Synthesis, characterization and antitumor activity of new ferrocene incorporated N,N′-disubstituted thioureas

Article abstract of DOI:10.1039/c2dt31570j

We report herein the synthesis, structural characterization and activity against human ovarian tumour models: A2780 (parent), A2780^cisR (resistant to cisplatin) and A2780^ZD0473R (resistant to the cisplatin analogue denoted as ZD0473)

Full text of DOI:10.1039/c2dt31570j

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Synthesis, characterization and antitumor activity of new ferrocene
incorporated N,N′-disubstituted thioureas†
Bhajan Lal,a,b Amin Badshah,*a Ataf Ali Altaf,a,c Muhammad Nawaz Tahir,d Shafiq Ullaha and Fazlul Huq*b
Received 16th July 2012, Accepted 19th September 2012
DOI: 10.1039/c2dt31570j
We report herein the synthesis, structural characterization and activity against human ovarian tumour
models: A2780 (parent), A2780cisR (resistant to cisplatin) and A2780ZD0473R (resistant to the cisplatin
analogue denoted as ZD0473) of two ferrocene incorporated N,N′-disubstituted thioureas {1-benzoyl-
3-(4-ferrocenylphenyl)thiourea, B16, and 1-acetyl-3-(4-ferrocenylphenyl)thiourea, B3}. Structural
characterization has been based on FT-IR, multinuclear (1H and 13C) NMR, elemental analysis and
single crystal X-ray diffractometry. Ferrocene-incorporated thioureas may present themselves as a new
class of metal-based tumour active compounds. The cyclic voltammetric measurements indicate that
B16 undergoes partial intercalation with the CT-DNAwhereas B3 undergoes only electrostatic interaction
with the same. Partial prevention of BamH1 digestion of pBR322 plasmid DNA that has been interacted
with high concentrations of both B16 and B3 indicates that even non-covalent interactions can induce
significant conformational changes in the DNA.
modes of interaction. The anticancer activity of ferrocene deriva-
tives is found to be dependent on the oxidation state of iron in
Introduction
Although platinum drugs cisplatin, carboplatin and oxaplatin are
routinely used in the clinic to kill cancerous cells, their use has
also been limited due to inherent and acquired resistance and the
presence of a number of dose-limiting side effects.1,2 The cyto-
toxic effect of the compounds is related to their interaction with
the DNA that ultimately inhibits the transcription and replication
resulting in cell death.3–9 The search for better metal-based
drugs having the ability to overcome problems of drug resistance
and side effects associated with platinum based chemotherapy
constitutes the foundation of bio-organometallic chemistry. Fer-
rocene and its derivatives have been excessively studied as
potential chemotherapeutics.10–12 The stability, electroactivity
and high spectroscopic activity of ferrocene based organometal-
lics make them promising candidates for many biological appli-
cations.13 The presence of the ferrocenyl moiety enhances
activity due to its reversible redox behaviour and increases cell
permeability due to its lipophilic nature.14 It has been reported
that when ferrocene was incorporated into tamoxifen, the anti-
cancer activity of the drug is enhanced.15 Ferrocene derivatives
may bind with the DNA via both covalent and noncovalent
the ferrocene moiety with some results indicating that the Fe(II)
ferrocenyl compound is more active than Fe(III) ones.16 The
results of the study on ferrocifen as one of the Fe(II) compounds
indicate that the ferrocifens act by changing the conformation of
the receptor protein.17 Binding of ferrocifen to ERβ is thought to
lead to its dimerization followed by attachment of the dimerized
species to a particular region of DNA. The electron transfer reac-
tion involving the ferrocenium ion in vivo or the ferrocifen–ERβ
complex may generate reactive oxygen species (ROS) such as
hydroxyl radicals (˙OH). ROS produced can cause damage to
DNA18 and may also be responsible for anticancer activity
through the formation of radical metabolites that bring about bio-
logical damage in the cancer cell.19
Thiourea-based complexes have also been screened for
various biological activities.20 The presence of the thiocarbonyl
moiety influences the lipophilicity/hydrophilicity and the elec-
tronic properties of the compounds affect the lability of the
leaving groups thus controlling the biochemical action.13 Several
thiourea derivatives have shown potential antitumour activity
because of their inhibitory response against receptor tyrosine
kinases (RTKs), protein tyrosine kinases (PTKs) and NADH
oxidase.21,22 Herein we report the synthesis, structural characteri-
zation, anticancer activity and extent of DNA binding of two
ferrocene incorporated N,N′-disubstituted thioureas.
aDepartment of Chemistry, Quaid-i-Azam University, Islamabad-45320,
Pakistan. E-mail: aminbadshah@yahoo.com; Fax: +92 51 9064 2241;
Tel: +92 51 9064 2131
bDiscipline of Biomedical Sciences, The University of Sydney, Sydney,
Australia. E-mail: Fazlul.Huq@sydney.edu.au; Fax: +61 2935 19520;
Tel: +61 2935 19522
Results and discussion
cDepartment of Chemistry, University of Malakand, Chakdara,
Dir (lower), Pakistan
Synthesis and characterization
dDepartment of Physics, University of Sargodha, Sargodha, Pakistan
†CCDC 889603–889604. For crystallographic data in CIF or other elec-
tronic format see DOI: 10.1039/c2dt31570j
Two ferrocene incorporated N,N′-disubstituted thioureas coded
as B16 and B3 have been synthesized by allowing 4-ferrocenyl
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aniline to react with freshly prepared acetyl and benzoyl isothio-
cyanates under a N2 atmosphere in dry acetone (Scheme 1). The
1130–1270 cm−1 is assigned to (CvS). The aliphatic and aro-
matic protons appeared at the normal range of 2850–3050 cm−1
.
compounds have been characterized based on FT-IR, 1H and 13
NMR, elemental analysis and single crystal XRD.
C
In 1H NMR spectra ferrocenyl protons appear at 4–5 ppm. In the
13C NMR spectra the (CvO) and (CvS) peaks appear at
160–185 ppm whereas ferrocenyl carbons appear at 60–90 ppm.
The aromatic and aliphatic protons and carbons appear in the
usual regions in 1H and 13C NMR.12,23
FT-IR spectra showed a broad peak for the N–H stretch in the
range of 3300–3430 cm−1 due to involvement of hydrogen
bonding and secondary bonding interactions that are confirmed
by the crystal structures. The peak in the range of
1650–1700 cm−1 is assigned to (CvO) while that at
Single crystal X-ray studies
Careful crystallization of B16 and B3 in ethyl acetate by slow
evaporation yielded orange crystals suitable for single crystal
X-ray diffraction analysis. Data pertaining to the data collection
and structure refinement are given in Table 1.
Table 2 provides a selection of some important interatomic
distances, bond angles and torsion angles. Fig. 1 and 2 represent
the molecular structures with the numbering scheme of B16 and
B3 respectively. The intra-molecular hydrogen bonding and
intermolecular interactions present in these compounds are sum-
marized in Table 2 and drawn in Fig. 3 and 4.
In B16, the rings A (C6–C10), B (C11–C16), C (H1A, N1,
C17, N2, C18 and O) and D (C19–C24) are planar with r.m.s.
deviations of 0.0016, 0.0043, 0.0416 and 0.0084 respectively.
All these four planes are on the same surface due to the extended
Scheme 1 Synthesis of ferrocenyl thioureas.
Table 1 Crystal data and structure refinement parameters for B16 and
B3
Crystal parameters
B16
B3
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
b (Å)
c (Å)
α (°)
β (°)
C24H20FeN2OS
440.34
296
0.71073
Monoclinic
P21/c
C19H18FeN2OS
378.27
296
0.71073
Triclinic
ˉ
P1
11.0296(4)
31.5720(13)
12.3030(5)
90
103.412(2)
90
8.0003(4)
11.0413(6)
19.8358(12)
81.868(4)
80.899(3)
86.194(4)
1710.98, 4
1.469
39 × 24 × 18
(h,k,l)max (9,13,23)
(h,k,l)min (−9, −13,
−23)
6192
1.011
Fig. 1 Molecular structure of B16.
γ (°)
V (Å3), Z
4167.4(3), 8
1.404
Density (g cm−3
)
Crystal size (mm3)
Index ranges
40 × 210 × 15
(h,k,l)max (14,42,16)
(h,k,l)min (−14, −42,
−16)
10 405
0.841
Total reflections
μ (mm−1
)
R indices (all data)
Final R indices
[I > 2σ(I)]
Goodness-of-fit
θ range (°)
0.1672
0.0580
0.1578
0.0585
0.966
1.29–28.33
0.969
2.10–25.25
Fig. 2 Molecular structure of B3.
Table 2 The intra-molecular and intermolecular hydrogen bond interactions in B16 and B3
Compound
X
H
Y
d(X–H) (Å)
d(H–Y) (Å)
d(X–Y) (Å)
∠(XHY) (°)
Intra-molecular hydrogen bonds
B16
B3
O1
O1
H1A
H1A
N1
N1
1.864(3)
1.925(4)
0.861(3)
0.859(5)
2.602(5)
2.648(6)
142.69(23)
140.99(29)
Intermolecular hydrogen bonds
B16
S
S
H20
H2A
C20
N2
2.993
2.571
0.929(4)
0.860(3)
3.269
3.376
98.95
156.46
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Fig. 5 Supramolecular structures of B16 mediated by secondary
bonding.
Fig. 3 Inter- and intra-molecular hydrogen bonding in B16.
Fig. 6 Supramolecular structures of B3 mediated by secondary
bonding.
Fig. 4 Inter- and intra-molecular hydrogen bonding in B3.
shown in Fig. 5 and 6. These kinds of secondary intermolecular
non-bonding interactions are considered to be an important
determinant of biological activities; compounds pertaining to
stronger nonbonding interactions are found to have a greater
ability to bind with macrobiological molecules such as proteins
and DNA.27 Among the compounds under study B16 is found to
have more secondary interactions that are evident from hydrogen
bonding data and supramolecular structure formation. Hence it is
expected that B16 will associate with DNA more strongly that
will translate into higher biological activities.
resonance over the twenty-three atoms in the molecule. The
existence of intermolecular hydrogen bonding (NH⋯O type) is
responsible for the planarity of six atoms forming ring C in B16
(Fig. 3). The interplanar angles between these planes are found
to be A/B = 19.04, A/C = 15.80, A/D = 12.93, B/C = 7.55,
B/D = 6.47, C/D = 4.71. In B3, the rings A (C6–C10), B (C11–
C16) and C (H1A, N1, C17, N2, C18 and O) are planar with
r.m.s. deviations of 0.0057, 0.0080 and 0.0174 respectively. In
the case of B3, three planes are on the same surface due to the
extended resonance over the sixteen atoms in the molecule. The
interplanar angles between these planes are found to be A/B
3.87, B/C 21.70, C/A 25.46. The existence of intra-molecular
hydrogen bonding (NH⋯O type) is responsible for the planarity
of six atoms forming ring C in B3 (Fig. 4). This type of intra-
molecular hydrogen bonding is well known for such kinds of
aroyl or acoyl substituted organic thioureas as we found in the
ferrocene analogues.24–26
The dihedral angle between rings A and B is larger in B16
than in B3, indicating the extended resonance among these rings
in B3 which makes them more planar. It may be due to the
attachment of the benzoyl group to the thiourea nitrogen which
extends resonance in a direction opposite to the ferrocene. The
acetyl group does not attract the electron charge density and
allows the ferrocene to gain resonance electrons charge density
and make the system more planar. The bond distances between
A and B rings (C6–C11 = 1.477(6) in B16 and C10–C11 =
1.456(8) in B3) also anticipate the same stronger bond in B3.
The two independent molecules exist in an asymmetric unit in
both structures (B16 and B3) which are connected alternately to
each other by the intermolecular hydrogen bonding of type
NH⋯O, NH⋯S and secondary non-covalent interactions
(π⋯H), to mediate a supramolecular structure for B16 and B3 as
Cytotoxicity
The cytotoxicity of B16 and B3 along with that for cisplatin was
screened against human ovarian cell lines: A2780 (parent),
A2780cisR (cisplatin-resistant type), A2780ZD0473R (ZD0473-
resistant type) by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) reduction assay. The IC50
values meaning the concentrations of compounds (B16, B3 and
cisplatin) required for 50% cell kill were determined and are
listed in Table 4. B16 is found to be more active than B3
although B16 and B3 are less active than cisplatin against all the
cell lines. However, B16 and B3 have lower resistance factors
(Rf) than cisplatin such that the compounds are equally or nearly
equally active against both the parent (A2780) and the resistant
(A2780cisR and A2780ZD0473R) cell lines. In contrast, cisplatin
has much lower activity against the resistant cell lines than the
parent cell line. The results indicate that at the level of their
activity, B16 and B3 have been better able to overcome mechan-
isms of resistance operating in the cell lines. One possible reason
for the difference in activity of B3 and B16 as compared to that
for cisplatin may lie in the difference in the nature of interaction
with the DNA.28 Whereas cisplatin binds covalently with
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nucleobases in the DNA, B16 is believed to undergo partial
intercalation in between nucleobases in the DNA and B3 under-
goes only electrostatic interaction with the same. In both B16
and B3, the antitumour effect is related to the oxidation state of
the central iron atom of the ferrocenyl group. The low oxidation
potential of B16 and B3 as compared to ferrocene supports the
idea that the ferrocenyl moiety may be easily oxidized to the
Fe3+ state. It is believed that it is mainly the Fe3+-ferrocenyl
cation that undergoes electrostatic interaction with the phosphate
backbone of the DNA. As noted earlier, the electron transfer
reaction involving Fe2+- and Fe3+-ferrocenyl species may gener-
ate ROS in vivo that cause damage to DNA.19,29
Fig. 8 Cyclic voltammograms of 1 mM B3 recorded at a 0.05 V s−1
potential sweep rate on a glassy carbon electrode at 298 K in the
absence and presence of 1 mL of increasing concentrations of CT-DNA
(10 μM, 20 μM and 60 μM) in a 20% aqueous DMSO buffer at pH 6.0;
supporting electrolyte 0.1 M TBAP.
DNA binding studies
Voltammetric studies
The cyclic voltammetric measurements of 1 mM of B16 and B3
were performed in the absence and presence of calf thymus
DNA (CT-DNA). On addition of increasing concentrations of
CT-DNA (1 mL of 20 μM, 40 μM and 60 μM) into 1 mM B16
and B3 (Fig. 7 and 8), the shift in anodic potential and drop in
ipa are observed. The drop in current is attributed to diffusion of
the drug into double helical DNA resulting in the formation of a
supramolecular complex. The shift in formal potential reveals
the mode of interaction between the drug and DNA.30 Intercala-
tion of the drug into the double helical structure of DNA causes
a positive shift in the formal potential whereas a negative shift in
the formal potential is indicative of the presence of electrostatic
interaction of the cationic drug with the anionic phosphate of the
DNA backbone. Thus the positive shift in formal potential
observed for B16 after the addition of CT-DNA can be seen to
indicate the occurrence of the intercalative mode of interaction.31
However the ferrocene moiety cannot intercalate into the double
helix because of its size but the remaining part of the molecule
(most likely phenyl rings) can intercalate. B3 shows a negative
shift in formal potential which is attributed to electrostatic inter-
actions between the drug and the DNA. The shift in formal
potential can be used to find out which form, ferrocene or
ferrocenium, interacts strongly with DNA. The binding ratio of
reduced and oxidized species calculated according to the follow-
ing equation32 is given in Table 5.
ꢀ
ꢁ
Kred
Eb° ꢀ Ef° ¼ 0:05916 log
Koxd
Eb° and Ef° are the formal potentials of the free and bound
forms of the drug respectively. The negative shift indicates that
in the case of B16 the oxidized form interacts strongly with
DNA whereas in the case of B3 it is the reduced form that inter-
acts more strongly with the DNA. The binding parameters calcu-
lated according to the following equation33 are given in Table 5.
1
Kð1 ꢀ AÞ
¼
ꢀ K
½DNAꢁ 1 ꢀ ði=i0Þ
In the above equation, K is the binding constant, i and i0 are
the peak currents with and without CT-DNA and A is the propor-
tionality constant. The plot of 1/[DNA] versus 1/(1 − i/i0) yields
binding constants30 that are given in Table 3. The binding free
energy change (−ΔG = RT ln K) of B16 and B3 in kJ mol−1 at
25 °C (Table 5) signifies the spontaneity of drug−DNA
interaction.34
Studies with pBR322 plasmid DNA
Untreated pBR322 plasmid DNA when electrophoresed for
40 min at room temperature gives two bands corresponding to
supercoiled form I and relaxed circular form II. When it is inter-
acted with increasing concentrations of cisplatin ranging from
5 μM to 80 μM, the mobility of both form I and form II bands is
found to increase – more so for form II than form I – so that the
distance of separation between the bands decreases. The change
in mobility of the bands indicates the occurrence of change in
DNA conformation believed to be brought about by covalent
binding of cisplatin with the DNA. It is known that cisplatin
Fig. 7 Cyclic voltammograms of 1 mM B16 recorded at a 0.05 V s−1
potential sweep rate on a glassy carbon electrode at 298 K in the
absence and presence of 1 mL of increasing concentrations of CT-DNA
(10 μM, 20 μM and 60 μM) in a 20% aqueous DMSO buffer at pH 6.0;
supporting electrolyte 0.1 M TBAP.
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Table 3 IC50 values and resistance factors for cisplatin, B16 and B3
Compounds A2780 IC50 (μM) A2780cisR IC50 (μM) IC50A2780cisR/IC50A2780 Rf A2780ZD0473R IC50 (μM) IC50A2780cisR/IC50A2780 Rf
Cisplatin
B16
B3
1.00 0.46
15.27 3.26
17.4 2.02
9.95 2.07
16.46 4.13
20.98 5.43
9.95
1.07
1.21
10.22 1.65
17.39 4.93
23.38 6.50
10.22
1.14
1.34
Table 4 The binding constant, Gibbs free energies and binding ratio of
reduced and oxidized species for B16 and B3
tetrabutylammonium perchlorate (TBAP) and all the other
chemicals were purchased from Sigma Aldrich and were used as
such. 4-Ferrocenyl-aniline was synthesized by the literature
reported method.37 Solvents such as ethanol, ethyl acetate,
acetone, diethyl ether, and petroleum ether were purified before
use according to the standard reported protocols. FT-IR and
multinuclear (1H and 13C) NMR spectra were obtained using a
Thermo Scientific Nicolet-6700 FTIR spectrometer and a
BRUKER AVANCE 300 MHz NMR spectrometer.
Compounds
Binding constant (M−1
)
−ΔG (kJ mol−1
)
Kred/Koxd
B16
B3
1.12 × 104
1.42 × 102
23.10
12.28
1.122
0.323
binds with the DNA to form mainly intrastrand bifunctional 1,2-
Pt(GG) and 1,2-Pt(AG) adducts that induce the bending of the
DNA strand at the binding site.35 When pBR322 plasmid DNA
is interacted with increasing concentrations of B16 and B3 for
40 min at room temperature, no change in intensity and mobility
of either the form I or form II band is observed, indicating that
the compounds cannot cause any change in DNA conformation,
in line with the idea that they do not bind covalently with the
DNA (Fig. 9).
General synthesis of thiourea
Potassium thiocyanate was dissolved in dried acetone; to this
solution acid chloride was added under a N2 atmosphere. 4-Ferro-
cenyl aniline was added to the resulting reaction mixture drop-
wise and stirred for 4 h. The reaction mixture was then poured
into ice cooled water and stirred well. The solid product
(thiourea) was filtered off and washed with deionized water. The
thioureas were dried in air and recrystallized in ethyl acetate.
BamH1 digestion
Untreated and undigested pBR322 plasmid DNA gave two
bands corresponding to forms I and II whereas untreated but
BamH1-digested pBR322 plasmid DNA gave a single band cor-
responding to form III. In the case of pBR322 plasmid DNA
interacted with increasing concentrations of cisplatin followed by
BamH1 digestion, three bands corresponding to forms I, II and
III are seen at all concentrations of the compound. In the case of
B16, only a single band corresponding to form III is seen at
5 μM. At higher concentrations of B16 also, a prominent form
III can be seen but along with two faint frontal bands. It is
believed that the frontal bands are due to DNA fragments pro-
duced rather than the form I. In the case of B3, three bands cor-
responding to forms I, II and III can be seen at all concentrations
ranging from 5 μM to 80 μM. The results indicate that cisplatin
is much more effective in preventing BamH1 digestion than B16
and B3. This is to be expected since BamH1 cuts the phospho-
diester bond between adjacent guanines (specifically it recog-
nizes G/GATTCC)36 and cisplatin is known to bind with DNA
forming an intrastrand bifunctional 1,2-Pt(GG) adduct that
causes significant distortion of the DNA strand close to the
binding site. Some prevention of BamH1 digestion by B16 and
B3 indicates that even non-covalent interactions can induce sig-
nificant conformational change in the DNA (especially at higher
concentrations) so that BamH1 partially fails to recognize the
sequence (Fig. 10).
Synthesis of 1-benzoyl-3-(4-ferrocenylphenyl) thiourea (B16)
Potassium thiocyanate (0.35 g, 3.6 mmol) was dissolved in dried
acetone. Benzoyl chloride (0.41 ml, 3.6 mmol) was added to the
resulting solution under a N2 atmosphere. 4-Ferrocenyl aniline
(1 g, 3.6 mmol) was added to the resulting reaction mixture and
stirred for 4 h. The reaction mixture was then poured into ice
cooled water and stirred well. The solid product was filtered off
and washed with deionized water. The 1-benzoyl-3-(4-ferro-
cenylphenyl) thiourea was dried in air and recrystallized in ethyl
acetate. Yield 73%, FTIR (ν cm−1): Fe-cp (481 cm−1), NH
(3422 cm−1), CvO (1661 cm−1), CvS (1137–1256 cm−1),
1
CvC Ar (1455–1592 cm−1), sp2 CH (3029.5 cm−1). H NMR
(300 MHz, DMSO): δ 12.646 (s, 1H, CSNH), 11.57 (s, 1H,
CONH), 7.99 (s, 4H, C6H4), 7.61 (m, 5H, C6H5), 4.81 (s, 2H,
C5H4), 4.36 (s, 2H, C5H4), 4.03 (s, 5H, C5H5) ppm., 13C NMR
(75 MHz, CDCl3): δ 178.73, 168.73, 137.61, 133.60, 132.61,
129.16, 128.92, 126.39, 124.44, 120.18, 84.62, 69.83, 69.47,
66.77 ppm. Elemental analysis Calc. (%) for C24H20FeN2OS:
C, 65.46; H, 4.58; N, 6.36; S, 7.28. Found (%): C, 65.32; H,
4.50; N, 6.70; S, 7.21.
Synthesis of 1-acetyl-3-(4-ferrocenylphenyl) thiourea (B3)
B3 was prepared by using the same method as for B16 except
using acetyl chloride (0.257 ml, 3.6 mmol) in place of benzoyl
chloride. Yield 67%, FTIR (ν cm−1): Fe-cp (481 cm−1), NH
(3309 cm−1), CvO (1692 cm−1), CvS (1140–1263 cm−1),
CvC Ar (1505–1593 cm−1), sp2 CH (3037 cm−1), sp3 CH
(2863 cm−1). 1H NMR (300 MHz, DMSO): δ 11.27 (s, 1H,
Experimental
Materials and methods
Ferrocene, 4-nitroaniline, sodium nitrite, potassium thiocyanate,
acid chlorides such as benzoyl chloride, acetyl chloride,
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Table 5 The intra-molecular and intermolecular hydrogen bond interactions in B16 and B3
Compound
X
H
Y
d(X–H) (Å)
d(H–Y) (Å)
d(X–Y) (Å)
∠(XHY) (°)
Intra-molecular hydrogen bonds
B16
B3
O1
O1
H1A
H1A
N1
N1
1.864(3)
1.925(4)
0.861(3)
0.859(5)
2.602(5)
2.648(6)
142.69(23)
140.99(29)
Intermolecular hydrogen bonds
B16
B3
S
S
S
S
H20
H2A
H2A
H2A
C20
N2
N2
N2
2.993
2.571
2.553
2.508
0.929(4)
0.860(3)
0.860(5)
0.860(5)
3.269
3.376
3.403
3.358
98.95
156.46
169.83
170.18
138.43, 131.92, 127.43, 126.69, 84.33, 70.07, 69.33, 66.73,
25.36 ppm. Elemental analysis Calc. (%) for C19H18FeN2OS:
C, 60.33; H, 4.80; N, 7.41; S, 8.48. Found (%): C, 60.71; H,
4.91; N, 7.13; S, 8.29.
X-ray structure analysis
X-ray measurements were made on a Bruker kappa APEXII
CCD diffractometer equipped with a graphite-monochromated
Mo-Kα (λ = 0.71073 Å) radiation source. Data collection used ω
scans, and a multi-scan absorption correction was applied. The
structure was solved by using the SHELXS97 (Sheldrick, 273
2008) program. The structure was refined by full matrix least-
squares techniques using SHELXL-97. The fractional coordi-
nates of B16 and B3 are presented in Table 3.
Fig. 9 Electrophoretograms applying to the interaction of pBR322
plasmid DNA with increasing concentrations of cisplatin, B16 and B3.
Lane P: untreated pBR322 plasmid DNA to serve as a control; lanes 1 to
5: pBR322 plasmid DNA interacted with increasing concentrations of
compounds (cisplatin, B16 and B3) (lane 1: 5 μM; lane 2: 10 μM; lane
3: 20 μM; lane 4: 40 μM; lane 5: 80 μM).
Cytotoxicity assays
The cytotoxicities of B16 and B3 were screened against human
ovarian cell lines: A2780 (parent), A2780cisR (cisplatin-resistant
type), A2780ZD0473R (ZD0473-resistant type) using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
reduction assay. Cisplatin was used as a control. Briefly between
4000 to 5000 cells were seeded into the wells of the flat-
bottomed 96-well culture plate in a 10% FCS/RPMI 1640 culture
medium. The plates were incubated for 24 h in a humidified
atmosphere (37 °C, 5% carbon dioxide in air, pH 7.4) to allow
the cells to attach. Drugs were added at four different concen-
trations to triplicate wells that were left in the incubator (37 °C,
5% carbon dioxide in air, pH 7.4) for 72 h. After the period, the
growth inhibition of cells was determined. 50 μL per well of
1 mg mL−1 freshly prepared MTT solution was added to each
plate. After 4 h, the formazan crystals formed in each well were
dissolved in 150 μL of DMSO and the absorbance of the result-
ing solution was read using a Bio-Rad Modal 3550 Microplate
Reader set at 550 nm.38
Fig. 10 Electrophoretograms applying to the interaction of pBR322
plasmid DNA with increasing concentrations of cisplatin, B16 and B3
followed by BamH1 digestion. Lane P: untreated and undigested
pBR322 plasmid DNA; lane B: untreated but BamH1-digested pBR322
plasmid DNA; lanes 1 to 5: pBR322 plasmid DNA interacted with
increasing concentrations of compounds (cisplatin, B16 and B3) (lane 1:
5 μM; lane 2: 10 μM; lane 3: 20 μM; lane 4: 40 μM; lane 5: 80 μM)
followed by BamH1 digestion.
DNA binding studies
Interaction with CT-DNA
Cyclic voltammetric (CV) measurements were performed in a
single compartment cell with a three electrode configuration
using an Eco Chemie Auto lab PGSTAT 12 potentiostat/galvano-
stat (Utrecht, The Netherlands) equipped with the electrochemi-
cal software package GPES 4.9. The three electrode system
CSNH), 10.39 (s, 1H, CONH), 7.92 (d, J = 7.5 Hz, 2H, C6H4),
7.44 (d, J = 7.5 Hz, 2H, C6H4), 4.82 (t, J = 1.8 Hz, 2H, C5H4),
4.36 (t, J = 1.8 Hz, 2H, C5H4), 4.02 (s, 5H, C5H5), 2.19 (s, 3H,
CH3) ppm. 13C NMR (75 MHz, CDCL3): δ 176.59, 171.27,
Dalton Trans.
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consisted of Ag/AgCl as the reference electrode, a platinum wire
of thickness 0.5 mm with an exposed end of 10 mm as the
counter electrode and a bare glassy carbon electrode (surface
area of 0.071 cm2) as the working electrode. The voltammogram
of a known volume of the test solution was recorded in the
absence of calf thymus DNA (CT-DNA) after flushing out
oxygen by purging with argon gas for 10 min just prior to each
experiment. The procedure was then repeated for systems with a
constant concentration of the drugs (1 mM) and varying concen-
trations of CT-DNA (20 μM to 60 μM). The working electrode
was cleaned after every electrochemical assay. The sodium salt
of calf thymus DNA purchased from Acros was used as
received. 1 mM stock solutions of B16 and B3 were prepared
in 20% aqueous ethanol (20% H2O : 80% ethanol) and all
sample solutions were buffered at pH 6 using a phosphate buffer
(0.1 M NaH2PO4 + 0.1 M NaOH). Methanol recrystallized tetra-
butylammonium perchlorate (TBAP) was used as the supporting
electrolyte. The stock solution of CT-DNA (200 μM) was pre-
pared by using doubly distilled water and stored at 4 °C.
The concentration of CT-DNA was determined by UV absor-
DNA whereas the less active (B3) undergoes only electrostatic
interaction.
Acknowledgements
Bhajan Lal is grateful to the Higher Education Commission
Pakistan for the award of an indigenous PhD scholarship com-
bined with an IRSIP scholarship. This research is partly funded
by Biomedical Science Research Initiative Grant and Biomedical
Science Cancer Research Donation Fund, School of Medical
Sciences, The University of Sydney.
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Dalton Trans.
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