Design and synthesis of 1,2,3-triazolo-phenanthrene hybrids as cytotoxic agents

Article abstract of DOI:10.1016/j.bmcl.2017.04.022

A series of new 1,2,3-triazolo-phenanthrene hybrids has been synthesized by employing Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. These compounds were evaluated for their in vitro cytotoxic potential against various human cancer cell line

Full text of DOI:10.1016/j.bmcl.2017.04.022

Accepted Manuscript
Design and synthesis of 1,2,3-triazolo-phenanthrene hybrids as cytotoxic agents
Niggula Praveen Kumar, Shalini Nekkanti, S. Sujana Kumari, Pankaj Sharma,
Nagula Shankaraiah
PII:
S0960-894X(17)30386-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.04.022
BMCL 24875
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
8 February 2017
24 March 2017
6 April 2017
Please cite this article as: Kumar, N.P., Nekkanti, S., Sujana Kumari, S., Sharma, P., Shankaraiah, N., Design and
synthesis of 1,2,3-triazolo-phenanthrene hybrids as cytotoxic agents, Bioorganic & Medicinal Chemistry Letters
(2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.04.022
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Bioorganic & Medicinal Chemistry Letters
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Design and synthesis of 1,2,3-triazolo-phenanthrene hybrids as cytotoxic agents
Niggula Praveen Kumar,a Shalini Nekkanti,a S. Sujana Kumari,b Pankaj Sharma,a Nagula Shankaraiaha*
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
ARTICLE INFO
ABSTRACT
Article history:
Received
A series of new 1,2,3-triazolo-phenanthrene hybrids has been synthesized by employing Cu(I)-
catalyzed azide-alkyne cycloaddition (CuAAC) reaction. These compounds were evaluated for
their in vitro cytotoxic potential against various human cancer cell lines viz. lung (A549),
prostate (PC-3 and DU145), gastric (HGC-27), cervical (HeLa), triple negative breast cancer
(MDA-MB-231, MDA-MB-453) and breast (BT-549, 4T1) cells. Among the tested compounds,
7d displayed highest cytotoxicity against DU145 cells with IC50 value of 1.5 0.09 µM. Further,
the cell cycle analysis shown that it blocks G0/G1 phase of the cell cycle in a dose dependent
manner. In order to determine the effect of compound on cell viability, phase contrast
microscopy, AO/EB, DAPI, DCFDA and JC-1 staining studies were performed. These studies
clearly indicated that the compound 7d inhibited the cell proliferation of DU145 cells. Relative
viscosity measurements and molecular docking studies indicated that these compounds bind to
DNA by intercalation.
Revised
Accepted
Available online
Keywords:
1,2,3-Triazoles
Phenanthrene
Cytotoxicity
Click reaction
DNA interaction
Heterocyclic scaffolds capable of DNA targeting are the most
promising agents in the anticancer drug discovery process.1
Intercalation is one of the major modes of drug-DNA interaction
that is exhibited by various potent natural and synthetic antitumor
agents such as doxorubicin, acridines, anthraquinones,
distamycins, and phenanthrene derivatives.2 Combilexins are a
class of DNA binding agents that exert their mode of action via
both minor groove binding and intercalation. They are capable of
strong binding with DNA than any other class of DNA binders
due to their dual mode of binding.3 Phenanthroindolizidine
alkaloids were originally isolated from plants of the
Asclepiadacea family,4 and are well-known to exhibit diverse
pharmacological activities such as antitumor,5 anti-
inflammatory,6 anti-microbial,7 antifungal,8 antiarthritis,9
antilupus,10 antiviral11 and anti-angiogenic activities.12 The most
noteworthy biological property among these is the intense
cytotoxic activity against various cancer cell lines, including
multidrug-resistant cell lines.13 This can be attributed to the
inhibition of the enzymes involved in DNA synthesis14 and
intercalation of the phenanthrene core between the DNA base
pairs.15 Tylophorine and tylocrebrine (Figure 1) are the
representative natural alkaloids from this class.
the bioisostere of amide and is capable of interacting with
biomolecular targets through hydrogen-bonding.17 This attractive
chromophore displays
a wide variety of activities like
antibacterial, antifungal, antiallergic, anti-HIV, antitubercular,
anticancer, antiviral, antimalarial and anticonvulsant profile.18
Additionally, it can also interact with DNA and acts as a
supporting motif for DNA targeting drugs.19 The Cu(I)-catalyzed
azide-alkyne cycloaddition or ‘click reaction’ can rapidly yield
bioactive molecules linked through 1,2,3-triazoles20 with high
atom economy and has been found wide range of applications in
combinatorial synthesis,21 bio-conjugate chemistry22 and material
science.23
OMe
2
OMe
OMe
MeO
MeO
3
1
MeO
H
14
13
H
N
4
R
N
N
N
13a
N
10
12
11
5
6
MeO
MeO
9
8
MeO
MeO
7
OMe
OMe
(-)-R- Tylophorine
Tylocrebrine
New 1,2,3-triazolo-phenanthrene
hybrids
Figure 1. Structures of Tylophorine, Tylocrebrine and the newly synthesized
C-9 linked 1,2,3-triazolo-phenanthrene hybrids.
On the other hand 1,2,3-triazoles are regarded as privileged
building blocks for the synthesis of bioconjugates because of
their high stability, selectivity and less adverse reactions.16 They
are highly stable under basic and acid hydrolysis including
oxidative and reductive conditions. Moreover, this heterocycle is
The Structure Activity Relationships (SARs) revealed that the
substitution of a polar pharmacophore at C9-position of the
phenanthrene ring is of prime importance for potential antitumor
activity.24 Hence, the incorporation of the polar 1,2,3-triazole ring
at C9-position might effectively enhance the antitumor activity.
As part of our scientific contributions toward bioactive
scaffolds,25 herein we have designed and synthesized a new series
of 1,2,3-triazolo-phenanthrene hybrids by using diverse azide and
alkyne building blocks. The newly synthesized compounds 7a–o
Authors to whom correspondence should be addressed. ∗Corresponding
author; Dr. Nagula Shankaraiah, E-mail: shankar@niperhyd.ac.in;
shankarnbs@gmail.com

were further evaluated for their in vitro cytotoxicity, cell growth
inhibition and DNA interaction.
lines with IC50 values of 14.8 0.37 µM and 19.2 0.46 µM
respectively. It could be easily observed from the in vitro
cytotoxicity data that the hybrids with napthalimide and
phthalimide groups at C9-position of triazolo-phenanthrene
scaffold elevated the biological response. On the other hand,
compound 7o with a triazolo-benzodioxolo substitution also
displayed a broad range of activities on all the tested cancer cell
lines indicating the importance of methylene dioxy group as a
bioactive scaffold. In view of the encouraging results, the most
active compound 7d was taken-up for further studies like cell
growth inhibition and DNA interaction.
The synthetic route for the preparation of 1,2,3-triazolo-
phenanthrene hybrids 7a–o was outlined in Scheme 1. The azide
partner
6
was synthesized from 2,3,6,7-tetramethoxy
phenanthren-9-yl methanol (4). The intermediate 4 was prepared
from the commercially available starting materials 1 and 2 via the
conventional four step sequence according to the reported
procedure.26
Initially,
Perkin
condensation
of
3,4-
dimethoxybenzaldehyde (2) and 3,4-dimethoxy phenyl acetic
acid (1) resulted in the formation of 2,3-bis(3,4-
dimethoxyphenyl)acrylic acid. The acid was converted into its
OMe
MeO
methyl ester
3
followed by m-CPBA/TFA mediated
HOOC
MeO
H
O
intramolecular oxidative cyclization to give the tricyclic
phenanthrene ester. The ester functionality was reduced to
alcohol 4 by LiAlH4. The alcohol 4 was mesylated by using
mesyl chloride in the presence of Et3N to give 5. As this
intermediate is highly unstable, then it was immediately taken-up
for azidation reaction by using NaN3, refluxing in DMF to afford
9-(azidomethyl)-2, 3, 6, 7-tetramethoxyphenanthrene (6). Next
for the synthesis of new 1,2,3-triazolo-phenanthrene hybrids,
different substituted alkyne building blocks i.e., N- and O-linked
alkynes were prepared from propargylation of substituted amines
and phenols respectively. Finally, we tailored these azides and
alkynes by employing ‘CuAAC’ reaction to obtain the new
hybrids 7a–o in overall 63–90% yields. All the newly
synthesized compounds 7a–o were characterized by IR, HRMS,
1H and 13C NMR spectroscopy.
i) Ac2O, Et3N
reflux, 20 h, 66%
+
OCH3
ii) SOCl2, MeOH
reflux, 5 h, 80%
MeO
O
OMe
OMe
MeO
2
1
OMe
3
i) TFA, m-CPBA
CH2Cl2, rt, 3 h, 80%
ii) LiAlH4, THF
0 oC-rt, 2 h, 90%
OMe
OMe
MeO
MeO
MeO
MeO
MsCl, Et3N,
OH
OMs
CH2Cl2, 4 h, 58%
OMe
OMe
5
4
The newly synthesized 1,2,3-triazolo-phenanthrene hybrids
7a–o were evaluated for their in vitro cytotoxicity on nine
different cancer cell lines such as lung (A549), prostate (PC-3
and DU145), breast (BT-549 and 4T1), gastric (HGC-27),
cervical (HeLa) and triple negative breast cancer (MDA-MB-231
and MDA-MB-453) by using 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay.27 The IC50 (µM)
values (concentration required to inhibit 50% of the tumor cells)
of the tested compounds and the reference drug 5-fluorouracil (5-
FU) are listed in Table 1. It is noticeable from the initial
screening that the hybrids 7c, 7d, 7j and 7o displayed broad
range of activity against majority of the tested human cancer cell
lines with IC50 values ranging from 1.5 0.09 µM to 18.9 0.81
µM (Table 1). Among them, one of the compounds 7d with a
napthalimide substitution at C9-position linked through 1,2,3-
triazolo moiety was found to be more active on all the cancer cell
lines except A549 and exhibited highest potency on DU145 cell
line (IC50 1.5 0.09 µM). It was also observed that, the derivatives
7j and 7o were active against A549 cells with IC50 values of
12.3 0.42 and 14.5 0.61 µM respectively. Compounds 7c, 7d,
7e, 7g, 7j, 7l and 7o displayed activity less than 20 µM in PC-3
cells, among which the compound 7j possessing 1,2,3-triazolo
methyl phthalimide substitution proved to be best with an IC50 of
7.12 0.73 µM, followed by compound 7c with a 1,2,3-triazolo
pthalimide substitution (IC50 7.6 0.32 µM) and then compound
7d having 1,2,3-triazolo napthalimide substitution (IC50
8.5 0.82 µM). Except 7e, 7g, 7h, and 7m, all other derivatives
were found to be active on HGC-27 cell line and 7d being the
most active (IC50 3.5 0.24 µM) followed by 7i with the
substitution of 4-bromo phenyloxy group (IC50 3.9 0.16 µM).
Derivatives 7b, 7c, 7h, and 7o showed moderate activity on
MDA-MB-231 triple negative breast cancer cells with IC50 values
ranging from 6.8 0.13 to 19.2 0.46 µM. Interestingly,
compound 7o with a triazolo-benzodioxolo substitution was
found to be active on most of the tested cancer cell lines.
Compound 7b with a trimethoxy phenyl substitution proved to be
selectively cytotoxic against HGC-27 and MDA-MB-231 cell
NaN3, DMF,
80 oC, 6 h, 63%
OMe
OMe
MeO
MeO
N
R
N
N
R
N3
CuSO4.5H2O
Na-ascorbate
MeO
6
tert-BuOH:H2O MeO
(1:1), rt, 3 h
7a-o
OMe
OMe
OCH3
O
O
H3CO
O
R =
O
CH2
N
OCH3
OCH3
N
HC
O
7c
CH2
O
7a
7b
7d
O
O
OH
N
OH
Br
7e
7g
7f
7h
7i
H3C
Br
O
CH3
O
O
O
O
N
CH3
H3C
Br
O
7m
7l
7j
7k
O
O
O
O
O
7o
7n
Scheme 1. Synthesis of various C9-linked 1,2,3-triazolo-phenanthrene
hybrids 7a–o.

Table 1. Cytotoxic activity (IC50 in µM)a of 1,2,3-triazolo-phenanthrene hybrids 7a–o
MDA-MB-
231h
MDA-MB-
453i
Compound
A549b
PC-3c
DU145d
HGC-27e
HeLaf
BT-549g
4T1j
7a
7b
7c
7d
7e
7f
>20
>20
>20
>20
>20
>20
7.6 0.21
14.8 0.37
7.5 0.18
5.1 0.36
>20
7.12 0.71
>20
>20
>20
>20
>20
7.3 0.52
>20
19.2 0.46
11.8 0.29
4.2 0.14
>20
>20
>20
>20
7.6 0.32
8.5 0.82
13.9 0.38
>20
2.6 0.34
1.5 0.09
>20
12.6 0.31
2.9 0.19
>20
>20
11.3 0.46
4.2 0.29
>20
>20
3.7 0.22
>20
5.1 0.37
>20
>20
>20
>20
14.2 0.29
>20
17.2 0.42
>20
>20
>20
>20
>20
7g
7h
7i
>20
12.5 0.51
>20
>20
>20
>20
>20
10.8 0.21
>20
>20
>20
>20
18.3 0.51
3.8 0.32
2.7 0.31
21.1 1.12
5.15 1.14
17.6 0.42
11.2 1.23
>20
8.2 0.21
>20
6.8 0.13
>20
13.5 0.43
>20
>20
>20
>20
3.9 0.16
3.5 0.24
9.6 0.25
3.6 0.13
>20
>20
7j
12.3 0.42
>20
7.12 0.73
>20
1.87 0.12
>20
3.9 0.31
14.3 0.21
>20
4.6 0.25
>20
6.5 0.41
>20
10 0.35
9.3 0.32
7.4 0.21
6.2 0.24
>20
7k
7l
>20
14.1 0.61
>20
>20
>20
>20
7m
7n
7o
>20
>20
12.4 0.26
>20
>20
12.6 0.25
>20
>20
>20
>20
9.1 0.22
9.5 0.41
>20
14.5 0.61
13.2 0.45
16.7 0.72
18.1 0.38
12.6 0.27
18.9 0.81
6.5 0.23
5-FUk
Antofinel
1.60 0.10
1.2 0.01
-
2.18 0.33
14.72 0.42
-
1.86 0.02
-
-
-
4.33 0.02
-
-
-
-
0.022 0.07
0.025 0.05
0.012 0.02
a50% inhibitory concentration after 48 h of compound treatment; bLung cancer cells; c, dprostate cancer cells; eGastric cancer cells; fcervical cancer cells;
k
l
h,itriple negative breast cancer; g, j Breast cancer cells; 5-fluorouracil: standard for MTT assay. antofine: reference compound.
Phase contrast microscopic studies were performed to know
whether the treatment with these compounds could lead to
reduced cell viability and apoptosis induction.28 DU145 prostate
cancer cells were treated with 2 µM, 4 µM and 8 µM
concentrations of active compound 7d. Cells were observed and
images were captured by phase contrast microscope. It can be
known from Figure 2 that the increased concentration of
compound led to the decreased viable cells in comparison to
control as observed by morphological changes such as cell
detachment and nuclear shrinkage.
and clearly showed the altered morphological characteristics such
as membrane blebbing, chromatin condensation, cell shrinkage
and apoptotic body formation, suggesting that the compound 7d
induced cell death on DU145 cells.
Figure 3. AO/EB staining in DU145 cells treated with various concentrations
of compound 7d. Apoptotic features such as apoptotic bodies, membrane
blebbing and dead cells were clearly observed.
DAPI (4’,6-diamidino-2-phenylindole) is a fluorescent stain
that effectively binds to A-T rich regions in DNA and reveals the
nuclear damage or chromatin condensation. DAPI permeates the
live cell membrane less efficiency. 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 was considered of
interest to study the effect of compound 7d on DU145 cells by
using DAPI staining.30 DAPI stains the nucleus bright blue by
forming a fluorescent complex with chromatin. As observed from
Figure 4, the nuclear structure of control cells was intact;
whereas compound 7d treated DU145 cells exhibited horse-shoe
shaped and condensed nuclei, indicating the characteristic
features of apoptosis induction.
Figure 2. Effect of compound 7d on morphology and cell viability of DU145
cells.
In order to distinguish the apoptotic, necrotic and live cells,
Acridine orange/ethidium bromide (AO/EB) fluorescent staining
assay was performed.29 AO permeates the intact cells and stain
the nuclei green, whereas EB penetrates only membrane
disintegrated cells and stain the nucleus red.
It can be observed from Figure 3 that the control cells showed
green colour due to their normal morphology. Fluorescence
microscopic images of cells treated with compound 7d at 8.0 µM

Majority of the cytotoxic compounds exhibit their growth
inhibitory potential by arresting specific phase of a cell cycle.
Thus blockade of cell cycle progression by cytotoxic agents has
been considered as an effective strategy to develop potential
chemotherapeutic agent. Therefore, we studied the effect of
compound 7d on distribution of cell population in different
phases of cell cycle by flow cytometry analysis.31 DU145 cells
were treated with compound 7d at 1 µM, 2 µM, 4 µM and 8 µM
for 24 h, and ethanol fixed cells were stained with propidium
iodide and further subjected to flow cytometry analysis. The
results from Figure 5 represents that the ratio of DU145 cells in
G0/G1 phase increased from 64.1% in control to 71.1% at 1 µM,
73.3% at 2 µM, 80.3% at 4 µM and 88.6% at 8 µM respectively
and simultaneous decreased number of cells in G2/M phase.
Therefore, these results were clearly indicated that compound 7d
exerts G0/G1 phase arrest in DU145 cells.
The generation of ROS is a typical characteristic of many
chemotherapeutic agents by initiating oxidative damage to the
mitochondrial permeability and membrane potential. Hence,
DCFDA staining method32 has been used to assess the generation
of intracellular reactive oxygen species (ROS) of compound 7d
on DU145 cells. The treatment of compound 7d for 6 h resulted
in enhanced DCFDA fluorescence in a dose dependent manner,
indicating the capability of compounds in accumulating ROS
(Figure 6). On the other hand, decreased fluorescence intensity
was observed when DU145 cells treated with N-acetyl cysteine
(NAC) prior to compound treatment thus indicating the
compounds induced cytotoxicity by ROS generation. Moreover,
H2O2 treatment of DU145 cells led to the increased fluorescence
when compared to the control due to the generation of radicals.
Figure 4. Nuclear morphology in DU145 cells stained with DAPI. DU145
cells treated with compound 7d for 24 h were stained with DAPI. The images
were captured with fluorescence microscope with a DAPI filter.
Figure 6. Effect of compound 7d on Reactive oxygen species (ROS) levels.
Dose dependent increment of fluorescence observed compared to control.
Each bar represents mean SD from three independent experiments.
Enhanced ROS generation can lead to oxidative stress thereby
cause loss in mitochondrial membrane potential. Therefore, it is
of high importance to study the effect of compound 7d on
mitochondrial membrane potential (DΨm). Lipophilic cationic
JC-1 dye was used to determine the membrane potential.33
Normal polarised mitochondria stains red due to formation of J-
aggregates, whereas depolarised mitochondria of apoptotic cells
stains green because of J-monomers. DU145 cells were treated
with 2 µM, 4 µM and 8 µM of compound 7d for 24 h and stained
with JC-1 dye. The results from Figure 7 clearly displayed the
dose dependent increase in the green fluorescence, thus
indicating the loss of mitochondrial membrane potential (DΨm)
by the compound 7d.
Figure 5 (A and B). Cell cycle analysis of DU145 cancer cells treated with
compound 7d for 24 h. The cell cycle distribution was analysed by using
propidium iodide staining method and analysed by flow cytometry. Each bar
represents mean SD from three independent experiments.

Molecular docking simulations of the synthesized compounds
were performed using the DNA duplex with the sequence
d(GAAGCTTC)2 using XP Glide 6.9 (Schrödinger 2015-4).35 The
perfectly planar insertion of the tricyclic phenanthrene ring
system between the G–C base pairs in the intercalation site was
observed in the docked poses for one of the best scored
derivatives, 7d (Figure 9). Further, the 1,2,3-triazole moiety
participates in hydrogen bonding as well as in orienting the
molecule so that the naphthalimide scaffold lies in the minor
groove. The docked poses were also stabilized electronically by
π–π stacking, dipole-dipole interactions and hydrogen-bonds
between the carbonyl oxygens of the naphthalimide and the DNA
base pairs in the minor groove. Such a dual mode of binding i.e.,
intercalation and minor groove binding, effectively enhances the
DNA binding affinity of these scaffolds. The results thus
obtained from docking studies were consistent with the in vitro
cytotoxicity as well as relative viscosity data.
Figure 7. (A) Effect of 7d on Mitochondrial membrane potential. DU145
cells were treated with 2 µM, 4 µM and 8 µM of 7d. Cells staining orange
(JC-1 aggregates) have a healthy DΨm, while cells staining green (JC-1
monomers) are cells with a disrupted DΨm. (B) Quantitative measurement of
fluorescence
intensity
(JC-1
aggregates)
was
measured
by
spectrofluorometry.
Figure 9. Side view (A) and top view (B) of the intercalation of 7d between
G–C base pairs of d(GAAGCTTC)2.
Relative viscosity experiments were performed for the most
potent compounds 7d and 7j to study their form of interaction
with DNA.34 A substantial increase in the viscosity of DNA
solutions can be observed upon intercalation of small molecules
like Ethidium bromide, between the base pairs of DNA; whereas
only slight change in viscosity occurs in case of groove binders
such as Hoechst 33258. The relative viscosity of the complex
solutions has increased considerably on gradually increasing the
concentration of the compounds 7d and 7j. This indicates that the
synthesized triazolo-phenanthrene hybrids bind to DNA by
intercalation. Among the hybrids, 7d showed equivalent increase
in viscosity to that of Ethidium bromide upon its interaction with
DNA, signifying considerable DNA binding affinity (Figure 8).
In conclusion, a new series of C9-linked 1,2,3-triazolo-
phenanthrene hybrids has been synthesized by employing ‘click’
reaction. These compounds were evaluated for their in vitro
cytotoxic potential against various human cancer cell lines viz.
lung (A549), prostate (PC-3 and DU145), breast (BT-549 and
4T1), gastric (HGC-27), cervical (HeLa) and triple negative
breast cancer (MDA-MB-231 and MDA-MB-453). From the
initial screening, it was observed that some of the compounds
were active on the tested cancer cell lines below 20 µM. The
cytotoxicity profile indicated that compound 7d and 7j showed
broad range of activity on majority of the cancer cell lines at
below 10 µM. The compound 7d on DU145 cells resulted G0/G1
phase of cell cycle arrest in a dose dependent manner. AO/EB,
DAPI staining, ROS study and JC-1 staining clearly showed the
distinctive apoptotic features indicating that the compound
inhibited cell proliferation by apoptosis. Further compounds 7d
and 7j led to increased viscosity suggesting that these compounds
bind to DNA by intercalation. Molecular docking study also
supports the DNA interaction of these compounds. Overall, the
current study established that these new 1,2,3-triazolo-
phenanthrene hybrids have the potential to be advanced as DNA
interactive cytotoxic agents for the treatment of prostate cancer.
Acknowledgments
N. P. K and S. N are thankful to DoP, Ministry of Chemicals
& Fertilizers, Govt. of India, for the award of NIPER fellowship.
Dr. N. Shankaraiah gratefully acknowledges SERB, DST, Govt.
of India for research grant (YSS-2015-001709).
Figure 8. Relative viscosity experiment of hybrids 7d and 7j with CT-DNA.
Ethidium Bromide and Hoechst 33258 were used as controls. Data represents
mean from three individual experiments.

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