A new series of benzoxazole-based SIRT1 modulators for targeted therapy of non-small-cell lung cancer

Article abstract of DOI:10.1002/ardp.202000235

In an attempt to identify potential anticancer agents for non-small-cell lung cancer (NSCLC) targeting sirtuin 1 (SIRT1), the synthesis of a new series of benzoxazoles (3a – i) was carried out through a facile and versatile synthetic route. The compounds were evaluated for their cytotoxic effects on A549 human lung adenocarcinoma and NIH/3T3 mouse embryonic fibroblast cells using the MTT assay. 2-[(5-Nitro-1H-benzimidazol-2-yl)thio]-N-(2-methylbenzoxazol-5-yl)acetamide (3e) and 2-[(5-chloro-1H-benzimidazol-2-yl)thio]-N-(2-methylbenzoxazol-5-yl)acetamide (3g) were the most potent and selective anticancer agents in this series against the A549 cell line, with IC₅₀ values of 46.66 ± 11.54 and 55.00 ± 5.00 μM, respectively. The flow cytometry-based apoptosis detection assay was performed to determine their effects on apoptosis in A549 cells. Both compounds induced apoptosis in a dose-dependent manner. The effects of compounds 3e and 3g on SIRT1 activity were determined. On the basis of in vitro studies, it was observed that compound 3g caused a significant decrease in SIRT1 levels in a dose-dependent manner, whereas compound 3e increased the SIRT1 levels. According to molecular docking studies, the substantial alteration in the type of action could be attributed to the difference between the interactions of compounds 3e and 3g with the same residues in the active site of SIRT1 (PDB code: 4IG9). On the basis of in silico ADME (absorption, distribution, metabolism, and excretion) studies, these compounds are predicted to possess favorable ADME profiles. According to the in vitro and in silico studies, compounds 3e and 3g, small-molecule SIRT1 modulators, were identified as potential orally bioavailable anticancer agents for the targeted therapy of NSCLC.

Full text of DOI:10.1002/ardp.202000235

Received: 9 July 2020
Revised: 21 August 2020
Accepted: 26 August 2020
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DOI: 10.1002/ardp.202000235
F U L L P A P E R
A new series of benzoxazole‐based SIRT1 modulators for
targeted therapy of non‐small‐cell lung cancer
Belgin Sever1
| Gülşen Akalın Çiftçi2
| Mehlika Dilek Altıntop1
1Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Anadolu University,
Eskişehir, Turkey
Abstract
In an attempt to identify potential anticancer agents for non‐small‐cell lung cancer
(NSCLC) targeting sirtuin 1 (SIRT1), the synthesis of a new series of benzoxazoles
(3a – i) was carried out through a facile and versatile synthetic route. The compounds
were evaluated for their cytotoxic effects on A549 human lung adenocarcinoma and
NIH/3T3 mouse embryonic fibroblast cells using the MTT assay. 2‐[(5‐Nitro‐1H‐
benzimidazol‐2‐yl)thio]‐N‐(2‐methylbenzoxazol‐5‐yl)acetamide (3e) and 2‐[(5‐chloro‐
1H‐benzimidazol‐2‐yl)thio]‐N‐(2‐methylbenzoxazol‐5‐yl)acetamide (3g) were the most
potent and selective anticancer agents in this series against the A549 cell line, with
IC50 values of 46.66 11.54 and 55.00 5.00 µM, respectively. The flow cytometry‐
based apoptosis detection assay was performed to determine their effects on apop-
tosis in A549 cells. Both compounds induced apoptosis in a dose‐dependent manner.
The effects of compounds 3e and 3g on SIRT1 activity were determined. On the basis
of in vitro studies, it was observed that compound 3g caused a significant decrease in
SIRT1 levels in a dose‐dependent manner, whereas compound 3e increased the SIRT1
levels. According to molecular docking studies, the substantial alteration in the type of
action could be attributed to the difference between the interactions of compounds
3e and 3g with the same residues in the active site of SIRT1 (PDB code: 4IG9). On the
basis of in silico ADME (absorption, distribution, metabolism, and excretion) studies,
these compounds are predicted to possess favorable ADME profiles. According to the
in vitro and in silico studies, compounds 3e and 3g, small‐molecule SIRT1 modulators,
were identified as potential orally bioavailable anticancer agents for the targeted
therapy of NSCLC.
2Department of Biochemistry, Faculty of
Pharmacy, Anadolu University,
Eskişehir, Turkey
Correspondence
Belgin Sever, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Anadolu
University, 26470 Eskişehir, Turkey.
Email: belginsever@anadolu.edu.tr
K E Y W O R D S
apoptosis, benzimidazole, benzoxazole, molecular docking, non‐small‐cell lung cancer, SIRT1
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1
INTRODUCTION
patients are diagnosed at advanced or metastatic stages (Stage III/IV)
when surgery is no longer
a therapeutic option; therefore,
Non‐small‐cell lung cancer (NSCLC), which accounts for almost 85%
of lung cancer (LC) cases, remains the leading cause of cancer‐related
mortality across the globe, with rates increasing in the past decade.
Despite recent advances in diagnosis and combined treatments
including surgical resection, radiotherapy, and chemotherapy, the
overall survival for NSCLC patients still remains poor. Surgical re-
section is still the best option for the treatment of NSCLC, but most
chemotherapy and radiotherapy gain prominence as important
therapeutic approaches for unresectable NSCLC. Platinum‐based
chemotherapeutic drugs often cause severe side effects, as these
agents destroy healthy cells as well as tumor cells. As a consequence,
targeted therapies acting on specific molecular targets have emerged
as promising therapeutic options for NSCLC to improve therapeutic
efficacy and overcome toxicity problems.[1‐5]
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Arch Pharm. 2021;354:e2000235.
wileyonlinelibrary.com/journal/ardp
© 2020 Deutsche Pharmazeutische Gesellschaft
https://doi.org/10.1002/ardp.202000235

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Sirtuins (SIRTs), NAD+‐dependent class III histone deacetylases,
have emerged as potential therapeutic targets due to their crucial roles
in diverse physiological and pathological processes such as gene tran-
scription, energy metabolism, lipid metabolism, insulin secretion, life-
span extension, neurodegeneration, age‐related disorders, obesity, heart
disease, inflammation, and cancer.[6,7] In mammals, there are seven
members (SIRT1–SIRT7) in the sirtuin family. In particular, SIRT1 has
been the most extensively studied member of this family. In recent
years, the dual role of SIRT1 in cancer has become a highly controversial
topic. SIRT1 acts as either a tumor suppressor or as an oncogenic factor,
depending on the cellular context or its targets in specific signaling
pathways or specific cancers. The modulation of SIRT1 activity by small‐
molecule activators or inhibitors stands out as a promising strategy for
the management of many types of cancer including NSCLC.[7‐13]
Heterocycles are common fragments of the vast majority of mar-
keted drugs. This is a reflection of their pivotal role in drug discovery
and development.[14,15] Among heterocycles, benzoxazoles occupy a
prominent place in contemporary medicinal chemistry as building blocks
for modulating affinity and/or selectivity of a ligand toward a particular
target through favorable interactions such as hydrogen bonds, cation–π,
π–π stacking, and hydrophobic interactions; thus, the benzoxazole core
is found in ligands targeting a plethora of receptors and enzymes in-
volved in the pathogenesis of many diseases including cancer. Due to
their important features, benzoxazole derivatives are suitable candi-
dates for targeted therapy of cancer.[16‐25] Besides, benzimidazole is a
hot topic of research in pharmaceutical industry as a privileged scaffold
due to its diverse therapeutic applications associated with its ability to
bind with a variety of enzymes and receptors via proper interactions
such as hydrogen bonds, ion–dipole, cation–π, π–π stacking, hydro-
phobic effect, the van der Waals force, and so on.[26‐36] Benzimidazole is
a structural isostere of indole and purine nuclei, and this core is also an
integral part of the structure of vitamin B12. In particular, benzimida-
zoles exhibit potent antitumor activity against various cancer cell lines
through different mechanisms including the inhibition of several crucial
targets such as epidermal growth factor receptor, vascular endothelial
growth factor receptor, platelet‐derived growth factor receptor, cyclin‐
dependent kinases, aurora kinases, mitogen‐activated protein kinases,
polo‐like kinases, casein kinases, checkpoint kinases, and sirtuins.[26‐35]
On the basis of the aforementioned findings, herein we described
the design and synthesis of a new series of benzoxazoles as potential
anticancer agents for targeted therapy of NSCLC and carried out in
silico and in vitro studies to elucidate their mode of action.
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2
RESULTS AND DISCUSSION
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2.1
Chemistry
The facile and efficient synthetic route for the preparation of the hi-
therto unreported benzoxazoles (3a – i) is depicted in Scheme 1.
SCHEME 1 The synthetic route for the
preparation of compounds 3a–i. Reagents and
conditions: (i) ClCOCH2Cl, TEA, toluene, rt; (ii)
(1) CS2, KOH, ethanol, reflux, 8 hr, (2)
CH3COOH; (iii) K2CO3, acetone, rt, 12 hr.
rt, room temperature

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TABLE 1 IC50 values of the compounds against A549 and
NIH/3T3 cells for 24 hr
3,336.85–3,269.34 cm−1. Aromatic and aliphatic C─H stretching bands
occurred at 3,134.33–3,001.24 cm−1 and 2,983.88–2,808.36 cm−1, re-
spectively. The N─H bending, C═N, and C═C stretching bands ap-
peared between 1,633.71 and 1,456.26 cm−1. In their 1H NMR spectra,
the signal due to the amide N─H proton appeared at 10.60–10.76 ppm
as a singlet. The S─CH2 protons gave rise to a singlet peak at
4.20–4.42 ppm. The signals due to the methyl protons at the 2nd po-
sition of the benzoxazole scaffold appeared at 2.60–2.65 ppm. The
signals due to the heteroaromatic protons were observed in the region
6.74–8.33 ppm. In the 1H NMR spectra of compounds 3e – i, the signal
due to the N─H proton of the benzimidazole core appeared at
12.56–12.87 ppm as a broad singlet. In their 13C NMR spectra,
the signal due to the amide C═O carbon was observed at
165.58–171.89 ppm. The S─CH2 carbon gave rise to a peak in the
region 32.81–37.71 ppm. The signal due to the methyl carbon at the
2nd position of the benzoxazole ring appeared at 14.09–14.13 ppm.
The HRMS data of all compounds were consistent with their proposed
structures.
IC50 (µM)
Compound
A549 cell line
350.00 70.71
>500
NIH/3T3 cell line
>500
SIa
3a
>1.43
ND
3b
>500
3c
253.33 87.37
>500
>500
>1.97
ND
3d
>500
3e
46.66 11.54
230.00 26.46
55.00 5.00
>500
383.33 5.77
440.00 52.92
>500
8.22
1.91
>9.09
ND
3f
3g
3h
>500
3i
>500
>500
ND
Cisplatin
27.00 1.73
ND
ND
Abbreviations: ND, not determined, SI, selectivity index.
aSI = IC50 for NIH/3T3 cells/IC50 for A549 cells.
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In vitro anticancer activity
Initially, 2‐chloro‐N‐(2‐methylbenzoxazol‐5‐yl)acetamide (1) was syn-
thesized via the reaction of 5‐amino‐2‐methylbenzoxazole with
chloroacetyl chloride in the presence of triethylamine (TEA). However,
the ring closure reaction of 4‐substituted‐o‐aminophenol derivatives
(2a – d) with carbon disulfide in the presence of potassium hydroxide
yielded 5‐substituted benzoxazole‐2‐thiol derivatives (3a – d)
(Scheme 1). Similarly, the ring closure reaction of 4‐substituted‐o‐
phenylenediamine derivatives (2e – i) with carbon disulfide in
the presence of potassium hydroxide afforded 5‐substituted
1H‐benzimidazole‐2‐thiol derivatives (3e – i). Finally, compounds 2a –
i were reacted with compound 1 in the presence of potassium car-
bonate to afford compounds 3a – i.
2.2
The MTT assay, a tetrazolium‐based colorimetric assay, was per-
formed to assess the cytotoxic effects of compounds 3a – i on the
A549 human lung adenocarcinoma cell line (Table 1). In order to
evaluate whether these compounds were toxic or nontoxic to normal
cells, their cytotoxic effects on NIH/3T3 mouse embryonic fibroblast
(normal) cells were investigated, and their selectivity index (SI) values
are presented in Table 1. Compounds 3a – d bearing two benzoxazole
moieties did not show any significant cytotoxic activity against A549
cells. In general, the replacement of the benzoxazole scaffold of
compounds 3a and 3c with the benzimidazole scaffold of compounds
3e and 3g led to a significant increase in potency. Compounds 3e and
3g exhibited a marked cytotoxic activity against the A549 cells, with
IC50 values of 46.66 11.54 and 55.0 5.0 µM, respectively, as
compared with cisplatin (IC50 = 27.0 1.73 µM). Additionally, the SI
values of compounds 3e and 3g were determined as 8.22 and >9.09,
respectively, indicating that their anticancer effects on A549 cells
were selective. The results pointed out the importance of the
The structures of compounds 3a – i were verified by different
spectroscopic techniques, namely ınfrared (IR), 1H nuclear magnetic
resonance (NMR), 13C NMR, and high‐resolution mass spectrometry
(HRMS). In the IR spectra of compounds 3a – i, the C═O stretching
vibration led to the formation of the characteristic amide I band at
1,675.15–1,660.71 cm−1. The N‐H stretching vibrations belonging to
the secondary amide group gave rise to the bands in the region
TABLE 2 Percentages of typical quadrant analysis of Annexin V‐FITC/PI flow cytometry of A549 cells treated with compounds 3e, 3g, and
cisplatin
Groups
Early apoptotic cells (%) Late apoptotic cells (%) Viable cells (%) Necrotic cells (%)
Control (untreated)
5.1
6.1
82.8
57.4
32.7
55.8
24.7
29.1
5.9
Cells treated with IC50/2 concentration of compound 3e 9.0
18.2
28.7
22.0
45.7
41.0
15.4
18.0
10.6
8.6
Cells treated with IC50 concentration of compound 3e
20.4
Cells treated with IC50/2 concentration of compound 3g 11.6
Cells treated with IC50 concentration of compound 3g
20.4
11.9
Cells treated with IC50 concentration of cisplatin
18.0
Note: A549 cells were cultured for 24 hr in a medium with IC50/2 and IC50 concentrations of compounds 3e and 3g and IC50 concentration of cisplatin.
At least 10,000 cells were analyzed per sample, and quadrant analysis was performed.

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FIGURE 1 A typical quadrant analysis of
Annexin V‐FITC/PI flow cytometric analysis of
A549 cells treated with different
concentrations of compounds 3e, 3g, and
cisplatin. At least 10,000 cells were analyzed
per sample, and quadrant analysis was
performed. Q1, Q2, Q3, and Q4 quadrants
represent necrosis, late apoptosis, viable cells,
and early apoptotic cells, respectively. A549
cells were cultured for 24 hr in a medium with
IC50/2 and IC50 concentrations of the
compounds. (a,b) Untreated control cells. (c,d)
Cells treated with IC50/2 and IC50
concentrations of compound 3e. (e,f) Cells
treated with IC50/2 and IC50 concentrations of
compound 3g. (g) Cells treated with IC50
concentration of cisplatin, respectively

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TABLE 3 The effects of compounds 3e, 3g, and cisplatin on SIRT1
activity
exposed to compounds 3e and 3g at IC50/2 and IC50 concentrations
were determined as 18.2%, 28.7%, 22.0%, and 45.7%, respectively
(Table 2 and Figure 1). However, the percentages of A549 cells un-
dergoing early and late apoptosis exposed to cisplatin were found as
11.9% and 41%, respectively. According to these findings, both com-
pounds induced apoptosis in a dose‐dependent manner. In particular,
compound 3g caused a more significant apoptotic activity than com-
pound 3e and cisplatin at IC50 concentration.
Compound
SIRT1 concentration (ng/ml)
3.12 0.03
Control
3e (23.33 µM)
3e (46.66 µM)
3g (27.5 µM)
3g (55 µM)
4.53 1.26
3.67 1.03
2.10 0.35
In an effort to provide insights into the mechanism underlying
their cytotoxic and apoptotic effects on NSCLC, the effects of com-
pounds 3e and 3g at IC50/2 and IC50 concentrations on SIRT1 activity
in the C6 rat glioma cell line were investigated using the enzyme‐
1.31 0.04
Cisplatin (13.5 µM)
Cisplatin (27 µM)
4.53 1.37
3.32 1.41
linked Immunosorbent assay (ELISA)‐based assay (Table
3 and
benzimidazole core as well as the substituent at position 5 of the
benzimidazole ring for anticancer activity against A549 cells. The
nitro and chloro substituents at the 5‐position of the benzimidazole
ring enhanced the cytotoxic activity against the A549 cell line.
Apoptosis is a physiological process that is essential for the re-
moval of damaged or redundant cells and maintenance of cellular
homeostasis. However, in cancerogenesis, deregulated apoptotic sig-
naling, particularly the activation of antiapoptotic systems, allows
cancer cells to escape from this crucial process, leading to uncontrolled
proliferation, which further results in tumor survival, therapeutic re-
sistance, and recurrence of cancer. Mounting evidence has revealed
that many anticancer agents induce apoptosis and related cell death
networks to eliminate tumor cells, and thus the induction of apoptosis
is of great importance for the management of many types of cancer
including NSCLC.[37‐41] In order to identify early and late apoptotic
cells, after 24‐hr incubation of A549 cells treated with compounds 3e
and 3g at IC50/2 and IC50 concentrations and cisplatin at IC50 con-
centration, the flow cytometry‐based apoptosis detection assay was
performed by means of Annexin V‐fluorescein isothiocyanate (FITC)/
propidium iodide (PI) staining. The percentages of A549 cells under-
going early apoptosis exposed to compounds 3e and 3g at IC50/2 and
IC50 concentrations were found to be 9.0%, 20.4%, 11.6%, and 20.4%,
respectively. The percentages of A549 cells undergoing late apoptosis
Figure 2). According to the assay, compound 3g decreased SIRT1 le-
vels in a dose‐dependent manner at 27.5 and 55 µM concentrations.
The SIRT1 activity inhibition value for compound 3g at IC50/2 con-
centration was found as 32.70%, whereas the SIRT1 activity inhibition
value for compound 3g at IC50 concentration was determined as
60.66%. However, compound 3e increased SIRT1 levels in the C6 rat
glioma cell line at 23.33 and 46.66 µM, similar to cisplatin. It can be
concluded that these compounds exhibit their anticancer activity
through different mechanisms of action. Compound 3g acts as a SIRT1
inhibitor, whereas compound 3e acts as a SIRT1 activator. According
to these results, the chloro substitution at the 5‐position of the
benzimidazole scaffold led to SIRT1 inhibition, whereas the nitro
substitution at position 5 of the benzimidazole ring resulted in SIRT1
activation. Taking into account the fact that SIRT1 acts as a double‐
edged sword in cancer,[7‐13] both agents could be beneficial for the
management of cancer.
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2.3
Molecular docking studies
Molecular docking studies were performed to elucidate the possible
binding modes of compound 3e (SIRT1 activator) and compound 3g
(SIRT1 inhibitor) in the active site of SIRT1 (PDB code: 4IG9) using
FIGURE 2 The effects of compounds 3e (a:
23.33 µM, b: 46.66 µM), 3g (a: 27.5 µM, b:
55 µM), and cisplatin (a: 13.5 µM, b: 27 µM) on
SIRT1 levels in the C6 rat glioma cell line.
*Significance from control group p < .05

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FIGURE 3 Docking poses (a) and interactions of compounds 3e (b) and 3g (c) in the active site of SIRT1 (PDB code: 4IG9)
Schrödinger's Maestro molecular modeling package. Molecular
docking studies indicated that compounds 3e and 3g showed high
affinity in the active site of SIRT1, as depicted in Figure 3. In the
active site of SIRT1, compound 3e was capable of forming hydrogen
bonds with Lys248 and Asp245 residues due to the presence of the
amide carbonyl group and the benzimidazole N─H moiety. The nitro
substituent at position 5 of the benzimidazole scaffold formed a
hydrogen bond with Asn241 and a salt bridge with Asp245. However,
compound 3g was capable of forming hydrogen bonds with Asn241
and Asp245 residues due to the presence of the amide carbonyl
group and the benzimidazole N─H moiety. The benzimidazole moiety
carrying a chloro substituent at the 5‐position formed cation–π in-
teractions with the Lys248 residue. In silico studies suggested that
the difference between the interactions of compounds 3e and 3g
with the same residues in the active site of SIRT1 might be re-
sponsible for the substantial alteration in the type of action (inhibi-
tion or activation).
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2.4
In silico absorption, distribution, metabolism,
and excretion (ADME) studies
The pharmacokinetic profiles of compounds 3a – i were predicted by
means of QikProp, a predictive ADME module within the Maestro
suite produced by Schrödinger (Table 4). According to in silico pre-
diction, the total solvent‐accessible surface area (SASA), the predicted
octanol/water partition coefficient (QPlogPo/w), the conformation‐
independent predicted aqueous solubility (CIQPlogS), and the

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TABLE 4 Predicted ADME profiles of
compounds 3a–i
Human oral Rule
absorption
of
fivec
Rule of
threec
b
Compound SASAa
QPlogPo/wa ClQPlogSa QPlogKhsaa
%
3a
3b
3c
3d
3e
3f
647.916 2.262
−5.109
−4.966
−5.296
−4.887
−5.268
−5.109
−5.433
−5.032
−5.068
−0.078
0.020
0.090
0.130
0.072
0.153
0.218
0.259
0.126
80.215
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
617.976 3.203
632.953 3.457
641.093 3.272
663.637 2.302
635.540 3.235
648.794 3.473
658.002 3.293
663.517 3.100
100.000
100.000
100.000
76.364
100.000
100.000
100.000
100.000
3g
3h
3i
aRecommended values for SASA: 300–1,000 Å2; QPlogPo/w: −2 to 6.5; CIQPlogS: −6.5 to 0.5;
QPlogKhsa: −1.5 to 1.5.
bPredicted human oral absorption on 0–100% scale. Human oral absorption higher than 80% is
considered to be high, whilst human oral absorption lower than 25% is considered to be poor.
cNumber of violations of Lipinski's rule of five. The rules are molecular weight of the molecule < 500,
QPlogPo/w < 5, hydrogen‐bond donor atoms ≤ 5, hydrogen‐bond acceptor atoms ≤ 10. Compounds
that obey these rules are considered as drug‐like molecules. Rule of three: Number of violations of
Jorgensen's rule of three. The three rules are QPlogS (predicted aqueous solubility) > −5.7,
QPPCaco > 22 nm/s, #Primary Metabolites < 7. Compounds with fewer (and preferably no) violations
of these rules are more likely to be orally available agents (Schrödinger Release 2016‐2; Schrödinger,
LLC, New York, NY).
predicted binding to human serum albumin (QPlogKhsa) values of all
compounds were within the optimum range. The human oral absorp-
tion percentages of the compounds were found to be in the range of
76.364–100.000%. All compounds comply with Lipinski's rule of five
and Jorgensen's rule of three. On the basis of these results, they are
expected to possess favorable oral bioavailability and drug‐like
properties.
responsible for the considerable alteration in the type of action (in-
hibition or activation). On the basis of in silico ADME studies, these
compounds are expected to possess favorable ADME profiles. Ac-
cording to in vitro and in silico studies, compounds 3e and 3g, small‐
molecule SIRT1 modulators in this series, stand out as potential or-
ally bioavailable anticancer agents for targeted therapy of NSCLC. In
the continuation of this study, further studies are needed to provide
insights into the mechanism underlying the alteration in their type of
action (SIRT1 inhibition or activation).
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3
CONCLUSIONS
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EXPERIMENTAL
In this paper, the synthesis of a new series of benzoxazoles was
carried out according to a facile and versatile synthetic procedure. All
compounds were evaluated for their cytotoxic effects on A549 and
NIH/3T3 cells. Compounds 3e and 3g were identified as the most
potent and selective anticancer agents against A549 cell line in this
series. These agents were also evaluated for their effects on apop-
tosis in the A549 cell line. The findings demonstrated that A549 cells
treated with both compounds underwent apoptosis. In particular,
compound 3g showed a more significant apoptotic activity than
compound 3e and cisplatin. In an attempt to elucidate their me-
chanism of action, the effects of compounds 3e and 3g at IC50/2 and
IC50 concentrations on SIRT1 activity in the C6 rat glioma cell line
were investigated. Compound 3g significantly inhibited SIRT1 in a
dose‐dependent manner, whereas compound 3e caused an increase
in SIRT1 levels. According to molecular docking studies, the differ-
ence between the interactions of compounds 3e and 3g with the
same residues in the active site of SIRT1 (PDB code: 4IG9) might be
4
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4.1
Chemistry
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4.1.1
General
The chemicals used in this study were procured from Acros Organics
(Geel, Belgium), Sigma‐Aldrich (St. Louis, MO), or Merck (Darmstadt,
Germany), and they were used without further purification. The
melting points (m.p.) of the synthesized compounds were determined
by an MP90 digital melting point apparatus (Mettler Toledo, OH) and
were uncorrected. IR spectra were recorded on an IRPrestige‐21
Fourier transform infrared spectrophotometer (Shimadzu, Tokyo,
Japan). 1H NMR and 13C NMR spectra were recorded on a Varian
Mercury 400 FT‐NMR spectrometer (Agilent, Palo Alto, CA) oper-
ating at 400 and 100 MHz, respectively. Chemical shifts were re-
ported in parts per million (ppm). Tetramethylsilane (TMS) was used

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as an internal standard. In the NMR spectra, the splitting patterns
were designated as follows: s: singlet; brs: broad singlet; d: doublet; t:
triplet; m: multiplet; dd: doublet of doublets. Coupling constants (J)
were reported in Hertz (Hz). HRMS spectra were recorded on an
LCMS‐IT‐TOF (Shimadzu) using the electrospray ionization (ESI)
technique. Thin‐layer chromatography (TLC) was performed on TLC
Silica gel 60 F254 aluminum sheets (Merck) to monitor the progress of
the chemical reactions and check the purity of the synthesized
compounds.
2‐[(5‐Nitrobenzoxazol‐2‐yl)thio]‐N‐(2‐methylbenzoxazol‐5‐yl)‐
acetamide (3a)
Yield: 85%. M.p.: 147.6°C. IR νmax (cm−1): 3,334.92 (N─H stretching),
3,076.46 (aromatic C─H stretching), 2,983.88, 2,929.87 (aliphatic C─H
stretching), 1,675.15 (C═O stretching), 1,625.99, 1,573.91, 1,517.98,
1,477.47 (N─H bending, C═N and C═C stretching), 1,435.04,
1,371.39, 1,332.81, 1,257.59, 1,220.94, 1,161.15, 1,116.78, 1,082.07
(C─H bending, C─N, C─O stretching and aromatic C─H in plane
bending), 929.69, 893.04, 823.60, 779.24, 748.38, 663.51 (aromatic
C─H out of plane bending and C─S stretching). 1H NMR (400 MHz,
DMSO‐d6) δ (ppm): 2.64 (s, 3H), 4.20 (s, 2H), 6.94 (d, J = 9.2 Hz, 1H),
7.44 (d, J = 8.0 Hz, 1H), 7.68–7.89 (m, 4H), 10.60 (s, 1H). 13C NMR
(100 MHz, DMSO‐d6) δ (ppm): 14.12 (CH3), 33.05 (CH2), 110.42 (CH),
116.53 (CH), 117.12 (CH), 119.60 (CH), 121.90 (CH), 125.11 (CH),
131.21 (C), 136.32 (C), 137.97 (C), 141.34 (C), 149.96 (C), 157.83 (C),
158.28 (C), 165.15 (C), 171.87 (C). HRMS (ESI) (m/z): [M + H]+ calcd.
for C17H12N4O5S: 385.0601, found: 385.0613.
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Information.
|
4.1.2
Synthesis of 2‐chloro‐N‐(2‐
methylbenzoxazol‐5‐yl)acetamide (1)
Chloroacetyl chloride (0.1 mol) was added dropwise, with stirring, to
a
mixture of 5‐amino‐2‐methylbenzoxazole (0.1 mol) and TEA
(0.1 mol) in toluene (50 ml) at 0–5°C. Upon completion of the reac-
tion, the solvent was evaporated using Rotavapor® R‐100 (Büchi,
Switzerland). The residue was washed with water and filtered. After
drying, the product was crystallized from ethanol.[42]
2‐[(5‐Fluorobenzoxazol‐2‐yl)thio]‐N‐(2‐methylbenzoxazol‐5‐yl)‐
acetamide (3b)
Yield: 78%. M.p. 142.1°C. IR νmax (cm−1): 3,269.34 (N─H stretching),
3,068.75 (aromatic C─H stretching), 2,978.09, 2,929.87 (aliphatic
C─H stretching), 1,675.15 (C═O stretching), 1,633.71, 1,608.63,
1,575.84, 1,477.47 (N─H bending, C═N and C═C stretching),
1,435.04, 1,369.46, 1,253.73, 1,174.65, 1,136.07, 1,091.71 (C─H
bending, C─N, C─O stretching and aromatic C─H in plane bending),
962.48, 933.55, 848.68, 808.17, 725.23, 663.51 (aromatic C─H out
of plane bending and C─S stretching). 1H NMR (400 MHz, DMSO‐d6)
δ (ppm): 2.65 (s, 3H), 4.22 (s, 2H), 6.83–6.87 (m, 1H), 7.36–7.38 (m,
1H), 7.43–7.81 (m, 3H), 8.01 (s, 1H), 10.64 (s, 1H). 13C NMR
(100 MHz, DMSO‐d6) δ (ppm): 14.13 (CH3), 32.93 (CH2), 108.09 (CH),
109.64 (d, J = 25.2 Hz, CH), 110.38 (CH), 110.60 (CH), 110.67 (CH),
116.49 (CH), 136.44 (C), 141.37 (C), 144.63 (C), 146.70 (C), 149.94
(C), 156.36 (C), 159.66 (C), 165.25 (C), 171.89 (C). HRMS (ESI) (m/z):
[M + H]+ calcd. for C17H12FN3O3S: 358.0656, found: 358.0658.
|
4.1.3
Synthesis of 5‐substituted benzoxazole‐2‐
thiol derivatives (2a – d)/5‐substituted 1H‐
benzimidazole‐2‐thiol derivatives (2e – i)
4‐Substituted‐o‐aminophenol/4‐substituted‐o‐phenylenediamine
(20 mmol) and 40 ml of absolute ethanol were added to
a
100 ml‐round bottom flask, and the content was stirred for 15 min. In
the meanwhile, potassium hydroxide pellets (20 mmol) were powdered
and transferred into the flask, followed by the addition of carbon
disulfide (15 ml). The mixture was refluxed for 8 hr. Upon completion
of the reaction, the solvent was evaporated using Rotavapor® R‐100
to yield a solid product. This product was carefully transferred into a
beaker and 75 ml of water was added. The solution was acidified with
acetic acid until a precipitate appeared. The precipitate was filtered
and then washed with water and dried. The product was crystallized
from ethanol.[43]
2‐[(5‐Chlorobenzoxazol‐2‐yl)thio]‐N‐(2‐methylbenzoxazol‐5‐yl)‐
acetamide (3c)
Yield: 84%. M.p. 120.4°C. IR νmax (cm−1): 3,336.85 (N─H stretching),
3,068.75 (aromatic C─H stretching), 2,976.16, 2,929.87 (aliphatic
C─H stretching), 1,674.21 (C═O stretching), 1,620.21, 1,597.06,
1,573.91, 1,477.47 (N─H bending, C═N and C═C stretching),
1,435.04, 1,365.60, 1,251.80, 1,172.72, 1,112.93, 1,082.07 (C─H
bending, C─N, C─O stretching and aromatic C─H in plane bending),
898.83, 869.90, 808.17, 779.24, 663.51 (aromatic C─H out of plane
bending and C – S stretching). 1H NMR (400 MHz, DMSO‐d6) δ (ppm):
2.64 (s, 3H), 4.21 (s, 2H), 6.81–6.87 (m, 2H), 6.96 (d, J = 8.4 Hz, 1H),
7.35–7.44 (m, 1H), 7.74–7.80 (m, 2H), 10.61 (s, 1H). 13C NMR
(100 MHz, DMSO‐d6) δ (ppm): 14.13 (CH3), 32.94 (CH2), 110.44 (CH),
117.52 (CH), 119.53 (CH), 120.94 (CH), 122.25 (CH), 124.47 (CH),
125.07 (C), 131.22 (C), 136.94 (C), 141.34 (C), 147.27 (C), 149.93 (C),
157.85 (C), 165.19 (C), 171.83 (C). HRMS (ESI) (m/z): [M + H]+ calcd.
for C17H12ClN3O3S: 374.0361, found: 374.0363.
|
4.1.4
Synthesis of 2‐[(5‐substituted benzoxazol/
benzimidazol‐2‐yl)thio]‐N‐(2‐methylbenzoxazol‐5‐yl)‐
acetamide derivatives (3a – i)
Compounds 2a – i (1.5 mmol) were treated with 2‐chloro‐N‐(2‐
methylbenzoxazol‐5‐yl)acetamide (1) (1.5 mmol) in the presence
of potassium carbonate (1.5 mmol) in acetone (25 ml) at room
temperature for 12 hr. Upon completion of the reaction, the
solvent was evaporated using Rotavapor® R‐100. The residue
was washed with water and dried. The product was crystallized
from ethanol.

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2‐[(5‐Methylbenzoxazol‐2‐yl)thio]‐N‐(2‐methylbenzoxazol‐5‐yl)‐
acetamide (3d)
J = 25.2 Hz, CH), 109.56 (2CH), 110.19 (2CH), 116.39 (CH), 135.51
(C), 141.35 (2C), 146.65 (C), 151.31 (C), 157.14 (C), 159.47 (C),
164.68 (C), 166.03 (C). HRMS (ESI) (m/z): [M + H]+ calcd. for
C17H13FN4O2S: 357.0816, found: 357.0819.
Yield: 81%. M.p. 112.9°C. IR vmax (cm−1): 3,321.42 (N─H stretching),
3,068.75 (aromatic C─H stretching), 2,972.31, 2,927.94 (aliphatic
C─H stretching), 1,674.21 (C═O stretching), 1,627.92, 1,595.13,
1,577.77, 1,475.54 (N─H bending, C═N and C═C stretching),
1,435.04, 1,357.89, 1,255.66, 1,203.58, 1,176.58, 1,155.36, 1,109.07
(C─H bending, C─N, C─O stretching and aromatic C─H in plane
bending), 931.62, 866.04, 831.32, 796.60, 680.87, 661.58 (aromatic
C─H out of plane bending and C─S stretching). 1H NMR (400 MHz,
DMSO‐d6) δ (ppm): 2.39 (s, 3H), 2.64 (s, 3H), 4.41 (s, 2H), 6.74 (t,
J = 8.4 Hz, 1H), 7.42–7.80 (m, 4H), 8.01 (d, J = 1.2 Hz, 1H), 10.60 (s,
1H). 13C NMR (100 MHz, DMSO‐d6) δ (ppm): 14.12 (CH3), 20.90
(CH3), 32.81 (CH2), 109.57 (CH), 110.25 (CH), 116.08 (CH), 116.48
(CH), 118.16 (CH), 125.13 (CH), 134.06 (C), 135.38 (C), 141.34 (C),
145.64 (C), 146.69 (C), 149.90 (C), 156.52 (C), 165.16 (C), 171.89 (C).
HRMS (ESI) (m/z): [M + H]+ calcd. for C18H15N3O3S: 354.0907, found:
354.0908.
2‐[(5‐Chloro‐1H‐benzimidazol‐2‐yl)thio]‐N‐(2‐methylbenzoxazol‐5‐
yl)acetamide (3g)
Yield: 85%. M.p. 239.5°C. IR νmax (cm−1): 3,300.20 (N─H stretching),
3,130.47, 3,062.96 (aromatic C─H stretching), 2,953.02, 2,916.37,
2,881.65 (aliphatic C─H stretching), 1,662.64 (C═O stretching),
1,625.99, 1,573.91, 1,541.12, 1,489.05, 1,456.26 (N─H bending, C═N
and C═C stretching), 1,435.04, 1,396.46, 1,348.24, 1,311.59,
1,276.88, 1,195.87, 1,182.36, 1,124.50, 1,058.92 (C─H bending,
C─N, C─O stretching and aromatic C─H in plane bending), 964.41,
941.26, 920.05, 896.90, 871.82, 817.82, 786.96, 721.38, 698.23,
661.58 (aromatic C─H out of plane bending and C─S stretching). 1H
NMR (400 MHz, DMSO‐d6) δ (ppm): 2.60 (s, 3H), 4.35 (s, 2H), 7.17
(dd, J = 1.6 Hz, 8.4 Hz, 1H), 7.45–7.49 (m, 2H), 7.54 (d, J = 1.2 Hz, 1H),
7.60 (d, J = 8.8 Hz, 1H), 8.03 (d, J = 1.6 Hz, 1H), 10.61 (s, 1H), 12.87
(brs, 1H). 13C NMR (100 MHz, DMSO‐d6) δ (ppm): 14.10 (CH3), 36.22
(CH2), 109.51 (CH), 110.20 (2CH), 116.37 (2CH), 121.60 (CH),
125.91 (C), 135.48 (2C), 141.32 (2C), 146.62 (C), 151.74 (C), 164.68
(C), 165.90 (C). HRMS (ESI) (m/z): [M + H]+ calcd. for C17H13ClN4O2S:
373.0521, found: 373.0537.
2‐[(5‐Nitro‐1H‐benzimidazol‐2‐yl)thio]‐N‐(2‐methylbenzoxazol‐5‐yl)
acetamide (3e)
Yield: 86%. M.p. 263.4°C. IR νmax (cm−1): 3,282.84 (N─H stretching),
3,068.75 (aromatic C─H stretching), 2,962.66, 2,916.37, 2,881.65
(aliphatic C─H stretching), 1,660.71 (C═O stretching), 1,620.21,
1,573.91, 1,537.27, 1,487.12 (N – H bending, C═N and C═C
stretching), 1,415.75, 1,381.03, 1,336.67, 1,278.81, 1,244.09,
1,192.01, 1,126.43, 1,064.71 (C─H bending, C─N, C─O stretching
and aromatic C─H in plane bending), 966.34, 939.33, 896.90, 871.82,
817.82, 738.74, 690.52, 669.30 (aromatic C – H out of plane bending
and C─S stretching). 1H NMR (400 MHz, DMSO‐d6) δ (ppm): 2.60 (s,
3H), 4.42 (s, 2H), 7.47 (dd, J = 1.6 Hz, 8.8 Hz, 1H), 7.61 (t, J = 8.4 Hz,
2H), 8.02 (d, J = 1.6 Hz, 1H), 8.08 (dd, J = 2.4 Hz, 8.8 Hz, 1H), 8.33 (d,
J = 2.4 Hz, 1H), 10.61 (s, 1H), 12.56 (brs, 1H). 13C NMR (100 MHz,
DMSO‐d6) δ (ppm): 14.10 (CH3), 36.25 (CH2), 109.53 (2CH), 110.20
(2CH), 116.39 (CH), 117.49 (CH), 135.44 (C), 141.31 (2C), 142.14 (C),
146.62 (2C ), 156.06 (C), 164.69 (C), 165.58 (C). HRMS (ESI) (m/z):
[M + H]+ calcd. for C17H13N5O4S: 384.0761, found: 384.0764.
2‐[(5‐Methyl‐1H‐benzimidazol‐2‐yl)thio]‐N‐(2‐methylbenzoxazol‐5‐
yl)acetamide (3h)
Yield: 81%. M.p. 204.7°C. IR νmax (cm−1): 3,307.92 (N─H stretching),
3,134.33, 3,061.03, 3,032.10 (aromatic C─H stretching), 2,960.73,
2,914.44 (aliphatic C─H stretching), 1,662.64 (C═O stretching),
1,625.99, 1,577.77, 1,539.20, 1,487.12 (N─H bending, C═N and C═C
stretching), 1,436.97, 1,409.96, 1,396.46, 1,381.03, 1,338.60,
1,311.59, 1,276.88, 1,197.79, 1,182.36, 1,124.50 (C─H bending,
C─N, C─O stretching and aromatic C─H in plane bending), 968.27,
943.19, 896.90, 873.75, 854.47, 833.25, 817.82, 786.96, 759.95,
721.38, 682.80, 663.51 (aromatic C─H out of plane bending and C─S
stretching). 1H NMR (400 MHz, DMSO‐d6) δ (ppm): 2.41 (s, 3H), 2.60
(s, 3H), 4.37 (s, 2H), 6.99 (d, J = 7.6 Hz, 1H), 7.32–7.61 (m, 4H), 8.10 (s,
1H), 10.76 (s, 1H), 12.63 (brs, 1H). 13C NMR (100 MHz, DMSO‐d6) δ
(ppm): 14.09 (CH3), 21.18 (CH3), 36.35 (CH2), 109.55 (CH), 110.19
(2CH), 116.36 (2CH), 122.87 (CH), 135.57 (2C), 141.39 (2C), 146.66
(2C), 149.28 (C), 164.65 (C), 166.28 (C). HRMS (ESI) (m/z): [M + H]+
calcd. for C18H16N4O2S: 353.1067, found: 353.1067.
2‐[(5‐Fluoro‐1H‐benzimidazol‐2‐yl)thio]‐N‐(2‐methylbenzoxazol‐5‐
yl)acetamide (3f)
Yield: 82%. M.p. 234.5°C. IR νmax (cm−1): 3,304.06 (N─H stretching),
3,091.89, 3,064.89 (aromatic C─H stretching), 2,914.44, 2,808.36
(aliphatic C─H stretching), 1,662.64 (C═O stretching), 1,625.99,
1,575.84, 1,537.27, 1,490.97 (N─H bending, C═N and C═C stretch-
ing), 1,435.04, 1,408.04, 1,396.46, 1,346.31, 1,311.59, 1,276.88,
1,263.37, 1,195.87, 1,182.36, 1,134.14, 1,103.28 (C─H bending,
C─N, C─O stretching and aromatic C─H in plane bending), 939.33,
896.90, 867.97, 817.82, 786.96, 767.67, 721.38, 690.52, 661.58
(aromatic C─H out of plane bending and C─S stretching). 1H NMR
(400 MHz, DMSO‐d6) δ (ppm): 2.60 (s, 3H), 4.33 (s, 2H), 7.00 (t,
J = 8.8 Hz, 1H), 7.31 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.60 (d,
J = 8.4 Hz, 1H), 8.02 (s, 1H), 10.60 (s, 1H), 12.81 (brs, 1H). 13C NMR
(100 MHz, DMSO‐d6) δ (ppm): 14.09 (CH3), 36.28 (CH2), 109.24 (d,
2‐[(5‐Methoxy‐1H‐benzimidazol‐2‐yl)thio]‐N‐(2‐methylbenzoxazol‐
5‐yl)acetamide (3i)
Yield: 83%. M.p. 177.7°C. IR νmax (cm−1): 3,304.06 (N─H stretching),
3,084.18, 3,001.24 (aromatic C─H stretching), 2,935.66, 2,837.29
(aliphatic C─H stretching), 1,672.28 (C═O stretching), 1,629.85,
1,568.13, 1,481.33 (N─H bending, C═N and C═C stretching),
1,431.18, 1,386.82, 1,355.96, 1,303.88, 1,269.16, 1,203.58, 1,178.51,
1,151.50, 1,111.00, 1,029.99 (C─H bending, C─N, C─O stretching
and aromatic C – H in plane bending), 981.77, 929.69, 848.68,

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SEVER ET AL.
|
798.53, 765.74, 690.52, 663.51 (aromatic C─H out of plane bending
and C─S stretching). 1H NMR (400 MHz, DMSO‐d6) δ (ppm): 2.60 (s,
3H), 3.79 (s, 3H), 4.29 (s, 2H), 6.79 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 7.01 (s,
1H), 7.39 (d, J = 8.8 Hz, 1H), 7.46 (dd, J = 2.0 Hz, 8.8 Hz, 1H), 7.60 (d,
J = 8.4 Hz, 1H), 8.03 (d, J = 2.0 Hz, 1H), 10.66 (s, 1H), 12.60 (brs, 1H).
13C NMR (100 MHz, DMSO‐d6) δ (ppm): 14.09 (CH3), 37.71 (CH2),
55.46 (CH3), 104.55 (CH), 109.63 (CH), 110.21 (2CH), 113.61 (CH),
116.47 (CH), 131.76 (C), 135.40 (C), 141.32 (2C), 146.67 (C), 153.84
(C), 158.66 (C), 165.14 (C), 167.02 (C). HRMS (ESI) (m/z): [M + H]+
calcd. for C18H16N4O3S: 369.1016, found: 369.1022.
24 hr. Annexin V staining protocol was applied according to the
manufacturer's instructions (BD Pharmingen) and analyzed by a BD
FACSAria flow cytometer using FACSDiva version 6.1.1 software (BD
Biosciences, San Jose, CA).[44]
|
4.2.4
SIRT1 activity detection by ELISA
C6 rat glioma cells were administrated by IC50 or IC50/2 concentra-
tions of compounds 3e and 3g and cisplatin for 24 hr. Cells were de-
tached with trypsin and then collected by centrifugation. After cells
were washed by 1× PBS for three times, cell lysates were prepared by
frozen cells at ≤−20°C. Freeze/thaw cycles were repeated for three
times. Then cells were centrifuged at 1,500g for 10 min at 2–8°C to
remove cellular debris. Rat Sirtuin‐1 activity protocol was applied
according to the manufacturer's instructions (USCN, Life Science Inc.,
Wuhan, China). Briefly, all standards, reagents, and samples were
prepared. Also, 100 µl standard or sample was added to each well and
incubated for 2 hr at 37°C, and 100 µl detection reagent A was added
and incubated for 1 hr at 37°C. Then solutions were aspirated and the
wells were washed three times. Then 100 µl detection reagent B was
added and incubated for 30 min at 37°C. The solutions were aspirated
and washed five times. Then, 90 µl substrate solution was added and
incubated for 15–25 min at 37°C. After 50 µl stop solution was added,
the plate was read at 450 nm with a microtiter plate spectro-
photometer (Bio‐Tek plate reader). Each concentration was repeated
in double wells. The results were expressed as ng/ml.[44]
|
4.2
Biochemistry
|
4.2.1
Cell culture and drug treatment
A549 Human lung adenocarcinoma (ATCC® CCL‐185™) and NIH/3T3
mouse embryonic fibroblast (ATCC® CRL‐1658™) cell lines were cul-
tured and drug treatments were carried out as previously described.[44]
|
4.2.2
MTT assay
The level of cellular 3‐(4,5‐dimethylthiazol‐2‐yl)−2,5‐diphenyltetrazolium
bromide (MTT; Sigma‐Aldrich, St. Louis, MO) reduction was quantified as
previously described in the literature,[45] with small modifications.[44]
Compounds 3a – i were investigated for their anticancer activity against
the A549 human lung adenocarcinoma cell line. NIH/3T3
mouse embryonic fibroblast cells were used to evaluate the selectivity of
the compounds.
|
4.3
Statistical analyses
After 24 hr of preincubation, compounds 3a – i and cisplatin
(positive control) were added to give a final concentration in the
range of 1–500 µM and the cells were incubated for 24 hr. At the end
of this period, MTT was added to a final concentration of 0.5 mg/ml
and the cells were incubated for 4 hr at 37°C. After the medium was
removed, the formazan crystals formed by MTT metabolism were
solubilized by the addition of 200 µl dimethyl sulfoxide (DMSO) to
each well and absorbance was read at 540 nm with a microtiter plate
spectrophotometer (Bio‐Tek Plate Reader, Winooski, VT). Each con-
centration was repeated in three wells. The half‐maximal inhibitory
concentration (IC50) values were defined as the drug concentrations
that reduced absorbance to 50% of control values. The SI values
Statistical Package for the Social Sciences (SPSS, Chicago, IL) for
Windows 15.0 was used for statistical analysis. Data were expressed
as mean SD. Comparisons were performed by one‐way analysis of
variance test for normally distributed continuous variables, and post-
hoc analyses of group differences were expressed by the Tukey test.
The p < .05 was considered as statistically significant in this study.
|
4.4
Molecular docking studies
Compounds 3e and 3g were docked to the active site of SIRT1. Li-
gands were prepared with energy minimization in ligand preparation
program of Schrödinger's Maestro molecular modeling package
(Schrödinger Release 2016‐2: Schrödinger, LLC, New York, NY) at
physiological pH (pH = 7.4) using Optimized Potential Liquid Simu-
lations (OPLS_2005) force field, and the crystal structure of SIRT1
was retrieved from Protein Data Bank server (PDB code: 4IG9) and
optimized for docking analysis in protein preparation module of
Schrödinger's Maestro molecular modeling package. In molecular
docking simulations, Glide/XP docking protocols were applied for the
prediction of the topologies of the compounds in the active site of the
target structure.[47]
were also calculated according to the following formula[46]
:
SI = IC50 for NIH/3T3 cell line/IC50 for A549 cellline.
|
4.2.3
Flow cytometric analyses of apoptosis
After the incubation of A549 cells with compounds 3e and 3g at
IC50/2 and IC50 concentrations and cisplatin at IC50 concentration,
phosphatidylserine externalization, which indicates early apoptosis,
was detected using FITC Annexin V Apoptosis Detection Kit (BD
Pharmingen, San Jose, CA) on a BD FACSAria flow cytometer for

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SEVER ET AL.
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|
4.5
In silico ADME studies
[25] A.‐M. M. E. Omar, O. M. AboulWafa, M. S. El‐Shoukrofy, M. E. Amr,
Bioorg. Chem. 2020, 96, 103593.
[26] L. Garuti, M. Roberti, G. Bottegoni, Curr. Med. Chem. 2014, 21, 2284.
[27] F. Fei, Z. Zhou, Expert Opin. Ther. Pat. 2013, 23, 1157.
[28] P. Singla, V. Luxami, K. Paul, RSC Adv. 2014, 4, 12422.
[29] O. O. Ajani, D. V. Aderohunmu, C. O. Ikpo, A. E. Adedapo, I. O.
Olanrewaju, Arch. Pharm. Chem. Life Sci. 2016, 349, 475.
[30] G. Yadav, S. Ganguly, Eur. J. Med. Chem. 2015, 97, 419.
[31] W. Akhtar, M. F. Khan, G. Verma, M. Shaquiquzzaman, M. A. Rizvi, S.
H. Mehdi, M. Akhter, M. M. Alam, Eur. J. Med. Chem. 2017, 126, 705.
[32] C. Karaaslan, Y. Duydu, A. Ustundag, C. O. Yalcin, B. Kaskatepe, H.
Goker, Med. Chem. 2019, 15, 287.
The QikProp module of Schrödinger's Molecular modeling package
(Schrödinger Release 2016‐2, QikProp, Schrödinger) was used to
predict the pharmacokinetic profiles of compounds 3a – i. On the
basis of Lipinski's rule of five and Jorgensen's rule of three, all
compounds were evaluated for their druglikeness and oral
bioavailability.
CONFLICT OF INTERESTS
[33] Y. K. Yoon, M. A. Ali, A. C. Wei, A. N. Shirazi, K. Parang, T. S. Choon,
Eur. J. Med. Chem. 2014, 83, 448.
The authors declare that there are no conflicts of interests.
[34] Y. K. Yoon, M. A. Ali, A. C. Wei, T. S. Choon, H. Osman, K. Parang, A.
N. Shirazi, Bioorg. Med. Chem. 2014, 22, 703.
ORCID
[35] Y. K. Yoon, H. Osman, T. S. Choon, Med. Chem. Commun. 2016, 7, 2094.
[36] M. O. Radwan, D. Takaya, R. Koga, K. Iwamaru, H. Tateishi, T. F. S. Ali,
A. Takaori‐Kondo, M. Otsuka, T. Honma, M. Fujita, Bioorg. Med. Chem.
2020, 28, 115409.
Belgin Sever
http://orcid.org/0000-0003-4847-9711
Gülşen Akalın Çiftçi
https://orcid.org/0000-0001-5098-8967
http://orcid.org/0000-0002-8159-663X
Mehlika Dilek Altıntop
[37] R. S. Y. Wong, J. Exp. Clin. Cancer Res. 2011, 30, 87.
[38] A. El‐Khattouti, D. Selimovic, Y. Haikel, M. Hassan, J. Cell Death 2013,
6, 37.
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How to cite this article: Sever B, Akalın Çiftçi G, Altıntop MD.
A new series of benzoxazole‐based SIRT1 modulators for
targeted therapy of non‐small‐cell lung cancer. Arch Pharm.
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