Synthesis, characterization and DNA binding studies of organoantimony(V) ferrocenyl benzoates

Article abstract of DOI:10.1016/j.jorganchem.2012.07.028

Organoantimony(V) ferrocenyl benzoates (OFBs) [p-(C₅H ₅FeC₅H₄)C₆H₄COO] ₂SbR₃ (R = C₆H₅, p-CH ₃C₆H₄) (3, 4) and [m-(C₅H ₅FeC₅H₄)C₆H₄COO] ₂SbR₃ (R = p-CH₃C₆H₄) (5) were synthesized and characterized by FT-IR, ¹H and ¹³C NMR, single crystal XRD and elemental analysis. The DNA interaction of the complexes (3-5) was investigated by UV-Vis spectroscopy and cyclic voltammetry (CV). The complex-DNA binding constant was found to vary in the sequence: K ₂ (4.80 × 10⁵) > K₁ (2.36 × 10⁴) > K₃ (1.87 × 10⁴) > K ₄ (6.61 × 10³) > K₅ (6.58 × 10³). The shift in peak potential, current and absorption maxima of OFBs in the presence of DNA revealed that CV coupled with UV-Vis spectroscopy could provide a convenient way to characterize complex-DNA interaction mechanism, a prerequisite for the design of new anticancer agents and understanding the molecular basis of their action.

Full text of DOI:10.1016/j.jorganchem.2012.07.028

Journal of Organometallic Chemistry 717 (2012) 1e8
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Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Synthesis, characterization and DNA binding studies of organoantimony(V)
ferrocenyl benzoates
,
a
a
a
Faiza Asghar a, Amin Badshah a , Afzal Shah , Muhammad Khawar Rauf , Muhammad Irshad Ali ,
*
Muhammad Nawaz Tahir b, Erum Nosheen a, Zia-ur-Rehman a, Rumana Qureshi a
a Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
b University of Sargodha, Department of Physics, Sargodha, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
Organoantimony(V) ferrocenyl benzoates (OFBs) [p-(C5H5FeC5H4)C6H4COO]2SbR3 (R ¼ C6H5, p-CH3C6H4)
(3, 4) and [m-(C5H5FeC5H4)C6H4COO]2SbR3 (R ¼ p-CH3C6H4) (5) were synthesized and characterized by
FT-IR, 1H and 13C NMR, single crystal XRD and elemental analysis. The DNA interaction of the complexes
(3e5) was investigated by UVeVis spectroscopy and cyclic voltammetry (CV). The complex-DNA binding
constant was found to vary in the sequence: K2 (4.80 ꢀ 105) > K1 (2.36 ꢀ 104) > K3 (1.87 ꢀ 104) > K4
(6.61 ꢀ 103) > K5 (6.58 ꢀ 103). The shift in peak potential, current and absorption maxima of OFBs in the
presence of DNA revealed that CV coupled with UVeVis spectroscopy could provide a convenient way to
characterize complex-DNA interaction mechanism, a prerequisite for the design of new anticancer agents
and understanding the molecular basis of their action.
Received 23 May 2012
Received in revised form
4 July 2012
Accepted 15 July 2012
Keywords:
Organoantimony(V) ferrocenyl benzoates
Single crystal XRD
Cyclic voltammetry
UVeVis spectroscopy
Complex-DNA interaction
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
of chemotherapy due to their efficient therapeutic index and
10-fold lower toxicity than trivalent analogues [17e19]. The major
Synthesis, structural aspects and antitumor activities of ferro-
cenyl benzoateeantimony complexes have been the subject of
intensive investigations [1e4]. Among the organometallic
compounds, ferrocene has captured the utmost attention for
conjugation with biomolecules due to its stability in aqueous
media, large variety of derivatization, facile oxidation and the
unique property of retaining simple one electron redox behavior
after the introduction of substituents at the cyclopentadienyl rings
[5e7]. Medicinal application of ferrocene is an active area of
research and many reports have shown that ferrocenyl derivatives
are endowed with interesting cytotoxic [8e10], antitumor [11,12],
antifungal [13], antimalarial [14] and DNA-cleaving activities [15].
Antimony was first introduced into medicinal treatment in 16th
century and gained a growing interest due to its emetic effect [16].
The organic derivatives of the element in both trivalent and
pentavalent states are known which leads to many different classes
of compounds that have various therapeutic and technological
applications. The pentavalent antimonials became the first choice
clinical use of organoantimonials is in the treatment of leishman-
iasis. Some organoantimony derivatives such as antimony tartrate
[20] and sodium stibogluconate [21,22] have been reported to
exhibit significant anti-cancer and anti-viral activities as well.
Deoxyribonucleic acid, DNA, is a molecule of great biological
significance since it contains all the genetic information for cellular
function. The interaction of small molecules to DNA is gaining wide
spread importance. This interaction may be summarized in three
ways: (a) electrostatic, (b) groove binding and (c) intercalation
[23,24]. A variety of analytical techniques have been used to study
the drug-DNA interactions such as luminescence [25], fluorescence
[26], UVevisible spectroscopy [27], and voltammetric methods
[28e30]. But the electrochemical methods are most widely used due
to their advantages like high sensitivity, cost effectivity, more reli-
ability and fast detection ability. UVeVis spectroscopic technique is
also best suited to ferrocenes owing to their intense colors. In this
paper, we have synthesized organoantimony derivatives of m-fer-
rocenylbenzoic acid and p-ferrocenylbenzoic acid (3e5), which
contain three activecenters, namely the organoantimony (V) moiety
and two ferrocenyl carboxylate groups. The synthesis and charac-
terization of compound (5) bis(p-ferrocenylbenzoato)tri(p-tolyl)
antimony(V) have already been reported by Yu et al. [1], but crys-
tallographic and electrochemical studies havenot yet been reported.
* Corresponding author. Tel.: þ92 5190642131; fax: þ92 5190642241.
E-mail addresses: aminbadshah@yahoo.com (A. Badshah), mkhawarrauf@
yahoo.co.uk (M.K. Rauf).
0022-328X/$ e see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jorganchem.2012.07.028

2
F. Asghar et al. / Journal of Organometallic Chemistry 717 (2012) 1e8
The main objective of the present work is to investigate the DNA
binding potential of these compounds by spectrophotometric and
electrochemical techniques. In addition we are interested in
studying the tunable redox and spectroscopic properties of the DNA
binding ferrocenes for the provision of useful insights about the
design of anticancer drugs and understanding of unexplored path-
ways by which such compounds exert their biochemical action.
modifications [1]. To a double neck round bottom flask sodium salts
of m-ferrocenyl benzoic acid (0.01 mol) or p-ferrocenyl benzoic acid
(0.01 mol) was added in 50 ml of chloroform and the reaction
mixture was allowed to stir for about an hour. Then (0.005 mol)
organo-antimony(V) was added and the suspension was refluxed
for 5e6 h. The progress of the reaction mixture was maintained by
TLC (chloroform/n-hexane 40:60). Precipitates of the salt formed
were filtered off and the filtrate was evaporated under vacuum.
Residue was dissolved in dichloromethane/chloroform and kept
for crystallization (Scheme 1). The synthesis and characterization
of compound (5) bis (p-ferrocenylbenzoato)tri( p-tolyl)anti-
mony(V) have already been reported by Yu et al. [1], but crystal-
lographic and electrochemical studies have not yet been reported.
2. Experimental
2.1. Materials and methods
All the manipulations were carried out under the specified
conditions of temperature. Solvents were distilled from the drying
agents and degassed before use.
2.2.1. Bis (m-ferrocenylbenzoato)triphenylantimony(V) (3)
NMR spectra were recorded on a Bruker ARX, 300 MHz spec-
trometer. 1H NMR (300 MHz): internal standard solvent CDCl3
(7.28 ppm from TMS): internal standard TMS; 13C NMR
(75.47 MHz): internal standard solvent CDCl3 (77.0 ppm from TMS):
internal standard TMS; the splitting of proton resonances in the
reported 1H NMR spectra are defined as s ¼ singlet, d ¼ doublet,
t ¼ triplet, q ¼ quartet and m ¼ complex pattern; coupling
constants are reported in Hz. FT-IR spectra were recorded as KBr
Quantities used were 3.10 g (0.01 mol) m-ferrocenyl benzoic
acid and 2.56 g (0.005 mol) organo-antimony(V); Yield 79%; Brown
solid; m.p. 190e192 ꢂC; FT-IR (KBr, cmꢁ1) 3021 (CeHaromatic), 1651
(C]O), 1600 (C]C), 534e582 (Sb-O), 482 (Fe-Cp), 443e461
(Sb-C); 1H NMR (300 MHz, CDCl3, 25 ꢂC)
d 4.03 (s, 10H, C5H5),
4.33 (t, 4H, C5H4, J ¼ 1.8 Hz), 4.65 (t, 4H, C5H4, 3J ¼ 1.8 Hz), 7.32 (d,
2H, ArH, 3J ¼ 7.8 Hz), 7.53e7.61 (m, 11H, ArH), 7.81 (d, 2H, ArH,
3J ¼ 7.8 Hz), 8.04 (s, 2H, ArH), 8.20 (dd, 6H, ArH, 3J ¼ 7.5 Hz,
pellets on
a
Bio-Rad Excalibur FT-IR Model FTS 3000 MX
4J ¼ 4.2 Hz); 13C NMR (75.47 MHz, CDCl3, 25 ꢂC)
d 66.7 (4C, meta on
(400e4000 cmꢁ1) and on ATR with Perkin Elmer System 2000
(200e500 cmꢁ1). The elemental analyses were performed using
a LECO-932 CHNS analyzer. The melting points were determined on
a Bio Cote SMP10- UK and are uncorrected. All solvents were
purified by distillation, dried under an atmosphere of nitrogen and
C5H4), 69.1 (4C, ortho on C5H4), 69.6 (10C, C5H5), 84.7 (2C, C5H4),
127.4 (2C, Ar), 127.6 (2C, Ar), 128.1 (2C, Ar), 129.5 (6C, Ar), 131.3 (3C,
Ar), 132.8 (2C, Ar), 133.9 (6C, Ar), 137.9 (2C, Ar), 139.4 (3C, ipso-C,
Ar), 170.1 (2C, C]O); Anal. Calcd. for C52H41Fe2O4Sb: C, 64.83; H,
4.29; Found: C, 64.78; H, 4.26.
ꢀ
stored over molecular sieves 4 A.
Commercial salmon sperm DNA was gifted by Prof. Dr.
Muhammad Siddiq. Its stock solution of 2.50 ꢀ 10ꢁ4 M was
prepared in doubly distilled water and stored at 4 ꢂC. The
concentration of the stock solution was determined from UV
2.2.2. Bis (m-ferrocenylbenzoato)tri(p-tolyl)antimony(V) (4)
Quantities used were 3.10 g (0.01 mol) m-ferrocenyl benzoic
acid and 2.77 g (0.005 mol) organo-antimony(V); Yield 82%; Brown
solid; m.p. 184e185 ꢂC; FT-IR (KBr, cmꢁ1) 3065e3092 (CeHaromatic),
2856e2922 (CeHaliphatic), 1643 (C]O), 1598 (C]C), 535e574
(SbeO), 481e491 (FeeCp), 447e456 (SbeC); 1H NMR (300 MHz,
absorbance at 260 nm using a molar extinction coefficient ( ) of
3
6600 Mꢁ1 cmꢁ1. A ratio of absorbance at 260 and 280 nm of (A260
A280) > 1.8 indicated protein free DNA.
/
CDCl3, 25 ꢂC)
d 2.41 (s, 9H, eCH3), 4.04 (s, 10H, C5H5), 4.34 (t, 4H,
Cyclic voltammetric experiments were performed using
m
Au-
meta on C5H4, J ¼ 1.8 Hz), 4.66 (t, 4H, ortho on C5H4, J ¼ 1.8 Hz),
7.21e7.33 (m, 2H, ArH), 7.36 (d, 2H, ArH, 3J ¼ 8.1 Hz), 7.59 (d, 2H,
ArH, 3J ¼ 7.8 Hz), 7.81 (d, 2H, ArH, 3J ¼ 7.8 Hz), 8.05 (s, 2H, ArH), 8.08
tolab running with GPES 4.9 software, Eco-Chemie, The
Netherlands. A glassy carbon (GC) (A ¼ 0.07 cm2) was used as
working electrode, a Pt wire served as counter electrode and
a saturated calomel electrode (SCE) was employed as the reference.
Before each experiment the surface of GCE was polished with
alumina powder followed by thorough rinsing with distilled water.
Electrochemical grade tetrabutylammonium fluoroborate (TBAFB)
was used as supporting electrolyte. All the voltammetric experi-
ments were conducted in a high purity nitrogen (99.995%) atmo-
sphere at room temperature (25 ꢃ 1 ꢂC).
(d, 6H, ArH, 3J ¼ 8.1 Hz); 13C NMR (75.47 MHz, CDCl3, 25 ꢂC)
d 21.6
(3C, eCH3), 66.7 (4C, meta on C5H4), 69.0 (4C, ortho on C5H4), 69.6
(10C, C5H5), 84.8 (2C, C5H4), 128.0 (3C, Ar), 128.3 (2C, Ar), 129.1 (2C,
Ar), 129.5 (2C, Ar), 130.3 (6C, Ar),133.2 (2C, Ar), 133.8 (6C, Ar), 134.2
(2C, Ar), 139.3 (2C, Ar), 141.5 (3C, ipso-C, Ar), 169.9 (2C, C]O); Anal.
Calcd. for C55H47Fe2O4Sb: C, 65.70; H, 4.71; Found: C, 65.63; H, 4.69.
2.2.3. Bis (p-ferrocenylbenzoato)tri(p-tolyl)antimony(V).chloroform
solvate (5) [1]
Quantities used were 3.10 g (0.01 mol) p-ferrocenyl benzoic acid
and 2.77 g (0.005 mol) organo-antimony(V); Yield 84%; Orange
solid; m.p. 205 ꢂC (dec); FT-IR (KBr, cmꢁ1) 3059 (CeHaromatic),
Absorption spectra were recorded on Shimadzu 1601 spectro-
photometer. First the spectra of the analytes were recorded in the
absence of DNA and then in the presence of different concentration
of DNA by keeping the volume and concentration of the compound
constant. Equal amount of DNA was added to both reference and
sample cells in order to avoid the appearance of its peak at about
260 nm where ferrocene also absorbs.
Chemicals ferrocene, m-nitroaniline, p-nitroaniline, sodium
nitrite, antimony(III) chloride, bromobenzene, p-bromotoluene and
solvents were purchased from SigmaeAldrich and used after
purification, where needed.
2923e2852 (CeHaliphatic). 1625 (C]O), 1605 (C]C), 566 (SbeO),
1
476 (FeeCp), 456 (SbeC); H NMR (300 MHz, CDCl3, 25 ꢂC)
d 2.40
(s, 9H, eCH3), 4.04 (s, 10H, C5H5), 4.37 (s, 4H, meta on C5H4), 4.68 (s,
4H, ortho on C5H4), 7.34 (d, 6H, ArH, 3J ¼ 8.1 Hz), 7.45 (d, 4H, ArH,
3J ¼ 8.4 Hz), 7.88 (d, 4H, ArH, 3J ¼ 8.4 Hz), 8.05 (d, 6H, ArH,
2.2. General procedure for the synthesis of organo-antimony (V)
ferrocenyl benzoates (3e5)
The organo-antimony (V) ferrocenyl benzoates have been
synthesized according to
a
reported method with some
Scheme 1. Synthesis of organoantimony(V) ferrocenyl benzoates (3e5).

F. Asghar et al. / Journal of Organometallic Chemistry 717 (2012) 1e8
3
3J ¼ 8.1 Hz); 13C NMR (75.47 MHz, CDCl3, 25 ꢂC)
d
21.6 (3C, eCH3),
(3) gives a doublet of a doublet for six protons and a multiplet
integrating for nine protons. The p-tolyl groups attached to anti-
mony (4, 5) appear as two doublets that each integrate for six
protons.
66.8 (4C, meta on C5H4), 67.9 (4C, ortho on C5H4), 69.5 (10C, C5H5),
83.9 (2C, C5H4), 125.4 (4C, Ar), 130.0 (4C, Ar), 130.2 (6C, Ar), 130.4
(2C, Ar), 133.8 (6C, Ar), 134.5 (3C, Ar), 141.3 (2C, Ar), 143.7 (3C, ipso-
C, Ar), 169.8 (2C, C]O); Anal. Calcd. for C56H48Cl3Fe2O4Sb: C, 59.80;
H, 4.30; Found: C,59.76; H,4.28.
In the 13C-NMR spectra of compounds (3e5) the carbonyl
carbon atoms appear in the narrow range of
ferrocenyl carbon atoms show signals in the region
The ipso-carbon of the (
5-C5H4) ring appears in the range of
83.9e84.8. The unsubstituted cyclopentadienyl (
5-C5H5) ring
gives an intense peak at 69.5e69.8, while for substituted cyclo-
pentadienyl (
5-C5H4) ring the ortho and meta carbon atoms have
chemical shifts in the region 66.7e69.5. In the case of meta
d
169.8e170.2. The
d
66.7e84.8.
2.3. Data collection and structural refinement of 5
h
d
h
Crystals of 5 was grown by the slow evaporation method in
toluene/chloroform (0.25:1). The reddish crystals were mounted in
a random orientation on a glass fiber and aligned on a Bruker kappa
d
h
d
APEXII CCD diffractometer using graphite-monochromated Mo-K
a
derivatives (3, 4) all the carbon atoms of the aromatic ring are non-
equivalent, therefore six signals are observed. While the para
substituted derivative (5) display four signals.
All the synthesized complexes were characterized by elemental
analysis using a CHNS analyzer. The results indicated that the ligand
to metal ratio in complexes is 1:2 which was further confirmed by
the crystal structure of the complex 5.
ꢀ
radiation (
l
¼ 0.71073 A). Data collection used
u scans, and a multi-
scan absorption correction was applied. The structure was solved
by using SHELXS97 program [31]. The hydrogen atoms were
generated by geometrical considerations. Final refinement on F2
carried out by full matrix least-squares techniques using SHELXL-
97. The disordered cyclopentadienyl’s were refined in two groups
as regular pentagons having equal occupancy ratio. The disordered
C atoms were refined as anisotropic with equal thermal parameters
in groups. The Cl atoms of the chloroform were refined over three
positions with occupancy ratio 0.60:0.20:0.20 and refined as
anisotropic with similar thermal parameters. Table 1 (suppl)
summarizes the crystal data and refinement values.
3.3. Structural studies
Product 5 grew in a monoclinic system with the P21/n space
group. The important geometric parameters are given in Table 1.
The complex adopts an approximate trigonal bipyramidal Sb
coordination environment with unidentate carboxylate ligands in
3. Results and discussion
ꢀ
the axial positions [SbeO: 2.114(3)e2.143(3) A, OeSbeO:
175.30(14)ꢂ] at trans orientation with respect to ferrocenyl
moiety. The three p-tolyl groups are in the equatorial plane and
show a distortion towards square-based pyramidal with a widening
of one of the trigonal angle [CeSbeC: 143.0(2)ꢂ]. The cyclo-
pentadienyl ring was coplanar with the adjacent phenyl ring
[dihedral angles 5.08 (18)]. The cyclopentadienyl ring A (C29eC33),
phenyl rings B (C23eC28) and C (C40eC45) are planar with r.m.s.
3.1. Chemistry
Two derivatives of ferrocene carboxylic acids (1, 2), m-ferrocenyl
benzoic acid (1) and p-ferrocenyl benzoic acid (2) were synthesized
by using a standard method [32]. By reacting these ligands with
organoantimony(V) complexes,
a series of hetero-bimetallic
complexes (3e5), namely: bis(m-ferrocenylbenzoato)tripheny-
lantimony(V) (3) bis(m-ferrocenylbenzoato)tri(p-tolyl)antimony(V)
(4) and bis(p-ferrocenylbenzoato)tri(p-tolyl)antimony(V) (5), were
also synthesized (Scheme 1). The purity of the ligands and
complexes was checked by TLC with Merck Kieselgel GF 254 Plates,
elemental analysis, 1H- and 13C-NMR (Bruker ARX, 300 MHz
spectrometer).
ꢀ
deviations of 0.0011, 0.0106 and 0.0037 A, respectively. The dihedral
angle between A/B is 5.08 (18)ꢂ which shows that central phenyl
ring is almost planar with attached cyclopentadienyl. The dihedral
angle between B/C is 11.07 (14)ꢂ. Three cyclopentadienyl rings of
ferrocene are disordered over two set of sites with equal occupancy
ratios. Selected bond lengths and bond angles are summarized in
Table 1 (Fig. 1).
3.2. Spectroscopic studies
3.4. Cyclic voltammetry of OFBs interacting with DNA
The ferrocene carboxylic acids and their antimony(V) complexes
(3e5) were synthesized in good yield, which were characterized by
different analytical techniques. The FT-IR spectra of the compounds
have been recorded in the range of 4000e400 cmꢁ1. In the spectra
of all the compounds no characteristic band was observed in the
The CV of 1 mM OFB (3) in the absence and presence of 2e20 mM
DNA at polished GCE has been shown in Fig. 2. The voltammogram
without DNA featured a couple of robust peaks in the potential
range of 0.0e1.5 V. The anodic and cathodic peaks appeared at 0.96
and 0.84 V versus SCE, while simple ferrocene was found to oxidize
at 0.52 V under the same conditions. The difference of 0.44 V in the
range of 3500e3300 cmꢁ1 due to
n(OH), indicating that deproto-
nation and coordination of antimony metal with carboxylate group
have occurred as was expected. The absorption bands for the
carbonyl groups are observed in the region 1651e1625 cmꢁ1. In
addition, the absorption frequencies due to SbeO bond are
observed at 532e582 cmꢁ1, while that for SbeC bond appears at
Table 1
ꢂ
ꢀ
Selected bond lengths (A) and angles ( ) for 5.
Sb(1)eC(1)
Sb(1)eC(15)
Sb(1)eO(3)
Sb(1)eC(8)
Sb(1)eO(2)
Fe(2)eC(33)
Fe(2)eC(32)
Fe(2)eC(31)
Fe(2)eC(29)
Fe(2)eC(30)
O(1)eC(22)
O(4)eC(39)
2.113(5)
2.106(5)
2.114(3)
2.114(4)
2.143(3)
2.018(5)
2.028(5)
2.036(6)
2.038(4)
2.044(6)
1.225(7)
1.217(7)
C(1)eSb(1)eC15
C(1)eSb(1)eO(3)
C(15)eSb(1)eO(3)
C(1)eSb(1)eC(8)
C15eSb(1)eC(8)
O3eSb(1)eC(8)
C(1)eSb(1)eO(2)
C(15)eSb(1)eO(2)
O(3)eSb(1)eO(2)
C(8)eSb(1)eO(2)
C(33)eFe(2)eC(32)
C(33)eFe(2)eC(31)
107.32(19)
90.16(15)
87.10(16)
143.0(2)
443e461 cmꢁ1
.
The 1H NMR spectra of complexes (3e5) show that the un-
substituted cyclopentadienyl (
5-C5H5) rings give a singlet of high
intensity at 4.0. In the case of substituted (
5-C5H4) ring the ortho
protons appear in the region 4.65e4.69 while the meta protons
occur in the range of 4.33e4.37. The splitting pattern for the meta
h
109.7(2)
d
h
90.48(15)
91.67(15)
88.22(17)
175.30(14)
90.68(15)
40.4(2)
d
d
derivatives (3, 4) consisted of a singlet, two doublets and a multi-
plet that each integrated for two protons. For the para substituted
derivative (5) the pattern observed was two doublets that each
integrate for four protons. The phenyl groups attached to antimony
67.8(2)

4
F. Asghar et al. / Journal of Organometallic Chemistry 717 (2012) 1e8
Fig. 1. Molecular diagram of 5 with non-hydrogen atoms represented by 30% probability boundary spheres and hydrogen atoms as spheres of arbitrary size. The CHCl3 solvent in the
lattice has also been shown.
oxidation potentials of 3 and ferrocene is attributed to the electron
withdrawing effect of benzoate group attached to the
cyclopentadienyl ring of ferrocene, which causes its oxidation
peak potentials of 3 shifted cathodically with the decrease in peak
currents. The substantial diminution in peak currents is attributed
to the formation of slowly diffusing 3-DNA supramolecular
complex due to which the concentration of the free drug (mainly
responsible for the transfer of current) is lowered.
difficult. The electrochemical signal at 0.96
V reflects the
oxidation of the ferrocenyl group of 3 to ferrocenium state, which
gets reduced to its neutral form upon scan reversal. The anodic
and cathodic peak current ratio (Ipa/Ipc) of 1 is suggestive of
reversible electrochemical process. In the presence of increasing
The DNA binding mode of a drug can be judged from the vari-
ation in formal potential. In general, positive shift in formal
potential is observed for intercalation of the drug into the double
helical structure of DNA [33], while negative shift is related to
electrostatic interaction of cationic drug with anionic phosphate of
DNA backbone [34]. So the obvious negative peak potential shift
(cathodic shift) in the CV behavior of 3 is related to the electrostatic
interaction of its positively charged ferrocenium state with the
negatively charged oxygen of DNA. Like 3 the CVs of 4 and 5 (see
Fig. 1 of Supplementary material) indicated the dominance of
electrostatic interaction with DNA. Such an interaction is expected
to induce perturbation in the normal functioning of DNA which
could presumably culminate in the damage of its replication
machinery and ultimate death of cancerous cell. The peak potential
shift in the cathodic direction further indicates that Fe (II) of 3 is
easier to oxidize in the presence of deoxyribonucleic acid because
its oxidized form is more strongly bound to DNA. For such a system,
where both forms of the drug interact with DNA, Scheme 2 can be
applied [35].
concentration (2e20
mM) of DNA, the oxidation and reduction
a
10
A
µ
f
Based upon the process discussed in Scheme 2, the following
equation is obtained [36]
0.25
0.5
0.75
1
1.25
1.5
Ebꢂ ꢁ Efꢂ ¼ 0:059 logðKred=Kox
Þ
(1)
E / V vs. SCE
Fig. 2. CVs of 1 mM 3 in the (a) absence and presence of (b) 2, (c) 8 (d) 12, (e) 16 and (f)
20 M DNA in 10% aqueous ethanol (10% H2O:90% ethanol).
: 100 mVsꢁ1, supporting
electrolyte: 0.1 M TBAFB.
where Eꢂf and Eꢂb are the formal potentials of 3 (II)/3 (III) couple in
the free and bound forms respectively.
m
y

F. Asghar et al. / Journal of Organometallic Chemistry 717 (2012) 1e8
5
The interaction of 3 with DNA can be described by the following
equation:
3 þ DNA43 ꢁ DNA
(2)
An equation for amperometric titrations can be deduced as [37,38],
Scheme 2. General redox process of the free and DNA bound 3.
logð1=½DNAꢄÞ ¼ log K þ logðI=ðIo ꢁ IÞÞ
(3)
where K is the binding constant, Io and I are the peak currents of the
drug in the absence and presence of DNA respectively.
For a shift of 40 mV caused by 1 mM 3 in the presence of 20 mM
The decrease in peak current of 3 in the presence of increasing
concentration of DNA was utilized for the evaluation of binding
constant. The plot of log (1/[DNA]) versus log (I/(Io ꢁ I)) (Fig. 4)
DNA (Fig. 2) a ratio of Kred/Kox was calculated as 0.21, which indi-
cates 4.76 times stronger interaction of the oxidized form of the
drug than the reduced form.
yielded K ¼ 1.87 ꢀ 104
M
ꢁ1, which is greater than the binding
The CVs of 1 and 2 showed a dramatically different behavior in
the presence of DNA. An increase in peak current of 1 along with
cathodic peak potential shift was noticed (Fig. 3A) which may be
related to the interaction of 1 with DNA in such a manner as to
cause exposition of its nitrogenous bases. As DNA bases have been
reported to oxidize in this region so their oxidation may have
contribution in peak current elevation. In contrast to other ferro-
cene derivatives, the cathodic peak potential of 2 registered anodic
shift along with peak current decay in the presence of DNA (Fig. 3B).
Such CV characteristics can be related to the intercalation of the
planar part of 2 into the stacked base pair pockets of deoxy-
ribonucleic acid.
constant (K ¼ 3.85 ꢀ 103
M
ꢁ1) of a reported ferrocene based
compound, p-nitrophenyl ferrocene [39].
The binding constants of 1, 2, 4 and 5 were also calculated
according to Eq. (3).
The binding constant varied in the sequence: K2
(4.80 ꢀ 105) > K1 (2.36 ꢀ 104) > K3 (1.87 ꢀ 104) > K4
(6.61 ꢀ103) > K5 (6.58 ꢀ 103). The greater DNA binding affinity of 2
can be related to the mixed binding mode including electrostatic
interaction (as verified by the negative shift of anodic peak in Fig. 3)
of the iron of ferrocenyl group with anionic phosphate backbone of
DNA and intercalation (as evidenced by the positive shift of
cathodic peak potential) of the planar benzoate moiety into the
base pair pockets of DNA. The comparatively high K value of 2
indicates its effectiveness as potential anticancer drug. The lower K
value of 5 than 2 can be attributed to the presence of two ferrocenyl
groups that block the planar moiety to intercalate. The same
attribution is responsible for the lower K values of 3 and 4 than 1.
The diffusion coefficients of the free and DNA bound ferrocene
derivatives were determined by using RandleseSevcik equation
A
b
a
5
A
µ
Ipa ¼ 2:69 ꢀ 105 n3=2ACD1=2n1=2
(4)
where Ipa is the anodic peak current in amperes, n is the number of
electrons transferred during oxidation, A is the geometric area of
the electrode in cm2, D is the diffusion coefficient in cm2 sꢁ1, C is the
bulk concentration of the reductant in mol cmꢁ3 and
rate in V sꢁ1 [40].
n is the scan
The results revealed that the values of diffusion coefficient of the
DNA-bound ferrocenes are lower as compared to free ferrocenes
due to the formation of slowly diffusing heavy drug-DNA adducts.
The values of diffusion coefficient varying in the sequence: D2
(9.93 ꢀ 10ꢁ5) > D3 (9.94 ꢀ 10ꢁ6) > D4 (6.41 ꢀ 10ꢁ6) > D1
(2.44 ꢀ 10ꢁ6) > D5 (1.10 ꢀ 10ꢁ7) are comparable with other
0.3
0.5
0.7
0.9
1.1
1.3
1.5
/ V vs. SCE
E
B
a
5.4
b
20
A
µ
y = 0.7329x + 4.2707
5.2
2
R = 0.9848
5
4
K = 1.87 x 10 M
-1
4.8
4.6
0.5
0.7
0.9
1.1
1.3
1.5
0.25
0.5
0.75
1
1.25
1.5
E / V vs. SCE
log (I /I o-I )
Fig. 3. CVs of 1 mM (A) 1 and (B) 2 in the (a) absence and (b) presence of 12
100 mV/s.
mM DNA at
Fig. 4. Plot of log (1/[DNA) vs. log (I/Io ꢁ I) for the determination of 3-DNA binding
constant.

6
F. Asghar et al. / Journal of Organometallic Chemistry 717 (2012) 1e8
ferrocene derivatives reported in literature [41e43]. The relatively
high D value of 2 may be due to its smaller molecular mass and
more electron deficient iron due to better electron withdrawing
ability of benzoate (attached at para position) from ferrocenyl
group. The greater D value of 3 than 4 and 5 support the idea that
molecules with smaller molecular mass diffuse quickly to the
electrode surface. Generally, drugs interacting with DNA by inter-
calative mode have an order of magnitude lower D than their free
forms while a slight decrease is observed for electrostatically
interacting drugs. Based upon this criterion, all the ferrocene
derivatives except 2 are suggested to interact with DNA via elec-
trostatic interaction as the dominant mode. The comparatively
larger difference in the D values of free and DNA bound 2 is
attributed to the involvement of intercalation. The CVs of 5 and the
plot between Ipa and n1/2 have been shown in supplementary
materials as Fig. 3. The linearity of the plot indicates that the
electrochemical process is diffusion controlled [44].
that the UVeVis signature can be modulated by the carboxyl
group attached at para and meta position.
The UVeVis spectra of 10e50
mM 5 can be seen in Fig. 4 of
supplementary materials. The absorption coefficients of all peaks
in the spectra of para and meta ferrocenyl derivatives were eval-
uated. The para derivatives showed four bands in UV and one in
visible region while the meta derivatives registered three enve-
lopes in the UV and one in the visible part of the spectrum. The
absorption coefficients varied from 3.84
ꢀ
104 to
6.60 ꢀ 102 Mꢁ1cmꢁ1. The values are in close agreement with those
reported for ferrocenyl chalcones [43]. The greater extinction
coefficient of 5 (1.87 ꢀ 103 Mꢁ1cmꢁ1) at 460 nm as compared to 4
(6.90 ꢀ 102 Mꢁ1cmꢁ1) at 451 nm is attributed to the more effective
electron withdrawing ability of eCOOH at para position thus
making the cyclopentadienyl ring to act as a weak donor. The poor
donating group capable of causing lower splitting of ded orbitals
of iron will result in higher probability of electronic transition at
longer wavelength. The interactions of organoantimony ferrocenyl
benzoates with DNA were examined by UVeVisible spectroscopy
in order to get some information about their mode of interaction
and binding strength. UVeVis absorption spectrometry is the most
frequently employed method for the detection of drug-DNA
interaction and assessment of binding mode due to its good
sensitivity, simplicity, reproducibility and versatility [45,46]. The
peak at 235 nm (Fig. 6), exhibiting blue shift in the presence of
increasing concentration of DNA can be attributed to the electro-
static interaction of the partial positive iron of 3 with the nega-
tively charged oxygen of DNA. While the peak at 279 nm
exhibiting hypochromism and slight red shift is indicative of
groove binding according to the criteria suggested for the mode of
interaction by the previous investigators [46]. Hence, complex 3
interacts with DNA by mixed binding mode (electrostatic and
groove binding) due to different interacting moieties. The hypo-
chromic effect is thought to be due to the interaction between the
electronic states of the binding chromophore and those of the
DNA bases [47]. It is expected that the strength of this electronic
interaction would decrease as the cube of the distance of separa-
tion between the chromophore and the DNA bases [48]. So, the
obviously large hypochromism observed in our experiments sug-
gested the close proximity of the chromophore of 3 to the DNA
3.5. Electronic absorption spectroscopy of OFBs interacting with DNA
The UVeVis spectra of 45
Fig. 5. Compound 5 registered five peaks at 234, 259, 296, 364 and
460 nm. The peak at 234 nm can be attributed to * tran-
sition in the unsubstituted CP ring. The signal at 259 nm may be
linked to n / * transition in CeOH due to better electron
donating effect of ferrocenyl group. The peak at 296 nm corre-
sponding to * transition is attributed to more conjugated
para cyclopentadienyl benzoate chromophore. The less intense
band at 364 nm can be linked to n / * transition of carbonyl
mM 4 and 5 have been depicted in
p
/ p
s
p
/ p
p
group and the least intense band at 460 nm may be due to ded
transition. The spectra of 2 also registered five peaks like 5 but
with different position and height. However, the spectrum of 4
recorded four (two intense and two broad) peaks. The more
intense peak at 234 nm can be corresponded to
p / p* transition
in both CP rings. The absence of peak at 259 nm in case of meta
directing groups may be due to the weak electron donating effect
of ferrocenyl group to excite n /
280 nm can presumably be associated with
less conjugated cyclopentadienyl benzoate group. The two broad
bands at longer wavelengths are attributable to n / * and ded
s
* transition. The peak at
p
/ p* transition in
p
bases. At the closest approach to DNA, the
p* orbital of the binding
transition in carbonyl group and iron respectively. The electronic
spectral responses of 1 and 3 were found in good agreement with
4. The differences in the absorption spectra of 5 and 4 demonstrate
moiety of 3 could couple with orbital of purine or pyrimidine.
p
The coupling
p* orbital may get partially filled by electrons, thus
1
1.8
5
1.5
1.2
0.8
0.6
0.4
0.2
0
a
0.9
4
d
0.6
0.3
0
225
275
325
375
425
225
300
375
450
525
600
wavelength/nm
wavelength/nm
Fig. 6. UVeVis spectra of 25
20.0 M DNA.
mM 3 in the (a) absence and (bed) presence of 4.0, 7.0 and
Fig. 5. UVeVis spectra of 45
m
M 4 and 5.
m

F. Asghar et al. / Journal of Organometallic Chemistry 717 (2012) 1e8
7
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
groove as a stacked dimer, so it is expected that the ferrocene
moiety of 2 can fit the groove when it binds to the DNA duplex in
a conformation similar to that of the conventional PIA dimers [53].
The suggested mixed binding mode (intercalation and groove
binding) will unwind the DNA helix at the interaction sites which
may lead to perturbation in its normal functioning that may
culminate in the death of cancerous cell.
The absence of red shift in the UVeVis spectra of 5 after the
addition of DNA (see Fig. 5 of supplementary materials) ruled out
the involvement of intercalation. The reason is not far to seek. The
planar part being sandwiched in two ferrocenyl groups may not
approach to the stacked base pairs. Hence, the UVeVis spectra of 5
evidenced slight electrostatic and dominant groove binding mode.
Since absorbance varied in the presence of ds.DNA, so based on
variation in absorption maxima the binding constant “K” of drug-
DNA complexation was determined according to WolfeeShimer
equation [54]:
c
a
225
250
275
300
325
350
wavelength/nm
Fig. 7. UVeVis spectra of 25
7.0 M DNA.
mM 1 in the (a) absence and (bec) presence of 3.0 and
m
½DNAꢄ
½DNAꢄ
1
3 B ꢁ
¼
þ
(5)
3
A ꢁ
3
3
B ꢁ
3
Kf ð
3 FÞ
F
F
decreasing the transition probabilities, and hence result in hypo-
chromism [49,50].
where
3
A
,
3
F
and
3
B
correspond to Aobserved/[M], the extinction
The UVeVis spectral characteristics of 4 with DNA were very
much similar to that of 3. However, the interesting observation for 1
in the presence of DNA was the increase in absorbance (hyper-
chromism) accompanied with very slight blue shift (Fig. 7). The
observed spectral effects can be explained by the electrostatic
interaction of 1 with the negatively charged oxygen causing
structural damage of DNA in the time domain of the experiment. F.
Arjmand and S. Shi have also reported that hyperchromic effect
arises mainly due to the binding of drug with DNA via electrostatic
attraction thereby causing a contraction and overall damage to the
secondary structure of DNA [51,52].
coefficient of the free drug, and the extinction coefficient of the
drug in the fully bound form, respectively. The binding constant
was obtained from the slope to intercept ratio of the plot of [DNA]/
(
3 Ae
3 F) versus [DNA].
The binding constant varied in the same sequence as revealed by
CV results. The highest K of 2 suggested its greater DNA binding
affinity than other ferrocenes.
4. Conclusion
Ferroceneeantimony complexes with tunable redox and spec-
troscopic properties were synthesized and characterized by elec-
trochemical and spectroscopic techniques with the aim of
investigating their potential anticancer activity. All the compounds
were found to follow one electron reversible redox process in
a diffusion controlled manner at a potential higher than simple
ferrocene. Hyperchromism, hypochromism, cathodic and anodic
peak potential shifts suggested OFBs to interact with DNA by
different modes due to variation in configuration. The binding
constant was found to vary in the sequence: K2 > K1 > K3 > K4 > K5.
The present study is expected to provide deep insights about the
understanding of unexplored mechanism by which such
compounds exert their biochemical action. The solid phase struc-
ture of the complex 5 adopts an approximate trigonal bipyramidal
Sb coordination environment with unidentate carboxylate ligands
in the axial positions at trans orientation with respect to ferrocenyl
moiety.
For the 2, stronger interaction with DNA was observed as shown
in Fig. 8. The peak at 227 nm due to
p / p* transition in the CP ring
after the addition of DNA got red shifted and almost disappeared
indicating drastic structural changes at this chromophore. The
substantial bathochromic shift can be attributed to the intercala-
tion of the planar part of 2 in to the double stranded DNA and the
diminution of the intensity can presumably be due to the shielding
of the ferrocenyl moiety by its probable fitting into the grooves of
DNA. Kohji et al. [53], have documented that the distance of
ꢀ
approximately 3.3 A, between the two Cp rings is close to the
typical distance of two aromatic rings in parallel-stacking geom-
etry. Because pyrrole-imidazole amides (PIA) can bind to the DNA
0.55
a
Acknowledgements
g
0.35
Dr. M. Khawar Rauf is thankful to Quaid-i-Azam University
Islamabad, Pakistan for Post-doctoral Fellowship and the grant of
funds to carry out this research work. Authors also acknowledge
the financial support from Higher Education Commission of
Pakistan.
0.15
Appendix A. Supplementary material
-0.05
Crystallographic data (excluding structure factors) for the
structures reported in this paper have been deposited to the
Cambridge Crystallographic Data Centre as supplementary publi-
cation no. CCDC-881520 for 5. Copies of available materials can be
obtained, free of charge, on application to the director, CCDC, 12
220
270
320
370
420
wavelength/nm
Fig. 8. UVeVis spectra of 25
5.0, 6.0 and 7.0 M DNA.
mM 2 in the (a) absence and (beg) presence of 2.0, 3.0, 4.0,
m

8
F. Asghar et al. / Journal of Organometallic Chemistry 717 (2012) 1e8
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