4-Phenyl quinoline derivatives as potential serotonin receptor ligands with antiproliferative activity

Article abstract of DOI:10.1016/j.ejmech.2017.05.002

Antagonists of signaling receptors are often effective non-toxic therapeutic agents. Over the years, there have been evidences describing the role of serotonin or 5-hydroxytryptamine (5-HT) in development of cancer. Although there are reports on the antip

Full text of DOI:10.1016/j.ejmech.2017.05.002

Accepted Manuscript
4-Phenyl quinoline derivatives as potential serotonin receptor ligands with
antiproliferative activity
Pranaya V. Joshi, Alim A. Sayed, Ameeta RaviKumar, Vedavati G. Puranik, Smita S.
Zinjarde
PII:
S0223-5234(17)30363-X
10.1016/j.ejmech.2017.05.002
EJMECH 9432
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 November 2016
Revised Date: 16 April 2017
Accepted Date: 1 May 2017
Please cite this article as: P.V. Joshi, A.A. Sayed, A. RaviKumar, V.G. Puranik, S.S. Zinjarde, 4-Phenyl
quinoline derivatives as potential serotonin receptor ligands with antiproliferative activity, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.05.002.
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4-Phenyl quinoline derivatives as potential serotonin receptor ligands with
antiproliferative activity
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Pranaya V. Joshia , Alim A. Sayedb , Ameeta RaviKumara , Vedavati G. Puranikc and Smita S.
Zinjardea,*
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a Institute of Bioinformatics and Biotechnology, Savitribai Phule Pune University, Pune 411007, India.
b Department of Chemistry, Abeda Inamdar Senior College, Azam Campus, Pune 411001, India.
c Centre for Material Characterization, CSIR-National Chemical Laboratory, Pune 411 008, India.
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Keywords:
Quinoline
Serotonin
Docking studies
Calcium flux
ERK
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ABSTRACT
Antagonists of signaling receptors are often effective non-toxic therapeutic agents. Over the
years, there have been evidences describing the role of serotonin or 5-hydroxytryptamine (5-HT)
in development of cancer. Although there are reports on the antiproliferative effects of some
serotonin receptor antagonists, there are very few investigations related to understanding their
structure-activity relationships. In this study, we report the screening of a library of 4-phenyl
quinoline derivatives for their antiproliferative activities. Preliminary docking studies indicated
that these ligands had the ability to bind to two of the serotonin receptors, 5-HT1B and 5-HT2B.
The results of the in silico experiments were validated by performing in vitro studies on MCF-7
breast cancer cell line. The ethylpiperazine derivatives showed maximum toxicity against this
cancer cell line. The compounds inhibited Calcium ion efflux (induced by serotonin) and ERK
activation. One of the most active 4-phenyl quinoline derivatives (H3a) also induced the
apoptosis, thereby, suggesting the use of this scaffold as a potential anticancer drug.
1. Introduction
Serotonin or 5-hydroxytryptamine (5-HT) is a monoamine neurotransmitter [1]. The activity of
serotonin is mediated through one of the seven receptors (5-HT1 - 5-HT7) that are present on
cells in various organs [2]. Serotonin is known to play a role in cancer progression by eliciting
mitogenic and angiogenic responses and antagonists towards certain serotonin receptors are

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known to display antiproliferative activity [3-8]. 5-HT has been associated with cell proliferation
in carcinomas of breast, prostate and hepatocellular tissues [9-11]. Deregulated expression of
certain 5-HT receptors results in the inhibition of apoptotic pathways [12]. In addition, malignant
tissues often show enhanced secretion of serotonin [13-15]. Binding of serotonin to 5-HT
receptors activates signal transduction pathways that involve phosphorylation of extracellular-
signal regulated kinases (ERK), eventually leading to cellular proliferation [16-18].
The crystal structures of two 5-HT receptors, namely 5-HT1B and 5-HT2B, have been determined
[19,20] and these details could be used to design target-based drugs. Although these two
receptors exhibit spatial and functional diversity, their orthosteric ligand binding sites show
structural similarities. Moreover, 5-HT1B and 5-HT2B are known to interact with different ligands
(5-HT, ergotamine, lysergic acid diethylamide, sumatriptan and norfenfluramine) in a similar
manner. The residues in the binding pockets of the 5-HT receptor family members are seen to be
conserved. In addition to the abovementioned ligands, compounds built on the quinoline scaffold
are also known to bind to 5-HT receptors [21].
Quinoline scaffold has been used to develop molecules with wide range of medicinal benefits
[22]. Anticancer drugs containing quinoline scaffold are able to counter neoplasmic progression
by inhibiting enzymes such as topoisomerases, telomerase, protein kinases, protein phosphatase
Cdc25, histone acetyltransferases and deacetylases [23]. Additionally, there are studies
elucidating the role of quinoline based compounds as agonists and antagonists of serotonin
targets [24-33]. In the current investigation, a series of thirty 4-phenyl quinoline (4-PQ)
derivatives (including nineteen new compounds: H1a, H3a, H3b, H5a, H5b, Cl3a, Cl3b, Cl5a,
Cl5b, and F1a-F5b) were synthesized. In silico docking studies were used to predict the binding
site and affinity of the compounds for 5-HT receptors. These compounds were evaluated for their
in vitro antiproliferative activity against MCF-7 breast cancer cell line that is known to express
5-HT2A receptors. The effects of one of the most active compounds (H3a) on calcium ion flux
and levels of ERK phosphorylation were determined to understand the mechanism of action.
2. Results and Discussion
2.1 Synthesis and single crystal X-ray diffraction studies
Some quipazine derivatives, particularly 4-phenyl quinolines, (with a phenyl group at the fourth
position of the quinoline nucleus) are known to display antagonist activities towards the 5-HT3

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receptor [30]. In literature there are reports on 4-phenyl quinoline derivatives being potential
antidepressants and antiulcer agents [27,34,35] We, in this study, have investigated their role as
antiproliferative agents.
In the present investigation, three series of 4-phenyl quinoline compounds with different
functionalizations – H (non-halogenated); Cl (Chlorine as R4); and F (Chlorine as R4; Fluorine as
R3), were synthesized with the aim of understanding their effect on cellular proliferation.
Modifications of the quinoline ring at the R1 position were based on the structure of quipazine, a
known 5-HT receptor agonist. Further, substitutions at R2 position (with methyl or phenyl
groups) were made to understand the effect of steric hindrance caused by these groups. Since
halogenated derivatives are suggested to enhance affinity of ligands towards biomolecules
[36,37], the R3 and R4 positions were halogenated with fluorine and chlorine. The general
procedure for synthesis of the thirty derivatives is illustrated in Scheme 1.
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Scheme 1. Synthesis of 4-phenyl quinoline derivatives.
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The synthesis was done in accordance with previously described procedure [38]. Briefly,
refluxing of commercially obtained amino benzophenones with sodium acetate and acetic
anhydride afforded analogous benzoylphenyl acetamides (BA1-BA3) with more than 85% yield.
Various heterocyclic amides (A1a-A5b) were prepared by complexing corresponding
commercially obtained heterocyclic amines with acyl chlorides (propionyl or phenacetyl). These
were reacted with the above synthesized benzoylphenyl acetamides (BA1-BA3) to give the thirty
4-phenyl quinoline (4-PQ) derivatives as listed in Table 1.
Table 1. Structural details of the 4-PQ derivatives and their binding affinities to
serotonin receptors [5-HT1B (PDB ID: 4IAR) and 5-HT2B (PDB ID: 4IB4)] obtained
from docking studies.
Compound
H1a
H1b
H2a
H2b
H3a
H3b
H4a
H4b
H5a
H5b
Cl1a
Cl1b
Cl2a
Cl2b
Cl3a
Cl3b
Cl4a
Cl4b
Cl5a
Cl5b
F1a
R1
CH2
CH2
NCH3
NCH3
NC2H5
NC2H5
O
R2
R3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
R4
5-HT1B
5-HT2B
-9.0
-9.2
-8.1
-8.8
-8.7
-8.8
-9.1
-9.2
-7.6
-8.3
-8.4
-8.5
-7.2
-7.7
-7.0
-6.8
-7.8
-8.1
-7.7
-6.7
-8.4
-9.1
-8.4
-7.3
-7.9
-8.4
-7.8
-7.6
-8.2
-7.9
CH3
C6H5
CH3
C6H5
CH3
C6H5
CH3
C6H5
CH3
C6H5
CH3
C6H5
CH3
C6H5
CH3
C6H5
CH3
C6H5
CH3
C6H5
CH3
C6H5
CH3
C6H5
CH3
C6H5
CH3
C6H5
CH3
C6H5
H
-9.7
H
-9.9
H
-9.9
H
-9.9
H
-10.3
-9.7
H
H
-9.4
O
H
-9.6
S
H
-9.2
S
H
-9.5
CH2
CH2
NCH3
NCH3
NC2H5
NC2H5
O
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
-10.0
-10.0
-9.5
-9.5
-9.5
-9.4
-9.5
O
-10.3
-9.6
S
S
-9.4
CH2
CH2
NCH3
NCH3
NC2H5
NC2H5
O
-10.3
-10.1
-9.6
F1b
F
F2a
F
F2b
F
-10.0
-9.7
F3a
F
F3b
F
-9.6
F4a
F
-9.7
F4b
O
F
-10.0
-9.8
F5a
S
F
F5b
S
F
-9.6
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The characterization of each of the synthesized compounds has been individually detailed in the
experimental section below. These compounds were purified by column chromatography,
crystallized from ethyl acetate by slow evaporation method and characterized by 1H NMR; FTIR
and ESI-MS. Their structures (except compound H2a) were determined by single crystal X-ray
diffraction analysis. Crystal data and structure refinement parameters have been listed in Table
S1 of supplementary material. The Oak Ridge Thermal-Ellipsoid Plot Program (ORTEP) plot of
a representative compound H3a is depicted in Figure 1. Crystallographic information files have
been submitted to the CCDC database (CCDC Numbers – 1499725-1499733; 289506-289509;
1499788-1499793 and 1499735-1499744).
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Figure 1. ORTEP diagram of compound H3a. Thermal ellipsoids are shown at 50% probability [Chlorine ion and
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water molecules have been omitted for clarity].
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2.2 Molecular Docking Studies
The crystal structures of the abovementioned compounds were docked on to serotonin receptors
5-HT1B and 5-HT2B (PDB ID: 4IAR [19], PDB ID: 4IB4 [20], respectively) to analyze their
mode of interactions. Crystallographic information files of these structures were converted into
PDB formats by using Mercury (3.5.1; CCDC, UK). Prior to docking studies, the co-crystallized
water molecules or chlorine ions were excluded from the compounds H3a, H3b, F3a and F3b. In
case of compound H2a, where crystal structure was not obtained, the energy minimized structure
(in PDB format) was generated using Maestro 10.0 (Schrödinger Inc., Academic License).
Docking experiments suggested the binding of all the thirty compounds in the orthosteric pocket
of 5-HT1B and 5-HT2B receptors with predicted binding affinities that were lower than -6.5
kcal/mol (Table 1).
The major type of interactions involved at the interface of the docked ligands and receptor
proteins were found to be π-π stacking and π-alkyl interactions. Figure 2 depicts the results

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obtained from the docking studies of a representative compound H3a (Insets in Figures 2a and
b). The amino acid residues Ile 130; Val 201; Ala 216; Phe 330; Phe 331 of the 5-HT1B receptor
(Figure 2a) and Asp 135; Phe 340; Leu 362 of the 5-HT2B receptor (Figure 2b) present in the
binding pocket were involved in the interaction. An earlier report also shows that the
abovementioned residues of 5-HT1B and 5-HT2B receptors are involved in interaction with the
known ligand ergotamine (PDB ID: 4IAR, PDB ID: 4IB4) [19,20]. On the basis of the observed
binding affinities, it was concluded that these compounds could be potential ligands of the
serotonin receptors with agonist or antagonist activities. There are reports on 5-HT antagonists
displaying growth inhibitory activity [39]. To understand the effect of these compounds on
growth of cancerous cells, in vitro studies were taken up.
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Figure 2. Interaction plots for compound H3a docked on (a) 5-HT1B and (b) 5-HT2B receptor proteins.
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2.3 In vitro antiproliferative studies
The antiproliferative activities of the compounds were validated by performing in vitro studies
using MCF-7 cell lines on the basis of the MTT assay (Figure 3). This cell line is known to

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display 5-HT2A receptor subtype [40]. From Figure 3 it is evident that the compounds display a
structure-activity relationship. Lower IC50 values were observed with compounds H2a, H3a and
H3b. These compounds had amino methyl or amino ethyl substitutions at the R1 and ethyl or
phenyl groups at the R2 positions. Within the Cl and F series of compounds, good activity was
also observed with the amino ethyl substitution. The comparatively lower activities displayed by
the Cl and F series may be accounted for by their bulkier side chains and possibly their low
permeability across cell membranes. When O and S were substituted at the R1 position, the
activity was found to decrease considerably.
The observed mean IC50 values of H2a, H3a and H3b in
µM (8.68 ± 0.94; 6.15 ± 1.55 and
7.48 ± 1.46, respectively) were comparable to that of cisplatin (5.67 ± 0.98
µ
M) under the
current experimental conditions. This is in agreement with the finding of another 5-HT1B
antagonist (SB224289 hydrochloride) being effective as an antiproliferative agent wherein,
similar activities were observed [41,42]. These compounds were tested on human embryonic
kidney (HEK293) cell line. The observed mean lethal doses (LD50) of the three most active
compounds were found to be more than 24
behavior towards normal cells.
µM (Figure S1.). This indicated their non-toxic
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Figure 3. Antiproliferative activity of the compounds against MCF-7 cell lines depicting IC50 values (µM) [error
bars represent mean standard deviation of three separate experiments performed in triplicates; P<0.0001, * for H
series; ^ for Cl series and # for F series]. Bonferroni correction was used to adjust for multiple comparisons during
post hoc analysis.
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The antiproliferative activities of the most active compounds (H2a, H3a and H3b) in the
presence of increasing concentrations of serotonin were also monitored by using the MTT assay.
It was hypothesized that with higher contents of serotonin, interactions of the ligands with
serotonin receptors would decrease. It was observed that with the ligands alone, at concentrations

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of 5 µM, cell survival was low indicating their antiproliferative activity (Supplementary Material
S2). In the presence of increasing concentration of serotonin, (5, 10 and 15 µM) better cell
survival was observed. The mitogenic effect of serotonin at increasing concentrations could be
masking the antiproliferative effects of the ligands.
All further studies related to calcium ion flux or ERK phosphorylation were performed with a
representative effective compound, namely H3a. The morphology of the MCF-7 cells after
treatment with compound H3a was observed after 72 h. Control cells showed a typical epithelial
morphology (Figure 4a). After incubation with the test compound, cells were found to be
detached and there was loss in cellular integrity (Figure 4b).
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Figure 4. Effect of compound H3a on MCF-7 cells. Morphological features of cells after 72 h (a) without treatment
(b) after treatment with the compound (5 µM) (c) intracellular Ca2+ ion flux in response to the presence of serotonin
(5 µM, grey line) and serotonin with H3a (5 µM each, black line) (d) Western blot analysis to study the effect of
H3a (5 µM) on ERK phosphorylation after treatment for 24 and 48 h.

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2.4 Calcium ion flux assay
It is a well established fact that activation of certain 5-HT receptors (such as 5-HT1, 5-HT2 or 5-
HT5A) by serotonin leads to cytoplasmic Ca2+ ion mobilization via the following pathway: 5-HT
receptor
receptor
‰
Phospholipase C
‰ diacylglycerol ‰ inositol trisphosphate ‰ inositol trisphosphate
‰
Ca2+ ion release [43,44]. In the presence of an antagonist, serotonin induced
alterations in intracellular Ca2+ ion levels be will be blocked [45].
In the current study, Ca2+ flux was measured to confirm the antagonist activity of compound
H3a. Ca2+ ion mobilization in response to the presence of serotonin and serotonin along with H3a
were measured by using the calcium binding dye Fluo-4AM (Fluo-4 Direct calcium assay kit,
F10471; Thermo Fisher Scientific). This dye on binding with free Ca2+ ions shows a
characteristic fluorescence (494 nm Excitation/ 516 Emission). In experiments that included only
serotonin, an increase in intracellular calcium ions resulted in higher fluorescence intensities
(Figure 4c, grey line). Quipazine, a known serotonin receptor agonist, showed similar activity
(data not shown). In the presence of H3a and serotonin there was a significant decrease in the
efflux of calcium ions (Figure 4c, black line). Compounds H2a and H3b showed similar decrease
in fluorescence intensities. On the basis of these results it was concluded that H3a was able to
inhibit serotonin induced calcium release and could be a potential serotonin receptor antagonist.
2.5 Western blot analysis
Previous reports have suggested that serotonin induces a pathway that phosphorylates ERK
[8,18,43,46]. ERK is also known to regulate the cell cycle by controlling the mitogenic pathway
and is thus being explored as an anticancer target [47,48]. To validate that growth inhibitory
activity of H3a was via the ERK pathway, the levels of phosphorylated ERK in MCF-7 cells
after 24 and 48 h of treatment were estimated by Western blot analysis and compared with
untreated control samples. Antibodies specific for ERK phosphorylation [Anti ERK 1
(pT22/pY204) + ERK 2 (pT185/pY187)] were used to detect these differences. Primary antibody
against
β-actin was used as protein loading control (Panel 3). Analysis of the immunoblot
(Figure 4d) confirmed that the basal level of ERK was unaffected (Panel 2). In contrast, in 48 h
treated cell lysates, there was more than 50% reduction in the levels of phosphorylated ERK
(Panel 1). These findings indicated that the compounds were able to inhibit ERK
phosphorylation and act as antiproliferative agents.

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2.6 Cell cycle analysis
ERK is implicated in regulation of cell cycle progression, in both G1/S and G2/M transitions of
the cell cycle [49]. To evaluate whether the compounds were able to arrest cell cycle or induce
apoptosis, cells were treated with H3a for 48 h, stained with propidium iodide (PI) and analyzed
by flow cytometry. No marked increase in the number of cells in any particular stage was
observed thereby ruling out the possibility of cell cycle arrest (results not shown). The results,
however, showed an increase in the sub-G0/G1 population. This indicated the presence of an
apoptotic cell population. To validate this, the treated cells were stained with PI and FITC
conjugated Annexin V. An increase in the FITC-Annexin V stained cells confirmed that H3a
treatment resulted in apoptotic cell death. Figures 5a and 5b are representative dot-plots of PI and
FITC stained control (untreated cells) and H3a treated cells, respectively.
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Figure 5. Flow cytometry analysis of MCF-7 cells stained with PI and FITC-Annexin V (a) Control cells (b) H3a (5
µM) treated cells for 48 h (c) Viability of control (black bars) and H3a treated (grey bars) cells [P1 = necrotic cells;
P2 = late-apoptotic cells; P3 = early-apoptotic cells; P4 = live cells]. The error bars represents standard deviation.
All experiments were repeated at least twice.

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Quantitative analysis of the flow cytometry is depicted in Figure 5c. Compared to control cells
(black bars), a distinct increase in the apoptotic cell population was observed in treated cells (P3
population, grey bars). Apoptosis is generally inhibited in cancer cells. This allows transformed
cells to escape regulatory mechanisms [50]. Agents such as H3a could induce cancerous cells to
undergo death by stimulating apoptotic pathways.
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3. Conclusion
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To the best of our knowledge, this is the first study that has investigated anticancer activities of
4-PQ compounds based on serotonin receptor ligands. Although docking studies indicated
interactions of all the compounds with the active sites of the 5-HT1B and 5-HT2B receptors, on
the basis of experimental studies it was concluded that the non-halogenated derivatives H2a, H3a
and H3b displayed effective anticancer activities that were comparable with cisplatin. A
representative compound, H3a, was able to inhibit calcium ion flux, ERK phosphorylation and
induced cell death via the apoptotic pathway. This combined effect of the compound makes it a
promising anticancer drug candidate.
4. Experimental part
4.1 General
All chemicals used for synthesis of ligands were purchased from Sigma Co. All solvents were of
analytical grade. 1H NMR were recorded at 200 MHz on BrukerAC-200 spectrometer (Equipped
with two solution probes: 5 mm H, C, P and F, QNP and 5mm H) and chemical shifts are
reported in
δ (ppm) relative to the internal CDCl3 reference. IR spectra were recorded on Bruker
Tensor 37 FTIR-ATR and are expressed in cm−1. High resolution mass spectra (HRMS) were
recorded in electrospray ionisation (ESI) mode on a Thermo Scientific Q-Exactive instrument
equipped with an Accela 1250 pump. The purity of all compounds were determined to be >95%
1
on the basis of H NMR and single crystal X-ray diffraction studies. Single crystals of the
column purified compounds were used for in vitro analysis. In case of compound H2a, where
single crystals were not obtained, column purified product was used. Fetal bovine serum (FBS),
Dead Cell Apoptosis Kit [with Annexin V Alexa Fluor 488 & Propidium Iodide (PI) (V13241)]
and Fluo-4 Direct calcium assay kit (F10472) were purchased from Invitrogen, ThermoFisher
Scientific. Other reagents and chemicals used for cell culture and in vitro assays were purchased

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from Hi-Media, India. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide]
and calcium assay results were recorded on a SpectraMax M5 multi-detection microplate reader
(Molecular Devices). Cell cycle analyses were done by flow cytometry using fluorescence-
activated cell sorting on FACSVerse (BD Biosciences).
4.2 Synthesis
The 4-phenyl quinoline analogs were prepared in three steps. (1) Amidation of heterocyclic
amines with acyl chlorides gave heterocyclic amides (A1 - A10). (2) Acetamido benzophenones
(BA1 - BA3) were prepared by acetamidylation of 2-aminobenzophenone using acetic anhydride
and sodium acetate. Finally, heterocyclic amides were reacted with acetamido benzophenones in
presence of phosphoryl chloride (POCl3).
4.2.1 1-(1-piperidyl)propan-1-one (A1a)
Piperidine (21.73 mL, 0.22 mol) was taken in chloroform (25 mL) and cooled in an ice bath.
Propionyl chloride (9.61 mL, 0.11 mol) was added in a drop-wise manner to the above solution
of the amine and the mixture was stirred for 30 min. The chloroform layer was initially worked
with 10% sodium carbonate solution, then with water and dried over sodium sulfate. After the
chloroform layer was retrieved, the pure product obtained was concentrated under reduced
pressure. Yield: 85%. 1H NMR (200 MHz, CDCl3):
δ = 3.4 (t, 2H); 3.3 (t, 2H); 2.2 (q, 2H); 1.4
(quin, 6H); 1.00 (t, 3H).
4.2.2 2-phenyl-1-(1-piperidyl)ethanone (A1b)
Piperidine (21.73 mL, 0.22 mol) was taken in chloroform (25 mL) and cooled in an ice bath.
Phenyl acetyl chloride (14.6 mL, 0.11 mol) was added drop-wise to the above solution of the
amine and the mixture was stirred for 30 min. The chloroform layer was initially worked with
10% sodium carbonate solution, then with water and dried over sodium sulfate. After the
chloroform layer was retrieved, the pure product obtained was concentrated under reduced
1
pressure. Yield: 90%. H NMR (200 MHz, CDCl3):
2H); 3.4 (t, 2H); 1.55 (quin, 4H); 1.35 (quin, 2H).
4.2.3 1-(4-methylpiperazin-1-yl)propan-1-one (A2a)
δ = 7.4 – 7.2 (m, 5H); 3.75 (s, 2H); 3.6 (t,
A2a was prepared following the same procedure used for the synthesis of A1a using 1-
methylpiperazine (24.4 mL, 0.22 mol). Yield: 87%. 1H NMR (200 MHz, CDCl3):
δ = 3.5 (t, 2H);
3.35 (t, 2H); 2.2 (t, 4H); 2.15 (q, 2H); 2.1 (s, 3H); 1.0(t, 3H).

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4.2.4 1-(4-methylpiperazin-1-yl)-2-phenyl-ethanone (A2b)
A2b was prepared following the same procedure used for the synthesis of A1b using 1-
1
methylpiperazine (24.4 mL, 0.22 mol). Yield: 86%. H NMR (200 MHz, CDCl3):
(m, 5H); 3.7 (s, 2H); 3.6 (t, 2H); 3.4 (t, 2H); 2.3 (t, 2H); 2.2 (t, 2H); 2.1 (s, 3H).
4.2.5 1-(4-ethylpiperazin-1-yl)propan-1-one (A3a)
δ = 7.3 – 7.2
A3a was prepared following the same procedure used for the synthesis of A1a using 1-
ethylpiperazine (27.94 mL, 0.22 mol). Yield: 85%. 1H NMR (200 MHz, CDCl3):
3.45 (t, 2H); 2.45 (t, 4H); 2.3 (q, 2H); 2.25 (q, 2H); 1.1 (t, 3H); 1.0 (t, 3H).
4.2.6 1-(4-ethylpiperazin-1-yl)-2-phenyl-ethanone (A3b)
δ = 3.6 (t, 2H);
A3b was prepared following the same procedure used for the synthesis of A1b using 1-
1
ethylpiperazine (27.94 mL, 0.22 mol). Yield: 84%. H NMR (200 MHz, CDCl3):
(m, 5H); 3.74 (s, 2H); 3.68 (t, 2H); 3.45 (t, 2H); 2.4 (t, 4H); 2.2 (q, 2H); 1.0 (t, 3H).
4.2.7 1-morpholinopropan-1-one (A4a)
δ = 7.4 – 7.2
A4a was prepared following the same procedure used for the synthesis of A1a using morpholine
(19.03 mL, 0.22 mol). Yield: 86%. 1H NMR (200 MHz, CDCl3):
δ = 3.65 (t, 4H); 3.55 (q, 2H);
3.25 (t, 4H); 1.15 (t, 3H).
4.2.8 2-phenyl-1-morpholino-ethanone (A4b)
A4b was prepared following the same procedure used for the synthesis of A1b using morpholine
(19.03 mL, 0.22 mol). Yield: 87%. 1H NMR (200 MHz, CDCl3):
δ = 7.2 – 7.35 (m, 5H); 3.71 (t,
4H); 3.61 (s, 2H); 3.45 (t, 2H); 3.4 (t, 2H).
4.2.9 1-thiomorpholinopropan-1-one (A5a)
A5a was prepared following the same procedure used for the synthesis of A1a using
1
thiomorpholine (22.13 mL, 0.22 mol). Yield: 88%. H NMR (200 MHz, CDCl3):
2H); 3.68 (t, 2H); 2.55 (t, 4H); 2.5 (q, 2H); 1.08 (t, 3H).
δ = 3.85 (t,
4.2.10 2-phenyl-1-thiomorpholino-ethanone (A5b)
A5b was prepared following the same procedure used for the synthesis of A1b using
thiomorpholine (22.13 mL, 0.22 mol). Yield: 92%. 1H NMR (200 MHz, CDCl3):
(m, 5H); 3.85 (t, 2H); 3.65 (s, 2H); 3.65 (t, 2H); 2.55 (t, 2H); 2.5 (t, 2H).
4.2.11 N-(2-benzoylphenyl)acetamide (BA1)
δ = 7.35 – 7.15
BA1 was prepared by heating mixture of 2-aminobenzophenone (1.98 g, 0.01 mol) with
anhydrous sodium acetate (0.91 g, 0.01mol) and acetic anhydride (4 mL) at 80°C and stirred for

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1 h. Upon cooling, the cold distilled water was added to the reaction mixture resulting in white
precipitate. This precipitate was dried, dissolved in ethanol and slowly evaporated to provide
pure single crystals of 2-acetamido benzophenones. Yield: 95%. 1H NMR (200 MHz, CDCl3):
δ
= 10.8 (br. s, NH); 8.6 – 7.4 (m, 9H); 2.2 (s, 3H).
4.2.12 N-(2-benzoyl-4-chloro-phenyl)acetamide (BA2)
BA2 was prepared following the same procedure used for the synthesis of BA1 using 2-amino-5-
chlorobenzophenone (2.31 g, 0.01 mol). Yield: 96%. 1H NMR (200 MHz, CDCl3):
δ = 10.6 (br.
s, NH); 8.6 – 7.5 (m, 8H); 2.2 (s, 3H).
4.2.13 N-[4-chloro-2-(2-fluorobenzoyl)phenyl] acetamide (BA3)
BA3 was prepared following the same procedure used for the synthesis of BA1 using 2-amino-5-
1
chloro-2
′
-fluorobenzophenone (2.4 g, 0.01 mol). Yield: 90%. H NMR (200 MHz, CDCl3):
δ
=
11.2 (br. s, NH); 8.8 – 7.3 (m, 7H); 2.3 (s, 3H).
4.2.14 3-methyl-4-phenyl-2-(1-piperidyl) quinoline (H1a)
To a solution of A1a (0.141 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture, BA1
(0.239 g, 0.001 mol) was added and was refluxed till the reactants were consumed (checked
using TLC up to 48 h). The cooled mixture was poured in 10% sodium carbonate solution (5
mL), warmed on water bath for 30 min and cooled. The product obtained was extracted with
chloroform (2 x 10 mL). The chloroform extract was washed with water (10 mL), recovered,
dried over anhydrous sodium sulfate and evaporated. The resulting residue was purified using
column chromatography (9:1; petroleum ether: ethyl acetate). The eluted product was evaporated
under reduced pressure giving white solid which was crystallized from ethyl acetate. Yield: 80%.
Melting Point (MP) = 130-132°C. FTIR (KBr): νmax = 3051.48; 2918.39; 1577.82; 1483.30;
1
1446.66; 1404.22; 1282.70. H NMR (200 MHz, CDCl3):
δ
= 7.95 (d, 1H); 7.7 – 7.25 (m, 8H);
= 162.39, 148.58, 145.67,
3.3 (t, 4H); 2.0 (quin, 6H); 1.5 (s, 3H). 13C NMR (101 MHz, CDCl3):
δ
138.31, 129.39, 128.49, 128.06, 127.54, 127.47, 125.69, 125.25, 123.69, 123.22, 51.22, 26.17,
24.66, 16.96 ppm. HRMS (ESI) calculated for C21H23N2 (M+H)+ 303.1856, found for 303.1850.
4.2.15 3,4-diphenyl-2-(1-piperidyl)quinoline (H1b)
To a solution of A1b (0.203 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture BA1
(0.239 g, 0.001 mol) was added and further following same procedure as for H1a afforded white

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solid which was crystallized from ethyl acetate. Yield: 77%. MP = 190-192°C. FTIR (KBr): νmax
= 3057.27; 2929.96; 1558.53; 1489.09; 1444.73; 1410.01. 1H NMR (200 MHz, CDCl3):
7.0 (m, 14H); 3.0 – 3.5 (quin, 4H); 1.5 – 1.2 (m, 6H). 13C NMR (101 MHz, CDCl3):
= 160.41,
δ = 7.9 –
δ
145.06, 137.84, 130.70, 130.13, 129.78, 129.75, 129.54, 129.00, 128.92, 127.61, 127.54, 126.87,
126.78, 126.47, 126.26, 125.23, 124.62, 50.28, 25.58, 24.46 ppm. HRMS (ESI) calculated for
C26H25N2 (M+H)+ 365.2012, found for 365.2007.
4.2.16 3-methyl-2-(4-methylpiperazin-1-yl)-4-phenyl-quinoline (H2a)
To a solution of A2a (0.156 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture BA1
(0.239 g, 0.001 mol) was added and further following same procedure as for H1a afforded white
solid which was crystallized from ethyl acetate. Yield: 73%. MP = 164-165°C. FTIR (KBr): νmax
= 3057.27; 2962.75; 1564.32; 1483.30; 1444.73; 1406.15; 1265.34. 1H NMR (200 MHz, CDCl3):
13
δ
= 7.85 – 7.15 (m, 8H); 3.15 (t, 4H); 2.35 (t, 4H); 2.25 (s, 3H); 2.10 (s, 3H). C NMR (101
MHz, CDCl3):
δ = 158.44, 149.18, 144.28, 138.63, 131.47, 130.55, 130.46, 129.75, 129.52,
129.15, 125.10, 124.62, 124.05, 55.28, 49.74, 46.31, 16.88 ppm. HRMS (ESI) calculated for
C21H24N3 (M+H)+ 317.4275, found for 317.4215.
4.2.17 2-(4-methylpiperazin-1-yl)-3,4-diphenyl-quinoline (H2b)
To a solution of A2b (0.156 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture BA1
(0.239 g, 0.001 mol) was added and further following same procedure as for H1a afforded white
solid which was crystallized from ethyl acetate. Yield: 75%. MP = 220-222°C. FTIR (KBr): νmax
1
= 3061.13; 3028.34; 2935.76; 1562.39; 1481.38; 1446.66; 1410.01; 1296.21. H NMR (200
MHz, CDCl3):
δ
= 7.95 (d, 1H); 7.60 – 7.0 (m, 13H); 3.3 (t, 4H); 2.3 (t, 4H); 2.2 (s, 3H). 13C
NMR (101 MHz, CDCl3):
δ
= 161.34, 150.39, 148.68, 140.17, 139.43, 132.76, 132.26, 131.17,
129.83, 129.75, 129.57, 129.12, 129.04, 128.60, 128.57, 126.99, 125.80, 57.00, 51.02, 48.21
ppm. HRMS (ESI) calculated for C26H26N3 (M+H)+ 380.2121 found for 380.2112.
4.2.18 2-(4-ethylpiperazin-1-yl)-3-methyl-4-phenyl-quinoline (H3a)
To a solution of A3a (0.171 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture BA1
(0.239 g, 0.001 mol) was added and further following same procedure as for H1a afforded white
solid which was crystallized from ethyl acetate. Yield: 79%. MP = 180-181°C. FTIR (KBr): νmax

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= 3055.35; 2928.04; 2885.61; 1577.82; 1491.02; 1446.66; 1408.08; 1290.42. H NMR (200
MHz, CDCl3): = 7.75 – 7.15 (m, 9H); 3.20 (t, 3H); 2.80 (t, 4H); 2.40 (q, 2H); 2.10 (S, 3H);
1.15 (t, 3H). 13C NMR (101 MHz, CDCl3):
= 161.44, 144.40, 143.53, 130.40, 130.24, 126.82,
δ
δ
125.49, 125.47, 125.26, 124.87, 124.83, 124.66, 53.21, 51.26, 47.07, 17.26, 9.85 ppm. HRMS
(ESI) calculated for C22H26N3 (M+H)+ 332.2121, found for 332.2117.
4.2.19 2-(4-ethylpiperazin-1-yl)-3,4-diphenyl-quinoline (H3b)
To a solution of A3b (0.171 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture BA1
(0.239 g, 0.001 mol) was added and further following same procedure as for H1a afforded white
solid which was crystallized from ethyl acetate. Yield: 74%. MP = 239-240°C. FTIR (KBr): νmax
1
= 3053.42; 2978.19; 2926.11; 1560.46; 1483.31; 1448.59; 1408.08; 1300.07. H NMR (200
MHz, CDCl3):
δ
= 8.00 (t, 1H); 7.60 (t, 1H); 7.35 – 7.00 (m, 12H); 3.75 (t, 2H); 3.65 (t, 2H);
= 156.95,
3.30 (t, 2H); 3.05 (q, 2H); 2.85 (t, 2H); 1.40 (t, 3H). 13C NMR (101 MHz, CDCl3):
δ
149.20, 145.95, 137.05, 136.61, 130.26, 129.95, 129.50, 128.15, 127.74, 127.54, 127.22, 127.06,
126.56, 126.48, 125.09, 124.57, 52.09, 50.46, 45.44, 8.95 ppm. HRMS (ESI) calculated for
C27H28N3 (M+H)+ 394.2278, found for 394.2272.
4.2.20 4-(3-methyl-4-phenyl-2-quinolyl) morpholine (H4a)
To a solution of A4a (0.143 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture BA1
(0.239 g, 0.001 mol) was added and further following same procedure as for H1a afforded white
solid which was crystallized from ethyl acetate. Yield: 76%. MP = 129-131°C. FTIR (KBr): νmax
= 3055.35; 3022.55; 2951.18; 2916.47; 2891.39; 2854.74; 1606.76; 1573.97; 1446.66; 1278.85.
13
1H NMR (200 MHz, CDCl3):
δ
= 7.7 – 7.1 (m, 9H); 4.0 (t, 4H); 3.4 (t, 4H); 1.5 (s, 3H). C
NMR (101 MHz, CDCl3):
δ
= 160.98, 149.06, 145.56, 138.01, 129.34, 128.55, 128.31, 127.69,
127.65, 125.77, 125.43, 124.13, 122.56, 67.07, 50.43, 16.94 ppm. HRMS (ESI) calculated for
C20H21N2O (M+H)+ 305.1648, found for 305.1642.
4.2.21 4-(3,4-diphenyl-2-quinolyl)morpholine (H4b)
To a solution of A4b (0.205 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture BA1
(0.239 g, 0.001 mol) was added and further following same procedure as for H1a afforded white
solid which was crystallized from ethyl acetate. Yield: 75%. MP = 200-201°C. FTIR (KBr): νmax

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= 3053.42; 3018.70; 2966.62; 2928.04; 1577.82; 1446.66; 1404.23; 1282.71. H NMR (200
MHz, CDCl3):
= 7.6 (t, 1H); 7.4 – 7.0 (m, 13H); 3.6 (t, 4H); 2.5 (t, 3H). 13C NMR (101 MHz,
CDCl3): = 161.04, 149.69, 145.32, 138.67, 136.13, 129.70, 129.40, 128.76, 128.69, 127.12,
δ
δ
127.08, 125.88, 125.72, 125.48, 125.11, 124.74, 123.47, 66.17, 49.67 ppm. HRMS (ESI)
calculated for C25H23N2O (M+H)+ 367.1805, found for 367.1795.
4.2.22 4-(3-methyl-4-phenyl-2-quinolyl) thiomorpholine (H5a)
To a solution of A5a (0.159 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture BA1
(0.239 g, 0.001 mol) was added and further following same procedure as for H1a afforded white
solid which was crystallized from ethyl acetate. Yield: 78%. MP = 141-142°C. FTIR (KBr): νmax
= 3061.13; 2985.90; 2953.11; 2901.03; 2831.59; 1575.89; 1491.02; 1446.66; 1406.15; 1228.69;
759.97. 1H NMR (200 MHz, CDCl3):
4H) ; 2.9 (t, 4H) ; 2.2 (s, 3H). 13C NMR (101 MHz, CDCl3):
δ
= 7.9 (d, 1H); 7.6 – 7.4 (m, 4H); 7.3 – 7.1 (m, 4H); 3.6 (t,
= 161.81, 137.99, 129.32, 129.23,
δ
128.55, 128.35, 127.82, 127.71, 127.52, 125.78, 125.36, 124.45, 124.21, 122.89, 52.39, 27.82,
16.94 ppm. HRMS (ESI) calculated for C20H21N2S (M+H)+ 321.1420, found for 321.1414.
4.2.23 4-(3,4-diphenyl-2-quinolyl)thiomorpholine (H5b)
To a solution of A5b (0.221 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture BA1
(0.239 g, 0.001 mol) was added and further following same procedure as for H1a afforded white
solid which was crystallized from ethyl acetate. Yield: 74%. MP = 216-218°C. FTIR (KBr): νmax
= 3055.34; 2953.11; 2904.89; 2874.03; 1556.60; 1485.23; 1446.66; 1408.08; 1305.85; 759.97.
1H NMR (200 MHz, CDCl3):
δ
= 7.40 – 7.00 (m, 14H); 3.50 (t, 4H); 2.45 (t, 4H). 13C NMR (101
MHz, CDCl3): = 159.95, 137.80, 137.13, 130.79, 130.44, 130.02, 129.21, 128.04, 127.76,
δ
127.66, 127.58, 127.39, 127.01, 126.54, 126.51, 124.96, 124.05, 51.86, 27.09 ppm. HRMS (ESI)
calculated for C25H23N2S (M+H)+ 383.1576, found for 383.1571.
4.2.24 6-chloro-3-methyl-4-phenyl-2-(1-piperidyl) quinoline (Cl1a)
General procedure for synthesis was same as for H1a. To a solution of A1a (0.141 mL, 0.001
mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001 mol) was added and the mixture was
stirred at room temperature for 24 h. To this mixture, BA2 (0.274 g, 0.001 mol) was added and
was refluxed till the reactants were consumed (checked using TLC up to 48 h). A pale yellow
solid was obtained which was crystallized from ethyl acetate. Yield: 84%. MP = 131-133°C.

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FTIR (KBr): νmax = 3037.99; 2922.25; 1570.11; 1479.45; 1280.78; 711.76. 1H NMR (200 MHz,
13
CDCl3):
δ
= 7.70 – 7.30 (m, 8H); 3.60 (t, 4H); 2.50 (quin, 6H); 1.70 (s, 3H). C NMR (101
MHz, CDCl3):
δ
= 162.49, 147.83, 144.12, 137.58, 129.31, 129.13, 129.03, 128.72, 127.89,
125.95, 124.55, 124.25, 51.14, 26.11, 24.63, 17.13 ppm. HRMS (ESI) calculated for C21H22ClN2
(M+H)+ 337.1466, found for 337.1466.
4.2.25 6-chloro-3,4-diphenyl-2-(1-piperidyl) quinoline (Cl1b)
To a solution of A1b (0.203 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture, BA2
(0.274 g, 0.001 mol) was added and was refluxed till the reactants were consumed (checked
using TLC up to 48 h). A pale yellow solid was obtained which was crystallized from ethyl
acetate. Yield: 81%. MP = 194-195°C. FTIR (KBr): νmax = 3061.13; 3028.34; 2933.83; 1558.54;
1
1411.94; 1261.49; 702.11. H NMR (200 MHz, CDCl3):
δ
= 7.90 – 7.00 (m, 13H); 3.40 (t, 4H);
2.50(quin, 4H). 13C NMR (101 MHz, CDCl3):
δ = 159.40, 146.55, 136.88, 135.73, 129.52,
129.02, 128.67, 127.86, 127.66, 127.35, 126.83, 126.66, 126.19, 125.49, 124.55, 124.30, 49.42,
24.53, 23.46 ppm. HRMS (ESI) calculated for C26H24ClN2 (M+H)+ 399.1623, found for
399.1618.
4.2.26 6-chloro-3-methyl-2-(4-methylpiperazin-1-yl)-4-phenyl-quinoline (Cl2a)
General procedure for synthesis was same as for H1a. To a solution of A2a (0.156 mL, 0.001
mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001 mol) was added and the mixture was
stirred at room temperature for 24 h. To this mixture, BA2 (0.274 g, 0.001 mol) was added and
was refluxed till the reactants were consumed (checked using TLC up to 48 h). A pale yellow
solid was obtained which was crystallized from ethyl acetate. Yield: 72%. MP = 228-229°C.
FTIR (KBr): νmax =3081.69; 3056.62; 3025.77; 2965.98; 2857.99; 1560.13; 1479.13; 1444.42;
1
1407.78; 1261.22; 761.74. H NMR (200 MHz, CDCl3):
δ
= 7.85 – 7.20 (m, 8H); 3.90 (t, 4H);
δ = 161.28, 148.06, 144.10, 137.44,
13
3.00 (t, 4H); 2.10 (s, 3H). C NMR (101 MHz, CDCl3):
129.33, 129.28, 129.22, 128.88, 128.74, 127.94, 126.02, 124.54, 123.63, 55.18, 49.60, 46.18,
17.25 ppm. HRMS (ESI) calculated for C21H23ClN3 (M+H)+ 352.1575, found for 352.1574.
4.2.27 6-chloro-2-(4-methylpiperazin-1-yl)-3,4-diphenyl-quinoline (Cl2b)
To a solution of A2b (0.219 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture, BA2
(0.274 g, 0.001 mol) was added and was refluxed till the reactants were consumed (checked

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using TLC up to 48 h). A pale yellow solid was obtained which was crystallized from ethyl
acetate. Yield: 78%. MP = 169-170°C. FTIR (KBr): νmax = 3062.41; 3031.55; 2969.84; 2904.27;
1
2879.20; 1562.06; 1475.28; 1444.42; 1405.85; 1290.14; 701.96. H NMR (200 MHz, CDCl3):
δ
= 7.85 – 6.90 (m, 13H); 3.30 (t, 4H); 2.35 (t, 4H); 2.30 (s, 3H). 13C NMR (101 MHz, CDCl3):
δ
=
159.22, 148.28, 146.56, 138.05, 137.31, 130.65, 130.14, 129.05, 127.71, 127.63, 127.45, 127.00,
126.92, 126.48, 126.45, 124.86, 123.69, 54.88, 48.89, 46.08, ppm. HRMS (ESI) calculated for
C26H25ClN3 (M+H)+ 414.1732, found for 414.1729.
4.2.28 6-chloro-2-(4-ethylpiperazin-1-yl)-3-methyl-4-phenyl-quinoline (Cl3a)
To a solution of A3a (0.171 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture, BA2
(0.274 g, 0.001 mol) was added and was refluxed till the reactants were consumed (checked
using TLC up to 48 h). A pale yellow solid was obtained which was crystallized from ethyl
acetate. Yield: 78%. MP = 183-184°C. FTIR (KBr): νmax 3075.90; 3048.91; 3014.19; 2969.84;
1
2923.56; 1579.41; 1481.06; 1448.28; 1450.85; 1261.22; 827.31. H NMR (200 MHz, CDCl3):
δ
13
= 7.90 – 7.40 (m, 8H); 3.50 (t, 4H); 2.85 (t, 4H); 2.15 (q, 2H); 2.10 (s, 3H); 1.25 (t, 3H). C
NMR (101 MHz, CDCl3):
δ = 161.29, 148.00, 144.13, 137.47, 129.28, 128.84, 128.75, 127.93,
126.00, 125.73, 124.76, 124.53, 124.15, 123.62, 52.89, 52.46, 49.65, 17.27, 11.97 ppm. HRMS
(ESI) calculated for C22H25ClN3 (M+H)+ 366.1732, found for 366.1732.
4.2.29 6-chloro-2-(4-ethylpiperazin-1-yl)-3,4-diphenyl-quinoline (Cl3b)
To a solution of A3b (0.232 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture, BA2
(0.274 g, 0.001 mol) was added and was refluxed till the reactants were consumed (checked
using TLC up to 48 h). A pale yellow solid was obtained which was crystallized from ethyl
acetate. Yield: 89%. MP = 242-243°C. FTIR (KBr): νmax = 3032.20; 2956.97; 1558.54; 1481.38;
1444.73; 1413.87; 1259.56; 704.04. 1H NMR (200 MHz, CDCl3):
δ
= 7.90 – 7.00 (m, 13H); 3.30
= 159.29, 147.48, 145.05,
13
(t, 4H); 2.50 (m, 6H); 1.10 (t, 3H). C NMR (101 MHz, CDCl3):
δ
137.73, 136.61, 130.45, 130.02, 129.68, 129.02, 128.96, 127.84, 127.79, 127.69, 127.21, 126.66,
125.59, 125.25, 52.54, 52.32, 48.80, 11.90 ppm. HRMS (ESI) calculated for C27H27ClN3 (M+H)+
428.1888, found for 428.1887.
4.2.30 4-(6-chloro-3-methyl-4-phenyl-2-quinolyl) morpholine (Cl4a)

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To a solution of A4a (0.143 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture, BA2
(0.274 g, 0.001 mol) was added and was refluxed till the reactants were consumed (checked
using TLC up to 48 h). A pale yellow solid was obtained which was crystallized from ethyl
acetate. Yield: 76%. MP = 132-133°C. FTIR (KBr): νmax = 3054.69; 3027.69; 2967.91; 2888.84;
1
2850.27; 1403.92; 1444.42; 1482.99; 1575.56; 1261.21; 825.38. H NMR (200 MHz, CDCl3):
δ
= 7.60 – 7.10 (m, 8H); 3.90 (t, 4H); 3.45 (t, 4H); 2.15 (s, 3H). 13C NMR (101 MHz, CDCl3):
δ
=
160.77, 158.31, 131.31, 130.54, 130.46, 129.39, 129.22, 125.83, 124.95, 124.58, 124.55, 124.35,
124.01, 66.99, 50.37, 16.64 ppm. HRMS (ESI) calculated for C20H20ClN2O (M+H)+ 339.1259,
found for 339.1255.
4.2.31 4-(6-chloro-3,4-diphenyl-2-quinolyl) morpholine (Cl4b)
To a solution of A4b (0.205 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture, BA2
(0.274 g, 0.001 mol) was added and was refluxed till the reactants were consumed (checked
using TLC up to 48 h). A pale yellow solid was obtained which was crystallized from ethyl
acetate. Yield: 75%. MP = 201-203°C. FTIR (KBr): νmax = 3058.55; 3021.91; 2965.98; 2927.41;
1
1577.49; 1552.42; 1481.06; 1452.14; 1407.78; 1265.07; 829.24. H NMR (200 MHz, CDCl3):
δ
13
= 8.00 – 7.00 (m, 13H); 3.55 (t, 4H); 3.15 (t, 4H). C NMR (101 MHz, CDCl3):
δ = 159.21,
147.74, 144.92, 137.44, 136.43, 130.42, 130.00, 129.85, 129.34, 129.03, 127.90, 127.88, 127.74,
127.32, 126.85, 125.73, 125.33, 66.64, 49.54 ppm. HRMS (ESI) calculated for C25H22ClN2O
(M+H)+ 401.1415, found for 401.1415.
4.2.32 4-(6-chloro-3-methyl-4-phenyl-2-quinolyl) thiomorpholine (Cl5a)
To a solution of A5a (0.159 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture, BA2
(0.274 g, 0.001 mol) was added and was refluxed till the reactants were consumed (checked
using TLC up to 48 h). A pale yellow solid was obtained which was crystallized from ethyl
acetate. Yield: 77%. MP = 141-143°C. FTIR (KBr): νmax = 3057.28; 2908.75; 1566.25; 1479.45;
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1406.15; 1286.56; 711.76. H NMR (200 MHz, CDCl3):
δ
= 7.65 – 7.10 (m, 8H); 3.75 (t, 4H);
δ = 161.94, 148.42, 143.86, 137.28,
13
2.85 (t, 4H); 2.15 (s, 3H). C NMR (101 MHz, CDCl3):
129.70, 129.23, 129.15, 129.05, 128.83, 128.78, 128.03, 126.08, 124.61, 123.94, 52.31, 27.75,
17.10 ppm. HRMS (ESI) calculated for C20H20ClN2S (M+H)+ 355.1030, found for 355.1025.

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4.2.33 4-(6-chloro-3,4-diphenyl-2-quinolyl) thiomorpholine (Cl5b)
To a solution of A5b (0.221 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture, BA2
(0.274 g, 0.001 mol) was added and was refluxed till the reactants were consumed (checked
using TLC up to 48 h). A pale yellow solid was obtained which was crystallized from ethyl
acetate. Yield: 74%. MP = 217-218°C. FTIR (KBr): νmax = 3059.20; 2964.69; 2910.68; 1562.39;
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1410.01; 1303.92; 704.04. H NMR (200 MHz, CDCl3):
δ = 7.70 – 6.95 (m, 13H); 3.65 (t, 4H);
13
2.40 (t, 4H). C NMR (101 MHz, CDCl3):
δ = 160.03, 147.99, 144.75, 137.45, 136.42, 130.59,
130.26, 129.91, 129.45, 128.95, 128.31, 128.13, 127.94, 127.87, 127.31, 126.76, 125.71, 125.31,
51.74, 27.01 ppm. HRMS (ESI) calculated for C25H22ClN2S (M+H)+ 417.1186, found for
417.1185.
4.2.34 6-chloro-4-(2-fluorophenyl)-3-methyl-2-(1-piperidyl)quinoline (F1a)
General procedure for synthesis was same as for H1a. To a solution of A1a (0.141 mL, 0.001
mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001 mol) was added and the mixture was
stirred at room temperature for 24 h. To this mixture, BA3 (0.292 g, 0.001 mol) was added and
was refluxed till the reactants were consumed (checked using TLC up to 48 h). A yellow solid
was obtained which was crystallized from ethyl acetate. Yield: 76%. MP = 134-136°C. FTIR
(KBr): νmax = 3047.63; 2924.18; 1579.75; 1483.30; 1442.80; 1402.29; 1259.55; 767.69. 1H NMR
(200 MHz, CDCl3):
δ
= 7.55 – 7.15 (m, 7H); 3.45 (t, 4H); 2.15 (s, 3H); 1.80 (quin, 4H); 1.70
= 162.19, 160.77, 158.32, 143.99, 141.63, 131.39,
13
(quin, 4H). C NMR (101 MHz, CDCl3):
δ
130.38, 130.31, 129.47, 128.98, 125.60, 124.73, 124.56, 124.48, 123.94, 116.27, 116.06, 51.18,
26.09, 24.57, 16.67 ppm. HRMS (ESI) calculated for C21H21ClFN2 (M+H)+ 355.1372, found for
355.1371.
4.2.35 6-chloro-4-(2-fluorophenyl)-3-phenyl-2-(1-piperidyl)quinoline (F1b)
To a solution of A1b (0.203 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture, BA3
(0.292 g, 0.001 mol) was added and was refluxed till the reactants were consumed (checked
using TLC up to 48 h). A yellow solid was obtained which was crystallized from ethyl acetate.
Yield: 74%. MP = 197-198°C. FTIR (KBr): νmax = 3061.13; 2989.76; 2933.82; 1562.39;
1
1483.30; 1417.72; 1261.48; 700.18. H NMR (200 MHz, CDCl3):
δ = 7.55 (d, 1H); 7.30 – 7.05
(m, 12H); 3.25 (t, 4H); 1.45 (quin, 2H); 1.35 (quin, 2H). 13C NMR (101 MHz, CDCl3):
δ =

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160.97, 160.48, 158.53, 144.97, 141.64, 137.94, 131.96, 131.92, 129.90, 129.57, 129.06, 128.98,
127.70, 126.93, 125.21, 124.75, 124.52, 123.90, 123.87, 115.58, 115.37, 50.43, 25.63, 24.57
ppm. HRMS (ESI) calculated for C26H23ClFN2 (M+H)+ 417.1528, found for 417.1525.
4.2.36 6-chloro-4-(2-fluorophenyl)-3-methyl-2-(4-methylpiperazin-1-yl)quinoline (F2a)
To a solution of A2a (0.156 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture, BA3
(0.292 g, 0.001 mol) was added and was refluxed till the reactants were consumed (checked
using TLC up to 48 h). A yellow solid was obtained which was crystallized from ethyl acetate.
Yield: 78%. MP = 170-172°C. FTIR (KBr): νmax = 3054.42; 2962.76; 2887.53; 1566.25;
1
1492.95; 1408.15; 1259.56; 831.35. H NMR (200 MHz, CDCl3):
δ
= 7.60 – 7.10 (m, 7H); 3.35
= 160.34,
(t, 4H); 2.90 (t, 4H); 2.40 (s, 3H); 2.10 (s, 3H). 13C NMR (101 MHz, CDCl3):
δ
159.99, 157.54, 143.38, 140.92, 130.60, 129.65, 128.85, 128.62, 128.25, 124.93, 124.19, 123.83,
123.72, 123.15, 115.51, 115.29, 54.37, 48.83, 45.40, 15.97 ppm. HRMS (ESI) calculated for
C21H22ClFN3 (M+H)+ 370.1481, found for 370.1475.
4.2.37 6-chloro-4-(2-fluorophenyl)-2-(4-methylpiperazin-1-yl)-3-phenyl-quinoline (F2b)
To a solution of A2b (0.219 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture, BA3
(0.292 g, 0.001 mol) was added and was refluxed till the reactants were consumed (checked
using TLC up to 48 h). A yellow solid was obtained which was crystallized from ethyl acetate.
Yield: 77%. MP = 227-228°C. FTIR (KBr): νmax = 3061.13; 2935.76; 1564.32; 1479.45;
1444.73; 1410.01; 1294.28; 696.33. 1H NMR (200 MHz, CDCl3):
δ
=7.40 – 7.15 (m, 12H); 3.65
δ = 159.21, 158.70, 148.26,
13
(t, 4H); 2.40 (t, 4H); 2.25 (s, 3H). C NMR (101 MHz, CDCl3):
146.55, 138.04, 137.31, 132.14, 130.64, 130.13, 129.04, 128.28, 127.70, 127.62, 126.44, 126.99,
126.90, 126.47, 126.44, 125.57, 124.85, 123.67, 116.89, 116.16, 54.88, 48.88, 46.08 ppm.
HRMS (ESI) calculated for C26H24ClFN3 (M+H)+ 432.1637, found for 432.1631.
4.2.38 6-chloro-2-(4-ethylpiperazin-1-yl)-4-(2-fluorophenyl)-3-methyl-quinoline (F3a)
To a solution of A3a (0.171 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture, BA3
(0.292 g, 0.001 mol) was added and was refluxed till the reactants were consumed (checked
using TLC up to 48 h). A yellow solid was obtained which was crystallized from ethyl acetate.
Yield: 74%. MP = 180-181°C. FTIR (KBr): νmax = 3053.42; 2968.55; 2929.97; 1577.82;

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1475.59; 1450.52; 1406.15; 1267.27; 769.62. H NMR (200 MHz, CDCl3):
δ
= 7.80 – 7.15 (m,
13
7H); 3.60 (t, 4H); 2.90 (t, 4H); 2.70 (q, 2H); 2.05 (s, 3H); 1.30 (t, 3H). C NMR (101 MHz,
CDCl3):
δ = 160.53, 159.02, 156.07, 143.60, 142.51, 131.13, 131.10, 130.68, 130.59, 130.46,
129.44, 125.93, 124.60, 124.56, 124.36, 123.96, 123.78, 116.26, 116.05, 52.30, 50.40, 46.11,
16.34, 8.96 ppm. HRMS (ESI) calculated for C22H24ClFN3 (M+H)+ 384.1637, found for
384.1632.
4.2.39 6-chloro-2-(4-ethylpiperazin-1-yl)-4-(2-fluorophenyl)-3-phenyl-quinoline (F3b)
To a solution of A3b (0.232 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture, BA3
(0.292 g, 0.001 mol) was added and was refluxed till the reactants were consumed (checked
using TLC up to 48 h). A yellow solid was obtained which was crystallized from ethyl acetate.
Yield: 77%. MP = 239-241°C. FTIR (KBr): νmax = 3041.84; 2937.68; 2879.81; 1577.82;
1
1558.53; 1489.09; 1446.66; 1415.79; 1305.85; 700.18. H NMR (200 MHz, CDCl3):
6.90 (m, 12H); 3.55 (t, 2H); 3.45 (t, 2H); 2.85 – 2.65 (m, 6H); 1.30 (s, 3H). 13C NMR (101 MHz,
δ = 7.90 –
CDCl3): = 159.12, 144.97, 141.78, 137.58, 131.80, 131.73, 129.88, 129.74, 129.26, 129.10,
δ
128.91, 127.89, 127.03, 125.27, 124.62, 124.29, 123.82, 123.76, 115.60, 115.17, 52.46, 52.30,
48.65, 11.80 ppm. HRMS (ESI) calculated for C27H26ClFN3 (M+H)+ 446.1794, found for
446.1794.
4.2.40 4-[6-chloro-4-(2-fluorophenyl)-3-methyl-2-quinolyl]morpholine (F4a)
To a solution of A4a (0.143 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture, BA3
(0.292 g, 0.001 mol) was added and was refluxed till the reactants were consumed (checked
using TLC up to 48 h). A yellow solid was obtained which was crystallized from ethyl acetate.
Yield: 79%. MP = 136-138°C. FTIR (KBr): νmax = 3047.63; 2960.83; 2910.68; 2885.61;
1
1577.82; 1483.31; 1446.66; 1259.56; 769.62. H NMR (200 MHz, CDCl3):
δ
δ
= 8.05 – 7.15 (m,
= 160.89, 160.69,
13
7H);3.95 (t, 4H); 3.40 (t, 4H); 2.20 (s, 3H). C NMR (101 MHz, CDCl3):
158.23, 143.99, 141.92, 131.28, 131.25, 130.46, 130.38, 129.13, 125.76, 124.89, 124.52, 124.48,
123.93, 116.27, 116.05, 66.93, 50.30, 16.56 ppm. HRMS (ESI) calculated for C20H19ClFN2O
(M+H)+ 357.1164, found for 357.1161.
4.2.41 4-[6-chloro-4-(2-fluorophenyl)-3-phenyl-2-quinolyl]morpholine (F4b)

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To a solution of A4b (0.205 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture, BA3
(0.292 g, 0.001 mol) was added and was refluxed till the reactants were consumed (checked
using TLC up to 48 h). A yellow solid was obtained which was crystallized from ethyl acetate.
Yield: 73%. MP = 203-204°C. FTIR (KBr): νmax = 3055.36; 3020.63; 2968.55; 2933.83;
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1577.82; 1487.17; 1450.51; 1286.56; 775.41. H NMR (200 MHz, CDCl3):
δ
= 7.60 – 6.90 (m,
12H); 3.55 (t, 4H) 3.22 (t, 4H). 13C NMR (101 MHz, CDCl3):
δ = 160.79, 159.07, 158.34,
144.82, 141.98, 137.30, 131.72, 131.69, 130.01, 129.70, 129.58, 129.10, 128.95, 127.86, 127.18,
125.40, 124.66, 124.38, 124.21, 123.83, 123.80, 115.52, 115.30, 66.60, 49.48 ppm. HRMS (ESI)
calculated for C25H21ClFN2O (M+H)+ ,419.1321 found for 419.1313.
4.2.42 4-[6-chloro-4-(2-fluorophenyl)-3-methyl-2-quinolyl]thiomorpholine (F5a)
To a solution of A5a (0.159 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture, BA3
(0.292 g, 0.001 mol) was added and was refluxed till the reactants were consumed (checked
using TLC up to 48 h). A yellow solid was obtained which was crystallized from ethyl acetate.
Yield: 71%. MP = 146-147°C. FTIR (KBr): νmax = 3059.20; 2962.75; 2910.68; 1575.89;
1
1554.68; 1481.38; 1444.73; 1410.01; 1265.34; 761.91. H NMR (200 MHz, CDCl3):
δ
= 8.00 –
7.50 (m, 7H); 3.90 (t, 4H); 3.55 (t, 4H); 2.10 (s, 3H). 13C NMR (101 MHz, CDCl3):
δ = 160.73,
158.28, 143.95, 142.03, 131.29, 130.52, 130.44, 129.99, 129.35, 129.20, 128.79, 125.78, 125.26,
124.57, 123.99, 116.32, 116.10, 52.32, 27.74, 16.62 ppm. HRMS (ESI) calculated for
C20H19ClFN2S (M+H)+ 373.0936, found for 373.0935.
4.2.43 4-[6-chloro-4-(2-fluorophenyl)-3-phenyl-2-quinolyl]thiomorpholine (F5b)
To a solution of A5b (0.221 mL, 0.001 mol) in dry chloroform (5 mL), POCl3 (0.153 mL, 0.001
mol) was added and the mixture was stirred at room temperature for 24 h. To this mixture, BA3
(0.292 g, 0.001 mol) was added and was refluxed till the reactants were consumed (checked
using TLC up to 48 h). A yellow solid was obtained which was crystallized from ethyl acetate.
Yield: 70%. MP = 220-221°C. FTIR (KBr): νmax = 3057.27; 3028.34; 2960.83; 2904.89;
1
2874.03; 1612.54; 1573.96; 1492.95; 1473.66; 1444.73; 1411.94; 1307.78; 827.49. H NMR
13
(200 MHz, CDCl3):
CDCl3):
129.16, 128.16, 127.93, 127.53, 127.24, 125.52, 124.80, 124.50, 124.33, 123.97, 123.93, 115.65,
δ
= 7.5 – 7.0 (m, 12H); 3.99 (t, 4H); 2.5 (t, 4H). C NMR (101 MHz,
δ
= 160.87, 160.01, 158.42, 137.46, 131.79, 130.21, 130.13, 130.01, 129.84, 129.65,

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115.44, 51.83, 27.13 ppm. HRMS (ESI) calculated for C25H21ClFN2S (M+H)+ 435.1093, found
for 435.1090.
4.3 Single crystal X-ray diffraction studies
The structures and conformation of the molecules were unambiguously determined by single
crystal X-ray diffraction studies. Crystallographic data for all ligands, besides H2a, were
collected on SMART APEX-II CCD using Mo-Kα radiation (λ = 0.7107 Å) to a maximum θ
range of 25.00°. The X-ray generator was operated at 50 kV and 30 mA and crystal to detector
distance of 5.00 cm was kept constant. Structure solutions and full matrix least-squares
refinements were done using SHELX-97 and data were corrected for Lorentzian, polarisation
and absorption effects [51]. Crystal data for all the ligands has been summarized in Table S1.
Crystallographic data (excluding structure factors) for all the available structures in this paper
have been deposited with the Cambridge Crystallographic Data Centre as supplementary
publication (CCDC Numbers - 1499725-1499733; 289506-289509; 1499788-1499793 and
1499735-1499744). The CCDC number can be used to obtain data free of charge from Data
Centre via www.ccdc.cam.ac.uk/data_request/cif OR https://summary.ccdc.cam.ac.uk/structure-
summary-form
4.4 Molecular Docking Studies
Docking studies were done by using structural coordinates of the ligands obtained from single
crystal X-ray diffraction studies with the available structures of serotonin receptors 5-HT1B (PDB
ID: 4IAR) [19] and 5-HT2B (PDB ID: 4IB4) [20], obtained from Research Collaboratory for
Structural Bioinformatics-Protein Data Bank (RCSB-PDB). In case of compound H2a, structure
was drawn and minimized using Schrödinger Maestro Version 10.0.013. AutoDock program ver.
4.2 and MGL Tools 1.5.6 were used for adding polar hydrogens and the preparation of input files
[52]. AutoDock VINA v1.1 e was used for docking simulation and calculations [53]. The final
files for the proteins were prepared by deleting agonist ergotamine with the receptors proteins.
The grid for docking was set around the active site to include the exterior surfaces of the helix
and extracellular topological domains of the receptor protein. The grid size of 40 x 26 x 44 points
(5-HT1B) and 42 x 42 x 40 points (5-HT2B) with a spacing of 1.0 Å between grid points was used.
The centre of the grids were fixed (x, y, z coordinates of 9.0, -17.0, 16.0 for 5-HT1B and 21, 22,

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16 for 5-HT2B) such that the grid enclosed the entire extracellular regions of the receptors. This
included the natural ligand binding pocket as well as the exterior surface of the receptors. Final
analysis and image generation was done using Accelrys Discovery Studio Visualizer
v4.1.0.14169.
4.5 In vitro antiproliferative activity assay
Human breast adenocarcinoma (MCF-7) and Human embryonic kidney (HEK293) cell lines
were obtained from National Center of Cell Sciences, Pune, India. The cells were cultured in
Dulbecco's Modified Eagle Medium (DMEM) with 10% FBS and antibiotic solution at 37°C in a
5% CO2 humidified atmosphere. To check the antiproliferative activity, cells were seeded in 96
well plates, at a density of 104 cells per well, and incubated for 24 h. The medium was replaced
and ligand solutions in DMSO were added at different concentrations (10 to 50 µM). Final
DMSO concentration was kept uniform at 0.5%. Control wells were kept with 0.5% DMSO
(vehicle control) under similar conditions. Morphological changes were recorded after 72 h of
treatment with H3a (5 µM).
After 72 h of incubation, 10 µL of MTT (5 mg mL-1) was added into the wells and incubated for
an additional time of 2 h. The formazen crystals formed due to the activity of live cells were
dissolved in 100 µL of DMSO and read at 570 nm. Control samples (without ligands) were
considered to be 100% viable.
Percentage viability was determined by using the formula: Percentage viability = (Reading of the
test samples x 100)/Reading of control samples
The percentage growth inhibition was calculated by using the formula: Growth inhibition = 100
– percent viability
All measurements are mean values of three experiments, performed in triplicates with error bars
indicating standard deviations. Statistical analysis was done by ANOVA at 0.05 level of
significance, post hoc analysis was used to compare results within the series and Bonferroni
correction was used for multiple comparisons.
To check the antiproliferative activity of the most active compounds (H2a, H3a and H3b) at a
fixed concentration (5 µM) in the presence of increasing concentrations of serotonin (0 to 25
µM), MTT assay was performed. The cells were seeded in 96 well plates, at a density of 104 cells

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per well, and incubated for 24 h. The medium was replaced and serotonin solutions (in PBS) at
different concentrations were added along with ligand solutions (in DMSO) at fixed
concentration. Final DMSO concentration was uniformly maintained at 0.5%. Control wells
included 0.5% DMSO (vehicle control) under similar conditions.
After 72 h of incubation, 10 µL of MTT (5 mg mL-1) was added into the wells and incubated for
an additional time of 2 h. The formazen crystals formed due to the activity of live cells were
dissolved in 100 µL of DMSO and read at 570 nm. Control samples (without ligands) were
considered to be 100% viable.
Percentage viability was determined by using the formula: Percentage viability = (Reading of the
test samples x 100)/Reading of control samples
4.6 Ca2+ flux assay
Cells were seeded in black 96 well plates (for fluorescence studies) and clear bottom plates (for
cell visualization) with 100
µ
L DMEM media (10% FBS), at a density of 104 cells per well, and
incubated at 37°C for 24 h or until attachment. After microscopic observations, 100
µ
L of 2x
Fluo-4AM dye (Cat No. F10472, Invitrogen) along with probenecid (prepared as per kit
instructions) was added to all wells and further incubated in the dark for 1 h at 37°C. The
intracellular Ca2+ flux was read at 495 nm (Ex) / 516 nm (Em) using Spectramax M5 multi-well
plate reader. After 10 basal readings, serotonin (positive control), quipazine (a known serotonin
receptor agonist) and serotonin with H3a (test) at final concentrations of 5 µM each were added.
Readings at an interval of 5 sec were taken for a period of 2 min and relative calcium flux values
were plotted for comparison.
4.7 Western blot analysis
MCF-7 cells were seeded in T-75 tissue culture flasks at a density of 106 cells per flask and
cultured as described above. After the cells reached 70-80% confluency, they were treated with 5
µ
M of H3a for 24 and 48 h, keeping the total DMSO concentration to 0.5%. Control cells were
treated with 0.5% DMSO for 48 h. After treatment, cells were trypsinized, neutralized and
washed twice with cold PBS and kept on ice or at 4°C. Total protein extracts were prepared by
using RIPA buffer containing protease and phosphates inhibitor cocktail (ab201119, Abcam).
Protein concentrations were determined by using the standard Bradford assay. For all the

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samples equal protein contents (40 µg) were subjected to sodium dodecyl sulfate polyacrylamide
gel electrophoresis (12%). The resolved protein bands were transferred onto nitrocellulose
membranes for Western blotting. The electrophoresis and membrane transfer efficiency were
monitored by using prestained protein standards. Membranes were serially immunoblotted with
mouse monoclonal antibodies for phospho-ERK (ab50011, Abcam); ERK (ab36991, Abcam)
and β-actin (A2228, Sigma) by removing the previously bound antibody with stripping buffer.
Each primary antibody was detected using horseradish peroxidase-labeled goat anti-mouse IgG
and ECL western blotting substrate kit (ab65623, Abcam) and visualized using C-DiGit Blot
Scanner (LI-COR).
4.8 Flow cytometry
To evaluate the effect of the compounds on cell cycle, MCF-7 cells were analyzed by
fluorescence activated cell sorter. MCF-7 cells were seeded (at a density of 105 cells) in a T-25
tissue culture flask and were treated with H3a (5 µM) for 48 h. Untreated controls samples were
maintained for comparison. After 48 h, cells were trypsinised, washed with cold PBS and fixed
in 3
ꢀmL ethanol (70%) at 4°C for 4 h and stored at -20°C. Prior to analysis, cells were washed
with cold PBS and stained with a solution of propidium iodide and RNase A (10
µ
g mL-1) at 4°C
for 20 min (Data not shown). Based on the above results, cells were stained with FITC
conjugated Annexin V and propidium iodide (V13241 - Alexa Fluor 488 Annexin V/Dead Cell
Apoptosis Kit, Thermo Fisher Scientific) at 37°C for 20 min to check for apoptosis. The staining
was done according to the protocol provided with the kit. Experiments were repeated at least
two times, and representative data are presented here.
Acknowledgments
The authors at Institute of Bioinformatics and Biotechnology, Savitribai Phule Pune University,
thank University Grants Commission (UGC), India for financial support under UPE phase II.
PVJ gratefully acknowledges UGC, India for Junior Research Fellowship.
Supplementary Material
Supplementary data associated with this article can be found, in the online version.
References and notes