Biological activity and molecular docking studies of some new quinolines as potent anticancer agents

Article abstract of DOI:10.1007/s12032-021-01530-w

Abstract: The objective of this study is to investigate the antiproliferative and cytotoxic properties and the?action mechanism of substituted quinoline and tetrahydroquinolines 3, 4, 5, 7, and 8 against rat glioblastoma (C6), human cervical cancer (HeLa), human adenocarcinoma (HT29) cancer cell lines by BrdU Cell Proliferation ELISA, Lactate Dehydrogenase, DNA laddering and Topoisomerase I assays. The results of the study showed that 6,8-dibromotetrahydroquinoline 3 possess in vitro antiproliferative activity against C6, HeLa, and HT29 cell lines while morpholine/piperazine substituted quinoline 7 and 8 showed selective antiproliferative activity on C6 cell line with IC₅₀ values 47.5 and 46.3?μg/mL, respectively. Moreover, 6,8-dibromoTHQ 3 caused DNA fragmentation while it did not inhibit the Topoisomerase I (Topo I) enzyme. On the other hand, compound?8 did not cause DNA laddering while 8 inhibited the Topo I enzyme. According to these results, 6,8-dibromoTHQ 3 stimulates apoptosis on the C6 cell line while 6,8-dibromo-3-morhonilylquinoline (8) inhibits the Topo I enzyme to cause antiproliferative activity. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s12032-021-01530-w

Medical Oncology (2021) 38:84
https://doi.org/10.1007/s12032-021-01530-w
ORIGINAL PAPER
Biological activity and molecular docking studies of some new
quinolines as potent anticancer agents
Tuğba Kul Köprülü1 · Salih Ökten2 · Vildan Enisoğlu Atalay3 · Şaban Tekin4 · Osman Çakmak5
Received: 1 May 2021 / Accepted: 4 June 2021 / Published online: 19 June 2021
© Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
The objective of this study is to investigate the antiproliferative and cytotoxic properties and the action mechanism of sub-
stituted quinoline and tetrahydroquinolines 3, 4, 5, 7, and 8 against rat glioblastoma (C6), human cervical cancer (HeLa),
human adenocarcinoma (HT29) cancer cell lines by BrdU Cell Proliferation ELISA, Lactate Dehydrogenase, DNA ladder-
ing and Topoisomerase I assays. The results of the study showed that 6,8-dibromotetrahydroquinoline 3 possess in vitro
antiproliferative activity against C6, HeLa, and HT29 cell lines while morpholine/piperazine substituted quinoline 7 and
8 showed selective antiproliferative activity on C6 cell line with IC50 values 47.5 and 46.3 µg/mL, respectively. Moreover,
6,8-dibromoTHQ 3 caused DNA fragmentation while it did not inhibit the Topoisomerase I (Topo I) enzyme. On the other
hand, compound 8 did not cause DNA laddering while 8 inhibited the Topo I enzyme. According to these results, 6,8-dibro-
moTHQ 3 stimulates apoptosis on the C6 cell line while 6,8-dibromo-3-morhonilylquinoline (8) inhibits the Topo I enzyme
to cause antiproliferative activity.
Graphic abstract
Keywords Quinoline · Tetrahydroquinoline · Anticancer activity · Cytotoxicity · Molecular docking
Introduction
* Tuğba Kul Köprülü
tugbakul.koprulu@sbu.edu.tr
The aromatic heterocycles display physicochemical proper-
ties with relevance in the design of new drug candidates.
Especially, nitrogen-containing heterocycles have drawn
* Salih Ökten
salihokten@kku.edu.tr
Extended author information available on the last page of the article
Vol.:(0123456789)
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84 Page 2 of 11
Medical Oncology (2021) 38:84
the attention for a long time due to their broad range of
biological and pharmacological properties [1, 2]. Among
heterocyclic compounds, quinolines play important roles
in the development of new drugs and are a very useful scaf-
fold for the design of promising versatile drug candidates
[3–5]. The bioactive quinolines substituted with diferent
groups exhibit diverse pharmacological properties such as
antimalarial [6], antibacterial [5, 7], antifungal [8], anti-
convulsant [9], anti-infammatory [10], antiprotozoal [11]
anticholinesterase [5, 12] and antiviral [13]. The quinoline
is also the efective structure for some important antican-
cer drug candidates [14–16]. Moreover, biologically natu-
ral, semi-synthetic, and synthetic active molecules based
on many quinoline scafolds have high antiproliferative
and antitumor properties through various mechanisms of
action such as cell cycle arrest, apoptosis, disruption of
cell migration, inhibition of angiogenesis [2, 15, 17, 18].
Several drugs containing the quinoline ring motif such as
Topotecan, Camptothecin, Irinotecan, and Belotecan are
used in clinical treatment as anticancer agents [19]. The
natural alkaloid Camptothecin and its semi-synthetic analog
Topotecan are two examples of cytotoxic quinoline with
the antitumor property through inhibition of DNA enzyme
topoisomerase I [20]. Numerous quinoline derivatives were
reported as anticancer agents through variable mechanisms
like tubulin inhibition [21], carbonic anhydrase inhibition
[22] cMet kinase inhibition [23], VEGFR inhibition [24],
increase in the protein expression of Bad, Bax and decrease
in Bcl-2, and PARP with apoptotic cell death [25].
cells. Glycosidase inhibitors also suppress the metastatic
potential of malignant cells by perturbing synthesis of the
correct carbohydrate arrangement [29]. On the other hand,
there are many connections between carbonic anhydrase
(CA) and cancer. Some CA isozymes are predominantly
found in cancer cells [30, 31] and these enzymes can help
maintain a physiological intracellular pH while simultane-
ously contributing to an acidic extracellular pH, leading to
tumor cell survival. Thus, this acidic pH promote tumor cell
invasion and metastasis [32]. CA inhibitors might provide
useful tools for controlling the pH (im)balance of tumor cells
and developing novel diagnostic or therapeutic applications
for the management of some tumors in hypoxic cancers [33].
In this study, it was aimed to determine the cytotoxic and
antiproliferative activities of recently synthesized morpholine/
piperazine/phenyl substituted quinoline derivatives [12], show-
ing signifcant inhibition against AChE, cytosolic carbonic
anhydrase (hCA I and hCA II) and α-Glu, against some cancer
cells and to reveal their mechanisms of action, due to that the
inhibition of metabolic enzymes has an important place in can-
cer research and quinolines display a broad range of biological
activity. In addition, the activity of the substituted quinoline
derivatives was supported by molecular docking.
Material and methods
Synthesis of the quinoline compounds
Due to many of the substituted quinolines that have
been used in medical applications, the synthesis of novel
quinoline derivatives has attracted a lot of attention from
researchers. Quinolines containing diferent groups have
been extensively studied by our group. As a result of the
studies, the potential anticancer activity properties of some
novel quinoline derivatives have been reported until date
by our group [1, 2, 5, 26].
This study was carried out with phenyl substituted tetrahy-
droquinoline and morpholinyl and piperazinyl quinoline
derivatives (5) reported in our previous papers [4, 12, 34,
35]. In brief, the synthesis of 6-bromo-1,2,3,4-tetrahydro-
quinoline (2) and 6,8-dibromo-1,2,3,4-tetrahydroquinoline
(3) via direct bromination in ionic conditions [27] and their
phenyl substituted derivatives (4 and 5, respectively) via
Suzuki Cross-coupling reactions were reported recently by
our previous publication [4]. Also, 3-morpholinyl- (7) and
3-piperazinyl-6,8-dibromoquinoline (8) were prepared by
treatment of 3,6,8-tribromoquinoline (6) and morpholine
or piperazine under microwave radiation according to our
reported procedure [12]. The isolated compounds (2–5, 7–8)
were fully characterized with a melting point, elemental
analysis, FT-IR, 1H NMR, 13C NMR, HMBC spectroscopy
in these papers [4, 12, 34, 35].
The inhibition of cholinergic and metabolic enzymes
(acetylcholinesterase, cytosolic carbonic anhydrase and
α-glucosidase enzymes) in the human body has an important
role in cancer treatment. The cytotoxic chemotherapeutic
agents have side efects like cholinergic syndromes. High
dose usage of CPT-11, Camptothecin derivative, lead to
cholinergic syndrome by direct interaction of the drug or
its metabolites with acetylcholinesterase (AChE). However,
in the literature, kinetic studies with Camptothecin bearing
4-piperidinopiperidine moiety indicated that this moiety
was primarily responsible for AChE inhibition with the, the
major determinant in the loss of enzyme activity [27]. More-
over, α-glucosidase in tumor cells is essential for the meta-
static process through the cellular interaction with collagen
type 1 and IV [28]. Metastasis was also inhibited by disturb-
ing the carbohydrate structure on the surface of neoplastic
Cell culture
The HeLa, HT29 and C6 cancer cell lines used in the study
were obtained from the American Type Culture Collection
(ATCC, Rockville, USA). HT29, HeLa, and C6 cell lines
were maintained in Dulbecco’s modifed eagle’s medium
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Medical Oncology (2021) 38:84
Page 3 of 11 84
(DMEM, Sigma-Aldrich), supplemented with 10% (v/v)
fetal bovine serum (Sigma-Aldrich, Germany) and 2% (v/v)
PenStrep solution (Sigma-Aldrich). At 80% confuence, cells
were detached from the fasks using 4 mL of trypsin–EDTA
(Sigma-Aldrich), centrifuged and the cell pellet resuspended
with 4 mL supplemented DMEM. Stock solutions of the
samples and 5-fuorouracil (5-FU) were prepared in sterile
dimethyl sulfoxide (DMSO) and diluted with DMEM. The
fnal concentration of dimethyl sulfoxide (DMSO, Sigma-
Aldrich) was kept below 1% in all tests. Tests were carried
out in triplicate for each experiment.
DNA laddering assay
The DNA laddering efect of the quinoline compounds was
determined using Gong’s method [36] HT29 cells (7.5×105
cells/well) were seeded in 25 cm2 culture fasks at 37 °C
in DMEM and allowed to attach overnight. After then, the
cells were treated with IC50 concentrations of 3, 7, and 8
compounds; the control group is represented by untreated
cells, incubated only with a cell culture medium. On the
second day, the cells are fxed using 70% ethanol, DNA is
extracted with 0.2 M phosphate-citrate bufer at pH 7.8, and
the extract is sequentially treated with RNase A and protein-
ase K and then subjected to electrophoresis.
BrdU cell proliferation ELISA (BCPE)
The antiproliferative efects of the quinolines were inves-
tigated against HT29, HeLa and C6 cancer cell lines using
BrdU Cell Proliferation ELISA kit (Roche, USA), a colori-
metric immunoassay based on BrdU incorporation into the
cellular DNA, according to manufacturer’s protocol. A Cell
suspension containing 3×103 cells in 100 µL was pipetted
into wells of 96-well cell culture plates (COSTAR, Corn-
ing, USA). The cells were treated with test compounds and
5-FU at fnal concentrations of 5, 10, 20, 30, 40, 50, 60,
and 75 µg/mL. The fnal volume of the wells was adjusted
to 200 µL by supplemented DMEM and was incubated at
37 °C with 5% CO2 overnight. Cells were exposed to BrdU
labeling reagent for 4 h followed by fxation in FixDenat
solution for 30 min. at room temperature. Then, cells were
cultured with 1:100 diluted anti-BrdU-POD for 90 min. at
room temperature, substrate solution was added to each well
and BrdU incorporation was measured at 450–650 nm using
a microplate reader (BioTek, Epoch). The IC50 of the test
and control compounds was calculated using XLft5 soft-
ware (IDBS) and expressed in μg/mL at 95% confdence
intervals.
Topoisomerase I inhibition
To investigate whether quinolines 3, 7, and 8 are topoisomer-
ase inhibitors, we tested these quinolines with a topoisomer-
ase I assay kit (TG1015-2; TopoGen, Buena Vista, CO).
The topoisomerase I assay is specifc for measuring Topo
I activity because it is based on the relaxation of super-
coiled pHOT1 plasmid DNA after incubation of the Topo
I enzyme. The supercoiled pHOT1 plasmid DNA was used
in 20 µL reaction bufer containing 1 unit of human Topo I
enzyme [37]. The IC50 concentrations of quinolines 3, 7, and
8 and positive control compound (Table 2), Camptothecin
(CPT) are added in the prepared reaction mixture, respec-
tively. After 30 min of incubation at 37 °C, the reaction was
stopped by adding equal volumes of stop solution. Then the
reaction mixtures were analyzed on a 1% agarose gel by
running 40 V for 60 min in TBE bufer. The gel was stained
with ethidium bromide (1 mg/mL) and observed under UV
illumination (UVP Biospectrum, Germany).
Wound healing assay
C6 cells were seeded at a density of 3.5×104 cells /well in a
culture-insert (ibidi culture-insert 2, ibidi GmbH, Germany)
well for wound healing assay. After allowing the cells to
attach overnight, the insert was removed and added 2 mL of
fresh DMEM. Cells were treated with IC50 concentration of 3
at 37 °C with 5% CO2 and then cells were taken photographed
using an inverted microscope (Leica DMIL, Germany).
Lactate dehydrogenase assay
The release of lactate dehydrogenase (LDH) is an indi-
cator of loss of membrane integrity and cell injury. LDH
assay was performed using LDH Cytotoxicity Detection Kit
(Roche, USA) standard protocols. Seed cells in a 96-well
fat-bottom microtiter plate at a density of 5000 cells/well in
100 μL of culture medium. The cells were treated with test
compounds and 5-FU at fnal concentrations of IC50. The
fnal volume of the wells was adjusted to 200 µL by sup-
plemented DMEM. The cells then were incubated at 37℃
with 5% CO2 overnight and were measured at 492–650 nm
using a microplate reader (BioTek, Epoch). The percentage
of LDH released was calculated as follows: (LDH activ-
ity in media) / (LDH activity in media+intracellular LDH
activity)×100.
Statistical analysis
Results are expressed as mean standard deviation (SD) for
at least three independent determinations. For the statisti-
cal analyses, the Statistical Package for Social Sciences was
used, and standard deviation, the P-value was calculated
using means, one‐way analysis of variance. P < 0.05 was
considered as signifcant diference.
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84 Page 4 of 11
Medical Oncology (2021) 38:84
quinolines were prepared according to our recent articles
[4, 12, 35]. 6-phenyl-1,2,3,4-tetrahydroquinoline (4) and
6,8-diphenyl-1,2,3,4-tetrahydroquinoline (5) were prepared
via Suzuki–Miyaura cross-coupling reactions between cor-
responding bromo tetrahydroquinolines (2, 3) and phenylbo-
ronic acid in presence of Pd-catalyst (Fig. 1) [4]. On the
other hand, the treatment of 3,6,8-tribromoquinoline (6) and
morpholine or piperazine under microwave radiation were
aforded 3-morpholinyl- (7) and 3-piperazinyl quinolines (8)
in moderate yields [35].
Geometry optimizations
In this study, a conformational search to determine the sta-
ble structure of the new synthesized fve compounds was
performed with a semi-empirical PM3 method [38] using
the program Spartan’04 [39]. The obtained most stable con-
former structures for all new synthesized molecules were
optimized with a semi-empirical PM6 method [40] using
the Gaussian09 suite of programs [41].
Molecular docking procedure
Anticancer activity studies
In all the molecular docking studies PDB ID: 5EG3 code
was selected as the crystal structure of PLCγ1, AutoDock4.2
[42, 43] and Auto Dock Tools (ADT) software were used to
calculate the binding energies in the docking studies. The
grid box was formed such that the active site amino acids of
the proteins were in the cube measured at 40×40×40 Å3,
each docking run was repeated 3 times.
The metabolic enzyme inhibition activities of 7 and 8 against
several metabolic enzymes (AChE, BChE, α-Glu, and hCA
II) were determined by Ellman’s method and esterase assay
[12]. The results show that these compounds signifcantly
inhibited metabolic enzymes in the nM concentration scale
[12]. These results encourage us to reveal antiproliferative
and cytotoxicities of substituted activities.
Results
Antiproliferative activities of compounds
Chemistry
Recent studies, reported by our research group displayed
antiproliferative activities of substituted quinolines against
certain cancer cell lines using sulforhodamine-B stain
(SRB), BCPE, and MTT assays [1, 2, 5, 14].
Due to that N-function heterocycles displayed a wide
range of pharmaceutical features, phenyl substituted tet-
rahydroquinolines and piperazine/morpholine substituted
Fig. 1 The structures of quino-
line analogues, 3, 4, 5, 7, and 8
N
H
1
Br2
Br
Br
PhB(OH)2
PhB(OH)2
Pd-cat
+
Pd-cat
N
H
N
H
N
H
N
H
Br
2
3
4
Br2
O
5
Br
Br
Br
N
Morpholine
DDQ
N
N
Br
Br
6
7
Piperazine
N
NH
Br
N
N
9
Br
8
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Medical Oncology (2021) 38:84
Page 5 of 11 84
3 showed high inhibition activity against all cancer
cell lines (HeLa, HT29, and C6) while 7 and 8 inhib-
ited significantly the proliferation of only C6 cell lines
in higher concentration (≤30 µg/mL) compared with the
control drug, 5-FU (Fig. 2). However, the other test com-
pounds 4 and 5 did not show any inhibition potency against
any cell lines (Fig. 2).
cancer cells in two dimensions. The ability of cancer cells to
metastasize makes them a clear target of anticancer drugs.
Wound healing assay was applied to observe the efect of 3
on cell migration and cells were photographed at the start of
the experiment (0 h) and 18 h later. It is observed that 3 has
not migration inhibitory efect (Fig. 5b).
Molecular docking studies
Cytotoxicity by LDH assay
Phospholipase C gamma 1 (PLCγ1) is overexpressed in
many metastatic tumors [44, 45]. The inhibition of PLCγ1 in
nude mice was reported to essentially suppress lung metas-
tasis [5, 45]. The small molecule binding site of the PLCγ1
complex is in the nSH2 domain that binds the phosphoryl-
ated tyrosine 766 (pV766) residue of the tyrosine kinase
domain of growth factors [5, 46]. As seen in Fig. 6, 3, 7,
and 8 suitably and favorably docks into the FGFR2 binding
site of PLCγ1.
It is an important situation for compounds having a high
antiproliferative activity to have minimal toxicity against
normal cells. Thus, the cytotoxic potentials of the com-
pounds were determined using a lactate dehydrogenase
(LDH) assay, indirectly demonstrating membrane damage
[5]. The LDH catalyzes the interconversion of pyruvate and
lactate and leaks from the cell inside to extracellular space
when the cell membrane is damaged.
The results of cytoplasmic LDH activity were showed
that the cytotoxicities of 3, 7, and 8 were lower, especially
compound 5 (8%), than 5-FU at their IC50 concentrations
against HT29 cell lines (Fig. 3). 3 and 8 have lower LDH
release percentages (27–33%) compared with the control
drug (34%) while 7 has higher cytotoxicity (62%) (Fig. 3).
On the other hand, 3 and 8 for C6 cell lines cause approxi-
mately 16–18% membrane damage at their IC50 concentra-
tions. However, 7 has a high LDH percentage (41%) against
C6 cell lines (Fig. 3).
Discussion
Quinoline derivatives are attributed to promising drug can-
didates showing biological activity in a wide range, espe-
cially anticancer activity [2, 5, 18, 35]. In a recent report
6,8-dibromo-tetrahydroquinoline (3), showed signifcant
antiproliferative activities against several cancer cell lines,
HeLa, Hep3B, MCF7 and HT29 with IC50 values ranging
4.3 and 21.8 µg/mL according to the MTT proliferation
assay [5]. Also, in the frst report of anticancer activity of
3. Its inhibitory activity of proliferation of HT29, C6, and
HeLa cancer cell lines was determined at 30 µg/mL and
higher concentrations by BCPE assay [14].
The evaluation of apoptotic potential,
topoisomerase I inhibition and cell migration
To evaluate the apoptotic potentials of 3, 7, and 8 on the
C6 cell line, the DNA laddering assay was used. DNA lad-
dering assay tests whether the mechanism of antiprolifera-
tive and cytotoxic activity of 3, 7, and 8 involved apoptosis
or not. Apoptotic DNA fragmentation of 3 was observed
as a DNA laddering pattern at 180–200 bp intervals indi-
cating the DNA damage (Fig. 5a). It was determined that
7 and 8 were not apoptotic, but 3 was induced apoptosis in
the C6 cell line.
In this study, the antiproliferativity of dibromotetrahyd-
roquinoline (3) was reworked against HeLa, HT29, and C6
cell lines to compare the proliferation inhibitory efects of
its corresponding derivatives bearing phenyl groups and to
investigate its action mechanism. According to BCPE assay
results, 6,8-dibromo-tetrahydroquinoline (3) inhibited the
proliferation of each three types of cancer cell lines at low
concentrations (IC50 values ranging 100.0 and 144.8 µM,
Table 2) compared with 5-FU. These results are confrmed
in previous reports [5, 14]. 6,8-diphenylTHQ 5, derived
from 6,8-dibromoTHQ 3 and 6-phenyltetrahydroquinoline
(5) derived from monobromo isomer of compound 3 did not
show any antiproliferative activity against tested cell lines
HeLa, HT29, and C6. However, the signifcant antiprolif-
erative efects of the aromatic form of 6,8-diphenylTHQ 5,
6,8-diphenylquinoline were reported against HeLa, HT29,
and C6 at IC50 values 77.6–119.5 µM [2, 26]. 9 has high
cytotoxicity (48–82%), except for HeLa (3%) [2]. On the
contrary, the cytoplasmic LDH assay showed that 6,8-dibro-
moTHQ (3) led to the release of LDH out of the cells at a low
Then, the inhibition activity of 3, 7, and 8 on Topo I
enzyme with relaxation assay was investigated on super-
coiled DNA using agarose gel electrophoresis. As illustrated
in Fig. 4, supercoiled pHOT1 DNA was relaxed by incuba-
tion with Topo I (Lane C1) while the control drug CPT,
a quinoline derivative, displays anticancer activity as an
inhibitor of DNA Topo I inhibited relaxation of supercoiled
DNA (Lane C2). 8 inhibited the Topo I enzyme (Lane 8).
However, 3 and 7 did not inhibit the Topo I enzyme.
Cell migration is key procedure involved in many bio-
logical processes and the wound healing assay is generally
performed to determine the growth and migration ability of
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84 Page 6 of 11
Medical Oncology (2021) 38:84
Fig. 2 The antiproliferative
activity of quinolines against
C6, HeLa, and HT29 cancer cell
lines in vitro using the BCPE
assay. The cells were treated
with eight diferent concentra-
tions of quinoline compounds
(P<0.05)
C6
100
80
60
40
20
0
5-FU
3
4
5
7
5
10
20
30
40
50
60
75
8
-20
Concentraꢀon (µg/mL)
HeLa
100
80
60
40
20
0
5-FU
3
4
5
7
8
5
10
20
30
40
50
60
75
-20
Concentraꢀon (µg/mL)
HT29
100
80
60
40
20
0
5-FU
3
4
5
7
8
5
10
20
30
40
50
60
75
-20
Concentraꢀon (µg/mL)
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Medical Oncology (2021) 38:84
Page 7 of 11 84
Fig. 3 The cytotoxic activ-
ity of 3, 7, and 8 at their IC50
concentrations on C6, HeLa,
and HT29. The LDH cytotoxic-
ity test showed a signifcant
increase (P<0.05) in the
cytotoxicity of cells. Percent
cytotoxicity was reported as
mean values SD of three
independent assays
3
7
HT29
HeLa
C6
8
5-FU
0
20
40
60
80
100
LDH release %
Fig. 4 The inhibition of human
topoisomerase I (topo I) by
3, 7, and 8. C1, supercoiled
DNA+Topo I; C2, super-
coiled DNA+Topo I+CPT;
CPT: camptothecin; Lane 3,
DNA+Topo I+compound 3;
Lane 7, DNA+Topo I+com-
pound 7; Lane 8, DNA+Topo
I+compound 8; R, relaxed
pHOT‐1 DNA marker; Rlx,
relaxed DNA; SC, supercoiled
DNA
percentage (9–33%), especially in the HT29 cell line (9%)
compared with 5-FU (45%). According to these results, it
can be concluded that phenyl groups bounded to 1,2,3,4-tet-
rahydroquinoline ring at C-6 or C-6 and C-8 dramatically
decrease the antiproliferative activity. On the other hand, the
phenyl derivative 5 can probably show high inhibition of cell
proliferation if this compound is aromatized.
In the LDH assay, 7 and 8 exhibited high cytotoxic efect
against C6, HeLa, and HT29 cancer cell lines (Fig. 3). The
results showed that 7 and 8, especially 7, signifcantly inhib-
ited C6 cells by BCPE assay through induction of necrotic
cell death.
The morpholine and piperazine substituted at C-3 qui-
noline derivatives (7 and 8) displayed an antiproliferative
efect against only the C6 cell line (IC50 values 125.2 and
128.7 µg/mL, respectively). The LDH assay displayed that
8 causes approximately 17% membrane damage at its IC50
concentration. However, morpholine substituted quinoline
led to cytotoxicity (42%, LDH release) at the C6 cell line.
If 7 and 8 are compared with 5-FU, the toxicity of 8 is very
close to the cytotoxicity value of 5-FU.
The results of DNA laddering, Topo I and cell migra-
tion assays show that 6,8-dibromoTHQ 3 caused DNA
fragmentation while it did not inhibit the Topo I enzyme
and cell migration. On the contrary, its aromatized from 9
bearing phenyl groups at C-6 and C-8 positions could not
induce the apoptosis detected by DNA laddering assay but
it inhibited Topo I enzyme [2]. According to these results,
6,8-dibromoTHQ 3 stimulates apoptosis on the C6 cell line
while 6,8-diphenylquinoline (9) inhibits the Topo I enzyme
to cause antiproliferative activity.
According to Table 1, the most interacted residues in the
active site of the PLCγ1 are VAL495, ALA515, LYS517,
GLU534, VAL564, TRY566, and LEU633, on the con-
trary MET538 aminoacid did not show enough interactions
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84 Page 8 of 11
Medical Oncology (2021) 38:84
with the molecules. On the other side, while the best bind-
ing energy was obtained for 3 with an energy value of
8.1 kcal mol−1, the weakest energy was obtained for 7 with
an energy value of 7.7 kcal mol−1 compound 7 gave low
binding energy due to weak interactions with all amino acids
except MET538. The binding of 3, 7, and 8 are highly likely
to prevent LEU484, VAL595, LYS517, GLU534, VAL564,
TRY566, ALA567, LEU533, and ALA643 the residues
FGFR2 to conduct its kinase activity on PLCγ1 (Table 1).
Therefore, 3, 7, and 8 could represent a potential drug can-
didate which should be further investigated for metastatic
cancer treatment.
Fig. 5 a Agarose gel electrophoresis for detecting DNA fragmen-
tation in C6 cell line (right). M: 1 kb DNA marker, C1 (control,
untreated C6); C2 (positive control, C6+CPT); Lane 3 (C6+com-
pound 3), Lane 7 (C6+compound 7), Lane 8 (C6+compound 8),
b Representation of the migration efect of compound 3 (left). (A)
Control, untreated C6 cells; (B) untreated C6 cells after 18 h (C) C6
cells+compound 3 (D) C6 cells+compound 3 after 18 h
Fig. 6 Docking poses of the active inhibitors of the target protein
Table 1 Binding Energy
Compound BE
LEU VAL ALA LYS GLU MET VAL TYR ALA LEU ALA
Values (BE) (Kcal mol−1) and
interacted aminoacid with the
new synthesized fve molecules
487
495
515
517 534
538
564
566
567
633
643
3
7
8
− 8.1
− 7.7
7.9
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
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Medical Oncology (2021) 38:84
Page 9 of 11 84
inhibitors. Arch Pharm. 2020;353: e2000086. https://doi.org/
10.1002/ardp.202000086.
Table 2 IC
50 (μM) values of compounds 3, 7, and 8
Compound
C6
HeLa
HT29
6. Modapa S, Tusi Z, Sridhar D, Kumar A, Siddiqi MI, Srivastava
K, Rizvi A, Tripathi R, Puri SK, Keshava GS, Shukla PK, Batra
S. Search for new pharmacophores for antimalarial activity. Part
I: synthesis and antimalarial activity of new 2-methyl-6-ureido-
4-quinolinamides. Bioorg Med Chem. 2009;17:203–21. https://
doi.org/10.1016/j.bmc.2008.11.021.
3
100.0 7.8
125.2 10.9
128.7 12.1
163.0 11.3
144.8 12.7
>1000
>1000
117.6 8.2
>1000
>1000
7
8
5-FU
469.6 37.6
501.2 45.4
7. Jin G, Li Z, Xiao F, Qi X, Sun X. Optimization of activity
localization of quinoline derivatives: Design, synthesis, and
dual evaluation of biological activity for potential antitumor and
antibacterial agents. Bioorg Chem. 2020;99: 103837. https://
doi.org/10.1016/j.bioorg.2020.103837.
Conclusion
8. Kumar S, Bawa S, Drabu S, Panda BP. Design and synthesis
of 2-chloroquinoline derivatives as non-azoles antimycotic
agents. Med Chem Res. 2011;20:1340–8. https://doi.org/10.
1007/s00044-010-9463-6.
Recently synthesized phenyl, morpholine, and piperazine
substituted quinoline and tetrahydroquinoline analogs were
tested for their antitumor activities and their mechanism of
action in vitro against three types of cancer cell lines. The
piperazine and morpholine substituted quinoline bromides
7 and 8 have selective antiproliferative efect against the C6
cell line with IC50 values 125.2 and 128.7 µM, respectively
(Table 2). While monophenyl- 4 and diphenyl- 5 tetrahyd-
roquinoline derivatives did not show any anticancer activity.
However, the starting material of diphenyl tetrahydroqui-
noline (5), 6,8-dibromotetrahydroquinoline 3 (IC50 values
ranging 100–144 µM in Table 2), and its aromatized form
9 have both signifcant antiproliferative efect and lower
cytotoxicities. Moreover, the docking study for anticancer
activity suggested that especially 3 represents a potential
antitumor drug candidate for metastatic cancer treatment due
to its potential to suppress the PLCγ1.
9. Guo LJ, Wei CX, Jia JH, Zhao LM, Quan ZS. Design and syn-
thesis of 5-alkoxy-[1,2,4]triazolo[4,3-a]quinoline derivatives
with anticonvulsant activity. Eur J Med Chem. 2009;44:954–8.
https://doi.org/10.1016/j.ejmech.2008.07.010.
10. Mukherjee S, Pal M. Medicinal chemistry of quinolines as
emerging anti-infammatory agents: an overview. Curr Med
Chem. 2013;20:4386–410. https://doi.org/10.2174/0929867311
3209990170.
11. Hochegger P, Faist J, Seebacher W, Saf R, Maser P, Kaiser M,
Weis R. Antiprotozoal activities of tetrazole-quinolines with
aminopiperidine linker. Med Chem. 2019;15:409–16. https://
doi.org/10.2174/1573406414666181015115101.
12. Çakmak O, Ökten S, Alımlı D, Ersanlı CC, Koçyiğit ÜM,
Taslimi P. Novel piperazine and morpholine substituted quino-
lines: selective synthesis through activation of 3,6,8-tribromo-
quinoline, characterization and their some metabolic enzymes
inhibition potentials. J Mol Struct. 2020;1220:1286662. https://
doi.org/10.1016/j.molstruc.2020.128666.
13. Guardia C, Stephens D, Dang H, Quijada M, Larionov O, Lleon-
art R. Antiviral activity of novel quinoline derivatives against
dengue virus serotype 2. Molecules. 2018;23:672–83. https://
doi.org/10.3390/molecules23030672.
Author contributions All authors were involved in research design and
data collection. Köprülü and Ökten conducted data analysis and drafted
manuscripts. Tekin and Çakmak revised the manuscript.
14. Ökten S, Çakmak O, Erenler R, Tekin Ş, Yüce Ö. Simple and
convenient preparation of novel 6,8-disubstituted quinoline
derivatives and their promising anticancer activities. Turk J
Chem. 2013;37:896–908. https://doi.org/10.3906/kim-1301-30.
15. Jain S, Chandra V, Jain PK, Pathak K, Pathak D, Vaidya A.
Comprehensive review on current developments of quinoline-
based anticancer agents. Arab J Chem. 2019;12:4920–46.
https://doi.org/10.1016/j.arabjc.2016.10.009.
Funding This study was supported fnancially by the Scientifc and
Technological Research Council of Turkey (TÜBİTAK; 112T394).
References
16. Afzal O, Kumar S, Haider MR, Ali MR, Kumar R, Jaggi M,
Bawa S. A review on anticancer potential of bioactive heterocy-
cle quinoline. Eur J Med Chem. 2015;97:871–910. https://doi.
org/10.1016/j.ejmech.2014.07.044.
1. Ökten S, Çakmak O, Tekin Ş. The SAR study of 6,8-disubsti-
tuted quinoline derivatives as anti cancer agents. Turk Clin Lab.
2017;8:152–9. https://doi.org/10.18663/tjcl.292058.
17. Köprülü TK, Tekin, Ökten S, Çınar M, Duman S, Çakmak O.
Detection of mechanism and anticancer activity of the new quin-
oline compounds MC20 and MC21. J Biotechnol. 2014;185:93.
https://doi.org/10.1016/j.jbiotec.2014.07.318.
2. Köprülü TK, Ökten S, Tekin Ş, Çakmak O. Biological evalu-
ation of some quinoline derivatives with diferent functional
groups as anticancer agents. J Biochem Mol Toxicol. 2019;33:
e22260. https://doi.org/10.1002/jbt.22260.
18. Ökten S, Şahin ÖY, Tekin Ş, Çakmak O. In vitro antiproliferative/
cytotoxic activity of novel quinoline compound SO-18 against
various cancer cell lines. J Biotechnol. 2014;185:106. https://doi.
org/10.1016/j.jbiotec.2014.07.359.
3. Solomon VR, Lee H. Quinoline as a privileged scafold in can-
cer drug discovery. Curr Med Chem. 2011;18:1488–508. https://
doi.org/10.2174/092986711795328382.
4. Ökten S. Synthesis of aryl substituted quinolines and tetrahy-
droquinolines through Suzuki-Miyaura coupling reactions. J
Chem Res. 2019;43:274–80. https://doi.org/10.1177/17475
19819861389.
19. Aydın A, Ökten S, Erkan S, Bulut M, Özcan E, Tutar A, Eren
T. In vitro anticancer and antibacterial activities of brominated
indenoquinoline amines supported with molecular docking and
MCDM. ChemistrySelect. 2021;6:3286–95. https://doi.org/10.
1002/slct.202004753.
5. Ökten S, Aydın A, Koçyiğit ÜM, Çakmak O, Erkan S, Andac
CA, Taslimi P, Gülçin İ. Quinoline-based promising antican-
cer and antibacterial agents, and some metabolic enzymes
20. Arafa RK, Hegazy GH, Piazza GA, Abadi AH. Synthesis and
in vitro antiproliferative efect of novel quinoline-based potential
1 3

84 Page 10 of 11
Medical Oncology (2021) 38:84
37. Ökten S, Erenler R, Köprülü TK, Tekin Ş. In vitro antiproliferative/
cytotoxic activity of 2,3’-biindole against various cancer cell lines.
Turk J Biol. 2015;39:15–22. https://doi.org/10.3906/biy-1402-60.
38. Stewart JJ. Application of the PM6 method to modeling the solid
state. J Mol Model. 2008;14:499–535. https://doi.org/10.1007/
s00894-008-0299-7.
anticancer agents. Eur J Med Chem. 2013;63:826–32. https://doi.
org/10.1016/j.ejmech.2013.03.008.
21. Alqasoumi SI, Al-Taweel AM, Alafeefy AM, Hamed MM,
Noaman E, Ghorab MM. Synthesis and biological evaluation of
2-amino-7,7-dimethyl 4-substituted-5-oxo-1-(3,4,5-trimethoxy)-
1,4,5,6,7,8-hexahydro-quinoline-3-carbonitrile derivatives as
potential cytotoxic agents. Bioorg Med Chem Lett. 2009;19:6939–
42. https://doi.org/10.1016/j.bmcl.2009.10.065.
39. Kong J, White CA, Krylov AI, Sherrill CD, Adamson RD, Fur-
lani TR, Lee MS, Lee AM, Gwaltney SR, Adams TR, Ochsen-
feld C, Gilbert ATB, Kedziora GS, Rassolov VA, Maurice DR,
Nair N, Shao Y, Besley NA, Maslen PE, Dombroski JP, Daschel
H, Zhang W, Korambath PP, Baker J, Byrd EFC, Voorhis TV,
Oumi M, Hirata S, Hsu CP, Ishikawa N, Florian J, Warshel A,
Johnson BG, Gill PGM, Gordon, MH, Pople JA. Q-Chem 2.0: a
high‐performance ab initio electronic structure program package.
J Comput Chem. 2000;21:1532–48. https://doi.org/10.1002/1096-
987X(200012)21:16<1532::AID-JCC10>3.0.CO;2-W.
22. Ghorab MM, Ragab FA, Heiba HI, Arafa RK, El-Hossary EM.
In vitro anticancer screening and radiosensitizing evaluation of
some new quinolines and pyrimido[4,5-b]quinolines bearing a
sulfonamide moiety. Eur J Med Chem. 2010;45:3677–84. https://
doi.org/10.1016/j.ejmech.2010.05.014.
23. Wang Y, Ai J, Wang Y, Chen Y, Wang L, Liu G, Geng M, Zhang
A. Synthesis and c-Met kinase inhibition of 3,5-disubstituted and
3,5,7-trisubstituted quinolines: Identifcation of 3-(4-acetylpipera-
zin-1-yl)-5-(3-nitrobenzylamino)-7- (trifuoromethyl)quinoline as
a novel anticancer agent. J Med Chem. 2011;54:2127–42. https://
doi.org/10.1021/jm101340q.
40. Stewart JJP. Application of the PM6 method to modeling pro-
teins. J Mol Model. 2009;15:765. https://doi.org/10.1007/
s00894-008-0420-y.
41. Frisch MJ, Trucks GW, Schlegel HB, Suzerain GE, Robb MA,
Cheeseman JR, Montgomery JA, Vreven T, Kudin KN, Burant JC,
Millam JM, Iyengar SS, Tomasi J, Barone V, Mennucci B, Cossi
M, Scalmani G, Rega N, Petersson GA, Nakatsuji H, Hada M,
Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima
T, Honda Y, Kitao O, Nakai H, Klene M, Li X, Knox JE, Hratch-
ian HP, Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts
R, Stratmann RE, Yazyev O, Austin AJ, Cammi R, Pomelli C,
Ochterski JW, Ayala PY, Morokuma K, Voth GA, Salvador P,
Dannenberg JJ, Zakrzewski VG, Dapprich S, Daniels AD, Strain
MC, Farkas O, Malick DK, Rabuck AD, Raghavachari K, Fores-
man JB, Ortiz JV, Cui Q, Baboul AG, Cliford S, Cioslowski J,
Stefanov B, Liu G, Liashenko A, Piskorz P, Komaromi I, Martin
RL, Fox DJ, Keith T, Al-Laham MA, Peng CY, Nanayakkara A,
Challacombe M, Gill PMW, Johnson B, Chen W, Wong MW,
Gonzalez C, Pople JA. Gaussian 09 (now Gaussian 16). Walling-
ford (CT): Gaussian Inc.; 2016.
24. Ghorab MM, Ragab FA, Heiba HI, Ghorab WM. Design and syn-
thesis of some novel quinoline derivatives as anticancer and radio-
sensitizing agents targeting VEGFR tyrosine kinase. J Heterocycl
Chem. 2011;48:1269–79. https://doi.org/10.1002/jhet.749.
25. Tseng C, Chen Y, Chung K, Wang C, Peng S, Cheng C, Tzeng C.
Synthesis and antiproliferative evaluation of 2,3-diarylquinoline
derivatives. Org Biomol Chem. 2011;9:3205–16. https://doi.org/
10.1039/c0ob01225d.
26. Ökten S, Çakmak O, Tekin Ş, Köprülü TK. A SAR Study: Evalu-
ation of bromo derivatives of 8-substituted quinolines as novel
anticancer agents. Lett Drug Des Discov. 2017;14(12):1415–24.
https://doi.org/10.2174/1570180814666170504150050.
27. Hyatt JL, Tsurkan L, Morton CL, Yoon KJ, Harel M, Brumshtein B,
Silman I, Sussman JL, Wadkins RM, Potter PM. Inhibition of acetyl-
cholinesterase by the anticancer prodrug CPT-11. Chem Biol Interact.
2005;157–158:247–52. https://doi.org/10.1016/j.cbi.2005.10.033.
28. Atsumi S, Nosaka C, Ochi Y, Iinuma H, Umezawa K. Inhibi-
tion of experimental metastasis by an alpha-glucosidase inhibitor,
1,6-epi-cyclophellitol. Cancer Res. 1993;53(20):4896–9.
29. Bernacki RJ, Niedhala MJ, Korytnyk W. Glycosidases in cancer
and invasion. Cancer Metastasis Rev. 1985;4(1):81–101. https://
doi.org/10.1007/BF00047738.
42. Cosconati S, Forli S, Perryman AL, Harris R, Goodsell DS, Olson
AJ. Virtual screening with AutoDock: theory and practice. Expert
Opin Drug Discov. 2010;5:597–607. https://doi.org/10.1517/
17460441.2010.484460.
43. Forli S, Olson AJ. A force feld with discrete displaceable waters
and desolvation entropy for hydrated ligand docking. J Med Chem.
2012;55:623–38. https://doi.org/10.1021/jm2005145.
30. Supuran CT, Scozzafava A. Carbonic anhydrase inhibitors and
their therapeutic potential. Expert Opin Ther Pat. 2000;10(5):575–
600. https://doi.org/10.1517/13543776.10.5.575.
44. Lattanzio R, Iezzi M, Sala G, Tinari N, Falasca M, Alberti S,
Buglioni S, Mottolese M, Perracchio L, Natali PG, Piantelli M.
PLC-gamma-1 phosphorylation status is prognostic of metastatic
risk in patients with early-stage Luminal-A and -B breast cancer
subtypes. BMC Cancer. 2019;19:747. https://doi.org/10.1186/
s12885-019-5949-x.
31. Thiry A, Dogné JM, Masereel B, Supuran CT. Targeting tumor-
associated carbonic anhydrase IX in cancer therapy. Trends Phar-
macol Sci. 2006;27(11):566–73. https://doi.org/10.1016/j.tips.2006.
09.002.
32. Singh S, Lomelino CL, Mboge MY, Frost SC, McKenna R. Can-
cer drug development of carbonic anhydrase ınhibitors beyond the
active site. Molecules. 2018;23(5):1045. https://doi.org/10.3390/
molecules23051045.
45. Sala G, Dituri F, Raimondi C, Previdi S, Mafucci T, Mazzoletti
M, Rossi C, Iezzi M, Lattanzio R, Piantelli M, Iacobelli S, Brog-
gini M, Falasca M. Phospholipase Cγ1 Is Required for Metasta-
sis Development and Progression. Cancer Res. 2008;68:10187.
https://doi.org/10.1158/0008-5472.CAN-08-1181.
33. Supuran CT. Therapeutic applications of the carbonic anhydrase
inhibitors. Therapy. 2007;4(3):355–78. https://doi.org/10.2217/
14750708.4.3.355.
46. Bunney TD, Esposito D, Mas-Droux C, Lamber E, Baxendale RW,
Martins M, Cole A, Svergun D, Driscoll PC, Katan M. Structural
and functional integration of the PLCγ interaction domains critical
for regulatory mechanisms and signaling deregulation. Structure.
2012;20:2062–75. https://doi.org/10.1016/j.str.2012.09.005.
34. Şahin A, Çakmak O, Demirtaş I, Ökten S, Tutar A. Efcent
and selective synthesis of quinoline derivatives. Tetrahedron.
2008;64:10068–74. https://doi.org/10.1016/j.tet.2008.08.018.
35. Çakmak O, Ökten S, Alımlı D, Saddiqa A, Ersanlı CC. Activation
of 6-bromoquinoline by nitration: synthesis of morpholinyl and
piperazinyl quinolines. ARKIVOC. 2018;3:362–74.
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
36. Gong J, Traganos F, Darzynkiewicz Z. A selective procedure for
DNA extraction from apoptotic cells applicable for gel electropho-
resis and fow cytometry. Anal Biochem. 1994;218:314–9. https://
doi.org/10.1006/abio.1994.1184.
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Authors and Afliations
Tuğba Kul Köprülü1 · Salih Ökten2 · Vildan Enisoğlu Atalay3 · Şaban Tekin4 · Osman Çakmak5
1
4
Hamidiye Vocational School of Health Services, Department
of Medical Services and Techniques, Medical Laboratory
Techniques, University of Health Sciences, Üsküdar,
Istanbul, Turkey
Hamidiye Faculty of Medicine, Department of Basic Medical
Sciences, Medical Biology, University of Health Sciences,
Üsküdar, Istanbul, Turkey
5
Faculty of Arts and Design, Department of Gastronomy,
Istanbul Rumeli University, Silivri, 34570 Istanbul, Turkey
2
3
Faculty of Education, Department of Maths and Science
Education, Division of Science Education, Kırıkkale
University, Yahşihan, Kırıkkale, Turkey
Health Services Vocational School, Department and Plant
and Animal Production, Medicinal and Aromatic Plants,
Üsküdar University, Üsküdar, Istanbul, Turkey
1 3