Robin Lescure

Malorie Privat

Jacques Pliquett

Aurélie Massot

Océane Baffroy

Benoit Busser

Pierre-Simon Bellaye

Bertrand Collin

Franck Denat

Ali Bettaïeb

Jacques Plique

Lucie Sancey

Catherine Paul
email: catherine.paul@u-bourgogne.fr

Christine Goze
email: christine.goze@u-bourgogne.fr

Ewen Bodio
email: ewen.bodio@u-bourgogne.fr

Near-infrared emitting fluorescent homobimetallic gold(I) complexes displaying promising in vitro and in vivo therapeutic properties

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Developing new therapeutic agents is a complex task. For a long time, many researchers have focused their research on the development of therapeutic agents with the highest cytotoxicity in vitro without worrying about pharmacokinetics or pharmacodynamics, which has often led to failurse, when the compounds were tested in vivo. Indeed, the ideal compound must not only be effective when it is in contact with its target, it must also be able to reach it without being degraded, accumulate at the right place in sufficient quantity, be safe for healthy tissues... The problem is that the different criteria to be improved to obtain an ideal compound are related to each other and fine tuning one by modifying the molecule can be detrimental to another one (e.g. increasing the stability of a product can decrease its therapeutic effect). It is therefore preferable to control as many parameters as possible to maximize the success of optimization of the therapeutic agent. In particular, knowing where the compound accumulates, in what quantity, by which routes it is eliminated, is crucial to rationalize the optimization. There are many techniques that can be used to do this follow-up, however, most of them are invasive and require numerous samples or animals. One more and more popular solution is the use of molecular imaging. Indeed, by grafting an imaging probe onto a therapeutic agent, it can be followed in real time and non-invasively and, thus, it is possible to collect numerous valuable information on the resulting trackable therapeutic agent thus formed (often also called theranostics). [START_REF] Yan | Recent progress on molecularly near-infrared fluorescent probes for chemotherapy and phototherapy[END_REF][START_REF] Velikyan | Radio)Theranostic Patient Management in Oncology Exemplified by Neuroendocrine Neoplasms[END_REF][START_REF] Pruis | The Added Value of Diagnostic and Theranostic PET Imaging for the Treatment of CNS Tumors[END_REF][START_REF] Pliquett | Design of a multifunctionalizable BODIPY platform for the facile elaboration of a large series of gold(I)-based optical theranostics[END_REF][START_REF] Yuan | Targeted Theranostic Platinum(IV) Prodrug with a Built-In Aggregation-Induced Emission Light-Up Apoptosis Sensor for Noninvasive Early Evaluation of Its Therapeutic Responses in Situ[END_REF][START_REF] Redrado | Theranostics Through the Synergistic Cooperation of Heterometallic Complexes[END_REF] Like us, different research groups have opted for optical fluorescence imaging. [START_REF] Yan | Recent progress on molecularly near-infrared fluorescent probes for chemotherapy and phototherapy[END_REF][START_REF] Pliquett | Design of a multifunctionalizable BODIPY platform for the facile elaboration of a large series of gold(I)-based optical theranostics[END_REF][START_REF] Bertrand | Metal-based BODIPY derivatives as multimodal tools for life sciences[END_REF][START_REF] Luengo | Bioactive Heterobimetallic Re(I)/Au(I) Complexes Containing Bidentate N-Heterocyclic Carbenes[END_REF][START_REF] Fernández-Moreira | Heterobimetallic Complexes for Theranostic Applications[END_REF][START_REF] Trommenschlager | Gold(I)-Coumarin-Caffeine-Based Complexes as New Potential Anti-Inflammatory and Anticancer Trackable Agents[END_REF][START_REF] Luengo | Trackable Metallodrugs Combining Luminescent Re(I) and Bioactive Au(I) Fragments[END_REF][START_REF] Wenzel | New Luminescent Polynuclear Metal Complexes with Anticancer Properties: Toward Structure-Activity Relationships[END_REF][START_REF] Dondaine | Coumarin-Phosphine-Based Smart Probes for Tracking Biologically Relevant Metal Complexes: From Theoretical to Biological Investigations[END_REF][START_REF] Bertrand | Development of trackable metal-based drugs: new generation of therapeutic agents[END_REF][START_REF] Visbal | Cytotoxicity and biodistribution studies of luminescent Au(I) and Ag(I) N-heterocyclic carbenes. Searching for new biological targets[END_REF][START_REF] Doulain | Towards the elaboration of new gold-based optical theranostics[END_REF][START_REF] Tasan | BODIPY-phosphane as a versatile tool for easy access to new metalbased theranostics[END_REF][START_REF] Citta | Fluorescent silver(i) and gold(i)-N-heterocyclic carbene complexes with cytotoxic properties: mechanistic insights[END_REF] For example, BODIPY fluorophores were used many times in vitro to track therapeutically relevant metals 2 such as Au, Cu, Ir, Os, Pt, Rh, Ru, Ti… Some examples are given on Figure 1 (the subject was reviewed recently [START_REF] Bertrand | Metal-based BODIPY derivatives as multimodal tools for life sciences[END_REF]). [START_REF] Pliquett | Design of a multifunctionalizable BODIPY platform for the facile elaboration of a large series of gold(I)-based optical theranostics[END_REF][START_REF] Redrado | Theranostics Through the Synergistic Cooperation of Heterometallic Complexes[END_REF][START_REF] Bertrand | Metal-based BODIPY derivatives as multimodal tools for life sciences[END_REF][START_REF] Gupta | Mitochondrial Localization of Highly Fluorescent and Photostable BODIPY-Based Ruthenium(II), Rhodium(III), and Iridium(III) Metal Complexes[END_REF] The choice of optical imaging for such a tracking could be explained by the fact that it is a versatile, sensitive, non-invasive, and non-ionizing technique, involving inexpensive equipment compared to most other imaging modalities and limited constraints. Its major drawback is its low penetrability of biological tissues, which means that today the monitoring of theranostic agents by optical imaging is mainly limited to studies on cells. The objective of the present study is to demonstrate that the use of recent and innovative fluorophores -such as aza-BODIPYs -allows such monitoring in vivo by optical imaging, while maintaining good anti-tumor activity of the therapeutic part. Our choice fell on a gold complex for the therapeutic part because unlike most of other metal ions, gold(I) does not quench fluorescence, and because of its therapeutic properties. [4,10,13,17,20] Indeed, gold complexes exhibit many biological properties: they can act as antibacterial, antiviral, antiparasitic agents, and they increasingly appear as a promising alternative to platinum complexes to fight cancer. [START_REF] Ceramella | From coins to cancer therapy: Gold, silver and copper complexes targeting human topoisomerases[END_REF][START_REF] Yeo | Gold-Based Medicine: A Paradigm Shift in Anti-Cancer Therapy?[END_REF][START_REF]Metallo-Drugs: Development and Action of Anticancer Agents[END_REF][START_REF] Bertrand | Gold(III) Complexes for Antitumor Applications: An Overview[END_REF][START_REF] Nardon | Gold Complexes for Therapeutic Purposes: an Updated Patent Review (2010-2015[END_REF][START_REF] Zou | Chemical biology of anticancer gold(III) and gold(I) complexes[END_REF][START_REF] Glišić | Gold complexes as antimicrobial agents: an overview of different biological activities in relation to the oxidation state of the gold ion and the ligand structure[END_REF] In particular, auranofin -clinically used for rheumatoid arthritis -has been repurposed recently as an anticancer drug. [START_REF] Zhang | Repurposing of auranofin: Thioredoxin reductase remains a primary target of the drug[END_REF][START_REF] Capparelli | Phase I Clinical Trial Results of Auranofin, a Novel Antiparasitic Agent[END_REF][START_REF] James | An investigation into the interactions of gold nanoparticles and anti-arthritic drugs with macrophages, and their reactivity towards thioredoxin reductase[END_REF][START_REF] Onodera | Potential Anticancer Activity of Auranofin[END_REF][START_REF] Simon | Screening trial with the coordinated gold compound auranofin using mouse lymphocyte leukemia P388[END_REF] Several clinical trials are testing its efficacy against recurrent epithelial ovarian, primary peritoneal, or fallopian tube cancer [NCT01747798, NCT03456700], lung cancer [NCT01737502], but also leukemia [NCT01419691]). [START_REF] Onodera | Potential Anticancer Activity of Auranofin[END_REF] Numerous targets have been reported for explaining the mechanism of action of gold complexes. [START_REF] Zou | Chemical biology of anticancer gold(III) and gold(I) complexes[END_REF] Concerning gold(I) complexes except topoisomerase, [START_REF] Ceramella | From coins to cancer therapy: Gold, silver and copper complexes targeting human topoisomerases[END_REF] most of the suggested targets of gold(I) complexes are enzymes, which are not localized in cell nuclei. This point is key because we are working on BODIPY and aza-BODIPY-based fluorescent platforms, which accumulate in the cell cytoplasm. [START_REF] Florès | In vitro and in vivo trackable titanocene-based complexes using optical imaging or SPECT[END_REF][34][START_REF] Pliquett | A Promising Family of Fluorescent Water-Soluble aza-BODIPY Dyes for in Vivo Molecular Imaging[END_REF] Among these enzymes, we can cite glutathione reductase, cysteine protease, and the most commonly studied are thioredoxin reductases (TrxRs). [START_REF] Trommenschlager | Gold(I)-BODIPY-imidazole bimetallic complexes as new potential anti-inflammatory and anticancer trackable agents[END_REF][START_REF] Bertrand | A golden future in medicinal inorganic chemistry: the promise of anticancer gold organometallic compounds[END_REF][START_REF] Ortego | Strong inhibition of thioredoxin reductase by highly cytotoxic gold(I) complexes. DNA binding studies[END_REF] All there enzymes contain thiols and/or selenol. The fact that gold complexes would target these enzymes is attributed to the strong binding affinity of gold ions for thiols. For this study, we decided to focus on aza-BODIPY platforms because we wanted to track gold complexes both in vitro and in vivo and aza-BODIPY dyes emit in the near infrared (NIR, 700 -900 nm for NIR I), an area of the electromagnetic spectrum where biological tissues are more transparent, scattering phenomenon is reduced, and the light toxicity is limited. Aza-BODIPY fluorophores are chemically and photochemically stable, exhibit good quantum yield and, unlike many fluorophores emitting in NIR, are easy to functionalize. Their main drawback, like most organic fluorophores, is their low solubility in water. However, we recently showed that the substitution of the fluorine atoms present on the boron atom of the aza-BODIPY core by ammonium arms made it possible to significantly improve the water-solubility of this type of fluorophores and to limit aggregation phenomena. [START_REF] Pliquett | A Promising Family of Fluorescent Water-Soluble aza-BODIPY Dyes for in Vivo Molecular Imaging[END_REF][START_REF] Godard | Water-Soluble Aza-BODIPYs: Biocompatible Organic Dyes for High Contrast In Vivo NIR-II Imaging[END_REF][START_REF] Kalot | Aza-BODIPY: A New Vector for Enhanced Theranostic Boron Neutron Capture Therapy Applications[END_REF] In addition, it is possible to use advantageously these arms to introduce different groups of interest, [START_REF] Pliquett | A Promising Family of Fluorescent Water-Soluble aza-BODIPY Dyes for in Vivo Molecular Imaging[END_REF][START_REF] Kalot | Aza-BODIPY: A New Vector for Enhanced Theranostic Boron Neutron Capture Therapy Applications[END_REF][START_REF] Godard | Water-Soluble Aza-BODIPYs: Biocompatible Organic Dyes for High Contrast In Vivo NIR-II Imaging[END_REF][START_REF] Florès | Aza-BODIPY Platform: Toward an Efficient Water-Soluble Bimodal Imaging Probe for MRI and Near-Infrared Fluorescence[END_REF] such as the two phosphines-gold, we decided to introduce as therapeutic moieties (Figure 2). Aza-BODIPYs have been used as trackable therapeutic agents for photodynamic therapy (PDT) [START_REF] Shi | Bioapplications of small molecule Aza-BODIPY: from rational structural design to in vivo investigations[END_REF][START_REF] Bodio | BODIPYS and aza-BODIPY derivatives as promising fluorophores for in vivo molecular imaging and theranostic applications[END_REF] or to monitor boron clusters for boron neutron capture therapy (BNCT). [START_REF] Godard | Water-Soluble Aza-BODIPYs: Biocompatible Organic Dyes for High Contrast In Vivo NIR-II Imaging[END_REF][START_REF] Kalot | Aza-BODIPY: A New Vector for Enhanced Theranostic Boron Neutron Capture Therapy Applications[END_REF] However, the previous study reporting an assay for tracking a cytotoxic metal complex -an analogue of carboplatin -led to the loss of anti-proliferative activity. [START_REF] Kitteringham | Development of a novel carboplatin like cytoplasmic trackable near infrared fluorophore conjugate via strainpromoted azide alkyne cycloaddition[END_REF] The present study will describe not only the synthesis and characterization of three innovative aza-BODIPY-based gold(I) complexes, but also the evaluation of their biological properties as well as their efficiency for in vitro imaging. Moreover, the in vivo anti-cancer activity of the most promising complex and its ability to be tracked in vivo by optical imaging was also investigated.

RESULTS AND DISCUSSION

Synthesis of gold-based complexes and photophysical characterization

The first step is the synthesis of the bis-acid platform Wazaby7 (Scheme 1). This platform was designed for in vivo applications due to its good water solubility and its excitation and emission in NIR (the presence of the methoxy group in para position of the aromatic ring allows a bathochromic shift in comparison to phenyl). Wazaby7 was synthesized in two steps, starting from the corresponding difluoro-aza-BODIPY 1, following the method we previously reported [START_REF] Pliquett | A Promising Family of Fluorescent Water-Soluble aza-BODIPY Dyes for in Vivo Molecular Imaging[END_REF]: substitution of the fluorine atoms of the boron center using the Grignard reagent of N,N-dimethylpropargylamine, leading to compound 2, followed by the quaternization of the amino groups. It is worth noting that Wazaby7 platform can be synthesized in less than five days in gram-scale. Gold(I) complexes were then introduced via peptidic coupling reaction with more than 80% yield to give azaBODI-Au-1 (Scheme 1). It should be noted that the chloride counter-anions present in Wazaby7 (due to the treatment with HCl at the end of the reaction), are partially replaced by PF6 -anions coming from the HBTU (Hexafluorophosphate Benzotriazole Tetramethyl Uronium) coupling agent. In order to have only chloride counter-anions, we subjected azaBODI-Au-1 to an anion exchange on resin that can be checked by ionic chromatography and 31 P-NMR (Figure 3). This first trackable therapeutic agent can be easily modified by reacting azaBODI-Au-1 with a thiol in the presence of a base. The reaction can be easily monitored by 31 P-NMR (Figure 4). Indeed, the substitution of the chlorido ligand by the sulfur derivative results in a downfield of the signal in 31 P-NMR, as well as a strong widening of the latter, as usually observed for such a ligand exchange. [START_REF] Doulain | Towards the elaboration of new gold-based optical theranostics[END_REF][START_REF] Barranco | Synthesis of the Ferrocene Derivative FcCHN(CH 2 ) 2 SH [Fc = (η 5 -C 5 H 5 )Fe(η 5 -C 5 H 4 )]: Reactivity toward (Phosphine)gold(I) Cations[END_REF][START_REF] Gutiérrez | Highly Cytotoxic Bioconjugated Gold(I) Complexes with Cysteine-Containing Dipeptides[END_REF] We chose two different thiols: one bearing a dimethylamino group and another one bearing a sulfonate. Our objective was to study the impact of the overall charge of the trackable therapeutic agent (or theranostic) on its biological properties. Indeed, it has been shown that it can have an impact on the cellular uptake, [START_REF] Miccoli | Aryloxy Triester Phosphoramidates as Phosphoserine Prodrugs: A Proof of Concept Study[END_REF] the mode of transport in the cells [START_REF] Park | Impact of molecular charge on GLUT-specific cellular uptake of glucose bioprobes and in vivo application of the glucose bioprobe, GB2-Cy3[END_REF], etc. At physiological pH, azaBODI-Au-1 possesses two positive charges (two ammonium arms), azaBODI-Au-2 can be charged 4+ (two ammonium arms and protonation of the two NMe2 groups brought by the thiolato ligand), and azaBODI-Au-3 is neutral (two ammonium arms and two negative charges brought by the two sulfonates). Another interest of exchanging the chlorido ligand of gold center by a thiolate is the fact that the resulting complexes will be more thermodynamically stable. [START_REF] Sadler | The Chemistry of Gold Drugs[END_REF] It will be interesting to investigate if this higher stability will improve the activity of the complexes or if it will be detrimental. The three gold(I)-aza-BODIPY complexes underwent ion exchange on resin to obtain chlorides as counter-anions. The photophysical properties of the three novel homo-bimetallic complexes azaBODI-Au-1, azaBODI-Au-2, and azaBODI-Au-3, as well as the corresponding bis-acid aza-BODIPY -Wazaby7 -were investigated (Table 1). As expected, the different aza-BODIPY derivatives display a maximal absorption wavelength around 694 nm, corresponding to the S0-S1 transition band and with a high coefficient absorption (between 60,000 and 70,000 M -1 .cm -1 ), and the second S0-S2 band appearing at around 450 nm for the three systems. All the different complexes possess a maximal fluorescence emission wavelength around 730 nm with relatively high quantum yields (Table 1). Moreover, we confirm the strong photobleaching resistance of our aza-BODIPY (under the same irradiation conditions, the brightness of azaBODI-Au-1 decreases of around 30% vs around 80% for ICG). Interestingly, the introduction of the two gold(I)-complexes as well as the ligand exchanges do not significantly affect the photophysical properties of the aza-BODIPYs, as we could observe previously. [START_REF] Pliquett | Design of a multifunctionalizable BODIPY platform for the facile elaboration of a large series of gold(I)-based optical theranostics[END_REF][START_REF] Doulain | Toward the elaboration of new gold-based optical theranostics for in vivo imaging[END_REF] The absorption and emission spectra are almost superimposable and the brightness is similar for all the compounds (Figure 5). The excitation spectra also match the absorption spectra for all the compounds, confirming the presence of one S1-S0 single emitting state. ,000 * reference: aza-BODIPY 1 φF = 36% in chloroform, 298 K, λexc = 620 nm [START_REF] Brouwer | Standards for photoluminescence quantum yield measurements in solution[END_REF] In vitro investigations The anti-proliferative properties of the three gold(I)-based aza-BODIPY were investigated in murine and human breast (4T1 and MDA-MB-231, respectively) or colon (CT26 and SW480, respectively) cancer cell lines, and on normal cell line (HMEC, human mammary epithelial cells) (Table 2). The aza-BODIPYs complexes were then compared with auranofin as a benchmark gold-based therapeutic (Table 2). AzaBODI-Au-1, azaBODI-Au-2, and azaBODI-Au-3 exhibit strong anti-proliferative activities. Indeed, even if auranofin displays slightly lower IC50 values, values below 10 µM are rare for metal complexes tethered to a fluorophore. In our previous studies, the gold(I)-based theranostics, whether the fluorophore was a coumarin, a BODIPY or another organic fluorophore, exhibited antiproliferative activities of the order of 30 to 80 µM. One hypothesis could be that the Wazaby platform carries two gold complexes, but this has to be confirmed as some studies show that the correlation is not obvious. [START_REF] Pliquett | Design of a multifunctionalizable BODIPY platform for the facile elaboration of a large series of gold(I)-based optical theranostics[END_REF] We can also notice that AzaBODI-Au-1 and azaBODI-Au-3 gave the same results, whereas azaBODI-Au-2 showed more limited activity on the 4T1, CT26, and SW480 cells. As a control, the anti-proliferative properties of compound 1 and Wazaby7 were investigated onto the four cancer cell lines and display IC50 > 100 µM, which highlights that the toxicity does not come from azaBODIPY backbone. It is also worth noting, that the three theranostics display comparable IC50 on healthy cell line and on cancer ones (healthy cells are known to be very sensitive), whereas auranofin is so toxic on HMEC that its IC50 is lower than 0.5 µM.

To go deeper into the mechanism of action of the different compounds, we measured the quantity of gold in the cells by ICP-MS after 4 hours of incubation of 5 μM of gold complexes (Table 3). It clearly appears that the complexes with chlorido ligands -azaBODI-Au-1 and auranofin -accumulate significantly less than those with thiolato ligands. If we keep in mind that there are two gold equivalents per azaBODI-Au-1 and only one for auranofin, we can consider that they accumulate in a similar way. In contrast, azaBODI-Au-3 accumulates significantly more than azaBODI-Au-2. It may be an impact of the global charge of the molecule, because, as indicated before and without considering the counter ions, azaBODI-Au-3 is globally neutral while azaBODI-Au-2 is charged 4+. Based on these results, it appears that the anti-proliferative properties of the compounds cannot be directly related to their ability to accumulate in cells. On the other hand, it seems that the "Au-Cl" complex is more active than the "Au-S" ones. Indeed, azaBODI-Au-1 accumulates 1.6 to 4.5 times less than azaBODI-Au-3, while exhibiting the same activities. As the compounds are fluorescent, it was also possible to determine their accumulation in cells by FACS analysis. This technique is less precise, but it is faster and less expensive than the ICP-MS assays. Thus, the cell uptake was confirmed by FACS analysis for azaBODI-Au-1, azaBODI-Au-2, and azaBODI-Au-3, but not for auranofin (Figure 6). The three compounds efficiently accumulated in a timedependent manner in the cells (Figure 6, Figure S18, and Figure S19). Indeed, the fluorescence intensity is already high at 15 min and gradually increases up to 24 hours. As stated before, the capacity of complexes to accumulate in cells is not sufficient to explain their anti-proliferative properties. We therefore decided to study their ability to inhibit the activity of thioredoxin reductases (TrxRs), a potential target of gold(I) complexes. The inhibition of these enzymes, upregulated in certain cancers and regulating oxidative stress, results in cell apoptosis. From a mechanistic point of view, the selenocysteine of these enzymes would replace the ligand "X" of the gold complex (chlorido or thiolato), which would induce the inhibition of TrxRs. We then evaluated the activity of the TrxRs after incubation of the CT26 cells in the presence of the gold complexes. At 5 µM, azaBODI-Au-1, azaBODI-Au-2, azaBODI-Au-3, and auranofin strongly inhibits TrxRs (Figure 7). In particular, the activity of TrxRs is almost undetectable for azaBODI-Au-1, whereas it is reduced by a factor of four to ten for the other complexes. Taking into account that azaBODI-Au-1 accumulates less in cells than azaBODI-Au-2 and azaBODI-Au-3, it is clear that its ability to inhibit TrxRs is much greater. This could partly explain why its relatively low accumulation is not correlated with a lower anti-proliferative activity. Such a difference in terms of inhibition of TrxRs can be explained, as mentioned above, by a difference in reactivity between the "Au-Cl" and "Au-S" bonds with respect to the enzyme. It can also be mentioned the accessibility of the gold center, which is easier in the case of azaBODI-Au-1 because the chlorido ligand is less bulky than those of azaBODI-Au-2 and azaBODI-Au-3. In addition, the azaBODI-Au-2 and azaBODI-Au-3 "X" ligands are either positively or negatively charged, which may hinder the approach of the enzyme. It should also be noted that even if azaBODI-Au-1 inhibits significantly more TrxRs than azaBODI-Au-3 into CT26 cell line, they display similar anti-proliferative properties, thus, it is not possible to make a strict correlation between inhibition of TrxRs and anti-proliferative properties. In order to complete this in vitro study, we took advantage of our compounds being trackable by optical imaging to visualize their localization in cells at different times. The experiments were conducted on human MDA-MB-231 cells (Figure 8A, Figure S15) and mouse 4T1 cells (Figure S16). The confocal imaging demonstrated a fast and efficient cell internalization of the three theranostics as well as aza-BODIPY 2 (Figure 8A, Figure S15, and figure S16). They mainly localized inside small cytoplasmic vesicles and close to the nucleus, at longer time they seem to accumulate in Golgi apparatus. One should note the absence of gold complexes inside the nucleus and the counterstaining experiment with a mito-tracker did not highlight a significant uptake of the azaBODI-Au-1 in mitochondria (Figure 8B).

A B

In vivo investigation

After these promising in vitro results, we decided to investigate the most promising complex in vivo.

Our choice fell on azaBODI-Au-1, which offered the best compromise between biological activity and imaging properties.

First, the stability of azaBODI-Au-1 was studied in DMSO, known to be a very coordinating solvent, and in the presence of phosphate buffer (PBS). This experiment was monitored by NMR (Figure 9 and Figures S11-S14). The 1 H-NMR enables to have information on the stability of the overall molecule and the 31 P-NMR enables to ensure the stability of the "P-Au" part. This follow-up, carried out over one week, shows without ambiguity that azaBODI-Au-1 is perfectly stable in DMSO and in the presence of PBS. The anticancer activity of azaBODI-Au-1 was then evaluated on BALB/c mice bearing syngenic colon CT26 flank tumors. Twice a week, azaBODI-Au-1 was injected intravenously into the mice at a concentration of 0.5 mg/kg. Interestingly, the decrease of the tumor growth was observed within a few days after the first injection and continued all along the study (Figure 10). It is worth noting that no sign of acute toxicity (no loss of weight, no signs of pain) was detected all along the treatment duration. These results are very good for a non-vectorized compound. Therefore, we wanted to get information on the distribution of azaBODI-Au-1 in order to see where it was accumulating. We tracked it by optical imaging with an excitation at 685 nm and a detection at 705 nm. Even if these filters are not optimal (maximum of emission of azaBODI-Au-1 is at ≈ 730 nm), azaBODI-Au-1 is perfectly detectable and trackable in real time for at least 24 h, which proves the good stability of our compound (Figure 11). Interestingly, the compound accumulated at the tumor site with a maximum around 4 hours after injection, which can explain the anticancer activity detected previously. Additionally, a strong accumulation in the liver can be noticed, which is usual for relatively lipophilic compounds, such as molecules containing organic fluorophores. It can be noted that the tumor-toskin ratio of the theranostic compound is relatively stable over time (Figure 12). The distribution profile (Figure 12B) indicated a major hepatic elimination, while a part of the small compound is washed out through the kidney route. This is in accordance with the relatively small size of azaBODI-Au-1, for which the hydrodynamic radius was evaluated at 1.2 nm, i.e. below the size threshold for the renal filtration. This suggests that the compound, once internalized inside the tumor, remains trapped in it, while it is gradually excreted from the liver and the kidneys. Moreover, the tumor-tomuscle ratio is almost 5 (Figure 12).

Conclusions

We reported the efficient synthesis of the first gold-based aza-BODIPY theranostics. The three homobimetallic complexes display interesting photophysical properties: absorption and emission maximal wavelengths in the NIR-I region, and good quantum yields up to ~30%. The in vitro results are very promising. The three gold-based aza-BODIPY have retained excellent anti-proliferative properties despite the presence of the fluorescence probe, which is not the case for the most of the reported optical based theranostic agents.[4,7,52-54] Indeed, they displayed IC50 in the micromolar range. They also showed a very good ability to inhibit TrxRs, in particular azaBODI-Au-1. In addition, the fluorescence properties of the aza-BODIPY platform were preserved and allowed a localization of the compounds by confocal imaging (they enter the cells and accumulate into cytoplasmic vesicles). Interestingly, the most active (cytotoxicity and TrxR inhibition) aza-BODIPY theranostic, azaBODI-Au-1, induced a very significant anti-cancer effect in vivo and can be tracked in vivo with optical imaging modalities. It is interesting to note that these very good results were obtained despite a limited accumulation of the anti-cancer agent within the tumor, the compound going mostly in the liver. This suggests that the grafting of a vector on this type of platform would dramatically increase their efficiency, which should be confirmed in next studies. All these results are very promising for future in vivo investigations and open the door of a novel class of powerful optical theranostic agents.

EXPERIMENTAL Material and methods

Unless stated otherwise reactions were carried out in HPLC grade solvents under normal atmosphere. Solvents were purchased from BioSolve. Dry solvents were non-stabilized, purchased from Carlo Erba and dried using a MB-SPS-800 (MBraun) or PureSolv-MD-5 (Inert®). Wazaby7 was synthesized adapting to the procedure, we reported previously. [START_REF] Florès | Aza-BODIPY Platform: Toward an Efficient Water-Soluble Bimodal Imaging Probe for MRI and Near-Infrared Fluorescence[END_REF][START_REF] Pliquett | A Promising Family of Fluorescent Water-Soluble aza-BODIPY Dyes for in Vivo Molecular Imaging[END_REF] All reagents were purchased from Sigma-Aldrich, Alfa Aesar, TCI or ACROS Organics™ and used as received without further purification. Column chromatography was carried out using alumina gel (Alfa Aesar; aluminum oxide, activated, neutral, Brockmann Grade II). Analytical thin-layer chromatography experiments were performed with Machery-Nagel Alugram Alox N/UV254 aluminum gel (precoated aluminum sheets, 0.2 mm thick). Reactions were monitored by thin-layer chromatography, RP-HPLC-MS and no-lock 31 P-NMR. ( 1 H, 13 C, 11 B, 31 P) were recorded at 300K (unless otherwise specified) on Bruker 500 Avance III or Bruker Avance III HD 600 MHz spectrometers (equipped with double resonance broad band probes). Chemical shifts are given relative to TMS ( 1 H, 13 C), BF3.Et2O ( 11 B) and H3PO4 ( 31 P) and were referenced to the residual solvent signal. High resolution mass spectra were recorded on a Thermo LTQ Orbitrap XL ESI-MS spectrometer. NMR and Mass-analyses were performed at the "Plateforme d'Analyse Chimique et de Synthèse Moléculaire de l'Université de Bourgogne" (PACSMUB).

Synthesis of the ligand precursor 4 Wazaby7

In a round-bottom flask, aza-BODIPY 2 (250 mg, 370 µmol, 1 eq) was dissolved in THF (35 mL) and deionized water (10 mL). Sodium bicarbonate (154 mg, 1.83 mmol, 5 eq) and 4-(bromomethyl)benzoic acid (173 mg, 80 µmol, 2.2 eq) were then added and the mixture was stirred at room temperature (15-20 °C) for 5 h. 4-(bromomethyl)benzoic acid was then removed via extraction using diethyl ether and water. The aqueous phase was concentrated under reduced pressure and HCl 3 M was added until precipitation. After centrifugation, the solid was dried under reduced pressure to finally give Wazaby7 as a green solid (270 mg, 68% yield).

HR-MS (ESI

AzaBODI-Au-1

In a round-bottom flask, Wazaby7 (45 mg, 40 µmol) was dissolved in anhydrous DMF (4 mL). A solution of HBTU (34 mg, 89 µmol, 2.2 eq) in anhydrous DMF (5 mL) was added, followed by an addition of DIPEA (56 µL, 323 µmol, 8 eq). After stirring at room temperature (≈ 20-25 °C) for 15 min, a solution of 2-(diphenylphosphino)ethylamine-aurichloride (37 mg, 80 µmol, 2 eq) in DMF (5 mL) was added to the mixture. After 4 h, TLC confirmed complete conversion of the reagent. The solvents were removed under reduced pressure and the crude product was purified on alumina column (DCM to DCM/MeOH 96/4). After removal of the solvents under reduced pressure, the product underwent ion exchange (Amberlite IRA-410, ACN/water 1:1). The solid was precipitated from DCM using diethyl ether to give azaBODI-Au-1 (67 mg, 82% yield) as a green solid.

Rf (alumina, DCM/MeOH 9:1) = 0.5. HR-MS (ESI): calcd for C88H84Au2BCl2N7O4P2-2HCl 2+ [M-2HCl] 2+ 883.76583 Th; found 883.76766 Th (error: 2.071 ppm). 1 H NMR (600 MHz, DMSO-d6): δ (ppm) = 2.77 (s, 12H), 3.05-3.09 (m, 4H), 3.50-3.55 (m, 4H), 3.77 (s, 6H), 3.98 (s, 4H), 4.21 (s, 4H), 7.14 (d, J = 8.5 Hz, 4H), 7.37 (d, J = 8.5 Hz, 4H), 7.48 (t, J = 7.3 Hz, 2H), 7.52-7.59 (m, 16H), 7.66 (s, 2H), 7.78 (ddd, J = 12.9, 7.5, 1.8 Hz, 8H), 7.82 (d, J = 7.9 Hz, 4H), 8.17 (d, J = 7.1 Hz, 4H), 8.45 (d, J = 8.5 Hz, 4H), 8.85 (t, J = 5.4 Hz, 2H, NH). 13

AzaBODI-Au-2

In a Schlenk tube, 2-(dimethylamino)ethanethiol hydrochloride (10 mg, 73 µmol, 2.1 eq) was solubilized in 15 mL of anhydrous DCM. Anhydrous DIPEA (60 µL, 10 eq.) was added and the suspension was stirred in the dark under argon at 20 °C for 10 minutes. In a second Schlenk tube, azaBODI-Au-1 (70 mg, 35 µmol) was dissolved in 35 mL of anhydrous DCM. The solution was stirred at 0 °C for 5 minutes. The suspension from the first Schlenk tube was then slowly added via canula to the second Schlenk tube in the dark at 0 °C. The mixture was then stirred at room temperature (≈ 20 °C) for 18 h. 2-(dimethylamino)ethanethiol hydrochloride (6 mg, 42 µmol, 1.2 eq.) and anhydrous DIPEA (60 µL, 10 eq.) dissolved in anhydrous DCM (2 mL) were added and the mixture was stirred for 2 additional hours. After completion (monitored by TLC), the solvents were removed under reduced pressure, and the residue was purified by alumina gel chromatography (eluent DCM to DCM/MeOH 97/3). Ion exchange was performed (Amberlite IRA-410, ACN/water 1:1) and after removal of the solvents under reduced pressure, the solid was precipitated from DCM using diethyl ether to give azaBODI-Au-2 (50 mg, 63% yield) as a green powder.

Rf (alumina, DCM/MeOH 9:1) = 0.3.

AzaBODI-Au-3

In a Schlenk tube, sodium 2-mercaptoethanesulfonate (5.4 mg, 33 µmol, 2.2 eq.) were suspended in 3.5 mL of degassed acetone and a solution of 0.1 M NaOH (150 µL, 1 eq.) were added. The suspension was stirred in the dark under argon at 10-15 °C for 10 minutes. In a second Schlenk tube, azaBODI-Au-1 (30 mg, 15 µmol) was solubilized in 4 mL of degassed acetone. The solution was stirred at 0 °C for 5 minutes. The suspension from the first Schlenk tube was then slowly added via canula to the second Schlenk tube in the dark. The mixture was then stirred at 12 °C for 3 h. After completion monitored by 31 P-NMR, the crude product was evaporated to dryness, solubilized in a dichloromethane/methanol mixture, then filtrated to remove uncolored particles. Ion exchange was performed (Amberlite IRA-410, ACN/water 1:1). The product was obtained after complete evaporation to give azaBODI-Au-3 (15 mg, 44% yield) as a green powder. HR-MS (ESI): calcd for C92H92Au2BN7O10P2S4-C2H4O3S2 2+ [M-C2H4O3S2] 2+ 954.75375 Th; found 954.751919 Th (error: -1.927 ppm). 1 H NMR (600 MHz, DMSO-d6): δ (ppm) = 2.68-2.73 (m, 4H), 2.77 (s, 12H), 3.01-3.06 (m, 4H), 3.06-3.12 (m, 4H), 3.52-3.60 (m, 4H), 3.73 (s, 6H), 4.02 (s, 4H), 4.22 (s, 4H), 7.17 (d, J = 8.5 Hz, 4H), 7.44 (d, J = 7.9 Hz, 4H), 7.48-7.57 (m, 18H), 7.67 (s, 2H), 7.76-7.84 (m, 8H), 7.91 (d, J = 7.8 Hz, 4H), 8.18 (d, J = 7.5 Hz, 4H), 8.48 (d, J = 8.4 Hz, 4H), 8.98 (s, 2H, NH). 13 C NMR (151 MHz, DMSO-d6): δ (ppm) = 14.0, 16.7, 18.0, 18.8, 19.9, 22.1, 23.8, 26.4-26.6(m), 28.4-29.1 (m), 30.4, 30.7, 31.3, 32.6, 33.7, 33.9, 35.1, 35.4, 48.6, 49.2, 51.3, 54.1, 54.9, 55.3, 55.7

FCS analysis (hydrodynamic radius)

FCS study was performed using a confocal laser-scanning microscope (LSM 710 Carl Zeiss, Jena, Germany) with a 40× water immersion C-Apochromat objective lens (numerical aperture (N.A.) = 1.2). The measurements were carried out at room temperature in 8-well Lab-Tek I chambered coverglass (Nalge Nunc International, Illkirch, France). The 633 nm He-Ne laser beam was focused into 20 μL solutions at 100 μm over the cover glass. The fluorescence emission was collected through a pinhole and a 650 nm-long pass filter. Photon counts were detected by an Avalanche PhotoDiode at 20 MHz for 30 seconds, and repeated 10 times in triplicates. The data evaluation was performed using the Zeiss FCS Fit software (Zeiss, Jena, Germany). The intensity autocorrelation curves were fitted using a free diffusion model with one component. A preliminary calibration step with Cy5 made it possible to evaluate the size of the confocal volume (≈1 fL). The hydrodynamic radius was calculated using the Stokes-Einstein equation.

Biological experimental procedures

Cell culture

Mouse and human breast (4T1 and MDA-MB-231 respectively) and colon (CT26 and SW480 respectively) cancer cell lines, were cultured in RPMI supplemented with 10 % FBS. All the cells were cultured at 37 °C under a humidified atmosphere containing 5 % CO2. The cells were detached by trypsin and re-seeded at least 1 day before any experiment.

In vitro confocal imaging

MDA-MB-231 and 4T1 cells were plated in 4-well Lab-Tek I Chamber slide (Nunc TM , Thermo Scientific) (50,000 cells in 500 µL of culture medium) and incubated overnight in RPMI without red phenol medium at 37 °C and 5% CO2. Uptake experiments were performed by incubating cells with the different theranostic compounds (5 µM) at 37 °C and 5 % CO2. For confocal imaging, live cells were scanned by confocal microscopy (Zeiss LMS510 microscope) using a 63x oil immersion objective of 1.2 numerical aperture. Hoechst 33342 was used to label the nucleus (0.5 µM), excited at 405 nm (2-5%) and its signal was collected from 410 to 499 nm. The theranostic compounds were excited at 633 nm (2% power) and the signal was collected from 640 to 747 nm. Images were processed with ImageJ software.

FACS experiment

Similar protocol was observed for FACS experiment, with a 15 min to 24 h incubation with 5 µM theranostic solution. After washing with PBS, the cells were observed using LSR II (Becton, Dickinson and Company, Pont de Claix, France). Cells were gated to exclude cell debris in the control group and 50,000 events were recorded in the gate for fluorescence intensity measurement.

Determination of cytotoxic properties

10 5 cells were seeded in 96-well flat-bottomed microplates (final volume 100 μL per well) and incubated for 24h to allow for cell adherence. 10 mM stock solutions were prepared by dissolving the compounds in DMSO. Fresh medium containing increasing concentrations of the compounds (ranging from 0.01 to 25 μM) was added and plates were incubated at 37 °C for 48 h. The cytotoxic activity of compounds and drug references was determined using the MTS assay (Promega®). The assay is based on the reduction of a tetrazolium compound to a colored formazan in the presence of an electron coupling reagent (phenazine ethosulfate; PES) and NADH/NADPH produced by metabolically active cells. Thereafter, 20 μL of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, Promega, Charbonnières, France) was added in each wells and absorbance at 490 nm was measured after 3h incubation at 37 °C. The resulting IC50 values were calculated using GraphPad Prism 5.0 software. Each treatment was performed in three independent experiments.

Thioredoxin reductase (TrxRs) activity assay

TrxRs assays were carried out on CT26 cells incubated during 24h in RPMI medium containing 10% FBS with 5 µM of azaBODI-Au-1, azaBODI-Au-2, azaBODI-Au-3 or auranofin. Cells were scratched from the bottom of the dish after a washing step in PBS. Cell pellets were resuspended in 50 µL of a lysis buffer CelLytic-M (Sigma-Aldrich) during 15 min, and centrifugated at 16,000g for 10 min at 4 °C. The supernatants were transferred to 96-well microplates for protein dosage. The protein concentrations in the cell homogenates were determined by the Lowry method thanks to a protein Assay Reagents Package Kit (Bio-Rad). From each homogenate 20 μg protein were collected for The TrxRs enzyme activity measurement with a commercially available kit (Sigma-Aldrich). The assay is based on the reduction of 5,5'-dithiobis(2-nitrobenzoic)acid(DTNB) with NADPH to 5-thio-2nitrobenzoic acid (TNB) by TrxRs. The yellow color product is measurable at 412 nm by spectrophotometry. The reaction was performed in a working buffer (100 mM potassium phosphate with 10 mM EDTA and 0.24 mM NADPH) in a final volume of 200 μL. This reaction was initiated by the addition of 6 μL DTNB (39.6 mg/mL) diluted in an assay buffer (6 M guanidine-HCl in 0.2 M Tris-HCl, pH 8.0). The absorbance at 412 nm was measured 18 times within 15 min on a Perkin Elmer Wallac 1420 Victor2 Microplate Reader. The same experiments were performed for each condition with a specific inhibitor of TrxRs. The TrxRs activity rate was calculated from the slope of absorbance at 412 nm versus time after the subtraction of the activity measured in presence of the inhibitor.

In vivo experiments guidelines

All experiments followed the guidelines of the Federation of European Animal Science Associations and were all approved by the Ministry of agriculture and food (France, Apafis number: #8782 and Apafis number: #15181) and by respectively the Ethics Committee of Grenoble Alpes University (Grenoble, France) and the Ethics Committee of Burgundy University (Dijon, France).

Determination of the anti-tumor effect

In order to test the potential anti-tumor effect of azaBODI-Au-1, BALB/c mice (8 weeks old, Charles River, France) were grafted by subcutaneous injection of murine colon cancer CT26 cells (5.10 5 cells). Seven days after tumor implantation (tumor volume around 50 mm 3 ), tumor-bearing mice were randomized into two groups, one received 0.5 mg/kg of azaBODI-Au-1 (in DMSO) and the other received the same quantity of DMSO in saline (Control) by intravenous injection (IV) in the vein tail. These IV injections were performed two times a week and tumor volume assessed three times per week. Mice whose tumor reached 1,500 mm 3 were euthanized due to ethical reasons.

In vivo optical imaging azaBODI-Au-1 distribution was determined on mice bearing sub-cutaneous 4T1 tumor cells. NMRI mice (6 weeks old, Janvier, France) were injected with 1.10 6 cells, and imaged when the tumors reached >300 mm 3 (i.e. 2 weeks). Mice were imaged before and after injection of 50 µg of azaBODI-Au-1, at 1, 2, 4 and 24 h using LI-COR Pearl Trilogy system (LI-COR, Germany) at 700 nm. Organs were imaged at the end of the experiment in the same conditions.

Conflicts of interest

"There are no conflicts to declare".

Figure 1 :

 1 Figure 1: Examples of metal-based BODIPY trackable therapeutics associated with their year of publication.

Figure 2 :

 2 Figure 2: Targeted theranostics and rationale for the chosen functionalizations.

Figure 3 :

 3 Figure 3: 31 P-NMR of azaBODI-Au-1 before (up) and after (bottom) anion exchange

Figure 4 :Scheme 1 :

 41 Figure 4: 31 P-NMR of azaBODI-Au-1 (red) and azaBODI-Au-1 (blue)

Figure 5 :

 5 Figure 5: Absorption and emission spectra of Wazaby7, azaBODI-Au-1, azaBODI-Au-2, and aza-BODI-Au-3 in DMSO at 25 °C (absorption and emission are normalized).

Figure 6 :

 6 Figure 6: Histograms of 4T1 cells incubated with azaBODI-Au-1, azaBODI-Au-2, and azaBODI-Au-3 at 5 µM from flow cytometry measurements. The fluorescence signal was collected using a band pass filter 730/40 nm.

Figure 7 :

 7 Figure 7: Normalized activity of TrxRs from CT26 cells treated with 5 µM of the azaBODI-Au-1, azaBODI-Au-2, azaBODI-Au-3, and auranofin during 24 h (n = 4). Results are expressed as mean % ± SEM, Student t-test: *p ≤ 0.05.

Figure 8 :

 8 Figure 8: A-Confocal microscopy imaging (merge) of MDA-MB-231 cells labelled with compound 2, azaBODI-Au-1, azaBODI-Au-2, and azaBODI-Au-3. Cells were incubated with the aza-BODIPY derivative (red) at 5 µM for 15 min, 1 h, 2 h, 4 h, and 24 h at 37 °C. The nuclei are counterstained with Hoechst 33342 (blue, fluorescent DNA dye). B-Confocal microscopy imaging (merged) of 4T1 cells labelled with azaBODI-Au-1. Mitochondria were labeled using Mito-tracker green (Life Technology), nuclei are counterstained with Hoechst 33342 (blue, fluorescent DNA dye), and azaBODI-Au-Cl (red) was incubated at 5 µM for 45 minutes.

Figure 9 :

 9 Figure 9: NMR stability study of azaBODI-Au-1 in DMSO/PBS solution.

Figure 10 :

 10 Figure 10: Anti-tumor effect of azaBODI-Au-1. Tumor-bearing BALB/c mice (n=6/group) were treated intravenously twice per week with 0.5 mg/kg of azaBODI-Au-1 (in DMSO) or with the same quantity of DMSO (control). Tumor growth was determined 3 times per week using a caliper. Results are expressed as means ± SEM, Two-way Anova: **p≤0.01.

Figure 11 :

 11 Figure 11: Distribution of azaBODI-Au-1. Tumor-bearing mice were injected intravenously and imaged before and 1, 2, 4 and 24 h after injection. The compound accumulated in the tumor area as indicated by the arrow, and was eliminated through the liver.

Figure 12 :

 12 Figure 12: Tumor-to-skin ratio (T/S) determined in vivo by optical imaging (left). Distribution profile of azaBODI-Au-1 in mice-bearing tumor, 24 h after intravenous administration (right).

  ): calcd for C60H56B1N5O62+ [M] 2+ 476.71563 Th; found 476.71604 Th (error: 0.860 ppm).1 H NMR (600 MHz, MeOD-d4): δ (ppm) = 2.87 (s, 12H), 3.83 (s, 6H), 3.95 (s, 4H), 4.26 (s, 4H), 7.15 (d, J = 8.7 Hz, 4H), 7.43-7.54 (m, 12H), 8.04 (d, J = 8.0 Hz, 4H), 8.15 (d, J = 7.4 Hz, 4H), 8.46 (d, J = 8.7 Hz, 4H). 13 C NMR (151 MHz, MeOD-d4): δ (ppm) = 50.7, 55.5, 56.2, 66.7, 88.4, 115.3, 121.3, 125.4, 128.9, 129.7, 129.8, 130.4, 130.7, 131.1, 131.5, 132.7, 133.4, 133.9, 134.0, 134.1, 134.5, 144.1, 144.2, 159.0, 163.7, 168.5. 11 B NMR (192 MHz, MeOD-d4): δ (ppm) = -12.36. LogP approximation: LogD = 1.36. Counter-anions have been checked by ion chromatography. Only chlorides were detected.

  C NMR (151 MHz, DMSO-d6): δ (ppm) = 14.0, 15.2, 18.8, 26.3 (d, JC-P = 37.8 Hz), 26.6, 30.4, 34.4, 35.5, 49.2, 53.8, 55.6, 64.8, 64.9, 87.1, 114.1, 120.8, 123.4, 124.9, 127.8, 128.9 (d, JC-P = 46.1 Hz), 128.9, 129.3, 129.4 (d, JC-P = 11.6 Hz), 130.1, 131.7, 132.1, 132.3, 132.7, 133.1 (d, JC-P = 13.1 Hz), 135.5, 139.2, 141.3, 142.3, 157.1, 161.7, 165.3. 31 P NMR (193 MHz, DMSO-d6): δ (ppm) = 24.6. LogP approximation: LogD = 3.81. Counter-anions have been checked by ion chromatography. Only chlorides were detected.

  1 H-NMR (600 MHz, 318 K, DMSO-d6): δ (ppm) = 2.08 (s, 12H), 2.45 (t, J = 5.5 Hz, 4H), 2.77 (s, 12H), 2.91 (t, J = 7.2 Hz, 4H), 2.98-3.02 (m, 4H), 3.52-3.58 (m, 4H), 3.70-3.80 (m, 6H), 3.97 (s, 4H), 4.19 (s, 4H), 7.00-8.82 (m, 50H, Harom, NH). 31 P-NMR (243 MHz, 328 K, DMSO-d6): δ (ppm) = 28.2 (broad). LogP approximation: LogD = 1.44. Counter-anions have been checked by ion chromatography. Only chlorides were detected.

  31 P-NMR (202 MHz, DMSO-d6): δ (ppm) = 25.4 (broad). Counter-anions have been checked by ion chromatography. Only chlorides were detected.

Table 1 :

 1 Main photophysical data of Wazaby7

, azaBODI-Au-1, azaBODI-Au-2, and azaBODI-Au-3 in

  DMSO at 298 K.Compoundλmax, abs (nm) λmax, em (nm) ελ,max (M -1 .cm -1 ) Φ* (%) Br (M -1 .cm -1 )

	Wazaby7	694	727	60,000	32	19,200
	azaBODI-Au-1	695	732	70,700	27	19,090
	azaBODI-Au-2	693	728	68,100	29	19,750
	azaBODI-Au-3	695	730	64,000	25	16

Table 2 :

 2 IC50 values (µM), determined by MTS assay at 48 h, of azaBODI

-Au-1, azaBODI-Au-2, azaBODI-Au-3, and

  auranofin on 4T1, MDA-MB-231, CT26, SW480, and HMEC cell lines.

	Compounds	4T1	MDA-MB-231	IC50±SEM (µM) CT26	SW480	HMEC
	azaBODI-Au-1	4.3±0.5	9.7±0.7	4.1±0.2	4.2±0.5	3.4±0.9
	azaBODI-Au-2	9.9±0.6	10.2±0.2	10.1±0.5	10.2±0.6	5.6±0.1
	azaBODI-Au-3	4.8±0.4	9.2±0.6	4.5±0.6	4.3±0.6	5.0±0.1
	auranofin	3.4±0.9	2.1±1.0	2.5±0.5	2.1±0.7	<0.5

Table 3 :

 3 Cellular uptake of azaBODI-

Au-1, azaBODI-Au-2, aza-BODI-Au-3, and

  auranofin in 4T1, MDA-MB-231, and CT26 cells treated or not (control) with 5 µM of metal complexes for 4 h at 37 °C analyzed by ICP-MS.

	Compounds	4T1	Gold uptake (mg Au / g protein) MDA-MB-231	CT26
	Untreated cells	< 0.5	< 0.5	< 0.5
	azaBODI-Au-1	15.3	7.2	13.3
	azaBODI-Au-2	24.1	11.9	15.9
	azaBODI-Au-3	29.6	32.6	21.8
	auranofin	10.5	3.0	3.7

Acknowledgements

The Ministère de l'Enseignement Supérieur et de la Recherche, the Centre National de la Recherche Scientifique (CNRS), the Conseil Régional de Bourgogne (PhD JCE grant # 2015-9205AAO033S04139 / BG0003203 and # 2018Y-08158), and the French Research National Agency (ANR) via project JCJC ''SPID" ANR-16-CE07-0020 and project JCJC "WazaBY" ANR-18-CE18-0012 are gratefully acknowledged. This work is part of the projects ''Pharmaco-imagerie et agents théranostiques" et ''Chimie durable, environnement et agroalimentaire" supported by the Université de Bourgogne and the Conseil Régional de Bourgogne through the Plan d'Actions Régional pour l'Innovation (PARI) and the European Union through the PO FEDER-FSE Bourgogne 2014/2020 programs. This work was also partially supported by grants from Ligue contre le Cancer, Comité de Côte d'Or. Prof. Anthony Romieu, FrenchBIC, GDR AIM, and OncoDesign® are acknowledged for fruitful discussion. Dr Picquet, Dr Bonnin, and Ms M.-J. Penouilh are gratefully acknowledged for HR-MS, NMR analyses, Dr Myriam Laly for ionic chromatography and Valérie Saint-Giorgio (zootechny center, UBFC)