Synthesis of benzimidazole derivatives as potent inhibitors for α-amylase and their molecular docking study in management of type-II diabetes

Article abstract of DOI:10.1016/j.molstruc.2021.130029

In the search of potent α-amylase inhibitors, we have synthesized seventeen derivatives of 2-mercaptobenzimidazole bearing sulfonamide (1–17) and evaluated for their α-amylase inhibitory potential. All synthesized compounds display a variable degree of α-amylase activity having IC₅₀ values ranging between 0.90 ± 0.05 to 11.20 ± 0.30 μM when compared with the standard drug acarbose having IC₅₀ value 1.70 ± 0.10 μM. Compound 1, 2, 11, 12 and 14 having IC₅₀ values 1.40 ± 0.10, 1.30 ± 0.05, 0.90 ± 0.05, 1.60 ± 0.05 and 1.60 ± 0.10 μM respectively were found many folds better than the standard drug acarbose. While others derivatives of the series showed good inhibitory potentials. All the synthesized compounds were characterized by HREI-MS, ¹H and ¹³C NMR spectroscopy. Structure activity relationship (SAR) has been established for all newly synthesized analogs. Binding interactions between ligands and active residues of the enzyme were confirmed through molecular docking study.

Full text of DOI:10.1016/j.molstruc.2021.130029

Journal of Molecular Structure 1232 (2021) 130029
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Synthesis of benzimidazole derivatives as potent inhibitors for
α-amylase and their molecular docking study in management of
type-II diabetes
Shafqat Hussaina, Muhammad Taha b,∗, Fazal Rahimc,∗, Shawkat Hayatc, Khalid Zamanc,
Naveed Iqbald, Manikandan Selvaraje, Muhammad Sajidf, Masroor Ahmad Bangeshc,
Fahad Khanc, Khalid Mohammed Khang, Nizam Uddinh, Syed Adnan Ali Shahi,j,
Muhammad Alik
a Department of Chemistry, University of Baltistan, Skardu, Kargil-Skardu Road, Hussainabad, Skardu, Gilgit-Baltistan, Pakistan
b Department of Clinical Pharmacy, Institute of Research and Medical Consultation (IRMC), Imran Abdulrahman Bin Faisal University, P.O. Box 1982, Damam
31441, Saudi Arabia
c Department of Chemistry, Hazara University, Mansehra-21300, Khyber Pakhtunkhwa, Pakistan
d Department of Chemistry, University of Poonch Rawalakot AJK, Pakistan
e Chemical Engineering Discipline, School of Engineering, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor, Malaysia
f Department of Biochemistry, Hazara University, Mansehra-21300, Khyber Pakhtunkhwa, Pakistan
g H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
h Department of Chemistry, University of Karachi, Karachi, 75270 Pakistan
i Faculty of Pharmacy, Universiti Teknologi MARA Cawangan Selangor Kampus Puncak Alam, 42300 Bandar Puncak Alam, Selangor D. E., Malaysia
j Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Universiti Teknologi MARA Cawangan Selangor Kampus Puncak Alam, 42300 Bandar
Puncak Alam, Selangor D. E., Malaysia
k Natural and Medical Sciences Research Center, University of Nizwa, Nizwa 616, Oman
a r t i c l e i n f o
a b s t r a c t
Article history:
In the search of potent α-amylase inhibitors, we have synthesized seventeen derivatives of 2-
mercaptobenzimidazole bearing sulfonamide (1–17) and evaluated for their α-amylase inhibitory poten-
tial. All synthesized compounds display a variable degree of α-amylase activity having IC50 values rang-
ing between 0.90 0.05 to 11.20 0.30 μM when compared with the standard drug acarbose having IC50
value 1.70 0.10 μM. Compound 1, 2, 11, 12 and 14 having IC50 values 1.40 0.10, 1.30 0.05, 0.90 0.05,
1.60 0.05 and 1.60 0.10 μM respectively were found many folds better than the standard drug acar-
bose. While others derivatives of the series showed good inhibitory potentials. All the synthesized com-
pounds were characterized by HREI-MS, 1H and 13 C NMR spectroscopy. Structure activity relationship
(SAR) has been established for all newly synthesized analogs. Binding interactions between ligands and
active residues of the enzyme were confirmed through molecular docking study.
Received 27 October 2020
Revised 24 January 2021
Accepted 25 January 2021
Available online 31 January 2021
Keywords:
Synthesis
Benzimidazole derivatives
Molecular docking study
α-amylase
SAR
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
synthesized to increase the stability, bioavailability and promising
biological profiles [2,3]. In view of their affinity towards enzymes
Benzimidazole is fused aromatic heterocyclic compound and a
privileged scaffold with active pharmacophore in medicinal chem-
istry. Until the present by moiety of choice it displayed versa-
tile character, and in nature N-ribosyldimethylbenzimidazol is the
most prominent compound which act as axial ligand in vitamin
B12 for cobalt [1]. Aimed at the structural modifications for bio-
logical potentials, various derivatives of benzimidazole have been
and proteins, medicinal chemist employed it as sub-structure in
drug designing and synthesis. Due to its chemotherapeutic val-
ues, benzimidazole and its analogs received much attention by
the researchers [4]. Benzimidazole derivatives proved to be use-
ful therapeutics such as diuretics [5], antimicrobial [6], anticancer
[7], antiprotozoal [8], antidiabetic [9], antioxidant [10], and an-
ticonvulsant [11], respectively. Moreover, many other benzimida-
zole derivatives were synthesized from 2-marcatobenzimidazole
(1H-benzo[d]imidazole-2-thiol), while these derivatives demon-
strated their biological activities such as antidiabetic [12], antiviral
[13] and antimicrobial [14], respectively.
∗
Correspondence author.
E-mail addresses: mtaha@iau.edu.sa (M. Taha), fazalstar@gmail.com (F. Rahim).
https://doi.org/10.1016/j.molstruc.2021.130029
0022-2860/© 2021 Elsevier B.V. All rights reserved.

S. Hussain, M. Taha, F. Rahim et al.
Journal of Molecular Structure 1232 (2021) 130029
Fig. 1. Current work with previously reported work.
Diabetes mellitus (DM) is chronic metabolic disorder charac-
terized by hyperglycemia ensuing by defects in insulin secretion
and insulin action [15]. The current estimate and forecast about
diabetic patient indicate that the figure may reach from 383 mil-
lion to 592 million by the year 2035 [16]. Defects in insulin secre-
tion and action prompt the glucose level in blood which particu-
larly damages the blood vessels and initiates some other compli-
cations like neuropathy, retinopathy, ulceration, cardiovascular and
nephropathy, respectively [17]. The detail innovative study showed
that postprandial hyperglycemia kept in normal level by inhibiting
intestinal hydrolyse enzyme α-amylase and α-glucosidase are the
practical solutions for glycemic control [18,19]
α-Amylase (E.C.3.2.1.1) is hydrolyse enzyme comprises of Ca+2
ion in its active pocket. It catalyzes the conversion of starch into
glucose and maltose by involving water molecule respectively.
Alpha-amylase catches considerable attention due to potential ca-
pability on attacking of α−1,4-glycosidic linkage by hydrolysis [20–
23]. Drugs such as voglibose, acarbose and miglitol have the func-
tion to inhibit the hydrolyzing ability of α-amylase enzyme and
consequently stopped the absorption of glucose. Although, these
drug found with some side effects and therefore to minimize its
adverse effect other antidiabetic agents are employed in combi-
nation during treatment [24–27]. In this context, medicinal and
synthetic chemists reported variety of heterocyclic scaffolds for
promising antidiabetic activity [28–31].
Based on our earlier reports on benzimidazole class of [40–42],
we have synthesized some novel benzimidazole derivatives bear-
ing sulfonamide moiety and evaluated them for their α-amylase
inhibitory activity in search potent candidates (Fig. 1).
2. Material and methods
All chemicals and reagents which used in this protocol were
purchased from sigma Aldrich. The purity of chemicals and
reagents which were employed for synthesis of intermediate and
benzimidazole derivatives were checked by TLC analysis. Moreover,
the structures of all derivatives were confirmed via spectroscopic
techniques like 1H NMR (500 MHz) and 13C NMR (125 MHz) spec-
troscopy respectively.
2.1. Synthesis of 2-((substituted-1H-benzo[d]imidazol-2-yl)thio)−1-
substituted-phenylethan-1-one
2-Mercaptobenzimidazole/6-methoxy-2-
mercaptobenzimidazole (5 mmol), different substituted phenacyl-
bromides (5 mmol) were reacted and stirred the mixture at room
temperature in acetone (20 ml) in the presence of potassium
carbonate which yielded intermediate product (I). The progress
of reaction was monitored with the help of TLC. After reaction
completion, reaction mixture was cooled at low temperature and
filtered the precipitate to achieve crude product. The product was
recrystallized in methanol to obtained pure product.
With continuing efforts, our group has reported numerous hete-
rocycles for potent therapeutics potentials [32–36] including some
potent antidiabetic agents as well [37–39]. In this manuscript,
2

S. Hussain, M. Taha, F. Rahim et al.
Journal of Molecular Structure 1232 (2021) 130029
2.2. Synthesis of substituted benzenesulfonohydrazide
148.89, 145.42, 140.33, 139.92, 134.97, 134.97, 133.54, 133.17, 132.2,
130.41, 129.36, 129.36, 129.25, 129.25, 129.11, 129.11, 128.7, 128.61,
128.61, 127.57, 127.19, 127.02, 127.02, 127.00, 114.19, 114.19, 32.51;
HREI-MS: m/z calcd for C27H21ClN4O2S2, [M]+ 532.0794; Found;
532.0778.
Various sulfonyl chlorides were treated (5 mmol) with equiva-
lent quantity of hydrazine hydrate and refluxed the reaction blend
in methanol (20 ml) to obtain substituted benzenesulfonohydrazide
as intermediate product (II). The progress of reaction was moni-
tored with the help of TLC. After reaction completion, the solvent
was evaporated under vacuum to obtain crude product. The prod-
uct was recrystallized in ethyl acetate to obtained pure product.
2.3.5. N’-(2-((1H-benzo[d]imidazol-2-yl)thio)−1-(4-bromophenyl)
ethylidene)−4-methoxy benzenesulfonohydrazide (5)
Yield: 68%; 1HNMR (500 MHz, DMSO-d6): δ 12.55 (s, 1H, NH),
11.99 (s, 1H, NH), 8.17 (d, J = 6.9 Hz, 2H, ArH), 7.93 (d, J = 6.9 Hz,
2H, ArH), 7.80 (d, J = 6.1 Hz, 2H, ArH), 7.59 (d, J = 7.4 Hz, 1H, ArH),
7.55 (t, J = 6.2 Hz, 2H, ArH), 7.47 (d, J = 6 Hz, 1H, ArH), 7.14 (d,
J = 6.4 Hz, 1H, ArH), 7.08 (dd, J = 1.9, 7.4, Hz, 1H, ArH), 5.41 (s, 2H,
CH2), 3.87 (s, 3H, O-Me). 13CNMR (125 MHz, DMSO-d6): δ 164.31,
157.33, 148.84, 145.41, 138.56, 138.56, 133.50, 129.27, 129.27, 129.11,
128.61, 128.61, 127.58, 127.58, 127.02, 127.02, 114.17, 114.17, 114.01,
114.01, 55.84, 32.52; HREI-MS: m/z calcd for C22H19BrN4O3S2, [M]+
530.0082; Found: 530.0068.
2.3. Synthesis of 2-marcaptobenzimidazole bearing sulfonamide
analogs (1–17)
Equivalent quantity of product (I) was reacted with intermedi-
ate product (II) and refluxed in methanol in the presence of acetic
acid to give 2-mercaptobenzimidazole bearing sulfonamide analogs
(1–17). The progress of reaction was monitored with the help of
TLC. After reaction completion, the solvent was allowed to evapo-
rate to achieve crude product. The crude product was washed with
hexane and ethyl acetate to obtained pure product
ꢀ
2.3.6. N’-(2-((1H-benzo[d]imidazol-2-yl)thio)−1-([1,1 -biphenyl]−4-
yl)ethylidene)−2-bromo benzenesulfonohydrazide (6)
2.3.1. N’-(2-((6-methoxy-1H-benzo[d]imidazol-2-yl)thio)−1-
Yield: 75%; 1HNMR (500 MHz, DMSO-d6): δ 11.46 (s, 1H, NH),
11.08 (s, 1H, NH), 8.03–7.99 (m, 3H, ArH), 7.80–7.76 (m, 3H, ArH),
7.52 (t, J = 6.8 Hz, 3H, ArH), 7.20–7.14 (m, 5H, ArH), 7.02–6.98
(m, 3H, ArH), 5.38 (s, 2H, CH2); 13CNMR (125 MHz, DMSO-d6): δ
157.37, 148.89, 145.42, 141.70, 139.92, 134.97, 134.97, 133.54, 132.5,
131.41, 129.36, 129.36, 129.25, 129.25, 129.12, 129.11, 129.11, 128.61,
128.61, 128.01, 127.57, 127.19, 127.02, 121.03, 114.19, 114.19, 32.50;
HREI-MS: m/z calcd for C27H21BrN4O2S2, [M]+ 576.0289; Found;
576.0274.
(3-nitrophenyl)ethylidene)−2- methylbenzenesulfonohydrazide (1)
Yield: 68%; 1HNMR (500 MHz, DMSO-d6): δ 12.51 (s, 1H, NH),
11.49 (s, 1H, NH), 8.53 (J = 1.2 Hz, 1H, Ar), 8.16 (J = 6.9 Hz, 2H,
ArH), 7.93 (d, J = 7.3 Hz, 2H, ArH), 7.80 (d, J = 6.8 Hz, 2H, ArH), 7.56
(m, 3H, ArH), 7.47 (d, J = 6.2 Hz, 1H, ArH), 7.12 (d, J = 7.5 Hz, 1H,
ArH), 7.05 (dd, J = 1.9, 6.8 Hz, 1H, ArH), 5.36 (s, 2H, CH2), 3.88
(s, 3H, O-Me), 2.09 (s, 3H, CH3). 13CNMR (125 MHz, DMSO-d6): δ
157.15, 156.42, 148.79, 145.37, 138.57, 134.57, 133.57, 131.28, 129.25,
129.25, 129.11, 128.60, 128.22, 127.02, 127.00, 125.80, 125.42,
114.06, 113.83, 96.16, 55.80, 30.64, 21.01; HREI-MS: m/z calcd for
C23H21N5O5S2, [M]+ 511.0984; Found: 511.0979.
ꢀ
2.3.7. N’-(2-((1H-benzo[d]imidazol-2-yl)thio)−1-([1,1 -biphenyl]−4-
yl)ethylidene)−4-methoxy benzenesulfonohydrazide (7)
Yield: 68 %; 1HNMR (500 MHz, DMSO-d6): δ 12.62 (s, 1H, NH),
11.98 (s, 1H, NH), 8.09 (d, J = 6.7 Hz, 2H, ArH), 7.98–7.95 (m, 3H,
ArH), 7.92–7.89 (m, 3H, ArH), 7.56–7.51 (m, 4H, ArH), 7.46–7.41 (m,
2H, ArH), 7.06 (d, J = 6.1 Hz, 1H, ArH), 7.02 (t, J = 6.7, Hz 1H, ArH),
5.34 (s, 2H, CH2), 3.81 (s, 3H, O-Me). 13CNMR (125 MHz, DMSO-
d6): δ 166.41,157.37, 148.89, 145.42, 139.92, 138.58, 134.97, 134.97,
133.54, 129.36, 129.36, 129.25, 129.25, 129.11, 129.11, 128.61,
128.61, 127.57, 127.19, 127.04, 127.04, 127.02, 127.02, 120.88, 120.88,
114.19, 114.19, 55.80, 32.50; HREI-MS: m/z calcd for C28H24N4O3S2,
[M]+ 528.1290; Found; 528.1277.
2.3.2. N’-(2-((6-methoxy-1H-benzo[d]imidazol-2-yl)thio)−1-
(4-nitrophenyl)ethylidene)−2-methylbenzenesulfonohydrazide (2)
Yield: 71%; 1HNMR (500 MHz, DMSO-d6): δ 12.49 (s, 1H, NH),
11.92 (s, 1H, NH), 8.76 (d, J = 6.7 Hz, 2H, Ar), 8.54 (dd, J = 1.5,
6.6,7 Hz, 2H, ArH), 7.92 (t, J = 6.6 Hz, 2H, ArH), 7.48 (d, J = 1.4 Hz,
1H, ArH), 7.06 (d, J = 6.2 Hz, 2H, ArH), 6.96 (dd J = 1.8, 7.2 Hz,
2H, ArH), 5.32 (s, 2H, CH2), 3.82 (s, 3H, O-Me), 2.09 (s, 3H,
CH3). 13CNMR (125 MHz, DMSO–d6): δ 157.15, 156.42, 151.03, 147.9,
145.37, 141.08, 138.57, 131.2, 129.25, 129.11, 128.60, 128.22, 128.11,
128.11, 127.61, 127.61, 127.50, 114.06, 113.83, 96.16, 55.8, 32.51,
21.02; HREI-MS: m/z calcd for C23H21N5O5S2, [M]+ 511.0984;
Found: 511.0970.
2.3.8. N’-(2-((1H-benzo[d]imidazol-2-yl)thio)−1-(4-bromophenyl)
ethylidene)−2-bromo benzenesulfonohydrazide (8)
Yield: 73%; 1HNMR (500 MHz, DMSO-d6): δ 12.03 (s, 1H, NH),
11.25 (s, 1H, NH), 8.20 (d, J = 7.2 Hz, 2H, ArH), 7.93 (d, J = 6.9 Hz,
2H, ArH), 7.80 (d, J = 6.2 Hz, 2H, ArH), 7.58 (m, 3H, ArH), 7.47 (t,
J = 6.1 Hz, 1H, ArH), 7.13 (d, J = 6.1 Hz, 1H, ArH), 7.07 (dd, J = 1.8,
7.2 Hz, 1H, ArH), 5.38 (s, 2H, CH2). 13CNMR (125 MHz, DMSO-d6): δ
157.33, 148.84, 145.41, 138.56, 138.56, 135.34, 133.50, 130.0, 129.53,
129.27, 129.27, 129.11, 128.61, 128.61, 128.11, 127.03, 127.03, 120.08,
114.17, 114.17, 32.50; HREI-MS: m/z calcd for C21H16 Br2N4O2S2,
[M]+ 577.9081; Found: 577.9078.
ꢀ
2.3.3. N’-(2-((1H-benzo[d]imidazol-2-yl)thio)−1-([1,1 -biphenyl]−4-
yl)ethylidene)−2-nitro benzenesulfonohydrazide (3)
Yield: 68%; 1HNMR (500 MHz, DMSO–d6): δ 11.58 (s, 1H, NH),
10.51 (s, 1H, NH), 8.78 (dd, J = 1.2, 7.4 Hz, 1H, ArH), 8.46(dd,
J = 6.6 Hz, 1H, ArH), 8.03 (t, J = 6.4 Hz, 1H, ArH), 7.48 (m, 5H,
ArH), 7.25 (m, 4H, ArH), 6.89 (m, 5H, Ar), 5.29 (s, 2H, CH2).
13CNMR (125 MHz, DMSO-d6): δ 157.37, 151.32, 148.89, 145.42,
139.92, 136.43, 134.97, 134.07, 133.54, 133.08, 129.36, 129.36,
129.25, 129.25, 129.11, 129.11, 128.61, 128.61, 128.05, 127.57, 127.19,
127.02, 127.02, 125.41, 114.19, 114.19, 32.50; HREI-MS: m/z calcd for
C27H21N5O4S2, [M]+ 543.1035; Found; 543.1023.
2.3.9. N’-(2-((1H-benzo[d]imidazol-2-yl)thio)−1-(4-methoxyphenyl)
ethylidene)−2-bromo benzenesulfonohydrazide (9)
Yield: 74%; 1HNMR (500 MHz, DMSO-d6): δ 9.50 (s, 2H, NH),
8.01 (d, J = 6.3 Hz, 2H, ArH), 7.78 (m, 2H, ArH), 7.66 (m, 2H, ArH),
7.50 (d, J = 6.7 Hz, 2H, ArH), 7.25 (m, 2H, ArH), 6.68 (m, 2H,
ArH), 5.19 (s, 2H, CH2), 3.88 (s, 3H, O-Me). 13CNMR (125 MHz,
DMSO-d6): δ 166.41, 157.33, 148.84, 145.41, 138.56, 138.56, 135.34,
130.02, 129.53, 129.11, 128.61, 128.61, 128.0, 127.03, 127.03, 120.06,
116.43, 116.43, 114.17, 114.17, 55.81, 32.53; HREI-MS: m/z calcd for
C22H19 BrN4O3S2, [M]+ 530.0082; Found; 530.0069
ꢀ
2.3.4. N’-(2-((1H-benzo[d]imidazol-2-yl)thio)−1-([1,1 -biphenyl]−4-
yl)ethylidene)−2-chloro benzenesulfonohydrazide (4)
Yield: 71%; 1HNMR (500 MHz, DMSO–d6): δ 12.34 (s, 1H, NH),
12.31 (s, 1H, NH), 7.62–7.58 (m, 4H, ArH), 7.45 (d, J = 7.2 Hz, 2H,
ArH), 7.43 (d, J = 7.2 Hz, 2H, Ar), 7.36 (d, J = 7.6 Hz, 2H, ArH), 6.98
(t, J = 7.6 Hz, 2H, ArH), 6.78 (t, J = 7.1 Hz, 2H, ArH), 6.67–6.63 (m,
3H, ArH) 4.63 (s, 2H, CH2). 13CNMR (125 MHz, DMSO–d6): δ 157.37,
3

S. Hussain, M. Taha, F. Rahim et al.
Journal of Molecular Structure 1232 (2021) 130029
2.3.10. N’-(2-((1H-benzo[d]imidazol-2-yl)thio)−1-(4-nitrophenyl)
ethylidene)−2-bromo benzenesulfonohydrazide (10)
2.3.15. N’-(2-((1H-benzo[d]imidazol-2-yl)thio)−1-(2,
5-dimethoxyphenyl)ethylidene)−2-nitro benzenesulfonohydrazide (15)
Yield: 73%; 1HNMR (500 MHz, DMSO-d6): δ 11.49 (s, 2H, NH),
8.00 (d, J = 7.1 Hz, 2H, ArH) 7.84 (d, J = 7.0 Hz, 2H, ArH), 7.53
(d, J = 7.4 Hz, 1H, ArH), 7.10 (d, J = 1.8 Hz, 1H, ArH), 7.00 (m,
4H, ArH), 6.51 (d, J = 8.6 Hz, 1H, ArH), 5.47 (s, 2H, CH2), 3.89
Yield: 69%; 1HNMR (500 MHz, DMSO-d6): δ 12.32 (s, 1H,
NH), 11.26 (s, 1H, NH), 8.78 (d, J = 2.0 Hz, 1H, ArH), 8.14 (d,
J = 6.6 Hz, 2H, ArH), 7.86 (d, J = 6.1 Hz, 1H, ArH), 7.51 (d, 1H,
J = 6.1 Hz, ArH), 7.38–7.32 (m, 2H, ArH), 7.08–7.02 (m, 4H, ArH),
6.69 (dd J = 1.9, 7.4 Hz, 1H, ArH), 6.47–6.42 (m 2H, ArH), 4.91 (s,
2H, CH2). 13CNMR (125 MHz, DMSO-d6): δ 157.33, 151.62, 148.79,
145.37, 138.57, 134.57, 133.57, 130.24, 129.25, 129.11, 128.60, 128.60,
128.22, 128.22, 127.96 127.02, 127.00. 119.00, 114.06, 113.83, 32.51;
HREI-MS: m/z calcd for C21H16 BrN5O4S2, [M]+ 544.9827; Found:
544.9811.
(s, 6H, O CH3). 13CNMR (125 MHz, DMSO-d6): δ 157.33, 154.4,
–
153.34, 150.21, 148.84, 145.5, 138.56, 138.56, 136.11, 133.22, 129.11,
127.13, 127.13, 123.61, 121.32, 119.71, 116.05, 115.34, 114.17, 114.17,
55.80, 55.07, 32.57; HREI-MS: m/z calcd for C23H21N5O6S2, [M]+
527.0933; Found; 527.0924.
2.3.16. N’-(2-((1H-benzo[d]imidazol-2-yl)thio)−1-
(4-nitrophenyl)ethylidene)−2-nitro benzenesulfonohydrazide (16)
Yield: 62%; 1HNMR (500 MHz, DMSO-d6): δ 11.19 (s, 1H, NH),
10.37 (s, 1H, NH), 8.25 (d, J = 7.4 Hz, 2H, ArH), 8.07 (d, J = 7.1 Hz, 2H,
ArH), 7.61 (d, J = 6.2 Hz, 2H, ArH), 7.51 (m, 3H, ArH), 7.39(dd, J = 1.6,
6.3 Hz, 1H, ArH), 6.89 (dd, J = 1.6, 6.6 Hz, 1H, ArH), 6.77 (dd, J = 1.3,
8.2 Hz, 1H, ArH), 5.48 (s, 2H, CH2). 13CNMR (125 MHz, DMSO-d6): δ
157.33, 150.41, 148.84, 145.41, 140.17, 138.56, 138.56, 134.05, 133.50,
129.27, 129.11, 129.11, 128.61, 127.58, 127.58, 127.02, 127.02, 123.6,
114.17, 114.17, 32.53; HREI-MS: m/z calcd for C21H16 N6O6S2, [M]+
512.0573; Found; 512.055.
2.3.11. N’-(2-((1H-benzo[d]imidazol-2-yl)thio)−1-(3-nitrophenyl)
ethylidene)−2,4-difluoro benzenesulfonohydrazide (11)
Yield: 65%; 1HNMR (500 MHz, DMSO-d6): δ 11.92 (s, 1H,
NH), 11.88 (s, 1H, NH), 8.48 (d, J = 1.4 Hz, 1H, ArH), 8.01 (dd
J = 1.2,8.2 Hz, 1H, ArH), 7.78 (dd, J = 1.6, 6.4 Hz, 1H, ArH), 7.51 (m,
1H, ArH), 7.46 (t, J = 6.2 Hz, 1H, ArH), 7.24 (dd, J = 1.9, 8.2 Hz,
1H, ArH), 6.91 (t, J = 7.2 Hz, 1H, ArH), 6.54 (m, 4H, ArH), 5.48 (s,
2H, CH2). 13CNMR (125 MHz, DMSO-d6): δ 167.98, 167.66, 157.33,
148.84, 138.56, 138.56, 133.50, 133.03, 130.31, 129.25, 129.25,
129.11, 127.21, 127.03, 127.03, 125.40, 114.17, 114.17, 109.95, 109.66,
32.51; HREI-MS: m/z calcd for C21H15F2N5O4S2, [M]+ 503.0534;
Found; 503.0518.
2.3.17. N’-(2-((1H-benzo[d]imidazol-2-yl)thio)−1-
(4-bromophenyl)ethylidene)−2-methyl benzenesulfonohydrazide (17)
Yield: 68%; 1HNMR (500 MHz, DMSO-d6): δ 12.52 (s, 1H, NH),
10.91 (s, 1H, NH), 8.22 (d, J = 7.8 Hz, 2H, ArH), 7.87 (d, J = 7.7 Hz,
2H, ArH), 7.76 (d, J = 6.2 Hz, 2H, ArH), 7.56 (dd, J = 1.4, 6.6 Hz,
1H, ArH), 7.49 (d, J = 6.8 Hz, 2H, ArH), 7.42 (dd J = 1.6, 7.4 Hz, 1H,
ArH), 7.11 (d, J = 6.9 Hz, 1H, ArH), 76.99 (dd J = 1.8, 8.2 Hz, 1H,
ArH), 5.48 (s, 2H, CH2), 2.25 (s, 3H, CH3). 13CNMR (125 MHz,
DMSO d6): δ 164.31, 157.33, 148.84, 139.21, 138.56, 138.56, 136.06,
133.50, 132.31, 131.61, 130.05, 129.27, 129.27, 129.11, 128.61, 128.61,
127.03, 127.03, 114.17, 114.17, 32.50, 21.07; HREI-MS: m/z calcd for
C22H19 BrN4O2S2, [M]+ 514.0133, Found; 514.0118.
ꢀ
2.3.12. N’-(1-([1,1 -biphenyl]−4-yl)−2-((6-methoxy-1H-benzo[d]
imidazol-2-yl)thio)ethylidene) −2,4-difluorobenzenesulfonohydrazide
(12)
Yield: 71%; 1HNMR (500 MHz, DMSO-d6): δ 12.66 (s, 1H, NH),
11.48 (s, 1H, NH), 8.31 (d, J = 7.2 Hz, 2H, ArH), 8.19 (m, 3H, ArH),
7.91 (m, 3H, ArH), 7.59 (m, 3H, ArH), 7.49 (dd, J = 1.6, 6.9 Hz, 2H,
ArH), 6.92 (d, J = 1.4 Hz, 1H, ArH), 6.88 (dd, J = 1.2, 8.3 Hz, 1H,
ArH), 5.47 (s, 2H, CH2) 3.88 (3H, O-Me). 13CNMR (125 MHz, DMSO-
d6): δ 167.98, 167.66, 157.37, 148.89, 145.42, 139.92, 134.97, 133.54,
130.30, 129.36, 129.36, 129.25, 129.25, 129.11, 129.11, 128.61,
128.61, 127.57, 127.19, 127.02, 127.02, 121.44, 110.39, 110.19, 105.95,
100.66, 55.8, 32.53; HREI-MS: m/z calcd for C28H22F2N4O3S2, [M]+
564.1101; Found; 564.1092.
3. Docking study protocol
Docking study was done targeting the crystal structure of amy-
lase (PDB ID: 4W93) [38] in order to reveal the binding modes of
synthesized derivatives (1–17). For the purpose of docking stud-
ies, protein preparation module in discovery studio 2018 (Dassault
systemes BIOVIA, USA) was used to optimize the crystal structure
of amylase [43]. From protein data bank (PDB), crystal structure
was retrieved and furthermore, structure was optimized by re-
moving co-factors, hetero-atoms and H2O molecule. Charges, hy-
drogen bond and missing atom were computed. Built and ligand
preparation module implemented in discovery studio 2018 (Das-
sault systems BIOVIA, USA) was used to prepared and optimized
the docking study of the synthesized analogs (1–17). Gold dock-
ing tool was used for the purpose of docking; ligand preparation
comprises stereochemistry, assigning bond order and various tau-
tomers. Moreover, by choosing centroid of complex ligand (Mont-
bretin A), receptor grid was produced around amylase active site.
2.3.13. N’-(2-((1H-benzo[d]imidazol-2-yl)thio)−1-(4-bromophenyl)
ethylidene)−2-nitro benzenesulfonohydrazide (13)
Yield: 69%; 1HNMR (500 MHz, DMSO-d6): δ 12.44 (s, 1H, NH),
12.19 (s, 1H, NH), 8.15 (d, J = 6.9 Hz, 2H, ArH) 7.92 (d, J = 6.9 Hz,
2H, ArH), 7.80 (d, J = 6.2 Hz, 2H, ArH), 7.60 (m, 3H, ArH), 7.47
(t, J = 6.1 Hz, 1H, ArH), 7.09 (d, J = 6.3 Hz, 1H, ArH), 7.00 (dd,
J = 1.7. 6.4 Hz, 1H, ArH), 5.46 (s, 2H, CH2). 13CNMR (125 MHz,
DMSO-d6): δ 157.33, 150.03, 148.84, 145.11, 138.56, 138.56, 135.02,
133.50, 132.82, 129.27, 129.27, 129.11, 129.11, 128.61, 128.61, 127.03,
127.03, 124.04, 114.17, 114.17, 32.57; HREI-MS: m/z calcd for
C21H16 BrN5O4S2, [M]+ 544.9827; Found; 544.9810.
˚
2.3.14. N’-(2-((1H-benzo[d]imidazol-2-yl)thio)−1-(3-nitrophenyl)
ethylidene)−2-nitro benzenesulfonohydrazide (14)
Active sites were defined with a radius of 12 A around Montbretin
A binding sites. By using Chem PLP scoring function, docking calcu-
lations were skilled [44]. By using Discover studio visualizer, dock-
ing result was further evaluated and each derivative, binding mode
was visually inspected.
Yield: 65%; 1HNMR (500 MHz, DMSO-d6): δ 10.79 (s, 1H, NH),
9.94 (s, 1H, NH), 8.16 (d, J = 6.9 Hz, 2H, ArH), 7.92 (d, J = 6.9 Hz,
2H, ArH) 7.79 (d, J = 6.2 Hz, 2H, ArH), 7.55 (m, 3H, ArH), 7.48 (t,
J = 6.1 Hz, 1H, ArH), 7.10 (d, J = 1.7 Hz, 1H, ArH), 7.00 (dd, J = 1.9,
6.3 Hz, 1H, ArH), 5.48 (s, 2H, CH2). 13CNMR (125 MHz, DMSO-d6):
δ 157.33, 150.04, 148.84, 145.53, 138.56, 138.56, 136.11, 135.03,
134.31, 133.01, 129.8, 129.8, 129.11, 127.02, 127.02, 126.54, 125.03,
123.60, 114.17, 114.17, 32.57; HREI-MS: m/z calcd for C21H16 N6O6S2,
[M]+ 512.0573; Found; 512.0561.
3.1. Alpha-amylase assay protocol
a-Amylase inhibition activity had been determined using as-
say slightly modified from Kwon et al. [45]. All compounds were
highly dissolved in dimethyl sufoxide (DMSO). A total of 40 μl
4

S. Hussain, M. Taha, F. Rahim et al.
Journal of Molecular Structure 1232 (2021) 130029
Table 1
of sample solution and 40 μl of 0.02 M sodium phosphate buffer
(pH 6.9 with 0.006 M sodium chloride) containing a-amylase so-
lution (Porcine pancreatic a-amylase) (0.5 mg/ml) were incubated
at 25 C for 10 min. Forty μl of a 1% starch solution in 0.02 M
sodium phosphate buffer (pH 6.9 with 0.006 M sodium chloride)
was then added to each tube every 5 s intervals. The reaction mix-
tures were then incubated at 25 C for another 10 min. The reaction
was stopped using 100 μl of dinitrosalicylic acid color reagent. The
test tubes were then incubated in a boiling water bath for 5 min
and cooled to room temperature. The reaction mixture was then
diluted after adding 900 μl distilled water and the absorbance was
measured at 540 nm.% inhibition was calculated using below for-
mula.
Different substituents of 2-mercaptobenzimidazole analogs and α-amylase in-
hibitory potential.
S.No
R
R1
R2
IC50
1
6-methoxy
3-Nitro
2-methyl
2-methyl
2-Nitro
1.40 0.10
1.30 0.05
2.40 1.0
2
6-methoxy
4-Nitro
3
–
4-phenyl
4-phenyl
4-bromo
4-phenyl
4-phenyl
4-bromo
4 -methoxy
3-Nitro
4
–
2-chloro
4 -methoxy
2-bromo
4 -methoxy
2-bromo
2-bromo
2-bromo
2,4-difloro
2,4-difloro
2-Nitro
2.80 0.10
7.80 0.30
9.30 0.30
4.30 0.20
11.20 0.30
4.10 0.20
8.60 0.30
0.90 0.05
1.60 0.05
8.40 0.10
1.60 0.10
2.30 0.10
3.30 0.10
8.76 0.05
1.70 0.10μM
5
–
6
–
7
–
8
–
9
–
10
–
11
–
3-Nitro
%inhibition = [(Ac - As)/Ac] × 100%
12
6-methoxy
4-phenyl
4-bromo
3-Nitro
13
–
–
–
–
–
Where:
14
2-Nitro
Ac = absorbance of the control.
15
2,5 -dimethoxy
4-Nitro
2-Nitro
As = absorbance of the sample.
16
2-Nitro
17
4-bromo
2-methyl
Acarbose
4. Results and discussion
4.1. Chemistry
varying degree of inhibitory potential with IC50 values ranging
between 0.90 0.05 to 11.20 0.30 μM when compared with the
standard acarbose having IC50 value 1.70 0.10 μM. Compound
1, 2, 11, 12 and 14 having IC50 values 1.40 0.10, 1.30 0.05,
0.90 0.05, 1.60 0.05 and 1.60 0.10 μM respectively were
displayed inhibitory activity than standard drug acarbose. The
remaining analogues also exhibited good α-amylase inhibition.
The limited SAR study revealed that the most potent com-
pound in the series is 11 having IC50 value 0.90 0.05 μM. This
is may be due to more electronegative substituents such as NO2
group on phenyl ring B and difluoro substituents on phenyl ring
C which create more polarity in derivative 11, and directly prompt
the binding interaction with active site of residues. By this ratio-
nality derivative 11 exhibited more potent potential.
Synthesis
of
2-mercaptobenzimidazole/6-metnoxy-2-
mercaptobenzimidazole derivatives (1–17) bearing sulfonamide
moiety were carried out in three steps.
Step-1:
2-mercaptobenzimidazole/6-metnoxy-2-
mercaptobenzimidazole, various substituted phenacylbromides
were dissolved in acetone (20 ml), followed by addition of catalytic
quantity of potassium carbonate and then stirred the reaction
mixture for 2 hrs at room temperature. Upon completion of the
reaction, reaction mixture was cooled at 10–15 ᵒC temperature and
filtered the precipitate which yielded intermediate (I) as a product
having white cotton like texture (Scheme 1) [46,47].
Step-2: Various substituted sulfonyl chlorides were reacted with
equivalent quantity of hydrazine hydrate in methanol and refluxed
the reaction blend for 3 hrs. Upon reaction completion, solvent
was evaporated under vacuum to yield intermediate product (II)
(Scheme 2).
SAR study also revealed that relocation of same substituent on
phenyl ring affects the inhibitory activity of derivatives. The effect
of this relocation of groups on inhibitory activity was found when
we compared the potential of derivative 1 (IC50 = 1.40 0.10 μM)
Step-3: Intermediate product (I) was reacted with intermedi-
ate product (II) in methanol, acidified the reaction medium by 3–7
drops of glacial acetic and then the mixture was refluxed for 4 hrs.
When the reaction was completed, precipitate formed in reaction
medium. These precipitates were filtered to afford benzimidazole
bearing sulfonamide moiety (1–17) (Scheme 3; Table 1).
with
2
(IC50 = 1.30 0.05 μM) (Fig. 2). Derivative
2 displayed
slightly better inhibitory activity than derivative 1, even they have
same basic skeleton but the difference found in their structure was
due to the position of NO2 on phenyl ring B. The same change in
activity was also found in derivatives 14 and 16 (Fig. 2). This clearly
indicates that electron withdrawing substituent like NO2 creates
more polarity within molecules when it relocate from one position
to other position on phenyl ring, and by increasing the polarity the
binding interactions of derivatives were also increased with active
site of an enzyme and therefore derivatives 1 displayed greater in-
hibitory activity than 2 . Similarly, derivatives 14 displayed greater
inhibitory activity than derivatives 16 due to more polarity index.
It was found through SAR study that some time less electroneg-
ative characters influences the efficacy of derivatives in enzyme
inhibition study because of their large sizes, which do not easily
coordinate with active site of an enzyme, and vice versa. By this
consideration derivatives 9 (IC50 = 4.10 0.20 μM) exhibited greater
potential than derivatives 8 (IC50 =11.20 0.30 μM) (Fig. 2), be-
cause derivatives 9 have 4-OMe on phenyl ring B and 2-Br on
phenyl ring C while derivative 8 have Br substituents on phenyl
ring B and C. This clearly indicates that declination in inhibitory
activity is due large size and less electronegative character of Br
atom.
4.2. Activity
4.2.1. In vitro alpha-amylase inhibitory activity
4.2.1.1. α-Amylase activity. Seventeen (17) derivatives of 2-
mercaptobenzimidazole bearing sulfonamide moety (1–17) were
evaluated for α-amylase inhibitory potential. All analogs displayed
Scheme 1. Synthesis of intermediate product (I).
Here, it was concluded from the SAR study that electron-
withdrawing substituents on phenyl ring deactivates the phenyl
ring via attracting pi electronic clouds, but induced polarity within
Scheme 2. Synthesis of intermediate product (II).
5

S. Hussain, M. Taha, F. Rahim et al.
Journal of Molecular Structure 1232 (2021) 130029
Scheme 3. Synthesis of 2-mercaptobenzimidazole bearing sulfonamide analogs (1–17).
Fig. 2. Inhibitory activity of derivatives, acarbose and their concentrations.
molecules and as result increased the binding interactions be-
tween derivatives (ligands) and active site of enzyme which di-
rectly increased the inhibitory activity of derivatives. This hy-
pothesis was further supported by comparison the inhibitory ac-
tivity of derivative 13 (IC50 = 8.40 0.10 μM) with derivative 17
(IC50 = 8.76 0.05 μM) (Fig. 2). Herein this approach, almost both
derivative have the same basic structure but derivative 13 have
2-NO2 substitution on phenyl ring C, while derivative 17 have 2-
CH3 substitution on phenyl ring C respectively. The slight better
inhibitory activity of derivative 13 was attributed due to 2-NO2
substitution which has electron with-drawing nature as compare
to 2-CH3 substitution in derivative 17 which has electron donating
nature. This comparison absolutely supports our hypothesis, which
proves that electron withdrawing substituents enhances the activ-
ity by increasing the polarity within/around the molecules respec-
tively.
case of our reported work carbon atom of S-CH2 group give peak
in the range of 33.5 and 34.1 to 34.5 ppm respectively [43,44]. This
rational study of present work based on proton and carbon-NMR
displayed great correlation with already reported work.
4.3. Docking
By using Gold molecular docking software, all synthesized
derivatives in this series were docked into active site of target en-
zyme amylase (pdb : 4W93). The key connections recognized by
˚
active analogs were within 4 A radius on the binding site of amy-
lase, were considered as most influential factor for activity. In this
series all 17 analogs were active with IC50 value ranging in be-
tween 0.90 - 11.20 μM. Here we reveal binding mode of four active
analogs (11, 2, 1 and 12). The interactive site and the binding mode
of standard acarbose was reported in our previous paper [48].
Compound 11 is most potent in this series and binding mode
exhibit that nitro group attached to the meta position of phenyl
ring of this compound is most influential in stabilizing the complex
and hydrogen bond is formed by this nitro group. The nitro group
has electrostatic interacts with ASP197 and HIS299, with ARG195
and GLU233. Also with TYR62 π-cation interaction formed as well
as C-cation interaction with TYR62. Moreover, with HIS201π- π
stacking, with LEU162 and LYS200 π-Alkyl interaction forms with
benzimidazole ring. the benzimidazole ring forms π- π stacking
with HIS201 and π- Alkyl interaction with LYS200 and LEU162. In
addition, the ring also forms hydrophobic contact with ILE235. The
other stretches of the molecule bearing fluoro group at ortho and
para of the phenyl ring forms π- π stacking with the TRP59 (Fig.
3a).
Although, among the series (1–17) all noticeable proton and
carbon peaks in the spectra were compared with literature, aim-
ing to confirm the exact range of their respective peaks. In this
context, the protons of S-CH2 group give singlet at 5.3 ppm, while
in literature S-CH2 proton give singlet at 4.6 to 4.9 ppm respec-
tively. Similarly within our synthesized derivatives the proton of
–
SO2NH N=C group give peak at 12.5 ppm but the same proton
–
of CONH N=C group in our reported work give peak at 12.1 to
12.7 ppm respectively. In the same way, carbon atom of S-C-N2H
in imidazole ring in our synthesized derivatives give peak at 148.7
to 148.7 but the carbon atom in the same S-C-N2H group in im-
idazole ring which we have already reported gives peak at 149.3
–
=
to 149.5 ppm respectively. Moreover, carbon of SCH2-C=N N NH
group give peak at 156.6 to 157.3 ppm, but the same type of car-
–
=
bon in SCH2-C=N N NH group in our reported work give peak in
164.3 to 164.3 ppm. The study about carbon atom of S-CH2 group
in our current work give peak at the range of 32.5 ppm, while in
In the case of compound 2, methoxy group at R2 behaves as
acceptor to form hydrogen bond with acidic residue on the other
hand R1 nitro group bearing phenyl ring forms π-sigma interac-
6

S. Hussain, M. Taha, F. Rahim et al.
Journal of Molecular Structure 1232 (2021) 130029
Fig. 3. Shows the binding interaction of the potent derivative in the α-amylase active site (a) compound 11 in brown stick, (b) compound 2 in gray stick(c) compound 1 in
yellow stick and (d) compound 12 blue stick. Key interacting residues are shown in greenish stick form and the hydrogen bond is represented by dashed green line and the
Carbon Hydrogen Bond and π-donor hydrogen bond are represented as pale green dashed line. The π- π are represented as magenta dashed line, electrostatic interaction
shown as orange dashed line, and the hydrophobic interaction are shown as pink dashed lines.
tion responsible for activity. In the case of compound 1 the nitro
group at the R2 position forms hydrogen bond with acidic residue
contribute for activity. Finally, the compound 12 the ortho-para flu-
oro moieties at R2 position have contact with acidic and aromatic
residue of 12 influencing in the activity. Likewise, the phenyl group
at R1 forms π - π stacking with histidine also plays role in activity
Fig. 3b show the binding mode of compound 2.
drogen bond with active residues such as Thr163 in compound 1
and Asp356 in compound 2 is shown as an example are impor-
tant for activity. Similarly, the presence of methyl at R2 position
stabilizes the complex further with the presence of hydrophobic
interactions as in 1 and 2. Finally, the presence of the nitro group
at R1 is highly preferred to increase the activity that is crucial for
hydrogen bonding and π-anion interaction as seen in compound
1, 2, 11 and 14. Collectively these sort of interaction predicted by
docking studies is key for the activity. On the other hand as seen
in the compound 13 and 17 the presence of bromo group at R1 po-
sition didn’t bring any interaction and therefore there was loss in
activity.
Fig. 3c represents the binding mode of the compound 1, a hy-
drogen bond is established between ARG195, ASN298, and HIS299
with nitro group attached to the phenyl ring and the same group
also forms electrostatic contact with ARG195 and ASP300. The
linker sulfonyl nitrogen forms hydrogen bond with ASP300. The
toluene group attached the sulfonyl group forms π- π stacking
and hydrophobic contact with HIS305. The methoxy attached to
the benzodiazole forms hydrogen bond with THR163 and π con-
tact with THY151.
5. Conclusion
In
the
present
work,
we
have
synthesized
2-
Finally, the binding mode orientation of the compound 14
shows that the benzonitro group forming hydrogen with ASP197
and ARG195 as well π-ionic interaction with TYR62, GLU233 and
ARG195. The benzonitro group forms hydrogen bond with HIS305
and the ring also forms π-anion contact with ASP300 and π-
alkyl contact. Next the sulfonyl nitrogen forms hydrogen bond with
ASP300. Next the nitro group forms hydrogen bond with TRP59
and the benzene group forms π- π stacking HIS305. These con-
tacts was influential to stabilize the complex and reflects in the
biological activity index.
marcaptobenzimidazole derivatives (1–17) bearing sulfonamide
moiety. The inhibitory activity derivatives against alpha-amylase
enzyme revealed that almost all derivatives of the series showed
good inhibitory activity in the presence of standard drug acarbose.
Some of the derivatives like 1, 2, 11, and 14 emerged with most
potent inhibitory activity as compared to standard drug. Moreover,
derivative 11 is the most potent derivative among the series
and exhibited many folds better potential than standard drug.
As derivatives 11 have NO2-group at 3 position on phenyl ring
B and 2/4-diflouro on phenyl ring, and these groups are very
electronegative which draw electronic density from phenyl rings
and at result increased the polarity of derivatives, due to which
The docking studies binding mode show that in the presence
of methoxy group at R position in compound 1, 2, forms a hy-
7

S. Hussain, M. Taha, F. Rahim et al.
Journal of Molecular Structure 1232 (2021) 130029
it strongly coordinated with active site of an enzyme as shown
in fig-2a, therefore exhibited better potential than standard drug.
Overall potential of the series displayed that it is most potent class
of alpha-amylase inhibitors, and may provide the base for further
investigation of alpha-amylase inhibitors in future in search of
lead candidates.
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Author statement
All authors equally contributed in the manuscript.
Declaration of Competing Interest
[24] I.G. Tamil, B. Dineshkumar, M. Nandhakumar, M. Senthilkumar, A. Mitra, In
vitro study on α-amylase inhibitory activity of an Indian medicinal plant, Phyl-
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None.
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Acknowledgement
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Authors would like to acknowledge Higher Education Commis-
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search Program for Universities under project No. 5721 & 5092.
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