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B. Lal et al.
perchlorate (TBAP), and all the other chemicals were obtained
from Sigma Aldrich and were used as such. Solvents such as
ethanol, ethyl acetate, acetone, diethyl ether, and petroleum ether
were purified before use according to the standard reported pro-
tocols. Synthesis of 3-ferrocenyl-aniline was based on a reported
method.[37,38] FT-IR spectra were obtained with a Thermo Sci-
entific Nicolet-6700 FTIR spectrometer and a BRUKER
AVANCE 300 MHz NMR spectrometer was used for obtaining
multinuclear (1H and 13C) NMR spectra. Cyclic voltammetric
measurements were performed using an Eco Chemie Auto
laboratory PGSTAT 12 potentiostat/galvanostat (Utrecht, The
Netharlands) with the electrochemical software package GPES
4.9. The gel pictures were captured by Eastman Kodak Co.,
Molecular Imaging Systems (Carestream Health Inc., Rochester,
New York, USA).
122.56, 122.37, 114.30, 84.67, 69.90, 69.51, 66.81, 56.08.
n
max/cmꢀ1 483 (Fe–cp), 3248 (NH), 1718 (C¼O), 1216–1251
(C¼S), 1472–1558 (C¼C Ar ), 3014 (sp2 CH), 2914 (sp3 CH).
Anal. Calc. for C25H22FeN2O2S: C 63.84, H 4.71, N 5.96,
S 6.82. Found: C 64.57, H 4.77, N 5.83, S 6.79 %.
1-(4-Methylbenzoyl)-3-(3-Ferrocenylphenyl) thiourea (3d)
Compound 3d was prepared by using the same method as that
for 3a except using 4-methylbenzoyl chloride (0.48 mL,
3.6 mmol) in place of benzoyl chloride. Yield: 71 %. dH
(300 MHz, [D6]DMSO) 12.66 (s, 1H, CSNH), 11.53 (s, 1H,
CONH), 7.94 (s, 1H, C6H4), 7.90 (d, J 7.2, 2H, C6H4), 7.44
(m, 3H, C6H4), 7.26 (d, J 7.8, 2H, C6H4), 4.78 (s, 2H,C5H4), 4.35
(s, 2H, C5H4), 4.06 (s, 5H, C5H5), 2.40 (s, 3H, CH3). dC
(75 MHz, [D6]DMSO) 179.63, 167.33, 143.23, 137.43, 130.29,
128.87, 127.45, 127.31, 124.56, 123.33, 122.77, 118.99, 84.66,
69.87, 69.54, 66.77, 20.22. nmax/cmꢀ1 475 (Fe–cp), 3354 (NH),
1668 (C¼O), 1159 (C¼S), 1504–1530 (C¼C Ar), 3116 (sp2
CH), 3026 (sp3 CH). Anal. Calc. for C25H22FeN2OS: C 66.09,
H 4.88, N 6.17, S 7.06. Found: C 66.13, H 4.93, N 6.69, S 7.12 %.
1-Benzoyl-3-(3-ferrocenylphenyl) Thiourea (3a)
To prepare the benzoyl isothiocyanate, potassium thiocyanate
(0.35 g, 3.6 mmol) was added under N2 atmosphere to the
solution of benzoyl chloride (0.42 mL, 3.6 mmol) in dried ace-
tone (100 mL). 3-Ferrocenyl aniline (1 g, 3.6 mmol) was added
to the resulting mixture and stirring maintained for 4 h. The
mixture was then poured into ice-cold water and stirred well.
The solid product was filtered off and washed with deionized
water to obtain 1-benzoyl-3-(3-ferrocenylphenyl) thiourea that
was dried in air. Yield 73 %; dH (300 MHz, [D6]DMSO) 12.62
(s, 1H, CSNH), 11.64 (s, 1H, CONH), 8.00 (d, J 7.5, 2H, C6H5),
7.92 (s, 1H, C6H4), 7.68 (m, 2H, C6H4), 7.55 (t, J 7.65, 2H,
C6H5), 7.44 (t, J 9, 1H, C6H4), 7.35 (d, J 7.8, 1H, C6H5), 4.79
(t, J 1.65, 2H,C5H4), 4.36 (t, J 1.65, 2H, C5H4),4.07 (s, 5H,
C5H5). dC (75 MHz, [D6]DMSO) 179.70, 168.76, 140.26,
138.57, 133.63, 132.64, 129.18, 129.11, 128.94, 124.34, 122.57,
122.38, 84.66, 69.91, 69.52, 66.94. nmax/cmꢀ1 3227 (NH), 3018
(sp2 CH ), 1739 (C¼O), 1487–1558 (C¼C Ar), 1139 (C¼S), 483
(Fe–cp). Anal. Calc. for C24H20FeN2OS: C 65.46, H 4.58,
N 6.36, S 7.28. Found: C 65.59, H 4.51, N 6.43, S 7.32 %.
X-Ray Structure Analysis
X-Ray measurements were made on a Bruker kappa APEXII
CCD diffractometer equipped with agraphite-monochromated
˚
MoKa radiation (l 0.71073 A) radiation source. Data collection
used v scans, and a multi-scan absorption correction was
applied. The structure was solved by using SHELXS97 program.
The structure was refined by full 276 matrix least-squares
techniques using SHELXL-97.
Cytotoxicity
The MTT reduction method was used to determine the IC50
values (meaning drug concentrations required for 50 % cell
death) of 3a–d against human ovarian tumour models: A2780
(parent), A2780cisR (resistant to cisplatin), and A2780ZD0473R
(resistant to cisplatin analogue denoted as ZD0473). The cells
were maintained and cultured in 10 % FCS/RPMI 1640 culture
medium. Depending upon the logarithmic growth of the cell
line, briefly 4000 to 6000 cells were sown in 100 mL per well in a
flat-bottomed 96-well culture plate. The 96-well culture plate
was incubated for 24 h at 378C in a humidified atmosphere to
allow cells to attach. Compounds 3a–d dissolved first in DMSO,
were serially diluted with the 10 % FCS/RPMI 1640 culture
medium. For a single treatment, four different concentrations of
drugs were added in triplicate and left to incubate under a
humidified atmosphere for 72 h at 378C. The inhibition of cell
growth was determined using the MTT assay. Four hours after
the addition of freshly prepared MTT solution (50 mL per well of
1 mg mLꢀ1), the yellow formazan crystals produced in each well
were dissolved in 150 mL of DMSO and read with a Bio-Rad
Modal 3550 Microplate Reader (BioRad Sydney Australia) set
at 550 nm.[39]
1-(4-Chlorobenzoyl)-3-(3-Ferrocenylphenyl) Thiourea (3b)
Compound 3b was prepared by using the same method as that
for 3a except using 4-chlorobenzoyl chloride (0.46 mL,
3.6 mmol) in place of benzoyl chloride. Yield: 67 %. dH
(300 MHz, [D6]C3D6O) 12.51 (s, 1H, CSNH), 11.75 (s, 1H,
CONH), 8.01 (d, J 8.4, 2H, C6H4), 7.92 (d, J 8.7, 2H, C6H4), 7.64
(m, 3H, C6H4), 7.33 (s, 1H, C6H4),4.79 (t, J 1.8, 2H, C5H4), 4.36
(t, J 1.65, 2H, C5H4), 4.07 (s, 5H, C5H5). dC (75 MHz, [D6]
C3D6O) 179.59, 167.25, 140.26, 138.46, 137.97, 132.94,
131.18, 131.09, 129.02, 128.94, 84.64, 69.90, 69.53, 66.93.
n
max/cmꢀ1 482 (Fe–cp), 3493 (NH), 1728 (C¼O), 1090–1253
(C¼S), 1481–1511 (C¼C Ar), 3084 (sp2 CH). Anal. Calc. for
C24H19ClFeN2OS: C 60.71, H 4.03, N 5.90, S 6.75. Found:
C 60.65, H 4.01, N 5.95, S 6.71 %.
1-(4-Methoxybenzoyl)-3-(3-Ferrocenylphenyl) thiourea (3c)
Interaction with pBR322 Plasmid DNA
Compound 3c was prepared by using the same method as that for
3a except using 4-methoxybenzoyl chloride (0.49 mL,
3.6 mmol) in place of benzoyl chloride. Yield: 69 %; dH
(300 MHz, [D6]DMSO) 12.72 (s, 1H, CSNH), 11.47 (s, 1H,
CONH), 8.04 (d, J 8.7, 2H, C6H4), 7.91 (s, 1H, C6H4), 7.43
(m, 2H, C6H4), 7.34 (d, J 7.8, 1H, C6H4), 7.08 (d, J 9, 2H, C6H4),
4.79 (t, J 1.5, 2H,C5H4), 4.36 (t, J 1.5, 2H, C5H4), 4.07 (s, 5H,
C5H5), 3.86 (s, 3H, OCH3). dC (75 MHz, [D6]DMSO) 179.83,
168.01, 163.71, 140.23, 138.60, 133.49, 130.07, 129.09, 124.31,
Agarose gel electrophoresis was carried out to obtain information
onany conformational changeinduced by3a–d to pBR322DNA.
To a fixed concentration of pBR322 plasmid DNA (1mL), an
increasing concentration (5to80mM)of 3a–d wereadded and the
mixture was kept on a shaking bath for 4 h at 378C. The reaction
was stopped by rapid cooling to ,08C followed by the addition
of 2 mL of the marker dye ethidium bromide (1 mg mLꢀ1).
The drug–DNA mixture was then loaded onto the 2 % gel and