Microwave-assisted one-pot synthesis of new phenanthrene fused-tetrahydrodibenzo-acridinones as potential cytotoxic and apoptosis inducing agents

Article abstract of DOI:10.1016/j.ejmech.2018.03.069

An expeditious microwave-assisted one-pot synthesis of new cytotoxic phenanthrene fused-tetrahydrodibenzo-acridinones has been successfully accomplished. This protocol offers wide substrate scope, catalyst-free synthesis, atom-economy, simple recrystallization, high yields, and ethanol was used as green solvent. These new compounds were tested for their in vitro cytotoxicity against cervical (HeLa), prostate (PC-3), fibrosarcoma (HT-1080), ovarian (SKOV-3) cancer cells, and were safer to normal (Hek-293T) kidney cell line. All the compounds have displayed significant cytotoxicity profile, among them 8m being the most potent compound with an IC₅₀ 0.24 ± 0.05 μM against SKOV-3 ovarian cancer cells. Flow cytometry analysis revealed that cells were blocked at the G2/M phase of the cell cycle. The effect of 8m on F-actin polymerisation was also studied. Hoechst staining clearly showed the decreased number of viable cells and indicated apoptosis progression. Compound 8m caused the collapse of mitochondrial membrane potential as observed via JC-1 staining and also enhanced the generation of reactive oxygen species. The increase of caspase-3 activation by 3.7 folds supported the strong apoptosis induction. In addition, an in vitro 3D-spheroid progression assay was performed with 8m that significantly suppressed the tumor cells.

Full text of DOI:10.1016/j.ejmech.2018.03.069

Accepted Manuscript
Microwave-assisted one-pot synthesis of new phenanthrene fused-tetrahydrodibenzo-
acridinones as potential cytotoxic and apoptosis inducing agents
Niggula Praveen Kumar, Pankaj Sharma, T. Srinivasa Reddy, Nagula Shankaraiah,
Suresh K. Bhargava, Ahmed Kamal
PII:
S0223-5234(18)30312-X
10.1016/j.ejmech.2018.03.069
EJMECH 10334
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 January 2018
Revised Date: 21 March 2018
Accepted Date: 22 March 2018
Please cite this article as: N.P. Kumar, P. Sharma, T.S. Reddy, N. Shankaraiah, S.K. Bhargava,
A. Kamal, Microwave-assisted one-pot synthesis of new phenanthrene fused-tetrahydrodibenzo-
acridinones as potential cytotoxic and apoptosis inducing agents, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.069.
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ACCEPTED MANUSCRIPT
Graphical abstract
Microwave-assisted one-pot synthesis of new phenanthrene fused-
tetrahydrodibenzo-acridinones as potential cytotoxic and apoptosis
inducing agents
a
a
c
a
Niggula Praveen Kumar, Pankaj Sharma, T. Srinivasa Reddy, * Nagula Shankaraiah, *
c
a,b
Suresh K. Bhargava, Ahmed Kamal *
a
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
b
School of Pharmaceutical Education and Research, Jamia Hamdard University, New Delhi
10062, India
1
c
Centre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science, RMIT
University, GPO BOX 2476, Melbourne-3001, Australia

ACCEPTED MANUSCRIPT
Microwave-assisted one-pot synthesis of new phenanthrene fused-
tetrahydrodibenzo-acridinones as potential cytotoxic and apoptosis
inducing agents
a
a
c
a
Niggula Praveen Kumar, Pankaj Sharma, T. Srinivasa Reddy, * Nagula Shankaraiah, *
c
a,b
Suresh K. Bhargava, Ahmed Kamal *
a
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
b
School of Pharmaceutical Education and Research, Jamia Hamdard University, New Delhi
10062, India
1
c
Centre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science, RMIT
University, GPO BOX 2476, Melbourne-3001, Australia
Abstract: An expeditious microwave-assisted one-pot synthesis of new cytotoxic
phenanthrene fused-tetrahydrodibenzo-acridinones has been successfully accomplished. This
protocol offers wide substrate scope, catalyst-free synthesis, atom-economy, simple
recrystallization, high yields, and ethanol was used as green solvent. These new compounds
were tested for their in vitro cytotoxicity against cervical (HeLa), prostate (PC-3),
fibrosarcoma (HT-1080), ovarian (SKOV-3) cancer cells, and were safer to normal (Hek-
2
93T) kidney cell line. All the compounds have displayed significant cytotoxicity profile,
among them 8m being the most potent compound with an IC50 0.24 ± 0.05 µM against
SKOV-3 ovarian cancer cells. Flow cytometry analysis revealed that cells were blocked at the
G2/M phase of the cell cycle. The effect of 8m on F-actin polymerisation was also studied.
Hoechst staining clearly showed the decreased number of viable cells and indicated apoptosis
progression. Compound 8m caused the collapse of mitochondrial membrane potential as
observed via JC-1 staining and also enhanced the generation of reactive oxygen species. The
increase of caspase-3 activation by 3.7 folds supported the strong apoptosis induction. In
addition, an in vitro 3D-spheroid progression assay was performed with 8m that significantly
suppressed the tumour cells.
Keywords:
Microwave
reaction,
Perkin-condensation,
phenanthrene
fused-
tetrahydrodibenzo-acridinones, cytotoxicity, 3D-spheroid inhibition, apoptosis.
∗
Corresponding authors: Dr Ahmed Kamal, E-mail: ahmedkamal@iict.res.in; Dr Nagula
Shankaraiah, E-mail: shankar@niperhyd.ac.in; shankarnbs@gmail.com; T. Srinivasa Reddy,
E-mail: srinivasareddy.telukutla@rmit.edu.au
.
1

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1
.0 Introduction
Cancer has been regarded as a life threatening disease to the mankind and the number of
cases has been enormously increasing in the developed countries [1]. Chemotherapy is the
effective treatment to combat cancer [2]. Cytotoxic agents that can induce apoptosis have an
invaluable role in cancer chemotherapy [3]. Most of the cytotoxic agents are either natural
origin or natural product derivatives [4]. Natural products such as phenanthrene alkaloids
mainly isolated from the plant species belonging to Asclepidaceae and Moraceae family. Due
to their diverse pharmacological profile they became the targets for their structural
modification [5]. They have been reported to posses anticancer [6], anti-microbial [7], anti-
inflammatory [8], anti-lupus [9] and anti-angiogenic [10] properties. These nitrogen-
containing alkaloids are not only recognised for their potent antiproliferative activity but also
significantly inhibit cancer cells resistant or cross resistant to most of the clinical drugs [11].
Phenanthrene alkaloids can induce cell apoptosis and inhibits the enzymes which involved in
cell division [12]. The structurally identical molecules are not functional mimics and elicit
action through binding to multiple targets [13]. These alkaloids contain a common
pentacyclic skeleton similar to phenanthrene ring fused with either six-membered
quinolizidine E ring or a five-membered indolizidine E ring [14]. Previous SAR studies
suggest that quinolizidine derivatives are highly potent when compared to indolizidine
containing phenanthrene derivatives [15]. Moreover, tylophorine, antofine and cryptopleurine
are some of the representative alkaloids having broad spectrum of activity from this class
[
16].
Similarly, podophyllotoxin is the natural tetralin lignan mainly present in the plants of
Podophyllum emodi and P. peltatum [17]. Efforts made to use podophyllotoxin as clinical
drug candidate were unsuccessful due to various side effects such as nausea and damage to
normal tissues [18]. However, its role as a lead in anticancer drug research has promoted the
development of potent chemotherapeutic agents like etoposide and teniposide which are
currently used for the treatment of various malignancies such as lymphoma, small cell lung
cancer, kaposis sarcoma and testicular carcinoma [19]. Due to their structural complexity in
the creation of four stereo centres at C ring, it is difficult task to prepare their analogues from
easily available raw materials [20]. However, the stereo chemical complexity can be
minimized by elimination of C-2 and C-3 stereogenic carbons [21]. In this context, a number
of medicinal chemists made considerable efforts towards the development of 4-aza
2

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podophyllotoxin (7) and its derivatives (Figure 1) to retain the similar cytotoxicity to that of
podophyllotoxin [22].
Next, microwave-assisted syntheses of bioactive scaffolds have gained prominent status
in the present medicinal chemistry research [23]. These reactions minimize the reaction time,
facilitate the reaction to proceed fast in a specific manner [24] and significantly reduce the
rate of decomposition of the final product when compared to conventional heating. Similarly,
one-pot protocols enable the chemists to access diverse array of bioactive molecules with
wide substrate scope by minimizing the synthetic steps, greater atom-economy and eliminate
the tedious work-up procedure. In addition, the reactions need to be performed by using
environmentally benign solvents from the green chemistry point of view [25]. Moreover,
exploring synthetic precursors to gain fast access to the diversified library of new scaffolds is
the emerging field wherein a single precursor can be utilized to produce different structural
analogues. A number of methods have been reported for the synthesis of azapodophyllotoxin
derivatives involving the multi-component reactions (MCRs) with aldehyde, amine and a
cyclic diketone; though the nature of the aromatic amine is the prerequisite to this class of
MCRs. However, some of the limitations have been observed for these methods including the
reactivity of various amines, higher temperatures, long reaction times and the use of additives
such as acetic acid, TFA, Et N, and L-proline [26]. In 2015, Jeedimalla and co-workers
3
successfully utilized various aromatic amines to access potent cytotoxic azapodophyllotoxin
derivatives through a multi-component reaction [27]. More recently, Kandil and co-workers
explored the commercially available 9-amino phenanthroline for the one-pot synthesis of
phenanthroline based azapodophyllotoxin derivatives which displayed potent cytotoxicity
[
28]. In this context, we have attempted to explore the tetramethoxy phenanthrene amine as
precursor for the one-pot synthesis of new cytotoxic phenanthrene fused-tetrahydrodibenzo-
acridinones and we anticipate that these new hybrids may show enhanced cytotoxicity
through apoptosis induction.
<Insert Figure 1 here>
2
2
.0 Results and discussion
.1 Chemistry
Phenanthrene fused-tetrahydrodibenzo-acridinones 8a–r were synthesized by a one-pot
method by involving 2,3,6,7-tetramethoxyphenanthren-9-amine (5), dimedone (6a) or
3

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cyclohexane dione (6b) with various substituted aromatic aldehydes 7a–r in refluxing EtOH
under microwave irradiation as shown in Scheme 1. Initially, Knoevenagel condensation of
cyclic diketone and aldehyde results in an intermediate which upon Michael addition with
amine followed by subsequent cyclization affords the desired phenanthrene fused-
dihydrodibenzo-quinolin-ones 8a–r. Herein, we have utilized the microwave-irradiation
technique to this one-pot process in order to increase the yields of the reaction and reduction
of the reaction time. Gratifyingly, the microwave heating remarkably decreased the reaction
time from 3 h (conventional heating) to 20 min (microwave) thus saving the time and
o
increasing the yields up to 91% (entry 6, Table 1). Microwave irradiation at 150 C for 20
min was the optimized reaction conditions which provided high reaction yields. Moreover,
this reaction did not require any base or catalyst and the electron with-drawing, electron
donating, heteroaromatic aldehydes and bicyclic aromatic aldehydes participated effectively
in the reaction. In order to make the protocol further environmental friendly, we have also
carried out the reaction in water, unfortunately the reaction did not proceed (entry 8, Table
1
). This may be due to the low solubility of phenanthrene amine in water. The desired final
products were collected by simple filtration followed by recrystallization and without using
any column chromatographic purification. 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 obtained by using six steps sequence according to the reported
procedure [29]. Initially, Perkin condensation of 2 with 1 affords 2,3-bis(3,4-
dimethoxyphenyl)acrylic acid which was further esterified and then intramolecular oxidative
cyclization with m-CPBA/TFA provides the corresponding ester 3. Later, this ester
functionality was converted to acyl hydrazide followed by diazotization using NaNO /HCl-
2
H O affords the acyl azide as a key intermediate. Upon Curtius rearrangement reaction, the
2
acyl azide is converted in to 2,3,6,7-tetramethoxyphenanthren-9-yl carbamate (4) in overall
6
7% yield. Finally, benzyl-carbamate was deprotected by using TBAF to get the
tetramethoxy phenanthrene amine (5) as key precursor for this one-pot method. All the
1
13
synthesized compounds (8a–r) were successfully characterized by H,
C NMR
1
spectroscopies and HRMS. The H NMR of compound 8a showed a sharp singlet of NH
proton at δ 9.05 ppm. The two doublets 2.19 and 1.99 correspond to the methylenic protons
adjacent to the dimethyl substitution. Protons of -CH - which is adjacent to carbonyl group
2
resonated as multiplet around 2.63 and 2.61 ppm. The two singlets of three protons each at δ
0
.98 and 0.79 belong to the dimethyl groups of cyclic ketone of ‘D’ ring. Proton attached to
tertiary carbon is detected as singlet at δ 5.65 ppm. Characteristic methoxy protons appeared
4

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1
3
as a singlet at δ 3.95–3.74 ppm. Similarly, in C NMR spectrum of compound 8a, the
carbonyl carbon appeared at δ 193.8 ppm. The two methyl carbons resonated around δ 26.8
and 29.6 ppm. The signal such as two -CH - of the ‘D’ ring appeared at δ 36.8 and 50.6 ppm.
2
Tertiary carbon showed a characteristic peak of δ 40.7 ppm and all the aromatic carbons
1
13
resonates around δ 151.4–103.6 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.
<Insert Table 1 here>
<Insert Scheme 1 here>
2
2
.2 Pharmacology
.2.1 In vitro cytotoxicity study
The new phenanthrene fused-tetrahydrodibenzo-acridinone derivatives 8a–r were tested
for their in vitro cytotoxic potential against selected human cancer cell lines of cervical
(
HeLa), prostate (PC-3), fibro sarcoma (HT-1080), ovarian (SKOV-3) and also a normal
Hek-293T) kidney cell line by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
(
bromide (MTT) assay [30]. The IC (µM) values (concentration required to inhibit 50% of
5
0
the cancer cells) of the tested compounds and the positive control drug cisplatin are listed in
Table 2. It was observed from the cytotoxicity results that derivatives 8l and 8m displayed
potent cytotoxicity (< 1 µM) against SKOV-3 ovarian cancer cell line with an IC value of
5
0
0
.61±0.08 and 0.24±0.05 µM respectively. The derivatives 8l and 8m were effective on all of
the tested cancer cell lines below 10 µM. In detailed analysis, majority of the compounds
such as 8i, 8k, 8l, 8m, 8o, 8q and 8r have showed significant cytotoxicity (< 10 µM) on the
entire tested cancer cell lines which are not having geminal methyl substitution in the
hexocyclic ‘D’ ring. Interestingly, one compound 8h having dimethyl substitution in ‘D’ ring
and a syringal group (3,5-dimethoxy-4-hydroxy substitution) as ‘E” ring displayed more
cytotoxicity (< 10 µM) on all cancer cells. All the new compounds were adequately active on
fibrosarcoma (HT-1080) and ovarian (SKOV-3) cancer cell lines. Except derivatives 8a, 8c,
8
d and 8j, remaining all derivatives was active on the cervical (HeLa) and prostate (PC-3)
cancer cell lines. Compound 8m with unsubstituted hexocyclic ‘D’ ring with a 3-
trifluoromethyl phenyl as ‘E’ ring proved to be the best potent compound among all and
displayed IC of 0.24±0.05 µM against SKOV-3 ovarian cancer cell line thus indicating the
5
0
crucial role of fluorine in binding to the biomolecular target. Compound 8i with a 3,4-
5

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methylene dioxy substitution on aromatic E ring was found to be active (< 10 µM) on all the
cancer cell lines with highest potency (IC 1.35±0.24 µM) on HeLa (cervical cancer) cells.
5
0
Compounds 8e, 8h, 8i, 8k, 8l, 8n, 8o, 8q and 8r displayed cytotoxicity below 10 µM against
PC-3 prostate cancer cells, among them compound 8r bearing bicyclic napthyl ‘E’ ring being
the most potent showed IC 5.31±0.23 µM. Further, cytotoxicity of all these compounds was
50
evaluated on normal cell line Hek-293T (human embryonic kidney) to determine the
selectivity index (ratio of cytotoxicity of normal cell line to that of cancer cell line) which is a
prerequisite for a lead chemotherapeutic agent. All the tested compounds displayed lower
cytotoxicities on normal kidney cells (Hek-293T) when compared to the cancer cells. Hence,
the compounds are safer towards the normal cells. The most potent compound showed IC50
value of 18.5±0.89 µM against (Hek-293T) and its selectivity index is approximately 77.0
against SKOV-3 ovarian cancer cells. After the careful analysis of cytotoxicity results, it
could be observed that methyl substituted ‘D’ ring analogues are comparatively less potent
than that of the derivatives with unsubstituted ‘D’ ring. On the other hand, phenyl ‘E’ ring is
favourable compared to that of the bicyclic and heterocyclic ‘E’ ring. Bulky electron donating
groups such as methoxy, methyl, dimethoxy and trimethoxy substitutions on the ‘E’ ring are
more favourable and displayed a greater cytotoxicity when compared to the halogen (Br and
Cl) containing derivatives. On the basis of all these observations, a general Structural
Activity Relation (SAR) was established and presented in Figure 2. Based on the interesting
cytotoxicity results, one of the potent compounds 8m was carried to further studies in detail
like cancer cell growth inhibition and apoptosis induction.
<Insert Table 2 here>
<Insert Figure 2 here>
2
.2.2 Effect on F-actin polymerisation
Membrane protrusions are responsible for the motility of cancer cells. Polymerisation of
actin filaments initiates the ordered arrangement of membrane protrusions and thereby
triggers cancer cell motility and stress fibre assembly [31]. Hence, it is very important to
study the effect of potent compound 8m on stress fibre formation and F-actin polymerisation.
The assembling of actin cytoskeleton in SKOV-3 cancer cells was studied by staining the
cells with rhodamine phalloidin, a red fluorescent dye which specifically binds the F-actin
proteins. SKOV-3 cells were treated with 0.1, 0.2 and 0.5 µM of compound 8m followed by
staining with rhodamine-phalloidin. Figure 3 clearly showed that control cells having
6

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numerous F-actin extensions and stress fibres where as compound 8m treatment led to the
stress fibre disruption around the nucleus and F-actin extensions were also not prominent.
This indicates that compound 8m inhibits the migratory potential of SKOV-3 cancer cells by
disrupting F-actin assembly.
<Insert Figure 3 here>
2
.2.3 Cell cycle analysis
Majority of the chemotherapeutic agents display their growth inhibitory potential by arresting
specific phase of the cell cycle [32]. Thus the blockade of cell cycle progression by cytotoxic
agents has a crucial role to develop into a potential chemotherapeutic agent. From in vitro
screening results, it is evident that compound 8m displayed potent cytotoxicity against
SKOV-3 ovarian cancer cells. Therefore, we studied the effect of compound 8m on
distribution of cells in various cell cycle phases by flow cytometric analysis [33]. SKOV-3
cells were treated with compound 8m at 0.1, 0.2, and 0.5 µM, for 24 h, and ethanol fixed
cells were stained with propidium iodide and further subjected to flow cytometry. As
observed from Figure 4A and 4B the ratio of cells in G2/M phase from 14.7% in control
raised to 22.6% at 0.1 µM, 33.5% at 0.2 µM, and 37.9% at 0.5 µM and simultaneous
decreased number of cells in G0/G1 phase. Therefore, these results clearly revealed that
compound 8m effectively exerts G2/M phase arrest on SKOV-3 cancer cells.
<Insert Figure 4 here>
2
.2.4 Hoechst staining
Condensation of chromatin and DNA fragmentation are the important characteristics of
apoptosis [34]. In order to know the apoptosis inducing ability of compound 8m on SKOV-3
cells, Hoechst nuclear staining was performed. Hoechst 33242 is a nuclear staining dye which
permeates through the cell wall and stains the live nucleus as light blue where as the
apoptotic cells are stained bright blue due to the presence of condensed chromatin. SKOV-3
cells were stained by Hoechst 33242 followed by treatment with the compound 8m at 0.1 ,
0
.2 and 0.5 µM. Fluorescence microscope was used to observe the altered morphological
changes in SKOV-3 cells. From Figure 5, it was clearly evident that control cells nuclei were
intact whereas compound 8m treated cells showed condensed chromatin, fragmented nuclei,
pyknotic and horse shoe shaped nuclei indicating the progress of apoptosis.
7

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<Insert Figure 5 here>
2
.2.5 Mitochondrial membrane potential staining
Further to investigate the apoptosis inducing ability of the compound 8m, mitochondrial
membrane potential alterations were analysed by using a fluorescent probe JC-1. This is a
lipophilic cationic dye that penetrates the cell membrane and accumulates in normally
respiring mitochondria [35]. In the control cells, the JC-1 accumulates in mitochondria and
stains high red fluorescence. However, in case of apoptotic cells, there is a collapsed
mitochondrial potential. Therefore, JC-1 exists in cell cytosol as a monomer and emits green
fluorescence. SKOV-3 cancer cells were treated with 8m at concentrations of 0.1, 0.2 , and
0
.5 µM respectively for 24 h and stained with JC-1 dye, untreated cells were used as control
and the results were depicted in Figure 6.
The J-aggregates and JC-1 monomer were excited at 585 nm and 514 nm respectively and
light emissions were recorded at 570-600 nm (red fluorescence) and 515-545 nm (green
fluorescence). From fluorescence microscopy (Figure 6), it was clearly observed that control
cells without compound 8m were normally red, while 8m treated cells showed strong green
fluorescence that indicates typical apoptotic morphology after 24 h. Therefore, it was clearly
evident from the JC-1 staining that active compound 8m induced apoptosis in SKOV-3
cancer cells.
<Insert Figure 6 here>
2
.2.6 Effect of 8m on reactive oxygen species (ROS) levels
The increased levels of ROS in the mitochondrion may lead to the oxidative damage to the
mitochondrial membrane and results in apoptosis [36]. Thus, we have used the DCFDA
staining method to study the extent of apoptosis through ROS generation. Carboxy-2, 7-
dichlorofluorescein diacetate (Carboxy-H DCFDA) dye which upon cleavage by intracellular
2
esterases oxidizes to highly fluorescent carboxy-2, 7-dichlorofluorescein (Carboxy-DCF).
This can be quantified by spectrofluorometer. As observed from Figure 7, increased ROS
production was observed in 8m treated cells that showed increased green fluorescence 128.5
%
at 0.1 µM, 150.3% at 0.2 µM and 204.76% at 0.5 µM when compared to control (100%).
Quantitative analysis by spectrofluorometry revealed that the ROS levels increased 2-times
higher when compared to that of control cells. This study suggests that the cytotoxicity of
compound also dependent on ROS production.
8

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<Insert Figure 7 here>
2
.2.7 Caspase-3 colorimetric assay
Apoptosis is a critical cellular process that regulates the balance between cell proliferation,
growth arrest and cell death. Several chemotherapeutics have been established to induce
apoptosis via a caspase-dependent pathway [37]. Caspases represent a family of cysteinyl
aspartate specific proteases and are responsible for induction of apoptosis. Caspase-3 is an
“effector” caspase associated with the initiation of cell death and is therefore an important
marker of the cells entry in to an apoptotic cascade pathway. The caspase-3 activity on
SKOV-3 cancer cells upon treating with 8m was measured calorimetrically at an absorbance
of 405 nm. As observed from Figure 8, caspase-3 activity (fold increase) was observed to be
1
.58 at 0.1 µM, 2.45 at 0.2 µM, and 3.78 at 0.5 µM respectively. Caspase activity in control
cells considered as 1 and the caspase activity was increased to 3.7 folds at 0.5 µM in
comparison to control. Therefore, the results clearly revealed that compound 8m effectively
induced caspase mediated cell apoptosis in SKOV-3 cancer cells.
<Insert Figure 8 here>
2
.2.8 Tumor spheroid growth inhibition
Mono layer culture cells are the most commonly used models to screen the antitumor activity
of new drugs. However, this model is devoid of dense extracellular-matrix, three
dimensionality, heterogeneity and penetration barriers observed in vivo in solid tumors. The
use of three dimensional (3D) models has been assumed as an effective approach to
overcome the limitations of the traditional two-dimensional (2D) systems. 3D spheroids are
good mimics of in vivo tumor environment in in vitro study and provide more reliable
information than 2D cultures [38]. In previous studies, we have studied the effectiveness of
compound 8m against SKOV-3 cells in 2D culture. Therefore, in order to know the potential
effect of compound 8m on solid tumors, we have developed 3D spheroids of SKOV-3 cancer
cells and treated with compound at 0.1, 0.2 and 0.5 µM concentrations for 72 h. From the
Figure 9, it was quite interesting to observe that the spheroid forming capability of the cells
treated with 0.5 µM of active compound is suppressed to the great extent after 3 days (72 h).
<Insert Figure 9 here>
9

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3
.0 Conclusion
In summary, the present work demonstrates the microwave-assisted one-pot synthesis of
phenanthrene fused-tetrahydrodibenzo-acridinone derivatives. The present synthetic strategy
provided quick assembly of three different components in to a single complex molecule. This
method also offered advantages like faster reaction times, high yields, wide substrate scope,
atom-economy, and eliminates the chromatographic purification process. Moreover, ethanol
was used as green solvent for this one-pot reaction. The synthesized compounds were
evaluated for their in vitro cytotoxic potential against various selected human cancer cell
lines and also a normal kidney cell line. From in vitro screening results, it was observed that
compound 8m displayed potent cytotoxicity against SKOV-3 ovarian cancer cells with an
IC value of 0.24±0.05 µM. Compound 8m induced disruption of F-actin filaments and
5
0
inhibited cancer cell motility. Flow cytometric analysis indicated that the compound blocked
the G2/M phase of the cell cycle. Moreover, the compound also leads to decrease in the
number of viable cells and increased apoptotic features as shown in case of Hoechst staining.
8
m also caused the collapse of mitochondrial membrane potential as evident from JC-1
staining and also enhanced the generation of reactive oxygen species. The compound has
increased the caspase-3 activation by 3.7 fold thereby supporting the strong apoptosis.
Moreover, 8m has greatly reduced the in vitro 3D-spheroid inhibition in SKOV-3 ovarian
cancer cells. Overall, all the studies indicated that these compounds are potentially inhibiting
the ovarian cancer cells and may be developed as potent chemotherapeutic agents for the
treatment of malignancies in the drug discovery.
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 solvent is using tetramethyl silane (TMS) as
3
1
13
the 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), dd (double doublet), t (triplet), q (quartet), and m (multiplet). Coupling
constant (J) values are reported in hertz (Hz). HRMS were determined with Agilent QTOF
1
0

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mass spectrometer 6540 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
o
Heidolph rotary evaporator below 45 C.
4
.1.1 General reaction procedure for the synthesis of phenanthrene fused-tetrahydrodibenzo-
acridinones 8a–r
A mixture of 2,3,6,7-tetramethoxy phenanthrene-9-amine (5, 0.28 mmol), cyclohexane dione
(6a, 0.28 mmol) or dimethyl cyclo hexane dione (6b, 0.28 mmol) and substituted aldehydes
o
(
7a–r, 0.28 mmol) in EtOH (3 mL) were heated through microwave irradiation at 150 C for
0
.3 h. After completion of the reaction, the reaction mixture was allowed to cool to room
temperature and the precipitated products were collected by vacuum filtration and washed
with water followed by recrystallization from ethanol to afford pure compounds 8a–r in 89-
1
13
9
5% yields. All the synthesized compounds were thoroughly characterized by H NMR, C
NMR and HRMS (ESI).
4
.1.1.1 Microwave irradiation experiments
Microwave irradiation was carried out in a monowave 300 single-mode microwave reactor
from Anton paar GmbH (Graz, Austria). The reaction temperature was monitored by an
external infra red (IR) sensor housed in the side walls of the microwave cavity measuring the
surface temperature of the reaction vessel. Reaction times refer to the hold time at the desired
set temperature and not to the total irradiation time. Reusable 10 mL G10 pyrex vials were
sealed with PEEK snap caps and standard PTFE coated silicone septa. Reaction cooling was
achieved by compressed air automatically after the heating period has elapsed.
4
.1.1.2
14-(4-Chlorophenyl)-2,3,6,7-tetramethoxy-11,11-dimethyl-10,11,12,14-
tetrahydrodibenzo[a,c]acridin-13(9H)-one (8a)
o
1
White solid, yield 92%; mp: 273-275 C; H NMR (500 MHz, DMSO-d ): δ 9.05 (s, 1H),
6
7
.93 (s, 1H), 7.87 (s, 1H), 7.78 (s, 1H), 7.20 (t, J = 8.08 Hz, 3H), 7.12 (d, J = 7.93 Hz, 2H),
.65 (s, 1H), 3.95 (s, 6H), 3.87 (s, 3H), 3.74 (s, 3H), 2.63-2.61 (m, 2H), 2.19 (d, J = 16.02
5
1
3
Hz, 1H), 1.99 (d, J = 15.86 Hz, 1H), 0.98 (s, 3H), 0.79 (s, 3H) ppm; C NMR (125 MHz,
DMSO-d ): δ 193.8, 151.4, 149.6, 149.4, 149.1, 147.9, 146.4, 130.4, 130.0, 128.7, 128.2,
6
1
24.7, 124.6, 122.1, 117.2, 112.8, 107.8, 105.2, 104.8, 104.5, 103.6, 56.7, 56.4, 56.3, 55.7,
1
1

ACCEPTED MANUSCRIPT
5
0.6, 40.7, 36.8, 32.6, 29.6, 26.8 ppm; HRMS (ESI): m/z calcd for C H ClNO 558.2042,
3
3
32
5
+
found 558.2050 [M+H] .
4
.1.1.3
14-(3,4-Dimethoxyphenyl)-2,3,6,7-tetramethoxy-11,11-dimethyl-10,11,12,14-
tetrahydrodibenzo[a,c]acridin-13(9H)-one (8b)
o
1
Yellow solid, yield 90%; mp: > 300 C; H NMR (500 MHz, DMSO-d ): δ 9.09 (s, 1H), 8.01
6
(
s, 1H), 7.95 (s, 1H), 7.86 (s, 1H), 7.39 (s, 1H), 7.04 (s, 1H), 6.69 (t, J = 8.54 Hz, 2H), 5.66
s, 1H), 4.03 (s, 6H), 3.96 (s, 3H), 3.85 (s, 3H), 3.62 (d, J = 13.42 Hz, 6H), 2.74-2.65 (m,
(
2
H), 2.27 (d, J = 16.02 Hz, 1H), 2.08 (d, J = 15.86 Hz, 1H), 1.07 (s, 3H), 0.91 (s, 3H) ppm;
1
3
C NMR (125 MHz, DMSO-d ): δ 193.9, 150.9, 149.4, 149.3, 149.1, 148.4, 147.8, 147.2,
6
1
1
40.4, 128.4, 124.9, 124.6, 122.1, 120.3, 117.2, 113.6, 112.8, 111.8, 108.5, 105.1, 104.9,
04.7, 103.4, 56.6, 56.5, 56.4, 56.3, 55.9, 55.8, 55.7, 50.7, 36.7, 32.6, 29.7, 26.8 ppm; HRMS
+
(
ESI): m/z calcd for C H NO 584.2643, found 584.2657 [M+H] .
3
5
37
7
4
.1.1.4
2,3,6,7-Tetramethoxy-11,11-dimethyl-14-(4-(trifluoromethyl)phenyl)-10,11,12,14-
tetrahydrodibenzo[a,c]acridin-13(9H)-one (8c)
o
1
Off white solid, yield 92%; mp: 186-188 C; H NMR (500 MHz, DMSO-d ): δ 9.19 (s, 1H),
6
8
3
.02 (s, 1H), 7.96 (s, 1H), 7.89 (s, 1H), 7.52 (s, 4H), 7.29 (s, 1H), 5.84 (s, 1H), 4.04 (s, 6H),
.96 (s, 3H), 3.83 (s, 3H), 2.74-2.69 (m, 2H), 2.28 (d, J = 15.86 Hz, 1H), 2.08 (d, J = 16.02
1
3
Hz, 1H), 1.08 (s, 3H), 0.87 (s, 3H) ppm; C NMR (125 MHz, DMSO-d ): δ 193.8, 151.8,
6
1
1
3
5
51.5, 149.6, 149.4, 149.2, 147.9, 129.0, 128.7, 126.8, 126.5, 125.8, 125.3, 124.8, 124.5,
23.7, 122.1, 117.0, 112.5, 107.5, 105.1, 104.9, 104.4, 103.5, 56.7, 56.4, 56.3, 55.7, 50.6,
7.5, 32.7, 29.5, 26.8 ppm; HRMS (ESI): m/z calcd for C H F NO 592.2305, found
3
4
32
3
5
+
92.2312 [M+H] .
4
.1.1.5
14-(4-Isopropylphenyl)-2,3,6,7-tetramethoxy-11,11-dimethyl-10,11,12,14-
tetrahydrodibenzo[a,c]acridin-13(9H)-one (8d)
o
1
Brown solid, yield 95%; mp: 175-177 C; H NMR (500 MHz, DMSO-d ): δ 9.09 (s, 1H),
6
8
.01 (s, 1H), 7.95 (s, 1H), 7.87 (s, 1H), 7.39 (s, 1H), 7.23 (d, J = 8.24 Hz, 2H), 7.00 (d, J =
.08 Hz, 2H), 5.68 (s, 1H), 4.04 (d, J = 2.13 Hz, 6H), 3.96 (s, 3H), 3.85 (s, 3H), 2.74-2.69
8
(m, 3H), 2.25 (d, J = 16.02 Hz, 1H), 2.09 (d, J = 16.02 Hz, 1H), 1.07 (d, J = 6.25 Hz, 9H),
1
3
0
.92 (s, 3H) ppm; C NMR (125 MHz, DMSO-d ): δ 193.8, 151.1, 149.5, 149.4, 149.0,
6
1
47.8, 145.8, 145.1, 128.4, 128.2, 126.1, 124.8, 124.5, 122.1, 117.2, 113.6, 108.4, 105.1,
1
2

ACCEPTED MANUSCRIPT
1
04.8, 104.7, 103.4, 56.6, 56.4, 56.3, 55.7, 50.7, 40.8, 36.8, 33.3, 32.7, 29.5, 27.2, 24.3, 24.1
+
ppm; HRMS (ESI): m/z calcd for C H NO 566.2901, found 566.2906 [M+H] .
3
6
39
5
4
.1.1.6
2,3,6,7-Tetramethoxy-11,11-dimethyl-14-(3,4,5-trimethoxyphenyl)-10,11,12,14-
tetrahydrodibenzo[a,c]acridin-13(9H)-one (8e)
o
1
Brown solid, yield 90%; mp: 254-256 C; H NMR (500 MHz, DMSO-d ): δ 9.15 (s, 1H),
6
8
.02 (s, 1H), 7.97 (s, 1H), 7.86 (s, 1H), 7.44 (s, 1H), 6.63 (s, 2H), 5.67 (s, 1H), 4.04 (d, J =
.57 Hz, 6H), 3.97 (s, 3H), 3.89 (s, 3H), 3.59 (s, 6H), 3.53 (s, 3H), 2.74-2.70 (m, 2H), 2.24
4
1
3
(
d, J = 16.02 Hz, 1H), 2.11 (d, J = 16.02 Hz, 1H), 1.08 (s, 3H), 0.95 (s, 3H) ppm; C NMR
(
125 MHz, DMSO-d ): δ 193.9, 152.7, 151.2, 149.5, 149.3, 149.1, 147.9, 143.5, 136.1,
6
1
5
28.4, 124.9, 124.6, 122.1, 117.2, 113.5, 108.3, 106.0, 105.1, 104.8, 104.7, 103.4, 60.3, 56.6,
6.4, 56.3, 56.2, 55.7, 50.7, 40.8, 37.5, 32.7, 29.7, 35.7 ppm; HRMS (ESI): m/z calcd for
+
C H NO 614.2748, found 614.2761 [M+H] .
36
39
8
4
.1.1.7
14-(4-Bromophenyl)-2,3,6,7-tetramethoxy-11,11-dimethyl-10,11,12,14-
tetrahydrodibenzo[a,c]acridin-13(9H)-one (8f)
o
1
White solid, yield 94%; mp: 193-195 C; H NMR (500 MHz, CDCl ): δ 7.83 (s, 1H), 7.72
3
(s, 1H), 7.30 (s, 1H), 7.26-7.21 (m, 5H), 5.86 (s, 1H), 4.12 (d, J = 7.01 Hz, 6H), 4.04 (s, 3H),
13
3
.89 (s, 3H), 2.56-2.52 (m, 2H), 2.34-2.24 (m, 2H), 1.10 (s, 3H), 0.94 (s, 3H) ppm; C
NMR (125 MHz, CDCl ): δ 194.8, 150.0, 149.3, 149.2, 147.9, 145.2, 131.1, 129.8, 127.2,
3
1
4
6
24.8, 122.1, 119.7, 116.3, 113.5, 109.1, 104.6, 104.0, 103.5, 100.7, 56.3, 56.1, 55.7, 50.5,
2.0, 37.0, 32.7, 29.4, 27.0 ppm; HRMS (ESI): m/z calcd for C H BrNO 602.1537, found
3
3
32
5
+
02.1541 [M+H] .
4
.1.1.8 14-(4-Hydroxy-3-methoxyphenyl)-2,3,6,7-tetramethoxy-11,11-dimethyl-10,11,12,14-
tetrahydrodibenzo[a,c]acridin-13(9H)-one (8g)
o
1
Yellow solid, yield 91%; mp: 285-287 C; H NMR (500 MHz, CDCl ): δ 7.87 (s, 1H), 7.75
3
(s, 1H), 7.45 (s, 1H), 7.20 (s, 1H), 7.11 (s, 1H), 6.95 (Brs, 1H), 6.69 (dd, J = 7.93, 24.26 Hz,
2
H), 5.87 (s, 1H), 5.45 (Brs, 1H), 4.14 (d, J = 9.91 Hz, 6H), 4.07 (s, 3H), 3.94 (s, 3H), 3.78
13
(
s, 3H), 2.56 (s, 2H), 2.35-2.27 (m, 2H), 1.12 (s, 3H), 0.99 (s, 3H) ppm; C NMR (125
MHz, CDCl ): δ 195.2, 149.3, 149.2, 149.1, 149.0, 147.8, 146.0, 143.5, 138.6, 127.1, 125.2,
3
1
5
24.7, 122.0, 120.7, 116.3, 114.2, 114.0, 111.2, 110.1, 105.0, 104.2, 103.5, 100.3, 56.3, 56.1,
5.8, 55.7, 50.8, 42.2, 36.8, 32.7, 29.6, 29.4, 27.1 ppm; HRMS (ESI): m/z calcd for
1
3

ACCEPTED MANUSCRIPT
+
C H NO 570.2486, found 570.2492 [M+H] .
34
35
7
4
1
.1.1.9
14-(4-Hydroxy-3,5-dimethoxyphenyl)-2,3,6,7-tetramethoxy-11,11-dimethyl-
0,11,12,14-tetrahydrodibenzo[a,c]acridin-13(9H)-one (8h)
o
1
Yellow solid, yield 90%; mp: 216-218 C; H NMR (500 MHz, CDCl ): δ 7.78 (s, 1H), 7.67
3
(s, 1H), 7.37 (s, 1H), 7.13 (s, 1H), 6.92 (Brs, 1H), 6.54 (s, 2H), 5.78 (s, 1H), 4.06 (d, J = 7.62
Hz, 6H), 3.98 (s, 3H), 3.86 (s, 3H), 3.61 (s, 6H), 2.48 (s, 2H), 2.22-2.10 (m, 2H), 1.02 (s,
1
3
3
1
1
H), 0.91 (s, 3H) ppm; C NMR (125 MHz, CDCl ): δ 195.1, 149.3, 149.2, 149.1, 149.0,
3
47.9, 146.7, 137.7, 132.9, 127.1, 125.3, 124.8, 122.0, 116.2, 114.0, 109.9, 105.3, 105.0,
04.2, 103.5, 100.4, 56.3, 56.2, 56.1, 56.0, 55.7, 50.7, 42.2, 37.2, 32.7, 29.4, 26.9 ppm;
+
HRMS (ESI): m/z calcd for C H NO 600.2592, found 600.2598 [M+H] .
3
5
37
8
4
.1.1.10
14-(Benzo[d][1,3]dioxol-5-yl)-2,3,6,7-tetramethoxy-11,11-dimethyl-10,11,12,14-
tetrahydrodibenzo[a,c]acridin-13(9H)-one (8i)
o
1
Off white solid, yield 89%; mp: 201-203 C; H NMR (500 MHz, DMSO-d ): δ 9.10 (s, 1H),
6
8.01 (s, 1H), 7.96 (s, 1H), 7.86 (s, 1H), 7.34 (s, 1H), 6.78 (s, 1H), 6.75 (d, J = 8.08 Hz, 1H),
6.67 (d, J = 8.08 Hz, 1H), 5.87 (s, 1H), 5.82 (s, 1H), 5.67 (s, 1H), 4.04 (d, J = 1.83 Hz, 6H),
3.96 (s, 3H), 3.84 (s, 3H), 2.72-2.68 (m, 2H), 2.26 (d, J = 16.02 Hz, 1H), 2.10 (d, J = 15.86
1
3
Hz, 1H), 1.07 (s, 3H), 0.91 (s, 3H) ppm; C NMR (125 MHz, DMSO-d ): δ 194.0, 151.1,
6
1
1
4
5
49.5, 149.4, 149.1, 147.9, 147.2, 145.3, 141.7, 128.5, 124.7, 124.6, 122.1, 121.1, 117.1,
13.4, 108.5, 108.3, 108.0, 105.1, 104.8, 104.7, 103.4, 101.0, 56.6, 56.4, 56.3, 55.7, 50.7,
0.7, 36.8, 32.6, 29.6, 26.9 ppm; HRMS (ESI): m/z calcd for C H NO 568.2330, found
3
4
33
7
+
68.2334 [M+H] .
4
.1.1.11
14-(3,4-Dimethoxyphenyl)-2,3,6,7-tetramethoxy-10,11,12,14-
tetrahydrodibenzo[a,c]acridin-13(9H)-one (8j)
o
1
Yellow solid, yield 92%; mp: 265-267 C; H NMR (500 MHz, CDCl ): δ 7.86 (s, 1H), 7.73
3
(s, 1H), 7.40 (s, 1H), 7.20 (s, 1H), 7.12 (d, J = 1.52 Hz, 1H), 6.99 (s, 1H), 6.73-6.71 (m, 1H),
6
.58 (d, J = 8.39 Hz, 1H), 5.92 (s, 1H), 4.13 (s, 3H), 4.09 (s, 3H), 4.05 (s, 3H), 3.90 (s, 3H),
13
3
.76 (s, 3H), 3.72 (s, 3H), 2.70 (s, 2H), 2.51-2.37 (m, 2H), 2.04-2.03 (m, 2H) ppm; C
NMR (125 MHz, CDCl ): δ 195.3, 151.1, 149.2, 149.1, 148.5, 147.8, 147.0, 139.5, 127.2,
3
1
5
25.2, 124.8, 122.0, 120.1, 116.3, 114.1, 112.0, 111.2, 110.8, 105.0, 104.1, 103.4, 100.6,
6.4, 56.1, 55.9, 55.7, 55.6, 37.0, 36.7, 28.5, 21.1 ppm; HRMS (ESI): m/z calcd for
1
4

ACCEPTED MANUSCRIPT
+
C H NO 556.2330, found 556.2330 [M+H] .
33
33
7
4
.1.1.12
2,3,6,7-Tetramethoxy-14-(p-tolyl)-10,11,12,14-tetrahydrodibenzo[a,c]acridin-
1
3(9H)-one (8k)
o
1
Yellow solid, yield 89%; mp: 290-292 C; H NMR (500 MHz, CDCl ): δ 7.84 (s, 1H), 7.71
3
(s, 1H), 7.41 (s, 1H), 7.27 (s, 1H), 7.25 (s, 1H), 7.15 (Brs, 1H), 6.92 (d, J = 7.78 Hz, 2H),
5
2
1
1
.91 (s, 1H), 4.12 (s, 3H), 4.09 (s, 3H), 4.03 (s, 3H), 3.89 (s, 3H), 2.72 (s, 2H), 2.49-2.35 (m,
1
3
H), 2.17 (s, 3H), 2.02 (s, 2H) ppm; C NMR (125 MHz, CDCl ): δ 195.1, 151.7, 149.2,
3
49.1, 147.8, 143.7, 135.3, 128.8, 128.1, 127.1, 125.1, 124.7, 122.1, 116.4, 114.5, 111.2,
04.9, 104.0, 103.4, 100.9, 56.4, 56.0, 55.7, 37.0, 36.9, 28.4, 21.1, 20.9 ppm; HRMS (ESI):
+
m/z calcd for C H NO 510.2275, found 510.2275 [M+H] .
3
2
31
5
4
.1.1.13
2,3,6,7-Tetramethoxy-14-(4-methoxyphenyl)-10,11,12,14-
tetrahydrodibenzo[a,c]acridin-13(9H)-one (8l)
o
1
Off white solid, yield 90%; mp: > 300 C; H NMR (500 MHz, CDCl ): δ 7.84 (s, 1H), 7.72
3
(s, 1H), 7.40 (s, 1H), 7.29 (s, 1H), 7.27 (s, 1H), 7.24 (s, 1H), 7.09 (s, 1H), 6.65 (d, J = 8.69
Hz, 2H), 5.90 (s, 1H), 4.12 (s, 3H), 4.09 (s, 3H), 4.04 (s, 3H), 3.89 (s, 3H), 3.65 (s, 3H),
1
3
2
.71-2.66 (m, 2H), 2.49-2.39 (m, 2H), 2.04-1.99 (m, 2H) ppm; C NMR (125 MHz,
CDCl ): δ 193.8, 156.3, 151.8, 148.0, 147.9, 147.7, 146.4, 138.7, 128.0, 127.2, 123.9, 123.4,
3
1
5
5
20.9, 116.2, 112.9, 112.2, 109.1, 103.7, 102.7, 102.6, 102.4, 102.3, 55.7, 55.1, 55.0, 54.6,
3.9, 36.0, 35.2, 26.5, 20.2 ppm; HRMS (ESI): m/z calcd for C H NO 526.2224, found
3
2
31
6
+
26.2227 [M+H] .
4
.1.1.14
2,3,6,7-Tetramethoxy-14-(3-(trifluoromethyl)phenyl)-10,11,12,14-
tetrahydrodibenzo[a,c]acridin-13(9H)-one (8m)
o
1
Yellow solid, yield 90%; mp: 279-281 C; H NMR (500 MHz, CDCl ): δ 7.84 (s, 1H), 7.72
3
(s, 1H), 7.69 (s, 1H), 7.56 (d, J = 7.62 Hz, 1H), 7.31 (d, J = 10.68 Hz, 2H), 7.25-7.21 (m,
2
2
H), 5.99 (s, 1H), 4.12 (s, 3H), 4.10 (s, 3H), 4.04 (s, 3H), 3.89 (s, 3H), 2.76-2.71 (m, 2H),
1
3
.49-2.40 (m, 2H), 2.07-1.99 (m, 2H) ppm; C NMR (125 MHz, CDCl ): δ 194.8, 152.9,
3
1
1
5
5
49.1, 148.9, 148.7, 148.0, 147.4, 131.6, 129.8, 129.5, 128.4, 128.2, 125.2, 124.7, 124.6,
24.6, 124.5, 123.0, 122.4, 122.3, 121.9, 116.9, 112.9, 109.4, 104.3, 103.5, 103.4, 103.1,
6.6, 55.9, 55.9, 55.4, 37.3, 36.8, 27.6, 21.0 ppm; HRMS (ESI): m/z calcd for C H F NO
5
3
2
28
3
+
64.1992, found 564.1998 [M+H] .
1
5

ACCEPTED MANUSCRIPT
4
.1.1.15
14-(4-Isopropylphenyl)-2,3,6,7-tetramethoxy-10,11,12,14-
tetrahydrodibenzo[a,c]acridin-13(9H)-one (8n)
o
1
Light green solid, yield 91%; mp: 199-201 C; H NMR (500 MHz, CDCl ): δ 7.84 (s, 1H),
3
7
.72 (s, 1H), 7.43 (s, 1H), 7.28 (d, J = 8.08 Hz, 2H), 7.24 (s, 1H), 7.12 (s, 1H), 6.95 (d, J =
.69 Hz, 2H), 5.92 (s, 1H), 4.12 (s, 3H), 4.08 (s, 3H), 4.04 (s, 3H), 3.89 (s, 3H), 2.75-2.70
8
1
3
(
m, 3H), 2.49-2.36 (m, 2H), 2.02-2.01 (m, 2H), 1.11 (dd, J = 2.28, 6.86 Hz, 6H), ppm; C
NMR (125 MHz, CDCl ): δ 195.4, 151.6, 149.1, 149.0, 147.7, 146.1, 144.0, 128.0, 127.2,
3
1
5
5
26.1, 125.2, 124.7, 122.0, 116.6, 114.5, 111.1, 105.1, 104.0, 103.3, 101.0, 56.3, 56.1, 56.0,
5.7, 37.1, 37.0, 33.5, 28.3, 23.8, 21.1 ppm; HRMS (ESI): m/z calcd for C H NO
5
3
4
35
+
38.2588, found 538.2592 [M+H] .
4
.1.1.16 2,3,6,7-Tetramethoxy-11,11-dimethyl-14-(thiophen-2-yl)-10,11,12,14-
tetrahydrodibenzo[a,c]acridin-13(9H)-one (8o)
o
1
Brown solid, yield 92%; mp: 270-272 C; H NMR (500 MHz, CDCl ): δ 7.86 (s, 1H), 7.77
3
(s, 1H), 7.48 (s, 1H), 7.25 (s, 1H), 6.96 (d, J = 4.57 Hz, 1H), 6.77 (s, 1H), 6.73 (t, J = 3.96
Hz, 1H), 6.26 (s, 1H), 4.14 (s, 3H), 4.08 (s, 6H), 3.94 (s, 3H), 2.61-2.57 (m, 2H), 2.54-2.37
13
(
m, 2H), 1.12 (s, 3H), 1.04 (s, 3H) ppm; C NMR (125 MHz, CDCl ): δ 194.7, 150.2, 150.0,
3
1
1
49.4, 149.3, 149.1, 148.0, 127.0, 126.2, 125.0, 124.8, 124.0, 123.4, 122.0, 116.3, 113.6,
09.0, 104.6, 104.0, 103.4, 100.7, 56.3, 56.1, 55.9, 50.5, 42.0, 32.7, 32.0, 29.5, 27.1 ppm;
+
HRMS (ESI): m/z calcd for C H NO S 530.1996, found 530.2005 [M+H] .
3
1
31
5
4
.1.1.17
2,3,6,7-Tetramethoxy-11,11-dimethyl-14-(3-methylthiophen-2-yl)-10,11,12,14-
tetrahydrodibenzo[a,c]acridin-13(9H)-one (8p)
o
1
Off white solid, yield 92%; mp: > 300 C; H NMR (500 MHz, CDCl ): δ 7.83 (s, 1H), 7.75
3
(
(
(
s, 1H), 7.42 (s, 1H), 7.25 (s, 1H), 6.86 (d, J = 5.03 Hz, 1H), 6.61 (d, J = 5.03 Hz, 1H), 6.17
s, 1H), 4.14-4.09 (m, 6H), 4.07 (s, 3H), 4.04 (s, 3H), 2.63 (s, 3H), 2.57 (s, 2H), 2.38-2.29
13
m, 2H), 1.11 (s, 3H), 1.02 (s, 3H) ppm; C NMR (125 MHz, CDCl ): δ 194.7, 149.4, 149.3,
3
1
1
49.2, 149.1, 147.9, 145.2, 131.4, 129.4, 126.6, 125.0, 124.7, 122.2, 122.0, 116.3, 114.8,
09.2, 104.2, 104.0, 103.6, 100.7, 56.3, 56.2, 56.1, 56.0, 42.1, 32.6, 30.8, 29.5, 27.0, 14.6
+
ppm; HRMS (ESI): m/z calcd for C H NO S 544.2152, found 544.2219 [M+H] .
3
2
33
5
4
.1.1.18
2,3,6,7-Tetramethoxy-14-(thiophen-2-yl)-10,11,12,14-
tetrahydrodibenzo[a,c]acridin-13(9H)-one (8q)
1
6

ACCEPTED MANUSCRIPT
o
1
Off white solid, yield 93%; mp: 258-260 C; H NMR (500 MHz, CDCl ): δ 7.84 (s, 1H),
3
7
.75 (s, 1H), 7.45 (s, 1H), 7.33 (s, 1H), 6.95 (d, J = 4.73 Hz, 1H), 6.74-6.70 (m, 2H), 6.26 (s,
1
2
1
1
H), 4.12 (s, 3H), 4.06 (Brs, 6H), 3.92 (s, 3H), 2.74-2.71 (m, 2H), 2.57-2.53 (m, 1H), 2.45-
1
3
.43 (m, 1H), 2.06 (s, 2H) ppm; C NMR (125 MHz, CDCl ): δ 195.1, 151.8, 150.4, 149.3,
3
49.2, 149.1, 147.9, 127.0, 126.2, 125.0, 124.8, 124.1, 123.5, 122.0, 116.3, 113.6, 110.3,
04.6, 104.0, 103.4, 100.8, 56.3, 56.0, 55.9, 36.9, 31.9, 28.3, 21.2 ppm; HRMS (ESI): m/z
+
calcd for C H NO S 502.1683, found 502.1685 [M+H] .
2
9
27
5
4
.1.1.19
2,3,6,7-Tetramethoxy-14-(naphthalen-1-yl)-10,11,12,14-
tetrahydrodibenzo[a,c]acridin-13(9H)-one (8r)
o
1
Yellow solid, yield 89%; mp: 284-286 C; H NMR (500 MHz, CDCl ): δ 9.53 (s, 1H), 7.82
3
(
s, 1H), 7.75 (d, J = 8.08 Hz, 1H), 7.66 (d, J = 10.98 Hz, 2H), 7.55 (d, J = 7.93 Hz, 1H), 7.42
t, J = 7.47 Hz, 1H), 7.34 (d, J = 7.01 Hz, 1H), 7.28 (s, 1H), 7.24 (s, 2H), 7.15 (t, J = 7.62 Hz,
(
1
H), 6.65 (s, 1H), 4.11 (s, 3H), 4.06 (s, 3H), 3.97 (s, 3H), 3.39 (s, 3H), 2.70 (s, 2H), 2.41-
1
3
2
1
1
5
5
.32 (m, 2H), 1.97-1.91 (m, 2H) ppm; C NMR (125 MHz, CDCl ): δ 195.6, 150.8, 149.2,
3
49.1, 149.0, 147.6, 145.3, 133.3, 130.8, 128.4, 127.5, 126.9, 126.8, 126.1, 125.8, 125.5,
25.3, 125.2, 124.6, 121.8, 116.8, 116.4, 112.1, 105.1, 104.1, 103.3, 100.6, 56.3, 56.1, 56.0,
5.8, 37.0, 32.7, 28.7, 20.9 ppm; HRMS (ESI): m/z calcd for C H NO 546.2275, found
3
5
31
5
+
46.2278 [M+H] .
4
.2 Biology
4
.2.1. Cell Cultures
Cervical (HeLa), prostate (PC-3), fibrosarcoma (HT-1080), ovarian (SKOV-3) and normal
kidney cells (HeK-293T) were procured from ATCC (American Type Cell culture
Collection), Maryland USA and were grown in suitable DMEM (Dulbecco modified Eagle
medium, Sigma), RPMI (Rosewell Park Memorial Institute, Sigma) supplemented with 10%
foetal bovine serum with 1X stabilized antibiotic-antimycotic solution (Sigma) in a CO2
incubator at 37 °C with 5% CO and 90% relative humidity. Cells were treated with 0.05%
2
trypsin/1mM EDTA solution for further passage.
4
.2.2 In vitro cytotoxic activity
MTT reduction assay was performed to determine the cytotoxicity for all the new compounds
4
8
a-r. 3x10 cells per well were seeded in 100 µL respective media, supplemented with 10%
1
7

ACCEPTED MANUSCRIPT
FBS in each well of 96-well microculture plates and incubated at 37 °C for 24 h, in a CO2
incubator. Samples were diluted to the required concentrations in culture medium, were
added to the wells with respective vehicle control. After incubating for 48 h, 100 µL MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (5 mg/mL) was added to all the
plates and allowed for 4 h incubation in dark. After 4h of incubation period, the MTT media
was removed and DMSO (100 µL) was added to each well to dissolve the formazan crystals.
The absorbance was recorded on a micro-plate reader at 570 nm wavelength and at a
reference wave length of 630 nm. The % inhibition was calculated as 100 –[(Mean OD of
treated cells x 100)/Mean OD of vehicle treated cells (DMSO)]. All the experiments and IC50
values were computed by using Probit Software.
4
.2.3 F-actin staining
6
SKOV-3 cells (1 × 10 cells/well) were grown on cover slips in 6 well plates for 24 h and
then incubated with 0.1 µM, 0.2 µM and 0.5 µM of compound 8m for 24 h. After the
compound treatment, cells were washed with PBS and fixed with 4% para-formaldehyde in
PBS. Cells were incubated with rhodamine-phalloidin for actin staining and Hoechst 33242
for nucleus staining. After washing thrice with PBS, cells were mounted with ProLong Gold
anti-fade reagent (Molecular Probes, Eugene, OR) on microscopic slide and were visualized
by confocal fluorescence microscopy (Nikon). Images were captured using 20X
magnification objective lenses.
4
.2.4 Cell cycle analysis
6
SKOV-3 cells (1 × 10 cells/well) in 6 well plate were treated with 0.1 µM, 0.2 µM and 0.5
µM of compound 8m for 24 h. Cells were collected by trypsinisation, washed with 150 mM
PBS and fixed with 70% ethanol for 30 min at 4 °C. After fixing, cells were again washed
with PBS and stained with 400 µL of propidium iodide staining buffer [PI (200 µg), Triton X
(100 µL), DNAse-free RNAse A (2 mg) in 10 mL of PBS] for 15 min in dark at room
temperature. The samples were then analyzed for propidium iodide fluorescence from 15,000
events by flow cytometry using BD Accuri C6 flow-cytometer.
4
.2.5 Hoechst staining
5
SKOV-3 ovarian cancer cells were grown on coverslips in a 6 well plates (5 x 10 cells/well)
and allowed to adhere for 24 h. The culture medium containing the compound 8m at 0.1, 0.2
and 0.5 µM concentrations were added to cells. After 24 h incubation, culture medium was
1
8

ACCEPTED MANUSCRIPT
o
removed; cells were washed with PBS and fixed with 4% formaldehyde solution at 4 C for
0 min. Then cells were washed twice with PBS and stained with Hoechst 33242 (5 µg/mL)
1
for 30 min at room temp. The excess dye was removed by washing twice with PBS and cell
suspension were mounted on slides and were examined for morphological changes under
fluorescence microscope using 350 nm excitation and 460 nm emission (Biorad,
magnification 20x).
4
.2.6 Measurement of mitochondrial membrane potential (DΨm)
5
SKOV-3 cells were cultured in 6 well plates at a density of 5 × 10 cells/mL and allowed to
adhere overnight. The cells were treated with 0.1, 0.2 and 0.5 µM of compound 8m for 24 h.
After 24 h treatment, the adherent cells were washed with PBS a solution of PBS containing
JC-1 (5 µg/mL, Sigma) was added to each well and further incubated at room temperature for
3
0 minutes. Cells were washed two times with PBS to remove excess dye and were
photographed in red and green channels using a fluorescence microscope at 20x
magnification (Biorad).
4
.2.7 Reactive oxygen Species
5
SKOV-3 cells (2×10 /well) in a 6 well plate were incubated with the 0.1, 0.2 and 0.5 µM
concentrations of the compound 8m for 24 h. After incubation, cells were harvested with
0
1
.05% trypsin-EDTA, washed with PBS and resuspended in 1 mL of PBS buffer containing
0 µM Carboxy-H DCFDA (Thermofischer) at room temperature in the dark for 15 min. The
2
excess dye was removed by washing with PBS, and the cells were immediately analyzed for
green fluorescence using a BDC6-accuri flow cytometer.
4
.2.8 Caspase-3 assay
Caspase-3 activity in SKOV-3 cells was monitored using a colorimetric Caspase-3 Assay Kit
5
(
Thermo Fischer, khz0022). SKOV-3 cells (2×10 cells/well) were treated with 0.1, 0.2 and
0
.5 µM concentrations of the compound 8m for 24 h. Cells were harvested with 0.05%
trypsin-EDTA and were lysed with lysis buffer (50 µL) for 20 min. Cell lysates were
centrifuged at 15000 rpm for 10 min at 4 °C and the supernatant was added into 96-well
plates. Final reaction buffer (50 µL) and caspase-3 substrate (Ac-DEVDpNA 5 µL) were then
added to each well and the plates were incubated at 37 °C for 2 h. Optical density of the
reactions in each well was measured at 405 nm with a plate reader (SpectraMax).
1
9

ACCEPTED MANUSCRIPT
4
.2.9 Spheroid inhibition assay
SKOV-3 cells (5000 cells/well) were seeded in 96 well Ultra Low Attachment round-bottom
plates and allowed to grow for 2 days to form spheroids. The spheroids were treated with 0.1,
0
.2, 0.5 µM concentrations of the gold complexes for a period of 72 h. The growth and
surface area of the spheroids were monitored using phase contrast microscopy after 72 h
treatment.
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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Tables/Figures/Scheme captions
Table 1. Optimization of the reaction conditions to phenanthrene fused-tetrahydrodibenzo-
acridinone 8a
a
Table 2. In vitro cytotoxic activity (IC in µM) of phenanthrene fused-dihydrodibenzo-
5
0
quinolin-ones 8a–r
Figure 1. Structures of phenanthrene alkaloids, azapodophyllotoxin derivatives and newly
designed molecules.
Figure 2. Structure activity relationship (SAR) of phenanthrene fused-tetrahydrodibenzo-
acridinone derivatives.
Figure 3. Effect of compound 8m on F-actin in SKOV-3 cells. The cells were treated with
0
.1, 0.2 and 0.5 µM concentrations of 8m. Compound 8m induced disruption of stress fibre
network. F-actin was stained with rhodamine phalloidin (red fluorescent dye). The blue
colour indicates Hoechst 33242 staining for nucleus.
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Figure 4. SKOV-3 ovarian cancer cells were treated with compound 8m for 24 h. (A) The
cell cycle distribution was analysed by using propidium iodide staining method and analysed
by flow cytometry (BDc6 accuri). (B) Data of 10,000 cells were collected for each data file.
The percentage of cell population in G0/G1, S and G2/M phase were calculated by using
BDc6accuri software
Figure 5. Compound 8m induced morphological changes in SKOV-3 cells observed by
Hoechst staining.
Figure 6. JC-1 mitochondrial staining of SKOV-3 cells after treatment with 0.1, 0.2 and 0.5
µM concentrations of 8m. DMSO treated cells were used as control.
Figure 7. Effect of 8m on reactive oxygen species. SKOV-3 cells were treated with 0.1, 0.2
and 0.5 µM of 8m, and incubated with Carboxy-H DCFDA. The DCF fluorescence was
2
quantified by using flowcytometer.
Figure 8. Effect of compound 8m on Caspase-3 activity; SKOV-3 cells were treated with
compound 8m and the activity of caspase-3 was determined using caspase-3 colorimetric
assay kit.
Figure 9. Compound 8m caused greater inhibition of tumor spheroid growth. An ultra low
attachment (ULA) 96-well round-bottomed plate was used to generate SKOV-3 tumor
spheroids.
Scheme 1. Microwave-assisted one-pot synthesis of various phenanthrene fused-
tetrahydrodibenzo-acridinones 8a–r.
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Tables/Figures/Schemes
Table 1. Optimization of the reaction conditions to phenanthrene fused-tetrahydrodibenzo-
acridinone 8a
o
b
entry
method
solvent
temperature ( C) time (min) yield (%)
1
2
3
4
5
6
7
8
.
.
.
.
.
.
.
.
conventional EtOH
conventional EtOH
80
180
300
30
61
65
69
77
80
91
91
-
80
MW
MW
MW
MW
MW
MW
EtOH
EtOH
EtOH
EtOH
EtOH
100
120
120
150
150
100
10
30
20
30
H O
2
12
a
Reaction conditions: 5 (0.28 mmol), 6 (0.28 mmol), 7 (0.28 mmol) and solvent (3 mL) .
b
Isolated yields. MW: microwave heating conditions.
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