(aminomethyl)naphthoquinone Mannich bases

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Novel platinum(II) complexes of 3-(aminomethyl)naphthoquinone Mannich bases: synthesis, crystal structure and cytotoxic activities† Amanda P. Neves,a Gustavo B. da Silva,a Maria D. Vargas,*a Carlos B. Pinheiro,b Lorenzo do C. Visentin,c Jos´e D. B. M. Filho,d Ana J. Ara´ujo,d Let´ıcia V. Costa-Lotufo,d Cl´audia Pessoad and Manoel O. de Moraesd

Downloaded by Virginia Commonwealth University on 06 November 2010 Published on 24 September 2010 on http://pubs.rsc.org | doi:10.1039/C0DT00572J

Received 28th May 2010, Accepted 21st July 2010 DOI: 10.1039/c0dt00572j The first examples of platinum(II) complexes of 3-(aminomethyl)naphthoquinone Mannich bases have been synthesised and their crystal structures are described. Neutral and charged complexes have been obtained, fully characterised and their cytotoxic activities have also been investigated. 3-[(R1 -amino)(pyridin-2-yl)methyl]-2-hydroxy-1,4-naphthoquinones (R1 = n-Bu, HL1; Bn, HL2; furfuryl, HL3; n-heptyl, HL4 and n-decyl, HL5) coordinate to platinum(II) through the two nitrogen atoms. The neutral complexes cis-[Pt(HL)Cl2 ] 1a–5a are analogous to cisplatin with the bidentate ligand HL and two chlorine atoms occupying cis positions. In the charged complexes cis-[Pt(L- )(NH3 )2 ]NO3 1b–5b the deprotonated form of the ligand L- also coordinates via the nitrogen atoms, and the other two positions around the platinum(II) ion are completed with NH3 ligands. The cytotoxic activities of all compounds have been tested for six different cancer cell lines: MDA-MB-435 (melanoma), HL-60 (promyelocytic leukaemia), HCT-8 (colon), SF-295 (brain), OVCAR-8 (ovary) and PC-3 (prostate). Proligands HL4 and HL5 have exhibited high activity against HL-60 (IC50 = 1.9 and 3.8 mmol L-1 , respectively), HCT-8 (IC50 = 1.6 and 1.7 mmol L-1 , respectively) and SF-295 (IC50 = 1.1 and 1.7 mmol L-1 , respectively). The chlorido complexes 1a–5a have shown high to moderate cytotoxic activities, complex 4a (R1 = n-heptyl) being more active than proligand HL4 against melanoma (IC50 = 6.4 and > 40 mmol L-1 , respectively) and more active than cisplatin against all tested cell lines. Among the amine charged complexes only 4b and 5b have exhibited significant cytotoxic activity against the tested cell lines, although they were only moderately active against the PC-3 cell line (IC50 = 29.9 and 15.6 mmol L-1 , respectively). In general the compounds with the longest carbon chains (R1 = n-heptyl and n-decyl) have exhibited the highest activities.

Introduction Platinum-based drugs have been extensively investigated since the first report of the tumour inhibiting properties of cisplatin [cis-diamminedichloridoplatinum(II)] by Rosenberg in 1969.1 This drug is widely used in chemotherapy to treat a variety of cancers.2 Despite its success in the treatment of certain tumours, cisplatin has shown severe side effects, such as nephrotoxicity and neurotoxicity, as well as the development of resistance, which have been limiting its effectiveness.3,4 Several “secondgeneration” drugs have been designed and shown, in some cases, improvement in the toxicity and/or potential use against cisplatinresistant tumours such as carboplatin (cis-diammine(cyclobutane1,1-dicarboxylate-O,O¢)platinum(II)) and oxaliplatin ([(1R,2R)-

a Instituto de Qu´ımica, Campus do Valonguinho, Universidade Federal Fluminense, 24020-150, Niter´oi, RJ, Brazil. E-mail: [email protected] b Departamento de F´ısica, ICEX, Universidade Federal de Minas Gerais, 31270-901, Belo Horizonte, MG, Brazil c Instituto de Qu´ımica, Universidade Federal do Rio de Janeiro, Ilha do Fund˜ao, 21945-970, Rio de Janeiro, RJ, Brazil d Departamento de Fisiologia e Farmacologia, Centro de Ciˆencias da Sa´ude, Universidade Federal do Cear´a, 60430-270, Fortaleza, CE, Brazil † Electronic supplementary information (ESI) available: 1 H, 13 C and 195 Pt NMR spectra and cyclic voltammograms (CVs). CCDC reference numbers 766250 and 766251. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c0dt00572j

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cyclohexane-1,2-diamine](ethanedioato-O,O¢)platinum(II)) which have achieved widespread clinical use.4,5 Although most of the early platinum drugs are based on the cisplatin structure, many non-traditional platinum compounds,6 including platinum(IV), trans and multinuclear compounds have been synthesised and shown promising results. For example, BBR3464, with two reactive platinum centres, exhibits high cytotoxicity and overcomes acquired cisplatin resistance.7 Even four decades after the discovery of cisplatin as an antitumour drug, the desire to find new platinum-based compounds exhibiting high cytotoxicity, low side effects and that are able to overcome clinical resistance to the currently used drugs still motivates research in this area.8 A wide variety of molecules have been attached to platinum(II), from simple amines9 to bioactive compounds, such as porphyrins, hormones and anthraquinones.10 Quinone derived compounds, such as naphthoquinones, are well known for their pharmacological activities.11,12a The quinone nucleus is present in many drugs, e.g. in anthracyclines, doxorubicin and mitomycin, which are used in the therapy of solid cancers.13 The cytotoxic activity of naphthoquinones is still not totally clear but studies have shown that it could be associated with factors such as inhibition of topoisomerase,14 intercalation on DNA15 and generation of reactive oxygen species (ROS) in a process known as quinone redox cycling.16 In this context, the design of complexes which have two biologically active fragments (ditopic Dalton Trans., 2010, 39, 10203–10216 | 10203

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Scheme 1

Chlorido 1a–5a and amino complexes 1b–5b of platinum(II) and 2-(aminomethyl)naphthoquinones.

complexes) can be an alternative approach to obtain compounds with improved cytotoxic activity.17 To our knowledge, there is no example in the literature of a 3-(aminomethyl)naphthoquinone platinum(II) complex, although copper complexes were recently described12 and a few platinum(II) complexes of aminonaphthoquinone-derivatives have been reported.18 We report herein the synthesis, crystal structures and the cytotoxic activities of a series of chlorido and amino platinum(II) complexes of the Mannich base proligands 3-[(R1 -amino)(pyridin2-yl)methyl]-2-hydroxy-1,4-naphthoquinone (R1 = n-Bu, HL1; Bn, HL2; furfuryl, HL3; n-heptyl, HL4 and n-decyl, HL5, Scheme 1).

Results and discussion Synthesis of the compounds Proligands 3-[(R1 -amino)(pyridin-2-yl)methyl]-2-hydroxy-1,4naphthoquinones (R1 = n-Bu, HL1; Bn, HL2; furfuryl, HL3; nheptyl, HL4 and n-decyl, HL5) were synthesised from the Mannich reactions19 of 2-hydroxy-1,4-naphthoquinone (lawsone) with the respective primary amines and 2-pyridinecarboxyaldehyde, in ethanol, under stirring, at room temperature (Scheme 2). According to analytical and spectroscopic data (see Experimental) the compounds were obtained in a pure state and thus did not need to be recrystallised after precipitation in solution. They are

Scheme 2 Synthesis of 3-(aminomethyl)naphthoquinones HL1–HL5 and their respective chlorido, cis-[Pt(HL1–5)Cl2 ] 1a–5a, and amino, cis-[Pt(L1-5)(NH3 )2 ]NO3 1b–5b, complexes.

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stable in the solid state, but undergo decomposition when kept in solution for long periods of time. The reactions of equimolar amounts of HL1–HL5 and cis[Pt(DMSO)2 Cl2 ]20 (Scheme 2) in dimethylformamide (DMF) yielded the chlorido complexes cis-[Pt(HL1–5)Cl2 ] 1a–5a as yellow solids. These analogues of cisplatin are stable in the solid state and in solution, insoluble in water, but very soluble in polar solvents, such as alcohols, acetone, DMSO and DMF. The amino complexes cis-[Pt(L1–5)(NH3 )2 ]NO3 1b–5b were obtained as orange powders from the reactions of the solid proligands HL1–HL5 with a solution of cis-[Pt(NH3 )2 (DMF)2 ](NO3 )2 21 in DMF (Scheme 2). These complexes are soluble in water and hygroscopic, with the exception of 4b, that is partially soluble in water, and 5b, insoluble in water due to the large carbon side chain (R1 = C10 H25 ). The proligands and complexes have been characterised by IR, UV-Vis and 1 H, 13 C and 195 Pt NMR spectroscopy, and the structures of 1a and 3b, determined by X-ray diffraction analyses. Description of the structures Single crystals of 1a and 3b were obtained by slow evaporation of their ethanol/isopropanol solutions. Compounds 1a and 3b crystallise in the monoclinic system in P21 /n and C2/c space groups, respectively. The asymmetric unit of 3b is composed by one cis-[PtL3)(NH3 )2 ]+ cation, one NO3 - counter ion and 3.5 H2 O molecules. The asymmetric unit of compound 1a is composed by two cis-[Pt(HL1)Cl2 ] molecules and one isopropanol solvent molecule. Differently from compound 1a the 2-(aminomethyl)naphthoquinone ligand of compound 3b is deprotonated. The molecular structures and numbering schemes for cis-[Pt(HL1)Cl2 ] and cis-[Pt(L3)(NH3 )2 ]NO3 are shown in Fig. 1 and 2, respectively. Details of the structure determinations and refinements are given in the Experimental. Selected bond lengths and angles are given in Table 1. In complex 1a, both symmetry independent platinum(II) ions (Pt1 and Pt2) are coordinated in a cis conformation to two chloride ions and to one chelating ligand HL1 which binds through the N2 of the pyridine ring and the aliphatic NH group, forming five membered quelate rings N1/C11/C12/N2/Pt1 and N3/C32/C31/N4/Pt2. The atoms N1, C11, C12, N2 and Pt1 are situated in a plane and the deviation of the fitted least-squares ˚ C11 being the plane through these atoms (r.m.s.) is about 0.106 A, ˚ most distant atom (-0.162(5) A). The atoms N3, C32, C31, N4 and Pt2 are also situated in a plane and the deviation of the fitted least-

Fig. 1 (a) View of the cis-[Pt(HL1)Cl2 ] 1a molecule with the labelling scheme shown in (3 4 18) direction. The second molecule in the asymmetric unit as well as the isopropanol solvent molecule were omitted for the sake of simplicity. (b) View of the asymmetric unit of the cis-[Pt(HL1)Cl2 ] structure (compound 1a) along a direction. Abnormal large displacement parameter values for C39 and C40 carbon atoms suggested that this group is disordered and they were refined with split positions. Atomic displacement ellipsoids at the 50% level. Hydrogen atoms are represented by open circles.

˚ C31 squares plane through these atoms (r.m.s.) is about 0.141 A, ˚ being the most distant atom (-0.208(7) A). The Pt–N and Pt–Cl

˚ and angles (◦ ) for cis-[Pt(HL1)Cl2 ] 1a and cis-[Pt(L3)(NH3 )2 ]NO3 3b in the crystals asymmetric units Table 1 Selected bond lengths (A) [Pt(HL1)Cl2 ] 1a Pt(1)–N(1) Pt(1)–N(2) Pt(1)–Cl(1) Pt(1)–Cl(2) N(1)–Pt(1)–N(2) N(1)–Pt(1)–Cl(1) N(2)–Pt(1)–Cl(1) N(1)–Pt(1)–Cl(2) N(2)–Pt(1)–Cl(2) Cl(1)–Pt(1)–Cl(2)

2.074(7) 2.008(8) 2.310(2) 2.313(3) 82.6(3) 176.2(2) 95.0(2) 93.1(2) 174.2(2) 89.56(9)

Pt(2)–N(3) Pt(2)–N(4) Pt(2)–Cl(3) Pt(2)–Cl(4) N(3)–Pt(2)–N(4) N(3)–Pt(2)–Cl(3) N(4)–Pt(2)–Cl(3) N(3)–Pt(2)–Cl(4) N(4)–Pt(2)–Cl(4) Cl(3)–Pt(2)–Cl(4)

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[Pt(L3)(NH3 )2 ]NO3 3b 2.073(7) 2.015(8) 2.301(2) 2.309(3) 83.0(3) 177.2(2) 94.2(2) 94.2(2) 176.2(2) 88.64(9)

Pt–N(1) Pt–N(2) Pt–N(3) Pt–N(4) N(1)–Pt–N(2) N(1)–Pt–N(3) N(2)–Pt–N(3) N(1)–Pt–N(4) N(2)–Pt–N(4) N(3)–Pt–N(4)

2.043(7) 2.031(7) 2.050(7) 2.040(7) 82.2(3) 179.2(3) 97.7(3) 94.2(3) 176.0(3) 85.8(3)

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parameters values for C39 and C40 carbon atoms of the HL1 ligand suggesting that this group is disordered. Thus, atoms C39 and C40 were refined with split positions (Fig. 1(b)). In the amino complex 3b, the platinum centre is surrounded by the quelate ligand in its deprotonated form L3- , coordinated through both Namine and Npyridine , and by two amino ligands. The charge of the cis-[Pt(L3- )(NH3 )2 ]+ monocation is neutralised by a NO3 - counter ion. The Pt–Namine and Pt–NH3 distances are in the normal range when compared to those observed for other platinum(II) complexes containing an N4 coordination environment.23 The atoms N1, N2, N3, N4 and Pt are situated in a plane and the deviation of the fitted least-squares plane ˚ N2 being the through these atoms (r.m.s.) is about 0.021 A, ˚ The packing of the molecules most distant atom (0.025(5) A). is assured by a complex 3D network of hydrogen bonds between cis-[Pt(L3)(NH3 )2 ]+ , NO3 - and H2 O molecules in which the latter occupy a cavity of approximately 7 A˚ of diameter observed in the structure projection along the c axis (Fig. 4). The structure refinements indicated that the H2 O molecules and NO3 - ions are all ordered.

Fig. 2 View of the asymmetric unit of the cis-[Pt(L3)(NH3 )2 ]NO3 3b structure with the labelling scheme. Crystallisation H2 O solvents (Ow) are also indicated despite the lack of precision in their hydrogen atoms positions. Atomic displacement ellipsoids at the 50% level. Hydrogen atoms are represented by open circles.

distance values can be considered typical and are similar to those found in other cis-[Pt(amine)2 Cl2 ] complexes.22 Strong hydrogen bonds between symmetry related naphthoquinone groups as well as between naphthoquinone and the isopropanol molecule assure the crystal packing (Fig. 3). Indeed, from the packing point of view, the two independent cis-[Pt(HL1)Cl2 ] molecules are different. Atom O2 makes hydrogen bonds towards the O7i atom (i = 1 - x, -y, 1 - z) of the isopropanol solvent molecule (O2–H2A ◊ ◊ ◊ O7i = ˚ whereas atom O5 bonds to atom O4ii (ii = 1 - x, 2.596(10) A) -1 - y, -z) from a symmetry related [Pt(HL1)Cl2 ] molecule (O5– ˚ nevertheless, as mentioned before, H5A ◊ ◊ ◊ O4ii = 2.673(10) A), packing does not change the Pt atoms coordination. The structure refinements of complex 1a indicated abnormal large displacement

Fig. 4 cis-[Pt(L3)(NH3 )2 ]NO3 3b structure projection along c direction. Grey areas indicate the channels in which only H2 O solvent molecules can be found. A complex 3D hydrogen bond network between cis-[Pt(L3)(NH3 )2 ]+ , NO3 - and H2 O assure the crystal packing.

Fig. 3 Packing of the structure of 1a indicating the most relevant hydrogen bonds between symmetry related cis-[Pt(HL1)Cl2 ] molecules and between cis-[Pt(HL1)Cl2 ] molecule and the isopropyl alcohol solvent molecule. Symmetry operations: i = 1 - x, -y, 1 - z; ii = 1 - x, -1 - y, -z; iii = x, -1 + y, -1 + z.

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Table 2 Electrochemical data at a scan rate of 100 mV s-1 obtained for HL1–HL5, 1a–5a and 1b–5b in CH3 OH + 0.1 mol L-1 n-Bu4 NClO4 , at 25 ◦ C

HL1 HL2 HL3 HL4 HL5 1a 2a 3a 4a 5a 1b 2b 3b 4b 5b

E pIc /V

E pIa /V

E pIIc /V

E pIIa /V

E pIIa a /V

E pIIIc /V

E pIIIa /V

DE p /V

— — -1.036 — — -0.652 -0.598 -0.640 -0.653 -0.639 — — — — —

— — -0.707 — — -0.021 -0.102 -0.068 -0.003 -0.066 — — — — —

— — -1.200 — — -1.158 -1.128 -1.145 -1.172 -1.151 — — — — —

— — -0.807 — — -1.117 -0.977 -1.009 -0.976 -0.981 — — — — —

— — — — — hidden -1.090 -0.917 hidden -0.912 — — — — —

-1.141 -1.156 — -1.183 -1.218 — — — — — -1.116 -1.068 (-0.848)c -1.067 -1.158 -1.146

-0.811 -0.755 — -0.816 -0.831 — — — — — -0.966 -0.848 -0.890 -0.923 -0.930

0.330 0.401 0.329 (0.393)b 0.367 0.387 0.631 (0.041)b 0.496 (0.151)b 0.572 (0.136)b 0.650 (0.196)b 0.573 (0.170)b 0.150 0.220 0.177 0.235 0.216

Data from voltammetric experiments in an anodic scan; potential values are reported vs. FcH/FcH+ . b Data in parentheses correspond to the DE p of the second process. c Data in parentheses correspond to peak IIIc* (Fig. S36, ESI†).

a

In both complexes, the coordination polyhedron around the platinum(II) ion corresponds to a square-planar geometry with only small angular distortions. Their unit cells contain a racemic mixture of S/R enantiomeric pairs due to chiral C11 carbon atom. General characterisation The FT-IR spectra of all compounds show similar profiles, with the O–H and N–H stretching bands appearing around 3400 and 3200 cm-1 , respectively, and the aliphatic and aromatic C–H bands, in the 3100–2800 cm-1 range. The carbonyl n CO absorptions appear as a single band around 1680 cm-1 in the ligands spectra, and as two bands around 1680 and 1645 cm-1 , in the spectra of the complexes. The electronic spectra, recorded in methanol, are characterised by one intense absorption around 270 nm attributed to the p–p* transitions of the naphthoquinone ring, and three ligand based transitions in the 330–495 nm range.12,24 Significant changes in the 1 H-NMR spectra of the proligands are observed upon coordination to the platinum(II) ion. The strongest evidence of complexation is the shift of hydrogen H14, in the ortho position to the pyridine nitrogen. This trend is observed in the spectra of all chlorido complexes (see Experimental and ESI†), when compared with the free ligands, e.g., this hydrogen appears at d 8.48 in the spectrum of HL1 and is shifted to d 9.42 in that of its chlorido complex 1a. In the spectra of some complexes it is also possible to observe the N–H hydrogen (as a doublet around d 6.4) and the coupling with its neighbour proton H11, which also appears as a doublet (Fig. S16, S22, S25, S28, S31, S40 and S43, ESI†). These observations indicate that coordination to the metal occurs through both nitrogen atoms. The other peaks were found in the expected regions. The 195 Pt NMR spectra of the chlorido complexes 1a–5a were measured in CDCl3 or DMF-d 7 and gave a single peak from d -2148 to -2113 ppm corresponding to a cis-N2 Cl2 environment.25 Those of the charged amino complexes 1b–5b were recorded in D2 O or DMF-d 7 , and yielded a single peak from d -2532 to -2510 ppm, confirming an N4 environment around the platinum atom.26 This journal is © The Royal Society of Chemistry 2010

Electrochemistry studies The electrochemical behaviour of proligands HL1–HL5 and their respective platinum complexes 1a–5a and 1b–5b was investigated in methanol solutions (at 1.0 ¥ 10-3 mol L-1 ) containing 0.1 mol L-1 n-Bu4 NClO4 as the supporting electrolyte, under an argon atmosphere. Electrochemical data are summarised in Table 2. The aim of this study was the search for possible correlations between redox potential (i.e. the ease of reduction represented by E pc , E 1 or 2 E redox ) and cytotoxic activity (given by IC50 values – see below). Few examples of positive correlations between the ease of reduction of naphthoquinones and biological activities have been described in the literature.27,11b In general, naphthoquinones exhibit two classical one-electron transfer processes corresponding to the conversion of quinone into semiquinone (process I – Scheme 3) and semiquinone into cathecol (process II – Scheme 3), in aprotic media.27,28 However, in protic solvents these systems can undergo two rapid one-electrontransfer processes, which are close enough to merge into one CV wave29–31 (process III – Scheme 3). The cyclic voltammograms (CVs) of the proligands showed one two-electron-transfer redox couple (except that of HL3, see below), attributed to the conversion of the quinone into cathecol29 and labeled IIIc (cathodic) and IIIa (anodic) in the voltammogram of HL1 in Fig. 5. Wave IIIc is associated with the formation of the respective double-oxidised species. This behaviour, usually observed for naphthoquinone systems in protic solvents, is explained by the fact that protic solvents are capable of making stronger hydrogenbonding interactions with the cathecol species than with the semiquinone radical, therefore favouring reduction to the cathecol species.29,30 In addition, if the hydrogen of the naphthoquinone OH group is abstracted, the negative charge is delocalized in the quinone ring. As a consequence the second redox process is so rapid that the two processes merge into a single one.28 In the absence of a protic solvent or delocalised negative charge effect, however, the two one-electron-transfer processes are slow and clearly observed, since it should be harder to add an electron to a species that is already negatively charged. Dalton Trans., 2010, 39, 10203–10216 | 10207

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Scheme 3 Redox behaviour of the 3-(aminomethyl)naphthoquinones (ANQ) in protic solvents: process I: conversion of the 2-(aminomethyl)naphthoquinone (ANQ) into semi-2-(aminomethyl)naphthoquinone (SANQ); process II: conversion of SANQ into aminomethyl-cathecol (ACAT); process III: conversion of the ANQ zwitterion into ACAT zwitterion.

processes approach electrochemical low reversibility at slow scan rates33 (Table 2). The CVs of the chlorido and amino platinum complexes 1a and 1b are shown in Fig. 6. For the chlorido complexes, coordination of the 3-(aminomethyl)naphthoquinone was expected to prevent intramolecular phenolic hydrogen deprotonation and therefore two one-electron-transfer redox processes were anticipated and indeed were observed according to the classical quinone redox behaviour.27–29,34

Fig. 5 Cyclic voltammograms of HL1 and HL3 obtained with a glassy carbon electrode (3 mm) in 0.1 mol L-1 n-Bu4 NClO4 /CH3 OH. The potential scan was initiated in the anodic direction. The cathodic (Ic, IIc, IIIc) and anodic (Ia, IIa, IIIa) peaks are indicated.

Considering that the 3-(aminomethyl)naphthoquinones HL1–HL5 may be found in their zwitterionic form in protic solvents,19 the kinetics of the redox processes is strongly influenced by the intramolecular phenolic hydrogen dissociation constant.32 The fact that only one CV wave is observed for compounds HL1, HL2, HL4 and HL5 and two one-electron transfer processes are clearly observed in the CV of HL3 (Fig. 5 and Table 2), indicates that the equilibrium between the zwitterionic and neutral species is shifted towards the neutral form in HL3,19 most probably due to a decrease in the basicity of the amine nitrogen as the result of a hydrogen-bonding interaction with the furfuryl oxygen. The observed DE p = E pa - E pc values are larger than 90 mV for all processes, at scan rates of 100 mV s-1 , indicating that the 10208 | Dalton Trans., 2010, 39, 10203–10216

Fig. 6 Cyclic voltammograms of complexes 1a and 1b obtained with a glassy carbon electrode (3 mm) in 0.1 mol L-1 n-Bu4 NClO4 /CH3 OH. The potential scan was initiated in the anodic direction. Both cathodic and anodic peaks are indicated.

For the amino complexes that contain a deprotonated 3-(aminomethyl)naphthoquinone ligand (supported by X-ray This journal is © The Royal Society of Chemistry 2010

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Fig. 7 (A) Cyclic voltammogram of 1b in the absence and presence of HCl(aq) . (B) Cyclic voltammogram of 1a in the absence and presence of NaOH(aq) . CVs were obtained with a glassy carbon electrode (3 mm) in 0.1 mol L-1 n-Bu4 NClO4 /CH3 OH. The potential scan was initiated in the anodic direction. Both cathodic and anodic peaks are indicated.

crystallographic data), only one two-electron-transfer redox process was expected and observed in all cases (Fig. 6).32 A comparison between the electrochemical data obtained for the proligands HL1–HL5 and the cationic amino platinum complexes 1b–5b shows a positive shift of the cathodic waves and a negative shift of the anodic waves upon complexation. This observation indicates that electron density is withdrawn from the negatively charged quinone nucleus by the platinum(II) cation via hydrogen bonding of the amine hydrogen with the phenoxide oxygen. As a consequence, the ligand becomes more susceptible to reduction which is expected to result in increased cytotoxic activity as the result of easier generation of reactive oxygen species (ROS).16 In addition, the redox processes become more reversible as shown by the decrease in the DE p values observed in the CVs of the complexes. The intermediate shoulders IIa*, present in the CVs of some of the chlorido complexes (Fig. 21, 24 and 30, ESI†), and IIIc*, in 2b CV (Fig. S36, ESI†), are related to the oxidation and reduction of hydrogen-bonded intermediates.28 The CV of the amino complex 1b was also taken in the presence of a proton source (Fig. 7A). As expected, the electrodic mechanism is strongly affected. The 3-(aminomethyl)naphthoquinone is protonated and two one-electron-transfer redox couples appear, in accord with the classical quinone electrochemistry behaviour.27 The two couples observed are indicated as Ic¢/Ia¢ and IIc¢/IIa¢ at potential values of -0.688/0.122 and -1.023/-0.729V vs. FcH/FcH+ , respectively. Considering the acidic nature of endocytic compartments such as endosomes (pH 5–6) and lysosomes (pH 4–5) this information may be valuable in the interpretation of cytotoxic activity data. The electrodic mechanism is also affected by the presence of OH- (Fig. 7B) that leads to deprotonation of the 2(aminomethyl)naphthoquinone in complex 1a and therefore, to the presence of one two-electron-transfer redox couple in the CV.32 This couple is indicated as IIIc¢ and IIIa¢ at potential values of -1.169 and -1.116 V vs. FcH/FcH+ , respectively. The negative shift of the waves and the presence of only one couple of waves confirm the influence of the hydroxyl proton on the electrodic reduction mechanism.27 This journal is © The Royal Society of Chemistry 2010

Biological studies The cytotoxicity of HL1–HL5, 1a–5a, 1b–5b and cisplatin, for comparison, has been investigated against six tumour cell lines: MDA-MB-435 (melanoma), HL-60 (promyelocytic leukaemia), HCT-8 (colon), SF-295 (brain), OVCAR-8 (ovary) and PC-3 (prostate). Doxorubicin was used as a positive control. The experimental data are presented in Table 3. According to the IC50 values, proligands HL1–HL5 exhibited moderate to good activities against the tested cell lines. Exceptions were: HL1 (R1 = n-Bu) that was inactive against melanoma, ovary, prostate and leukaemia; HL2 (R1 = Bn) and HL3 (R1 = furfuryl) that were inactive against melanoma and prostate, and HL4 (R = n-heptyl) and HL5 (R = n-decyl) that were inactive and little active against melanoma, respectively. In contrast, compounds HL4 and HL5 exhibited high activities against the other cell lines tested, with IC50 values around 1.5 mmol L-1 against human colon (HCT-8) and human brain (SF-295) cell lines. These data indicate clearly that activity depends strongly on the nature of R1 , the highest values being observed, in all cases, for the derivatives containing long aliphatic carbon chains (n-heptyl, HL4, and n-decyl, HL5). Interestingly, the differences in the equilibrium constant between the zwitterionic and neutral species observed for HL3 ¥ other proligands showed no effect in the IC50 values. The chlorido complexes 1a–5a were also active against the tested cell lines, with the exception of 1a that was inactive against prostate, and 2a and 3a, inactive against prostate and ovary cell lines. In contrast, the charged complexes 1b–5b were inactive against all cells tested, except 4b and 5b, that exhibited low and moderate activities (IC50 = 29.9 and 15.6 mmol L-1 , respectively) against the prostate cell line. In general, the chlorido complexes exhibited lower activity when compared with their respective proligands, however, chlorido complexes 1a–5a were more active against melanoma than proligands HL1–HL5, respectively. For instance, the increase in the activity of HL4, against melanoma, after coordination with platinum(II) was quite pronounced (IC50 > 40 mmol L-1 for HL4 and 6.4 mmol L-1 for 4a). Furthermore, complexes 4a and 5a (IC50 = 6.4 and Dalton Trans., 2010, 39, 10203–10216 | 10209

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Table 3 IC50 values in mmol L-1 of HL1–HL5, 1a–5a and 1b–5b, after 72 h incubation, compared to cisplatin

HL1 HL2 HL3 HL4 HL5 1a 2a 3a 4a 5a 1b 2b 3b 4b 5b cisplatin Doxorubicin

MDA-MB-435

HL-60

HCT-8

SF-295

OVCAR-8

PC-3

>40 >40 >40 >40 23.6 (19.2–28.6) 19.7 (17.9–25.6) 21.1 (17.3–25.6) 27.6 (24.6–30.8) 6.4 (5.1–7.8) 9.0 (7.4–11.1) >40 >40 >40 >40 >40 15.3 (11.3–20.7) 0.86 (0.62–1.2)

>40 4.7 (3.7–5.9) 5.3 (3.9–6.9) 1.9 (1.3–2.6) 3.8 (2.8–5.3) 7.1 (2.8–17.9) 9.4 (4.2–21.2) 19.5 (16.4–23.2) 5.7 (3.3–10.2) 12.5 (9.5–16.6) >40 >40 >40 >40 >40 28.6 (21.3–39.0) 0.04 (0.02–0.04)

12.0 (9.2–15.7) 2.3 (1.9–2.7) 3.9 (2.5–5.8) 1.6 (1.3–1.9) 1.7 (1.4–1.9) 21.0 (17.9–24.9) 20.7 (13.7–31.3) 29.7 (26.5–31.8) 11.5 (10.1–13.3) 15.9 (14.0–18.2) >40 >40 >40 >40 >40 12.3 (9.3–16.7) 0.07 (0.05–0.09)

9.0 (7.7–11.9) 1.4 (1.1–1.9) 2.5 (1.9–3.3) 1.1 (1.0–1.3) 1.7 (1.2–1.9) 17.4 (13.9–21.9) 25.8 (21.2–31.3) 26.2 (21.4–31.8) 7.9 (7.1–8.7) 11.8 (10.0–14.0) >40 >40 >40 >40 >40 26.7 (17.3–41.0) 0.42 (0.35–0.46)

>40 5.7 (5.1–6.2) 16.9 (13.0–22.2) 5.5 (5.0–6.0) 4.0 (2.4–5.5) 21.4 (18.6–24.7) >40 >40 14.3 (13.2–15.8) 22.9 (19.4–27.5) >40 >40 >40 >40 >40 >40 0.44 (0.3–0.52)

>40 >40 >40 7.7 (5.3–10.8) 3.3 (2.1–4.8) >40 >40 >40 23.0 (19.4–27.2) 28.0 (22.7–34.5) >40 >40 >40 29.9 (20.2–45.8) 15.6 (12.8–19.0) >40 0.44 (0.39–0.50)

Data are presented with 95% of confidence interval for MDA-MB-435 (melanoma), HL-60 (promyelocytic leukaemia), HCT-8 (colon), SF-295 (brain), OVCAR-8 (ovary) and PC-3 (prostate) cancer cells lines. Doxorubicin was used as a positive control. Experiments were performed in triplicate.

9.0 mmol L-1 , respectively) exhibited higher activity than cisplatin (IC50 = 15.3 mmol L-1 ). If the cytotoxicity of these compounds resulted only from oxidative stress and therefore were based only on their redox potential values, the cisplatin analogues 1a–5a would be expected to show the highest cytotoxicity considering that these compounds are more easily reduced than the proligands HL1–HL5 and the cationic complexes 1b–5b (Table 2). However, a direct correlation between the cyclic voltammetry results and the IC50 values could not be established. Certainly other factors are involved in the biological activity of the platinum complexes, which are directly related with the uptake of the compounds such as lipophilicity, solubility and membrane permeability that depend on the nature of R1 , and also reactivity, i.e., interaction with DNA. The nature of the leaving group as well as the type of interaction of platinum(II) complexes with DNA have been related to their cytotoxic activities.35 It is known that chlorido complexes analogous to cisplatin interact with DNA through covalent binding after replacement of the chloride ions.36 Amino complexes, however, are inert37 and therefore bind to DNA exclusively through noncovalent interactions38,39 i.e. electrostatic interactions and hydrogen bonding, which are weaker than the covalent bonds exhibited by cisplatin analogues.38 Finally, increase in the cytotoxic activity of the compounds was observed upon increase of the R1 carbon chain length. Indeed, only compounds with a large carbon chain (R1 ≥ 7) exhibited significant activity against the PC-3 cell line. One can speculate that a large organic portion enhances cellular penetration through the membranes to the nucleus, as previously observed.40

Experimental General Considerations Solvents (Vetec), 2-hydroxy-1,4-naphthoquinone (Aldrich), 2pyridinecarboxyaldehyde (Aldrich), butylamine (Aldrich), benzylamine (Aldrich), furfurylamine (Aldrich) and K2 PtCl4 10210 | Dalton Trans., 2010, 39, 10203–10216

(Aldrich) were used as supplied. Dimethylformamide (DMF) (Vetec) was distilled prior to use. cis-[Pt(DMSO)2 Cl2 ],20 cis[Pt(DMF)2 (NH3 )2 ](NO3 )2 21 and cisplatin41 were prepared as described in the literature. Microanalyses were performed using a Perkin-Elmer CHN 2400 microanalyser at Central Anal´ıtica - Instituto de Qu´ımica, Universidade Federal de S˜ao Paulo, Brazil. Melting points were obtained with a Mel-Temp II, Laboratory Devices - USA apparatus and are uncorrected. IR spectra (KBr pellets) were recorded on a FTIR Spectrum One (Perkin Elmer) spectrophotometer. 1 H NMR (300 MHz) and 13 C NMR (75 MHz) spectra were recorded with a Varian Unit Plus spectrometer in CDCl3 , D2 O, CD3 OD or DMSO-d 6 ; chemical shifts are reported in parts per million (ppm) relative to an internal standard of Me4 Si. The hydrogen signals were attributed through coupling constant values and 1 H ¥ 1 H – COSY experiments. 195 Pt NMR (64 MHz) spectra were recorded on a Bruker spectrometer at Universidade Federal de Juiz de Fora, Brazil, in CDCl3 , D2 O or DMF-d 7 . X-Ray data collection ´ was performed at the Laboratorio de Cristalografia (LabCri) of Universidade Federal de Minas Gerais (UFMG). Electronic spectra were taken on a Cary 50 (Varian) spectrophotometer using spectroscopic grade solvents (Tedia Brazil) in 10-4 mol L-1 solutions. Cyclic voltammograms were carried out with a BAS Epsilon potentiostat–galvanostat system at room temperature in methanol (spectroscopic grade – Tedia Brazil) solutions of the compounds (at 1.0 ¥ 10-3 mol L-1 ) containing 0.1 mol L-1 nBu4 NClO4 (Aldrich) as the supporting electrolyte, under an argon atmosphere. The electrochemical cell was a conventional one with three electrodes: Ag/Ag+ was used as the reference electrode, a platinum wire as the auxiliary electrode and glassy carbon as the working electrode. The spectra were obtained at 100 mV s-1 scan rate. The ferrocene/ferrocenium (FcH/FcH+ ) couple was used as the internal standard. (E 1/2 372 mV vs. Ag/Ag+ ; E 1/2 0.40 V vs. NHE).42 For the studies in the presence of HCl(aq) and NaOH(aq) , the conditions were the same as those described above but for the sequential addition of 50 mL aliquots of HCl 0.02 mol L-1 (or NaOH). Pure Argon was bubbled through the electrolytic solution to remove oxygen in all experiments. This journal is © The Royal Society of Chemistry 2010

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Synthesis of pro-ligands HL1–HL5. The novel Mannich bases HL1–HL5 were synthesised according to the methodology described recently in the literature.12 General procedure: to a suspension of lawsone (870 mg, 5 mmol) in ethanol (15 mL), kept under stirring at 22 ◦ C, were added the amine (5.5 mL) and, after about 5 min, 2-pyridinecarboxyaldehyde (0.574 mL, 6 mmol). The mixture was stirred at this temperature for 5 h, the bright orange solid was filtered, extensively washed with ethanol and dried under vacuum. For the synthesis of HL4, it was necessary to use half the amount of ethanol in order to obtain a precipitate. 3-[(Butylamino)(pyridin-2-yl)methyl]-2-hydroxy-1,4-naphthoquinone, HL1. Yield: 1.18 g (70%). m.p. 142–143 ◦ C (dec.). IR (KBr): n = 3446 (O–H), 3148 (N–H), 2964 (C–H), 2932 (C–H), 2865 (C–H), 1682 (C O), 1590 (C C/C N), 1560 (C C), 1520 (d N–H), 1472 (C C), 1372 (C C), 1277 (C–O/C–N), 1219 (C– O/C–N). 1 H NMR (300 MHz, CDCl3 ): d 8.48 (ddd, J = 4.9, 1.7, 0.9 Hz, 1H, H14), 7.84–7.79 (m, 2H, H5/H8), 7.59 (td, J = 7.4, 1.7 Hz, 1H, H16), 7.48 (td, J = 7.5, 1.5 Hz, 1H, H6 or H7), 7.42– 7.35 (m, 2H, H7 or H6/H17), 7.17 (ddd, J = 7.4, 4.9, 0.8 Hz, 1H, H15), 5.75 (s, 1H, H11), 3.35–3.23 (m, 1H, H19), 3.18–3.05 (m, 1H, H19¢), 1.99–1.84 (m, 2H, H20), 1.58–1.43 (m, 2H, H21), 0.99 (t, J = 7.3 Hz, 3H, H22). 13 C {1 H} NMR (75 MHz, DMSO-d 6 ): d 184.4, 178.7, 171.4, 157.4, 147.8, 137.4, 134.9, 133.9, 131.8, 131.0, 125.6, 125.3, 122.8, 121.5, 109.9, 57.6, 44.9, 27.7, 19.4, 13.6 ppm. Found: C, 71.3; H, 5.9; N, 8.4. Calc. for C20 H20 N2 O3 : C, 71.4; H, 6.0; N, 8.3%. UV-Vis [CH3 OH; l/nm (log e)]: 270 (4.28), 330 (3.48), 440 (3.57), 485 (3.46). 3-[(Benzylamino)(pyridin-2-yl)methyl]-2-hydroxy-1,4-naphthoquinone, HL2. Yield: 1.57 g (85%). m.p. 140–141 ◦ C (dec.). IR (KBr): n = 3447 (O–H/N–H), 3065 (C–H), 2949 (C–H), 1684 (C O), 1611 (C C/C N), 1590 (C C/C N), 1557 (C C), 1519 (d N–H), 1470 (C C), 1389 (C C), 1272 (C–O/C–N), 1261 (C–O/C–N). 1 H NMR (300 MHz, DMSO-d 6 ): d 8.66 (ddd, J = 4.8, 1.7, 1.2 Hz, 1H, H14), 8.06 (dd, J = 7.6, 1.4 Hz, 1H, H5 or H8), 7.96 (dd, J = 7.6, 1.4 Hz, 1H, H8 or H5), 7.86 (td, J = 7.7, 1.7 Hz, 1H, H16), 7.84 (td, J = 7.6, 1.4 Hz, 1H, H6 or H7), 7.72 (td, J = 7.6, 1.4 Hz, 1H, H7 or H6), 7.60–7.47 (m, 5H, Ph), 7.47–7.40 (m, 2H, H15/H17), 5.82 (s, 1H, H11), 4.37 (d, J = 13.0 Hz, 1H, H19), 4.25 (d, J = 13.0 Hz, 1H, H19¢). 13 C {1 H} NMR (75 MHz, DMSO-d 6 ): d 184.4, 179.2, 171.6, 157.2, 148.0, 137.8, 135.0, 134.1, 132.7, 132.0, 131.3, 130.4, 129.2, 128.9, 125.8, 125.5, 123.2, 122.0, 109.9, 57.8, 48.9 ppm. Found: C, 74.1; H, 5.1; N, 7.4. Calc. for C23 H18 N2 O3 : C, 74.6; H, 4.9; N, 7.6%. UV-Vis [CH3 OH; l/nm (log e)]: 270 (4.44), 330 (3.68), 440 (3.76), 495 (3.60). 3 - [(Furfurylamino) (pyridin - 2 - yl) methyl] - 2 - hydroxy - 1, 4 naphthoquinone, HL3. Yield: 1.51 g (84%). m.p. 128–129 ◦ C (dec.). IR (KBr): n = 3446 (O–H), 3059 (C–H), 2950 (C–H), 1681 (C O), 1612 (C C/C N), 1589 (C C/C N), 1567 (C C), 1521 (d N–H), 1471 (C C), 1385 (C C), 1275 (C–O/C–N), 1262 (C–O/C–N). 1 H NMR (300 MHz, DMSO-d 6 ): d 8.66 (ddd, J = 4.8, 1.7, 1.2 Hz, 1H, H14), 8.04 (ddd, J = 7.5, 1.4, 0.4 Hz, 1H, H5 or H8), 7.95 (ddd, J = 7.6, 1.4, 0.4 Hz, 1H, H5 or H8), 7.85 (td, J = 7.8, 1.8 Hz, 1H, H16), 7.83 (td, J = 7.5, 1.4 Hz, 1H, H6 or H7), 7.78 (dd, J = 1.8, 0.8 Hz, 1H, H23), 7.77 (td, J = 7.5, 1.4 Hz, 1H, H7 or H6), 7.46–7.40 (m, 2H, H15/H17), 6.65 (dd, J = 3.3, 0.8 Hz, 1H, H21), 6.57 (dd, J = 3.3, 1.8 Hz, 1H, H22), 5.84 (s, 1H, H11), 4.44 (d, J = 14.5 Hz, 1H, H19), 4.35 (d, J = 14.5 Hz, 1H, H19¢). This journal is © The Royal Society of Chemistry 2010

C {1 H} NMR (75 MHz, DMSO-d 6 ): d 184.2, 178.7, 171.1, 156.9, 147.7, 146.1, 144.0, 137.4, 134.8, 133.8, 131.7, 130.9, 125.5, 125.2, 122.8, 121.6, 112.1, 111.0, 109.6, 57.3, 55.9 ppm. Found: C, 70.2; H, 4.7; N, 7.3. Calc. for C21 H16 N2 O4 : C, 70.0; H, 4.5; N, 7.8%. UV-Vis [CH3 OH; l/nm (log e)]: 270 (4.38), 330 (3.64), 440 (3.71), 493 (3.55). 13

3-[(Heptylamino)(pyridin-2-yl)methyl]-2-hydroxy-1,4-naphthoquinone, HL4. Yield: 1.60 g (85%). m.p. 109–110 ◦ C. IR (KBr): n = 3424 (O–H), 3052 (C–H), 2927 (C–H), 2857 (C–H), 1678 (C O), 1590 (C C/C N), 1525 (d N–H), 1466 (C C), 1372 (C C), 1273 (C–O/C–N). 1 H NMR (300 MHz, CDCl3 ): d 8.47 (d, J = 4.7 Hz, 1H, H14), 7.82–7.77 (m, 2H, H5/H8), 7.59 (td, J = 7.6, 1.7 Hz, 1H, H16), 7.46 (td, J = 7.5, 1.2 Hz, 1H, H6 or H7), 7.40–7.35 (m, 2H, H7 or H6/H17), 7.16 (dd, J = 7.6, 4.7 Hz, 1H, H15), 5.73 (s, 1H, H11), 3.32–3.24 (m, 1H, H19), 3.13–3.06 (m, 1H, H19¢), 1.92 (q, J = 7.6 Hz, 2H, H20), 1.49–1.41 (m, 2H, H21), 1.39–1.32 (m, 2H, H22), 1.31–1.25 (m, 4H, H23/H24), 0.87 (t, J = 6.9 Hz, 3H, H25). 13 C {1 H} NMR (75 MHz, CDCl3 ): d 184.7, 180.9, 170.5, 155.5, 147.6, 137.2, 134.1, 133.5, 131.1, 130.8, 126.0, 125.3, 122.6, 122.2, 111.4, 58.6, 46.9, 31.4, 28.6, 26.6, 26.4, 22.4, 13.9 ppm. Found: C, 72.8; H, 7.1; N, 7.4. Calc. for C23 H26 N2 O3 : C, 73.0; H, 6.9; N, 7.4%. UV-Vis [CH3 OH; l/nm (log e)]: 269 (4.48), 332 (3.88), 440 (3.91), 488 (3.82). 3-[(Decylamino)(pyridin-2-yl)methyl]-2-hydroxy-1,4-naphthoquinone, HL5. Yield: 1.57 g (75%). m.p. 141–142 ◦ C (dec.). IR (KBr): n = 3430 (O–H), 3126 (N–H), 2926 (C–H), 2855 (C–H), 1682 (C O), 1585 (C C/C N), 1514 (d N–H), 1434 (C C), 1389 (C C), 1273 (C–O/C–N), 1233 (C–O/C–N). 1 H NMR (300 MHz, CDCl3 ): d 8.46 (ddd, J = 4.9, 1.4, 0.8 Hz, 1H, H14), 7.77 (dd, J = 7.6, 0.8 Hz, 1H, H5 or H8), 7.75 (dd, J = 7.6, 0.8 Hz, 1H, H8 or H5), 7.57 (td, J = 7.7, 1.7 Hz, 1H, H16), 7.43 (td, J = 7.6, 1.3 Hz, 1H, H6 or H7), 7.38–7.33 (m, 2H, H7 or H6/H17), 7.14 (dd, J = 7.7, 4.9 Hz, 1H, H15), 5.73 (s, 1H, H11), 3.30–3.24 (m, 1H, H19), 3.11–3.05 (m, 1H, H19¢), 1.93 (q, J = 7.6 Hz, 2H, H20), 1.44 (q, J = 7.6 Hz, 2H, H21), 1.37–1.19 (m, 12H, H22–H27), 0.87 (t, J = 7.0 Hz, 3H, H28). 13 C {1 H} NMR (75 MHz, DMSO-d 6 ): d 184.7, 180.8, 170.4, 155.5, 147.6, 137.2, 134.0, 133.5, 131.1, 130.8, 126.0, 125.2, 122.6, 122.2, 111.4, 58.7, 46.9, 31.7, 29.3, 29.2, 29.1, 28.9, 26.6, 26.4, 22.5, 13.9 ppm. Found: C, 74.1, H, 7.4, N, 6.5. Calc. for C26 H32 N2 O3 : C, 74.3; H, 7.7; N, 6.7%. UV-Vis [CH3 OH; l/nm (log e)]: 270 (4.47), 334 (3.73), 438 (3.78), 492 (3.62). Synthesis of the chlorido platinum complexes 1a–5a. To a suspension of the proligand (HL1–HL5, 0.50 mmol) in 5 mL of DMF, was added cis-[Pt(DMSO)2 Cl2 ] (232 mg, 0.55 mmol). The orange reaction mixture was stirred at room temperature for 48 h in the dark. The resulting orange solution was evaporated under reduced pressure. The orange oily product was washed with water to obtain a yellow solid which was isolated by filtration. Complexes 1a, 4a and 5a were recrystallised from a mixture of ethanol/petroleum ether or isopropanol/petroleum ether while 2a and 3a were purified by recrystallisation in CHCl3 . [Pt(HL1)Cl2 ] 1a. Yield: 180 mg (60%). m.p. 188–190 ◦ C. IR (KBr): n = 3393 (O–H/N–H), 2957 (C–H), 2958 (C–H), 2930 (C–H), 2870 (C–H), 1680 (C O), 1641 (C O), 1590 (C C/C N), 1528 (d N–H), 1478 (C C), 1459 (C C), 1374 (C C), 1351 (C C), 1274 (C–O/C–N), 1227 (C–O/C–N). 1 H NMR (300 MHz, CDCl3 ): d 9.42 (d, J = 6.1 Hz, 1H, H14), 8.14 Dalton Trans., 2010, 39, 10203–10216 | 10211

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(d, J = 7.6 Hz, 2H, H5/H8), 7.88–7.73 (m, 2H, H16/H6 or H7), 7.75 (td, J = 7.6, 1.4 Hz, 1H, H7 or H6), 7.29 (t, J = 6.1, 1H, H15), 7.09 (d, J = 7.9 Hz, 1H, H17), 6.26 (d, J = 8.7 Hz, 1H, NH), 5.81 (d, J = 8.7 Hz, 1H, H11), 3.40–3.30 (m, 1H, H19), 2.46–2.35 (m, 1H, H19¢), 1.95–1.76 (m, 2H, H20), 1.42–1.24 (m, 2H, H21), 0.89 (t, J = 7.3 Hz, 3H, H22). 195 Pt NMR (64 MHz, CDCl3 ): d -2142 ppm. Found: C, 40.4; H, 3.5; N, 4.5. Calc. for C20 H20 N2 O3 Cl2 Pt: C, 39.9; H, 3.4; N, 4.7%. UV-Vis [CH3 OH; l/nm (log e)]: 261 (4.37), 348 (3.26), 416 (3.21), 442 (2.93). [Pt(HL2)Cl2 ] 2a. Yield: 260 mg (82%). m.p. > 243 ◦ C (dec.). IR (KBr): n = 3347 (N–H), 3190 (N–H), 3110 (N–H), 3069 (C–H), 1679 (C O), 1649 (C O), 1632 (C C/C N), 1592 (C C/C N), 1577 (C C), 1476 (C C), 1454 (C C), 1363 (C C), 1351 (C C), 1284 (C–O/C–N), 1213 (C–O/C–N). 1 H NMR (300 MHz, DMF-d7 ): d 9.37 (d, J = 5.7 Hz, 1H, H14), 8.23 (d, J = 7.1 Hz, 2H, H5/H8), 8.05 (d, J = 7.4 Hz, 1H, Ph), 8.01–7.97 (m, 2H, Ph), 7.91 (td, J = 7.5, 1.3 Hz, 1H, H16), 7.86 (td, J = 7.4, 1.2 Hz, 1H, Ph), 7.47–7.43 (m, 2H, H15, Ph), 7.43–7.39 (m, 1H, NH), 7.33–7.24 (m, 3H, H17/H6/H7), 5.74 (d, J = 7.6 Hz, 1H, H11), 4.77 (dd, J = 13.4, 4.2 Hz, 1H, H19), 4.14 (dd, J = 13.4, 2.6 Hz, 1H, H19¢). 195 Pt NMR (64 MHz, DMF-d 7 ): d -2113 ppm. Found: C, 42.8; H, 2.8; N, 4.2. Calc. for C23 H18 N2 O3 Cl2 Pt·0.4H2 O: C, 42.9; H, 2.9; N, 4.4. UV-Vis [CH3 OH; l/nm (log e)]: 266 (4.26), 349 (3.43), 433 (3.46), 480 (3.35). [Pt(HL3)Cl2 ] 3a. Yield: 241 mg (77%). m.p. > 225 ◦ C (dec.). IR (KBr): n = 3445 (O–H), 3220 (N–H), 3116 (N–H), 2923 (C– H), 2851 (C–H), 1674 (C O), 1646 (C O), 1592 (C C/C N), 1533 (d N–H), 1478 (C C), 1372 (C C), 1275 (C–O/C–N), 1225 (C–O/C–N). 1 H NMR (300 MHz, DMF-d 7 ): d 9.40 (d, J = 5.9 Hz, 1H, H14), 8.08–8.00 (m, 3H, H5/H8/H6 or H7), 7.91 (td, J = 7.4, 1.5 Hz, 1H, H16), 7.87 (td, J = 7.4, 1.4 Hz, 1H, H7 or H6), 7.55 (d, J = 8.0 Hz, 1H, H17), 7.51 (t, J = 6.7 Hz, 1H, H15), 7.42 (dd, J = 1.8, 0.7 Hz, 1H, H23), 7.17–7.12 (m, 1H, NH), 6.89 (d, J = 3.2 Hz, 1H, H21), 6.29 (dd, J = 3.2, 1.8 Hz, 1H, H22), 5.88 (d, J = 8.0 Hz, 1H, H11), 4.63 (dd, J = 14.6, 6.1 Hz, 1H, H19), 4.51 (dd, J = 14.6, 2.4 Hz, 1H, H19¢). 195 Pt NMR (64 MHz, DMF-d 7 ): d -2120 ppm. Found: C, 40.5; H, 2.6; N, 4.4. Calc. for C21 H16 N2 O4 Cl2 Pt: C, 40.3; H, 2.6, N, 4.5. UV-Vis [CH3 OH; l/nm (log e)]: 272 (4.37), 343 (3.68), 443 (3.75), 481 (3.24). [Pt(HL4)Cl2 ] 4a. Yield: 284 mg (79%). m.p. 138–140 ◦ C. IR (KBr): n = 3195 (N–H), 2953 (C–H), 2925 (C–H), 2854 (C– H), 1680 (C O), 1642 (C O), 1591 (C C/C N), 1534 (d N–H), 1479 (C C), 1459 (C C), 1383 (C C), 1370 (C C), 1351 (C C), 1276 (C–O/C–N), 1224 (C–O/C–N). 1 H NMR (300 MHz, CDCl3 ): d 9.44 (d, J = 5.6 Hz, 1H, H14), 8.16 (dd, J = 7.4, 0.9 Hz, 1H, H5 or H8), 8.15 (dd, J = 7.6, 1.1 Hz, 1H, H8 or H5), 7.87–7.81 (m, 2H, H16/H6 or H7), 7.77 (td, J = 7.6, 1.1 Hz, 1H, H7 or H6), 7.30 (t, J = 6.8, 1H, H15), 7.07 (d, J = 8.0 Hz, 1H, H17), 6.27 (d, J = 8.7 Hz, 1H, NH), 5.82 (d, J = 8.7 Hz, 1H, H11), 3.36–3.33 (m, 1H, H19), 2.44–2.37 (m, 1H, H19¢), 1.92–1.82 (m, 2H, H20), 1.35–1.17 (m, 8H, H21-H24), 0.83 (t, J = 6.9 Hz, 3H, H25). 195 Pt NMR (64 MHz, CDCl3 ): d -2147 ppm. Found: C, 43.4; H, 4.6; N, 4.0. Calc. for C23 H26 N2 O3 Cl2 Pt·CH3 CH2 OH: C, 43.5; H, 4.7; N, 4.1%. UV-Vis [CH3 OH; l/nm (log e)]: 271 (4.54), 334 (3.72), 431 (3.78), 481 (3.28). [Pt(HL5)Cl2 ] 5a. Yield: 240 mg (70%). m.p. 136–137 ◦ C. IR (KBr): n = 3144 (N–H), 2923 (C–H), 2852 (C–H), 1679 (C O), 10212 | Dalton Trans., 2010, 39, 10203–10216

1644 (C O), 1591 (C C/C N), 1538 (d N–H), 1522 (C C), 1459 (C C), 1374 (C C), 1351 (C C), 1275 (C–O/C–N), 1228 (C–O/C–N). 1 H NMR (300 MHz, CDCl3 ): d 9.43 (d, J = 5.4 Hz, 1H, H14), 8.15 (d, J = 7.7 Hz, 2H, H5/H8), 7.88–7.80 (m, 2H, H16/H6 or H7), 7.76 (t, J = 7.7 Hz, 1H, H7 or H6), 7.29 (t, J = 6.8, 1H, H15), 7.07 (d, J = 8.0 Hz, 1H, H17), 6.29 (d, J = 8.6 Hz, 1H, NH), 5.83 (d, J = 8.6 Hz, 1H, H11), 3.38–3.30 (m, 1H, H19), 2.45–2.35 (m, 1H, H19¢), 1.92–1.82 (m, 2H, H20), 1.35–1.15 (m, 14H, H21–H27), 0.84 (t, J = 7.0 Hz, 3H, H28). 195 Pt NMR (64 MHz, CDCl3 ): d -2148 ppm. Found. C, 43.1; H, 4.5; N 3.8. Calc. for C26 H32 N2 O3 Cl2 Pt·1.7H2 O: C, 43.5; H, 5.0; N, 3.9%. UVVis [CH3 OH; l/nm (log e)]: 271 (4.34), 338 (3.69), 436 (3.73), 476 (3.13). Synthesis of the amino platinum complexes 1b–5b. The proligand HL1–HL3 (1.1 mmol) was added as a solid to a previously prepared solution of cis-[Pt(NH3 )2 (DMF)2 ](NO3 )2 21 (1 mmol) in 10 mL of DMF and the orange reaction mixture was left stirring at room temperature in the dark. After 72 h the resulting orange solution was evaporated under vacuum and the product, extracted with water. After evaporation of the water, the complex was obtained as an orange solid after precipitation in a mixture of ethanol/petroleum ether. Same procedure was followed for the synthesis of complexes 4a and 5a, except for the amount of proligands HL4 and HL5 (0.8 mmol). After 72 h, the DMF was evaporated and the complexes were obtained as orange solids after addition of water to the residue. The compounds were extensively washed with water, filtered off and dried under vacuum. [Pt(L1)(NH3 )2 ]NO3 , 1b. Yield: 180 mg (29%). m.p. > 221 ◦ C (dec.). IR (KBr): n = 3484 (O–H), 3163 (N–H), 2958 (C–H), 2928 (C–H), 2871 (C–H), 1676 (C O), 1612 (C C/C N), 1587 (C C/C N), 1533 (d N–H), 1484 (C C), 1373 (C C/N O), 1333 (C C), 1280 (C–O/C–N), 1242 (C–O/C–N). 1 H NMR (300 MHz, D2 O): d 8.33 (d, J = 6.1 Hz, 1H, H14), 8.03 (td, J = 8.0, 1.3 Hz, 1H, H16), 7.89 (dd, J = 7.6, 1.3 Hz, 1H, H5 or H8), 7.84 (dd, J = 7.6, 1.3 Hz, 1H, H8 or H5), 7.73 (td, J = 7.6, 1.3 Hz, 1H, H7 or H6), 7.63 (td, J = 7.6, 1.3 Hz, 1H, H6 or H7), 7.46 (t, J = 6.1 Hz, 1H, H15), 7.38 (d, J = 8.0 Hz, 1H, H17), 6.50 (d, J = 6.4 Hz, 1H, NH), 5.80 (d, J = 6.4 Hz, 1H, H11), 2.93–2.84 (m, 2H, H19), 1.96–1.83 (m, 2H, H20), 1.44–1.28 (m, 2H, H21), 0.88 (t, J = 7.4 Hz, 3H, H22). 195 Pt NMR (64 MHz, D2 O): d -2530 ppm. Found: C, 36.7; H, 4.0; N, 10.6. Calc. for C20 H25 N4 O3 Pt·1.5H2 O: C, 36.8; H, 4.3; N, 10.7%. UV-Vis [CH3 OH; l/nm (log e)]: 268 (4.41), 328 (3.63), 434 (3.65), 472 (3.61). [Pt(L2)(NH3 )2 ]NO3 , 2b. Yield: 250 mg (38%). m.p. > 158 ◦ C (dec.). IR (KBr): n = 3458 (O–H), 3147 (N–H), 1678 (C O), 1643 (C O), 1614 (C C/C N), 1587 (C C/C N), 1520 (d N–H), 1318 (C C/N O), 1275 (C–O/C–N), 1234 (C–O/C–N). 1 H NMR (300 MHz, D2 O): d 8.21 (d, J = 6.4 Hz, 1H, H14), 7,91 (td, J = 8.0, 1.2 Hz, 1H, H16), 7.70–7.65 (m, 2H, H5/H8), 7.61 (td, J = 7.4, 1.4 Hz, 1H, H6 or H7), 7.39–7.50 (m, 1H, H7 or H6/3H (Ph)), 7.34 (t, J = 6.4 Hz, 1H, H15), 7.20 (d, J = 8.0 Hz, 1H, H17), 7.08–6.99 (m, 2H, Ph), 5.70 (s, 1H, H11), 4.15 (d, J = 18.6 Hz, 2H, H19). 195 Pt NMR (64 MHz, D2 O): d -2510 ppm. Found: C, 38.3; H, 3.9; N, 10.3. Anal. Calc. for C23 H23 N5 O6 Pt·3H2 O: C, 38.7; H, 4.1; N, 9.8%. UV-Vis [CH3 OH; l/nm (log e)]: 266 (4.35), 332 (3.68), 431 (3.66), 491 (3.58). This journal is © The Royal Society of Chemistry 2010

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[Pt(L3)(NH3 )2 ]NO3 , 3b. Yield: 170 mg (26%). m.p. > 193 ◦ C (dec.). IR (KBr): n = 3452 (O–H), 3160 (N–H), 1680 (C O), 1642 (C O), 1633 (C C/C N), 1613 (C C/C N), 1587 (C C/C N), 1527 (d N–H), 1477 (C C), 1376 (C C/N O), 1335 (C C), 1277 (C–O/C–N), 1236 (C–O/C–N). 1 H NMR (300 MHz, D2 O): d 8.22 (d, J = 6.2 Hz, 1H, H14), 7.97–7.86 (m, 3H, H16/H5 or H8/H23), 7.80 (d, J = 7.6 Hz, 1H, H8 or H5), 7.71 (t, J = 7.6 Hz, 1H, H6 or H7), 7.61 (t, J = 7.6 Hz, 1H, H7 or H6), 7.36 (t, J = 6.2 Hz, 1H, H15), 7.27–7.19 (m, 1H, H17), 6.51–6.47 (m, 1H, H21), 6.19 (dd, J = 4.8, 2.5 Hz, 1H, H22), 5.69 (s, 1H, H11), 4.17–4.12 (m, 2H, H19). 195 Pt NMR (64 MHz, DMF-d 7 ): d -2532 ppm. Found: C, 36.3; H, 3.3; N, 10.7. Calc. for C21 H21 N5 O7 Pt·2H2 O: C, 36.7; H, 3.7; N, 10.2%. UV-Vis [CH3 OH; l/nm (log e)]: 267 (4.18), 331 (3.60), 430 (3.54), 486 (3.45). [Pt(L4)(NH3 )2 ]NO3 , 4b. Yield: 180 mg (34%). m.p. > 217 ◦ C (dec.). IR (KBr): n = 3438 (O–H), 3176 (N–H), 2954 (C–H), 2925 (C–H), 2855 (C–H), 1680 (C O), 1643 (C O), 1634 (C C/C N), 1614 (C C/C N), 1587 (C C/C N), 1527 (d N–H), 1477 (C C), 1380 (C C N-1 O), 1276 (C–O/C–N), 1236 (C–O/C–N) cm-1 . 1 H NMR (300 MHz, CD3 OD): d 8.40 (d, J = 5.7 Hz, 1H, H14), 8.04 (td, J = 7.8, 1.2 Hz, 1H, H16), 7.94 (d, J = 7.5 Hz, 1H, H5 or H8), 7.85 (d, J = 7.5 Hz, 1H, H8 or H5), 7.68 (td, J = 7.5, 1.0 Hz, 1H, H7 or H6), 7.55 (td, J = 7.5, 1.0 Hz, 1H, H6 or H7), 7.46–7.40 (m, 2H, H15/H17), 6.75 (d, J = 5.2 Hz, 1H, NH), 5.81 (d, J = 5.2 Hz, 1H, H11), 2.97–2.86 (m, 2H, H19), 2.00–1.85 (m, 2H, H20), 1.43–1.20 (m, 8H, H21–H24), 0.85 (t, J = 6.9 Hz, 3H, H25). 195 Pt NMR (64 MHz, DMF-d 7 ): d -2522 ppm. Found. C, 41.5; H, 4.5; N, 10.2. Calc. for C23 H31 N5 O6 Pt: C, 41.3; H, 4.7; N, 10.5. UV-Vis [CH3 OH; l/nm (log e)]: 266 (4.43), 331 (3.92), 440 (3.88), 495 (3.82). [Pt(L5)(NH3 )2 ]NO3 , 5b. Yield: 283 mg (50%). m.p. > 171 ◦ C (dec.). IR (KBr): 3438 (O–H), 3163 (N–H), 2953 (C–H), 2923 (C– H), 2853 (C–H), 1679 (C O), 1645 (C O), 1634 (C C/C N), 1613 (C C/C N), 1587 (C C/C N), 1528 (d N–H), 1476 (C C), 1378 (C C/N O), 1276 (C–O/C–N), 1236 (C–O/C– N) cm-1 . 1 H NMR (300 MHz, CD3 OD): d 8.39 (d, J = 5.7 Hz, 1H, H14), 8.03 (t, J = 7.8 Hz, 1H, H16), 7.94 (d, J = 7.6 Hz, 1H, H5 or H8), 7.85 (d, J = 7.6 Hz, 1H, H8 or H5), 7.68 (t, J = 7.6 Hz, 1H, H7 or H6), 7.55 (t, J = 7.6, 1.0 Hz, 1H, H6 or H7), 7.45–7.39 (m, 2H, H15/H17), 6.74 (d, J = 5.0 Hz, 1H, NH), 5.80 (d, J = 5.0 Hz, 1H, H11), 2.96–2.85 (m, 2H, H19), 2.02–1.85 (m, 2H, H20), 1.43–1.17 (m, 14H, H21-H27), 0.87 (t, J = 7.0 Hz, 3H, H28). 195 Pt NMR (64 MHz, DMF-d 7 ): d -2521 ppm. Found. C, 43.9; H, 5.4; N, 9.8. Calc. for C26 H37 N5 O6 Pt: C, 43.9; H, 5.3; N, 9.9%. UV-Vis [CH3 OH; l/nm (log e)]: 269 (4.60), 328 (3.89), 437 (3.85), 482 (3.74). X-Ray crystallography† X-Ray diffraction data collections were performed on a Oxford-Diffraction GEMINI CCD diffractometer using mirror˚ at 120 K. monochromatised Cu-Ka radiation (l = 1.54184 A) Final unit cell parameters were based on the fitting of all reflections positions integrated during the data reduction. Data Integration and scaling of the reflections were performed with the CrysalisPRO suite.43 For compound 3b an analytical numeric absorption correction was performed using a multifaceted crystal model based on expressions derived by Clark and Reid,44 whereas for compound This journal is © The Royal Society of Chemistry 2010

1a an empirical absorption correction was carried out using intensity measurements implemented in SCALE3 ABSPACK scaling algorithm.43 The structures were solved by direct methods using the SHELXS45 program. The positions of all atoms could be unambiguously assigned on consecutive difference Fourier maps. Refinements were performed using SHELXL45 based on F 2 through full-matrix least square routine. All but hydrogen atoms were refined with anisotropic atomic displacement parameters. Hydrogen atoms were added in the structure in their typical distances to the neighbour atoms and were further refined according to the riding model.46 Fourier difference maps of both compounds present quite high peaks around the platinum atoms. The height of these peaks was not influenced by the choice of parameters in the absorption model correction neither by the ionisation number of the platinum atoms and indeed similar structural features were observed in many platinum structures investigated recently.47 Final structure refinement of compound 1a was performed considering split positions for carbon atoms C39 and C40. The C–C distances and the atomic displacement parameters of the split atoms were restrained in order to keep the correct molecule geometry. The high noise level in the final electron density maps due the peaks around the platinum atoms made the placement of the hydrogen atoms around the H2 O molecules a quite complicated task. In particular, the hydrogen atoms of these molecules in compound 3b were added manually in the structure and then refined according to riding model with anti-bumping constraints what did not provide a satisfactory structural model as far as the H2 O hydrogen bonds is concerned. Crystal structure and refinement data for compounds 1a and 3b are summarised in Table 4. Biological studies (a) Cells and culture conditions. The human cell lines used in this work were HL-60 (promyelocytic leukaemia), HCT-8 (colon), MDA-MB-435 (melanoma) and SF-295 (brain), OVCAR8 (ovary), PC3 (prostate), which were all obtained from the National Cancer Institute (Bethesda, MD, USA). The cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mmol L-1 glutamine, 100 U/mL penicillin, and 100 mg mL-1 streptomycin at 37 ◦ C with 5% CO2 . (b) Proliferation assays. The cytotoxicity of the compounds and cisplatin41 were tested against HL-60 (promyelocytic leukaemia), HCT-8 (colon), MDA-MB-435 (melanoma), SF-295 (brain), OVCAR-8 (ovary) and PC3 (prostate) cell lines. For all experiments, cells were plated in 96-well plates (105 cells/well for adherent cells or 0.5 ¥ 105 cells/well for suspended cells in 100 mL of medium). After 24 h, the compounds (0.06 to 77.0 mmol L-1 ) dissolved in 1% DMSO were added to each well using a high-throughput screening system (Biomek 3000 - Beckman Coulter, Inc. Fullerton, California, EUA), and the cultures were incubated for 72 h. Doxorubicin (Zodiac) was used as a positive control. Control groups received the same amount of DMSO. Tumour cell growth was quantified by the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazolium bromide (MTT) to a purple formazan product.48 At the end of the incubation, the plates were centrifuged and Dalton Trans., 2010, 39, 10203–10216 | 10213

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Table 4 Crystal data and structure refinement for 3b and 1a Identification code

3b

1a

Empirical formula Formula weight Temperature/K Wavelength/A˚ Crystal system Space group Unit cell dimensions a/A˚ b/A˚ c/A˚ a/◦ b/◦ g /◦ Volume/A˚ 3 Z Density (calculated)/Mg m-3 Absorption coefficient/mm-1 F(000) Crystal size/mm3 Theta range for data collection/◦ Index ranges

C21 H28 N5 O10.50 Pt 713.5 120(2) 1.54178 Monoclinic C2/c

C43 H48 Cl4 N4 O7 Pt2 1264.83 120(2) 1.54184 Monoclinc P21 /n

31.7768(12) 9.4926(2) 19.6173(14) 90 122.01 90 5018.0(4) 8 1.87 11.058 2808 0.13 ¥ 0.06 ¥ 0.04 3.28 to 66.26 -37 < = h < = 36 -11 < = k < = 9 -23 < = l < = 23 11 925 4197 [R(int) = 0.0337] 95.3% 0.673 and 0.391 Full-matrix least-squares on F 2 4197/7/339 1.057 R1 = 0.0490 wR2 = 0.1313 R1 = 0.0616 wR2 = 0.1405 3.764 and -0.774

16.5463(6) 13.2528(5) 20.6428(8) 90 103.106(4) 90 4408.7(3) 4 1.9 14.378 2448 0.10 ¥ 0.09 ¥ 0.03 3.10 to 62.65 -19 < = h < = 18 -14 < = k < = 13 -23 < = l < = 23 18 998 6877 [R(int) = 0.0495] 97.4% 0.672 and 0.327 Full-matrix least-squares on F 2 6877/28/549 1.006 R1 = 0.0509 wR2 = 0.1307 R1 = 0.0798 wR2 = 0.1452 2.686 and -1.356

Reflections collected Independent reflections Completeness to q = 66.26◦ /62.65◦ Max. and min. transmission Refinement method Data/restraints/parameters Goodness-of-fit on F 2 Final R indices [I > 2s(I)] R indices (all data) Largest diff. peak and hole/e A˚ -3

the medium was replaced with fresh medium (150 mL) containing MTT (0.5 mg mL-1 ). Three hours later, the plates were centrifuged, the MTT formazan product was dissolved in 150 mL DMSO, and the absorbance was measured using a multiplate reader (Spectra Count, Packard, Ontario, Canada). The drug effect was quantified as the percentage of the control absorbance of the reduced dye at 595 nm.

Conclusions In summary, we have synthesised and fully characterised five novel 3-(aminomethyl)naphthoquinones HL1–5 and their chloridocis-[Pt(HL1–5)Cl2 ] and amino cis-[Pt(L3)(NH3 )2 ]NO3 platinum complexes, and evaluated their cytotoxicity against MDA-MB435, HL-60, HCT-8, SF-295, OVCAR-8 and PC-3 cancer cell lines. Spectroscopic and voltammetric studies have evidenced that the 3(aminomethyl)naphthoquinone ligand is present in the protonated HL form in the chlorido complexes and in the deprotonated form in the amino-complexes. Furthermore, X-ray diffraction studies have confirmed that in both cases these ligands coordinate through the nitrogen atoms forming a five membered chelate ring around the platinum(II) ions which exhibit square planar geometry. The results of the cytotoxic activity have demonstrated that proligands and chlorido complexes exhibit high to moderate activities, whereas the amino complexes are mostly inactive. Lipophilicity is an important factor to be considered, since compounds with 10214 | Dalton Trans., 2010, 39, 10203–10216

large organic side chains showed the highest IC50 values, and amino cationic complexes were generally inactive. Poor activity of the amino compounds compared to the chlorido complexes may be associated to the different types of interactions complexesDNA-cisplatin type, covalent, for the chlorido complexes and noncovalent for the amino cations. Detailed biophysical and cellular studies on interaction with DNA and cellular uptake are under way, as well as the investigation of the platinum(IV) analogues aiming at complexes with better IC50 values.

Acknowledgements The authors thank the Brazilian agencies ‘Conselho Na´ cional de Desenvolvimento Cient´ıfico e Tecnologico’ (CNPq), ‘Coordenac¸a˜ o de Aperfeic¸oamento de Pessoal de N´ıvel Superior’ (CAPES) and ‘Fundac¸a˜ o de Amparo a` Pesquisa do Estado do Rio de Janeiro’ (FAPERJ) for financial support. Pronex-FAPERJ (grant number E-26/171.512/2006) is acknowledged. M. D. Vargas and A. P. Neves are recipients of CNPq research fellowships. We also thank Dr Ana Paula S. Fontes of Universidade Federal de Juiz de Fora (UFJF) for the 195 Pt NMR spectra.

References 1 B. Rosenberg, L. VanCamp, J. E. Trosko and V. H. Mansour, Nature, 1969, 222, 385–386.

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