ProtectiWe Effects of Cu(II) Complexes against Free Radicals
brane permeability, nontoxicity, and cost-effectiveness.6 The
search for SOD mimics yielded a variety of chelates of
transition metals such as copper.7-9 In this study, we have
published several papers describing the SOD mimic activity
of copper complexes with N-substituted sulfonamides.10-15
Other pharmacological properties have been reported in detail
for a large number of metal complexes of sulfonamides.16
Scheme 1. N-Substituted Sulfonamides
Numerous indirect assays, such as the nitro blue tetrazo-
lium (NBT) or cytochrome c or brazilin assays, have been
used in attempts to measure the SOD activity of putative
SOD mimics.17-21 However these assays, which typically rely
on a spectrophotometric change of a redox indicator to
measure superoxide levels, cannot kinetically distinguish
between a catalytic dismutation of superoxide and a sto-
ichiometric interaction of superoxide with the putative SOD
mimic. The direct methods to determine the SOD activity,
pulse radiolysis, and stopped-flow kinetic analysis help
elucidate if the complexes are a true catalyst or not, but the
concentration of superoxide anions is higher than that
observed in vivo. In fact, several SOD mimics which showed
very efficient SOD-like activity in vitro failed to do so in
vivo.22-25 Thus far, none of the assays currently employed
can exactly replicate the environment encountered by these
SOD mimics in the cellular moiety. As a consequence we
have proposed a new method based on the protection of the
complexes against oxidative stress over three different strains
of Saccharomyces cereVisiae.11
As a continuation of our research, we describe the
synthesis, structural determination, and spectroscopic proper-
ties of three mononuclear copper complexes with N-
substituted sulfonamides (Scheme 1). A study of the SOD
mimetic activity in vitro and the protection against reactive
oxygen species in vivo of these compounds and two dinuclear
complexes previously synthesized26 is reported.
2. Experimental Section
Materials and Methods. Reagents and solvents were com-
mercially available and were used without further purification.
Elemental analyses (C, N, H, S) were performed on a Carlo Erba
AAS instrument. IR spectra (KBr disks) were obtained using a
Mattson Satellite FT-IR in the range 4000-400 cm-1. Fast atomic
bombardment (FAB) mass spectra were obtained on a VG Autospec
spectrometer with 3-nitrobenzyl alcohol as a matrix. The electro-
spray mass spectra, in positive mode (ESI+), of the compounds
dissolved in dmso:EtOH [1:4] were obtained from an ESQUIRE
3000 Plus (Bruker) ion trap mass spectrometer. Diffuse reflectance
spectra (Nujol mulls) of the complexes were recorded on a
Shimadzu UV-2101 PC spectrophotometer. Electronic paramagnetic
resonance (EPR) spectra obtained at the X-band frequency at room
temperature with a Bruker ELEXSYS spectrometer.
Synthesis of the Ligands. N-2-(4-Methylphenylsulfamoyl)-6-
nitro-benzothiazole (HL1). A mixture containing 1 g of 2-amino-
6-nitrobenzothiazole and 2.5 g of toluene-4-sulfonyl chloride in 6
mL of pyridine was heated at reflux for 1 h. Then, it was added to
10 mL of cold water and stirred for several minutes. A solid was
obtained and it was purified using ethanol.27 Data for compound
N-2-(4-sulfamoyl)-6-nitro-benzothiazole (HL1) (1.786 g, 74%)
found: C, 56.75; H, 2.96; N, 12.03; S, 17.11. C14H11N2S2O2Cl
(6) Dowling, E. J.; Chander, C. L.; Claxson, A. W.; Lillie, C.; Blake, D.
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U. Inorg. Chim. Acta 1994, 47, 853.
(8) Pierre, J. L.; Chautemps, P.; Refaif, S.; Beguin, C.; Marzouki, A. E.;
Serratricel, G. J. Am. Chem. Soc. 1995, 117, 1965.
(9) Batinic´-Haberle, I.; Spasojevic´, I.; Stevens, R. D.; Hambright, P.;
Pedatsur, N.; Okado-Matsumoto, A.; Fridovich, I. Dalton Trans. 2004,
1696.
(10) Gonza´lez-AÄ lvarez, M.; Alzuet, G.; Borra´s, J.; Mac´ıas, B.; Montejo, J.
M.; Garc´ıa-Granda, S. Z. Anorg. Allg. Chem. 2003, 629, 112.
(11) Gonza´lez-Alvarez, M.; Alzuet, G.; Borra´s, J.; del Castillo-Agudo, L.;
Montejo-Bernardo, J. M.; Garc´ıa-Granda, S. J. Biol. Inorg. Chem.
2003, 8, 112.
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Granda, S.; Montejo, J. M. J. Inorg. Biochem. 2004, 98, 189.
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Granda, S. J. Inorg. Biochem. 1995, 60, 219.
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Trans. 1996, 2239.
(15) Casanova, J.; Alzuet, G.; Borra´s, J.; Ferrer, S.; LaTorre, J. A.; Ram´ırez,
J. Inorg. Chim. Acta 2000, 304, 170.
(16) Borra´s, J.; Alzuet, G.; Ferrer, S.; Supuran, C. T. In Metal complexes
of heterocyclic Sulfonamides as Carbonic Anhydrase Inhibitors in
Carbonic Anhydrase. Its Inhibitors and ActiVators; Supuran, C. T.,
Scozzafava, A., Conway, J., Eds.; CRC Press: Boca Raton, 2004.
(17) Beauchamp, C.; Fridovich, I. Anal. Biochem. 1971, 44, 276.
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1992, 267, 86.
(19) Kahn, C. R.; White, M. F. J. Clin. InVest. 1988, 82, 1151.
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(21) Fisher, A. E. O.; Maxwell, S. C.; Naughton, D. P. Inorg. Chem.
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requires C, 56.93; H, 3.14; N, 12.04; S, 16.32, M 349. νjmax/cm-1
:
1550 (thiazole); 1314, 1151 (SO2); 955 (S-N). FAB: m/z 350 (M+).
Solid UV-vis (λmax/nm): 371, 509(sh).
N-2-(Phenylsulfamoyl)-6-chloro-benzothiazole (HL2) and N-2-
(4-Methylphenylsulfamoyl)-6-chloro-benzothiazole (HL3). These
ligands were obtained and characterized as previously reported.10,12
Synthesis of the Complexes. [Cu(L1)2(NH3)2]‚2MeOH (1),
[Cu(L2)2(NH3)2] (2), and [Cu(L3)2(NH3)2] (3). We dissolved 1
mmol of the corresponding benzothiazolesulfonamide in 30 mL of
methanol plus 2 mL of aqueous NH3 (30%). This solution was
added dropwise to 20 mL of a methanolic solution containing 1
mmol of Cu(NO3)2‚3H2O. The resulting mixture was stirred for
several hours. A violet solid was formed and removed by filtration.
After a few days, black crystals for 1 and 3 and violet crystals for
2 were obtained from the filtrate after slow evaporation at room
(23) Riley, D. P. Chem. ReV. 1999, 99, 2573.
(24) Goldstein, S.; Czapski, G. Free Radical Res. Commun. 1991, 12, 13.
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G., Ed.; Elsevier Science Publishers: New York, 1986.
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Inorganic Chemistry, Vol. 44, No. 25, 2005 9425