Synthesis of substituted phenanthrene-9-benzimidazole conjugates: Cytotoxicity evaluation and apoptosis inducing studies

Article abstract of DOI:10.1016/j.ejmech.2017.09.006

A series of new phenanthrene-9-benzimidazole conjugates has been synthesized by condensing phenanthrene aldehydes with various substituted o-phenylenediamines. The title compounds were evaluated for their in vitro cytotoxic potential against various human

Full text of DOI:10.1016/j.ejmech.2017.09.006

Accepted Manuscript
Synthesis of substituted phenanthrene-9-benzimidazole conjugates: Cytotoxicity
evaluation and apoptosis inducing studies
Niggula Praveen Kumar, Pankaj Sharma, S. Sujana Kumari, Umarani Brahma, Shalini
Nekkanti, Nagula Shankaraiah, Ahmed Kamal
PII:
S0223-5234(17)30688-8
10.1016/j.ejmech.2017.09.006
EJMECH 9721
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 June 2017
Revised Date: 21 August 2017
Accepted Date: 4 September 2017
Please cite this article as: N.P. Kumar, P. Sharma, S.S. Kumari, U. Brahma, S. Nekkanti, N.
Shankaraiah, A. Kamal, Synthesis of substituted phenanthrene-9-benzimidazole conjugates: Cytotoxicity
evaluation and apoptosis inducing studies, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.09.006.
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ACCEPTED MANUSCRIPT
Synthesis of substituted phenanthrene-9-benzimidazole conjugates:
Cytotoxicity evaluation and apoptosis inducing studies
Niggula Praveen Kumara, Pankaj Sharmaa, S. Sujana Kumarib, Umarani Brahmab, Shalini
Nekkantia, Nagula Shankaraiaha,*, Ahmed Kamala,*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
bDepartment of Pharmacology and Toxicology, National Institute of Pharmaceutical
Education and Research (NIPER), Hyderabad 500 037, India
Graphical Abstract

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Synthesis of substituted phenanthrene-9-benzimidazole conjugates:
Cytotoxicity evaluation and apoptosis inducing studies
Niggula Praveen Kumara, Pankaj Sharmaa, S. Sujana Kumarib, Umarani Brahmab, Shalini
Nekkantia, Nagula Shankaraiaha,*, Ahmed Kamala,*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
bDepartment of Pharmacology and Toxicology, National Institute of Pharmaceutical
Education and Research (NIPER), Hyderabad 500 037, India
Abstract: A series of new phenanthrene-9-benzimidazole conjugates has been synthesized by
condensing phenanthrene aldehydes with various substituted o-phenylenediamines. The title
compounds were evaluated for their in vitro cytotoxic potential against various human cancer
cell lines like breast (BT-549), prostate (PC-3 and DU145), triple negative breast cancer
(MDA-MB-453), and human colon cancer (HCT-116 and HCT-15) cells. Among the tested
compounds, 10o displayed significant in vitro cytotoxic activity against PC-3 prostate cancer
cells with an IC50 value of 6.32±0.09 µM. Further, the cell cycle analysis indicated that it
blocks G2/M phase of the cell cycle in a dose dependent manner. In order to determine the
effect of the compound 10o on cell viability; phase contrast microscopy, AO/EB staining,
DAPI staining, and DCFDA staining studies were performed. In these studies, apoptotic
features were clearly observed indicating that the compound inhibited cell proliferation by
apoptosis. JC-1 staining and annexin binding assays indicated the extent of apoptosis in PC-3
cells. Further, relative viscosity measurements and molecular docking studies indicated that
these compounds bind to DNA by intercalation.
Keywords: Benzimidazoles, phenanthrene, Perkin condensation, cytotoxicity, apoptosis,
DNA interaction, molecular modeling.
Corresponding authors: Dr Ahmed Kamal, E-mail: ahmedkamal@iict.res.in; Dr Nagula
Shankaraiah, E-mail: shankar@niperhyd.ac.in
1. Introduction
Cancer is one of the lethal diseases worldwide and characterized by uncontrolled cell
proliferation, metastasis and resistance to apoptotic signals [1]. Chemotherapeutic drugs that
target the cell division, angiogenesis and apoptotic pathways are emerging as effective tools
to combat cancer in the present drug discovery [2]. However, due to their off-target effects
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and enhanced drug resistance in cancer cells, there is a demand to identify and synthesize
novel efficacious and innocuous pharmacophores as chemotherapeutic agents [3]. DNA
replication plays a crucial role in cancer cell division and planar cytotoxic agents such as
doxorubicin, acridines, anthraquinones, distamycins, and phenanthrene derivatives are well
known for targeting DNA [4]. In this context, the development of new molecular scaffolds
that can act on DNA and induce apoptosis is of great importance [5]. Among the natural
product derivatives, phenanthroindolizidine alkaloids from the Asclepiadacea [6] family
represent a unique chemical class and are well established for their antitumor [7],
antiangiogenic [8], antilupus [9], antiarthritis [10], antifungal [11], antimicrobial [12], and
antiviral activities [13]. The most intriguing biological property among these alkaloids is their
cytotoxic potential against various cancer cell lines [14]. This is due to the intercalation of the
phenanthrene core between the DNA base pairs [15] and also inhibition of the enzymes
involved in DNA synthesis [16]. Tylophorine (Figure 1) is one of the representative alkaloids
of this class.
On the other hand, benzimidazole, a nitrogen containing heterocyclic is regarded as
the “Master Key” as it is the vital pharmacophore in numerous bioactive agents targeting
various proteins [17]. N-Ribosyl-dimethyl benzimidazole exists naturally and serves as an
axial ligand for cobalt in vitamin B12 [18]. Benzimidazoles are structural isosteres of
nucleobases owing to their fused nitrogen nuclei [19]. They are capable of interacting with
biomolecular targets and exhibit numerous activities such as antiproliferative [20], anti-
inflammatory [21], antiallergic [22], antibacterial [23] and antiviral [24] properties.
Furthermore, recent studies revealed that some of the naturally existing and synthetic
benzimidazoles are potent anticancer agents and their activity is due to their interaction with
the DNA double helix [25]. Abonia et al reported the quinolinone-benzimidazoles (Figure 1)
as potential antitumour agents [26]. A few benzimidazoles tethered to planar napthalimide
rings were also established as drug leads [27]. Telmisartan possessing an N-benzylated
benzimidazole core is reported to induce early apoptosis in the prostate cancer cells [28].
Moreover, in medicinal chemistry, functionalization of the known active scaffolds based on
SAR to access diverse bioactive molecules is of high rationale.
The earlier reports reveal that the presence of polar moieties at C9-position of the
planar phenanthrene skeleton is of great importance for their cytotoxic activity [29].
Tethering of polar benzimidazole nucleus at the C9-position might effectively enhance the
antitumor activity. In continuation of our research dedicated to bioactive scaffolds [30],
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herein we have designed and synthesized a series of new phenanthrene-9-benzimidazole
conjugates. The newly synthesized compounds 10a–q were further evaluated for their in vitro
cytotoxicity, and cell growth inhibition studies.
<Insert Figure 1 here>
2. Results and discussion
2.1 Chemistry
The synthetic strategy for the preparation of phenanthrene-9-benzimidazole conjugates 10a–q
was depicted in Scheme 1. Various substituted o-phenylenediamines 9a–l were condensed
with phenanthrene-9-carbaldehydes (5a,b). The aldehyde partners 5a and 5b were
synthesized from the commercially available starting materials via the conventional five step
sequence [31]. Initially, Perkin condensation of the substituted benzaldehydes 2a,b and 3,4-
dimethoxy phenyl acetic acid 1 resulted in the formation of substituted diphenyl acrylic acids.
Next, the acid was converted into its methyl ester 3 followed by m-CPBA/TFA mediated
intramolecular oxidative cyclization to give the tricyclic phenanthrene esters 4a,b. However,
in case of R = H this reaction was carried in the presence of FeCl3 and m-CPBA. Further, the
ester functionality was reduced to alcohol by LiAlH4. The alcohols were oxidized by using
Dess-Martin periodinane reagent to give the respective phenanthrene-9-carbaldehydes 5a,b.
For the synthesis of N-substituted o-phenylenediamines, 4-chloro-2-nitro aniline was initially
benzylated using benzyl bromides and later the nitro group was reduced to amino
functionality by stannous chloride dihydrate as shown in Scheme 2. Finally, the title
compounds 10a–q were synthesized by refluxing phenanthrene-9-carbaldehydes and o-
phenylenediamines in ethanol in the presence of sodium metabisulphite. All the newly
synthesized compounds 10a–q were characterized by HRMS, 1H and 13C NMR spectroscopy.
<Insert Scheme 1 here>
<Insert Scheme 2 here>
2.2 Pharmacology
2.2.1 Cytotoxic activity
The newly synthesized phenanthrene-9-benzimidazole conjugates 10a–q were screened for
their in vitro cytotoxicity on different human cancer cell lines such as breast (BT-549),
prostate (PC-3 and DU145), triple negative breast cancer (MDA-MB-453), and human colon
cancer
(HCT-116
and
HCT-15)
by
using
3-(4,5-dimethylthiazol-2-yl)-2,5-
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diphenyltetrazolium bromide (MTT) assay [32]. The IC50 (µM) values (concentration
required to inhibit 50% of the tumor cells) of the tested compounds and the positive controls,
5-fluorouracil and nocodazole are listed in Table 1. It is noticeable from the preliminary
screening that a majority of the tested compounds displayed cytotoxicity less than 20 µM on
the PC-3 prostate cancer cell line. The compound 10o with a fluoro substitution on
benzimidazole nucleus was found to show the highest potency on PC-3 cell line (IC50
6.32±0.09 µM). 10d, 10e, 10g, 10k, and 10l displayed cytotoxic activities against a majority
of the cancer cell lines with IC50 values ranging from 8.34 ± 0.15 µM to 17.3 ± 0.31 µM
(Table 2). It was observed that, only the derivatives 10e, 10g and 10n were active against the
HCT-116 colon cancer cells with IC50 values of 9.92 ± 0.82, 8.91 ± 0.72 and 11.3 ± 0.46 µM
respectively. Compounds 10b, 10c, 10e, 10g, 10k, 10l, 10o, and 10p displayed IC50 values
less than 20 µM in the MDA-MB-453 breast cancer cells, among which, the compound 10o
possessing fluoro group as R3 substituent proved to be best with an IC50 of 6.61 ± 0.9 µM.
This is followed by the compound 10p with a benzyl substituted nitrogen atom and a chloro
substitution on benzimidazole aromatic ring displayed IC50 7.20 ± 0.18 µM and then
compound 10c, a chloro benzimidazole having a free NH (IC50 9.42 ± 0.14 µM). Derivatives
10d and 10q showed activity less than 10 µM on the BT-549 breast cancer cell line with 10q
(R3 = Br, X = N) being the most active (IC50 7.91 ± 0.23 µM) followed by 10d (R3 = F, IC50
10.8 ± 0.06 µM). Interestingly, the compound 10g with a methoxy benzimidazole substitution
at the C9-position was found to be active on all the cell lines except DU-145 with the most
potent activity (IC50 8.91 ± 0.72 µM) on HCT-116 colon cancer cell line. The derivatives 10d,
10e, 10g, 10k, 10l, and 10p displayed cytotoxicity against HCT-15 colon cancer cells with
IC50 values ranging from 8.34 ± 0.15 µM to 15.6 ± 0.16 µM. Compound 10f with R3 =
COOCH3 substitution proved to be moderately active against the PC-3 and DU-145 prostate
cancer cell lines with IC50 values of 16.7 ± 0.72 µM and 17.2 ± 0.42 µM, respectively. It
could be easily observed from the in vitro cytotoxicity data that these new derivatives with
electro negative substituents like fluoro and chloro benzimidazoles at the C9-position of
phenanthrene scaffold augmented the biological response. The electronegative nucleus of
these groups connected to carbon atom with-draws electrons from other parts of the molecule,
thus strongly polarizing the bond and may enhance interaction with biomolecular targets [33].
Based on the encouraging results, one of the most active compounds 10o was taken-up for
further examining the mechanism of cancer cell growth inhibition and DNA viscosity study.
<Insert Table 1 here>
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2.2.2 Morphology studies using microscopy
Microscopic examination was performed to know whether the treatment with these
compounds could lead to reduced cancer cell viability [34]. PC-3 prostate cancer cells were
treated with 2.5, 5.0 and 10.0 µM concentrations of the active compound 10o. Cells were
observed and the images were captured by a phase contrast microscope. It is clearly observed
from Figure 2 that the increased concentration of the compound 10o led to the decreased
number of viable cells in comparison to control, as observed by significant morphological
changes such as nuclear shrinkage and cell detachment. This indicates that the compounds
induced distinctive morphological changes on PC-3 cells.
<Insert Figure 2 here>
2.2.3 Acridine orange-ethidium bromide (AO-EB) staining
Acridine orange/ethidium bromide (AO/EB) fluorescent staining assay was performed to
differentiate between live cells, necrotic and apoptotic cells [35]. AO readily diffuses through
the intact cell membrane and stains the nuclei green, whereas EB enters only the membrane
disintegrated cells and stains the nucleus red. Figure 3 shows green colour in case of control
cells due to their normal morphology. Fluorescence microscopic images of cells treated with
10.0 µM of the compound 10o clearly showed the altered morphological characteristics such
as chromatin condensation, apoptotic body formation, cell shrinkage and membrane blebbing
suggesting that the compound 10o induced cell death on PC-3 cells.
<Insert Figure 3 here>
2.2.4 DAPI staining
DAPI (4’,6-diamidino-2-phenylindole) is a fluorescent stain that reveals the chromatin
condensation or nuclear damage through effectively binding to A-T rich regions of DNA.
DAPI permeates the intact cell membrane with less efficiency when compare to damaged cell
membrane. Therefore, the intensity of staining in live cells is less whereas apoptotic cells are
stained bright due to the presence of condensed nucleus which is a typical apoptotic
characteristic. Therefore, it is considered of our interest to examine the effect of the
compound 10o on PC-3 cells by using DAPI staining [36]. This technique is useful to
distinguish live cells from apoptotic cells based on their nuclear morphology. DAPI forms
fluorescent complex with chromatin there by stains the nucleus bright blue. As observed from
Figure 4, the nuclear structure of control cells was intact, whereas compound 10o treated PC-
3 cells exhibited apoptotic features such as horse-shoe shaped and condensed nuclei.
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<Insert Figure 4 here>
2.2.5 Cell cycle analysis
Many of the cytotoxic agents display their growth inhibitory potential by arresting the cell
cycle at a specific point. Thus, the blockade of cell cycle progression by cytotoxic
compounds has an important role in the action of a potential chemotherapeutic agent. From in
vitro screening, it is apparent that the compound 10o displayed significant anticancer activity
against PC-3 prostate cancer cells. Therefore, we studied the effect of compound 10o on
distribution of the cell population in different phases of cell cycle by flow cytometry analysis
[37]. PC-3 cells were treated with compound 10o at 2.5 µM, 5.0 µM and 10.0 µM for 24 h,
and the cells were fixed by ethanol, stained with propidium iodide and further analyzed by
flow cytometry. The results from Figure 5 shows that the ratio of PC-3 cells in G2/M phase
from 25.9% in control increased to 27.8% at 2.5 µM, 29.7% at 5.0 µM and 44.1% at 10.0 µM
respectively and simultaneous decreased number of cell population in G0/G1 phase.
Therefore, these result clearly indicated that compound 10o arrests G2/M phase of cell cycle
in PC-3 cells.
<Insert Figure 5 here>
2.2.6 Measurement of reactive oxygen species (ROS)
The production of ROS is a typical characteristic of many cytotoxic agents by initiating
oxidative damage to the mitochondrial membrane potential and permeability. Hence, DCFDA
staining method [38] has been used to assess the generation of intracellular ROS on PC-3
cells by the compound 10o. PC-3 cells were treated with compound 10o at 2.5 µM, 5.0 µM
and 10.0 µM for 6 h, which resulted in enhanced DCFDA fluorescence in a dose dependent
manner, indicating the accumulation of ROS (Figure 6). On the other hand, decreased
fluorescence intensity was observed when PC-3 cells were treated with N-acetyl cysteine
(NAC) prior to the compound treatment. This indicated the decreased free radical production
by NAC, thereby suggesting that the compounds induced cytotoxicity by ROS generation.
Moreover, H2O2 treatment of PC-3 cells led to the increased fluorescence when compared to
the control due to the generation of radicals.
<Insert Figure 6 here>
2.2.7 Analysis of mitochondrial membrane potential (DΨm)
Enhanced ROS generation can cause oxidative stress there by leading to altered
mitochondrial membrane potential. Therefore, JC-1 staining was performed to study the
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effect of the compound 10o on mitochondrial membrane potential (DΨm). The lipophilic
cationic JC-1 dye was used to stain the mitochondria [39]. Normal polarised mitochondria
stains red due to the formation of J-aggregates, whereas the depolarised mitochondria of
apoptotic cells stains green because of J-monomers. PC-3 cells were treated with 2.5, 5.0 and
10.0 µM of compound 10o for 24 h and stained with JC-1 dye. Flow-cyometric analysis of
the treated cells clearly displayed an increase in depolarised cell population (P3) from 22.8 of
control to 33.35, 67.42 and 95.64, respectively in a concentration dependent manner (Figure
7). Thus, the results indicate loss of mitochondrial membrane potential (DΨm) by the
compound 10o.
<Insert Figure 7 here>
2.2.8 AnnexinV-FITC/Propidium iodide dual staining assay
Annexin V-FITC/propidium iodide dual staining assay [40] was performed to determine the
extent of apoptosis induced by the compound 10o on PC-3 cells. This assay facilitates the
detection of necrotic cells (Q1-UL; AV-/PI+), live cells (Q1-LL; AV-/PI-), early apoptotic
cells (Q1-LR; AV+/PI-), and late apoptotic cells (Q1- UR; AV+/PI+). As observed from
Figure 8, the percentage of total apoptotic cells (early and late apoptotic cells) increased to
71.96% after treatment with 10 µM concentration of 10o for 24 h, in comparison to the
control (22.68%) cells. The percentage of early and late apoptotic cells comparatively
increased with an increase in the concentration of compound 10o, which indicates the
induction of dose dependent apoptosis in PC-3 cancer cells.
<Insert Figure 8 here>
2.2.9 Relative viscosity experiment
Relative viscosity experiments provide the evidence for the mode of interaction of the most
potent compounds 10n and 10o with DNA [41]. Intercalation of molecules sandwiched
between the DNA base pairs results in a major enhancement in the viscosity of DNA
solutions; but a minor alteration in viscosity is shown by groove binders or DNA surface
binders. Interestingly, covalent DNA-binders reduce the relative viscosity of DNA solutions.
At this juncture, we have used Ethidium Bromide, Doxorubicin and Hoechst 33258 as
controls. Ethidium bromide and Doxorubicin, the intercalators increased the relative viscosity
strongly by lengthening the DNA strands; while relative viscosity increased only a little in
case of Hoechst 33258, a minor groove binder. On gradually increasing the concentration of
the compounds 10n and 10o, there was a noticeable increase in the relative viscosity of the
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complex solutions, which suggests DNA intercalation of these derivatives. Upon interaction
with DNA, 10o produced an increase in viscosity comparable to that of Ethidium bromide
and Doxorubicin, indicating strong DNA binding affinity (Figure 9). The data obtained from
viscosity studies are consistent with the results obtained from the in vitro cytotoxicity studies.
<Insert Figure 9 here>
2.2.10 Molecular docking
Molecular docking simulations were performed into the DNA duplex d(GAAGCTTC)2 using
XP Glide 6.9 (Schrödinger 2015-4). The docked poses for the top scored compounds 10n and
10o, shown in Figure 10, depict comfortable insertion of the phenanthrene ring between the
G–C base pairs. Interestingly, the benzimidazole ring lies at right angles to the planar
phenanthrene ring system due to steric effects and orients itself perfectly in the minor groove.
The docked poses were stabilized electronically by π–π stacking, dipole-dipole interactions
and formation of hydrogen-bonds between the NH-hydrogen of benzimidazole and the DNA
base pairs. The results obtained from molecular docking studies were consistent with the in
vitro cytotoxicity as well as relative viscosity data.
<Insert Figure 10 here>
3. Conclusion
In conclusion, a new series of C9-tethered phenanthrene benzimidazoles were synthesized
and these compounds were evaluated for their in vitro cytotoxic potential against various
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 cytotoxicity profile indicated that the most active compound 10o showed broad range of
activity on three of the tested cell lines with a remarkable IC50 value of 6.32 ± 0.09 µM on
PC-3 prostate cancer cells. The compound 10o blocked the cell cycle progression at G2/M
phase in a dose dependent manner. Moreover, the compound also led to a decrease in the
number of viable cells and increased apoptotic features as shown in case of phase contrast
microscopy, AO/EB and DAPI staining. JC-1 staining and annexin binding assays indicated
the dose-dependent apoptosis by compound 10o on PC-3 cells. Further, increasing the
concentrations of compounds 10n and 10o lead to increased viscosity suggesting that these
compounds binds to DNA by intercalation. Molecular docking study also supports the DNA
interaction of these new compounds. Overall, the current study established that these
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phenanthren-9-benzimidazole conjugates have the potential to be advanced as DNA
interactive cytotoxic agents for the treatment of prostate cancer.
4. Experimental protocols
4.1 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 CDCl3 and DMSO-d6 solvent using tetramethyl silane
1
(TMS) as the internal standard. Chemical shifts for H and 13C 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 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 rotary evaporator below 45 oC.
4.1.1 2,3,6,7-tetramethoxyphenanthrene-9-carbaldehyde (5a).
o
1
Yellow solid, yield 76%; mp: 183-184 C; H NMR (500 MHz, CDCl3): δ 10.24 (s, 1H),
8.94 (s, 1H), 8.02 (s, 1H), 7.75 (s, 1H), 7.73 (s, 1H), 7.30 (s, 1H), 4.15 (s, 3H), 4.12 (s, 3H),
13
4.09 (s, 3H), 4.05 (s, 3H) ppm; C NMR (125 MHz, CDCl3): δ 193.8, 152.0, 150.0, 149.3,
149.0, 139.1, 127.9, 127.8, 124.9, 124.5, 122.9, 109.3, 106.1, 102.5, 102.4, 56.0, 55.9, 55.9,
55.8 ppm; HRMS (ESI): m/z calcd for C19H18O5 327.1227, found 327.1210 [M + H]+.
4.1.2 3,6,7-trimethoxyphenanthrene-9-carbaldehyde (5b).
Colourless solid, yield 71%; mp: 169-171oC; 1H NMR (500 MHz, CDCl3): δ 10.19 (s, 1H),
8.92 (s, 1H), 8.01 (s, 1H), 7.89 (d, J = 8.69 Hz, 1H), 7.78 (s, 1H), 7.75 (s, 1H), 7.23-7.21 (m,
13
1H), 4.08 (d, J = 1.67 Hz, 6H), 4.03 (s, 3H) ppm; C NMR (125 MHz, CDCl3): δ 193.8,
161.0, 150.5, 149.2, 140.3, 134.1, 132.1, 127.4, 124.7, 124.2, 123.8, 116.0, 106.2, 103.9,
103.0, 55.9, 55.8, 55.5 ppm; HRMS (ESI): m/z calcd for C18H16O4 297.1121, found 297.1115
[M + H]+.
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4.1.3 General reaction procedure for benzylation of 4-chloro-2-nitro aniline 8a–b
The mixture of 4-chloro-2-nitroaniline 6 (1.0 mmol) and benzyl bromides 7a, 7b (1.2 mmol)
in water (4 mL) was refluxed at 100oC for 2h. After completion of the reaction as monitored
by TLC, the reaction mixture was cooled and extracted with ethylacetate. The organic layer
was dried over sodium sulphate and concentrated under vaccum. The obtained crude solid
was further purified by column chromatography by using ethyl acetate/n-hexane to afford the
1
pure compounds 8a–b. All the synthesized compounds were characterized by H NMR and
13C NMR spectroscopy.
4.1.3.1 N-benzyl-4-chloro-2-nitroaniline (8a).
Red solid, yield 83%; mp: 78-80 oC; 1H NMR (500 MHz, DMSO-d6): δ 8.73 (t, J = 6.10 Hz,
1H), 8.06 (d, J = 2.59 Hz, 1H), 7.49-7.47 (m, 1H), 7.35-7.31 (m, 4H), 7.26-7.23 (m, 1H),
13
6.94 (d, J = 9.30 Hz, 1H), 4.64 (d, J = 6.25 Hz, 2H) ppm; C NMR (125 MHz, DMSO-d6): δ
143.6, 138.0, 136.0, 131.1, 128.5, 127.0, 126.7, 124.8, 118.4, 116.9, 45.6 ppm.
4.1.3.2 4-chloro-N-(3,5-dimethylbenzyl)-2-nitroaniline (8b).
1
brown liquid, yield 80%; H NMR (500 MHz, DMSO-d6): δ 8.67 (t, J = 5.95 Hz, 1H), 8.04
(d, J = 2.59 Hz, 1H), 7.48-7.46 (m, 1H), 6.93 (s, 2H), 6.91 (d, J = 9.30 Hz, 1H), 6.86 (s, 1H),
4.53 (d, J = 6.10 Hz, 2H), 2.21 (s, 6H) ppm; 13C NMR (125 MHz, DMSO-d6): δ 143.7, 137.9,
137.5, 136.0, 131.0, 128.5, 124.8, 124.4, 118.4, 116.9, 45.7, 20.8 ppm.
4.1.4 General reaction procedure for Substituted O-phenylene diamines 9k–l
To a mixture of benzyl nitro anilines 8a, 8b (1.0 mmol) in ethanol was added stannous
chloride dihydrate (4.0 mmol) and the reaction was stirred at room temperature for 12 h.
After completion of the reaction as monitored by TLC, sodium bicarbonate solution was
added and extracted with ethyl acetate. The organic layer was dried over sodium sulphate and
concentrated under vaccum. The obtained crude solid was further purified by column
chromatography by using ethyl acetate/n-hexane to afford the pure compounds 9k–l. All the
synthesized compounds were characterized by 1H NMR and 13C NMR spectroscopy.
4.1.4.1 N1-benzyl-4-chlorobenzene-1,2-diamine (9k).
1
Orange liquid, yield 70%; H NMR (500 MHz, DMSO-d6): δ 7.35-7.29 (m, 4H), 7.23-7.17
(m, 1H), 6.56 (d, J = 2.44 Hz, 1H), 6.38-6.36 (m, 1H), 6.27 (d, J = 8.39 Hz, 1H), 5.23 (t, J =
13
5.79 Hz, 1H), 4.91 (s, 2H), 4.28 (d, J = 5.79 Hz, 2H) ppm; C NMR (125 MHz, DMSO-d6):
δ 139.8, 136.9, 134.1, 128.1, 127.0, 126.5, 120.3, 115.9, 112.8, 110.8, 46.7 ppm.
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4.1.4.2 4-chloro-N1-(3,5-dimethylbenzyl)benzene-1,2-diamine (9l).
Yellow liquid, yield 68%; 1H NMR (500 MHz, DMSO-d6): δ 6.95 (s, 2H), 6.84 (s, 1H), 6.60
(s, 1H), 6.41 (d, J = 8.39 Hz, 1H), 6.28 (d, J = 8.39 Hz, 1H), 5.15 (t, J = 5.79 Hz, 1H), 4.90
13
(s, 2H), 2.23 (s, 6H) ppm; C NMR (125 MHz, DMSO-d6): δ 139.8, 137.1, 136.9, 134.4,
128.1, 124.8, 120.3, 116.1, 113.0, 110.8, 46.9, 20.9 ppm.
4.1.5 General reaction procedure for the synthesis of compounds 10a–q
To an aqueous solution of Na2S2O5 (1.5 mmol), was added the corresponding o-phenylene
diamine (9a-l) (1.0 mmol) followed by a solution of phenanthrene aldehydes 5a,b (1.0 mmol)
in ethanol and the reaction mixture was stirred at 70 oC. After completion of the reaction, the
o
reaction mixture was cooled; water was added and left to stand for 1 h at 0 C to obtain the
precipitate. The obtained crude solid was filtered and further purified by column
chromatography by using ethyl acetate/n-hexane to afford the pure compounds 10a–q. All the
1
synthesized compounds were characterized by HRMS (ESI), H NMR and 13C NMR
spectroscopy.
4.1.5.1 2-(2,3,6,7-Tetramethoxyphenanthren-9-yl)-1H-benzo[d]imidazole (10a).
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1
Off white solid, yield 85%; mp: 210-212 C; H NMR (500 MHz, DMSO-d6): δ 12.92 (s,
1H), 8.80 (s, 1H), 8.17 (s, 1H), 8.11 (s, 1H), 8.07 (s, 1H), 7.77 (d, J = 7.62 Hz, 1H), 7.58 (d, J
= 7.47 Hz, 1H), 7.47 (s, 1H), 7.28-7.22 (m, 2H), 4.08 (s, 6H), 3.94 (s, 3H), 3.87 (s, 3H) ppm;
13C NMR (125 MHz, DMSO-d6): δ 152.0, 150.1, 149.0, 148.8, 148.5, 143.8, 134.2, 126.3,
124.9, 124.8, 124.7, 123.3, 123.2, 122.4, 121.3, 118.9, 111.1, 108.6, 107.5, 103.8, 103.5,
55.9, 55.8, 55.4, 55.1 ppm; HRMS (ESI): m/z calcd for C25H22N2O4 415.1652, found
415.1665 [M + H]+.
4.1.5.2 6-Bromo-2-(2,3,6,7-tetramethoxyphenanthren-9-yl)-1H-benzo[d]imidazole (10b).
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1
White solid, yield 82%; mp: 157-159 C; H NMR (500 MHz, DMSO-d6): δ 13.14 (d, J =
18.9 Hz, 1H), 8.74 (s, 1H), 8.18 (s, 1H), 8.11 (s, 1H), 8.07 (s, 1H), 7.55 (d, J = 8.54 Hz, 1H),
7.73 (d, J = 8.39 Hz, 1H), 7.48 (d, J = 2.74 Hz, 1H), 7.41-7.36 (m, 1H), 4.08 (d, J = 2.44 Hz,
13
6H), 3.94 (s, 3H), 3.87 (s, 3H) ppm; C NMR (125 MHz, DMSO-d6): δ 153.4, 150.2, 149.1,
148.8, 148.6, 145.2, 133.4, 126.6, 124.9, 124.8, 124.7, 123.0, 122.6, 121.2, 120.6, 113.6,
112.9, 108.7, 107.3, 103.8, 103.5, 55.9, 55.8, 55.4, 55.1 ppm; HRMS (ESI): m/z calcd for
C25H21BrN2O4 493.0957, found 493.0766 [M + H]+.
4.1.5.3 6-Chloro-2-(2,3,6,7-tetramethoxyphenanthren-9-yl)-1H-benzo[d]imidazole (10c).
11

ACCEPTED MANUSCRIPT
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1
Off white solid, yield 77%; mp: 225-227 C; H NMR (300 MHz, CDCl3 + DMSO-d6): δ
8.25 (s, 1H), 7.86 (s, 1H), 7.72-7.65 (m, 4H), 7.28-7.25 (m, 1H), 7.10 (s, 1H), 4.13 (s, 3H),
13
4.11 (s, 3H), 3.99 (s, 3H), 3.91 (s, 3H) ppm; C NMR (125 MHz, CDCl3+ DMSO-d6): δ
153.2, 149.6, 148.5, 148.3, 148.2, 126.9, 126.4, 124.6, 124.5, 124.3, 123.1, 122.8, 122.0,
121.1, 115.4, 114.4, 113.6, 107.9, 106.6, 102.2, 102.0, 55.5, 55.4, 55.3, 55.1 ppm; HRMS
(ESI): m/z calcd for C25H21ClN2O4 449.1263, found 449.1261 [M + H]+.
4.1.5.4 6-Fluoro-2-(2,3,6,7-tetramethoxyphenanthren-9-yl)-1H-benzo[d]imidazole (10d).
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1
Light brown solid, yield 76%; mp: 229-231 C; H NMR (300 MHz, DMSO-d6): δ 8.53 (s,
1H), 7.98 (s, 1H), 7.85 (d, J = 11.51 Hz, 2H), 7.75 (s, 1H), 7.31-7.22 (m, 2H), 6.95 (t, J =
13
9.61 Hz, 1H), 4.07 (s, 6H), 3.94 (s, 3H), 3.90 (s, 3H) ppm; C NMR (125 MHz, DMSO-d6 +
CDCl3): δ 158.0, 153.6, 150.4, 149.3, 149.1, 148.8, 126.7, 125.2, 125.1, 123.4, 123.1, 110.4,
110.2, 108.9, 107.4, 103.7, 103.4, 56.1, 56.0, 55.8, 55.5 ppm; HRMS (ESI): m/z calcd for
C25H21FN2O4 433.1558, found 433.1571 [M + H]+.
4.1.5.5 2-(2,3,6,7-Tetramethoxyphenanthren-9-yl)-1H-benzo[d]imidazole-6-carboxylic acid
(10e).
Brown solid, yield 62%; mp: 240-242 oC; 1H NMR (500 MHz, DMSO-d6): δ 13.25 (s, 1H),
8.83-8.74 (m, 1H), 8.36-8.17 (m, 2H), 8.12 (s, 1H), 8.08 (s, 1H), 7.96-7.63 (m, 2H), 7.50 (d, J
13
= 11.29 Hz, 1H), 4.08 (s, 6H), 3.94 (s, 3H), 3.89 (s, 3H) ppm; C NMR (125 MHz, DMSO-
d6): δ 167.9, 153.9, 150.3, 149.1, 148.9, 148.6, 137.5, 126.8, 125.0, 124.9, 124.7, 124.2,
123.9, 123.0, 122.8, 122.5, 120.7, 110.9, 108.7, 107.3, 103.8, 103.5, 55.9, 55.8, 55.4, 55.1
ppm; HRMS (ESI): m/z calcd for C26H22N2O6 459.1551, found 459.1552 [M + H]+.
4.1.5.6
Methyl
2-(2,3,6,7-tetramethoxyphenanthren-9-yl)-1H-benzo[d]imidazole-6-
carboxylate (10f).
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1
Brown solid, yield 60%; mp: 165-167 C; H NMR (500 MHz, DMSO-d6): δ 8.78 (s, 1H),
8.29-8.22 (m, 2H), 8.12 (s, 1H), 8.08 (s, 1H), 7.92 (d, J = 8.39 Hz, 1H), 7.76-7.74 (m, 1H),
7.49 (s, 1H), 4.09 (s, 6H), 3.94 (s, 3H), 3.90 (d, J = 4.73 Hz, 6H), ppm; 13C NMR (125 MHz,
DMSO-d6): δ 166.7, 154.6, 150.3, 149.1, 148.9, 148.6, 131.7, 126.8, 125.0, 124.8, 124.7,
123.2, 123.0, 122.3, 108.7, 107.2, 106.5, 105.4, 103.8, 103.5, 55.9, 55.8, 55.4, 55.1, 51.9
ppm; HRMS (ESI): m/z calcd for C27H24N2O6 473.1707, found 473.1715 [M + H]+.
4.1.5.7 6-Methoxy-2-(2,3,6,7-tetramethoxyphenanthren-9-yl)-1H-benzo[d]imidazole (10g).
Brown solid, yield 78%; mp: 219-211 oC; 1H NMR (500 MHz, DMSO-d6): δ 12.78 (s, 1H),
12

ACCEPTED MANUSCRIPT
8.81 (s, 1H), 8.13 (s, 1H), 8.10 (s, 1H), 8.06 (s, 1H), 7.65-7.45 (m, 2H), 7.27-7.01 (m, 1H),
13
6.88 (d, J = 8.54 Hz, 1H), 4.07 (s, 6H), 3.93 (s, 3H), 3.87 (s, 3H), 3.83 (s, 3H) ppm; C
NMR (125 MHz, DMSO-d6): δ 150.0, 149.0, 148.8, 148.5, 126.0, 124.8, 124.6, 123.3, 123.2,
108.6, 107.6, 103.8, 103.5, 55.9, 55.8, 55.5, 55.4, 55.2 ppm; HRMS (ESI): m/z calcd for
C26H24N2O5 445.1758, found 445.1774 [M + H]+.
4.1.5.8 6-Methyl-2-(2,3,6,7-tetramethoxyphenanthren-9-yl)-1H-benzo[d]imidazole (10h).
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1
white solid, yield 80%; mp: 221-223 C; H NMR (500 MHz, DMSO-d6): δ 8.75 (s, 1H),
8.14 (s, 1H), 8.10 (s, 1H), 8.06 (s, 1H), 7.56 (d, J = 8.08 Hz, 1H), 7.47-7.45 (m, 2H), 7.09-
13
7.08 (m, 1H), 4.07 (s, 6H), 3.93(s, 3H), 3.87(s, 3H), 2.46(s, 3H) ppm; C NMR (125 MHz,
CDCl3+DMSO-d6): δ 151.6, 149.6, 148.6, 148.4, 148.3, 131.5, 126.2, 124.7, 124.4, 123.5,
123.4, 123.3, 114.1, 108.1, 106.9, 102.4, 102.2, 55.6, 55.5, 55.4, 55.1, 21.2 ppm; HRMS
(ESI): m/z calcd for C26H24N2O4 429.1809, found 429.1822 [M + H]+.
4.1.5.9 5,7-Dimethyl-2-(2,3,6,7-tetramethoxyphenanthren-9-yl)-1H-benzo[d]imidazole (10i).
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1
Off white solid, yield 83%; mp: 268-270 C; H NMR (500 MHz, DMSO-d6 ): δ 12.71 (s,
1H) 9.17-8.81 (m, 1H), 8.19 (s, 1H), 8.10 (s, 1H), 8.07 (s, 1H), 7.47 (s, 1H), 7.25-7.22 (m,
13
1H), 6.87 (s, 1H), 4.08 (s, 6H), 3.94 (s, 3H), 3.89 (s, 3H), 2.58 (s, 3H), 2.42 (s, 3H) ppm; C
NMR (125 MHz, DMSO-d6): δ 151.0, 150.0, 148.9, 148.8, 148.4, 131.2, 127.4, 126.0, 125.9,
124.8, 124.6, 124.2, 123.6, 123.3, 123.1, 108.6, 107.7, 105.2, 103.7, 103.5, 55.9, 55.7, 55.4,
54.9, 21.2, 16.5 ppm; HRMS (ESI): m/z calcd for C27H26N2O4 443.1965, found 443.1975 [M
+ H]+.
4.1.5.10
6-Bromo-2-(2,3,6,7-tetramethoxyphenanthren-9-yl)-1H-imidazo[4,5-b]pyridine
(10j).
o
1
Brown solid, yield 71%; mp: 211-213 C; H NMR (300 MHz, DMSO-d6): δ 10.24 (s, 1H),
8.93 (s, 1H), 8.08 (s, 1H), 7.81 (d, J = 7.74 Hz, 2H), 7.50-7.36 (m, 2H), 4.16 (d, J = 9.25 Hz,
13
6H), 4.07 (d, J = 6.04 Hz, 6H), ppm; C NMR (125 MHz, DMSO-d6 +CDCl3): δ 159.3,
155.1, 149.3, 148.9, 143.8, 131.6, 130.7, 127.9, 124.6, 124.2, 124.0, 121.5, 116.4, 112.7,
107.3, 104.0, 103.8, 55.7, 55.6, 55.4, 55.2 ppm; HRMS (ESI): m/z calcd for C24H20BrN3O4
494.0710, found 494.0713 [M + H]+.
4.1.5.11 1-Benzyl-5-chloro-2-(2,3,6,7-tetramethoxyphenanthren-9-yl)-1H-benzo[d]imidazole
(10k).
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1
Off white solid, yield 81%; mp: 234-236 C; H NMR (500 MHz, CDCl3): δ 7.90 (d, J =
13

ACCEPTED MANUSCRIPT
1.22 Hz, 1H), 7.86 (s, 1H), 7.83 (s, 1H), 7.63 (s, 1H), 7.28-7.24 (m, 2H), 7.22-7.18 (m, 3H),
7.15 (s, 1H), 7.08 (s, 1H), 6.94-6.92 (m, 2H), 5.26 (s, 2H), 4.15 (d, J = 6.86 Hz, 6H), 4.00 (s,
13
3H), 3.77 (s, 3H) ppm; C NMR (125 MHz, CDCl3): δ 154.6, 150.3, 149.5, 149.1, 149.1,
144.0, 135.7, 133.7, 128.7, 128.1, 127.8, 127.5, 126.5, 125.4, 125.0, 124.9, 124.8, 123.4,
123.0, 119.8, 111.4, 108.5, 106.0, 102.9, 102.6, 56.1, 56.0, 55.9, 55.7, 48.5 ppm; HRMS
(ESI): m/z calcd for C32H27ClN2O4 539.1732, found 539.1762 [M + H]+.
4.1.5.12
5-Chloro-1-(3,5-dimethylbenzyl)-2-(2,3,6,7-tetramethoxyphenanthren-9-yl)-1H-
benzo[d]imidazole (10l).
White solid, yield 79%; mp: 175-177 oC; 1H NMR (500 MHz, CDCl3 ): δ 7.89-7.78 (m, 3H),
7.62 (s, 1H), 7.34-7.27 (m, 2H), 7.10 (s, 2H), 6.81 (s, 1H), 6.49 (s, 2H), 5.16 (s, 2H), 4.16 (s,
13
3H), 4.14 (s, 3H), 4.00 (s, 3H), 3.75 (s, 3H), 2.11 (s, 6H) ppm; C NMR (125 MHz, CDCl3):
δ 154.5, 150.2, 149.4, 149.0, 144.0, 139.2, 138.2, 135.5, 133.8, 129.4, 127.9, 127.4, 125.3,
125.0, 124.8, 123.3, 123.3, 119.7, 111.4, 109.3, 108.5, 106.0, 102.8, 102.5, 56.0, 55.9, 55.8,
55.6, 48.4, 21.1 ppm; HRMS (ESI): m/z calcd for C34H31ClN2O4 567.2045, found 567.2036
[M + H]+.
4.1.5.13 2-(3,6,7-Trimethoxyphenanthren-9-yl)-1H-benzo[d]imidazole (10m).
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1
White solid, yield 80%; mp: 247-249 C; H NMR (300 MHz, DMSO-d6): δ 8.53 (s, 1H)
8.02 (s, 1H), 7.89 (s, 1H), 7.82 (d, J = 5.66 Hz, 1H), 7.77-7.73 (m, 2H), 7.54 (s, 1H), 7.31-
13
7.27 (m, 2H), 7.22-7.19 (m, 1H), 4.13 (s, 3H), 4.05 (s, 3H), 3.95 (s, 3H) ppm; C NMR (125
MHz, CDCl3+DMSO-d6): δ 159.0, 151.9, 149.3, 148.9, 131.3, 130.6, 126.9, 124.6, 124.5,
124.4, 122.7, 121.9, 116.5, 107.6, 104.2, 104.0, 55.8, 55.5, 55.2 ppm; HRMS (ESI): m/z calcd
for C24H20N2O3 385.1547, found 385.1548 [M + H]+.
4.1.5.14 6-Chloro-2-(3,6,7-trimethoxyphenanthren-9-yl)-1H-benzo[d]imidazole (10n).
White solid, yield 82%; mp: 213-215 oC; 1H NMR (500 MHz, DMSO-d6): δ 8.81 (d, J = 4.42
Hz, 1H), 8.22 (s, 1H) 8.19 (s, 1H), 8.15 (s, 1H), 7.99 (d, J = 8.85 Hz, 1H), 7.82-7.76 (m,
1H), 7.59 (d, J = 8.85 Hz, 1H), 7.32-7.24 (m, 2H), 4.08 (s, 3H), 4.04 (s, 3H), 3.89 (s, 3H)
13
ppm; C NMR (125 MHz, CDCl3+DMSO-d6): δ 159.0, 153.1, 149.3, 148.9, 131.4, 130.5,
127.2, 126.4, 124.6, 124.3, 124.1, 122.1, 122.0, 116.4, 107.3, 104.1, 103.8, 55.7, 55.4, 55.1
ppm; HRMS (ESI): m/z calcd for C24H19ClN2O3 419.1157, found 419.1150 [M + H]+.
4.1.5.15 6-Fluoro-2-(3,6,7-trimethoxyphenanthren-9-yl)-1H-benzo[d]imidazole (10o).
o
1
Yellow solid, yield 75%; mp: 256-258 C; H NMR (300 MHz, DMSO-d6): δ 8.50 (s, 1H)
14

ACCEPTED MANUSCRIPT
8.01 (s, 1H), 7.89-7.78 (m, 4H), 7.48 (s, 1H), 7.23-7.20 (m, 1H), 7.07-7.00 (m, 1H), 4.13 (s,
3H), 4.05 (s, 3H), 3.97 (s, 3H) ppm; 13C NMR (125 MHz, CDCl3+DMSO-d6): δ 159.7, 158.4,
157.8, 152.9, 148.8, 148.3, 130.9, 130.0, 126.9, 124.1, 124.0, 122.2, 115.4, 115.0, 110.0,
109.7, 106.6, 102.8, 102.6, 100.5, 100.4, 55.3, 55.0, 54.8 ppm; HRMS (ESI): m/z calcd for
C24H19FN2O3 403.1452, found 403.1452 [M + H]+.
4.1.5.16
1-Benzyl-5-chloro-2-(3,6,7-trimethoxyphenanthren-9-yl)-1H-benzo[d]imidazole
(10p).
White solid, yield 83%; mp: 201-203 oC; 1H NMR (500 MHz, CDCl3): δ 7.94-7.93 (m, 2H),
7.88 (d, J = 2.13 Hz, 1H), 7.70 (s, 1H), 7.68 (d, J = 8.85 Hz, 1H), 7.30-7.27 (m, 2H), 7.21-
7.17 (m, 4H), 7.15 (s, 1H), 6.93-6.91 (m, 2H), 5.27 (s, 2H), 4.13 (s, 3H), 4.04 (s, 3H), 3.79 (s,
13
3H) ppm; C NMR (125 MHz, CDCl3): δ 152.1, 150.9, 150.0, 148.9, 148.8, 148.4, 140.0,
138.8, 134.0, 132.7, 131.2, 130.3, 127.4, 125.9, 124.8, 124.6, 124.2, 123.6, 123.3, 123.1,
108.6, 107.7, 105.2, 103.9, 103.7, 103.5, 55.9, 55.7, 55.4, 54.9 ppm; HRMS (ESI): m/z calcd
for C31H25ClN2O3 509.1626, found 509.1621 [M + H]+.
4.1.5.17 6-Bromo-2-(3,6,7-trimethoxyphenanthren-9-yl)-1H-imidazo[4,5-b]pyridine (10q).
o
1
Off white solid, yield 72%; mp: 206-208 C; H NMR (500 MHz, DMSO-d6): δ 8.78 (s,
1H), 8.47 (s, 1H), 8.29-8.15 (m, 4H), 7.99 (d, J = 8.85 Hz, 1H), 7.33-7.30 (m, 1H), 4.08 (s,
13
3H), 4.05 (s, 3H), 3.89 (s, 3H) ppm; C NMR (125 MHz, DMSO-d6): δ 150.3, 149.1, 148.9,
148.6, 126.8, 125.0, 124.9, 124.7, 124.2, 123.9, 122.5, 120.7, 118.2, 112.9, 110.9, 108.7,
107.3, 103.8, 103.5, 55.9, 55.8, 55.1 ppm; HRMS (ESI): m/z calcd for C23H18BrN3O3
464.0604, found 464.0607 [M + H]+.
4.2 Biology
4.2.1. Cell Cultures
Cells were procured from National Centre for Cell Science (NCCS) Pune, India and stocks
were maintained under sterile conditions. Breast ( BT-549), triple negative breast (MDA-
MB-453), prostate (PC-3 and DU145) and colon (HCT-116 and HCT-15) cancer cells were
grown in tissue culture flasks in RPMI 1640 medium, Sigma, MEM (Minimum Essential
Medium, Sigma) or DMEM (Dulbecco modified Eagle medium, Sigma) supplemented with
10% fetal bovine serum with 1X stabilized antibiotic-antimycotic solution (Sigma) in a CO2
incubator at 37 °C with 90% relative humidity and 5% CO2.
4.2.2 MTT assay
15

ACCEPTED MANUSCRIPT
MTT assay was performed to determine the anticancer activity for all the new compounds
10a-q. 1x104 cells per well were seeded in 100 µL respective media, supplemented with 10%
FBS in each well of 96-well microculture plates and incubated for 24 h, at 37 °C in a CO2
incubator. Compounds, diluted to the required concentrations in culture medium, were added
to the wells with respective vehicle control. After 48 h of incubation, 100 µL MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (5 mg/mL) was added to all the
plates and incubated for 4 h. Then, the supernatant was carefully decanted from each well,
formazon crystals were dissolved in 100 µL of DMSO and absorbance at 570 nm
wavelengths was recorded.
4.2.3 Morphology studies using phase contrast microscopy
PC-3 cells were plated in 6 well culture plates with a density of 1x105 cells/mL and allowed
to adhere for overnight. Cells were incubated with different concentrations of 10o. After 48 h,
cells were observed for morphological changes and phase contrast microscope (Nikon) was
used to capture the images.
4.2.4 Acridine orange–ethidium bromide (AO–EB) staining
PC-3 cells were plated at a concentration of 1×106 cell/ml and treated with increased
concentration of compound 10o. Plates were incubated at 37 °C in an atmosphere of 5% CO2
for 48 h. 10 µL of fluorescent dyes containing Acridine Orange (AO) and Ethidium Bromide
(EB) added into each well in equal volumes (10 ꢀg/mL) respectively and after 10 min the
cells were visualized under fluorescence microscope (Nikon, Inc. Japan) with excitation (488
nm) and emission (550 nm) at 200x magnification.
4.2.5 DAPI staining
DAPI staining was performed to observe alterations in Nuclear morphology. After treatment
with 10o for 48 h in PC-3 cells, 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.
4.2.6 Cell cycle analysis
The distribution of the cell population through different phases of cell cycle was analysed by
Flow cytometric analysis (FACS). PC-3 cells were incubated with 10o at different
concentrations like 2.5 µM, 5.0 µM, and 10 µM for 24 h. control and 10o treated cells were
16

ACCEPTED MANUSCRIPT
harvested, washed with PBS, fixed in 70% 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
for 30 min at 37°C in dark, and about 10000 events were analyzed by flow cytometer.
4.2.7 Measurement of reactive oxygen species (ROS)
PC-3 cells were plated in 24 well plates at a density of (1 x 106 cells/mL) and allowed to
adher for overnight. Then the cells were treated with 2.5 µM, 5.0 µM and 10.0 µM of active
compound 10o for 24 h. The media was replaced with culture medium containing DCFDA
dye (10 µM) and incubated for 30 min dark. The fluorescence intensity from samples was
analysed by spectrofluorometer at an excitation and emission wavelength of 488 and 525 nm,
respectively.
4.2.8 Analysis of mitochondrial membrane potential (DΨm)
PC-3 cells (1 x 106 cells/mL) were plated in 6 well plates and allowed to adhere for
overnight. The cells were incubated with 2.5 µM, 5.0 µM and 10 µM concentrations of the
compound 10o for 24 h. cells were collected, washed with PBS and resuspended in solution
of JC-1 (2.5 µg/mL) and allowed for incubation at 310o C for 45 min. The cells were washed
twice with PBS and cells were trypsinized, centrifuged and analyzed by flow cytometer (BD
FACSVerseTM, USA).
4.2.9 Annexin V-FITC/Propidium iodide dual staining assay
The Annexin V-FITC/Propidium iodide dual staining assay was performed using PC-3 cells.
To quantify the percentage of apoptotic cells, PC-3 cells (1 x 106 mL per well) were plated in
six-well culture plates and allowed to grow for 24 h. after treatment with increasing
concentrations of compound 10o (2.5, 5.0 and 10 µM) for 24 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-FITC, and then in 300 µL1 x binding
buffer containing 5 µL Propidium iodide (PI) for 5 min at room temp in the dark. After 15
min of incubation, cells were analyzed for apoptosis using flow-cytometer (BD
FACSVerseTM, USA).
4.2.10 Relative Viscosity measurements
The viscosities of the complexes were determined by the Rolling-ball Viscometer, Lovis
2000 M/ME (Anton Paar GmbH, Graz, Austria), based on the falling ball principle. The
temperature was controlled to ±0.005 K by a built in Peltier thermostat. A calibrated glass
17

ACCEPTED MANUSCRIPT
capillary (1.59 mm) with a steel ball was filled with the sample for measuring the ball falling
time at angles in the range of 20o to 70o. The ball falling time and densities were used to
o
estimate kinematic as well as dynamic viscosities at 25 C. In each measurement, the
uncertainty of the viscosity is 0.006 mPas. Titrations were performed for each derivative
(5µM), while it was added to CT-DNA solution (50 µM). 5µM of Ethidium bromide,
Doxorubicin and Hoechst 33258 solution was used as controls in the experiment. DNA
solution was prepared in 100 mM Tris-HCl (pH 7.0). Data were presented as (ŋ/ŋo)1/3 versus
the ratio of the concentration of the hybrid to CT-DNA, where ŋ is the viscosity of CT-DNA
in the presence of hybrid and ŋ0 is the viscosity of CT-DNA alone.
4.2.11 Molecular modelling studies
The title compounds were built using molecular builder of Maestro 10.4, prepared using
Ligprep 3.6 and geometrically minimized with Macromodel 11.0 based on OPLS-2005 force
field followed by conformational analysis. Truncated Newton Conjugate Gradient (TNCG)
minimization method was used with 500 iterations and convergence threshold of 0.05 kJ/mol.
The DNA duplex was prepared using protein preparation wizard. The Glide XP 6.9 algorithm
was employed using a grid box volume of 10x 10x 10 Å.
Acknowledgements
N.P.K. and P.S 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).
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Tables/Figures/Scheme captions
Table 1. In vitro cytotoxic activity (IC50 in µM)a of phenanthrene-9-benzimidazole
conjugates 10a–q
Figure 1. Structures of phenanthrene alkaloid, benzimidazole containing cytotoxic
compounds and the designed phenanthrene-9 benzimidazole conjugates.
Figure 2. Phase contrast imaging: Effect of compound 10o on the viability of PC-3 cells.
Figure 3. PC-3 cells were treated with various concentrations of the compound 10o and
stained with AO/EB. Apoptotic charecteristics such as apoptotic bodies, membrane blebbing
and dead cells were clearly observed.s
Figure 4. Nuclear morphology on PC-3 cells stained with DAPI. PC-3 cells treated with
compound 10o for 24 h were stained with DAPI. The images were captured with
fluorescence microscope with a DAPI filter.ss
Figure 5 (A and B). Cell cycle analysis of PC-3 cancer cells treated with 10o for 24 h. The
cell cycle distribution was analysed by flow cytometry using propidium iodide staining
method.
Figure 6. Effect of the compound 10o on Reactive oxygen species (ROS) levels. Dose
dependent increment of fluorescence was observed compared to control.
Figure 7. Effect of 10o on the Mitochondrial membrane potential (DΨm). PC-3 cells were
treated with 2.5 µM, 5.0 µM and 10.0 µM of 10o, incubated with JC-1 and analysed by flow
cytometer (BD FACSVerseTM, USA).
Figure 8. Annexin binding assay for the detection of apoptosis. The compound 10o treated
cells were stained with Annexin V-FITC/PI and analysed for apoptosis. 10,000 cells from
each sample were analysed by flowcytometry. The percentage of cells positive for Annexin
22

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V-FITC and/or Propidium iodide is represented inside the quadrants. Cells in the upper left
quadrant (Q1-UL; AV-/PI+): necrotic cells; lower left quadrant (Q1-LL; AV-/PI-): live cells;
lower right quadrant (Q1-LR; AV+/PI-): early apoptotic cells and upper right quadrant (Q1-
UR; AV+/PI +): late apoptotic cells.
Figure 9. Relative viscosity experiment of hybrids 10n and 10o with CT-DNA. Ethidium
Bromide and Hoechst 33258 were used as controls. Data represents average from three
individual experiments.
Figure 10. Side view (A,C) and top view (B,D) of the intercalation of 10n (yellow) and 10o
(green), respectively between G–C base pairs of d(GAAGCTTC)2.
Scheme 1. Synthesis of different phenanthrene-9-benzimidazole conjugates 10a–q.
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Tables/Figures/Schemes
Table 1. In vitro cytotoxic activity (IC50 in µM)a of phenanthrene-9-benzimidazole
conjugates 10a–q
MDA-MB-
Compound
BT-549b
HCT-116d
PC-3e
DU145f
HCT-15g
453c
>20
10a
10b
>20
>20
>20
>20
12.8±0.24
15.7±0.11
18.9±0.31
14.6±0.19
10.2±0.25
16.7±0.72
13.1±0.42
>20
18.9±0.81
>20
>20
>20
15.1±0.16
9.42±0.14
>20
10c
>20
>20
>20
>20
10d
10.8±0.06
>20
>20
>20
9.42±0.21
8.34±0.15
>20
10e
10.6±0.32
>20
9.92±0.82
>20
12.7±0.19
17.2±0.42
>20
10f
>20
10g
11.5±0.41
>20
9.98±0.95
>20
8.91±0.72
>20
10.15±0.2
>20
10h
8.21±0.5
>20
10i
7.15±0.9
>20
>20
>20
18.3±0.51
>20
>20
10j
>20
>20
10±0.35
>20
>20
10k
>20
17.3±0.31
16.5±0.15
>20
>20
11.5±0.17
12.8±0.08
>20
10.31±0.4
15.6±0.16
>20
10l
>20
>20
>20
10m
10n
11.4±0.35
14.2±0.26
13.2±0.24
>20
>20
17.2±0.42
>20
>20
11.3±0.46
>20
7.24±0.98
6.32±0.09
>20
>20
10o
6.61±0.9
7.20±0.18
>20
>20
>20
10p
>20
>20
14.1±0.61
>20
10q
7.91±0.23
-
>20
11.3±0.46
1.84±0.1
1.2±0.01
>20
Nocodazoleh
5-FUi
0.91±0.07
-
1.06±0.21
8.62±0.87
1.14±0.05
2.18±0.33
-
-
-
Doxorubicinj
-
1.99±1.75
1.91±0.13
-
1.01±0.09
0.19±0.01
b
c
a50% inhibitory concentration after 48 h of compound treatment; Breast cancer cells; triple negative breast cancer cells; d,gColon cancer
cells; e, fprostate cancer cells; hnocodazole, i5-fluorouracil: positive control, jdoxorubicin: standard drug.
24

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OMe
2
3
MeO
MeO
O
O
1
H
14
13a
10
13
11
H
4
N
NH
N
O
12
N
5
N
N
O
N
9
N
OH
6
S
8
7
OMe
Nocodazole
Telmisartan
(-)-R- Tylophorine
R
MeO
MeO
OH
N
HN
R1
N
NH
N
N
O
NH
HN
N
R2
Cl
OMe
Designed phenanthrene-9-benzimidazoles
N
HOECHST 33258
quionolinone benzimidazoles
N
Figure 1. Structures of phenanthrene alkaloid, benzimidazole containing cytotoxic
compounds and the designed phenanthrene-9 benzimidazole conjugates.
Figure 2. Phase contrast imaging: Effect of compound 10o on the viability of PC-3 cells.
25

ACCEPTED MANUSCRIPT
Figure 3. PC-3 cells were treated with various concentrations of the compound 10o and
stained with AO/EB. Apoptotic charecteristics such as apoptotic bodies, membrane blebbing
and dead cells were clearly observed.
Figure 4. Nuclear morphology on PC-3 cells stained with DAPI. PC-3 cells treated with
compound 10o for 24 h were stained with DAPI. The images were captured with
fluorescence microscope with a DAPI filter.
26

ACCEPTED MANUSCRIPT
Figure 5 (A and B). Cell cycle analysis of PC-3 cancer cells treated with 10o for 24 h. The
cell cycle distribution was analysed by flow cytometry using propidium iodide staining
method.
27

ACCEPTED MANUSCRIPT
Figure 6. Effect of the compound 10o on Reactive oxygen species (ROS) levels. Dose
dependent increment of fluorescence was observed compared to control.
Figure 7. Effect of 10o on the Mitochondrial membrane potential (DΨm). PC-3 cells were
treated with 2.5 µM, 5.0 µM and 10.0 µM of 10o, incubated with JC-1 and analysed by flow
cytometer (BD FACSVerseTM, USA).
28