Design and synthesis of DNA-intercalative naphthalimide-benzothiazole/cinnamide derivatives: cytotoxicity evaluation and topoisomerase-IIα inhibition

Article abstract of DOI:10.1039/c8md00395e

A new series of different naphthalimide-benzothiazole/cinnamide derivatives were designed, synthesized and tested for their in vitro cytotoxicity on selected human cancer cell lines. Among them, derivatives 4a and 4b with the 6-aminobenzothiazole ring and 5g with the cinnamide ring displayed potent cytotoxic activity against colon (IC₅₀: 3.715 and 3.467 μM) and lung cancer (IC₅₀: 4.074 and 3.890 μM) cell lines when compared to amonafide (IC₅₀: 5.459 and 7.762 μM). Later, the DNA binding studies for these selected derivatives (by CD, UV/vis, fluorescence spectroscopy, DNA viscosity, and molecular docking) suggested that these new derivatives significantly intercalate between two strands of DNA. In addition, the most potent derivatives 4a and 4b were also found to inhibit DNA topoisomerase-II.

Full text of DOI:10.1039/c8md00395e

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RESEARCH ARTICLE
Design and synthesis of DNA-intercalative
naphthalimide-benzothiazole/cinnamide deriva-
tives: cytotoxicity evaluation and topoisomerase-
IIα inhibition†
Cite this: DOI: 10.1039/c8md00395e
c
N. Sankara Rao,ab Narayana Nagesh,
V. Lakshma Nayak,a Satish Sunkari,ab
*
Ramya Tokala, d Gaddam Kiranmai,c Phanindranath Regur,c
abe
d and Ahmed Kamal
*
*
Nagula Shankaraiah
A new series of different naphthalimide-benzothiazole/cinnamide derivatives were designed, synthesized
and tested for their in vitro cytotoxicity on selected human cancer cell lines. Among them, derivatives 4a
and 4b with the 6-aminobenzothiazole ring and 5g with the cinnamide ring displayed potent cytotoxic ac-
tivity against colon (IC50: 3.715 and 3.467 μM) and lung cancer (IC50: 4.074 and 3.890 μM) cell lines when
compared to amonafide (IC50: 5.459 and 7.762 μM). Later, the DNA binding studies for these selected de-
rivatives (by CD, UV/vis, fluorescence spectroscopy, DNA viscosity, and molecular docking) suggested that
these new derivatives significantly intercalate between two strands of DNA. In addition, the most potent
derivatives 4a and 4b were also found to inhibit DNA topoisomerase-II.
Received 13th August 2018,
Accepted 1st November 2018
DOI: 10.1039/c8md00395e
rsc.li/medchemcomm
A naphthalimide (NI) core is the most well-known unit
that exhibits DNA-intercalating properties and topoisomerase
Introduction
Nowadays, cancer is considered to be one of the most common
diseases which causes death and has always been an unsolved
problem worldwide. The development of selective, efficient and
safer drugs for cancer chemotherapy remains a high priority
and an unmet medical need. Therefore, discovery of new anti-
cancer agents with improved pharmacological profiles includ-
ing selectivity remains a fascinating challenge in the field of
cancer chemotherapy. DNA plays an important role in the pro-
cess of cell growth and division in a controlled manner, and
consequently this macromolecule continues to be one of the
main molecular targets in the design of antineoplastic agents.1
Moreover, DNA topoisomerase (topo) is recognized as an im-
portant cellular target in anticancer drug discovery.2–6
II inhibition,7 and considered as one of the most versatile
fluorophore units owing to its unique photophysical proper-
ties.8 These intercalators form and stabilize a ternary drug–
DNA–topoisomerase complex and it has been proposed that
these kinds of derivatives bind at the protein/DNA interface
in such a way that the planar ring system intercalates into
DNA, while the side chains would interact with the enzyme.9
The well-known DNA-interactive agents such as amonafide
(1) and mitonafide (2, Fig. 1) were the most potent
naphthalimide-based topo-II inhibitors.10–12 In particular,
amonafide showed potent antitumor activity against ad-
vanced breast cancer, but apparently, its clinical development
was regrettably terminated due to its poor therapeutic index
or dose-limiting bone marrow toxicity.13,14 Accordingly, exten-
sive efforts including the modification of the side chain, the
aromatic ring system, and the substituents on the ring have
been attempted to search for more selective naphthalimide
derivatives to improve the potency and reduce the adverse ef-
fects.15,16 Braña et al. have designed and synthesized a series
of heterocyclic fused naphthalimide derivatives as better cyto-
toxic agents than amonafide.17 Our research group has also
made significant attempts at synthesizing naphthalimide de-
rivatives to enhance cytotoxicity.18 As a continuation of our
efforts to develop effective anticancer agents, we designed
and synthesized a new series of naphthalimide-based deriva-
tives containing 2-aminobenzothiazole, 6-aminobenzothiazole
a Medicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500 007, India. E-mail: ahmedkamal@iict.res.in
b Academy of Scientific and Innovative Research. (AcSIR), CSIR-Indian Institute of
Chemical Technology, Hyderabad 500 007, India
c CSIR-Centre for Cellular and Molecular Biology, Hyderabad 500007, India
d Department of Medicinal Chemistry, National Institute of Pharmaceutical
Education and Research (NIPER), Hyderabad 500 037, India.
E-mail: shankar@niperhyd.ac.in
e School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi,
110062, India
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8md00395e
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Fig. 1 Lead naphthalimides (1 and 2) and newly designed naphthalimide derivatives 3a–e, 4a,b and 5a–m.
and cinnamides (Fig. 1), linked through a piperazine spacer
for establishing a better structure–activity relationship (SAR).
Most of these derivatives from this series exhibited superior
anticancer activities in comparison with amonafide against
human cancer cell lines and also acted as topoisomerase-II
inhibitors.
more potent forms, several derivatives of the compound were
designed and synthesized. The newly synthesized derivatives
3a–e, 4a,b and 5a–m were evaluated for their in vitro cytotoxic
activity against a panel of three human cancer cell lines,
namely HT-29 (colon), A549 (lung), and MCF-7 (breast), by
employing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-
lium bromide (MTT) assay.19 These cell lines were procured
from the National Centre for Cell Sciences (NCCS, Pune, In-
dia (ESI)†). The assay was performed thrice, and the average
of the cytotoxicity data, expressed as IC50 values, is summa-
rized in Table 1 and compared with that of amonafide as the
positive control. The screening results demonstrated that
these derivatives displayed promising cytotoxic activity with
IC50 values ranging from 3.46 to 34.15 μM against different
cancer cell lines compared with the parent compound 1. The
most active derivatives 4a and 4b showed IC50 values ranging
from 3.46–5.08 and 3.71–7.90 μM, respectively, on the tested
cancer cell lines. The antiproliferative activity of the hybrid
derivatives can be further characterized in terms of their sub-
stitution patterns. In particular, compounds 3b and 3e with
electron-donating groups (6-OMe and 5,6-dimethyl) on the
phenyl ring of benzothiazole show significant cytotoxic activ-
ity compared to amonafide. The cytotoxicity data (Table 1)
clearly indicate that 5g,j are more potent analogs that ex-
hibit significant antiproliferative activity with IC50 values
ranging from 3.80–4.78 and 4.17–6.39 μM against both the
HT-29 and A549 cancer cell lines. It is observed that com-
pound 5f with the 4-N,N-dimethyl cinnamic acid moiety is
more active against the HT-29 cancer cell line, whereas 5l
with the 3,5-difluoro cinnamic acid moiety is more active
against the A549 cancer cell line. Overall, these results dem-
onstrated that the amide derivatives achieved with
6-aminobenzothiazoles showed better activity compared to
2-aminobenzothiazoles. All the synthesized derivatives were
Results and discussion
Chemistry
The synthesis of
newly designed
naphthalimide-
amidobenzothiazole derivatives 3a–e, 4a–b and 5a–m is
depicted in Scheme 1. Naphthalimide ethylpiperazine (8) was
prepared in good yield by the reaction of 1,8-naphthalic anhy-
dride (6) with amino ethyl piperazine (7) in ethanol under re-
flux. Then naphthalimide ethylpiperazine (8) was esterified
using K2CO3 in dry DMF with α-bromoethyl acetate to furnish
naphthalimide ester (9). Hydrolysis of this ester with LiOH–
H2O afforded naphthalimide acid (10). Derivatives 3a–e were
successfully synthesized by the coupling of naphthalimide
acid (10) with different 2-aminobenzothiazoles 11a–e in the
presence of EDCI/HOBt and obtained in good yields. Simi-
larly, other derivatives 4a,b were synthesized using
6-aminobenzothiazoles 12a,b, as shown in Scheme 1. Later,
another series of new naphthalimide-cinnamide derivatives
5a–m was also synthesized, as shown in Scheme 1. These am-
ide derivatives were obtained by the acid amine coupling re-
action of naphthalimide ethylpiperazine (8) with different
trans-cinnamic acids 13a–m by using EDCI/HOBt and
obtained in good yields.
Biological studies
In vitro cytotoxicity evaluation. In an effort to define a
structure–activity relationship (SAR) for 1 and to identify
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Scheme 1 Synthesis of naphthalimide-amidobenzothiazole derivatives 3a–e, 4a,b and naphthalimide-cinnamide derivatives 5a–m.
selectively more potent in the HT-29 and A549 cell lines
compared to MCF-7.
bition with GI50 values ranging from 1.38–43.3 and 1.19–14.1
μM, respectively.
Further, the most potent derivatives 4a and 4b from this
series were selected for the NCI-60 cell line anticancer screen-
ing program by the National Cancer Institute (NCI), USA. Af-
ter preliminary screening on the tumor cell lines, these deriv-
atives were tested for five dose concentrations on a panel of
59 human tumor cell lines derived from 9 different cancer
types: leukemia, non-small lung, colon, CNS, melanoma,
ovarian, renal, prostate and breast. The results expressed as
GI50 values for the test compounds are illustrated in the ESI
as Table S1.† The five dose assay20 results revealed that these
two compounds possess significant anticancer activity
against different cancer cell lines with GI50 values in the
range of 1.19–43.3 μM. The most active compounds 4a and
4b in the MTT assay retained their activity in the NCI screen-
ing data. These compounds exhibited promising growth inhi-
DNA binding affinity studies
Circular dichroism (CD). Circular dichroism studies were
performed in order to analyze the interaction of DNA with
small molecules which indicated changes that are likely to
take place with respect to the DNA conformation. DNA is a
biological macromolecule, which is responsible for cell prolif-
eration and several other biological functions. Hence, the
interaction and changes in the DNA topology will provide suf-
ficient information on the nature of DNA–complex interac-
tions and conformational changes in the derivatives binding
to DNA. The CD spectrum was recorded while keeping the
CT-DNA concentration constant (15 μM) and increasing the
compound concentration from 15 μM up to 30 μM. The
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Table 1 In vitro cytotoxicity (IC50 values in μM) data of naphthalimide
derivatives 3a–e, 4a,b and 5a–m against a panel of human cancer cellsa
DNA. In the present study, addition of 4a,b and 5a,b deriva-
tives to CT-DNA results in positive CD, indicating continuous
hyperchromicity even at 1 : 1 and 1 : 2 stoichiometry of DNA :
compound. This suggests the stabilization of the DNA struc-
ture on compound interaction.21 The intensities of the nega-
tive band did not alter much; this indicates less effect on
DNA helicity during interaction. 4a,b and 5g exhibited lower
intensity with negative ICD in the region starting from 300
nm to 400 nm. The ICD exhibited by 4a,b and 5g derivatives
on interaction with CT-DNA in the 300 nm to 350 nm region
was shown separately in the inset. This indicates that the 4a,
b and 5g molecules possibly interact with and stabilize CT-
DNA. Having relatively less effect on the derivatives, the
intensity of the positive CD band was noticed when 5a was
added to CT-DNA, whereas 5g has brought a moderate
change in the positive CD band. The appearance of negative
ICD in 4a,b and 5g indicates that the derivatives may be
intercalating between the two strands of DNA. The CD spec-
tra of 4a,b, 5a and 5g on interaction with CT DNA are shown
in Fig. 2.
Compound
HT-29b
A549c
MCF-7d
3a
3b
3c
3d
3e
4a
4b
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
10.85 0.1
04.22 0.2
11.10 0.2
04.07 0.1
04.98 0.2
03.72 0.3
03.47 0.2
16.04 0.3
15.60 0.2
12.00 0.2
16.96 0.1
23.94 0.2
04.01 0.2
03.80 0.1
10.29 0.2
17.41 0.1
04.18 0.2
13.42 0.1
08.39 0.2
13.81 0.1
05.46 0.1
14.79 0.3
26.47 0.5
07.94 0.6
17.36 0.3
15.04 0.2
12.64 0.4
07.91 0.4
05.08 0.3
22.95 0.4
34.15 0.4
28.85 0.3
26.36 0.2
28.65 0.3
13.12 0.4
18.00 0.3
13.69 0.2
23.48 0.4
11.24 0.3
28.29 0.4
13.66 0.5
19.50 0.4
4.05 0.3
04.76
0
16.55 0.3
08.07 0.3
06.20 0.3
4.074 0.3
03.89 0.3
21.92 0.3
21.72 0.3
19.44 0.2
19.74 0.3
27.20 0.4
08.26 0.3
04.79 0.3
14.15 0.3
16.17 0.3
06.39 0.2
18.40 0.3
06.41 0.3
14.80 0.3
07.76 0.2
5m
Std
a
50% growth inhibitory concentration and the values are the average
b
of three individual experiments after 48 h of drug treatment. Colon
UV-visible spectral study
cancer – HT-29. c Lung cancer – A549. d Breast cancer – MCF-7.
These studies indicate the possible changes that occur in the
CT-DNA topology during the course of interaction of the com-
pounds with CT-DNA. The UV-vis spectral studies conducted
with CT-DNA and 4a exhibited prominent absorption bands
at 244 nm and 347 nm. Addition of 4a to 25 μM CT-DNA in
an equal amount showed a continuous decrease in the inten-
sity of the absorption bands. Of 244 nm and 347 nm
wavelength scan was done from 240 nm to 400 nm. The CD
spectrum of CT-DNA exhibits a positive band at 275 nm and
a negative band at 245 nm. These indicate π–π base stacking
and right-hand helicity, the characteristic profile of B-form
Fig. 2 The CD spectra of CT-DNA (240 to 400 nm) in 100 mM TE (pH 7.0, 25 °C). 15 μM and 30 μM 4a (A), 4b (B), 5g (C) and 5a (D) were added to
15 μM CT-DNA prepared in 100 mM TE (pH 7.0). The arrows indicate the direction in which the CD peaks move after the interaction of 4a,b, 5a
and 5g with CT-DNA. The ICD observed in the wavelength range starting from 300 nm to 350 nm is shown separately in the inset.
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absorption peaks, the absorption band at 244 nm exhibited a
slight bathochromic shift from 244 nm to 246 nm (about 2
nm red shift). Further, an isosbestic point was noticed at 248
nm and 299 nm when DNA interacted with 4a. On the other
hand, 4b exhibited absorption bands at 235 nm and 345 nm.
Similarly, the absorption band of 4b exhibited
hypochromicity with the addition of DNA. The
hypochromicity of the complex's absorption band is more evi-
dent at 345 nm than 235 nm. 5g exhibited absorption bands
at 231 nm and 356 nm. The UV-vis absorption band at 231
nm was more evident compared to those of 4a and 4b. Con-
tinuous hypochromicity without much bathochromic shift of
the absorption bands in the case of 4b and 5g and a slight
bathochromic shift in the case of 4a on the addition of DNA
and the CD results indicate that these derivatives probably
bind to DNA and they may intercalate with CT-DNA.22 A effect
is noticed in the UV-vis spectra of CT-DNA-5a. The Kd values
obtained when 4a,b, 5g and 5a complexes interact with CT-
DNA are 132 μM, 121 μM, 136 μM and 144 μM, respectively.
The UV-vis titration data were fitted to eqn (1) to obtain the
dissociation constant (Kd) taking into account the total
amount of the 1 : 1 ratio of the derivative : CT-DNA complex
formed, independent of the complex binding site(s). The Kd
monitored even at lower concentrations. The emission spec-
tra of 4a and 4b show a prominent peak at 407 nm, whereas
5a and 5g exhibit emission maxima at 586 nm. On continu-
ous addition of CT-DNA to 4a,b, 5a and 5g, the emission peak
intensity increased gradually. It was noticed that upon addi-
tion of CT-DNA, the fluorescence emission intensities of 5a
and 5g exhibited a higher increase when compared to those
of 4a and 4b. From these results, we speculate that all the de-
rivatives may come close to the DNA bases possibly through
an intercalative mode of interaction.24 The increase in emis-
sion intensity of a peak can be attributed to the non-
radioactive energy transfer between the excited fluorescent
derivatives and DNA bases, wherein these derivatives are the
donors and the DNA bases are the acceptors. Their spectra
are shown in Fig. 4.
Viscosity studies
The relative viscosity of DNA generally increases when a mole-
cule intercalates with DNA and this enhanced viscosity would
be due to the increase in the axial length of the DNA helix on
the molecule's intercalation. Hence, intercalation of a molecule
results in an increase of the viscosity of DNA solutions.25 Mean-
while, reduction in the relative viscosity is typically observed
with molecules binding covalently to DNA.26 Moreover, the vis-
cosity does not change or show a slight change when molecules
bind to the surface of DNA.27 According to the spectroscopic
studies, the active derivatives 4a,b and 5g interact with CT-
DNA; viscosity studies were also carried out to confirm the
intercalating nature of these derivatives with CT DNA. These
studies demonstrated that the viscosity of the DNA solution in-
creased upon the addition of these derivatives, wherein the
change in viscosity was larger in the case of 4b compared to 4a.
Similarly, between 5a and 5g, derivative 5a has a weaker inter-
action with CT-DNA compared to 5g. Therefore, this study also
values were calculated using the formula Kd
DNA]ijderivative]/[CT-DNA derivative].
= [CT-
The results obtained from the UV-vis spectral data reveal
that 4a,b and 5g interact with DNA possibly through interca-
lation between the two strands of CT-DNA. The UV-vis spectra
obtained for 4a,b, 5a and 5g are shown in Fig. 3.
Fluorescence spectroscopy
Fluorescence titration is widely used to understand the type
of interaction between DNA and small fluorescent molecules.
This method is also useful for studying the electronic envi-
ronment around the DNA–complex.23 Since 4a,b, 5a and 5g
are fluorescent substances, their interaction with DNA can be
Fig. 4 Fluorescence emission spectra of compounds 4a,b, 5a and 5g
(10 μM) titrated with increasing concentrations of CT-DNA (in multiples
of 0.5 μM). (A) 4a, (B) 4b, (C) 5a and (D) 5g. 10 μM 4a,b, 5a and 5g de-
rivatives were taken in the fluorescence cuvette and titrated with in-
creasing concentrations of CT-DNA (in multiples of 0.5 μM). The ar-
rows indicate the direction in which the absorption bands are moving.
Fig. 3 UV-vis absorption spectra of compounds 4a,b, 5a and 5g (25
μM) in 100 mM TE (pH 7.0, 25 °C). (A) 4a, (B) 4b, (C) 5a and (D) 5g. 25
μM 4a,b, 5a and 5g solution and 25 μM CT DNA were prepared in 100
mM TE (pH 7.0). The arrows indicate the direction in which the
absorption bands are moving.
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indicates that among the derivatives, 4a and 4b have stronger
interaction with CT-DNA which is closely followed by 5g.
Among the derivatives studied, 5a has the weakest interaction
with CT-DNA. The viscosity data indicate that among the deriv-
atives studied, 4a,b and 5g derivatives may intercalate with CT-
DNA. In the present study, the interaction between CT-DNA
and ethidium bromide (EtBr), a well-known intercalator, was
considered as the control. The viscosity of DNA increased con-
tinuously with the addition of ethidium bromide (EtBr), due to
the intercalation of EtBr with CT-DNA. The graph plotted
between (η/ηo)1/3 and compound/CT-DNA is shown in Fig. 5.
Fig. 6 Topo II inhibition assay. To about 200–300 ng of kDNA, about
5 units of Topo II was added and incubated at 37 °C for 30 minutes.
Lane 1- kDNA alone; lane 2- kDNA and topo II; lane 3- kDNA, topo II
and amonafide; lane 4- kDNA, topo II and 4b; lane 5- kDNA, topo II
and 4a; lane 6- kDNA, topo II and 5g; lane 7- kDNA, topo II and 5a.
5a) is lower compared to that of the other derivatives.
Among the derivatives considered, 5a exhibited moderately
higher cytotoxicity in the MTT assay. Considering this as-
pect, the 5a derivative was considered as the negative con-
trol throughout this study. The topo II inhibition by 5a was
comparatively lower when compared to that by 4a,b and 5g.
These results support the cytotoxicity data, where 4a,b and
5g showed maximum cytotoxicity compared to 5a. This sug-
gests that a ternary complex is not formed with kDNA in
the absence of any derivatives and with topo II alone as
depicted in lanes 1 and 2, respectively, in Fig. 6. The forma-
tion of the ternary complex is also an indication that these
derivatives intercalate with DNA. From these studies, it is
evident that all the derivatives (mainly 4a,b and 5g) interca-
late with DNA and interfere with the catalytic activity of the
topo II enzyme-like amonafide and thus they function as
catalytic inhibitory compounds.
Topoisomerase II inhibition assay
Topoisomerases control the topology of DNA and these en-
zymes play a significant role in DNA replication, transcrip-
tion,
segregation
and
recombination.28
Type
I
topoisomerases (topo I) make a single nick in the DNA
strand, while type II topoisomerases (topo II) make similar
nicks on both the strands of DNA. Topo II is a nuclear en-
zyme that influences the DNA topology by catalyzing the
DNA cleavage and by relegating the phosphodiester bonds;
as a result, topo II initiates multiple recombination/repair
pathways that trigger cell death.29 Topo II inhibition studies
were carried out using a topo II drug screening kit (TG
1009, Topogen, USA). Catenated DNA and topo II enzyme (5
units) were incubated in the presence of 10 μM amonafide
for 30 min (lane 3 in Fig. 6), which is considered as a posi-
tive control in the present experiment. The experimental re-
sults revealed that decatenation did not take place (by
forming minicircles) and the ternary complex (kDNA + topo
II and amonafide complex) remained close to the origin.
The studies, when repeated with catenated kDNA (200–300
ng) in the presence of 10 μM 4b,a, 5a and 5g (lanes 4, 5, 6
and 7, respectively, in Fig. 6), revealed less formation of
decatenated DNA and most of the DNA in the presence of
4a,b and 5g derivatives did not enter the gel. But with 5a,
the formation of the ternary complex (kDNA + topo II and
Molecular modelling studies
Most of the naphthalimide-containing derivatives show their
anticancer activity by intercalation into DNA30 and through
inhibiting topoisomerase enzymes. In order to understand
their binding poses and interaction patterns, the most active
molecules 4a and 4b in the present study were selected for
molecular docking studies. All the coordinates of the protein
crystal structures were obtained from the RCSB Protein Data
Bank. Energy minimizations and necessary corrections of the
proteins and DNA were performed by using the protein prep-
aration wizard of Maestro version 11.1 (Schrödinger 2017-
1).31 These docking studies were performed using Glide 7.4
(Schrödinger 2017-1).
Docking studies were performed for the representative de-
rivatives 4a and 4b at the ATP binding site of topoisomerase-
IIα (PDB ID: 1ZXN).32 The results revealed that these deriva-
tives were well accommodated in the binding site of topo-
isomerase-IIα. Further, for ligands 4a and 4b some common
interactions similar to those of the co-crystallized ligand were
observed (Fig. 7). In naphthalimide derivative 4a, the nitro-
gen of benzothiazole shows hydrogen-bonding interaction
with Asn120, and hydrophobic interactions are observed with
Ala92, Ile125, Pro126, Val137, Leu140, Ile141, Phe142, and
Ile217. Meanwhile, in 4b, the naphthalimide ring forms π–π
stacking interactions with Hie130, hydrogen-bonding
Fig. 5 Change in the CT-DNA (150 μM) viscosity before and after the
addition of 4a,b, 5a and 5g (15 μM). Variation in the viscosity of CT-
DNA (150 μM) on addition of ethidium bromide (15 μM) which was
considered as the control.
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Fig. 7 (a) Binding pose of 4a in topoisomerase-IIα. (b) Binding pose of 4b in topoisomerase-IIα.
interactions with Ser148 and Asn150 and hydrophobic con- Conclusion
tacts with Ile125, Pro126, Val137, Leu140, Ile141, Phe142,
In conclusion, a series of new naphthalimides were designed
and synthesized and their cytotoxic activities were evaluated.
The majority of these naphthalimides potentially inhibited the
growth of HT-29 and A549 cancer cell lines selectively. Interest-
ingly, hybrid derivatives 4a and 4b which have an amide bond
at the 6-position displayed promising cytotoxic activity com-
pared to the reference drug amonafide. We also investigated
the most active derivatives 4a and 4b for their antitumor mech-
anism of action, which potentially inhibit DNA topo II. More-
over, the DNA interactions of the representative derivatives
were studied by CD, UV-visible and fluorescence spectroscopy
demonstrating that they intercalate with DNA.
Tyr151, Tyr165, and Ala167. DNA docking was also performed
in the DNA duplex dIJGAAGCTTC)2 (PDB ID: 1NAB)33 using XP
Glide 7.4 (Schrödinger 2017-1). The docked pose for the top
scored derivatives 4a and 4b showed intercalation of the
naphthalimide ring between the G–C base pairs of DNA,
whereas the piperazine linker and benzothiazole were ex-
tended out to the minor groove (Fig. 8). These molecules
were well stabilized in the active site of DNA by
hydrogen-bonding and π–π stacking interactions between
piperazine and benzothiazole motifs with DNA base pairs,
respectively.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The authors thank CSIR-New Delhi for the award of senior re-
search. We acknowledge funding received from the project
entitled “Affordable Cancer Therapeutics” (CSC0301) under
the XIIth five year plan. Dr N. Shankaraiah gratefully acknowl-
edges SERB, DST, Govt. of India for the research grant (YSS-
2015-001709).
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Fig. 8 Side view (A) and top view (B) of the intercalation of 4a and
side view (C) and top view (D) of the intercalation of 4b between the
G–C base pairs of dIJGAAGCTTC)2.
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