Synthesis, DNA binding and antibacterial activity of metal(II) complexes of a benzimidazole Schiff base

Article abstract of DOI:10.1016/j.poly.2018.10.020

The benzimidazole derivative (E)-2-((4-(1H-benzo[d]imidazol-2-yl)phenylimino)methyl)-4-bromo phenol and the corresponding Zn(II), Ni(II), Cu(II), and Pd(II) complexes were prepared. The synthesized ligand and complexes were fully characterized and the ligand structure confirmed by single crystal X-ray diffraction analysis. The ability of this compound and its complexes to bind to DNA was first investigated with DNA thermal denaturation experiments, in general showing weak interactions. Additionally, UV–Vis absorption spectroscopy was used to assess the binding to DNA and the corresponding binding constants (K_b) were calculated suggesting an intercalative mode of binding for the benzimidazole ligand. All compounds were screened for their antibacterial activity and the Ni(II) complex showed promising results against all bacterial strains (Gram positive and Gram negative) while the rest of the compounds showed activity against selective strains.

Full text of DOI:10.1016/j.poly.2018.10.020

Accepted Manuscript
Synthesis, DNA binding and antibacterial activity of metal(II) complexes of a
benzimidazole Schiff base
Khalid Mahmood, Waleed Hashmi, Hammad Ismail, Bushra Mirza, Brendan
Twamley, Zareen Akhter, Isabel Rozas, Robert J. Baker
PII:
S0277-5387(18)30656-9
https://doi.org/10.1016/j.poly.2018.10.020
POLY 13494
DOI:
Reference:
Polyhedron
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 August 2018
3 October 2018
5 October 2018
Please cite this article as: K. Mahmood, W. Hashmi, H. Ismail, B. Mirza, B. Twamley, Z. Akhter, I. Rozas, R.J.
Baker, Synthesis, DNA binding and antibacterial activity of metal(II) complexes of a benzimidazole Schiff base,
Polyhedron (2018), doi: https://doi.org/10.1016/j.poly.2018.10.020
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Synthesis, DNA binding and antibacterial activity of
metal(II) complexes of a benzimidazole Schiff base
Khalid Mahmood,[a] Waleed Hashmi,[b] Hammad Ismail,[c] Bushra Mirza,[b] Brendan Twamley,[d]
Zareen Akhter,*[a] Isabel Rozas,*[d] and Robert J. Baker*[d]
[a] Department of Chemistry, Quaid-i-Azam University of Islamabad, Pakistan
[b] Department of Biochemistry, Quaid-i-Azam University of Islamabad, Pakistan
[c] Department of Biochemistry and Biotechnology, University of Gujrat, Pakistan
[d] School of Chemistry, Trinity College Dublin, The University of Dublin, Dublin 2, Ireland
*Corresponding authors:
Zareen Akhter, E-mail: zareen_a@qau.edu.pk
Isabel Rozas, E-mail: rozasi@tcd.ie
Robert J. Baker, E-mail: bakerrj@tcd.ie
1

Abstract
The benzimidazole derivative (E)-2-((4-(1H-benzo[d]imidazol-2-yl)phenylimino)methyl)-4-bromo
phenol and the corresponding Zn(II), Ni(II), Cu(II), and Pd(II) complexes were prepared. The
synthesized ligand and complexes were fully characterized and the ligand structure confirmed by
single crystal X-ray diffraction analysis. The ability of this compound and its complexes to bind to
DNA was first investigated with DNA thermal denaturation experiments, in general showing weak
interactions. Additionally, UV-vis absorption spectroscopy was used to assess the binding to DNA and
the corresponding binding constants (Kb) were calculated suggesting an intercalative mode of binding
for the benzimidazole ligand. All compounds were screened for their antibacterial activity and the
Ni(II) complex showed promising results against all bacterial strains (Gram positive and Gram
negative) while the rest of the compounds showed activity against selective strains.
Graphical Abstract
Synopsis
Several metal complexes of a benzimidazole Schiff base were prepared and characterised showing
moderate DNA binding as intercalators and interesting activity against Gram positive and negative
bacteria.
Keywords: benzimidazole • Schiff base • metallodrug • DNA-interaction • antibacterial
2

1. Introduction
Considering the lack of new antibiotics in the market, there is a high risk of an uncontrolled spread of
resistant pathogens. The increase in the incidence of chronic infections represents an added problem
because many of these diseases are untreatable due to resistance. There are many possible targets in
bacteria, and one that has recently attracted much attention is DNA [1].
Thus, many DNA binding drugs have shown antimicrobial activity [2]. For example, the DNA
intercalator echinomycin is more effective against clinical isolates of methicillin-resistant
Staphylococcus aureus (MRSA) than the clinically used vancomycin not only in vitro but also in vivo in
mice [3]. Additionally, derivatives of the DNA binder metallo-complex [CoL3]2+ containing 2,2-
bipyridine, 1,10-phenanthroline, imidazole, methylimidazole, ethylimidazole or dimethylimidazole,
have been reported to show antibacterial activity against Escherichia coli, Salmonella typhimurium, S.
aureus or Enterococcus faecalis, among others [4,5].
With the aim of avoiding antibacterial resistance, the FDA has approved broad-spectrum antibiotics
such as the Cu complexes of lomefloxacin [6] or ciprofloxacin [7], which act by inhibiting DNA gyrase
activity and have demonstrated activity against both Gram positive and Gram negative bacteria, with
MIC values around 0.3–2.6 μM. Another important DNA-binding agent is the minor groove binder
pentamidine, which has been clinically used since the 1940s against a number of protozoal diseases.
It was found that metals can really enhance the antibacterial activity of groove binders and, thus,
while pentamidine analogue bis-(3-(4-dimethylaminophenyl)allylidene)-1,2-diaminoethane shows
MIC values of 250 and 125 μg mL-1 for B. subtilis and E. coli, respectively, its Zn(II) complex has MIC
values around 0.49 μg mL-1 for both bacteria [8].
3

Regarding suitable ligands for this type of potential metallodrugs, the benzimidazole heterocycle
(Figure 1), which is found in commercial drugs such as omeprazole, mebendazole, and astemizole [9],
is present in many biologically important DNA minor groove binding molecules such as Hoechst-
33258 and Hoechst-33342 [10], as well as in several antimicrobials [11]. This type of derivative is also
reported as mitochondria targeting photocytotoxic agent [12]. Furthermore, benzimidazole based
compounds are important for their applications as anti-inflammatory, antioxidant, antiulcers,
antihypertensives, antiviral, antifungals, antihistaminics and antiparasitic activities [13]. In addition,
benzimidazoles can form stable complexes with different transition metals and their coordination
chemistry continues receiving huge attention not only because of their interesting spectral, magnetic
and structural properties [14], but also because metal complexes of benzimidazoles and its
derivatives have shown therapeutic applications such as anticancer agents, antioxidant and enzyme
inhibitors [15].
Figure 1. Structures of benzimidazole, azomethine (Schiff base) functionality, compound 1 and
metallocomplexes [M= Zn(II), Pd(II), Ni(II), Cu(II)] proposed in this study.
A brief literature summary on the biological applications of (benz)imidazole Schiff base metal
complexes has been recently reported [16]. Thus, it is stated that Ag(I), Zn(II), Sn(II) and Pd(II)
complexes of these Schiff bases result in effective in vitro antimicrobial, antifungal and antibacterial
activities [17]. Moreover, the azomethine functionality (-HC=N-) in the mentioned Schiff bases (Figure
1) seems to be responsible for the various biological activities of these compounds, for instance as
4

antibacterial [18,19], antifungal [20,21], anti-inflammatory [22] or anticancer/antitumor agents [23-
25]. Additionally, it has been found that these biological activities are enhanced in their metal(II)
complexes [26-30].
Keeping in mind the urgent need for new antibiotics and taking into account the suitability of
benzimidazoles and Schiff bases as ligands in antibacterial metallodrugs, we present in this paper the
preparation, biophysical and biochemical investigation of a new family of metal complexes of
compound 1 (Figure 1). The effect on the biological activity of different metals such as Zn(II), Pd(II),
Ni(II) and Cu(II), belonging to different groups and rows of the Periodic Table, was explored based on
the bioactivity of related complexes. Thus, Zn(II), Pd(II), Ni(II) and Cu(II) derivatives have shown
activity as antibacterial, antifungal and anticancer agents as mentioned before [6-8,15-17,26-30].
Additionally, the drug-DNA interaction was investigated by UV-vis spectroscopy titration and DNA
thermal denaturation experiments as well as their antimicrobial activities against a number of Gram
positive and Gram negative bacteria.
2. Material and Methods
2.1. General Information
All chemicals including solvents and reactant materials were purchased from Sigma Aldrich and were
used without further purification. Salmon testes DNA [(st)DNA] was purchased from Sigma Aldrich (St
Louis, MO, USA) and has an extinction coefficient ε260= 6600 M-1 cm-1. Infrared (IR) spectra were
1
13
recorded on Perkin Elmer Spectrum One spectrometer in 4000-400 cm-1 region. H and C{1H} NMR
spectra were obtained on a Bruker Avance 400 and Bruker DPX 400, and were referenced to the
1
residual H and 13C resonances of the solvent used. High resolution mass spectrometry analysis were
carried out using a Premier Waters Maldi-quadrupole time-of-flight (Q-TOF) mass spectrometer
5

equipped with Z-spray electrospray ionization (ESI) and matrix assisted laser desorption ionization
(MALDI) sources.
2.2. Synthesis
2.2.1. Preparation of 4-(1H-benzo[d]imidazole-2-yl)aniline (2)
In a two neck round bottom flask (250 mL) equipped with magnetic stirrer and reflux condenser, o-
phenylenediamine and 4-aminobezoic acid were refluxed in polyphosphoric acid, in a 1:1 molar ratio.
The reaction mixture was heated at 130 °C for 3 h. After the completion of reaction the thick liquid
mixture was poured onto crushed ice, neutralized with 4N NaOH solution and precipitates were
formed. The precipitate containing compound 2 was removed from the mother liquor by filtration
and was used immediately for the preparation of compound 1.
2.2.2. Preparation of (E)-2-((4-(1H-benzo[d]imidazol-2-yl)phenylimino) methyl)-4-bromophenol (1)
[31]
Compound 1 was synthesized by condensing 4-(1H-benzo[d]imidazole-2-yl)benzenamine (2) (0.21 g,
1 mmol) and 5-bromosalicylaldehyde (0.2 g, 1 mmol) in dry ethanol under reflux for 6 hours. The
resultant orange coloured precipitate was collected by filtration, washed with hot ethanol and
recrystallized from THF. Yield: 81%. M.p.: 165 °C (Lit.: 161-165 °C [29]). IR (, cm-1): 3451 (O-H), 3337
(N-H), 1619 (azomethine-C=N), 1558 (imidazole-C=N), 1436 (imidazole-C-N), 1262 (C-O). 1H NMR (400
13
MHz, DMSO-d6) δ (ppm): 6.96-8.29 (m, Ar), 8.86 (s, CH=N), 11.68 (bs, s, OH), 12.91 (s, NH). C{1H}
NMR (400 MHz, DMSO-d6) δ (ppm): 111.3-145.7 (Ar C), 162.0 (CH=N), 149.9 (imidazole CH=N). Anal.
Calc. for C20H14N3OBr: C, 61.24; H, 3.60; N, 10.71. Found: C, 61.27; H; 3.59, N; 10.69. HRMS (m/z)
calculated for C20H15N3OBr [M+H+] 392.0398, found 392.0394.
2.2.3. Preparation of the Zn(II) complex of compound 1 (3)
6

In a two neck round bottom flask (100 mL) equipped with magnetic stirrer and reflux condenser, a
hot ethanolic solution of 1 (1 mmol) and a solution of Zn(CH3COO)2 • 2H2O (0.5 mmol) were mixed in
a 2:1 molar ratio and refluxed for 4 hours. A bright yellow precipitate was collected by filtration,
washed with hot ethanol and recrystallized from THF. Yield: 73%. M.p.: >300 oC. IR (, cm-1): 3367 (N-
H), 1606 (azomethine-C=N), 1545 (imidazole-C=N), 1449 (imidazole-C-N), 1306 (C-O), 580 (M-O), 460
(M-N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 6.68-8.29 (m, Ar), 9.06 (s, CH=N), 13.06 (s, NH). 13C{1H}
NMR (400 MHz, DMSO-d6) δ (ppm): 112.34-145.64 (Ar C), 165.7 (CH=N), 150.91 (imidazole CH=N).
Anal. Calc. for C40H26N6O2ZnBr2: C, 56.66; H, 3.09; N, 9.91; Zn, 7.71. Found: C, 56.59; H, 3.03; N, 9.92;
Zn, 7.72. HRMS (m/z) calculated for C40H26N6O2ZnBr2 [M+H+] 847.9834, found 847.9817.
2.2.4. Preparation of the Pd(II) complex of compound 1 (4)
Compound 1 (1 mmol) was dissolved in hot ethanol in a two neck round bottom flask (100 mL)
equipped with magnetic stirrer and reflux condenser. Then, a solution of Pd(CH3COO)2 (0.5 mmol)
was added dropwise and the mixture refluxed for 3 hours. A dark brown precipitate was collected by
filtration, washed with hot ethanol and recrystallized from THF. Yield: 65%. M.p.: >300 °C. IR (,
cm-1): 3356 (N-H), 1588 (azomethine-C=N), 1559 (imidazole-C=N), 1440 (imidazole-C-N), 1314 (C-O),
1
567 (M-O), 442 (M-N). H NMR (400 MHz, DMSO-d6) δ (ppm): 6.97-8.29 (m, Ar), 9.046 (s, CH=N),
12.97 (s, NH). 13C{1H} NMR (400 MHz, DMSO-d6) δ (ppm): 114.2-133.2 (Ar C), 164.1 (azomethine
CH=N), 150.0 (imidazole CH=N). Anal. Calc. for C40H26N6O2PdBr2: C, 54.05; H, 2.95; N, 9.45; Pd, 11.97.
Found: C, 54.16; H, 3.18; N, 9.51; Pd, 11.99. HRMS (m/z) calculated for C40H27N6O2PdBr2 [M+H+]
886.9597, found 886.9602.
2.2.5. Preparation of the Ni(II) complex of compound 1 (5)
In a two neck round bottom flask (100 mL) equipped with magnetic stirrer and reflux condenser,
ethanolic solutions of compound 1 (1 mmol) and Ni(CH3COO)2 • 4H2O (0.5 mmol) were mixed in 2:1
molar ratio and refluxed for 3 hours. An orange precipitate was collected by filtration, washed with
7

hot ethanol and crystallized from THF. Yield: 60%. M.p.: 285 °C. IR (, cm-1): 3365 (N-H), 1597
(azomethine-C=N), 1531 (imidazole-C=N), 1445 (imidazole-C-N), 1286 (C-O), 553 (M-O), 436 (M-N). 1H
13
NMR (400 MHz, DMSO-d6) δ (ppm): 6.79-8.68 (m, Ar) 9.08(s, CH=N), 13.17(s, NH) C{1H} NMR (400
MHz, DMSO-d6) δ (ppm): 115.28-135.53 (Ar, C), 170.14 (CH=N), 151.06 (imidazole CH=N). Anal. Calc.
For C40H26N6O2NiBr2: C, 57.11; H, 3.12; N, 9.99; Ni, 6.98. Found: C, 57.47; H, 3.23; N, 10.33; Ni, 6.91.
HRMS (m/z) calculated for C40H28N6O2NiBr2 [M+H+] 843.1935, found 843.1912.
2.2.6. Preparation of the Cu(II) complex of compound 1 (6)
In a two neck round bottom flask (100 mL) equipped with magnetic stirrer and reflux condenser,
ethanolic solutions of compound 1 (1 mmol) and Cu(CH3COO)2 • 2H2O (0.5 mmol) were mixed and
refluxed for 6 hours. A greenish yellow precipitate was collected by filtration washed with hot
ethanol and crystallized from THF. Yield: 80%. M.p.: 223 °C. IR (, cm-1): 3349 (N-H), 1595
(azomethine-C=N), 1515 (imidazole-C=N), 1442 (imidazole-C-N), 580 (M-O), 420 (M-N). Anal. Calc. for
C40H26N6O2CuBr2: C, 56.79; H, 3.10; N, 9.93; Cu, 7.51. Found (%): C, 56.65; H, 3.02; N, 9.91; Cu, 7.46.
HRMS (m/z) calculated for C40H28N6O2CuBr2 [M+H+] 844.9936, found 844.9901.
2.3. Biophysical experiments
2.3.1. DNA thermal denaturation
Thermal melting experiments were conducted with Perkin Elmer Lambda 35 UV-VIS spectrometer,
(TempLAB software). Temperatures were monitored with a thermistor inserted into a 1 mL quartz
cuvette containing the same volume of water as in the sample cells. Absorbance changes at λ = 260
nm were monitored from a range of 30 to 90 °C in a gradient of 1 °C per min and measuring every
0.2 °C. A quartz cell with a 1 cm path length was filled with a 1 mL solution of (st)DNA or (st)DNA-
ligand complex in DMSO:water mixture in 30:70 ratios by %volume, respectively. The (st)DNA
8

oligomer (100 μM) and the ligand solution (10 μM) were prepared in a phosphate buffer (0.01 M
K2HPO4/KH2PO4), adjusted to pH 7 and syringe filtered, so that a ligand to DNA base ratio of 0.1 was
obtained. Experiments were performed immediately after solutions were prepared to avoid
decomposition. The difference in melting temperature (ΔTm, °C) was calculated as factor of stability
and the Tm was defined as the mid-point of transition temperature.
2.3.2. Absorption spectroscopic studies
The absorption spectroscopic and the thermal denaturation studies to assess the interaction
between the ligands (compound 1 and its metal complexes) and DNA was carried out using Perkin
Elmer Lambda 35 UV-vis spectrometer equipped with temperature control unit and temp lab
software. For the titrations in the absorption spectroscopic study, stock solutions (500 µM) of Schiff
base 1 and the four metal complexes were prepared in DMSO and for further dilutions (100 µM)
deionized water was used. Salmon testes DNA [(st)DNA] was prepared in Tris-HCl buffer solution (0.6
M HCl) and 50 mM NaCl with pH = 7.33 and this buffered (st)DNA was filtered using syringe filtration
method to make it protein free. DNA and ligand solution were incubated for 5 min before recording
the absorption spectrum. A fixed amount (1000 µL) of the ligand (100 µM) was titrated with
increasing quantity (10 to 80 µL) of DNA. The intrinsic binding constants or Kb (a measure of the
strength of interaction between (st)DNA and the chromophores of the ligand) were calculated from
the Benesi-Hildebrand equation [32].
A0
εG
εG
1
=
+
×
A−A0
εH−G−εG
εH−G−εG
퐾푏[DNA]
where [DNA] represents the concentration of (st)DNA in base molarity; A0 and A is the absorbance of
the free drug and the drug in the presence of DNA, respectively; and εG and εH-G are the absorption
coefficients of the drug and drug-DNA complex, respectively. Thus, the intrinsic binding constant Kb
can be calculated from the intercept to slope ratio of A0/A-A0 vs. 1/[DNA] plots [33] .
9

2.4. X-Ray crystallographic analysis
A suitable crystal of ligand 1 was selected and data was collected on Bruker D8 Quest ECO
diffractometer. The sample was mounted on a MiTeGen micromount and data was collected at
100(2) K using an Oxford Cryostream. Bruker APEX software was used to collect and reduce the data,
determine the space group, solve and refine the structures [34]. Absorption corrections were applied
using SADABS 2014 [35]. The structure was solved with the XT structure solution program [36] using
Intrinsic Phasing and refined with the XL refinement package [37] using Least Squares minimization in
Olex2 [38]. All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were assigned to
calculated positions using a riding model with appropriately fixed isotropic thermal parameters.
Parameters and refinements for data collection are summarized in Table S1 (Supporting
Information). CCDC 1566746 contains the supplementary crystallographic data for this paper. This
data can be obtained free of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
2.5. Biochemistry
2.5.1. Antibacterial disc diffusion assays
The antibacterial activity of the compounds was screened against Escherichia coli, Enterobacter
aerogenes and Micrococcus luteus. The bacteria were cultured in nutrient broth under controlled
conditions of temperature (37 °C) for 24 hours. From this cultured medium, 1% broth containing
about 106 colony-forming units (CFU/mL) of bacterial strain was transferred to the nutrient agar
medium at 45 °C and then poured into the sterile petri plates. When the medium was solidified,
about 5 μL of the test compound (40 mg mL-1 in DMSO) was poured on sterile paper discs of 4 mm
size and placed on nutrient agar plates respectively. In each plate the standard antibacterial drug
kanamycin (1 mg mL-1) served as a positive control and DMSO served as negative control. For each
bacterial strain, the plates were prepared in triplicate. These plates were incubated for 24 hours at
37 °C with compounds 1, 3-6 (200 µg mL-1). The antibacterial activity was determined by measuring
10

the diameter of the zone of complete inhibition in mm when the concentration of the compounds
was 200 µg mL-1. Downstream calculations were done for the compounds which showed activity at
this concentration (inhibition zone ≥10 mm) by making dilutions (200 µg mL-1, 100 µg mL-1, 50 µg
mL-1, 25 µg mL-1, 12.5 µg mL-1, 6.25 µg mL-1, 3.12 µg mL-1). Then, minimum inhibitory concentration
(MIC) values for compounds 1, 3-6, defined as the lowest concentration of the compound that
inhibits visible growth, and were calculated from the inhibition zone diameters via linear regression.
All determinations were performed in triplicate.
3. Results and Discussion
3.1. Synthesis and characterization
The preparation of compound 1 had been reported in the literature by Cernatescu and Comanita [31]
starting from precursor 2; however, we have carried out the preparation of compound 1 following a
slightly different method. First, precursor 2 was synthesized by the condensation of 2-aminoaniline
and p-aminobenzoic acid in the presence of polyphosphoric acid at 130 °C (Scheme 1).
Scheme 1. Synthesis of 1 and its metal complexes
11

Then, without further purification, compound 2 was reacted with 4-bromo-2-hydroxybenzaldehyde
in a reflux of ethanol forming compound 1 in an excellent yield (81%) without the need of acetic acid
as a catalyst, as it had been previously reported [31], since traces of phosphoric acid from the
previous step are present. The four new metal complexes 3-6 were successfully prepared by the
reaction of 1 and the corresponding metal(II) acetate in refluxing ethanol (Scheme 1), in good to
excellent yields (60-80%). All compounds prepared are stable under normal atmospheric conditions
and soluble in organic solvents such as DMSO or DMF, whilst 4 and 6 are also partially soluble in
ethanol. All were fully spectroscopically and analytically characterized.
The infrared spectrum of 1 showed a broad band at 3451 cm-1 assigned to the phenolic OH group and
a low intensity band at 3377 cm-1 due to the N-H group of the imidazole ring. Strong bands at 1619,
1558 and 1436 cm-1 are assigned to the azomethine group (C=N), imidazole azomethine group (C=N)
and imidazole C-N bond, respectively [39] . The IR spectra of the metal complexes showed a loss of
the phenolic OH band and an increase in the (C-O) at 1286-1319 cm-1, indicative of deprotonation
and formation of a M-O bond. The bands associated with the imidazole fragment do not shift
12

indicating that this moiety does not coordinate to the metal centre. However, a shift of the C=N band
associated with the azomethine group is observed, signifying its participation in bonding to the
metal. The (M-O) and (M-N) bands in the 530-580 and 420-471 cm-1 region also confirm metal
bonding.
1
Both H and 13C{1H} NMR spectroscopy confirm binding of the ligand to the diamagnetic metal
centres. The phenolic proton in 1 (H = 11.68 ppm) is no longer present whilst there is a down-field
shift of the azomethine proton (H = 8.86 ppm in 1 to H ~ 9.05 ppm in 3 and 4), attributable to
13
electron transfer from the Schiff base N atom to the metal ion [40]. Correspondingly, in the C{1H}
NMR spectra the azomethine carbon shifts from C = 162 ppm in 1 to C = 165.7 ppm in 2 and 3.
3.2. Single crystal X-Ray analysis
A single crystal of 1·MeOH was obtained by the slow evaporation of a methanol solution under
normal atmospheric conditions and the ordered arrangement of atoms in the molecule is shown in
Figure 2a. Parameters and refinements for data collection are summarized in Table S1 (Supporting
Information). This structure validates the spectroscopic data previously recorded. Metric parameters
are normal and selected bond lengths and angles are presented in Table 1.
Table 1. Selected bond lengths (Å) and angles (°) for compound 1
Bond length
1.322(5)
Bond angle
N(6)-C(7)
C(7)-N(8)
O(24)-C(19)-C(20)
O(24)-C(19)-C(18)
C(17)-N(16)-C(13)
N(16)-C(17)-C(18)
C(7)-N(8)-C(9)
118.0(4)
122.6(3)
123.4(3)
120.1(4)
107.3(3)
1.351(5)
1.410(5)
1.284(5)
1.337(5)
0.8400
C(13)-N(16)
N(16)-C(17)
C(19)-O(24)
O(24)-H(24)
13

It can be observed that all aromatic systems and the azomethine group are in the same plane. The
most obvious feature is the strong intra and inter molecular hydrogen bonding formed by the
hydroxyl groups in 1 and MeOH, e.g. O24-H24…N16 (dO…N = 2.586(4) Å), O26-H26…N6 (dO…N =
2.698(4) Å, see Figure 2b). Further intermolecular interactions are seen by the amide N8-H8…O26
(dN…O = 2.733(4) Å).
(a)
(b)
(c)
Figure 2. (a) Asymmetric unit of ligand 1 showing partial atom numbering and displacement ellipsoids
(50% probability level). Dashed lines indicate H-bonding interactions; (b) intra/inter-molecular
interactions (Br1…C20 = 3.998(4); Br1…C1 = 3.563(4) Å, O26…N6 = 2.698(4) Å, N8…O26 = 2.733(4) Å,
O24…N16 = 2.586(4) Å); and (c) packing diagram of 1·MeOH.
Moreover, intermolecular interactions such as π-π (between phenyl rings, 3.68 Å with a shift of 1.49
Å) and Br1…C20 and Br1…C1 [3.998(4) and 3.563(4) Å, see Figure 2b] are observed. Additionally,
MeOH hydrogen bonding described above and other non-conventional hydrogen bonds [C15…O26,
dC…O = 3.452(5) Å] are combined to order the molecule in sheets as shown by the packing diagram in
14

Figure 2c. The ligand 1 adopted a P-1 space group symmetry with unit cell dimensions of a =
7.1625(5), b = 11.1249(9), c = 12.0359(9) Å and cell angles of α = 96.415(3),  = 96.576(3),  =
105.598(3)°.
3.3. DNA binding studies: DNA thermal denaturation
Thermal denaturation experiments were employed, as detailed in our previous work [41], to assess
the binding affinities of the compounds prepared to unspecific salmon testes DNA [(st)DNA]. It is
well-known that when the temperature of a double stranded DNA solution increases, the double
helix splits because of the dissociation of the hydrogen bonds between the base pairs. Accordingly,
we can define the Tm melting temperature as that where half the bases dissociate from each other
detected at 260 nm [42]. Depending on the magnitude of the interaction between a ligand and DNA,
this Tm will shift (∆Tm) indicating the strength of the interaction. Thus, Tm values of (st)DNA in the
presence of compound 1 and its metal complexes 3-6 were obtained; the increments (ΔTm) are
collated in Table 2 and the plots are shown in Figure 3.
The ΔTm value obtained after the addition of each ligand to (st)DNA indicates that, even though the
values are small, 1 and its metal complexes bind to DNA. The free compound 1 as well as its Zn(II)
and Ni(II) complexes present similar values of ΔTm indicating a similar binding strength; in the case of
the Pd(II) and Cu(II) complexes smaller values of ΔTm are obtained indicating a weaker interaction.
Therefore, the ∆Tm values meassured for the uncoordinated compound 1, and the Zn(II) and Ni(II)
o
complexes, 3 and 5 (3.3-3.2 C) are the largest of this set, indicative of medium DNA binding.
Considering that unspecific (st)DNA was used for these experiments, no specific information on the
mode of binding to DNA can be extracted from the results; however, since the three ΔTm values are
very similar it could be deduced that the binding is driven by the ligand itself and the corresponding
cations do not highly influence the interaction.
15

Table 2. Increments on (st)DNA thermal denaturation (ΔTm, °C), binding constants (Kb, M−1) and
isobestic points (nm) found for compound 1 and metal complexes 3-6.
ΔTm (°C)[a]
Kb (M-1)[b]
Isobestic points (nm)
1
3.3
3.2
2.3
3.2
2.8
1.47 ×105
9.8 ×104
6.0 ×103
9.5 ×104
3.1 ×104
338
346
-
3 [Zn(II)]
4 [Pd(II)]
5 [Ni(II)]
6 [Cu(II)]
347
353
Proflavin[42]
-
1.60 ×105
-
[a] The increment in DNA thermal melting (ΔTm, °C) was measured in unspecific salmon
testes DNA. Experiments were run in phosphate buffer (10 mM, pH 7). Thermal denaturation
for natural (st)DNA = 68.5 °C. [b] Calculated from the A0/A-A0 vs. 1/[DNA] plots of the UV-vis
DNA titrations results.
Figure 3. Thermal melting plots for (dark blue) (st)DNA (100 µM) in the presence of (red) compound
1, (green) complex 3 [Zn(II)], (purple) complex 4 [Pd(II)], (light blue) complex 5 [Ni(II)] and (orange)
complex 6 [Cu(II)].
3.4. DNA binding studies: UV-visible absorption spectroscopy
It is well known that UV-vis spectroscopy is a very useful technique to evaluate the binding ability of
small molecules to DNA and it can provide information about the binding mode [43]. Thus, we have
carried out UV-vis titration experiments with 1 and the metal complexes 3-6 by adding aliquots of
(st)DNA. The interaction between any ligand and DNA is evident from the shifting either in
16

absorbance (hypochromic, hyperchromic) or in λmax (hypsochromic, bathochromic). Intercalation of a
ligand between the DNA base pairs results in the coupling of the π* orbital of the intercalator with
the DNA base pairs, reducing the π→π* transition energy, and thus causing a red shift
(bathochromism). Moreover, if the coupling π* orbital is partially filled with electrons it decreases
the transition probabilities and a hypochromic shift in the absorbance can be observed [44].
Therefore, the presence of hypochromic and bathochromic shifts in the resulting UV-vis spectra of
the titration of a ligand with (st)DNA indicates that such a ligand is an intercalator. The absorption
spectra of the titration of 1, 3-6 with (st)DNA are shown in Figure 4 and all the calculated Kb and
isobestic points (nm) are presented in Table 2.
When the concentration of (st)DNA was increased a shift in absorbance as well as in λmax was
observed for most of the compounds. The absorption titration spectrum for DNA binding with 1 is
shown in Figure 4a. Two isobestic points are observed, one at 338 and the other at 423 nm. The
appearance of an isobestic point at 338 nm (Table 2) exemplifies that there are two chemical species,
one being the free compound 1 and the other the DNA-1 complex. Furthermore, a hypochromic shift
in absorbance is observed along with a small bathochromic shift (∆λ = 15 nm) to λmax = 371 nm
indicating the disappearance of the free compound 1 and the formation of the DNA-1 complex.
Hence, the λmax bathochromic shift and hypochromic shift in absorbance suggest that compound 1 is
an intercalator. The absorption spectra for the titration of 3-6 are shown in Figures 4b-e. Notably, the
second isobestic point of 1 (at λmax 423 nm) does not appear in the metal complexes. Among the
complexes, Pd(II) complex 4 showed not only the smallest Kb value, but also neither a significant shift
in absorbance or λmax nor isobestic point indicating lack of interaction with DNA in agreement with
the DNA thermal denaturation results.
17

Figure 4. Changes in the UV-vis spectra of (a) compound 1 (b) complex Zn(II)-1 (3) (c) complex Pd(II)-1
(4) (d) complex Ni(II)-1 (5) and (e) complex Cu(II)-1 (6), having concentration 1×10-4 M, as a function
of increasing quantities of (st)DNA (10-80 µM) in 50 mM Tris-HCl/NaCl buffer (pH= 7.35) at 25 °C. The
arrows indicate the decrease in absorbance with increase in DNA concentration. Inset plots show
A0/A-A0 vs. 1/[DNA] for the calculation of the corresponding intrinsic binding constant Kb.
18

The DNA binding constants for complexes 3, 5 and 6 (Table 2) indicate a stronger interaction than
that of Pd(II) complex 4, but weaker than for the free ligand 1, which Kb compares well with that of
the known intercalator proflavin [42]. As in the case of the uncoordinated ligand, isobestic points are
observed for the DNA interaction with both complexes 3 and 5 (Table 2) indicating the existence of
two species, the free metal complex (3 or 5) and DNA-complexes. The titration spectra of 3 shows a
λmax bathochromic shift and hypochromic shift in absorbance (Figure 3b) similar to 1 and, hence, DNA
intercalation is expected. In the case of the Ni(II) complex 5, the titration UV-vis spectra shows that a
decrease in absorbance occurs with the addition of DNA aliquots (hypochromic effect); however, a
hypsochromic effect is observed for the λmax
.
Summarizing, the strongest interaction with (st)DNA was exhibited by the ligand 1 in agreement with
the DNA thermal denaturation experiments. The corresponding absorption spectra showed the
appearance of an isobestic point indicating the formation of a DNA-1 complex as well as a
hypochromic effect along with a hypsochromic shift (∆λ = 14 nm) to λmax = 369 nm (Table 2). These
results suggest that ligand 1 and complex 3 bind to DNA by intercalation mode while the absorption
spectra of complexes 5 and 6 show binding to DNA by multiple modes but not intercalation [44].
The experimental results clearly indicate that compounds 1, 3, 5 and 6 interact with DNA since the
distinct hypochromic effect suggests the formation of DNA-ligand complexes. The lack of DNA
binding of the Pd(II) complex 4 could be attributed to the large atomic radius of the Pd(II) core (86
pm [45]) that hinders potential intercalation or minor groove binding.
Compound 1 and its Zn(II) complex (3) bind to DNA by intercalation with what could be justified on
the one hand by the planarity of the Schiff base helped by the intramolecular hydrogen bond formed
between the imino N atom and the phenolic OH (see Fig. 1), and on the other hand, the tetrahedral
configuration of the Zn(II) complex, which allows for the intercalation of one of the ligand molecules
19

in between base pairs while the other stays perpendicular probably establishing extra interactions
with the DNA strands. On the contrary, complexes 5 [Ni(II)] and 6 [Cu(II)], seem to interact with DNA
by a different mode of binding. Typically, Ni(II) forms square planar complexes with Schiff base
ligands (confirmed by the diamagnetic nature of 5) and Cu(II) favours square planar geometries
because of its d9 electronic configuration [46]; hence, even though intercalation of one of the ligand
moieties could still be allowed, no extra interaction of the second moiety with the DNA strands’
phosphates would be expected. Moreover, under the oxidizing extracellular environment, copper
exists as Cu(II), but in the reducing conditions inside the cell, it probably exists as Cu(I); that means
that the metal core will have a larger radius that could interfere with the DNA binding. Additionally,
Cu(II) complexes can induce reactive oxygen species (ROS) [47] damaging DNA.
3.5. Antibacterial activity
Compounds 1-6 were screened against three pathogenic bacterial strains: one Gram positive
bacteria, Micrococcus luteus (ATCC 10240) and two Gram negative bacteria Escherichia coli (ATCC
15224) and Enterobacter aerogenes (ATTCC 13048) by applying the disc diffusion method [48,49].
Both Gram positive and negative bacteria were chosen to screen a broad antibiotic spectrum. The
inhibition zone (measured in mm) and MIC (μg mL-1) values obtained from triplicate measurements
are shown in Table 3.
Even though less active than the positive control (kanamycin), in general, these compounds show
antibacterial activity against both the Gram positive and Gram negative bacteria, with the exception
of the Pd(II) and Cu(II) complexes (4 and 6) which were inactive against E. coli and E. aerogenes,
respectively (Table 3). The metal complexes were more active against bacteria than the ligand 1.
Chelation of metal ions with Schiff base ligands enhances the lipophilicity of the ligand due to
delocalization of π-electrons over the whole chelate system [50]. This enhanced lipophilicity
20

increases the penetration of the complexes through the lipid membranes into the cells facilitating
the interaction with DNA.
Table 3. Antibacterial activities against Micrococcus luteus, Escherichia coli and Enterobacter
aerogenes were measured in triplicate and expressed as inhibition zone (mm) and absolute MIC (µg
mL-1) values.
Inhibition zone (mm)
Absolute MIC (µg mL-1)
M. luteus
E. coli
E. aerogenes
M. luteus
E. coli
E. aerogenes
1
6.0 ±0.2
11.0 ±0.8
9.0 ±0.5
17.0 ±1.2
18.9 ±1.5
-
9.0 ±0.9
13.6 ±0.8
12.0 ±1.0
13.0 ±1.2
-
166.7 ±4.5
90.9 ±3.9
111.1±5.0
58.9 ±2.2
67.1 ±3.1
6.8 ±0.5
60.2 ±2.6
52.9 ±3.1
-
111.1 ±5.0
73.9 ±3.7
81.7 ±1.9
76.9 ±4.6
-
3
4
5
16.8 ±1.1
14.9 ±1.0
24.6 ±0.6
18.8 ±1.6
11.8 ±0.9
21.2 ±0.5
53.0 ±4.4
84.4 ±4.8
8.4 ±0.4
6
K[a]
22.4 ±0.4
7.5 ±0.2
[a] Kanamycin.
The MIC values obtained for the antibacterial activity of the Ni(II) and Zn(II) complexes against E. coli
and E. aerogenes follow a similar trend (Table 3); however the Cu(II) complex shows lower activity
(for E. Coli) or no activity at all (E. aerogenes). Nevertheless, the results obtained for the antibacterial
activities against M. luteus show that both the complexes 5 and 6 have very similar profile with
better antibacterial activity than the Zn(II) complex. Hence, their biological activity seems to be
dependent on the bacteria used.
Comparing the DNA binding and biological results obtained, we observe that even though the
uncoordinated ligand (1) exhibits the best DNA binding, this is not reflected in its antibiotic activity
maybe because this compound is not lipophilic enough to reach the nucleus of these
microorganisms. The poor binding to DNA observed for complex 6 does not account for the relatively
21

good activity achieved against M. luteus and E. Coli. On the contrary, in the case of Zn(II) and Ni(II)
complexes (3 and 5) a good correlation is observed between the DNA binding (Kb and ΔTm values)
and the MIC values obtained with E. coli and E. aerogenes.
Summarising, the best antibacterial results were obtained with the Ni(II) complex 5 in agreement
with a good binding to DNA. The Zn(II) complex 3 shows a good antibacterial profile against the three
organisms studied also in agreement to the DNA binding observed. Finally, Cu(II) complex 6 shows
promising antibacterial activity against M. luteus, though not related to its poor DNA binding. Some
studies have shown that the antibacterial properties of Cu(II) complexes could result from damaging
the bacterial membrane inhibiting the expression of some extracellular proteins [51].
In order to have a preliminary assessment of the toxicity of these compounds, the brine shrimp
lethality assay was performed at three different concentrations in triplicate along with DMSO as
negative control and doxorubicin (LD50 value 7.3 µg mL-1) as positive control (Table S2). The results
showed that most of the compounds are, in principle, non-toxic with LD50 > 200 µg mL-1. Only one
compound (complex 6) showed a LD50 value of 192.3 µg mL-1 which represents slight toxicity. These
findings suggest that the synthesized compounds are non-toxic against shrimp.
4. Conclusions
An imidazole ring containing Schiff base ligand with ‘O’ and ‘N’ donor atoms and its metal complexes
with different transition metals were successfully synthesized and fully characterized by different
spectroscopic techniques.
DNA binding activity of all compounds was examined by thermal denaturation experiments and UV-
vis absorption spectroscopy titrations. From the results obtained it can be concluded that the ligand
22

as well as most of its metal complexes have the ability to bind to (st)DNA by intercalation (compound
1 and its Zn(II) complex) or by other modes of binding (Ni(II) and Cu(II) complexes). Among all the
compounds the free ligand is the stronger DNA binder while the Pd(II) complex is the weakest DNA
binder.
Antimicrobial activity assays were also performed on both Gram positive and Gram negative bacteria
and the results indicate that Ni(II), Zn(II) and Cu(II) complexes were good to moderate antibacterial
agents with no toxicity in the brine shrimp assay with the exception of the Cu(II) complex which was
slightly toxic.
Appendix A. Supplementary data
CCDC 1566746 contains the supplementary crystallographic data for compound 1. These data can be
obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the
Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-
1
336-033; or e-mail: deposit@ccdc.cam.ac.uk. Additionally, supplementary data containing H and
13C{1H} NMR, IR and HRMS spectra of the compounds/complexes presented in this article is also
available.
Acknowledgements
We thank the HEC Pakistan for funding this work.
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