Synthesis, cytotoxicity, and molecular docking of substituted 3-(2-methylbenzofuran-3-yl)-5-(phenoxymethyl)-1,2,4-oxadiazoles

Article abstract of DOI:10.1002/ardp.202000006

A series of new benzofuran/oxadiazole hybrids (8a–n) was synthesized from 2H-chromene-3-carbonitriles (3a–c) through the multistep synthetic methodology, and these hybrids are known to exhibit anticancer activities. All the compounds were evaluated for their in vitro cytotoxicity against the HCT116 and MIA PaCa2 cell lines. Compounds 6a (IC₅₀: 9.71 ± 1.9 μM), 6b (IC₅₀: 7.48 ± 0.6 μM), and 6c (IC₅₀: 3.27 ± 1.1 μM) displayed a significant cytotoxic activity, whereas compounds 8d and 8e exhibited good activity against both cell lines. The depletion of glycogen synthase kinase-3β (GSK3β) induces apoptosis through the inhibition of basal NF-κB activity in HCT116 colon cancer cells and MIA PaCa2 pancreatic cancer cells. Molecular docking of compounds 6a, 6b, 6c, 8d, and 8e with GSK3β demonstrated the best binding affinity, correlating with the biological activity assay. Furthermore, the structure–activity relationship of these novel compounds reveals promising features for their use in anticancer therapy.

Full text of DOI:10.1002/ardp.202000006

Received: 7 January 2020
Revised: 24 March 2020
Accepted: 27 March 2020
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DOI: 10.1002/ardp.202000006
F U L L P A P E R
Synthesis, cytotoxicity, and molecular docking of substituted
3‐(2‐methylbenzofuran‐3‐yl)‐5‐(phenoxymethyl)‐1,2,
4‐oxadiazoles
Sudhakar Mokenapelli1
Jayaprakash R. Yerrabelli1
Someswar R. Sagurthi2
| Gangadhar Thalari1 | Naveen Vadiyaala1
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| Vamshi K. Irlapati2 | Neelima Gorityala2
| Prasad R. Chitneni1
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1Natural Products Laboratory, Department of
Chemistry, Osmania University, Hyderabad,
Telangana, India
Abstract
A series of new benzofuran/oxadiazole hybrids (8a–n) was synthesized from
2H‐chromene‐3‐carbonitriles (3a–c) through the multistep synthetic methodology,
and these hybrids are known to exhibit anticancer activities. All the compounds
were evaluated for their in vitro cytotoxicity against the HCT116 and MIA PaCa2
cell lines. Compounds 6a (IC50: 9.71 1.9 μM), 6b (IC50: 7.48 0.6 μM), and 6c
(IC50: 3.27 1.1 μM) displayed a significant cytotoxic activity, whereas compounds
8d and 8e exhibited good activity against both cell lines. The depletion of glycogen
synthase kinase‐3β (GSK3β) induces apoptosis through the inhibition of basal NF‐κB
activity in HCT116 colon cancer cells and MIA PaCa2 pancreatic cancer cells.
Molecular docking of compounds 6a, 6b, 6c, 8d, and 8e with GSK3β demonstrated
the best binding affinity, correlating with the biological activity assay. Furthermore,
the structure–activity relationship of these novel compounds reveals promising
features for their use in anticancer therapy.
2Department of Genetics and Biotechnology,
Osmania University, Hyderabad, Telangana,
India
Correspondence
Prasad R. Chitneni, Natural Products
Laboratory, Department of Chemistry,
Osmania University, Hyderabad,
Telangana 500007, India.
Email: prasadraoou@yahoo.com
Funding information
University Grants Commission,
Grant/Award Number: 124192
K E Y W O R D S
1, 2, 4‐oxadiazole, benzofuran, cytotoxicity, docking studies, glycogen synthase kinase‐3β
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1
INTRODUCTION
have been widely investigated for selective biological and phar-
macological properties.[13,14] 1,2,4‐Oxadiazoles are the most pop-
The benzofuran scaffold has been considered as a significant moi-
ety in drug design and development since few years due to its
progressive chemotherapeutic and physiological properties, and
wide existence in nature.[1] Synthetic and natural benzofurans
and its derivatives displayed numerous biological activities such
as antimicrobial,[2] antibacterial,[3] anti‐inflammatory,[4] anti-
diabetic,[5] antitumor,[6] and antineophobic activities.[7] Recently,
U.S. FDA accepted drugs containing benzofuran derivatives.[8,9]
Natural products with benzofuran core showed potential cyto-
toxicity.[10] Besides, synthetic benzofuran derivatives signify a vital
source of antiproliferative agents against several tumor cell
lines.[11,12] Benzofurans with substituents at C‐2 and C‐3 positions
ular scaffolds in material science, medical chemistry, and
pharmaceuticals.[15] Furthermore, the oxadiazole scaffold pos-
sesses hydrolytic, metabolic stability, and enhanced pharmacoki-
netic properties.[16] 1,2,4‐Oxadiazoles have been defined as
bioisosteres of amides and esters.[17] These are promising scaffolds
of biologically active compounds, isolated from natural pro-
ducts.[18] Researchers mainly focused on the synthesis of 1,2,4‐
oxadiazole derivatives in medicinal chemistry[19] due to their re-
markable biological activities, such as anticancer,[20] antibiotic,[21]
antifungal,[22] antioxidant,[23] anti‐inflammatory,[24] and antic-
onvulsant activities.[25] Recently, several 1,2,4‐oxadiazole deriva-
tives were reported having anticancer activities.[26,27] Particularly,
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Arch Pharm. 2020;e2000006.
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3,5‐disubstituted‐1,2,4‐oxadiazoles were reported to have poten-
tial anticancer activities and apoptosis against various cancer cell
lines.[28–32] These results established that 3,5‐disubstituted‐1,2,4‐
oxadiazoles are potential anticancer motifs. Herein, we designed
and synthesized novel benzofuran‐based 3,5‐disubstituted‐1,2,4‐
oxadiazole hybrids and evaluated their cytotoxic activities against
the HCT116 (colon cancer) and MIA PaCa2 (pancreatic cancer) cell
lines.
2H‐chromene‐3‐carbonitriles 3a–c.[33] Sodium azide‐intervened
catalyst‐free ring contraction reaction of compounds 3a–c in di-
methyl sulfoxide (DMSO) at 160°C temperature converted the
pyran ring into the furan ring to yield 2‐methylbenzofuran‐3‐
carbonitriles 4a–c.[34] Furthermore, compounds 4a–c treated with
hydroxylamine hydrochloride in the presence of triethylamine and
ethanol at reflux temperature gave the corresponding N‐hydroxy‐
2‐methylbenzofuran‐3‐carboximidamides 5a–c. Moreover, chlor-
oacetyl chloride reacted with 5a–c in tetrahydrofuran (THF) under
reflux condition to yield corresponding 5‐(chloromethyl)‐3‐(2‐
methylbenzofuran‐3‐yl)‐1,2,4‐oxadiazoles 6a–c; subsequently,
substituted phenols 7a–e were treated with 6a–c in the presence
of potassium carbonate in acetone at reflux temperature to yield
the corresponding 3‐(2‐methylbenzofuran‐3yl)‐5‐[(substituted
phenoxy)methyl]‐1,2,4‐oxadiazole derivatives 8a–n in good
yields. All the synthesized compounds were characterized by
1H nuclear magnetic resonance (NMR), 13C NMR, electrospray
ionization–high‐resolution mass spectrometry (ESI–HRMS),
and electrospray ionization–mass spectrometry (ESI–MS).
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2
RESULTS AND DISCUSSION
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2.1
Chemistry
The synthetic route of novel benzofuran‐1,2,4‐oxadiazole
hybrids, 8a–n, is outlined in Scheme 1. Initially, substituted
salicylaldehydes 1a–c reacted with acrylonitrile (2) in
the presence of 1,4‐diazabicyclo[2.2.2]octane (DABCO) under
Baylis–Hillman reaction condition to give corresponding
SCHEME 1 Synthesis of compounds 8a–n. Reagents and conditions: (i) DABCO, 80°C, 12 hr; (ii) NaN3, DMSO, 160°C, 30 min; (iii)
NH2OH·HCl, TEA, EtOH, reflux, 6 hr; (iv) chloroacetyl chloride, THF, reflux, 6 hr; (v) K2CO3, acetone, reflux, 1 hr. DABCO, 1,4‐diazabicyclo
[2.2.2]octane; DMSO, dimethyl sulfoxide; EtOH, ethyl alcohol; TEA, triethylamine; THF, tetrahydrofuran

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atmospheric pressure. The structure of 8b was confirmed by single‐
crystal X‐ray diffraction analysis (Figure 1). The crystallographic data
of 8b are reported in Table 1.
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2.3
Cytotoxic activity
All the synthesized compounds were tested for their cytotoxic activity
against two cancerous cell lines, HCT116 (colon cancer) and MIA PaCa2
(pancreatic cancer). Compounds 6a, 6b, 6c, 8d, and 8e showed <50%
growth consistent with the positive control (paclitaxel), as shown in
Figures 2 and 3. Comparatively, compounds 6a, 6b, and 6c demon-
strated notable cytotoxicity than 8d and 8e. IC50 values of all the
compounds are shown in Table 2. Compounds 6a, 6b, 6c, 8d, and 8e
FIGURE 1 Molecular structure of compound 8b
Furthermore, the structure of compound 8b was confirmed by
X‐ray crystallography, as shown in Figure 1.
reported
a significant cytotoxic activity, of which compound 6c
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2.2
X‐ray crystallography
exhibited potential cytotoxicity against both cell lines.
A single crystal was grown by the slow evaporation of the solution
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Molecular docking studies
of compound 8b in dichloromethane at room temperature and
2.4
Glycogen synthase kinase‐3β (GSK3β) is a multifunctional serine/
threonine kinase that plays a crucial role in the regulation of several
signaling pathways, including cellular processes such as cell cycle,
inflammation, and cell proliferation. GSK3β has been reported
in the literature as one of the potential therapeutic targets for diseases
such as diabetes, cancer, cardiac disease, Alzheimer's, and other cen-
tral nervous system disorders.[35,36] The depletion of GSK3β induces
apoptosis through the inhibition of basal NF‐κB activity in HCT116
colon cancer cells[37] and MIA PaCa2 pancreatic cancer cells.[38] NF‐κB
activation is known to promote human cancer progression.[39] GSK3β
positively regulates NF‐kB binding to its target gene promoters and
activates a subset of antiapoptotic (XIAP, Bcl‐2) and proliferation (cyclin
D1) genes, leading to cancer cell proliferation and survival.[40] Hence,
the inhibition of GSK3β could be effective in the treatment of a wide
variety of cancers. The interaction of all the synthesized compounds
with GSK3β was studied by molecular docking; results are shown in
Table 3. Compounds 6a–c and 8a–n displayed significant MolDock
scores greater than −100 when docked with GSK3β (1H8F), indicating
a good interaction. Conversely, compounds 6a, 6b, 6c, 8d, and 8e
exhibited potent cytotoxicity against HCT116 and MIA PaCa2 cell
lines. The MolDock score, Rerank score, and interacting residues for
6a, 6b, 6c, 8d, and 8e are listed in Table 4. Three‐ and two‐dimensional
figures are shown in Figures 4 and 5, respectively. Oxadiazole rings of
6a and 6b compounds form a hydrogen bond with Phe67. Further-
more, benzofuran and oxadiazole rings participate in various π inter-
actions with Val70, Lys85, and Val87 residues. Compound 6c formed a
hydrogen bond with Asn95 and carbon–hydrogen bond with Arg96
and Glu97. π interactions were also seen with Lys85, Phe67, and
Val87. In compound 8d, hydrogen bond interactions were observed
with Ser66, Asn95, and Arg96, along with carbon–hydrogen bonding
with Phe67 and Glu97. Compound 8d also displayed π bonding with
Phe67, Arg96, and Glu97. Compound 8e showed hydrogen bond in-
teractions with amino acid residues Ser66 and Asn95, along with
TABLE 1 Crystallographic data of compound 8b
Compound
CCDC
Formula
Mw
8b
1991479
C18H13N2O3Cl
340.75
Crystal system
Space group
T (K)
Triclinic
P1
290 K
a (Å)
7.769 (3)
b (Å)
8.044 (2)
c (Å)
12.906 (4)
α (°)
85.430 (12)
β (°)
80.205 (13)
γ (°)
88.778 (12)
Z
2
V (Å3)
792.2 (4)
Dcalc (g/cm3)
1.429
µ (mm−1
)
0.260
Total reflns
Unique reflns
Observed reflns
R1[I > 2σ(I)]
wR2 (all)
4,172
4,075
2,713
0.3048
0.1058
GOF
1.030
Diffractometer
Bruker D8 Venture Photon III detector
Abbreviation: CCDC, Cambridge Crystallographic Data Centre; GOF,
goodness of fit.

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FIGURE 2 Cell proliferation of HCT116 cells 72 hr after treatment with synthesized compounds at 10‐μM concentration. p = <.0001
indicates the significance of the data. The p value summary was represented as "****" after comparison of each test with DMSO control. DMSO,
dimethyl sulfoxide
carbon–hydrogen bond interactions with Phe67, Arg96, and Glu97.
Additionally, π interactions were also observed with Val87 and Arg96.
All these compounds were found to have an optimum orientation with
key catalytic residues such as Lys85, Glu97, and Arg96, suggesting
that these hits may serve as potential cytotoxic inhibitors.
Arg96, Phe67, Val70, Val87, and Asn95. Furthermore, oxadiazole ring
and chlorine interact with Lys85, which is the critical residue for the
catalytic activity of the enzyme. However, 6c showed least IC50 values,
3.27 and 5.34 μM in HCT116 and MIA PaCa2, respectively, due to the
hydrogen bond interaction of bromine with Arg96, in addition to
chlorine interaction with Lys85. Hence, electronegative group substitu-
tion of bromine at N‐5 position of benzofuran ring confers enhanced
activity. The phenyl substitutions of oxadiazole ring with different groups
(halogen, benzyl, phenyl, and heteroaryl) have resulted in an increase and
decrease of activity. In compounds 6a, 6b, and 6c, the electronegative
group substitution of chlorine contributed to enhanced activity, but
substitution with other groups like phenoxy at the same position in
compounds 8a–n resulted in a decreased activity in spite of high dock
scores, probably due to strong electron‐withdrawing property of chlor-
ine in compounds 6a–c than phenoxyl group in compounds 8a–n.
However, a biological activity was observed in 8d as the oxygen atom of
oxadiazole ring and the oxygen atom of nitro group formed hydrogen
bonds with the residues Asn95, Arg96, Glu97, and Ser66, respectively. In
compound 8e, oxadiazole ring and methoxy group interacted with Asn95
and Ser66 residues through hydrogen bonds. Thus, these studies reveal
that oxadiazole ring upon substitution with highly electronegative atoms
or heteroaryl groups imparts increased cytotoxicity. Benzofuran ring and
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2.5
Structure–activity relationship (SAR) studies
All the synthesized compounds were analyzed; 17 compounds displayed
a significant interaction with GSK3β protein, which is revealed by a
higher, negative binding free energy, as shown in Table 3. Among these
synthesized derivatives, 6a, 6b, and 6c showed potential cytotoxicity
when tested against the HCT116 and MIA PaCa2 cell lines, in addition to
compounds 8d and 8e that showed significant cytotoxicity against
HCT116 and MIA PaCa2 cells individually. Compounds 6a, 6b, 6c, 8d,
and 8e exhibited significant IC50 values. Docking studies of experimen-
tally identified compounds showed a fairly good correlation between
binding free energy and IC50 values against GSK3β. The study reveals
that compounds 6a, 6b, and 6c with minimum IC50 values can serve as
potential inhibitors, as the benzofuran ring and nitrogen of oxadiazole
interact with key active site residues of an enzyme such as Lys85, Glu97,
FIGURE 3 Cell proliferation of MIA PaCa2 cells 72 hr after treatment with synthesized compounds at 10‐μM concentration. p = <.0001
indicates the significance of the data. The p value summary was represented as "****" after comparison of each test with DMSO control. DMSO,
dimethyl sulfoxide

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5‐(chloromethyl)‐3‐substituted‐1,2,4‐oxadiazole played an important
role in strengthening the interactions between synthesized derivatives
and target GSK3β protein.
TABLE 3 Molecular docking results of the compounds in chain A
of glycogen synthase kinase‐3β (PDB ID: 1H8F)
Rerank
Ligand
Paclitaxel
3a
MolDock score (kcal/mol)
−164.14
−71.03
score (kcal/mol)
−101.40
−64.14
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3
CONCLUSION
3b
3c
−76.80
−67.06
In the present study, we developed a simple and efficient protocol for
the synthesis of a novel series of benzofuran‐1,2,4‐oxadiazole hybrids
(8a–n) from 2H‐chromene‐3‐carbonitriles (3a–c) via key intermediate
−74.49
−63.61
4a
−79.77
−65.71
4b
4c
−85.08
−75.58
−86.15
−70.42
TABLE 2 MTT assay in HCT116 and MIA PaCa2 cell lines after
24‐hr treatment with synthesized compounds
5a
−85.02
−71.53
5b
5c
−94.14
−75.30
Sl.No.
1
Compound
HCT116
MIA PaCa2
No activity
No activity
No activity
>100 μM
−89.36
−73.82
3a
3b
3c
No activity
No activity
No activity
>100 μM
6a
−109.54
−113.87
−117.14
−133.82
−139.43
−130.21
−142.07
−140.79
−136.32
−143.58
−137.98
−144.12
−136.14
−134.34
−141.85
−140.26
−145.97
−137.69
−85.21
2
6b
6c
−83.83
3
−90.03
4
4a
4b
4c
8a
−107.84
−109.98
−89.59
5
>100 μM
>100 μM
8b
8c
6
>100 μM
>100 μM
7
5a
5b
5c
No activity
>100 μM
No activity
>100 μM
8d
8e
−116.79
−106.05
−106.42
−104.91
−110.09
−109.11
−97.98
8
9
>100 μM
>100 μM
8f
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
6a
6b
6c
11.27 1.3 μM
7.48 0.6 μM
3.27 1.1 μM
No activity
No activity
No activity
11.12 1.8 μM
No activity
>100 μM
9.71 1.9 μM
8.31 0.9 μM
5.34 1.2 μM
No activity
No activity
No activity
No activity
16.20 1.7 μM
>100 μM
8g
8h
8i
8a
8b
8c
8j
8k
−99.87
8l
−106.44
−96.81
8d
8e
8f
8m
8n
8o
−118.44
−106.62
8g
8h
8i
>100 μM
>100 μM
>100 μM
>100 μM
>100 μM
>100 μM
8j
>100 μM
>100 μM
2‐methylbenzofuran‐3‐carbonitriles (4a–c). All the synthesized com-
pounds were tested for their cytotoxicity against HCT116 and MIA
PaCa2 cells. Compounds 6a, 6b, and 6c demonstrated potential cyto-
toxicity against both cell lines, whereas compounds 8d and 8e
exhibited significant cytotoxicity against the HCT116 and MIA PaCa2
cell lines, respectively. Furthermore, molecular docking and SAR studies
explained binding affinities of benzofuran, oxadiazole, and heteroaryl
rings with GSK3β protein. Thus, these results indicate that benzofuran
and 3,5‐disubstituted‐1,2,4‐oxadiazole scaffolds play a crucial role in
imparting potential cytotoxicity. Further research would validate these
molecules as potent anticancer agents.
8k
8l
No activity
No activity
>100 μM
No activity
No activity
>100 μM
8m
8n
Paclitaxel
>100 μM
>100 μM
3.9 μM
4.2 μM
Note: Values are expressed as the mean standard error of the mean
of three experiments. Values shown in bold possess a significant
cytotoxic activity.
Abbreviation: MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium
bromide.

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TABLE 4 Docking score results of top compounds with interacting amino acid residues in the active site of glycogen synthase kinase 3β (PDB
ID:1H8F)
MolDock score
(kcal/mol)
Rerank score
(kcal/mol)
Ligand
Paclitaxel
6a
Interacting residues
−164.14
−106.98
−113.87
−111.12
−142.07
−140.79
−101.40
−81.47
−83.83
−79.43
−116.79
−106.05
Gln185, Asp200, Lys183, Cys218
Phe67, Val87, Lys85, Val70
6b
Phe67, Val87, Lys85, Val70
6c
Asn95, Phe67, Val87, Lys85, Glu97, Arg96
Ser66, Phe67, Val87, Asn95, Arg96, Glu97
Ser66, Phe67, Val87, Asn95, Arg96, Glu97
8d
8e
(t, J = 7.5 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 4.80 (s, 2H). 13C NMR
(101 MHz, CDCl3) δ 154.3, 138.8, 132.7, 128.4, 122.4, 120.0, 116.6,
116.4, 103.3, 64.3. ESI–HRMS m/z calcd for C10H7NO 158.0606 [M
+H]+, found 158.0600.
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4
EXPERIMENTAL
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4.1
Chemistry
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4.1.1
General
8‐Methoxy‐2H‐chromene‐3‐carbonitrile (3b)
Unless otherwise specified, all solvents and reagents were obtained
from commercial suppliers. Solvents were purified as per the
procedures given in the Textbook of Practical Organic Chemistry by
Vogel (6th edition). All reactions were performed under nitrogen
atmosphere unless otherwise noted. Column chromatography was
performed using Merck silica gel 60–120 mesh. NMR spectra were
recorded on a Bruker Avance III spectrometer at 400 and 101 MHz.
Tetramethylsilane was used as the internal standard, and chemical
shift (δ) was reported in parts per million (ppm). Multiplicity of NMR
signals were described as a singlet (s), doublet (d), doublet of doublet
(dd), triplet of doublet (td), and multiplet (m), and coupling constant
was denoted by J (in Hz). Mass spectral analysis was accomplished
using electrospray ionization (ESI) technique.
Yield: 69%, yellow solid, mp. 102–104°C. 1H NMR (400 MHz, CDCl3)
δ 7.16 (t, J = 1.3 Hz, 1H), 6.94 (d, J = 1.8 Hz, 1H), 6.92 (s, 1H), 6.74 (dd,
J = 5.4, 3.7 Hz, 1H), 4.86 (s, 2H), 3.88 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 148.0, 143.2, 138.8, 122.2, 120.7, 120.3, 116.3, 115.3,
103.4, 64.5, 56.1. ESI–HRMS m/z calcd for C11H9NO2 188.0711 [M
+H]+, found 188.0714.
6‐Bromo‐2H‐chromene‐3‐carbonitrile (3c)
Yield: 63%, white solid, mp. 148–150°C. 1H NMR (400 MHz, CDCl3) δ
7.36 (dd, J = 8.7, 2.4 Hz, 1H), 7.23 (d, J = 2.4 Hz, 1H), 7.10 (s, 1H), 6.76
(d, J = 8.7 Hz, 1H), 4.82 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 153.2,
137.4, 135.2, 130.6, 121.6, 118.4, 115.9, 114.4, 104.8, 64.4.
ESI–HRMS m/z calcd for C10H6BrNO 235.9711 [M+H]+, found
235.9715.
The original spectra of the investigated compounds, together
with their InChI keys and some biological activity data, are provided
as Supporting Information Data.
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4.1.3
General procedure for the synthesis of 2‐
methylbenzofuran‐3‐carbonitriles (4a–c)
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4.1.2
General procedure for the synthesis of
2H‐chromene‐3‐carbonitriles (3a–c)
Sodium azide (3.971 g, 61.084 mmol) was added to a stirred solution
of 2H‐chromene‐3‐carbonitrile (3a) (8 g, 50.903 mmol) in DMSO
(20 ml), and then the reaction mixture was heated to 160°C for
30 min. After the completion of the reaction, it was monitored by
thin‐layer chromatography (TLC), and the reaction mixture was
quenched with ice water (200 ml) and extracted with ethyl acetate
(300 ml). The organic layer was dried over anhydrous Na2SO4,
purified by column chromatography using 60–120 mesh silica gel,
and eluted with EtOAc/hexane (1:10) to give 2‐methylbenzofuran‐3‐
carbonitrile (4a) as a white solid (4.8 g).
DABCO (1.837 g, 16.377 mmol) was added to a solution of O‐salicylal
dehyde (1a) (10.0 g, 81.886 mmol) in acrylonitrile (2) (100 ml) and stirred
for 12 hr at 80°C. After completion of the reaction, the excess acryloni-
trile was removed under reduced pressure, and the resultant crude
product was dissolved in ethyl acetate (EtOAc; 500 ml) and washed with
5% dil. HCl. The organic layer was washed with brine solution, dried
over anhydrous Na2SO4, purified by column chromatography using
60–120 mesh silica gel, and eluted with EtOAc/hexane (1:10) to give
2H‐chromene‐3‐carbonitrile (3a) as a yellow solid (8.5 g).
2‐Methylbenzofuran‐3‐carbonitrile (4a)
2H‐Chromene‐3‐carbonitrile (3a)
Yield: 65%, off‐white solid, mp. 51–53°C. 1H NMR (400 MHz, CDCl3)
δ 7.30–7.24 (m, 1H), 7.16 (s, 1H), 7.10 (dd, J = 7.5, 1.5 Hz, 1H), 6.97
Yield: 75%, white solid, mp. 148–150°C. 1H NMR (400 MHz, CDCl3) δ
7.62–7.57 (m, 1H), 7.48–7.45 (m, 1H), 7.36–7.31 (m, 2H), 2.65 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 164.8, 153.7, 125.5, 124.3, 119.5,

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FIGURE 4 Three‐dimensional structure view of molecular docking of 6a, 6b, 6c, 8d, and 8e (green sticks) with human GSK3β protein (gray
lines). GSK3β, glycogen synthase kinase‐3β

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FIGURE 5 Two‐dimensional receptor–ligand interactions of paclitaxel, 6a, 6b, 6c, 8d, and 8e at the active site of the GSK3β protein. The
oxadiazole ring of 6a and 6b shows a hydrogen bond (green dotted lines) interaction with Phe67, and the oxadiazole ring of 8d and 8e shows a
hydrogen bond interaction with Asn95. GSK3β, glycogen synthase kinase‐3β

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113.3, 111.4, 91.3, 13.8. ESI–HRMS m/z calcd for C10H7NO 158.0606
112.3, 13.7. ESI–HRMS m/z calcd for C10H9BrN2O2 268.9925 [M
[M+H]+, found 158.0600.
+H]+, found 268.9989.
7‐Methoxy‐2‐methylbenzofuran‐3‐carbonitrile (4b)
Yield: 56%, white solid, mp. 151–153°C. 1H NMR (400 MHz, CDCl3) δ
7.29–7.25 (m, 1H), 7.18 (dd, J = 7.9, 1.0 Hz, 1H), 6.86 (dd, J = 8.0,
0.9 Hz, 1H), 4.01 (s, 3H), 2.67 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
164.7, 145.1, 127.6, 125.2, 113.3, 111.5, 107.5, 91.7, 56.1, 13.8.
ESI–HRMS m/z calcd for C11H9NO2 188.0711 [M+H]+, found
188.0715.
4.1.5
General procedure for the synthesis of
|
5‐(chloromethyl)‐3‐[(2‐methylbenzofuran‐3‐yl)]‐
1,2,4‐oxadiazoles (6a–c)
A
mixture of N′‐hydroxy‐2‐methylbenzofuran‐3‐carboximidamide
(5a) (3.0 g, 15.773 mmol) in 30 ml of THF was cooled to 10°C and
chloroacetyl chloride (1.50 ml, 18.927 mmol) was added dropwise;
the mixture was then refluxed for 6 hr. The progress of reaction was
monitored by TLC, and after the completion of the reaction, excess
THF and chloroacetyl chloride were removed under reduced pres-
sure, dissolved in ethyl acetate, and washed with water. Organic
layer was dried over anhydrous Na2SO4, filtered, and evaporated.
The crude compound was subjected to column purification with n‐
hexane to yield a colorless solid (6a).
5‐Bromo‐2‐methylbenzofuran‐3‐carbonitrile (4c)
Yield: 55%, white solid, mp. 180–182°C. 1H NMR (400 MHz,
CDCl3) δ 7.74 (d, J = 1.9 Hz, 1H), 7.45 (dd, J = 8.8, 2.0 Hz, 1H), 7.35
(d, J = 8.7 Hz, 1H), 2.67 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
166.0, 152.4, 128.6, 127.8, 122.3, 117.6, 112.9, 91.0, 13.9.
ESI–HRMS m/z calcd for C10H6BrNO 235.9711 [M+H]+, found
235.9715.
5‐(Chloromethyl)‐3‐[(2‐methylbenzofuran‐3‐yl)]‐1,2,4‐
oxadiazole (6a)
|
4.1.4
General procedure for the synthesis
of N‐hydroxy‐2‐methylbenzofuran‐3‐
carboximidamides (5a–c)
Yield: 65%, white solid, mp. 208–210°C. 1H NMR (400 MHz, CDCl3) δ
8.12–8.07 (m, 1H), 7.48–7.43 (m, 1H), 7.34–7.29 (m, 2H), 4.76 (s, 2H),
2.84 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 173.4, 164.7, 158.8, 154.0,
126.0, 124.5, 123.6, 121.6, 110.7, 104.1, 33.3, 14.2. ESI–HRMS m/z
calcd for C12H9ClN2O2 249.0431 [M+H]+, found 249.0437.
A mixture of 2‐methylbenzofuran‐3‐carbonitrile (4a) (4 g, 25.450 mmol),
hydroxylamine hydrochloride (2.122 g, 30.540 mmol), and 10 ml of
triethylamine in 50 ml of ethanol was refluxed for 6 hr. The progress of
the reaction was monitored by TLC, and after the completion of the
reaction, the excess ethanol and triethylamine were removed under
reduced pressure. The obtained brown solid was placed in water (20 ml),
and the solid precipitate was separated by filtration, washed with water,
and dried at 50°C to give amidoxime as a brown solid.
5‐(Chloromethyl)‐3‐[(7‐methoxy‐2‐methylbenzofuran‐3‐yl)]‐1,2,
4‐oxadiazole (6b)
Yield: 59%, white solid, mp. 212–214°C. 1H NMR (400 MHz, CDCl3) δ
7.69 (dd, J = 7.9, 0.6 Hz, 1H), 7.28–7.24 (m, 1H), 6.84 (d, J = 8.0 Hz,
1H), 4.77 (s, 2H), 4.03 (s, 3H), 2.86 (s, 3H).13C NMR (101 MHz,
CDCl3) δ 173.4, 164.6, 158.8, 144.8, 143.2, 127.6, 124.4, 113.9,
106.7, 104.5, 56.0, 33.3, 14.3. ESI–HRMS m/z calcd for
C13H11ClN2O3 279.0536 [M+H]+, found 279.0543.
N‐Hydroxy‐2‐methylbenzofuran‐3‐carboximidamide (5a)
Yield: 85%, brown solid, mp. 188–190°C. 1H NMR (400 MHz,
CDCl3) δ 7.70 (dd, J = 6.1, 2.7 Hz, 1H), 7.41 (dd, J = 6.5, 2.3 Hz,
1H), 7.26–7.22 (m, 1H), 4.92 (s, 2H), 2.61 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 155.0, 153.8, 147.7, 126.9, 124.0, 123.1,
119.9, 110.9, 109.1, 13.6. ESI–HRMS m/z calcd for C10H10N2O2
191.0820 [M+H]+, found 191.0951.
3‐(5‐Bromo‐2‐methylbenzofuran‐3‐yl)‐5‐[(chloromethyl)]‐1,2,4‐
oxadiazole (6c)
Yield: 61%, white solid, mp. 215–217°C. 1H NMR (400 MHz, CDCl3) δ
8.23 (d, J = 2.0 Hz, 1H), 7.40 (dd, J = 8.7, 2.0 Hz, 1H), 7.31 (d,
J = 8.6 Hz, 1H), 4.77 (s, 2H), 2.83 (s, 3H).13C NMR (101 MHz, CDCl3) δ
173.6, 164.2, 159.9, 152.8, 127.9, 127.5, 124.3, 116.8, 112.2, 103.8,
33.2, 14.3. ESI–HRMS m/z calcd for C12H8BrClN2O2 326.9536 [M
+H]+, found 326.9543.
N‐Hydroxy‐7‐methoxy‐2‐methylbenzofuran‐3‐carboximidamide (5b)
Yield: 80%, white solid, mp. 193–195°C. 1H NMR (400 MHz, CDCl3) δ
7.25–7.22 (m, 2H), 6.83 (dd, J = 5.9, 2.9 Hz, 1H), 6.00 (s, 2H), 4.02 (s,
3H), 2.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 166.1, 161.1, 145.2,
142.8, 127.2, 124.5, 123.8, 112.3, 111.5, 106.4, 56.0, 14.1. ESI–HRMS
m/z calcd for C11H12N2O3 221.0926 [M+H]+, found 221.1008.
|
4.1.6
General procedure for the synthesis
of substituted 3‐(2‐methylbenzofuran‐3‐yl)‐5‐
[(phenoxymethyl)]‐1,2,4‐oxadiazoles (8a–n)
5‐Bromo‐N‐hydroxy‐2‐methylbenzofuran‐3‐carboximidamide (5c)
Yield: 83%, white solid, mp. 205–207°C. 1H NMR (400 MHz, CDCl3) δ
7.86 (dd, J = 12.1, 1.9 Hz, 1H), 7.35 (dd, J = 8.7, 2.0 Hz, 1H), 7.27 (d,
J = 3.2 Hz, 1H), 4.88 (s, 1H), 2.62 (s, 3H). 13C NMR (101 MHz, CDCl3)
δ 156.2, 152.6, 128.8, 127.3, 126.9, 122.9, 122.4, 116.9, 116.2, 112.7,
A stirred solution of 5‐(chloromethyl)‐3‐[(2‐methylbenzofuran‐3‐yl)]‐
1,2,4‐oxadiazole (6a) (0.150 g, 0.603 mmol), phenol (7a) (0.062 g,
0.663 mmol), and anhydrous K2CO3 (0.100 g, 0.723 mmol) in acetone

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(20 ml) was refluxed for 1 hr. The progress of reaction was monitored
by TLC, and after the completion of the reaction, acetone was
removed under reduced pressure, dissolved in ethyl acetate, and
washed with water (50 ml). The organic layer was dried over anhy-
drous Na2SO4, filtered, and evaporated. The crude compound was
purified with column chromatography by using 5% EtOAc in n‐
hexane to yield compound 8a.
124.5, 124.4, 123.6, 123.5, 121.7, 121.6, 114.9, 110.7, 110.7, 104.3,
61.4, 33.3, 14.2. ESI–HRMS m/z calcd for C19H16N2O4 337.1188 [M
+H]+, found 337.1196.
3‐(7‐Methoxy‐2‐methylbenzofuran‐3‐yl)‐5‐(phenoxymethyl)‐1,2,4‐
oxadiazole (8f)
Yield: 80%, white solid, mp. 205–207°C. 1H NMR (400 MHz, CDCl3) δ
7.70 (d, J = 8.4 Hz, 1H), 7.36–7.30 (m, 2H), 7.27–7.24 (m, 1H),
7.07–7.01 (m, 3H), 6.84 (d, J = 7.9 Hz, 1H), 5.37 (s, 2H), 4.02 (s, 3H),
2.86 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 173.9, 164.3, 158.7, 157.6,
144.8, 129.7, 127.7, 124.3, 122.3, 114.9, 113.9, 106.6, 104.6, 61.1,
56.0, 14.3. ESI–HRMS m/z calcd for C19H16N2O4 337.1188 [M+H]+,
found 337.1196.
3‐(2‐Methylbenzofuran‐3‐yl)‐5‐[(phenoxymethyl)]‐1,2,
4‐oxadiazole (8a)
Yield: 81%, white solid, mp. 210–212°C. 1H NMR (400 MHz, CDCl3)
δ 8.13–8.08 (m, 1H), 7.45 (td, J = 4.5, 3.4, 2.6 Hz, 1H), 7.35–7.28
(m, 4H), 7.07–7.01 (m, 3H), 5.35 (s, 2H), 2.83 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 173.9, 164.4, 158.7, 157.6, 154.0, 129.7, 126.1,
124.5, 123.6, 122.3, 121.7, 114.9, 110.7, 104.2, 61.1, 14.3. ESI–HRMS
m/z calcd for C18H14N2O3 307.1082 [M+H]+, found 307.1086.
5‐[(2‐Chlorophenoxy)methyl]‐3‐(7‐methoxy‐2‐methylbenzofuran‐3‐
yl)‐1,2,4‐oxadiazole (8 g)
Yield: 84%, white solid, mp. 209–211°C. 1H NMR (400 MHz, CDCl3) δ
7.69 (d, J = 7.9 Hz, 1H), 7.41 (d, J = 7.2 Hz, 1H), 7.27–7.20 (m, 2H),
7.09 (d, J = 8.0 Hz, 1H), 7.00 (t, J = 7.4 Hz, 1H), 6.83 (d, J = 7.9 Hz, 1H),
5.43 (s, 2H), 4.02 (s, 3H), 2.85 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
173.4, 164.3, 158.7, 153.3, 144.8, 143.2, 130.8, 127.8, 124.3, 123.46,
115.0, 113.9, 106.7, 104.6, 62.2, 56.0, 14.3. ESI–HRMS m/z calcd for
C19H15ClN2O4 371.0798 [M+H]+, found 371.0806.
5‐[(2‐Chlorophenoxy)methyl]‐3‐[(2‐methylbenzofuran‐3‐yl)]‐1,2,4‐
oxadiazole (8b)
Yield: 85%, white solid, mp. 210–212°C. 1H NMR (400 MHz, CDCl3) δ
8.12–8.08 (m, 1H), 7.48–7.44 (m, 1H), 7.42 (dd, J = 7.9, 1.6 Hz, 1H),
7.33–7.29 (m, 2H), 7.24–7.20 (m, 1H), 7.09 (dd, J = 8.3, 1.3 Hz, 1H),
7.00 (td, J = 7.7, 1.4 Hz, 1H), 5.44 (s, 2H), 2.84 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 173.4, 164.4, 158.7, 154.0, 153.3, 130.8, 127.8,
126.0, 124.5, 123.9, 123.6, 123.4, 121.6, 115.0, 110.7, 104.1, 62.3,
14.3. ESI–HRMS m/z calcd for C18H13ClN2O3 341.0693 [M+H]+,
found 341.0698.
5‐[(4‐Bromophenoxy)methyl]‐3‐(7‐methoxy‐2‐methylbenzofuran‐3‐
yl)‐1,2,4‐oxadiazole (8h)
Yield: 88%, white solid, mp. 212–214°C. 1H NMR (400 MHz, CDCl3) δ
7.68 (dd, J = 7.8, 0.6 Hz, 1H), 7.45–7.40 (m, 2H), 7.25 (dd, J = 10.2,
5.7 Hz, 1H), 6.96–6.90 (m, 2H), 6.84 (d, J = 7.8 Hz, 1H), 5.34 (s, 2H),
4.03 (s, 3H), 2.85 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 173.4, 164.4,
158.7, 156.7, 144.8, 132.6, 127.7, 124.4, 116.8, 113.9, 106.7, 104.5,
61.2, 56.0, 14.3. ESI–HRMS m/z calcd for C19H15BrN2O4 415.0293
[M+H]+, found 415.0298.
5‐[(4‐Bromophenoxy)methyl]‐3‐[(2‐methylbenzofuran‐3‐yl)]‐1,2,4‐
oxadiazole (8c)
Yield: 88%, white solid, mp. 215–217°C. 1H NMR (400 MHz, CDCl3) δ
8.11–8.07 (m, 1H), 7.48–7.40 (m, 3H), 7.34–7.30 (m, 2H), 6.95–6.91
(m, 2H), 5.34 (s, 2H), 2.83 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
173.4, 164.4, 158.7, 156.7, 154.0, 132.6, 126.0, 124.5, 123.6, 121.6,
116.8, 114.7, 110.7, 104.1, 61.2, 14.3. ESI–HRMS m/z calcd for
C18H13BrN2O3 385.0188 [M+H]+, found 385.0201.
3‐(7‐Methoxy‐2‐methylbenzofuran‐3‐yl)‐5‐[(4‐methoxyphenoxy)‐
methyl]‐1,2,4‐oxadiazole (8i)
Yield: 70%, white solid, mp. 212–214°C. 1H NMR (400 MHz, CDCl3) δ
7.69 (dd, J = 7.9, 0.7 Hz, 1H), 7.27–7.23 (m, 1H), 7.11 (d, J = 8.3 Hz,
2H), 6.95–6.92 (m, 2H), 6.83 (d, J = 7.8 Hz, 1H), 5.33 (s, 2H), 4.02 (s,
3H), 2.85 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 174.1, 164.3, 158.6,
155.6, 144.7, 143.2, 131.7, 130.1, 127.7, 124.3, 114.8, 113.9, 106.6,
104.7, 61.3, 56.0, 20.5, 14.3. ESI–HRMS m/z calcd for C20H18N2O5
367.1294 [M+H]+, found 367.1298.
3‐(2‐Methylbenzofuran‐3‐yl)‐5‐[(4‐nitrophenoxy)methyl]‐1,2,4‐
oxadiazole (8d)
Yield: 65%, white solid, mp. 208–210°C. 1H NMR (400 MHz, CDCl3) δ
8.28–8.22 (m, 2H), 8.10–8.06 (m, 1H), 7.50–7.44 (m, 1H), 7.35–7.30
(m, 2H), 7.16–7.10 (m, 2H), 5.47 (s, 2H), 2.83 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 172.5, 164.5, 162.1, 158.8, 154.0, 142.7, 126.0,
124.6, 123.7, 121.5, 114.9, 110.8, 104.0, 61.1, 14.3. ESI–HRMS m/z
calcd for C18H13N3O5 352.0963 [M+H]+, found 352.0984.
3‐(5‐Bromo‐2‐methylbenzofuran‐3‐yl)‐5‐(phenoxymethyl)‐1,2,4‐
oxadiazole (8j)
5‐[(4‐Methoxyphenoxy)methyl]‐3‐(2‐methylbenzofuran‐3‐yl)‐1,2,4‐
oxadiazole (8e)
Yield: 75%, white solid, mp. 207–209°C. 1H NMR (400 MHz, CDCl3) δ
8.10 (s, 2H), 7.44 (s, 2H), 7.11 (d, J = 7.6 Hz, 2H), 6.93 (d, J = 7.9 Hz,
2H), 5.32 (s, 2H), 4.74 (s, 3H), 2.83 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 174.0, 173.4, 164.7, 164.3, 158.7, 158.6, 154.0, 130.1,
Yield: 84%, white solid, mp. 208–210°C. 1H NMR (400 MHz, CDCl3) δ
8.24 (d, J = 2.0 Hz, 1H), 7.40 (dd, J = 8.7, 2.0 Hz, 1H), 7.36–7.30 (m,
3H), 7.07–7.02 (m, 3H), 5.38 (s, 2H), 2.83 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 174.1, 163.9, 159.8, 157.5, 152.8, 129.7, 128.0, 127.4,
124.4, 122.4, 116.8, 114.9, 112.2, 103.9, 61.0, 14.3. ESI–HRMS m/z
calcd for C18H13BrN2O3 385.0188 [M+H]+, found 385.0193.

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|
Cytotoxic assay
3‐(5‐Bromo‐2‐methylbenzofuran‐3‐yl)‐5‐[(2‐chlorophenoxy)methyl]‐
1,2,4‐oxadiazole (8k)
4.2.2
Yield: 87%, white solid, mp. 212–214°C. 1H NMR (400 MHz, CDCl3) δ
8.23 (d, J = 2.0 Hz, 1H), 7.45–7.38 (m, 2H), 7.31 (d, J = 8.6 Hz, 1H),
7.25 (td, J = 8.3, 6.1, 1.6 Hz, 1H), 7.10 (dd, J = 8.3, 1.3 Hz, 1H), 7.01 (td,
J = 7.7, 1.3 Hz, 1H), 5.45 (s, 2H), 2.82 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 173.6, 163.9, 159.9, 152.8, 130.8, 127.8, 127.4, 124.3,
123.5, 116.8, 115.0, 112.2, 103.8, 62.2, 14.3. ESI–HRMS m/z calcd for
C18H12BrClN2O3 418.9798 [M+H]+, found 418.9803.
MTT assay (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium
bromide) was used to determine the cell proliferation of the cell
lines after treatment with test compounds.[42] The log‐phase cells
were trypsinized, counted, and seeded into 96‐well plates in pre-
determined numbers on the basis of the cell line type. After 24 hr of
recovery period, the cells were treated with test compounds at a final
concentration of 10 µM along with DMSO control and positive con-
trol (paclitaxel). The treated cells were processed after 72 hr end-
point by adding 40‐μl MTT solution (5 mg/ml stock prepared in
phosphate‐buffered saline). Incubated for 2 hr at 37°C in CO2 in-
cubator, followed by centrifugation at 500g for 5 min and aspiration
of the growth media, 200 μl of DMSO was added to each well to
solubilize the insoluble purple formazan crystals that were formed
due to cleavage of MTT by cellular mitochondrial dehydrogenases.
The plates were subjected to shaking on a plate shaker to ensure
complete solubilization. The final absorbance was read at 540 nm
with a reference filter of 690 nm. The percentage cell viability or %
growth was determined relative to DMSO control. IC50 values were
generated using 5‐dose treatment and finally analyzed with Graph-
Pad Prism version 6.0.
3‐(5‐Bromo‐2‐methylbenzofuran‐3‐yl)‐5‐[(4‐bromophenoxy)methyl]‐
1,2,4‐oxadiazole (8l)
Yield: 84%, white solid, mp. 215–217°C. 1H NMR (400 MHz, CDCl3) δ
8.22 (d, J = 2.0 Hz, 1H), 7.44–7.40 (m, 2H), 7.39 (d, J = 2.0 Hz, 1H),
7.33–7.29 (m, 1H), 6.95–6.91 (m, 2H), 5.34 (s, 2H), 2.81 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 173.6, 163.9, 159.9, 156.6, 152.8, 132.6,
127.9, 127.4, 124.3, 116.8, 114.8, 112.2, 103.8, 61.2, 14.3. ESI–HRMS
m/z calcd for C18H12Br2N2O3 462.9293 [M+H]+, found 462.9298.
3‐(5‐Bromo‐2‐methylbenzofuran‐3‐yl)‐5‐[(4‐nitrophenoxy)methyl]‐
1,2,4‐oxadiazole (8m)
Yield: 73%, white solid, mp. 212–214°C. 1H NMR (400 MHz, CDCl3) δ
8.28–8.24 (m, 2H), 8.21 (d, J = 1.9 Hz, 1H), 8.15 (d, J = 9.1 Hz, 1H),
7.41 (dd, J = 8.7, 2.0 Hz, 1H), 7.17–7.12 (m, 2H), 5.49 (s, 2H), 2.82
(s, 3H). 13C NMR (101 MHz, CDCl3) δ 172.8, 164.0, 162.1, 160.0,
152.8, 142.7, 127.8, 127.5, 126.2, 126.0, 116.9, 115.6, 114.9, 103.7,
61.1, 14.3. ESI–HRMS m/z calcd for C18H12BrN3O5 430.0038 [M
+H]+, found 430.0045.
|
4.3
Docking studies
The interaction of GSK3β protein with substituted 3‐(2‐methylbenzo
furan‐3‐yl)‐5‐(phenoxymethyl)‐1,2,4‐oxadiazoles was studied using
Molegro Virtual Docker (MVD 2012.5.5).[43] Docking was performed
at the potential active site of GSK3β protein with synthesized deri-
vatives and paclitaxel. The structures of all the synthesized compounds
were generated using MarvinSketch 5.6.0.2. (1998–2011, Copyright ©
ChemAxon Ltd.), cleaned in 3D, and saved in .pdb format for docking
studies. The PDB file of the target protein was downloaded from RCSB
PDB (www.rcsb.org), GSK3β (PDB ID:1H8F). During docking, the
protein and ligands were prepared by assigning bonds, bond orders,
charges, explicit hydrogen, and flexible torsions in ligands if they were
missing. The search algorithm was taken as MolDock SE and the
number of runs was taken as 10; maximum iterations were 2,000, with
a population size of 50 and with an energy threshold of 100. Further
investigations of the binding interactions of the most active docked
compounds were performed using BIOVIA Discovery Studio 2017 R2
Client. To obtain more potent compounds as inhibitors, the optimal
binding mode of a molecule to the active site of macromolecule was
interpreted.
3‐(5‐Bromo‐2‐methylbenzofuran‐3‐yl)‐5‐[(4‐methoxyphenoxy)‐
methyl]‐1,2,4‐oxadiazole (8n)
Yield: 86%, white solid, mp. 209–211°C. 1H NMR (400 MHz, CDCl3) δ
8.25 (d, J = 2.0 Hz, 1H), 7.40 (dd, J = 8.7, 2.0 Hz, 1H), 7.31 (d,
J = 8.7 Hz, 1H), 7.13 (d, J = 8.2 Hz, 2H), 6.98–6.92 (m, 2H), 5.35 (s, 2H),
2.83 (s, 3H), 2.30 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 174.3, 163.8,
159.8, 155.5, 152.8, 131.8, 130.1, 127.4, 124.4, 116.8, 114.9, 112.1,
103.9, 61.3, 20.5, 14.3. ESI–HRMS m/z calcd for C19H15BrN2O4
413.0293 [M+H]+, found 413.0298.
|
4.2
Biology
|
4.2.1
Maintenance of cell line model systems
In this study, two cancer cell lines were used, namely HCT116 and
MIA PaCa2, which represent colon and pancreatic cancers, respec-
tively. Both the cell lines were maintained as monolayer cultures and
continuously observed under phase‐contrast microscope to ensure
contamination‐free cells. The cell lines were grown in Dulbecco's
modified Eagles's medium supplemented with 10% (v/v) of fetal
bovine serum and 1× concentration of PenStrep antibiotic (Gibco).
The cells were maintained in 5% CO2 incubator supplemented with
37°C humidification.[41]
ACKNOWLEDGMENTS
Sudhakar Mokenapelli acknowledges UGC, New Delhi, India, for
providing UGC‐SRF fellowship, head of Department of Chemistry
and CFRD Osmania University, Hyderabad, India, for providing
facilities and Advanced Centre of Research in High Energy Mate-
rials (ACRHEM), and the University of Hyderabad for X‐ray crys-
tallography facility.

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CONFLICT OF INTERESTS
[21] E. Spink, D. Ding, Z. Peng, J. Med. Chem. 2015, 58, 1380.
[22] J. N. Sangshetti, D. B. Shinde, Eur. J. Med. Chem. 2011, 46, 1040.
[23] J. G. Leal, A. C. Sauer, J. C. P. Mayer, New J. Chem. 2017, 41, 5875.
[24] M. Farooqui, R. Bora, C. R. Patil, Eur. J. Med. Chem. 2009, 44, 794.
[25] M. M. Khanaposhtani, M. Shabani, M. Faizi, Eur. J. Med. Chem. 2016,
112, 91.
The authors declare that there are no conflicts of interests.
ORCID
Sudhakar Mokenapelli
Jayaprakash R. Yerrabelli
Someswar R. Sagurthi
http://orcid.org/0000-0001-9575-4830
http://orcid.org/0000-0003-4387-8334
http://orcid.org/0000-0003-1939-0849
[26] W. Kemnitzer, J. Kuemmerle, H. Z. Zhang, Bioorg. Med. Chem. Lett.
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