Materials and methods
All chemicals and enzymes (α-amylase and α-glucosidase enzyme) used in this study were purchase from Sigma Aldrich. Avance Bruker 500 MHz has been used for carrying out 1H-NMR and 13C–NMR. FTIR (PerkinElmer) was employed to study the functional groups of the compounds. HR-MS were determined on Agilent 6330 Ion Trap using positive/negative mode. Elemental analysis was performed using PerkinElmer instrument. Melting point was recorded on Stuart (SMP-10) melting point apparatus. Pre-coated silica gel aluminum foils (Germany) were utilized for execution of thin layer chromatography (TLC). UV lamp was used for visualizing chromatograms.
α-Amylase inhibitory activity
The inhibition of α-amylase was established by the methods described earlier [21, 22]. Incubation of 500 µL of test sample (1–100 µg/mL) along with 500 µL of α-amylase (0.5 mg/mL in phosphate buffer; 0.2 mM maintained at pH 6.9) was carried out for 10 min at 25 °C. After pre-incubation, 1% starch solution was added (500 μL, in 0.2 mM phosphate buffer maintained at pH 6.9) and incubated at 25 °C for another 10 min. The reaction was brought to arrest by using 1 mL of di-nitro-salicylic acid color reagent. The tubes were afterwards refluxed for 5 min and then cooled at ambient temperature. Resulting solutions, after diluted with 10 mL of distilled water, were analyzed at 540 nm by recording absorbance [23]. Acarbose was used as the standard drug.
The percentage of inhibition was estimated by employing the formula;
$$ \%_{\text{Inhibition}} = \left( {{\text{A}}_{\text{Control}} - {\text{A}}_{\text{Sample}} } \right)/{\text{A}}_{\text{Control}} \times 100. $$
The optimum concentration needed to hydrolyze the α-amylase by 50% (IC50 values) was computed via non-linear regression plot of % inhibition at x axis and concentrations at y axis, with the help of Graph Pad Prism Software (Ver. 5).
α-Glucosidase activity
The inhibition of α-glucosidase was established with the help of the modified version of the published technique [24]. α-glucosidase solution was prepared by dissolving 1 mg in 100 mL phosphate buffer (pH 6.8) comprising of 200 mg bovine serum albumin. 10 μL of sample at variable concentrations (1 to 100 μg/mL) was pre-mixed with 490 μL phosphate buffer (pH 6.8), and to this reaction mixture was added 250 μL of 5 mM p-nitrophenyl α-d-glucopyranoside and preincubated at 37 °C. After 5 min, addition of 250 μL α-glucosidase (0.15 unit/mL) was done with further incubation at 37 °C for 15 min. The inhibition was concluded by adding 2000 μL of Na2CO3 (200 mM). α-Glucosidase activity was computed at 400 nm on Shimadzu 265 UV–Vis spectrophotometer (Japan) by computing the amount of p-nitrophenol released from p-NPG, using acarbose as positive control. The optimum concentration needed to hydrolyze 50% of α-glucosidase was defined as the IC50 value.
The percentage of inhibition was estimated by employing the formula;
$$ \%_{\text{Inhibition}} = \left( {{\text{A}}_{\text{Control}} - {\text{A}}_{\text{Sample}} } \right)/{\text{A}}_{\text{Control}} \times 100. $$
Statistical analysis
The concentration required to inhibit the α-amylase by 50% (IC50 values) were computed via non-linear regression plot between percentage inhibition at the x axis and concentrations at y axis, with the help of Graph Pad Prism Software (Ver. 5).
Computational docking methodology
Computational docking studies of α-amylase and α-glucosidase
With the aim of revealing the binding properties of the all the 5-amino-nicotinic acid derivatives in α-amylase (pdb id: 4w93) [25] and in α-glucosidase (pdb id: 3top) [26], molecular docking studies were done using CDOCKER implemented in Discovery studio.CDOCKER is a grid-based method of molecular docking that make use of CHARMM force field [27]. The Montbretin A binding site in α-amylase crystal structure was defined as binding site for docking the compounds. Similarly the acarbose binding site in α-glucosidase crystal structure was defied as the binding site to dock the compounds. Prior to docking both enzymes and the 5-amino-nicotinic acid derivatives were structurally optimized by adding hydrogen and atom valencies were satisfied so that atoms are properly typed. After the binding site sphere was defined, docking calculation was subsequently done. Top 10 binding pose were opted for prediction and results were analysed using Discovery studio visualizer.
General procedure for synthesis of 5-amino-nicotinic acid derivatives
1 mmol of 5-amino-nicotinic acid was weighed and transfer into 50 mL round bottomed flask, then 1.2 mmol of phenyl isothiocyanate (Table 1) was added, followed by the addition of 10 mL of chloroform. The reaction mixture was left overnight with stirring and was monitored by TLC. After the reaction was completed, the product was transferred into beakers and evaporated at room temperature. Diethyl ether was used to wash the solid product.
5-(3-Phenylthioureido)pyridine-3-carboxylic acid (1)
Yield: 76%; M.p.: > 300 °C; FTIR (ATR, cm−1): 3332 (N–H), 3190 (Ar–CH), 1654 (C=O), 1586 (C–N), 1488 (C=C),1330 (C=S); 1HNMR (500 MHz, DMSO-d6): δ 12.16 (s, 1H, NH), 11.50 (s, 1H, NH), 8.86 (s, 1H, H-2), 8.62 (s, 1H, H-6), 7.69 (s, 1H, H-4), 7.06–6.94 (m, 5H, H-2′to H-5′), 3.60 (br. s, 1H, OH); 13CNMR (125 MHz, DMSO-d6): δ 179.6 (C=S), 169.0 (C=O), 141.2 (C2), 140.5 (C6), 137.0 (C1′), 134.2 (C5), 129.0 (C3′) 129.0 (C5′), 128.4 (C3), 126.4 (C2′), 126.4 (C6′), 124.3 (C4′), 122.8 (C4); HR-MS for C13H11N3O2S calculated 273.0572 and found 273.0549; Anal. calcd. for C13H11N3O2S: C, 57.13; H, 4.06; N, 15.37;O, 11.71; S, 11.73; found: C, 57.11; H, 4.05; N, 15.36; O, 11.70; S, 11.72.
5-(3-(4-Chlorophenyl)thioureido)pyridine-3-carboxylic acid (2)
Yield: 72%; M.p.: > 300 °C; FTIR (ATR, cm−1): 3334 (N–H), 3191 (Ar–CH), 1655 (C=O), 1587 (C–N), 1469 (C=C), 1331(C=S), 785(C–Cl); 1HNMR (500 MHz, DMSO-d6): δ 11.50 (s, 1H, NH), 11.30 (s, 1H, NH), 8.85 (s, 1H, H-2), 8.60 (s, 1H, H-6), 7.62 (s, 1H, H-4), 7.03 (d, J = 8.0 Hz, 2H, H-2′, H-6′), 6.40 (d, J = 8.0 Hz, H-3′, H-5′), 3.58 (br. s, 1H, OH); 13CNMR (125 MHz, DMSO-d6): δ 179.5 (C=S), 169.0 (C=O), 141.1 (C2), 140.0 (C6), 135.0 (C1′), 134.3 (C5), 130.1(C4′), 129.0 (C3′), 129.0 (C5′), 128.8 (C3), 127.5 (C 2′), 127.5 (C6′), 123.3 (C4); HR-MS for C13H10ClN3O2S calculated 307.0182 and found 307.0171; Anal. calcd. for C13H10ClN3O2S: C, 50.73; H, 3.28; N, 13.65; O, 10.40; S, 10.42. Found: C, 50.72; H, 3.26; N, 13.64; O, 10.39; S, 10.41.
5-(3-p-Tolylthioureido)pyridine-3-carboxylic acid (3)
Yield: 79%; M.p.: > 300 °C; FTIR (ATR, cm−1): 3334 (N–H), 3191 (Ar–CH), 1655 (C=O), 1586 (C–N), 1468 (C=C), 1331 (C=S). HR-MS; 1HNMR (500 MHz, DMSO-d6): δ 11.80 (s, 1H, NH), 11.10 (s, 1H, NH), 8.87 (s, 1H, H-2), 8.61(s, 1H, H-6), 7.61 (s, 1H, H-4), 6.80 (d, J = 8.5 Hz, 2H, H-3′, H-5′), 6.32 (d, J = 8.5 Hz, 2H, H-2′, H-6′), 3.62 (br. s, 1H, OH), 2.46 (s, 3H, CH3); 13CNMR (125 MHz, DMSO-d6): δ 179.3 (C=S), 169.3 (C=O), 141.2 (C2), 140.2 (C6), 134.3 (C4′), 134.2 (C1′), 134.0 (C5), 129.2 (C3′), 129.2 (C5′), 128.5 (C3), 127.0 (C2′), 127.0 (C6′), 123.1 (C4), 24.2 (CH3); for C14H13N3O2S calculated 287.0728 and found 287.0717; Anal. calcd. for C14H13N3O2S: C, 58.52; H, 4.56; N, 14.62; O, 11.14; S, 11.16 found: C, 58.50; H, 4.55; N, 14.60; O, 11.13; S, 11.15.
5-(3-(4-Fluorophenyl)thioureido)pyridine-3-carboxylic acid (4)
Yield: 75%; M.p.: > 300 °C; FTIR (ATR, cm−1): 3334 (N–H), 3191 (Ar–CH), 1655 (C=O), 1586 (C–N), 1469(C=C), 1331 (C=S), 785 (C-F); 1HNMR (500 MHz, DMSO-d6): δ 11.50 (s, 1H, NH), 11.15 (s, 1H, NH), 8.86 (s, 1H, H-2), 8.62 (s, 1H, H-6), 7.62 (s, 1H, H-4), 6.72 (d, J = 7.5 Hz, 2H, H-3′, H-5′), 6.40 (t, J = 8.5 Hz, 2H, H-2′, H-6′), 3.62 (br. s, 1H, OH); 13CNMR (125 MHz, DMSO-d6): δ 179.7 (C=S), 169.0 (C=O), 158.5 (C4′), 140.5 (C2), 140.0 (C6), 134.3 (C5), 132.1 (C1′), 128.8 (C2′), 128.8 (C6′), 128.3 (C3), 115.6 (C3′), 115.6 (C5′), 123.2 (C4); HR-MS for C13H10FN3O2S calculated 291.0478 and found 291.0463; Anal. calcd. for C13H10FN3O2S: C, 53.60; H, 3.46; N, 14.42; O, 10.98; S, 11.01; found: C, 53.59; H, 3.44; N, 14.41; O, 10.97; S, 11.01.
5-(3-(4-Nitrophenyl) thioureido) pyridine-3-carboxylic acid (5)
Yield: 69%; M.p.: > 300 °C; FTIR (ATR, cm−1): 3333 (N–H), 3190 (Ar–CH), 1654 (C=O), 1586 (C–N), 1468(C=C), 1330 (C=S);1HNMR (500 MHz, DMSO-d6): δ11.42 (s, 1H, NH), 11.02 (s, 1H, NH), 8.86 (s, 1H, H-2), 8.60 (s, 1H, H-6), 7.90 (d, J = 8.0 Hz, 2H, H-3′, H-5′), 7.58 (s, 1H, H-4), 6.68 (d, J = 8.0 Hz, 2H, H-2′, H-6′), 3.60 (br. s, 1H, OH); 13CNMR (125 MHz, DMSO-d6): δ 179.6 (C=S), 169.2 (C=O), 144.5 (C4′), 143.4 (C1′), 140.5 (C2), 140.0 (C6), 133.9 (C5), 128.9 (C3), 127.2 (C2′), 127.2 (C6′), 121.2 (C3′), 121.2 (C5′), 123.1 (C4);HR-MS for C13H10N4O4S calculated 318.0423 and found 318.0416; Anal. calcd. for C13H10N4O4S: C, 49.05; H, 3.17; N, 17.60; O, 20.11; S, 10.07; found: C, 49.03; H, 3.16; N, 17.58; O, 20.10; S, 10.06.
5-(3-(4-Methoxyphenyl) thioureido) pyridine-3-carboxylic acid (6)
Yield: 77%; M.p.: > 300 °C; FTIR (ATR, cm−1): 3333 (N–H), 3190 (Ar–CH), 1654 (C=O), 1586 (C–N), 1468(C=C), 1330(C=S),1089(Ar–O–C); 1HNMR (500 MHz, DMSO-d6): δ 11.42 (s, 1H, NH), 11.05 (s, 1H, NH), 8.86 (s, 1H, H-2), 8.60 (s, 1H, H-6), 7.59 (s, 1H, H-4), 6.52 (d, J = 8.0 Hz, 2H, H-3′, H-5′), 6.37 (d, J = 8.0 Hz, 2H, H-2′, H-6′), 3.80 (s, 3H, OCH3) 3.60 (br. s, 1H, OH); 13CNMR (125 MHz, DMSO-d6): δ 179.6 (C=S), 169.0 (C=O), 156.4 (C4′), 140.5 (C2), 140.0 (C6), 134.2 (C5), 129.2 (C1′), 128.9 (C3), 127.3 (C2′), 127.3 (C6′), 123.1 (C4), 114.2 (C3′), 114.2 (C5′), 55.6 (CH3); HR-MS for C14H13N3O3S calculated 303.0678 and found 303.0667; Anal. calcd. for C14H13N3O3S: C, 55.43; H, 4.32; N, 13.85; O, 15.82; S, 10.57; found: C, 55.42; H, 4.31; N, 13.83; O, 15.81; S, 10.56.
5-(3-(4-Bromophenyl) thioureido) pyridine-3-carboxylic acid (7)
Yield: 79%; M.p.: > 300 °C; FTIR (ATR, cm−1): 3333 (N–H), 3190 (Ar–CH), 1654 (C=O), 1586 (C–N), 1468 (C=C), 1331(C=S) 785(C–Br);1HNMR (500 MHz, DMSO-d6): δ11.70 (s, 1H, NH), 11.30 (s, 1H, NH), 8.80 (s, 1H, H-2), 8.62 (s, 1H, H-6), 7.70 (s, 1H, H-4), 7.15 (d, J = 8.0 Hz, 2H, H-3′, H-5′), 6.38 (d, J = 8.0 Hz, 2H, H-2′, H-6′), 3.64 (br. s, 1H, OH); 13CNMR (125 MHz, DMSO-d6): δ 179.6 (C=S), 169.3 (C=O), 140.3 (C2), 140.1 (C6), 136.0 (C1′), 134.2 (C5), 132.1 (C3′), 132.1 (C5′), 128.6 (C3), 128.5 (C2′), 128.5 (C6′), 123.1 (C4), 119.2 (C4′); HR-MSfor C13H10BrN3O2S calculated 350.9677 and found 350.9653; Anal. calcd. for C13H10BrN3O2S: C, 44.33; H, 2.86; N, 11.93; O, 9.09; S, 9.10; found: C, 44.32; H, 2.84; N, 11.92; O, 9.08; S, 9.08.
5-(3-(4-(Trifluoromethyl)phenyl) thioureido) pyridine-3-carboxylic acid (8)
Yield: 72%; M.p.: > 300 °C; FTIR (ATR, cm−1): 3333 (N–H), 3190 (Ar–CH), 1654 (C=O), 1586 (C–N), 1468(C=C), 1330(C=S),785(C-F); 1HNMR (500 MHz, DMSO-d6): δ 11.62 (s, 1H, NH), 11.32 (s, 1H, NH), 8.72 (s, 1H, H-2), 8.65 (s, 1H, H-6), 7.67 (s, 1H, H-4), 7.15 (d, J = 8.0 Hz, 2H, H-3′, H-5′), 6.38 (d, J = 8.0 Hz, 2H, H-2′, H-6′), 3.68 (br. s, 1H, OH); 13CNMR (125 MHz, DMSO-d6): δ 179.6 (C=S), 169.3 (C=O), 140.6 (C2), 140.1 (C6), 140.0 (C1′), 134.2 (C5), 128.8 (C3), 127.3 (CF3), 126.7 (C2′), 126.7 (C6′), 125.5 (C3′), 125.5 (C5′), 124.1 (C4′), 123.1 (C4); HR-MS for C14H10F3N3O2S calculated 341.0446 and found 341.0432; Anal. calcd. for C14H10F3N3O2S: C, 49.27; H, 2.95; F, 16.70; N, 12.31; O, 9.38; S, 9.39; found: C, 49.26; H, 2.93; F, 16.69; N, 12.30; O, 9.37; S, 9.37.
5-(3-(3-Chloro-4-methylphenyl) thioureido) pyridine-3-carboxylic acid (9)
Yield: 68%; M.p.: > 300 °C; FTIR (ATR, cm−1): 3333 (N–H), 3190 (Ar–CH), 1654 (C=O), 1586 (C–N), 1468(C=C), 1330(C=S), 785(C–Cl); 1HNMR (500 MHz, DMSO-d6): δ 11.43 (s, 1H, NH), 11.23 (s, 1H, NH), 8.72 (s, 1H, H-2), 8.69 (s, 1H, H-6), 7.61 (s, 1H, H-4), 6.74 (d, J = 7.5 Hz, 1H, H-5′), 6.37 (s, 1H, H-2′), 6.21 (d, J = 8.0 Hz, 1H, H-6′), 3.64 (br. s, 1H, OH), 2.38 (s, 3H, CH3); 13CNMR (125 MHz, DMSO-d6): δ 179.8 (C=S), 169.5 (C=O), 140.5 (C2), 140.2 (C6), 135.6 (C1′), 134.6 (C3′), 134.1 (C5), 132.1 (C4′), 130.5 (C5′), 128.9 (C3), 126.7 (C2′), 124.7 (C6′), 123.1 (C4), 15.6 (CH3); HR-MS for C14H12ClN3O2S calculated 321.0339 and found 321.0327; Anal. calcd. for C14H12ClN3O2S: C, 52.26; H, 3.76; N, 13.06; O, 9.94; S, 9.96; found: C, 52.25; H, 3.75; N, 13.04; O, 9.92; S, 9.95.
5-(3-(2-Fluorophenyl) thioureido) pyridine-3-carboxylic acid (10)
Yield: 79%; M.p.: > 300 °C; FTIR (ATR, cm−1): 3333 (N–H), 3191 (Ar–CH), 1655 (C=O), 1586 (C–N), 1469(C=C), 1331(C=S), 785(C-F); 1HNMR (500 MHz, DMSO-d6): δ 11.96 (s, 1H, NH), 11.38 (s, 1H, NH), 8.74 (s, 1H, H-2), 8.65 (s, 1H, H-6), 7.67 (s, 1H, H-4), 6.75–6.70 (m, 2H, H-2′, H-5′), 6.63–6.60 (m,1H, H-4′), 6.41 (d, J = 7.0 Hz, 1H, H-6′), 3.66 (br. s, 1H, OH); 13CNMR (125 MHz, DMSO-d6): δ 179.8 (C=S), 169.4 (C=O), 167.6 (C2′), 140.3 (C2), 140.1 (C6), 134.1 (C5), 128.8 (C3), 128.2 (C6′), 126.4 (C4′), 124.6 (C5′), 123.3 (C4), 120.3 (C1′), 115.6 (C3); HR-MS for C13H10FN3O2S calculated 291.0478 and found 291.0464; Anal. calcd. for C13H10FN3O2S: C, 53.60; H, 3.46; N, 14.42; O, 10.98; S, 11.01; found: C, 53.58; H, 3.44; N, 14.41; O, 10.97; S, 11.01.
5-(3-(3-Fluorophenyl) thioureido) pyridine-3-carboxylic acid (11)
Yield: 75%; M.p.: > 300 °C; FTIR (ATR, cm−1): 3331 (N–H), 3191 (Ar–CH), 1655 (C=O), 1587 (C–N), 1468(C=C), 1332(C=S), 785(C-F); 1HNMR (500 MHz, DMSO-d6): δ 11.92 (s, 1H, NH), 11.34 (s, 1H, NH), 8.75 (s, 1H, H-2), 8.64 (s, 1H, H-6), 7.65 (s,1H, H-4), 6.96–6.92 (m, 1H, H-5′), 6.35–6.30 (m, 2H, H-4′, H-6′), 6.21 (d, J = 7.5 Hz, 1H, H-1′), 3.68 (br. s, 1H, OH); 13CNMR (125 MHz, DMSO-d6): δ 179.8 (C=S), 169.4 (C=O), 163.1 (C3′), 140.3 (C2), 140.1 (C6), 138.6 (C1′), 134.1 (C5), 130.5 (C5′), 128.4 (C3), 123.1 (C4), 122.0 (C6′), 115.4 (C2′), 111.3 (C4′); HR-MS for C13H10FN3O2S calculated 291.0478 and found 291.0458; Anal. calcd. for C13H10FN3O2S: C, 53.60; H, 3.46; N, 14.42; O, 10.98; S, 11.01; found: C, 53.59; H, 3.45 N, 14.41; O, 10.97; S, 11.01.
5-(3-(2-Bromophenyl) thioureido) pyridine-3-carboxylic acid (12)
Yield: 71; M.p.: > 300 °C; FTIR (ATR, cm−1): 3333 (N–H), 3191 (Ar–CH), 1655 (C=O), 1586 (C–N), 1468(C=C), 1331(C=S), 785(C–Br); 1HNMR (500 MHz, DMSO-d6): δ 11.72 (s, 1H, NH), 11.31 (s, 1H, NH), 8.70 (s, 1H, H-2), 8.61 (s, 1H, H-6), 7.63 (s,1H, H-4), 7.03 (t, J = 7.0 Hz, 1H, H-3), 6.98–6.95 (m, 1H, H-5), 6.45–6.40 (m, 2H, H-4, H-6), 3.64 (br. s, 1H, OH);13CNMR (125 MHz, DMSO-d6): δ 179.5 (C=S), 169.3 (C=O), 140.3 (C2), 140.1 (C6), 137.4 (C1′), 134.3 (C5), 132.2 (C3′), 128.9 (C3), 128.6 (C6′), 128.0 (C5′), 127.4 (C2′), 127.1 (C4′), 123.4 (C4); HR-MS for C13H10BrN3O2S calculated 350.9677 and found 350.9677; Anal. calcd. for C13H11N3O2S: C, 44.33; H, 2.86; N, 11.93; O, 9.09; S, 9.10; found: C, 44.31; H, 2.85; N, 11.91; O, 9.07; S, 9.09.
5-(3-(3-Bromophenyl) thioureido) pyridine-3-carboxylic acid (13)
Yield: 73%; M.p.: > 300 °C; FTIR (ATR, cm−1): 3337 (N–H), 3191 (Ar–CH), 1655 (C=O), 1586 (C–N), 1468(C=C), 1331(C=S), 785(C–Br); 1HNMR (500 MHz, DMSO-d6): δ 11.78 (s, 1H, NH), 11.30 (s, 1H, NH), 8.72 (s, 1H, H-2), 8.60 (s, 1H, H-6), 7.62 (s, 1H, H-4), 6.88–6.85 (m, 1H, H-5′), 6.76–6.73 (m, 1H, H-4′), 6.62 (d, J = 2.0 Hz, 1H, H-2′), 6.42 (t, J = 7.5 Hz, 1H, H-6′), 3.66 (br. s, 1H, OH);13CNMR (125 MHz, DMSO-d6): δ 179.5 (C=S), 169.3 (C=O), 140.3 (C2), 140.1 (C6), 139.4 (C1′), 134.3 (C5), 131.2 (C5′), 128.9 (C3), 127.5 (C4′), 125.7 (C6′), 125.3 (C2′), 123.6 (C3′), 123.4 (C4); HR-MS for C13H10BrN3O2S calculated 447.0927 and found 447.0911; Anal. calcd. for C13H10BrN3O2S: C, 44.33; H, 2.86; 11.93; O, 9.09; S, 9.10; found: C, 44.32; H, 2.85; N, 11.92; O, 9.08; S, 9.08.