Atomistic and Molecular Level Portrayal of DNA – 1,8 – Napthalimides

interaction
R. Radhikaa, , R. Shankara,*, S. Vijayakumarb,
a
Department of Physics, Bharathiar University, Coimbatore 641 046, Tamilnadu, India
b
Department of Medical Physics, Bharathiar University, Coimbatore 641 046,Tamilnadu, India
E-mail: rshankar@buc.edu.in

Abstract
The present work reports the physicochemical interaction between DNA and 1,8 – Napthalimide
drugs to enlighten the field of drug discovery. Molecular dynamics studies of 1,8 – Napthalimides
interacted DNA revealed distortions and destabilization of the DNA. Among the drugs (Amonafide
Azonafide and NNM-25), NNM-25 is found to have a strong interaction with DNA in both gas and
water phases by using ONIOM method through investigating deformation and interaction energies. The
reason of the higher interaction energy is attributed to the higher deformation of DNA during the
interaction with NNM-25. Further, complex is investigated by quantum chemical method to find the
detailed observation of the direct Interaction of the 1,8 – Napthalimides with the DNA base pairs. NBO
analysis is done to understand the charge transfer mechanism of 1,8 – Napthalimides interacted DNA
and also it is observed that highly stabilized complexes are found to have the highest interaction
energies. The Density functional theory (DFT) is used to calculate the chemical reactivity and site
selectivity of the molecular system to explore the results that may call for further experimental studies.

Keywords : DNA, Cancer, Naphthalimide, ONIOM, Quantum chemical calculations

1 Introduction

Naphthalimides are used in both biochemical and chemical applications including potent
intercalating agents. Few naphthalimide derivatives have entered into clinical trials due to their anti-
cancer activity, while many derivatives have been used as fluorescent probing of cells and in effective
inhibition of enveloped viruses in blood and blood products [1]. 1,8 – Naphthalimide shows its cytotoxic
activity through interaction with the DNA by intercalation against various cell lines such as murine and
human cancer cell lines [2]. Hepatocellular carcinoma (HCC) is among the third leading causes of
cancer deaths [3]. 1,8 - Naphthalimide shows enhanced anticancer activity against Hepatocellular
carcinoma, breast cancer, leukemia, and melanoma. Though, many reports revealed that the alkylating
agent and naphthalimide conjugates are more effective than the naphthalimides and the detailed
molecular mechanism of antitumor activity remains dimness up to now [4]. Amonafide is the leading
member of the 1,8 –Naphthalimide family that results in protein-associated single-strand breaks
mediated by inhibition of the DNA nicking closing enzyme topoisomerase [5]. Amonafide acts as a
Topo II poison by perturbing the cleavage-religation equilibrium, which results in accumulation of
DNA-Topo II covalent adducts [6]. Arresting the religation step after DNA cleavage generates of
double-strand breaks. Due to the dose-limiting bone marrow toxicity, amonafide is failed to enter Phase
III that leads to anemia, thrombocytopenia and leucopenia [7]. Hence, the development of new 1,8 -
Naphthalimides as DNA targeting is a fast growing area that will result such new derivatives entering
into clinical trials. The introduction of such derivatives targeting DNA can lead the major effects on the
electronic properties in the drug interacted DNA with the subsequent influence on the structural,
photochemical, chemical and spectroscopic properties. Azonfide is an important compound in the 1,8–
naphthalimide family where the naphthalene ring is substituted by an anthracene moiety show enhanced
anticancer activity compared to amonafide [8]. The mode of action comprised of protein-associated
single-strand breaks by involving inhibition of topoisomerase II [9]. Previous studies show that 1,8 –
Naphthalimide derivative NNM-25 shows increased antitumour activity with the low systemic
toxicity against Hepatocellular carcinoma compared to amonafide [10]. The mode of action of this
compound is found to induce cell cycle arrest at G2/M boundary and trigger apoptosis [2]. To the best of
our knowledge, there is no detailed theoretical study that relates the binding mode and the activity of the
complexes that remain a challenging problem in biophysics. Hence in the present work theoretical studies
of DNA and 1,8 – Napthalimide drugs have been done to find the structure-activity relationships.
Through the structural studies, it is possible to find the activity of the drug and also to design new drugs
with high selectivity and low toxicity. In the past few decades, investigation on the non-covalent
interactions in the bio molecules such as DNA and protein are of particular interest of some research
groups [11,12]. Since Hydrogen bond (H-bond) takes an important role in the stability of DNA & RNA,
ligand reception, protein folding and many other biologically significant processes, the H-bond study
takes an important role in all the biological significant processes [13,14]. Recently, Singh et al., reported
the influence of C-H…O interaction and -stacking on photoluminescence on a dinuclear silver (I), a
mononuclear zinc (II) and a cadmium (II) formylbenzoate complex possessing N-(4-pyridylmethyl)-1,8-
naphthalimide [15]. Since H-bond plays an important role in the stability of DNA and investigation on
the non-covalent interactions in the bio molecules such as DNA and protein are of particular interest of
some research groups [11,14]. Recently, Singh et al., reported the influence of C-H…O interaction and
-stacking on photoluminescence on a dinuclear silver (I), a mononuclear zinc (II) and a cadmium (II)
formylbenzoate complex possessing N-(4-pyridylmethyl)-1,8-naphthalimide [15]. Though many other
groups have reported about the interaction phenomenon of DNA with 1,8 – Napthalimide drugs, still
detailed theoretical investigation is elusive [16,17]. Hence, in the current investigation, various
theoretical methods, such as ONIOM, quantum chemical as well as molecular dynamics are used to
understand the interaction mechanism of octamer duplex DNA,d(CCTGGTCC).d(GGACCAGG) with
the anticancer drugs amonafide, azonafide, and 3-nitro-1,8naphthalimide conjugated with nitro mustard
(NNM-25). These results will be useful to predict the mechanism of such complexes that will enlighten
the field of drug discovery.

2 Computational details
2.1 Molecular dynamics simulation
Molecular dynamics simulation is executed for the isolated octamer duplex DNA and the 1,8-
naphthalimides interacted DNA complexes by using Amber (ff03) force field [18] implemented in
YASARA software with periodic cell boundaries [19,20]. YASARA changes bond orders, adds
hydrogens automatically as well as changes the charge distribution of the molecules based on the
environment we considered [21,22]. To replicate the better experimental values, force field Amber 03 is
used [23,24]. Particle Mesh Ewald algorithm is used to treat the Long-range interactions (cut off value
=12 Å) [25,26]. To avoid the long-range interactions, large dimensions of the cells (21 x 21 x 21 Å &
60.06 x 60.06 x 60.06 Å) are used for all the complexes. To solvate all the complexes, TIP3P model
(total density = 0.977 g l -1) is used [27]. Counter ions (Na+ and Cl-) were placed randomly in various
electrostatic potential to neutralize the complexes. The steepest descent algorithm is used to obtain the
energy minimized structures to remove all the unpleasant Van der Waals interactions [28].

2.2 ONIOM method

ONIOM (Our own N‐layer integrated molecular orbital molecular mechanics) is emerged as best
method to study larger biomolecular systems such as DNA and protein [29,-30]. Since, the DNA
structure is too large to study using Density functional method, ONIOM method is chosen to investigate
the whole structure. In this method, both two methods QM and MM will be combined with one another
to study the molecular system. In the present work, we used M06-2X and MPWB1K for the high level
layer (GC:CC) and rest of the system is optimized with amber force field. Both gas and water phases
have been considered in all our calculations as implemented in the Gaussian 09 package [31]. ONIOM
technique is used to study 1,8 – Napthalimide drug interacted DNA complex, in which main interactive
parts have been treated with higher level methods M06-2X and MPWB1K with the basis set 6-31+g(d,p)
and the remained parts have been considered with Amber force field [32-34]. In addition to that to
examine the solvent effects, self consistent reaction field (SCRF) is used with the default method
‘polarizable continuum model’ (PCM) [31]. To compare the interaction of all the three drugs, the
deformation energy ( D) of the DNA is calculated with the formula:
D = E (DNA)Complex - E (DNA) Isolated
For E (DNA)Complex, we made single point energy calculation for each residue of DNA
interacting with 1,8 – Napthalimide drugs. E (DNA) Isolated is the energy of the optimized structure of the
DNA at the same level of theory. To observe the interaction between the DNA and 1,8 – a detailed
manner, high level layer d(GG : CC) and its interacted parts have been extracted and quantum chemical
method is used.

2.3 Quantum chemical method

Isolated DNA structure d(GG:CC) and 1,8 – Napthalimides interacted DNA are optimized by
using M06-2X/6-31+G(d,p) method. The vibrational frequency calculations have been performed at the
same level of theory, and it has been confirmed that the structures are on real minima without imaginary
frequencies. The interaction energies and NBO analysis were performed.The many body analysis have
been performed, by partitioning the interaction energy (ΔE) into two and five body interactions,
 ΔETOTAL=E(ABCDE) -[E(A) + E(B) + E(C) + E(D) + E(E)]

Δ5E(ABCDE)=ΔETOTAL-[Δ2E(AB)+Δ2E(AC) + Δ2E(BC)+Δ2E(AD)+ Δ2E(AE)

+Δ2E(BD) +Δ2E(BE)+Δ2E(CD)+ Δ2E(CE)+Δ2E(DE)]

Δ (AB) = [ ( ) + ( )]

Where, E(ABCDE) is the total energy of drug-interacting base pairs, E(A), E(B), E(C), E(D), and E(E) are the
total energies of single base pair or drug and is the total energy of any two interacting molecules
(base pairs or base pairs with a drug).In addition to that, reactivity and the toxicity values of the drug
interacted complexes were observed by using chemical reactivity analysis.

3 Results and Discussion

3.1 Molecular dynamics analysis
The structure of the DNA sequence d(CCTGGTCC) .d (GGACCAGG) is taken from protein
data bank (pdb code: 1AU5) and used as an initial structure [35]. The initial structure is subjected to
energy minimization of the system by using the steepest descent algorithm followed by MD simulation
[28]. During the simulation process to mimic exact DNA environment, counter ions and water medium
are added. Molecular dynamics simulation is performed for the 1,8-naphthalimides interacted DNA
complexes for 100 ns by applying Amber 03 force field and NNM-25 interacted DNA structures
obtained are given in Figure 1. Azonafide as well as amonafide interacted DNA structures are given in
Figure S1and S2. From the figures, it is observed that structures are stabilized through π-π stacking as
well as H-bond interactions. RMSD for the back bone atoms in the DNA is calculated for 0–100 ns
simulation and displayed in Figure 2 with the 10 ns time interval to explore the influence of 1,8 –
Napthalimides on the stability of the DNA.
Figure 1a. Conformational changes NNM-25 interacted DNA structures at (a) 10ns (b) 20ns (c)
30ns (d) 40ns (e) 50ns (f) 60ns (g) 70ns (h) 80ns (i) 90ns (j) 100ns .

/
(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)

Figure 1b. Conformational changes NNM-25 interacted DNA structures at (a) 10ns (b) 20ns
(c)30ns (d) 40ns (e) 50ns (f) 60ns (g) 70ns (h) 80ns (i) 90ns (j) 100ns

(a) (b) (c)

 (d) (e) (f)

(g) (h) (i)

(j)

From Figure 2, A, B, C and D represent isolated DNA, amonafide interacted DNA, azonafide
interacted DNA and NNM-25 interacted DNA respectively. Because of the initial relaxation &
conformational adjustment, isolated DNA shows high fluctuations from 0 nm to 0.55 nm during the
simulation and finally gets stabilized beyond 88000 ps. In the case of amonafide interacted DNA,
backbone RMSD values are lower till 15000 ps followed by a sudden increase to about 0.6 nm at 40000
ps to 68000 ps and finally became stable beyond 70000 ps.While considering the azonafide interacted
DNA, during the starting of the simulation, RMSD values are found to be slightly super imposed with
the isolated DNA till 18000 ps.
 Figure 2. RMSD of the isolated and 1,8–Napthalimides interacted DNA

In the case of NNM-25 interacted DNA, though the RMSD is slightly found to be coincide with
the isolated DNA till 20000 ps and beyond it some structural deviation is occurred and finally structure
is stabilized well beyond 60000 ps to the end of the simulation. During the course of simulation, due to
the interaction between DNA and 1,8 – Napthalimide, fluctuation is observed in the backbone RMSD
which results from the free movement of backbone residues. Thus the structures are stabilized through
π-π stacking as well as H-bond interactions and the variations of the RMSD could be related with the
formation of bonds between them. NNM-25 interacted DNA shows significant deviation from the
isolated DNA. This variation is due to the formation of bonds between DNA and drugs.Hence, it is
observed that interaction of the 1,8 – Napthalimides highly stabilize DNA during the simulation. In
addition to that stabilized structures are to be used in the QM/MM study to observe the atomic level
properties.

3.2 ONIOM analysis

ONIOM (QM/MM) method is used for the most stable 1,8 – Napthalimides interacted DNA
complexes obtained from the molecular dynamic simulation to investigate the electronic interaction of such
complexes in two different environments including gas phase and water phase. To optimize the complexes,
1,8 – Napthalimides and DNA, d(GG).d(CC) are considered as high level layer and the remaining parts
are considered as low level layer respectively. In the case of high level layer, two different level of
theories M06-2X and MPWB1K are used along with 6-31+G(d,p) basis set and Amber force field is
used in the low level layer.
 3.2.1 Deformation energy
Deformation energies of the DNA with 1,8 – Napthalimides are calculated by using M06-2X
and MPWB1K level of theories as high level and amber force field as low level in both gas and water
phases and given in Table 1 to know how much distortion is occurred in the complexed DNA compared
with the isolated DNA. From Table 1, the deformation energies of DNA that are complexed with
amonafide, azonafide and NNM-25 are 11.21, 10.11 & 18.93 kcal/mol and 6.73, -18.20& 8.18 kcal/mol
in the corresponding gas phase and water phase respectively at M06-2X method. In the case of
MPWB1K method, deformation energies are obtained as 7.30, 10.97 & 16.83 kcal/mol (gas phase) and -
21.62, 4.34 & 10.55 kcal/mol (water phase) for the DNA complexed with amonafide, azonafide and
NNM-25 respectively. The deformation order of the DNA interacted with 1,8 – Napthalimides in both
phases is,
NNM-25_DNA > Azonafied_ DNA > Amonafide_ DNA

From the results, it is found that highest deformation energies are noted for DNA interacted with
NNM-25and the second highest deformation energies are obtained for DNA interacted with Azonafide.

Table 1. Deformation energies of DNA during interaction with 1,8 – Napthalimides (Complex A, B
& C represent Amonafide interacted DNA, Azonafide interacted DNA and NNM-25 interacted
DNA respectively)

Deformation energy D (kcal/mol)

ONIOM(M06-2X/6-31G**:AMBER) ONIOM(MPWB1K/6-31G**:AMBER)

Complex
Gas phase Water phase Gas phase Water phase

DNA DNA DNA DNA DNA DNA DNA DNA

D D D D
Complex Isolated Complex Isolated Complex Isolated Complex Isolated

A -2031.58 -2031.60 11.21 -2031.57 -2031.58 6.73 -2031.45 -2031.47 7.30 -2031.50 -2031.46 -21.62

B -2031.58 -2031.60 10.11 -2031.61 -2031.58 -18.20 -2031.45 -2031.46 10.97 -2031.46 -2031.46 4.34

C -2031.57 -2031.60 18.93 -2031.57 -2031.58 8.18 -2031.44 -2031.46 16.83 -2031.45 -2031.46 10.55
 These highest deformation energies are observed due to the disruption of H-bond between d(GG)
and d(CC) base pairs. In addition to that, it is found that deformation energy is higher in the gas phase
than that of the water phase due to the fact that1,8 – Napthalimides interacted DNA complexes interact
with water molecules that prevent the deformation of the DNA in the corresponding complexes
compared to the gas phase.

3.2.2 Interaction energy

The interaction energies of 1,8 – Napthalimides interacted with DNA complexes are calculated by
using ONIOM method by using methods ONIOM(M06-2X/6-31G** : AMBER) and
ONIOM(MPWB1K/6-31G** : AMBER) in both gas and water phases and tabulated in Table 2.

Table 2. Interaction energy ( ) of DNA during interaction with 1,8 – Napthalimides (Complex A,
B & C represent amonafide interacted DNA, azonafide interacted DNA and NNM-25 interacted
DNA respectively)

ONIOM(M06-2X/6-31G**:AMBER) ONIOM(MPWB1K/6-31G**:AMBER)

Gas phase Water phase Gas phase Water phase

Com
plex DNA- DNA Drug DNA- DNA Drug DNA- DNA Drug DNA- DNA Drug
Drug Complex complex (kcal/ Drug Complex complex (kcal/ Drug Complex complex (kcal/ Drug Complex complex (kcal/
Complex mol) Complex mol) Complex mol) Complex mol)

A -2966.0 -2031.6 -934.4 -46.5 -2966.1 -2031.6 - 934.5 -21.1 -2965.8 -2031.5 -934.3 -23.8 -2965.8 -2031.5 -934.3 -13.8
B -3064.2 -2031.7 -1032.6 -46.5 -3064.2 -2031.6 -1032.6 -35.2 -3064.0 -2031.5 -1032.5 -24.7 -3064.1 -2031.5 -1032.6 -20.4
C -4191.4 -2031.6 -2159.7 -50.8 -4191.4 -2031.6 -2159.8 -37.4 -4191.2 -2031.4 -2159.7 -31.9 -4191.2 -2031.5 -2159.7 -27.2

From Table 2, the interaction energies of DNA complexed with amonafide, azonafide and NNM-
25 are -46.5, -46.5&-50.8 kcal/mol and -21.1, -35.2 &-37.4 kcal/mol in the corresponding gas phase and
water phase respectively at M06-2X method. In the case of MPWB1K method, interaction energies are
obtained as -23.8, -24.7&-31.9 kcal/mol (gas phase) and -13.8, -20.4 &-27.2 kcal/mol (water phase) for
the DNA complexed with amonafide, azonafide and NNM-25 respectively. Observed results agree well
with the previous theoretical studies on amonafide and azonafide interacted with DNA [36]. It is found
that NNM-25 had a strong interaction with DNA in both gas and water phases. The reason of the higher
interaction energy is attributed to the higher deformation of DNA during the interaction with NNM-25.
In addition to that, it is also confirmed that interaction energy of the complex with DNA decreases in the
presence of water molecules compared to the gas phase. The reason for the lower interaction energy in
water phase can be attributed to the lower deformation of the 1,8 – Napthalimides interacted DNA
complexes and the effect of electrostatic field of the water that polarizes the wave function of the
complexes which leads to repulsive interaction between DNA and 1,8 – Napthalimides.Though we got
the interaction energies of all the 1,8 – Napthalimides interacted DNA complexes, the values are not
accounting for the direct interaction of the 1,8 – Napthalimides with the base pairs but also would be
accounting for the energy penalty associated with the disruption of the DNA structure in order for the
drug to fit in its binding position. Hence to find the detailed observation of the direct Interaction of the
1,8 – Napthalimides with the DNA base pairs, high level layers (GCGC tetrads with drug) have been
separately extracted and further investigated by quantum chemical method.

3.3 Quantum chemical studies

The interaction of 1,8 – Napthalimides with the tetrads are presented in the current work by using
DFT. The geometries are optimized with M06-2X/6-31+G** method and depicted in Figure 3.

Figure 3. Optimized structures of (a) Amonafide interacted DNA (b) Azonafide interacted DNA
and (C) NNM-25 interacted DNA

(a) (b) (c)

3.3.1 Interaction Energy

To design new potential DNA-binding anticancer molecules, interaction energy calculation takes
an important role to shed the light on specificity and energetics. In the present work, many body analysis
for the 1,8-naphthalimides interacted tetrads are given in Table 3.The negative interaction energy values
results from the high binding potential of 1,8-naphthalimides with tetrads that is energetically favored in
an anhydrous environment.
Table 3. Interaction energy of drugs (Amonafide,Azonafide, NNM-25) with GCGC tetrad

Interaction energy (kcal/mol)

Base Pair
GCGC -Amonafide GCGC –Azonafide GCGC -NNM-25

∆E(TOTAL) -40.08 -55.29 -60.20

Δ2E(G1+G2) -0.80 -0.67 -6.10
Δ2E(G1+C2) -0.26 -0.80 -2.31
Δ2E(G2+C2) -0.04 -0.84 -2.21
Δ2E(G1+C1) -0.01 -0.70 -0.70
Δ2E (G2+C1) -4.94 -3.77 -3.77
Δ2E(C2+C1) -2.02 -3.76 -2.76
Δ2E (G1+ drug) -9.13 -9.14 -9.14
Δ2E (G2+ drug) -16.86 -11.2 -5.49
Δ2E(C1+drug) -8.05 -5.18 -5.18
Δ2E(C2+drug) -5.65 -8.88 -7.44
∆5E(ABCDE) 7.68 -10.35 -15.10

Among the 1,8-naphthalimides interacted tetrads, NNM-25 interacted GCGC tetrad is found to
have highest interaction energy of -60.20 kcal/mol followed by azonafide interacted tetrad (-55.29
kcal/mol), whereas amonafide interacted tetrad is observed with the least interaction energy of -40.08 kcal/mol.
The order of the interaction energy of the complexes is found to be

GCGC -NNM-25 > GCGC-Azonafide > GCGC –Amonafide

Interaction energy reflects the influence of 1, 8-naphthalimides with the GCGC tetrad through
that could be observed from Figure 3. From Table 4, it observed that strong H-bonds are found to be
associated with higher interaction energy of NNM-25.In the case of NNM-25 interacted GCGC tetrad,
two body interaction energies are found to be -9.14, -5.49, -5.18 and -7.44 kcal/mol respectively for the
corresponding base pairs G1_NNM-25, G2_NNM-25, C1_NNM-25 and C2_NNM-25. Many-body
interaction plays a significant role of around 30% of the total cluster binding energy that arise from the
H-bond interactions [37]. Five body interaction energy values (∆5E) of the complexes GCGC-NNM-25,
GCGC–Azonafide and GCGC–Amonafide are found to be -15.10, -10.35 and 7.68 kcal/mol
respectively. Due to five body effects of the complexes GCGC-NNM-25 and GCGC–Azonafide,
negative sign values (-15.10 and -10.35 kcal/mol) are obtained that represent the stabilization of the 1,8-
naphthalimides interacted tetrads. Thus the strong contributions of the GCGC-NNM-25 complex leads
to the high interaction energy of -60.20 kcal/mol followed by GCGC –Azonafide (-55.29 kcal/mol). The
positive value of the GCGC –Amonafide (7.68 kcal/mol) complex represents the least interaction
between the tetrad and the drug amonafide. The drug molecule NNM-25 is found to have highest
interaction with the tetrads that induce cell cycle arrest at G2/M boundary and trigger apoptosis.

3.3.2 Natural bonding orbital analysis

To observe charge transfer mechanism during the complex formation, Natural Bonding Orbital
analysis is performed [38,39]. In this analysis, stabilization energies E(2) and NBO occupation numbers
for the anti-bonds lone pairs [N(Y) and σ* (X-H)] have been observed and reported in Table 4.
In this analysis, charge transfer is observed through the elongation and contraction of the H-bond
by using NBO analysis. The stabilization energies of N-H…N bonds in Amonafide, azonafide and
NNM-25 are found to be in the range of 23.9 to 24.7 kcal/mol, 25.1 to 26.6 kcal/mol and 28.81 kcal/mol
respectively as well as N-H…O bonds are observed between 7.0 to 12.1 kcal/mol, 6.8 to 8.2 kcal/mol
and 4.9 to 8.4 kcal/mol respectively. These stabilization energy values indicate the strength of the
individual H-bond. In the N-H…N bonds, donor covalent bond lengthening is occurred in amonafide,
azonafied and NNM-25. In the case of Amonafide, N22-H28…N34 & N7-H13…N46 bonds with the
respective hydrogen bond lengths of 1.890 Å & 1.900Å are occurred. N6-H12…N45 and N21-
H27…N33 bonds with the respective H-bond lengths of 1.856 and 1.897Å are observed in azonafide
and in the case of NNM-25, N7-H13…N46 (1.866 Å) bond is occurred.
Electron density transfers to the N-H anti bonding orbitals (N22-H28, N7-H13, N6-H12, N21-
H27 and N7-H13) with least H-bond lengths result better stabilization energies of 24.721, 23.930,
26.621, 25.100 and 28.811 kcal/mol. In the N-H…O bonds, donor covalent bond lengthening has
occurred in amonafide, azonafide and NNM-25. In the case of amonafide, N9-H214…O45, N24-
H29…O33, N36-H39…..O21and N48-H51….O6 bonds with the respective hydrogen bond lengths of
1.860, 1.872, 1.908 and 1.855 Å are occurred. N23-H28…O32, N8-H13…O44, N47-H50…O99 and
N35-H38…O20 bonds with the corresponding hydrogen bond lengths of 1.813, 1.886, 1.927 and
1.874Å are observed in azonafide and in the case of NNM-25, N36-H39…O56, N48-H52…O33, N48-
H51…O6 and N9-H14….O45 (1.946,1.893, 1.890 and1.832 Å) bond is occurred.
 Table 4. NBO analysis of 1,8 – Napthalimides interacted DNA complexes

Bond Bond angle X-H X-H…X N (Y) σ*(X-H) E(2)

Bonding type length
(°) (Å) (°) (a.u) (a.u) kcal/mol
(Å)

GCGC -Amonafide
N9-H14...O45 1.930 176.6 1.018 119.5 1.960(1.965)* 0.040(0.032)* 12.1
N24-H29....O33 1.946 174.2 1.017 117.9 1.960(1.965) 0.038(0.038) 9.5
N36-H39...O21 1.803 171.9 1.034 118.6 1.964(1.959) 0.043(0.052) 7.0
N22-H28...N34 1.911 177.5 1.030 119.2 1.868(1.965) 0.064(0.064) 24.7
N7-H13...N46 1.911 171.9 1.033 119.9 1.868(1.864) 0.064(0.068) 23.9
N48-H51...O6 1.786 174.9 1.031 116.9 1.962(1.961) 0.046(0.053) 8.8

GCGC - Azonafide
N23-H28...O32 1.885 176.1 1.021 120.7 1.958(1.963) 0.045(0.038) 8.2
N8-H13...O44 1.960 167.9 1.020 116.3 1.962(1.965) 0.003(0.032) 6.9
N6-H12...N45 1.877 4.49 1.032 119.9 1.865(1.864) 0.069(0.068) 26.6
N47-H50…O99 1.854 160.4 1.021 116.4 1.965(1.961) 0.041(0.053) 6.8
N21-H27…N33 1.913 176.9 1.033 114.3 1.867(1.868) 0.065(0.064) 25.1
N35-H38…O20 1.796 177.0 1.029 118.8 1.963(1.959) 0.044(0.052) 8.0

GCGC - NNM-25
N36-H39…O56 1.878 166.2 1.020 118.4 1.964(1.959) 0.032(0.052) 8.4
N48-H52...O33 1.992 158.9 1.014 117.5 1.963(1.963) 0.036(0.038) 4.9
N48-H51...O6 1.797 170.4 1.024 118.7 1.953(1.961) 0.043(0.053) 7.3
N7-H13...N46 1.861 177.8 1.041 115.1 1.861(1.864) 0.073(0.068) 28.8
N9-H14…O45 1.833 177.2 1.025 121.8 1.963(1.965) 0.044(0.032) 7.5

Electron density transfers to the N-H anti bonding orbitals (N9-H14, N24-H29, N36-H39, N48-
H-bond lengths result better second-order perturbation energies of 12.160, 9.461, 7.011, 8.840,
8.161,6.990,6.781, 8.010, 8.380,7.260 and7.542 kcal/mol. Hence the high stabilization energies of the
N-H…O and N-H…N bonds lead to the strong interaction of the above anticancer drugs with the DNA.
Especially highly stabilized complexes (azonafide and NNM-25 interacted DNA) are found to have the
highest interaction energies than that of amonafide interacted DNA.

3.3.3 Chemical Reactivity

The DFT method is used to calculate the chemical reactivity and site selectivity of the molecular
system by using M06-2X/6-31+G (d,p). The Frontier molecular orbitals are explained the eventual
charge transfer interaction with in the molecule, electronegativity (χ), chemical potential (µ), global
hardness (η), global hardness (s) and global electrophilicity (ω) were calculated using below equation
and the results are shown in Table 5.
 χ=-1/2(ELUMO+EHOMO) (1)

µ=-χ=1/2(ELUMO+EHOMO) (2)

η=1/2(ELUMO-EHOMO) (3)

s=1/2η (4)

ω=µ2/2η (5)

The inverse value of the global harness is designated as the softness as follows:
σ=1/η (6)

Electrophilicity index is one of the most important quantum chemical descriptors in describing toxicity
of various, pollutants in terms of their reactivity and site selectivity. Also, the electrophilicity properly
quantifies the biological activity of drug-receptor interaction. The calculated HOMO and LUMO values
of the compound amonafide and azonafide, NNM-25 are -0.243 & -0.485 eV, -6.784 & -0.785 eV and -
6.918 & -0.584 eV respectively.
In the case of of 1,8 – Napthalimides interacted DNA, while considering Frontier molecular orbitals,
HOMO is localized over the base pairs and LUMO is distributed over the 1,8 – Napthalimides and is
given in Figure 4.
The higher values of chemical hardness and softness of the NNM-25 (3.167 eV) and azonafide
(3.018 eV) leads to the high bioactivity of the molecules via intermolecular charge transfer. From the
chemical reactivity parameters, it is observed that the drugs are reacted well with the DNA to stabilize
the system.
Figure 4. Frontier molecular orbitals involved in the interaction (a) Amonafide interacted DNA
(b) Azonafide interacted DNA and (C) NNM-25 interacted DNA

HOMO LUMO
(a)

(b)

(c )
Table 5. The chemical reactivity of the 1,8 – Napthalimides interacted DNA complexes

Amonafide Azonafide NNM-25

PARAMETERS
(eV) (eV) (eV)
HOMO energy -0.2425 -6.7835 -6.9180
LUMO energy -0.4846 -0.7485 -0.5847
HOMO-LUMO energy gap 0.4848 7.1577 7.5022
Global Hardness 0.1212 3.0175 3.1666
Global Softness 0.0606 1.5087 1.5833
Chemical potential 0.1326 7.5321 7.5026
Electronegativity 0.3635 3.7660 3.7513
Electrophilicity 0.0001 8.5594 8.9122

4 Conclusion
We have investigated the deformation and interaction energies of 1,8 – Napthalimides interacted
DNA complexes by using ONIOM method in which stabilized structures are obtained through molecular
dynamics simulation. High level layers are extracted separately and quantum chemical method is
employed. Isolated and 1,8 – Napthalimides interacted DNA are optimized using M06-2X method with
the 6-31+G(d,p) basis set. The optimized geometries of the isolated DNA are almost planar whereas the
geometries of the drug interacted complexes deviate from planarity. From the chemical reactivity
parameters, it is observed that the drugs are reacted well with the DNA to stabilize the system. From the
NBO analysis, it is observed that the high stabilization energies of the N-H…N & N-H…O bonds lead
to the strong interaction of the above anticancer drugs with the DNA. From the values, it is observed that
the highest stabilization energy of 28.811 kcal/mol is observed for the NNM-25 interacted DNA which
favours the strong interaction. The total interacting energy (ΔE) and many body interacting energies
(Δ5E) have been calculated for drug interacted DNA base pairs. The five body interaction energies of
amonafide, azonafide and NNM-25 are -8.477 kcal/mol, -10.203 kcal/mol and -15.008 kcal/mol. The
drug NNM-25 strongly interacts with DNA that leads to apoptosis in the cancer cells.
Supplementary:

Figure S1 Conformational changes Azonafide interacted DNA structures at (a) 10ns (b) 20ns
(c)30ns (d) 40ns (e) 50ns (f) 60ns (g) 70ns (h) 80ns (i) 90ns (j) 100ns

(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)

Figure S2 Conformational changes Amonafide interacted DNA structures at (a) 10ns (b) 20ns
(c)30ns (d) 40ns (e) 50ns (f) 60ns (g) 70ns (h) 80ns (i) 90ns (j) 100ns

(a) (b) (c) (d) (e)

 (f) (g) (h) (i) (j)

Acknowledgement
The author R. Shankar, acknowledge RUSA 2.0-BCTRC official memorandum number-
IQAC/RUSA 2.0/PA/2021/1 dated January 08, 2021 funding for establish the computational facilities in
Molecular Simulation Laboratory, Bharathiar University, Coimbatore – 46, India.

Author contributions
All the authors have read and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

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