Co(ii), Ni(ii) and Cu(ii) complexes with phenylthiazole and thiosemicarbazone-derived ligands: Synthesis, structure and cytotoxic effects

Article abstract of DOI:10.1039/c4nj00977k

Complexes of Co(ii), Ni(ii) and Cu(ii) with 2-(3,5-dimethyl-1H-pyrazole-1-yl)-4-phenyl-1,3-thiazole (a) and 3,5-dimethyl-1H-pyrazole-1-carbothioamide (b) ligands were synthesized. The crystal and molecular structures of four of them were determined by the X-ray diffraction method. In the complexes with ligand a the metal atom was bound via two nitrogen atoms, whereas ligand b interacted through the nitrogen and sulfur atoms. Comparing the coordination modes observed for both studied ligands, the thiocarbamoyl sulfur atom can participate in metal binding and in bridge formation, whereas the thiazole (aromatic) sulfur atom is not involved in the coordination. This effect seems to enhance (at least partially) conformational flexibility. Hydrogen bonds played a principal role in the crystal network formation of the analyzed compounds. The cytotoxic activity of the complexes against HL-60 and NALM-6 leukemia cells and the WM-115 melanoma cell line was also measured. The copper(ii) complex with the thiosemicarbazone ligand (7b) exhibited relatively high cytotoxicity towards all tested tumor cells. The cytotoxicity of copper(ii) complexes 7b and 9b against the melanoma WM-115 cells was over three times higher than that of cisplatin. This journal is

Full text of DOI:10.1039/c4nj00977k

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Co(II), Ni(II) and Cu(II) complexes with
phenylthiazole and thiosemicarbazone-derived
Cite this: New J. Chem., 2014,
ligands: synthesis, structure and cytotoxic effects†
3
8, 5349
a
b
c
c
Marta Sobiesiak,* Tadeusz Muzioł, Marek Rozalski, Urszula Krajewska and
Elzbieta Budzisz
d
Complexes of Co(II), Ni(II) and Cu(II) with 2-(3,5-dimethyl-1H-pyrazole-1-yl)-4-phenyl-1,3-thiazole (a)
and 3,5-dimethyl-1H-pyrazole-1-carbothioamide (b) ligands were synthesized. The crystal and molecular
structures of four of them were determined by the X-ray diffraction method. In the complexes with ligand
a the metal atom was bound via two nitrogen atoms, whereas ligand b interacted through the nitrogen and
sulfur atoms. Comparing the coordination modes observed for both studied ligands, the thiocarbamoyl sulfur
atom can participate in metal binding and in bridge formation, whereas the thiazole (aromatic) sulfur atom is
not involved in the coordination. This effect seems to enhance (at least partially) conformational flexibility.
Hydrogen bonds played a principal role in the crystal network formation of the analyzed compounds. The
cytotoxic activity of the complexes against HL-60 and NALM-6 leukemia cells and the WM-115 melanoma
cell line was also measured. The copper(II) complex with the thiosemicarbazone ligand (7b) exhibited
relatively high cytotoxicity towards all tested tumor cells. The cytotoxicity of copper(II) complexes 7b and 9b
against the melanoma WM-115 cells was over three times higher than that of cisplatin.
Received (in Victoria, Australia)
2th June 2014,
Accepted 22nd August 2014
1
DOI: 10.1039/c4nj00977k
www.rsc.org/njc
Thiosemicarbazone ligands are especially interesting because
of their mixed hard–soft donor character, diverse coordination
Introduction
4
5
Transition metal complexes with N,N- and N,S-donor ligands modes and various applications, including antiviral, anti-
6
–8
9–11
12–14
have attracted remarkable attention because of their interesting cancer,
antitumor,
antibacterial,
anti-inflammatory
1
5–17
chemical and biological properties. Some of these compounds and antiamoebic
activities. Thiazole-containing chelating
induce apoptosis by activation of DNA fragmentation in cancerous agents and their metal derivatives have also attracted notable
1
cell lines. Other complexes change the structure of DNA or interest over the years because of their various biological
2
18–20
21–23
destroy the cytoskeleton. It is well known that N and S atoms activities
play important roles in the coordination of metal ions at active Among the metal complexes, many copper(II) complexes with
sites of numerous metallobiomolecules. The pharmacological N,N- and N,S-donors ligands have been proposed as promising
including anticancer potency.
3
2
4
properties of metal complexes depend on the properties cytotoxic agents on the basis of in vitro assays. Some of these
of either the metal or the ligand. In many cases, addition of complexes with bidentate k N,S-thiosemicarbazone ligands
2
metal ions to an organic ligand can lead to the increase of its exhibited high antiproliferative activity (at 1 to 10 mM concen-
biological activity.
tration) in the HeLa (human cervical carcinoma) cancer cell line.
Pyrazole–pyridine ligands and substituted bis(pyrazolyl)alkanes
with N,N copper-coordinating abilities formed Cu(II) complexes
with high cytotoxicity against HT1080 (human fibrosarcoma)
a
Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun,
Faculty of Pharmacy, Department of Inorganic and Analytical Chemistry,
dr. A. Jurasza 2, 85-094 Bydgoszcz, Poland. E-mail: marta.sobiesiak@cm.umk.pl;
Fax: +48 (52) 585 38 04; Tel: +48 (52) 585 38 03
24
cells.
b
In our previous paper we described the synthesis of thiose-
micarbazone derivatives and their complexes with copper(II)
ions. The copper(II) complex with 3,5-dimethyl-1H-pyrazole-1-
carbothioamide showed a 10-fold higher cytotoxicity towards
the melanoma A375 cell line than the reference cisplatin. This
complex was able to modify the structure of plasmid DNA by
nicking the DNA form I into forms II and III and this feature
Nicolaus Copernicus University, Faculty of Chemistry, Gagarina 7, 87-100 Torun,
Poland
c
Department of Pharmaceutical Biochemistry, Faculty of Pharmacy,
Medical University of Lodz, Muszynski 1 Str., 90-151 Lodz, Poland
d
Department of Cosmetic Raw Materials Chemistry, Faculty of Pharmacy,
Medical University of Lodz, Muszynski 1 Str., 90-151 Lodz, Poland
†
Electronic supplementary information (ESI) available. CCDC 988739 (1a),
88743 (6a), 989127 (8a) and 988741 (5b). For ESI and crystallographic data in
9
2
5
CIF or other electronic format see DOI: 10.1039/c4nj00977k
may be a predominant factor for the biological activity.
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The ability of 3,5-dimethyl-1H-pyrazole-1-carbothioamide to of CoCl
complex several transition metal ions has been previously tested. the ligand coordinates to the metal in the same way as in the
It has been reported that the molecular structure of the resulting compounds described by Radosavljevi ´c Evans et al. In the
NJC
2
ꢁ6H
2
O and CuCl
2
ꢁ3H
2
O with the pyrazole derivative (b),
2
6
complexes depends primarily on the characteristic bonding ethyl acetate–methanol solution of CoCl and CuCl , the large,
2
2
preferences of the central metal ion and on the nature of the moderately ‘‘soft’’ Co(II) and Cu(II) cations preferentially bond
2
6,27
anion.
complexes with 4-phenylthiazole derivatives generated from Sz ´e cs ´e nyi et al. reported the structures of ligand b with
-(3,5-dimethyl-1H-pyrazole-1-yl)-4-phenyl-1,3-thiazole. nickel(II) salts in which Ni(II) preferentially binds to the ‘‘hard’’
We examined two types of N,N- and N,S-donor chelators and base N atom of the thioamide group (carrying a deprotonated
here we present the synthesis and structural characterization of NH function), resulting in N,N coordination of the ligand. In
some new cobalt(II), nickel(II) and copper(II) complexes with this work we obtained nickel(II) compounds wherein chelation
-(3,5-dimethyl-1H-pyrazole-1-yl)-4-phenyl-1,3-thiazole (ligand a), of 3,5-dimethyl-1-thiocarboxamide pyrazole occurs through a
However, there is no report on the synthesis of metal to the ‘‘soft’’ S atom, resulting in N,S coordination. M ´e sz ´a ros
2
7
2
2
2
and with 3,5-dimethyl-1H-pyrazole-1-carbothioamide (ligand b). ‘‘soft’’ base S atom of the thioamide group. Ligand (b) reacted
The resulting complexes were evaluated for their cytotoxic activity in ethyl acetate–methanol solutions with metal(II) chloride
against three tumor cell lines.
hexahydrate or metal(II) nitrate hexahydrate in a 1 : 1 molar
3 2
ratio to yield [MLCl ] complexes 1b, 4b, and 7b or [ML(NO ) ]
2
complex 8b. Nickel(II) nitrate hexahydrate and ligand b formed
complex 5b in a molar ratio of 2 : 1, irrespective of the L : M
substrate molar ratio (1 : 1 or 2 : 1). The reaction of ligand b with
cobalt(II) perchlorate hexahydrate in ethyl acetate solution
formed complex 3b in a molar ratio of 2 : 1. However, complexes
Results and discussion
Chemistry
The 4-phenylthiazole ligand (a) and the thiosemicarbazone
ligand (b) (Fig. 1) were synthesized according to the procedure
described in the Experimental section. The general method
employed to prepare the final compounds is outlined in Scheme 1.
Both ligands in the reactions with metal(II) salts yielded complexes
6b and 9b (ligand b and nickel(II) or copper(II) perchlorate
hexahydrate) were formed in a molar ratio of 3 : 1, irrespective
of the L : M substrate molar ratio (2 : 1 or 3 : 1). The molecular
structure of a representative complex [NiL
was determined by X-ray crystallography.
2 3 3
(NO )](NO ) (Fig. 5)
1a–9a and 1b–9b as solids.
In the complexes with ligand a, the metals ions were coordi-
nated by the two nitrogen atoms of the ligand, while the sulfur
atom did not take part in the coordination. The 4-phenylthiazole
compound reacted in ethyl acetate–methanol solutions with
metal(II) chloride hexahydrate or metal(II) nitrate hexahydrate
Neither cobalt(II) nitrate and ligand a nor nickel(II) chloride
and ligand b created the expected complexes.
Characterization of ligands and metal complexes
Comparative analysis of the infrared spectra of the complexes
and the metal-free ligands has revealed that the absorption
bands characteristic of the stretching vibrations of the pyrazole
in a 1 : 1 molar ratio to yield [MLCl ] complexes 1a and 7a or
2
[ML(NO
3 2
) ] complexes 2a and 5a. Surprisingly, copper(II) nitrate
trihydrate and this ligand formed an ionic complex 8a with
ꢀ
1
ꢀ1
+
2ꢀ
n(CQN) group shifted from 1603 cm
and 1600 cm for
general structure [CuL NO ] [Cu(NO ) ] . Ligand a with metal(II)
2 3 2 3 4
ꢀ1
ligands a and b to higher frequencies at 1626–1647 cm for
their complexes. This observation can be explained by the
participation of the nitrogen atom of pyrazole in the coordination
perchlorate hexahydrate in ethyl acetate solution formed com-
plexes 3a, 6a, and 9a, in a molar ratio of 2 : 1.
The course of complex formation by thiosemicarbazone is
anion-dependent and may be explained on the basis of Pearson’s
theory, taking into account the steric factors. The thiosemi-
carbazone derivative (b) has three potential coordination sites:
the nitrogen atom of the pyrazole ring and the nitrogen and
sulfur atoms of the thiocarbamoyl group. That ligand may act
as a neutral bi- or monodentate moiety. In our experiments,
ligand b interacted with the metal ions (in metal(II) chlorides,
metal(II) nitrates or metal(II) perchlorates) as a bidentate
N,S-donor to form the corresponding five-membered chelate
rings. In the Co(II) and Cu(II) complexes, obtained by a reaction
ꢀ1
with metal ions in complexes. The band in the 1502–1511 cm
2
8
region of complexes with ligand a has been assigned to n(CQN)
of the thiazole moiety. For these complexes, this characteristic
ꢀ
1
band shifted by 23–32 cm to higher frequency in comparison
ꢀ
1
with the spectrum of the ligand (ca. 1479 cm ). The infrared
spectra of ligand a and its complexes (1a–9a) showed no changes
in the absorption bands attributed to the vibrations of the n(CQS)
2
6
ꢀ1
group in the 685–700 cm region. These data indicate that
ligand–metal bonds in these complexes involved the nitrogen
atoms and not the sulfur atoms of the thiazole ring.
ꢀ
1
ꢀ1
The characteristic bands at 3083–3107 cm and 2976–2995 cm
of the methyl group of ligands a and b and their complexes
were assigned to n(C–H) vibration, respectively.
The FTIR spectra of ligand b and its complexes exhibited
ꢀ1
strong bands at 3400–3100 cm due to n(NH ) vibration of the
2
primary amide group. The NH2 bands in the spectrum of
ꢀ
1
ꢀ1
2
the ligand appeared at 3388 cm (NH stretch), 3241 cm
ꢀ1
27
(
asymmetric stretch) and 3134 cm
An intense strong band at 880 cm and a moderate one at
(symmetric stretch).
ꢀ1
Fig. 1 The structures of the investigated ligands a and b.
5
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Scheme 1 Synthesis and proposed structures of the Co(II), Ni(II), Cu(II) complexes 1a–9a, 1b–9b with ligands a and b.
ꢀ
1
ꢀ1
8
08 cm in the spectrum of this ligand (the n(CQS) vibration) 810–845 cm (medium) showed that complexes 2a, 5a, 8a,
ꢀ1
ꢀ1
ꢀ
9
shifted to lower frequencies (863–812 cm and 783–750 cm
,
5b and 8b include the NO
3
ions in their structure.
respectively) in the spectra of 1b–9b complexes, suggesting
the sulfur atom of {CQS to be a possible site of metal
coordination.
The chemical shifts and intensities of the signals in the
H NMR spectra of ligands a and b were very much similar to
those in the spectra recorded for the corresponding nickel(II)
1
Complexes 3a, 6a, 9a, 3b, 6b, and 9b showed intense bands complexes 5a, 6a, and 4b–6b. The spectra were recorded in
ꢀ
1
at 1120–1085 cm due to the involvement of the perchlorate DMSO-d
6
and are presented in the Experimental section.
The ligands and complexes showed signals in the range of
characteristic of uncoordinated perchlorate ions. The presence d 2.15–2.80 ppm for the methyl groups and d 5.92–6.29 ppm for
ꢀ
1
ion, and a sharp band of medium intensity at 629–623 cm
ꢀ1
of two bands between 1379–1383 cm (very strong) and at the proton of the pyrazole ring. In the spectrum of ligand a and
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2
+
+
its complexes we observed a signal at d B 7.80 ppm for the [CuL
2 2 3
] and [CuL NO ] ions (observed in positive ion mode) and
2ꢀ
ꢀ
proton from the thiazole moiety and at d 7.30–7.90 ppm for the the [Cu(NO
3
) ]
4
3
and [Cu(NO ) ] ions (observed in negative ion
2
protons from the phenyl group. The amine protons from ligand mode). The most structurally informative fragment ions in the
b were seen as two singlets at d 9.13 and 9.54 ppm for ligand 5a positive-ion mode of complexes 1a–9a, 1b–9b are presented in
and at d 8.86–9.10 ppm and d 9.27–9.51 ppm for complexes Table S1 (ESI†).
4
b–6b. The presence of two singlets for the two NH
may indicate some involvement in hydrogen bonding. Although
these protons are not directly involved in metal coordination, Herein we report the structures of [Co(a)Cl2] (1a), cis-
their signals tend to be shielded, perhaps due to inductive [Ni(a) (H O) ](ClO (6a), [Cu(a) (NO )] [Cu(NO ] (8a) and
NO ](NO ꢁ0.4H O (5b). The details of the refine-
)ꢁ0.9OEt
The H NMR spectra of cobalt(II) and copper(II) complexes ment of all compounds reported in this paper are presented in
2
protons
Molecular structures of 1a, 6a, 8a and 5b
2
2
2
4
)
2
2
3
2
3 4
)
7
effects.
[Ni(b)
2
3
3
2
2
1
could not be measured due to their paramagnetism.
Table 1.
[Co(a)Cl ] (1a) crystallized in the monoclinic P2 /n space group
The metal complexes have been investigated by mass spectro-
2
1
metry. The parent ions of complexes have not been observed in with all atoms in general positions and the whole molecule given
the mass spectra. However, in the FAB-MS spectra of all complex by the formulae in the asymmetric unit (Fig. 2). In (1) the
+
compounds the ions attributable to [LM] (L = a, b, M = Co(II), coordination sphere around the Co atom adopts a significantly
Ni(II), Cu(II)) were observed. The molecular ions corresponding to distorted tetrahedral environment. The coordination sphere
+
compounds [L
2
M] have been found for complexes 3a, 6a, 9a, 3b, consists of two nitrogen atoms and two chlorine atoms. The
+
5
b, and 6b. Additionally, [LMCl] ions for 1a, 7a, 1b, 4b, and 7b, N1–Co1–N10 angle is very small [80.28(7)1], whereas all other
+
+
as well as [LMNO ] ions for 2a, 5a, 5b, and 8b and [LMClO ]
ions for 3a, 6a, 9a, and 3b were found. Furthermore, [L MNO ]
and [L MClO ] ions were observed in the FAB-MS spectra of longer [2.0782(18) Å] by 0.07 Å. Both chlorine atoms are bound
2 4
angles exceed 1101 (Table S2, ESI†). Co1–N1 is the shortest
[2.0087(19) Å] in the coordination sphere and Co1–N10 is
3
4
+
2
3
+
coordination complexes 2a, 5a, 8a, 5b, 8b and 6a, 3b, 6b, 9b, to the Co atom at the same distance within experimental error
respectively. The FAB-MS spectra of complexes 1b–9b presented [2.2064(6) and 2.2090(6) Å]. All rings are flat with maximal
ion peaks at (m/z) 256, corresponding to L + 1H. Additionally, deviation for the chelate ring reaching 0.01 Å and are coplanar
mass spectrometry showed the presence of [CuL
ions 8a. This is evidenced by the fact that FAB produced [CuL]
2
NO
3
]
2
[Cu(NO
3
)
4
]
with the biggest angle 1.241 between the pyrazole and the
chelate rings. It is interesting that also the phenyl ring remains
+
Table 1 Crystal data and structure refinement for (1a), (6a), (8a) and (5b)
Parameters
1a
6a
8a
5b
Empirical formula
Formula weight
Temperature [K]
C
14
H
13Cl
2
3
CoN S
C
28
H
30Cl
2
N
6
NiO10
S
2
C
56
H
52Cu
3
N
18
O
18
S
4
15.6 27.8 8 2
C H N NiO7.3S
567.09
293(2)
385.16
293(2)
804.31
293(2)
1584.02
293(2)
Wavelength [Å]
Crystal system, space group
Unit cell dimensions [Å] and [1] a = 8.9069(4)
b = 14.7753(6)
0.71073
Monoclinic, P2(1)/n
0.71073
0.71073
Orthorhombic, Pbcn
a = 19.8159(6)
b = 13.1908(6)
c = 25.0863(9)
0.71073
Monoclinic, C2/c
a = 24.0372(10)
b = 9.6174(3)
c = 16.0976(7)
b = 113.255(5)
3419.0(2)
4, 1.563
0.909
1656
Monoclinic, P2(1)/n
a = 16.6552(6)
b = 14.1497(5)
c = 23.1221(10)
b = 104.258(4)
5281.2(4)
8, 1.426
0.943
2366
c = 12.3744(5)
b = 102.793(4)
1588.07(12)
4, 1.611
1.543
3
Volume [Å ]
6557.2(4)
4, 1.605
1.176
3236
ꢀ
ꢀ
3
Z, calculated density [mg m
Absorption coefficient [mm
F(000)
]
1
]
780
Crystal size [mm]
Theta range for data collection [1] 2.18 to 26.37
Limiting indices
0.55 ꢂ 0.28 ꢂ 0.18
0.98 ꢂ 0.58 ꢂ 0.27
0.26 ꢂ 0.17 ꢂ 0.15
2.02 to 26.37
ꢀ7 r h r 24
ꢀ10 r k r 16
ꢀ20 r l r 31
0.34 ꢂ 0.26 ꢂ 0.13
2.32 to 25.03
ꢀ16 r h r 19
ꢀ7 r k r 16
ꢀ27 r l r 19
17 920/9384 [R(int) =
0.0792]
2.31 to 26.37
ꢀ11 r h r 11
ꢀ30 r h r 14
ꢀ10 r k r 12
ꢀ17 r l r 20
ꢀ
18 r k r 15
6 r l r 15
ꢀ
Reflections collected/unique
5674/3246 [R(int) = 0.0362] 5751/3494 [R(int) = 0.0849] 16 524/6694 [R(int) =
0.0534]
Completeness to theta
Absorption correction
Max. and min. transmission
Refinement method
26.371 99.9%
Numerical
0.7686 and 0.4841
26.371 99.8%
Numerical
0.7913 and 0.4694
26.371 99.7%
Numerical
0.8433 and 0.7496
25.031 99.9%
Numerical
0.8872 and 0.7399
Full-matrix least-squares Full-matrix least-squares Full-matrix least-squares Full-matrix least-squares
2
2
2
2
on F
on F
on F
on F
Data/restraints/parameters
Goodness-of-fit on F
Final R indices [I 4 2s(I)]
R indices (all data)
3246/0/192
3494/0/231
6694/0/450
9384/6/635
2
0.873
1.068
0.942
1.042
a
b
b
a
b
b
a
b
b
a
b
b
R
R
1
= 0.0320, wR
= 0.0403, wR
2
= 0.0920 R
1
= 0.0458, wR
= 0.0528, wR
2
2
= 0.1279 R
1
= 0.0459, wR
= 0.1010, wR
2
2
= 0.1021 R
1
= 0.0720, wR
= 0.1445, wR
2
= 0.2057
= 0.2381
a
1
a
a
a
2
= 0.0963 R
1
= 0.1320 R
1
= 0.1230 R
1
2
Extinction coefficient
—
0.0008(4)
0.706 and ꢀ0.440
—
—
ꢀ
3
Largest diff. peak and hole [e Å ] 0.327 and ꢀ0.312
0.810 and ꢀ0.893
0.460 and ꢀ0.430
P||F
| ꢀ |F
||/P
|F
0
|. wR
= [ w(F
P
ꢀ F
) / (w(F
P
) )]
.
a
b
2
2 2
2 2 1/2
R
1
=
0
c
2
0
c
0
5
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Fig. 2 Molecular structure of complex 1a with the numbering scheme
and the displacement ellipsoids drawn at the 20% probability level.
coplanar with these three rings and it forms angles not exceed-
ing 9.121. It happens often that such phenyl rings are signifi-
cantly twisted. In this case the conformation of the organic
ligand results from the packing revealing all aromatic rings in
the ac layer and the intramolecular hydrogen bond is formed
between H92 and N10 atoms. Hence, the separation between
the layers is maintained by chloride ions. As a consequence,
the Co–Co distance is much bigger inside one layer (at least
Fig. 3 Molecule of cis-[Ni(a)
scheme and the thermal ellipsoids at 20% probability. For the clarity of
the figure the perchlorate ion is omitted.
2
(H
2
O)
2
](ClO
4
)
2
(6a) with the numbering
In the packing (Fig. S2, ESI†) we observe well-ordered
8.907 Å) than between layers (the shortest distance between the structure in all three dimensions with significant separation of
layers is 7.504 Å). Therefore, the molecules inside one layer interact at least 9.341 Å between nickel(II). Due to the distances between the
weakly creating only two hydrogen bonds: H41C–Cl3(1 + x, y, z) metal ions we describe this packing as bc layers with the angle of
and H41B–Cl2(1 + x, y, z). In the crystal network of (1a) the p–p only 61.701 between three closest nickel(II) ions. Hence, the nickel
interactions dominate between the systems being fairly coplanar ions in the layer are arranged in almost perfect rhombuses with
(
Fig. S1, ESI†).
the sides being 9.341(1 ꢀ x, ꢀy, 1 ꢀ z) Å and 9.617(x, 1 + y, z) Å
cis-[Ni(b) (H
2
2
O)
2
](ClO
4
)
2
(6a) crystallized in the monoclinic long and the shorter diagonal of 9.412(1 ꢀ x, 1 ꢀ y, 1 ꢀ z) Å,
C2/c space group with the nickel ion positioned at the twofold whereas the distance between the adjacent layers exceeds 11 Å.
axis. In the asymmetric unit there are the nickel ion, the organic In the crystal network formation numerous p–p interactions
ligand, one coordinated water molecule and a perchlorate anion play the main role, among them a system of stacking inter-
(
Fig. 3). The nickel ion is found in an octahedral environment actions composed of four rings: C91(x, y, z)–S7(x, y, z)–S7(1 ꢀ x,
with four nitrogen atoms from organic ligands (both N1 nitrogen 1 ꢀ y, 1 ꢀ z)–C91(1 ꢀ x, 1 ꢀ y, 1 ꢀ z). Hence, the intermolecular
atoms are found in trans positions) and two oxygen atoms from interactions between two S7 rings are capped by two C91 phenyl
water molecules found in cis positions. In the nickel coordi- rings inclined only by 4.361 and forming intramolecular stack-
nation sphere, Ni–N bonds are 2.091(2) Å for Ni1–N1 and ing interaction. Additionally, the closest molecules interact via
2.1417(18) Å for Ni1–N10, whereas Ni–O bonds are much a hydrogen bond network created by both coordinated O4 water
shorter being 2.0692(17) Å long (Table S2, ESI†). The valence molecules and four perchlorate ions revealing the importance of
angles deviate from 78.25(7) to 104.39(7)1 and from 170.11(7) to these ions. They fasten the network participating in hydrogen
1
76.51(9)1 with the smallest angles close to 801 found for the bonds as well as Cl–Oꢁ ꢁ ꢁp interactions.
five-membered chelate rings. [Cu(a) (NO )] [Cu(NO ] (8a) crystallized in the orthorhombic
All aromatic rings remain flat with rms deviation reaching Pbcn space group with a Cu2 atom positioned at the b twofold
2
3
2
3 4
)
+
0
0
.003 Å, whereas the chelate ring is slightly folded with rms of axis. Hence, in the asymmetric unit there is one [Cu(a)
2 3
(NO )]
.032 Å. The angle between S7 and N1 rings of the ligand moiety and the Cu2 atom with two nitrate anions (Fig. 4a and b).
molecule reaches 14.521 showing that the fused ring system in this Cu1 atoms exist in a square pyramidal environment with the
2
9
molecule is quite folded. The C91 phenyl ring is significantly trigonal distortion parameter t = 0.20 and the coordination
inclined up to 69.671 to other rings. This conformation of the sphere is composed of four nitrogen atoms from two organic
ligand enables numerous strong (due to mutual orientation) p–p ligands as well as one oxygen atom from the nitrate ion. The
interactions between two ligands coordinated to the same nickel(II) Cu–N bonds range from 1.982(3) to 2.275(3) Å (Table S2, ESI†).
ion. Apart from that C21–H21CꢁꢁꢁO4(ꢀx + 1, y, ꢀz + 1/2) and an The longest value occurring for the apical atom reveals elongation
intramolecular C41–H41BꢁꢁꢁS7 hydrogen bond are formed.
due to the Jahn–Teller effect. The length of 1.988(3) Å for the
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Fig. 4 Molecule of [Cu(a)
2
(NO
3
)]
2
[Cu(NO
3
)
4
] (8a) with the numbering scheme and the thermal ellipsoids at 20% probability. In the asymmetric unit there
+
2ꢀ
is one [Cu(a)
2
(NO
3
)] cation (a) and Cu2 copper cation with two nitrate anions from the [Cu(NO
3
)
4
]
unit (b).
Cu1–O3 bond is similar to the values observed for Cu1–N1
and Cu1–N11 bonds, whereas the forth basal Cu–N bond is
elongated by 0.06 Å [Cu–N20: 2.045(3) Å]. In the coordination
sphere valence angles fall in two ranges: from 77.50(11) to
108.42(12)1 and from 161.67(12) to 173.98(12)1. In the second
moiety the Cu2 ion is surrounded by four nitrate anions mono-
dentately coordinated, which form a significantly distorted
tetrahedral environment with Cu–N distances of 1.975(4) and
1
9
.984(4) Å and the angles falling into two ranges: close to
01 and 1581.
All aromatic rings remain flat with rms deviation reaching
.014 Å for the S17 ring. The chelate rings are much more folded
0
with rms 0.084 and 0.057 Å for N11 and N1 rings, respectively.
All fused rings in one ligand are rather flat with the biggest angles
Fig. 5 Crystal network of (8a) along the a axis. For clarity of the figure
of 17.7 and 14.01 created for S17 and S7 ligands, respectively. all hydrogen atoms are omitted. CuT denotes tetrahedral copper(II) ions
coordinated by four nitrates.
In complex 8a both phenyl rings are significantly twisted like
the fused rings with an angle close to 501 and 401 for S17 and S7
ligands, respectively. The angle formed between S7 and S17
molecules is 52.201 indicating that the orientation of both ligands molecule (0.8 occupancy). Both nickel ions are found in an
is significantly tilted. Several intramolecular p–p interactions octahedral environment with two nitrogen and two sulfur
determine such mutual orientation of these rings. Additionally, atoms from organic ligands and two oxygen atoms from nitrate
C–Hꢁ ꢁ ꢁp interactions and hydrogen bonds between the methyl anions. The oxygen atoms occupy cis positions and are trans to
groups and O3 or sulfur atoms were detected.
the sulfur atoms. Hence, both nitrogen atoms forming the
The packing viewed along the a axis reveals ab layers of shortest bonds to the nickel ion are found in trans positions.
0
+
2ꢀ
[
Cu(L ) (NO )] cations, which are separated by layers of [Cu(NO ) ]
The Ni–N bonds range from 2.033(5) Å for Ni1–N11 to 2.070(6) Å
2
3
3 4
anions (Fig. 5). In the network we observe strong interactions, for Ni1–N1 bonds (Table S2, ESI†). The Ni–O bonds are longer
which are assured mainly by hydrogen bonds and p–p inter- and fall in a very broad range from 2.100(5) to 2.212(6) Å. The
2ꢀ
actions. Despite the separation of cation layers by [Cu(NO
3
)
4
]
longest bonds are created by the sulfur atoms, which partici-
anions, they form direct p–p interactions between C9A(x, y, z) pate in coordination instead of the N10 atoms. They range from
and C9G(x, 1 ꢀ y, ꢀ1/2 + z) phenyl rings. However, interlayer 2.333(3) to 2.374(2) Å. However, N10 atoms are not aromatic as
2
ꢀ
0
moieties in the L ligand and bear two hydrogen atoms (in our model we
interactions are transferred mainly by [Cu(NO
3
)
4
]
involved in hydrogen bonds and N32–O32ꢁ ꢁ ꢁN1(x, y, z) pyrazole found three positions with 0.67 occupancy each). The coordina-
ring interactions. tion spheres reveal significant distortions as suggested by the
Ni(b) NO ](NO )ꢁ0.9OEt ꢁ0.4H O (5b) crystallized in the mono- valence angles falling into two ranges: from 59.6(2)1 to 104.1(2)1
[
2
3
3
2
2
1
clinic P2 /n space group with two nickel-containing moieties and from 153.0(2)1 to 178.7(2)1 for the Ni1 atom and from
in the asymmetric unit (Fig. 6a and b). Additionally in the 59.7(2)1 to 105.14(9)1 and from 155.2(2)1 to 176.1(3)1 for the Ni2
asymmetric unit there are two nitrate ions, two diethyl ether coordination sphere. 104.1(2)1 and 105.14(9)1 were found for
molecules (one of them occurs with 0.8 occupancy) and a water S–Ni–S angles. Among trans positioned atoms the biggest values
5
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Paper
Fig. 6 Molecule of [Ni(b)
2
NO
3
](NO
3
)ꢁ0.9OEt
2
ꢁ0.4H
2
O (5b) with the thermal ellipsoids at 20% probability. Only two Ni units are presented with the
numbering scheme (a, b) whereas the non-coordinated nitrates, diethyl ether and water molecules are omitted for clarity of the picture.
were found for the N–Ni–N angles, whereas the O–Ni–S angles between the non-coordinated amine group and also non-
are smaller revealing larger deviation from 1801. The smallest coordinated nitrate anions. Hence, we can hypothesize that these
angle occurs for bidentately coordinated nitrate anions, which are anions are very important for the crystal network formation,
involved in the formation of the four-membered chelate rings. The because they are involved in the interactions with nickel subunits
small size of this anion does not permit for the appropriate from the adjacent layers. Similarly, an ether molecule also
occupation of two adjacent positions in the regular octahedron mediates interaction between the adjacent layers (Fig. S3, ESI†).
and the deformation of the octahedral nickel environment is a
In this paper we report structures of complexes containing
consequence of the smaller size and the rigid structure of the two related ligands. In all compounds reported in this paper
nitrate anion. All aromatic rings remain flat with rms deviation the organic ligand coordinate via the N1 pyrazole nitrogen atom
reaching 0.012 Å, whereas the chelate ring is significantly folded and in three cases also via the N10 thiazole nitrogen atom. We
with rms up to 0.141 Å. Pyrazole and chelate rings are not coplanar observe that the thiazole sulfur atom is not involved in the
and are inclined at an angle ranging from 10.9(4)1 to 13.2(4)1. This coordination while the aliphatic sulfur exerts high affinity towards
results in the shift of N10 from the chelate ring plane, which reveals nickel as evidenced in complex 5b. For M–N1 metal–pyrazole
a very broad range: from 0.0543 Å for N10 to ꢀ0.6830 Å for N40. bonds the distances range from 1.982(3) Å for the copper complex
The angles between the organic ligands and the coordinated nitrate to 2.091(2) Å for the nickel complex (6a). In general, we observe
anion bound to the same nickel ion are almost perpendicular with that these distances are usually close to 2 Å, but differ quite
values being close to 80–901. Two organic ligands coordinated to significantly for both nickel complexes. The M–N10 distance
the same nickel ion are inclined by approximately 601.
varies from 2.045(3) to 2.275(3) Å and both extreme values were
In the packing we observe ab layers with nickel ions from found for the copper complex. This bond is usually about
adjacent layers separated at least by 11.574 Å. Every layer is 0.05–0.07 Å longer than the metal–pyrazole nitrogen bond
composed of both nickel units. In a single layer there are and only for the Cu1–N10 bond in complex 8a this elongation
almost equally distributed nickel units. Every nickel unit is is much bigger reaching 0.3 Å. It is worth noticing that in the
surrounded by six other moieties with Ni–Ni distances ranging reported structures we found two kinds of nitrate coordination:
from 7.802 to 8.783 Å. The Ni1 block is surrounded by two other monodentately in complex 8a and bidentately in 5b. In the latter
Ni1 units and four Ni2 units, whereas Ni2 is surrounded by two case the small dimensions resulted in significant distortions of
Ni2 and four Ni1 units. Every layer is composed of alternately the coordination sphere geometry.
arranged Ni2 and Ni1 zigzags with Ni2–Ni2 and Ni1–Ni1 distances
of 7.802 and 7.854 Å, respectively. These values correspond to the with cobalt, copper, nickel, zinc, cadmium and palladium ions.
There are several interesting papers reporting related ligands
30,31
shortest intermetallic distances in the crystal network and they are Due to the diversity of the known structures in terms of metal as
slightly shorter for Ni2–Ni2 contacts due to a slightly bigger value well as ligands we decided to discuss these structures together and
of the Ni2–Ni2–Ni2 angle (130.12 vs. 128.511). In the crystal to compare with compounds reported in this paper. Bolos et al.
packing the interactions the inside ab layer are transmitted directly reported the structure of a copper complex with 2-amino-5-methyl-
between nickel moieties via numerous p–p interactions mainly 1,3-thiazole, which is related to ligand a because it possesses a
32
created by the edge-to-face oriented pyrazole rings. Additionally, thiazole ring. It is coordinated monodentately via the aromatic
there are N–Oꢁꢁꢁp interactions occurring in Ni1 and Ni2 zigzags. nitrogen atom with Cu–Nthiazole bonds being 1.945(6) and
Moreover, this network reveals a robust network of hydrogen 2.063(7) Å. The former bond is much shorter than the corre-
bonds. However, there are only a few examples of direct interac- sponding bond found in 8a. Other copper complexes are known
2
6,30
tions between nickel subunits and only in one layer, whereas with ligands related to the b ligand.
nickel moieties from adjacent layers do not interact directly. Such nated L ligand in two complexes formulated as [Cu
hydrogen bonds are created by C–H bonds of the pyrazole ring or [Cu(NO (L) ] (L = 3,5-dimethyl-1H-pyrazole-1-carboxamidine)
In the bidentately coordi-
2
(NCS) (L) ] and
4
2
3
)
2
2
3
0
its methyl substituents and the coordinated nitrate anions. Apart the sulfur atom is substituted by a nitrogen atom. The Cu–N1
from that, there are numerous N–Hꢁ ꢁ ꢁO interactions created distances [1.983(3) and 2.0221(14) Å] are similar to those in our
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copper complex. In contrast, Cu–N3pyrazole distances [1.952(3) on the crystal network and possible interactions. For complexes
and 1.9457(15) Å] are significantly shorter than Cu–N10 bonds 1b, 6a and 8a, coordinated via N10 nitrogen from the aromatic
observed in complex 8a with the N10 thiazole nitrogen atom ring, we observe smaller angles for the N1 pyrazole and chelate
involved in the coordination. In two copper complexes two rings. For 5b bearing only the pyrazole and chelate rings these
molecules of the b ligand were bound to metal ions via nitrogen angles are slightly bigger. Therefore, the lack of an additional
2
6
and sulfur atoms. The copper atom was found in trigonal phenyl ring and of the thiazole ring results in two important
bipyramidal or tetrahedral environments and Cu–N distances consequences: (i) the sulfur atom participates in the coordination
differed being 1.984(4) Å in the pentacoordinated complex and of the metal ion, (ii) the lack of the thiazole ring allows for bigger
2.023(3) Å in the tetrahedral one. These distances are similar to conformational flexibility, as presented by a bigger value of the
Cu–N1 bond lengths in 8a. However, Radosavljevi ´c et al. showed dihedral angle created between the N1 pyrazole and chelate rings.
3
4
that the sulfur atom may be involved in the bridging of two This is confirmed by the results obtained by Radosavljevi ´c et al.
00
00
copper ions and the Cu–S bond lengths depend on the binding for Cd L Cl (L = 3,5-dimethyl-1H-pyrazole-1-carbothioamide).
2
2
4
mode. They are 2.308(1) Å in the pentacoordinated complex, They showed that the pyrazole and chelate rings remain coplanar
whereas an elongation is observed [2.341(1) and 2.522(1) Å] if the with an angle of 19.8(5)1 being significantly bigger than in our
0
0
00
00
26
sulfur atom is involved in bridge formation. We found coordina- structures. Similarly, for [Cu
2
L
2
Cl
4
], [Cu
2
L
2
Br
2
] and [Co
2
L
2 4
Cl ]
tion via sulfur only in the case of nickel complex 5b with Ni–S complexes these values were 151, 241 and 16.31, respectively. The
0
00
bond lengths similar to the values observed for bridging Cu–S mutual conformation of L or L ligands in complexes described by
bonds. Radosavljevi ´c et al. reported the structure of 3,5-dimethyl- the 1 : 2 metal : pyrazole ligand stoichiometric ratio usually ranges
1
-thiocarboxamidopyrazole complexed with nickel(II) in a square from 50–601 in the reported structures. The values observed for the
1
2
planar environment of four nitrogen atoms bound at an average structures of [ZnL (H O)]ꢁ2DMSO and [ZnL (H O)]ꢁ2DMSO
2
2
2
2
3
3
1
2
distance of 1.902 Å. In octahedral complexes 6a and 5b the (L and L are 5-oxo-1H-pyrazole-1-carbothioamide derivatives)
Ni–N1 bond lengths range from 2.033(5) to 2.091(2) Å, so they are 51.541 and 56.801, being very similar to those observed for
35
36
are remarkably longer than those described in the literature. the reported structures. Casas et al. presented three complexes
Moreover, there are four nitrogen atoms in the coordination formulated as [ZnL
4
2
2
2
5
2
2
]ꢁ0.5H O[CdL
(H
2
O)]ꢁ2DMSO[CdL (DMSO)]ꢁ
sphere in 6a, but Cu–Npyrazole distances are significantly shorter DMSO with these angles being +781, +55.91 and 221, respectively.
than the Cu–Nthiazole bonds. In the cobalt(II) complex with two These data show that two ligands being pyrazole derivatives can
molecules of the b ligand the metal ion was found in a trigonal adopt a mutual orientation in a very broad range. In our copper
bipyramidal environment. The metal ion is coordinated via and nickel complexes, there are two pyrazole ligands in the
nitrogen and sulfur atoms of the b ligand with the Co–N distance coordination sphere and they are rather strongly twisted. Both
of 2.108(1) Å which is much longer than the Co–N1 bonds in 1a. fused systems in the reported compounds were found inclined at
We evaluated dihedral angles between the rings in all ligands an angle 68.921 (6a), 52.201 (8a), 43.591 and 63.941 (5b) revealing a
present in the four reported compounds (Table S3, ESI†). Only in significant twist.
26
the cobalt(II) complex 1a all rings are nearly coplanar and even the
phenyl group remains in the plane of three other rings (very small
Biological assay
rms for these rings). In three other structures the rings are not so The cytotoxicity of the ligands (a, b) and their cobalt(II),
perfectly coplanar. The most important deviation was found for nickel(II) and copper(II) complexes 1a–9a, 1b–9b was measured
the phenyl rings, in 6a and 8a, which are twisted by 52–701 towards against human melanoma WM-115 cells as well as leukemia
the pyrazole ring due to numerous intramolecular p–p interactions promyelocytic HL-60 and lymphoblastic NALM-6 cells. Cisplatin
3
7
and hydrogen bonds. In the zinc(II) structure, these rings are and carboplatin were used as the reference compounds. Cells
31
twisted by ꢀ5.6(2)1 and 27.4(2)1. This difference results from were exposed to a broad range of drug concentrations for
steric hindrance between both phenyl rings coming from the 48 h and cell viability was analyzed by MTT assay. IC50 values
neighboring molecules. These authors also found that in the of ligands and their complexes are shown in Table 2.
molecules neighboring along the c axis both phenyl rings are
The ligand a was inactive in the case of WM-115 cells but
perpendicularly oriented [88.2(2)1]. In our compounds, the biggest expressed moderate cytotoxicity against NALM-6 and HL-60
values of dihedral angles between the C91 phenyl ring and leukemia cells. The ligand b was not toxic to the tested tumor
pyrazole, chelate or thiazole rings (55–701) were found for nickel cell lines. In both cases, the coordination of a metal to the
complex 6a. Hence, it is conceivable that this observation might be ligand increased their cytotoxic activity.
attributed to the coordination number. The higher number for
The highest cytotoxic activity has been observed for cobalt(II)
nickel (6) than for copper (5) results in bigger steric hindrance complexes 3a, 1b, 3b and copper(II) complexes 8a, 7b against
forcing bigger twist of phenyl rings. Moreover, in the reported NALM-6 cells (IC50 coefficient in the range from 5.4 to 16.7 mM),
compounds the N1 chelate and sulfur rings form angles of at least and for copper(II) complexes 8a, 7b, 9b against the melanoma
12.541, which differ significantly from 0.771 found for 1a. The WM-115 cell line (IC50 coefficient in the range from 4.9 to 20.6 mM).
adjacent rings, despite being fused, can form angles up to 14.711. It should also be mentioned that significant cytotoxicity (IC50
For complexes 6a and 8a the angles between the N1 pyrazole and coefficient in the range from 11.5 to 20.6 mM) among the metal
S7 rings range from 14.01 to 17.71. Hence, we observe some complexes with 2-(3,5-dimethyl-1H-pyrazole-1-yl)-4-phenyl-1,3-
flexibility of this ligand, which adopts conformation dependent thiazole (a) against the all cell lines was exhibited by ionic
5
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Table 2 Cytotoxicity of the ligands and their metal(II) complexes 1a–9a, Conclusions
1
b–9b expressed as the IC50 values (in mM)
In this paper we have described the synthesis of novel cobalt(II),
nickel(II) and copper(II) complexes with N,N-donor phenyl-
thiazole and with an N,S-donor thiosemicarbazone-derived
a
IC50 [mM]
Compound
HL-60
NALM-6
WM-115
a
82.6 ꢃ 3.5
48.4 ꢃ 3.4
55.9 ꢃ 0.6
46.6 ꢃ 2.8
64.3 ꢃ 1.6
47.1 ꢃ 3.8
37.9 ꢃ 2.1
11.5 ꢃ 0.9
45.4 ꢃ 3.6
583.9 ꢃ 84.3
65.0 ꢃ 3.1
47.5 ꢃ 1.67
60.7 ꢃ 1.45
50.6 ꢃ 3.8
53.1 ꢃ 0.9
5.3 ꢃ 0.2
82.1 ꢃ 1.6
29.8 ꢃ 5.5
36.4 ꢃ 1.8
16.1 ꢃ 1.1
61.1 ꢃ 1.4
50.4 ꢃ 1.4
54.1 ꢃ 2.9
16.7 ꢃ 0.5
54.1 ꢃ 4.4
569.3 ꢃ 21.9
9.1 ꢃ 0.2
945.0 ꢃ 44.4 ligand. The structures of these complexes were confirmed by
1
2
3
5
6
7
8
9
a
a
a
a
a
a
a
a
289.1 ꢃ 52.6
spectral and elemental analysis. The molecular structures of four
277.5 ꢃ 25.7
complexes 1a, 6a, 8a and 5b were confirmed by X-ray analysis. We
78.1 ꢃ 5.9
434.3 ꢃ 44.8 found that the M–N10 bonds are usually by 0.05–0.07 Å longer
251.3 ꢃ 28.4
than the metal–pyrazole nitrogen bond, but for complex 8a this
elongation is much bigger (0.3 Å). In the reported structures two
kinds of nitrate coordination were found: the monodentate
in 8a and bidentate in 5b. We observed the influence of the
coordination number and of substituents on the conformation
64.7 ꢃ 3.2
20.6 ꢃ 2.4
60.7 ꢃ 2.5
b
41000
1
3
4
5
6
7
8
9
b
78.1 ꢃ 15.2
66.0 ꢃ 3.5
b
b
b
b
b
b
b
8.9 ꢃ 0.2
63.1 ꢃ 2.2
51.6 ꢃ 1.6
52.7 ꢃ 0.6
5.4 ꢃ 0.2
184.9 ꢃ 22.8 of the complexes. In all analyzed complexes hydrogen bonds
74.7 ꢃ 6.8
played a principal role in the crystal network formation. Having
compared the coordination modes observed for both studied
74.9 ꢃ 3.9
4.9 ꢃ 0.2
53.3 ꢃ 3.0
56.1 ꢃ 7.2
0.8 ꢃ 0.1
50.3 ꢃ 5.3
56.7 ꢃ 3.0
0.7 ꢃ 0.3
35.4 ꢃ 4.2
5.5 ꢃ 0.6
ligands we have concluded that aliphatic sulfur can participate
in metal binding and sometimes even in bridge formation,
whereas thiazole sulfur is not involved in the coordination. At
least partially this effect can be ascribed to bigger conforma-
tional flexibility.
b
Cisplatin
Carboplatin
18.2 ꢃ 4.3
422.2 ꢃ 50.2
b
4.3 ꢃ 1.3
0.7 ꢃ 0.2
a
IC50 – concentration of a tested compound required to reduce the
fraction of surviving cells to 50% of that observed in the control, non-
treated cells. Mean values of IC50 (in mM) ꢃ S.D. from 4 experiments are
A cytotoxic study demonstrates that the coordination com-
pounds of copper(II) with the both ligands were more effective
against the skin melanoma WM-115 cells when compared to
cobalt(II) and nickel(II) compounds. It should be noted that
higher cytotoxic activity was observed in WM-115 cultures treated
with copper(II) complexes containing the N,S-donor thiosemi-
carbazone ligand than in those treated with copper(II) complexes
containing the N,N-donor phenylthiazole ligand. A Cu(II) complex
of 3,5-dimethyl-1H-pyrazole-1-carbothioamide (7b) exhibits the
highest cytotoxic activity towards all tested cell lines. Cytotoxic
effectiveness of this complex and of another copper(II) complex
with thiosemicarbazone-derived ligand (9b) against melanoma
WM-115 cells was about four times higher than that of cisplatin.
Unfortunately, 7b complex also showed a high toxic effect to the
noncancerogenic HUVECs.
b
38
presented. See the literature.
copper(II) complex 8a. Copper(II) complex 7b (with 3,5-dimethyl-
H-pyrazole-1-carbothioamide) exhibited the highest cytotoxic
activity against all the tested cell lines (IC50 coefficient in the
range from 4.9 to 5.4 mM). This compound with an IC50 of
.3 mM expressed similar activity against leukemia promyelocytic
HL-60 cells and was almost four times more effective (IC50 = 4.9 mM)
in the case of melanoma WM-115 cells when compared to
cisplatin (IC50 18.2 mM).
The presence of the carbothioamide moiety in the
structure improved the activity of these metal complexes
significantly, compared to the corresponding compounds with
the 4-phenylthiazole-derived ligand in the case of melanoma
WM-115 cells.
1
5
Preliminary experiments also showed promising cytotoxic
activity of copper(II) compound 8a. Its activity was the greatest
among the complexes with the 4-phenylthiazole derivative, which
could be ascribed to its different ionic structure. Therefore, more
experimental work is necessary to confirm the cytotoxic behavior
and the mechanism of action of this copper(II) complex.
All the metal(II) complexes, besides copper complexes with
the thiosemicarbazone-derived ligand (7b–9b), showed higher
cytotoxic activity to both acute leukemia HL-60 and NALM-6 cell
lines than against the melanoma cells, whereas these copper
compounds, especially compound 9b, exhibited selective cyto-
toxicity against melanoma WM-115 cells.
The lowest activity (IC50 4 184 mM) against WM-115 cells
showed cobalt(II) and nickel(II) complexes 1a, 2a, 5a, 4b. However,
the cytotoxicity of almost all the metal(II) complexes was higher
than that of carboplatin against these tumor cells.
Experimental
Materials and methods
All substances were used without further purification. Metal(II)
We also decided to examine the cytotoxicity of the more chloride, nitrate and perchlorate salts were purchased from
promising compounds (1b, 3b, 7b) against normal human Aldrich. DMSO-d solvents for NMR spectroscopy were obtained
6
umbilical vein endothelial cells (HUVECs). Analysis of the from Dr Glaser AG Basel. Solvents for synthesis (acetonitrile,
cytotoxicity data revealed that 1b and 3b presented higher dichloromethane, diethyl ether, dimethylformamide, ethyl acetate,
effectiveness against NALM-6 leukemia cells compared to the and methanol) were reagent grade or better and were dried
3
8
normal HUVECs (IC50 = 73.0 mM and IC50 = 38.6 mM respectively). according to standard protocols. The melting points were
The cytotoxic activity of 7b against healthy cells was also high determined using an Electrothermal 1A9100 apparatus and
1
with IC50 = 5.1 mM.
they are uncorrected. The H NMR spectra were registered
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at 300 MHz on a Varian Mercury spectrometer. The IR spectra were acetate (5 ml). The reaction solution was stirred for 12 hours
recorded on KBr pellets on an FT-IR-8400S Shimadzu spectro- at room temperature. The resulting maroon solid 2a was
photometer. The FAB-MS data were determined on a Finnigan obtained by the diffusion of diethyl ether into the mixture.
+
Matt 95 mass spectrometer (NBA, Cs gun operating at 13 keV). Yield: (35.0 mg, 25.5%), mp dec. 4282.3 1C. Anal. calcd for 2a
ꢀ
1
For the new compounds elemental analyses (C, H and N) were C H N SCoN O ꢁ1.5C H O ꢁ0.5H O (579.36 g mol ) C 41.24%;
1
4
13
3
2
6
4
8
2
2
performed using a Perkin Elmer PE 2400 CHNS analyzer.
H, 4.52%; N, 12.09%. Found: C, 40.89%; H, 4.42%; N, 12.26%.
FTIR (KBr cm ): 3367br, 3098m, 1736m, 1647w, 1576m, 1506s,
379s, 1011w, 845m, 698m. MS-FAB (m/z): 256.0 (25%) [a] , 313.1
ꢀ1
Synthesis of the ligands
+
1
+
+
Synthesis of ligand 2-(3,5-dimethyl-1H-pyrazole-1-yl)-4-phenyl- (15%) [(a)Co] , 376.1 (100%) [(a)CoNO
3
] .
1
,3-thiazole (a) was based on the method described previously Cobalt(II) complex (3a). Cobalt(II) perchlorate hexahydrate
3
9
with some modifications. Acetylacetone (480 mg, 4.8 mmol) Co(ClO ) ꢁ6H O (43.9 mg, 0.12 mmol) was added dropwise at
4
2
2
and 2-hydrazino-4-phenylthiazole (920 mg, 4.8 mmol) were room temperature to a stirred solution of ligand a (61.3 mg,
stirred in anhydrous methanol (7 ml) and catalytic amounts 0.24 mmol) in ethyl acetate (8 ml). The reaction solution was
of p-toluenesulfonic acid were added to a stirred solution. The stirred for 24 hours at room temperature. The resulting maroon
reaction was monitored by thin-layer chromatography (TLC) solid 3a was obtained by the diffusion of diethyl ether into the
using a silica gel on aluminum sheets with fluorescent dye mixture. Yield: (81.2 mg, 84%), mp 299.8–302.3 1C. Anal. calcd
ꢀ
1
F254 and a 20/80 mixture of acetone/chloroform as the mobile for 3a C28
phase. After 3 h of refluxing, the concentration of the solution 3.76%; N, 10.45%. Found: C, 41.89%; H, 3.23%; N, 10.33%.
to half of its initial volume gave a solid that was filtered out FTIR (KBr cm ): 3363br, 3100m, 1630m, 1578s, 1503s, 1116s,
and dried under reduced pressure. Yield: 969 mg, 85.0%, 1026s, 689m, 629m. MS-FAB (m/z): 256.2 (80%) [a] , 313.1 (74%)
H
26
N
6
S
2
CoCl
2
O
8
ꢁ2H
2
O (804.45 g mol ) C, 41.81%; H,
ꢀ
1
+
ꢀ
1
+
+
+
mp 102.2–103.5 1C. FTIR (KBr cm ): 3083s, 1603m, 1573s, [(a)Co] , 413.2 (96%) [(a)CoClO ] , 569.3 (52%) [(a) Co] , 669.3
4
2
1
+
1
(
(
539s, 1479m, 1026m, 685m. H NMR (DMSO-d
s, 3H, C(3)–CH pyrazole) 2.80 (s, 3H, C(5)–CH
s, 1H C(4) pyrazole), 7.27 (m, 1H, phenyl), 7.35 (m, 2H, phenyl), Ni(NO
6
) d (ppm): 2.32 (100%) [(a)
pyrazole), 6.03 Nickel(II) complex (5a). Nickel(II) nitrate hexahydrate
ꢁ6H O (74.5 mg, 0.26 mmol) was dissolved in 2 ml of
methanol and was added dropwise at room temperature to a
The 3,5-dimethyl-1H-pyrazole-1-carbothioamide (b) was pre- stirred solution of ligand a (65.4 mg, 0.26 mmol) in ethyl
2 4
CoClO ] .
3
3
3
)
2
2
7
.77 (s, 1H, thiazole), 7.90 (m, 2H, phenyl).
2
5
pared as described elsewhere.
Hydrazinecarbothioamide acetate (8 ml). The reaction solution was stirred for 24 hours
(1.2 g, 13.2 mmol) was dissolved in HCl (80 ml, 0.05 M) and at room temperature. A light green-blue crystalline product was
pentano-2,4-dione (1.3 ml, 13.2 mmol) was added dropwise to obtained by the diffusion of diethyl ether into the mixture.
the stirred mixture. After stirring for 2 h and 3 h incubation at Yield: (79.0 mg, 39%), mp 153.6–155.2 1C. Anal. calcd for 5a
ꢀ
1
room temperature, a white precipitate was filtered off, washed
C
14
H
13
N
3
SNiN
2
O
6
ꢁCH
3
OH (470.03 g mol ) C, 38.33%; H,
with water and dried under reduced pressure. Yield: (1.52 g, 3.65%; N, 14.90%. Found: C, 38.31%; H, 3.41%; N, 14.66%.
5.0%), mp 92.3–93.7 1C. FT-IR (KBr, cm ): 3241s, 3134s, FTIR (KBr cm ): 3353br, 3104m, 1580m, 1536w, 1511s, 1383s,
976w, 1600s, 1576s, 1341m, 880s, 808m. H NMR (700 MHz, 1022m, 825m, 687m. H NMR (DMSO-d ) d (ppm): 2.15 (s, 3H,
6
ꢀ
1
ꢀ1
8
2
1
1
DMSO-d ) d (ppm): 2.17 (d, 6H, C(3)–CH , C(5)–CH pyrazole), C(3)–CH pyrazole); 2.67 (s, 3H, C(5)–CH pyrazole); 6.17 (s, 1H,
6
3
3
3
3
3
6
.19 (s, 1H, C(4)pyrazole), 9.13 (s, 1H, NH ), 9.54 (s, 1H, NH ). C(4) pyrazole); 7.30 (t, 1H, C(phenyl), JHH = 7.7 Hz), 7.40
2
2
3
(
(
t, 2H, C(phenyl) J
d, 2H, C(phenyl), JHH = 7.7 Hz). MS-FAB (m/z): 256.2 (5%) [a] ,
= 7.7 Hz), 7.80 (s, 1H, C thiazole); 7.90
HH
Synthesis of the complexes
3
+
+
+
Caution! Perchlorate salts are potentially explosive and were 314.0 [(a)Ni] , 375.1 (100%) [(a)NiNO
handled only in small quantities with care. Nickel(II) complex (6a). Nickel(II) perchlorate hexahydrate
Cobalt(II) complex (1a). Cobalt(II) chloride hexahydrate Ni(ClO O (44.5 mg, 0.12 mmol) was dissolved in 2 ml of
ꢁ6H
CoCl O (48.5 mg, 0.2 mmol) was dissolved in 1.0 ml of methanol (1.0 ml) and was added dropwise at room tempera-
3
] .
4
)
2
2
2
ꢁ6H
2
methanol and was added dropwise at room temperature to a ture to a stirred solution of ligand a (62.2 mg, 0.24 mmol) in
stirred solution of ligand a (52.0 mg, 0.2 mmol) in ethyl acetate ethyl acetate (10 ml). A green-blue crystalline product was
(5.0 ml). The reaction solution was stirred for 12 hours at room obtained by the diffusion of diethyl ether into the mixture.
temperature. The resulting green crystals of 1a were obtained by Yield: (68.6.0 mg, 70.0%), mp dec. 4299.1 1C. Anal. calcd for
ꢀ
1
the diffusion of diethyl ether into the mixture. Yield: (57.8 mg, 6a C28
H
26
N
6
S
2
NiCl
2
O
8
ꢁ2H
2
O (804.19 g mol ) C, 41.82%; H,
6
2
9%), mp 296.5–298.5 1C. Anal. calcd for 1a C14
H
13
N
3
SCoCl
2
ꢁ
3.76%; N, 10.45%. Found: C, 41.69%; H, 3.46%; N, 10.45%.
O (421.15 g mol ) C, 39.93%; H, 4.07%; N, 9.98%; found: FTIR (KBr cm ): 3390br, 3084m, 1654m, 1582s, 1538s, 1505s,
C, 40.09%; H, 4.22%; N, 10.10%. FTIR (KBr cm ): 3091w, 1031s, 700m, 1085s, 623m. H NMR (DMSO-d
ꢀ
1
ꢀ1
H
2
ꢀ
1
1
6
) d (ppm): 2.16
1
623w, 1574m, 1502s, 1020w, 686m. MS-FAB (m/z): 256.1 (10%) (s, 3H, C(3)–CH pyrazole); 2.67 (s, 3H, C(5)–CH pyrazole); 6.17
3
3
+
+
+
3
[a] , 313.1 (90%) [(a)Co] , 349.1 (100%) [(a)CoCl] .
(s, 1H, C(4) pyrazole); 7.30 (t, 1H, C(phenyl), J = 7.0 Hz), 7.41
HH
3
Cobalt(II) complex (2a). Cobalt(II) nitrate hexahydrate (t, 2H, C(phenyl), J
= 7.0 Hz); 7.81 (s, 1H, C(thiazole)); 7.89
HH
3
+
Co(NO
3
)
2
ꢁ6H
2
O (68.9 mg, 0.24 mmol) was dissolved in 1 ml of (d, 2H, C(phenyl), JHH = 7.0 Hz). MS-FAB (m/z): 256.0 (10%) [a] ,
+
+
methanol and was added dropwise at room temperature to a 313.1 (50%) [(a)Ni] , 412.1 (80%) [(a)NiClO
stirred solution of ligand a (60.4 mg, 0.24 mmol) in ethyl [(a) Ni] , 667.1 (100%) [(a) NiClO ] .
2 2 4
4
] , 568.1 (25%)
+
+
5
358 | New J. Chem., 2014, 38, 5349--5361
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NJC
Copper(II) complex (7a). Copper(II) chloride dihydrate CuCl
Paper
2
ꢁ2H
2
O
filtered off and dried. Yield: (55.5 mg, 84%), mp dec. 4130 1C.
ꢀ
1
(54.9 mg, 0.2 mmol) was dissolved in 1.0 ml of methanol and was Anal. calcd for 3b C12
H
18
N
6
S
2
CoCl
2 8
O ꢁ2H
2
O (604.19 g mol ) C,
added dropwise at room temperature to a stirred solution of 23.86%; H, 3.67%; N, 13.91%. Found: C, 23.94%; H, 3.59%; N,
ligand a (50.5 mg, 0.2 mmol) in ethyl acetate (10.0 ml). The 13.66%. FTIR (KBr, cm ): 3418br, 3253s, 3142s, 2927w, 1634s,
stirring was continued for 24 hours at room temperature and the 1596s, 1343s, 1114s, 860s, 777s, 624m. MS-FAB (m/z): 213.0 (18%)
ꢀ
1
+
+
+
4 2
reaction mixture was left standing overnight. A yellow micro- [(b)Co] , 313.0 (55%) [(b)CoClO ] , 368.0 (100%) [(b) Co] , 468.0
ꢀ
+
crystalline product was obtained, filtered off and dried in vacuum. (18%) [(b)
Yield: (73.0 mg, 71%), mp 163.9–166.0 1C. Anal. calcd for 7a Nickel(II) complex (4b). Nickel(II) chloride anh. NiCl
O (461.79 g mol ) C, 36.41%; H, 4.58%; N, 0.38 mmol) was dissolved in 0.5 ml of methanol and was added
2 4
CoClO ] .
2
(49.58 mg,
ꢀ
1
C
14
H
13
N
3
SCoCl
ꢁ4H
2 2
ꢀ
1
9
3
2
.10%; found: C, 36.80%; H, 4.98%; N, 9.69%. FTIR (KBr cm ): dropwise at room temperature to a stirred solution of ligand b
366br, 3091m, 1623m, 1574s, 1502s, 1020m, 686w. MS-FAB (m/z): (59.1 mg, 0.38 mmol) in ethyl acetate (5.0 ml). The reaction
+
+
+
56.1 (35%) [a] , 318.0 (100%) [(a)Cu] , 353.0 (75%) [(a)CuCl] .
Copper(II) complex (8a). Copper(II) nitrate trihydrate resulting orange-brown crystals of 4b were obtained by the diffu-
Cu(NO O (22.3 mg, 0.09 mmol) was dissolved in 0.5 ml of sion of diethyl ether into the mixture. Yield: (74.5 mg, 69%), mp
ꢁ3H
methanol and was added dropwise at room temperature to a stirred 145.8–146.75 1C. Anal. calcd for 4b C
solution of ligand a (23.6 mg, 0.09 mmol) in ethyl acetate (5 ml). The C, 25.31%; H, 3.19%; N, 14.75%; found: C, 25.29%; H, 2.93%; N,
solution was stirred for 12 hours at room temperature. The
3
)
2
2
ꢀ
1
6 9 3 2
H N SNiCl (284.88 g mol )
ꢀ
1
reaction solution was stirred for 12 hours at room temperature. The 14.25%. FTIR (KBr, cm ): 3364s, 3254s, 3074m, 2995w, 1626s,
resulting dark green solid 8a was obtained by the diffusion of diethyl 1616s, 1588s, 1339s, 863s, 779m. H NMR (DMSO-d
1
6
) d (ppm):
ether into the mixture. Yield: (35.0 mg, 72%), mp 155.2–156.6 1C. 1.90 (s, 3H, C(3)–CH pyrazole), 2.40, (s, 3H, C(5)–CH pyrazole);
3
3
ꢀ
1
Anal. calcd for 8a C H N O S Cu (1583.80 g mol ) C 42.47%; 5.92 (s, 1H, C(4) pyrazole); 8.86 (s, 1H, NH ); 9.27 (s, 1H, NH ).
H, 3.31%; N, 15.92%. Found: C, 42.35%; H, 3.41%; N, 15.46%. FTIR MS-FAB (m/z): 213.0 (40%) [(b)Ni] , 249.0 (100%) [(b)NiCl] .
5
6
52 18 18 4
3
2
2
+
+
ꢀ
1
(
KBr cm ): 3107w, 1635w, 1573s, 1506s, 1381s, 1009m, 829m, 699w.
MS-FAB(+) (m/z): 318.2 (100%) [(a)Cu] , 573.3 (30%) [(a)
Nickel(II) complex (5b). Nickel(II) nitrate hexahydrate
2
O (104.7 mg, 0.36 mmol) was dissolved in 2 ml of
methanol and was added dropwise at room temperature to a
+
+
2
Cu] , 637.3 Ni(NO
3
)
2
ꢁ6H
+
2ꢀ
(
3
5%) [(a)
2
CuNO
3
] . MS-FAB(ꢀ) (m/z): 186.8 (100%) [Cu(NO
3
)
4
]
,
ꢀ
11.9 (10%) [Cu(NO
3
)
2
] .
stirred solution of ligand b (54.5 mg, 0.36 mmol) in ethyl acetate
Copper(II) complex (9a). Copper(II) perchlorate hexahydrate (6 ml). The reaction solution was stirred for 12 hours at room
Cu(ClO ) ꢁ6H O (40.2 mg, 0.11 mmol) was dissolved in 1.0 ml of temperature. A dark green crystalline product was obtained by the
4
2
2
methanol and was added dropwise at room temperature to a diffusion of diethyl ether into the mixture. Yield: (69.8 mg, 26%),
stirred solution of ligand a (56.1 mg, 0.22 mmol) in ethyl acetate mp dec. 4171.6 1C. Anal. calcd for 5b C H N S NiN O ꢁ0.5H O
1
2
18
6
2
2
6
2
ꢀ
1
(
10 ml). The reaction solution was stirred for 24 hours at room (502.29 g mol ) C, 28.69%; H, 3.81%; N, 22.31%. Found: C,
temperature. The resulting maroon solid 9a was obtained by the 28.66%; H, 3.24%; N, 21.84%. FTIR (KBr, cm ): 3286br, 3249s,
ꢀ
1
diffusion of diethyl ether into the mixture. Yield: (64.0 mg, 84%), 3114s, 2976w, 1641s, 1586s, 1383s, 1348s, 863s, 834m, 783m.
1
mp 233.2–235.3 1C. Anal. calcd for 9a C28
(
H
26
N
6
S
2
CuCl
2
O
8
ꢁ0.5H
2
O
H NMR (DMSO-d
6
) d (ppm): 2.14 (s, 3H, C(3)–CH
pyrazole); 6.16 (s, 1H, C(4) pyrazole);
3.01%; H, 3.89%; N, 10.32%. FTIR (KBr cm ): 3395br, 3105m, 9.11 (s, 1H, NH ); 9.52 (s, 1H, NH ). MS-FAB (m/z): 213.0 (20%)
3
pyrazole);
ꢀ
1
782.02 g mol ) C, 43.01%; H, 3.48%; N, 10.75%. Found: C, 2.63 (s, 3H, C(5)–CH
3
ꢀ
1
4
1
2
5
2
2
+
+
+
574m, 1533w, 1507s, 1108m, 1015s, 701w, 623m. MS-FAB (m/z): [(b)Ni] , 275.0 (100%) [(b)NiNO ] , 368.0 (64%) [(b) Ni] , 430.2
56.1 (35%) [a] , 318.1 (100%) [(a)Cu] , 418.1 (5%) [(a)CuClO ] , (48%) [(b)NiNO ] .
3
2
+
+
+
+
4
3
+
74.3 (30%) [(a)
Cobalt(II) complex (1b). Cobalt(II) chloride hexahydrate Ni(ClO
CoCl O (95.5 mg, 0.4 mmol) was dissolved in 0.5 ml of of methanol (1.0 ml) and was added dropwise at room tempera-
methanol and was added dropwise at room temperature to a ture to a stirred solution of ligand b (42.2 mg, 0.27 mmol) in ethyl
stirred solution of ligand b (62.3 mg, 0.4 mmol) in ethyl acetate acetate (4.0 ml). A green crystalline product was obtained by the
2
Cu] .
Nickel(II) complex (6b). Nickel(II) perchlorate hexahydrate
4 2
)
ꢁ6H O (49.5 mg, 0.135 mmol) was dissolved in 1.0 ml
2
2
ꢁ6H
2
(
7.0 ml). The stirring was continued for half an hour at room diffusion of diethyl ether into the mixture. Yield: (35.0 mg, 53.4%),
temperature and the reaction mixture was left standing overnight. mp dec. 4168.1 1C. Anal. calcd for 6b C H N S NiCl O
2 8
18
27
9
3
ꢀ
1
A blue micro-crystalline product was obtained, filtered off and (723.44 g mol ) C, 29.87%; H, 3.33%; N, 17.25%. Found: C,
dried in vacuum. Yield: (52.5 mg, 55%), mp 180.0–182.1 1C. Anal. 29.80%; H, 3.76%; N, 17.40%. FTIR (KBr, cm ): 3202s, 3119s,
ꢀ
1
ꢀ
1
1
calcd for 1b C
4.75%; found: C, 25.48%; H, 2.84%; N, 14.38%. FTIR (KBr, cm ): (DMSO-d
264s, 3155m, 2991w, 1627s, 1591s, 1341s, 842s, 779m. MS-FAB C(5)–CH
6 9 3 2
H N SCoCl (285.0 g mol ) C, 25.29%; H, 3.19%; N, 2984w, 1627s, 1589s, 1355s, 1103s, 875s, 776m, 623m. H NMR
ꢀ1
1
3
6
) d (ppm): 2.14 (s, 3H, C(3)–CH
3
pyrazole); 2.62 (s, 3H,
);
2
9.51 (s, 1H, NH ). MS-FAB (m/z): 213.0 (3%) [(b)Ni] , 367.0 (100%)
3
pyrazole); 6.16 (s, 1H, C(4) pyrazole); 9.10 (s, 1H, NH
2
+
+
+
(m/z): 249.0 (45%) [Co(b)Cl] , 404.1 (23%) [Co(b)
2
Cl] .
+
+
Cobalt(II) complex (3b). Cobalt(II) perchlorate hexahydrate [(b) Ni] , 467.0 (20%) [(b) NiClO ] .
2
2
4
Co(ClO ) ꢁ6H O (84.0 mg, 0.23 mmol) was added at room
Copper(II) complex (7b). This complex was synthesized
4
2
2
26
temperature to a stirred solution of ligand b (71.2 mg, according to the literature with some modifications. Copper(II)
.46 mmol) in ethyl acetate (7 ml). The reaction solution was chloride dihydrate CuCl O (85.2 mg, 0.5 mmol) was dissolved
0
ꢁ2H
2 2
stirred for 12 hours at room temperature and was left standing in a mixture of ethyl acetate (4.0 ml) and methanol (0.5 ml) and
overnight. The resulting pink micro-crystalline product was was added dropwise at room temperature to a stirred solution of
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Paper
NJC
ligand b (77.6 mg, 0.5 mmol) in ethyl acetate (5.0 ml). The and incubation was continued for 2 h. MTT – formazan crystals
reaction solution was stirred for 24 hours at room temperature. The were dissolved in 20% SDS and 50% DMF at pH 4.7 and absor-
resulting gray-green solid product of 7b was obtained by the diffu- bance was read at 562 and 630 nm on an ELISA – plate reader (ELX
sion of diethyl ether into the mixture. Yield: (128.00 mg, 88%), mp 800, Bio-Tek, USA). The values of IC50 (the concentration of the test
ꢀ
1
1
64.8–166.2 1C. Anal. calcd for 7b C H N SCuCl (289.68 g mol ) C, compound required to reduce the cells survival fraction to 50% of
6 9 3 2
2
4.88%; H, 3.13%; N, 14.51%; found: C, 24.72%; H, 3.14%; N, the control) were calculated from concentration-response curves
ꢀ1
14.48%. FTIR (KBr cm ): 3296s, 3106m, 1621s, 1588m, 1346s, and used as a measure of cellular sensitivity to a given treatment.
9
99m, 847m. Other details of the synthesis, and characterization Stock solutions of ligands, metal complexes, carboplatin and
25
of this complex were described in our previous article.
cisplatin were freshly prepared in DMSO and diluted with
Copper(II) complex (8b). Copper(II) nitrate trihydrate complete culture medium to obtain a concentration range from
ꢀ
7
ꢀ3
ꢀ7
ꢀ4
Cu(NO ) ꢁ3H O (56.9 mg, 0.24 mmol) was dissolved in 2 ml of 10 –10 M for ligands a, b and from 10 –5 ꢂ 10 M for
3
2
2
methanol and was added dropwise at room temperature to a metal complexes. DMSO concentration never exceeded 0.2%
stirred solution of ligand b (73.0 mg, 0.48 mmol) in ethyl and had no influence on cell growth. As control, cultured cells
acetate (12 ml). The stirring was continued for 2 hours at room were grown in the absence of drugs.
temperature and the reaction mixture was left standing over-
night. A brown-yellow micro-crystalline product was obtained, collected on Oxford Sapphire using a CCD area detector. All
filtered off and dried in vacuum. Yield: (70.5 mg, 60%), mp structures were solved by direct methods and refined by full-
X-ray measurements. Diffraction data of all complexes were
4
0
2
41,42
1
35.2–136.6 1C. Anal. calcd for 8b C12
H
18
N
6
S
2
CuN
2
O
6
ꢁC
4
H
8
O
2
ꢁ
matrix least-squares techniques on F with SHELXL program.
ꢀ
1
3.5H O (649.04 g mol ) C, 29.61%; H, 5.12%; N, 17.26%. The numerical absorption correction was applied for all crystals
2
ꢀ
1
5
Found: C, 29.74%; H, 5.66%; N, 17.16%. FTIR (KBr, cm ): (RED171 package of programs, Oxford Diffraction, 2000 ).
3
1
462br, 3284s, 3146m, 3109w, 2983w, 1734m, 1573s, 1383s,
Heavy atoms were refined with anisotropic thermal displace-
+
350s, 810m, 750m. MS-FAB (m/z): 218.0 (20%) [(b)Cu] , 281.1 ment parameters. Positions of hydrogen atoms attached to
+
+
(
100%) [(b)CuNO
Copper(II) complex (9b). Copper(II) perchlorate hexahydrate positions of hydrogen atoms attached to oxygen or nitrogen
Cu(ClO O (60.9 mg, 0.22 mmol) was dissolved in 1.0 ml of atoms were found from electron density synthesis. All hydrogen
ꢁ6H
3
] , 437.2 (48%) [(b)CuNO
3
] .
carbon atoms were assigned at calculated positions, whereas
4
)
2
2
methanol and was added dropwise at room temperature to a stirred atoms were refined with isotropic thermal displacement para-
solution of ligand b (68.0 mg, 0.44 mmol) in ethyl acetate (7.0 ml). meters fixed to a value of 20% or 50% higher than those of the
The stirring was continued for half hour at room temperature and corresponding C, N or O atoms. For compound 5b several
the reaction mixture was left standing overnight. A brown-green geometrical restraints (DFIX) were used for O73 and O83 diethyl
micro-crystalline product was obtained, filtered off and dried in ether molecules. In the final model of this compound there is a
vacuum. Yield: (51.7 mg, 43%), mp 159.9–160.5 1C. Anal. calcd for lack of hydrogen atoms attached to the O51 water molecule.
ꢀ
1
9b C18
H
27
N
9
S
3
CuCl
2
O
8
ꢁC
4
H
8
O
2
ꢁ3H
2
O (870.09 g mol ) C, 30.37%; Additionally, twining (twofold axis) was detected for this struc-
4
3
H, 4.75%; N, 14.49%. Found: C, 30.05%; H, 4.29%; N, 14.57%. FTIR ture using ROTAX program and the refinement process was
ꢀ
1
(KBr, cm ): 3524br, 3288s, 3149m, 3113m, 2983w, 1716m, 1620m, carried out using TWIN and BASF commands. In complex 8a the
1
574s, 1348s, 1120s, 812s, 750m, 623m. MS-FAB (m/z): 219.0 (3%) extinction coefficient was refined revealing a value of 0.0008.
2+
+
44
45
[(b)Cu] , 473.1 (20%) [(b) CuClO ] .
All figures were prepared in DIAMOND and ORTEP-3. The
results of data collections and refinement have been sum-
marized in Table 1.
2
4
Cells and cytotoxicity assay
Cell cultures. Human skin melanoma WM-115 cells as well as
human leukemia promyelocytic HL-60 and lymphoblastic NALM-6
cell lines were used. Leukemia cells were cultured in RPMI 1640
medium supplemented with 10% fetal bovine serum and anti-
CCDC 988739 (1a), 988743 (6a), 989127 (8a), and 988741 (5b).
Acknowledgements
biotic (gentamicin, at 37 1C under a 5% CO /95% air atmosphere).
2
Financial support from Collegium Medicum in Bydgoszcz,
Department of Inorganic and Analytical Chemistry, grant No 411,
the Medical University of Ł o´ d ´z , grant No. 503/3-066-02/503-01
to E. Budzisz) and grant no 503/3-015-02/503-01 (to M. Rozalski)
is gratefully acknowledged.
For melanoma WM-115 cells Dulbecco’s minimal essential
medium (DMEM) instead of RPMI 1640 was used. In some
cases normal human umbilical vein endothelial cells (HUVECs)
were also used. HUVECs and all reagents for cell culture were
purchased from Cascade Biologics (Portland, Oregon, USA).
The HUVECs were cultured according to the manufacturer’s
instructions and the cells underwent 3e8 passages. Cells were
(
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