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 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 (30 mL) 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 [25].
Z-5-(4-hydroxy-3-methoxybenzylidene) rhodanine-3-acetic acid (2a)
Yield: 75.0%. M.p.: 144 °C [25].
(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 (20 ml) 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 [26].
(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, 400 MHz 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, 100 MHz, 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, 400 MHz 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 = 8.4 Hz, J = 2.7 Hz ArH), 4.72 (s, 2H, CH2-CO), 3.84 (s, 3H, OCH3), 3.05 (t, 4H, J = 7.2 Hz Morpholine H), 1.19 (t, 4H, J = 7.2 Hz Morpholine H), 13CNMR (DMSO-d6, 100 MHz, 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, 400 MHz 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 = 8.4 Hz, J = 2.7 Hz), 7.29–7.48 (m, 4H, ArH), 5.52 (s, 2H, CH2-CO), 3.82 (s, 3H, OCH3), 13CNMR (DMSO-d6, 100 MHz, 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, 400 MHz 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 = 8.4 Hz, J = 2.7 Hz, ArH), 7.29–7.48 (m, 5H, ArH), 4.75 (s, 2H, CH2-CO), 3.83 (s, 3H, OCH3), 13CNMR (DMSO-d6, 100 MHz, 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, 400 MHz 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, 100 MHz, 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, 400 MHz δ ppm) 7.85 (s, 1H, vinylic H), 7.64 (d, 2H, J = 8.7 Hz ArH), 7.13 (d, 2H, J = 8.7 Hz ArH), 4.73 (s, 2H, CH2-CO), 3.84 (s, 3H, OCH3), 3.79 (t, 4H, J = 7.5 Hz Morpholine H), 1.19 (t, 4H, J = 7.5 Hz Morpholine H), 13CNMR (DMSO-d6, 100 MHz, 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 (3 g)
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, 400 MHz 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 = 8.4 Hz, J = 2.7 Hz, ArH), 4.72 (s, 2H, CH2-CO), 3.83 (d, 3H, OCH3), 3.04 (m, 4H, Pyrrolidine H),1.19 (t, 4H, J = 7.5 Hz, Pyrrolidine H), 13CNMR (DMSO-d6, 100 MHz, 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 ELIZA 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 [27].
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 [28].
ALR1 Enzyme inhibition assay
The assay was performed on ELIZA (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 [29].
ALR2 Enzyme inhibition assay
The assay was performed on ELIZA (Bio-Tek ELx800™ 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 [30].
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