Facile synthesis of C6-substituted benz[4,5]imidazo[1,2-a]quinoxaline derivatives and their anticancer evaluation

Article abstract of DOI:10.1002/ardp.202000393

Cancer remains a leading cause of death worldwide, resulting in continuous efforts to discover and develop highly efficacious anticancer drugs. High-throughput screening of heterocyclic compound libraries is one of the promising approaches that provided several new lead molecules with a novel mechanism of action. On the basis of the promising anticancer potential of imidazoquinoxaline as well as the structurally similar imidazoquinoline-derived scaffold, we prepared a set of C6-substituted benzimidazo[1,2-a]quinoxaline derivatives via two novel synthetic routes using commercially available starting materials, with good to excellent yields and evaluated their anticancer activity against the NCI-60 cancer cell lines. The one-dose (10 μM) anticancer screening of the synthesized compounds in the NCI-60 cell line panel revealed that the substituents have a significant role in the activity. In particular, the indole (7f), imidazole (7g),?and benzimidazole (7h) derivatives showed significant activity against the triple-negative breast cancer cell line, MDA-MB-468. The lead compounds also exhibited notable IC₅₀ values against another breast cancer cell line, MCF-7. Furthermore, it was observed that these compounds were relatively nontoxic to normal cell lines: HEK293 (human embryonic kidney cell line) and MCF12A (nontumorigenic human breast epithelial cell line). The IC₅₀ values against healthy cells were at least 5- to 11-fold higher, offering a new class of heterocycles that can be further developed as promising therapeutics for cancer treatment.

Full text of DOI:10.1002/ardp.202000393

Received: 16 October 2020
Revised: 17 February 2021
Accepted: 19 February 2021
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DOI: 10.1002/ardp.202000393
F U L L P A P E R
Facile synthesis of C6‐substituted benz[4,5]imidazo[1,2‐a]
quinoxaline derivatives and their anticancer evaluation
Rahul Singh1 | Ravinder Kumar1 | Mallesh Pandrala2 | Parleen Kaur1
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Saloni Gupta3 | Dhanir Tailor2 | Sanjay V. Malhotra2 | Deepak B. Salunke1,4
1Department of Chemistry and Centre for
Advanced Studies in Chemistry, Panjab
University, Chandigarh, India
Abstract
Cancer remains a leading cause of death worldwide, resulting in continuous efforts
to discover and develop highly efficacious anticancer drugs. High‐throughput
screening of heterocyclic compound libraries is one of the promising approaches
that provided several new lead molecules with a novel mechanism of action. On the
basis of the promising anticancer potential of imidazoquinoxaline as well as the
structurally similar imidazoquinoline‐derived scaffold, we prepared a set of
C6‐substituted benzimidazo[1,2‐a]quinoxaline derivatives via two novel synthetic
routes using commercially available starting materials, with good to excellent yields
and evaluated their anticancer activity against the NCI‐60 cancer cell lines. The one‐
dose (10 µM) anticancer screening of the synthesized compounds in the NCI‐60 cell
line panel revealed that the substituents have a significant role in the activity. In
particular, the indole (7f), imidazole (7g), and benzimidazole (7h) derivatives showed
significant activity against the triple‐negative breast cancer cell line, MDA‐MB‐468.
The lead compounds also exhibited notable IC50 values against another breast
cancer cell line, MCF‐7. Furthermore, it was observed that these compounds were
relatively nontoxic to normal cell lines: HEK293 (human embryonic kidney cell line)
and MCF12A (nontumorigenic human breast epithelial cell line). The IC50 values
against healthy cells were at least 5‐ to 11‐fold higher, offering a new class of
heterocycles that can be further developed as promising therapeutics for cancer
treatment.
2Department of Cell, Developmental and
Cancer Biology, Center for Experimental
Therapeutics, Knight Cancer Institute, Oregon
Health and Science University, Portland,
Oregon, USA
3Department of Human Biology, University of
Toronto, St. George Campus, Toronto,
Ontario, Canada
4National Interdisciplinary Centre of Vaccine,
Immunotherapeutics and Antimicrobials,
Panjab University, Chandigarh, India
Correspondence
Sanjay V. Malhotra, Department of Cell,
Developmental and Cancer Biology, Center
for Experimental Therapeutics, Knight Cancer
Institute, Oregon Health and Science
University, Portland, OR 97201, USA.
Email: malhotsa@ohsu.edu
Deepak B. Salunke, Department of Chemistry
and Centre for Advanced Studies in
Chemistry, Panjab University, Chandigarh
160014, India.
Email: salunke@pu.ac.in
Funding information
Department of Biotechnology, Ministry of
Science and Technology, New Delhi, India,
Grant/Award Number: BT/RLF/Re‐entry/
16/2013
K E Y W O R D S
anticancer agents, benzimidazole, breast cancer cell line, heterocycles, MDA‐MB‐468, NCI‐60,
quinoxaline
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1
INTRODUCTION
demonstrate a need for the development of effective treatment for
cancer patients. Tremendous efforts have been invested in both
Despite several advances toward the discovery of oncology drugs
helping many patients to live longer,[1] cancer remains a leading
cause of death, with an estimated 18.1 million new cases and 9.6
million deaths worldwide in 2018.[2,3] These statistics clearly
academia and the pharmaceutical industry for decades to discover
and develop highly efficacious cancer drugs that have negligible side
effects. Several new approaches such as nanomedicine, extracellular
vesicles, natural antioxidants, immunotherapy, gene therapy, thermal
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Arch Pharm. 2021;e2000393.
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ablation, radiomics, pathomics, and targeted therapy are being in-
vestigated to find a better treatment.[4] Similarly, high‐throughput
screening of new heterocyclic compound libraries to find new scaf-
folds and/or modification of the structure of the existing drugs to
identify new cancer therapeutics are used routinely to develop po-
tentially effective anticancer agents.[5,6]
imidazoquinoxaline core by incorporation of functional groups
drastically affected the activity observed for EAPB02203 and
EAPB0503; therefore, we hypothesized that the modification/elon-
gation of this heterocyclic core structure could also impact the ac-
tivity. Inspired by the applications of imidazoquinoxaline containing
therapeutic agents, we envision that chemical modifications of this
core structure could provide suitable physicochemical properties
required for their biological activities; therefore, developing
structure–activity relationship (SAR) around the benzimidazo[1,2‐a]
quinoxaline chemotype (7, Figure 1) would be a feasible strategy.
Heterocyclic skeletons appear in a wide range of pharmaceu-
ticals. More than 60% of drugs that were approved by the US Food
and Drugs Administration (FDA) between 2010 and 2015 contained
at least one heterocyclic ring.[7] Among various heterocycles,
imidazoquinoxaline[8–10] and the structurally similar imidazoquino-
line core structure have drawn our attention, as these structures
were observed in various bioactive compounds and approved drugs.
For example, imiquimod (1, Figure 1) is the first member of a new
class of immune response modifiers that simulates a localized im-
mune response, and it was approved by the FDA in 1997 to treat
infectious skin conditions.[11–13] Imiquimod was also reported to
show a promising in vivo anticancer activity in mice bearing tumors
(colon carcinomas, melanomas, lung sarcomas, mammary and bladder
carcinomas).[14] Another immune response modifier, resiquimod (2),
an analog of imiquimod, has shown similar potency like
imiquimod.[13] The imidazoquinoxaline analog, BMS‐345541 (3),
showed significant cytotoxicity on melanoma.[15] Furthermore,
EAPB0203 (4) showed a significant in‐vitro activity (IC50 = 1.57 µM)
against A375 cells, a human melanoma cell line, and was found 45
times more effective than imiquimod.[16] Chemical modifications of 4
led to EAPB02303 (5), which showed excellent anticancer activity
(IC50 = 10 nM) against A375 cells.[17] In addition, EAPB0503 (6), an
analog of EAPB0203, demonstrated excellent cell growth inhibition
on human chronic myeloid leukemia (CML) cell lines. Most important,
EAPB0503 inhibited the proliferation of imatinib‐resistant CML cells,
offering promising therapeutic modalities.[18] Modification of the
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
Very few approaches to synthesize benzimidazo[1,2‐a]quinoxaline
scaffolds are known in the literature. In 2015, Sunke et al.[19] used a
two‐step strategy that involved quinoxaline‐directed Pd (or Ru)‐
catalyzed ortho C–H alkenylation, followed by subsequent hyperva-
lent iodine‐promoted intramolecular ortho C–H cycloamination of
initially synthesized N‐arylquinoxalin‐2‐amine derivatives. Huang
et al.[20] synthesized benzimidazo[1,2‐a]quinoxaline derivatives via
copper‐catalyzed Ullman coupling between 2‐(chloro)‐1H‐benzo[d]
imidazole and N‐(2‐iodophenyl)‐4‐methylbenzenesulfonamide, but it
suffers from the lack of facile routes to generate reactants. In 2014,
Timothy et al.[21] developed a visible light‐induced radical coupling
strategy to produce the benzimidazo[1,2‐a]quinoxaline framework,
which demands the preparation of 1‐(2‐isocyanophenyl)‐1H‐benzo[d]
imidazole via a tedious synthetic procedure, costly iridium complex
[fac‐Ir(ppy)3] (as photocatalyst) and phenyliodine(III) dicyclohex-
anecarboxylate. Reeves et al.[22] reported a copper‐catalyzed C–N
FIGURE 1 Chemical structures of
imidazoquinoxaline analogs of major biological
significance

SINGH ET AL.
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bond formation via cascade intramolecular dehydrative cyclization
reaction between 2‐formylbenzimidazole and 2‐iodoaniline to syn-
thesize the benzimidazo[1,2‐a]quinoxaline scaffold. Given the lim-
itation of these methods (lack of readily available starting materials,
expensive and tedious synthetic process), a novel method to syn-
thesize the benzimidazo[1,2‐a]quinoxaline chemotype is highly de-
sirable. We optimized two novel routes to synthesize benzimidazo
[1,2‐a]quinoxaline from commercially available starting materials in
good to excellent yields (Scheme 1) and evaluated their anticancer
activity against the NCI‐60 cancer cell lines.
SCHEME 2 Synthesis of compounds 7a–i. Reagents and
conditions: (a) DMF, POCl3, 0–110°C, 4 h; (b) for 7a, NaOMe, MeOH,
60°C, 2 h; for 7b–h, benzylamine or butylamine or octylamine or
pyrrole or indole or imidazole or benzimidazole, DMSO, K2CO3,
60–80°C, 3–6 h; for 7i, guanidine, NaH, 1,4‐dioxane, 70°C, 3 h
In the first approach (Route I), o‐phenylenediamine 8 was re-
acted with methyl 2,2,2‐trichloroacetimidate at 0°C in the presence
of acetic acid to generate 2‐(trichloromethyl)‐1H‐benzo[d]imidazole
9, which on treatment with K2CO3 in methanol resulted in the for-
mation of methyl‐1H‐benzo[d]imidazole‐2‐carboxylate 10 in 90%
yield. Introduction of the methyl ester functionality was confirmed
by the observation of a peak at 1731 cm−1 in the Fourier transform
infrared (FT‐IR) spectra and a singlet that represents three protons
at 3.95 δ ppm in 1H NMR (nuclear magnetic resonance). Compound
10 was then coupled with 1‐fluoro‐2‐nitrobenzene via a nucleophilic
aromatic substitution reaction using K2CO3 as a base to produce
compound 11 as a pale‐yellow solid in good yield. This reaction also
worked well with other bases such as Et3N or Na2CO3 or Cs2CO3.
The nitro reduction using zinc dust and ammonium chloride at 0°C
resulted in the formation of a new product, and within 30 min, a new
spot was observed on thin‐layer chromatography (TLC). As expected,
the in‐situ generated primary amine underwent intramolecular cy-
clization to furnish compound 12 in 70% yield (confirmed by NMR
and mass spectrometry). The nitro group reduction in compound 11
using Pd/C in the presence of hydrogen gas also resulted in the
formation of the desired cyclic product 12 without much improve-
ment in the yield.
In the second approach (Route II), a commercially available
benzimidazole was reacted with 1‐fluoro‐2‐nitrobenzene in the
presence of K2CO3 and dimethyl sulfoxide (DMSO) to produce
2‐nitro‐phenyl‐benzimidazole intermediate 13, which was reduced
using zinc dust and ammonium chloride to obtain 14. Compound 14
was then reacted with 1,1‐carbonyldiimidazole in o‐xylene at 140°C
to furnish the desired compound 12 in 75% yield. Our initial ap-
proach wherein compound 12 was treated with two equivalents of
POCl3 (neat) at refluxing condition resulted in charring of the
starting material after 1 h of heating. However, increasing the
amount of POCl3 (10 equivalents) allowed the reaction mixture to
reflux for 3 h, which resulted in the formation of the desired product,
as observed on TLC. Although the TLC showed complete consump-
tion of the reactant within 3 h, the basic workup at low temperature,
followed by a column chromatographic purification, resulted in a
very low yield of the desired product. Finally, as depicted in
Scheme 2, reaction of compound 12 with 10 equivalents of a 3:1
mixture of POCl3 and DMF at 110°C yielded 80% of the desired
compound 15.
SCHEME 1 The two novel routes to synthesize benzimidazo[1,2‐a]quinoxaline. Reagents and conditions: (a) CCl3C(═NH)OCH3, CH3COOH,
0°C to rt, 5–8 h; (b) K2CO3, MeOH, reflux, 16 h; (c) 1‐fluoro‐2‐nitrobenzene, K2CO3, DMSO, 60°C, 8 h; (d) NH4Cl, Zn, 0°C, 30 min; (e) CDI,
o‐dichlorobenzene, 140°C, 4 h

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The chloro functionality in compound 15 was then replaced by a
wide array of substituents using sodium methoxide, aliphatic amines,
nitrogen‐containing aromatic heterocycles (such as pyrrole, indole,
imidazole, and benzimidazole), and guanidine. Using appropriate
conditions and the procedure shown in Scheme 2, a library of
benzimidazo[1,2‐a]quinoxaline analogs was synthesized in about 83%
yield. The final compounds were then purified by column chroma-
tography using 230–400 mesh silica gel. The products were con-
firmed by IR, 1H NMR, 13C NMR, and mass spectrometric analysis.
The purity of the synthesized library was determined by high‐
performance liquid chromatography (HPLC).
shown a significant activity against breast cancer cell lines, especially
against triple‐negative breast cancer (TNBC), MDA‐MB‐468. Breast
cancer is the most commonly diagnosed cancer and the second
leading cause of death in women,[27] with the highest mortality rate
in TNBC. TNBC can be treated with chemotherapy (adriamycin,
cyclophosphamide, paclitaxel, docetaxel, and their combinations) and
it is more likely to recur than the other subtypes of breast cancer,[28]
warranting the development of new therapeutics specifically tar-
geting this breast cancer subtype. A very interesting SAR was ob-
served in preliminary studies. It was observed that replacing the C‐6
chloride functionality by a methoxy group resulted in loss of activity
(7a did not show any comparable activity as 15 against the four cell
lines, which were sensitive to 15). Furthermore, replacing the
chloride group with aliphatic amines was also ineffective (compounds
7b, 7c, and 7d were less potent than 15). The activity was retained in
compounds that were synthesized by substituting the chloride group
with nitrogen‐containing heterocyclic rings. Moreover, the activity
was increased by increasing the number of nitrogen atoms in the
heterocyclic ring. For example, replacing the chloride group with
pyrrole (7e) did not show any significant inhibition against the NCI‐
60 cell lines, but the structurally similar imidazole analog (7g), which
has two nitrogen atoms in the ring, exhibited improved efficacies
against several cell lines. 7g showed an improved activity over 15,
which inhibited more than 70% of NCI‐H522 cell growth, whereas 15
inhibited around 48% cells. The inhibitory effect of compound 15
was also observed on other non‐small‐cell lung cancer cells such as
HOP‐62 and NCI‐H460. Furthermore, 7g inhibited more than 70% of
NCI‐H522 and SK‐OV‐3 cells and inhibited more than 50% of SF‐
268, SNB‐19 (CNS cancer), OVCAR‐8, NCI/ADR‐RES, and BT‐549
(breast cancer). Interestingly, although 7g showed an improved ac-
tivity as compared with 15, it did not inhibit the growth of
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2.2
Biological studies
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2.2.1
Cell viability
The series of benzimidazo[1,2‐a]quinoxaline compounds shown in
Figure 2 was screened through the NCI‐60 panel of cell lines.[23–26]
The panel is organized into nine subpanels representing diverse
histologies: leukemia, melanoma, lung, colon, kidney, ovary, prostate,
breast, and central nervous system. The two‐stage screening process
started with the evaluation of all the compounds against the 60
human tumor cell lines at a single dose of 10.0 µM; cisplatin was used
as a positive control. The output was processed and is reported as a
percentage inhibition heatmap in Figure S1.
NCI‐60 data suggested that compound 15 showed significant
inhibition against several cell lines. It inhibited 48% of NCI‐H522
(non‐small‐cell lung cancer), 62% of OVCAR‐3 (ovarian cancer), 59%
and 70% of MCF‐7 and MDA‐MB‐468 (breast cancer) cell lines, re-
spectively. The results were encouraging, as compound 15 had
FIGURE 2 A library of benzimidazo[1,2‐a]quinoxalines

SINGH ET AL.
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MDA‐MB‐468. In contrast, replacing the chloride group with benzi-
midazole (7h) inhibited more than 90% of MCF‐7 and MDA‐MB‐468
(breast cancer) cells, but showed no effect on NCI‐H522. Further-
more, 7h inhibited 79% of HCC‐2998 and 50% of HCT‐15, HT29, and
KM12 (colon cancer) cells. Replacing chloride with guanidine (7i)
further improved the activity; most of the cells tested were inhibited
significantly at the given concentration. It is worth noting that 7i
inhibited more than 90% of MOLT‐4 (leukemia), NCI‐H460, and
MCF‐7 (breast cancer) cell growth. In addition, it also inhibited more
than 75% of the growth of most of the cells that were tested in this
panel. Overall, the activity of the benzimidazoquinoxaline scaffold
was increased by introducing nitrogen groups on the core moiety and
was found to be related to the increasing number of nitrogen atoms.
It was also observed that the structural modifications resulted in the
variable activity of the compounds against different cell lines.
Although the benzimidazoquinoxaline analogs showed a sig-
nificant anticancer activity, confirmation about the selective toxicity
of these compounds toward the cancer cell lines was critical. To
determine this, we used HEK293 cells (an embryonic human kidney
cell line) and tested all the compounds at 10 μM concentration
(Figure S1). It was observed that, except 7i, all compounds were
found to be relatively nontoxic and inhibited the growth of HEK293
cells only moderately (0–25%) at the tested concentration. Com-
pound 7i inhibited the growth of the maximum number of cancer cell
lines in the NCI‐60 panel, but it also showed high toxicity against
HEK‐293 at 10 μM.
TABLE 1 The IC50 values of the selected benzimidazoquinoxalines
(15, 7f, 7g, 7h, and 7i) against MDA‐MB‐231 and MDA‐MB‐468
(triple‐negative breast cancer cell lines), MCF7 (breast cancer cell line),
and MCF12A (non‐tumorigenic human breast epithelial cell line)
Cell lines (IC50 in µM)
MDA‐
MDA‐
MB‐468
S. No. Compound MB‐231
MCF‐7 MCF12A
1
2
3
4
5
6
15
49.97
15.88
25.57
16.8
24.09
17.28
5.24
3.2
26.74
7.85
18.77
2.59
1.55
4.23
138.1
44.28
45.32
32.07
1.04
–
7f
7g
7h
7i
14.62
3.62
4.78
1.43
Cisplatin
Note: IC50 is the concentration (μM) of the compound required to inhibit
cell viability by 50%. Cisplatin was used as a positive control.
activity at least against one of the breast cancer cell lines. The ac-
tivity of these compounds may be due to the replacement of the
chloride group by a nitrogen‐containing heterocyclic ring. The deri-
vatives in which the heterocyclic ring contains two nitrogen atom‐
containing heterocycles attached to the benzimidazoquinoxaline
core exhibited a substantial activity against two breast cancer cell
lines. Therefore, we investigated the efficacies of these compounds
against two TNBC cell lines, MDA‐MB‐231 and MDA‐MB‐468. In
addition, the MCF‐7 cell line and also a nontumor breast epithelial
cell line MCF‐12A (Figure 3) were also included in this study. The cell
The one‐dose (10 μM) anticancer screening of compounds 7a–i
revealed that substituents significantly influence the activity. Among
the synthesized compounds, 7f, 7g, 7h, and 15 showed a significant
FIGURE 3 Percent cell viability, measured using an MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay, of two
TNBC (triple‐negative breast cancer) (MDA‐MB‐231 and MDA‐MB‐468), MCF7, and a nontumor breast (MCF12A) cell line after 48 h of
treatment with 7f, 7g, 7h, 15, and 7i. The percent viability was calculated with respect to dimethyl sulfoxide vehicle control‐treated cells, with
100% viability indicating the same number of viable cells as for the control sample and 0% viability indicating complete cell death

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lines were treated with the imidazoquinoxaline analogs to evaluate
their IC50 values, and the results are summarized in Table 1.
Interestingly, compounds 7f and 7h exhibited notable IC50 va-
lues against the MCF‐7 cell line, that is, inhibiting 50% growth of the
cells at 7.85 and 2.59 μM concentrations, respectively. In addition,
compounds 7g and 7h exhibited significant IC50 values against MDA‐
MB‐468, inhibiting 50% cell growth at 5.24 and 3.2 µM, respectively.
However, 7g and 7h were found to possess poor activity against
another TMBC cell line, MDA‐MB‐231. Compound 7i showed
IC50 values of 4.78 and 1.55 µM against MDA‐MB‐468 and MCF7,
respectively, but it was also found to be toxic against HEK293
(a normal cell line) as well as MCF12A, a nontumorigenic human
breast epithelial cell line (IC50 of 1.04 µM). Furthermore, the selected
HITs (7f, 7g, and 7h) were nontoxic to the normal cell line, and the
IC50 value against healthy cells was at least 5‐ to 11‐fold higher,
suggesting the possibility of these HITs for further development as
promising therapeutics for cancer treatment.
iodine. Compounds were purified by flash column chromatography
using 230–400 mesh silica gel, where the eluents used for column
chromatography were ethyl acetate/hexanes or MeOH/di-
chloromethane systems. 1H and 13C spectra (see the Supporting In-
formation) were recorded on a Bruker Avance‐II 400 or 500 MHz
spectrophotometer in CDCl3 and/or DMSO‐d6. Chemical shifts are
reported in parts per million (ppm), relative to tetramethylsilane as
the internal standard. The following abbreviations were used in re-
porting spectra: s (singlet), d (doublet), t (triplet), m (multiple), and br
(broad). The purity of synthesized compounds was confirmed using
HPLC. IR spectra were recorded on a Thermo Scientific Nicolet 50
FT‐IR system. High‐resolution mass spectrometry was carried out
using a Waters Q‐TOF Micromass System. MDA‐MB‐231, MDA‐MB‐
468, MCF7, and MCF12A cells were purchased from ATCC, USA.
MDA‐MB‐231, MDA‐MB‐468, and MCF7 cells were cultured
in DMEM media supplemented with 10% FBS and 1%
antibiotic–antimycotic solution at 37°C with 5% CO2. MCF12 cells
were cultured in mammary epithelial cell growth media (MEGM Kit
Catalog No. CC‐3150; Lonza) at 37°C with 5% CO2.
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3
CONCLUSIONS
The InChI codes of the investigated compounds, together with some
biological activity data, are provided as Supporting Information.
We developed a facile route to synthesize a library of C6‐substituted
benzimidazo[1,2‐a]quinoxaline analogs in high yields using commercially
available cheap raw materials. The preliminary anticancer screening on
the NCI‐60 panel has identified new compounds that showed significant
activity against several cancer cell lines. In particular, 7f and 7h exhibited
notable activities against MCF‐7 breast cancer cell lines, whereas 7g and
7h demonstrated significant activity against a TNBC cell line, MDA‐MB‐
468. These compounds were also observed to be nontoxic to normal
cells, providing a scope to explore more analogs of this class to discover
more potent anticancer drugs.
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4.1.2
Synthesis and characterization of the
compounds
Methyl‐1H‐benzo[d]imidazole‐2‐carboxylate (10)
To a solution of o‐phenyldiamine 8 (250 mg, 2.31 mmol) in acetic acid
(3 ml), methyl 2,2,2‐trichloroacetimidate (315 µl, 2.54 mmol) was
added dropwise at 0°C with vigorous stirring, and the final reaction
mixture was stirred at room temperature for 5 h. After completion of
the reaction (monitored by TLC), crushed ice was added into the
reaction mixture and stirred for 20 min further. The off‐white pre-
cipitate formed was filtered, washed with water (3 × 100 ml), and
dried in an oven to get 2,2,2‐trichloromethyl benzimidazole 9 in 80%
yield (430 mg). Rf = 0.80 (3%, MeOH‐DCM); 1H NMR (400 MHz,
DMSO‐d6) δ 13.43 (s, 1H), 7.67 (s, 2H), and 7.35 (dd, J = 6.1, 3.2 Hz,
2H); 13C NMR (101 MHz, DMSO‐d6) δ 151.1, 138.8, 124.3, 116.7,
and 89.3. To a solution of compound 9 (2.2 g, 9.21 mmol) in 50 ml of
methanol, potassium carbonate (2.5 g, 18.42 mmol) was added, and
the reaction mixture was refluxed at 80°C for 16 h. After the com-
pletion of the reaction, the solvent was evaporated and the product
was extracted in ethyl acetate. The organic layer was then dried over
anhydrous Na2SO4 and evaporated at reduced pressure to afford
methyl‐1H‐benzo[d]imidazole‐2‐carboxylate 10 as an off‐white solid.
Yield: 90% (1.46 g); Rf = 0.25 (4%, MeOH‐DCM); (Neat, ν cm−1): 1731,
1504, 1439, 1260, 1205, 1022, and 745; 1H NMR (500 MHz,
DMSO‐d6) δ 13.52 (s, 1H), 7.70 (s, 2H), 7.33 (s 2H), and 3.94 (s, 3H);
13C NMR (125 MHz, DMSO‐d6) δ 159.8, 142.9, 141.6, 134.5, 125.1,
123.2, 120.9, 112.8, and 52.7; high‐resolution mass spectrometry
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4
EXPERIMENTAL
Chemistry
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4.1
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4.1.1
General
Commercially available reagents and solvents were purchased from
various sources and used without further purification. Reagents such
as methyl‐2,2,2‐trichloroacetimidate and octylamine were obtained
from Sigma‐Aldrich, whereas 1‐fluoro‐2‐nitrobenzene, sodium
methoxide, guanidine hydrochloride, sodium hydride, butylamine,
imidazole, indole, pyrrole, sodium sulfate, phosphoryl chloride, and
dimethylformamide (DMF) were purchased from Avra Synthesis.
Benzimidazole, benzylamine, and 1,1ʹ‐carbonyldiimidazole were ob-
tained from Spectrochem, Qualikems, and CDH, respectively. Alter-
natively, zinc dust, potassium carbonate, and ammonium chloride
were obtained from Fisher Scientific, and sodium carbonate, DMSO,
and dioxane were obtained from Finar Chemicals. TLC was per-
formed on Merck silica gel F254 aluminum sheets and visualized
under UV light at 254 or 360 nm or by staining with ninhydrin or
(HRMS) m/z: Calculated for C9H8N2NaO2 [M+Na]+ 199.04780,
found 199.04875; calculated for C9H9N2O2 [M+H]+ 177.06585,
+
+
found 177.06750.

SINGH ET AL.
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Methyl 1‐(2‐nitrophenyl)‐1H‐benzo[d]imidazole‐2‐carboxylate (11)
To a solution of 1‐fluoro‐2‐nitrobenzene (1.0 g, 7.085 mmol) in
DMSO (10 ml), compound 10 (1.37 g,7.79 mmol) and K2CO3 (1.95 g,
14.18 mmol) were added, and the reaction mixture was stirred at
60°C for 8 h. After the completion of the reaction (monitored by
TLC), crushed ice was added, and the mixture was stirred for another
30 min. The precipitate formed was filtered, dried, and purified using
flash column chromatography to produce 1‐(2‐nitrophenyl)‐1H‐
benzo[d]imidazole‐2‐carboxylate 11 as a pale‐yellow solid. Yield:
50% (1.05 g); Rf = 0.45 (5%, MeOH/DCM); IR (neat, ν cm−1): 3043,
2958, 2864, 1720, 1607, 1502, 1441, 1349, 1219, 848, and 745; 1H
NMR (500 MHz, CDCl3) δ 8.31 (dd, J = 8.5, 1.5 Hz, 1H), 7.98–7.97 (m,
1H), 7.85 (ddd, J = 8, 7.5, 1.5 Hz, 1H), 7.77–7.74 (m, 1H), 7.51 (dd,
J = 8, 1.5 Hz, 1H), 7.44–7.37 (m, 2H), 7.02 (dd, J = 7.5, 1.5 Hz, 1H), and
7.92 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 159.7, 145.7, 141.9,
140.8, 137.0, 134.4, 130.6, 130.5, 130.4, 110.6, and 53.1. Electro-
2‐(1H‐Benzo[d]imidazol‐1‐yl)aniline (14)
To a heterogeneous solution of 13 (0.4 g, 1.35 mmol) and Zn dust
(0.9 g, 13.46 mmol) at 0°C, ammonium chloride (0.72 g, 13.46 mmol)
was added in portions, and the resultant mixture was stirred for
30 min at 0°C. Upon completion of the reaction (monitored by TLC,
ninhydrin stain), Zn was filtered out on a celite bed and washed with
copious amounts of methanol (100 ml). The methanol layer was
evaporated at reduced pressure and the solid obtained was ex-
tracted in dichloromethane. The organic layer was dried over anhy-
drous Na2SO4 and evaporated to yield compound 14 as an off‐white
solid. Yield 90% (0.25 g). Rf = 0.50 (5%, MeOH‐DCM). IR (neat,
ν cm−1): 3342, 1634, 149, 1472, 1306, 1278, 1231, 1157, and 747;
1H NMR (500 MHz, CDCl3) δ 8.16 (s, 1H), 7.97 (d, J = 7.8 Hz, 1H),
7.38–7.30 (m, 3H), 7.28–7.26 (m, 1H), 7.18 (dd, J = 7.8, 1.4 Hz, 1H),
6.94–6.86 (m, 2H), and 3.64 (s, 2H); 13C NMR (126 MHz, CDCl3)
δ 143.7, 142.8, 133.9, 130.6, 128.3, 124.1, 123.3, 120.9, 120.4, 118.9,
116.8, and 111.2.
spray ionization‐mass spectrometry (ESI‐MS) m/z: Calculated for
+
C
15H12N3O4 [M+H]+ 298.0822, found 298.3420.
Benzo[4,5]imidazo[1,2‐a]quinoxalin‐6(5H)‐one (12) via Route II
To a solution of 14 (3.5 g, 16.7 mmol) in o‐dichlorobenzene (3 ml),
1,1‐carbonylimidazole (4.1 g, 25 mmol) was added, and the reaction
mixture was stirred at 140°C for 4 h. After the completion of the
reaction, the reaction mixture was cooled to 0°C and further diluted
with hexanes (80 ml) to precipitate the desired product. The pre-
cipitate obtained was further washed with hexanes (100 ml), fol-
lowed by 500 ml of water, and dried in an oven at 80°C to furnish
product 12 in 75% yield (2.94 g). The analytical data obtained were
similar to that obtained earlier from Route I.
Benzo[4,5]imidazo[1,2‐a]quinoxalin‐6(5H)‐one (12) via Route I
In a heterogeneous solution of 11 (1.6 g, 5.40 mmol) and Zn dust
(3.5 g, 54.05 mmol) in methanol (20 ml), ammonium chloride (2.8 g,
54.05 mmol) was added in portions at 0°C (reaction was exothermic).
The resultant reaction mixture was then stirred at 0°C for 30 min,
and after the completion of the reaction (monitored by TLC, ninhy-
drin stain), Zn dust was filtered out on a bed of celite and washed
with methanol (500 ml). The methanol layer was evaporated and the
solid obtained was washed with water (500 ml) and dried in an oven
to get compound 12 as a creamy white solid. Yield: 70% (0.89 g);
Rf = 0.40 (5%, MeOH‐DCM); IR (neat, ν cm−1): 1683, 1446, 1388, 747,
and 730; 1H NMR (500 MHz, DMSO‐d6) δ 12.18 (s, 1H), 8.64 (d,
J = 8.4 Hz, 1H), 8.57 (d, J = 7.9 Hz, 1H), 8.03 (d, J = 7.7 Hz, 1H),
7.66–7.56 (m, 2H), and 7.49–7.38 (m, 3H); 13C NMR (126 MHz,
DMSO‐d6) δ 153.3, 143.1, 140.9, 131.0, 128.2, 126.0, 125.7, 124.9,
123.8, 123.5, 121.6, 116.8, 115.8, and 114.9; ESI‐MS m/z: Calculated
for C14H10N3O+ [M+H]+ 236.1, found 236.1.
6‐Chlorobenzo[4,5]imidazo[1,2‐a]quinoxaline (15)
To a suspension of 12 (700 mg, 2.97 mmol) in DMF (0.8 ml) at 0°C,
POCl3 (2.45 ml, 29.70 mmol) was added dropwise with vigorous
stirring. The reaction mixture was then stirred at 110°C for 4 h. After
the completion of the reaction (monitored by TLC), the reaction
mixture was cooled at room temperature and was added in portions
to an ice‐cold solution of saturated sodium bicarbonate with vigor-
ous shaking (reaction was exothermic). The product formed was
extracted in ethyl acetate, and the combined ethyl acetate extract
was dried over anhydrous Na2SO4, evaporated, and purified using
column chromatography to afford chloro product 15 as an off‐white
solid. Yield 70% (0.53 g); Rf = 0.45 (30%, ethyl acetate/hexanes); IR
(neat, ν cm−1): 1507, 1461, 1388, 1081, 843, and 748; 1H NMR
(500 MHz, CDCl3) δ 8.52 (dd, J = 8.4, 1.0 Hz, 1H), 8.43–8.40 (m, 1H),
8.23–8.21 (m, 1H), 8.13 (dd, J = 8.1, 1.4 Hz, 1H), 7.83–7.80 (m, 1H),
and 7.69–7.63 (m, 3H); 13C NMR (126 MHz, CDCl3) δ 144.7, 144.0,
1‐(2‐Nitrophenyl)‐1H‐benzo[d]imidazole (13)
To a solution of 1‐fluoro‐2‐nitrobenzene (2.0 g, 14.17 mmol) in
DMSO (14 ml), benzimidazole (1.9 g,17.01 mmol) and K2CO3 (3.9 g,
28.36 mmol) were added, and the reaction mixture was stirred at
60°C for 8 h. After the completion of the reaction (monitored by
TLC), crushed ice was added, and the precipitate formed was filtered
out and dried in an oven to give 1‐(2‐nitrophenyl)‐1H‐benzo[d]imi-
dazole 13 as a pale yellow solid. Yield: 95% (3.22 g); Rf = 0.55 (5%,
MeOH‐DCM); IR (neat, ν cm−1): 3081, 1526, 1347, and 743; 1H NMR
(500 MHz, CDCl3) δ 8.15 (dd, J = 8.2, 1.4 Hz, 1H), 8.02 (s, 1H), 7.88 (d,
J = 7.9 Hz, 1H), 7.82 (ddd, J = 8, 7.5, 1.5 Hz, 1H), 7.70 (ddd, J = 8.0, 7.5,
1 Hz, 1H), 7.58 (dd, J = 7.9, 1.3 Hz, 1H), 7.38–7.28 (m, 2H), and 7.14
(d, J = 7.9 Hz, 1H); 13C NMR (126 MHz, CDCl3) δ 145.7, 143.4, 142.5,
134.4, 134.3, 130.0, 129.8, 129.4, 126.2, 124.2, 123.2, 120.8, and
139.0, 134.6, 131.3, 130.6, 130.2, 129.6, 126.4, 126.3, 125.8, 122.9,
+
114.9, and 114.4; HRMS m/z: Calculated for
C
14H9ClN3 [M
+H]+ 253.0480, found 253.0484.
6‐Methoxybenzo[4,5]imidazo[1,2‐a]quinoxaline (7a)
To a solution of 15 (80 mg, 0.28 mmol) in methanol (5 ml) under
nitrogen atmosphere, sodium methoxide (90 mg, 1.65 mmol) was
added in one portion at room temperature. The reaction mixture was
+
109.3; ESI‐MS m/z: Calculated for C13H10N3O2 [M+H]+ 240.1,
found 240.1.

8 of 10
SINGH ET AL.
|
stirred at 60°C for 2 h. After the completion of the reaction, solvent
was evaporated on a rotatory evaporator to get crude reaction
mixture, which was purified by column chromatography to obtain
pure product as an off‐white solid. Yield: 46% (32 mg); Rf = 0.30
(ethyl acetate/hexanes, 30%); IR (neat, ν cm−1): 1564, 1525, 1437,
1396, 1315, 1282, 1227, 1004, 980, 919, and 741; 1H NMR
(500 MHz, CDCl3) δ 8.43 (dd, J = 8.2, 1.1 Hz, 1H), 8.41–8.37 (m, 1H),
8.16–8.12 (m, 1H), 7.94 (dd, J = 8.0, 1.5 Hz, 1H), 7.62–7.59 (m, 3H),
7.55–7.52 (m, 1H), and 4.33 (s, 3H); 13C NMR (126 MHz, CDCl3) δ
153.7, 144.0, 136.4, 134.3, 131.0, 128.7, 128.5, 126.6, 125.8, 125.3,
124.8, 122.4, 114.6, 114.0, and 54.3; HRMS m/z: Calculated for
3.76–3.70 (m, 2H), 1.82–1.75 (m, 2H), 1.53–1.45 (m, 2H), 1.42–1.28
(m, 8H), and 0.89 (t, J = 6.9 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ
147.9, 143.5, 137.1, 131.6, 127.4, 126.9, 125.8, 125.0, 124.3, 123.6,
121.3, 114.6, 114.4, 77.5, 77.4, 77.2, 76.8, 41.0, 32.00, 29.8, 29.6,
+
29.5, 29.4, 27.3, 22.8, and 14.2; HRMS m/z: Calculated for C22H27N4
[M+H]+ 347.2230, found 347.2229.
6‐(1H‐Pyrrol‐1‐yl)benzo[4,5]imidazo[1,2‐a]quinoxaline (7e)
White powder. Yield: 40% (0.031 g); Rf = 0.76 (30%, ethyl acetate/
hexanes); IR (neat, ν cm−1): 1513, 1473, 1411, 1386, 1386, 1360,
1328, 1101, 731, and 618; 1H NMR (500 MHz, CDCl3) δ 8.58
(t, J = 2.5 Hz, 2H), 8.302 (dd, J = 8.5, 1 Hz, 1H), 8.28 (dd, J = 7, 2 Hz,
1H), 8.08 (dd, J = 6.7, 2.3 Hz, 1H), 7.95 (dd, J = 8.0, 1.4 Hz, 1H),
7.58–7.51 (m, 3H), 7.49–7.46 (m, 1H), and 6.54–6.42 (t, J = 2.5 Hz,
2H); 13C NMR (126 MHz, CDCl3) δ 143.8, 141.9, 136.2, 134.2, 130.4,
129.6, 128.6, 127.8, 125.9, 125.6, 125.1, 122.4, 121.7, 114.3, 114.2,
C
15H12N3O+ [M+H]+ 250.0975, found 250.0976.
N‐Benzylbenzo[4,5]imidazo[1,2‐a]quinoxalin‐6‐amine (7b)
To a solution of 15 (70 mg, 0.27 mmol) in DMSO (2 ml), K2CO3
(75 mg, 0.55 mmol), and benzylamine (40 µl, 0.36 mmol) were added.
The reaction mixture was then stirred at 60°C for 4 h. After the com-
pletion of the reaction, the mixture was cooled to room temperature.
Then, crushed ice was added and stirred at room temperature for 2 h.
The precipitate obtained was filtered, washed with water, and dried in
an oven to obtain a crude product. The crude product was purified by
silica gel column chromatography using ethyl acetate/hexanes as eluent
to obtain compound 7b as an off‐white solid. Yield: 70% (61 mg); Rf =
0.6 (30%, ethyl acetate/hexanes); IR (neat, ν cm−1): 3222, 3031, 2917,
1556, 1503, 1458, 1397, 1311, 1245, 739, and 728; 1H NMR
(500 MHz, CDCl3) δ 8.38–8.33 (m, 2H), 8.01–7.97 (m, 1H), 7.84
(dd, J = 7.8, 1.7 Hz, 1H), 7.58–7.54 (m, 2H), 7.50–7.41 (m, 4H), 7.38–7.35
(m, 2H), 7.31–7.28 (m, 1H), 6.73 (t, J = 5.3 Hz, 1H), and 4.96
(d, J = 5.7 Hz, 2H); 13C NMR (126 MHz, CDCl3) δ 138.5, 136.9, 136.9,
131.7, 128.8, 128.4, 127.7, 127.6, 127.2, 125.9, 125.1, 124.4, 124.0,
and 112.0; HRMS m/z: Calculated for
C
18H12N4Na+ [M
+Na]+ 307.0954, found 307.0961.
6‐(1H‐Indol‐1‐yl)benzo[4,5]imidazo[1,2‐a]quinoxaline (7f)
Pale yellow solid. Yield = 40% (0.036 g); Rf = 0.70 (30%, ethyl acetate/
hexanes); IR (neat, ν cm−1): 1535, 1510, 1444, 1403, 1357, 1329,
1186, and 739; 1H NMR (400 MHz, CDCl3) δ 9.42 (d, J = 3.7 Hz, 1H),
8.95 (dd, J = 8.3, 0.6 Hz, 1H), 8.48 (dd, J = 8.3, 1.1 Hz, 1H), 8.44–8.40
(m, 1H), 8.17–8.12 (m, 2H), 7.70–7.58 (m, 5H), 7.42–7.38 (m, 1H),
7.32–7.29 (m, 1H), and 6.82 (dd, J = 3.7, 0.6 Hz, 1H); 13C NMR
(101 MHz, CDCl3) δ 143.8, 143.5, 136.8, 136.6, 134.5, 130.9, 130.7,
129.6, 129.5, 128.5, 127.8, 126.1, 125.7, 125.2, 123.8, 122.9, 122.4,
121.0, 116.7, 114.5, 114.4, and 107.1; HRMS m/z: Calculated for
+
C
22H15N4 [M+H]+ 335.1291, found 335.1291.
+
121.4, 114.7, 114.4, and 45.1; HRMS m/z: Calculated for C21H17N4
[M+H]+ 325.1448, found 325.1450.
6‐(1H‐Imidazol‐1‐yl)benzo[4,5]imidazo[1,2‐a]quinoxaline (7g)
Compounds 7c–h were synthesized following similar procedures
as mentioned for 7b, with the appropriate amines.
Off‐white solid. Yield = 83% (64 mg); Rf = 0.4 (30%, ethyl acetate/
hexanes); IR (neat, ν cm−1): 1519, 1506, 1447, 1409, 1339, 1194,
970, and 740; 1H NMR (500 MHz, CDCl3) δ 9.61 (s, 1H), 8.60
(t, J = 1.3 Hz, 1H), 8.54–8.52 (m, 1H), 8.48–8.43 (m, 1H), 8.20–8.17
(m, 1H), 8.12 (dd, J = 8.1, 1.4 Hz, 1H), 7.78–7.75 (m, 1H), 7.70–7.62
(m, 3H), and 7.30 (br, 1H); 13C NMR (126 MHz, CDCl3) δ 144.0,
140.4, 138.7, 135.6, 133.8, 130.7, 130.4, 130.2, 129.2, 129.1, 126.3,
126.2, 125.7, 122.7, 118.1, 114.7, and 114.4; HRMS m/z: Calculated
N‐Butylbenzo[4,5]imidazo[1,2‐a]quinoxalin‐6‐amine (7c)
Off‐white solid. Yield: 64% (50 mg); Rf = 0.7 (30%, ethyl acetate/
hexanes); IR (neat, ν cm−1): 3362, 2966, 2864, 1548, 1563, 1507,
1401, 1229, 1008, and 742; 1H NMR (500 MHz, CDCl3) δ 8.39–8.35
(m, 1H), 8.33 (dd, J = 8.1, 1.4 Hz, 1H), 8.04–8.00 (m, 1H), 7.82
(dd, J = 7.9, 1.2 Hz, 1H), 7.59–7.53 (m, 2H), 7.46–7.38 (m, 2H), 6.42
(s, 1H), 3.75 (dd, J = 16, 5.6 Hz, 2H) 1.81–1.75 (m, 2H), 1.57–1.50
(m, 2H), and 1.01 (t, J = 7.4 Hz, 3H); 13C NMR (126 MHz, CDCl3)
δ 148.0, 143.6, 137.1, 131.7, 127.4, 127.0, 125.9, 125.1, 124.3, 123.7,
121.3, 114.7, 114.5, 40.7, 31.7, 20.4, and 14.0; HRMS m/z: Calculated
+
for C17H12N5 [M+H]+ 286.1087, found 286.1093.
6‐(1H‐Benzo[d]imidazol‐1‐yl)benzo[4,5]imidazo[1,2‐a]‐
quinoxaline (7h)
Off‐white solid. Yield: 67% (61 mg); Rf = 0.5 (30%, ethyl acetate/
hexanes); IR (neat, ν cm−1): 1518, 1473, 1416, 1219, 1099, 835, 762,
and 749; 1H NMR (400 MHz, CDCl3) δ 10.37 (s, 1H), 8.83–8.80
(m, 1H), 8.41 (dd, J = 8.3, 1.0 Hz, 1H), 8.37–8.32 (m, 1H), 8.13–8.09
(m, 2H), 7.91 (dd, J = 6.8, 1.9 Hz, 1H), 7.68–7.53 (m, 4H), and 7.48–7.40
(m, 2H); 13C NMR (101 MHz, CDCl3) δ 144.8, 144.1, 143.7, 141.7,
135.7, 133.9, 132.7, 130.6, 129.9, 128.9, 128.5, 126.2, 126.0, 125.6,
124.8, 124.5, 122.5, 120.6, 116.7, 114.6, and 114.3; HRMS m/z:
+
for C18H19N4 [M+H]+ 291.1604, found 291.1609.
N‐Octylbenzo[4,5]imidazo[1,2‐a]quinoxalin‐6‐amine (7d)
Off‐white solid. Yield 80% (75 mg); Rf = 0.8 (30%, ethyl acetate/
hexanes); IR (neat, ν cm−1): 3415, 2917, 2847, 1567, 1401, 1499,
1401, 1270, 1241, 1229, and 740; 1H NMR (400 MHz, CDCl3) δ
8.35–8.28 (m, 2H), 8.01–7.99 (m, 1H), 7.80 (dd, J = 7.9, 1.6 Hz, 1H),
7.57–7.50 (m, 2H), 7.45–7.35 (m, 2H), 6.44 (t, J = 5.2 Hz, 1H),
+
Calculated for C21H14N5 [M+H]+ 336.1244, found 336.1246.

SINGH ET AL.
9 of 10
|
1‐(Benzo[4, 5]imidazo[1,2‐a]quinoxalin‐6‐yl)guanidine (7i)
to attach for 24 h. The cells were treated with respective compounds at
a concentration of 10 µM in fresh media for 48 h. Then, 50 µl of MTT
reagent prepared in phosphate‐buffered saline was added to each well
and incubated for 60–90 min. Media and MTT reagents were removed
and MTT formazan crystals were dissolved in 100 µl of DMSO. Ab-
sorbance was recorded at 370 nm using a multimode plate reader.
Statistical analysis and IC50 calculations were conducted with GraphPad
Prism 8 software. Quantitative data are presented as mean SD. Cis-
platin was used as a control to verify the assay conditions. The IC50
values for MCF‐7, MDA‐MB‐231, and MDA‐MB‐468 were found to be
4.23, 3.62, and 1.43 µM, respectively. These IC50 values match with the
reported values within acceptable error limits.[29]
Under nitrogen atmosphere, at room temperature, 60% sodium hy-
dride in mineral oil (355 mg, 8.90 mmol) was added to a stirred
mixture of guanidine hydrochloride (1.69 g, 17.8 mmol) in freshly
dried 1,4‐dioxane (5 ml). The reaction mixture was heated to 70°C and
stirred for 10 min. After cooling the reaction mixture to room tem-
perature, a solution of 15 (169 mg, 1.78 mmol) in 1,4‐dioxane (2 ml) was
added portion‐wise. The resultant reaction mixture was then stirred at
70°C for 3 h. After the completion of the reaction, the solvent was
evaporated on a rotatory evaporator to get a crude material, which was
purified by column chromatography using neutral alumina to furnish
pure product as an off‐white solid. Yield: 73% (120 mg); Rf = 0.2 (15%,
MeOH/DCM); IR (neat, ν cm−1): 3289, 1614, 1493, 1466, 1330, 1256,
and 738; 1H NMR (500 MHz, DMSO‐d6) δ 8.67–8.62 (m, 1H), 8.58–8.53
(m, 1H), 7.98–7.95 (m, 1H), 7.71–7.69 (m, 1H), 7.57–7.53 (m, 2H), and
7.47–7.41 (m, 2H); 13C NMR (126 MHz, DMSO‐d6) δ 160.5, 154.5,
143.5, 141.1, 135.5, 130.6, 126.5, 126.3, 125.3, 124.4, 124.1, 123.9,
ACKNOWLEDGMENTS
The authors are thankful to the NCI Developmental Therapeutics
Program for the 60 cell line screen of compounds described in this
paper. Deepak B. Salunke is thankful to DBT New Delhi for the
award of the Ramalingaswami Fellowship (BT/RLF/Re‐entry/16/
2013). Rahul Singh (09/135(0718)/2015‐EMR‐I) and Ravinder
Kumar are thankful to CSIR, New Delhi, and UGC, New Delhi, re-
spectively, for the award of Senior Research Fellowships.
+
121.1, 114.8, and 114.7; HRMS m/z: Calculated for C15H13N6 [M
+H]+ 277.1196, found 277.1197.
|
4.2
Biological experiments
ORCID
|
4.2.1
NCI‐60 panel assay and COMPARE analysis
Deepak B. Salunke
https://orcid.org/0000-0002-1241-9146
Growth inhibition experiments were performed at the NCI. Details of
the methodology are available at http://dtp.nci.nih.gov/branches/
btb/ivclsp.html. Briefly, the panel was organized into nine subpanels
representing diverse histologies: leukemia, non‐small‐cell lung can-
cer, colon cancer, CNS (central nervous system) cancer, melanoma,
ovarian cancer, renal cancer, prostate, and breast cancer. The cells
were grown in a supplemented RPMI 1640 medium for 24 h. A so-
lution of test compounds in DMSO was added and incubated with
cells at 10 μM. The assay was terminated by the addition of cold
trichloroacetic acid, and the cells were fixed and stained with sul-
forhodamine B. The bound stain was solubilized, and the absorbance
was recorded on an automated plate reader. The one‐dose data of all
the compounds are reported as a mean graph of the percent growth
versus control. The number reported for the one‐dose assay is
growth relative to the no‐drug control and relative to the number of
cells at time zero. This allows detection of both growth inhibition
(values between 0 and 100) and lethality (values less than 0). For
example, a value of 100 means no growth inhibition. A value of 40
would mean 60% growth inhibition. A value of 0 means no net
growth over the course of the experiment. A value of −40 would
mean 40% lethality. A value of −100 means all cells are dead.
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|
4.2.2
Cell viability assay
Cell viability was determined using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐
diphenyltetrazolium bromide (MTT) assay using a standard protocol. In
brief, 5000 cells were plated in each well of a 96‐well plate and allowed

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How to cite this article: R. Singh, R. Kumar, M. Pandrala,
P. Kaur, S. Gupta, D. Tailor, S. V. Malhotra, D. B. Salunke,
Arch. Pharm. 2021, e2000393.
https://doi.org/10.1002/ardp.202000393