Zinc(II) complexes with novel 1,3-thiazine/pyrazole derivative ligands: Synthesis, structural characterization and effect of coordination on the phagocytic activity of human neutrophils

Article abstract of DOI:10.1016/j.poly.2011.07.014

The new ligands 2-(1-pyrazolil)-1,3-thiazine (PzTz), 2-(3,5-dimethyl-1- pyrazolil)-1,3-thiazine (DMPzTz) and 2-(3,5-diphenyl-1-pyrazolil)-1,3-thiazine (DPhPzTz) and the complexes [ZnCl₂(H₂O)(PzTz)] (1), [ZnCl₂(DMPzTz)] (2)

Full text of DOI:10.1016/j.poly.2011.07.014

Polyhedron 30 (2011) 2627–2636
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Zinc(II) complexes with novel 1,3-thiazine/pyrazole derivative ligands:
Synthesis, structural characterization and effect of coordination on the
phagocytic activity of human neutrophils
b
a
P. Torres-García a, E. Viñuelas-Zahínos a, F. Luna-Giles a, , J. Espino , F.J. Barros-García
⇑
a Departamento de Química Orgánica e Inorgánica, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain
b 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:
The new ligands 2-(1-pyrazolil)-1,3-thiazine (PzTz), 2-(3,5-dimethyl-1-pyrazolil)-1,3-thiazine (DMPzTz)
and 2-(3,5-diphenyl-1-pyrazolil)-1,3-thiazine (DPhPzTz) and the complexes [ZnCl2(H2O)(PzTz)] (1),
[ZnCl2(DMPzTz)] (2) and [ZnCl2(DPhPzTz)] (3) have been isolated and then characterized by elemental
analysis, IR spectra and UV–Vis spectroscopy. Besides, the crystal structure of ligands PzTz and DPhPzTz
and complexes 1–3 have been determined by single-crystal X-ray diffraction. In 1, the geometry around
the Zn(II) atom can be considered a highly distorted trigonal bipyramid, with the metallic atom bonded to
two chlorine atoms, one water molecule and one bidentate PzTz ligand. In 2 and 3, the environment
around the metal ion can be described as a distorted tetrahedron with the zinc atom coordinated to
one bidentate organic ligand molecule and two chloro ligands. In addition, the phagocytic function of
human neutrophils treated with complexes 1–3, their organic ligands and ZnCl2 has been evaluated.
The activity of cells enhanced in samples treated with 1, 2 and 3 with respect to the ones to which the
inorganic salt, PzTz, DMPzTz or DPhPzTz were added.
Received 5 May 2011
Accepted 15 July 2011
Available online 23 July 2011
Keywords:
Thiazine
Pyrazole
Zinc(II) complexes
Crystal structures
Phagocytosis
Human neutrophils
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Zn(II) ion which acts on channels of the neutrophil membrane
[6]. Likewise, by its antioxidant capacity, zinc contributes to the
Human neutrophil polymorphonuclear leukocytes play an
important role in the innate immune system (cell-mediated
defense system) since these cells are able to recognize invading
microorganism, migrate to the infected area, incorporate the
pathogens into their cytoplasm and degrade them using a chemical
process named respiratory burst, by means of which reactive oxy-
gen intermediates, such as superoxide anions, hydrogen peroxide
or hydroxyl radicals, are formed. The generation of these reactive
species, that are responsible for killing phagocytosed microorgan-
ism, is controlled by several enzymes, including NADPH-oxidase,
superoxide dismutase, peroxidase and catalase [1,2].
protection of cells from the damaging effects of reactive oxygen
radicals produced during immune activation [5].
It is well-known that some coordination compounds can inter-
fere with in vitro phagocytosis, either inhibiting it, as it is the case
of certain diclofenac transition metal complexes [7], or improving
it, like copper(II) and ruthenium(II) complexes [8]. Additionally, it
has been proved that some zinc(II) [9], cadmium(II) [10] and cop-
per(II) [11] complexes with several thiazoline or thiazine deriva-
tive ligands are able to improve the phagocytic activity of human
neutrophils with respect to the untreated samples (control) and
the samples treated with the organic ligands.
Zinc is an essential trace element for many biological functions,
including immune functions [3]. A large number of enzymes de-
pend on zinc for their catalytic activity (e.g. alcohol dehydroge-
nase, RNA polymerases, NADPH-oxidase, superoxide dismutase)
and a deficiency of zinc is associated with loss of the enzymatic
activity and impaired immune responses like phagocytic function
In this paper we present the synthesis of new ligands
2-(1-pyrazolyl)-1,3-thiazine (PzTz), 2-(3,5-dimethyl-1-pyrazolyl)-
1,3-thiazine (DMPzTz) and 2-(3,5-diphenyl-1-pyrazolyl)-1,3-thia-
zine (DPhPzTz), whose structures contain pyrazole and 1,3-thia-
zine rings (see Scheme 1). These heterocycles are present in
several natural and synthetic products with antimicrobial, analge-
sic and antiinflammatory properties, examples being celecoxib
[12] and the cephalosporins [13]. The ligands have been character-
ized by elemental analysis, IR spectroscopy, 1H and 13C NMR
spectroscopy and, in the case of PzTz and DPhPzTz, by single
crystal X-ray diffraction. Besides, their complexes with Zn(II) have
been obtained and structurally characterized by elemental
ꢀ
[4,5]. Thus, the generation of superoxide anion O2 from O2 in
phagocytes by means of NADPH oxidase activity is regulated by
⇑
Corresponding author. Tel.: +34 924 289 397; fax: +34 924 271 149.
E-mail address: pacoluna@unex.es (F. Luna-Giles).
0277-5387/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2011.07.014

2628
P. Torres-García et al. / Polyhedron 30 (2011) 2627–2636
a DMSO solution of the organic ligands and the complexes at a con-
centration of 10ꢀ5 M were measured on a Shimadzu UV-3101 PC
spectrophotometer using a 10 mm quartz cell.
2.2. Synthesis of the ligands
2.2.1. Synthesis of 2-(1-pyrazolyl)-1,3-thiazine (PzTz)
Sodium hydride 60% (0.600 g, 0.017 mol) was added to a solu-
tion of pyrazole (0.695 g, 0.010 mol) in toluene (50 mL). The mix-
ture was stirred at room temperature for 3 h. Then a solution of
3-chloropropylisothiocyanate (1.490 g, 0.010 mol) in toluene
(5 mL) was added and the reaction mixture was refluxed for 4 h
and cooled. After the addition of methanol (5 mL) the solvent
was removed in vacuo. Water was added to the residue and the or-
ganic phase was extracted with chloroform, dried with anhydrous
Na2SO4 and the solvent evaporated. The resulting oil was recrystal-
lized in diethyl ether/petroleum ether 1:1 v/v, obtaining colorless
crystals (0.930 g, 55.6%) that were filtered and air-dried. Anal. Calc.
for C7H9N3S: C, 50.27; H, 5.42; N, 25.13; S, 19.17. Found: C, 50.49;
H, 5.50; N, 25.24; S, 19.46%. 1H NMR (400 MHz, CDCl3, 25 °C):
C(7)H, d = 8.17 ppm (d, J = 2.8 Hz, 1H); C(5)H, d = 7.60 ppm
(d, J = 0.8 Hz, 1H); C(6)H, d = 6.34 ppm (m, J = 1.5 Hz, 1H); CH2–N,
d = 3.81 ppm (t, J = 5.6 Hz, 2H); CH2–S, d = 3.11 ppm (t, J = 6.0 Hz,
2H); CH2, d = 1.93 ppm (m, J = 5.7 Hz, 2H); 1H NMR (400 MHz,
DMSO-d6, 25 °C): C(7)H, d = 8.30 ppm (d, J = 2.8 Hz, 1H); C(5)H,
d = 7.68 ppm (s, 1H); C(6)H, d = 6.48 ppm (d, J = 1.6 Hz, 1H);
CH2–N, d = 3.77 ppm (t, J = 5.4 Hz, 2H); CH2–S, d = 3.15 ppm
(t, J = 5.8 Hz, 2H); CH2, d = 1.84 ppm (m, J = 5.6 Hz, 2H); 13C NMR
(100 MHz, CDCl3, 25 °C): d = 146.8 ppm C(1); 141.0 ppm C(5);
125.7 ppm C(7); 107.4 ppm C(6); 46.0 ppm C(2), 26.2 ppm C(4),
19.7 ppm C(3). IR(KBr): thiazine ring vibrations 1635 [
932, 915, 884, 779, 620, 580, 525, 423 cmꢀ1; pyrazole ring
vibrations: 1510, 1419, 1386, 1327, 995 cmꢀ1
m(C@N)],
Scheme 1. Synthesis of ligands and zinc(II) complexes.
.
analysis, IR spectroscopy, X-ray diffraction, UV–Vis spectroscopy
and mass spectroscopy. In addition, the influence of the coordina-
tion to Zn(II) in the phagocytic activity of human neutrophils was
investigated and the results were compared with those obtained
for the zinc(II) complex with ligand 2-(3,4-dichlorophenyl)imino-
N-(4H-5,6-dihydro-1,3-thiazin-2-yl)tetrahydro-1,3-thiazine (TzTz),
previously reported [9], motivated by the structural resemblance
between the complexes studied in this work and [ZnCl2(TzTz)],
with the objective of determine whether the type of organic ligand
has any influence in the biological activity.
2.2.2. Synthesis of 2-(3,5-dimethyl-1-pyrazolyl)-1,3-thiazine
(DMPzTz)
3,5-Dimethylpyrazole (0.961 g, 0.010 mol) was dissolved in
toluene (50 mL) and sodium hydride 60% (0.600 g, 0.017 mol)
was added. After stirring the mixture for 3 h at room temperature,
a solution of 3-chloropropylisothiocyanate (1.490 g, 0.010 mol) in
toluene (5 mL) was added. After this, the reaction mixture was
heated to reflux for 4 h, cooled, and methanol (5 mL) was added.
The solvent was removed under reduced pressure and the residue
was washed with water. The organic phase was extracted with
chloroform, dried over anhydrous sodium sulfate and treated with
activated carbon. After filtration and evaporation a pale yellow oil
is obtained (1.18 g, 60.4%). Anal. Calc. for C9H13N3S: C, 59.32; H,
7.19; N, 23.06; S, 17.60. Found: C, 59.01; H, 7.12; N, 22.69; S,
17.31%. 1H NMR (400 MHz, CDCl3, 25 °C): C(6)H, d = 5.88 ppm (s,
1H); CH2–N, d = 3.82 ppm (t, J = 5.6 Hz, 2H); CH2–S, d = 3.09 ppm
(t, J = 6.0 Hz, 2H); C(8)H3, d = 2.43 ppm (s, 3H); C(9)H3,
d = 2.21 ppm (s, 3H); CH2, d = 1.90 ppm (m, J = 5.8 Hz, 2H); 1H
NMR (400 MHz, DMSO-d6, 25 °C): C(6)H, d = 6.03 ppm (s, 1H);
CH2–N, d = 3.77 ppm (t, J = 5.6 Hz, 2H); CH2–S, d = 3.10 ppm (t,
J = 5.6 Hz, 2H); C(8)H3, d = 2.40 ppm (s, 3H); C(9)H3, d = 2.12 ppm
(s, 3H); CH2, d = 1.81 ppm (m, J = 5.6 Hz, 2H); 13C NMR (100 MHz,
CDCl3, 25 °C): d = 148.8 ppm C(5); 147.4 ppm C(1); 141.0 ppm
C(7); 108.7 ppm C(6); 46.1 ppm C(2), 26.7 ppm C(4), 19.7 ppm
C(3); 14.1 ppm C(8); 13.4 ppm C(9). IR(KBr): thiazine ring vibra-
2. Materials and methods
2.1. General procedures
All reagents were commercial grade material and used without
any further purification. 3-Chloropropylisothiocyanate was ob-
tained as previously reported [14]. Chemical analyses of carbon,
hydrogen, nitrogen and sulfur were performed by microanalytical
methods using a Leco CHNS-932 microanalyser. IR spectra were re-
corded on a Termo IR-300 spectrophotometer, from KBr pellets in
the 4000–370 cmꢀ1 range and on a Perkin-Elmer FT-IR 1700ꢁ
spectrophotometer, from Nujol mull in the 500–150 cmꢀ1 range.
1H NMR spectra for the organic ligands and their complexes at
400 MHz in CDCl3 and DMSO-d6, and 13C NMR spectra for the li-
gands in CDCl3 at 100 MHz were obtained with Bruker AM 400
instrument. Mass spectra for 1–3 were performed in a water–
DMSO 9:1 v/v mixture. An API ion trap spectrometer (Finnigan
LCQ Advantage Max, Thermo Electron Corporation) was used to
introduce the samples by syringe pump into the MS detector in
the ESI/MS. The MS conditions were: sheath gas flow rate: 10;
aux/sweep gas flow rate: 5; spray voltage: 4.5 kV; capillary tem-
perature: 250 °C, capillary voltage: 10.00 V. Electronic spectra in
tions 1639 [
m
(C@N)], 960, 881, 862, 750, 736, 586 cmꢀ1; pyrazole
ring vibrations: 1566, 1411, 1375, 1315, 981 cmꢀ1
.
2.2.3. Synthesis of 2-(3,5-diphenyl-1-pyrazolyl)-1,3-thiazine
(DPhPzTz)
A solution of 3,5-diphenylpyrazole (2.200 g, 0.010 mol) in tolu-
ene (50 mL) was treated with sodium hydride 60% (0.600 g,

P. Torres-García et al. / Polyhedron 30 (2011) 2627–2636
2629
0.0170 mol) and the resulting mixture was stirred 3 h at room tem-
perature. Then, a solution of 3-chloropropylisothicyanate (1.490 g,
0.010 mol) in toluene (5 mL) was added. The mixture was refluxed
for 4 h and cooled. After addition of 5 mL of methanol, the organic
solvent was removed in vacuo and water was added. Extraction
with chloroform, drying over anhydrous sodium sulfate and con-
centration gave a yellow oil. The product was purified by flash
chromatography, using a mixture dichloromethane/diethyl ether
20:1 v/v. The early fractions of the chromatography contained
DPhPzTz, which was recrystallized in diethyl ether/petroleum
ether 1:1 v/v, yielding colorless crystals (1.750 g, 54.8%). Anal. Calc.
for C19H17N3S: C, 71.44; H, 5.33; N, 13.15; S, 10.04. Found: C, 71.33;
H, 5.20; N, 12.80; S, 9.68%. 1H NMR (400 MHz, CDCl3, 25 °C): C(9)H,
C(13)H, d = 7.90 ppm (d, J = 7.6 Hz, 2H); C(19)H, C(15)H,
d = 7.50 ppm (d, J = 7.6 Hz, 2H); C(10–12)H, C(16–18)H, d = 7.44–
7.33 ppm (m, 6H); C(6)H, d = 6.73 ppm (s, 1H); CH2–N,
d = 3.69 ppm (t, J = 5.4 Hz, 2H); CH2–S, d = 3.18 ppm (t, J = 5.8 Hz,
2H); CH2, d = 1.92 ppm (m, J = 5.7 Hz, 2H); 1H NMR (400 MHz,
DMSO-d6, 25 °C): C(9)H, C(13)H, d = 7.89 ppm (d, J = 8.4 Hz, 2H);
C(10–12)H, C(15–19)H, d = 7.52–7.37 ppm (m, 8H); C(6)H,
d = 7.11 ppm (s, 1H); CH2–N, d = 3.59 ppm (t, J = 5.6 Hz, 2H); CH2–
S, d = 3.24 ppm (t, J = 5.6 Hz, 2H); CH2, d = 1.84 ppm (m, J = 5.1 Hz,
2H); 13C NMR (100 MHz, CDCl3, 25 °C): d = 151.9 ppm C(1);
147.7 ppm C(5); 144.6 ppm C(7); 132.4 ppm C(14); 130.8 ppm
C(8); 128.0–128.8 ppm (eight signals) C(9), C(10), C(12), C(13),
C(15), C(16), C(18), C(19); 126.0 ppm C(17); 99.9 ppm C(6);
46.8 ppm C(2), 27.1 ppm C(4), 19.3 ppm C(3). IR(KBr): thiazine ring
2.3.3. Preparation of [ZnCl2(DPhPzTz] (3)
This complex was isolated by adding a methanol solution
(3 mL) of ZnCl2 (0.043 g, 0.3 mmol) to another methanol solution
(15 mL) of DPhPzTz (0.100 g, 0.3 mmol), being obtained colorless
crystals after a slow evaporation of the solution at room tempera-
ture (0.112 g, 78.6%). The crystals were filtered, washed with cold
ether and air dried. Anal. Calc. for C19H17Cl2N3SZn: C, 50.07; H,
3.76; N, 9.22; S, 7.04. Found: C, 49.88; H, 4.08; N, 9.23; S, 6.95%.
1H NMR (400 MHz, DMSO-d6, 25 °C): C(9)H, C(13)H, d = 7.89 ppm
(d, J = 8.0 Hz, 2H); C(10–12)H, C(15–19)H, d = 7.52–7.37 ppm (m,
8H); C(6)H, d = 7.11 ppm (s, 1H); CH2–N, d = 3.59 ppm (t,
J = 5.4 Hz, 2H); CH2–S, d = 3.24 ppm (t, J = 5.8 Hz, 2H); CH2,
d = 1.83 ppm (m, J = 5.5 Hz, 2H); IR(KBr): thiazine ring vibrations
1635 [m
(C@N)], 946, 916, 877, 757, 613, 599, 536, 487 cmꢀ1; pyra-
zole ring vibrations: 1548, 1406, 1303, 998 cmꢀ1
.
2.4. Crystal structures determination
X-ray diffraction measurements were performed using a Bruker
APEX or a Bruker SMART CCD diffractometer with Mo K radiation
a
(k = 0.71073 Å, graphite monochromator). The first 50 frames were
measured at the end of the data collection to monitor instrument
and crystal stability. Absorption correction were applied using the
program SADABS [15]. The structures were solved by direct methods
and subsequent Fourier differences using the SHELXS-97 [16] pro-
gram and refined by full-matrix least-squares on F2 with SHEXL-97
[17], included in WINGX package [18], assuming anisotropic
vibrations 1639 [m(C@N)], 945, 921, 871, 761, 617, 605, 534,
461 cmꢀ1; pyrazole ring vibrations: 1548, 1406, 1303, 998 cmꢀ1
.
Table 1
Crystal data and structure refinement for ligands PzTz and DPhPzTz.
PzTz
DPhPzTz
2.3. Preparation of the complexes
Crystal shape
Color
prism
prism
colorless
0.46 ꢁ 0.43 ꢁ 0.21
C7H9N3S
colorless
0.55 ꢁ 0.37 ꢁ 0.27
C19H17N3S
319.42
monoclinic
P21/n
2.3.1. Preparation of [ZnCl2(H2O)(PzTz] (1)
Size (mm)
Chemical formula
Formula weight
Crystal system
Space group
Unit cell dimensions
a (Å)
This complex was prepared by reacting an ethanol 96% solution
(3 mL) of ZnCl2 (0.081 g, 0.6 mmol) with an ethanol 96% solution
(5 mL) of PzTz (0.100 g, 0.6 mmol). The resulting solution was
surrounded with ether using a liquid-vapor diffusion method to
obtain colorless crystals (0.106 g, 55.5%). The crystals were fil-
trated, washed with cold ether and air dried. Anal. Calc. for
C7H11Cl2N3OSZn: C, 26.15; H, 3.45; N, 13.07; S, 9.97. Found: C,
26.54; H, 3.30; N, 13.31; S, 10.18%. 1H NMR (400 MHz, DMSO-d6,
25 °C): C(7)H, d = 8.30 ppm (d, J = 2.8 Hz, 1H); C(5)H, d = 7.68 ppm
(s, 1H); C(6)H, d = 6.48 ppm (d, J = 1.5 Hz, 1H); CH2–N, d =
3.77 ppm (t, J = 5.6 Hz, 2H); CH2–S, d = 3.15 ppm (t, J = 5.8 Hz,
2H); CH2, d = 1.85 ppm (m, J = 5.6 Hz, 2H); IR(KBr): thiazine ring
167.23
triclinic
ꢀ
P1
9.661(1)
9.708(1)
17.478(1)
75.854(2)
76.596(2)
89.904(2)
1543.5(1)
8
100(1)
1.439
0.351
704
9.141(1)
37.458(2)
9.655(1)
b (Å)
c (Å)
a
(°)
b (°)
101.203(2)
c
(°)
Cell volume (Å3)
3243.1(3)
8
100(1)
1.308
0.202
1344
Z
T (K)
Dcalc (g cmꢀ3
)
vibrations 1625 [m(C@N)], 954, 917, 889, 790, 777, 646, 607, 530,
(mmꢀ1
)
418 cmꢀ1; pyrazole ring vibrations: 1525, 1396, 1332, 997 cmꢀ1
.
l
F(0 0 0)
h Range
Index ranges
1.2–26.0
ꢀ11 6 h 6 11,
ꢀ11 6 k 6 11, 0 6 l 6 21
6024
1.1–26.0
ꢀ11 6 h 6 11,
0 6 k 6 46, 0 6 l 6 11
6386
2.3.2. Preparation of [ZnCl2(DMPzTz] (2)
Independent
ZnCl2 (0.070 g, 0.5 mmol) dissolved in methanol (3 mL) was
added to methanol solution (5 mL) of DMPzTz (0.100 g,
reflections
Observed reflections
a
5061
4967
0.5 mmol). The resulting solution was allowed to evaporate slowly
at room temperature. After a few days, colorless crystals of consid-
erable size were isolated from the solution (0.119 g, 70.1%). Crys-
tals were separated by filtration, washed with cold ether and
finally air dried. Anal. Calc. for C9H13Cl2N3SZn: C, 32.59; H, 3.92;
N, 12.67; S, 9.66. Found: C, 32.44; H, 3.73; N, 12.52; S, 9.36%. 1H
NMR (400 MHz, DMSO-d6, 25 °C): C(6)H, d = 6.03 ppm (s, 1H);
CH2–N, d = 3.77 ppm (t, J = 5.2 Hz, 2H); CH2–S, d = 3.10 ppm
(t, J = 5.6 Hz, 2H); C(8)H3, d = 2.40 ppm (s, 3H); C(9)H3,
d = 2.12 ppm (s, 3H); CH2, d = 1.81 ppm (m, J = 5.2 Hz, 2H); IR(KBr):
[F > 4.0r(F)]
Data completeness
Maximum/minimum
transmission
No. of reflection
parameters
0.994
0.930/0.855
1
0.947/0.897
397
415
R [F > 4.0
r
(F)]a
(F)]b
0.032
0.076
0.042
0.091
0.981
0.355, ꢀ0.312
wR [F > 4.0
r
Goodness-of-fit (GOF)c 1.038
qmax
,
qmin (e Åꢀ3
)
0.265, ꢀ0.383
P
P
a
R ¼ jFoj ꢀ jFcjj= jFoj.
R ¼
n
h
i
h
io
n
1=2
P
P
2
2
wðF2o ꢀ F2c Þ
=
wðF2oÞ
.
b
thiazine ring vibrations 1597 [
626, 593, 545, 439 cmꢀ1; pyrazole ring vibrations: 1564, 1417,
1380, 1317, 989 cmꢀ1
m(C@N)], 975, 904, 862, 748, 694,
h
i
o
1=2
ꢀ
ꢁ
P
2
wðF2o ꢀ F2c Þ = Nrf ln s ꢀ Nparams
.
c
The goodness-of-fit (GOF) equals
.

2630
P. Torres-García et al. / Polyhedron 30 (2011) 2627–2636
Table 2
Crystal data and structure refinement for compounds 1–3.
1
2
3
Crystal shape
Color
prism
colorless
prism
colorless
prism
colorless
Size (mm)
Chemical formula
Formula weight
Crystal system
Space group
Unit cell dimensions
a (Å)
0.20 ꢁ 0.16 ꢁ 0.10
C7H11Cl2N3OSZn
321.52
0.39 ꢁ 0.33 ꢁ 0.24
C9H13Cl2N3SZn
331.55
0.50 ꢁ 0.28 ꢁ 0.27
C19H17Cl2N3SZn
455.69
monoclinic
Cc
triclinic
triclinic
ꢀ
ꢀ
P1
P1
8.415(2)
8.449(2)
8.849(2)
8.728(1)
9.103(1)
9.330(1)
10.213(1)
15.117(1)
12.244(1)
b (Å)
c (Å)
a
(°)
69.288(4)
88.471(2)
b (°)
79.737(4)
74.254(2)
95.306(2)
c
(°)
79.630(4)
574.4(2)
67.540(2)
656.9(1)
Cell volume (Å3)
1882.2(2)
Z
2
2
4
T (K)
110(2)
1.859
2.76
100(2)
1.676
2.41
100(1)
1.608
1.71
Dcalc (g cmꢀ3
)
l
(mmꢀ1
)
F(0 0 0)
324
336
928
h Range
2.5–26.4
2.3–26.0
0.5–26.0
Index range
ꢀ10 6 h 6 10, ꢀ9 6 k 6 10, 0 6 l 6 11
ꢀ10 6 h 6 10, ꢀ11 6 k 6 11, 0 6 l 6 11
ꢀ12 6 h 6 12, 0 6 k 6 18, ꢀ15 6 l 6 15
Independent reflections
Observed reflections [F > 4.0
Data completeness
2342
2587
3352
r(F)]
2096 [F > 4.0
0.994
r(F)]
2421 [F > 4.0
0.999
r(F)]
3225 [F > 4.0
1
r(F)]
Maximum/minimum transmission
No. of reflection parameters
0.761/0.598
144
0.024
0.058
1.041
0.595/0.453
147
0.020
0.046
1.055
0.636/0.568
235
0.018
0.038
0.985
R [F > 4.0
r
(F)]a
(F)]b
wR [F > 4.0
r
Goodness-of-fit (GOF)c
qmax
,
qmin (e Åꢀ3
)
0.353, ꢀ0.385
0.286, ꢀ0.328
0.194, ꢀ0.245
P
P
a
R ¼ jjFoj ꢀ jFcjj= jFoj.
n
h
i
h
io
n
1=2
P
P
2
2
wðF2o ꢀ F2c Þ
=
wðF2oÞ
.
b
R ¼
h
i
o
1=2
ꢀ
ꢁ
P
2
wðF2o ꢀ F2c Þ = Nrf ln s ꢀ Nparams
.
c
The goodness-of-fit (GOF) equals
displacement parameters for non-hydrogen atoms, except for dis-
ordered atoms with isotropic displacement parameters. The crystal
structure of the ligand DPhPzTz presented a dynamic disorder
affecting the C(22) atom (equivalent to the C(4) atom in Scheme 1
for other independent molecule) that was modellized using two
sets of positions. For this procedure the occupancy of two alternate
orientations were constrained to unity. The refined occupancy was
83% and 17% for C(22A) and C(22B), respectively. All hydrogen
atoms attached to carbon atoms were positioned geometrically,
with Uiso values derived from Ueq values of the corresponding car-
bon atoms. However, hydrogen atoms of the water molecule were
detected by Fourier differences and were refined with fixed O–H
and H–H distances (0.957(3) and 1.513(3) Å, respectively). Graphi-
cal representations of the molecular structures were generated
using ORTEP3 [19] and Mercury [20] for Windows. Experimental
details of the crystal structure determinations are listed in Tables
1 and 2.
and the pellet resuspended. Cells were counted in a Neubauer
haemocytometer under phase contrast microscopy at 40ꢁ, and
the suspension was adjusted to 5 ꢁ 105 neutrophils/mL.
2.6. Cell viability assay
Metabolic activity of cells was evaluated using
a MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay. The cells were plated in 96-multiwell plates at a density of
105 cells/well and treated with the organic ligands, complexes
1–3 and ZnCl2 (in DMSO solutions) in different doses (100 nM, 1,
10 and 100
lM) for 30 min at 37 °C. After the treatment period,
MTT (5 mg/mL, 5
lL) was added to each well and incubated for
60 min at 37 °C. After the incubation period, the media were re-
moved and DMSO was added to solubilize the dark blue formazan
crystals, and optical density was measured in triplicate in an auto-
matic microplate reader (Tecan Infinite M200) at a test wavelength
of 490 nm and reference wavelength of 650 nm. The data are
presented as variation above control (untreated samples).
2.5. Isolation of neutrophils
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. 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
2.7. Phagocytosis of latex beads
Aliquots of 200 lL of Hank’s cellular suspension were put into
the wells of plastic macrophague migration inhibition factor
(MIF)-type plaques (AFORA, Spain), following the method
described by Rodríguez et al. [21]. After 30 min incubation at
37 °C in a stove, the adhered monolayer was washed with PBS at
37 °C. Then, 200 L of Hank’s medium, 20 L of latex beads (1.1 ꢁ
10ꢀ3 mm, diluted to 1% in PBS; Sigma) and 3 L of ligand, salt or
complex 1 ꢁ 10ꢀ4 M, yielding
a
final concentration of 1.3 ꢁ
10ꢀ3 mM (3 L of DMSO for control) were added, following by

P. Torres-García et al. / Polyhedron 30 (2011) 2627–2636
2631
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 hematoxylin (five passes). The plaques
were carefully rinsed with tap water and dried, followed by count-
ing under oil-immersion phase-contrast microscopy at 100ꢁ.
The number of particles ingested per 100 neutrophils was ex-
pressed as the latex-bead phagocytosis index (P.I.). The percentage
of cells that had phagocytized at least one latex bead was ex-
pressed as the phagocytosis percentage (P.P.). The ratio P.I./P.P.
was calculated, giving the phagocytosis efficiency (P.E.). Results
were referred to 100% of the control to avoid the dispersion due
to the great variability of patients’ cells phagocytic activity.
2.8. Statistical analysis
The results of the biological study were analyzed using the
ANOVA-Scheffe’s F-test. P < 0.05 was considered statistically signif-
icant. Data were expressed as the mean (X) standard error (SE) of
six experiments, performed in duplicate.
3. Results and discussion
3.1. Description of the crystal structures
The single-crystal X-ray diffraction analysis revealed that PzTz
crystallizes in the triclinic system with four independent mole-
cules in the asymmetric unit cell, meanwhile the crystal of
DPhPzTz is developed into an monoclinic lattice with two com-
pound molecules per unit cell. A diagram of the molecular struc-
ture of one molecule with the atom labeling is displayed in Fig. 1
(in the case of PzTz) and Fig. 2 (for DPhPzTz). Likewise, selected
interatomic distances and angles are given in Table 3.
Fig. 2. Crystal structure of one independent molecule of DPhPzTz. The thermal
ellipsoids are plotted at the 50% probability level.
Table 3
Selected bond lengths (Å) and angles (°) for ligands PzTz and DPhPzTz.
PzTz
DPhPzTz
PzTz
DPhPzTz
N(1)–C(1)
S(1)–C(4)
C(2)–C(3)
N(2)–C(1)
N(3)–C(5)
C(6)–C(7)
1.261(2) 1.254(2) S(1)–C(1)
1.816(2) 1.828(2) C(3)–C(4)
1.517(2) 1.512(3) N(1)–C(2)
1.427(2) 1.432(2) N(2)–N(3)
1.326(2) 1.334(2) C(5)–C(6)
1.366(2) 1.369(3) N(2)–C(7)
1.758(2) 1.762(2)
1.516(2) 1.516(3)
1.466(2) 1.469(2)
1.366(2) 1.366(2)
1.403(2) 1.410(4)
1.362(2) 1.367(2)
In both ligands, in the S(1)–C(1)–N(1)–C(2)–C(3)–C(4) ring (and
the equivalent rings in the rest of the independent molecules in the
S(1)–C(1)–N(1) 131.8(1) 131.9(2) C(1)–N(1)–C(2) 121.4(1) 119.8(2)
N(1)–C(2)–C(3) 116.5(1) 114.4(2) C(2)–C(3)–C(4) 111.9(1) 111.5(2)
S(1)–C(4)–C(3) 112.1(1) 112.3(1) C(1)–S(1)–C(4) 98.1(1)
99.6(1)
N(1)–C(1)–N(2) 116.3(1) 117.3(2) S(1)–C(1)–N(2) 111.8(1) 111.0(1)
C(1)–N(2)–C(7) 126.5(1) 128.0(2) N(3)–N(2)–C(1) 121.0(1) 119.2(1)
N(3)–N(2)–C(7) 112.4(1) 112.5(2) N(2)–N(3)–C(5) 103.7(1) 104.5(1)
N(3)–C(5)–C(6) 112.4(1) 111.0(2) C(5)–C(6)–C(7) 105.1(2) 106.2(2)
N(2)–C(7)–C(6) 106.4(2) 105.8(2)
Table 4
Selected bond lengths (Å), angles (°) and hydrogen bond parameters for compounds
1–3.
1
2
3
Zn–Cl(1)
Zn–Cl(2)
Zn–N(1)
Zn–N(3)
Zn–O(1w)
2.254(1)
2.296(1)
2.200(2)
2.072(2)
2.170(2)
2.218(1)
2.203(1)
2.050(1)
2.034(1)
2.213(1)
2.195(1)
2.035(2)
2.058(2)
Cl(1)–Zn–Cl(2)
Cl(1)–Zn–N(1)
Cl(1)–Zn–N(3)
N(1)–Zn–Cl(2)
N(3)–Zn–Cl(2)
N(1)–Zn–N(3)
O(1w)–Zn–Cl(1)
O(1w)–Zn–Cl(2)
O(1w)–Zn–N(1)
O(1w)–Zn–N(3)
109.0(1)
98.1(1)
137.6(1)
99.5(1)
113.4(1)
75.1(1)
94.5(1)
97.3(1)
154.5(1)
80.6(1)
113.8(1)
121.9(1)
108.4(1)
107.0(1)
123.6(1)
78.5(1)
120.6(1)
110.4(1)
110.1(1)
112.8(1)
116.7(1)
78.6(1)
D–Hꢂ ꢂ ꢂA
position of A
Aꢂ ꢂ ꢂD (Å)
Aꢂ ꢂ ꢂH–D (°)
1
O(1w)–H(1w)ꢂ ꢂ ꢂCl(2)
ꢀx + 1, ꢀy, ꢀz + 1
ꢀx, ꢀy + 1, ꢀz
2.209(20)
2.277(22)
169(2)
157(2)
Fig. 1. Crystal structure of one independent molecule of PzTz. The thermal
ellipsoids are plotted at the 50% probability level.
O(1w)–H(2w)ꢂ ꢂ ꢂCl(1)

2632
P. Torres-García et al. / Polyhedron 30 (2011) 2627–2636
unit cell) the short endocyclic C@N bond is accompanied by a high
S–C–N angle and a large S–C(sp2) bond. This fact is characteristic of
a 5,6-dihydro-4H-1,3-thiazine ring [22].
On the other hand, selected bond distances and angles for com-
plex 1 are shown in Table 4. Fig. 3 shows an ORTEP diagram of the
molecular structure. In this complex the central zinc ion is five-
coordinated by two chloro ligands, one water molecule and two
nitrogen atoms from PzTz ligand. In order to determine the geom-
etry around the metallic ion the methods proposed by Addison et
al. [23] and by Muetterties and Guggemberger [24] have been ap-
the a axis is formed (Fig. 4). These chains are linked to each other
through van der Waals forces.
Conversely, the structure of complexes 2 and 3 consists of dis-
crete neutral monomeric units in which the environment around
the zinc(II) atom may be described as a distorted tetrahedron. In
both complexes the metallic atom is coordinated to two chloro li-
gands, one thiazinic nitrogen and one pyrazolic nitrogen. A dia-
gram of the molecular structures and the atom numbering
system used is represented in Figs. 5 and 6, while the most relevant
bond lengths and angles are listed in Table 4.
plied. The values obtained for parameters
s
and
D
were 0.76 and
The difference in the coordination index of the five-coordinated
complex 1 and the four-coordinated complexes 2 and 3 could be a
consequence of the absence of voluminous substituents in the pyr-
azole ring of the organic ligand PzTz, which permits the coordina-
tion of a higher number of ligands to the metallic ion. Additionally,
the distortion of the tetrahedral geometry was expected to be high-
er in 3 than in 2, since the steric effects caused by the phenyl sub-
stituents should be stronger than the hindrance induced by methyl
groups. However, there are not big differences in the distortion of
the coordination geometry (dihedral angle between planes Cl(1)–
Zn–Cl(2) and N(1)–Zn–N(3) = 77.1° in 2, 88.4° in 3) probably due
to the fact that the phenyl rings can rotate through C(5)–C(8)
and C(7)–C(14) bonds and adopt a position that minimizes the
steric hindrance.
0.44, respectively, which indicates that the coordination geometry
can be described as a highly distorted trigonal bipyramid. The
equatorial plane is constituted by atoms Cl(1), Cl(2) and N(3),
while the axial positions are occupied by atoms N(1) and O(1w).
The crystal structure of this complex is stabilized by a hydro-
gen-bond network where the oxygen atom of the water molecule
acts as donor of hydrogen while the chloro ligands of neighbor
molecules act as acceptors, in such a way that a zigzag chain along
Finally, as it can be observed in Figs. 3 and 6, one remarkable
difference in complexes 1 and 3 with respect to the structure of
their corresponding free ligands PzTz and DPhPzTz is the different
degree of rotation of the thiazine ring around C(1)–N(2) bond,
which permits the coordination through the thiazine and pyrazole
nitrogen atoms [torsion angle N(1)–C(1)–N(2)–N(3) = 165.3° in
PzTz, 3.9° in 1; 123.6° in DPhPzTz, 7.9° in 3].
3.2. IR spectra
The IR spectra of complexes 1–3 showed a strong absorption
corresponding to the thiazine ring
m(C@N) vibration (at
1625 cmꢀ1 in 1, 1597 cmꢀ1 in 2 and 1635 cmꢀ1 in 3). These bands
are shifted negatively relative to the uncoordinated thiazine ring of
the respective ligands (1635 cmꢀ1 in PzTz, 1639 cmꢀ1 in DMPzTz
and DPhPzTz). Likewise, the stretching vibrations corresponding
to the pyrazole ring shifted to higher wavenumbers compared with
those for the respective free ligands. From these facts it can be de-
duced coordination via the thiazine and pyrazole nitrogen atoms of
the ligands [25–27].
In the low-frequency region, some bands due to metal–ligand
stretching vibrations are observed. Tentative assignments are
listed in Table 5. These bands have been assigned in good agree-
Fig. 3. Crystal structure of 1. The thermal ellipsoids are plotted at the 50%
probability level.
ment with the literature data. In this way, m(Zn–Cl) vibrations are
Fig. 4. Hydrogen bonds in the crystal of 1.

P. Torres-García et al. / Polyhedron 30 (2011) 2627–2636
2633
detected in the range 332–214 cmꢀ1 [9,28–30]. Likewise,
(Zn–Npyrazole) vibration is registered in several zinc(II) complexes
3.3. Mass spectra
m
between 198 and 252 cmꢀ1 [31,32], whereas
m(Zn–Nthiazine) vibra-
The mass spectra for 1–3 show signals at m/z values corre-
sponding to Zn(II)/organic ligand coordinated species. Thus, for 1
a signal at m/z 168 is due to the (PzTz+) ligand, which is also the
base peak of the spectrum. Similarly, the signal at m/z 269 is
tion are assigned in the range 210–250 cmꢀ1 in complexes with
this type of bond [9,28]. Finally, the Zn–Owater stretching vibration
is usually detected over 300 cmꢀ1 [33,34].
Fig. 5. Crystal structure of 2. The thermal ellipsoids are plotted at the 50% probability level.
Fig. 6. Crystal structure of 3. The thermal ellipsoids are plotted at the 50% probability level.

2634
P. Torres-García et al. / Polyhedron 30 (2011) 2627–2636
assigned to the [ZnCl(PzTz)]+ fragment, and the signal correspond-
Table 5
Far IR bands for zinc complexes (in cmꢀ1).
ing to [ZnCl2(H2O)(PzTz)]+ appears at m/z 322. In the case of 2, the
presence of signals at m/z 196 (corresponding to DMPzTz+) and 332
(assignable to [ZnCl2(DMPzTz)]+) are registered. Finally, in the
spectrum of 3 there is one peak at m/z 320, which corresponds at
the ligand (DPhPzTz+), and another one at m/z 386, which can be
assigned to the [Zn(DPhPzTz)]+ fragment. Besides, no signals corre-
sponding to Zn(II)/DMSO species have been detected. One may
1
2
3
m
m
m
m
(Zn–Clterminal
(Zn–Npyrazole
(Zn–Nthiazine
(Zn–OH2)
)
)
308, 291
235
336, 318
245
220
337, 313
244
209
)
220
308
Fig. 7. Cell viability of human neutrophils incubated in the presence of PzTz (A), 1 (B), DMPzTz (C), 2 (D), DPhPzTz (E), 3 (F) and ZnCl2 (G). Each value represents X SE of six
determinations performed in triplicate. ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001.

P. Torres-García et al. / Polyhedron 30 (2011) 2627–2636
2635
hence deduce from these observations that the coordination of
Zn(II) to the organic ligands is maintained in these conditions.
no statistically significant differences have been found in the bio-
logical activity of cells treated with the three Zn(II) complexes.
With respect to the results obtained for the zinc(II) complex
[ZnCl2(TzTz)], previously reported [9] (see Fig. 8), this latter com-
pound shows a significantly greater phagocytosis percentage than
complexes 1–3 and a significantly lower phagocytic efficiency than
2, while the phagocytosis index does not present significant differ-
ences among all complexes. Therefore, it could be deduced that in
[ZnCl2(TzTz)] the phagocytosis promoting effect (reflected in the
phagocytosis index) is due mainly to active phagocytes number in-
crease (phagocytosis percentage), while in 2 the main factor is the
greater efficiency of these active cells phagocytizing antigenic par-
ticles (phagocytic efficiency). This different biologic behavior could
3.4. UV–Vis spectra
The UV–Vis spectra of the ligands, the Zn(II) complexes and
ZnCl2 have been recorded in DMSO solution at a concentration of
10ꢀ5 M. The spectra of complexes 1–3 are very similar to those
of their respective organic ligands. In PzTz and 1 a single band at
257 nm (
for 1) is detected. In DMPzTz and 2 a maximum at 257 nm
= 9400 L cmꢀ1 molꢀ1 for DMPzTz and 14320 L cmꢀ1 molꢀ1 for
e
= 10380 L cmꢀ1 molꢀ1 for PzTz and 8090 L cmꢀ1 molꢀ1
(
e
2) with a shoulder at 277 nm (e
= 3750 L cmꢀ1 molꢀ1 for DMPzTz
and 6120 L cmꢀ1 molꢀ1 for 2) is registered, whereas the spectra
of DPhPzTz and 3 consist of a broad band with a maximum at
257 nm (
e
= 12340 L cmꢀ1 molꢀ1) for DPhPzTz and at 259 nm
(e
= 20000 L cmꢀ1 molꢀ1) for 3. Finally, in the spectrum of the
inorganic salt a maximum is observed at 275 nm (e = 6650 L
cmꢀ1 molꢀ1). This band is not registered in the spectra of 1–3,
and so it can be deduced that the structure of the complexes is
maintained in DMSO solution.
3.5. Cell viability
The cell viability of human neutrophils when treated with the
three organic ligands, complexes 1–3 and ZnCl2 have been studied
at concentrations of 100 nM, 1, 10 and 100 lM. Fig. 7 shows the re-
sults obtained. As can be observed in this figure, cell viability is not
strictly dose-dependent. These data confirm that there is not any
significant difference in cell viability before and after treatment
at a concentration below 10 lM, according to Scheffe F-test, except
in the case of complex 2, ligand DPhPzTz and ZnCl2, which present
significant fall at a concentration of 100 nM. In all cases, the cell
viability with respect to the control sample at a concentration of
1 lM was above 85%. This suggest that the treatment with PzTz,
DMPzTz, DPhPzTz, 1, 2, 3 and ZnCl2 does not cause any cellular
damage under the work conditions and during the time our exper-
iments lasted. Therefore, the optimal concentration chosen to per-
form the phagocytosis studies reported in this work was 1 lM.
3.6. Study of the phagocytic function
The capacity of neutrophils to ingest latex beads, expressed as
the phagocytosis index (Fig. 8A) increases as consequence of the
treatment of neutrophils with our compounds. This growth is sta-
tistically significant (P < 0.001) in the case of cells treated with 1, 2
and 3 in comparison with the control sample and with the sample
treated with the Zn(II) salt. Likewise, the samples treated with zinc
complexes 1–3 present a statistically significant growth (P < 0.01
or P < 0.001) with respect to their respective organic ligands.
Regarding the phagocytic percentage (Fig. 8B) no significant differ-
ences have been found in samples treated with complexes with re-
spect to control and the samples treated with the organic ligands
and the inorganic salt. Finally, in the case of phagocytic efficiency
(Fig. 8C), this is significantly higher in cells treated with 1–3
(P < 0.001) compared with control and ZnCl2. Besides, this param-
eter is also significantly greater than the corresponding ligands in
all complexes.
From these results it is demonstrated that coordination to zin-
c(II) improves neutrophils function, enhancing the non-specific im-
mune response, since phagocytosis index and phagocytic efficiency
are higher in the case of samples treated with 1, 2 and 3 with re-
spect to the samples to which the organic ligands or the Zn(II) salt
were added. However, substituents in the organic ligands seem to
have no influence in the phagocyting capacity of neutrophils, since
Fig. 8. Phagocytosis index (A), phagocytosis percentage (B) and phagocytic
efficiency (C) variations of human neutrophils treated with ZnCl2, PzTz, DMPzTz,
DPhPzTz, 1, 2, 3 and [ZnCl2(TzTz)] and incubated in the presence of latex beads.
Each value represents X SE of six determinations performed in duplicate. ⁄P < 0.05;
⁄⁄P < 0.01; ⁄⁄⁄P < 0.001.

2636
P. Torres-García et al. / Polyhedron 30 (2011) 2627–2636
[9] F.J. Barros-García, A. Bernalte-García, A.M. Lozano Vila, F. Luna-Giles, J.A.
Pariente, R. Pedrero-Marín, A.B. Rodríguez, J. Inorg. Biochem. 100 (2006) 1861.
[10] E. Viñuelas-Zahínos, F. Luna-Giles, P. Torres-García, A.B. Rodríguez, A.
Bernalte-García, Inorg. Chim. Acta 366 (2011) 373.
[11] A.M. Lozano-Vila, F. Luna-Giles, E. Viñuelas-Zahínos, F.L. Cumbrera, A.L. Ortiz,
F.J. Barros-García, A.B. Rodríguez, Inorg. Chim. Acta 365 (2011) 282.
[12] T.D. Pennig, J.J. Talley, S.R. Bertenshaw, J.S. Carter, P.W. Collins, S. Docter, M.J.
Graneto, L.F. Lee, J.W. Malecha, J.M. Miyashiro, R.S. Rogers, D.J. Rogier, S.S. Yu,
G.D. Anderson, E.G. Burton, J.N. Cogburn, S.A. Gregory, C.M. Koboldt, W.E.
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be related to the structural differences between [ZnCl2(TzTz)] and
the three complexes studied in this work (e.g. the size of the che-
late ring), which are a consequence of the presence of structurally
different ligands as TzTz on one hand, and PzTz, DMPzTz and
DPhPzTz on the other hand. These differences could affect the
assimilation of the compound by the cell and, by extension, the
influence of the complex in the cell biologic activity.
[13] S.H. Kim, H. Son, G. Nam, D.Y. Chi, J.H. Kim, Bioorg. Med. Chem. Lett. 10 (2000)
1143.
Acknowledgements
[14] H.W. Schubert, O. Behner, Arch. Pharm. 301 (1968) 750.
[15] Bruker AXS Inc., SADABS, Version 2.03 Bruker AXS Inc., Madison, WI, 2001.
[16] M. Sheldrick, SHELXS-97, Program for Crystal Structures Solution, University of
Göttingen, Germany, 1997.
[17] M. Sheldrick, SHELXL-97, Program for Crystal Structures Refinement, University
of Göttingen, Germany, 1997.
The authors would like to thank the Junta de Extremadura (III
PRI + D + I) and the FEDER (Project PRI08A022) for financial sup-
port. J. Espino is beneficiary of a grant from MEC (AP2009-0753).
[18] L.J. Farrugia, J. Appl. Crystallogr. 32 (1999) 837.
[19] L.J. Farrugia, J. Appl. Crystallogr. 30 (1997) 565.
Appendix A. Supplementary data
[20] C.F. Macrae, I.J. Bruno, J.A. Chisholm, P.R. Edgington, P. McCabe, E. Pidcock, L.
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[23] A.W. Addison, T.N. Rao, J. Reedijk, J. Van Rijn, G.C. Verschoor, J. Chem. Soc.,
Dalton Trans. (1984) 1349.
[24] E.L. Muetterties, J. Guggemberger, J. Am. Chem. Soc. 96 (1974) 1748.
[25] M. Shakir, S.P. Vakey, F. Firdaus, P.S. Hameed, Polyhedron 13 (1994) 2319.
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[28] A. Bernalte-García, F.J. Barros-García, F.J. Higes-Rolando, F. Luna-Giles,
Polyhedron 20 (2001) 3315.
CCDC 819474, 819475, 819476, 819477 and 819478 contain the
supplementary crystallographic data for this paper. 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.
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