Toward a novel metal-based chemotherapy against tropical diseases. 6. Synthesis and characterization of new copper(II) and gold(I) clotrimazole and ketoconazole complexes and evaluation of their activity against Trypanosoma cruzi.

Article abstract of DOI:10.1021/ic0103087

The complexes [Cu(CTZ)(4)]Cl(2).2H(2)O (1), [Cu(CTZ)Cl(2)](2) (2), [Cu(KTZ)(3)Cl(2)] (3), and [Cu(KTZ)Cl(2)](2).2H(2)O (4) were prepared by reaction of CuCl(2) with CTZ and KTZ (where CTZ = 1-[[(2-chlorophenyl)diphenyl]methyl]-1H-imidazole and KTZ = cis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine), respectively, in acetonitrile at different ligand to metal molar ratios. Gold complexes [Au(PPh(3))(CTZ)]PF(6) (5) and [Au(PPh(3))(KTZ)]PF(6).H(2)O (6) were synthesized by reaction of AuClPPh(3), with KPF(6) and CTZ or KTZ in acetonitrile. All the new compounds were characterized by NMR spectroscopy and microanalytical methods, and for the paramagnetic species EPR spectroscopy and DC magnetic susceptibility measurements were also employed. The solid-state structure of 1 has been determined by X-ray crystallography. 1 crystallizes in the triclinic space group P(-)1, with a = 12.773(2) A, b = 15.326(4) A, c = 11.641(2) A, V = 1957.4(7) A(3), Z = 1, and D(calcd) = 1.284 g/cm(3). The structure refinement converged at R1 = 0.0731 and wR2 = 0.1962. Complex 1 displayed a square-planar structure typical for tetrakis(imidazole)copper(II) complexes. The new compounds were tested for in vitro activity against cultures of epimastigotes of Trypanosoma cruzi, the causative agent of Chagas disease. At concentrations equivalent to 10(-6) M of total CTZ or KTZ (in DMSO) all the complexes exhibited significantly higher growth inhibitory activity than their respective parental compounds.

Full text of DOI:10.1021/ic0103087

Inorg. Chem. 2001, 40, 6879-6884
6879
Toward a Novel Metal-Based Chemotherapy against Tropical Diseases. 6. Synthesis and
Characterization of New Copper(II) and Gold(I) Clotrimazole and Ketoconazole Complexes
and Evaluation of Their Activity against Trypanosoma cruzi
Maribel Navarro,*,† Efren J. Cisneros-Fajardo,† Teresa Lehmann,†
Roberto A. Sa´nchez-Delgado,*,† Reinaldo Atencio,† Pedro Silva,‡ Renee Lira,§ and
Julio A. Urbina§
Chemistry, Physics, and Biophysics and Biochemistry Centers, Instituto Venezolano de Investigaciones
Cient´ıficas (IVIC), Apartado 21827, Caracas 1020-A, Venezuela
ReceiVed March 21, 2001
The complexes [Cu(CTZ)4]Cl2‚2H2O (1), [Cu(CTZ)Cl2]2 (2), [Cu(KTZ)3Cl2] (3), and [Cu(KTZ)Cl2]2‚2H2O (4)
were prepared by reaction of CuCl2 with CTZ and KTZ (where CTZ ) 1-[[(2-chlorophenyl)diphenyl]methyl]-
1H-imidazole and KTZ ) cis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-
4-yl]methoxy]phenyl]piperazine), respectively, in acetonitrile at different ligand to metal molar ratios. Gold
complexes [Au(PPh3)(CTZ)]PF6 (5) and [Au(PPh3)(KTZ)]PF6‚H2O (6) were synthesized by reaction of AuClPPh3,
with KPF6 and CTZ or KTZ in acetonitrile. All the new compounds were characterized by NMR spectroscopy
and microanalytical methods, and for the paramagnetic species EPR spectroscopy and DC magnetic susceptibility
measurements were also employed. The solid-state structure of 1 has been determined by X-ray crystallography.
1 crystallizes in the triclinic space group P1h, with a ) 12.773(2) Å, b ) 15.326(4) Å, c ) 11.641(2) Å, V )
1957.4(7) Å3, Z ) 1, and Dcalcd ) 1.284 g/cm3. The structure refinement converged at R1 ) 0.0731 and wR2 )
0.1962. Complex 1 displayed a square-planar structure typical for tetrakis(imidazole)copper(II) complexes. The
new compounds were tested for in vitro activity against cultures of epimastigotes of Trypanosoma cruzi, the
causative agent of Chagas disease. At concentrations equivalent to 10-6 M of total CTZ or KTZ (in DMSO) all
the complexes exhibited significantly higher growth inhibitory activity than their respective parental compounds.
Introduction
In our search for new drugs that combine a high activity with
a low toxicity, we have been using a novel strategy based on
the fact that coordination of organic compounds such as CTZ
and KTZ, with known anti T. cruzi activity, to transition metals,
such as Ru, Rh, Cu, Au, and Pt, may result in a remarkable
enhancement of the biological activity.3
Trypanosoma cruzi is the causative agent of Chagas disease,
an illness which currently afflicts over 17 million people in Latin
America and ranks as the third largest parasitic disease
worldwide after malaria and schistosomiasis.1 Despite recent
achievements in the understanding of the biochemistry of T.
cruzi,2 specific chemotherapy of Chagas disease is still very
unsatisfactory as currently available drugs have very limited
activity in the prevalent chronic phase of the disease and have
common toxic side effects, which can lead to discontinuation
of treatment.1,2
* Corresponding authors. Phone: (58-212) 5041642. Fax: (58-212)
5041350. E-mail address: mnavarro@ivic.ve.
† Chemistry Center.
‡ Physics Center.
§ Biophysics and Biochemistry Center.
(1) World Bank, World DeVelopment Report 1993: InVesting in Health;
Oxford University Press: Oxford, U.K., 1993.
(2) (a) Urbina, J. A.; Payares, G.; Molina, J.; Sanoja, C.; Liendo, A.;
Lazardi, K.; Piras, M. M.; Piras, R.; Pe´rez, N.; Wincker, P.; Ryley J.
F.; Science 1996, 273, 969. (b) Urbina, J. A.; Lazardi, K.; Aguirre,
T.; Piras, M. M.; Piras, R. Antimicrob. Agents Chemother. 1998, 32,
1237. (c) Lazardi, K.; Urbina, J. A.; De Souza, W. Antimicrob. Agents
Chemother. 1990, 34, 2097. (d) Urbina, J. A.; Lazardi, K.; Aguirre,
M.; Piras, M. M.; Piras, R. Antimicrob. Agents Chemother. 1991, 35,
730. (e) Urbina, J. A.; Lazardi, K.; Marchan, E.; Visbal, G.; Aguirre,
M.; Piras, M. M.; Piras, R.; Maldonado, R. A.; Payares, G.; De Souza,
W. Antimicrob. Agents Chemother. 1993, 7, 580. (f) Maldonado, R.
A.; Molina, J.; Payares, G.; Urbina, J. A. Antimicrob. Agents
Chemother. 1993, 37, 1353. (g) Urbina, J. A.; Vivas, J.; Visbal, G.;
Contreras, L. M.; Mol. Biochem. Parasitol. 1995, 73, 199. (h) Urbina,
J. A.; Vivas, J.; Lazardi, K.; Molina, J.; Payares, G.; Piras, M. M.;
Piras, R. Chemotherapy 1996, 42, 1294.
In this paper, we report a new series of copper and gold
complexes of these two ligands. Various copper complexes have
10.1021/ic0103087 CCC: $20.00 © 2001 American Chemical Society
Published on Web 12/06/2001

6880 Inorganic Chemistry, Vol. 40, No. 27, 2001
Navarro et al.
respect to Cu). Anal. Calcd for C88.66H69.32Cl7.32N8Cu: C, 67.82; H,
4.42; N, 7.14. Found: C, 67.76; H, 5.08; N, 6.69. IR (cm-1): ν(CdC)
1492, ν(CdN) 1443. λmax (CHCl3, 10-3 M) ) 290 nm, 741 nm.
been reported to inhibit bacterial, fungal, yeast, algal, myco-
plasma, and viral growth.4 Copper derivatives have also been
employed in the treatment of inflamation.5 Gold complexes in
turn have long been used in the treatment of various other
diseases such as rheumatoid arthritis (aurothiomalate and
aurothioglucose complexes) and tuberculosis (cyanide and
thiosulfate derivatives).6 The complex auranofin [(s)-2,3,4,5-
tetraacetyl-1-â-O-thioglucose(trietilphosphine)gold(I)] has also
proved to be active against P388 lukemia.7 In contrast, the
potential of copper and gold derivatives as antiparasitic agents
has so far been little explored.6f,9
In our laboratory we have found that copper-chloroquine8
and gold-chloroquine9 complexes are active against malaria
parasites. Continuing our efforts to develop new anti T. cruzi
agents, we now report the coordination of CTZ and KTZ to
copper and gold ions to yield new Cu(II)-CTZ and Cu(II)-
KTZ as well as Au(I)-CTZ and Au(I)-KTZ complexes. The
characterization of these complexes was achieved through NMR,
EPR, ødc, IR, UV-vis, elemental analysis, and, in the case of
1, also an X-ray diffraction study. The biological activities of
these complexes against the epimastigote form of T. cruzi were
also evaluated indicating that all of them inhibit the proliferation
of the parasite. The results are compared with other previously
reported metal-CTZ, and -KTZ complexes.3
[Cu(CTZ)Cl2]2 (2). CuCl2 (52 mg; 0.387 mmol) was dissolved in
acetonitrile (15 mL), CTZ (131 mg; 0.380 mmol) was added to the
solution which was stirred at room temperature for 1 h. The volume of
the solvent was reduced until precipitation began, and the mixture was
allowed to stand overnight at -10 °C, after which the green solid was
obtained was filtered off, washed with diethyl ether, and dried under
vacuum (yield, 123 mg, 66%). Anal. Calcd for C44H34Cl6N4Cu2: C,
55.06; H, 3.55; N, 5.84. Found: C, 55.29; H, 3.72; N, 5.65. IR (cm-1):
ν(CdC) 1491, ν(CdN) 1448. λmax (CHCl3, 10-3 M) ) 241 nm, 293
1
nm. H NMR (CDCl3; δ, ppm): 46.5 (s, 1H); 40.1 (s, 1H); 38.0 (s,
1H).
[Cu(KTZ)3Cl2] (3). CuCl2 (50 mg; 0.370 mmol) was dissolved in
acetonitrile (15 mL). KTZ (810 mg; 1.53 mmol) was added to the
solution which was stirred at room temperature under a nitrogen
atmosphere for 1 h. The volume of the solution was reduced by 70%.
Ethanol was added, and the mixture was allowed to stand overnight at
-10 °C. The blue solid obtained was filtered off, washed with diethyl
ether, and dried under vacuum (yield, 414 mg, 65%). Anal. Calcd for
C78H84Cl4N12O12Cu: C, 53.50; H, 5.14; N, 9.60. Found: C, 53.65; H,
4.97; N, 9.49. IR (cm-1): ν(CdC) 1510, ν(CdN) 1439. λmax (CHCl3,
10-3 M) ) 299, 656, 796 nm.
[Cu(KTZ)Cl2]2·2H2O (4). CuCl2 (22 mg; 0.15 mmol) was dissolved
in freshly distilled acetonitrile (10 mL). KTZ (80 mg; 0.15 mmol) was
added to the solution, which was stirred and kept under reflux in a
nitrogen atmosphere for 1 h. The solvent was removed under vacuum,
and the red solid obtained was redissolved in acetone (3 mL). The
solution was filtered and precipitated with diethyl ether, and the mixture
was allowed to stand overnight at -10 °C. The red solid obtained was
filtered off, washed with ether diethyl, and dried under vacuum (yield,
63 mg, 61%). Anal. Calcd for C52H56Cl4N8O8Cu2‚2H2O: C, 45.64; H,
4.39; N, 8.19. Found: C, 45.44; H, 4.05; N, 7.87. IR (cm-1): ν(CdC)
Experimental Section
General Procedure. Solvents of analytical grade were distilled from
appropriate drying agents prior to use. CTZ, KTZ, AuClPPh3, and CuCl2
were used as obtained without purification. Elemental analyses were
performed at the Chemistry Center (IVIC) on a Fisons analyzer EA
1108. IR spectra were recorded on a Nicolet 5DCX FT spectrometer,
while UV-vis spectra were recorded on a HP 8453 diode array
instrument using chloroform solutions (10-3 M) of the complexes.
[Cu(CTZ)4]Cl2·0.66CH2Cl2 (1). CuCl2 (332 mg; 2.47 mmol) was
dissolved in acetonitrile (35 mL), and CTZ (1.629 mg; 4.97 mmol)
was added to the solution which was stirred under reflux in a nitrogen
atmosphere for 1 h. The volume of the solvent was reduced until
precipitation began, and the mixture was allowed to stand overnight at
-10 °C, after which the blue solid obtained was filtered off, washed
with diethyl ether, and dried under vacuum (yield, 1550 mg, 41% with
1
1510, ν(CdN) 1435. λmax (CHCl3, 10-3 M) ) 262 nm, 485 nm. H
NMR (CDCl3; δ, ppm): 46.5 (s, 1H); 39.8 (s, 1H); 37.0 (s, 1H).
[Au(CTZ)(PPh3)]PF6 (5). A suspension of AuClPPh3 (100 mg; 0.20
mmol) in acetonitrile (25 mL) was refluxed under nitrogen until
complete dissolution. KPF6 (273 mg; 1.48 mmol) was added, and
refluxing was continued for 2 h. CTZ (108 mg; 0.31 mmol) was added.
The mixture was stirred and refluxed for 24 h and then cooled to room
temperature and filtered through Celite. The volume of the solvent was
reduced under a nitrogen stream, and diethyl ether was added until the
solution became turbid. On cooling of the sample to -10 °C overnight,
a white product precipitated. It was filtered off, washed with water
and diethyl ether, and dried under vacuum (yield, 46 mg, 24%). Anal.
Calcd for C40H32ClF6N2P2Au: C, 47.0; H, 3.92; N, 2.74. Found: C,
46.9; H, 3.34; N, 2.73. IR (cm-1): ν(CdC)1500, ν(CdN) 1435, ν(P-
(3) (a) Sa´nchez-Delgado, R. A.; Lazardi, K.; Rinco´n, L.; Urbina, J. A.;
Hubert, A. J.; Noels, A. F. J. Med. Chem. 1993, 36, 2041. (b) Sa´nchez-
Delgado, R. A.; Navarro, M.; Lazardi, K.; Atencio, R.; Capparelli,
M.; Vargas, F.; Urbina, J. A.; Bouillez, A.; Hubert, A. J.; Noels, A.
F. Inorg. Chim. Acta 1998, 275-276, 528-540. (c) Navarro, M.;
Lehmann, T.; Cisneros-Fajardo, E. J.; Fuentes, A.; Sanchez-Delgado,
R. A.; Silva, P.; Urbina, J. A. Polyhedron 2000, 19, 2319-2325.
(4) Carcelli, M.; Mazza, P.; Pelizzi, C.; Zani, F. J. Inorg. Biochem. 1995,
15, 188-222.
(5) (a) Adams, S.; Cobb, R. Prog. Med. Chem. 1976, 5, 59-138. (b)
Sorenson, J. R. Inflamatory Diseases and Cooper, 1st ed.; Humana
Press: Clifton, NJ, 1982. (c) Sorenson, J. R. J. Prog. Med. Chem.
1989, 26, 437. (d) Sorenson, J. R. J. A Physiological Basis for
Pharmacological ActiVities of cooper complexes: An hypothesis;
Humana Press: Clifton, NJ, 1987; pp 3-16, 43, 57. (g) West, D. X.;
Thientanavanich, I.; Liberta, A. E. Trans. Met. Chem. 1995, 20, 303.
(h) Liberta, A. E.; West, D. X. BioMetals 1992, 5, 121.
(6) (a) Farrell, N. Transition Metal Complexes as Drugs and Chemo-
therapeutic Agents. In Catalysis by Metal Complexes; James, B. R.R,
Ugo, R., Eds.; Kluwer: Dordrecht, The Netherlands, 1989; Chapter
11 and references therein. (b) Rhodes, M. D.; Sadler, P. J.; Scawen,
M. D.; Silver, S. J. Inorg. Biochem. 1992, 46, 129. (c) Brown, D. H.;
Smith, W. E. Chem. Soc. ReV. 1980, 9, 217. (d) Cofer, M. T., Shaw,
C. F., III. Inorg. Chem. 1986, 25, 333. (e) Aaseth, J.; Haugen, M.;
Forre, O. Analyst 1998, 123, 3-6. (f) Shaw, C. F., III. Chem. ReV.
1999, 99, 2589.
1
F) 843. λmax (CHCl3, 10-4 M) ) 248 nm. H NMR (CDCl3; δ, ppm):
7.91 (s, 1H); 7.54 (m, 15H, PPh3); 7.36 (m, 9H); 7.13 (m, 4H); 7.01
(m, 2H). 31P {1H}NMR (CDCl3; δ, ppm): 27.9 (s, PPh3); 146 (hep,
PF6).
[Au(KTZ)(PPh3)]PF6·2H2O (6). A suspension of AuClPPh3 (155
mg; 0.31 mmol) in acetonitrile (25 mL) was refluxed under nitrogen
until complete dissolution. KPF6 (230 mg; 1.25 mmol) was added, and
refluxing was continued for 2 h. KTZ (166 mg; 0.31 mmol) was added,
and the mixture was stirred and refluxed for 24 h, cooled to room
temperature, and then filtered through Celite. The solution was dried,
and the solid was dissolved in acetone (3 mL). The solution was filtered,
and a white solid was made to precipitate by the addition of diethyl
ether; then it was filtered off, washed with water and diethyl ether,
and dried under vacuum (yield, 221 mg, 59%). Anal. Calcd for C44H43-
Cl2F6N4P2Au‚2H2O: C, 45.1; H, 3.67; N, 4.78. Found: C, 45.0; H,
3.87; N, 4.37. IR (cm-1): ν(CdC) 1517, ν(CdN) 1445. λmax (CHCl3,
10-4 M) ) 250, 298 nm. 1H NMR (CDCl3; δ, ppm): 8.24 (s, 1H); 7.62
(d, J ) 9.0 Hz, 1H); 7.54 (m, 15H, PPh3); 7.43 (d, J ) 2.1 Hz, 1H);
7.28 (m, 1H); 7.20 (s, 1H); 7.06 (s, 1H); 6.77 (AA′XX′, 4H); 4.59
(AB, 2H); 4.33 (m, 1H); 3.75 (m, 6H); 3.52 (m, 2H); 2.85 (m, 4H);
2.10 (s, 3H); 1.67 (s, H2O). 31P {1H}NMR (CDCl3; δ, ppm): 28.2 (s,
PPh3); 146 (hep, PF6).
(7) Mirabelli, C. K.; Johnson, R. K.; Song, C. M.; Fancette L.; Muirhead,
K.; Crooke, S. T. Cancer Res. 1985, 45, 32.
(8) Navarro, M.; Sa´nchez-Delgado, R. A.; Pe´rez H. Unpublished results.
(9) Navarro, M.; Pe´rez, H.; Sa´nchez-Delgado, R. A. J. Med. Chem. 1997,
40, 1937.

Metal-Based Chemotherapy against Tropical Diseases
Inorganic Chemistry, Vol. 40, No. 27, 2001 6881
Table 1. Crystallographic Data for 1
empirical formula
Magnetic Measurements. EPR spectra were measured at room
temperature in a Varian E-line spectrometer working in the X-band
(ν ) 9.3 GHz) with a homemade cylindrical cavity and a coaxial
microwave coupler for the X-band. Experimental conditions (microwave
power and modulation field) were adjusted to avoid saturation of signal
intensity. DC magnetic susceptibility measurements were carried out
in a computerized Faraday balance in the temperature range 76 e T e
250 K; diamagnetic corrections of constituent atoms and temperature-
independent paramagnetism were taken into account in our results. The
C88.66H69.32Cl7.32CuN8
fw
1585.8
cryst color, habit
cryst dimens (mm)
cryst syst
blue-purple, irregular
0.43 × 0.30 × 0.16
triclinic
no. of reflcns used for unit
cell determn (2θ range)
space group
formula units/unit cell
a (Å)
25 (20-31°)
P1h
1
-1
øm vs T curve is obtained from the experiment, and the µeff is
calculated from the Curie constant C of the Cure-Weiss law10
12.773(2)
b (Å)
15.326(4)
c (Å)
11.641(2)
C
ø )
R (deg)
110.43(2)
T - Θ
â (deg)
100.67(2)
γ (deg)
66.62(2)
V (Å3)
1957.4(7)
where
Dcalcd (g/cm3)
F000
1.345
Ng2µB2S(S + 1)
819
µ(Mo KR) (cm-1
difractometer
radiation (λ, Å)
T (K)
)
5.34
2
C )
µeff ≡ g2µB2S(S + 1)
3KB
Rigaku AFC7S
Mo KR (0.710 69)
293 (2)
From this
2θ range (deg)
range of h, k, l
no. of reflcns measd
no. of unique reflcns
no. of obsd reflcns
no. of params varied
weighting scheme
1.5 e 2θ e 25
0 f 15, 16 f 18, 13 f 13
µeff ) 2.827
C
x
M
7236
6896
3967
N is the number of paramagnetic species per gram of sample, KB is the
Boltzman constant, CM is the molar Curie constant, and Θ is the Curie
temperature.
476
[(σF0)2 + (0.1088P)2 + 0.5276P ]-1
,
where P ) (F02 + 2Fc2)/3
NMR Spectroscopy. NMR spectra were obtained in DMSO-d6
solutions in a Bruker AVANCE500 spectrometer, 1H NMR shifts being
recorded relative to residual resonance in the deuterated solvent. For
the paramagnetic complexes the NMR spectra were obtained using a
90ï pulse of 10 µs. An inversion-recovery pulse sequence (180ï -τ-
90ï-Aq) was used to obtain nonselective proton longitudinal relaxation
times (T1) with the carrier frequency set at different positions to ensure
the validity of the measurements. Signal: noise ratios were improved
by applying a line-broadening factor of 30 Hz to the FID prior to Fourier
transformation.
S(GOF)
1.173
R1a [I > 2σ(I)]
wR2b [I > 2σ(I)]
largest feature final
0.0706
0.1783
0.002
diff map (e Å-3
)
a R1 ) ∑||Fo| - |Fc||/∑|Fo|. b wR2 ) {∑[w(Fo2 - Fc2)2]/∑[w(Fo2)2]}1/2.
displacement parameters and listings of atomic coordinates of the
hydrogen atoms, anisotropic displacement parameters, and structure
factors are available as Supporting Information.
X-ray Crystallography. A blue-purple irregular crystal of dimen-
sions 0.43 × 0.30 × 0.16 mm of 1 was mounted on a Rigaku AFC7S
diffractometer with graphite-monochromated Mo KR radiation. Cell
constants and an orientation matrix for data collection were obtained
from 25 centered reflections (range: 20° < 2θ < 31°). Data were
collected at rt (room temperature) using the ω-2θ scan technique to a
maximum 2θ value of 50°. A total of 3 standard reflections were
measured for orientation and intensity control. No decay was noticed.
Intensity data were corrected for Lorentz-polarization effects, and a
semiempirical absorption correction was carried out on Ψ-scan
measurements.11 Of the 7236 reflections that were collected, 6896 were
unique. The structure was solved by direct methods12 and expanded
using Fourier techniques. The full-matrix least-squares refinement13 was
based on F2. All non-hydrogen atoms were refined anisotropically,
except for those of the disordered dichloromethane solvent molecule,
which was refined constraining the isotropic displacement parameters
to be the same (0.12 Å2) for the three non-hydrogen atoms. Preliminary
refinement of the occupancy of this molecule converged to ap-
proximately 33%, so it was fixed to this value for subsequent
refinements. Hydrogen atoms for this molecule were not included.
Atomic scattering factors were those reported by Cromer and Waber14
with anomalous dispersion correction.15 Crystallographic details are
collected in Table 1. Final atomic coordinates with equivalent isotropic
Biological Tests against Epimastigotes of T. cruzi. Tests were
conducted by following a previously reported method.16 The EP stock
of the epimastigote17 form of T. cruzi was used throughout this study.
The epimastigotes were cultured in liver infusion-tryptose medium
supplemented with 10% calf serum at 28 °C with strong stirring (120
rpm) as previously described. The cultures were initiated with a cell
density of 2 × 106 epimastigotes/mL. CTZ, KTZ, and compounds 1-6
were added as DMSO solutions (all concentrations corresponding to
10-6 M of the free CTZ or KTZ ligand) when the culture reached a
cellular density of 107 epimastigotes/mL. Parasite proliferation was
followed daily by the use of an electronic particle counter (model ZBI;
counter Electronics, Inc, Hialeah, FL) and by direct counting with an
hemacytometer. Controls (nontreated) cultures contained an equivalent
amount of the solvent used to deliver the experimental compounds to
the growth medium (1% v/v of dimethyl sulfoxide); at this concentration
the solvent had no effects on cell viability or proliferation.
Safety Note. Handling of live T. cruzi was done according to
established guidelines18
Results and Discussion
Synthesis and Characterization of Copper Complexes. The
copper complexes were synthesized from CuCl2 by reaction with
(10) McMillan, J. A. Electron Paramagnetism Chemical Series; Organiza-
tion of American States (OAS): Washington, DC, 1975; No. 14.
(11) North, A. T. C.; Phillips, D. C.; Mathews, F. S. Acta Crystallogr.,
Sect. A 1968, 24, 351.
(12) TEXSAN: Single-Crystal Structure Analysis Software, version 1.6;
Molecular Structure: The Woodlands, TX, 1993.
(16) Lazardi, K.; Urbina, J. A.; De Sousa, W. Antimicrob. Agents
Chemother. 1990, 34, 2097.
(17) Epimastigote: one of the proliferative stages of the parasite, equivalent
to that present in its insect vector. Although this form is noninfective
for vertebrates, it has many biochemical similarities with the infective
(intracellular amastigote) form present in vertebrates and is commonly
used in preliminary drug testing against this organism.
(13) Sheldrick, G. M. SHELXTL, release 5.03; Siemens Analytical X-ray
Instruments Inc.: Madison, WI, 1994.
(14) Cromer, D. T.; Waber, J. T. Acta Crystallogr. 1965, 18, 104.
(15) International Tables of Crystallography; Kynoch: Birmingham,
England, 1974; Vol. 4.
(18) Hudson, L.; F. Grover, W. E.; Gutteridge, R. A.; Klein, W.; Peters,
R. A.; Neal, M. A.; Miles M. T.; Scott, R.; Nourish and B. P. Ager.
Trans. R. Soc. Trop. Med. Hyg. 1983, 77, 416-419.

6882 Inorganic Chemistry, Vol. 40, No. 27, 2001
Navarro et al.
Figure 1. EPR spectrum for [Cu(KTZ)3Cl2] showing the spectral
parameters. The inset shows the broadened EPR spectrum for [Cu-
(KTZ)Cl2]2.
Table 2. EPR Spectroscopic Data for the Cu(II) Complexes
complex
g|
g
A (G)
µ
eff (µB)
[Cu(CTZ)4 ]Cl2 (1)
[Cu(CTZ)Cl2]2 (2)
[Cu(KTZ)3Cl2] (3)
[Cu(KTZ)Cl2]2 (4)
2.25
2.25
2.26
2.27
2.05
2.06
2.05
2.08
85
141
166
128
2.10
2.12
0.64
1.92
Figure 2. 1H NMR spectra of the copper complexes 1-4. In these
spectra it is noteworthy the presence of signals at very low field for 2
and 4 (dimeric complexes), while 1 and 3 (monomeric complexes) do
not show sharp isotropically shifted signals.
CTZ or KTZ at different molar ratios. Compounds 1 and 3 were
obtained by using an excess of the ligand, while 2 and 4 were
obtained with a 1:1 copper to ligand molar ratio.
EPR Spectra and Magnetic Moment Data. The EPR
spectra of powdered complexes 1-4 exhibit resolved features
isotropically shifted signals; this is in agreement with mono-
nuclear Cu(II) structures and coherent with the magnetic and
EPR data. The extremely large line widths of the NMR signals
derived from the paramagnetic protons in these complexes make
their detection impossible.21,22 On the other hand, the spectra
of complexes 2 and 4 exhibit reasonably sharp, isotropically
shifted signals. The most likely reason for that is the presence
in these complexes of two paramagnetic Cu(II) centers in each
complex, which is known to produce effective relaxation
mechanisms.23-25 The NMR spectrum of 2 displays isotropically
shifted signals at 46.5 ppm (T1 ) 3.97 ms), 40.2 ppm (T1 )
5.77 ms), and 38.0 ppm (T1 ) 32.5 ms). Similarly, the
isotropically shifted signals from the protons in 4 are located
at 46.5, 39.8, and 37.1 ppm with relaxation times of 4.0, 5.8,
and 18.0 ms, respectively. Since most probably in these
complexes the KTZ and CTZ ligands bind to the Cu(II) ions
through N3, the signals located at 46.5 and 40.2 ppm for 2 and
46.5 and 39.8 ppm for 4 can be tentatively assigned to the H2
and/or H4 protons, on the basis of NMR spectra of other
dicopper(II) complexes containing imidazole ligands reported
previously.21 Unfortunately, due to the similar location of these
protons with respect to the metal centers in 2 and 4 definitive
assignments of these signals to specific protons cannot be
performed without deuteration of the CTZ and KTZ ligands in
the H2 and H4 positions. On the other hand, the signals at 38.0
ppm (2) and 37.0 ppm (4) can be confidently assigned to the
H5 protons in CTZ and KTZ, respectively. This imidazole
proton is the one located furthest away from the Cu(II) centers,
and therefore, it is the least affected by the paramagnetic
influence of the metal center; as a result, the H5 proton
resonances are the least broadened which is reflected in their
relatively long relaxation times (32.5 ms in 2 and 18.0 ms in
4).
with g > g , consistent with a tetragonally elongated structure;
||
a representative set of spectra, corresponding to 3 and 4, is
shown in Figure 1, and the EPR parameters for all compounds
are shown in Table 2. All g values and spectra are characteristic
of Cu(II) with axial symmetry.19
Complexes 1 and 3 show well-resolved signals due to the
hyperfine splitting. For complex 1, the low value of the
hyperfine-coupling constant is indicative of the low electron
density at the copper site and the freedom of movement in the
copper plane. Additionally, the low intensity of the hyperfine
signals can be taken as evidence of the unpaired electron
spending a long time in the copper plane. The EPR spectrum
of 3, in turn, shows several well-defined narrow signals due to
hyperfine splitting, which, in addition to the values obtained
for the parameters of the spectrum, leads us to propose a
monomeric structure for both of these compounds, as was
confirmed by X-ray diffraction for 1 (vide infra). The EPR
spectra for 2 and 4 showed a set of broadened hyperfine signals
and an increment of the g values; this is an evidence of an
increase dipolar interaction between paramagnetic ions and
suggests the presence of more than 1 copper atom/molecule in
these compounds.20
All the complexes obey the Curie-Weiss law in the tem-
perature range 76 < T < 250 K, and the values for µeff are
shown in Table 2. The µeff values obtained for 1 and 3 are 2.10
and 0.64, respectively. Complex 1 shows a normal value of µeff
for Cu(II); however, the µeff value for 3 is very low, which can
be taken as indicative of a strong diamagnetic shielding from
the environment (KTZ) around the metal ion. The µeff values
obtained for 2 and 4 are 2.12 and 1.92, also normal for Cu(II)
complexes. These results are in good agreement with those
obtained from NMR experiments.
NMR Spectra. The NMR spectra of complexes 1-4 are
shown in Figure 2. Complexes 1 and 3 do not show sharp
(21) Maekawa, M.; Kitagawa, S.; Munakata, M.; Masuda, H. Inorg. Chem.
1989, 28, 1904.
(22) Bertini, I.; Turano, P.; Vila, A. J. J. Chem. ReV. 1993, 98, 2833.
(23) Satcher, J. H.; Balch, A. L. Inorg. Chem. 1995, 34, 3371.
(24) Holz, R. C.; Brink, J. M.; Gobena, F. T.; O’Connor, C. J. Inorg. Chem.
1994, 33, 6086.
(25) Kitajima, N.; Fijusawa, K.; Fujimoto, C.; Moro-oko, Y.; Hashimoto,
S.; Kitagawa, T.; Toriumi, K.; Tatsumi, K.; Nakamura, A. J. Am.
Chem. Soc. 1992, 114, 1277.
(19) (a) Sushil, K.; Misra; Chunzheng, W. Magn. Reson. ReV. 1990, 14,
157. (b) Newton, L. D. F.; Yoshitaka, G. J. Mol. Struct. (THEOCHEM).
1995, 335, 175.
(20) (a) Boas, J. F.; Pilbrow, J. R. J. Chem. Soc. A 1969, 721. (b) Bellitto,
C.; Bigoli, F.; Deplano, P.; Mercuri M. L.; Pellinghelli, M. A.; Staulo,
G.; Trogo, E. F. Inorg. Chem. 1994, 33, 3005.

Metal-Based Chemotherapy against Tropical Diseases
Inorganic Chemistry, Vol. 40, No. 27, 2001 6883
structural features are similar to those reported for tetragonal
27
[Cu(1,4,5-tmi)4][ClO4]2 (tmi ) trimethylimidazole). In the
crystal structure of 1 two chloride anions are symmetrically
located in both apical positions of discrete Cu(CTZ)42+ cations
at a Cu‚‚‚Cl distance of 2.998 Å suggesting a weak interaction
between these two atoms. This Cu‚‚‚Cl distance is longer than
the Cu-Cl bond distances (2.250 Å) previously reported for
four-coordinate Cu(II) complexes.28
Synthesis and Characterization of Gold Complexes. Two
new gold complexes [Au(PPh3)(CTZ)]PF6 (5) and [Au(PPh3)-
(KTZ)]PF6‚H2O (6) were synthesized by reaction of AuClPPh3
with KPF6 in acetonitrile to remove the coordinated chloride,
after which the CTZ or KTZ ligand was added. We have
previously employed a similar procedure for the preparation of
[Au(PPh3)(CQ)]PF6 (CQ ) chloroquine).9
These compounds were obtained as air stable white solids
whose elemental analyses are in agreement with the molecular
formula [Au(PPh3)(L)]PF6 (L ) CTZ or KTZ). The infrared
spectrum of complex 5 showed the signals corresponding to
the aromatic C-H stretching at 3050 cm-1, the CdN stretching
at 1500 cm-1, and the CdC stretching at 1453 cm-1. The IR
spectrum of complex 6 showed signals corresponding to the
Figure 3. Molecular diagram (35% displacement ellipsoids) of the
cation of 1 showing the atom-numbering scheme. Prime atom labels
are generated by a center of symmetry.
stretching vibrations: C-H (aromatic) at 3150 cm-1
;
C-H(aliphatic) at 2920 cm-1; CdO at 1643 cm-1; CdC at
1517 cm-1; CdN at 1445 cm-1; C-O-Ar at 1200 cm-1. All
these signals are slightly shifted with respect to those shown
by the free ligands. The IR spectra also showed the characteristic
Table 3. Selected Bond Distances (Å) and Angles (deg) for 1
Cu-N(1)
2.013(4) N(3)-C(23)
1.996(4) N(3)-C(25)
1.316(7)
1.382(7)
1.377(7)
1.486(7)
1.344(7)
1.344(7)
1.335(8)
Cu-N(3)
signal due to the P-F stretching at 843 cm-1 for the PF6
counterion.
-
Cu-Cl
2.998
N(4)-C(24)
N(1)-C(1)
1.297(7) N(4)-C(26)
1.373(7) N(4)-C(23)
1.381(7) C(2)-C(3)
1.491(6) C(24)-C(25)
1.357(6)
The 31P{1H} NMR spectra for [Au(PPh3)(CTZ)]PF6 and [Au-
(PPh3)(KTZ)]PF6‚H2O exhibit singlet signals corresponding to
PPh3 at 27.9 ppm and at 28.3 ppm, respectively, which are 3.1
and 2.7 ppm shifted to highfield with respect to the chemical
shift in the parental gold compound (AuClPPh3). These spectra
N(1)-C(3)
N(2)-C(2)
N(2)-C(4)
N(2)-C(1)
N(1)-Cu-N(3′)
N(1)-Cu-N(3)
N(1)-C(1)-N(3)
N(2)-C(2)-C(3)
88.0(2)
92.0(2)
111.8(5)
107.6(5)
C(1)-N(1)-C(3)
C(1)-N(2)-C(2)
C(1)-N(2)-C(4)
C(2)-C(3)-N(1)
C(2)-N(2)-C(4)
106.8(4)
105.3(4)
130.0(4)
108.5(5)
124.7(5)
-
also showed the characteristic PF6 signal, as a heptuplet
centered at 145.5 ppm with a coupling constant JP-F ) 713
Hz. The 1H NMR chemical shifts of the imidazole protons H2,
H4, and H5, which are displaced downfield with respect to the
free ligand, are shown in Table 4. This indicates that CTZ or
KTZ are coordinated to gold in both complexes 5 and 6 through
the imidazole nitrogen atom of each ligand. Additionally, the
relative integral of the signals for the phenyl protons of PPh3
in these spectra are in agreement with a PPh3:L ratio of 1:1 in
each complex. The structures of 5 and 6 that correspond to a
14-electron configuration for Au(I) are most probably in the
typical linear coordination geometry29 which has been previously
associated with biological activity.6,7
Biological Activity. The effect of the new complexes 1-6
on the proliferation of in vitro cultures of the epimastigote form
of T. cruzi was evaluated as described in the Experimental
Section; Table 5 summarizes the results of such tests. Since
the number of CTZ or KTZ ligands vary from one complex to
another, to allow direct comparisons, the experiments were
N(3)-C(23)-N(4) 111.6(5)
N(4)-C(24)-C(25) 107.2(5)
Cu-N(1)-C(1)
Cu-N(1)-C(3)
Cu-N(3)-C(23)
Cu-N(3)-C(25)
C(23)-N(4)-C(24) 106.3(5)
124.7(4)
C(23)-N(4)-C(26) 130.0(5)
127.25(4) C(24)-C(25)-N(3) 109.4(5)
128.7(4)
125.8(4)
C(24)-N(4)-C(26) 123.6(4)
C(25)-N(3)-C(23) 105.5(5)
Relative integration of the isotropically shifted signals in the
NMR spectra of 2 and 4 indicates that all of them are generated
by the same number of protons. The results of the elemental
analysis performed on 2 and 4 suggest that each complex
contains two CTZ (complex 2) or two KTZ (complex 4) ligands
in their structures. Therefore, these ligands must be located in
equivalent positions with respect to the metal centers in
complexes 2 and 4.
Crystal Structure of [Cu(CTZ)4]Cl2. A molecular diagram
for the cation of compound 1 is shown in Figure 3, while
selected bond distances and angles are collected in Table 3.
The crystal structure shows (apart from the solvent molecule)
2+
half of the Cu(CTZ)4 cation and a chloride anion as crystal-
(26) (a) Farida, A.; Goodgame, D. M. L.; Goodgame, M.; Rayner-Canham
G. W.; Skapski, C. A. Chem. Commun. 1968, 757-61. (b) McFadden,
D. L.; McPhail, A. T.; Garner, C. D.; Mabbs, F. E. J. Chem. Soc.,
Dalton Trans. 1976, 47-52.
(27) Bernarducci, E.; Bharadwaj, P. K.; Krogh-Jespersen, K.; Potenza, J.
A.; Schugar, H. J. J. Am. Chem. Soc. 1983, 105, 3860.
(28) (a) Biagini Cingi, M.; Bigoli F.; Lanfranchi, M.; Pellinghelli M. A.;
Vera, A.; Buluggiu, E. J. Chem. Soc., Dalton Trans. 1992, 21, 3145.
(b) Raithby, P. R.; Shields, G. P.; Allen, F. H.; Samuel Mortherwell,
W. D. Acta Crystallogr., Sect. B 2000, 56, 444-454.
(29) Shroder, M.; Stephenson, T. A. In ComprehensiVe Coordination
Chemistry; Wilkinson, G., Gillard, R. D., McCleverty, J. A., Eds.;
Pergamon: Oxford, U.K., 1987; Vol. 2, Chapter 15.
lographically independent molecular fragments in the asym-
metric unit. The copper located in a center of symmetry and
four nitrogen atoms lie in the same plane leading to a perfect
planar configuration. However, the bond angles N(1)-Cu-N(3)
[92.0(2)°] and N(1)-Cu-N(3′) [88.0(2)°] show a small devia-
tion from the square arrangement. The bond distances Cu-N(1)
2.013(4) Å and Cu-N(3) 1.996(4) Å are typical for tetrakis-
(imidazole)copper(II) complexes.26 The dihedral angles between
the N1C1N2C2C3 and N3C23N4C24C25 imidazole planes and
the equatorial CuN4 unit are 77.1 and 77.8°, respectively. These

6884 Inorganic Chemistry, Vol. 40, No. 27, 2001
Navarro et al.
1
Table 4. Selected H and 31P NMR Data for Gold Complexes
1H NMR [δ/ppm (∆δ)]
31P NMR [δ/ppm (∆δ)] for PPh3
compd
H2
H4
H5
[Au(CTZ)PPh3]PF6
[Au(KTZ)PPh3]PF6
7.91 (0.47)
8.25 (0.77)
7.36 (0.33)
7.20 (0.25)
7.01 (0.28)
7.06 (0.13)
27.9 (3.1)
28.2 (2.8)
phosphine oxide which is known to be ineffective.30 The
possibility that in our case the CTZ and KTZ ligands are merely
been protected against some deactivation process by the metal
ions cannot be entirely ruled out, but it seems less likely, since
the difference in activity with respect to the free ligands in this
case and other previously reported examples3 are notable.
Moreover, preliminary studies on the possible mechanisms of
action of these metal-azole complexes,3b as well as further
experiments which are currently in progress in our laboratories,
indicate a complex reaction scheme involving multistep hydro-
lytic processes, coupled with ligand exchange reactions leading
to strong interactions of the metal ions with the DNA of the
parasite. Hopefully these studies will shed more light on the
influence of the metal and the structure of the complexes on
the biological activity and, thus, allow us to improve the
potential of these novel agents in a rational manner.
Table 5. Effect of CTZ, KTZ, and Their Cu and Au Complexes on
the Proliferation of the Epimastigotes of T. Cruzia
compd
% inhibition
CTZ
KTZ
0
39
66
67
73
69
66
71
[Cu(CTZ)4]Cl2 (1)
[Cu(CTZ)Cl2]2 (2)
[Cu(KTZ)3]Cl2 (3)
[Cu(KTZ)Cl2]2 (4)
[Au(CTZ)PPh3]PF6 (5)
[Au(KTZ)PPh3]PF6 (6)
a Complexes were used as DMSO solutions of 1 µM (concentration
based on the ligands). For details, see Experimental Section.
conducted by use of DMSO solutions of the complexes
providing in each case a total concentration of 10-6 M of CTZ
or KTZ in the growth medium. In general, the activities of the
new metal derivatives were all considerably higher than those
exhibited by the free ligands. The marked enhancement in the
efficacy of CTZ by complexation to metals is demonstrated by
the fact that, at 10-6 M, no effect on growth was observed with
the free ligand, while the same concentration of the imidazole
bound to copper or gold in the complexes induced over 66%
growth inhibition. In the case of KTZ, the activity increases
from 39% inhibition for the free ligand to over 70% inhibition
for the complexes. These results can be considered as important
improvements to previously known activities against T. cruzi,
and they are consistent also with our published data with
ruthenium complexes which provide growth inhibition of up to
82% at the same concentration.3a It is interesting to note also
that the enhancement of the activity of the free ligands reported
in Table 5 is not markedly influenced by the number of ligands
attached to a single metal center, to the monomeric or dimeric
nature of the copper complexes, or even to the nature of the
metal ion.
Conclusion
Here we have reported the synthesis and characterization of
new Cu(II)-CTZ and Cu(II)-KTZ as well as Au(I)-CTZ and
Au(I)-KTZ complexes. The characterization of these complexes
was achieved through NMR, EPR, IR, UV-vis, elemental
analysis, and X-ray diffraction. The biological activity of these
complexes against the epimastigote form of T. cruzi was also
evaluated indicating that all of them exhibit improved activity
on parasite proliferation when compared with the free ligands.
Acknowledgment. CONICIT is gratefully acknowledged for
the acquisition of the NMR spectrometer through the National
NMR Laboratory at IVIC and for funding to M.N. through
Project S1-98000945 and to R.A. through Project S1-96001062.
This work was also supported by the UNDP/World Bank/World
Health Organization Program for Research and Training in
Tropical Diseases (Grant 9990201 to J.A.U.).
We cannot at this moment offer a satisfactory explanation
for this observation, but these results are reminiscent of the
situation found for 1,2-bis(diphenylphosphino)ethane complexes
of Cu, Ag, and Au, all of which display anticancer activities
very similar to each other and to that of the free diphosphine;
this has been interpreted in terms of the phosphine ligand itself
being the anticancer agent, while the metal ions would be acting
as protectors of the ligand against oxidation to the corresponding
Supporting Information Available: X-ray data in the form of CIF
files. This material is available free of charge via the Internet at
http://pubs.acs.org.
IC0103087
(30) Lippard, S. J. In Bioinorganic Chemistry; Bertini, I., Gray, H. B.,
Lippard, S. J., Valentine, J. S., Eds.; University Science Books,
Sausalito, CA, 1994; Chapter 9, p 520.