Ugi efficient synthesis, biological evaluation and molecular docking of coumarin-quinoline hybrids as apoptotic agents through mitochondria-related pathways

Article abstract of DOI:10.1016/j.bioorg.2019.103147

Ugi reaction was a reliable procedure for the synthesis of new coumarin-quinoline frameworks. Excellent yields, mild reaction conditions and easily available and inexpensive starting materials are advantages of this protocol. Cytotoxic effects of fourteen products were investigated in A2780 human ovarian cancer cells. Two synthesized compounds (L11 and L12) exhibited more anti-cancer activity than other derivatives with IC₅₀ values of 0.042 mmol/L and 0.102 mmol/L, respectively and were thus selected for further studies. Apoptosis was induced through the intrinsic pathway by activating caspase 9 and ended at the executioner pathway of caspase 3. Measurement of intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were also carried out for both of them. Further studies on a mechanism by Real Time-PCR and Western blot analysis were performed for anti-apoptotic proteins Bcl-2 and survivin both in mRNA and protein level relating to the untreated A2780 cells. The treatment of A2780 cells with compound L11 significantly (P-value ≤ 0.05) induced apoptosis by down-regulation of Bcl-2 and survivin both in mRNA and protein level via a single dose (0.042 mmol/L), as well as activation of caspase 9 and 3, loss of MMP, and high ROS. Accordingly, findings supported the first report under which the pro-apoptotic activity of compound L11 as an apoptosis-inducing agent was related to mitochondrial-mediated dysfunction signaling pathways. Molecular docking supports experimental outcomes. Evidently, coumarin-quinoline scaffolds are potentially favorable options for further assessment as influential chemotherapeutic agents for the future.

Full text of DOI:10.1016/j.bioorg.2019.103147

Accepted Manuscript
Ugi efficient synthesis, biological evaluation and molecular docking of cou-
marin-quinoline hybrids as apoptotic agents through mitochondria-related path-
ways
Salman Taheri, Maryam Nazifi, Mahboubeh Mansourian, Leila Hosseinzadeh,
Yalda Shokoohiniad
PII:
S0045-2068(19)30496-1
https://doi.org/10.1016/j.bioorg.2019.103147
103147
DOI:
Article Number:
Reference:
YBIOO 103147
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
30 March 2019
20 July 2019
22 July 2019
Please cite this article as: S. Taheri, M. Nazifi, M. Mansourian, L. Hosseinzadeh, Y. Shokoohiniad, Ugi efficient
synthesis, biological evaluation and molecular docking of coumarin-quinoline hybrids as apoptotic agents through
mitochondria-related pathways, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103147
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JOURNAL PRE-PROOF
Ugi efficient synthesis, biological evaluation and molecular docking of
coumarin-quinoline hybrids as apoptotic agents through mitochondria-related
pathways
a
a
b,c*
d*
Salman Taheri , Maryam Nazifi , Mahboubeh Mansourian , Leila Hosseinzadeh , Yalda
Shokoohiniadd,e
aChemistry & Chemical Engineering Research Center of Iran, P.O. Box 14335-186, Tehran, Iran
bMedicinal Plants Research Center, Yasuj University of Medical Sciences, Yasuj, Iran
cDepartment of Pharmacology, Faculty of Medicine, Yasuj University of Medical Sciences, Yasuj,
Iran
dPharmaceutical Sciences Research Center, School of Pharmacy, Kermanshah University of
Medical Sciences, Kermanshah, Iran
eRic Scalzo Botanical Research Institute, Southwest College of Naturopathic Medicine, Tempe,
AZ, USA
*
Corresponding author (Mahboubeh Mansourian): E-mail: m.mansourian@yums.ac.ir.
Tel: +98 74 33346070; Fax: +98 74 33346071. ORCID ID: https://orcid.org/0000-0001-5419-
026.
2
E-mail address: taheri@ccerci.ac.ir (S. Taheri).
E-mail address:lhosseinzadeh90@yahoo.com (L. Hosseinzadeh).
E-mail address: yshokoohinia@gmail.com (Y. Shokoohinia).
E-mail address: maryam.nazifi1991@gmail.com (M. Nazifi).
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ABSTRACT
Ugi reaction was a reliable procedure for the synthesis of new coumarin-quinoline frameworks.
Excellent yields, mild reaction conditions and easily available and inexpensive starting materials
are advantages of this protocol. Cytotoxic effects of fourteen products were investigated in A2780
human ovarian cancer cells. Two synthesized compounds (L11 and L12) exhibited more anti-
cancer activity than other derivatives with IC values of 0.042 mmol/L and 0.102 mmol/L,
5
0
respectively and were thus selected for further studies. Apoptosis was induced through the intrinsic
pathway by activating caspase 9 and ended at the executioner pathway of caspase 3. Measurement
of intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP)
were also carried out for both of them. Further studies on a mechanism by Real Time-PCR and
Western blot analysis were performed for anti-apoptotic proteins Bcl-2 and survivin both in mRNA
and protein level relating to the untreated A2780 cells. The treatment of A2780 cells with
compound L11 significantly (P-value ≤ 0.05) induced apoptosis by down-regulation of Bcl-2 and
survivin both in mRNA and protein level via a single dose (0.042 mmol/L), as well as activation
of caspase 9 and 3, loss of MMP, and high ROS. Accordingly, findings supported the first report
under which the pro-apoptotic activity of compound L11 as an apoptosis-inducing agent was
related to mitochondrial-mediated dysfunction signaling pathways. Molecular docking supports
experimental outcomes. Evidently, coumarin-quinoline scaffolds are potentially favorable options
for further assessment as influential chemotherapeutic agents for the future.
Keywords: Coumarin-quinoline hybrids; Apoptosis; Mitochondria-related pathways; Bcl-2;
Survivin; Cancer
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1
. Introduction
Cancer as a global health problem has progressed dramatically in recent years, likely to reach
more than 13.1 million deaths by 2030 [1, 2]. Ovarian cancer is a leading gynecologic malignancy.
Cisplatin-based chemotherapy is the mainstay for treatment after surgical intervention of the
ovarian cancer. The occurrence of chemo-resistance is a major challenge for fighting ovarian
cancer [3, 4].
Finding high-quality compounds is an important task in the step-by-step development of multi-
target drug discovery (MTDD). This strategy only uses a chemical compound against the multi-
target activity. This useful strategy in pharmaceutical chemistry is a way to reduce the likelihood
of drug resistance, toxicity, difficulty dosage, drug interactions problems, or increase the patient
satisfaction to exhibit more favorable pharmacokinetics and pharmacodynamics [5, 6]. A hybrid
molecule with the participation of two groups (or more) of pharmacophores in a molecular
structure may form a new single multifunctional entity. The connection of multi-target drugs is
challenging whilst being an opportunity and innovative to moderate numerous directed activities
against numerous single receptors to create competent synergistic impact or in the form of dual
behaving drugs [7]. The use of a wide range of information obtained from bioinformatics,
structural biology, chemistry and pharmacology can be useful for discovering/designing and
synthesizing new hybrid molecules with diverse bioavailability [5, 8].
A preferred method to synthesize of the desired products with maximum efficiency includes
the fewest possible steps using commercial available and environmentally friendly starting
materials [9]. One-pot multi-component reactions with ease of implementation protocols, no need
for isolation of intermediates, and high-speed have gotten much attention for the construction of a
wide type of organic scaffolds [10-12].
Hybrid anti-cancer drugs such as lapatinib and sunitinib have remarkable advantages over
conventional anti-cancer drugs due to their improved pharmacodynamic profiles [8]. Further
research is essential to gain a better understanding of anti-cancer activities of these hybrids which
may lead to design of new therapies that may improve patients' survival. Coumarin-quinoline
hybrids are derivatives with different mechanisms of action [6, 8, 13, 14].
Among heterocyclic compounds, quinoline derivatives are pharmacologically important [8,
1
5, 16]. Quinoline is the core of several synthetic drug compounds, such as chloroquine, that is
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used as an anti-malarial agent [17]. Quinoline derivatives have remarkable biological properties
such as anti-tumor [13, 16, 18, 19], in the breast cancer [13, 18], cervical cancer [20], lung cancer
[19, 20], and pancreatic cancer [20]. Coumarin derivatives, on the other hand, have medical
benefits such as anti-microbial [21], anti-tuberculosis [22], anti-inflammatory [23, 24], anti-
Alzheimer [14], anti-coagulants [25], anti-influenza [26], anti-HIV [27], and anti-cancer [7, 23,
2
8, 29] activities [6, 8]. Hybridizing the coumarin nucleus with other moieties has created new
molecules with improved anti-cancer activity profiles [6, 7, 23, 29]. Coupling of different
coumarin derivatives with different type of heterocyclic compounds such as quinoline [14],
triazole [30], chalcone [23], indole [29], triazine [27], and α-lipoic acid [24] has produced novel
hybrid molecules [6]. Also, a molecular hybridization approach with quinoline, which is based on
triazole [30], pyrazole–quinoline–pyridine [15], coumarin [14], triazine [31], and some
miscellaneous structures play important roles in the development of novel molecules for the
remedy of numerous multifactorial diseases [8].
Despite a great number of studies of cancers, precise mechanisms of drug resistance remained
elusive. It is generally believed that chemotherapeutic drugs like cisplatin reduce cancer cells by
inducing programmed cell death (apoptosis) [3]. One of the vital cellular procedures for
multicellular organisms’ development and homeostasis is apoptosis [32]. During apoptosis, there
are changes within cell morphology, namely membrane blebbing and the creation of apoptotic
bodies as cell shrinkage, nuclear fragmentation, chromatin condensation, and DNA fragmentation.
The regulation of apoptotic pathways has been investigated in numerous pharmaceutical studies
[33-35]. Malignant disorders of the apoptotic process could directly or indirectly lead to many
human diseases including cancer and neuro-degeneration such as Alzheimer’s disease [32]. The
inefficiency of apoptosis is a vital trait which permitting cancer cells to escape from death.
Apoptotic signaling pathway activation is an anti-cancer drug target in cytotoxicity induction [13].
Apoptosis is regulated by the complex protein interaction networks which its phases are dependent
on a subset of caspases (Cysteine-dependent ASPartyl specific proteASES) [33]. Human caspases
with a clear role in apoptosis, are divided into two categories; the initiator caspases (caspase 2, 8,
9
, and 10) and the effector caspases (caspase 3, 6, and 7) [36]. The outcome of every cell is
eventually depended on the cross-talking of pro- or anti-apoptotic signaling pathways [37]. Anti-
apoptotic B-cell lymphoma-2 (Bcl-2) family regulates mitochondria-mediated apoptosis pathways
[34, 35]. Overexpression of Bcl-2 in A2780 human ovarian cancer cells leads to the resistance to
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cisplatin and is associated with the tumor cell resistance to chemotherapeutic agents by preventing
an apoptotic response [3, 38, 39]. Survivin cannot be traced within differentiated normal tissues
whilst it exhibits overexpression within the majority of human neoplasms [40, 41]. The expression
of survivin at high levels is associated with more intense diseases, a reduction of tumor cell
apoptosis, an increase of resistivity to chemotherapeutic agents and a shortened survival period
[42]. Down-regulation of survivin, and the induction of apoptosis by activation of caspases 8, 9,
and 3 are other effects of quinoline compounds [13, 18, 19], but no significant changes of Bcl-2
anti-apoptotic protein level were observed [13, 19]. The use of a molecular hybridization strategy
is highly suitable for introducing more potent molecules to simultaneously target more than a
pathogenic mechanism [8].
Therefore, a series of hybrids with the coumarin-quinoline scaffolds as anti-cancer drug entities
have been designed and synthesized based on Ugi reaction. It seems that the synthesized
compounds, which have proper structures for the placement of pharmacophore groups in suitable
spatial positions in enzymatic activities, may be useful for inhibiting activities of Bcl-2 and
survivin toward inducing apoptosis. To this end, several important pathways of apoptosis were
investigated. First, the screening and cytotoxic effects of hybrid compounds in the A2780 cancer
cell line was performed for fourteen synthesized compounds (Table 1). The impact of the best
compounds was measured on activities of caspase 9 and 3 as intrinsic and execution pathways,
respectively. In addition, the production of intracellular reactive oxygen species (ROS) as well as
mitochondrial membrane potential (MMP) were also evaluated as essential markers of apoptosis.
Expression of survivin and Bcl-2 as key regulators of apoptosis were also evaluated both in gen
and protein levels after cell treatment with the best compound by Real-time-PCR and the Western
blot analysis. The results of theoretical and laboratory methods have accelerated the understanding
of the interaction of molecules [43]. To achieve this goal in this project, a binding site of the best
inducer of apoptosis on Bcl-2 and survivin was investigated by molecular docking along with the
Western blot analysis.
2
2
. Results and discussion
.1. Chemistry
One of the most famous multi-component reactions is the Ugi four-component reaction which
involves condensation of aldehydes, amines, isocyanides, and carboxylic acids [44]. Heterocyclic
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compounds play important roles in developing a new class of structural entities for pharmaceutical
applications [6, 8, 29]. These compounds indicate diverse biological activities owing to their
unique ability to copy structures of peptides and reversibly bind to proteins [29]. The present study
aimed to combine the quinoline skeleton with the coumarin skeleton using alkyldiamide chains
with different motifs to extract inhibitory potentials on anti-apoptotic proteins Bcl-2 and survivin
(Table 1). In the present study, coumarin-3-carboxylic acid (2-oxo-2H-benzopyran-3- carboxylic
acids) and 2-chloroquinoline 3- carbaldehyde used as raw materials in the presence of amines and
isocyanide derivatives through a Ugi reaction.
A simple synthetic method developed for the efficient preparation of quinoline-coumarin
derivatives using an Ugi four-component reaction involving coumarin-3-carboxylic acid, various
derivatives of 2-chloroquinoline-3-carbaldehydes, cyclohexyl isocyanide and amines in methanol
1
(
see Scheme 1). All the synthesized compounds were fully characterized by FT-IR, H NMR, and
13C NMR spectral data (Supplementary 1). CHN elemental analysis was performed using an
analysator Flash EA 1112 from Thermo Quest. The purities of compounds were confirmed by
analytical high performance liquid chromatography (HPLC). Chromatograms were obtained using
KNAUER on 18 reverse phase column (250×4.6 mm, Eurospher 100-5 C18) with
water/acetonitrile (20:80 v/v) as solvent and the flow rate at 1.0 mL/min with UV detection at 257
nm (Supplementary 2).
R2
O
O
O
O
O
NH2
H
N
O
OH
H
C
+
NH
+
+
N
O
N
Cl
R3
O
Cl
R1
R3
N
R2
R1
1
2
L1-L14
Scheme 1. Efficient synthesis of quinoline-coumarin scaffolds using an Ugi reaction.
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O
H
NH2
R2
O
N
Cl
R1
R2
O
H
N
O
O
O
NH
OH
C
O
Cl
N
R3
N
O
R1
R3
Area
%)e
Product
R
1
R
2
R
3
Yield (%)a
88
RTd
6.58
7.38
7.43
7.65
8.80
8.43
9.18
7.67
9.02
9.18
10.90
12.47
5.63
8.75
(
b
100.00
95.47
L1
L2
H
H
H
H
6 11
C H
7
8
8
7
7
7
8
6
8
8
9
5
8
5
9
4
5
6
9
1
8
0
p-CH
3
C
C
C
C
C
C
C
C
C
C
C
6
6
6
6
6
6
6
6
6
6
6
H
H
H
H
H
H
H
H
H
H
H
11
100.00
97.35
L3
m-CH
3
11
11
11
11
11
11
11
11
11
11
L4
7-Me
7-Me
7-Me
6-Me
6-Me
H
100.00
100.00
100.00
97.24
L5
p-CH
3
L6
m-CH
3
L7
p-CH
H
3
L8
100.00
100.00
96.63
L9
5,8-Dimethyl
6-Me
p-CH
m-CH
H
3
L10
L11
L12
L13
3
5,8-Dimethyl
5,8-Dimethyl
H
95.98
m-CH
H
3
t-Buc
t-Bu
78
97.11
8
1
100.00
L14
5,8-Dimethyl
H
a
Isolated yield
Cyclohexyl isocyanide
b
c
tert-butyl isocyanide
d
Retention Time (HPLC)
Area Percent (HPLC)
e
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Table 1. Formation of diamide Ugi reaction compounds (L1-L14)
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2
2
.2.
Biological evaluation
.2.1. Evaluation of the cytotoxic activity against A2780 cells by MTT assay
In the present study, cytotoxic effects of fourteen novel coumarin-quinoline structures on
A2780 human cancer cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
MTT) assay were investigated (Supplementary 3). In the MTT assay, doxorubicin was chosen as
(
a positive control (Supplementary 4). It must be noted that doxorubicin has a potent anti-
proliferative effect against the growth of A2780 cells (IC =6.1 µM). A2780 cells were treated
5
0
with various concentrations of hybrids (0-100 μg/mL) for 24 h and the cell viability was evaluated
by MTT growth inhibition assay. Compounds, L11 and L12, were significantly more cytotoxic
than other derivatives with IC50 values of 25 µg/mL (0.042 mmol/L) and 62 µg/mL (0.102
mmol/L), respectively and were thus selected for further studies (Fig. 1). In addition, the cytotoxic
effect of the most potent compounds L11 and L12 were assayed in PC12 cells as a model of the
normal cell (Supplementary 5). A glance at the figure S.5 reveals that the L11 and L12 did not
have any significant cytotoxic effect on the PC12 cells.
Fig. 1. Cytotoxic effects of L11 and L12 in A2780 cell line. The cytotoxic effect of the compounds
was evaluated by MTT assay. Data presented as mean ± S.E.M (n = 3).
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2
.2.2. Caspase 9 and 3 activity assay
The induction of apoptosis is conducted through activation in reaction to various cell death
stimulus through intrinsic (mitochondrial) and/or extrinsic (death receptor) pathways [13, 35].
Activation of the intrinsic pathway as a response to a several stressing conditions, namely oxidative
stress and DNA damage induced mitochondrial protein release e.g. cytochrome c (Cyt c) from
intermembrane space into cytosol. Cyt c with other compounds e.g. ATP (or dATP) and Apaf-1
(apoptosis protease activating factor) creates a multimeric complex namely apoptosome which
stimulates the activation of caspase 9. The stimulated caspase 9 activates and cleaves effector
caspases e.g. caspase 7, 6 and 3 [13, 40]. It is common knowledge that caspase 3 is the main
executioner within the apoptotic procedure, and its stimulation causes cell death stages through
proteolytic dismantling of extensive cellular components and pro-apoptotic factors’ activation [35,
4
5].
The results showed that compounds L11 and L12, especially L11, dramatically increased the
activity of caspase 3 in addition to cytotoxic effect in A2780 cells (Fig. 2). However, L12 does not
effect on the activity of caspase 9. Therefore, L11, a coumarin-quinoline hybrid, induced the
cytotoxicity via activation of both caspase 9 and 3 in A2780 ovarian cancer cells in a single dose
(
at IC value 0.042 mmol/L) by more than 1.4-1.8 fold, following a 24 h treatment. It is indicated
50
that the induction of apoptosis occurred via the intrinsic and execution pathways. The results were
consistent with previous literature [13, 28, 31, 35]. Manohar et al. synthesized novel molecular
hybrids of 4-aminoquinoline and triazine as potential anti-cancer agents [31]. Among their
synthesized products, two compounds had significantly activity against NCI 60 human tumor cell
lines. Although, their representative compounds presented high activation of caspase 9, but none
of them caused statistically significant activation of effector caspases 3 and 7 [31]. Furthermore,
caspase 9 and 3 are prominent signaling proteins corresponding to the mitochondria-mediated
apoptosis pathway [35].
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Fig. 2. Involvement of activation of caspases 9 and 3 in the induction of apoptosis by potent
compounds on A2780 human ovarian cancer cells. Data presented as mean ± S.E.M. * P-value <
0
.05, *** P-value < 0.001 vs. control.
1
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2
.2.3. Measurement of mitochondrial membrane potential
Mitochondrial function, a key parameter of cell health, can be assessed by monitoring
changes in MMP [19]. As the cells’ energy center, mitochondria are associated to cell apoptotic
events. Mitochondrial dysfunction is a prominent apoptosis trait and includes procedures e.g.
MMP reduction and mitochondrial permeability transition [19]. Changes in MMP in A2780 cells
was detected for mitochondrial toxicity by evaluating the effects of L11 and L12 on MMP.
As shown in Fig. 3, after exposure to 0.042 mmol/L and 0.102 mmol/L of compounds L11
and L12 for 24 h, only L11 notably showed lower fluorescence, and reduced the MMP of treated
A2780 cells. Consistent with another study [19], compound L11 induced the disturbance of MMP
induced the apoptosis of A2780 cells through a mitochondria-mediated pathway. Amidst cancer
cells’ mediating apoptosis via mitochondrial pathway, the mitochondria discharged Cyt C within
cytosol followed by apoptosis factors’ induction and caspases activation [34, 35].
Fig. 3. The impact of compounds concerning mitochondrial membrane potential (MMP) collapse
determined via Rhodamine 123. Data showed as mean± S.E.M for three individual tests. **P-value
<
0. 01 vs. control.
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2
.2.4. Determination of intracellular reactive oxygen species level
ROS play the role of secondary messenger in regard to cellular signaling and associated to
cell death and survival. Extreme ROS levels can lead to mitochondrial dysfunction and consequent
cancer cells’ apoptosis through mitochondrial dysfunction, MMP loss, Cyt C discharge,
executioner caspases 3/9 cleavage and alterations in Bcl-2 family proteins levels [35]. The
microscopic fluorescence imaging was conducted to evaluate whether compounds L11 and L12
induces ROS accumulation in A2780 cells. A2780 cells were incubated with IC concentrations
5
0
of compounds, L11 and L12 (0.042 mmol/L and 0.102 mmol/L, respectively). It is evident from
Fig 4 that treatment using L11 led to increased dihydrodichlorofluorescein (DCF) fluorescence
intensity in comparison to control cells that were not subjected to treatment. Imaging evaluations
showed that compound L11 actively produced ROS and caused apoptosis induction in A2780
ovarian cancer cells through ROS-mediated mitochondrial dysfunction signaling pathway in the
form of intrinsic routes. Based on the study conducted by Wang et al. [35], the aforementioned
results indicated that compound L11 stimulated the creation and aggregation of intracellular ROS
within A2780 cells.
Fig. 4. The effect of potent compounds on the ROS generation in A2780 cell line. Data presented
as the mean ± S.E.M of three separate experiments (n = 4). ***P-value < 0.001 vs. control.
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2
.2.5. Assessment of Bcl-2 and survivin mRNA expression by Real-time-PCR
Caspases are regulated by many cellular processes. The suppression of caspase activity
within tumor cells occurs in the presence of specific members of the Inhibitor of Apoptosis
Proteins (IAP) protein family [33]. In other words, the IAPs as negative apoptosis regulator
proteins endogenously antagonize the cell death by inhibiting enzymatic activities of mature
caspases [46]. Furthermore, IAPs can also inhibit caspases 3 and 7 directly, and thus block
downstream apoptotic events [3]. As the smallest IAPs member, survivin plays a prominent role
in the inhibition of apoptosis and the division of cells. Survivin can be specific to terminal effector
cell death protease, caspases 3 and 7 through BIR (Baculovirus IAP Repeat) domain causing
hindrance in protease behavior and resulting apoptosis [40, 47]. Survivin is verified as a cancer
therapeutic target [48]. Researches imply that high survivin expression may enable it as a selective
target carried via chemo-protective agents which induce apoptosis, namely within cancer cells.
Hindered or decreased survivin expression alongside various cytotoxic agents may be useful for
the treatment of cancer [40]. In addition, higher Bcl-2 expression levels within cancer cells stops
apoptosis and causes tumor proliferation. Enhanced Bcl-2 expression is documented in numerous
tumor cells of a drug-resistant nature [49].
Based on these results, in addition to Bcl-2, survivin as a member of the IAP family is over-
expressed in A2780 cancer cells (Fig. 5). A2780 cells were treated at certain concentration of L11
(0.042 mmol/L) and vehicle (Control group) for 24 h and 48 h for inhibition assessment of Bcl-2
and survivin mRNA level. The over-expression of Bcl-2 and survivin genes may contribute to the
apoptotic inhibition in A2780 cells and the development of multidrug-resistance of human ovarian
cancer [3, 38-40]. The present data indicated that the expression of anti-apoptotic genes including
Bcl-2 and survivin was significantly down-regulated after the treatment of A2780 cells with L11
after 24 h and 48 h, respectively (P-value ≤ 0.05), while it was not significantly changed after 24
h for survivin in a time-independent manner. The decrement in mRNA levels of Bcl-2 and survivin,
which were decreased due to the L11 treatment in A2780 cells, indicated the sensitivity towards
the L11 treatment (Fig. 5), suggesting that Bcl-2 and survivin levels could regulate the caspase
activation in A2780 cells. Therefore, the apoptosis induction was also confirmed at mRNA levels
of Bcl-2 and survivin proteins.
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In regard to numerous tumor cell lines, the existence of survivin is associated to apoptosis
resistance which corresponds to malignancy enhancement [45, 50]. In vitro researches showed the
survivin hindrance increased or restored chemo-agents’ cytotoxicity [45, 51]. Animal researches
exhibited profound effectiveness towards xenografts through adenoviral strategy targeted to
survivin [45, 52]. Despite the apoptosis inhibition nature of survivin and Bcl-2, they are active via
different routes in cell apoptosis regulation. The mitochondrial pathways are mainly inhibited by
the anti-apoptotic protein Bcl-2 [45, 53], whilst survivin directly prevents and stimulation of
processing of effector caspase 7 and 3 which typically act downstream of the signaling pathways
[45, 47] which implies the induction of cell apoptosis by compound L11 via different pathways.
1
.2
1
0
0
0
0
.8
.6
.4
.2
0
*
*
bcl-2
Survivin
Control
24 h
48 h
Fig.5. Effects of compound L11 on the Bcl-2 and survivin mRNA expression in A2780 cells with
β-actin as a housekeeping control. The cells were treated with L11 (0.042 mmol/L) and vehicle
(Control group) for 24 and 48 h. Data represented as meanꢀ±ꢀS.E.M; *P-value ≤ 0.05 statistically
significant compared to control group.
2
.2.6. Western blot analysis
Anti-apoptotic proteins’ expression that includes survivin and Bcl-2 upon A2780 cells’
treatment via compound L11 for a period of 48 h was conducted through Western blot analysis.
The proteins’ Bcl-2 family is an integral regulator concerning with an intrinsic apoptosis pathway.
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Such associated proteins share a minimum of one to four homologous areas named Bcl homology
(BH) domains (BH1 to BH4) which regulate the competency of Bcl-2 proteins for the purpose of
binding to one another to create hetero and homodimers. The BH domain is involved at numerous
levels for the functioning of such proteins pertaining to cell survival and cell death [37, 54]. The
Bcl-2 protein is a member of the Bcl-2 family and is bonded to the external mitochondria surface
as well as the surface of endoplasmic reticulum whilst separating pro-apoptotic members e.g. Bax
from mitochondria and averts apoptotic death of cells [55]. Enhanced Bcl-2 expression is
documented in numerous tumor cells of a drug-resistant nature [49].
In the present study, apoptosis-connected cellular processes were studied by the Western blot.
Based on results, in addition to Bcl-2, survivin is over-expressed in A2780 cancer cells (Fig. 6).
Changes in the protein expression of Bcl-2 and survivin (anti-apoptotic) was evaluated under the
influence of compound L11. The A2780 cells, which were treated with a certain concentration
(0.042 mmol/L) of L11, exhibited the widespread and effective induction of apoptosis after 48 h.
As shown in Fig. 6, the immunoblot results confirmed that levels of Bcl-2 expression in cell line
significantly (P-value ≤ 0.05) decreased by the treatment with compound L11 compared to control
cells (Fig. 6). Interestingly, the expression of survivin was also reduced in A2780 cells after the
treatment with L11 (P-value ≤ 0.05). Thus, it was found that compound L11 inhibited Bcl-2 and
survivin activities with the IC value of 0.042 mmol/L by the Western blot analysis.
5
0
L11 might intervene with the expression of survivin and Bcl-2 proteins or more likely activated
degradation pathways of proteins. In regard to tumor cells, apoptosis may be activated through the
stimulation of molecules that are upstream of apoptosis signaling or through the hindrance of anti-
apoptotic parameters [13, 35]. Researches on intrinsic apoptotic pathways showed that L11 may
induce intrinsic checkpoint protein caspase 9 whilst decreasing transcription level and the
expression of anti-apoptotic Bcl-2 and survivin. The induction of apoptosis by L11 was achieved
by down- regulation of level expression of genes and proteins Bcl-2 and survivin, and activation
of caspase 9 and 3.
On the other hand, our findings indicated that compound L11 induced apoptosis by regulating
Bcl-2, survivin, caspase 9 and 3, loss of MMP, and high ROS. The Bcl-2 protein is predominantly
located on the mitochondria where the critical decision point, Cyt C release, is regulated. In regard
to cancer cells, they inhibit the formation of a pore within the mitochondrial membrane and Cyt C
discharge, thus preventing caspase 9 and 3 induction in downstream molecules [35]. According to
1
6

JOURNAL PRE-PROOF
prior suggestions, Bcl-2 protein expression reduction may cause MMP to collapse whilst
accumulating intracellular ROS leads to cell apoptosis through caspase 3 and 9 activation. The
anti-proliferative impact of the compound L11 was attributed to numerous processes, namely
apoptosis activation and the expression of apoptosis-relevant proteins and genes. Consistent with
other studies [19, 35], the present results supported that the apoptosis induction in A2780 cancer
cells by compound L11 was related to mitochondrial dysfunction signaling pathways as an
intrinsic pathway.
Fig. 6. Survivin and (B) Bcl-2 protein levels were assayed in cell lysates by Western blotting.
Semi-quantitative data measured by imageJ software for survivin and Bcl-2 bands were
normalized by the intensities of respective β-actin. *P-value ≤ 0.05 statistically significant
compared to control group.
2
.2.7. Molecular docking studies
The hybrid molecules have multiple biological activities, different or dual modes of action,
modified selectivity profile, and reduced unfavorable side effects due to an involvement of
pharmacophores in a molecular scaffold [5, 6]. According to bioinformatics studies on possible
pathways of apoptosis induction, in the present project attempts to determine the theoretical
binding site and the binding energy of compound L11 on Bcl-2 and survivin by molecular docking
along with Western blotting analysis.
IAPs such as survivin can bind to caspases which are essential proteins in apoptosis, and
subsequently deactivate their activity. After the apoptotic stimulus, Second Mitochondria-derived
Activator of Caspases/ Direct IAP-binding protein with low pI (Smac/DIABLO) protein as a
deactivator for apoptosis inhibitors is released from the inner mitochondrial membrane into the
cytosol, and thus endogenously antagonizes functions of the inhibitory activity of IAPs on
1
7

JOURNAL PRE-PROOF
caspases. Some synthetic compounds, which are called Smac-mimetics, can kill malignant cells
by inhibiting IAPs. This specific binding of Smac-mimetics, as single agents, to IAPs cause
caspases to be released from Smac, remove the IAP-mediated inhibition of caspases, thereby
promoting apoptosis in a subset of human cancer cells [33, 46, 48, 56]. The development of Smac-
mimetics as novel anti-cancer drugs increases the utility, so that several compounds targeting IAPs
are developed in clinical trials [56]. Analysis on molecular docking proved that survivin BIR
domain binding to inhibitors may cause the discharge of caspases from survivin and bolster
apoptosis as with prior documented results by others [40, 46, 57]. As an example, survivin blockers
can conduct binding to survivin BIR domain within a common binding site such as Smac/DIABLO
with sufficient energy for binding [40, 46, 57].
Starting from such conceptual design for further investigation of such hypothesis in parallel
with biochemical studies about the apoptosis induction, molecular docking studies were conducted
in details. Therefore, the evaluation of compound L11 as a promising Smac-mimetic to BIR
domain of survivin potentially was carried out via molecular docking. However, continued efforts
to prove the ability of L11 targeting both survivin and Bcl-2-regulated apoptosis as well as further
in vitro studies should be conducted to provide more evidence on the anti-cancer effect of L11
according to previous studies [4, 7, 29, 40].
As shown in Fig. 7, some Bcl-2 amino acids including Phe104, Tyr108, Phe112, Met115,
Gln118, Leu119, Val133, Glu136, and Phe153 are predicated to be within 4.0 Å of compound L11
for forming intermolecular hydrophobic interactions. In addition, L11 established a strong
hydrogen bond with Tyr108 of Bcl-2 at a distance 2.79 Å. Based on L11 inhibition constant
(
K =401.24 nM), it is favorable to interact with free binding energy -8.73 kcal/mol and electrostatic
i
force -0.07 kcal/mol. Tyr108 and Gln118, Bcl-2 amino acids are typical residues which entail
interaction with Bcl-2 blockers. It is noteworthy that Gln118 enables a robust interaction with
BAX peptide [4, 29]. As antagonists such as indole-coumarin hybrid small molecules were
synthesized and designed as prospective molecules with targeting Bcl-2 due to their anti-cancer
properties [7, 29].
Based on estimated inhibition constant of L11 (K =1.40 µM) with the binding free energy
i
(-7.98 kcal/mol), it is appropriate interaction with survivin. Hydrogen bonds adopt the role of
stabilizers for ligand-protein interactions. A pair of hydrogen bonds with NH group of Arg108 and
coumarin moiety oxygen carbonyl at distances 3.05 Å and 3.31 Å have vital responsibilities in
1
8

JOURNAL PRE-PROOF
survivin-L11 complex stabilization as shown in Fig 7. Electrostatic force between L11 and
survivin was -0.14 kcal/mol which entailed appropriate interaction concerning proteins and drugs
[40]. It was reported that residues Pro8, Ala9, Pro12, Ile44, Gln56, Cys57, Phe58, phe61, and
Lys112 of survivin were responsible for intermolecular hydrophobic interactions. Survivin protein
possesses an individual N-terminal BIR domain from amino acids 15 to 89 as well as long C-
terminal a-helix of amino acids 100 to 140 within its formation. Zinc ion in the survivin structure
is placed at BIR domain center and has a vital role for BIR domain stabilization. Such domain is
integral in inhibiting caspase behavior [41, 47].
Fig. 7. Schematic interaction of the best docking resulting from AutoDock software presented by
LigPlot software for (A) Bcl-2 and (B) Survivin. In this figure, compound L11 exposure is blue-
highlighted. Hydrogen bonding is in green and van der Waals interactions are in red circulars.
The predicted binding conformations revealed that compound L11 had worthy inhibition
potential with two electron donating groups (EDG) on quinoline ring without any substation on
the aryl group and alkyldiamide chain. It was observed that the number of substituents and their
respective positions on the aryl group and alkyldiamide chain also affected the orientation and
binding pattern of compounds in the binding pocket of enzyme. The slight difference in potential
1
9

JOURNAL PRE-PROOF
was mainly affected by the position of substituent; and in some cases, the number of substituent
also plays a role.
Findings from biochemical assays are in line with in silico docking researches for the
prediction of potential anti-cancer behavior for such coumarin-quinoline hybrid. Docking
researches present favorable information on compound L11 binding affinity and aided in the
understanding of interactions. Because of the high occurrence of unfavorable consequences
activated through most present drugs, compound L11 seems prospective to design enhanced
selective therapeutic agents for the purpose of fighting cancer.
The findings of this study implied that novel coumarin-quinoline hybrids are prospective
options for extended studies and may be presented as influential agents concerning chemotherapy.
Extensive tests are required to analyze more detailed processes of compound L11 activity.
Additionally, it must be ascertained whether L11 may also target other inhibitor apoptosis protein
members, namely XIAP, cIAP2 and cIAP1 as well as other apoptosis-regulating proteins, namely
Bcl-2 family that includes BAX or on other cell lines.
3
. Conclusion
The present study introduced fourteen novel analogs of coumarin-quinoline hybrids with
cytotoxic activity in the A2780 human ovarian cancer cells. Furthermore, L11 and L12 were
introduced as the most active compounds compared with other compounds after evaluating its
cytotoxic effects. Several methods were also performed including cell viability, induction of
apoptosis, MMP assay, determination of intracellular ROS, caspase 9 and 3 activity assay. Since
the applied compound L11 in the present study had special biological properties, the expression
of survivin and Bcl-2 as key regulators of apoptosis was also evaluated both at gen and protein
levels after the cell treatment with compound L11. The molecular docking study was performed
to understand the binding interaction of the most active analog.
The present study initially indicated that L11 and L12 as cytotoxic compounds could
induce apoptosis in A2780 cell line. The synergistic effect of compound L11 on apoptosis
induction might be due to the regulation of anti-apoptotic agents including down-regulation of Bcl-
2
and survivin and activation of caspase 9 and 3. Further examinations indicated that compound
L11 increased ROS levels, reduced MMP, and induced apoptosis in A2780 cells through the
intrinsic mitochondrial pathway. Therefore, it was possible that a compound L11 may be a
2
0

JOURNAL PRE-PROOF
sufficient agent in ovarian cancer therapy. The results can aid in designing novel synthetic
coumarin-quinoline derivatives of competent cytotoxic behavior to induce apoptosis within A2780
cancer cells.
4
4
. Experimental
.1. Materials and methods
All reagents and chemical solvents in the present study were purchased from Merck, Fluka
and Acros, and were used without any purification. Nuclear magnetic resonance (NMR) spectra
were accomplished in CDCl on Bruker AC 400 spectrometer operating at 500 MHz/125 MHz for
3
1
13
H NMR and C NMR, respectively. Chemical shifts (δ) were reported in ppm, downfield from
internal TMS standard. Precoated silica gel (E. Merck Kiesegel 60F254, layer thickness 0.25 mm)
was applied for the thin layer chromatography (TLC).
4
4
.2.
Synthesize and characterization
.2.1. Synthesis of coumarin-3-carboxylic acid (1)
In a round-bottomed flask, salicylaldehyde (10 mmol) and Meldrum’s acid (12 mmol) in
water (20 mL) were heated at reflux under stirring for 10 h. The reaction mixture was cooled and
filtered on Büchner funnel. Further purification was done using the crystallization in ethyacetate.
(
Yield = 95%).
.2.2. Synthesis of 2-chloroquinoline-3-carbaldehydes (2)
-Chloroquinoline-3-carbaldehydes (2) were synthesized from acetanilides 1 via a
Vilsmeier- Haack reaction (Scheme 2).
4
2
O
O
i) POCl / DMF
R
3
H
Me
HN
ii) 80-90°C,5h or Mw
N
Cl
R
1
2
R= alkyl, alkoxy, halo
Scheme 2. Vilsmeier-Haak reaction
4
.2.3. General procedure for the synthesis of chromene-3-carboxamide (L1-L14)
2
1

JOURNAL PRE-PROOF
The reaction between 2-chloroquinoline-3-carbaldehydes and amine in methanol, imine
was first created, and then coumarin-3-carboxylic acid was added to the reaction and stirring was
continued for 30 min. Imine intermediate was prepared for attacking by the nucleophilic
isocyanides. The resulting solution was stirred at room temperature for 4−6 h. The reaction was
completed after 4–6 h. The reaction mixture was cooled and filtered on Büchner funne (Yields:
6
9-90%). Scheme 3 shows a mechanism for the synthesis of chromene-3-carboxamide.
Cl
O
O
NH2
N
OH
H
N
R2
+
N
R1
+
O
O
R1
N
Cl
R2
Cl
Cl
O
O
N
O
R3
R2
N
H
R2
N
H
N
C
+
R1
1 +
R
O
O
R3
O
O
R1
O
N
O
R1
HN
N
O
R2
NH
N
Cl
N
Cl
R3
O
O
R2
O
O
H
N
O
NH
O
Cl
N
R1
Scheme 3. Proposed mechanism for the synthesis of chromene-3-carboxamide
4
.2.3.1.
N-(1-(2-chloroquinolin-3-yl)-2-(cyclohexylamino)-2-oxoethyl)-2-oxo-N-
phenyl-2H-chromene-3-carboxamide (L1)
-1
White Solid, Yield: 88%. m.p: 199-202 °C. IR (KBr) (νmax, cm ): 3280 (NH), 2930, 1728 (C=O),
1
1
656 (C=O), 755; H NMR (500 MHz, CDCl ): δ :1.28-1.33 (m, 1H, CH), 1.39-1.64 (m, 4H,
3
H
CH), 1.71-1.73 (m, 1H, CH), 1.82-1.84 (m, 1H, CH), 1.90-1.93 (m, 1H, CH), 2.08-2.10 (m, 1H,
CH), 2.26-2.28(m, 1H, CH), 4.06-4.12 (m, 1H, CH), 6.93 (t, 1H, J=10Hz, Ar-H), 6.98-7.06 (m,
2
H, Ar-H), 7.09 (s, 1H, Ar-H), 7.20 (brs, 2H, NH+Ar-H ), 7.26 (d, 1H, J=10Hz, Ar-H), 7.34 (t,
2
2

JOURNAL PRE-PROOF
1
H, J=5Hz, Ar-H), 7.51 (t, 1H, J=10Hz, Ar-H), 7.56-7.59 (m, 2H, Ar-H), 7.70 (t, 2H, J=5Hz, Ar-
1
3
H), 7.91 (d, 1H, J=5Hz, Ar-H), 8.05-8.09 (m, 2H, Ar-H), 8.10 (s, 1H, Ar-H). C NMR (125 MHz,
CDCl ): δ : 25.4, 25.6, 25.8, 33.1, 33.6, 49.9, 62.6, 117.2, 118.3, 125.5, 126.1, 126.8, 127.1, 127.5,
3
C
1
28.34 (2C), 128.39, 128.8, 129.1, 129.3, 131.3, 133.4, 138.3, 142.4, 143.4, 147.3, 151.3, 154.1,
1
59.1, 165.8, 167.0. Anal. Calcd. for C H ClN O : C, 70.02; H, 4.99; N, 7.42. Found: C, 70.42;
3
3
28
3
4
H, 5.01; N, 7.63.
4
.2.3.2.
N-(1-(2-chloroquinolin-3-yl)-2-(cyclohexylamino)-2-oxoethyl)-2-oxo-N-(p-
tolyl)-2H-chromene-3-carboxamide (L2)
-1
White Solid, Yield: 75%. m.p: 251-255 °C. IR (KBr) (νmax, cm ): 3292 (NH), 2930, 1735 (C=O),
1
1
685 (C=O), 1656 (C=O), 750; H NMR (500 MHz, CDCl ): δ :1.15-1.26 (m, 1H, CH), 1.35-1.57
3
H
(m, 4H, CH), 1.66-1.68 (m, 1H, CH), 1.74-1.77 (m, 1H, CH), 1.85-1.88 (m, 1H, CH), 1.98 (s, 3H,
CH ), 2.03-2.05 (m, 1H, CH), 2.16-2.20 (m, 1H, CH), 4.03-4.06 (m, 1H, CH), 6.72-6.74 (m, 2H,
3
Ar-H), 6.95 (s, 1H, Ar-H), 7.03(brs, 1H, NH+CH), 7.21 (d, 1H, J=5Hz, Ar-H), 7.29 (t, 1H, J=
5
Hz, Ar-H), 7.46 (t, 1H, J=10Hz, Ar-H), 7.50-7.52 (m, 2H, Ar-H), 7.63-7.68 (m, 2H, Ar-H), 7.87
1
3
(
d, 1H, J=10Hz, Ar-H), 7.97-8.05 (m, 2H, Ar-H), 8.08 (s, 1H, Ar-H). C NMR (125 MHz, CDCl ):
3
δC: 21.2, 25.4, 25.6, 25.8, 33.1, 33.6, 49.9, 62.6, 117.2, 118.4, 125.5, 126.2, 126.9, 127.0, 127.4,
1
1
5
28.4, 129.1, 129.8 (2C), 131.2, 133.2, 135.8, 138.7, 142.4, 143.2, 147.3, 151.4, 154.0, 159.1,
65.9, 167.1. Anal. Calcd. for C H ClN O : C, 70.40; H, 5.21; N, 7.24. Found: C, 70.83; H,
3
4
30
3
4
.23; N, 7.49.
4
.2.3.3.
N-(1-(2-chloroquinolin-3-yl)-2-(cyclohexylamino)-2-oxoethyl)-2-oxo-N-(m-
tolyl)-2H-chromene-3-carboxamide (L3)
-1
White Solid, Yield: 88%. m.p: 230-232 °C. IR (KBr) (νmax, cm ):3279 (NH), 2928, 1723 (C=O),
1
1
1
2
6
658 (C=O), 755; H NMR (500 MHz, CDCl ): δ :1.24-1.29 (m, 1H, CH), 1.37-1.59 (m, 4H, CH),
3
H
.68-1.71 (m, 1H, CH), 1.80-1.82 (m, 1H, CH), 1.87-1.90 (m, 1H, CH), 2.03 (s, 3H, CH ), 2.07-
3
.10 (m, 1H, CH), 2.22-2.31 (m, 1H, CH), 4.04-4.10 (m, 1H, CH), 6.71 (d, 1H, J=5Hz, Ar-H),
.83-6.86 (m, 1H, Ar-H), 6.96 (s, 1H, Ar-H), 7.01 ( brs, 2H, NH+OH ), 7.24 (d, 1H, J=10Hz, Ar-
H), 7.31 (t, 1H, J=10Hz, Ar-H), 7.48 (t, 1H, J=10Hz, Ar-H), 7.52-7.55 (m, 2H, Ar-H), 7.66-7.70
(m, 2H, Ar-H), 7.89 (d, 1H, J=5Hz, Ar-H), 7.95 (d, 1H, J=10Hz, Ar-H), 7.98 (s, 1H, Ar-H), 8.11
1
3
(
s, 1H, Ar-H). C NMR (125 MHz, CDCl ): δ :21.4, 25.4, 25.6, 25.8 ,33.1, 33.6, 49.9, 62.7, 117.2,
3
C
2
3

JOURNAL PRE-PROOF
1
1
18.4, 125.5, 126.2, 126.4, 126.9, 127.5, 128.31, 128.36, 128.8, 129.0, 129.5, 129.7, 131.3, 133.3,
35.8, 138.3, 139.1, 142.4, 143.1, 147.3, 151.4, 154.0, 159.1, 165.7, 167.0. Anal. Calcd. for
C H ClN O : C, 70.40; H, 5.21; N, 7.24. Found: C, 70.84; H, 5.24; N, 7.51.
3
4
30
3
4
4
.2.3.4.
N-(1-(2-chloro-7-methylquinolin-3-yl)-2-(cyclohexylamino)-2-oxoethyl)-2-
oxo-N-phenyl-2H-chromene-3-carboxamide (L4)
-1
White Solid, Yield: 85%. m.p: 225-226 °C. IR (KBr) (νmax, cm ):3284 (NH), 2928, 1723 (C=O),
1
1
1
2
6
652 (C=O), 757; H NMR (500 MHz, CDCl ): δ :1.27-1.31 (m, 1H, CH), 1.35-1.59 (m, 3H, CH),
3
H
.62-1.71 (m, 2H, CH), 1.80-1,82 (m, 1H, CH), 1.88-1.90 (m, 1H, CH), 2.05-2.08 (m, 1H, CH),
.23-2.25 (m, 1H, CH), 2.51 (s, 3H, CH ), 4.06-4.08 (m, 1H, CH), 6.91 (t, 1H, J= 5Hz, Ar-H),
3
.98-6.99 (m, 3H, Ar-H), 7.19 (brs, 1H, NH), 7.23 (d, 1H, J= 10Hz, Ar-H), 7.29-7.32 (m, 3H, Ar-
H), 7.53-7.57 (m, 3H, Ar-H), 7.66 (s, 1H, Ar-H), 7.94 (d, 1H, J= 5Hz, Ar-H), 8.01 (d, 2H, J=10Hz,
1
3
Ar-H). C NMR (125 MHz, CDCl ): δ : 22.3, 25.4, 25.6, 25.8, 33.1, 33.6, 49.9, 62.6, 117.2, 118.3,
3
C
1
24.9, 125.5, 125.8, 126.1, 127.4, 127.9 (2C), 128.8, 129.1, 129.4, 129.8, 133.3, 138.2, 138.7,
42.0, 143.3, 147.6, 151.3, 154.1, 159.1, 165.8, 167.1. Anal. Calcd. for C H ClN O : C, 70.40;
1
3
4
30
3
4
H, 5.21; N, 7.24. Found: C, 70.85; H, 5.22; N, 7.48.
4
.2.3.5.
N-(1-(2-chloro-7-methylquinolin-3-yl)-2-(cyclohexylamino)-2-oxoethyl)-2-
oxo-N-(p-tolyl)-2H-chromene-3-carboxamide (L5)
-1
White Solid, Yield: 79%. m.p: 262-264 °C. IR (KBr) (νmax, cm ):3269 (NH), 2929, 1722 (C=O),
1
1
686 (C=O), 1644 (C=O), 758; H NMR (500 MHz, CDCl ): δ :1.26-1.31 (m, 1H, CH), 1.37-1.60
3
H
(
m, 4H, CH), 1.68-1.71 (m, 1H, CH), 1.80-1.81 (m, 1H, CH), 1.88-1.90 (m, 1H, CH), 2.00 (s, 3H,
CH ), 2.05-2.07 (m, 1H, CH), 2.23-2.25 (m, 1H, CH), 2.50 (m, 3H, CH ), 4.04-4.09 (m, 1H, CH),
3
3
6
.76 (d, 2H, J=10Hz, Ar-H), 6.97 (s, 1H, Ar-H), 7.06 (brs, 2H, NH+Ar-H), 7.24 (d, 1H, J=10Hz,
Ar-H), 7.30-7.32 (m, 2H, Ar-H), 7.53-7.54 (m, 2H, Ar-H), 7.58 (d, 1H, J=10Hz, Ar-H), 7.65 (s,
1
3
1
2
1
1
5
H, Ar-H), 8.00 (s, 2H, Ar-H), 8.03 (s, 1H, Ar-H). C NMR (125 MHz, CDCl ): δ :21.2, 22.3,
3 C
5.4, 25.6, 25.8, 33.1, 33.6, 49.8, 62.6, 117.2, 118.4, 125.0, 125.5, 125.9, 126.3, 127.4, 127.9,
29.1, 129.72, 129.78 (2C), 133.2, 135.5, 138.7, 141.9, 142.0, 143.1, 147.6, 151.3, 154.0, 159.1,
65.9, 167.2. Anal. Calcd. for C H ClN O : C, 70.76; H, 5.43; N, 7.07. Found: C, 71.08; H,
3
5
32
3
4
.45; N, 7.29.
2
4

JOURNAL PRE-PROOF
4
.2.3.6.
N-(1-(2-chloro-7-methylquinolin-3-yl)-2-(cyclohexylamino)-2-oxoethyl)-2-
oxo-N-(m-tolyl)-2H-chromene-3-carboxamide (L6)
-1
White Solid, Yield: 74%. m.p: 203-205 °C. IR (KBr) (νmax, cm ):3282 (NH), 2929, 1738 (C=O),
1
1
682 (C=O), 1661 (C=O), 747. H NMR (500 MHz, CDCl ): δ :1.27-1.31 (m, 1H, CH), 1.36-1.60
3
H
(
m, 4H, CH), 1.68-1.71 (m, 1H, CH), 1.80-1.82 (m, 1H, CH), 1.88-1.90 (m, 1H, CH), 2.04 (s, 3H,
CH ), 2.05-2.07 (m, 1H, CH), 2.22-2.25 (m, 1H, CH), 2.51 (s, 3H, CH ), 4.06-4.08 (m, 1H, CH),
3
3
6
7
.71 (d, 1H, J=5Hz, Ar-H), 6.80-6.84 (m, 1H, Ar-H), 6.96 (s, 2H, Ar-H), 7.02 ( brs, 1H, NH ),
.24 (d, 1H, J=5Hz, Ar-H), 7.30-7.32 (m, 2H, Ar-H), 7.53-7.56 (m, 2H, Ar-H), 7.58 (d, 1H, J=5Hz,
1
3
Ar-H), 7.66 (s, 1H, Ar-H), 7.95 (d, 1H, J=5Hz, Ar-H), 7.99 (s, 1H, Ar-H), 8.04 (s, 1H, Ar-H). C
NMR (125 MHz, CDCl ): δ :21.4, 22.3, 25.4, 25.6, 25.8, 33.1, 33.6, 49.8, 62.7, 117.2, 118.4,
3
C
1
1
24.9, 125.5, 125.9, 126.2, 126.4, 127.4, 127.8, 128.8, 129.0, 129.5, 129.7, 131.3, 133.2, 138.2,
39.0, 142.01, 142.05, 143.1, 147.6, 151.3, 154.0, 159.1, 165.7, 167.1. Anal. Calcd. for
C H ClN O : C, 70.76; H, 5.43; N, 7.07. Found: C, 71.39; H, 5.81; N, 7.12.
3
6
35
3
4
4
.2.3.7.
N-(1-(2-chloro-6-methylquinolin-3-yl)-2-(cyclohexylamino)-2-oxoethyl)-2-
oxo-N-(p-tolyl)-2H-chromene-3-carbox amide (L7)
-1
White Solid, Yield: 75%. m.p: 248-250 °C. IR (KBr) (νmax, cm ):3282 (NH), 2929, 1738 (C=O),
1
1
680 (C=O), 1661 (C=O), 747; H NMR (500 MHz, CDCl ): δ :1.25-1.30 (m, 1H, CH), 1.36-1.58
3
H
(
m, 4H, CH), 1.67-1.70 (m, 1H, CH), 1.79-1.81 (m, 1H, CH), 1.87-1.89 (m, 1H, CH), 1.99 (s, 3H,
CH ), 2.05-2.07 (m, 1H, CH), 2.22-2.24 (m, 1H, CH), 2.46 (s, 3H, CH ), 4.03-4.09 (m, 1H, CH),
3
3
6
.76 (d, 2H, J=5Hz, Ar-H), 6.94 (s, 1H, Ar-H), 7.07 (brs, 2H, NH+Ar-H), 7.22 (d, 1H, J=5Hz, Ar-
H), 7.26-7.31 (m, 1H, Ar-H), 7.46-7.48 (m, 2H, Ar-H), 7.52 (t, 2H, J=5Hz, Ar-H), 7.76 (d, 1H,
1
3
J=5Hz, Ar-H), 8.00 (d, 3H, J=5Hz, Ar-H). C NMR (125 MHz, CDCl ): δ :21.2, 21.9, 25.4, 25.6,
3
C
2
5.8, 33.1, 33.6, 49.8, 62.6, 117.1, 118.4, 125.4, 126.2, 126.8, 126.9, 127.2, 127.9, 129.1, 129.7
2C), 130.3, 133.2, 133.5, 135.5, 138.6, 141.6, 143.2, 145.9, 150.4, 154.0, 159.1, 165.9, 167.2.
Anal. Calcd. for C H ClN O : C, 70.76; H, 5.43; N, 7.07. Found: C, 71.36; H, 5.82; N, 7.15.
(
3
6
35
3
4
4
.2.3.8.
N-(1-(2-chloro-6-methylquinolin-3-yl)-2-(cyclohexylamino)-2-oxoethyl)-2-
oxo-N-phenyl-2H-chromene-3-carboxamide (L8)
-1
White Solid, Yield: 86%. m.p: 254-256 °C. IR (KBr) (νmax, cm ): 3282 (NH), 2931, 1736 (C=O),
1
1
682 (C=O), 1659 (C=O), 749; H NMR (500 MHz, CDCl ): δ :1.25-1.30 (m, 1H, CH), 1.36-1.58
3
H
2
5

JOURNAL PRE-PROOF
(m, 4H, CH), 1.68-1.70 (m, 1H, CH), 1.79-1.81 (m, 1H, CH), 1.87-1.89 (m, 1H, CH), 2.05-2.07
(
m, 1H, CH), 2.22-2.25 (m, 1H, CH), 2.46 (s, 3H, CH ), 4.03-4.09 (m, 1H, CH), 6.90 (t, 1H, J=5Hz,
3
Ar-H), 6.97-6.99 (m, 3H, Ar-H+NH), 7.21 (d, 3H, J=5Hz, Ar-H), 7.28-7.31 (m, 1H, Ar-H), 7.44
(s, 1H, Ar-H), 7.47 (d, 1H, J=5Hz, Ar-H), 7.51-7.52 (m, 1H, Ar-H), 7.54 (d, 1H, J=10Hz, Ar-H),
1
3
7
.75 (d, 1H, J=5Hz, Ar-H), 7.97-7.98 (d, 1H, J=10Hz, Ar-H), 8.01 (d, 2H, J=5Hz, Ar-H). C
NMR (125 MHz, CDCl ): δ 21.9, 25.4, 25.6, 25.8, 33.1, 33.6, 49.9, 62.6, 117.2, 118.3, 125.5,
3
C:
1
26.0, 126.7, 126.9, 127.1, 127.9, 128.8, 129.1, 129.4 (2C), 133.3, 133.6, 137.5, 138.3, 141.6,
43.3, 145.9, 150.4, 154.0, 159.1, 165.8, 167.1. Anal. Calcd. for C H ClN O : C, 70.40; H, 5.21;
1
3
4
30
3
4
N, 7.24. Found: C, 70.83 H, 5.24; N, 7.43.
4
.2.3.9.
N-(1-(2-chloro-5,8-dimethylquinolin-3-yl)-2-(cyclohexylamino)-2-oxoethyl)-
-oxo-N-(p-tolyl)-2H-chromene-3-carboxamide (L9)
2
-1
White Solid, Yield: 69%. m.p: 292-294 °C. IR (KBr) (νmax, cm ):3270 (NH), 2931, 1730 (C=O),
1
1
1
2
651 (C=O), 759; H NMR (500 MHz, CDCl ): δ :1.26-1.31 (m, 1H, CH),1.37-1.43 (m, 2H, CH),
3
H
.45-1.58 (m, 2H, CH), 1.68-1.75 (m, 1H, CH), 1.79-1.89 (m, 2H, CH), 2.05 (s, 3H, CH ), 2.06-
3
.07 (m, 1H, CH), 2.20-2.23 (m, 1H, CH), 2.50 (s, 3H, CH ), 2.66 (s, 3H, CH ), 4.02-4.08 (m, 1H,
3
3
CH), 6.80 (d, 2H, J=5Hz, Ar-H), 6.97 (s, 1H, Ar-H), 7.08 (brs, 2H, Ar-H+NH), 7.20 (d, 1H, J=5Hz,
Ar-H), 7.24 (d, 1H, J=5Hz, Ar-H), 7.29-7.32 (m, 1H, Ar-H), 7.40 (d, 1H, J=5Hz, Ar-H), 7.52-7.55
1
3
(
m, 2H, Ar-H), 7.67-7.69 (d, 1H, J=5Hz, Ar-H), 7.95 (s, 1H, Ar-H), 8.21 (s, 1H, Ar-H). C NMR
125 MHz, CDCl ): δ 18.0, 18.7, 21.2, 25.4, 25.5, 25.9, 33.0, 33.5, 49.8, 62.8, 117.2, 118.4, 125.4,
(
3
C:
1
25.7, 126.3, 126.4, 127.5, 129.0, 129.3, 129.8, 131.0, 133.11, 133.18, 134.4, 135.8, 138.8, 139.2,
42.8, 147.1, 149.9, 154.1, 159.0, 165.9, 167.3. Anal. Calcd. for C H ClN O : C, 71.10; H, 5.64;
1
3
6
34
3
4
N, 6.91. Found: C, 71.75; H, 6.01; N, 6.97.
4
.2.3.10.
N-(1-(2-chloro-6-methylquinolin-3-yl)-2-(cyclohexylamino)-2-oxoethyl)-2-
oxo-N-(m-tolyl)-2H-chromene-3-carboxamide (L10)
-1
White Solid, Yield: 81%. m.p: 272-274 °C. IR (KBr) (νmax, cm ):3290 (NH), 2931, 1738 (C=O),
1
1
1
2
661 (C=O), 749; H NMR (500 MHz, CDCl ): δ 1.26-1.35 (m, 1H, CH), 1.37-1.41 (m, 2H, CH),
3
H:
.45-1.61 (m, 2H, CH), 1.68-1.71 (m, 1H, CH), 1.80-1.82 (m, 1H, CH), 1.88-1.90 (m, 1H, CH),
.05 (s, 3H, CH ), 2.06-2.07 (m, 1H, CH), 2.19-2.24 (m, 1H, CH), 2.48 (s, 3H, CH ), 4.06-4.08
3
3
(m, 1H, CH), 6.72 (d, 1H, J=5Hz, Ar-H), 6.83-6.85 (m, 1H, Ar-H), 6.93 (s, 1H, Ar-H), 6.97-7.00
2
6

JOURNAL PRE-PROOF
(brs, 1H, Ar-H), 7.03 (brs, 1H, N-H), 7.23 (d, 1H, J=10Hz, Ar-H), 7.30 (t, 1H, J=5Hz, Ar-H), 7.46
(s,
1H,
Ar-H),
7.50
(d,
1H,
J=10Hz,
Ar-H),
7.54-7.55
(m,
1
3
7
.90 (d, 1H, J=5Hz ,Ar-H), 7.98 (s, 1H, Ar-H), 8.02 (s, 1H, Ar-H). C NMR (125 MHz, CDCl ):
3
δC: 21.4, 21.9, 25.4, 25.6, 25.8, 33.1, 33.6, 49.8, 62.8, 117.2, 118.4, 125.5, 126.2, 126.4, 126.7,
1
1
7
26.9, 127.1, 127.9, 128.8, 129.0, 129.5, 129.8, 133.2, 133.6, 137.5, 138.3, 139.0, 141.7, 143.1,
45.9, 150.4, 154.0, 159.1, 165.7, 167.1. Anal. Calcd. for C H ClN O : C, 70.76; H, 5.43; N,
3
5
32
3
4
.07. Found: C, 70.38; H, 5.81; N, 7.13.
4
.2.3.11.
N-(1-(2-chloro-5,8-dimethylquinolin-3-yl)-2-(cyclohexylamino)-2-oxo ethyl)-2-
oxo-N-phenyl-2H-chromene-3-carboxamide (L11)
-1
White Solid, Yield: 88%. m.p: 273-275 °C. IR (KBr) (νmax, cm ): 3261 (NH), 2930, 1724 (C=O),
1
1
1
2
655 (C=O), 761; H NMR (500 MHz, CDCl ): δ 1.26-1.35 (m, 1H, CH), 1.35-1.59 (m, 4H, CH),
3
H:
.68-1.73 (m, 1H, CH), 1.79-1.82 (m, 1H, CH), 1.87-1.89 (m, 1H, CH), 2.05-2.08 (m, 1H, CH),
.20-2.24 (m, 1H, CH), 2.50 (s, 3H, CH ), 2.66 (s, 3H, CH ), 4.03-4.09 (m, 1H, CH), 6.94-7.03
3
3
(m, 4H, NH+Ar-H), 7.19 (d, 2H, J=5Hz, Ar-H), 7.24 (d, 2H, J=10Hz, Ar-H), 7.29-7.32 (m, 1H,
Ar-H), 7.40 (d, 1H, J=5Hz, Ar-H), 7.52-7.56 (m, 2H, Ar-H), 7.66 (d, 1H, J=5Hz, Ar-H), 7.97 (s,
1
3
1
3
1
H, Ar-H), 8.22 (s, 1H, Ar-H). C NMR (125 MHz, CDCl ): δ 18.0, 18.7, 25.4, 25.5, 25.9, 33.0,
3 C:
3.5, 49.8, 62.8, 117.2, 118.3, 125.4, 125.6, 126.1, 126.4, 127.6, 128.9, 129.0, 129.1, 129.6, 131.1,
33.0, 133.2, 134.4, 138.5, 139.2, 143.0, 147.1, 149.8, 154.1, 159.0, 165.8, 167.3. Anal. Calcd. for
C H ClN O : C, 70.76; H, 5.43; N, 7.07. Found: C, 71.11; H, 5.45; N, 7.25.
3
5
32
3
4
4
.2.3.12.
N-(1-(2-chloro-5,8-dimethylquinolin-3-yl)-2-(cyclohexylamino)-2-oxoethyl)-2-
oxo-N-(m-tolyl)-2H-chromene-3-carboxamide (L12)
-1
White Solid, Yield: 90%. m.p: 235-236 °C IR (KBr) (νmax, cm ):3281 (NH), 2928, 1724 (C=O),
1
1
1
2
657 (C=O), 762; H NMR (500 MHz, CDCl ): δ 1.25-1.28 (m, 1H, CH), 1.38-1.45 (m, 2H, CH),
3
H:
.48-1.56 (m, 2H, CH), 1.67-1.70 (m, 1H, CH), 1.79-1.88 (m, 2H, CH), 2.07 (s, 3H, CH ), 2.08-
3
.10 (m, 1H, CH), 2.18-2.20 (m, 1H, CH), 2.50 (s, 3H, CH ), 2.64 (s, 3H, CH ), 4.03-4.07 (m, 1H,
3
3
CH), 6.74 (d, 1H, J=10Hz, Ar-H), 6.85 (s, 1H, Ar-H), 6.96 (s, 2H, Ar-H), 7.09 (brs, 1H, N-H),
.18 (d, 1H, J=5Hz, Ar-H), 7.23 (d, 1H, J=5Hz, Ar-H), 7.28-7.30 (m, 1H, Ar-H), 7.39 (d, 1H,
J=10Hz, Ar-H), 7.51-7.53 (m, 2H, Ar-H), 7.68 (s, 1H, Ar-H), 7.96 (s, 1H, Ar-H), 8.22 (s, 1H, Ar-
7
1
3
H). C NMR (125 MHz, CDCl ): δ 18.0, 18.7, 21.4, 25.4, 25.5, 25.8, 33.0, 33.5, 49.8, 62.9, 117.1,
3
C:
2
7

JOURNAL PRE-PROOF
1
1
18.4, 125.4, 125.7, 126.2, 126.3, 126.7, 127.6, 128.8, 129.0, 129.5, 129.8, 131.1, 132.9, 133.2,
34.4, 138.5, 139.0, 139.2, 142.9, 147.0, 149.9, 154.0, 159.0, 165.8, 167.3. Anal. Calcd. for
C H ClN O : C, 71.10; H, 5.64; N, 6.91. Found: C, 71.52; H, 5.67; N, 7.17.
3
6
34
3
4
4
.2.3.13.
N-(2-(tert-butylamino)-1-(2-chloroquinolin-3-yl)-2-oxoethyl)-2-oxo-N-phenyl-
H-chromene-3-carboxamide (L13)
2
-1
White Solid, Yield: 78%. m.p: 186-188 °C. IR (KBr) (νmax, cm ): 3274 (NH), 2976, 1728 (C=O),
1
1
1
664 (C=O), 758; H NMR (500 MHz, CDCl ): δ 1.56 (s, 9H, CH), 6.85 (s, 1H, Ar-H), 6.92 (t,
3
H:
H, J=5Hz, Ar-H), 6.98-6.99 (m, 2H, Ar-H), 7.18-7.30 (m, 4H, Ar-H+NH), 7.48-7.49 (m, 4H, Ar-
H), 7.65 (t, 1H, J=10Hz, Ar-H), 7.70 (d, 1H, J=10Hz, Ar-H), 7.87 (d, 1H, J=5Hz, Ar-H), 7.95 (s,
1
3
1
1
1
6
H, Ar-H), 8.16 (s, 1H, ArH). C NMR (125 MHz, CDCl ): δ : 29.1 (3 (CH )), 52.8, 62.8, 117.1,
3 C 3
18.3, 125.4, 125.9, 126.9, 127.0, 127.5, 128.2, 128.4, 128.8, 129.0, 129.1, 129.5, 131.4, 133.2,
38.5, 142.1, 143.1, 147.3, 151.3, 154.1, 158.6, 165.9, 167.3. Anal. Calcd. for C H ClN O : C,
3
1
26
3
4
8.95; H, 4.85; N, 7.78. Found: C, 69.41; H, 4.88, N, 7.95.
4
.2.3.14.
N-(2-(tert-butylamino)-1-(2-chloro-5,8-dimethylquinolin-3-yl)-2-oxoethyl)-2-
oxo-N-phenyl-2H-chromene-3-carboxamide (L14)
-1
White Solid, Yield: 81%. m.p: 180-182 °C. IR (KBr) (νmax, cm ): 3340 (NH), 2925, 1716 (C=O),
1
1
3
662 (C=O), 757; H NMR (500 MHz, CDCl ): δ :1.56 (s, 9H, CH), 2.51 (s, 3H, CH ), 2.66 (s,
3
H
3
H, CH ), 6.82 (s, 1H, Ar-H), 6.97 (t, 1H, J=5Hz, Ar-H), 7.03 (t, 2H, J=5Hz, Ar-H), 7.15 (s, 1H,
3
NH), 7.20-7.24 (m, 3H, Ar-H), 7.28-7.31 (m, 2H, Ar-H), 7.41 (d, 1H, J=5Hz, Ar-H), 7.50-7.54
1
3
(
m, 2H, Ar-H), 7.92 (s, 1H, Ar-H), 8.29 (s, 1H, Ar-H). C NMR (125 MHz, CDCl ): δ : 18.0,
3 C
1
1
1
5
8.6, 29.0 (3 (CH )), 52.7 (2 (CH )), 63.3, 117.1, 118.3, 125.3, 125.8, 126.1, 126.4, 127.7, 128.9,
3 3
29.0, 129.1, 129.7, 131.2, 133.0, 133.1, 134.4, 138.8, 138.9, 142.8, 147.1, 149.8, 154.1, 158.6,
65.9, 167.6. Anal. Calcd. for C H ClN O : C, 69.77; H, 5.32; N, 7.40. Found: C, 70.19; H,
3
3
30
3
4
.34, N, 7.64.
2
8

JOURNAL PRE-PROOF
Biological studies
4
4
.2.4. Antiproliferative activity
.2.4.1. Cell culture
The aforementioned A2780 cells were subjected to culturing using Dulbecco’s adjusted
Eagle’s medium (DMEM-F12) including 10% (v/v) fetal bovine serum (FBS), 100 U/mL
penicillin and 100 mg/mL streptomycin. Upon 2 to 3 days, the medium was changed and subjected
to sub-culturing as soon as the density of the cell population achieved 70% to 80% confluence.
4
.2.4.2.
Cell viability assay
MTT assay was performed on A2780 and PC12 cells in order to evaluate the cytotoxicity
of compounds [29, 38]. Doxorubicin was used as a positive control. Based on the manufacturer's
protocol, the cells were cultured onto a 96-well cell culture plates at the density of 15000 cells/well.
After 24 h, various concentrations of compounds (0-100 μg/mL) were added to wells following by
the incubation for 24 h. Subsequently, 20 µL of 5.0 mg/mL MTT solution was put into each well;
and the cells were incubated for an additional 4 h. For the next stage, the medium was omitted
prior to the dissolving of the purple formazan crystals within DMSO. An ELISA plate reader was
used to determine the absorbance with test wavelength (570 nm) and reference wavelength (630
nm) to acquire simple signal i.e. OD570-OD630.
4
.2.5. Caspase 9 and 3 activity assay
Caspase 9 and 3 activities were determined using the sigma colorimetric caspase kit. To
this end, A2780 cells were seeded in a 12-well plate and incubated for 24 h. 0.042 mmol/L and
0
2
.102 mmol/L of L11 and L12, respectively were then added to wells and incubated for the next
4 h. Thereafter, the treated cells were centrifuged at 1200 rpm for 5 min and were lysed in 15 μL
of the cell lysis buffer included with the kit. The extraction of protein content of cells was achieved
by the centrifugation of lysates at 16000-20000 rpm and 4 °C for 15 min. The substrate reaction
buffer containing caspase 3 substrate and caspase 9 substrate was added to the supernatant and
then incubated for 2 h at 37 °C. The absorbance was then read at 405 nm by means of a plate reader
(BioTek, H1M).
4
.2.6. Measurement of mitochondrial membrane potential
2
9