Synthesis, chemical characterisation, and DNA binding studies of ferrocene-incorporated selenoureas

Article abstract of DOI:10.1071/CH12570

In this article we have presented the synthesis, chemical characterisation (by NMR and FTIR spectroscopy, atomic absorption spectrophotometry, elemental analysis, and single crystal X-ray diffraction), electrochemistry, and DNA binding studies (with cycli

Full text of DOI:10.1071/CH12570

CSIRO PUBLISHING
Aust. J. Chem. 2013, 66, 626–634
Full Paper
http://dx.doi.org/10.1071/CH12570
Synthesis, Chemical Characterisation, and DNA Binding
Studies of Ferrocene-Incorporated Selenoureas
A
A C
,
B
Raja Azadar Hussain, Amin Badshah,
Muhammad Nawaz Tahir,
A
A
Bhajan Lal, and Inayat Ali Khan
A
Coordination Chemistry Laboratory, Department of Chemistry, Quaid-i-Azam
University, Islamabad 45320, Pakistan.
B
Department of Physics, University of Sargodha, Punjab, Pakistan.
C
Corresponding author. Email: aminbadshah@yahoo.com
In this article we have presented the synthesis, chemical characterisation (by NMR and FTIR spectroscopy, atomic
absorption spectrophotometry, elemental analysis, and single crystal X-ray diffraction), electrochemistry, and DNA
binding studies (with cyclic voltammetry, viscometry, and UV-vis spectroscopy) of six new ferrocene incorporated
selenoureas. All the six compounds interact electrostatically with DNA which was evident by a negative shift in the cyclic
voltammetry peak potential of the drug–DNA adduct relative to the free drug. The drug–DNA binding constant was
calculated by a decrease in peak current after the addition of DNA to the free drug. We have also reported binding site sizes
and diffusion coefficients of the synthesised compounds.
Manuscript received: 4 January 2013.
Manuscript accepted: 5 February 2013.
Published online: 12 March 2013.
Barton et al.[15]). In another publication, unsymmetric sele-
noureas and selenoureidopeptides were prepared by the reac-
tion of isoselenocyanates with different aromatic amines.[16]
Andaloussi and Mohr have published the synthesis of trityl
isoselenocyanates from trityl chloride and potassium
selenocyanate (KSeCN).[17,18] Trityl isoselenocyanate has
colourless needle-like crystals which decompose in the pres-
ence of light and give a red coloured product.[18] Decomposi-
tion also occurs in dichloromethane and acetone. Crystal
structure analysis proved that the compound was indeed iso-
selenocyanate and not the selenocyanate as was suggested by
Rheinboldt and de Campos.[19] Selenium derivatives find their
applications in antioxidant defence enzymes (reduction of
hydroperoxides-GPx mimics, reduction of peroxinitriles, and
lipid peroxidation), enzyme inhibition (nitric oxide synthase
inhibitors, ionosine monophosphate dehydrogenase inhibitors,
lipoxygenase inhibitors, urdpase and thymidylate synthase
inhibitors, and tyrosine kinase and iodothyronine deiodinase
inhibitors), photochemotherapeutic agents, antitumour agents,
anti-infective drugs (antiviral, antifungal, and antibacterial),
cytokine inducers/immunomodulators, and antihypertensive
and cardiotonic agents.[20]
Meanwhile, ferrocene derivatives have been extensively
evaluated for their antibiotic, antimalarial, and anticancer activ-
ities.[21] It has now been established that ferrocene has antineo-
plastic activity but little is known about the mechanism of its
antitumour effect. In a quest to know the mode of interaction and
magnitude of its DNA binding constant, different techniques
like cyclic voltammetry (CV), UV-vis spectroscopy,[22] visco-
metry,[23] and luminescence[24] have been used. Among these,
electrochemical methods are more useful due to their high
sensitivity, cost effectiveness, low detection limit, reliability,
Introduction
In 1937 Irwin B. Douglass reported[1] the synthesis of acylse-
lenoureas from corresponding isoselenocyanates, although he
was not sure about the intermediates of this reaction, i.e. sele-
nocyanate, isoselenocyanate, or an equilibrium mixture of the
two. In 1956 selenopurines and selenopyrimidines were pre-
pared[2] with the help of selenourea and sodium hydroselenide.
Early developments on this area were slow because selenium
was long considered as an absolute biological poison until
Schwarz and Foltz in 1957 identified it as a micronutrient for
bacteria, mammals, and birds.[3] Preparation of the selenoureas
from thioureas was reported in good yield by the reaction
of 1,1,3,3-tetramethyl-2-thiourea[4] with methyl iodide and
hydroselenide in ethanol solution. In 1973 two bacterial
enzymes, formate dehydrogenase and glycine reductase, were
reported to contain selenium.[5,6] After the findings that orga-
noselenium derivatives are far less toxic than inorganic seleni-
um species, curiosity developed in their synthesis for use in
enzymology and bioorganic chemistry.[7]
Selenoureas can also be synthesised by a three component
condensation reaction[8] of triethylorthoformate, selenium,
and amines (both primary and secondary) in a solvent free
process at 180–1908C. This route has an advantage over the
previously used methods in that the reagents used in this
route are comparatively less toxic and thermally stable,
i.e. hydrogen selenide,[9] carbon diselenide,[10,11] bis(trimethyl-
silyl) selenide,[12] or isoselenocyanate[13] are more hazardous
than metallic selenium which has low volatility and is
easily accessible. O-Unprotected selenoureas of gluco and
manno configurations were organised[14] by the reaction of
b-D-glycopyranosylamines with phenyl isoselenocyanates
in aqueous pyridine (a modification of the procedure of
Journal compilation Ó CSIRO 2013
www.publish.csiro.au/journals/ajc

Ferrocene-Incorporated Selenoureas
627
NO2
Fe
COOH
Fe
Fe
2.36 ꢃ 104 Mꢂ1
4.8 ꢃ 105 Mꢂ1
1 (m-COOH)
2 (p-COOH)
(C5H5FeC5H4COO)2SbR3
1.87 ꢃ 104 Mꢂ1
6.61 ꢃ 103 Mꢂ1
6.58 ꢃ 103 Mꢂ1
3.45 ꢃ 102 Mꢂ1
3.85 ꢃ 103 Mꢂ1
3 (R ꢀ ꢂC6H5 and m-COO)
4 (R ꢀ p-CH3C6H4 and m-COO)
5 (R ꢀ p-CH3C6H4 and p-COO)
Chart 1. DNA binding constants for recently reported ferrocence derivatives.
ꢁ
5°C
Fe
HCl
O2N
N
NClꢂ
O2N
O2N
NH2
NaNO2
ꢁ
PTC
Diethyl ether
ꢁ
NO2
N
NClꢂ
ꢁ
Fe
Pd Charcoal
Hydrazine
NO2
NH2
Fe
Part 1
Fe
δꢂ
Se
Cl
R
δꢁ
C
R
ꢁ ꢂ
KSeCN
Acetone
δꢂ
O
C
N
C
Se
ꢁ
δꢁ
C
N
O
δꢂ
O
δꢂ
R
O
C
Se
R
Acetone
NH2
ꢁ
N
C
N
H
R
N
C
Se
Fe
Fe
H
O
δꢂ
F
F
Br
,
Br
F
Br
,
R ꢀ
,
,
,
Scheme 1. Synthetic scheme for selenoureas (PTC: phase transfer catalyst (hexadecyltrimethyl-ammonium bromide)).
and fast analysis. The attachment of different substituents to the
cyclopentadiene (Cp) moieties of ferrocene change its spectro-
scopic, biological, and redox behaviour which in turn alters
the mode of interaction and magnitude of the DNA binding
constant.[25] Protonated ferrocene has a binding constant of
3.45 ꢀ 102 Mꢁ1 while the binding constant for some other
ferrocene derivatives have been given in Chart 1.[24,25] Inspired
from synthetic chemistry and biological applications of sele-
noureas along with ferrocene incorporated derivatives we have
combined both functionalities, i.e. ferrocene and selenoureas, in
ferrocene-incorporated selenoureas for the first time.
single crystal X-ray structures for three compounds (P2Br,
P2F, and P3F) and all the compounds have been completely
characterised with multinuclear NMR spectroscopy, FTIR spec-
troscopy, elemental analysis, and atomic absorption spectropho-
tometry (AAS). Cyclic voltammetry (CV) measurements were
used to determine the DNA binding constant (K), diffusion
coefficient (Do), and binding site size (s). DNA binding constant
values of all the synthesised compounds are stronger than that
previously reported for protonated ferrocene,[25] which shows
that the selenourea moiety in the side chain is playing a role in
the magnitude of the DNA binding constant.
This paper presents the synthesis (Scheme 1), chemical
characterisation, and DNA binding studies of six new ferrocene-
incorporated selenoureas namely: 1-(2-bromobenzoyl)-3-(4-
ferrocenylphenyl)selenourea (P2Br), 1-(3-bromobenzoyl)-3-
(4-ferrocenylphenyl)selenourea (P3Br), 1-(4-bromobenzoyl)-3-
(4-ferrocenylphenyl)selenourea (P4Br), 1-(2-fluorobenzoyl)-3-
(4-ferrocenylphenyl)selenourea (P2F), 1-(3-fluorobenzoyl)-3-
(4-ferrocenylphenyl)selenourea (P3F), and 1-(4-fluorobenzoyl)-
3-(4-ferrocenylphenyl)selenourea (P4F). We have provided
Results and Discussion
All the compounds are orange in colour and have been synthe-
sised in 71–83 % yield. Possible impurities, i.e. unreacted 4-
ferrocenylaniline and by-products like amides, carboxylic acids,
and KCl are easily removed from the products on the basis of
their different solubilities in water and common organic sol-
vents. 1H and 13C NMR spectra of the five compounds (P2Br,

628
R. A. Hussain et al.
Table 1. Crystal data of 1-(2-bromobenzoyl)-3-(4-ferrocenylphenyl)selenourea (P2Br), 1-(2-fluorobenzoyl)-3-(4-ferrocenylphenyl)selenourea
(P2F), and 1-(3-fluorobenzoyl)-3-(4-ferrocenylphenyl)selenourea (P3F)
Parameter
P2Br
P2F
P3F
CCDC No.
897146
908933
897147
Empirical formula
Formula weight
Temperature [K]
C24H19BrFeN2OSe
566.13
C24H19FFeN2OSe
C24H21FFeN2O2Se
505.22
523.24
296
296
296
˚
Wavelength [A]
0.71073
0.71073
0.71073
˚
a [A]
˚
b [A]
˚
c [A]
7.3853 (3)
12.0283 (5)
13.7635 (5)
111.9210 (1)
105.255 (2)
93.452 (2)
1077.000
Triclinic
7.2128 (2)
15.7656 (6)
22.7520 (5)
11.7030 (4)
12.9592 (3)
11.9683 (4)
a [deg]
b [deg]
g [deg]
90
90
105.175(1)
104.958 (2)
90
90
3
˚
Volume [A ]
2052.52 (9)
2133.38 (13)
Crystal System
Space group
Monoclinic
Monoclinic
P-1
P21/n
P21/c
Z
2
4
4
Density (calculated) [Mg m3]
1.746
1.635
1.629
Index Ranges
ꢁ7 # h # 9, ꢁ14 # k # 14,
ꢁ16 # l # 16
4.26
ꢁ8 # h # 8, ꢁ24 # k # 28,
ꢁ15 # l # 15
2.54
ꢁ18 # h # 19, ꢁ14 # k # 13,
ꢁ14 # l # 10
2.45
Absorption coefficient (m) [mmꢁ1
F(000)
Goodness-of-fit on F2 (S)
]
560
1016
1056
1.04
1.03
1.04
R Factor [%]
˚
Largest differences peak and hole [e A
R1 0.036, wR2 0.092
0.72 and ꢁ0.59
R1 0.036, wR2 0.091
0.64 and ꢁ0.85
R1 0.043, wR2 0.123
0.52 and ꢁ0.81
ꢁ3
]
P3Br, P2F, P3F, and P4F) were recorded in deuterated acetone/
benzene and that of P4Br was run in d6-DMSO because of its low
solubility in acetone. There are three types of protons in all the
compounds, i.e. –NH protons, aromatic protons, and the protons
a Bruker kappa APEXII CCD diffractometer using a graphite
˚
monochromator having MoKa radiation (l 0.71073 A) at 296 K.
The structures were solved by direct methods and refined by
full-matrix least-squares against F2 of data using SHELXL97
software.[26] Basic crystal data and a description of the dif-
fraction experiment are given in Table 1.
1
of ferrocene. In the H NMR spectra, all the compounds pro-
vided two signals for their two NH protons between 9–14 ppm as
a singlet and aromatic protons appeared as a multiplet between
7–8 ppm. The five protons of the unsubstituted Cp ring of fer-
rocene yielded an intense signal at ,4 ppm and the substituted
Cp ring provided two triplets downfield from the singlet of the
unsubstituted Cp ring. In the 13C NMR spectra, the carbon
attached to selenium provided a weak signal around ,180 ppm
and the carbonyl carbon appeared at 165–170 ppm for all the
compounds. Carbons of the unsubstituted Cp ring yielded one
signal and the substituted Cp ring gave three signals, two of them
were between 60–70 ppm, and one signal for the substituted
carbon of the Cp ring was visible at ,84 ppm for all the syn-
thesised compounds. Aromatic carbons were visible in a region
between 120–140 ppm.
In the FTIR spectra, the –NH signal of all the selenoureas
gave a broad band above 3200 cmꢁ1 owing to intramolecular
hydrogen bonding with the oxygen of the carbonyl. Just
above 3000 cmꢁ1 an Ar–H stretch was evident for all the
compounds. The carbonyl group appeared as an intense band
at 1655–1665 cmꢁ1 and C¼Se was available between 1050–
1250 cmꢁ1 for all the compounds.
An ORTEP diagram of P2Br is given in Fig. 1 whereas
selected bond lengths and bond angles are summarised in Table
S1 (Supplementary Material). The structure reveals that the
phenyl ring attached to the Cp ring of ferrocene is slightly
tilted out of the plane of the Cp moiety and the phenyl ring
attached to the carbonyl group is almost at a right angle to it.
The angle between Se–C(17)–N(1) is 128.348 and Se–C(17)–
N(2) is 117.788. Fig. S1 (Supplementary Material) shows the
presence of intramolecular hydrogen bonding between O(1)
and H(1A).
An ORTEP diagram of P2F is given in Fig. 2 whereas
selected bond lengths and bond angles have been given in Table
S2 (Supplementary Material). A major difference between P2Br
and P2F is that, in P2F, the phenyl ring attached to the Cp ring of
ferrocene is less tilted and the phenyl ring attached to the
carbonyl carbon is tilted in the opposite direction as compared
with phenyl attached to the Cp ring. Intramolecular hydrogen
bonding is similar in both the structures but the hydrogens on
the Cp rings of P2F are completely eclipsed (Fig. S2 in the
Supplementary Material) whereas in case of P2Br the hydrogens
of the two rings are not exactly eclipsed.
Elemental analysis showed that calculated and found per-
centages of carbon, hydrogen, nitrogen, and iron are in a close
agreement with each other in all the compounds. The percen-
tages of carbon, hydrogen, and nitrogen were determined with a
CHNS analyser and that of Fe by AAS.
An ORTEP diagram of P3F is given in Fig. 3 whereas
selected bond lengths and bond angles have been given in Table
S3 (Supplementary Material). This crystal structure has a water
molecule in the lattice which was used as a non-solvent for
crystallisation. Hydrogens of the Cp ring are completely
eclipsed and the phenyl ring attached to the carbonyl has some
alignment with the Cp ring of ferrocene (Fig. S3 in the
Supplementary Material) opposed to that observed in P2Br.
Crystallography
Suitable single crystals of all the three compounds were
mounted on a glass fibre and the intensity data were collected on

Ferrocene-Incorporated Selenoureas
629
Br1
H23
Se1
C17
N2
H13
C24
C23
H2A
C18
C12
C13
H12
H9
C9
C22
H22
C14
C8
C10
C11
N1
C19
H8
C7
C6
C20
C21
H1A
C16
C15
O1
H21
H7
H6
H2
H15
Fe1
H20
H16
C1
H3
H1
C2
C5
C3
H5
C4
H4
Fig. 1. Molecular diagram of 1-(2-bromobenzoyl)-3-(4-ferrocenylphenyl)selenourea (P2Br) with ellipsoid displacement,
non-hydrogen atoms are represented by 30 % probability boundary spheres and hydrogen atoms are spheres of arbitrary size.
H9
H8
C8
H12
C12
C13
H13
O1
C18
H24
C10
H1
C9
C6
C23
H23
C22
C24
C20
C11
H1A
C17
C19
C7
H7
C14
N1
H22
C16
H16
H6
H2
C15
H15
Fe1
C1
N2
H2A
C21
H21
F1
C5
H5
Se1
C2
C3
H3
C4
H4
Fig. 2. Molecular diagram of 1-(2-fluorobenzoyl)-3-(4-ferrocenylphenyl)selenourea (P2F) with ellipsoid displacement, non-hydrogen atoms
are represented by 30 % probability boundary spheres and hydrogen atoms are spheres of arbitrary size.
Se1
H9
H12
C17
H13
C14
C12
C13
C8
H20
C20
C11
H8
H7
F1
H2A
C18
C9
H6
C16
N2
C10
N1
C6
C7
Fe1
C15
H1A
C22
H22
H16
H5
H1
H15
C21
C24
C19
O1
C5
H2
C1
C2
H24
C23
H23
H2D
C3
H2C
H3
O2
C4
H4
Fig. 3. Molecular diagram of 1-(3-fluorobenzoyl)-3-(4-ferrocenylphenyl)selenourea (P3F) with ellipsoid displacement, non-
hydrogen atoms are represented by 30 % probability boundary spheres and hydrogen atoms are spheres of arbitrary size.
DNA Binding Studies
peak potential provided information about the drug–DNA bind-
ing constant and mode of interaction, respectively. The changes
in peak current of the free drug by the addition of varying con-
centrations of DNA was used to evaluate the drug–DNA binding
constant with the help of the following equation:[27]
In CV studies a three electrode system was used consisting
of working (platinum disc electrode with a geometric area of
0.071cm2), reference (Ag/AgCl), and auxiliary electrodes
(platinum electrode with geometric area much greater than
working electrode). CV data of selected synthesised compounds
have been given in Figs 4 and 5. Changes in peak current and
logð1=½DNAꢂÞ ¼ log K þ logðI=Io ꢁ IÞ
ð1Þ

630
R. A. Hussain et al.
0 μM DNA
2 μM DNA
4 μM DNA
6 μM DNA
8 μM DNA
10 μM DNA
5.8
5.7
5.6
5.5
5.4
5.3
5.2
5.1
5.0
4.9
(a)
(b)
y ꢀ 1.2344x ꢁ 4.6758
R2 ꢀ 0.9969
0.0075
0.0055
0.0035
0.0015
ꢂ0.0005
ꢂ0.0025
ꢂ0.0045
K ꢀ 4.7402 ꢃ 104 Mꢂ1
ꢂ0.1
0.1
0.3
0.5
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
log(I/Io–I)
E [V] vs. Ag/AgCl
0.6
0.5
0.4
0.3
0.2
0.1
0
16
14
12
10
8
(d)
(c)
6
0 μm
2 μm
4
2
0
0.2
0
2
4
6
8
10
12
0.4
0.6
0.8
1/2 [(V sꢂ1 1/2
) ]
Conc. DNA [μM]
ν
Fig. 4. (a) Cyclic voltammograms of 1 mM 1-(3-fluorobenzoyl)-3-(4-ferrocenylphenyl)selenourea (P3F) with 1 mL of 1.5 M KCl as a
supporting electrolyte in the absence and presence of 2–10 mM DNA showing a decrease in I from Io and a concentration dependent negative
shift in potential showing electrostatic interactions. (b) Plot of log (I/Io ꢁ I) versus log (1/[DNA]) for determination of binding constant.
(c) Plots of I versus v1/2, for the determination of diffusion coefficients of free drug P3F (0 mM DNA) and 2 mM DNA bound drug. (d) Plot of
Cb/Cf versus [DNA] in mM for determination of binding site size of 2–10 mM DNA concentrations. See text for definitions of symbols and
equations used.
(a)
0.013
(b)
0.013
0.008
0.003
0.008
0.003
0 μM DNA
20 μM DNA
0 μM DNA
5 μM DNA
10 μM DNA
20 μM DNA
20.002
20.007
20.012
20.002
20.007
20.012
30 μM DNA
40 μM DNA
50 μM DNA
20.1
0.1
0.3
0.5
20.1
0.1
0.3
0.5
E [V] vs. Ag/AgCl
E [V] vs. Ag/AgCl
Fig. 5. (a) Cyclic voltammograms of 1 mM P3Br with 1 mL of 1.5 M KCl as a supporting electrolyte in the absence and presence
of 5–20 mM DNA showing a decrease in I from Io and a concentration dependent negative shift in potential showing electrostatic
interactions. (b) Cyclic voltammograms of 1 mM P3Br with 1 mL of 1.5 M KCl as supporting electrolyte in the absence and
presence of 20–50 mM DNA showing an increase in I from 20–50 mM DNA. See text for definitions of symbols and equations used.
where K is the binding constant and Io and I are the peak currents
of the free drug and DNA-bound drug, respectively. It should be
clear that Io is the current measured from a cyclic voltammo-
gram which was run without the addition of DNA to the drug. I is
the current measured from another cyclic voltammogram when
a certain concentration of DNA was added to the solution.
For the measurement of binding site size (s) the following
equation[28] was used:
Cb=Cf ¼ Kf½free base pairsꢂ=sg
ð2Þ

Ferrocene-Incorporated Selenoureas
631
Table 2. Important parameters for redox behaviour and DNA binding studies
P2Br: 1-(2-bromobenzoyl)-3-(4-ferrocenylphenyl)selenourea; P3Br: 1-(3-bromobenzoyl)-3-(4-ferrocenylphenyl)selenourea, P4Br: 1-(4-bromobenzoyl)-3-
(4-ferrocenylphenyl)selenourea, P2F: 1-(2-fluorobenzoyl)-3-(4-ferrocenylphenyl)selenourea, P3F: 1-(3-fluorobenzoyl)-3-(4-ferrocenylphenyl)selenourea,
and P4F: 1-(4-fluorobenzoyl)-3-(4-ferrocenylphenyl)selenourea. K is the DNA binding constant, Do are diffusion coefficients, and s is the binding site size
given in number of base pairs (bp)
Compounds
K [Mꢁ1
]
Do [cm2 sꢁ1] free drug
Do [cm2 sꢁ1] drug–DNA
s [bp]
P3F
4.7 ꢀ 104
4.56 ꢀ 104
4.952 ꢀ 104
1.3 ꢀ 103
4.13 (ꢅ 0.02) ꢀ 10ꢁ6
3.28 (ꢅ 0.04) ꢀ 10ꢁ6
3.44 (ꢅ 0.13) ꢀ 10ꢁ6
1.54 (ꢅ 0.71) ꢀ 10ꢁ7
3.54 (ꢅ 0.11) ꢀ 10ꢁ6
5.03 (ꢅ 0.20) ꢀ 10ꢁ6
3.90 (ꢅ 0.04) ꢀ 10ꢁ6
8.29 (ꢅ 0.11) ꢀ 10ꢁ7
1.63 (ꢅ 0.09) ꢀ 10ꢁ6
7.40 (ꢅ 1.04) ꢀ 10ꢁ7
1.18 (ꢅ 0.23) ꢀ 10ꢁ6
3.93 (ꢅ 0.38) ꢀ 10ꢁ6
0.462
0.204
0.558
0.703
0.965
0.106
P3Br
P4F
P4Br
P2F
7.425 ꢀ 103
1.192 ꢀ 103
P2Br
where s is the binding site size in terms of base pair, K is the
binding constant, Cf is the concentration of free species, and Cb
represents the concentration of drug–DNA bound species.
Considering the concentration of DNA in terms of nucleotide
phosphate, the concentration of DNA base pairs is taken as
[DNA base pair]/2 and Eqn 2 is written as:
50, 121, 161, and 150 mV. The decrease in peak current is
attributed to the formation of a slow diffusing drug–DNA adduct
which is responsible for the consumption of free drug, mainly
active for conduction of current, and a substantial cathodic
shift in peak potential is due to the strong electrostatic inter-
action of the positively charged drug with the negatively
charged phosphate backbone of DNA.[32] By increasing the
concentration from 2–8 mM DNA, the extent of electrostatic
interaction increases but after addition of 10 mM DNA, it slightly
decreases, i.e. the concentration dependent cathodic shift
stops (Fig. 4a). The decrease in peak current as a consequence
of varying DNA concentrations gave drug–DNA binding con-
stant of 4.7402 ꢀ 104 Mꢁ1 by a plot of log (1/[DNA]) versus
log (I/Io ꢁ I) according to Eqn 1 (Fig. 4b). A decrease in
diffusion coefficient (Do) for 2 mM drug–DNA adduct with
reference to the free drug (0 mM DNA) indicated that the
drug–DNA adduct is slow diffusing as compared with the free
drug (Fig. 4c). The Do of the free drug was found to be
4.13 (ꢅ 0.02) ꢀ 10ꢁ6 cm2 sꢁ1 and that of 2 mM drug–DNA
adduct was 3.90 (ꢅ 0.04) ꢀ 10ꢁ6 cm2 sꢁ1. The binding site size
was found to be 0.462 bp with the help of Eqn 3 by a plot of Cb/Cf
versus DNA/mM (Fig. 4d) and such a small value of s indicates
the dominance of electrostatic interactions which are responsi-
ble for the disturbance of normal DNA functions and replication
mechanism.
Cb=Cf ¼ Kf½DNA base pairꢂ=2sg
ð3Þ
The value of Cb/Cf is equal[29] to (Io ꢁ I/I) which are the
values of experimental peak currents.
For the determination of the diffusion coefficient of free and
DNA-bound drug, the following form of the Randles–Sevcik
equation[30] was used:
Ipa ¼ 2:69 ꢀ 105n3=2A Co ꢃ Do1=2v1=2
ð4Þ
where Ipa is the anodic peak current in ampere, Co* is the
reductants’s concentration in mol cmꢁ3, v is the scan rate in
voltsꢁ1, A is the geometric area of the electrode in cm2, n is
the number of electrons involved in the process, and Do is the
diffusion coefficient in cm2 sꢁ1. Eqn 4 is used for reversible
electrochemical processes and for our synthesised selenoureas
reversibility was determined using the following criteria:[31]
ꢄ Absence of a shift in peak potential with a variation in scan
P3Br gave interesting behaviour in the sense that its Ipa
values decreased by the addition of 5–20 mM DNA (similar to
P3F) but 30–50 mM DNA addition caused an increase in Ipa with
reference to 20 mM DNA (Fig. 5a, b). According to the literature,
at a higher concentration of DNA there are chances for the
formation of 8-oxoguanine from damaged guanine bases of
DNA. This 8-oxoguanine is itself electroactive and provides
an oxidation peak at ,0.4 V. This is the region where oxidation
of our P3Br is also in progress. We expect that 8-oxoguanine
along with our compound (P3Br) may have increased the peak
current.[33] Moreover, the report of Cavazzoni et al. also
provides evidence of DNA damage by 5-benzylidene-hydantoin
(Fig. S5 in the Supplementary Material).[34] Calculated para-
meters have been summarised in Table 2. Moreover, for P3Br
the change in Ipa was not readable for lower concentrations of
DNA, i.e. 2 mM DNA, therefore we have used higher DNA
concentrations.
rate.
ꢄ DEp ¼ Epc ꢁ Epa for a one electron system gives a value of
almost 57 mV.
ꢄ Anodic and cathodic peak current ratios (Ipa/Ipc) were
about one.
meta-Substituted Derivatives
We first of all discuss the DNA binding studies of meta-
substituted derivatives (P3F and P3Br) in detail. P3F yielded a
couple of well defined redox peaks within a potential window of
0–0.6 V using a constant concentration of 1 mM P3F and
varying concentrations of DNA with P3F in the presence of
1 mL of 1.5 M KCl aqueous solution as a supporting electrolyte
at a platinum disc electrode. The reversibility of the process is
evident by the consistency of voltage with the variation of scan
rate (Fig. S4 in the Supplementary Material). Addition of 2 mM
DNA solution to P3F decreases the Ipa (anodic peak current)
from 7.88 ꢀ 10ꢁ3 to 6.47 ꢀ 10ꢁ3 mA with a cathodic shift in
peak potential of 10 mV. DNA solutions of 4–10 mM interacted
with P3F with a decrease in peak current and a cathodic shift of
para-Substituted Derivatives
P4F and P4Br provided two well defined redox peaks in a
potential window of 0–0.6 V by using similar experimental

632
R. A. Hussain et al.
E0
f
FeII
FeIII
ꢁ
1eꢂ
Se
C
O
C
Kox
Kred
N
H
N
H
R
ꢁFe
E0
b
FeII
DNA
FeIII
DNA
ꢁ
1eꢂ
Scheme 2. General redox process of free and bound ferrocene incorporated selenoureas. A molecule in the
centre shows that FeIII form is resonance stabilised.[20]
Pure DNA 50 μM
2.5 μL P3F
5 μL P3F
7.5 μL P3F
10 μL P3F
8
7
6
5
4
3
2
1
0
(b)
(a)
y ꢀ 53.578x ꢁ 0.7128
R2 ꢀ 0.9852
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
12.5 μL P3F
15 μL P3F
17.5 μL P3F
20 μL P3F
22.5 μL P3F
25 μL P3F
27.5 μL P3F
30 μL P3F
K ꢀ 1.33 ꢃ 104 Mꢂ1
0.02
0.04
0.06
0.08
0.10
200
250
300
1/conc. P3F [μM–1
]
λ [nm]
Fig. 6. (a) Plot of absorbance versus wavelength (nm) showing a hyperchromic shift. (b) Plot of (Ao/A ꢁ Ao) versus 1/[P3F] (P3F ¼ 1-(3-
fluorobenzoyl)-3-(4-ferrocenylphenyl)selenourea) (mM)ꢁ1 for the estimation of drug–DNA binding constant. See text for definitions of symbols
and equations used.
conditions as were used for meta-substituted derivatives. Elec-
trostatic interactions were indicated by a cathodic shift in the
peak potential after the addition of DNA to the free drug but
unlike P3F this electrostatic interaction had no proportionality
with increasing concentrations of DNA.
different 20mL batches of 2.5 mM drug were added in the UV cell
having a constant concentration of DNA (70 mM). Pure DNA
provided a signal at 260 nm and with the addition of different
concentrations of the drug, a well defined hyperchromic shift in
the absorbance values confirmed the presence of electrostatic
interactions. A DNA binding constant of 1.33 ꢀ 104 Mꢁ1 for P3F
is in very close agreement with the values determined from CV
(Fig. 6).
Fig. 7 shows a representative plot of relative specific viscos-
ity versus [P3F]/[DNA]. A decrease in relative specific viscosity
with increasing concentration of the drug once again confirms
the presence of electrostatic interactions between the drug
and DNA.
ortho-Substituted Derivatives
P2Br and P2F also showed electrostatic interactions with
binding constants of 1.192 ꢀ 103 and 7.425 ꢀ 103 Mꢁ1, respec-
tively. Table 2 provides other important information for these
two compounds.
The electrochemical behaviour of the synthesised ferrocene-
incorporated selenoureas demonstrates that there is a shift in
both the oxidation and reduction peaks after the addition of
DNA. For this type of electrochemical behaviour Scheme 2 can
be applied. A cathodic shift in all the compounds indicates that
FeII of the ferrocene moiety is easier to oxidise in the presence of
DNA because its oxidised form is more easily bound to DNA
than its reduced form (neutral form). On the basis of the process
discussed in Scheme 2 the following equation is obtained:
Experimental
Materials and Methods
Melting points were determined in a capillary tube using an
Electro-Thermal melting point apparatus model MP-D Mita-
mura Riken Kogyo (Japan). Infrared spectra were measured on a
Thermoscientific NICOLET 6700 FTIR. 1H and 13C NMR
spectra were recorded on a Jeol JNM-LA 500 FT-NMR. Si
(CH3)4 was used as an internal reference. The elemental analysis
was performed using a LECO-932 CHNS analyser while the Fe
concentrations were determined on an Atomic Absorption
Spectrophotometer Perkin Elmer 2380.
Ebo ꢁ Efo ¼ 0:059 logðKred=Koxi
Þ
ð5Þ
where Ebo and Efo are the formal potentials of the FeII/FeIII couple
in the free and bound form, respectively.
CV measurements were equally supported by UV-vis spec-
troscopy and viscometry. InUV-visspectroscopic measurements,
Commercially available salmon sperm DNA was solubilised
in doubly distilled water to prepare a stock solution of

Ferrocene-Incorporated Selenoureas
633
1.00
0.95
0.90
0.85
0.80
0.75
0.70
0.65
0.60
into the reaction mixture. The reaction mixture was stirred under
reflux for a further 6 h and monitored by thin-layer chromato-
graphy (TLC). An orange coloured product obtained in the
solution state was mixed with cold water to remove the impuri-
ties and give the product in the solid state with 72 % yield. The
product was washed with n-hexane and recrystallised in an
acetone/n-hexane mixture. Mp 1408C (dec.). dH (acetone) 13.04
(s, 1H), 11.03 (s, 1H) 7.85–7.49 (m, 8H), 4.82 (t, J 2.1, 2H), 4.38
(t, J 1.8, 2H), 4.07 (s, 5H). dC (acetone) 179.2, 164.3, 138.5,
136.5, 136.1, 133.1, 132.4, 132.1, 129.3, 127.7, 126.4, 124.1,
84.4, 69.4, 69.3, 68.7. nmax /cmꢁ1 3310–3296 (b), 3101, 1662,
1592, 1532, 1283, 1150. Anal. Calc. for C24H19N2SeFeOBr:
C 50.93, N 4.94, H 3.35, Fe 9.86. Found: C 50.69, N 4.87,
H 3.28, Fe 9.74 %.
0.03
0.08
0.13
0.18
0.23
Synthesis of 1-(3-Bromobenzoyl)-3-(4-ferrocenylphenyl)
selenourea (P3Br)
[P3F]/[DNA]
Fig. 7. Effect of increasing concentrations of 1-(3-fluorobenzoyl)-3-
(4-ferrocenylphenyl)selenourea (P3F) on relative specific viscosity at
25 ꢅ 18C. [DNA] ¼ 70 mM and [P3F] ¼ 2.5–12.5 mM.
P3Br was obtained by mixing 0.3 g (0.00208 mol) of KSeCN
with 0.27 mL (0.00208 mol) of 3-bromobenzoyl chloride in
acetone[1] following the same procedure was used for P2Br.
Yield 74 %. Mp 1478C (dec.). dH (acetone) 11.00 (s, 1H), 9.70
(s, 1H) 8.39–7.37 (m, 8H), 4.78 (t, J 1.8, 2H), 4.33 (t, J 1.5, 2H),
4.07 (s, 5H). dC (acetone) 179.4, 162.1, 137.6, 135.8, 134.2,
133.8, 132.1, 131.1, 130.7, 127.7, 126.5, 124.6, 84.0, 69.4, 69.3,
68.9. nmax /cmꢁ1 3310–3164 (b), 3002, 1662, 1598, 1528, 1242,
1140. Anal. Calc. for C24H19N2SeFeOBr: C 50.93, N 4.94,
H 3.35, Fe 9.86. Found: C 50.81, N 4.79, H 3.31, Fe 9.77 %.
6 ꢀ 10ꢁ4 M from which working concentrations of DNA were
prepared. The concentration of the stock solution was
measured by UV absorbance at 260 nm using an epsilon value
of 6600 Mꢁ1 cmꢁ1. This DNA was protein free because A260
A280 . 1.8.[24]
/
CV was performed on a Biologic SP-300 Cyclic Voltmeter
running with EC-Laboratory Express V 5.40 software, Japan.
Before every reading the working electrode was polished with
alumina powder and rinsed with distilled water. Analytical
grade KCl was used as a supporting electrolyte and nitrogen
gas (99.9 %) was purged through the mixture to avoid interfer-
ence by oxygen.
Absorption spectra were recorded on a Shimadzu 1800
Spectrophotometer. First the spectra of the pure DNA solution
were recorded in the absence of drug and then in the presence of
different concentrations of drug. An equal amount of drug was
added to both reference and sample cells in order to avoid the
appearance of its peak at 260 nm where ferrocene also absorbs
(252 nm).
Synthesis of 1-(4-Bromobenzoyl)-3-(4-ferrocenylphenyl)
selenourea (P4Br)
P4Br was obtained by mixing 0.3 g (0.00208 mol) of KSeCN
with 0.27 mL (0.00208 mol) of 4-bromobenzoyl chloride in
acetone[1] following the same procedure used for P2Br. Yield
83 %. Mp 1458C (dec.). dH (DMSO) 11.13 (s, 1H), 10.72 (s, 1H)
7.97–7.38 (m, 8H), 4.76 (t, J 2.1, 2H), 4.33 (t, J 1.8, 2H), 4.02
(s, 5H). dC (DMSO) 178.3, 163.4, 137.1, 135.0, 134.6, 133.8,
131.1, 130.9, 127.6, 125.0, 84.4, 69.3, 69.1, 68.8. nmax /cmꢁ1
3210–3250 (b), 3022, 1655, 1587, 1540, 1238, 1117. Anal. Calc.
for C24H19N2SeFeOBr: C 50.93, N 4.94, H 3.35, Fe 9.86. Found:
C 50.90, N 4.68, H 3.29, Fe 9.83 %.
A Ubbelohde Viscometer was used for viscosity measure-
ments at room temperature (25 ꢅ 18C). Flow time was measured
with a digital stopwatch. Measurements were made in triplicate
for the measurement of average flow time. Data were presented
as relative specific viscosity (Z/Zo) versus the binding ratio
([P3F]/[DNA]) where Z is the viscosity of DNA in the presence
of complex and Zo is the viscosity of DNA alone.
Ferrocene, para-nitro-aniline, sodium nitrite, diethyl ether,
acetone, DMSO, Pd-charcoal, carboxylic acid chlorides, and
hydrazine were purchased from Sigma Aldrich. para-Ferrocenyl
aniline was synthesised by a procedure reported by our group
previously.[35]
Synthesis of 1-(2-Fluorobenzoyl)-3-(4-ferrocenylphenyl)
selenourea (P2F)
P2F was synthesised by mixing 0.3 g (0.00208 mol) of
KSeCN with 0.25 mL (0.00208 mol) of 2-fluorobenzoyl chlo-
ride in acetone[1] following the same procedure used for P2Br.
Yield 76 %. Mp 1448C (dec.). dH (acetone) 13.10 (s, 1H), 10.90
(s, 1H) 8.06–7.37 (m, 8H), 4.81 (t, J 2.1, 2H), 4.38 (t, J 2.1, 2H),
4.07 (s, 5H). dC (acetone) 179.8, 162.0, 140.5, 139.1, 135.5,
131.4, 128.8, 125.2, 124.7, 122.18, 121.8, 116.4, 84.4, 69.4,
69.1, 68.8. nmax /cmꢁ1 3410–3395 (b), 3107, 1660, 1587, 1512,
1279, 1128. Anal. Calc. for C24H19N2SeFeOF: C 57.14, N 5.50,
H 3.80, Fe 11.07. Found: C 56.99, N 5.41, H 3.74, Fe 10.98 %.
Synthesis
Synthesis of 1-(2-Bromobenzoyl)-3-(4-ferrocenylphenyl)
selenourea (P2Br)
Synthesis of 1-(3-Fluorobenzoyl)-3-(4-ferrocenylphenyl)
selenourea (P3F)
P2Br was synthesised by mixing 0.3 g (0.00208 mol) of
KSeCN with 0.27 mL (0.00208 mol) of 2-bromobenzoyl chlo-
ride in acetone.[1] The reaction was carried out in a two necked
round bottom flask for 3 h under constant stirring. After the
formation of a yellow coloured product with a suspension of
KCl, 0.576 g (0.00208 mol) of 4-ferrocenylaniline was added
P3F was synthesised by mixing 0.3 g (0.00208 mol) of
KSeCN with 0.25 mL (0.00208 mol) of 3-fluorobenzoyl chlo-
ride in acetone[1] following the same procedure used for P2Br.
Yield 71 %. Mp 1388C (dec.). dH (acetone) 13.20 (s, 1H), 10.70
(s, 1H) 7.88–7.55 (m, 8H), 4.81 (t, J 1.8, 2H), 4.33 (t, J 1.8, 2H),
4.05 (s, 5H). dC (acetone) 179.0, 165.2, 140.55, 131.1, 130.9,

634
R. A. Hussain et al.
130.8, 128.7, 126.4, 126.0, 124.3, 120.1, 119.7, 84.3, 69.4, 69.3,
69.1. nmax /cmꢁ1 3200–3400 (b), 3030, 1651, 1593, 1524, 1257,
1143. Anal. Calc. for C24H19N2SeFeOF: C 57.14, N 5.50,
H 3.80, Fe 11.07. Found: C 57.11, N 5.47, H 3.74, Fe 11.00 %.
[4] D. L. Klayman, R. J. Shine, Chem. Commun. 1968, 137, 372.
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[5] J. R. Andreesen, L. Ljungdahl, J. Bacteriol. 1973, 116, 867.
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Synthesis of 1-(4-Fluorobenzoyl)-3-(4-ferrocenylphenyl)
selenourea (P4F)
P4F was synthesised by mixing 0.3 g (0.00208 mol) of
KSeCN with 0.24 mL (0.00208 mol) of 4-fluorobenzoyl chlo-
ride in acetone[1] following the same procedure used for P2Br.
Yield 73 %. Mp 1518C (dec.). dH (acetone) 10.91 (s, 1H), 9.90
(s, 1H) 8.26–7.26 (m, 8H), 4.75 (t, J 1.8, 2H), 4.33 (t, J 1.8, 2H),
4.05 (s, 5H). dC (acetone) 179.0, 169.1, 137.8, 136.7, 131.08,
130.8, 129.8, 126.7, 126.1, 125.7, 84.1, 69.8, 69.7, 69.6.
n
1167. Anal. Calc. for C24H19N2SeFeOF: C 57.14, N 5.50,
H 3.80, Fe 11.07. Found: C 57.06, N 5.42, H 3.77, Fe 11.02 %.
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ꢄ Ferrocene and selenourea functionalities have been success-
fully combined in six ferrocene-incorporated selenoureas for
the first time by the reaction of para-ferrocenyl aniline with
different carboxylic acid chlorides substituted with halogens
in a one-pot procedure.
ꢄ Estimated DNA binding constant values for the synthesised
compounds are in the range of 103–104 Mꢁ1 which are
comparable or even better than most of the ferrocene deriva-
tives. Because of good binding behaviour with DNA these
compounds may be evaluated for their anticancer activities in
future.
ꢄ The synthesised ferrocene-incorporated selenoureas are sol-
uble in most non-polar (benzene, toluene) and polar (ethanol,
methanol, DMSO, acetonitrile) solvents. This will enhance
their possible applications not only in drug development (due
to enhanced lipophilicity) but also in material sciences (e.g. as
single source precursors for nanosized FeSe production and
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Supplementary Material
Supplementary material containing cyclic voltammetric and
crystallographic data are available on the Journal’s website.
Crystallographic data for the structures reported in this paper
have also been deposited at the Cambridge Crystallographic
Data Center as supplementary publication numbers CCDC Nos
897146, 897147, and 908933. Copies of the data are available
free of charge at deposit@ccdc.cam.ac.uk.
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Acknowledgement
This work was supported by the Higher Education Commission, Islamabad,
Pakistan. The authors thank Professor Afzal Shah for his help with
electrochemistry.
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