Design and synthesis of newer N-benzimidazol-2yl benzamide analogues as allosteric activators of human glucokinase

Article abstract of DOI:10.1007/s00044-020-02697-z

Allosteric activators of human glucokinase (GK) had revealed significant hypoglycemic effects for therapy of type-2 diabetes (T2D) in animal as well as human models. Some newer N-benzimidazol-2yl substituted benzamide analogues were prepared and assessed for activation of GK accompanied by molecular docking investigations for predicting the bonding interactions of these derivatives with the residues in allosteric site of GK protein. Amongst the derivatives synthesized, compounds 2 and 7 strongly increased catalytic action of GK (GK activation fold >2.0 in comparison to control) in vitro. The results of in-vitro testing were supported by the molecular docking investigations of these analogues with GK protein’s allosteric site residues (showed appreciable H-bond interactions with Arg63 residue of GK). Derivatives investigated in present study afforded few lead compounds for the discovery of harmless and strong allosteric GK activating compounds for treating T2D.

Full text of DOI:10.1007/s00044-020-02697-z

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:760–770
https://doi.org/10.1007/s00044-020-02697-z
ORIGINAL RESEARCH
Design and synthesis of newer N-benzimidazol-2yl benzamide
analogues as allosteric activators of human glucokinase
1
Sukhbir Singh1 Sandeep Arora1 Ervon Dhalio1 Neelam Sharma1 Kunal Arora1 Ajmer Singh Grewal
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Received: 28 September 2020 / Accepted: 22 December 2020 / Published online: 13 January 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021
Abstract
Allosteric activators of human glucokinase (GK) had revealed significant hypoglycemic effects for therapy of type-2 diabetes
(T2D) in animal as well as human models. Some newer N-benzimidazol-2yl substituted benzamide analogues were prepared and
assessed for activation of GK accompanied by molecular docking investigations for predicting the bonding interactions of these
derivatives with the residues in allosteric site of GK protein. Amongst the derivatives synthesized, compounds 2 and 7 strongly
increased catalytic action of GK (GK activation fold >2.0 in comparison to control) in vitro. The results of in-vitro testing were
supported by the molecular docking investigations of these analogues with GK protein’s allosteric site residues (showed
appreciable H-bond interactions with Arg63 residue of GK). Derivatives investigated in present study afforded few lead
compounds for the discovery of harmless and strong allosteric GK activating compounds for treating T2D.
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Keywords Allosteric Benzamides Benzimidazoles Glucokinase Docking GK assay
Introduction
effective hypoglycemic agents having distinct mechanism of
action at molecular level which could be used as single drug
with improved safety [6, 7]. Glucokinase (GK) is a cytosolic
enzyme that is primarily expressed in pancreatic β-cells and
liver hepatocytes; as well as lifts up glucose transformation to
glucose-6-phosphate with the aid of adenosinetriphosphate
(ATP) [8, 9]. In beta-cells of pancreas gland, GK regulates
glucose-instigated discharge of insulin and in liver hepato-
cytes of liver; it commands the breakdown of sugars. GK acts
as an emergent medication focus for treatment and manage-
ment of T2D due to its key function in controlling sugar
breakdown. Small molecule activators of human GK are the
unique class of therapeutically useful agents that allosterically
activate GK and illustrate their plasma sugar lowering
potential [6, 9–12]. Several GK activators had been pro-
gressed into clinical trials (phase II) including AZD6370,
AZD1656, MK-0941, Piragliatin, and AMG151; even though
strong decrease in blood sugar was observed, potential
adverse reactions were also reported, such as hypoglycemia
and elevated levels of triglycerides. Literature survey revealed
that most of the drug discovery and development research
associated with allosteric activators of human GK were
mainly focused on the substituted benzamide analogues
(specially bearing a hetero-aromatic ring connected to the
benzamide NH- atom) probably owing towards their corre-
sponding alignment outline and bonding interactions with the
residues of allosteric binding site of GK protein [4, 6, 13–27].
Type-2 diabetes (T2D) is a chronic disease of the food
metabolic pathways owing to decreased insulin action
resulting in hyperglycemia and prevalent amongst most of the
patients suffering from diabetes [1–3]. Although ample types
of oral antidiabetic agents are existing to be used for the
management of T2D, no individual antidiabetic agent is
valuable in attaining persistent homeostasis of plasma sugar
within usual physiological range in majority of the persons
suffering from T2D. Owing to the above points, these days
doctors advise combination of hypoglycemic agents at an
initial phase of T2D treatment. Additionally, overdose of
hypoglycemic drugs may possibly result in serious hypogly-
cemia triggering brutal adverse reactions, and patients gen-
erally require urgent medical treatment [2, 4, 5]. Now-a-days,
medicinal chemistry scientists are aiming at designing newer
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02697-z) contains supplementary
material, which is available to authorized users.
* Ajmer Singh Grewal
ajmergrewal2007@gmail.com
1
Chitkara College of Pharmacy, Chitkara University,
Rajpura, Punjab, India

Medicinal Chemistry Research (2021) 30:760–770
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Based on the above facts few newer N-benzimidazol-2-yl
benzamide analogues were proposed as potential activators of
human GK enzyme.
(CH str., Aromatic), 1642.58 (Carbonyl C=O str., Benza-
mide), 1558.12 (NH bend, Aromatic amine), 1463.36 (C=N
str., Aromatic, Benzimidazol-2-yl), 1417.54 (C=C str.,
Aromatic), 1296.70 (SO2 asym. str., SO2NH), 1100.00
(C–N str., Benzimidazol-2-yl), 1076.13 (SO2 sym. str.,
1
Materials and methods
SO2NH, Sulphonamide), 752.08 (CH bend, Aromatic); H-
NMR (δ ppm, DMSO-d6): 12.68 (s, 1H, NH, CONH),
8.02–8.64 (m, 4H, CH, C2, C4, C5 and C6 of C6H4CO),
7.36–8.46 (m, 4H, CH, Benzimidazol-2-yl), 6.38–7.19 (m,
5H, CH of C2, C3, C4, C5 and C6 of C6H5), 3.00 (s, 1H, NH,
Benzimidazol-2-yl), 2.58 (s, 1H, NH, SO2NH); 13C-NMR
(δ ppm, DMSO-d6): 168.67 (C=N), 164.15 (C=O), 151.78
(C), 136.16 (C), 134.89 (C), 131.78 (C), 130.13 (CH),
129.17 (CH), 128.41 (C), 128.02 (CH), 125.23 (CH),
123.67 (CH), 120.15 (CH), 119.34 (CH), 117.97 (CH),
115.42 (CH).
Chemicals were acquired from reputed companies such as
Spectrochem, SRL, S.D. Fine-Chem, Merck, Fisher Scientific
and Sigma-Aldrich etc., and employed without purification.
Melting points of the synthesized molecules were estimated by
uncorrected Veego VMP-D melting point device. Silica Gel-G
TLC was used for monitoring the progress of chemical reac-
tion. Shimadzu FTIR spectrophotometer using potassium
bromide pellet process was utilized for recording IR spectra.
‘Avance-II (Bruker) 400 MHz NMR spectrophotometer’ was
employed for taking 1H-NMR and 13C-NMR spectra
employing appropriate dutereated solvents. Results were
represented in ‘parts per million’ (‘δ’, ppm) downfield to ‘Si
(CH3)4’ (internal reference).
N-(1H-Benzimidazol-2-yl)-3-[(2-chloro-4-nitrophenyl)
sulfamoyl]benzamide (2)
Yellowish brown solid; Yield: 78%; Mp (°C) 162–165;
FTIR (KBr Pellets) ν cm−1: 3836.20 (NH str., CONH),
3446.91 (NH str., SO2-NH), 2928.28 (CH str., Aromatic),
1641.34 (C=O str., CONH), 1632.23 (NH bend, Ar–NH),
1551.35 (C=N str., Aromatic), 1464.11 (NO2 sym. str.,
NO2), 1413.94 (NO2 asym. str., NO2), 1299.66 (SO2 asym.
str., SO2NH), 1100.00 (C–N str., Benzimidazol-2-yl),
1079.66 (SO2 sym. str., SO2NH), 684.36 (C–Cl str., Aro-
General procedure for preparation of designed
molecules
Dry benzoic acid (1 mmol) was transferred to a flat base
flask fixed on a magnetic agitator at constant temperature
around 10 °C. Excess of HClSO3 (8.0 mL) was added
carefully and observed to avoid any escape. When all the
acid was liquefied and the exothermic response terminated,
the flat bottom flask was heated at 70–80 °C using water
bath for 2 h followed by cooling. The materials of flask were
poured to crushed ice (150 g) cautiously with stirring and
crystals of 3-(chlorosulphonyl)benzoic acid were filtered
employing vacuum subsequent to cold water wash followed
by air drying. The precipitates prepared above (1 mmol)
were then reacted with the corresponding aliphatic and
aromatic amines (1 mmol) under reflux using acetone till
completion of reaction (observed using silica gel G TLC)
following cooling and drying of the precipitates. The dif-
ferent sulphonamides prepared above (1 mmol) were
refluxed for 3 h with sulfinyl chloride (1 mmol) and to
receive equivalent acid chlorides excess sulfinyl chloride
was withdrawn. Acid chlorides prepared above (1 mmol)
were refluxed with 2-aminobenzimidazole (1.5 mmol). The
end products (compounds 1–10) obtained by the evaporation
of solvent were purified using recrystallization from ethanol
[28–30].
1
matic); H-NMR (δ ppm, DMSO-d6): 9.28 (s, 1H, NH,
CONH), 8.67–8.43 (m, 4H, CH, C6H4CO), 7.34–7.91 (m,
4H, CH, Benzimidazol-2-yl), 7.08–7.24 (m, 3H, CH,
C6H3ClNO2), 4.48 (s, 1H, NH, Benzimidazol-2-yl), 4.05 (s,
1H, NH, SO2NH); 13C-NMR (δ ppm, DMSO-d6): 169.78
(C=N), 164.58 (C=O), 147.46 (C), 139.06 (C), 137.12 (C),
135.03 (C), 134.45 (C), 132.08 (CH), 129.12 (CH), 128.69
(C), 127.58 (CH), 126.47 (C), 125.07 (CH), 124.17 (CH),
119.58 (CH), 118.14 (CH), 114.36 (CH), 112.94 (CH).
N-(1H-Benzimidazol-2-yl)-3-(benzylsulfamoyl)benzamide (3)
Light brown solid; Yield: 62%; Mp (°C) 160–163; FTIR
(KBr Pellets) ν cm−1: 3755.80 (NH str., Benzamide), 3448.08
(NH str., SO2NH), 2996.40 (CH str., Aromatic), 2912.85 (CH
str., Aliphatic), 1659.53 (C=O str., CONH), 1429.38 (NH
bend, Ar–NH), 1311.51 (SO2 asym. str., SO2NH), 1100.00
(C–N str., Benzimidazol-2-yl), 1025.25 (SO2 sym. str.,
1
SO2NH), 696.02 (CH bend, Aromatic); H-NMR (δ ppm,
DMSO-d6): 10.93 (s, 1H, NH, CONH), 8.20–8.51 (m, 4H,
CH, C6H4CO), 7.70–8.14 (m, 4H, CH, Benzimidazol-2-yl),
7.12–7.58 (m, 5H, CH, C6 of C6H5), 6.64 (t, 1H, NH, Sul-
phonamide), 2.54 (s, 1H, NH, Benzimidazol-2-yl), 2.51 (d,
1H, CH, CH2); 13C-NMR (δ ppm, DMSO-d6): 175.18
(C=N), 164.76 (C=O), 153.46 (C), 141.24 (C), 140.15 (C),
N-(1H-Benzimidazol-2-yl)-3-(phenylsulfamoyl)benzamide (1)
Pale white solid; Yield: 72%; Mp (°C) 158–161; FTIR (KBr
Pellets) ν cm−1: 3867.78 (NH str., CONH), 3737.50 (NH
str., Benzamide), 3432.08.46 (NH str., SO2NH), 2973.38

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Medicinal Chemistry Research (2021) 30:760–770
135.14 (C), 135.03 (C), 132.26 (CH), 131.92 (CH), 128.08
(CH), 124.14 (CH), 122.16 (CH), 120.94 (CH), 119.04 (CH),
116.68 (CH), 45.88 (CH2).
Sulphonamide), 1100.00 (C–N str., Benzimidazol-2-yl),
850.55 (CH bend, Aromatic); 1H-NMR (δ ppm, DMSO-d6):
10.27 (s, 1H, NH, CONH), 8.01–8.46 (m, 4H, CH,
C6H4CO), 7.34–8.02 (m, 4H, CH, Benzimidazol-2-yl),
7.04–7.39 (m, 4H, CH, C6H4CH3), 2.57 (s, 1H, NH, Ben-
zimidazol-2-yl), 2.18 (s, 1H, NH, SO2NH), 2.14 (s, 3H,
Methyl); 13C-NMR (δ ppm, DMSO-d6): 175.16 (C=N),
165.26 (C=O), 151.42 (C), 139.55 (C), 138.06 (C), 136.32
(C), 134.87 (CH), 133.34 (C), 129.67 (C), 129.12 (CH),
126.55 (CH), 125.12 (CH), 123.56 (CH), 121.68 (CH),
117.36 (CH), 17.32 (CH3).
N-(1H-Benzimidazol-2-yl)-3-(butylsulfamoyl)benzamide (4)
Light brown solid; Yield: 59%; Mp (°C) 156–158; FTIR
(KBr Pellets) ν cm−1: 3754.38 (NH str., CONH, Benza-
mide), 3448.26 (NH str., SO2NH, Sulphonamide), 2930.77
(CH str., Aromatic), 2962.66 (CH str., Aliphatic), 1643.43
(C=O str., CONH, Benzamide), 1554.13 (NH bend,
Ar–NH), 1464.82 (C=C str., Aromatic), 1415.35 (SO2
asym. str., SO2NH, Sulphonamide), 1100.00 (C–N str.,
N-(1H-Benzimidazol-2-yl)-3-[(4-bromophenyl)sulfamoyl]
benzamide (7)
1
Benzimidazol-2-yl), 1076.78 (SO2 sym. str., SO2NH); H-
NMR (δ ppm, DMSO-d6): 8.59 (s, 1H, NH, CONH),
7.34–7.88 (m, 4H, CH, C6H4CO), 7.05–7.86 (m, 4H, CH,
Benzimidazol-2-yl), 2.54 (t, 1H, NH, Sulphonamide), 2.50
(s, 1H, NH, Benzimidazol-2-yl), 1.31 (m, 9H, Butyl); 13C-
NMR (δ ppm, DMSO-d6): 175.18 (C=N), 165.29 (C=O),
151.49 (C), 139.65 (C), 135.26 (C), 135.02 (C), 131.07
(CH), 128.44 (CH), 127.68 (CH), 124.59 (CH), 119.28
(CH), 118.93 (CH), 118.02 (CH), 115.02 (CH), 45.38
(CH2), 25.58 (CH2), 21.07 (CH2), 15.08 (CH3).
Grayish black solid; Yield: 74%; Mp (°C) 158–160; FTIR
(KBr Pellets) ν cm−1: 3837.14 (NH str., Benzamide),
3732.98 (NH str., Benzamide), 3441.64 (NH str., SO2NH,
Sulphonamide), 2974.87 (CH str., Aromatic carbon),
1641.67 (Carbonyl str., Benzamide), 1553.91 (NH bend,
Aromatic amine), 1464.33 (C=N str., Aromatic), 1415.88
(SO2 asym. str., SO2NH), 1296.76 (SO2 sym. str., SO2NH),
1100.00 (C–N str., Benzimidazol-2-yl), 809.70 (CH bend,
Aromatic), 753.82 (C–Br str., Aromatic); 1H-NMR (δ ppm,
DMSO-d6): 9.49 (s, 1H, NH, CONH), 7.78–8.66 (m, 4H,
CH, C6H4CO), 7.14–7.92 (m, 4H, CH, Benzimidazol-2-yl),
6.20–7.44 (m, 4H, CH, C6H4Br), 4.48 (s, 1H, NH, Benzi-
midazol-2-yl), 4.02 (s, 1H, NH, SO2NH); 13C-NMR (δ
ppm, DMSO-d6): 175.28 (C=N), 165.82 (C=O), 152.47
(C), 142.05 (C), 139.10 (C), 136.97 (C), 136.26 (C), 132.98
(C), 132.02 (CH), 130.36 (CH), 130.01 (CH), 129.67 (CH),
127.12 (CH), 124.84 (CH), 122.04 (CH), 120.18 (CH),
116.45 (CH).
N-(1H-benzimidazol-2-yl)-3-(methylsulfamoyl)benzamide (5)
Dark brown solid; Yield: 52%; Mp (°C) 148–152; FTIR
(KBr Pellets) ν cm−1: 3798.48 (NH str., CONH), 3448.44
(NH str., SO2NH), 3017.57 (CH str., Aromatic), 2966.14
(CH str., Alkyl), 1654.21 (Carbonyl str., CONH), 1598.09
(NH bend, Aromatic amine), 1544.68 (C=N str., Aromatic),
1388.45 (SO2 asym. str., SO2NH), 1189.77 (SO2 sym. str.,
SO2NH), 1100.00 (C–N str., Benzimidazol-2-yl), 789.65
1
(CH bend, Aromatic); H-NMR (δ ppm, DMSO-d6): 8.60
(s, 1H, NH, CONH), 7.49–7.98 (m, 4H, CH, C6H4CO),
7.10–7.58 (m, 4H, CH, C4, C5, C6 and C7 of Benzimidazol-
2-yl), 5.20 (t, 1H, NH, Sulphonamide), 2.54 (s, 1H, NH,
Benzimidazol-2-yl), 2.51 (s, 3H, Methyl); 13C-NMR (δ
ppm, DMSO-d6): 174.27 (C=N), 165.16 (C=O), 152.86
(C), 141.06 (C), 138.84 (C), 135.38 (CH), 133.08 (C),
132.66 (CH), 127.58 (CH), 126.05 (CH), 122.98 (CH),
120.94 (CH), 118.18 (CH), 117.08 (CH), 31.14 (CH3).
N-(1H-Benzimidazol-2-yl)-3-[(4-methylphenyl)sulfamoyl]
benzamide (8)
Light brown solid; Yield: 63%; Mp (°C) 160–163; FTIR
(KBr Pellets) ν cm−1: 3870.59 (NH str., Benzamide),
3755.40 (NH str., CONH), 3452.66 (NH str., SO2NH),
2997.49 (CH str., Aromatic), 1708.27 (C=O str., Amide),
1429.03 (NO2 sym. str., Aromatic nitro group), 1362.55
(NO2 asym. str., Aromatic nitro group), 1323.98 (SO2 asym.
str., SO2NH), 1223.02 (SO2 sym. str., SO2NH), 1100.00
(C–N str., Benzimidazol-2-yl), 696.02 (CH bend, Aro-
N-(1H-Benzimidazol-2-yl)-3-[(2-methylphenyl)sulfamoyl]
benzamide (6)
1
matic); H-NMR (δ ppm, DMSO-d6): 7.95 (s, 1H, NH,
Light brown solid; Yield: 60%; Mp (°C) 162–165; FTIR
(KBr Pellets) ν cm−1: 3791.96 (NH str., CONH, Benza-
mide), 3456.56 (NH str., SO2NH, Sulphonamide), 3013.67
(CH str., Aromatic), 2912.67 (CH str., Aliphatic), 1667.25
(NH str., SO2NH), 1604.66 (NH bend, Aromatic amine),
1578.56 (C=N str., Aromatic), 1345.34 (SO2 asym. str.,
SO2NH, Sulphonamide), 1103.78 (SO2 sym. str., SO2NH,
CONH), 7.16–7.90 (m, 4H, CH, C6H4CO), 7.26–7.84 (m,
4H, CH, Benzimidazol-2-yl), 7.16–7.88 (m, 4H, CH,
C6H4NO2), 5.00 (s, 1H, NH, Benzimidazol-2-yl), 2.50 (s,
1H, NH, Sulphonamide); 13C-NMR (δ ppm, DMSO-d6):
174.63 (C=N), 164.94 (C=O), 151.68 (C), 142.21 (C),
139.04 (C), 138.14 (C), 135.28 (C), 132.76 (C), 132.12
(CH), 128.69 (CH), 125.36 (CH), 122.09 (CH), 120.01

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763
Fig. 1 Procedure used for docking of designed analogues in the allosteric site of GK protein
(CH), 119.25 (CH), 118.42 (CH), 117.68 (CH), 115.18
(CH), 24.14 (CH3).
Aromatic), 1441.52 (NO2 asym. str.), 1415.35 (CH bend,
Aliphatic), 1300.62 (SO2 asym. str., SO2NH), 1100.00
(C–N str., Aromatic), 1076.79 (SO2 sym. str., SO2NH),
717.52 (CH bend, Aromatic); 1H-NMR (δ ppm, DMSO-d6):
10.47 (s, 1H, NH, CONH), 7.89–8.32 (m, 4H, CH,
C6H4CO), 7.39–7.56 (m, 4H, CH, Benzimidazol-2-yl),
7.20–7.50 (m, 4H, CH, C6H4CH3), 2.50 (s, 1H, NH, Ben-
zimidazol-2-yl); 13C-NMR (δ ppm, DMSO-d6): 175.56
(C=N), 165.08 (C=O), 153.84 (C), 143.80 (C), 139.46 (C),
138.86 (C), 137.47 (CH), 135.31 (C), 133.94 (C), 131.08
(CH), 129.95 (CH), 129.18 (CH), 126.02 (CH), 122.91
N-(1H-Benzimidazol-2-yl)-3-[(4-nitrophenyl)sulfamoyl]
benzamide (9)
Pale yellow solid; Yield: 74%; Mp (°C) 165–168; FTIR
(KBr Pellets) ν cm−1: 3868.16 (NH str., CONH), 3754.28
(NH str., CONH), 3448.36 (NH str., SO2NH), 2930.77 (CH
str., Aromatic), 1643.31 (C=O str., CONH), 1553.03 (NH
bend), 1524.42 (NO2 sym. str.), 1464.83 (C=N str.,

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Scheme 1 General scheme
employed for synthesis of N-
benzimidazol-2-yl benzamide
analogues. Reagents and
’
conditions: a ‘HClSO3 ; b ‘NH2-
R’, reflux; c ‘SO2Cl’, reflux;
d 2-Amino
benzimidazole, reflux
(CH), 121.27 (CH), 120.67 (CH), 119.37 (CH),
116.62 (CH).
(0.25 mM), 10 mM dextrose (10 mM), KCl (25 mM),
MgCl2 (1 mM), 1,4-dithio-D-threitol (1 mM), 1 mM nico-
tinamide adenine dinucleotide, 1 mM ATP, G6PDH (2.5 U/
mL), 0.5 µg GK, and derivatives to be tested (10 µM).
Readings were taken at 340 nm following nurture time of
3 min and GK activation was computed in comparison to
DMSO (activation of GK by DMSO alone was treated as
100%). All the results were represented as mean (n = 3)
standard deviation (S.D.). The in vitro GK assay data for
test groups was statistically analyzed by one-way ANOVA
for comparison and significance from control group (value
of p < 0.05) using GraphPad Prism (GraphPad Software
Inc.) [22, 24, 33, 34].
N-(1H-benzimidazol-2-yl)-3-(propylsulfamoyl)benzamide (10)
Light brown solid; Yield: 48%; Mp (°C) 155–158; FTIR
(KBr Pellets) ν cm−1: 3450.06 (NH str., CONH), 2996.68
(NH str., SO2NH), 2912.98 (CH str.), 1689.51 (C=O str.,
CONH), 1428.92 (NH bend), 1311.73 (C=N str., Aro-
matic), 1100.00 (C–N str., Benzimidazol-2-yl), 1023.65
(SO2 asym. str., SO2NH), 950.47 (SO2 sym. str., SO2NH),
696.27 (CH bend, Aromatic); 1H-NMR (δ ppm, DMSO-d6):
10.25 (s, 1H, NH, CONH), 8.37–8.71 (s, 3H, CH,
C6H3CO), 7.56–7.94 (m, 4H, CH, Benzimidazol-2-yl), 2.61
(s, 1H, NH, Benzimidazol-2-yl), 2.58 (s, 1H, NH, SO2NH),
2.44 (m, 2H, Methylene), 2.05 (m, 2H, Methylene), 1.27 (t,
3H, methyl); 13C-NMR (δ ppm, DMSO-d6): 174.24
(C=N), 165.02 (C=O), 151.33 (C), 139.00 (C), 136.88 (C),
134.65 (C), 130.34 (CH), 129.17 (CH), 125.37 (CH),
124.92 (CH), 120.49 (CH), 118.78 (CH), 118.02 (CH),
117.33 (CH), 46.04 (CH2), 32.48 (CH2), 12.44 (CH3).
Molecular docking investigations
AutoDock Vina and AutoDock Tools were used for
executing molecular docking of all the analogues in the
allosteric location of the GK (PDB ID: ‘3IMX’) [35, 36].
The protocol followed for docking of designed analogues
with GK is presented in Fig. 1 as previously reported
[22, 24, 26, 30, 37, 38].
In silico estimation of pharmacokinetic properties
In silico prediction of toxicity
The designed molecules were analyzed for the prediction of
pharmacokinetics using FAF-Drugs4 server; as well as
accessed for their “drug likeness” potential utilizing
Lipinski’s rule [27, 31, 32].
All samples were screened in silico to estimate the potential
toxicity of such substances using online computer software
pkCSM [39–41].
In vitro GK assay
Results and discussion
GK activation potential of all the derivatives was assessed
employing a combined response with glucose-6-phosphate
dehydrogenase (G6PDH) using spectrometry. All the sam-
ples were made using DMSO and the in-vitro GK test was
done in an ultimate quantity of ‘2000 µL’ comprising of 4-
(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4)
Chemistry
3-(Chlorosulphonyl)-benzoic acid acquired through chlor-
osulphonation of benzoic acid was reacted with different
amine derivatives to obtain various sulphonamide analogues

Medicinal Chemistry Research (2021) 30:760–770
765
Table 1 Physicochemical characteristics of the prepared N-benzimidazol-2-yl benzamide analogues
Compound
1
R
Mol. formula
M. Pt. (ºC)
Rf*
% Yield
-C6H5
C20H16N4O3S
158-161
0.61
72
+O
O–
C20H14ClN5O5S
162-165
0.53
78
2
Cl
N
-CH2C6H4
-C4H9
C21H18N4O3S
C18H20N4O3S
C15H24N4O3S
160-163
156-158
148-152
0.62
0.73
0.57
62
59
52
3
4
5
-CH3
C21H18N4O3S
C20H15BrN4O3S
C21H18N4O3S
162-165
158-160
160-163
0.78
0.47
0.72
60
74
63
6
7
8
H3C
Br
CH3
+O
N
C20H15N5O5S
C17H18N4O3S
165-168
155-158
0.68
0.67
74
48
9
O–
-C3H7
10
aTLC; mobile phase: Toluene: Ethyl acetate (7:3)
which were then reacted with SO2Cl followed by reaction
with 2-aminobenzimidazole to synthesize the desired com-
pounds (Scheme 1) in good yield (Table 1).
benzimidazol-2-yl scaffold in synthesized compounds. In 13C-
NMR pattern of the prepared molecules signals around δ
170 ppm indicated the existence of C=N bond in these deri-
vatives and signals about δ 165 ppm illustrated occurrence of
amide C=O bond therefore supporting development of ben-
zamide linkage in produced derivatives. The FTIR spectrum
of manufactured N-benzimidazol-2-yl substituted benzamide
analogues demonstrated the occurrence of characteristics
‘stretching frequencies’ at 3300–3200, above 3000, 1400-
1300/1200-1100 (asymmetric) and 3400–3100 cm−1 which
correspond to amide NH, aromatic CH, SO2, sulphonamide
NH functional groups respectively, therefore supporting the
linkage of amide (CONH) and sulphonamide (SO2–NH)
groups in developed molecules. Furthermore, the presence of
carbonyl C=O stretching (1700–1600 cm−1) and NH-bending
(1600 cm−1) vibrations in spectrum of molecules signified the
occurrence of amide carbonyl and aromatic NH-functional
group (Fig. 2).
A singlet signal comparable to one CONH proton at around
δ 10 ppm was observed in the proton NMR spectra of the
synthesized molecules verifying the existence of ‘benzamide
link’ in synthesized compounds. The detection of singlet
signal for one ‘NH proton’ of SO2NH functional moiety was
recorded ~2.5 ppm, demonstrating the development of sul-
phonamide analogues by an interaction of sulphonyl chloride
compounds with the respective amines. The presence of a
singlet, two doublets and a triplet signal about δ 8 ppm
referring to 1H-atoms at C2, C4, C5 and C6; correspondingly of
the benzoic acid-derived phenyl moiety verified that the
‘benzamide link’ and the ‘sulphonamide linkage’ were meta to
one another i.e., alienated by C2. Two doublets were recorded
in the proton-NMR spectra of prepared compounds in
7–8 ppm range, apart from 2 ‘triplet signals’ pointing to four
1H-atoms (corresponding to aromatic CH), demonstrating that
2-aminobenzimidazole was combined with benzoyl chloride
for development of benzamide derivatives. Occurrence of
Prediction of ADME properties
singlet signal at
benzimidazol-2-yl
δ
5 ppm corresponding to NH of
ADME parameters such as ‘molecular weight’ (MW), ‘par-
tition coefficient’ (log P), distribution coefficient (log D),
further confirmed presence of

766
Medicinal Chemistry Research (2021) 30:760–770
Fig. 2 GK activity (GK fold
activation) of the synthesized
derivatives (at 10 µM
concentration). Values were
presented as mean S.D. (n = 3)
and data was significantly
dissimilar compared to control
(p < 0.05)
Table 2 Predicted ADME
properties of the designed
compounds
Compound
MW
log P
log D
log Sw
tPSA
HBA
HBD
Solubility
NRB
1
2
3
4
5
6
7
8
9
10
392.43
471.87
406.46
372.44
330.36
406.46
471.33
406.46
437.43
358.41
3.30
3.64
3.12
2.99
1.74
3.54
3.87
3.54
3.01
2.63
3.38
3.55
3.59
3.19
1.86
3.90
4.14
3.92
3.21
2.74
−4.44
−5.07
−4.33
−3.88
−3.09
−4.66
−5.29
−4.66
−4.46
−3.65
112.33
161.72
112.33
112.33
112.33
112.33
112.33
112.33
158.15
112.33
4
6
4
4
4
4
4
4
6
4
3
3
3
3
3
3
3
3
3
3
4623.29
2974.48
5372.02
7705.53
15085.68
3859.76
2377.48
3859.76
5077.05
9341.62
4
5
5
6
3
4
5
4
5
5
water solubility (log Sw), ‘topological polar surface area’
(tPSA), ‘H-bond acceptors’ (HBA), ‘H-bond donors’ (HBD),
solubility (mg/L) and ‘number of rotatable bonds’ (NRB)
were predicted for all the designed analogues. All of the
designed analogues showed good pharmacokinetic para-
meters for oral bioavailability (Table 2) and drug-like prop-
erties as described using “Lipinski’s rule of five” (i.e., MW <
500 Da; log P < 5; HBA ≤ 10 and HBD: ≤ 5). All the
designed molecules showed MW (330–472 Da), log P value
(1.70–3.87), HBA (4–6) and HBD (3) within the range of
ideal orally bioavailable drug candidate.
Amongst the synthesized analogues, compounds bearing
N-(2-chloro-4-nitrophenyl) sulphonamide moiety (2) and
N-(4-bromophenyl) sulphonamide moiety (7) exhibited
highest GK activity (GK activation fold of 2.11 and 2.07,
respectively compared to control). Analogues having N-
phenyl, N-2-methylphenyl and N-2- nitrophenyl sulphona-
mide moiety (1, 6 and 9) demonstrated 1.82, 1.81 and 1.78-
fold GK activation, correspondingly. Synthesized com-
pounds having N-4-methylphenyl and N-methyl sulphona-
mide moiety (8 and 10, respectively) exhibited moderate
GK activity (1.67 and 1.64-fold GK activation, respec-
tively). Analogues bearing N-benzyl and N-butyl sulpho-
namide moiety (3 and 4, respectively) exhibited ~1.5 times
activation in comparison to control. Derivative having N-
methyl sulphonamide moiety (5) showed lowest GK fold
activation. Outcomes of the GK test demonstrated that
replacement of substituted phenyl moiety substituted to
SO2NH resulted in improved GK activity compared to those
having alkyl group as can be observed from the GK acti-
vation potential of compounds 2, and 7. Substitution of
aromatic moiety attached to SO2NH with alkyl chains have
decreased the capacity for GK activation compared to
In vitro GK assay
The GK catalytic effect of the newly prepared compounds
was assessed spectrometrically by calculating the absor-
bance around 340 nm through coupled interaction with
G6PDH. Amongst the tested derivatives, two molecules
(compounds 2 and 7) demonstrated peak GK activation
(fold activation exceeding 2.0 in contrasts to control). Other
compounds demonstrated modest activation (1.4 to 1.8-fold
compared to control) of GK enzyme (Fig. 2).

Medicinal Chemistry Research (2021) 30:760–770
Table 3 Binding interactions
and docking score (ΔG) of the
docked designed derivatives
767
Ligand H-bond interactions
Residues Distance (Å)
Residues involved in hydrophobic interactions
ΔG
1
2
Arg63
Arg63
Arg63
Arg63
Arg63
Arg63
Arg63
Arg63
Arg63
Ser69
3.0, 3.0
3.1, 2.9
4.2, 3.8
3.1, 3.0
3.2, 3.0
3.0, 3.0
2.9, 2.9
3.1, 3.0
3.0, 3.0
3.1, 3.0
Pro66, Pro99, Ile159, Ile211, Tyr214, Ala454, Val455
−10.4
Pro66, Pro99, Ile159, Met 210, Ile211, Tyr214, Ala454, Val455 −10.9
3
Pro66, Ile159, Ile211, Tyr214, Ala454, Val455
−9.1
−9.0
−8.9
−0.0
−10.8
−9.9
−10.0
−9.5
4
Pro66, Ile159, Ile211, Tyr214, Ala454, Val455
5
Pro66, Ile159, Met 210, Ile211, Tyr214, Ala454, Val455
Pro66, Pro99, Ile159, Ile211, Tyr214, Ala454, Val455
Pro66, Pro99, Ile159, Ile211, Tyr214, Ala454
6
7
8
Pro66, Pro99, Ile159, Met 210, Ile211, Tyr214, Ala454
Pro66, Pro99, Ile159, Met 210, Ile211, Tyr214, Ala454
Pro66, Pro99, Ile159, Met 210, Ile211, Tyr214, Ala454
9
10
Fig. 3 Superposition of the docked poses of compounds 1, 2, 6 and 7 (yellow) on docked pose of the PDB ligand of 3IMX (gray) in the allosteric
binding site of GK protein
compounds 4, 5 and 10 with aromatic moiety as shown by
GK activity.
GK (PDB ID: 3IMX). The reference GK activator (PDB
entry: 3IMX) developed a similar linking sequence and
transposed on bonding fashion of co-crystallized GK activator
with ΔG of −9.0 kcal/mol validating accuracy of the docking
methodology used. Most of the docked ligands showed
appreciable linking in allosteric location of GK as established
by examining their linkage interactions and ΔG of the para-
mount docked facades (Table 3). These compounds displayed
In silico docking investigations
In silico molecular docking investigations were performed to
discover the connection and linking behaviors of developed
molecules using AutoDock Vina in the allosteric location of

768
Medicinal Chemistry Research (2021) 30:760–770
Fig. 4 Best docked poses displaying binding interactions of the analogues 1, 2, 6 and 7 with allosteric site residues of GK protein
strong H-bond interactions between ‘–NH’ of benzamide and
‘amide carbonyl’ of Arg63; and ‘N’ of the benzimidazol-2-yl
ring and ‘amide –NH’ of Arg63 in GK protein’s allosteric
position.
Super-positioning of the docking conformations of ana-
logues 1, 2, 6 and 7, on that of reference ligand in the
allosteric site of GK demonstrated that these molecules had
the analogous binding and orientation arrangement in the
allosteric site of GK enzyme as that of the x-ray crystallized
effector (PDB entry: 3IMX) supporting the outcomes of
in vitro GK test for these compounds (Fig. 3).
The docked conformations of analogues 1, 2, 6 and 7
demonstrated strong hydrogen bond interactions between ‘N’
of benzimidazol-2-yl group and amide –NH of Arg63 resi-
dues; and ‘benzamide –NH’ group and ‘backbone carbonyl’
of Arg63 in the allosteric location of GK with bond length in
the range 2.9–3.1 Å; and 2.9–3.0 Å, respectively. Overall, the
benzimidazol-2-yl moiety bonded to the benzamide ‘NH’ of
these compounds protruded in the hydrophobic cavity dis-
playing connections with the Val455 and Lys459 amino acid
residues, along with Pro66 and Ile159 amino acid residues,
aromatic moiety parceled in the cavity composed of Met210,
Tyr214 and Val455 residues (Fig. 4) (Table 3).
In silico prediction of toxicity
The possible toxicity (mutagenic, cardiotoxicity, acute
toxicity, hepatotoxicity, skin irritation, and chronic toxicity)
for the optimized compounds was accessed using pkCSM
online platform. Conferring to the results represented in
Table 4; some of the compounds showed little toxicity
probability. Mutagenicity was predicted for all the synthe-
sized compounds. Cardiotoxicity (inhibition of hERG-I and
hERG-II) was predicted for compounds 1 and 3. Hepato-
toxicity was predicted for compounds 1, 3 and 8. In this
perspective, the initial evaluation performed in silico using
pkCMS online platform, can supplement forthcoming stu-
dies related to the safety of these compounds.
Conclusions
In summary, a series of novel N-benzimidazol-2-yl benza-
mide analogues were designed and prepared using structure-
based drug design approach. Among these newly identified
derivatives, analogues 2 and 7 unveiled maximum GK
activation potential (>2.0-fold increase in GK catalytic

Medicinal Chemistry Research (2021) 30:760–770
Table 4 Toxicity prediction for
the optimized compounds
obtained using pkCSM
769
Compound Mutagenicitya Cardio-
Acute
Chronic
toxicityd
Hepato-
toxicity
Skin
irritation
Max.
toxicityb
toxicityc
tolerated
dosee
1
2
3
4
5
6
7
8
9
10
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
No
No
No
No
No
No
No
2.491
2.461
2.494
2.137
2.038
2.447
2.410
2.483
2.409
2.085
2.516
3.181
2.575
2.592
2.613
2.865
2.774
3.224
2.824
2.586
Yes
No
Yes
No
No
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
0.331
0.391
0.315
0.326
0.348
0.399
0.373
0.350
0.374
0.335
aMutagenicity was accessed using “AMES” test
bCardiotoxicity was accessed using hERG-I and hERG-II inhibition
cAcute Toxicity: “Oral rat acute toxicity (LD50 in mol/kg)”
dChronic Toxicity: “Oral rat chronic toxicity (log mg/kg_bw/day)”
eMax. Tolerated Dose (Human): “log mg/kg/day”
activity in vitro). Outcomes of the in-vitro test were found to
be analogous to the in-silico docking investigations with the
GK enzyme. These analogues followed the ‘Lipinski’s rule
of five’ for the “drug-like” characteristics. These newly
developed compounds might assist in finding the lead ana-
logues for discovery of strong and harmless activators of GK
for T2D handling and management.
the treatment of metabolic syndrome. Med Chem. 2016;12:03–21.
https://doi.org/10.2174/1573406411666150525105826
6. Grewal AS, Sekhon BS, Lather V. Recent updates on glucoki-
nase activators for the treatment of type 2 diabetes mellitus.
Mini Rev Med Chem. 2014;14:585–602. https://doi.org/10.
2174/1389557514666140722082713
7. Grewal AS, Bhardwaj S, Pandita D, Lather V, Sekhon BS. Updates
on aldose reductase inhibitors for management of diabetic com-
plications and non-diabetic diseases. Mini Rev Med Chem.
2016;16:120–162. https://doi.org/10.2174/1389557515666150909
143737
8. Pal M. Medicinal chemistry approaches for glucokinase activation
to treat type 2 diabetes. Curr Med Chem. 2009;16:3858–3874.
https://doi.org/10.2174/092986709789177993
Acknowledgements The authors are thankful to Chitkara College of
Pharmacy, Chitkara University, Punjab for their support and encour-
agement for this research work.
9. Coghlan M, Leighton B. Glucokinase activators in diabetes
management. Expert Opin Investig Drugs. 2008;17:145–167.
https://doi.org/10.1517/13543784.17.2.145
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
10. Perseghin G. Exploring the in vivo mechanisms of action of
glucokinase activators in type 2 diabetes. J Clin Endocrinol
Metab. 2010;95:4871–4873. https://doi.org/10.1210/jc.2010-2049
11. Matschinsky FM, Zelent B, Doliba N, Li C, Vanderkooi JM, Naji
A, et al. Glucokinase activators for diabetes therapy. Diabetes
Care. 2011;34:S236–43. https://doi.org/10.2337/dc11-s236
12. Grewal AS, Lather V, Charaya N, Sharma N, Singh S, Kairys V.
Recent developments in medicinal chemistry of allosteric activa-
tors of human glucokinase for type 2 diabetes mellitus ther-
apeutics. Curr Pharm Des. 2020;26:2510–2552. https://doi.org/10.
2174/1381612826666200414163148
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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