Synthesis, DNA binding, cellular DNA lesion and cytotoxicity of a series of new benzimidazole-based Schiff base copper(II) complexes

Article abstract of DOI:10.1039/c5dt02880a

A series of new benzimidazole containing compounds 2-((1-R-1-H-benzimidazol-2-yl)phenyl-imino)naphthol HL^1-3 (R = methyl, ethyl or propyl, respectively) have been synthesized by Schiff base condensation of 2-(1-R-1-H-benzo[d]imidazol-2-yl)anili

Full text of DOI:10.1039/c5dt02880a

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Trans., 2015, DOI: 10.1039/C5DT02880A.
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Synthesis, DNA binding, cellular DNA lesion and cytotoxicity of a
series of new benzimidazole-based Schiff base copper(II) complexes
a
a
b
a
b
Anup Paul, * Sellamuthu Anbu, Gunjan Sharma, Maxim L. Kuznetsov, Biplob Koch, *
a
a
M.Fátima C. Guedes da Silva, * Armando J. L. Pombeiro *
The synthesis, characterization and biological evaluation of a series of new benzimidazole-
based Schiff base copper(II) complexes are described. One of the compounds has a higher
cytotoxic effect than cisplatin on lung cancer (A-549) cell line.

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Synthesis, DNA binding, cellular DNA lesion and cytotoxicity of a series of
new benzimidazole-based Schiff base copper(II) complexes
a
a
b
a
b
Anup Paul, * Sellamuthu Anbu, Gunjan Sharma, Maxim L. Kuznetsov, Biplob Koch, * M.
a
a
Fátima C. Guedes da Silva, * Armando J. L. Pombeiro *
aCentro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001
Lisboa. Portugal.
bDepartments of Zoology, Faculty of Science, Banaras Hindu University, Varanasi - 221 005 (U.P.) India.
Abstract
A series of new benzoimidazole containing compounds (2-(1-R-1-H-benzimidazol-2-
yl)phenyl)imino)methyl) naphthol) HL1-3 (R = methyl, ethyl or propyl, respectively) has
been synthesized by Schiff base condensation of 2-(1-R-1-H-benzo[d]imidazol-2-yl)aniline
and 2-hydroxy-1-naphthaldehyde. The reactions of HL1-3 with Cu(NO
)
.2.5H O led to the
3
2
2
corresponding copper(II) complexes [Cu(L)(NO )] 1-3. All the compounds were
3
characterized by conventional analytical techniques and, for 1 and 3, also by single-crystal
X-ray analysis. The interactions of complexes 1-3 with calf thymus DNA were studied by
absorption and fluorescence spectroscopic techniques and the calculated binding constant
5
−1
5
−1
(K ) are in the range of 3.5 × 10 M - 3.2 × 10 M . Complexes 1-3 effectively bind DNA
b
through intercalative mode, as proved by molecular docking studies. The binding affinity of
the complexes decreases with the size increase in the N-alkyl substituent, in the order of 1
>
2 > 3, which is also in accord with the calculated LUMOcomplex energies. They show
substantial in vitro cytotoxic effect against human lung (A-549), breast (MDA-MB-231) and
cervical (HeLa) cancer cell lines. Complex 1 exhibits a significant inhibitory effect on
1

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proliferation of the A-549 cancer cells. The antiproliferative efficacy of 1 has also been
analysed by DNA fragmentation assay, fluorescence activated cell sorting (FACS) and
nuclear morphology using fluorescence microscope. The possible mode of apoptosis
pathway of 1 has also been evaluated by reactive oxygen species (ROS) generation study.
*
Corresponding
authors.
E-mail:
kanupual@gmail.com,
kochbiplob@gmail.com,
fatima.guedes@tecnico.ulisboa.pt, pombeiro@tecnico.ulisboa.pt,
Introduction
The majority of drugs used for cancer treatment are cytotoxic (cell-killing) interfering in
some way with the operation of the DNA cells. The serendipitous discovery of cisplatin1
has revolutionized the cancer treatment and many other platinum based drugs have been
2
3
discovered. However, side-effects associated with these drugs restrict their wider use.
Thus, in pursuit of overcoming these issues associated with Pt based anticancer drugs, non-
platinum metal complexes are being explored, particularly those capable of binding DNA
4
effectively. Copper(II) complexes are regarded as promising and have attracted
considerable attention owing to their capability of interacting directly with DNA/nuclear
proteins.5-7
On the other hand, benzimidazole-based Schiff base metal complexes possess good
8
antibacterial, antimalarial and antifungal activities. Several 2-(2-aminophenyl)-1-H-
benzimidazole derived qunizoline based metal complexes have been utilized as fluorescent
9
probes for intracellular imaging. Moreover, benzimidazole-based platinum complexes
2

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have been extensively studied and the mechanism of cytotoxic properties has been
discussed with regard to the development of new antitumor agents.10
However, benzimidazole-based Schiff base copper(II) complexes for DNA binding
11
and cytotoxic properties are scarce. Furthermore, the benzimidazole-based ligands are
promising systems for the development of biologically active metal complexes, due to the
structural similarity of the benzimidazole nucleus with natural compounds such as
vitamin-B12 derivatives and purine bases. In particular, the non-planar nature of
benzimidazole ligands, their flexibility and bulkiness affect the kinetics and cytotoxic
properties of the corresponding metal complexes. In addition, benzimidazole-based ligands
can possess hydroxyl and/or N-H moieties which can facilitate DNA cleavage in cancer
cells.12
Thus, in light of our continuing investigation for developing new metal-based
chemotherapeutic agents, we have designed a new series of N-alkyl substituted
benzimidazole-based Schiff base copper(II) complexes [Cu(L)(NO )] and investigated their
3
DNA interaction and in vitro cytotoxicity properties against human lung (A-549) cancer cell
line. Furthermore, the action mechanism and apoptotic pathway of one of these complexes
was investigated from the morphologic changes using fluorescence staining, fluorescence
activated cell sorting (FACS), DNA fragmentation and by reactive oxygen species (ROS)
generation studies.
Experimental Section
Materials and Physical methods
3

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The reagents for the syntheses of the ligands and Cu(NO
3 2
) .2.5H
2
O were obtained from
commercial sources and used as received without further purifications. Solvents were
dried and distilled prior to their use following standard procedures.13 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and RPMI-1640 were
purchased from Hi-Media, ethidium bromide (EB) from Acros Organics, Portugal. Calf
Thymus (CT) DNA was purchased from Bangalore Genie (India). H (300 MHz) and 13C
1
(
75.45 MHz) NMR spectra were run on a Bruker 300 MHz spectrometer using
tetramethylsilane [Si(CH ] as internal reference. IR and electronic absorption spectra
3 4
)
were recorded on Bruker Alpha Fourier transform infrared (FT-IR) spectrometer and
Perkin-Elmer LAMBDA 750 UV-visible spectrophotometer, respectively. Fluorescence
emission studies were carried out on LS-55 fluorescence spectrometer (Perkin-Elmer, UK).
The excitation and emission slit widths were set at 4.5 nm and the temperature was
maintained by recycling water using a peltier system. Electrospray ionization mass
spectrometric data (ESI-MS) were acquired in methanol on a Bruker Micro TOF QII, and
THERMO Finnigan LCQ Advantage Max ion trap Mass Spectrometer.
Syntheses
Synthesis of 2-(1-methyl-1-H-benzo[d]imidazol-2-yl)aniline (P1)
To a solution of 2-(2-aminophenyl)-1-H-benzimidazole (209 mg, 1 mmol) in dry DMF (5
mL) sodium hydride (36 mg, 1.5 mmol; 60% dispersion in mineral oil, prewashed with
hexane) was added in fractions, iodomethane (171 mg, 1.2 mmol) in dry DMF (5 mL) was
added dropwise over 15 minutes and the reaction mixture stirred for 24 h. Completion of
the reaction was monitored by TLC and, after filtration, the solvent was removed under
4

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reduced pressure. The residue thus obtained was partitioned between ethyl acetate and
water, the combined organic layers were washed with water, brine solution and dried over
Na
2
SO . Decantation followed by removal of the solvent under reduced pressure afforded
4
the crude product which was further purified by silica gel column chromatography (EtOAc:
Hexane; 1:9) to afford the desired product. Yield: 70% . Anal. Calcd. for C14
H
13
3
N : C, 75.31;
1
H, 5.87; N, 18.82 Found C, 75.41; H, 5.92; N, 18.72 %. H NMR (CDCl
3
, 300 MHz, δ ppm):
), 5.04 (s, 2H,
, 75.45 MHz, δ ppm): 155.21, 148.32, 145.10, 135.45, 130.34, 126.73,
7
.79 (s, Ar-H), 6.76-7.25 (m, Ar-H), 6.80 (d, J = 6.2, Ar-H), 3.83 (s, 3H, CH
3
13
NH
2
). C NMR (CDCl
3
1
22.14, 121.34, 120.52, 117.89, 112.32, 109.72, 31.27. ESI-MS in CH OH: Calcd. for [(M +
3
+
H)] : m/z 224.1, found 224.0.
Synthesis of 2-(1-ethyl-1H-benzo[d]imidazol-2-yl)aniline (P2)
It was prepared following the above procedure for P1 but using iodoethane (188 mg, 1.2
mmol) in place of iodomethane. Yield: 65%. Microanalytical data: C15
H
15
N
3
, Calcd. : C,
, 300 MHz, δ
), 4.28 (q,
, 75.45 MHz, δ ppm): 154.81, 148.62, 143.50,
1
7
5.92; H, 6.37; N, 17.71%. Found C, 75.82; H, 6.28; N, 17.81%. H NMR (CDCl
3
ppm): 7.83 (s, Ar-H), 7.44-7.28 (m, Ar-H), 6.87 (d, J = 6.2, Ar-H), 5.06 (s, 2H, NH
2
13
2H, -CH
2
), 1.28 (t, 3H, -CH
3
). C NMR (CDCl
3
136.45, 129.84, 125.13, 123.52, 120.75, 119.10, 118.91, 111.52, 109.82, 40.27. 16.21. ESI-
+
MS in CH
3
OH: Calcd. for [(M + H)] : m/z 238.1, found 238.0.
Synthesis of 2-(1-propyl-1-H-benzo[d]imidazol-2-yl)aniline (P3)
It was prepared following the above procedure for P1 but using iodopropane (204 mg, 1.2
mmol) in place of iodomethane. Yield: 60%. Microanalytical data: C16
H
17
N
3
, Calcd. : C,
1
7
6.46; H, 6.82; N, 16.72%. Found C, 76.50; H, 6.78; N, 16.76%. H NMR (CDCl
3
, 300 MHz, δ
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ppm): 7.82 (s, Ar-H), 7.44-7.28 (m, Ar-H), 6.87 (d, J = 6.2, Ar-H), 5.01 (s, 2H, NH
2
), 4.19 (t,
2
1
1
2
H, -CH
2
), 3.2 (sep, 2H, -CH
2
), 0.87 (t, 3H, -CH
3
), 13C NMR (CDCl
3
, 75.45 MHz, δ ppm):
53.21, 146.36, 142.51, 134.25, 129.54, 126.33, 123.00, 119.84, 119.92, 116.27, 110.12,
+
09.12, 51.65, 24.34, 11.25. ESI-MS in CH
52.0.
3
OH: Calcd. for [(M + H)] : m/z 252.1, found
Synthesis of 2-(1-methyl-1-H-benzimidazol-2-yl)phenyl)imino)methyl) naphthol (H
1
L )
It was synthesized by following the general procedure described in the literature.12b To an
ethanolic solution (50 mL) of P1 (224 mg, 1 mmol), 2-hydroxy-1-naphthaldehyde
dissolved in ethanol (5 ml, 122 mg, 1 mmol) and a catalytic amount of acetic acid were
added and the reaction mixture refluxed for 5 h. After completion of the reaction
(
monitored by TLC), the volume of the reaction mixture was reduced to ca. 10 mL. Diethyl
ether (5 mL) was added to it and the solution left undisturbed at -4 °C. After 4-5 days, light
yellow needle shaped crystals appeared which were separated by filtration, washed with
diethyl ether and dried in air. Yield: 75%. Microanalytical data: C25
H, 5.07; N, 11.13%. Found C, 79.80; H, 4.98; N, 10.98%. IR (KBr pellets, cm ): νC=N: 1634. H
NMR (CDCl , 300 MHz, δ ppm): 14.43 (s, -OH), 9.41 (s, C(H)=N), 7.95 (d, J = 8.5 Hz, Ar-H),
.88 (s, Ar-H), 7.78 (d, J = 9.1 Hz, Ar-H), 7.68 (m, Ar-H), 7.53 (s, Ar-H), 7.50 (s, Ar-H), 7.45 (s,
H
19 3
N 0, Calcd. : C, 79.55;
-1
1
3
7
1
3
Ar-H), 7.40 (s, Ar-H), 7.34 (m, Ar-H), 7.01 (d, J = 9.1 Hz, Ar-H), 3.71 (s, 3H, -CH
CDCl , 75.45 MHz, δ ppm): 166.30, 158.87, 136.15, 132.34, 131.48, 129.22, 128.07, 127.58,
26.65, 123.67, 123.02, 120.54, 119.84, 119.54, 119.17, 109.75, 30.95. ESI-MS in CH OH:
3
). C NMR
(
3
1
3
+
Calcd. for [(M + H)] : m/z 378.44, found 379.0.
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Synthesis of 2-(1-ethyl-1-H-benzimidazol-2-yl)phenyl)imino)methyl)phenol (HL2)
1
It was prepared following the above procedure for HL but using P2 (238 mg, 1 mmol) and
2
-hydroxy-1-naphthaldehyde (122 mg, 1 mmol). Yield: 60%. Microanalytical data:
0, Calcd. : C, 79.77; H, 5.41; N, 10.73%. Found C, 79.89; H, 5.50; N, 10.79%. IR (KBr
C
26
H
N
21 3
-1
1
pellets, cm ): νC=N: 1618. H NMR (CDCl
.94 (d, J = 8.5 Hz, Ar-H), 7.87 (s, Ar-H), 7.76 (d, J = 9.1 Hz, Ar-H), 7.68 (m, Ar-H), 7.54 (s, Ar-
H), 7.50 (s, Ar-H), 7.43(s, Ar-H), 7.39 (s, Ar-H), 7.34 (m, Ar-H), 6.97 (d, J = 9.1 Hz, Ar-H),
3
, 300 MHz, δ ppm): 14.37 (s, -OH), 9.42 (s, C(H)=N),
7
1
3
4
1
1
.18 (q, 2H, -CH
2
), 1.29 (t, 3H, -CH
3
). C NMR (CDCl , 75.45 MHz, δ ppm): 164.78, 159.10,
3
51. 83, 146.85, 136.09, 132.91, 131.77, 129.18, 128.06, 127.56, 126.61, 123.65, 123.65,
OH: Calcd. for [(M + H)]+
19.71, 119.27, 110.18, 107.04, 106.74, 39.34, 14.99. ESI-MS in CH
3
:
m/z 392.46, found 392.89.
Synthesis of 2-(1-propyl-1-H-benzimidazol-2-yl)phenyl)imino)methyl)phenol (HL3)
1
It was prepared following the above procedure for HL but using P3 (406 mg, 1 mmol) and
2
-hydroxy-1-naphthaldehyde (122 mg, 1 mmol). Yield: 40%. Microanalytical data:
0, Calcd. : C, 79.97; H, 5.72; N, 10.36%. Found C, 79.88; H, 5.67; N, 10.48%. IR (KBr
C
27
H
N
23 3
-1
1
pellets, cm ): νC=N: 1614. H NMR (CDCl
.91 (d, J = 8.5 Hz, Ar-H), 7.88 (s, Ar-H), 7.75 (d, J = 9.1 Hz, Ar-H), 7.65 (m, Ar-H), 7.53 (s, Ar-
H), 7.49 (s, Ar-H), 7.43 (d, J = 9.1 Hz, Ar-H), 7.37 (s, Ar-H), 7.32 (m, Ar-H), 6.96 (d, J = 9.1 Hz,
3
, 300 MHz, δ ppm): 14.38 (s, -OH), 9.40 (s, C(H)=N),
7
13
2
Ar-H), 4.13 (q, 2H, -CH ), 1.71 (sep, 2H, -CH
2
), 0.74 (t, 3H, -CH
3
). C NMR (CDCl , 75.45
3
MHz, δ ppm): 165.76, 158.82, 151.57, 146.83, 143.23, 131.35, 129.18, 128.02, 123.61,
122.61, 122.30, 120.47, 119.95, 119.55, 110.32, 109.20, 46.08, 22.78, 11.23. ESI-MS in
+
CH
3
OH: Calcd. for [(M + H)] : m/z 406.49, found 407.0.
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1
Synthesis of [Cu(L )(NO )] (1)
3
A methanolic solution (2 mL) of CuNO
3
∙2.5H O (32 mg, 0.13 mmol) was slowly layered
2
1
upon a chloroform solution of HL (50 mg, 0.13 mmol) and left undisturbed. After 3-4 days
an olive-green microcrystalline product deposited which was separated by filtration,
washed with diethyl ether and dried at room temperature. Yield: 45%. Microanalytical
data: C25
H
18CuN
4
0 , Calcd. : C, 59.82; H, 3.61; N, 11.16%. Found C, 60.02; H, 3.69; N, 11.29%.
4
-1
IR (KBr pellet, cm ): ν 1614 (C=N), 1471vs (N-O), 1246s (N-O). ESI-MS in CH
3
OH: Calcd. for
1
+
-1
-1
4
[
CuL ] : m/z 439.1, found 439.0. λmax, nm (ε, M cm ) in DMF: 327 (0.9 × 10 ), 426 (0.6 ×
1
04).
2
Synthesis of [Cu(L )(NO )] (2)
3
3
CuNO ∙2.5H
2
O (30 mg, 0.13 mmol) dissolved in methanol (2 mL) was added dropwise to a
2
methanolic solution (2 mL) of HL (50 mg, 0.13 mmol) and the reaction mixture stirred for
1
h at room temperature. The resulting solution was filtered and left for slow evaporation.
After a few days, an olive-green microcrystalline product deposited and was separated by
filtration, washed with diethyl ether and dried at room temperature. Yield: 50%.
Microanalytical data: C26
H
20CuN
4
0 , Calcd. : C, 60.52; H, 3.91; N, 10.86 %. Found C, 60.58; H,
4
-1
4
.01; N, 10.95%. IR (KBr pellet, cm ): ν 1614 (C=N), 1471vs (N-O), 1246s (N-O). ESI-MS in
2
+
-1
-1
CH
3
OH: Calcd. for [CuL ] : m/z 453.0, found 453.1. λmax, nm (ε, M cm ) in DMF: 330 (1.5 ×
4
4
1
0 ), 426 (1.1 × 10 ).
3
Synthesis of [Cu(L )(NO
3
)] (3)
Compound 3 was prepared in an analogous procedure to that described for 1, using
O (30 mg, 0.13 mmol) and HL3 (50 mg, 0.13 mmol). Yield: 40%.
3
CuNO ∙2.5H
2
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Microanalytical data: C27
H
22CuN
4
0 , Calcd. : C, 61.18; H, 4.18; N, 10.57%. Found C, 61.26; H,
4
-1
4
.11; N, 10.65%. (KBr pellets, cm ): ν 1614 (C=N), 1481vs (N-O), 1246s (N-O). ESI-MS in
3
+
-1
-1
CH
3
OH: Calcd. for [CuL ] : m/z 467.1, found 467.1. λmax, nm (ε, M cm ) in DMF: 332 (2.1 ×
4
4
1
0 ), 430 (1.4 × 10 ).
X-ray Structure Determination
Crystals of 1 and 3 suitable for single crystal X-ray analyses were obtained from slow
evaporation of methanol-chloroform or methanol solutions of the compounds. The crystals
were mounted in nylon loops and measured at 296 K. Intensity data were collected using a
Bruker APEX-II PHOTON 100 diffractometer with graphite monochromated Mo Kα (λ =
0.71069 nm) radiation. Data were collected using phi and omega scans of 0.5° per frame
and a full sphere of data was obtained. Cell parameters were retrieved using Bruker
SMART15a software and refined using Bruker SAINT14a on all the observed reflections.
Absorption corrections were applied using SADABS.14a Structure was solved by direct
methods by using SIR97 software14b and refined with SHELXL-2013.14c Calculations were
performed using the WinGX System-Version 1.80.03.14d All non-hydrogen atoms were
refined anisotropically. The hydrogen atoms bonded to carbon atoms were included in the
model at geometrically calculated positions and refined using a riding model. Uiso(H) were
defined as 1.2Ueq of the parent carbon atoms for phenyl and methylene residues and 1.5Ueq
of the parent carbon atoms for the methyl groups. Crystallographic data and refinement
parameters are summarized in Table 1 and a comparison of bond distances and angles are
presented in Table 2. For compound 2 the very low quality of the structure prevents its
discussion.
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DNA Interaction Studies
Electronic absorption titration studies were carried out using a fixed concentration of
compounds in DMF solution (1%) with increasing concentration of CT-DNA in 5 mM Tris
15
HCl/50 mM NaCl buffer, pH 7.5, following an earlier procedure. The purity of the CT-DNA
was verified by electronic absorption studies following the ratio of absorbances at 260 and
280 nm, which was 1.9 and suggested that the DNA was sufficiently free of protein. The
DNA concentration per nucleotide was determined by examining the molar extinction
coefficient of 6600 M−1 cm−1 at 260 nm. An analogous method was used for emission
studies also. EB displacement experiments were performed by monitoring the changes in
fluorescence intensity at excitation (λex = 525 nm) and emission (λem = 603 nm)
wavelengths, after aliquot additions of compounds to the 5 mM Tris HCl/50 mM NaCl
buffer, pH 7.5, of EB bounded DNA (EB-DNA).
DFT calculations and Molecular Docking
The full geometry optimization of complexes 1–3 has been carried out at the DFT level of
16
17
theory using the M06-2X functional with the help of the Gaussian-09 program package.
The relativistic Stuttgart pseudopotential that described 10 core electrons (MDF10) and the
18
appropriate contracted basis set were employed for the Cu atom while the standard basis
set 6-31+G** was applied for all other atoms. No symmetry operations have been applied for
any of the structures calculated. The Hessian matrix was calculated analytically for the
optimized structures in order to prove the location of correct minima (no imaginary
frequencies).
1
0

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Molecular docking studies on 1-3 have been performed using HEX 8.0.0 software and
visualization of the docked systems has been performed using Discovery Studio 3.5
software. The crystal structure of B-DNA (PDB ID: 1BNA) and human serum albumin (PDB
ID: 1HA2) were obtained from the protein data bank (http://www.rcsb.org./pdb). The
initial study of the DNA-complex interaction was carried out by docking of compounds 1-3
with DNA (PDB ID: 1BNA). For this, the whole DNA crystal structure was considered as
active site to avoid biasness towards either DNA minor or major grooves during docking
study. The parameters used for docking include: correlation type shape only, FFT mode 3D,
grid dimension 0.6, receptor range 180, ligand range 180, twist range 360, distance range
40. The coordinates of complexes 1-3 were taken from DFT optimized structures,
converted to the PDB format using Mercury software.
Cell Viability and Proliferation Assay/MTT Assays
Human lung carcinoma (A-549), cervical carcinoma (HeLa) and, breast adenocarcinoma
(
MDA-MB 231) cell lines were used to determine the anticancer and cytotoxic activity in
this study. Cell viability was checked by MTT assay which is a colorimetric assay based on
the conversion of the yellow tetrazolium salt MTT to purple formazan crystals after
19
reaction with mitochondrial dehydrogenase of metabolically active cells. Stock solutions
of complexes 1-3 were prepared in DMSO and diluted with DMEM (Dulbecco's Modified
Eagle's Medium) (the final concentration of DMSO did not exceeded 0.1% v/v). The cell
lines were maintained in complete DMEM (culture medium consisting 10% fetal bovine
serum, supplemented with 20 mM L-glutamine, 100 units/mL penicillin and 100 μg/mL
4
streptomycin) at 37 °C in a humidified atmosphere of 5% CO
2
. Briefly, 1x10 lung A-549 or
1
1

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HeLa or MDA-MB 231 cancer cells were seeded in three different 96 well cell culture plate.
After 24 h, the culture medium was replaced by fresh medium containing various
concentrations of 1-3 and incubated further for 24 h in a 5% CO humidified atmosphere.
2
The medium was then removed and 100 µL of fresh medium and 10 μL of 5 mg/mL MTT in
phosphate buffered saline (PBS, pH 7.4) was added to each well for an additional 2 h.
Finally, DMSO was added to dissolve the MTT formazan crystals followed by incubation for
30 mins. The absorbance of samples was measured at 570 nm with an ELISA plate reader.
Cytotoxicity effect was revealed as the percentage of treated cells relative to untreated cells
at A570 nm. Percent control was calculated using the following formula:
%
Control = [Mean O. D. of Drug treated well / Mean O. D. of control well] × 100
Fluorescence staining of A-549 cells
Cells were seeded in 96 well plates and kept for 24 at 37 °C in a humidified atmosphere of
5
% CO . After that, cells were treated with different concentration of complex 1 for 24 h.
2
Subsequently, cells were washed with 1x PBS and stained with Hoechst 33342 (10µg/mL)
and propidium iodide (5µg/mL) solution. Cells were then washed with PBS and images
were taken in fluorescent microscope (Evos FL, Life technologies) in red and blue channel.
DNA fragmentation assay
DNA was isolated from control and treated A-549 cells according to the procedure
20
5
described by Kuo et al. Briefly, 1 × 10 lung cancer cells were seeded in 6 well plates and
kept at 37 °C in a humidified atmosphere of 5% CO and allowed to adhere overnight. Cells
2
were incubated with different concentrations of complex 1 (15, 18 and 21 μM) for 24 h in
DMEM with 10% FBS. After 24 h, cells were washed and lysed [20 mM Tris-Cl (pH-7.5),
1
2

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0.15 M NaCl, 1 mM EDTA, 0.5% SDS] at 4 °C for 30 min and centrifuged (3,000 rpm for 10
min) at room temperature, the supernatant was collected, and 25 μL Proteinase K (stock 20
mg/mL) was added to it followed by incubation for 2 h at 37 °C. It was again centrifuged at
0
1
2,000 rpm for 30 min at 4 C and supernatant was collected in another tube, DNA was
precipitated by addition of chilled absolute ethanol and 0.1 M NaCl. It was then collected by
centrifugation and washed thoroughly with 70% ethanol; the pellet was dried and
dissolved in autoclaved double distilled water. DNA samples were prepared in a loading
solution (0.25% bromophenol blue, 0.25% xylene cyanol FF and 30% glycerol) in 1:5 ratio
and 10 μL of samples loaded in each well of 1.8% agarose gel containing 0.5 μg/mL
ethidium bromide. Electrophoresis was carried out in TAE buffer for 2 h. The DNA bands in
gel were photographed in a Gel documentation system (G: BOX, SYNGENE).
Cell cycle analysis
5
1
× 10 lung cancer cell lines were seeded in 6 well plates as described above, then treated
with different concentrations of complex 1 for 24 h in DMEM with 10% FBS. Cells were
then washed, trypsinised and fixed in ice cold 70% ethanol and kept overnight at -20 °C.
Then the cells were washed twice in PBS, 500 µL of staining solution [960 µL of 0.1% (v/v)
Triton x-100 solution, 20 µL of PI (1 mg/mL) solution and 20 µL of RNase (10 mg/mL in
PBS)] were added in each sample and incubated for 30 min at room temperature in the
dark. After the staining procedure, the samples were evaluated by a flow cytometer.
ROS generation assay
Intracellular reactive oxygen species level was assessed using DCFH-DA in an inverted
fluorescence microscope. Briefly, A-549 cells were seeded in 96 well plates at a density of
1
3

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4
1
x10 cells per well and treated with different concentrations of complex 1 based on IC50
value for 24 h at 37 °C in a humidified atmosphere of 5% CO . The cells were then exposed
2
to 10 μM of DCFH-DA solubilized in ethanol for 30 min. The samples were subjected to
inverted fluorescence microscopy and DCF fluorescence was detected and photographed.
Results and Discussion
Synthesis and Characterization
1
2
3
Syntheses of HL , HL and HL has been achieved by condensation of N-alkyl substituted 2-
aminophenylbenzimidazole (P1, P2 and P3: Scheme S1, ESI) with 2-hydroxy-1-
naphthaldehyde in a 1:1 molar ratio in ethanol with a catalytic amount of acetic acid under
1
13
refluxing conditions (Scheme S2, ESI). Their H and C NMR spectra are detailed in the
experimental section and relevant ones are presented in Figs. S1-S6, ESI. The mononuclear
1
2
3
copper(II) complexes [Cu(L)(NO
reaction of the corresponding HL1-3 with Cu(NO
MeOH:CHCl or MeOH, as depicted in Scheme 1.
3
)] (1, L = L ; 2, L = L ; 3, L = L ) were prepared by the
3
).2.5H O in a 1:1 molar ratio, in
2
3
1
4

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Scheme 1 Syntheses of complexes 1-3.
These complexes are crystalline, non-hygroscopic solids, stable at room temperature, and
soluble in MeOH, DMF, DMSO and partially soluble in water. They have been characterized
by elemental analyses, FT-IR and ESI-MS spectral methods. The molecular structures of 1
and 3 have further been determined by single crystal X-ray crystallography.
The IR spectra of the free ligands HL1-3 clearly show ν(C=N) at ca. 1633, 1618, and
-1
1
614 cm , respectively. In the complexes 1-3, the ν(C=N) vibration is downshifted to a
-1
-1
lower frequency by ca. 6-19 cm , appearing at ca. 1614, 1612 and 1608 cm , respectively.
The presence of the coordinated nitrate is indicated by the bands observed for
-1
antisymmetric and symmetric stretching at ca. 1471-1481 and ca. 1246 cm , respectively.
-1
The difference between the stretching N–O vibration bands (ca. 230 cm ) confirms the
bidentate coordination of the nitrate ion.21 The ESI mass spectra of 1-3 display the ion
peaks at m/z 439.1, 453.1 and 467.1, respectively, assignable to the corresponding [ML]+
ions (Figs. S7-S9, ESI).
The electronic absorption spectra of the complexes in DMF (experimental section)
show peaks in the UV region at 327 (1), 330 (2) and 332 (3) nm which are attributed to
π → π transitions of the coordinated ligands.22 In addition, broad and strong bands
observed at 426 nm (1 and 2) and 430 nm (3) are assigned to the ligand-to-metal charge
transfer transition.22
*
1
5

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Further, in order to monitor the stability of the complexes 1-3 in DMF and in DMSO
solutions, UV/vis spectra were run for a period of 48 h. No changes were observed on the
initial spectra of the solutions along that period, suggesting that the complexes under study
are stable in both DMF and DMSO solutions (Fig. S10, ESI).
Crystal Structure
The single crystal X-ray diffraction studies of 1 and 3 indicate that they crystallize in the
orthorhombic Pbca and triclinic P-1 space groups, respectively. The molecular
structures of 1 and 3 are shown in Figs. 1(a) and 1(b). The asymmetric units of the
compounds consist of one (1) or two (3) independent complex molecules with the same
coordination environment around the copper centres. In 3, a significant difference is
observed not only in the orientation of the propyl side groups but also in the angle between
the least square planes of the two sets of fused rings in each molecule (51.38º and 56.61º
for the Cu1 and Cu2 containing molecules, respectively). A superposition of the molecules
showing this distinction is shown in Fig. S11, ESI. The copper(II) cation in 1 and 3 presents
a N
2
O
3
coordination environment which deviates from a regular square pyramid as a result
-O-chelation of the nitrate anions [τ5 = 0.10 (Cu1; in 1 and 3) and 0.31 (Cu2 in
)]. Those dissimilar values of 5 in 3 may be associated with the steric influence from
of the 
2
23
3
propyl groups, the highest value concerning the molecule where this moiety is nearly in the
plane of its benzimidazole group. The metals are involved in two fused 6-membered
CuC
3
NO and CuC
3
N metallacycles. While the former is roughly planar with the metal cation
2
at a distance of 0.360 (1) or 0.376 and 0.031 Å (3; Cu1 and Cu2, in this order) from the
least-square plane defined by the non-metal atoms, the latter adopts a boat conformation.
1
6

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The Cu-N bond distances range from 1.926(6) to 1.980(6) Å, while the Cu-O lengths
involving the organic ligand assume values of 1.862(5) or 1.868(5) Å. This distance is
significantly shorter than those involving the nitrate anion (values between 1.990(6) and
2.497(7) Å).
(a)
(b)
Fig. 1. X-ray molecular structures of compounds 1 and 3 with atom numbering scheme.
For 3, only one of the complex molecules is shown.
On the other hand, because of the difficulty in obtaining high-quality crystals of compound
2
, despite repeated attempts, the quality of the X-ray results for 2 is less than optimal, and
thus prevented its discussion. However, it appears to be sufficient to indicate that the
compound 2 also possesses a N coordination environment as that of 1 and 3, with a
O
2 3
distorted square pyramidal geometry. A representation of the structure of 2 and unit cell
parameters are given in Fig. S12, ESI.
DNA interaction studies
Electronic absorption titration
1
7

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DNA is an important cellular target of many metallodrugs for the treatment of multiple
pathologies, including cancer, and plays a relevant role in unrevealing the potential of
metal binding sites with either electron rich nucleobases or phosphate oxygen atoms of the
24
DNA duplex. Thus the binding ability of the complexes 1-3 with CT DNA is characterized
by measuring the effects on UV spectroscopy. Absorption spectroscopic titration
-5
experiments of the complexes 1-3 (1.7 × 10 M) in 5 mM TrisHCl/50 mM NaCl buffer at pH
7.5, were performed using a fixed metal concentration to which increasing amounts of the
DNA stock solution (0-12 µM) was gradually added. The binding of the complexes to duplex
DNA led to a decrease in the absorption intensities with a slight red shift in the absorption
spectra (Fig. 2 and Figs S13-14, ESI). To compare quantitatively the affinity of the
complexes towards DNA, the binding constant (K ) of each complexes to CT DNA was
b
determined by monitoring the changes of absorbance at 327, 328 and 330 nm, respectively,
with increasing concentration of DNA.
-5
Fig. 2. (a) Absorption spectra of complex 1 (1.7 × 10 M) in the absence and presence of
increasing amounts of CT-DNA (0–12 µM) at 25 °C in 5 mM Tris-HCl/50 mM NaCl buffer
1
8

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(
pH 7.5). Arrow shows the absorbance changing upon increasing DNA concentrations. (b)
The non-linear fit of (ε - ε )/(ε - ε ) vs. [DNA].
a
f
b
f
The absorption titration profiles of 1-3 clearly suggest intercalation between these
complexes and DNA, because in general intercalation is associated to hypochromism with
red-shifts, involving a stacking interaction between the aromatic chromophore of
25
complexes and the base pairs of DNA. Further, on the basis of the hypochromism effect in
the complexes, we can also ascertain that their modes of interaction are similar.
Further, to evaluate the binding strength of the complexes with CT DNA, the equilibrium
binding constants (K
linear fit of the plot of (ε
method and using the expression of Bard and coworkers (equation 1):
b
) and binding site size (s, per base pair) were determined from a non-
a
- ε )/(ε - ε ) vs. [DNA] applying the McGhee-von Hippel (MvH)
f
b
f
26
2
2C
½
(ε
a
- ε
f
)/(ε
b
- ε
f
) = (b - (b - 2K
b
t
[DNA]
t
/s) )/2K
b
C
t
(1)
where, b= 1 + K
is the binding constant, C
fitting constant giving an estimate of the binding site size (in base pairs) of the metal
complex interacting with the DNA, ε , ε and ε are, respectively, the molar extinction
b
C
t
+ K
b
[DNA]/2s,
K
b
t
is the total concentration of the metal complex, and s is the
f
a
b
coefficients of the free complex in solution, complex bound to DNA at a definite
concentration and the complex in completely bound form with CT DNA.
The calculated intrinsic equilibrium binding constant (K ) of complexes 1-3 were found to
b
5
−1
5
−1
5
be 3.45(±0.23) × 10 M (s = 0.43), 3.33 (±0.18) × 10 M (s = 0.35) and 3.15 (±0.16) × 10
M−1 (s = 0.33), respectively, and are comparable to those of earlier reported copper(II)
1
9

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complexes.22,27,28 These values found for our complexes, although higher, are of the same
2
9
5
–1
order of magnitude of the classical intercalator EB (K = 1.23 (s = 0.07) 10 M ), which
b
further suggests the binding of the complexes to DNA through an intercalative mode. This
intercalation ability of the complexes 1-3 can be associated to the presence of the
naphthalene moiety in the ligand.30 Further, the relatively stronger binding affinity of
complex 1 compared to those of 2 and 3 can be due to the presence of the least bulky N-
alkyl moiety in the ligand framework, the binding affinity progressively decreasing with the
size increase in the N-alkyl substituent, in the order of 1 > 2 > 3.
Ethidium Bromide (EB) Displacement Assays
Further to substantiate the effective mode of binding of the complexes 1-3 to DNA, via the
31
EB displacement technique, competitive binding studies have been performed for those
complexes using EB bound to DNA (EB-DNA) in 5 mM Tris-HCl/50 mM NaCl buffer at pH
7.5. The emission spectra of EB and EB-DNA in absence and presence of the complexes (0-
30 μM) are shown in Fig. 3a, and Figs. S15-S16, ESI. The addition of complex 1-3 (0- 30 μM)
to EB-DNA (λem = 603 nm) leads to quenching of the emission intensity by 82, 77 and 68%,
respectively relative to the initial fluorescence intensity. A steady decrease in the emission
intensity of EB-DNA, upon addition of complexes 1-3, suggests displacement of the EB,
which is the characteristic feature of intercalators. Further, quenching strength (K
complexes towards EB-DNA has been calculated using Stern-Volmer equation shown below
equation 2):
q
) of
(
F0/F = K
[Q] + 1
(2)
q
2
0

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0
where, F and F correspond to the fluorescence intensities in the absence and presence of
complexes, respectively, and [Q] represents the ratio of total concentration of complexes to
that of CT-DNA. K
q
is a linear Stern–Volmer quenching constant, which can be obtained
0
from the linear regression of F /F with [Q]. The calculated K
q
values for the complexes 1-3
5
5
5
are 3.3 × 10 , 2.8 × 10 and 1.8 × 10 , respectively.
Fig. 3. Emission spectra from EB bound to the DNA in the absence (---) and in the presence
of 1 (a). [EB] = 3.3 μM, [DNA] = 3.3 μM, [1] = 0-30 μM. Arrow shows changes in the
emission intensity upon addition of increasing complex concentration. Stern-Volmer plots
of the EB-DNA fluorescence titration for complexes 1-3 (b).
Further, the apparent DNA binding constants can also be used to evaluate the three main
6
-1
DNA-binding modes; a value above 10 M is an indication of intercalation (for example, EB
4
5
-1
and daunomycin), while values in the range of 10 -10 M imply the groove binding
mode.32 Thus to rationalize the DNA binding interaction of the complexes 1-3, apparent
binding constants (Kapp) were calculated using equation 3:
KEB[EB] = Kapp[complex]
(3)
2
1

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where [complex] is the concentration value at 50% reduction in the fluorescence intensity
7
-1
of EB, KEB (1.0 × 10 M ) is the DNA binding constant of EB, and [EB] is the concentration of
6
6
6
-1
EB (3.3 μM). The Kapp values for 1-3 are found to be 5.5 × 10 , 3.33 × 10 and 3.00 × 10 M ,
respectively. From the above data, it is clear that 1 replaces EB more effectively than the
other complexes, which is in accord with the results obtained from electronic absorption
studies. Although the observed Kapp values for complexes 1-3 are lower than the binding
7
–1 33
constant of the classical intercalators and metallointercalators (10 M ), from the
observed quenching and binding parameters we can ascertain a possible intercalative
mode of binding of the complexes to DNA.
Quantum Chemical calculations
Geometry optimization of complexes 1–3 has been undertaken at the DFT level of theory to
obtain equilibrium structures suitable for the molecular docking studies and to reveal the
composition and energies of the frontier molecular orbitals (MOs) of the complexes. X-ray
structures of 1 and 3 were used as the starting geometries for the optimization procedure.
The general structure of the complexes is preserved as a result of the geometry
optimization. The calculated structural parameters are in good agreement with the
experimental X-ray data often falling within 3-interval of the experimental data (Table S1,
ESI).
The highest doubly occupied MO (HOMO) of the complexes is mostly centered at atoms of
the naphthalene moiety of the ligand, whereas the lowest unoccupied MO (LUMO) is
strongly delocalized between atoms of the ligands (Figs. 4a-b). The spin density is localized
mostly at the Cu atom (0.75 e) with small contributions coming from the donor N(1), N(2),
2
2

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O(1) and O(3) atoms of the ligands and NO –
(up to 0.08 e) reflecting some spin
3
delocalization between Cu and the O and N atoms (Fig. 4c).
Fig. 4. Plots of the HOMO (a) and LUMO (b) of complex 1. (c) Calculated spin density
distribution in complex 1.
In the interaction of DNA with complex 1, the former acts as an electron donor and
the latter serves as an electron acceptor. Hence, in terms of the frontier MO theory, such an
interaction should be determined by the LUMO energy of the complex. The calculated
LUMOcomplex energies slightly increases from 1 to 3 (–1.59 eV for 1, –1.55 eV for 2 and –1.54
eV for 3) correlating with the binding energy estimated by the molecular docking
calculations and with the cytotoxicity of complexes (vide infra). At the same time, the
differences of the LUMO energies is rather small, and other factors may also affect the
relative biological activity of these compounds (e.g., electrostatic and van der Waals
interactions and steric effects imposed by the varying N-alkyl substituent at the imidazole
ring).
2
3

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Molecular Docking with DNA
The designing of molecules can lead to the recognition of specific sequence and structures
of nucleic acids and plays an important role in the development of new chemotherapeutic
drugs. Molecular docking studies have played a very significant role in understanding the
34
mechanistic pathway of DNA-drug interactions, by placing a molecule into the binding
site of the DNA and delivering the visual representation of the binding of the drugs to DNA,
which can substantiate the spectroscopic results. Thus, in order to confirm and rationalize
the observed spectroscopic results and to get a further insight into the intercalation ability,
the complexes 1–3 were successively docked within the DNA duplex of sequence
d(CGCGAATTCGCG)
2
dodecamer (PDB ID: 1BNA). The resulting docking experiments
revealed that the complexes under study interact with B-DNA (PDB ID: 1BNA) to the G-C
35
rich intercalation site of the minor groove (Fig. 5a, 1; Fig. S17a, 2; Fig. S18a, 3, ESI) with
-1
relative binding energy of -341.83 (1), -302.5 (2) and -301.9 (3) kJ mol . The intercalation
ability of these complexes can be due to the presence of the planar naphthalene moiety in
the ligand framework which forms π−π staking with the two DNA flanking bases. These
interactions are considered to be the main driving force of the intercalation binding mode
of the complexes under study and are further stabilized by van der Waals, hydrophobic
contacts and hydrogen bonding.36
2
4

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(a)
(b)
Fig. 5. Molecular docked model of compound 1 with DNA (PDB ID: 1BNA). DG:Guanine, DC:
Cytosine, DA: Adenine
To assess the possible role of the N-alkyl moiety in the ligand framework of the complexes
in their DNA binding we monitored the hydrogen bonding interaction of the energetically
favorable conformation of the docked model for our complexes. For complex 1, the
interaction established two hydrogen bonds involving hydrogen atoms of the N-methyl
group and oxygen atom of DG-24 (2.33 Å) and DC-23 (1.61 Å) (Fig. 5b). As for complex 2,
the binding was stabilized by two hydrogen bonds: hydrogen bonding between hydrogen
atoms of the N-ethyl group and oxygen atom of DG-24 (2.66 and 2.88 Å) (Fig. S17b, ESI),
whereas only one such interaction was observed for 3 involving an hydrogen atom of the
N-propyl group and oxygen atom of DG-24 (1.99 Å) (Fig. S18b, ESI). Thus, it can be
2
5

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concluded that the presence of N-methyl group reinforces the hydrogen bonding between
complex 1 and the DNA, consequently stabilizing the system formed by the DNA and this
complex, thereby increasing the binding affinity. The greater binding affinity (1 > 2 > 3)
obtained from molecular docking studies is in agreement with the experimental results
acquired from electronic and fluorescence studies.
Cytotoxicity studies
MTT assay
The effectiveness of Schiff base copper(II) complexes to suppress cell growth and promote
37
apoptosis has been well documented. The in vitro cell culture studies provide valuable
information for the screening of chemotherapeutic agents and preliminary data for further
relative studies. Thus, cytotoxicities (IC50 values; Table 3; Figs. S19-S21, ESI) of complexes
1-3 against three different human cancer cell lines, viz. A-549 (lung carcinoma), MDA-MB-
231 (breast cancer) and HeLa (cervical cancer) cancer cells, were evaluated through the
19
loss of cell viability using the MTT assay. Complex 1 exhibits a significant inhibitory effect
on proliferation of the A-549 cancer cells and is more potent than the widely used drug
cisplatin (IC50 = 27.2 ± 1.71 µM) under similar experimental condition.19
Table 3: IC50 values of complexes 1-3 against A-549, MDA-MB-231 and HeLa cancer cells (n =
4
).
Incubation time (24 h)
IC50 (µM)
Compounds
A-549
MDA-MB-231
HeLa
1
2
3
16.7 ± 0.29
24.0 ± 1.99
29.1 ± 1.12
29.58 ± 1.17
31.92 ± 0.73
46.03 ± 1.12
32.66 ± 0.58
33.79 ± 0.36
45.27 ± 0.65
2
6

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Further, the IC50 values of complex 1 in all the cell lines under study are smaller than those
of the related complexes 2 and 3, suggesting that the N-methyl group of benzimidazole unit
renders it more cytotoxic to the cells than the N-ethyl or N-propyl substituents. This is in
accord with the DNA binding propensity order of the complexes 1-3.
Since there are no biological data reported for the benzimidazole Schiff base
copper(II) complexes on A-549 cancer cell line, the IC50 values of complexes 1-3 have been
compared with other related Schiff base copper(II) complexes reported recently (Table
38
4
). The in vitro cytotoxicity of our complexes (for A-549) is higher. Moreover, since the in
vitro cytotoxicity of the complex 1 was found to be the highest one for A-549 among our
complexes, all further analysis were carried out using this cell line.
Table 4: Comparison of cytotoxicities [IC50 (µM)] of benzimidazole-based copper(II)
complexes with related ones.
IC50 (µM)
A-549
Compounds
Reference
This paper
1
2
3
16.7 ± 0.29
24.0 ± 1.99
29.1 ± 1.12
This paper
This paper
[
Cu L
Hydroxybenzylidene)isonicotinohydrazide]
Cu L ] [L = (4-Hydroxy-3-
2
] [L = (2-
4
75
38a
[
2
methoxybenzylidene)-N-
92
38a
methylisonicotinohydrazide)]
[
CuL
methylisonicotinohydrazide)]
CuL ] [ L = 2-(((4-
phenoxyphenyl)imino)methyl)phenol ]
CuLCl].CH OH [ L = {2-ethyl-2-((2-
hydroxybenzylideneamino)}propane-1,3-diol)]
2
] [ L =
(4-Methoxybenzylidene)-N-
<50
>50
>50
38a
38b
38c
[
2
[
3
2
7

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Nuclear Morphology using Fluorescence Microscope
Chromatin condensation and fragmentation of nucleus is one of the major properties of
apoptotic cell death. Thus, to determine the type of cell death (apoptosis or necrosis)
caused by complex 1 in A-549 cell line, DNA binding dyes, Hoechst 33342/propidium
iodide (PI) were used (Fig. 6). In control cells, a uniform level of blue fluorescence nucleus
was seen which indicates that almost all cells are alive and healthy. However, for treated
cells, early apoptotic cells with deep blue fluorescence (yellow circle), late apoptotic cells
with fragmented nucleus (blue circle) and necrotic cells with red fluorescence (red circle)
were
clearly
observed
upon
increasing
concentration
of
the
drug
administration (15 and 18 µM). These results clearly suggest that the
complex under investigation favors the apoptotic mode of cell death in lung cancer (A-549)
cells.
Fig. 6.
Images showing morphological changes of complex 1 in lung cancer A-549 cells detected
with dual staining of Hoechst 33342/PI. Yellow circle indicates early apoptotic cells, blue
circle indicates late apoptotic cells and red circle indicates necrotic cells.
2
8