Synthesis of New 1,2,3-Triazolo-naphthalimide/phthalimide Conjugates via ‘Click’ Reaction: DNA intercalation and cytotoxic studies

Article abstract of DOI:10.21577/0103-5053.20180111

Cancer is a complex disease which involves abnormalities of multiple cellular pathways. Current chemotherapeutic drugs are mainly designed to target the DNA and cell division. Therefore, in the present study, we have synthesized a new series of 1,2,3-triazolo-naphthalimide/phthalimide conjugates and evaluated their in vitro cytotoxicity against selected human cancer cells. Among the tested compounds, one of them displayed notable cytotoxic activity against A549 lung cancer cells with an IC₅₀ (half maximal inhibitory concentration) value of 7.6 ± 0.78 μM. To determine the effect of this compound on cell viability, acridine orange/ethidium bromide (AO/EB) and 4’,6-diamidino-2-phenylindole (DAPI) staining studies were performed. These apoptotic features were clearly indicating that the compound inhibited cell proliferation by apoptosis. Further, relative viscosity measurements and molecular docking studies with the most three active compounds indicated that these new compounds bind to DNA by intercalation.

Full text of DOI:10.21577/0103-5053.20180111

http://dx.doi.org/10.21577/0103-5053.20180111
J. Braz. Chem. Soc., Vol. 00, No. 00, 1-8, 2018
Printed in Brazil - ©2018 Sociedade Brasileira de Química
Article
Synthesis of New 1,2,3-Triazolo-naphthalimide/phthalimide Conjugates
via ‘Click’ Reaction: DNA Intercalation and Cytotoxic Studies
Nagula Shankaraiah,*,a Niggula P. Kumar,a Ramya Tokala,a Bulusu S. Gayatri,a
Venu Tallab and Leonardo S. Santos*,c
aDepartment of Medicinal Chemistry,
National Institute of Pharmaceutical Education and Research (NIPER),
500 037 Hyderabad, India
bDepartment of Pharmacology and Toxicology,
National Institute of Pharmaceutical Education and Research (NIPER),
500 037 Hyderabad, India
cLaboratory of Asymmetric Synthesis, Chemistry Institute of Natural Resources,
University of Talca, P.O. Box 747, Talca, Chile
Cancer is a complex disease which involves abnormalities of multiple cellular pathways. Current
chemotherapeutic drugs are mainly designed to target the DNA and cell division. Therefore, in
the present study, we have synthesized a new series of 1,2,3-triazolo-naphthalimide/phthalimide
conjugates and evaluated their in vitro cytotoxicity against selected human cancer cells. Among
the tested compounds, one of them displayed notable cytotoxic activity against A549 lung cancer
cells with an IC50 (half maximal inhibitory concentration) value of 7.6 0.78 µM. To determine
the effect of this compound on cell viability, acridine orange/ethidium bromide (AO/EB) and
4’,6-diamidino-2-phenylindole (DAPI) staining studies were performed. These apoptotic features
were clearly indicating that the compound inhibited cell proliferation by apoptosis. Further, relative
viscosity measurements and molecular docking studies with most three active compounds indicated
that these new compounds bind to DNA by intercalation.
Keywords: naphthalimides, 1,2,3-triazoles, DNA intercalation, click reaction
Introduction
highly proliferative tissues, the DNA becomes one of the
most promising biological targets to develop antitumor
agents.4 DNA replication has an invaluable role in cancer
cell division, hence polycyclic planar molecules such
as doxorubicin, acridines, anthraquinones, distamycins,
naphthalimides and phenanthrene derivatives are well
recognized as DNA targeting antitumor agents.5
Naphthalimides are aromatic heterocycles with profound
biological significance and they serve as core scaffold
for many anti-tumor, anti-inflammatory, antidepressant,
antiprotozoal and antiviral compounds.6 Planarity is the
most important prerequisite for DNA intercalation and
also facilitates embedding into DNA base pairs.7 Owing to
their tricyclic planar structure, naphthalimide is primarily
responsible for its intercalation with DNA to perturb the
cellular events, thereby prevents cancer cell divison.8
Representative examples of naphthalimide based molecules
such as mononaphthalimide (i.e., amonafide, Figure 1) and
Cancer is considered as one of the lethal diseases
worldwide and the incidence of cancer has been rising
in major regions of the world with predicted substantive
increase to 19.3 million by the year 2025.1 Although the
scientificadvancesfocusedonfindingexactpathophysiology
of the disease and tremendous efforts have been made on
early detection of cancer, the overall mortality rate has not
declined.2 In this connection, there is an immense need
to design and synthesize effective new chemotherapeutic
agents to combat cancer. Due to complexity with redundant
and robust biological networks, cancer cells exhibits
uncontrolled cell proliferation, metastasis and also
become resistant to apoptotic signals.3 As cancer cells are
*e-mail: shankarnbs@gmail.com, shankar@niperhyd.ac.in;
lssantos@utalca.cl

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Synthesis of New 1,2,3-Triazolo-naphthalimide/phthalimide Conjugates via ‘Click’ Reaction
J. Braz. Chem. Soc.
bisnaphthalimides (i.e., elinafide) have reached the clinical
trials, but due to central neurotoxicity and limited efficacy
in solid tumors, the clinical development was regrettably
terminated.9 However, their structural modification lead
to potent antitumor agents with improved efficacy and
toxicological profile.10 In 2005, Cholody et al.11 investigated
asymmetrical bi-functional antitumor agents by conjugating
imidazoacridone moiety to the naphthalimide core. This
imidazoacridone linked nitro naphthalimide conjugate
efficiently interacted with DNA and induction of
apoptosis. Moreover, naphthalimides conjugated with
pyrrolo[2,1-c][1,4]benzodiazepines through a pyrazine
moiety with an alkane spacer has also been proved to
increase the DNA binding affinity and anticancer activity.12
In the dimeric form of intercalators such as
bisphenanthridines, bisnaphthalimides and bisphenazine
carboxamides have also displayed potent antitumor
potential. The promising in vivo activities of drugs
such as bisnafide (bisnaphthalimide)13 and WP631
(bisanthracyclines)14 have stimulated to search for novel
bifunctional DNA ligands. The basic principle behind the
design of dimers is to enhance sequence recognition and
DNA binding affinity. However, in some instances, the
second chromophore does not play the role of intercalator,
instead it can serve as a hook to trap DNA binding proteins
and cofactors.
anti-allergic, antifungal, antibacterial, antitubercular, anti-
HIV, anticancer, antimalarial and antiviral properties.16
Fascinatingly, it can interact with DNA and also acts
as a supporting motif for DNA targeting drugs.17
1,2,3-Triazoles tethered to various bioactive molecules
such as β-carbolines and phenanthrenes also resulted
in enhanced antitumor potential through effective DNA
intercalation.18 Quian and co-workers19 have reported
that 1,8-naphthalimide substituted triazole derivatives
could enhance the cytotoxicity and improves the affinity
towards calf thymus (CT)-DNA. The enhanced cytotoxicity
has been attributed to the presence of triazole nucleus.
Prompted by the above reports, in the present study we have
designed and synthesized a library of 1,2,3-triazolo based
naphthalimide/phthalimide conjugates and evaluated their
in vitro cytotoxicity and DNA intercalating capabilities.
The CuI-catalyzed 1,3-dipolar cycloaddition of
azide and alkynes (CuAAC) or ‘click chemistry’ can
rapidly yield bioactive molecules linked through
1,2,3-triazole.20 CuI catalyst allows the cycloaddition to
provide 1,4-regioselectivity in 1,2,3-triazole ring formation.
Currently, the most widely used reaction conditions for
the 1,4-regiospecific method is the use of an organic
solvent, such as t-BuOH or CH2Cl2, water, CuSO4.5H2O
and sodium ascorbate. Despite the numerous advantages
of the CuAAC reactions, the organic azides employed
therein are hazardous and their handling is very risky.21
In this context, efficient protocols that eliminate the
handling of organic azides are highly preferable.22 In
continuation of our earlier efforts dedicated to triazole
chemistry towards the synthesis of diverse bioactive
molecules,23 recently, we have also reported a novel,
simple and efficient bis[(tetrabutylammonium)di-µ-iodo-
diiododicuprate(I)] catalyzed one-pot, three component
1,3-dipolar cycloaddition of alkyl/benzyl bromides with
On the other hand, 1,2,3-triazoles are considered as
privileged building blocks in the context of bioconjugates.
They are highly stable under basic and acid hydrolysis
including oxidative and reductive conditions. Moreover,
this heterocycle is the bioisostere of amide and is
capable of interacting with biomolecular targets through
hydrogen-bonding.15 This scaffold is well recognized
for their multitude of biological properties such as
anti-malarial, anti-leishmanial, anti-trypanosomal,
Figure 1. Structures of naphthalimide, triazole containing derivatives and newly designed 1,2,3-triazolo-naphthalimide conjugates.

Vol. 00, No. 00, 2018
Shankaraiah et al.
3
sodium azide and alkynes for the regioselective synthesis of
1,4-disubstituted 1,2,3-triazoles in water.24 Herein we have
applied this efficient protocol for the synthesis of a variety
of diversely substituted 1,2,3-triazolo-naphthalimide/
phthalimide conjugates. The advantages of this one-
pot reaction are the use of water as solvent and in situ
generation of azides from alkyl halides and thus handling
of hazardous azides is avoided. The newly synthesized
compounds were evaluated for their in vitro cytotoxicity
and DNA binding affinity.
provide the corresponding 1,4-disubstituted 1,2,3-triazoles.
Bis[(tetrabutylammonium)di-µ-iodo-diiododicuprate(I)]
was used as a CuI catalyst to promote this reaction in
water as reaction medium. The catalyst enhanced the water
solubility of the alkyl halides. In the presence of sodium
azide, naphthalimide and phthalimide azides are formed
in situ from N-alkylated naphthalimides and phthalimides,
respectively. Finally, different alkyne partners undergo
CuI-catalyzed azide-alkyne cycloaddition (CuAAC)
reaction with various naphthalimide/phthalimide azides
to give diverse 1,2,3-triazolo-naphthalimide/phthalimide
conjugates 6a-x and 7a-c.
Results and Discussion
Chemistry
Pharmacology
The syntheses of 1,2,3-triazole based naphthalimide/
phthalimide conjugates 6a-x, and 7a-c have been achieved
by a two-step reaction as depicted in Scheme 1. Initially,
alkylation followed by one-pot three component reaction
affords the desired 1,4-disubstituted-1,2,3-triazoles
in excellent yields. At first, 1,8-naphthalimide (1) and
phthalimide (2) were alkylated by using various dibromo
alkane spacers (like 1,3-dibromo propane, 1,4-dibromo
butane, 1,5-dibromo pentane and 1,6-dibromo hexane) in
the presence of K2CO3 and dimethylformamide (DMF)
was used as solvent, which resulted in the formation of
various N-alkylated naphthalimides 3a-c and phthalimide 4
precursors. Simultaneously, various N- and O-linked
alkyne partners were also prepared by propargylation of
substituted amines and phenols, respectively. Finally, one-
pot three component 1,3-dipolar cycloaddition reaction was
performed with various synthesized alkyne partners 5a-i to
Cytotoxic activity
The newly synthesized 1,2,3-triazolo-naphthalimide/
phthalimide conjugates were evaluated for their in vitro
cytotoxicity against different cancer cell lines such as
lung (A549), prostate (PC-3), breast (MCF-7), cervical
cancer (HeLa) and a normal cell line (RPE1) by using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay.25 Cell lines were treated with the
compounds at 50 µM concentration for 48 h and growth
inhibition percentage was noted. Among them, some of
the representative compounds 6b, 6c, 6d, 6j, 6k, 6p, 6u,
6v and 7a displayed cytotoxicity above 50% at 50 µM and
were selected to screen for the dose dependent responses
and IC50 (concentration required to inhibit 50% of the
tumor cells) values for these active compounds were
reported. The IC50 (µM) values of the active compounds
and the reference drug doxorubicin are listed in Table 1.
Scheme 1. Synthetic strategy for the preparation of 1,2,3-triazolo-naphthalimide/phthalimide conjugates 6a-x, 7a-c.

4
Synthesis of New 1,2,3-Triazolo-naphthalimide/phthalimide Conjugates via ‘Click’ Reaction
J. Braz. Chem. Soc.
It is observed from the cytotoxicity results that derivatives
containing planar phthalimide or naphthalimide nucleus
in their structures showed moderate cytotoxicity and
compound 6c, possessing naphthalimide and triazole cores
linked through a 4 carbon linker, displayed good cytotoxic
activity on A549 lung cancer cells with an IC50 value of
7.6 0.78 µM (Table 1). Some of the tested compounds
6d, 6k, 6u, and 7a displayed moderate cytotoxicity against
the cervical and prostate cancer cells. From careful analysis
of IC50 values, it was observed that most of the dimers
containing phthalimide or naphthalimide scaffolds linked
through various triazole linkers were adequately active
against the cancer cell lines. Further, cytotoxicity of the
most active compounds was also tested on normal cell line
(RPE1). Interestingly, most of the compounds were non-
toxic to the normal cells and the most active compound 6c
showed selectivity index (ratio of cytotoxicity of normal
cell line to that of cancer cells) of 6 folds approximately.
Based on the observed cytotoxicity results, one of the active
compounds 6c was taken-up for examining the mechanism
of cancer cell growth inhibition through cell staining
techniques. Additionally, to prove the DNA interaction
of these planar molecules, we have also investigated the
relative viscosity alterations of these new molecules 6c, 6d,
6p and performed molecular docking studies to explain the
possible mode of interaction with DNA.
It can be observed from Figure 2 that the green color of the
control cells is due to their normal morphology. From the
Figure, it was clear that the cell viability is decreased in
concentration dependent manner. Fluorescence microscopic
images of the cells treated with 20 µM of compound 6c
clearly showed the altered morphological characteristics,
suggesting that the compound 6c induced cell death in
A549 cells.
DAPI staining
DAPI (4’,6-diamidino-2-phenylindole) is a fluorescent
stain that effectively binds toA-T rich regions in DNA and
reveals the nuclear damage. DAPI permeates the live cell
membrane with less efficiency. Therefore, the intensity of
staining in live cells is lesser, whereas apoptotic cells are
stained bright due to the presence of condensed nucleus.
Therefore, we have examined the effect of compound 6c
on A549 cells by using DAPI staining.27 This technique
differentiates dead and live cells based on nuclear
morphology. DAPI stains the nucleus bright blue by
forming a fluorescent complex with chromatin.As observed
from Figure 3, the nuclear structure of control cells was
intact whereas compound 6c treated A549 cells exhibited
condensed nuclei, indicating the characteristic features of
cancer cell death.
Relative viscosity study
Acridine orange/ethidium bromide (AO/EB) staining
Acridine orange/ethidium bromide (AO/EB) fluorescent
staining assay was performed to differentiate the live,
apoptotic, and necrotic cells.26 AO penetrates the healthy
cells and stains the nuclei green, whereas EB enters only
into membrane disintegrated cells and stain the nucleus red.
Relative viscosity studies are helpful to predict the
interactions of tested compounds with DNA. These
studies were performed for the active compounds 6c, 6d,
and 6p along with ethidium bromide, Hoechst 33258,
and doxorubicin to study their interactions with DNA by
measuring the changes in relative viscosity. Intercalation
Table 1. Cytotoxic activity of some of the 1,2,3-triazolo based naphthalimide/phthalimide conjugates
IC50a / µM
Compound
A549b
MCF-7c
> 50
PC-3d
> 50
HeLae
> 50
RPE1f
72.7 0.88
46.5 1.31
67.9 0.57
−
6b
21.3 1.43
7.6 0.78
17.0 1.39
27.9 1.53
24.1 0.52
19.1 1.26
> 50
6c
> 50
> 50
> 50
6d
> 50
> 50
44.8 2.34
> 50
6j
> 50
> 50
6k
> 50
> 50
33.6 3.85
> 50
89.1 0.04
83.6 0.47
−
6p
> 50
> 50
6u
> 50
34.2 2.65
> 50
> 50
6v
41.7 2.79
25.1 1.83
1.88 0.56
> 50
> 50
−
7a
> 50
45.1 3.28
0.08 0.001
> 50
−
Doxorubicing
10.9 1.76
0.36 0.02
13.9 1.10
a50% inhibitory concentration after 48 h of treatment with compound; blung (A549); cbreast (MCF-7); dprostate (PC-3); ecervical (HeLa) cancer cell lines;
fnormal retinal cells (RPE1); greference compound.

Vol. 00, No. 00, 2018
Shankaraiah et al.
5
Figure 2. AO/EB staining in A549 cells treated with various concentrations of compound 6c. Apoptotic features and dead cells were observed.
Figure 3. DAPI staining: A549 cells treated with compound 6c and stained with DAPI. The images were captured with fluorescence microscope with a
DAPI filter.
of small molecules like ethidium bromide between the
base pairs of DNA leads to the substantial increase
in viscosity; whereas a minimal change in viscosity
is observed with groove binders like Hoechst 33258.
Doxorubicin is a well-known intercalator, which shows
a rapid increase in viscosity down the concentration. The
addition of these compounds 6c, 6d, and 6p to CT-DNA
results in a gradual increase of viscosity in concentration

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Synthesis of New 1,2,3-Triazolo-naphthalimide/phthalimide Conjugates via ‘Click’ Reaction
J. Braz. Chem. Soc.
depending manner. This infers that the naphthalimide-
triazole congeners bind to DNA by intercalation. The
Figure 4 shows a graph plotted between concentration
(compound/CT-DNA) and viscosity ((η/η0)1/3) on x and
y-axis, respectively, with ethidium bromide, Hoechst
33258 and doxorubicin as standards.
Molecular docking
Molecular docking studies were performed for the
most active compounds 6c, 6d and 6p using duplex DNA
(PDB: 209D)28 by XP Glide 7.4 (Schrödinger 2017-1)29
with default settings (Figure 5). The perfect planar insertion
of the naphthalimide ring between G-C base pairs in the
site of intercalation was observed. Further, the triazole and
alkyl chain acts as the linker and orients the complementary
substitution (naphthalimide in 6c and 6p and phthalimide
moiety in 6p) into the minor groove. Moreover, the docked
poses were stabilized by π-π stacking interactions of
naphthalimide ring and hydrogen-bonding interactions
between the carbonyl oxygens of naphthalimide towards
base pairs of DNA in the minor groove. Thereby, dual mode
of binding with DNA (DNA intercalation and minor groove
binding) was observed with these compounds supporting
their efficacy as DNA interacting agents. These results
Figure 4. Graphical representation of relative viscosity studies using
compounds 6c, 6d and 6p with doxorubicin, Hoechst-33258, and ethidium
bromide as reference standards.
were compatible with in vitro cytotoxicity and relative
viscosity studies.
Conclusions
In the present work, a new series of 1,2,3-triazolo-
naphthalimide/phthalimide conjugates were synthesized
by employing CuI complex. This protocol involved
Figure 5. Side view (a) and top view (b) of 6c; side view (c) and top view (d) of 6d; side view (e) and top view (f) of 6p, interaction with duplex DNA.
DNA is represented as ribbon in blue color and compounds 6c, 6d and 6p are represented in yellow, green and orange color, respectively.

Vol. 00, No. 00, 2018
Shankaraiah et al.
7
three component 1,3-dipolar cycloaddition of alkyl/
benzyl bromides, sodium azide and alkynes. The
synthesized compounds were evaluated for their in vitro
cytotoxic potential against lung (A549), prostate (PC-3),
breast (MCF-7) and cervical cancer (HeLa) cell lines.
Compound 6c displayed IC50 of 7.6 0.78 µM against
A549 lung cancer cells. Compound 6c also caused the
death of cancer cells and induced apoptosis in concentration
dependent manner as observed from DAPI and AO/EB
staining. Further, relative viscosity studies indicated that
increase in concentration of compounds 6c, 6d and 6p
proportionally enhanced viscosity suggesting that these
compounds binds to DNA by intercalation. Molecular
docking studies also supported the DNA interaction of
these compounds. Overall, the current study established
that these derivatives have the potential to be advanced as
DNA interactive agents for the treatment of lung cancer.
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Supplementary Information
1
Spectroscopic data and H and 13C NMR spectra
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1743.
of 6a-x and 7a-c are available free of charge at
http://jbcs.sbq.org.br as PDF file.
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Acknowledgments
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We are thankful to DoP, Ministry of Chemicals &
Fertilizers, Government of India, New Delhi, for the award
of NIPER fellowship. Dr N. Shankaraiah is thankful to
SERB, DST, Government of India for the start-up grant
(YSS-2015-001709). Dr Santos thanks FONDECYT
(1180084) and PIEI-Utalca (Qui-Bio) for supporting
research activities.
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Submitted: March 23, 2018
Published online: June 11, 2018
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