Preliminary investigation of anticancer activity by determining the DNA binding and antioxidant potency of new ferrocene incorporated N,N',N'-trisubstituted phenylguanidines

Article abstract of DOI:10.1016/j.saa.2013.08.033

Six new bioactive ferrocene based phenylguanidines were successively synthesized and characterized by means of various analytical techniques like elemental analysis, FT-IR, multinuclear (¹H and ¹³C) NMR, UV-Vis spectroscopy and cyclic voltammetry. The interaction of compounds with DNA was investigated by spectroscopic and cyclic voltammetric measurements. The interaction was found to be the electrostatic and the binding constants values were impressively larger. Compounds f-1, f-2, f-3 have slight larger binding constant values ranging from 0.8 × 10⁵ to 2.4 × 10 ⁵ as compared to g-1, g-2 and g-3 ranging from 7.6 × 10 ⁴ to 1.1 × 10⁵ which is most probably due to the presence of ferrocene at para position where the delocalization of electrons is maximum. Antioxidant activity was determined by UV-Vis spectrophotometer by using DPPH as a free radical. All the compounds exhibit good antioxidant activity and the results so obtained support the structure activity relationship.

Full text of DOI:10.1016/j.saa.2013.08.033

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 264–269
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Preliminary investigation of anticancer activity by determining
the DNA binding and antioxidant potency of new ferrocene
incorporated N,N0,N00-trisubstituted phenylguanidines
a
c
b
Rukhsana Gul a, Amin Badshah b, , Azim Khan , Asif Junaid , Muhammad Khawar Rauf
⇑
a Department of Chemistry, Gomal University, Dera Ismail Khan 29050, Pakistan
b Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
c Department of Michrobiology, Kohat University of Science and Technology, Kohat 26000, Pakistan
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The DNA binding potency of the synthesized guanidines (1–6) were determined by UV–Vis spectropho-
tometer and cyclic voltammeter by keeping the concentration of the compounds constant (A) while vary-
ing the amount of DNA (B–H). All the compounds showed electrostatic mode of interaction with DNA
having impressively high binding constants values (9.9 ꢁ 104–1.3 ꢁ 105 (Mꢂ1)). The compounds also
showed good antioxidant activities. These two parameters are helpful to design new anticancer drugs.
ꢀ
ꢀ
New ferrocenyl phenylguanidines.
Preliminary investigation of
anticancer potency.
DNA binding studies by UV and CV.
High binding constant values
(Kb = 105).
ꢀ
ꢀ
ꢀ
Good free radical scavengers.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 June 2013
Received in revised form 30 July 2013
Accepted 2 August 2013
Available online 13 August 2013
Six new bioactive ferrocene based phenylguanidines were successively synthesized and characterized by
means of various analytical techniques like elemental analysis, FT-IR, multinuclear (1H and 13C) NMR,
UV–Vis spectroscopy and cyclic voltammetry. The interaction of compounds with DNA was investigated
by spectroscopic and cyclic voltammetric measurements. The interaction was found to be the electro-
static and the binding constants values were impressively larger. Compounds f-1, f-2, f-3 have slight lar-
ger binding constant values ranging from 0.8 ꢁ 105 to 2.4 ꢁ 105 as compared to g-1, g-2 and g-3 ranging
from 7.6 ꢁ 104 to 1.1 ꢁ 105 which is most probably due to the presence of ferrocene at para position
where the delocalization of electrons is maximum. Antioxidant activity was determined by UV–Vis spec-
trophotometer by using DPPH as a free radical. All the compounds exhibit good antioxidant activity and
the results so obtained support the structure activity relationship.
Keywords:
Trisubstituted ferrocenyl phenylguanidines
DNA binding
Antioxidant activity
Ó 2013 Elsevier B.V. All rights reserved.
Introduction
Guanidines are the organic compounds containing three nitro-
gens with a central carbon atom and exhibits diverse biological
⇑
Corresponding author. Tel.: +92 5190642131; fax: +92 512873869.
E-mail address: aminbadshah@yahoo.com (A. Badshah).
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.08.033

R. Gul et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 264–269
265
applications [1–3]. Nitrogens of the guanidines have the ability to
donate electron pair so they can be used as super bases. The guan-
idines having electron donating groups attached with guanidine
moiety have strong basic character due to the more availability
of lone pair for donation, so such type of guanidines are less soluble
in lipids [4–7]. The introduction of electron withdrawing or conju-
gated substituents on nitrogen atoms can change the biological
properties of the guanidines by making these compound more
lipophylic or less basic vice versa. The synthesis of more lipophylic
guanidines is a field of interest for so many years because the lipo-
phylic compounds can easily migrate across the cell membrane.
A number of guanidinium compounds have been synthesized
which showed strong non-covalent interactions with anionic
groups or with DNA through hydrogen bonding and electrostatic
interactions due to the involvement of three nitrogens guanidine
core thus possesses anticancer activity [8–10]. Scott and his co-
workers have studied the biological activities of some variolin
and meriolin. Varioline has been found to have modest activities
against leukemia cell line. Meriolin (b) displays considerabaly en-
hanced cyclin-dependent kinase inhibition and cytotoxisity [11].
Some naturally occurring guanidines were screened for their
nuclease activity and exhibit cytotoxic properties [12]. Incorpora-
tion of ferrocenyl group into organic compounds often enhances
the biological action of the resulting products, like ferrocene deriv-
atives of tamoxifen called ferrocifen showed pronounced activity
against tamoxifen resistant breast cancer cells [13]. The low
cytotoxicity of ferrocene in biological systems, lipophilicity, stabil-
of thickness 0.5 mm with an exposed end of 10 mm as the counter
electrode and a platinum disc as working electrode. UV-Absorption
spectra were measured with a UV–Vis spectrometer; Shimadzu
1800. Herring sperm DNA were purchased from Acros Organics UK.
Procedure for the synthesis of compounds (1–6)
The syntheses of the ferrocenyl phenylguanidines were
achieved in four steps (Scheme-1 suppl). In the first step; nitrophe-
nylferrocene (a and b) were made by the coupling of ferrocene
with diazonium salts of nitroaniline using phase transfer catalyst
[20]. In the second step; these nitro phenyl ferrocene were reduced
into ferrocenylaniline (c and d) using palladium on charcoal and
hydrazine as reducing agent [21]. In the third step three different
chloro-substituted thioureas (e) were synthesized by the coupling
of substituted aniline with thiocynates in acetone [22]. In the
fourth step; the benzoylphenylthiourea (e) were mixed with the
ferrocenyl aniline (c and d) in dimethyl formamide (DMF) in equi-
molar ratio with two equivalents of triethylamine (Et3N). The tem-
perature was maintained below 5 °C using an ice bath and one
equivalent of mercuric chloride (HgCl2) was added to the reaction
mixture with vigorous stirring. The ice bath was removed after
30 min while the stirring continued overnight. The progress of
the reaction was monitored by thin layer chromatography (TLC) till
the completion of reaction. Chloroform (CHCl3, 20 ml) was added
to the reaction mixture and the suspension was filtered through
a sintered glass funnel to remove the mercuric sulfide (HgS) resi-
due. The solvents from filtrate were evaporated under reduced
pressure and residue was re-dissolved in dichloromethane (CH2Cl2,
20 ml), washed with water (4 ꢁ 30 ml) and dried the organic phase
over anhydrous magnesium sulfate (MgSO4). The solvent was
evaporated and residue was purified by column chromatography
to afford ferrocenyl phenylguanidines (f and g) [23]. Solid and li-
quid state characterization data is given in Supplemental part.
ity and the
p-conjugated system make ferrocene derivatives as
decent candidates for investigation of their biological applications
[14–17].
Keeping in view, it was thought worthwhile to synthesizing
some ferrocene based trisubstituted phenylguanidines having elec-
tron withdrawing groups in the form of chlorine, in order to in-
crease the binding potency of the compounds with DNA and
make them more free radical scavengers. Before going to expensive
cell line study it was decided to preliminary explore their antican-
cer potency of the compounds by determining the DNA interaction
and antioxidant activity, because ‘The control over DNA can control
different cancerous cell growth’ [18] and the free radicals are
among the major causes of cancer [19].
Results and discussion
Elemental analysis of the compounds (1–6) was in good agree-
ment with the calculated values which shows that the compounds
are sufficiently pure in bulk. In the FT-IR spectra two peaks, a sharp
and a weak, were observed for NAH bonds in the range 3366–
3266 cmꢂ1. The C@N stretching frequency in all compounds were
observed in the range of 1589–1555 cmꢂ1 which is an intermediate
between single and double bond, indicating conjugation between
all three nitrogen atoms of the guanidine moiety. A sharp C@O
stretch was also observed in the range of 1683–1667 cmꢂ1and a
characteristic peak for FeAC associated with ferrocene group was
observed in the range of 479–468 cmꢂ1 for the synthesized
compounds.
Experimental
Materials and apparatus
Ferrocene, 3-nitroaniline, 4-nitroaniline, sodium nitrate, hex-
adecyltrimetyl ammonium bromide, palladium on charcoal and
hydrazine, benzoic acid, thionyl chloride, NH4SCN, 2,4-dichloroan-
iline, 3,4-dichloroaniline, 3,5-dichloroaniline and mercury (II)
chloride were obtained from Fluka, Switzerland. Analytical grade
solvents like diethyl ether, triethylamine, dimethylformamide
(DMF), acetone, ethanol and dimethylsulfoxide (DMSO) were pur-
chased from Merck, Germany and used as supplied. The elemental
analyses were performed on a LECO-183 CHNS analyzer. The
melting temperature was determined on a Bio Cote SMP 10-UK
and reported uncorrected. The solid state Fourier transform infra-
red spectrum was recorded on Bio-Rad Excalibur FT-IR Model FTS
3000 MX. 1H and 13C NMR spectra were obtained on a Bruker
300 MHz NMR spectrophotometer in CDCl3 using tetra methyl
silane as an internal reference. Cyclic voltammetric measurements
were performed using Biologsp 300 Potentiostate. Tetrabutylam-
moniumperchlorate (TBAP) having 99% purity, supplied by Fluka
was used as an electrolyte. Measurements were carried out in a
single compartment cell with a three electrode configuration,
consisting of Ag/AgCl as a reference electrode, a thin platinum wire
1H and 13C NMR spectra of the compounds were recorded
relative to TMS as reference standard. All the NMR spectra were
taken in CDCl3 at room temperature. In 1H NMR, the ferrocenyl
protons appeared in the range of 4.1–4.8 ppm as three set of
signals (singlet, triplet, triplet) with relative integral values of
(5:2:2) protons [24]. NAH protons in all compounds appeared as
broad singlet in range 10.4–11.6 ppm. All the other aromatic and
non-aromatic protons appeared in their specific regions. In 13C
NMR spectra, the most down field signals appeared in the range
of 176–180 ppm are attributed to the most deshielded carbon of
the carbonyl group where as the guanidine carbon (CN3) appeared
relatively up-field in the range of 156–161 ppm. In all the
compounds (1–6), ferrocenyl carbon appeared at 65–84 ppm as
four signals with the relative intensities 5:2:2:1 of carbons. All
the other aromatic and non-aromatic carbon appeared in their
specific regions.

266
R. Gul et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 264–269
In UV–Vis spectroscopy all the compounds gave four bands,
purity of DNA (free from bound protein) was assessed from the ra-
tio of absorbance at 260 and 280 nm. The A260/A280 ratio was
1.80–1.90, showing that the DNA was sufficiently free from pro-
tein. At first the spectra of the compound before the addition of
DNA were recorded by taking solvent in the reference cell and
solution of the compound in the sample cell. Then the spectra were
recorded by adding different concentration of DNA in solutions
having constant concentration of the compound. The whole exper-
iment was carried out by keeping the volume and concentration of
the compound constant while varying the amount of DNA.
Peaks 1 and 4 were not much affected by the addition of DNA,
just a decrease in the absorption take place. Peak 2 showed a slight
red shift and 3 exhibited a blue shift. Both the peaks showed pro-
nounced hypochromic effect (Fig. 2) by the incremental addition of
DNA. In general a red shift is an indication of intercalation, in
which planer groups approaches the nitrogenous bases of DNA
three strong bands in the wavelength range of 200–285 nm while
a weak signal in visible region at about 450 nm (Fig. 1). The high
absorptivity bands in the UV-region of spectrum can be assigned
to the
p–
p⁄ transition of aromatic phenyl ring and C@N chromo-
phore. In literature it has been reported that the UV–Vis spectra
of ferrocene derivatives give two absorption bands originating
from ferrocene moiety. The following three spin-allowed ligand
field transitions are expected: 1A1g ? a1E1g
, , and
1A1g ? 1E2g
1A1g ? b1E1g. The first two transitions are unresolved and give rise
to the band at about 450 nm and the third transition is responsible
for the signal at 325 nm. Both bands are weak owing to the La-
porte-forbidden d–d character of ligand field transitions [25]. So
the weak bands in the test compounds may be assigned to the fer-
rocene based d–d transitions.
The electrochemical properties of the presented compounds
were investigated by CV on a glassy carbon electrode in 20% aque-
ous ethanol, with supporting electrolyte 0.1 M TBAP, in the concen-
and
p-stacking takes place between the p electrons of drug and
nitrogenous bases of DNA which lead to a decrease in the energy
gap between the highest (HUMO) and the lowest (LUMO) molecu-
lar orbitals. In present case a very minute bathochromic shift was
observed which may be due to partial intercalation or grove bind-
ing as classical intercalation lead to a shift of P15 nm.
The hypochromic effect and slight blue shift of peak 3 suggests
the electrostatic interaction of its corresponding chromophore
with DNA [27]. Thus it was suggested that the compound may
interact with DNA by mixed mode of binding (electrostatic and
grove binding). In compound 4 the peak 3 also showed a slight
red shift so here the grove binding may be dominant over
electrostatic interaction (Fig. 3). Further clues about the mode of
tration of 40 lM vs. standard Ag/AgCl, in the cathodic direction
from 0.0 V to +0.80 V at the scan rate of 100 mV sꢂ1. Ferrocene
moiety is well known (for its derivatives) to undergo easily one-
electron oxidation to the ferrocenium ion in a reversible manner.
The anodic peaks of compounds appeared in the range of 0.551–
0.585 V with corresponding cathodic peaks ranging 0.365–0.399
respectively. For simple ferrocene, oxidation peak was observed
at 0.518 V under the same conditions. The result reveals that the
electrochemical behavior of the oxidizing moiety of ferrocene can
be modulated by changing the electronic properties of the cyclo-
pentadienyl ring. The slight change in the redox behavior of the
studied compounds than pure ferrocene is attributed to the elec-
tron donating effect of NH in the presence of electronegative chlo-
rine atoms. This group makes the oxidation slightly difficult than
ferrocene is evidenced by a positive shift for compounds. In gen-
eral, the inductive effect works up to three or four bonds, however,
the minor change in the position of redox peak due to chloro group
suggests that the three nitrogens are in conjugation with each
other.
DNA binding studies
By UV–Vis spectrophotometer
Stock solutions of the compounds were prepared in 30% aque-
ous ethanol. DNA stock solution was also prepared. The concentra-
tion of DNA was determined spectrophotometrically at 260 nm
using molar extinction coefficient
e
= 6600 Mꢂ1 cmꢂ1 [26]. The
Fig. 2. UV–Vis spectrum of compound 1 in the absence (A) and presence of (B 30; C
40; D 50; E 60; F 70; G 80; H 90 lM) of DNA.
Fig. 3. UV–Vis spectrum of compound 6 in the absence (A) and presence of (B 30; C
40; D 50; E 60; F 70; G 80; H 90; I 100; J 110 M) of DNA.
Fig. 1. UV–Vis spectrum of ferroceney phenylguanidine 2.
l

R. Gul et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 264–269
267
Table 1
hydrogen bonding with DNA as compared to ferrocenyl anilines
having a single NH [28].
Binding constant values for ferrocenyl guanidines (1–6), simple phenylguanidines and
ferrocenyl anilines.
Compounds
Binding
Binding
By cyclic voltammetry
constant
constant
Stock solutions of the compounds of known concentrations
were prepared in 30% aqueous dimethyl sulfoxide (DMSO). The
voltammogram of the compounds solution was recorded after
flushing out oxygen by purging with argon gas for 10 min just prior
to each experiment. The procedure was then repeated for the sys-
tems with a constant concentration of the tested compound and
varying concentration of DNA [29]. The working electrode was
cleaned after every electrochemical assay.
The binding potency of the compounds with DNA was also
checked on cyclic voltammeter to find further clues about binding
mode. By the addition of DNA, the anodic peak is shifted in the neg-
ative going direction and Ipa is also dropped by 20%. The substantial
diminution in peak current is attributed to the formation of the
slowly diffusing compound–DNA supramolecular complex due to
which concentration of the free compound, mainly responsible
for the transfer of current is lowered. The mode of compound–
DNA interaction can be judged from the variation in peak potential
(Figs. 4 and 5).
In general, the positive shift called anodic shift in formal poten-
tial is caused by the intercalation of the drug into the double heli-
cal structure of DNA, while negative shift is observed for the
electrostatic interaction of the cationic drug DNA backbone [29].
The obvious negative peak potential shift in the CV behavior of
the compounds by the addition of DNA is attributed to the electro-
static interaction between the compound and the DNA. On the sim-
ilar lines, the negative peak potential shift called cathodic shift in
the CV behavior of all studied guanidines is related to the electro-
static interaction of positively charged ferrocenium state with the
negatively charged DNA.
(Mꢂ1) (UV)
(Mꢂ1) (CV)
f-1
f-2
f-3
g-1
g-2
g-3
1.22 ꢁ 105
1.6 ꢁ 105
0.8 ꢁ 105
9.9 ꢁ 104
0.1 ꢁ 105
9.9 ꢁ 104
1.6 ꢁ 103
7.8 ꢁ 103
1.31 ꢁ 105
2.4 ꢁ 105
1.1 ꢁ 105
1.1 ꢁ 105
0.84 ꢁ 105
7.61 ꢁ 104
5.6 ꢁ 103
1.4 ꢁ 104
N,N0-diphenyl-N00-benzoylguanidine (A)
N-phenyl-N0-(2,4-dichlorophenyl)-N00-
benzoylguanidine (B)
4-Ferrocenyl aniline [24]
3-Ferrocenyl aniline [30]
2.02 ꢁ 103
9.3 ꢁ 103
3.85 ꢁ 103
–
interaction can be obtained from the binding const values and also
from the cyclic voltammetric measurements.
Based upon the decrease in absorbance at the kmax, the values of
DNA binding constant for these compounds have been calculated
according to the following host guest equation I [27].
Ao
eG
ꢂ
eG
eG eHꢂG
1
¼
þ
ð1Þ
A ꢂ Ao eHꢂG
ꢂ
eG k½DNAꢃ
where Ao and A is the absorbance of the free compound and com-
pound–DNA adduct respectively and, eG and eH-G is the molar
extinction coefficient of the free compound and compound–DNA
adduct respectively. The slope to intercept ratio of the plot Ao/
(A ꢂ Ao) versus 1/[DNA] yields the binding constants of these com-
pounds and are reported in Table 1.
Iron of ferrocene is easier to oxidize in the presence of DNA be-
cause its oxidized form is more strongly bound to DNA than its re-
duced form (neutral form). This can also be explained by equation
II [29].
The binding constant values calculated for the ferrocenyl phe-
nylguanidines were compared with the guanidines themselves
having no ferrocene which were found to be lower than the pre-
sented compounds. Hence, the presence of ferrocene is concluded
to enhance the DNA binding activity of guanidines. This may be
due to the fact that in the presence of ferrocene the delocalization
of lone pair of nitrogen extended to cyclopentadienyl ring of ferro-
cene and the nitrogen become more polarized, stable and favorable
for electrostatically bind with negatively charged DNA (Scheme 2).
The binding constants results showed that the compounds having
ferrocene at para-position have slight larger binding ability as com-
pared to the meta-ferrocenyl phenylguanidines. This may be due to
more delocalization of electron when the cyclopentadienyl ring of
ferrocene is at para position. The binding constant values were also
compared with the ferrocenyl anilines (c and d). High binding
constant values revealed that the guanidine core having three
nitrogens is more likely to bind electrostatically or through
ꢀ
ꢁ
Kred
Koxi
E0b ꢂ E0f ¼ 0:059 log
ð2Þ
where Ef0 and Eb0 are the formal potentials of the ferrocenyl phenyl-
guanidines couple in the free and bound forms respectively. For a
shift of ꢂ17 mV caused by the addition of 40 M DNA into 3 mM
compound 1, a ratio of Kred/Koxi was calculated as 0.37, which indi-
cates the stronger interaction of the oxidized form of the compound
with DNA than the reduced form.
Based upon the decayin peak current of all the studied com-
pounds by the addition of different concentrations of DNA (30–
60-lM, B–E) the binding constant values were calculated accord-
ing to the following equation.
1
Kð1 ꢂ AÞ
¼
ð3Þ
½DNAꢃ 1 ꢂ ðI=I0Þ
where K is binding constant, I and I0 are the peak currents with and
without DNA and A is proportionality constant. The plot of 1/[DNA]
versus 1/(1 ꢂ I/I0) yielded binding constant values (Table 1). For the
determination of binding site size the following equation was used.
ꢂ
ꢃ
Cb
Cf
free base pairs
¼ K
ð4Þ
s
where s is the binding site size in terms of base pairs. Measuring the
concentration of DNA in terms of base pairs, the concentration of
the base pairs can be expressed as.
ꢂ
ꢃ
Cb
Cf
DNA
2s
¼ K
ð5Þ
Scheme 2. Increase in conjugation due to the presence of ferrocene at para
position.

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R. Gul et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 264–269
Fig. 6. In vitro Antioxidant assay chart of synthesized ferrocenyl phenylguanidines
(1–6) with
comparison to ascorbic acid (standard) and N,N0-diphenyl-N00-
a
benzoylguanidine (A).
Fig. 4. Voltammogram of 3 mM compound 1 in the absence (A) and presence of (B
20; C 40; D 60; E 80 lM) of DNA.
Scheme 3. Extended resonance due to the presence of ferrocene and electroneg-
ative chlorine atoms.
constant DPPH concentration were prepared in ethanol and
buffered at pH 6. At first the spectra of the DPPH solution before
the addition of compound was recorded by taking solvent in the
reference cell and solution of the analyte in the sample cell. Then
the spectra were recorded for different concentration of com-
pounds in solutions having constant concentration of the DPPH.
The whole experiment was carried out by keeping the volume
and concentration of the DPPH constant while varying the amount
of compounds concentration. The procedure was repeated by using
the ascorbic acid as standard.
Fig. 5. Voltammogram of 3 mM compound 3 in the absence (A) and presence of (B
20; C 40; D 60; E 80; F 100 M) of DNA.
l
Cf and Cb denote the concentration of the free and DNA-bound
species respectively. The Cb/Cf ratio was determined by the equation
given below.
Antioxidants had the ability to trap the free radicals. Highly
reactive free radicals and oxygen species are being found in
biological systems from different sources. These free radicals are
responsible for the oxidation of nucleic acids, proteins, lipids or
DNA and thus antioxidant inhibits the oxidative mechanisms
leading to degenerative diseases. The synthesized ferrocenyl
phenylguanidines have been analyzed by the DPPH assay. Antiox-
idants react with DPPH and can produce 1,1-diphenyl-2-picryl-
hydrazine. Due to odd electron, DPPH gives a strong absorption
band at 517 nm. In the presence of a free radical tested samples
and this assay is useful as a primary screening system. The activity
of tested compound (1–6) is reported here as IC50 values (Fig. 6).
Guanidines are the compound having two NH protons, and having
the ability to scavenge the free radical by losing the protons. After
trapping the free radical, the compound itself becomes a free
radical, which may be stabilized by the extended resonance due
to the presence of ferrocene and chloro substituents on phenyl ring
or by the formation of a dimer of guanidines. Results revealed that
all the compounds have slight high activity as compared to
plenylguanidines without ferrocene. This may be due to resonance
Cb I0 ꢂ I
¼
ð6Þ
Cf
I
where I and I0 represent the peak currents of the drug in the pres-
ence and absence of DNA. Putting the value of K as calculated
according to the equation III, the binding site size of 0.88 bp was ob-
tained from the plot of Cb/Cf versus [DNA]. The small value of s indi-
cates a dominant electrostatic interaction of ferrocenyl phenyl
guanidine 1 with DNA.
Antioxidant activity
Free radical scavenging activity (antioxidant activity) of the
ferrocenyl phenylguanidines (1–6) was performed by using
167 lM concentrations of 1,1-diphenyl-2-picrylhydrazyl (DPPH)
in ethanol [30–32]. The absorbance of DPPH without any additions
was stable over 30 min. Ethanol was purified and dried before use
according to the standard protocol. Test samples of 3.125, 6.25,
12.5, 25, 50, 100 lg/ml concentrations of the compounds with

R. Gul et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 264–269
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The work presented in the present paper describes the synthesis
of the six new ferrocene incorporated phenylguanidines and their
structural characterization by means of different analytical tech-
niques. High binding constant values and good antioxidant activity
reveal that the incorporation of ferrocene and electronegative
chlorine atoms can enhance the biological activities of guanidines
by making the compounds stable and more lipophylic. High binding
constant values and good antioxidant activities are those parame-
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