Synthesis, structural characterization, DNA binding and antioxidant potency of new ferrocene incorporated acyl ureas

Article abstract of DOI:10.1016/j.jorganchem.2015.08.010

In this article, we have reported the synthesis of three new ferrocene incorporated ureas namely; 1-(4-Chlorobenzoyl)-3-(4-ferrocenylphenyl)urea (P4Cl), 1-(3-Chlorobenzoyl)-3-(4-ferrocenyl phenyl)urea (P3Cl) and 1-(2-Chlorobenzoyl)-3-(4-ferrocenylphenyl)urea (P2Cl). All new compounds were unambiguously characterized by common analytical techniques (NMR, FT-IR, AAS, CHNS and molecular docking). Furthermore, single crystal XRD analysis was done for 1-(3-Chlorobenzoyl)-3-(4-ferrocenylphenyl)urea. DNA binding is a pre-requisite for a compound to be used as an antitumor agent. The DNA binding study was done by cyclic voltammetry, UV-vis spectroscopy and viscometry. The drug-DNA binding constant was found to vary in the sequence: K_P2Cl (6.056 × 10⁴ M^-1) > K_P4Cl (5.713 × 10⁴ M^-1) > K_P3Cl (4.631 × 10⁴ M^-1). Diffusion coefficient of the drug-DNA adduct for all the compounds is lower than the free drug, indicating that drug-DNA adduct is of higher molecular weight and slow diffusing as compared to the free drug. Small binding site size of 0.440 (P3Cl), 0.585 (P4Cl) and 0.673 (P2Cl) base pairs is consistent with electrostatic interaction. All the compounds exhibited good antioxidant activities with IC₅₀ values of 82, 136 and 54 μM for P4Cl, P3Cl and P2Cl respectively, against DPPH.

Full text of DOI:10.1016/j.jorganchem.2015.08.010

Accepted Manuscript
Synthesis, structural characterization, DNA binding and antioxidant potency of new
ferrocene incorporated acyl ureas
Faiza Asghar, Amin Badshah, Raja Azadar Hussain, Manzar Sohail, Kamran Akbar,
Ian Sydney Butler
PII:
S0022-328X(15)30113-3
10.1016/j.jorganchem.2015.08.010
JOM 19198
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 3 April 2015
Revised Date: 5 August 2015
Accepted Date: 16 August 2015
Please cite this article as: F. Asghar, A. Badshah, R.A. Hussain, M. Sohail, K. Akbar, I.S.
Butler, Synthesis, structural characterization, DNA binding and antioxidant potency of new
ferrocene incorporated acyl ureas, Journal of Organometallic Chemistry (2015), doi: 10.1016/
j.jorganchem.2015.08.010.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, structural characterization, DNA binding and antioxidant potency of new
ferrocene incorporated acyl ureas
a,b
a
a
c
Faiza Asghar , Amin Badshah *, Raja Azadar Hussain , Manzar Sohail , Kamran
a
b
Akbar Ian Sydney Butler
,
a*. Corresponding Author: Coordination Chemistry Laboratory, Department of Chemistry,
Quaid- i-Azam University (45320) Islamabad, Pakistan. aminbadshah@yahoo.com
a. Coordination Chemistry Laboratory, Department of Chemistry, Quaid-i-Azam University
(45320) Islamabad, Pakistan.
b. Department of Chemistry, McGill University, Montreal, QC, Canada H3A 2K6
c. Faculty of Science, Health, Education and Engineering ML12, University of the Sunshine
Coast, Australia.
Abstract:
In this article, we have reported the synthesis of three new ferrocene incorporated ureas namely;
1
-(4-Chlorobenzoyl)-3-(4-ferrocenylphenyl)urea (P4Cl), 1-(3-Chlorobenzoyl)-3-(4-ferrocenyl
phenyl)urea (P3Cl) and 1-(2-Chlorobenzoyl)-3-(4-ferrocenylphenyl)urea (P2Cl). All new
compounds were unambiguously characterized by common analytical techniques (NMR, FT-IR,
AAS, CHNS and molecular docking). Furthermore, single crystal XRD analysis was done for 1-
(3-Chlorobenzoyl)-3-(4-ferrocenylphenyl)urea. DNA binding is a pre-requisite for a compound
to be used as an antitumor agent. The DNA binding study was done by cyclic voltammetry, UV-
vis spectroscopy and viscometry. The drug-DNA binding constant was found to vary in the
4
-1
4
-1
4
-1
sequence: KP2Cl (6.056 x 10 M ) > K
(5.713 x 10 M ) > K
(4.631 x 10 M ). Diffusion
P4Cl
P3Cl
coefficient of the drug-DNA adduct for all the compounds is lower than the free drug, indicating
that drug-DNA adduct is of higher molecular weight and slow diffusing as compared to the free
drug. Small binding site size of 0.440 (P3Cl), 0.585 (P4Cl) and 0.673 (P2Cl) base pairs is
consistent with electrostatic interaction. All the compounds exhibited good antioxidant activities
with IC values of 82, 136 and 54 ꢀM for P4Cl, P3Cl and P2Cl respectively, against DPPH.
5
0
Key words: DNA binding constant, diffusion coefficients, desulphurization, binding site size.
1
.
Introduction
In the area of tumor treatment, interaction of different possible drugs with DNA is
improving day by day. DNA is an important biological molecule which contains all the genetic
1

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information for the proper functions of the cells. Drug-DNA interactions may be categorized as:
a) electrostatic, b) groove binding and c) intercalation. It is believed that if we are able to have a
check on the uncontrolled proliferation of the cells through any of the above mentioned
interactions with DNA then we will be able to control the cancer [1]. Cisplatin is a potent
antineoplastic agent but has some negative side effects. In search of the compounds which
should be active like cisplatin but with minimal or no side effects, different classes of the
compounds have been under investigation. In this struggle tremendous attention has been given
to the ferrocene and its derivatives. Ferrocene [2], nitrophenyl ferrocene [3], antimony
derivatives of ferrocene [1] and thiourea [4] and selenourea derivatives of ferrocene [5-7] have
been evaluated for their interactions with DNA. It has been observed that almost all the
derivatives of ferrocene have higher drug-DNA binding constant than simple positively charged
2
-1
ferrocene moiety (3.45 x 10 M ) although when these derivatives are not coupled with
ferrocene then they are themselves less active for DNA. Therefore it is need of the time to
explore such derivatives which may orient themselves with DNA in such a way that they can
control the proliferation of infected cells. Generally ferrocene derivatives interact
electrostatically with DNA but partial intercalation also have been reported [4]. Use of ferrocene
moiety has multifold advantages in term of its anticancer, antibiotic and antimalarial activities
[8]. Moreover, it enhances the lipophilicity and is a beautiful electrochemical marker during
cyclic voltammetric studies. Without the use of ferrocene it is difficult to study the DNA binding
of ureas with cyclic voltammetry as their electrochemical signals are very weak. Different
techniques (UV-vis, fluorescence, luminescence, laser light scattering) have been used for the
evaluation of drug-DNA binding constant but electrochemical methods are cost effective, highly
sensitive and have fast detection [2].
On the other hand, ureas have huge applications as antiproleferative agents [9], anticancer
agents (renal cancer, colon cancer, lungs cancer, prostate cancer and breast cancer) [10], anti
platelets aggregatives, antifungal and antibacterial agents [11]. Inspired from the applications of
ferrocenes and ureas separately we have combined them in a new class i.e. ferrocene
incorporated ureas. We have presented in this paper, synthesis, structural characterization and
DNA binding study of three new ferrocene based ureas. Single crystal X-ray structure for P3Cl is
1
also provided. All the compounds have been completely characterized with multinuclear ( H and
1
3
C) NMR, FT-IR, elemental analysis and atomic absorption spectrophotometry (AAS). Drug-
2

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DNA binding has been determined with cyclic voltammetry (CV) and mode of interaction has
further been confirmed with UV-vis spectroscopy, molecular docking and viscometry. We have
also reported their antioxidant activity in addition.
2
.
Expermental
2
.1
Materials and Methods
Melting points were determined in a capillary tube using electro-thermal melting point
apparatus model MP-D Mitamura Riken Kogyo (Japan). Infrared spectra were taken on
1
13
Thermoscientific NICOLET 6700 FTIR. H and C NMR measurements were recorded on a
Bruker AV500 MHz spectrometer in DMSO. Si(CH ) was used as internal reference. The
3
4
elemental analysis were performed using a LECO-932 CHNS analyzer while the Fe
concentrations were determined on an Atomic Absorption Spectrophotometer Perkin Elmer
2
380.
Commercial salmon DNA was solubalized in doubly distilled water to prepare a stock
-4
solution of 6 x 10 M from which working concentrations of DNA were prepared. Concentration
-
of stock solution was measured by UV absorbance at 260 nm using an epsilon value of 6600 M
1
-1
cm . This DNA was protein free because A260/A280 > 1.8.
Cyclic voltammetry was performed on Biologic SP-300 cyclic voltammeter running with
EC-Lab Express V 5.40 software, Japan. Before every reading working electrode was polished
with alumina powder and rinsed with distilled water. Analytical grade TBAP
(Tertiarybutylammonium perchlorate) was used as supporting electrolyte and nitrogen gas (99.9
%) was purged through the mixture to avoid interference of oxygen.
The Ubbelohde viscometer was used for viscosity measurements at room temperature
(25 ± 1 ͦC ). Flow time was measured with a digital stopwatch. Flow time measurements were
made in triplicate for the measurement of average flow time. Data were presented as relative
specific viscosity (η/η ), vs. binding ratio ([Drug]/[DNA]) where η is the viscosity of DNA in the
o
presence of complex and η is the viscosity of DNA alone.
o
Docking studies were carried out using Autodock (Version 4.2) docking software
[
12]. Structure of B-DNA dodecamer d(CGCGAATTCGCG) (1BNA) was taken from
2
3

ACCEPTED MANUSCRIPT
protein data bank (PDB) [13] while Crystallographic Information File of P3Cl was used as
ligand for subsequent docking. Essential hydrogen atoms and Gasteiger charges were added
with the aid of Auto-Dock tools (ADT). The grid size was set to 64, 64 and 116 along the x, y
and z-axes respectively. The center of the grid was set to 14.98, 20.976 and 8.807. After DNA
was enclosed in the grid defined with 0.375 Å spacing, the grid map was calculated using the
AutoGrid program. Docking to macromolecule was performed using an empirical free energy
function and Lamarckian Genetic Algorithm, with an initial population of 50 randomly placed
5
individuals, a maximum number of 2 x 10 energy evaluations, a mutation rate of 0.02, and a
crossover rate of 0.80 were given. P3Cl molecule was allowed to move within a specified
region to achieve the lowest energy conformation while B-DNA dodecamer was kept
rigid during docking.
The reducing abilities of P4Cl, P3Cl and P2Cl were determined with the help of 1,1-
diphenyl-2-picrylhydrazyl in DMSO to produce 1,1-diphenyl-2-picrylhydrazine. Decrease in the
absorption of 1,1-diphenyl-2-picrylhydrazyl (DPPH) was monitored to calculate % age
scavenging according to the following formula [7, 14]:
Scavenging Activity (%) = Aₒ-A/Aₒ x 100
Where Aₒ is the absorbance of free DPPH and A is the absorption of DPPH-drug mixture with
increasing concentration of drug. To a solution of DPPH (3.9 mg of DPPH in 100 mL DMSO)
were added the increasing concentrations (27 ꢀM) of drug. The decrease in absorption of DPPH
was monitored after 30 minutes at wavelength of 517 nm with the help of spectrophotometer. All
the readings were taken in triplicate and average of all the readings were used.
Ferrocene, Paranitro aniline, sodium nitrite, diethyl ether, acetone, DMSO, Pd-charcoal,
hydrazine, KSCN, NaOH, HgCl and acid chlorides such as 4-chlorobenzoyl chloride, 3-
2
chlorobenzoyl chloride and 2-chlorobenzoyl chloride were obtained from commercial sources
(
Sigma Aldrich/Fluka) and used as received. Para ferrocenyl aniline and ferrocene incorporated
N, N-disubstituted thioureas were synthesized by a procedure reported by our group previously
Part 1 Scheme 1) [4, 15].
(
2
.2
General procedure for synthesis of ferrocene incorporated ureas
To the solution of ferrocene incorporated N, N-disubsituted thioureas in 20 ml DMF,
mercuric chloride was introduced in 1:1 molar ratio. The reaction mixture was stirred for 30 min,
4

ACCEPTED MANUSCRIPT
afterwards 3 ml of 100 mM NaOH(aq) was added dropwise with constant magnetic stirring and
the suspension was allowed to reflux for about 8 hrs. Progress of the reaction was examined by
thin layer chromatography (TLC). On completion of the reaction black precipitates of HgS were
filtered off and the filtrate was then poured into ice cold water and stirred well in order to remove
water soluble impurities. Solid product was separated by filtration, washed with deionized water
and recrystallized from ethanol (Scheme 1).
2
.2.1 1-(4-Chlorobenzoyl)-3-(4-ferrocenylphenyl)urea (P4C1)
Quantities used were 0.5 g (0.00105 mol) 1-(4-Chlorobenzoyl)-3-(4-ferrocenylphenyl)
thiourea, 0.29 g (0.00105 mol) HgCl and 3 ml of 100 mM NaOH(aq). Yield 74 %; Yellow solid;
2
-1
m.p. 210
C=C), 484 (Fe-Cp); H NMR (500 MHz, DMSO-d , ppm) δ 11.20 (s, 1H, NH), 10.81 (s, 1H,
NH), 7.94 (d, 2H, ArH), 7.38 (d, 4H, ArH), 7.09 (d, 2H, ArH), 4.70 (s, 2H, C ), 4.29 (s, 2H,
C H ), 4.02 (s, 5H, C H ); C NMR (125.81 MHz, DMSO-d , ppm) δ 167.2, 160.5, 137.9,
ͦC ; FT-IR (powder, cm ): 3232 (N-H), 3035 (C-Haromatic), 1696 (C=O), 1474-1586
1
6
(
5
H
4
13
6
5
4
5
5
1
32.9, 131.1, 130.8, 129.1, 128.9, 127.6, 122.4, 84.9, 69.7, 69.2, 66.4; Elemental Analysis Calc.
(
%) for C24 : C, 62.83; H, 4.18; N, 6.10; Fe, 12.17. Found (%): C, 62.78; H, 4.12; N,
2 2
H19ClFeN O
6
.16; Fe, 12.26.
2
.2.2 1-(3-chlorobenzoyl)-3-(4-ferrocenylphenyl)urea (P3Cl)
Quantities used were 0.5 g (0.00105 mol) 1-(3-Chlorobenzoyl)-3-(4-ferrocenylphenyl)
thiourea, 0.29 g (0.00105 mol) HgCl and 3 ml of 100 mM NaOH(aq). Yield 71 %; Brown solid;
2
-1
m.p. 185
458-1592 (C=C), 483 (Fe-Cp); H NMR (500 MHz, DMSO-d , ppm) δ 11.12 (s.1H, NH), 10.68
s,1H, NH), 8.09 (s, 1H, ArH), 7.98 (d, 2H, J = 7.5 Hz, ArH), 7.74 (d, 2H, J = 8.0 Hz, ArH),
ͦC (decompose); FT-IR (powder, cm ): 3249 (N-H), 3082 (C-Haromatic), 1694 (C=O),
1
6
1
(
1
3
7
5 4 5 4 5 5
.61-7.37 (m, 3H, ArH), 4.76 (s, 2H, C H ), 4.33 (s, 2H, C H ), 4.08 (s, 5H, C H ); C NMR
6
(
125.81 MHz, DMSO-d , ppm) δ 167.8, 161.3, 135.8, 134.4, 133.8, 131.0, 128.6, 127.5, 126.8,
1
4
20.4, 86.8, 69.8, 69.2, 66.5; Elemental Analysis Calc. (%) for C H ClFeN O : C, 62.83; H,
24 19 2 2
.18; N, 6.10; Fe, 12.17. Found (%): C, 62.86; H, 4.14; N, 6.13; Fe, 12.19.
2
.2.3 1-(2-Chlorobenzoyl)-3-(4-ferrocenylphenyl)urea (P2C1)
5

ACCEPTED MANUSCRIPT
Quantities used were 0.5 g (0.00105 mol) 1-(2-Chlorobenzoyl)-3-(4-ferrocenylphenyl)
thiourea, 0.29 g (0.00105 mol) HgCl and 3 ml of 100 mM NaOH(aq). Yield 66 %; Orange solid;
2
-1
m.p. 198
ͦC ; FT-IR (powder, cm ): 3243 (N-H), 3089 (C-Haromatic), 1688 (C=O), 1471-1600
1
6
(
C=C), 482 (Fe-Cp); H NMR (500 MHz, DMSO-d , ppm) δ 11.21 (s, 1H, NH), 10.41 (s, 1H,
13
NH), 7.63-7.46 (m, 8H, ArH), 4.77 (s, 2H, C H ), 4.33 (s, 2H, C H ), 4.03 (s, 5H, C H ); C
5
4
5
4
5
5
6
NMR (125.81 MHz, DMSO-d , ppm) δ 171.2, 160.6, 135.8, 135.0, 132.4, 130.2, 129.6, 127.7,
26.8, 120.4, 83.4, 69.8, 69.2, 66.5; Elemental Analysis Cal. (%) for C H ClFeN O : C, 62.83;
1
2
4
19
2
2
H, 4.18; N, 6.10; Fe, 12.17. Found (%): C, 62.79; H, 4.21; N, 6.08; Fe, 12.20.
3
.
Results and Discussion
3
.1
Chemistry
Treatment of ferrocene based thioureas with alkaline mercuric chloride in DMF yielded
the respective ureas. The targeted compounds were synthesized by the replacement of sulfur with
2
+
oxygen, [16] in the presence of alkaline Hg as sulfur capturing agent that yielded ureas in good
-
yield. NaOH was used as an alkali which provides OH anion that attacks on thio-carbon of
thiourea. The proposed mechanism similar to that of guanylation is shown in Scheme 3 [17].
3
.2
Spectroscopic Analysis
In the FT-IR spectra of synthesized compounds the two –NH protons gave a broad signal
-1
between 3250-3228 cm owing to intra and intermolecular hydrogen bonding. The stretch due to
-1
Aromatic-H’s was evident just above 3000 cm
,
while the carbonyl group appeared as an intense
-1
band at 1696–1688 cm . All the compounds (P4Cl, P3Cl and P2Cl) have basically three types of
1
protons i.e. protons of ferrocene, protons of –NH and aromatic protons. In the H NMR spectra,
all the compounds displayed two singlets for the two –NH protons. –NH proton which is present
between two carbonyl carbons is maximum deshielded and provides a singlet at ~11 ppm. The
second –NH which is attached to the phenyl ring is comparatively less deshielded, therefore it
appears as a singlet at ~10 ppm. Five protons of unsubstituted cyclopentadiene (Cp) ring of
ferrocene yielded an intense signal at ~4 ppm and substituted Cp provided two singlets
downfield from the singlet of unsubstituted Cp ring. Aromatic protons were visible between 8-7
1
3
ppm. In C NMR maximum downfield carbon was between the two –NH groups with a value of
1
71-167 ppm. The other carbonyl carbon appeared at ~161 ppm because it was comparatively
6

ACCEPTED MANUSCRIPT
less deshielded. The Aromatic-C appears between 143-117 ppm. Unsubstituted Cp carbons of
ferrocene gave an intense singlet whereas substituted Cp provided three peaks i.e. the ipso
carbon appeared between 87-83 ppm and the other two signals were apparent between 70-66
ppm.
Elemental analysis of all the compounds showed that the calculated and found values for
carbon, hydrogen, nitrogen and iron are in close agreement with each other. The carbon,
hydrogen and nitrogen percentages were determined with a CHNS analyser and that of Fe by
AAS.
3
.3
Crystallography
Diffraction data of P3Cl were collected at 100 (2) K on beamline MX1 at the Australian
Synchrotron (λ = 0.71703 Å) [18]. The data reduction and indexing of diffraction pattern was
performed by XDS software [19]. The structures were solved by direct methods and refined by
2
full-matrix least squares against F of data using SHELXL97 (Sheldrick, 1997) software. All
non-hydrogen atoms were refined with anisotropic displacement parameters. Basic crystal data
and description of diffraction experiment are given in Table 1. ORTEP of P3Cl is given in Figure
1
a whereas selected bond lengths and bond angles have been given in Table 2. The structure
reveals that the phenyl ring attached with Cp of ferrocene is not exactly in plane with Cp moiety
and is tilted at an angle of 30 whereas terminal chloro substitutes phenyl ring is at an angle of
3.37 from the plane of Cp moiety. Hydrogens of the ferrocene are staggered and O (1) has an
ͦ
5
ͦ
intramolecular hydrogen bond with the H (2) whereas intermolecular hydrogen bond is between
O (2) of one molecule and H (1) of the other molecule (Figure 1b). A partial interaction between
H (4) and O (2) is also visible in the structure (Figure 1b). Packing of the molecule reveals that it
exists as a monomer and ferrocene moieties of two neighboring molecules are at a maximum
distance from each other with trans orientation (Figure 1c).
3
.4
Cyclic Voltammetry
Cyclic volammetric studies were carried out with a setup having three electrodes system
2
-1
i.e. working (platinum disc electrode with a geometric area of 0.071 cm s ), reference (saturated
calomel electrode i.e. SCE) and auxiliary electrodes (platinum electrode with geometric area
much greater than working electrode). Changes in the peak current provided information about
7

ACCEPTED MANUSCRIPT
DNA binding constant whereas the variation in the peak potential were useful for the
determination of mode of interaction of drug with DNA. Drug-DNA binding constant was
determined with the help of following equation [20]:
log (1/[DNA]) = log K + log (I/I
o
- I)
Eq 1
o
Where K is the binding constant and I and I are the peak currents of free drug and DNA bound
drug respectively. Binding site size was calculated with the help of following equation [21]:
C /C = K[free base pairs]/s
Eq 2
b
f
Where s is the binding site size in terms of base pair, K is the binding constant, C
b
is the
concentration of free species and C represents concentration of drug-DNA bound species. If we
f
consider the concentration of DNA in terms of nucleotide phosphate, then the concentration of
DNA base pairs will be equal to [DNA]/2 and Eq 2 will be written as:
C /C = K[DNA]/2s
Eq 3
b
f
and the value of C /C is equal [19] to (I - I/I) which are the values of experimental peak
b
f
o
currents. The diffusion coefficient of free drug and DNA-bound drug provides best information
about the molecular mass of drug-DNA adduct. Following form of Randles-Sevcik Equation [22,
2
3] was used for the values of diffusion coefficients:
5
3/2
*
o
1/2 1/2
I = 2.99 x 10 n A C D
o
v
Eq 4
pa
*
o
-3
Where Ipa is the anodic peak current C
is the reductant’s concentration in molcm , A is the
2
geometric area of the electrode in cm , n is the number of electrons involved in the process, D is
o
2
-1
the diffusion coefficient in cm s .
Consistency of voltage at different scan rates by a plot of current (mA) vs. potentials (E/V
vs. SCE) for all the three compounds i.e P3Cl, P4Cl and P2Cl (Figure 2a) favours 100 %
reversible electrochemical process. P3Cl provides a couple of well defined redox peaks with an
oxidation maxima at 0.522 V and a reduction maxima at 0.404 V. For the calculation of drug-
DNA binding constant, changes in oxidation peak were monitored with successive addition of 2-
8
ꢀM DNA solutions (Figure 2b). With the addition of 2 ꢀM DNA in cyclic voltammetric cell
there is a 26 mV shift towards the negative potential with a decrease in the peak current. This
8

ACCEPTED MANUSCRIPT
negative shift in the peak potential conforms electrostatic mode of interaction of positively
charged P3Cl with a negatively charged phosphate backbone of the DNA. Decrease in peak
current with successive addition of DNA was used to determine the P3Cl-DNA binding constant
4
-1
of 4.631 x 10 M with the help of Eq 1 (Figure 2d). This value of binding constant is far better
2
-1
than protonated ferrocene (3.45 x 10 M ) and is comparabale with many of the recently
reported ferrocene derivatives (Scheme 2) which shows that urea moiety is playing its part in the
-7
2 -1
enhancement of binding constant. The diffusion coefficient of P3Cl-DNA is 6.47 x 10 cm s
-7
2 -1
which is far less than the diffusion coefficient of free P3Cl (7.44 x 10 cm s ). This indicates
that the drug-DNA adduct is of higher molecular mass and slow diffusing as compared to the
free drug (Figure 2c). Binding site of 0.440 bp was calculated with the help of Eq. 2 (Figure 2e).
This small value of binding site size is an evidence for electrostatic mode of interaction.
P4Cl also provides a couple of well defined redox peaks in the potential region of -0.2–
4
-1
0
.9 V which provided the DNA binding constant of 5.713 x 10 M (Figure 3a). A negative shift
in potential and a decrease in peak current on successive addition of DNA suggests an
electrostatic interaction of P4Cl with DNA. P4Cl-DNA adduct has a lower diffusion coefficient
-7
2 -1
-7
2 -1
(
4.42 x 10 cm s ) than the free P4Cl (6.55 x 10 cm s ), which is indicative for the formation
of slow diffusing P4Cl-DNA adduct.
4
-1
P2Cl also behaves similar to P3Cl and P4Cl with binding constant of 6.056 x 10 M ,
which is less than P3Cl and P4Cl. As mentioned above P2Cl-DNA adduct has lower diffusion
-7
2 -1
-7
2 -1
coefficient (2.80 x 10 cm s ) than free P2Cl (5.47 x 10 cm s ). P2Cl also binds
electrostatically with DNA (Figure 3b). Binding site size varies in following order: P2Cl (0.673
bp) > P4Cl (0.585 bp) > P3Cl (0.440 bp), such small values of s confirms electrostatic
interaction.
3
.5
UV-vis Spectroscopy
The results obtained from CV were equally supported by UV-vis spectroscopy in which a
prominent hypochromism and a slight blue shift of the drug-DNA adducts relative to the free
4
drug confirms the electrostatic interactions (Figure 4a). A DNA binding constant of 5.004 x 10
-1
M for P3Cl is in very close agreement with the values obtained from CV (Figure 4b) [6].
3
.6 Viscometry
9

ACCEPTED MANUSCRIPT
Mode of interaction was further confirmed with the help of viscometry as well. Figure 5
shows representative plot of relative viscosity (η/η ) against [P3Cl]/[DNA] concentrations. P3Cl
o
shows a decrease in relative viscosity with increasing binding ratio which is a characteristic for
4
-1
electrostatic interactions [6]. DNA binding constant of 7.8752 x 10 M is in agreement with the
values of binding constant determined with CV.
3
.7
Molecular Docking
Figure 6a shows the representative docked conformation of P3Cl with DNA, having
lowest binding energy, suggested by Autodock while Figure 6b represents the surface view of
the same docked conformation. It is evident from Figure 6b that ferrocenyl moiety of the docked
P3Cl is in close contact with oxygen attached to sugar-phosphate backbone of DNA which in
turns suggest electrostatic force of interaction between Iron and oxygen of sugar-phosphate
backbone. Figure 6c shows the close-up view of the atoms of DNA which are interacting with
the surface of P3Cl and it can be seen that oxygen of sugars-phosphate back bone, present
between deoxyadenosine (DA)-18 and DA17, is interacting with ferrocenyl moiety,
electrostatically. It can also be seen that, there is also hydrogen bond present between one of the
oxygen of P3Cl and hydrogen attached to nitrogen of DA5 [6, 24, 25].
3
.8
DPPH Scavenging activity
DPPH provides a strong absorption band at 517 nm due to its odd electron. When any
antioxidant reacts with DPPH it produces 1,1-diphenyl-2-picrylhydrazine. As a result the band
intensity of DPPH decreases. This changes the color of DPPH and a corresponding decrease in
the absorption. Figure 7 shows a representative plot of absorbance versus wavelength for P3Cl.
IC50 values of 82, 136 and 54 ꢀM were determined for P3Cl, P4Cl and P2Cl respectively.
4
.
Conclusions
Synthesis of anticipated compounds was accomplished as a result of deprotection of
ferrocene incorporated thioureas to corresponding oxo analogue. Characterization by different
techniques confirms the formation of targeted ureas. Synthesized ferrocene incorporated ureas
show electrostatic mode of interaction with DNA and a decent binding constant value. Their
antioxidant activity is also reasonable with reference to ascorbic acid.
10

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Acknowledgements
Faiza Asghar is highly obliged to the Higher Education Commission (HEC) of Pakistan for
providing scholarship under the program 5000 Indigenous PhD Scholarships at Quaid-i-Azam
University in Islamabad and for a 6-month scholarship under the International Research Support
Initiative program (IRSIP) as a Graduate Research Trainee in the Department of Chemistry,
McGill University, Montreal, Quebec, Canada.
References
[1] F. Asghar, A. Badshah, A. Shah, M.K. Rauf, M.I. Ali, M.N. Tahir, E. Nosheen, R. Zia ur, R.
Qureshi, J. Organomet. Chem. 717 (2012) 1-8.
[
2] A. Shah, R. Qureshi, N.K. Janjua, S. Haque, S. Ahmad, Anal. Sci. 24 (2008) 1437-1441.
3] A. Shah, M. Zaheer, R. Qureshi, Z. Akhter, M. Faizan Nazar, Spectrochim. Acta, Part A:
[
Molecular and Biomolecular Spectroscopy, 75 (2010) 1082-1087.
4] B. Lal, A. Badshah, A.A. Altaf, M.N. Tahir, S. Ullah, F. Huq, Dalton Trans. 41 (2012)
4643-14650.
[
1
[5] R.A. Hussain, A. Badshah, M. Sohail, B. Lal, K. Akbar, J. Mol. Struct. 1048 (2013) 367-374.
[6] R.A. Hussain, A. Badshah, M.N. Tahir, B. Lal, I.A. Khan, Aust. J. Chem. 66 (2013) 626-634.
[7] R.A. Hussain, A. Badshah, M. Sohail, B. Lal, A.A. Altaf, Inorg. Chim. Acta. 402 (2013) 133-
1
39.
8] B. Lal, A. Badshah, A.A. Altaf, N. Khan, S. Ullah, Appl. Organomet. Chem. 25 (2011) 843-
55.
[
8
[
(
[
[
9] M. Radić Stojković, S. Marczi, L. Glavaš-Obrovac, I. Piantanida, Eur. J. Med. Chem. 45
2010) 3281-3292.
10] E.L. Luzina, A.V. Popov, Eur. J. Med. Chem. 45 (2010) 5507-5512.
11] P. Ventosa-Andrés, Á.M. Valdivielso, I. Pappos, M.T. García-López, N.E. Tsopanoglou, R.
Herranz, Eur. J. Med. Chem. 58 (2012) 98-111.
12] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J. Olson,
J. Comput. Chem. 30 (2009) 2785-2791.
13] H.R. Drew, R.M. Wing, T. Takano, C. Broka, S. Tanaka, K. Itakura, R.E. Dickerson,
Proceedings of the National Academy of Sciences, 78 (1981) 2179-2183.
14] (a) S. Zouari, M. Ketata, N. Boudhrioua, E. Ammar. Ind. Crop Prod. 41 (2013) 172-178;
[
[
[
11

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(b) A. Choudhary, R. Sharma, M. Nagar, M. Mohsin, H. Meena, J. Chil. Chem. Soc. 56 (2011)
9
11–917.
15] S. Ali, A. Badshah, A.A. Ataf, B. Lal, K.M. Khan, Med. Chem. Res. 22(7) (2013) 3154-
159.
[
3
[
[
[
[
16] S.G. Davies, A.A. Mortlock, Tetrahedron Lett. 32 (1991) 4791.
17] C. Levallet, J. Lerpiniere, S.Y. Ko, Tetrahedron. 53 (1997) 5291.
18] Analytical Chemistry, 61 (1989) 1309A-1309A.
19] T.M. McPhillips, S.E. McPhillips, H.-J. Chiu, A.E. Cohen, A.M. Deacon, P.J. Ellis, E.
Garman, A. Gonzalez, N.K. Sauter, R.P. Phizackerley, S.M. Soltis, P. Kuhn, J. Synchrotron.
Radiat. 9 (2002) 401-406.
[
[
[
[
[
[
20] Q. Feng, N.-Q. Li, Y.-Y. Jiang, Anal. Chim. Acta. 344 (1997) 97-104.
21] M. Aslanoglu, G. Ayne, Anal. Bioanal. Chem. 380 (2004) 658-663.
22] A. Oliveira-Brett, J. Piedade, L. Silva, V. Diculescu, Anal. Biochem. 332 (2004) 321-329.
23] A.M. Oliveira-Brett, V. Diculescu, J.A.P. Piedade, Bioelectrochemistry, 55 (2002) 61-62.
24] V. Namasivayam, R. Günther, Chem. Biol. Drug Des. 70 (2007) 475-484.
25] F. Österberg, G.M. Morris, M.F. Sanner, A.J. Olson, D.S. Goodsell, Proteins, 46 (2002) 34-
4
0.
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Scheme 1. Synthetic scheme for ferrocene incorporated acyl ureas.
13

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Scheme 2. Drug-DNA binding constants for ferrocene derivatives.
14

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Scheme 3. Proposed mechanism for formation of ferrocene incorporated acyl ureas.
15

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Table 1. Crystal Data of P3Cl.
P3Cl
Empirical formula
Formula weight
Temperature (K)
Wavelength Å
a [Å]
C24 H ClFeN2O2
19
458.7
100 K
0.71073
7.987 (2)
9.9530 (12)
12.6520 (6)
81.935 (4)°
89.228 (6)°
79.397 (15)°
978.7 (3)
Triclinic
P-1
b [Å]
c [Å]
α [deg]
β [deg]
γ [deg]
3
Volume Å
Crystal System
Space group
Z
2
Density (calculated)
1.557g/cm3
10<=h<=10, -12<=k<=12, -16<=l<=16
-
Index Ranges
Absorption coefficient (µ)
F(000)
0.932 mm-1
472
2
(S)
1.058
0.638 and -0.879 e.Å-3
Goodness-of-fit on F
Largest diff. peak and hole
Final R indices [I>2sigma(I)] R1 = 0.0520, wR2 = 0.1152
R indices (all data) R1 = 0.0782, wR2 = 0.1379

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Table 2. Selected Bond lengths [Å] and angles [°] for P3Cl.
C(8)-N(1)
1.406(4)
1.406(4)
1.406(4)
1.406(4)
1.406(4)
1.406(4)
1.406(4)
1.406(4)
0.88(4)
C(8)-N(1)
C(8)-N(1)
C(8)-N(1)
C(8)-N(1)
C(8)-N(1)
C(8)-N(1)
C(8)-N(1)
N(1)-H(1N)
N(2)-H(2N)
O(1)-C(7)-N(1)
O(1)-C(7)-C(3)
N(1)-C(7)-C(3)
O(2)-C(8)-N(2)
C(7)-N(1)-C(8)
0.83(5)
123.0(3)
121.3(3)
115.7(3)
125.2(3)
128.5(3)
C(7)-N(1)-H(1N) 123(3)
C(8)-N(1)-H(1N) 108(3)
C(8)-N(2)-C(9)
126.2(3)
C(8)-N(2)-H(2N) 116(3)
C(9)-N(2)-H(2N) 118(3)
C(7)-N(1)-C(8)
128.5(3)
2

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Figure 1. (a) Molecular diagram of P3Cl with ellipsoid displacement, non-hydrogen atoms
represented by 30 % probability boundary spheres and hydrogen atoms are sphere of arbitrary size.
(b) Hydrogen bonding in P3Cl. c) Packing of P3Cl.

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Figure 2. (a) Representative plots of Current vs. Potential/V (SCE) at different scan rates for P3Cl.
(b) Cyclic voltammograms of 1 mM P3Cl with 1 mL of 0.5 M TBAP as supporting electrolyte in
the absence and presence of 2-8 µM DNA showing a decrease in I from I and a concentration
o
dependent –ve shift in potential showing electrostatic interactions. (c) Representative plot of current
½
vs. (V/s) , for the determination of diffusion coefficient of free P3Cl and P3Cl-2 µM DNA. (d)
Representative plot of log (I/I -I) vs. log (1/[DNA] for determination of binding constant of P3Cl.
o
(e) Plot of C
b f
/C vs. [DNA]/µM for determination of binding site size of 2-8 µM DNA
concentrations (P3Cl).

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Figure 3. (a) Cyclic voltammograms of 1 mM P4Cl with 1 mL of 0.5 M TBAP as supporting
electrolyte in the absence and presence of 2-8 µM DNA showing a decrease in I from I and a
o
concentration dependent –ve shift in potential showing electrostatic interactions. (b) Cyclic
voltammograms of 1 mM P2Cl with 1 mL of 0.5 M TBAP as supporting electrolyte in the absence
and presence of 2-8 µM DNA.

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Figure 4. (a) Representative plots of absorbance versus wavelength of P3Cl with increasing
concentration of DNA (7.5-37.5 µM). (b) Plot of A /A-A versus 1/[DNA] for determination of
o
o
DNA binding constant of P3Cl.

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Figure 5. Representative plot of relative viscosity vs. [Drug]/[DNA] for determination of mode of
interaction.

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Figure 6. (a) Representative docked conformation of P3Cl with 1BNA, here P3Cl is in green color
while 1BNA can be seen as ribbon structure. (b) Surface view of docked P3Cl with 1-BNA (Color
code. Grey-carbon, Red- Oxygen, and Blue-Nitrogen). (c) Three-dimensional model showing
interactions of P3Cl with the DNA.

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Figure 7. Representative plots of absorbance versus wavelength of P3Cl for DPPH scavenging
activity.

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•
•
We have synthesized three new ferrocene incorporated acyl ureas.
All the compounds were characterized by NMR, FT-IR, AAS, CHNS and single crystal
XRD.
•
•
CV, UV-vis spectroscopy, viscometry and molecular docking were used for DNA
binding studies.
IC values of 82, 136 and 54 µM for P4Cl, P3Cl and P2Cl respectively, were observed
5
0
against DPPH.

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Single crystal structure of ferrocene incorporated acyl ureas, their spectroscopic,
electrochemical, antioxidant and molecular docking studies.

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checkCIF/PLATON report
Structure factors have been supplied for datablock(s) I
No syntax errors found.
CIF dictionary
Interpreting this report
Datablock: I
Bond precision: C-C = 0.0062 A
Wavelength=0.71073
Cell:
a=7.987(2)
b=9.9530(12)
c=12.6520(6)
alpha=81.935(4)
beta=89.228(6)
gamma=79.397(15)
Temperature: 100 K
Calculated
Reported
Volume
Space group
Hall group
978.7(3)
P -1
-P 1
978.7(3)
P -1
-P 1
Moiety formula C24 H19 Cl Fe N2 O2
C20 H20 Fe1 O4 Se1
Sum formula
Mr
Dx,g cm-3
Z
C24 H19 Cl Fe N2 O2
C24 H19 Cl Fe N2 O2
458.71
1.557
2
458.71
1.557
2
Mu (mm-1)
F000
F000’
h,k,lmax
Nref
0.925
472.0
473.13
10,12,16
4342
0.932
472.0
10,12,16
4342
Tmin,Tmax
Tmin’
Correction method= Not given
Data completeness= 1.000
Theta(max)= 27.150
wR2(reflections)= 0.1379( 4020)
R(reflections)= 0.0520( 3066)
S = 1.058
Npar= 348
The following ALERTS were generated. Each ALERT has the format
test-name_ALERT_alert-type_alert-level.
Click on the hyperlinks for more details of the test.
Alert level A
PLAT029_ALERT_3_A _diffrn_measured_fraction_theta_full Low .......
0.926
Author Response: Data were collected on a synchrotron

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Alert level C
PLAT222_ALERT_3_C Large Non-Solvent
H
Uiso(max)/Uiso(min) ..
4.6 Ratio
0.012 AngSq
0.020 AngSq
0.027 AngSq
0.0062 Ang
PLAT245_ALERT_2_C U(iso) H5
PLAT245_ALERT_2_C U(iso) H10
PLAT245_ALERT_2_C U(iso) H21
Smaller than U(eq) C5
Smaller than U(eq) C10
Smaller than U(eq) C21
by ...
by ...
by ...
PLAT341_ALERT_3_C Low Bond Precision on C-C Bonds ...............
Alert level G
FORMU01_ALERT_1_G There is a discrepancy between the atom counts in the
_
chemical_formula_sum and _chemical_formula_moiety. This is
usually due to the moiety formula being in the wrong format.
Atom count from _chemical_formula_sum: C24 H19 Cl1 Fe1 N2 O2
Atom count from _chemical_formula_moiety:C20 H20 Fe1 O4 Se1
ABSMU_01 Radiation type not identified. Calculation of
_exptl_absorpt_correction_mu not performed.
PLAT002_ALERT_2_G Number of Distance or Angle Restraints on AtSite
PLAT005_ALERT_5_G No _iucr_refine_instructions_details in CIF ...
PLAT042_ALERT_1_G Calc. and Reported MoietyFormula Strings Differ
PLAT164_ALERT_4_G Nr. of Refined C-H H-Atoms in Heavy-Atom Struct.
4
?
?
17
PLAT232_ALERT_2_G Hirshfeld Test Diff (M-X) Fe1
PLAT860_ALERT_3_G Note: Number of Least-Squares Restraints .......
PLAT984_ALERT_1_G The Fe-f’= 0.346 Deviates from the B&C-Value
-- C17
..
6.5 su
2
0.348
1
0
5
8
ALERT level A = Most likely a serious problem - resolve or explain
ALERT level B = A potentially serious problem, consider carefully
ALERT level C = Check. Ensure it is not caused by an omission or oversight
ALERT level G = General information/check it is not something unexpected
3
5
4
1
1
ALERT type 1 CIF construction/syntax error, inconsistent or missing data
ALERT type 2 Indicator that the structure model may be wrong or deficient
ALERT type 3 Indicator that the structure quality may be low
ALERT type 4 Improvement, methodology, query or suggestion
ALERT type 5 Informative message, check
checkCIF publication errors
Alert level A
PUBL004_ALERT_1_A The contact author’s name and address are missing,
_
publ_contact_author_name and _publ_contact_author_address.
PUBL005_ALERT_1_A _publ_contact_author_email, _publ_contact_author_fax and
publ_contact_author_phone are all missing.
_
At least one of these should be present.
PUBL006_ALERT_1_A _publ_requested_journal is missing
e.g. ’Acta Crystallographica Section C’
PUBL008_ALERT_1_A _publ_section_title is missing. Title of paper.
PUBL009_ALERT_1_A _publ_author_name is missing. List of author(s) name(s).
PUBL010_ALERT_1_A _publ_author_address is missing. Author(s) address(es).
PUBL012_ALERT_1_A _publ_section_abstract is missing.
Abstract of paper in English.
Alert level G
PUBL013_ALERT_1_G The _publ_section_comment (discussion of study) is