Structure, photophysics, electrochemistry, DFT calculation, and in-vitro antioxidant activity of coumarin Schiff base complexes of Group 6 metal carbonyls

Article abstract of DOI:10.1016/j.jinorgbio.2010.04.013

N-[(2-Pyridyl)methyliden]-6-coumarin (L) is synthesized by the condensation of 6-aminocoumarin and pyridine-2-carboxaldehyde. Group-6 tetracarbonyl complexes, [M(CO)₄(L)] (M = Cr, Mo, and W) are synthesized and characterized by mass spectrometry and NMR, FT-IR and UV-visible spectroscopy. X-ray crystal structure of [Cr(CO)₄(L)] shows N(pyridine), N(imine) chelation to chromium(0). A supramolecular chain is formed by C-H?O and π?π interactions. The ligand and the complexes are fluorescent. Cyclic voltammetry of the complexes exhibit quasireversible M(I)/M(0) redox couple. The complexes exhibit potential antioxidant property both in cell free and in-vitro studies and highest activity is observed to [W(CO)₄(L)]. Density functional theory (DFT) computation has been performed to correlate with the electronic configuration, composition of wave functions with the UV-visible spectra and redox properties.

Full text of DOI:10.1016/j.jinorgbio.2010.04.013

Journal of Inorganic Biochemistry 105 (2011) 577–588
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Structure, photophysics, electrochemistry, DFT calculation, and in-vitro antioxidant
activity of coumarin Schiff base complexes of Group 6 metal carbonyls
Papia Datta a, Ambika Prasad Mukhopadhyay a, Prasenjit Manna b, Edward R.T. Tiekink c,
Parames Chandra Sil b, Chittaranjan Sinha a,
⁎
a
Department of Chemistry, Inorganic Chemistry Section, Jadavpur University, Kolkata 700032, India
Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata 700054, India
Department of Chemistry, University of Malaya, Kuala Lumpur 50603, Malaysia
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
N-[(2-Pyridyl)methyliden]-6-coumarin (L) is synthesized by the condensation of 6-aminocoumarin and
pyridine-2-carboxaldehyde. Group-6 tetracarbonyl complexes, [M(CO)4(L)] (M=Cr, Mo, and W) are
synthesized and characterized by mass spectrometry and NMR, FT–IR and UV-visible spectroscopy. X-ray
crystal structure of [Cr(CO)4(L)] shows N(pyridine), N(imine) chelation to chromium(0). A supramolecular
chain is formed by C–H⋯O and π⋯π interactions. The ligand and the complexes are fluorescent. Cyclic
voltammetry of the complexes exhibit quasireversible M(I)/M(0) redox couple. The complexes exhibit
potential antioxidant property both in cell free and in-vitro studies and highest activity is observed to [W
(CO)4(L)]. Density functional theory (DFT) computation has been performed to correlate with the electronic
configuration, composition of wave functions with the UV-visible spectra and redox properties.
© 2010 Elsevier Inc. All rights reserved.
Received 30 December 2009
Received in revised form 24 April 2010
Accepted 30 April 2010
Available online 15 May 2010
Keywords:
Group 6 metal carbonyls
Coumarin Schiff base
Structure
Photophysics
DFT
Antioxidant activity
1. Introduction
energy. Carbon monoxide is well known π-acidic molecule which may
easily regulate energy levels and has been used in many cases for the
Coumarin is a phytochemical [1] found in many plants such as
Tonka bean, lavender, sweet clover grass, licorice, strawberries,
apricots, cherries, and cinnamon. Coumarin derivatives have been
proven to function as anti-coagulants [2], antibacterial agents [3],
antifungal agents [4], and biological inhibitors [5], chemotherapeutics
[6,7] and as bio-analytical reagents [8]. They are useful antioxidants
and show antitumour activity [9] and cytotoxicity [10–15].
Considerable effort has now been given to the functionalisation of
coumarin so that metal–coumarin complexes may be synthesized
towards the development of artificial photosynthetic systems, chemical
sensors and molecular level devices [16]. We are interested to anchor
diimine (–N C–C N–) function to coumarin backbone so that the
molecule may act as N, N-chelating agent and could bind metal ions.
Considering molecular efficiency of polypyridine system [17,18] we are
interested to condense pyridine-2-carboxaldehyde and 6-aminocou-
marin to synthesise N-[(2-pyridyl)methyliden]-6-coumarin (L).
Towards the development of effective fluorescent molecules the
complexes of low energy metal-to-ligand charge transfer (MLCT)
excited states are important. Presence of π-acidic co-ligands in the
metal-complex unit has potential efficiency to monitor the MLCT
preparation of luminescent complexes [19,20]. For the last few years
we are engaged in the study of metal–carbonyl complexes of Group 6
and Group 8 metals with different azoimidazoles [21,22] and diimine
functions [23]. Usually metal carbonyls are toxic at vapor phase due to
dissociation into free toxic metal and CO [24]. In continuation of our
interest herein we wish to report the Group 6 metal carbonyl
complexes of N-[(2-pyridyl)methyliden]-6-coumarin (L) (Scheme 1).
The structure of one of the complexes is confirmed by single crystal X-
ray diffraction study and the photophysical and redox properties are
described hereunder. The complexes exhibit solvatochromism and,
antioxidant activity better than those of free ligand and hexacarbonyl
precursor (M(CO)6). The properties are rationalized by density
functional theory calculation.
2. Experimental
2.1. Materials and measurements
M(CO)6 (M=Cr, Mo, and W) and pyridine-2-carboxaldehyde
were purchased from Aldrich Chemical Co. THF and dichloromethane
were dried before use [25]. Coumarin was purchased from S. D. Fine
Chem. Ltd., Boisar. Dinitrogen was purified by bubbling through an
alkaline pyrogallol solution. All other chemicals and solvents were
of reagent grade and were used without further purification.
⁎
Corresponding author. Fax: +91 33 2414 6584.
E-mail address: c_r_sinha@yahoo.com (C. Sinha).
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.04.013

578
P. Datta et al. / Journal of Inorganic Biochemistry 105 (2011) 577–588
Scheme 1. Synthetic outline for the preparation of ligand and complexes.
Commercially available SRL silica gel (60–120 mesh) was used for
column chromatography.
thoroughly with cold water (10 mL×10) and dried over CaCl2 and
recrystallized from acetic acid. Yield, 9.2 g, 88%. Mp, 185 2 °C;
microanalytical data, Found: C, 56.45; H, 2.67; and N, 7.28%.
Calculated for C9H5NO4: C, 56.54; H, 2.62; and N, 7.33%. UV-visible
spectra (λmax(CH3CN)/nm; ε/103 M−1 cm−1) 327 (1.80), 314 (2.13),
268 (6.93), and 259 (9.51); IR, νmax/cm−1 1751, 1620, 1564, 1536,
1480, and 1436; 1H NMR (300 MHz, CD3CN) δ 8.54 (1H, s (singlet)),
8.38 (1H, d (doublet), 7.5 Hz), 7.98 (1H, d, 7.5 Hz), 7.49 (1H, d,
7.0 Hz), and 6.55 (1H, d, 8.0 Hz).
Microanalytical data (C, H, and N) were collected on Perkin Elmer
2400 CHNS/O elemental analyzer. Spectroscopic data were obtained
using the following instruments: UV-visible (UV-Vis) spectra by
Perkin Elmer UV-Vis spectrophotometer model Lambda 25; FT–IR
spectra (KBr disk, 4000–450 cm−1) by Perkin Elmer FT–IR spectro-
photometer model RX-1; the 1H NMR spectra by Bruker (AC)
300 MHz FTNMR spectrometer; FAB-MS was collected from JEOL-
JMS 600. Electrochemical measurements were performed using
computer-controlled PAR model 250 VersaStat electrochemical
instruments using a Pt-disk working electrode and Pt-wire auxiliary
electrode under inert (dry N2) environment in CH3CN. The solution
was IR compensated and the results were collected at 298 K. The
reported results were referenced to Ag/AgCl in CH3CN and were
uncorrected for junction potential. [n-Bu4N][ClO4] was used as
supporting electrolyte. Emission was examined by LS 55 Perkin
Elmer spectrofluorimeter at room temperature (298 K) in methanol
solution under degassed condition. The fluorescence quantum yield of
the complexes was determined using carbazole as a reference with a
known ϕR of 0.42 in C6H6 [26].
2.1.1.2. Synthesis of 6-aminocoumarin (Step-2). Reduction of 6-
nitrocoumarin was done using iron powder and ammonium chloride
in water. 6-Nitrocoumarin (8 g, 41.9 mmol) in water (150 mL) was
treated with Fe-powder (20 g) and ammonium chloride (2.6 g,
48.6 mmol). The mixture was kept in water bath for 2 h with stirring.
A dark brown precipitate was obtained which was then extracted
with acetone. Evaporation of acetone yielded silky yellow precipitate
of 6-aminocoumarin (mp 73 °C). It was then recrystallised from dil
Table 1
Crystallographic parameters of [Cr(CO)4(L)] (1).
Fluorescence lifetimes were measured using a time-resolved
spectrofluorimeter from IBH, UK. The instrument uses a picosecond
diode laser (NanoLed-05, 370 nm) as the excitation source and works
on the principle of time-correlated single photon counting [27] The
instrument responses function is ∼230 ps at FWHM. To eliminate
depolarization effects on the fluorescence decays, measurements
were done with magic angle geometry (54.7°) for the excitation and
emission polarizers. The observed decays of coumarin Schiff base
ligand and its metal carbonyl complexes fitted well with a bi-
exponential function [28].
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
b (Å)
c (Å)
α (°)
C19 H10 Cr N2 O6
414.29
Triclinic
P −1
7.3483(15)
9.1846(18)
13.120(3)
80.93(3)
81.58(3)
79.59(3)
853.7(3)
β (°)
γ (°)
V(Å3)
ρ (calculated) (g cm−3
)
1.612
Z
T(K)
2
2.1.1. Synthesis of N-[(2-pyridyl)methyliden]-6-coumarin (L)
There are three steps in the preparation of ligand (Scheme 1): nitro
coumarin (step 1), aminocoumarin (step-2) and condensation with
pyridine-2-carboxaldehyde (step-3).
173(2)
0.712
9216
3220
Absorption coefficient (mm−1
Total reflection collected
Unique reflections
Refined parameters
hkl range
)
253
−9≤h≤8; −11≤k≤11; −15≤l≤16
2.1.1.1. Synthesis of 6-nitrocoumarin (Step-1). Coumarin was nitrated
with mixed acid in ice bath. Coumarin (8 g, 54.8 mmol) was dissolved
in conc. H2SO4 (40 mL) and temperature was maintained at −5 °C
and then 16 mL mixed acid (HNO3 and H2SO4 (conc.) in 1:3 volume
ratio) was added. The mixture was stirred keeping at room
temperature for 1 h and then ice was added to it. A white precipitate
of 6-nitrocoumarin was obtained. It was then filtered and washed
θ range (°)
2.3–26.0
0.042
0.103
1.08
Ra (IN2σ (I))
wRb
GOFc
a
R=Σ||Fo|−|Fc||/Σ|Fo|.
b
wR={Σ[w(F2o −F2c )2]/Σ[w(F2o)2]}1/2
;
w= [σ2(Fo)2 +(0.0481P)2 + 0.5210P]− 1
,
where P=(F2o +2Fc2)/3.

P. Datta et al. / Journal of Inorganic Biochemistry 105 (2011) 577–588
579
was obtained. Yield 90%, 0.698 g; mp 152 2 °C; Microanalytical data,
Found: C, 72.07; H, 4.04; and N, 11.16%. Calculated for C15H10N2O2: C,
72.0; H, 4.0; and N, 11.2%.
ꢀ
ꢁ
IR νmax = cm−1 1714ðCOOÞ; 1629ðC = NÞ; 1581; 1566; 1472;
1437 ðC = CÞ:MS m= z = 249 Mþ
ꢀ
ꢁ
2.1.2. Synthesis of [M(CO)4(L)] (M=Cr(1), Mo (2) and W (3))
The compounds were prepared following same general procedure.
Detail of synthesis of [Mo(CO)4(L)] is given herewith.
To Mo(CO)6 (50 mg, 0.189 mmol) in dry tetrahydrofuran (15 mL)
was added Me3NO (30 mg, 0.40 mmol) and the solution was stirred
for 45 min. To the resulting yellow solution N-[(2-pyridyl)methyli-
den]-6-coumarin (47 mg, 0.189 mmol) was added and stirred for
another 5 h. The colour of the solution changes to deep purple. The
solvent was evaporated under low pressure and the crude product
was passed down a neutral Al2O3 column. A blue-violet portion is
eluted with 1:10 acetonitrile–benzene solution. The complex is
obtained as a blue crystalline solid in 90% yield. Microanalytical data:
Found: C; 49.70; H, 2.13; and N, 6.03%. Calculated for [Mo(CO)4(L)] (2),
C
19H10N2O6Mo, C, 49.79; H, 2.18; and N, 6.11%. IR, (νmax/cm−1) 2019,
1920, 1877, 1815 (CO); 1727(COO), and 1615 (C N). FAB-mass
spectrometry: M+, 457; (M–CO)+, 429; (M–2CO)+, 421; (M–3CO)+
,
373; and (M–4CO)+, 345.
For [Cr(CO)4(L)] (1); microanalytical data: Found: C, 55.01; H,
2.34; and N, 6.66%. Anal cal for [Cr(CO)4(L)], C19H10N2O6Cr: C, 55.07;
H, 2.42; and N, 6.76%. IR, (νmax/cm−1) ν(CO), 2011, 1910, 1878, 1814;
ν(COO) 1710, and ν(C N), 1615. FAB-mass spectrometry: M+, 413;
(M–CO)+, 385; (M–2CO)+, 357; (M–3CO)+, 329; and (M–4CO)+
,
301.
For [W(CO)4(L)] (3); microanalytical data: Found: C, 41.80; H,
1.80; and N, 5.25%. Anal cal for [W(CO)4(L)], C19H10N2O6W: C, 41.77;
H, 1.83; and N, 5.13%. IR (νmax/cm−1): ν(CO) , 2006, 1923, 1898, 1830,
and ν(COO) 1713. FAB-mass spectrometry: M+, 545; (M–CO)+, 517;
(M–2CO)+, 489; (M–3CO)+, 461; and (M–4CO)+,433.
Fig. 1. (a) X-ray structure of [Cr(CO)4(L)], (b) packing view of the molecule.
2.2. Synthesis and formulation
HCl solution as 6-aminocoumarin hydrochloride. Yield, 5.1 g, 76%.
mpN260 °C; microanalytical data, Found C, 67.12; H, 4.25; and N,
8.65%. Calculated for C9H7NO2: C, 67.08; H, 4.35; and N, 8.70%. UV-
visible (λmax(CH3CN)/nm; ε/103 M−1 cm−1) 370 (3.54), 280 (11.43),
253 (21.44); IR, (νmax/cm−1): 3409, 3329, 1705, 1635, 1570, 1490,
and 1451; 1H NMR (300 MHz, CD3CN) δ 7.71 (1H, d, 7.5 Hz), 7.09 (1H,
d, 7.5 Hz), 6.88 (1H, d, 7.5 Hz), 6.77 (1H, s), 6.30 (1H, d, 8.0 Hz), and
4.26 (2H, s).
Nitration of coumarin and its subsequent reduction to 6-
aminocoumarin has been carried out by Fe-powder/NH4Cl. The
condensation of pyridine-2-carboxaldehyde with 6-aminocoumarin
in alcohol under refluxing condition has isolated straw yellow
crystalline compound. Infrared spectra of 6-aminocoumarin shows
two ν(NH2) bands at 3329 and 3409 cm−1 which is eliminated in the
condensation product and a new band appears at 1629 cm−1 that is
corresponding to ν(C N) along with lactone ν(COO) at 1714 cm−1
.
2.1.1.3. N-[(2-pyridyl)methyliden]-6-coumarin (L) (Step-3). 6-Amino-
coumarine (0.5 g, 3.1 mmol) and pyridine-2-carboxaldehyde
(0.26 mL, 3.1 mmol) were taken in dry methanol (15 mL) and it was
refluxed for 8 h. After evaporation straw colour crystalline compound
Absorption spectra show intense band at 337, 280 and 241 nm. These
are assigned to n→π* and π→π* transitions. The structural
characterisation has been carried out by 1H and 13C NMR spectral
data. The CH N shows singlet signal in 1H NMR spectrum at 8.64 ppm.
Table 2
Selected bond parameters of [Cr(CO)4(L)].
Bond length (Å)
Experimental value
Theoretical value
Bond angle (°)
Experimental value
Theoretical value
Cr–N(9)
2.109(2)
2.1221(19)
1.914(3)
1.841(3)
1.838(3)
1.893(3)
1.292(3)
2.1109
2.1053
1.8924
1.8462
1.8512
1.8914
1.3137
N(9)–Cr–N(12)
N(9)–Cr–C(20)
N(9)–Cr–C(22)
N(9)–Cr–C(24)
N(9)–Cr–C(26)
N(12)–Cr–C(20)
N12–Cr–C22
N(12)–Cr–C(26)
C(22)–Cr–C(24)
C(20)–Cr–C(26)
76.35(8)
91.84(9)
76.9096
91.8464
94.3540
96.9057
91.6595
90.4489
171.1181
93.7292
91.8253
175.0639
Cr–N(12)
Cr–C(20)
Cr–C(22)
Cr–C(24)
Cr–C(26)
N12–C(13)
98.59(10)
172.48(9)
92.59(10)
94.92(9)
174.80(9)
93.45(9)
88.68(11)
171.25(10)

580
P. Datta et al. / Journal of Inorganic Biochemistry 105 (2011) 577–588
Table 4
Solvent dependence of metal-to-ligand charge transfer absorption maxima of [M(CO)4
(L)] complexes at 298 K (M=Cr, Mo, and W).
Solvent
EMLCT(Kcal mol−1
)
ET
(30)
[Cr(CO)4(L)]
[Mo(CO)4(L)]
[W(CO)4(L)]
Toluene
Benzene
Tetrahydrofuran
Chloroform
Dichloromethane
Acetone
Acetonitrile
Dimethylformamide
Dimethylsulphoxide
46.8
47.04
47.5
48.21
48.45
49.34
52.74
53.63
52.18
49.65
49.98
50.69
51.71
51.71
52.74
56.61
57.52
55.96
49.03
49.24
49.78
50.69
50.71
51.49
54.98
55.83
54.36
33.9
34.3
37.4
39.1
40.7
42.2
45.6
43.2
45.1
AFC12/Saturn724 CCD fitted with Mo Kα radiation so that θmax was
26.5°. Data processing and empirical absorption correction were
accomplished with the programs Crystal Clear [29] and ABSCOR [30],
respectively. Crystallographic refinement data and selected geometric
parameters are collected in Table 1. The structure was solved by
heavy-atom methods and followed by successive Fourier and
difference Fourier syntheses. Full matrix least squares refinements
on F2 were carried out using SHELXL-97 [31,32] with anisotropic
displacement parameters for all non-hydrogen atoms. Hydrogen
atoms were constrained to ride on the respective carbon or nitrogen
atoms with isotropic displacement parameters equal to 1.2 times the
equivalent isotropic displacement of their parent atom in all cases of
aromatic units. The structure was drawn with ORTEP [33] at the 50%
probability level and hydrogen bonding 1-D chain was drawn with the
DIAMOND program with arbitrary spheres [34]. Data manipulation
and analysis was with teXsan [35].
Fig. 2. Absorption spectra of [Cr(CO)4(L)] (A), [Mo(CO)4(L)] (B), [W(CO)4(L)] (C) in
dichloromethane and fluorescence spectra of [Cr(CO)4(L)] (a), [Mo(CO)4(L)] (b) and
[W(CO)4(L)] (c) in methanol. Inset figure shows absorption spectra at higher wave-
length region.
Pyridyl protons appear at δ 7.4–8.8 ppm while coumarin protons
appear at δ 6.4 to 8.2 ppm. In 13C NMR spectra carbon atom of lactone
carbonyl and excocyclic imine group comes in most downfield region
i.e. 161.7 and 153.08 ppm respectively. The other NMR values lie in
120–150 ppm region.
The reaction of N-[((2-pyridyl)methyliden)-6-aminocoumarin]
(L) and M(CO)6 (M=Cr, Mo, and W) in stirring condition in dry
THF at room temperature in presence of two equivalent Me3NO under
nitrogen environment for a period of 6 h has synthesized [M(CO)4(L)]
in 80–90% yield. The ligand is N, N′ chelating system where N and N′
refer to N(pyridyl) and N(imine) donor centres, respectively. The
composition of the complexes, cis-[M(CO)4(L)] (M=Cr (1); Mo (2),
and W (3)) has been supported by microanalytical and spectroscopic
data.
2.4. Computational methods
All computations were performed using the Gaussian03 (G03) [36]
software package running under Windows. The Becke's three-
parameter hybrid exchange functional and the Lee–Yang–Parr
nonlocal correlation functional [37] (B3LYP) was used throughout
this computation. Elements except chromium were assigned a 6-31G
basis set in our calculations. For chromium, molybdenum and
tungsten the Los Alamos effective core potential plus double zeta
(LanL2DZ) [38] basis set were employed. The geometric structures of
the ligand and the complexes in the ground state (S0) were fully
optimized at the B3LYP level. Geometry optimization was carried out
from the geometry obtained from the crystal structure without any
symmetry constraints. In all cases, vibrational frequencies were
calculated to ensure that optimized geometries represented local
minima. Using the respective optimized S0 geometries we employed
The complexes are soluble in hydrocarbons, alcohols, CH3CN,
CHCl3, CH2Cl2, dimethyl formamide (DMF), DMSO etc. Solution of
cis-[M(CO)4(L)] is blue-violet in benzene but in DMF solution it is
orange-red (solvatochromism is given below). They are non-electrolyte
and diamagnetic.
2.3. X-ray crystallographic data collection and refinement of [Cr(CO)4(L)]
The crystals were grown by diffusion of CH2Cl2 solution of the
complex into hexane layer at low temperature (10–12 °C). Intensity
data for the crystal of [Cr(CO)4(L)]was measured at 173 K on a Rigaku
Table 3
UV-visiblea, fluorescencea, spectral, life time and cyclic voltammetricc data.
Computed
UV-visible dataa
max/nm (10−3ε/M−1 cm−1
λex/
nm
λem/
nm
ϕb
τf
(ns)
χ2
kr ×10−9
(s−1
knr ×10−9
(s−1
Cyclic voltammetric datac
λ
)
)
)
EM, V (ΔEp, mV)c
EL, V (ΔEp, mV)c
L
337(8.895),280 (23.99),
241 (22.85)
337
327
337
331
481
486
484
482
0.0152
0.192
5.41
0.92
0.0028
0.0183
0.0079
0.0044
0.182
0.077
0.084
0.080
−0.40 (130)
[Cr(CO)4(L)]
[Mo(CO)4(L)]
[W(CO)4(L)]
592(1.841), 327(18.22),
279(37.41), 230(27.99)
554(1.319), 337(6.433),
281(16.77), 242(25.01)
565(1.457), 331(12.81),
280(29.04), 234(28.52)
10.5
10.9
11.9
1.002
0.978
1.09
0.67 (100)
0.81 (130)
0.94 (120)
−0.48 (120), −1.25d
−0.45 (160)
0.0858
0.0528
−0.43 (100)
a
In CH2Cl2.
Quantum yield.
b
c
In acetonitrile; Pt-working electrode, Ag/AgCl, Cl− reference, Pt-auxiliary electrode; [n-Bu4N](ClO4) supporting electrolyte, scan rate 50 mV/s; metal oxidation EM=0.5 (Epa+Epc), V
for M(I)/M(0) couple in unit of V, ΔEp=|Epa−Epc|, mV; Epa (anodic-peak-potential).
d
Epc (cathodic peak potential).

P. Datta et al. / Journal of Inorganic Biochemistry 105 (2011) 577–588
581
Fig. 3. Bi-exponential decay profiles of L (a) [Cr(CO)4(L)] (b), [Mo(CO)4 (L)] (c), [W(CO)4(L)] (d) in dichloromethane. Excitation is carried out at 370 nm.
time dependent density functional theory (TD-DFT) at the B3LYP level
to predict their absorptions characteristics. The excitation energies
were calculated by the TD-DFT approach. To correlate the exper-
imental solvatochromism phenomenon with the calculated results,
we performed TD-DFT calculations of the low lying excitation at the
singlet optimized geometry, including solvation effect by means of the
nonequilibrium implementation of the polarizable continuum model
(PCM) [39] as in the experimental conditions, the chosen solvents are
benzene and dimethyl sulphoxide.
formaldehyde produced was detected spectrophotometrically at
412 nm. All determinations were performed six times.
The superoxide radical scavenging activity was measured follow-
ing the method of Siddhuraju and Becker [42]. The reaction mixture
contained 0.1 M phosphate buffer, pH 7.4, 150 μM nitroblue tetrazo-
lium (NBT), 60 μM phenazine methosulphate (PMT), 468 μM NADH
and different concentrations of the compounds. The mixture was
incubated in the dark for 10 min at 25 °C and the absorbance was read
at 560 nm. Results were expressed as percentage inhibition of the
superoxide radicals. All determinations were performed six times.
2.5. Radical scavenging activity
2.5.1. In cell free system
The compounds had shows radical scavenging activity and were
investigated spectrophotometrically using 1,1-diphenyl-2-picrylhy-
drazyl (DPPH) [40], hydroxyl (OH·) [41], superoxide (O−2 ) [42] and
nitroxyl radical (NO·) [43] radicals. The DPPH radical is a stable free
radical (due to extensive delocalization of the unpaired electron)
having λmax at 517 nm. When this compound abstracts a hydrogen
radical from external source then absorption vanishes due to the
absence of free electron delocalization. Various concentrations of the
experimental compounds were added to solution of DPPH in
methanol (125 μM, 2 mL) and in each case the final volume was
made up to 4 mL with water. The solution was shaken and incubated
at 37 °C for 30 min in the dark. The decrease in absorbance of DPPH
was measured at 517 nm. Percent inhibition was calculated by
comparing the absorbance values of control and test samples.
The hydroxyl radical scavenging activities of the compounds have
been investigated following the method of Nash [41]. In-vitro
hydroxyl radicals were generated by Fe3+/ascorbic acid system. The
detection of hydroxyl radicals was carried out by measuring the
amount of formaldehyde produced from the oxidation of DMSO. The
Fig. 4. Cyclic voltammogram of [Cr(CO)4(L)] (–), [Mo(CO)4(L)] ( – – – ) and [W(CO)4
(L)]. (– –) in MeCN using Pt-disk working and Pt-wire auxiliary electrodes and
reference to Ag/AgCl electrode at 298 K.

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P. Datta et al. / Journal of Inorganic Biochemistry 105 (2011) 577–588
The nitric oxide radical scavenging activities of the compounds
2.5.2.2. Experimental setup. All experiments were performed using
1 mL of hepatocytes suspension (∼2×106 cells) in each. The hepato-
cytes were kept in culture medium only was served as normal control.
The toxin control was prepared by incubating the hepatocytes with
NaAsO2 (1 mM) for 2 h. Stock solutions of compounds were prepared
by dissolving 1 mg of each compound in 100 μL of DMSO separately
and the solution was diluted up to 1 mL by adding water. To
investigate the antioxidant role of the compounds, the hepatocyte
suspensions were incubated with NaAsO2 (1 mM) and the com-
pounds (100 μg/mL) simultaneously for 2 h. All the incubations were
performed at 37 °C with gentle shaking.
were determined following the method of Green et al. [43]. The
absorbance of the chromophore formed was read at 540 nm. Results
were expressed as percentage inhibition of nitric oxide. All determi-
nations were performed six times.
2.5.2. In-vitro study
2.5.2.1. Isolation of primary hepatocyte. Hepatocytes were isolated
from mice liver by literature method [44] with some modifications.
The animals were anaesthetized, sacrificed and livers were collected.
After collection, the organs were extensively perfused in situ in
phosphate buffer saline to get rid of blood and irrigated in a buffer
containing HEPES [4-(2-hydroxyethyl)-1-piperazineethane sulfonic
acid] (10 mM), KCl (3 mM), NaCl (130 mM), NaH2PO4–H2O (1 mM)
and glucose (10 mM) pH 7.4 and incubated with a second buffer
containing CaCl2 (5 mM), 0.05% collagenase type I mixed with the
buffer previously described for about 45 min at 37 °C. The liver sample
was then passed through wide bore syringe, filtered, centrifuged and
the pellet was suspended in DMEM [Dulbecco's modified eagle's
medium] containing 10% FBS [Fetal bovine serum] and the suspension
was adjusted to obtain ∼2×106 cells/mL.
2.5.2.3. Cell viability assessment. Cell viability assessment has been
measured by MTT [3-(4,5-Dimethyl-2-thiazolyl)-2,5- diphenyl-2H-
tetrazolium bromide] reduction assay following the reported method
[45].
2.5.2.4. Measurement of intracellular ROS production. The intracellular
ROS production was measured by using 2, 7-dichlorofluorescein
diacetate (DCF-DA) as a probe following the method of LeBel and
Bondy [46] as modified by Kim et al. [47]. Briefly, an aliquot of
∼2×106 cells were incubated with DCF-DA (5 μM) for 1 h at 37 °C in
Fig. 5. Surface plots of the molecular orbitals of L and [Cr(CO)4(L)] along with the orbital composition.

P. Datta et al. / Journal of Inorganic Biochemistry 105 (2011) 577–588
583
Table 5
Energies of electronic transitions of L with oscillator strength greater than 0.03 calculated by TD-DFT in gas phase.
λ/nm
f a
Major contribution
Assignmentb
L
359
0.4775
0.0472
0.5472
H→L (65%)
n(Cm)→π*(Py)
π(Py)→π*(Py)
n(Cm)→π*(Cm)
H-2→L (13%)
H→L+1 (64%)
H-1→L (11%)
H-4→L (36%),
H-3→L (36%)
332
279
[n(Cm)→π*(Py), π(Cm)→π*(Py)]
[π(Cm)→π*(Cm), π(Cm)→π*(Py)]
π(Py)→π*(Cm), π(Cm)→π*(Cm)
π(Cm)→π*(Cm), π(Cm)→π*(Py)
[n(Cm)→π*(Py), π(Cm)→π*(Py)]
π(Cm)→π*(Cm)
[π(Cm)→π*(Cm), π(Cm)→π*(Py)]
π(Cm)→π*(Py)
[π(Cm)→π*(Cm), π(Cm)→π*(Py)]
π(Py)→π*(Py)
273
257
251
232
0.2595
0.0334
0.143
H-1→L (26%)
H-1→L+1 (57%)
H-4→L (27%),
H→L+2 (56%)
H-4→L (41%)
H-2→L (16%)
H-6→L (50%)
H→L+2 (20%)
0.0466
π(Py)→π*(Cm), π(Py)→π*(Py)
π(Cm)→π*(Py)
[Cr(CO)4(L)]
558
477
359
315
0.0011
0.1129
0.3064
0.0382
0.0382
0.04
HOMO→LUMO (70%)
H-2→L (66%),
H-3→L (65%),
H-4→L (49%), H-3→L+1 (16%)
H-2→L+4 (24%), H-2→L+6 (35%), H-1→L+8 (26%)
H-2→L+2 (36%),
H-2→L+7 (33%)
H-3→L+1 (60%)
MLCT (Major) (Cr→CSB)+XLCT (CO→CSB)
MLCT (Cr→CSB)+XLCT (CO→CSB)
ILCT ( CSB→CSB)
ILCT ( CSB→CSB)
309
305
MXCT+XXCT+MMCT
MLCT+XLCT+ILCT
MLCT+MXCT+XLCT+XXCT
ILCT ( CSB→CSB)
ILCT (CSB→CSB)
LXCT+LMCT
299
268
259
0.2345
0.3805
0.0255
H-4→L+1 (53%), H-3→L+1 (9%)
H-3→L+3 (58%)
H-3→L+2 (32%)
ILCT
256
253
0.0464
0.0287
H-5→L (17%),
H→L+9 (28%), H-2→L+9 (18%)
H-3→L+2 (44%), H-7→L (12%)
ILCT
MXCT+XXCT+MMCT
ILCT (CSB→CSB)
H=HOMO, L=LUMO, L+1=LUMO+1 etc.
a
Oscillator strength.
Dominant excitation, Cm=Coumarin part, Py=Pyridine-imine part.
b
the dark. After treatment, the cells were immediately washed and
resuspended in PBS. The fluorescence emitted was measured at
525 nm. The fluorescence intensity was expressed as percent
fluorescence intensity.
structure in the literature containing a [Cr(CO)4(R)] core, R=4-
HOC6H4–N CH–C5H4N [50]. Here, the comparable geometric para-
meters follow the same trends as outlined above for [Cr(CO)4(L)] and
the conformation of the diimine ligand is again twisted. The crystal
packing in [Cr(CO)4(L)] is dominated by C–H⋯O and π⋯π interactions.1
A supramolecular 1-D chain is evident mediated by C–H⋯O interac-
tions involving the carbonyl-O(25) and O(21) atoms giving rise to 20-
membered [OCCrNC5H]2 and 12-membered [OCCrNCH]2 rings, re-
spectively, as illustrated in Fig. 1b. This arrangement facilitates the
formation of π⋯π interactions between the aromatic residues so that
the distance between the ring centroids of the (O(1), C(2), C(3), C(4),
C(9), C(10)) and (C(5), C(6), C(7), C(8), C(9), C(10))i aromatic rings is
3.66(15)Å; symmetry operation i: 2-x, -y, -z. These chains are
connected into layers via further π⋯π interactions occurring between
the same rings but external to the chain; ring centroid dis-
tance=3.692(15)Å for symmetry operation ii: 1-x, -y, -z. Layers
stack along (0, −1, 1) and are connected via C–H⋯O interactions,
involving the carbonyl-O(11) and O(27) atoms.
2.5.2.5. Assay of intracellular GSH/GSSG. Reduced glutathione (GSH)
level was measured following the literature protocol [48] by using
DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)] [(Ellman's reagent)] as
the key reagent. Oxidized glutathione GSSG contents in the hepatic
tissues of the experimental and normal animals were determined
following the method of Hissin and Hilf [49].
3. Results and discussion
3.1. Molecular structure
Full structural characterization of [Cr(CO)4(L)] is afforded by a
single crystal X-ray diffraction study. The molecular structure is
represented in Fig. 1a, and metric parameters are given in Table 2. The
coordination geometry for the central Cr atom is based on an
octahedron defined by a cis-C4N2 donor set. The Cr–Ccarbonyl bond
distances fall in two quite distinct ranges, i.e. 1.838(3) to 1.841(3)Å
and 1.893(3) to 1.914(3)Å, with the shorter distances having the
carbonyl-C atoms occupying positions trans to the N-donor atoms
derived from the diimine ligand. The maximum distortion from the
ideal octahedral geometry is manifested in the acute N(9)–Cr–N(12)
chelate angle of 76.35(8)°. The diimine ligand is not planar as seen in
the magnitude of the C(5)–C(6)–N(12)–C(13) torsion angle of 43.3
(3)°. The twist in the N(12)–C(6) bond probably occurs so as to
minimize steric interactions between components of the Cr(CO)4
moiety and the C5–C10 aromatic ring. There is only one closely related
3.2. Spectroscopic characterization
3.2.1. FT–IR and NMR spectra
The complexes show four ν(CO) stretching bands at 1814–
2019 cm−1 region [51]. The lactone stretching ν(COO) comes at
1
Intermolecular interactions in (1): C(3)–H(3)…O(25)i = 2.53 Å, C(3)–H(3)
…O(25)i = 3.442(3) Å, angle at H(3) = 160° for i: 2-x, -y, -z; C(18)–H(18)...
O(21)iii = 2.54 Å, C(18)...O(21)iii =3.430(3)Å, angle at H(18)= 156° for iii: 1-x,
1-y, 1-z; C(16)–H(16)...O(11)iv=2.39 Å, C(16)...O(11)iv=3.330(3)Å, angle at H(16)=
172° for iv: −1+x, y, 1+z; and C(4)–H(4)...O27v=2.46 Å, C(4)...O(27)v=3.321(3)Å,
angle at H(4)=152° for v: 1+x, −1+y, z.

584
P. Datta et al. / Journal of Inorganic Biochemistry 105 (2011) 577–588
Fig. 6. DPPH radical scavenging activities of the compounds, “*” sign indicates the maximum percentage of inhibition of the respective compounds.
1710–1727 cm−1 region. A moderately strong band appears at 1615–
1620 cm−1 and are assigned to ν(C N). Due to complexation C N
stretching is shifted about 10 cm−1 lower region compared to free
ligand value (1629 cm−1). The mass fragmentation by FAB-MS of the
complexes support the expected fragmentation pattern of M+, (M–
CO)+, (M–2CO)+, and (M–3CO)+ etc.
changing the solvent from benzene to dimethylsulphoxide (DMSO)
the MLCT maxima experience 68–75 nm blue shift. The correlation of
the energy of the MLCT band with Richard's ET(30) parameter [52,53]
is shown in Table 4 (Supplementary materials, Figs. S1 and S2). With
increasing solvent polarity the MLCT band is blue shifted and is
described negative solvatochromism which is exhibited by [M(CO)4
(α-diimine)] (M=Cr, Mo, and W) type of complexes [54]. The
efficiency of solvation depends largely on hydrogen bonding ability,
polarity, etc that's why the solvatochromism does not follow exactly
linear relationship. The superior solvation of the dipolar ground state
than that of the less dipolar excited state may be the reason for
negative solvatochromism.
The 1H NMR spectra were recorded in CD3CN and assigned on
comparing with free ligand value. Pyridine protons (13-H to 16-H)
experiences shift by 0.02–0.16 ppm while coumarin protons (3,4-H;
5-H; 7-H; and 8-H) do not exhibit significant shifting. Immine proton
(–CH N–) appears as a singlet at 8.64–8.66 ppm (see Supplementary
material, Table S1).
The 13C NMR spectra of L (in CDCl3) and [M(CO)4(L)] (1–3) (in
CD3CN) are assigned on comparing the spectra of free ligand and
complexes, and following the intensity of the lines. The DEFT study
has also distinguished the primary, secondary, tertiary and quaternary
C-centres. There are three quaternary C-centres (C-9, C-10, and C-12),
eleven secondary C-centres (C-2, C-3, C-4, C-5, C-6, C-7, C-8, C-11, C-
13, C-14, C-15, and C-16). The resonance generally appears within the
range of 118–200 ppm (Supplementary material, Table S1). Data
reveal that resonances are shifted to higher δ value on going from free
ligand to the metal–carbonyl complexes. Some of the 13C NMR
resonances are closely spaced values which reflect their resemblances
in chemical and electronic environment. C of CO in the complexes
usually appear at most downfield side (N199 ppm) that is due to high
π-acidity of CO.
Photoluminescence study of the ligand and complexes are carried
out at room temperature in methanol (Fig. 2). The ligand and the
complexes exhibit fluorescence when they are excited at π–π* band.
The maximum wavelength of emission is obtained at 481–486 nm
region [55,56]. The complexes exhibit weak emission when excited at
MLCT maxima (N450 nm) and so we do not proceed further. The
fluorescence quantum yield of the complexes at π–π* (327–337 nm)
band are higher (ϕ=0.05–0.19) than that of the ligand (ϕ=0.015).
The mechanism of fluorescence enhancement for the coumarin
derivatives is believed to work through photo induced electron
transfer (PET). In PET, the fluorophore fails to fluoresce or fluoresces
very weakly because the excited state is quenched by electron
transfer, unless the relative energies of the fluorophore are perturbed.
Heteroatom containing fluorophores develop partial charges due to
internal charge transfer (ICT) and, interaction with charged groups
can affect its energy. It has been reported that the metal ions can
enhance or quench the fluorescence emission of pyridine containing
compounds [57,58] depending on the strength interaction between
ligand field potential and metal ion. In the absence of metal ions the
fluorescence of ligand is probably quenched by the occurrence of PET
process due to the presence of a lone pair on nitrogen atoms and
vibrational relaxation of the molecule. In the complexes, the PET
process is effectively reduced and physically strained due to chelation
and the π-acidic CO molecule helps to populate the excited states
[19,20]. It may be the reason for increased fluorescence quantum yield
3.2.2. Absorption and emission spectra
The complexes are blue-violet in benzene solution. They show
absorption in 200–600 nm region (Fig. 2). The high intense transitions
(ε, 104) at shorter wavelength region (327–337, 279–281, and 230–
242 nm) are also present in the free ligand due to n–π* and π–π*
transitions (Table 3). A band is observed at longer wavelength region
(N500 nm) which is absent in free ligand and can be attributed metal-
to-ligand charge transfer (MLCT) band. The energy of MLCT maxima
follows the order: [Mo(CO)4(L)] (2)N[W(CO)4(L)] (3) N [Cr(CO)4(L)]
(1). The MLCT transition is susceptible to solvent polarity. On

P. Datta et al. / Journal of Inorganic Biochemistry 105 (2011) 577–588
585
100
80
60
40
20
0
Fig. 8. Preventive effects of the metal carbonyl on the arsenic induced reduction in cell
viability.
3.2.3. Electrochemistry
The electrochemical properties of the complexes were investigat-
ed by cyclic voltammetry in 0.1 M [n-Bu4N][ClO4] in acetonitrile
solution at scan rate 50 mV S−1. All CV data were collected under
nitrogen environment and potential are expressed with reference to
Ag/AgCl. Results are collected in Table 3. The voltammogram of the
complexes are shown in Fig. 4. The nature of voltammogram does not
change with scan rate (50–200 mV S−1). The compounds show one
oxidative response positive to reference electrode and one or two
reduction negative to this reference (Ag/AgCl) in the potential range
of 2.0 to −2.0 V The oxidation is primarily metal-centred electron
extraction and is quasireversible in nature. It is compared with similar
types of α-diimine complexes [63] and [M(CO)4(2-PAP)] (2-PAP=2-
(phenylazo)pyridine) [64]. There may be two consecutive reductive
couples to diimine chelating function. We observe one quasireversible
(ΔEp N100 mV) and one irreversible reduction for [Cr(CO)4(L)] while
two other complexes [M(CO)4(L)] (M=Mo, and W) show only one
reductive response. The reduction is referred to electron accommo-
dation to π* MO of chelated diimine function.
3.3. DFT calculations
Fig. 7. Radical scavenging activities of (a) [Cr(CO)4(L)], (b) [Mo(CO)4(L)] and (c ) [W
(CO)4(L)] using superoxide, hydroxyl and nitrosyl radicals.
The calculated structure correlates well with the experimental X-
ray structure. The Cr(1)–N(9) bond is 0.01 Å longer and Cr(1)–N(12)
bond is 0.017 Å shorter than the experimental data. The Cr–CO bond
distances also vary ∼0.002–0.02 Å range. The chelated bond angle, i.e.
N(12)–Cr(1)–N(9) is 0.6° lower than the experimental data. DFT
calculations of the ligand and [Cr(CO)4 L] has been done using the
optimized geometry of the respective compounds. The energy of
HOMO (Highest Occupied Molecular Orbital) of L is 0.47 eV lower
than the corresponding Cr-complex, whereas the energy of LUMO
(Lowest Unoccupied Molecular Orbital) of L is 0.89 eV higher than the
[Cr(CO)4(L)] . The surface plots of the frontier orbitals of L and [Cr
(CO)4(L)] are shown in Fig. 5. The HOMO of the ligand is mainly
constituted by coumarin part (70%) and the LUMO is constituted
mainly by imino-pyridine part (82%). In [Cr(CO)4(L)] the HOMO is
constituted by 66% metal along with significant contribution from
carbonyl group. But the LUMO is dominated by ligand function (91%).
In [Cr(CO)4(L)] HOMO, HOMO-1, HOMO-2 are energetically closer to
each other (EHOMO, −5.94 eV, EHomo-1, −6.07 eV, EHomo-2, 6.16 eV)
(Supplementary data, Table S3).
of the complexes than ligand. Intersystem crossing due to spin-orbit
coupling introduced by the metal centre may also influence the
emission intensity. This enhances fluorescence intensity upon
coordination to metal ion.
Life time data of the complexes are taken in dichloromethane
when excited at 370 nm. The fluorescence decay curve was deconvo-
luted with respect to the lamp profile. The observed florescence decay
fits with bi-exponential nature for the complexes (Fig. 3). We have
used mean fluorescence life time (τf =a1τ1 +a2τ2 where a1 and a2 are
relative amplitudes of decay process) to compare excited state
stability of the complexes. The radiative and non-radiative rate
constants (kr and knr) are calculated and data show usual higher knr
value than kr (Table 3). The emission feature of these complexes may
be due to participation of strongly π-acidic CO [19,20,59,60]. High π-
acidic, strong field ancillary CO may increase d–d transition in
combination with the incorporation of third-row metal which also
increases the metal–ligand bonding [54,61,62] and are contributing to
maintain population at excited state and hence the emission property.
The lifetime of the complexes are almost double than the ligand.
In the calculated spectrum four peaks are assigned in 230–600 nm
region. Longer wavelength transition (λ∼550–590 nm) is thought to
be MLCT transition type but TD-DFT calculation reveals that it is a
mixture of MLCT and XLCT (X=CO) [54]. The transition obtained at
327–337 nm regions is of intraligand charge transfer type and other

586
P. Datta et al. / Journal of Inorganic Biochemistry 105 (2011) 577–588
Fig. 9. The intracellular ROS production was detected by DCF-DA method. Left panel represents the images under fluorescence microscope (original magnification ×400), Right panel
B represents the DCF fluorescence measured using a spectrofluorimeter.
two high energy transitions are also found to be intraligand charge
transfer type and mixture of MXCT, XXCT and MMCT (Table 5).
The solvent dependence of MLCT band has been supported by TD-
DFT computation when the calculation is carried out in different
solvent. For example, the absorption maxima of the MLCT band
obtained experimentally in nonpolar solvent like benzene and highly
polar solvent like dimethylsulphoxide are 608 nm and 533 nm,
respectively and the calculated transition found to be at 610 nm and
530 nm respectively.
DFT data suggested that the HOMO of the complexes comprises of
mainly by metal (62–66%) with some contribution from CO (33–37%).
Thus the oxidation is rightly explained as oxidation of metal M(0) to
M(+1) while the oxidation of CO to CO2 demands large amount of
energy that is incapable to support by the redox condition. On the
other hand ligand contributes N85% to constitute LUMO and thus the
reduction may be referred as electron accommodation to π* orbitals of
diimine group.
radicals vanishes rapidly at a particular wavelength. It has been
observed that the complexes showed maximum percentage of
inhibition when they were incubated at
a concentration of
∼0.03 mg/mL, ∼0.30 mg/mL and 0.01 mg/mL for [Cr(CO)4(L)], [Mo
(CO)4(L)] and [W(CO)4(L)] respectively with almost all radical
solutions. On the other hand coumarin and the [M(CO)6] precursors
possess very poor scavenging property and the activity slightly
increases in free ligand, L (all data are not shown), and the activities
are found to be maximum in the complexes. Hence from these figures
(Figs. 6 and 7) it can be attributed that the complexes possess potent
free radical scavenging activity. The radicals are either oxidized or
reduced by scavengers. In complexes metal ions present in M(0)
formal oxidation state. They may be oxidized as evident from cyclic
voltammetry and act as reducing scavenger. The radical scavenging
activity in cell free system is examined with reference to hydroxyl
radical, superoxide and nitric oxide. In these cases the effect of [M
(CO)4(L)] on radical scavenging activity show superior to [M(CO)4(L)]
than that of free L and [M(CO)6]. Free ligand is inefficient to supply
reducing equivalent. [M(CO)6] is incapable due to very strong π-
acidity of CO. DFT computations of L and metal complexes also
indirectly support this conjecture. Thus electron density distributed
on the complexes may be responsible to show antioxidant activity
[65]; W being heaviest member in Group 6 elements shows highest
scavenging ability. It is also reflected from the energy of HOMO that is
highest in the series for [W(CO)4(L)] (−5.74 eV) (see Supplementary
data, Table S3).
3.4. Antioxidant property
3.4.1. In cell free system
The radical scavenging activity of the compounds in cell free
system is examined with reference to DPPH, superoxide (O−2 ),
hydroxyl (OH·) and nitroxyl (NO·) radicals. The effects of the
compounds have been shown in Figs. 6 and 7. The plots show that
with increase in concentration of the test samples the intensity of
3.4.2. In-vitro study
During cellular aerobic metabolism oxygen free radical and other
ROS (reactive oxygen species) including super oxide anion (O−2 ), H2O2
and hydroxyl radical (OH.) are generated via incomplete reaction of
oxygen as well as exposure from environmental agents, radiation and
redox cycling agents [66,67]. ROS, at low concentration have been
implicated in the regulation of various physiological processes such as
proliferation [68], differentiation [69], apoptosis [70] and senescence
[71]. But at high concentration ROS are responsible for considerable
tissue damage including DNA damage, lipid peroxidation and protein
damage [72]. Oxidative stress occurs when the production of ROS
exceeds the natural antioxidant defense capacity. During oxidative
insult production of excessive reactive oxygen species (ROS) destroys
the balance between pro-oxidant and antioxidant systems. In recent
years the mechanism of arsenic induced toxicity as well as
carcinogenesis has been under intense investigation. Earlier reports
[73–76] showed a strong link between the formations of reactive
oxygen species (ROS), reactive nitrogen species (RNS) and arsenic
Fig. 10. Effects of the metal carbonyl on the reduction in GSH/GSSG.

P. Datta et al. / Journal of Inorganic Biochemistry 105 (2011) 577–588
587
induced oxidative damage to membrane lipids, intracellular proteins
and DNA.
and stabilizes M(0) oxidation state. The ligands as well as complexes
are highly fluorescent. The complexes show high efficient π–π*
emission. All the complexes possess very good antioxidant activity
both in cell free system and in-vitro. [W(CO)4(L)] exhibits the
strongest free radical scavenging activity.
In the present study intracellular ROS production was estimated by
using DCF-DA as a probe. DCF-DA diffuses through the cell membrane
where it is enzymatically de-acetylated by intracellular esterases to
the more hydrophilic nonfluorescent reduced dye DCF. In the
presence of reactive oxygen metabolites, nonfluorescent DCFH rapidly
oxidized to highly fluorescent product DCF. In our study we observed
that arsenic intoxication increased the rate of DCF (dichlorofluor-
escin) formation, which is an indicator of intracellular ROS produc-
tion. Simultaneous treatment with complexes prevented the arsenic
induced intracellular ROS production.
Increased production of ROS caused cell death via initiating the
peroxidation of membrane polyunsaturated fatty acids (PUFA), DNA
fragmentation etc. In our study we found that arsenic exposure caused
reduction in cell viability and that could be prevented by the
treatment with the complexes.
Acknowledgements
Financial support from the Council of Scientific and Industrial
Research (CSIR) and Department of Science & Technology, New Delhi
are gratefully acknowledged. We are thankful to Prof. Nitin Chatto-
padhyay, Jadavpur University for lifetime experiments.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jinorgbio.2010.04.013.
Maintenance of normal cellular functions largely depends on the
efficiency of the defense mechanisms against free radical mediated
oxidative stress. Thiols are considered to be the non-enzymatic
antioxidants providing cellular defense against toxin mediated
oxidative injury in the brain tissue. GSH, the most abundant non
protein thiol in the brain tissues, maintains the intracellular redox
status against pro-oxidative stress by detoxifying various xenobiotics
as well as by scavenging free radicals. During the metabolic action of
GSH, its sulfhydryl group becomes oxidized resulting with the
formation of corresponding disulfide compound, GSSG (oxidized
form). Thus depletion of GSH content is associated with an increase in
GSSG concentration resulting with the depletion in GSH/GSSG ratio. In
the present study, significant decreases in the GSH/GSSG ratio have
been observed in the arsenic treated cells. Simultaneous treatment
with the complexes prevented the arsenic induced reduction in GSH/
GSSG ratio.
In Fig. 8 it has been observed that incubation of the hepatocytes
with NaAsO2 increased the production of intracellular reactive oxygen
species (ROS) which caused reduction in cell viability (Fig. 9) and the
ratio of GSH to GSSG (Fig. 10). Incubation of the complexes with
NaAsO2 prevented the increased production of ROS and its adverse
effects due to their radical scavenging and antioxidant property.
Among them [W(CO)4(L)] showed significantly better antioxidant
property. So far literature is concerned the antioxidant property of
Group 6 metal–carbonyl diimine complexes is reported first time in
this work.
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