Synthesis and structural characterization of two new copper(II) complexes with thiazoline derivative ligands: Influence of the coordination on the phagocytic activity of human neutrophils

Article abstract of DOI:10.1016/j.ica.2010.09.031

Two new copper(II) complexes dichloro[2-(3,4-dichlorophenyl)imine-κN- (2-thiazolin-κN-2-yl)thiazolidine]copper(II) [CuCl₂(TdTn)] (1) and dichloro[2-(3,4-dichlorophenyl)imine-κN-(2-thiazolin-κN-2-yl) tetrahydrothiazine]copper(II) [CuCl₂(TzTn)] (2) were synthesized, then characterized by elemental analysis, UV-Vis-NIR diffuse reflectance, electron paramagnetic resonance (EPR) spectroscopy, magnetic susceptibility, infrared spectroscopy, and finally their crystal structures determined by X-ray diffractometry. The structural determination of [CuCl₂(TzTn)] (2) was made by conventional single-crystal diffractometry, whereas the procedure followed to resolve the crystal structure of [CuCl₂(TdTn)] (1) by means powder diffractometry using direct-space methods with a 'Monte-Carlo/parallel tempering' search algorithm. A final refinement of the crystal structure was performed using the Rietveld method. It was found that the environment around the copper(II) ion for both complexes can be described as having a distorted tetrahedral geometry, with the metallic atom coordinated with two chlorides, one imine nitrogen and one thiazoline nitrogen. The biological activity of the complexes, inorganic salt and their ligands has been evaluated by examining their phagocytic activity on human neutrophils. This activity enhances in the case of the samples treated with [CuCl₂(TdTn)] (1) and [CuCl₂(TzTn)] (2) with respect to the ones to which CuCl ₂, TdTn or TzTn was added.

Full text of DOI:10.1016/j.ica.2010.09.031

Inorganica Chimica Acta 365 (2011) 282–289
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis and structural characterization of two new copper(II) complexes with
thiazoline derivative ligands: Influence of the coordination on the phagocytic
activity of human neutrophils
a
b
c
a
A.M. Lozano-Vila a, F. Luna-Giles a, , E. Viñuelas-Zahínos , F.L. Cumbrera , A.L. Ortiz , F.J. Barros-García ,
⇑
A.B. Rodríguez d
a Departamento de Química Orgánica e Inorgánica, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain
b Departamento de Física, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain
c Departamento de Ingeniería Mecánica, Energética y de los Materiales, Escuela de Ingenierías Industriales, Universidad de Extremadura, 06071 Badajoz, Spain
d Departamento de Fisiología, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new copper(II) complexes dichloro[2-(3,4-dichlorophenyl)imine-
dine]copper(II) [CuCl2(TdTn)] (1) and dichloro[2-(3,4-dichlorophenyl)imine-
j
N-(2-thiazolin-
j
N-2-yl)thiazoli-
Received 20 May 2010
Received in revised form 15 September
2010
Accepted 20 September 2010
Available online 29 September 2010
j
N-(2-thiazolin-j
N-2-yl)tet-
rahydrothiazine]copper(II) [CuCl2(TzTn)] (2) were synthesized, then characterized by elemental analysis,
UV–Vis–NIR diffuse reflectance, electron paramagnetic resonance (EPR) spectroscopy, magnetic suscep-
tibility, infrared spectroscopy, and finally their crystal structures determined by X-ray diffractometry.
The structural determination of [CuCl2(TzTn)] (2) was made by conventional single-crystal diffractome-
try, whereas the procedure followed to resolve the crystal structure of [CuCl2(TdTn)] (1) by means pow-
der diffractometry using direct-space methods with a ‘Monte–Carlo/parallel tempering’ search algorithm.
A final refinement of the crystal structure was performed using the Rietveld method. It was found that the
environment around the copper(II) ion for both complexes can be described as having a distorted tetra-
hedral geometry, with the metallic atom coordinated with two chlorides, one imine nitrogen and one
thiazoline nitrogen. The biological activity of the complexes, inorganic salt and their ligands has been
evaluated by examining their phagocytic activity on human neutrophils. This activity enhances in the
case of the samples treated with [CuCl2(TdTn)] (1) and [CuCl2(TzTn)] (2) with respect to the ones to which
CuCl2, TdTn or TzTn was added.
Keywords:
Tetrahydrothiazine
Thiazoline
Thiazolidine
Copper(II) complexes
Human neutrophils
Phagocytosis
Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction
infections in the respiratory tract of infants [3]. Copper deficiency
reduces both the number of neutrophils in human peripheral blood
Phagocytosis is one of the principal processes by which the in-
nate immune system eliminates invading microorganisms or for-
eign materials. One type of phagocytes is the neutrophils which
are able to recognize pathogens, migrate to the site of invasion, en-
gulf the particles or microorganisms, and kill or degrade them by
means of a specialized mechanism termed the respiratory burst.
This mechanism is characterized by the generation of reactive oxy-
gen intermediates such as superoxide anion, hydroxyl radical,
hydrogen peroxide, and singlet oxygen. These reactive oxygen spe-
cies, which are responsible for killing phagocytosed organisms, are
controlled by various enzymes, including NADPH-oxidase, super-
oxide dismutase, peroxidase, and catalase [1,2].
and their ability to generate superoxide anion and kill ingested
microorganisms [4]. While the immunity dysfunction produced
by copper deficiency is resolved by copper supplementation, an ex-
cess of copper may have adverse effects on the immune system,
including phagocytic activity [5].
A few years ago, a copper complex, [Cu(KTZ)3Cl2], in which the
KTZ ligand is the antimycotic ketoconazole [6], was reported to en-
hance the phagocytic capacity activity of human neutrophils. How-
ever, the crystal structure of this copper complex was not
investigated by X-ray diffractometry [7], probably because it could
not be crystallized with the required size and quality for conven-
tional single-crystal diffractometry. Indeed, this is unfortunately
the case for many substances of chemical and biological interest.
In these situations, the crystal structure can be determined by
the alternative method of X-ray powder diffractometry (XRPD).
In the present work, we report the novel synthesis and character-
ization of two new copper(II) complexes, one with the organic ligand
[2-(3,4-dichlorophenyl)imine-N-(2-thiazolin-2-yl)thiazolidine]
Copper plays an important role in the development and mainte-
nance of the immune system, and its deficiency increases suscep-
tibility to such infections as Menkes’s disease which causes
⇑
Corresponding author. Tel./fax: +34 924289397.
E-mail address: pacoluna@unex.es (F. Luna-Giles).
0020-1693/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2010.09.031

A.M. Lozano-Vila et al. / Inorganica Chimica Acta 365 (2011) 282–289
283
Scheme 1.
(TdTn) and a second one with [2-(3,4-dichlorophenyl)imine-N-(2-
thiazolin-2-yl)tetrahydrothiazine (TzTn), whose structures contain
thiazoline, thiazolidine, and 1,3-thiazine rings (see Scheme 1). These
heterocycles form part of natural biologically-active products with
antibiotic properties, examples being micacocidin [8,9], the penicil-
lins [10], and the cephalosporins [11]. In addition to this structural
study, we also investigated the influence of the coordination on
the phagocytic activity of human neutrophils, motivated by the fact
that the coordination of zinc(II) with TdTn has been shown to
improve the phagocytosis stimulating effect of TdTn [12], an exam-
ple of the biological activity of a compound being enhanced by
coordination or chelation with metal ions.
rated by filtration, washed with cold ether and air-dried. Anal. Calc.
for C12H11Cl4CuN3S2: C, 30.88; H, 2.38; N, 9.00; S, 13.74. Found: C,
30.98; H, 2.65; N, 8.70; S, 14.02%. IR (KBr): m(C@N) 1614, (thiazo-
line ring vibrations) 1538, 1000, 953, 773, 724, 633, 589, 534,
444 (thiazolidine ring vibrations) 1032, 916, 827, 700, 673, 484,
408 cmꢀ1
.
2.3. Preparation of [CuCl2(TzTn)] (2)
The [CuCl2(TzTn)] complex was isolated from an ethanolic (96%)
solution (1 mL) of CuCl2ꢁ2H2O (49.2 mg, 0.29 mmol) that was
added to an ethanol–water solution (15 mL) of TzTn (100 mg,
0.29 mmol). The resulting solution was allowed to evaporate
slowly at room temperature. After a few days, green crystals were
isolated from the solution (94 mg, 67.7%). The crystals were first
separated by filtration, then washed with cold ether and finally
air-dried. Anal. Calc. for C13H13Cl4CuN3S2: C, 32.50; H, 2.73; N,
8.74; S, 13.30. Found: C, 32.35; H, 2.78; N, 8.85; S, 12.98%. IR
2. Experimental
2.1. General procedures
All reagents were commercial grade material and were used
without further purification. The ligands [2-(3,4-dichlorophenyl)
imino-N-(2-thiazolin-2-yl)thiazolidine] (TdTn) and [2-(3,4-dichloro-
phenyl)imino-N-(2-thiazolin-2-yl)tetrahydrothiazine (TzTn) were
synthesized according to reported procedures [13] and [14],
respectively.
(KBr): m(C@N) 1594 (thiazoline ring vibrations) 1524, 962, 947,
778, 757, 721, 647, 604, 551, 455, 444 (tetrahydro-1,3-thiazine ring
vibrations) 1027, 911, 836, 828, 695, 674, 659, 587, 400 cmꢀ1
.
2.4. Isolation of neutrophils
Chemical analyses of carbon, hydrogen, nitrogen, and sulphur
were performed by microanalytical methods using a Leco CHNS-
932 microanalyser. IR spectra were recorded on a Perkin–Elmer
FT-IR 1720 spectrophotometer, from KBr pellets in the 4000–
370 cmꢀ1 range and on a Perkin–Elmer FT-IR 1700X spectropho-
tometer, from polyethylene pellets in the 500–150 cmꢀ1 range.
Solid state electronic spectra for complexes in the 200–1500 nm
range were obtained from pellets of the samples, using a Shimadzu
UV-3101 PC spectrophotometer and BaSO4 as a reference. Magnetic
susceptibility measurements were performed on polycrystalline
samples using a magnetometer with pendulum MANICS DSM8,
equipped with helium continuous-flow cryostat and an electromag-
netometer DRUSCH EAF 16 UE. Data were corrected for temperature
independent paramagnetism and diamagnetic contributions, which
were estimated from the Pascal constants. EPR spectra were re-
corded at room temperature in solid state and at 77 K in frozen
DMSO employing a BRUKER ESP-300E spectrometer using the X
band of microwave.
Neutrophils were isolated by adding in thin glass tubes and in
the following order, 2.5 mL of 1119 Histopaque (Sigma–Aldrich)
separating medium, 2.5 mL of 1077 Histopaque (Sigma–Aldrich)
separating medium and 2.5 mL of human peripheral blood from
six healthy volunteers who were not treated with antibiotics, anti-
inflammatories or anticoagulants, in order to not affect cell adher-
ence (this was not necessary for cell viability). The samples were
then centrifuged at 600g for 15 min at room temperature. The halo
of neutrophils was withdrawn using a glass Pasteur pipette and
washed with PBS medium in plastic tubes. The supernatant was
discarded and the tube was beaten so that cells detached. Then,
2 mL of Hank’s medium was added and the pellet resuspended.
Cells were counted in a Neubauer haemocytometer under phase
contrast microscopy at 40ꢂ, and the suspension was adjusted to
5 ꢂ 105 neutrophils/mL.
2.5. Cell viability
2.2. Preparation of [CuCl2(TdTn)] (1)
To study the cell viability and the phagocytosis of latex beads
the tested substances were dissolved in H2O:DMSO (9:1). Cell via-
bility was assessed using calcein as fluorescent probe. For calcein
loading, cells were incubated for 45 min with 1 M calcein AM at
37 °C and centrifuged 10 min at 480g, resuspending the pellet in
fresh Na-HEPES buffer. Aliquots of 2 mL of the cell suspension were
treated with 1 ꢂ 10ꢀ3 mM CuCl2, TdTn, TzTn, complexes 1 and 2 for
30 min at room temperature, then cells were centrifuged and
This complex was isolated from a freshly prepared ethanolic
(96%) solution (1 mL) of CuCl2ꢁ2H2O (51.3 mg, 0.30 mmol) that
was added to an ethanol solution (15 mL) of TdTn (100 mg,
0.30 mmol). The resulting solution was allowed to evaporate
slowly at room temperature. After a few days, green crystals were
isolated from the solution (82 mg, 58.4%). The crystals were sepa-

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A.M. Lozano-Vila et al. / Inorganica Chimica Acta 365 (2011) 282–289
Table 1
resuspended in fresh buffer. Finally, the calcein fluorescence
remaining in the cells after treatments with these substances
was recorded using a Shimadzu RF-5301-PC fluorescence spectro-
photometer. Samples were excited at 490 nm and the resulting
fluorescence was measured at 510 nm.
Crystal data, data collection and refinement details for TzTn and 2.
TzTn
2
Crystal shape
Colour
prism
colourless
prism
green
Size (mm)
0.58 ꢂ 0.38 ꢂ 0.35
0.42 ꢂ 0.36 ꢂ 0.32
C13H13Cl4CuN3S2
480.72
monoclinic
P21/c
Chemical formula
Formula weight
Crystal system
Space group
Unit cell dimensions
a (Å)
C13H13Cl2N3S2
2.6. Phagocytosis of latex beads
346.28
triclinic
Aliquots of 200 lL of Hank’s cellular suspension were put into
ꢀ
P1
the wells of plastic macrophage migration inhibition factor
(MIF)-type plaques (AFORA, Spain), following the method
described by Rodríguez et al. [15]. After 30 min incubation at
37 °C in a stove, the adhered monolayer was washed with PBS at
10.967(1)
12.163(1)
12.829(1)
85.505(1)
80.517(1)
64.147(1)
1519.0(2)
4
9.873(1)
17.386(1)
10.386(1)
b (Å)
c (Å)
a
(°)
37 °C. Then, 200 lL of Hank’s medium, 20 lL of latex beads
b (°)
96.361(1)
(1.1 ꢂ 10ꢀ3 mm, diluted to 1% in DMSO; Sigma) and 3
lL of ligand
a final concentration of
c
(°)
Cell volume (Å3)
1771.8(2)
4
1.802
or complex 1 ꢂ 10ꢀ4 M, yielding
Z
1.3 ꢂ 10ꢀ3 mM (3
lL of H2O:DMSO (9:1) for control) were added,
Dcalc (g cmꢀ3
)
1.5514
0.694
following by another 30 min incubation at 37 °C in a stove. Finally,
the samples were washed with PBS, fixed with methanol 5 min and
stained with eosin (five passes) and haematoxylin (five passes).
The plaques were carefully rinsed with tap water and dried,
followed by counting under oil-immersion phase-contrast micros-
copy at 100ꢂ.
l
(mmꢀ1
)
2.071
F(0 0 0)
2h range
Index ranges
712
964
3.2–56.7
ꢀ14 6 h 6 14,
ꢀ15 6 k 6 15
ꢀ16 6 l 6 16
6880
4.2–58.3
ꢀ13 6 h 6 13,
ꢀ22 6 k 6 22,
ꢀ14 6 l 6 14
3923
Independent
reflections
Observed reflections 5706[F > 4.0
Maximum/minimum 0.7832/0.7289
transmission
Number of refined
parameters
The number of particles ingested per 100 neutrophils was
expressed as the latex-bead phagocytosis index (P.I.). The percent-
age of cells that had phagocytosed at least one latex bead was
expressed as the phagocytosis percentage (P.P.). The ratio P.I./P.P.
was calculated, giving the phagocytosis efficiency (P.E.).
r(F)]
3552[F > 4.0 r(F)]
0.5571/0.4767
358
208
R [I > 2
r
(I)]a
(I)]b
0.049
0.125
1.034
0.030
0.077
1.102
wR [I > 2
r
2.7. Statistical analysis
GOFc
qmaximum, qminimum
0.652, ꢀ0.583
0.756, ꢀ0.472
(e Åꢀ3
)
The results of the biological study were analyzed using the AN-
OVA-Scheffe’s F-test. P < 0.05 was considered statistically signifi-
cant. Data were expressed as the mean (X) standard error (SE)
of six experiments, performed in duplicate. In the phagocytosis
studies, results were referred to 100% of the control to avoid the
dispersion due to the great variability of patients’ cells phagocytic
activity.
a
R ¼
R ¼
R
jjFoj ꢀ jFcjj=
R
jFoj.
ꢄ
ꢂ
ꢃ
ꢂ
ꢃꢅ
ꢄ
1=2
ꢀ
ꢁ
ꢀ
ꢁ
2
2
F2o ꢀ F2c
=
R
w
Fo2
.
b
R
w
ꢂ
ꢃ
ꢅ
1=2
ꢀ
ꢁ
2
À
Á
The goodness-of-fit (GOF) equals
R
w
F2o ꢀ F2c
= Nrf ln s ꢀ Nparams
.
c
2.8. Crystal structure determinations
and 0.41(1) for C(6B2) in second one. All hydrogen atoms attached
to carbon and nitrogen atoms were positioned geometrically, with
Uiso values derived from Ueq values of the corresponding carbon
and nitrogen atoms. Diagrams were generated using ORTEP3 for
Windows [20]. A summary of the crystal data, experimental details
and refinement results are listed in Table 1.
Crystals of the TzTn ligand and complex 2 with the appropriate
size and quality for X-ray single-crystal diffractometry were iso-
lated and used for the structural analysis. Unfortunately, this was
not possible for complex 1 and therefore their crystal structure
was determined by X-ray powder diffractometry.
The XRD pattern for the structural determination of complex 1
was obtained via conventional powder diffractometry. Before data
collection, the powder particles resulting from the synthesis of
complex 1 were ground ultra-fine to avoid texture and graininess
effects in the XRPD pattern. The resulting powder was then loaded
into the holder (3-mm thick), and pressed slightly with a glass slide
to ensure a flat surface and thus the absence of instrumental shift
in the position of the XRPD peaks. Table 2 gives the specific exper-
imental and set-up conditions used. The determination of the crys-
tal structure of complex 1 was performed from its XRPD pattern by
successive stages of molecular modeling, direct-space methods,
and Rietveld refinement. Firstly, the most stable conformation of
the isolated molecule was obtained by molecular modeling with
a semi-empirical quantum-chemical model using the HYPERCHEM
program [21]. Subsequently, a first estimate of the crystal structure
was obtained by means of direct-space methods with Monte Carlo
algorithms and the ‘‘parallel tempering” formulation using the FOX
program [22]. This was done using as input the orthorhombic unit
cell and space group (Pbcn) determined by indexing and analyzing
the first 30 XRD peaks with the CRYSFIRE and CHECKCELL programs
[23,24]. Lastly, the so-determined crystal structure was refined
The experimental details of the crystal structure determination,
including crystal data, data collection, structure determination,
and refinement are listed in Table 1 for the ligand and complex
2. X-ray single-crystal diffraction measurements were performed
using a Bruker SMART CCD diffractometer with Mo K
a radiation
(k = 0.71073 Å, graphite monochromator). The first fifty frames
were measured at the end of data collection to monitor instrument
and crystal stability. The data were empirically corrected for
absorption and other effects using the SADABS [16] program. The
structures were solved by direct methods and subsequent Fourier
differences using the SHELXS-97 [17] program and refined by full-
matrix least-squares on F2 with SHELXL-97 [18], included in WINGX
[19] package assuming anisotropic displacement parameters for
non-hydrogen atoms, except for C(3A), C(2B) and C(6B) disordered
atoms with isotropic displacement parameters. In the structure of
TzTn, the thiazoline ring of both independent molecules and the
thiazine ring of second one show a disorder affecting C(3A),
C(2B) and C(6B) atoms, being the value of the refined occupancy
0.57(1) for C(3A) and 0.43(1) for C(3A2) in first independent mol-
ecule and 0.48(2) for C(2B), 0.52(2) for C(2B2), 0.59(1) for C(6B)

A.M. Lozano-Vila et al. / Inorganica Chimica Acta 365 (2011) 282–289
285
Table 2
Details of the data collection, structure solution and refinement for 1.
Colour
Green
C12H11Cl4CuN3S2
Chemical formula
Crystal system
Space group
orthorhombic
Pbcn
Unit cell dimensions
a (Å)
17.88840(14)
b (Å)
8.91130(8)
c (Å)
23.59260(23)
3760.87(5)
Cell volume (Å3)
Z
8
2h range
5–100
Step scan increment (°), counting time (s)
Number of reflections
Number of atoms (H excluded)
Number of global parameters refined
Number of profile parameters refined
Number of structural parameters refined
Rwp, Rp, v2r
0.02, 20
2046
22
117
10
55
0.091, 0.064, 8.51
Fig. 2. The atom numbering scheme for two molecules on TzTn constituting the
asymmetric unit of the structure. The thermal ellipsoids are plotted at the 50%
probability level.
S(1A) = 118.0(2)°; N(1B)–C(1B)–S(1B) = 118.1(2)°] and relatively
long SII–C(sp2) bonds [S(1A)–C(1A) = 1.767(2) Å; S(1B)–C(1B) =
1.773(3) Å], which are characteristic of a thiazoline ring [29]. In
contrast, in the S(2)–C(4)–N(2)–C(5)–C(6)–C(7) rings, the
endocyclic C–N bonds [C(4A)–N(2A) = 1.387(3) Å; C(4B)–N(2B) =
1.383(3) Å] are longer to the exocyclic C@N bonds [C(4A)–
N(3A) = 1.266(3) Å; C(4B)–N(3B) = 1.266(3) Å], the S–C(sp2) bonds
[S(2A)–C(4A) = 1.763(2) Å; S(2B)-C(4B) = 1.763(2) Å] and the S-C-
Fig. 1. Plot output from the Rietveld analysis of the X-ray diffraction pattern
corresponding to 1. Points are the experimental X-ray diffraction data; the solid line
is the calculated pattern; the residuals are plotted at the bottom.
N
angles [S(2A)–C(4A)–N(2A) = 121,4(2)°; S(2B)–C(4B)–N(2B) =
by the Rietveld method using the FULLPROF program [25]. The Riet-
veld refinement, which was made by first taking the molecules
to be rigid bodies and then replacing the rigid-body constraints
by soft restraints on the atom–atom angles and distances, included
the typical crystallographic, profile, and instrumental aspects
[14,26–28]. The residuals at the completion of the Rietveld
refinement were Rwp = 0.091 and Rp = 0.064, and the refined
cell parameters were a = 17.88840(14) Å, b = 8.91130(8) Å, and
c = 23.59260(23) Å. Fig. 1 shows the Rietveld refinement plot out-
put and Table 2 lists the most representative crystallographic
parameters of this structure.
121,1(2)°]; are lower to those found in a 1,3-thiazine ring [12]
which we seems indicate an imine-tetrahydro-1,3-thiazine form.
It is known that the imine tautomers can exist as two geomet-
rical isomers, syn (Z) and anti (E), but in both crystals of two
ligands, only Z isomers have been observed.
We discuss next the crystal structure of copper complexes,
although our interpretation of complex 1 should be treated with
caution since the crystallographic data were determined from
powder diffractometry.
The structure of copper complexes consists of discrete neutral
monomeric units in which the environment around the copper(II)
atom may be described as a distorted tetrahedral geometry. In both
complexes, the metallic atom is coordinated by two chloride
atoms, one imine nitrogen and one thiazoline nitrogen. A diagram
of the molecular structures and the atom numbering system used
is shown in Figs. 3 and 4. The most relevant bond lengths and an-
gles are given in Table 4.
The Cu–Cl bond lengths [Cu–Cl(1) and Cu–Cl(2) in 1; Cu–Cl(2)
in 2] are slightly shorter than the calculated average value
[2.248(34) Å] for 291 tetrahedral copper(II) complexes with a
Cl2N2 coordination environment around Cu(II) ion, obtained with
CONQUEST software [30] from the Cambridge Structural Database
(CSD, Version v5.31, Feb. 2010) [31]. However, the Cu–Cl(1) distance
in 2 is slightly longer than the aforementioned mean value.
3. Results and discussion
3.1. Description of the crystal structures
The single crystal X-ray diffraction analysis has revealed that
TzTn crystallizes in the triclinic system with two independent mol-
ecules in the asymmetric unit cell. The overall view and labeling of
the atoms in two independent molecules are displayed in Fig. 2.
Table 3 lists the selected bond lengths and angles.
In the S(1)–C(1)–N(1)–C(2)–C(3) rings, the short endocyclic
C@N bonds [C(1A)–N(1A) = 1.267(3) Å; C(1B)-N(1B) = 1.265(3) Å]
are accompanied by high N–C–S angles [N(1A)–C(1A)–

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Table 3
Table 4
Selected bond lengths (Å), angles (°) and hydrogen-bond parameters for TzTn.
Selected bond lengths (Å) and angles (°) for 1 and 2.
S(1A)–C(1A)
S(2A)–C(4A)
N(1A)–C(1A)
N(2A)–C(1A)
N(2A)–C(4A)
N(3A)–C(4A)
N(3A)–C(8A)
1.767(2)
1.763(2)
1.267(3)
1.399(3)
1.387(3)
1.266(3)
1.412(3)
S(1B)–C(1B)
S(2B)–C(4B)
N(1B)–C(1B)
N(2B)–C(1B)
N(2B)–C(4B)
N(3B)–C(4B)
N(3B)–C(8B)
1.773(3)
1.763(2)
1.265(3)
1.397(3)
1.383(3)
1.266(3)
1.406(3)
1
2
Cu–Cl(1)
Cu–Cl(2)
Cu–N(1)
Cu–N(3)
2.180(5)
2.188(2)
1.914(8)
1.932(16)
2.316(1)
2.197(1)
1.949(2)
2.046(2)
Cl(1)–Cu–Cl(2)
N(3)–Cu–Cl(1)
Cl(2)–Cu–N(3)
N(1)–Cu–Cl(1)
Cl(2)–Cu–N(1)
N(3)–Cu-N(1)
97.2(2)
143.1(9)
99.1(6)
94.8(3)
137.0(4)
95.5(11)
98.3(1)
145.6(1)
95.3(1)
99.6(1)
144.7(1)
86.8(1)
C(1A)–N(1A)–C(2A)
C(1A)–N(2A)–C(4A)
N(1A)–C(1A)–S(1A)
N(1A)–C(1A)–N(2A)
N(2A)–C(1A)–S(1A)
N(2A)–C(4A)–S(2A)
N(3A)–C(4A)–N(2A)
C(4A)–N(3A)–C(8A)
119.9(2)
121.0(2)
118.0(2)
119.8(2)
122.2(2)
121.3(2)
119.4(2)
122.2(2)
C(1B)–N(1B)–C(2B)
C(1B)–N(2B)–C(4B)
N(1B)–C(1B)–S(1B)
N(1B)–C(1B)–N(2B)
N(2B)–C(1B)–S(1B)
N(2B)–C(4B)–S(2B)
N(3B)–C(4B)–N(2B)
C(4B)–N(3B)–C(8B)
106.3(4)
121.6(2)
118.1(2)
119.6(2)
122.3(2)
121.0(3)
120.1(2)
122.6(2)
in 2 is larger than in 1, being indicated by the ligand–metal–ligand
bite angles [that vary between 143.1(9)° and 94.8(3)° in 1 and
145.6(1)° and 86.8(1)° in 2] and by the dihedral angle between
the planes Cl(1)–Cu–Cl(2) y N(1)–Cu–N(3) are [54.6° in 1 and
48.3° in 2].
As it can be seen in Figs. 3 and 4, thiazoline rings rotate around
C(1)–N(2) bond in such a way that the N(1) and N(3) atoms are on
the same side in order to coordinate to copper(II) ion. This is
demonstrated by the values of the correspondent torsion angles:
S(1)–C(1)–N(2)–C(5) = 7.3(1)° in 1, ꢀ175.6(2)° in TdTn [12]; S(1)–
C(1)–N(2)–C(5) = 6.5° in 2; 177.9° and ꢀ178.2° in unit A and B of
TzTn, respectively.
At last, there has been an increase of the distance N(3)–C(7) (in
1) and N(3)–C(8) (in 2) with respect to the respective ligand: N(3)–
C(7) = 1.421(2) Å in TdTn [12], 1.455(1) Å in 1; N(3)–C(8) =
1.413(4) Å and 1.403(4) Å in unit A and B of TzTn, respectively,
1.418(3) Å in 2. This could be due to the lost of electronic density
of the imine nitrogen atom when coordinates the metallic ion.
Fig. 3. Molecular structure of 1 showing the atom-numbering scheme. The thermal
ellipsoids are drawn at a 50% level.
3.2. Electronic spectra
The electronic reflectance spectra of the copper(II) complexes
are very close which is a consequence of molecular structure
similarity. They were consisted of two strong bands at 35710 and
24940 cmꢀ1 for complex 1 and at 32360 and 24270 cmꢀ1 for com-
plex 2. For both compounds the bands registered around
34000 cmꢀ1 are assigned to the ligand’s
p ? p* transition [14,32]
whereas the bands registered near to 24000 cmꢀ1 are assigned to
the chlorine-to-copper change transfer transition [33]. There were
also two d–d bands of equal intensity at 10640 and 14770 cmꢀ1 for
1 and at 10170 and 14430 cmꢀ1 for 2, which are consistent with a
compressed tetrahedral geometry [34,35] in accord with the dihe-
dral angle values between N–Cu–N and Cl–Cu–Cl obtained means
X-ray diffraction data.
For a distorted tetrahedral symmetry flattening towards xy
plane, the energetic d orbitals sequence would be dxy > dxz, dyz > d-
x2–y2 > dz2 [36]. However, in complexes with bidentate ligands the
distortion is not uniaxial and all the degeneracy is removed. For
these copper(II) complexes with a CuCl2N2 chromophore group
and C2v symmetry (if only the atoms bonded to the metal are con-
sidered) 2E(D) state splits to 2A1 and 2A2 terms whereas 2T2(D)
state splits to A1, B1 and B2 and the energetic d orbital sequence
is dx2–y2 > dxy > dxz > dz2 > dyz [37,38]. Thus the bands centered at
10 640 cmꢀ1 for 1 and 10 170 cmꢀ1 for 2 correspond to 2A2(E)-
[(dxy)1] 2A1(T2)[(dx2–y2)1] and 2B1(T2)[(dxz)1] 2A1(T2)[(dx2–y2)1]
Fig. 4. Molecular structure of 2 showing the atom-numbering scheme. The thermal
ellipsoids are drawn at a 50% level.
Likewise, the Cu–Nimine bond distances are similar than the mean
value [1.965(63) Å] calculated for 121 tetrahedral copper(II) com-
plexes in CSD [31]. The Cu–Nthiazoline bond distances are slightly
shorter than the corresponding mean value [1.963(39) Å] found
for 18 copper(II) complexes with Cu–Nthiazoline bonds in CSD [31].
The distortion of the tetrahedral environment of the copper(II) ion
transitions and the bands at 14 470 cmꢀ1 for
1 and at
14 430 cmꢀ1 for 2 correspond to 2A1(E)[(dz2)1] 2A1(T2)[(dx2–y2)1]
and 2B2(T2)[(dyz)1] 2A1(T2)[(dx2–y2)1] transitions.
Several authors have suggested a correlation between the spin
allowed transitions caused by the ligand field splitting of a
tetrahedral environment to square planar and the dihedral angle

A.M. Lozano-Vila et al. / Inorganica Chimica Acta 365 (2011) 282–289
287
Table 5
EPR parameters and far IR bands of copper complexes.
Compound
EPR parameters
Solid (298 K)
Far IR bands (cmꢀ1
)
DMSO (77 K)
g||
giso
g||
g ?
g ?
A||a
G
m(Cu–Clterminal
)
m
(Cu–Nimine
)
m(Cu–Nthiazoline)
1
2
2.080
2.295
2.065
134
4.54
320
301
337
314
350
286
2.266
2.055
2.294
2.065
135
4.52
337
281
a
Units: ꢂ10ꢀ4 cmꢀ1
.
subtended by the ligands bonded at copper [39–41]. As the spectra
of our compounds presents only two bands, we can to deduce that
the difference of position between them increase with the dihedral
angle. The higher separation between d–d transitions of complex 2
suggests a higher distortion degree of tetrahedral geometry, which
is in good accord with the X-ray structural determination data.
numbers compared with that for the respective free ligand. From
all this mentioned can be deduced a coordination via the imine
nitrogens of the ligands [32,47–50].
In the low-frequency region, the C1 symmetry of 1 and 2 cations
predicts the appearance of four bands assignable to metal–ligand
stretching vibrations. Tentative assignments are listed in Table 5.
These bands have been assigned in good agreement with literature
data. In this way,
complexes with the chromophore group CuCl2N2 in the range
320–254 cmꢀ1 [51]. Likewise,
(Cu–Nimine) vibration is registered
in several copper(II) complexes between 365 and 278 cmꢀ1 [47–
m(Cu–Cl) vibrations are detected in copper(II)
3.3. Magnetic susceptibility measurements
m
The observed molar magnetic susceptibility for complexes 1
and 2 was corrected for diamagnetism and temperature-indepen-
dent paramagnetism to provide the fully corrected magnetic mo-
ments at room temperature of 2.19 and 2.02 BM, respectively.
These values can be considered in the range 1.75–2.20 BM typical
for mononuclear copper(II) complexes without Cu–Cu interactions,
regardless of the stereochemistry and independently of tempera-
ture, except at extremely low temperatures (<5 K) [42].
49,52,53], whereas m(Cu–Nthiazoline) vibration are assigned between
253 and 222 cmꢀ1 in complexes with this type of bonds [52–57].
3.6. Cell viability
The results obtained confirm that there is not any significant fall
between the calcein fluorescence remaining in the cells before and
after treatment, according to Scheffe F-test. This suggests that the
treatment with CuCl2, TdTn, TzTn, 1 and 2 does not cause any cel-
lular damage under the work conditions and during the time our
experiments lasted. The percentage of cells that were viable after
the different treatments were: 99.6 0.4 for CuCl2, 84.0 4.9 for
TdTn, 84.7 6.8 for TzTn, 83.4 2.2 for 1, 99.4 0.5 for 2 and
87.5 4.9 for the control sample H2O:DMSO (9:1). Each value rep-
resents the mean standard error of six determinations performed
in duplicate.
3.4. EPR data
The EPR spectra of 1 and 2 were recorded in the X-band using
the 100 kHz field modulation for polycrystalline samples at 298 K
and for samples in DMSO solution at 77 K.
The EPR parameters of copper(II) complexes are presented in
Table 5.
The spectrum of 1 in the solid state at 298 K shows an isotropic
profile (giso = 2.08). Such an isotropic spectrum gives no informa-
tion on the electronic ground state of the copper(II) ion present
in the compound. However, the spectrum of 2 at same temperature
is typically axial with well-defined g|| and g ? values.
3.7. Study of the phagocytic function
With respect to the EPR spectra of 1 and 2 in frozen DMSO at
77 K these show the presence of four hyperfine lines, which are
characteristic of monomer copper(II) complexes [33] (no superhy-
perfine structure is observed). Likewise, the geometric parameter G
[G = (g|| ꢀ 2)/(g\ ꢀ 2)] in both complexes is found to be in the
range 3.5–5.0, indicating a negligible exchange coupling among
magnetically no equivalent copper(II) ions in the unit cell
[43,44]. Moreover, the g values in the solid state at room temper-
ature and in frozen solution for complex 2 are not much different
from each other, hence the geometry around the copper ion is
unaffected on cooling the solution to liquid nitrogen temperature.
The trend exhibited by the g values (g|| > g\ > 2.0023) are con-
sistent with a (dx2 ꢀ y2)1 ground state of a compressed tetrahedral
environment with a chromophore group CuCl2N2 [45,46].
The capacity of neutrophils to ingest latex beads, expressed as
the phagocytosis index (Fig. 5A), increases as consequence of the
treatment of neutrophils with our compounds, except with TzTn.
This growth is statistically significant (P < 0.05) in the case of cells
treated with 1 in comparison with control and the sample treated
with TdTn. Likewise, for cells treated with 2 the growth is statisti-
cally significant (P < 0.05 or P < 0.001) in comparison with control,
the sample treated with CuCl2 and the sample treated with TzTn.
Likewise, the phagocytosis percentage (Fig. 5B) is significantly
higher (P < 0.05) in the case of cells treated with 1 with respect
to control and CuCl2, which means that there are more active cells.
Besides, this parameter is also significantly greater (P < 0.001) than
control and TzTn in the case of the sample treated with TdTn. Final-
ly, in the case of phagocytic efficiency for complexes 1 and 2
(Fig. 5C), this is higher in treated neutrophils. This percentage is
significantly greater (P < 0.05 or P < 0.001) than the control values
in the case of the samples treated with 1 (too with samples treated
with CuCl2 and TdTn) and with 2 (too with samples treated with
CuCl2, TzTn and 1).
3.5. IR spectra
The IR spectra of complexes
1 and 2 showed a strong
m
(C@N)imine absorption (at 1614 cmꢀ1 in 1 and at 1594 cmꢀ1 in
2). These bands are shifted negatively relative to the uncoordinated
imine function of the respective ligand [14,32]. Likewise, the in-
With respect to results obtained for similar zinc(II) complexes
previously reported [12], cells treated with copper(II) complexes
show a higher biological activity. Thus, cells treated with complex
2 have a significantly higher phagocytosis index and phagocytic
plane stretching vibrations corresponding to W1[m(C@N)] skeletal
vibration of the heterocyclic ring were also shifted to lower wave-

288
A.M. Lozano-Vila et al. / Inorganica Chimica Acta 365 (2011) 282–289
with 2 and 1 with respect to the ones to which CuCl2, TdTn or TzTn
was added.
Acknowledgements
We would like to thank the Junta de Extremadura (III PRI+D+I)
and the FEDER [Project PRI08A022] for financial support.
Appendix A. Supplementary material
CCDC 691948, 726711 and 726712 contain the supplementary
crystallographic data for CuCl2(TdTn), CuCl2(TzTn) and TzTn,
respectively. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif. Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/j.ica.2010.
09.031.
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