Synthesis, Characterization and Cytotoxicity Evaluation of New Biimidazole Palladium(II) Complexes with Thioureas

The reactions between [PdCl2(tmbiimH2)]·H2O (1) {tmbiimH2 = 2,2’-bis(4,5-dimethylimidazole)} and thiourea (tu), N-methylthiourea (mtu), N-phenylthiourea (ptu), N,N’-dimethylthiourea (dmtu) or N,N’-diphenylthiourea (dptu) in the 1:2 molar ratio resulted in the compounds [PdL2(tmbiimH2)]Cl2·nH2O {L = tu (2), mtu (3), ptu (4), dmtu (5) and dptu (6)}, which were characterized by elemental analyses, infrared (IR), and 1H NMR spectroscopies and conductivity measurements. The IR spectra of 1–6 were consistent with the presence of chelating tmbimH2 ligand. All compounds and cisplatin were tested in vitro by MTT assay for their cytotoxicity against three murine cancer cell lines: mammary adenocarcinoma (LM3), lung adenocarcinoma (LP07) and mouse fibroblast (L929) cells. Relating the series of compounds to their biological activities we found compound 6 as the most promising of them. Keyword: Palladium(II); 2,2’-bis(4,5-dimethylimidazole); Thioureas; Cytotoxicity; Cancer


Introduction
] In this context, numerous cisplatin analogues have been synthesized by modifying the nature of the leaving groups and the carrier ligands. 5Nevertheless, such derivatives generally have shown similar DNA-binding modes which often result in a similar spectrum of activity.Therefore, one conceivable strategy to achieve a spectrum of activity distinct to that of cisplatin involves the development of agents capable of producing cytotoxicity through new types of DNA interaction. 6t is well-established that metal-based molecules are able to interact non-covalently with DNA by means of a non-specific (mainly electrostatic) binding along the DNA exterior, a specific groove binding and intercalation. 7Particularly, much effort has been directed towards the design of square-planar complexes of the type [M(N-N)L 2 ] 2+ (M = Pd, Pt) incorporating chelating planar aromatic heterocycles with extended π-systems (N-N) such as 2,2'-bipyri-Franchi et al.: Synthesis, Characterization and Cytotoxicity Evaluation ... dine, 1,10-phenanthroline, and kinetically less labile ligands (L), e.g.][10] These compounds are relatively inert toward possible competitive covalent interactions and display the suitable shape for DNA intercalation.
Specifically, 2,2'-biimidazoles are N,N-donor ligands which can be used to obtain new metal complexes able to interact non-covalently to DNA as they can act as neutral bidentate ligands depending upon its protonation state. 11etal-based complexes containing neutral chelating 2,2'-biimidazole-type ligands have attracted considerable interest due to their externally-directed pair of N-H groups which are frequently involved in hydrogen bonding with solvent molecules and counterions 12 .Indumathy and co-workers 13 reported that the complexes [Co(N-N) 2 (H 2 biim)] 3+ (N-N = 2,2'-bipyridine, 1,10-phenanthroline) interact with DNA through the groove via hydrogen bonding due to presence of -NH in the ancillary ligand 2,2'-biimidazole.
Inspired by the ability of 4,5-dialkylimidazoles in extracting metal complexes into hydrophobic or hydrophilic solvents, 14 Stringfield and co-workers 15 have employed 2,2'-bis(4,5-dimethylimidazole), tmbiimH 2 , as a carrier ligand in order to facilitate transport of metal complexes across cell membranes.We assumed that the introduction of 2,2'-bis(4,5-dimethylimidazole) in the structure of Pd(II) complexes may improve the membrane penetration by increasing their lipid solubility and, consequently, resulting in an enhancement of the cytotoxicity.

1. Materials and Measurements
The syntheses were performed at room temperature.Commercial reagents and solvents were employed without further purification.The starting material Na 2 [PdCl 4 ] was prepared as previously described. 22lemental analyses (C, N and H) were performed on an EA1110-CHNS-O microanalyzer from CE-Instruments.Infrared spectra were recorded on a Nicolet Impact 400 spectrophotometer in the spectral range 4000-400 cm -l (KBr pellets).Conductivities were measured with a Digimed-DM-31 conductimeter using 1 × 10 -3 mol L -1 solutions in methanol. 1

3. Compounds
Test solutions of the compounds (1000 µmol L -1 ) were freshly prepared by dissolving the substances in 50 µL of DMSO and completing with 4950 µL of the culture medium.Afterwards, the tested compounds were diluted in a culture medium to reach the desired concentrations ranging from 10 to 300 µmol L -1 .The DMSO solvent did not reveal any cytotoxic activity in the tested concentrations.Cisplatin (commercial compound from Sigma) was employed as the standard antitumor drug.

3. 3. MTT Assay
For the cytotoxicity evaluation, 200.0 µL samples of L929, LM3 and LP07 cells (5×10 4 cell mL -1 , adjusted in MEM) were added to each well of a 96-well tissue culture plate and then preincubated in the absence of the compounds for 24 h to allow adaptation of the cells prior to the addition of the test agents.Afterwards, the supernatants were removed and 200.0 µL solutions of the compounds in concentrations ranging from 10 to 300 µmol L -1 or 200.0 µL of MEM-Complete as cell control of viability was added to each well.The effects of the compounds towards the cells were determined 24 h after the culture incubation.After that, the supernatants were removed and 100.0 µL solutions of [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], MTT, were added in each well containing the samples. 24The MTT assay was performed and the plates were incubated for 3 h.Then, the absorbances were measured and the cytotoxic midpoint value, i.e. the concentration of the chemical agent needed to reduce the spectrophotometric absorbance to 50%, was determined by linear regression analysis with 95% of confidence limits.The IC 50 was defined as the medium of three independent experiments through the equation of graphic line obtained (Microcal Origin 8.0 ™ ).Triplicates tests were performed for each concentration of each compound.

Results and Discussion
The precursor Na Franchi et al.: Synthesis, Characterization and Cytotoxicity Evaluation ... air-stable powders and exhibit a red brownish color.The molar conductivities of complexes 2-6 in methanol are between 185-214 Ω -1 cm 2 mol -1 and is in agreement with their 1:2 electrolytic character. 25The low solubility of 1 in methanol (and other non-coordinating solvents) has precluded measurements of its molar conductivity.Analytical results are in agreement with their proposed formulae (Table 1).

1. IR and NMR Studies
The neutral bidentate chelating coordination mode of tmbiimH 2 was clearly evidenced in the IR spectrum of 1. Firstly, the presence of an intense νN-H absorption at 3227 cm -1 is indicative of the neutral character of the imidazolyl ligand.According to the literature, the shift of νN-H absorption to higher energies in the IR spectrum of 1 compared to the position found in that of the free tm-bimH 2 (~3000 cm -1 ) is typical of neutral bidentate chelating coordination mode. 26Second, the decrease in intensity and shift to lower frequency of the band attributed to the νC=N and in-plane N-H bending mode (δN-H) in 1 (1594 cm -1 ), compared with that of the ligand (1604 cm -1 ), is also an evidence of the chelating coordination mode of tmbiimH 2 .The presence of water of hydration was detected by the appearance of its characteristic absorptions at 3485 cm -1 (νO-H) and 1660 cm -1 (δHOH).
IR spectra of compounds 2-6 exhibited a very broad continuum band over the spectral range of 3560-2500 cm -1 assigned to the vibrations of water molecules, counterions and coordinated ligands involved in hydrogen bonding interactions.It is important to point out that the expected νC=N band of the neutral bidentate chelating tmbiimH 2 ligand was observed in IR spectra of compounds 2 to 6.
][29][30] The shift of νCN and νCS absorptions is frequently used as diagnosis for S-coordination.Firstly, the intense νCN absorption at 1475 cm -1 (tu), 1556 cm -1 (mtu), 1463 cm -1 (ptu), 1560 cm -1 (dmtu) and 1326 cm -1 (dptu) observable in the IR spectra of the ligands, [30][31][32][33][34][35] decreased in intensity and shifted to 1504 cm -1 (2), 1576 cm -1 (3), 1448 cm -1 (4), 1593 cm -1 (5) after coordination.In 6, the νCN shift has small displacement.1][32][33][34][35] These spectroscopic modifications clearly indicated an increase of the double bond character of the CN bond and a weakening of the C=S bond, being consistent with S-bonding of thiourea-type ligands in 2-6. 36ccording to the literature, 15 one singlet at 2.19 ppm is observed in the 1 H NMR spectrum of the free tmbiimH 2 .The appearance of this single signal indicates that Me 4,4' and Me 5,5' must be magnetically equivalent, possibly due to the rapid migration of the nitrogen atom's protons. 37 1H NMR spectra of freshly prepared samples of 1, 2 and 3 showed the presence of one single peak at ca. 2.20 ppm (Table 2), which may indicate that the tmbiimH 2 is totally dissociated in DMSO-d 6 .This behavior has also been observed in other 2,2'-bisimidazolyl-based metal complexes in DMSO solutions. 37n the other hand, in the 1 H NMR spectrum of 4, two singlet resonances of equal integrated area were observed at 2.23 and 1.19 ppm and assigned to chemically inequivalent tmbiimH 2 ring methyl groups (Me 4,4' , Me 5,5' ), in agreement with the bidentate chelating coordination mode of tmbiimH 2 ligand.Over a period of time, these signals attributed to the mononuclear compound in solution decrease in intensity with the appearance and increase in intensity of one single peak at 2.19 ppm, suggesting that the dissociation rate of the tmbiimH 2 ligand in 4, in DM-SO-d 6 , is relatively slower that observed for 1-3.This finding could be probably related to the expected decrease of the rate of substitution reactions in square planar complexes due to the presence of more sterically demanding N-phenylthiourea ligands, increasing the difficulty encountered by the entering ligand in binding to the metal center during an associative substitution process. 38n 5, two signals groups were observed.The first group show one signal in 2.23 ppm, assigned to chemically equivalent tmbiimH 2 ring methyl groups (Me 4,4' , Me 5,5' ) for free ligand in solution and two singlet resonances of equal integrated area in 2.11 and 1.20 ppm, assigned to chemically inequivalent tmbiimH 2 ring methyl groups (Me 4,4' , Me 5,5' ) for coordinated ligand.The same manner as 4, over a period of time, the signals attributed to the mononuclear compound in solution (2.11 and 1.20 ppm) decrease in intensity with the appearance and increase in intensity of one single peak at 2.23 ppm.The second group shows one signal in 3.43 ppm and two signals in 3.67 and 3.65 ppm, assigned for methyl groups for coordinated N,N'-dimethylthiourea ligand. 39he 1 H NMR spectrum of 6 were obtained in DM-SO-d 6 solution and only one signal in 2.20 ppm assigned to chemically equivalent tmbiimH 2 ring methyl groups (Me 4,4' , Me 5,5' ) for free ligand in solution.
In short, when solubilized, all compounds showed a possible dynamic equilibrium between the partial exit of the coordinated ligands and the coordination of solvent molecules (such as H 2 O, present in the composition of the compounds themselves, or the deuterated solvent itself).Even when spectra were obtained immediately after solubilization and with times oscillating between 1 h and 48 h, these same behaviors were noticed, even when other deuterated solvents were used.However, due to the low resolution of the spectra obtained in other solvents, we chose to maintain the data presented in DMSO-d 6 , since the compounds were appreciably more soluble in this solvent, compared to the other deuterated solvents used.
Electronic delocalisation in a copper-(1-phenylthiourea) complex, 40 which has a thiourea-derivated ligand, as well as our compounds, also seems to corroborate us for a dynamics of exchange processes.

2. Cytotoxic Activities Against Murine Tumor Cell Lines
The cytotoxic activities of the palladium(II) complexes 1-6 were tested against murine mammary adenocarcinoma (LM3), lung adenocarcinoma (LP07) and mouse fibrosarcoma (L929) cell lines.Cells were exposed to a range of drug concentrations (300-10 µmol L -1 ) for 24 h and cell viability was analyzed by MTT assay.IC 50 values (the concentration that inhibited in 50% the cellular proliferation) are presented in Table 3.The cytotoxicity data of cisplatin against the selected tumor cell lines were used for comparison purposes. 16,41ompounds 1-6 showed no drug response at drug concentrations < 300 µmol L -1 against LP07 cells, and thus they were considered inactive.After treatment of LM3 cells with compounds 1-6, it was observed that the replacement of two chlorido by two thiourea (1 → 2), two N-methylthiourea ligands (1 → 3) or two N,N'-methylthiourea ligands (1 → 5) did not result in any increase in the cytotoxic activity towards LM3 cell line.On the other hand, 4, containing the sterically demanding N-phenylthiourea ligand, was ca. 2 fold more active than compounds 1-3 and 5, and approximately 4 times less active than cis-   With respect to the cytotoxic effects on L929 cells, a progressive increase on the cytotoxic activity of Pd(II) complexes was noticed according to the ancillary ligand bulkiness of substituents on thiourea moiety, following the order tu < mtu < dmtu < ptu < dptu.Probably, a lipophilic effect is prevalent for this series of compounds when H atoms are substituted by methyl and phenyl groups.Compound 6 not only showed the highest cytotoxic activity against L929 cell line (IC 50 value of 7.3 µmol L -1 ) among all tested compounds, but also it was more active than cisplatin (65.3 µmol L -1 ). 41ur findings agree well with those described by Marverti and co-workers, 8 in which it was verified that the cytotoxicity of the metallointercalators [Pt(bpy)L 2 ]Cl 2 (bpy = 2,2´-bipyridine; L = thioureas) was dependent on the structure of thiourea substituents.

Conclusions
The synthesis, structural and spectroscopic characterization, as well as the biological activity of palladium(II) compounds containing 2,2'-bis(4,5-dimethylimidazole) and thiourea-type ligands were described in this work.Conductivity data in methanol were in agreement with a 1:2 electrolyte nature for compounds 2-6.The IR data of 1-6 were consistent with the presence of chelating tmbiimH 2 ligand and S-coordination of thioureas.NMR studies on compounds 1-3 and 6 in DMSO-d 6 indicated that tmbiimH 2 ligand is completely dissociated.On contrary, the dissociation rate of the tmbiimH 2 in 4 and 5 is slower than that observed for 1-3.The distinct behavior of 4 in solution may be responsible for the maintenance of its structural integrity long enough to reach the pharmacological targets as well as for its highest cytotoxicity against LM3 and L929 cell lines, when compared to compounds 1-3.The substituent groups in thiourea-type ligands are directly related to the increase in citotoxicity.
The good cytotoxicity presented by compound 6 deserves considerable attention, which presents us the challenge of finding better conditions of stability for it in solution, either by drug delivery systems or structural modifications to fulfill with greater success its action in the pharmacological targets.

2 [ 1 :
Scheme 1: General representation for the synthesis of the complexes (water of hydration is omitted).

Table 1 .
Analytical and physicochemical data for the compounds 1-6.

Table 3 .
Cytotoxicity (IC 50 ) of the coordination compounds 1-6 and cisplatin against murine LM3, L929 and LP07 cell lines. .The compound 6 showed drug response at drug concentrations 8.9 µmol L -1 , approximately 3 times more active than cisplatin.In this case, the presence to more sterically ligand (N,N'-diphenylthiourea) increases cytotoxic activity against this cell line.