Journal of Molecular Structure 1292 (2023) 136184

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Journal of Molecular Structure

journal homepage: www.elsevier.com/locate/molstr

Exploration of quinoxaline-benzimidazole hybrids as apoptosis-inducing

agents and tubulin polymerisation inhibitors
Ojaswitha Ommi a, Shrilekha Chilvery b, Priyanka Sudhir Dhopat a, Anamika Sharma b,
Harshada Anil Bhalerao c, Srinivas Reddy Dannaram c, Srinivas Nanduri a, Rajesh Sonti c, *,
Chandraiah Godugu b, *, Venkata Madhavi Yaddanapudi a, *
a
Department of Chemical Sciences, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad, Telangana 500037, India
b
Department of Biological Sciences, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad, Telangana 500037, India
c
Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad, Telangana 500037, India

A R T I C L E I N F O A B S T R A C T

Keywords: We herein report the design and synthesis of a new class of quinoxaline-benzimidazole hybrids based on the
Quinoxaline molecular hybridization concept and evaluation of their in vitro cytotoxicity profile against human cancer cell
Benzimidazole lines, i.e., colorectal, lung, skin, and mouse melanoma cell line. Among all the screened compounds, compound
Tubulin polymerisation
5l (2-(1-(3, 4-dichlorobenzyl)-1H-benzo[d]imidazol-2-yl)quinoxaline) exhibited potent cytotoxicity against lung
Cytotoxicity
Apoptosis
cancer cell line (A549) with an IC50 of 4.37 ± 0.09 µM with a cytospecificity towards cancer cells. Further, AO/
Molecular docking EB, DAPI, DCFDA, and JC-1 staining techniques confirmed that 5l induced apoptosis in the A549 cell line. In
addition, 5l in annexin V-FITC/PI assay induced early apoptosis. The clonogenic assay revealed that 5l inhibited
colony formation in a dose-dependent manner. Cell cycle distribution analysis unveiled that 5l caused the arrest
of G2/M phase of cell cycle. Moreover, 5l inhibited tubulin polymerization with an IC50 value of < 2.19 µM.
Docking studies revealed that 5l has a prominent binding affinity towards colchicine binding site of α/β- tubulin
with good protein-ligand interactions with a binding energy of -45.139 kcal/mol. In silico ADME/T prediction
studies disclosed the favourable drug-like properties of 5l.

1. Introduction inhibitors with diverse heterocyclic backbones, mainly those targeting

Cancer prevails as the primary cause of death globally, amounting to
nearly 10 million deaths in 2020 and an estimated 1958,310 new cancer
cases and 609,820 cancer deaths to occur in 2023 [1–3]. Furthermore, in
2040, an increase in cancer cases of ~29.5 million per year and cancer
related deaths to 16.4 million is predicted [3]. Despite chemotherapy
being the most offered treatment option, it suffers complications such as
off-target side effects and drug resistance [4–6]. Therefore, developing
newer antineoplastic agents with improved potency, high target speci­
ficity, and least resistance is required [7,8]. Microtubules are regarded
as one of the critical targets among the several cancer-inhibiting targets
since they are crucial for cell division and functionality. Microtubules
are polymers comprised of heterodimers of α- and β- tubulins in a
head-to-tail fashion [9]. They maintain dynamic equilibrium through
continuous polymerization and depolymerization, and any disruption in
this process leads to cell apoptosis [10]. Tubulin polymerization
colchicine binding site, have attracted much attention because of their
least susceptibility to p-gp mediated drug resistance [11–16].
Quinoxalines are proven heterocyclic rings that demonstrated anti­
cancer potential through various mechanisms (kinase, tubulin poly­
merisation, and topoisomerase inhibitors) [17–21] Some of the
quinoxaline based tubulin polymerisation inhibitors (A-C) are given in
Fig. 1 [18,22–25]. Also, discrete pharmacological activities of qui­
noxalines are well documented [26–29]. Furthermore, quinoxaline
moiety is part of several FDA-approved drugs and a few ongoing clinical
trials with diverse therapeutic activities [30]. On the other hand,
benzimidazole, a bioisostere of purine, has attained “privileged scaffold”
status owing to its presence in many breakthrough drugs with unique
pharmacological profile [31–36]. There have been tremendous reports
manifesting the anticancer profile of benzimidazole hybrids [36–40].
Nocodazole D [41,42], Denibulin E [43,44], and BAL27862 F [45]
(Fig. 1) are among the several benzimidazole-based anticancer drugs

* Corresponding authors.
E-mail addresses: rajesh.sonti@niperhyd.ac.in (R. Sonti), chandra.niperhyd@gov.in (C. Godugu), yvmadhavi.niperhyd@gov.in (V.M. Yaddanapudi).

https://doi.org/10.1016/j.molstruc.2023.136184
Received 15 May 2023; Received in revised form 5 July 2023; Accepted 9 July 2023
Available online 10 July 2023
0022-2860/© 2023 Elsevier B.V. All rights reserved.
O. Ommi et al.
that act via tubulin polymerization inhibition [39]. Molecules bearing
N-benzylated benzimidazoles have been explored extensively for their
anticancer properties. Telmisartan G, a well-established antihyperten­
sive drug containing an N-benzylated benzimidazole core, is reported to
exhibit anticancer activity [46]. Tsung-Chieh Shih et al., demonstrated
the synergistic activity of LLS30 H(N-benzylated benzimidazole deriv­
ative) in combination with docetaxel and its ability to inhibit the pro­
gression of prostate cancer cells in vivo [47,48]. On the other hand,
Triazole, a bioisostere of amide and many others, offers metabolic sta­
bility and H-bond forming capacity to the molecule and is known to
exhibit anticancer activity on different cancer cell lines when linked to
benzimidazoles [49–52]. Goud et al., reported the anticancer potential
of Benzimidazole-triazole hybrids, and the potent compound I has
shown significant growth inhibition against lung cancer (A549) cells
with an IC50 value of 0.63 ± 0.21 µM [53]. β-amino alcohols are one of
the important structural components in many potential therapeutics (e.
g., Ethambutol, Phenylephrine, and Propranolol, to name a few). They
are known to exhibit various biological activites such as antimicrobial
[54,55], antimalarial [56,57], antihypertensive [58,59], anticancer
[60–64] and as α- and β-adrenergic agonists [65]. Baker, J.R., and group
reported anticancer properties of aminoalchol acrylonitriles, among
which compound J showed selectivity towards lung cancer cell line
(Fig. 1) [64].
Thus, considering the potential antiproliferative effects of quinoxa­
line and benzimidazole moieties as tubulin polymerization inhibitors,
we designed quinoxaline-benzimidazole hybrids based on the molecular
hybridization concept (Fig. 2) by incorporating 3 different fragments (N-
Fig. 1. Representative examples of quinoxaline and benzimidazole containing tubulin polymerization inhibitors (A–F) and reported anticancer agents containing N-
benzyl (G, H), triazole (I), and amino alcohol (J) motifs.
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Journal of Molecular Structure 1292 (2023) 136184
benzyl, triazole, and amino alcohol) with a primary goal of finding
potent molecules with an improved therapeutic profile. Molecular hy­
bridization involves the amalgamation of two or more pharmacologi­
cally essential subunits into a single architecture with improved activity
than the parent pharmacophores [45].
2. Results and discussion
2.1. Chemistry
A library of N-substituted 2-(1H-benzo[d]imidazol-2-yl)quinoxaline
derivatives comprising 32 compounds was synthesized as outlined in
Scheme 1. Condensation between o-phenylenediamine 1 and pyr­
uvaldehyde(methylglyoxal) in water gave the intermediate methyl­
quinoxaline 2 with 85% yield [66,67]. Oxidation of intermediate 2 using
selenium dioxide in the presence of a catalytic amount of tert‑butyl
hydroperoxide resulted in quinoxaline-2-aldehyde 3 in excellent yields
[67]. Condensation of thus obtained aldehyde 3, with o-phenylenedi­
amine using sodium metabisulphite via the formation of the
aldehyde-bisulfite adduct, afforded the key intermediate 4 in good yield
[68,69]. Synthesis of N-benzylated derivatives 5a-q was achieved by
treating 4 with various substituted benzyl halides using sodium hy­
droxide as a base in DMF at room temperature with excellent yields of
90–95%. N-propargylated intermediate 6 was prepared by the reaction
of 4 with propargyl bromide employing sodium hydroxide as the base in
acetone upon heating [70,71]. A series of aromatic azides were syn­
thesized from the respective amines following reported procedures using
O. Ommi et al.
Fig. 2. Illustration of design rationale: Representative examples of quinoxaline (A–C) and benzimidazole (D, E) containing tubulin polymerization inhibitors and
design of quinoxaline-benzimidazole hybrids (5a-q, 8a-l, and 9a-f) via molecular hybridization approach.
sodium nitrite in aqueous HCl to give diazonium salt, which is later
displaced by sodium azide [72]. Cycloaddition of the alkyne component
6 with different aromatic azides in the presence of copper sulfate and
sodium ascorbate in DMF-water solvent mixture at room temperature
yielded the respective triazole derivatives 8a-i in 85–90% yields [70].
The reaction of intermediate 4 with epichlorohydrin using sodium hy­
droxide as a base in acetone upon heating accomplished intermediate 7
with 82% yield [73]. Further, the ring opening of epoxide with various
aliphatic amines in DMF under microwave irradiation resulted in the
formation of amino alcohol derivatives, 9a-f, in 60–80% yields [74,75].
2.2. NMR study
Structures of the key intermediate (4, 6, and 7) and final compounds
(5a-q, 8a-i and 9a-g) were confirmed by 1H, 13C NMR and HRMS. The
aromatic carbons (C–H) of the representative compound i.e., 4 (inter­
mediate) are specifically and unambiguously assigned. On compound 4,
we have performed selective 1D ROESY by irradiating the NH (C1)
resonance that resulted in an ROE to the D4. A 1D selective TOCSY on
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Journal of Molecular Structure 1292 (2023) 136184
the D4 proton gave all the scalar coupled proton network of the D ring.
Further, 2D 1H–13C HSQC provided the aryl carbon assignments of the A,
B and D rings. The 1D 13C spectra of the analogous compounds are
assigned easily by stacking the spectra and identifying merged signals
through compound 4, 5 h and 5l (Fig. 3).
2.3. Pharmacology
2.3.1. In vitro cytotoxicity study
The synthesized N-substituted 2-(1H-benzo[d]imidazol-2-yl)qui­
noxaline derivatives 5a-q, 8a-i, and 9a-f along with the standard
colchicine were evaluated for their in vitro cytotoxicity potential against
a panel of human cancer cell lines such as HCT116 (Human Colorectal
Carcinoma), A549 (Human Lung Adenocarcinoma), SK-ML (Human
Melanoma) along with one mouse cancer cell line B16F10 (Murine
Melanoma) by employing MTT (3-(4,5-dimethylthiazol-2-yl)− 2,5-
diphenyltetrazolium bromide) assay. The IC50 values from the in vitro
cytotoxicity study are presented in Table 1.
Detailed investigation of these results revealed that N-benzylated
O. Ommi et al.
Scheme 1. Synthetic route to quinoxaline-benzimidazole hybrids 5a-q, 8a-i and 9a-f.
series 5a-q exhibited more promising results than the triazole 8a-i and
amino alcohol 9a-f derivatives. Dihalo benzyl derivatives 5f, 5 g, and 5l
displayed IC50 of < 50 µM against all tested cancer cell lines, particularly
compound 5l showed the highest cytotoxicity of IC50 4.37 ± 0.09 µM
against the A549 cell line. Also, compound 5l exhibited cytotoxicity
against two other tested cancer cell lines, HCT116 and SK-ML, with IC50
values of 7.13 ± 0.19 µM and 6.25 ± 0.59 µM, respectively. Interest­
ingly, the IC50 value of 5l against two regular human cell lines, HEK-293
(Human Embryonic Kidney) and HaCaT (Human Keratinocyte) was
found to be 16.98 ± 0.41 µM and 16.52 ± 0.23 µM, respectively. On the
A549 cell line, the IC50 value was 4.37 ± 0.09, indicating cytospecificity
of compound 5l towards cancer cell lines.
The results disclose that most benzyl derivatives with halogens
(electron-withdrawing groups) demonstrated better activity than those
substituted with electron-donating groups (Me, OMe, iPr) or without any
substitution. Compound 5k with trifluoromethyl group expressed se­
lective cytotoxicity against two cancer cell lines HCT116 (IC50: 11.94 ±
0.76 µM), A549 (IC50: 10.32 ± 1.41 µM). Compounds 5c (NO2), 5j (CN)
displayed poor or no activity. Notably, triazole 8a-i and amino alcohol
derivatives 9b-e exhibited moderate to low cytotoxicity against the
tested cancer cell lines. However, compound 9a retained its cytotoxicity
with < 20 µM in all the tested cancer cell lines. Compound 9f showed
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Journal of Molecular Structure 1292 (2023) 136184
significant activity in the SK-ML cell line with an IC50 value of 7.61 ±
0.50 µM. These observations are summarized in Fig. 4.
Several reports have described the cytotoxic properties of quinoxa­
line, benzimidazole-based compounds. In our study, a combination of
quinoxaline and N-benzylated benzimidazole resulted in compounds
with good cytotoxicity. 3,4-diCl phenyl substitution has been found in
some anticancer agents such as H (LLS30) [47,48] and J [64] (Fig. 1),
wherein 3,4-dihalobenzyl substitution has shown excellent cytotoxic
activity, which can be correlated to our lead compound 5l. Based on the
observed cytotoxicity results, 5l was chosen for further studies in the
A549 cell line.
2.3.2. Determination of apoptosis
The compound 5l was further evaluated for apoptosis induction
ability by performing various morphological and quantitative assays on
the A549 cell line.
Phase contrast microscopy. Initially, A549 cells were treated with
different concentrations of compound 5l (2.19, 4.37, and 8.74 µM) for
48 h and observed for morphological changes under a phase contrast
microscope. Compound 5l displayed characteristic apoptotic features
like cell shrinkage, disruption of the cell membrane, and decrease in the
O. Ommi et al.
Fig. 3. (a) 1D 1H 500 MHz NMR spectra in DMSO‑d6; (i) 1H NMR spectrum of compound 4. (ii) 1H NMR selective TOCSY spectrum illustrating the irradiation of the
D4 peak of compound 4. (iii) 1H NMR selective ROESY spectrum illustrating the irradiation of C1 peak of compound 4. (b) 2D 1H − 13C HSQC spectrum of the
aromatic region of compound-4. (c) 1D 13C overlay spectra of compound 4, 5H and 5 L illustrating the assignments of aryl carbons.
number of viable cells in a dose-dependent fashion, of which images
were captured and presented in Fig. 5.
Acridine orange/Ethidium bromide (AO/EB) staining. AO/EB assay is a
dual staining technique to differentiate between live, apoptotic, and
necrotic cells. Acridine orange enters cells with the intact cell membrane
(live and early apoptotic cells) and stains green. In contrast, ethidium
bromide stains the cells whose cell membrane integrity is vanished (late
apoptotic and necrotic cells) as orange or red [75]. Results from this
assay revealed that control cells exhibited normal morphology and
appeared green in color. Nevertheless, cells treated with compound 5l
displayed apoptotic features like cell shrinkage, membrane blebbing,
chromatin condensation, and the red appearance of late apoptotic and
necrotic cells (Fig. 6).
4,6-diamino-2-phenylindole (DAPI) staining. DAPI is a fluorescent dye
that binds to AT-rich DNA regions and visualizes chromatin condensa­
tion or nuclear damage. Upon staining by DAPI, normal cells appear
light blue, whereas apoptotic cells develop bright blue color [76]. In the
current study, horse-shoe-shaped, bright, fragmented nuclei formation
can be seen in compound 5l-treated A549 cells, indicating apoptosis;
however, control cells showed intact nuclei (Fig. 7).
2′,7′-Dichlorofluorescein diacetate (DCFDA) staining. DCFDA stain de­
termines the compound’s ability to generate intracellular reactive oxy­
gen species (ROS), thereby inducing apoptosis. DCFDA oxidizes to 2′,7′-
Dichlorofluorescein in the presence of reactive oxygen species and
shows green fluorescence [77]. A549 cells were treated with varied
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Journal of Molecular Structure 1292 (2023) 136184
concentrations of compound 5l and studied for apoptosis using DCFDA
stain. As evident from the results (Fig. 8), the intensity of green fluo­
rescence is directly proportional to the compound’s concentration,
implying that dose-dependent induction of apoptosis in the A549 cell
line by compound 5l.
2.3.3. Flow cytometry analysis
Effect on mitochondrial membrane potential(DΨ m). Mitochondrial mem­
brane potential (DΨm) is the driving force for the synthesis of ATP in
mitochondria and for the transport of charged compounds that are
crucial for cell viability. Hence, maintenance of stability DΨm is a pre­
requisite for healthy cell functionality [78]. Loss of DΨm leads to
increased ROS levels and oxidative stress in mitochondria, eventually
leading to cell death, which occurs during apoptosis. JC-1 dye stains
normal polarized mitochondria possessing J-aggregates red in color,
whereas depolarized mitochondria with J-monomers of apoptotic cells
appear green. The effect of compound 5l at different concentrations
(2.19, 4.37, and 8.74 µM) on DΨm in A549 cells was investigated using
membrane-permeate JC-1 dye. The results revealed a significant in­
crease in green fluorescence (depolarised cells-J monomers) in a
dose-dependent manner from 2.19 to 8.74 µM, indicating apoptosis
associated with mitochondria (Fig. 9).
Annexin V-FITC/Propidium iodide dual staining. Annexin V-FITC/Propi­
dium iodide (PI) assay is used for the quantification of apoptosis and also
to identify the apoptosis phase. Annexin V binds to externalized phos­
phatidyl serine in apoptotic cells with intact cell membranes and stains
O. Ommi et al. Journal of Molecular Structure 1292 (2023) 136184

Table 1 them positively. On the contrary, PIbinds to apoptotic and necrotic cells
In-vitro cytotoxicity data (IC50 values in µM)a for the synthesized quinoxaline- whose membrane integrity is lost. A549 cells were treated with varied
benzimidazole hybrids 5a-q, 8a-i, and 9a-f against different cancer cell lines. concentrations (2.19, 4.37, and 8.74 µM) of compound 5l and stained
Compound HCT116b A549c SK-MLd B16F10e with Annexin V-FITC/PI. The cells at different stages were represented
5a >50 32.09±6.01 >50 >50
on four quadrant graph, such as live cells (LL; FITC-/PI-), early apoptotic
5b 27.44±4.12 23.27±1.97 >50 36.53±1.06 cells (LR; FITC+/PI-), late apoptotic cells (UR; FITC+/PI+), and necrotic
5c >50 >50 >50 >50 cells (UL; FITC-/PI +). Results from Fig. 10 indicate early apoptosis, late
5d 31.7 ± 3.61 29.38±0.91 44.86±2.19 31.08±7.67 apoptosis, and necrosis induction at concentrations 2.19 and 4.37 µM by
5e 42.85±6.14
>50 >50 >50
compound 5l. Compound 5l promotes significant early apoptosis in
5f 27.10±2.17 18.69±0.08 18.75±1.31 34.78±2.20
5g 12.75±0.56 23.77±2.56 24.42±0.18 28.60±0.93 A549 cells at 2.19 µM. However, the increased cocnentration led to only
5h 16.97±0.96 >50 >50 >50 a slight increase in early apoptosis.
5i >50 24.11±0.29 >50 >50
5j 35.10±0.53 >50 >50 >50
Cell cycle analysis. Cell cycle analysis was performed on A549 cells after
5k 11.94±0.76 10.32±1.41 >50 >50
5l 7.13±0.19 4.37±0.09 6.25±0.59 16.64±1.51 treatment with compound 5l (2.19, 4.37, and 8.74 µM) for 48 h using
5m 20.95±0.70 >50 >50 20.17±0.18 flow cytometry to determine the phase of cell cycle arrest. Results from
5n 35.94±1.77 >50 >50 >50 Fig. 11 showed that exponential increase in cells at the G2/M phase after
5o 14.70±0.28 23.50±0.35 >50 17.13±0.11 treatment with compound 5l (69.2% at 2.19 µM and 90.9% at 4.37 µM)
5p 23.24±0.93 21.64±0.49
compared to the untreated control group (57.8%), indicating G2/M
>50 >50
5q >50 >50 >50 >50
8a >50 18.76±0.63 >50 33.85±2.76 phase of cell cycle arrest.
8b >50 38.88±3.93 >50 25.77±0.53
8c >50 22.61±0.36 47.6 ± 3.74 37.65±5.09 2.3.4. Clonogenic assay
8d 12.78±0.77 22.11±0.09 27.45±0.03 23.03±0.38
Clonogenic assay reveals the proliferative ability of a single cell to
8e >50 33.02±2.62 37.58±3.79 24.45±1.17
8f 40.86±2.85 >50 >50 >50 form colonies. Herein, the media containing A549 cells was treated with
8g 16.31±0.42 >50 >50 49.33±8.22 different concentrations of compound 5l (2.19, 4.37, and 8.74 µM) and
8h 25.90±7.26 23.07±1.33 50.39±2.98 >50 observed for the formation of colonies after one week. The cells were
8i >50 43.56±4.97 >50 >50 stained with crystal violet for the visualization of colonies. The results
9a 15.99±0.88 11.80±0.02 11.66±0.01 8.73±0.19
showed an apparent reduction in the number of colonies after one week
9b >50 >50 >50 >50
9c >50 >50 >50 >50 in a concentration-dependent manner (Fig. 12).
9d >50 >50 >50 >50
9e >50 >50 30.43±4.97 39.01±5.30 2.3.5. Effect on tubulin polymerization
9f 11.28±0.00 7.61±0.50 18.2 ± 0.23
>50
Compound 5l induced cell cycle arrest at the G2/M phase, typically
Colchicinef 0.55±1.86 1.03±1.37 11.08±1.77 1.70±0.55
associated with tubulin polymerization inhibition. Moreover, it is clear
a
50% inhibition concentration determined after 48 h of compound treatment; from the literature that quinoxaline and benzimidazole-based de­
Data indicate the mean values ± SEM of three independent experiments. rivatives show anticancer activity via tubulin polymerization inhibition.
b
Colorectal carcinoma,. Therefore, the potent compound 5l was evaluated for in vitro enzyme
c
Lung adenocarcinoma,.
d assay to determine the possible mechanism of action and to observe
Melanoma,.
e effects on cellular microtubules. The assay was performed by monitoring
Mouse melanoma, and.
f
Reference compound. changes in excitation wavelength (350 nm) at 37 οC for 1 h. The assay
was carried out at different concentrations (2.19, 4.37, and 8.74 µM) by
taking colchicine (1.04 µM) and paclitaxel (3 µM) as positive and
negative controls, respectively. Interestingly, compound 5l efficiently

Fig. 4. Structure-activity relationships (SAR) developed based on cytotoxic evaluation of compounds.

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O. Ommi et al. Journal of Molecular Structure 1292 (2023) 136184

Fig. 5. Morphological changes observed in A549 cells treated with compound 5l at three different concentrations (2.19, 4.37, and 8.74 µM) in comparison with the
untreated control and standard colchicine (1.03 µM) for 48 h.

Fig. 6. AO/EB dual staining in A549 cells treated with compound 5l at three different concentrations (2.19, 4.37, and 8.74 µM) compared with the untreated control
and standard colchicine (1.03 µM) for 48 h. Compound 5l treated cells exhibited apoptotic body formation, membrane blebbing, and cell shrinkage.

inhibited tubulin polymerization (50% inhibition at all the tested con­
centrations) with an IC50 value of <2.19 µM in comparison to untreated
control, DMSO (Fig. 13). Further, we also performed an immunofluo­
rescence assay to investigate the effect of 5l on tubulin expression at
IC50 concentration compared with colchicine and paclitaxel as stan­
dards. As shown in Fig. 14, tubulin expression was suppressed by 5l and
colchicine compared to the control, whereas the opposite trend was
observed with the paclitaxel. These results suggested the inhibition of
tubulin polymerization on treatment with 5l, supporting the in vitro
enzyme assay results.
displayed a hydrogen bond interaction with the ASN101. The phenyl
ring of the quinoxaline showed π-cation interaction with the LYS254. In
addition, several hydrophobic interactions were noticed between com­
pound 5l and the active site residues THR179, ALA180, LYS352,
ALA250, LYS254, LEU248, and ALA354. These interactions stabilize the
binding of the potent compound 5l to the colchicine binding pocket of
tubulin, thereby supporting the in vitro enzyme inhibition assay
(Fig. 15).
2.5. In-silico ADME/T and prime MM/GBSA binding energy studies

2.4. Molecular docking
The most potent compound, 5l was docked with tubulin-colchicine-
soblidotin: stathmin-like domain complex (PDB ID: 3E22) [51,79,80] to
determine the binding mode and type of interactions with the tubulin
using Schrödinger Release, 2021 [81]. The nitrogen atom at the 4th
position of the quinoxaline ring acted as a hydrogen bond acceptor and
QikProp program of Schrödinger software was used to study the
drug-likeness of the most potent compound 5l in terms of ADME/T
properties, and the results are given in Table 2. In silico studies revealed
that compound 5l follows Lipinski’s rule of five, and its physicochemical
descriptor values are in the recommended range [82]. Further,
MM-GBSA (Molecular mechanics generalized born surface area) bind­
ing free energy calculations confirmed that compound 5l has excellent

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O. Ommi et al. Journal of Molecular Structure 1292 (2023) 136184

Fig. 7. Nuclear morphological changes in A549 cell line after DAPI staining. A549 cells were treated with compound 5l at three different concentrations (2.19, 4.37,
and 8.74 µM) compared to the untreated control and standard colchicine (1.03 µM) for 48 h. White arrows represent membrane blebbing and fragmentation of nuclei,
and red arrows represent horse-shoe-shaped nuclei.

Fig. 8. DCFDA staining in A549 cells treated with compound 5l at three different concentrations (2.19, 4.37, and 8.74 µM) compared with the untreated control and
standard colchicine (1.03 µM) for 48 h. Compound 5l treated cells showed a dose-dependent increase in the green fluorescence compared to the control indicating
ROS-mediated apoptosis induction.

binding capability with a binding energy value of − 45.139 kcal/mol.
3. Conclusion
In conclusion, a new series of quinoxaline-benzimidazole hybrids
were designed, synthesized, and evaluated for their cytotoxicity against
human colorectal, lung, and skin cancer cell lines and one mouse mel­
anoma cell line by using an MTT assay. Among these hybrids, compound
5l exhibited the highest cytotoxicity on all the tested human cancer cell
lines. Compound 5l exhibited potent cytotoxicity on the A549 cell line
(IC50: 4.37±0.09 µM) and four-fold selectivity upon regular human cell
lines (IC50 in HEK-293 and HaCaT was found to be 16.98±0.41 µM and
16.52±0.23 µM, respectively). Different staining techniques (AO/EB,
DAPI, and DCFDA) demonstrated induction of apoptosis by compound
5l in A549 cell lines with various apoptotic features like alteration in cell
size/shape, formation of horse-shoe shaped nuclei, as well as frag­
mented bright nuclei. Further, flow cytometric analysis revealed that
compound 5l triggered ROS generation, disrupting mitochondrial
membrane potential and the G2/M phase of cell cycle arrest. The
enzyme-based assay revealed that compound 5l inhibited tubulin
polymerization. Molecular modeling studies confirmed the binding of
compound 5l at the colchicine binding site of tubulin with a binding
energy of − 45.139 kcal/mol. Hence, 5l generated out of molecular
hybridistion technique with excellent anticancer activity could be
considered for further development in the discovery of potent anti-
cancer drugs.
4. Experimental section
All the chemicals, reagents, and solvents were obtained from com­
mercial providers and were used as such without any further purifica­
tion. Reactions were monitored using TLC-MERCK pre-coated silica gel
60-F254 (0.5 mm) aluminum plates under UV light. Compounds were
purified by column chromatography using silica of 60–120 mesh. 1H and
13
C NMR spectra were recorded on Bruker Avance 500 MHz spectrom­
eter using tetramethyl silane (TMS) as the internal standard and by
making samples in CDCl3 or DMSO‑d6. Chemical shifts are reported in
ppm and referenced to TMS (δ 0.00 for 1H NMR and 13C NMR) or sol­
vents used for NMR recording CDCl3 (δ 7.26 for 1H NMR and 77.2 13C
NMR) or DMSO‑d6 (δ 2.50 for 1H NMR and 39.5 for 13C NMR). Spin

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O. Ommi et al. Journal of Molecular Structure 1292 (2023) 136184

Fig. 9. Effect on mitochondrial membrane potential. JC-1 staining in A549 cells treated with compound 5l at three different concentrations (2.19, 4.37, and 8.74 µM)
in comparison with the untreated control and standard colchicine (1.03 µM) for 48 h. Compared to the control, a dose-dependent increase in the green fluorescence
(J-monomers) and a decrease in the red fluorescence (J-aggregates) of compound 5l was observed.

Fig. 10. (A) Effect of compound 5l on apoptosis at three different concentrations (2.19, 4.37, and 8.74 µM) compared with the untreated control and standard for 48
h using annexin V-FITC/PI dual staining technique. (B) Statistical representation of percentage live, apoptotic and necrotic cells post-treatment with compound 5l,
colchicine, and control. Data was represented as the Mean ± SEM (n = 3) and analysed by one-way ANOVA following Tukey’s multiple comparison test. ***P <
0.001; **p<0.001; *p<0.05 vs. Control.

multiplicities for 1H NMR are described as s (singlet), brs (broad singlet),
d (doublet), dd (double doublet), t (triplet), and m (multiplet). Coupling
constant values are reported in hertz (Hz). For fluorine-containing
compounds, the carbon-fluorine coupling is denoted as JCF. Pulse pro­
grams from BRUKER standard library were used to record the experi­
ments. All processing and analysis for compounds 4, 5H and 5 L were
done using Bruker TopSpin 3.6.3 software. HRMS was recorded on an
Agilent quadrupole-time-of-flight (QTOF) mass spectrometer 6540 se­
ries instrument and was performed in electrospray ionization (ESI)
techniques at 70 eV. The melting point was taken using the Stuart R
SMP30 apparatus. Microwave reactions were conducted in Monowave
300 single-mode microwave reactor (make-Anton Paar GmbH). Micro­
wave reactions were performed in SiC10 Silicon Carbide reaction vessels
(10 mL) or reusable Pyrex vials (30 mL).
4.1. Chemistry
4.1.1. General procedure for the synthesis of intermediate, 2-(1H-Benzo[d]
imidazol-2-yl)quinoxaline, 3
To a solution of o-phenylene diamine, 1 (1 equiv.) in water was
added pyruvaldehyde (1 equiv.) at room temperature and stirred for 30
min. After completion (checked by TLC), the reaction mixture was
extracted with EtOAc. Combined organic layers were washed with brine,
dried over Na2SO4, and finally evaporated to give methyl quinoxaline 2
as a brown oil (85%) which is used as such in the next step without any
further purification. Crude methyl quinoxaline was dissolved in dioxane,
and selenium dioxide (1.5 equiv.) was added, followed by a catalytic
amount of TBHP. The reaction mixture was refluxed for 1.5 h. Thereafter
the solvent was evaporated from the reaction mixture, and ethyl acetate
was added and subjected to celite filtration. Obtained filtrate was then

9
O. Ommi et al. Journal of Molecular Structure 1292 (2023) 136184

Fig. 11. (A) Effect of different concentrations of compound 5l (2.19, 4.37, and 8.74 µM) on A549 cell cycle progression in comparison with the untreated control,
standard, and colchicine treated cells. (B) Graphical representation of quantification of cells at different phases of cell cycle. Data was represented as the Mean ± SEM
(n = 3) and analysed by one-way ANOVA following Tukey’s multiple comparison test. ***P < 0.00 vs. Control.

Fig. 12. (A) Effect of compound 5l (2.19, 4.37, and 8.74 µM) on colony forming ability of the A549 cell line. (B) & (C) Statistical representation of the effect of the
compound 5l on colony forming ability of the A549 cell line. Data was represented as the Mean ± SEM (n = 3) and analysed by one-way ANOVA following Tukey’s
multiple comparison test. ***P < 0.001; *p<0.05 vs. Control.

washed with brine, dried over Na2SO4, and concentrated under a vac­
uum. The residue was further purified by column chromatography to
give the intermediate aldehyde 3 in 90% yield. A mixture of aldehyde 3
(1 equiv.), o-phenylene diamine (1 equiv.), and Na2S2O5 (1 equiv.) in
DMF was stirred at rt for 30 mins. Upon completion, the reaction
mixture was poured onto crushed ice, and the resulting precipitate was
collected using vacuum filtration and recrystallized using methanol to
give the intermediate 4 in 85% yield.
Light brown solid; yield 85%; mp: 191–192 ◦ C; 1H NMR (500 MHz,
DMSO‑d6) δ 13.46 (s, 1H), 9.82 (s, 1H), 8.23 – 8.16 (m, 2H), 7.98 – 7.91
(m, 2H), 7.82 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.34 (t, J =
7.0 Hz, 1H), 7.29 (t, J = 7.0 Hz, 1H); 13C NMR (125 MHz, DMSO‑d6) δ

10
O. Ommi et al. Journal of Molecular Structure 1292 (2023) 136184

Fig. 13. (A) Effect of compound 5l (2.19, 4.37, and 8.74 µM) on the tubulin polymerization. Colchicine and paclitaxel were used as positive and negativecontrols,
respectively. (B) Percentage inhibition of tubulin by compound 5l at various concentrations.

Fig. 14. Microscopic images of immunofluorescence-labeled tubulin in A549 cells treated with compound 5l at IC50 concentration (4.37 µM) showed decrease in
protein expression as colchicine compared to paclitaxel. Tubulin was stained red and nuclei was stained blue.

Fig. 15. (A) 3D interaction of compound 5l at the colchicine binding site of tubulin protein (PDB ID: 3E22); The yellow dotted line indicates H-bonding, and the
green dotted line indicates π-cation interaction. (B) 2D interaction of compound 5l, π-cation interaction is represented as red arrow, and hydrogen bonding is shown
with magneta arrow.

148.72, 144.02, 143.93, 143.55, 141.92, 140.99, 135.14, 131.14,
130.74, 129.20, 128.87, 124.17, 122.43, 119.83, 112.35;HRMS-QTOF
(ESI): m/z calcd. for [M + H]+ C15H11N4 247.0983; found 247.0972.
4.1.2. General procedure for the synthesis of 2-(1-(Substituted benzyl)−
1H-benzo[d]imidazol-2-yl)quinoxalines, 5a-q
To a solution of 2-(1H-benzo[d]imidazol-2-yl)quinoxaline, 4 (1
equiv.) in DMF was added substituted benzyl chloride (1 equiv.)
followed by the addition of NaOH pellet(1 equiv.). The reaction mixture
was stirred at rt for 5 h or till the TLC showed the disappearance of
starting material. Afterward, the reaction mixture was poured onto
crushed ice. Thus the formed precipitate was isolated by vacuum
filtration and subsequently purified by column chromatography eluting
at EtOAc: Hexane 3: 7.

11
O. Ommi et al. Journal of Molecular Structure 1292 (2023) 136184

Table 2 (125 MHz, DMSO‑d6) δ 147.35, 146.04, 145.01, 142.77, 141.59,

QikProp prediction of drug-likeness and physicochemical properties of potent 140.55, 137.79, 137.30, 131.93(2C), 131.63, 131.59, 129.71, 129.50
compound 5l. (2C), 129.48, 125.13, 123.76, 120.81, 120.75, 111.83, 48.43; HRMS-
S. Descriptors Recommended Compound QTOF (ESI): m/z calcd. for [M + H]+ C22H16BrN4 415.0558; found
No. range 5l 415.0547
1. Molecular weight 130–725 405.285
2. H-bond donors 0–6 0 2-(1-(3-Chlorobenzyl)− 1H-benzo[d]imidazol-2-yl)quinoxaline (5e). Yel­
3. H-bond acceptors 2–20 3.5 low solid; yield 92%; mp: 195–197 ◦ C; 1H NMR (500 MHz, CDCl3) δ
4. QP logP (O/W) (predicted octanol/ − 2.0 to 6.5 6.265
water partition coefficient)
10.05 (s, 1H), 8.18 – 8.14 (m, 1H), 8.02 – 7.97 (m, 2H), 7.83 – 7.76 (m,
5. SASA (Total solvent accessible surface 300–1000 698.987 2H), 7.46 – 7.37 (m, 3H), 7.30 (s, 1H), 7.23 – 7.18 (m, 2H), 7.09 (d, J =
area in Å2) 6.5 Hz, 1H), 6.25 (s, 2H); 13C NMR (125 MHz, CDCl3) δ 147.15, 145.96,
6. CNS − 2 to +2 1 144.43, 142.41, 141.87, 140.76, 139.19, 136.81, 134.68, 130.79,
7. QP log HERG (predicted IC50 value for <− 5 − 7.27
130.61, 130.07, 129.49, 129.37, 127.85, 126.98, 125.04, 124.80,
blockage of HERG K+ channels.)
8. QP log BB (predicted brain/blood − 3 to 1.2 0.277 123.78, 120.73, 110.52, 48.81; HRMS-QTOF (ESI): m/z calcd. for [M +
partition coefficient) H]+ C22H16ClN4 371.1063; found 371.1048
9. QP Caco (predicted apparent Caco-2 <25 poor, >500 3736.537
cell permeability in nm s− 1) great
2-(1-(2,4-Dichlorobenzyl)− 1H-benzo[d]imidazol-2-yl)quinoxaline (5f).
10. Dipole moment 1 – 12.5 3.247
11. QP logKhsa (prediction of binding to − 1.5 to 1.5 1.126 Beige solid; yield 95%; mp: 180–182 ◦ C; 1H NMR (500 MHz, CDCl3) δ
human serum albumin) 10.05 (s, 1H), 8.13 (dd, J = 8.1, 1.4 Hz, 1H), 8.02 (dd, J = 6.8, 1.6 Hz,
12. QP Polrz (predicted polarizability in 13–70 45.83 1H), 7.92 – 7.88 (m, 1H), 7.81 – 7.73 (m, 2H), 7.51 (d, J = 2.1 Hz, 1H),
cubic angstroms) 7.45 – 7.37 (m, 3H), 6.98 (dd, J = 8.4, 2.1 Hz, 1H), 6.49 (d, J = 8.4 Hz,
13. QP logkp (predicted skin permeability) − 8 to − 1 − 0.446
14. Human oral absorption 1–3 1
1H), 6.28 (s, 2H); 13C NMR (125 MHz, CDCl3) δ 147.09, 145.69, 143.99,
15. Rule of five 4 1 142.08, 141.88, 140.79, 136.67, 133.76, 133.52, 132.63, 130.93,
16. TPSA/ PSA(Van der Waals surface area 7.0–200.0 36.582 130.67, 129.56, 129.41, 129.36, 127.80, 127.61, 125.31, 124.09,
of polar nitrogen and oxygen atoms 120.76, 110.22, 46.89; HRMS-QTOF (ESI): m/z calcd. for [M + H]+
and carbonyl carbon atoms.
C22H15Cl2N4 405.0673; found 405.0656
17. Number of rotatable bonds 0–15 2
18. Binding energy (-kcal/mol) − 45.139
2-(1-(2-Chloro-4-fluorobenzyl)− 1H-benzo[d]imidazol-2-yl)quinoxaline (5
g). Orange solid; yield 92%; mp: 184–186 ◦ C; 1H NMR (500 MHz,
2-(1-(4-Isopropylbenzyl)− 1H-benzo[d]imidazol-2-yl)quinoxaline (5a). CDCl3) δ 10.04 (s, 1H), 8.15 – 8.12 (m, 1H), 8.02 – 7.99 (m, 1H), 7.92 –
Off-white solid; yield 90%; mp: 175–177 ◦ C; 1H NMR (500 MHz, CDCl3) 7.89 (m, 1H), 7.80 – 7.73 (m, 2H), 7.45 – 7.41 (m, 1H), 7.45 – 7.41 (m,
δ 10.03 (s, 1H), 8.18 – 8.14 (m, 1H), 8.07 – 8.03 (m, 1H), 7.98 – 7.95 (m, 1H), 7.41 – 7.38 (m, 2H), 7.24 (dd, J = 8.3, 2.6 Hz, 1H), 6.73 (td, J = 8.4,
1H), 7.83 – 7.76 (m, 2H), 7.51 – 7.46 (m, 1H), 7.40 – 7.35 (m, 2H), 7.18 2.6 Hz, 1H), 6.55 (dd, J = 8.7, 5.9 Hz, 1H), 6.28 (s, 2H); 13C NMR (125
(d, J = 8.2 Hz, 2H), 7.11 (d, J = 8.2 Hz, 2H), 6.28 (s, 2H), 2.88 – 2.78 (m, MHz, CDCl3) δ 161.66 (d, J = 249.6 Hz), 147.12, 145.74, 144.03,
1H), 1.18 (s, 3H), 1.16 (s, 4H); 13C NMR (125 MHz, DMSO‑d6) δ 147.94, 142.06, 141.90, 140.82, 136.70, 132.62, 132.54, 130.88, 130.73 (d, J =
147.36, 146.13, 145.27, 142.87, 141.63, 140.59, 137.38, 135.52, 3.5 Hz), 130.62, 129.48 (d, J = 13.9 Hz), 128.06 (d, J = 8.8 Hz), 125.26,
131.53(2C), 129.70, 129.49, 127.37(2C), 126.92(2C), 124.95, 123.58, 124.05, 120.72, 116.99 (d, J = 25.0 Hz), 114.52 (d, J = 21.2 Hz),
120.69, 112.02, 48.60, 33.42, 24.15; HRMS-QTOF (ESI): m/z calcd. for 110.29, 46.75; HRMS-QTOF (ESI): m/z calcd. for [M + H]+ C22H15ClFN4
[M + H]+ C25H23N4 379.1922; found 379.1925. 389.0969; found 389.0958

2-(1-(4-Fluorobenzyl)− 1H-benzo[d]imidazol-2-yl)quinoxaline (5b). Off- 2-(1-Benzyl-1H-benzo[d]imidazol-2-yl)quinoxaline (5 h). Yellow solid;

white solid; yield 92%; mp: 209–211 ◦ C; 1H NMR (500 MHz, CDCl3) δ
10.04 (s, 1H), 8.19 – 8.15 (m, 1H), 8.02 – 7.96 (m, 2H), 7.83 – 7.77 (m,
2H), 7.47 – 7.43 (m, 1H), 7.41 – 7.38 (m, 2H), 7.26 – 7.22 (m, 3H), 6.96
(t, J = 8.7 Hz, 2H), 6.25 (s, 2H); 13C NMR (125 MHz, DMSO‑d6) δ 161.79
(d, J = 243.3 Hz), 147.29, 146.08, 145.07, 142.77, 141.57, 140.54,
137.27, 134.47(d, J = 2.8 Hz), 131.62, 131.59, 129.72, 129.47(d, J =
2.8 Hz, 2C), 129.39, 125.08, 123.72, 120.72, 115.83 (d, J = 21.5 Hz,
2C), 111.92, 48.21; HRMS-QTOF (ESI): m/z calcd. for [M + H]+
C22H16FN4 355.1359; found 355.1363
2-(1-(4-Nitrobenzyl)− 1H-benzo[d]imidazol-2-yl)quinoxaline (5c). Yel­
low solid; yield 90%; mp: 192–194 ◦ C; 1H NMR (500 MHz, CDCl3) δ
10.05 (s, 1H), 8.16 (d, J = 8.7 Hz, 3H), 8.01 (d, J = 7.1 Hz, 1H), 7.88 (dd,
J = 8.1, 1.4 Hz, 1H), 7.83 – 7.75 (m, 2H), 7.46 – 7.39 (m, 4H), 7.39 (s,
1H), 6.37 (s, 2H); 13C NMR (125 MHz, DMSO‑d6) δ 147.41, 147.13,
146.36, 145.96, 144.81, 142.74, 141.56, 140.49, 137.34, 131.67,
131.59, 129.69, 129.46, 128.28(2C), 125.30, 124.27(2C), 123.92,
120.84, 111.70, 48.77; HRMS-QTOF (ESI): m/z calcd. for [M + H]+
C22H16N5O2382.1304; found 382.1307
yield 90%; mp: 195–197 ◦ C; 1H NMR (500 MHz, CDCl3) δ 10.09 (s, 1H),
8.20 – 8.17 (m, 1H), 8.06 – 8.00 (m, 2H), 7.85 – 7.78 (m, 2H), 7.51 –
7.48 (m, 1H), 7.45 – 7.39 (m, 2H), 7.31 – 7.29 (m, 1H), 7.28 – 7.22 (m,
4H), 6.33 (s, 2H); 13C NMR (125 MHz, DMSO‑d6) δ 146.86, 145.58,
144.64, 142.28, 141.06, 140.05, 137.79, 136.93, 131.09, 131.07,
129.21, 128.99, 128.54(2C), 127.25, 126.77(2C), 124.54, 123.17,
120.21, 111.49, 48.41; HRMS-QTOF (ESI): m/z calcd. for [M + H]+
C22H17N4 337.1453; found 337.1448
2-(1-(4-(Tert‑butyl)benzyl)− 1H-benzo[d]imidazol-2-yl)quinoxaline (5i).
Off-white solid; yield 93%; mp: 220–223 ◦ C; 1H NMR (500 MHz, CDCl3)
δ 10.00 (s, 1H), 8.17 – 8.13 (m, 1H), 8.07 – 8.03 (m, 1H), 7.96 – 7.91 (m,
1H), 7.81 – 7.75 (m, 2H), 7.49 – 7.45 (m, 1H), 7.38 – 7.33 (m, 2H), 7.26
(d, J = 8.2 Hz, 2H), 7.19 (d, J = 8.4 Hz, 2H), 6.27 (s, 2H), 1.23 (s, 9H);
13
C NMR (125 MHz, DMSO‑d6) δ 150.21, 147.28, 146.15, 145.28,
142.82, 141.61, 140.57, 137.34, 135.16, 131.59(2C), 129.75, 129.50,
127.15(2C), 125.81(2C), 124.98, 123.62, 120.69, 112.09, 48.45, 34.60,
31.48; HRMS-QTOF (ESI): m/z calcd. for [M + H]+ C26H25N4 393.2079;
found 393.2067

2-(1-(4-Bromobenzyl)− 1H-benzo[d]imidazol-2-yl)quinoxaline (5d). 4-((2-(Quinoxalin-2-yl)− 1H-benzo[d]imidazol-1-yl)methyl)benzonitrile

White solid; yield 93%; mp: 245–247 ◦ C; 1H NMR (500 MHz, CDCl3) δ (5j). Cream solid; yield 95%; mp: 232–234 ◦ C; 1H NMR (500 MHz,
10.02 (s, 1H), 8.17 – 8.14 (m, 1H), 7.98 – 7.96 (m, 2H), 7.82 – 7.74 (m, DMSO‑d6) δ 9.88 (s, 1H), 8.18 – 8.13 (m, 1H), 8.05 – 8.00 (m, 1H), 7.95
2H), 7.43 – 7.36 (m, 5H), 7.12 (d, J = 8.5 Hz, 2H), 6.22 (s, 2H); 13C NMR – 7.89 (m, 3H), 7.76 (t, J = 7.2 Hz, 3H), 7.46 – 7.38 (m, 4H), 6.40 (s, 2H);

12
O. Ommi et al.
13
C NMR (125 MHz, DMSO‑d6) δ 147.39, 145.98, 144.86, 144.25,
142.72, 141.56, 140.48, 137.34, 133.03(2C), 131.66, 131.59, 129.67,
129.47, 128.05(2C), 125.26, 123.89, 120.81, 119.12, 111.72, 110.46,
48.86; HRMS-QTOF (ESI): m/z calcd. for [M + H]+ C23H16N5 362.1405;
found 362.1416
2-(1-(4-(Trifluoromethyl)benzyl)− 1H-benzo[d]imidazol-2-yl)quinoxaline
(5k). Off-white solid; yield 90%; mp: 197–199 ◦ C; 1H NMR (500 MHz,
DMSO‑d6) δ 9.89 (s, 1H), 8.20 – 8.12 (m, 1H), 8.07 – 8.00 (m, 1H), 7.92
(d, J = 5.5 Hz, 3H), 7.75 (d, J = 7.7 Hz, 1H), 7.67 (d, J = 8.1 Hz, 2H),
7.48 (d, J = 8.0 Hz, 2H), 7.45 – 7.36 (m, 2H), 6.41 (s, 2H); 13C NMR
(125 MHz, DMSO‑d6) δ 147.38, 146.01, 144.93, 143.21, 142.76,
141.57, 140.50, 137.34, 131.62, 131.60 129.66, 129.47, 128.31 (d, J =
31.7 Hz), 127.90, 125.97 (q, J = 3.8 Hz, 4C), 125.22, 123.83, 120.79,
111.76, 48.70; HRMS-QTOF (ESI): m/z calcd. for [M + H]+ C23H16F3N4
405.1327; found 405.1315
2-(1-(3,4-Dichlorobenzyl)− 1H-benzo[d]imidazol-2-yl)quinoxaline (5l).
Cream solid; yield 92%; mp: 214–216 ◦ C; 1H NMR (500 MHz, DMSO‑d6)
δ 9.88 (s, 1H), 8.19 – 8.15 (m, 1H), 8.12 – 8.07 (m, 1H), 7.96 – 7.93 (m,
1H), 7.91 (d, J = 7.6 Hz, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.69 (d, J = 2.0
Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.46 – 7.37 (m, 2H), 7.19 (dd, J = 8.4,
2.0 Hz, 1H), 6.31 (s, 2H); 13C NMR (125 MHz, DMSO‑d6) δ 146.89,
145.61, 144.44, 142.26, 141.13, 140.04, 139.14, 136.77, 131.21,
131.15, 131.09, 130.84, 129.86, 129.19, 129.12, 129.04, 127.10,
124.80, 123.42, 120.34, 111.31, 47.51; HRMS-QTOF (ESI): m/z calcd.
for [M + H]+ C22H15Cl2N4 405.0673; found 405.0651
2-(1-(4-Methoxybenzyl)− 1H-benzo[d]imidazol-2-yl)quinoxaline (5 m).
Off-white solid; yield 90%; mp: 187–189 ◦ C; 1H NMR (500 MHz,
DMSO‑d6) δ 9.87 (s, 1H), 8.20 – 8.14 (m, 2H), 7.97 – 7.92 (m, 2H), 7.87
(d, J = 8.1 Hz, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.42 – 7.33 (m, 1H), 7.25 (d,
J = 8.8 Hz, 1H), 6.81 (d, J = 8.8 Hz, 1H), 6.27 (s, 2H), 3.64 (s, 3H); 13C
NMR (125 MHz, DMSO‑d6) δ 158.96, 147.24, 146.11, 145.20, 142.81,
141.56, 140.55, 137.28, 131.54(2C), 130.08, 129.71, 129.47, 128.78
(2C), 124.91, 123.57, 120.66, 114.40(2C), 112.03, 55.42, 48.25; HRMS-
QTOF (ESI): m/z calcd. for [M + H]+ C23H19N4O 367.1558; found
367.1543
2-(1-(4-Methylbenzyl)− 1H-benzo[d]imidazol-2-yl)quinoxaline (5n). Off-
white solid; yield 91%; mp: 177–179 ◦ C; 1H NMR (500 MHz, DMSO‑d6) δ
9.87 (s, 1H), 8.19 – 8.11 (m, 2H), 7.96 – 7.91 (m, 2H), 7.88 (d, J = 7.4
Hz, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.42 – 7.34 (m, 1H), 7.16 (d, J = 8.1
Hz, 2H), 7.06 (d, J = 8.0 Hz, 2H), 6.30 (s, 2H), 2.19 (s, 3H); 13C NMR
(125 MHz, DMSO‑d6) δ 147.33, 146.09, 145.19, 142.78, 141.56,
140.57, 137.39, 136.93, 135.21, 131.60(2C), 129.73, 129.57(2C),
129.49, 127.27(2C), 124.98, 123.62, 120.68, 112.02, 48.63, 21.04;
HRMS-QTOF (ESI): m/z calcd. for [M + H]+ C23H19N4 351.1609; found
351.1593
2-(1-(3-Fluorobenzyl)− 1H-benzo[d]imidazol-2-yl)quinoxaline (5o). Off-
white solid; yield 92%; mp: 252–254 ◦ C; 1H NMR (500 MHz, DMSO‑d6) δ
9.88 (s, 1H), 8.19 – 8.15 (m, 1H), 8.12 – 8.08 (m, 1H), 7.96 – 7.92 (m,
2H), 7.91 (d, J = 7.6 Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.45 – 7.36 (m,
1H), 7.32 (td, J = 8.1, 6.2 Hz, 1H), 7.17 (d, J = 10.1 Hz, 1H), 7.07 – 7.02
(m, 2H), 6.35 (s, 2H); 13C NMR (125 MHz, DMSO‑d6) δ 162.66 (d, J =
243.5 Hz),147.35, 146.08, 145.02, 142.73, 141.57, 141.28, 141.23,
140.52, 137.32, 131.62 (d, J = 5.1 Hz), 131.09 (d, J = 8.3 Hz), 129.67,
129.49, 125.16, 123.79, 123.20 (d, J = 2.5 Hz), 120.75, 114.58 (d, J =
20.9 Hz), 114.21 (d, J = 22.1 Hz), 111.86, 48.49; HRMS-QTOF (ESI): m/
z calcd. for [M + H]+ C22H16FN4 355.1359; found 355.1342
2-(1-(2-Fluorobenzyl)− 1H-benzo[d]imidazol-2-yl)quinoxaline (5p). Yel­
low solid; yield 90%; mp: 188–190 ◦ C; 1H NMR (500 MHz, DMSO‑d6) δ
9.85 (s, 1H), 8.17 – 8.12 (m, 1H), 8.02 – 7.98 (m, 1H), 7.94 – 7.89 (m,
13
Journal of Molecular Structure 1292 (2023) 136184
3H), 7.77 (d, J = 7.8 Hz, 1H), 7.45 – 7.36 (m, 2H), 7.26 (dd, J = 9.1, 3.1
Hz, 2H), 7.02 – 6.97 (m, 1H), 6.84 (t, J = 7.7 Hz, 1H), 6.35 (s, 2H); 13C
NMR (125 MHz, DMSO‑d6) δ 160.17 (d, J = 244.5 Hz), 147.45, 145.92,
144.99, 142.65, 141.52, 140.51, 137.49, 131.62(2C), 129.75 (d, J = 8.2
Hz), 129.48 (d, J = 3.6 Hz), 128.46 (d, J = 4.1 Hz) 125.39 (d, J = 14.2
Hz), 125.18, 125.12, 125.10, 123.78, 120.77, 115.83 (d, J = 21.0 Hz),
111.66, 43.32; HRMS-QTOF (ESI): m/z calcd. for [M + H]+ C22H16FN4
355.1359; found 355.1353
2-(1-(3-Methylbenzyl)− 1H-benzo[d]imidazol-2-yl)quinoxaline (5q).
Beige solid; yield 92%; mp: 173–175 ◦ C; 1H NMR (500 MHz, DMSO‑d6) δ
9.87 (s, 1H), 8.18 – 8.10 (m, 2H), 7.95 – 7.91 (m, 2H), 7.89 (d, J = 7.4
Hz, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.42 – 7.34 (m, 1H), 7.13 (dd, J = 9.7,
5.3 Hz, 2H), 7.00 (d, J = 7.9 Hz, 2H), 6.29 (s, 2H), 2.18 (s, 3H); 13C NMR
(125 MHz, DMSO‑d6) δ 147.41, 146.09, 145.16, 142.78, 141.56,
140.56, 138.18, 138.12, 137.43, 131.58(2C), 129.68, 129.49, 128.93,
128.41, 127.89, 125.00, 124.31, 123.63, 120.69, 111.99, 48.88, 21.42;
HRMS-QTOF (ESI): m/z calcd. for [M + H]+ C23H19N4 351.1609; found
351.1600
4.1.3. 2-(1-(Prop-2-yn-1-yl)− 1H-benzo[d]imidazol-2-yl)quinoxaline, 6
A stirred solution of intermediate 3 (1 equiv.), NaOH (1 equiv.) and
propargyl bromide (1.2 equiv.) in acetone were refluxed for 2 h. On
completion, acetone was evaporated from the reaction mixture, to
which ice-cold water was added later. The solid intermediate was
filtered, extensively washed with ice-cold water, and dried under vac­
uum which is then subjected to column chromartography to obtain
propargylated intermediate 6 as a white solid (65% yield).
White solid; yield 65%; mp: 160–163 ◦ C; 1H NMR (500 MHz,
DMSO‑d6) δ 9.90 (s, 1H), 8.34 – 8.29 (m, 1H), 8.27 – 8.24 (m, 1H), 8.08
– 8.00 (m, 2H), 7.94 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 8.1 Hz, 1H), 7.53 (t,
J = 8.1 Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H), 6.05 (d, J = 2.4 Hz, 2H); 13C
NMR (125 MHz, DMSO‑d6) δ 146.77, 145.91, 144.98, 142.63, 141.67,
140.59, 136.61, 131.74, 131.67, 129.90, 129.52, 125.10, 123.86,
120.72, 111.83, 79.49, 75.79, 35.74; HRMS-QTOF (ESI): m/z calcd. for
[M + H]+ C18H13N4 285.1140; found 285.1106
4.1.4. General procedure for the synthesis of 2-(1-((1-(Substituted
phenyl)− 1H-1,2,3-triazol-4-yl)methyl)− 1H-benzo[d]imidazol-2-yl)
quinoxaline, 8a-h
2-(1-(prop‑2-yn-1-yl)− 1H-benzo[d]imidazol-2-yl)quinoxaline 6 (1
equiv.), and appropriate azide (1 equiv.) were dissolved in a mixture of
DMF: H2O (10: 0.5) followed by addition of CuSO4⋅5H2O (0.2 equiv.)
and sodium ascorbate (0.4 equiv.). The resulting reaction mixture was
stirred for 15 h at rt. After completion, the reaction mixture was poured
onto crushed ice and filtered using vacuum filtration. Obtained solids
were further purified by column chromatography eluting at EtOAc:
Hexane 4: 6 to afford the target derivatives 8a-h in 85–90% yields.
2-(1-((1-(3,4,5-Trimethoxyphenyl)− 1H-1,2,3-triazol-4-yl)methyl)− 1H-
benzo[d]imidazol-2-yl)quinoxaline (8a). Off-white solid; yield 85%; mp:
212–214 ◦ C; 1H NMR (500 MHz, CDCl3) δ 10.08 (s, 1H), 8.22 – 8.18 (m,
1H), 8.12 (dd, J = 6.6, 2.9 Hz, 1H), 7.96 (d, J = 7.9 Hz, 1H), 7.91 (s, 1H),
7.87 – 7.82 (m, 2H), 7.80 (d, J = 8.3 Hz, 1H), 7.46 (t, J = 7.2 Hz, 1H),
7.41 (t, J = 7.1 Hz, 1H), 6.81 (s, 2H), 6.45 (s, 2H), 3.83 (s, 4H); 13C NMR
(125 MHz, DMSO‑d6) δ 153.86(2C), 147.30, 146.08, 145.21, 144.84,
142.75, 141.62, 140.65, 137.90, 137.10, 132.83, 131.61, 131.55,
129.93, 129.48, 124.96, 123.69, 122.35, 120.62, 112.06, 98.85(2C),
60.62(2C), 56.72, 41.36; HRMS-QTOF (ESI): m/z calcd. for [M + H]+
C27H24N7O3 494.1940; found 494.1932
2-(1-((1-(4-Chlorophenyl)− 1H-1,2,3-triazol-4-yl)methyl)− 1H-benzo[d]
imidazol-2-yl)quinoxaline (8b). Light-brown solid; yield 88%; mp:
243–245 ◦ C; 1H NMR (500 MHz, DMSO‑d6) δ 9.90 (s, 1H), 8.81 (s, 1H),
8.29 – 8.24 (m, 1H), 8.22 – 8.16 (m, 1H), 7.98 – 7.93 (m, 2H), 7.89 (dd,
O. Ommi et al.
J = 12.9, 8.1 Hz, 1H), 7.84 (d, J = 8.9 Hz, 1H), 7.59 (d, J = 8.9 Hz, 1H),
7.44 (t, J = 8.1 Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 6.48 (s, 2H); 13C NMR
(125 MHz, DMSO‑d6) δ 147.20, 146.08, 145.23, 145.23, 142.75,
141.64, 140.69, 137.07, 135.73, 133.44, 131.60, 131.55, 130.20(2C),
129.98, 129.46, 124.97, 123.71, 122.33(2C), 122.14, 120.59, 112.09,
41.25; HRMS-QTOF (ESI): m/z calcd. for [M + H]+ C24H17ClN7
438.1234; found 438.1257
2-(1-((1-(4-Fluorophenyl)− 1H-1,2,3-triazol-4-yl)methyl)− 1H-benzo[d]
imidazol-2-yl)quinoxaline (8c). Cream solid; yield 90%; mp: 209–211 ◦ C;
1
H NMR (500 MHz, DMSO‑d6) δ 9.90 (s, 1H), 8.77 (s, 1H), 8.29 – 8.25
(m, 1H), 8.21 – 8.17 (m, 1H), 7.98 – 7.93 (m, 2H), 7.89 (dd, J = 16.8,
8.1 Hz, 2H), 7.86 – 7.82 (m, 1H), 7.44 (t, J = 8.2 Hz, 1H), 7.41 – 7.35 (m,
3H), 6.47 (s, 2H); 13C NMR (125 MHz, DMSO‑d6) δ 162.07 (d, J = 245.4
Hz), 147.12, 146.08, 145.21, 145.07, 142.74, 141.62, 140.66, 137.05,
133.49, 131.51 (d, J = 3.8 Hz, 2C), 129.98, 129.44, 124.92, 123.66,
122.98 (d, J = 8.8 Hz, 2C), 122.31, 120.58, 117.05 (d, J = 23.3 Hz, 2C),
112.10, 41.23; HRMS-QTOF (ESI): m/z calcd. for [M + H]+ C24H17FN7
422.1529; found 422.1557.
2-(1-((1-(4-Nitrophenyl)− 1H-1,2,3-triazol-4-yl)methyl)− 1H-benzo[d]imi­
dazol-2-yl)quinoxaline (8d). Pale yellow solid; yield 86%; mp: 262–264
◦
C; 1H NMR (500 MHz, DMSO‑d6) δ 9.65 (s, 1H), 8.72 (s, 1H), 8.12 (d, J
= 9.1 Hz, 2H), 8.00 (dd, J = 6.4, 3.2 Hz, 1H), 7.94 (dd, J = 6.4, 3.2 Hz,
1H), 7.88 (d, J = 9.1 Hz, 2H), 7.74 – 7.67 (m, 2H), 7.64 (t, J = 8.5 Hz,
2H), 7.20 (t, J = 7.5 Hz, 1H), 7.13 (t, J = 7.5 Hz, 1H), 6.25 (s, 2H); 13C
NMR (125 MHz, DMSO‑d6) δ 147.19, 147.16, 146.02, 145.77, 145.15,
142.69, 141.61, 141.13, 140.67, 137.02, 131.67, 131.61, 129.98,
129.44, 125.91(2C), 125.05, 123.79, 122.46, 121.18(2C), 120.59,
112.03, 41.23; HRMS-QTOF (ESI): m/z calcd. for [M + H]+ C24H17N8O2
449.1474; found 449.1469
2-(1-((1-(4-Methoxyphenyl)− 1H-1,2,3-triazol-4-yl)methyl)− 1H-benzo[d]
imidazol-2-yl)quinoxaline (8e). Off-white solid; yield 85%; mp: 214–216
◦
C; 1H NMR (500 MHz, DMSO‑d6) δ 9.89 (s, 1H), 8.68 (s, 1H), 8.30 –
8.25 (m, 1H), 8.21 – 8.17 (m, 1H), 7.98 – 7.93 (m, 2H), 7.91 (d, J = 8.1
Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 9.1 Hz, 1H), 7.44 (t, J =
7.6 Hz, 1H), 7.37 (t, J = 7.2 Hz, 1H), 7.05 (d, J = 9.1 Hz, 1H), 6.45 (s,
2H), 3.78 (s, 3H); 13C NMR (125 MHz, DMSO‑d6) δ 159.71, 147.16,
146.12, 145.27, 144.75, 142.74, 141.63, 140.68, 137.07, 131.56,
131.54, 130.35, 129.98, 129.47, 124.91, 123.66, 122.28(2C), 122.04,
120.57, 115.22(2C), 112.16, 55.99, 41.25; HRMS-QTOF (ESI): m/z
calcd. for [M + H]+ C25H20N7O 434.1729; found 434.1755
2-(1-((1-(3-Chlorophenyl)− 1H-1,2,3-triazol-4-yl)methyl)− 1H-benzo[d]
imidazol-2-yl)quinoxaline (8f). yellow solid; yield 87%; mp: 197–199 ◦ C;
1
H NMR (500 MHz, DMSO‑d6) δ 9.89 (s, 1H), 8.84 (s, 1H), 8.27 – 8.22
(m, 1H), 8.20 – 8.16 (m, 1H), 7.96 – 7.94 (m, 1H), 7.93 (dd, J = 4.1, 2.1
Hz, 2H), 7.90 – 7.86 (m, 2H), 7.82 (d, J = 11.1 Hz, 1H), 7.54 (t, J = 8.1
Hz, 1H), 7.51 – 7.47 (m, 1H), 7.44 (t, J = 8.1 Hz, 1H), 7.37 (t, J = 8.1 Hz,
1H), 6.47 (s, 2H); 13C NMR (125 MHz, DMSO‑d6) δ 147.11, 146.05,
145.29, 145.18, 142.75, 141.61, 140.65, 137.95, 137.04, 134.53,
131.91, 131.51, 131.45, 129.97, 129.42, 128.90, 124.93, 123.65,
122.20, 120.59, 120.32, 119.15, 112.03, 41.31; HRMS-QTOF (ESI): m/z
calcd. for [M + H]+ C24H17ClN7 438.1234; found 438.1247.
2-(1-((1-(4-Bromo-3-(trifluoromethyl)phenyl)− 1H-1,2,3-triazol-4-yl)
methyl)− 1H-benzo[d]imidazol-2-yl)quinoxaline (8 g). Brown solid; yield
85%; mp: 164–166 ◦ C; 1H NMR (500 MHz, DMSO‑d6) δ 9.89 (s, 1H),
8.94 (s, 1H), 8.24 (dd, J = 6.2, 3.4 Hz, 1H), 8.21 (s, 1H), 8.18 (dd, J =
6.2, 3.4 Hz, 1H), 8.09 – 8.02 (m, 2H), 7.94 (dd, J = 6.3, 3.4 Hz, 2H), 7.88
(d, J = 8.2 Hz, 2H), 7.44 (t, J = 7.6 Hz, 1H), 7.37 (t, J = 7.6 Hz, 1H), 6.48
(s, 2H); 13C NMR (125 MHz, DMSO‑d6) δ 147.17, 146.05, 145.56,
145.17, 142.74, 141.63, 140.66, 137.02, 136.32, 131.61, 131.52,
130.04 (d, J = 31.2 Hz), 130.00, 129.45, 125.87, 124.99, 123.90,
14
Journal of Molecular Structure 1292 (2023) 136184
123.72, 122.49, 121.72, 120.61, 120.15 (q, J = 5.4 Hz), 118.97, 112.04,
41.33; HRMS-QTOF (ESI): m/z calcd. for [M + H]+ C25H16BrF3N7
550.0602; found 550.0606
2-(1-((1-(3,5-Difluorophenyl)− 1H-1,2,3-triazol-4-yl)methyl)− 1H-benzo
[d]imidazol-2-yl)quinoxaline (8 h)*. Yellow solid; yield 88%; mp:
232–234 ◦ C; 1H NMR (500 MHz, DMSO‑d6) δ 9.88 (s, 1H), 8.82 (s, 1H),
8.25 – 8.20 (m, 1H), 8.18 – 8.14 (m, 1H), 7.95 – 7.90 (m, 2H), 7.87 (d, J
= 8.2 Hz, 2H), 7.67 (dd, J = 8.0, 2.0 Hz, 1H), 7.43 (t, J = 8.1 Hz, 1H),
7.39 – 7.31 (m, 2H), 6.46 (s, 2H); 13C NMR (125 MHz, DMSO‑d6) δ
163.20 (d, J = 246.9 Hz), 163.08 (d, J = 246.9 Hz), 147.11, 146.03,
145.57, 145.15, 142.75, 141.62, 140.65, 138.65 (q, J = 13.2 Hz),
137.03, 131.54, 131.48, 130.00, 129.42, 124.96, 123.69, 122.31,
120.60, 112.00, 104.53, 104.32 (d, J = 8.2 Hz, 2C), 41.33; HRMS-QTOF
(ESI): m/z calcd. for [M + H]+ C24H16F2N7 440.1435; found 440.1445.
2-(1-((1-(3,4-Dimethoxyphenyl)− 1H-1,2,3-triazol-4-yl)methyl)− 1H-
benzo[d]imidazol-2-yl)quinoxaline (8i). Cream solid; yield 88%; mp:
234–236 ◦ C; 1H NMR (500 MHz, DMSO‑d6) δ 9.89 (s, 1H), 8.80 (s, 1H),
8.23 (d, J = 35.6 Hz, 2H), 7.92 (d, J = 33.6 Hz, 4H), 7.41 (d, J = 30.4 Hz,
2H), 6.97 (s, 2H), 6.56 (s, 1H), 6.46 (s, 2H), 3.77 (s, 6H); 13C NMR (125
MHz, DMSO‑d6) δ 161.56(2C), 147.22, 146.09, 145.23, 144.95, 142.75,
141.62, 140.66, 138.44, 137.09, 131.57, 131.52, 129.94, 129.47,
124.94, 123.67, 122.20, 120.60, 112.08, 100.62, 98.96(2C), 56.14(2C),
41.32; HRMS-QTOF (ESI): m/z calcd. for [M + H]+ C26H22N7O2
464.1835; found 464.1836
4.1.5. General procedure for the synthesis of 2-(1-(Oxiran-2-ylmethyl)−
1H-benzo[d]imidazol-2-yl)quinoxaline, 7
A mixture of 2-(1H-benzo[d]imidazol-2-yl)quinoxaline 4 (1 equiv.),
epichlorohydrin (2 equiv.), and sodium hydroxide (1 equiv.) in acetone
was refluxed for 30 min. Upon completion, the solvent was evaporated
from the reaction mixture to which ice-cold water was added and
filtered using vacuum filtration to obtain the epoxy intermediate 7 as a
yellow solid in 82% yield.
Cream solid; yield 82%; mp: 155–157 ◦ C; 1H NMR (500 MHz,
DMSO‑d6) δ 9.83 (s, 1H), 8.24 – 8.16 (m, 2H), 7.99 – 7.93 (m, 2H), 7.85
(d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H),
7.36 (t, J = 7.1 Hz, 1H), 5.44 (dd, J = 15.0, 3.3 Hz, 1H), 4.92 (dd, J =
15.0, 5.8 Hz, 1H), 3.64 – 3.60 (m, 1H), 2.81 (dd, J = 5.0, 4.1 Hz, 1H),
2.70 (dd, J = 5.0, 2.6 Hz, 1H); 13C NMR (125 MHz, DMSO‑d6) δ 147.70,
146.16, 145.23, 142.63, 141.61, 140.62, 137.64, 131.56, 131.52,
129.89, 129.48, 124.80, 123.55, 120.48, 112.21, 51.34, 47.47, 45.49;
HRMS-QTOF (ESI): m/z calcd. for [M + H]+ C18H15N4O 303.1245;
found 303.1248
4.1.6. General procedure for the synthesis of 1-(Substituted amino)− 3-(2-
(quinoxalin-2-yl)− 1H-benzo[d]imidazol-1-yl) propan-2-ol, 9a-f
A mixture of epoxide (1 equiv.) and an excess of aliphatic amines (up
to 3 equiv.) in DMF were stirred in a closed microwave vessel at 180 ◦ C
for 20 mins. After completion, the reaction mixture was cooled to room
temperature, to which water was added and extracted with ethyl ace­
tate. The organic layer was washed with brine, dried over Na2SO4, and
concentrated under reduced pressure. The residue was then purified by
column chromatography using methanol and DCM (2: 8) as the eluent to
give the target derivatives 9a-g in 60 - 80% yields.
1-Morpholino-3-(2-(quinoxalin-2-yl)− 1H-benzo[d]imidazol-1-yl)propan-
2-ol (9a). Light yellow solid; yield 80%; mp: 239–241 ◦ C; 1H NMR (500
MHz, DMSO‑d6) δ 9.80 (s, 1H), 8.19 (d, J = 8.1 Hz, 2H), 8.01 – 7.93 (m,
2H), 7.83 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.39 (t, J = 7.6
Hz, 1H), 7.33 (t, J = 7.5 Hz, 1H), 5.13 (dd, J = 14.2, 3.7 Hz, 1H), 4.91 (d,
J = 5.3 Hz, 1H), 4.83 (dd, J = 14.1, 8.3 Hz, 1H), 4.22 – 4.15 (m, 1H),
3.46 – 3.41 (m, 3H), 2.49 – 2.40 (m, 2H), 2.32 (s, 4H); 13C NMR (125
MHz, DMSO‑d6) δ 148.10, 146.36, 145.79, 142.71, 141.50, 140.63,
O. Ommi et al.
138.03, 131.52, 131.47, 129.70, 129.52, 124.32, 123.13, 120.29,
112.69, 67.71, 66.50(2C), 63.16, 54.47(2C), 50.25; HRMS-QTOF (ESI):
m/z calcd. for [M + H]+ C22H24N5O2 390.1930; found 390.1939
1-(Piperazin-1-yl)− 3-(2-(quinoxalin-2-yl)− 1H-benzo[d]imidazol-1-yl)
propan-2-ol (9b). Off-white solid; yield 80%; mp: 170–172 ◦ C; 1H NMR
(500 MHz, DMSO‑d6) δ 9.81 (s, 1H), 8.22 – 8.17 (m, 2H), 8.01 – 7.95 (m,
2H), 7.94 (s, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.40
(t, J = 8.1 Hz, 1H), 7.33 (t, J = 8.1 Hz, 1H), 5.12 (dd, J = 14.2, 3.9 Hz,
1H), 4.93 (d, J = 5.3 Hz, 1H), 4.84 (dd, J = 14.1, 8.4 Hz, 1H), 4.25 – 4.17
(m, 1H), 3.29 – 3.19 (m, 4H), 2.56 – 2.53 (m, 1H), 2.49 – 2.45 (m, 1H),
2.36 (t, J = 4.9 Hz, 2H), 2.31 (t, J = 5.1 Hz, 2H); 13C NMR (125 MHz,
DMSO‑d6) δ 147.62, 145.90, 145.30, 142.23, 141.04, 140.17, 137.57,
131.09, 131.02, 129.25, 129.06, 123.89, 122.69, 119.84, 112.23, 67.49,
62.05, 53.95, 52.81, 49.79, 44.65, 38.85; HRMS-QTOF (ESI): m/z calcd.
for [M + H]+ C22H25N6O 389.2089; found 389.2087
1-(Piperidin-1-yl)− 3-(2-(quinoxalin-2-yl)− 1H-benzo[d]imidazol-1-yl)
propan-2-ol (9c). Cream solid; yield 75%; mp: 194–195 ◦ C; 1H NMR
(500 MHz, DMSO‑d6) δ 9.80 (s, 1H), 8.21 – 8.18 (m, 2H), 8.00 – 7.93 (m,
2H), 7.83 (d, J = 7.9 Hz, 1H), 7.77 (d, J = 8.1 Hz, 1H), 7.42 – 7.37 (m,
1H), 7.35 – 7.30 (m, 1H), 5.14 (dd, J = 14.2, 3.9 Hz, 1H), 4.78 (dd, J =
14.1, 8.3 Hz, 2H), 4.15 (s, 1H), 2.45 – 2.35 (m, 2H), 2.28 (s, 4H), 1.40 –
1.34 (m, 4H), 1.33 – 1.28 (m, 2H); 13C NMR (125 MHz, DMSO‑d6) δ
148.16, 146.38, 145.84, 142.74, 141.52, 140.65, 138.06, 131.45,
131.43, 129.67, 129.52, 124.27, 123.10, 120.29, 112.63, 67.83, 63.70,
55.27(2C), 50.43, 25.95(2C), 24.37; HRMS-QTOF (ESI): m/z calcd. for
[M + H]+ C23H26N5O 388.2137; found 388.2176
1-(Propylamino)− 3-(2-(quinoxalin-2-yl)− 1H-benzo[d]imidazol-1-yl)
propan-2-ol (9d). White solid; yield 70%; mp: 224–226 ◦ C; 1H NMR
(500 MHz, DMSO‑d6) δ 9.87 (s, 1H), 8.49 (s, 1H), 8.35 – 8.31 (m, 1H),
8.22 – 8.18 (m, 1H), 8.02 – 7.95 (m, 2H), 7.86 (dd, J = 16.8, 8.1 Hz, 2H),
7.46 – 7.41 (m, 1H), 7.39 – 7.35 (m, 1H), 5.91 (d, J = 5.4 Hz, 1H), 5.09
(dd, J = 14.2, 4.3 Hz, 1H), 4.90 (dd, J = 14.2, 8.1 Hz, 1H), 4.50 (s, 1H),
3.26 (dd, J = 12.6, 2.4 Hz, 1H), 3.12 (dd, J = 12.6, 10.1 Hz, 1H), 2.88 (t,
J = 7.9 Hz, 1H), 1.66 – 1.55 (m, 2H), 0.87 (t, J = 7.4 Hz, 3H); 13C NMR
(125 MHz, DMSO‑d6) δ 147.78, 146.28, 145.34, 142.72, 141.58,
140.60, 137.93, 131.60, 131.43, 130.13, 129.45, 124.67, 123.47,
120.52, 112.40, 66.99, 50.22, 49.55, 49.19, 19.27, 11.32; HRMS-QTOF
(ESI): m/z calcd. for [M + H]+ C21H24N5O 362.1980; found 362.2023
1-(Pentylamino)− 3-(2-(quinoxalin-2-yl)− 1H-benzo[d]imidazol-1-yl)
propan-2-ol(9e). White solid; yield 60%; mp: 180–182 ◦ C; 1H NMR (500
MHz, DMSO‑d6) δ 9.81 (s, 1H), 8.24 – 8.18 (m, 2H), 7.99 – 7.93 (m, 2H),
7.82 (dd, J = 18.1, 8.0 Hz, 2H), 7.42 – 7.37 (m, 1H), 7.36 – 7.32 (m, 1H),
5.10 (dd, J = 14.1, 4.0 Hz, 1H), 4.83 (dd, J = 14.1, 8.2 Hz, 1H), 4.16 –
4.11 (m, 1H), 2.71 (dd, J = 11.9, 4.6 Hz, 1H), 2.62 (dd, J = 11.9, 6.7 Hz,
1H), 2.49 – 2.46 (m, 2H), 1.90 (s, 2H), 1.40 – 1.34 (m, 2H), 1.26 – 1.20
(m, 4H), 0.84 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz, DMSO‑d6) δ
148.01, 146.30, 145.64, 142.70, 141.52, 140.66, 137.98, 131.46,
131.40, 129.80, 129.48, 124.36, 123.19, 120.33, 112.63, 69.87, 53.65,
50.05, 49.82, 29.45, 22.51, 21.66, 14.39; HRMS-QTOF (ESI): m/z calcd.
for [M + H]+ C23H28N5O 390.2293; found 390.2344
1-(Octylamino)− 3-(2-(quinoxalin-2-yl)− 1H-benzo[d]imidazol-1-yl)
propan-2-ol (9f). White solid; yield 60%; mp: 193–195 ◦ C; 1H NMR (500
MHz, DMSO‑d6) δ 9.87 (s, 1H), 8.45 (s, 1H), 8.35 – 8.32 (m, 1H), 8.23 –
8.18 (m, 1H), 8.01 – 7.95 (m, 2H), 7.86 (dd, J = 13.7, 8.1 Hz, 2H), 7.47 –
7.42 (m, 1H), 7.39 – 7.35 (m, 1H), 5.87 (s, 1H), 5.09 (dd, J = 14.2, 4.2
Hz, 1H), 4.89 (dd, J = 14.2, 8.0 Hz, 1H), 4.49 (s, 1H), 3.25 (dd, J = 12.6,
2.3 Hz, 1H), 3.11 (dd, J = 12.6, 10.1 Hz, 1H), 2.88 (t, J = 8.0 Hz, 2H),
1.65 – 1.50 (m, 2H), 1.25 (dd, J = 14.9, 7.8 Hz, 4H), 1.22 (s, 6H), 0.85 (t,
J = 6.9 Hz, 3H); 13C NMR (125 MHz, DMSO‑d6) δ 147.75, 146.27,
145.34, 142.69, 141.56, 140.59, 137.92, 131.62, 131.42, 130.14,
15
Journal of Molecular Structure 1292 (2023) 136184
129.46, 124.69, 123.48, 120.51, 112.43, 66.99, 50.18, 49.55, 47.53,
31.57, 28.85(2C), 26.34, 25.67, 22.50, 14.40; HRMS-QTOF (ESI): m/z
calcd. for [M + H]+ C26H34N5O 432.2763; found 432.2771
4.2. Pharmacology
4.2.1. Cell culture
The HCT116 (Human Colorectal Carcinoma), A549 (Human Lung
Adenocarcinoma), SK-ML (Human Melanoma), B16F10 (Murine Mela­
noma), HEK-293 (Human Embryonic Kidney), and HaCaT (Human
Keratinocyte) cell lines, were obtained from National centre for Cell
Science, Pune, India. Cells were maintained in appropriate media
enriched with 10% fetal bovine serum (FBS) and stabilized with 1%
antibiotic-antimycotic solution (Sigma-Aldrich). Cells were incubated at
37 ◦ C and subcultured after attaining 80–90% confluency using 0.25%
trypsin/1 mM EDTA solution for further passage. 10 mM stock solution
was prepared and diluted with respective media to obtain the required
concentrations for both compounds and standard by dissolving in
DMSO.
4.2.2. MTT assay
MTT assay is a colorimetric, sensitive, and reliable study used to
determine cell viability. Metabolically active cells reduce MTT 3-(4,5-
dimethylthiazol-2-yl)− 2,5-diphenyl tetrazolium bromide) into insol­
uble formazan, which is measured as the level of absorbance denoting
the viability of cells. Initially, cells were seeded in 96-well plates with
the medium at a density of 1000–4000 per well in 100 µl and incubated
overnight to grow and adhere. Then, the old media was replaced with
fresh media with different compound concentrations and incubated for
48 h. The treatment media was removed, and 100 µl of MTT (0.5 mg/
mL) was added and incubated at 37 ◦ C for 4 h. Then the supernatant was
removed, and formazan crystals formed were solubilized by the addition
of 100 µL of DMSO. The quantity of formazan was measured using a
multimode plate reader (Perkin Elmer, USA) at 570 nM wavelength.
4.2.3. Phase contrast microscopy
A549 cells were plated at a concentration of 40,000–50,000 cells/
well in a 24-well plate, and different concentrations of compound 5l
were added. Treated plates were incubated in a CO2 incubator for 48 h.
Later, cells were visualized using a microscope (Nikon, Inc. Japan)
under a bright field and captured the observed morphological changes at
20X magnification.
4.2.4. Acridine orange/ethidium bromide (AO/EB) staining
A549 cells were plated at a concentration of 40,000–50,000cells/
well in a 24-well plate, and different concentrations of compound 5l
were added. Treated plates were incubated in a CO2 incubator for 48 h,
followed by the addition of 10 µL of Acridine Orange and Ethidium
Bromide of 10 μg/ml each was added respectively. Cells were visualized
using a fluorescence microscope (Nikon, Inc. Japan with excitation (488
nm) and emission (550 nm) and images were captured at 20X
magnification.
4.2.5. 4,6-diamino-2-phenylindole (DAPI) staining
A549 cells were plated at a concentration of 40,000–50,000 cells/
well in a 24-well plate, and different concentrations of compound 5l
were added. Treated plates were incubated in a CO2 incubator for 48 h,
followed by washing with PBS and fixed with 4% paraformaldehyde for
15 min. After the removal of Paraformaldehyde, cells were per­
meabilized with 0.1% Triton X for 15 min and stained with DAPI(10 µg/
mL). Later, the staining solution was removed, and the wells were
washed with PBS. Cells were visualized using a fluorescence microscope
(Nikon, Inc. Japan) with excitation at 359 nm and emission at 461 nm
using a DAPI filter at 20X magnifications.
O. Ommi et al.
4.2.6. 2′,7′-Dichlorofluorescein diacetate (DCFDA) staining
A549 cells were plated at a concentration of 40,000–50,000cells/
well in a 24-well plate, and different concentrations of compound 5l
were added. Treated plates were incubated in a CO2 incubator for 48 h
and stained with DCFDA (10 µM) for 30 min at 37 ◦ C. Later, the staining
solution was removed, and wells were washed with PBS to remove the
excess dye. Further, cells were visualized using a fluorescence micro­
scope (Nikon, Inc. Japan) with excitation at 498 nm and emission at 530
nm and captured images at 20X magnifications.
4.2.7. Effect on mitochondrial membrane potential(DΨ m)
A549 cells were plated at 40,000–50,000 cells/well in a 24-well
plate, and different concentrations of compound 5l were added.
Treated plates were incubated in a CO2 incubator for 48 h and stained
with 5 μg/mL of 5,5′,6,6′-Tetrachloro-1,1′,3,3′ tetraethyl benzimidazole
carbocyanine iodide (JC1 dye) for 30 min at 37 ◦ C. Later, the staining
solution was removed, and the wells were washed with PBS to remove
the excess dye.. Further, fluorescence images were captured using a
fluorescence microscope at 20X magnification (Nikon, Inc. Japan).
4.2.8. Annexin V-FITC/Propidium iodide dual staining
A549 cells were plated at a concentration of 1 × 106 cells/well in a 6-
well plate, and different concentrations of compound 5l were added.
Treated plates were incubated in a CO2 incubator for 48 h, and cells were
trypsinized using trypsin-EDTA, followed by twice washing with ice-
cold PBS. Cells were centrifuged to obtain a pellet. Then the superna­
tant was removed from each tube, and the cells pellet was resuspended
in a 1 X binding buffer containing 5 μL and 1 μL of FITC-conjugated
annexin V and PI, respectively. The tubes were incubated for 15 min
in the dark as per the manufacturer’s protocol of the Apoptosis Detection
Kit (Invitrogen). Later, 400 μL of 1x binding buffer was added, and cells
were kept on ice for flow cytometric analysis using a flow cytometer (BD
FACSVerseTM, USA).
4.2.9. Cell cycle analysis
A549 cells were plated at a concentration of 1 × 106 cells/well in a 6-
well plate, and different concentrations of compound 5l were added.
Treated plates were incubated in a CO2 incubator for 48 h, and cells were
trypsinized using trypsin-EDTA, followed by twice washing with ice-
cold PBS. Cells were centrifuged and then fixed in 70% ethanol and
stored at 4 ◦ C overnight. Fixed cells were further centrifuged, and the
cells pellet was washed with cell cycle buffer before being stained with
Enzyme A and nuclear dye for 30 min at 37 ◦ C in the dark. Further, flow
cytometric analysis was performed using a flow cytometer (BD FACS­
VerseTM, USA).
4.2.10. Effect on tubulin polymerization
To examine the effect of compound 5l on tubulin inhibition, a
fluorescence-based in vitro tubulin polymerization assay was executed
based on the manufacturer’s protocol (Cytoskeleton, Inc. BK011).
Colchicine was used as a standard in the assay at 1.03 µM final con­
centration, and 2.19, 4.37, and 8.74 µM concentrations of compound 5l
were used for the assay. The 50% tubulin polymerization inhibitory
concentration of 5l was calculated based on the assay result.
4.2.11. Immunofluorescence assay
A549 cells were treated with compound 5l (4.37 µM) in a 4-cham­
bered slide for 48 h. cells were washed in phosphate-buffered saline,
fixed with 4% paraformaldehyde, and permeabilized using 0.1% triton-
X. After blocking with 0.3% bovine serum albumin, the primary anti­
body for beta-tubulin (1:200; Cell Signal Technology) and fluorescence-
labeled-secondary antibody was subsequently added to the cells. Cell
nuclei were stained by DAPI. Images were acquired with a Leica Laser
Scanning Spectral Confocal Microscope (Leica, Germany) with overlays
from separate images of tubulin (red) and nuclei (blue). For comparison,
we also included standards Colchicine (1.04 µM) and paclitaxel (3 µM)
16
Journal of Molecular Structure 1292 (2023) 136184
along with the 5l
5. Molecular docking
Molecular docking study was performed using the glide docking
module of the Schrödinger suite release 2021 [81]. The crystal co­
ordinates of α,β-tubulin subunits (PDB: 3E22) were obtained from the
RCSB. The protein was prepared using the Protein Preparation Wizard of
the Schrödinger suite by adding hydrogens, bond orders were assigned,
and water molecules were removed within 5 Å around the co-crystal.
The protein structure was minimized using the OPLS-5 force field. The
grid for docking was generated by taking the centroid of the co-crystal
and validated by re-docking of co-crystal to an RMSD of 0.92. Com­
pound 5l was drawn and prepared using LigPrep, and was docked using
the GLIDE module in the standard precision mode.
Author contributions
Ojaswitha Ommi: conceptualization, design, optimization, synthesis,
data analysis & interpretation and writing – original draft.
Shrilekha chilvery: biological studies
Priyanka Sudhir Dhopat: Synthesis
Anamika sharma: biological studies
Harshada Anil Bhalerao and Srinivas Reddy Dannaram: NMR and
mass studies
Srinivas Nanduri: Writing-review & editing
Rajesh Sonti: Supervision, writing – review & editing
Chandraiah Godugu: Supervision, Resources
Venkata Madhavi Yaddanapudi: Project administrator,
Supervision, Writing-review & editing
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
No data was used for the research described in the article.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.molstruc.2023.136184.
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