Combining imaging and anticancer properties with new heterobimetallic Pt(ii)/M(i) (M = Re, ^99mTc) complexes.

View Article Online View Journal Dalton Transactions An international journal of inorganic chemistry Accepted Manuscript This article can be cited before page numbers have been issued, to do this please use: L. Quental, P. Raposinho, F. Mendes, I. Santos, C. Navarro-Ranninger, A. Alvarez-Valdés, H. huang, H. Chao, R. Rubbiani, G. Gasser, A. Gomez Quiroga and A. Paulo, Dalton Trans., 2017, DOI: 10.1039/C7DT00043J. This is an Accepted Manuscript, which has been through the Volume 45Number 17 January 2016Pages 1–398 Dalton Royal Society of Chemistry peer review process and has been accepted for publication. Transactions Accepted Manuscripts are published online shortly after An international journal of inorganic chemistry www.rsc.org/dalton acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. 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In no PJEfr offi A a s m P ceE ie pe R w hn to T er . kxHtruapcpti,o On mofa sr uKlf. aFtaer hfrao met awl.ater using a Zr-metal–organic event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. rsc.li/dalton Please do not adjust margins Page 1 of 15 Dalton Transactions Journal Name ARTICLE Combining Imaging and Anticancer Properties with New t Heterobimetallic Pt(II)/M(I) (M = Re, 99mTc) Complexes p i r R A e cc c e e p iv t e e d d 0 0 0 0 t t h h J J a a n n u u a a r r y y 2 2 0 0 x x x x , Letícia Quentala, Paula Raposinhoa, Filipa Mendesa, Isabel Santosa, Carmen Navarro-Ranningerb, c Amparo Alvarez-Valdesb, Huaiyi Huangc, d, Hui Chaod, Riccardo Rubbianic, Gilles Gassere,*, s DOI: 10.1039/x0xx00000x Adoración G. Quirogab,*, António Pauloa,* u www.rsc.org/ In this article, we report on the development of new metal-based anticancer agents with imaging, chemotherapeutic and n photosensitizing properties. Hence, a new heterobimetallic complex (Pt-LQ-Re) was prepared by connecting a non- a conventional trans-chlorido Pt(II) complex to a photoactive Re tricarbonyl unit (LQ-Re), which can be replaced by 99mTc to M allow for in vivo imaging. We describe the photophysical and tbiological properties of the new complexes, in the dark and upon light irradiation (DNA interaction, cellular localization and uptake, and cytotoxicity). Furthemore, planar scintigraphic d images of mice injected with Pt-LQ-Tc clearly showed that the radioactive compound is taken up by the excretory system organs, namely liver and kidneys, without significant retention in other tissues. All in all, the strategy of conjugating a e chemotherapeutic compound with a PDT photosensitizer endows the resulting complexes with an intrinsic cytotoxic t p activity in the dark, driven by the non-classical platinum core, and a selective activity upon light irradiation. Most importantly, the possibility of integrating a SPECT imaging radiometal (99mTc) in the structure of these new e heterobimetallic complexes might allow for in vivo non-invasive visualization of their tumoral accumulation, a crucial issue c to predict therapeutic outcomes. c A (1O ) from endogenous 3O and/or other reactive oxygen 2 2 Introduction species (ROS), mediated by a photosensitizer. PDT is, for s example, used for the treatment of certain malignant tissues. Anticancer therapeutic approaches relying on a single n Compared with other conventional anticancer treatments, PDT therapeutic modality (e.g. chemotherapy, external has many advantages, namely spatial and temporal control o radiotherapy, etc.) and/or involving the administration of a single drug (e.g. chemotherapeutic, radiosensitizer, etc.) very and the possibility of repeated doses. 1, 2 i At a first glance, the more obvious and straightforward t often do not lead to a satisfactory curative outcome, mostly c strategy for a dual chemo/PDT therapeutic approach is based due to acquired chemo- and radio-resistance or the a on the administration of two individually different occurrence of undesired side effects. Different strategies have s chemotherapeutic and photosensitizing agents already in been applied over the past few years to overcome these clinical use. However, the two individual agents will necessarily n drawbacks, including the design of more selective and target- show different pharmacokinetics and different tumor targeting a specific anticancer drugs or the combination of different independent therapeutic modalities. An example of such ability. To circumvent these drawbacks, the merging of the r cytotoxic and photosensitizing units into the same chemical T combination may involve chemotherapy and photodynamic therapy (PDT).1, 2 PDT is a clinically approved technique, which entity is an attractive alternative that has been investigated using mainly porphyrin derivatives as the photosensitizer n involves light-induced generation of cytotoxic singlet oxygen unit.1, 2 Importantly, the exploration of novel metal-based o “hybrid” anticancer agents, acting at the same time as a t cytotoxic drug and as PDT photosensitizer, might open new l a avenues in the design of this type of agents, profiting from the D variety of structural motifs and the diversity of physicochemical properties and biological characteristics exhibited by metal complexes. In the last few years, research interests of several groups have been focused on two types of compounds that can be used as tools to achieve “hybrid” metal-based drugs: a) organometallic Re(I)/Tc(I) compounds to obtain photosensitizers for PDT and radiopharmaceuticals for nuclear imaging or radionuclide This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 1 Please do not adjust margins .65:51:71 7102/10/52 no ogeiD naS - ainrofilaC fo ytisrevinU yb dedaolnwoD .7102 yraunaJ 52 no dehsilbuP View Article Online DOI: 10.1039/C7DT00043J Please do not adjust margins Dalton Transactions Page 2 of 15 ARTICLE Journal Name therapy 1, 3-6 and b) platinum compounds to obtain powerful analysis of their structures, they were not expected to be anticancer agents.7 These two classes of compounds can also active against cancer cells. However, they were shown to be allow for the development of novel agents combining highly cytotoxic.27 diagnostic and therapeutic tools (theranostic agents), which In this study, we decided to follow the idea of multifunctional has emerged as a new approach to fight cancer over the last metal-based compounds that combine therapeutic and decade and, in consequence, became a major goal in diagnosis in the same chemical entity, to obtain new medicinal/medical research.1 anticancer drugs suitable for a theranostic approach. For this The interest in the use of Re(I) tricarbonyl complexes as purpose, we developed new heterobimetallic complexes with photosensitizers for PDT purposes is relatively recent.3, 6, 8, 9 a trans-chlorido Pt(II) moiety and a Re(I)/99mTc(I) tricarbonyl Re(I) complexes have been mainly explored as luminescent core. The rationale behind this concept is the central role of t p probes because of their photophysical properties, like long Pt(II) complexes in the development of chemotherapeutic lifetimes, polarized emission and large Stoke’s shift.10-15 In drugs, the importance of 99mTc for in vivo tumor imaging and i r particular, those having the core fac-[Re(CO) 3 ]+ with ligands the possibility of using the Re(CO) 3 core in PDT. c derived from N,N’-bis[(quinolin-2-yl)methyl]amine are Based on our results that showed that some non-classical s endowed with luminescent properties that might allow for trans-Pt(II) complexes with isopropylamine and pyridine u visualization of their intracellular trafficking by fluorescence derivatives are active in cisplatin-resistant cell lines,27, 28 we microscopy.3, 8 On the other hand, the 99mTc analogues of selected this type of compounds to synthesize the new n Re(CO) complexes can be easily prepared (based on their Pt(II)/M(I) (M= Re, 99mTc) heterobimetallic complexes. For the a 3 chemical similarities) to perform in vivo radioimaging, bridging corresponding organometallic moiety, we have focused on M the gap between cellular, animal and human imaging studies.8, N,N’-bis[(quinolin-2-yl)methyl]amine M(CO) derivatives 3 13-17 99mTc is employed to obtain a variety of containing a n-propylamine pendant arm because this class of d radiopharmaceuticals that are in clinical use for early detection compounds can present remarkable phototoxic properties e of several diseases by means of Single Photon Emission and, importantly, since the cold Re atom can be efficiently Computed Tomography (SPECT).18, 19 The longstanding and replaced with 99mTc, as mentioned above.3, 8, 13-17 We expected t p unsurpassed success of 99mTc in medical imaging is due to its to obtain complexes with cytotoxic activity, PDT e commercial availability and its optimal features for imaging, photosensitizing properties and, more importantly, allowing in among other factors. These include a short half-life of 6.02 h vivo imaging. To assemble this new family of complexes, our c that is long enough to allow for the preparation of the strategy involved 4-picolinic acid as a bridging element c radiopharmaceuticals by 99mTc-labelling of commercially between the Pt(II) metallic core and the bis(quinoline)amine A available freeze-dried kits20 and the in vivo accumulation in the moiety, which can react with the Re(I)/99mTc(I) tricarbonyl core target tissue/organ, but it is sufficiently short to avoid a long to afford the aforementioned organometallic compounds. s term exposition of the patient to the radiation dose. Herein, we report the synthesis and characterization of the n In summary, organometallic M(I) (M=99mTc or Re) complexes resulting heterobimetallic Pt(II)/M(I) (M=Re, 99mTc) complexes, o have useful and unique features for biomedical applications: i) as well as the study of their photophysical properties and their chemical robustness of the fac-[M(CO) ]+ core; ii) well-studied in vitro and in vivo biological evaluation (cytotoxicity, i 3 t coordination chemistry with a variety of chelating ligands photocytotoxicity, cell uptake assays, and biodistribution in c including targeting molecules 17, 21-23; iii) possible application in normal mice). a the design of metal-based cancer theranostic agents where Re s complexes can be incorporated into a cytotoxic or Results and discussion n photosensitizing entity that will exert a therapeutic effect, while the 99mTc congeners are part of the correspondent Chemical synthesis: ligands and non-radioactive complexes (Re a imaging tool for in vivo assessment of tumor accumulation.24-26 and Pt) r Due to its radioactive properties, 99mTc can only be obtained in T As indicated in Scheme 1, two different approaches were small amounts (< 1 µM) that are well below the minimum investigated to obtain the final heterobimetallic compound Pt- n threshold concentrations that are required for a biological response. In brief, isostructural Re and 99mTc complexes allow LQ-Re: o Approach 1) amidation reaction between trans- for a theranostic approach of cancer, where the Re complexes t exert the therapeutic effects and the 99mTc congeners are used [PtCl 2 (isopropylamine)(4-picolinic acid)] (trans-[PtCl 2 ipa(pic)]) a l and fac-tricarbonyl(N’,N’-bis(quinolin-2-ylmethyl)propane-1,3- solely for imaging/diagnostic purposes. D diamine)rhenium(I) (LQ-Re) and Platinum compounds, namely cisplatin, carboplatin and Approach 2) initial coupling of the bifunctional chelator N’,N’- oxaliplatin, are used in the clinic as powerful anticancer drugs. bis(quinolin-2-ylmethyl)propane-1,3-diamine (LQ) to trans- However, they are responsible for severe side effects and [PtCl ipa(pic)], followed by complexation of the fac-[Re(CO) ]+ there is undoubtedly a growing need for novel compounds of 2 3 core by the resulting conjugate Pt-LQ. the same or higher efficiency with reduced side effects. Non- Approach 1. This approach was envisaged to avoid the conventional drug design grew as an interesting alternative to involvement of the free bis(quinoline) chelator (LQ) in fulfill this need, and trans-chlorido Pt(II) complexes appeared competitive coordination reactions with the Pt(II) core, as one of these non-classical examples. Interestingly, from the 2 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .65:51:71 7102/10/52 no ogeiD naS - ainrofilaC fo ytisrevinU yb dedaolnwoD .7102 yraunaJ 52 no dehsilbuP View Article Online DOI: 10.1039/C7DT00043J Please do not adjust margins Page 3 of 15 Dalton Transactions Journal Name ARTICLE allowing therefore for a more controlled process to obtain the presence of a single Pt(II) species displaying a PtCl 2 desired Pt-Re heterobimetallic complex. This approach starts environment, as discussed in more detail below. with the synthesis of LQ-Re that should be subsequently applied in the amide coupling with trans-[PtCl ipa(pic)]. Approach 2. In the second approach, it was taken into 2 Initially, the synthesis of LQ-Re was attempted by reacting a consideration that the radioactive congener of Pt-LQ-Re, i.e. methanol solution of LQ with a stoichiometric amount of Pt-LQ-Tc, should be obtained in a similar manner, i.e. by [Re(CO) (H O) ]Br. This synthetic method was considered due reacting Pt-LQ with fac-[99mTc(CO) (H O) ]+ (see below). Due to 3 2 3 3 2 3 to the highest reactivity of [Re(CO) (H O) ]Br compared with the similar chemical behavior of Re(I) and Tc(I) tricarbonyl 3 2 3 more “classical” pentacarbonyl precursors, like [Re(CO) X] (X = complexes, the study of the reaction of Pt-LQ with 5 Cl, Br).29 This enhanced reactivity reflects the high lability of [Re(CO) (H O) ]Br to obtain Pt-LQ-Re will give important hints t 3 2 3 p the coordinated water molecules and allows for the use of about the possibility of synthesizing the radioactive congener milder conditions to synthesize Re(I) tricarbonyl complexes Pt-LQ-Tc at the low concentrations of 99mTc (<10-6M, no carrier i r with tridentate chelators. The reaction of LQ with added level) that are characteristic of 99mTc- c [Re(CO) (H O) ]Br led to the desired complex, which was radiopharmaceuticals. 3 2 3 s obtained in high yield (83 %) with bromide as the counter-ion To obtain the Pt-LQ intermediate, LQ was reacted with the u ([LQ-Re]Br). The coupling of [LQ-Re]Br to trans-[PtCl ipa(pic)] NHS-activated of trans-[PtCl ipa(pic)]) (Scheme 1). Pt-LQ was 2 2 proceeded well upon activation of the free carboxylic acid of recovered as an orange oil in moderate yield (58 %) after semi- n the later with N-hydroxysuccinimide (NHS). However, the preparative HPLC purification. Thereafter, the ability of Pt-LQ a amide coupling reaction was followed by metathesis reactions to coordinate the fac-[Re(CO) 3 ]+ core was investigated. For this M involving the Pt(II) core that led to the replacement of the purpose, the reaction of Pt-LQ with [Re(CO) (H O) ]Br was first 3 2 3 coordinated chloride by bromide with formation of a mixture studied. [Re(CO) (H O) ]+ readily reacts with Pt-LQ, as 3 2 3 d of heterobimetallic complexes: the desired Pt-LQ-Re and the indicated by HPLC analysis of the reaction mixture. However congeners presenting the trans-[PtClBr(ipa)(pic)] and trans- the complexation of fac-[Re(CO) ]+ by Pt-LQ is followed by the e 3 [PtBr (ipa)(pic)] units. The formation of these three replacement of the coordinated chlorides by bromide with the t 2 p heterobimetallic complexes was corroborated by ESI-MS formation of a mixture of heterobimetallic Pt(II) halides e analysis of one aliquot of the reaction mixture and by the containing the trans-[Pt(X)(Y)ipa(pic)] (X=Y=Cl; X=Cl, Y=Br; presence of three signals in its 195Pt NMR spectrum (Fig. S1). X=Y=Br) units, as indicated by ESI-MS analysis. Purification of c the different Pt(II) complexes in this mixture by HPLC was c attempted, but without success. Finally, as shown in Scheme 1, A Pt-LQ was reacted with [Re(CO) Cl] in refluxing THF. This 5 synthetic method allowed for the synthesis of the chloride salt s of Pt-LQ-Re in moderate yield (29 %). n Pt-LQ-Re and all the new compounds involved in this study o were analyzed by different spectroscopic techniques (IR and 1H, 13C and 195Pt-NMR) and mass spectrometry, allowing for i t the unambiguous identification of their chemical structures c (see experimental section for detail procedures and a characterization). Concisely, the 1H-NMR and IR data collected s for LQ-Re confirmed the proposed formulation and are n consistent with those reported recently for similar bis(quinoline) Re(I) tricarbonyl complexes.2, 30 In particular, the a methylenic H-4 protons (see Scheme 1 for atom numbering) r give rise to a double doublet in the 1H-NMR spectrum of LQ- T Re, consistent with an AB spin pattern and with the n coordination to the Re(I) centre that renders these protons Scheme 1. Synthesis of Pt-LQ-Re. Approach 1: i) DIPEA, NHS, EDC, DMF, 16 o diastereotopic. The IR spectrum of LQ-Re shows intense h, r.t. (η=19 %). Approach 2: ii) DIPEA, NHS, EDC, DCM, 16 h (η=58 %); iii) absorption bands in the range 1897-2018 cm-1 that are easily t Re(CO)Cl,THF, o.n., r.t. (η=29 %). 5 l assigned to the v(C≡O) stretching modes by comparison with a These results prompted us to synthesize LQ-Re by reacting the values previously reported for such bands in other related D [Re(CO) Cl] with LQ in refluxing THF. This synthetic method Re(I) tricarbonyl complexes.30, 31 5 gave [LQ-Re]Cl in high yield (69 %), after HPLC purification to The 1H-NMR spectrum of Pt-LQ confirms the linkage of LQ to remove any unreacted starting materials. As shown in Scheme the picolinic acid of the original trans complex without release 1, the coupling reaction of [LQ-Re]Cl with trans-[PtCl ipa(pic)] of the ligands coordinated to Pt(II). The spectrum shows the 2 afforded the desired Pt-LQ-Re compound, which was obtained presence of the signals of the aliphatic protons of as a light orange oil, after HPLC purification, with a relatively isopropylamine at 3.39 and 1.39 ppm. The spectrum also low yield of 19 %. In this case, the 195Pt NMR and ESI-MS shows the presence of aromatic signals arising from the spectra of the isolated Pt-LQ-Re were consistent with the protons of the functionalized picolinic acid (appearing at 7.37 This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3 Please do not adjust margins .65:51:71 7102/10/52 no ogeiD naS - ainrofilaC fo ytisrevinU yb dedaolnwoD .7102 yraunaJ 52 no dehsilbuP View Article Online DOI: 10.1039/C7DT00043J Please do not adjust margins Dalton Transactions Page 4 of 15 ARTICLE Journal Name and 8.75 ppm) and bis(quinoline) moiety (twelve aromatic Re surrogates, as exemplified for Pt-LQ-Tc in Figure S4.A. For protons at 7.60-8.22 ppm), as well as aliphatic signals due to this purpose, HPLC-purified samples of these 99mTc-containing the methylenic protons of the N-propyl amine pendant arm. compounds were used. The in vitro and in vivo studies The IR spectrum of Pt-LQ-Re shows intense v(C≡O) bands reported below for LQ-Tc and Pt-LQ-Tc were also always between 1975-2025 cm-1, which confirms the presence of the performed using HPLC-purified samples of these compounds. fac-[Re(CO) ]+ core in this heterobimetallic complex. 3 Consistently, the 1H-NMR spectrum of Pt-LQ-Re corresponds almost to the sum of the spectra of Pt-LQ and LQ-Re, reflecting N the assembly of the two metal cores (Figures S2 and S3). The H2N N N N CO most striking difference is in the chemical shifts of the LQ H2N N 99mTc CO t i N CO p aromatic protons of picolinic acid, which are significantly high- field shifted in the case of Pt-LQ if compared with Pt-LQ-Re OH2 i (7.37 and 8.75 ppm vs 7.92 and 8.98 ppm, respectively). This H2O 99mTc OH2 LQ-Tc c r shift towards high-field is possibly caused by the ring current OC CO CO shielding effects of the intramolecular π-π stacking ii H2 C N l Pt C N l H N CO u s interactions between the picolinic acid aromatic ring and the N N 99mTc CO dangling quinolone aromatic rings. The metalation of NH2 Cl N CO n Pt O f b o is r ( q th u i i s n o re li a n s e o ) n h , i t n h d e e r c s h e s m uc i h ca i l n s t h ra if m ts o o le f c t u h la e r p π ic − o π li n s i t c a c a k c i i n d g i n a n P d t- , Cl N O H N N N N Pt-LQ-Tc M a Pt-LQ LQ-Re are almost coincident with those of the parental complex trans-[PtCl ipa(pic)].27 Finally, the 195Pt-NMR spectra Scheme 2. Synthesis of the 99mTc complexes. i) 0.9 % NaCl, 50 oC, 1 h, pH=5 2 (η=50 %); ii) 0.9 % NaCl, 60 oC, 1 h, pH=5 (η>90 %). d of solutions of Pt-LQ and Pt-LQ-Re exhibited only one signal at e similar chemical shifts consistent with the proposed The in vitro evaluation of LQ-Tc and Pt-LQ-Tc comprised the coordination spheres (-2098.39 and -2098.20 ppm, measurement of their lipophilicity and the study of their t p respectively).27, 32 1H and 13C-NMR spectra assignment were stability in different media (PBS, human serum, cell culture e double checked by two-dimensional experiments using homo- medium) with relevance for the biological studies that were and heteronuclear techniques, such as DEPT-135, 1H-1H COSY performed for these compounds and for the Re congeners (LQ- c and 1H-13C HSQC. The ESI-MS spectra of LQ-Re, Pt-LQ and Pt- Re of Pt-LQ-Re). c LQ-Re showed peaks and isotopic patterns consistent with the The study of the stability of the complexes LQ-Tc and Pt-LQ-Tc A expected molecular ions in the presence of the different challenging media was performed by co-incubating each tested compound with the s Synthesis, characterization and in vitro evaluation of 99mTc desired medium at 37 ºC for different intervals of time, n complexes followed by radio-HPLC analysis of the reaction mixtures. Both o The synthesis of the 99mTc complexes, namely LQ-Tc and Pt-LQ- compounds exhibited a high stability under all evaluated i Tc, was performed in aqueous saline solution at pH≈5 by challenging conditions, without formation of new t reaction of fac-[99mTc(CO) (H O) ]+ with LQ (5x10-4 M final radiochemical species until 6 h of incubation, as exemplified c 3 2 3 concentration) and Pt-LQ (10-3 M final concentration) for 60 for Pt-LQ-Tc, in the presence of human serum (Figure S4.B). a min, at 50 ºC and 60 ºC, respectively (Scheme 2). To our Overall, the in vitro evaluation of Pt-LQ-Tc has shown that it is s knowledge, Pt-LQ-Tc is the first reported example of a 99mTc/Pt a quite robust complex, under several biological conditions. It n heterobimetallic complex. Its synthesis was achieved in is reasonable to assume that the same is valid for the congener a reasonably high radiochemical yield (> 90 %) after appropriate Pt-LQ-Re, that displays the same Pt(II) core and a fac- optimization of the reaction conditions, such as ligand [Re(CO) 3 ]+ core chemically related to fac-[Tc(CO) 3 ]+. T r concentration, pH and temperature. Noticeably, the presence The lipophilicity of LQ-Tc and Pt-LQ-Tc was assessed by of the Pt(II) complex had a positive influence on the measurement of the respective log Po/Pw values (n- n radiolabeling reaction, as Pt-LQ-Tc was typically obtained in octanol/0.1 M PBS, pH = 7.4) using the multiple back- o higher radiochemical yield than LQ-Tc (90 % vs 50 % yield). extraction method. The obtained log Po/Pw values of 0.57 ± t However, the synthesis of Pt-LQ-Tc is strongly pH-dependent. 0.02 and 1.44 ± 0.04, respectively, show that both compounds l When the reactions were carried out at neutral pH the have a lipophilic character. Other cationic lipophilic complexes a formation of two major radioactive species was observed in described in the literature with favorable cell uptake have log D addition to the desired complex that was formed, under these P o /P w in the same range of values, e.g. the promising conditions, only with ca. 30 % yield. By performing the reaction myocardial agent 99mTc-TMEOP (0.61 ± 0.04).33 Pt-LQ-Tc is at pH 5, the formation of these two radiochemical impurities, more lipophilic than LQ-Tc, due to the introduction of a neutral resulting most probably from hydrolysis processes involving Pt(II) core that contains coordinated organic ligands. By the trans-PtCl core, was almost eliminated. contrast, the heterobimetallic Pt-LQ-Tc is much more lipophilic 2 The chemical identity of LQ-Tc and Pt-LQ-Tc was ascertained than Pt drugs in clinical use, like cisplatin, carboplatin or by comparison of their HPLC profiles with the corresponding oxaliplatin, which display log P o /P w values ranging between - 2,5 and -1.5. 34, 35 4 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .65:51:71 7102/10/52 no ogeiD naS - ainrofilaC fo ytisrevinU yb dedaolnwoD .7102 yraunaJ 52 no dehsilbuP View Article Online DOI: 10.1039/C7DT00043J Please do not adjust margins Page 5 of 15 Dalton Transactions Journal Name ARTICLE Singlet oxygen sensitization Photophysical properties A quantitative evaluation of the singlet oxygen 1O (1∆) 2 g N,N’-bis[(quinolin-2-yl)methyl]amine Re(I) complexes have production upon irradiation at 350 nm was performed to favorable properties as luminescent probes.3, 8 Therefore, to assess the potential of these complexes as photosensitizers in assess if the new heterobimetallic complexes would present PDT. An indirect method was used in both PBS (with 10 mM similar characteristics, the luminescence properties of the histidine) and acetonitrile (with 12 mM imidazole) solutions. different complexes were studied and are summarized in Table 1O can react with an imidazole derivative to form a trans- 2 1; the respective electronic and emission spectra are annular peroxide adduct, which is able to quench the presented in Figures S5 and S6. absorbance of p-nitrosodimethyl aniline (RNO).3, 40, 41 The 1O 2 t All complexes showed a weak absorption in the visible region production quantum yields were evaluated by comparison p (400-800 nm). The absorbance between 200-250 nm was with a reference molecule phenalenone (Φ(1∆) = 95 %).41 The g i almost the same for the three compounds, suggesting that it is results obtained are shown in Table 1. LQ-Re exhibited the r induced by the ligand. LQ-Re exhibited a UV absorbance highest singlet oxygen generation efficiency among the three c between 280–350 nm with a maximum at 322 nm (Figure S5). compounds, both in PBS and acetonitrile. The quantum yields s These features are comparable with structurally similar Re(I) in acetonitrile were higher than those obtained in PBS u complexes 36 and are the result of a metal-to-ligand charge- solution. These results were comparable with previously n transfer (MLCT: d (Re)→π*(ligand)).37 As expected, the reported data on a very similar complex.3 Interestingly, the π a absorbance spectrum of Pt-LQ-Re was found to be a mixture introduction of a platinum(II) complex to the structure of LQ- of the absorbance spectra of LQ-Re and Pt-LQ. Re decreased the singlet oxygen generation efficiency of LQ- M Re, particularly in the case of the values obtained in acetonitrile. These values are even lower than the ones d Table 1. Photophysical properties of the complexes at room temperature observed for Pt-LQ. e Quantum 1O2 t Excitation Emission Lifetime Quantum Yield p Compound Yield a λex/nm bλem/nm bΦem /% τ /ns (Φ(1∆g) / % Dark- and photo-toxicity e PBS/Acetonitrile The dark and photo-toxicity of the complexes LQ-Re, Pt-LQ and LQ-Re 350 452, 575 0.28, 0.17 708 22 / 65 Pt-LQ-Re was evaluated on several human cell lines, namely on c Pt-LQ 350 600 0.11 704 15 / 24 c tumoral - A2780 (epithelial ovarian cancer, cisplatin sensitive), Pt-LQ-Re 350 449 0.14 509 11 / 19 A2780R (epithelial ovarian cancer, cisplatin resistant) and HeLa A ameasured in PBS solution. bmeasured in DMSO, by comparison with the emission (epithelial cervical cancer), as well as on non-tumoral MRC-5 of [Ru(bipy)3]Cl2 in aerated water (Φem = 0.040). (fibroblast) cell lines (Table 2). s Initial studies were performed with incubation of the n Upon excitation at 350 nm, LQ-Re showed two distinct complexes for 48 h in the dark to compare their toxicity with a o emission bands, typical of Re(I) complexes (Figure S6).38 The known chemotherapeutic drug, namely cisplatin. LQ-Re i exhibited extremely low dark toxicity in all cell lines studied in t high-energy transition (430 nm) was assigned to ligand- c this work. On the contrary, Pt-LQ was found to be the most centered fluorescence, whilst the lower energy transition (560 nm) originated from a 3MLCT phosphorescence state. Pt-LQ cytotoxic complex in the dark with IC 50 values in the low a micromolar range. The heterobimetallic Pt-LQ-Re complex s presented only one emission peak at 430 nm, which comes from the LQ ligand. The heterobimetallic Pt-LQ-Re complex exhibited moderate dark cytotoxicity against all cancer cell n lines. Of note, all three complexes exhibited lower cytotoxicity showed a broad emission band between 400-650 nm. a than cisplatin on the non-tumoral MRC-5 cells. Unlike all other Luminescence lifetimes were also evaluated (Table 1) and for r LQ-Re the value obtained was similar to the values of other compounds, Pt-LQ-Re displayed a similar cytotoxic activity T rhenium tricarbonyl bisquinoline complexes.37 The lifetime of against the A2780 and A2780R cell lines, showing that this heterobimetallic complex can significantly circumvent cisplatin n Pt-LQ was almost identical to the one of LQ-Re (around 700 cross-resistance. ns), however when the two metals are present the lifetime o As anticipated, decreasing the exposure time of the cells to the falls down to 509 ns. t The luminescence quantum yields (Φ ) were evaluated to complexes from 48 h to 4 h followed by 44 h incubation with l em fresh medium reduced significantly their cytotoxicity (Table 2, a understand the behavior of the compounds in the excited condition b). Upon light irradiation (350 nm, 2.58 J cm-2), LQ- D state. The Φ for all complexes was evaluated in DMSO (Table em 1) and compared with [Ru(bipy) ]2+ in water (Φ = 4.0 %).39 Re exhibited the highest improvement in phototoxicity toward 3 em A2780 cancer cells with an interesting photo-index (PI) of 18.6, The quantum yields of all complexes were smaller than 1 % while a lower PI of 10 was observed on the A2780R and HeLa (Table 1). In the case of Pt-LQ-Re and LQ-Re, these results are cell lines. These PI values are in agreement with the values in agreement with the data obtained for other Re(I) tricarbonyl complexes described in the literature.3, 12 reported by some of us for similar Re(I) tricarbonyl derivatives or with Photofrin3, 42 Based on the singlet oxygen assay, one would expect that the Pt-LQ-Re complex should not exhibit This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 5 Please do not adjust margins .65:51:71 7102/10/52 no ogeiD naS - ainrofilaC fo ytisrevinU yb dedaolnwoD .7102 yraunaJ 52 no dehsilbuP View Article Online DOI: 10.1039/C7DT00043J Please do not adjust margins Dalton Transactions Page 6 of 15 ARTICLE Journal Name significant phototoxicity in cancer cell lines. In line with this consistent with the results described below for the reasoning, the PI value of 6.7 presented by Pt-LQ-Re in the quantification of cellular uptake based on ICP-MS. A2780 cell line is quite lower than the one displayed by LQ-Re The non-visualization of the fluorescence signal of cellular (PI 18.6), although being comparable to the PI of Pt-LQ. In the accumulation of LQ-Re can be due to luminescence quenching other cells lines analyzed Pt-LQ and Pt-LQ-Re also display processes that the compound undergoes once inside the cell, similar PI values. as reported for related Re(I) tricarbonyl complexes.43,38 Our results reinforce the idea that for some luminescent compounds their true cellular accumulation and distribution can be distorted, as the intensity of the luminescent signal emitted from inside the cell can be depleted or enhanced upon t Table 2. IC 50 values (µµµµM) and phototoxic index (PI). interaction of the compounds with biological molecules that p are present in the different cell compartments. i LQ-Re Pt-LQ Pt-LQ-Re Cisplatin r c MRC-5 (non-tumoral) s 48 ha 121± 10.1 14.3± 3.0 22.0± 5.3 8.4± 2.1 u A2780 n 48 ha 46.1± 6.5 9.1± 1.8 28.7± 4.2 1.7± 0.5 4 hb 145 ± 9.5 30.6 ± 5.5 124 ± 8.3 8.1 ± 2.4 a 4 h + irradiationc 7.8 ± 1.6 4.3 ± 0.8 18.4 ± 5.2 9.3 ± 2.1 M PI 18.6 7.1 6.7 n.ad A2780R d 48 ha 198 ± 20.5 25.6 ± 4.6 27.8 ± 4.7 9.5 ± 2.3 e 4 hb >200 55.6 ± 7.4 67.8 ± 10.6 28.9 ± 5.3 4 h + irradiationc 19.3 ± 2.1 7.5 ± 1.4 16.5 ± 2.7 27.5 ± 3.1 t p PI >10 7.4 4.1 n.ad e HeLa 48 ha 155 ± 22 12.6 ± 4.6 42.8 ± 4.7 10.2 ± 2.3 c 4 hb >200 34.8 ± 4.1 77.8 ± 8.4 32.4 ± 5.7 c 4 h + irradiationc 20.1 ± 6.5 5.5 ± 0.9 13.5 ± 4.1 35.2 ± 4.6 A PI >10 6.3 5.7 n.ad a48 h incubation with complexes; b4 h incubation with complexes and then 44 h s incubation with fresh medium; c4 h incubation with complexes, followed by 10 n min irradiation at 350 nm (2.58 J cm-2) and then 44 h incubation with fresh o medium; dNot applicable i t c Figure 1. Live fluorescence microscopy of Hela cells treated with 20 Cellular uptake and subcellular distribution a μM of each compound for 2 h. Confocal microscopy s The cellular uptake efficiency of each complex plays an Quantification of cellular uptake and subcellular distribution n important role in the cytotoxicity profile and needs to be taken Cellular uptake studies in A2780 and A2780cisR cells were a into account to interpret the dark and photo-toxicity data performed to evaluate the amount of LQ-Re, Pt-LQ and Pt-LQ- r presented above. To visualize the cellular localization of the Re that enters into the cells and that localizes in the nucleus. T compounds in living cells, confocal fluorescence microscopy Cells were incubated with complexes at 5 µM for 24 h and the n studies were performed. For this purpose, HeLa cells were cytoplasmic and nuclear fractions were isolated and analyzed incubated with the corresponding compounds (20 µM) for 2 h. by ICP-MS to quantify the presence of the corresponding o As shown in Figure 1, the signal of LQ-Re was too weak to be metals (Pt and/or Re). t observed. It has been previously reported that the analogue As can be seen in Figure 2, the most striking difference is the a l complex having a 4-butylenic amine pendant arm instead of a much lower ability of Pt-LQ-Re to cross the cell membrane and D 4-propylenic amine one, was observed exclusively in the to reach the cytoplasm and nuclear compartment if compared cytoplasm, using however a much higher concentration (100 with LQ-Re and Pt-LQ. For the Pt-containing compounds, it can µM).36 be also concluded from these results that the neutral complex Under the same experimental conditions as the one used for Pt-LQ has a better cellular uptake than the positively charged LQ-Re, Pt-LQ-Re homogeneously distributed throughout the complex Pt-LQ-Re. This agrees with the cytotoxicity assay, cell, although the signal was still very weak. Pt-LQ was found which showed that Pt-LQ exhibited the highest dark to accumulate mostly in the cytoplasm. With the exception of cytotoxicity of these two compounds. Most probably, the the LQ-Re, the confocal fluorescence imaging results are lower cellular uptake of the heterobimetallic complexes Pt-LQ- 6 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .65:51:71 7102/10/52 no ogeiD naS - ainrofilaC fo ytisrevinU yb dedaolnwoD .7102 yraunaJ 52 no dehsilbuP View Article Online DOI: 10.1039/C7DT00043J Please do not adjust margins Page 7 of 15 Dalton Transactions Journal Name ARTICLE Re reflects its increased size and molecular weight, in 0.2). One likely explanation of this behavior is that upon light comparison with the corresponding LQ- Re and Pt-LQ units. irradiation there is singlet oxygen production and relative t p i r oxidative damage, which leads to DNA single strand breaks c (SSBs). s Figure 2. Subcellular distribution of LQ-Re, Pt-LQ and Pt-LQ-Re in A2780 Figure 3. Agarose gel electrophoresis of pBR322 plasmid treated with Pt-LQ- cells and in A2780 cisR cells after 24 h of incubation with 5 µM of each Re and cisplatin a) in the dark and b) after irradiation. For both gels lane 1- u complex. Data expressed as metal (nmol Re or Pt) content in nucleus or C DNA plasmid control; lanes 2 to 6 - DNA incubated with Pt-LQ-Re at ri 0.01 n cytoplasm per million cells. to 0.2 and lanes 7 to 12 - DNA incubated with cisplatin at ri 0.01 to 0.2. Ri is the ratio metal complex:DNA base pairs. OC –open circular and CCC a covalently closed circular. M DNA interaction As can be clearly observed in the plasmid DNA interaction The experiments reported above have shown that our Pt- assay, upon light irradiation for higher concentrations, the d containing compounds (Pt-LQ and Pt-LQ-Re) are moderately supercoiled isoform of the plasmid was almost completely e cytotoxic to cancer cells and that irradiation enhances their converted to open circular isoform (Figure 3). To characterize if t activity. It is important to highlight that the Pt starting material the in vitro oxidative damage to the DNA was also observed in p itself (picolinic complex) is not cytotoxic,27 although its living cells, we have also performed pulse field gel e oxidation to Pt(IV) afforded an active complex.44 All in all, the electrophoresis of A2780 cells treated with 20 µM of Pt-LQ-Re c functionalization of the Pt fragment with LQ seems to be a key in the dark and upon light irradiation at 350 nm for 10 min c factor in the cytotoxicity observed. (2.58 J cm-2). It is possible to observe only negligible double A To have a first insight on the mechanism of action of the new strand breaks (DSBs) and no extended DNA fragmentation on heterobimetallic Pt/Re complex, we have studied the binding A2780 cancer cells upon treatment with the target complex, s effect of Pt-LQ-Re on DNA tertiary structure models. This was confirming that the oxidative damage produced by Pt-LQ-Re n done by assessing its capacity to alter the electrophoretic on DNA leads mainly to single strand breaks (see Figure S7). mobility of the covalently closed circular (ccc) and open Moreover, it was not possible to detect any platination by ICP- o circular (oc) isoforms of a model plasmid such as pBR322. PtII MS analysis of the DNA extracted from the cells treated with i complexes and in particular cisplatin45 have been widely Pt-LQ-Re, in the dark or under irradiation. This agrees with the t c reported to produce changes in both plasmid DNA isoforms: results of the gel electrophoresis experiments and suggests a reducing the ccc mobility (via unwinding) and increasing the oc once again that the cytotoxic effect of Pt-LQ-Re at the DNA s mobility until both reach a co-migration point.46 Figure 3 level is related to its photosensitization and relative shows the results of the experiments performed with production of 1O 2 . n increasing amounts of Pt-LQ-Re and cisplatin (as control) with a the plasmid pBR322 simultaneously in two different Biodistribution and Imaging r conditions: a) maintaining the samples at 37 ºC in the dark; b) Finally, we have performed biodistribution and imaging studies T irradiating the sample for 4 h at 350 nm (32.40 J.cm-2)) and of Pt-LQ-Tc in normal mice, to prove the theranostic n then maintaining it in the dark at 37 °C. capabilities of this type of heterobimetallic complexes. To In the dark, Pt-LQ-Re, contrary to cisplatin, does not alter the perform these studies, Pt-LQ-Tc was injected into Balb/C mice. o mobility of the plasmid. Cisplatin, on the other hand, altered The animals were euthanized 1 h and 4 h after administration. t both forms that finally co-migrate at ri=0.02 (ri = molar ratio Samples of blood and urine were collected and analyzed by a l Pt/nucleotide), as previously reported.45 radio-HPLC after adequate treatment, as detailed in the D After irradiation of Pt-LQ-Re for 30 min (4.05 J.cm-2), 2 h (16.20 experimental section, to have a further insight on the Pt-LQ-Tc J.cm-2) (data not shown) and 4 h (32.40 J.cm-2), we observed stability in biological milieu. These studies revealed that the some differences in the mobility and amount of the plasmid circulating radioactivity in the serum corresponds mainly to DNA isoforms (lines 2 to 6). Pt-LQ-Re only slightly altered the intact Pt-LQ-Tc at least at 1 h post injection (p.i) (Fig. S8). This mobility of the supercoiled form ccc (lines 3 to 5, experiment encouraging result points out that the congener Pt-LQ-Re b), showing that DNA platination does not occur in an might also have a favourable stability in the bloodstream. In appreciable extent. However, the ccc isoform is almost fully contrast, one major metabolite was found in the urine in converted to oc at the highest tested concentration (line 6, ri = addition to intact Pt-LQ-Tc (Fig. S8). This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 7 Please do not adjust margins .65:51:71 7102/10/52 no ogeiD naS - ainrofilaC fo ytisrevinU yb dedaolnwoD .7102 yraunaJ 52 no dehsilbuP View Article Online DOI: 10.1039/C7DT00043J Please do not adjust margins Dalton Transactions Page 8 of 15 ARTICLE Journal Name General procedures All chemicals and solvents were of reagent grade and were used without purification unless stated otherwise. Solvents were dried and distilled prior to use according to described procedures.47 Unless stated otherwise, the syntheses of the ligands and complexes were not carried under a nitrogen L atmosphere, or using standard Schlenk techniques and dry solvents; the work-up procedures were all performed under K air. The platinum complex trans-[PtCl (4-picolinic 2 t acid)(isopropylamine)] and the starting material fac- p [Re(CO) 3 (H 2 O) 3 ]Br were synthesized as described elsewhere.27, i 29 Na[99mTcO ] was eluted from a commercial 99Mo/99mTc r 4 c generator, using a 0.9 % saline solution. 1H and 13C NMR spectra were recorded on a Varian Unity 300 s MHz and a Brucker DRX 500spectrometer, and 195Pt NMR u spectra was recorded on a Varian Unity 500 MHz and a Brucker n Figure 4. Planar scintigraphic image of a Balb/C mice injected with Pt-LQ-Tc Advance II-HD Nanobay 300 spectrometer; 1H and 13C chemical at 1 h p.i. (L = liver; K= kidney). a shifts are given in ppm and were referenced to the residual M The biodistribution data obtained for Pt-LQ-Tc, at 1 h and 4 h solvent resonances relative to SiMe 4 . 195Pt chemical shifts were p.i. times, are presented in the Supplementary Information assigned using a solution of K 2 [PtCl 4 ] in saturated aqueous KCl (Table S1 and Fig. S9). Pt-LQ-Tc has a slow clearance from the as the external reference. The shift for K 2 PtCl 4 was adjusted to d blood stream (3.9 ± 1.0 % ID/g, 4 h p.i) with an important -1628 ppm from Na 2 PtCl 6 (δ=0 ppm). The NMR samples were e uptake in excretory organs (liver and kidney). The lipophilic prepared in CDCl 3 or CD 3 OD. The spectra were assigned with t nature of this radioactive heterobimetallic compound justifies the help of 2D experiments (1H–1H correlation spectroscopy, p its very slow excretion, involving most probably the and 1H–13C heteronuclear single quantum coherence). The e hepatobiliary route. In particular, the accumulation of spectra recorded for the monitoring of the Pt-LQ-Re reaction c radioactivity in the stomach is rather low (< 2 %/organ) with GMP were obtained in D 2 O:MeOD (3:1) using c showing that the 99mTc core of this heterobimetallic Pt(II)/Tc(I) trimethylsilyl propanoic acid as an internal reference. A compound resists in vivo to oxidation towards pertechnetate. IR spectra were recorded in the range 4000–400 cm-1 as KBr or The biodistribution properties of Pt-LQ-Tc were further CsI pellets on a Bruker Tensor 27 spectrometer. Electrospray s corroborated by imaging studies that allowed a clear ionization mass spectrometry (ESI-MS) was performed using a n visualization of the accumulation of radioactivity in the Bruker HCT electrospray ionization quadrupole ion trap mass excretory organs, namely liver and kidney, as shown in Figure spectrometer. o 4. Thin layer chromatography (TLC) was done on Merck silica gel i t 60 F254 plates. Column chromatography was performed with c silica gel 60 (Merck). Radioactivity measurements were done a Conclusions using an ionization chamber Aloka, Curiemeter IGC-3 or a γ- s counter Berthold, LB 2111. HPLC analysis of the ligands, Re, Pt The development of novel anticancer agents with imaging, and 99mTc complexes, was performed on a Perkin-Elmer LC n chemotherapeutic and photosensitizing properties within a pump 200 coupled to a LC 290 tunable UV–vis detector and to a single molecule is highly sought. In this article, we demonstrated that a new heterobimetallic complex containing a Berthold LB-507A radiometric detector, using an analytic r Supelco Discovery C18 reversed-phase column with a pre- T platinum and rhenium (Pt-LQ-Re) had indeed photosensitizing column, 25 cm x 4.6 mm, 5 µm, with a flow rate of 1 mL.min-1; properties due to the presence of the rhenium core and an UV detection, 254 nm. HPLC purification of the Re and Pt n intrinsic cytotoxic activity in the dark due to the presence of a complexes was performed in a HPLC system of Waters 2535 o non-classical platinum core. Importantly, it could be shown that the Re tricarbonyl unit in Pt-LQ-Re could be replaced by Quaternary Gradient, using a semipreparative Supelco C18 t 99mTc to allow for in vivo imaging and, moreover, that the reversed-phase column, 25 cm x 10 mm, 10 µm, with a flow a l rate of 2 mL.min-1. Eluents: aqueous 0.9 % NaCl solution, B: “cold” complex could overcome cisplatin-resistance. Overall, D MeOH. The HPLC analysis was done with gradient elution, the biological results of this study are quite appealing, using the following methods: justifying further studies for a better understanding of the Method 1, 0–3 min, 100 % A; 3–3.1 min, 100 %–75 % A; 3.1–9 biological effects induced by this new class of heterobimetallic min, 75 % A; 9–9.1 min 75 %–66 % A; 9.1–20 min, 66 %–0 % A; complexes. 20–25 min, 0 % A; 25–25.1 min, 0 %–100 % A; 25.1–30 min, 100 % A. Experimental Section Method 2, 0–3 min, 100 % A; 3–3.1 min, 100 %–75 % A; 3.1–9 min, 75 % A; 9–9.1 min 75 %–66 % A; 9.1–15 min, 66 %–0 % A; 8 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .65:51:71 7102/10/52 no ogeiD naS - ainrofilaC fo ytisrevinU yb dedaolnwoD .7102 yraunaJ 52 no dehsilbuP View Article Online DOI: 10.1039/C7DT00043J Please do not adjust margins Page 9 of 15 Dalton Transactions Journal Name ARTICLE 15–25 min, 0 % A; 25–25.1 min, 0 %–100 % A; 25.1–30 min, 136.61 (CH-Arom); 147.58 (Cq-Arom); 160.33 (Cq-Arom); ESI- 100 % A. MS (+) C H N (356.5) (m/z) ( %): 357.4 [M+H]+ (100). 23 24 4 Method 3, 0–3 min, 100 % A; 3–3.1 min, 100 %–75 % A; 3.1–9 min, 75 % A; 9–9.1 min 75 %–66 % A; 9.1–20 min, 66 %–0 % A; Synthesis of the Re complex of N’,N’-bis(quinolin-2- 20–30 min, 0 % A; 30–30.1 min, 0 %–100 % A; 30.1–35 min, ylmethyl)propane-1,3-diamine ([LQ-Re]X, X = Cl, Br) 100 % A. Method 4, 0–3 min, 100 % A; 3–3.1 min, 100 %–75 % A; 3.1–9 LQ-Re was synthesized by two different approaches (method A min, 75 % A; 9–9.1 min 75 %–66 % A; 9.1–24 min, 66 %–0 % A; and method B), as described below. 24–29 min, 0 % A; 29–29.1 min, 0 %–100 % A; 29.1–30 min, Method A (X=Br) 100 % A. To a solution of LQ (123 mg, 0.350 mmol) in MeOH (22.0 mL) t p was added [Re(CO) (H O) ]Br (143 mg, 0.350 mmol) and the 3 2 3 Synthesis and characterization of chelators, Re(I) and Pt(II) resulting solution refluxed overnight. After solvent i r complexes evaporation, the solid obtained was washed with water, c redissolved in MeOH and centrifuged at 4000 rpm for 10 s Synthesis of tert-butyl-3-(bis(quinolin-2- minutes. The solid was discarded and the liquid was dried ylmethyl)amino)propylcarbamate (LQ-Boc) u under reduced pressure. The residue was then washed with A solution of 2-quinolinecarboxaldehyde (189 mg, 1.200 mmol) diethyl ether and chloroform, to give LQ-Re as a dark orange n in dichloroethane (2.0 mL, DCE) was added at 0ºC, under oil (182 mg, 0.290 mmol, η=83 %). a nitrogen, to a solution of N-Boc-1,3-propanediamine (0.1 mL, Method B (X=Cl) M 0.570 mmol) and Na(OAc) BH (302 mg, 1.430 mmol) in DCE To a solution of LQ (50 mg, 0.140 mmol) in THF (10.0 mL), was 3 (6.0 mL). The mixture was stirred at room temperature for 4h. added Re(CO) Cl (51 mg, 0.140 mmol) and refluxed overnight. 5 d After evaporation of DCE, the reaction mixture was quenched After this time, the solvent was removed under reduced e with water and extracted with chloroform. The combined pressure and the desired product was purified by organic phases were dried over Na SO , concentrated under semipreparative HPLC (Method 2) to give a dark orange oil (60 t 2 4 p reduced pressure and purified through a pad of silica gel mg, 0.096 mmol, η=69 %). (eluent: CHCl (100-95 %)/MeOH (0-5 %)). LQ-Boc was R-HPLC (Method 2)=26.6 min; 1H-NMR (300 MHz, e 3 t obtained as a dark orange oil after removal of the solvent from MeOD+CDCl ): δ = 2.49 (m, 2H, H-2); 3.21 (t, 2H, H-1); 4.07 (dd, c 3 the collected fractions (111 mg, 0.240 mmol, η=42 %). 2H, H-3); 5.32 (dd, 4H, H-4); 7.74 (dd, 4H, Ar); 7.91 (dd, 2H, Ar); c R f (10 % MeOH/DCM)=0.29; R time -HPLC (Method 1)=22.0 min; 8.05 (d, 2H, Ar); 8.56 (dd, 4H, Ar); 13C-NMR (300 MHz, A 1H-NMR (300 MHz, CDCl ): δ = 1.38 (s, 9H, Boc); 1.76 (m, 2H, MeOD+CDCl ): δ = 25.06 (C-2); 37.92 (C-1); 65.49 (C-3); 69.53 3 3 H-2); 2.72 (t, 2H, H-3); 3.18 (m, 2H, H-1); 4.00 (s, 4H, H-4); 5.78 (C-4); 120.78 (CH-Arom); 129.24 (CH-Arom); 129.33 (CH- s (br s, 1H, NH-Boc); 7.50 (dd, 2H, Ar); 7.66 (dd, 4H, Ar); 7.77 (d, Arom); 129.56 (Cq-Arom); 130.77 (CH-Arom); 134.03 (CH- n 2H, Ar); 8.10 (dd, 4H, Ar); 13C-NMR (300 MHz, CDCl ): δ = 29.42 Arom); 142.86 (CH-Arom); 147.98 (Cq-Arom); 165.78 (Cq- 3 o (3 CH -Boc); 30.27 (C-2); 39.81 (C-1); 53.40 (C-3); 61.57 (C-4); Arom); 195.20 (2xC=O); 196.79 (C=O); ESI-MS (+) C H N O Re 3 26 24 4 3 79.33 (Cq-Boc); 121.78 (CH-Arom); 126.95 (CH-Arom); 127.96 (627.70) (m/z) ( %): 627.30 (100), 625.3 (67), 628.3 (35), 626.3 i t (Cq-Arom); 128.11 (CH-Arom); 128.45 (Cq-Arom); 129.59 (CH- (11), 629.3 (6); IR ν (CsI)/cm-1(C≡O) 2018, 1897. c max Arom); 130.14 (CH-Arom); 137.23 (CH-Arom); 148.06 (C=O a Boc); 156.67 (Cq-Arom); ESI-MS (+) C H N O (456.3) (m/z) ( 28 32 4 2 s %): 457.3[M+H]+ (100), 479.3 [M+Na]+ (29). Synthesis of mono ((4-(3-(bis(quinolin-2- ylmethyl)amino)propylcarbamoyl) pyridinium-1- n Synthesis of N’,N’-bis(quinolin-2-ylmethyl) propane-1,3-diamine yl)(isopropylammonio)platinum(II)) dichloride (Pt-LQ) a (LQ) N-hydroxysuccinimide (17 mg, 0.130 mmol) was added to a r T LQ-Boc (179 mg, 0.380 mmol) was dissolved in MeOH (12.0 solution of t-[PtCl (4-picolinic acid)(isopropylamine)] (47 mg, 2 mL), and HCl 37 % (2.1 mL) was added, at 0º, dropwise. The 0.110 mmol) in DCM (14.0 mL). The solution was stirred for 15 n mixture was stirred at room temperature for 24h. The pH was minutes, and then 1-ethyl-3-(3-dimethylaminopropyl) o adjusted to 10-12 with aqueous NaOH. After evaporation of carbodiimide (26 mg, 0.130 mmol) was added. After stirring for the MeOH under reduced pressure, the mixture was extracted 16 h at room temperature, the solution was evaporated to t l with chloroform. The combined organic phases were dried dryness and 5 mL of water were added. The resulting mixture a over Na SO and concentrated under reduced pressure to give was filtrated to separate the urea by-product and the resulting 2 4 D LQ as a brown oil (131.0 mg, 0.370 mmol, η=97 %). mixture was evaporated to dryness. The activated ester was R(100 % MeOH)=0.13; R-HPLC (Method 1)=18.0 min; 1H-NMR redissolved in dry DMF (3.5 mL), and added to a solution of LQ f t (300 MHz, CDCl ): δ = 1.74 (m, 2H, H-2); 2.69 (t, 2H, H-3); 2.76 (75 mg, 0.210 mmol), DIPEA (the minimal volume to reach 3 (t, 2H, H-1); 4.01 (s, 4H, H-4); 7.57 (dd, 2H, Ar); 7.69 (dd, 4H, pH>8) and dry DMF (6.0 mL). The reaction mixture was stirred Ar); 7.76 (d, 2H, Ar); 8.12 (dd, 4H, Ar); 13C-NMR (300 MHz, during 22 h, under nitrogen atmosphere, at room CDCl ): δ = 29.89 (C-2); 40.07 (C-1); 52.25 (C-3); 61.38 (C-4); temperature. 3 121.07 (CH-Arom); 126.31 (CH-Arom); 127.36 (Cq-Arom); After solvent evaporation, the remaining oil was purified by 127.59 (CH-Arom); 128.98 (CH-Arom); 129.53 (CH-Arom); semi-preparative HPLC (Method 3); the collected fraction was This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 9 Please do not adjust margins .65:51:71 7102/10/52 no ogeiD naS - ainrofilaC fo ytisrevinU yb dedaolnwoD .7102 yraunaJ 52 no dehsilbuP View Article Online DOI: 10.1039/C7DT00043J Please do not adjust margins Dalton Transactions Page 10 of 15 ARTICLE Journal Name dried under vacuum to give Pt-LQ as an orange oil (49 mg, MHz, MeOD): δ = -2098.20; ESI-MS (+) C H Cl N O PtRe 35 36 2 6 4 0.062 mmol, η=58 %). (1056.89) (m/z) ( %): 1057.1 (100), 1056.3 (73), 1058.1 (62), R-HPLC (Method 1)= 21.3 min; 1H-NMR (300 MHz, MeOD): δ = 1059.1 (57), 1055.4 (55), 1060.1 (21), 1053.7 (16); IR ν (KBr)/ t max 1.39 (d, 6H, H-6); 1.93 (m, 2H, H-2); 2.73 (t, 2H, H-3); 3.39 (m, cm-1 (C=O) 1675; (C≡O) 1975, 2025; (NH) 3450. 1H, H-5); 3.46 (t, 2H, H-1); 4.04 (s, 4H, H-4); 7.37 (d, 2H, H-8); 7.60 (dd, 2H, Ar); 7.74 (dd, 4H, Ar); 7.85 (d, 2H, Ar); 7.95 (d, 2H, Synthesis and in vitro evaluation of the 99mTc complexes: LQ-Tc Ar); 8.22 (d, 2H, Ar); 8.75 (d, 2H, H-7); 13C-NMR (300 MHz, and Pt-LQ-Tc MeOD): δ = 23.78 (C-6); 26.57 (C-2); 38.40 (C-1); 49.42 (C-5); Synthesis 52.48 (C-3); 61.75 (C-4); 122.31 (CH-Arom); 123.33 (C-8); 127.90 (CH-Arom); 128.56 (CH-Arom); 128.79 (Cq); 129.00 (CH- The radioactive precursor fac-[99mTc(CO) (H O) ]+ was t 3 2 3 Arom); 131.00 (Cq); 131.14 (CH-Arom); 138.69 (CH-Arom); prepared by addition of 3 mL of Na[99mTcO ] to a mixture of p 4 148.04 (Cq); 149.18 (C=O); 153.90 (C-7); 161.60 (Cq); 195Pt- potassium boranocarbonate (5 mg), sodium tartrate (7 mg) i r NMR (400 MHz, MeOD): δ = -2098.39; ESI-MS (+) and sodium tetraborate (7 mg). After heating at 100 ºC for 30 c C H Cl N OPt (786.20) (m/z) (%): 787.3 (100), 786.5 (77), min, the pH was adjusted to 5. Then, in a nitrogen-purged glass 32 36 2 6 s 788.2 (70), 789.2 (63), 785.6 (57), 790.2 (21), 791.1 (15), 792.1 vial, 0.8 mL of fac-[99mTc(CO) (H O) ]+ was added to 0.8 mL of a 3 2 3 u (4); IR ν (KBr)/cm-1 (CO) 1643; (NH) 3429. ethanolic solution of the compounds Pt-LQ (10-3M) or Pt-LQ max (10-2M). The reaction mixtures were then heated for 60 min at n Synthesis of the Re complex of mono ((4-(3-(bis (quinolin-2- 50°C. After cooling to room temperature, a ylmethyl) amino) propylcarbamoyl) pyridinium-1-yl) Prior to their in vitro evaluation, the 99mTc complexes were M (isopropylammonio) platinum(II)) dichloride (Pt-LQ-Re) submitted to HPLC purification using a Supelco Discovery RP- Pt-LQ-Re was synthesized by two different approaches column, using a gradient elution (Method 4) with a flow rate of d (method A and method B), as described below. 1 mL.min-1. The solvent from the collected fractions was e Method A evaporated under a stream of nitrogen and the residue N-hydroxysuccinimide (13 mg, 0.090 mmol) was added to a redissolved in NaCl 0.9 % to obtain the desired radioactive t p solution of t-[PtCl (4-picolinic acid)(isopropylamine)] (35 mg, concentration. The purified complexes were analyzed by RP- 2 e 0.080 mmol) in DCM (10.0 mL). The solution was stirred for 15 HPLC and their chemical identity ascertained by HPLC co- minutes, and then 1-ethyl-3-(3-dimethylaminopropyl) injection with the Re counterparts. c carbodiimide (19 mg, 0.090 mmol) was added. After stirring for R-HPLC (Method 4): LQ-Tc, 19.3 min; Pt-LQ-Tc, 26.4 min. c t 16 h at room temperature, the solution was evaporated to A Partition coefficient measurements dryness and 5 mL of water were added. The resulting mixture was filtrated to separate the urea by-product and the resulting The log P o/w values of the 99mTc complexes were determined by s mixture was evaporated to dryness. The activated ester was the “shake flask” method.48 A mixture of octanol (1.0 mL) and n redissolved in dry DMF (3.0 mL), and added to a solution of 0.1 M PBS pH = 7.4 (1.0 mL) was stirred vigorously, followed by o [LQ-Re]Cl (53 mg, 0.080 mmol), DIPEA (the minimal volume to the addition of 25 µL of the aqueous solutions of each reach pH>8) in dry DMF (5.0 mL). The reaction mixture was complex. The mixtures were vortexed and centrifuged (3000 i t stirred overnight at room temperature. rpm, 10 min, RT) to allow phase separation. Aliquots of 25 µL c After solvent evaporation, the remaining oil was purified by of the octanol and PBS phases were counted in a gamma a semi-preparative HPLC (Method 3), and the collected fraction counter. The partition coefficient (P ) was calculated by o/w s was dried under vacuum to give Pt-LQ-Re as a light orange oil dividing the number of counts of the octanol phase by those n (16 mg, 0.015 mmol, η=19 %). from the PBS phase, and the results expressed as log D . o/w a Method B To a solution of Pt-LQ (30 mg, 0.038 mmol) in THF (6.0 mL) was In vitro stability assays r T added [Re(CO) Cl] (14 mg, 0.038 mmol) and the resulting Aliquots (0.1 mL) of Pt-LQ-Tc solutions were added to 1 mL of 5 solution was refluxed overnight. After this time, the solvent the different challenging media (0.1 M PBS, pH 7.4; human n was removed under reduced pressure and the desired product serum; cell culture medium) and incubated at 37 ºC for 2, 4, 6 o was purified by HPLC (Method 3) to give a light orange oil (11 and 24 h, except in the case of cell culture medium where the mg, 0.011 mmol, η=29 %). incubation was performed only during 4 h. After incubation, t R t -HPLC (Method 2)= 25.3 min; 1H-NMR (300 MHz, MeOD): δ = 0.4 mL aliquots of the human serum and cell culture medium a l 1.43 (d, 6H, H-6); 2.44 (m, 2H, H-2); 3.57 (m, 1H, H-5); 3.69 (t, mixtures were treated with 0.8 mL of EtOH to precipitate the D 2H, H-1); 4.06 (dd, 2H, H-3); 5.24 (dd, 4H, H-4); 7.65 (d, 2H, Ar); proteins. Samples were centrifuged at 3000 rpm for 5 min, and 7.76 (m, 4H, Ar + H-8); 7.92 (dd, 2H, Ar); 8.07 (d, 2H, Ar); 8.57 aliquots of the supernatant were analyzed by RP-HPLC. For the (dd, 4H, Ar); 8.98 (d, 2H, H-7); 13C-NMR (300 MHz, MeOD): δ = challenging experiment in PBS, aliquots of the reaction mixture 23.90 (C-6); 27.17 (C-2); 38.60 (C-1); 47.83 (C-5); 67.03 (C-3); were directly injected in the HPLC without any treatment. 69,85 (C-4); 120.81 (CH-Arom); 124.04 (C-8); 129.51 (2xCH- Arom); 129.87 (CH-Arom); 131.04 (2xCq); 134.14 (CH-Arom); Photophysical measurements 143.01 (CH-Arom); 144.24 (Cq); 148.25 (C=O pic); 154.97 (C-7); 166.29 (Cq); 193.36 (C=O); 195.01 (2xC=O); 195Pt-NMR (400 10 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .65:51:71 7102/10/52 no ogeiD naS - ainrofilaC fo ytisrevinU yb dedaolnwoD .7102 yraunaJ 52 no dehsilbuP View Article Online DOI: 10.1039/C7DT00043J Please do not adjust margins Page 11 of 15 Dalton Transactions Journal Name ARTICLE UV/Vis absorption spectra Φ = Φ *S /S *I /I (1) sample ref sample ref ref sample I=I *(1 - 10-A ) (2) UV/Vis absorption spectra and extinction coefficients were 0 λ I (absorbance correction factor) was obtained with Equation obtained on a Varian Cary 50 Scan UV/vis spectrophotometer (2), where I is the light intensity of the irradiation source in using standard quartz cells with 1 cm path length. 0 the irradiation interval and A is the absorbance of the sample λ Emission spectra at wavelength λ. Emission spectra were recorded on an Edinburgh Instrument FLSP920 spectrometer equipped with a 450 W Xenon lamp, Cellular Studies double monochromators for the excitation and emission t pathways, and a red-sensitive photomultiplier (PMT-R928) as Cell Culture p detector. The emission spectra were fully corrected by using Human ovarian epithelial cancer A2780 (cisplatin sensitive) i the standard corrections supplied by the manufacturer for the and A2780R(acquired cisplatin resistance) cell lines were r spectral power of the excitation source and the sensitivity of c maintained in RPMI1640 Medium. Human cervical carcinoma the detector. s cells (HeLa) were grown in DMEM containing GlutaMax Iand normal lung fibroblast cell line (MRC-5) was cultured in F-10 u Quantum yields medium. All culture media were supplemented with 10 % n The quantum yields were measured by use of an integrating heat-inactivated foetal bovine serum (FBS) and 1 % a sphere with an Edinburgh Instrument FLSP920 spectrometer. penicillin/streptomycin antibiotic solution. All culture media The absorbance of the samples was kept below 0.1 to avoid M and supplements were from Gibco. inner filter effects, except for the concentration-dependent measurements, and all measurements were carried out at 293 Dark- and photo-toxicity d K. A fluorometric cell viability assay using resazurin (Promocell e Luminescence lifetime GmbH) was used to compare the cytotoxicity of the complexes t in the dark and upon UV irradiation. A2780 and A2780R cell p The luminescence lifetimes were measured by using µF900 lines were plated in triplicates in 96-well plates at a density of e pulsed 60W xenon microsecond flash amp with a repetition 4000 cells per well in 100 µL, which MRC-5 cell lines was plated c rate of 100 Hz and a multichannel scaling module. The in triplicates in 96-well plates at a density of 7500 cells per well c emission was collected at right angles to the excitation source in 100 µL, 24 h prior to treatment. Cells were then treated with with the emission wavelength selected by using a double A increasing concentrations of compounds for 48 h. For grated monochromator and detected by a R928-P PMT. The phototoxicity studies, cells were treated for 4 h with increasing instrument response function (IRF) was measured by using the s concentrations of the compounds in the dark. After that, the blank solvent as scattering sample and setting the n medium was removed and replaced by fresh culture medium monochromator at the emission wavelength of the excitation prior to 10 min irradiation at 350 nm (2.58 J cm-2). After 44 h in o beam. The resulting intensity decay is a convolution of the the incubator, the medium was replaced by 100mL complete i luminescence decay with the IRF and iterative convolution of medium containing resazurin (final concentration 0.2 mg mL-1). t c the IRF with a decay function and non-linear least-squares After 4 h incubation at 37 °C, fluorescence of the highly red analysis was used to analyze the convoluted data. a fluorescent resorufin product was quantified at 590 nm s Indirect evaluation of singlet oxygen 49 emission with 540 nm excitation wavelength in a SpectraMax M5 microplate reader. Light doses were evaluated with a n An air-saturated acetonitrile solution, containing the complex Gigahertz Optic X1-1 optometer. a (OD=0.1 at irradiation wavelength), p-nitrosodimethyl aniline r (RNO, 24 µM), imidazole (12 mM) or an air-saturated PBS Cellular uptake and distribution by ICP-MS T buffer solution, containing the complex (OD = 0.1 at irradiation To assess the uptake of complexes and their intracellular wavelength), RNO (20 µM), histidine (10 mM) were irradiated distribution, A2780 and A2780cisR cells were exposed to the n in a luminescence quartz cuvette at 350 or 420 nm in a RPR100 complexes and the Pt and Re content in the nucleus and o Rayonet chamber reactor (Southern New England Ultraviolet cytoplasm analyzed by ICP-MS. t Company) complete with six lamps, at different time intervals. Cells were seeded into 24-well tissue culture plates at a l The absorbance of the solution was then evaluated. Plots of a density of 200000 and 400000 cells for A2780 and A2780cisR, variations in absorbance at 440 nm in PBS or at 420 nm in D respectively, and allowed to attach overnight at 37 ºC. acetonitrile (A –A, where A is the absorbance before 0 0 Adherent cells were incubated with each complex (5 µM) for irradiation) versus the irradiation times for each sample were 24 h at 37 ºC. Then, cells were washed with PBS and detached prepared and the slope of the linear regression was calculated from plates with trypsin. In suspension, cells were counted, (S ). As a reference compound, phenalenone ( Φ (1O ) = sample ref 2 twice washed with cold PBS, and centrifuged (800 rpm, 2 min, 95 %) was used in both methods, to obtain S Equation (1) ref 4 ºC) to obtain a pellet. In order to disrupt the cellular was applied to calculate the singlet oxygen quantum yields membrane, the pellet was ressuspended in lysis buffer (10 mM (Φ ) for every sample: sample Tris, 1.5 mM MgCL , 140 mM NaCl, pH 8.0-8.3) with Nonidet 2 This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 11 Please do not adjust margins .65:51:71 7102/10/52 no ogeiD naS - ainrofilaC fo ytisrevinU yb dedaolnwoD .7102 yraunaJ 52 no dehsilbuP View Article Online DOI: 10.1039/C7DT00043J Please do not adjust margins Dalton Transactions Page 12 of 15 ARTICLE Journal Name P40 (0,02 %). After 15 min incubation on ice, the suspension In vivo stability was centrifuged at 1300 g for 2 min at 4 ºC and nuclear The in vivo stability of Pt-LQ-Tc was evaluated in urine and fraction (pellet) separated from the cytoplasmic fraction serum by HPLC analysis, using method 4, at 1 h (p.i.). The urine (supernatant). was collected at the time of sacrifice and analyzed by HPLC. The Pt content for complexes Pt-LQ and Pt-LQ-Re and the Re Blood was collected from mice and immediately centrifuged content for complexes LQ-Re and Pt-LQ-Re, in the two for 5 min at 3000 rpm, and the serum was separated. Aliquots fractions was measured, after digestion (with ultrapure HNO 3 of 0.1 mL of serum were treated with 0.2 mL of EtOH to (65 %), H O , and HCl, evaporated, and ressuspended in 2 2 precipitate the proteins. Samples were centrifuged at 3000 ultrapure water to obtain a 2.0 % (v/v) nitric acid solution, by rpm, for 10 min, and the supernatant was collected and ICP-MS on a ICP-MS NexION 300xx PerkinElmer instrument, t analyzed by RP-HPLC. with 187Rhenium used as internal standard. p i Confocal fluorescence microscopy studies r DNA interaction studies c Cellular localization of the compounds was also assessed by s Plasmid DNA interaction studies fluorescence microscopy using HeLa cells. Cells were grown on u 35 mm Cellview glass bottom dishes (Greiner) in 3 mL The DNA interaction studies were performed in a total volume complete medium at a density of 1 × 105 cells per mL and of 20 µL. The DNA stock was purchased to Gencust at a n incubated for 2 h with the complexes at a concentration of 20 concentration of 0.5 µg/µL in phosphate buffer 50 mM (pH = a μM. Cells were washed with 1× PBS prior to imaging by 7.4). Stock solutions of the complexes were prepared in DMSO M confocal microscopy using a CLSM Leica SP5 Mid UV-VIS Leica at 5 mM. The 20 µL containing 0.125 µg/µL of DNA-pBR322 in microscope. All the complexes were excited at 355 nm and the 10 mM Tris-HCl (pH 7.6) and 1mM EDTA, were incubated with d emission above 420 nm was recorded. the platinum compounds at ri values ranging from 0.05 to 0.2 e (defined as the molar ratio Pt/nucleotide). The samples were incubated simultaneously at dark and with light at 350 nm t Animal Studies during 4 h (32.4 J.cm-2) and afterwards all together at dark and p e 37 °C in a thermoshaker for 24 h. After the incubation 2 µL of a Biodistribution and imaging studies loading dye containing 50% glycerol, 0.25% bromophenol blue c All animal experiments were performed in compliance with and 0.25% Xylene cyanol was added. The total of the sample c Portuguese regulations for animal treatment. The animals was loaded in the agarose gel (1,2 % p/v) and the A were housed in a temperature- and humidity-controlled room electrophoresis was carried out for a period of 150 min. with a 12 h light/12 h dark schedule. Biodistribution of the approximately at 70V in a TAE 1x (Tris-acetate/EDTA) buffer. s radiocomplex was estimated in healthy Balb/C mice (8-10 After electrophoresis, the gel was immersed in 200 mL of n weeks old). Animals were intravenously injected into the retro- Millipore water containing 10 mL from a 10 mg/mL stock orbital sinus with the radiolabelled complex (3-10 MBq) o solution of ethidium bromide for 30 min to stain the DNA. diluted in 100 µL of PBS pH 7.2. Mice were sacrificed by Finally, the stained gel was analysed with a UVITEC Cambridge i t cervical dislocation at 1h and 4 h postinjection (p.i.). The with a UVIDOC HD2. c administered dose and the radioactivity in the sacrificed The irradiation of the samples was performed in carrousel a animals were measured in a dose calibrator (Aloka, Curiemeter device inside a photo-activator (Luzchem Research, Inc. Option s IGC-3, Tokyo, Japan or Capintec CRC-15W, Ramsey, USA). The 1) at 350 nm. In addition, the experiment was repeated twice. difference between the radioactivity in the injected and n sacrificed animals was assumed to be due to excretion. Tissues DNA Platination Studies a of interest were dissected, rinsed to remove excess blood, A2780 ovarian cancer cells were cultured to 70% confluency, r weighed, and their radioactivity was measured using a gamma T the medium was replaced with medium containing 20 µM of counter (Berthold, LB2111, Germany). The uptake in the the target metal complex and the cells further incubated for 4 tissues of interest was calculated and expressed as a n h. After the treatment the cells were washed with PBS and percentage of the injected radioactivity dose per gram of o new fresh complex-free medium was added. Then, one batch tissue (% ID/g). For blood, bone, muscle and skin, total activity of cells was further processed while a second batch was t was estimated assuming that these organs constitute 6 %, 10 l irradiated for 10 min at 350 nm in a Rayonet Irradiation a %, 40 % and 15 % of the total body weight, respectively. Urine Chamber (2.58 J/cm2) and then further processed. The cell D was also collected and pooled together at the time of sacrifice. pellets were collected per trypsinization and further For imaging, the animals were injected into the tail vein with centrifugation at 650 g, 4 °C for 5 minutes (5910R, Eppendorf) Pt-LQ-Tc (5 MBq) and sacrificed at 1 h p.i.. A set of static and washed two times with ice-cold PBS. DNA was extracted images (256 × 256 matrix, Zoom 2, 2 min) was acquired, by using a DNA Extraction kit (illustra BACC1, GE Healthcare) placing the animals over a γ-camera (GE 400AC; Maxicamera, following the manufacturer procedure. The content of nucleic Milwaukie, USA) coupled with a high-resolution parallel acid was quantify via 260/280 nm absorbance ratio in a Cary60 collimator and controlled with GENIE Acquisition computer. UV-Vis spectrophotometer (Agilent Technologies), equipped with a ND-1000 nanodrop system (Witec AG). The DNA 12 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .65:51:71 7102/10/52 no ogeiD naS - ainrofilaC fo ytisrevinU yb dedaolnwoD .7102 yraunaJ 52 no dehsilbuP View Article Online DOI: 10.1039/C7DT00043J Please do not adjust margins Page 13 of 15 Dalton Transactions Journal Name ARTICLE aliquots were then lyophilized in an Alpha 2-4 LD plus MED/0233/2012 and FCT Investigator grant to FMendes), the (CHRIST). The obtained samples were chemically digested for Swiss National Science Foundation (Professorships N° 24 h in a 10% aqua regia solution and further diluted to PP00P2_133568 and PP00P2_157545 to G.G), the University of achieve a 2% solution that was injected in ICP-MS. Zurich (G.G), the Stiftung für wissenschaftliche Forschung of ICP-MS measurements were performed on an Agilent QQQ the University of Zurich (G.G.), the COST Action CM1105 (G.G, 8800 Triple quad ICP-MS spectrometer (Agilent Technologies) A.Q. and A.P.), the UBS Promedica Stiftung (R.R., G.G.), the with a ASX200 autosampler (Agilent Technologies), equipped Forschungskredit of the University of Zurich (R.R.), the Novartis with standard nickel cones and a “micro-mist” quartz nebulizer Jubilee Foundation (R.R. G.G.), the PSL Excellence Chair fed with 0.3 ml/min analytic flow (as a 2% HNO aqueous Program Grant (ANR-10-IDEX-0001-02 PSL to G.G.), the 973 3 solution). Platinum was measured against a Pt single element Program (No. 2015CB856301), the National Science t p standard (Merck 1703410100) and verified by a control Foundation of China (No. 21471164), the China Scholarships (Agilent5188-6524 PA Tuning 2). Platinum content of the Council (Grant No. 201506380026 to H.H.), and the MINECO i r samples was determined by means of an 8-step serial dilution (Spain Governmental Agency) GRANT SAF-2012-34424 and c in the range between 0 and 200 ppb in Pt (R>0.99) with a CTQ-2015-68779-R. The authors would also like to thank Célia s background equivalent concentration of BEC: 11.8 ppt and a Fernandes for the Mass Spectrometry analyses, which was u detection limit of DL: 5.4 ppt. The isotope Pt194 (32.97% carried out on a QITMS instrument, acquired with the support abundance) Pt195 (33.83% abundance) was evaluated in “no- of the Programa Nacional de Reequipamento Científico n gas” and “He-gas” mode. Spiking the samples with 1% (Contract REDE/1503/REM/2005- ITN) of FCT and is part of a methanol (to account for eventual carbon content from the RNEM-Rede Nacional de Espectrometria de Massa. Ana Graça M biological samples) resulted in equivalent values within error is acknowledged for the gamma camera imaging studies with ranges. A solution of Indium (500 ppb) and Tungsten (500 ppb) Pt-LQ-Tc injected BALB/C female mice d was used as internal standard. The results are expressed as ppb Pt / sample50, 51. e Notes and references t Pulse Field Gel Electrophoresis Studies p e A2780 ovarian cancer cells were cultured to sub-confluency, 1. T. Gianferrara, C. Spagnul, R. Alberto, G. Gasser, S. Ferrari, V. the medium was replaced with medium containing with Pierroz, A. Bergamo and E. Alessio, ChemMedChem, 2014, 9, c vehicle alone (DMF), cisplatin (cPt, 10 µM), camptothecin 1231-1237. c (CMPT 2.5 µM) or with the target metal complex (20 µM) and 2. A. Naik, R. Rubbiani, G. Gasser and B. Spingler, Angew Chem A the cells further incubated for 4 h. After the treatment the Int Ed Engl, 2014, 53, 6938-6941. cells were washed with PBS and new fresh complex-free 3. A. Leonidova, V. Pierroz, R. Rubbiani, J. Heier, S. Ferrari and s medium was added. Then, one batch of cells was further G. Gasser, Dalton Trans, 2014, 43, 4287-4294. n processed while a second batch was irradiated for 10 min at 4. T. Esteves, F. Marques, A. Paulo, J. Rino, P. Nanda, C. J. Smith o 350 nm in a Rayonet Irradiation Chamber (2.58 J/cm2) and and I. Santos, J Biol Inorg Chem, 2011, 16, 1141-1153. then further processed. The cell pellets were collected per 5. G. Mion, T. Gianferrara, A. Bergamo, G. Gasser, V. Pierroz, R. i t trypsinization and further centrifugation at 650 g, 4°C for 5 Rubbiani, R. Vilar, A. Leczkowska and E. Alessio, ChemMedChem, c minutes (5910R, Eppendorf) and washed two times with ice- 2015, 10, 1901-1914. a cold PBS. Agarose plugs of 106 cells in low melting point 6. A. Leonidova and G. Gasser, ACS Chem Biol, 2014, 9, 2180- s agarose (LMP) were prepared in a disposable plug mold (Bio- 2193. n Rad). Plugs were incubated in lysis buffer (100mM EDTA, 1% 7. T. C. Johnstone, K. Suntharalingam and S. J. Lippard, Chem. a (w/v) sodium lauryl sarcosyl, 0.2% (w/v) sodium deoxycholate, Rev., 2016. 1 mg/ml1 proteinase K) at 37 °C for 72 h, and washed three 8. I. Kitanovic, S. Can, H. Alborzinia, A. Kitanovic, V. Pierroz, A. r T times in 20 mM Tris–HCl pH 8.0, 50 mM EDTA before loading Leonidova, A. Pinto, B. Spingler, S. Ferrari, R. Molteni, A. Steffen, onto an agarose gel. Electrophoresis was performed for 23 h at N. Metzler-Nolte, S. Wolfl and G. Gasser, Chemistry, 2014, 20, n 14 °C in 0.9% (w/v) Pulse Field Certified Agarose (Bio-Rad CHEF 2496-2507. o DR III apparatus) containing 0.5% Tris-borate/EDTA buffer. The 9. A. Kastl, S. Dieckmann, K. Wahler, T. Volker, L. Kastl, A. L. gel was post-stained with ethidium bromide (EtBr) for 30 min Merkel, A. Vultur, B. Shannan, K. Harms, M. Ocker, W. J. Parak, t l and analyzed using an Alpha Innotech Imaging system and the M. Herlyn and E. Meggers, ChemMedChem, 2013, 8, 924-927. a band intensity calculated in multiple band mode. The 10. S. James, K. P. Maresca, J. W. Babich, J. F. Valliant, L. Doering D experiments were repeated and one of the two sets was and J. Zubieta, Bioconjug Chem, 2006, 17, 590-596. depicted52, 53. 11. K. A. Stephenson, S. R. Banerjee, T. Besanger, O. O. Sogbein, M. K. Levadala, N. McFarlane, J. A. Lemon, D. R. Boreham, K. P. Maresca, J. D. Brennan, J. W. Babich, J. Zubieta and J. F. Valliant, Acknowledgements J. Am. Chem. Soc., 2004, 126, 8598-8599. This work was financially supported by the Portuguese 12. L. Wei, J. W. Babich, W. Ouellette and J. Zubieta, Inorg Chem, Fundação para a Ciência e Tecnologia (projects PTDC/QUI- 2006, 45, 3057-3066. QUI/114139/2009, UID/Multi/04349/2013 and EXCL/QEQ- This journal is © The Royal Society of Chemistry 20xx J. 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