New supramolecular ferrocene incorporated N,N'-disubstituted thioureas: Synthesis, characterization, DNA binding, and antioxidant studies

Article abstract of DOI:10.1080/00958972.2014.934819

Ferrocene incorporated N,N'-disubstituted thioureas (S1-S6) were synthesized by allowing 4-ferrocenyl-3-methylaniline to react with freshly prepared aliphatic isothiocyanates and were characterized by using different analytical techniques. Based on single

Full text of DOI:10.1080/00958972.2014.934819

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New supramolecular ferrocene
incorporated N,N′-disubstituted
thioureas: synthesis, characterization,
DNA binding, and antioxidant studies
a
a
b
Shabeeb Hussain , Amin Badshah , Bhajan Lal , Raja Azadar
a
a
c
d
Hussain , Shafqat Ali , Muhammad Nawaz Tahir & Ataf Ali Altaf
a
Department of Chemistry, Quaid-i-Azam University, Islamabad,
Pakistan
b
Department of Chemistry, Shah Abdul Latif University, Khairpur,
Pakistan
Click for updates
c
Department of Physics, University of Sargodha, Sargodha,
Pakistan
d
Department of Chemistry, Bahauddin Zakariya University,
Sahiwal, Pakistan
Accepted author version posted online: 17 Jun 2014.Published
online: 15 Jul 2014.
To cite this article: Shabeeb Hussain, Amin Badshah, Bhajan Lal, Raja Azadar Hussain, Shafqat Ali,
Muhammad Nawaz Tahir & Ataf Ali Altaf (2014) New supramolecular ferrocene incorporated N,N′-
disubstituted thioureas: synthesis, characterization, DNA binding, and antioxidant studies, Journal
of Coordination Chemistry, 67:12, 2148-2159, DOI: 10.1080/00958972.2014.934819
To link to this article: http://dx.doi.org/10.1080/00958972.2014.934819
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Journal of Coordination Chemistry, 2014
Vol. 67, No. 12, 2148–2159, http://dx.doi.org/10.1080/00958972.2014.934819
New supramolecular ferrocene incorporated N,N′-disubstituted
thioureas: synthesis, characterization, DNA binding, and
antioxidant studies
SHABEEB HUSSAIN†, AMIN BADSHAH*†, BHAJAN LAL*‡,
RAJA AZADAR HUSSAIN†, SHAFQAT ALI†, MUHAMMAD NAWAZ TAHIR§ and
ATAF ALI ALTAF¶
†
Department of Chemistry, Quaid-i-Azam University, Islamabad, Pakistan
Department of Chemistry, Shah Abdul Latif University, Khairpur, Pakistan
Department of Physics, University of Sargodha, Sargodha, Pakistan
Department of Chemistry, Bahauddin Zakariya University, Sahiwal, Pakistan
‡
§
¶
(
Received 25 March 2014; accepted 4 May 2014)
This research article describes the synthesis, structural characterization, electro-
chemistry, nature and extent of binding with the DNA and free radical scavenging
activity of new ferrocene based N, N′-disubstituted thioureas.
Ferrocene incorporated N,N′-disubstituted thioureas (S1–S6) were synthesized by allowing 4-ferr-
ocenyl-3-methylaniline to react with freshly prepared aliphatic isothiocyanates and were character-
ized by using different analytical techniques. Based on single-crystal X-ray analysis compound S1
shows supramolecular structure mediated by secondary bonding interactions, intermolecular hydro-
gen bonding NH–O, NH–S, and secondary non-covalent interactions (π–H). Voltammetric measure-
ments were used to study their redox behavior and DNA binding of the compounds. Different
binding parameters like binding constants, binding energies, and diffusion coefficients (D
culated for further insight on the affinity with DNA. All the compounds show electrostatic mode of
interactions. The D of molecule-DNA adducts were found to be lower than that of free molecule.
o
) were cal-
o
−
1
Compounds S1–S6 having 50% inhibition values less than 37.89 μg mL indicate that they may
have value as antioxidants to be used as therapeutic agents.
Keywords: Ferrocene; Crystal structure; Electrochemistry; DNA binding; Antioxidant activity
*
Corresponding authors. Email: aminbadshah@yahoo.com (A. Badshah); bhajanqau@yahoo.com (B. Lal)
©
2014 Taylor & Francis

Ferrocene incorporated thioureas
2149
1. Introduction
Ferrocene is of interest because of its neutral behavior, stability, and biologically non-toxic
effect [1]. Ferrocene and its derivatives have applications as antitumor [2, 3], antimalarial
[
4], antifungal [5], cytotoxic effects [6, 7], and DNA cleavage activity [8]. An important
application of ferrocenyl derivatives is against cancer. Tamoxifen and hydroxytamoxifen are
routinely used for the treatment of breast cancer but long-term exposure of clinically avail-
able drugs lead to side effects such as development of resistance to the drug, increase in risk
of blood clotting in the lungs, and non-effectiveness in hormone-independent tumors [9].
The ferrocifens, which are analogous compounds of tamoxifen with a ferrocenyl moiety,
have greater activity against both hormone-dependent and hormone-independent breast can-
cer cells [10]. The significant increase in activity by incorporation of the ferrocene in basic
structure of drug is due to increase in lipophilicity and the ferrocene moiety itself.
Some ferrocenyl derivatives like polyphenolic compounds (1,1-bis(4-hydroxyphenyl)-
2
-ferrocenyl-but-1-ene) have greater anticancer activity than 4-hydroxytamoxifen in hor-
mone-dependent and hormone-independent breast cancer [11, 12]. The anticancer activities
2
+
of ferrocenyl derivatives generally depend upon the oxidation state of iron (Fe) with Fe
3
+
ferrocenyl derivatives more active than Fe derivatives by changing the conformation of
receptor protein [13–15]. According to Osella et al. [16], anticancer activity arises due to
generation of oxygen active radicals by reduction of ferrocenium ion. It means that the good
redox properties of ferrocenyl moiety will ultimately exhibit enhanced activity. The antican-
cer activity of compounds is due to the interaction with DNA by which the physical and
chemical nature of DNA alters. The interaction of drug with DNA can be studied by using
different analytical techniques, among which electrochemical methods are used because of
reliability, high sensitivity and selectivity, and low cost [17, 18]. The interaction of the drug
with DNA may be covalent or non-covalent (electrostatic, groove binding, and intercala-
tion) that can be probed by substantial change in peak potential and change in peak current
after addition of different concentration of DNA.
Antioxidants have ability to trap free radicals. Highly reactive free radicals and oxygen
species (such as hydro peroxides, peroxides, and lipids peroxyl) are found in biological sys-
tems from different sources. These free radicals are responsible for oxidation of nucleic
acids, proteins, lipids, or DNA and can initiate degenerative disease and thus antioxidant
inhibit the oxidative mechanisms that lead to these kinds of diseases [19]. Some ferrocene-
based thioureas, selenoureas, guanidines, amides, and bimetallics from our research group
have been screened as potential DNA binders and have shown good antitumor and antioxi-
dant activities [20–25]. Herein, we report the synthesis, structural characterization, nature,
and extent of binding with the DNA and free radical scavenging activity of new ferrocene-
based N,N′-disubstituted thioureas (S1–S6).
2
2
. Experimental
.1. Materials and methods
Ferrocene, 3-methyl-4-nitroaniline, potassium thiocyanate, sodium nitrite, acetyl chloride,
isobutyryl, phenylacetyl, dichloroacetyl, propionyl, pivaloyl chloride, tetrabutylammonium
perchlorate (TBAP), and hydrochloric acid were purchased from Sigma Aldrich and used as

2
150
S. Hussain et al.
such. Ethanol, methanol, acetone, diethyl ether, and petroleum ether were purified before
use according to the standard reported protocols [26, 27].
2
.2. Synthesis of N-acetyl-N′3-(4-ferrocenyl-3-methylbenzyl) thiourea (S1)
To the solution of acetyl chloride (0.302 mL, 4.25 mM) in dried acetone (100 mL), potassium
thiocyanate (0.414 g, 4.25 mM) was added to prepare the acetyl isothiocyanate under N2.
4
-Ferrocenyl-3-methyl aniline (1 g, 4.25 mM) was added to the resulting reaction mixture
and kept stirring for 4 h. The reaction mixture was then poured into the ice-cooled water and
stirred well. The solid product was filtered off and washed with deionized water. 1-Acetyl-3
(4-ferrocenyl-3-methylbenzyl) thiourea was dried in air. Yield: 74%. δH (300 MHz, [D6]
C D O) 12.65 (s, 1H, CSNH), 10.31 (s, 1H, CONH), 7.77 (d, J 8.4 Hz, 1H, C H ), 7.65 (d, J
3
6
6 3
8
.4 Hz, 1H, C H ), 7.42 (s, 1H, C H ), 4.56 (s, 2H, C H ), 4.34 (s, 2H, C H ), 4.15 (s, 5H,
6 3 6 3 5 4 5 4
C H ), 2.35 (s, 3H, CH ), 2.10 (s, 3H, CH ), ppm. δ (75 MHz, [D ]DMSO) 179.13, 172.47,
5
5
3
3
C
6
1
36.00, 135.65,132.17, 130.53, 125.00, 120.58, 86.55, 69.34, 69.22, 68.02, 28.21, 20.61
−
1
−1
2
−1
3
−1
ppm. υmax/cm NH (3200–3400 cm ), sp C–H (3080 cm ), sp C–H (2905 cm ), C=O
−
1
−1
−1
−1
(1650–1700 cm ), C=C Ar (1513–1598 cm ), C=S (1130–1250 cm ), Fe–Cp (485 cm ).
Elemental analysis Calcd (%) for C H FeN OS: C, 61.23; H, 5.14; N, 7.14; S, 8.17. Found
2
0
20
2
(%): C, 61.59; H, 5.41; N, 7.43; S, 8.32.
2
.3. Synthesis of N-isobutyryl-N′-(4-ferrocenyl-3-methylbenzyl) thiourea (S2)
Compound S2 was prepared using the same method as for S1 except using isobutyryl chlo-
ride (0.445 mL, 4.25 mM) in place of acetyl chloride. Yield: 79%. δH (300 MHz, [D6]
DMSO) 12.54 (s, 1H, CSNH), 11.45 (s, 1H, CONH), 7.68 (d, J 8.4 Hz, 1H, C H ), 7.54 (d,
6
3
J 8.4 Hz, 1H, C H ), 7.37 (s, 1H, C H ), 4.57 (s, 2H, C H ), 4.35 (s, 2H, C H ), 4.13 (s,
6
3
6
3
5
4
5 4
5
H, C H ), 2.75 (m, 1H, C H ), 2.35 (s, 3H, CH ), 1.15 (d, J 6.6 Hz, 6H, C H ) ppm. δ
5 5 3 7 3 3 7 C
(
8
(
(
75 MHz, [D ]DMSO) 179.13, 171.23, 136.03, 135.90, 135.64, 130.50, 124.99, 120.52,
6
−
1
−1
2
6.57, 69.53, 69.34, 68.02, 35.41, 20.59, 18.43 ppm. υmax/cm NH (3262 cm ), sp C–H
−
1
3
−1
−1
−1
3080 cm ), sp C–H (2905 cm ), C=O (1693 cm ), C=C Ar (1513–1598 cm ), C=S
−
1
−1
1154–1227 cm ), Fe–Cp (485 cm ). Elemental analysis Calcd (%) for C H FeN OS:
2
2
24
2
C, 61.23; H, 5.14; N, 7.14; S, 8.17. Found (%): C, 61.46; H, 5.25; N, 7.56; S, 8.37.
2
.4. Synthesis of N-phenylacetyl- N′-(4-ferrocenyl-3-methylbenzyl) thiourea (S3)
Compound S3 was prepared using the same method as for S1 except using phenylacetyl
chloride (0.562 mL, 4.25 mM) in place of acetyl chloride. Yield: 67%. δ (300 MHz, [D ]
H
6
DMSO) 12.40 (s, 1H, CSNH), 11.69 (s, 1H, CONH), 7.32–7.25 (m, 9H), 4.51 (d, 2H,
C H ), 4.30 (s, 2H, C H ), 4.18 (s, 5H, C H ), 4.09 (s, 2H, CH ), 2.31 (s, 3H, CH3), ppm.
5
4
5
4
5
5
2
δ (75 MHz, [D ]DMSO) 178.25, 169.13, 135.59, 135.21, 133.24, 130.08, 130.45, 129.24,
C
6
−
1
1
25.5, 124.18, 120.65, 116.45, 84.53, 69.79, 69.64, 68.03, 52.88, 21.02 ppm. υmax/cm
−
1
2
−1
3
−1
−1
NH (3252 cm ), sp C–H (3064 cm ), sp C–H (2869 cm ), C=O (1688 cm ), C=C Ar
1540–1570 cm ), C=S (1133–1257 cm ), Fe–Cp (485 cm ). Elemental analysis Calcd
%) for C H FeN OS: C, 66.67; H, 5.16; N, 5.98; S, 6.85. Found (%): C, 66.86; H, 5.35;
−
1
−1
−1
(
(
2
6
24
2
N, 6.06; S, 6.96.

Ferrocene incorporated thioureas
2151
2.5. Synthesis of N-dichloroacetyl-N′-(4-ferrocenyl-3-methylbenzyl) thiourea (S4)
Compound S4 was prepared using the same method as for S1 except using dichloroacetyl
chloride (0.408 mL, 4.25 mM) in place of acetyl chloride. Yield: 69%. δ (300 MHz, [D ]
H
6
DMSO) 12.71 (s, 1H, CSNH), 10.97 (s, 1H, CONH), 7.60–7.30 (m, 2H, C H ), 7.27 (s,
6
3
1
5
1
H, C H ), 5.92 (s, 1H, CH), 4.55 (d, J 12 2H, C H ), 4.35 (d, J 9 Hz,2H, C H ), 4.14 (s,
6 3 5 4 5 4
H, C H ), 2.34 (s, 3H, CH3) ppm. δ (75 MHz, [D ]DMSO) 178.52, 174.35, 134.63,
5
5
C
6
32.13, 132.15, 126.15, 121.165 120.05, 86.32, 70.05, 69.88, 69.52, 66.98, 20.71 ppm.
−1
-1
2
−1
3
−1
−1
υmax/cm NH (3302 cm ), sp C–H (3124 cm ), sp C–H (2952 cm ), C=O (1692 cm ),
C=C Ar (1524–1591 cm ), C=S (1112–1265 cm ), Fe–Cp (484 cm ). Elemental analysis
−
1
−1
−1
Calcd (%) for C H Cl FeN OS: C, 61.23; H, 5.14; N, 7.14; S, 8.17. Found (%): C,
2
0
18
2
2
6
1.09; H, 5.33; N, 7.07; S, 8.01.
2
.6. Synthesis of N-propionyl-N′-(4-ferrocenyl-3-methylbenzyl) thiourea (S5)
Compound S5 was prepared using the same method as for S1 except using propionyl chlo-
ride (0.371 mL, 4.25 mM) in place of acetyl chloride. Yield: 79%. δ (300 MHz, [D ]DMSO)
H
6
1
2.62 (s, 1H, CSNH), 11.59 (s, 1H, CONH), 7.73 (d, J 8.7 Hz, 1H, C H ), 7.66 (d, J 8.7 Hz,
6 3
1
H, C H ), 7.38 (s, 1H, C H ), 4.59 (s, 2H, C H ), 4.36 (s, 2H, C H ), 4.15 (s, 5H,
6
3
6
3
5
4
5 4
C H ),2.91 (t, J 8.1 Hz, 2H, C H ), 2.37 (s, 3H, CH ), 1.09 (q, J 8.1 Hz, 3H, C H ) ppm. δ
C
5
5
2
5
3
2
5
(
8
(
(
75 MHz, [D ]DMSO) 172.63, 169.04, 137.13, 135.41, 135.16, 132.33, 126.19, 118.32,
6
−
1
−1
2
5.35, 69.84, 69.52, 67.91, 40.94, 20.57, 10.13 ppm. υmax/cm NH (3242 cm ), sp C–H
−
1
3
−1
−1
−1
3056 cm ), sp C–H (2912 cm ), C=O (1674 cm ), C=C Ar (1531–1565 cm ), C=S
−
1
−1
1122–1256 cm ), Fe–Cp (482 cm ). Elemental analysis Calcd (%) for C H FeN OS: C,
21 22 2
6
2.08; H, 5.46; N, 6.89; S, 7.89. Found (%): C, 62.19; H, 5.23; N, 6.97; S, 7.95.
2
.7. Synthesis of N-pivaloyl-N′-(4-ferrocenyl-3-methylbenzyl) thiourea (S6)
Compound S6 was prepared using the same method as for S1 except using pivaloyl chloride
0.522 mL, 4.25 mM) in place of acetyl chloride. Yield: 73%. δH (300 MHz, [D ]DMSO)
(
6
1
2.65 (s, 1H, CSNH), 10.73 (s, 1H, CONH), 7.68–7.29 (m, 3H, C H ), 4.56 (s, 2H, C H ),
6 3 5 4
4
.32 (s, 2H, C H ), 4.16 (s, 5H, C H ), 2.35 (s, 3H, CH ), 1.15 (d, J 6.6 Hz, 9H, C H ) ppm.
5
4
5
5
3
3 9
δ (75 MHz, [D ]DMSO) 176.42, 169.19, 135.73, 135.07, 134.91, 129.85, 125.24, 122.02,
C
6
−
1
−1
2
8
4.24, 69.71, 69.52, 67.72, 52.87, 29.01, 20.63 ppm. υmax/cm NH (3315 cm ), sp C–H
−1
3
−1
−1
−1
(
(
3102 cm ), sp C–H (2921 cm ), C=O (1669 cm ), C=C Ar (1516–1591 cm ), C=S
1131–1262 cm ), Fe–Cp (483 cm ). Elemental analysis Calcd (%) for C H FeN OS: C,
−
1
−1
23 26 2
6
3.60; H, 6.03; N, 6.45; S, 7.38. Found (%): C, 63.49; H, 6.13; N, 6.37; S, 7.45.
2
.8. Cyclic voltammetry and DNA binding studies
The interaction of the drug with DNA was studied by Eco Chemie Auto lab PGSTAT 12
potentiostat/galvanostat (Utrecht, The Netherlands). The investigation of binding ability of
drug with DNA was made by using the three electrode system having platinum disk as
working electrode, Ag/AgCl as reference electrode and platinum wire as counter electrode.
The surface of the working electrode was cleaned and polished each time accompanied by

2
152
S. Hussain et al.
rinsing with doubly distilled water. The voltammograms were recorded in absence and pres-
−
1
ence of CT-DNA (2, 4, and 6 μM) at different scan rates (25–300 mV s ) to probe the elec-
trochemical nature and DNA binding affinities of S1–S6 in 90% aqueous DMSO.
2
.9. Antioxidant activity
The antioxidant activities of S1–S6 were determined by using DPPH. The sample solutions
were prepared by keeping constant concentration of DPPH (165 μM) and increasing con-
−1
centration of compounds (3.1, 6.2, 12.5, 25, 50, 100 μg mL ) in ethanol and buffered at
pH 6 (0.1 M NaH PO + 0.1 M NaOH). All these mixtures were kept in the dark for 30 min
2
4
and absorbance was measured at 517 nm in dim light using a Shimadzu 1800 UV–vis spec-
trophotometer. From the mean value of three readings, % inhibition was calculated by using
following equation [28].
A ꢀ A
0
s
Scavenging activityð%Þ ¼
ꢁ 100
(1)
A
0
3
3
. Results and discussion
.1. Synthesis and spectroscopic analysis
Synthetic pathway for the synthesis of six new ferrocene-incorporated N,N′-disubstituted
thioureas (S1–S6) is sketched in scheme 1.
The compounds have been synthesized by reacting 4-ferrocenyl-3-methyl aniline with
freshly prepared isothiocyanates under N in dry acetone. The compounds were structurally
2
characterized by using different analytical techniques. In FT-IR spectra N–H gave a broad
−
1
band at 3400–3200 cm . The broadening of the band is due to intermolecular and intramo-
lecular hydrogen bonding and secondary bonding interaction which can be confirmed in
−
1
crystal structure of S1. The diagnostic band at 1700–1650 cm corresponds to the carbonyl
−
1
1
while thiocarbonyl appeared at 1250–1130 cm . In H NMR spectra ferrocenyl protons
1
3
appeared at 4–5 ppm, however, NH protons appeared above 11.45 ppm. In the C NMR
spectra, the (C=O) and (C=S) peaks appeared between 169 and 180 ppm, whereas ferroce-
nyl carbons appear at 60–90 ppm. The aromatic and aliphatic protons and carbons appear in
1
13
the usual regions in H and C NMR [29, 30].
3
.2. Single-crystal X-ray studies of S1
Careful crystallization of S1 in acetone yielded orange crystals suitable for single-crystal X-
ray diffraction analysis. Data pertaining to the data collection and structure refinement shows
that S1 was crystallized in the triclinic crystal system with P-1 space group. Multi-scan
absorption correction method was used. Crystal parameters were calculated at 296 K using
Mo K (λ = 0.71073) radiations with empirical formula C H FeN OS, unit cell dimensions
α
20 20
2
a = 7.4316(10) Å, b = 10.0064(16) Å, c = 12.3340(18) Å, α = 91.180(6)°, β = 103.806(5)°,
3
3
γ = 101.334(6)°, Mr = 392.30, volume = 871.1(2) Å , Z = 2, density = 1.496 g/cm ,
3
F (0 0 0) = 408.0, crystal size = 0.12 × 0.15 × 0.25 mm , index ranges (h, k, l)
4), (h, k, l)min = (−8, −12, −14), and total reflections = 3235, μ = 0.996 mm ,
= (8, 12,
max
−
1
1
θmax = 25.50°, R = 0.0502 and wR = 0.1204.
2

Ferrocene incorporated thioureas
2153
Scheme 1. Synthetic scheme for ferrocene-incorporated N,N′-disubstituted thioureas (S1–S6).
Table 1 provides a selection of important inter atomic distances, bond angles, and torsion
angles. Figure 1 represents the molecular structures with numbering scheme of S1. The
intramolecular hydrogen bonding and intermolecular interactions present in title compound
are summarized in table 2 and drawn in figure 2.
The rings A (C6–C10), B (C11–C16), and C (H1A, N1, C18, N2, C19, and O) in S1 are
planar with RMS deviation of 0.0035, 0.0054, and 0.0267 Å, respectively. All these three
planes are on the same surface due to the extended resonance over the 17 atoms in the mol-
ecule. The bond distance between A and B rings [C10–C11 = 1.485(7)], less than the nor-
mal C–C single bond distance 1.54 Å) supports resonance over these rings in S1.
Intermolecular hydrogen bonding (NH–O type) is responsible for the planarity of six atoms
forming ring C in S1 (figure 2). The interplanar angles between these planes are A/B =
3
9.88°, A/C = 28.36°, and B/C = 12.93°. This type of intramolecular hydrogen bonding is
well known for such aroyl or acoyl substituted organic thioureas as we found in the ferro-
cene analogs [31, 32].
Two independent molecules exist in an asymmetric unit which are connected alternately
by intermolecular NH–S hydrogen bonding and secondary non-covalent interactions (π–H),
to mediate a supramolecular structure as shown in figure 3. These intermolecular non-bond-
ing secondary interactions have great importance for biological activities. Literature studies

2
154
S. Hussain et al.
Table 1. Selective interatomic distances, bond angles, and torsion angles in S1.
Bond lengths (Å)
Bond angles (°)
Fe1–C8
C8–C9
2.033(7)
1.411(6)
1.485(7)
1.503(6)
1.383(7)
1.416(7)
1.335(6)
1.397(7)
1.636(7)
1.378(7)
1.209(7)
0.860(4)
2.034(8)
1.376(8)
1.503(6)
0.861(4)
0.930(6)
0.930(4)
0.960(5)
C4–Fe1–C5
C5–Fe1–C9
C9–C10–C11
C10–C11–C12
C13–C14–N1
C14–N1–C18
N1–C18–N2
N1–C18–S1
O1–C19–N2
40.59(21)
127.30(19)
123.54(34)
119.24(34)
125.43(34)
131.16(34)
114.08(33)
127.61(31)
122.88(40)
118.30(29)
C10–C11
C16–C17
C15–C16
C14–N1
N1–C18
C18–N2
C18–S1
N2–C19
C19–O1
N1–H1A
Fe1–C5
S1–C18–N2
Torsion angles (°)
C9–C10–C11–C12
C12–C11–C10–C6
C6–C10–C11–C16
C15–C14–N1–C18
C14–N1–C18–S1
C14–N1–C18–N2
C18–N2–C19–O1
C18–N2–C19–C20
−38.34(55)
142.20(42)
−41.38(62)
−172.26(38)
4.27(63)
−176.62(36)
−7.24(66)
172.59(37)
C2–C3
C17–C16
N2–H2A
C5–H5
C13–H13
C20–H20A
Figure 1. Molecular structure of S1.
Table 2. The intramolecular and intermolecular hydrogen bonds interactions in S1.
H-bonding
X
H
Y
d(X–H) (Å)
(H–Y) (Å)
d(X–Y) (Å)
<(XHY) (°)
Intramolecular
Intermolecular
N1
N2
H1a
H2a
O1
S1
0.860(4)
0.861(4)
1.888(8)
2.511(3)
2.619(12)
3.354(3)
141.96(23)
166.86(5)
demonstrate that compounds with stronger non-bonding interactions have more ability to
bind with macro-biological molecules likes proteins and DNA [33–35]. So, we were expect-
ing good affiliation of S1 with DNA.

Ferrocene incorporated thioureas
2155
Figure 2. Inter- and intramolecular hydrogen bonding in S1.
Figure 3. Supramolecular structures of S1 mediated by secondary bonding interactions.
3.3. Voltammetric measurements and DNA binding studies
The interaction of the drug with DNA and investigation of binding ability of drug with
DNA were made by voltammetric measurements. The voltammograms were recorded in
absence and presence of DNA at different scan rates to probe the electrochemical nature
and DNA binding affinities of S1–S6 in 90% aqueous DMSO. The electrochemical and
DNA binding of S1 is discussed briefly (figures 4 and 5) here, whereas the electrochemical
and DNA binding data of other compounds are tabulated in table 3. The cyclic voltammo-
gram of 1 mM S1 {N-acetyl-N′-(4-ferrocenyl-3-methylbenzyl) thioureas} revealed a couple
of well-defined redox peaks from 0 to 0.8 V. Compound S1 has an oxidation maximum at
0
.582 V and reduction maximum at 0.343 V in a quasi-reversible electrochemical process.
The quasi reversibility of the process was evident by non-consistency of potential at differ-
−
1
ent scan rates (25, 50, 75, 100, 200, and 300 mV s ), the ratio of anodic and cathodic cur-
rent was not unity and the difference between the oxidation and reduction maxima (E =
p
E − E ) was 0.225 V, which confirms the quasi reversible nature of the system. The num-
pa
pc
2
+
3+
ber of electrons transferred is one by oxidation of Fe of the ferrocenyl moiety to Fe in
the forward scan and in the reverse scan reduces to Fe . On addition of 2 μM of calf
thymus DNA (CT-DNA) to S1, there is a decrease in peak current from 4.45 × 10 to
.89 × 10 mA with a negative shift in oxidation peak potential of 20 and 10 mV for the
2
+
−
3
−
3
3

2
156
S. Hussain et al.
Figure 4. (a) Representative cyclic voltammograms of 1 mM S1 recorded at different potential sweep rates
−
1
(
25–300 mV s ) on glassy carbon electrode at 298 K in 10% aqueous DMSO buffer at pH 6.0; supporting electro-
1/2
lyte 0.1 M TBAP (b) I vs. υ plots of 1 mM S1 in the absence of DNA (–) and presence of 0.6 μM DNA (–) at
scan rates ranging from 25 to 300 mV s under the experimental condition.
p
−1
−
1
Figure 5. Representative cyclic voltammograms of 1 mM S1 recorded at 100 mV s potential sweep rate on plati-
num disk electrode at 298 K in the absence and presence of increasing concentration of CT-DNA (2, 4 and 6 μM)
in 10% aqueous DMSO buffer at pH 6.0, supporting electrolyte 0.1 M TBAP.
reduction peak. This negative shift in peak potential indicates that the product of oxidized
specie of S1 have more binding ability with DNA than to reduced species. The ferrocenyl
2
+
3+
derivatives involve Fe and Fe ; electron transfer may generate ROS in vivo that cause
damage to DNA [36]. The decrease in the current by addition of DNA is due to decrease in
the number of free molecules due to formation of S1-DNA complex which is equally
supported by the idea that heavy molecules diffuse slowly. The cathodic shift in peak is due
to electrostatic interaction of the drug with anionic phosphate backbone of double helical
DNA [21]. The diffusion coefficients calculated by using equations (2) and (3) are
−
7
2
−1
−7
2 −1
2
.66 × 10 cm s for DNA-free drug and 1.47 × 10 cm s for S1-DNA adduct.

Ferrocene incorporated thioureas
2157
Table 3. The change in oxidation and reduction maxima, formal potential, binding constant, and Gibbs free ener-
gies (−ΔG) for S1–S6, ferrocene, and 4-nitrophenyl ferrocene.
2
−1
2 −1
ΔE
p
=
(Epc
+
Binding
constant (M
Binding energy
D
o
[cm s
]
D
o
[cm s
]
−
1
−1
Compounds
Epa − Epc
E
pa)/2
)
(kJ M
)
free drug
drug-DNA
3
−7
−7
S1
S2
S3
S4
S5
S6
0.22
0.22
0.24
0.20
0.22
0.21
0.46
0.35
0.50
0.26
0.41
0.45
2.03 × 102
18.87
12.83
11.88
24.79
25.55
23.29
14.47
20.45
2.66 × 10
4.38 × 10
1.47 × 10
2.88 × 10
−7
−7
1.77 × 10
2
A
A
1.21 × 104
–
–
A
A
2.21 × 10
–
–
4
−7
−7
−9
−5
−7
−7
3.01 × 104
2.84 × 10
4.16 × 10
1.34 × 10
1.03 × 10
1.53 × 10
3.95 × 10
9.54 × 10
7.51 × 10
1.21 × 10
3.45 × 103
A
A
2
−10
−5
Ferrocene [38]
–
0.18
–
0.52
4
-Nitrophenyl
3.85 × 10
ferrocene [39]
A
Not found/calculated.
Ipa ¼ 2:69 ꢁ 10 n ACo ꢂ D1=2t1=2
5
3=2
(2)
(3)
E
pa
ꢀ Epa=2 ¼ 47:7=ða nÞ
a
The low diffusion coefficient of S1-DNA adduct compared to S1 confirmed the interac-
tion of the drug molecule with DNA. Further addition of 4 and 6 μM DNA changed the
−
3
−3
−3
peak current from 4.45 × 10 to 3.73 × 10 mA and 3.63 × 10 mA, respectively. The
negative shift in peak potential attributed to the electrostatic mode of interaction can be
2+
3+
explained as Fe /Fe of the ferrocenyl moiety interacted with negatively charged phos-
phate backbone of the double helix structure of DNA. The binding constant is calculated by
plot of log (I/I − I) versus log (1/[DNA]) [37]. I is the current which is observed at E for
o
o
pa
DNA-free drug while I is the current at Epa for drug-DNA adduct. The electrochemical
parameters, binding constants, and binding energies are listed in table 3. From the data
listed in table 3, the values of binding constants obtained for compounds are fairly impres-
sive. The binding constant of simple ferrocene and 4-nitrophenyl ferrocene is less than S4,
S5, and S6 which reflects that the linker part of ferrocene also plays a very important role
4
towards the DNA binding affinities. Compounds having binding constants of 10 may
potentially be used as non-covalent DNA binders [38, 39].
3
.4. Antioxidant activity
Free radical scavenging activities of S1–S6 along with ascorbic acid were examined by using
,1-diphenyl-2-picryl-hydrazyl (DPPH) assay in ethanol. A deep violet solution of DPPH
1
gives a strong absorption band at 517 nm which is attributed to its odd electron. As this elec-
tron is paired up by a free radical scavenger, the absorbance decreases in accordance with the
number of electrons taken up. The decrease in absorbance at 517 nm (figure 6) is used to
evaluate the antioxidant potency of compounds [40]. Ascorbic acid was used as standard and
−
1
was found to have IC50 of 10.12 μg mL . The free radical scavenging profiles of S1–S6 were
fairly impressive. The trend in antioxidant activity obtained by using the DPPH method
−
1
showed S1 had the highest DPPH free radical scavenging activity (IC = 15.66 μg mL )
5
0
−
1
followed by S5 (IC50 = 18.22 μg mL ) whereas S6 showed the lowest activity

2
158
S. Hussain et al.
Figure 6. Representative UV–vis absorption spectra of 165 μM DPPH in the absence and presence of increasing
−
1
concentrations of S1 and S2 (3.125–100 μg mL ).
−
1
−1
(
IC50 = 37.89 μg mL ). S2, S3, and S4 have IC values 28.70, 23.08, and 24.15 μg mL
50
which revealed that substituents decrease antioxidant activity, but having IC less than
5
0
−1
3
7.89 μg mL proved that these compounds have value as antioxidant.
4
. Conclusion
Six new ferrocene incorporated N,N′-disubstituted thioureas (S1–S6) were synthesized and
characterized. The compounds were screened for their potency as DNA binders and antioxi-
dants. The behavior of the series of compounds may suggest electrostatic mode of interac-
tions with DNA as revealed by voltammetric measurements [41]. S1 has free radical
scavenging activity among all screened compounds as revealed by DPPH assay.
Supplementary material
Crystallographic data for the structure reported in this article have been deposited to the
Cambridge Crystallographic Data Center as supplementary publication number CCDC-
9
01281. Copies of the data will be available free of charge at deposit@ccdc.cam.ac.uk.
Acknowledgements
The authors are grateful to Quaid-i-Azam University and Higher Education Commission
(HEC) Islamabad [grant number PD-IPFP/HRD/HEC/2013/30240], Pakistan.
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