www.elsevier.

nl/locate/ica

Inorganica Chimica Acta 318 (2001) 207– 211

Note

Tuning up superoxide dismutase activity of copper complex of

salicylaldehyde semicarbazone by heterocyclic bases pyridine and
N-methyl imidazole
Jayendra Patole a, Sabari Dutta a, Subhash Padhye a,*, Ekkehard Sinn b
a
Department of Chemistry, Uni6ersity of Pune, Ganeshkhind Road, Pune 411007, India
b
Department of Chemistry, Uni6ersity of Missouri-Rolla, Rolla, MO 65409, USA
Received 13 November 2000; accepted 28 February 2001

Abstract

The copper(II) complexes of the Schiff base salicylaldehyde semicarbazone (SALSC) have been prepared and structurally
characterized. The compound possesses a distorted square planar geometry where the metal atom lies slightly below the ligand
donor atom plane and exhibits a longer Cu–Cl bond distance (2.226 A, ). The superoxide dismutase activity of the compound can
be synergistically enhanced by the addition of heterocyclic bases. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Crystal structures; Superoxide dismutase; Copper complexes; Semicarbazone complexes

1. Introduction tion and subsequent short lifetimes in the biological

Superoxide dismutase (SOD) is an antioxidant en-
zyme known to protect cells from the toxic effects of
superoxide ion by its dismutation into dioxygen and
hydrogen peroxide in biological systems [1]. Three basic
types of SOD’s are known, viz. Cu – Zn – SOD, Mn –
SOD and Fe –SOD respectively of which Cu – Zn –SOD
is found in higher organisms, especially mammals [2].
In recent years a large number of studies have indicated
that imbalance or deficiency of such antioxidant en-
zymes can lead to several diseases and disorders includ-
ing diabetes, ischmia, cataract, Parkinson’s disease,
cancers and many others [3,4]. Consequently supple-
mentation with antioxidant enzymes is thought to be
beneficial in treating such disorders. However, adminis-
tration of these enzymes through oral or intraperitonial
routes is severely restricted due to their rapid degrada-
* Corresponding author. Tel.: + 91-20-5656061; fax: +91-20-
5651728.
E-mail address: sbpadhye@chem.unipune.ernet.in (S. Padhye).
systems [5]. Attempts are, therefore, undertaken in
many laboratories to prepare low molecular weight
mimics of the antioxidant enzymes especially SOD with
good radical scavenging activities.
Amongst the large number of compounds that have
been reported so far as SOD mimics include the schiff
base ligands derived from various aldehydes and ke-
tones. These include ligands derived from 2-pyridyl
ketones of the type, [Cu(apy)L] (ClO4), (where apy=
2-acetyl pyridine and L=N-ethylene(2-acetylpyridine-
imine)) and [Cu(appn)] (ClO4)2·H2O and {[Cu(bppn)]-
(ClO4)2}2 (where appn =N,N%-propylenebis(2-actoyl-
pyridineiminato) and bppn= N,N%-propylenebis(2-ben-
zoylpyirdineiminato)) [6]. Among the most successful
compounds possessing SOD activity are imidazole
bridged copper complexes of which [Cu2(bpzbiap)Cl3] is
the best SOD mimic reported so far [7] due to their
close resemblance to native Cu –Zn –SOD. Recently
SOD activity of the planar complex, [Cu(II)(aspirinate)2
(DMSO)] was shown to be enhanced considerably on
addition of bases like N-methyl imidazole and pyridine
[8].

0020-1693/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 2 0 - 1 6 9 3 ( 0 1 ) 0 0 4 1 6 - 9
208 J. Patole et al. / Inorganica Chimica Acta 318 (2001) 207–211
In the present communication, we describe a single
X-ray crystal structure of the copper complex of sali-
cylaldehyde semicarbazone along with its SOD activity
and show that the activity could be tuned further by
the addition of heterocyclic bases.
2. Experimental
2.1. Materials
Salicylaldehyde and semicarbazide hydrochloride
were products of CDH (Mumbai, India) and Quali-
gens (Mumbai, India) respectively. All other solvents
and chemicals employed were of analytical grade and
were used as supplied.
2.2. Physical measurements
Elemental analyses were performed at the Micro
analytical Laboratory, University of Pune. IR spectra
were recorded as Nujol mulls on Perkin– Elmer 283-B
spectrophotometer. The electronic spectra were
recorded on a Spectronic Genesys 2 Spectrophotome-
ter using 10 mm rectangular matched quartz cells. The
room temperature magnetic susceptibilities of the solid
samples were measured on a Faraday-type magnetic
balances with magnetic field of 8000 G using Hg[-
Co(SCN)4] as the calibrant, while correcting their
molecular susceptibilities for atomic diamagnetism us-
ing Pascal’s constants [9]. Cyclic Voltammetric mea-
surements were performed with a Bio-Analytical
System’s model BAS CV-27 coupled with a X– Y
recorder in DMSO using 0.1 M tetraethyl ammonium
perchlorate (TEAP) as the supporting electrolyte. The
three-electrode system used for the measurements con-
sisted of a glassy carbon working electrode, a plat-
inum wire auxiliary electrode and a saturated calomel
reference electrode. All solutions were degassed with
nitrogen for 15 min prior to the measurements. The
ESR spectra were recorded in DMSO at 77 K on
Varian X-band spectrophotometer (Model E line cen-
tury series, no. E-112) at RSIC, IIT Powai, Mumbai,
India.
2.3. Synthesis
2.3.1. Salicylaldehyde semicarbazone (SALSC)
To an aqueous solution containing semicarbazide
hydrochloride (1 g, 0.008 mol) and sodium acetate (1.5
g, 0.01 mol), salicylaldehyde (0.5 g, 0.004 mol) was
added with stirring resulting in a white precipitate
immediately. The stirring was continued for half an
hour after which the precipitate was filtered, washed
with distilled water and finally dried in vacuum. Anal.
Calc. for SALSC: C, 53.63; H, 5.06; N, 23.48. Found:
C, 53.36; H, 5.11; N, 23.38%. IR (Nujol): w(OH) 3454
cm − 1, w(CO) 1669 cm − 1, w(CN) 1618 cm − 1.
2.3.2. [Cu(SALSC)Cl] ·2H2O
An aqueous solution of CuCl2·3H2O (0.285 g, 0.001
mol) was mixed with an ethanolic solution of SALSC
(0.3 g, 0.001 mol) slowly with stirring and was refluxed
on the water bath for 2 h and then set aside to attain
the ambient temperature when a dark green product
was obtained. Crystals suitable for X-ray measure-
ments of this product were obtained by a slow evapo-
ration of its solution in ethanol+ water (60:40 v/v)
mixture. Anal. Calc. for CuC8H12O4N3Cl: C, 32.42; H,
3.38; Cu, 20.28; N, 13.40. Found: C, 32.09; H, 3.97;
Cu, 20.21; N, 13.26%. IR (Nujol) w(CO) 1652 cm − 1,
w(CN) 1602 cm − 1.
2.4. X-ray structure determination
A summary of crystal data, intensity collection and
structure refinement is given in Table 1. A black pris-
matic crystal of the copper complex having dimensions
0.40× 0.35× 0.20 mm3 mounted on the glass fibre was
used for unit cell and structure determination. Mea-
surements were carried out using the …-scan method
on a Rigaku AFCS diffractometer [10] using graphite
monochromated Mo Ka radiation (u=0.71069 A, ) and
were corrected for Lorentz-polarization effects and ab-
sorption.1 The intensities of three standard reflections
measured after every 150 reflections showed no greater
variation than those expected from Poisson statistics.
The structure was solved from Patterson and Fourier
difference maps. All non-hydrogen atoms were refined
anisotropically by full-matrix least-squares (F). Neu-
tral atom scattering factors were taken from Cromer
and Weber. Anomalous dispersion effects were in-
cluded in Fcalc; the values for Df and Df % were those of
Cromer [11].
2.5. SOD acti6ities
The SOD activity of the copper compound was de-
termined spectrophotometrically by the NBT assay
employing KO− ’ as the source of superoxide radical
2
[12]. One unit of SOD activity was defined as the
concentration of the test substance required for 50%
inhibition of NBT reduction by the superoxide (IC50
value) anion.
1
An empirical absorption correction, based on azimuthal scans of
several reflections, was applied which resulted in transmission factors
ranging from 0.71 to 1.41.
 J. Patole et al. / Inorganica Chimica Acta 318 (2001) 207–211 209

Table 1 Table 2
Selected crystallographic data for [Cu(SALSC)Cl]·2H2O Selected bond lengths (A, ) and bond angles (°) for
[Cu(SALSC)Cl]·2H2O
Empirical formula CuClO4N3C8H12
Formula weight 313.2 Bond lengths
Crystal color black Cu–Cl 2.226(2) Cu–O(1) 1.970(3)
Habit prism Cu–N(1) 1.935(3) Cu–O(2) 1.970(3)
Dimensions (mm3) 0.40×0.35×0.20 O(2)–C(8) 1.266(3)
Number of refections in unit 25(30.4–43.1°)
Bond angles
cell calc. (2q range)
Cl–Cu–O(1) 93.4(1) Cl–Cu–O(2) 93.3(1)
Unit cell dimensions
N(1)–Cu–O(1) 92.6(1) N(1)–Cu–O(2) 80.6(1)
a (A, ) 8.518(3)
b (A, ) 10.115(5)
c (A, ) 6.905(3)
h (°) 92.48(48)
i (°) 96.60(3)
k (°) 79.43(3) 3. Results and discussion
V (A, 3) 580.8(9)
Space group P1(
Z 2 The interaction of cupric chloride with tridendate
Dcalc (g cm−3) 1.79 SALSC ligand yields a dark green, neutral complex
F000 740 having a structural composition of [Cu(SALSC)Cl]·
v(Mo Ka) (cm−1) 21.2
Diffractometer Rigaku AFC6S
2H2O as revealed from its elemental analyses. The
Radiation Mo Ka (u, A, ) 0.71069 compound shows magnetic moment of 1.66 BM, which
Temperature (°C) 20 is close to the spin only value for the planar molecule.
Scan type, rate (° min−1) …–2q, 8 (in …)
2qmax (°) 54.2 (to h= 10, k=12, l =8)
Number of reflections measured total 2716, unique 2542
3.1. Crystal structure of [Cu(SALSC)Cl] ·2H2O
(Rint = 0.024)
Structure solution heavy atom (Patterson) The molecular structure of the complex is shown in
Refinement 0.01 Fig. 1. Selected bond distances and bond angles are
Take-off angle (°), detector 6.0, 6.0 mm horizontal and
listed in Table 2. The crystal structure consists of a
aperture vertical
Crystal to detector distance 40 neutral, mononuclear four-coordinate copper(II) com-
(cm) plex with the SALSC ligand providing N,O,O donor
Scan width (°) 1.84+0.30 tan q atom set while the fourth position is being occupied by
Corrections Lorentz-polarization, absorption
the chloride ion. The overall coordination geometry of
(trans, factors: 0.71–1.41)
Residuals: R; Rw, 0.042; 0.043, 3.19 the compound is distorted square planar where the
goodness-of-fit metal atom lies 0.02 A, below the ligand donor atom
Max., min. peak in final 0.50, −0.78 plane consisting of C(l)–O(1)– O(2)–N(1) atoms. The
difference map (e A, −3)
five and six-membered chelate rings within the copper
complex are inclined to each other by a dihedral angle
of 3°. Amongst the various isomeric forms possible for
the semicarbazone ligand, the E isomer is preferred
during present copper complexation.
The Cu –Cl bond distance is the longest (2.226 A, ) in
the complex as compared to Cu–Cl bond distance
found (2.208 A, ) for the copper complex of salicylalde-
hyde benzoylhydrazone ligand [13], but is shorter than
found in case of salicylaldehyde acetylhydrazone cop-
per (2.6235 A, ) compound [14]. All other bond dis-
tances, viz. Cu–N(1), Cu–O(1) and Cu–O(2) resemble
closely with those reported earlier for the complex,
[Cu(II)(SBH)Cl] (where SBH=Salicylaldehyde ben-
zoylhydrazone). The C(8)–O(2) bond length of 1.266(4)
A, approaches the value expected for a formal CO
bond distance [15] indicating that the ligand is in the
‘keto’ form in the complex. The bond angles around
Fig. 1. ORTEP diagram of [Cu(SALSC)Cl]·2H2O showing the number- copper deviate from 90° confirming a distorted square
ing scheme used. planar geometry for the compound.
210 J. Patole et al. / Inorganica Chimica Acta 318 (2001) 207–211

Table 3

Compound A×10−4 cm−1 g f a

(cm) References

CuAITSC 139 2.360 170 [19]

CuATSC 144 2.147 149 [19]
CuMFuTSC 146 2.151 147 [19]
CuPyTSC 158 2.180 138 [19]
CuPTSC 169 2.191 130 [19]
CuGTSC 173 2.184 126 [19]
CuSaTSC 193 2.190 113 [19]
CuRibTSC 188 2.125 113 [19]
[Cu(SALSC)Cl] 156 2.280 146 present work
[Cu(SALSC)]+pyridine 150 2.264 151 present work
[Cu(SALSC)]+N-methyl imidazole 150 2.261 150 present work

a
f =g/A.

3.2. Electronic absorption spectra
The copper compound shows a strong ligand to
metal charge transfer (LMCT) band at 26 500 cm − 1 in
its electronic spectrum which on addition of pyridine
and N-methyl imidazole solvents gets shifted to 26 700
and 28 000 cm − 1, respectively, due to the ligand field
effects of the added bases. The metal-based 2B2g “ 2A2g
(d – d) transition can be seen at 13 800 cm − 1 in the
parent compound [16], which undergoes a hyp-
sochromic shift on addition of pyridine (14 900) and
N-methyl imidazole bases (16 000), respectively, due to
distortion of the complex from square planar towards
tetrahedral geometry [17]. The precipitation of the chlo-
ride ions on addition of AgNO3 solution indicates their
displacement from the coordination sphere on addition
of these bases. However, no precipitation is observed in
absence of these bases indicating coordinated halides in
the parent compound.
3.3. Cyclic 6oltammetry
An irreversible peak seen in the cyclic voltamogram
of the parent ligand at − 1.0 V is due to the reduction
of azomethine linkage [18], which is retained on com-
plexation. A fully reversible Cu(II)/Cu(I) redox couple
is observed at + 0.40 V in the present compound. On
addition of pyridine or N-methyl imidazole bases it is
shifted to + 0.18 and −0.04 V, respectively, as a result
of the distortion effected by these axial ligands in the
planar geometry of the complex.
3.4. EPR spectroscopy
The EPR spectrum of the copper compound at 77 K
in DMSO is typical of a distorted square planar geome-
try. On addition of the heterocyclic bases the spectra is
modified to accommodate the distortion introduced by
the distortion factor f (given by g/A) and these are
included in Table 3. Their values are compared with
analogous copper compounds as compiled by Cao et al.
[19] and can be used as a caveat for predicting SOD
activity for the synthetic mimics.
3.5. SOD acti6ity
The SOD activity measured for the present copper
complex by the method of NBT reduction yields a
linear relationship between the concentration of the
copper compound and the subsequent inhibition of
superoxide ions. The IC50 value for the copper com-
pound is found to be 0.3 mM, which is comparable
with that found for the complex [Cu (as-
pirinite)2(DMSO)2] [8]. The parent ligand as well as the
nitrogen bases pyridine and N-methyl imidazole has no
activity at all. The addition of the nitrogen bases en-
hances the SOD activity considerably from 26 to 44%
in case of added pyridine and to 67% in case of
N-methyl imidazole which is perhaps reflective of the
distortion introduced in the square planar geometry of
the parent complex by the added bases as also revealed
in their EPR spectra (Fig. 2).
Fig. 2. The effect of added nitrogen bases on % inhibition of SOD
activity by copper complex. (1) [Cu(SALSC)Cl], (2) [Cu(SALSC)-
these ligands. The g and A were calculated along with Cl]+pyridine and (3) [Cu(SALSC)Cl] + N-methyl imidazole.
 J. Patole et al. / Inorganica Chimica Acta 318 (2001) 207–211
Acknowledgements
J.P. would like to thank University of Pune (India)
for the financial assistance while S.P. acknowledges
visitorship assistance from the British Council.
References
[1] T.C. Pederson, S.D. Aust, Biochem. Biophys. Res. Commun. 52
(1973) 1071.
[2] Y. Mizushima, R. Igarshi, C.G. Wermuth, N. Koga, H. Kinig,
B.W. Metcalf, Medicinal Chemistry for the 21st Century, Black-
well Scientific Publications, Oxford, 1992, p. 331.
[3] G. Tabbi, T. Nauser, W.H. Koppenol, J. Reedijk, Eur. J. Inorg.
Chem. (1998) 1939.
[4] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and
Medicine, 2nd edn., Clarendon Press, Oxford, 1989.
[5] A.E. Liczmanski, H.-J. Hartmann, U. Weser, Bull. Pol. Acad.
Sci. Biol. Sci. 42 (1994) 291.
[6] A.-M. Liu, R.-G. Xiong, X.-Z. You, Polyhedron 16 (1997) 119.
[7] G. Tabbi, W.L. Driessen, J. Reedijk, R.P. Bonomo, N. Veld-
man, A.L. Spek, Inorg. Chem. 36 (1997) 1168.
211
[8] R.G. Bhirud, T.S. Srivastava, Inorg. Chim. Acta 173 (1990) 121.
[9] W.E. Hatfield, in: E.A. Boudreaux, L.N. Mulay (Eds.), Theory
and Applications of Molecular Paramagnetism, Wiley, New
York, 1976, p. 491.
[10] J.R. Backhouse, H.M. Lowe, E. Sinn, S. Woodward, J. Chem.
Soc., Dalton Trans. (1995) 1489.
[11] D.T. Cromer, J.T. Waber, International Tables for X-ray Crys-
tallography, Table 2.3.1, vol. IV, The Kynoch Press, Birming-
ham, UK, 1974.
[12] R.L. Arudi, A.O. Allen, B.H.J. Bielski, FEBS Lett. 135 (1981)
265.
[13] A.A. Alejandro, T.B. Murphy, D.K. Johnson, N.J. Rose, V.
Schonaker, Inorg. Chim. Acta 67 (1982) L25.
[14] E.W. Ainscough, A.M. Brodie, J.D. Dobbs, J.M. Waters, Inorg.
Chim. Acta 267 (1998) 27.
[15] International Tables For X-ray Crystallography, vol. III,
Kynoch Press, Birmingham, UK, 1974, p. 276.
[16] B.N. Figgis, Introduction to Ligand Fields, Interscience, New
York, 1966, p. 316.
[17] H. Yokoi, A.E. Addison, Inorg. Chem. 16 (1977) 1341.
[18] A.S. Kumbhar, S.B. Padhye, D.X. West, A.E. Liberta, Transi-
tion Met. Chem. 16 (1991) 276.
[19] A. Diaz, R. Cao, A. Fargoso, I. Sanchez, Inorg. Chem. Com-
mun. 2 (1999) 358.