Structure / Activity of PtII / N , N-Disubstituted-N ’-acylthiourea Complexes : Anti-Tumor and Anti-Mycobacterium tuberculosis Activities

The syntheses, characterization, cytotoxicity against tumor cells and anti-Mycobacterium tuberculosis activity assays of Pt/PPh3/N,N-disubstituted-N’-acylthioureas complexes with general formulae [Pt(PPh3)2(L)]PF6, PPh3 = triphenylphosphine; L = N,N-disubstituted-N’-acylthiourea, are here reported. The complexes were characterized by elemental analysis, molar conductivity, infrared (IR), nuclear magnetic resonance (NMR) (H, C{H} and P{H}) spectroscopy. The P{H} NMR data are consistent with the presence of two PPh3 ligands cis to each other position, and one N,N-disubstituted-N’-acylthiourea coordinated to the metal through O and S, in a chelate form. The structures of the complexes were determined by X-ray crystallography, forming distorted square-planar structures. The complexes were tested in human cell lines carcinomas and also screened with respect to their anti-Mycobacterium tuberculosis activity (H37RvATCC 27294). It was found that complexes with N,N-disubstituted-N’-acylthiourea containing open and small chains as R2 groups show higher cytotoxic and higher anti-Mycobacterium tuberculosis activity than those containing rings in this position.


Introduction
Among the most effective agents for the treatment of cancer, there are some metallodrugs based on platinum(II).3][4][5][6][7][8][9] Thus, in an attempt to overcome the drawbacks of cisplatin and derivatives (severe toxicity, drug resistance and poor oral bioavailability), the development of platinum-based drugs have progressed to the newest generation of drugs, such as satraplatin, picoplatin and the multinuclear platinum complex BBR3464 (triplatin).In this context, platinum(II) complexes of the type [Pt(L)Cl(DMSO)] (L = acylthiourea ligand, R1-C(O)NHC(S)NR2; R' = aryl, NR2 = amine; DMSO = dimethylsulfoxide) were prepared by Sacht et al. [10][11][12] for their biological and chemical evaluation.The acylthiourea group, after deprotonation of the amide moiety (NHCO), can act as a bidentate chelating ligand, coordinating to platinum through the oxygen and sulfur donor atoms.
][15] In previous works, [16][17][18][19][20] some of us synthesized and determined the structures of some ligands related to those here described, and their corresponding complexes with Co II , Cu II , Ni II , Pd II and Pt II were also studied, which contain a thiourea derivative as a bidentate ligand, in different environments.In the present work, we studied the syntheses, characterization, cytotoxicity and anti-Mycobacterium tuberculosis activity of new platinum(II) complexes containing PPh 3 and N,N-disubstituted-N'-acylthioureas as ligands.The N,N-disubstituted-N'-acylthioureas used as ligands were synthesized by the procedure previously reported. 21Scheme 1 shows the pathway for the synthesis of the Pt II complexes, which were obtained by reacting methanolic solutions of acylthioureas with the precursor, dichloro-bis(triphenylphosphine)platinum(II).
The complexes were obtained by a nucleophilic substitution reaction of the two chlorido ligands from the precursor [PtCl 2 (PPh 3 ) 2 ], by the acylthiourea ligands.For the formation of the platinum(II) complexes the loss of the hydrogen atom of the acylthioureido group of the ligands occurs (see Scheme 1). 16ytotoxic studies realized on DU-145 (human prostate tumor cells) and MDA-MB-231 (human breast tumor cells) tumor cell lines have shown that certain palladium(II) complexes with the acylthiourea ligands exhibit cytotoxicity with antiproliferative effects being dependent on the nature or the type of the substituent at the acylthiourea ligand. 20Recently there have been efforts to design non-classical platinum-acylthiourea complexes, due to their antifungal activity and inhibitory activities against viruses.Thus, here we investigate the cytotoxicity of the complexes against MDA-MB-231 and DU-145 tumor cells, and their anti-Mycobacterium tuberculosis activity, with the aim of evaluating a possible influence of R1 and R2 in the cytotoxicity and in the anti-mycobacterial activity of the complexes. 8In this work three series of N,N-disubstituted-N'-acylthioureas were synthesized where R1 = phenyl, furoyl group or thiophenyl group.

Material and measurements
The dichloro-bis(triphenylphosphine)platinum(II) complex was obtained from Sigma.All reagents were purchased with reagent grade and used without further purification.Solvents were dried and used freshly distilled, unless otherwise specifically indicated.Thin layer chromatography (TLC) was performed on 0.25 mm silica gel pre-coated plastic sheets (40/80mm) (Polygram_SIL G/ UV254, Macherey& Nagel, Düren, Germany) using Caution benzene/methanol (9:1) as eluent.
The infrared (IR) spectra of the compounds were recorded on a Fourier transform infrared (FTIR) Bomem-Michelson 102 spectrometer in the 4000-200 cm −1 region using CsI pellets.Conductivity values were obtained using 1.0 mM solutions of complexes in CH 2 Cl 2 , using a Meter Lab CDM2300 instrument. 1 H, 31 P{ 1 H} and 13 C{ 1 H} nuclear magnetic resonance (NMR) were recorded on a Bruker DRX 400 MHz, internally referenced to tetramethylsilane (TMS), chemical shift (d), multiplicity (m), spin-spin coupling constant (J), integral (I).CDCl 3 was used as a solvent unless mentioned.The

Syntheses of N,N-disubstituted-N'-acylthioureas
The N,N-disubstituted-N'-acylthiourea ligands L (1-12)  used in this work were synthesized by the procedure previously reported, and the identity of the products was confirmed by comparing their 1 H and 13 C{ 1 H} NMR data with those reported in the literature. 21The groups R1 and R2 of the ligands are shown in Figure 1.

Synthesis of the complexes
The complexes were obtained as previously described for similar Pd II complexes, from direct reactions of the precursors, [PtCl 2 (PPh 3 ) 2 ], with the N,N-disubstituted-N'-acylthioureas, in methanol solutions. 20The complexes were separated from the reaction mixtures as white crystalline solids.Filtration and further washing with hot water and hot hexane were enough to afford pure compounds, in about 80% yields.Thus, the general procedure for the syntheses of the complexes is described: a solution of [PtCl 2 (PPh 3 ) 2 ] 1580 mg (2 mmol) in 5 mL of methanol was added dropwise to a solution of the corresponding N,N-dialkyl-N'-acylthiourea (2 mmol), dissolved in 30 mL of the same solvent, and 368 mg (2 mmol) of KPF 6 .The reaction was heated under magnetic stirring at 80 °C, for 2 h.The reaction mixture was left in the refrigerator overnight.The white solids obtained were filtered off and washed, successively, with hot water and hot hexane (3 × 20 mL).The obtained compounds are stable in DMSO solutions for at least five days, as it was showed by 31 P{ 1 H} NMR experiments.After this time the spectra of the complexes were the same, when compared with those recorded using fresh solutions.
The 1 H, 13 C{ 1 H} and 31 P{ 1 H} NMR data, the elemental analyses, melting point temperature (mp) and molar conductivity (Λ m , 1.0 × 10 −3 M in CH 2 Cl 2 ) for the complexes (1-12) are listed below and the other data used for the characterization of the complexes are in the text (the multiplicity of signals in the 13 C{ 1 H} NMR due the coupling C-P).

Crystal structure determination
Single crystals suitable for X-ray diffraction were obtained by slow evaporation of CHCl 3 :n-hexane (3:1) solutions of some of the complexes (3, 6, 9 and 10).Diffraction data were collected on an Enraf-Nonius Kappa-CCD diffractometer with graphite-monochromated MoKα radiation (λ = 0.71073 Å).The final unit cell parameters were based on all reflections.Data collections were performed using the COLLECT program; 22 integration and scaling of the reflections were performed with the HKL Denzo-Scalepack system of programs. 23Absorption corrections were carried out using the Gaussian method. 24The structures were solved by direct methods with SHELXS-97. 25The models were refined by full-matrix least-squares on F 2 by means of SHELXL-97. 26The projection views of the structures were prepared using ORTEP-3 for Windows. 27Hydrogen atoms were stereochemically positioned and refined with the riding model.Data collections and experimental details are summarized in Table 1.Relevant interatomic bond lengths and angles are listed in Table 2.

Cell culture assay
In vitro cytotoxicity assays on cultured human tumor cell lines still represent the standard method for the initial screening of antitumor agents.Thus, as a first step in assessing the pharmacological properties of the new platinum(II) complexes, they were assayed against human breast tumor cell line MDA-MB-231 (ATCC: HTB-26), human prostate tumor cells DU-145 (ATCC: HTB-81) and against the L929 non-tumor cell line (ATCC: CCL 1).The cells MDA-MB-231 and L929 were routinely maintained in Dulbecco's modified Eagle's medium  28 MTT solution (0.5 mg mL -1 ) was added to the cell cultures and incubated for 3 h, after which 100 μL of isopropanol was added to dissolve the precipitated formazan crystals.The conversion of MTT to formazan by metabolically viable cells was monitored in an automated microplate reader at 540 nm.The percentage of cell viability was calculated by dividing the average absorbance of the cells treated with the test compounds by that of the control; then cell viability percentage was plotted against drug concentration (logarithmic scale) to determine the IC 50 (drug concentration at which 50% of the cells are viable in relation to the control), with the error estimated from the average of 3 trials.

Anti-mycobacterial activity assay
The anti-Mycobacterium tuberculosis activity of the compounds was determined by the REMA (resazurin microtiter assay) method. 29Stock solutions of the tested compounds were prepared in DMSO and diluted in Middlebrook 7H9 broth (Difco) supplemented with oleic acid, albumin, dextrose and catalase (OADC), performed by Precision XS (Biotek ® ) to obtain the final drug concentration range of 0.09-25 μg mL −1 .Isoniazid was dissolved in distilled water and rifampicin in DMSO, and both were used as standard drugs.A suspension of MTB H37Rv ATCC 27294 was cultured in Middlebrook 7H9 broth supplemented with OADC and 0.05% Tween 80.The cultures were frozen at −80 °C in aliquots.After two days the CFU per mL (colony formation unit per mL) of an aliquot was determined.The concentrations were adjusted by 5 × 10 5 CFU per mL and 100 μL of the inoculum were added to each well of a 96-well microplate together with 100 μL of the compounds.Samples were set up in triplicate.The plates were incubated for 7 days at 37 °C.Resazurin (solubilized in water) was added (30 μL of 0.01%).The fluorescence of the wells was read after 24 h with a Cytation 3 (Biotek ® ).The MIC (minimum inhibitory concentration) was defined as the lowest concentration resulting in 90% inhibition of MTB growth.
The data from the infrared spectra shown in the Experimental section, suggest the formation of [Pt(PPh 3 ) 2 (Ln)]PF 6 complexes (1-12), where L is an anionic ligand, formed by the deprotonation of the N,N-disubstituted-N'-acylthiourea during its coordination to the platinum.Thus, typical NH stretching vibrations in the IR spectra of the free ligands in the range of 3050-3260 cm −1 as broad and strong absorptions, disappears after their coordinations to the metal, while the ν(C-N) bands, present in the complexes at 1574-1586 cm −1 , are absent in the free ligands, suggesting the formation of heterocyclics, according to Scheme 1. Also, comparing the band of the C=O group present in the free ligands with the infrared spectra of the complexes, there is a decreasing of the νC-O stretching vibration frequency, which is in agreement with the literature. 30Thus, it is reasonable to assign the bands in the range 1415-1523 cm −1 to the coordinated C-O group, since the ν(C=O) bands in the free ligands are at about 1680 cm −1 .The absorptions at 815-878 cm −1 in the spectra of the free bases, N,N-disubstituted-N'-acylthioureas, attributed to the ν(C-S) stretching vibrations, shift to the 745-769 cm −1 range in the complexes spectra.The infrared spectra show 3015-3019 νC-H (PPh 3 ), 842-845 ν(P-F), 694-697 νP-Ph (Ph 3 -P-Ph 3 ), 531-552 ν(Pt-P).6][37] Thus, from the IR data it is possible to suggest that the N,N-disubstituted-N'-acylthioureas are attached to the metal through the oxygen and sulfur, in a chelating form.
The type of the N,N-dialkyl-acylthiourea ligands used in this work are reported in the literature, 38 and it was found that for a series of hydrophilic N,N-dialkyl-N-aroylthioureas, the acid dissociation constants, pK a (NH) have been found to range from 7.5 to 10.9 in water-dioxane mixtures.
In the complexes (1-12) there are two coordinated triphenylphosphine ligands, in cis position, according to the 31 P{ 1 H} NMR data, and their molar conductivities show that the complexes are cationic species. 39he NMR ( 1 H and 13 C{ 1 H}) spectroscopy was used to speculate about the molecular structures of the complexes, and a comparative analysis on the basis of the spectroscopic data corresponding to both, free and coordinated ligands with the metallic ion, was performed.The 1 H NMR data for the complexes (1-12) are given in the Experimental section.The 1 H NMR spectra of the free ligands show basically three sets of well-separated signals corresponding to their R1, R2 substituents and to the NH proton.The signals of the NH protons appear as a broad singlet in the region between 8.55 and 8.80 ppm, 21 and after the coordination of the ligands to the metal, these signals disappear. 40,41he aromatic and the nitrogen substituents protons in the coordinated ligands were slightly downfield shifted when compared to the chemical shift of the free ligands.The aromatic protons appear as a complex pattern in the region d 8.17-5.74ppm (given in Figure 2 and Figures S1-S8 of the Supplementary Information) in comparison to similar compounds previously reported in the literature.Interestingly, heteronuclear multiple bond coherence (HMBC) NMR experiments showed that the chemical shifts of the carbon bonded to the sulfur atom in the coordinated ligands are at higher field than the carbon bonded to the oxygen atom, unlike the observed for the free ligands, where carbon from the C=O group is at higher field than the carbon bonded to the sulfur atom (see Figure 3). 21is, probably, is due to the delocalization of the electron density towards the sulfur atom after the deprotonation of the secondary amide of the ligand.
The precursor [PtCl 2 (PPh 3 ) 2 ], in CH 2 Cl 2 solution, shows a singlet peak for phosphorous atoms, at 13.72 ppm, in its 31 P{ 1 H} NMR spectrum, therefore the complexes here reported show two doublets, at about 21.08 and 9.83 ppm, indicating the presence of two magnetically different phosphorus atoms coordinated to the platinum(II) ion, and the four satellite signals of the platinum(II).Thus, in this case it is possible to assign the more shielded chemical shift to the phosphorus atom trans to the sulfur, since this atom is a better donor than the oxygen, and more likely the chloride ligand.The multiple signals of the PF 6 − is in the region -(138-158) ppm (Figure 4 and Figures S12-S22 of the Supplementary Information) in comparison to similar compounds previously reported in the literature. 43he structures of complexes 3, 6, 8 and 10, were determined by X-ray diffraction analysis.Their ORTEP views are in Figure 5 and the selected bond lengths (Å) and angles (°) for the complexes are listed in Table 2.The X-ray structures of the complexes confirmed that the N,N-disubstituted-N'-acylthioureas are coordinated to the central ion Pt II as bidentate ligands, by the oxygen and sulfur atoms, and there are two PPh 3 ligands, which are also in the cis fashion, as previously suggested by IR spectroscopy.In all complexes, the Pt II ion is nearly planar, in a fourfold environment.For the complexes synthesized in this work, the C-S bond distance is 1.734 Å (average), longer than the C-S bond distance of neutral species (1.661-1.676Å). 16,21,44,45 For free N,N-disubstituted-N'-acylthioureas, the C-O bonds lengths of 1.214-1.215Å indicate double-bond character, whereas the C-N bonds, as single one, are at about 1.373-1.412Å. 44,45 As a result of acylthiourea coordination to the metal, the bond lengths present significant C-S and C-O lengthening and C-N shortening (see Figure 5, Table 2), which is an evidence of resonance effect, as above mentioned in the 13 C{ 1 H} NMR experiment discussion, as a consequence of the deprotonation of the nitrogen atom N-H, which is between the carbonyl and thiocarbonyl groups.
The distances for the C-S, C-N and C-O bonds in the chelate rings, listed in Table 2, are the characteristic of single and double bond lengths, respectively. 45able 3 lists the ligand and complex concentrations that produce 50% of growth inhibition (IC 50 , μmol L -1 ) against DU-145 (human prostate tumor cells), MDA-MB-231 (human breast tumor cells) and against the L929 cell line (mouse healthy cell line).The new platinum(II) complexes, the free ligands, and the precursor [PtCl 2 (PPh 3 ) 2 ] were tested against the tumor cells and L929 cells.For comparison, the cytotoxicity of cisplatin and of [PtCl 2 (PPh 3 ) 2 ] was also evaluated, under the same experimental conditions.The IC 50 values, calculated from the dose-survival curves generated by the MTT assays obtained after drug treatment, are shown in Table 3.As can be seen from Table 3, for all compounds, the IC 50 are very high against the L929 (non-tumor cell line), indicative of selectivity towards tumor cells.
Overall, the complexes are more active against the MDA-MB 231 tumor cells, and the most promising complexes are 1 (N,N-dimethyl-N'-benzoylthiourea), 2 (N,N-diethyl-N'-benzoylthiourea), 6 (N,N-diethyl-N'-furoylthiourea), and 10 (N,N-dimethyl-N'-thiophenylthiourea).It is interesting to observe that the ligands in these complexes are those of open chain as R2 groups when compared with other complexes.Thus, it may be that is the steric factor that better define the cytotoxicity of the complexes, mainly against the MDA-MB-231 tumor cells line.Thus, the size of the R2 group in the ligands plays a important role in the activity of the complexes.In this case, probably, the low volume of these complexes can facilitate their entrance in the cell, making them more active than the other ones.
The compounds were also investigated for their in vitro anti-mycobacterial activity against Mycobacterial tuberculosis H37Rv strains by the MABA methodology.The MICs found for the platinum complexes, free ligands and ethambutol are shown in Table 4.
Also, as can be seen from these data, the complexes 1, 2, 6 and 10 present lower values of MICs supporting the above correlation of structure/activity for these complexes.Work is ongoing in our laboratory, with new N,N-disubstituted-N'-acylthioureas, to check this hypothesis.
According to the anti-mycobacterial activity assays, compounds 1 and 10 exhibited promising activity, with MIC values of 2.81 and 2.78 μM, respectively.The results indicate that the complex 1 and 10, which have methyl groups (R2 groups), have stronger in vitro activity than that of ethambutol (MIC 5.62 μM), which is clinically used as a firstline drug in several schemes of conventional tuberculosis treatment.Complexes 2 and 3 (MIC of 8.13 and 7.87 μM, respectively) were also active, but to a lesser extent.Thus,  MIC values for free ligands (25 μg mL -1 ) were several times higher than those observed for the respective complexes.

Conclusions
A novel series of Pt II complexes with N,N-disubstituted-N'-acylthiourea as bidentate ligands was here synthesized and characterized.The X-ray crystallographic characterization of the Pt II complexes with N,N-disubstituted-N'-acylthioureas as bidentate ligands shows that these ligands coordinate with the metal through the oxygen and sulfur atoms.In this work, there are three series of complexes: for the first one, the complexes 1-5, R1 is the phenyl group; for the second it is the furoyl group (6-9); and for the other one, it is the thiophenyl group (10-12).In general, the complexes present better results for MDA-MB-231 tumor cells than for DU-145 tumor cells, and in this case the most promising complexes are those for which R2 groups are open and have small chains, suggesting that this is due to their low steric hindrance, which allows the complex penetrate easier to the cells, acting more effectively.Additionally, antimicrobial activity assays of the new complexes provided evidence that the complexes 1 and 10 are potential agents against mycobacterial infections, specifically against M. tuberculosis H37Rv.

Figure 1 .
Figure 1.Structures of R1 and R2 for the N,N-disubstituted-N'-acylthioureas used as ligands in this work.
31 P{ 1 H} shifts are reported in relation to H 3 PO 4 , 85%.2D heteronuclear single quantum coherence (HSQC) NMR experiments were performed in order to unequivocally assign the C=O and C=S signals of the complexes.Partial elemental analyses were carried out by the Department of Chemistry of the Federal University of São Carlos, in an instrument of CHNS staff EA 1108 of the FISONS.

Table 1 .
Crystal data and refinement parameters for complexes 3

Table 2 .
Selected bond lengths and angles for the complexes 3, 6, 8 and 10

Table 4 .
MIC values of anti-mycobacterial activity of platinum complexes and reference drug a All ligands Ln present MIC > 25 μg mL -1 .