New 3-(1H-benzo[d]imidazol-2-yl)quinolin-2(1H)-one-based triazole derivatives: Design, synthesis, and biological evaluation as antiproliferative and apoptosis-inducing agents

Article abstract of DOI:10.1002/ardp.202100074

A series of 1,2,3-triazole derivatives based on the quinoline–benzimidazole hybrid scaffold was designed, synthesized, and screened against a panel of NCI-60 humanoid cancer cell lines for in vitro cytotoxicity evaluation, which revealed that compound Q6 was the most potent cytotoxic agent with excellent GI₅₀, TGI, and LC₅₀ values on multiple cancer cell lines. Q6 was tested further on the BT-474 breast cancer line to evaluate the mechanism of action. Preliminary screening studies based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay revealed that compound Q6 had an excellent antiproliferative effect against human breast cancer cells, BT-474, with IC₅₀ values of 0.59 ± 0.01 μM. The detailed study based on the acridine orange/ethidium bromide staining (AO/EB) and the 4′,6-diamidino-2-phenylindole (DAPI) assay suggested that the antiproliferative activity shown was due to the induction of apoptosis on exposure to Q6. Further, DCFDA staining showed the generation of reactive oxygen species, altering the mitochondrial potential and leading to the initiation of apoptosis. This was further supported by JC-1 staining, indicating that this scaffold can contribute to the development of more potent derivatives.

Full text of DOI:10.1002/ardp.202100074

Received: 22 February 2021
Revised: 12 July 2021
Accepted: 15 July 2021
|
|
DOI: 10.1002/ardp.202100074
F U L L P A P E R
New 3‐(1H‐benzo[d]imidazol‐2‐yl)quinolin‐2(1H)‐one‐based
triazole derivatives: Design, synthesis, and biological
evaluation as antiproliferative and apoptosis‐inducing agents
Nikhil B. Gaikwad1
Nanduri Srinivas1
| Sapana Bansode2 | Shankar Biradar1 | Mayuri Ban1
| Chandraiah Godugu2 | Venkata M. Yaddanapudi1
|
1Department of Chemical Sciences, National
Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad, Telangana,
India
Abstract
A series of 1,2,3‐triazole derivatives based on the quinoline–benzimidazole hybrid
scaffold was designed, synthesized, and screened against a panel of NCI‐60 huma-
noid cancer cell lines for in vitro cytotoxicity evaluation, which revealed that com-
pound Q6 was the most potent cytotoxic agent with excellent GI50, TGI, and LC50
values on multiple cancer cell lines. Q6 was tested further on the BT‐474 breast
cancer line to evaluate the mechanism of action. Preliminary screening studies based
on the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay revealed
that compound Q6 had an excellent antiproliferative effect against human breast
cancer cells, BT‐474, with IC50 values of 0.59 0.01 μM. The detailed study based
on the acridine orange/ethidium bromide staining (AO/EB) and the 4′,6‐diamidino‐
2‐phenylindole (DAPI) assay suggested that the antiproliferative activity shown was
due to the induction of apoptosis on exposure to Q6. Further, DCFDA staining
showed the generation of reactive oxygen species, altering the mitochondrial po-
tential and leading to the initiation of apoptosis. This was further supported by JC‐1
staining, indicating that this scaffold can contribute to the development of more
potent derivatives.
2Department of Biological Sciences, National
Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad, Telangana,
India
Correspondence
Venkata M. Yaddanapudi, Department of
Chemical Sciences, National Institute of
Pharmaceutical Education and Research
(NIPER), Hyderabad 500037, Telangana, India.
Email: yssmadhavi@gmail.com
K E Y W O R D S
1,2,3‐triazole, antiproliferative agent, apoptosis induction, quinoline
|
1
INTRODUCTION
have been reported to induce apoptosis, namely, DNA‐damaging
agents (e.g., chemical, heat shock, radiation), reactive nitrogen spe-
Cancer, a noncommunicable disease, is the second‐largest leading
cause of death as per WHO; it caused 9.6 million deaths in 2018,
suggesting that one out of six deaths is due to cancer.[1] The primary
symptoms of cancer are uncontrolled growth/division and impaired/
inappropriate apoptosis. Apoptosis is a natural process that brings
about programmed death to the cells that are genetically damaged
and beyond repair (such as cancer cells in which the genetic material
is damaged due to environmental factors or mutation). Many factors
cies, reactive oxygen species (ROS), hypoxia, and viral infection.[2,3]
The mitochondria are found to be a major contributor to ROS
production, which has a strong association with the apoptotic
pathways.[4] The elevated levels of the ROS lead to the augmentation
of oxidative stress, which further alters the mitochondrial membrane
potential (ΔΨm), and the mitochondrial dysfunction induces
apoptosis.[5] Studies suggested that the cancer cells have a higher
amount of ROS compared with normal cells and using this function
© 2021 Deutsche Pharmazeutische Gesellschaft
|
Arch. Pharm. 2021;e2100074.
wileyonlinelibrary.com/journal/ardp
1 of 15
https://doi.org/10.1002/ardp.202100074

2 of 15
GAIKWAD ET AL.
|
and ability to discriminate cellular ROS levels between the cancer cell
and normal cell may provide us with an avenue toward obtaining a
cancer‐specific apoptosis inducer.[6] Thus, any chemical agent that
can further increase the oxidative stress in cancer cells exceeding the
tolerable cut‐off point may lead to a set of events such as DNA
damage, lipid damage, membrane disruption, and finally tumor cell
death.[7] Thus, many of the current anticancer agents cause cellular
modifications that ultimately result in apoptosis. Hence, targeting
apoptosis has become an emerging trend in cancer research,[8] and
many of the current anticancer agents act through this mechanism.
A thorough literature search for new apoptosis inducers led us to
dovitinib (a multikinase inhibitor and apoptosis inducer). This
2‐oxo‐quinoline‐based anticancer drug is known to induce apoptosis
as well as autophagy, which showed potent antiproliferative activity
against breast cancer cells.[9] Since the discovery of the dovitinib, the
2‐oxo‐quinoline‐benzimidazole scaffold (Figure 1) has received at-
tention from researchers working on anticancer therapeutics owing
to its apoptosis‐inducing property, making it a good scaffold for an-
ticancer activity. Despite the promising activity, this drug was with-
drawn from the market due to the toxicity observed in the clinical
trial. Thus, many researchers, while working on the optimization of
this scaffold, have reported on molecules leading to ROS generation,
followed by apoptosis induction.[10–12] In a related finding, Han
et al.[13] reported exploration of the effects of substitutions on the
benzimidazole moiety on the 2‐oxo‐quinoline scaffold, which showed
potent VEGFR‐2 inhibition activity, the best molecule being the
FIGURE 1 Triazole‐, benzimidazole‐, and quinoline‐based molecules with anticancer and apoptotic induction properties and molecular
hybridization of the benzimidazole–quinoline framework with 1,2,3‐triazole

GAIKWAD ET AL.
3 of 15
|
isonicotinamide derivative I (Figure 1). In a similar study, Kuang
et al.[10] reported antitumor agents based on the 3‐benzoimidazol‐2‐
yl‐quinolin‐2‐one scaffold, among which compound II was the best,
with antitumor activity exerted by apoptosis induction through ROS
generation. These results provided insight into the scaffold and un-
veiled the potential opportunities for its further development.
Additionally, 1,2,3‐triazole has gained the status of a privileged
scaffold from a medicinal chemistry perspective, owing to its diverse
spectrum of activity such as anticancer,[14,15] antifungal,[16]
antibacterial,[17] anti‐HIV,[18] anticonvulsant,[19] anti‐inflammatory,[20]
and antitubercular activities.[21] The physicochemical parameters of the
triazole ring such as rigidity of the scaffold, high dipole nature, hetero-
cyclic nature, and hydrogen bonding impart usefulness. The most im-
portant aspect is that triazole can be used as a linker to join two
bioactive scaffolds to generate a more active hybrid derivative than the
parent scaffolds.[22] Many of the triazole‐based scaffolds with antic-
ancer activity were found to act mainly by triggering ROS production[23]
and induction of apoptosis by inhibiting topoisomerase II (compound
III)[24,25] (Figure 1). Cavalcanti Chipoline et al.[26] reported triazole‐based
naphthoquinone derivatives (IV) as cytotoxic agents that act by the
induction of microtubule disorganization. Sulfonamide‐based anticancer
agents were reported by Jia et al.[27] acting on the Wnt/β‐catenin/
GSK3β pathway, quinoline‐based triazole derivatives (V) were reported
by Sonego et al.[28] that exhibited cytotoxic activity through cell
cycle arrest in the G0/G1 phase and apoptosis induction.[28]
Antiproliferative agents based on the triazole fused 2‐mercapto
benzimidazole scaffold (VI) were reported by Andrade et al.[15] that
showed significant apoptosis induction potential against the glio-
blastoma cell line (GBM).[15] In a similar finding, STAT3 inhibitors re-
ported by Ríos‐Malváez et al.[29] based on the salicylic acid tethered
triazoles (VII) showed potent antiproliferative activity. Importantly, it is
conceivable that the combination of the triazole scaffold with well‐
established pharmacophores may lead to more potent therapeutic
agents. This hypothesis was also reinforced by reports based on the
acridinone‐,[30] podophyllotoxin‐,[25] and isatin‐based scaffolds[31]
(Figure 1), wherein the triazole‐incorporated moiety was found to elicit
better activity than their corresponding lead molecules.
The reports on drug–receptor interactions of dovitinib empha-
sized the strong hydrogen‐bonding interactions of 2‐oxo‐quinoline
(–C═O–, –NH–) with the receptor protein,[32] yielding a hydrophilic
nature that may be linked to its toxicity. In an attempt to overcome
the limitations of dovitinib, we envisioned that a fused scaffold of the
2‐oxo‐quinoline‐benzimidazole and 1,2,3‐triazole would lead to a
more potent pharmacophore (Figure 1). The rationale behind this
design was to minimize the pharmacophoric features aiding toxicities
and incorporate pharmacophorically competent structural moieties
like benzimidazole owing to the fact that interactions from benzimi-
dazole –NH– were vital for the activity. Finally, to test our hypoth-
esis, synthesis of the designed pharmacophore was carried out and
evaluation of their antiproliferative and apoptosis induction
profiles in the BT474 breast cancer cells was performed. To the best
of our knowledge, 1,2,3‐triazole derivatives of 2‐oxo‐quinoline‐
benzimidazole have not been reported to date.
|
2
RESULTS AND DISCUSSION
Chemistry
|
2.1
The designed scaffold was synthesized as shown in Scheme 1. The
two key intermediates 3‐(1H‐benzo[d]imidazol‐2‐yl)‐1‐(prop‐2‐yn‐1‐
yl)quinolin‐2(1H)‐one and substituted azides were synthesized using
SCHEME 1 Synthetic pathway for the preparation of quinoline–benzimidazole‐linked triazole derivatives

4 of 15
GAIKWAD ET AL.
|
the conventional synthetic methods. The 3‐(1H‐benzo[d]imidazol‐2‐
yl)‐1‐(prop‐2‐yn‐1‐yl)quinolin‐2(1H)‐one intermediate was synthe-
sized starting with the Vilsmeier–Haack formylation reaction on the
commercially available acetanilide, which yields 2‐chloroquinoline‐3‐
carbaldehyde.[33] The obtained aldehyde was hydrolyzed using dilute
HCl and heated to 60°C for about 5 h, resulting in quinoline‐2‐one‐3‐
aldehyde 1. Further, 1 was subjected to propargylation in the pre-
sence of 2 equiv. of the inorganic base in dimethylformamide (DMF)
solvent, which yielded our primary intermediate 2.
aromatic protons were observed at 7.26, 7.43, 7.71, 7.85, and
8.10 ppm. Likewise, interpretation of 13C NMR spectra of compound
Q9 shows the existence of the quinolinone group by the presence of
a singlet at 160.4 ppm that belongs to the amide group. The me-
thylene (–CH2–) carbon of the triazole–quinoline linker and the
benzylic group was observed at 38.7 and 53 ppm, respectively. The
aliphatic carbons of the morpholine ring were observed at 48.6 and
66.5 ppm. The remaining aromatic carbons were observed between
113.2 and 151.3 ppm. All the other designed derivatives were also
characterized by 1H, 13C NMR, and HRMS spectroscopic methods
and were found in accordance with the depicted structures as pre-
sented in Section 4. The HRMS electrospray ionization (ESI) data of
each compound represented as [M+H]+ or [M+Na]+ peaks comply
with their precise molecular formula.
The substituted benzyl azides were synthesized from the com-
mercially available reagents using the reported procedure. Sub-
stituted benzyl alcohols, upon treatment with PBr3 in DCM, lead to
corresponding benzyl bromides (2a–e). Finally, the substituted benzyl
bromides, when treated with the sodium azide in acetonitrile along
with two drops of water on stirring, resulted in benzyl azides 6.
The final derivatives Q1–Q11 were synthesized by a click reaction
using the [3+2] azide–alkyne cycloaddition reaction using copper(I)Io-
dide in DMF, leading to the formation of the 1,4‐cyclization product
exclusively. For compounds Q10–Q11, the required 2‐azido‐N‐
phenylacetamide was synthesized from 2‐bromo‐N‐phenylacetamide
using the same conditions as those used for benzyl azide synthesis. 2‐
Bromo‐N‐phenylacetamide was obtained by the acetylation of aniline
(Scheme 2) using bromoacetyl bromide in the presence of the
base in DCM. All the synthesized derivatives were characterized using
1H, 13C spectroscopy, and high‐resolution mass spectrometry (HRMS)
techniques.
|
2.2
Pharmacology/biology
|
2.2.1
NCI‐60 cell line screening
The synthesized derivatives were screened against the panel of
NCI‐60 cancer cell lines for evaluation of their in vitro anti-
proliferative activity. The primary results were obtained as the mean
growth percentage graph of the respective compound (at a fixed
concentration of 10 μM) against each cell line. In the second stage of
the screening (five‐dose studies), compounds were screened at five
different concentrations of 0.01, 0.1, 1, 10, and 100 μM. Based on
the five‐dose studies, the GI50, TGI, and LC50 were calculated for
the active compound. (Detailed graphs and results are presented in
the Supporting Information.) Thus, the primary results of all the
compounds were found to be promising, and two derivatives Q2 and
Q6 were selected by NCI for the five‐dose studies, which inferred
that compound Q6 has promising anticancer activity on the central
nervous system (CNS), leukemia, melanoma, ovarian, and breast
cancer cell lines (Table 1). Other derivatives Q1–Q11 showed good
to moderate growth inhibition in major cancer cell line groups. All the
designed derivatives were synthesized and a structure–activity re-
lationship was assigned. The details of the structure to activity as-
sessment based on the percentage growth‐inhibitory studies carried
out by NCI are discussed in a later section. These results suggest the
The analytical data were found in compliance with the designed
structures presented in Section 4. As a representative compound, the
characterization of one of the potent derivatives Q9 is discussed
here. The 1H NMR spectra of the Q9 showed a characteristic singlet
peak of the –NH– proton of the benzimidazole moiety at 12.76 ppm,
along with the singlet proton of the quinoline 4th position, and the
methylene linker connecting the triazole moiety to the quinoline were
observed as singlets at 9.21 and 5.45 ppm, respectively, confirming
the formation of the designed framework. Further, the morpholine
methylene protons were observed as a triplet at 3.75 and 3.11 ppm.
The benzylic methylene proton was observed at 5.75 ppm. The tria-
zole benzylic phenyl ring protons were observed as doublets at 7.23
and 6.93 ppm and the triazole –CH– was observed as a singlet at
8.07 ppm. Apart from these characteristic peaks, the remaining
SCHEME 2 Synthetic pathway for the
preparation of quinoline–benzimidazole‐linked
acetamide triazole derivatives Q10 and Q11

GAIKWAD ET AL.
5 of 15
|
TABLE 1 Percentages of growth (G%) were induced by the synthesized compounds at 10 μM concentration against the panel of the NCI‐60
cell line
Growth percent
Cancer
Cell line
Q1
Q2
Q3
Q4
Q5
Q6
22.63
Q7
Q8
Q9
Q10
65.19
Q11
63.58
Leukemia
CCRF‐CEM
HL‐60(TB)
K‐562
95.41
23.4
0.03
96.44 102.13
99.69 100.31
98.17 103.09
88.19 101.84
90.94 105.49
81.26 104.43
113.22
97.29
106.3
98.98
94.07
94.85
102.8
92.02
100.3
109.8
95.45
88.58
94.29
97.31
87.7
7.75
9.91
88.73 103.7
67.45
30.69
32.5
72.02
83.63
38.94
68.15
63.58
17.74
19.88
16.68
30.18
102.94
96.23
82.1
100.0
98.97
80.39
71.7
MOLT‐4
RPMI‐8226
SR
17.89
16.14
21.75
87.64
94.72
79.02
114.38
92.89
12.4
11.5
NSCLC
A549/ATCC
EKVX
88.33
93.08
79.24
92.23
87.61
92.06
88.3
11.4
31.44
21.41
34.93
23.53
19.37
−10.6
19.55
92.51
90.22
90.74
100.3
89.13
95.51
88.74
76.58
94.9
99.59
96.14
111.53
92.8
96.9
101.66
96.42
117.75
91.13
96.68
103.42
85.68
9.33
30.76
9.29
88.3
100.1
92.07
101.3
88.67
97.86
84.92
79.71
55.57
99.12
82.35
83.25
83.93
86.43
56.34
76.1
74.78
89.73
16.29
75.9
15.41
74.82
35.07
11.26
56.89
52.3
46.97
94.76
73.92
66.82
76.4
HOP‐62
HOP‐92
−2.19
4.71
NCI‐H23
NCI‐H322M
NCI‐H460
NCI‐H522
76.46
68.75
55.28
28.04
97.88
99.99
92.02
17.99
1.94
97.77
90.8
6.86
76.87
−1.1
45.09
39.38
Colon cancer
COLO 205
HCC‐2998
HCT‐116
HCT‐15
HT29
118.8
106.3
18.17
110.7
104.7
96.92
80.1
118.98
111.62
99.11
111.7
105.84
101.76
100.64
108.32
102
13.5
−11.7
5.1
105.9
107.6
73.65
104.5
91.22
99.36
95.72
105.2
98.6
83.71
93.24
83.6
53.97
78.29
18.23
19.66
0.45
102.4
100.9
52.84
75.05
97.29
76.22
71.27
−14.47
88.12 −13.51
63.67
66.39
88.94
90.56
44.74
94.02
104.3
95.64
89.86
16.9
2.52
109.98
11.09
4.85
78.63
56.3
99.74 116.21
98.08 106.81
KM12
9.74
4.44
87.26
70.72
45.48
37.49
SW‐620
22.06
87.84
98.76
90.96
92.21
97.01
16.92
CNS cancer
SF‐268
SF‐295
SF‐539
SNB‐19
SNB‐75
U251
83.2
29.38
1.67
96.88
100.43
81.8
2.39
1.99
79.01
106.4
65.09
79.17
58.21
88.25
69.24
92.94
65.62
84.23
66.69
91.96
22.62
95.38
52.6
45.24
32.86
4.13
74.19
78.29
74.46
73.17
59.25
90.51
100.1
98.66 103.15
26.75 −55.89
85.86 21.79
54.86 −19.19
78.96
87.96
85.21
76.05
82.76
56.46
−28.58
22.83
−74.04
12.66
93.6
47.96
54.81
82.63
100.22
5.39 −41.17
90.85
14.18
97.02 102.27
64.29
37.94
Melanoma
LOX IMVI
MALME‐3M
M14
80.65
76.85
12.75
12.8
89.3
88.3
95.95
89.16
97.5
95.11
96.52
108.54
108.47
110.5
107.5
96.32
98.65
88.74
18.92
24.65
−50.3
−79.5
28.04
50.07
5.92
93.39
65.99
89.47
88.45
103.6
94.44
73.7
62.38
62.14
89.05
56.65
93.55
71.91
83.66
87.51
75.4
78.68
64.2
12.75
5.45
61.81
101.1
82.46
92.35
94.06
84.48
83.52
95.38
67.3
89.29 −20.71
88.77 −37.02
102.0
80.1
38.39
15.09
94.15
1.94
MDA‐MB‐435
SK‐MEL‐2
SK‐MEL‐28
SK‐MEL‐5
UACC‐257
UACC‐62
94.12 103.9
102.6 113.08
76.57
86.95
76.23
71.89
78.7
105.6
93.14
72.14
99.08
64.41
35.96
45.14
6.69
99.98 107.11
91.03 103.02
0.03
49.94
17.18
92.87
72.18
95.27
93.64
46.86
23.18
91.07
85.91
56.83
22.06
79.21
Ovarian cancer
IGROV1
65.74
104.8
77.57
107.5
85.7
9.95
3.02
75.65
103.2
86.59
113.8
87.62
110.63
99.27
26.99
−32.4
0.61
80.17
113.4
96.71
105.5
76.94
81.43
109.5
81.9
81.8
40.01
83.51
27.91
19.78
69.92
87.01
92.22
86.2
OVCAR‐3
OVCAR‐4
OVCAR‐5
OVCAR‐8
NCI/ADR‐RES
SK‐OV‐3
43.54
−20.47
28.46
31.66
29.83
82.85 104.01
55.15
107.3
84.9
6.82 −21.98
102.8
91.73
93.62
99.74
93.31
95.42
98.95
98.94
95.21
19.08
23.88
−2.32
34.9
77.34
37.42
68.76
89.07
78.18
50.35
40.6
97.81
83.7
79.85
103.6
100.11
107.28
67.2
95.24
69.29
108.2
64.95
(Continues)

6 of 15
GAIKWAD ET AL.
|
TABLE 1 (Continued)
Growth percent
Q1 Q2
Cancer
Cell line
Q3
Q4
96.43
Q5
Q6
Q7
79.58
Q8
93.07
Q9
Q10
Q11
Renal cancer
786‐0
84.75 −23.24
69.08 −14.49
101.8
105.46
109.46
91.54
89.65
99.7
−50.1
−27.3
35.01
21.69
−28.4
18.72
33.91
18.17
57.07 −20.25
80.62
86
A498
92.53 112.42
119.2
78.03
89.63
33.87
81.63
109.2
77.47
72.24
84.45
92.29
79.4
92.37
35.73
77.26
35.65
49.74
92.67
67.01
12.23
77.39
13.09
12.04
30.96
ACHN
CAKI‐1
RXF 393
SN12C
TK‐10
UO‐31
77.1
14.96
86.2
86.75
105.0
91.67
111.2
78.05
91.39
88.09
55.6
88.07
32.87
69.1
72.84 −25.48
103.74
96.97
90.27
68.82
88.2
82.34
134.5
14.23
21.67
97.26
114.71
68.52
59.74
116.0
92.96
117.22
70.08
66.56 −18.87
74.81 −65.68
63.66
Prostate cancer
Breast cancer
PC‐3
88.83
22.01
97.37 108.14
83.63 104.72
104.93
106.68
17.03
99.36
104.5
53.97
53.97
65.89
36.87
36.63
59.3
DU‐145
87.75 −23.51
−59.5
18.12
75.01
MCF7
76.64
56.22
72.85
90.63
93.72
91.92
14.51
4.33
75.81
74.58
90.48
94.77
96.7
92.74
70.35
82.76
93.37
106.5
93.27
86.32
88.6
17.07
1.9
84.23
51.75
74.89
68.18
72.26
76.14
56.77
26.15
71.73
96.98
79.47
82.22
88.94
86.58
MDA‐MB‐231
HS 578T
BT‐549
71.48 −21.02
−6.42
22.35
42.28
4.78
98.81
83.48
99.59
96.56
−9.86
−12.2
29.72
13.65
74.65
45.85
71.39
64.28
19.25
13.29
66.36
14.06
86.62 109.1
T‐47D
103.9
96.83
83.31
65.81
MDA‐MB‐468
86.05
Note: The compounds Q2 and Q6 were the most potent molecules from the series and were selected for the five‐dose studies; thus the G% values
corresponding to these compounds are presented in boldface.
potential activity of these compounds. Further mechanistic assays
were carried out to understand the mechanism of action.
apoptosis induced by the compound Q6. Thus, to further investigate
the apoptosis induction property of compound Q6, detailed in-
vestigations involving acridine orange–ethidium bromide (AO–EB)
staining, ROS generation, EB staining, and study of changes in the
MMP were carried out. The results of these studies are discussed in
the next sections.
|
2.2.2
Antiproliferative activity
Compound Q6 was selected for further evaluation to determine the
apoptosis induction potential and the cytotoxic activity against the
human breast cancer line (BT‐474). Inhibitory concentration (IC50), a
concentration at which the proliferation of 50% cells was inhibited
after exposure to the test compound, was determined using the MTT
assay. Compound Q6 showed excellent GI50 on the multiple cell line;
the MTT assay on the BT‐474 cell line showed the potential cyto-
toxicity of this derivative on breast cancer cells with an IC50 of
0.59 0.01 μM.
|
2.2.4
Clonogenic assay
To evaluate the inhibitory effect of compound Q6 on the colony‐
forming capability of BT‐474 cells, the clonogenic assay was per-
formed. BT‐474 cells were allowed to grow in a six‐well plate and
treated with compound Q6. The obtained results clearly showed that
there was a dose‐dependent growth‐inhibitory effect of compound
Q6 on BT‐474 cells at 0.25, 0.5, and 1 μM concentrations. Moreover,
compound Q6 completely inhibited the colony‐forming capability of
single cancer cells at 1 μM, as there were no visible colonies in the
wells as shown in Figure 3.
|
2.2.3
Morphological observations using phase‐
contrast microscopy
To study the morphological effects caused by compound Q6, BT‐474
|
breast cancer cells were exposed to compound Q6 in
a
2.2.5
AO–EB staining
concentration‐dependent manner and were observed under a phase‐
contrast microscope. After exposure to the different concentrations,
that is, 0.25, 0.5, and 1 μM of compound Q6, the BT‐474 cells
showed morphological changes such as reduced number of viable
cells, cell wall deformation, and cell shrinkage, whereas these dis-
tinctive morphological topographies were absent in the control cells
(Figure 2). These physical changes caused were attributed to
The AO/EB staining was carried out for the assessment of the
apoptosis‐inducing effect of compound Q6 on the BT‐474 cells.
When treated with the AO, both intact and damaged cell membrane
nuclei get stained and emit green fluorescence. In contrast, the EB
stains only the damaged cell nucleus membrane and emits red
fluorescence. Thus, the viable cells can be detected with an evenly

GAIKWAD ET AL.
7 of 15
|
FIGURE 2 Morphological changes observed in BT‐474 breast cancer cells after treatment with compound Q6 for 24 h. Images were
captured at ×200 magnification. The scale bar in the image indicates 100 μm
FIGURE 3 Clonogenic assay was performed with compound Q6. BT‐474 cells were treated with Q6 at concentrations of 0.25, 0.5, and 1 μM
compared with the control (untreated cells). Data presented as mean SEM (n = 3). $$$ P < 0.001 are significantly different from the normal
versus apoptotic cells
green nucleus and orange‐stained cytoplasm. Due to the presence of
the dense chromatin, early apoptotic cells were found to show in-
tense green nuclei. In contrast, in late apoptotic cells, regions of the
condensed chromatin nucleus showed a bright orange color. Thus,
both control BT‐474 cells and cells treated with compound Q6 at
0.25, 0.5, and 1.0 μM were stained with AO–EB and were evaluated
under a fluorescence microscope. Control BT‐474 cells showed a
normal morphology, whereas the apoptosis‐induced cells showed
different characteristics such as fragmentation of nuclei with
horseshoe‐shaped structures as shown in Figure 4.
fluorescence that can be visualized using the microscope. Then,
the nuclear damage and chromatin condensation due to compound
Q6 at 0.25, 0.5, and 1.0 μM concentrations in BT‐474 cells were
detected using DAPI staining. Staining was performed according to
the known protocol. The results from Figure 5 clearly illustrated
that the nuclear structures of control BT‐474 cells were found to
be intact, whereas the BT‐474 cell treated with compound Q6
showed condensed, fragmented, pyknotic, and horseshoe‐shaped
nuclei.
|
2.2.7
Effect on the MMP (ΔΨm)
|
2.2.6
4′,6‐Diamidino‐2‐phenylindole (DAPI)
staining
Loss of the MMP or damage to its integrity is an early event in the
process of apoptotic induction. Furthermore, the evaluation of ROS‐
induced alteration in MMP in BT‐474 cells was carried out using the
JC‐1 staining method, as any alteration in MMP (ΔѰm) is associated
with one of the major pathways for cellular death. JC‐1 is a fluor-
escent dye that imparts a red color as a monomer, while, in apoptotic
cells, the depolarized mitochondria become aggregated and are vi-
sualized in green color. Thus, dissipation in ΔΨm can be observed by
DAPI is a nuclear staining technique, which is used to assess nuclear
damage or chromatin condensation. DAPI reagent binds strongly with
the minor groove of double‐stranded DNA to adenine–thymine
clusters which lead to the bright color fluorescence. Thus, the
apoptosis‐induced cells have the characteristic condensed nucleus
that binds with the DAPI stain and illuminates as bright

8 of 15
GAIKWAD ET AL.
|
FIGURE 4 Acridine orange/ethidium bromide staining of compound Q6. BT‐474 cells were treated with Q6 at concentrations of 0.25, 0.5,
and 1 μM and compared with the control (untreated cells). Images were captured at ×200 magnification. The blue scale bar in the image indicates
100 μm
FIGURE 5 4′,6‐Diamidino‐2‐phenylindole (DAPI) staining of compound Q6. BT‐474 cells were treated with Q6 at concentrations of 0.25,
0.5, and 1 μM compared with the control (untreated cells). Images were captured at ×200 magnification. The blue scale bar in the image indicates
100 μm
|
the shift in the green fluorescence intensity using a flow cytometer.
Treatment of BT‐474 cells for 24 h showed increased green color as
can be seen in Figure 6, indicating the alteration of MMP (ΔѰm) by
different concentrations of compound Q6. These results collectively
suggest the induction of apoptosis in BT‐474 cells through the
collapse of ΔΨm.
2.2.9
Detection of intracellular ROS by DCFH‐DA
It has been identified that the increase in intracellular ROS levels is
associated with the induction of apoptosis in cancer cells. In-
tracellular ROS levels were assessed using the DCFH‐DA staining
method. BT‐474 cells were treated with compound Q6 at 0.25, 0.5,
and 1 μM for 6 h. The results showed an increase in the intensity of
green fluorescence in a dose‐dependent manner when equated with
the control, which indicates the induction or an increase in in-
tracellular ROS production by compound Q6 (Figure 8). This indicates
that the cytotoxicity profile shown by compound Q6 could be due to
the induction of intracellular ROS generation.
|
2.2.8
Annexin V‐FITC/propidium iodide (PI)
staining assay
To calculate the percentage of cells undergoing apoptosis by
compound Q6, the Annexin V‐FITC/PI staining assay was per-
formed according to known procedures.[34] This assay enables the
identification of live cells (Q1‐LL; AV−/PI−), early apoptotic cells
(Q1‐LR; AV+/PI−), late apoptotic cells (Q1‐UR; AV+/PI+), and
necrotic cells (Q1‐UL; AV−/PI+). BT‐474 cells were treated with
0.5 and 1 μM of compound Q6 for 24 h and then stained with
Annexin V‐FITC/PI. The results from Figure 7 suggested that the
percentage of total apoptotic cells (early and late apoptotic cells)
increased from 2.56% (control) to 5.25% (0.5 μM) and 14.96%
(1 μM), respectively. Therefore, the percentage increase in early
and late apoptotic cells is from 2.56% to 14.96%. Thus, an
approximate sixfold increase in apoptosis indicates that compound
Q6 induced apoptosis in BT‐474 cancer cells in a dose‐dependent
manner.
|
2.2.10
SAR interpretation
Compound Q6 was evaluated completely by screening against the
NCI‐60 cell line panel by single‐dose studies and five‐dose study
protocols, which showed the significant antiproliferative activity of
compound Q6. To identify the mechanism of the given derivative,
various staining assays (AO–EB staining, DCFH‐DA staining, DAPI
staining, Annexin V‐FITC/PI staining assay, and JC‐1 staining for
evaluating the effect of Q6 on the MMP [ΔΨm], etc.) on the BT‐474
cell line were performed, which indicated that compound Q6 induced
apoptosis on the BT‐474 cell line. Although the present studies un-
derscore the significance of developing extensive SAR based on Q6,

GAIKWAD ET AL.
9 of 15
|
FIGURE 6 Effect of compound Q6 on the mitochondrial membrane potential. BT‐474 cells were treated with Q6 at concentrations of 0.25,
0.5, and 1 μM and were compared with the control (untreated cells)
FIGURE 7 Effect of compound Q6 on the apoptotic cell death in BT‐474 cells. Compound Q6‐treated cells were stained with Annexin
V‐FITC/propidium iodide and analyzed for apoptosis using a flow cytometer. A total of 10,000 cells from each sample were analyzed via flow
cytometry
FIGURE 8 Determination of reactive oxygen species (ROS) generation in BT‐474 cells. Fluorescence images were obtained from the cells
treated with compound Q6 for 24 h and then observed the production of ROS by DCFH‐DA. Control cells (BT‐474), Q6 (0.25 μM), Q6 (0.5 μM),
and Q6 (1 μM). The concentration‐dependent increase in the bright green fluorescence as compared with untreated cells indicated that
compound Q6 induced an increase in intracellular ROS in BT‐474 cells in a concentration‐dependent manner. Mechanistically, the elevation in
the ROS level in the BT‐474 cells leading to apoptosis could be the potential reason for the cytotoxic potential of compound Q6
while the inputs based on the NCI data can be utilized to potentially
also a phenyl acetamido‐1,2,3‐triazole derivative lacking the chloro
group, which has shown moderate growth inhibition on breast cancer
and other cell lines. This indicates the importance of the chloro
substitution on the phenyl ring. To substantiate this, other halo
groups bearing benzyl triazole derivatives such as Q2 (para‐fluoro),
Q3 (meta‐chloro), and Q4 (para‐bromo) were evaluated; notably, the
para‐fluorobenzyl triazole showed good growth inhibition on the
colon, melanoma, renal, and breast cancer cell lines, which indicates
gain insights into the SAR of the 3‐(1H‐benzo[d]imidazol‐2‐yl)‐1‐[(1‐
benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]quinolin‐2(1H)‐one
derivative.
The second derivative selected for the five‐dose studies was com-
pound Q10, which is a 2‐chlorophenyl acetamido‐1,2,3‐triazole de-
rivative that also showed excellent GI50 on the breast cancer cell line
in the micromolar range, and similar results were observed on the
melanoma and renal cancer cell lines. Interestingly, compound Q11 is

10 of 15
GAIKWAD ET AL.
|
that the position, as well as the nature of the halo group, are essential
for the activity. The bromo derivative did not show any significant
growth inhibition in the foremost group of cell lines. Also, the meta‐
chloro derivative showed moderate growth inhibition, suggesting the
effects of chloro and fluoro groups on the activity. Next, the methoxy
and methyl derivatives were studied. Q6, which has already been
evaluated as a promising apoptosis inducer in this study, is a tri-
methoxy derivative, and to understand the effect of the methoxy
group, the dimethoxy derivative Q5 (3,4‐dimethoxy) was examined,
which showed complete loss of potency. In contrast, the para‐methyl
derivative Q7 showed better growth inhibition than Q5. To assess
the effect of the nitrogen at the para‐position, compounds Q8 (para‐
NO2) and Q9 (para‐morpholino) were evaluated, which suggested
that the morpholino group had a significant growth‐inhibitory effect
on renal, ovarian, prostate, and breast cancer cell lines.
TABLE 2 In silico physicochemical properties of compound Q6
ADME parametersa
Q6
Standard range
CNS
−1
–2 (inactive) to +2 (active)
concern below –5
<25 poor to >500 great
–3.0 to 1.2
QPlog HERG
QPP Caco
−7.31
1205.69
−0.931
605.56
−1.054
5
QP logBB
QPP MDCK
QP log Kp
<25 poor to >500 great
–8.0 to –1.0
Metab
1 to 8
QP log Khsa
% Human oral absorption
0.63
–1.5 to 1.5
87.17
>80% is high and <25 poor
Abbreviations: ADME, absorption, distribution, metabolism, and
excretion; CNS, central nervous system.
aCalculated using the Qikprop module of the Schrodinger suite. Standard
range is as given in the Qikprop module, which is calculated based on 95%
of known approved drugs.
|
2.3
In silico absorption, distribution, metabolism,
and excretion (ADME) studies
After successful evaluation of the antiproliferative activity of com-
pound Q6, it is important to understand the physicochemical
properties of the designed molecules to consider it as a potential
lead molecule. The ADME parameters of designed quinoline triazole
hybrids were calculated using the Qikprop module of the Schrö-
dinger suite. All the projected drug likeliness properties of these
derivatives were found to be within the standard range. QPPCaco is
a descriptor to predict the Caco‐2 cell permeability of a molecule in
nm/s (>500 indicates good absorption and <25 indicates poor
absorption). Similarly, for the target molecule to become CNS‐
active, it should have a CNS value higher than or equal to 2 on a –2
(inactive) to +2 (active) scale; likewise, Log BB values determine the
distribution of the compound in the brain. If the compound has a
log BB value of more than 0.3, it will cross the blood–brain barrier,
and if it is less than −1.0, those compounds will be weakly dis-
tributed to the brain. Furthermore, the QPlogHERG value denotes
the predicted IC50 value for the blockage of HERG K+ channels.
Designed compounds demonstrated good predicted pharmacologi-
cal properties like the HERG K+ channel (HERG K+), QPlogKhsa,
which predicts binding to human serum albumin, QPPMDCK, which
predicts MDCK cell permeability, and QPlogKp, which predicts the
skin permeability of the molecule. Also, the percent absorption
values of the majority of the compounds were found to be in the
range of 85–100% (Table S1). None of the designed derivatives
violated the rule of three and the rule of five. Values of predicted
descriptors are presented in Table S1. The observed ADME values
of major descriptors of all the designed compounds were found to
be within the standard approved limits,[35–37] which suggests that
these molecules can be used for further biological evaluation to
develop potential antiproliferative agents. The detailed ADME
evaluation of these molecules is presented in the Supporting
Information, and the ADME values of compound Q6 are presented
in Table 2.
|
3
CONCLUSION
In conclusion, a series of substituted triazole derivatives based on the
3‐(1H‐benzo[d]imidazol‐2‐yl)quinolin‐2(1H)‐one scaffold were syn-
thesized and assessed for their in vitro cytotoxicity potential against
the NCI‐60 cell line panel of humanoid cancers. This primary eva-
luation guided us to compound Q6, a potential cytotoxic agent. The
MTT assay estimated the IC50 of the compound Q6 to be 0.59 μM on
the breast cancer cell line, that is, BT‐474. Further, the morphological
studies performed indicated the apoptotic characteristics in the cells
treated with Q6, suggesting apoptosis as a possible primary me-
chanism of action for these compounds, which was confirmed by the
DAPI and AO/EB assays. Further, DCF‐DA suggested an increase in
intracellular ROS levels and JC‐1 staining, suggesting the alteration in
the MMP. This indicates that the plausible mechanism of action is
apoptosis initiated by a surge in the intracellular ROS, leading to a
change in the MMP, which finally induces apoptosis in cancer cells.
Also, the in silico ADME studies suggested that these compounds
may have excellent druggability characteristics. Thus, compound Q6
could be a potential lead in the development of new and more potent
anticancer agents.
|
4
EXPERIMENTAL
Chemistry
|
4.1
|
4.1.1
General
All the chemicals, reagents, and starting materials were acquired from
commercially available providers and were used as received. The
monitoring of reactions was performed by thin‐layer chromatography

GAIKWAD ET AL.
11 of 15
|
(TLC; Merck pre‐coated silica gel 60‐F254 aluminum plates) under
UV light. 1H and 13C NMR spectra were performed on a Bruker
Avance 500 MHz spectrometer using tetramethylsilane (TMS) as the
internal standard, and chemical shifts are reported in ppm. Chemical
shifts are referenced to TMS (δ 0.00 for 1H NMR and 13C NMR),
dimethyl sulfoxide (DMSO)‐d6 (δ 2.50 for 1H NMR and 39.5 for 13C
NMR) or CDCl3 (δ 7.26 for 1H NMR and δ 77.0 for 13C NMR). Spin
multiplicities are reported as s (singlet), brs (broad singlet), d (doublet),
dd (double doublet), t (triplet), and m (multiplet). Coupling constant (J)
values are reported in hertz (Hz). HRMS spectrometry data were
performed with an Agilent QTOF mass spectrometer 6540 series
instrument and were obtained in the ESI technique mode at 70 eV.
Melting points were recorded using the Stuart® SMP30 apparatus
and are uncorrected. Column chromatography was performed
using a silica gel 60–120 or 100–200 mesh.
ArH), 5.73 (s, 2H, –CH2–), 5.50 (s, 2H, –CH2–), 5.03 (s, 2H, –CH2–);
13C NMR (126 MHz, DMSO‐d6) δ 160.4, 159.0, 148.0, 143.3, 139.3,
139.3, 137.9, 137.3, 132.4, 130.7, 130.4, 128.9, 128.3, 128.2, 124.0,
123.4, 122.6, 120.7, 120.5, 119.9, 115.7, 115.1, 114.7, 69.7, 53.2,
38.7; HRMS–QTOF MS/MS: m/z [M+H]+ calcd. for C33H27N6O2:
539.2195; found, 539.2195.
3‐(1H‐Benzo[d]imidazol‐2‐yl)‐1‐{[1‐(4‐fluorobenzyl)‐1H‐1,2,3‐
triazol‐4‐yl]methyl}quinolin‐2(1H)‐one (Q2)
Yield 51%; MP: 281–283°C; 1H NMR (500 MHz, DMSO‐d6) δ 12.72
(s, 1H, –NH–), 9.17 (s, 1H, ArH), 8.10 (s, 1H, ArH), 8.06 (d,
J = 6.4 Hz, 1H, ArH), 7.80 (d, J = 8.8 Hz, 1H, ArH), 7.71 (t, J = 6.6 Hz,
2H, ArH), 7.67 (dd, J = 6.1, 3.2 Hz, 1H, ArH), 7.40–7.34 (m, 3H,
ArH), 7.25–7.19 (m, 3H, ArH), 7.17 (d, J = 8.8 Hz, 1H, ArH), 5.73 (s,
2H, –CH2–), 5.53 (s, 2H, –CH2–); 13C NMR (126 MHz, DMSO‐d6) δ
163.3, 160.4, 148.0, 143.4, 143.3, 139.4, 139.3, 135.0, 132.6,
132.6, 132.5, 130.8, 130.7, 130.7, 123.9, 123.5, 122.8, 122.4,
120.5, 119.9, 118.8, 116.1, 115.9, 115.7, 113.2, 52.5, 38.7;
HRMS–QTOF MS/MS: m/z [M+H]+ calcd. for C26H20N6OF:
451.1683; found, 451.1680.
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Information.
|
4.1.2
General procedure for the synthesis of
3‐[1H‐benzo(d)imidazol‐2‐yl]‐1‐(prop‐2‐yn‐1‐yl)‐
quinolin‐2(1H)‐one (3)
3‐(1H‐Benzo[d]imidazol‐2‐yl)‐1‐{[1‐(3‐chlorobenzyl)‐1H‐1,2,3‐
triazol‐4‐yl]methyl}quinolin‐2(1H)‐one (Q3)
A mixture of 2‐oxo‐1‐(prop‐2‐yn‐1‐yl)‐3‐formyl‐1,2‐dihydroquinoline
(2) (1 mmol) and o‐phenylenediamine (1 mmol) was dissolved in glacial
acetic acid (10 ml). The resulting mixture was stirred at reflux 80°C
for 5 h. The reaction was monitored using a TLC. After completion of
the reaction, the reaction mixture was cooled to room temperature
(rt) and poured on crushed ice and the resulting precipitate was
collected using vacuum filtration. The residue obtained was dried and
purified using column chromatography (hexane/ethyl acetate), ob-
taining compound 3 with 60% yield.
Yield 55%; MP: 224–226°C; 1H NMR (500 MHz, DMSO‐d6) δ 12.51
(s, 1H, –NH–), 9.26 (s, 1H, ArH), 8.17 (dd, J = 14.5, 6.9 Hz, 3H, ArH),
8.10 (dd, J = 8.4 Hz, 2H, ArH), 7.84 (t, J = 7.1 Hz, 1H, ArH), 7.75
(d, J = 8.4 Hz, 1H, ArH), 7.69–7.53 (m, 2H, ArH), 7.47 (dt, J = 11.2,
7.4 Hz, 3H, ArH), 7.45 (d, 1H, ArH), 5.31 (s, 2H, –CH2–), 3.35 (s, 2H,
–CH2–); 13C NMR (126 MHz, DMSO‐d6) δ 161.3, 152.1, 152.1,
139.1, 138.8, 136.7, 133.2, 132.7, 131.4, 130.2, 126.9, 126.8, 125.6,
125.4, 123.8, 123.2, 122.8, 122.7, 122.7, 122.6, 122.5, 120.2, 115.8,
115.7, 79.1, 75.5; HRMS–QTOF MS/MS: m/z [M+H]+ calcd. for
C26H20N6OCl: 467.1387; found, 467.1387.
|
4.1.3
General procedure for the synthesis of
3‐(1H‐Benzo[d]imidazol‐2‐yl)‐1‐{[1‐(4‐bromobenzyl)‐1H‐1,2,3‐
triazol‐4‐yl]methyl}quinoline‐2(1H)‐one (Q4)
triazole derivatives (Q1–Q11)
Yield 55%; MP: 276–278°C; 1H NMR (500 MHz, DMSO‐d6) δ 12.73
(s, 1H, –NH–), 9.17 (s, 1H, ArH), 8.11 (s, 1H, ArH), 8.06
(d, J = 7.8 Hz, 1H, ArH), 7.80 (d, J = 8.6 Hz, 1H, ArH), 7.74–7.67
(m, 3H, ArH), 7.56 (d, J = 8.0 Hz, 2H, ArH), 7.39 (t, J = 7.5 Hz, 1H,
ArH), 7.23 (t, J = 6.5 Hz, 4H, ArH), 5.73 (s, 2H, –CH2–), 5.53 (s, 2H,
–CH2–); 13C NMR (126 MHz, DMSO‐d6) δ 160.7, 148.0, 143.4,
141.8, 139.4, 139.3, 135.8, 135.1, 132.5, 132.1, 130.7, 129.0,
126.9, 124.1, 123.5, 123.2, 121.9, 120.5, 119.9, 115.7, 52.5, 38.6;
HRMS–QTOF MS/MS: m/z [M+H]+ calcd. for C26H20N6OBr:
513.0862; found, 513.0867.
A mixture of compound 3 (1 mmol) and the substituted azide
(1 mmol) was dissolved in DMF, to which a catalytic amount of
CuI was added and stirred at rt for 5–7 h. After completion of the
reaction indicated by the TLC, the reaction mixture was poured on
crushed ice and the resulting precipitate was collected using
vacuum filtration. The residue obtained was dried and purified
using column chromatography (hexane/ethyl acetate), obtaining a
pure triazole derivative.
3‐(1H‐Benzo[d]imidazol‐2‐yl)‐1‐{[1‐(4‐(benzyloxy)benzyl)‐1H‐1,2,3‐
triazol‐4‐yl]methyl}quinolin‐2(1H)‐one (Q1)
3‐(1H‐Benzo[d]imidazol‐2‐yl)‐1‐{[1‐(3,4‐dimethoxybenzyl)‐1H‐
1,2,3‐triazol‐4‐yl]methyl}quinolin‐2(1H)‐one (Q5)
Yield 65%; MP: 284–286°C; 1H NMR (500 MHz, DMSO‐d6) δ 12.71
(s, 1H, –NH–), 9.16 (s, 1H, ArH), 8.06 (d, J = 7.6 Hz, 2H, ArH), 7.81
(d, J = 8.6 Hz, 1H, ArH), 7.73–7.69 (m, 2H, ArH), 7.67 (dd, J = 5.9,
3.2 Hz, 1H, ArH), 7.38 (t, J = 7.4 Hz, 1H, ArH), 7.22 (dd, J = 6.1, 3.2 Hz,
Yield 65%; MP: 228–230°C; 1H NMR (500 MHz, DMSO‐d6) δ 12.73
(s, 1H, –NH–), 9.17 (s, 1H, ArH), 8.11 (s, 1H, ArH), 8.05 (dd, J = 7.9,
1.5 Hz, 1H, ArH), 7.80 (d, J = 8.6 Hz, 1H, ArH), 7.70 (td, J = 6.4, 5.7,
2.8 Hz, 3H, ArH), 7.41–7.21 (m, 9H, ArH), 6.96 (dd, J = 8.1, 2.6 Hz,
1H, ArH), 6.91 (t, J = 2.0 Hz, 1H, ArH), 6.84 (dt, J = 7.6, 1.1 Hz, 1H,

12 of 15
GAIKWAD ET AL.
|
2H, ArH), 6.93–6.89 (m, 2H, ArH), 6.84 (d, J = 8.2 Hz, 1H, ArH), 5.72
(s, 2H, –CH2–), 5.44 (s, 2H, –CH2–), 3.71 (s, 3H, –OCH3), 3.66 (s, 3H,
–OCH3); 13C NMR (126 MHz, DMSO‐d6) δ 160.4, 149.2, 148.0,
143.3, 139.3, 139.3, 132.4, 130.7, 128.6, 123.7, 123.4, 122.8, 122.4,
121.1, 120.5, 119.9, 118.8, 115.7, 113.2, 112.5, 112.4, 56.0, 55.9,
53.2, 38.6; HRMS–QTOF MS/MS: m/z [M+H]+ calcd. for
CH2), 3.15–3.07 (m, 4H, morpholine‐CH2); 13C NMR (126 MHz,
DMSO‐d6) δ 160.4, 151.4, 148.0, 143.2, 139.3, 139.3, 137.7, 135.0,
132.4, 130.7, 129.6, 126.5, 123.9, 123.5, 123.4, 123.3, 122.8, 122.4,
120.5, 119.9, 118.8, 115.7, 115.4, 113.2, 66.5, 53.0, 48.6, 38.7;
HRMS–QTOF MS/MS: m/z [M+H]+ calcd. for C30H28N7O2:
518.2305; found, 517.2304.
C28H25N6O3: 493.1988; found, 493.1994.
2‐(4‐{[3‐(1H‐Benzo[d]imidazol‐2‐yl)‐2‐oxoquinolin‐1(2H)‐yl]
methyl}‐1H‐1,2,3‐triazol‐1‐yl)‐N‐(2‐chlorophenyl)acetamide (Q10)
Yield 52%; MP: 246–248°C; 1H NMR (500 MHz, DMSO‐d6) δ 12.76
(s, 1H, –NH–), 9.99 (s, 1H, ArH), 9.18 (s, 1H, ArH), 8.15–8.04 (m, 2H,
ArH), 7.87 (d, J = 8.6 Hz, 1H, ArH), 7.80–7.63 (m, 4H, ArH), 7.50 (dd,
J = 8.1, 1.5 Hz, 1H, ArH), 7.39 (t, J = 7.5 Hz, 1H, ArH), 7.32 (td, J = 7.7,
1.5 Hz, 1H, ArH), 7.21 (ddd, J = 11.9, 6.3, 2.3 Hz, 3H, ArH), 5.78
(s, 2H, –CH2–), 5.39 (s, 2H, –CH2–); 13C NMR (126 MHz, DMSO‐d6) δ
165.2, 162.1, 148.7, 144.7, 142.8, 139.3 138.1, 134.6, 132.5, 130.7,
130.0, 128.0, 127.2, 126.7, 126.3, 125.7, 123.5, 122.8, 122.6, 120.6,
115.8, 113.2, 52.4, 38.6; HRMS–QTOF MS/MS: m/z [M+H]+ calcd.
for C27H20N7O2Cl: 510.1445; found, 510.1442.
3‐(1H‐Benzo[d]imidazol‐2‐yl)‐1‐((1‐(2,3,4‐trimethoxybenzyl)‐1H‐
1,2,3‐triazol‐4‐yl)methyl)quinolin‐2(1H)‐one (Q6)
Yield 65%; MP: 262–264°C; 1H NMR (500 MHz, DMSO‐d6) δ 12.72
(s, 1H, –NH–), 9.16 (s, 1H, ArH), 8.13 (s, 1H, ArH), 8.05 (dd, J = 7.9,
1.6 Hz, 1H, ArH), 7.81 (d, J = 8.6 Hz, 1H, ArH), 7.73–7.67 (m, 3H,
ArH), 7.38 (t, J = 7.5 Hz, 1H, ArH), 7.25–7.19 (m, 2H, ArH), 6.59
(s, 2H, ArH), 5.75 (s, 2H, –CH2–), 5.45 (s, 2H, –CH2–), 3.66 (s, 6H,
–OCH3), 3.61 (s, 3H, –OCH3); 13C NMR (126 MHz, DMSO‐d6) δ
160.4, 153.4, 148.0, 143.3, 139.4, 139.3, 137.8, 134.4, 132.4, 131.8,
130.7, 127.6, 124.0, 123.4, 122.7, 120.5, 119.9, 115.7, 105.9, 60.4,
56.3, 53.4, 38.6; HRMS–QTOF MS/MS: m/z [M+H]+ calcd. for
C29H27N6O4: 523.2093; found, 523.2099.
2‐(4‐{[3‐(1H‐Benzo[d]imidazol‐2‐yl)‐2‐oxoquinolin‐1(2H)‐yl]ethyl}‐
1H‐1,2,3‐triazol‐1‐yl)‐N‐phenylacetamide (Q11)
3‐(1H‐Benzo[d]imidazol‐2‐yl)‐1‐{[1‐(4‐methylbenzyl)‐1H‐1,2,3‐
triazol‐4‐yl]methyl}quinolin‐2(1H)‐one (Q7)
Yield 62%; MP: 234–236°C; 1H NMR (500 MHz, DMSO‐d6) δ 10.46
(s, 1H, –NH–), 9.19 (s, 1H, ArH), 8.12 (s, 1H, ArH), 8.06 (d, J = 6.4 Hz,
1H, ArH), 7.89 (d, J = 8.6 Hz, 1H, ArH), 7.78–7.70 (m, 3H, ArH), 7.55
(d, J = 8.0 Hz, 2H, ArH), 7.41 (t, J = 7.5 Hz, 1H, ArH), 7.31 (t, J = 7.8 Hz,
2H, ArH), 7.25 (dd, J = 6.1, 3.2 Hz, 2H, ArH), 7.07 (t, J = 7.4 Hz, 1H,
ArH), 5.78 (s, 2H, –CH2–), 5.29 (s, 2H, –CH2–); 13C NMR (126 MHz,
DMSO‐d6) δ 164.6, 160.2, 147.7, 142.7, 139.7, 139.4, 138.8, 132.7,
130.8, 129.3, 125.7, 124.2, 123.5, 123.0, 120.4, 119.6, 119.4, 115.8,
52.7, 38.6; HRMS–QTOF MS/MS: m/z [M+H]+ calcd. for
C27H20N7O2Cl: 475.1757; found, 475.1747.
Yield 54%; MP: 272–274°C; 1H NMR (500 MHz, DMSO‐d6) δ 12.71
(s, 1H, –NH–), 9.16 (s, 1H, ArH), 8.05 (s, 2H, ArH), 7.80 (d, J = 8.7 Hz, 1H,
ArH), 7.75–7.65 (m, 3H, ArH), 7.38 (t, J = 7.6 Hz, 1H, ArH), 7.28–7.11
(m, 5H, ArH), 5.72 (s, 2H, –CH2–), 5.48 (s, 2H, –CH2–), 2.26 (s, 3H, –CH3);
13C NMR (126 MHz, DMSO‐d6) δ 160.4, 148.0, 143.3, 139.3, 139.3,
137.9, 134.9, 133.3, 132.4, 130.7, 129.7, 128.4, 123.8, 123.4, 122.5,
120.5, 119.9, 118.8, 115.7, 53.1, 38.7, 21.1; HRMS–QTOF MS/MS: m/z
[M+H]+ calcd. for C27H23N6O: 447.1933; found, 447.1931.
3‐(1H‐Benzo[d]imidazol‐2‐yl)‐1‐{[1‐(4‐nitrobenzyl)‐1H‐1,2,3‐
triazol‐4‐yl]methyl}quinolin‐2(1H)‐one (Q8)
Yield 69%; MP: 256–258°C; 1H NMR (500 MHz, DMSO‐d6) δ 9.21
(s, 1H, ArH), 8.21 (d, J = 8.3 Hz, 3H, ArH), 8.06 (d, J = 7.6 Hz, 1H),
7.94–7.69 (m, 4H, ArH), 7.50 (d, J = 8.3 Hz, 2H, ArH), 7.40
(t, J = 7.4 Hz, 1H, ArH), 7.25 (s, 2H, ArH), 5.77 (s, 2H, –CH2–), 5.73
(s, 2H, –CH2–); 13C NMR (126 MHz, DMSO‐d6) δ 164.0, 147.7, 146.1,
143.7, 143.5, 140.3, 139.6, 132.8, 130.7, 129.5, 128.9, 125.6, 124.5,
124.4, 124.4, 123.6, 123.0, 120.5, 116.2, 113.3, 52.4, 39.0;
HRMS–QTOF MS/MS: m/z [M+H]+ calcd. for C26H20N7O3:
478.1628; found, 478.1625.
4.2
Pharmacological/biological assays
|
|
4.2.1
Cell culture
The human breast adenocarcinoma (BT‐474), triple‐negative breast
cancer (MDA‐MB‐231), lung adenocarcinoma (A549), and mouse
melanoma (B16F10) cells were obtained from the National Centre for
Cell Science (NCCS). Dulbecco's modified Eagle's medium (DMEM),
Rosewell Park Memorial Institute medium 1640 (RPMI‐1640), and
3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT)
were purchased from Sigma‐Aldrich. Fetal bovine serum (FBS),
penicillin–streptomycin mixture, and trypsin–EDTA were obtained
from Gibco. Cells were grown in respective media supplemented with
10% FBS and 1% penicillin–streptomycin mixture and allowed to grow
in an incubator maintained at 37°C with 90% humidity and 5% CO2.
The cells were treated with compounds dissolved in DMSO, while
untreated controls were exposed to corresponding volumes of DMSO.
3‐(1H‐Benzo[d]imidazol‐2‐yl)‐1‐{[1‐(4‐morpholinobenzyl)‐1H‐1,2,3‐
triazol‐4‐yl]methyl}quinolin‐2(1H)‐one (Q9)
Yield 57%; MP: 240–242°C; 1H NMR (500 MHz, DMSO‐d6) δ 12.76
(s, 1H, –NH–), 9.21 (s, 1H, ArH), 8.11–8.05 (m, 2H, ArH), 7.85
(d, J = 8.5 Hz, 1H), 7.78–7.71 (m, 3H, ArH), 7.43 (t, J = 7.5 Hz, 1H,
ArH), 7.28–7.22 (m, 4H, ArH), 6.93 (d, J = 8.9 Hz, 2H, ArH), 5.75
(s, 2H, –CH2–), 5.45 (s, 2H, –CH2–), 3.79–3.71 (m, 4H, morpholine‐

GAIKWAD ET AL.
13 of 15
|
|
4.2.2
MTT assay
converted into a highly fluorescent derivative when oxidized by in-
tracellular ROS. BT‐474 cells were plated in a 12‐well plate and al-
lowed to grow for 24 h. Cells were treated with an increasing
concentration of compound Q6 (0.25, 0.5, and 1 μM) for 4 h. After
4 h of incubation, the treatment medium was discarded and cells
were washed with PBS, followed by incubation of cells with DCFH‐
DA (10 μM) at 37°C for 20 min. The oxidation of DCFH‐DA to
fluorescent DCF mediated by intracellular ROS was observed under a
fluorescent microscope (Nikon) with excitation (498 nm) and emis-
sion (530 nm) wavelength.
The cytotoxicity of synthesized compounds was evaluated using the
MTT assay, which involves the conversion of yellow color MTT into
purple formazan crystals by live cells.[38] Briefly, 3000–4000 cells
were seeded in 100 μl of DMEM supplemented with 10% BS and 1%
antibiotic–antimycotic mixture in each well of a 96‐well microtiter
plate and incubated at 37°C and a 5% CO2 environment in an in-
cubator. The seeded cells were exposed to gradient concentrations
(0.625–20 μM) of compounds along with the proper vehicle control
for 48 h. After 48 h of incubation, the media were discarded and cells
were washed twice with PBS. Hundred microliters of MTT (0.5 mg/
ml) was added to each well and incubated for the next 4 h at 37°C.
Subsequently, the supernatant was discarded and formazan crystals
were dissolved in 200 μl of DMSO. The absorbance was determined
at 570 nm using a spectrophotometer (Molecular Devices). The clo-
nogenic assay (colony formation assay) is an in vitro cell survival assay
technique that is based on the ability of a single cell to grow and form
colonies. The assay represents a valuable technique to assess the
retained capacity of cells after treatment with cytotoxic agents, to
eventually produce more progeny. The clonogenic assay was per-
formed to assess the effect of compounds on the colony‐forming
capability of BT‐474 cells as described by Franken et al., with slight
modifications. Briefly, the cells were plated at a density of 200 cells/
well in a six‐well plate, followed by their exposure to different con-
centrations of compound Q6 for 24 h. Subsequently, cells were al-
lowed to grow for 14 days after the addition of fresh media. The
growth medium was changed with a fresh medium every third day to
supplement the cells with the required nutrients and growth factors.
After 14 days, grown colonies were fixed and stained with crystal
violet (1% in methanol) and the number of stained colonies was
counted.
|
4.2.5
AO/EB staining
The AO/EB staining method was used to visualize changes in cel-
lular morphology such as chromatin condensation and apoptotic
body formation, which are characteristic features of cells un-
dergoing apoptosis.[40] BT‐474 cells were grown in a 24‐well
plate and treated with a gradient concentration of compound Q6
(0.25, 0.5, and 1 μM) for 24 h at 37°C in an atmosphere of 5%
CO2. After 24 h of treatment, cells were washed with PBS after
discarding treatment medium and were stained with AO/EB
(10 μg/ml each), followed by observation under a fluorescent
microscope (Nikon) with excitation (488 nm) and emission
(550 nm) at ×200 magnification.
|
4.2.6
Measurement of the MMP (ΔѰm)
Loss of the MMP (ΔѰm) is implicated as one of the major pathways
for cellular death as maintenance of the MMP (ΔѰm) is crucial to
sustaining mitochondrial integrity and its function.[41] To in-
vestigate the loss of the MMP (ΔѰm), BT‐474 cells (1 × 105 cells/
well) were plated in a six‐well plate and allowed to grow for 24 h.
The cells were then incubated with different concentrations (0.25,
0.5, and 1 μM) of compound Q6 for 24 h. Subsequently, cells were
harvested, washed with PBS, and resuspended in PBS containing
JC‐1 dye (5 μg/ml) and incubated in the dark for 30 min. Cells were
washed with PBS twice and observed under a fluorescent micro-
scope (Nikon).
|
4.2.3
DAPI staining
Breast cancer cells (1 × 105 cells/well) were grown in 12‐well plates
and treated with compound Q6 (0.25, 0.5, and 1 μM) for 48 h. Then,
cells were washed, fixed (with 4% paraformaldehyde), and permea-
bilized (with 0.1% Triton X). Cells were stained with DAPI (10 μg/ml),
and apoptotic changes such as nuclear differentiation and con-
densation were observed using a fluorescence microscope with ex-
citation/emission at 359/461 nm wavelength by a DAPI filter at ×200
magnification.
|
4.2.7
Annexin V‐FITC/PI staining
To determine the extent of apoptosis and necrosis, cells were stained
with annexin V conjugated with FITC and PI using an Apoptosis
Detection Kit (Invitrogen) according to the manufacturer's protocol.
Annexin V has a high affinity for the phosphatidylserine moiety ex-
posed on the outer membrane of apoptotic cells, while PI, which is
membrane impermeable, binds to cells with a ruptured membrane
(late apoptotic, necrotic cells).[42] BT‐474 cells (1 × 105 cells/well)
were seeded in a six‐well plate and were exposed to different con-
centrations (0.25, 0.5, and 1 μM) of compound Q6. After 24 h of
|
4.2.4
Detection of intracellular ROS by DCF‐DA
Increased intracellular levels of ROS have been implicated as playing
major role in the induction of apoptosis in cancer cells.[39]
a
To measure the levels of intracellular ROS induced by the
test compound, an oxidant‐sensitive fluorescent probe, 2ʹ,7ʹ‐
dichlorofluorescein diacetate (DCFH‐DA), was used, which is

14 of 15
GAIKWAD ET AL.
|
incubation, cells were harvested using trypsin–EDTA and washed
twice with PBS, and resuspended in 100 μl of 1× binding buffer. Next,
5 μl of FITC‐conjugated annexin V and 1 μl of PI were added to it and
incubated for 15 min in the dark. After the addition of 400 μl of 1×
binding buffer, cells were kept on ice and immediately analyzed using
a flow cytometer (BD FACSVerse™) for detection of the population
of cells undergoing apoptosis. The cell population was divided into
live (annexin V‐negative, PI‐negative), early apoptotic (annexin
V‐positive, PI‐negative), late apoptotic (annexin V‐positive,
PI‐positive), and necrotic (annexin‐V‐negative, PI‐positive) cells
after counting a minimum of 10,000 events.
ORCID
Nikhil B. Gaikwad
Nanduri Srinivas
http://orcid.org/0000-0002-1103-3786
http://orcid.org/0000-0002-6671-2022
Venkata M. Yaddanapudi
http://orcid.org/0000-0002-9813-8694
REFERENCES
[1] WHO, Vol. 2020, 2020. https://www.who.int/news-room/fact-
sheets/detail/cancer. Accessed December 8, 2020.
[2] S. Fulda, A. M. Gorman, O. Hori, A. Samali, Int. J. Cell Biol. 2010,
2010, 214074.
[3] M. O. Hengartner, Nature 2000, 407(6805), 770.
[4] (a) L. Jin, L. Dai, M. Ji, H. Wang, Bioorg. Chem. 2019, 85, 179.; (b)
J. Lopez, S. W. G. Tait, Br. J. Cancer 2015, 112(6), 957.
[5] (a) Y. Wang, C. Wu, Q. Zhang, Y. Shan, W. Gu, S. Wang, Bioorg.
Chem. 2019, 84, 468.; (b) M. Redza‐Dutordoir, D. A. Averill‐Bates,
Biochim. Biophys. Acta, Mol. Cell Res. 2016, 1863(12), 2977.
[6] (a) W. Liang, J. Cui, K. Zhang, H. Xi, A. Cai, J. Li, Y. Gao, C. Hu, Y. Liu,
Y. Lu, N. Wang, X. Wu, B. Wei, L. Chen, Oncotarget 2017, 8(65),
109094.; (b) T. Ma, Q.‐S. Ma, B. Yu, H.‐M. Liu, Eur. J. Med. Chem.
2019, 174, 76.
|
4.2.8
Clonogenic assay
The clonogenic assay (colony formation assay) is an in vitro cell
survival assay technique that is based on the ability of a single cell to
grow and form colonies. The assay represents a valuable technique to
assess the retained capacity of cells after treatment with cytotoxic
agents, to eventually produce a large quantity of progeny. The clo-
nogenic assay was performed to assess the effect of compounds on
the colony‐forming capability of BT‐474 cells as described by
Franken et al., with slight modifications.[43] Briefly, the cells were
plated at a density of 200 cells/well in a six‐well plate, followed by
their exposure to different concentrations of compound Q6 for 24 h.
Subsequently, cells were allowed to grow for 14 days after the ad-
dition of fresh media. The growth medium was changed with a fresh
medium every third day to supplement the cells with the required
nutrients and growth factors. After 14 days, grown colonies were
fixed and stained with crystal violet.
[7] J. Qian, Z. Xu, C. Meng, J. Liu, P.‐L. Hsu, Y. Li, W. Zhu, Y. Yang,
S. L. Morris‐Natschke, K.‐H. Lee, Y. Zhang, Y. Ling, Eur. J. Med.
Chem. 2020, 204, 112610.
[8] American Cancer Society, Vol. 2020. https://www.cancer.org/
cancer/cancer-basics/history-of-cancer/cancer-treatment-targeted-
therapy.html. Accessed December 9, 2020.
[9] Y.‐H. Chiu, Y.‐Y. Lee, K.‐C. Huang, C.‐C. Liu, C.‐S. Lin, J. Oncol.
2019, 2019.
[10] W.‐B. Kuang, R.‐Z. Huang, J.‐L. Qin, X. Lu, Q.‐P. Qin, B.‐Q. Zou,
Z.‐F. Chen, H. Liang, Y. Zhang, Eur. J. Med. Chem. 2018, 157, 139.
[11] Y.‐C. Yu, W.‐B. Kuang, R.‐Z. Huang, Y.‐L. Fang, Y. Zhang, Z.‐F.
Chen, X.‐L. Ma, MedChemComm 2017, 8(6), 1158.
[12] P. Netto Batalha, M. C. Bastos Vieira de Souza, E. Peña‐Cabrera,
D. Cruz Cruz, F. D. Costa Santos Boechat, Curr. Pharm. Des. 2016,
22(39), 6009.
[13] S.‐Y. Han, J. W. Choi, J. Yang, C. H. Chae, J. Lee, H. Jung, K. Lee,
J. D. Ha, H. R. Kim, S. Y. Cho, Bioorg. Med. Chem. Lett. 2012, 22(8),
2837.
|
4.3
In silico ADME studies
[14] A. Bistrović, L. Krstulović, A. Harej, P. Grbčić, M. Sedić, S. Koštrun,
S. K. Pavelić, M. Bajić, S. Raić‐Malić, Eur. J. Med. Chem. 2018, 143,
1616.
All the designed molecules to be evaluated for the ADME properties
were drawn using ChemDraw software. Drawn structures were
minimized using the LigPrep module of Schrodinger Software, and
ADMET properties of the synthesized compounds were calculated
using the Qikprop program (QikProp, version 6.5; Schrödinger, LLC).
The compounds were also evaluated for compliance with Lipinski's
rule of five.
[15] P. Andrade, A. F. Dias, F. Figueiró, F. C. Torres, D. F. Kawano,
A. M. O. Battastini, I. Carvalho, C. H. T. P. da Silva, J. M. Campos,
Future Med. Chem. 2020, 12(8), 689.
[16] Z. Jiang, J. Gu, C. Wang, S. Wang, N. Liu, Y. Jiang, G. Dong, Y. Wang,
Y. Liu, J. Yao, Z. Miao, W. Zhang, C. Sheng, Eur. J. Med. Chem. 2014,
82, 490.
[17] (a) B. Zhang, Eur. J. Med. Chem. 2019, 168, 357.; (b) R. Kant,
V. Singh, G. Nath, S. K. Awasthi, A. Agarwal, Eur. J. Med. Chem.
2016, 124, 218.
ACKNOWLEDGMENTS
[18] T. G. G. Daniel, M. L. S. Thiago, M. M. A. Viviane, F. F. Vitor,
C. S. de Fernando, Med. Chem. 2018, 14(3), 242.
The authors thank and acknowledge NIPER Hyderabad and the De-
partment of Pharmaceuticals, Ministry of Chemicals & Fertilizers,
Government of India, for providing funds, scientific and instrumental
support, and are also very grateful to the National Cancer Institute
(NCI) Bethesda, MD, USA, for carrying out in vitro cytotoxicity stu-
dies on NCI‐60 cell lines. The manuscript has a NIPER‐H acknowl-
edgmet number‐NIPER‐H/2021/186.
[19] (a) P. K. Kadaba, J. Med. Chem. 1988, 31(1), 196.; (b) S. Ulloora,
R. Shabaraya, A. V. Adhikari, Bioorg. Med. Chem. Lett. 2013, 23(11),
3368.
[20] S. Shafi, M. Mahboob Alam, N. Mulakayala, C. Mulakayala,
G. Vanaja, A. M. Kalle, R. Pallu, M. S. Alam, Eur. J. Med. Chem. 2012,
49, 324.
[21] (a) A. Aziz Ali, D. Gogoi, A. K. Chaliha, A. K. Buragohain, P. Trivedi,
P. J. Saikia, P. S. Gehlot, A. Kumar, V. Chaturvedi, D. Sarma, Bioorg.
Med. Chem. Lett. 2017, 27(16), 3698.; (b) M. H. Shaikh,
D. D. Subhedar, L. Nawale, D. Sarkar, F. A. K. Kalam Khan,
J. N. Sangshetti, B. B. Shingate, MedChemComm 2015, 6(6), 1104.
CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.

GAIKWAD ET AL.
15 of 15
|
[22] V. Abbot, P. Sharma, S. Dhiman, M. N. Noolvi, H. M. Patel,
V. Bhardwaj, RSC Adv. 2017, 7(45), 28313.
[35] P. W. L. Jorgensen, QikProp 3.5 User Manual, Vol. Section 3.6,
2012, p. 20.
[23] H. F. Ashour, L. A. Abou‐zeid, M. A. A. El‐Sayed, K. B. Selim, Eur.
J. Med. Chem. 2020, 189, 112062.
[36] A. Dagan‐Wiener, I. Nissim, N. Ben Abu, G. Borgonovo, A. Bassoli,
M. Y. Niv, Sci. Rep. 2017, 7(1), 12074.
[24] T. Srinivasa Reddy, H. Kulhari, V. Ganga Reddy, A. V. Subba Rao,
V. Bansal, A. Kamal, R. Shukla, Org. Biomol. Chem. 2015, 13(40), 10136.
[25] V. G. Reddy, S. R. Bonam, T. S. Reddy, R. Akunuri, V. G. M. Naidu,
V. L. Nayak, S. K. Bhargava, H. M. S. Kumar, P. Srihari, A. Kamal, Eur.
J. Med. Chem. 2018, 144, 595.
[37] D.‐F. Wang, O. Wiest, P. Helquist, H.‐Y. Lan‐Hargest, N. L. Wiech,
Bioorg. Med. Chem. Lett. 2004, 14(3), 707.
[38] G. S. Mani, S. P. Shaik, Y. Tangella, S. Bale, C. Godugu, A. Kamal,
Org. Biomol. Chem. 2017, 15(32), 6780.
[39] D. Trachootham, J. Alexandre, P. Huang, Nat. Rev. Drug Discov.
2009, 8(7), 579.
[26] I. Cavalcanti Chipoline, A. C. Carvalho da Fonseca,
G. Ribeiro Machado da Costa, M. Pereira de Souza, V. Won‐Held
Rabelo, L. N. de Queiroz, T. L. Ferraz de Souza,
E. Cardozo Paes de Almeida, P. Alvarez Abreu, B. Pontes,
V. F. Ferreira, F. de Carvalho da Silva, B. K. Robbs, Bioorg. Chem.
2020, 101, 103984.
[27] Y. Jia, M. Li, Y. Cao, W. Feng, X. Li, W. Xue, H. Shi, Drug Des., Dev.
Ther. 2020, 14, 207.
[28] M. S. Sonego, N. V. Segatto, L. Damé, M. Fronza, C. B.
Gomes, T. L. Oliveira, F. K. Seixas, L. Savegnago,
K. M. Schachtschneider, D. Alves, T. Collares, Invest. New Drugs
2020, 38(4), 1020.
[40] S. Kasibhatla, G. P. Amarante‐Mendes, D. Finucane, T. Brunner,
E. Bossy‐Wetzel, D. R. Green, Cold Spring Harbor Protocols 2006,
2006(3), pdb.prot4493.
[41] R. A. Gottlieb, FEBS Lett. 2000, 482(1–2), 6.
[42] P. Sharma, D. Thummuri, T. S. Reddy, K. R. Senwar, V. Naidu,
G. Srinivasulu, S. K. Bharghava, N. Shankaraiah, Eur. J. Med. Chem.
2016, 122, 584.
[43] N. A. P. Franken, H. M. Rodermond, J. Stap, J. Haveman,
C. van Bree, Nat. Protoc. 2006, 1(5), 2315.
[29] Z. G. Ríos‐Malváez, M.‐A. Cano‐Herrera, J. C. Dávila‐Becerril,
G. Mondragón‐Solórzano, M. T. Ramírez‐Apan, D. Morales‐Morales,
J. Barroso‐Flores, J. G. Santillán‐Benítez, M. V. B. Unnamatla,
M. A. García‐Eleno, N. González‐Rivas, E. Cuevas‐Yañez, J. Mol.
Struct. 2021, 1225, 129149.
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the
supporting information tab for this article.
[30] M. Mohammadi‐Khanaposhtani, M. Safavi, R. Sabourian,
M. Mahdavi, M. Pordeli, M. Saeedi, S. K. Ardestani, A. Foroumadi,
A. Shafiee, T. Akbarzadeh, Mol. Divers. 2015, 19(4), 787.
[31] B. Yu, S.‐Q. Wang, P.‐P. Qi, D.‐X. Yang, K. Tang, H.‐M. Liu, Eur.
J. Med. Chem. 2016, 124, 350.
How to cite this article: N. B. Gaikwad, S. Bansode, S. Biradar,
M. Ban, N. Srinivas, C. Godugu, V. M. Yaddanapudi. New
3‐(1H‐benzo[d]imidazol‐2‐yl)quinolin‐2(1H)‐one‐based
triazole derivatives: Design, synthesis, and biological
evaluation as antiproliferative and apoptosis‐inducing agents.
Arch. Pharm. 2021;e2100074.
[32] (a) W. Cheng, M. Wang, X. Tian, X. Zhang, Eur. J. Med. Chem. 2017,
126, 476.; (b) G. Liang, Z. Liu, J. Wu, Y. Cai, X. Li, Trends Pharmacol.
Sci. 2012, 33(10), 531.
[33] S. Pal, S. Durgadas, S. B. Nallapati, K. Mukkanti, R. Kapavarapu,
C. L. T. Meda, K. V. L. Parsa, M. Pal, Bioorg. Med. Chem. Lett. 2011,
21(21), 6573.
https://doi.org/10.1002/ardp.202100074
[34] L. J. Browne, C. Gude, H. Rodriguez, R. E. Steele, A. Bhatnager,
J. Med. Chem. 1991, 34(2), 725.