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F. Asghar et al. / Inorganica Chimica Acta 439 (2016) 82–91
2.2.1. 1-(4-Bromobenzoyl)-3-(4-ferrocenylphenyl)urea (1)
Quantities used were 0.54 g (0.00107 mol) 1-(4-bromoben-
zoyl)-3-(4-ferrocenylphenyl) thiourea, 0.29 g (0.00107 mol) HgCl2
stock solution was measured by UV absorbance at 260 nm using
an epsilon value of 6600 Mꢁ1 cmꢁ1. For electrochemical measure-
ments, a known concentration of the test solution was kept in an
electrochemical cell and the voltammogram was recorded in the
absence of DNA. The procedure was then repeated for systems with
a constant concentration of the drug and varying concentrations of
DNA. The working electrode was polished with alumina powder
and rinsed with distilled water before every reading.
and 3 ml of 100 mM NaOH(aq). Yield 75%; Brown solid;
m.p. 173 °C; FT-IR (powder, cmꢁ1): 3338–3249 (NAH), 3091
(CAHaromatic), 1697 (C@O), 1579 (C@C), 484 (FeACp); 1H NMR
(500 MHz, DMSO-d6, ppm) d 11.67 (s, 1H, NH), 10.40 (s, 1H, NH),
7.87 (d, 2H, J = 8.0 Hz, ArH), 7.72 (d, 2H, J = 8.5 Hz, ArH), 7.20 (d,
2H, J = 8.0 Hz, ArH), 6.51 (d, 2H, J = 8.5 Hz, ArH), 4.58 (s, 2H,
C5H4), 4.21 (s, 2H, C5H4), 3.98 (s, 5H, C5H5); 13C NMR
(125.81 MHz, DMSO-d6, ppm) d 167.1, 161.8, 147.5, 132.2, 131.8,
127.1, 125.7, 114.4, 87.5, 69.5, 68.2, 65.5; Elemental Anal. Calc.
for C24H19BrFeN2O2: C, 57.28; H, 3.80; N, 5.58; Fe, 11.09. Found:
C, 57.31; H, 3.77; N, 5.53; Fe, 11.08%.
2.4.2. UV–Vis spectrophotometry
Absorption spectra were measured on a Shimadzu 1800 UV–Vis
spectrophotometer. The absorption spectrum of a known concen-
tration of the drug was recorded without DNA. The spectroscopic
response was then monitored for the same amount of drug on
addition of small aliquots of DNA solution. All samples were
allowed to equilibrate for 15 min prior to each spectroscopic
measurement.
2.2.2. 1-(3-Bromobenzoyl)-3-(4-ferrocenylphenyl)urea (2)
Quantities used were 0.54 g (0.00107 mol) 1-(3-bromoben-
zoyl)-3-(4-ferrocenylphenyl) thiourea, 0.29 g (0.00107 mol) HgCl2
and 3 ml of 100 mM NaOH(aq). Yield 71%; Brown solid; m.p.
194 °C; FT-IR (powder, cmꢁ1): 3334–3244 (NAH), 3086
(CAHaromatic), 1686 (C@O), 1591 (C@C), 483 (FeACp); 1H NMR
(500 MHz, DMSO-d6, ppm) d 11.05 (s, 1H, NH), 10.47 (s, 1H, NH),
8.05 (s, 1H, ArH), 7.78 (d, 2H, J = 8.0 Hz, ArH), 7.64 (t, 2H,
J = 7.5 Hz, ArH), 7.41 (d, 1H, J = 8.5 Hz, ArH), 7.20 (d, 2H,
J = 8.0 Hz), 4.93 (s, 2H, C5H4), 4.40 (s, 2H, C5H4), 4.09 (s, 5H,
C5H5); 13C NMR (125.81 MHz, DMSO-d6, ppm) d 166.8, 160.6,
143.4, 137.4, 129.3, 127.7, 126.8, 125.3, 120.4, 116.9, 83.6, 70.4,
70.0, 67.2; Elemental Anal. Calc. for C24H19BrFeN2O2: C, 57.28; H,
3.80; N, 5.58; Fe, 11.09. Found: C, 57.26; H, 3.77; N, 5.55; Fe,
11.14%.
2.5. DPPH scavenging activity
The reducing abilities of the compounds 1–3 were determined
with the help of 1,1-diphenyl-2-picrylhydrazyl (DPPH) in DMSO
to produce 1,1-diphenyl-2-picrylhydrazine. The decrease in the
absorption of DPPH was monitored to calculate % age scavenging
according to the following formula [10,25]:
Scavenging Activity ð%Þ ¼ Ao ꢁ A=Ao ꢀ 100
where Ao is the absorbance of free DPPH and A is the absorption of
DPPH–drug mixture with an increasing concentration of drug. To a
solution of DPPH (3.9 mg of DPPH in 100 ml DMSO) were added the
increasing concentrations (12.5 lg/mL) of drug. The decrease in
absorption of DPPH was monitored spectrophotometrically after
30 min at a wavelength of 517 nm. All the readings were taken in
triplicate and the average of the readings was used.
2.2.3. 1-(2-Bromobenzoyl)-3-(4-ferrocenylphenyl)urea (3)
Quantities used were 0.54 g (0.00107 mol) 1-(2-bromoben-
zoyl)-3-(4-ferrocenylphenyl) thiourea, 0.29 g (0.00107 mol) HgCl2
and 3 ml of 100 mM NaOH(aq). Yield 62%; Yellow solid; m.p.
210 °C; FT-IR (powder, cmꢁ1): 3299–3230 (NAH), 3091
(CAHaromatic), 1695 (C@O), 1589 (C@C), 477 (FeACp); 1H NMR
(500 MHz, DMSO-d6, ppm) d 11.20 (s, 1H, NH), 10.42 (s, 1H, NH),
7.74–7.40 (m, 8H, ArH), 4.77 (s, 2H, C5H4), 4.33 (s, 2H, C5H4),
4.03 (s, 5H, C5H5); 13C NMR (125.81 MHz, DMSO-d6, ppm) d
169.9, 160.1, 137.3, 135.8, 135.0, 133.2, 132.5, 129.4, 128.1,
126.8, 120.3, 119.1, 85.0, 69.8, 69.2, 66.5; Elemental Anal. Calc.
for C24H19BrFeN2O2: C, 57.28; H, 3.80; N, 5.58; Fe, 11.09. Found:
C, 57.25; H, 3.84; N, 5.61; Fe, 11.07%.
2.6. DFT study
Computational studies were carried out for calculating the
EHOMO and ELUMO orbitals of the compounds using DFT RB3LYP
and 3-21G basis set. The density functional method (DFT) was
used because of its simplicity and less time consumption [26].
Molecules were first optimized using DFT and energy calculations
were performed on the optimized structures. Gaussian 03 W soft-
ware was used for calculations.
2.7. Protein kinase inhibition assay
2.3. X-ray structure analysis
The kinase inhibition assay was performed using Streptomyces
85E strain according to the previously described protocol [27] with
slight modification. The microorganisms under examination were
first refreshed in a sterile Trypton soy broth (Merck, Germany)
for 24–48 h and then applied to petri plates containing ISP4 mini-
mal medium. Then, 6-mm Whatman filter paper discs soaked with
X-ray data for compound 2 were collected on a Bruker Kappa
APEXII CCD diffractometer equipped with a graphite-monochro-
mated Mo K
a (k = 0.71073 Å) radiation source. Data collection used
x
scans, and a multi-scan absorption correction was applied. The
structure was solved using SHELXS-97 program. Final refinement
on F2 was carried out by full-matrix least squares using SHELXL-97
software [24].
5 lL of each test sample (20 mg/mL DMSO) were employed on
freshly seeded plates. Incubating the plates at 28 °C for 72 h was
done to permit the growth of hyphae. The clear or bald zones
around the disc, which indicate hyphae formation inhibition, were
measured to the nearest mm with the help of a Vernier caliper.
Surfactin served as the positive control, while DMSO impregnated
discs were set as the negative control in order to confirm the non-
toxic effect of DMSO.
2.4. DNA binding studies
2.4.1. Cyclic voltammetry
Voltammetric experiments were performed using a Biologic
SP-300 voltammeter running with EC-Lab Express V 5.40 software,
Japan. Analytical grade TBAP (tertiarybutylammonium perchlo-
rate) was used as supporting electrolyte and N2 gas (99.9%) was
purged through the mixture to avoid any interference from oxygen.
Commercial salmon DNA was solubilized in doubly distilled water
to prepare a stock solution of 6 ꢀ 10ꢁ4 M from which working
concentrations of DNA were prepared. The concentration of the
2.8. Antimicrobial assay
2.8.1. Antibacterial study
Antibacterial activities of the synthesized ureas 1–3 were tested
against five representative, gram-positive (Staphylococcus aureus,