Inorganica Chimica Acta 362 (2009) 4891–4898

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Inorganica Chimica Acta

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Copper(II) complexes as superoxide dismutase mimics: Synthesis,characterization, crystal structure and bioactivity of copper(II) complexes

R.N. Patel *, K.K. Shukla, Anurag Singh, M. Choudhary, U.K. Chauhan 1, S. Dwivedi 1

Department of chemistry, A.P.S. University, Rewa, MP 486003, India

a r t i c l e i n f o

Article history:Received 26 March 2009Received in revised form 25 June 2009Accepted 21 July 2009Available online 8 August 2009

Keywords:Copper(II) complexesX-band EPRSuperoxide dismutase (SOD) activity

0020-1693/$ - see front matter � 2009 Elsevier B.V. Adoi:10.1016/j.ica.2009.07.037

* Corresponding author. Tel.: +91 7662 231373; faxE-mail address: rnp64@yahoo.co.uk (R.N. Patel).

1 School of Environment Biology, A.P.S. University, R

a b s t r a c t

Two new copper(II) complexes of the type [Cu(L)X2), where L = (E)-N-phenyl-2-[phenyl (pyridine-2-yl)methylene]hydrazinecarboxamide X = Cl/Br have been synthesized and characterized by elementalanalyses, FAB (fast atomic bombardment) magnetic measurements, electronic absorption, conductivitymeasurements cyclic voltammetry (CV) and Electron paramagnetic resonance (epr) spectroscopy. Thestructures of these complexes determined by single crystal X-ray crystallography show a distorted squarebased pyramidal (DSBP) geometry around copper(II) metal center. The distorted CuN2OX (X = Cl/Br) basalplane in them is comprised of two nitrogen and one oxygen atoms of the meridionally coordinated ligandand a chloride or bromide ion and axial position is occupied by other halide ion. The epr spectra of thesecomplexes in frozen solutions of DMSO showed a signal at g ca. 2. The trend in g-value (g|| > g\ > 2.00)suggest that the unpaired electron on copper(II) has dx2�y2 character. Biological activities in terms ofsuperoxide dismutase (SOD) and antimicrobial properties of copper(II) complexes have also been mea-sured. The superoxide dismutase activity reveals that these two complexes catalyze the fast dispropor-tionation of superoxide in DMSO solution.

� 2009 Elsevier B.V. All rights reserved.

1. Introduction

The synthesis of low-molecular weight copper(II) complexesmimicking the superoxide dismutase (SOD) activity [1–4], providemodels for metalloproteins active sites and lend insight toward thedesign of new catalysts. Copper is a biologically relevant elementand many enzymes that depend on copper for their activity havebeen identified. Among these complexes, copper(II) complexesare known to play a significant role in naturally occurring biologi-cal systems [5] like (Cu,Zn–SOD) superoxide dismutase. Superox-ide dismutase (SOD) which can destroy the superoxide veryrapidly, is nature’s agent for protection of the organism from thisradical burden. In fact native SOD enzymes have been shown inmany studies to exhibit protection in animal models of inflamma-tory diseases [6]. In a variety of scenarios, therapeutic dosage ofadditional SOD enzyme has shown promise, but from a numberof view points synthetic metal complexes offer considerable prom-ise as SOD catalysts for pharmaceutical applications. Depending onthe metal at the active center, there are three general types of SODenzymes, namely Cu,Zn–SOD and Mn–SOD in mammalian systemsNi–SOD and Fe–SOD in bacterial systems. These SODs dispropor-tionate the toxic O2

� radical to molecular oxygen and hydrogen

ll rights reserved.

: +91 7662 230819.

ewa, MP 486003, India.

peroxide [7–9]. All SODs employ the two step ping-pong mecha-nism shown in Eqs. (1) and (2), where M is the redox active metalcenter capable of both oxidizing and reducing superoxide.

Mox þ O�2 !Mred þ O2 ð1ÞMred þ O�2 þ 2Hþ !Mox þH2O2 ð2Þ

The presence of copper in the active site of Cu,Zn–SOD has ledmany groups to search for stable, non toxic, low-molecular weightcomplexes of this metal that have SOD activity (functional model)and could be substituted for SOD in clinical applications withdesirable qualities being cell permeability, low cost and non-immunogenicity [10]. Since copper is a most relevant metal forthe design of synthetic SOD catalysts, our school [11–18] also em-barked on an effect in designing of some structural and functionalCu, Cu–SOD and Cu, Zn–SOD models. (E)-N-phenyl-2-[phenyl (pyr-idine-2-yl)methylene]hydrazinecarboxamide is a tidentate NNOdonar ligand (Scheme 1) with donar groups suitable placed forforming 2 five-membered chelate rings.

The mononuclear complexes 1 and 2 show penta-coordinatedcopper(II) ion with distorted square pyramidal (DSBP) coordinationgeometry. Herein we report the synthesis, structure, spectroscopic,magnetic measurement, cyclic voltammetry and superoxide dis-mutase (SOD) activity of two new copper(II) complexes of the type[Cu(L)X2] where L is a simple tridentate asymmetric ligand and X ishalogen (Cl and Br) atoms.

NH

O

NHN

N

Scheme 1.

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2. Experimental

2.1. Materials

Copper(II) chloride dihydrate was purchased from S.d. fine-chemicals, India. All other chemicals used were of synthetic gradeand used without further purification.

2.2. Physical measurements

2.2.1. FAB mass spectraFAB mass spectra were recorded on a JEOL SX 102/DA 6000

mass spectrometer using (6 kV, 10 mA) as the FAB gas. The acceler-ating voltage was 10 kV and the spectra were recorded at roomtemperature (RT) with m-nitrobenzoyl alcohol as the matrix.

2.2.2. Magnetic measurementsMagnetic susceptibility measurements were made on a Gouy

balance using a mercury(II) tetrathiocynato cobaltate(II) as cali-brating agent (vg = 16.44 � 10�6 c.g.s. units). The molar suscepti-bilities were corrected for the diamagnetism of the constituentatoms by using Pascal constants.

2.2.3. SpectrometryUV–Vis spectra were recorded at 25 �C on a Shimadzu UV–Vis

recording spectrophotometer UV-1601 in quartz cells. IR spectrawere recorded in KBr medium on a Perkin–Elmer 783 spectropho-tometer. X-band (�9.4 GHz) epr spectra were recorded with a Vari-on E-line Century Series Spectrometer equipped with a dual cavityand operating at X-band with 100 kHz modulation frequency atroom temperature and at 77 K. TCNE was used as field marker.The frozen solution at 77 K used for epr spectra were in3 � 10�3 M of DMSO solution. The epr parameters for copper(II)complexes were determined accurately using simulation program[19].

2.2.4. ElectrochemistryCyclic voltammetry was carried out with a BAS-100 Epsilon

electrochemical analyzer having an electrochemical cell with athree-electrode system. Ag/AgCl was used as a reference electrode,glassy carbon as working electrode and platinum wire as an auxil-iary electrode. NaClO4 (0.1 M) was used as supporting electrolyteand DMSO as solvent. All measurements were carried out at298 K under a nitrogen atmosphere.

2.2.5. BioactivityAntimicrobial (antibacterial) and superoxide dismutase (SOD)

activities were evaluated using the following methods.

2.2.6. SOD activityThe in vitro SOD activity was measured using alkaline DMSO as

a source of superoxide radical (O2�) and nitrobluetetrazolium chlo-

ride (NBT) as O2� scavenger [20,21]. In general, 400 ll sample to be

assayed was added to a solution containing 2.1 ml of 0.2 M potas-

sium phosphate buffer (pH 8.6) and 1 ml of 56 lM of alkalineDMSO solution was added while string. The absorbance was thenmonitered at 540 nm against a sample prepared under similar con-dition except NaOH was absent in DMSO. A unit of superoxide dis-mutase (SOD) activity is the concentration of complex, whichcauses 50% inhibition of alkaline DMSO mediated reduction ofnitrobluetetrazolium chloride (NBT).

2.2.7. Antibacterial activity measurementsThe in vitro antimicrobial (antibacterial) activities of these com-

plexes were tested using paper disc diffusion method [22,23] thechosen strains were G(+) Staphylococcus and G(�) Proteus vulgaris,Streptococcus, Pseudomonas and Escherichia coli. The liquid mediumcontaining the bacterial subcultures was autoclaved for 20 min at121 �C and at 15 lb pressure before inoculation. The bacteria werethen cultured for 24 h at 36 �C in an incubator. Nutrient agar waspoured into a plate and allowed to solidify. The test compounds(DMSO solutions) were added dropwise to a 10 mm diameter filterpaper disc placed at the center of each agar plate. The plates werethen kept at 5 �C for 1 h then transferred to an incubator main-tained at 36 �C. The width of the growth inhibition zone aroundthe disc was measured after 24 h incubation. Four replicas weremade for each treatment.

2.3. Synthesis

2.3.1. Synthesis of ligand (E)-N-phenyl-2-[phenyl (pyridine-2-yl)methylene]hydrazinecarboxamide (L)

The Schiff base was prepared by condensation of 2-benzoylpyr-idine and 4-phenyl semicarbazide. A solution of 4-phenylsemicar-bazide (10.0 mmol, 1.512 g) in 10 mL ethanol was reflexed with aethanolic solution of 2-benzoylpyridine (10.0 mmol, 1.832 g) con-tinuously for 6 h then 1–2 drops of acetic acid was added. On cool-ing the solution at room temperature pale yellow crystalsseparated which were filtered and washed with methanol. Anal.Calc. for C19H16N4O(L): C, 72.06; H, 5.06; N, 17.70. Found: C,72.08; H, 5.08; N, 17.72%.

2.3.2. Synthesis of [Cu(L)Cl2] (1)To a methanolic solution (20 mL) of copper(II) chloride dihy-

drate (1.0 mmol, 0.170 g) a solution of L (1.0 mmol, 0.316 g) inmethanol was added with constant stirring at ambient temp. Theblue crystal suitable for single crystal X-ray diffraction were recov-ered from mother liquor Anal. Calc. for C19H16Cl2CuN4O (1): C,50.58; H, 3.55; N, 12.42. Found: C, 50.55; H, 3.52; N, 12.45%. FABMass (m/z). Calc.: 451. Found: 450.80.

2.3.3. Synthesis of [Cu(L)Br2] (2)To a methanolic solution (20 mL) of copper(II) bromide

(1.0 mmol, 0.223 g) solution of L (1.0 mmol, 0.316 g) in methanolwas added and stirred well for 1 h. The red crystals suitable for sin-gle crystal X-ray diffraction were recorded from mother liquorAnal. Calc. for C19H16Br2CuN4O (2): C, 42.24; H, 2.965; N, 10.37.Found: C, 42.22; H, 2.93; N, 10.39%. FAB Mass (m/z) Calc.: 540.Found: 539.72.

2.3.4. Crystal structure determinationSingle crystal suitable for X-ray analysis for the complexes

[Cu(L)Cl2] 1 and [Cu (L)Br2] 2 were grown from the slow evapora-tion of the reaction mixtures at room temperature. Single crystalssuitable for single crystal X-ray of 1, and 2 were mounted on a glassfibre and used for data collection. Crystal data were collected onEnraf–Nonius MACH3 diffractometer using graphite monochroma-tized Mo Ka radiation (k = 0.71073 Å). The crystal orientation, cellrefinement and intensity measurements were made using the pro-gram CAD-4PC performing W-scan measurements. The structures

Fig. 2. Projection view of [Cu(L)Br2] (2).

R.N. Patel et al. / Inorganica Chimica Acta 362 (2009) 4891–4898 4893

were solved by direct method using the program SHELXS-97 [24] andrefined by full-matrix least-square techniques against F2 usingSHELXL-97 [25]. All non-hydrogen atoms were refined anisotropi-cally. All the hydrogen atoms were geometrically fixed and allowedto refine using a riding model.

3. Results and discussion

3.1. Synthesis and characterization

The preparation of complexes 1 and 2 can be achieved via the1:1 reaction of respective cupric chloride dihydrate with L in meth-anol. In the same manner complex 2 was obtained using anhydrouscupric bromide instead of cupric chloride dihydrate this procedureenabled us to isolate the complex 1 and 2.

Both complexes are soluble in water, acetonitrile, methanol,and DMF but sparingly soluble in dichloromethane and benzeneand most of the organic solvents. These complexes were character-ized using elemental analysis and further characterized by FAB+

mass spectrometry. The molar conductivity are values in DMSOsolution are very low (i.e. 12 X�1 cm�1 mol�1 for 1 and 9 X�1 cm�1

mol�1 for 2), indicating the non electrolyte nature [26] of thecomplexes.

3.2. Crystal structures

Complexes 1 and 2 have been characterized by the single crystalX-ray diffraction technique. The ORTEP views of the complexes areshown in Figs. 1 and 2. Crystallographic data and structure refine-ment parameters are given in Table 1 and selected bond length andbond angles are listed in Tables 2 and 3 The structures of the thesecomplexes are quite similar. The geometry around copper(II) ioncan be best described as trigonal bipyramidal distorted squarebased pyramid (TBSBP) as indicated by the value of the trigonal in-dex s = 0.14 for 1 and 0.25 for 2 (s = (b � a)/60), where a and b arethe largest coordination angles. The value of s is 1 for perfect trigo-nal bipyramidal geometry and is zero for a perfect square pyrami-dal geometry [27]. All the three donor atoms two nitrogen and oneoxygen atoms (N1N2O1) of the meridionally coordinated ligandoccupy the three corners of the square plane and a chloride(Cl�)/bromide (Br�) at the fourth one, the remaining chloride/bro-mide ions occupying the axial position. As expected the Cu–Cl and

Fig. 1. Projection view of [Cu(L)Cl2] (1).

Cu–Br bond lengths are the longest in the coordination sphere[Cu(1)–Cl(1) = 2.205(3) Å, Cu(1)–Cl(2) = 2.5138(16) Å, for 1 andCu(1)–Br(1) = 2.3488(5) Å, Cu(1)–Br(2) = 2.6544(4) Å for 2] andare comparable to those reported for other mononuclear dihalocopper(II) complexes [28–30]. The Cu–Npy bond distances 2.024and 2.018 Å in complex 1 and 2, respectively are similar and canbe viewed as strongly coordinated to the metal [31] and are alsoin the range of Cu–Npy distance determined for other mononuclearcopper(II) complexes 2. The C(6)–N(2) bond distances (1.291(10) Åfor complex 1 and 1.281(4) Å for complex 2) are close to the theo-retically predicted value of a double bond C=N confirms the forma-tion of the Schiff base.

In complex 2, the molecules are packed in the lattice in an or-dered manner along a/b/c axis. The intermolecular N(3)–H(3 N)� � �Br(2), N(4)–H(4 N)� � �Br(2) and C(2)–H(2)� � �Br(2) as wellas the intramolecular C(1)–H(1)� � �Br(2) and C(19)–H(19)� � �O(1)hydrogen bonds (Table 4) of the neighboring molecules stabilizethe distorted square based pyramidal geometry around copper(II).However, no significant hydrogen bonding interactions are foundin the packing for complex 1. The main difference in the packingis shown by a weak interactions in the crystal structure of complex2 not present in 1.

3.3. Magnetic moment measurements

The magnetic moment measurements in the solid state showthat present complexes are paramagnetic at room temperature.The observed magnetic moments of these complexes are 1.78and 1.89 BM for complexes 1 and 2 respectively. These values sug-gest planarity in copper(II) complexes and characteristics of d9

electronic configuration of Cu(II). These values are quite close tothe values expected for copper(II) complexes [32,33] and is in fairagreement with the spin only system S = 1/2.

3.4. Epr spectra

Epr spectra of complexes 1 and 2 were recorded in polycrystal-line and in solution. Representative epr spectra are shown in Figs. 3and 4. Derived epr parameters are given in Table 5. The epr spectraof polycrystalline samples recorded at room and liquid nitrogen

Table 1Crystal data and structure refinement for complexes [Cu (L)2](Cl)2 1, [Cu(L)2](Br)2 2.

Empirical formula C19H16Cl2CuN4O C19H16Br2CuN4OFormula weight 450.80 539.725Temperature (K) 150(2) 150(2)Wavelength (Å) 0.71073 0.71073Crystal system triclinic monoclinicSpace group P�1 CcUnit cell dimensionsa (Å) 9.2657(10) 11.6199(3)b (Å) 9.7851(7) 13.3819(2)c (Å) 11.5208(16) 13.4763(2)a (�) 104.268(9) 90b (�) 102.792(10) 114.631(2)c (�) 99.696(7) 90V (Å3) 959.32(18) 1904.85(6)Dcalc (Mg/m3) 1.561 1.882Z 2 4Absorption coefficient (mm�1) 1.433 5.359F(0 0 0) 458 1060Crystal size (mm) 0.27 � 0.22 � 0.17 0.33 � 0.27 � 0.21h range for data collection (�) 3.31–25.00 3.33–24.98Maximum and minimum transmission 0.7927 and 0.6982 0.3991 and 0.2708Goodness-of-fit (GOF) on F2 1.187 1.033Final R indices [I > 2r(I)] R1 = 0.0777, wR2 = 0.1686 R1 = 0.0176, wR2 = 0.0424R indices (all data) R1 = 0.0953, wR2 = 0.1765 R1 = 0.0193, wR2 = 0.0428

Table 2Selected bond length (Å) and bond angels (�) of complex 1.

Cu(1)–N(2) 1.965(6) Cu(1)–O(1) 2.081(5)Cu(1)–N(1) 2.024(9) Cu(1)–Cl(1) 2.205(3)Cu(1)–Cl(2) 2.5138(16) O(1)–C(13) 1.248(8)N(1)–C(1) 1.344(10) N(1)–C(5) 1.345(14)N(2)–C(6) 1.291(10) N(2)–N(3) 1.358(7)N(3)–C(13) 1.359(10) N(4)–C(13) 1.342(8)N(4)–C(14) 1.402(10)N(2)–Cu(1)–N(1) 78.5(3) C(5)–N(1)–Cu(1) 114.4(5)N(2)–Cu(1)–O(1) 78.3(2) C(6)–N(2)–Cu(1) 120.4(5)N(1)–Cu(1)–O(1) 154.2(3) N(3)–N(2)–Cu(1) 114.6(5)N(2)–Cu(1)–Cl(1) 162.37(16) N(1)–Cu(1)–Cl(1) 99.0(3)O(1)–Cu(1)–Cl(1) 99.59(17) N(1)–Cu(1)–Cl(2) 98.75(18)N(2)–Cu(1)–Cl(2) 96.85(15) O(1)–Cu(1)–Cl(2) 95.24(13)C(13)–O(1)–Cu(1) 111.8(5) Cl(1)–Cu(1)–Cl(2) 100.78(7)C(1)–N(1)–Cu(1) 125.3(10)

Table 3Selected bond length (Å) and bond angles (�) of complex 2.

Cu(1)–N(2) 1.975(3) Cu(1)–O(1) 2.043(2)Cu(1)–N(1) 2.018(3) Cu(1)–Br(1) 2.3488(5)Cu(1)–Br(2) 2.6554(4) O(1)–C(13) 1.243(4)N(1)–C(1) 1.333(4) N(1)–C(5) 1.345(4)N(2)–C(6) 1.281(4) N(2)–N(3) 1.351(4)N(3)–C(13) 1.379(4) N(4)–C(13) 1.326(4)N(4)–C(14) 1.414(4)N(2)–Cu(1)–N(1) 78.66(10) N(2)–Cu(1)–O(1) 78.21(9)C(6)–N(2)–Cu(1) 119.9(2) N(3)–N(2)–Cu(1) 114.6(2)N(1)–Cu(1)–O(1) 153.96(10) N(2)–Cu(1)–Br(1) 163.00(8)N(1)–Cu(1)–Br(1) 100.02(8) N(2)–Cu(1)–Br(2) 92.80(8)O(1)–Cu(1)–Br(1) 98.60(6) Br(1)–Cu(1)–Br(2) 104.016(16)N(1)–Cu(1)–Br(2) 101.58(7) C(13)–O(1)–Cu(1) 112.9(2)O(1)–Cu(1)–Br(2) 91.33(6) C(5)–N(1)–Cu(1) 114.2(2)C(1)–N(1)–Cu(1) 127.0(2)

Fig. 3. EPR spectra of [Cu(L)Cl2] (1) at 298 K.

Table 4Hydrogen bonding interactions for Cu(II) complexes 1 (Å and �).

D–H� � �A D–H (Å) H� � �A (Å) D� � �A (Å) \D–H� � �A (�)

1 N(3)–H(3 N)� � �Br(2) 0.74(4) 2.92(4) 3.523(3) 140(3)1 N(4)–H(4 N)� � �Br(2) 0.92(4) 2.34(4) 3.245(3) 168(3)Intra 1 C(1)–H(1)� � �Br(1) 0.95 2.91 3.489(3) 1201 C(2)–H(2)� � �Br(2) 0.95 2.93 3.708(3) 140Intra1 C(19)–H(19)� � �O(1) 0.95 2.28 2.886(4) 121

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temperature are in agreement with geometry observed in the X-ray analysis. There is no apparent difference in the epr spectral fea-tures or in spectral parameters. In polycrystalline state, the eprspectra are slightly orthorhombic with gx < gy < and gz (Table 5).In solution both complexes show an axial symmetry (g|| > g\) witha dx2�y2 ground state in copper(II) located in the square basedgeometries [34,35]. The g|| (2.23 for 1 and 2.24 for 2) and A||

(0.0135 for 1 and 0.0121 cm�1 for 2) values observed for these

complexes fall in the range for CuN3O chromophore [36,37] inthe g|| versus A|| plot [27]. This kind of observations suggests thatthe weak axial and even the equatorial chloride ions are possibly

Fig. 4. EPR spectra of [Cu(L)Cl2] (1) in DMSO (0.003 mol�1 dm�3) at 77 K.

Table 5Epr spectral parameters of the copper(II) complexes.

Epr parameter 1 2

Polycrystalline state (298 K)gz 2.110 2.139gy 2.048 2.061gx 2.022 2.020giso/ave 2.06 2.073

DMSO (77 K)g|| 2.233 2.240g\ 2.069 2.076AG (G) 145 130G 3.377 3.156a2 0.676 0.647b2 1.044 1.090c2 1.123 1.220K\ 0.758 0.792K|| 0.705 0.710f 165 185

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replaced by solvent molecules [34] to form the CuN3O chromo-phore in solution. The g|| and A|| values for complex 2 are higherand lower, respectively, than for 1 is retained even in solution,which is consistent with the ligand field spectral results and singlecrystal X-ray structures. The geometric parameter G, which is mea-sure of the exchange of interaction between the copper centers in apolycrystalline solid has been calculated. According to Hathaway[38,39] if G > 4 the exchange interaction is negligible and if G < 4,indicates exchange interaction. The value of G for present com-plexes are 3.38 for 1 and 3.16 for 2 indicating exchange interactionin solid state.

The EPR parameters and d–d transition energies were used toevaluate the bonding parameter a2, b2 and c2, which may be re-garded as measure of the covalency of the in-plane r bondingand the in-plane p- and out-of-plane p bonding, respectively.The in-plane r bonding parameter a2 was calculated by usingthe expression [40]:

a2 ¼ ðAjj=0:036Þ þ ðgjj � 2:0023Þ þ 3=7ðg? � 2:0023Þ þ 0:04

The orbital reduction factors K|| and K\ were estimated from theexpression [41].

K2jj ¼ ðgjj � 2:0023ÞEd-d=8k0

K2? ¼ ðg? � 2:0023ÞEd-d=2k0

where K|| = a2b2, K\ = a2c2 and k0 represents the one electron spin–orbit coupling constant for the free ion, equal to �828 cm�1. Signif-icant information about the nature of bonding in the copper(II)com-plexes can be derived from the magnitude of K|| and K\. In case ofpure r bonding K|| � K\ � 0.77 whereas K|| < K\ implies consider-able in-plane bonding, while for out-of-plane bonding, K|| > K\ Inall the present copper(II) complexes, it is observed that K|| < K\

which indicates the presence of significant in-plane bonding. Theevaluated values of a2, b2 and c2 of the complexes are consistentwith both strong in-plane r and in-plane p bonding. The computedvalue of a2 and b2 (Table 5) are compared with other copper(II)complexes while are ionic in nature [42]. Therefore present com-plexes may be regarded as ionic complexes. The empirical factorf = g||/A|| cm�1 is an index of tetragonal distortions and its valuemay vary from 105–135 for small to extreme distortions in squareplanar complexes and it depends on the nature of the coordinatedatoms [43]. The f values of these complexes are found 165 for 1and 185 for 2, indicating significant distortion from planarity.

3.5. Ligand field spectra

The room temperature ligand field spectra of these complexeshave been recorded in 100% DMSO solution. The electronic spectraof these two complexes are very similar to each other and show alow energy ligand field (LF) band (705 ± 5 nm) and a high energyligand bands (300 and 310 nm) n ? p of pyridine ring in agree-ment with its X-ray structural analysis [44]. The complex alsoshow a band at 400 nm due to Cl–Cu charge transfer band .The li-gand field band energy of complex 1 is lower than that for 2 due tothe higher electronegativity of Cl atoms than Br atoms.

3.6. IR spectra

The IR spectra of the complexes also give expected band shapesand positions. The (>C@N) band of the ligand at 1623 cm�1 is foundto be slightly shifted to lower energies (1624 for 1 and 1618 cm�1

for 2) in the spectra of complexes, indicating the coordination viathe azomethine nitrogen (C@N) [45,46] The bands in the regions508–535 and 280–332 cm�1 are attributed to the (Cu–N) an-d)(Cu–X) (X = Cl/Br) band, respectively [47]. Vibrations at455 cm�1 (weak) can be attributed to M–O bonds [48].

3.7. Electrochemical studies

The electrochemical properties of the two complexes have beenstudied by cyclic voltammetry (CV) under a nitrogen atmospherein DMSO solution in the potential range +1.00 to �9.00 V versusAg/AgCl reference and the results are presented in Table 6. A rep-resentative CV is shown in Fig. 5. The cyclic voltammograms ofthese complexes exhibit only one irreversible CuII/CuI redox couple[49]. The difference in CuII/CuI redox potential between complexes1 and 2 demonstrates that the coordination environments aroundthe copper(II) ions of the two complexes are different and afford asubstantial explanation for the difference in SOD activity of them.It is known that an adequate CuII/CuI redox potential for effectivecatalysis of superoxide radical must be required between�0.405 V versus SCE for O2/O2

� +0.645 V versus SCE for O2�/

H2O2. Though it is inappropriate to compare the E0 values with

Table 7Superoxide dismutase activity of some copper (II) complexes.

S. No. Complex IC50 (lmol dm�1) Ref.

1 Native SOD 0.04 502 [Cu(L*)Cl2] 0.09 512 [Cu(PMDT)(H2O)](ClO4)2 149 523 [Cu(PMDT)(ImH)](ClO4)2 125 524 [Cu(glygly)]�3H2O 132 205 [Cu(glygly)(phen)]�3H2O 32 206 [Cu(glygly)(bipy)]�3H2O 25 207 [Cu(PMDT)(bipy)](ClO4)2 105 118 [Cu(PMDT)(phen)]�(ClO4)2 111 119 [Cu(SAA)H2O] 63 5310 [Cu(SAA)(MeImH)] 35 5311 [Cu(L)Cl2] 22 this work12 [Cu(L)Br2] 18 this work

PMDT = N,N,N0 ,N00 ,N00-pentamethyldiethylenetriamine; glygly = glycylglycine; SAA =salicylidineanthranilic acid, L* = dichloro[(4,5-dihydroimidazole-2-yl)-1H-benzimi-dazole-N,N0].

Table 8Antibmicrobial activity of copper(II) complexes.

Complex (mM) Diameter of inhibition zone(in mm)

Streptococcus aureus E. Coli

[Cu(L)Cl2] 15 6 610 8 1215 10 2020 16 30

[Cu(L)Br2)] 25 4 810 8 1615 10 2020 20 30

Fig. 5. Cyclic voltammogram (0.003 mol�1 dm�3) of [Cu(L)Cl2] (1) in DMSO (0.1 MNaClO4 as supporting electrolyte, scan rate: 200 mV/s).

Table 6Cyclic voltammetric data for 1 mM solution of the Cu(II) complexes in DMSO containing 0.1 M NaClO4 as supporting electrolytes.

Scan rate (mV/s) Epc (mV) Ipc (lA) Epa (mV) Ipa (lA) DEp (mV) E0’ (mV) Ipa/Ipc (lA)

[Cu(L)(Cl)2]100 14.0 6.384 526 1.2543 512 270 0.196200 5 7.0985 549 1.2390 544 277 0.175

[Cu(L)(Br)2]100 132 7.3152 934 20.7279 802 533 2.833200 107 10.8400 951 27.4113 844 529 2.529

DEp = Epa � Epc; E�0 = (Epa + Epc)/2.

4896 R.N. Patel et al. / Inorganica Chimica Acta 362 (2009) 4891–4898

the reduction potentials of the two complexes in DMSO should bein the allowed range of an SOD mimic, since the obtained results ofIC50 shows that both of them have relatively low activity. Tworeduction waves at high negative potential are assigned to thereduction of ligand.

3.8. Superoxide dismutase activity

Biological activities (SOD mimetic activities) of the complexeshave also been measured. The SOD mimetic activities of the pres-ent complexes were examined by the NBT assay [20,21] followingkinetically the reduction of NBT to MF+ at 560 nm. Superoxide wasenzymatically supplied from alkaline DMSO. The count fractioncausing 50% inhibition of NBT reduction is called IC50. Concentra-tion equivalent to one unit of SOD activity (IC50 values), togetherwith the IC50 value of native SOD are given in Table 7. Obtained re-sults of IC50 for present complexes remain in the range 20 ± 2 lM.The observed IC50 values of the present complexes are comparablewith the various reported values for the copper(II) complexes[11,20], but are less active than the native SOD. The difference inIC50 values for two complexes should be ascribed to the evidencediscrepancy in the electronegativity of halo atoms. The good activ-ities of two complexes may be attributed to the flexible L ligand,which is able to accommodate the geometrical change from CuII

to CuI, specially the two labile halogen atoms, which are proposed

to be easily substituted by the substrate O2�

, in the catalytic pro-cess, just like the O2

�, in place of H2O bound to copper site in the

mechanism of dismutation of O2� by native SOD. Complex 2 shows

lower IC50, and exhibits higher SOD activity than other mononu-clear complexes except for two systems including native SOD sofor reported [50–53]. Important factors for SOD like activity are amedium strength donar and the ability of the ligand to accommo-date the reduced copper(I) in a tetrahedral-like environment [54–56].

This reasonable correlation allow us to propose that the higherdissociation of this complex lead of the transition DSBP geometryto the distorted square planer (like tetrahedral), a structure whichis more accessible to the reactions with superoxide [57].

3.9. Antimicrobial activity of complexes against pathogens

Present complexes were evaluated against different species ofbacteria as the test organism in an antimicrobial study. Antimicro-bial assessment of the complexes was tested as a function of con-centration of theses complexes and results are presented in Table8. Four concentration of the complexes were taken, i.e. 5 mM,

0

5

10

15

20

25

30

35

2015105

Concentration (mM)

Dia

met

er o

f in

hib

itio

n z

on

e(m

m)

E.Coli

S.aureus

Fig. 6. Antibmicrobial activity of [Cu(L)Cl2] (1).

0

5

10

15

20

25

30

35

2015105

Concentration (mM)

Dia

met

er o

f in

hib

itio

n z

on

e( m

m)

S.aureus

E.Coli

Fig. 7. Antibmicrobial activity of [Cu(L)Br2] (2).

R.N. Patel et al. / Inorganica Chimica Acta 362 (2009) 4891–4898 4897

10 mM, 15 mM, and 20 mM. Paper disc were prepared and dippedwith the help of these different solution. The susceptibility of the

certain strains of bacteria towards the present copper(II) com-plexes were determined by measuring the size of inhibition diam-eter. The growth inhibitory effects were observed against thefollowing bacterial pathogens, E. coli, Staphylococcus aureus and Sal-monella typhi. Complexes 1 and 2 were tested for their antimicro-bial activity. Both complexes are effective against two micro-organisms, i.e. E. coli and S. aureus. Both the Bacteria are pathogensfor humans, which causes dysentery and food poisoning, respec-tively. Results of these antimicrobial assessments of complexesare presented in Table 8. The area of zone of inhibition is less inthe concentration of 5 mM in both micro-organisms and more in20 mM concentration (Figs. 6 and 7). This kind of observation issuggestive of that both complexes are effective against both patho-gens. In case of complex 1 diameter of inhibition zone (30 mm) ishighest for E. coli. It was noted that complex 1 is more effectiveagainst E. coli than the S. aureus. Similar observations were foundfor complex 2. Both complexes are also tested in S. typhi whichcauses typhoid in humans and the results showed that both com-plexes are also effective against this pathogens. Similar antimicro-bial results were reported by Tarafder et al. [58] and also by ourschool [15,59] on simple copper(II) binary and ternary complexes.

4. Conclusion

Our aim when beginning this work was to design low-molecu-lar weight copper(II) complexes as potential SOD mimics. The crys-tal structures of these complexes have been successfullydetermined and are turn out to be CuN3OCl2 or CuN3OBr2 trigonalbipyramidal distorted square based pyramid (TBSBP) geometryaround copper(II) metal centers. These two complexes exhibit sig-nificant SOD activity being the superior catalysts.

In conclusion a combined magnetic and spectroscopic approachis useful not only to give the complete characterization of the com-plexes, but also to correlate their structural features with biologicalactivity. These complexes can be considered as good model for SODactivity.

Acknowledgements

Our grateful thanks are due to the National Diffraction Facility,X-ray Division, and RSIC (SAIF), IIT Mumbai for single crystal datacollection and epr measurements, respectively. The Head RSIC(SAIF), Central Drug Research Institute, Lucknow is also thankfullyacknowledged for providing analytical and spectral facilities.

Appendix A. Supplementary material

CCDC 698930 and 698931 contain the supplementary crystallo-graphic data for [Cu(L)Cl2] and [Cu(L)Br2)]. These data can be ob-tained free of charge from The Cambridge Crystallographic DataCentre via www.ccdc.cam.ac.uk/data_request/cif. Supplementarydata associated with this article can be found, in the online version,at doi:10.1016/j.ica.2009.07.037.

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