Comparison of protective effects against reactive oxygen species of mononuclear and dinuclear Cu(II) Complexes with N-substituted benzothiazolesulfonamides

Article abstract of DOI:10.1021/ic050110c

Copper(II) complexes of N-benzothiazolesulfonamides (HL¹ = N-2-(4-methylphenylsulfamoyl)-6-nitro-benzothiazole, HL² = N-2-(phenylsulfamoyl)-6-chloro-benzothiazole, and HL³= N-2-(4-methylphenylsulfamoyl)-6-chloro-benzothiaz

Full text of DOI:10.1021/ic050110c

Inorg. Chem. 2005, 44, 9424−9433
Comparison of Protective Effects against Reactive Oxygen Species of
Mononuclear and Dinuclear Cu(II) Complexes with N-Substituted
Benzothiazolesulfonamides
Marta Gonza´lez-AÄ
lvarez,† Gloria Alzuet,† Joaqu´ın Borra´s,*,† Lucas del Castillo Agudo,‡
Santiago Garc´ıa-Granda,§ and Jose´ Manuel Montejo-Bernardo§
Departamento de Qu´ımica Inorga´nica and Departamento de Microbiolog´ıa y Ecolog´ıa,
Facultad de Farmacia, UniVersitat de Vale`ncia, AVda. Vicent Andre´s Estelle´s s/n,
46100 Burjassot, Spain, and Departamento de Qu´ımica-F´ısica y Anal´ıtica, UniVersidad de OViedo,
AVda. Julia´n ClaVer´ıa 8, 33006 OViedo, Spain
Received January 24, 2005
Copper(II) complexes of N-benzothiazolesulfonamides (HL1
HL2 N-2-(phenylsulfamoyl)-6-chloro-benzothiazole, and HL3
)
)
N-2-(4-methylphenylsulfamoyl)-6-nitro-benzothiazole,
N-2-(4-methylphenylsulfamoyl)-6-chloro-benzothiazole)
)
with ammonia have been synthesized and characterized. The crystal structures of the [Cu(L1)2(NH3)2]
‚
2MeOH (1),
[Cu(L2)2(NH3)2] (2), and [Cu(L3)2(NH3)2] (3) compounds have been determined. Compounds 1 and 2 present a
distorted square planar geometry. In both compounds the metal ion is coordinated by two benzothiazole N atoms
from two sulfonamidate anions and two NH3 molecules. Complex 3 is distorted square-pyramidal. The Cu(II) ion
is linked to the benzothiazole N and sulfonamidate O atoms of one of the ligands, the benzothiazole N of another
sulfonamidate anion, and two ammonia N atoms. We have tested the superoxide dismutase (SOD)-like activity of
the compounds and compared it with that of two dinuclear compounds [Cu2(L4)2(OCH3)2(NH3)2] (4) and [Cu2(L4)2-
(OCH3)2(dmso)2] (5) (HL4
dimeric complexes are better SOD mimics than the monomeric ones. We have also assayed the protective action
provided by the compounds against reactive oxygen species over sod1 mutant of Saccharomyces cerevisiae. In
) N-2-(phenylsulfamoyl)-4-methyl-benzothiazole). In vitro indirect assays show that the
∆
contrast to the in vitro results, the mononuclear compounds were more protective to SOD-deficient S. cerevisiae
strains than the dinuclear complexes.
1. Introduction
Free radicals represent one of the main causes of both
cellular damage and numerous degenerative diseases.1,2
reacts with hydrogen peroxide (H2O2) and nitric oxide
radicals to generate the extremely reactive species hydroxyl
radical (HO•) and peroxynitrite (ONOO-), respectively.4 The
first line of cellular defense is the superoxide dismutases
(SODs), the metalloenzymes which catalyze the dismutation
of superoxide anions into hydrogen peroxide and dioxygen.
Increasing intracellular levels of SOD or administering
exogenous SOD has been investigated for use in protection
against oxidative injury, but the results were disappointing
because, as a protein, SOD has a number of delivery and
stability shortcomings.5 To circumvent such limitations, there
is considerable interest in developing synthetic SOD mimics
that have low molecular weight, biological stability, mem-
A
predominant cellular free radical is an oxygen-derived
species, superoxide anion (O2•-), that is formed by the
leakage of high-energy electrons along mitochondrial electron
transport chains and by a variety of cytosolic and membrane-
bound enzymes, including xanthine oxidase and cytochrome
•-
P450 complexes.3 O2 mediates direct cell damage and
* E-mail: Joaquin.Borras@uv.es. Telephone: 00 34 963544530. Fax:
00 34 963544960.
† Departamento de Qu´ımica Inorga´nica, Universitat de Vale`ncia.
‡ Departamento de Microbiolog´ıa y Ecolog´ıa, Universitat de Vale`ncia.
§ Departamento de Qu´ımica-F´ısica y Anal´ıtica, Universidad de Oviedo.
(1) Haliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and
Medicine, 3rd ed.; Oxford University: New York, 1999.
(2) Simonian, N. A.; Coyle, J. T. Annu. ReV. Pharmacol. Toxicol. 1996,
36, 83.
(4) Beckman, J. S.; Koppenol, W. H. Am. J. Physiol. 1996, 271C, 424.
(5) Weiss, R. H.; Riley, D. P. Therapeutic Aspects of Manganese(II)-
based Superoxide Dismutase Mimics. In Uses of Inorganic chemistry
in Medicine; Farrell N. P, Ed; Royal Society of Chemistry:Cambridge,
1999.
(3) Fridovich, I. Annu. ReV. Pharmacol. Toxicol. 1993, 23, 239.
9424 Inorganic Chemistry, Vol. 44, No. 25, 2005
10.1021/ic050110c CCC: $30.25
© 2005 American Chemical Society
Published on Web 10/22/2005

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.
R. Free Radical Res. Commun. 1993, 18, 291.
(7) Mu¨ller, J.; Felix, K.; Maichle, C.; Lengfelder, E.; Strahle, I.; Weser,
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.
(12) Gonza´lez-AÄ lvarez, M.; Alzuet, G.; Borra´s, J.; del Castillo, L.; Garc´ıa-
Granda, S.; Montejo, J. M. J. Inorg. Biochem. 2004, 98, 189.
(13) Casanova, J.; Alzuet, G.; Borra´s, J.; LaTorre, J.; Sanau, M.; Garc´ıa-
Granda, S. J. Inorg. Biochem. 1995, 60, 219.
(14) Casanova, J.; Alzuet, G.; Borra´s, J.; Carugo, O. J. Chem. Soc., Dalton
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.
(18) Lienhard, G. E.; Slot, J. W.; James, D. E.; Mueckler, M. M. Sci. Am.
1992, 267, 86.
(19) Kahn, C. R.; White, M. F. J. Clin. InVest. 1988, 82, 1151.
(20) White, M. F.; Shaleson, S. E.; Keutmann, H.; Kahn, C. R. J. Biol.
Chem. 1988, 263, 2969.
(21) Fisher, A. E. O.; Maxwell, S. C.; Naughton, D. P. Inorg. Chem.
Commun. 2003, 6, 1205.
(22) Riley, D. P.; Rivers, W. J.; Weiss, R. H. Anal. Biochem. 1991, 196,
344.
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.
(25) Aranovitch, J.; Samuni, A.; Godinger, A.; Czapski, G. In Superoxide
and superoxide dismutase in chemistry, biology and medicine; Rotilio
G., Ed.; Elsevier Science Publishers: New York, 1986.
(26) Gonza´lez-AÄ lvarez, M.; Alzuet, G.; Borra´s, J.; Garc´ıa-Granda, S.;
Montejo, J. M. J. Inorg. Biochem. 2003, 96, 443.
(27) Vogel, A. I. Longman Scientific & Technical, 1989.
Inorganic Chemistry, Vol. 44, No. 25, 2005 9425

Gonza´lez-AÄ lvarez et al.
Table 1. Crystal Data and Structure Refinement for
temperature. Crystals of 1, 2, and 3 were isolated by filtration,
washed with methanol, and dried under vacuum.
[Cu(L1)2(NH3)2]‚2MeOH (1), [Cu(L2)2(NH3)2] (2), and
[Cu(L3)2(NH3)2] (3)
Complex 1 (0.282 g, 65.8%) found: C, 41.72; N, 13.10; H, 4.00;
S, 15.02. C30H34CuN8O10S4 requires C, 41.97; N, 13.06; H, 3.99;
S, 14.94. νmax/cm-1: 1455 (thiazole); 1284, 1143-1126 (SO2); 973
(S-N). Solid UV-vis (λmax/nm): 600. ESI-MS gives a peak at
m/z ) 858. Conductivity measurements in dmso:EtOH (1:4)
solutions: 34.5 µS/cm.
Complex 2 (0.178 g, 47.9%) found: C, 41.91; N, 11.30; H, 2.99;
S, 17.41. C26H22Cl2CuN6O4S4 requires C, 41.90; N, 11.27; H, 2.97;
S, 17.21. νmax/cm-1: 1486-1453d (thiazole); 1255, 1139 (SO2);
974 (S-N). Solid UV-vis (λmax/nm): 312, 559, 684(sh). ESI-MS
gives a peak at m/z ) 771. Conductivity measurements in dmso:
EtOH (1:4) solutions: 30.9 µS/cm.
Complex 3 (0.206 g, 53.5%) found: C, 43.44; N, 10.83; H, 3.46;
S, 16.96. C28H26Cl2CuN6O4S4 requires C, 43.49; N, 10.86; H, 3.38;
S, 16.58.νmax/cm-1: 1483-1451d (thiazole); 1275, 1137 (SO2); 974
(S-N). Solid UV-vis (λmax/nm): 318, 576, 660sh. ESI-MS gives
a peak at m/z ) 745. Conductivity measurements in dmso:EtOH
(1:4) solutions: 24.7 µS/cm.
1
2
3
empirical formula C30H34CuN8- C26H22Cl2CuN6- C28H26Cl2CuN6-
O10S4
858.43
triclinic
P1h
8.041(1)
8.176(1)
14.451(1)
90.904(1)
115.877(1)
92.568(1)
912.51(17)
1
O4S4
745.18
monoclinic
Pa
12.533(1)
12.203(1)
10.466(1)
O4S4
770.95
monoclinic
Pc
12.704(1)
12.275(1)
10.570(1)
formula weight
crystal system
space group
a, Å
b, Å
c, Å
R (deg)
â (deg)
γ (deg)
V, Å3
111.62(1)
105.879(3)
1488.1(2)
2
1585.4(2)
2
1.54184
5.375
Z
λ, Å
1.54184
3.561
1.562
200(2)
0.0474
0.1160
1.54184
5.703
1.663
200(2)
0.0446
0.1216
µ, mm-1
F
calcd, g/cm3
T, K
R1
1.607
150(2)
0.0759
0.1944
wR2
[Cu2(L4)2(OCH3)2(NH3)2] (4) and [Cu2(L4)2(OCH3)2(dmso)2]
(5) [HL4) N-2-(4-Methylbenzothiazole)benzenesulfonamide].
These compounds were obtained and characterized as previously
reported.26
non-hydrogen atoms were anisotropically refined. All hydrogen
atoms were considered as ideal and geometrically placed. All
hydrogen atoms were isotropically refined. Final cycle of full-matrix
least-squares refinement based on 2560 reflections and 243
parameters for complex 1, 2022 reflections and 389 parameters for
complex 2, or 2455 reflections and 407 parameters for complex 3
converged to a final value of R1 (F2 > 2σ(F2)) ) 0.0474 (complex
1), 0.0456 (complex 2), or 0.0759 (complex 3); wR2(F2 > 2σ(F2))
) 0.1160 (1), 0.1222 (2), and 0.1944 (3); R1(F2) ) 0.0601 (1),
0.0486 (2), and 0.774 (3); wR2(F2) ) 0.1252 (1), 0.1237 (2), and
0.1956 (3). Final difference Fourier maps showed no peaks higher
than 0.304 e Å-3 for compound 1, 1.028 e Å-3 for compound 2, or
1.150 e Å-3 for compound 3 nor deeper than -0478 e Å-3 (1),
-0.875 e Å-3 (2), or -0.604 e Å-3 (3).
For the three complexes atomic scattering factors were taken
from the International Tables for Crystallography (Vol. C).32
Geometrical calculations were made using PARST.33 The
crystallographic plots were made using PLATON.34 All calculations
were performed at the University of Oviedo on the Scientific
Computer Center and X-ray group computers.
X-ray Data Collection. [Cu(L1)2(NH3)2]‚2MeOH (1), [Cu-
(L2)2(NH3)2] (2), and [Cu(L3)2(NH3)2] (3). A black crystal, size
0.30 × 0.25 × 0.10 mm, triclinic, space group P1h (determined from
the systematic absences) for complex 1, a black crystal, size 0.22
× 0.16 × 0.10 mm, monoclinic, space group Pa for complex 2, or
a dark violet crystal, size 0.17 × 0.15 × 0.12 mm, monoclinic,
space group Pc for complex 3 was used for the data collection.
Data collection was performed at 200(2) K on a Nonius KappaCCD
single-crystal diffractometer, using Cu-KR radiation (λ ) 1.5418
Å). Crystal-detector distance was fixed at 29 mm, and a total of
760 (1), 670 (2), or 766 (3) images were collected using the
oscillation method (both f and w oscillations were necessary to fill
the Ewald Sphere), with 2° oscillation and 40 s (1 and 2) or 150 s
(3) exposure time per image. Data collection strategy was calculated
with the program Collect.28 Data reduction and cell refinement were
performed with the programs DENZO and SCALEPACK.29 A total
of 7312 (1), 10995 (2), or 13350 (3) reflections were collected
between θ ) 2° and 70°. Unit cell dimensions were determined
from 1286 (1), 2022 (2), or 2212 (3) reflections. Multiple measured
reflections were averaged, Rmerge ) 0.052, resulting in 2568 unique
reflections (hkl range -0 < h < 8, -9 < k < 8, 0 < l < 17), of
In Vitro Evaluation of SOD-like Activity. SOD-like activity
of the complexes 1-5 was determined by using the inhibition by
•-
the complexes of the reaction of O2 with nitro blue tetrazolium
(NBT), following the method published by Oberley and Spitz with
slight modifications.35 Xanthine (1.5 × 10-4 M) and xanthine
oxidase in 50 mM potassium phosphate buffer, pH ) 7.8, are used
to generate a reproducible and constant flux of superoxide anions.
The rate of reduction of NBT (5.6 × 10-5 M) to blue formazane
was followed spectrophotometrically at 560 nm. Data in the absence
of the complex were used as a reference. The rate of NBT reduction
which 2010 are observed with I > 2σ(I) for complex 1, Rmerge
)
0.064, resulting in 2022 unique reflections (hkl range 0 < h < 12,
0 < k < 14, -12 < l < 12), of which 1882 are observed with I >
2σ(I) for complex 2 or Rmerge ) 0.068, resulting in 2455 unique
reflections (hkl range -14 < h < 14, 0 < k < 14, 0 < l < 12), of
which 2353 are observed with I > 2σ(I) for complex 3. Final
mosaicity was 0.659(4)° (1), 0.596(4)° (2), or 0.624(3)° (3). Data
completeness was 98.7% (1 and (2) or 96.0% (3). Intensity-error
ratio for all reflections was 379.4:15.8 (1), 397.4:18.5 (2), or 479.0:
25.8 (3).
A summary of crystallographic data for complexes 1-3 is
collected in Table 1.
Structure Refinement. Crystal structures of 1-3 were solved
by Patterson methods, using the program DIRDIF-96.30 Anisotropic
least-squares refinement was carried out using SHELXL-97.31 All
(30) Beurskens, P. T.; Beurskens, G.; Bosman, W. P.; de Gelder, R.; Garc´ıa-
Granda, S.; Gould, R. O.; Israe¨land, R.; Smits, J. M. M. The DIRDIF-
96 Program System; Crystallography Laboratory, University of
Nijmegen: The Netherlands, 1996.
(31) Sheldrick, G. M. SHELXL-97, A computer program for refinement of
crystal structures; University of Gottingen, 1997.
(32) International Tables for X-ray Crystallography; Kynoch Press: Bir-
mingham (Present distributor: Kluwer Academic Publishers: Dor-
drecht, The Netherlands), 1974
(33) Nardelli, M. Comput. Chem. 1983, 7, 95.
(34) Spek, A. L. PLATON, A Multipurpose Crystallographic Tool; Utrecht
University: Utrecht, The Netherlands, 2000.
(35) Oberley, L. W.; Spitz, D. R. In Handbook of Methods for Oxygen
Radicals Research; Greenwald, R. A., Ed.; CRC Press: Boca Raton,
FL, 1986; p 217.
(28) Nonius, B. V. Collect 1997-2000.
(29) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307.
9426 Inorganic Chemistry, Vol. 44, No. 25, 2005

ProtectiWe Effects of Cu(II) Complexes against Free Radicals
Scheme 2. Diameter of Inhibition around the Paper Disks Containing
Menadione and Hydrogen Peroxide
was progressively inhibited after the addition of copper complex
solutions at increasing concentrations prepared in 50 mM Tris-HCl
buffer (pH ) 7.8) to the system. The percentage inhibition of the
NBT reduction was used as a measure of the SOD activity of the
compounds. In a typical experiment 0.1 mL of xanthine oxidase
was added to 0.8 mL of a solution containing 0.69 mL of potassium
phosphate buffer (pH ) 7.8), 0.025 mL of NBT, and 0.085 mL of
xanthine. The enzymatic system was also tested against a possible
inactivation caused by the copper(II) complexes. To evaluate if the
complexes affect the generation of superoxide anions by directly
interacting with the enzymatic system, the formation of uric acid
from xanthine was followed at 310 nm. The inhibition percentage
of enzyme activity was subtracted from that of NBT. The
concentration of complex required to yield 50% inhibition of NBT
reduction (named IC50) was determined from a plot of percentage
inhibition versus complex concentration. The IC50 data have been
calculated from the mean of three independent measurements.
In Vivo Evaluation of the SOD-like Activity. The in vivo SOD-
like activity of the complexes 1-5 was evaluated over three
different strains of S. cereVisiae: W303-1A, wild type (MATa
ade2-1 ura3-1 his3-11 trp1 leu2-3 leu2-112 can1-100), ATCC96687,
strain defective in the cytoplasmatic copper-dependent SOD (MATa
ura3-52 trp1-289 his3-∆1 leu2-3 leu2-112 sod1::URA3), and
ATCC96688, strain defective in the mitochondrial manganese
dependent SOD (MATa ura3-52 trp1-289 his3-∆1 leu2-3 leu2-112
sod2::TRP1). The strains were obtained from American Type
Culture Collection (ATCC). The protective action of the complexes
against free radicals produced by respiration and against free radicals
generated by menadione or H2O2 was determined.
were incubated at 28 °C for 72 h. Diameters of the inhibition area
(zone without cell growing around the disks) were measured and
were taken as a quantitative estimate of the protective action (see
Scheme 2). The final data have been calculated as the media of
three independent measurements.
Toxicity Assays. Before examination of the protective action
of the compounds against free radicals, their putative toxicity over
the wild, ∆sod1, and ∆sod2 strains of S cereVisiae was determined
following the procedure previously described.11
3. Results and Discussion
3.1. Crystal Structure. The crystal structures with the
atomic numbering scheme of compounds 1, 2, and 3 are
shown in Figures 1, 2, and 3, respectively. Significant bond
lengths and angles are listed in Table 2. In [Cu(L1)2(NH3)2]‚
2MeOH (1) the copper ion is four-coordinated in a slightly
distorted square planar environment. The copper(II) is linked
to the benzothiazole N2 and N2#1 atoms from two sulfon-
amidate anions and to the N4 and N4#1 atoms from two
NH3 molecules. The Cu-Nbenzothiazole distances are 1.975(3)
Å and the Cu-Nammonia ones are 2.003(3) Å. The benzothia-
zole nitrogen atoms are in trans position. The bond angles
in the CuN4 chromophore are nearly regular, ranging from
89.52(12)° to 90.48(12)°. Tetrahedrality, for any tetracoor-
dinate copper complexes, can be characterized by the angle
subtended by two planes, each encompassing the copper and
two adjacent donor atoms.37 For strictly square planar
complexes with D4h symmetry, the tetrahedrality is 0°. For
tetrahedral complexes with D2d symmetry, the tetrahedrality
equals 90°. The value of the dihedral angle for complex 1 is
0°, indicating that the complex geometry is square planar.
Two uncoordinated methanol molecules stabilize the
crystal structure through moderate intermolecular hydrogen
bonds38 between the ammonia nitrogen and the methanol
oxygen [N4‚‚‚O5 3.0228 Å, 142.70°], and between the
methanol oxygen and the sulfonamide oxygen [O5‚‚‚O4
2.8273 Å, 159.32°]. Also, there are some weak hydrogen
bonds between the ammonia nitrogen and the oxygen of the
NO2 group.
Evaluation of the Protective Effect of the Complexes against
Free Radicals Produced by Respiration. Solutions of the com-
plexes 1-5 (30, 50, 70, or 90 µM) in dmso:EtOH (1:4) were added
to 15 mL of melted YPgly medium (1% yeast extract, 2% peptone,
2% glycerol, 1.5% agar, and 2% ethanol), which was kept at 45
°C. Media were poured in Petri plates and allow to solidify at room
temperature. Cell cultures were grown aerobically overnight at 28
°C in stirred liquid YPD reach medium (1% yeast extract, 2%
peptone, and 2% glucose). Drops of 5 µL of the three strains at
decreasing dilutions (10-2, 10-3, 10-4, 10-5, and 10-6) obtained
from an overnight culture were poured in the Petri dishes and were
incubated at 28 °C for 4 days. Untreated cultures were incubated
in parallel. The increased growth of cells found in the presence of
the copper(II) compounds compared with cell growth in the absence
of the complexes was taken as a qualitative estimate of the
protective effect based on serial dilution drops assays. Experiments
were performed three times.
Evaluation of the Protective Effect of the Complexes against
Free Radicals Produced by Oxidative Agent. Yeast cells were
grown in YPD reach medium (1% yeast extract, 2% peptone, and
2% glucose). Solid media contained 1.5% agar. Cell density from
overnight cultures was determined by cell counting in a “Nebauer”
hematimeter. A total of 106 cells were resuspended in 15 mL of
melted solid YPD media, which was kept at 45 °C. Solutions of
the complexes in dmso:EtOH (1:4) at increasing concentrations (30,
50, or 70 µM) were added to the growth medium. Cell suspensions
were poured in Petri dishes and allow to solidify at room
temperature. Paper disks 6 mm in diameter (Antibiotica test
Bla¨ttchen) containing 15 µL of a 40 mM menadione solution in
ethanol or 5 µL of H2O2 (35%) for wild and ∆sod2 strains were
placed over the media. For ∆sod1 strain the paper disks contained
5 µL of a 5 mM menadione solution in ethanol or 5 µL of H2O2
(17.5%). Differences in the amounts of oxidants used depended
on the differences in sensibility of the different strains.36 Cultures
The geometrical environment of the metal ion in [Cu(L2)2-
(NH3)2] (2) is distorted square planar. Cu(II) is coordinated
by four nitrogen atoms: two ammonia nitrogen atoms and
(36) Jamienson, D. J.; Rivers, S. L.; Stephen, W. S. Microbiology 1994,
140, 3277.
(37) Battaglia, L. P.; Bonamartini-Corradi, A.; Marcotrigiano, G.; Menabue,
L.; Pellacani, G. C. Inorg. Chem. 1979, 18, 148.
(38) Jeffrey, G. A. An Introduction to Hydrogen Bonding; Oxford University
Press: Oxford, 1997.
Inorganic Chemistry, Vol. 44, No. 25, 2005 9427

Gonza´lez-AÄ lvarez et al.
Table 2. Selected Bond Lengths (Å) and Angles (°) for [Cu(L1)2(NH3)2]‚2MeOH (1), [Cu(L2)2(NH3)2] (2), and [Cu(L3)2(NH3)2] (3)a
[Cu(L1)2(NH3)2]‚2MeOH [Cu(L2)2(NH3)2] [Cu(L3)2(NH3)2]
Cu1-N2
1.975(3)
1.975(7)
2.003(3)
Cu1-N1
2.014(7)
2.029(7)
2.082(8)
2.217(8)
Cu1-N5
Cu1-N4
Cu1-N2
Cu1-N6
Cu1-O1
N5-Cu1-N4
N5-Cu1-N2
N4-Cu1-N2
N5-Cu1-N6
N4-Cu1-N6
N2-Cu1-N6
N5-Cu1-O1
N4-Cu1-O1
N2-Cu1-O1
N6-Cu1-O1
2.000(9)
2.000(10)
2.023(9)
2.024(8)
2.269(7)
90.0(4)
Cu1-N2#1
Cu1-N4#1
Cu1-N4
Cu1-N21
Cu1-N32
Cu1-N31
N2-Cu1-N2#1
N2-Cu1-N4#1
N2#1-Cu1-N4#1
N2-Cu1-N4
N2#1-Cu1-N4
N4#1-Cu1-N4
180.0
N1-Cu1-N21
N1-Cu1-N32
N21-Cu1-N32
N1-Cu1-N31
N21-Cu1-N31
N32-Cu1-N31
171.8(3)
89.52(12)
90.48(12)
90.48(12)
89.52(12)
180.0
89.7(3)
93.0(3)
90.8(3)
85.4(3)
171.2(3)
89.2(4)
172.5(4)
170.1(4)
88.8(4)
90.7(4)
86.9(3)
101.7(3)
85.7(3)
103.0(3)
a Symmetry transformations #1 -x, -y, -z.
Figure 1. ORTEP drawing of the [Cu(L1)2(NH3)2]‚2MeOH (1).
Figure 2. ORTEP drawing of the [Cu(L2)2(NH3)2] (2).
two benzothiazole nitrogen atoms from the two sulfonamidate
ligands. The Cu(II)-Nammonia coordinative bonds are signifi-
cantly different [2.217(8) and 2.082(8) Å]. The Cu-
Nbenzothiazole lengths [2.014(7) and 2.029(7) Å] are slightly
longer than those obtained for the compound described
before. Distortion of the ideal square-planar geometry can
be inferred from the bond angles that deviate from 90° [85.4-
(3)- 93.9(3)]. The tetrahedrality value of 11.55° suggests a
slight distortion from the square-planar geometry. In the
crystal packing, moderate hydrogen bonds between the
ammonia nitrogen and the sulfonamide nitrogen [N3‚‚‚N2,
3.3186 Å, 140.35°] and between the ammonia nitrogen and
the sulfonamide oxygen [N32‚‚‚O22, 3.0127 Å, 169.61°]
stabilize the structure. The crystal structure of [Cu(L3)2-
(NH3)2] (3) contains a CuN4O entity in slightly distorted
square-pyramidal geometry. In the basal plane the copper-
(II) ion is coordinated by two benzothiazole nitrogen atoms
from two different sulfonamidate ligands at 2.000(9) and
2.000(10) Å and by two ammonia nitrogen atoms at 2.023-
(9) and 2.024(8) Å. The axial site is occupied by a
sulfonamide oxygen atom of one of the sulfonamidate ions
at much longer distance, 2.269(7) Å. The Cu-Nbenzothiazole
lengths are similar and comparable to those found in 1, 2,
and other related compounds.10,12 The cis N-Cu-N angles
in the equatorial plane that range from 88.8(4)° to 90.7(4)°
do not deviate significantly from the ideal ones. The τ value
Figure 3. ORTEP drawing of the [Cu(L3)2(NH3)2] (3).
of 0.04 [τ ) (R - â/60)]39 indicates a small distortion from
a regular square-pyramidal geometry. The T5 parameter
(T ) mean in-plane Cu-L bond distance/mean out-of-plane
Cu-L bond distance)40 has a value of 0.88.
(39) Addison, A. W.; Rao, T. N.; Reedijk, J.; van Rijn, J.; Verschoor, C.
G. J. Chem. Soc., Dalton Trans. 1984, 1349.
9428 Inorganic Chemistry, Vol. 44, No. 25, 2005

ProtectiWe Effects of Cu(II) Complexes against Free Radicals
In complexes 1 and 2 the sulfonamidate anions behave as
monodentate ligands through the benzothiazole nitrogen
atom. By contrast, the sulfonamidate anions in complex 3
present two different coordination behaviors. One sulfon-
amidate ion acts as a monodentate ligand through the
benzothiazole nitrogen and the other as a bidentate ligand
via the benzothiazole nitrogen and the sulfonamide oxygen.
According to the coordination, the distance S1-O1 (1.472-
(8) Å) is markedly longer than the S1-O2 one (1.421(8)
Å). The same behavior of the sulfonamide as monodentate
and bidentate ligands has been found in the related complexes
[Cu(L2)2(dmso)2] and [Cu(L3)2(dmso)2].12
The electrospray data of these solutions show the molec-
ular peak and another peak with a molecular mass corre-
sponding to a species with one ligand and solvent molecule
such as [CuL(NH3)2(solvent)n]+. Moreover, the ESI-MS
spectra of the dinuclear compounds 4 and 5 in dmso:EtOH
(1:4) were obtained. The spectra display peaks at m/z ) 830
and 952, respectively, corresponding to the molecular ones
showing that the complex entities remain in solution.
3.3. Magnetic Properties and EPR Spectra. The room-
temperature magnetic moments of complexes 1 (µeff ) 1.75
µB), 2 (µeff ) 1.73 µB), and 3 (µeff ) 1.78 µB) are consistent
with the presence of a single unpaired electron.
3.2. Spectroscopic Properties. The IR spectra of 1, 2,
and 3 present a similar pattern. With respect to the IR spectra
of the ligands the following modifications are found: (i) the
band corresponding to the stretching vibration of the thiazole
ring is markedly shifted from 1550 cm-1 in the free ligands
to 1455 cm-1 in the complexes, (ii) the ν(SO2)as and ν(SO2)s
bands in the complexes are shifted to lower frequencies, and
(iii) the characteristic band corresponding to the ν(S-N)
appears near 975 cm-1, changed ca. 15-20 cm-1 to higher
frequencies. In general, the IR spectra are similar to those
of other copper N-sulfonamide compounds.10-12,26 Indepen-
dently of the coordination behavior of the ligands, changes
of the IR characteristic bands are found to be the same
because the modifications are mainly due to the sulfonamide
deprotonation.
The diffuse reflectance spectrum of complex 1 shows a
broad and intense band at about 400 nm and a shoulder at
about 600 nm, that correspond to a ligand-to-metal charge-
transfer (LMCT) and d-d transitions, respectively. The
spectrum agrees well with the coordination polyhedron
shown in the crystal structure. The reflectance spectra of
complexes 2 and 3 show a shoulder at 407 (complex 2) and
at 415 (complex 3) nm and a broad band at 556 (complex
2) and at 568 (complex 3) nm that can be attributed to a
LMCT and to a d-d transition, respectively. It is noteworthy
that both complexes present similar spectra despite their
coordination geometry that is distorted square planar in
complex 2 and distorted square-pyramidal in complex 3. This
is probably because of the large Cu-Osulfonamidate bond
distance. So, the fifth coordination atom (Osulfonamidate) has a
slight influence. The difference between the spectra of
complexes 1 and 2 must be due to the high square planar
distortion of the latter as is shown in the crystal structures.
The UV-vis spectra of the three complexes in dmso:EtOH
(1:4) solutions used in the biological assays show a very
weak d-d band at about 600 nm similar to that found in
diffuse reflectance spectra.
The polycrystalline X-band EPR spectrum at room tem-
perature of complex 1 is axial. The EPR parameters obtained
by simulation42 are g| ) 2.27, g ) 2.05, and A| ) 172 ×
10-4 cm-1. The quotient g|/A| is a measure for the degree of
tetrahedral distortion. This quotient ranges from ca. 105 to
135 cm for square planar structures.43 The g|/A| value of 132
cm of compound 1 agrees well with the almost regular square
planar geometry found in the crystal structure.
For complex 2 the EPR spectrum is rhombic. The
parameters calculated by simulation, g3 ) 2.20, g2 ) 2.06,
and g1 ) 2.03, correlate well with the geometrical distortion
found in the crystal structure. The R value44 [R ) (g2 - g1/
g3 - g2)] of 0.2 indicates that the unpaired electron is in the
2
2
dx -y orbital.
The axial spectrum of complex 3 presents values of g| )
2.22 and g ) 2.05, which are characteristic of the geometry
deduced from crystallographic data.
3.4. SOD-like Activity. In Vitro Superoxide Dismutase-
like Activity. The superoxide dismutase activity of the
monomeric complexes 1-3 and the dimeric complexes [Cu2-
(L4)2(OCH3)2(NH3)2] (4) and [Cu2(L4)2(OCH3)2(dmso)2] (5)
[HL4 ) N-2-(4-methylbenzothiazole)benzenesulfonamide]
previously reported26 was assayed by their ability to inhibit
the reduction of nitro blue tetrazolium. By way of compari-
son, the activity of CuCl2‚2H2O was assayed under the same
conditions. The superoxide scavenging data indicate that
complexes 1-3 exhibit high activity, similar to those of
complexes with related benzothiazole sulfonamide ligands
and higher than the activity of copper sulfathiazole complexes
(Table 3).10-15 Their IC50 values are 0.244((0.041), 0.181-
((0.002), and 0.182((0.010) µM, respectively. Complexes
4 and 5 clearly show higher superoxide dismutase activity
[IC50 ) 0.103((0.007) and 0.059((0.001] µM, respectively)
compared with the other three complexes. It is interesting
to note that the activity exhibited by complexes 4 and 5 is
only 17 and 10 times, respectively, less than that of the native
enzyme. These results seem to indicate that the dimer nature
increases the ability of the compounds to dismutate the
superoxide radical. In fact, the most active compounds in
vitro found in the literature are dinuclear complexes.45 In
The conductivity measurements of the three complexes
in dmso:EtOH (1:4) solution suggest that the three complexes
are partially dissociated, showing conductivity values be-
tween nonelectrolyte and 1:1 electrolyte types.41 These values
are indicative of a mixture of the complex and a species in
which one of the ligands was dissociated.
(42) WINEPR-Simfonia. 1.25; Bruker Analytik GmgH: Karlsruhe, FRG,
1994-1996.
(43) Sakaguchi, U.; Addison, W. A. J. Chem. Soc., Dalton Trans. 1978,
600.
(44) Hathaway, B. J.; Duggan, M.; Murphy, A.; Mullane, J.; Power, C.;
Walsh, A.; Walsh, B. Coord. Chem. ReV. 1981, 36, 267.
(40) Matovic´, Z. D.; Pelosi, G.; Ianelli, S.; Ponticelli, G.; Radanovic´, D.
D.; Radanovic´, D. J. Inorg. Chim. Acta 1998, 268, 221.
(41) Geary, Coord. Chem. ReV. 1971, 7, 81.
Inorganic Chemistry, Vol. 44, No. 25, 2005 9429

Gonza´lez-AÄ lvarez et al.
Table 3. IC50 (µM) Values of Copper Complexesa
determined by a previously reported method.11 The results
show that complexes do not present significant toxicity at
the assayed concentrations (30, 50, 70, and 90 µM) (data
not shown).
complex
IC50
ref.
[Cu(L1)2(NH3)2]‚MeOH (1)
[Cu(L2)2(NH3)2] (2)
[Cu(L3)2(NH3)2] (3)
[Cu2(L4)2(OCH3)2(NH3)2] (4)
[Cu2(L4)2(OCH3)2(dmso)2] (5)
[Cu(L2)2(dmso)2]
0.244
0.181
0.182
0.103
0.059
0.188
0.344
0.146
0.172
1.310
2.510
0.664
0.742
1.03
this work
this work
this work
this work
this work
12
To quantify the in vivo SOD-like activity of the Cu(II)
complexes we have employed a method recently developed
by our research group, based on the protection provided by
the compounds against free radicals over S. cereVisiae strains
(the copper defective ∆sod1 mutant and the manganese
defective ∆sod2 mutant).11 The protective effect of the
complexes against both free radicals produced in the respira-
tion and against free radicals generated by oxidative agents
(hydrogen peroxide or menadione) has been tested.
Complex Protection against Endogenous Free Radicals.
A low level of superoxide radicals is constantly generated
by aerobic respiration. The electron-transport chain of
mitochondria, which is meant to escort four electrons to
molecular oxygen to form water, occasionally leaks a single
electron and it is a constant source of radicals. Free radical
reactions produced death in cells due to their ability to
damage a large number of cellular constituents, especially
the defective SOD cells which are more sensitive.
[Cu(en)2](L3)2
10
11
11
13
13
14
15
15
[Cu(L4)2(py)2]
[Cu(L5)2(py)2]
[Cu(stz)(py)3Cl]
[Cu(Hstz)(MeOH)Cl2]
[Cu(stz)2(Him)2]MeOH
[Cu(stz)2(4,4-dmHim)2]
[Cu(stz)2(1,2-dmHim)2]
[Cu(stz)2(4-mHim)2]
Cu2Zn2SOD
0.586
0.006
15
11
a Abbreviations: HL1 ) N-2-(4-methylphenylsulfamoyl)-6-nitro-ben-
zothiazole; HL2 ) N-2-(phenylsulfamoyl)-6-chloro-benzothiazole; HL3 )
N-2-(4-methylphenylsulfamoyl)-6-chloro-benzothiazole; HL4 ) N-2-(phen-
ylsulfamoyl)-4-methyl-benzothiazole; HL5 ) N-2-(naphthalenesulfamoyl)-
6-nitro-benzothiazole; Hstz ) sulfathiazole (4-amino-N-2-thiazolylben-
zenesulfonamide); Him ) imidazole; 4,4-dmHim ) 4,4-dimethylimidazoline;
1,2-dmHim ) 1,2 dimethylimidazole; 4-mHim ) 4-methylimidazole;
py ) pyridine.
this sense, nuclearity could play an important role in the
SOD-like activity exhibited by our complexes in vitro. The
IC50 values of the five complexes are lower than that of
Cu(II) [IC50 ) 0.450((0.015) µM], indicating that their
activity is not due to a dissociation of the complexes.
It has been found that a more distorted geometry leads to
higher SOD activity.46 In good agreement with this, complex
1 (that has the less distorted copper environment) presents
the lower activity.
To evaluate the protective effect of complexes 1-5 against
free radicals generated in respiration, three strains of S.
cereVisiae, wild, ∆sod1, and ∆sod2, were grown in a medium
with glycerol, which is a nonfermentable carbon source for
S. cereVisiae. Figure 4 shows the growth of dilutions 10-2,
10-3, 10-4, 10-5, and 10-6 (obtained from an overnight
culture) of the three strains treated with increasing concentra-
tions of complexes 1-5.
As expected, the growth of the wild strain is not
significantly modified by the complexes because this strain
keeps both mitochondrial MnSOD and cytoplasmatic Cu2-
Zn2SOD enzymes (Figure 4). Similar behavior is observed
in the ∆sod2 strain because this strain contains the cyto-
plasmatic Cu2Zn2SOD enzyme.
All the complexes are able to protect the ∆sod1 strain.
The protective effect of the complexes on the ∆sod1 strain
is found to be significant and much higher than the copper
salt protective effect. In particular, 70 and 90 µM concentra-
tions of complex 1 produce an almost complete recuperation
of ∆sod1 strain and its growth is indistinguishable from that
of the wild strain. The activity of complexes 2-5 (similar
to that of the previously reported complexes)11,12 is lower
than that of complex 1 but higher than the CuCl2 activity. It
is remarkable that the protective effect of the dimeric
complexes 4 and 5 is not higher than that of the monomeric
ones, although their activities, determined by the indirect
method, were more elevated.
From these results we can conclude that the five complexes
are able to penetrate into the cells and protect them against
the free radicals produced in respiration processes by
compensation for the Cu2Zn2SOD. In particular, complex 1,
which is the most effective, can be considered a promising
protective agent against toxicity of oxidative stress.
Complex Protection against Exogenous Free Radicals.
To evaluate the protective effect of complexes 1-5 against
exogenous free radicals, the ∆sod1 strain was grown in a
Also, it is remarkable that, despite the different coordina-
tion polyhedra of compounds 2 and 3, their IC50 values are
almost coincident. The similar SOD activity of the complexes
can be explained considering that both complexes present
limited steric hindrance and vacant coordination sites that
•-
permit the access and the successful binding of the O2
radical. Moreover, a favorable response of π-electrons of
the aromatic side chain could stabilize the Cu-O2•- interac-
tions. Furthermore, the binding of the sulfonamide to copper-
(II) is important to obtain complexes with high SOD activity
because previous related complexes10 in which the sulfon-
amide acts as a contra-ion presented lower activity than the
complexes presented in this work (Table 3).
In Vivo SOD-like Activity. As has been shown the
synthesized complexes exhibit high SOD activity measured
in vitro. However, the ability to dismutate the superoxide
anions in vitro is not enough to be a SOD mimic in vivo.
Assays with mutants of the yeast S. cereVisiae were carried
out to determine the SOD activity in a biological system.
Previously, the presence of the complexes in solution was
confirmed by ESI-MS as described in section 3.2.
Before evaluation of the in vivo superoxide dismutase
mimetic activity of the complexes, their putative toxicity was
(45) Tabbi, G.; Driessen, W. L.; Reedijk, J.; Bonomo, R. P.; Veldman, N.;
Spek, A. L. Inorg. Chem. 1997, 36, 1168.
(46) Bonomo, R. P.; Conte, E.; Impellizzeri, G.; Pappalardo, G.; Purrello,
R.; Rizzarelli, E. J. Chem. Soc., Dalton Trans. 1996, 3093.
9430 Inorganic Chemistry, Vol. 44, No. 25, 2005

ProtectiWe Effects of Cu(II) Complexes against Free Radicals
Figure 4. Effect of complexes 1-5, and CuCl2 on the growth of the Saccharomyces cereVisiae against free radicals produced by respiration. In each panel
the first column represents a wild strain; the second column represents the ∆sod1 mutant; the third column represents the ∆sod2 mutant. In each column;
strains are shown at decreasing dilutions: 10-2, 10-3, 10-4, 10-5, and 10-6
.
culture containing glucose. In this medium the initial
metabolic pathway is fermentation. The source of free
radicals is the oxidative stress inducers, menadione or H2O2.
Menadione (paper disks at the top of each Petri dish) (see
This result clearly suggests that the complexes provide
protection against the oxidative stress generated by both
menadione and H2O2. This protective effect does not depend
significantly on complex concentration. The protective action
•-
•-
Scheme 2) generates O2 and H2O2 (paper disks at the
against O2 produced by menadione is higher than the
•
•
bottom) produces OH. The results are given in Figure 5
protection against OH generated by H2O2. Of special
which shows the growth inhibition area (black region around
the disk) of the ∆sod1 strain produced by the oxidative agents
with different concentrations of complex 1-5. By compari-
son, both the wild and the ∆sod2 mutant strains, which share
the copper-dependent SOD, were also studied (data not
shown).
In the presence of the five complexes, a growth increase
is observed for the ∆sod1 strain (smaller diameter of the
inhibition area) at 30, 50, and 70 µM complex concentrations.
relevance is that complexes 4 and 5 do not have a higher
activity than the other complexes assayed, in contrast to the
expectation from their IC50 values obtained in vitro.
These assays have been performed with CuCl2 under the
same conditions. In the presence of CuCl2 it is possible to
observe a reduction of the diameter of the inhibition area
for the ∆sod1 strain but this reduction is lower than that in
the presence of the complexes. These results are summarized
in Figure 6 that represent the percent of reduction of the
Inorganic Chemistry, Vol. 44, No. 25, 2005 9431

Gonza´lez-AÄ lvarez et al.
Figure 5. Effect of complexes 1-5 on the growth of the ∆sod1 mutant against free radicals produced by menadione (paper disk at the top of each Petri
dish) and H2O2 (paper disk at the bottom of each Petri dish). For each photograph, 1 is the control; 2 is the 30 µM complex; 3 is the 50 µM complex; 4 is
the 70 µM complex.
diameter of the growth inhibition area produced by the five
complexes at 50 µM and by CuCl2 at the same concentration
when the oxidative stress is produced by menadione. It is
possible to deduce that the protective effect of the complexes
is higher than the protection produced by the copper salt.
Also, it is noteworthy that the reduction of the inhibition
diameter produced by complex 2 or 3 is double that of
copper(II).
strains (data not shown), since both strains have the copper-
dependent SOD.
The results obtained from these assays clearly demonstrate
that, at least in the range of the concentrations tested, the
complexes 1-5 show SOD-like activity in vivo higher than
that of Cu(II). The protective action of 1-5 is similar to
that shown by previously reported complexes with related
sulfonamide ligands.11,12
As expected, no protection effect of the five complexes
or CuCl2 was observed in either the wild type or the ∆sod2
In conclusion, the dimer complexes are more effective than
the monomer ones in vitro. However, the in vivo activity of
9432 Inorganic Chemistry, Vol. 44, No. 25, 2005

ProtectiWe Effects of Cu(II) Complexes against Free Radicals
cellular structures, enzymatic activity, detoxifying mecha-
nisms, etc.) must be involved in the activity of our
complexes. In particular, we consider that the lower activity
of the dinuclear compounds in respect to the mononuclear
ones is due to their bulky size that hinders them, to some
extent, from crossing the cellular membrane.
Acknowledgment. J.B. and G.A. acknowledge financial
support from the Spanish CICYT (BQU2001-3173-C02-01
and CTQ2004-03735/BQU). M.G.-A. wishes to thank
Fundacio´n Ana y Jose´ Royo from Valencia (Spain) for a
postdoctoral fellowship. J.M.M.-B. and S.G.-G. are grateful
for financial support from CICYT (BQU2003-0593).
Figure 6. Percentage reduction of the diameter of the growth inhibition
area of 50 µM complexes 1-5 (dark color), and CuCl2 (light color) when
oxidative stress was produced by menadione.
Supporting Information Available: CIF file for the structure
of the compound. This material is available free of charge via the
Internet at http://pubs.acs.org.
the dimer complexes is lower than that of the monomer ones.
Therefore, it could be deduced that in vivo some physiologi-
cal factors (lipophility, ability of the complexes to cross the
IC050110C
Inorganic Chemistry, Vol. 44, No. 25, 2005 9433