Polyhedron 52 (2013) 1096–1102

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Polyhedron

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Synthesis, structure, spectroscopic studies and cytotoxic effect of novelpalladium(II) complexes with 2-formylpyridine-4-Nethyl-thiosemicarbazone:Potential antitumour agents

Dimitra Kovala-Demertzi a,⇑, Anastasia Galani a, John R. Miller b, Christopher S. Frampton b,Mavroudis A. Demertzis a

a Department of Chemistry, Inorganic and Analytical Chemistry, University of Ioannina, 45110 Ioannina, Greeceb Department of Biological and Chemical Sciences, University of Essex, X-Ray Crystallography, Colchester CO4 3SQ, UK

a r t i c l e i n f o a b s t r a c t

Article history:Available online 11 July 2012

Dedicated to Alfred Werner on the 100thAnniversary of his Nobel Prize in Chemistryin 1913.

Keywords:ThiosemicarbazonesPalladium complexesCrystal structuresSpectroscopic studiesAntiproliferative activity

0277-5387/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.poly.2012.06.068

⇑ Corresponding author. Tel.: +30 2651008425; faxE-mail address: dkovala@cc.uoi.gr (D. Kovala-Dem

New palladium(II) complexes have been synthesized by the reaction of Pd(II) with 2-formylpyridine-4-Nethyl-thiosemicarbazone, HFo4NEt, 1. The complexes [Pd(Fo4NEt)Cl], 2, [Pd(H2Fo4NEt)(Fo4NEt)Cl2], 3and [Pd(Fo4NEt)2], 4 have been characterised by elemental analyses and spectroscopic studies. The crys-tal structure of the complex [Pd(H2Fo4NEt)(Fo4NEt)Cl2], 3 and the protonated ligand [(H2Fo4NEt)Cl], 5have been solved by single-crystal X-ray diffraction. The cytotoxic activities of 1–4 have been evaluatedfor antiproliferative activity in vitro against the cells of three human cancer cell lines: MCF-7 (humanbreast cancer cell line), T24 (bladder cancer cell line), A-549 (non-small cell lung carcinoma) and a mouseL-929 (a fibroblast-like cell line cloned from strain L). The compound 3 display IC50 values in a lM rangebetter than that of the antitumor drug cis-platin against MCF-7 and T-24 cell lines and is considered asagent with potential antitumor activity candidates for further stages of screening in vitro and/or in vivo.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Thiosemicarbazones possess a wide range of biological activitydepending on the parent aldehyde or ketone. Heterocyclic thio-semicarbazones are important because of their possible beneficialbiological activity [1]. Tsc’s are among the most potent inhibitorsof ribonucleotide reductase, RR, enzyme. RR catalyses the synthesisof deoxyribonucleotides from their ribonucleotide precursors andas such is responsible for maintaining a balanced supply of thedeoxyribonucleotides required for DNA synthesis and repair.Strong positive correlation has been established between RR activ-ity and the rate of replication of cancer cells [2]. The chemistry oftransition metal complexes of thiosemicarbazone, Tsc’s, has beenreceiving considerable attention largely because their broad profileof pharmacological activity affords a diverse variety of compoundswith different activities [3–5]. Also, complexes of palladium(II)with Tsc’s were used for first time as effective catalysts for theHeck and Suzuki-Miyaura cross-coupling reactions [6,7].

The combination of Tsc’s with agents like platinum(II) or palla-dium(II) that damage DNA produces synergistic inhibition of tu-mour growth and may lead to improvements in the effectivenessof cancer chemotherapy regimens [8–10]. Cis-platin has for a long

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: +30 2651008791.ertzi).

time been of major significance in cancer therapy. The clinical suc-cess of cis-platin is limited by significant side effects and acquiredor intrinsic resistance. Therefore, much attention has focused ondesigning new platinum compounds with improved pharmacolog-ical properties and a broader range of antitumor activity. Severalderivatives of this parent drug have been tested clinically and sev-eral platinum complexes are currently in clinical trials. Strategiesfor developing new platinum anticancer agents include the incor-poration of carrier groups that can target tumor cells with highspecificity [11–15]. Also, of interest is to develop platinum com-plexes that bind to DNA in a fundamentally different manner thancis-platin in an attempt to overcome the resistance pathways. Thegoal of reducing toxic side effects while maintaining therapeuticalefficacy can be accomplished by improving the solubility of thecomplexes, by slowing down degradation processes throughshielding of the platinum with bulky ligands, and by increasingmembrane permeability with more lipophilic ligands. We havesynthesized metal complexes of Tsc’s, which are active antitumour,antimicrobial and antiviral agents. Synthetic, spectroscopic andbiological studies have been carried out in order to obtain informa-tion on structure–activity relationships for systems involvingpalladium(II), platinum(II) atoms.

The present work is an extension of previously studiedplatinum(II), palladium(II) and zinc(II) complexes of thiosemicar-bazones with potential interesting biological activities [10,16–18].

D. Kovala-Demertzi et al. / Polyhedron 52 (2013) 1096–1102 1097

The present paper includes synthesis and spectral characterisationof the newly prepared palladium(II) complexes with HFo4Nethyl.To our knowledge, this is the first report of a thiosemicarbazonemetal complex with a monodeprotonated and a monoprotonated li-gand. The cytotoxic activities of 1–4 have been evaluated for anti-proliferative activity in vitro against the cells of three humancancer cell lines: MCF-7 (human breast cancer cell line), T24 (blad-der cancer cell line), A-549 (non-small cell lung carcinoma) and amouse L-929 (a fibroblast-like cell line cloned from strain L).

2. Experimental

2.1. General and instrumental

All reagents were commercially available (Aldrich or Merck)and used as supplied. All solvents were of A.R. grade and were usedas received for the synthetic work. Melting points (m.p.) weredetermined in open capillaries and are uncorrected. Infrared spec-tra were recorded on a Perkin Elmer Spectrum GX Fourier trans-form spectrophotometer using KBr pellets (4000–400 cm�1) andNujol mulls dispersed between polyethylene disks (400–40 cm�1). UV–Vis spectra were acquired with a JASCO V-570 spec-trophotometer UV–Vis/NIR. The 1H NMR and 13C NMR spectrawere recorded on a Bruker AC-250 spectrometer operating at250.13 and 62.90 MHz for 1H and 13C acquisitions, respectively.The splitting of proton resonances in the reported 1H NMR spectrais defined as s = singlet, d = doublet, t = triplet, and m = multiplet.The chemical shifts are reported in ppm for 1H and 13C NMR. Sam-ples were dissolved in d6-dimethylsulfoxide and spectra were ob-tained at room temperature (298 K) with the signal of free d6-dimethylsulfoxide (at 2.50 ppm 1H NMR, 39.5 ppm 13C NMR) as areference. Elemental analyses were carried out by the micro ana-lytical service of the University of Ioannina, Greece.

2.2. Preparation of the ligand and palladium(II) complexes

2.2.1. Synthesis of the ligand HFo4NEtThe heterocyclic thiosemicarbazone, 2-formylpyridine-4N-

ethyl-thiosemicarbazone (1), HFo4NEt, was prepared as describedin the literature [19]. M.p.: 202–205 �C. IR (cm�1): 3283s, 3130m,m(NH); 2965ms; m(CH2), 1587s, 1538mw, m(C„N); 1058s, m(NN);877ms, m(C„S). UV–Vis for 1 (CH3CN) k/nm (e/L mol�1 cm�1) 317(12400), 234 (2740). 1H NMR (DMSO-d6), d (ppm): 8.52 (d, 1H,C(1)H); 7.32 (t, 1H, C(2)H); 7.80 (t, 1H, C(3)H); 7.80 (d, 1H, C(4)H);8.042 (s, 1H, C(4)H); 8.69 (s, 1H, NH); 3.55 (m, 2H, C(8)H2); 1.11 (t,3H, C(9)H3). 13C NMR, 150.2 (C1); 121.0 (C2); 142.9 (C3); 124.9(C4); 154.2 (C5); 137.3 (C6); 177.8 (C7); 41.2 (C8); 15.4 (C9).

2.2.2. Synthesis of [Pd(HFo4NEt)Cl] (2)To a solution of HFo4NEt (0.302 g, 1.3 mmol) in methanol

(10 mL) was added lithium tetrachloropalladate(II), Li2PdCl4, pre-pared in situ from PdCl2 and LiCl (PdCl2, 0.266 g, 1.5 mmol) inmethanol (15 mL). The reaction mixture was stirred for 24 h atRT. The orange powder was filtered off, washed with cold methanoland cold ether, recrystallised from methanol and dried in vacuumover silica gel for 1 h, at 90 �C. Yield: 30%. M.p.: 254 �C. Anal. Calc.for C9H11N4SPdCl (MW: 208.27): C, 31.0; H, 3.2; N, 16.0; S, 9.2.Found: 31.2; H, 3.3; N, 15.8; S, 9.0%. IR (cm�1): 3289s, m(NH);2978ms, m(CH2); 1578s, 1562mw, m(C„C), m(C„N); 1079ms,m(NN); 851s, m(C„S); 383mw, m(Pd–S); 340ms, m(Pd–Cl); 450ms,m(Pd–N); 259ms, m(PdNpy). UV–Vis for 2 (CH3CN) k/nm (e/L mol�1 cm�1) 459 (2180), 425 (2320), 371 (10400), 350 (10000).1H NMR (DMSO-d6), d (ppm): 8.50 (d, 1H, C(1)H); 7.63 (t, 1H,C(2)H); 8.19 (t, 1H, C(3)H); 7.79 (d, 1H, C(4)H); 8.02 (s, 1H,C(4)H); 3.38 (q, 2H, C(8)H2); 1.15 (t, 3H, C(9)H3). 13C NMR, 148.9

(C1); 126.0 (C2); 141.7 (C3); 126.4 (C4); 158.9 (C5); 147.9 (C6);179.8 (C7); 42.4 (C8); 15.2 (C9).

2.2.3. Synthesis of [Pd(Fo4NEt)(H2Fo4NEt)Cl2]�H2O (3)To a solution of HFo4NEt (0.743 g, 3.2 mmol) in methanol

(10 mL) was added lithium tetrachloropalladate(II), Li2PdCl4, pre-pared in situ from PdCl2 and LiCl (PdCl2, 0.266 g, 1.5 mmol) in meth-anol (15 mL). The pH of the solution was adjusted to 1.0–2.0 by theaddition of drops of 1 N HCl and the reaction mixture was stirred for24 h at room temperature and then left in the refrigerator for 1 day.The yellow-orange powder was recrystallised from methanol andwas filtered off, washed with cold methanol and ether and driedin vacuo over silica gel, they were redried at 70 �C in vacuo overP4O10. D.p. 208 �C. Yield: 30%. Elemental analyses were consistentwith the stoichiometry C18H24N8S2PdCl2H2O (MW: 611.85): Calc.:C, 35.3; H, 4.3; N, 18.3; S, 10.5. Found: C, 35.0; H, 4.3; N, 18.4; S,10.3%. IR (cm�1): 3289s, 3208m, m(NH); 2977m, m(CH2); 1576s,1544mw, m(C„N); 1094ms, m(NN); 851s, m(C„S); 397mw, 386ms,m(Pd–S); 445ms, m(Pd–N); 257ms, m(PdNpy). UV–Vis for 3 (CH3CN)k/nm (e/L mol�1 cm�1) 460 (1940), 367 (27200), 343sh (33400).1H NMR (DMSO-d6), d (ppm): 8.67 (d, 1H, C(1)H); 7.55 (t, 1H,C(2)H); 7.69 (t, 1H, C(3)H); 8.44 (d, 1H, C(4)H); 8.16, 7.94 (s, 1H,C(6)H); 3.24, 3.41 (m, 2H, C(8)H2); 1.13, 1.01(t, 3H, C(9)H3). 13CNMR, 147.8 (C1); 129.3 (C2); 141.7 (C3); 126.6 (C4); 148.9 (C5);147.8 (C6); 177.8 (C7); 41.4 (C8); 15.3 (C9). Slow crystallisation of3 from a fresh solution of MeOH/MeCN yielded pale yellow crystalsof [H2Fo4NEt)Cl] (5) and wine red crystals of [Pd(Fo4NEt)(H2Fo4-NEt)Cl2]�H2O (3). The crystals were selected after manual separa-tion and the X-ray structure of them was solved.

2.2.4. Synthesis of [Pd(HFo4NEt)2] (4)The complex was prepared from aqueous solution of K2PdCl4

and methanolic solution of HFo4NEt in pH 8 following the proce-dure described in the literature [20]. Yield: 50%. D.p.: 165 �C. Ele-mental analyses are consistent with C18H22N8S2Pd: Calc.: C, 41.5;H, 4.2; N, 21.5; S, 12.3. Found: C, 41.8; H, 4.3; N, 21.3; S, 12.1%.IR (cm�1): 3284vs, 3124m, m(NH), 2966m, m(CH2), 1587s,1538mw, m(C„N); 1090ms, m(NN); 769s, 749m m(C„S); 383m,375ms m(Pd–S); 436m, m(Pd–N); 285ms m(PdNpy). UV–Vis for 4(CH3CN) k/nm (e/L mol�1 cm�1) 467 (1140), 369 (8200), 315(24000). 1H NMR (CHD3): 8.65 (d, 1H, C(1)H); 7.67 (t, 1H, C(2)H);8.49 (t, 1H, C(3)H); 7.79 (d, 1H, C(4)H); 8.20 (s, 1H, C(4)H); 3.56(m, 2H, C(8)H2); 1.11 (t, 3H, C(14)H3). 13C NMR, 150.2 (C1); 121.1(C2); 137.3 (C3); 124.9 (C4); 154.2 (C5); 142.9 (C6); 177.8 (C7);41.9 (C8); 15.4 (C9).

2.3. X-ray crystallography

Crystals of yellow 3 and wine-red 5 were obtained by slowevaporation of a fresh MeOH–MeCN solution of 3 after manual sep-aration. Intensity data were collected at 123 K on a Bruker SMART1 K CCD diffractometer equipped with an Oxford CryosystemsCryostream cooler. Data reduction were performed with the pro-gram SAINT V4.05. Area detector scaling and absorption correctionswere performed by SADABS. Lorentz and polarisation effects werecorrected. All non-hydrogen atoms were refined anisotropicallyon F by full matrix least squares. All calculations were performedwith SHELXTL [21,22]. All non-hydrogen atoms were anisotropic.During the structure determination and refinement of 5 all thehydrogen atoms were found in Fourier difference syntheses andit proved possible to refine their geometric and (isotropic) thermalparameters. During the refinement process all aromatic and N-at-tached hydrogen atoms for 3 were detected in Fourier differencesyntheses; ultimately those attached to nitrogen atoms were re-fined isotropically whilst those attached to carbon atoms wereplaced in riding positions. In the later refinement stages of 3 the

Table 2Experimental bond distances (Å) and angles for 5 and 3.

5 3

S–C(7) 1.6786(14) Pd–N(2) 1.974(4)N(1)–C(1) 1.3371(19) Pd–N(1) 2.074(4)N(1)–C(5) 1.3597(18) Pd–S(1) 2.2551(13)N(2)–C(6) 1.2831(18) Pd–S(2) 2.3242(14)N(2)–N(3) 1.3536(16) S(1)–C(7) 1.771(5)N(3)–C(7) 1.3749(18) N(1)–C(1) 1.329(6)N(4)–C(7) 1.3257(18) N(1)–C(5) 1.369(6)N(4)–C(8) 1.4623(19) N(2)–C(6) 1.284(6)

N(2)–N(3) 1.376(5)N(3)–C(7) 1.316(6)N(4)–C(7) 1.331(6)S(2)–C(16) 1.724(5)N(5)–C(14) 1.352(6)N(6)–C(15) 1.274(6)N(6)–N(7) 1.364(6)

C(1)–N(1)–C(5) 122.39(13) N(7)–C(16) 1.357(6)C(6)–N(2)–N(3) 117.69(12) N(8)–C(16) 1.306(6)N(2)–N(3)–C(7) 119.89(11)C(7)–N(4)–C(8) 123.09(12) N(2)–Pd–N(1) 80.71(15)N(1)–C(1)–C(2) 120.66(15) N(2)–Pd–S(1) 84.13(11)N(4)–C(7)–N(3) 117.09(12) N(1)–Pd–S(1) 164.83(12)N(4)–C(7)–S 125.41(11) N(2)–Pd–S(2) 171.62(12)N(3)–C(7)–S 117.49(10) N(1)–Pd–S(2) 98.66(12)N(4)–C(8)–C(9) 114.07(13) S(1)–Pd–S(2) 96.29(5)

1098 D. Kovala-Demertzi et al. / Polyhedron 52 (2013) 1096–1102

Fourier difference synthesis showed a peak in lattice space, distantfrom any heavy atom. As it was stronger than the peaks round thepalladium, it was assigned to solvent of crystallisation, modelled asan oxygen atom of fractional site occupation and labelled O(1) inthe data and figures. The formula was adjusted accordingly. TheO(1) site is 2.649(2) Å from N(3) and may be held to it by hydro-gen-bonding. Molecular graphics were performed from PLATON2001[23]. A summary of the crystallographic data is collected in Table 1,and the important bond distances and angles are collected inTable 2.

2.4. Antiproliferative assay in vitro

2.4.1. CompoundsTest solutions of the compounds tested (1 mg/mL) were pre-

pared by dissolving the substance in 100 ll of DMSO completedwith 900 ll of tissue culture medium. Afterwards, the tested com-pounds were diluted in culture medium to reach the final concen-trations of 100, 40, 10, 1, and 0.1 ng/ll. The solvent (DMSO) in thehighest concentration used in test did not reveal any cytotoxicactivity.

2.4.2. CellsThe established in vitro human cancer cell line MCF-7 (human

breast cancer cell line), T24 (bladder cancer cell line) and a mouseL-929 (a fibroblast-like cell line cloned from strain L) were applied.The cell lines are maintained in the Cell Culture Collection of theUniversity of Ioannina. Twenty-four hours before addition of thetested agents, the cells were plated in 96-well plates at a densityof 104 cells per well. The T-24 and MCF-7 cells were cultured inthe D-MEM (modified Eagle’s medium) medium supplementedwith 1% antibiotic and 10% foetal calf serum. L-929 cells weregrown in Hepes-buffered RPMI 1640 medium supplemented with10% foetal calf serum, penicillin (50 U/mL) and streptomycin

Table 1Crystallographic data and structure refinement results for compounds 5 and 3.

5 3

Empirical formula C9H13N4S1Cl1 C18H24N8O0.19PdS2Cl2

Formula weight 244.74 596.87Crystal system orthorhombic monoclinicSpace group Pbca P2(1)/nUnit cell dimensionsa (Å) 7.0881(11) 7.5367(15)b (Å) 13.526(2) 24.697(5)c (Å) 23.965(4) 12.793(3)a (�) 90 90b (�) 90 97.938(4)c (�) 90 90Volume (Å3) 2297.7(6) 2358.5(8)Z, calculated density

(mg m�3)8, 1.415 4, 1.681

Absorption coefficient(mm�1)

0.487 1.215

F(000) 1024 1206Crystal size (mm) 0.10 � 0.10 � 0.35 0.10 � 0.10 � 0.35Theta range for data

collection (�)1.70–26.37 1.65–26.37

Limiting indices �8 6 h 6 8,�16 6 k 6 16,�29 6 l 6 29

�9 6 h 6 9,�30 6 k 6 30,�15 6 l 6 15

Reflections collected/unique

18396/2351[Rint = 0.0312]

20061/4828[Rint = 0.0652]

Data/restraints/parameters 2351/0/188 828/0/303Goodness-of-fit on F2 1.070 0.936Final R indices [I > 2r(I)] R1 = 0.0302,

wR2 = 0.0808R1 = 0.0459,wR2 = 0.1008

Largest diff. peak and hole(e �3)

0.290 and �0.357 1.740 and �1.478

(50 mg/mL). A-549 cells were grown in F-12 K Ham’s medium sup-plemented with 1% glutamine, 1% antibiotic/antimycotic, 2% NaH-CO3 and 10% foetal calf serum. The cell cultures were maintained at37 �C in humid atmosphere saturated with 5% CO2. Cell numberwas counted by the Trypan blue dye exclusion method. MCF-7,L-929 and A-549 cells were determined by the sulforhodamine Bassay [24], while T24 cells by the MTT assay [25]. The in vitro testswere performed as described previously [26,27].

3. Results and discussion

3.1. Synthetic aspects

HFo4NEt, 1, was synthesized by the condensation of formyl pyr-idine, ethyl-isothiocyanate and hydrazine. The Pd(II) complex[PdCl(Fo4NEt)], 2 was prepared by reacting lithium tetrachloropal-ladate, Li2PdCl4, prepared in situ from PdCl2 and LiCl with the 1 inMeOH solution in a molar ratio of 1:1. It has been found that pal-ladium(II) promotes deprotonation of thiosemicarbazones in thepresence of methanolic solutions. The reaction of Li2PdCl4 with 1in methanolic solution and an apparent pH value of 1–2 gave thecomplex [Pd(H2Fo4NEt)(Fo4NEt)Cl2], 3. One solvated water mole-cule was detected in the crystal structure of 3. The reaction ofK2PdCl4 with HFo4NEt in 1:2 molar ratio in aqueous/methanolicsolution and an apparent pH value of 8.5 gave the complex[Pd(Fo4NEt)2], 4, without solvated molecules. Slow crystallisationof 3 from a fresh solution of MeOH/MeCN yielded wine red crystalsof 3 and yellow crystals of [H2Fo4NEt]Cl, 5. Probably a partialtransformation of [Pd(H2Fo4NEt)(Fo4NEt)Cl2], 3 takes place withthe time in MeOH/MeCN solution according to the reaction.

½PdðH2Fo4NEtÞðFo4NEtÞCl2� ! ½PdClðFo4NEtÞ� ð2Þþ ½H2Fo4NEt�Cl ð5Þ

The formula of these Pd(II) complexes was confirmed by ele-mental analysis, spectroscopic studies and for 3 and 5 by X-rayanalysis. The stoichiometries of the complexes indicate that Pd(II)is coordinated by the singly charged anion in 2 and 4. The com-plexes of Pd(II) were prepared according to the reactions (1)–(3),in methanolic or aqueous solutions in the pH range of 1–8,Scheme 1.

D. Kovala-Demertzi et al. / Polyhedron 52 (2013) 1096–1102 1099

Li2PdCl4 þHFo4NEt���!CH3OH ½PdClðFo4NEtÞ� þ 2Cl� þHCl ð1Þ

Li2PdCl4 þ 2HFo4NEt����!CH3OH ½PdðH2Fo4NEtÞðFo4NEtÞCl2� þ 2Cl� ð2Þ

K2PdCl4 þ 2HFo4NEtþ 2NH4OH����������!CH3OH==H2 O=pH 8½PdðFo4NEtÞ2� þ 2KCl

þ 2NH4Clþ 2H2O ð3Þ

3.2. Structural characterisation

2-Formylpyridine-4Nethylthiosemicarbazone chloride [H2Fo4Nethyl]Cl, 5 consists of a cation, [C9H13N4S]+, combined with achloride anion. The molecular structure is depicted in Fig. 1 and se-lected geometrical parameters are shown in Table 1. Protonation ofthe cation [H2Fo4Nethyl]+ occurs at the pyridyl nitrogen, N(1), sothat N(1), N(3) and N(4) are protonated. The cation is co-planar ex-cept for the ethyl group, C(8)–C(9), confirming the high degree ofconjugation shown by the bond lengths, bond angles and torsionangles. The largest deviation from the least-squares plane is0.209(1) Å for the sulfur atom. The cation has the configurationZEEEZ about the sequence of bonds C(5)–C(6)–N(2)–N(3)–C(7)–N(4). The chloride ion is bifurcated and forms three hydrogenbonds to three protonated nitrogen atoms and lies only0.292(2) Å from the least-squares plane mentioned above. Two ofthe three hydrogen bonds are with N(1) and N(4) of the cation.The bite angle at Cl(1) is 113.5(3)�. The third hydrogen bond isfrom N(3) of a second cation related to the first one by a b-glide.The Cl(1) atom participates in inter and intra hydrogen bondsand a four-membered ring Cl(1)–HN(1)–N(2)–HN(4) is formed.All three protonated nitrogen atoms are thus involved in hydro-gen-bonding to a chloride ion, and the structure thereby buildsinto a zig-zag chain of alternate cations and anions and a supramo-lecular assembly, Fig. 2.

An ORTEP diagram of the molecular structure of [Pd(H2Fo4NEt)(Fo4NEt)Cl2] is shown in Fig. 3. Complex 3 exhibits one monoprot-onated and one protonated ligand. The palladium atom is in asquare planar environment, being attached to two cis-sulfur andtwo cis-nitrogen atoms. One of the thiosemicarbazone ligands istridentate (N,N,S), in the deprotonated, formally anionic, form.The other ligand is monodentate, bound to the palladium by sulfur(S2). The tridentate ligand deviates slight from planarity. Thedisplacement from coplanarity is indicated by the dihedral anglebetween the pyridyl ring and the plane defined by the five-mem-bered chelate ring Pd–S–C–N–N, and between the pyridyl ringand the plane defined by Pd–N–C–C–N, 1.8(2)� and 2.0(2)�, respec-tively. The maximum deviation from the least-squares of the tri-

Scheme 1. The reaction scheme for synthesis of 1–4.

dentate ligand framework atoms is 0.193(2) Å for S(2). Thedihedral angle between the two ligand least squares planes is68.70(4)�, arising from torsions along Pd–S(2) and S(2)–C(16).The tridentate ligand shows a Z, E, Z configuration about the bondsC(1)–C(5), C(6)–N(2) and N(3)–C(8) for the donor centres nitrogen,nitrogen and sulfur, respectively, while the monodentate oneshows an E, E, Z configuration for the same atoms. This monoden-tate ligand is remarkable in being geometrically very similar to thecation in 5, formally in a positively charged state, carrying protonsat N(5), N(7) and N(8). For these reasons the complex is formulatedas [Pd(H2L)(L)]2+[Cl]2

�. The two Pd–S distances in 3 are different2.255(2) for the tridentate and 2.324(2) Å for the monodentateprotonated ligand. The stronger coordination of the metal to theazomethine nitrogen compared to the pyridyl nitrogen is attrib-uted to its higher basicity. The Pd–N bond distances are compara-ble to those found in other square planar palladium(II) complexes[16–18]. The negative charge of the deprotonated ligand is delocal-ized over the thiosemicarbazonatomoiety, and the S–C bond dis-tances are consistent with increased single-bond charactersimilar to the complexes of other metal ions. The azomethine C–N distances also indicate a reduction in double bond characterfor the tridentate ligand, while both thioamide C–N distances indi-cate increased double bond character. The chloride ions are held inthe lattice by hydrogen bonding to nitrogen atoms. The chlorideions Cl(1) and Cl(2) are bifurcated and three hydrogen bonds areformed. Cl(2) is hydrogen-bonded to N(7), to N(4) of the proton-ated ligand and to C(3) of the deprotonated ligand in a neighbour-ing molecule. It is positioned on the z-axis of the co-ordinationplane round palladium, distant 3.597(1) Å from the metal atom.This distance is too long to be considered as a bond but it is never-theless close enough for there to be some interaction with the me-tal d2

z orbital. Cl(1) is also bifurcated and forms three hydrogenbonds bonded to N(5) and N(8) of the protonated thiosemicarba-zone in a four-membered chelate ring system and to C(6) of themonodeprotonated ligand in a neighbouring molecule. A peak inlattice space, distant from any heavy atom, was found. As it wasstronger than the peaks round the palladium, it was assigned tosolvent of crystallisation, modelled as an oxygen atom of fractionalsite occupation and labelled O(1) in the data and the formula wasadjusted accordingly. The O(1) site is 2.649(2) Å from N(3) andmay be held to it by hydrogen-bonding. There is a weak interactionbetween the metal atom and the N(7), and its attached hydrogen,H(7N), the metal–hydrogen distance being 3.04 Å. The orientationof the monodentate ligand seems to be determining by the minimi-sation of steric interactions at the out-of-plane position of the me-tal as well as by possible weak long-range interactions with themetal. It might be possible that there is a weak ‘agostic’ bondbetween Pd and H(7N) in 3 [28]. The crystal packing is determinedby inter and intra molecular bonds and leads to aggregation and toa supramolecular structure (Fig. 4 and Table 3).

Fig. 1. Perspective view of [H2Fo4Nethyl]Cl, 5 showing the atomic numberingscheme.

Fig. 2. A view of the extended network of [H2Fo4Nethyl]Cl, 5 along the a axis.

Fig. 3. Perspective view of [Pd(H2Fo4NEt)(Fo4NEt)Cl2], 3 showing the atomicnumbering scheme.

Fig. 4. A view of the extended network of of [Pd(H2Fo4NEt)(Fo4NEt)Cl2], 3 along thea axis.

Table 3Inter- and intra-molecular hydrogen bonds for 3 and 5. D and A refers to donor andacceptor, respectively.

D H A D� � �A H� � �A D–H� � �A

Inter- and intra-molecular hydrogen bonds for 5N(1) H(1N) N(2) 2.7011 2.38 102N(4) H(4N) N(2) 2.6568 2.28 109N(1)i H(1N) Cl 3.0581 2.24 1570

N(3)ii H(3N) Cl 3.2223 2.38 165N(4)i H(4N) Cl 3.2998 2.54 1560

C(9)iii H(9B) S 3.7558 2.81 160

Inter- and intra-molecular hydrogen bonds for 3N(5) H(5N) N(6) 2.6761 2.32 101N(8) H(8N) N(6) 2.6159 2.23 106N(7) H(7N) Cl(2) 3.0158 2.16 164C(15) H(15) Cl(2) 3.5047 2.75 137C(3)iv H(3) Cl(2) 3.7111 2.77 169N(4)v H(4N) Cl(2) 3.1624 2.34 178N(5)vi H(5N) Cl(1) 3.0115 2.11 156N(8)vi H(8N) Cl(1) 3.1059 2.29 153C(6)vii H(6) Cl(1) 3.5112 2.73 140

Symmetry transformations: i, 1 � x, 1/2 + y, 1/2 � z; ii, �1/2 + x, y, 1/2 � z; iii, 1/2 � x, 1/2 + y, z; iv, 1/2 + x, 1/2 � y, �1/2 + z; v, �1 + x, y, z; vi, 1 � x, �y, 1 � z; vii,�1/2 + x, 1/2 � y, �1/2 + z.

1100 D. Kovala-Demertzi et al. / Polyhedron 52 (2013) 1096–1102

3.3. Spectroscopy

3.3.1. Infrared spectroscopyThe thioamide band, which contains considerable m(CS) charac-

ter, was less intense in the complexes and found at a lower fre-quency, suggesting coordination of the metal through sulfur for2–4. Coordination of the thiolato S-atom was further indicated bythe presence of a band at ca. 370–390 cm�1 assignable to m(Pd–S). The coordination of the azomethine N-atom to the Pd(II) centrewas suggested in the IR spectra of complexes 2–4, relative to thoseof the free ligands, by a shift of the m(C@N) band to lower frequen-cies, along with the occurrence of a m(N–N) band to higher fre-quency. Furthermore, the presence of a band at ca. 450–460 cm�1

was assigned to m(Pd–N). The band at ca. 260–280 cm�1 was as-signed to m(Pd–Npy) for 2–4 [16–18].

3.3.2. UV–Vis absorption spectraThe electronic spectra of complexes 2–4 were indicative of

square-planar geometries. In the visible region, three spin allowed(d–d) transitions can be expected. However, strong charge-transfer(CT) transitions interfered preventing the observation of all thesepredicted bands. The very intense band at about 460 nm is assign-able to a combination of sulfur ? Pd(II), nitrogen (pyridyl) ? Pd(II)charge transfer (L(p) ? MCT) and Pd(II) d–d bands. The strongband at about 370 nm is assignable to a combination of metal–li-gand charge transfer (M ? LCT) and d–d bands [16–18,26,27].The electronic absorption spectra of 1–4 in (DMF and CH3CN solu-tion) exhibit bands at ca 310 nm and are due to n ? p⁄ transitionsand are associated with the azomethine functions of the thiosem-icarbazone moieties [26,27].

3.4. Pharmacology antiproliferative activity in vitro

Pt(II) complexes of 2-formyl and 2-acetylpyridine-4Nethyl-thio-semicarbazones, HFo4Net and HAc4Net, showed cytotoxicity andwere found to be able to overcome the cisplatin resistance ofA2780/Cp8 cells [16]. Pt(II) or Pd(II) complexes with 2-acetylpyri-dine-4Nethyl-thiosemicarbazone, HAc4Et were tested in a panel

Table 4The in vitro activity of 1 and its Pd(II) complexes and related Zn(II), Pd(II) and Pt(II)complexes [17,18,26,27] (expressed as IC50 (lM) against MCF-7, T-24, L-929 and A-549 cancer cell lines).

Compounds MCF-7 T-24 L-929 A-549

HFoTscb <0.55 2.55 inactiveHAcTscb 0.55 2.06 2.16[HFo4Nethyl] 1a 4.88 10.3 43.1 27.7HAc4CyclHexyld 0.36 10.10 3.31 11.10HFo4Nhexime 2.23 74.32 2.67 27.31HAc4Nhexime 5.02 11.80 3.04 6.55HFo4Npypipec 2.24 3.77 7.96 1.75HAc4Npypipec 2.70 2.64 7.84 16.6[ZnCl2(HFoTsc)]�H2Ob 3.29 1.73 inactive[ZnCl2(HAcTsc)] b 1.36 1.06 1.57[ZnCl2(Fo4Npypipe)] c <0.21 9.79 1.60 10.6[ZnCl2(Ac4Npypipe)] c 2.20 0.96 19.90 8.08[Pd(Fo4Nethyl)Cl] 2a 104.1 72.5 69.3 216.7[Pd(H2Fo4Nethyl)(Fo4Nethyl)Cl2]

3a8.42 5.88 8.33 21.6

[Pd(Fo4Nethyl)2] 4a 14.52 7.59 4.72 13.6PdCl(Fo4Npypipe)] c 83.9 4.89 214.01 12.8[PdCl(Ac4Npypipe)] c 3.75 5.2 160.05 113.0[PtCl(Ac4CyclHexyl)] d 0.63 9.17 6.87 6.06[Pt(Ac4CyclHexyl)]2

d <0.13 0.37 0.21 1.17[Pt(Fo4hexim)Cl] e 20.30 19.83 37.70 89.30[Pt(Ac4hexim)Cl] e 25.70 12.83 77.10 6.92ZnCl2�1.5H2O 605.30 >605.30 734.05 281.55K2PdCl4 306.05 >306.05 >306.05 >306.05Cis-platin 8.00 41.7 0.70 1.53

a This work.b Ref. [26], where HFoTsc and HAcTsc are 2-formyl and 2-acetylpyridinethiose-

micarbazones, respectively.c Ref. [17], where HFo4Npypipe and HAc4Npypipe are 2-formyl and 2-acety-

lpyridineN(4)-1-(2-pyridyl)-piperazinyl thiosemicarbazone, respectively.d Ref. [27], where HAc4CyclHexyl is 2-acetylpyridine-4-cyclohexyl-

thiosemicarbazone.e Ref. [18], where HFo4Nhexim and HAc4Nhexim are 2-formyl and 2-acetyl

pyridine 4N-hexamethyleneiminyl.

D. Kovala-Demertzi et al. / Polyhedron 52 (2013) 1096–1102 1101

of human tumor cell lines of different origins (breast, colon, andovary cancers), and cisplatin-refractory/resistant cell lines andwere found to exhibit very remarkable growth inhibitory activitieswith mean IC50 values of 0.9–0.5 nM and support the hypothesisthat both [Pt(Ac4Et)2] and [Pd(Ac4Et)2] complexes can be charac-terised by cellular pharmacological properties distinctly differentfrom those of cisplatin [10]. Also, Zn(II) complexes of 2-formyl and2-acetylpyridinethiosemicarbazones, HFoTsc and HAcTsc, respec-tively, were found active against the MCF-7 (human breast cancercell line), T24 (bladder cancer cell line) and a mouse L-929 (a fibro-blast-like cell line cloned from strain L) [26]. The acute toxicity andantitumor activity were evaluated on leukaemia P388-bearing micefor the complexes of Pt(II) with 2-formyl and 2-acetylpyridine-4N-hexamethyleneiminyl, HFo4Nhexim and HAc4Nhexim, respec-tively. The complex [Pt(Fo4Nhexim)Cl] was found to afford five tosix cures against leukaemia P388. The in vivo results of the antitu-mor activity show the two platinum complexes as very effectivechemotherapeutic antileukaemic agents [18].

Compounds 1–4 were tested for their antiproliferative activityin vitro against the cells of three human cancer cell lines: MCF-7(human breast cancer cell line), T24 (bladder cancer cell line), A-549 (non-small cell lung carcinoma) and a mouse fibroblast L-929 cell line. The results of cytotoxic activity in vitro are expressedas IC50 – the concentration of compound (in lM) that inhibits aproliferation rate of the tumor cells by 50% as compared to controluntreated cells, Table 4.

The ligand 1 is in the same lM range compared to cis-platinagainst four cell lines, less cytotoxic against L-929 and A-549 celllines and more cytotoxic against MCF-7 and T-24 cancer cell lines.Complex 2 and also ZnCl2 and K2PdCl4 exhibit very poor cytotoxicactivity in all these four lines. The palladium complex 3 is less

cytotoxic against A-549 and L-929 cell lines and in the same lMrange compared to cis-platin. The IC50 values for 3 against MCF-7and T-24 cell lines are 8.42 and 5.88 lM, respectively, and againstA-549 and L-929 cell lines are 8.33 and 21.6 lM, respectively.

Complex 3 is seven times more active than cisplatin against T-24 cell line. IC50 values for 4 against MCF-7 and T-24 cell lines are14.52 and 7.59 lM, respectively, and against A-549 and L-929 celllines are 4.72 and 13.6 lM, respectively. Complex 4 is 5.4 timesmore active than cis-platin against T-24 cell line. Complex 3 is moreactive of all complexes against MCF-7 and T-24 cancer cell lines.Selectivity was exhibited from the palladium(II) complexes 3 and4, which were found active against MCF-7 and T-24 cancer celllines. The mentioned evident differences in the antiproliferativeaction of the ligand and its palladium(II) complexes indicate thatthe Pd(II) complexes really exist under the condition of the biolog-ical tests [29].

From Table 4, we can see that the highest activity is presentedby the two zinc(II) complexes with bulky substituents. The thio-semicarbazones HFoTsc and HAcTsc and the Zinc(II) complexeswith bulky substituents exhibit highest selectivity and activityagainst MCF-7 and T-24 cancer cell lines.

4. Conclusions

We have described the synthesis, spectral and structural charac-terisation of new palladium(II) complexes [Pd(Fo4NEt)Cl], 2,[Pd(H2Fo4NEt)(Fo4NEt)Cl2], 3 and [Pd(Fo4NEt)2], 4 with 2-formyl-pyridine-4-Nethyl-thiosemicarbazone, HFo4NEt, 1. The thiosemi-carbazone can bind to Pd(II) as tridentate N,N,S-donors at 2–4and as monodentate S-donor at 3 and 4 palladium(II) complexes.Partial transformation of 3 takes place with the time in MeOH/MeCN solution and gives 3 and the protonated ligand [(H2Fo4-NEt)Cl], 5. The formula of these palladium(II) complexes 2–4 wereconfirmed by elemental analysis, spectroscopic studies and byX-ray analysis. The crystal structure of 3 and 5 were determinedby X-ray crystallography. Complex 3 exhibits the palladium atomin a square planar environment being attached to two cis-sulfurand two cis-nitrogen atoms. One of the thiosemicarbazone ligandsis tridentate (N,N,S), in the mono-deprotonated form and the otherligand is monodentate, bound to the palladium by sulfur (S2), in themono-protonated form. The chloride ions Cl(1) and Cl(2) are bifur-cated and three hydrogen bonds are formed. The crystal packing isdetermined by inter and intra molecular bonds and leads to aggre-gation and to a supramolecular structure. Compound 5 consists of acation, [C9H13N4S]+, combined with a chloride anion, Cl(1). The Cl(1)atom participates in inter and intra hydrogen bonds and the struc-ture thereby builds into a zig-zag chain of alternate cations and an-ions. Selectivity was observed for complexes 3 and 4, which werefound especially active against MCF-7 and T-24 cancer cell lines.The most active of all was found the palladium complex 3 complex.Interestingly enough, complexes 3 and 4 were found to be more po-tent cytotoxic agent against cancer cells T-24 than the prevalentbenchmark metallodrug, cis-platin, under the same experimentalconditions measured by us. Compound 3 is considered as agentwith potential antitumor activity, and can therefore be candidatesfor further stages of screening in vitro and/or in vivo.

Acknowledgement

We thank the NMR center of the University of Ioannina.

Appendix A. Supplementary data

CCDC 293437 and 293438 contain the supplementary crystallo-graphic data for 5 and 3, respectively. These data can be obtained

1102 D. Kovala-Demertzi et al. / Polyhedron 52 (2013) 1096–1102

free of charge via http://www.ccdc.cam.ac.uk/conts/retriev-ing.html, or from the Cambridge Crystallographic Data Centre, 12Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033;or e-mail: deposit@ccdc.cam.ac.uk.

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