Novel 3-(Aminomethyl)naphthoquinone Mannich Base-Platinum(IV) Complexes: Synthesis, Characterization, Electrochemical and Cytotoxic Studies

Três novos complexos de platina(IV), cis,cis,trans-[Pt(HL1-3)Cl2(OH)2] 1b-3b (HL = 2-hidroxi3-[(R-amino)(piridin-2-il)metil]-1,4-naftoquinona, R = n-butil, HL1; n-heptil, HL2 and n-decil, HL3) foram obtidos através da oxidação dos respectivos precursores cis-[Pt(HL1-3)Cl2] 1a-3a. Estudos de voltametria cíclica de 1b-3b em MeCN mostraram o processo redox quase reversível do íon naftoquinonato (NQO−, i.e., L−) e o processo irreversível atribuído à redução do par Pt/Pt, em potenciais aproximadamente 400 mV menos negativos que no precursor da cisplatina cis,cis,trans-[Pt(NH3)2Cl2(OH)2]. Propõe-se que este processo Pt /Pt seja favorecido, em 1b-3b, por uma interação de hidrogênio entre o grupo 2-hidroxil da naftoquinona e um ligante hidroxil axial. Estudos de citotoxicidade contra quatro linhagens de células tumorais humanas mostraram que, em geral, os derivados de platina(IV) e platina(II) exibem o mesmo perfil citotóxico, além de serem mais ativos que a cisplatina. Valores de CI50 in vitro mais baixos foram observados para 2b-3b, cujos ligantes possuem os maiores grupos R (HL2-HL3), sendo, portanto, mais lipofílicos. Além disto, os valores semelhantes de CI50 dos complexos análogos de platina(II) e platina(IV) devem-se à rápida redução de 1b-3b in vitro para gerar 1a-3a.


Introduction
Cisplatin (cis-diaminodichloro-platinum(II)) is a well known antineoplastic agent 1 that acts mainly through DNA binding, interrupting replication and transcription processes and consequently inducing apoptosis. 2Even though cisplatin has been currently used in cancer treatment, side effects such as nephrotoxicity and acquired resistance have limited its effectiveness and motivated the search for new Pt-based drugs able to overcome these drawbacks. 3,4ne of the problems associated with platinum(II) complexes, including cisplatin, is their deactivation by sulfur containing species, 5 which prevents their uptake into the cell and DNA binding.As an alternative, octahedral low spin d 6 platinum(IV) complexes of the type cis,cis,trans-[Pt(AA')Cl 2 L 2 ] (AA' = amine or diamine carrier ligands, L = axial ligands), which have slow ligand exchange rates, have been designed aiming to avoid side reactions in the bloodstream. 6These complexes are believed to be activated by reduction to platinum(II) complexes in vivo by reductants, such as glutathione, ascorbic acid and cystein, with loss of the axial L ligands, the resulting species exerting their effects in a similar manner to cisplatin and analogous compounds. 7he advantage of the platinum(IV) complexes is their relative inertia, compared to the platinum(II) counterparts, which allows them to be administrated orally. 7Two factors are critical for their activation: the Pt 4+ /Pt 2+ reduction potential of the complex and the rate of reduction, 6 and both can be tuned by judicious choice of the carrier and axial ligands, the latter playing an important role in the pharmacokinetic properties of these complexes. 7,8[10] Furthermore, axial and carrier ligand steric hindrance have also been found to influence both the reduction potential and rate of reduction. 8,9Recent evidences of multiple reduction pathways for cis,cis,trans-[Pt(AA')Cl 2 L 2 ] and cis,trans-[PtCl 2 (mpy)(NH 3 )L 2 ] (mpy = 2-methylpyridine) with axial L = acetate ligands have indicated that the mechanism proposed for the reduction of platinum(IV) complexes may not be as simple as commonly assumed. 6,11ne of the strategies to enhance the cytotoxic activity of platinum(IV) complexes has been the incorporation of biologically relevant fragments to their axial positions, 12,13 since these ligands are frequently released upon reduction of the platinum(IV) center.For instance, increased cellular uptake has been achieved by tethering nanocarriers, e.g., carbon nanotubes, to axial carboxylate ligands, which prolong the circulation time of these drugs in the bloodstream. 14,15DNA-Au-NP (NP = nanoparticles) tethered to a platinum(IV) complex via the carboxylate ligand were also successful carrying vehicles 16 and most recently, a platinum(IV) complex containing carboxylate axial ligands was covalently incorporated into cross-linked micelles allowing slow drug release. 17he use of ligands possessing recognized cytotoxic activity in the design of novel platinum drugs has also been described. 18Our interest in hybrid platinum-3-(aminomethyl) naphthoquinone complexes stems from the fact that both platinum complexes and aminonaphthoquinones are active fragments, and therefore compounds with enhanced antineoplastic activity might be anticipated. 19Cisplatin and analogous compounds exert their cytotoxicity by DNA platination, leading to apoptosis. 20The mechanism of action of naphthoquinone derivatives is still not totally clear but it is known that they can act via inhibition of topoisomerases, 21 generation of reactive oxygen species (ROS) by quinone redox cycle 22 and/or bioalkylation. 23t was reported that hybrid platinum(II) complexes of 3-(aminomethyl)naphthoquinones, 1a-3a, show moderate to high activity against selected human cancer cell lines with IC 50 values lower than cisplatin. 24Recently, a series of biophysical and cellular studies with these compounds have been undertaken. 25Complexes 1a-3a bind covalently to DNA model bases, inducing cellular DNA strand breaks.The same behavior has been observed in vitro.However, they are still less active than the pro-ligands HL1-HL3.For this reason, with the aim of improving the cytotoxicity of the platinum(II) compounds 1a-3a, we have synthesized their platinum(IV) analogues.In this work, we describe the synthesis, characterization, electrochemical and cytotoxic studies of the novel complexes [Pt(HL)Cl 2 (OH) 2 ] 1b-3b, shown in Scheme 1.

Material and methods
All chemicals were used without further purification.2 -H y d r o x y -1 , 4 -n a p h t h o q u i n o n e , 2 -p y r i d i n ecarboxaldehyde, butylamine, heptylamine, decylamine and K 2 PtCl 4 were purchased from Aldrich and H 2 O 2 , NaOH and solvents were acquired from Vetec.HL1-3 (orange powders), 26 cis-[Pt(DMSO) 2 Cl 2 ], 27 1a-3a (yellow powders) 23 and cis,cis,trans-[Pt(NH 3 ) 2 Cl 2 (OH) 2 ] 28 were prepared as described in the literature and their identity was confirmed by melting point measurements and 1  recorded on a Nicolet FTIR Magna 760 spectrophotometer using CsI pellets and Raman experiments were carried out on a Bruker FT-Raman MultiRAM using the 1064 nm line of a Nd:YAG laser and a Ge detector operating at liquid nitrogen temperature.Electrospray ionization mass spectroscopy (ESI-MS) spectra were acquired on a quadrupole time-of-flight mass spectrometer (QTOF spectrometer) (Micromass, Manchester, U.K.) at Instituto de Química, UFRJ, Rio de Janeiro State, Brazil.The mass spectrometry (MS) parameters were set to scan from 90 to 1000 m/z using positive detection.The dry gas flow was set to 50.0 L min −1 , the dry temperature to 120 °C and the flow injection rate to 4.0 µL min −1 .All measurements were performed using MeCN and formic acid.Cyclic voltammetry was carried out with a BASi EPSILON potentiostat/galvanostat system at room temperature, in 5 mL of MeCN (anhydrosolv grade-Tedia Brazil) solutions of the compounds (1.0 × 10 −3 mol L −1 ) containing 0.1 mol L −1 n-Bu 4 NClO 4 as the supporting electrolyte under an argon atmosphere.A conventional three-electrode cell was used to carry out these experiments: a glassy carbon electrode as the working electrode, a platinum wire as counterelectrode and Ag/AgCl (0.1 mol L -1 n-Bu 4 NClO 4 /MeCN) was used as the reference electrode.The scan rate was in the 0.100-0.200V s −1 range and the experiments were carried out in the cathodic branch.The ferrocene/ferrocenium (FcH/FcH + ) couple was used as internal reference. 29or the studies in the presence of NaOH (aq) and HCl (aq) , the conditions were the same as those described above but for the sequential addition of 50 µL aliquots of 0.02 mol L −1 NaOH and 20 µL aliquots of 0.05 mol L −1 HCl.Pure argon gas was bubbled through the electrolytic solution to remove oxygen in all experiments.
For a suspension of the platinum(II) complexes 1a-3a (0.8-0.95 mmol) in acetone was added 1.0 mL of H 2 O 2 (10 mmol).The solution was heated under reflux for 7 h.The solvent was removed under vacuum and the residue was washed with isopropanol or petroleum ether to yield lightyellow powders, which were isolated by centrifugation and dried under vacuum.

Proliferation assays
For all experiments cells were plated in 96-well plates (0.7 × 10 5 cells/well for adherent cells or 0.3 × 10 5 cells/well for suspended cells).After 24 h the compounds (0.66 to 77.0 µmol L −1 ) dissolved in 1% DMSO were added to each well using a high-throughput screening system (Biomek 3000-Beckman Coulter, Inc. Fullerton, California, USA) and the cultures were incubated for 72 h.Doxorubicin hydrochloride (Sigma Aldrich) was used as a positive control.Control groups received the same amount of DMSO.Tumor cell growth was quantified by the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to the purple formazan product. 31At the end of the incubation, the plates were centrifuged and the medium was replaced with fresh medium (200 µL) containing MTT 10%.Three hours later, the plates were centrifuged (3000 rpm/10 min), the MTT formazan product was dissolved in 150 µL 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.
Elemental analyses provided the expected chemical formulae, [Pt(HL1-3)Cl 2 (OH) 2 ], also supported by the ESI mass spectra which exhibited the molecular ion peaks for 1b-3b (Figures S1-S3).A broad band around 3450 cm −1 in the infrared spectra of all compounds (Figures S4-S7) confirms the presence of water, as suggested by the elemental analyses data.
Differently from complexes 1a-3a, 24 single crystals of 1b-3b suitable for X-ray diffraction studies could not be grown under a variety of conditions, however, IR and Raman spectroscopic studies are in accord with the structure proposed for complexes cis,trans-dichloridodihydroxide 1b-3b, illustrated in Scheme 1, with two axially coordinated hydroxide ligands.
IR and Raman spectra of the platinum(IV) complexes are similar in terms of wavenumber values (Figures S4-S9), indicating the absence of a symmetry center in these compounds, as expected.Unfortunately, the Raman spectrum of 1b was seriously disturbed by fluorescence and thus is not presented.Special attention has been paid to the region characterized by metal-ligand vibrations, which is essential to distinguish between square planar platinum(II) and octahedral platinum(IV) complexes.Thus, far-IR and Raman spectra of HL1-3 were initially acquired.3][34] These features were used to analyze and compare the Raman spectra of the platinum(II) and platinum(IV) complexes.
Two very well resolved bands can be observed at 321 and 334 cm −1 for complex 2a, and at 331 and 346 cm −1 for complex 2b (Figure 1).These bands have been assigned to the symmetric and asymmetric Cl-Pt-Cl stretches, respectively.It is worth stressing that the bands observed for 2b are upshifted by ca. 10 cm −1 , as compared to 2a.Our result finds support in the work published by Giandomenico et al., who have interpreted such a shift as being due to full oxidation of the platinum atom. 32However, the range usually reported for Pt-Cl stretches is larger than 10 cm −1 , so that the trend observed herein is not sufficient to distinguish between the two complexes.On the other hand, the 567 cm −1 band in the spectrum of 2b is a determining factor for such distinction since it is not present in the spectrum of 2a and has been commonly assigned to the Pt 4+ -O vibration.One can also observe, in Figure 1, that the new band is slightly disturbed by another one at 584 cm −1 , which is due to the aromatic ring breathing mode of the HL2.It is possible that the asymmetric O-Pt 4+ -O stretching vibration is either hidden under this band envelope, or is not Raman-active.This latter possibility seems pertinent if one recognizes the existence of a linear O-Pt-O moiety, as illustrated in Scheme 1.Unfortunately, the 1035 cm −1 band, attributed to the Pt-O-H bending mode 34 is blurred by another band associated to the ligand.
The Pt-N (amine) stretching modes are typically observed near the Pt-O vibrations. 35Indeed, the mixture of these modes has been also considered by Miller et al. 34 with respect to the band at 567 cm −1 .Although the Pt-N vibration has not been observed, coordination to the metal through the N atom was indirectly accompanied in this work by the spectral changes exhibited at the 1523 cm −1 band, which is ascribed to the C-N-H bending mode of the ligand. 24It is upshifted by ca. 10 cm −1 for platinum(II) complexes and ca.30 cm −1 for platinum(IV) complexes, suggesting that the Pt 4+ ion significantly affects the electronic density of this oscillator.Information on the Pt-N (pyridine) vibration could not be obtained due to the overlapping of bands at 230 and 260 cm −1 , and 1700-1580 cm −1 .

Electrochemical studies
The biological activity of platinum(IV) complexes depends on their being reduced to platinum(II) species and for this reason the redox potential of those species is believed to be an important parameter in their cytotoxic activity. 7][38] Cyclic voltammetry studies have shown that 2-hydroxy-1,4-naphthoquinone derivatives undergo two main redox processes in aprotic medium, the first one corresponding to an irreversible (Ic/Ia), and the second one to a quasi-reversible processes (IIc/IIa).Other peaks and shoulders in the cyclic voltammograms (CVs) of this class of compounds have been associated to concentration dependent processes due to the presence of the hydroxyl proton: (i) self-protonation reactions (intramolecular proton transfer) and (ii) hydrogen-bonding which stabilizes electron generated intermediates. 39,40ndeed in alkaline medium (in MeCN and in the presence of n-Bu 4 NOH), 36 only one reversible redox couple (IIc/IIa) at more negative potential relative to the original first one has been observed and attributed to the bielectronic instead of double-redox process of the naphthoquinone.
Attempts have been made at correlating cytotoxicity (IC 50 or LD 50 values) with the first or second redox E 1/2 processes of naphthoquinone derivatives. 41Correlations between LD 50 values and the second E 1/2 process were drawn recently for a series of 2-hydroxy-1,4-naphthoquinones. 39ecause of the very limited aqueous solubility of platinum(II) complexes 1a-3a and platinum(IV) complexes 1b-3b the CVs were obtained in MeCN solutions (1.0 × 10 −3 mol L −1 ) containing 0.1 mol L −1 n-Bu 4 NClO 4 as the supporting electrolyte, under argon atmosphere.The cyclic voltammetry of cis,cis,trans-[Pt(NH 3 ) 2 Cl 2 (OH) 2 ] (A) was also performed in MeCN (Figure S10) in order to evaluate the effect of the naphthoquinone coordination on the Pt 4+ /Pt 2+ redox process.Electrochemical data are summarized in Table 1.The CVs of the platinum(II) complexes (Figures 2a and  S11-S13) exhibit two main redox pairs, Ic/Ia and IIc/IIa, which correspond to irreversible and quasi-reversible processes, respectively.The first reduction process is related to the formation of the seminaphthoquinone (NQOH − , i.e., HL − -Ic/Ia) and the second, to the electron reduction of the respective anion (NQO − , i.e., L − -IIc/IIa).This indicates that the naphthoquinone ligands HL1-3 are indeed protonated.The two additional shoulders (Ic' and Ia') due to selfprotonation reactions and/or hydrogen bonding processes are also observed. 39,40n the CVs of all three platinum(IV) complexes 1b-3b (Figures 2b and S14-S16), an irreversible peak (IIIc) attributed to the reduction of Pt 4+ /Pt 2+ is observed, 8,9 which is absent in the CVs of the analogous platinum(II) complexes 1a-3a (Figure 2a).Unexpectedly only one couple of peaks (IIc and IIa) is observed, which has been attributed to the quasi-reversible naphthoquinonate (NQO − , i.e., L − ) redox process. 36,39The absence of the expected two redox processes present in the CVs of the analogous 1a-3a platinum(II) complexes could be explained by the formation of a hydrogen bonded species, 42,43 involving the naphthoquinone 2-hydroxyl group and an axially coordinated hydroxide ligand (Scheme 2), which would shift the first naphthoquinone reduction process to negative potential.Platinum(IV) reduction with concomitant loss of the axial hydroxide ligands would lead to the deprotonation of the coordinated Mannich bases HL1-3, as illustrated in Scheme 2 to yield [Pt(L1 − -3 − )Cl 2 ].
The result of the cyclic voltammetry experiment of 2b under the same conditions, except for the addition of 3.0 equiv. of HCl, evidences two redox processes (Ic/Ia and IIc/IIa, Figure S17), thus confirming that the hydroxide ion is indeed responsible for the deprotonation of the 2-hydroxynaphthoquinones.The cathodic potential values (Ep Ic = −0.67 and Ep IIc = −1.60V) are the same as those observed for the analogous platinum(II) complex 2a (Table 1), however the anodic potentials were strongly affected by the presence of the acid: Ep Ia = −0.06V (2a, Ep Ia = −0.31V) and Ep IIa could not be clearly assigned (Figure S17).The Pt 4+ /Pt 2+ irreversible reduction process (Ep IIIc ) is still observed at −1.22 V The Pt 4+ /Pt 2+ reduction values for 1b-3b (Table 1, Ep IIIc = −1.15,−1.18 and −1.18 V vs FcH + /FcH, respectively), in MeCN, are much less negative than for cis,cis,trans-[Pt(NH 3 ) 2 Cl 2 (OH) 2 ] (A), which has been measured in the same solvent (−1.60 V vs FcH + /FcH), thus showing that coordination of the Mannich bases favors the Pt 4+ /Pt 2+ reduction, as compared to amine ligands.The π-acceptor character of the HL1-HL3 pyridyl group and the steric hindrance of these ligands certainty contribute to this behavior. 8,9Moreover, the intramolecular hydrogen bond interaction in 1b-3b proposed above would destabilize the Pt-OH bond, thus making reduction of 1b-3b easier than for complex A. As expected, increasing the size of the R 1 group in the HL1-HL3 ligands (R 1 = n-butyl, n-heptyl, n-decyl, respectively) does not cause appreciable shifts in the Pt 4+ /Pt 2+ reduction potentials of complexes 1b-3b.
To evaluate whether coordination affects the electrochemical behavior of the pro-ligands, cyclic voltammetry of HL1-HL3 was performed in MeCN (Figure S18 and Table 2) and compared with the CVs of 1a-3a, whose naphthoquinone ligand is protonated.
The CVs of HL1-HL3 in pure MeCN (Figure S18, Table 2) are similar to those previously described for analogous Mannich bases in DMF. 40Comparison of these data (Table 2) with those obtained for 1a-3a (Figure 2a, Table 1) indicates that upon coordination to platinum(II) the HL1-HL3 reduction processes occur at more positive potentials (positive shifts of about 600 mV are observed for the first redox pair Ic/Ia), but are less reversible.It seems reasonable to suggest that intramolecular O-H … NHR 1 (CHR) hydrogen-bonding occurs in the free ligands, which presumably allows for electron density transfer to the quinone enone system. 42,43This process would be hindered by HL1-HL3 coordination to platinum(II), with the consequent positive shift of the reduction potentials, as observed.
The CVs of the deprotonated species L1 − -L3 − , generated in situ by adding 1.6 equiv. of 0.02 mol L −1 NaOH (aq) (i.e., 400 µL of water) 44,45 to the MeCN solutions of HL1-HL3 (Figure S19), were measured and compared with the data obtained for the platinum(IV) complexes 1b-3b measured in MeCN in the presence of the same amount of water (Figure 3, Table 2).
The CVs of the deprotonated Mannich bases L1 − -L3 − show the expected quasi-reversible pair of peaks (IIc and IIa) related to the formation of NQO − (i.e., L − ). 45,44This pair of peaks undergoes slight positive shift upon coordination, in complexes 1b-3b (Figure 3b, Table 2), under similar conditions, i.e., in the presence of the same amount of water, which also lead to broadening of the complexes' CVs, probably due to decrease in solubility.The irreversible peak (IIIc) attributed to the reduction of Pt 4+ /Pt 2+ is also positively shifted (70-100 mV) in the presence of water (Figures 2b, 3b and Tables 1, 2).These data indicate that water also stabilizes the coordinated reduced aminonaphthoquinone species.

Cytotoxic assays
The cytotoxicity of the platinum(IV) complexes 1b-3b has been investigated against four tumor cell lines: MDA-MB-435 (melanoma), HL-60 (promyelocytic   3, together with the data previously reported for complexes 1a-3a and HL1-HL3 for comparison. As observed for the pro-ligands (HL1-HL3) and the platinum(II) complexes (1a-3a) 24 (Table 3), the size of the side chain (R 1 ) plays an important role in the cytotoxicity of the platinum(IV) complexes, as indicated by the much lower activity of 1b (R 1 = n-Bu) when compared with its longer carbon chain analogues 2b (R 1 = n-hexyl) and 3b (R 1 = n-decyl).Our recent accumulation studies 25 confirm that increase of the carbon chain length of R 1 causes significant enhancement of cellular accumulation of complexes 1a-3a, consequently contributing to the highest cytotoxicity being exhibited by the n-heptyl and n-decyl derivatives.
As discussed previously, the increase of the R 1 carbon chain length from 1b to 3b does not alter significantly the Pt 4+ /Pt 2+ reduction potential, which means that the differences observed in the cytotoxicity of these complexes are not influenced by the platinum(IV) reduction in the cells.
One of the potential advantages of the platinum(IV) prodrugs, when compared with the platinum(II) compounds, is their relative inertness, which prevents deactivation of the drug by undesirable substitution reactions with plasma proteins. 14,15,46,47This allows a greater proportion of the drug to reach its target intact before undergoing reduction, and may result in increased cytotoxicity.
In this work, we have observed that the platinum(IV) complexes 1b-3b were only slightly more active than their platinum(II) counterparts 1a-3a, except in the case of 3b, that was almost twice as active as its precursor 3a against MDA-MB-435 (IC 50 = 5.5 and 9.0 µmol L −1 , respectively)  and also more active than 3a against HL-60 and HCT-8 tumor cell lines.Thus, the very similar cytotoxic profile exhibited by the complexes studied in this work may indicate that 1b-3b undergo rapid in vitro reduction.It is accepted 46 that the rate of reduction depends on both the rate of electron transfer from the reductant to the platinum(IV) center and on how easily the two Pt-ligand bonds are broken.In the cases of 1b-3b, one of the Pt−OH bonds would be weakened by formation of the hydrogen bonded species proposed above (Scheme 2).Also, fast electron transfer to the platinum(IV) atom has been postulated to occur in complexes containing axial hydroxide ligands, which are capable of forming an inner sphere bridge with the reductant (ascorbate).Thus, both factors would contribute to the formation of platinum(II) derivatives 1a-3a outside the cell.

Conclusions
Spectroscopic and electrochemical studies have been successfully used to structurally characterize three novel aminomethylnaphthoquinone cis,trans-dichloridodihydroxide platinum(IV) complexes, 1b-3b.The cytotoxicity results have indicated that the hydroxide ligands in the axial position of these platinum(IV) complexes have not led to an appreciable improvement of the cytotoxicity compared to the platinum(II) precursors 1a-3a, probably due to fast reduction of the platinum(IV).A possible intramolecular H−bond between the OH − axial ligand and the hydroxyl group at position 2 of the naphthoquinone ring may lead to the weakening of the Pt 4+ −OH bond, thus favoring the reduction.Aiming to improve the cytotoxicity of these aminomethylnaphthoquinone platinum(IV) complexes, the synthesis of novel analogues of 1b-3b containing different axial ligands capable of tuning the reduction potentials of the platinum(IV) is underway.

Table 1 .
Electrochemical data obtained for