Sulfamic acid promoted one-pot synthesis of phenanthrene fused-dihydrodibenzo-quinolinones: Anticancer activity, tubulin polymerization inhibition and apoptosis inducing studies

Article abstract of DOI:10.1016/j.bmc.2018.02.050

A facile one-pot method for the synthesis of new phenanthrene fused-dihydrodibenzo-quinolinone derivatives has been successfully accomplished by employing sulfamic acid as catalyst. These new compounds were evaluated for their in vitro cytotoxic potential against human lung (A549), prostate (PC-3 and DU145), breast (MCF-7) and colon (HT-29 and HCT-116) cancer cell lines. Among all the tested compounds, one of the derivatives 8p showed good anti-proliferative activity against A549 lung cancer cell line with an IC₅₀ of 3.17 ± 0.52 μM. Flow cytometric analyses revealed that compound 8p arrested both Sub G1 and G2/M phases of cell cycle in a dose dependent manner. The compound 8p also displayed significant inhibition of tubulin polymerization and disruption of microtubule network (IC₅₀ of 5.15 ± 0.15 μM). Molecular docking studies revealed that compound 8p efficiently interacted with critical amino acid Cys241 of the α/β-tubulin by a hydrogen bond (S–H…O = 2.4 ?). Further, the effect of 8p on cell viability was also studied by AO/EB, DCFDA and DAPI staining. The apoptotic characteristic features revealed that 8p inhibited cell proliferation effectively through apoptosis by inducing the ROS generation. Analysis of mitochondrial membrane potential through JC-1 staining and annexin V binding assay indicated the extent of apoptosis in A549 cancer cells.

Full text of DOI:10.1016/j.bmc.2018.02.050

Accepted Manuscript
Sulfamic acid promoted one-pot synthesis of phenanthrene fused-dihydrodi-
benzo-quinolinones: Anticancer activity, tubulin polymerization inhibition and
apoptosis inducing studies
Niggula Praveen Kumar, Sowjanya Thatikonda, Ramya Tokala, S. Sujana
Kumari, Uppu Jaya Lakshmi, Chandraiah Godugu, Nagula Shankaraiah, Ahmed
Kamal
PII:
S0968-0896(17)32484-7
https://doi.org/10.1016/j.bmc.2018.02.050
BMC 14236
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
25 December 2017
21 February 2018
28 February 2018
Please cite this article as: Kumar, N.P., Thatikonda, S., Tokala, R., Kumari, S.S., Lakshmi, U.J., Godugu, C.,
Shankaraiah, N., Kamal, A., Sulfamic acid promoted one-pot synthesis of phenanthrene fused-dihydrodibenzo-
quinolinones: Anticancer activity, tubulin polymerization inhibition and apoptosis inducing studies, Bioorganic &
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.02.050
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Sulfamic acid promoted one-pot synthesis of phenanthrene fused-
dihydrodibenzo-quinolinones: Anticancer activity, tubulin polymerization
inhibition and apoptosis inducing studies
a
c
a
b
Niggula Praveen Kumar, Sowjanya Thatikonda, Ramya Tokala, S. Sujana Kumari, Uppu
b
c
a
a
Jaya Lakshmi, Chandraiah Godugu, * Nagula Shankaraiah, * Ahmed Kamal *
a
b
Department of Medicinal Chemistry, Department of Pharmacology and Toxicology,
c
Department of Regulatory Toxicology, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
Abstract
A facile one-pot method for the synthesis of new phenanthrene fused-dihydrodibenzo-
quinolinone derivatives has been successfully accomplished by employing sulfamic acid as
catalyst. These new compounds were evaluated for their in vitro cytotoxic potential against
human lung (A549), prostate (PC-3 and DU145), breast (MCF-7) and colon (HT-29 and
HCT-116) cancer cell lines. Among all the tested compounds, one of the derivatives 8p
showed good anti-proliferative activity against A549 lung cancer cell line with an IC50 of
3
.17 ± 0.52 µM. Flow cytometric analyses revealed that compound 8p arrested both Sub G1
and G2/M phases of cell cycle in a dose dependent manner. The compound 8p also displayed
significant inhibition of tubulin polymerization and disruption of microtubule network (IC50
of 5.15±0.15 µM). Molecular docking studies revealed that compound 8p efficiently
interacted with critical amino acid Cys241 of the α/β-tubulin by a hydrogen bond (S–H…..O
=
2.4 Å). Further, the effect of 8p on cell viability was also studied by AO/EB, DCFDA and
DAPI staining. The apoptotic characteristic features revealed that 8p inhibited cell
proliferation effectively through apoptosis by inducing the ROS generation. Analysis of
mitochondrial membrane potential through JC-1 staining and annexin V binding assay
indicated the extent of apoptosis in A549 cancer cells.
Keywords: Phenanthrene fused-dihydrodibenzo-quinolinones, Perkin condensation, tubulin
polymerization, one-pot reaction, apoptosis.
Corresponding authors: Dr Nagula Shankaraiah, Tel.: (+91) 040 23073740; Fax: (+91) 040
2
3073751; E-mail: shankar@niperhyd.ac.in; Dr Ahmed Kamal, Tel.: +91-40-27193157; fax:
91-40-27193189; E-mail: ahmedkamal@iict.res.in; Dr. Chandraiah Godugu, E-mail:
chandra.niperhyd@gov.in
+
1

1
.0 Introduction
Cancer has been considered as one of the life threatening diseases worldwide [1]. In spite of
tremendous development in the chemotherapeutic drugs, finding efficacious new cytotoxic
drugs with minimum off target effects is the active area of research [2]. The principle target
for most of the antitumor agents is either apoptosis or cell division [3]. Natural products have
been an invaluable source of new chemotherapeutics [4]. Cyanchum, Pergularia, Tylophora
and some other genera from the Asclepiadacea family serves as natural source for diverse
pentacyclic derivatives popularly known as phenanthroindolizidine alkaloids [5]. These
nitrogen-containing alkaloids are well distinguished for their broad range of antitumor [6],
anti-inflammatory [7], antilupus [8], anti-angiogenic [9], antiarthritis [10], anti-microbial
[
11], antiviral [12] and antifungal properties [13]. Profound cytotoxic profile of these
alkaloids makes them as targets for structure activity modification [14]. Tylophorine was the
representative alkaloid of this class, (Figure 1) which showed strong growth inhibitory
potential on a large number of cancer cells by arresting the cell cycle at G2/M phase [15].
Additionally, these derivatives were also reported to induce apoptosis and inhibit the
enzymes involved in the nucleic acid synthesis [16].
On the other hand, podophyllotoxin is a naturally occurring cytotoxic aryl tetralin lignan
reported as well-known potent tubulin polymerization inhibitor [17]. Its semi-synthetic
derivatives such as etoposide and teniposide are among the frontline chemotherapeutic drugs
and are used against various malignancies especially testicular carcinoma, small cell lung
cancer, lymphoma and kaposis sarcoma [18]. Further, many research groups have been
reported the synthesis of 4-azapodophyllotoxin (Figure 1) and its derivatives retaining
mitotic phase arrest and anti-tubulin properties comparable to that of podophyllotoxin [19]. In
this connection, we rationally designed these new compounds resembling both tylophorine
and 4-azapodophyllotoxin in their structures, which may display enhanced cytotoxicity
through G2/M phase arrest and apoptosis induction.
Multi-component reactions serve as an effective tool to synthesize diverse range of bioactive
molecules by involving minimum number of synthetic steps and improving atom economy
[
20]. Moreover, in medicinal chemistry point of view, exploring synthetic precursors to
obtain biologically active molecules is gaining prominent status [21]. More recently
commercially available precursor, 9-amino phenanthroline has been successfully utilized to
synthesize phenanthroline based azapodophyllotoxin mimics (Figure 1). These derivatives
2

displayed potent cytotoxicity and tubulin polymerization inhibition properties [22]. Inspired
by these significant contributions and in continuation of our earlier efforts dedicated to
bioactive podophyllotoxin derivatives, [23] herein, we wish to present a one-pot protocol for
the synthesis of modified analogues of azapodophyllotoxin devoid of “D ring”. A new series
of phenanthrene fused-dihydrodibenzo-quinolinone derivatives has been synthesized and
evaluated for their in vitro cytotoxic potential and apoptosis inducing ability. Moreover, we
have also explored their tubulin polymerization inhibition capability of these newly
synthesized azapodophyllotoxin mimics.
Figure 1. Structures of tylophorine, podophyllotoxin, azapodophyllotoxin derivatives and
newly designed phenanthrene fused-dihydrodibenzo-quinolinones.
2
2
.0 Results and discussion
.1 Chemistry
Phenanthrene fused-dihydrodibenzo-quinolinones 8a–r were synthesized by a one-pot
reaction by using 2,3,6,7-tetramethoxyphenanthren-9-amine (5), Meldrums acid (6) with
various aromatic aldehydes 7a–r and catalytic amount of sulfamic acid in refluxing ethanol
as depicted in Scheme 1. Initially, Knoevenagel condensation between Meldrums acid and
aldehyde results in the formation of arylidene meldrums acid intermediate followed by the
Michael addition of amine and subsequent dehydration, cyclization by loss of CO and
2
acetone affords phenanthrene fused-dihydrodibenzo-quinolinones [24]. Herein, we have also
3

explored the substrate scope of phenanthrene amine. To our interest, multi-component
protocol was optimised by using catalytic amount of sulfamic acid. However, the reaction
was also proceeded in absence of Lewis acid, but the yields obtained were not satisfactory.
Further to study the feasibility of the reaction, different Lewis acids such as TFA, acetic acid,
and bases such as piperidine and Et N have been evaluated. Sulfamic acid in ethanol solvent
3
was proved to be the best for this conversion with improved yields (Table 1). All the
electron-donating, electron-withdrawing, heteroaromatic aldehydes and bicyclic aldehyde
participated effectively in the reaction. The key amine precursor 5 was synthesized from
benzyl 2,3,6,7-tetramethoxyphenanthren-9-yl carbamate (4) in good yield as shown in
Scheme 1. Compound 4 was prepared from the commercially available starting materials via
the conventional six step sequence according to the documented procedure [25]. Initially,
Perkin condensation of 3,4-dimethoxybenzaldehyde (2) and 3,4-dimethoxy phenyl acetic acid
(
1) resulted in the formation of 2,3-bis(3,4-dimethoxyphenyl)acrylic acid. The acid was
converted into its methyl ester followed by m-CPBA/TFA mediated intramolecular oxidative
cyclization [26] to give the tricyclic phenanthrene ester 3. This ester functionality was
converted into acyl hydrazide with hydrazine hydrate followed by diazotization reaction
using NaNO
,3,6,7-tetramethoxyphenanthren-9-yl carbamate (4) by Curtius rearrangement. Finally,
benzyl-carbamate was deprotected with TBAF to give the tetramethoxy phenanthrene amine
2
/HCl-H O affords corresponding acyl azide. This acyl azide is converted to
2
2
1
13
(
5). All the synthesized compounds 8a–r were characterized by HRMS, H and C NMR
1
spectroscopy. The H NMR of compound 8a showed a sharp singlet at δ 9.83 ppm
representing the typical NH proton. The two signals, multiplet at 3.34 ppm and doublet at
2
.99 corresponds to -CH
2
- of the amido hexocyclic C ring. Proton attached to tertiary carbon
is detected as doublet at δ 4.90 ppm. Characteristic methoxy protons appeared as a singlet of
1
3
three protons around δ 3.29–4.13 ppm. In C NMR spectrum of compound 8a the carbonyl
carbon appeared at δ 170.8 ppm. The methoxy substituted carbons resonated around δ 148.1–
1
49.5 ppm. The signal attributed to the -CH
2
- of the C ring appeared at δ 39.3 ppm. Tertiary
carbon showed a characteristic peak of δ 39.2 ppm and all the aromatic carbons resonates
1
13
around δ 101.7–141.3 ppm. Similar pattern was observed in H NMR and C NMR of all the
+
final compounds 8b–r. The HRMS (ESI) of all the derivatives showed an [M + H] peak
equivalent to their corresponding molecular formula.
4

a
Table 1. Optimization of the reaction conditions for the one-pot synthesis of 8a
o
b
entry
reagent
solvent
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
temperature ( C)
time (h)
yield (%)
1
2
3
4
5
6
7
8
-
-
TFA
TFA
80
80
80
80
80
80
80
60
3
57
59
61
54
68
72
89
-
12
12
12
12
12
3
Et
3
N
piperidine
NH
NH
2
SO
SO
3
H
H
2
3
H
2
O
12
a
Reaction conditions: 5 (0.22 mmol), 6 (0.22 mmol), 7 (0.22 mmol), catalyst or base (20 mol%) and solvent (3 mL) .
Isolated yields.
b
5

Scheme 1. One-pot synthesis of new phenanthrene fused-dihydrodibenzo-quinolinones (8a-r).
2
2
.2 Biology
.2.1 In vitro cytotoxic activity
The newly synthesized phenanthrene fused-dihydrodibenzo-quinolinones 8a–r were screened
for their in vitro cytotoxicity against different human cancer cell lines such as lung (A549),
6

prostate (PC-3 and DU145), breast (MCF-7) and colon (HT 29 and HCT-116) by using 3-
(
(
4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [27]. The IC50
µM) values (concentration required to inhibit 50% of the cancer cells) of the tested
compounds and the standard drugs etoposide and podophyllotoxin are listed in Table 2. It is
observed from the initial screening that derivatives 8e and 8p displayed broad range of
activity against all the tested cancer cell lines with an IC50 values ranging from 3.17±0.52 µM
to 19.89±0.21 µM (Table 2). Compound 8p with 3,4-methylenedioxy substitution on
aromatic E ring was found to be most active on all the cancer cell lines particularly on A549
cancer cells (IC50 3.17±0.52 µM). With close analysis of IC50 data, it was observed that most
of the compounds were not adequately active against MCF7 (breast cancer) cells except 8e
and 8p with an IC50 values of 12.23±0.51 and 9.59±0.16 µM, respectively. All the
compounds (except 8a, 8b, 8f, 8i, 8l, 8n and 8r) displayed significant activity less than 10
µM on A549 (lung cancer) cells with 8p being the most potent (IC50 3.17±0.52 µM) followed
by derivative 8g (IC50 4.09±0.48 µM) with a 3,4- dimethoxy substitution on E ring. Majority
of the tested compounds (except 8i, 8j, 8m, 8o and 8q) were active against DU145 prostate
cancer cell line, with derivative 8c possessing trifluoromethyl substitution on E ring being the
most active (IC50 6.19±0.09 µM). Derivatives such as 8c, 8e, 8h, 8i, 8p and 8q, were found to
be active on PC-3 prostate cancer cell line, while rest of them were inactive on this particular
cell line at 20 µM. Compound 8o with a heterocyclic thiophene E ring showed cytotoxicity
on three of the tested cancer cells with an IC50 values between 8.50±0.11 and 18.94±0.32 µM.
However, 8n with a methyl thiophene substitution was active only on DU145 (prostate) and
A549 (lung) cancer cell lines. Most of the compounds (except 8a, 8b, 8f, 8l, 8n and 8q) were
active against the two tested colon cancer cell lines. The most active compound against these
cell lines 8p followed by 8g bearing the 3,4-dimethoxy substitution (HCT-116, IC50 value:
8
.12±0.04 µM). It could be observed from the in vitro cytotoxicity studies that derivatives
containing 3,4-disubstitution and oxygen hetero atom in their E ring such as methylenedioxy
8p), 3,4-dimethoxy (8g) and 3,4-dihydroxy (8i) showed improved biological response. On
(
the other hand, fluorine substituted analogues such as 8c (3-trifluoromethyl) and 8e (4-fluoro)
were also displayed remarkable activity on lung (A549) and prostate (DU145) cancer cells.
Moreover, sulphur containing heterocyclic E ring was also displayed improved cytotoxicity
as the compound 8o with a thiophene ring displayed IC50 value of 8.50±0.11 µM on A549
lung cancer cells. Based on the cytotoxicity results, the most active compounds 8p was taken-
up for further studies towards the mechanism of cancer cell growth inhibition and tubulin
polymerization inhibition.
7

a
Table 2. In vitro cytotoxic activity (IC50 in µM) of phenanthrene fused-dihydrodibenzo
quinolinones 8a–r
b
c
d
e
f
g
Compound
PC-3
DU145
MCF7
>20
A549
>20
HT29
>20
HCT-116
>20
8
8
a
>20
>20
18.84±0.12
18.49±0.27
6.19±0.09
18.2±0.02
11.1±0.1
16.92±0.08
13.12±0.02
17.02±0.07
>20
b
>20
>20
>20
>20
8
c
18.49±0.27
>20
>20
5.03±0.09 18.71±0.13 15.42±0.05
8
d
>20
8.84±0.03 16.01±0.09
>20
16.2±0.1
>20
8
e
19.89±0.21
>20
12.23±0.51 6.29±0.09
17.4±0.10
>20
8
f
>20
>20
16.71±0.06
8
8
g
>20
4.09±0.48 12.21±0.04 8.12±0.04
h
19.09±0.06
19.79±0.12
>20
>20
>20
8.94±0.32 16.71±0.13
11.58±0.05 10.3±0.04
>20
8
i
12.6±0.14
8
j
>20
>20
7.16±0.18 15.54±0.10 16.5±0.12
8
8
k
l
>20
>20
19.0±0.11
18.91±0.21
>20
>20
>20
6.49±0.09 13.78±0.08
>20 >20
14.2±0.3
>20
8
m
>20
>20
7.78±0.08 17.85±0.12 13.4±0.21
15.4±0.25 >20 >20
8.50±0.11 18.94±0.32 13.8±0.45
8
8
n
o
p
q
>20
>20
19.59±0.08
>20
>20
>20
8
8
12.11±0.03
18.40±0.06
-
17.51±0.17
>20
9.59±0.16
>20
3.17±0.52
7.95±0.31
7.84±0.03
>20
7.61±0.21
>20
8
r
16.1±0.17
2.13±0.13
>20
14.69±0.39 18.37±0.20 17.16±0.1
h
Etoposide
Podophyllotoxin
5.4±0.2
2.03±0.05
-
3.25±0.09
1.8±0.1
2.6±0.07
i
0.07±0.003 0.063±0.008
0.09±0.009
-
-
a
b,c
d
e
f,g
5
0% inhibitory concentration after 48 h of compound treatment; prostate cancer cells; breast cancer cells; lung cancer cells; Colon
h
i
cancer cells; Etoposide: positive control; podophyllotoxin: reference drug.
2
.2.2 Apoptosis induction studies
a. DAPI nuclear staining
DAPI (4’,6-diamidino-2-phenylindole) is a fluorescent stain that rigidly binds to A-T rich
sequence of nucleic acid and reveals the nuclear damage through chromatin condensation.
DAPI penetrates the intact cell membrane with less efficiency when compared to
disintegrated cell membrane. Therefore, intensity of stained live cells is less in comparison to
apoptotic cells. Apoptotic features such as condensed nuclei make them to stain brighter.
Hence, it is of great importance to know the effect of compound 8p on A549 cells by using
DAPI staining [28]. Moreover, this nuclear morphology technique differentiates the live cells
from apoptotic cells. DAPI forms fluorescent complex with chromatin there by stains the
nucleus bright blue. As observed from Figure 2, the nuclear structure of control cells was
intact, whereas compound 8p treated A549 cells showed condensed and horse-shoe shaped
nuclei indicating the extent of apoptosis.
8

Figure 2. Nuclear morphology in A549 cells stained with DAPI. A549 cancer cells treated
with compound 8p and stained with DAPI. The images were taken with fluorescence
microscope with a DAPI filter.
b. Acridine orange–ethidium bromide (AO–EB) staining
Live cells, apoptotic and necrotic cells can be differentiated by performing Acridine
orange/ethidium bromide (AO/EB) fluorescent staining assay [29]. AO readily diffuses the
normal cell membrane and stains the nuclei green, whereas EB penetrates only membrane
disintegrated cells and smear the nucleus in red. In Figure 3 green colour of the control cells
is due to their normal morphology. Fluorescence microscopic images of cells treated with
1
0.0 µM of compound 8p clearly shows the altered morphological characteristics such as cell
shrinkage, chromatin condensation, membrane blebbing and apoptotic body formation,
suggesting that the compound 8p induced cell death in A549 lung tumor cells.
9

Figure 3. A549 cells were treated with increased concentrations of compound 8p and stained
with AO/EB. Decreased number of viable cells and apoptotic characteristics were observed at
2
00X magnification.
c. Morphological studies
Microscopic observation could reveal the effect of compounds on cell viability. A549 cancer
cells were treated with 1.25, 2.5, 5.0 and 10.0 µM concentrations of active compound 8p.
Cells were observed for morphological alterations and images were captured by using phase
contrast microscope. It is clearly observed from Figure 4, as the concentration of the
compound increases there is a clear decrease in cell viability with significant morphological
changes such as cell shrinkage and detachment were observed. This indicates the compound
induced distinctive morphological changes in A549 lung cancer cells.
10

Figure 4. Phase contrast imaging: A549 cells were treated with different concentrations of
compound 8p and observed for their morphological changes at 200X magnification.
d. Effect of compound 8p on reactive oxygen species (ROS)
Most of the cytotoxic compounds induce apoptosis by the generation of ROS, where it
initiates oxidative damage to the mitochondrial membrane potential and permeability. Hence,
to assess the extent of ROS generation by compound 8p in A549 cells, DCFDA staining
method [30] was performed. A549 cells were treated with compound 8p resulted in enhanced
DCFDA fluorescence in a dose dependent manner, indicating the capability of compound in
accumulating ROS (Figure 5). Whereas decrease in fluorescence intensity was observed in
case of N-acetyl cysteine (NAC) treated A549 cells, thus indicating the decreased free radical
production by NAC, which suggest one of the mechanism of cytotoxicity is by elevating ROS
levels. On the other hand, increased fluorescence was observed with the positive control
H
2
2
O due to the generation of radicals.
11

Figure 5. Effect of compound 8p on Reactive Oxygen Species (ROS) levels. Dose dependent
increment of fluorescence observed compared to control. Data presented as mean ± SEM
(
n=3). **P<0.01 and ***P<0.001 versus control.
d. Analysis of mitochondrial membrane potential (ΔΨm)
Increased ROS generation could lead to oxidative stress thereby causes alterations in
mitochondrial membrane potential. Therefore, it is considered of interest to observe the effect
of compound 8p on mitochondrial membrane potential (ΔΨm). JC-1, a lipophilic cationic dye
was used to stain the mitochondria [31]. Normal polarised mitochondria stains in red due to
formation of J-aggregates, whereas depolarised mitochondria of apoptotic cells stains in
green because of J-monomers. A549 cells were treated with 1.25, 2.5, 5.0 and 10.0 µM of
compound 8p for 24 h and stained with JC-1 dye. Flow cytometric analysis showed increase
in depolarised cell population (P3) from control 8.31±3.72 to 79.80±4.54% at 10.0 µM in
12

concentration dependent manner (Figure 6). Thus, the results indicate decreased
mitochondrial membrane potential (ΔΨm) in A549 cells.
Figure 6 (A and B). Effect of 8p on mitochondrial membrane potential (ΔΨm). A549 cells
were treated with 1.25, 2.5, 5.0 and 10.0 µM of 8p, incubated with JC-1 stain and analysed
TM
by flow cytometer (BD FACSVerse , USA). Data presented as mean ± SEM (n=3).
*
*P<0.01 versus control.
13

e. AnnexinV /Propidium iodide dual staining assay
Annexin V/propidium iodide dual staining assay was performed to determine the extent of
apoptosis induced by the compound 8p on A549 cells. This assay facilitates the detection of
necrotic cells (Q1-NC; AV-/PI+), live cells (Q2-LC; AV-/PI-), late apoptotic cells (Q3- LA;
AV+/PI+), and early apoptotic cells (Q4- EA; AV+/PI-). As observed from Figure 7, the
percentage of total apoptotic cells (early and late apoptotic cells) increased to 52.53% after
treatment with 10 µM concentration of 8p, in comparison to the control (5.98%) cells. The
percentage of early and late apoptotic cells comparatively increased with an increase in the
concentration of compound 8p, which indicates the induction of dose dependent apoptosis in
A549 cancer cells.
Figure 7. Annexin binding assay for the detection of apoptosis. The compound 8p treated
cells were stained with Annexin V/PI and analysed for apoptosis. 10,000 cells from each
sample were analysed by flowcytometry. The percentage of cells positive for Annexin V/
Propidium iodide is represented inside the quadrants.
2
.2.3 Cell cycle analysis
Arresting specific phase of the cell cycle is the characteristic feature of most of the cytotoxic
compounds. Thus blockade of cell cycle progression by cytotoxic agents has a vital role to
develop effective chemotherapeutic agents. From in vitro screening data, it was observed that
the compound 8p displayed significant cytotoxic potential against A549 lung cancer cells.
14

Therefore, we studied the effect of compound 8p on distribution of cell population in each
phases of cell cycle by flow cytometry analysis [32]. A549 cells were treated with compound
8
p at 1.25, 2.5, 5.0 and 10.0 µM for 48 h, and the cells were fixed using ethanol, stained with
propidium iodide and analyzed by flow cytometry. The results from Figure 8 shows that the
compound 8p arrested A549 cells in G2/M phase from 34.25±5.75% (control) to
6
1.20±3.32% (10.0 µM), In addition, significant Sub G1 phase arrest is also observed in
concentration dependant manner from 4.52±2.47% in control to 31.22±4.03% at 10.0 µM.
Figure 8 (A and B). Cell cycle analysis of A549 lung cancer cells treated with 8p for 48 h.
The cell cycle distribution was analysed by flow cytometry using propidium iodide staining
method. Data presented as mean ± SEM (n=3). **P<0.01 and ****P<0.0001 versus control
#
###
$$$
Sub G1 population;
P<0.0001 versus control G0/G1 population;
P<0.001 and
$
$$$
P<0.0001 versus control G2/M population.
15

2
.2.4 Effect on tubulin polymerization
As these new compounds have structural resemblance to the podophyllotoxin, it was
considered of pertinent to know their effect on microtubule system. The compound 8p was
investigated for its tubulin polymerization inhibitory potential in a cell-free in vitro assay
[
33]. The inhibition of tubulin assembly is most of the times associated with G2/M cell cycle
arrest in most of the cancer cells. The compound 8p also displayed G2/M cell cycle arrest.
Hence, we studied the ability of compound 8p to inhibit tubulin polymerization by
monitoring the increase in fluorescence emission at 440 nm (excitation wavelength is 360
o
nm) for 1 h at 37 C (Figure 9). In this study, podophyllotoxin and paclitaxel, were used as
reference standards at 5 µM and 3 µM concentrations respectively. Compound 8p was used
at final concentrations of 10, 5, 2.5, 1.25 µM respectively. The experiment was performed in
duplicates. The IC50 value was calculated from the drug concentration required to inhibit 50%
of tubulin assembly compared to control. Interestingly, the compound 8p displayed potent
tubulin polymerization inhibition with an IC50 value of 5.15±0.15µM.
Figure 9. Effect of compound 8p on the tubulin polymerization: tubulin polymerization was
monitored by the increase in fluorescence at 360 nm (excitation) and 440 nm (emission) for 1
o
h at 37 C.
16

Figure 10. % Dose-response inhibition of tubulin polymerization by compound 8p at final
concentrations of 10, 5, 2.5, 1.25 µM. Podophyllotoxin and paclitaxel, were used as reference
standards. Data expressed as mean ± SEM (n=3). *P<0.05 and ****P<0.0001 versus control.
2
.2.5 Molecular docking
To understand the mode of binding with tubulin, active compound 8p was docked into the
podophyllotoxin binding site of α/β-tubulin (PDB ID: 1SA1) located at the interface of the
α,β subunits [34]. Co-crystallized ligand podophyllotoxin was re-docked into tubulin so as to
compare the interaction pattern. The podophyllotoxin interacted strongly with the critical
amino acid residues Cys241 and Leu252 in the binding site of tubulin. The interaction with
Cys241 is attributed due to the hydrogen-bonding with oxygen atom corresponding to the
trimethoxy ‘E’ ring of the podophyllotoxin. Therefore, this amino acid residue is critical and
has important role in binding to modified podophyllotoxin derivatives retaining ‘E’ ring in
their structure. Interestingly, compound 8p displayed strong hydrogen-bond interactions with
Cys241, Lys254, Asn258, and Gln11 amino acid residues of the binding site (Figure 11).
Oxygen of methylene dioxy ‘E’ ring of the compound 8p interacted strongly with critical
amino acid Cys241 by a hydrogen-bond (S–H…..O = 2.4 Å). Compound 8p also exhibited
strong hydrophobic interactions with Val177, Ala180, Tyr224, Cys241, Leu248, Ala250,
Ala316, Ala317, Val318 and Ala354. Moreover, the oxygen atom of amide carbonyl
corresponding to hexocyclic ‘C’ ring formed a hydrogen-bonding with the side chain of
Asn258 (N–H…..O = 2.6 Å). Additionally, the two oxygen atoms of the methoxy groups
substituted on the planar phenanthrene system formed hydrogen-bonding with side chain of
Lys254 (N–H…..O = 1.8 Å) and Gln11(N–H…..O = 3.4 Å). Cys241 played a crucial role
during the protein-ligand binding process, possibly explaining the stronger inhibitory activity
17

of compound 8p. All these binding modes were responsible to the hypothesis that these new
azapodophyllotoxin derivatives exhibit efficient binding in to the podophyllotoxin binding
site on tubulin and thus displaying antitubulin activity.
Figure 11. a) Docking model of the most potent compound 8p (magenta colour stick) and b)
its ligand-protein interactions in the podophyllotoxin binding site of α/β-tubulin (PDB ID:
1
SA1). The red dashed lines represent hydrogen bonds.
3
.0 Conclusion
In conclusion, a new series of phenanthrene fused-dihydrodibenzo-quinolinone derivatives
were synthesized and evaluated for their in vitro cytotoxic potential against various selected
human cancer cell lines. From the initial screening, it was observed that some of the
derivatives were active on most of the tested cancer cell lines with IC50 values below 20 µM.
The in vitro screening results indicated that most active compound 8p showed broad range of
activity on all the tested cell lines with a significant IC50 value of 3.17±0.52 µM on A549
lung cancer cells. The cell cycle analysis indicated that it blocked both Sub G1 and G2/M
phase of the cell cycle. Moreover, the compound also decreased viable cells by inducing
apoptotic changes as shown in case of AO/EB and DAPI staining. JC-1 and Annexin-V/PI
staining revealed the dose dependent apoptosis induction by 8p in lung cancer cells with
elevated ROS generation. Further, compound 8p effectively led to the inhibition of tubulin
18

polymerization by increasing the concentrations. Molecular docking studies also supported
the possible mode of binding to the α/β-tubulin. Overall, the current study indicates that these
new azapodophyllotoxin mimics have the potential to be advanced as microtubule targeting
cytotoxic agents for the treatment of cancer.
4
4
.0 Experimental protocols
.1 Chemistry
All reagents and solvents were obtained from commercial suppliers and were used without
further purification. Analytical thin layer chromatography (TLC) was performed on MERCK
precoated silica gel 60-F254 (0.5 mm) aluminum plates. Visualization of the spots on TLC
1
13
plates was achieved by UV light. H and C NMR spectra were recorded on Bruker 500
MHz making a solution of samples in CDCl
3
solvent using tetramethyl silane (TMS) as the
1
13
internal standard. Chemical shifts for H and C are reported in parts per million (ppm)
downfield from tetra methyl silane. Spin multiplicities are described as s (singlet), bs (broad
singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). Coupling constant (J) values
are reported in hertz (Hz). HRMS were determined with Agilent QTOF mass spectrometer
6
540 series instrument. Wherever required, column chromatography was performed using
silica gel (60-120 or 100-200) or neutral alumina. The reactions wherever anhydrous
conditions required are carried under nitrogen positive pressure using freshly distilled
solvents. All evaporation of solvents was carried out under reduced pressure on Heidolph
o
rotary evaporator below 45 C.
4
.1.1 General reaction procedure for the synthesis of phenanthrene fused-dihydrodibenzo-
quinolin-ones 8a–r
A mixture of 2,3,6,7-tetramethoxy phenanthrene-9-amine (5, 0.22 mmol), Meldrums acid (6,
0
.22 mmol) and substituted aldehydes (7a–r, 0.22 mmol), and sulfamic acid (20 mol%) in
EtOH (3 mL) was refluxed for 3–5 h. After the completion of the reaction, the reaction
mixture was allowed to cool to room temperature and the precipitated products was collected
by vacuum filtration and washed with water and followed by recrystallization from ethanol to
afford pure compounds 8a–r in 78-93% yields. All the synthesized compounds were
1
13
thoroughly characterized by H NMR, C NMR and HRMS (ESI).
4
.1.1.1 6,7,10,11-Tetramethoxy-4-phenyl-3,4-dihydrodibenzo[f,h]quinolin-2(1H)-one (8a)
19

o
1
Light brown solid, yield 89%; mp: >300 C; H NMR (500 MHz, CDCl
3
): δ 9.83 (s, 1H),
.84 (s, 1H), 7.79 (s, 1H), 7.51 (s, 1H), 7.22 (t, J = 8.24 Hz, 2H), 7.16 (t, J = 7.32 Hz, 4H),
.90 (d, J = 7.01 Hz, 1H), 4.13 (s, 3H), 4.08 (s, 3H), 3.99 (s, 3H), 3.85 (s, 3H), 3.34-3.29 (m,
7
4
1
1
1
1
3
H), 2.99 (d, J = 15.71 Hz, 1H) ppm; C NMR (125 MHz, CDCl ): δ 170.8, 149.5, 149.4,
3
49.3, 148.1, 141.3, 129.3, 129.0 (2C), 127.1, 127.0 (2C), 124.9, 124.3, 122.0, 117.3, 114.7,
04.1, 103.6, 103.5, 101.7, 56.2, 56.1(2C), 55.6, 39.3, 39.2 ppm; HRMS (ESI): m/z calcd for
+
C
27
H25NO
5
444.1805, found 444.1808 [M+H] .
4
.1.1.2 4-(3-Bromophenyl)-6,7,10,11-tetramethoxy-3,4-dihydrodibenzo[f,h]quinolin-2(1H)-
one (8b)
o
1
Light brown solid, yield 92%; mp: 271-273 C; H NMR (500 MHz, CDCl
3
): δ 9.70 (s, 1H),
.83 (s, 1H), 7.79 (s, 1H), 7.48 (s, 1H), 7.34-7.30 (m, 2H), 7.10-7.08 (m, 3H), 4.86 (d, J =
.32 Hz, 1H), 4.13 (s, 3H), 4.09 (s, 3H), 4.01 (s, 3H), 3.87 (s, 3H), 3.33-3.29 (m, 1H), 2.96
7
7
1
3
(
d, J = 15.86 Hz, 1H) ppm; C NMR (125 MHz, CDCl
3
): δ 170.4, 149.6, 149.5, 149.4,
1
1
48.2, 143.8, 130.6, 130.4, 130.1, 129.5, 125.7, 125.0, 124.1, 123.1, 122.0, 117.2, 113.7,
03.8, 103.7, 103.4, 101.8, 56.2, 56.1, 56.0, 55.7, 39.2, 38.8 ppm; HRMS (ESI): m/z calcd for
+
C
27
H
24BrNO
5
522.0911, found 522.0904 [M+H] .
4
.1.1.3
6,7,10,11-Tetramethoxy-4-(3-(trifluoromethyl)phenyl)-3,4-
dihydrodibenzo[f,h]quinolin-2(1H)-one (8c)
o
1
Off white solid, yield 90%; mp: 290-292 C; H NMR (500 MHz, CDCl
3
): δ 9.77 (s, 1H),
7
2
3
1
1
.84 (s, 1H), 7.80 (s, 1H), 7.56 (s, 1H), 7.51 (s, 1H), 7.47 (d, J = 7.47 Hz, 1H), 7.32-7.27 (m,
H), 7.07 (s, 1H), 4.95 (d, J = 7.32 Hz, 1H), 4.14 (s, 3H), 4.08 (s, 3H), 4.00 (s, 3H), 3.85 (s,
1
3
H), 3.38-3.33 (m, 1H), 2.98 (d, J = 15.71 Hz, 1H) ppm; C NMR (125 MHz, CDCl ): δ
3
70.3, 149.6, 149.5, 149.4, 148.2, 142.6, 131.4 (d, JC-F = 31.7 Hz), 130.4, 129.7, 129.6, 125.1,
24.9 (d, JC-F = 272.4 Hz), 124.1 (d, JC-F = 3.6 Hz), 124.0, 123.9, 122.0, 117.2, 113.6, 103.7
(
2C), 103.5, 101.9, 56.1 (3C), 55.6, 39.1, 39.0 ppm; HRMS (ESI): m/z calcd for C28
H
24
F
3
NO
5
+
5
12.1679, found 512.1680 [M+H] .
4
2
.1.1.4
4-(4-Isopropylphenyl)-6,7,10,11-tetramethoxy-3,4-dihydrodibenzo[f,h]quinolin-
(1H)-one (8d)
o
1
Off white solid, yield 89%; mp: 295-297 C; H NMR (500 MHz, CDCl
3
): δ 9.85 (s, 1H),
7
.83 (s, 1H), 7.78 (s, 1H), 7.53 (s, 1H), 7.19 (s, 1H), 7.08 (dd, J = 8.24, 21.66 Hz, 4H), 4.86
20

(
d, J = 7.17 Hz, 1H), 4.13 (s, 3H), 4.08 (s, 3H), 3.99 (s, 3H), 3.87 (s, 3H), 3.31-3.26 (m, 1H),
1
3
2
.97 (d, J = 15.71 Hz, 1H), 2.81-2.75 (m, 1H), 1.15 (d, J = 6.86 Hz, 6H) ppm; C NMR (125
): δ 171.2, 149.4, 149.3, 149.2, 147.9, 147.5, 138.7, 129.3, 127.0 (2C), 126.8
2C), 124.7, 124.4, 121.9, 117.4, 115.0, 104.2, 103.5, 103.3, 102.0, 56.2, 56.1, 56.0, 55.6,
MHz, CDCl
3
(
3
4
9.3, 38.8, 33.5, 23.8 (2C) ppm; HRMS (ESI): m/z calcd for C30
H
31NO 486.2275, found
5
+
86.2274 [M+H] .
4
.1.1.5 4-(4-Fluorophenyl)-6,7,10,11-tetramethoxy-3,4-dihydrodibenzo[f,h]quinolin-2(1H)-
one (8e)
o
1
Light brown solid, yield 90%; mp: >300 C; H NMR (500 MHz, CDCl
.83 (s, 1H), 7.79 (s, 1H), 7.51 (s, 1H), 7.13-7.09 (m, 3H), 6.88 (t, J = 8.54 Hz, 2H), 4.88 (d,
J = 7.17 Hz, 1H), 4.13 (s, 3H), 4.08 (s, 3H), 4.01 (s, 3H), 3.85 (s, 3H), 3.38-3.32 (m, 1H),
3
): δ 9.82 (s, 1H),
7
1
3
2
1
1
5
4
.94 (d, J = 15.71 Hz, 1H) ppm; C NMR (125 MHz, CDCl ): δ 170.7, 162.7, 160.8, 149.5,
3
49.4, 149.3, 148.1, 137.0, 136.9 (d, JC-F = 3.6 Hz), 129.4, 128.6 (d, JC-F = 8.1 Hz), 124.9,
24.1 (d, JC-F = 236.1Hz), 117.3, 115.9, 115.7, 114.4, 103.8, 103.6, 103.4, 101.9, 56.2, 56.1,
6.0, 55.6, 39.4, 38.4 ppm; HRMS (ESI): m/z calcd for C27
H
24FNO 462.1711, found
5
+
62.1714 [M+H] .
4
.1.1.6 6,7,10,11-Tetramethoxy-4-(p-tolyl)-3,4-dihydrodibenzo[f,h]quinolin-2(1H)-one (8f)
o
1
Off white solid, yield 93%; mp: >300 C; H NMR (500 MHz, CDCl
s, 1H), 7.78 (s, 1H), 7.55-7.53 (m, 1H), 7.16 (s, 1H), 7.06-6.99 (m, 4H), 4.85 (d, J = 7.17
Hz, 1H), 4.13 (s, 3H), 4.07 (s, 3H), 3.98 (s, 3H), 3.86 (s, 3H), 3.26-3.13 (m, 1H), 2.95 (d, J =
3
): δ 9.93 (s, 1H), 7.83
(
1
3
1
1
1
5.56 Hz, 1H), 2.23 (s, 3H) ppm; C NMR (125 MHz, CDCl ): δ 171.1, 149.4, 149.3, 149.2,
3
47.9, 138.3, 136.6, 129.6 (2C), 129.4, 126.8 (2C), 124.7, 124.4, 121.9, 117.4, 114.9, 104.0,
03.4, 103.3, 102.0, 56.2, 56.1, 56.0, 55.6, 39.4, 38.4, 20.9 ppm; HRMS (ESI): m/z calcd for
+
C
28
H27NO
5
458.1962, found 458.1964 [M+H] .
4
2
.1.1.7
4-(3,4-Dimethoxyphenyl)-6,7,10,11-tetramethoxy-3,4-dihydrodibenzo[f,h]quinolin-
(1H)-one (8g)
o
1
White solid, yield 87%; mp: 229-230 C; H NMR (500 MHz, CDCl
3
): δ 9.86 (s, 1H), 7.84
(
s, 1H), 7.79 (s, 1H), 7.53 (s, 1H), 7.17 (s, 1H), 6.77 (s, 1H), 6.66-6.62 (m, 2H), 4.84 (d, J =
6
3
.86 Hz, 1H), 4.14 (s, 3H), 4.08 (s, 3H), 4.02 (s, 3H), 3.87 (s, 3H), 3.75 (s, 3H), 3.72 (s, 3H),
1
3
.30-3.25 (m, 1H), 2.98 (d, J = 15.56 Hz, 1H) ppm; C NMR (125 MHz, CDCl ): δ 171.1,
3
21

1
1
49.4, 149.3, 149.2, 149.1, 148.1, 148.0, 133.8, 129.3, 124.8, 124.3, 121.9, 119.1, 117.3,
15.0, 111.4, 110.3, 104.0, 103.5, 103.4, 101.9, 56.1, 56.0 (2C), 55.8, 55.7, 55.6, 39.4, 38.8
+
ppm; HRMS (ESI): m/z calcd for C29
H29NO
7
504.2017, found 504.2012 [M+H] .
4
.1.1.8 6,7,10,11-Tetramethoxy-4-(4-methoxyphenyl)-3,4-dihydrodibenzo[f,h]quinolin-2(1H)-
one (8h)
o
1
Yellow solid, yield 81%; mp: 297-299 C; H NMR (500 MHz, CDCl
s, 1H), 7.78 (s, 1H), 7.52 (s, 1H), 7.15 (s, 1H), 7.08 (d, J = 8.08 Hz, 2H), 6.74 (d, J = 8.24
Hz, 2H), 4.84 (d, J = 6.40 Hz, 1H), 4.13 (s, 3H), 4.08 (s, 3H), 4.00 (s, 3H), 3.86 (s, 3H), 3.68
3
): δ 9.86 (s, 1H), 7.83
(
1
3
(
s, 3H), 3.29-3.25 (m, 1H), 2.95 (d, J = 15.56 Hz, 1H) ppm; C NMR (125 MHz, CDCl
3
): δ
1
1
70.9, 158.5, 149.5, 149.4, 149.3, 148.0, 133.2, 129.2, 128.0 (2C), 124.8, 124.3, 121.9, 117.3,
15.1, 114.3, 114.1, 104.1, 103.5, 103.4, 101.7, 56.2, 56.1, 56.0, 55.6, 55.1, 39.5, 38.4 ppm;
+
HRMS (ESI): m/z calcd for C28
H27NO
6
474.1911, found 474.1914 [M+H] .
4
2
.1.1.9
4-(3,4-Dihydroxyphenyl)-6,7,10,11-tetramethoxy-3,4-dihydrodibenzo[f,h]quinolin-
(1H)-one (8i)
o
1
Light brown solid, yield 81%; mp: >300 C; H NMR (500 MHz, DMSO-d
H), 8.52 (Br, s, 2H), 7.92 (s, 1H), 7.87 (s, 1H), 7.75 (s, 1H), 7.16 (s, 1H), 6.61 (d, J = 8.08
Hz, 1H), 6.54 (d, J = 8.08 Hz, 1H), 6.47 (s, 1H), 4.74 (d, J = 7.17 Hz, 1H), 4.04 (s, 3H), 3.99
6
): δ 10.35 (s,
1
1
3
(
d, J = 3.05 Hz, 6H), 3.77 (s, 3H), 3.10-3.06 (m, 1H), 2.67 (d, J = 15.41 Hz, 1H) ppm; C
NMR (125 MHz, CDCl +DMSO-d ): δ 169.4, 148.7, 148.5, 148.4, 147.3, 144.8, 143.7,
32.9, 129.6, 124.1, 124.0, 121.0, 117.7, 117.1, 115.4, 114.7, 114.0, 104.0, 103.8, 103.6,
3
6
1
1
4
03.5, 102.8, 55.8, 55.7, 55.6, 55.0, 37.6 ppm; HRMS (ESI): m/z calcd for C27
H
25NO
7
+
76.1704, found 476.1710 [M+H] .
4
.1.1.10 4-(4-Bromophenyl)-6,7,10,11-tetramethoxy-3,4-dihydrodibenzo[f,h]quinolin-2(1H)-
one (8j)
o
1
White solid, yield 88%; mp: >300 C; H NMR (500 MHz, CDCl
3
): δ 10.03 (s, 1H), 7.83 (s,
H), 7.79 (s, 1H), 7.53 (s, 1H), 7.33 (d, J = 8.54 Hz, 2H), 7.07 (s, 1H), 7.04 (d, J = 8.39 Hz,
H), 4.86 (d, J = 7.17 Hz, 1H), 4.13 (s, 3H), 4.08 (s, 3H), 4.02 (s, 3H), 3.85 (s, 3H), 3.34-
1
2
3
1
1
1
3
.30 (m, 1H), 2.95 (d, J = 15.71 Hz, 1H) ppm; C NMR (125 MHz, CDCl ): δ 170.6, 149.5,
3
49.4 (2C), 148.1, 140.3, 132.1 (2C), 129.5, 128.8 (2C), 124.9, 124.0, 122.0, 121.0, 117.2,
14.0, 103.7, 103.6, 103.4, 101.9, 56.2, 56.1, 56.0, 55.6, 39.1, 38.6 ppm; HRMS (ESI): m/z
+
calcd for C27
H
24BrNO
5
522.0911, found 522.0904 [M+H] .
22

4
.1.1.11 4-(4-Chlorophenyl)-6,7,10,11-tetramethoxy-3,4-dihydrodibenzo[f,h]quinolin-2(1H)-
one (8k)
o
1
Off white solid, yield 86%; mp: >300 C; H NMR (500 MHz, CDCl
3
): δ 9.85 (s, 1H), 7.84
s, 1H), 7.79 (s, 1H), 7.50 (s, 1H), 7.18 (d, J = 8.54 Hz, 2H), 7.09 (d, J = 9.46 Hz, 3H), 4.87
d, J = 7.01 Hz, 1H), 4.13 (s, 3H), 4.08 (s, 3H), 4.02 (s, 3H), 3.85 (s, 3H), 3.33-3.29 (m, 1H),
(
(
1
3
2
1
1
.96 (d, J = 15.71 Hz, 1H) ppm; C NMR (125 MHz, CDCl ): δ 170.7, 149.5, 149.4, 149.3,
3
48.1, 139.7, 132.9, 129.4, 129.1 (2C), 128.4 (2C), 124.9, 124.0, 122.0, 117.2, 114.0, 103.8,
03.6, 103.4, 102.0, 56.2, 56.1, 56.0, 55.6, 39.2, 38.5 ppm; HRMS (ESI): m/z calcd for
+
C
27
H
24ClNO
5
478.1416, found 478.1419 [M+H] .
4
2
.1.1.12 6,7,10,11-Tetramethoxy-4-(3-methoxyphenyl)-3,4-dihydrodibenzo[f,h]quinolin-
(1H)-one (8l)
o
1
Yellow solid, yield 85%; mp: 288-290 C; H NMR (500 MHz, CDCl
3
): δ 9.44 (s, 1H), 7.83
s, 1H), 7.78 (s, 1H), 7.42 (s, 1H), 7.15 (t, J = 7.32 Hz, 2H), 6.78 (d, J = 7.62 Hz, 1H), 6.71
d, J = 7.78 Hz, 2H), 4.86 (d, J = 7.01 Hz, 1H), 4.13 (s, 3H), 4.08 (s, 3H), 4.01 (s, 3H), 3.86
(
(
(
1
3
s, 3H), 3.66 (s, 3H), 3.30-3.26 (m, 1H), 2.99 (d, J = 15.8 Hz, 1H) ppm; C NMR (125
): δ 170.8, 160.0, 149.5, 149.4, 149.3, 148.1, 143.0, 130.0, 129.2, 124.9, 124.4,
22.0, 119.4, 117.2, 114.6, 113.6, 111.6, 104.1, 103.6, 103.5, 101.6, 56.2, 56.1, 56.0, 55.7,
MHz, CDCl
3
1
5
5.0, 39.2 (2C) ppm; HRMS (ESI): m/z calcd for C28
H
27NO 474.1911, found 474.1914
6
+
[
M+H] .
4
2
.1.1.13 4-(3,4-Difluorophenyl)-6,7,10,11-tetramethoxy-3,4-dihydrodibenzo[f,h]quinolin-
(1H)-one (8m)
o
1
Off white solid, yield 87%; mp: 290-292 C; H NMR (500 MHz, CDCl
3
): δ 9.64 (s, 1H),
.84 (s, 1H), 7.80 (s, 1H), 7.48 (s, 1H), 7.05 (s, 1H), 7.01 (t, J = 9.46 Hz, 1H), 6.94-6.89 (m,
H), 4.87 (d, J = 7.01 Hz, 1H), 4.14 (s, 3H), 4.09 (s, 3H), 4.04 (s, 3H), 3.86 (s, 3H), 3.32-
7
2
3
1
1
3
.27 (m, 1H), 2.94 (d, J = 15.86 Hz, 1H) ppm; C NMR (125 MHz, CDCl
3
): δ 170.2, 149.7,
49.6, 149.5, 148.3, 138.2 (q, JC-F = 4.5 Hz), 129.3, 125.1, 123.9 (d, JC-F = 236.1 Hz), 123.0
(
q, JC-F = 3.6 Hz), 122.1, 117.9, 117.7, 117.1, 116.2, 116.1, 113.7, 103.8, 103.7, 103.6, 101.6,
5
6.3, 56.2, 56.1, 55.7, 39.2, 38.3 ppm; HRMS (ESI): m/z calcd for C27
H
23
F
2
NO 480.1617,
5
+
found 480.1622 [M+H] .
4
.1.1.14 6,7,10,11-Tetramethoxy-4-(3-methylthiophen-2-yl)-3,4-dihydrodibenzo[f,h]quinolin-
23

2
(1H)-one (8n)
o
1
Light brown solid, yield 90%; mp: >300 C; H NMR (500 MHz, CDCl
3
): δ 9.65 (s, 1H),
.83 (s, 1H), 7.78 (s, 1H), 7.49 (s, 1H), 7.10 (s, 1H), 6.88 (d, J = 5.03 Hz, 1H), 6.80 (d, J =
.03 Hz, 1H), 5.17 (d, J = 6.86 Hz, 1H), 4.13 (s, 3H), 4.07 (s, 3H), 4.03 (s, 3H), 3.90 (s, 3H),
7
5
3
1
3
.28-3.24 (m, 1H), 2.91 (d, J = 15.56 Hz, 1H), 2.45 (s, 3H) ppm; C NMR (125 MHz,
): δ 170.3, 149.5, 149.3 (2C), 148.1, 138.9, 131.8, 130.0, 128.9, 126.9, 125.0, 123.9,
22.9, 121.9, 117.2, 115.5, 103.6, 103.5, 101.7, 56.2 (2C), 56.0, 55.5, 38.6, 33.1, 13.7 ppm;
CDCl
3
1
+
HRMS (ESI): m/z calcd for C26
H
25NO S 464.1526, found 464.1522 [M+H] .
5
4
.1.1.15 6,7,10,11-Tetramethoxy-4-(thiophen-2-yl)-3,4-dihydrodibenzo[f,h]quinolin-2(1H)-
one (8o)
o
1
Off white solid, yield 92%; mp: >300 C; H NMR (500 MHz, CDCl
s, 1H), 7.81 (s, 1H), 7.53 (s, 1H), 7.30 (s, 1H), 7.08 (d, J = 5.03 Hz, 1H), 6.78 (t, J = 3.96
Hz, 1H), 6.73 (d, J = 3.20 Hz, 1H), 5.14 (d, J = 6.40 Hz, 1H), 4.13 (s, 3H), 4.10 (s, 3H), 4.07
3
): δ 9.80 (s, 1H), 7.83
(
1
3
(
s, 3H), 3.95 (s, 3H), 3.29-3.25 (m, 1H), 3.11 (d, J = 15.71 Hz, 1H) ppm; C NMR (125
MHz, CDCl ): δ 170.6, 149.6, 149.5, 149.4, 148.1, 144.6, 128.7, 127.0, 124.9, 124.6, 124.3,
23.9, 121.9, 117.2, 115.3, 103.9, 103.6, 103.4, 101.6, 56.2, 56.1, 56.0, 55.8, 39.3, 34.4 ppm;
3
1
+
HRMS (ESI): m/z calcd for C25
H
23NO S 450.1370, found 450.1365 [M+H] .
5
4
.1.1.16
4-(Benzo[d][1,3]dioxol-5-yl)-6,7,10,11-tetramethoxy-3,4-
dihydrodibenzo[f,h]quinolin-2(1H)-one (8p)
o
1
Light brown solid, yield 80%; mp: 284-286 C; H NMR (500 MHz, CDCl
3
): δ 9.84 (s, 1H),
.83 (s, 1H), 7.79 (s, 1H), 7.52 (s, 1H), 7.14 (s, 1H), 6.65 (s, 2H), 6.61 (s, 1H), 5.83 (d, J =
.42 Hz, 2H), 4.82 (d, J = 7.17 Hz, 1H), 4.13 (s, 3H), 4.08 (s, 3H), 4.02 (s, 3H), 3.88 (s, 3H),
7
4
3
1
1
1
3
.29-3.25 (m, 1H), 2.92 (d, J = 15.56 Hz, 1H) ppm; C NMR (125 MHz, CDCl ): δ 170.9,
3
49.4, 149.3, 149.2, 149.1, 148.0, 146.5, 135.2, 129.4, 124.8, 124.2, 121.9, 120.1, 117.3,
14.7, 108.6, 107.4, 104.0, 103.5, 103.4, 101.9, 100.9, 56.2, 56.1, 56.0, 55.7, 39.6, 38.9 ppm;
+
HRMS (ESI): m/z calcd for C28
H25NO
7
488.1704, found 488.1705 [M+H] .
4
.1.1.17
4-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-6,7,10,11-tetramethoxy-3,4-
dihydrodibenzo[f,h]quinolin-2(1H)-one (8q)
o
1
Light brown solid, yield 80%; mp: 287-289 C; H NMR (500 MHz, DMSO-d
6
): δ 10.27 (s,
1
H), 7.84-7.80 (m, 3H), 7.71 (t, J = 4.42 Hz, 1H), 7.14-7.11 (m, 1H), 6.65-6.59 (m, 1H),
24

6
3
.52-6.50 (m, 1H), 4.74 (d, J = 5.95 Hz, 1H), 4.09-4.07 (m, 3H), 4.05 (d, J = 5.79 Hz, 3H),
1
3
.99 (t, J = 6.25 Hz, 6H), 3.80-3.77 (m, 3H), 3.11-3.06 (m, 2H), 2.73-2.69 (m, 1H) ppm; C
): δ 169.5, 149.0, 148.8, 148.6, 147.5, 143.1, 142.0, 135.4, 129.7,
24.3, 123.8, 121.1, 119.7, 117.0, 116.9, 115.4, 114.7, 104.5, 104.3, 104.1, 103.1, 63.9, 63.8,
NMR (125 MHz, DMSO-d
6
1
5
5.9, 55.8, 55.7, 55.2, 38.9, 37.0 ppm; HRMS (ESI): m/z calcd for C29
H
27NO 502.1860,
7
+
found 502.1856 [M+H] .
4
2
.1.1.18
6,7,10,11-Tetramethoxy-4-(naphthalen-1-yl)-3,4-dihydrodibenzo[f,h]quinolin-
(1H)-one (8r)
o
1
Yellow solid, yield 78%; mp: >300 C; H NMR (500 MHz, CDCl
3
+DMSO-d
6
): δ 10.00 (s,
1
H), 8.43 (d, J = 8.39 Hz, 1H), 7.94 (d, J = 7.93 Hz, 1H), 7.87 (s, 1H), 7.79 (s, 1H), 7.70 (t, J
=
7.78 Hz, 3H), 7.58 (t, J = 7.47 Hz, 1H), 7.14 (t, J = 7.62 Hz, 1H), 6.96-6.94 (m, 2H), 5.71
1
3
(
d, J = 7.62 Hz, 1H), 4.15 (s, 3H), 4.05 (d, J = 4.27 Hz, 6H), 3.48-3.36 (m, 5H) ppm; C
NMR (125 MHz, CDCl +DMSO-d ): δ 169.3, 149.1, 148.7, 148.6, 147.5, 137.2, 133.8,
30.8, 130.0, 129.0, 127.3, 126.7, 125.7, 125.4, 124.4, 124.0, 123.8, 122.6, 121.3, 117.0,
3
6
1
1
14.4, 104.5, 104.2, 104.1, 103.2, 55.9, 55.8 (2C), 54.6, 38.0, 33.7 ppm; HRMS (ESI): m/z
+
calcd for C31
H27NO
5
494.1962, found 494.1964 [M+H] .
4
.2 Biology
.2.1. Cell Cultures
4
Cells were procured from National Centre for Cell Science (NCCS) Pune, India and stocks
were maintained under sterile conditions. lung (A549), prostate (PC-3 and DU145), breast
(
MCF-7) and colon (HT 29 and HCT-116) cancer cells were grown in tissue culture flasks in
DMEM (Dulbecco modified Eagle medium, Sigma), MEM (Minimum Essential Medium,
Sigma) or RPMI 1640 medium, Sigma, supplemented with 10% fetal bovine serum with 1X
stabilized antibiotic-antimycotic solution (Sigma) in a CO
and 90% relative humidity.
2
incubator at 37 °C with 5% CO
2
4
.2.2 In vitro cytotoxic activity
MTT assay was performed to determine the cytotoxicity for all the new compounds 8a-r.
4
1
x10 cells per well were seeded in 100 µL respective media, supplemented with 10% FBS in
each well of 96-well microculture plates and incubated at 37 °C for 24 h, in a CO
2
incubator.
Samples were diluted to the required concentrations in culture medium, were added to the
25

wells with respective vehicle control. After incubating for 48 h, 100 µL MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (0.5 mg/mL) was added to all the
plates and allowed for 4 h incubation. Then, the supernatant was carefully decanted from
each well, formazon crystals were dissolved in 200 µL of DMSO and at 570 nm wavelength
absorbance was recorded.
4
.2.3 DAPI nuclear staining
DAPI staining was performed to observe morphological changes in nucleus. After treatment
of A549 cells with 8p for 48 h, cells were washed with PBS and solubilized with 0.1%
Tween 20 for 10 min followed by staining with 1 µM DAPI. Control and treated cells were
observed with fluorescence microscope (Model: Nikon, Japan) with excitation at 359 nm and
emission at 461 nm using DAPI filter at 200X magnification.
4
.2.4 Acridine orange–ethidium bromide (AO–EB) staining
6
A549 cells were plated at a concentration of 1×10 cell/ml and treated with different
concentrations of compound 8p. Plates were incubated at 37 °C in an atmosphere of 5% CO
for 48 h. 10 µL of fluorescent dyes containing Acridine Orange (AO) and Ethidium Bromide
EB) added into each well in equal volumes (10 µ g/mL) respectively and after 10 min the
2
(
cells were visualized under fluorescence microscope (Nikon, Inc. Japan) with excitation (488
nm) and emission (550 nm) at 200x magnification.
4
.2.5 Morphological studies
5
A549 cells with a density of 1x10 cells/mL were plated in 6 well culture plates and allowed
to adhere for overnight. Cells were incubated with various concentrations of 8p. After 48 h,
cells were observed for morphological changes and images were captured by using phase
contrast microscope (Nikon).
4
.2.6 Effect on reactive oxygen species (ROS)
6
A549 cells with a density of (1 x 10 cells/mL) were plated in 24 well plates and allowed to
adhere for overnight. Then the cells were treated with 1.25, 2.5, 5.0 and 10.0 µM compound
8
p for 24 h. The media was replaced with culture medium containing DCFDA dye (10 µM)
and incubated in dark for 30 min. The intensity of fluorescence from samples was determined
26

by multimode plate reader at an excitation and emission wavelength of 488 and 525 nm,
respectively and the images were captured by fluorescent microscope.
4
.2.7 Analysis of mitochondrial membrane potential (ΔΨm)
6
A549 cells (1 x 10 cells/mL) were plated in 6 well plates and allowed to adhere for
overnight. The cells were incubated with 1.25, 2.5, 5.0 and 10 µM concentrations of 8p for
2
4 h. cells were collected, washed with PBS and resuspended in solution of JC-1 (2.5
o
µg/mL) and allowed for incubation at 37 C for 10 min. The cells were washed twice with
PBS and cells were trypsinized, centrifuged and analyzed by flow cytometer (BD
TM
FACSVerse , USA).
4
.2.8 Annexin V/Propidium iodide dual staining assay
The Annexin V/Propidium iodide dual staining assay was performed using A549 cells. To
6
quantify the percentage of apoptotic cells, A549 cells (1 x 10 mL per well) were plated in
six-well culture plates and allowed to grow for 24 h. After treatment with the compound 8p
(
2.5, 5.0 and 10 µM) for 48 h, cells were collected by trypsinisation. The collected cells were
washed twice with ice-cold PBS, then incubated with 200 µL1 x binding buffer containing 5
µL Annexin V-Alexa flour 488, and then in 300 µL1 x binding buffer containing 5 µL
Propidium iodide (PI) for 15 min at room temp in the dark. After incubation, cells were
analyzed for apoptosis using flow-cytometer.
4
.2.9 Cell cycle analysis
The accumulation of cells in various phases of cell cycle was analysed by Flow cytometric
analysis (FACS). A549 cells were incubated with 8p at different concentrations 1.25, 2.5, 5.0,
and 10 µM for 48 h. control and 8p treated cells were harvested, washed with PBS, fixed in
7
0% ethanol and stained with propidium iodide (50 µg/mL sigma aldrich) in the presence of
RNase A (20 µg/mL) containing 0.1% Triton X-100 at 37°C for 30 min in dark, and about
1
0000 events were analyzed by flow cytometer.
4
.2.10 Effect on tubulin polymerization
Tubulin polymerization kit was procured from Cytoskeleton, Inc. (BK011). To study the
effect of compound 8p, fluorescence based in vitro tubulin polymerization assay was
27

performed following the manufacturer's protocol. The reaction mixture having porcine brain
tissue (2 mg/mL) in 80 mM PIPES at pH 6.9, 2.0 mM MgCl , 0.5 mM EGTA,1.0 mM GTP
2
and glycerol in the presence and absence of test compound 8p (final concentration of 10 µM)
was prepared and added to each well of 96-well plate. Tubulin polymerization was followed
by a time dependent increase in fluorescence due to the insertion of a
fluorescence reporter into microtubules as polymerization takes place. Spectramax M4 Multi
mode Micro plate Detection System was used to measure Fluorescence emission at 440 nm
(
excitation wavelength is 360 nm). podophyllotoxin was used as positive control in the assay
at 5µM final concentration. The IC50 value was calculated from the drug concentration
required for inhibiting 50% of tubulin assembly compared to control.
4
.2.11 Molecular docking
The crystal co-ordinates of α,β-tubulin subunits were retrieved from the protein data bank
PDB ID: 1SA1). The 3D structure of compound 8p was drawn on Maestro Molecule Builder
(
of Schrödinger. The molecule was optimised using OPLS_2005 force field in LigPrep
module of Schrödinger. Docking procedure was performed according to the standard protocol
implemented in maestro software, version 9.9 and the compound 8p was docked into the α,β-
tubulin interphase. The ligand –protein complex was analysed for interactions and 3D pose of
most active compound 8p was imaged using Schrödinger and PyMOL v0.99.
Acknowledgements
Authors are thankful to DoP, Ministry of Chemicals & Fertilizers, Govt. of India, New Delhi,
for the award of NIPER fellowship. Dr. N. Shankaraiah thankful to SERB, DST, Govt. of
India for the start-up grant (YSS-2015-001709).
5
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