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Orthopalladated acetophenone oxime compounds bearing thioamides as ligands: Synthesis, structure and cytotoxic evaluation
Inorganica Chimica Acta 486 (2019) 617–624
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Inorganica Chimica Acta journal homepage: www.elsevier.com/locate/ica
Research paper
Orthopalladated acetophenone oxime compounds bearing thioamides as ligands: Synthesis, structure and cytotoxic evaluation
T
Ronan F.F. de Souzaa, , Gislaine A. da Cunhaa, José C.M. Pereiraa, Daniel M. Garciab, Claudia Bincolettob, Renata N. Gotoc, Andréia M. Leopoldinoc, Isabel C. da Silvad, Fernando R. Pavand, Victor M. Deflone, Eduardo T. de Almeidaf, Antônio E. Mauroa, ⁎ Adelino V.G. Nettoa, ⁎
a
UNESP – Univ Estadual Paulista, Instituto de Química, Departamento de Química Geral e Inorgânica, 14800-060 Araraquara, SP, Brazil UNIFESP – Univ Federal de São Paulo, Escola Paulista de Medicina, Departamento de Farmacologia, 04044-020 São Paulo, SP, Brazil USP – Univ de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, 14040-903 Ribeirão Preto, SP, Brazil d UNESP – Univ Estadual Paulista, Faculdade de Ciências Farmacêuticas, 14800-903 Araraquara, SP, Brazil e USP – Univ de São Paulo, Instituto de Química de São Carlos, 13560-970 São Carlos, SP, Brazil f UNIFAL – Univ Federal de Alfenas, Instituto de Química, 37130-001 Alfenas, MG, Brazil b c
ARTICLE INFO
ABSTRACT
Keywords: Cyclopalladated complex Oximes Cytotoxicity Thioamides
Cleavage reactions involving the halide-bridged [Pd(μ-Cl)(C2,N-aphox)]2 precursor (aphox = acetophenone oxime) with thioamides, in the 1:2 molar ratio, yielded mononuclear cyclopalladated of the type [Pd(C2,Naphox)(Cl)(L) {L = thiourea (1); N-methylthiourea (2); N,N’-dimethylthiourea (3); N-phenylthiourea (4); N,N’diphenylthiourea (5); thioacetamide (6) and benzothioamide (7)} which were characterized by elemental analyses, infrared and 1H- and 13C{1H}-NMR spectroscopies. The crystal and molecular structures of 1, 3 and 6 were determined by single-crystal X-ray diffraction studies. The cytotoxicity of the cyclopalladated compounds has been evaluated against a panel of murine {breast (4T1) and melanoma (B16F10-Nex2)} and human {melanoma (A2058, SK-Mel-110 and SK-Mel-05), oral squamous cell carcinoma (Cal27), hepatocellular carcinoma (HepG2)} cancer cell lines. All studied compounds were cytotoxic for the seven cancer cell lines studied and in general, most cyclopalladated compounds obtained in this work were more active than cisplatin to the seven tumor cell lines evaluated.
1. Introduction The bioinorganic and medicinal chemistry of palladium(II) compounds has experienced spectacular advances in the past years since many of them have been considered as alternative candidates to the classical platinum anticancer agents [1–6]. The structural and thermodynamic analogy between Pd(II) and Pt(II) is rather limited. Although they both share a d8 configuration, their reactivity is quite different, being palladium complexes more labile than platinum, with an exchange rate about 105 times higher [6]. Ligand dissociation in aqueous medium gives very reactive Pd(II) species, which can be toxic and ineffective with respect to Pt(II) analogues because they can be easily inactivated before reaching the cellular targets. For these reasons an accurate choice of the ligands on the Pd(II) centre has to be made in order to achieve enough kinetic stability for being a good drug in therapy [4]. ⁎
Within this context, N-donor ligands such as benzylamines and imines have been extensively employed in cyclopalladation reactions, affording chelated organopalladium(II) compounds with promising in vitro and in vivo antitumor effects [5–9]. These compounds exhibit different cell death mechanism of cytotoxicity compared to that of cisplatin and, therefore, they display a distinct spectrum of activity and toxicity. For instance, Barbosa et al. [10] have described that the cyclopalladated [Pd(C2,N-dmpa)(dppf)]Cl compound {C2,N-dmpa = (S)(-)-N,N-dimethyl-1-phenethylamine; dppf = 1,1′-bis(diphenylphosphine)ferrocene} induces apoptosis in K562 leukemia cells, mainly due to lysosomal membrane permeabilization, suggesting that lysosomes are its main target. In addition, toxicological studies showed that such compound produces no changes in red blood cells morphology and no detectable lesions in the liver and kidney of the treated mice 14 days after of its administration. Oximes are also another important class of N-donor ligands widely
Corresponding authors. E-mail addresses: [email protected] (R.F.F. de Souza), [email protected] (A.V.G. Netto).
https://doi.org/10.1016/j.ica.2018.11.022 Received 21 August 2018; Received in revised form 14 November 2018; Accepted 18 November 2018 Available online 20 November 2018 0020-1693/ © 2018 Elsevier B.V. All rights reserved.
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used in cyclopalladation reactions [11–17]. Palladacycles derived from oximes have attracted considerable attention due to their important role as catalysts for CeC bond forming reactions [18], for degradation of thiophosphates pesticides [19], as a source of palladium nanoparticles [20] and as mimetics of hydrolytic metalloenzymes [21]. The well-known stability of these cyclopalladated compounds towards air, moisture, and temperature [22] may also be of interest under the medicinal inorganic chemistry point of view. The use of orthometallated oximes as non-labile chelating ligand may represent an attractive strategy to increase the kinetic stability of these complexes in the biological media. Nevertheless, the study of cytotoxic profile of palladacycles derived from oximes remains unknown in the literature. In the past few years, we have investigated the cytotoxic effect of the compounds [Pd(C2,N-dmba)(X)(tu)] (C2,N-dmba = N,N-dimethylbenzylamine; tu = thiourea; X = halides) on a mammary murine tumor cell line [23,24]. This study has motivated us to explore the antitumor potential of new analogues obtained by the replacement of C2,N-dmba moiety by orthopalladated acetophenone oxime (C2,Naphox). In pursuing our interest in the field of coordination and biological chemistry of Pd(II) compounds [25–29], we report herein the synthesis and characterization of new palladacycles of the type [Pd(C2,N-aphox) (Cl)(L)] {L = thiourea (1); N-methylthiourea (2); N,N’-dimethylthiourea (3); N-phenylthiourea (4); N,N’-diphenylthiourea (5); thioacetamide (6) and benzothioamide (7)}, (Chart 1). The cytotoxicity of compounds 1–7 has been evaluated against a panel of murine {breast (4 T1) and melanoma (B16F10-Nex2)} and human {oral squamous cell carcinoma (Cal27), melanomas (A2058, SK-Mel-110, SK-Mel-05) and liver (HepG2)} cell lines.
Fig. 1. Numbering scheme of the compounds of the type [Pd(C2.N-aphox)(Cl) (L)] {L = tu (1), mtu (2), dmtu (3), ptu (4), dptu (5), taa (6) and tbz (7)}.
deuterated acetone (CD3)2CO (Aldrich). 2.3. General synthesis of [Pd(C2,N-aphox)(Cl)(L)] complexes All complexes were obtained by similar methodology to that reported by Moro et al. [24]. Briefly, a 10.0 mL methanol solution of the suitable thiourea/thioamide (1.80 mmol) is added dropwise to a suspension containing the complex [Pd(C2,N-aphox)(μ-Cl)]2 (0.90 mmol) in a 1:1 mixture of acetone:methanol (25.0 mL, 25 °C). The resulting yellow solution was stirred for 1 h. Afterwards, the solvent was removed under reduced pressure and the resulting solid was washed thoroughly with an 1:1 ethanol:water mixture (3 × 2.0 mL) and npentane (1 × 2.0 mL) and dried in vacuo to afford compounds of the type [Pd(C2,N-aphox)(Cl)(L)]. Fig. 1 shows the numbering scheme for the [Pd(C2,N-aphox)(Cl)(L)] complexes.
2. Experimental 2.1. Materials
2.3.1. [Pd(C2,N-aphox)(Cl)(tu)] (1) Brown solid. Yield: 82%. M.p. > 149.6 °C (dec.). Anal. Calc. for C9H12ClN3OPdS (%): C 30.7; H 3.43; N 11.9; Found. C 31.0; H 3.49; N 11.5. FT-IR (cm−1, KBr): 3362 s (νOH); 3288 m (νNH); 1647 w (νC = N); 1615 s (δNH); 1496 m (νCN); 1434 s (δasCH3); 1044 m (νNO); 1017 m (βCH); 751 s (γCH); 716 m (νC = S). 1H NMR in acetone-d6 {multiplicity, integration, assignment, J (Hz)}: δ 2.30 {s, 3H, H8}; δ 7.01 {td, 1H, H3, J32 = J34 = 7.4 and J35 = 1.6}; δ 7.08 {td, 1H, H4, J43 = J45 = 7.4 and J42 = 1.1}; δ 7.24 {dd, 1H, H5, J54 = 7.4 and J53 = 1.6}; δ 7.38 {dd, 1H, H2, J23 = 7.4 and J24 = 1.1}; δ 7.68 – 8.24 {br, 4H, NH2 + NH2}; δ 10.56 {s, 1H, H9}. 13C{1H} NMR in acetone-d6 (assignment): δ 11.00 (C8); δ 125.54 (C4); δ 126.48 (C5); δ 129.17 (C3); δ 132.34 (C2); δ 144.47 (C6); δ 151.36 (C1); 165.62 (C7); 180.39 (C]S).
The reagents thiourea (Merck), N-methylthiourea (Aldrich), N,N’dimethylthiourea (Aldrich), N-phenylthiourea (Merck), N,N’-diphenylthiourea (Merck), thioacetamide (Merck), benzothioamide (Aldrich), acetophenone oxime (Aldrich), N,N-dimethylbenzylamine (Aldrich), PdCl2 (Aldrich) were employed without further purification. Acetone, methanol, chloroform and n-pentane of analytical purity were purchased from Merck. 2.2. Physical measurements Elemental analysis of carbon, hydrogen, and nitrogen was performed on Elemental Analyzer, Leco Instruments LTD – TruSpec model CHNS-O. IR spectra were recorded on a Nicolet IS5 Thermo Scientific spectrophotometer in the spectra range 4000–400 cm−1 using the KBr pellets. 1H- and 13C{1H}-NMR spectra were obtained on the BRUKER Multinuclear Spectrometers, model Fourier 300, operating at 300 MHz. All compounds of the type [Pd(C2,N-aphox)(Cl)(L)] were solubilized in
2.3.2. [Pd(C2,N-aphox)(Cl)(mtu)] (2) Brown solid. Yield: 56%. M.p. > 126.1 °C (dec.). Anal. Calc. for C10H14ClN3OPdS (%): C 32.8; H 3.85; N 11.5; Found. C 32.8; H 3.98; N 11.1. FT-IR (cm−1, KBr): 3434 m (νOH); 3219 s (νNH); 1573 s (νCN); 1479 s (δNH); 1441 s (δasCH3); 1049 s (νNO); 1020 s (βCH); 754 s (γCH). 1H NMR in acetone-d6 {multiplicity, integration, assignment, J (Hz)}: δ 2.30 {s, 3H, H8}; δ 2.99 {s, 3H, CH3}; δ 7.00 {d, 1H, H3, J32 = J34 = 7.4}; δ 7.08 {t, 1H, H4, J43 = J45 = 7.4}; δ 7.24 {dd, 1H, H5, J54 = 7.4 and J53 = 1.3}; δ 7.37 {d, 1H, H2, J23 = 7.4}; δ 7.50–8.61 {br, 3H, NH2 + NH}; δ 10.53 {s, 1H, H9}. 13C{1H} NMR in acetone‑d6 (assignment): δ 10.99 (C8); δ 125.52 (C4); δ 126.47 (C5); δ 129.16 (C3); δ 132.35 (C2), δ 144.44 (C6); δ 151.38 (C1); δ 165.60 (C7); δ 176.88 (C]S). 2.3.3. [Pd(C2,N-aphox)(Cl)(dmtu)] (3) Yellow solid. Yield: 68%. M.p. > 184.8 °C (dec.). Anal. Calc. for C11H16ClN3OPdS (%): C 34.7; H 4.24; N 11.0; Found. C 35.2; H 4.31; N 10.9. FT-IR (cm−1, KBr): 3340 s (νOH); 3245 s (νNH); 1641 w (νC = N);
Chart 1. Palladacycles of the type [Pd(C2,N-aphox)(Cl)(L)] {L = tu (1), mtu (2), dmtu (3), ptu (4), dptu (5), taa (6) and tbz (7)}. 618
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1586 s (νCN); 1510 s (δNH); 1438 m (δasCH3); 1049 m (νNO); 1019 s (βCH); 750 s (γCH); 700 w (νC = S). 1H NMR in acetone‑d6 {multiplicity, integration, assignment, J (Hz)}: δ 2.30 {s, 3H, H8}; δ 2.91 {s, 3H, CH3}; δ 3.22 {s, 3H, CH3}; δ 7.03 {td, 1H, H3, J32 = J34 = 7.5 and J35 = 1.6}; δ 7.08 {td, 1H, H4, J43 = J45 = 7.4 and J42 = 1.0}; δ 7.24 {dd, 1H, H5, J54 = 7.4 and J53 = 1.6}; δ 7.47 {dd, 1H, H2, J23 = 7.6 and J24 = 1.7}; δ 7.50 {br, 1H, NH}; δ 8.79 {br, 1H, NH}; δ 10.53 {s, 1H, H9}. 13C{1H} NMR in acetone-d6 (assignment): δ 11.01 (C8); δ 32.23 (CLig.); δ 125.57 (C4); δ 126.50 (C5); δ 129.29 (C3); δ 132.72 (C2); δ 144.47 (C6); δ 151.54 (C1); δ 165.68 (C7); δ 176.69 (C]S).
1Har.}; δ 8.04 {m; 2H; 2Har.}; δ 10.11 {br, 1H, NH}; δ 10.46 {s, 1H, H9}; δ 10.86 {br, 1H, NH}. 13C{1H} NMR in acetone-d6 (assignment): δ 11.13 (C8); δ 120.77 (CLig.); δ 125.98 (C4); δ 126.97 (C5); δ 129.71 (C3); δ 132.31 (C2); δ 134.11 (CLig.); δ 137.37 (CLig.); δ 144.46 (C6); δ 151.36 (C1); δ 166.44 (C7); δ 197.61 (C]S). 2.4. Single-crystal X-ray diffraction studies Single crystals for X-ray crystallography of 1, 3 and 6 were obtained by slow solvent evaporation (acetone) of their saturated solutions. Xray diffraction data for 1, 3 and 6 were collected on a BRUKER APEX II Duo diffractometer (Mo-Kα graphite-monochromated radiation, λ = 0.71073 Å) at 23 °C and a multi-scan absorption correction was applied. The structures were solved and refined with the SHELX programs [30,31]. The hydrogen atoms positions were restrained to their parent atoms applying the standard riding model parameters of SHELXL [31].
2.3.4. [Pd(C2,N-aphox)(Cl)(ptu)] (4) Reddish brown solid. Yield: 76%. M.p. > 120.1 °C (dec.). Anal. Calc. for C15H16ClN3OPdS (%): C 42.1; H 3.76; N 9.81; Found. C 42.6; H 4.02; N 9.45. FT-IR (cm−1, KBr): 3430 sh (νOH); 3289 m (νNH); 1611 s (δNH); 1496 m (νCN); 1436 m (δasCH3); 1050 m (νNO); 1020 m (βCH); 749 s (γCH). 1H NMR in acetone-d6 {multiplicity, integration, assignment, J (Hz)}: δ 2.31 {s, 3H, H8}; δ 7.02 {td, 1H, H3, J32 = J34 = 7.4 and J35 = 1.7}; δ 7.10 {td, 1H, H4, J43 = J45 = 7.4 and J42 = 1.3}; δ 7.24 {dd, H, H5, J54 = 7.4 and J53 = 1.7}; δ 7.38 {m, 4H, H2 + 3Har.}; δ 7.50 {m; 2H; 2Har.}; δ 7.65–8.79 {br, 2H, NH2}; δ 10.32 {br, 1H, NH}; δ 10.48 {s, 1H, H9}. 13C{1H} NMR in acetone-d6 (assignment): δ 11.04 (C8); δ 125.67 (CLig.); δ 125.94 (C4); δ 126.63 (C5); δ 128.47 (CLig.); δ 129.29 (C3); δ 130.84 (CLig.); δ 132.41 (C2); δ 137.96 (CLig.); δ 144.46 (C6); δ 151.44 (C1); δ 165.90 (C7); δ 177.09 (C]S).
2.5. Cell lines and culture conditions The B16F10-Nex2 (murine melanoma), A2058 (human melanoma), and 4 T1 (mammary carcinoma) cells were generously donated by Dr. Elaine G Rodrigues, from the Experimental Oncology Unit, EPMUNIFESP). The SK-Mel-110 and SK-Mel-05 human melanomas were gently donated by Dr. Mauro Piacentini from Università degli Studi di Roma Tor Vergata, Rome Italy. The Cal27 (oral squamous cell carcinoma) and HepG2 (hepatocellular carcinoma) cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA) All cell lines studied (SK-Mel-110, SK-Mel-05, B16F10-Nex2, A2058, 4 T1, Cal27 and HepG2) were cultured in monolayer in RPMI-1640 medium (Gibco®, USA) or DMEM (Sigma-Aldrich, St. Louis, MO) contains 10% fetal bovine serum (Gibco®, USA), antibiotics and antimycotics (Sigma-Aldrich), in a humidified condition of 95% air and 5% CO2 at 37 °C. The cells were trypsinized using 0.01% trypsin in 1 mM EDTA (Gibco®, USA). Viable of adherent cells was counted after 24 h using Trypan blue dye exclusion method (0.04%). After culturing, the cells were treated with the complexes 1–7 (1.0 to 200 µM) for 24 or 48 h.
2.3.5. [Pd(C2,N-aphox)(Cl)(dptu)] (5) Reddish brown solid. Yield: 73%. M.p. > 139.3 °C (dec.). Anal. Calc. For C21H20ClN3OPdS (%): C 50.0; H 4.00; N 8.33; Found. C 50.1; H 4.15; N 8.16%. FT-IR (cm−1, KBr): 3436 m (νOH); 3336 m (νNH); 1513 s (δNH); 1437 sh (δasCH3); 1050 m (νNO); 1018 m (βCH); 900 w (νC = S); 755 s (γCH). 1H NMR in acetone-d6 {multiplicity, integration, assignment, J (Hz)}: δ 2.30 {s, 3H, H8}; δ 6.97 {td, 1H, H3, J32 = J34 = 7.4 and J35 = 1.6}; δ 7.08 {td, 1H, H4, J43 = J45 = 7.4 and J42 = 1.0}; δ 7.25 {m, 2H, H5 + Har.}; δ 7.35 {m, 2H, H2 + Har.}; δ 7.47 {m; 9H; NH + 8Har.}; δ 10.20 {br, 1H, NH}; δ 10.43 {s, 1H, H9}. 13 C{1H} NMR in acetone-d6 (assignment): δ 11.04 (C8); δ 119.35 (CLig.); δ 122.87 (CLig.); δ 125.71 (C4); δ 126.77 (C5); δ 128.29 (CLig.); δ 129.41 (C3); δ 129.54 (CLig.); δ 130.29 (CLig.); δ 132.83 (C2); δ 137.96 (CLig.); δ 144.40 (C6); δ 151.68 (C1); δ 166.05 (C7); δ 176.56 (C]S).
2.6. Cytotoxicity assays
2.3.6. [Pd(C2,N-aphox)(Cl)(taa)] (6) Dark brown solid. Yield: 77%. M.p. > 127.2 °C (dec.). Anal. Calc. For C10H13ClN2OPdS (%): C 34.2; H 3.73; N 7.98; Found. C 34.4; H 3.85; N 7.79. FT-IR (cm−1, KBr): 3416 sh (νOH); 3257 m (νNH); 1618 s (δNH); 1449 sh (δasCH3); 1429 m (νCN); 1045 s (νNO); 1017 s (βCH); 751 s (γCH); 713 s (νC = S).). 1H NMR in acetone-d6 {multiplicity, integration, assignment, J (Hz)}: δ 2.32 {s, 3H, H8}; δ 2.67 {s, 3H, CH3}; δ 7.06 {td, 1H, H3, J32 = J34 = 7.3 ans J35 = 1.8}; δ 7.11 {td, 1H, H4, J43 = J45 = 7.3 and J42 = 1.3}; δ 7.25 {dd, 1H, H5, J54 = 7.3 and J53 = 1.8}; δ 7.35 {dd, 1H, H2, J23 = 7.3 and J24 = 1.3}; δ 9.88 {br, 1H, NH}; δ 10.53 {s, 1H, H9}; δ 10.51 {br, 1H, NH}. 13C{1H} NMR in acetone-d6 (assignment): δ 11.06 (C8); δ 125.84 (C4); δ 126.84 (C5); δ 129.56 (C3); δ 132.11 (C2); δ 144.44 (C6); δ 151.19 (C1); δ 166.15 (C7); δ 201.03 (C]S).
Immediately before to be used, the complexes were dissolved in high purity DMSO at 1 mM as stock solution. To obtain the final assay concentrations, aliquot parts of these solutions were diluted in the suitable culture medium and the final concentration of DMSO (lower than 1%) did not reveal any cytotoxic activity. To assess 4 T1, B16F10-Nex2, A2058, SK-Mel-110 and SK-Mel-05 tumor cell lines viability the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used [32]. Briefly, 2.0x104 cells were plated into 96-well flat microplates incubated with different concentrations of the indicated complexes for 24 h. It was added 10 μg/ μL of MTT and incubated for 4 h, then 150 µL of solubilizing solution (10% SDS in 0.01 M HCl) was added to each well overnight to solubilize the formazan crystals. The absorbance was measured at 595 nm. A resazurin reduction assay was used to investigate cytotoxicity of the complexes toward Cal27 and HepG2 tumor cells. The assay is based on reduction of the indicator dye, resazurin, to the highly fluorescent resorufin by viable cells. Nonviable cells rapidly lose the metabolic capacity to reduce resazurin and thus do not produce a fluorescent signal [33,34]. Briefly, 2.5 × 104 cells were culture into a 96-well plate and treated with the complexes 1–7 in different concentrations for 24 h to HepG2 and 48 h to Cal27. After the incubation, the medium was removed, and resazurin 0.01 mg/mL in DMEM (Sigma-Aldrich) was added to each well, and the plates were incubated at 37 °C, under 5% CO2 for 4 h. The fluorescence was measured in a microplate fluorimeter under the excitation wavelength of 530 nm and an emission wavelength
2.3.7. [Pd(C2,N-aphox)(Cl)(tbz)] (7) White solid. Yield: 70%. M.p. > 143.9 °C (dec.). Anal. Calc. for C15H15ClN2OPdS (%): C 43.6; H 3.66; N 6.78; Found. C 44.3; H 3.77; N 6.58. FT-IR (cm−1, KBr): 3416 sh (νOH); 3280 m (νNH); 1629 s (δNH); 1466 m (νCN); 1439 m (δasCH3); 1044 s (νNO); 1019 s (βCH); 874 s (νC = S); 747 s (γCH). 1H NMR in acetone‑d6 {multiplicity, integration, assignment, J (Hz)}: δ 2.32 {s, 3H, H8}; δ 7.08 {td, 1H, H3, J32 = J34 = 7.3 and J35 = 1.8}; δ 7.14 {td, 1H, H4, J43 = J45 = 7.3 and J42 = 1.4}; δ 7.30 {dd, 1H, H5, J54 = 7.3 and J53 = 1.8}; δ 7.47 {dd, 1H, H2, J23 = 7.3 and J24 = 1.4}; δ 7.59 {m; 2H; 2Har.}; δ 7.71 {m; 1H; 619
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Scheme 1. Synthesis of the compounds of the type [Pd(C2.N-aphox)(Cl)(L)] {L = tu (1), mtu (2), dmtu (3), ptu (4), dptu (5), taa (6) and tbz (7)}.
of 590 nm. Three experiments in triplicate were performed for each cell line and the IC50 value was calculated using the GraphPad Prism 5 software. 3. Results and discussion The starting complex [Pd(C2,N-aphox)(μ-Cl)]2 was prepared from transcyclometallation reaction involving [Pd(C2,N-dmba)(μ-Cl)]2 and acetophenone oxime (aphox) [16]. The bridge-splitting reaction of [Pd (C2,N-aphox)(μ-Cl)]2 with thioureas/thioamides (L) in a 1:2 molar ratio at room temperature afforded neutral mononuclear compounds of the type [Pd(C2,N-aphox)(Cl)(L)], as depicted in Scheme 1. Complexes 1–7 are air and light stable solids, soluble in acetone, DMF and DMSO and show colour solutions that varies from light yellow to dark orange. The elemental analysis results for compounds 1–7 agree well the empirical formula [Pd(C2,N-aphox)(Cl)(L)]. All compounds have been characterized by infrared (IR), 1H- and 13C{1H}-NMR spectroscopies. Complexes 1, 3, and 6 have been characterized by X-ray crystallography. The results obtained from these techniques are discussed as follows.
Fig. 3. An ORTEP representation of the molecular structure of the cyclopalladated compound [Pd(C2,N-aphox)(Cl)(dmtu)] (3).
3.1. Single-crystal X-ray diffraction studies Molecular structures of 1, 3, and 6 were established by single crystal X-ray crystallography and ORTEP representations of compounds with the atom labelling scheme are shown in Figs. 2–4, respectively. Crystal data are listed in Table 1 and selected interatomic bond distances and angles with their estimated standard deviations are shown in Table 2. Cyclometallated compounds 1, 3, and 6 have similar mononuclear molecular structures. The palladium centre adopts a distorted square-
Fig. 4. An ORTEP representation of the molecular structure of the cyclopalladated compound [Pd(C2,N-aphox)(Cl)(taa)] (6).
planar coordination in which the orthometallated acetophenone oxime ligand acts as chelating ligand through the imine N1 and aromatic C1 atoms to yield a five-membered ring. All compounds showed comparable acute N1–Pd–C1 bite angle imposed by the chelating C2,N-aphox ligand (79.54–79.95°) which agree well with those values found for similar five-membered orthopalladated oximes [16,25,35]. The sulfur atom from thiourea/thioamide ligand and the imine N1 atom are in a
Fig. 2. An ORTEP representation of the molecular structure of the cyclopalladated compound [Pd(C2,N-aphox)(Cl)(tu)] (1). 620
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Table 1 Crystal data and structural refinement for [Pd(C2,N-aphox)(Cl)(tu)] (1), [Pd(C2,N-aphox)(Cl)(dmtu)] (3) and [Pd(C2,N-aphox)(Cl)(taa)] (6). Empirical formula
C9H12ClN3OPdS (1)
C11H16ClN3OPdS (3)
C10H13ClN2OPdS (6)
Formula weight Temperature (K) Crystal system Space group
352.13 296(2) Orthorhombic P212121
380.18 296(2) Monoclínic C2/c
351.13 296(2) Triclinic
P1
Unit cell dimensions a (Å) b (Å) c (Å) α (°) β (°) γ (°)
7.05730(10) 11.1431(2) 15.6977(2) – – –
21.3481(9) 12.0602(5) 13.6945(11) – 126.1920(10) –
5.7239(2) 9.4913(4) 11.8695(5) 81.439(2) 77.3220(10) 85,528(2)
1234.47(3) 4 1.895 1.870 696 0.530 × 0.090 × 0.080 2.595–62.429 −8 ≤ h ≤ 6 −13 ≤ k ≤ 13 −15 ≤ l ≤ 19 8545 2540 (0.0202) 99.8 0.7454 and 0.5706 2540/0/148 1.080 R1 = 0.0149 wR2 = 0.0361 R1 = 0.0154 wR2 = 0.0364 0.295 and −0.267
2845.5(3) 8 1.775 1.630 1520 0.280 × 0.130 × 0.110 2.061–26.466 −26 ≤ h ≤ 26 −15 ≤ k ≤ 15 −17 ≤ l ≤ 16 17,958 2932 (0.0281) 100.0 0.7454 and 0.5694 2932/0/168 1.098 R1 = 0.0180 wR2 = 0.0439 R1 = 0.0195 wR2 = 0.0454 0.299 and −0.562
621.42(4) 2 1.877 1.855 348 0.650 × 0.150 × 0.060 1.775–26.383 −7 ≤ h ≤ 7 −11 ≤ k ≤ 11 −14 ≤ l ≤ 14 17,726 2543 (0.0256) 100.0 0.7454 and 0.6397 2543/0/148 1.127 R1 = 0.0179 wR2 = 0.0459 R1 = 0.0190 wR2 = 0.0472 0.526 and −0.236
Volume (Å3) Z Density calculated (g cm−3) Absorption coefficient (mm−1) F (0 0 0) Crystal size (mm) Θ range for collected (°) Index ranges Reflections collected Independent reflections (Rint) Completeness to Θ = 25.242° (%) Max. and min. transmission Data/Restraints/Parameters Goodness-of-fit on F2 Final R indices [I > 2σ(I)] R indices (all data) Largest difference peak and hole (e Å3)
found in [PdCl(C2,N-dmba)(tu)] (2.314(1) Å) [24] but comparable to those values obtained for [Pd(phen)(tu)2](NO3)2 (2.270–2.293 Å), in which Pd–S bonds are trans to N(sp2) atoms [36].
Table 2 Selected geometric parameters for [Pd(C2,N-aphox)(Cl)(tu)] (1), [Pd(C2,Naphox)(Cl)(dmtu)] (3) and [Pd(C2,N-aphox)(Cl)(taa)] (6). 1
3
6
Bond lenght (Å) Pd–N1 Pd–C1 Pd–Cl1 Pd–S1 C(9)–S(1) C(9)–N(2) C(9)–N(3)
2.031(2) 1.998(3) 2.4689(7) 2.3029(8) 1.716(3) 1.309(4) 1.314(4)
2.0259(14) 1.9935(19) 2.4503(5) 2.3063(5) 1.7252(18) 1.325(2) 1.324(2)
2.0319(17) 1.9949(19) 2.4494(5) 2.2887(5) 1.690(2) 1.305(3) –
Bond angle (°) N1—Pd—C1 N1—Pd—Cl1 N1—Pd—S1 S1—Pd—Cl S1—Pd—Cl1 C1—Pd—Cl1
79.75(10) 88.93(6) 170.48(8) 91.74(8) 99.62(3) 168.63(8)
79.61(7) 88.78(5) 172.68(4) 94.77(5) 96.844(18) 168.38(5)
79.87(7) 90.84(5) 174.87(5) 96.11(6) 93.15(2) 170.70(6)
3.2. IR and NMR spectra IR results give important evidences about the occurrence of the orthometallation reaction on acetophenone oxime. The most useful bands are those resulting from the C]N and NeO stretching of the oxime group, and CeH out-of-plane deformation vibrations (γCH) of the aromatic ring. The IR spectrum of the acetophenone oxime (aphox) exhibits bands at 1680 and 921 cm−1, attributed to νC = N and νNO vibrational modes, respectively [37]. The appearance of two intense γCH absorptions at 757 and 691 cm−1 supports the monosubstituted nature of the benzene ring in aphox [38]. Upon cyclopalladation, relevant spectral changes are observed. For instance, in the IR spectrum of the [Pd(C2,N-aphox)(μ-Cl)]2 precursor, the νC = N band is shifted 44 cm−1 to lower frequencies compared to the aphox ligand, being consistent with coordination of the imine nitrogen atom [17]. In addition, the shift of ca. 100 cm−1 to higher frequencies of the νNeO agrees well with the expected strengthening of the NeO bond when oximes are N-coordinated [39,40]. The presence of a sharp νOH absorption at 3430 cm−1 also indicates that the oxime coordination takes place through the nitrogen atom. The number of γCH bands is also used as diagnosis for orthopalladation of acetophenone oxime. In this case, the appearance of only one intense γCH band at 752 cm−1 for [Pd(C2,Naphox)(μ-Cl)]2, instead of two γCH absorptions for monosubstituted benzene, strongly supports the o-di-substitution of aphox [38]. Besides the bands of orthometallated acetophenone oxime, the IR spectra of mononuclear compounds 1–7 also revealed the presence of the typical absorptions of thioamide-type ligands. In all cases, thioureas and thioamides coordinate via S atom in 1–7. Complexation through S
trans arrangement with a N1ePdeS1 bond angle of 170.40–174.92°. The remaining coordination site is occupied by a chlorido ligand transpositioned to the carbopalladated C1 atom. The Pd–C1 bond distances in 1, 3 and 6 (1.9915–2.0010 Å) are characteristic of the Pd–C(sp2) bonds observed in related cyclopalladated compounds [16,25,35]. In all three compounds, the Pd–Cl bond lengths (2.4489–2.4696 Å) are quite similar and slightly longer than the upper limit of the expected interval of PdeCl bond distances (2.37–2.45 Å) [16,25]. Such stretching of the PdeCl bond has already been observed in similar orthopalladated oximes because of intramolecular OH⋯Cl hydrogen bonding [16]. In the present case, chlorido ligands in 1, 3 and 6 take part in intramolecular OH⋯Cl and/or intermolecular NH⋯Cl hydrogen bonds. The PdeS1 distances of 2.2882–2.3068 Å are slightly shorter than that 621
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atom induces shifting and intensity changes in IR bands with considerable contribution from νCN and νC = S modes [23,41]. The shift to low frequency of the νC = S band together with a high frequency displacement of νCN absorption, when compared with those of the free ligands [42–48], suggests the S-coordination [23]. Hydrogen and carbon atoms in 1–7 were fully assigned from the connectivity observed in HSQC and HMBC NMR spectra. The 1H NMR spectra of l-7 exhibits only one set of signals, which suggests that the presence of only one geometrical isomer in solution. The appearance of only 4 aromatic resonances at, approximately, 7.40 (H2, dd, J23 ≅ 7.4 and J24 ≅ 1.2 Hz), 7.02 (H3, td, J32 = J34 ≅ 7.4 and J35 ≅ 1.7 Hz), 7.10 (H4, td, J43 = J45 ≅ 7.4 and J42 ≅ 1.2 Hz) and 7.25 ppm (H5, dd, J54 ≅ 7.4 and J53 ≅ 1.7 Hz) is consistent with the presence of the C2,Northopalladated aphox moiety in the molecular structures of 1–7. NMR spectroscopy also unequivocally allows detecting the S-coordination of thioamides in 1–7. Significant downfield shifts are observed for NH and eNH2 resonances as a consequence of the enhanced double bond character of CeN bond which hampers the rotation about CeN bond together with a weakening of C]S bond on S-coordination of thioamides [23,49,50]. In the 1H NMR spectra of cyclopalladated compounds bearing unsubstituted thiourea (1), thioacetamide (6) and benzothioamide (7), the NH2 protons are split into two broad signals probably due to internal and external NeH environments [50,51]. In the case of coordinated N-methylthiourea (mtu), two geometric isomers (syn and anti) can be observed at low temperatures (210 K) depending on the orientation of the methyl group with respect to the S atom [49,50]. In the case of the compound [Pd(C2,N-aphox)(Cl)(mtu)] (2), the appearance of a set of broadened signal over the 7.50–8.61 ppm range, attributed to NH2 and NH groups, together with two broadened singlets centered at 2.99 ppm (N-methyl group) suggests the presence of two exchanging conformers in solution at room temperature. The 1H NMR spectrum of [Pd(C2,N-aphox)(Cl)(dmtu)] (3) displays two broadened NH signals at 7.50 and 8.79 ppm together with two resonances at 2.91 and 3.22 ppm ascribed to N-methyl groups, indicating that dmtu ligand adopts a syn–anti arrangement. Such finding has already been observed for other Pd(II) and Pt(II) complexes containing symmetrically substituted N,N’-di-alkyl thioureas [49,50]. Regarding the 1H NMR spectra of [Pd(C2,N-aphox)(Cl)(ptu)] (4) and [Pd (C2,N-aphox)(Cl)(dptu)] (5), one NH signal is clearly detected at ca. 10.2–10.3 ppm, but the remaining NH signal is masked by the intense aromatic multiplets. 13 C{1H}-NMR spectra of 1–7 showed that the signal of the azomethine eC]N atom is shifted to ca. 10 ppm downfield with respect to the free aphox (156.00 ppm) [11]. This deshielding supports the coordination through the iminic nitrogen in all cyclopalladated compounds. The existence of C2,N-orthopalladated aphox moiety is confirmed by the appearance of its six 13C signals at, approximately, 151.40 (C1), 132.45 (C2), 129.40 (C3), 125.70 (C4), 126.65 (C5) and 144.45 ppm (C6). The additional quaternary 13C signal attributed to the metalated carbon (C1) is downfield shifted relative to the free ligand (126.00 ppm) [11,51], confirming the cyclopalladation of aphox in 1–7. The 13C signals of the methyl substituents of coordinated thioamides in 2, 3 and 6 are masked by the 13CD3 signal of acetone-d6 (29.84 ppm) as can be confirmed by the HSQC NMR spectra where protons of the methyl groups are correlating with a signal under that acetone-d6 signal. In the 13C{1H}-NMR spectra of complexes 4, 5 and 7, some aromatic carbons of the phenyl substituents of thioamides absorb at the same frequency, hindering their distinction in the spectra. The S-coordination of thioamide ligands in 1–7 is evidenced in all compounds by the upfield shift of the 13C = S signal relative to the free ligand, as a result of the weakening of C]S bond upon PdeS bond formation [26,49,50].
needed to inhibit 50% of the cellular proliferation) of complexes 1–7 with cisplatin against murine {breast (4T1), melanoma (B16F10Nex2)}, human {melanomas (A2058, SK-Mel-110 and SK-Mel-05), oral adenosquamous carcinoma (Cal27), liver (Hep-G2)} cancer cell lines. It can be seen in Table 3 that compounds 1–7 showed different cytotoxicity pattern depending on the tumor cell line assayed. In most of the cases, a significant number of compounds showed IC50 values lower or like those of cisplatin. From the inspection of cytotoxicity results against murine (4 T1, B16F10-Nex2), human melanoma (A2058, SK-Mel-110 and SK-Mmel-05) and hepatocellular carcinoma (HepG2) cell lines, some interesting trends can be noticed. In general, cyclopalladated compounds containing thioureas/thioamides with phenyl substituents (compounds 4, 5 and 7) tend to be more active than their counterparts bearing thiourea/N-methylthiourea/thioacetamide (1, 2 and 6). Compound [Pd(C2,N-aphox)(Cl)(dmtu)] (3), where dmtu = N,N’-dimethylthiourea, exhibited an intermediate character, being as active as 4, 5 and 7 depending on the tested cell line. This finding suggests that lipophilicity plays an important role in the cytotoxicity. Among them, compound [Pd(C2,N-aphox)(Cl)(tbz)] (7) displayed the best cytotoxic profile, showing a greater potency than cisplatin in most of these cell lines. In addition, the cytotoxicity of the complexes to human SK-Mel-110 melanoma cells is of interest as this cell line contains p53 mutations and is very resistant to apoptosis induction [53]. Regarding the cytotoxicity results obtained against oral adenosquamous carcinoma cell line (Cal27), no structure–activity relationship can be rationalized. Compound [Pd(C2,N-aphox)(Cl)(dmtu)] (3) showed the highest potency, with a IC50 value of 29.21 ± 1.29 μM. At this point, one could ask why a significant number of cyclopalladated compounds described here is more active than cisplatin, especially those bearing thioureas/thioamides with phenyl substituents in 4, 5 and 7. We assumed that such difference could be explained, at least in part, on the basis of kinetics considerations. It is well established that mechanism of ligand exchange in square-planar Pd and Pt compounds is associative, whose rate can be decreased/inhibited by steric crowding at the reaction centre [54,55]. Therefore, the presence of bulky substituents in the ancillary S-donor ligands in 4, 5 and 7, together with the chelating C2,N-aphox ligand, is expected to enhance their kinetic stability on the biological media and, consequently, their structures would remain intact long enough to interact with the pharmacological target(s). In addition, it is worth mentioning that cyclopalladated compounds exhibit distinct cell death mechanisms when compared to that of cisplatin. While DNA is considered to be the main target of cisplatin, a growing body of evidence has demonstrated that cyclopalladated compounds exert their cytotoxic effects by targeting mitochondria [9] or lysosomes [56]. Nevertheless, further studies are required in order to confirm this assumption. 4. Conclusions In conclusion, a family of seven new palladium compounds containing orthometallated acetophenone oxime and S-based molecules as co-ligands has been successfully synthesized, and the molecular structures of three of them have been determined. Cleavage reactions involving [Pd(C2,N-aphox)(μ-Cl)]2 and thioamides demonstrated to be an efficient method to obtain mononuclear compounds of the type [Pd (C2,N-aphox)(Cl)(L)] in moderate to good yields. The cytotoxicity profile of cyclometallated compounds 1–7 has been dependent on the tumor cell line evaluated. Under the structure-activity relationship point of view, it was noticed that the compounds which contain phenyl substituents at the ancillary S-donor ligands exhibit lower IC50 values towards murine (4 T1, B16F10-Nex2), human melanoma (A2058, SK-Mel-110 and SK-Mel-05) and hepatocellular carcinoma (HepG2) cell lines. In general, the cyclopalladated compounds obtained in this work demonstrated to be more active than cisplatin to seven tumor cell lines, opening new perspectives for the development of new metal-based anticancer agents.
3.3. Cytotoxic activities of complexes (1–7) against tumor cell lines studied Table 3 exhibits the comparative IC50 values (the concentration 622
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Table 3 Cytotoxicity activities (IC50, µM) of the compounds 1–7 against murine {breast (4 T1) and melanoma (B16F10-Nex2)} and human {melanoma (A2058, SK-Mel-110, SK-Mel-05), oral adenosquamous carcinoma (Cal27) and liver (HepG2)} cancer cell lines. Compound
1 2 3 4 5 6 7 Cisplatin a
Cell lines 4 T1
B16F10-nex2
A2058
SK-Mel-110
SK-Mel-05
Cal27
84.62 ± 2.14 56.76 ± 3.54 47.17 ± 2.61 38.68 ± 3.57 45.87 ± 0.74 53.41 ± 2.97 34.70 ± 2.54 108.54 ± 15.78
68.42 54.47 43.97 33.07 50.35 43.69 25.92 23.14
192.00 ± 59.92 189.49 ± 19.87 93.03 ± 20.29 89.72 ± 15.82 82.23 ± 2.46 107.15 ± 16.77 65.05 ± 7.34 111.85 ± 27.42
> 100 102.05 ± 4.95 66.79 ± 12.1 60.54 ± 1.88 61.18 ± 2.00 80.85 ± 3.87 51.05 ± 4.14 85.00 ± 11.03
137.42 ± 10.11 88.39 ± 2.86 72.49 ± 2.25 50.05 ± 7.47 59.20 ± 2.13 88.55 ± 8.04 49.57 ± 2.97 78.24 ± 5.83
73.14 51.66 29.21 55.06 47.79 77.70 69.45 –
± ± ± ± ± ± ± ±
5.77 4.30 7.23 3.77 5.02 6.07 3.52 1.27
HepG2 ± ± ± ± ± ± ±
3.88 6.44 1.29 0.47 2.57 2.17 3.57
41.48 41.42 31.59 27.72 15.92 44.56 33.32 60.30
± ± ± ± ± ± ± ±
4.02 2.29 0.81 1.00 3.84 6.03 5.88 15.10a
Ref. [52].
Acknowledgments
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Research paper
Orthopalladated acetophenone oxime compounds bearing thioamides as ligands: Synthesis, structure and cytotoxic evaluation
T
Ronan F.F. de Souzaa, , Gislaine A. da Cunhaa, José C.M. Pereiraa, Daniel M. Garciab, Claudia Bincolettob, Renata N. Gotoc, Andréia M. Leopoldinoc, Isabel C. da Silvad, Fernando R. Pavand, Victor M. Deflone, Eduardo T. de Almeidaf, Antônio E. Mauroa, ⁎ Adelino V.G. Nettoa, ⁎
a
UNESP – Univ Estadual Paulista, Instituto de Química, Departamento de Química Geral e Inorgânica, 14800-060 Araraquara, SP, Brazil UNIFESP – Univ Federal de São Paulo, Escola Paulista de Medicina, Departamento de Farmacologia, 04044-020 São Paulo, SP, Brazil USP – Univ de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, 14040-903 Ribeirão Preto, SP, Brazil d UNESP – Univ Estadual Paulista, Faculdade de Ciências Farmacêuticas, 14800-903 Araraquara, SP, Brazil e USP – Univ de São Paulo, Instituto de Química de São Carlos, 13560-970 São Carlos, SP, Brazil f UNIFAL – Univ Federal de Alfenas, Instituto de Química, 37130-001 Alfenas, MG, Brazil b c
ARTICLE INFO
ABSTRACT
Keywords: Cyclopalladated complex Oximes Cytotoxicity Thioamides
Cleavage reactions involving the halide-bridged [Pd(μ-Cl)(C2,N-aphox)]2 precursor (aphox = acetophenone oxime) with thioamides, in the 1:2 molar ratio, yielded mononuclear cyclopalladated of the type [Pd(C2,Naphox)(Cl)(L) {L = thiourea (1); N-methylthiourea (2); N,N’-dimethylthiourea (3); N-phenylthiourea (4); N,N’diphenylthiourea (5); thioacetamide (6) and benzothioamide (7)} which were characterized by elemental analyses, infrared and 1H- and 13C{1H}-NMR spectroscopies. The crystal and molecular structures of 1, 3 and 6 were determined by single-crystal X-ray diffraction studies. The cytotoxicity of the cyclopalladated compounds has been evaluated against a panel of murine {breast (4T1) and melanoma (B16F10-Nex2)} and human {melanoma (A2058, SK-Mel-110 and SK-Mel-05), oral squamous cell carcinoma (Cal27), hepatocellular carcinoma (HepG2)} cancer cell lines. All studied compounds were cytotoxic for the seven cancer cell lines studied and in general, most cyclopalladated compounds obtained in this work were more active than cisplatin to the seven tumor cell lines evaluated.
1. Introduction The bioinorganic and medicinal chemistry of palladium(II) compounds has experienced spectacular advances in the past years since many of them have been considered as alternative candidates to the classical platinum anticancer agents [1–6]. The structural and thermodynamic analogy between Pd(II) and Pt(II) is rather limited. Although they both share a d8 configuration, their reactivity is quite different, being palladium complexes more labile than platinum, with an exchange rate about 105 times higher [6]. Ligand dissociation in aqueous medium gives very reactive Pd(II) species, which can be toxic and ineffective with respect to Pt(II) analogues because they can be easily inactivated before reaching the cellular targets. For these reasons an accurate choice of the ligands on the Pd(II) centre has to be made in order to achieve enough kinetic stability for being a good drug in therapy [4]. ⁎
Within this context, N-donor ligands such as benzylamines and imines have been extensively employed in cyclopalladation reactions, affording chelated organopalladium(II) compounds with promising in vitro and in vivo antitumor effects [5–9]. These compounds exhibit different cell death mechanism of cytotoxicity compared to that of cisplatin and, therefore, they display a distinct spectrum of activity and toxicity. For instance, Barbosa et al. [10] have described that the cyclopalladated [Pd(C2,N-dmpa)(dppf)]Cl compound {C2,N-dmpa = (S)(-)-N,N-dimethyl-1-phenethylamine; dppf = 1,1′-bis(diphenylphosphine)ferrocene} induces apoptosis in K562 leukemia cells, mainly due to lysosomal membrane permeabilization, suggesting that lysosomes are its main target. In addition, toxicological studies showed that such compound produces no changes in red blood cells morphology and no detectable lesions in the liver and kidney of the treated mice 14 days after of its administration. Oximes are also another important class of N-donor ligands widely
Corresponding authors. E-mail addresses: [email protected] (R.F.F. de Souza), [email protected] (A.V.G. Netto).
https://doi.org/10.1016/j.ica.2018.11.022 Received 21 August 2018; Received in revised form 14 November 2018; Accepted 18 November 2018 Available online 20 November 2018 0020-1693/ © 2018 Elsevier B.V. All rights reserved.
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used in cyclopalladation reactions [11–17]. Palladacycles derived from oximes have attracted considerable attention due to their important role as catalysts for CeC bond forming reactions [18], for degradation of thiophosphates pesticides [19], as a source of palladium nanoparticles [20] and as mimetics of hydrolytic metalloenzymes [21]. The well-known stability of these cyclopalladated compounds towards air, moisture, and temperature [22] may also be of interest under the medicinal inorganic chemistry point of view. The use of orthometallated oximes as non-labile chelating ligand may represent an attractive strategy to increase the kinetic stability of these complexes in the biological media. Nevertheless, the study of cytotoxic profile of palladacycles derived from oximes remains unknown in the literature. In the past few years, we have investigated the cytotoxic effect of the compounds [Pd(C2,N-dmba)(X)(tu)] (C2,N-dmba = N,N-dimethylbenzylamine; tu = thiourea; X = halides) on a mammary murine tumor cell line [23,24]. This study has motivated us to explore the antitumor potential of new analogues obtained by the replacement of C2,N-dmba moiety by orthopalladated acetophenone oxime (C2,Naphox). In pursuing our interest in the field of coordination and biological chemistry of Pd(II) compounds [25–29], we report herein the synthesis and characterization of new palladacycles of the type [Pd(C2,N-aphox) (Cl)(L)] {L = thiourea (1); N-methylthiourea (2); N,N’-dimethylthiourea (3); N-phenylthiourea (4); N,N’-diphenylthiourea (5); thioacetamide (6) and benzothioamide (7)}, (Chart 1). The cytotoxicity of compounds 1–7 has been evaluated against a panel of murine {breast (4 T1) and melanoma (B16F10-Nex2)} and human {oral squamous cell carcinoma (Cal27), melanomas (A2058, SK-Mel-110, SK-Mel-05) and liver (HepG2)} cell lines.
Fig. 1. Numbering scheme of the compounds of the type [Pd(C2.N-aphox)(Cl) (L)] {L = tu (1), mtu (2), dmtu (3), ptu (4), dptu (5), taa (6) and tbz (7)}.
deuterated acetone (CD3)2CO (Aldrich). 2.3. General synthesis of [Pd(C2,N-aphox)(Cl)(L)] complexes All complexes were obtained by similar methodology to that reported by Moro et al. [24]. Briefly, a 10.0 mL methanol solution of the suitable thiourea/thioamide (1.80 mmol) is added dropwise to a suspension containing the complex [Pd(C2,N-aphox)(μ-Cl)]2 (0.90 mmol) in a 1:1 mixture of acetone:methanol (25.0 mL, 25 °C). The resulting yellow solution was stirred for 1 h. Afterwards, the solvent was removed under reduced pressure and the resulting solid was washed thoroughly with an 1:1 ethanol:water mixture (3 × 2.0 mL) and npentane (1 × 2.0 mL) and dried in vacuo to afford compounds of the type [Pd(C2,N-aphox)(Cl)(L)]. Fig. 1 shows the numbering scheme for the [Pd(C2,N-aphox)(Cl)(L)] complexes.
2. Experimental 2.1. Materials
2.3.1. [Pd(C2,N-aphox)(Cl)(tu)] (1) Brown solid. Yield: 82%. M.p. > 149.6 °C (dec.). Anal. Calc. for C9H12ClN3OPdS (%): C 30.7; H 3.43; N 11.9; Found. C 31.0; H 3.49; N 11.5. FT-IR (cm−1, KBr): 3362 s (νOH); 3288 m (νNH); 1647 w (νC = N); 1615 s (δNH); 1496 m (νCN); 1434 s (δasCH3); 1044 m (νNO); 1017 m (βCH); 751 s (γCH); 716 m (νC = S). 1H NMR in acetone-d6 {multiplicity, integration, assignment, J (Hz)}: δ 2.30 {s, 3H, H8}; δ 7.01 {td, 1H, H3, J32 = J34 = 7.4 and J35 = 1.6}; δ 7.08 {td, 1H, H4, J43 = J45 = 7.4 and J42 = 1.1}; δ 7.24 {dd, 1H, H5, J54 = 7.4 and J53 = 1.6}; δ 7.38 {dd, 1H, H2, J23 = 7.4 and J24 = 1.1}; δ 7.68 – 8.24 {br, 4H, NH2 + NH2}; δ 10.56 {s, 1H, H9}. 13C{1H} NMR in acetone-d6 (assignment): δ 11.00 (C8); δ 125.54 (C4); δ 126.48 (C5); δ 129.17 (C3); δ 132.34 (C2); δ 144.47 (C6); δ 151.36 (C1); 165.62 (C7); 180.39 (C]S).
The reagents thiourea (Merck), N-methylthiourea (Aldrich), N,N’dimethylthiourea (Aldrich), N-phenylthiourea (Merck), N,N’-diphenylthiourea (Merck), thioacetamide (Merck), benzothioamide (Aldrich), acetophenone oxime (Aldrich), N,N-dimethylbenzylamine (Aldrich), PdCl2 (Aldrich) were employed without further purification. Acetone, methanol, chloroform and n-pentane of analytical purity were purchased from Merck. 2.2. Physical measurements Elemental analysis of carbon, hydrogen, and nitrogen was performed on Elemental Analyzer, Leco Instruments LTD – TruSpec model CHNS-O. IR spectra were recorded on a Nicolet IS5 Thermo Scientific spectrophotometer in the spectra range 4000–400 cm−1 using the KBr pellets. 1H- and 13C{1H}-NMR spectra were obtained on the BRUKER Multinuclear Spectrometers, model Fourier 300, operating at 300 MHz. All compounds of the type [Pd(C2,N-aphox)(Cl)(L)] were solubilized in
2.3.2. [Pd(C2,N-aphox)(Cl)(mtu)] (2) Brown solid. Yield: 56%. M.p. > 126.1 °C (dec.). Anal. Calc. for C10H14ClN3OPdS (%): C 32.8; H 3.85; N 11.5; Found. C 32.8; H 3.98; N 11.1. FT-IR (cm−1, KBr): 3434 m (νOH); 3219 s (νNH); 1573 s (νCN); 1479 s (δNH); 1441 s (δasCH3); 1049 s (νNO); 1020 s (βCH); 754 s (γCH). 1H NMR in acetone-d6 {multiplicity, integration, assignment, J (Hz)}: δ 2.30 {s, 3H, H8}; δ 2.99 {s, 3H, CH3}; δ 7.00 {d, 1H, H3, J32 = J34 = 7.4}; δ 7.08 {t, 1H, H4, J43 = J45 = 7.4}; δ 7.24 {dd, 1H, H5, J54 = 7.4 and J53 = 1.3}; δ 7.37 {d, 1H, H2, J23 = 7.4}; δ 7.50–8.61 {br, 3H, NH2 + NH}; δ 10.53 {s, 1H, H9}. 13C{1H} NMR in acetone‑d6 (assignment): δ 10.99 (C8); δ 125.52 (C4); δ 126.47 (C5); δ 129.16 (C3); δ 132.35 (C2), δ 144.44 (C6); δ 151.38 (C1); δ 165.60 (C7); δ 176.88 (C]S). 2.3.3. [Pd(C2,N-aphox)(Cl)(dmtu)] (3) Yellow solid. Yield: 68%. M.p. > 184.8 °C (dec.). Anal. Calc. for C11H16ClN3OPdS (%): C 34.7; H 4.24; N 11.0; Found. C 35.2; H 4.31; N 10.9. FT-IR (cm−1, KBr): 3340 s (νOH); 3245 s (νNH); 1641 w (νC = N);
Chart 1. Palladacycles of the type [Pd(C2,N-aphox)(Cl)(L)] {L = tu (1), mtu (2), dmtu (3), ptu (4), dptu (5), taa (6) and tbz (7)}. 618
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1586 s (νCN); 1510 s (δNH); 1438 m (δasCH3); 1049 m (νNO); 1019 s (βCH); 750 s (γCH); 700 w (νC = S). 1H NMR in acetone‑d6 {multiplicity, integration, assignment, J (Hz)}: δ 2.30 {s, 3H, H8}; δ 2.91 {s, 3H, CH3}; δ 3.22 {s, 3H, CH3}; δ 7.03 {td, 1H, H3, J32 = J34 = 7.5 and J35 = 1.6}; δ 7.08 {td, 1H, H4, J43 = J45 = 7.4 and J42 = 1.0}; δ 7.24 {dd, 1H, H5, J54 = 7.4 and J53 = 1.6}; δ 7.47 {dd, 1H, H2, J23 = 7.6 and J24 = 1.7}; δ 7.50 {br, 1H, NH}; δ 8.79 {br, 1H, NH}; δ 10.53 {s, 1H, H9}. 13C{1H} NMR in acetone-d6 (assignment): δ 11.01 (C8); δ 32.23 (CLig.); δ 125.57 (C4); δ 126.50 (C5); δ 129.29 (C3); δ 132.72 (C2); δ 144.47 (C6); δ 151.54 (C1); δ 165.68 (C7); δ 176.69 (C]S).
1Har.}; δ 8.04 {m; 2H; 2Har.}; δ 10.11 {br, 1H, NH}; δ 10.46 {s, 1H, H9}; δ 10.86 {br, 1H, NH}. 13C{1H} NMR in acetone-d6 (assignment): δ 11.13 (C8); δ 120.77 (CLig.); δ 125.98 (C4); δ 126.97 (C5); δ 129.71 (C3); δ 132.31 (C2); δ 134.11 (CLig.); δ 137.37 (CLig.); δ 144.46 (C6); δ 151.36 (C1); δ 166.44 (C7); δ 197.61 (C]S). 2.4. Single-crystal X-ray diffraction studies Single crystals for X-ray crystallography of 1, 3 and 6 were obtained by slow solvent evaporation (acetone) of their saturated solutions. Xray diffraction data for 1, 3 and 6 were collected on a BRUKER APEX II Duo diffractometer (Mo-Kα graphite-monochromated radiation, λ = 0.71073 Å) at 23 °C and a multi-scan absorption correction was applied. The structures were solved and refined with the SHELX programs [30,31]. The hydrogen atoms positions were restrained to their parent atoms applying the standard riding model parameters of SHELXL [31].
2.3.4. [Pd(C2,N-aphox)(Cl)(ptu)] (4) Reddish brown solid. Yield: 76%. M.p. > 120.1 °C (dec.). Anal. Calc. for C15H16ClN3OPdS (%): C 42.1; H 3.76; N 9.81; Found. C 42.6; H 4.02; N 9.45. FT-IR (cm−1, KBr): 3430 sh (νOH); 3289 m (νNH); 1611 s (δNH); 1496 m (νCN); 1436 m (δasCH3); 1050 m (νNO); 1020 m (βCH); 749 s (γCH). 1H NMR in acetone-d6 {multiplicity, integration, assignment, J (Hz)}: δ 2.31 {s, 3H, H8}; δ 7.02 {td, 1H, H3, J32 = J34 = 7.4 and J35 = 1.7}; δ 7.10 {td, 1H, H4, J43 = J45 = 7.4 and J42 = 1.3}; δ 7.24 {dd, H, H5, J54 = 7.4 and J53 = 1.7}; δ 7.38 {m, 4H, H2 + 3Har.}; δ 7.50 {m; 2H; 2Har.}; δ 7.65–8.79 {br, 2H, NH2}; δ 10.32 {br, 1H, NH}; δ 10.48 {s, 1H, H9}. 13C{1H} NMR in acetone-d6 (assignment): δ 11.04 (C8); δ 125.67 (CLig.); δ 125.94 (C4); δ 126.63 (C5); δ 128.47 (CLig.); δ 129.29 (C3); δ 130.84 (CLig.); δ 132.41 (C2); δ 137.96 (CLig.); δ 144.46 (C6); δ 151.44 (C1); δ 165.90 (C7); δ 177.09 (C]S).
2.5. Cell lines and culture conditions The B16F10-Nex2 (murine melanoma), A2058 (human melanoma), and 4 T1 (mammary carcinoma) cells were generously donated by Dr. Elaine G Rodrigues, from the Experimental Oncology Unit, EPMUNIFESP). The SK-Mel-110 and SK-Mel-05 human melanomas were gently donated by Dr. Mauro Piacentini from Università degli Studi di Roma Tor Vergata, Rome Italy. The Cal27 (oral squamous cell carcinoma) and HepG2 (hepatocellular carcinoma) cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA) All cell lines studied (SK-Mel-110, SK-Mel-05, B16F10-Nex2, A2058, 4 T1, Cal27 and HepG2) were cultured in monolayer in RPMI-1640 medium (Gibco®, USA) or DMEM (Sigma-Aldrich, St. Louis, MO) contains 10% fetal bovine serum (Gibco®, USA), antibiotics and antimycotics (Sigma-Aldrich), in a humidified condition of 95% air and 5% CO2 at 37 °C. The cells were trypsinized using 0.01% trypsin in 1 mM EDTA (Gibco®, USA). Viable of adherent cells was counted after 24 h using Trypan blue dye exclusion method (0.04%). After culturing, the cells were treated with the complexes 1–7 (1.0 to 200 µM) for 24 or 48 h.
2.3.5. [Pd(C2,N-aphox)(Cl)(dptu)] (5) Reddish brown solid. Yield: 73%. M.p. > 139.3 °C (dec.). Anal. Calc. For C21H20ClN3OPdS (%): C 50.0; H 4.00; N 8.33; Found. C 50.1; H 4.15; N 8.16%. FT-IR (cm−1, KBr): 3436 m (νOH); 3336 m (νNH); 1513 s (δNH); 1437 sh (δasCH3); 1050 m (νNO); 1018 m (βCH); 900 w (νC = S); 755 s (γCH). 1H NMR in acetone-d6 {multiplicity, integration, assignment, J (Hz)}: δ 2.30 {s, 3H, H8}; δ 6.97 {td, 1H, H3, J32 = J34 = 7.4 and J35 = 1.6}; δ 7.08 {td, 1H, H4, J43 = J45 = 7.4 and J42 = 1.0}; δ 7.25 {m, 2H, H5 + Har.}; δ 7.35 {m, 2H, H2 + Har.}; δ 7.47 {m; 9H; NH + 8Har.}; δ 10.20 {br, 1H, NH}; δ 10.43 {s, 1H, H9}. 13 C{1H} NMR in acetone-d6 (assignment): δ 11.04 (C8); δ 119.35 (CLig.); δ 122.87 (CLig.); δ 125.71 (C4); δ 126.77 (C5); δ 128.29 (CLig.); δ 129.41 (C3); δ 129.54 (CLig.); δ 130.29 (CLig.); δ 132.83 (C2); δ 137.96 (CLig.); δ 144.40 (C6); δ 151.68 (C1); δ 166.05 (C7); δ 176.56 (C]S).
2.6. Cytotoxicity assays
2.3.6. [Pd(C2,N-aphox)(Cl)(taa)] (6) Dark brown solid. Yield: 77%. M.p. > 127.2 °C (dec.). Anal. Calc. For C10H13ClN2OPdS (%): C 34.2; H 3.73; N 7.98; Found. C 34.4; H 3.85; N 7.79. FT-IR (cm−1, KBr): 3416 sh (νOH); 3257 m (νNH); 1618 s (δNH); 1449 sh (δasCH3); 1429 m (νCN); 1045 s (νNO); 1017 s (βCH); 751 s (γCH); 713 s (νC = S).). 1H NMR in acetone-d6 {multiplicity, integration, assignment, J (Hz)}: δ 2.32 {s, 3H, H8}; δ 2.67 {s, 3H, CH3}; δ 7.06 {td, 1H, H3, J32 = J34 = 7.3 ans J35 = 1.8}; δ 7.11 {td, 1H, H4, J43 = J45 = 7.3 and J42 = 1.3}; δ 7.25 {dd, 1H, H5, J54 = 7.3 and J53 = 1.8}; δ 7.35 {dd, 1H, H2, J23 = 7.3 and J24 = 1.3}; δ 9.88 {br, 1H, NH}; δ 10.53 {s, 1H, H9}; δ 10.51 {br, 1H, NH}. 13C{1H} NMR in acetone-d6 (assignment): δ 11.06 (C8); δ 125.84 (C4); δ 126.84 (C5); δ 129.56 (C3); δ 132.11 (C2); δ 144.44 (C6); δ 151.19 (C1); δ 166.15 (C7); δ 201.03 (C]S).
Immediately before to be used, the complexes were dissolved in high purity DMSO at 1 mM as stock solution. To obtain the final assay concentrations, aliquot parts of these solutions were diluted in the suitable culture medium and the final concentration of DMSO (lower than 1%) did not reveal any cytotoxic activity. To assess 4 T1, B16F10-Nex2, A2058, SK-Mel-110 and SK-Mel-05 tumor cell lines viability the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used [32]. Briefly, 2.0x104 cells were plated into 96-well flat microplates incubated with different concentrations of the indicated complexes for 24 h. It was added 10 μg/ μL of MTT and incubated for 4 h, then 150 µL of solubilizing solution (10% SDS in 0.01 M HCl) was added to each well overnight to solubilize the formazan crystals. The absorbance was measured at 595 nm. A resazurin reduction assay was used to investigate cytotoxicity of the complexes toward Cal27 and HepG2 tumor cells. The assay is based on reduction of the indicator dye, resazurin, to the highly fluorescent resorufin by viable cells. Nonviable cells rapidly lose the metabolic capacity to reduce resazurin and thus do not produce a fluorescent signal [33,34]. Briefly, 2.5 × 104 cells were culture into a 96-well plate and treated with the complexes 1–7 in different concentrations for 24 h to HepG2 and 48 h to Cal27. After the incubation, the medium was removed, and resazurin 0.01 mg/mL in DMEM (Sigma-Aldrich) was added to each well, and the plates were incubated at 37 °C, under 5% CO2 for 4 h. The fluorescence was measured in a microplate fluorimeter under the excitation wavelength of 530 nm and an emission wavelength
2.3.7. [Pd(C2,N-aphox)(Cl)(tbz)] (7) White solid. Yield: 70%. M.p. > 143.9 °C (dec.). Anal. Calc. for C15H15ClN2OPdS (%): C 43.6; H 3.66; N 6.78; Found. C 44.3; H 3.77; N 6.58. FT-IR (cm−1, KBr): 3416 sh (νOH); 3280 m (νNH); 1629 s (δNH); 1466 m (νCN); 1439 m (δasCH3); 1044 s (νNO); 1019 s (βCH); 874 s (νC = S); 747 s (γCH). 1H NMR in acetone‑d6 {multiplicity, integration, assignment, J (Hz)}: δ 2.32 {s, 3H, H8}; δ 7.08 {td, 1H, H3, J32 = J34 = 7.3 and J35 = 1.8}; δ 7.14 {td, 1H, H4, J43 = J45 = 7.3 and J42 = 1.4}; δ 7.30 {dd, 1H, H5, J54 = 7.3 and J53 = 1.8}; δ 7.47 {dd, 1H, H2, J23 = 7.3 and J24 = 1.4}; δ 7.59 {m; 2H; 2Har.}; δ 7.71 {m; 1H; 619
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Scheme 1. Synthesis of the compounds of the type [Pd(C2.N-aphox)(Cl)(L)] {L = tu (1), mtu (2), dmtu (3), ptu (4), dptu (5), taa (6) and tbz (7)}.
of 590 nm. Three experiments in triplicate were performed for each cell line and the IC50 value was calculated using the GraphPad Prism 5 software. 3. Results and discussion The starting complex [Pd(C2,N-aphox)(μ-Cl)]2 was prepared from transcyclometallation reaction involving [Pd(C2,N-dmba)(μ-Cl)]2 and acetophenone oxime (aphox) [16]. The bridge-splitting reaction of [Pd (C2,N-aphox)(μ-Cl)]2 with thioureas/thioamides (L) in a 1:2 molar ratio at room temperature afforded neutral mononuclear compounds of the type [Pd(C2,N-aphox)(Cl)(L)], as depicted in Scheme 1. Complexes 1–7 are air and light stable solids, soluble in acetone, DMF and DMSO and show colour solutions that varies from light yellow to dark orange. The elemental analysis results for compounds 1–7 agree well the empirical formula [Pd(C2,N-aphox)(Cl)(L)]. All compounds have been characterized by infrared (IR), 1H- and 13C{1H}-NMR spectroscopies. Complexes 1, 3, and 6 have been characterized by X-ray crystallography. The results obtained from these techniques are discussed as follows.
Fig. 3. An ORTEP representation of the molecular structure of the cyclopalladated compound [Pd(C2,N-aphox)(Cl)(dmtu)] (3).
3.1. Single-crystal X-ray diffraction studies Molecular structures of 1, 3, and 6 were established by single crystal X-ray crystallography and ORTEP representations of compounds with the atom labelling scheme are shown in Figs. 2–4, respectively. Crystal data are listed in Table 1 and selected interatomic bond distances and angles with their estimated standard deviations are shown in Table 2. Cyclometallated compounds 1, 3, and 6 have similar mononuclear molecular structures. The palladium centre adopts a distorted square-
Fig. 4. An ORTEP representation of the molecular structure of the cyclopalladated compound [Pd(C2,N-aphox)(Cl)(taa)] (6).
planar coordination in which the orthometallated acetophenone oxime ligand acts as chelating ligand through the imine N1 and aromatic C1 atoms to yield a five-membered ring. All compounds showed comparable acute N1–Pd–C1 bite angle imposed by the chelating C2,N-aphox ligand (79.54–79.95°) which agree well with those values found for similar five-membered orthopalladated oximes [16,25,35]. The sulfur atom from thiourea/thioamide ligand and the imine N1 atom are in a
Fig. 2. An ORTEP representation of the molecular structure of the cyclopalladated compound [Pd(C2,N-aphox)(Cl)(tu)] (1). 620
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Table 1 Crystal data and structural refinement for [Pd(C2,N-aphox)(Cl)(tu)] (1), [Pd(C2,N-aphox)(Cl)(dmtu)] (3) and [Pd(C2,N-aphox)(Cl)(taa)] (6). Empirical formula
C9H12ClN3OPdS (1)
C11H16ClN3OPdS (3)
C10H13ClN2OPdS (6)
Formula weight Temperature (K) Crystal system Space group
352.13 296(2) Orthorhombic P212121
380.18 296(2) Monoclínic C2/c
351.13 296(2) Triclinic
P1
Unit cell dimensions a (Å) b (Å) c (Å) α (°) β (°) γ (°)
7.05730(10) 11.1431(2) 15.6977(2) – – –
21.3481(9) 12.0602(5) 13.6945(11) – 126.1920(10) –
5.7239(2) 9.4913(4) 11.8695(5) 81.439(2) 77.3220(10) 85,528(2)
1234.47(3) 4 1.895 1.870 696 0.530 × 0.090 × 0.080 2.595–62.429 −8 ≤ h ≤ 6 −13 ≤ k ≤ 13 −15 ≤ l ≤ 19 8545 2540 (0.0202) 99.8 0.7454 and 0.5706 2540/0/148 1.080 R1 = 0.0149 wR2 = 0.0361 R1 = 0.0154 wR2 = 0.0364 0.295 and −0.267
2845.5(3) 8 1.775 1.630 1520 0.280 × 0.130 × 0.110 2.061–26.466 −26 ≤ h ≤ 26 −15 ≤ k ≤ 15 −17 ≤ l ≤ 16 17,958 2932 (0.0281) 100.0 0.7454 and 0.5694 2932/0/168 1.098 R1 = 0.0180 wR2 = 0.0439 R1 = 0.0195 wR2 = 0.0454 0.299 and −0.562
621.42(4) 2 1.877 1.855 348 0.650 × 0.150 × 0.060 1.775–26.383 −7 ≤ h ≤ 7 −11 ≤ k ≤ 11 −14 ≤ l ≤ 14 17,726 2543 (0.0256) 100.0 0.7454 and 0.6397 2543/0/148 1.127 R1 = 0.0179 wR2 = 0.0459 R1 = 0.0190 wR2 = 0.0472 0.526 and −0.236
Volume (Å3) Z Density calculated (g cm−3) Absorption coefficient (mm−1) F (0 0 0) Crystal size (mm) Θ range for collected (°) Index ranges Reflections collected Independent reflections (Rint) Completeness to Θ = 25.242° (%) Max. and min. transmission Data/Restraints/Parameters Goodness-of-fit on F2 Final R indices [I > 2σ(I)] R indices (all data) Largest difference peak and hole (e Å3)
found in [PdCl(C2,N-dmba)(tu)] (2.314(1) Å) [24] but comparable to those values obtained for [Pd(phen)(tu)2](NO3)2 (2.270–2.293 Å), in which Pd–S bonds are trans to N(sp2) atoms [36].
Table 2 Selected geometric parameters for [Pd(C2,N-aphox)(Cl)(tu)] (1), [Pd(C2,Naphox)(Cl)(dmtu)] (3) and [Pd(C2,N-aphox)(Cl)(taa)] (6). 1
3
6
Bond lenght (Å) Pd–N1 Pd–C1 Pd–Cl1 Pd–S1 C(9)–S(1) C(9)–N(2) C(9)–N(3)
2.031(2) 1.998(3) 2.4689(7) 2.3029(8) 1.716(3) 1.309(4) 1.314(4)
2.0259(14) 1.9935(19) 2.4503(5) 2.3063(5) 1.7252(18) 1.325(2) 1.324(2)
2.0319(17) 1.9949(19) 2.4494(5) 2.2887(5) 1.690(2) 1.305(3) –
Bond angle (°) N1—Pd—C1 N1—Pd—Cl1 N1—Pd—S1 S1—Pd—Cl S1—Pd—Cl1 C1—Pd—Cl1
79.75(10) 88.93(6) 170.48(8) 91.74(8) 99.62(3) 168.63(8)
79.61(7) 88.78(5) 172.68(4) 94.77(5) 96.844(18) 168.38(5)
79.87(7) 90.84(5) 174.87(5) 96.11(6) 93.15(2) 170.70(6)
3.2. IR and NMR spectra IR results give important evidences about the occurrence of the orthometallation reaction on acetophenone oxime. The most useful bands are those resulting from the C]N and NeO stretching of the oxime group, and CeH out-of-plane deformation vibrations (γCH) of the aromatic ring. The IR spectrum of the acetophenone oxime (aphox) exhibits bands at 1680 and 921 cm−1, attributed to νC = N and νNO vibrational modes, respectively [37]. The appearance of two intense γCH absorptions at 757 and 691 cm−1 supports the monosubstituted nature of the benzene ring in aphox [38]. Upon cyclopalladation, relevant spectral changes are observed. For instance, in the IR spectrum of the [Pd(C2,N-aphox)(μ-Cl)]2 precursor, the νC = N band is shifted 44 cm−1 to lower frequencies compared to the aphox ligand, being consistent with coordination of the imine nitrogen atom [17]. In addition, the shift of ca. 100 cm−1 to higher frequencies of the νNeO agrees well with the expected strengthening of the NeO bond when oximes are N-coordinated [39,40]. The presence of a sharp νOH absorption at 3430 cm−1 also indicates that the oxime coordination takes place through the nitrogen atom. The number of γCH bands is also used as diagnosis for orthopalladation of acetophenone oxime. In this case, the appearance of only one intense γCH band at 752 cm−1 for [Pd(C2,Naphox)(μ-Cl)]2, instead of two γCH absorptions for monosubstituted benzene, strongly supports the o-di-substitution of aphox [38]. Besides the bands of orthometallated acetophenone oxime, the IR spectra of mononuclear compounds 1–7 also revealed the presence of the typical absorptions of thioamide-type ligands. In all cases, thioureas and thioamides coordinate via S atom in 1–7. Complexation through S
trans arrangement with a N1ePdeS1 bond angle of 170.40–174.92°. The remaining coordination site is occupied by a chlorido ligand transpositioned to the carbopalladated C1 atom. The Pd–C1 bond distances in 1, 3 and 6 (1.9915–2.0010 Å) are characteristic of the Pd–C(sp2) bonds observed in related cyclopalladated compounds [16,25,35]. In all three compounds, the Pd–Cl bond lengths (2.4489–2.4696 Å) are quite similar and slightly longer than the upper limit of the expected interval of PdeCl bond distances (2.37–2.45 Å) [16,25]. Such stretching of the PdeCl bond has already been observed in similar orthopalladated oximes because of intramolecular OH⋯Cl hydrogen bonding [16]. In the present case, chlorido ligands in 1, 3 and 6 take part in intramolecular OH⋯Cl and/or intermolecular NH⋯Cl hydrogen bonds. The PdeS1 distances of 2.2882–2.3068 Å are slightly shorter than that 621
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atom induces shifting and intensity changes in IR bands with considerable contribution from νCN and νC = S modes [23,41]. The shift to low frequency of the νC = S band together with a high frequency displacement of νCN absorption, when compared with those of the free ligands [42–48], suggests the S-coordination [23]. Hydrogen and carbon atoms in 1–7 were fully assigned from the connectivity observed in HSQC and HMBC NMR spectra. The 1H NMR spectra of l-7 exhibits only one set of signals, which suggests that the presence of only one geometrical isomer in solution. The appearance of only 4 aromatic resonances at, approximately, 7.40 (H2, dd, J23 ≅ 7.4 and J24 ≅ 1.2 Hz), 7.02 (H3, td, J32 = J34 ≅ 7.4 and J35 ≅ 1.7 Hz), 7.10 (H4, td, J43 = J45 ≅ 7.4 and J42 ≅ 1.2 Hz) and 7.25 ppm (H5, dd, J54 ≅ 7.4 and J53 ≅ 1.7 Hz) is consistent with the presence of the C2,Northopalladated aphox moiety in the molecular structures of 1–7. NMR spectroscopy also unequivocally allows detecting the S-coordination of thioamides in 1–7. Significant downfield shifts are observed for NH and eNH2 resonances as a consequence of the enhanced double bond character of CeN bond which hampers the rotation about CeN bond together with a weakening of C]S bond on S-coordination of thioamides [23,49,50]. In the 1H NMR spectra of cyclopalladated compounds bearing unsubstituted thiourea (1), thioacetamide (6) and benzothioamide (7), the NH2 protons are split into two broad signals probably due to internal and external NeH environments [50,51]. In the case of coordinated N-methylthiourea (mtu), two geometric isomers (syn and anti) can be observed at low temperatures (210 K) depending on the orientation of the methyl group with respect to the S atom [49,50]. In the case of the compound [Pd(C2,N-aphox)(Cl)(mtu)] (2), the appearance of a set of broadened signal over the 7.50–8.61 ppm range, attributed to NH2 and NH groups, together with two broadened singlets centered at 2.99 ppm (N-methyl group) suggests the presence of two exchanging conformers in solution at room temperature. The 1H NMR spectrum of [Pd(C2,N-aphox)(Cl)(dmtu)] (3) displays two broadened NH signals at 7.50 and 8.79 ppm together with two resonances at 2.91 and 3.22 ppm ascribed to N-methyl groups, indicating that dmtu ligand adopts a syn–anti arrangement. Such finding has already been observed for other Pd(II) and Pt(II) complexes containing symmetrically substituted N,N’-di-alkyl thioureas [49,50]. Regarding the 1H NMR spectra of [Pd(C2,N-aphox)(Cl)(ptu)] (4) and [Pd (C2,N-aphox)(Cl)(dptu)] (5), one NH signal is clearly detected at ca. 10.2–10.3 ppm, but the remaining NH signal is masked by the intense aromatic multiplets. 13 C{1H}-NMR spectra of 1–7 showed that the signal of the azomethine eC]N atom is shifted to ca. 10 ppm downfield with respect to the free aphox (156.00 ppm) [11]. This deshielding supports the coordination through the iminic nitrogen in all cyclopalladated compounds. The existence of C2,N-orthopalladated aphox moiety is confirmed by the appearance of its six 13C signals at, approximately, 151.40 (C1), 132.45 (C2), 129.40 (C3), 125.70 (C4), 126.65 (C5) and 144.45 ppm (C6). The additional quaternary 13C signal attributed to the metalated carbon (C1) is downfield shifted relative to the free ligand (126.00 ppm) [11,51], confirming the cyclopalladation of aphox in 1–7. The 13C signals of the methyl substituents of coordinated thioamides in 2, 3 and 6 are masked by the 13CD3 signal of acetone-d6 (29.84 ppm) as can be confirmed by the HSQC NMR spectra where protons of the methyl groups are correlating with a signal under that acetone-d6 signal. In the 13C{1H}-NMR spectra of complexes 4, 5 and 7, some aromatic carbons of the phenyl substituents of thioamides absorb at the same frequency, hindering their distinction in the spectra. The S-coordination of thioamide ligands in 1–7 is evidenced in all compounds by the upfield shift of the 13C = S signal relative to the free ligand, as a result of the weakening of C]S bond upon PdeS bond formation [26,49,50].
needed to inhibit 50% of the cellular proliferation) of complexes 1–7 with cisplatin against murine {breast (4T1), melanoma (B16F10Nex2)}, human {melanomas (A2058, SK-Mel-110 and SK-Mel-05), oral adenosquamous carcinoma (Cal27), liver (Hep-G2)} cancer cell lines. It can be seen in Table 3 that compounds 1–7 showed different cytotoxicity pattern depending on the tumor cell line assayed. In most of the cases, a significant number of compounds showed IC50 values lower or like those of cisplatin. From the inspection of cytotoxicity results against murine (4 T1, B16F10-Nex2), human melanoma (A2058, SK-Mel-110 and SK-Mmel-05) and hepatocellular carcinoma (HepG2) cell lines, some interesting trends can be noticed. In general, cyclopalladated compounds containing thioureas/thioamides with phenyl substituents (compounds 4, 5 and 7) tend to be more active than their counterparts bearing thiourea/N-methylthiourea/thioacetamide (1, 2 and 6). Compound [Pd(C2,N-aphox)(Cl)(dmtu)] (3), where dmtu = N,N’-dimethylthiourea, exhibited an intermediate character, being as active as 4, 5 and 7 depending on the tested cell line. This finding suggests that lipophilicity plays an important role in the cytotoxicity. Among them, compound [Pd(C2,N-aphox)(Cl)(tbz)] (7) displayed the best cytotoxic profile, showing a greater potency than cisplatin in most of these cell lines. In addition, the cytotoxicity of the complexes to human SK-Mel-110 melanoma cells is of interest as this cell line contains p53 mutations and is very resistant to apoptosis induction [53]. Regarding the cytotoxicity results obtained against oral adenosquamous carcinoma cell line (Cal27), no structure–activity relationship can be rationalized. Compound [Pd(C2,N-aphox)(Cl)(dmtu)] (3) showed the highest potency, with a IC50 value of 29.21 ± 1.29 μM. At this point, one could ask why a significant number of cyclopalladated compounds described here is more active than cisplatin, especially those bearing thioureas/thioamides with phenyl substituents in 4, 5 and 7. We assumed that such difference could be explained, at least in part, on the basis of kinetics considerations. It is well established that mechanism of ligand exchange in square-planar Pd and Pt compounds is associative, whose rate can be decreased/inhibited by steric crowding at the reaction centre [54,55]. Therefore, the presence of bulky substituents in the ancillary S-donor ligands in 4, 5 and 7, together with the chelating C2,N-aphox ligand, is expected to enhance their kinetic stability on the biological media and, consequently, their structures would remain intact long enough to interact with the pharmacological target(s). In addition, it is worth mentioning that cyclopalladated compounds exhibit distinct cell death mechanisms when compared to that of cisplatin. While DNA is considered to be the main target of cisplatin, a growing body of evidence has demonstrated that cyclopalladated compounds exert their cytotoxic effects by targeting mitochondria [9] or lysosomes [56]. Nevertheless, further studies are required in order to confirm this assumption. 4. Conclusions In conclusion, a family of seven new palladium compounds containing orthometallated acetophenone oxime and S-based molecules as co-ligands has been successfully synthesized, and the molecular structures of three of them have been determined. Cleavage reactions involving [Pd(C2,N-aphox)(μ-Cl)]2 and thioamides demonstrated to be an efficient method to obtain mononuclear compounds of the type [Pd (C2,N-aphox)(Cl)(L)] in moderate to good yields. The cytotoxicity profile of cyclometallated compounds 1–7 has been dependent on the tumor cell line evaluated. Under the structure-activity relationship point of view, it was noticed that the compounds which contain phenyl substituents at the ancillary S-donor ligands exhibit lower IC50 values towards murine (4 T1, B16F10-Nex2), human melanoma (A2058, SK-Mel-110 and SK-Mel-05) and hepatocellular carcinoma (HepG2) cell lines. In general, the cyclopalladated compounds obtained in this work demonstrated to be more active than cisplatin to seven tumor cell lines, opening new perspectives for the development of new metal-based anticancer agents.
3.3. Cytotoxic activities of complexes (1–7) against tumor cell lines studied Table 3 exhibits the comparative IC50 values (the concentration 622
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Table 3 Cytotoxicity activities (IC50, µM) of the compounds 1–7 against murine {breast (4 T1) and melanoma (B16F10-Nex2)} and human {melanoma (A2058, SK-Mel-110, SK-Mel-05), oral adenosquamous carcinoma (Cal27) and liver (HepG2)} cancer cell lines. Compound
1 2 3 4 5 6 7 Cisplatin a
Cell lines 4 T1
B16F10-nex2
A2058
SK-Mel-110
SK-Mel-05
Cal27
84.62 ± 2.14 56.76 ± 3.54 47.17 ± 2.61 38.68 ± 3.57 45.87 ± 0.74 53.41 ± 2.97 34.70 ± 2.54 108.54 ± 15.78
68.42 54.47 43.97 33.07 50.35 43.69 25.92 23.14
192.00 ± 59.92 189.49 ± 19.87 93.03 ± 20.29 89.72 ± 15.82 82.23 ± 2.46 107.15 ± 16.77 65.05 ± 7.34 111.85 ± 27.42
> 100 102.05 ± 4.95 66.79 ± 12.1 60.54 ± 1.88 61.18 ± 2.00 80.85 ± 3.87 51.05 ± 4.14 85.00 ± 11.03
137.42 ± 10.11 88.39 ± 2.86 72.49 ± 2.25 50.05 ± 7.47 59.20 ± 2.13 88.55 ± 8.04 49.57 ± 2.97 78.24 ± 5.83
73.14 51.66 29.21 55.06 47.79 77.70 69.45 –
± ± ± ± ± ± ± ±
5.77 4.30 7.23 3.77 5.02 6.07 3.52 1.27
HepG2 ± ± ± ± ± ± ±
3.88 6.44 1.29 0.47 2.57 2.17 3.57
41.48 41.42 31.59 27.72 15.92 44.56 33.32 60.30
± ± ± ± ± ± ± ±
4.02 2.29 0.81 1.00 3.84 6.03 5.88 15.10a
Ref. [52].
Acknowledgments
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