Bioorganic Chemistry 75 (2017) 224–234

Contents lists available at ScienceDirect

Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg

Synthesis and bioelectrochemical behavior of aromatic amines

Muhammad Shabbir a, Zareen Akhter a,⇑, Iqbal Ahmad b, Safeer Ahmed a,⇑, Michael Bolte c, Vickie McKee d
a
Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
b
Department of Chemistry, Allama Iqbal Open University, Islamabad 44000, Pakistan
c
Institut für Anorganische Chemie, J.W. Goethe-Universität Frankfurt, Max-Von-Laue-Strasse 7, Frankfurt/Main 60438, Germany
d
School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland

a r t i c l e i n f o a b s t r a c t

Article history: Four aromatic amines 1-amino-4-phenoxybenzene (A1), 4-(4-aminophenyloxy) biphenyl (A2), 1-(4-
Received 7 May 2017 aminophenoxy) naphthalene (A3) and 2-(4-aminophenoxy) naphthalene (A4) were synthesized and char-
Revised 21 September 2017 acterized by elemental, spectroscopic (FTIR, NMR), mass spectrometric and single crystal X-ray diffrac-
Accepted 2 October 2017
tion methods. The compounds crystallized in monoclinic crystal system with space group P21.
Available online 4 October 2017
Intermolecular hydrogen bonds were observed between the amine group and amine/ether acceptors of
neighboring molecules. Electrochemical investigations were done using cyclic voltammetry (CV), square
Keywords:
wave voltammetry (SWV) and differential pulse voltammetry (DPV). CV studies showed that oxidation of
Aromatics amines
Crystal structures
aromatic amines takes place at about 0.9 V (vs. Ag/AgCl) and the electron transfer (ET) process has irre-
Electrochemistry versible nature. After first scan reactive intermediate were generated electrochemically and some other
Drug-DNA interaction study cathodic and anodic peaks also appeared in the succeeding scans. DPV study revealed that ET process is
accompanied by one electron. DNA binding study of aromatic amines was performed by CV and UV–vis-
ible spectroscopy. These investigations revealed groove binding mode of interaction of aromatic amines
with DNA.
Ó 2017 Elsevier Inc. All rights reserved.

1. Introduction b-naphthylamine and 4-amino diphenyl. Various drugs have been

Aromatic amines, one of the largest class of compounds utilized
by the chemical industry, are produced by catalytic reduction of
nitroaromatics [1]. It is a versatile class of organic compounds
which ranges from monocyclic to highly complex conjugated poly-
cyclic or heterocyclic aromatic structures and multiple sub-
stituents. As aromatic amines take part in many chemical
reactions therefore they are used for the syntheses of drugs, pesti-
cides, plastics, azodyes, Schiff bases, zeolites, polyimides, polya-
mides, stationary phase for HPLC, epoxy resins, paints, pigments,
rubber accelerators, as a catalyst for the cross linking of polyester,
stabilizers for phenolic resins coagulants, antiknock additives for
gasoline and diesel fuel. They are also widely used in some
metal-coating multifunctional compositions for motor, transmis-
sion and industrial oils. Some aromatic amines are used in cosmet-
ics, textile and rubber industries [2,3]. Simplest aromatic amine
aniline is converted into sulfanilic acid which is an important
precursor of sulpha drugs. Besides their numerous applications
some of them are considered to be carcinogenic like benzidine,
⇑ Corresponding authors.
E-mail addresses: zareenakhter@yahoo.com (Z. Akhter), safeerad@qau.edu.pk
(S. Ahmed).
designed earlier to combat different diseases like cancer [4,5]. Can-
cer cells rapidly divide and synthesize new DNA. Cytotoxic drugs
work by interfering with DNA. DNA-binding agents are currently
the most effective drugs used [6]. The interaction of drugs with
DNA is the central aspect of biological studies in pharmaceutical
development and drug discovery processes [7]. Structural proper-
ties of DNA, origin of diseases, mechanistic action of antitumor
and antiviral drugs are helpful to design new and more efficient
DNA targeted drugs [8]. A drug can interact with DNA either cova-
lently (intercalation) or non-covalently (groove binding). Non-
covalent interaction of drugs with DNA occurs at minor or major
groove. When DNA binds with minor groove, walls of the groove
get interacted with the drug and hydrogen bonding establishes
or electrostatic interactions occur with the phosphate backbone
and the bases while major groove establishes hydrogen bonding
with drug [9].
The pharmacological investigations like brine shrimp cytotoxic-
ity, potato disc antitumor, antibacterial, antifungal, DPPH free rad-
ical scavenging and DNA damage studies of the synthesized
aromatic amines were reported by our research group earlier
[10]. In the present study we extended our research to explore
their drug-DNA binding behavior by cyclic voltammetry and
UV–visible spectroscopy. For this purpose amine compounds

https://doi.org/10.1016/j.bioorg.2017.10.002
0045-2068/Ó 2017 Elsevier Inc. All rights reserved.
 M. Shabbir et al. / Bioorganic Chemistry 75 (2017) 224–234 225

Scheme 1. Synthesis of aromatic amines.

4-phenoxybenzenamine (A1), 4-(naphthalen-1-yloxy)benzena (A1-A4) were characterized by elemental analysis, spectroscopic

mine (A3),4-(naphthalen-2-yloxy)benzenamine (A4) and struc- (FTIR, NMR), mass spectrometric and single crystal X-ray diffrac-
turally similar 4-(4-aminophenyloxy) biphenyl (A2) [11] were syn- tion studies. The redox drug-DNA interaction aspect of the com-
thesized. Crystal structures of three aromatic amines (A1, A3 & A4) pounds was investigated by cyclic voltammetric and UV–visible
were grown in ethanol solution and studied by single crystal X-ray spectroscopic techniques.
diffraction analysis.
2.1. Spectral characterization
2. Results and discussion
2.1.1. FTIR spectroscopy
The absorption bands of the nitro functionality at 1506–1509
Four aromatic amines were prepared (Scheme 1) and character-
and 1338–1342 cm
1 corresponding to symmetric and asymmetric
ized. The synthesized compounds appeared crystalline, non-
stretches of -NO2 group disappeared after reduction, in the FTIR
hygroscopic, insoluble in water and soluble in common organic
spectra and typical NAH stretching bands in the region 3391–
solvents (acetone, ethanol, DMSO, DMF etc.) The aromatic amines
3468(asymmetric) and 3315–3372(symmetric) cm
1 appeared
because of NAH stretching bands. The CAH stretching frequency
Table 1 of aromatic groups are shifted slightly higher due to the loss of
1
HNMR data of aromatic amines (ppm). inter molecular association of the nitro group after reduction in
Compound H ortho to ANH2 Rest of aromatic protons amines. The FTIR spectra of aromatic amines (A1, A2, A3 and A4)
ANH2 exhibited characteristic broad absorption bands at 1225–1242
A1 6.54 (d, 2H, 4.45
0 0
7.35 (m, 2H, H7,7 ), 7.05 (m, 3H, H6,6 ,8), cm
1 due to CAOAC and 1592–1622 cm
1 because of C@C of aro-
0
H2,2 , J = 9.2 (s, H)
0
6.85 (d, 2H, H3,3 , J = 8.9 Hz) matic rings [10,12].
Hz)
0
A 2 6.67 (d, 2H, 4.53 7.65 (m, 2H, H10,10 ), 7.57 (m, 2H,
0
H2,2 , J = 9.0 (s, H)
0 0
H7,7 ), 7.48 (m, 2H, H11,11 ), 7.35 (m, 2.1.2. 1H &13C NMR spectral analysis
0 1
Hz) 1H, H12), 7.17 (m, 2H, H6,6 ), 6.92 (d, H NMR spectra of all the compounds of this series showed a
0
2H, H3,3 , J = 8.7 Hz) two proton singlet near 4.50 d(ppm) corresponding to primary aro-
A 6.50 (d, 2H, 4.31 7.95(m, 1H, H8), 7.65(m, 1H, H5), 7.28
3
0 matic amine protons indicating the conversion of nitro groups into
H13,13 , J = 8.8 (s, H) (m, 3H, H4,6,7), 7.12 (m, 1H, H3), 6.74
Hz) (m, 3H, H2,12,12 )
0 amines. It is also confirmed by the upfield shift of aromatic protons
A4 6.45 (d, 2H, 4.54 7.53 (m, 2H, H1,8), 7.45 (m, 1H, H5), due to electron donating effect of amino group (Table 1). For
0
H13,13 , J = 8.8 (s, H) 7.25 (m, 2H, H6,7), 6.89 (m, 2H, H2,4), nitroaromatics, signals for protons present at ortho position of
0
Hz) 6.62 (m, 2H, H12,12 ) nitro group appeared in the downfield region i.e. at 8.25 ppm.

Fig. 1. Synthesized aromatic amines (A1- A4).

226 M. Shabbir et al. / Bioorganic Chemistry 75 (2017) 224–234

However in case of aromatic amines, these protons resonated
upfield at 6.50 ppm. The rest of aromatic protons gave their char-
acteristic signals ranging from 7.95 to 6.65 ppm as shown in Table 1
according to numbering scheme shown in Fig. 1.
13
C NMR spectroscopic studies also confirmed the formation of
all the aromatic amines. In A1- A4, the carbon atoms of the aromatic
rings attached directly to the oxygen atoms of ether linkage were
most deshielded due to highest electronegativity of oxygen atom,
showing downfield signals at 159.44–155.25 ppm. Then signals
for carbon atom attached with nitrogen (CAN) were observed
around 4.50 ppm. Remaining aromatic carbons showed signals
ranging from 135 to 110 ppm. Data for 13C NMR is given in Table 2
and numbering scheme is shown in Fig. 1.
2.1.3. Mass spectral studies
The mass spectral data of the aromatic amines confirmed their
formation as molecular ion peaks were obtained at (m/z) 185 for
A1, 261 for A2, 235 for A3 and A4 respectively. The mass spectral
data of the compounds displayed molecular ions as base peaks
(Fig. S1).
2.1.4. X-ray structure determination
Suitable single crystals of aromatic amines (A1, A3, A4) were
mounted on Bruker Apex II CCD diffractometer or a STOE-IPDS II
diffractometer using MoKk radiation (k = 0.71073 Å) and the mole-
cules are shown in Figs. 2–4. The structures were solved by direct
methods using the program SHELXS and refined against F2 with
full-matrix least-squares techniques using the program SHELXL-
2016 [13]. All the non-hydrogen atoms were refined using aniso-
tropic atomic displacement parameters and hydrogen atoms
bonded to carbon were inserted at calculated positions using a rid-
ing model. Hydrogen atoms bonded to nitrogen were located from
difference maps and their coordinates freely refined. Parameters
for data collection and refinement are summarized in Table 3.
Important bond lengths and bond angles for compounds A1, A3
and A4 are given in Table 4. Hydrogen bonds [Å and °] in A1, A3
and A4 are shown in Table 5.
Compound A1 (Fig. 2(a)) crystallizes in the chiral space group
P21, however the absolute structure has not been determined.
The interplanar angle between the two aromatic rings is 81.34
(5)°. The molecules are linked into a 2D sheet structure by rather

Table 2
13
C NMR data of aromatic amines (ppm).

Compound CAOAC CAN C ortho to ANH2 Rest of aromatic carbons

A1 159.44(C5), 145.93 (C4) 140.75 (C1) 115.33 (C2,20 ) 130.12 (C7,70 ), 122.17 (C8), 118.47(2C, C3,30 ), 116.85 (C6,60 )
A2 163.10(C5), 153.25 (C4) 141.67 (C1) 116.57 (C2,20 ) 134.7 (C9), 128.52 (C8), 127.83 (C11,110 ), 126.45 (C10,100 ), 120.84 (C3,30 ),
118.46(C6,60 )
A3 155.25(C1), 146.39(C11) 146.07(C14) 115.40 (13,130 ) 134.83(C10), 128.10 (C5), 127.11 (C6), 126.50 (C7), 125.77 (C9,C3), 121.94 (C8),
121.85 (C4), 121.31 (C12,120 ), 110.10 (C2)
A4 157.49(C3), 146.17 (C11) 145.81(C14) 118.73 (C13,130 ) 134.43 (C10), 130.20 (C1), 129.48(C9),128.02(C8),127.23(C6),126.98(C5),
124.56(C7),121.59(C12,120 ),119.24(C2), 110.85 (C4)

Fig. 2. (a) Crystal structure of compound A1 (b) H-bonds in compound A1.

 M. Shabbir et al. / Bioorganic Chemistry 75 (2017) 224–234 227

Fig. 3. (a) Crystal structure of compound A3 (b) H-bonds in compound A3.

long H-bonds between the amine group and amine/ether acceptors
of neighboring molecules as shown in Fig. 2(b).
The dihedral angle between the two aromatic moieties is 78.9°.
The crystal structure is stabilized by NAH. . .N and NAH. . .O hydro-
gen bonds connecting the molecules to sheets parallel to the ab
plane (Fig. 3).
The dihedral angle between the two aromatic moieties is 83.4°.
The crystal structure is stabilized by NAH. . .N and NAH. . .O hydro-
gen bonds connecting the molecules to sheets parallel to the ab
plane (Fig. 4).
2.2. Electrochemical studies
Electrochemical behavior of aromatic amines (A1- A4) was
investigated on platinum electrode employing cyclic voltammetry
(CV), differential pulse voltammetry (DPV) and square wave
voltammetry (SWV) techniques.
2.2.1. Cyclic voltammetry and square wave voltammetry
Electrochemical behavior of four different aromatic amines (A1-
A4) was explored on glassy carbon electrode (GCE) first using CV.
228 M. Shabbir et al. / Bioorganic Chemistry 75 (2017) 224–234

Fig. 4. (a). Crystal structure of compound A4 (b) H-bonds in compound A4.

The CV response of each compound (1 mM) was observed in argon
saturated DMSO/H2O (9:1) solution containing 0.1 M tetrabuty-
lammonium tetrafluoroborate (TBATFB) as a supporting electrolyte
at potential scan rate of 100 mVs
1 at 25 °C. The cyclic voltammo-
grams of the aromatic amines are presented in Fig. 5. During first
scan only single oxidation peak (1a), which appears in the range
of 0.83–0.90 V, was found without the corresponding cathodic
peak on the backward sweep which reveals the irreversible oxida-
tion process. A new pair of redox peaks (2a and 1c) was observed in
the range from 0.13 V to 0.56 V in the successive cycles. Peak 1a can
be obviously ascribed to the oxidation of aromatic amine, which
gives birth to cation radicals [14]. Consequently, the peak 1c is
the intermediate produced from the cation radical generated in
the first anodic scan and not the original aromatic amine. This is
further confirmed from the appearance of peak 2a in second scan
which was absent in first scan. A small hump around 0 V in case
of A2 and A4 also confirm the generation of electrochemically
active intermediates in the reverse scan. The behavior could be
attributed to the electropolymerization which fouls the surface of
electrode and same has been indicated in the following experi-
ments. In case of aniline oxidation occurs at potential >1 V and
here shift towards less positive potential is attributed to the elec-
tron donating effect of oxy-aromatic substituents attached to the
aniline system [15]. Further it was observed that the current of
peak 1a decreases gradually for successive scans, from which we
can infer that the oxidation product of the first anodic scan get
deposited on the electrode surface, which prevents aromatic
amines from further electroxididation. Peak 3 in curve a is a
 M. Shabbir et al. / Bioorganic Chemistry 75 (2017) 224–234 229

Table 3 takes place immediately because of high electrochemical activity

Crystal data and structure refinements for compounds A1, A3 and A4. of cation radical. The dimer could act as an active center for the fur-
A1 A3 A4 ther growth of the polymeric chains [20].
Empirical formula C12 H11 N O C16H13NO C16H13NO
Polyaromatic amine produced by the electrochemical oxidation
Formula weight 185.22 235.27 235.27 with a unique structure, fairly high conductivity, environmental
Temperature (K) 150(2) 173(2) 173(2) stability, high electrochemical activity, electrocatalytic and
Wavelength (Å) 0.71073 0.71073 0.71073 electro-coloring properties, and a potential of applying to a variety
Crystal system Monoclinic Monoclinic Monoclinic
of fields, has been applied widely [21–23]. But there is still much
Space group P21 P21 P21
controversy on the whole mechanism. The products of the reaction
Unit cell
are the most important to comprehend the electrochemical behav-
a (Å) 7.8976(5) 8.2226(9) 7.8082(9)
b (Å) 5.6272(4) 6.0188(5) 5.7961(9) ior of aromatic amines. So far, in the anodic oxidation of aromatic
c (Å) 10.6804(7) 12.1231(13) 13.3583(15) amines, the electrogenerated cation radicals and dications formed
(°) 91.5960(10) 94.814(9) 91.347(9) were found to undergo a variety of coupling pathways. There are
Volume (Å3) 474.47(5) 253.57(2) 604.39(14)
three primary anodic coupling modes i.e. tail-to-tail, head-to-tail,
Z 2 2 2
D (calc) (Mg/m3)1.549 Mg/ 1.296 1.307 1.293
and head-to-head couplings to generate benzidines, diphenylami-
m3 nes, and hydrazobenzenes, respectively [15]. These pathways
Abs. coeff. (mm
1)0.841 0.083 0.082 0.081 strongly depend upon the conditions such as nature of the med-
mm
1 ium, pH and concentration of solution, substituent effect, initial
F(0 0 0)1176 196 248 248
potential of scan and current density in electrolysis.
Crystal size (mm3)0.37  0.42  0.27 0.35  0.32  0.32  0.30 
0.23  0.05 mm  0.21 0.28 0.29 Also CV experiments were performed at different scan rates to
Crystal descriptionorange Colourless White White further explore the electron transfer process. For all the aromatic
block triangular amines, it was observed that anodic peak current of peak 1a
Reflections collected 5508 5791 3431 increases linearly with square root of scan rates as shown in
Independent refl (Rint) 6196 1557 1497 (0.0300) 1239 (0.0353)
[R (int) = 0.0322] (0.0217)
Fig. 6 and follows the following Randles-Sevcik equation
Goodness on F21.042 1.023 1.049 1.063
R1, wR2 [I > 2r (I)] 0.0337, 0.0304,0.0811 0.0281,0.0728
ip ¼ ð2:99  105 ÞnðanÞ1=2 ACD1=2 a1=2 ð1Þ
0.0904
R1, wR2 (all data)R1 = 0.0353, 0.0317,0.0818 0.0311,0.0743
0.0646, wR2 = 0.1275 0.0928 where a is the charge transfer coefficient, ip is peak current, A is
the surface area of the working electrode, n is number of electrons
K: kelvin temperature; Å: angstrom; Å3: volume; Z: number of chemical formula
units per unit cell; D: density; F: structure factor; R: reliability factor. involved in the redox process, C is the bulk concentration of the
analyte, and D is the diffusion coefficient of the molecule. Diffusion
coefficients werecalculated from the slope of anodic peak current
dehydrogenated peak, corresponding to the deprotonation arising (1a) vs. square root of scan rate to be 2.78  10
5, 3.16  10
6,
from the coupling process of cation radicals. For the whole reaction 1.5  10
8 and 1.41  10
5 for A1, A2, A3 and A4 respectively.
the electrochemical oxidation of aromatic amines is in line with Irreversible nature of oxidation process of aromatic amines was
ECE pathway. The electrochemical properties of aromatic amines further assessed by SWV. SW voltammograms of A1- A4 are pre-
have been studied previously [16–19] and similar conclusions sented in Fig. 7(A–D). The complete absence of cathodic peak in
were drawn. After the formation of radical cations generated from SW voltammograms of all the aromatic amines reveal the irre-
the anodic oxidation of aromatic amines, a dimerization process versible nature of oxidation process.

Table 4
Important bond lengths and bond angles for compounds A1, A3 and A4.

A1 A3 A4
N(1)-C(10) 1.4093(2) N(1)-C(4) 1.4237(19) N(1)-C(4) 1.418(2)
O(1)-C(1) 1.3961(17) O(1)-C(1) 1.4027(17) O(1)-C(1) 1.4045(17)
O(1)-C(7) 1.3931(15) O(1)-C(11) 1.4037(19) O(1)-C(11) 1.400(2)
C(7)-O(1)-C(1) 117.94 (11) C(11)-O(1)-C(1) 117.88(11) C(11)-O(1)-C(1) 117.20 (12)
C(2)-C(1)-O(1) 117.86(13) C(2)-C(1)-O(1) 123.55(14) C(2)-C(1)-O(1) 122.38(16)
C(6)-C(1)-O(1) 120.39(15) C(6)-C(1)-O(1) 116.11(14) C(6)-C(1)-O(1) 117.12(14)
C(8)-C(7)-O(1) 123.17(13) O(1)-C(11)-C(20) 119.49(14) O(1)-C(11)-C(20) 117.24(14)
C(12)-C(7)-O(1) 116.11(12) O(1)-C(11)-C(12) 118.49(13) O(1)-C(11)-C(12) 120.94(17)

Table 5
Hydrogen bonds [Å and °] in A1, A3 and A4.

D-H. . .A D(D-H) D(H. . .A) D(D. . .A) <(DHA)

A1
N(1)-H(1A). . .O(1)#1 0.90(2) 2.25(2) 3.1385(17) 167(2)
N(1)-H(1B). . .N(1)#2 0.87(2) 2.34(2) 3.2042(13) 168(2)
A3
N(1)-H(1A). . .O(1)#1 0.92(2) 2.47(2) 3.3485(18) 159(2)
N(1)-H(1B). . .N(1)#2 0.93(3) 2.49(3) 3.3878(13) 163.9(19)
A4
N(1)-H(1A). . .O(1)#1 0.93(2) 2.20(2) 3.1254(19) 171.4(18)
N(1)-H(1B). . .N(1)#2 0.90(3) 2.37(3) 3.2617(15) 166.6(18)
230 M. Shabbir et al. / Bioorganic Chemistry 75 (2017) 224–234

Fig. 5. Cyclic voltammograms of compounds A1- A4 (1 mM each) recorded at GCE in their argon saturated DMSO/H2O (9:1) + 0.1M TBATFB solution at 100 m Vs
1 scan rate at
25°C.

Fig. 6. Plots of ip vs. square root of scan rate for the determination of values of diffusion coefficient for A1- A4.

Fig. 7. Square wave voltammograms of compounds A1 (A), A2 (B), A3 (C) and A4 (D) (1 mM each) recorded at GCE in argon saturated DMSO/H2O (9:1) + 0.1M TBAP solution at
25°C, Esw = 5 mV frequency = 10 Hz. Pulse amplitude = 25 mV. The symbols are; It– total current, If – forward current, Ib –backward current.
 M. Shabbir et al. / Bioorganic Chemistry 75 (2017) 224–234 231

Fig. 8. Differential pulse voltammograms of the aromatic amines with 1 mM each recorded at GCE in argon saturated DMSO/H2O (9:1) + 0.1M TBATFB solution at 25 °C.

Fig. 9. Cyclic voltammograms of 2 mM aqueous-DMSO (1: 9) of A1(A), A2 (B), A3 (C) and A4(D)(─) without DNA, ( ) in the presence of 1.32 mM DNA, ( )2.62 mM DNA and
( )3.90 mM DNA on glassy carbon electrode at scan rate of 100 mV/s.

2.2.2. Differential pulse voltammetry
DP voltammograms of the studied aromatic amines are pre-
sented in Fig. 8. Peak currents are in the same order i.e. A1 > A4 >
A2 > A3 as detected in CV studies. The order is in accordance to
the Do values. The facile ET process in A1 is supportive from its sim-
ple and planner structure. On the other hand, sluggish ET process
in A3 might be attributed to structural effects which cause a hin-
drance to the electrooxidation. The observed W1/2 values (200
mV) of all the four aromatic amines suggests more than ET process
and involvement of intermediates as a close look indicates two
peaks partially superimposed on each other. So, it can be inferred
that one electron is involved in the oxidation process, although
the value is quite larger than the theoretical value of 90 mV for
electron process involving one electron and might be due to
uncompensated solution resistance.
2.3. DNA binding study
2.3.1. DNA binding study by cyclic voltammetry
Cyclic voltammetry is an excellent tool to study the drug-DNA
interaction behavior and here CV has been used to analyse the
DNA binding response of aromatic amines. The CVs of compounds
(A1- A4) have been recorded for blank compounds and along with
different amounts of DNA (1.32, 2.62 and 3.90 mM). The shift in val-
ues of peak potentials and peak currents of the compounds (Fig. 9)
are indicative of compound-DNA adduct formation. It has been
noticed for all the compounds that with increasing concentration
of DNA, there is slight shift in peak position towards more positive
potential. However, there is decrease in the peak current values for
all the compounds with increasing concentration of DNA. This
observed behavior indicates that aromatic amines are interacting
with DNA through its grooves. The magnitudes of diffusion coeffi-
cients for free compounds and compound-DNA adduct have been
calculated using the Eq. (1). The values of binding constant (K)
were calculated by following equation:
1=½DNA ¼ Kð1
AÞ=ð1
I=I0 Þ
K ð2Þ
where A is an empirical constant, K is binding constant, Io is peak
current for free compound and I is peak current for compound-
DNA adduct.
The number of binding sites (s) have been calculated from
equation:
232 M. Shabbir et al. / Bioorganic Chemistry 75 (2017) 224–234

Table 6
The drug-DNA interaction electrochemical parameters of compounds on glassy carbon electrode vs. Ag/AgCl in aqueous DMSO (1:9) solution at 50 m Vs
1 scan rate at 25°C.

Compound Do(cm2s
1)(without DNA) Do(cm2s
1)(with DNA) K (M
1) s(bp)

5
5 4
A1 2.78  10 1.72  10 8.01  10 0.32
A2 3.16  10
6 6.49  10
7 3.66  103 0.01
A3 1.50  10
8 1.12  10
8 2.38  104 0.05
A4 1.41  10
5 6.59  10
7 7.30  104 0.01

DNA: Deoxyribonucleic acid; Do: diffusion coefficient; K: complex stability constant; s: number of binding sites.

Fig. 10. UV–visible spectra of A1 (A) and A4 (B) recorded in the absence and presence of different concentrations of DNA and plots of A0/A-A0 vs. 1/ [DNA] for A1 (C) and A4 (D).

Cb =Cf ¼ Kf½DNA=2sg
where Cf and Cb are the concentrations of free compound and
compound-DNA adduct respectively. The Cb/Cf can be calculated
from Cb/Cf = (Io
I)/I [12,24,25].
The values of diffusion coefficient for all the free compounds are
greater than their respective compound-DNA adducts (Table 6) and
are attributed to slow diffusing property of compound-DNA
adduct. Moreover, the values of binding constant (K) for aromatic
amines are in the range of 2.38  104–8.01  104, while number
of binding sites are in the range of 0.01–0.32.
2.3.2. DNA binding study by UV–visible spectroscopy
DNA interaction response of aromatic amines has also been
recorded by UV–visible spectroscopy. UV–visible absorption spec-
tra of all the amines were recorded for their fixed concentration
(1.0  10
4 M) in the absence and presence of different concentra-
tions of DNA and representative spectra of A1 and A4 are presented
in Fig. 10(A and B) respectively. It has been observed for all the
ð3Þ aromatic amines that with increase in concentration of DNA, inten-
sity of absorption band also increases which is indicative of groove
binding interaction [26] and is according to the DNA binding
results obtained from CV studies.
The values of binding constant for all the aromatic amines from
UV–visible spectroscopic data have been calculated from the fol-
lowing well known Benesi-Hildebrand equation [27]
A0 eG eG 1
¼ þ  ð4Þ
A
A0 eH
G
eG eH
G
eG K½DNA
where k is binding constant, A0 is absorbance of compound (drug) in
the absence of DNA, A is absorbance of drug-DNA adduct, eG is
molar extinction coefficient of free compound and eH-G is
molar extinction coefficient of drug-DNA adduct. The values of
binding constant for all aromatic amines have been calculated by
slope values of A0/A-A0 vs. 1/[DNA] plots and are found almost
same calculated from CV data. The values of K are in the order of
A1 > A4 > A2 > A3.
 M. Shabbir et al. / Bioorganic Chemistry 75 (2017) 224–234
3. Conclusions
Four aromatic amines 1-amino-4-phenoxybenzene (A1), 4-(4-
aminophenyloxy) biphenyl (A2), 1-(4-aminophenoxy) naphthalene
(A3) and 2-(4-aminophenoxy) naphthalene (A4) were synthesized
and characterized by elemental, spectroscopic (FTIR, NMR), mass
spectrometric and single crystal X-ray diffraction methods. The
compounds crystallized in monoclinic crystal system with space
group P21. Intermolecular hydrogen bonds were observed between
the amine group and amine/ether acceptors of neighboring mole-
cules. Electrochemical study revealed the irreversible oxidation
process involving one electron. DNA binding studies (CV &UV–vis-
ible) suggest that aromatic amines interact with DNA via groove
binding.
4. Experimental
4.1. Chemistry
The nitroaromatics (1-nitro-4-phenoxybenzene, 4–4(nitro-
phenyloxy) biphenyl, 1-(4-nitrophenoxy) naphthalene and 2-
(4nitrophenoxy) naphthalene) employed for the synthesis of aro-
matic amines are already reported in our earlier report [12]. Pd/C
(5%) and hydrazine monohydrate were purchased from aldrich
and used as such. Melting points were determined using Gallen
Kamp apparatus. Infrared measurements (4000–400 cm
1) were
performed on thermoscientific NICOLET 6700 FTIR spectropho-
tometer. Multinuclear (1H and 13C NMR) spectra were recorded
in solution on Bruker ARX 300 MHz using tetramethylsilane
(TMS) as internal reference. GC-MS spectra were recorded in
methanol on GC 6890 N with MS 5973 in presence of helium gas.
Single crystal X-ray data was collected on a Bruker Apex II CCD
diffractometer. Electrochemical studies (cyclic voltammetry (CV),
differential pulse voltammetry (DPV) and square wave voltamme-
try (SWV) were carried out using Eco Chemie Autolab PGSTAT 302
potentiostat/galvanostat operated through GPES 4.9 software
(Utrecht, The Netherlands). Details are given in our earlier report
[12]. UV–visible spectra were recorded with a UV–visible Spec-
trometer Lambda 35 (Perkin Elmer) in the range 200–400 nm.
Solutions were prepared in UV–grade ethanol.
4.2. General procedure for the synthesis of aromatic amines
The synthetic details followed are reported earlier in our paper
[10].
4.2.1. 1-amino-4-phenoxybenzene (A1)
1-amino-4-phenoxybenzene (AI) was synthesized by using 2.00
g (9.30 mmol) 1-nitro-4-phenoxybenzene, 5.00 mL hydrazine
monohydrate and 0.05 g Pd/C.
Color: white, Yield 79%, m. p. 83 °C. FTIR:(t /cm
1) 3391 (asym)
3315(sym) (NAH), 1597(aromatic C@C), 1225 (CAOAC), 1H NMR
0 0
(300 MHz, CDCl3, d ppm): 7.35 (m, 2H, H7,7 ), 7.05 (m, 3H, H6,6 ,8),
3,30 2,20
6.85 (d, 2H, H , J = 8.9 Hz), 6.54 (d, 2H, H , J = 9.2 Hz) (H ortho
to -NH2) 4.45(2H, s, NH2), 13C NMR (75 MHz, CDCl3, d ppm):
159.44–116.85(12 aromatic carbons), MS (m/z): 185(M +) CHN
found (calcd.) for C12H11NO: C: 77.02 (77.84), H: 5.87 (5.95), N:
5.83 (5.75).
4.2.2. 4–4(aminophenyloxy) biphenyl (A2)
4–4(aminophenyloxy) biphenyl (A2) was prepared by taking
2.00 g 4-(4-nitrophenyloxy) biphenyl (7.66 mmol), 5.00 mL hydra-
zine monohydrate and 0.050 g Pd/C.
Color: white, Yield 88%, m. p. 103 °C. FTIR:(t /cm
1)3468 (asym)
3372 (sym) (NAH), 1611 (aromatic C@C), 1232 (CAOAC), 1H NMR
233
0 0
(300 MHz, CDCl3, d ppm): 7.65 (m, 2H, H10,10 ), 7.57 (m, 2H, H7,7 ),
0 0
7.48 (m, 2H, H11,11 ), 7.35 (m, 1H, HH12), 7.17 (m, 2H, H6,6 ), 6.92 (d,
3,30 2,20
2H, H , J = 8.7 Hz), 6.67 (d, 2H, H , J = 9.0 Hz) (H ortho to ANH2)
4.53(2H, s, NH2), 13C NMR (75 MHz, CDCl3, d ppm): 163.10–118.46
(18 aromatic carbons), MS (m/z): 261 (M +) CHN found (calcd.) for
C18H15NO: C: 82.23 (82.76), H: 5.45 (5.74), N: 5.42 (5.36).
4.2.3. 1-(4-aminophenoxy) naphthalene (A3)
1-(4-aminophenoxy) naphthalene (A3) was manufactured by
taking 2.00 g (6.94 mmol) 1-(4-nitrophenoxy) naphthalene, 5.00
mL hydrazine monohydrate and 0.05 g Pd/C.
Color: light brown, Yield 74%, m. p. 55 °C, FTIR:(t/cm
1) 3410
(asym) 3327(sym) (NAH), 1592 (aromatic C@C), 1242 (CAOAC),
1
H NMR (300 MHz, CDCl3, d ppm): 7.95(m, 1H, H8), 7.65(m, 1H,
0
H5), 7.28 (m, 3H, H4,6,7), 7.12 (m, 1H, H3), 6.74 (m, 3H, H2,12,12 ),
13,130
6.50 (d, 2H, H , J = 8.8 Hz) (H ortho to ANH2),4.31 (2H, s,
NH2), 13C NMR (75 MHz, CDCl3, d ppm): 155.25–110.10 (16 aro-
matic carbons), MS (m/z): 235(M +) CHN found (calcd.) for
C16H13NO: C: 81.84 (81.07), H: 5.59 (5.53), N: 5.94 (5.96).
4.2.4. 2-(4-aminophenoxy) naphthalene (A4)
2-(4-aminophenoxy) naphthalene (A4) was prepared by using
2.00 g (6.94 mmol) 2-(4-nitrophenoxy) naphthalene, 5.00 mL
hydrazine monohydrate and 0.05 g Pd/C.
Color: reddish brown, Yield 75%, m. p. 116 °C. FTIR:(t /cm
1)
3393 (asym) 3323(sym) (NAH), 1622 (aromatic C@C), 1232
(CAOAC), 1H NMR (300 MHz, CDCl3, d ppm): 7.53 (m, 2H, H1,8),
7.45 (m, 1H, H5), 7.25 (m, 2H, H6,7), 6.89 (m, 2H, H2,4), 6.62 (m,
0 0
2H, H12,12 ), 6.45 (d, 2H, H13,13 , J = 8.8 Hz) (H ortho to ANH2),
13
4.54 (2H, s, NH2); C NMR (75 MHz, CDCl3, d ppm): 157.49–
110.85 (16 aromatic carbons). MS (m/z): 235(M +) CHN found
(calcd.) for C16H13NO: C: 81.44 (81.07), H: 5.51 (5.53), N: 5.92
(5.96).
Declaration of interest
The authors have declared no conflict of interest.
Acknowledgements
The authors are highly grateful to Chemistry departments of
Quaid-I-Azam University Islamabad, Pakistan, Institut für Anorgan-
ische Chemie, J.W. Goethe-Universität Frankfurt, Germany, School
of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9,
Ireland for providing technical support and laboratory facilities.
Appendix A. Supplementary material
CCDC 1538782, 782134 and 782132 for A1, A3 and A4 respec-
tively contain the supplementary crystallographic data for this
paper. These data can be obtained free of charge from The Cam-
bridge Crystallographic Data Centre via www.ccdc.cam.ac.
uk/data_request/cif. Supplementary data associated with this arti-
cle can be found, in the online version, at https://doi.org/10.1016/j.
bioorg.2017.10.002.
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