luminescent ruthenium complexes for theranostic applications

Luminescent Ruthenium Complexes for Theranostic ApplicationsCarolina R. Cardoso,† Marcia V. S. Lima,† Juliana Cheleski,‡ Erica J. Peterson,§ Tiago Venancio,†

Nicholas P. Farrell,*,§,∥ and Rose M. Carlos*,†

†Departamento de Química, Universidade Federal de Sao Carlos, Sao Carlos, Sao Paulo CP 676, 13565-905, Brazil‡Instituto de Física de Sao Carlos, Universidade de Sao Paulo, Sao Carlos, Sao Paulo 13560-970, Brazil§Goodwin Laboratory, Massey Cancer Center, Virginia Commonwealth University, 401 College Street, Richmond, Virginia 23298,United States∥Department of Chemistry, Virginia Commonwealth University, 1001 W. Main Street, Richmond, Virginia 23284-2006, United States

*S Supporting Information

ABSTRACT: The water-soluble and visible luminescent complexes cis-[Ru(L-L)2(L)2]2+ where L-L = 2,2-bipyridine and 1,10-

phenanthroline and L= imidazole, 1-methylimidazole, and histamine have been synthesized and characterized by spectroscopictechniques. Spectroscopic (circular dichroism, saturation transfer difference NMR, and diffusion ordered spectroscopy NMR)and isothermal titration calorimetry studies indicate binding of cis-[Ru(phen)2(ImH)2]

2+ and human serum albumin occurs vianoncovalent interactions with Kb = 9.8 × 104 mol−1 L, ΔH = −11.5 ± 0.1 kcal mol−1, and TΔS = −4.46 ± 0.3 kcal mol−1. Highuptake of the complex into HCT116 cells was detected by luminescent confocal microscopy. Cytotoxicity of cis-[Ru(phen)2(ImH)2]

2+ against proliferation of HCT116p53+/+ and HCT116p53−/− shows IC50 values of 0.1 and 0.7 μmolL−1. Flow cytometry and western blot indicate RuphenImH mediates cell cycle arrest in the G1 phase in both cells and is moreprominent in p53+/+. The complex activates proapoptotic PARP in p53−/−, but not in p53+/+. A cytostatic mechanism based onquantification of the number of cells during the time period of incubation is suggested.

■ INTRODUCTION

The antitumor properties of ruthenium complexes haveattracted much attention primarily because some of themhave shown favorable pharmacological profiles in vitro and invivo in different models including platinum-resistant celllines.1−11 The Ru(III) complexes NAMI-A, ([Him][trans-RuCl4(DMSO)(im)], im = imidazole), KP1019, ([Hind]-[trans-[RuCl4(ind)2], ind = indazole), and its Na+ analogue(KP1339) have been intensively investigated. NAMI-A andKP1339 are currently in clinical trials.12−14 The axial ligandsplay a fundamental role on the pharmacological properties ofthese complexes.15−20 For example, upon intravenous admin-istration, both complexes bind to human serum albumin(HSA).21−26 KP1019 interacts with the hydrophobic domainsof HSA in a noncovalent manner whereas NAMI-A bindsstrongly to HSA. The binding differences may play animportant role in determining the pharmacologic propertiesand efficacy of these agents.27−31 Indeed, the looser associationof KP1019 results in higher cellular absorption of KP1019compared to NAMI-A.27−31 Moreover, KP1019 is active in

primary cancers and induces apoptosis in the colorectal tumorcell lines SW480 and HT29, being more active in these cellsthan the clinically used drugs 5-fluorouracil and cisplatin,32,33

whereas NAMI-A is antiangiogenic and shows antimetastaticactivity in secondary tumors.34,35

Importantly, the cytotoxicity of cisplatin is mainly correlatedto DNA binding while the biologic targets of NAMI-A andKP1019 have not yet been totally elucidated, as bothcompounds are able to target DNA and proteins, suggestingdifferent, or multiple, pathways from those for cisplatin.36−38

Both complexes are in fact prodrugs and in biological mediumare reduced to Ru(II) by reductant molecules such asglutathione, cysteine, and ascorbic acid.39−43 The manymechanistic studies support the “activation by reduction”hypothesis where by reduction of Ru(III) complex by redoxbiomolecules gives the Ru(II) active species that attacks thetarget cells.44 In accord, electrochemical experiments have

Received: March 19, 2014Published: May 15, 2014

Article

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© 2014 American Chemical Society 4906 dx.doi.org/10.1021/jm5005946 | J. Med. Chem. 2014, 57, 4906−4915

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shown that, in the hypoxic and acid medium environment ofsolid tumors, the Ru(III) complexes are reduced to Ru(II).45−47

In view of these properties, there is wide interest in Ru(II)complexes as antitumor agents with both Werner-type andorganometallic compounds currently in development.48−51

Many Ru(II) compounds containing chelate ligands such aspolypyridine ligands have been assayed and show interestingpreclinical results.52,53 Moreover, the photophysical propertiesof polypyridine Ru(II) complexes open wide possibilities ofusing molecular systems in biological applications such ascellular accumulation, trafficking and biodistribution, andcharacterization of cancer cells.54,55 The key features aremetal-to-ligand charge transfer (MLCT) absorption andemission of light at wavelengths in the visible to near-infraredregion for depth penetration in the tissue, large Stokes shifts tominimize the incident light scattering, and long-lived emissionthat often increases the viability of the energy transfer processto a biologic target. In this respect, the luminescence ofantitumor candidates based on Ru(II) polypyridine complexeshas been used to determine the cellular localization and uptakeof the complex.55 Small changes in the nature of the ligands andoxidation state of ruthenium can have a major effect on theproperties of the complex tuning both the photophysicalproperties as well as the pharmacologic properties of thecomplex.These findings encouraged us to study the cytotoxicity and

antitumor activity and diagnosis of a new Ru(II) series ofgeneral formula cis-[Ru(L-L)2(L)2]

2+ where L-L = 2,2-bipyridine (bpy) and 1,10-phenanthroline (phen) and L=imidazole (ImH), 1- methylimidazole (1MeIm), and histamine(Him) (Figure 1). Imidazole is an electron-rich ligand thatbehaves as a strong σ donor toward the Ru(II) atom. A

coordinated imidazole is a stronger hydrogen-bond donor thana free imidazole, as the pKa of coordinated imidazole exceedsthat of free imidazole (∼7.0). A small environmental changecan thus substantially change the hydrogen-bond donating oraccepting properties of the imidazole ligand.56−58 Hydrogenbonds are essential in many biological and enzymatic processesand may play a role in defining anticancer activities.59,47

Furthermore, the rigid structure of the {Ru(phen)2}2+ moiety

provides stability to the complex and π−π stacking interactionswith aromatic residues found in the hydrophobic region ofproteins.The {Ru(phen)2}

2+ moiety also provides intense MLCTabsorptions enabling design of a luminescent complex as apossible molecular biological probe in human cells, tissue, andorganisms. This paper reports on the synthesis, character-ization, and biological properties of cis-[Ru(L-L)2(L)2]

2+ withspecial emphasis on the cis-[Ru(phen)2(ImH/Im-CH3)2]

2+ pair(Figure 1). The binding interactions with HSA wereinvestigated using isothermal titration calorimetry (ITC),circular dichroism (CD), saturation transfer difference (STD),and diffusion ordered spectroscopy (DOSY) techniques. Thecomplexes were assayed in vitro for cell proliferation inhibitoryactivity in human colon and ovarian tumors. The cell uptakestudies were monitored by fluorescent imaging of fixedHCT116 colon tumor cell lines with complexes. The cellularmechanism of action was investigated by assaying theproapoptotic p53, caspase-3, and PARP by western blot andflow cytometry. The results indicate an interesting profile ofbiological activity with weak binding to HSA, indications thatboth cytotoxic and cytostatic mechanisms contribute toproliferation inhibition while the lead compound cis-[Ru-(phen)2(ImH)2]

2+ maintains luminescence in vitro allowingcellular localization and distribution studies.

■ RESULTS AND DISCUSSIONSynthesis and Characterization. The complexes were

synthesized by reacting cis-[Ru(L-L)2Cl2]60 with 2 equiv of L,

or 1 equiv of L-L in the case of histamine, in H2O/EtOH (1:1).All complexes were isolated as hexafluorophosphate salts, andtheir composition and structure were confirmed from CHNanalysis and NMR spectroscopy. We prepared the complex asPF6 counterion for characterization purposes. The counterionwas replaced by chloride to increases the solubility ofcomplexes in aqueous solution. A sample of complex wasdissolved in acetone, and an excess of tetrabutylammoniumchloride (N(n-Bu)4Cl) was added. The precipitate formedimmediately was filtered, rinsed with ether to remove the excessof N(n-Bu)4Cl, and dried. Preliminary cytotoxicity studies (seebelow) indicated the pair of Ru−phen complexes to be of mostinterest; therefore, detailed studies were carried out on thesecompounds.The geometries of the phenanthroline complexes were

density functional theory (DFT) optimized using B3LYP/LANL2DZ including solvent using the PCM model andacetonitrile. Selected bond lengths and angles for the optimizedgeometry in CH3CN are given in Table S1 (SupportingInformation) and Figure S1 (Supporting Information). Theyreproduce the X-ray crystal structural data of related rutheniumα-diimine complexes quite closely. The planar configuration ofthe phen ligand and the dihedral angles between the plane ofthe phen and imidazole ligand provide hydrogen-bonding andπ−π stacking interactions that may stabilize the complex withproteins, nucleic acids, and DNA.

Figure 1. Structures of NAMI-A, KP1019, and the Ru(II) complexesstudied in this work.

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dx.doi.org/10.1021/jm5005946 | J. Med. Chem. 2014, 57, 4906−49154907

1H NMR Spectroscopy. The 1H NMR spectra in D2O for thecomplexes RuphenImH and Ruphen1MeIm exhibit eightdifferent signals for the asymmetric phenanthroline in theregion 7.40−9.40 ppm and three signals for the imidazoleligand in the region 6.80−7.70 ppm, Figure S2 (SupportingInformation). Complex RuphenImH exhibits signals at 7.70,6.90, and 7.00 ppm corresponding to the protons Hγ,γ′, Hβ,β′,and Hα,α′ of the imidazole ring, respectively. The 1H NMRspectrum of complex Ruphen1MeIm exhibits three signals at7.60, 6.90, and 6.85 ppm corresponding to the protons Hγ,γ′,Hβ,β′, and Hα,α′ of the imidazole ring and one signal at 3.45ppm (Hδ,δ′) assigned to the methyl group.Electrochemistry. Figure S3 (Supporting Information)

shows the cyclic voltammograms of the two RuIIphencomplexes in DMF at a platinum electrode versus Fc+/Fc(100 mV s−1), which exhibit a quasireversible metal-basedredox RuII/III couple at E1

/2 = 1.19 V for RuphenImH and E1/2 =

1.15 V for Ruphen1MeIm. In accord, Figure S4 (SupportingInformation) shows that the anodic peak current (ipa)

increases with the square root of the scan rate (ν1/2), but it is

not quite linear. By using the Tafel plot,61 Figure S5

(Supporting Information), the numbers of electrons involvedin the electrochemical reaction were found to be 1.3 and 0.92for RuphenImH and Ruphen1MeIm, respectively.

Absorption and Emission Spectrum. The UV−visible(UV−vis) absorption spectrum of complexes in aqueoussolution are characterized by intense UV absorption bandsmostly π−π* in origin and a broad and intense absorption withmaximum at 490 nm (ε = 7550 mol−1 L cm−1) in the visibleregion, Figure 2A.The emission spectra of the complexes are similar in both

spectral structure and position suggesting a 3MLCT emittingstate, Figure 2B. For both complexes, the Stokes shift was large,∼5500 cm−1, and the phosphorescence at 660 nm was fit as asingle exponential decay with lifetimes of 239 and 211 ns forcomplexes Ruphen1MeIm and RuphenImH, respectively. TheUV−vis and emissive characteristics of complexes are retainedin an aqueous buffer solution (Tris/HCl, pH 7.4) in theabsence and in the presence of HSA.

Stability of Complexes. The complexes are stable in thedark both in the solid state and in aqueous solution. The 1HNMR spectra of complexes RuphenImH and Ruphen1MeIm inD2O at pD 7.8 (phosphate buffer) remain unchanged for at

Figure 2. (A) UV−vis absorption spectrum of RuphenImH in aqueous solution in the visible region; insert: UV−vis in the region 200−600 nm. (B)(right) Absorption and (left) emission (normalized) (λexc = 490 nm) spectra.

Figure 3. (A) RuphenImH binding to HSA determined by ITC. The upper panel shows the raw data of binding and the bottom panel shows theintegrated interaction heat. (B) CD spectra of HSA + RuphenImH at varying concentrations.



least three days, Figure S6A (Supporting Information). Thechemical stability of the complex as a function of the pD ofsolution was also investigated by 1H NMR. D2O solutions ofthe complex at pD 2.6, 4.6, 7.5, 9.7, and 12.6 were prepared,and the spectrum was taken at appropriate time intervals.Figure S6B (Supporting Information) indicates that thecomplexes are quite stable toward hydrolysis and that theimidazole ligand remains coordinated to the metal center at pDvalues from 2.6 to 12.6. Only at pD 12.6, formation of theimidazolate species is observed where the extent ofdeprotonation is 15%.HSA−Complex Interaction. The interaction of Ruphe-

nImH and Ruphen1MeIm with HSA was studied by ITC,NMR-STD, NMR-DOSY, and CD.Figure 3A shows the calorimetric data for the titration of 523

μmol L−1 of the RuphenImH into 30 μmol L−1 of HSA at pH7.4 buffer solution at 37 °C. The binding isotherms (heatchange versus RuphenImH/HSA molar ratio) were obtainedfrom the integration of raw data, and the best fit was obtainedusing a single binding site model, with Kb = 9.8 × 104 mol−1 L.The analysis of data indicates that the interaction betweenRuphenImH and HSA is enthalpy driven, exothermic, ΔH =−11.5 ± 0.1 kcalmol−1, and shows an unfavorable bindingentropy, TΔS = −4.46 ± 0.3 kcal mol−1. The weak bindingaffinity for RuphenImH to HSA suggests the formation ofnoncovalent interactions, observed by other methods such asCD, STD-NMR, and DOSY-NMR analyses. Given that theITC signal for the Ruphen1MeIm complex binding to the HSAshows no interaction, it is likely that hydrogen-bond andelectrostatic interactions contribute to the RuphenImH−HSAbinding reaction. The unfavorable entropy contributionindicates a less pronounced effect of release of water ofhydration and counterions during complex binding (hydro-phobic effect). The CD spectrum monitored in the region ofHSA absorption, Figure 3B, also supports this conclusion. Nochanges in the secondary structure of HSA were observed forconcentrations as high as 25 μM of complex, Figure 3B.

Figure 4 shows the STD-NMR spectrum obtained for theinteraction of RuphenImH with HSA. The signals exhibited inthe STD-NMR spectrum come from the interaction of theprotons of the complex, which are interacting with protein sitesless than 5 Å distant.62 The intensities of the signals increase asthe distance reduces, and this behavior is related to the strengthof the interaction, being possible to obtain an epitope map, aspointed in the structures of the complexes. The epitope mapdemonstrates that all the protons from the complex interactwith the protein, with the imidazole protons exhibiting a greaterinteraction with HSA compared with those on phenanthrolines.Presumably, a hydrogen bond from the imidazole may interactwith the protein allowing closer approach of the vicinalhydrogens. This hydrogen bond is itself difficult to observebecause of the exchange with deuterium from solvent (D2O),where the signal vanishes as expected. The STD-NMRexperiments therefore confirm the demonstration from theITC experiments that the complex−protein interactionpresents a polar character where the imidazole ligand exhibitsa higher polarity when compared with phenanthroline ligand.In this context, DOSY-NMR experiments were also

performed, and a small shift in the diffusion coefficient of thecomplex in the presence of protein (average D = 4.36 × 10−10

m2 s−1) was obtained when compared with free complex (D =4.78 × 10−10 m2 s−1), and free protein D = 7.22 × 10−11 m2 s−1,Figure S7A, B (Supporting Information). It is interesting tonote that the free complex exhibits the same D value for all thesignals (D = 4.78 × 10−10 m2 s−1). However, when the proteinis present, two different diffusion coefficients are observed.According to the STD-NMR data, the protons with weakerinteraction had a diffusion coefficient of 4.57 × 10−10 m2 s−1,whereas those ones with a stronger interaction (H2, H2′, α, α′,and β, β′) are less mobile and thus exhibit slow diffusion (D =4.16 × 10−10 m2 s−1).These results are in agreement with those obtained by STD-

NMR, which showed that the protons from imidazole (α, α′and β, β′) and also the (H2 and H2′) from phenanthroline are

Figure 4. of f resonance and STD spectra of RuphenImH in the presence of HSA.



the ones that present the highest STD-effect (66.66%, 100.00%,and 51.11%). The studies on the interaction between thecomplex Ruphen1MeIm and HSA were also evaluated by STD-NMR, but due to the weaker interaction, the signals of the STDexperiment were too small to determine the percentages(Figure S8, Supporting Information).In summary, the results indicate that the weak HSA−

RuphenImH interaction involves hydrogen bonding from theimidazole ligand and π−π interactions from phen to the HSAprotein.Biological Studies. Cell Proliferation Assay. The inhib-

ition of cell proliferation in ovarian tumor A2780 and theisogenic HCT116 p53+/+ and HCT116 p53−/− human coloncancer cell lines by the complexes was evaluated using the MTTassay, Table 1. For comparison purposes, cisplatin was also

assayed under the same experimental conditions. The IC50values for all complexes were comparable to that of cisplatin.Among the Ru(II) complexes, complex RuphenImH is themost interesting compound, and the higher activity in p53−/−

than p53+/+ is of special interest. The greater activity of bothRuphenImH and RubpyImH complexes compared to theirRuphen1MeIm and Rubpy1MeIm analogues confirms ourproposal that the hydrogen bond on the imidazole ligand has animportant impact on the cytotoxicity of these complexes. Giventhis initial survey and the responses of RuphenImH, we focusedon the mechanism of action studies on this complex, describedin the next section.Cell Cycle Arrest and Apoptosis Assays with Complex

RuphenImH. Figure 5 shows the flow cytometry results for cellcycle analysis of HCT116 p53+/+ and HCT116 p53−/− cellstreated with 20 μmol L−1 complex RuphenImH and incubatedfor 24 and 48 h. The complex induces a robust G1 arrest, as themajority of the cell population accumulates in the G1, orgrowth phase. This effect coincides with a reduction in thenumber of cells in the S phase, or synthesis phase, when theDNA is copied. The cell cycle arrest shows to be p53−/−

dependent, as p53+/+ cells arrest upon treatment with thecomplex, while p53−/− cells do not.We investigated the effect of RuphenImH on the cleavage

and activation of the proapoptotic proteins p53, PARP, andcaspase 3. The samples were prepared from cells treated with orwithout RuphenImH (20 μmol L−1) and subject to westernblot analysis. The blot was probed with β-actin to control forequal gel loading and cisplatin as a positive control. The cellswere lysed after 48 and 72 h of treatment.Western blot analysis revealed that treatment of HCT116

with the complex leads to induction of p53 in HCT116 p53+/+

cells, Figure 6. p53 induction classically leads to cell cycle arrestand is consistent with the G1 arrest observed during cell cycleanalysis. Caspase-3 and Parp-1 cleavage are not observed in

HCT116 p53+/+ cells, indicating the apoptosis may not becausative of the observed cell inhibitory effects. Interestingly,Parp-1 cleavage is observed in HCT116 p53−/− cells. Thisdifference may be a result of the inability of p53−/− cells to elicitan arrest allowing for evaluation and repair, the resultingdamage thereby inducing an apoptotic cascade. In light of theseresults, it is probable that the primary mechanism of actioninvolves a cytostatic pathway. Indeed, as can be seen in Table 2,the number of viable HCT116 cells does not changesignificantly in both p53+/+ and p53−/− cells over time whencompared to the control, confirming a cytostatic effect ofRuphenImH.

Table 1. IC50 Values for Ruthenium Complexes andCisplatin in Cell Lines HCT116 p53+/+ and HCT116 p53−/−

IC50 (μmol L−1)

complex HCT116 (p53+/+) HCT116 (p53−/−) A2780

RuphenImH 0.72 ± 0.18 0.10 ± 0.03 1.30 ± 1.0Ruphen1MeIm 4.00 ± 0.76 5.13 ± 0.45 6.92 ± 0.79RubpyImH 1.02 ± 0.22 0.53 ± 0.16 8.24 ± 1.09Rubpy1MeIm 6.52 ± 0.72 0.70 ± 0.19 6.52 ± 0.84RuphenBiHim 1.65 ± 0.87 1.04 ± 0.12 1.65 ± 0.69cisplatin 9.15 ± 1.18 6.00 ± 0.78 1.30 ± 0.17

Figure 5. Cell cycle analysis of p53+/+ and p53−/− cells after 24 and 48h of exposure to complex RuphenImH.

Figure 6. Western blot analysis for PARP, caspase 3, P53, and β-actinwith incubation of RuphenImH complex and cisplatin for 48 and 70 h.

Table 2. Proliferation of HCT116 Cells with IncubationTime of RuphenImH (20 μmol L−1)

time (h)

30 50 77

HCT116 p53+/+ (104 cells/mL)control 120 190 320RuphenImH 37 42 45

HCT116 p53−/− (104 cells/mL)control 96 120 160RuphenImH 30 32 35



Cell Uptake. Figure 7 shows the luminescent microscopyimages for RuphenImH and Ruphen1MeIm in HCT116 p53+/+

cells treated for 24 h with 20 μmol L−1 concentration ofcomplex, which is the value of IC70. The cells were marked withDAPI as a nuclear stain. As shown in Figure 7, the complexeswere transported into the interior of the cell and located bothin the cytoplasm and in the nucleus. We note that, in principle,substitution and/or redox processes could occur during theuptake experiments, in which case the absorption spectrumwould correspond with the spectrum of the aquo/hydroxo orRu(III) product. Since these species are not emissive, it isconcluded that they are not being formed during the uptakeexperiment.

■ CONCLUSIONS

The series cis-[Ru(phen/bipy)2(L)2]2+ adds to the diversity of

Ru(II) chemotypes available for comparative study anddevelopment. These features include feasibility of synthesis,water solubility, and luminescence in the near IR region that ismaintained in vitro allowing cellular localization and distribu-tion studies. The high inhibition of HCT116 p53−/− related top53+/+ by cis-[Ru(phen)2(ImH)2]

2+ is of significance, given thatalmost 70% of colon rectal tumor cells and 50% of all tumorcells present the mutations in the p53 gene. The interestingprofile of biological activity with weak binding to HSA allowingfree perfusion to tissue while the inhibition of cellularproliferation probably involves a combination of both cytostaticand cytotoxic mechanisms.

■ EXPERIMENTAL SECTIONGeneral Methods. Reagents were purchased from Aldrich and

used without further purification. The elemental analysis shows thatthe complexes are greater than 95% pure. 1H NMR analyses were usedto confirm the purity of all compounds.Materials. HSA was purchased from Sigma−Aldrich. The HSA

solutions were prepared in 0.1 mmol L−1 phosphate buffer of pH 7.4considering the molecular weight of 66.5 kDa. The primary antibodiesused were p53 (cell signaling, no. 9282), total caspase 3 (cell-signaling,9662), total Parp (cell signaling, no. 9541), and β-actin (Abcam,ab8226).

Synthesis of cis-[Ru(phen)2(ImH)2](PF6)2 (RuphenImH) andcis-[Ru(phen)2(1MeIm)2](PF6)2 (Ruphen1MeIm). cis-RuCl2(phen)2(0.187 mmol) was dissolved in a 1:1 EtOH/H2O mixture (10 mL),and an amount of ImH/CH3−Im (0.375 mmol) ligand was added.The solution was stirred under nitrogen atmosphere for 8 h underreflux. A stoichiometric amount of NH4PF6 was added to precipitatethe complex. The dark red precipitate was filtered, recrystallized with a1:1 EtOH/H2O mixture, and washed with water and ethanol undervacuum.

RuphenImH, Yield: 79%. Anal. Calcd for C30H24F12N8P2Ru: C,40.59; H, 2.72; N, 12.62. Found: C, 39.21; H, 2.88; N, 12.03.

Ruphen1MeIm, Yield: 69%. Anal. Calcd for C32H28F12N8P2Ru: C,41.97; H, 3.08; N, 12.23. Found: C, 44.02; H, 3.21; N, 11.81.

Synthesis of cis-[Ru(bpy)2(ImH)2](PF6)2 (RubpyImH) and cis-[Ru(bpy)2(1MeIm)2](PF6)2 (Rubpy1MeIm). cis-RuCl2(bpy)2 (0.206mmol) was dissolved in a 1:1 EtOH/H2O mixture (10 mL), and anamount of ImH/CH3−Im (0.412 mmol) ligand was added. Thesolution was stirred under nitrogen atmosphere for 8 h under reflux. Astoichiometric amount of NH4PF6 was added to precipitate thecomplex. The dark red precipitate was filtered, recrystallized with a 1:1EtOH/H2O mixture, and washed with water and ethanol undervacuum.

RubpyImH, Yield: 70%. 1H NMR (D2O): δ 8.91 (H1,H1′ d), 8.24(H8,H8′, d), 8.16 (H7,H7′, d), 7.95 (H4,H4′, d),7.90 (H5,H5′, d),7.73 (H3,H3′, t), 7.54 (H6,H6′,Hγ,Hγ′, m), 7.14 (H2,H2′, t), 6.97(Hβ,Hβ′, s), 6.74 (Hα,Hα′, s) ppm. Anal. Calcd for C26H24F12N8P2Ru:C, 37.10; H, 2.88; N, 13.34. Found: C, 36.32; H, 3.10; N, 13.21.

Rubpy1MeIm, Yield: 65%. 1H NMR (D2O): δ 8.90 (H1, H1′, d),8.26 (H8, H8′, d), 8.18 (H7, H7′, d), 7.95 (H4, H4′, t),7.87 (H5, H5′,d), 7.73 (H3, H3′, t), 7.56 (H6, H6′, t), 7.44 (Hγ, Hγ′, s), 7.13 (H2,H2′, t), 6.89 (Hβ, Hβ′, s), 6.69 (Hα, Hα′, s), 3.47 (Hδ, Hδ′, s) ppm.Anal. Calcd for C28H28F12N8P2Ru: C, 38.76; H, 3.25; N, 12.96. Found:C, 38.07; H, 3.41; N, 13.00.

Synthesis of cis-[Ru(phen)2(BiHim)](PF6)2 (RuphenBiHim).This complex was prepared according to the literature procedure.63

ITC Studies. Titration experiments were carried out on an ITCinstrument (VP-ITC, MicroCal).64 Reaction cells (1.43 mL) werefilled with solutions and equilibrated at 37 °C. After this equilibration,an additional delay period was allowed to generate the baseline used inthe subsequent data analyses. Solutions were degassed by use of avacuum degasser (ThermoVac, MicroCal) for 5 min prior to anyexperimental run. Stirring speed was maintained at 307 rpm, and heatflow (μcal s−1) was recorded as a function of time. The ligandRuphenImH (523 μmol L−1) was titrated into a cell containing theprotein HSA (30 μmol L−1). Protein and ligand containing solutionswere prepared in the same buffer (0.1 mol L−1 Na2HPO4, 0.1 mol L

−1

NaH2PO4, 2% DMSO, pH 7.40). The experiment was followed by 30injections of 8 μL at 180 s intervals. The heat of dilution, measured bythe injection RuphenImH into the same buffer assay solution, wassubtracted from each titration to obtain the net reaction heat value.The heat of RuphenImH dilution in buffer is less than 0.8 μcal/s. Datawere recorded and analyzed using Origin (version 7, OriginLab)software. ITC curves were fitted using a one-site independent modelfor measuring thermodynamics parameters. The shape of the bindingisotherm changed according to the product of the binding constantand the target concentration through the so-called c value, which isdefined as Kb[M]n (or [M]n/Kd), where Kb is the binding constant,[M] is the macromolecule concentration, and n the number ofinteraction sites.65 For accurate determination of binding constants, a cvalue between 1 and 1000 is recommended.64,66 In conformity withthis recommendation, the c value obtained was 3.

CD Spectroscopy. The interaction was monitored by far-UV CDspectroscopy over a wavelength range 200−250 nm, using a J-715Jasco spectropolarimeter. CD spectra were measured from samples in0.1 cm quartz cuvettes and were the average of 16 accumulations. Theprotein concentration was 3 μmol L−1 in phosphate buffer (0.1 molL−1 Na2HPO4 and 0.1 mol L

−1 NaH2PO4, pH 7.40). The complex wasincubated for 24 h with HSA before measurement. The concentrationsof the complex were 3 and 25 μmol L−1.

Figure 7. Fluorescent confocal imaging for complexes RuphenImHand Ruphen1MeIm in the HCT116 p53+/+ cell line.



NMR Experiments. All the NMR experiments were conducted in aBruker Avance III 600 MHz spectrometer, by employing a 1H {13C,15N} TCI triple resonance cryogenic cooled probehead, equipped witha z-gradient coil producing a nominal maximum gradient of 53.5 Gcm−1. To simulate the biological medium, all the experiments were runat 37 °C.STD-NMR. Sample for STD-NMR experiments were prepared with

a protein HSA concentration of 9 × 10−6 mol L−1 and the complexesRuphenImH and Ruphen1MeIm concentration of 1 × 10−3 mol L−1

dissolved in a solution of 297 μL of buffer (2.03 mmol L−1 Na2HPO4and 0.437 mmol L−1 NaH2PO4, pD 7.8) in D2O (99%) and 3 μLDMSO (1%). A recovery time of 4 s of acquisition time by collecting64 k points were used. 256 scans were averaged for both on- and off-resonance experiments, which were run by irradiating at 20 000 and385 Hz, respectively. A train of 2000 Gaussian pulses of 1 ms eachwith a 4 μs delay between pulses was used, with a total saturation timeof 2 s. The STD effect, ASTD, can be calculated using the equationbelow:

= ×AI

I[L][P]STD

STD

0

T

Where [L]T is the total ligand concentration, [P] is the proteinconcentration, ISTD is the peak intensity of the STD NMR spectra, andI0 is the intensity of the peaks in the 1H off-resonance spectra.DOSY-NMR. The pulsed field gradient (PFG) stimulated spin−echo

(STE) experiment was used with a pulse field gradient length of 1.3 ms(little delta) and a diffusion delay (big delta) of 60 ms. An acquisitiontime of 1.8 s (32 k points) were used for acquiring a sweep window of7211.54 Hz. Sixteen experiments were recorded with gradient intensitylinearly sampled from 2% to 98%, and 16 scans were averaged for eachexperiment; the recovering time used was 2 s. For the DOSYexperiment, the sample preparation was the same as in the STDexperiment but here using a protein concentration of 5 × 10−5 mol L−1

in order to visualize not only the protein signals and its diffusioncoefficient but also an effective interaction between complex andprotein.Cell Lines. HCT116 p53+/+ and HCT116 53−/− cells lines were

cultured in RPMI 1640 (Invitrogen), supplemented with 10% calfserum (Atlanta Biologicals) and 1% penicillin/streptomycin (Invi-trogen). Cells were maintained in logarithmic growth as a monolayerin T75 culture flasks at 37 °C in a humidified atmosphere containing5% CO2.Cellular Growth Inhibition. To measure growth inhibition using

the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay, cells were seeded in 96-well plates at 5 × 103

cells/well and allowed to attach overnight. RPMI 1640 media wasremoved, and 100 μL of each compound was added after serialdilution to treatment concentrations of 200−0.0002 μmol L−1 inquadruplicate wells and exposed to cells for 72 h. Plates were thenwashed with PBS, and 100 μL of 5 mg/mL MTT solution was added.MTT solution was incubated with cells for 3 h at 37 °C. Afterincubation, MTT solution was removed, and 100 μL of DMSO wasadded. Quantification of cell growth in treated and control wells wasthen assessed by measurement of absorbance at 595 nm. IC50 valueswere determined by using the standard curve analysis of Origin.Confocal Laser Scanning Microscopy. HCT116 p53+/+ cells

were seeded in 8-well chamber slides (Lab-Tekll Chamber slide) for24 h. The cells were treated with complex (20 μmol L−1) for 24 h.After treatment, slides were washed 3 times with ice-cold PBS andfixed with 3% paraformaldehyde. Paraformaldehyde was removed, andcells were washed again 3 times with ice-cold PBS and allowed to dry.Slides were then mounted with Vectashield mounting mediacontaining DAPI. Fluorescence was observed by confocal laserscanning microscopy (Zeiss LSM 510).Cell Cycle Arrest. HCT116 p53+/+ and HCT116 p53−/− cells were

seeded in Petri dishes for 24 h. The cells were treated with complex(20 μmol L−1) for 24 and 48 h. After treatment, the cells weredetached using trypsin (Gibco), washed twice with PBS, andsuspended in propidium iodide staining solution (50 μg mL−1

propidium iodide, 0.1% Triton-X in PBS) containing RNase A (200U mL−1). Cell cycle analysis was done after 24 h incubation on aFACSort flow cytometer (Becton Dickinson).

Western Blot Analysis. Following PPC treatments, both floatingand adherent cells were harvested, washed once with ice-cold PBS, andpelleted (10 000 rpm, 5 min, 4 °C). The pellets were then lysed inSDS lysis buffer (62.5 mmol L−1 Tris−HCl, pH 7.5, 5% glycerol, 4%SDS, 4% complete protease inhibitor (Roche) and 5% BME). Proteinconcentrations were determined by the Bradford assay and transferredto PVDF membrane (350 mA, 2h, 4 °C). The membrane was blockedin 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 for 30 min. The membranes were then probed with the primaryantibodies in blocking buffer overnight at 4 °C, followed by asecondary antibody for 1 h at room temperature. Chemiluminescentprotein bands were visualized on X-ray films.

Computational Details. Geometry-optimized structure wasobtained using Gaussian 09, revision B.01, C.0167 employing DFTcalculations, using the hybrid B3LYP exchange-correlation functional68

and the LanL2DZ basis.69−71 The solvent (acetonitrile) was includedusing the polarized continuum model (PCM).72,73 A tight convergence(10−8 au) was used for all DFT calculations.

■ ASSOCIATED CONTENT*S Supporting InformationOptimized structure of the complex RuphenImH; bond lengthsand angles for the optimized structure of RuphenImH; 1HNMR spectra in D2O of complexes RuphenImH andRuphen1MeIm; cyclic voltammetry to complex RuphenImHand Ruphen1MeIm in water solution; plots of anodic peak

current as a function of ν1/2 and log j versus E for RuphenImH

and Ruphen1MeIm; 1H NMR spectra in different time and pHfor the complex RuphenImH; DOSY-NMR for the complexRuphenImH and RuphenImH in the presence of HSA; andSTD-NMR spectrum for Ruphen1MeIm in the presence ofHSA. This material is available free of charge via the Internet athttp://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Authors*(N.P.F.) Phone: (804) 828-6320; e-mail: [email protected].*(R.M.C.) Phone: (+55) 16-3351-8780; e-mail: [email protected] ContributionsAll authors contributed equally.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSLuciana Vizotto from UFSCar for NMR measurements, Prof.Ana Paula Araujo and Dr. Julio Cesar Pissuti Damalio fromUSP-Sao Carlos for CD measurements, and Prof. Amando S.Ito from USP-RP for emission lifetime measurements. FAPESP(proc. 2009/08218-0) and CAPES for the grants andfellowships. Supported in part by RO1CA-78754 to N.F.

■ ABBREVIATIONS USEDITC, isothermal titration calorimetry; CD, circular dichroism;STD, saturation transfer difference; DOSY, diffusion orderedspectroscopy; DFT, density functional theory

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http://pubs.acs.org

mailto:[email protected]



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