Synthesis, X-ray structures and cytotoxic activity of platinum(II), palladium(II) and copper(II) complexes with chelating ligands

Article abstract of DOI:10.1016/j.ejmech.2010.02.050

Here we present the synthesis of the new chelating ligands 1-benzothiazol-2-yl-5-(2-hydroxyphenyl)-3-methyl-1H-pyrazole-4-carboxylic acid methyl ester (2a) and 1-(6-chloropyridazin-3-yl)-5-(2-hydroxyphenyl)-3-methyl-1H-pyrazole-4-carboxylic acid methyl es

Full text of DOI:10.1016/j.ejmech.2010.02.050

European Journal of Medicinal Chemistry 45 (2010) 2613e2621
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, X-ray structures and cytotoxic activity of platinum(II), palladium(II)
and copper(II) complexes with chelating ligands
,
a
b
b
c
Elzbieta Budzisz a , Magdalena Miernicka , Ingo-Peter Lorenz , Peter Mayer , Ewa Balcerczak ,
*
Urszula Krajewska c, Marek Rozalski c
a Department of Cosmetic Raw Materials Chemistry, Faculty of Pharmacy, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland
b Department of Chemistry and Biochemistry, Ludwig-Maximilians-University, Butenandtstr. 5-13 (D), D-81377 Munich, Germany
c Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Here we present the synthesis of the new chelating ligands 1-benzothiazol-2-yl-5-(2-hydroxyphenyl)-3-
methyl-1H-pyrazole-4-carboxylic acid methyl ester (2a) and 1-(6-chloropyridazin-3-yl)-5-(2-hydrox-
yphenyl)-3-methyl-1H-pyrazole-4-carboxylic acid methyl ester (2b), obtained in the reaction of
2-methyl-4-oxo-4H-chromene-3-carboxylic acid methyl ester (1) with hydrazine derivatives. These
ligands 2a and 2b create solid complexes with Pt(II) (4a, 4b), Pd(II) (5a, 5b) and Cu(II) (6a, 7a, 8a, 8b and
9b) metal ions or can be cyclized to 1-benzothiazol-2-yl-3-methyl-1H-chromeno[4,3-c]pyrazol-4-one
(3a) or 1-(6-chloropyridazin-3-yl)-3-methyl-1H-chromeno[4,3-c]pyrazol-4-one (3b). The crystal and
molecular structures of ligand 2a, its Cu(II) complexes 6a and 7a were determined by X-ray diffraction
method. Cytotoxic activity of the ligands 2a and 2b and their complexes 4a, 4b, 5a, 5a, 6a, 8a, 8b and 9b,
and modulation of expression of BAX and P53 genes are also shown.
Received 29 July 2009
Received in revised form
27 January 2010
Accepted 20 February 2010
Available online 1 March 2010
Keywords:
Synthesis
X-ray structure
Cytotoxic effect
Expression of BAX/P53
Pyrazole ligands
Metal(II) complexes
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
many heterocyclic compounds that can act as N-donor ligands, such
as pyrazoles, pyridazine, pyridines [9,10], piperidine, piperazine,
During the last decades, a growing interest in transition metal
complexes has been stimulated by their potential applications as
anti-neoplastic agents. We have been investigating the synthesis,
X-ray structures, cytotoxic effect and structures-activity relation-
ship of metal ions complexes with different ligands [1e3]. Special
attention has been paid to pyrazoles and related N-containing
heterocyclic derivatives, which play an important role in many
biological processes due to their coordination ability with metal
ions. Platinumesulfur interactions in the human body influence
uptake, excretion, DNA binding, the toxicity of platinum
compounds and drug resistance of cancer cells. Knowledge of these
interactions is very helpful for the synthesis of new platinum
anticancer drugs [4]. A modification of cisplatin structure can be
connected with the displacement of ammonia with more stable
N- and S- donor ligands. The best known of these N,S-donor ligands
are: thiosemicarbazones [5], thiohydrazides [6], aminoacids
(cysteine, methionine) [7] and thiazole [8]. Beside them, there are
picoline [11] and imidazoles [12]. Pyrazole-derived ligands have
attracted scientists' attention over the last decades because of their
interesting coordination chemistry, unusual structural features and
remarkable physical and chemical properties [13]. Pyrazoles are
potential imidazole mimics and therefore can serve in the devel-
opment of ligand systems that resemble active sites of metal-
loenzymes [14]. Although a large number of complexes containing
pyrazole ligands have been synthesized, the design and synthesis of
novel pyrazole-containing complexes by varying the nature of the
reactants and synthetic conditions are still under investigation [15].
The anticancer activity of many drugs involving cisplatin is
often related to the modulation of expression of some genes
engaged in programmed cell death e apoptosis. Among them is
BAX e a proapoptotic member of the Bcl-2 family of genes. The Bax
(Bcl-2-associated protein) protein forms a heterodimer with Bcl-2
and functions as an apoptotic activator. Overexpressed BAX
accelerates apoptotic death induced by cytokine deprivation in an
IL-3-dependent cell line. Overexpressed BAX also counters the
death-repressor activity of Bcl-2 [16]. The expression of the BAX
gene is regulated by the tumor suppressor P53 and has been shown
to be involved in P53-induced apoptosis. P53 is central to many of
* Corresponding author. Tel.: þ48 42 677 91 25; fax: þ48 42 678 83 98.
E-mail address: elora@ich.pharm.am.lodz.pl (E. Budzisz).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.050

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E. Budzisz et al. / European Journal of Medicinal Chemistry 45 (2010) 2613e2621
the cell's anticancer mechanisms. It can induce growth arrest,
apoptosis and is responsible for genetic stability and inhibition of
angiogenesis [17,18]. Pharmacological manipulation of BAX and
P53 expression, which enhances their proapoptotic effect, has
implications for many diseases involving apoptosis such as cancer.
Novel metal-based complexes containing metals such as palla-
dium(II), copper(II), ruthenium(III), gold(III) and rhodium(III) have
been reported with promising chemotherapeutic potential and
varied mechanisms of action in comparison with the platinum-
based drugs [19]. In our previous papers we described the synthesis
of 5-(2-hydroxyphenyl)-3-methyl-1-(2-pyridinyl)-1H-pyrazol-4-
carboxylic acid methyl ester and its complexes with platinum(II),
palladium(II) and copper(II) ions [20,21]. These complexes showed
lower cytotoxicity than cisplatin and the platinum(II) and copper
(II) complexes were found to be more efficient in the induction of
leukemia cell death than the palladium(II) complex.
conditions such as triethylamine or in high temperature to form
1-benzothiazol-2-yl-3-methyl-1H-chromeno[4,3-c]pyrazol-4-one
(3a) and 1-(6-chloropyridazin-3-yl)-3-methyl-1H-chromeno[4,3-c]
pyrazol-4-one (3b), respectively. Thermogravimetric analysis of the
copper(II) complex described in our recent paper [21] confirmed
detachment of the methanol molecule and DMF. The detachment of
the methanol molecule could suggest the cyclization of the
pyrazole ligand to the coumarin derivative. According to these data,
we successfully performed the thermal cyclization at 150 ꢀC of the
pyrazole ligands 2a and 2b to their coumarin derivatives 3a and 3b,
in order to compare the cytotoxic activity of pyrazole derivatives 2a
and 2b and coumarin derivatives 3a and 3b. Unfortunately,
compound 3a, with its very poor solubility in any solvents, could
not be used to synthesize the complexes. Despite the fact that the
ligand 3b has better solubility, it does not react with metal salts and
does not produce complexes.
As a part of our systematic investigation here we present the
synthesis of new chelating ligands 1-benzothiazol-2-yl-5-(2-
hydroxyphenyl)-3-methyl-1H-pyrazole-4-carboxylic acid methyl
ester (2a) and 1-(6-chloropyridazin-3-yl)-5-(2-hydroxyphenyl)-3-
methyl-1H-pyrazole-4-carboxylic acid methyl ester (2b). In
comparison with the ligand's structure described in our previous
paper [2] the pyridinyl group was replaced by a benzothiazoyl group
in the ligand's 2a structure to check if a sulfur atom can take part in
the coordination of the metals' ions or alter the cytotoxic activity. In
this paper we also present the cyclization of ligands 2a and 2b to very
low soluble 1-benzothiazol-2-yl-3-methyl-1H-chromeno[4,3-c]pyr-
azol-4-one (3a) and 1-(6-chloropyridazin-3-yl)-3-methyl-1H-chro-
meno[4,3-c]pyrazol-4-one (3b), synthesis of ligands 2a or 2b solid
complexes with platinum(II) (4a, 4b), palladium (II) (5a, 5b) and
copper(II) (6a, 7a, 8a, 8b and 9b), X-ray structures of ligand 2a and its
copper(II) complexes 6a and 7a, cytotoxic activity of ligands 2a and
2b and their complexes performed on leukemia and melanoma cell
lines, and the modulation of expression of BAX and P53 genes by two
chosen complexes: Pt(II) 4a and Cu(II) 6a.
The ligands 2a and 2b in the reactions with the metals' salts
(potassium tetrachloridoplatinate(II), bis(benzonitrile)palladium
(II) chloride, copper(II) chloride dihydrate, copper(II) perchlorate
hexahydrate) create solid complexes 4a, b, 5a, b, 6a, 7a, 8a, 8b and
9b (Schemes 2 and 3). The metals' ions are coordinated by the two
nitrogen atoms of the ligands 2a and 2b. The sulfur atom does not
take part in the coordination. Recrystallization of the copper(II)
complex 6a by the slow diffusion of diethyl ether to DMF gave the
pentacoordinated copper(II) complex 7a.
2.2. Structural studies
2.2.1. Spectroscopic characterization of ligands and complexes
The IR-bands at 3416e3056 cmꢁ1 in the spectra of the ligands 2a
and 2b and their complexes 4a, 4b, 5a, 5b, 6a, 7a, 8a, 8b, 9b are
assigned to the hydroxy group of the phenyl ring. We could not
observe this band in the spectra of compounds 3a and 3b, because
they do not have the hydroxy group in their structures. The bands at
1617e1476 cmꢁ1 in the spectra of the ligands 2a and 2b are assigned
to the aromatic group of the phenyl ring. In the spectrum of ligand 3a
the bands assigned to the aromatic group of the chromene are in
wider range of frequencies (1609e1566 cmꢁ1) and in the spectrum
of ligand 3b, in lower frequencies (1562e1450 cmꢁ1). The charac-
teristic bands at 3103e2915 cmꢁ1 of the methyl group of the ligands
2a, 2b, 3a, 3b and complexes 4a, 4b, 5a, 5b, 6a, 7a, 8a, 8b, 9b are
assigned to CeH vibration. The bands at 1718 cmꢁ1 and 1706 cmꢁ1 in
the spectra of ligands 2a and 2b, respectively, which were assigned
to the C]O vibrations, shift to higher energies for the complexes 4a,
4b, 5a, 5b, 6a, 7a, 8a, 8b, 9b (1727e1715 cmꢁ1) and especially for the
compounds 3a (1736 cmꢁ1) and 3b (1742 cmꢁ1). The bands at
1109 cmꢁ1 and 1094 cmꢁ1 in the spectra of the ligands 2a and 2b,
2. Results and discussion
2.1. Preparation of the ligands and their complexes
In the reaction of 2-methyl-4-oxo-4H-chromene-3-carboxylic
acid methyl ester (1) with 2-hydrazinobenzothiazole or 3-chloro-6-
hydrazino-pyridazine in ethanolic solution we have obtained the
new ligands 2a and 2b, respectively (Scheme 1). This reaction
proceeds according to the general mechanism described for the
reaction of chromones with nitrogen nucleophiles [22] such as
hydrazines [23]. Compounds 2a and 2b easily cyclize under basic
OH
O
O
O
O
CH3
Et3N
+
NH2NHR
CO2CH3
CH3
CO2CH3
CH3
N
N
N
R
N
R
2a, 2b
3a, 3b
1
N
S
2a, 3a R =
2b, 3b R =
Cl
N
N
Scheme 1. Synthesis of the ligands 2a, 2b and 3a, 3b.

E. Budzisz et al. / European Journal of Medicinal Chemistry 45 (2010) 2613e2621
2615
OH
spectra of 8a and 8b are characteristic for non-coordinated
perchlorate ions [24].
K2PtCl4
CO2CH3
CH3
2a, 2b
The 1H NMR spectra of ligands 2a, 2b, 3b and complexes 4a
and 5a show signals in the range of
d 2.50e2.60 ppm for the
Pd(PhCN)2Cl2
N
N
methyl groups of pyrazole ring, 3.59e3.70 ppm for the methoxy
groups and 6.82e8.05 ppm for phenyl groups. In the spectrum of
R
M
complex 4a we observe the signal at
d 9.64 ppm for the hydroxy
Cl
Cl
group. In the spectrum of ligand 2a the signal for the hydroxy
group was observed in the aromatic region. The 1H NMR spectra
of copper(II) complexes could not be measured due to their
paramagnetism [25].
N
4a, 5a R =
(2a)
(2b)
S
N
N
In the 13C NMR spectra of ligand 2a and 2b, signals for carbon
4b, 5b
R =
Cl
atoms of sp3 hybridization are the most characteristic. Ligand 2a
shows a signal at
methyl group of pyrazole ring and ligand 2a e at
ligand 2b e at 51.93 ppm for the methoxy group [26].
d
14.65 ppm, ligand 2b e at
d
14.68 ppm for the
Scheme 2. Synthesis of the pyrazole derivatives Pt(II) and Pd(II) complexes 4a,
4b e 5a, 5b.
d
51.85 ppm,
d
respectively, are assigned to the methoxy group. This band shifts to
higher energy for the compound 3a (1134 cmꢁ1) and to lower energy
for the compound 3b (1053 cmꢁ1). The new bands at 550e426 cmꢁ1
in the spectra of complexes 4a, 5a, 5b, 6a, 7a, 9b correspond to the
metalenitrogen vibration involving the nitrogen atoms of the pyr-
azole ring. The bands observed at 1117,1106, 623 and 621 cmꢁ1 in the
2.2.2. Conductivity measurements
The conductivity data of the studied copper(II) complexes in
ethanol at 298 K indicate that the complexes 6a, 7a and 9b are non-
electrolyte compounds, but complexes 8a and 8b possess ionic
structure. Therefore chloride is bonded to the copper(II) ion, while
the perchlorate ion is outside the metal coordination sphere.
OH
OH
CO2CH3
CO2CH3
DMF/Et2O
N
S
N
S
CH3
N
CH3
H
N
N
Cu
N
Cu
Cl
Cl
O
Cl
Cl
Cl
Cl
Cu
N
N
N
7a
CH3
N
H3C
H3C
S
2+
OH
H3CO2C
CO2CH3
CH3
HO
6a
N
CuCl2 x 2H2O
N
R
OH
Cu
-
2ClO4
CO2CH3
CH3
Cu(ClO4)2 x 6H2O
R
N
H3C
N
N
R
N
H3CO2C
2a, 2b
HO
8a, 8b
CuCl2 x 2H2O
OH
CO2CH3
N
CH3
Cl
N
N
Cl
N
Cl
Cu
N
Cl
N
N
H3C
N
H3CO2C
HO
9b
Scheme 3. Synthesis of the pyrazole derivatives Cu(II) complexes 6a, 7a, 8a, 8b and 9b.

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E. Budzisz et al. / European Journal of Medicinal Chemistry 45 (2010) 2613e2621
2.2.3. X-ray structure of ligand 2a and its copper(II) complexes 6a
and 7a
(Cl1eCueN1 128.85(10), Cl1eCueCl2a 131.24(5) and N1eCueCl2a
98.28(9)ꢀ). The smallest angles around Cu(II) are the bite angle
N1eCueN3 77.25(13)ꢀ and the angle between both chlorido
bridges (Cl2eCueCl2a 87.34(4)ꢀ). Due to this bonding situation, the
trans-apical angle Cl2eCueN3 is somewhat bent (175.61(10)ꢀ).
The geometry at Cu(II) in 7a is a distorted trigonal bipyramidal
with two chlorido ligands and N3 of the pyrazole part of 2a in
equatorial positions. The N1-atom of the benzothiazole part and O4
of the coordinated solvent molecule DMF occupy the apical posi-
tions. Both bond lengths CueCl differ somewhat (CueCl1 2.369(2),
The crystal data and details of structure refinement of ligand 2a
and its complexes 6a and 7a are given in Table 1. The basic
framework of ligand 2a (Fig. 1) is almost planar. The torsion angle
between the benzothiazole and pyrazole ring comes to only 6ꢀ.
Even the substituents CH3 and CH3OC(O) at C9 and C10 lie within
the pyrazole plane, their dihedral angles are less than 2ꢀ. Only the
phenyl substituent is turned out of the plane at 81ꢀ in an almost
perpendicular orientation. This might be due to sterical reasons of
the hydroxy group at C13 and its formation of a hydrogen bridge
between O13 of 2a and N3 of an adjoining 2a (O3/H83/N3i 3.008
ꢀ
CueCl2 2.291(2) A), whereas those of CueN differ substantially
ꢀ
(CueN1 2.011(5), CueN3 2.198(5)A). This is noteworthy because N1
ꢀ
ꢀ
(9) A, 149.00 ). All bond lengths and angles of 2a lie in the normal
range. This applies also for the second molecule 2a found within
the cell. It is noteworthy that in both cases N1 (N4) of the pyrazole
ring and N3 (N6) of the benzothiazole system lie on the same side of
2a indicating their favourable bidentate ligand function.
lies in the apical position with normally longer bonds than in the
equatorial one. This may be due to the better p-acceptor character
of N1 in benzothiazole compared to N3 of substituted pyrazole. The
distance CueO4 1.953(5) is found in the normal range of DMF
complexes of Cu(II). The trigonal plane with the sum of
angles ¼ 360ꢀ around Cu possesses two very similar small-bond
angles (Cl1eCueCl2 115.99(7), Cl1eCueN3 116.49(16)ꢀ) and
a rather enlarged angle Cl2eCueN3 (127.50(15)ꢀ). The bite angle
N1eCueN3 of the chelate ligand 2a is naturally the smallest around
Cu and found to be 78.36(19)ꢀ. The further angles between the
equatorial and axial atoms are found more or less at 90ꢀ
(N3eCueO4 88.901(19), N1eCueCl1 93.25(16)ꢀ) except the angle
N1eCueCl2 97.49(16)ꢀ. This results from the bent axis through N1,
Cu, O4 which is bent with the angle 166.77(19)ꢀ towards the ligand
2a because of the steric demand of ligand DMF.
In contrast to the monomeric complex 7a (Fig. 3), complex 6a
(Fig. 2) is a dimer exhibiting a distorted trigonal bipyramidal
configuration at both Cu(II) centers connected by two asymmetrical
chlorido bridges. The chlorido ligands Cl1 (terminal) and Cl2a
(bridging) and the N1 of benzothiazole form the trigonal plane and
the N3 of pyrazole together with the second chlorido bridge Cl2 lie
in axial positions. Both m2-Cl bridges are asymmetrical with two
different CueCl distances (CueCl2 2.2714(13) and CueCl2a 2.5010
ꢀ
(13)A); the distance to the terminal Cl1 atom is the shortest (2.2341
ꢀ
(13) A). The CueN distances are also quite different, CueN1 2.170(3)
ꢀ
and CueN3 1.989(3) A, where the last one in axial position is again
All the other bonds and angles within the ligand in 7a are the
same as found in free 2a. The dihedral angle, however, between the
planes of the pyrazole and the phenyl ring has changed to 72.18ꢀ.
The triazametallacycle shows a slight envelope conformation with
an angle of 5ꢀ along the N1/N3 axis.
shorter as in the Cu-complex 7a because of the better
p-acceptor
character of the pyrazole part. As in 7a, the trigonal plane is rather
undistorted with a sum of angles of 358ꢀ, but different angles
2.3. Biological assays
Table 1
Crystal data and details of structure refinement of ligand 2a and complexes 6a and
7aef.
2.3.1. Cytotoxicity studies
The cytotoxicity of ligands 2a and 2b and their metal complexes
4a,b, 5a,b, 6a, 8a,b, 9b was assayed against melanoma WM-115
cells as well as leukemia promyelocytic HL-60 and lymphoblastic
NALM-6 cells. Cisplatin and carboplatin were used as the reference
compounds. Cells were exposed to a broad range of drug concen-
trations (10ꢁ7 to 10ꢁ3 M) for 48 h and cell viability was analyzed by
MTT assay. IC50 values are presented in Table 2. Metal complexes 4a,
b, 5a,b, 6a, 8a,b, 9b exhibited rather moderate cytotoxicity to both
acute leukemia HL-60 and NALM-6 cell lines although complex 8a
was more active. Relatively high cytotoxic activity was observed for
complexes 6a and 8a in the case of skin melanoma WM-115 cells.
The cytotoxic effectiveness of these compounds with an IC50 of
Compound
2a
6a
7a
Formula
C19H15N3O3S
365.407
C44H42Cl4Cu2N6O8S2 C22H22Cl2CuN4O4S
1115.875
Mr/g molꢁ1
572.952
Crystal size/mm
Crystal system
Space group
0.16 ꢂ 0.12 ꢂ 0.03 0.20 ꢂ 0.09 ꢂ 0.02
0.19 ꢂ 0.16 ꢂ 0.04
Monoclinic
P21
Monoclinic
P21/n
8.42230(10)
30.0030(6)
9.7527(2)
90
106.0611(11)
90
2368.26(7)
2
1.56485(5)
1.271
14380
0.0300
0.0320
Triclinic
P1bar
ꢀ
a/A
11.7434(5)
6.9352(3)
21.0770(10)
90
103.001(2)
90
9.6420(3)
11.1059(3)
11.8270(4)
94.9177(18)
106.0409(19)
94.3708(17)
1206.06(6)
2
1.57774(8)
1.251
7241
0.0270
0.0394
ꢀ
b/A
ꢀ
c/A
ꢀ
ꢀ
a
b
g
/
/
ꢀ
/
3
ꢀ
V/A
1672.57(13)
4
Z
calc. density/g cmꢁ3 1.45113(11)
17.4
cisplatin (18.2
(422.2 M). It should be noted that melanomas often characterize
mM (6a) and 8.2
mM (8a) was comparable or higher than that of
m
/mmꢁ1
0.219
10874
0.0533
0.0917
mM) and was much higher than that of carboplatin
Refls. measured
Rint
m
themselves by low susceptibility to many chemotherapeutics.
Mean
s(I)/I
q
range
3.44e25.36
4840
0.1117, 3.2270
3.14e25.34
3646
0.0219, 7.4375
3.23e24.00
3316
0.0238, 10.5702
Observed refls.
x, y (weighting
scheme)
2.3.2. The effect of complexes 4a and 6a on BAX and P53 gene
expression
In this study, using real-time PCR we evaluated the effect of
complexes 4a and 6a on BAX and P53 gene expression in WM-115
melanoma cells after 5 and 48 h exposure. The Cu(II) complex 6a
was chosen for this study because of the best cytotoxic activity on
WM-115 cell lines, and Pt(II) complex 4a was chosen as a structural
analog of complex 6a. It was observed that the platinum(II)
complex 4a caused distinct overexpression of BAX after a relatively
short exposure time (5 h) (Fig. 4A). Overexpression of the P53 gene
was less pronounced. The expression of BAX and P53 genes was
relatively decreased after 48 h. Various patterns of BAX and P53
Refls in refinement 6056
4326
302
0
0.0509
0.1159
1.124
0.001
1.155
3746
311
0
0.0598
0.1566
1.137
0.001
1.357
Parameters
Restraints
473
1
R (Fobs
)
0.0892
0.2402
1.101
0.001
0.875
Rw (F2)
S
Shift/errormax
Max electron
density/e A
ꢀꢁ3
Min electron
density/e A
ꢁ0.412
ꢁ0.664
ꢁ0.467
ꢀꢁ3

E. Budzisz et al. / European Journal of Medicinal Chemistry 45 (2010) 2613e2621
2617
Fig. 1. Molecular structure of ligand 2a.
gene expression were observed in melanoma cells exposed to the
copper(II) complex 6a. Complex 6a did not induce the expression of
both genes although a weak growing tendency for P53 after 48 h
was seen (Fig. 4B). Obtained results may suggest that proapoptotic,
antiproliferative and cytotoxic pathways triggered by complexes 4a
and 6a are different.
a molar ratio of 2:1: the non-electrolyte complex 9b in and ionic
complex 8b. The structures of the ligands and their metal
complexes were confirmed by spectral and elemental analyses.
Additionally, the molecular structures of the ligand 2a and its Cu(II)
complexes 6a and 7a were confirmed by X-ray analysis. Cytotox-
icity studies revealed the high effectiveness of Cu(II) complexes 6a
and 8a for skin melanoma WM-115 cells at the level comparable to
that of cisplatin for complex 6a and two times higher for complex
8a. Therefore, compound 6a and its structural analog e Pt(II)
complex 4a e were chosen for BAX and P53 gene expression study.
3. Conclusion
The highly substituted pyrazoles 2a and 2b were synthesized
and used as ligands for the synthesis of novel platinum(II), palla-
dium(II) and copper(II) complexes (4a, 4b, 5a, 5a, 6a, 8a, 8b and 9b).
The cyclized derivatives 3a and 3b of the ligands 2a and 2b did not
create complexes. Ligand 2a formed two copper(II) complexes:
non-electrolyte copper(II) complex 6a in molar ratio 1:1, which
after recrystallization by slow diffusion of diethyl ether to DMF,
gave the pentacoordinated complex 7a, and the ionic complex 8a in
a molar ratio of 2:1. Ligand 2b formed two copper(II) complexes in
4. Experimental
4.1. Chemistry
All substances were used without further purification.
Potassium tetrachloridoplatinate(II), bis(benzonitrile)palladium
(II) chloride, copper(II) chloride dihydrate and copper(II)
Fig. 2. Molecular structure of complex 6a.

2618
E. Budzisz et al. / European Journal of Medicinal Chemistry 45 (2010) 2613e2621
4.1.1. Synthesis of the ligands
4.1.1.1. 1-Benzothiazol-2-yl-5-(2-hydroxyphenyl)-3-methyl-1H-pyr-
azole-4-carboxylic acid methyl ester (2a). 2-Hydrazinobenzothia-
zole (826 mg, 5 mmol) and p-toluensulfonic acid as catalyst were
added at room temperature to a solution of 2-methyl-4-oxo-4H-
chromene-3-carboxylic acid methyl ester 1 (1.09 g, 5 mmol) in
EtOH (20 mL). The mixture was refluxed for 4 h. The solid white
product was filtered off, washed with ethanol (2 mL) and dried.
Yield: 1.61 g (50%), mp: 235e237 ꢀC. IR(KBr)
3063(CeCH3), 1718(C]O), 1617, 1570, 1522(C]C, phenyl), 1109
(OeCH3). 1H NMR(CDCl3)
(ppm): 2.59(s, 3H, eCH3), 3.69(s, 3H,
eOCH3), 6.98e7.79(m, 8H, ArH). 13C NMR(CDCl3)
(ppm): 14.65
n
(cmꢁ1): 3333(CeOH),
d
d
(eCH3), 51.85(OeCH3), 115.83, 118.44; 119.34; 121.15; 121.46;
123.20; 126.77; 131.99; 132.44; 133.55; 144.57; 149.66; 154.09;
154.98; 159.48; 163.93 (C]O). MS-EI (m/z): 365 (65%); 333 (100%).
Anal. found: C, 62.27; H, 3.80; N, 11.38. Calc for C19H15N3O3S
(365.397): C, 62.45; H, 4.14; N, 11.50.
4.1.1.2. 1-(6-Chloropyridazin-3-yl)-5-(2-hydroxyphenyl)-3-methyl-
1H-pyrazole-4-carboxylic acid methyl ester (2b). 3-Chloro-6-
hydrazinopyridazine (360 mg, 2.5 mmol) and p-toluensulfonic acid
as catalyst were added at room temperature to a solution of 2-
Fig. 3. Molecular structure of complex 7a.
methyl-4-oxo-4H-chromene-3-carboxylic acid methyl ester
1
(550 mg, 2.5 mmol) in methanol (20 mL). The mixture was refluxed
for 2 h. The solid white product was filtered off, washed with
methanol (0.5 mL) and dried. Yield: 554 mg (64%), mp: 147e148 ꢀC.
perchlorate hexahydrate were purchased from Aldrich. Chloro-
form-d and DMSO-d6 solvents for NMR spectroscopy were
obtained from Dr. Glaser AG, Basel. Solvents for synthesis
(chloroform, diethyl ether, methanol) were reagent grade or
better and were dried according to standard protocols [27]. The
melting points were determined using a Buchi B-540 apparatus
and are uncorrected. The IR spectra were recorded on a Pye-
Unicam 200G Spectrophotometer in KBr. The 1H NMR spectra
were registered at 300 MHz on a Varian Mercury spectrometer.
The MS data were obtained on a LKB 2091 mass spectrometer
(70 eV ionisation energy). The MS-FAB data were determined on
Finnigan Matt 95 mass spectrometer (NBA, Csþ gun operating at
13 keV). For the new compounds, satisfactory elemental analyses
were obtained using a Perkin Elmer PE 2400 CHNS analyser.
2-Methyl-4-oxo-4H-chromene-3-carboxylic acid methyl ester (1)
was prepared as described elsewhere [28]. Conductivity experi-
ments with the complexes studied (in 1 mmol dmꢁ3 ethanolic
solution) were carried out in an Elmetron CC-41 (specific
IR(KBr)
1577, 1476 (C]C, phenyl), 1094 (CeOeC). 1H NMR(CDCl3),
2.59(s, 3H, eCH3), 3.70 (s, 3H, eOCH3), 6.89e7.36 (m, 4H, ArH), 7.58
n
(cmꢁ1): 3416 (CeOH), 1706 (C]O), 1690 (C]N), 1613,
d
(ppm):
3
3
(d, 1H, ^CH, JHH ¼ 9.125 Hz), 7.80 (d, 1H, ^CH, JHH ¼ 9.125 Hz).
13C NMR (CDCl3),
d
(ppm): 14.68(eCH3), 51.93(OeCH3), 115.48,
118.61; 118.96; 121.01; 125.31; 130.67; 131.71; 144.64; 153.88;
154.54; 154.59; 155.76; 164.31 (C]O). MS-EI (m/z): 344 (3.54%);
327 (100%); 311 (35.39%). Anal. found: C, 55.17; H, 3.28; N, 16.19.
Calc for C16H13N4O3Cl (344.745): C, 55.74; H, 3.80; N, 16.25.
4.1.1.3. 1-Benzothiazol-2-yl-3-methyl-1H-chromeno[4,3-c]pyrazol-4-
one (3a). Triethylamine (100 mg, 0.14 mL, 1 mmol) was added at
room temperature to a suspension of 1-benzothiazol-2-yl-5-(2-
hydroxyphenyl)-3-methyl-1H-pyrazole-4-carboxylic acid methyl
ester (2a) (365 mg, 1 mmol) in EtOH (10 ml). The mixture was
refluxed for 1.5 h and cooled at 4 ꢀC for 24 h. The solid white
product was filtered off and dried. Yield: 250.0 mg (75%), mp:
conductivity ¼ 21
mS/cm at 298 K).
277e279 ꢀC. IR(KBr)
n
(cmꢁ1): 3069(CeCH3), 1736(C]O), 1609,
1580, 1556(C]C, chromene), 1134(CeOeC). MS-DEI (m/z): 333
(100%). Anal. found: C, 64.89; H, 3.37; N,12.59. Calc for C18H11N3O3S
(333.355): C, 64.85; H, 3.33; N, 12.61.
Table 2
Cytotoxic activity of ligands 2 and 2a and their metal complexes 4a, b, 5a, b, 6a, 8a, b,
9b, cisplatin and carboplatin to HL-60, NALM-6 and WM-115 cells from at least 3
experiments.
4.1.1.4. 1-(6-Chloropyridazin-3-yl)-3-methyl-1H-chromeno[4,3-c]
pyrazol-4-one (3b). Triethylamine (100 mg, 0.14 mL, 1 mmol)
was added at room temperature to a suspension of 1-(6-chlor-
opyridazin-3-yl)-5-(2-hydroxyphenyl)-3-methyl-1H-pyrazole-4-
carboxylic acid methyl ester (2b) (344.7 mg, 1 mmol) in EtOH
(10 ml). The mixture was refluxed for 1 h. The solid white product
was filtered off and dried. Yield: 200.0 mg (64%), mp: 246e247 ꢀC.
IR (KBr)
1511, 1450 (C]C, phenyl), 1053 (CeOeC). 1H NMR (DMSO),
d (ppm): 2.60 (s, 3H, CeCH3); 7.26e7.75 (m, 4H, ArH); 8.34 (d, 2H,
Compound
Cytotoxicity (
HL-60
m
M)a
NALM-6
WM-115
2a
2b
4a
4b
5a
5b
6a
8a
> 1000
74.0 ꢃ 12.9
183.4 ꢃ 33.2
49.7 ꢃ 5.4
59.8 ꢃ 4.8
77.2 ꢃ 6.2
117.3 ꢃ 7.3
32.6 ꢃ 2.9
12.04 ꢃ 2.35
55.2 ꢃ 4.5
57.4 ꢃ 7.3
0.7 ꢃ 0.3
55.1 ꢃ 8.2
439.8 ꢃ 50.9
66.8 ꢃ 2.3
63.8 ꢃ 4.8
72.7 ꢃ 3.9
148.0 ꢃ 37.1
17.4 ꢃ 3.0
8.2 ꢃ 0.2
228.6 ꢃ 46.9
332.2 ꢃ 30.0
52.5 ꢃ 2.8
230.3 ꢃ 35.7
71.9 ꢃ 6.6
51.9 ꢃ 3.4
8.31 ꢃ 0.67
47.6 ꢃ 5.5
52.2 ꢃ 2.8
0.8 ꢃ 0.1
n
(cmꢁ1): 3069 (CeCH3), 1742 (C]O), 1612 (C]N), 1562,
8b
9b
50.0 ꢃ 7.6
54.8 ꢃ 4.6
18.2 ꢃ 4.3
422.2 ꢃ 50.2
eCH]CHe). MS-EI (m/z): 311 (100%). Anal. found: C, 57.20; H,
2.39; N, 17.92. Calc for C15H9N4O2Cl (312.716): C, 57.61; H, 2.90; N,
17.92.
Cisplatin
Carboplatin
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
4.1.2. Synthesis of the complexes
4.1.2.1. Pt(II) complex 4a. The aqueous solution of K2[PtCl4]
(82.9 mg, 0.1997 mmol, in 2 mL) was slowly added dropwise to the
of IC50 (in
presented.
m
M) ꢃS.D. from 2 to 4 experiments, each performed five times, are

E. Budzisz et al. / European Journal of Medicinal Chemistry 45 (2010) 2613e2621
2619
6a
5h
48h
4a
B
A
80
70
60
50
40
30
20
10
0
1,2
1,0
bax
p53
bax
48h
0,8
0,6
0,4
0,2
0,0
5h
bax
bax
bax
p53
bax
p53
p53
bax
p53
p53
p53
bax p53
control
1X IC 50
5X IC 50
1X IC 50
control
1X IC 50
5X IC 50
1X IC 50
Fig. 4. Effect of 4a complex (A) and 6a complex (B) on BAX (white box) and P53 (black box) gene expression at the level of mRNA in WM-115 melanoma cells as measured by real-
time PCR. Melanoma cells were exposed to 4a and 6a at concentration equal to IC50 and 5 ꢂ IC50 for 5 and 48 h.
methanolic solution of the ligand 2a (73 mg, 0.1997 mmol, in
10 mL). The mixture was stirred at room temperature for 1 h. The
yellow solid that precipitated after 24 h was filtered off, washed
with water and diethyl ether and dried to yield complex 4a. Yield:
450 (0.05%), [Pd(L)]2þ; 345 (50%), L. Anal. found: C, 35.84; H, 2.24;
N, 10.25. Calc. for C16H13N4O3 Cl3Pd þ H2O (522.071): C, 35.58; H,
2.43; N, 10.37.
68.11 mg (54%), dec.: 380 ꢀC. IR (KBr)
n
(cmꢁ1): 3397 (CeOH), 2951
4.1.2.5. Cu(II) complex 6a. A methanolic solution of CuCl2 ꢂ 2H2O
(34.0 mg, 0.1997 mmol, in 1 mL) was slowly added dropwise to
a solution of ligand 2a in ethyl acetate (73.0 mg, 0.1997 mmol, in
10 mL). The mixture was stirred at room temperature for 24 h. The
resulting yellow solid was filtered off and dried. Yield: 84.0 mg
(CeCH3), 1720 (C]O), 1617, 1571, 1521 (C]C, phenyl), 1108
(OeCH3), 462 (PteN). 1HNMR (DMSO-d6),
d
(ppm): 2.50 (s, 3H,
eCH3), 3.59 (s, 3H, eOCH3), 6.82e8.05 (m, 8H, ArH), 9.64 (s,1H,OH).
MS-FAB (m/z): 631 (0.13%), Pt(L)Cl2; 595 (0.7%), [Pt(L)Cl]þ; 559
(0.1%), [Pt(L)]2þ; 366 (40%), L. Anal. found: C, 36.54; H, 2.70; N, 6.60.
Calc. for C19H15N3O3SPtCl2 (631.37): C, 36.14; H, 2.39; N, 6.65.
(84%), dec.: 230 ꢀC. IR (KBr)
1719 (C]O), 1612, 1591, 1561 (C]C, phenyl), 1110 (OeCH3), 536
(CueN). MS-FAB (m/z): 463 (3.4%), [Cu(L)Cl]þ; 428 (5.0%), [Cu(L)]2þ
n
(cmꢁ1): 3271 (CeOH), 2976 (CeCH3),
.
4.1.2.2. Pt(II) complex 4b. The aqueous solution of K2[PtCl4]
(62.3 mg, 0.15 mmol, in 2 mL) was slowly added dropwise to the
methanolic solution of the ligand 2b (51.7 mg, 0.15 mmol, in 10 mL).
The mixture was stirred at room temperature for 24 h. The orange
solid that precipitated after 24 h was filtered off, washed with
water and diethyl ether and dried to yield complex 4b. Yield: 68 mg
Anal. found: C, 45.24; H, 3.13; N, 8.22;. Calc. for C19H15N3O3SCuCl2
(499.84): C, 45.65; H, 3.02; N, 8.41.
4.1.2.6. Cu(II) complex 7a. Recrystallization done by the diffusion
of diethyl ether into a DMF solution of 6a gave complex 7. dec.:
260 ꢀC. IR (KBr) (cmꢁ1): 3204 (CeOH), 2953 (CeCH3), 1719 (C]O),
n
(54%). IR (KBr)
1613, 1573, 1439 (C]N), 1101 (CeOeC). 1HNMR (DMSO-d6),
(ppm): 2.95 (s, 3H, eCH3), 3.57 (s, 3H, eOCH3), 6.70e8.12(m, 8H,
n
(cmꢁ1): 3402(CeOH), 2952 (CeCH3), 1722 (C]O),
1643 (C]O, DMF), 1576, 1519 (C]C, phenyl), 1189 (CeOeC),
1096 (OeCH3), 537 (CueN). MS-FAB (m/z): 590 (3%),
CuLCl2 þ DMF þ H2O; 428 (100%), [Cu(L)]2þ. Anal. found: C, 44.63;
H, 3.56; N, 9.47;. Calc. for C22H122N4O4SCuCl2 þ H2O(590.962): C,
44.70; H, 4.09; N, 9.48.
d
3
ArH), 9.54 (s, 1H, OH), 8.37 (d, 1H, ^CH, JHH ¼ 9 Hz), 8.47 (d, 1H,
3
^CH, JHH ¼ 9 Hz). MS-FAB (m/z): 609 (85%), Pt(L)Cl2; 573 (100%),
[Pt(L)Cl]þ. Anal. found: C, 31.95; H, 1.97; N, 9.50. Calc. for
C16H13N4O3Cl3Pt (610.731): C, 31.46; H, 2.15; N, 9.17.
4.1.2.7. Cu(II) complex 8a. A methanolic solution of Cu
(ClO4)2 ꢂ 6H2O (37 mg, 0.1 mmol, in 2 mL) was slowly added
dropwise to a solution of ligand 2a in ethyl acetate (73 mg,
0.2 mmol, in 10 mL). The mixture was stirred at room temperature
for 24 h. The resulting green solid was obtained by the diffusion of
diethyl ether into the mixture, then filtered off, washed with
diethyl ether and dried. Yield: 57 mg (57%), mp: 235e239 ꢀC. IR
4.1.2.3. Pd(II) complex 5a. A solution of (C6H5CN)2PdCl2 (76.6 mg,
0.1997 mmol, in 3 mL chloroform þ 3 mL methanol) was slowly
added dropwise to a chloroform solution of ligand 2a (73.0 mg,
0.1997 mmol, in 5 mL). The mixture was stirred at room tempera-
ture. After 24 h the resulting yellow crystals were filtered off,
washed with water and diethyl ether and dried. Yield: 57.5 mg
(KBr) n
(cmꢁ1): 3371 (CeOH), 2956 (CeCH3), 1715 (C]O), 1615 (C]
(53%), dec.: 370 ꢀC. IR (KBr)
1723 (C]O), 1618, 1516, 1491 (C]C, phenyl), 1103 (CeOeC), 426
(PdeN). 1HNMR (DMSO-d6),
(ppm): 2.50 (s, 3H, eCH3), 3.59 (s, 3H,
n
(cmꢁ1): 3406 (CeOH), 2957 (CeCH3),
N), 1593, 1568, 1518 (C]C), 1117 (ClOꢁ4 ), 1052 (CeOeC), 621 (ClO4ꢁ).
MS-FAB (m/z): 794 (45%), [Cu(L)2]2þ; 429 (100%), [Cu(L)]2þ. Anal.
found: C, 44.04; H, 3.23; N, 7.73; Calc. for C38H30N6O14 S2Cl2Cu
(993.238): C, 45.39; H, 3.04; N, 8.46.
d
eOCH3), 6.82e8.02(m, 8H, ArH), 9.64 (s, 1H, OH). MS-FAB (m/z):
542 (0.05%), Pd(L)Cl2; 507 (0.18%), [Pd(L)Cl]þ; 470 (0.26%), [Pd
(L)]2þ
.
Anal. found: C, 40.27; H, 2.37; N, 7.20. Calc. for
4.1.2.8. Cu(II) complex 8b. A methanolic solution of Cu
(ClO4)2 ꢂ 6H2O (27.8 mg, 0.075 mmol, in 1 mL) was slowly added
dropwise to a solution of ligand 2b in ethyl acetate (51.7 mg,
0.15 mmol, in 10 mL). The mixture was stirred at room temperature
for 48 h. The resulting green solid was filtered off and dried. Yield:
C19H15N3O3SPdCl2 þ H2O (560.74): C, 40.69; H, 3.05; N, 7.49.
4.1.2.4. Pd(II) complex 5b. A solution of (C6H5CN)2PdCl2 (57.5 mg,
0.15 mmol, in 3 mL chloroform þ 3 mL methanol) was slowly added
dropwise to a solution of ligand 2b (51.7 mg, 0.15 mmol, in 10 mL
chloroform þ 10 mL methanol). The mixture was stirred at room
temperature. After 24 h the resulting brown solid was filtered off,
washed with water and diethyl ether and dried. Yield: 44.9 mg
26.3 mg (48%), dec.: 221 ꢀC. IR (KBr)
n
(cmꢁ1): 3407 (CeOH), 2957
(CeCH3), 1726 (C]O), 1696 (C]N), 1613, 1575, 1474 (C]C), 1106
(ClOꢁ4 ), 1052 (CeOeC), 623 (ClOꢁ4 ). MS-FAB (m/z): 753 (45%), [Cu
(L)2]2þ; 407 (100%), [Cu(L)]2þ. Anal. found: C, 40.03; H, 2.76; N,
11.28;. Calc. for C32H26N8O14Cl4Cu (951.939): C, 40.37; H, 2.75;
N, 11.77.
(55%). IR (KBr)
1617, 1574, 1447 (C]N), 1100 (CeOeC), 550 (PdeN). MS-FAB (m/z):
n
(cmꢁ1): 3387 (CeOH), 2923 (CeCH3), 1727 (C]O),

2620
E. Budzisz et al. / European Journal of Medicinal Chemistry 45 (2010) 2613e2621
4.1.2.9. Cu(II) complex 9b. A methanolic solution of CuCl2 ꢂ 2H2O
(25.6 mg, 0.15 mmol, in 3 mL) was slowly added dropwise to
a solution of ligand 2b in ethyl acetate (51.7 mg, 0.15 mmol, in
10 mL). The mixture was stirred at room temperature for 1.5 h. The
resulting yellow solid was filtered off and dried. Yield: 43 mg (69%),
PCR reaction was conducted according to protocol “AccuTaqÔ LA
DNA Polymerase Kit” (Sigma, Germany). The reaction mixture for
PCR amplification consisted of a cDNA template, 0.5
primer, 10 ꢂ AccuTaq Buffer, 0.5 U of AccuTaq LA DNA Polymerase
Mix, 0.2 mM each dNTP, water to a final volume of 20 l. Negative
mM of each
m
dec.: 212 ꢀC. IR (KBr)
n
(cmꢁ1): 3056 (CeOH), 2915 (CeCH3) 1717
control was included in each experiment (sample without a cDNA
template). The primers sequences for both genes were planned by
using software Primer3: WWW primer tool (http://biotools.
umassmed.edu/bioapps/primer3_www.cgi). The BAX primer set
50-CAGCTCTGAGCAGATCATGAAGACA-30 (forward) and 50-GCCCAT-
CTTCTTCCAGATGGTGAG-30 (reverse), and the P53 primer set
50-CACATGACGGAGGTTGTGAG-30 (forward) and 50- CATAGGGCAC-
CACCACACTA-30 (reverse) were used.
(C]O), 1591 (C]N), 1569, 1476 (C]C), 1104 (CeOeC), 546 (CueN).
MS-FAB (m/z): 753 (45%), [Cu(L)2]2þ; 407 (100%), [Cu(L)]2þ. Anal.
found: C, 46.26; H, 2.59; N, 13.55;. Calc. for C32H26N8O6Cl4Cu
(823.939): C, 46.64; H, 3.18; N, 13.60.
4.2. Biological assays
4.2.1. Cell cultures
Real-time PCR reactions were done using MX3005PÔ System
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 (Invitrogen, U.K.) supplemented with 10% fetal bovine
(STRATAGEN). The BAX gene and a housekeeping gene,
(ACTB) were amplified in parallel for each sample during the same
PCR. -Actin was utilized as an internal positive control and as
a normalizer for expression data correction. For the BAX and P53 the
b-actin
b
serum and antibiotics (100
mg/ml streptomycin and 100 U/ml
same primer sets as above were used and for the b
-actin 50-
penicillin). For melanoma WM-115 cells Dulbecco's minimal
essential medium (DMEM) instead of RPMI 1640 was used. Cells
were grown at 37 ꢀC in a humidified atmosphere of 5% CO2 in air.
GTGGGGCGCCCCAGGCACCA-30 (forward) and 50-CTCCTTAATGT-
CACGCACGATTTC-30 (reverse). For each PCR run, a reaction mixture
was prepared consisting of 12.5
ReadyMixÔ (Sigma), 0.5 l forward primer (final concentration
0.2 M), 0.5 l reverse primer, 9 l nuclease-free water and 2.5
m
l SYBRÒ Green JumpStartÔ Taq
m
4.2.2. Cytotoxicity assay by MTT
m
m
m
ml
Cytotoxicity of ligands 2a and 2b, their complexes 4a, b, 5a, b,
6a, 8a, b, 9b, carboplatin and cisplatin was determined by the MTT
template cDNA. The thermal cycling conditions comprised an initial
denaturation step at 95 ꢀC for 3 min, 35 cycles at 94 ꢀC for 15 s,
59 ꢀC(BAX)/57 ꢀC(P53) for 15 s and 72 ꢀC for 45 s and a final
extension step at 72 ꢀC for 3 min. After reaction, a melting curve
was performed to confirm reaction specificity. Experiments for all
samples were performed in triplicate. The relative level of BAX and
P53 expression was calculated as described previously [31].
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide,
Sigma, USA] assay as described [29]. After 46 h of incubation with
drugs, the cells were briefly treated with the MTT reagent and
incubation was continued for 2 h. MTT e formazan crystals were
dissolved in 20% SDS and 50% DMF at pH 4.7 and absorbance was
read at 562 and 630 nm on an ELISA e plate reader (ELX 800, Bio-
Tek, USA). The values of IC50 (the concentration of the test
compound required to reduce the cells survival fraction to 50% of
the control) were calculated from concentration-response curves
and used as a measure of cellar sensitivity to a given treatment.
Ligands, metal complexes, carboplatin and cisplatin were tested for
their cytotoxicity in a final concentration of 10ꢁ7e10ꢁ3 M. As
a control, cultured cells were grown in the absence of drugs. Data
4.3. X-ray structure determinations of 2a, 6a and 7a
X-ray data were collected at 200 K with MoK
¼ 0.71073 A) with a Nonius KappaCCD diffractometer equipped
a
radiation
ꢀ
(l
with a rotating anode. The structures were solved by direct
methods [32] and refined with SHELXL-97 by full-matrix least-
squares on F2 [33]. All non-hydrogen atoms were refined aniso-
tropically. The crystal data and X-ray details are given in Table 1.
Further details are available from the Crystallographic Data Centre
under the depository numbers CCDC 727843 (2a), 727845 (6a) and
727844 (7a). Copies of the data can be obtained free of charge
upon application to CCDC, 12, Union Road, Cambridge CB2 1EZ, U.K.,
E-mail: deposit@ccdc.cam.ac.uk.
points represent means from at least
3 experiments each
performed at 5 repeats ꢃ S.D.
4.2.3. Analysis of BAX and P53 gene expression in melanoma WM
115 cells exposed to drugs
Melanoma WM-115 cells treated with 4a and 6a (in two
concentrations: 1 ꢂ IC50 and 5 ꢂ IC50) were used for analysis of BAX
and P53 gene expression by real-time PCR. After 5 and 48 h of
incubation with the tested compound, the cells were spun down,
washed twice with cold 0.01 M phosphate buffer (pH 7.0)
containing 0.9% NaCl. Cell pellets were than used for RNA
extraction.
Acknowledgements
Financial support from grant No. 507-13-041 to Prof. Budzisz
and grant No. 503-3015-2 to the Department of Pharmaceutical
Biochemistry at the Medical University of Lodz are gratefully
acknowledged.
4.2.3.1. RNA extraction and cDNA synthesis. Total RNA was isolated
by Total RNA prep plus Minicolumn Kit (A&A Biotechnology,
Poland) based on a method described earlier [30]. RNA concen-
tration and its quality were determined by the measurement of
absorbance at 260 and 280 nm.
To obtain cDNA, reverse transcription reaction was done using
Enhanced Avian HS RT-PCR-100 Kit (Sigma, Germany) according to
the manufacturer's instruction using 400 ng of total RNA per
reaction.
References
[1] E. Budzisz, I.-P. Lorenz, P. Mayer, P. Paneth, L. Szatkowski, U. Krajewska,
M. Rozalski, M. Miernicka, N J. Chem. 32 (2008) 2238e2244.
[2] E. Budzisz, M. Miernicka, I.-P. Lorenz, P. Mayer, U. Krajewska, M. Rozalski,
Polyhedron 28 (2009) 637e645.
[3] M. Grazul, E. Budzisz, Coord. Chem. Rev. 253 (2009) 2588e2598.
[4] X. Wang, Z. Guo, Anti-Cancer Agents Med. Chem. 7 (2007) 19e34.
[5] A.E. Liberta, D.X. West, BioMetals 5 (1992) 121e126.
[6] N.K. Singh, S.T. Singh, N. Singh, A. Shriviastav, BioMetals 16 (2003) 471e477.
[7] C. Rothenburger, M. Galanski, V.B. Arion, H. Görls, W. Weigand, B.K. Keppler,
Eur. J. Inorg. Chem. 18 (2006) 3746e3752.
[8] J. Kasparkova, O. Novakova, V. Marini, Y. Najajreh, D. Gibson, J.M. Perez,
V. Brabec, J. Biol. Chem. 278 (2003) 47516e47525.
4.2.3.2. Real-time PCR analysis of BAX and P53 genes. In all cases,
before the quantitative analysis of gene expression by real-time
PCR reaction, the parameters were checked using qualitative PCR.

E. Budzisz et al. / European Journal of Medicinal Chemistry 45 (2010) 2613e2621
2621
[9] D. Kovala-Demertzi, A. Papageorgiou, L. Papathanasis, A. Alexandratos,
P. Dalezis, J.R. Miller, M.A. Demertzis, Eur. J. Med. Chem. 44 (2009) 1296e1302.
[10] D. Kovala-Demertzi, M.A. Demertzis, E. Filiou, A.A. Pantazaki, P.N. Yaday, J.
R. Miller, Y. Zheng, D.A. Kariakidis, Biometals 16 (2003) 411e418.
[11] J. Kasparkova, V. Marini, Y. Najajreh, D. Gibson, V. Brabec, Biochemistry 42
(2003) 6321e6332.
[20] A. Kufelnicki, M. Wozniczka, L. Checinska, M. Miernicka, B. Budzisz, Poly-
hedron 26 (2007) 2589e2596.
[21] M. Miernicka, A. Szulawska, M. Czyz, I.-P. Lorenz, P. Mayer, B. Karwowski,
E. Budzisz, J. Inorg. Biochem. 102 (2008) 157e165.
[22] K. Kostka, Chem. Anal. 14 (1969) 1145e1160.
[23] S. Kerrison, P.J. Sadler, Chem. Commun. 23 (1977) 861e863.
[24] B. Stuart, Infrared Spectroscopy e Fundamentals and Applications. Wiley,
Chichester, UK, 2004, pp. 45e111.
[12] F. Saczewski, E. Dziemidowicz-Borys, P.J. Bednarski, M. Gdaniec, Arch. Pharm.
Chem. Life Sci. 340 (2007) 333e338.
[13] R. Mukherjee, Coord. Chem. Rev. 203 (2000) 151e218.
[14] T.N. Sorrell, Tetrahedron 45 (1989) 3e68.
[25] R.S. Macomber, A Complete Introduction to Modern NMR Spectroscopy. Wiley,
New York, USA, 1998, pp. 68e87.
[15] M. Viciano-Chumillas, S. Tanase, G. Aromí, J.M.M. Smits, R. de Gelder, X. Solans,
E. Bouwman, J. Reedijk, Eur. J. Inorg. Chem. 18 (2007) 2635e2640.
[16] G.Z. Rassidakis, L.J. Medeiros, T.J. McDonnell, S. Viviani, V. Bonfante, G. Nadali,
T.P. Vassilakopoulos, R. Giardini, M. Chilosi, C. Kittas, A.M. Gianni,
G. Bonadonna, G. Pizzolo, G.A. Pangalis, F. Cabanillas, A.H. Sarris, Clin. Cancer
Res. 8 (2002) 488e493.
[17] S. Haupt, M. Berger, Z. Goldberg, Y. Haupt, J. Cell Sci. 116 (2003) 4077e4085.
[18] F. Kayaselcuk, T.Z. Nursal, A. Polat, T. Noyan, S. Yildirim, A. Tarim, G. Seydioglu,
J. Exp. Clin. Cancer Res. 23 (2004) 105e112.
[26] R.S. Macomber, A Complete Introduction to Modern NMR Spectroscopy. Wiley,
New York, USA, 1998, pp. 88e103.
[27] J.T. Wrobel, Preparatyka
i elementy syntezy organicznej. PWN, Warsaw,
Poland, 1983, 9e52.
[28] G.M. Coppola, R.W. Dodsworth, Synthesis 75 (1981) 523e524.
[29] E. Budzisz, U. Krajewska, M. Rozalski, Pol. J. Pharmacol. 56 (2004) 473e478.
ꢁ
[30] P. Chomczynski, N. Sacchi, Anal. Biochem. 162 (1987) 156e159.
[31] K.J. Livak, T.D. Schmittgen, Methods 25 (2001) 402e408.
[32] A. Altomare, M.C. Burla, M. Camalli, G.L. Cascarano, C. Giacovazzo,
A. Guagliardi, A.G.G. Moliterni, G. Polidori, R. Spagna, J. Appl. Crystallogr. 32
(1999) 115e119.
[19] M. Gielen, E.R.T. Tiekink, in: Metallotherapeutic Drugs and Metal-based
Diagnostic Agents. The Use of Metals in Medicine, Wiley, Chichester, UK, 2005,
p. 39.
[33] G.M. Sheldrick. SHELXL-97 97-2 Version. University of Göttingen.