Design, synthesis, in vitro and in silico evaluation of a new series of oxadiazole-based anticancer agents as potential Akt and FAK inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.06.049

In the current work, new 1,3,4-oxadiazole derivatives were synthesized and investigated for their cytotoxic effects on A549 human lung adenocarcinoma, C6 rat glioma and NIH/3T3 mouse embryonic fibroblast cell lines. Compounds 2, 6 and 9 were found to be the most potent anticancer agents against A549 and C6 cell lines and therefore their effects on apoptosis, caspase-3 activation, Akt, FAK, mitochondrial membrane potential and ultrastructural morphological changes were evaluated. N-(5-Nitrothiazol-2-yl)-2-[[5-[((5,6,7,8-tetrahydronaphthalen-2-yl)oxy)methyl]-1,3,4-oxadiazol-2-yl]thio]acetamide (9) increased early and late apoptotic cell population in A549 and C6 cells more than cisplatin and caused more mitochondrial membrane depolarization in both cell lines than cisplatin. On the other hand, N-(6-methoxybenzothiazol-2-yl)-2-[[5-[((5,6,7,8-tetrahydronaphthalen-2-yl)oxy)methyl]-1,3,4-oxadiazol-2-yl]thio]acetamide (6) caused higher caspase-3 activation than cisplatin in both cell lines. Compound 6 showed significant Akt inhibitory activity in both cell lines. Moreover, compound 6 significantly inhibited FAK (Phospho-Tyr397) activity in C6 cell line. Molecular docking simulations demonstrated that compound 6 fitted into the active sites of Akt and FAK with high affinity and substrate-specific interactions. Furthermore, compounds 2, 6 and 9 caused apoptotic morphological changes in both cell lines obtained from micrographs by transmission electron microscopy. A computational study for the prediction of ADME properties of all compounds was also performed. These compounds did not violate Lipinski's rule, making them potential orally bioavailable anticancer agents.

Full text of DOI:10.1016/j.ejmech.2018.06.049

Accepted Manuscript
Design, synthesis, in vitro and in silico evaluation of a new series of oxadiazole-based
anticancer agents as potential Akt and FAK inhibitors
Mehlika Dilek Altıntop, Belgin Sever, Gülşen Akalın Çiftçi, Gülhan Turan-Zitouni, Zafer
Asım Kaplancıklı, Ahmet Özdemir
PII:
S0223-5234(18)30540-3
10.1016/j.ejmech.2018.06.049
EJMECH 10519
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 April 2018
Revised Date: 19 June 2018
Accepted Date: 21 June 2018
Please cite this article as: M.D. Altıntop, B. Sever, Güş.Akalı. Çiftçi, Gü. Turan-Zitouni, Zafer.Ası.
Kaplancıklı, A. Özdemir, Design, synthesis, in vitro and in silico evaluation of a new series of oxadiazole-
based anticancer agents as potential Akt and FAK inhibitors, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.06.049.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis, in vitro and in silico evaluation of a new series of oxadiazole-based
anticancer agents as potential Akt and FAK inhibitors
a
b
a
Mehlika Dilek Altıntop a,*, Belgin Sever , Gülşen Akalın Çiftçi , Gülhan Turan-Zitouni ,
Zafer Asım Kaplancıklı a, Ahmet Özdemir a
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University,
26470 Eskişehir, Turkey
b
Department of Biochemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir,
Turkey
* Corresponding author.
E-mail address: mdaltintop@anadolu.edu.tr (M.D. Altıntop).
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ABSTRACT
In the current work, new 1,3,4-oxadiazole derivatives were synthesized and investigated for
their cytotoxic effects on A549 human lung adenocarcinoma, C6 rat glioma and NIH/3T3
mouse embryonic fibroblast cell lines. Compounds 2, 6 and 9 were found to be the most
potent anticancer agents against A549 and C6 cell lines and therefore their effects on
apoptosis, caspase-3 activation, Akt, FAK, mitochondrial membrane potential and
ultrastructural morphological changes were evaluated. N-(5-Nitrothiazol-2-yl)-2-[[5-
[((5,6,7,8-tetrahydronaphthalen-2-yl)oxy)methyl]-1,3,4-oxadiazol-2-yl]thio]acetamide
(9)
increased early and late apoptotic cell population in A549 and C6 cells more than cisplatin
and caused more mitochondrial membrane depolarization in both cell lines than cisplatin. On
the other hand, N-(6-methoxybenzothiazol-2-yl)-2-[[5-[((5,6,7,8-tetrahydronaphthalen-2-
yl)oxy)methyl]-1,3,4-oxadiazol-2-yl]thio]acetamide (6) caused higher caspase-3 activation
than cisplatin in both cell lines. Compound 6 showed significant Akt inhibitory activity in
both cell lines. Moreover, compound 6 significantly inhibited FAK (Phospho-Tyr397) activity
in C6 cell line. Molecular docking simulations demonstrated that compound 6 fitted into
the active sites of Akt and FAK with high affinity and substrate-specific interactions.
Furthermore, compounds 2, 6 and 9 caused apoptotic morphological changes in both cell lines
obtained from micrographs by transmission electron microscopy. A computational study for
the prediction of ADME properties of all compounds was also performed. These compounds
did not violate Lipinski’s rule, making them potential orally bioavailable anticancer agents.
Keywords: Apoptosis, Akt, Benzothiazole, Cancer, FAK, Oxadiazole, Thiazole.
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1. Introduction
Cancer is one of the most formidable afflictions throughout the world [1,2]. By 2030,
the annual number of new cancer diagnoses is projected to be 21 million worldwide, with 17
million patients dying of cancer every year and 75 million people living with cancer
diagnoses [3].
Over the years, the design of chemotherapy has become increasingly sophisticated due
to resistance to existing anticancer drugs and lack of selectivity towards tumor cells [4-6]. In
an attempt to enhance efficacy and selectivity, targeted cancer therapies are designed to
inhibit the growth, progression, and spread of cancer by interfering with specific molecular
targets [7].
Akt pathway is one of the most frequently deregulated signaling pathways in human
cancers. Akt, also known as protein kinase B (PKB), is overexpressed or activated in a variety
of human cancers, including gliomas, lung, breast, ovarian, gastric and pancreatic carcinomas.
Inhibition of Akt signaling results in induction of apoptosis and inhibition of tumor growth.
As a result, inhibition of Akt has long been an attractive therapeutic approach in oncology and
extensive efforts have been devoted to the discovery of new potent and selective anticancer
drugs targeting Akt [7-11]. On the other hand, focal adhesion kinase (FAK, also known as
PTK2) has been recognised as a key regulator of growth factor receptor- and integrin-
mediated signals governing fundamental processes in healthy and cancer cells. FAK is
activated and/or overexpressed in a variety of human cancers, and promotes tumor
progression and metastasis. For this reason, FAK has emerged as an outstanding therapeutic
target for cancer, and several FAK inhibitors have been developed and are being tested in
clinical phase trials [12-16].
In the last few decades, oxadiazoles have been the subject of considerable research
owing to their metabolic profile and ability to engage in hydrogen bonding with receptor site.
Moreover, oxadiazoles have been frequently used in drug-like molecules as bioisosters of
esters and amides [17,18]. Recent studies have pointed out the importance of oxadiazole
scaffold in the field of cancer research. 1,3,4-Oxadiazole derivatives have been reported to
show potent antitumor activity against different cancer cell lines through the inhibition of
different enzymes and growth factors including focal adhesion kinase (FAK), telomerase,
histone deacetylase (HDAC), methionine aminopeptidase (MetAP), thymidylate synthase
(TS), glycogen synthase kinase-3 (GSK), epidermal growth factor (EGF) and vascular
endothelial growth factor (VEGF). Among these derivatives, zibotentan is an anticancer drug
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candidate in late stage clinical trials (Fig. 1). Due to the significant medicinal importance of
oxadiazoles as anticancer agents, oxadiazole has emerged as a promising lead structure for the
generation of new active anticancer agents and in the process of anticancer drug development
[19,20]. On the other hand, the myriad spectrum of therapeutic applications associated with
thiazoles and benzothiazoles has encouraged medicinal chemists to synthesize a large number
of new therapeutic agents [21-30]. In particular, the clinical efficacy of tiazofurin, bleomycins
(BLMs) and dasatinib (Fig. 2) has pointed out the medicinal significance of thiazole scaffold
in the field of current cancer research [21-25]. Recent patents have indicated that thiazole
derivatives show potent antitumor activity against different cancer cell lines through the
inhibition of kinases, pro-matrix metalloproteinase activation, signal transducer and activator
of transcription 3 (STAT3), Bcl-2 family, HDACs [23]. In recent years, benzothiazole has
emerged as a privileged scaffold for anticancer drug discovery. Therefore, antitumor effects
of benzothiazole derivatives on different cancer cells have been extensively studied and these
studies have led to the discovery of clinical candidates such as Phortress (Fig. 2) [25-30].
Prompted by the afore-mentioned findings and in the continuation of our ongoing
research on oxadiazoles related to their anticancer activity [31], herein we described the
design, synthesis of a new series of thiazole/benzothiazole-based oxadiazole derivatives (Fig.
3) and investigated their cytotoxic activity against A549 human lung adenocarcinoma, C6 rat
glioma and NIH/3T3 mouse embryonic fibroblast cell lines. The most effective compounds
were evaluated for their effects on apoptosis, caspase-3 activation, Akt, FAK, mitochondrial
membrane potential and morphological changes in TEM micrographs. The most potent Akt
and FAK inhibitor in this series was also analyzed for molecular docking interactions in the
active sites of Akt (PDB code: 3OW4) and FAK (PDB code: 5TO8), respectively.
Furthermore, a computational study was carried out to determine the physicochemical
parameters of all compounds for the evaluation of their compliance to Lipinski’s rule of five.
2. Results and Discussion
The synthesis of oxadiazole derivatives (1-9) followed the general pathway outlined in
Scheme 1. Initially, 2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]acetic acid (A) was
synthesized via the reaction of 5,6,7,8-tetrahydronaphthalen-2-ol with chloroacetic acid in the
presence of sodium hydroxide. The treatment of compound A with ethanol afforded ethyl 2-
[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]acetate (B). The reaction of the ester with hydrazine
hydrate gave 2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]acetohydrazide (C). 5-[[(5,6,7,8-
Tetrahydronaphthalen-2-yl)oxy]methyl]-1,3,4-oxadiazole-2(3H)-thione (D) was obtained via
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the ring closure reaction of compound C with carbon disulfide in the presence of potassium
hydroxide. On the other hand, 2-chloro-N-(thiazol/benzothiazol-2-yl)acetamide derivatives
were synthesized via the reaction of 2-aminothiazoles/2-aminobenzothiazoles with
chloroacetyl chloride in the presence of triethylamine (TEA). In the last step, the nucleophilic
substitution reaction of compound D with 2-chloro-N-(thiazol/benzothiazol-2-yl)acetamides
in the presence of potassium carbonate afforded the target compounds (1-9). IR, 1H NMR, 13C
NMR, and mass spectral data were in agreement with the proposed structures of compounds
1-9.
In the research for new anticancer agents, the most common screening methods are the
screening tests against a panel of different cancer cell lines. In this study, MTT assay was
carried out to determine the cytotoxic effects of the compounds on A549 human lung
adenocarcinoma and C6 rat glioma cell lines (Table 1). Compounds 1, 2, 4, 5, 6, 7 and 9
exhibited more significant cytotoxic activity against C6 cells than A549 cells, whereas
compounds 3 and 8 were more effective on A549 cells than C6 cells.
Compound 6 was found to be the most promising anticancer agent in this series due to
its significant inhibitory effects on C6 and A549 cell lines with IC50 values of 4.63±0.85 µM
and 39.33±4.04 µM, respectively. This compound was also more effective than cisplatin on
both cell lines. This outcome indicated that the methoxy substituent at the 6th position of the
benzothiazole ring significantly enhanced anticancer activity against both cell lines.
Compounds 9 and 2 exhibited notable cytotoxic activity against C6 cell line with IC50
values of 66.67±12.58 and 89.33±14.47 µM, respectively. On the other hand, compounds 2
and 9 also showed anticancer activity against A549 cell line with IC50 values of 91.67±7.64
and 115±5 µM, respectively.
MTT assay revealed that the chloro substituent at the 6th position of the benzothiazole
ring decreased anticancer activity, whereas other substituents increased anticancer activity
against C6 cell line. The ethoxy substituent at the 6th position of the benzothiazole ring
decreased anticancer activity, whilst other substituents increased anticancer activity against
A549 cell line.
Considering the anticancer effects of thiazole-substituted compounds 8 and 9 on A549
and C6 cell lines, it can be concluded that the nitro substituent at the 5th position of the
thiazole ring increased anticancer activity against both cancer cells.
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Toxicity to host cells is an important characteristic to assess the safety of drug
candidates early in the drug discovery process. In order to evaluate whether the compounds
were toxic or non-toxic to healthy cells, the viability of NIH/3T3 mouse embryonic fibroblast
cells exposed to each compound was assessed using MTT assay (Table 1). The selectivity
index (SI) values of the compounds were also determined to compare the selectivity of the
compounds (Table 2). Compounds 2, 6 and 9 were chosen for further studies due to their
notable and selective antitumor effects on both cancer cells.
After 24 h incubation period, the apoptotic effects of compounds 2, 6 and 9 were
analyzed for A549 and C6 cells based on Annexin V-PI binding capacities in flow cytometry
(Figs. 4 and 5). Following flow cytometric analyses, the early and late apoptotic effects of
compounds 2, 6 and 9 (for IC50 doses) on C6 cell line were determined as 11.9%, 5.0% and
12.3%, while their viabilities were determined as 84.9%, 92.3%, 80.8%, respectively (Table 3,
Fig. 4). On the other hand, the early and late apoptotic effects of compounds 2, 6 and 9 (for
IC50 doses) on A549 cell line were determined as 17.0%, 12.2% and 25.3%, whereas their
viabilities were determined as 79.1%, 71.7%, and 67.8%, respectively (Table 3, Fig. 5).
According to these findings, compounds 2 and 9 (11.9 and 12.3%) showed similar apoptotic
effects on C6 cells compared to cisplatin (11.4%). Compound 9 was also the most effective
apoptotic compound (25.3%) on A549 cells compared to cisplatin (17.8%).
In order to investigate the effects of compounds 2, 6 and 9 on mitochondrial
membrane potential (MMP) of A549 and C6 cells, the cells were incubated by IC50
concentrations of these compounds for 24 hours (Figs. 6 and 7, Table 4).
Compounds 2, 6 and 9 caused higher disturbance on mitochondrial membrane
potential in C6 cells than A549 cells. Mitochondrial membrane polarized C6 cell percentages
of compounds 2, 6, 9 and cisplatin (for IC50 doses) were determined as 32.6, 24.4, 0.6 and
45.1, while mitochondrial membrane depolarized C6 cell percentages of compounds 2, 6, 9
and cisplatin (for IC50 doses) were determined as 60.1, 74.4, 98.0 and 55.0, respectively (Fig.
6 and Table 4). On the other hand, mitochondrial membrane polarized A549 cell percentages
of compounds 2, 6, 9 and cisplatin (for IC50 doses) were determined as 78.2, 69.8, 2.4 and
90.0, whilst mitochondrial membrane depolarized A549 cell percentages of compounds 2, 6, 9
and cisplatin (for IC50 doses) were determined as 21.0, 27.9, 95.5 and 10.0, respectively (Fig.
7 and Table 4). According to these findings, compounds 2, 6 and 9 were more effective than
cisplatin on the depolarization of mitochondrial membrane in A549 and C6 cells.
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Due to the key role of caspase-3 activation in the initiation of cellular events during
early apoptotic process [32,33], the effects of compounds 2, 6 and 9 on caspase-3 activation
were determined. Caspase-3 positive cell percentages of compounds 2, 6, 9 and cisplatin (for
IC50 doses) were determined as 11.7, 33.9, 11.3 and 9.6, whereas caspase-3 negative cell
percentages of compounds 2, 6, 9 and cisplatin (for IC50 doses) were determined as 90.8, 69.8,
90.1 and 91.6 respectively, on C6 cell line (Fig. 8 and Table 5). On the other hand, caspase-3
(+) cell percentages of compounds 2, 6, 9 and cisplatin (for IC50 doses) were determined as
7.1, 25.0, 5.4 and 10.5, whilst caspase-3 (-) cell percentages of compounds 2, 6, 9 and
cisplatin (for IC50 doses) were determined as 93.5, 76.4, 95.1, and 90.4, respectively, on A549
cell line (Fig. 9 and Table 5). These findings indicated that compound 6 was the most
effective agent on caspase-3 activation in both cell lines.
As a consequence of the central role of Akt signaling in cancer [7-11], compounds 2, 6
and 9 were investigated for their inhibitory effects on Akt activity (Table 6). Compound 6
inhibited Akt activity with an IC50 value of 2.60±0.17 µM in C6 cell line more than cisplatin
(IC50= 39.00±1.41 µM) and GSK690693 (IC50= 6.97±0.06 µM). Compound 6 also showed
significant Akt inhibitory activity with an IC50 value of 27.50±0.71 µM in A549 cell line
when compared with cisplatin (IC50= 34.00±1.73 µM) and GSK690693 (IC50= 17.33±2.87
µM). On the other hand, compound 9 inhibited Akt kinase activity (IC50= 44.50±3.53 µM) in
C6 cell line similar to cisplatin. On the other hand, the significance of 1,3,4-oxadiazoles as
FAK inhibitors [17-20] prompted us to evaluate the inhibitory effects of compounds 2, 6, 9 on
FAK (Phospho-Tyr397) activity (Table 7). According to the assay, compound 6 showed
significant FAK (Phospho-Tyr397) inhibitory activity with an IC50 value of 19.50±2.12 µM in
C6 cell line when compared with cisplatin (IC50= 59.00±1.41 µM). However, compounds 2
and 9 did not cause significant FAK inhibition on C6 cells. This outcome pointed out the
importance of the methoxy substituent at the 6th position of the benzothiazole ring for Akt and
FAK inhibitory activities.
Molecular docking studies were performed to rationalize the observations of the
biological activity of compound 6 to elucidate the possible binding modes of this compound
in the active site of Akt when compared with GSK690693, a potent pan-AKT kinase inhibitor
(PDB code: 3OW4) [34]. The docking results of compound 6 suggested that π-π interactions
and hydrogen bonds were responsible for the observed affinity in the active site of Akt (Fig.
10). 6-Methoxybenzothiazole moiety of compound 6 presented π-π interactions with Arg4,
Phe442 residues and H-bond with Asp439 residue. As compound 6 notably inhibited FAK, it
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was also docked to the active site of FAK (PDB code: 5TO8) [35]. Molecular docking results
indicated that 6-methoxybenzothiazole moiety of compound 6 formed π-π stacking with
Phe568 residue and its acetamido group and oxadiazole moiety presented salt-bridge
formation and π-cation with Lys457 residue in the active site of FAK (Fig. 11). The docking
results of compound 6 in the active site of Akt and FAK pointed out that the presence of 6-
methoxybenzothiazole moiety of this compound enhanced its inhibitory effects on these
enzymes. Due to its high potency as a caspase-3 activator in this series, compound 6 was also
analyzed for molecular docking interactions in the active site of caspase-3 (PDB codes: 4EHA
and 4QTX) [36, 37]. Docking results of compound 6 in the active site of caspase-3 also
indicated that compound 6 showed good binding affinity. 6-Methoxybenzothiazole,
oxadiazole and tetrahydronaphthalene moieties of compound 6 presented π-π stacking and H-
bonds with Arg207, Thr62 and Phe256 residues, respectively. However, the acetamido group
of compound 6 was engaged in salt-bridge formation and H-bond with Arg207 in the active
site of caspase-3 (Fig. 12). The docking score, glide gscore and glide emodel results of
compound 6 were also given in Table 8.
The apoptosis results were consistent with TEM micrographs. At IC50 concentrations
of compounds 2, 6 and 9, the shape of the treated cells became rounded, the cellular
organization was disrupted, cell shrinkage, DNA condensation were observed and the
membranes of organelles were sharply damaged indicating morphological changes of
apoptosis in both cell lines (Figs. 13 and 14). The large vacuoles in the cytoplasm containing
degraded cellular material also increased and the membranes were slightly damaged and
nucleus fragmentation in A549 cell line incubated with compound 6 was shown (Fig. 13c).
Furthermore, compound 6 significantly induced apoptotic morphological alterations causing
chromatin condensation, large vacuoles and sharply damaged membranes in C6 cell line (Fig.
14c).
As a part of this study, Molinspiration software was used to determine their
physicochemical parameters (log P, TPSA, nrotb, molecular weight, number of hydrogen
bond donors and acceptors, molecular volume) for the evaluation of the compliance of the
compounds to Lipinski’s rule of five [38]. This rule states that most “drug like” molecules
have log P ≤ 5, molecular weight ≤ 500, number of hydrogen bond acceptors ≤ 10, and
number of hydrogen bond donors ≤ 5. Compounds violating more than one of these rules may
have bioavailability problems [38-41]. According to in silico studies, compound 3 only
violated one parameter of Lipinski’s rule of five, whereas other compounds did not violate
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Lipinski’s rule (Table 9). On the basis of Lipinski’s rule of five, they were expected to have
good oral bioavailability.
3. Conclusion
In conclusion, new thiazole/benzothiazole-based 1,3,4-oxadiazole derivatives were
synthesized and evaluated for their cytotoxic effects on A549, C6 and NIH/3T3 cell lines.
Compounds 2, 6 and 9 were evaluated for their effects on apoptosis, caspase-3 activation,
Akt, FAK, MMP and ultrastructural morphological changes.
In general, the compounds displayed more potent inhibitory effects on C6 cells than
A549 cells. Compounds 2, 6 and 9 were the most effective anticancer agents on A549 and C6
cell lines. In particular, compound 6 was identified as the most promising anticancer agent
due to its significant antitumor effects on both cancer cell lines. Compound 6 increased
caspase-3 cell population more than cisplatin on A549 and C6 cell lines. Compound 6 also
showed significant Akt and FAK inhibitory activities. Docking studies confirmed that
compound 6 showed high affinity to the active site of Akt comparing with GSK690693. This
compound was able to form π-π interactions and hydrogen bond with proper residues.
Docking results also confirmed that compound 6 formed reasonable interactions with Lys457
and Phe568 residues in the active site of FAK. Overall, the docking results were
fundamentally in agreement with the biological data. Although all compounds caused
apoptotic morphological alterations, compound 6 caused more significant morphological
changes than other compounds on both cell lines. According to the in vitro and in silico
studies, compound 6 stands out as a promising orally bioavailable anticancer drug candidate
for further in vivo studies.
4. Experimental
4.1. Chemistry
All reagents were purchased from commercial suppliers and were used without further
purification. The melting points (M.p.) of the compounds were determined on an
Electrothermal 9100 melting point apparatus (Weiss-Gallenkamp, Loughborough, UK) and
are uncorrected. IR spectra were recorded on an IRPrestige-21 Fourier Transform Infrared
1
spectrophotometer (Shimadzu, Tokyo, Japan). H NMR spectra were recorded on a Bruker
spectrometer (Bruker, Billerica, MA, USA), whereas 13C NMR spectra were recorded on a
Varian Mercury-400 FT-NMR spectrometer (Agilent, Palo Alto, CA, USA). Mass spectra
were recorded on a Shimadzu LCMS-IT-TOF system (Shimadzu, Kyoto, Japan). Thin Layer
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Chromatography (TLC) was performed on TLC Silica gel 60 F254 aluminium sheets (Merck,
Darmstadt, Germany) to check the purity of the compounds.
4.1.1. General procedure for the synthesis of the compounds
4.1.1.1. 2-[(5,6,7,8-Tetrahydronaphthalen-2-yl)oxy]acetic acid (A)
Sodium 2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]acetate was obtained via the
reaction of 5,6,7,8-tetrahydro-2-naphthol (0.05 mol) with chloroacetic acid (0.05 mol) in
NaOH solution (0.12 mol in 25 mL water) under reflux for 6 h. Then the salt solution was
acidified with sulphuric acid until compound A was precipitated. The solid was filtered off,
washed with water and dried. The product was crystallized from ethanol [42].
4.1.1.2. Ethyl 2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]acetate (B)
A solution of (5,6,7,8-tetrahydronaphthalen-2-yl)oxyacetic acid (A) (0.04 mol) in
ethanol (70 mL) in the presence of concentrated sulfuric acid (1 mL) was refluxed for 6 h.
The obtained ester was poured into distilled water. The ester layer was extracted with ether.
Then, the ether layer was washed with sodium bicarbonate solution and water. Ether layer
was extracted again and dried with anhydrous sodium sulphate. Finally, organic ether phase
was evaporated to obtain the product [42].
4.1.1.3. 2-[(5,6,7,8-Tetrahydronaphthalen-2-yl)oxy]acetohydrazide (C)
A mixture of ethyl 2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]acetate (B) (0.03 mol)
and hydrazine hydrate (0.06 mol) in ethanol (50 mL) was refluxed for 2 h and the resulting
solid product was filtered and dried. The product was crystallized from ethanol [42].
4.1.1.4. 5-[[(5,6,7,8-Tetrahydronaphthalen-2-yl)oxy]methyl]-1,3,4-oxadiazole-2(3H)-thione
(D)
A mixture of [(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]acetohydrazide (C) (0.025 mol)
and carbon disulfide (0.03 mol) in the presence of potassium hydroxide (0.025 mol) in ethanol
(50 mL) was refluxed for 6 h. The solution was cooled and acidified with hydrochloric acid
solution. The solid was filtered off, washed with water and dried. The product was
crystallized from ethanol.
4.1.1.5. 2-Chloro-N-(aryl)acetamides
Chloroacetyl chloride (0.03 mol) was added dropwise with stirring to a mixture of
aromatic amine (0.025 mol) and TEA (0.025 mol) in toluene (50 mL) at 0-5 °C. The solvent
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was evaporated under reduced pressure. The residue was washed with water to remove TEA
and crystallized from ethanol. The products were crystallized from ethanol [43].
4.1.1.6. N-(Aryl)-2-[[5-[((5,6,7,8-tetrahydronaphthalen-2-yl)oxy)methyl]-1,3,4-oxadiazol-2-
yl]thio]acetamide (1-9)
A mixture of 5-[[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]methyl]-1,3,4-oxadiazole-
2(3H)-thione (D) (0.0015 mol) and appropriate 2-chloro-N-(aryl)acetamide (0.0015 mol) in
acetone (25 mL) was stirred at room temperature for 8 h in the presence of potassium
carbonate (0.0015 mol). The solvent was evaporated under reduced pressure. The residue was
washed with water and crystallized from ethanol.
4.1.1.6.1.
N-(Benzothiazol-2-yl)-2-[[5-[((5,6,7,8-tetrahydronaphthalen-2-yl)oxy)methyl]-
1,3,4-oxadiazol-2-yl]thio]acetamide (1)
Yield: 85%. M.p. 214-215 °C.
IR νmax (cm-1): 3174.83, 3138.18 (N-H stretching), 3061.03 (aromatic C-H stretching),
2964.59, 2929.87, 2860.43 (aliphatic C-H stretching), 1681.93 (amide C=O stretching),
1598.99, 1562.34, 1500.62, 1481.33 (N-H bending, C=N and C=C stretching), 1438.90,
1381.03 (C-H bending), 1325.10, 1259.52, 1232.51, 1165.00, 1122.57, 1028.06, 1012.63 (C-
N, C-O stretching and aromatic C-H in plane bending), 987.55, 869.90, 823.60, 792.74,
758.02, 690.52, 671.23 (aromatic C-H out of plane bending and C-S stretching).
1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.68 (s, 4H), 2.62 (d, J= 16 Hz, 4H), 4.47 (s, 2H),
5.30 (s, 2H), 6.71-6.75 (m, 2H), 6.93 (d, J= 8.5 Hz, 1H), 7.33 (t, J= 7.5 Hz, 1H), 7.46 (t, J=
7.5 Hz, 8.0 Hz, 1H), 7.78 (d, J= 8.0 Hz, 1H), 7.99 (d, J= 8.0 Hz, 1H), 12.79 (s, 1H).
13C NMR (100 MHz, DMSO-d6) δ (ppm): 22.67 (d, J= 26.6 Hz, 2CH2), 27.91 (CH2), 28.95
(CH2), 35.65 (CH2), 59.40 (CH2), 114.67 (2CH), 120.66 (CH), 121.74 (CH), 123.73 (CH),
126.20 (CH), 129.79 (CH), 129.94 (C), 131.44 (C), 137.82 (C), 148.41 (C), 154.95 (C),
157.64 (C), 164.01 (C), 164.06 (C), 166.21 (C).
HRMS (ESI) (m/z): [M+H]+ calcd. for C22H20N4O3S2: 453.1050, found: 453.1040.
4.1.1.6.2.
N-(6-Fluorobenzothiazol-2-yl)-2-[[5-[((5,6,7,8-tetrahydronaphthalen-2-
yl)oxy)methyl]-1,3,4-oxadiazol-2-yl]thio]acetamide (2)
Yield: 81%. M.p. 219-220 °C.
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IR νmax (cm-1): 3188.33 (N-H stretching), 3091.89 (aromatic C-H stretching), 2931.80,
2879.72 (aliphatic C-H stretching), 1681.93 (amide C=O stretching), 1610.56, 1570.06,
1500.62, 1481.33, 1456.26 (N-H bending, C=N and C=C stretching), 1381.03 (C-H bending),
1336.67, 1259.52, 1246.02, 1197.79, 1163.08, 1120.64, 1055.06, 1037.70, 1002.98 (C-N, C-O
stretching and aromatic C-H in plane bending), 977.91, 912.33, 891.11, 854.47, 794.67,
746.45, 692.44 (aromatic C-H out of plane bending and C-S stretching).
1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.67-1.68 (m, 4H), 2.62 (d, J= 15.5 Hz, 4H), 4.47
(s, 2H), 5.30 (s, 2H), 6.70-6.75 (m, 2H), 6.93 (d, J= 8.5 Hz, 1H), 7.31 (td, J= 3.0 Hz, 2.5 Hz,
1H), 7.79 (dd, J= 9.0 Hz, J= 5.0 Hz, 1H), 7.91 (dd, J= 8.75 Hz, J= 3.0 Hz, 2.5 Hz, 1H), 12.82
(brs, 1H).
13C NMR (100 MHz, DMSO-d6) δ (ppm): 22.60 (d, J= 26.7 Hz, 2CH2), 27.85 (CH2), 28.89
(CH2), 35.55 (CH2), 59.42 (CH2), 108.10 (d, J= 27.5 Hz, CH), 112.46 (CH), 114.24 (d, J=
24.3 Hz, CH), 114.67 (CH), 121.74 (d, J= 9.1 Hz, CH), 129.71 (CH), 129.93 (C), 132.67 (d,
J= 11.4 Hz, C), 137.78 (C), 145.12 (C), 154.93 (C), 157.53 (d, J= 8.4 Hz, C), 159.88 (C),
163.94 (C), 163.98 (C), 166.24 (C).
HRMS (ESI) (m/z): [M+H]+ calcd. for C22H19FN4O3S2: 471.0955, found: 471.0940.
4.1.1.6.3.
N-(6-Chlorobenzothiazol-2-yl)-2-[[5-[((5,6,7,8-tetrahydronaphthalen-2-
yl)oxy)methyl]-1,3,4-oxadiazol-2-yl]thio]acetamide (3)
Yield: 88%. M.p. 208-209 °C.
IR νmax (cm-1): 3174.83, 3138.18 (N-H stretching), 3080.32, 3055.24 (aromatic C-H
stretching), 2929.87, 2858.51 (aliphatic C-H stretching), 1681.93 (amide C=O stretching),
1602.85, 1568.13, 1500.62, 1471.69 (N-H bending, C=N and C=C stretching), 1438.90,
1382.96 (C-H bending), 1336.67, 1263.37, 1232.51, 1170.79, 1122.57, 1097.50, 1056.99,
1035.77, 1002.98 (C-N, C-O stretching and aromatic C-H in plane bending), 987.55, 975.98,
866.04, 808.17, 761.88, 688.59 (aromatic C-H out of plane bending and C-S stretching).
1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.68 (s, 4H), 2.62 (d, J= 10.5 Hz, 4H), 4.46 (s, 2H),
5.30 (s, 2H), 6.70-6.74 (m, 2H), 6.93 (d, J= 7.5 Hz, 1H), 7.48 (d, J= 7.0 Hz, 2H), 8.14 (s, 1H),
12.88 (s, 1H).
13C NMR (100 MHz, DMSO-d6) δ (ppm): 22.66 (d, J= 26.7 Hz, 2CH2), 27.91 (CH2), 28.94
(CH2), 35.59 (CH2), 59.39 (CH2), 112.46 (CH), 114.65 (CH), 121.46 (CH), 121.87 (CH),
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126.55 (CH), 127.81 (C), 129.77 (CH), 129.92 (C), 133.14 (C), 137.80 (C), 147.31 (C),
154.95 (C), 158.47 (C), 164.01 (2C), 166.42 (C).
HRMS (ESI) (m/z): [M+H]+ calcd. for C22H19ClN4O3S2: 487.0660, found: 487.0652.
4.1.1.6.4.
N-(6-Nitrobenzothiazol-2-yl)-2-[[5-[((5,6,7,8-tetrahydronaphthalen-2-
yl)oxy)methyl]-1,3,4-oxadiazol-2-yl]thio]acetamide (4)
Yield: 90%. M.p. 212-213 °C.
IR νmax (cm-1): 3172.90, 3138.18 (N-H gstretching), 3080.32, 3049.46 (aromatic C-H
stretching), 2926.01, 2858.51 (aliphatic C-H stretching), 1695.43 (amide C=O stretching),
1610.56, 1575.84, 1558.48, 1521.84, 1500.62, 1467.83 (N-H bending, NO2, C=N ve C=C
stretching), 1446.61, 1381.03 (C-H bending), 1334.74, 1269.16, 1232.51, 1166.93, 1124.50,
1058.92, 1039.63 (C-N, C-O stretching and aromatic C-H in plane bending), 985.62, 902.69,
821.68, 786.96, 742.59, 719.45, 682.80 (aromatic C-H out of plane bending and C-S
stretching).
1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.66 (s, 4H), 2.60 (d, J= 16.5 Hz, 4H), 4.50 (s, 2H),
5.29 (s, 2H), 6.70-6.73 (m, 2H), 6.91 (d, J= 8.5 Hz, 1H), 7.91 (d, J= 8.5 Hz, 1H), 8.28 (d, J=
8.5 Hz, 1H), 9.04 (s, 1H), 13.19 (s, 1H).
13C NMR (100 MHz, DMSO-d6) δ (ppm): 22.66 (d, J= 26.7 Hz, 2CH2), 27.90 (CH2), 28.95
(CH2), 35.69 (CH2), 59.39 (CH2), 112.44 (CH), 114.62 (CH), 119.02 (CH), 120.74 (CH),
121.74 (CH), 129.76 (CH), 129.91 (C), 132.19 (C), 137.79 (C), 143.07 (C), 153.31 (C),
154.94 (C), 163.16 (C), 163.98 (C), 164.05 (C), 167.02 (C).
HRMS (ESI) (m/z): [M+H]+ calcd. for C22H19N5O5S2: 498.0900, found: 498.0883.
4.1.1.6.5.
N-(6-Methylbenzothiazol-2-yl)-2-[[5-[((5,6,7,8-tetrahydronaphthalen-2-
yl)oxy)methyl]-1,3,4-oxadiazol-2-yl]thio]acetamide (5)
Yield: 77%. M.p. 218-219 °C.
IR νmax (cm-1): 3184.48, 3155.54 (N-H stretching), 3072.60 (aromatic C-H stretching),
2972.31, 2933.73, 2860.43 (aliphatic C-H stretching), 1683.86 (amide C=O stretching),
1608.63, 1573.91, 1556.55, 1500.62, 1479.40, 1460.11 (N-H bending, C=N and C=C
stretching), 1381.03 (C-H bending), 1330.88, 1261.45, 1230.58, 1165.00, 1120.64, 1056.99,
1033.85, 1004.91 (C-N, C-O stretching and aromatic C-H in plane bending), 989.48, 819.75,
792.74, 746.45, 690.52, 665.44 (aromatic C-H out of plane bending and C-S stretching).
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1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.68 (s, 4H), 2.42 (s, 3H), 2.63 (d, J= 14.5 Hz, 4H),
4.45 (s, 2H), 5.30 (s, 2H), 6.71-6.75 (m, 2H), 6.94 (d, J= 8.0 Hz, 1H), 7.27 (d, J= 7.5 Hz, 1H),
7.66 (d, J= 8.0 Hz, 1H), 7.77 (s, 1H), 12.70 (s, 1H).
13C NMR (100 MHz, DMSO-d6) δ (ppm): 20.86 (CH3), 22.59 (d, J= 26.7 Hz, 2CH2), 27.84
(CH2), 28.88 (CH2), 35.57 (CH2), 59.42 (CH2), 112.46 (CH), 114.69 (CH), 120.21 (CH),
121.21 (CH), 127.44 (CH), 129.70 (CH), 129.93 (C), 131.54 (C), 133.17 (C), 137.77 (C),
154.93 (2C), 157.64 (C), 163.95 (2C), 166.42 (C).
HRMS (ESI) (m/z): [M+H]+ calcd. for C23H22N4O3S2: 467.1206, found: 467.1190.
4.1.1.6.6.
N-(6-Methoxybenzothiazol-2-yl)-2-[[5-[((5,6,7,8-tetrahydronaphthalen-2-
yl)oxy)methyl]-1,3,4-oxadiazol-2-yl]thio]acetamide (6)
Yield: 79%. M.p. 207-208 °C.
IR νmax (cm-1): 3192.19 (N-H stretching), 3089.96 (aromatic C-H stretching), 2927.94
(aliphatic C-H stretching), 1681.93 (amide C=O stretching), 1610.56, 1579.70, 1562.34,
1500.62, 1469.76 (N-H bending, C=N and C=C stretching), 1429.25, 1382.96 (C-H bending),
1338.60, 1257.59, 1224.80, 1170.79, 1122.57, 1056.99, 1028.06, 1002.98 (C-N, C-O
stretching and aromatic C-H in plane bending), 821.68, 796.60, 748.38, 692.44 (aromatic C-H
out of plane bending and C-S stretching).
1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.68 (s, 4H), 2.62 (d, J= 15.5 Hz, 4H), 3.81 (s, 3H),
4.44 (s, 2H), 5.30 (s, 2H), 6.71-6.75 (m, 2H), 6.94 (d, J= 8.5 Hz, 1H), 7.04-7.06 (m, 1H),
7.57-7.58 (m, 1H), 7.67 (d, J= 8.5 Hz, 1H), 12.65 (s, 1H).
13C NMR (100 MHz, DMSO-d6) δ (ppm): 22.63 (d, J= 26.0 Hz, 2CH2), 27.88 (CH2), 28.92
(CH2), 35.56 (CH2), 55.59 (CH3), 59.41 (CH2), 104.71 (CH), 112.48 (CH), 114.68 (CH),
114.99 (CH), 121.24 (CH), 129.75 (CH), 129.94 (C), 132.76 (C), 137.80 (C), 142.48 (C),
154.94 (C), 155.51 (C), 156.25 (C), 163.97 (C), 164.01 (C), 165.82 (C).
HRMS (ESI) (m/z): [M+H]+ calcd. for C23H22N4O4S2: 483.1155, found: 483.1139.
4.1.1.6.7.
N-(6-Ethoxybenzothiazol-2-yl)-2-[[5-[((5,6,7,8-tetrahydronaphthalen-2-
yl)oxy)methyl]-1,3,4-oxadiazol-2-yl]thio]acetamide (7)
Yield: 78%. M.p. 216-217 °C.
IR νmax (cm-1): 3196.05 (N-H stretching), 3088.03 (aromatic C-H stretching), 2983.88,
2929.87, 2881.65 (aliphatic C-H stretching), 1685.79 (amide C=O stretching), 1610.56,
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1560.41, 1502.55, 1479.40, 1458.18 (N-H bending, C=N and C=C stretching), 1386.82 (C-H
bending), 1342.46, 1263.37, 1249.87, 1228.66, 1161.15, 1111.00, 1060.85, 1029.99 (C-N, C-
O stretching and aromatic C-H in plane bending), 999.13, 941.26, 891.11, 850.61, 808.17,
748.38, 694.37, 665.44 (aromatic C-H out of plane bending and C-S stretching).
1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.11 (t, J= 7.0 Hz, 3H), 1.43 (s, 4H), 2.38 (d, J=
15.5 Hz, 4H), 3.82 (q, J= 7.0 Hz, 2H), 4.18 (s, 2H), 5.05 (s, 2H), 6.47-6.50 (m, 2H), 6.69 (d,
J= 8.0 Hz, 1H), 6.77-6.79 (m, 1H), 7.29-7.30 (m, 1H), 7.40 (d, J= 8.5 Hz, 1H), 12.38 (brs,
1H).
13C NMR (100 MHz, DMSO-d6) δ (ppm): 14.59 (CH3), 22.61 (d, J= 26.6 Hz, 2CH2), 27.86
(CH2), 28.89 (CH2), 35.65 (CH2), 59.42 (CH2), 63.58 (CH2), 105.00 (CH), 112.48 (CH),
114.69 (CH), 115.28 (CH), 121.16 (CH), 129.72 (CH), 129.93 (C), 132.76 (C), 137.79 (C),
142.44 (C), 154.94 (C), 155.40 (C), 155.73 (C), 163.93 (C), 164.01 (C), 165.89 (C).
HRMS (ESI) (m/z): [M+H]+ calcd. for C24H24N4O4S2: 497.1312, found: 497.1299.
4.1.1.6.8.
N-(Thiazol-2-yl)-2-[[5-[((5,6,7,8-tetrahydronaphthalen-2-yl)oxy)methyl]-1,3,4-
oxadiazol-2-yl]thio]acetamide (8)
Yield: 83%. M.p. 204-205 °C.
IR νmax (cm-1): 3199.91 (N-H stretching), 3072.60 (aromatic C-H stretching), 2931.80,
2850.79, 2752.42 (aliphatic C-H stretching), 1691.57 (amide C=O stretching), 1591.27,
1502.55, 1463.97 (N-H bending, C=N and C=C stretching), 1436.97, 1382.96 (C-H bending),
1332.81, 1309.67, 1267.23, 1246.02, 1232.51, 1170.79, 1161.15, 1107.14, 1049.28, 1029.99
(C-N, C-O stretching and aromatic C-H in plane bending), 972.12, 929.69, 823.60, 813.96,
800.46, 775.38, 729.09, 694.37, 623.01 (aromatic C-H out of plane bending and C-S
stretching).
1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.69 (s, 4H), 2.65 (d, J= 21.0 Hz, 4H), 4.40 (s, 2H),
5.30 (s, 2H), 6.73-6.76 (m, 2H), 6.96 (d, J= 8.5 Hz, 1H), 7.26 (d, J= 3.5 Hz, 1H), 7.51 (d, J=
3.5 Hz, 1H), 12.53 (s, 1H).
13C NMR (100 MHz, DMSO-d6) δ (ppm): 23.16 (d, J= 26.7 Hz, 2CH2), 28.41 (CH2), 29.45
(CH2), 35.85 (CH2), 59.92 (CH2), 113.03 (CH), 114.34 (CH), 115.21 (CH), 130.28 (CH),
130.47 (C), 137.79 (CH), 138.32 (C), 155.45 (C), 158.18 (C), 164.45 (C), 164.55 (C), 165.53
(C).
HRMS (ESI) (m/z): [M+H]+ calcd. for C18H18N4O3S2: 403.0893, found: 403.0887.
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4.1.1.6.9.
N-(5-Nitrothiazol-2-yl)-2-[[5-[((5,6,7,8-tetrahydronaphthalen-2-yl)oxy)methyl]-
1,3,4-oxadiazol-2-yl]thio]acetamide (9)
Yield: 82%. M.p. 181-182 °C.
IR νmax (cm-1): 3157.47 (N-H stretching), 2929.87, 2854.65 (aliphatic C-H stretching),
1691.57 (amide C=O stretching), 1593.20, 1573.91, 1519.91, 1475.54 (N-H bending, NO2,
C=N and C=C stretching), 1421.54, 1346.31 (C-H bending), 1301.95, 1255.66, 1168.86,
1122.57, 1055.06, 1037.70 (C-N, C-O stretching and aromatic C-H in plane bending), 966.34,
902.69, 864.11, 823.60, 802.39, 731.02, 694.37 (aromatic C-H out of plane bending and C-S
stretching).
1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.69 (t, J= 3 Hz, 4H), 2.64 (d, J= 21.5 Hz, 4H), 4.39
(s, 2H), 5.30 (s, 2H), 6.72-6.76 (m, 2H), 6.95 (d, J= 8.5 Hz, 1H), 8.58 (s, 1H), 13.19 (s, 1H).
13C NMR (100 MHz, DMSO-d6) δ (ppm): 22.69 (d, J= 26.7 Hz, 2CH2), 27.94 (CH2), 28.98
(CH2), 36.72 (CH2), 59.45 (CH2), 112.54 (CH), 114.71 (CH), 129.80 (CH), 129.99 (C),
137.85 (C), 140.27 (C), 143.59 (CH), 154.99 (C), 163.90 (C), 164.30 (C), 165.39 (C), 169.26
(C).
HRMS (ESI) (m/z): [M+H]+ calcd. for C18H17N5O5S2: 448.0744, found: 448.0738.
4.2. Biochemistry
4.2.1. Cell culture and drug treatment
The cell lines used in this work were obtained from American Type Culture Collection
(ATCC). C6 Rat glioma, A549 human lung adenocarcinoma, and NIH/3T3 mouse embryonic
fibroblast cells were cultured 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, USA) reduction was quantified as previously
described in the literature [44] with small modifications [45]. Cisplatin and GSK690693 were
used as positive controls. Selectivity index (SI) values were also calculated according to the
formula [46] below:
SI= IC50 for normal cell line / IC50 for cancerous cell line
4.2.3. Flow cytometric analyses of apoptosis
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After the incubation of A549 and C6 cells with compounds 2, 6, 9 and cisplatin at IC50
concentrations, phosphatidylserine externalization, which indicates early apoptosis, was
measured by FITC Annexin V apoptosis detection kit (BD Pharmingen, San Jose, CA, USA)
on a BD FACSAria flow cytometer for 24 h. Annexin V staining protocol was applied
according to the manufacturer’s instructions (BD Pharmingen, San Jose, CA, USA). The data
were analyzed by a BD FACSAria flow cytometer using FACSDiva version 6.1.1 software.
4.2.4. Analysis of mitochondrial membrane potential (JC1) by flow cytometry
The cells were seeded in six-well plates at a density of 105 cells/mL, and the IC50
doses of compounds 2, 6, 9 and cisplatin were added to cells. The cells were incubated in 5%
CO2 air-conditioned atmosphere at 37 °C. After 24 h of incubation, mitochondrial membrane
potential protocol was applied according to the manufacturer’s instructions (BD Pharmingen,
San Jose, CA, USA) and analyzed by a BD FACSAria flow cytometer using FACSDiva
version 6.1.1 software.
4.2.5. Flow cytometric analyses of caspase-3
After A549 and C6 cells were incubated with compounds 2, 6, 9 and cisplatin at IC50
concentrations for 24 h, caspase-3 activity measurement protocol was applied according to
manufacturer’s instructions (BD Pharmingen, San Jose, CA, USA). In brief, the cells were
washed with cold phosphate buffer solution (PBS) 1X cells and incubated with 0.5 mL perm
lyse solution for 30 min at room temperature in the dark. Pellets were washed twice with 0.5
mL perm wash buffer. Cells were resuspended in 100 µL perm wash buffer, and 10 µL
caspase-3 antibody was added for 20 min at room temperature in the dark. At least 10,000
cells were counted for each sample and cells were analyzed by flow cytometry using
FACSDiva version 6.1.1 software.
4.2.6. Inhibition of Akt enzyme
After 10,000 cells/well were incubated with compounds 2, 6, 9 and cisplatin at IC50
concentrations for 24 h, in cell ELISA colorimetric Akt activity protocol was applied
according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA).
Briefly, the media was removed and 100 µL of 4% formaldehyde was added to each well. The
plate was incubated in a fume hood at room temperature for 15 minutes. Formaldehyde was
removed and plate was washed twice with 100 µL/well of 1X TBS. 1X TBS was removed,
100 µL/well of 1X permeabilization buffer was added and incubated for 15 minutes at room
temperature. Permeabilization buffer was removed and plate was washed once with 100
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µL/well of 1X TBS. 1X TBS was removed, 100 µL/well quenching solution was added and
incubated at room temperature for 20 minutes. Quenching Solution was removed and plate
was washed once with 100 µL/well of 1X TBS. Then, 1X TBS was removed and 100 µL/well
of blocking buffer was added and incubated at room temperature for 30 minutes. After
blocking buffer was removed, 50 µL/well of primary antibody was added. A plate sealer was
applied and incubated overnight at 4 °C. The primary antibody solution was removed and
plate was washed three times with 100 µL/well of 1X wash buffer. After wash buffer was
removed, 100 µL/well of diluted HRP conjugate was added and incubated for 30 minutes at
room temperature. Wash buffer was removed and 100 µL/well of TMB substrate was added.
Then plate was incubated at room temperature, protected from light. 100 µL/well of TMB
stop solution was added and the absorbance was measured at 450 nm within 30 minutes of
stopping the reaction. The experiment was performed in triplicate wells. The values of blank
wells were subtracted from each well of treated and control cells. Percent Akt activity was
defined as the relative absorbance of treated versus untreated control cells. IC50 values were
determined from graphics using three different concentrations.
4.2.7. FAK (Phospho-Tyr397) Activity Detection by ELISA
The effects of compounds 2, 6, 9 and cisplatin on FAK (Phospho-Tyr397) activity
were determined by Colorimetric Cell-Based ELISA FAK (Phospho-Tyr397) activity kit
(Aviva Systems Biology, San Diego, USA). C6 cells were administrated by IC50
concentrations of compound 2, 6, 9 and cisplatin for 24 hours. Cells were rinsed with 200 µL
of 1 x TBS twice. Cells were fixed by incubating with 100 µL of fixing solution for 20
minutes at room temperature by 4% formaldehyde. Fixing solution was removed and the plate
was washed 3 times with 200 µL 1 x wash buffer for five minutes each time with gentle
shaking on the orbital shaker. Then, 100 µL quenching buffer was added and incubated for 20
minutes at room temperature. Plate was washed 3 times with 1 x wash buffer for 5 minutes at
a time. Then, 200 µl of blocking buffer was added and incubated for 1 hour at room
temperature and washed 3 times with 200 µL of 1 x wash buffer for 5 minutes at a time. 50
µL of 1 x primary antibodies (Anti-FAK (Phospho-Tyr397) antibody, Anti-FAK antibody
and/or Anti-GAPDH antibody) was added to the corresponding wells, covered with parafilm
and incubated for 16 hours (overnight) at 4 °C. Then plate was washed 3 times with 200 µL of
1x wash buffer for 5 minutes at a time. 50 µL of 1 x secondary antibodies (HRP-Conjugated
anti-rabbit IgG and/or HRP-conjugated anti-mouse IgG) was added to corresponding wells
and incubated for 1.5 hours at room temperature with gentle shaking on the shaker. Plate was
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washed 3 times with 200 µL of 1x wash buffer for 5 minutes at a time, and 50 µL of ready-to-
use substrate was added to each well and incubated for 30 minutes at room temperature in the
dark with gentle shaking on the shaker. 50 µL of stop solution was added to each well and
read OD at 450 nm immediately using an ELx808-IU BioTek apparatus. Every concentration
was repeated in double wells. The OD450 values obtained for the phosphorylated FAK was
normalized using the OD450 values obtained for GAPDH. IC50 values were determined from
graphics using three different concentrations.
4.2.8. Transmission Electron Microscopy (TEM)
TEM (TEM FEI Tecnai BioTWIN; Hillsboro, OR, USA) was processed for
ultrastructral changes. C6 and A549 cells grown in DMEM and RPMI, respectively, were
incubated with compounds 2, 6, 9 and cisplatin at IC50 values for 24 h. They were then fixed
with 2.5% glutaraldehyde in 0.1 M PBS at pH 7.4 and left in PBS overnight at 4 °C. After
being embedded in agar and post fixation in 2% osmium tetroxide, cells were dehydrated in
graded ethanol: 70, 90, 96 and 100%. Then cells were embedded in EPON 812 epoxy (Sigma
Aldrich, Seelze, Germany) and sectioned on ultramicrotome (Leica EM UC6; Wetzlar,
Germany).
4.2.9. Statistical analyses
Statistical Package for the Social Sciences (SPSS) for Windows 15.0 was used for
statistical analysis. Data was expressed as Mean ± SD. Comparisons were performed by one
way ANOVA test for normally distributed continuous variables and post hoc analyses of
group differences were expressed by the Tukey test.
4.2.10. Molecular docking studies
Compound 6 was docked to the active site of Akt together with GSK690693, FAK and
caspase-3. Ligands 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, USA) at physiological pH (pH= 7.4) using Optimized
Potential Liquid Simulations (OPLS_2005) force field and crystal structures of Akt, FAK and
caspase-3 were retrieved from Protein Data Bank server (PDB codes: 3OW4 for Akt; 5TO8
for FAK; 4EHA and 4QTX for caspase-3) 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
topologies of compound 6 and GSK690693 in the active sites of target structures [34-37].
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4.2.11. Molinspiration calculations
The physicochemical parameters (log P, TPSA, nrotb, molecular weight, number of
hydrogen bond donors and acceptors, molecular volume) of compounds 1-9 were calculated
using Molinspiration software [37-40].
Acknowledgements
This study was supported by Anadolu University Scientific Research Projects
Commission under the grant no: 1402S054.
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Table 1. IC50 values of the compounds against A549, C6 and NIH/3T3 cells for 24 h.
IC50 (µM)
Compound
C6 cell line
153.33±35.12
89.33±14.47
236.67±23.10
98.33±12.58
108.33±16.07
4.63±0.85
A549 cell line
246.67±50.33
91.67±7.64
NIH/3T3 cell line
96.67±12.58
291.67±59.23
246.67±50.58
335.67±74.54
>403.00
1
2
263.33±47.36
233.33±20.82
210.00±10.00
39.33±4.04
3
4
5
94.33±7.51
>403.00
6
93.33±15.27
213.33±35.12
66.67±12.58
40.67±3.79
283.33±15.28
228.33±44.81
115.00±5.00
70.67±1.15
7
>403.00
8
9
>403.00
>403.00
Cisplatin
14.5±3.54
105.33±5.03
ND
GSK690693
ND: Not Determined.

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Table 2. SI values of the compounds.
SI values*
Compound
C6 cell line
0.63
A549 cell line
0.39
1
3.27
3.18
2
1.04
0.94
3
3.41
1.44
4
3.72
1.92
5
20.37
4.32
2.40
6
1.42
7
1.89
1.76
8
9
6.04
3.50
9.91
5.70
Cisplatin
*
SI= IC50 for normal cell line / IC50 for cancerous cell line. IC50 > 403.00 was accepted as 403.00.

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Table 3. Percents of typical quadrant analysis of annexin V-FITC/propidium iodide flow
cytometry of C6 and A549 cells treated with the compounds.
C6 cell line
A549 cell line
Groups
Early and late
Early and late
Viability %
Viability %
apoptotic cells %
apoptotic cells %
Control
8.0
11.9
5.0
90.0
84.9
92.3
80.8
81.7
7.1
89.0
79.1
71.7
67.8
78.6
Compound 2 treated cells
Compound 6 treated cells
Compound 9 treated cells
Cisplatin treated cells
17.0
12.2
25.3
17.8
12.3
11.4
C6 and A549 cells were cultured for 24 h in medium with IC50 values of compounds. At least 10.000 cells were
analyzed per sample, and quadrant analysis was performed.

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Table 4. The effects of the compounds on mitochondrial membrane potential of C6 and A549
cells for 24 h.
C6 cell line
A549 cell line
P4 (%)
P2 (%)
80.0
32.6
24.4
0.6
P3 (%)
19.9
P5 (%)
9.8
Control
90.4
78.2
69.8
2.4
Compound 2 treated cells
Compound 6 treated cells
Compound 9 treated cells
Cisplatin treated cells
60.1
21.0
27.9
95.5
10.0
74.4
98.0
45.1
55.0
90.0

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Table 5. Percents of typical quadrant analysis of caspase-3 positive/negative C6 and A549
cells treated with the compounds.
C6 cell line
A549 cell line
Caspase-3 (-)
Caspase-3 (+)
cells %
Caspase-3 (-)
Caspase-3 (+)
cells %
Groups
cells %
cells %
Control
96.4
4.8
11.7
33.9
11.3
9.6
98.1
2.1
7.1
Compound 2 treated cells
Compound 6 treated cells
Compound 9 treated cells
Cisplatin treated cells
90.8
93.5
69.8
76.4
25.0
5.4
90.1
95.1
91.6
90.4
10.5
C6 and A549 cells were cultured for 24 h in medium with IC50 values of compounds. At least 10.000 cells were
analyzed per sample, and quadrant analysis was performed.

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Table 6. Akt inhibitory effects of the compounds.
IC50 (µM)
Compound
C6 cell line
165.00±5.00
2.60±0.17
A549 cell line
98.33±2.89
27.50±0.71
138.33±45.37
34.00±1.73
17.33±2.87
2
6
9
44.50±3.53
39.00±1.41
6.97±0.06
Cisplatin
GSK690693