Synthesis, characterization, biological screenings and interaction with calf thymus DNA of a novel azomethine 3-((3,5-dimethylphenylimino)methyl) benzene-1,2-diol

Article abstract of DOI:10.1016/j.saa.2012.03.068

The novel azomethine, 3-((3,5-dimethylphenylimino)methyl)benzene-1,2-diol (HL) was synthesized and characterized by elemental analysis, FT-IR, ¹H, ¹³C NMR spectroscopy and single crystal analysis. The title compound has been screened

Full text of DOI:10.1016/j.saa.2012.03.068

Spectrochimica Acta Part A 94 (2012) 134–142
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis, characterization, biological screenings and interaction with calf
thymus DNA of a novel azomethine
3-((3,5-dimethylphenylimino)methyl)benzene-1,2-diol
Muhammad Sirajuddina, Saqib Alia,∗, Naseer Ali Shahb, Muhammad Rashid Khanb,
Muhammad Nawaz Tahirc
a Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
b Department of Biochemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
c Department of Physics, University of Sargodha, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
The novel azomethine, 3-((3,5-dimethylphenylimino)methyl)benzene-1,2-diol (HL) was synthesized and
characterized by elemental analysis, FT-IR, 1H, 13C NMR spectroscopy and single crystal analysis. The
title compound has been screened for its biological activities including enzymatic study, antibacterial,
antifungal, cytotoxicity, antioxidant and interaction with CTDNA, and showed remarkable activities in
each area of research. The titled compound interacts with DNA via two binding modes: intercalation
and groove binding. In intercalation the compound inserts itself into the base pairs of DNA and the
compound–DNA complex is stabilized by ␲–␲ stacking. Interaction via groove binding may be due to
hydrogen bonding to bases, typically to N3 of adenine and O2 of thymine. The synthesized compound
was also found to be an effective antioxidant of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and gives
percent inhibition (%I) of 90.7 at a concentration level of 31.3 ␮g/mL.
Received 1 February 2012
Received in revised form 13 March 2012
Accepted 21 March 2012
Keywords:
Schiff base
Crystal structure
CTDNA interaction
Antibacterial activity
Antifungal activity
Cytotoxicity
© 2012 Elsevier B.V. All rights reserved.
DPPH
1. Introduction
base (TSB) complexes of cobalt(II) have been used extensively
as macrocycle models, while asymmetric complexes are required
Azomethines (Schiff bases) have been widely studied as
they possess many interesting features, including photochromic
and thermochromic properties, proton transfer tautomeric equi-
libria, biological and pharmacological activities, as well as
suitability for analytical applications [1]. They show biological
applications including antibacterial [2–7], antifungal [4–7] and
antitumor activity [8]. Diamino tetradentate azomethines, e.g., N,N-
bis(1-naphthaldimine)-o-phenylenediamine, and their metal com-
plexes, e.g., lanthanide metal complexes [LnL(NO3)2(H2O)x](NO3)
{Ln(III) = Nd, Dy, Sm, Pr, Gd, Tb, La and Er, L = N,N-bis(1-
naphthaldimine)-o-phenylenediamine, X = 0 for Nd, Sm, 1 for La,
Gd, Pr, Nd, Dy, and 2 for Tb} [9] have been used as biological mod-
els to understand the structures of biomolecules and biological
processes. For example transition metals occur in metalloenzyme
bound to a macrocycle such as a heme ring or to donor atoms of
peptide chains usually in a distorted environment, as in hemery-
thrin (Fe2) or hemocyanin (Cu2). Symmetric tetradentate Schiff
to model the irregular binding of peptides [10]. The Schiff bases
are found to act as tetradentate ligands using N2O2 donor set
of atoms leading to a square-planar geometry for the complexes
around all the metal ions [11]. The use of Schiff base as neutral
carriers have been reported as ion selective electrodes for determi-
nation of metal cations such as copper(II), mercury(II), nickel(II),
silver(II), lead(II), cobalt(III) gadolinium(III), yttrium(III), dyspro-
sium(III) [12]. Azomethines ligands are considered as “privileged
ligands” due to their ease of preparation and their use as fluoro-
genic agent, pesticides, herbicidal agents, blocking agents, as well
as in catalysis [13,14].
Design and synthesis of novel and potent DNA-targeted com-
pounds have important theoretical significance and application
value in chemistry, biology and medicine fields. Such compounds
include DNA-alkylating agents, DNA groove binders and DNA
intercalators. DNA-intercalating molecules are those which inter-
calate DNA base pairs via electron-deficient planar chromophores
with flexible side chains. These specially designed groups confer
DNA sequence selectivity and allow aromatic heterocycles to
position at proper sites or interact with topoisomerases so as to
interfere with DNA replication and transcription [15,16]. Acridine,
∗
Corresponding author. Tel.: +92 51 90642130; fax: +92 51 90642241.
E-mail address: drsa54@yahoo.com (S. Ali).
1386-1425/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.03.068

M. Sirajuddin et al. / Spectrochimica Acta Part A 94 (2012) 134–142
135
naphthodiazine and anthraquinone are all famous intercalating
matrixes [17]. DNA binding is the critical step for DNA activity [10].
the DNA is sufficiently free from protein [19]. The DNA concentra-
tion was determined via absorption spectroscopy using the molar
absorption coefficient of 6600 M−1 cm−1 (260 nm) for CTDNA [20]
and was found to be 2.37 × 10−5 M. The compound was dissolved
in 80% ethanol at a concentration of 0.392 mM. The UV absorption
titrations were performed by keeping the concentration of the com-
pound fixed while varying the CTDNA concentration. Equivalent
solutions of CTDNA were added to the complex and reference solu-
tions to eliminate the absorbance of DNA itself. Compound-CTDNA
solutions were allowed to incubate for 30 min at room tempera-
ture before measurements were made. Absorption spectra were
recorded using cuvettes of 1 cm path length at room temperature
(25 1 ◦C).
In
the
recent
study
we
synthesized
3-((3,5-
dimethylphenylimino)methyl)benzene-1,2-diol
successfully
and characterized it by various techniques. Biological activities
including DNA interaction, enzymatic study, antibacterial, anti-
fungal, cytotoxic and antioxidant activities of the titled compound
were studied. We have found that the titled compound show
remarkable activities in each area of research.
2. Experimental
2.1. Materials
2.2.2. Viscosity measurements
Reagents, 3,5-dimethylaniline and 2,3-dihydroxybenzaldehyde
were obtained from Aldrich (USA) and were used without further
purification. Sodium salt of CTDNA (Arcos) was used as received. All
the solvents purchased from E. Merck (Germany) were dried before
use according to the literature procedure [18].
Viscosity measurements were carried out using Ubbelohde vis-
cometer at room temperature (25 1 ◦C). Flow time was measured
with a digital stopwatch. Each sample was measured three times
and an average flow time was calculated. Data were presented as
relative viscosity, (ꢀ/ꢀo)1/3, vs. binding ratio ([HL]/[DNA]) where ꢀ
is the viscosity of DNA in the presence of complex and ꢀo is the
viscosity of DNA alone. Viscosity values were calculated from the
observed flow time of DNA containing solution (to), ꢀ = t − to [21].
2.2. Physical measurements
The melting point was determined in a capillary tube using a
Gallenkamp (U.K.) electrothermal melting point apparatus. IR spec-
trum in the range of 4000–400 cm−1 was obtained on a Thermo
Nicolet-6700 FT-IR Spectrophotometer. Microanalysis was done
using a Leco CHNS 932 apparatus. 1H and 13C NMR were recorded
on a Bruker-300 MHz FT-NMR Spectrometer, using CDCl3 as an
internal reference [1H (CDCl3) = 7.25 and 13C (CDCl3) = 77]. Chem-
ical shifts are given in ppm and coupling constants (J) values are
given in Hz. The multiplicities of signals in 1H NMR are given with
chemical shifts (s = singlet, d = doublet, t = triplet, m = multiplet).
The absorption spectrum was measured on a Shimadzu 1800
UV–Visible Spectrophotometer. The electrical conductance was
measured on a WTW Series Inolab Cond 720. The GC–MS spectrum
was taken on a gas chromatograph, model GC-6890N coupled with
mass spectrometer, model MS-5973 MSD (mass selective detec-
tor). Separation was performed on a capillary column DB-5MS
(30 m × 0.32 mm, 0.25 ␮m of film thickness). The mass Spectrom-
eter coupled with GC was set to scan in the range of m/z 50–550
with electron impact (EI) mode of ionization. The X-ray diffraction
data were collected on a Bruker SMART APEX CCD diffractometer,
equipped with a 4 K CCD detector set 60.0 mm from the crystal.
The crystals were cooled to 100 1 K using the Bruker KRYOFLEX
low temperature device and intensity measurements were per-
formed using graphite monochromated Mo-K␣ radiation from a
sealed ceramic diffraction tube (SIEMENS). Generator settings were
50 kV/40 mA. The structure was solved by Patterson methods and
extension of the model was accomplished by direct methods using
the program DIRDIF or SIR2004. Final refinement on F2 carried out
by full-matrix least squares techniques using SHELXL-97, a mod-
ified version of the program PLUTO (preparation of illustrations)
and PLATON package.
2.2.3. Cyclic voltammetry
Voltammetric experiments were carried out using a ␮Autolab
running with GPES 4.9 software, Eco-Chemie, Utrecht, the
Netherlands. Measurements were carried out using a glassy car-
bon working electrode (GCE) with a geometric area of 0.071 cm2, a
Pt wire counter electrode and a saturated calomel reference elec-
trode (SCE), in one-compartment electrochemical cell. The GCE was
cleaned by polishing with 1 ␮m alumina paste and rinsed with
water before each experiment. Stock solution 3 mM of HL was pre-
pared in 10% aqueous DMSO at pH = 7.0 (phosphate buffer). All
experiments were done at room temperature (25 1 ◦C).
2.2.4. Enzymatic activity
2.2.4.1. Assay of alkaline phosphatase inhibition. The inhibition of
alkaline phosphatase was assayed by monitoring the rate of hydrol-
ysis of p-nitrophenyl phosphate at 25 ◦C in 0.1 M Na2CO3 NaHCO3
(sodium carbonate–bicarbonate) buffer (pH = 10.1) [22,23]. The
enzyme catalyzes the hydrolysis of phosphate monoesters result-
ing in the formation of inorganic phosphate and an alcohol. The
identity of the alcohol varies depending on the specific phos-
phatase. The assay of alkaline phosphatase activity takes advantage
of the fact that the enzyme is non-specific and utilizes the non-
biological substrate p-nitrophenyl phosphate (colorless) to give
yellow colored p-nitrophenol upon hydrolysis which helps to mon-
itor the reaction as shown in Scheme 1 [23].
Stock solution of 50 ␮M inhibitor (HL) in 1 mL DMSO was pre-
pared at room temperature. The buffer and substrate were mixed
in 1:4 ratios to make the reagent solution. Then from the reagent
solution 2000 ␮L (2 mL) was taken in the cell and to which 40 ␮L
enzyme and varying concentrations of the inhibitor were added.
The spectrum of the alkaline phosphatase in the presence and
absence of inhibitor was measured using UV–Visible Spectropho-
tometer. The release of yellow colored p-nitrophenol chromophore
was monitored at 405 nm wavelength. Enzyme activity has been
expressed as the ␮M of p-nitrophenol released per min for 5 min
for each concentration of the inhibitor and then take their average
value. The inhibition of enzyme by inhibitor was calculated by the
following formula [24]:
2.2.1. DNA interaction study by UV–Visible spectroscopy
CTDNA (20 mg) was dissolved and stirred for overnight in double
deionized water (pH = 7.0) and kept at 4 ◦C for less than 4 days. The
nucleotide to protein (N/P) ratio of ∼1.9 was obtained from the ratio
of absorbance at 260 and 280 nm (A260/A280 = 1.9), indicating that
(ꢁA405 nm/ min Test − ꢁA405 nm/ min Blank) total VmL(reagent + enzyme + inhibitor) × D.F.
18.5 × VmL of enzyme taken
Units/mL enzyme =

136
M. Sirajuddin et al. / Spectrochimica Acta Part A 94 (2012) 134–142
O
P
O
Alkaline
phosphatase
O2N
OH
HO
O
+
+ H O
O
O2N
O
P
2
O
O
p-nitrophenyl phosphate
p-nitrophennol
(colorless)
(yellow colored)
Scheme 1.
2.2.7. Cytotoxicity
D.F. = dilution factor; 18.5 = millimolar extinction coefficient of p-
nitrophenol at 405 nm.
Cytotoxicity was studied by the brine-shrimp lethality assay
method [25,26]. Brine-shrimp (Artemia salina) eggs were hatched in
artificial sea water (3.8 g sea salt/L) at room temperature (22–29 ◦C).
After two days these shrimps were transferred to vials contain-
ing 5 mL of artificial sea water (10 shrimps per vial) with 10, 100
and 1000 ␮g/mL final concentrations of each compound taken from
their stock solutions of 12 mg/mL in DMSO. After 24 h number of
surviving shrimps was counted. Data were analyzed with a bio-
stat 2009 computer programme (Probit analysis) to determine LD50
values.
By the addition of increasing amounts of inhibitor the activity
of the enzyme decreased and at higher concentration it was almost
completely inhibited as shown in Fig. 7.
2.2.5. Antibacterial assay
The synthesized compound was tested against six bacte-
rial strains: two Gram-positive [Micrococcus luteus (ATCC10240)
and Staphylococcus aureus (ATCC6538) and four Gram-negative
[Escherichia coli (ATCC15224), Pseudomonas aeruginosa (CM0559),
Bordetella bronchiseptica (ATCC4617) and Klebsiella pneumonia
(MTCC618)]. The agar well-diffusion method was used for the
determination of antibacterial activity [25]. Broth culture (0.75 mL)
containing ca. 106 colony forming units (CFU) per mL of the test
strain was added to 75 mL of nutrient agar medium at 45 ◦C, mixed
well, and then poured into a 14 cm sterile petri plate. The media was
allowed to solidify, and 8 mm wells were dug with a sterile metal-
lic borer. Then a DMSO solution of test sample (100 ␮L) at 1 mg/mL
was added to the respective wells. DMSO served as negative control,
and the standard antibacterial drugs Roxithromycin (1 mg/mL) and
Cefixime (1 mg/mL) were used as positive control. Triplicate plates
of each bacterial strain were prepared which were incubated aero-
bically at 37 ◦C for 24 h. The activity was determined by measuring
the diameter of zone showing complete inhibition (mm).
2.2.8. Scavenging effect on 2,2-diphenyl-1-picryhydrazyl (DPPH)
The solution of 2,2-diphenyl-1-picryhydrazyl (DPPH) radical
was obtained by dissolving 3.94 mg of DPPH in 100 mL methanol.
To 2.8 mL of the methanolic solutions of DPPH, 0.2 mL of HL
solution with different concentration ranging from 0.0625 mg/mL
to 1.0 mg/mL was added [27–29]. After 10 min the decrease in
absorption was measured at 517 nm of DPPH using UV–Visible
Spectrophotometer. The actual decrease in absorption was mea-
sured against that of the control and the percentage inhibition was
calculated. The same experiment was carried out on ascorbic acid
which is known antioxidant. All test and analysis were run in trip-
licates and the results obtained were averaged.
2.3. Synthesis
3-((3,5-dimethylphenylimino)methyl)benzene-1,2-diol (HL)
2.2.6. Antifungal assay
Antifungal activity against five fungal strains [Fusarium monil-
iformis, Aspergillus niger, Fusarium solani, Mucor species and
Aspergillus fumigatus] was determined by using agar tube dilution
method [25]. Screw caped test tubes containing sabouraud dex-
trose agar (SDA) medium (4 mL) were autoclaved at 121 ◦C for
15 min. Tubes were allowed to cool at 50 ◦C and non solidified
SDA was loaded with 66.6 ␮L of compound from the stock solu-
tion (12 mg/mL in DMSO) to make 200 ␮g/mL final concentration.
Tubes were then allowed to solidify in slanting position at room
temperature. Each tube was inoculated with 4 mm diameter piece
of inoculum from seven days old fungal culture. The media sup-
plemented with DMSO and Turbinafine (200 ␮L/mL) were used as
negative and positive control, respectively. The tubes were incu-
bated at 28 ◦C for 7 days and growth was determined by measuring
linear growth (mm) and growth inhibition was calculated with ref-
erence to the negative control.
Stoichiometric amounts of 3,5-dimethylamine and 2,3-
dihydroxybenzaldehyde (5 mmol of each) were added to freshly
dried toluene. The mixture was refluxed for 3–4 h and the water
formed was removed by using Dean and Stark apparatus. The
reaction mixture volume was reduced to one-third of its original
and left for crystallization at room temperature. The dark red
crystals of HL suitable for a single crystal analysis were isolated
from the mother liquor and dried. The chemical reaction is shown
in Scheme 2.
Yield: 85%, m.p.: 134–135 ◦C, Mol. Wt.: 241, Anal. Calc. for
C15H15NO2: C, 74.67; H, 6.27; N, 5.81; Found: C, 74.60; H, 6.30;
N, 5.80%, IR (cm−1): ꢂ 1604 (C N), 1405 and 1589 (Ar C C), 3421
(free OH), 3341 (H-bonded OH), 1205–1357 (Phen. C O Str. vib.),
1H NMR (CDCl3, ppm): 2.39 (s, 6H, H1, H1ꢀ), 6.98 (s, 1H, H3), 6.95
(s, 2H, H4, H4ꢀ), 8.60 (s, 1H, H6), 7.03–7.07 (dd, 1H, H9), 6.8–6.85 (t,
1H, H10, 3J[1H–1H] = 7.8), 6.97–6.98 (d, H11), 5.85 (s, OHa), 14.08
1'
CH3
CH3
2'
9
4'
4
10
Toluene, Reflux
6
5
7
11
12
O
CH
+
3
1
NH2
N
CH
-H2O
8
2
CH3
b
a
OH
OH
CH3
OH
OH
Scheme 2.

M. Sirajuddin et al. / Spectrochimica Acta Part A 94 (2012) 134–142
137
CH3
CH3
+
.
-OH.
N
CH
N
CH
+.
m/z = 241
m/z = 224
CH3
OH
OH
CH3
OH
+.
+.
N
CH
CH3
CH3
OH
OH
m/z = 105
m/z = 136
.
-CH2
+
.
.
+
-C2H2
-CH2
C4H3
m/z = 51
+.
CH2
m/z = 65
m/z = 91
.
-CH2
+.
m/z = 77
Scheme 3.
(s, OHb, N· · ·H O); 13C NMR (CDCl3, ppm): 21.3 (2C, C1, C1ꢀ), 139.3
(2C, C2, C2ꢀ), 128.9 (1C, C3), 118.7 (2C, C4, C4ꢀ), 159.1 (1C, C5),
161.4 (1C, C6), 118.1 (1C, C7), 152.1 (1C, C8), 123.3 (1C, C9), 122.8
(1C, C10), 119 (1C, C11), 146.9 (1C, C12); Molar conductance in
80% ethanol (ꢃm, S cm2 mol−1): 16 at 25 ◦C, Solubility: chloroform,
toluene, ethanol, methanol and DMSO.
The GC spectrum contains only a single peak. The compound
showed a molecular ion peak (parent peak) of maximum intensity
with m/z = 241. The initial fragmentation is due to the loss of a
hydroxyl group giving ion peak at m/z = 224 which is of second
maximum intensity. The molecular ion peak undergoes two major
fragmentation pathways with m/z = 105 and 136. The former under-
goes cleavage to give ion peak at m/z = 91 by the elimination of
methylene group (CH2) and then to give ion peak at m/z = 77 which
can be attributed to the phenyl radical ion. The fragment of m/z = 91
eliminates C2H2 group to give ion peak at m/z = 65 with another
rout which then gives ion peak at m/z = 51 by the elimination of
CH2 group.
3. Results and discussion
The azomethine is prepared as described in the experimental
part, crystallized, dried and subjected to elemental analyses. The
results of elemental analyses (C, H, N) obtained are in good agree-
ment with those calculated for the suggested formula. The sharp
melting point indicates the purity of the synthesized azomethine.
The structure of azomethine (HL) is given in Scheme 1 which is
also confirmed by IR, 1H and 13C NMR spectra and single crystal
analysis. The electron impact mass spectrum of HL is recorded and
fragmentation pattern is given in Scheme 3.
3.2. IR spectroscopy
In the IR spectrum of HL a sharp band observed at 3421 cm−1 is
assigned to free (ꢂ O H) while a broad band observed at 3341 cm−1
is assigned to hydrogen bonded (ꢂ O H). A strong band attributable
to (ꢂ C N) is observed at 1604 cm−1. The stretching vibration of
phenolic C O band occurs at 1205–1357. The stretching vibration
bands of aliphatic and aromatic C H group are observed at 2914
and 3040 cm−1, respectively. A CH3 bending vibration is observed
3.1. Mass spectrometry
The possible suggested molecular ion fragments are given in
Scheme 3, while its GC–MS spectrum is shown in Fig. 1. In Fig. 1 (A)
represent the GC spectrum while (B) represent the MS spectrum.
at 1389 cm−1
.

138
M. Sirajuddin et al. / Spectrochimica Acta Part A 94 (2012) 134–142
Fig. 1. Total ion chromatogram of HL.
3.3. NMR spectroscopy
The NMR spectrum of HL was recorded in chloroform (CDCl3).
The chemical shifts of the different types of protons and carbons
are given in experimental part. The formation of HL was supported
by the appearance of a sharp singlet at 8.60 ppm, corresponding
to the azomethine proton (
N CH ). One hydroxyl proton gives a
singlet at 5.85 ppm (OHa) while the other at 14.08 ppm (OHb). This
downfield shifting of OHb is due to strong intramolecular O H· · ·N
hydrogen bonding, to which the chemical shift of the proton is very
sensitive. In the 13C NMR spectrum the peak at 161.4, belongs to
the azomethine carbon (C-6). The remaining peaks are described in
the same positions as calculated by incremental methods [30].
3.4. Crystal structure of HL
The
molecular
structure
of
3-((3,5-
dimethylphenylimino)methyl)benzene-1,2-diol (HL) is shown
in Fig. 2 while crystal data, selected bond distances and angles
Fig. 2. ORTEP drawing of HL with atomic numbering scheme.

M. Sirajuddin et al. / Spectrochimica Acta Part A 94 (2012) 134–142
139
Table 1
Crystal data and structure refinement parameters for HL.
Table 3
Hydrogen-bond geometry (Å,◦) for ligand HL.
Formula
Formula weight
Crystal system
Space group
a (Å)
C15H15NO2
241.28
D
H· · ·A
D
H
H· · ·A
D· · ·A
D
H· · ·A
N1 H1· · ·O1
O2 H2· · ·O1
O2 H2· · ·O1
0.8600
0.8500
0.8500
1.8300
2.2600
1.9800
2.5465(16)
2.6974(15)
2.7527(13)
140.00
113.00
151.00
Triclinic
¯
P1 (No. 2)
7.5598(10)
7.6837(10)
11.4815(16)
74.433(4)
75.663(5)
85.707(5)
622.43(15)
2
Symmetry codes: (i) 1 − x, 2 − y, −z; (ii) 2 − x, 1 − y, −z; (iii) x, 1 + y, −1 + z.
b (Å)
c (Å)
˛ (◦)
ˇ (◦)
ꢄ (◦)
V (Å3)
Z
d (g cm−3
)
1.287
0.086
256
ꢅ (Mo K␣) (mm−1
F(0 0 0)
)
Crystal habit/size (mm)
T (K)
Needle/0.26 × 0.14 × 0.12
296(2)
Radiation (Å) (Mo K␣)
0.71073
2.75–25.25
2229
ꢆ Min–Max (◦)
Total reflections
Tot., Uniq. Data, R(int)
Observed data [I > 0.0 sigma(I)]
Nref, Npar
2229, 1670, 0.0256
1670
2229, 157
w = 1/[ꢇ2(Fo)2 + (0.0560P)2 + 0.1512P]
where P = [(Fo)2 + 2(Fc)2]/3
R, wR2, S
0.0463, 0.1083, 1.047
0.00, 0.00
Max. and Av. shift/error
Min. and Max. Resd. Dens. [e/Å3]
Goodness-of-fit
−0.198, 0.162
1.044
Table 2
Fig. 4. CV behavior of 3 mM sample in 10% aqueous DMSO at 100 mV/s, 0.1 M KCl
as supporting electrolyte and pH 7.0 phosphate buffer.
Selected bond lengths (Å) and bond angles (◦) for ligand HL.
Bond lengths
O1 C2
N1 C8
C1 C7
C1 C6
1.2986(18)
1.4132(19)
1.416(2)
1.407(2)
1.355(2)
1.383(2)
O2 C3
N1 C7
C1 C2
C2 C3
C8 C9
C10 C15
1.3516(19)
1.2961(19)
1.415(2)
1.422(2)
1.381(2)
1.502(2)
3.5. Cyclic voltammetry
For exploring the binding mode between the HL and CTDNA,
the CV behaviors of the HL (3 mM) without and with CTDNA were
studied with the scan rate of 100 mV/s and the results are shown
in Fig. 4. It can be seen from Fig. 5, that in the absence of DNA (pure
sample), the 1st voltammogram started in the positive direction,
one oxidation peak was observed showing that HL is oxidizable
in these conditions. No corresponding peak was observed in the
reverse scan which indicated that the oxidation product of HL is
C3 C4
C11 C12
Bond angles
C7 N1 C8
C6 C1 C7
O1 C2 C1
C1 C2 C3
C2 C3 C4
C3 C4 C5
N1 C8 C13
127.95(12)
120.57(14)
123.29(14)
117.80(14)
120.49(15)
121.33(16)
116.29(13)
C2 C1 C7
C2 C1 C6
O1 C2 C3
O2 C3 C4
O2 C3 C2
N1 C7 C1
C13 C12 C14
119.34(14)
120.08(14)
118.91(14)
120.92(15)
118.59(14)
121.90(14)
120.07(13)
are given in Tables 1 and 2, respectively. The compound exists as
a monomer. The two hydroxyl groups are cis to each other. The
hydroxyl H atom is involved in an intramolecular interaction with
the imine N atom as shown in Fig. 3. Details of hydrogen bonds are
given in Table 3.
Fig. 5. Absorption spectra of 0.04 mM HL in the absence (a) and presence of 0.13 ␮M
(b), 0.26 ␮M (c), 0.39 ␮M (d), 0.52 ␮M DNA (e). The arrow direction indicates increas-
ing concentrations of DNA. Insite graph is the plot of A0/(A − A0) vs. 1/[DNA] for
the determination of binding constant and Gibb’s free energy of HL-DNA Adduct at
ꢈ1 = 267 nm and ꢈ2 = 288 nm.
Fig. 3. The molecular packing of HL viewed along the b-axis. The dotted lines show
the intramolecular H-bonding.

140
M. Sirajuddin et al. / Spectrochimica Acta Part A 94 (2012) 134–142
not reducible. The attribution is strengthened by the appearance of
irreversible anodic peak in the same CV region of guanine due to
the oxidation of
N CH group as reported by the earlier elec-
trochemists [31,32]. The formal potential (Eoꢀ) was found to be
0.596 V. In the presence of DNA (60 ␮M and 120 ␮M) the voltam-
metric peak currents decreased apparently, suggesting that there
exists an interaction between the HL and CTDNA [33,34]. Bard has
reported three kinds of modes for small molecules binding to DNA
[35], if E1/2 shifted to more negative value when small molecules
interacted with DNA; the interaction mode was electrostatic bind-
ing. On the other hand, if E1/2 shifted to more positive value, the
interaction mode was intercalative binding. Therefore, it could be
concluded that the HL could bind to DNA by intercalation because
E1/2 has shifted to positive value as shown in Fig. 4. In addition,
considering the structure of the HL which has aromatic rings, we
may deduce that it may bond to CTDNA more dominantly via an
intercalation mode.
3.6. Conductometry
Fig. 6. Effects of increasing amount of HL on relative viscosity of CT-DNA at
25 0.1 ◦C. [DNA] = 2.37 × 10−5 M.
The conductance of HL in 80% ethanol at 25 ◦C falls in the range
of 16 S cm2 mol−1, suggest its non electrolytic nature [32].
the drug–DNA complex, respectively. The association constants
were obtained from the intercept-to-slope ratios of A0/(A − A0) vs.
1/[DNA] plots. The Gibb’s free energy (ꢁG) was determined from
the equation:
3.7. Absorption spectral features of CTDNA binding
The drug–DNA interaction can be carried out by UV–Visible
absorption spectroscopy by monitoring the changes in the absorp-
tion properties of the drug or the DNA molecules. Drug–DNA
interactions can be studied by comparison of UV–Visible absorp-
tion spectra of the free drug and drug–DNA complexes, which are
usually different. Drug binding with DNA through intercalation
usually results in hypochromism and bathochromism. Because of
the intercalative mode involving a stacking interaction between
an aromatic chromophore and the base pair of DNA, the extent of
the hypochromism commonly parallels the intercalative binding
strength [36,37].
The absorption spectrum of HL in the absence (a) and pres-
ence (b–e) of DNA is shown in Fig. 5. There exist two bands, one
at 267 nm and other at 288 nm for free HL, respectively. With the
addition of increasing concentration of DNA, the transition band
at 267 exhibit hypochromism of 6.3%, 13%, 20.3%, 32.8% and 41%
as well as hypsochromic shift (blue shift) of ∼3.5 nm while at
288 nm exhibit hypochromism of 2.1%, 3.7%, 4.4%, 6.7% and 11.6%
as well as bathochromic shift (red shift) of ∼7 nm, respectively.
These spectral characteristics suggest that the HL might bind to
DNA by an intercalative mode as well as by groove binding. After
intercalating the base pairs of DNA, the ␲* orbital of the inter-
calated ligand could couple with ␲ orbital of base pairs, thus
decreasing the ␲–␲* transition energy, and further resulting in
the bathochromism. On the other hand, the coupling of a ␲ orbital
with partially filled electrons decreases the transition probabilities
hence results hypochromic shift. Since hypochromism due to ␲–␲*
stacking interactions may appear in the case of the intercalative
binding mode, while bathochromism (red-shift) may be observed
when the DNA duplex is stabilized [38]. After 24 h, the spectrum
was again taken and obtained the same results which conforms
the stability of drug–DNA complex. Based upon the variation in
absorbance, the intrinsic binding constant of the compound with
DNA were determined according to Benesi–Hildebrand equation
[36]:
ꢁG = −RT ln K
where R is general gas constant (8.314 J K−1 mol−1) and T is the
temperature (298 K).
Binding constants and Gibb’s free energies were calculated for
ꢈ1 = 267 nm and ꢈ2 = 288 nm. The binding constants were found
to be 5.9 × 105 M−1 and 1.1 × 106 M−1 for ꢈ1 and ꢈ2, respec-
tively, while the Gibb’s free energies were −14.3 kJ mol−1 and
−14.97 kJ mol−1, respectively. Also it shows to some extent inter-
action with the minor grooves (ꢈ2) which may be due the hydrogen
bonding to bases, typically to N3 of adenine and O2 of thymine [39]
that results in bathochromism. The interaction of the drug with
DNA is a spontaneous process as shown by the negative values of
ꢁG.
3.8. Viscosity measurements
To further clarify the binding modes of the HL with DNA, viscos-
ity measurements were carried out. Hydrodynamic measurements
that are sensitive to length change (i.e., viscosity) are regarded
as the least ambiguous and the most critical tests of the binding
model in solution. A classical intercalation model resulted in the
lengthening of the DNA helix as the base pairs were separated to
accommodate the binding complex, leading to an increase in DNA
viscosity. In contrast, a partial, non-classical intercalation could
bend (or kink) the DNA helix and reduce its effective length and,
concomitantly, its viscosity. There is a marked effect of HL on the
viscosity of CT-DNA as shown in Fig. 6. With an increasing amount
of HL, the relative viscosity of DNA increases while the increasing
amount of HL further increases the effective length of DNA which
supports that HL bind through intercalation mode but with differ-
ent affinity, i.e., also show some affinity for binding with grooves of
DNA through hydrogen bonding. However, strong binding is pre-
sumably due to intercalation with DNA [40]. As shown in Fig. 6, the
viscosity increases with the addition of the increasing amount of
HL, therefore, intercalative mode, as a dominant mode, has selected
for the interaction of HL with DNA. Since the viscosity first remains
almost constant and then increases with increasing concentration
A0
εG
εG
1
=
+
×
A − A0
εH–G − εG
εH–G − εG
K[DNA]
where K is the association/binding constant, A0 and A are the
absorbances of the drug and its complex with DNA, respectively,
and εG and εH–G are the absorption coefficients of the drug and

M. Sirajuddin et al. / Spectrochimica Acta Part A 94 (2012) 134–142
141
Table 4
Antibacterial activity of HL.
Compound
Average zone of inhibition (mm)
Staphylococcus aureus
Klebsiella pneumoniae
Micrococcus luteus
Pseudomonas aeruginosa
Bordetella bronchiseptica
Escherichia coli
HL
20
36
22
23
26
21
20
35
30
14.2
20
16
20
35
30
16
26
22
Roxythromycin
Cefixime
MIC (␮g/mL)
500
250
1.95
62.5
500
250
Concentration: 1 mg/mL of DMSO. Reference drugs, Roxythromycin and Cefixime 1 mg/mL.
Table 4. Roxithromycin and Cefixime were used as standard drugs
in these assays. Criteria for activity is based on inhibition zone
(mm); inhibition zone more than 20 mm shows significant activ-
ity, for 18–20 mm inhibition activity is good, 15–17 mm is low,
and below 11–14 mm is non-significant activity. The antibacterial
studies demonstrated that HL has activity toward tested bacte-
ria. The values obtained for activity of HL against S. aureus and
M. luteus, K. pneumonia and B. bronchiseptica falls in category of
significant activity while against Pseudomonas and E. coli showed
low to non significant activity. HL was also subjected to antifun-
gal activity against five fungal strains [F. moniliformis, A. niger, F.
solani, Mucor species and A. fumigatus] by using Agar tube dilution
method. The results are shown in Table 5. Terbinafine was used as
standard drug in this assay. Criteria for activity is based on per-
cent growth inhibition; more than 70% growth inhibition shows
significant activity, 60–70% inhibition activity is good, 50–60% inhi-
bition activity is moderate and below 50% inhibition activity is
non-significant. The investigation shows that HL has significant to
moderate activity against four strains while non significant activity
was shown only against flavus [25,26]. Since Gram-negative bacte-
ria are considered a quantitative microbiological method testing
beneficial and important drugs in both clinical and experimental
tumor chemotherapy [43], therefore the synthesized compound
can be used for such purposes. Its anticancer and antitumor activi-
ties are under experiments and soon we will report them.
Fig. 7. % inhibition of the ALP enzyme vs. concentration of the HL (␮M).
of HL, which indicates that groove binding also contribute to some
extent.
The cytotoxicity was studied by the brine-shrimp bioassay
lethality method [25,26]. The LD50 data show HL is toxic with LD50
value in the range of 1.7598 ␮g/mL in comparison to reference drug,
MS-222 (Tricaine methanesulfonate) with LD50 value of 4.3 ␮g/mL.
The data are given in Table 6.
3.9. Enzymatic study
The remarkable activity of the ligand may be due to OH group,
which can play an important role in the enzymatic activity [41] and
the imine group may impart in the elucidation of the mechanism
of transformation reaction in biological system [42]. The results are
shown in Fig. 7.
3.11. DPPH scavenging activity
3.10. Antimicrobial activity and cytotoxicity
The model of the scavenging of the stable DPPH radical is exten-
sively used to evaluate antioxidant activities in less time than
other methods. The reducing abilities of the HL were determined
by their interaction with the free stable radical 1,1-diphenyl-2-
picryl-hydrazyl (DPPH) at different concentrations. Antioxidants
can react with DPPH and produce 1,1-diphenyl-2-picryl-hydrazine
[44]. Due to its odd electron DPPH gives a strong absorption band at
517 nm appearing a deep violet color [45]. As this electron becomes
paired off in the presence of a free radical scavenger, the absorp-
tion disappears and the resulting decolorization is stoichiometric
with respect to the number of electrons taken up. The change of
absorbance produced in this reaction is reviewed to evaluate the
In vitro biological screening tests of the synthesized compound
(HL) were carried out for antibacterial, antifungal, cytotoxic-
ity. The antibacterial activity was tested against six bacterial
strains: two Gram-positive [M. luteus (ATCC10240) and S. aureus
(ATCC6538) and four Gram-negative [E. coli (ATCC15224), P.
aeruginosa (CM0559), B. bronchiseptica (ATCC4617) and K. pneu-
monia (MTCC618)]. The agar well-diffusion method was used in
these assays and each experiment was performed in triplicate.
Readings of the zone of inhibition represents the mean value of
three readings with standard deviation (SD), which are shown in
Table 5
Antifungal activity of HL.
Compound
Mean value of percent growth inhibition SD
Aspergillus flavus
Fusarium solani
Aspergillus niger
Mucor species
Aspergillus fumigates
HL
Terbinafine
30 0.045
100
71 0.056
100
65 0.035
100
63 0.040
100
61 0.038
100
In vitro agar tube dilution method, concentration: 200 ␮g/mL of DMSO. % inhibition of fungal growth = 100 − gt/gc × 100. gt = linear growth in test (mm) and gc = linear growth
in vehicle control (mm).

142
M. Sirajuddin et al. / Spectrochimica Acta Part A 94 (2012) 134–142
Acknowledgment
The authors gratefully acknowledged Mr. Shafiq Ullah Marwat,
Quaid-i-Azam University Islamabad, Pakistan, for his help in the
cyclic voltammetry.
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b,c
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× 100
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4. Supplementary material
Crystallographic data for the structure reported in this paper has
been deposited with the Cambridge Crystallographic Data Centre,
CCDC 836396 for HL. Copies of this information may be obtained
free of charge from The Director, CCDC, 12, Union Road, Cambridge
CB2 1EZ [Fax: +44 1223 336 033] or deposit@ccdc.cam.ac.uk or
http://www.ccdc.cam.ac.uk.
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