Synthesis, in vitro and computational studies of novel glycosyl-1, 2, 3-1H-triazolyl methyl benzamide derivatives as potential α-glucosidase inhibitory activity

Article abstract of DOI:10.1016/j.bioorg.2021.104687

A series of novel glycosyl-1,2,3-1H-triazolyl methyl benzamide analogues were synthesized by the unambiguous strategy and evaluated for α-glucosidase inhibitory activity. Glycosyl benzamide exhibited a dose-dependent inhibition of α-glucosidase activity.

Full text of DOI:10.1016/j.bioorg.2021.104687

Bioorganic Chemistry 109 (2021) 104687
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, in vitro and computational studies of novel glycosyl-1, 2,
3-1H-triazolyl methyl benzamide derivatives as potential α-glucosidase
inhibitory activity
,
,*
Akhilesh Kumar Shuklaa, Manoj Kumar Shrivashb c, Anwesh Pandeyc, Jyoti Pandeya
a Department of Chemistry, Babasaheb Bhimrao Ambedkar University, Lucknow, Uttar Pradesh 226025, India
b Department of Applied Sciences, Indian Institute Information Technology Allahabad, India
c Special Centre for Molecular Medicine, JNU, New Delhi 110067, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of novel glycosyl-1,2,3-1H-triazolyl methyl benzamide analogues were synthesized by the unambiguous
strategy and evaluated for -glucosidase inhibitory activity. Glycosyl benzamide exhibited a dose-dependent
inhibition of -glucosidase activity. The In-vitro
Benzamide
Click reaction
α
α
α
-glucosidase inhibition activity results indicated that all the
α
-Glucosidase activity
synthesized triazolyl methyl benzamide compounds (IC50 values ranging from 25.3 0.8 to 118.5 5.3
exhibited more inhibitory activity in comparison with the standard drug acarbose (IC50 = 750.0 12.5
μM)
Type-2 diabetes
μM).
In-silico-study
Among all, the 3 deacetylated glycosyl methyl benzamide derivatives (4c, 4d and 4f) showed promising
α
-glucosidase enzyme inhibitory activities with IC50 value 25.3 0.8, 26.1 1.5 and 30.6 2.1 respectively.
Furthermore, these compounds were subjected to molecular docking and molecular dynamics simulation studies.
The molecular docking studies were performed between (PDB ID: 3A4A) target protein and these synthesized
molecules. The compounds displayed good docking energies in the range of ꢀ 7.5 to ꢀ 7.8 Kcal/mol. This work
could be used as an initial approach in identifying potential novel molecules with the promising activity of type-2
diabetes mellitus.
1. Introduction
generation of small heterocycles in search of lead molecules[11,12].
They can introduce multiple functionalities in restricted steps. Ac-
Diabetes mellitus (DM) is a chronic disease of global commons,
which tends to have problems with adherence [1,2]. Adherence with
medication, diet, and exercise, and blood glucose self–monitoring is
quite challenging [3]. Due to consumption of carbohydrate-enriched
diet, a metabolic and heterogeneous disorder causing high blood-
glucose level, leading to hyperglycaemia [4]. As per the Global Report
on Diabetes (2018) by the World Health Organization, diabetes was the
seventh major cause of death in 2016[5,6]. Carbohydrates form the
largest group of naturally occurring compounds in nature and also
crucial due to their pivotal role in medicinal chemistry and drug dis-
covery[7–9]. They are also known for their essential function in devel-
opment, recognition, growth, function and survival of living cells and
organisms. Many drug molecules including several antibiotics and few
anti-diabetic medicines contain terminal sugar moieties which are
necessary for their biological action [10]. Sugar molecules are benefitted
with immense structural diversity which makes it appropriate for library
cording to estimation, there are more than 194 million people are
suffering from diabetes globally that this will increase to 333 million by
2025 [5]. α-Glucosidase is a membrane-bound enzyme that catalyzes the
hydrolysis of the glycosidic bond at the non-reducing terminal of the
sugar, resulting in the release of free glucose into the digestive tract.
They are responsible for glucose absorption and increase glucose con-
centration in blood. Inhibition of α-glucosidase could diminish the rate
of carbohydrates assimilation and concealment of postprandial hyper-
glycaemia [13]. Along these, -glucosidase has been regarded as the
significant objective for the discovery and development of novel anti-
diabetic drugs. As of now, there are three -glucosidase inhibitors like
α
α
acarbose, voglibose, and miglitol, which have been utilized in clinical
for the treatment of type-2 diabetes mellitus[14,15]. It is proven in
literature regularly that the presence of sugar functionality in drug-like
molecules alter the bioavailability and efficacy of molecules drastically
(Fig. 1). Hence Medicinal Chemists have a tremendous burden of
* Corresponding author.
E-mail address: jyotipandey@bbau.ac.in (J. Pandey).
https://doi.org/10.1016/j.bioorg.2021.104687
Received 14 November 2020; Received in revised form 22 December 2020; Accepted 21 January 2021
Available online 2 February 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

A.K. Shukla et al.
Bioorganic Chemistry 109 (2021) 104687
Fig. 1. Potential glycosidase inhibitors synthesised using ’click chemistry’: glucosidase 1, galactoside 2, acarbose mimic 3 and MetAP2 II inhibitors 4.
activities such as antifungal[25],antitubercular[26,27] anticancer[28],
anti-HIV[29,30] antibacterial[25] antiviral[31] anti-alzheimer[32]anti-
mycobacterial[33]and glycosidase inhibitors[34–36]
Keeping the above facts on priority we were prompted to synthesize a
novel series of glycohybrids triazoles, consisting of benzamides, triazole
and sugars and their inhibition potential was studied against
sidase enzyme in a quest for new anti-diabetic agents (Fig. 2).
α-gluco-
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of 2-amino-benzamide-4(1H)-ones
Fig. 2. Designing structure for novel glycosyl benzamides.
2-Amino-N-propargyl benzamides (2a and 2b) were prepared from
commercially available anthranilic acid (1a) and 2-hydroxy-benzoic
acid (1b) following earlier reported protocols[37] as shown in Scheme
1. The structures were established based on their spectroscopic data
[38]. The flask was degassed and then filled with N2 (balloon), and HOBt
(1 mmol), 4- dimethyl aminopyridine (DMAP, 1 mmol) were added to
the reaction mixture. Then N, N’-Diisopropylcarbodiimide (DIPC, 1
mmol) was added to the reaction mixture in a dropwise manner. After 10
mins, propargyl amine (1.1 mmol) was added and the reaction was
stirred overnight at ambient temperature to get the desired 2-amino-N-
(prop-2-yn-1-yl) benzamide (2a) 2-hydroxy-N-(prop-2-yn-1-yl) benza-
mide (2b) in 89% yield (Scheme 1).
developing new molecules with anti-diabetic potential. We hypothesize
to couple terminal sugar moiety with aglycon pharmacophore to get
better pharmacological property.
Benzamide moiety is very significant class of nitrogen heterocycles
and is considered as privileged structure in drug discovery owing to their
important roles as key building blocks in the synthesis of many drugs
[16]. This heterocyclic nucleus is linked with diverse range of phar-
macological activities such as antihypertensive[17], antibacterial[18],
anti-inflammatory[19], anticancer[20], analgesic[21],antihistamine
[22], CNS stimulant[23] and antidiabetic activities[24].Hence we tried
to couple the benzamide nucleus with terminal sugar moieties by
applying a suitable and biologically significant linker. 1,2,3-Triazoles
are also an essential scaffold in drug discovery and development as
several molecules with this moiety exhibited important biological
The structure of the product 2a was established by their spectro-
scopic data. ESIMS of the compound displays m/z = 175 as [M + H]+
peak corresponding to its molecular formulae C10H11N2O. In IR spec-
trum, characteristic absorption peaks observed at 3372 cmꢀ 1 for amine,
3019 cmꢀ 1 for –NH, 1648 cmꢀ 1 for carbonyl (–NHC = O). In the 1H NMR
Scheme 1. Synthesis of 2-amino-N-propargyl benzamide derivatives.
2

A.K. Shukla et al.
Bioorganic Chemistry 109 (2021) 104687
stir at room temperature overnight. The reaction mixture was quenched
with water, and the aqueous layer was extracted with dichloromethane.
The combined organic extracts were washed with aqueous copper sul-
phate. The organic layer was then dried over magnesium sulphate,
filtered, and concentrated under reduced pressure. The residue was
purified by column chromatography to afford the desired 2-(4-methyl-
phenylsulfonamido)-N-(prop-2-yn-1-yl) benzamide (2c) in 79% yield
(Scheme 2).
2.1.3. Synthesis of glycosyl triazolyl benzamide-4(3H)-ones
The glycosyl azides(I and II) were prepared from commercially
available glucose and galactose following the methods already reported
[40] in the literature as shown in Scheme 3. The structures were
established based on their spectroscopic data. These were identical in all
respects to those reported earlier[41,42].
Scheme 2. Synthesis of 2-(4-methylphenylsulfonamido)-N-(prop-2-yn-1-yl)
benzamide derivative.
spectrum, the two exchangeable NH2 protons were observed at δ 5.43
(bs, 2H, –NH2) and the amide –NH proton was visible at δ 6.25 (bs, 1H,
–NH) while the alkynyl proton was visible at δ 2.16 besides other usual
protons at their usual chemical shift. In 13C NMR spectrum, the peaks at
δ 168.8 accounted for the amide group carbon (–NH-CO-) along with
other usual signals. Similarly, the reaction of 2-hydroxy-benzoic acid
(1b) with propargyl amine the above reaction conditions led to the
formation of 2-hydroxy-N-(prop-2-yn-1-yl) benzamide (2b) in 88% yield
(Scheme 1).
The strategy for the synthesis of glycosyl-triazolyl benzamide-4(3H)-
ones is depicted in Scheme 4. Having the 2-phenyl-3-propargyl-benza-
mide-4(1H)-ones (2-c) and glycosyl azides (I and II) in our hand the
CuAAC reactions were performed in t-BuOH/H2O (1:1) using equimolar
quantities of the reagents, CuSO4⋅5H2O (10 mol%) and sodium ascor-
bate (20 mol%) at ambient temperature to afford epimeric mixtures of
peracetylglycosyl-triazolylbenzamide-4(1H)-ones (3a-f) in good yields
(Scheme 4, Table 1). Propargyl benzamides and glycosyl azides selec-
tively gave only one regioisomer, 1,4-disubstituted triazole via 1,3-
dipolar cycloaddition reaction.
2.1.2. Synthesis of 2-(4-methylphenylsulfonamido)-N-(prop-2-yn-1-yl)
benzamide (2c)
The Zemplen deacetylation of the above peracetylated glycosyl tri-
azolyl quinazoline-4(3H)-ones (3a-f) with NaOMe/MeOH at room
temperature led to the formation of the deacetylated glycosyl triazolyl
quinazoline-4(3H)-ones (4a-f) respectively in good yields (Scheme 4,
Table 2).
2-(4-methylphenylsulfonamido)-N-(prop-2-yn-1-yl) benzamide (2c)
were prepared by following earlier reported protocols[39] as shown in
Scheme 2. To a solution of the 2-amino-N-(prop-2-yn-1-yl) benzamide
(2a, 1.0 equiv) in pyridine (1 M) at room temperature, p-toluenesulfonyl
chloride (1.05 equiv) was added. The reaction mixture was allowed to
Scheme 3. Synthesis of the glycosyl azides.
Scheme 4. Synthesis of glycosyl benzamide-4(3H)-ones.
3

A.K. Shukla et al.
Bioorganic Chemistry 109 (2021) 104687
Table 1
Table 2
Peracetylatedglycosyltriazolyl 2-amino-benzamide-4(3H)-ones (4a-f) from
glycosyl azides and 2-amino-benzamide-4(1H)-ones.
Synthesized deacetylated compounds 4a-f from per acetylated glycosyl triazolyl
quinazoline-4(3H)-ones (3a-f).
Entry
2-amino-
Glycosyl
Azide
Peracetylatedglycosylbenzamide-4
Yield
Entry
1
Peracetylated substrates
Deacetylated products
Yield (%)a
benzamide-
4(1H)-ones
(3H)-one
(%)a
3a
94
1
2a
2a
2b
2b
2c
2c
I
80
78
77
75
73
71
4a
4b
4c
4d
3a
3b
3c
3d
3e
3f
2
3
4
5
3b
3c
3d
3e
3f
92
93
91
92
90
2
3
4
5
II
I
II
I
4e
4f
6
6
II
a
Isolated yield as the pure product.
a
Isolated yield as a pure product.
organic extracts were washed with brine (50 mL), dried over sodium
sulphate, filtered, concentrated, and purified by column chromatog-
raphy (1:1 EtOAc: Hexanes) to afford desired compounds 2a-b.
3. Experimental section
Commercially available reagent grade synthetic compounds were
utilized as received. Every reaction was monitored by TLC on E. Merck
Kieselgel 60 F254, with detection by UV light, spraying 20% aq KMnO4
solution as well as spraying with 4% ethanolic H2SO4. Column chro-
matography was performed on Silica Gel (60–120 mesh, E. Merck). IR
spectra were recorded as thin films or in KBr solution with a Per-
kin–Elmer Spectrum RX-1 (4000–450 cmꢀ 1) spectrophotometer. 1H and
13C NMR spectra were recorded on Bruker DRX 400 MHz, and 100 MHz
instruments, respectively, in CDCl3 and DMSO‑d6. The chemical shift
values are reported in ppm concerning TMS (tetramethylsilane) as the
internal reference, unless otherwise generally expressed; s (singlet),
d (doublet), t (triplet), dd (double doublet), m (multiplet); J in Hertz. ESI
mass spectra were performed utilizing a Quattro II (Micromass) instru-
ment. Optical rotations were estimated in a 1.0-dm tube with a Rudolf
Autopol III and Horiba polarimeters in CHCl3 and MeOH.
3.2. 2-amino-N-(prop-2-yn-1-yl) benzamide (2a)
It was obtained by the reaction of anthranilic acid 1a (5.0 g, 36.49
mmol) with HOBt (4.92 g, 36.49 mmol), DMAP (4.45 g, 36.49 mmol),
DIPC (5.67 mL, 36.49 mmol) and propargyl amine (2.56 mL, 40.14
mmol) in dry DMF (20 mL) in 89% yield (5.65 g) as a white solid; mp
87 ◦C; IR (νmax, cmꢀ 1): 3372, 3019, 1648, 1586, 1403, 1215 and 757.1H
NMR (400 MHz, CDCl3 + CCl4): δ 7.24 (m, 1H, Ar-H), 7.12 (m, 1H, Ar-
H), 6.57 (m, 2H, Ar-H), 6.25 (bs, 1H, –NH), 5.43 (bs, 2H, –NH2), 4.10
(m, 2H, –CH2), 2.16 (m, 1H, –CH). 13C NMR (100 MHz, CDCl3 + CCl4): δ
–
168.8 (C O), 148.9 (Ar-C), 132.6 (Ar-C), 127.2 (Ar-C), 117.3 (Ar-C),
–
116.5 (Ar-C), 114.9 (Ar-C), 79.7, 71.7 (–CH), 29.4 (–CH2). M.F:
C10H11N2O [M + H]+; ESIMS: m/z 175.
3.3. 2-hydroxy-N-(prop-2-yn-1-yl) benzamide (2b)
3.1. General procedure for the compounds 2a-b
It was obtained by the reaction of salicylic acid 1b (5.0 g, 34.70
mmol) with HOBt (4.88 g, 34.70 mmol), DMAP (4.41 g, 34.70 mmol),
DIPC (5.32 mL, 34.70 mmol) and propargyl amine (2.46 mL, 34.12
mmol) in dry DMF (20 mL) in 88% yield (5.42 g) as a white solid; mp
Anthranilic acid/2-hydroxybenzoic acid (1a, 1b, 1 mmol) was added
to a 100 mL round bottom flask in dry N, N-dimethylformamide (20 mL)
equipped with ice. The flask was degassed, then filled with N2 (balloon),
and HOBt (1 mmol), 4- dimethyl aminopyridine (DMAP, 1 mmol) added
to the reaction mixture. Then N, N’-Diisopropylcarbodiimide (DIPC, 1
mmol) was added to the solution in dropwise. After 10 mins, propargyl
amine (1.1 mmol) was added to the reaction mixture and stirred over-
night at ambient temperature. The crude mixture was taken up in water
(50 mL) and extracted with ethyl acetate (3 × 40 mL). The combined
86–88 C; IR (νmax, cmꢀ 1): 3372, 3019, 1648, 1586, 1403, 1215 and
◦
757.1H NMR (400 MHz, CDCl3 + CCl4): δ 7.24 (m, 1H, Ar-H), 7.12 (m,
1H, Ar-H), 6.57 (m, 2H, Ar-H), 6.25 (bs, 1H, –NH), 5.43 (bs, 2H, –NH2),
4.10 (m, 2H, –CH2), 2.16 (m, 1H, –CH). 13C NMR (100 MHz, CDCl3
+
–
CCl ): δ 168.8 (C O), 148.9 (Ar-C), 132.6 (Ar-C), 127.2 (Ar-C), 117.3
–
4
(Ar-C), 116.5 (Ar-C), 114.9 (Ar-C), 79.7, 71.7 (–CH), 29.4 (–CH2). M.F:
4

A.K. Shukla et al.
Bioorganic Chemistry 109 (2021) 104687
C10H9NO2 [M + H]+; ESIMS: m/z 175.
5.25 (m, 1H, H-4), 4.74 (m, 2H, –CH2), 4.24–4.09 (m, 2H, H-5, H-6 ),
2.22 (s, 3H, –OCOCH3), 2.03 (s, 3H, –OCOCH3), 2.00 (s, 3H, –OCOCH3),
1.87 (s, 3H, –OCOCH3).13C NMR (100 MHz, DMSO‑d6): δ 170.0, 169.5,
169.2, 168.4(4 × –COCH3), 160.8(–CO), 145.4(Ar-C), 133.8(Ar-C),
128.0(Ar-C), 122.4(Ar-C), 118.6(Ar-C), 117.6(Ar-C), 115.2(Ar-C), 96.0
(C-1′), 84.4(C-5′), 79.0(C-3′), 78.6(C-4′), 73.9(C-2′), 72.8(C-6′), 70.5,
′
́
́
3.4. 2-(4-methylphenylsulfonamido)-N-(prop-2-yn-1-yl)benzamide (2c)
It was obtained by the reaction the 2-amino-N-(prop-2-yn-1-yl)
benzamide (2a, 2.0 g, 13.28 mmol) in pyridine (5 mL) at room tem-
perature with p-toluenesulfonyl chloride (5.05 mL) wad added. The
reaction mixture was allowed to stir at room temperature overnight and
extracted with DCM to give the crude product, which was purified by
column chromatography (20% EtOAc/Hexane) to give the title com-
pound (2c) in79% yield (g) as a white solid; mp 87 ◦C; IR (νmax, cmꢀ 1):
3376, 3028, 1644, 1307, 1215, 1173, 907 and 669.1H NMR (400 MHz,
CDCl3 + CCl4): δ7.76 (bs, 1H, –NH), 7.54 (m, 2H, Ar-H), 7.32 (m, 2H, Ar-
H), 7.12 (m, 2H, Ar-H), 6.94(m, 2H, Ar-H), 4.10 (m, 2H, –CH2), 2.36 (m,
67.9, 61.9, 40.3(–CH2), 39.8, 34.8,20.8, 20.6, 20.5, 20.2(4
×
–OCOCH3); HRMS: Calcd. Accurate mass for (C24H28N4O11): 549.1755.
Found 549.1792 [M + H]+.
3.8. 2-hydroxy-N-[(1-(1′-deoxy-2′,3′,4′,6′-tetra-O-acetyl-β-D-
galactopyranose-1′-yl)]-1H-1, 2, 3-triazol-4-yl)methyl-benzamide (3d)
It was obtained by the reaction of 2b (0.53 g, 1.85 mmol), azido
sugar II (0.73 g, 1.89 mmol), CuSO4⋅5H2O (0.042 g, 0.16 mmol) and
sodium ascorbate (0.071 g, 0.35 mmol) in 1:1 tert-Butanol-water (40
3H, –CH3), 2.16 (m, 1H, –CH). 13C NMR (100 MHz, CDCl3 + CCl4): δ
–
169.3 (C O), 149.7 (Ar-C), 132.8 (Ar-C), 127.2 (2C, Ar-C), 118.3 (2C,
–
Ar-C), 117.5 (Ar-C), 115.9 (Ar-C), 79.7, 71.7 (–CH), 29.3 (–CH2), 21.6
mL) in 75% yield (1.07 g) as a yellow solid; mp 120–122 ◦C; IR (νmax
,
(–CH3);M.F: C17H16N2O3S [M + H]+; ESIMS: m/z 329.09.
cmꢀ 1): 3434, 2381, 1625, 1537, 1226, 1217, 1184, 753 and 659.1H
NMR (400 MHz, CDCl3): δ 12.18(m, 1H, –NH), 7.90 (s, 1H, triazolyl-H),
7.53 (m, 1H, Ar-H), 7.47 (m, 1H, Ar-H), 7.37 (m, 2H, Ar-H),6.82 (m, 1H,
3.5. 2-amino-N-[(1-(1′-deoxy-2′, 3′, 4′, 6′-tetra-O-acetyl-β-D-
glucopyranos-1′-yl)]-1H-1, 2, 3-triazol-4-yl) methyl-benzamide (3a)
–OH), 5.83(d, J = 9.2 Hz, 1H, H-1), 5.52–5.47 (m, 2H, H-2, H-3), 5.26
́
́
́
′
́
́
(m, 1H, H-4), 4.78 (m, 2H, –CH2), 4.24–4.09 (m, 2H, H-5, H-6 ), 2.22 (s,
3H, –OCOCH3), 2.03 (s, 3H, –OCOCH3), 2.00 (s, 3H, –OCOCH3), 1.87 (s,
3H, –OCOCH3).13C NMR (100 MHz, CDCl3): δ 169.9, 169.7, 168.4,
168.7(4 × –COCH3), 161.7(–CO), 144.8(Ar-C), 134.1(Ar-C), 125.8(Ar-
C), 121.3(Ar-C), 118.5(Ar-C), 114.0(Ar-C), 86.3(C-1′), 77.2(C-5′), 76.9
(C-3′), 74.0(C-4′), 70.6(C-2′), 68.0(C-6′), 34.6(–CH2), 20.5, 20.5, 20.4,
20.1(4 × –OCOCH3); HRMS: Calcd. Accurate mass for (C24H28N4O11):
549.1755 Found 549.1796 [M + H]+.
It was obtained by the reaction of 2a (0.50 g, 1.89 mmol), azido
sugar I (0.72 g, 1.89 mmol), CuSO4⋅5H2O (0.042 g, 0.16 mmol) and
sodium ascorbate (0.071 g, 0.35 mmol) in 1:1 tert-Butanol-water (40
◦
mL) in 80% yield (1.03 g) as a white solid; mp 106–108 C; IR (νmax
,
cmꢀ 1): 3290, 2349, 1607, 1523, 1245, 1217, 1182, 758 and 669.1H
NMR (400 MHz, CDCl3 + CCl4): δ 7.83 (s, 1H, triazolyl-H), 7.35 (m, 1H,
Ar-H), 7.26 (m, 1H, Ar-H), 6.81 (bs, 1H, –NH), 6.66 (m, 2H, Ar-H), 5.82
́
́
́
́
(d, J = 9.2 Hz, 1H, H-1), 5.52–5.47 (m, 2H, H-2, H-3), 5.25 (m, 1H, H-4),
′
3.9. 4-methyl-N-[(1-(1′-deoxy-β-D-glucopyranos-1′-yl)]-1H-1,2,3-
́
4.74 (m, 2H, –CH2), 4.24–4.09 (m, 2H, H-5, H-6 ), 2.22 (s, 3H,
–OCOCH3), 2.03 (s, 3H, –OCOCH3), 2.00 (s, 3H, –OCOCH3), 1.87 (s, 3H,
–OCOCH3).13C NMR (100 MHz, DMSO‑d6): δ 170.4, 170.3, 169.9, 168.9,
150.2, 146.3, 132.3, 128.6, 122.7, 116.8, 115.0, 114.6, 84.6, 73.4, 70.9,
68.0, 67.8, 62.0, 34.8, 20.9, 20.8, 20.7, 20.4; HRMS: Calcd. Accurate
mass for (C24H29N5O10): 548.1914. Found 548.1956 [M + H]+.
triazol-4-yl)methyl-benzenesulphonamide (3e)
It was obtained by the reaction of 2c (0.1 g, 0.66 mmol), azido sugar I
(0.25 g, 0.66 mmol), CuSO4⋅5H2O (0.02 g, 0.06 mmol) and sodium
ascorbate (0.03 g, 0.13 mmol) in 1:1 tert-butanol-water (20 mL) in 73%
yield (0.32 g) as a white solid; m.p. 86–88 C;IR (νmax, cmꢀ 1): 3245,
◦
3.6. 2-amino-N-[(1-(1′-deoxy-2′, 3′, 4′, 6′-tetra-O-acetyl-β-D-
2371, 1658, 1510, 1309, 1227, 1169 and 768.1H NMR (400 MHz,
DMSO‑d6): δ 10.74 (s, 1H, –NH), 7.91 (s, 1H, triazolyl-H), 7.61 (m, 3H,
Ar-H), 7.40 (m, 2H, Ar-H), 7.26 (m, 2H, Ar-H), 7.11 (m, 2H, Ar-H), 6.98
galactopyranose-1′-yl)]-1H-1, 2, 3-triazol-4-yl) methyl)-benzamide (3b)
́
́
It was obtained by the reaction of 2a (0.50 g, 1.89 mmol), azido
sugar II (0.72 g, 1.89 mmol), CuSO4⋅5H2O (0.042 g, 0.16 mmol) and
sodium ascorbate (0.071 g, 0.35 mmol) in 1:1 tert-Butanol-water (40
(m, 2H, Ar-H), 5.88 (d, J = 9.1 Hz, 1H, H-1),5.48–5.42 (m, 2H, H-2, H-
́
́
́
3), 5.26 (m, 1H, H-4), 4.60 (m, 2H, –CH2),4.29–4.12 (m, 2H, H-5, H-
6′),2.32 (s, 3H, –OCOCH3), 2.06(s, 3H, –OCOCH3), 2.03(s, 3H,
–OCOCH3), 1.86(s, 3H, –OCOCH3).13C NMR (100 MHz, CDCl3) δ 170.2,
169.7, 169.0, 168.6, 168.3, 143.3, 139.0, 136.6, 132.6, 129.4, 127.2,
127.0, 123.3, 121.0, 120.9, 96.1, 85.8, 77.2, 76.9, 76.6, 75.2, 72.6, 70.4,
67.6, 61.3, 35.1, 21.4, 20.5, 20.4, 20.1. HRMS: Calcd. Accurate mass for
(C31H35N5O12S): 702.2003. Found 702.2042 [M + H]+.
mL) in 78% yield (1.08 g) as a yellow solid; mp 118–120 ◦C; IR (νmax
,
cmꢀ 1): 3292, 2365, 1610, 1535, 1230, 1216, 1184, 771 and 669.1H
NMR (400 MHz, CDCl3 + CCl4): δ 7.88 (s, 1H, triazolyl-H), 7.37 (m, 1H,
Ar-H), 7.20 (m, 1H, Ar-H), 6.91 (bs, 1H, –NH), 6.66 (m, 2H, Ar-H), 5.82
́
́
́
́
(d, J = 9.2 Hz, 1H, H-1), 5.52–5.47 (m, 2H, H-2, H-3), 5.25 (m, 1H, H-4),
′
́
4.74 (m, 2H, –CH2), 4.24–4.09 (m, 2H, H-5, H-6 ), 2.22 (s, 3H,
–OCOCH3), 2.03 (s, 3H, –OCOCH3), 2.00 (s, 3H, –OCOCH3), 1.87 (s, 3H,
–OCOCH3).13C NMR (100 MHz, DMSO‑d6): δ 170.4, 170.3, 169.9, 168.9,
150.2, 146.3, 132.3, 128.6, 122.7, 116.8, 115.0, 114.6, 84.6, 73.4, 70.9,
68.0, 67.8, 62.0, 34.8, 20.9, 20.8, 20.7, 20.4; HRMS: Calcd. Accurate
mass for (C24H29N5O10): 548.1914. Found 548.1952 [M + H]+.
3.10. 4-methyl-N-[(1-(1′-deoxy-β-D-galactopyranose-1′-yl)]-1H-1,2,3-
triazol-4-yl)methyl-benzenesulphonamide (3f)
It was obtained by the reaction of 2c (0.1 g, 0.66 mmol), azido sugar
II (0.25 g, 0.66 mmol), CuSO4⋅5H2O (0.02 g, 0.06 mmol) and sodium
ascorbate (0.03 g, 0.13 mmol) in 1:1 tert-butanol-water (20 mL) in 71%
3.7. 2-hydroxy-N-[(1-(1′-deoxy-2′, 3′, 4′, 6′-tetra-O-acetyl-β-D-
yield (0.30 g) as a white solid; m.p. 86–88 C;IR (νmax, cmꢀ 1): 3242,
◦
glucopyranos-1′-yl)]-1H-1,2,3-triazol-4′-yl)methyl-benzamide (3c)
2374, 1661, 1512, 1307, 1228, 1163 and 768.1H NMR (400 MHz,
DMSO‑d6): δ δ 10.74 (s, 1H, –NH), 7.94 (s, 1H, triazolyl-H), 7.64 (m, 3H,
Ar-H), 7.43 (m, 2H, Ar-H), 7.26 (m, 2H, Ar-H), 7.13 (m, 2H, Ar-H), 6.99
It was obtained by the reaction of 2b (0.53 g, 1.85 mmol), azido
sugar I (0.73 g, 1.89 mmol), CuSO4⋅5H2O (0.042 g, 0.16 mmol) and
́
́
(m, 2H, Ar-H), 5.85 (d, J = 9.1 Hz, 1H, H-1),5.57–5.52 (m, 2H, H-2, H-
′
́
́
́
sodium ascorbate (0.071 g, 0.35 mmol) in 1:1 tert-Butanol-water (40
3), 5.26 (m, 1H, H-4), 4.63 (m, 2H, –CH2), 4.25–4.19 (m, 2H, H-5, H-6 ),
2.32 (s, 3H, –OCOCH3), 2.23 (s, 3H, –OCOCH3), 2.01 (s, 3H, –OCOCH3),
1.88 (s, 3H, –OCOCH3).13C NMR (100 MHz, CDCl3) δ 170.0, 169.8,
169.5, 168.8(4 × –COCH3), 168.3(–CO), 144.4 (Ar-C), 143.3 (Ar-C),
139.0 (Ar-C), 136.7 (Ar-C), 132.6 (Ar-C), 129.4 (Ar-C), 127.3 (Ar-C),
127.2 (Ar-C), 127.0, 123.3, 121.4, 121.0, 121.0, 96.1, 86.3, 77.2, 76.9,
◦
mL) in 77% yield (1.06 g) as a white solid; mp 111–113 C; IR (νmax
,
cmꢀ 1): 3449, 2395, 1620, 1534, 1230, 1218, 1185 and 754.1H NMR
(400 MHz, DMSO‑d6): δ 12.40 (m, 1H, –NH), 9.22 (s, 1H, triazolyl-H),
7.37 (m, 1H, Ar-H), 7.20 (m, 1H, Ar-H), 6.66 (m, 2H, Ar-H),6.20 (m,
́
́
́
1H, –OH), 5.82 (d, J = 9.2 Hz, 1H, H-1), 5.52–5.47 (m, 2H, H-2, H-3),
5

A.K. Shukla et al.
Bioorganic Chemistry 109 (2021) 104687
76.6, 74.1, 70.7, 68.0, 66.7, 61.0, 42.1, 35.1, 23.5, 21.4, 20.1, 20.5,
20.4, 20.2. HRMS: Calcd. Accurate mass for (C31H35N5O12S): 702.2003.
Found 702.2045 [M + H]+.
Table 3
α
-Glucosidase inhibitory activity (10 µM) of the synthesized benzamide
analogues.
S.No.
Code
%
α-Glucosidase inhibition
IC50
(
μ
M)a
3.11. 2-amino-N-[(1-(1′-deoxy-β-D-glucopyranos-1′-yl)]-1H-1,2,3-
1.
3a
5.18
6.21
22.7
26.9
15.7
17.3
35.7
36.9
39.7
40.6
24.6
42.9
53.4
112.4 ± 6.3
118.5 ± 5.3
104.3 ± 6.0
107.6 ± 7.1
109.7 ± 2.8
113.2 ± 1.0
48.5 ± 1.5
52.6 ± 2.1
25.3 0.8
26.1 1.5
43.7 ± 1.8
30.6 2.1
750.0 ± 12.5
triazol-4-yl)methyl-benzamide (4a)
2.
3b
3.
3c
4.
3d
It was obtained by treating the acetylated compound 3a (0.40 g,
5.
3e
0.63 mmol) with NaOMe in methanol, in 94% yield (0.27 g) as a white
6.
3f
solid;; mp 83–85 C;[
α
]
0.4 (c 0.1, CH3OH); IR (νmax, cmꢀ 1): 3411,
25
D
◦
7.
4a
3019, 1638, 1215 and 758.1H NMR (400 MHz, DMSO‑d6): δ 8.74 (m, 1H,
–NH), 8.08 (s, 1H, triazolyl-H), 7.53 (d, J = 6.9 Hz, 1H, Ar-H), 7.16 (m,
1H, Ar-H,), 6.70 (d, J = 8.0 Hz, 1H, Ar-H), 6.53 (t, J = 7.7 Hz, 1H, Ar-H),
8.
4b
9.
4c
10.
11.
12.
13.
4d
4e
́
6.41 (bs, 2H, –NH2), 5.51 (d, J = 9.3 Hz, 1H, H-1), 5.38 (m, 1H, –OH),
4f
5.26 (m, 1H, –OH), 5.17 (m, 1H, –OH),4.68 (m, 1H, –OH), 4.48 (m, 2H,
Acarbose
–CH2), 3.78–3.59 (m, 4H, H-2′, H-3′, H-4′, H-5′), 3.25 (m, 2H, H-6′).13C
a
Data expressed as the mean of ± S.E. of at least 3 different experiments.
–
–
NMR (100 MHz, DMSO‑d ): δ 169.3 (C O), 150.1 (Ar-C), 145.8 (Ar-C),
132.3 (Ar-C), 128.6 (Ar-6C), 122.4(Ar-C), 116.8 (Ar-C), 115.1 (Ar-C),
4.09 (m, 3H, 3 × –OH), 3.75–3.34 (m, 5H, H-2 , H-3 , H-4, H-6), 13C
NMR (100 MHz, DMSO‑d6): δ 169.2(–CO), 160.3(Ar-C), 144.9(Ar-C),
134.2(Ar-C), 128.4(Ar-C), 122.2(Ar-C), 119.1(Ar-C), 117.8(Ar-C), 115.7
(Ar-C), 88.5(C-1′), 78.8(C-5′), 74.1(C-3′), 69.7(C-4′), 68.9(C-2′), 60.8(C-
6′), 40.6(–CH2);HRMS: Calcd. Accurate mass for (C16H20N4O7):
381.1332. Found 381.1372 [M + H]+.
′
′
́
́
́
́
́
́
́
114.5 (Ar-C), 87.8 (C-1), 80.3 (C-5), 77.4 (C-3), 72.4 (C-4), 69.9 (C-2),
́
61.1 (C-6), 34.8 (–CH2). HRMS: Calcd. Accurate mass for (C16H21N5O6):
380.1492. Found 380.1531 [M + H]+.
3.12. 2-amino-N-[(1-(1′-deoxy-β-D-galactopyranose-1′-yl)]-1H-1,2,3-
triazol-4-yl)methyl-benzamide(4b)
3.15. 4-methyl-N-[(1-(1′-deoxy-β-D-glucopyranos-1′-yl)]-1H-1,2,3-
It was obtained by treating the acetylated compound 3b (0.40 g,
triazol-4-yl)methyl-benzenesulphonamide (4e)
0.63 mmol) with NaOMe in methanol, in 92% yield (0.26 g) as a white
solid; mp 64–66 ◦C;[
α
]
1.3 (c 0.1, CH3OH); IR (νmax, cmꢀ 1): 3400,
25
D
3019, 1644, 1215 and 769.1H NMR (400 MHz, DMSO‑d6): δ 8.77 (m, 1H,
It was obtained by treating the acetylated compound 3e (0.40 g,
0.59 mmol) with NaOMe in methanol in 92% yield (0.26 g) as a white
–NH), 8.04 (s, 1H, triazolyl-H), 7.53 (m, 1H, Ar-H), 7.16 (m, 1H, Ar-H),
25
́
solid; mp 88–90 ◦C;[
α
]
D
– 1.2 (c 0.1, CH3OH); IR (νmax, cmꢀ 1): 3408,
6.70 (m, 1H, Ar-H), 6.53 (m, 1H, Ar-H), 5.47 (d, J = 9.1 Hz, 1H, H-1),
3019, 1753, 1644, 1215 and 769.1H NMR (400 MHz, DMSO‑d6): δ 11.56
(s, 1H, –NH), 9.33 (s, 1H, triazolyl-H), 8.17 (s, 1H, Ar-H), 7.74 (m, 1H,
Ar-H), 7.63 (m, 2H, Ar-H), 7.46 (m, 2H, Ar-H), 7.30 (m, 2H, Ar-H),7.11
4.48 (m, 2H, –CH2), 4.02 (m, 2H, H-5′, –OH), 3.75 (m, 3H, 3 × –OH),
3.70 (m, 4H, H-2 , H-3 , H-4, H-6), 3.17 (bs, 2H, –NH2).13C NMR (100
′
′
́
́
–
–
MHz, DMSO‑d ): δ 169.3 (C O), 150.1 (Ar-C), 145.9 (Ar-C), 132.3,
6
́
(d, J = 9.1 Hz, 1H, H-1), 5.55 (m, 2H, –CH2), 5.52 (m, 1H, –OH), 4.50
128.6 (Ar-C), 122.0, 116.9 (Ar-C), 115.1 (Ar-C), 114.5 (Ar-C), 88.4 (C-
′
́
́
́
́
́
(m, 2H, H-5′, –OH), 3.80 (m, 3H, 3 × –OH), 3.76–3.67 (m, 4H, H-2′, H-
1), 78.8 (C-5), 74.1 (C-3), 69.8 (C-4), 68.9 (C-2), 60.8 (C-6 ), 34.9.
HRMS: Calcd. Accurate mass for (C16H21N5O6): 380.1492. Found
380.1528 [M + H]+.
3 , H-4, H-6),2.31 (s, 3H, –CH3).13C NMR (100 MHz, DMSO) δ 168.5,
144.6, 144.3, 138.8, 136.0, 133.1, 130.3, 128.9, 127.2, 123.8, 122.7,
119.9, 87.8, 80.3, 77.4, 72.4, 70.0, 40.4, 40.2, 40.0, 39.8, 39.8, 39.5,
39.3, 39.1, 35.1, 21.4. HRMS: Calcd. Accurate mass for (C23H27N5O8S):
534.1580. Found 534.1621 [M + H]+.
′
́
́
3.13. 2-hydroxy-N-[(1-(1′- deoxy-β-D-glucopyranos-1′-yl)]-1H-1,2,3-
triazol-4-yl)methyl-benzamide (4c)
3.16. 4-methyl-N-[(1-(1′-deoxy-β-D-galactopyranose-1′-yl)]-1H-1,2,3-
It was obtained by treating the acetylated compound 3c (0.40 g, 0.60
triazol-4-yl)methyl-benzenesulphonamide (4f).
mmol) with NaOMe in methanol in 93% yield (0.27 g) as a white solid;
25
mp 84–86 ◦C;[
α
]
D
– 2.4 (c 0.1, CH3OH); IR (νmax, cmꢀ 1): 3670, 3401,
3018, 1644, 1215 and 757.1H NMR (400 MHz, DMSO‑d6): δ 8.74 (m, 1H,
–NH), 7.94 (s, 1H, triazolyl-H), 7.53 (m, 1H, Ar-H), 7.15 (m, 1H, Ar-H),
6.72 (m,1H, Ar-H), 6.52 (m, 1H, Ar-H), 6.43 (bs, 1H, –OH), 5.88 (d, J =
It was obtained by treating the acetylated compound 3f (0.40 g, 0.59
mmol) with NaOMe in methanol in 90% yield (0.25 g) as a white solid;
mp 83–85 ◦C;[
α]
– 5.4 (c 0.1, CH3OH); IR (νmax, cmꢀ 1): 3408, 3019,
25
D
1753, 1644, 1215 and 769.1H NMR (400 MHz, DMSO‑d6): δ 11.60 (s,
1H, –NH), 9.34 (s, 1H, triazolyl-H), 8.15 (s, 1H, Ar-H), 7.75 (m, 1H, Ar-
H), 7.63 (m, 2H, Ar-H), 7.51 (m, 2H, Ar-H), 7.31 (m, 2H, Ar-H), 7.13 (d,
́
9.3 Hz, 1H, H-1), 5.38 (m, 1H, –OH), 5.26 (m, 1H, –OH), 5.17 (m, 1H,
–OH),4.68 (m, 1H, –OH), 4.48 (m, 2H, –CH2), 3.78–3.59 (m, 3H, H-2′, H-
3′, H-4′), 3.25 (m, 2H, H-5′, H-6′)13C NMR (100 MHz, DMSO‑d6): δ 169.2
–
–
́
J = 9.1 Hz, 1H, H-1), 5.51 (m, 2H, –CH2), 4.51 (m, 1H, –OH), 4.08 (m,
(C O), 150.1(Ar-C), 145.8(Ar-C), 132.2(Ar-C), 128.6(Ar-C), 123.8(Ar-
2H, H-5′, –OH), 3.78 (m, 3H, 3 × –OH), 3.57–3.45 (m, 4H, H-2′, H-3′, H-
C), 116.8(Ar-C), 114.7(Ar-C), 111.2, 104.9, 85.4, 79.6, 74.0, 49.2,
34.9, 27.0, 26.HRMS: Calcd. Accurate mass for (C18H23N5O5):
390.1699. Found 390.1735 [M + H]+.
4, H-6),2.31 (s, 3H, –CH3).13C NMR (100 MHz, DMSO) δ 168.5, 144.6,
144.3, 138.9, 136.1, 133.1, 130.3, 128.9, 127.2, 123.8, 122.3, 120.6,
120.0, 88.5, 78.8, 74.2, 69.8, 68.8, 60.8, 49.0, 40.5, 40.3, 40.1, 39.9,
39.7, 39.5, 39.3, 35.2, 23.7, 21.4. HRMS: Calcd. Accurate mass for
(C23H27N5O8S): 534.1580. Found 534.1618 [M + H]+.
́
́
3.14. 2-hydroxy-N-[(1-(1′-deoxy-β-D-galactopyranose-1′-yl)]-1H-1,2,3-
triazol-4-yl)methyl-benzamide (4d)
It was obtained by treating the acetylated compound 3d (0.40 g,
4. Biology
0.58 mmol) with NaOMe in methanol in 91% yield (0.26 g) as a yellow
25
solid; mp 73–75 ◦C;[
α
]
D
– 1.6 (c 0.1, CH3OH); IR (νmax, cmꢀ 1): 3408,
The compounds synthesized glycosyl 1,2,3-1H-triazolyl methyl
3019, 1753, 1644, 1215 and 769.1H NMR (400 MHz, DMSO‑d6): δ 12.43
(s, 1H, –NH), 9.35 (s, 1H, triazolyl-H), 8.13 (s, 1H, Ar-H), 7.90 (m, 1H,
Ar-H), 7.43 (m, 1H, Ar-H), 7.39 (m, 1H, Ar-H), 6.92 (d, J = 9.1 Hz, 1H,
benzamide derivatives were evaluated against the α-glucosidase purified
from rat intestine, purified rat liver following earlier protocols [43,44].
The In-vitro -glucosidase inhibition activity results indicated that all the
synthesized compounds (IC50 values ranging from 25.3 ± 0.8 to 118.5 ±
α
′
́
H-1), 5.48 (m, 2H, –CH2), 5.21 (m, 1H, –OH), 4.59 (m, 2H, H-5 , –OH),
6

A.K. Shukla et al.
Bioorganic Chemistry 109 (2021) 104687
Fig 3. Figure showing chemical structures of the selected active molecules.
5.3
μ
M) exhibited more inhibitory activity in comparison to standard
6. Computational methodology
drug acarbose (IC50 = 750.0 ± 12.5
μ
M). The percent inhibitory activity
of screened compounds against
α
-glucosidase enzyme and IC50value of
6.1. System selection and preparation
all the compounds have been shown in Table 3 and evidently, most of
the triazolo-methyl benzamide derivative with deacetylated sugar unit
The lead molecules (Mol-4c, Mol-4d & Mol-4f) that had good bio-
logical activities were selected for computational studies. Their chemi-
cal structures are shown in (Fig. 3). The protein, 3A4A [45] was
obtained from Protein Data Bank [46]. Water molecules, from selected
protein, were removed using UCSF Chimera before the start of docking
calculations [47].
displayed better α-enzyme inhibition than that of analogues with acet-
ylated sugar unit. The deacetylated glycosyl methyl benzamide deriva-
tive (4c, 4d and 4f) IC50 value 25.3 0.8,26.1 1.5 and 30.6 2.1
respectively.
5. Preparation of
α-Glucosidase from rat intestinal mucosa
6.2. Molecular docking calculations
α
-Glucosidase was prepared according to a slight modification of the
procedure reported earlier [43,44]. The intestine of male albino rats (CF
strain average body weight 200 ± 20 g) was excised opened and the
mucosa was collected and pooled. A 10% homogenate was prepared in
150 mM KCl using Potter Elvejhem glass homogeniser fitted with Teflon
pestle. The homogenate was centrifuged at 1000g for 15 min and the
supernatant was decanted and stored at 4 ◦C. The supernatant was
dialyzed at 4 ◦C against 50 mM Tris–HCl buffer pH 7.0 with two to three
changes of buffer. The dialyzed supernatant was saturated with
ammonium sulphate to a final concentration of 30%. The sample was
kept at 4 ◦C overnight and then centrifuged to collect the precipitate and
the supernatant separately. The 30% ammonium sulphate saturated
supernatant was further saturated to 60% with ammonium sulphate.
Again the precipitate and supernatant were separated by centrifugation.
Finally, the 60% ammonium sulphate saturated supernatant was further
saturated to 100% with a further addition of ammonium sulphate. The
precipitate and supernatant were once again separated and all the
Molecular Docking is a widely used computational technique that
predicts the binding of potent inhibitors in the vicinity of the target
macromolecules [48,49]. The ability to determine the binding affinity
and main interacting protein residues are the key objectives of molec-
ular docking of protein–ligand complexes [50]. Here, the docking
¨
calculation was carried out using Schrodinger’s GLIDE module [51]. The
docked pose with the least binding affinity was extracted and aligned
with the receptor for further analysis.
6.3. Molecular dynamics simulation
MDS is a widely used computational technique to study protein
folding, unwinding and other conformational changes including com-
plex stability, over a specified period. Its applications have gained
utmost importance due to lack of experimental resources [52]. GRO-
MACS 5.1.1 (Groningen Machine for Chemical Simulations) software
package [53] was used to carry out the molecular dynamics simulations
in the current research work. A total of two ligand–protein complexes
were created, after docking simulations for molecular dynamic simula-
tions viz., (Mol-4c, Mol-4d & Mol-4f) (Fig. 4). These ligand–protein
complexes were put to for 10000 ps (10 ns) time scale simulation. There
are many studies for the comparison of force fields for the nucleic acids
but GROMOS54A7 force fields [54] seems to be good for nucleic acid
simulation due to the presence of specific topologies for the terminal
nucleotides. The topology for the selected ligands was obtained using
PRODRG web server [55]. The protein–ligand complexes were solvated
samples were analysed for α-glucosidase activity using p-nitrophenyl-
α
-D-glucopyranoside as substrate. When it was observed that the
enzyme activity is maximum in 60–100% ammonium sulphate precipi-
tate it was stored at 4 ◦C and used as a source of enzyme for studying the
effect of test compounds on α-glucosidase inhibition.
Fig 4. Figure showing best docked posed protein–ligand complexes.
7

A.K. Shukla et al.
Bioorganic Chemistry 109 (2021) 104687
Fig 5. Figure showing different interactions in best docked posed protein–ligand complexes.
7. Results and discussion
Table 4
Table representing various docking results.
This study was aimed to complement the experimental results and
observations obtained, through means of computer simulations. It also
included the identification of new leads, targeting the ligand-binding
affinity, structural stability and applicability for the experimentally
active ligands with protein. The results obtained through computer
simulations are summarized and discussed as follows:
S. No.
Molecule
3A4A
Docking Score
Glide Score
(kcal/mol)
1.
2.
3.
Mol-4c
Mol-4d
Mol-4f
ꢀ 7.526
ꢀ 7.526
ꢀ 7.865
ꢀ 7.555
ꢀ 7.555
ꢀ 7.893
7.1. Molecular docking
in an octahedral box using the TIP3P water model at 298 K [56]. Counter
ions were then added to the solvated box containing the protein–ligand
complex by randomly replacing the water molecules to neutralize the
system. Particle Mesh Ewald (PME) was used to handle long-range
electrostatic interactions in periodic boundary conditions [57]. Energy
minimization of the whole system was carried out in 25,000 steps using
Steepest Descent leap- Frog Integration Method followed by NVT
ensemble equilibration at a constant temperature of 300 K for the 50 s
using Berendsen thermostat [58]. The system was then equilibrated with
NPT ensemble at a constant pressure of 1 atm in 25,000 steps using
steepest descent leap-frog integrator [59]. Particle Mesh Ewald (PME)
was used to handle long-range electrostatic interactions in periodic
boundary conditions and all the bonds involving hydrogen atoms were
constrained using the LINCS algorithm [60]. Graphs were plotted using
XMgrace software [61].
The selected active ligands (Mol-4c, Mol-4d & Mol-4f) were docked
to 3A4A protein in search of the interacting residues against the best
docked posed complexes (Fig. 5). The docking results, corresponding to
the selected protein sequences are summarized below in table 4. Various
studies have been done to understand the docking studies of ligand-
bound biological macromolecules [62]. Docking calculations revealed
that all three biologically active molecules had comparable docking
energies and Glide scores. And this result validates the fact that exper-
imentally active molecules have good as well as comparable docking &
Glide scores.
The key amino acid residues that are involved in the docking cal-
culations are discussed in the forthcoming hydrogen bonding analysis
section.
7.2. Hydrogen bonding analysis
Following Fig. 6 & Fig. 7 represent the binding site and corre-
sponding H-donor/acceptor clouded regions near those hydrogen
Fig 6. H-interaction sites for Mol-4c, Mol-4d & Mol-4f with 3A4A.
8

A.K. Shukla et al.
Bioorganic Chemistry 109 (2021) 104687
Fig 7. H-interaction cloud for Mol-4c, Mol-4d & Mol-4f with 3A4A.
Table 5
The following table represents the donor and the acceptor amino acid residues
involved in the formation of a hydrogen bond between the PROTEIN and ligand
atoms.
S.
Complex
No of H-
bonds
Interacting Species in H-
Bonding
H-Bond Length
(Å)
No.
Formed
1
2
3
3A4A + Mol-
8
8
8
ARG315:HE - LIG900:
2.609662
1.638967
1.775453
2.119318
2.823075
2.716957
1.948873
1.629865
4c
O6
LIG900:H16 - GLU411:
OE2
LIG900:H15 - GLU411:
OE2
ARG315:H - LIG900:N2
LIG900:H9 - HIS280:
NE2
THR310:HG1 - LIG900:
O1
LIG900:H1 - SER311:O
LIG900:H10 - PRO312:
O
3A4A + Mol-
LIG900:H16 - GLU411:
1.638967
1.775453
2.609662
2.119318
2.716957
1.948873
2.823075
1.629865
4d
OE2
LIG900:H15 - GLU411:
OE2
ARG315:HE - LIG900:
Fig 8. Figure depicting variation in energy of the system.
O6
ARG315:H - LIG900:N2
THR310:HG1 - LIG900:
bonding sites, using Discovery Studio Visualizer [63]. This data gives us
more detailed information about the docking results and the existence of
H-bonds and key amino acid residues in the formation of H-bonds.
Table 5 gives information about the donor and the acceptor amino acid
residues involved in the formation of the hydrogen bond between the
protein and ligand atoms, along with their respective bond lengths.
O1
LIG900:H1 - SER311:O
LIG900:H9 - HIS280:
NE2
LIG900:H10 - PRO312:
O
3A4A + Mol-
LYS156:HZ2 - LIG900:
2.104894
1.967705
1.774475
2.820494
2.454007
2.839473
3.062884
2.161487
4f
O4
LIG900:H11 - LEU313:O
7.3. Molecular dynamics simulation
LIG900:H1 - ASP242:
OD2
Structural stability of biomolecules under a specifically mimicked
environment for a pre-defined period can be studied via molecular dy-
namics simulations. Such studies are of significant importance in the
structure and dynamics of biomolecules owing to less experimental
costs. Here in this study, various parameters were studied and analyzed
for their biological validation and are discussed as follows:
LIG900:H9 - A:PRO312:
O
ARG315:H - LIG900:N2
GLN279:HE21 - LIG900:
O8
GLN279:HE22 - LIG900:
O8
LIG900:H15 - GLU411:
OE2
7.3.1. Energy variations-
The energy variations obtained from molecular dynamics simulation
of 3A4A for a time scale of 10000 ps are shown in Fig. 8. The variations
in the energies of all the two ligand–protein complexes are comparable.
9

A.K. Shukla et al.
Bioorganic Chemistry 109 (2021) 104687
Fig 11. RMSD for protein–ligand complexes.
Fig 9. Fig representing variations for Radius of Gyration.
Fig 12. RMSF for protein–ligand complexes.
be seen from the figure that mol-4c could form an average of 7H-bonds,
mol-4d could form an average of 8H-bonds whereas the maximum
number of H-bonds formed by mol-4f is 9. This suggests the stronger
interaction between all the three protein–ligand complexes and hence
gets validated from the docking results also.
7.3.4. Root mean square deviation-
Fig 10. Variation in No. of H-bonds for protein–ligand complexes.
The RMSD is treated as the measure of conformational stability. The
plots for RMSD of all the ligand- PROTEIN complexes are represented in
Fig. 11. From the RMSD graph shown below, it can be seen that for Mol-
4c & Mol-4d the RMSD range is between 10 ~ 10.4 Å whereas for Mol-4f
the RMSD range lies between 10.4 ~ 10.8 Å; but the RMSD for Mol-4f
shows stabilizing variations towards the end of the simulations and
hence claim its stability than Mol-4c & Mol-4d.
And hence the confirmation of stable complex formation requires
analysis from various other perspectives viz., interactions and confor-
mational stabilities, etc. of protein–ligand complexes.
7.3.2. Variation in radius of Gyration-
Radii of Gyration values are determined t understand the dynamic
stability and compactness of protein–ligand complexes. The variation in
radius of gyration of protein–ligand complexes can be seen from Fig. 9.
The avg. radiuses of gyration for all the three protein–ligand complexes
lie between 2.425 nm and 2.5 nm resp. These variation results reveal
that protein sequence remains compact for the whole 10000 ps molec-
ular dynamics simulation and hence confirms the stability of the
complexes.
7.3.5. Root mean square Fluctuation-
RMSF give us information about the fluctuation of each amino acid
along with the fluctuations in the flexible regions of the protein during
molecular dynamics simulation. The graphs shown below in Fig. 12
suggest that all the three protein–ligand complexes had comparable
RMSF and therefore they contribute significantly towards the stability of
the complexes. Here this range lies between 0.1 ~ 0.4 Å.
7.3.3. Variation in number of hydrogen bonds-
8. Conclusion
Hydrogen bonds play a crucial role in determining the binding af-
finity as well as binding specificity in protein–ligand interactions.
Fig. 10 depicts the average number of hydrogen bonds being formed
during the molecular dynamics trajectory for 10000 ps. Further, it can
In conclusion, we have synthesized novel glycosyl methyl benzamide
analogues with 1,4-regioselectivity employing the well-known CuAAC
10

A.K. Shukla et al.
Bioorganic Chemistry 109 (2021) 104687
benzamides and phenylacetamides as antimicrobial agents, Bioorg. Med. Chem. 15
(2007) 2032–2044.
reaction of the propylated benzamides with different sugar azides in the
ambient condition in very good yields. These compounds were evalu-
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N-[4-(alkyl) cyclohexyl]-substituted benzamides with anti-inflammatory and
analgesic activities, Il Farmaco 54 (1999) 524–532.
ated for α-glucosidase enzyme inhibitory activity and three compounds
4f, 4d and4c exhibited 42.9%, 40.6%, and 39.7%inhibition, respectively
as compared to standard drug acarbose having 53.4% inhibition of the
enzyme. Additionally, the docking results generated best docked posed
complexes and proved to validate the findings of the experiments and
complimented them very well. Further, when these docked complexes,
when subjected to molecular dynamics, the results obtained also vali-
dated the findings of the docking & experimental results. Therefore, it
can be concluded that computational techniques confirmed the stability
of the active molecules and hence add to the validation of the experi-
mental results. Thus, these glycosyl methyl benzamide analogues hold
potential to be developed as antidiabetic agents.
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Declaration of Competing Interest
[25] X.-L. Wang, K. Wan, C.-H. Zhou, Synthesis of novel sulfanilamide-derived 1, 2, 3-
triazoles and their evaluation for antibacterial and antifungal activities, Eur. J.
Med. Chem. 45 (2010) 4631–4639.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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The authors AP and MKS are thankful to ICMR RA fellowship, Govt.
of India, New Delhi and AKS is thankful to University-UGC fellowship
for financial assistance and Dr Abhinay Pandey for providing biological
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