E.T. Souza et al. / Journal of Inorganic Biochemistry 105 (2011) 1767–1773
1769
718.30 g mol-1) calc. (found): C, 47.84 (47.93); H, 4.01 (4.21); N,
11.96 (11.70)%. ΛM =138 Ω-1 mol-1 cm2 (electrolyte 1:1 in metha-
nol) [30]. 1H NMR (DMSO-d6, 500 MHz, d=doublet, m=multiplet,
formazan crystals into 200 μL of DMSO, and the cell viability was deter-
mined by absorbance measurements at 570 nm. The amounts of surviv-
ing cells, compared to those of the untreated controls were determined.
t=triplet, bd=broad doublet, bt=broad triplet):
δ 9.07 (d,
J=5.2), 8.06 (d, J=2.6), 8.00-7.90 (m), 7.76-7.71 (m), 7.70-7.64
(m), 7.54 (d, J=7.8), 7.42 (d, J=7.3), 7.18 (t, J=6.2), 6.92 (d,
J=8.8), 6.76 (d, J=9.3), 4.57 (bd, J=13.5), 3.97 (bt, J=14.5), 3.89
(bt, J=15.0), 3.43-3.32 (m), 3.12 (bd, J=15.0), 2.99-2.91 (m), 2.71
(d, J=11.5), 2.13-2.00 (m).
2.4. X-ray crystal structure determination
Suitable single crystals for X-ray diffraction of complexes 2, 3 and
4 were obtained by slow evaporation of methanol:acetonitrile (3:1),
ethanol and acetone solutions, respectively. A remarkable character-
istic of complex 4 is the expressive solvent lost when crystalline
sample is removed from the crystallization solution. In order to keep
the crystals stable during the X-ray diffraction experiment the sample
of complex 4 had to be fast frozen. The procedure systematically
twinned the samples, thus both X-ray diffraction measurements and,
consequently, the structural refinements are limited by the poor
crystalline sample quality.
Single crystals X-ray diffraction data of 2 and 3 were collected
using a Bruker Kappa CCD equipped graphite-monochromatized
MoKα radiation (0.71073 Å) at room temperature. The unit cell
parameters were determined and refined using all reflections [32]
and the data integration and scaling were realized with EVALLCCD
program [33]. Absorption corrections were performed by multi-scan
method implemented in SADABS program [34]. X-ray diffraction
data collection for complex 4 was performed on an Oxford-Diffraction
GEMINI diffractometer using graphite-Enhance Source MoKα radia-
tion (λ=0.7107 Å) at 130 K. Data integration and scaling of the re-
flections were performed with the Crysalis suite [35]. Final unit cell
parameters were based on the fitting of all reflections positions. Ana-
lytical absorption corrections of the diffracted intensities were per-
formed using Crysalis suite [36]. Program XPREP [37] was used for all
data reduction.
[Co(L3)2]ClO4•½CH3OH, 3. Brown single crystals of complex 3 suit-
able for X-ray analysis were obtained by recrystallization of the precip-
itate in pure ethanol. Yield: 285 mg, 40%. Decomposition point: 260 °C.
FTIR (KBr, cm-1): ν(C-HAr/C-HAlf), 3589 – 3422; ν(C=N/C=CAr
)
,
1623/1550-1450; ν(C-O) 1280; ν(Cl-O), 1109 and δ(CHAr ), 765. Ele-
mental analysis: calc. for 28H26ClCoN4O6•½CH3OH (FW:
C
624.94 g mol-1) calc. (found): C, 54.77 (55.23); H, 4.52 (4.30); N, 8.97
(9.20)%. ΛM=137.6 Ω-1 mol-1 cm2 (electrolyte 1:1 in acetonitrile)
[30]. 1H NMR (DMSO-d6, 500 MHz, bs=broad singlet, d=doublet,
t=triplet, m=multiplet): δ 8.23 (1H, bs), 7.96 (1H, d, J=5.8), 7.89
(1H, t, J=7.7), 7.46 (1H, d, J=7.4), 7.30-7.24 (2H, m), 6.97 (1H, t,
J=7.0), 6.59 (1H, d, J=8.2), 6.48 (1H, t, J=6.7), 4.45 (1H, bs), 4.20
(1H, bs), 3.71 (1H, bs), 2.60 (1H, bs). 13 C NMR (DMSO-d6, 125 MHz):
δ 168.8 (CH), 164.4 (C), 162.9 (C), 155.41 (CH), 140.8 (CH), 135.0
(CH), 134.2 (CH), 126.5 (CH), 124.4 (CH), 121.2 (CH), 120.1 (C), 115.8
(CH), 53.8 (CH2), 34.5 (CH2).
[Co(L4)2]ClO4•5H2O, 4. Single crystals of complex 4 were obtained
by recrystallization in pure acetone which yielded dark red single
crystals suitable for X-ray analysis. Yield: 236 mg, 30%. Decomposi-
tion point: 268 °C. FTIR (KBr, cm-1): ν(C-HAr/C-HAlf), 3078–2861;
ν(C=N/C=CAr
) 1633/1556-1449; ν(N=O), 1312; ν(C-O) 1248;
,
ν(Cl-O), 1110 and δ(CHAr ), 756. Elemental analysis: calc. for C28H24-
ClCoN6O10•5H2O (FW: 807.03 g mol-1
)
calc. (found): C, 42.60
The structures of all complexes were solved by direct methods
using the SHELXS [37] program. For each compound, the positions of
all atoms could be unambiguously assigned on consecutive difference
Fourier maps. Solvent and counter ions molecules were observed in
the structure of all compounds. Refinements were performed using
SHELXL [37] based on F2 through full-matrix least square routine. All
but hydrogen atoms and perchlorate counter ion in complex 4 were
refined with anisotropic atomic displacement parameters. The hydro-
gen atoms in the compounds were added in the structure in idealized
positions and further refined according to the riding model [38]. Com-
plexes 2, 3 and 4 cartoons are represented in Figs. 2, 3 and 4, respec-
tively. Solvents and counter ions were omitted for the sake of clarity.
The unit cell of complex 2 is composed of two molecules of [Co(L2)2]
•ClO4 with slight differences between them. In complex 3, the per-
chlorate counter ion is disordered over two positions. In complex 4,
two acetone solvent molecules and a disordered perchlorate counter
ion were observed in a large structural cavity (Fig. S2). The abnormal
high peaks close to the Co atom, high R-values and goodness of fit
parameters evidence complex 4 refinement problems. The crystallo-
graphic data for complexes 2 – 4 are given in Table 1, and the relevant
structural parameters are listed in Table 2.
(42.62); H, 4.27 (4.34); N, 10.57 (10.65)%. ΛM =88.1 Ω-1 mol-1 cm2
(electrolyte 1:1 in methanol) [29]. 1H NMR (DMSO-d6, 500 MHz,
bs=broad singlet, m=multiplet, d=doublet, t=triplet) Fig. S1): δ
8.60 (1H, bs), 8.51 (1H, bs), 8.09-8.00 (2H, m), 7.80 (1H, d, J=9.1),
7.57 (1H, d, J=8.1), 7.40 (1H, t, J=7.0), 6.60 (1H, d, J=9.1), 4.49
(1H, bs), 4.21 (1H, bs), 3.47 (1H, bs), 2.17 (1H, bs). 13 C NMR
(DMSO-d6, 125 MHz): δ 171.9 (CH), 170.6 (C), 165.0 (C), 157.1
(CH), 143.1 (CH), 138.3 (C), 133.2 (CH), 130.8 (CH), 128.9 (CH),
126.7 (CH), 123.5 (CH), 121.8 (C), 55.0 (CH2), 35.6 (CH2).
2.3. In vitro cytotoxicity assays
Biological properties were assessed for ligands (HL1 – HL4) and
complexes (1 – 4) which were assayed against the tumor cell line
B16F10 (murine melanoma). The cell line was cultured using the
Roswell Park Memorial Institute (RPMI) adjusted to contain 1.5 g L-1
sodium bicarbonate, 4.5 g L-1 glucose at pH 7.0. All cells were kept
in a humidified atmosphere with 5% CO2 at 37 °C, supplemented
with 10% fetal bovine serum and 1% of antibiotic/antimycotic. After
reaching confluence, the cells were removed from the flasks using
trypsin/EDTA (1×) and counted for the experiments. To evaluate the
cytotoxic activity of compounds, cellular viability was determined by
the MTT test [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolim
bromide], a colorimetric assay determined by the mitochondrial-
dependent reduction of the soluble yellow tetrazolium salt to blue
formazan crystals [31]. The cells were seeded into a 96-well plate
(1.5×104 cells per well) in 200 μL of the appropriate complete
medium 24 h prior to the beginning of the experiment. The stock solu-
tions of the compounds were prepared in sterile DMSO and directly
diluted into the medium in order to achieve different final concentra-
tions, with a final concentration of maximum 2% DMSO (vehicle).
Forty-eight hours after the addition of the complex or the vehicle,
MTT (0.5 g L-1) was added and the cells were incubated for a period of
3 h. The optical density was measured after dissolving the blue
3. Results and discussion
3.1. Syntheses and characterization
All ligands were synthesized by direct condensation of the appro-
priate para-substituted salicylaldehyde with 2-2(aminoethylpyri-
dine) according to previously reported methodologies [27,28].
Infrared and 1H NMR spectroscopies, melting points and elemental
analysis were used to confirm the purity of the ligands. Complexes
2 – 4 were prepared by the same methodology employed for complex
1, and were isolated as single crystals by recrystallization in appropri-
ate solvents. In order to guarantee the purity of the samples, only
single crystals were used in the analyses of all complexes. Initially, the