Nội dung

(2019) 13:2 Grewal et al. BMC Chemistry https://doi.org/10.1186/s13065-019-0532-8 RESEARCH ARTICLE BMC Chemistry Open Access Design, synthesis and evaluation of novel 3,5‑disubstituted benzamide derivatives as allosteric glucokinase activators Ajmer Singh Grewal1,2, Rajeev Kharb3, Deo Nandan Prasad4, Jagdeep Singh Dua4 and Viney Lather3,5* Abstract Glucokinase (GK) is the key enzyme expressed in β-cells of pancreas and liver hepatocytes and helps in the maintenance of blood glucose levels in normal range. Activators of GK are the novel category of drug candidates which activate GK enzyme allosterically and show their antidiabetic activity. A new series of 3,5-disubstituted benzamide analogues was designed, synthesized and evaluated as GK activators by in vitro assay as well as in silico docking studies followed by evaluation of antihyperglycemic activity in animal model. Amongst the synthesized derivatives, compounds 5c, 5f, 5i, 6c, 6e and 6h displayed excellent in vitro GK activation. Compounds 6c and 6e exhibited highest antihyperglycemic activity in oral glucose tolerance test in animal model. Compound 6e displayed most significant antihyperglycemic activity and comparable to that of standard drug in animal studies. In addition, antihyperglycemic activity of the synthesized molecules was further supported by the in silico docking studies of the synthesized derivatives in the allosteric site of GK protein. Keywords: Antidiabetic activity, Benzamides, Glucokinase, GK activators, In silico, In vitro assay, OGTT​ Introduction Diabetes mellitus, often simply known as diabetes is a long-lasting disorder of food metabolism characterized by hyperglycemia due to defect in insulin secretion, insulin function or together resulting in vascular and tissue damage leading to a variety of complications of diabetes related to kidney, eyes and nerves [1–5]. Type 2 diabetes (T2D) affects more than 90% of all the diabetic patients, and is a long-term malady of food metabolism bring about due to declined insulin function [6, 7]. Despite the fact that various types of oral hypoglycaemic drugs are existing intended for the T2D management, in majority of T2D patients no single drug is helpful in achieving long-term management of blood glucose under normal physiological range. Owing to the above reason, currently physicians prescribe combination of antidiabetic drugs at an early stage for the therapeutics of T2D. Moreover, *Correspondence: vinnylather@gmail.com 3 Amity Institute of Pharmacy, Amity University, Noida, U.P 201303, India Full list of author information is available at the end of the article overdose of antihyperglycemic agents may lead to severe hypoglycaemia resulting in brutal toxic and side effects, and patients usually need urgent therapeutic treatment [7]. The medicinal chemists are currently working on discovering new potent antidiabetic medicines having pharmacologically different mechanism of action which can be used for single drug therapy of T2D with better safety profile. Results from numerous latest reports, including promising clinical information, have indicated that small-molecule allosteric glucokinase (GK) activators can be used to acquire these aims [8–10]. GK accelerates the breakdown of glucose to glucose-6-phosphate in presence of adenosine triphosphate (ATP) in cytoplasm and helps in the maintenance of the normal blood glucose levels in humans. GK enzyme is expressed primarily in β-cells of the pancreas and liver hepatocyte [9–11]. GK controls glucose-stimulated insulin secretion in β-cells of pancreas and metabolism of sugars in liver hepatocyte cells. GK is a promising target for the therapeutic management of T2D patients as it plays a major function in the regulation of carbohydrate breakdown. GK activators © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Grewal et al. BMC Chemistry (2019) 13:2 are the novel class of therapeutic agents which activate GK enzyme and show their hypoglycemic activity [8, 12–14]. A wide variety of small molecule derivatives including benzamide analogues [15–29], carboxamide derivatives [30–35], acrylamide derivatives [36], benzimidazole analogues [37, 38], quinazolines derivatives [39], thiazole derivatives [40], pyrimidine derivatives [41], and urea derivatives [42, 43] were developed in last decade to act as effective allosteric GK activators with potential antidiabetic effects. Several GK activators had been advanced to phase II clinical trials including Piragliatin, AZD6370, AZD1656, MK-0941, and AMG151; potent glucose lowering efficacy had been reported, and potential burdens have also been observed, including hypoglycaemia and increased triglyceride levels [11]. Various types of benzamide derivatives were reported as potent GK activators and the maximum drug discovery and development programmes linked to benzamide GK activators were primarily focused on the 3,5-disubstituted benzamide derivatives [16, 20–26, 44–51] possibly due to their orientation in the allosteric site and complementary binding pattern with the allosteric site amino acid residues of GK protein. In view of the significant contribution of the GK activators in the management of various diabetic disorders and the potential of 3,5-disubstituted benzamides as allosteric GK activators, we had made an attempt to design and synthesize some novel GK activators based on 3,5-disubstituted benzamide scaffold. The substitutions on benzamide nucleus were carried out in such a way that appreciable H-bond and hydrophobic interactions of the designed molecules with the amino acid residues in the allosteric site of GK protein can be achieved based on pharmacophoric requirements for binding of the ligands with GK protein (Fig. 1). Materials and methods All the chemicals, reagents, solvents and proteins required during research work were procured from SRL Pvt. Ltd. (Thane), Spectrochem Pvt. Ltd. (Thane), SigmaAldrich (Bangalore), Merck Pvt. Ltd. (Mumbai), S.D. Fine-Chem Ltd. (Mumbai), LOBA Chemie (Mumbai) and Fisher Scientific (Mumbai) etc., and used as such. Veego VMP-D melting point apparatus was used for melting point determination of synthesized derivatives. Thin layer chromatography (TLC) was used for monitoring the reaction completion using silica gel-G. Shimadzu IR affinity FTIR spectrophotometer (KBr pellet technique) was used for recording IR spectra. BrukerAvance II 300 MHz NMR spectrophotometer was used for recording 1H Nuclear magnetic resonance (1H-NMR) and 13CNMR spectra using DMSO-d6 as solvent and presented in parts per million (δ, ppm) downfield from internal Page 2 of 14 Pi-Pi interaction with Tyr214 O Hydrophobic interaction Y with Val455, Ala456 and Lys459 X N H SO 2 HN R 1 R 2 (C)n N Hydrophobic interaction with Tyr214 and Ile211 H-bond with Arg63 Hydrophobic interaction with Met210, Met235 and Val62 Fig. 1 General structure of the newly designed 3,5-disubstituted benzamide GK activators and possible binding interaction with GK protein standard (tetramethylsilane). Mass spectra were recorded on Waters Q-TOF Micromass spectrometer (ESI–MS). Synthesis of sulfamoyl benzamide derivatives Dry 3-nitrobenzoic acid (0.01 mol) was taken in a round bottom flask fixed with a magnetic stirrer and the temperature was kept constant between 10 and 15 °C using cold water bath. Chlorosulphonic acid (8.0 mL) was introduced cautiously and checked to substantiate no leakage. After acid had been dissolved and the exothermic reaction had been over, the reaction flask was heated on water bath at 70–80 °C for 2 h to complete the reaction followed by cooling the flask. The contents of flask were added to 150 g crushed ice with stirring to break the lumps and precipitates of 3-(chlorosulphonyl)-5-nitrobenzoic acid were filtered under vacuum followed by washing with cold water and air dried. The product obtained above (0.01 mol) was refluxed with commercially available amines (0.01 mol) in chloroform until reaction completion as monitored by TLC on silica gel G followed by cooling and precipitates of respective sulphonamides were dried. The various sulphonamides (0.01 mol) were refluxed with thionyl chloride (0.01 mol) for 3 h and excess ­SOCl2 was distilled off to get the respective benzoyl chlorides. Benzoyl chloride (1 mmol) obtained above was refluxed with 2-aminopyrimidine (1.5 mmol) and 2-aminothiazole (1.5 mmol) in chloroform. The final product (benzamide derivatives) received after the evaporation of acetone was purified by recrystallization using ethyl alcohol [24, 26, 52]. 3‑Nitro‑5‑(phenylsulfamoyl)‑N‑(pyrimidin‑2‑yl)benzamide (5a) Black solid; yield—56%; Mp (°C) 140–142; 1H-NMR (δ ppm, DMSO-d6): 8.85 (s, 1H, NH, CO–NH), 8.04–8.46 (s, 3H, CH, ­C2, ­C4 and ­C6 of ­C6H3CO), 6.95–8.45 (m, 3H, CH, ­ C4, ­C5 and ­ C6 of pyrimidin-2-yl), 6.44–7.67 Grewal et al. BMC Chemistry (2019) 13:2 (m, 5H, ­C2, ­C3, ­C4, ­C5 and ­C6 of C ­ 6H5), 2.33 (s, 1H, NH, ­SO2NH); IR (KBr Pellets) ν cm−1: 3328.21 (NH str., CO– NH), 3257.77 (NH str., ­SO2–NH), 3012.81 (CH str., aromatic), 1670.35 (C=O str., CO–NH), 1624.06 (NH bend, Ar–NH), 1579.70 (C=N str.), 1533.48 ­(NO2 sym. str.), 1483.26 (C=C str., aromatic), 1409.96 ­(NO2 asym. str.), 1352.10 ­(SO2 asym. str., ­SO2–NH), 1141.86 ­(SO2 sym. str., ­SO2–NH), 717.52 (CH bend, aromatic). 3‑[(2‑Chlorophenyl)sulfamoyl]‑5‑nitro‑N‑(pyrimidin‑2‑yl) benzamide (5b) Reddish brown solid; yield—63%; Mp (°C) 157–158; 1 H-NMR (δ ppm, DMSO-d6): 8.69 (s, 1H, NH, CO– NH), 8.21–8.60 (s, 3H, CH, ­C2, ­C4 and ­C6 of C ­ 6H3CO), 6.95–8.45 (m, 3H, CH, C ­ 4, ­C5 and ­C6 of pyrimidin-2-yl), 6.57–7.40 (m, 4H, ­C3, ­C4, ­C5 and ­C6 of ­C6H4Cl), 2.50 (s, 1H, NH, S ­ O2NH); 13C-NMR (δ ppm, DMSO-d6): 165.94 (C=O), 157.57 (C), 156.61 (CH), 148.34 (C), 135.83 (C), 132.93 (CH), 128.32 (C), 127.78 (CH), 124.13 (CH), 110.31 (CH); IR (KBr pellets) ν cm−1: 3328.21 (NH str., CO–NH), 3257.77 (NH str., ­SO2–NH), 3012.81 (CH str., aromatic), 1708.93 (C=N str.), 1674.21 (C=O str., CO– NH), 1614.42 (NH bend, Ar–NH), 1529.55 ­(NO2 sym. str.), 1409.96 (­NO2 asym. str.), 1350.17 (­SO2 asym. str., ­SO2–NH), 1236.37 (­SO2 sym. str., ­SO2–NH); MS (ESI TOF) m/z for ­C17H12ClN5O5S [M+H]+ Calcd 434.032, found 434.087. 3‑[(3‑Chlorophenyl)sulfamoyl]‑5‑nitro‑N‑(pyrimidin‑2‑yl) benzamide (5c) Reddish brown solid; yield—62%; Mp (°C) 161–162; 1 H-NMR (δ ppm, DMSO-d6): 8.61 (s, 1H, NH, CO– NH), 8.12–8.45 (s, 3H, CH, ­C2, ­C4 and ­C6 of C ­ 6H3CO), 6.88–8.02 (m, 3H, CH, C ­ 4, ­C5 and ­C6 of pyrimidin-2-yl), 6.23–7.17 (m, 4H, ­C2, ­C4, ­C5 and ­C6 of ­C6H4Cl), 2.54 (s, 1H, NH, ­SO2NH); IR (KBr pellets) ν cm−1: 3360.00 (NH str., CO–NH), 3331.07 (NH str., ­SO2–NH), 3086.11 (CH str., aromatic), 1703.14 (C=N str.), 1674.21 (C=O str., CO–NH), 1614.42 (NH bend, Ar–NH), 1529.55 (­NO2 sym. str.), 1448.54 (­NO2 asym. str.), 1352.10 ­(SO2 asym. str., ­SO2–NH), 1149.57 (­SO2 sym. str., ­SO2–NH), 785.03 (C–Cl str., aromatic), 713.66 (CH bend, aromatic). 3‑[(4‑Chlorophenyl)sulfamoyl]‑5‑nitro‑N‑(pyrimidin‑2‑yl) benzamide (5d) Light brown solid; yield—61%; Mp (°C) 165–167; 1HNMR (δ ppm, DMSO-d6): 8.74 (s, 1H, NH, CO–NH), 8.18–8.62 (s, 3H, CH, C ­ 2, ­C4 and C ­ 6 of C ­ 6H3CO), 6.92– 8.46 (m, 3H, CH, C ­ 4, ­C5 and C ­ 6 of pyrimidin-2-yl), 6.52– 7.44 (m, 4H, ­C2, ­C3, ­C5 and C ­ 6 of ­C6H4Cl), 2.50 (s, 1H, NH, ­SO2NH); IR (KBr pellets) ν cm−1: 3363.86 (NH str., CO–NH), 3182.55 (NH str., ­SO2–NH), 3086.11 (CH str., Page 3 of 14 Aromatic), 1697.36 (C = N str.), 1674.21 (C=O str., CO– NH), 1616.35 (NH bend, Ar–NH), 1531.48 ­(NO2 sym. str.), 1354.03 (­NO2 asym. str.), 1311.59 (­SO2 asym. str., ­SO2–NH), 1143.79 (­ SO2 sym. str., ­SO2–NH). 3‑Nitro‑5‑[(2‑nitrophenyl)sulfamoyl]‑N‑(pyrimidin‑2‑yl) benzamide (5e) Yellow solid; yield—68%; Mp (°C) 155–156; 1H-NMR (δ ppm, DMSO-d6): 8.72 (s, 1H, NH, CO–NH), 8.14–8.56 (s, 3H, CH, ­C2, ­C4 and ­C6 of ­C6H3CO), 6.78–8.34 (m, 3H, CH, ­C4, ­C5 and ­C6 of pyrimidin-2-yl), 6.34–7.44 (m, 4H, ­C3, ­C4, ­C5 and ­C6 of ­C6H4NO2), 2.54 (s, 1H, NH, ­SO2NH); IR (KBr pellets) ν cm−1: 3317.57 (NH str., CO– NH), 3157.46 (NH str., ­SO2–NH), 3091.89 (CH str., aromatic), 1674.21 (C=O str., CO–NH), 1674.21 (C=N str.), 1620.21 (NH bend, Ar–NH), 1525.69 (­NO2 sym. str.), 1483.26 (C=C str., Aromatic), 1440–83 (­NO2 asym. str.), 1311.59 ­(SO2 asym. str., ­SO2-NH), 1157.29 ­(SO2 sym. str., ­SO2–NH), 713.66 (CH bend, aromatic). 3‑Nitro‑5‑[(3‑nitrophenyl)sulfamoyl]‑N‑(pyrimidin‑2‑yl) benzamide (5f) Pale yellow solid; yield—65%; Mp (°C) 158–160; 1H-NMR (δ ppm, DMSO-d6): 8.68 (s, 1H, NH, CO–NH), 8.26–8.56 (s, 3H, CH, ­C2, ­C4 and ­C6 of ­C6H3CO), 6.82–8.42 (m, 3H, CH, ­C4, ­C5 and ­C6 of pyrimidin-2-yl), 6.72–7.42 (m, 4H, ­C2, ­C4, ­C5 and ­C6 of ­C6H4NO2), 2.53 (s, 1H, NH, ­SO2NH); 13 C-NMR (δ ppm, DMSO-d6): 166.42 (C=O), 136.06 (C), 157.92 (C), 157.42 (CH), 148.62 (C), 148.12 (C), 136.18 (C), 130.86 (CH), 127.02 (CH), 124.74 (CH), 111.12 (CH), 110.39 (CH), 108.19 (CH); IR (KBr pellets) ν cm−1: 3358.07 (NH str., CO–NH), 3180.62 (NH str., ­SO2–NH), 3093.82 (CH str., aromatic), 1674.21 (C=O str., CO–NH), 1624.06 (NH bend, Ar–NH), 1531.48 (­NO2 sym. str.), 1448.54 ­(NO2 asym. str.), 1352.10 (­SO2 asym. str., S ­ O2– NH), 1153.43 ­(SO2 sym. str., S ­ O2–NH), 719.45 (CH bend, aromatic); MS (ESI TOF) m/z for ­C17H12N6O7S [M+H]+ Calcd 445.056, found 445.016. 3‑Nitro‑5‑[(4‑nitrophenyl)sulfamoyl]‑N‑(pyrimidin‑2‑yl) benzamide (5g) Yellow solid; yield—74%; Mp (°C) 152–154; 1H-NMR (δ ppm, DMSO-d6): 8.98 (s, 1H, NH, CO–NH), 8.26–8.68 (s, 3H, CH, ­C2, ­C4 and ­C6 of ­C6H3CO), 7.04–8.33 (m, 3H, CH, ­C4, ­C5 and ­C6 of pyrimidin-2-yl), 6.45–7.34 (m, 4H, ­C2, ­C3, ­C5 and ­C6 of ­C6H4NO2), 2.52 (s, 1H, NH, ­SO2NH); IR (KBr pellets) ν cm−1: 3471.87 (NH str., CO– NH), 3360.00 (NH str., ­SO2–NH), 3089.96 (CH str., aromatic), 1674.21 (C=O str., CO–NH), 1624.06 (NH bend, Ar–NH), 1612.49 (C=N str.), 1531.48 ­(NO2 sym. str.), 1479.40 (C=C str., aromatic), 1352.10 ­(NO2 asym. str.), Grewal et al. BMC Chemistry (2019) 13:2 Page 4 of 14 1311.59 ­(SO2 asym. str., S ­ O2-NH), 1151.50 ­(SO2 sym. str., ­SO2–NH), 711.73 (CH bend, aromatic). 3‑(Benzylsulfamoyl)‑5‑nitro‑N‑(pyrimidin‑2‑yl)benzamide (5h) Light brown solid; yield—76%; Mp (°C) 138–140; 1HNMR (δ ppm, DMSO-d6): 8.63 (s, 1H, NH, CO–NH), 8.01–8.31 (s, 3H, CH, C ­ 2, ­C4 and C ­ 6 of C ­ 6H3CO), 6.88– 8.34 (m, 3H, CH, C ­ 4, ­C5 and C ­ 6 of pyrimidin-2-yl), 6.51– 7.42 (m, 5H, C ­ 2, ­C3, ­C4, ­C5 and ­C6 of ­C6H5), 4.34 (s, 2H, CH of C ­ H2), 2.56 (s, 1H, NH, S ­ O2NH); FTIR (KBr pellets) ν cm−1: 3377.36 (NH str., CO–NH), 3307.92 (NH str., ­SO2–NH), 3024.38 (CH str., aromatic), 2926.01 (CH str., alkyl), 1712.43 (C=O str., CO–NH), 1624.06 (NH bend, Ar–NH), 1523.71 ­(NO2 sym. str.), 1471.69 (­NO2 asym. str.), 1350.17 ­(SO2 asym. str., ­SO2–NH), 1122.57 ­(SO2 sym. str., ­SO2–NH), 709.80 (CH bend, aromatic). 3‑(Ethylsulfamoyl)‑5‑nitro‑N‑(pyrimidin‑2‑yl)benzamide (5i) White solid; yield—69%; Mp (°C) 147–148; 1H-NMR (δ ppm, DMSO-d6): 8.68 (s, 1H, NH, CO–NH), 8.22– 8.44 (s, 3H, CH, ­C2, ­C4 and ­C6 of ­C6H3CO), 6.51–7.82 (m, 3H, CH, ­C4, ­C5 and ­C6 of pyrimidin-2-yl), 7.62 (s, 1H, NH, S ­ O2NH), 2.48 (s, 3H, CH, ­CH3), 1.12 (t, 3H, CH of ­CH3); 13C-NMR (δ ppm, DMSO-d6): 169.92 (C=O), 165.10 (C), 164.95 (CH), 148.12 (C), 136.76 (C), 135.32 (C), 126.92 (CH), 124.72 (CH), 108.12 (CH), 40.01 ­(CH2); IR (KBr pellets) ν cm−1: 3381.21 (NH str., CO–NH), 3311.78 (NH str., S ­ O2–NH), 3086.11 (CH str., Aromatic), 2924.09 (CH str., alkyl), 1660.71 (C=O str., CO–NH), 1606.70 (NH bend, Ar–NH), 1529.55 (­NO2 sym. str.), 1400 (C=C str., aromatic), 1477.47 ­ (NO2 asym. str.), 1352.10 ­(SO2 asym. str., ­SO2–NH), 1141.86 (­SO2 sym. str., ­SO2–NH), 713.66 (CH bend, aromatic); MS (ESI TOF) m/z for C ­ 13H13N5O5S [M+H]+ Calcd 352.071, found 353.024. 3‑Nitro‑5‑(phenylsulfamoyl)‑N‑(1,3‑thiazol‑2‑yl)benzamide (6a) 1 Dark brown solid; yield—76%; Mp (°C) 154–160; HNMR (δ ppm, DMSO-d6): 8.68 (s, 1H, NH, CO–NH), 8.24–8.48 (s, 3H, CH, ­C2, ­C4 and ­C6 of ­C6H3CO), 7.56 (d, 1H, CH, C ­ 5 of thiazol-2-yl), 7.34 (d, 1H, CH, C ­ 4 of thiazol-2-yl), 6.68–7.34 (m, 5H, CH of C ­ 6H5), 2.54 (s, 1H, NH, ­SO2NH); IR (KBr pellets) ν cm−1: 3371.57 (NH str., CO–NH), 3311.78 (NH str., ­SO2–NH), 3089.96 (CH str., Aromatic), 1724.43 (C=O str., CO–NH), 1624.06 (NH bend, Ar–NH), 1531.48 (­NO2 sym. str.), 1392.61 (­NO2 asym. str.), 1350.17 (­SO2 asym. str., S ­ O2-NH), 1143.79 ­(SO2 sym. str., ­SO2–NH), 715.81 (CH bend, aromatic), 653.87 (C–S str., aromatic). 3‑[(2‑Chlorophenyl)sulfamoyl]‑5‑nitro‑N‑(1,3‑thiazol‑2‑yl) benzamide (6b) White solid; yield—66%; Mp (°C) 181–182; 1H-NMR (δ ppm, DMSO-d6): 8.72 (s, 1H, NH, CO–NH), 8.18–8.45 (s, 3H, CH, ­C2, ­C4 and ­C6 of ­C6H3CO), 6.91–8.32 (m, 3H, CH, ­C4, ­C5 and ­C6 of pyrimidin-2-yl), 6.35–7.24 (m, 4H, ­C3, ­C4, ­C5 and ­C6 of ­C6H4Cl), 2.52 (s, 1H, NH, ­SO2NH); IR (KBr pellets) ν cm−1: 3371.57 (NH str., CO–NH), 3311.78 (NH str., S ­ O2–NH), 3089.96 (CH str., aromatic), 1720.50 (C=O str., CO–NH), 1701.22 (C=N str.), 1631.78 (NH bend, Ar–NH), 1523.76 (­NO2 sym. str.), 1473.62 (­NO2 asym. str.), 1350.17 (­SO2 asym. str., S ­ O2–NH), 1143.79 ­(SO2 sym. str., ­SO2–NH), 790.81 (C–Cl str.), 713.66 (CH bend, aromatic), 655.80 (C–S str., aromatic). 3‑[(3‑Chlorophenyl)sulfamoyl]‑5‑nitro‑N‑(1,3‑thiazol‑2‑yl) benzamide (6c) Greenish solid; yield—59%; Mp (°C) 189–191; 1H-NMR (δ ppm, DMSO-d6): 9.22 (s, 1H, NH, CO–NH), 8.32–8.64 (s, 3H, CH, ­C2, ­C4 and ­C6 of ­C6H3CO), 7.58 (d, 1H, CH, ­C5 of thiazol-2-yl), 7.38 (d, 1H, CH, C ­ 4 of thiazol-2-yl), 6.56–7.10 (m, 4H, CH of ­C2, ­C4, ­C5 and ­C6 of ­C6H4Cl), 2.51 (s, 1H, NH, S ­ O2NH); 13C-NMR (δ ppm, DMSO-d6): 170.02 (C=O), 165.18 (C), 148.22 (C), 136.42 (C), 135.04 (C), 134.72 (C), 132.92 (CH), 130.12 (CH), 126.03 (CH), 125.95 (CH), 124.42 (CH), 118.72 (CH), 117.18 (CH), 109.12 (CH); IR (KBr pellets) ν cm−1: 3354.21 (NH str., CO–NH), 3284.77 (NH str., ­SO2–NH), 3091.89 (CH str., aromatic), 1720.50 (C=O str., CO–NH), 1631.78 (NH bend, Ar–NH), 1531.48 (­NO2 sym. str.), 1442.75 (­NO2 asym. str.), 1350.17 (­SO2 asym. str., S ­ O2–NH), 1143.79 ­(SO2 sym. str., ­SO2–NH), 786.96 (C–Cl str.), 717.52 (CH bend, aromatic), 651.94 (C–S str., aromatic), MS (ESI TOF) m/z for ­C16H11ClN4O5S2 [M+H]+ Calcd 438.993, found 439.012. 3‑[(4‑Chlorophenyl)sulfamoyl]‑5‑nitro‑N‑(1,3‑thiazol‑2‑yl) benzamide (6d) Dark brown solid; yield—77%; Mp (°C) 185–186; 1HNMR (δ ppm, DMSO-d6): 8.66 (s, 1H, NH, CO–NH), 8.16–8.48 (s, 3H, CH, C ­ 2, ­C4 and C ­ 6 of C ­ 6H3CO), 6.90– 8.40 (m, 3H, CH, C ­ 4, ­C5 and C ­ 6 of pyrimidin-2-yl), 6.46– 7.28 (m, 4H, ­C2, ­C3, ­C5 and C ­ 6 of ­C6H4Cl), 2.46 (s, 1H, NH, ­SO2NH); IR (KBr pellets) ν cm−1: 3354.21 (NH str., CO–NH), 3284.77 (NH str., ­SO2–NH), 3091.89 (CH str., aromatic), 1726.29 (C=O str., CO–NH), 1658.78 (NH bend, Ar–NH), 1608.63 (C=N str.), 1529.55 (­NO2 sym. str.), 1442.75 (­NO2 asym. str.), 1352.10 (­SO2 asym. str., ­SO2–NH), 1141.86 ­(SO2 sym. str., ­SO2–NH), 790.81 (C– Cl str.), 713.66 (CH bend, aromatic), 650.01 (C–S str., aromatic). Grewal et al. BMC Chemistry (2019) 13:2 3‑Nitro‑5‑[(2‑nitrophenyl)sulfamoyl]‑N‑(1,3‑thiazol‑2‑yl) benzamide (6e) Yellowish brown solid; yield—72%; Mp (°C) 180–182; 1 H-NMR (δ ppm, DMSO-d6): 8.98 (s, 1H, NH, CO– NH), 8.34–8.76 (s, 3H, CH, ­C2, ­C4 and ­C6 of C ­ 6H3CO), 7.56 (d, 1H, CH, ­C5 of thiazol-2-yl), 7.28 (d, 1H, CH, ­C4 of thiazol-2-yl), 6.68–8.10 (m, 4H, CH of ­C3, ­C4, ­C5 and ­C6 of ­C6H4NO2), 2.50 (s, 1H, NH, ­SO2NH); 13C-NMR (δ ppm, DMSO-d6): 165.02 (C=O), 167.61 (C), 148.23 (C), 142.53 (C), 136.77 (C), 134.62 (C), 132.44 (CH), 128.90 (CH), 128.79 (CH), 119.42 (CH), 115.28 (CH), 107.78 (CH); IR (KBr Pellets) ν c­m−1: 3498.87 (NH str., CO– NH), 3385.07 (NH str., ­SO2-NH), 3089.96 (CH str., aromatic), 1687.71 (C=O str., CO–NH), 1622.13 (NH bend, Ar–NH), 1608.63 (C=N str.), 1529.55 ­(NO2 sym. str.), 1438.90 ­(NO2 asym. str.), 1348.24 (­SO2 asym. str., S ­ O 2– NH), 1145.72 (­ SO2 sym. str., S ­ O2–NH), 713.66 (CH bend, aromatic), MS (ESI TOF) m/z for C ­ 16H11N5O7S2 [M+H]+ Calcd 450.017, found 450.283. 3‑Nitro‑5‑[(3‑nitrophenyl)sulfamoyl]‑N‑(1,3‑thiazol‑2‑yl) benzamide (6f) Dark yellow solid; yield—68%; Mp (°C) 176–177; 1H-NMR (δ ppm, DMSO-d6): 8.64 (s, 1H, NH, CO–NH), 8.18–8.56 (s, 3H, CH, ­C2, ­C4 and ­C6 of ­C6H3CO), 6.92–8.48 (m, 3H, CH, ­C4, ­C5 and C ­ 6 of pyrimidin-2-yl), 6.53–7.42 (m, 4H, C ­ 3, ­C4, ­C5 and ­C6 of ­C6H4NO2), 2.53 (s, 1H, NH, S ­ O2NH); IR (KBr pellets) ν cm−1: 3275.13 (NH str., CO–NH), 3091.89 (NH str., ­SO2–NH), 2962.66 (CH str., aromatic), 1718.58 (C=O str., CO–NH), 1627.92 (NH bend, Ar–NH), 1529.55 (­NO2 sym. str.), 1444.68 ­(NO2 asym. str.), 1350.17 ­(SO2 asym. str., ­SO2–NH), 1141.86 (­SO2 sym. str., S ­ O2–NH), 719.45 (CH bend, aromatic), 661.58 (C–S str., aromatic). 3‑Nitro‑5‑[(4‑nitrophenyl)sulfamoyl]‑N‑(1,3‑thiazol‑2‑yl) benzamide (6g) Reddish brown solid; yield—59%; Mp (°C) 182–183; 1HNMR (δ ppm, DMSO-d6): 8.88 (s, 1H, NH, CO–NH), 8.54–8.78 (s, 3H, CH, C ­ 2, ­C4 and ­C6 of C ­ 6H3CO), 7.51 (d, 1H, CH, C ­ 5 of thiazol-2-yl), 7.22 (d, 1H, CH, ­C4 of thiazol-2-yl), 6.92–8.06 (m, 4H, CH of ­C2, ­C3, ­C5 and ­C6 of ­C6H4NO2), 2.58 (s, 1H, NH, S ­ O2NH); IR (KBr pellets) ν cm−1: 3352.28 (NH str., CO–NH), 3238.48 (NH str., ­SO2–NH), 3096.75 (CH str., aromatic), 1674.21 (C=O str., CO–NH), 1631.78 (NH bend, Ar–NH), 1531.48 ­(NO2 sym. str.), 1438.90 ­(NO2 asym. str.), 1348.24 ­(SO2 asym. str., ­SO2–NH), 1143.79 ­(SO2 sym. str., ­SO2–NH), 711.73 (CH bend, aromatic), 626.87 (C–S str., aromatic). 3‑(Benzylsulfamoyl)‑5‑nitro‑N‑(1,3‑thiazol‑2‑yl)benzamide (6h) Orange solid; yield—53%; Mp (°C) 164–166; 1H-NMR (δ ppm, DMSO-d6): 9.89 (s, 1H, NH, CO–NH), 8.18–8.20 (s, Page 5 of 14 3H, CH, ­C2, ­C4 and ­C6 of ­C6H3CO), 7.27 (d, 1H, CH, C ­5 of thiazol-2-yl), 7.05 (d, 1H, CH, ­C4 of thiazol-2-yl), 7.05– 7.26 (m, 4H, CH of C ­ 2, ­C3, ­C4, ­C5 and C ­ 6 of ­C6H5), 7.26 (s, 1H, NH, ­SO2NH), 4.81 (s, 2H, CH of ­CH2); 13C-NMR (δ ppm, DMSO-d6): 170.69 (C=O), 167.61 (C), 153.09 (C), 140.57 (C), 139.27 (C), 137.69 (C), 132.54 (CH), 128.95 (CH), 128.79 (CH), 115.28 (CH), 45.34 (­CH2); IR (KBr pellets) ν cm−1: 3361.93 (NH str., CO–NH), 3186.40 (NH str., S ­ O2-NH), 3020.53 (CH str., aromatic), 2864.29 (CH str., alkyl), 1666.50 (C=O str., CO–NH), 1614.42 (NH bend, Ar–NH), 1531.48 (­NO2 sym. str.), 1473.48 ­(NO2 asym. str.), 1352.10 (­SO2 asym. str., ­ SO2–NH), 1118.71 ­(SO2 sym. str., ­SO2–NH), 709.80 (CH bend, aromatic), 663.51 (C-S str., aromatic), MS (ESI TOF) m/z for ­C17H14N4O5S2 [M+H]+ Calcd 419.047, found 420.156. 3‑(Ethylsulfamoyl)‑5‑nitro‑N‑(1,3‑thiazol‑2‑yl)benzamide (6i) Light brown solid; yield—48%; Mp (°C) 162–163; 1HNMR (δ ppm, DMSO-d6): 8.58 (s, 1H, NH, CO–NH), 8.21–8.60 (s, 3H, CH, C ­ 2, ­C4 and C ­ 6 of C ­ 6H3CO), 6.95– 8.45 (m, 3H, CH, ­C4, ­C5 and C ­ 6 of pyrimidin-2-yl), 7.52 (s, 1H, NH, ­SO2NH), 2.58 (s, 3H, CH, ­CH3), 1.35 (t, 3H, CH of ­CH3); IR (KBr pellets) ν cm−1: 3315.63 (NH str., CO–NH), 3205.69 (NH str., S ­ O2-NH), 3020.53 (CH str., aromatic), 2856.58 (CH str., Alkyl), 1674.21 (C=O str., CO–NH), 1662.64 (C=N str.), 1618.28 (NH bend, Ar– NH), 1521.84 ­(NO2 sym. str.), 1483.26 ­(NO2 asym. str.), 1350.17 ­(SO2 asym. str., ­SO2–NH), 1145.72 ­(SO2 sym. str., ­SO2–NH), 715.59 (CH bend, aromatic). In vitro enzyme assay The GK activity of the synthesized compounds was evaluated using a coupled reaction with glucose-6-phosphate dehydrogenase (G-6-PDH) spectrometrically [26, 53– 55]. All the compounds were prepared in dimethyl sulfoxide (DMSO) and the assay was performed in a final volume of 2000 µL containing 2-(4-(2-hydroxyethyl) piperazin-1-yl)ethanesulfonic acid (25 mM, pH 7.4), glucose (10 mM), potassium chloride (25 mM), magnesium chloride (1 mM), dithiothreitol (1 mM), ATP (1 mM), NAD (1 mM), G-6-PDH (2.5 U/mL), GK (0.5 µg), and compounds under investigation (10 µM). Absorbance was measured at 340 nm after 3 min incubation period and GK activation fold by the synthesized compounds and GK fold activation was calculated compared to control (GK activation by the control (i.e., DMSO only) was considered as 100%). Docking studies Docking simulations were performed in the allosteric site of GK protein with Glide 5.8 module of Schrödinger Suite 2012 extra precision mode [56–58]. The X-ray crystallographic information of GK protein with the Grewal et al. BMC Chemistry (2019) 13:2 Page 6 of 14 allosteric activator was obtained from RCSB protein data bank. After studying a numbers of entries, the best entry (PDB code: 3IMX) was selected based on resolution and was used as the docking model. The 2D structures for the designed ligands were drawn in MarvinSketch and transformed to 3D with LigPrep 2.5 with OPLS2005 force field [59]. An analogous docking method was used for the molecular docking of the synthesized derivatives as described in detail in earlier publications using Glide and the ligand poses with most favorable docking score (Glide score and Glide energy) were selected [24, 25]. The binding interactions of the ligands with GK protein were analysed further for the docked poses of the ligands using PyMOL (the PyMOL molecular graphics system, Schrödinger, LLC). Oral glucose tolerance test (OGTT) Healthy Sprague–Dawley rats (150–200 g) procured from Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar and kept at controlled room temperature and fed with the normal pellet diet and water ad libitum, prior to the dietary manipulation. Consent was taken from Institutional Animal Ethics Committee to conduct this study (Approval No. JCDMCOP/IAEC/07/15/30). Based on the results of in vitro GK assay and docking studies, selected synthesized derivatives (5b, 5f, 5i, 6c, 6e and 6h) were evaluated in rat OGTT model. Rats were divided into different groups containing six animals in each group and all the rats were fasted overnight for at least 8 h before experiment. Control group was administered vehicle only (5% DMSO, p.o.), standard group was Results and discussion Chemistry The synthetic pathway used for the preparation of 3,5-disubstituted benzamide derivatives is highlighted in Scheme 1. In brief, 3-(chlorosulphonyl)-5-nitrobenzoic acid (2) was prepared by chlorosulphonation of 3-nitrobenzoic acid (1) and the resultant product was reacted with amines to get the desired sulphonamide derivatives (3). The sulphonamides obtained above were reacted with ­SOCl2 to get respective benzoyl chlorides (4) which were then reacted with available heteroaromatic amines to obtain the final products (5a–5i and 6a–6i). The physiochemical properties of the synthesized compounds are presented in Table 1. The synthesis as well as purity of synthesized derivatives was ensured by single spot TLC and was further established by their consistent FTIR, 1H and 13C NMR, and mass spectra. The 1H-NMR spectra of the synthesized benzamide derivatives showed the singlet signal equivalent to one proton of CONH functional group at around δ 9–10 ppm O O O 2N administered metformin (30 mg/kg, p.o.), and test groups were administered compounds 5b, 5f, 5i, 6c, 6e and 6h (50 mg/kg, p.o.). All the animals were loaded with glucose (3 g/kg, p.o.) 30 min after drug administration. Blood samples were collected just prior to drug administration, and 0, 30, 60, 90 and 120 min after oral glucose administration. Blood glucose level was measured immediately and glucose area under curve (AUC) was calculated from the data (from 0 to 2 h). The OGTT assay results were statistically analyzed by two-way ANOVA [26, 27, 60]. OH O 2N (a) (1) O OH O S (2) (b) O 2N O Cl OH O S (3) O NH R (c) N O O 2N NH O S O NH R (5a-5i) O N (d) O 2N O Cl O S O NH R (4) (e) O 2N N NH O S S O NH R (6a-6i) Scheme 1 Synthetic route followed for 3,5-disubstituted benzamide derivatives. Reagents and conditions: (a) chlorosulphonic acid, 80 °C, 2 h; (b) ­NH2-R, chloroform, reflux; c thionyl chloride, chloroform, reflux; (d) 2-aminopyrimidine, chloroform, reflux; (e) 2-aminothiazole, chloroform, reflux Grewal et al. BMC Chemistry (2019) 13:2 Page 7 of 14 Table 1 Physicochemical properties and GK activity of the synthesized 3,5-disubstituted benzamide derivatives Molecular Formula M. Pt. (°C) Raf Compound R % Yield 5a –C6H5 C17H13N5O5S 140–142 0.62 56 5b 2-ClC6H4– C17H12ClN5O5S 157–158 0.37 63 5c 3-ClC6H4– C17H12ClN5O5S 161–162 0.53 62 5d 4-ClC6H4– C17H12ClN5O5S 165–167 0.57 61 5e 2-NO2C6H4– C17H12N6O7S 155–156 0.69 68 5f 3-NO2C6H4– C17H12N6O7S 158–160 0.72 65 5g 4-NO2C6H4– C17H12N6O7S 152–154 0.69 74 5h –CH2C6H5 C18H15N5O5S 138–140 0.62 76 5i –C2H5 C13H13N5O5S 147–148 0.56 69 6a –C6H5 C16H12N4O5S2 154–160 0.75 76 6b 2-ClC6H4– C16H11ClN4O5S2 181–182 0.56 66 6c 3-ClC6H4– C16H11ClN4O5S2 189–191 0.64 59 6d 4-ClC6H4– C16H11ClN4O5S2 185–186 0.62 77 6e 2-NO2C6H4– C16H11N5O7S2 180–182 0.38 72 6f 3-NO2C6H4– C16H11N5O7S2 176–177 0.48 68 6g 4-NO2C6H4– C16H11N5O7S2 182–183 0.42 59 6h –CH2C6H5 C17H14N4O5S2 164–166 0.55 53 6i –C2H5 C12H12N4O5S2 162–163 0.58 48 a TLC mobile phase: Benzene: Ethyl acetate (7:3) b All the values are mean of three measurements ± SD (as GK fold activation at 10 µM concentration compared to control i.e., DMSO only) confirming the formation of amide linkage and presence of singlet signal for one NH proton of S ­ O2NH functional group at around δ 2.5 ppm confirmed the formation of sulphonamides in the synthesized benzamide derivatives. The presence of three singlet signals at around δ 8 ppm belonging to the protons at ­C2, ­C4 and ­C6 of the phenyl ring derived from 3-nitrobenzoic acid (metanitro benzoic acid) confirmed that the amide bond, sulphonamide linkage and N ­ O2 group were placed meta to each other i.e., separated by C ­ 2, ­C4 and ­C6. Due to this reason, the protons of ­C2, ­C4 and ­C6 of the phenyl ring had shown singlet signals at such a high chemical shift value. In the 1H-NMR spectra of compounds 5a–5i, two doublet signals and one triplet signal corresponding to three aromatic CH protons were observed at around δ 8 ppm confirming the presence of pyrimidin-2-yl ring in the structure of these benzamide derivatives. In the 1 H-NMR spectra of compounds 6a–6i, two doublet signals corresponding to two aromatic CH protons were observed at around δ 7–8 ppm which confirmed that 2-amino-4-thiazol was reacted with nitro benzoyl chloride derivatives for the synthesis of respective benzamide derivatives. In the 1H-NMR spectra of compound 5i, a multiplet signal for the alkyl protons in the range δ 2.5– 4.0 ppm corresponding to methylene group (–CH2–) and a doublet signal in the range δ 1.8–2.5 ppm corresponding to protons of the methyl group confirmed the presence of ethyl group attached to ­SO2NH– in the structure GK ­activityb 1.43 ± 0.06 1.74 ± 0.08 1.34 ± 0.05 1.36 ± 0.03 1.49 ± 0.07 1.89 ± 0.06 1.65 ± 0.04 1.20 ± 0.06 1.68 ± 0.04 1.65 ± 0.06 1.59 ± 0.08 2.11 ± 0.05 1.55 ± 0.08 2.11 ± 0.09 1.37 ± 0.07 1.24 ± 0.08 1.99 ± 0.07 1.08 ± 0.09 of these compounds. In 1H-NMR spectrum of compound 35, a doublet signal for the alkyl protons around δ 4.8 ppm and a multiplet signal around δ 6–8 ppm corresponding to five aromatic CH protons of the phenyl ring confirmed the presence of benzyl ring attached to ­SO2NH– in the structure of these compounds. In the 1HNMR spectra of compound 6a, two doublet signals and three triplet signals equivalent to five aromatic CH protons were observed at around δ 7 ppm which depicted that unsubstituted phenyl ring attached to ­SO2NH was present in the structure of these compounds. In the 1HNMR spectra of compound 5b two doublet signals and two triplet signals equivalent to four aromatic CH protons were observed in the range δ 7.1–7.6 ppm depicting the presence of 2-chlorophenyl ring attached to ­SO2NH in the structure of these compounds. In the 1H-NMR spectrum of compound 6c one singlet signal, two doublet signals and one triplet signal equivalent to four aromatic CH protons were observed in the range δ 6.8–7.3 ppm indicating the presence of 3-chlorophenyl ring attached to ­SO2NH. In the 1H-NMR spectrum of compound 6e two doublet signals and two triplet signals equivalent to four aromatic CH protons were observed in the range δ 6.6–8.1 ppm depicting the presence of 2-nitrophenyl ring attached to S ­ O2NH. In the 1H-NMR spectrum of compound 5f one singlet signal, two doublet signals and one triplet signal equivalent to four aromatic CH protons were observed indicating the presence of 3-nitrophenyl Grewal et al. BMC Chemistry (2019) 13:2 ring attached to S ­ O2NH. In the 1H-NMR spectrum of compound 6g, four doublet signals corresponding to four aromatic CH protons were observed in the range δ 6.9–8.1 ppm indicating the presence of 4-nitrophenyl ring attached to S ­ O2NH. In the 13C-NMR spectra of synthesized compounds, singlet signal equivalent to carbonyl (C=O) carbon was observed at around δ 165– 170 ppm indicating the presence of amide linkage (i.e., CO–NH) in the structure of the synthesized benzamide derivatives. In the 13C-NMR spectra of synthesized compounds, singlet signal for carbon at around δ 152 ppm confirming the presence of nitro group in the structure of synthesized compounds. In the 13C-NMR spectra of compounds 5b, 5f and 5i; three singlet signals at around δ 157 ppm corresponding to C ­ 2, ­C5 and ­C6 of pyrimidine ring and one singlet signal at around δ 120 ppm were observed indicating the presence of pyrimidin-2-yl ring attached to CONH group in the structure of these compounds. A singlet signal at around δ 165 ppm corresponding to ­C2, δ 132 ppm corresponding to C ­ 4 and δ 109 ppm corresponding to C ­ 5 in the 13C-NMR spectra of compounds 6c, 6e and 6h depicted the presence of thiazol-2-yl ring attached to CONH group in the structure of these benzamide derivatives. In the 13C-NMR spectra of compound 5i singlet signals corresponding to C ­ H2 and ­CH3 at around δ 40 ppm and δ 14 ppm indicated the presence of ethyl group attached to S ­ O2NH group in the structure of compound 5i. In the 13C-NMR spectrum of compound 6h, a singlet signal corresponding to ­CH2 at around δ 45 ppm was observed indicating the presence methylene group (–CH2–) attached to S ­ O2NH. The IR spectra of the synthesized benzamide derivatives showed the presence of amide NH-stretching vibrations at around 3300–3200 cm−1; aromatic CH-stretching vibrations above 3000 cm−1; ­SO2 asymmetric and symmetric stretching vibrations at around 1400–1300 cm−1 and 1200–1100 cm−1 respectively; and sulphonamide NH-stretching vibrations in the range 3400–3100 cm−1, thus supporting the fact that an amide linkage (CO–NH) and a sulphonamide functional group ­(SO2–NH) were present in the structure of the synthesized molecules. In the IR spectra of the synthesized molecules, C=O stretching vibrations in the range 1700–1600 cm−1 indicated the presence of amide carbonyl functional group in the structure of the synthesized benzamide derivatives. The NH-bending vibrations at around 1600 cm−1 were present in the IR spectra of the synthesized molecules confirming the presence of aromatic NH-functional group in the structure of the synthesized molecules. In the IR spectra of the synthesized molecules, CH-bending vibrations at around 800–700 cm−1 confirmed the presence of aromatic ring in the structure of molecules. The IR spectra of the synthesized benzamide derivatives Page 8 of 14 showed the presence of ­NO2 symmetric and asymmetric stretching vibrations around 1600–1500 cm−1 and 1400–1300 cm−1 respectively supporting the presence of nitro functional group in the structure of all the synthesized molecules. In the IR spectra of compounds 5a–5i, presence of C=N stretching vibrations at around 1700– 1600 cm−1 depicted the occurrence of pyrimidine ring in these compounds. The IR spectra of compounds, 5h, 5i, 6h and 6i showed aliphatic CH-stretching vibrations in the range 3000–2800 cm−1 confirming the presence of aliphatic group (e.g., ethyl or benzyl) in the structure of these compounds. The IR spectra of compounds 28–36 showed the asymmetric stretching vibrations at around 1650–1600 cm−1 and stretching vibrations around 700–600 cm−1 corresponding to aromatic C–S depicting the presence of thiazole ring in the structure of these compounds. In vitro enzyme activity The results of the in vitro GK assay (fold activation compared to control i.e., DMSO only) are presented in Table 1. Among the synthesized derivatives tested in vitro, compounds 5b, 5f, 5i, 6c, 6e and 6h showed maximum fold activation (in the range 1.7–2.1 compared to control) of GK enzyme. Compounds 5e, 5g, 6a, 6b and 6d showed moderate fold activation (around than 1.5 compared to control) of GK enzyme. Compounds 5a, 5c, 5d, 5h, 6f and 6g demonstrated lower fold activation (around 1.25) of GK enzyme compared to that of control. Compound 6i was found to be inactive in the in vitro GK assay. The results of in vitro GK assay depicted that introduction of the thiazol-2-yl ring attached to CONH resulted in increased GK activity (most potent) compared to compounds bearing pyrimidine-2-yl ring (compounds 6c and 6e). Amongst the compounds bearing pyrimidin2-yl ring, the derivative bearing N-3-nitrophenyl sulphonamide group (compound 5f) demonstrated highest GK fold activation of 1.89. The N-thiazol-2-yl substituted benzamide derivatives bearing N-3-chlorophenyl and N-2-nitrophenyl substituted sulphonamide group (compounds 6c and 6e) displayed highest GK fold activation of 2.11 compared to control. The results of in vitro GK assay demonstrated that replacement of the aromatic ring attached to sulphonamide NH with alkyl group such as ethyl (compound 6i) led to reduced GK activity compared to the compounds bearing substituted aromatic ring attached to sulphonamide NH. Molecular docking studies Lead optimization of the synthesized derivatives was done via calculation of drug-likeness properties (log P, mol. wt., hydrogen bond acceptors (HBA), and hydrogen bond donors (HBD) and all the synthesized derivatives Grewal et al. BMC Chemistry (2019) 13:2 Page 9 of 14 Table 2 Molecular properties, docking score and Glide energy of the synthesized 3,5-disubstituted benzamide molecules Compound M. Wt.a log ­Pa HBAa HBDa 5a 399.38 2.25 7 2 5b 433.82 2.85 7 2 5c 433.82 2.85 7 2 5d 433.82 2.85 7 2 5e 444.38 2.19 9 2 5f 444.38 2.19 9 2 5g 444.38 2.19 9 2 5h 413.41 2.31 7 2 5i 351.34 0.95 7 2 6a 404.42 2.84 6 2 6b 438.86 3.44 6 2 6c 438.86 3.44 6 2 6d 438.86 3.44 6 2 6e 449.41 2.78 8 2 6f 449.41 2.78 8 2 6g 449.41 2.78 8 2 6h 418.44 2.97 6 2 6i 356.37 1.54 6 2 a Glide score Glide energy − 9.59 − 45.95 − 11.23 − 50.77 − 11.26 − 9.97 − 10.20 − 10.37 − 9.91 − 10.69 − 10.12 − 11.22 − 9.96 − 10.98 − 10.86 − 10.35 − 9.86 − 10.31 − 11.11 − 9.62 − 53.63 − 43.86 − 51.40 − 57.62 − 56.42 − 56.89 − 50.57 − 48.59 − 53.62 − 53.01 − 38.77 − 56.93 − 56.19 − 56.48 − 57.19 − 47.28 Mol. Wt., LogP, HBA, and HBD were calculated using MarvinSketch (Marvin 15.9.21, 2015, ChemAxon, Budapest, Hungary) showed appreciable drug-like properties as ascertained using Lipinski’s rule of 5 (Table 2). The docking studies were performed using Glide in the allosteric site of GK protein (PDB entry: 3IMX) and validated by docking of 3IMX ligand in the allosteric site. The designed GK activators were docked in the allosteric binding site comprising of Arg63, Tyr215, Met210, Tyr214, Val452 and Val455 residues. Glide score and Glide energy of the synthesized derivatives are presented in Table 2. All the synthesized 3,5-disubstituted benzamide derivatives showed significant binding in the allosteric site as determined by analysing the H-bond and hydrophobic interactions of the selected best docked poses. Based on their most favourable Glide score, lowest Glide energy (kcal/mol) and binding interactions with allosteric site residues of GK protein, compounds 5b, 5f, 5i, 6c, 6e and 6h were further analyzed in details using PyMOL to explore the binding mode and docking interactions of the designed molecules with the amino acid residues in the allosteric site of GK protein. An overlay of docked poses of the selected compounds 5b, 5f, 5i, 6c, 6e and 6h with that of 3IMX ligand showed that these compounds had the similar orientation and binding pattern in the allosteric binding site of GK enzyme as that of co-crystallized ligand (Fig. 2). All the selected molecules were found to bind to an allosteric pocket of GK protein, which is about 20 Å remote from the glucose binding site [61]. The pyrimidin-2-yl group attached to benzamide nucleus of compounds 5b, 5f and 5i protruded in the hydrophobic pocket showing the interactions with Val455, Ala456, and Lys459 of the R13 helix, as well as Pro66 of connecting region I and Ile159 of the large domain, phenyl ring packs between Tyr214, Met210 and Val455 whereas the substituted aryl group of sulphonamide oriented into the hydrophobic pocket comprising residues Leu451, Val 455, Tyr215, and Trp99. The thiazol-2-yl group attached to benzamide nucleus of compounds 6c, 6e and 6h protruded in the hydrophobic pocket showing the interactions with Val455, Ala456, and Lys459 of the R13 helix, as well as Pro66 of connecting region I and Ile159 of the large domain, the phenyl ring was found to pack between Tyr214, Met210 and Val455 whereas the substituted aryl group of sulphonamide oriented into the hydrophobic pocket comprising residues Leu451, Val 455, Tyr215, and Trp99. Docked pose showing H-bond interactions of the selected compounds 5b, 5f, 5i, 6c, 6e and 6h in the allosteric binding site of GK protein is presented in Fig. 3. The selected 3,5-disubstituted benzamide displayed similar H-bonding interactions with amino acid residues in the allosteric binding site of GK protein as that of the cocrystallized ligand (PDB ID: 3IMX). The docked pose of compound 5b showed the H-bond interaction between the ‘1-N’ of pyrimidin-2-yl and benzamide NH with NH and backbone carbonyl of Arg63 residue on GK protein with H-bond distance of 2.8 Å and 3.0 Å respectively. Compound 5f showed the H-bond interaction between the ‘1-N’ of pyrimidin-2-yl and benzamide NH with NH Grewal et al. BMC Chemistry (2019) 13:2 Page 10 of 14 Fig. 2 Superimpose of the docked poses for compounds 5b, 5f, 5i, 6c, 6e and 6h (red sticks) with that of co-crystallized ligand (PDB ID: 3IMX, black sticks) in the allosteric site of GK protein Fig. 3 Docked poses showing H-bond interactions for compound 5b, 5f, 5i, 6c, 6e and 6h in the allosteric binding site of GK protein and backbone carbonyl of Arg63 residue on GK protein with H-bond distance of 2.9 Å and 2.8 Å respectively. Compound 5i showed the H-bond interaction between the ‘1-N’ of pyrimidin-2-yl and benzamide NH with NH and backbone carbonyl of Arg63 residue on GK protein with H-bond distance of 2.9 Å and 2.8 Å respectively. Compound 6c showed the H-bond interaction between the ‘3-N’ of thiazol-2-yl and benzamide NH with NH