Synthesis, structure, protolytic properties, alkylating and cytotoxic activity of novel platinum(II) and palladium(II) complexes with pyrazole-derived ligands

Article abstract of DOI:10.1002/ejic.200700139

Novel platinum(II) and palladium(II) complexes [PtCl₂(HL ¹)] (3a), [NH₂CH₃)₂][PtCl ₂(L¹)] (3b), [PdCl₂(HL¹)] (4a), [NH₂(CH₃)₂][PdCl₂(L¹)] (4b) and [PdCl(CH₃CN)(L¹)] (4c), where HL¹ = 4-(2-hydroxybenzoyl)-2-(pyridin-2-yl)-1H-pyrazol-3-ol, have been synthesised as potential anticancer compounds. Protonation constants of the ligand were determined by pH-metric titration (log K₁ = 7.74 ± 0.03; log K₂ = 2.85 ± 0.05). The solid-state structures of complexes 3b and 4c have been determined by X-ray diffraction, showing sguare-planar coordination geometry of Pt^II and Pd^II ions. In addition, the compounds have been characterised by IR, ¹H NMR and FAB mass spectrometry. The results of preliminary studies on the cytotoxic activity in vitro against HL-60 and NALM-6 leukaemia cell lines are also reported and the IC₅₀ values compared with those of the metal-free ligand and cisplatin. Cytotoxic evaluation revealed that Pt^II and Pd ^II complexes were active in the micromolar concentration range. Wiley-VCH Verlag GmbH & Co. KGaA, 2007.

Full text of DOI:10.1002/ejic.200700139

FULL PAPER
DOI: 10.1002/ejic.200700139
Synthesis, Structure, Protolytic Properties, Alkylating and Cytotoxic Activity
of Novel Platinum(II) and Palladium(II) Complexes with Pyrazole-Derived
Ligands
Elzbieta Budzisz,*[a] Magdalena Malecka,[b] Bernhard K. Keppler,[c] Vladimir B. Arion,[c]
Grzegorz Andrijewski,[d] Urszula Krajewska,[e] and Marek Rozalski[e]
Keywords: Metal complexes / Platinum / Palladium / Pyrazole ligands / Antitumor agents / Alkylating and cytotoxic
activity
Novel platinum(II) and palladium(II) complexes [PtCl2(HL1)]
(3a), [NH2(CH3)2][PtCl2(L1)] (3b), [PdCl2(HL1)] (4a),
[NH2(CH3)2][PdCl2(L1)] (4b) and [PdCl(CH3CN)(L1)] (4c),
where HL1 = 4-(2-hydroxybenzoyl)-2-(pyridin-2-yl)-1H-pyr-
azol-3-ol, have been synthesised as potential anticancer com-
pounds. Protonation constants of the ligand were determined
addition, the compounds have been characterised by IR, 1H
NMR and FAB mass spectrometry. The results of preliminary
studies on the cytotoxic activity in vitro against HL-60 and
NALM-6 leukaemia cell lines are also reported and the IC50
values compared with those of the metal-free ligand and cis-
platin. Cytotoxic evaluation revealed that PtII and PdII com-
plexes were active in the micromolar concentration range.
by pH-metric titration (logK1
= 7.74Ϯ0.03; logK2 =
2.85Ϯ0.05). The solid-state structures of complexes 3b and
4c have been determined by X-ray diffraction, showing
square-planar coordination geometry of PtII and PdII ions. In
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
ticancer drugs.[6] Simple structural analogues of cisplatin
were found to possess no advantages because of similar
mechanism of action, structure of DNA adducts, toxicity
profile, adverse effects and tumour cross-resistance.[7] Re-
cently, attention has been focused on inert, nonleaving li-
gands, whose steric and electronic features may affect the
mechanism of substitution at the metal centre, DNA bind-
ing and drug metabolism. It is believed that complexes con-
taining bulky ligands are kinetically and thermodynami-
cally more stable than simple cisplatin analogues.[8] Taking
into account the structural analogy between PtII and PdII
complexes, a variety of studies on PdII complexes have been
performed, including on their cytotoxicity[9] and antitu-
mour activity.[10–12] Palladium complexes with β-carboline
alkaloids,[13–15] pyrazoles[16] and DMSO[17] were tested
against solid tumour cell lines, and in some cases exhibited
remarkable activity. It was suggested that their biological
activity depends on the nature of the ligand, the type of
counterion used and the configuration of the complex.[18]
For many years the research in our laboratory has been
directed towards the synthesis of chromone (benzo-γ-pyr-
one) and coumarin (benzo-α-pyrone) derivatives and the
study of their reactivity towards nucleophilic reagents con-
taining nitrogen, oxygen, sulfur and carbon.[19] In particu-
lar, in protolytic solvents at room temperature chromones
have been converted efficiently into enamine-type com-
pounds with a typical lemon-yellow colour.[20,21] The chro-
mones and coumarins react with hydrazines to afford pyr-
Introduction
Cisplatin [diamminedichloroplatinum, cis-PtCl2(NH3)2]
is known as a DNA-modifying agent with strong anticancer
potency.[1–3] Despite its wide clinical application, cisplatin
causes many serious side effects, such as nephrotoxicity,
ototoxicity, peripheral neuropathy and allergy.[4] In ad-
dition, the therapeutic efficacy of cisplatin is limited by in-
herent or treatment-induced resistance of tumour cell sub-
populations.[5] Therefore, various platinum(II) and palladi-
um(II) complexes with nitrogen-containing ligands were
evaluated in the search for less toxic and more selective an-
[a] Department of Cosmetic Raw Materials Chemistry, Faculty of
Pharmacy, Medical University of Lodz,
Muszynski 1, 90-151 Lodz, Poland
Fax: +48-42-678-83-98
E-mail: elora@ich.pharm.am.lodz.pl
[b] Department of Crystallography and Crystal Chemistry, Univer-
sity of Lodz,
Pomorska 149/153, 90-236 Lodz, Poland
E-mail: malecka@uni.lodz.pl
[c] Institute of Inorganic Chemistry, University of Vienna,
Währingerstrasse 42, 1090 Vienna, Austria
E-mail: bernhard.keppler@univie.ac.at
[d] Department of Inorganic Chemistry, University of Lodz,
Narutowicza 68, 90-136 Lodz, Poland
E-mail: grzegorz@chemul.uni.lodz.pl
[e] Department of Pharmaceutical Biochemistry, Faculty of Phar-
macy, Medical University of Lodz,
Muszynski 1, 90-151 Lodz, Poland
E-mail: mrozalski@pharm.am.lodz.pl
Supporting information for this article is available on the
WWW under http://www.eurjic.org or from the author.
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Cytotoxic PtII and PdII Complexes
FULL PAPER
azole derivatives.[22] A class of pyrazole-containing palladi- of HL1 presumably proceeds by an attack of 2-hydrazi-
um(II) complexes has been reported to possess antibacte- nopyridine on the carbonyl group (C-3 position in couma-
rial,[23] antidepressant,[24] monoamine oxidase inhibiting[25] rin), followed by the nucleophilic attack of the hydrazine
and antitumour activity.[26,27]
nitrogen on the carbonyl atom at C-2 position (Scheme 3).
In addition, both type of compounds (chromones and
coumarins) exhibit interesting biological properties,[28,29] in-
cluding anticonvulsant,[30] antimicrobial,[31] spasmolytic,[32]
antioxidant[33] and antitumour activities.[34] The metal com-
plexes with coumarin as a ligand reveal anticoagulant ac-
tion[35,36] and possess antiproliferative activity.[37,38] No-
tably, complexes with cerium(III), zirconium(IV), cop-
per(II), zinc(II), bismuth(III) and cadmium(II) show pro-
nounced cytotoxic activity in vitro.[39,40] The rare earth
metal complexes of coumarin derivatives endowed with an-
ticoagulant and anti-HIV activity were also documen-
ted.[41–43] Current efforts are focused on developing metal-
based drugs with improved clinical effectiveness, reduced
general toxicity and a broader spectrum of activity.
Herein we report the synthesis of a novel pyrazole ligand
HL1 by the reaction of coumarin with 2-hydrazinopyridine.
Protonation constants of the ligand were determined by
pH-metric titration. This ligand, as will be shown below,
forms cis complexes with platinum(II) and palladium(II),
which are often more cytotoxic than the trans isomers.[44]
The structures of two PtII and PdII complexes determined
by X-ray diffraction are reported. The results of preliminary
studies on the alkylating and in vitro cytotoxic activity
against HL-60 and NALM-6 leukaemia cell lines are also
discussed.
Scheme 2. Possible tautomeric forms of the ligand HL1.
Scheme 3. Stepwise formation of HL1.
The ligand has been characterised by elemental analysis,
Results and Discussion
1
IR spectroscopy, H and 13C NMR spectroscopy and mass
Preparation of the Ligands
spectrometry (see Experimental Section).
Pyrazole-based chelating ligands have attracted our at-
tention, because of their ability to form cis-configured com-
plexes. The anticancer activity of a metal complex depends
not only on the metal itself, but also on its oxidation state,
on the number and type of bound ligands, and the coordi-
nation geometry of the complex.[45]
By reaction of 3-acetyl-4-hydroxycoumarin (2), which
was obtained from methyl ester 1, with 2-hydrazinopyridine
in a 1:1 molar ratio in refluxing methanol, we obtained 4-
(2-hydroxybenzoyl)-2-(pyridin-2-yl)-1H-pyrazol-3-ol (HL1)
in 62% yield (Scheme 1).
Synthesis of Complexes
The synthesised ligand HL1 behaves as a strong bidentate
chelating agent forming a five-membered metallocycle
through coordination of the pyrazole and the pyridine ni-
trogen atoms to the corresponding metal ion. A review of
the coordination chemistry of acylpyrazolone derivatives
has been recently published.[46]
Complexes [MIICl2(HL1)], where M = Pt (3a) and Pd
(4a) (Scheme 4), were prepared at room temperature by
mixing equimolar amounts of HL1 and potassium tetra-
chloroplatinate(II) or potassium tetrachloropalladate(II),
respectively, in water/methanol. Reactions of K2[PtCl4] or
K2[PdCl4] with HL1 in 1:1 molar ratio in dimethylform-
amide (DMF) produced (after two weeks) the complexes
[NH2(CH3)2][MCl2(L1)], where M = Pt (3b) and Pd (4b).
The cation [NH2(CH3)2]+ has been presumably formed by
light-induced decomposition of DMF and protonation of
the arising dimethylamine.[47] The complex [PdCl(CH3CN)-
Scheme 1. Synthesis of the ligand HL1.
Three different tautomeric forms (A, B and C) can be (L1)] (4c) resulted from the reaction of [PdCl2(C6H5CN)2]
envisaged for HL1 (Scheme 2). Of these, B and C are stabi- with HL1 in 1:1 molar ratio in chloroform/methanol (2:1),
lised by intramolecular hydrogen bonding. The formation followed by recrystallisation from acetonitrile/diethyl ether.
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E. Budzisz et al.
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The Protonation Constants of the Ligand HL1
The potentiometric measurements performed within the
pH range 2.5–11.0 showed that under titration with KOH,
the ligand HL1 acts as a weak acid with a protonation con-
stant of about 108. These acidic properties can be attributed
to the hydroxy group attached to the aromatic ring. Its acid-
ity, slightly higher than that observed for phenols, is proba-
bly enhanced by the vicinity of the carbonyl group. In the
presence of a strong acid the ligand HL1 acts as a weak
base. However, its basicity is small and, within accessible
range of concentrations, it was possible to determine
potentiometrically only one additional protonation con-
stant. Supposedly, it refers to the equilibrium of proton-
ation of the pyridine nitrogen atom, which is expected to
have a higher affinity for the proton than the pyrazole nitro-
gen atom.
Thus, the stepwise protonation constants of logK1
=
7.74Ϯ0.03 and logK2 = 2.85Ϯ0.05 were calculated from
titration curves. This implies that, under physiological con-
ditions, the nitrogen atoms of the ligand present in solution
are unprotonated.
X-ray Crystallography
Single crystal X-ray diffraction studies of compound 3b
were undertaken to elucidate the coordination sphere of
platinum(II) and the tautomeric form of the ligand. The
molecular structure of 3b with the atom numbering scheme
is shown in Figure 1. Selected bond lengths and angles are
given in Table 1. The asymmetric unit consists of one
[PtCl2(L1)]– anion and one [NH2(CH3)2]+ cation. The coor-
dination environment of PtII atom is planar, formed by two
Scheme 4. The library of novel complexes prepared.
The synthesised complexes 3a and 4a are sparingly solu- nitrogen atoms and two chloride ligands. The PtN2Cl2
ble in water, acetone and ethanol, but well soluble in “square” adopts a cis configuration. The geometry about
DMSO and DMF.
the Pt atom deviates from the ideal square plane: the atoms
The composition and the structure of the complexes pre- N1, N3 and Cl1, Cl2 form angles smaller than 90° with the
1
pared were confirmed by elemental analyses, IR spectra, H Pt atom, and consequently the N3–Pt–Cl2 and N1–Pt–Cl1
NMR spectra, mass spectrometry measurements and X-ray angles are larger than 90°. The distribution of electron den-
diffraction for compounds 3b and 4c. Some characteristic IR sity over the ligand backbone and the presence of a
vibrations for these complexes are given in Table S1 (see Sup- countercation indicate that HL1 acts as a monodeproton-
porting Information). Significant differences in the IR spectra ated ligand and adopts the tautomeric form C (Scheme 2).
of complexes 3 and 4 were observed in comparison with that
The crystal lattice is stabilised by a net of intermolecular
of ligand HL1. The latter shows a strong band at 1660 cm–1 and intramolecular hydrogen bonds. The atom O1 partici-
due to ν(C=O). This band is shifted by 14–32 cm–1 to lower pates in two O–H···O hydrogen bonds: intramolecular O1–
frequencies in the spectra of metal complexes. Moderately H5···O2, which closes an extra six-membered ring, and
strong bands at 355, 334 and 328 cm–1 can be attributed to intermolecular O5–H5···O1(i). In addition, N4–H16···O3 in-
ν(M–Cl) of the complexes. Stretching vibrations of the M–N teraction is of note. The atom N4 is also involved in hydro-
bonds were observed at 420 cm–1. The FAB mass spectrum gen bonds to the Cl atoms (see Table S2).
of 3a recorded in positive-ion mode contains a peak at m/z
The packing of the molecules in the crystal lattice is sta-
563 due to the [M + H]+ ion and a strong peak at m/z 528 bilised by C–H···π interactions. Atom C11 is involved in a
attributed to [M – Cl]+. Analogously, the spectrum of 4a weak C–H···π intermolecular interaction with the centroid
shows two peaks at m/z 473 and 438, which were assigned to of the ring 3 (N3/C12/C13/C14/C15) [symmetry code (ii):
[M + H]+ and [M – Cl]+, correspondingly. For complex 4c 2 – x, 1 – y, –z] and C18 interacts with Cg4, where Cg4 is
we have observed a peak at m/z 478 due to [M + H]+. The the centroid of ring C1–C6 (more details in Table S3). A
purity of complexes 3b and 4b was confirmed by elemental number of π–π stacking interactions are observed in the
analysis.
crystal lattice (see Figure S2).
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Cytotoxic PtII and PdII Complexes
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Biological Characterisation
Alkylating Activity
Alkylating agents belong to the first class of cytostatics
used for chemotherapy.[48] Under in vivo conditions they
alkylate nucleophilic centres of nucleobases and amino ac-
ids, resulting either in cleavage or cross-linking of double-
stranded DNA molecules or proteins. Such cleavage de-
stroys DNA, while covalent cross-links prevent unwinding
of nucleic acids, which is functionally important in replica-
tion and transcription processes.[49] Cisplatin and carbopla-
tin are the nonclassical alkylating agents. These drugs inter-
act with cellular DNA according to the same mecha-
nism[50,51] by metallation of nucleobases of both DNA
strands.
An effective drug concentration for carboplatin is 4–7-
fold higher as compared to cisplatin, and the former is also
less toxic.[52] The “alkylating” activities of the ligand HL1
and of the complexes 3a and 4a were determined by the
Preussmann test.[53] The level of “alkylation” of 4-(4-ni-
trobenzyl)pyridine (NBP) was quantified spectrophotomet-
rically at 560 nm. The molar absorbances (A) for complexes
3a and 4a were 0.303 and 0.069, respectively, compared
with 0.047 for HL1 (Table 2). Interestingly, cisplatin in this
test shows A = 0.300[54] and carboplatin alkylates NBP giv-
ing A = 0.231. According to the Preussmann scale (see foot-
note to Table 2), the complex 3a and both reference com-
pounds cisplatin and carboplatin exhibit moderate alkylat-
ing activity in this test.
Figure 1. ORTEP drawing of 3b (top) and of the first independent
molecule of 4c (bottom) with thermal ellipsoids depicted at 50%
probability level.
Table 2. Alkylating activity of substituted pyrazoles and their PtII
and PdII complexes.
Compound
ε [–1 cm–1]
Absorbance[a]
λmax = 560 nm
Alkylation
activity[b]
Compound 4c crystallised in the orthorhombic space
group Pca21 with four crystallographically independent
molecules of [PdCl(CH3CN)(L1)] in the unit cell. In all four
molecules the organic species acts as a monodeprotonated
bidentate ligand coordinating to the Pd ion through two
nitrogen atoms with formation of a five-membered metallo-
cycle. The square-planar coordination geometry of each
PdII ion is completed by one chlorido ligand and one
CH3CN molecule. The bond lengths in the coordination
polyhedron of all four independent molecules are given for
comparison in Table 1. The bond lengths C6–O2 of
1.243(13), C10–O1 of 1.240(12) and C16–O3 of 1.343(13) Å
clearly indicate the stabilisation of tautomer C in the com-
plex (Scheme 2). The same tendency in bond length distri-
HL1
47.3
303.4
69.0
300.0
231.2
0.0473
0.3034
0.0690
0.3000
0.2312
–
++
+
++
++
3a
4a
Cisplatin[c]
Carboplatin
[a] Means from 3 determinations. [b] According to ref.[53]: (–) A Ͻ
0.05, (+) A = 0.05–0.1, (++) A = 0.1–0.5, (+++) A Ͼ 0.5. [c] Ac-
cording to ref.[54]
.
Cytotoxicity Assay
Cytotoxicity assays for complexes 3a and 4a were per-
bution is observed in three other crystallographically inde- formed on human acute leukaemia HL-60 and NALM-6
pendent molecules (Table 1).
cell lines (Figure 2). Cisplatin and carboplatin were used as
Table 1. Selected bond lengths [Å] for compounds 3b and 4c.
Structure 3b
Structure 4c
Molecule 1A
Molecule 1B
Molecule 1C
Molecule 1D
Pt–N1
Pt–N3
Pt–Cl2
Pt–Cl1
2.019(4)
2.033(3)
2.302(2)
2.303(2)
Pd1–N1
2.022(9)
2.022(9)
2.014(10)
2.297(3)
Pd2–N5
Pd2–N7
Pd2–N8
Pd2–Cl2
2.001(10)
2.007(10)
1.989(10)
2.286(3)
Pd3–N9 2.019(10)
Pd4–N13
2.003(10)
2.007(10)
1.979(10)
2.294(3)
Pd1–N3
Pd1–N4
Pd1–Cl1
Pd3–N11
Pd3–N12
Pd3–Cl3
2.024(9)
1.972(11)
2.285(3)
Pd4–N15
Pd4–N16
Pd4–Cl4
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E. Budzisz et al.
FULL PAPER
the reference compounds. Both metal complexes exhibited
relatively high cytotoxic activity with the IC50 values in the
micromolar range. Their cytotoxic effectiveness was nearly
the same as that for carboplatin and only 5 (3a) or 10 (4a)
times lower in comparison to cisplatin, a well-established
therapeutic agent used for the treatment of leukaemia
(Table 3). The most potent was the platinum(II) complex
3a, with an IC50 of 4.7 and 3.9 µ for HL-60 and NALM-
6 cells, respectively, in line with alkylating activity towards
NBP.
Conclusions
In this paper we have described the synthesis of a new
chelating pyrazole ligand HL1, which is characterised by
two protonation constants determined by pH-metric ti-
tration (logK1 = 7.74Ϯ0.03; logK2 = 2.85Ϯ0.05). The li-
gand forms neutral complexes of the type [MCl2(HL1)],
where M = Pt (3a) and Pd (4a) in aqueous methanol. In
DMF solutions the ionic species [NH2(CH3)2][PtCl2(L1)]
with M = Pt (3b) and Pd (4b) have been isolated. Starting
from [PdCl2(C6H5CN)2] and HL1, and using acetonitrile for
recrystallisation, the complex [PdCl(CH3CN)(L1)] (4c) has
been prepared. X-ray diffraction studies of 3b and 4c
showed that the ligand in both square-planar complexes
acts as a monodeprotonated one, adopting the tautomeric
form C.
Complexes 3a and 4a exhibit in vitro antiproliferative ac-
tivity in HL-60 and NALM-6 leukaemia cells, with the
cytotoxicity IC50 values similar to those for carboplatin.
The sequence in activity against HL-60 and NALM-6 leu-
kaemia cells is as follows 3a Ͼ 4a Ͼ HL1. The alkylating
activity of the platinum compound 3a was comparable with
those of cisplatin and carboplatin.
Experimental Section
Materials: All substances were used without further purification.
Potassium tetrachloroplatinate(II) and potassium tetrachloropalla-
date(II) were purchased from Aldrich. CDCl3 and [D6]DMSO sol-
vents for NMR spectroscopy were obtained from Dr. Glaser AG,
Basel, Switzerland. Solvents for synthesis (toluene, methanol, di-
methylformamide, acetone) were reagent-grade or better and were
dried according to standard protocols.[55] The melting points were
determined using an Electrothermal 1A9100 apparatus and they
are uncorrected. The IR spectra were recorded with a Pye-Unicam
200G Spectrophotometer in KBr or CsI pellets. The 1H NMR spec-
tra 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 FAB mass spectra were recorded
with a Finnigan Matt 95 mass spectrometer (NBA, Cs+ gun op-
erating at 13 keV). For the new compounds satisfactory elemental
analyses (Ϯ0.3% of the calculated values) were obtained using a
Perkin–Elmer PE 2400 CHNS analyser. Methyl 2-methyl-4-oxo-
4H-chromene-3-carboxylate (1)[56] and 3-acetyl-4-hydroxychromen-
2-one (2)[57,58] were prepared as described in the literature.
Figure 2. A: Survival curves for HL-60 and B: NALM-6 leukaemia
cells exposed for 48 h to tested compounds HL1, 3a and 4a. Each
point represents the mean of four independent determinations.
Standard deviations are excluded for clarity and do not exceed 20%
of the mean value for each point.
Table 3. IC50 values [µ] for HL1 and complexes 3a and 4a.
[a]
Compound
IC50
HL-60
NALM-6
HL1
3a
4a
Cisplatin
Carboplatin
29.9Ϯ8.3
4.7Ϯ0.6
7.0Ϯ0.5
0.8Ϯ0.1
4.3Ϯ1.3
30.0Ϯ5.3
3.9Ϯ0.6
8.3Ϯ0.3
0.7Ϯ0.3
0.7Ϯ0.2
4-(2-Hydroxybenzoyl)-2-pyridin-2-yl-1H-pyrazol-3-ol (HL1): 2-Hy-
drazinopyridine (0.13 g, 1.15 mmol) in methanol (5 mL) was added
to a solution of 2 (0.24 g, 1.15 mmol) in methanol (15 mL) and the
mixture was refluxed for 1 h. The solid crude product formed was
filtered off, dried in air and recrystallised from ethanol as a white
solid. Yield 0.21 g (62%); m.p. 197–198 °C, Rf = 0.78 (methanol/
[a] IC50 – concentration of a tested compound required to reduce
the fraction of surviving cells to 50% of that observed in the con-
trol, nontreated cells. Mean values of IC50 (in µ)ϮS.D. from four
experiments are presented.
chloroform, v/v 1:1). IR (KBr): ν = 3428 (OH), 1735 (C=O), 1630,
˜
1589 (C=N), 1458 (C=C) cm–1. 1H NMR (300 MHz, CDCl3,
25 °C): δ = 2.47 (s, 3 H, CH3), 6.88 (t, J = 6.15 Hz, 1 H), 6.99 (d,
J = 2 Hz, 1 H), 7.22–7.26 (m, 2 H), 7.46–7.49 (m, 1 H), 7.86 (t, J
= 6.35 Hz, 1 H), 7.96 (d, J = 3.4 Hz, 1 H), 8.26 (dt, 1 H, Ar) ppm
(see Figure S1a, Supporting Information). 13C NMR (75.4 MHz,
[D6]DMSO, 25 °C): δ = 14.45, 103.18, 112.22, 116.25, 117.89,
119.79, 123.23, 131.33, 133.17, 141.71, 143.67 153.66, 158.77,
160.91, 190.92 ppm (Figure S1b). EI-MS: m/z (%) = 295 (51) [M+],
Comparison of the data in Table 3 clearly shows that the
in vitro antiproliferative activity of complexes 3a and 4a
exceeds that of uncomplexed ligand HL1. This strongly sup-
ports the view that cytotoxicity can be quite safely ascribed
to the presence of the metal centre.
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Cytotoxic PtII and PdII Complexes
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175 (100%), 79 (7%). C16H13N3O3·1/3H2O (301.29): calcd. C 63.78,
H 4.57, N 13.94; found C 63.93, H 4.33, N 13.60.
samples at (25.0Ϯ0.1) °C. The total ligand concentration in each
sample was 2.859 mmoldm–3. The samples contained also
3 mmoldm–3 HClO4 and 0.05 moldm–3 NaClO4. Owing to very
low solubility in pure water, a mixed 70% (v/v) ethanol/water sol-
vent was used. The titrations were carried out with carbonate-free
KOH solutions of known concentration (1 moldm–3) using an au-
toburette. The drop volume during these titrations was 1 µL. The
experimental value of pKw = 15.50Ϯ0.03 was obtained from our
acid–base calibration in the mixed solvent. The pH was measured
with a combined glass electrode ESAgP-301 WM (Eurosensor)
connected to computer-aided universal electrochemical meter
EMU0,[59] produced by the Technical University of Wrocław, Po-
land. The same equipment was also used to control the autoburette,
as well as for the data acquisition and their introductory treatment.
The electrode was calibrated by titration of 0.01  HCl (in 70%
ethanol) with 1  KOH at (25Ϯ0.1) °C. Stepwise protonation con-
stants were calculated with our own program based on algorithm
PKAS.[60,61]
Pt Complex 3a: A solution of K2PtCl4 (0.04 g, 0.10 mmol) in water
(5 mL) was added dropwise to
a
solution of HL1 (0.03 g,
0.10 mmol) in methanol (5 mL). The reaction mixture was stirred
at room temperature for 48 h. The solvent was removed under re-
duced pressure at room temperature to half of the initial volume.
After 3 h the precipitated yellow solid was filtered off, washed with
methanol/water, 1:1 and dried under reduced pressure over CaCl2.
Yield 38.2 mg (68%); m.p. 315–317 °C. IR spectrum in CsI, se-
lected bands: ν = 3428 ν(OH); 1631 (C=O), 1593 (C=N), 1460
˜
(C=C), 420 (Pt–N), 384 (Pt–Cl) cm–1. 1H NMR (300 MHz, [D6]-
DMSO, 25 °C): δ = 2.71 (s, 3 H, CH3), 6.79–6.86 (m, 2 H), 7.22 (t,
1 H), 7.34 (t, 1 H), 7.82 (d, 1 H), 8.02 (t, 1 H), 8.42 (d, 1 H), 8.78
(d, 1 H) ppm. FAB-MS: m/z (%) = 563. C16H13Cl2N3O3Pt (561.26):
calcd. C 34.24, H 2.33, N 7.48; found C 34.43, H 2.16, N 7.46.
Pt Complex 3b: A solution of K2[PtCl4] (0.04 g, 0.10 mmol) in
DMF (10 mL) was added dropwise to a solution of HL1 (0.03 g,
0.10 mmol) in DMF (10 mL). The mixture was stirred at room tem-
perature for 24 h. The KCl formed was removed by filtration and
the diethyl ether was allowed to diffuse into a DMF solution of
the complex. After two weeks the yellow solid was filtered off,
washed with diethyl ether and dried in air. The single crystals suit-
able for X-ray analyses were obtained directly from the synthesis.
Yield 27.28 mg (45%); m.p. 251–253 °C. C18H20Cl2N4O3Pt
(606.34): calcd. C 35.65, H 3.32, N 9.24; found C 35.72, H 3.41, N
9.23.
X-ray Crystallographic Data: X-ray diffraction data for yellow and
red single crystals of 3b and 4c, respectively, were collected at 193 K
on a STOE IPDS diffractometer and at 120 K on a Nonius Kappa
CCD diffractometer. The unit cell parameters were determined
from all reflection data in θ ranges 2.26–28.08° and 2.71–24.73°.
11713 and 11440 intensities were collected using graphite-mono-
chromated Mo-Kα radiation. 4316 and 11440 independent reflec-
tions were measured in the ranges –9 Յ h Յ 9, –13 Յ k Յ 13, –18
Յ l Յ 18, and –51 Յ h Յ 51, –8 Յ k Յ 8, –24 Յ l Յ 24, respectively
for 3b and 4c. In the data-reduction step, intensities were corrected
for Lorentz and polarisation effect[62] for 3b and using Denzo-SMN
software[63] for 4c. The structure was solved by direct methods
using the SHELXS86[64] program and refined by full-matrix least-
squares calculation on F2 using SHELXL97.[65] Non-hydrogen
atoms were refined with anisotropic displacement parameters. The
H atoms were placed in calculated positions and allowed to ride.
The molecular geometry was calculated by PLATON.[66] The draw-
ings were made by PLATON and are presented in Figure 1. The
crystal data and details of data collection for both compounds are
given in Table 4.
Pd Complex 4a: A solution of K2[PdCl4] (0.09 g, 0.27 mmol) in
water (3 mL) was added dropwise to a stirred solution of HL1
(0.08 g, 0.27 mmol) in methanol (15 mL). A yellow solid formed
immediately; the suspension was stirred for a further 30 min, then
the solid was filtered off, washed with cold water, cold methanol
and diethyl ether, and dried in air. Yield 93.15 mg (73%); m.p. 284–
285 °C. IR spectrum in CsI, selected bands: ν = 3457 (OH); 1654
˜
(C=O), 1593 (C=N), 1475(C=C), 420 (Pd–N), 338 (Pd–Cl) cm–1.
1H NMR (300 MHz, [D6]DMSO, 25 °C): δ = 2.41 (s, 3 H, CH3),
6.77–6.87 (m, 2 H), 7.20 (t, 1 H), 7.34 (t, 1 H), 7.81 (d, 1 H), 8.02
(t, 1 H), 8.42 (d, 1 H), 8.73 (d, 1 H) ppm (Figure S1c). FAB-MS:
m/z (%) 473. C16H13Cl2N3O3Pd·H2O (490.626): calcd. C 39.16, H
3.08, N 8.56; found C 38.96, H 3.11, N 8.63.
CCDC-622068 (for 3b) and -622067 (for 4c) contain the supple-
mentary crystallographic data for this paper. These data can be
obtained free of charge from The Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Pd Complex 4b: A solution of K2[PdCl4] (0.03 g, 0.10 mmol) in
DMF (10 mL) was added dropwise to a solution of HL1 (0.03 g,
0.10 mmol) in DMF (10 mL). The mixture was stirred at room tem-
perature for 48 h. The KCl formed was removed by filtration and
the diethyl ether was allowed to diffuse into a DMF solution of
the complex. After two weeks the yellow solid was filtered off,
washed with diethyl ether and dried in air. Yield 27.3 mg (45%);
m.p. 253–254 °C. C18H20Cl2N4O3Pd (606.34) calcd. C 35.65, H
3.32, N 9.24; found C 35.72, H 3.41, N 9.23.
Biological Measurements
Determination of Alkylating Properties: The test compound
(0.005 mmol) was dissolved in 2-methoxyethanol (1 mL) and a
solution of 4-(4-nitrobenzyl)pyridine (NBP) in 2-methoxyethanol
(5% solution, 1 mL) was added. The sample was heated at
(100Ϯ0.5) °C for 1 h and then quickly cooled to 20 °C. 2-Methoxy-
ethanol (2.5 mL) and piperidine (0.5 mL) were added to the sample
to give a total volume of 5 mL. The final concentration of the test
compound was 1ϫ10–3 . After 90 s the absorbance was measured
at λ560 nm in a glass cell (1 cm). 2-Methoxyethanol was used as a
reference solvent.
Pd Complex 4c: A suspension of [Pd(C6H5CN)2Cl2] (0.10 g,
0.25 mmol) in chloroform (5 mL) was added to a stirred solution
of HL1 (0.074 g, 0.25 mmol) in methanol/chloroform (1:1) (5 mL).
The product precipitated immediately from the reaction mixture.
Stirring was continued for 30 min and the complex was filtered off,
washed with cold water, cold methanol and diethyl ether, and dried
in air. Recrystallisation from acetonitrile/diethyl ether (1:1) af-
forded a red crystalline product 4c. Yield 53.7 mg (45%); m.p.
276.4–278.0 °C. FAB-MS: m/z (%) 478. C18H15ClN4O3Pd
(477.198): calcd. C 45.30, H 3.17, N 11.74; found C 45.35, H 2.98,
N 11.83.
Cell and Cytotoxicity Assay: The cytotoxicity was determined in
the human leukaemia promyelocytic HL-60 and lymphoblastic
NALM-6 cell lines. Cells were cultured in RPMI 1640 medium sup-
plemented with 10% foetal calf serum and antibiotics (penicillin
(100 U/mL) and streptomycin (100 µg/mL) in 5% CO2/95% air. Ex-
ponentially growing cells were seeded at 3ϫ105 per well of a 24-
well plate (Nunc), and cells were then exposed to the compounds
for 48 h. Stock solutions were prepared freshly in DMSO, then
Potentiometric Measurements: The protonation constants of HL1
were determined by pH-metric titrations of five identical 10-mL dilutions from 10–3 to 10–7  in complete culture medium were
Eur. J. Inorg. Chem. 2007, 3728–3735
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
3733

E. Budzisz et al.
FULL PAPER
Table 4. Crystal data and details of data collection for 3b and 4c.
C16H12Cl2N3O3Pt·C2H6NH2 (3b)
C18H15ClN4PdO3 (4c)
Formula mass
Space group
606.37
P1
477.19
Pca21
¯
a [Å]
b [Å]
c [Å]
α
7.633(2)
10.431(2)
13.763(2)
105.90(2)
93.17(2)
110.98(2)
969.6(2)
2
44.112(9)
7.519(2)
21.226(4)
β
γ
V [Å3]
7040(3)
16
Z
λ [Å]
0.71073
2.077
0.50ϫ0.25ϫ0.06
7.539
0.0391
0.0808
0.71073
1.801
0.10ϫ0.08ϫ0.04
12.34
0.0699
0.1667
1.128
ρcalcd. [gcm–3]
Crystal size [mm3]
µ [cm–1]
[a]
R1
[b]
wR2
Gof[c]
0.981
[a] R1 = Σ||Fo| – |Fc||/Σ|Fo|. [b] wR2 = {Σ[w(Fo – Fc ) ]/Σ[w(Fo2)2]}1/2. [c] Gof = {Σ[w(Fo – Fc ) ]/(n – p)}1/2, where n is the number of
2
2 2
2
2 2
reflections and p is the total number of parameters refined.
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Acknowledgments
Financial support from the Medical University of Łódz´ (grant nos.
502-13-629 and 503-3066-2 to E. B. and 503-315-2 to the Depart-
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© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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3735