Coordination to copper(II) strongly enhances the in vitro antimicrobial activity of pyridine-derived N(4)-tolyl thiosemicarbazones

Article abstract of DOI:10.1016/j.poly.2007.03.002

Five copper(II) complexes with N(4)-ortho, N(4)-meta and N(4)-para-tolyl thiosemicarbazones derived from 2-formyl and 2-acetylpyridine were obtained and thoroughly characterized. The crystal structure of N(4)-meta-tolyl-2-acetylpyridine thiosemicarbazone (H2Ac4mT) was determined, as well as that of its copper(II) complex [Cu(2Ac4mT)Cl], which contains an anionic ligand and a chloride in the coordination sphere of the metal. The in vitro antimicrobial activities of all thiosemicarbazones and their copper(II) complexes were tested against Salmonella typhimurium and Candida albicans. Upon coordination a substantial decrease in the minimum inhibitory concentration, from 225 to 1478 μmol L^-1 for the thiosemicarbazones to 5-30 μmol L^-1 for the complexes was observe against the growth of Salmonella typhimurium and from 0.7-26 to 0.3-7 μmol L^-1 against the growth of C. albicans, suggesting that complexation to copper(II) could be an interesting strategy of dose reduction.

Full text of DOI:10.1016/j.poly.2007.03.002

Polyhedron 26 (2007) 3263–3270
www.elsevier.com/locate/poly
Coordination to copper(II) strongly enhances the in vitro
antimicrobial activity of pyridine-derived N(4)-tolyl thiosemicarbazones
a
a
b
b
Isolda C. Mendes , Juliana P. Moreira , Antonio S. Mangrich , Solange P. Balena ,
c
a,
*
Bernardo L. Rodrigues , Heloisa Beraldo
a
Departamento de Quı´mica, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, Brazil
b
Departamento de Quı´mica, Universidade Federal do Parana´, 81531-970 Curitiba, Brazil
c
Instituto de Fı´sica, Universidade de Sa˜o Paulo, Sa˜o Carlos, Brazil
Received 12 January 2007; accepted 2 March 2007
Available online 12 March 2007
Abstract
Five copper(II) complexes with N(4)-ortho, N(4)-meta and N(4)-para-tolyl thiosemicarbazones derived from 2-formyl and 2-acetyl-
pyridine were obtained and thoroughly characterized. The crystal structure of N(4)-meta-tolyl-2-acetylpyridine thiosemicarbazone
(H2Ac4mT) was determined, as well as that of its copper(II) complex [Cu(2Ac4mT)Cl], which contains an anionic ligand and a chloride
in the coordination sphere of the metal. The in vitro antimicrobial activities of all thiosemicarbazones and their copper(II) complexes
were tested against Salmonella typhimurium and Candida albicans. Upon coordination a substantial decrease in the minimum inhibitory
concentration, from 225 to 1478 lmol Lꢀ1 for the thiosemicarbazones to 5–30 lmol Lꢀ1 for the complexes was observe against the
growth of Salmonella typhimurium and from 0.7–26 to 0.3–7 lmol Lꢀ1 against the growth of C. albicans, suggesting that complexation
to copper(II) could be an interesting strategy of dose reduction.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Thiosemicarbazones; Copper(II) complexes; Crystal structures; Antimicrobial activity; Salmonella typhimurium; Candida albicans; Dose
reduction
1. Introduction
ones has been demonstrated in clinical isolates of bacteria
[1,4]. Significant activity has been found toward gram posi-
tive bacilli but poor antibacterial activity has been shown
toward gram negative cultures [1,4,5]. In addition, the
in vitro inhibitory activity of 2-acetylpyridine-derived thi-
osemicarbazones has been demonstrated against clinically
significant bacterial cultures, including isolates with known
antibiotic resistance [1,4,5]. A variety of metal complexes of
these ligands has also shown to present potent antibacterial
activity [1]. Less attention has been given to 2-benzoylpyri-
dine-derived thiosemicarbazones.
Thiosemicarbazones present a wide range of pharmaco-
logical applications as anticancer, antiviral and
antimicrobial agents [1–3]. The biological properties of
thiosemicarbazones are often related to metal coordina-
tion. Firstly, lipophilicity, which controls the rate of entry
into the cell, is modified by coordination. Also, the metal
complex can be more active than the free ligand, or the
metal complex can be a vehicle for activation of the ligand
as the cytotoxic agent [1–3].
The antimicrobial action of thiosemicarbazones against
a variety of bacteria and fungi has been investigated. The
activity of 2-formyl and 2-acetylpyridine thiosemicarbaz-
Some 2-formyl and 3-formylpyridine thiosemicarbazones
and their metal complexes were tested against Candida albi-
cans and Apergillus fumigatus. In addition, N(4)-alkyl and
N(4)-dialkyl-2-acetylpyridine thiosemicarbazones showed
marked inhibitory activity of A. niger growth. Moreover,
metallocene derivatives of titanium and zirconium with
*
Corresponding author. Tel.: +55 31 3499 5740; fax: +55 31 3499 5700.
E-mail address: hberaldo@ufmg.br (H. Beraldo).
0277-5387/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2007.03.002

3264
I.C. Mendes et al. / Polyhedron 26 (2007) 3263–3270
thiosemicarbazones derived from 2-acetylpyridine, 2-acety-
lfuran, 2-acetylthiophene and 2-acetylnaphtalene were
tested against a number of pathogenic fungi and bacteria.
All the complexes were more active against the studied
organisms than the free thiosemicarbazones [1].
It has been demonstrated that the presence of a bulky
group at N(4) strongly improves the biological activity of
thiosemicarbazones [2,3], probably due to an increase in
lipophilicity.
problems [13]. Therefore, preparation of new antimicrobi-
als with activity in low doses is extremely important.
In the present work, we studied the activity of N(4)-
ortho-, N(4)-meta- and N(4)-para-2-formyl (hereafter
named H2Fo4oT, H2Fo4mT, H2Fo4pT), 2-acetyl
(H2Ac4oT, H2Ac4mT, H2Ac4pT) and 2-benzoylpyridine
thiosemicarbazones (H2Bz4oT, H2Bz4mT, H2Bz4pT)
(see Fig. 1) and their copper(II) complexes against the
growth of Salmonella typhimurium and C. albicans.
In previous works, we started an evaluation of the
antimicrobial and cytotoxic activities of pyridine-derived
thiosemicarbazones and their metal complexes. N(4)-alkyl-
and N(4)-phenyl-substituted 2-benzoylpyridine thiosemic-
arbazones have been prepared, as well as their palladium(II),
tin(IV) and iron(II) complexes [6–8].
Taking into consideration that copper(II) complexes
have found possible medical uses in the treatment of many
diseases including microbial infections [2,3,9] we recently
prepared N(4)-tolyl thiosemicarbazones derived from
2-benzoylpyridine as well as their copper(II) complexes.
Both ligands and complexes proved to be active against
the growth of C. albicans with low values of minimum
inhibitory concentration. The activity is improved upon
coordination to copper [10].
Salmonella sp. is a gram-negative facultative rod-shaped
bacterium which causes infections about 12–24 h following
ingestion of contaminated food. Strains of Salmonella
which are resistant to a range of antimicrobials, including
first-choice agents for the treatment of humans have
emerged, and are threatening to become a serious public
health problem [11]. The same occurs with strains of fungi.
Among the azole drugs used against infections caused by
C. albicans, fluconazole and itraconazole exhibit satisfac-
tory safety and efficacy. However, the emergence of clinical
resistance during long-term therapy is well known [12].
Resistance results from the use of antimicrobials both in
humans and animal husbandry. Multi-drug resistance to
critically important antimicrobials is compounding the
2. Experimental
2.1. Apparatus
Partial elemental analyses were performed on a Perkin
Elmer CHN 2400 analyzer. Infrared spectra were recorded
on a Perkin Elmer FT-IR Spectrum GX spectrometer using
CsI/nujol; an YSI model 31 conductivity bridge was
employed for molar conductivity measurements. Magnetic
susceptibility measurements were carried out on a Johnson
Matthey MSB/AUTO balance. Electron paramagnetic res-
onance (EPR) spectra of samples in frozen DMF solution
were obtained in quartz tubes of 3 mm of internal diameter
at liquid N2 temperature (77 K) on a Bruker ESP300E
equipment with modulation frequency of 100 kHz operat-
ing at 9.5 GHz (X-band). Spectra simulations were per-
formed using the WINSIMFONIA Bruker package.
Crystal structures of H2Ac4mT and its copper complex
[Cu(2Ac4mT)Cl] (see below) were investigated using sin-
gle-crystal X-ray diffractometry. Data have been collected
at room temperature in a Kappa CCD diffractometer,
˚
Mo Ka monochromated radiation, k = 0.71073 A. Crystal
data, data collection procedure, structure determination
methods and refinement results are summarized in Table 1.
The structures were solved by direct methods and refined
on F2 by full-matrix least-squares using the SHELX 97
[14,15] program in a WINGX system [16,17]. Although some
of the hydrogen atoms could be identified in a Fourier dif-
ference map, in the final model they were geometrically
positioned and refined using a riding model. All non-H
atoms were refined anisotropically.
2.2. Synthesis of the copper(II) complexes with 2-formyl-
and 2-acetylpyridine N(4)-tolyl thiosemicarbazones
The thiosemicarbazones were prepared as described in
the literature [18]. The copper(II) complexes were obtained
by refluxing an ethanol solution (25 mL) of the desired thi-
osemicarbazone (3 mmol) with CuCl2 Æ 2H2O (Aldrich) in
1:1 ligand-to-metal molar ratio for 5 h. The resulting solids
were filtered then washed with ethanol followed by diethyl
ether, and dried in vacuo. Crystals of H2Ac4mT were
grown by slow diffusion from ethanol solution and crystals
of its copper(II) complex from a 1:9 DMSO/acetone
solution.
Fig. 1. General structure of N(4)-ortho-, N(4)-meta- and N(4)-para-tolyl 2-
formyl 2-acetyl and 2-benzoylpyridine thiosemicarbazones, E and Z
configurations.
The copper(II) complexes will be hereafter numbered as
follows: [Cu(H2Fo4oT)Cl2] (1), [Cu(2Fo4mT)Cl] (2),

I.C. Mendes et al. / Polyhedron 26 (2007) 3263–3270
3265
UVsolution: kmax (cmꢀ1): 30960 (sh), 27030 (4.40), 16520
(2.28). UVsolid: kmax (cmꢀ1): 31230, 27160, 23652, 22543,
16920. IR (CsI/nujol, cmꢀ1): m(NH) 3332 m(C@N) +
m(C@C) 1607–1538, m(C@S) 782, q(py) 601, m(CuN) 416,
m (CuNpy) 360, m(CuS) 330, m(CuCl) 307. Molar conductiv-
ity (1 · 10ꢀ3 mol Lꢀ1 DMF): 15 Xꢀ1 cm2 molꢀ1. Effective
magnetic moment = 1.76 BM. Yield: 88%.
Table 1
Crystal data and structure refinement for H2Ac4mT and [Cu(2Ac4mT)Cl]
(5)
Empirical formula
C
15H16N4S
C15H15N4SCuCl
Crystal size (mm)
Molecular weight (g molꢀ1
Crystal system, space group
0.14 · 0.10 · 0.04 0.13 · 0.09 · 0.02
284.38
monoclinic,
P21/c
)
382.36
ꢀ
triclinic, P1
Chloro(N(4)-o-tolyl-2-acetylpyridine-thiosemicarbazo-
nato)copper(II)[Cu(2Ac4oT)Cl] (4): Green solid; Anal.
Calc. for CuC15H13N4SCl: C, 47.12; H, 3.95; N, 14.65.
Lattice parameters
˚
a(A)
10.3812(4)
5.7074(2)
24.606(1)
90
100.059(2)
90
1435.5(1)
4
600
7.8130(3)
8.1716(3)
12.8239(5)
100.925(3)
100.400(3)
97.327(2)
779.52(5)
2
˚
b (A)
˚
Found: C, 46.92; H, 3.94; N, 14.35%; FW: 382.37 g molꢀ1
.
c (A)
a (ꢁ)
b (ꢁ)
c (ꢁ)
UVsolution: kmax (cmꢀ1): 32467 (sh), 27624 (4.33), 24213
(4.43), 16393 (2.67). UVsolid: kmax (cmꢀ1): 33512, 27770,
22868, 15818. IR (CsI/nujol, cmꢀ1): m(NH) 3429
m(C@N) + m(C@C) 1602–1536, m(C@S) 763, q(py) 604,
m(CuN) 460, m(CuNpy) 346, m(CuS) 329, m(CuCl) 308. Molar
3
˚
V (A )
Z
F(000)
390
1.629
1.706
Dcalc (g cmꢀ3
)
1.316
0.211
conductivity (1 · 10ꢀ3 mol Lꢀ1 DMF): 56 Xꢀ1 cm2 molꢀ1
Effective magnetic moment = 1.96 BM. Yield: 90%.
.
Absorption coefficient (mmꢀ1
)
h Range for data collection (ꢁ)
Limiting indices
2.37–25.05
ꢀ12 6 h 6 12,
ꢀ6 6 k 6 6,
ꢀ29 6 l 6 29
19929
2536 [0.0874]
99.7
none
3.31–25.00
ꢀ9 6 h P 9,
ꢀ9 6 k P 9,
ꢀ15 6 l P 15
11681
2750 [0.1036]
99.8
analytical
199/0
R1 = 0.0441,
wR2 = 0.0946
R1 = 0.0809,
wR2 = 0.1108
0.995
Chloro(N(4)-m-tolyl-2-acetylpyridine-thiosemicarbazo-
nato)copper(II)[Cu(2Ac4mT)Cl] (5): Green solid; Anal.
Calc. for CuC15H13N4SCl: C, 47.12; H, 3.95; N, 14.65.
Reflections collected
Found: C, 47.52; H, 3.90; N, 14.29%; FW: 382.37 g molꢀ1
.
Reflections unique [Rint
Completeness to h (%)
Absorption correction
Parameters/restraints
]
UVsolution: kmax (cmꢀ1): 31150 (4.53), 27170 (sh), 24750
(4.41), 16530 (2.47). UVsolid: kmax (cmꢀ1): 32062, 27330,
23277, 19810, 16958. IR (CsI/nujol, cmꢀ1): m(NH) 3320
m(C@N) + m(C@C) 1604–1547, m(C@S) 765, q(py) 610,
m(CuN) 449, m(CuNpy) 374, m(CuS) 337, m(CuCl) 318. Molar
182/0
Final R indices [I > 2r(I)]
R1 = 0.0488,
wR2 = 0.1100
R1 = 0.0898,
wR2 = 0.1356
1.030
R indices (all data)
conductivity (1 · 10ꢀ3 mol Lꢀ1 DMF): 55 Xꢀ1 cm2 molꢀ1
Effective magnetic moment = 1.85 BM. Yield: 82%.
.
Goodness-of-fit on F2
Largest difference in peak and
0.231 and ꢀ0.247 0.376 and ꢀ0.433
Chloro(N(4)-p-tolyl-2-acetylpyridine-thiosemicarbazo-
nato)copper(II)[Cu(2Ac4pT)Cl] (6): Green solid; Anal.
Calc. for CuC15H13N4SCl: C, 47.12; H, 3.95; N, 14.65.
ꢀ3
˚
hole (e A
)
Found: C, 47.07; H, 3.97; N, 14.37%; FW: 382.37 g molꢀ1
.
[Cu(2Fo4pT)Cl] (3), [Cu(2Ac4oT)Cl] (4), [Cu(2Ac4mT)Cl]
(5), [Cu(2Ac4pT)Cl] (6), [Cu(H2Bz4oT)Cl2] (7), [[Cu(H2-
Bz4mT)Cl2] (8), [Cu(H2Bz4pT)Cl2] (9) (see Section 3 for
the discussion of the proposed formulations). Complex 1,
previously reported by other authors [18] and complexes
7–9, recently prepared by us [10] are listed in the present
work because this is the first investigation on their antibac-
terial activity.
UVsolution: kmax (cmꢀ1): 31250 (4.47), 26600 (sh), 24570
(4.40), 16650 (2.51). UVsolid: kmax (cmꢀ1): 31450, 25710,
22830, 17240. IR (CsI/nujol, cmꢀ1): m(NH) 3322
m(C@N) + m(C@C) 1604–1541, m(C@S) 757, q(py) 608,
m(CuN) 446, m(CuNpy) 360, m(CuS) 341, m(CuCl) 317. Molar
conductivity (1 · 10ꢀ3 mol Lꢀ1, DMF): 37 Xꢀ1 cm2 molꢀ1
.
Effective magnetic moment = 2.03 BM. Yield: 87%.
Chloro(N(4)-m-tolyl-2-formylpyridine-thiosemicarbazo-
nato)copper(II) [Cu(2Fo4mT)Cl] (2): Green solid; Anal.
Calc. for CuC14H11N4SCl: C, 45.65; H, 3.56; N, 15.20.
2.3. Antibacterial activity
Antibacterial activity was evaluated by minimum inhib-
itory concentration (MIC) using the macro dilution test
[19–21]. S. typhimurium ATCC13311 stored in Brain Heart
Infusion (BHI) broth was subcultured for testing in the
same medium and grown at 37 ꢁC. Then the bacterial cells
were suspended, according to the McFarland protocol in
saline solution [21], to produce a suspension of about
105 CFU mLꢀ1 (colony-forming units per mL). Serial dilu-
tions of the test compounds, previously dissolved in
dimethyl sulfoxide (DMSO), were prepared in test tubes
to final concentrations of 512, 256, 128, 64, 32, 16, 8, 4,
2, and 1 lg mLꢀ1; 100 lL of a 24-h-old-inoculum was
added to each tube. The MIC, defined as the lowest con-
centration of the test compound, which inhibits the visible
Found: C, 45.60; H, 3.42; N, 14.94%; FW: 368.34 g molꢀ1
.
UVsolution: kmax (cmꢀ1): 30860 (sh), 27780 (sh), 24210
(4.40), 16304 (2.20). UVsolid: kmax (cmꢀ1): 27770, 22568,
19305, 16703. IR (CsI/nujol, cmꢀ1): m(NH) 3325
m(C@N) + m(C@C) 1607–1547, m(C@S) 760, q(py) 624,
m(CuN) 456, m(CuNpy) 343, m(CuS) 333, m(CuCl) 311. Molar
conductivity
(1 · 10ꢀ3 mol Lꢀ1
,
dimethylformamide,
DMF): 13 Xꢀ1 cm2 molꢀ1. Effective magnetic moment =
1.73 (BM). Yield: 84%.
Chloro(N(4)-p-tolyl-2-formylpyridine-thiosemicarbazo-
nato)copper(II) [Cu(2Fo4pT)Cl] (3): Green solid; Anal.
Calc. for CuC14H11N4SCl: C, 45.65; H, 3.56; N, 15.20.
Found: C, 45.58; H, 3.45; N, 14.99%; FW: 368.34 g molꢀ1
.

3266
I.C. Mendes et al. / Polyhedron 26 (2007) 3263–3270
ꢀ
growth after 20 h, was determined visually after incubation
for 20 h at 37 ꢁC. Tests using chloramphenicol as reference
and DMSO as negative control were carried out in parallel.
All tests were performed in triplicate with full agreement
between results.
As previously, antifungal activity was evaluated by min-
imum inhibitory concentration (MIC) using the macro
dilution test [19–21]. C. albicans (ATCC 18804) stored in
Sabouraud broth, was subcultured for testing in the same
medium and grown at 37 ꢁC. Then the yeast cells were sus-
pended, according to the McFarland protocol in saline
the triclinic system, P1, both with one molecule in the
asymmetric unit. Selected intra-molecular bond distances
and angles are given in Table 2; hydrogen bond distances
and angles are detailed in Table 3 and angles between
planes in Table 4. The asymmetric units are shown in per-
spective in Figs. 2 and 3.
H2Ac4mT adopts the EE configuration in relation to
the C7–N2 and C8–N3 bonds (see Fig. 1). In complex 5
the ligand adopts the EZ, configuration, to match the
sterical requirements of coordination through the Npy–
N–S chelating system. The C8–N3 bond distance goes
˚
˚
solution [21], to produce
a
suspension of about
from 1.359(3) A in the free base to 1.311(5) A in the com-
plex, due to deprotonation at N3 and formation of new
predominantly double bond. The C8–S bond changes
105 CFU mLꢀ1. Serial dilutions of the test compounds,
previously dissolved in dimethyl sulfoxide (DMSO), were
prepared in test tubes to final concentrations of 512, 256,
˚
˚
from 1.671(3) A in H2Ac4mT to 1.749(4) A in complex
5 in agreement with deprotonation at N3 and formation
of a thiolate bond. Significant modifications have also
been observed in the bond angles upon coordination
(see Table 2). For example, the N2–N3–C8 bond angle
changes from 118.5(2)ꢁ in the free base to 111.6(3)ꢁ in
the complex, and N3–C8–S goes from 120.3(2)ꢁ to
125.2(3)ꢁ (see Table 2).
128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.3 and 0.1 lg mLꢀ1
.
100 lL of a 24-h-old inoculum was added to each tube.
The MIC was determined visually after incubation for
18 h at 37 ꢁC and DMSO was used as negative control.
All tests were performed in triplicate.
3. Results and discussion
The bond distances and angles in 5 are comparable to
those related in the literature for copper(II) complexes con-
taining anionic thiosemicarbazones [23].
Microanalyses suggest the formation of [CuLCl] for
complexes 2–6, in which the thiosemicarbazones coordi-
nate as anionic ligands. The molar conductivity data reveal
that the complexes are non-electrolytes [22], in accordance
with the proposed formulations. The values of magnetic
moments in the 1.73–2.03 BM range are close to the calcu-
lated value of 1.73 BM, characteristic of the presence of
one unpaired electron as in copper(II) complexes.
Complexes 1 and 7–9 obtained previously [18,10] were
formulated as [Cu(HL)Cl2], in which the thiosemicarbaz-
ones coordinate as neutral ligands. Once again the com-
plexes are non-electrolytes, in agreement with the
presence of two chlorides in the metal coordination sphere.
In H2Ac4mT N3-H forms an intermolecular hydrogen
bond with a sulfur of an adjacent molecule, N3–H. . .S1
(ꢀx, ꢀy + 1, ꢀz + 1). In [Cu(2Ac4mT)Cl] (5) N4 is hydro-
gen bonded to a thiolate sulfur of a second adjacent mole-
cule forming dimers (see Fig. 4); N4–Hꢁ ꢁ ꢁS (ꢀx + 1,
˚
ꢀy + 1, ꢀz + 2), d(D–H) = 2.68 A, \DHA = 173.4ꢁ and
˚
d(Dꢁ ꢁ ꢁA) 3.539(3) A (Table 3).
The C7–N2–N3–C8S1–N4 skeleton is approximately
planar in the structures of both H2Ac4mT and complex
˚
5, with mean deviation from planarity of 0.0591 A in the
˚
free thiosemicarbazone and 0.0132 A in 5.
The angle between the pyridine plane and the thiosemi-
carbazone moiety is 17.2ꢁ in H2Ac4mT and 2.6ꢁ in
complex 5. Similarly, the angle between the thiosemicarba-
zone chain and the tolyl ring is 65.4ꢁ in H2Ac4mT and 3.2ꢁ
in 5, due to the almost planar structure of the complex (see
Table 4).
3.1. Structural characterization of H2Ac4mT and
[Cu(2Ac4mT)Cl] (5)
The structure of H2Ac4mT was refined in the mono-
clinic system, P21/c and that of [Cu(2Ac4mT)Cl] (5) in
Table 2
Selected bond distances and angles for H2Ac4mT and [Cu(2Ac4mT)Cl] (5)
˚
˚
Atoms
H2Ac4mT (A)
5 (A)
Atoms
H2Ac4mT (ꢁ)
5 (ꢁ)
S1–C8
N2–C7
N2–N3
N3–C8
N4–C8
N4–C9
Cu–N1
Cu–N2
Cu–S1
Cu–Cl1
1.671(3)
1.289(3)
1.381(3)
1.359(3)
1.339(3)
1.429(3)
1.749(4)
1.299(5)
1.380(4)
1.311(5)
1.353(5)
1.419(5)
2.012(3)
1.960(3)
2.253(1)
2.211(1)
N2–N3–C8
N3–C8–S1
N4–C8–N3
N4–C8–S1
C7–N2–N3
C8–N4–C9
N2–Cu–N1
N2–Cu–S1
N2–Cu–Cl1
N1–Cu–Cl1
N1–Cu–S1
Cl1–Cu–S1
118.5(2)
120.3(2)
114.8(2)
124.9(2)
119.0(2)
125.7(2)
111.6(3)
125.2(3)
119.3(4)
115.3(3)
118.3 (3)
131.2(3)
80.5(1)
84.12(9)
175.2(1)
97.8(1)
164.6(1)
97.56(4)

I.C. Mendes et al. / Polyhedron 26 (2007) 3263–3270
3267
Table 3
Hydrogen bonds parameters for H2Ac4mT and [Cu(2Ac4mT)Cl] (5)
Compound
D–Hꢁ ꢁ ꢁA
d (D–H)
d(Hꢁ ꢁ ꢁA)
DHA
d(Dꢁ ꢁ ꢁA)
H2Ac4mT
[Cu(2Ac4mT)Cl]
N3–Hꢁ ꢁ ꢁS1 [ꢀx, ꢀy + 1, ꢀz + 1]
0.86
0.86
2.78
2.68
174.7
173.4
3.642(2)
3.539(3)
N4–Hꢁ ꢁ ꢁS1 [ꢀx + 1, ꢀy + 1, ꢀz + 2]
Table 4
Angles between planes in the structures of H2Ac4mT and
[Cu(2Ac4mT)Cl] (5)
Atoms defining the plane
Deviation from the Angle between
˚
plane (A)
planes (ꢁ)
HAc4mT
5
H2Ac4mT
5
N1–C2–C3–C4–C5–C6
C7–N2–N3–C8–S1–N4
C7–N2–N3–C8–S1–N4
0.0021
0.0591
0.0591
0.0016 17.2(2)
0.0132
0.0132 65.39(8)
0.0068
2.6(2)
3.2(2)
C9–C10–C11–C12–C13–C14 0.0031
Fig. 3. Molecular plot of [Cu(2Ac4mT)Cl] (5) showing the labeling
scheme of the non-H atoms with displacement ellipsoids at the 50%
probability level.
Fig. 2. Molecular plot of H2Ac4mT showing the labeling scheme of the
non-H atoms with displacement ellipsoids at the 50% probability level.
3.2. Infrared spectra
The m(C@C) + m(C@N) composed mode observed in the
1608–1564 cmꢀ1 in the spectra of the thiosemicarbazones
shifts 1607–1538 cmꢀ1 in the spectra of the complexes, indi-
cating coordination of the azomethine nitrogen N2 [6–
8,10].
The m(C@S) absorption at 870–821 cmꢀ1 in the spectra
of the uncomplexed thiosemicarbazones is observed at
782–757 cmꢀ1 in those of the complexes, in accordance
with coordination through a thiolate sulfur. The 88–
64 cmꢀ1 shift observed upon complexation is compatible
with coordination of an anionic thiosemicarbazone in all
cases [6–9]. The pyridine in-plane deformation mode at
639–595 cmꢀ1 in the spectra of the ligands shifts to 624–
601 cmꢀ1 in those of the complexes, suggesting coordina-
Fig. 4. Molecular packing of [Cu(2Ac4mT)Cl] (complex 5) showing the
presence of dimers.
tion of the heteroaromatic nitrogen [6–8,10]. In addition,
new absorptions at 460–416 cmꢀ1 and 374–343 cmꢀ1 have
been attributed to m(Cu–N) and m(Cu–Npy), respectively,

3268
I.C. Mendes et al. / Polyhedron 26 (2007) 3263–3270
and bands in the 341–329 cmꢀ1 range have been assigned
to m(Cu–S). An absorption at 318–308 cmꢀ1 has been
attributed to m(Cu–Cl) [23,24].
The infrared data for the complexes indicate coordina-
tion of the thiosemicarbazones through the Npy–N–S che-
lating system.
dral distortion to the chromospheres [25]. The values of
the g =A ratio (Table 5) indicate nearly square-planar
k
k
environments for complexes 2–6, some with small distor-
tions, which is in good agreement with the X-ray deter-
mined structure for complex 5. The relatively low g
values of the studied compounds are characteristic of an
N enriched environment around the Cu(II) ions [26].
An interesting feature of the spectra is the appearance of
nitrogen super-hyperfine lines (five lines) in the g^ region,
which generally mirror the number of nitrogen donors
(2nI + 1, where I = 1 for nitrogen and n is the number of
nitrogen atoms in the same chemical environment). The
five lines correspond to two nitrogen donor atoms in the
equatorial positions. The AN^ values, around 16 ·
10ꢀ4 cmꢀ1 are in agreement with the imine/amine N coor-
dination to the Cu(II) ions [27].
3.3. Electronic spectra
In the electronic spectra of the thiosemicarbazones
(DMSO) the p–p* absorptions at ca. 37000 cmꢀ1 are not
significantly modified upon complexation. The absorption
at 31153–30395 cmꢀ1 in the spectra of the free bases was
attributed to n–p* transitions associated to the azomethine
and thioamide [6–8,10] functions which overlap under the
same envelope. In the spectra of complexes three absorp-
tions were observed at 31250–30860 cmꢀ1
, 27780–
26600 cmꢀ1 and 24750–24210 cmꢀ1. The first two were
assigned to n–p* transitions of the azomethine and thioam-
ide functions, and the latter to N–CuII and S–CuII ligand-
to-metal charge transfer transitions [10,24]. Ligand field
transitions were found at 16650–16304 cmꢀ1 [10,24]. The
electronic spectra of the solids are not significantly differ-
ent, particularly in the d–d region.
3.5. Antimicrobial activity
Table 6 lists the minimum inhibitory concentration
(MIC) of the thiosemicarbazones and their copper(II) com-
plexes against the growth of S. typhimurium and Table 7
lists the MIC against C. albicans. Included in this section
are values previously obtained for 2-benzoylpyridine
N(4)-tolyl thiosemicarbazones and their copper(II) com-
plexes [10].
3.4. ESR spectra
For the antibacterial action, MIC values in the 225–
1478 lmol Lꢀ1 range were found for the thiosemicarbaz-
ones. Upon coordination, a substantial decrease in MIC
was observed, with values in the 5–30 lmol Lꢀ1 range.
Among the thiosemicarbazones, the lowest values of MIC
were found for the 2-acetylpyridine derivatives, but in the
complexes the lowest values were found for those with
the 2-benzoylpyridine-derived thiosemicarbazones (7–9).
The most significant decrease upon coordination occurred
for these same complexes (Table 6). Interestingly, in the lat-
ter a neutral ligand is attached to the metal center, along
with two chloride ions, forming species with coordination
number five and with geometries which are different from
that of complexes 2–6.
Fig. 5 shows the experimental EPR spectra in frozen
DMF solution, liquid nitrogen (77 K), for the studied com-
plexes. The Hamiltonian parameters obtained from the
spectra, together with the d–d ligand field transitions, are
resumed in Table 5.
The g values are in agreement with the relation
gk > g? > 2, indicating the presence of dx2ꢀy2 ground states.
The g =A ratio can be used as a convenient empirical index
k
k
of distortion from square-planar structure. This value
ranges from ca. 105 to 135 cm for square-planar structure,
and this quotient increases on the introduction of tetrahe-
To our knowledge this is one of the few cases of thiose-
micarbazones’ activity against gram negative bacilli, which
indicates new possibilities of applications for this class of
compounds.
Much lower values of MIC were obtained for the thiose-
micarbazones against C. albicans (0.5–26 lmol Lꢀ1) (Table
7), indicating selectivity. Upon coordination a decrease of
the MIC values was observed as well, although not as sig-
nificant as in the case of the antibacterial action, probably
because the free thiosemicarbazones were already very
active.
On complexation modifications of lipophilicity can
occur [1,2]. Also, the rigid structure of the ligand in the
complex could facilitate its interaction with the biological
target, so that the metal complex may be a vehicle for acti-
vation of the ligand as the antimicrobial agent. Since the
copper salt presents antimicrobial action as well, a syner-
Fig. 5. X-band EPR spectra of [Cu(2Fo4mT)Cl] (2), [Cu(2Fo4pT)Cl] (3),
[Cu(2Ac4oT)Cl] (4), [Cu(2Ac4mT)Cl] (5) and [Cu(2Ac4pT)Cl] (6) in DMF
at 77 K (Inset: five perpendicular super-hyperfine interaction lines –
AN^ ꢂ 16 · 10ꢀ4 cmꢀ1 – of two 14N (I = 1) atoms with the Cu(II) ions
from each complex).

I.C. Mendes et al. / Polyhedron 26 (2007) 3263–3270
3269
Table 5
Ligand field transitions in the electronic spectrum of frozen DMF solution, at liquid N2 temperature (77 K), parameters for the copper(II) complexes of
N(4)-tolyl 2-formyl and N(4)-tolyl 2-acetylpyridine thiosemicarbazones
a
a
a
Compound
g
A
k
g =A
g^
A^
AN^
DE(d–d)b
k
k
k
[Cu(2Fo4mT)Cl] (2)
[Cu(2Fo4pT)Cl] (3)
[Cu(2Ac4oT)Cl] (4)
[Cu(2Ac4mT)Cl] (5)
[Cu(2Ac4pT)Cl] (6)
2.1850
2.1830
2.1745
2.1740
2.1745
177
177.5
182
162
184
123
122
119
134
118
2.0435
2.0435
2.0480
2.0460
2.0460
22
22
15
22
20
17
17
16
16
16
16703
16920
15818
16958
17240
a
A values in 10ꢀ4 cmꢀ1 units.
DE(d–d) transitions, in cmꢀ1 units, of the solid compounds.
b
amphotericin (4.4 lmol Lꢀ1). As already mentioned, resis-
tance to used drugs has increased due to the increased
and indiscriminate use of antibiotics and antifungal agents
in the treatment of humans and animals [13].
Table 6
Minimum inhibitory concentration (MIC) against Salmonella typhimurium
ATCC13311 for N(4)-tolyl 2-formyl, N(4)-tolyl 2-acetyl and N(4)-tolyl 2-
benzoylpyridine thiosemicarbazones and their copper(II) complexes
Compounds
MIC
Complexes
MIC
These results suggest that coordination of the studied
thiosemicarbazones with copper(II) could be an interesting
strategy for preparing new antimicrobial agents which
could be used against clinically significant bacteria and
fungi cultures presenting resistance. In the case of the anti-
fungal action, the free N(4)-tolyl thiosemicarbazones could
be promising drug candidates, as well as their copper(II)
complexes.
(lmol Lꢀ1
)
(lmol Lꢀ1
)
H2Fo4oT
H2Fo4mT
H2Fo4pT
H2Ac4oT
H2Ac4mT
H2Ac4pT
H2Bz4oT
959
959
966
890
225
450
1478
[Cu(HL1)Cl2] (1)
[Cu(2Fo4mT)Cl] (2)
[Cu(2Fo4pT)Cl] (3)
[Cu(2Ac4oT)Cl] (4)
[Cu(2Ac4mT)Cl] (5)
[Cu(2Ac4pT)Cl] (6)
[Cu(H2Bz4oT)Cl2]
(7)
[Cu(H2Bz4mT)Cl2]
(8)
[Cu(H2Bz4pT)Cl2]
(9)
30
24
20
21
15
13
12
Structure–activity relationship analyses are presently
underway in our laboratory as well as an investigation on
the mechanisms of action of the studied compounds.
H2Bz4mT
985
985
12
5
8
H2Bz4pT
Cloramphenicol
CuCl2 Æ 2H2O
2663
4. Supplementary material
CCDC 632461 and 632460 contain the supplementary
crystallographic data for H2Ac4mT and [Cu(2Ac4mT)Cl]
(5). These data can be obtained free of charge via http://
www.ccdc.cam.ac.uk/conts/retrieving.html, or from the
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or
e-mail: deposit@ccdc.cam.ac.uk.
Table 7
Minimum inhibitory concentration (MIC) against Candida albicans ATCC
18804 for N(4)-tolyl 2-formyl, N(4)-tolyl 2-acetyl and N(4)-tolyl 2-
benzoylpyridine thiosemicarbazones and their copper(II) complexes
Compounds
MIC
(lmol Lꢀ1
Complexes
MIC
(lmol Lꢀ1
)
)
H2Fo4oT
H2Fo4mT
H2Fo4pT
H2Ac4oT
H2Ac4mT
H2Ac4pT
H2Bz4oT
3
3
3
14
5
26
6
[Cu(HL1)Cl2] (1)
[Cu(2Fo4mT)Cl] (2)
[Cu(2Fo4pT)Cl] (3)
[Cu(2Ac4oT)Cl] (4)
[Cu(2Ac4mT)Cl] (5)
[Cu(2Ac4pT)Cl] (6)
[Cu(H2Bz4oT)Cl2]
(7)
4
3
2
7
4
4
2
Acknowledgements
This work was supported by CNPq and Instituto do
´
Mileˆnio-Inovac¸ao e Desenvolvimento de Novos Farmacos
˜
e Medicamentos (IM-INOFAR, Proc. CNPq 420015/05-1).
H2Bz4mT
23
[Cu(H2Bz4mT)Cl2]
(8)
[Cu(H2Bz4pT)Cl2]
(9)
0.5
0.3
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0.7
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