N-pyridin-2-yl benzamide analogues as allosteric activators of glucokinase: Design, synthesis, in vitro, in silico and in vivo evaluation

Article abstract of DOI:10.1111/cbdd.13423

Glucokinase (GK) is the key enzyme controlling levels of blood glucose under normal physiological range, and GK activators are emerging class of drug candidates with promising hypoglycaemic activity. The current study was planned to design, synthesize and evaluate novel N-pyridin-2-yl benzamide analogues as allosteric activators of GK. A novel series of N-pyridin-2-yl benzamide analogues were synthesized starting from 3-nitrobenzoic acid and evaluated in vitro for GK activation followed by in silico studies to predict the binding interactions of the designed molecules with GK protein. The selected synthesized molecules (compounds 5b, 5c, 5e, 5g, 5h and 6d) which displayed excellent GK activity (GK fold activation around 2) in GK assay and appreciable binding interaction with GK in docking studies were further evaluated for their antihyperglycaemic potential using oral glucose tolerance test (OGTT) in rats. Amongst the compounds tested in vivo (OGTT assay) for antihyperglycaemic potential, compounds 5c, 5e and 5g displayed significant reduction in blood glucose levels. Compound 5e displayed most significant antidiabetic activity and comparable to that of standard drug in animal studies. The N-pyridin-2-yl benzamide analogues discovered in the current study can provide some lead molecules for the development of potent oral GK activators with minimum side-effects for the management of type 2 diabetes.

Full text of DOI:10.1111/cbdd.13423

DR VINEY LATHER (Orcid ID : 0000-0002-7323-3142)
Article type : Research Article
N-Pyridin-2-yl Benzamide Analogues as Allosteric Activators of Glucokinase: Design, Synthesis, In Vitro,
In Silico and In Vivo Evaluation
Ajmer Singh Grewal1, 2, Rajeev Kharb3, Deo Nandan Prasad4, Jagdeep Singh Dua4, Viney Lather3,5*
2
1Chitkara College of Pharmacy, Chitkara University, Rajpura, 140401, Punjab, India; I. K. Gujral Punjab
Technical University, Jalandhar, 144601, Punjab, India; 3Amity Institute of Pharmacy, Amity University, Noida,
4
5
201313, U.P., India; Shivalik College of Pharmacy, Naya-Nangal, 140126, Punjab, India; Jan Nayak Ch.
Devi Lal Memorial College of Pharmacy, Sirsa, 125055, Haryana, India
*Corresponding author: Prof. (Dr.) Viney Lather; Amity Institute of Pharmacy, Amity University, Noida,
201303, U.P., India; E-mail: vinnylather@gmail.com.
Abstract: Glucokinase (GK) is the key enzyme controlling levels of blood glucose under normal physiological
range and GK activators are emerging class of drug candidates with promising hypoglycaemic activity. The
current study was planned to design, synthesize and evaluate novel N-pyridin-2-yl benzamide analogues as
allosteric activators of GK. A novel series of N-pyridin-2-yl benzamide analogues were synthesized starting
from 3-nitrobenzoic acid and evaluated in vitro for GK activation followed by in silico studies to predict the
binding interactions of the designed molecules with GK protein. The selected synthesized molecules
(compounds 5b, 5c, 5e, 5g, 5h and 6d) which displayed excellent GK activity (GK fold activation around 2) in
GK assay and appreciable binding interaction with GK in docking studies were further evaluated for their
antihyperglycemic potential using oral glucose tolerance test (OGTT) in rats. Amongst the compounds tested in
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doi: 10.1111/cbdd.13423
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vivo (OGTT assay) for antihyperglycemic potential, compounds 5c, 5e and 5g displayed significant reduction in
blood glucose levels. Compound 5e displayed most significant antidiabetic activity and comparable to that of
standard drug in animal studies. The N-pyridin-2-yl benzamide analogues discovered in current study can
provide some lead molecules for the development of potent oral GK activators with minimum side effects for
the management of type 2 diabetes.
Introduction
Type 2 diabetes (T2D) is a long-term disorder of food metabolism caused due to reduced action of insulin,
characterized by hyperglycaemia and affects majority of the diabetic patients [1-2]. 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, overdose of antihyperglycemic agents may lead to severe
hypoglycaemia resulting in brutal toxic and side effects, and patients usually need urgent therapeutic treatment
[1]. Currently, medicinal chemists are focusing on synthesizing novel potent antihyperglycemic drugs with
biologically different mechanism of action which can be utilized as single drug therapy with better safety.
Results from various latest studies together with promising clinical reports, have indicated that allosteric
activators of glucokinase (GK) could be used as antidiabetic drugs with superior potency and safety [3-6].
GK is a cytoplasmic enzyme which accelerates the breakdown of glucose to glucose-6-phosphate in presence of
ATP and helps in the maintenance of the normal blood glucose levels in humans. GK enzyme is expressed
primarily in β-cells in pancreas and liver hepatocyte [4, 6-7]. In pancreatic β-cells, GK controls glucose-
stimulated insulin release and in liver hepatocyte cells, it controls the metabolism of carbohydrates. GK is an
emerging drug target for T2D therapeutics owing to its major role in the regulation of sugar metabolism. GK
activators are the novel category of therapeutic agents which activate GK allosterically and demonstrate their
hypoglycaemic potential [3, 8-10]. A broad variety of chemically different classes of compounds including
substituted benzamide analogues [11-20], carboxamide derivatives like acetamides, butanamides and other [21-
26], acrylamides [27], heterocyclic compounds like benzimidazole derivatives [28-29], quinazolines derivatives
[30], thiazole derivatives [31], pyrimidine derivatives [32], and substituted urea compounds [33-34] were
developed recently to act as potent GK activators with antidiabetic effects. Several GK activators had been
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advanced to phase II clinical trials including Piragliatin, AZD6370, AZD1656, MK-0941, and AMG151;
although potent reduction of blood glucose efficacy had been reported, possible adverse effects had also been
observed, including hypoglycaemia and raised triglyceride levels. The maximum drug discovery and
development programmes linked with GK activators were primarily cantered on the substituted benzamide
derivatives possibly due to their complementary orientation pattern and binding interactions with the allosteric
site of the GK enzyme [5]. Based on the significance of GK activators in T2D therapeutics and the potential of
benzamide scaffold for GK activation, some newer N-pyridin-2-yl substituted sulfamoyl benzamide derivatives
were designed as allosteric GK activators. General structure of the designed N-pyridin-2-yl benzamide
derivatives and the possible interactions with residues in allosteric site of GK protein are presented in Figure 1.
Materials and Methods
Chemicals and reagents were procured from SRL Pvt. Ltd., Spectrochem Pvt. Ltd., Sigma-Aldrich, Merck Pvt.
Ltd., and Fisher Scientific etc., and used as such. Veego VMP-D melting point apparatus was used for
determination of melting point of synthesized derivatives. Shimadzu IR affinity FTIR spectrophotometer (KBr
pellet technique) was used for recording IR spectra. BrukerAvance II NMR spectrophotometer was used for
1
recording H-Nuclear magnetic resonance (1H-NMR) and 13C-NMR spectra using DMSO-d6 as solvent and
presented in parts per million (δ, ppm) downfield from internal standard (tetramethylsilane).
Synthesis of N-pyridin-2-yl benzamide derivatives (Scheme 1)
Dry 3-nitrobenzoic acid (0.01 mol) was taken in a round bottom flask fixed with a magnetic stirrer and the
temperature was maintained between 10 and 15 ºC using cold water bath. Chlorosulphonic acid (8.0 mL) was
introduced cautiously and checked to substantiate no leakage. After whole acid had been dissolved and the
exothermic reaction had been over, the reaction flask was heated on water bath at 70-80 ℃ 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
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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. The different benzoyl chlorides (0.01 mol) and pyridin-2-amines
(0.015 mol) were refluxed in chloroform and the final products were recrystallized using ethanol [18, 20, 35].
3-Nitro-5-(phenylsulfamoyl)-N-(pyridin-2-yl)benzamide (5a): Pale white solid; Yield- 72%; Mp (°C) 148-150;
Rf- 0.71 (Benzene: Ethyl acetate); IR (KBr Pellets) ν cm-1: 3317.57 (NH str., CO-NH), 3157.46 (NH str., SO2-
NH), 3091.89 (CH str.), 1674.21 (C=O str.), 1620.21 (NH bend), 1608.43 (C=N str.), 1533.41 (NO2 sym. str.),
1483.26 (C=C str.), 1392.61 (NO2 asym. str.), 1348.24 (SO2 asym. str.), 1155.36 (SO2 sym. str.), 717.52 (CH
bend).
3-[(2-Chlorophenyl)sulfamoyl]-5-nitro-N-(pyridin-2-yl)benzamide (5b): Pale white solid; Yield- 69%; Mp (°C)
168-169; Rf- 0.71 (Benzene: Ethyl acetate); IR (KBr Pellets) ν cm-1: 3319.49 (NH str., CO-NH), 3088.03 (NH
str., SO2-NH), 2926.01 (CH str.), 1674.81 (C=O str.), 1608.43 (C=N str.), 1529.55 (NO2 sym. str.), 1483.26
(C=C str.), 1392.61 (NO2 asym. str.), 1350.17 (SO2 asym. str.), 1139.93 (SO2 sym. str.), 717.52 (CH bend); 1H-
NMR (δ ppm, 300 MHz, DMSO-d6): 8.70 (s, 1H, NH, CO-NH), 8.32-8.45 (s, 3H, CH, C2, C4 and C6 of
C6H3CO), 6.52-8.30 (m, 4H, CH, C3, C4, C5 and C6 of Pyridin-2-yl), 7.11-7.62 (m, 4H, CH of C3, C4, C5 and C6
of C6H4Cl), 2.52 (s, 1H, NH, SO2NH); 13C-NMR (δ ppm, 100 MHz, DMSO-d6): 166.56 (C=O), 157.62 (C),
148.12 (C), 145.02 (CH), 138.92 (C), 135.52 (C), 133.38 (CH), 133.38 (CH), 130.78 (CH), 126.88 (CH), 126.78
(CH), 126.78 (CH), 126.68 (C), 126.56 (C), 125.58 (CH), 123.28 (CH), 123.26 (CH), 112.12 (CH), 110.10
(CH); HRMS (ESI TOF) m/z for C18H13ClN4O5S [M+H]+ Calcd 432.029, Found 432.067.
3-[(3-Chlorophenyl)sulfamoyl]-5-nitro-N-(pyridin-2-yl)benzamide (5c): Grayish black solid; Yield- 68%; Mp
(°C) 174-179; Rf- 0.71 (Benzene: Ethyl acetate); IR (KBr Pellets) ν cm-1: 3352.28 (NH str., CO-NH), 3155.84
(NH str., SO2-NH), 3097.68 (CH str.), 1666.50 (C=O str.), 1622.13 (NH bend), 1533.41 (NO2 sym. str.),
1394.53 (NO2 asym. str.), 1348.24 (SO2 asym. str.), 1155.36 (SO2 sym. str.), 719.45 (CH bend), 692.44 (C-Cl
str.); 1H-NMR (δ ppm, 300 MHz, DMSO-d6): 9.89 (s, 1H, NH, CO-NH), 8.32-8.45 (s, 3H, CH, C2, C4 and C6 of
C6H3CO), 6.00-8.20 (m, 4H, CH, C3, C4, C5 and C6 of Pyridin-2-yl), 6.89-7.27 (m, 4H, CH of C2, C4, C5 and C6
of C6H4Cl), 4.81 (s, 1H, NH, SO2NH); 13C-NMR (δ ppm, 100 MHz, DMSO-d6): 170.88 (C=O), 153.07 (C),
153.02 (C), 152.80 (CH), 140.92 (C), 140.62 (C), 137.99 (CH), 137.93 (C), 135.71 (C), 132.48 (CH), 128.94
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(CH), 128.79 (CH), 123.96 (CH), 120.44 (CH), 119.18 (CH), 118.18 (CH), 117.58 (CH), 116.88 (CH); HRMS
(ESI TOF) m/z for C18H13ClN4O5S [M+H]+ Calcd 432.031, Found 432.014.
3-[(4-Chlorophenyl)sulfamoyl]-5-nitro-N-(pyridin-2-yl)benzamide (5d): Light brown solid; Yield- 62%; Mp
(°C) 169-171; Rf- 0.71 (Benzene: Ethyl acetate); IR (KBr Pellets) ν cm-1: 3323.45 (NH str., CO-NH), 3149.76
(NH str., SO2-NH), 3088.03 (CH str.), 2968.45 (CH str.), 16770.35 (C=O str.), 1622.13 (NH bend), 1529.55
(NO2 sym. str.), 1483.26 (C=C str.), 1390.68 (NO2 asym. str.), 1350.17 (SO2 asym. str.), 1138.00 (SO2 sym.
str.), 717.52 (CH bend), 690.52 (C-Cl str.).
3-Nitro-5-[(2-nitrophenyl)sulfamoyl]-N-(pyridin-2-yl)benzamide (5e): Pale yellow solid; Yield- 68%; Mp (°C)
162-164; Rf- 0.71 (Benzene: Ethyl acetate); IR (KBr Pellets) ν cm-1: 3691.91 (NH str., CO-NH), 3442.94 (NH
str., SO2-NH), 3095.75 (CH str.), 1674.21 (C=O str., CO-NH), 1624.06 (NH bend, Ar-NH), 1579.70 (C=N str.),
1529.55 (NO2 sym. str.), 1384.89 (NO2 asym. str.), 1348.24 (SO2 asym. str.), 1172.72 (SO2 sym. str.), 717.52
1
(CH bend); H-NMR (δ ppm, 300 MHz, DMSO-d6): 8.81 (s, 1H, NH, CO-NH), 8.32-8.45 (s, 3H, CH, C2, C4
and C6 of C6H3CO), 6.58-8.44 (m, 4H, CH, C3, C4, C5 and C6 of Pyridin-2-yl), 6.64-7.88 (m, 4H, CH of C3, C4,
C5 and C6 of C6H4NO2), 2.51 (s, 1H, NH, SO2NH); 13C-NMR (δ ppm, 100 MHz, DMSO-d6): 166.36 (C=O),
155.43 (C), 148.30 (C), 148.11 (CH), 144.04 (C), 135.81 (C), 137.54 (CH), 135.26 (CH), 134.41 (C), 126.74
(CH), 125.80 (CH), 115.91 (CH), 119.64 (CH), 114.31 (CH); HRMS (ESI TOF) m/z for C18H13N5O7S [M+H]+
Calcd 443.053, Found 444.016.
3-Nitro-5-[(3-nitrophenyl)sulfamoyl]-N-(pyridin-2-yl)benzamide (5f): Brownish yellow solid; Yield- 67%; Mp
(°C) 161-162; Rf- 0.71 (Benzene: Ethyl acetate); IR (KBr Pellets) ν cm-1: 3332.99 (NH str., CO-NH), 3153.61
(NH str., SO2-NH), 3093.82 (CH str.), 1670.35 (C=O str.), 1622.13 (NH bend), 1529.55 (NO2 sym. str.),
1481.33 (C=N str.), 1388.75 (NO2 asym. str.), 1350.17 (SO2 asym. str.), 1143.79 (SO2 sym. str.), 721.38 (CH
bend).
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3-Nitro-5-[(4-nitrophenyl)sulfamoyl]-N-(pyridin-2-yl)benzamide (5g): Yellow solid; Yield- 73%; Mp (°C)
158-160; Rf- 0.71 (Benzene: Ethyl acetate); IR (KBr Pellets) ν cm-1: 3346.50 (NH str., CO-NH), 3172.90 (NH
str., SO2-NH), 2972.31 (CH str.), 1668.43 (C=O str.), 1625.99 (NH bend), 1533.41 (NO2 sym. str.), 1404.18
1
(NO2 asym. str.), 1344.38 (SO2 asym. str.), 1170.79 (SO2 sym. str.), 717.52 (CH bend); H-NMR (δ ppm, 300
MHz, DMSO-d6): 8.63 (s, 1H, NH, CO-NH), 8.32-8.60 (s, 3H, CH, C2, C4 and C6 of C6H3CO), 6.59-8.44 (m,
4H, CH, C3, C4, C5 and C6 of Pyridin-2-yl), 6.79-7.94 (m, 4H, CH of C2, C3, C5 and C6 of C6H4NO2), 2.50 (s,
1H, NH, SO2NH); 13C-NMR (δ ppm, 100 MHz, DMSO-d6): 166.04 (C=O), 156.18 (C), 155.02 (C), 148.28
(CH), 143.89 (C), 136.87 (CH), 136.03 (CH), 135.73 (C), 130.79 (CH), 127.65 (CH), 126.78 (CH), 124.10
(CH), 113.54 (CH), 112.82 (CH), 112.42 (CH); HRMS (ESI TOF) m/z for C18H13N5O7S [M+H]+ Calcd
443.042, Found 443.064.
3-(Benzylsulfamoyl)-5-nitro-N-(pyridin-2-yl)benzamide (5h): Light brown solid; Yield- 62%; Mp (°C) 144-
146; Rf- 0.71 (Benzene: Ethyl acetate); IR (KBr Pellets) ν cm-1: 3348.42 (NH str., CO-NH), 3149.76 (NH str.,
SO2-NH), 3026.26 (CH str.), 2962.66 (CH str., Alkyl), 1670.35 (C=O str., CO-NH), 1622.13 (NH bend),
1533.41 (NO2 sym. str.), 1479.40 (CH bend), 1390.68 (NO2 asym. str.), 1350.17 (SO2 asym. str.), 1153.43 (SO2
1
sym. str.), 717.52 (CH bend); H-NMR (δ ppm, 300 MHz, DMSO-d6): 9.89 (s, 1H, NH, CO-NH), 7.27-8.20 (s,
3H, CH, C2, C4 and C6 of C6H3CO), 6.89-8.18 (m, 4H, CH, C3, C4, C5 and C6 of Pyridin-2-yl), 6.00-7.27 (m, 5H,
CH of C2, C3, C4, C5 and C6 of C6H4NO2), 7.25 (s, 1H, NH, SO2NH), 4.81 (d, 1H, CH, CH2); 13C-NMR (δ ppm,
100 MHz, DMSO-d6): 170.92 (C=O), 160.93 (C), 153.08 (C), 147.28 (CH), 143.95 (C), 140.57 (C), 138.12
(CH), 135.70 (C), 128.93 (CH), 128.77 (CH), 117.25 (CH), 117.12 (CH), 45.36 (CH2); HRMS (ESI TOF) m/z
for C19H16N4O5S [M+H]+ Calcd 412.084, Found 412.039.
3-(Ethylsulfamoyl)-5-nitro-N-(pyridin-2-yl)benzamide (5i): Pale white solid; Yield- 69%; Mp (°C) 151-153; Rf-
0.71 (Benzene: Ethyl acetate); IR (KBr Pellets) ν cm-1: 3687.90 (NH str., CO-NH), 3350.35 (NH str., SO2-NH),
3095.75 (CH str.), 2926.01 (CH str.), 1674.21 (C=O str.), 1624.06 (NH bend), 1533.41 (NO2 sym. str.), 1442.75
(CH bend), 1394.53 (NO2 asym. str.), 1350.17 (SO2 asym. str.), 1138.00 (SO2 sym. str.), 719.45 (CH bend).
3-Nitro-5-(phenylsulfamoyl)-N-(4-methylpyridin-2-yl)benzamide (6a): Red solid; Yield- 58%; Mp (°C) 149-
150; Rf- 0.71 (Benzene: Ethyl acetate); IR (KBr Pellets) ν cm-1: 3361.93 (NH str., CO-NH), 3188.33 (NH str.,
SO2-NH), 3088.03 (CH str.), 2922.16 (CH str.), 1664.57 (C=O str.), 1612.49 (NH bend), 1527.62 (NO2 sym.
str.), 1481.33 (CH bend), 1352.10 (NO2 asym. str.), 1311.59 (SO2 asym. str.), 1151.50 (SO2 sym. str.), 711.73
1
(CH bend); H-NMR (δ ppm, 300 MHz, DMSO-d6): 8.76 (s, 1H, NH, CO-NH), 8.32-8.45 (s, 3H, CH, C2, C4
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and C6 of C6H3CO), 6.52-8.30 (m, 3H, CH, C3, C5 and C6 of Pyridin-2-yl), 7.11-7.62 (m, 4H, CH of C2, C3, C4,
C5 and C6 of C6H5), 2.52 (s, 1H, NH, SO2NH), 2.36 (s, 3H, CH, CH3).
3-[(2-Chlorophenyl)sulfamoyl]-5-nitro-N-(4-methylpyridin-2-yl)benzamide (6b): Pale white solid; Yield- 78%;
Mp (°C) 144-145; Rf- 0.71 (Benzene: Ethyl acetate); IR (KBr Pellets) ν cm-1: 3361.93 (NH str., CO-NH),
3188.33 (NH str., SO2-NH), 3088.03 (CH str.), 2922.16 (CH str.), 1726.29 (C=O str.), 1676.14 (NH bend),
1525.69 (NO2 sym. str.), 1481.33 (CH bend), 1386.82 (NO2 asym. str.), 1348.24 (SO2 asym. str.), 1232.51 (SO2
sym. str.).
3-[(3-Chlorophenyl)sulfamoyl]-5-nitro-N-(4-methylpyridin-2-yl)benzamide (6c): Dark brown solid; Yield-
58%; Mp (°C) 178-180; Rf- 0.71 (Benzene: Ethyl acetate); IR (KBr Pellets) ν cm-1: 3329.00 (NH str., CO-NH),
3319.49 (NH str., SO2-NH), 3089.96 (CH str.), 2924.09 (CH str.), 1662.64 (C=O str., CO-NH), 1624.06 (NH
bend), 1533.41 (NO2 sym. str.), 1533.41 (C=N str.), 1481.33 (C=C str.), 1440.33 (CH bend), 1417.68 (NO2
asym. str.), 1352.10 (SO2 asym. str.), 1141.86 (SO2 sym. str.), 715.59 (CH bend), 690.52 (C-Cl str.).
3-[(4-Chlorophenyl)sulfamoyl]-5-nitro-N-(4-methylpyridin-2-yl)benzamide (6d): Brown solid; Yield- 64%;
Mp (°C) 167-168; Rf- 0.71 (Benzene: Ethyl acetate); IR (KBr Pellets) ν cm-1: 3446.79 (NH str., CO-NH),
3321.42 (NH str., SO2-NH), 3080.32 (CH str.), 2926.01 (CH str.), 1670.35 (C=O str.), 1624.06 (NH bend),
1525.69 (NO2 sym. str.), 1487.12 (C=C str.), 1436.97 (CH bend), 1488.75 (NO2 asym. str.), 1350.17 (SO2 asym.
1
str.), 1138.00 (SO2 sym. str.), 717.52 (CH bend); 638.44 (C-Cl str.); H-NMR (δ ppm, 300 MHz, DMSO-d6):
9.89 (s, 1H, NH, CO-NH), 7.21-8.20 (s, 3H, CH, C2, C4 and C6 of C6H3CO), 6.18-8.18 (m, 3H, CH, C3, C5 and
C6 of Pyridin-2-yl), 6.89-7.27 (m, 4H, CH of C2, C3, C5 and C6 of C6H4Cl), 4.81 (s, 1H, NH, SO2NH); 13C-NMR
(δ ppm, 100 MHz, DMSO-d6): 170.85 (C), 160.76 (C), 159.39 (C), 143.02 (C), 123.96 (CH), 153.07 (C), 152.80
(CH), 140.92 (C), 135.71 (C), 128.94 (CH), 120.44 (CH), 120.11 (CH), 116.88 (CH), 26.56 (CH3); HRMS (ESI
TOF) m/z for C19H15ClN4O5S [M+H]+ Calcd 446.053; Found 446.032.
3-Nitro-5-[(2-nitrophenyl)sulfamoyl]-N-(4-methylpyridin-2-yl)benzamide (6e): Yellow solid; Yield- 70%; Mp
(°C) 157-159; Rf- 0.71 (Benzene: Ethyl acetate); IR (KBr Pellets) ν cm-1: 3477.66 (NH str., CO-NH), 3360.00
(NH str., SO2-NH), 3089.96 (CH str.), 2926.01 (CH str.), 1674.21 (C=O str.), 1625.99 (NH bend), 1525.69 (NO2
sym. str.), 1388.75 (NO2 asym. str.), 1350.17 (SO2 asym. str.), 1251.80 (SO2 sym. str.), 719.45 (CH bend).
3-Nitro-5-[(3-nitrophenyl)sulfamoyl]-N-(4-methylpyridin-2-yl)benzamide (6f): Yellow solid; Yield- 71%; Mp
(°C) 153-155; Rf- 0.71 (Benzene: Ethyl acetate); IR (KBr Pellets) ν cm-1: 3325.28 (NH str., CO-NH), 3167.12
(NH str., SO2-NH), 3093.82 (CH str.), 2924.09 (CH str.), 1664.57 (C=O str.), 1624.06 (NH bend), 1531.48 (NO2
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sym. str.), 1483.26 (C=C str.), 1442.74 (NO2 asym. str.), 1352.10 (SO2 asym. str.), 1141.86 (SO2 sym. str.),
1
723.31 (CH bend); H-NMR (δ ppm, 300 MHz, DMSO-d6): 8.74 (s, 1H, NH, CO-NH), 7.76-8.22 (s, 3H, CH,
C2, C4 and C6 of C6H3CO), 6.59-7.89 (m, 3H, CH, C3, C5 and C6 of Pyridin-2-yl), 6.56-7.86 (m, 4H, CH of C2,
C4, C5 and C6 of C6H4NO2), 2.50 (s, 1H, NH, SO2NH), 2.32 (s, 3H, CH, CH3).
3-Nitro-5-[(4-nitrophenyl)sulfamoyl]-N-(4-methylpyridin-2-yl)benzamide (6g): Yellow solid; Yield- 74%; Mp
(°C) 162-164; Rf- 0.71 (Benzene: Ethyl acetate); IR (KBr Pellets) ν cm-1: 3444.87 (NH str., CO-NH), 3323.35
(NH str., SO2-NH), 2929.87 (CH str.), 2856.58 (CH str.), 1662.64 (C=O str.), 1627.92 (NH bend), 1531.48 (NO2
sym. str.), 1348.24 (NO2 asym. str.), 1305.81 (SO2 asym. str.), 1111.00 (SO2 sym. str.).
3-(Benzylsulfamoyl)-5-nitro-N-(4-methylpyridin-2-yl)benzamide (6h): Light brown solid; Yield- 63%; Mp (°C)
148-150; Rf- 0.71 (Benzene: Ethyl acetate); IR (KBr Pellets) ν cm-1: 3327.21 (NH str., CO-NH), 3165.19 (NH
str., SO2-NH), 3088.03 (CH str.), 2960.73 (CH str.), 1624.06 (NH bend), 1533.41 (NO2 sym. str.), 1481.33
(C=C str.), 1448.54 (NO2 asym. str.), 1352.10 (SO2 asym. str.), 1141.86 (SO2 sym. str.), 715.59 (CH bend).
3-(Ethylsulfamoyl)-N-(4-methylpyridin-2-yl)-5-nitrobenzamide (6i): Orange solid; Yield- 52%; Mp (°C) 153-
154; Rf- 0.71 (Benzene: Ethyl acetate); IR (KBr Pellets) ν cm-1: 3446.79 (NH str., CO-NH), 3323.35 (NH str.,
SO2-NH), 3165.19 (CH str.), 2926.01 (CH str.), 1670.35 (C=O str.), 1627.92 (NH bend), 1529.55 (NO2 sym.
1
str.), 1350.17 (NO2 asym. str.), 1230.58 (SO2 asym. str.), 1138.00 (SO2 sym. str.); H-NMR (δ ppm, 300 MHz,
DMSO-d6): 8.74 (s, 1H, NH, CO-NH), 7.76-8.22 (s, 3H, CH, C2, C4 and C6 of C6H3CO), 6.52-7.88 (m, 3H, CH,
C3, C5 and C6 of Pyridin-2-yl), 7.68 (s, 1H, NH, SO2NH), 3.36 (m, 2H, CH of CH2), 2.32 (s, 3H, CH, CH3), 1.08
(t, 3H, CH of CH3).
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. 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), KCl (25 mM), MgCl2 (1
mM), dithiothreitol (1 mM), adenosine triphosphate (1 mM), NAD (1 mM), G-6-PDH (2.5 U/mL), GK (0.5 µg),
and test compounds (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 with control
(GK activation by DMSO only was considered as 100%) [20, 36-38].
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Docking studies
Docking simulations were performed in the allosteric site of GK protein with Glide 5.8 module of Schrödinger
Suite 2012 with extra precision mode [39-41]. The X-ray crystallographic information of GK protein with the
allosteric activator was obtained from 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 [42]. A similar docking methodology 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 were selected [18-19]. The binding interactions were analysed further for the docked poses of the
ligands using PyMOL [20].
Oral glucose tolerance test (OGTT)
Sprague-Dawley rats (150-200 g) were procured from Lala Lajpat Rai University of Veterinary and Animal
Sciences, Hisar and kept at controlled room temperature (22 ± 2 ºC) and humidity (55 ± 5%). All the rats were
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 and in silico studies, selected synthesized derivatives (5b, 5c, 5e, 5g, 5h and 6d) were
evaluated in rat OGTT. Rats were divided into different groups containing six animals in each group and all
animals were fasted overnight for at least 8 h before treatment. Control group was administered vehicle only
(5% DMSO, p.o.), standard group was administered metformin (30 mg/kg, p.o.), and test groups were
administered compounds 5b, 5c, 5e, 5g, 5h and 6d (50 mg/kg, p.o.). All the animals were administered with
glucose (3 g/kg, p.o.) 30 min after drug administration. Blood samples were collected by puncturing the retro
orbital sinus just prior to drug administration, and 0, 30, 60, 90, 120 min after glucose administration. Serum
glucose level was measured immediately by using glucose estimation kit. Glucose area under curve (AUC) was
calculated from the data (from 0 h to 120 h). The OGTT assay results were statistically analyzed by two-way
ANOVA [20, 43].
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Results and discussion
Chemistry
The general synthetic pathway followed for the preparation of pyridin-2-yl benzamide derivatives is given in
Scheme 1. 3-nitrobenzoic acid (1) was chlorosulphonated to obtain 3-(chlorosulphonyl)-5-nitrobenzoic acid (2)
followed by reacting with amines to obtain respective sulphonamides (3). The sulphonamides were reacted with
SOCl2 to get respective benzoyl chlorides (4) which were then reacted with 2-aminopyridine and 2-amino-4-
methylpyridine to obtain the final products (5a-5i and 6a-6i, respectively). 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 spectra.
1
The H-NMR spectra of the synthesized benzamide derivatives contained a singlet signal corresponding to one
proton of CO-NH around δ 9-10 ppm verifying the amide linkage formation and occurrence of singlet signal
equivalent to one NH proton of SO2NH around δ 2.5 ppm established the sulphonamides formation in the
synthesized benzamide derivatives. The presence of three singlet signals around δ 8 ppm belonging to the
protons at C2, C4 and C6 of the aryl ring derived from 3-nitrobenzoic acid (meta-nitro benzoic acid) indicated
1
that the CONH bond, SO2NH bond and NO2 group were placed meta to each other. In the H-NMR spectra of
compounds 5a-5i, two doublet signals and two triplet signals corresponding to four aromatic CH protons were
1
observed in the range δ 7-8 ppm which confirmed the presence of pyridin-2-yl ring. In the H-NMR spectra of
compounds 6a-6i, two doublet signals and one singlet signal corresponding to three aromatic CH protons were
observed around δ 7-8 ppm which confirmed the presence of 4-methylpyridin-2-yl. The presence of methyl
group attached to C4 of pyridine ring in compounds 6a-6i was further confirmed by the presence of a singlet
1
signal corresponding to methyl protons in the H-NMR spectra of these compounds. In the 13C-NMR spectra of
synthesized compounds, singlet signal equivalent to carbonyl carbon was observed around δ 165-170 ppm
indicating the presence of amide linkage in the structure of the synthesized benzamide derivatives. In the 13C-
NMR spectra of synthesized compounds singlet signal for carbon around δ 152 ppm confirming the presence of
nitro group. The 13C-NMR spectra of synthesized compounds showed singlet signal around δ 151 ppm
corresponding to C2 carbon of pyridine-2-yl ring indicating the presence of pyridine ring attached to CONH
group in the structure of these compounds. The signal for C4 of pyridine ring around δ 158 ppm and signal
corresponding to methyl carbon around δ 26 ppm in the 13C-NMR spectrum of compound 6d indicated the
presence of methyl group attached to pyridine ring. The IR spectra of the synthesized benzamide derivatives
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showed the presence of amide NH- stretching vibrations around 3300-3200 cm-1; aromatic CH stretching
vibrations above 3000 cm-1; SO2 asymmetric and symmetric stretching vibrations 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 and a sulphonamide functional group were present in the structure of
the synthesized molecules. The NH- bending vibrations around 1600 cm-1 were present in the IR spectra of the
synthesized molecules confirming the presence of aromatic NH functional group. The IR spectra of the
synthesized benzamide derivatives showed 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 the synthesized compounds, presence of C=N
stretching vibrations around 1700-1600 cm-1 depicted the occurrence of pyridine ring in these compounds.
In vitro GK activity
The results of the in vitro GK assay (fold activation) are presented in Table 1. Amongst the synthesized
derivatives, compounds 5b, 5c, 5e, 5g, 5h and 6d showed maximum GK activation in the in vitro GK assay
(fold activation around 2 compared to control). Compounds 5a, 5d, 6a, 6g and 6h showed moderate fold
activation (around 1.5 compared to control) of GK enzyme. Compounds 5f, 6b, 6c, 6e and 6f showed lower GK
activation (around 1.25) compared to that of control. Compounds 5i and 6i were found to be inactive in the GK
assay. The results of in vitro GK assay depicted that substitution of the pyridine-2-yl ring attached to CONH
with 4-methyl group resulted in decreased GK activity compared to unsubstituted pyridine-2-yl ring. Amongst
the compounds bearing 4-methylpyridin-2-yl ring only compound 6d showed good GK activation (fold
activation of 1.92) compared to compounds bearing unsubstituted pyridine-2-yl ring attached to benzamide
nucleus. The N-pyridin-2-yl substituted benzamide derivative bearing N-2-nitrophenyl sulphonamide group
(compound 5e) displayed highest GK fold activation of 2.07. The N-pyridin-2-yl substituted benzamide
derivatives bearing N-3-clorophenyl and N-benzyl substituted sulphonamide group (compounds 5c and 5h)
displayed potent GK fold activation of 2.02 and 2.01, respectively. The N-pyridin-2-yl substituted benzamide
derivatives bearing N-2-clorophenyl and N-4-nitrophenyl substituted sulphonamide group (compounds 5b and
5g) displayed good GK fold activation of 1.96 and 1.95, respectively compared to control. The results of in vitro
GK assay demonstrated that replacement of the aromatic ring attached to sulphonamide NH with alkyl group
(compounds 5i and 6i) led to decreased GK activity compared to compounds bearing aromatic ring attached to
sulphonamide NH.
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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). Almost all the synthesized
derivatives showed drug like properties as established using Lipinski’s rule of five (Table 1). 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 1. The docking studies of these molecules suggested a
complimentary fit in the allosteric site of GK protein. On the basis of their Glide score, lowest Glide energy
(kcal/mol) and docking interactions, compounds 5c, 5e and 5h 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.
A superimpose of the docked poses of compounds 5c, 5e and 5h with that of 3IMX ligand showed that these
compounds had the similar orientation and binding pattern in the allosteric site of GK protein as that of
reference ligand (Figure 2a). The pyridin-2-yl group of compounds 5c, 5e and 5h projected in the hydrophobic
pocket demonstrating 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 of benzamide scaffold protruded between
Tyr214, Met210 and Val455 whereas the substituted aryl group of SO2NH projected into the hydrophobic
pocket consisting of residues Leu451, Tyr215, and Trp99 (Figure 2a). The docked pose of the selected
compounds (5c, 5e and 5h) displayed the H-bond interaction between the amide NH and ‘N’ of pyridin-2-yl
with backbone carbonyl (C=O) and NH of Arg63 residue on GK protein with H-bond distance of 2.9 Å and 2.9
Å, 3.0 Å and 2.9 Å, and 3.0 Å and 2.9 Å respectively for compounds 5c, 5e and 5h (Figures 2b, 2c and 2d). The
molecular docking of the designed N-pyridin-2-yl benzamide derivatives in the allosteric binding site of GK
protein helped in predicting that the designed N-pyridin-2-yl benzamide derivatives could act as potent
activators of GK enzyme.
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In vivo antihyperglycemic activity
Based on screening by in vitro GK assay and docking studies selected compounds (5b, 5c, 5e, 5g, 5h and 6d)
were further evaluated for their glucose lowering effects by means of rat OGTT assay using metformin as
standard antidiabetic drug. The results of antihyperglycemic activity were measured as blood glucose levels
(mg/dL) at different time intervals and glucose AUC represented in Figure 3. The results of antihyperglycemic
activity assay depicted that amongst compounds tested for OGTT assay, compounds 5c, 5e and 5h were found
to be highly active with compound 5e having superior efficacy than compounds 5c and 5h in OGTT assay.
Compound 5e was almost equipotent to standard drug at 30 and 60 min and decreased blood glucose levels
equivalent to that of standard at 120 min interval. Compound 5e was found to reduce significantly glucose AUC
compared to control and analogues to that of standard. Compounds 5c and 5h displayed appreciable reduction in
blood glucose levels compared to that of standard drug in OGTT assay. The results of antihyperglycemic
activity assay indicated that the compounds 5c, 5e and 5h followed the similar pattern in blood glucose lowering
as that of metformin. Compounds 5b, 5g and 6d were found to be fairly effective in the in vivo assay compared
to standard drug metformin. All the compounds tested for antihyperglycemic activity reduced blood glucose in
safe range at time interval of 120 during OGTT assay (i.e., no hypoglycaemic effect was observed during assay
period). The results of antihyperglycemic activity assay showed that substitution with electron withdrawing
groups like chloro and nitro at phenyl ring attached to sulphonamide led to better antihyperglycemic activity
which can be seen from in vivo results of compounds 5c and 5e. These antihyperglycemic activity results were
in accordance to the results reported for acrylamide derivatives [27]. Further the results of GK activity assay
indicated that replacement of substituted phenyl ring at sulphonamide NH with alkyl groups such as ethyl
resulted in decreased activity as observed for compounds 5i and 6i. The results of antihyperglycemic activity
assay indicated that substitution of pyridine-2-yl ring attached to benzamide nucleus with electron donating
groups such as methyl resulted in reduced antihyperglycemic activity as observed for compound 6d.
Conclusion
Based on the pharmacophoric characteristics necessary for binding of GK activators with GK protein,
benzamide moiety was selected for the design of novel N-pyridin-2-yl benzamide analogues via substitution at
CONH linker and introduction of SO2NH group at the aromatic ring. Amongst the synthesized derivatives,
compounds 5b, 5c, 5e, 5g, 5h and 6d displayed appreciable GK activity in the in vitro enzymatic assay and
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appreciable binding interaction with Arg63 of allosteric site of GK protein in docking studies. Amongst the
derivatives tested in vivo for their antihyperglycemic activity (OGTT), compounds 5c, 5e and 5h showed better
activity in antihyperglycemic studies. The results of the in vivo antihyperglycemic activity assay were in parallel
to that of the enzyme assay and docking studies. These newly discovered molecules can serve as the starting hits
for the development of safe, potent and orally active GK activators for diabetic disorders.
Acknowledgements
The authors are thankful to I. K. Gujral Punjab Technical University, Jalandhar (Punjab); Management, JCD
Vidyapeeth, Sirsa (Haryana) and Shivalik College of Pharmacy, Nangal (Punjab) for their support and
encouragement for this research work.
Conflict of Interest
The authors declare no conflict of interest.
Figure 1: Pharmacophoric requirements and general structure of the designed N-pyridin-2-yl benzamide GK
activators.
Scheme 1: Synthetic route for the N-pyridin-2-yl benzamide derivatives. Reagents and Conditions: (a) HClO3,
stir, 10-15 ºC, heat, 80 ºC, 2 h; (b) NH2-R, chloroform, reflux; (c) SOCl2, chloroform, reflux, 3 h; (d) 2-
Aminopyridines, chloroform, reflux.
Figure 2: (a) Superimpose of the docked poses for compounds 5c (red stick), 5e (yellow stick) and 5h (blue
stick) with that of PDB Ligand 3IMX (black stick); (b) Docked pose showing H-bond interactions for
compound 5c; (c) compound 5e; (d) compound 5h in the allosteric binding site of GK protein.
Figure 3: (a) Effect of selected compounds on blood glucose levels at specified time intervals in rat OGTT; (b)
Glucose AUC reduction exhibited by the selected compounds in rat OGTT model. All the values are mean of six
*
measurements ± SD. Data were significantly different compared to control group (p < 0.05), **Data were not
significantly different from control group.
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Table 1: Molecular properties, GK activity, docking score and Glide energy of the synthesized molecules.
M.
log
P*
GK
Glide
score
Glide
energy
Compound Mol. Formula
HBA* HBD*
Wt.*
activity#
C18H14N4O5S
398.39
3.00
3.52
3.52
3.52
2.96
2.96
2.96
3.10
1.67
3.47
3.99
3.99
3.99
3.43
3.43
3.43
3.57
2.13
6
6
6
6
8
8
8
6
6
6
6
6
6
8
8
8
6
6
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1.42 ± 0.03
1.96 ± 0.06
2.02 ± 0.07
1.49 ± 0.05
2.07 ± 0.04
1.33 ± 0.04
1.95 ± 0.07
2.01 ± 0.08
1.04 ± 0.10
1.35 ± 0.07
1.26 ± 0.04
1.14 ± 0.06
1.92 ± 0.08
1.11 ± 0.07
1.19 ± 0.04
1.30 ± 0.06
1.51 ± 0.10
1.02 ± 0.08
-9.57
-48.21
-47.21
-52.69
-52.85
-49.70
-55.93
-48.37
-55.01
-56.34
-56.47
-56.48
-55.66
-57.17
-50.74
-51.10
-52.49
-50.04
-49.99
5a
5b
5c
5d
5e
5f
C18H13ClN4O5S 432.84
C18H13ClN4O5S 432.84
C18H13ClN4O5S 432.84
-8.75
-11.16
-10.54
-11.26
-7.51
C18H13N5O7S
C18H13N5O7S
C18H13N5O7S
C19H16N4O5S
C14H14N4O5S
C19H16N4O5S
443.39
443.39
443.39
412.12
350.35
412.42
-7.75
5g
5h
5i
-11.12
-11.14
-9.92
6a
6b
6c
6d
6e
6f
C19H15ClN4O5S 446.86
C19H15ClN4O5S 446.86
C19H15ClN4O5S 446.86
-10.05
-11.11
-9.48
C19H15N5O7S
C19H15N5O7S
C19H15N5O7S
C20H18N4O5S
C15H16N4O5S
457.42
457.42
457.42
426.45
364.38
-11.79
-10.12
-10.12
-7.63
6g
6h
6i
-9.43
*Mol. Wt., Log P, HBA, and HBD were calculated using MarvinSketch (2015); #All the values are mean of
three measurements ± SD (as GK fold activation at 10 µM concentration).
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.