Synthesis, characterization, biological and electrochemical evaluation of novel ether based on donor bidentate Schiff bases

Article abstract of DOI:10.1016/j.molstruc.2016.03.008

Four novel ON donor Schiff bases (E)-2-((4-phenoxyphenylimino)methyl)phenol (HL1), (E)-2-((4-(4-biphenyloxy)phenylimino)methyl)phenol(HL2), (E)-2-((4-(naphthalen-1-yloxy) phenylimino)methyl)phenol(HL₃)and(E)-2-((4-(2-naphthoxy)phenylimino)methy

Full text of DOI:10.1016/j.molstruc.2016.03.008

Accepted Manuscript
Synthesis, characterization, biological and electrochemical evaluation of novel ether
based ON donor bidentate Schiff bases
Muhammad Shabbir, Zareen Akhter, Iqbal Ahmad, Safeer Ahmed, Hammad Ismail,
Bushra Mirza, Vickie McKee, Michael Bolte
PII:
S0022-2860(16)30203-4
10.1016/j.molstruc.2016.03.008
MOLSTR 22318
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 2 January 2016
Revised Date: 2 March 2016
Accepted Date: 2 March 2016
Please cite this article as: M. Shabbir, Z. Akhter, I. Ahmad, S. Ahmed, H. Ismail, B. Mirza, V.
McKee, M. Bolte, Synthesis, characterization, biological and electrochemical evaluation of novel
ether based ON donor bidentate Schiff bases, Journal of Molecular Structure (2016), doi: 10.1016/
j.molstruc.2016.03.008.
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Synthesis, characterization, biological and electrochemical evaluation of novel
ether based ON donor bidentate Schiff bases
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Muhammad Shabbira, Zareen Akhtera, Iqbal Ahmada, Safeer Ahmeda, Hammad Ismailb, Bushra
Mirzab, Vickie McKeec, Michael Bolted
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aDepartment of Chemistry, Quaid-i-Azam University, Islamabad-45320. Pakistan.
bDepartment of Biochemistry, Quaid-i-Azam University, Islamabad-45320. Pakistan.
cSchool of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland.
dInstitutfür Anorganische Chemie, J.W. Goethe-Universität Frankfurt, Max-Von-Laue-Strasse 7,
Frankfurt/Main 60438, Germany.
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Corresponding author
Telephone +92 051 90642111; fax: +92 051 9064224
E-mail address: zareenakhter@yahoo.com, zareen_a@qau.edu.pk
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Abstract
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Four novel ON donor Schiff bases (E)-2-((4-phenoxyphenylimino)methyl)phenol (HL1), (E)-2-
((4-(4-biphenyloxy)phenylimino)methyl)phenol(HL2),(E)-2-((4-(naphthalen-1-yloxy)
phenylimino)methyl)phenol(HL3)and(E)-2-((4-(2-naphthoxy)phenylimino)methyl)phenol
(HL4)have been synthesized and characterized by various spectroscopic, analytical and electro-
analytical techniques. Single crystal X-ray diffraction analysis of Schiff base (HL3) revealed that
phenol and anthracene rings are inclined at 30.25(9)° and 89.64(4)° to the central phenyl ring,
respectively. Intra and inter molecular interactions are observed in single crystal analysis of HL3
Intramolecular interactions are hydrogen bonding but most of the intermolecular interactions are
of the C-H…π type .There is a bit of π…π stacking between the anthracene groups. Only
compounds (HL1) and (HL3) have been investigated for the biological activities due to slight
solubility of (HL2) and (HL4) in DMSO. The results of brine shrimp cytotoxicity assay indicated
LD50 values <1ꢀg/ml showing significant antitumor activity with IC50 values 14.20 and 4.54
µg/ml respectively. The compounds were highly active in protecting DNA against hydroxyl free
radicals in concentration dependent manner. Voltammetric results indicated the one electron
irreversible oxidation product is formed due to hydroxyl moiety and the process is diffusion
controlled. On exposing to DNA environment the electrooxidised product developed electrostatic
linkage and groove binding intercalation while consuming the DNA concentration substantially.
The binding strength was quantitative in terms of drug-DNA binding of the order of 104 M-1.
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Keywords: Schiff bases, electrochemical studies, pharmacological studies, DNA-interaction
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1. Introduction
Schiff bases are most widely used organic compounds that coordinate to metal ions via
azomethine nitrogen and have a wide variety of applications in many fields including analytical,
biological, and inorganic chemistry. In azomethine derivatives, the C=N linkage is present in
various natural (ancistrocladidine have antimalarial activity), natural-derived (chitosin-derived
Schiff bases have antifungal activity), and non-natural compounds which is essential for
biological activity[1].Several azomethines possess remarkable antibacterial, antifungal,
anticancer and diuretic activities.The nitrogen atom of azomethine may be involved in the
formation of a hydrogen bond with the active centers of cell constituents and interferes in normal
cell processes[2]. Apart from biological activities, they have found applications in many other
fields such as intermediates in organic synthesis, dyes, pigments, polymer stabilizers, corrosion
inhibitors, fungicidal, agrochemical, analytical chemistry, electrical conductivity, magnetism,
host guest chemistry, ion exchange, nonlinear optics and catalysis [3-10]. Schiff bases have
played an important role in the development of coordination chemistry and inorganic
biochemistry as well. They have been used for the synthesis of a number of biologically and
industrially active compounds like formazans, 4-thiazolidinines, benzoxazines, and so forth, via
ring closure, cycloaddition and replacement reactions [11].
Particularly salicyaldehyde-Schiff bases derived from salicyaldehyde and primary amines have
recently acquired a considerable importance due to their promising biological properties. Such
Schiff bases are found to be a versatile pharmacophore for design and development of various
bioactive lead compounds.These Schiff bases may act as bidentate-O, N and a tridentate -O, O,
N donor ligand etc[12]. which can be employed for the synthesis of various coordination
complexes.Cambridge structural database have shown that about 42% of Schiff bases
synthesized worldwide are derived from salicylaldehyde based aldehydes and almost 68% of
Schiff base complexes are synthesized from such ligands [13]. A very useful application of such
new compounds is that they specially target DNA molecule with significant potential and thus
can be proposed as drugs. Among other techniques the voltammetric methods have established a
prominent role to study drug-DNA interaction due to high sensitivity, selectivity, versatility and
fast detection ability in addition to cost effectiveness. Monitoring of the reaction of interest at the
electrode surface helps to elucidate the mechanism of drug- DNA interactions [14-16].
In the present work (E) -2- ((4-phenoxyphenylimino) methyl) phenol (HL1), (E)-2-((4-(4-
biphenyloxy)phenylimino)methyl)phenol(HL2),(E)-2-((4-(naphthalen-1-yloxy)phenylimino)
methyl)phenol(HL3)and(E)-2-((4-(2-naphthoxy)phenylimino)methyl)phenol(HL4)were
synthesised and characterized successfully by various spectroscopic, analytical, advanced
electro-analytical techniques and single crystal analysis. As Schiff bases are biological active in
nature therefore synthesised compounds (HL1-HL4) were studied to acquire valuable information
about their role in cellular vicinities. Biological studies (cytotoxic, antitumor and inhibition of
hydroxyl (OH) free radical induced DNA damage assay) were proceeded to screen the
pharmacological importance of the compounds. Voltammetric studies were carried to investigate
the redox behaviour and to identify the electrophoric centres in the compounds. Further, the
interactions with the DNA and the binding mode of the synthesised compounds were also
investigated.
2. Experimental

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2.1. Materials and methods
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Solvents used were purified by standard distillation procedure and drying methods [17].The
Schiff bases (HL1 –HL4) were prepared by condensation reactions of salicylaldehyde and
corresponding aromatic amines [18] already reported by our research group in ethanol following
the reported method [19-20].The progress as well as purity of products was checked by thin layer
chromatography on pre-coated Kieselgel 60HF TLC plates. Elemental analysis was carried out
on a CHNS 932 (Leco-USA) elemental analyzer. Melting points were determined, using a MPD
Mitamura Riken Kogyo (Japan) electrothermal melting point apparatus. FTIR spectra were
recorded on a Thermoscientific (USA) Nicolet 6700 spectrometer in the frequency range of
4000-400 cm-1.1H NMR (300MHz) and13C NMR (75MHz) spectra were recorded on a Bruker
NMR Spectrometer.
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Single crystal of Schiff base (HL3) was obtained by the slow evaporation of ethanol from the
mother liquor at room temperature. X-ray data were collected at 150(2) K on a Bruker Apex II
CCD diffractometer using MoKα radiation (λ = 0.71073Å). The structure was solved by direct
methods [21] and refined on F2 using all the reflections [22].All the non-hydrogen atoms were
refined using anisotropic atomic displacement parameters and hydrogen atoms bonded to carbon
were inserted at calculated positions using a riding model. The phenolic proton was located and
its coordinates refined. Parameters for data collection and refinement are summarised in Table 1.
The bioactive nature of the synthesized compounds was evaluated by brine shrimp lethality
assay[23].Brine shrimp (Artemiasalina) eggs (Ocean Star Inc., USA) were hatched in shallow
rectangular dish (22×32 cm) filled with prepared seawater (34 g/l) under constant aeration for 48
h at room temperature. After 24 h, phototropicnauplii (brine shrimp larvae) were shifted to glass
vial by Pasteur pipette and 25 µl of the each stock solution (0.1 g/ml, 1 g/ml, and 10 g/ml) of
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the test compound was added .The volume of test compounds from their stock solutions was
raised up to 5 ml of artificial seawater with 10, 1, 0.5, 0.25, 0.125 and 0.0625 µgml-1 final
concentration. Three replicates were prepared for each concentration. The vials were maintained
under illumination at room temperature. After 24 h of incubation survivors were observed and
LD50 (Lethal Dose that killed 50% of shrimps) was calculated by using Finny (1971) software
[24].
Antitumor activity of the synthesized compounds was checked by executing modified potato disc
antitumor assay [25].Inoculum with three different concentrations (1000, 100 and10 g/ml) was
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prepared containing 48 h bacterial culture of Agrobacterium tumefaciens(At 10). Red-skinned
potatoes were surface sterilized in 0.1% HgCl2 solution and potato discs of size 8mm×4mm were
prepared with the help of sterilized cork borer. Ten discs were placed on the agar plates along
with 50 µl of inoculum on the surface of each disc. After 21 days of incubation at 28°C, discs
were stained with Lugol solution (10% KI & 5% I2) and tumors were counted on each disc. The
tumor inhibition was calculated by formula, (Percentage inhibition = 100 – average number of
tumors of sample/ average number of tumors of negative control × 100).
Antioxidant or prooxidant activity of the synthesized compounds was assessed by DNA damage
assay [26].Plasmid DNA (pBR322 Fermentas) with a concentration of 0.5 ꢀg/3ꢀl was treated
with three different concentrations of test samples (1000, 100, and 10 ppm).Fenton reaction was
induced by addition of 30% H2O2 (4 ꢀl) and 2 mM FeSO4 (3 ꢀl) into the reaction mixture. Three
controls, untreated pBR322 DNA as negative, DNA treated with compound (C+P), DNA treated
with 2 mM FeSO4 and 30% H2O2 as positive control were run simultaneously. Each reaction
mixture was incubated at 37ºC for an hour. After incubation, the sample was loaded on a 0.9%

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agarose gel and was visualized with Doc-IT (VWR). Estimation of antioxidant or pro-oxidant
effects on DNA was estimated on the basis of percentage increase or loss of a super-coiled
monomer, compared with the control.
Voltammetric studies (cyclic voltammetry (CV), differential pulse voltammetry (DPV) and
square wave voltammetry (SWV)) were conducted by using Eco Chemie Autolab PGSTAT 302
potentiostat/galvanostat (Utrecht, the Netherlands) with software version GPES 4.9.The
conventional three-electrode cell with Ag/AgCl(aq) as a reference, home-made platinum wire as
a counter electrode and a glassy carbon electrode (GCE) with surface area of 0.09 as a working
electrode was employed. The active surface area of GCE was polished with 0.25 m alumina
paste on a nylon buffing pad followed by rinsing with distilled water and DMSO. The
electrochemical behaviour of each compound (2mM) was investigated in argon saturated
H2O/DMF (1:9,v:v) solution containing 0.1M tetrabutylammoniumtetrafluroborate
(TBATFB).Drug-DNA interaction studies by CV were carried out in aqueous-DMF mixture
(1/9,v/v) at a scan rate of 100 mV/s by the sequential addition of DNA.
2.2 General procedure for the synthesis of ON donor Schiff bases (HL1-HL4)
ON donor Schiff bases (HL1-HL4) were synthesized by adopting the following general
procedure:
In a pre-backed two necked round bottom flask (250 ml) equipped with magnetic stirrer and
reflux condenser, the solution of salicyaldehyde was taken in dried ethanol. To this, solution of
corresponding amine in dried ethanol was added drop wise with constant stirring. The reaction
mixture was refluxed for 8 h under inert conditions using nitrogen. The progress of the reaction
was monitored by TLC. After the completion of the reaction the ethanol was removed by rotary
evaporator [27].The solid product was recrystallized from ethanol (scheme 1).
O
Ethanol
R
NH2
H
C
R
N
C
H2O
H
Reflux, 8h
HO
HO
O
O
R=
O
O
HL1
HL2
HL3
HL4
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Scheme1: Synthesis of Schiff bases (HL1-HL4)
2.2.1. (E)-2-((4-phenoxyphenylimino)methyl)phenol (HL1)

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(E)-2-((4-phenoxyphenylimino)methyl)phenol (HL1) was manufactured by refluxing equimolar
quantities of 1-amino-4-phenoxybenzene 1.85 g (10 mmol) and 1.05 ml (10 mmol) 2-hydroxy
benzaldehyde.
Color: yellow, Yield 90%, melting point 910C. FTIR: ( υ /cm-1) 3405 (−OH), 1603, (-CH=N),1H
NMR (300 MHz, CDCl3, δ ppm):13.30 (s, 1 H, -OH), 8.65 (s, 1 H, -CH=N), 7.43-6.94 (13 H, m,
13
Ar- H)., C NMR (75 MHz ,CDCl3, δ ppm): 162.13 (-C=N), 160.24-117.15 (18 C, Ar-C), MS
(m/z): 289(M +) CHN found (calcd.) for C19H15NO2 : C: 78.79 (78.89), H: 5.73 (5.19), N: 4.72
(4.84).
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2.2.2. (E)-2-((4-(4-biphenyloxy)phenylimino)methyl)phenol (HL2)
(E)-2-((4-(4-biphenyloxy)phenylimino)methyl)phenol (HL2) was synthesized by refluxing
equimolar quantities of 4-4(aminophenyloxy) biphenyl 2.61 g (10 mmol) and 1.05 ml (10 mmol)
2-hydroxy benzaldehyde.
Color: light yellow, Yield 92%, melting point 1730C. FTIR: ( υ /cm-1) 3400 (−OH), 1601 (-
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CH=N), H NMR (300 MHz, CDCl3, δ ppm):13.30 (s, 1 H, -OH),8.66 (s, 1 H, -CH=N), 7.63-
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6.95 (17 H, m, Ar- H), C NMR (75 MHz, CDCl3, δ ppm): 161.73 (-C=N), 160.15-117.28 (24
C, Ar-C), MS (m/z): 365 (M +) CHN found (calcd.) for C25H19NO2 : C: 82.15 (82.19), H: 5.58
(5.20), N: 3.94 (3.84).
2.2.3. (E) - 2-((4-(naphthalen-1-yloxy)phenylimino)methyl)phenol (HL3)
(E) - 2-((4-(naphthale,n-1-yloxy)phenylimino)methyl)phenol (HL3) was prepared by taking
equimolar quantities of 1-(4-aminophenoxy) naphthalene 2.35 g (10 mmol) and 1.05 ml (10
mmol) 2-hydroxy benzaldehyde.
Color: yellow, Yield 87%, melting point 1050C. FTIR: ( υ /cm-1) 3412 (−OH), 1613 (-CH=N), 1H
NMR (300 MHz, CDCl3, δ ppm): 13.32, (s, 1 H, -OH), 8.65 (s, 1 H, -CH=N), 8.23-6.94 (15 H,
m, Ar- H), 13C NMR (75 MHz, CDCl3, δ ppm): 162.27 (-C=N), 159.01-114.72 (22 C, Ar-C), MS
(m/z): 339(M +) CHN found (calcd.) for C23H17NO2 : C: 81.69 (81.41), H: 5.06 (5.01), N: 4.06
(4.13).
2.2.4. (E)-2-((4-(2-naphthoxy)phenylimino)methyl)phenol (HL4)
(E)-2-((4-(2-naphthoxy)phenylimino)methyl)phenol (HL4) was synthesized by using equimolar
quantities of 2-(4-aminophenoxy) naphthalene 2.35g (10 mmol) and 1.05 ml (10mmol) 2-
hydroxy benzaldehyde.
0
Color: yellow, Yield 93%, melting point 120 C. FTIR :( υ /cm-1) 3408 (-OH), 1610 (-CH=N),
1H NMR (300 MHz, CDCl3, δ ppm):13.30 (s, 1H, -OH),8.68 (s, 1 H ,-CH=N), 7.90-6.96 (15H,
m, Ar- H), 13C NMR (75 MHz , CDCl3, δ ppm): 162.25 (-C=N), 161.08-114.27 (22 C, Ar-C),
MS (m/z): 339(M +) CHN found (calcd.) for C23H17NO2 : C: 81.39 (81.41), H: 5.75 (5.01), N:
4.06 (4.13).
3 Results and Discussion

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Schiff bases (HL1-HL4) have been successfully synthesised in a single step as described in
experimental section and were characterised by spectroscopic, analytical, voltammetric and X-
ray diffraction analysis. They are air stable under ambient conditions and soluble in common
organic solvents such as C2H5OH, CH2Cl2, CHCl3, DMSO, and DMF. The synthesized Schiff
bases (HL1-HL4) were tested for biological, voltammetric and DNA interaction studies by cyclic
voltammetrically.
3.1 Spectral characterization
The structures of the synthesised compounds were established by means of spectral studies
(FTIR, 1H NMR, 13C NMR and mass spectrometry), elemental analysis, and single crystal X-ray
diffraction studies.
The FTIR spectral analysis of Schiff bases indicated the presence of all expected
functionalities.Schiff bases having O-hydroxy group on an aldehyde can form intramolecular
hydrogen bonding which affects ν(OH) vibration by shifting towards lower frequency with
broadening. The extent of shift depends on the strength of hydrogen bonding.The FTIR spectra
of these compounds exhibited the strong absorptions bands in the region of 1601-1613 cm-1 due
to azomethine bond (CH=N).The hydroxyl groups (-OH) of the Schiff bases showed broad
absorption bands in the region of 3400-3412 cm-1.The disappearance of peaks in the region
3391-3468 (asymmetric) and 3315-3372 (symmetric) cm-1 for –NH2 moiety confirmed the
conversion of amino group into azomethine bond. They also exhibited absorption bands of C-O-
C vibrations in the region of 1244-1260 cm-1 [28]. The FTIR spectra of Schiff bases as presented
in Fig. S1(a-d).
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The H NMR and13C NMR spectra of all the Schiff bases are consistent with the proposed
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molecular structures. The H NMR spectra showed the characteristic azomethine singlet in the
region of 8.68-8.65 ppm. Phenyl protons were present in all the compounds which were verified
by the appearance of multiplets in the range of 7.43-6.94 (HL1), 7.63-6.95 (HL2), 8.23-6.94
(HL3) and 7.90-6.96 (HL4) ppm, according to the substituents attached. The resonance signals
observed around 13.30-12.14 were assigned to phenolic proton as shown in Fig. S2(a-d).
13C NMR spectra of Schiff bases show all the characteristic signals of azomethine and aromatic
carbon atoms in their respective ranges of chemical shift values. Azomethine carbon resonated
around 162 ppm. The resonance signals observed around 160, 155-150 and 141 ppm were
assigned to C–OH, C-O-C and C-N carbons respectively. The rest of the peaks from 146-110
ppm were due to aromatic carbon atoms of Schiff bases as displayed in Fig. S3(a-d).
The mass spectral data of the Schiff bases justified their formation as their molecular ion peaks
were obtained at (m/z) 289, 365, 339 and 339 respectively as revealed in Fig. S4(a-d).
3.2 X- ray structure analysis of HL3
The single crystals of Schiff base (HL3) for X-ray crystallographic analysis were grown by the
slow evaporation of its ethanolic solution at room temperature. A single suitable crystal was
selected for X-ray diffraction analysis and was mounted on Bruker Apex II CCD diffractometer.
The crystal was kept at 150(2)K during analysis. A perspective diagram of HL3 is shown in Fig.
1. There are no unusual bond lengths; the phenol and anthracene rings are inclined at 30.23(8)°
and 89.64(4)° to the central phenyl ring, respectively and the phenol group makes an
intramolecular hydrogen bond to the imine nitrogen ()1 – N1, 2.5591(18)Å) .There is some π…π

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stacking between anthracene groups, but most of the intermolecular interactions are of the C-H…π
type as shown in Fig. 2.
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Fig.1 Perspective diagram of HL3 showing 50% probability ellipsoids.
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Fig. 2 Intermolecular interactions in HL3
Crystal data and refinement details are listed in Table 1. Selected bond lengths and angles for
HL3 are summarized in Table 2 and hydrogen bonding parameters in Table 3.
Table 1.Crystal data and structure refinement for HL3
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Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
C23 H17 N O2
339.38
150(2) K
0.71073 Å
Monoclinic
P21/c
Unit cell dimensions
a = 13.3952(11)Å
b = 8.8205(7)Å
c = 14.7042(12)Å
α= 90°.
β= 100.9600(10)°
γ= 90°.
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Volume
Z
1705.6(2)Å3
4
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Density (calculated)
1.322 Mg/m3
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Absorption coefficient
F(000)
0.084 mm-1
712
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Crystal size
Crystal description
Theta range for data collection
Index ranges
Reflections collected
Independent reflections
Completeness to theta = 28.32°
Absorption correction
Max. and min. transmission
0.44 × 0.30 × 0.04mm3
Yellow plate
1.55 to 26.40°
-16<=h<=16, -11<=k<=11, -18<=l<=18
14712
3497 [R(int) = 0.0439]
100.0%
Semi-empirical from equivalents
0.9966 and 0.9638
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Refinement method
Full-matrix least-squares on F2
3497 / 0 / 235
Data / restraints / parameters
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Goodness-of-fit on F2
Final R indices [I>2sigma(I)]
R indices (all data)
0.993
R1 = 0.0423, wR2 = 0.0975
R1 = 0.0752, wR2 = 0.1127
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Largest diff. peak and hole
0.264 and -0.173 e.Å-3
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K: kelvin temperature; Å: angstrom; Å3: volume; Z: number of chemical formula units per unit
cell; D: density; F: structure factor; R: reliability factor
Table 2.Selected bond lengths [Å] and angles [°] for HL3
Bond
Length Measurement(Å) Bond angle
Angle measurement(°)
118.46(16)
O(1)-C(1)
C(7)-N(1)
N(1)-C(8)
C(11)-O(2)
O(2)-C(14)
1.356(2)
1.286(2)
1.415(2)
1.387(2)
1.399(2)
O(1)-C(1)-C(2)
O(1)-C(1)-C(6)
N(1)-C(7)-C(6)
C(11)-O(2)-C(14)
C(15)-C(14)-O(2)
121.05(15)
120.79(16)
117.31(13)
120.58(18)
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Table 3.Hydrogen bonds for HL3 [Å and °].
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D-H...A
O(1)-H(1)...N(1)
d(D-H)
0.84
d(H...A)
1.81
d(D...A)
2.5613(18)
<(DHA)
147.6
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3.3. Pharmacological studies
3.3.1. Brine shrimp cytotoxic assay
Brine shrimp cytotoxicity assay is a pre-screen test to observe the bioactive nature of
compounds. The compounds HL1 and HL3 showed significant activity against brine shrimp
nauplii to be highly active with LD50 values 0.18 and 0.06 ꢀg/ml respectively. This cytotoxic
action of a drug is simply provided by disturbing the basic mechanisms concerned with mitotic
activity, cell growth, function and differentiation (Table 4).
Table 4. Results of cytotoxicity, potato disc antitumor and inhibition of hydroxyl free radical
induced DNA damage assays.
Compound Cytotoxic
activity
Antitumor activity
DNA protection activity
IC50
percentage inhibition± SD
10(µg/ml) 100(µg/ml) 1000(µg/ml)
LD50value
(µg/ml)
10(µg/ml)
100(µg/ml) 1000(µg/ml)
(µg/ml)
14.20
4.54
0.18
0.06
+
+
+++
+++
+++
+++
HL1
HL3
40±1.8
55±1.4
82.5±0.8 95±0.4
75±1.3 100±0
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DNA: Deoxyribonucleic acid; LD50: lethal dose 50 or median lethal dose; SD: standard
deviation; IC50: half maximal inhibitory concentration
3.3.2. Potato disc antitumor assay
The compounds were screened for possible antitumor activity by using potato disc antitumor
assay and results are summarized in Table 4. The results of compounds HL1 and HL3 showed
significant antitumor activity with IC50 values 14.20 and 4.51 µg/ml respectively. It is thought
that these compounds at different doses may be used against different cancer-chemopreventive
models and may appear to contrive safer drugs for future.
3.3.3. DNA protection assay
To check the antioxidant and pro-oxidant behavior of compounds free radical induced DNA
•
damage assay was performed. With the attack of OH produced from the Fenton reaction,
supercoiled plasmid DNA is broken into two forms, including open circular (OC) and linear
form (linear). By analyzing the intensity of bands formed on 1% agarose gel, results are noted
and tabulated (Fig.3). The compounds HL1and HL3 showed moderate protection at 1000 µg/ml
and 100 µg/ml concentrations; while slight protection at 10 µg/ml concentration. So, it was
concluded that these compounds showed DNA protection in concentration dependent manner i.e.
DNA protection increased with the increase in concentration of the test compound.
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L
P
X
1
2
3
4
5
6
7
8
Open circular DNA
Super coiled DNA
Linear DNA
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Fig.3 Effect of compounds HL1and HL2 on pBR322 plasmid DNA [L = 1Kb ladder, P = pBR322
plasmid, X = pBR322 plasmid treated with FeSO4 and H2O2 (positive control), 1 (C+P) =
pBR322 plasmid + 1000 µg/ml of HL1 control for the pro-oxidant effect of the compound on
DNA, 2 = plasmid + 1000 µg/ml of HL1+ FeSO4 + H2O2, 3 =plasmid + 100 µg/ml of HL1+
FeSO4 + H2O2, 4 = plasmid + 10 µg/ml of HL1 + FeSO4 + H2O2, 5 = pBR322 plasmid + 1000
µg/ml of HL2; control for the pro-oxidant effect of the compound on DNA, 6 = plasmid + 1000
µg/ml of HL2+ FeSO4 + H2O2, 7 = plasmid + 100 µg/ml of HL2+ FeSO4 + H2O2, 8= plasmid + 10
µg/ml of HL2+ FeSO4 + H2O2
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3.2 Voltammetric studies.
Electrochemical behaviour of the Schiff bases (HL1-HL4, 2mM) was investigated by employing
three voltammetry techniques (CV, DPV and SWV) in oxygen free atmosphere in DMF solution.
The cyclic voltammograms are displayed in Fig. 4. During anodic scan only one oxidation peak
was observed for all the four Schiff bases, which appears in the range of 0.815 to 0.844 V,
without corresponding reduction peak on the backward scan. This describes the irreversible
oxidation of OH moiety present on the Schiff base molecule. To inspect the adsorption of the
oxidation product, which is likely in such type of compounds, the successive scans were
recorded at constant potential scan rate (100 mVs-1). A systematic decrease in peak current was
observed in following cycles for all the compounds revealing adsorption of oxidation product
while hindering the further electron transfer process on the surface of working electrode. The
GCE was very carefully renewed each time for a new compound. While increasing the scan rate
for all the Schiff bases, it was found that oxidation peak current increases linearly with square
root of scan rates as shown in Fig. 5 and follows the Randles-Sevcik equation of an irreversible
process. The details about the equation and parameters are given in our earlier papers[29-
32].The diffusion coefficients evaluated from the slope of anodic peak current vs. square root of
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scan rate were found to be 4.65 × 10-6, 3.18 × 10-7, 5.15 ×10-10and 1.55 × 10-7for HL1, HL2, HL3
and HL4 respectively. The values depict that the ET process is diffusion controlled.

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Fig.4 Cyclic voltammograms of Schiff bases HL1, HL2, HL3and HL4 (2mM each) recorded in
their argon saturated H2O/DMF (1/ 9; v/v) + 0.1M TBATFB solution at 100 m Vs-1(Vs-1)1/2scan
rate at 25oC.
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Fig. 5 Plots of anodic peak current (ipa) with square root of scan rate v1/2/ (Vs-1)1/2for HL1, HL2,
HL3 and HL4.
To further confirm the irreversibility of the ET process SWV was run. The resultant
voltammograms of HL1, HL2, HL3 and HL4 are shown in Fig. 6 (A-D). The absence of reduction
current in all the four cases evidently indicates the complete irreversible nature of ET process as
shown in CV results.
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Fig.6 Square wave voltammograms of compound, HL1(a), HL2(b), HL3(c) and HL4(d) (2 mM
each) recorded at GCE in argon saturated H2O/DMF (1/ 9; v / v) + 0.1 M TBATFB solution at
25oC, Esw =5 mV frequency = 10 Hz . Pulse amplitude =25mV. The symbols are; It– total
current, If – forward current, Ib –backward current.
DP voltammograms of the Schiff bases, displayed in Fig. 7, were recorded to estimate the
number of electrons involved in the electrooxidation. The recorded peak currents are in the order
of HL1> HL3> HL2>HL4 same as observed in CV studies indicating the enough sensitivity of CV
for these compounds in the given time scale. The full peak width at half peak current(W1/2)
values are 122, 88, 117 and 88 mV for HL1, HL2, HL3and HL4,respectively and are close to the
theoretical value of 90 mV for ET process involving one electron only. Hence, it is concluded on
the basis of voltammetry results that there is single electrophore in the studied Schiff bases
offering one ET irreversible diffusion controlled oxidation process. Corollary of this result is that
the electro-oxidised species will act as a good nucleophile for the DNA molecules.

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Fig.7 Differential pulse voltammograms of the Schiff bases with 2 mM each recorded in argon
saturated H2O /DMF(1/9; v /v) + 0.1M TBATFB solution at 25oC.
4 DNA-drug interaction studies
The CV was employed to envisage the role of Schiff bases as drug molecules against DNA
degradation. The characteristic parameters of the peak potential and peak current of a compound
are prone to change on addition of DNA if the former is ‘active’. The variation in peak current
with varying concentration of DNA at fixed concentration of a Schiff base was utilized to
determine the quantitative binding parameters (Fig. 8, Table 4) whereas the shift in peak
potential was used to get an idea about the modes of interaction.
The values of binding constant (K) were calculated from equation 1 which determines the
strength of binding of a molecule to the DNA.
1/ [DNA] = K (1-A) / (1-I/I0) – K
(1) [33]
Where A is an empirical constant, I0 and I are peak currents in the absence and presence of DNA,
respectively.
From Fig. 8 and the tabulated data (Table 4) it is evident that on addition of first increment of
DNA (5.44 µM) to the electro-oxidised product of the Schiff base there is a marked decrease in
the peak current which is about two third of the original peak current. To ensure that this
decrease is not due to adsorption of the molecule on the electrode surface, the GCE surface was
renewed each time before running the next increment of DNA. On the other hand the addition of
DNA caused a very small negative shift in the peak potential even up to the 16.01µM DNA. The
negative shift in peak potential is revealing the electrostatic interaction between the
electrooxidised electrophile of Schiff base and the negative phosphate component of DNA
molecules. The result is in agreement to that conferred above in DPV analysis of the compounds.
The sequential drop in peak current has attributed the decrease in free drug concentration due to
formation of slow diffusing Schiff base-DNA adducts. Formation of this slow diffusing complex

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is also evident from lowering in diffusion coefficient values upon the addition of DNA as
compared to free compounds as shown in Table 4. The binding constant values given in Table 4
are in the range of the order of ≈ 104 M-1. These moderately high values (K = 3.6 x 104 M-1for
cisplatin[34]) suggest that ON donor Schiff bases are efficient DNA binders.
As a whole it can be safely interpreted from the results that the investigated Schiff bases possess
sufficient potential to be pursed as drug molecules whereas they were found interacting with
DNA while being consumed chemically and partially due to electrostatic linkage.
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439
Fig.8 Cyclic voltammograms of 2mM H2O/DMF (1/ 9; v/v) of HL1 (a), HL2 (b), HL3 (c) and HL4
(d) (▬) without DNA, (▬) in the presence of 5.44 µM DNA, (▬) 10.78 µM DNA and (▬)
16.01 µM DNA on glassy carbon electrode at scan rate of 100 mV/s.
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Table 5. The drug-DNA interaction electrochemical parameters of compounds on glassy carbon
electrode vs. Ag/AgCl in H2O/DMF (1/ 9; v/v) solution at 50 mVs-1 scan rate at 25oC.
Compound
In the absence of DNA
In the Presence of DNA
-1
Ep (V)
Do(cm2s-1)
Ep (V)
Do(cm s )
K (M-1)
2
4.65 ×10-6
3.18 × 10-7
5.15 × 10-10
1.55 × 10-7
2.25 ×10-6
2.27 × 10-7
1.62 × 10-10
1.12 × 10-7
2.04 × 104
1.77 × 104
4.16 × 104
3.06 × 104
0.844
0.815
0.838
0.821
0.833
0.809
0.833
0.809
HL1
HL2
HL3
HL4
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DNA: Deoxyribonucleic acid; Do: diffusion coefficient; Ep: peak potential; K: complex stability
constant.
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4 Conclusions
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Four novel ON donor Schiff bases (HL1-HL4) have been successfully synthesized and
characterized by various spectroscopic, analytical and electro-analytical techniques. Single
crystal X-ray diffraction analysis of Schiff base (HL3) reveals intra (hydrogen bonding) and inter
molecular interactions(C-H…π type, π…π stacking between anthracene groups).The biological
findings showed significant activity in inhibiting the tumour formation by A. tumefaciens on
potato discs. These compounds also have potential to prevent H2O2 induced oxidative damage to
pBR322 DNA. Overall these compounds displayed a variant degree of activity in all the assays,
further investigations are required to explore the exact mechanism of biological activities which
may be helpful to explore new and alternative chemotherapeutic agent(s) in clinical implications.
The compounds were found to respond irreversible one electron diffusion controlled
electrooxidation on glassy carbon electrode. Further, they were found to interact to DNA in
electrooxidised form through combined mode of groove binding and electrostatically with
sufficiently good strength to be pursued further as drug molecules.
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Acknowledgements
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The authors are highly grateful to Chemistry and Biochemistry Departments of Quaid-I-Azam
University Islamabad, Pakistan and Institutfür Anorganische Chemie, J. W. Goethe-Universität
Frankfurt, Germany for providing technical support and laboratory facilities.
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Highlights
•
•
Four novel ON donor Schiff bases were synthesized with yields around 90%.
Exhibited strong •OH scavenging activity in concentration dependent manner.
•
•
Voltammetry revealed irreversible single electron oxidation with Do ~10-10-10-6 cm2s-1.
Anticancerous nature, intercalating with DNA having binding constants of 104 M-s1.