Biologically active: Halo -substituted ferrocenyl thioureas: Synthesis, spectroscopic characterization, and DFT calculations

Article abstract of DOI:10.1039/c8nj00483h

In our search for new therapeutic agents, ferrocene-based thioureas (M1-M9) were successively synthesized and characterized by various analytical techniques like FT-IR, Raman, CHNS, AAS, and multinuclear (¹H and ¹³C) NMR. The interac

Full text of DOI:10.1039/c8nj00483h

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The role of atropisomers on the photo-reactivity and fatigue of
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Biologically active haloꢀsubstituted ferrocenyl thioureas: Synthesis, spectroscopic
characterization, and DFT calculations
Faiza Asghara*, b, c, Sadaf Ranab, Saira Fatimab, Amin Badshahb*,
Bhajan Lald, Ian S. Butlerc
*. Corresponding Authors:
Department of Chemistry, University of Wah, Quaid Avenue, Wah (47000), Pakistan.
faiza994@hotmail.com
Department of Chemistry, QuaidꢀiꢀAzam University, Islamabad (45320), Pakistan.
aminbadshah@yahoo.com
a. Department of Chemistry, University of Wah, Quaid Avenue, Wah (47000), Pakistan.
b. Department of Chemistry, QuaidꢀiꢀAzam University, Islamabad (45320), Pakistan.
c. Department of Chemistry, McGill University, Montreal, QC, Canada H3A 2K6.
d. Institute of Chemistry, Shah Abdul Latif University, Khairpurꢀ66020, Pakistan.
Abstract
In our search for new therapeutic agents, ferroceneꢀbased thioureas (M1ꢀM9) were
successively synthesized and characterized by various analytical techniques like: FTꢀIR,
Raman, CHNS, AAS, and multinuclear (1H and 13C) NMR. The interaction of the compounds
with DNA was investigated by electrochemical and spectroscopic measurements. These
complexes exhibited impressive binding constants ranging from 9.75 x 103 to 5.71 x 104 Mꢀ1.
The diffusion coefficients of the drugꢀDNA adducts are lower than is that for the free drug
indicating the formation of a high molecular weight complex that diffuses slowly towards the
electrode. The cathodic peak potential shift in the cyclic voltammetric behavior of the
synthesized complexes by the addition of DNA is attributable to the electrostatic interactions
between the compound and DNA, an indicator of the oxidizable behavior of the compounds
in the presence of negative environment of DNA. The energy of frontier molecular orbitals
(EHOMO and ELUMO) and the Mulliken charge distribution on the optimized structures was
determined using DFT/B3LYP method combined with 6ꢀ31G(d,p) basis set in DMSO. The
theoretically calculated HOMO and LUMO energies (HOMOꢀLUMO band gap) based on
DFT study correlate well with the electrochemically determined redox potentials
(electrochemical band gap), having a correlation coefficient of 0.953. The synthesized
thioureas were preliminary screened for their in vitro antimicrobial, and cytotoxic activities.
However, the most significant antitumor activity was found for M3 with IC50 value of
1.87 ꢁM. These compounds played a significant role in arresting microbial growth. The
computational measurements were found to be in close agreement with the outcomes of
biological activities.
1

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Keywords: Ferrocene; DNA binding; DFT study; Anticancer activity; Antimicrobial.
1.
Introduction
Interest in metal complexes with therapeutic applications has developed at a
tremendous rate since the discovery of cisplatin as an effective antitumor agent.1,2 Nowadays,
the most promising nonꢀplatinum cytotoxic drugs are evolving from the arena of bioꢀ
organometallics.3,4 Metallocenes contain an entire class of numerous transition metals such
as: V(II), Cr(II), Co(II), Ru(II) and Ni(II), nevertheless ferrocene still serves the foremost
motivation of research chiefly due to its notable stability and ease of preparation.5 Ferrocene
is also a particularly attractive candidate for integration into biomolecules due to its
aromaticity, redox properties, and low toxicity. Ferrocifen, the ferrocene analogue of
hydroxytamoxifen shows enhanced inhibitory effect against hormoneꢀdependent and
independent breast cancer cells.6,7 The anticancer activities of ferrocenyl derivatives
generally depend upon the oxidation state of iron (Fe), with Fe2+ derivatives more active than
Fe3+ derivatives, by changing the conformation of receptor protein.8,9 According to Osella et
al.,10 anticancer activity arises due to generation of oxygen active radicals by the reduction of
ferrocenium ion. The cytotoxicity of compounds is due to the interaction with DNA by which
the physical and chemical nature of DNA alters.11
Thioureas display comprehensive range of bioꢀactivities comprising: antiviral,12,13
antibacterial,14 antifungal,15 antitubercular,16 herbicidal,17 insecticidal,18 and pharmacoꢀ
dynamic properties,19 act as corrosion inhibitors, free radical scavengers, and are polymer
constituents.20,21 Acyl thiourea derivatives are wellꢀrecognized for their pharmacological
potential such as: antiꢀinflammatory, bactericidal, fungicidal, and plant growth regulatory
activity.22,23 One of the chief concerns associated with thiourea derivatives is a high dose is
required for the treatment due to their low lipophilicity, which restrains their efficiency and
imparts lot of lethal side effects.24 This problem can be overwhelmed by introducing a
lipophilic moiety such as ferrocene in the thiourea structure, which will minimize the side
effects due to its enhanced lipid soluble potential.25
The primary characteristic of the contemporary pharmaceutical investigation and
development demands presenting novel candidates for several biological targets. To further
explore the prominence of ferroceneꢀincorporated thioureas in medicinal chemistry, herein
we report the synthesis, structural characterization, and in vitro biological studies of some
2

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new N,N´ꢀdisubstituted ferrocenyl thioureas for their DNA binding efficiency, antimicrobial,
and anticancer properties. Moreover, the DFT study was also accomplished on these
structures to theoretically determine the energy of frontier molecular orbitals, vibrational
frequencies, and the Mulliken charge distribution on the molecular structures.
2.
Experimental
2.1
Materials and methods
Melting points were determined in a capillary tube using an electroꢀthermal melting
point apparatus model MPꢀD Mitamura Riken Kogyo (Japan). Infrared spectra were taken on
1
a Thermoscientific NICOLET 6700 FTꢀIR instrument. Hꢀ and 13CꢀNMR data were recorded
on a Bruker AV500 MHz spectrometer in DMSO. Si(CH3)4 was used as an internal reference.
Raman spectra (± 1 cmꢀ1) were measured on an InVia Renishaw spectrometer, using argonꢀ
ion (514.5 nm) and nearꢀinfrared diode (785 nm) lasers. The Renishaw WiRE 2.0 software
was used for the Raman data acquisition and spectral manipulation. The elemental analyses
were performed using a LECOꢀ932 CHNS analyzer, while the Fe concentrations were
determined on an atomic absorption spectrophotometer Perkin Elmer 2380.
Ferrocene, 2ꢀmethoxyꢀ5ꢀnitroaniline, sodium nitrite, diethyl ether, acetone, Zn dust,
DMSO, ammonium formate, KSCN, and acid chlorides such as: oꢀfluorobenzoyl chloride,
mꢀfluorobenzoyl chloride, pꢀfluorobenzoyl chloride, oꢀchlorobenzoyl chloride, mꢀchloro
benzoyl chloride, pꢀchlorobenzoyl chloride, oꢀbromobenzoyl chloride, mꢀbromobenzoyl
chloride, and pꢀbromo benzoyl chloride were obtained from commercial sources (Sigma
Aldrich/Fluka) and used as received. 4ꢀmethoxyꢀ3ꢀferrocenylaniline was synthesized by a
procedure reported by our group previously (Step 1, Scheme 1).26,27
2.2
General procedure for the synthesis of ferroceneꢀbased acyl thioureas (M1ꢀM9)
The synthesis of ferrocene incorporated thioureas was accomplished by adopting the
method reported previously by our group with some modifications.28 To the suspension of
potassium thiocyanate (10 mmol) in dry acetone (50ꢀ60 mL), different acid chlorides (10
mmol) were introduced under an inert atmosphere (N2) leading to the preparation of the
respective isothiocyanates. 4ꢀmethoxyꢀ3ꢀferrocenylaniline (10 mmol) was then added to the
resulting reaction mixtures, which were then stirred for 12 h. Afterwards, the reaction
mixtures were transferred to ice cold water and stirred well in order to remove water soluble
3

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impurities. The solid products were separated by filtration and washed with deionized water.
The residues were then dissolved in dichloromethane/chloroform and kept for crystallization
(Scheme 1).
Scheme 1. Synthetic scheme for ferroceneꢀbased thioureas.
2.2.1 1ꢀ(2ꢀfluorobenzoyl)ꢀ3ꢀ(4ꢀmethoxyꢀ3ꢀferrocenylphenyl)thiourea (M1)
Quantities used were 2.92 g (10 mmol) 4ꢀmethoxyꢀ3ꢀferrocenylaniline, 0.98 g (10
mmol) potassium thiocyanate, and 1.20 ml (10 mmol) of 2ꢀfluorobenzoyl chloride. Yield
77%; Brown solid; m.p. 204 °C; FTꢀIR and Raman (powder, cmꢀ1): 3392ꢀ3227 (NꢀH), 3079,
3025 (CꢀHaromatic), 2946, 2910 (CꢀHaliphatic), 1663, 1671 (C=O), 1602, 1593 (C=C), 1257ꢀ1138
1
cmꢀ1 (C=S), 481, 483 (FeꢀCp); HꢀNMR (500 MHz, DMSOꢀd6, ppm) δ 12.26 (s, 1H, NH),
11.52 (s, 1H, NH), 7.98ꢀ7.35 (m, 7H, ArH), 4.65 (s, 2H, C5H4), 4.36 (s, 2H, C5H4), 4.16 (s,
5H, C5H5), 3.55 (s, 3H, OCH3); 13CꢀNMR (125.81 MHz, DMSOꢀd6, ppm) δ 177.5, 169.7,
162.6, 140.8, 138.9, 136.4, 134.5, 132.1, 127.6, 124.9, 122.7, 118.8, 116.6, 86.7, 70.2, 69.3,
67.9, 57.4; Elemental anal. Calcd. (%) for C25H21FFeN2O2S: C, 61.52; H, 4.35; N, 5.78; S,
6.56; Fe, 11.41. Found (%): C, 61.41; H, 4.26; N, 5.69; S, 6.64; Fe, 11.32.
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2.2.2 1ꢀ(3ꢀfluorobenzoyl)ꢀ3ꢀ(4ꢀmethoxyꢀ3ꢀferrocenylphenyl)thiourea (M2)
Quantities used were 2.92 g (10 mmol) 4ꢀmethoxyꢀ3ꢀferrocenylaniline, 0.98 g (10
mmol) potassium thiocyanate, and 1.22 ml (10 mmol) of 3ꢀfluorobenzoyl chloride. Yield
70%; Brown solid; m.p. 197 °C; FTꢀIR and Raman (powder, cmꢀ1): 3360ꢀ3217 (NꢀH), 3074,
3059 (CꢀHaromatic), 2935, 2952 (CꢀHaliphatic), 1665, 1674 (C=O), 1590, 1598 (C=C), 1261ꢀ1143
1
cmꢀ1 (C=S), 478, 482 (FeꢀCp); HꢀNMR (500 MHz, DMSOꢀd6, ppm) δ 12.33 (s, 1H, NH),
11.60 (s, 1H, NH), 7.92ꢀ7.31 (m, 7H, ArH), 4.73 (s, 2H, C5H4), 4.40 (s, 2H, C5H4), 4.09 (s,
5H, C5H5), 3.74 (s, 3H, OCH3); 13CꢀNMR (125.81 MHz, DMSOꢀd6, ppm) δ 178.4, 167.5,
162.8, 141.1, 138.7, 136.5, 134.7, 132.4, 128.2, 123.4, 121.3, 117.5, 115.8, 87.0, 70.7, 69.5,
67.2, 56.5; Elemental anal. Calcd. (%) for C25H21FFeN2O2S: C, 61.52; H, 4.35; N, 5.78; S,
6.56; Fe, 11.41. Found (%): C, 61.60; H, 4.33; N, 5.72; S, 6.47; Fe, 11.49.
2.2.3 1ꢀ(4ꢀfluorobenzoyl)ꢀ3ꢀ(4ꢀmethoxyꢀ3ꢀferrocenylphenyl)thiourea (M3)
Quantities used were 2.92 g (10 mmol) 4ꢀmethoxyꢀ3ꢀferrocenylaniline, 0.98 g (10
mmol) potassium thiocyanate, and 1.18 ml (10 mmol) of 4ꢀfluorobenzoyl chloride. Yield
67%; Brown solid; m.p. 188 °C; FTꢀIR and Raman (powder, cmꢀ1): 3374ꢀ3233 (NꢀH), 3086,
3061 (CꢀHaromatic), 2931, 2941 (CꢀHaliphatic), 1676, 1665 (C=O), 1591, 1588 (C=C), 1268ꢀ1147
1
cmꢀ1 (C=S), 483, 473 (FeꢀCp); HꢀNMR (500 MHz, DMSOꢀd6, ppm) δ 12.75 (s, 1H, NH),
11.67 (s, 1H, NH), 8.01ꢀ7.46 (m, 7H, ArH), 4.66 (s, 2H, C5H4), 4.35 (s, 2H, C5H4), 4.06 (s,
5H, C5H5), 3.68 (s, 3H, OCH3); 13CꢀNMR (125.81 MHz, DMSOꢀd6, ppm) δ 179.1, 165.4,
161.6, 140.5, 138.7, 135.9, 133.4, 132.2, 129.5, 125.4, 122.3, 117.9, 115.7, 86.6, 70.2, 69.7,
66.5, 55.1; Elemental anal. Calcd. (%) for C25H21FFeN2O2S: C, 61.52; H, 4.35; N, 5.78; S,
6.56; Fe, 11.41. Found (%): C, 61.44; H, 4.23; N, 5.71; S, 6.66; Fe, 11.35.
2.2.4 1ꢀ(2ꢀchlorobenzoyl)ꢀ3ꢀ(4ꢀmethoxyꢀ3ꢀferrocenylphenyl)thiourea (M4)
Quantities used were 2.92 g (10 mmol) 4ꢀmethoxyꢀ3ꢀferrocenylaniline, 0.98 g (10
mmol) potassium thiocyanate, and 1.26 ml (10 mmol) of 2ꢀchlorobenzoyl chloride. Yield
74%; Brown solid; m.p. 213 °C; FTꢀIR and Raman (powder, cmꢀ1): 3388ꢀ3212 (NꢀH), 3076,
3091 (CꢀHaromatic), 2927, 2936 (CꢀHaliphatic), 1668, 1661 (C=O), 1593, 1596 (C=C), 1265ꢀ1132
1
cmꢀ1 (C=S), 475, 486 (FeꢀCp); HꢀNMR (500 MHz, DMSOꢀd6, ppm) δ 12.33 (s, 1H, NH),
11.61 (s, 1H, NH), 7.88ꢀ7.36 (m, 7H, ArH), 4.68 (s, 2H, C5H4), 4.41 (s, 2H, C5H4), 4.13 (s,
5H, C5H5), 3.77 (s, 3H, OCH3); 13CꢀNMR (125.81 MHz, DMSOꢀd6, ppm) δ 178.2, 168.3,
139.3, 137.6, 134.7, 133.9, 131.6, 129.3, 125.1, 121.8, 117.2, 115.6, 86.2, 69.8, 68.4, 66.2,
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56.3; Elemental anal. Calcd. (%) for C25H21ClFeN2O2S: C, 59.46; H, 4.16; N, 5.53; S, 6.31;
Fe, 11.08. Found (%): C, 59.36; H, 4.08; N, 5.62; S, 6.23; Fe, 11.01.
2.2.5 1ꢀ(3ꢀchlorobenzoyl)ꢀ3ꢀ(4ꢀmethoxyꢀ3ꢀferrocenylphenyl)thiourea (M5)
Quantities used were 2.92 g (10 mmol) 4ꢀmethoxyꢀ3ꢀferrocenylaniline, 0.98 g (10
mmol) potassium thiocyanate, and 1.28 ml (10 mmol) of 3ꢀchlorobenzoyl chloride. Yield
66%; Brown solid; m.p. 195 °C; FTꢀIR and Raman (powder, cmꢀ1): 3364ꢀ3204 (NꢀH), 3088,
3054 (CꢀHaromatic), 2951, 2944 (CꢀHaliphatic), 1672, 1664 (C=O), 1595, 1591 (C=C), 1260ꢀ1136
1
cmꢀ1 (C=S), 485, 478 (FeꢀCp); HꢀNMR (500 MHz, DMSOꢀd6, ppm) δ 12.42 (s, 1H, NH),
11.57 (s, 1H, NH), 7.81ꢀ7.50 (m, 7H, ArH), 4.72 (s, 2H, C5H4), 4.33 (s, 2H, C5H4), 4.11 (s,
5H, C5H5), 3.59 (s, 3H, OCH3); 13CꢀNMR (125.81 MHz, DMSOꢀd6, ppm) δ 177.1, 167.0
140.6, 138.7, 135.8, 132.3, 130.9, 129.1, 125.8, 122.4, 116.6, 114.3, 86.5, 70.3, 69.7, 66.8,
54.8; Elemental anal. Calcd. (%) for C25H21ClFeN2O2S: C, 59.46; H, 4.16; N, 5.53; S, 6.31;
Fe, 11.08. Found (%): C, 59.54; H, 4.25; N, 5.44; S, 6.39; Fe, 11.17.
2.2.6 1ꢀ(4ꢀchlorobenzoyl)ꢀ3ꢀ(4ꢀmethoxyꢀ3ꢀferrocenylphenyl)thiourea (M6)
Quantities used were 2.92 g (10 mmol) 4ꢀmethoxyꢀ3ꢀferrocenylaniline, 0.98 g (10
mmol) potassium thiocyanate, and 1.30 ml (10 mmol) of 4ꢀchlorobenzoyl chloride. Yield
75%; Brown solid; m.p. 192 °C; FTꢀIR and Raman (powder, cmꢀ1): 3391ꢀ3223 (NꢀH), 3055,
3083 (CꢀHaromatic), 2949, 2934 (CꢀHaliphatic), 1669, 1672 (C=O), 1587, 1596 (C=C), 1253ꢀ1154
1
cmꢀ1 (C=S), 481, 474 (FeꢀCp); HꢀNMR (500 MHz, DMSOꢀd6, ppm) δ 12.52 (s, 1H, NH),
11.73 (s, 1H, NH), 7.77ꢀ7.48 (m, 7H, ArH), 4.77 (s, 2H, C5H4), 4.45 (s, 2H, C5H4), 4.16 (s,
5H, C5H5), 3.83 (s, 3H, OCH3); 13CꢀNMR (125.81 MHz, DMSOꢀd6, ppm) δ 179.9, 168.7,
141.5, 139.3, 136.9, 134.4, 133.0, 131.7, 126.8, 123.5, 118.2, 116.1, 87.2, 70.7, 69.6, 66.8,
58.5; Elemental anal. Calcd. (%) for C25H21ClFeN2O2S: C, 59.46; H, 4.16; N, 5.53; S, 6.31;
Fe, 11.08. Found (%): C, 59.37; H, 4.05; N, 5.59; S, 6.25; Fe, 11.05.
2.2.7 1ꢀ(2ꢀbromobenzoyl)ꢀ3ꢀ(4ꢀmethoxyꢀ3ꢀferrocenylphenyl)thiourea (M7)
Quantities used were 2.92 g (10 mmol) 4ꢀmethoxyꢀ3ꢀferrocenylaniline, 0.98 g (10
mmol) potassium thiocyanate, and 1.30 ml (10 mmol) of 2ꢀbromobenzoyl chloride. Yield
63%; Brown solid; m.p. 206 °C; FTꢀIR and Raman (powder, cmꢀ1): 3382ꢀ3219 (NꢀH), 3064,
3081 (CꢀHaromatic), 2944, 2926 (CꢀHaliphatic), 1674, 1663 (C=O), 1594, 1589 (C=C), 1266ꢀ1125
1
cmꢀ1 (C=S), 488, 479 (FeꢀCp); HꢀNMR (500 MHz, DMSOꢀd6, ppm) δ 12.29 (s, 1H, NH),
11.06 (s, 1H, NH), 7.93ꢀ7.45 (m, 7H, ArH), 4.66 (s, 2H, C5H4), 4.40 (s, 2H, C5H4), 4.13 (s,
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5H, C5H5), 3.74 (s, 3H, OCH3); 13CꢀNMR (125.81 MHz, DMSOꢀd6, ppm) δ 178.7, 168.2,
138.7, 136.5, 134.8, 133.6, 132.5, 131.6, 129.4, 127.7, 122.2, 118.3, 87.5, 70.6, 69.4, 68.5,
56.8; Elemental anal. Calcd. (%) for C25H21BrFeN2O2S: C, 54.64; H, 3.91; N, 5.13; S, 5.89;
Fe, 10.19. Found (%): C, 54.73; H, 3.98; N, 5.02; S, 5.80; Fe, 10.27.
2.2.8 1ꢀ(3ꢀbromobenzoyl)ꢀ3ꢀ(4ꢀmethoxyꢀ3ꢀferrocenylphenyl)thiourea (M8)
Quantities used were 2.92 g (10 mmol) 4ꢀmethoxyꢀ3ꢀferrocenylaniline, 0.98 g (10
mmol) potassium thiocyanate, and 1.32 ml (10 mmol) of 3ꢀbromobenzoyl chloride. Yield
67%; Brown solid; m.p. 192 °C; FTꢀIR and Raman (powder, cmꢀ1): 3374ꢀ3206 (NꢀH), 3077,
3051 (CꢀHaromatic), 2940, 2954 (CꢀHaliphatic), 1677, 1671 (C=O), 1598, 1593 (C=C), 1262ꢀ1147
1
cmꢀ1 (C=S), 484, 477 (FeꢀCp); HꢀNMR (500 MHz, DMSOꢀd6, ppm) δ 12.18 (s, 1H, NH),
11.53 (s, 1H, NH), 7.94ꢀ7.39 (m, 7H, ArH), 4.58 (s, 2H, C5H4), 4.26 (s, 2H, C5H4), 4.08 (s,
5H, C5H5), 3.67 (s, 3H, OCH3); 13CꢀNMR (125.81 MHz, DMSOꢀd6, ppm) δ 180.2, 166.4,
141.5, 138.9, 136.4, 134.6, 132.2, 130.6, 128.3, 125.7, 122.5, 118.2, 86.8, 70.5, 69.7, 68.9,
57.7; Elemental anal. Calcd. (%) for C25H21BrFeN2O2S: C, 54.64; H, 3.91; N, 5.13; S, 5.89;
Fe, 10.19. Found (%): C, 54.57; H, 3.82; N, 5.20; S, 5.95; Fe, 10.12.
2.2.9 1ꢀ(4ꢀbromobenzoyl)ꢀ3ꢀ(4ꢀmethoxyꢀ3ꢀferrocenylphenyl)thiourea (M9)
Quantities used were 2.92 g (10 mmol) 4ꢀmethoxyꢀ3ꢀferrocenylaniline, 0.98 g (10
mmol) potassium thiocyanate, and 1.32 ml (10 mmol) of 4ꢀbromobenzoyl chloride. Yield
75%; Orange solid; m.p. 186 °C; FTꢀIR and Raman (powder, cmꢀ1): 3386ꢀ3209 (NꢀH), 3067,
3044 (CꢀHaromatic), 2936, 2948 (CꢀHaliphatic), 1667, 1661 (C=O), 1592, 1602 (C=C), 1255ꢀ1139
1
cmꢀ1 (C=S), 482, 485 (FeꢀCp); HꢀNMR (500 MHz, DMSOꢀd6, ppm) δ 12.47 (s, 1H, NH),
11.74 (s, 1H, NH), 7.90ꢀ7.42 (m, 7H, ArH), 4.68 (s, 2H, C5H4), 4.36 (s, 2H, C5H4), 4.17 (s,
5H, C5H5), 3.79 (s, 3H, OCH3); 13CꢀNMR (125.81 MHz, DMSOꢀd6, ppm) δ 178.8, 167.7,
140.1, 137.5, 135.8, 132.6, 130.5, 128.9, 125.6, 122.8, 120.7, 118.5, 86.9, 70.7, 69.8, 67.6,
53.8; Elemental anal. Calcd. (%) for C25H21BrFeN2O2S: C, 54.64; H, 3.91; N, 5.13; S, 5.89;
Fe, 10.19. Found (%): C, 54.69; H, 3.96; N, 5.05; S, 5.77; Fe, 10.29.
2.3
2.3.1 Cyclic voltammetry
Voltammetric experiments were performed using a Biologic SPꢀ300 voltammeter
DNA binding studies
running on ECꢀLab Express V 5.40 software, Japan. Analytical grade TBAP (Tertiarybutyl
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ammonium perchlorate) was used as supporting electrolyte and N2 gas (99.9 %) was purged
through the mixture to avoid any interference from oxygen. Commercial salmon sperm DNA
(SSꢀDNA) obtained from Sigma Aldrich (Cat. No. 74782) was solubilized in doubly distilled
water to prepare a stock solution of 6 x 10ꢀ4 M from which working concentrations of DNA
were prepared. The concentration of the stock solution was measured by UV absorbance at
260 nm using an epsilon value of 6600 Mꢀ1 cmꢀ1. This DNA was protein free because
A260/A280 > 1.8. For electrochemical measurements, 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 each measurement.29
2.3.2 UVꢀVis spectrophotometry
Absorption spectra were recorded on a Shimadzu 1800 UVꢀVis spectrophotometer.
The absorption spectrum of a known concentration 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.29 Based on the variation in absorption maxima (λmax) in the
presence of DNA, the binding constant “K” of drugꢀDNA complex was calculated using the
BenesiꢀHildebrand equation:30
Eq 1
where Ao and A are the absorbance of the free compound and of the compoundꢀDNA
complex, ƐG and ƐHꢀG corresponds to the molar extinction coefficient of the free drug, and the
extinction coefficient of the drug in the fully bound form, respectively.31
2.4
DFT calculations
Computational studies were carried out in order to calculate the energies of the
frontier molecular orbitals (EHOMO and ELUMO), vibrational frequencies, and the Mulliken
charge distribution on the molecular structures using the DFT/B3LYP method combined with
6ꢀ31G(d,p) basis set in DMSO. The density functional theory (DFT) was used because of its
simplicity and less time consumption compared to more sophisticated calculations.32 The
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structures of the molecules were first optimized using DFT and then energy calculations were
performed on the optimized structures. Gaussian 03W software was used for calculations.
2.5
2.5.1 Antibacterial study
Antibacterial activities of the synthesized ferrocenyl compounds were determined
Antimicrobial assay
against five representative, gramꢀpositive (S. aureus, S. typhimurium, M. luteus) and gramꢀ
negative (B. bronchiseptica and E. aerogenes) bacterial strains, by the disc diffusion
method.33,34 Prior to use, the bacterial isolates under investigation were first cultured in a
nutrient broth for 18 h and standardized to 0.5 McFarland turbidity scale (106 cfu mLꢀ1). The
nutrient agar medium, which was prepared by adding 2.3 g nutrient agar (Merck) to 100 mL
o
distilled water at pH 7.0, was autoclaved and then cooled down to 45 C and seeded. Petri
dishes in triplicate were prepared by introducing 75 mL of seeded nutrient agar medium and
allowed to solidify. Wells were bored into the agar using a sterile 6 mm diameter cork borer.
Approximately, 100 ꢁL of the test compounds were infused into the wells, which were
o
permitted to stand at room temperature for about 2 h and then incubated at 37 C. Controls
were set in parallel in which the respective solvents were used to fill the well. Subsequent to
o
the incubation of plates at 37 C for 24 h, the diameter of zone of inhibition was measured.
The effects were compared with penicillin (positive control) at a concentration of 1.0 mg/mL.
The relative percentage inhibition of the tested compounds with respect to positive control
was calculated by using the following formula:
Relative percentage inhibition of the test compound = 100 × (XꢀY)/(ZꢀY)
Eq 2
where X is the total area of inhibition of the test sample, Y is the total area of inhibition of the
solvent and Z is the total area of inhibition of the standard drug.
2.5.2 Antifungal study
The sensitivity of the synthesized complexes was tested against three different fungal
strains, namely F. moniliforme, A. fumigatus and A. flavus using the agar tube dilution
method.33,34 Sample preparation was done by dissolving 2 mg of the compound in 1.0 mL of
DMSO. In order to prepare the culture media, 6.5 g of sabouraud dextrose agar was dissolved
in 100 mL distilled water (pH = 5.6). Next, 10 mL of the sabouraud dextrose agar (Merck)
was introduced into screwꢀcapped tubes or cottonꢀplugged test tubes and autoclaved at 121
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°C for 21 min. The tubes were cooled to 50 °C and sabouraud dextrose agar was loaded with
70 µL of the compound taken from stock solution. The tubes containing the media were then
solidified in a slanting position at room temperature. For each fungal strain, three slants of
test compound were prepared. Tubes comprising the solidified media and test compounds
were infused with a 4ꢀmm diameter piece of inoculum, taken from a 7ꢀday old culture of
fungus. One test tube of each compound was prepared, which was used for positive control.
Slants without compound were used for negative control. The test tubes were incubated at 28
°C for 7 days. During incubation, the cultures were examined twice a week. Readings were
taken by measuring the linear length (mm) of the fungus in slant and growth inhibition was
calculated with reference to the control. Percentage inhibition of fungal growth for each
concentration of the compound was determined by using following formula:
Percentage inhibition of fungal growth = 100 – Linear growth in test (cm) × 100
Eq 3
Linear growth in control (cm)
2.6
Cytotoxic screening
The growth inhibitory effect of the synthesized complexes on human breast cancer
cell line MCFꢀ7 (ATCC# HTBꢀ22), and breast nonꢀcancerous cells MCFꢀ10A (ATCC# CRLꢀ
10317) was assessed by use of the MTT (3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyl
tetrazolium bromide) assay, in which the metabolically active cells reduce MTT to yield a
DMSO soluble formazan product that can be analyzed calorimetrically.35,36 The cells were
maintained and cultured in RPMI 1640 culture medium supplemented with 10% FBS (fetal
bovine serum). Cisplatin was used as a control. In brief, 1 x 104 cells were seeded in triplicate
into the wells of the flat bottomed 96ꢀwell culture plates. The plates were incubated at 37 °C
for 24 h in a humidified atmosphere (5% carbon dioxide in air, pH 7.4) to permit the cells to
attach. The samples were then added at different concentrations (0ꢀ100 µM) to triplicate
wells. After 72 h of incubation, the cells were exposed to 20 ꢁL of MTT (5 mg/mL) and
incubated for 4 h at 37 °C for further cultivation. The yellow formazan crystals thus produced
in each well were solubilized in 200 ꢁL of DMSO and the absorbance of the resulting
solution was read at 570 nm using a microplate reader (Labsystems Multiskan MS). The
sensitivity of the compounds M1ꢀM9 against the subjected cell lines was expressed in terms
of IC50 values (drug concentration that resulted in 50% reduction of cell growth), calculated
on the regression line where the absorbance values at 570 nm were plotted against logarithm
of drug concentration. The experiment was performed in triplicate.
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3.
Results and discussion
Chemistry and spectroscopic study
The synthesis of ferroceneꢀcontaining acyl thioureas (M1ꢀM9) was accomplished by
3.1
allowing the ferrocenyl anilines to react with freshly prepared isothiocyanates under an inert
(N2) atmosphere in dry acetone (Scheme 1).
The FTꢀIR and Raman spectra of the compounds M1ꢀM9 indicated that the main
vibrational stretching modes were observed between 3392ꢀ3204 (NꢀH), 3091ꢀ3025 (Cꢀ
H
aromatic), 2954ꢀ2926 (CꢀHaliphatic), 1677ꢀ1663 (C=O), 1602ꢀ1587 (C=C), 1268ꢀ1125 cmꢀ1
(C=S), and 488ꢀ473 (FeꢀCp), respectively. The disappearance of the characteristic band for
isothiocyanate (N=C=S) in the region of 2000 cmꢀ1 and the appearance of a thiocarbonyl
(C=S) band in the range of 1300ꢀ1100 cmꢀ1 confirms the formation of thioureas (M1ꢀM9), as
a result of coupling of 4ꢀmethoxyꢀ3ꢀferrocenylaniline with freshly prepared isothiocyanates.
A broad signal between 3392ꢀ3204 cmꢀ1 due to the NꢀH stretch is attributable to intraꢀ and
intermolecular hydrogen bonding. The carbonyl group appeared as an intense band at 1676ꢀ
1661 cmꢀ1, while all the other bands are evident in their normal characteristic regions.
1
The HꢀNMR data for the synthesized thioureas (M1ꢀM9) indicate that the –NH
hydrogen resonates considerably downfield from other resonances in the spectra at around
12.75ꢀ12.18 and 11.74ꢀ11.06 ppm. In most cases, the formation of thioureas results in slight
downfield chemical shifts of the aromatic and ferrocenyl protons compared to 4ꢀmethoxyꢀ3ꢀ
ferrocenyl aniline, which is obvious as the neighboring thiocarbonyl moiety is also attracting
1
electron density. The HꢀNMR spectra show that the unsubstituted cyclopentadienyl (η5ꢀ
C5H5) ring of ferrocene yielded an intense singlet at ~4 ppm. In the case of substituted (η5ꢀ
C5H4) ring, the ortho protons appear in the region of 4.77ꢀ4.58 ppm, while the meta protons
occur in the range of 4.45ꢀ4.26 ppm. Aromatic protons were visible between 8ꢀ7 ppm. The
13CꢀNMR spectral data also justifies the coupling of ferrocenyl aniline with respective
isothiocyanates yielding the ferrocene incorporated acyl thioureas (M1ꢀM9). The chemical
shifts of the carbons of the CSNH and CONH groups of the thioureas resonate around 180.2ꢀ
177.1 and 169.7ꢀ165.4 ppm, respectively.
Elemental analyses (CHNS) of all the compounds showed that the calculated and
found values for carbon, hydrogen, nitrogen, and iron are in good agreement with each other,
which establishes that the compounds are amply pure in bulk.
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3.2
DNA binding studies
3.2.1 Cyclic voltammetry (CV)
A cyclic voltammetric study was executed with the aim of understanding the redox
behavior and the DNA binding affinities of the synthesized compounds. Electrochemical
measurements were undertaken using a threeꢀelectrode system, i.e., working (platinum disc
electrode with a geometric area of 0.071 cm2), reference (saturated calomel electrode i.e.
SCE) and auxiliary electrodes (platinum electrode with geometric area much greater than the
working electrode). Variation in the peak current provided information about the DNA
binding constants, whereas the mode of interaction of compound with DNA can be judged
from the shift in the peak potential. DrugꢀDNA binding constants were determined with the
help of following equation:37
log (1/[DNA]) = log K + log {I/(Io ꢀ I)}
Eq 5
where K is the binding constant, I and Io are the peak currents with and without DNA. For the
determination of binding site size the following equation was used:38
Cb/Cf = K [free base pairs]/s
Eq 6
where s is the binding site size in terms of base pair, K is the binding constant, Cf represents
the concentration of free species and Cb denotes the concentration of drugꢀDNA bound
species. Considering the concentration of DNA in terms of nucleotide phosphate, the
concentration of DNA base pair will be taken as [DNA base pair]/2 and Eq 6 can be written
as:37
Cb/Cf = K [DNA base pairs]/2s
Eq 7
and the value of Cb/Cf is equal to (IoꢀI)/I, which are the values of experimental peak currents.
The diffusion coefficient of free drug and DNAꢀbound drug provides the best information
about the molecular mass of the drugꢀDNA adduct. The following form of the Randlesꢀ
Sevcik equation was used for calculating the diffusion coefficients:39,40
5
1/2
Ipa = 2.99 × 10 n (α n) A Co Do1/2 ʋ1/2
Eq 8
*
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where Ipa is the anodic peak current, Co is the concentration of the reductant in mol Lꢀ1, A is
the geometric area of the electrode in cm2, ʋ is the scan rate in V/s, α is the transfer
coefficient, n is the number of electrons involved in the process, Do is the diffusion
coefficient in cm2 sꢀ1.
*
The synthesized ferroceneꢀbased compounds display similar electrochemical behavior
with two wellꢀdefined and stable redox peaks in the potential range of ꢀ0.2ꢀ1.0 V. Figure 1
shows the DNA binding study of a representative compound (M3) with CV. The consistency
of the voltage at different scan rates from the plots of current (mA) vs. potential (E/V vs.
SCE) for the compound favors a quasiꢀreversible electrochemical process (Figure 1a). The
voltammogram of M3 indicates an oxidation maximum at 0.377 V and a reduction maximum
at 0.253 V. With the addition of 3ꢀ12 ꢁM DNA, a negative shift in the peak potential and a
decay in the peak current is observed. This negative shift in potential justifies the probability
of an electrostatic mode of interaction of the positively charged M3 with the anionic
phosphate backbone of DNA. The decrease in current is attributed to the formation of a high
molecular weight M3ꢀDNA adduct, which diffuses comparatively slowly, thus causing a
reduction in peak current (Figure 1b). The diffusion coefficient of M3ꢀDNA adduct is 4.53 x
10ꢀ7 cm2 sꢀ1, and this is far less than the diffusion coefficient of the free M3 (6.66 x 10ꢀ7 cm2 sꢀ
1). This result indicates the slow diffusion of the high molecular weight M3ꢀDNA adduct as
compared to the free M3 (Figure 1c). The binding constant (5.713 x 104 Mꢀ1), and binding
site size (0.532 bp) were calculated using Eqs. 5 and 6, respectively (Figure 1d and 1e).
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Figure 1. (a) Representative plots of Current vs. Potential/V (SCE) at different scan rates for
M3. (b) Cyclic voltammogram of 1 mM M3 with 1 mL of 0.5 M TBAP as supporting
electrolyte in the absence and presence of 3ꢀ12 µM DNA showing a decrease in I from Io and
a concentration dependent ꢀve shift in potential showing electrostatic interactions. (c)
Representative plot of current vs. (V/s)1/2, for the determination of diffusion coefficient of
free M3 and M3ꢀ3 µM DNA. (d) Representative plot of log (I/IoꢀI) vs. log (1/[DNA] for
determination of binding constant of M3. (e) Plot of Cb/Cf vs. [DNA]/ꢁM for determination
of binding site size of 3ꢀ12 µM DNA concentrations M3.
The important DNA binding parameters of the compounds studied are listed in Table
1. These binding constant values are far better than protonated ferrocene (3.45 x 102 Mꢀ1) and
are comparable with many of the recently reported ferrocene derivatives,40ꢀ43 which shows
that the thiourea moiety is playing its part in the enhancement of binding constant. The
synthesized ferrocenyl derivatives are believed to undergo electrostatic interaction of
positively charged ferrocenium state with the negatively charged oxygen of DNA. The
phenyl group present in the structure is also capable of forming πꢀH bonding (πꢀstacking)
with DNA bases. This may be due to the existence of amide in the structure that could
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interact with DNA bases via hydrogenꢀbonding, as previously reported for benzamides,44
ferrocenyl urea, thiourea and guanidine derivatives.45,46
Table 1. Important parameters for redox behavior and DNA binding studies (CV and UV).
CV
UV
Code Binding Constant Do [cm2sꢀ1] Do [cm2sꢀ1] Binding site Binding Constant
K [Mꢀ1]
Free drug drugꢀDNA size ‘s’ [bp]
K [Mꢀ1]
4.87 x 104
4.25 x 104
5.71 x 104
3.84 x 104
2.92 x 104
2.21 x 104
1.93 x 104
9.75 x 103
1.36 x 104
1.48 x 10ꢀ7 1.03 x 10ꢀ7
3.43 x 10ꢀ7 2.84 x 10ꢀ7
6.66 x 10ꢀ7 4.53 x 10ꢀ7
5.25 x 10ꢀ7 2.44 x 10ꢀ7
6.87 x 10ꢀ7 4.13 x 10ꢀ7
2.67 x 10ꢀ7 1.67 x 10ꢀ7
5.26 x 10ꢀ7 3.58 x 10ꢀ7
4.28 x 10ꢀ7 2.03 x 10ꢀ7
7.94 x 10ꢀ7 4.41 x 10ꢀ7
0.505
0.338
0.532
0.303
0.278
0.217
0.196
0.105
0.177
4.93 x 104
3.27 x 104
5.00 x 104
3.15 x 104
3.07 x 104
2.58 x 104
2.26 x 104
1.04 x 104
1.98 x 104
M1
M2
M3
M4
M5
M6
M7
M8
M9
3.2.2
UVꢀVis spectroscopy
The results obtained from CV were equally supported by UVꢀVis spectroscopy
confirming the electrostatic interactions of the compounds with DNA. The characteristic UV
spectrum of the compound M3 gave two peaks in the UV region. One at 244 nm is due to a
πꢀπ* transition in the cyclopentadienyl ring (Cp) of ferrocene and the other at 286 nm is
attributed to the πꢀπ* transition of electrons in the phenyl chromophore. Following the
substantial addition of DNA, the UVꢀVis spectra of the compounds showed a decrease in
absorption with a slight blue shift (hypochromism) (Figure 2a). The hypochromic effect is
thought to be due to the interaction between the electronic states of the binding chromophore
and those of the DNA bases.29,47 It is likely that the strength of this electronic interaction
would decline as the cube of the distance of separation between the chromophore and the
DNA bases.48 So, the noticeable hypochromism observed in our experiments proposed the
close vicinity of the chromophore of synthesized complexes to the DNA bases. At the closest
approach to DNA, the π* orbital of the binding moiety of compounds could couple with π
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orbital of purine or pyrimidine. The coupling π* orbital may get partially filled by electrons,
thus decreasing the transition probabilities, and hence result in hypochromism.49,50 A binding
constant of 5.004 x 104 Mꢀ1 for M3 was determined from the Figure 2b.
Figure 2. (a) Representative plots of absorbance vs. wavelength of 25 µM M3 in ethanol with
increasing concentration of DNA (2ꢀ12 µM). (b) Plot of Ao/AꢀAo vs. 1/[DNA] for
determination of DNA binding constant of M3.
3.3
DFT study
Computational calculations were performed for these electroactive compounds in
order to complement the experimental outcomes of cyclic voltammetry and infraꢀred
spectroscopy. The redox potentials of the compounds measured from cyclic voltammetry
revealed an analogous trend to that anticipated from the DFT work.
Figure 3a and 3b shows the representative graphical demonstration of the HOMO and
LUMO orbitals of M3. The HOMO orbitals are localized on the ferrocene moiety (Fe metal),
whereas, the LUMO orbitals are extended over the thiourea functionality (containing electron
withdrawing N, and O atoms), and the fluoroꢀsubstituted phenyl ring. Generally, the energy
values of HOMO/LUMO and their energy gap reflect the chemical activity of the molecule.
The HOMO orbitals indicate the preferred site for the oxidation of electroactive species,
whereas the LUMO orbitals correspond to the probable site for the reduction of optimized
compound. HOMO as an electron donor represents the ability to donate an electron, while
LUMO as an electron acceptor indicates the ability to receive an electron.51,52 The HOMOꢀ
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LUMO energy gap reveals the intramolecular charge transfer (ICT) interaction occurs within
the molecule.
The ease of reduction of the compounds was found to vary as: M3 > M1 > M2 > M4
> M5 > M6 > M7 > M9 > M8. A similar trend was acquired from the ELUMO values, i.e., the
highest reduction potential and most negative ELUMO value of M3 corresponds to easiest
reduction.53ꢀ55 A more negative ELUMO favors addition of electrons as the energies of the
orbitals are reduced.
Figure 3. (a) Representative graphical demonstration of HOMO of M3. (b) Representative
graphical representation of LUMO of M3.
The EHOMO values acquired from DFT were compared with the oxidation potentials
obtained from the CV measurements. The oxidation potentials observed experimentally for
compounds M1ꢀM9 fluctuate as: M8 > M9 > M7 > M6 > M5 > M4 > M2 > M1 > M3. This
observation is supported from the DFT study by comparing the EHOMO values, which is less
negative for M8, representing its ease of oxidation as compared to other compounds (Figure
4 and Table 2). The HOMOꢀLUMO energy gaps were observed to be in close agreement with
the electrochemical band gaps (correlation coefficient = 0.953), computed from the difference
between the oxidation and reduction potential of the species (Eredox = Eoxi ꢀ Ered) (Table 2 and
Figure 5).
DFTꢀbased measurements also facilitated us to ascertain the Mulliken charges on
these molecular structures to determine the sites which are prone to electrophilic or
nucleophilic attack.56 Figure 6a and 6b demonstrates the graphical description of Mulliken
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charge distribution on the representative compound M3. The investigated molecule M3 has
several possible sites for electrophilic (the electrophilic sites are most electronegative and are
represented as red color), and nucleophilic attack (the nucleophilic sites are most positive and
are represented as green color).
Table 2. EHOMO and ELUMO values obtained through DFT calculations.
Sample
Code
EHOMO
(eV)
ELUMO
(eV)
HOMOꢀLUMO Electrochemical
Band Gap (eV)
Band Gap (V)
ꢀ0.4365
ꢀ0.4129
ꢀ0.4586
ꢀ0.3903
ꢀ0.3561
ꢀ0.3216
ꢀ0.3025
ꢀ0.2572
ꢀ0.2686
ꢀ0.3341
ꢀ0.3022
ꢀ0.3657
ꢀ0.2715
ꢀ0.2304
ꢀ0.1823
ꢀ0.1637
ꢀ0.1029
ꢀ0.1230
0.1024
0.1107
0.0929
0.1188
0.1257
0.1323
0.1388
0.1543
0.1456
0.1296
0.1331
0.1260
0.1384
0.1432
0.1489
0.1552
0.1736
0.1608
M1
M2
M3
M4
M5
M6
M7
M8
M9
Figure 4. Comparison of oxidation and reduction potentials of 1 mM ferrocenyl thioureas
M1ꢀM9 in DMSO recorded at a scan rate of 200 mV/s.
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Figure 5. Linear correlation graph between electrochemical band gap and HOMOꢀLUMO
band gap. (Correlation coefficient = 0.953)
Figure 6. (a) Graphical representation of Mulliken charges on the molecular structure M3 (by
numbers). (b) Mulliken charge distribution on the optimized molecule M3 designated by
color change on the atoms (red for negative charge and blue for positive charge).
The vibrational frequencies determined experimentally are compared with those
obtained theoretically from DFT calculations employing the B3LYP/6ꢀ31G(d,p) method for
the optimized geometry of the compounds (M1ꢀM9). Some important calculated and
experimental vibrational frequencies along with their vibrational assignments are listed in
Table 3. Vibrational frequencies were scaled by the typical factor 0.96. The theoretical
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vibrational frequencies and the observed spectroscopic data illustrate a linear relationship as
presented in the Figure 7 (correlation coefficient = 0.994). The largest difference between the
calculated and observed frequencies is 20 cmꢀ1. The experimental and calculated IR spectra
for M3 are shown in Figure S1 and S2 (ESI file).
Table 3. Vibrational frequencies (for selected vibrations only) of ferrocenyl thioureas (M1ꢀM9)
calculated at the B3LYP/6ꢀ31G(d,p) level compared with the experimental data.
Calculated and Experimental IR Vibrational Frequencies (cmꢀ1)
Assignment
υ NꢀH
M1
M2
M3
M4
M5
M6
M7
M8
M9
3379ꢀ3215 3348ꢀ3197 3395ꢀ3287 3375ꢀ3226 3352ꢀ3219 3380ꢀ3206 3397ꢀ3231 3389ꢀ3216 3364ꢀ3225
(Cal) (Cal) (Cal) (Cal) (Cal) (Cal) (Cal) (Cal) (Cal)
3392ꢀ3227 3360ꢀ3217 3374ꢀ3233 3388ꢀ3212 3364ꢀ3204 3391ꢀ3223 3382ꢀ3219 3374ꢀ3206 3386ꢀ3209
(Exp)
(Exp) (Exp) (Exp) (Exp) (Exp) (Exp) (Exp) (Exp)
3038 (Cal) 3056 (Cal) 3071 (Cal) 3060 (Cal) 3072 (Cal) 3069 (Cal) 3047 (Cal) 3093 (Cal) 3082 (Cal)
3025 (Exp) 3074 (Exp) 3086 (Exp) 3076 (Exp) 3088 (Exp) 3055 (Exp) 3064 (Exp) 3077 (Exp) 3067 (Exp)
υ CꢀHaromatic
2962 (Cal) 2954 (Cal) 2948 (Cal) 2940 (Cal) 2970 (Cal) 2937 (Cal) 2965 (Cal) 2932 (Cal) 2951 (Cal)
2946 (Exp) 2935 (Exp) 2931 (Exp) 2927 (Exp) 2951 (Exp) 2949 (Exp) 2944 (Exp) 2940 (Exp) 2936 (Exp)
υ CꢀHaliphatic
1674 (Cal) 1652 (Cal) 1664 (Cal) 1675 (Cal) 1660 (Cal) 1657 (Cal) 1663 (Cal) 1668 (Cal) 1655 (Cal)
1663 (Exp) 1665 (Exp) 1676 (Exp) 1668 (Exp) 1672 (Exp) 1669 (Exp) 1674 (Exp) 1677 (Exp) 1667 (Exp)
υ C=O
υ C=C
1609 (Cal) 1598 (Cal) 1603 (Cal) 1602 (Cal) 1599 (Cal) 1604 (Cal) 1601 (Cal) 1589 (Cal) 1597 (Cal)
1602 (Exp) 1590 (Exp) 1591 (Exp) 1593 (Exp) 1595 (Exp) 1587 (Exp) 1594 (Exp) 1598 (Exp) 1592 (Exp)
1567 (Cal) 1549 (Cal) 1563 (Cal) 1553 (Cal) 1545 (Cal) 1572 (Cal) 1547 (Cal) 1551 (Cal) 1542 (Cal)
1553 (Exp) 1562 (Exp) 1546 (Exp) 1567 (Exp) 1560 (Exp) 1558 (Exp) 1564 (Exp) 1568 (Exp) 1557 (Exp)
β NꢀH
1460 (Cal) 1449 (Cal) 1445 (Cal) 1438 (Cal) 1435 (Cal) 1447 (Cal) 1451 (Cal) 1440 (Cal) 1446 (Cal)
1446 (Exp) 1438 (Exp) 1456 (Exp) 1453 (Exp) 1442 (Exp) 1458 (Exp) 1435 (Exp) 1449 (Exp) 1455 (Exp)
δ CꢀHaliphatic
Cal = calculated frequency; Exp = experimental frequency; υ = stretching; δ = deformation; β = inꢀ
plane bending; γ = out of plane bending; ω = wagging; ρ = rocking.
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Table 3. Continued……
Calculated and Experimental IR Vibrational Frequencies (cmꢀ1)
M2 M3 M4 M5 M6 M7
Assignment
M1
M8
M9
1244ꢀ1131 1250ꢀ1135 1288ꢀ1190 1248ꢀ1125 1251ꢀ1128 1264ꢀ1142 1257ꢀ1149 1245ꢀ1124 1270ꢀ1150
(Cal) (Cal) (Cal) (Cal) (Cal) (Cal) (Cal) (Cal) (Cal)
υ C=S
1257ꢀ1138 1261ꢀ1143 1268ꢀ1147 1265ꢀ1132 1260ꢀ1136 1253ꢀ1154 1266ꢀ1125 1262ꢀ1147 1255ꢀ1139
(Exp) (Exp) (Exp) (Exp) (Exp) (Exp) (Exp) (Exp) (Exp)
1064 (Cal) 1048 (Cal) 1046 (Cal) 1061 (Cal) 1046 (Cal) 1067 (Cal) 1063 (Cal) 1044 (Cal) 1050 (Cal)
1052 (Exp) 1040 (Exp) 1068 (Exp) 1043 (Exp) 1058 (Exp) 1050 (Exp) 1054 (Exp) 1053 (Exp) 1061 (Exp)
ω CꢀHaliphatic
1010 (Cal) 1014 (Cal) 1012 (Cal) 1029 (Cal) 1015 (Cal) 1009 (Cal) 1028 (Cal) 1017 (Cal) 1012 (Cal)
1023 (Exp) 1019 (Exp) 1025 (Exp) 1020 (Exp) 1027 (Exp) 1024 (Exp) 1013 (Exp) 1002 (Exp) 1025 (Exp)
ρ CꢀHaliphatic
980 (Cal)
967 (Exp)
985 (Cal)
972 (Exp)
973 (Cal)
984 (Exp)
989 (Cal)
980 (Exp)
981 (Cal)
976 (Exp)
962 (Cal)
984 (Exp)
977 (Cal)
960 (Exp)
958 (Cal)
975 (Exp)
990 (Cal)
979 (Exp)
β CꢀHaromatic
856 (Cal)
847 (Exp)
840 (Cal)
859 (Exp)
831 (Cal)
853 (Exp)
863 (Cal)
854 (Exp)
850 (Cal)
837 (Exp)
853 (Cal)
842 (Exp)
860 (Cal)
849 (Exp)
851 (Cal)
846 (Exp)
867 (Cal)
858 (Exp)
γ CꢀHaromatic
795 (Cal)
776 (Exp)
777 (Cal)
784 (Exp)
786 (Cal)
770 (Exp)
798 (Cal)
786 (Exp)
792 (Cal)
783 (Exp)
776 (Cal)
789 (Exp)
785 (Cal)
774 (Exp)
791 (Cal)
779 (Exp)
785 (Cal)
772 (Exp)
β C=O
γ NꢀH
γ C=O
713 (Cal)
704 (Exp)
719 (Cal)
711 (Exp)
718 (Cal)
723 (Exp)
708 (Cal)
717 (Exp)
720 (Cal)
712 (Exp)
705 (Cal)
716 (Exp)
718 (Cal)
701 (Exp)
710 (Cal)
721 (Exp)
720 (Cal)
725 (Exp)
638 (Cal)
647 (Exp)
630 (Cal)
642 (Exp)
644 (Cal)
635 (Exp)
648 (Cal)
640 (Exp)
645 (Cal)
637 (Exp)
633 (Cal)
641 (Exp)
636 (Cal)
646 (Exp)
630 (Cal)
639 (Exp)
644 (Cal)
635(Exp)
471 (Cal)
481 (Exp)
485 (Cal)
478 (Exp)
482 (Cal)
470 (Exp)
487 (Cal)
475 (Exp)
472 (Cal)
485 (Exp)
475 (Cal)
481 (Exp)
473 (Cal)
488 (Exp)
468 (Cal)
484 (Exp)
470 (Cal)
482 (Exp)
υ FeꢀCp
Cal = calculated frequency; Exp = experimental frequency; υ = stretching; δ = deformation; β = inꢀ
plane bending; γ = out of plane bending; ω = wagging; ρ = rocking.
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Figure 7. Linear correlation graph between the experimental and calculated vibrational
frequencies of a representative compound M3. (Correlation coefficient = 0.994)
3.4
Antimicrobial studies
These compounds were subjected against representative bacterial and fungal strains
and the results are summarized in Table 4 and 5, respectively. Penicillin and terbinafin were
used as standard antimicrobial drugs and DMSO was selected as a negative control in order
to determine its nonꢀtoxic effect. The zone of inhibition values represent the mean value of
the three readings with standard deviation. The fluoro derivatives M1, M2, and M3 showed
more potent antimicrobial activity as compared to the chloro and bromo containing
molecules. Moreover, the ortho substituted compounds were found to be biologically more
active as compared to their meta and para derivatives, possibly due to the prominent effect of
an electronegative group (X) at the ortho position than at the meta and para positions
(inductive effect works up to three or four bonds). Due to electron withdrawing effect, a
decrease in the basicity of NH and increase in lipophilicity may occur.41 The decrease in
basicity is due to less availability of the lone pair of electron to hydrogen bond with water in
the living cells (Scheme 2a and 2b).
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Scheme 2. (a) Hydrophilicity of ferrocenyl thioureas. (b) Decrease in hydrophilicity and
increase in lipophilicity by an electronꢀwithdrawing group (where X = F, Cl, and Br).
Table 4. In vitro antibacterial activity of synthesized ferrocenyl thioureas.
Sample Staphylococcus Salmonella
Micrococcus
luteus
Bordetella
bronchiseptica
Enterobacter
aerogenes
Code
aureus
typhimurium
21.4 ± 0.33a
18.6 ± 0.60
19.5 ± 0.45
18.1 ± 0.67
17.5 ± 0.33
17.0 ± 0.60
16.4 ± 0.77
14.7 ± 0.30
15.8 ± 0.45
26.7 ± 0.33
18.8 ± 0.67
17.5 ± 0.30
18.0 ± 0.44
17.3 ± 0.37
16.8 ± 0.53
16.2 ± 0.70
15.6 ± 0.63
14.4 ± 0.33
15.1 ± 0.77
26.3 ± 0.33
20.2 ± 0.70
18.3 ± 0.58
18.9 ± 0.33
16.5 ± 0.67
15.7 ± 0.30
14.9 ± 0.33
14.2 ± 0.72
13.0 ± 0.55
13.6 ± 0.58
27.3 ± 0.33
16.4 ± 0.30
14.2 ± 0.70
15.3 ± 0.58
13.7 ± 0.55
13.2 ± 0.65
12.6 ± 0.45
12.0 ± 0.60
10.8 ± 0.77
11.3 ± 0.33
23.0 ± 0.58
16.0 ± 0.58
14.9 ± 0.33
15.8 ± 0.67
14.0 ± 0.44
13.4 ± 0.57
12.5 ± 0.60
11.6 ± 0.73
09.9 ± 0.58
10.5 ± 0.33
20.3 ± 0.67
M1
M2
M3
M4
M5
M6
M7
M8
M9
PC
PC = Penicillin (1 mg/mL) was used as standard drug (positive control), while DMSO was
used as negative control.
a Zone of inhibition in mm.
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Table 5. In vitro antifungal assay of synthesized ferrocenyl derivatives and the standard drug.
Sample Code Fusarium moniliforme Aspergillus fumigatus Aspergillus flavus
3.46 ± 0.03a
3.78 ± 0.07
2.65 ± 0.33
4.35 ± 0.03
5.53 ± 0.33
4.69 ± 0.09
5.96 ± 0.03
3.17 ± 0.70
7.94 ± 0.09
0.77 ± 0.07
10.3 ± 0.3
64b
67
75
57
46
53
42
30
23
92
0
3.55 ± 0.33
3.84 ± 0.06
2.89 ± 0.09
4.21 ± 0.33
5.50 ± 0.07
5.04 ± 0.63
5.45 ± 0.09
6.19 ± 0.67
6.85 ± 0.33
0.89 ± 0.03
10.7 ± 0.03
66
63
73
59
55
50
47
40
33
92
0
2.97 ± 0.33
2.25 ± 0.03
1.57 ± 0.07
3.29 ± 0.33
4.27 ± 0.06
4.15 ± 0.09
4.59 ± 0.33
5.17 ± 0.03
5.85 ± 0.67
0.83 ± 0.01
10.3 ± 0.3
72
79
86
68
56
60
54
49
43
92
0
M1
M2
M3
M4
M5
M6
M7
M8
M9
PC
NC
PC = Terbinafin (1 mg/ml) was used as standard drug (positive control), while DMSO
was used as negative control (NC).
a Fungal growth (cm)
b Zone of inhibition (%)
3.5
Cytotoxicity assay
The anticancer activity of the synthesized complexes along with that for cisplatin
(used as control) were assessed against MCFꢀ7 carcinoma cells and also towards the nonꢀ
cancerous cell line MCFꢀ10A, using the MTT reduction assay. From the 50% inhibitory
concentration (IC50) data listed in Table 6, it can be seen that the screened compounds reveal
substantial action against cancerous cells, though much less than cisplatin. However, these
ferrocenyl derivatives employ fewer toxic effects in normal cells. One plausible cause for the
variance in action as related to that for cisplatin may lie in the difference in the approach of
interaction with the DNA.57 Cisplatin interacts covalently with nitrogenous bases in the DNA,
while the synthesized complexes are assumed to experience electrostatic interactions as
designated by electrochemical and spectroscopic studies. It is obvious from the Figure 8 that
the tested compounds inhibited the growth of tumor cells in a doseꢀdependent fashion.
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Table 6. Cytotoxicity against human tumor and normal cells, after 72 h of incubation.
IC50 (µM)
Sample Code
MCFꢀ7
MCFꢀ10A
1.55 ± 1.82
2.56 ± 1.01
3.24 ± 0.64
1.87 ± 1.07
4.02 ± 0.96
4.45 ± 1.25
4.99 ± 0.76
5.20 ± 1.19
5.91 ± 0.55
5.73 ± 1.47
17.3 ± 0.59
16.6 ± 1.27
15.9 ± 0.74
17.0 ± 1.62
16.2 ± 0.95
15.7 ± 1.33
14.9 ± 0.88
13.8 ± 0.64
12.7 ± 0.97
13.3 ± 1.54
Cisplatin
M1
M2
M3
M4
M5
M6
M7
M8
M9
The results of the cytotoxicity assays indicate that those complexes bearing fluoro
moiety on the phenyl ring show enhanced cell growth inhibitory effects as compared to the
chloro and bromo containing molecules. This indicates that the presence of an electronꢀ
withdrawing group increases the polarity of these compounds, which in turn augments their
lipophilicity and helps the molecules to interact or penetrate more through the cell membrane,
thereby enhancing their cytotoxic action However, no particular cytotoxicity trend was
observed by changing the position of the substituents from ortho to para on the benzoyl
group.
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Figure 8. Cell viability (%) of cancerous and nonꢀcancerous cells at various concentrations of
(a) M3, (b) M5, and (c) M7, after 72 h of incubation.
The results of anticancer activity were found to be in close agreement with the DFT
calculations. The anticancer activity of ferrocene derivatives is found to be dependent on the
oxidation state of iron in the ferrocene moiety with some results indicating that the reduced
Fe(II) ferrocenyl compound is more active than oxidized Fe(III) ones.58 In the present study,
a more negative ELUMO and a small HOMOꢀLUMO band gap of M3 favors the addition of
electrons as the energies of the orbitals are reduced. Whereas, the complex M8 has the least
negative EHOMO value and a large HOMOꢀLUMO band gap, which represents its ease of
oxidation as compared to the other compounds.27,45 Thus, M3 complex with most negative
ELUMO and a small band gap favors the reduced form Fe(II) (more bioactive) in the ferrocene
moiety, thus M3 shows the highest growth inhibitory effect against the tumor cells.
4.
Conclusions
Ferroceneꢀmodified thioureas were synthesized and structurally characterized by
various spectroscopic techniques, which validated the purity of these compounds. Moreover,
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DNA binding potentiality of the synthesized complexes was explored as a probable
mechanism for the anticancer activity using DNA association constants evaluated by
electrochemical and spectroscopic methods. These compounds have also been demonstrated
to be promising candidates for inhibiting the microbial and tumor growth. The computational
calculations based on DFT/B3LYP study correlate well with the cytotoxicity results. Thus,
the insertion of a ferrocenyl moiety into thioureas has proved to be a significant approach to
enhancing their therapeutic properties by augmenting their lipophilic character.
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
Faiza Asghar is highly obliged to the Higher Education Commission (HEC) of Pakistan for
providing a scholarship under the program 5000 Indigenous PhD Scholarships at Quaidꢀiꢀ
Azam University, Islamabad and for a 6ꢀmonth scholarship under the International Research
Support Initiative Program (IRSIP) as a Graduate Research Trainee in the Department of
Chemistry, McGill University, Montreal, Quebec, Canada.
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