Palladium(II) complexes bearing thiosemicarbazone and phosphines as inhibitors of DNA-Topoisomerase II enzyme: Synthesis, characterizations and biological studies

Journal Pre-proofs Palladium(II) Complexes Bearing Thiosemicarbazone and Phosphines as inhibitors of DNA-Topoisomerase II enzyme: Synthesis, Characterizations and Biological Studies Mauro Almeida Lima, Vinicius A. Costa, Mariane Araújo Franco, Gabriela P. de Oliveira, Victor M. Deflon, Fillipe Vieira Rocha PII: DOI: Reference:

S1387-7003(19)30602-1 https://doi.org/10.1016/j.inoche.2019.107708 INOCHE 107708

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Inorganic Chemistry Communications

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9 July 2019 3 October 2019 27 November 2019

Please cite this article as: M. Almeida Lima, V.A. Costa, M. Araújo Franco, G.P. de Oliveira, V.M. Deflon, F. Vieira Rocha, Palladium(II) Complexes Bearing Thiosemicarbazone and Phosphines as inhibitors of DNATopoisomerase II enzyme: Synthesis, Characterizations and Biological Studies, Inorganic Chemistry Communications (2019), doi: https://doi.org/10.1016/j.inoche.2019.107708

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Palladium(II) Complexes Bearing Thiosemicarbazone and Phosphines as inhibitors of DNA-Topoisomerase II enzyme: Synthesis, Characterizations and Biological Studies. Mauro Almeida Lima1, Vinicius A. Costa1, Mariane Araújo Franco1, Gabriela P. de Oliveira1, Victor M. Deflon2, Fillipe Vieira Rocha1* 1

Departamento de Química, Universidade Federal de São Carlos, São Carlos, São Paulo CEP 13.565-905, Brazil Instituto de Química de São Carlos, Universidade de São Paulo, Avenida Trabalhador São-Carlense, 400, São Carlos, São Paulo CEP 13566-590, Brazil 2

*Corresponding Author: Fillipe V. Rocha E-Mail: [email protected]

Abstract Four new palladium(II) complexes bearing thiosemicarbazone and phosphines as ligands were synthesized and fully characterized by IR, NMR, UV-Vis spectroscopies, elemental analysis and X-ray diffraction. The metal complexes were tested towards five cell lines, including, tumor and non-tumor cells. Cytotoxicity of the palladium(II) complexes overcame cisplatin. Compounds did not interacted with the DNA. However, palladium(II) complexes showed an inhibition of action of the Topoisomerase II enzyme at lower concentrations than did etoposide, a standard drug. Keywords: palladium(II), topoisomerase II, cytotoxicity; spectroscopy; X-ray diffactoin The Topoisomerase IIα (Topo II) enzyme is one of the major pharmacological targets of the anticancer agents[1][2][3]. Topo II is present as an over expression in some kinds of cell lines [4]. Thiosemicarbazones (TSCs) have structural features that maximize the noncovalent interactions in the activity site of the enzyme[5][6]. In general, the coordination of the TSCs with a metal ion improves their biological activity[7][8][9]. Zeglis et al. synthesized several Cu(II) complexes bearing thiosemicarbazones. All of the complexes were more active than their free ligands against two tumor cell lines and in the topoisomerase II inhibition assay. The increased activity was attributed to the squareplanar geometry that was imposed by the metallic centers[10]. Other authors have found the same behavior[11][12][13]. The Pd(II) ion naturally assume a square-

planar geometry in their coordination compounds, besides, in the lats years, Pd(II) complexes have been studied for their anticancer activities, due to their similarity with the Pt(II) metal complexes [14][15][16]. The presence of chelated ligands in coordination sphere as TSCs has been showing encouraging results [17][18]. An incorporation of voluminous phosphines in metal compounds is a strategy that has been adopted by many researchers. [19][20][21][22]. The phosphine groups have been able to establish a more favorable interaction between the metal complexes and the hydrophobic pockets of some enzymes[23][24]. In this work, we synthesized, characterized and evaluated the cytotoxicity and interaction with biomolecules of four new Pd(II) complexes of the type [PdCl(TSC_CC)(P)] [TSC_CC = N-methyl-3-phenylprop-2-em-1ylidene)hydrazinecarbothioamide; P = triphenylphosphine (PPh3) (1); tri(ptolyl)phosphine (Pp-T) (2); tri(otolyl)phosphine (Po-T) (3); tris(4fluorophenyl)phosphine] (Pp-4F) (4). The molecular structure of the compounds 2 and 3 were determined by X-ray crystallography. The precursor [PdCl2(MeCN)2] reacted with equimolar amount of the TSC_CC and the appropriate phosphine ligand to afford 1-4 complexes. The complexes were air-stable powders and they were yellow to orange in color. The molar conductivities of all of the complexes in acetonitrile were between 16 and 35 µS mol-1 cm-2 (Supplementary material), which werein agreement with their nonelectrolytic nature [25]. The structures of 1-4 were confirmed by IR, UV-Vis, NMR and elemental analyzes

2 (Supplementary material). The most significant difference in the IR spectra between the TSC-CCs and the metal complexes was a shift to the lower frequencies of the band which was attributed to the C=S stretching of 60 cm-1. A characteristic bond of the P-C stretching was observed at 1095 cm-1, which confirmed the presence of the phosphine ligand (Table S5). In fact, only one signal in 31P NMR was observed around 28 ppm, due the phosphorus atom trans to a nitrogen atom (Figure S5). The 1H NMR data shown a downfield displacement to the olefinic hydrogen and N(1)H-, due to the withdrawal effects of the metal centers after complexation. Besides, signals in the range of 7.51-7.80 were observed in the complexes spectra attributable to the -phenyl groups of the phosphine ligands (Table S1). In the UV/Vis spectra, two bands common to all complexes were observed: the band centered between 300-350 nm corresponded to the π–π* intra-ligand electron transition; the band 350-420 nm was attributed to the combination of the S-Pd, Cl-Pd charge transfer and palladium(II) d-d transictions (Figure S6). Single crystals suitable for the X-ray diffraction studies were obtained for complexes 2 and 3 by a slow evaporation of the solvents of the saturated solution [acetonitrile:chloroform (1:1)]. The ORTEP representation with the atom labeling scheme is illustrated in Figure 1. The Pd(II) compounds adopts a distorted square-planar geometry, with interatomic bond angles that deviated slightly from 90°. The bond lengths and the angles werein the expected range [26] (Table S3 and Table S4). TSC_CC act as an anionic bidentate ligand, which led to a cis coordination through S1 and N2 atoms to form a five-membered ring. The remaining binding sites were occupied by a chloride ion that were coordinated trans to the S1 atom and with a phosphine ligand coordinated trans to the N2 atom[27]. In the structure of compound 3, it was possible to observe the presence of acetonitrile as a crystallization solvent.

Figure 1: The single crystal structure of complex 2 and complex 3.

Changes in the bond lengths of the TSC_CC ligand were observed after the coordination to the metal centers. There occurred a significant lengthening of the CS distances of 1.688 to 1.764 (2) and 1.760 (3)Å. Consequently, the bond of C(1)–N(2) became shortened [from 1.371 to 1.292 (2) and 1.299 (3), respectively]. These modifications were indicative of an electronic density redistribution on the ligand upon the coordination. In vitro cytotoxic activity of ligand TSC_CC, phosphines, PdCl2, [PdCl2(MeCN)2] and complexes 1–4 was determined towards three tumor cells (MDA-MB-231: human breast adenocarcinoma; A549: tumor lung cancer cells; DU-145: tumor prostate cancer cells). Additional, the compounds 1-4 were tested against two non-tumor cells (MRC-5 fetal lung fibroblast-like cells; PNT-2 non-tumor prostate cells). The corresponding IC50 values were compared to cisplatin (Figure 2). The thiosemicarbazone and phosphines ligands not showed drug response at concentrations < 50 µM in the tested cell lines. The same results were found for the PdCl2 salt and precursor complex [Pd(MeCN)2Cl2 (Table S6). Analysing the data in the Figure 2 and Table S7, excluding the results against A549 cell line, all the complexes were more cytotoxic than cisplatin[28][29][30].

Figure 2: IC50 values (µmolL-1) for the the complexes 1-4 and cisplatin.

3 The IC50 values showed a significant preference of the palladium (II) complexes to the breast cancer cells towards lung (A549) and prostate (DU145) cancer cells. Compounds 1-4 exhibited remarkable cytotoxicity against MDA-MB-231, exhibited values between 3.4 to 0.56 µM, on average, 20 fold more active than cisplatin. Further, a significant SI values were found comparing the cytotoxicity between the breast cancer cell line and the non-tumor cell lines, MRC5 and PNT-2. Highlighting, the complexes 1 and 4 with SI values bigger than 4.8 in both situations (Table S7). In fact, literature indicate a SI value greater than 3.0 as a promissory compound[31][32]. Typical titration curves for the complex 4 at a constant concentration (2.0x10-5 M) in the presence of different amounts of the DNA (0 – 30 M) were performed (Figure S8). The absorption spectra indicated that the complex did not interact with DNA by a strong pathway (intercalate or covalent-bond), once none significant changes were observed. The Kb value (1.7x102 M) presented by complex 4 corroborate with this hypothesis, due this value has been considerably lower than value found for the ethidium bromide (Kb = 1x106 M) [33]. The influence of complexes 1 and 4 on the structure of the supercoiled DNA was determined by their ability to modify the mobility of the pBR322 plasmid in a gel electrophoresis assay. It is well known that upon an interaction between a DNA structure and complexes, it produces different forms with a slower migration [34]. In the Figure 3 was possible to observed that under the presented experimental conditions, compounds 1 and 4 did not interact with the DNA structure. Only cisplatin cause a relaxation of the plasmidial DNA structure (line 2).

Figure 3: The plasmid incubation with complexes 1-4 and cisplatin after 24 h. Line 1: plasmid/DMSO (3.3%). Line 2: Cisplatin. Line 3: 1 (5 µM). Line 4: 1 (12.5 µM). Line 5: 2 (5 µM). Line 6: 2 (1.25µM). Line 7: 3 (5 µM). Line 8: 3 (12.5 µM).Line 9: 4 (5 µM). Line 10: 4 (12.5 µM).

Figure 4: The effects of complexes 1–4 on the activities of Topo II. Line 1: plasmid. Line 2: Topo II and plasmid. Line 3: 1 (5 µM). Line 4: 1 (12.5 µM). Line 5: 2 (5 µM). Line 6: 2 (1.25µM). Line 7: 3 (5 µM). Line 8: 3 (12.5 µM).Line 9: 4 (5 µM). Line 10: 4 (12.5 µM).

How the metal compounds showed a high activity towards the breast cancer cell line, Topo II became a potential target, due their overexpression in this kind of cancer cells. Topo II is recognize to produce a relaxation on the DNA structure that can observed in an electrophoresis assay. Therefore, in a enzymatic inhibition assay it is extremely important that the metal compounds not affect the mobility of supercoiled plasmid. Thus, any displacement in the Topo II assay will be essentially an interaction between the metal compounds and the enzyme. Inhibitory effect of the complexes 1-4 in the action of Topoisomerase II are showed in Figure 4. Even with the similarity between the compound structures, only complexes 1 and 4 showed a total inhibition of the Topo II enzyme at 12.5 µM. Interestingly the same compounds that presented the lower IC50 values against the breast cell line. These results indicate a possible relationship between the high cytotoxicity on MDA-MB-231 cells and enzymatic inhibition. However, more studies are necessary to confirm this relationship. Furthermore, the ability of compounds 1 and 4 to inhibit the relaxation of the DNA was three fold more potent than etoposide (35 μM), an usual Topo II inhibitor [35]. These results have suggested that the use of thiosemicarbazone and phosphine groups as ligands was an interesting strategy to synthesize new complexes with a good cytotoxicity and capacity to inhibit Topo II enzyme. Biological profile towards the five cell lines tested indicate a different mode of action between cisplatin and our complexes. Cisplatin shown better results in lung and

4 prostate cancer cell lines, Pd(II) compounds were more effective against breast cancer cells. It seems that a eletronic density withdrawal group in the aromatic ring of the phosphine ligand increase the cytotoxicity of this kind of palladium(II) compounds, due the best results presented by compound 4. Conflicts of Interest The authors declare that there are no conflicts of interest regarding the publication of this paper. Acknowledgments This work was sponsored by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2016/04201-9; FAPESP 2019/11242-1), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Finance Code 001. References [1] J. M. Fortune, N. Osheroff, Topoisomerase II as a target for anticancer drugs: Wen enzymes stop being nice, Progress in Nucleic Acid Research and Molecular Biology. 64 (2000) 221-253. https://doi.org/10.1016/S0079-6603(00)640060 [2] Y. Pommier, E. Leo, H. Zhang, C. Marchand, DNA Topoisomerase and their poisonin by anticancer and antibacterial drugs, Chemistry & Biology. 17 (2010) 421-433. https://doi.org/10.1016/j.chembiol.2010.04.01 2 [3] W. Chen, J. Qiu, Y. Shen, Topoisomerase Iiα, rather than Iiβ, is a promising target in development of anti-cancer drugs, Drug. Discov. Ther. 6 (2012) 230-237. https://doi.org/10.5582/ddt.2012.v6.5.230 [4] A. M. Fry, C.M. Chresta, S. M. Davies, M. C. Walker, A. L. Harris, J. A. Hartley, J. R. W. Masters, I. D. Hickson, Relationship between topoisomerase II level and chemosensitivity in human tumor cell lines, Cancer Res. 51 (1991) 6592-6595. [5] H. Huang, Q. Chen, X. Ku, L. Meng, L. Lin, X. Wang, C. Zhu, Y. Wang, Z. Chen, M. Li, H. Jiang, K. Chen, J. Ding, H. Liu, A series

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Highlights 

Synthesis and characterization of 4 new palladium(II) complexes;

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Crystal structure of 2 compounds;

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High cytotoxicity of all compounds;

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Compounds did not interact with DNA;

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Compounds inhibit the action of Human Topoisomerase IIα;

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Declaration

The authors declare that there are no conflicts of interest regarding the publication of this paper. Yours sincerely

Fillipe Vieira Rocha on behalf of all the authors of the article referred to above

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