (thiazolidine-C), 48.5, 48.5, 23.7, 23.7 (pyrrolidine-C) 169.0 (CO), 134.7 (CH), 58.2 (OCH3), 51.3 (CH2), Anal. Calcd. For C17H18N2O3S2: C, 56.28; H, 5.00; N, 7.72; S, 17.65; Found: C, 56.31; H, 4.99; N, 7.70; S, 17.67.
Enzyme inhibition studies
All required chemicals used in the enzyme extraction procedure were of high analytical grade. Enzyme inhibitory assay was performed on ELISA microplate reader at 340 nm and 96 well-plates were used for the sample analysis. Micropipettes from Gilson were used for sample loading. Sodium-D-glucoronate and D,L-glyceraldehyde were used as substrates along with a cofactor i.e. NADPH (nicotinamide adenine dinucleotide phosphate) from Sigma Aldrich.
Extraction and purification of Aldehyde reductase (ALR1)
Aldehyde reductase enzyme was extracted from lamb kidney and the cortical part was separated carefully. The cortex was homogenized in triple volume of extraction buffer (2.0 mM EDTA, 0.25 M sucrose, 10 mM sodium phosphate and 2.5 mM β-mercaptoethanol at pH 7.2). The homogenate was centrifuged at 12,000 rpm at 4 °C for 30 min, the insoluble precipitates were discarded and the supernatant was saturated with 40%, 50% and 75% ammonium sulfate respectively and after each addition the solution was centrifuged at 12000 rpm at 4°C for 30 min, each time the pallet was discarded and for the last saturation the supernatant was taken and dialyzed overnight in extraction buffer. Next day the protein content was calculated via Bradford method and the crude aldehyde reductase was stored at -80°C [30].
Extraction and Purification of Aldose reductase (ALR2)
The enzyme aldose reductase was extracted from calf lenses. 200-300 g lenses were added to triple volume of cold water and homogenized for 20 min. The homogenate was then centrifuged at 10,000 rpm for 15 min at 4°C. The insoluble precipitates were discarded and the supernatant was saturated with 70% ammonium sulfate and after centrifugation at 10,000 rpm at 4°C for 15 min the supernatant was dialyzed overnight and the protein contents was calculated via Bradford method and the crude aldose reductase was stored at -80°C [31].
ALR1 Enzyme inhibition assay
The assay was performed on ELISA (Bio-Tek ELx800TM Instrument, Inc. USA) based spectrophotometric analysis in 96 well plate. The assay mixture was composed of 20 µL buffer (100 mM potassium dihydrogen phosphate pH 6.2), 10 µL test compound (1 mM), 70 µL enzyme and incubated for 10 min at 37°C followed by addition of 40 µL Glucoronate 50 mM (as a substrate) and 50 µL (0.5 mM) NADPH (nicotinamide adenine dinucleotide phosphate) as a co-factor. After 30 min incubation optical density was measured at 340 nm. Valproic acid was used as a positive control for ALR1 [32].
ALR2 Enzyme inhibition assay
The assay was performed on ELISA (Bio-Tek ELx800TM Instrument, Inc. USA) based spectrophotometric analysis in 96 well plate. The assay mixture was composed of 20 µL buffer (100 mM potassium dihydrogen phosphate pH 6.2), 10 µL test compound (1 mM), 70 µL enzyme and incubated for 10 min at 37°C followed by addition of 40 µL substrate (DL-glyceraldehyde 50 mM for ALR2) and 50 µL NADPH (0.5 mM) (nicotinamide adenine dinucleotide phosphate) as a co-factor. After 30 min incubation optical density was measured at 340 nm. Sulindac was used as positive control for ALR2 [33].
Results were analyzed by graph pad prism® software to calculate IC50 and percentage inhibition was calculated by the following formula
% Inhibition= [100-(Absorbance test well/Absorbance control)] x 100
Molecular Docking studies
For docking studies, crystal structures of human aldose reductase (PDB ID: 1US0) [33] and aldehyde reductase (PDB ID: 3FX4) [25] were used. Structures of the tested compounds were drawn by MOE builder tool [34] and optimization was achieved using MMFF94x forcefield [35]. Afterwards the energy minimization of the target proteins was carried out by Molecular Operating Environment [36]. LeadIT (BioSolveIT GmbH, Germany) [37] was used to perform docking analysis of the prepared ligands inside the respective receptors. Load Receptor Utility of the LeadIT software was used to load the receptors. Active pocket of the proteins for docking analysis was identified by keeping the amino acid residues in 10.0 Å radius and keeping co-factor (NADPH) within ALR1 and ALR2. Values of the amino acid flips and water handling were kept as by default. Once docking analysis was completed, the possible interactions of ligands with receptor proteins were inspected for studying the possible interactions using HYDE assessment [37]. Discovery Studio Visualizer was used to perform visualize the interactions of ligand and receptors [38].
Molecular Dynamics Simulations
All MD simulations were carried out using the PMEMD (Particle Mesh Ewald Molecular Dynamics) module of AMBER 18 simulation package [39]. The crystallographic structures of ALR1 (3FX4) and ALR2 (1US0) were downloaded from RCSB protein databank, the protein structures were protonated by using the H++ webserver [40]. Each complex system was solvated in a cubic box of water molecules. The ff14SB4 [41] force filed was used for proteins while for ligands the second-generation of the General Amber Force Filed (GAFF2) [42] with AM1-BCC charges and TIP3P [43] for water molecules. The GAFF parameters and coordinates were generated by using the antechamber [44] and xleap modules of AMBER 18 simulation package. To neutralize the total charge of each system only sodium as a counter ion was added with parameters from Joung and Cheatham [45]. For post-simulation inspection, VMD was used [46].
For equilibration each system was minimized with 500 steps of steepest decent minimization with 2.0 kcalmol-1 restrains on the heavy atoms of protein. Then the systems were heated for 100 ps from 10 to 300 K temperature using 2.0 kcalmol-1 as a restraint on protein in NVT ensemble with Langevin thermostat [47] and collision frequency is 5 ps-1. After that 20 ns MD simulation was carried out using Berendsen barostat [48] and time constant was 2 ps in NPT ensemble. Moreover 100 ps equilibration was done in NPT ensemble without any restrain. For data collection 20 ns MD simulation have been carried out in NPT ensemble without restrain with time step of 2 fs. The temperature and pressure during these MD simulations is 300 K and 1 bar respectively. Furthermore, SHAKE protocol was used involving all hydrogen bonded atoms, 10 Å non-bonded cut-off and particle mesh Ewald (PME) methodology [49] to calculate the long-range electrostatic interactions with periodic boundary condition was used. Finally, the trajectories of these MD simulations were analyzed using CPPTRAJ [50] module of AMBER.