Metal-NHC Complexes for Anti-Cancer Applications: Gold(I) for Antimitochondrial Activity and Iridium(III) for

Photodynamic Therapy

In this work of thesis, several groups of novel NHC-based gold(I) complexes containing aliphatic and aromatic amino-side arms with interesting potential in biomedical applications have been synthesized and fully characterized. Also, a series of iridium(III) complexes containing NHC ligands with pronounced PDT anticancer activities has been prepared and fully characterized.

The first group represents a family of cationic bis(NHC)-gold(I) complexes containing aliphatic amino-side arms. These complexes have been synthesized and investigated for their antiproliferative activities towards four human cancer cell lines and the non-cancerous MDCK cell line. In this series, the lipophilicity correlates directly with the cytotoxic activity against cancer cells.

The second family of compounds concerns cationic gold(I) bis(NHC) complexes containing aromatic amino-side arms. The in vitro cytotoxicity of these complexes and proligands on the representative PC-3 prostate and T24 bladder cancer cell lines has been evaluated. All these complexes show higher Log P values (lipophilicity) than the first series of complexes, and in line with this higher cytotoxicity, nevertheless too high lipophilicity can also lead to lower selectivity.

In order to develop a drug candidate with optimized activity and selectivity, we designed and synthesized the third family of cationic gold(I) bis(NHC) complexes. The Log P values of this series were between the first series and the second series. The lower cytotoxicity towards non-cancerous NIH3T3 cells was found for this series of complexes whereas they also displayed less activities towards cancer cells than the second series.

The mechanistic studies on two gold complexes by monitoring the cellular uptake showed the rapid cellular accumulation of the intact gold bis(NHC) and a good bioavailability, in good agreement with the antiproliferative activity of these two

[Abstract] Chen ZHANG, PhD thesis complexes. Moreover, both complexes inhibit TrxR, a common target for gold(I) complexes. The cell death induced by these two complexes was ROS-dependent. Besides anticancer activities, we also tested gold(I) mono-NHC complexes for other biomedical applications in parasite disease Leishmaniasis. They were screened in vitro against both promastigote and axenic amastigote forms of L. infantum. Moreover, their cytotoxicity was evaluated on the murine J774A.1 macrophages in order to determine their selectivity of action.

Another topic of this thesis concerns iridium(III)-NHC complexes. Three families of theranostic iridium(III)-NHC complexes were prepared and characterized. The in vitro cytotoxicity of all the complexes against PC-3 prostate, T24 bladder cancer cells and non-cancerous NIH3T3 cells was evaluated. Moreover, all complexes are theranostic agents, and the confocal microscopy experiments of one complex showed that it can be quickly and effectively taken up into PC-3 cells and specifically localize into mitochondria. Interestingly, these complexes can act as efficient photosensitizers. The cytotoxicity of these complexes was increased substantially upon 365 nm light irradiation, which suggested the high potential to be mitochondria-targeting theranostic anticancer agents for photodynamic therapy.

[Ré sumé ] Chen ZHANG, thè se PhD

Complexes mé talliques à ligands NHC pour des applications anticancer: Or(I) pour l'activité antimitochondriale et

Iridium(III) pour la thé rapie photodynamique

Dans ce travail de thè se, plusieurs groupes de nouveaux complexes d'or(I) à base de carbè nes N-hé té rocyclique (NHC) contenant des bras amino-aliphatiques et aromatiques avec un potentiel inté ressant dans des applications biomé dicales ont é té synthé tisé s et entiè rement caracté risé s. En outre, une sé rie de complexes d'iridium (III) contenant des ligands NHC avec des activité s anticancé reuses prononcé es pour une application en thé rapie photodynamique, a é té pré paré e et entiè rement caracté risé e.

Le premier groupe repré sente une famille de complexes cationiques or(I) bis(NHC) contenant des bras laté raux amino-aliphatiques. Ces complexes ont é té synthé tisé s et é tudié s pour leurs activité s antiprolifé ratives vis-à -vis de quatre ligné es cellulaires cancé reuses humaines et de la ligné e cellulaire non cancé reuse MDCK. Dans cette sé rie, la lipophilie est directement lié e à l'activité cytotoxique contre les cellules cancé reuses.

La deuxiè me famille de composé s concerne les complexes cationiques or(I) bis(NHC) contenant des bras laté raux amino-aromatiques. La cytotoxicité in vitro de ces complexes et de leurs proligands sur les ligné es cellulaires repré sentatives du cancer de la prostate PC-3 et de la vessie T24 a é té é valué e. Tous ces complexes pré sentent des valeurs de Log P (lipophilie) supé rieures à celles de la premiè re sé rie de complexes en accord avec leur cytotoxicité plus é levé e, mais une lipophilie trop é levé e peut é galement conduire à une sé lectivité plus faible.

Afin de dé velopper un candidat-mé dicament avec une activité et une sé lectivité optimisé es, nous avons conç u et synthé tisé la troisiè me famille de complexes cationiques or(I) bis(NHC). Les valeurs de log P de cette sé rie se situent entre la premiè re sé rie et la deuxiè me sé rie. Ces complexes moins lipophiles sont moins cytotoxiques envers les ligné es cellulaires saines (NIH3T3) et montre des activité s anticancé reuses un peu plus faibles sur les cellules PC-3 que la deuxiè me sé rie, avec né anmoins des valeurs de GI50 dans la gamme du nanomolaire.

. Metals in medicine

Medicinal inorganic chemistry is a developing field, and novel therapeutic and diagnostic metal complexes have a great impact on medical treatment.

Metal complexes are very important for our health both endogenously and exogenously. Exogenous metal complexes can be administrated to our body in a controlled way, as the case of therapeutic agents, or in an uncontrolled way, as by air or pollutants. It is remarkable that the therapeutic agents or pollutants are directed towards the endogenous metal targets or require metal coordination to act. The biological activity of pharmaceuticals depends on the nature and oxidation state of metal and its ligands. Two main factors, thermodynamic stability and selectivity, are very important in the design of metal complexes for medical applications. In 1890, the first well-defined metal-based drug potassium dicyanoaurate with formula K[Au(CN)2] as antibacterial was discovered. Since 1940, gold complexes have been used for the treatment of arthritic disorder in humans. At the end of last century, gold complexes such as gold sodium thiomalate and gold thioglucose used to treat arthritis were administered by injection. It was discovered, however, that orally-administered auranofin (Fig. 1.1.2) was equally effective against rheumatoid arthritis. It is notable that the coordination of gold by thiolate appears to be a natural process in the body. [2][3] Fig 1 .1.2 Structures and names of some metal-based drugs.

The cytotoxicity of cisplatin has drastically promoted the development of treatment of certain types of cancer. 4 Cisplatin and carboplatin (Fig 1.1.2) are used to treat various types of cancers such as testicular, ovarian, lung and bladder cancer.

Oxaliplatin (Fig 1.1.2) was the first drug which could avoid the cisplatin resistance of colon carcinoma. 5 The ultimate target for Pt(II) drugs is DNA and certain platinated However, the use of cisplatin in clinic trials is limited by 1) the resistance observed in several types of cancer cells, 2) the increase of resistance after continuous treatment and 3) the high toxicity against normal cells, which means strong side effects. 7 Therefore, there are more interests in the development of platinum drugs against a wide range of types of cancer cells, especially cisplatin-resistant cancer cells.

Although platinum complexes have been the most detailedly investigated, research has been extended to other metal-based drugs, such as gold, silver, copper, palladium, rhodium, iridium and ruthenium.

N-heterocyclic carbene metal complexes in medicine

The last decade has witnessed the dramatic development in the field of metal complexes of N-heterocyclic carbenes (NHCs). 8 NHCs are a class of electron donating ligand, which form strong σ bonds to metal ions in both high and low oxidation states. Some similarities between NHC and phosphine ligands have been noted and NHCs have been used to replace phosphines, which are sensitive to oxygen, to prepare catalysts. 9 Nowadays, metal-NHC complexes appear to be a more and more popular field of research in the development of metal-based drugs because of their high stability and the ease of NHC derivatization (Fig 1 .2.1). 8b, 8d, 10 At the beginning, most of the researchers focused on the 1,3-nitrogen substituents to do modulations. [START_REF] Baker | Cationic, linear Au(I) N-heterocyclic carbene complexes: synthesis, structure and anti-mitochondrial activity[END_REF] Then substituents at 4-and 5-positions Here, we will talk about Ag, Ru, Rh and Cu complexes bearing NHC ligands, and then introduction of gold NHC complexes will be presented in a special section.

Silver complexes: Silver complexes have been noted as antimicrobial agents for many years and nowadays they also display anti-cancer activity. 13 The first Ag(I)-NHC complexes with antimicrobial activity was published by Youngs' group.

In this report by Youngs and co-workers, [START_REF] Melaiye | Formation of water-soluble pincer silver(I)carbene complexes: a novel antimicrobial agent[END_REF] the synthesis of two pyridine-linked pincer silver NHC complexes 1a and 1b (Fig 1 .2.2) is presented. 1a and 1b were active against E. coli, S. aureus and P. aeruginosa, showing minimum inhibitor concentration (MIC) values much lower than that of silver nitrate, which is the appropriate reference for this type of application. 2). Complex 2a is not stable enough to study its activity, but the IC50 values of complexes 2b and 2c were in the same range as cisplatin against ovarian (OVCAR-3) and breast (MB157) cancer cell in vitro. [START_REF] Medvetz | Anticancer activity of Ag(I) N-heterocyclic carbene complexes derived from 4,5-dichloro-1H-imidazole[END_REF] On the other hand, dimeric complexes 2d and 2e just showed 10-fold less activity than cisplatin against lung (H460) cancer cell and no significant effect against cervical (HeLa) cancer cell. [START_REF] Siciliano | Synthesis and anticancer properties of gold(I) and silver(I) N-heterocyclic carbene complexes[END_REF] Ruthenium complexes: Group 8 metal complexes based on ruthenium have arisen interest in the research of metal-based anticancer agents. Two ruthenium complexes NAMI-A and KP1019 (Fig 1.2.3) have entered phase II clinical trials, and the former as an antimetastatic agent. 17 They are less toxic than platinum-based drugs and capable of overcoming the resistance of cisplatin-resistant cancer cells. Ott and co-workers reported a series of ruthenium complexes (3a-3d, Fig 1 .2.4) of the type (p-cymene)(NHC)RuCl2 and studied their behavior in biological tests. [START_REF] Oehninger | Evaluation of arene ruthenium(II) N-heterocyclic carbene complexes as organometallics interacting with thiol and selenol containing biomolecules[END_REF] These compounds interact with biologically relevant thiols and selenols, which lead to the inhibition of enzymes like thioredoxin reductase (TrxR) and cathepsin B (catB).

One of the complexes (3d) showed pronounced antiproliferative effects with low IC50 values against breast cancer cells (MCF-7, 2.07 μM) and colon adenocarcinoma cells ( HT-29, 2.40 μM). The inhibition of tumor cell growth was accompanied by a perturbation of metabolic parameters such as cellular respiration. Besides anticancer activity, rhodium complexes can also be taken as antimicrobial agents. Cetinkaya and co-workers were the first to investigate the in vitro antimicrobial activities of Rh(I)-NHC complexes. [START_REF] Çetinkaya | Antimicrobial activity of carbene complexes of rhodium(I) and ruthenium(II)[END_REF] The rhodium complexes 6-9 (Fig 1 .2.5) were evaluated against E. coli, S. aureus, Enterococcus faecalis and P.

aeruginosa. Of all these compounds, 6a and 6c were found to be the most effective against both Gram-positive bacteria with MIC values of 5 μg/ml. No significant inhibition was observed against Gram-negative bacteria.

Copper complexes: Copper is an essential element for most aerobic organisms, and it has a long history for medical applications. Copper, which is important for the function of several enzymes and proteins and is involved in many biological pathways, is maybe less toxic than non-essential elements. [START_REF] Santini | Advances in copper complexes as anticancer agents[END_REF] Two types of novel NHC-copper(I) bromide derivatives 10a-10j (Fig 1. 3.2. [START_REF] Bindoli | Thioredoxin reductase: a target for gold compounds acting as potential anticancer drugs[END_REF] The mitochondrial respiratory chain produces superoxide anion that becomes hydrogen peroxide and oxidizes thioredoxin in a reaction mediated by peroxiredoxin (Prx).

However, TrxR, inhibited by gold complexes, is no longer able to reduce back the oxidized thioredoxin that accumulates together with hydrogen peroxide and both act on several different mitochondrial targets leading to the opening of the mitochondrial permeability transition pore or an increase of the permeability of outer membrane, and therefore causing the release of proapoptotic factors.

Mitochondria targeting mode of action:

Mitochondria play a major role in the regulation of apoptosis (cell death) and in diseases characterized by abnormal apoptotic responses such as cancer. Apoptosis is a form of cell death characterized by activation of caspases that cleave multiple targets in the cell. [START_REF] Barnard | Targeting the mitochondrial cell death pathway with gold compounds[END_REF] In vertebrate cells, the main form of apoptosis proceeds through the mitochondrial pathway, and the critical event in the process is mitochondrial outer membrane permeabilization (MOMP), which represents the "point of no return" of cell death. [START_REF] Green | The pathophysiology of mitochondrial cell death[END_REF] The mechanisms responsible for MOMP during apoptosis remain controversial, but there are two classes of mechanism that have been described, and each of them may function under different circumstances (Fig 1.3.3). [START_REF] Green | Pharmacological manipulation of cell death: clinical applications in sight?[END_REF] On one hand, MOMP can occur as a consequence of the mitochondrial permeability transition (MPT). [START_REF] Kokoszka | The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore[END_REF] The opening of MPT involves the adenosine nuclear transporter (ANT) in the inner mitochondrial membrane, and the voltage-dependent anion channels (VDACs) in the outer membrane. On the other hand, MOMP appears to be mediated by members of the Bcl-2 family of apoptosis-regulating proteins acting directly on the outer mitochondrial membrane. [START_REF] Wei | Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death[END_REF] The proapoptotic multidomain member Bax or Bak, leading to the release of cytochrome c from mitochondria, are vital for MOMP to occur during apoptosis. [START_REF] Barnard | Targeting the mitochondrial cell death pathway with gold compounds[END_REF] Chen and co-workers found that compared with living cells, carcinoma cells have higher mitochondrial membrane potential (ΔΨm). [START_REF] Chen | Mitochondrial membrane potential in living cells[END_REF] 2 0 0 4 2 0 0 5 2 0 0 6 2 0 0 7 2 0 0 8 2 0 0 9 2 0 1 0 2 0 1 1 2 0 1 2 2 0 1 3 2 0 1 4 2 0 1 5 2 0 1 6 2 0 1 7 2 0 1 0 4 2 0 0 5 2 0 0 6 2 0 0 7 2 0 0 8 2 0 0 9 2 0 1 0 2 0 1 1 2 0 1 2 2 0 1 3 2 0 1 4 2 0 1 5 2 0 1 6 2 0 1 7 2 0 1 It has been previously reported that gold(III) complexes are not very stable under physiological environments, and Au(III) can be reduced to Au(I) by thiols and thioethers containing cysteine residues in proteins and peptides. [START_REF] Shaw | Gold-based therapeutic agents[END_REF] However, by taking advantage of the donating property of NHC ligands and the ease of pharmaco-modulation, Che's group previously reported a panel of stable cyclometalated gold(III) complexes 12a-g containing NHC ligands (Fig 1.3.5). [START_REF] Fung | Cyclometalated Gold(III) Complexes Containing N-Heterocyclic Carbene Ligands Engage Multiple Anti-Cancer Molecular Targets[END_REF] These complexes show potent and selective cytotoxicity against cancer cells and in vivo anti-tumor activities in mice bearing xenografts of HeLa (cervical) and NCI-H460 (lung) cancer cells. Furthermore, they studied about the mechanism of action, and they found the specific engagement of Au(III) NHC complexes with various cellular targets, such as heat shock proteins (HSP60), vimentin, nucleophosmin (NPM) and Y-box binding protein 1 (YB-1). The gold(III) NHC complex (14, Fig 1.3.7) was prepared by Haque and co-workers. [START_REF] Haque | Synthesis, crystal structures, and in vitro anticancer properties of new N-heterocyclic carbene (NHC) silver(I)-and gold(I)/(III)-complexes: a rare example of silver(I)-NHC complex involved in redox transmetallation[END_REF] Haque and co-workers (14) and by Huynh and co-workers (15a-15e).

Huynh and co-workers reported a series of Au(III) mono-, homobis-and heterobis-(NHC) complexes (15a-15e, Fig 1.3.7). [START_REF] Sivaram | Syntheses, characterizations, and a preliminary comparative cytotoxicity study of gold(I) and gold(III) complexes bearing benzimidazole-and pyrazole-derived N-heterocyclic carbenes[END_REF] The cytotoxic activities of these complexes against NCI-H1666 (non-small cell lung cancer) cells were tested using cisplatin as a reference drug (IC50 = 2.51 μM). In terms of benzimidazole moiety, the neutral monocarbene complex 15a (IC50 > 10 μM) was found to be less active than cisplatin, and displayed much lower activities as compared to the cationic homo The group of Berners-Price was a pioneer in the research of gold(I) complexes containing NHC ligands as anticancer agents. They reported a series of dinuclear Au(I) -NHC complexes (16a-16g, Fig 1.3.8). [START_REF] Barnard | Mitochondrial permeability transition induced by dinuclear gold(I)-carbene complexes: potential new antimitochondrial antitumour agents[END_REF] These complexes were studied for their ability to induce mitochondrial membrane permeability (MMP) in isolated rat liver mitochondria. The rates and levels of uptake of these complexes into mitochondria were evaluated by measuring mitochondrial gold levels using inductively coupled plasma optical emission spectroscopy. The mechanism by which MMP is induced by these Au(I)-NHC complexes was not a purely function of the level of gold accumulation, but it may involve disruption of the function of a specific enzyme or interaction with a mitochondrial permeability transition pore (MPT) component. Another dinuclear NHC complex (17, Fig 1.3.9) was obtained through a transmetalation route by Goite, Castro and co-workers. [START_REF] Sanchez | Novel silver(I)-and gold(I)-N-heterocyclic carbene complexes. Synthesis, characterization and evaluation of biological activity against tumor cells[END_REF] Complex 17 was found to interact with DNA mainly through noncovalent interaction as π-π stacking and produced conformational changes in the structure of DNA. Sulphorhodamine-B (SRB) assay was used to distinguish between a cytostatic effect (reduction in cell proliferation) and a cytotoxic effect (decrease of the number of viable cells). Complex (prostate carcinoma) and HT29 (colon carcinoma) cell lines (GI50 = 0.6, 1.3 and 2.3 μM, respectively), while it showed a low cytotoxic effect on all cancer cell lines with a LC50 (50 % cytotoxicity) lower than 30 μM.

Che and co-workers reported three dinuclear complexes (18a-18c, Fig -workers (18a-18c), and by Sun and co-workers (19a-19b).

Various new gold(I) pyrrolidinedithiocarbamato complexes (19a-19b, Fig 1.3.9) bearing NHC ligands were prepared by Sun and co-workers. A dinuclear gold(I) complex 19a with a bidentate carbene ligand displayed a potent cytotoxic (IC50 value of 0.83 μM) and antimigratory activity in vitro towards cisplatin-resistant ovarian cancer cells A2780cis. Due to the rigid scaffold, the complex 19a enabled zinc(II)-based metal-organic framework (Zn-MOF) to be used as a carrier to improve its cellular uptake and release under physiological conditions. Notably, the biocompatible Zn-MOF didn't exhibit cytotoxicity to the cancer cells, whereas the survival percentage of A2780cis cancer cells decreased significantly with increasing incubation time of 19a@Zn-MOF. 42 1.3. Moreover, this selective inhibition of TrxR over structural related enzymes like glutathione reductase (GR) and glutathione peroxidase (GPx) indicated that covalent interaction with selenium was relevant for the mechanism of drug action. [START_REF] Rubbiani | Comparative in vitro evaluation of N-heterocyclic carbene gold(I) complexes of the benzimidazolylidene type[END_REF] To study the mechanism of cell death induced by gold(I) NHC complexes, Wölfl and co-workers reported an organometallic complex of Au(I)(NHC)(phosphane) type (21, Fig 1.3.10). Strong cytotoxicity in vitro was found in various cancer cells, associated with irreversible loss of mitochondrial membrane potential as well as oxidative stress due to the inhibition of TrxR, leading to apoptotic cell death. Růžička and co-workers. [START_REF] Turek | 1, 2, 4-Triazole-based N-heterocyclic carbene complexes of gold(I): synthesis, characterization and biological activity[END_REF] The antiproliferative activities of these complexes were screened in vitro against various cancer cell lines, such as HepG-2 (liver cancer), HeLa S3 (cervical cancer), HL-60 and CCRF-CEM (leukemia) as compared to cisplatin and auranofin. It was shown that complexes 25a and 25b with chloride and bromide were more active than the complex 25c with iodide and cisplatin, and slightly less active than auranofin against HeLa S3 and HL-60 cell lines, but more effective than auranofin against HepG-2. In addition, the apoptotic properties of these complexes were confirmed using cell cycle analysis where they induced an obvious increase in sub-G1 population by around 50% compared to the non-treated control.

All complexes also had some effects on cell cycle distribution by slightly increasing the percentage of G2/M phase, suggesting the mechanism of antiproliferative activities of the tested complexes may differ from that of auranofin.

Mohr, Casini, Rigobello and co-workers reported a gold-NHC complex bearing fluorescent anthracenyl group (26, Fig 1.3.12). [START_REF] Citta | Fluorescent silver(I) and gold(I)-N-heterocyclic carbene complexes with cytotoxic properties: mechanistic insights[END_REF] Complex 26 was evaluated for the cytotoxic activity in vitro against cancer and normal cells. It showed no selectivity between cancer cells (A2780s and A2780R) and normal cell (HEK-293T). Both on isolated enzyme and in cell extracts, this complex exhibited a pronounced TrxR inhibition compared with related enzyme GR. From the fluorescence microscopy experiments, the cellular distribution displayed a larger diffusion of these molecules in tumor cells when compared to normal cells.

Similarly, Au(I)-NHC complex 27 (Fig 1 .3.12) with a fluorescent coumarin group was evaluated for its cellular uptake and subcellular localization in the PC-3 human prostate cancer cell line by Gautier and co-workers. This complex was obtained by the combination of "auto-click" reaction with the transmetallation route. After incubation with complex 27, the fluorescence was found in the cytoplasm, not in the nucleus.

Moreover, it could be specifically localized in mitochondria. [START_REF] Ibrahim | Auto-click'functionalization for diversified copper(I) and gold(I) NHCs[END_REF] Fig 1 .3.12 Gold NHC complexes bearing chromophores reported by Casini and co-workers ( 26), Gautier and co-workers ( 27) and Gimeno and co-workers (28a-28f).

Gimeno and co-workers reported a series of gold(I)-NHC complexes bearing a fluorescent acridine group (28a-28f, Fig 1.3.12). [START_REF] Visbal | Cytotoxicity and biodistribution studies of luminescent Au(I) and Ag(I) N-heterocyclic carbenes. Searching for new biological targets[END_REF] Their cytotoxicity against A549

(human liver cancer) and Miapaca2 (pancreatic carcinoma) revealed thiolate-Au-NHCs had higher antiproliferative activities in vitro as compared to the corresponding chloride derivatives. In particular, complexes 28e and 28f with the tetra-O-acetyl-1-thio-β-D-glucopyranoside ligand displayed even better anticancer activities, especially in Miapaca2 cells reaching IC50 values of 2.8 μM and 3.4 μM, respectively. Furthermore, flow cytometry assay showed that apoptotic pathway was the main mechanism for cell death. From the fluorescent microscopy experiments, it was shown that these complexes were mainly located into lysosomes, and small amout of them have been accumulated in the nucleus.

A series of gold(I) complexes bearing a glucopyranoside-incorporated NHC ligand were reported by D'Amora and co-workers (29a-29c, Fig 1.3.13). [START_REF] Cucciolito | Sugar-Incorporated N-Heterocyclic-Carbene-Containing Gold(I) Complexes: Synthesis, Characterization, and Cytotoxic Evaluation[END_REF] Different secondary ligands (chloride and phosphane) were coordinated to the gold atom, and these complexes were evaluated for their cytotoxicity in vitro against various cancer cell lines, such as PC-3 (prostate cancer), A375 (malignant melanoma), U-2 OS (osteoblast) and MCF-7 (breast carcinoma) as compared to cisplatin. Among the entire series, complex 29a and 29b showed an EC50 (half maximal effective concentration) against PC-3 comparable with cisplatin, nevertheless, the complex 29c was inactive.

Fig 1.3.13 Gold NHC complexes reported by Amora and co-workers (29a-29c) and reported by Richeter and co-workers (30a-30c).

Richeter and co-workers reported several Au(I) complexes bearing porphyrins-incorporated NHC ligands (30a-30c, Fig 1.3.13). Photodynamic properties of these complexes to kill MCF-7 (human breast cancer) were investigated. The Gold(I) for Antimitochondrial Activity and Iridium(III) for Photodynamic Therapy 25 results showed that these complexes 30a-30c were not effective in the dark whereas upon irradiation photocytotoxicity was observed, not only due to the photodynamic properties of porphyrin but also due to the peripheral Au(I) complex losing its thiolato ligand upon irradiation. (HMEC). These complexes showed the selectivity between cancer cells and normal cells, and the degree of selectivity was related with the lipophilicity of these complexes. Furthermore, it was shown that complex 31a induced selective cell death through a mitochondrial apoptotic pathway. TrxR activity was inhibited by 50 % with 5 μM of complex 31a, whereas no inhibition of glutaredoxin (GR) activity was found.

Therefore, TrxR plays an important role in cytotoxicity and its inhibition may lead to the apoptosis in breast cancer cells. [START_REF] Hickey | Mitochondria-targeted chemotherapeutics: the rational design of gold(I) N-heterocyclic carbene complexes that are selectively toxic to cancer cells and target protein selenols in preference to thiols[END_REF] Fig 1 .3.14 Bis-NHC Au(I) complexes reported by Berner-Price and co-workers (31a-31d) and by Gust and co-workers (32a-32d).

Gust and co-workers reported a series of cationic bis [1,3-diethyl-4,5diarylimidazol-2-ylidene] gold(I) complexes (32a-32d, Fig 1.3.14). [START_REF] Liu | Synthesis, characterization, and in vitro studies of bis [1,3-diethyl-4,5-diarylimidazol-2-ylidene] gold(I/III) complexes[END_REF] The antiproliferative activities in vitro against MCF-7 (breast carcinoma), MDA-MB-231 (breast carcinoma) and HT-29 (colon carcinoma) cells were evaluated as compared to cisplatin and fluorouracil (5-FU). Among all the complexes, 32b being the most cytotoxic compound, gave a pronounced IC50 value of 0.1 μM, which was 10-fold higher than that of cisplatin and 5-FU. However, the mode of action remained unclear.

Thioredoxin reductase (TrxR), the estrogen receptor (ER) and the cyclooxygenase enzymes (COX), which may be considered as targets for the drug design, can be excluded.

In the past few years, gold complexes bearing NHC ligands have drawn more and more attention. However, only gold mono(NHC) and homo-bis(NHC) complexes have been reported, whereas gold complexes with different kinds of NHC ligands were barely investigated. Triggered by the interest on heteroleptic complexes, Huynh and co-workers reported a series of cationic gold(I) complexes bearing pyrazole-derived NHC and 1, 3-disubstitued benzimidazole-derived NHC ligands (33a-33h, Fig 1.3.15). The cytotoxicity of complexes 33a-33h was evaluated in vitro against NCI-H1666 (non-small cell lung cancer cell line) as compared with cisplatin.

Among the entire series, complex 33a showed the best effective antiproliferative activity against NCI-H1666, with an IC50 value of 0.241 μM, and most of the complexes were more active than cisplatin. [START_REF] Sivaram | Cationic gold(I) heteroleptic complexes bearing a pyrazole-derived N-heterocyclic carbene: syntheses, characterizations, and cytotoxic activities[END_REF] Fig 1 .3.15 Bis-NHC Au(I) complexes reported by Huynh and co-workers (33a-33h) and by Mao and co-workers (34a, 34b).

Mao and co-workers reported two gold(I) complexes bearing NHCs derived from cyclophanes as potential anticancer agents (34a and 34b, Fig 1.3.15). [START_REF] Li | Antitumor properties and mechanisms of mitochondria-targeted Ag(I) and Au(I) complexes containing N-heterocyclic carbenes derived from cyclophanes[END_REF] and by Gornitzka, Hemmert and co-workers (36a-36d).

Four novel gold(I) complexes bearing aliphatic amino-functionalized NHC ligands (36a-36d, Fig 1.3.16) were synthesized, characterized and evaluated for their cytotoxicity in vitro on PC-3 prostate cancer cell line by Gornitzka, Hemmert and co-workers. The lipophilicity (log P) of these complexes was in good agreement with the antiproliferative activities. The most active complex 36d exhibited pronounced cytotoxicity against five different kinds of cell lines (PC-3 prostate cancer, MCF-7 breast cancer, U87 brain cancer, A549 lung cancer and Hep3B liver cancer) with GI50

values ranging from 400 nM to 1 μM, whereas it was insensitive to normal primary human umbilical vein endothelial cells (HUVECs) with an GI50 value over 5 μM, suggesting a selectivity between cancer cells and healthy cells. [START_REF] Boselli | Synthesis, structures, and selective toxicity to cancer cells of gold(I) complexes involving N-heterocyclic carbene ligands[END_REF] In order to extend the structural diversity of coordinated NHC ligands, Ott and co-workers prepared gold(I) complex 37 (Fig Arambula et al. (39a-39c) and by

Casini and co-workers (40).

To study about the interaction of the biscarbene gold(I) complex and DNA sequence, [Au(9-methylcaffein-8-ylidene)2]BF4 (40, Fig 1.3.18) To achieve an approach of targeting antioxidant pathway, gold(I) complexes bearing 1, 4-naphthoquinone-based NHC ligands (42a and 42b, Fig 1.3.19) were designed and synthesized by Arambula and co-workers and their cytotoxic effects were tested in vitro against a panel of human cancer cell lines. Complex 42a, containing two NHC-quinone groups, was found to show much higher antiproliferative activities than the corresponding mono(NHC)-Au complex. In addition, A549 cells treated with complex 42a, which was found to localize in mitochondria, led to an increase of ROS production. Furthermore, complex 42a was found to induce cell death via apoptotic pathway, and it was shown to be effective in zebrafish bearing A549 xenografts. [START_REF] Mccall | Dual targeting of the cancer antioxidant network with 1,4-naphthoquinone fused Gold(I) N-heterocyclic carbene complexes[END_REF] 1.3.5. Polynuclear NHC complexes as theranostic agents

The first example concerning the luminescence studies of the intracellular distribution of biologically active NHC complexes was reported by Berners-Price and co-workers in 2006. [START_REF] Barnard | Luminescence Studies of the Intracellular Distribution of a Dinuclear Gold(I) N-Heterocyclic Carbene Complex[END_REF] Two dinuclear gold(I) complexes (43-cis and 43-trans, Fig 1.3.20) bearing bidentate NHC ligands were designed to support Au-Au interactions of a predetermined length, which led to native luminescence of these complexes. Incorporation of organic chromophores within the carbene structure has been a

straightforward method in fluorescence confocal microscopy technologies, leading to the development of theranostic agents. In the previous paragraph, several NHC-metal complexes with organic chromophores have been mentioned about their anticancer activities, and additionally, they displayed luminescence that enabled them to study cellular distribution. Complex 34a and 34b (Fig 1.3.15) reported by Mao and co-workers contained fluorescent anthracene groups incorporated in the cyclic bis(NHC) ligands. [START_REF] Li | Antitumor properties and mechanisms of mitochondria-targeted Ag(I) and Au(I) complexes containing N-heterocyclic carbenes derived from cyclophanes[END_REF] These complexes were excited at 405 nm and blue emission could be observed on the confocal microscopy. Colocalization assays with organelle-specific stains, such as MitoTracker green, manifested these complexes were localized in mitochondria and not in lysosomes (Fig 1.3.22). Another way to make a bioactive molecule luminescence consists of the attachment of an organometallic dye. Gornitzka, Hemmert and co-workers reported three heterobimetallic gold(I)-ruthenium(II) complexes containing heteroditopic bipyridine-based NHC ligands (44a-44c, Fig 1.3.23). [START_REF] Boselli | Synthesis, Structures, and Biological Studies of Heterobimetallic Au(I)-Ru(II) Complexes Involving N-Heterocyclic Carbene-Based Multidentate Ligands[END_REF] Then in vitro cytotoxic, antileishmanial and antimalarial activities of these complexes were evaluated, and all these complexes showed moderate effects due to the presence of Ru(bpy)3 moiety.

The fluorescent confocal microscopy experiments indicated that 44b was localized in the cytoplasm. Experiments using MitoTracker green for mitochondria labeling and DAPI for nuclei labeling demonstrated that no colocalization could be found in mitochondria or in nucleus (Fig 1.3.24). 46) and Tacke and co-workers (47).

A series of gold(I) bis-carbene complexes (45a-45f, Fig 1.3.25) with NHC ligands derived from the plant metabolite combretastatin A-4 (CA-4) was designed and synthesized by Schobert and co-workers. [START_REF] Muenzner | Gold(I) Biscarbene Complexes Derived from Vascular-Disrupting Combretastatin A-4 Address Different Targets and Show Antimetastatic Potential[END_REF] The in vivo anticancer activity and tolerance of gold complexes 45d and 45f were investigated in a Balb/c mouse xenograft model of highly metastatic B16-F10 mouse melanoma cells. Two weeks after continuous implantation of 200,000 cells in mice, complexes 45d and 45f were repeatedly administered at 15 mg per kg body weight for two consecutive days. The results showed that this treatment was well tolerated by the mice, leading to a slight decrease in body weight, and noticeable tumor regression.

Another gold bis(NHC) complex (46, Fig 1.3.25) was tested in vivo on mice bearing the B16-F10 mouse melanoma by Dinda, Saha and co-workers. [START_REF] Nandy | Gold(I) N-heterocyclic carbene complex inhibits mouse melanoma growth by p53 upregulation[END_REF] Complex 46

was administered at the dosages of 5 and 10 mg/kg body weight per day from 15 th day onward since the day of injection of B16-F10 cells in the mice till the 23 rd day.

Dose-dependent decrease in the tumor size was observed following treatment with complex 46, whereas body weight remained nearly constant.

The two neural Au(I) complexes 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene gold(I) chloride 47a (Fig 1.3.25) andits 2',3',4',6 Generally, absorption of light (photons) leads to a transform from a ground state (singlet state) of PS into an excited state (triplet state) via an excited singlet state.

Then two kinds of relaxation reactions can occur (Fig 1.4.2). Firstly, the PS in triplet state can transfer its energy to molecular oxygen ( 3 O2) leading to singlet oxygen ( 1 O2), which is the major cytotoxic agent involved in photodynamic therapy (type II process).

Alternatively, the triplet state PS can react directly with a substrate, such as the cell membrane or a molecule, and transfer a hydrogen atom or electron to form radicals.

These radicals interact with oxygen to produce oxidation products ( 1 O2), which is a type I process. 73 known or strongly suspected bladder carcinoma. [START_REF] Jocham | Improved detection and treatment of bladder cancer using hexaminolevulinate imaging: a prospective, phase III multicenter study[END_REF] It is a pro-drug with no photochemical activity that will enter the body and initiate a series of biochemical reaction in the cells after irradiation leading to the formation of PS drug, hypericin, a powerful PS. This is a polycyclic aromatic compound belonging to the class of quinone derivatives used for fluorescence diagnosis of bladder carcinoma. [START_REF] D'hallewin | Fluorescence detection of flat bladder carcinoma in situ after intravesical instillation of hypericin[END_REF] Hypericin has drawn a lot of attention in its possible use in the treatment of bladder cancer. It causes damage to the urothelial tumor, however, there are 2 to 5% of tumor cells that can survive after photodynamic treatment, which leads to the regrowth of tumors after a prolonged period of time.

Various researchers in the field of coordination chemistry have recognized the importance of metals in medicine and have made significant progress towards the development of unique organometallic PSs. [START_REF] Mari | Combination of Ru(II) complexes and light: new frontiers in cancer therapy[END_REF] Most of the metal complexes proposed as PSs are ruthenium polypyridyl complexes, the Ru(II) complex TLD1433 (a Ru(II) From the confocal microscopy experiments, it was shown that complex 48a accumulated in the cytoplasm, particularly in mitochondria. The results confirmed by ICP-MS showed an accumulation of 67% of Ru taken up into mitochondria for complex 48a, and by comparison, the cellular uptake for complex 48b was more diffuse. Therefore, improving the cellular uptake of the complexes may help to increase the photodynamic activity and selectivity against cancer cells. 81 cm 4 s photon -1 ), which were much larger than that of tetraphenylporphyrin (H2TPP, σ = 2.2 GM). In addition, OP photocytotoxicity of these complexes towards HeLa monolayer cells was evaluated. Complexes 49a-49c exhibited similar phototoxic activity with IC50 values of 12.4-15.5 μM, whereas 49d displayed the lowest IC50 value of 3.5 μM, having a PI value of over 28. Furthermore, two-photon photocytotoxicity against 3D multicellular spheroids (MCSs) was assessed. After laser irradiation, complex 49a-49d displayed a very effective inhibition of MCSs growth.

Dyad derived from 2-(1-pyrenyl)-1H-imidazo[4,5-f][1,10]phenanthroline, Fig 1.
Remarkably, complex 49d demonstrated the lowest IC50 value of 1.9 μM in TP PDT (λirr = 830 nm) for 3D MCSs, suggesting this cationic ruthenium(II) complex as a mitochondria-targeting TP PDT agent. [START_REF] Liu | Ruthenium(II) polypyridyl complexes as mitochondria-targeted two-photon photodynamic anticancer agents[END_REF] 1.4.3. Iridium(III) complexes as photodynamic anticancer agents Iridium(III) complexes as PSs were studied due to their easy synthesis, air and moisture stability. Furthermore, heteroleptic Ir(III) complexes with bipyridyl and cyclometalated ligands have displayed high photostability due to high metal-centered state and ease of modulation of HOMO and LUMO energy levels. Based on this work, Mao and co-workers reported a second generation of mixed-ligand phosphorescent iridium complexes (51a-51d, Fig 1.4.6). [START_REF] Zheng | Mixed-ligand iridium(III) complexes as photodynamic anticancer agents[END_REF] These complexes showed high luminescence quantum yields, long lifetimes and high aqueous photostability. Since complexes 51b and 51d with phenyl-pyridine (ppy) ligands were more lipophilic than 51a and 51c with bipyridine (bpy) ligands, they can be taken up into A549 (lung carcinoma) cells more effectively. The intracellular phosphorescence intensity of 51b at pH roughly mimicking the lysosomal environment (5.0) was much stronger than that at pH in the physiological condition (7.4). This result manifested that the selective lysosomal targeting property of 51b originated from its pH-dependent emission. However, 51d showed similar phosphorescence intensity both in acidic and physiological condition, and it can specifically target mitochondria. Notably, complex 51b displayed the best PDT activity against various cell lines with a photocytotoxicity index (PI) ranging from 12.7 to 54.1. From the study of mechanism, it was shown that 51b and 51d induced apoptotic cell death through caspase-and ROS-dependent pathways. The results revealed the potential of these iridium(III) complexes as novel photodynamic anticancer agents.

Zhao and co-workers developed two PSs based on iridium(III) complexes (52a and 52b, Fig 1.4.7), which specifically target the mitochondria and lysosomes. The singlet oxygen quantum yields (ΦΔ) of 52a and 52b were 0.17 and 0.21, respectively, which revealed their potentials as PDT agents. The phototoxic activities of these complexes under normoxia (normal oxygen condition) and hypoxia (low oxygen condition) were evaluated. The results indicated that mitochondria-targeting complex 52a had a better PDT activity than lysosome-targeted complex 52b, especially under hypoxia condition, which was an advantage for PDT applications in hypoxic solid tumors. One explanation was that the complex 52a displayed a more efficient inhibition of mitochondrial respiration leading to a higher mitochondria oxygen concentration. 85 To study the detailed mechanism of protein inactivation triggered by ROS, Kwon and co-workers reported four Ir(III) complexes (53a-53d, Fig 1.4.7). [START_REF] Nam | Endoplasmic Reticulum-localized iridium(III) complexes as efficient photodynamic therapy agents via protein modifications[END_REF] From MTT tests, it was shown that the complexes 53c and 53d had potentials to be PDT agents at low concentration upon low-energy light irradiation (1 J cm -2 ), due to their relatively high ROS generation ability. In addition, 53c could induce cell death upon TP irradiation (860 nm). Furthermore, two kinds of protein modifications (protein-protein cross-linking and protein oxidation) were verified by mass spectrometry (MS). In living cells, these modifications induced by these Ir(III) complexes were found at both endoplasmic reticulum and mitochondria, leading to an obvious effect for cancer cell death.

To avoid the problem of aggregation-induced fluorescence quenching and reduced photocytotoxicity, the group of Chao designed a series of iridium(III) complexes (54a-54c, Fig 1.4.8) which had aggregation-induced emission (AIE) properties. As an AIE-active agent, the phosphorescence quantum yields (ΦPL) of complex 54a increased from 0.001 in DMSO to 0.044 in 90% water-DMSO. All these complexes exhibited more efficient ROS generation in a 90% water-DMSO mixture. The cellular uptake and intracellular distribution experiments revealed that iridium complexes selectively accumulated in mitochondria. Furthermore, all the complexes showed low toxicity towards HeLa and LO2 cell lines, whereas after OP-PDT, the cytotoxicity increased drastically. Interestingly, all these complexes were less toxic towards noncancerous LO2 cell lines. Notably, complex 54a was found to be the most effective against HeLa cells with an IC50 value of 0.4 μM and a PI value of 75.

Additionally, complex 54a, which exhibited the highest TP absorbing cross-section (214 GM in 90% DMSO-water), the highest ROS generation ability and the most impressive lethality towards 3D multicellular tumor spheroids (MCTS), was a promising candidate for TP-PDT. 87 In the same group, they reported a series of novel cyclometalated iridium(III) complexes acting as co-drugs (55a-55d, Fig 1.4.8) with dichloroacetate (DCA) to obtain enhanced PDT anticancer activities. The 1 O2 quantum yield values of these Ir(III) complexes were ranging from 0.53 to 0.74, comparable to that of H2TPP. The confocal imaging experiments displayed that all these complexes could accumulate in mitochondria to a high degree. Complexes 55a-55c including DCA moiety induced obvious decrease of mitochondrial membrane potential (MMP) as a result of efficient cellular uptake and successful release of DCA, while 55d and DCA caused slight changes. It was shown that obvious enhancement of anticancer activities was found for 55a-55c after OP irradiation, and they manifested lower toxicity against LO2 than other cancerous cells, whereas for DCA monotherapy and 55d without DCA moiety, no discrepancy occurred. Furthermore, after TP irradiation, complexes 55a-55c displayed significant inhibition of MCTSs growth. In particular, 55a was found to be the most potent towards both monolayer cells and hypoxia MCTSs, being a promising candidate for TP-PDT. [START_REF] Liu | Enhanced cancer therapy by the marriage of metabolic alteration and mitochondrial-targeted photodynamic therapy using cyclometalated Ir(III) complexes[END_REF] Weinstein, Bryant and coworkers reported two cationic iridium(III) complexes bearing bisbenzimidazole ligands (56a and 56b, Fig 1.4.9). [START_REF] Mckenzie | Metal Complexes for Two-Photon Photodynamic Therapy: A Cyclometallated Iridium Complex Induces Two-Photon Photosensitization of Cancer Cells under Near-IR Light[END_REF] The emission quantum yields of complexes 56a and 56b were 0.33 and 0.24, respectively, and complex 56a had a remarkable TP cross section of 112 GM at 760 nm. Clonogenic assay in HeLa cells was made to evaluate the cytotoxicity of these complexes. Both complexes displayed high photocytotoxicity, with LC50 (50 % lethal concentration) values of 0.3 and 0.5 μM, respectively. Notably, complex 56a exhibited low dark cytotoxicity, with a phototoxic index (PI) value of over 333, suggesting its potential as a PDT agent. The mechanistic studies revealed that ROS production and apoptosis were the mode of action of cell death. Furthermore, TP excitation at 760 nm in 56a-treated HeLa cells showed high photocytotoxicity with light power of 25 mW (2720 J cm -2 ) whereas no cell death was found without the complex. (liver carcinoma) cells under dark and UV light condition was evaluated, and the results revealed 57b displayed less dark toxicity whereas the obvious decrease of cell viability occurred under UV light irradiation. In addition, from the intracellular distribution experiments, it was shown that complex 57a was located in nuclear region, while the others accumulated in mitochondria. Furthermore, the potential of 57a and 57b as candidates for TP-PDT agents was determined on mice with planted solid tumors (4T1 mammary carcinoma cells). Interestingly, when treated with complex 57a, the tumor growth was significantly inhibited, comparable with the commercial PDT agent Chlorin e6 (Ce6).

A series of biscyclometalated Ir(III) complexes bearing borondipyrromethene (BODIPY)-based ancillary ligands (58a-58c, Fig 1.4.10) was designed by Martinez, Ortiz and co-workers. [START_REF] Palao | Acetylacetonate BODIPY-Biscyclometalated Iridium(III) Complexes: Effective Strategy towards Smarter Fluorescent Photosensitizer Agents[END_REF] Complexes 58a and 58b with BODIPY groups directly linked to acetylacetonate displayed higher molar absorption coefficient, stronger emission quantum yield and more singlet oxygen production (ΦΔ = 0.86 and 0.60, respectively) upon visible light irradiation than 58c (ΦΔ = 0.51, only upon UV light). Additionally, the dark and photocytotoxicity of complexes 58a and 58b were tested in HeLa tumor cells. The results revealed that when irradiated by green light (for 58a) and red light (for 58b), both complexes induced remarkable photocytotoxicity, whereas no dark toxicity was found for them. Notably, 58a exhibited much better photocytotoxicity than 58b, with an IC50 value of 50 nM. system of the polypyridyl ligand on cytotoxicity against various cancer cells. The less lipophilic complexes 59a and 59b with lower cellular uptake displayed higher cytotoxicity than complexes 59c and 59d against A2780 (ovarian carcinoma), HeLa (cervical carcinoma) and MCF-7 (breast carcinoma) cell lines with IC50 values ranging from 0.5 μM to 1.8 μM. Moreover, 59a and 59b were more efficient than cisplatin and showed better selectivity between cancer cells and normal cells (MCF-10A noncancerous breast cells) with SI values of 3.7 and 6.4, respectively.

Mechanistic studies revealed that these iridium complexes involved inhibition of protein translation targeting endoplasmic reticulum (ER) and induced cell death through apoptotic pathway. Furthermore, the phototoxic activities against HeLa cells of all these complexes were evaluated, and the results demonstrated that upon visible light (420 nm) irradiation, the toxicity was significantly enhanced with IC50 values ranging from 0.044 μM to 0.866 μM due to a remarkable increase of intracellular ROS production. 92 2. NHC-gold complexes containing aliphatic amino-side arms

Introduction

In this chapter, the synthesis and the characterization of a family of NHC-gold(I) complexes containing aliphatic amino-side arms and the related anticancer activities will be discussed.

Our group reported the synthesis and the biological investigation of a series of

gold(I) complexes (II-1a to II-1d, Fig 2.1) containing two 1-[2-(diethylamino)ethyl]
imidazolydene ligands. These complexes have been tested for their cytotoxic activities against PC-3 cells. It was shown that the lipophilicity of these complexes was in good agreement with the observed biological activities. [START_REF] Boselli | Synthesis, structures, and selective toxicity to cancer cells of gold(I) complexes involving N-heterocyclic carbene ligands[END_REF] The most active complex II-1d

displayed remarkable cytotoxicity against five cancer cell lines (PC-3 prostate cancer, MCF-7 breast cancer, U87 brain cancer, A549 lung cancer and Hep3B liver cancer) with GI50 values ranging from 400 nM to 1 μM, whereas for HUVECs (normal primary human umbilical vein endothelial cells), it exhibited low activity even at the highest concentration of 5 μM, which suggested a good selectivity of II-1d. The cationic charge and the lipophilic properties of complex II-1d are typical characteristic for delocalized lipophilic cations (DLCs). It was previously reported that DLCs can pass through the lipid bilayer and selectively accumulate in mitochondria of carcinoma cells as the consequence of elevated mitochondrial membrane potential (ΔΨm) in tumor cells. [START_REF] Hickey | Mitochondria-targeted chemotherapeutics: the rational design of gold(I) N-heterocyclic carbene complexes that are selectively toxic to cancer cells and target protein selenols in preference to thiols[END_REF] A huge number of DLCs have shown remarkable anticancer activities, and the selectivity between cancer cells and normal cells can be modulated by changing the log P values of DLCs. In the previous work, we fixed the nitrogen-containing side arm but differ in their non-functionalized groups, namely, methyl, butyl, benzyl and mesityl. Based on previous biological activities of gold complexes, we designed a new series of complexes using benzyl group on one side of the azolium ring and varied the amine on the other side (Fig 2 .2) in order to extend the SAR study.

Synthesis and characterization

The carbene precursors (II-2a to II-2d,

II-2a

Cl II-3a 40

II-2b

Br II-3b 50

II-2c

Cl II-3c 84

II-2d

Cl II-3d 84 From the comparison between the spectra, the success of the reaction can be readily proved (Fig 2 .4). The characteristic signal related to H2 proton of the imidazolium salt was absent in the 1 H NMR spectrum of the complex accompanied by a significant shift of other peaks. These experimental evidences can be observed for all the other compounds. In all case, the integration fits with the number of protons of the analyzed compounds.

Molecular structures

Crystals of compounds II-3a and II-3c have been obtained by slow gas-phase diffusion of diethyl ether into a concentrated solution of the complexes in acetonitrile.

Crystals of compounds II-3b and II-3d suitable for X-ray diffraction analysis were obtained by slow evaporation from a methanol solution of these complexes. The cationic gold(I) bis(NHC) complexes II-3a and II-3b were found to display potent antiproliferative activities against cancer cell lines, with GI50 values ranging from 0.91 to 4.8 μM. The most active complex resulted to be II-3b with GI50 values of around 0.9 μM towards two kinds of ovarian cancer cell lines (A2780 and A2780cis). These results are indicative of a bioactivity of these complexes for cancer cells dependent on the nature of the groups on the NHC ligands. Importantly, this series of cationic gold(I) complexes (II-3a-II-3d) showed much higher cytotoxicity to ovarian carcinoma cell lines than to hepatocellular carcinoma ones. However, it should be mentioned that the commercial drug Sorafenib currently used to treat hepatocarcinoma exhibited a high IC50 value of 6.4 μM against HepG-2 cell line. [START_REF] Rangwala | Differential effects of arsenic trioxide on chemosensitization in human hepatic tumor and stellate cell lines[END_REF] Remarkably, complexes II-3a and II-3b were found to be very effective towards 61 cisplatin-resistant ovarian cell line A2780cis, suggesting that these complexes are able to overcome the resistance to cisplatin with a resistant ratio of around 10. The GI50 values represent the concentrations of the complexes and compounds causing 50% inhibition of cellular growth. Data are expressed as mean ± standard error µM and are resulted from at least three independent experiments.

In this series of cationic gold(I) bis(NHC) complexes, the two complexes II-3c and II-3d containing a morpholine moiety and an aliphatic amine, respectively, were found to be inactive against the cancer cell lines. All the complexes II-3a to II-3d

showed a lower cytotoxic activity towards the healthy MDCK cells in comparison to cancer cells. The IC50 value of complex II-3b against a non-cancerous origin derived cell line MDCK was found to be 5.9 μM. In comparison to the A2780 cell line, II-3b exhibited a 6.5-fold selectivity towards cancerous cells than normal cells.

In order to study SAR, lipophilicity of the gold complexes has been determined by using a shake-flask method. [START_REF] Albert | The Physico-Chemical Basis of Therapy: Selective Toxicity[END_REF] Stock solutions of these four compounds (50 μM) were prepared in the aqueous phase (previously saturated with n-octanol) and aliquots (1 mL) of each of these stock solutions were then added to an equal volume of the n-octanol phase (previously saturated with water). The resultant biphasic solutions were mixed for 1 h and then centrifuged (3000 × g, 5 min) to separate the phases. The concentrations of these complexes in the organic and aqueous phases were then determined using UV absorbance spectroscopy (260 nm). Log P was defined as the logarithm of the ratio of the concentrations of the studied complex in the organic and In conclusion, these data show that complex II-3b is active against a panel of cancer cell lines whereas reduced cytotoxicity is found towards healthy cells, which suggests that this complex is a good candidate as an anticancer agent with good prerequisites for in vivo tests.

3. NHC-gold complexes containing aromatic amino-side arms 3.1. Introduction In this chapter, the synthesis and the characterization of a family of NHC-gold(I) complexes containing amino groups linked to the NHC skeleton by an aromatic spacer and the related anticancer activities will be discussed. Recently, we have demonstrated that some mononuclear lipophilic cationic gold(I) bis(NHC) complexes containing aliphatic or aromatic functionalized arms displayed in vitro potent and selective anticancer activities on several human cancer cell lines. [START_REF] Boselli | Synthesis, structures, and selective toxicity to cancer cells of gold(I) complexes involving N-heterocyclic carbene ligands[END_REF][START_REF] Zhang | Cationic and Neutral N-Heterocyclic Carbene Gold(I) Complexes: Cytotoxicity, NCI-60 Screening, Cellular Uptake, Inhibition of Mammalian Thioredoxin Reductase, and Reactive Oxygen Species Formation[END_REF] In chapter 2, we discussed about one class of our ligands characterized by a

Synthesis and characterization

Firstly, we wanted to use the same synthetic route presented in chapter 2 to obtain the imidazolium salts. In the case of III-2a, we started from 1-benzylimidazole to react with 4-bromo-N,N-dimethylaniline under different conditions (Fig 3 .2). In spite of the high temperature and the addition of catalysts, no formation of the imidazolium salt III-2a could be observed. ranging from 76 to 95%. They were classically characterized by 1 H and 13 C NMR spectroscopy, mass spectrometry and elemental analysis. The most notable features in the 1 H and 13 C NMR spectra of the imidazolium salts are the resonances for imidazolium protons (H2) located at 8. 33-10.36 ppm, and the corresponding imidazolium carbons (C2) in the range of 135. 22-136.09 ppm. In the solid state, III-3b forms dimers with Au-Au distances of 3.45 Å (Au1-Au1) and 3.64 Å (Au2-Au2), respectively. One is in the range for aurophilic interaction (aurophilic interaction can occur when the distance between two gold atoms is less than 3.6 Å, around twice the van der Waals radius of gold atom), [START_REF] Schmidbaur | High-carat gold compounds[END_REF] the other one not.

Also in this case, the anions are located in channels following direction b. [°]: Au1-C1 2.024( 16), Au1-C21 2.018( 14), C1-Au1-C21 171.9( 6), Au1-Au1

3.309 (2).

In

Antiproliferative activities

We firstly evaluated the in vitro cytotoxicity of the cationic gold(I) bis(NHC) In order to study about the SAR, we have determined the lipophilicity of the gold complexes III-3a to III-3d using a shake-flask method. 8b The obtained results were reported in Table 3.1. In our group, we have previously reported that lipophilic mononuclear cationic gold(I) bis(NHC) complexes containing amino groups on NHC scaffold were highly cytotoxic and selective. For example, a cationic gold(I) bis(NHC) complex involving a 1-[2-(diethylamino)ethyl] group on one side and a mesitylene group on the other side of the NHC ligands, with a Log P value of 1.13, showed GI50 values of 400 nM against PC-3 prostate cancer cells and > 5 μM on normal primary human umbilical vein endothelial cells (HUVECs). [START_REF] Boselli | Synthesis, structures, and selective toxicity to cancer cells of gold(I) complexes involving N-heterocyclic carbene ligands[END_REF] The corresponding benzyl containing complex had a Log P of 0.3 and showed a GI50 value of 1.44 μM on PC-3 prostate cancer cells.

By replacing the aliphatic linker of this complex by an aromatic one (complex III-3b),

the Log P value increased to 0.9 and the GI50 value on PC-3 prostate cancer cells decreased to 30 nM. In the case of complex III-3c, the log P is about 1 and the GI50 value on HepG-2 cells is 640 nM, whereas the corresponding aliphatic connected pyrrole system display a log P value of 0.3 and an GI50 value on HepG-2 cells of 3.1 μM. [START_REF] Zhang | Cationic and Neutral N-Heterocyclic Carbene Gold(I) Complexes: Cytotoxicity, NCI-60 Screening, Cellular Uptake, Inhibition of Mammalian Thioredoxin Reductase, and Reactive Oxygen Species Formation[END_REF] In summary, the Log P values of the tested complexes reflect the high lipophilicity of these complexes in good agreement with the obtained antiproliferative activities, whereas too high lipophilicity may also lead to the loss of selectivity. were calculated from dose-response curves obtained by nonlinear regression analysis for each cell line (Table 3.2). The GI50 values of complex III-3c were in the range of 0.12 μM to 0.25 μM for all tested cancer cell lines except for HepG-2 with a GI50 value of 0.64 μM. It should be pointed out that hepatocarcinoma is notoriously refractory to chemotherapy and even sorafenib, a commercial drug to treat hepotocarcinoma, displays a high GI50 value of 6.4 μM on HepG-2 cell line. [START_REF] Rangwala | Differential effects of arsenic trioxide on chemosensitization in human hepatic tumor and stellate cell lines[END_REF] In contrast, the cytotoxicity of complex III-3c towards MC3T3 osteoblasts and NIH3T3 fibroblasts was low with GI50 values of 900 nM and days (for PC-3) and 6 days (for T24) of cultivation.

Concentration of III-3c (M)

Optimization of gold(I) bis(NHC) complexes

After increasing the lipophilicity of cationic gold(I) complexes, better anticancer activities were achieved. However, this series of gold complexes containing aromatic side-arms are mainly toxic towards non-cancerous cells, such as MC3T3 and NIH3T3.

The lack of selectivity between healthy and cancer cells triggers us to do some pharmaco-modulation to optimize the system for gold(I) complexes.

It has been previously reported by our group that gold(I) NHC complexes containing quinoline and thioanisole functionalized groups displayed better cytotoxicity than the complexes bearing a benzyl moiety against a panel of cancer cell lines with GI50 values ranging from 0.11 μM to 0.67 μM. [START_REF] Zhang | Cationic and Neutral N-Heterocyclic Carbene Gold(I) Complexes: Cytotoxicity, NCI-60 Screening, Cellular Uptake, Inhibition of Mammalian Thioredoxin Reductase, and Reactive Oxygen Species Formation[END_REF] Therefore, we replaced In order to investigate the antiproliferative activities of this novel series of gold(I) cationic bis(NHC) complexes III-6a to III-6d, they were screened in vitro against PC-3 human prostate cancer cells and non-cancerous NIH3T3 murine fibroblast cells.

This work has been done in collaboration with Dr. Olivier Cuvillier (IPBS, France), who supervised me to make the biological measurements. Results of the preliminary cytotoxic activities are presented in Table 3.3.

The GI50 values reported express the mean of three independent experiments analyzed 72 h after treatment. Auranofin, a commercial drug for anti-arthritis, was used as a reference. Gold complexes III-6a to III-6c were more cytotoxic against PC-3 than auranofin with GI50 values ranging from 0.53 to 0.71 μM. Interestingly, all the complexes III-6a to III-6c induced less cytotoxicity than auranofin against non-cancerous NIH3T3 cells, which exhibited a higher selectivity between cancer cells and normal cells than auronofin. In order to study the SAR of these gold(I) complexes, lipophilicity of this series of gold complexes has been measured. The obtained results are reported in Table 3.3. All the complexes displayed pronounced cytotoxicity with GI50 values ranging from 0.53 μM to 0.71 μM. However, no linear correlation between cytotoxic activity and lipophilicity could be observed. Compared with the family of gold(I) complexes containing aromatic amino side-arm (III-3a to III-3d), this series of compounds III-6a to III-6b showed less lipophilicity and less cytotoxicity towards healthy cell lines (NIH3T3) although they displayed lower anticancer activities than III-3a to III-3d against the tested cancer cell lines (PC-3). In addition, all these complexes exhibited higher antiproliferative activities towards cancer cells than the complexes bearing aliphatic amino side-arm (II-3a to II-3d).

In summary, lipophilicity plays an important role in cytotoxic activity. Nevertheless, higher lipophilicity may lead to a better anticancer activity, but it can also trigger higher cytotoxicity against healthy cells, resulting in less selectivity. Therefore, the further optimization by pharmaco-modulation will be done to develop some gold complexes with high anticancer activity and low toxicity towards healthy cells.

Mechanistic studies of gold(I) complexes (III-3c and IV-1)

In this chapter, we will discuss about the mechanism of action of Au(I)-NHC complexes, such as cellular uptake, inhibition of isolated TrxR and cellular reducing agents, and reactive oxygen species (ROS) generation.

In chapter 3, the gold complex (III-3c, Fig 4 .1) was found to be the most effective and selective one among all the tested compounds. In addition, the cationic bis(NHC) complex (IV-1, Fig 4 .1), [START_REF] Zhang | Cationic and Neutral N-Heterocyclic Carbene Gold(I) Complexes: Cytotoxicity, NCI-60 Screening, Cellular Uptake, Inhibition of Mammalian Thioredoxin Reductase, and Reactive Oxygen Species Formation[END_REF] which was previously synthesized by Dr. Catherine Hemmert in our group, showed also high cytotoxic potency and selectivity. Therefore, they were chosen for mechanistic studies. Relationship between high cytotoxicity and intracellular accumulation is reported in previous literature. 100 Therefore, we evaluate the cellular uptake of complex III-3c in PC-3 cells by a method based on gold quantification by inductive coupled plasma atomic emission spectroscopy (ICP-AES). The measurements were performed under serum-free cell culture medium in order to avoid protein binding. For this purpose, cells were incubated with a 2 μM solution of complex III-3c for up to 4 h at 37 o C and 5 % CO2, and the gold and protein contents of the cells were determined by ICP-AES and Bradford method, respectively. As shown in For the gold complex IV-1, the same method was used. HepG-2 cells were incubated with a 2 μM solution of complex IV-1 for 1, 2 and 4 h at 37 o C and 5% CO2, and the gold and protein levels of the cells were determined by ICP-AES and the Bradford method, respectively. Complex IV-1 showed a cellular gold level of 0.63 μg/mg protein after 1 h of exposure, which then increased over the total incubation period of 4 h to 3.01 μg/mg protein. This result manifested a fast cellular uptake of complex IV-1, in accordance with cytotoxic activity of the complex.

The in vitro cytotoxicity of complex IV-1 towards HepG-2 cells has been tested, giving a GI50 value of 0.46 μM. In addition, it has been discussed in chapter 3 that complex III-3c induces an anticancer activity on HepG-2 cells with a GI50 value of 0.64 μM. From the Fig 4 .2, it is shown that complex IV-1 induces more cellular uptake than complex III-3c. Therefore, there is a certain correlation between cellular uptake and in vitro cytotoxic activity. Higher cellular uptake may lead to better cytotoxicity.

Inhibition of mammalian TrxR

The ubiquitous selenoenzyme TrxR is considered as one of the common targets for gold complexes, as gold displays a high infinity to selenol groups. [START_REF] Bindoli | Thioredoxin reductase: a target for gold compounds acting as potential anticancer drugs[END_REF]101 TrxR is a NADPH-dependent flavoprotein responsible for cell homeostasis regulation and inhibition of TrxR could lead to apoptosis through a mitochondrial pathway. 8d, 102 However, if gold displays a high affinity towards thiol and selenol groups, the inhibition potency of TrxR is influenced by the N-substituted groups of the NHC ligands and cannot be always correlated to the in vitro cytotoxicity. For example, Ott and co-workers reported a series of 20 structural diverse Au-NHC complexes (general structure: IV-2, Fig 4 .3) with the aim of identifying the relationship between TrxR inhibition, antiproliferative activity and structural features of compounds. [START_REF] Rubbiani | TrxR inhibition and antiproliferative activities of structurally diverse gold N-heterocyclic carbene complexes[END_REF] Most of the tested complexes displayed cytotoxic activity towards HT-29 colon adenocarcinoma cells with GI50 values in the low micromolar range. The most active complex was IV-2a with a GI50 value of 2.8 μM. However, the IC50 value of this complex towards isolated TrxR was just 5.08 μM. Therefore, a direct correlation between the cytotoxicity of complexes and the inhibition of TrxR could not be reached. Recently, a novel series of cationic gold(I) bis(NHC) complexes (IV-3a to IV-3d, Fig 4.6) designed by Ott and co-workers displayed high cytotoxicity against cancer cells, whereas they just showed moderate to low inhibitory activities towards isolated mammalian TrxR enzyme with IC50 values ranging from 16.3 to 127.8 μM. [START_REF] Schmidt | Biscarbene gold(I) complexes: structure-activity-relationships regarding antibacterial effects, cytotoxicity, TrxR inhibition and cellular bioavailability[END_REF] It has been previously reported that neutral mono(NHC) gold(I) complexes were more effective TrxR inhibitors than their cationic bis(NHC) analogues, which can be illustrated in the case of 1,3-diethlbenzimidazol-2-ylidene NHC ligand. [START_REF] Rubbiani | Comparative in vitro evaluation of N-heterocyclic carbene gold(I) complexes of the benzimidazolylidene type[END_REF] The Au(I)

Effects on intracellular ROS levels

Generation of reactive oxygen species (ROS) is a key characteristic of apoptotic cell death. ROS are mostly the products of the physiological mitochondrial cell metabolism and are involved in cellular redox homeostasis. Their formation may perturb the cellular antioxidant defense system. Au(I)-NHC complexes have been reported to generate ROS, which may be due to the inhibition of the TrxR, one of the antioxidant enzymes present in the mammalian cell together with catalase, glutathione peroxidase and superoxide dismutase that carry out the removal of ROS from the cells. [START_REF] Rubbiani | Comparative in vitro evaluation of N-heterocyclic carbene gold(I) complexes of the benzimidazolylidene type[END_REF]104 In order to examine whether ROS played a role in cytotoxicity induced by complex IV-1, we used N-acetylcysteine (NAC), a ROS scavenger, to study its effect on cell viability. For this purpose, HepG-2 cells were pretreated with NAC at different concentrations (2 mM, 5 mM and 10 mM) for 1 h before the gold complex (1 μM) was added. After treatment, the results showed that NAC substantially dose-dependently reduced the ROS accumulation and cytotoxicity of complex IV-1 in HepG-2 cells (Fig 4.7). These data suggested cytotoxicity induced by IV-1 was ROS-dependent, which is in line with other previously reported gold complexes. Moreover, the effect of complex III-3c on the ROS levels in PC-3 cells was determined by the nitroblue tetrazolium chloride (NBT) and 3,3'-diaminobenzidine (DAB) assays. DAB is oxidized by H2O2 in the presence of peroxidases and produces reddish brown precipitate. NBT reacts with O2 -. to form a dark blue insoluble formazan compound. 105 For DAB assays, PC-3 cells were seeded in 24-well plates.

After 24 h of incubation at 37 o C and 5% CO2, the cell culture medium was replaced by fresh medium containing gold complex III-3c (1 μM), and the plates were incubated for different period of time (0 h, 3 h, 6 h and 24 h). 500 μL of 3,3'-diaminobenzidine solution (DAB, 2.5 mM) in potassium phosphate buffer were added to the plates after removal of medium. After 4 h of incubation, ROS generation was quantified by spectrophotometry with a microplate reader at 405 nm absorbance.

For NBT assays, the PC-3 cells were plated in 24-well plates and the tested complex III-3c was added at concentration of 1 μM after 24 h of incubation at 37 o C and 5%

CO2. Then a 0.1% solution of NBT (250 μL per well) in PBS was added to the cells after removal of the medium at different time point (0 h, 3 h, 6 h and 24 h). After another 4 h of incubation, the supernatant NBT solution was discarded and the reduced formazan was dissolved equentially in 360 μL of 2 M KOH followed by 400 μL of DMSO and read at 620 nm using KOH/DMSO as a blank.

As shown in Fig 4 .9, after 3 h of incubation in PC-3 cells, complex III-3c at concentration of 1 μM induced around 30 % increase of O2 -. levels and 15 % elevation of H2O2 production compared with untreated control cells. However, with the increase of time, the ROS production decreased to the same level as the controls perhaps due to cell death. Nevertheless, none of these drugs are ideal for treatment due to their high toxicity, resistance issues, prohibitive prices, long treatment regimen and mode of administration. 110 Until now, there is no available vaccine for humans, however, dogs can be preventively treated by the CaniLeish® (Europe), Leishmune® or Therefore, the need for the discovery and development of more efficient drugs with reduced side effects in their treatment against Leishmania is in high demand for health researchers. Furthermore, cytotoxicity was assessed on the murine J774A.1 macrophages to determine the selectivity of action. The highest selectivity index close to 10 has been observed for the neutral complex V-1b. This complex displays an antipromastigotes activity 38 times and 3 times higher than the one of amphotericin B and pentamidine, respectively, but 5.8 times lower than the one of miltefosine, and an antiamatisgotes activity 4.3 times lower than the one of miltefosine also. Overall, SAR can be brought out for this family of gold complexes. Firstly, the results evidenced the key role of the gold cation in antileishmanial activities against L. infantum promastigotes and intracellular amastigotes. Secondly, the charge of the complex constitutes an important factor for the selectivity: the neutral gold complex V-1b can be considered as the hit molecule in this study. reported from our group.

Synthesis and Characterization

I participated in this study with the synthesis of complexes V-2e and V-2f. The synthesis and characterization of proligands II-2b and II-2c have been mentioned in Chapter 2, Section 2.2. are too long for aurophilic interactions, so these two complexes present isolated units without strong intermolecular interactions.

Antileishmanial activity

The antileishmanial activity of complexes V-2a to V-2i were screened in vitro on both the promastigote and the axenic amastigote stages of L. infantum by determining their inhibitory concentration 50% (IC50), in collaboration with Pr. Alexis Valentin and Dr. Sandra Bougeade Delmas (IRD-Pharma-DEV, Toulouse). These complexes were also evaluated in vitro for their cytotoxicity, measured by cytotoxic concentration 50% (CC50) on J774A.1 macrophages, giving access to selectivity index (SI = CC50/IC50).

The results were summarized in Table 5.1. Except complex V-2e, all gold(I) complexes were active against promastigote and axenic amatisgote forms of L. infantum, with IC50 values ranging from 2.52 to 11.16 μM and from 0.19 to 2.17 μM, respectively. In terms of antipromastigote activity, the NHC-Au(I)-Cl complexes were rather toxic for the most part, with SI values between 0.24 to 1.78, except the complex V-2d with a SI value of 3.09. Complexes V-2a to V-2d and V-2f to V-2h were more efficient than miltefosine (IC50 = 20.26 μM) and show the same level of selectivity as that of pentamidine (SI = 0.40) and miltefosine (SI = 2.30). Moreover, the gold(I)-NHC complexes were more active against the axenic amastigote form of L. infantum when compared to their antipromastigote activity and five of them were selective against axenic parasites with SI values ranging from 9.14 to 40.29. b Amphotericin B, Miltefosine and Pentamidine were used as antileishmanial compounds of reference.

c Doxorubicin was used as positive control of cytotoxicity.

These complexes can be separated into two families. One family of complexes (V-2a to V-2c, V-2g) used in this study contained a quinoline moiety on one side of the azolium ring and several aliphatic or aromatic groups on the other side, namely methyl (V-2g), benzyl (V-2a), mesityl (V-2b) and thioanisole (V-2c). Concerning this series, it should be noted that all these complexes showed strong activities against amatisgotes with IC50 values between 0.68 and 1.17 μM. This means that the antiamastigote activity was not affected by the nature of the second subtituent on the NHC. However, it is the second substituent that affects the toxicity against J774A.1 macrophages. The most lipophilic complex of this series, the mesityl substituted V-2b, gave the lowest CC50 value of 3.69 μM and the lowest selectivity index of 5.4 and the less lipophilic one, the methyl substituted V-2g, shows a high CC50 value of 10.76 μM giving a selectivity index of 15.23, which represents the second best value of all the complexes tested in this study. Complexes V-2a and V-2c showed also very good selectivity indexes of 9.60 and 9.20, respectively. As observed for the complex V-2g, the bipyridylmethyl substituted complex V-2h displayed a low SI value of 1.64, due to its high toxicity against J774A.1 macrophages (CC50 value of 0.66 μM).

Another family of complexes (V-2a, V-2d to V-2f and V-2i) concerned benzyl substituted ligands with complex V-2a being the common point with the quinoline series. From the table 5.1, we can see that both the morpholine containing complex V-2e and pyrrole bearing complex V-2f showed moderate activities against amatisgotes with IC50 values of 11.07 and 2.17 μM and low SI values of 2.93 and 4.29, respectively. Moreover, the thioanisole substituted complex V-2i displayed very similar activity (IC50 = 0.88 μM) and selectivity (SI = 9.14) as obtained for the quinoline/benzyl complex V-2a and the thioanisole/quinoline complex V-2c.

Interestingly, complex V-2d showed the lowest IC50 value (0.19 μM) and the highest SI value (40.29) of all the tested complexes and was the lead-complex of this pharmaco-modulation work.

Another important aspect in the development for antileishmanial drug is capacity to differentiate between promastigote and amastigote stages. It should be noted that the reference drugs Amphotericin B and Pentamidine have SI (Pro/Ama) values around 1,

and Miltefosine shows a high SI value of 31, due to its low antipromastigote activity.

All the tested complexes display high SI (Pro/Ama) values ranging from 4.76 to 13.66, with the best value for the most active complex V-2d.

In conclusion, a new family of five neutral gold(I) complexes based on functionalized NHCs has been synthesized and fully characterized. Together with four previously described ones, they were screened in vitro against both promatigote and axenic amastigote forms of L. infantum. Moreover, their cytotoxicity was evaluated on the murine J774A.1 macrophages in order to determine their selectivity of action.

From this study, 7 of 9 gold(I) NHC complexes displayed good antiamastigote activities with IC50 values ranging from 0.19 to 1.17 μM. In particular, the most active one, bearing simple mesityl and benzyl substituents, represents a promising metallodrug showing selectivity of action against the pathological relevant form of Leishmania.

6. Iridium(III) NHC complexes as theranostic agents with potential for photodynamic therapy (PDT) 6.1. Introduction In this chapter, the synthesis and characterization of eight cyclometalated iridium(III) complexes containing NHC ligands, their photophysical properties and related photodynamic anticancer activities will be discussed.

In the literature, only a limited number of iridium(III) bis(NHC) complexes for an application as photosensitizers (PSs) have been reported. Mao and co-workers Following these interesting results, the same group reported four cyclometalated iridium(III) complexes with bisNHC ligands (VI-2a to V-2d, Fig 6 .1) as mitochondria-targeting photodynamic anticancer agents. 115 In this series, they did not change the bisNHC ligands, whereas they did some pharmaco-modulation by using two different kinds of C^N ligands like phenylquinoline and pyridylbenzothiophene.

These complexes VI-2a to VI-2d displayed higher cytotoxicity than cisplatin against A549 and A549R with IC50 values spanning from 1.2 to 5.6 μM. The antiproliferative activities of these complexes were correlated with the lipophilicity and their cellular uptake abilities. Complex VI-2a entered A549 cells, and it was localized in mitochondria. Mechanistic studies showed that these complexes can induce a series of events associated with cell death containing mitochondrial depolarization, ROS production, activation of caspase and apoptosis. Due to their high singlet oxygen quantum yields ranging from 0.32 to 0.62, the phototoxic activities of these complexes upon 450 nm LED light irradiation were tested. The IC50 values of VI-2a

to V-2d decreased very significantly after irradiation, and the PI values of VI-2a were over 500 towards A549 and A549R cells, VI-2a being a potential visible light PDT agent.

Synthesis and characterization

The objective of the project was the preparation of luminescent cyclometalated iridium(III) NHC complexes as theranostic agents with PDT activity. The The bis(imidazolium) salts VI-3a to VI-3g were synthesized by two methods (Fig 6.3). Method 1: firstly, 1,1'-methylene bis(1H-imidazole) was obtained using modified protocols of Tejeda. 116 Then it was reacted with 2.2 equivalents of alkyl halides, such as 2-bromopropane (a),

) piperidine hydrochloride (d) to obtain the carbene precursors with yields ranging from 34% to 71%. In the case of 4-bromothioanisole, we failed to obtain the carbene precursor using the same conditions. Method 2: the substituted imidazoles were achieved by reacting imidazole with various alkyl and aryl halides like 4-bromothioanisole (e), benzyl chloride (f) and 1-bromo-2-methylpropane (g).

Following that, they were reacted with an excess of dibromomethane or dibromoethane to obtain the desired proligands with yields ranging from 44% to 90%. The proligands VI-3a to VI-3g have been characterized by 1 H, 13 C NMR spectroscopy, elementary analysis and mass spectrometry (MS). The most notable feature in the 1 H and 13 C NMR spectra of imidazolium salts are the resonances for imidazolium protons (H2) located between 9.06-10.31 ppm and the corresponding imidazolium carbons (C2) in the range of 136. 97-139.38 ppm. The mass spectra (MS) of the proligands exhibit the classical peaks corresponding to [M-X -] + cations. However, we failed to get some bis(imidazolium) salts (VI-5a to VI-5c, Fig 6.7) and iridium complexes (VI-7a and VI-7b, Fig 6.7). For the proligands VI-5a and VI-5c, almost no reaction occurred even under toluene reflux condition. In the case of VI-5b, 1 H NMR spectra showed some by-products, such as mono and bis-substituted imidazolium salts. In addition, we managed to obtain the bis(imidazolium) salts VI-6a

and VI-6b through the previously described method (Fig 6 .3). Nevertheless, we failed to get the two corresponding iridium complexes VI-7a and VI-7b, probably due to the steric hindrance of the two bulky substituents (mesityl and quinoline) directly connected with the NHCs. 12), N6-Ir1-N5 167.82 (10), C14-Ir1-N5 79.47(12), C25-Ir1-C8 97.75(12), N6-Ir1-C8 94.16(12), C14-Ir1-C8 173.25(13), N5-Ir1-C8 94.99(12), C25-Ir1-C1 177.74(13), N6-Ir1-C1 98.53(11), C14-Ir1-C1 92.22(12), N5-Ir1-C1 90.43(11), C8-Ir1-C1 83.91(12). 2), C16-Ir1-N5 80.00 (11), C16-Ir1-C27 84.74(11), N5-Ir1-C27 94.39(12), C16-Ir1-N6 91.27(11), N5-Ir1-N6 169.93(10), C27-Ir1-N6 79.69(12), C16-Ir1-C1 98.77(12), N5-Ir1-C1 92.36(11), C27-Ir1-C1 172.84(12), N6-Ir1-C1 93.95(11), C16-Ir1-C9 175.17 (12), N5-Ir1-C9 96.41(10), C27-Ir1-C9 92.34(12), N6-Ir1-C9 91.99 (10), C1-Ir1-C9 84.56(12). Selected distances [Å] and angles [°]:Ir1-C1 2.113( 8), Ir1-C12 2.086 (10), Ir1-C22 2.055( 8), Ir1-C33 2.066( 9), Ir1-N5 2.055( 8), Ir1-N6 2.056 (7), C22-Ir1-N5 80.2(3) , C22-Ir1-N6 92.4(3), N5-Ir1-N6 171.5(3), C22-Ir1-C33 81.1(3), N5-Ir1-C33 95.4(4), N6-Ir1-C33 79.3(3), C22-Ir1-C12 100.4(3), N5-Ir1-C12 89.1(3), N6-Ir1-C12 96.4(3), C33-Ir1-C12 175.5(4), C22-Ir1-C1 172.8(3), N5-Ir1-C1 95.0(3), N6-Ir1-C1 91.9(3 9), N6-Ir1-N5 172.41(8), C28-Ir1-C17 80.25(9), N6-Ir1-C17 98.14( 9), N5-Ir1-C17 78.80 (10), C28-Ir1-C10 100.25(9), N6-Ir1-C10 90.65(9), N5-Ir1-C10 92.33(9), C17-Ir1-C10 171.12(10), C28-Ir1-C1 163.02(9), N6-Ir1-C1 98.16(9), N5-Ir1-C1 88.43(9), C17-Ir1-C1 83.40(9), C10-Ir1-C1 96.61(9).

All here presented iridium structures show some common features: the iridium atom is coordinated by a bis-NHC ligand and two phenylpyridine ligands and the four carbon atoms and the two nitrogen atoms linked to iridium form a distorted octahedral geometry. It is notable that the carbene carbon atoms are in trans positions to the deprotonated carbon atoms of the ppy ligands and the two nitrogen atoms are in all cases also in trans positions related to the central iridium. The strong absorption bands below 300 nm correspond to spin-allowed 1 π→π* electronic ligand-centered LC transitions. The structureless bands at 300-360 nm were attributed to phenyl-to-pyridine ligand-to-ligand charge transfer (LLCT) and Ir-to-phenylpyridine metal-to-ligand charge transfer (MLCT). The lowest-lying bands in the visible region over 360 nm can be assigned to both singlet and triplet MLCT transitions. 119 No significant difference in the absorption spectra of complexes VI-4a

to VI-4h can be found in CH2Cl2 solution. The photophysical data are summaried in Table 6.1. The molar attenuation coefficient also known as the molar extinction coefficient (Ɛ) is a measurement of how strongly a compound attenuates light at a certain wavelength. From the table 6.1, it was shown that the molar attenuation coefficients of complexes VI-4a to VI-4h at 360 nm were ranging from 6159 to 17552 M -1 cm -1 .

In addition, the relative emission quantum yields (ΦPL) of these complexes at room temperature were measured by using quinine sulfate in 1 N H2SO4 as a reference standard (ΦPL = 0.55). 120 For the C1 system (VI-4a to VI-4e), the quantum yields were in the range of ΦPL = 1.1 -1.6 % in CH2Cl2 solution; and for C2 and C0 system (VI-4f to VI-4h), just 0.2 % (Table 6.1).

The emission lifetime refers to the average time that the compound stays in its excited state before emitting a photon. Table 6.1 showed that the excited-state lifetimes of complexes VI-4a to VI-4e are in the range of 166. 4-190.7 ns.

In vitro cytotoxicity

The in vitro cytotoxicity of the tested complexes VI-4a to VI-4h was evaluated against two cancer cells PC-3 (human prostate), T24 (human bladder) and the non-cancerous NIH3T3 (murine fibroblasts) using colorimetric MTT tests after 48 h of treatment (Table 6.2). The iridium precursor [IrCl(ppy)2]2 and auranofin were used as references. These results are summarized in Table 6.2. All the complexes except VI-4d containing piperidine substituents displayed higher cytotoxicity than auranofin against the two tested cancer cell lines (PC-3 and T24) with GI50 values ranging from 0.25 to 0.95 μM. To study the SAR, the lipophilicity of complexes VI-4a to VI-4h

was determined by a shake-flask method (Table 6.2). 100 Unfortunately, no significant relationship between lipophilicity and anticancer activity was found for our iridium(III) complexes. However, the lowest cytotoxicity of complex VI-4d could be attributed to its lowest log P value. The two most cytotoxic complexes VI-4f and VI-4h with GI50 values ranging from 0.25 to 0.29 μM against PC-3 and T24 are among the most lipophilic complexes. GI50 values correspond to the concentration of complexes causing 50 % inhibition of cell growth. Data were obtained from three independent experiments.

All the iridium(III) complexes were evaluated in vitro for their cytotoxicity towards healthy NIH3T3 cells, giving access to the selectivity index (SI, ratio of GI50 values towards NIH3T3 to that towards PC-3). From the results (Table 6.2), complexes VI-4a to VI-4h showed some selectivities for cancer cells since they were found to be less active against non-cancerous NIH3T3 cells than cancer cells (T24 and PC-3).

Notably, compared with auranofin (SI value around 1), iridium complexes VI-4a to VI-4h exhibited better selectivity with SI values ranging from 4.5 to 17.3. The iridium precursor was less cytotoxic than all these complexes against all the tested cell lines.

Therefore, comparison the anticancer activity of iridium complexes VI-4a to VI-4h

with that of the precursor showed clearly the importance of the presence of NHC ligands. Gold(I) for Antimitochondrial Activity and Iridium(III) for Photodynamic Therapy 126 6.6. In vitro photodynamic activities Iridium complexes with long-lived excited states can be used as effective singlet oxygen ( 1 O2) sensitizers. 121 1 O2 is considered to be the main cytotoxic agent in photodynamic therapy (PDT). 122 Subsequently, the photocytotoxicity of complexes VI-4a to VI-4h was tested against PC-3 and T24 cancer cells. Cells were treated with increasing concentrations of the complexes (from 0.01 μM to 20 μM) for 4 h in the dark. After that, the medium was removed and replaced by fresh culture medium prior to 10 min irradiation at 365 nm light (4 W). Cell viability was determined by MTT assay 44 h after the end of irradiation. The phototoxic index (PI) was calculated as the ratio of GI50 values in the dark to those upon light irradiation. The results showed that GI50 values of complexes VI-4a to VI-4e (C1 system) upon light irradiation were decreased by 1.8-to 5.2-fold as compared with the values obtained in the dark except complex VI-4d (Table 6.3). However, no significant difference in cytotoxicity was found for cancer cells treated with complex VI-4f to VI-4h (C2 and C0 systems), iridium precursor and auranofin in the presence and absence of light. Notably, among all the complexes, VI-4a displayed the most potent photodynamic anticancer activity with phototoxic index of 5.2 and 2.6 for PC-3 and T24, respectively. These preliminary results suggested that these complexes may be used as potent photodynamic anticancer agents. In addition, the effects of different incubation times on the photocytotoxicity of iridium complexes was evaluated. PC-3 and T24 cells were treated with iridium complexes VI-4a and VI-4e for 12 h, and then irradiated at 365 nm light for 10 min.

Cell viability was determined by MTT assays 36 h after the end of irradiation. From Fig 6.16 and Fig 6.17, it was shown that GI50 values of complex VI-4a towards PC-3 and T24 cells upon light irradiation were 0.092 μM and 0.17 μM, respectively, which is in the same level as previous measurements obtained for 4 h of incubation. The same phenomenon was found for complex VI-4e (GI50 ≈ 0.20 μM and 0.29 μM towards PC-3 and T24 cells, respectively), suggesting that the different incubation 128 times had no effects on the photocytotoxicity of our iridium complexes. Furthermore, in vitro toxicity of iridium complex VI-4a against A549 and HepG-2 cells upon 365 nm irradiation was measured. For this purpose, cells were treated with increasing concentrations of the complexes (from 0.01 μM to 1 μM) for 4 h in the dark. After that, the medium was removed and replaced by fresh culture medium prior to 10 min irradiation at 365 nm light (4 W). Cell viability was determined by MTT assay 44 h after the end of irradiation. In the case of A549 cells, the GI50 value in the dark was 0.65 μM, and after light irradiation, the toxicity was increased by four times with a GI50 value of 0.16 μM. Nevertheless, the toxicity in the dark and photocytotoxicity towards HepG-2 cells were decreased significantly with GI50 values in the micromolar range. Moreover, they exhibited better selectivity between cancer cells (PC-3 and T24) and normal cells (NIH3T3) than auranofin. Therefore, the charge on the iridium complexes has no effect on their antiproliferative activity and selevtivity. Notably, complex VI-4a can be rapidly and efficiently taken up into PC-3 cells and specifically localize into mitochondria. Interestingly, complexes VI-4a to VI-4e (C1 system) can act as efficient photosensitizers. The cytotoxicity of VI-4a to VI-4e against PC-3 and T24 was increased substantially upon irradiation at 365 nm. Therefore, our preliminary studies demonstrated that these iridium(III) complexes had high potential to be mitochondria-targeted theranostic and photodynamic anticancer agents.

Conclusions and perspectives

In this work, three series of new N-heterocyclic carbene (NHC) gold(I) complexes containing functionalized nitrogen groups bridged to the NHC by aliphatic or aromatic linkers and one series of original cationic and neutral cyclometalated iridium(III) complexes bearing bisNHC ligands or a monoNHC have been synthesized, fully characterized and studied for their anticancer activities.

The first family consists of four cationic Au I (NHC)2 complexes (II-3a to II-3d)

containing nitrogen groups connected to the NHC by aliphatic side arms. These complexes were investigated for their in vitro antiproliferative activity towards four human cancer cell lines, including ovarian carcinoma (A2780 and its cisplatin-resistant variant A2780cis), hepatocellular carcinoma (HepG-2 and HepAD38) and the non-cancerous Madin-Darby canine kidney epithelial (MDCK) using MTT assays. Two complexes displayed potent anticancer activity and exhibited higher cytotoxicity against ovarian carcinoma than hepatocellular carcinoma. Notably, they were found to be very effective towards A2780cis, when compared to the reference drug cisplatin. Importantly, these complexes showed low cytotoxicity towards the healthy MDCK cells, demonstrating selectivity for cancer cells.

Regarding structure-activity relationship (SAR), the higher lipophilicity found for the complex II-3b correlates with the higher cytotoxic activity and selectivity against cancer cells, which is a common feature for delocalized lipophilic cations (DLCs). 11a

In order to increase the lipophilicity of our complexes, we performed Mechanistic studies on complexes III-3c were conducted. Bioavailability has been evidenced by a fast cellular uptake. Moreover, this complex inhibited effectively TrxR, a common target for gold(I) complexes, and its cytotoxicity towards PC-3, T24 and HepG-2 cells was found to be ROS-dependent.

Overall, the biological results showed that some of our lipophilic cationic gold(I) complexes can be considered as good candidates with good prerequisites for in vivo tests, in order to provide some conclusive evidence and to pursue the development of gold-based anticancer agents.

Besides anticancer activities, we also tested neutral Au I (NHC)Cl complexes V-2a to V-2i for other biomedical applications in parasite disease leishmaniasis. These complexes were screened in vitro against both promastigote and axenic amastigote forms of L. infantum. Moreover, their cytotoxicity was evaluated on the murine J774A.1 macrophages in order to determine their selectivity of action. 7 of 9 gold(I) complexes displayed good antiamastigote activities with IC50 values ranging from 0.19 to 1.17 μM. In particular, the most active complex V-2d, bearing simple mesityl and benzyl substituents, represents a promising metallodrug showing selectivity of action against the pathological relevant form of Leishmania.

Another axe of research was focused on the synthesis and characterization of new Ir III (ppy)2(NHC) complexes as theranostic agents with potential for photodynamic therapy (PDT). These complexes can be separated to three families: -4h). After excitation at 360 nm, the cationic complexes showed similar blue-green emission with a maximum wavelength of around 500 nm, while the neutral complex displayed two emission maxima at 425 nm and 535 nm.

The in vitro cytotoxicity of these complexes was evaluated on the representative To gain further mechanistic insights, we have performed confocal microscopy experiments by treating PC-3 cells with the most active C1-linked complex VI-4a.

The results showed that this complex can be quickly and effectively taken up into PC-3 cells and specifically localize into mitochondria. Moreover, these complexes can act as efficient photosensitizers. For this purpose, some preliminary in vitro experiments in the dark and upon UV light irradiation have been made. The cytotoxicity of these complexes was increased substantially upon 365 nm light irradiation, which suggested their potential as a mitochondria-targeting theranostic anticancer agents.

In the near future, it would be interesting to improve iridium complexes to become activable upon two-photon (TP) irradiation in the near IR for deeper penetration of tissues. For this purpose, conjugation of chromophores to the iridium complexes can help to extend π-conjugated systems to achieve NIR emission. Subsequently, the addition of a vector to the PS that target receptors expressed on specific cancer cells will lead to the better selectivity towards cancer cells with limited side effects to normal cells. The vector could be a variety of biomolecules such as antibodies, proteins and peptide etc.

Concerning our cationic Au I (NHC)2 complexes, some of them displayed very interesting anticancer activities and high selectivity. It would be very interesting to modify these complexes by vector-incorporation to become more selective.

Another possibility is the combination of gold and iridium complexes by designing heterobimetallic complexes. Such theranostic complexes with rich photophysical properties of the iridium unit will help to understand some mechanistic aspects, and the activation with the specific wavelength will enable these organometallic complexes to execute dual modes of action, TrxR deactivation by the gold and direct ROS production by the iridium.

Metal-NHC Complexes for Anti-Cancer Applications: Gold(I) for Antimitochondrial Activity and Iridium(III) for Photodynamic Therapy 136 Experimental section

General information

Unless stated, all reactions were performed under air. All reagents were used as received from commercial suppliers. Reactions involving silver compounds were performed with the exclusion of light. 1 H (300 or 400 MHz) and 13 C NMR spectra ( 75or 100 MHz) were recorded at 298 K on Bruker AV300 or Bruker AV400 spectrometers in CDCl3, CD3CN, CD3OD and DMSO-d6 as solvants. All chemical shifts for 1 H and 13 C are relative to TMS using 1 H (residual) or 13 C chemical shifts of the solvent as a secondary standard. The temperature was set at 298 K. All the 1 H and 13C signals were assigned on the basis of chemical shifts, spin-spin coupling constants, splitting patterns and signal intensities, and by using 1 H-1 H COSY45, 1 H- 13 C HSQC, 1 H- 13 C HMBC, 13 C and 1 H experiments. Gradient-enhanced 1 H COSY45 experiments were realized including 2 scans per increment. 1 H- 13 C correlation spectra using a gradient-enhanced HSQC sequence (delay was optimised for 1 JCH of 145 Hz) were obtained with 2 scans per increment. Gradient-enhanced HMBC experiments were performed allowing 62.5 ms for long-range coupling evolution (8 scans were accumulated). Typically, 1024 t2 data points were collected for 256 t1 increments.

Elemental analyses were carried out by the "Service de Micro-analyse du Laboratoire 1.1. Preparation of imidazolium salts 1.1.1. 1-Benzyl-3-[2-(piperidine-4-yl)ethyl]-1H-imidazol-3-ium chloride hydrochloride (II-2a)

In a pressure flask, 1-(2-chloroethyl)piperidine hydrochloride (290 mg, 1. 58 mmol) and benzylimidazole (300 mg, 1.9 mmol) were dissolved in 10 mL of dry MeCN. The mixture was stirred at 80 °C for 48 h. After cooling to room temperature, the precipitate formed was filtered, washed with Et2O and dried under vacuum to give a white solid (370 mg, 60% yield). Anal. Calcd. For C17H25Cl2N3: C,59.65;H,7.36;N,12.28. Found: C,59.44;H,7.58;N,12.18. 1 H NMR (300 MHz, DMSO-d6): δ 11.47 (s, 1H, HCl), 9.50 (s, 1H, H2), 7.92 (s, 1H, H5), 7.82 (s, 1H, H4), 7. 49-7.39 (m, 5H, benzyl), 5.44 (s, 2H, H6), 4.73 (t, 2H, J= 6 Hz, H7), 3. 62-3.40 (m, 4H, H10), 2. 97-2.87 (m, 2H, H8), 1. 92-1.67 (m, 6H, H11, H12). 13 4.1.6. 1-Benzyl-3-[3-(3-benzyl-1H-imidazol-3-ium-1-yl)propyl]-1H-imidazol-3

-ium dibromide (VI-3f)

A mixture of imidazole (6.201 g, 91 mmol), K2CO3 (15.420 g, 112 mmol) in 60 mL of DMF was strirred for 20 min, then benzyl chloride (12.6 mL, 109 mmol) was slowly added to the solution. The reaction mixture was stirred at 50 °C for 2 h. After cooling to room temperature, water (100 mL) was added. The aqueous layer was extracted twice with EtOAc (100 mL) and the combined organic layers were washed with water and brine and dried over Na2SO4. The solvent was evaporated to give

Measure of lipophilicity

The octanol-water partition coefficients (P) of all the complexes were determined using a shake-flask method. [START_REF] Albert | The Physico-Chemical Basis of Therapy: Selective Toxicity[END_REF] Water (100 mL, distilled after milli-Q purification) and n-octanol (100 mL, Sigma Aldrich, ACS spectrophotometric grade, ≥ 99%) were shaken together for 24 h to allow saturation of both phases. Stock solutions of the compounds (50 µM) were prepared in the aqueous phase. Aliquots (2 mL) of each of these stock solutions were then added to an equal volume of the n-octanol phase. The resultant biphasic solution was mixed for 15 min and then centrifuged (3000 tr/min, 5 min) to separate the phases. The concentrations of the complex in the organic and aqueous phases were then determined using UV absorbance spectroscopy (260 nm).

The blank is the water saturated to n-octanol. Log P was defined as logarithm of the ratio of the concentrations of the complex in the organic and aqueous phases (values reported are the means of at least three separated determinations).

𝐿𝑜𝑔 𝑃 = [𝐼𝑟] (𝑜𝑟𝑔)

[𝐼𝑟] (𝑎𝑞)

Confocal microscopy

The fluorescence of the complexes was evaluated using a Zeiss LSM 710

Cell viability assay

The MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used to determine cell death as originally described by Mosmann [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF] and modified by Cuvillier et al. 127 Briefly, cells were seeded 5000 cells/well in 24-well The cellular metal uptake was determined as described according to a previously described method. [START_REF] Rubbiani | Comparative in vitro evaluation of N-heterocyclic carbene gold(I) complexes of the benzimidazolylidene type[END_REF] Briefly, for the cellular uptake into HepG-2 human liver cancer cells, the cells were cultured until at least 80% confluence in 75 cm 2 flasks. Stock solutions of gold complexes in sterile DMSO were prepared and diluted with cell culture medium to a final concentration of 2 μM (freshly prepared, final DMSO concentration of 0.1 % v/v). The cell culture medium was replaced by the fresh medium containing the gold complexes, and the flasks were incubated at 37 °C and 5%

CO2 for 1, 2 and 4 h. After the desired incubation period, the cellular uptake was stopped by removing the cell culture medium. The cells were washed with PBS (10 mL), and then the pellets were collected after 10 min of trypsinization (0.05% trypsin-EDTA solution (10 mL) and centrifugation (room temperature; 1,500 rpm, 5 min). Cells were resuspended in Mili-Q water (1 mL) and lysed by sonication for 30 s. nm at 1 min intervals for 10 measurements. The increase of TNB concentration over time followed a linear tendency (r 2 ≥ 0.99), and the enzymatic activities were calculated as the slopes (increase in absorbance per second) thereof. Non-interference with the assay components was confirmed by a negative control experiment using an enzyme-free test compound. IC50 values were calculated as the concentration of the compound decreasing the enzymatic activity of the untreated control by 50%.

Cellular activity of reducing agents present in cells

The cellular activity of reducing agents present in the cell was determined according to a previously described method. 128 Cells were counted and seeded at 2x10 5 per well in 6-well plates and incubated for 24 h. Gold complexes were freshly dissolved as stock solution in sterile DMSO, and the stock solution was diluted with cell culture medium to graded concentrations (final DMSO concentration of 1 % v/v).

The cell culture medium was replaced by fresh medium containing gold complexes, and the plates were incubated for 1 h at 37 °C and 5% CO2. Afterwards, the cells were washed with PBS for three times, and 100 μL of cold lysis buffer (50 mM phosphate buffer, pH 7.4, 1 mM EDTA, 0.1 % Triton-X 100) were added to the plates. Cell lysis was carried out on ice for 5 min and the cell lysates were collected and stored at -80 °C. The protein contents of lysates were determined by the Bradford method.

Then cell lysates (10 μg) were added to 100 μL of reaction buffer (50 mM phosphate buffer, pH 7.4, 1 mM EDTA and 0.2 mM NADPH). Finally, 5,5 '-dithiobis (2-nitrobenzoic acid) (DTNB, final concentration of 3 mM) was added to initiate the reaction and the cellular activity of reducing agents was determined as increases in O.D. 410 nM in 10 min. All the results were calculated from data obtained in three independent experiments.

Measurement of intracellular reactive oxygen species (ROS)

For N-acetyl-cysteine (NAC) and reduced glutathione (GSH) treatment, the cells were pre-treated with different concentrations of NAC and GSH (2, 5 and 10 mM) for 1 h, then gold complexes (1 μM) were added for an additional 24 h. After incubation, cells were incubated at 37 °C and 5% CO2 with 25 μL MTT solution (5 mg/mL; Sigma-Aldrich) in 24-well plates for approximately 3 h. The cytotoxicity was determined as described above.

The cellular ROS generation was shown by the increase of fluorescence intensity of DCF according to a previously reported protocol. 129 Briefly, PC-3, T24 and HepG-2 cells were seeded at a density of 50,000 cells/well in 24-well v/v). After 24 h of incubation at 37 o C and 5% CO2, the cell culture medium was replaced by fresh medium containing gold complexes, and the plates were incubated for different period of time (0 h, 3 h, 6 h and 24 h). 250 μL of nitroblue tetrazolium solution (NBT, 1 mg/mL) in PBS were added to the plates after removal of medium.

After 4 h of incubation, the supernatant NBT solution was discarded. Then the reduced formazan was dissolved by 360 μL of 2 M KOH followed by 400 μL of DMSO and quantified by spectrophotometry with a microplate reader at 620 nm absorbance using KOH/DMSO as a blank. All the results were calculated from data obtained in three independent experiments. 5.9. Measurement of intracellular hydrogen peroxide (H 2 O 2 )

Cells were counted and seeded at 50,000 per well in 24-well plates. The compounds were freshly dissolved as stock solution in sterile DMSO, and the stock solution was diluted with cell culture medium to graded concentrations. (final DMSO concentration of 0.5 % v/v). After 24 h of incubation at 37 o C and 5% CO2, the cell culture medium was replaced by fresh medium containing gold complexes, and the plates were incubated for different period of time (0 h, 3 h, 6 h and 24 h). 500 μL of 3,3'-diaminobenzidine solution (DAB, 2.5 mM) in potassium phosphate buffer were added to the plates after removal of medium. After 4 h of incubation, ROS generation was quantified by spectrophotometry with a microplate reader at 405 nm absorbance.

All the results were calculated from data obtained in three independent experiments.

Antileishmanial evaluation

Leishmania species used in this study were Leishmania infantum MHOM/MA/67/ITMAP-263 (CNR Leishmania, Montpellier, France) expressing luciferase activity.

Antileishmanial activity on promatisgotes

The effects of the tested compounds on the growth of L. infantum promastigotes were assessed by Luciferase Assay like previously described. 113a Briefly, promastigotes in log-phase in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM l-glutamine and antibiotics (100U/mL penicillin, 100 μg/mL streptomycin and 50 μg/mL geneticin), were incubated at a density of 10 6 parasites/mL in sterile 96-well plates with various concentrations of compounds dissolved in DMSO (final concentration less than 0.5% v/v), in duplicate. Appropriate controls treated by DMSO, amphotericin B, miltefosine and pentamidine (reference drugs purchased from Sigma Aldrich) were added to each set of experiments. After a 72 h of incubation period at 24 °C, each plate-well was then microscope-examined for detecting possible precipitate formation. To estimate the luciferase activity of promastigotes, 80 μl of each well are transferred in white 96-well plates, Steady Glow reagent (Promega) was added according to manufacturer's instructions, and plates were incubated for 2 min. The luminescence was measured in Microbeta Luminescence Counter (PerkinElmer).

Inhibitory concentration 50% (IC50) was defined as the concentration of drug required to inhibit by 50% the metabolic activity of L. infantum promastigotes compared to the control. IC50 were calculated by non-linear regression analysis processed on dose-response curves, using TableCurve 2D V5 software. IC50 values represent the mean value calculated from three independent experiments.

Antileishmanial activity on axenic amastigotes

Here, we described a new method to generate axenic amastigotes. For this, L. Infantum promastigotes in logarithmic phase were centrifuged at 900 g for 10 min, the supernatant was removed carefully and was replaced by the same volume of RPMI 1640 complete medium at pH 5.4 and incubated for 24 h at 24 °C. The acidified promastigotes were incubated 24 h at 37 °C in a ventilated flask. Then promastigotes were altered to amastigotes form.

The effects of the tested compounds on the growth of L. infantum axenic amastigotes were assessed in the following way. L. Infantum amastigotes were incubated at a density of 1.10 6 parasites/mL in sterile 96-well

[

  Ré sumé ] Chen ZHANG, thè se PhD Les é tudes mécanistiques sur deux complexes d'or(I) ont été réalisées. Les mesures d'absorption cellulaire ont montré une accumulation cellulaire rapide et une bonne biodisponibilité des complexes, en accord avec l'activité antiprolifé rative de ces deux complexes. De plus, les deux complexes inhibent la thioré doxine reductase (TrxR), une cible commune pour les complexes d'or(I). La mort cellulaire induite par ces deux complexes est dépendante des espèces réactives de l'oxygène. En plus des activité s anticancé reuses, nous avons é galement testé des complexes d'or(I) mono-NHC pour une autre application biomé dicale, la leishmaniose, maladie parsitaire. Ils ont é té testé s in vitro sur les formes promastigotes et amastigotes axé niques de L. infantum. De plus, leur cytotoxicité a é té é valué e sur les macrophages murins J774A.1 afin de dé terminer leur sé lectivité d'action. Un autre sujet de cette thèse concerne les complexes d'iridium(III)-NHC. Trois familles de complexes ont é té pré paré es et caracté risé es. La cytotoxicité in vitro de tous les complexes sur les cellules cancé reuses de la prostate PC-3 et de la vessie T24, et les cellules non cancé reuses NIH3T3 a é té é valué e. De plus, tous les complexes sont des agents thé ranostiques, et les expé riences de microscopie confocale d'un complexe ont montré qu'il pouvait ê tre rapidement et efficacement absorbé dans les cellules PC-3 et se localiser spé cifiquement dans les mitochondries. De maniè re inté ressante, ces complexes peuvent agir comme des photosensibilisateurs efficaces. La cytotoxicité de ces complexes a é té augmenté e substantiellement aprè s une irradiation lumineuse de 365 nm, ce qui suggè re le potentiel é levé de ces agents anticancé reux thé ranostiques ciblant les mitochondries pour la thé rapie photodynamique.5.11. Cytotoxicity evaluation ................................................................................... 5.12. Photodynamic activity .................................................................................... Bibliography ..........................................................................................................................

1

 1

Fig 1 . 1 . 1

 111 Fig 1.1.1 Essential elements.

6 Fig 1 . 1 . 3

 6113 Fig 1.1.3 Mechanism of cisplatin.

10 Fig 1 . 2 . 1

 10121 Fig 1.2.1 Scheme of different types of NHC ligands.

Fig 1 . 2

 12 Fig 1.2.2 NHC-Ag(I) silver complexes as antimicrobial (1a and 1b) and anticancer (2a-2e) agents reported by Youngs and co-workers.

Fig 1 . 2 . 3

 123 Fig 1.2.3 Chemical structure of NAMI-A and KP1019.

Fig 1 . 2 . 4

 124 Fig 1.2.4 Structure of NHC-Ru complexes as anticancer agents.

25 Fig 1 . 3 . 1

 25131 Fig 1.2.6 Structure of NHC-Cu complexes as anticancer agents.

Fig 1 . 3 . 2

 132 Fig 1.3.2 Model depicting the mechanism of action of cell death induced by gold complexes as proposed by Bindoli and co-workers. 26

Fig 1 . 3 . 3

 133 Fig 1.3.3 Mechanism for MOMP during apoptosis. 27

  Recent researches in mitochondria result in the elevated interest in the development of delocalized lipophilic cations (DLCs). They can readily pass through the lipid bilayer and selectively accumulate in mitochondria of carcinoma cells as the consequence of elevated ΔΨm in tumor cells. A huge variety of DLCs have shown pronounced anti-tumor activities, and the selectivity of cancer cells over normal cells can be modulated by changing the lipophilicity of DLCs. 33

Fig 1 . 3 . 4

 134 Fig 1.3.4 Number of articles (left) and citations (right) per year on the topic of "gold + anticancer + NHC or carbene". Data were obtained from Web of Science.

Fig 1 . 3 . 5

 135 Fig 1.3.5 Au(III)-NHC complexes reported by Che and co-workers.

Fig 1 . 3 . 8

 138 Fig 1.3.8 Dinuclear gold(I)-NHC complexes reported byBerners-Price and co-workers (16a-16g).

3 .

 3 Mononuclear gold(I) mono-NHC complexes as anticancer agents After the development of auranofin (Fig 1.3.10) as an antirheumatic drug and the discovery of its antiproliferative activity, gold(I) complexes start to play a vital role in metal-based anticancer drugs. Ott and co-workers previously reported a series of gold(I) NHC complexes (20a-20c, Fig 1.3.10) with 1,3-diethybenzimidazol-2-ylidene NHC ligands. These complexes of type NHC-Au-L (L= Cl, NHC, PPh3) were evaluated as potent TrxR inhibitors with remarkable antiproliferative activities in vitro against a wide range of tumor cells. Of the entire series, complex 20a with chloride substituent showed a strong and selective inhibition of TrxR, similar to auranofin.

44 Fig 1 . 3 . 23 )

 441323 Fig 1.3.10 Gold(I) mono-NHC complexes reported by Ott and co-workers(20, 22, 23) and reported by Wölfl and co-workers(21).

52

 52

1. 3 . 4 .

 34 Bis-NHC gold(I) complexes as anticancer agents The first discovery of mitochondria targeting property of bis-NHC gold(I) complexes was made by Berners-Price and co-workers. A series of Au(I) bis-NHC complexes functionalized with different alkyl substituents (31a-31d, Fig 1.3.14) was investigated about their cytotoxicity in vitro against two tumorigenic breast cell lines (MDA-MB-231, MDA-MB-468) and normal human mammary epithelial cells

Fig 1 . 3 .

 13 Fig 1.3.20 Polynuclear NHC complexes reported by Berners-Price and co-workers (43-cis and 43-trans).

Fig 1 . 3 .

 13 Fig 1.3.22 Determination of intercellular localization of compound by confocal microscopy (63 * oil-immersion objective lens). 34a and 34b were excited at 405 nm (blue). MitoTracker green was excited at 488 nm (green).[START_REF] Li | Antitumor properties and mechanisms of mitochondria-targeted Ag(I) and Au(I) complexes containing N-heterocyclic carbenes derived from cyclophanes[END_REF]

Fig 1 . 3 . 66 1. 3 . 6 . 67 Fig 1 . 3 .

 1366366713 Fig 1.3.24 Fluorescence and transmission images of the cells treated with 44b (in red) and labeled with MitoTracker (in green, bottom) or DAPI (cyan, top).[START_REF] Boselli | Synthesis, Structures, and Biological Studies of Heterobimetallic Au(I)-Ru(II) Complexes Involving N-Heterocyclic Carbene-Based Multidentate Ligands[END_REF]

72 Fig 1 . 4 . 1

 72141 Photodynamic therapy (PDT), an emerging light-activated treatment for cancer, has been successfully used for skin cancer for over 100 years.[START_REF] Moan | An outline of the hundred-year history of PDT[END_REF] PDT requires three essential non-toxic components, namely, light, a photosensitizer (PS) and molecular oxygen (Fig1.4.1). Reactive oxygen species (ROS), such as singlet oxygen and free radicals, are generated by the energy transfer between the excited state of PS and the ground state of molecular oxygen, leading to cellular toxicity.[START_REF] Dolmans | Photodynamic therapy for cancer[END_REF]

Fig 1 . 4 . 2

 142 Fig 1.4.2 Photosensitization process illustrated by a modified Jablonski diagram.

Fig 1 . 4 . 3

 143 Fig 1.4.3 Photosensitizers in clinical PDT.

4 . 4 ) 80 Fig 1 . 4 . 4 Fig 1 . 4 . 5 )

 4480144145 Fig 1.4.4 Structure of TLD1433.

Fig 1 . 4 . 5

 145 Fig 1.4.5 Ruthenium(II) complexes as PSs reported by Gasser and co-workers (48a and 48b) and by Chao andco-workers (49a-49d).Since the irradiation wavelength of the one-photon (OP) PSs for PDT was visible light, which had limited tissue penetration, further work was done to develop novel ruthenium two-photon (TP) PSs that were active in the red-to-NIR region. Chao and co-workers developed four ruthenium(II) polypyridyl complexes (49a-49d,Fig 1.4.5), which acted as mitochondria-targeting TP PDT agents. These complexes 49a-49d displayed remarkable singlet oxygen quantum yield (0.74-0.81) and two-photon absorption cross section (σ2) values were ranged from 124 to 198 GM (1 GM =10 -50

  79b

Four 83 Fig 1 . 4 . 6

 83146 Fig 1.4.6 Iridium(III) complexes as PSs reported by Mao and co-workers (50a-50d, 51a-51d).

Fig 1 . 4 . 7

 147 Fig 1.4.7 Iridium(III) complexes as PSs reported by Zhao and co-workers (52a and 52b) and by Kwon and co-workers (53a-53d).

Fig 1 . 4 . 8

 148 Fig 1.4.8 Iridium(III) complexes as PSs reported by Chao and co-workers (54a-54c, 55a-55d).

Fig 1 . 4 . 9

 149 Fig 1.4.9 Iridium(III) complexes as PSs reported by Weinstein, Bryant and co-workers (56a and 56b) and by Tian et al. (57a-57e).

Fig 1 . 4 .

 14 Fig 1.4.10 Iridium(III) complexes as PSs reported by Martinez, Ortiz and co-workers (58a-58c) and by Ruiz and coworkers (59a-59e).

Fig 2 . 1

 21 Fig 2.1 Bis-NHC Au(I) complexes reported by our group (II-1a to II-1d).

Fig 2 . 2

 22 Fig 2.2 Scheme of the NHC gold(I) complexes used in this study.

  Fig 2.3) were readily obtained after a quaternization step of 1-benzylimidazole with 1-(2-chloroethyl) piperidine hydrochloride, 1-(2-bromoethyl)-1H-pyrrole, 4-(2-chloroethyl)morpholine hydrochloride and 3-chloro-N,N-dimethylpropylamine hydrochloride, respectively, in aceronitrile at 80 o C with moderate yields ranging from 33 to 76%. The most notable features in the 1 H and 13 C NMR spectra of the imidazolium salts are the resonances for imidazolium protons (H2) located at 8.76-9.51 ppm and the corresponding imidazolium carbons (C2) in the range of 136.89-137.72 ppm. The resonance of the hydrochloride proton is comprised between 11.24 and 11.47 ppm. The high resolution mass spectra of the proligands exhibit the classical peak correponding to 53 [M-Cl --HCl] + for II-2a, II-2c and II-2d, and [M-Br -] + for II-2b.

Fig 2 . 3

 23 Fig 2.3 Synthesis of carbene precusors (II-2a to II-2d) and gold(I) complexes (II-3a to II-3d).

Fig 2 .

 2 Fig 2.4 1 H NMR spectra of II-2a (top) and II-3a (down).

Fig 2 .

 2 Fig 2.5 13 C NMR spectrum of II-3a.

Fig 2 . 6 Fig 2 . 7 Fig 2 . 8

 262728 Fig 2.6 Molecular structure of II-3a in the solid state. The cationic part is depicted on the left side at a 30% level thermal ellipsoid plot. For clarity only one of two independent cations is shown and H-atoms, non-coordinating anions and a disorder of one piperidine group are omitted. The arrangement of the two independent cations is shown on the right side. Selected bond distances [Å] and angles [°]: Au1-C1 2.020(9), Au1-C18 1.995(9),C1-Au1-C18 176.5(4).

Fig 2 . 9

 29 Fig 2.9 Molecular structure of II-3c in the solid state. The cationic part is depicted on the left side at a 50% level thermal ellipsoid plot. For clarity only one of four independent cations is shown, H-atoms, non-coordinating anions and a disorder of one morpholine group are omitted. The arrangement of the cations in the solid state is shown on the right side. Selected bond distances [Å] and angles [°]: Au3-C652.014(7), Au3-C81 2.015(7),C65-Au3-C81 174.8(3).

Fig 2 .

 2 Fig 2.10 Crystal packing of II-3c.

Fig 2 .

 2 Fig 2.11 Molecular structure of II-3d in the solid state. The cationic part is depicted on the left side at a 50% level thermal ellipsoid plot. For clarity H-atoms and non-coordinating anions are omitted. The crystal packing is shown on the right side. Selected bond distances [Å] and angles [°]: Au1-C1 2.012(2), C1-Au1-C1 180.0.

Fig 3 . 1

 31 Fig 3.1 Sheme of the NHC gold(I) complexes used in this study.

  nitrogen containing group connected by aliphatic linkers to the NHC skeleton. By measuring the Log P values of the complexes II-3a to II-3d, we confirmed the direct relationship between cytotoxicity and lipophilicity. Herein, we present the synthesis and antiproliferative screening of an optimized series of complexes(Fig 3.1). Our pharmaco-modulation consisted in the replacement of the aliphatic linker by an aromatic linker in order to increase the lipophilicity.

Fig 3 . 2

 32 Fig 3.2 Attempts to synthesize the proligand III-2a.

Fig 3 . 3

 33 Fig 3.3 Attempts to synthesize the substituted imidazoles III-1c and III-1d.

Fig 3 . 4 Fig 3 . 5

 3435 Fig 3.4 Synthesis of imidazolium salts III-2a to III-2d and gold(I) complexes III-3a to III-3d.

Fig 3 . 6 Fig 3 . 7 Fig 3 . 8

 363738 Fig 3.6 Crystal packing of III-3a with view along b.

Fig 3 . 9 Fig 3 . 10

 39310 Fig 3.9 Crystal packing of III-3b with view along b.

Fig 3 . 11

 311 Fig 3.11 Dimeric arrangement of III-3c in the solid state. Left side shows the view perpendicular to the Au-Au bond and the on the right the view along the Au-Au bond is depicted.

Fig 3 . 13 Fig 3 . 14 Fig 3 . 15

 313314315 Fig 3.13 Cell viability of PC-3 prostate cancer cells treated for 72 h with increasing concentrations of gold complexes III-3a to III-3d, proligands III-2c and III-2d and auranofin.

Fig 3 . 16 Fig 3 .

 3163 Fig 3.16 Cell viability of PC-3, T24, HepG-2, A549, U-2 OS, MCF-7, MC3T3 and NIH3T3 treated for 72 h with increasing concentrations of gold complex III-3c.

1. 3 Fig 3 . 18 Fig 3 .

 33183 Fig 3.18 Cell viability ofPC-3 (left) and T24 (right) cells treated with increasing concentrations of gold complex III-3c for the indicated times.

Figure 3 .

 3 Figure 3.20 Colony formation in PC-3 (top) and T24 (bottom) cells treated with increasing concentrations of complex III-3c and stained with crystal violet after 8

Fig 3 . 21

 321 Fig 3.21 Synthesis of imidazolium salts (III-5a to III-5d) and gold(I) complexes (III-6a to III-6d).

Fig 3 . 22 Fig 3 . 23

 322323 Fig 3.22 Molecule structure of III-6c in the solid state. On the left side the cationic part is depicted as a 50% level thermal ellipsoid plot. On the right side the dimeric arrangement is shown. For clarity H-atoms, non-coordinating anion, acetonitrile and disordered water molecules are omitted. Selected bond distances [Å] and angles [°]: Au1-C1 2.023(2), Au1-C19 2.023(2), C1-Au1-C19 177.20(7),Au1-Au1 3.424.

Fig 3 . 24 Fig 3 . 25 7 Å.

 3243257 Fig 3.24 Molecule structure of III-6d in the solid state. The cationic part is depicted as a 50% level thermal ellipsoid plot. For clarity H-atoms, non-coordinating anion and a disordered methanol molecule are omitted. Selected bond distances [Å] and angles [°]: Au1-C1 2.022(5), Au1-C20 2.018(5), C1-Au1-C20 179.1(2), Au1-Au1 3.715.

Fig 4 . 1

 41 Fig 4.1 Structures of NHC gold(I) complexes III-3c and IV-1 used in this study.

Fig 4 . 2 ,Figure 4 . 2

 4242 Figure 4.2 Gold uptake by PC-3 cells exposed to 2 μM of III-3c (left) and by HepG-2 cells exposed to 2 μM of IV-1 (right).

Figure 4 . 3

 43 Figure 4.3 Structures of gold(I) NHC complexes (IV-2) reported by Ott and co-workers.

Figure 4 . 4 Figure 4 . 5

 4445 Figure 4.4 IC50 values of complex III-3c towards isolated TrxR (left) and reducing agents in PC-3 cells (right).

  bis(NHC) complex IV-4b (IC50 = 4.89 μM) was found to be less active compared with the mono(NHC) complex IV-4a (IC50 = 0.36 μM) towards isolated TrxR. Generally, the neutral complexes are less stable in solution and particularly in physiological medium because of the lability of the halide, which generates a vacant site on the gold and allow a fast coordination with selenocysteine residue of the TrxR. Overall, as Metal-NHC Complexes for Anti-Cancer Applications: Gold(I) for Antimitochondrial Activity and Iridium(III) for Photodynamic Therapy 92 discussed in the introduction section, the TrxR appears to be a common relevant target among others shared for this kind of gold complexes.

Figure 4 . 6

 46 Figure 4.6 Structures of gold(I) NHC complexes (IV-3 and IV-4) reported by Ott and co-workers.

Figure 4 . 7 Figure 4 . 8

 4748 Figure 4.7 Effects of NAC at different concentrations on IV-1-induced cytotoxicity. Data are presented as means ± SEM of three independent experiments. *p<0.05, **p<0.005, ***p<0.001, compared with the cell viability of complex IV-1 alone.

Figure 4 . 9 Figure 4 . 5 .

 4945 Figure 4.9 ROS levels in PC-3 cells as determined by NBT assays (left) and DAB assays (right).

Figure 5 . 1

 51 Figure 5.1 Scheme of the life cycle of Leishmania. 109

Figure 5 . 2

 52 Figure 5.2 Commercial drugs currently used against leishmaniasis.

Fig 5 . 3 Fig 5 . 4

 5354 Fig 5.3 NHC-Au(I) complexes as antileishmanial agents (V-1a to V-1e) reported in our group.

Fig 5 . 6

 56 Fig 5.6 Crystal structure of V-2e depicted at 50% level. Hydrogen atoms have been omitted for clarity. Selected bond lengths [Å] and angles [°]: Au-C1 1.992(7), Au-Cl 2.284(2), C1-Au-Cl 179.4(2).

Figure 5 . 7

 57 Figure 5.7 Crystal structure of V-2f depicted at 50% level. Hydrogen atoms have been omitted for clarity. Selected bond lengths [Å] and angles [°]: Au-C1 1.982(4), Au-Cl 2.309(1), C1-Au-Cl 177.6(2).

  photodynamic anticancer agents.114 All these complexes displayed better cytotoxicity than cisplatin and their precursor [IrCl(ppy)2]2 against various cancer cells with IC50 values ranging from 1.0 to 10.4 μM, and they were less toxic against normal LO2 cells than against cancer cells with IC50 values ranging from 8.0 to 17.3 μM. In addition, by measuring the lipophilicity of these complexes, the direct relationship between log P and antiproliferative activity was obtained. Complexes VI-1a to VI-1c could be taken up into HeLa cells very efficiently, and particularly accumulate in mitochondria. Mechanistic studies demonstrated that complexes VI-1a to VI-1c induced cell death in HeLa cells through apoptotic pathway involving ROS generation, release of cytochrome c and activation of caspase. Furthermore, the photocytotoxicity of all the complexes were tested against HeLa, A549 and cisplatin-resistant A549R cells upon 365 nm light irradiation. Of note, the cytotoxicity of complexes VI-1a to VI-1c against various cancer cells was enhanced significantly upon irradiation with PI values ranging from 9 to 3488, which suggests that these iridium(III) complexes have potential to be utilized as photodynamic therapeutic agents.

Fig 6 . 1

 61 Fig 6.1 Iridium(III)-bisNHC complexes as PSs reported by Mao and co-workers (VI-1a to VI-1c and VI-2a to VI-2d).

Fig 6. 2 showsFig 6 . 2

 262 Fig 6.2 Three families of iridium NHC complexes. R and R' will be defined later.

Fig 6 . 3

 63 Fig 6.3 Two synthetic approaches to obtain proligands VI-3a to VI-3g.

Fig 6 . 4

 64 Fig 6.4 Synthesis of iridium(III) NHC complexes VI-4a to VI-4h.

Fig 6 . 7 Fig 6 . 8

 6768 Fig 6.7 Attempts to synthesize the bis(imidazolium) salts (VI-5a to VI-5c) and the iridium complexes (VI-7a and VI-7b).

Fig 6 . 9

 69 Fig 6.9 Molecule structure of VI-4b in the solid state. Hydrogen atoms, disorder of the side arms and non-coordinating anions are omitted for clarity. Selected distances [Å] and angles [°]:Ir1-C1 2.107(3), Ir1-C9 2.130(3), Ir1-C16 2.049(3), Ir1-C27 2.052(3), Ir1-N5 2.051(2), Ir1-N6 2.058(2), C16-Ir1-N5 80.00(11), C16-Ir1-C27

Fig 6 .

 6 Fig 6.10 Molecule structure of VI-4c in the solid state. Hydrogen atoms,

Fig 6 .Fig 6 .

 66 Fig 6.11 Molecule structure of VI-4f in the solid state. Only one of two cations presented in the asummetric unit is shown. Hydrogen atoms, anions, disordered ether molecules are omitted and ppy ligands are simplified for clarity. Selected distances [Å] and angles [°]: Ir-C1 2.087(10), Ir-C13 2.161(9), Ir-C23 2.049(8), Ir-C34 2.043(9), Ir-N5 2.041(7), Ir-N62.027(7), N6-Ir1-N5 172.7(3), N6-Ir1-C34 80.4(3), N5-Ir1-C34 92.7(3), N6-Ir1-C23 96.9(3), N5-Ir1-C23 79.1(3), C34-Ir1-C23 77.3(3), N6-Ir1-C1 95.9(3), N5-Ir1-C1 90.1(3), C34-Ir1-C1 165.0(3), C23-Ir1-C1 88.8(3), N6-Ir1-C13 88.7(3),N5-Ir1-C13 94.9(3),C34-Ir1-C13 99.0(3),C23-Ir1-C13 172.7(3),C1-Ir1-C13 95.4(3).

Fig 6 . 13

 613 Fig 6.13 Normalized absorption (red) and emission (blue) spectra of complex VI-4a in CH2Cl2 solution.

Fig 6 .

 6 Fig 6.14 Normalized emission spectra of iridium complexes VI-4a to VI-4h in CH2Cl2 solution.

  Phototoxic index (PI) is the ratio of GI50 values in the dark to those upon light irradiation. Cells were treated with the complexes for 4 h and then irradiated at 365 nm light (4 W) for 10 min.

Fig 6 .Fig 6 .

 66 Fig 6.15 Dose-response curves for the in vitro photocytotoxicity of iridium complexes (VI-4a and VI-4e) towards PC-3 cells.

Fig 6 .

 6 Fig 6.17 Dose-response curves for the in vitro photocytotoxicity of iridium complex VI-4a towards A549 cells in the dark (left) and upon irradiation (right).

Fig 6 .

 6 Fig 6.18 Dose-response curves for the in vitro photocytotoxicity of iridium complex VI-4a towards HepG-2 cells in the dark (left) and upon irradiation (right).

  pharmaco-modulation to synthesize a second family of four cationic Au I (NHC)2 complexes (III-3a to III-3d) containing nitrogen groups linked to the NHC by an aromatic spacer. The in vitro cytotoxicity of these lipophilic complexes and their proligands III-2c and III-2d were evaluated on the representative PC-3 prostate and T24 bladder cancer cell lines and non-cancerous MC3T3 osteoblasts by colorimetric MTT assays. Whereas the carbene precusors were inactive, all the complexes exhibited high cytotoxicity with GI50 values in the nanomolar range, but unfortunatedly, less selectivity for cancer cells was found for three of them (III-3a, III-3b and III-3d). These results highlight that high lipophilicity of these complexes is in good agreement with the obtained cytotoxic activity, nevertheless too high lipophilicity can also lead to poor selectivity. Accordingly, complex III-3c was selected for further in vitro screening on six cancer cell lines (prostate PC-3, bladder T24, lung A549, bone U-2 OS, liver HepG-2 and breast MCF-7) and two non-cancerous cell lines (MC3T3 osteoblasts and NIH3T3 fibroblasts). The obtained GI50 values of these complexes were in the nanomolar range, suggesting that this complex was highly cytotoxic towards various cancer cells with restricted effects on healthy cells. Clonogenic assays manifested the impressive cytotoxicity of this complex by inhibiting significantly the number of PC-3 and T24 colonies at low concentration.Taking into account the anticancer activity and selectivity of the two first series, we designed and synthesized an optimized third series of four Au I (NHC)2 complexes (III-6a to III-6d) with an intermediate lipophilicity between the first two. These complexes were tested in vitro for their antiproliferative effects on human prostate cancer cell line PC-3 and non-cancerous NIH3T3 fibroblasts. These four complexes displayed high and specific cytotoxic activity towards cancer cells.

human PC- 3

 3 prostate and T24 bladder cancer cell lines and non-cancerous NIH3T3 fibroblasts by colorimetric MTT assays. The iridium precursor [IrCl(ppy)2]2 was less active than all these complexes against the two tested cancer cells, demonstrating the critical role of NHC ligands. The complexes with Log P values beween -0.06 and 0.86 (VI-4a to VI-4c and VI-4e to VI-4h) gave very good GI50 values ranging from 0.25 to 0.95 μM for the two cancer cell lines, while one complex VI-4d with a Log P value of -0.4 possessed GI50 values of 3.8 and 3.2 μM, respectively. The selectivity of the seven active complexes evaluated by the ratio of GI50 values of NIH3T3 to those of the two cancer cell lines varies between 4.5 and 17.3, suggesting moderate to good selectivity.

deFig 1 . 1

 11 Fig 1.1 General numbering for NMR attribution.

Å - 3 .2

 3 collected at low temperature using oil-coated shock-cooled crystals at 100(2) K on a Bruker-AXS APEX II diffractometer with MoKa radiation (λ = 0.71073 Å). The structures were solved by direct methods 123 and all non hydrogen atoms were refined anisotropically using the least-squares method on F 2 .124 The absolute structure parameters have been refined using the Crystallographic data for II-3a,II-3b, II-3c, II-3d, III-3a, III-3b, III-3c, V-2e, V-2f, VI-4a, VI-4b, VI-4c, VI-4f and VI-4g: II-3a: C34H46.5AuN7O3.5, Mr = 806.25, crystal size = 0.40 x 0.10 x 0.02 mm 3 , triclinic, space group P1, a = 10.684(2) Å, b = 16.145(2) Å, c = 21.292(2) Å, α = 79.396(3)°, β = 77.511(3)°, γ = 77.607(3)°, V = 3465.6(7) Å 3 , Z = 4, 48878 reflections collected, 9811 unique reflections (Rint = 0.0526), R1 = 0.0538, wR2 = 0.1089 [I>2σ(I)], R1 = 0.0674, wR2 = 0.1140 (all data), residual electron density = 2.641 e II-3b: C32H34AuN7O3, Mr = 761.63, crystal size = 0.20 x 0.10 x 0.05 mm 3 , triclinic, space group P1, a = 10.835(1) Å, b = 11.485(1) Å, c = 12.716(1) Å, α = 76.585(4)°, β = 77.134(4)°, γ = 84.412(4)°, V = 1498.7(2) Å 3 , Z = 2, 48878 reflections collected, 9811 unique reflections (Rint = 0.0526), R1 = 0.0491, wR2 = 0.1271 [I>2σ(I)], R1 = 0.0586, wR2 = 0.1338 (all data), residual electron density = 2.345 e Å -3 . II-3c: C32H42AuN7O5, Mr = 801.69, crystal size = 0.30 x 0.30 x 0.20 mm 3 , monoclinic, space group P21, a = 10.984(1) Å, b = 30.847(3) Å, c = 19.590(2) Å, group C2/c, a = 17.575(1) Å, b = 21.290(1) Å, c = 9.851(1) Å, β = 121.108(2)°, V = 3155.7(2) Å 3 , Z = 4, 31004 reflections collected, 4798 unique reflections (Rint = 0.0480), R1 = 0.0173, wR2 = 0.0359 [I>2σ(I)], R1 = 0.0338, wR2 = 0.0402 (all data), residual electron density = 0.692 e Å -3 . 2. Photophysical measurements UV/Vis absorption spectra were recorded at RT on solutions contained in quartz cuvettes (optical pathlength 1 cm, Hellma®) with a PerkinElmer®, UV/VIS/NIR spectrophotometer Lambda 950 and a PerkinElmer® ditector; UV/VIS/NIR Accessory 2D Detector Module. Fluorescence and phosphorescence emission spectra were obtained with a HORIBA JOBIN YVON, fluromax-4 and spectrofluorometer. Luminescence quantum yields were measured according to the method of Demas and Crosby, 126 on solution samples at RT. The quantum yield is defined as: Where Φ 𝑆 is the quantum yield of the sample, Φ 𝑅 is the know quantum yield of the reference standard. At the area subtended by the emission band (on a wavelength scale) and η is the refractive index of the solvent used for the preparation of the solution. As (related to the sample) and Ar (related to the reference) must be excitation wavelength. Different standards were selected depending on the spectral region of the interest: POPOP in hexane (Φ = 0.97) and QUININNE in water (Φ = 0.54). Fluorescence lifetime measurements were performed by an Edinburgh FLS920 spectrofluorimeter equipped with a TCC900 card for data acquisition in time-correlated single-photon counting experiments (0.5 ns time resolution) with a D2 lamp and an LDH-P-C-405 pulsed diode laser. The estimated experimental error is 2 nm on the band maximum. (5% on the lifetime, 10% on the fluorescence quantum yield).

  NLO-Meta Confocal microscope. PC-3 cells were cultured in6-well plates and on glass slides (180,000 cells per well) for 18 h before treated with complexes at different concentrations (0.25 μM, 0.5 μM and 1 μM). After 24 hours, a MitoTracker deep red probe was incubated on live cells at a final concentration of 200 nM during 15 min at 37 o C in a 5 % CO2 humidified incubator and then observed in two channels 440-544 nm for complex VI-4a (two-photon excitation at 720 nm) and 644-700 nm for MitoTracker (excitation at 633 nm) at the same time. PC-3 and lung carcinoma A549 cell lines were obtained from DSMZ (Braunschweig, Germany). Human bladder cancer T24, osteosarcoma U-2 OS, breast cancerMCF-7, hepatocarcinoma HepG-2, mouse osteoblast MC3T3 and fibroblast NIH3T3 cell lines were fromATCC-LGC Standards (Molsheim, France).PC-3, HepG-2 and A549 cells were cultured in RPMI 1640, T24,MCF-7, U-2 OS and NIH3T3 in DMEM, and MC3T3 in MEM containing 10% fetal bovine serum and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streotimycin) at 37 o C in 5% CO2 humidified incubators.

5 . 4 .

 54 plates for various cell lines (PC-3, T24, A549,MCF-7, HepG-2, U-2 OS, MC3T3 and NIH3T3) and allowed to attach overnight. All of the complexes were dissolved in DMSO. The concentration of the complexes was calculated according to the elemental composition of the complexes determined by the elemental analyses. Media in the presence of the tested complexes were added and serially diluted to various concentrations (from 5 μM to 0.01 μM). The maximum concentration of DMSO in media did not exceed 0.5 % (v/v). After 24 h, 48 h or 72 h of treatment, cells were incubated at 37 o C and 5% CO2 with 25 μL MTT solution (5 mg/mL;Sigma-Aldrich) in24-well plates for approximately 3 h. After solubilization with 500 μL of lysis buffer (DMSO), formazan was quantified by spectrophotometry with a microplate reader at 570 nm absorbance. The GI50 values corresponding to the concentration that caused 50% inhibition of cell proliferation were calculated from dose-response curves obtained by nonlinear regression analysis (Prism 5, Graphpad Software). All the results were calculated from data obtained in three independent experiments.5.3. Clonogenic assayPC-3 and T24 cells were counted and seeded (500 cells/well) to 6-well plates. Cells were allowed to attach overnight and then treated with different concentrations of gold complexes (0.01 μM,0.1 μM, 0.25 μM, 0.5 μM, 1 μM, 5 μM). After incubation for an additional 6 days for T24 and 8 days for PC-3, the cells were washed with PBS (2 mL), fixed with methanol (1 mL) for 20 min and stained with 0.5% crystal violet (2 mL) for 30 min. The relative survival was calculated from the number of single cells that formed colonies of more than 50 cells.Metal-NHC Complexes for Anti-Cancer Applications: Gold(I) for Antimitochondrial Activity and Iridium(III) for Photodynamic Therapy 181 Cellular uptake studies

  The protein contents of lysates were determined by the Bradford method. 100 μL of samples were digested in 300 μL of 70 % HNO3 at 70 °C for 2 h and room temperature overnight, then diluted 1:100 in ultrapure water for inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis. Results were calculated from the data obtained from three independent experiments and are expressed as microgram of gold per milligram of protein.5.5. Inhibition of mammalian TrxRTo determine the inhibition of mammalian TrxR, an established microplate reader-based assay was performed with minor modifications. 101b For this propose, commercially available rat liver TrxR (from Sigma-Aldrich) was used and diluted with distilled water to achieve 3.5 U/mL. Gold complexes were freshly dissolved as stock solution in sterile DMSO. 25 μL aliquot of the enzyme and either 25 μL potassium phosphate buffer (pH 7.0) containing gold complexes in graded concentrations or 25 μL buffer without the complex but DMSO (positive control) were added. The resulting solution (final DMSO concentration of 0.5 % v/v) was incubated at 37 °C for 75 min with moderate shaking in a 96-well plate. To each well, 225 μL of the reaction mixture (1.0 mL reaction mixture consists of 500 μL 100 mM potassium phosphate buffer pH 7.0, 80 μL 100 mM EDTA solution pH 7.5, 20 μL 0.2 % BSA, 100 μL of a 20 mM NADPH and 300 μL distilled water) was added and the reaction was initiated immediately by adding 25 μL of 20 mM DTNB solution. After thorough mixing, the formation of TNB was monitored by a microplate reader at 405

  plates for 24 h. Cells were washed with PBS buffer and stained with DCFH-DA (final concentration 20 μM) for 45 min. Then cells were washed with PBS buffer and the culture medium without phenol red containing gold complexes was added to the cells. The fluorescence intensity of DCF (excitation/emission, 485/535 nm) was measured by fluorescence microplate reader at different time points. Data are presented as means ± SEM of three independent experiments. *p<0.05, **p<0.005, ***p<0.001, compared with ROS generation at 0 h. 5.8. Measurement of intracellular superoxide anion (O 2 -. ) To determine reactive oxygen species (ROS) generation, an established microplate-reader-based assay was performed with minor modifications. 130 Cells were counted and seeded at 50,000 per well in 24-well plates. The compounds were freshly dissolved as stock solution in sterile DMSO, and the stock solution was diluted with cell culture medium to graded concentrations. (final DMSO concentration of 0.5 %

  plates with various concentrations of compounds dissolved in DMSO (final concentration less than 0.5% v/v), in duplicate.Appropriate controls treated by DMSO, amphotericin B, miltefosine and pentamidine (reference drugs purchased from Sigma Aldrich) were added to each set of experiments.After a 48 h incubation period at 37 °C, each plate-well was then microscope-examined for detecting possible precipitate formation. To estimate the luciferase activity of promastigotes, 80 μl of each well are transferred in white 96-well plates, Steady Glow reagent (Promega) was added according to manufacturer's instructions, and plates were incubated for 2 min. The luminescence was measured in Microbeta Luminescence Counter (PerkinElmer). Inhibitory concentration 50% (IC50) was defined as the concentration of drug required to inhibit by 50% the metabolic activity of L. infantum amastigotes compared to the control. IC50 were calculated by non-linear regression analysis processed on dose-response curves, using TableCurve 2D V5 software. IC50 values represent the mean value calculated from three independent experiments. fresh culture medium prior to 10 min irradiation at 365 nm (4 W). After 44 hours of incubation, cells were incubated at 37 o C and 5% CO2 with 25 μL MTT solution (5 mg/mL; Sigma-Aldrich) in24-well plates for approximately 3 h. After solubilization with 500 μL of lysis buffer (DMSO), formazan was quantified by spectrophotometry with a microplate reader at 570 nm absorbance. The GI50 values corresponding to the concentration that caused 50% inhibition of cell proliferation were calculated from dose-response curves obtained by nonlinear regression analysis (Prism 5, Graphpad Software). All the results were calculated from data obtained in three independent experiments.131

  Complex 14 displayed remarkable antiproliferative activities against

	MCF-7 (breast carcinoma), PC-3 (prostate carcinoma) and U937 (histiocytic leukemia)
	cells in the nanomolar range with IC50 values of 0.31 nM, 0.34 nM and 190 nM,
	respectively. Moreover, human colorectal normal cells (CCD-18Co) were used to
	determine the cytotoxic selectivity of the compound towards cancer cells and its
	safety towards normal cells. Selectivity index (SI) results revealed that complex 14
	exhibited high selectivity towards all tested cancer cells with SI values of 57096,
	52095 and 93 for MCF-7, PC-3 and U937, respectively.

Fig 1.3.7 Au(III) complexes reported by

Table 2

 2

	.1 Cytotoxic activity of gold(I) NHC complexes expressed as GI50 (μM) values
	towards selected cancer and normal cell lines as determined by MTT assay (72 h).
	complex A2780cis	A2780	HepG-2	HepAD38 MDCK	Log P
	II-3c	44±11	28±9	>50	>50	>50	-0.46±0.02
	II-3d	20±3	11±2	>50	>50	>50	-0.23±0.06
	II-3a	2.8±0.6	2.1±0.4	4.8±1.5	4.5±1.7	10±3	-0.01±0.01
	II-3b	0.97±0.11 0.91±0.23	3.1±1.1	1.1±0.2	5.9±1.5 0.29±.0.02
	cisplatin	34±2.40	3.2±0.61 4.31±1.10	12±1.3	27±3.8

Table 3 .

 3 1 Lipophilicity for complexes III-3a to III-3d expressed as Log P.

	Complex	Log P
	III-3a	1.256±0.047
	III-3b	0.926±0.030
	III-3c	1.033±0.060
	III-3d	0.501±0.024

Table 3

 3

		.2 GI50 values of complex III-3c after 72h.
		GI50 values of complex III-3c after 72h
	Origin	Cell line	GI50
	Prostate cancer	PC-3	0.25 μM
	Bladder cancer	T24	0.24 μM
	Liver cancer	HepG-2	0.64 μM
	Lung cancer	A549	0.23 μM
	Osteosarcoma	U-2 OS	0.24 μM
	Breast cancer	MCF-7	0.12 μM
	Osteoblast	MC3T3	0.90 μM
	Fibroblast	NIH3T3	1.30 μM

Table 3

 3

	.3 GI50 values of gold (I) complexes III-6a to III-6d and auronofin against
		PC-3 and NIH3T3 after 72h.
	Complex	PC-3 (μM)	NIH3T3 (μM)	Log P
	III-6d	0.55	1.97	0.168±0.011
	III-6c	0.70	3.00	0.479±0.014
	III-6a	0.71	2.70	0.549±0.009
	III-6b	0.53	1.85	0.843±0.060
	Auranofin	1.05	1.80

Table 5 . 1

 51 In vitro antileishmanial activity and cytotoxicity of complexes V-2a to V-2i.

	Compound	L. infantum	L. infantum	J774A.1	Selectivity	Selectivity	Selectivity
		promastigotes	axenic	CC50 (µM) a	index Pro	index Ama	index
		IC50 (µM) a	amastigotes		CC50/IC50 (Pro)	CC50/IC50	Pro/Ama
			IC50 (µM) a			(Ama)	IC50 (Pro)/IC50
							(Ama)
	V-2a	11.16±0.15	1.17±0.21	11.20±2.07	1.00	9.60	9.54
	V-2b	4.68±0.61	0.68±0.02	3.69±0.73	0.79	5.40	6.88
	V-2c	8.27±0.15	0.73±0.09	6.72±0.17	0.81	9.20	11.33
	V-2d	2.52±0.51	0.19±0.04	7.79±1.14	3.09	40.29	13.26
	V-2e	57.03±0.46	11.07±1.04	32.39±1.22	0.57	2.93	5.15
	V-2f	10.32±1.29	2.17±0.14	9.30±076	0.90	4.29	4.76
	V-2g	6.05±0.17	0.71±0.05	10.76±2.23	1.78	15.23	8.52
	V-2h	2.78±0.28	0.40±0.13	0.66±0.08	0.24	1.64	6.95
	V-2i	6.76±0.72	0.88±0.30	8.04±1.14	1.19	9.14	7.68
	AmphotericinB b	0.04±0.01	0.05±0.01	1.31±0.09	34.47	24.11	0.80
	Pentamidine b	1.64±0.03	1.50±0.21	0.65±0.17	0.40	0.43	1.09
	Miltefosine b	20.46±0.72	0.66±0.03	47.13±1.79	2.30	71.78	31.00
	Doxorubicin c			0.047±0.001

a Mean of three independent experiments.

Table 6 .1 Photophysical data of iridium complexes VI-4a to VI-4h. Complex Medium ε (M -1 cm -1 ) ΦPL(%) τ1/ns

 6

	VI-4a	CH2Cl2	17552	1.2	190.7
	VI-4b	CH2Cl2	8937	1.6	166.4
	VI-4c	CH2Cl2	6159	1.6	179.7
	VI-4d	CH2Cl2	8174	1.6	187.0
	VI-4e	CH2Cl2	12474	1.1	167.4
	VI-4f	CH2Cl2	6799	0.2	___
	VI-4g	CH2Cl2	7381	0.2	___
	VI-4h	CH2Cl2	10544	0.2	___

Table 6 .

 6 2 Cytotoxic activity of iridium(III) NHC complexes expressed as GI50 (μM) values towards selected cancer and normal cell lines as determined by MTT assay (48 h).

	Complex	PC-3	T24	NIH3T3	Log P
	VI-4a	0.48	0.31	3.3	-0.063±0.032
	VI-4b	0.51	0.38	3.5	0.842±0.222
	VI-4c	0.41	0.43	7.1	0.581±0.047
	VI-4d	3.8	3.2	> 20	-0.407±0.012
	VI-4e	0.71	0.95	8.5	0.035±0.080
	VI-4f	0.25	0.29	2.5	0.847±0.116
	VI-4g	0.91	0.91	4.1	0.519±0.061
	VI-4h	0.27	0.25	2.7	0.861±0.009
	[IrCl(ppy)2]2	> 20	14.8	>20
	Auranofin	1.2	1.0	1.3

Table 6 .

 6 3 GI50 values (μM) and phototoxic indexes of the tested complexes (VI-4a to VI-4h) towards different cell lines.

	Metal-NHC Complexes for Anti-Cancer Applications:
	Gold(I) for Antimitochondrial Activity and Iridium(III) for Photodynamic Therapy

  Gold(I) for Antimitochondrial Activity and Iridium(III) for Photodynamic Therapy 146 H2), 7.82 (s, 1H, H5), 7.76-7.71 (m, 4H, H8/H9), 7.45 (t, 2H, J = 2.4 Hz, H11), 7.16 (s, 1H, H4), 6.30 (t, 2H, J = 2.4 Hz, H12). A mixture ofIII-1c (200 mg, 0.96 mmol) and benzyl chloride (133 mg, 1.05 mmol) in MeCN was stirred in a closed pressure flask at 80 °C for 10 h. After cooling down to room temperature, the solution was After cooling to room temperature, distilled water was added to the reaction and the crude was extracted with EtOAc. The organic layer was washed with brine and dried over Na2SO4. After filtration and evaporation of the solvent under reduced pressure, the residue was purified by column chromatography on silica gel with a mixture of CH2Cl2 and MeOH (30:1) as eluent to give Under nitrogen and in the dark, a Schlenk tube was charged with II-2c (100 mg, 0.29 mmol) in 6 mL ofMeCN and Ag2O (67.3 mg, 0.29 mmol). The reaction mixture was stirred at 40 °C for 24 h. After that, Au(SMe2)Cl (102.5 mg, 0.348 mmol) was added and the mixture was stirred for 4 h. The solution was filtered through a pad of Imidazole(3.24 g, 47.6 mmol),4-bromothioanisole (4.83 g, 23.8 mmol), K2CO3(3.29 g, 23.8 mmol) and a catalytic amount of CuSO4 were stirred in a closed pressure flask at 205 °C for 10 h. After cooling to room temperature, the crude product was extracted by CH2Cl2, filtered and evaporated to dryness. The resulting residue was

	Metal-NHC Complexes for Anti-Cancer Applications:
	Gold(I) for Antimitochondrial Activity and Iridium(III) for Photodynamic Therapy Gold(I) for Antimitochondrial Activity and Iridium(III) for Photodynamic Therapy Gold(I) for Antimitochondrial Activity and Iridium(III) for Photodynamic Therapy
	1.1.2. 1-Benzyl-3-[2-(1H-pyrrol-1-yl)ethyl]-1H-imidazol-3-ium bromide (II-2b) 6.87; N, 12.06. 1 H NMR (400 MHz, CD3OD): δ 9.25 (s, 1H, H2), 7.81 (s, 1H, H5), 7.71 (s, 1H, H4), 7.53-7.45 (m, 5H, benzyl), 5.46 (s, 2H, H6), 4.79 (t, 2H, J= 6.8 Hz, CH2Cl2 and MeOH (30:1) as eluent to give N,N-diethyl-4-(1H-imidazol-1-yl)aniline bromide (III-5a) (III-1b) as a brown solid (1.43 g, 90% yield). 1 H NMR (400 MHz, CDCl3): δ 7.73 (s, 2.1.5. 3-(2-(1H-Pyrrol-1-yl)ethyl)-1-(4-(methylthio)phenyl)-1H-imidazol-3-ium 3. Neutral gold(I) mono(NHC) complexes 3.2. Complex V-2f 4. Iridium(III) NHC complexes
	H7), 4.01 (s, 4H, H11), 3.76 (t, 2H, J= 6.8 Hz, H8), 3.50 (s, 4H, H10). 13 C NMR (100 1H, H2), 7.21 (d, 2H, J = 9.2 Hz, H8), 7.18-7.17 (m, 2H, H4/H5), 6.72 (d, 2H, J = 9.2
	MHz, CD3OD): δ 137.1 (1C, C2), 133.3 (1C, benzyl), 129.1 (2C, benzyl), 129.0 (1C, Hz, H9), 3.40 (q, 4H, J = 7.2 Hz, H11), 1.21 (t, 6H, J = 7.2 Hz, H12). A mixture of
	benzyl), 128.7 (2C, benzyl), 123.0 (1C, C5), 122.7 (1C, C4), 63.3 (2C, C11), 55.0 (1C, III-1b (300 mg, 1.39 mmol) and benzyl chloride (194 mg, 1.53 mmol) in MeCN was evaporated to dryness. The residue was purified by column chromatography on silica 4-[4-(1H-imidazol-1-yl)phenyl]morpholine (III-1d) as a yellow solid (200 mg, 30% washed three times with Et2O (20 mL) to give 1-(4-methylthiophenyl)-1H-imidazole
	C6), 53.1 (1C, C7), 52.2 (2C, C10), 42.9 (1C, C8). HRMS (ES+): calcd. for stirred in a closed pressure flask at 80 °C for 10 h. After cooling down to room gel with a mixture of CH2Cl2 and MeOH (10:1) as eluent to give III-2c as a yellow yield). 1 H NMR (400 MHz, DMSO-d6): δ 8.13 (s, 1H, H2), 7.66 (s, 1H, H5), as a grey solid (3.6 g, 80% yield). 1 H NMR (400 MHz, CDCl3): δ 7.84 (t, 1H, J= 1.2 Fig 3.1 General numbering for NMR attribution. In a pressure flask, 1-(2-bromoethyl)-1H-pyrrole (220 mg, 1.26 mmol) and C16H22N3O 272.1763; found 272.1771 temperature, the solution was evaporated to dryness. The residue was purified by solid (306 mg, 95% yield). Anal. Calcd. For C20H18ClN3: C, 71.53; H, 5.40; N, 12.51. 7.76-7.71 (d, 2H, J = 8.8 Hz, H8), 7.45 (m, 3H, H4/H9), 3.75 (t, 4H, J = 4.8 Hz, H12), Imidazole (3.24 g, 47.6 mmol), 4-bromothioanisole (4.83 g, 23.8 mmol), K2CO3 3.1. Complex V-2e Hz, H2), 7.38-7.32 (m, 4H, HSMePh), 7.27 (t, 1H, J= 1.2 Hz, H5), 7.22 (t, 1H, J= 1.2
	benzylimidazole (200 mg, 1.26 mmol) were stirred in 10 mL of dry MeCN at 80 °C column chromatography on silica gel with a mixture of CH2Cl2 and MeOH (10:1) as Found: C, 71.57; H, 5.38; N, 12.48. 1 H NMR (400 MHz, DMSO-d6): δ 10.36 (s, 1H, 3.14 (t, 4H, J = 4.8 Hz, H11). HRMS (ES + ): calcd. for C13H16N3O 230.1293; found (3.29 g, 23.8 mmol) and a catalytic amount of CuSO4 were stirred in a closed pressure Under nitrogen and in the dark, a Schlenk tube was charged with II-2b (100 mg, Hz, H4), 2.54 (s, 3H, HSMePh). 1-(4-(methylthiophenyl)-1H-imidazole (200 mg, 1.05
	for 72 h. After cooling to room temperature, the solvent was evaporated to give a brown paste. The paste was dissolved in CH2Cl2 and precipitated with Et2O. The eluent to give III-2b a highly hydroscopic yellow solid (443 mg, 93% yield). Anal. H2), 8.41 (s, 1H, H5), 8.10 (s, 1H, H4), 7.94-7.89 (m, 4H, H8/H9), 7.59-7.43 (m, 7H, 230.1293. A mixture of III-1d (150 mg, 0.65 mmol) and benzyl chloride (121.5 mg, flask at 205 °C for 10 h. After cooling to room temperature, the crude product was 0.30 mmol) in 6 mL of MeCN and Ag2O (69.7 mg, 0.30 mmol). The reaction mixture mmol) was dissolved in 2 mL of dibromomethane, and the reaction mixture was 1.1.4. 1-[2-Dimethyl)propyl]-benzylimidazolium chloride hydrochloride (II-2d) Calcd. For C20H24ClN3: C, 70.26; H, 7.08; N, 12.29. Found: C, 70.47; H, 7.28; N, benzyl/H11), 6.33 (s, 2H, H12), 5.56 (s, 2H, H6). 13 C NMR (100 MHz, DMSO-d6): δ 0.96 mmol) in MeCN was stirred in a closed pressure flask at 80 °C for 10 h. After extracted by CH2Cl2, filtered and evaporated to dryness. The resulting residue was was stirred at 40 °C for 24 h. After that, Au(SMe2)Cl (106.4 mg, 0.36 mmol) was refluxed for 10 h. After cooling down, the precipitate formed was filtered, washed
	precipitate was filtered, washed with Et2O and dried under vacuum to give a brown 12.48. 1 H NMR (400 MHz, DMSO-d6): δ 9.98 (s, 1H, H2), 8.19 (s, 1H, H5), 8.00 (s, 140.9 (1C, C10), 136.1 (1C, C2), 135.0 (1C, benzyl), 131.9 (1C, C7), 129.4 (2C, cooling down to room temperature, the solution was evaporated to dryness. The washed three times with Et2O (20 mL) to give 1-(4-methylthiophenyl)-1H-imidazole added and the mixture was stirred for 4 h. The solution was filtered through a pad of Fig 4.1 General numbering for NMR attribution. with Et2O and dried under vacuum to obtain a white solid (255 mg, 44% yield). Anal.
	solid (320 mg, 76% yield). Anal. Calcd. For C16H18BrN3: C, 57.84; H, 5.46; N, 12.65. 1H, H4), 7.55-7.51 (m, 4H, benzyl/H8), 7.47-7.40 (m, 3H, benzyl), 6.81 (d, 2H, J = benzyl), 129.3 (1C, benzyl), 129.0 (2C, benzyl), 123.7 (2C, C8), 123.6 (1C, C5), residue was purified by column chromatography on silica gel with a mixture of celite and the solvent was removed under reduced pressure to give a grey solid (105 Calcd. For C21H22Br2N4S2: C, 45.50; H, 4.00; N, 10.11. Found: C, 45.29; H, 3.93; N, (III-4a) as a grey solid (3.6 g, 80% yield). 1 H NMR (400 MHz, CDCl3): δ 7.84 (t, 1H, Found: C, 57.70; H, 5.33; N, 12.58. 1 H NMR (400 MHz, DMSO-d6): δ 8.87 (s, 1H, H2), 7.76 (s, 1H, H5), 7.56 (s, 1H, H4), 7.43-7.27 (m, 5H, Bn), 6.61 (t, 2H, J= 2.0 Hz, In a pressure flask, 3-chloro-N,N-dimethylpropylamine hydrochloride (300 mg, 9.2 Hz, H9), 5.50 (s, 2H, H6), 3.40 (q, 4H, J = 7.2 Hz, H11), 1.11 (t, 6H, J= 7.2 Hz, H12). 13 C NMR (100 MHz, DMSO-d6): δ 148.5 (1C, C10), 135.2 (1C, C2), 135.1 122.1 (1C, C4), 120.4 (2C, C9), 119.6 (2C, C11), 112.7 (2C, C12), 52.8 (1C, C6). HRMS (ES + ): calcd. for C20H18N3 300.1501; found 300.1500. CH2Cl2 and MeOH (10:1) as eluent to give III-2d as a highly hydroscopic yellow solid (200 mg, 91 % yield). Anal. Calcd. For C20H22ClN3O: C, 67.50; H, 6.23; N, J= 1.2 Hz, H2), 7.38-7.32 (m, 4H, HSMePh), 7.27 (t, 1H, J= 1.2 Hz, H5), 7.22 (t, 1H, J= 1.2 Hz, H4), 2.54 (s, 3H, HSMePh). In a pressure flask, a mixture of III-4a (200 mg, mg, 72% yield). Anal. Calcd. For C16H17AuClN3: C, 39.73; H, 3.54; N, 8.69. Found: C, 39.81; H, 3.56; N, 8.58. 1 H NMR (400 MHz, CD3CN): δ 7.41-7.36 (m, 3H, benzyl), 10.50. 1 H NMR (400 MHz, DMSO-d6): δ 10.31 (s, 2H, H2), 8.42 (s, 2H, H5), 8.38 (s, 4.1. Preparation of imidazolium salts 2H, H4), 7.76 (d, 4H, J= 8.8 Hz, HSMePh), 7.56 (d, 4H, J= 8.8 Hz, HSMePh), 6.90 (s, 2H,
	C NMR (75 MHz, DMSO-d6): δ 137.7 (1C, C2), 135.0 (1C, benzyl), 129.4 (2C, benzyl), 129.2 (1C, benzyl), 128.9 (2C, benzyl), 123.3 (1C, C5), 123.2 (1C, C4), 54.6 (1C, C6), 52.8 (2C, C10), 52.5 (1C, C7), 43.5 (1C, C8), 22.4 (2C, C11), 21.7 (1C, C12). HRMS (ES + ): calcd. for C17H24N3 270.1970; found 270.1975. H10), 5.97 (t, 2H, J= 2.0 Hz, H11), 5.38 (s, 2H, H6), 4.55 (t, 2H, J= 5.2 Hz, H7), 4.36 (t, 2H, J= 5.2 Hz, H8). 13 C NMR (100 MHz, DMSO-d6): δ 136.9 (1C, C2), 135.1 (1C, Bn), 129.4 (2C, Bn), 129.1 (1C, Bn), 128.6 (2C, Bn), 123.3 (1C, C5), 123.0 (1C, C4), 121.0 (2C, C10), 108.8 (2C, C11), 52.2 (1C, C6), 50.6 (1C, C7), 48.4 (1C, C8). (1C,benzyl), 129.4 (2C, benzyl), 129.2 (1C, benzyl), 128.9 (2C, benzyl), 123.6 (2C, C8), 123.3 (1C, C7), 123.1 (1C, C5), 122.2 (1C, C4), 112.0 (2C, C9), 52.6 (1C, C6), 44.3 (2C, C11), 12.7 (2C, C12). HRMS (ES + ): calcd. for C20H24N3 306.1970; found 306.1967. 2.1.4. 3-Benzyl-1-[4-(morpholin-4-yl)phenyl]-1H-imidazol-3-ium chloride (III-2d) 7.29-7.27 (m, 2H, benzyl), 7.06 (s, 1H, H5), 6.79 (s, 1H, H4), 6.53 (t, 2H, J= 2 Hz, H10), 6.02 (t, 2H, J= 2 Hz, H11), 5.33 (s, 4H, H6), 4.49 (t, 2H, J= 6 Hz, H7), 4.35 (t, 2H, J= 6 Hz, H8). 13 C NMR (100 MHz, CD3CN): δ 170.7 (1C, C2), 136.5 (1C, 4.1.1. 1,1'-Methylenebis(3-isopropyl-1H-imidazol-3-ium) bromide (VI-3a) H6), 2.56 (s, 6H, HSMePh). 13 C NMR (100 MHz, DMSO-d6): δ 141.9 (2C, CSMePh), 137.6 (2C, C2), 131.6 (2C, CSMePh), 127.2 (4C, CSMePh), 123.4 (2C, C5), 122.9 (4C, CSMePh), 122.1 (2C, C4), 58.9 (1C, C6), 14.9 (2C, CSMePh). MS (ES + ): calcd. for 11.81. 3.76 (t, 4H, J = 4.8 Hz, H12), 3.22 (t, 4H, J = 4.8 Hz, H11). 13 C NMR (100 MHz, celite and the solvent was removed under reduced pressure to give a pale-yellow solid benzyl), 128.8 (2C, benzyl), 128.2 (1C, benzyl), 127.5 (2C, benzyl), 121.6 (1C, C5), C21H22BrN4S2 473.05; found 473.25 [M-Br -] + . Calcd. for C21H21N4S2 393.12; found was dissolved in CH2Cl2 and precipitated with Et2O. The precipitate was filtered, HRMS (ES + ): calcd. for C16H18N3 252.1501; found 252.1496. 1.1.3. 1-Benzyl-3-(2-morpholinoethyl)-1H-imidazol-3-ium chloride hydrochloride (II-2c) In a pressure flask, 4-(2-chloroethyl)morpholine hydrochlodide (585 mg, 3.14 mmol) was suspended in 10 mL of dry MeCN. The mixture was heated at 80 °C and After cooling to room temperature, the precipitate formed was filtered, washed by Na2SO4. After filtration and evaporation of the solvent under reduced pressure, the 8.8 Hz), 3.86 (t, 4H, J = 4.8 Hz), 3.12 (t, 4H, J = 4.8 Hz). Imidazole (275.6 mg, 4.05 39.61; H, 5.63; N, 14.21. Found: C, 39.45; H, 5.54; N, 14.05. 1 H NMR (400 MHz, mmol) was slowly added to the solution. The mixture was stirred for 72 h at 80 °C. extracted with EtOAc. The organic layer was washed with brine and dried over 85% yield). 1 H NMR (400 MHz, CDCl3): δ 7.37 (d, 2H, J = 8.8 Hz), 6.78 (d, 2H, J = vacuum to obtain a white solid (50 mg, 38% yield). Anal. Calcd. For C13H22Br2N4: C, stirred until the solid was completely dissolved. Then benzylimidazole (745 mg, 4.71 Anal. Calcd. For C15H23N3Cl2: C, 56.97; H, 7.33; N, 13.29. Found: C, 57.09; H, 7.38; N, 13.31. 1 H NMR (400 MHz, DMSO-d6): δ 11.24 (s, 1H, HCl), 9.51 (s, 1H, H2), 7.90 (s, 1H, H5), 7.86 (s, 1H, H4), 7.46-7.40 (m, 5H, benzyl), 5.46 (s, 2H, H6), 4.35 (t, 2H, J= 7.2 Hz, H7), 3.06 (t, 2H, J= 7.2 Hz, H9), 2.73 (s, 6H, H11), 2.32-2.25 (m, 2H, H8). 13 C NMR (100 MHz, DMSO-d6): δ 137.0 (1C, C2), 135.2 (1C, benzyl), 129.5 (2C, benzyl), 129.2 (1C, benzyl), 128.8 (2C, benzyl), 123.3 (1C, C5), 123.1 (1C, C4), 53.4 (1C, C6), 52.4 (1C, C7), 46.7 (1C, C9), 42.4 (2C, C11), 24.7 (1C, C8). HRMS (ES + ): calcd. for C15H22N3 244.1814; found 244.1823. 2.1.3. Benzyl-1-(1H-pyrrol-1-yl)-1H-imidazol-3-ium chloride (III-2c) Imidazole (268.5 mg, 3.94 mmol), 1-(4-chlorophenyl)-1H-pyrrole (500 mg, 2.81 mmol), K3PO4 (1.19 g, 5.62 mmol) and CuI (107 mg, 0.56 mmol) in DMF were placed in a closed pressure flask, then stirred at 160 °C for 3 days. After cooling to room temperature, distilled water was added to the reaction and the crude was mmol) in EtOAc (20 mL) was added hydrobromic acid (48 %, 1.85 g, 5.5 mmol) at 60 °C under air. The mixture was stirred at 60 °C, then potassium carbonate (1 g) was added to the solution and the mixture was stirred for another 0.5 h. After cooling down to room temperature and evaporation of the solvent under vacuum, the residue EtOAc (10:1) as eluent to give 4-(4-bromophenyl)morpholine as a white solid (1.03 g, Fig 2.2 General numbering for NMR attribution. temperature, the precipitate formed was filtered, washed with Et2O and dried under 284.1219. was purified by column chromatography on silica gel with a mixture of hexane and C16H21AuN3O 468.1350; found 468.1351. and the reaction mixture was refluxed for 48 h. After cooling down to room 48.4 (1C, C7), 15.0 (1C, CSMePh). HRMS (ES + ): calcd. for C16H18N3S 284.1221; found bis(1H-imidazole) (50 mg, 0.34 mmol) was dissolved in 2 mL of 2-bromopropane, 122.7 (2C, CSMePh), 121.5 (1C, C4), 121.2 (2C, C9), 108.9 (2C, C10), 50.9 (1C, C6), C6), 54.3 (1C, C7), 53.4 (2C, C10), 47.8 (1C, C8). HRMS (ES + ): calcd. for (s, 2H, H2), 7.14 (s, 2H, H5), 7.14 (s, 2H, H4), 6.02 (s, 2H, H6). 1,1'-Methylene CSMePh), 135.8 (1C, C2), 131.8 (1C, CSMePh), 127.2 (2C, CSMePh), 123.7 (1C, C5), (1C, Bn), 127.6 (2C, Bn), 122.1 (1C, C5), 121.1 (1C, C4), 66.2 (2C, C11), 58.1 (1C, o C to yield a white product (885 mg, 60 % yield). 1 H NMR (400 MHz, CDCl3): δ 7.67 6 Hz, H7), 2.55 (s, 3H, HSMePh). 13 C NMR (100 MHz, DMSO-d6): δ 141.5 (1C, NMR (100 MHz, CD3CN): δ 170.5 (1C, C2), 136.7 (1C, Bn), 128.8 (2C, Bn), 128.2 was obtained. Purification of the crude product was performed by sublimation at 180 HSMePh), 6.72 (s, 1H, H9), 6.01 (s, 1H, H10), 4.61 (t, 2H, J= 6 Hz, H6), 4.36 (t, 2H, J= To a mixture of 4-phenylmorpholine (816 mg, 5 mmol) and DMSO (429 mg, 5.5 DMSO-d6): δ 152.1 (1C, C10), 135.4 (1C, C2), 135.2 (1C, benzyl), 129.4 (2C, benzyl), 129.2 (1C, benzyl), 129.0 (2C, benzyl), 126.4 (1C, C7), 123.4 (1C, C5), 123.1 (2C, H8), 122.1 (1C, C4), 115.6 (2C, H9), 66.4 (2C, C12), 52.6 (1C, C6), 48.2 (2C, C11). 1H, H5), 7.66 (s, 1H, H4), 7.62 (d, 2H, J= 8.4 Hz, HSMePh), 7.51 (d, 2H, J= 8.4 Hz, liquid was slowly added dibromomethane (0.7 mL, 10 mmol). After 5 hours, a solid H7), 3.63 (t, 4H, J= 4 Hz, H11), 2.84 (s, 2H, H8), 2.57 (t, 4H, J= 4 Hz, H10). 13 C HRMS (ES + ): calcd. for C20H22N3O 320.1763; found 320.1761. washed with Et2O and dried under vacuum to give III-5a as a brown solid (154 mg, 40% yield). Anal. Calcd. For C16H18BrN3S: C, 52.75; H, 4.98; N, 11.53. Found: C, benzyl), 7.26 (s, 1H, H5), 7.18 (s, 1H, H4), 5.39 (s, 2H, H6), 4.31 (t, 2H, J= 5.6 Hz, hydroxide (2.24 g, 40 mmol) and stirred for 1 h at room temperature. To the resulting 52.82; H, 4.86; N, 11.66. 1 H NMR (400 MHz, DMSO-d6): δ 9.41(s, 1H, H2), 8.25 (s, (140 mg, 90% yield). Anal. Calcd. For C16H21AuClN3O: C, 38.15; H, 4.20; N, 8.34. tetrabutylammonium bromide (193 mg, 0.6 mmol), and powdered potassium Found: C, 38.21; H, 4.26; N, 8.28. 1 H NMR (400 MHz, CD3CN): δ 7.43-7.35 (m, 5H, 120.6 (1C, C4), 117.3 (2C, C10), 108.5 (2C, C11), 54.3 (1C, C6), 52.2 (1C, C7), 49.3 (1C, C8). HRMS (ES + ): calcd. for C16H17AuN3 448.1088; found 448.1097. A three-necked flask was charged with a mixture of imidazole (1.36 g, 20 mmol), 393.33 [M-2Br-H + ] + .
	138 139 158

CH2Cl2 and dried under vacuum to give a pale-yellow solid (700 mg, 76% yield).

Anal. Calcd. For C16H23Cl2N3O:

C, 55.82; H, 6.73; N, 12.21. Found: C, 55.64; H, 1.90 mmol)

was suspended in 10 mL of dry MeCN. After the solid was completely dissolved, benzylimidazole (450 mg, 2.

85 mmol)

was slowly added to the solution.

The reaction mixture was stirred for 72 h at 80 °C. After cooling to room temperature, the solvent was removed under vacuum to give a pale-yellow paste. The paste was dissolved in MeCN and precipitated with Et2O. The precipitate was filtered, washed with Et2O and dried under vacuum to give a pale-yellow solid (200 mg, 33% yield).

residue was purified by column chromatography on silica gel with a mixture of CH2Cl2 and MeOH (30:1) as eluent to give 1-(1H-pyrrol-1-yl)-1H-imidazole (III-1c) as a yellow solid (300 mg, 51% yield).

1

H NMR (400 MHz,

DMSO-d6

): δ 8.33 (s, 1H, mmol),

4-(4-bromophenyl)

morpholine (700 mg, 2.89 mmol), K3PO4 (1.2 g, 5.65 mmol) and CuI (110 mg, 0.56 mmol) in DMF were placed to a closed pressure flask, then stirred at 160 °C for 3 days. Found:

C, 67.57; H, 6.38; N, 11.58. 1

H NMR (400 MHz,

DMSO-d6

): δ 10.15 (s, 1H, H2), 8.27 (s, 1H, H5), 8.04 (s, 1H, H4), 7.65

(d, 2H, J = 8.8 Hz, H8), 7.55 (m, 2H, benzyl)

, 7.

47-7.40 (m, 3H, benzyl), 7.15 (d, 2H

, J = 8.8 Hz, H9), 5.52 (s, 2H, H6), 1.05 mmol) and 1-(2-bromoethyl)-1H-pyrrole (183 mg, 1.05 mmol) was dissolved in 10 mL of dry MeCN. The mixture was stirred at 80 °C for 72 h. After cooling to room temperature, the solvent was removed by vacuum to give a brown paste. The paste

DMSO-d6

): δ 9.68 (s, 2H, H2), 8.12 (s, 2H, H5), 8.06 (s, 2H, H4), 6.67 (s, 2H, H6), 4.

74-4.68 (m, 2H, H7), 1.51 (d, 12H, J= 6.4 Hz, H8)

.

13

C NMR (100 MHz,

DMSO-d6

): δ 137.0 (2C, C2), 122.9 (2C, C5), 121.8 (2C, C4),

58.5 (1C, C6), 53.3 (2C, C7), 22.6

values for NHC-Au-X (X = halide). [START_REF] Lin | Coinage Metal-N-Heterocyclic Carbene Complexes[END_REF] The elemental analysis of the gold(I) complexes correspond to the general formula AuLCl and the HRMS exhibit the classical peaks corresponding to the cationic fragments [M-Cl -] + for compounds V-2e and V-2f.

Molecular structure

Crystals of V-2e suitable for X-ray diffraction analysis were obtained by slow evaporation from an acetonitrile solution of this compound (Fig 5.6).

Cytotoxicity evaluation

The evaluation of the tested molecules cytotoxicity by MTT assay on the J774A.1 cell line (mouse macrophage cell line, Sigma-Aldrich) was done according like previously described. 113a Briefly, cells (5 × 10 4 cells/mL) in 100 μL of complete medium, [DMEM high Glucose supplemented with 10% fetal calf serum (FCS), 2 mM l-glutamine and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin)] were seeded into each well of 96-well plates and incubated at 37 °C and 5% CO2. After a 24 h incubation, 100 μL of medium with various product concentrations and appropriate controls treated by DMSO, amphotericin B, miltefosine, doxorubicine and pentamidine (reference drugs purchased from Sigma Aldrich) were added and the plates were incubated for 72 h at 37 °C and 5% CO2. Each plate-well was then microscope-examined for detecting possible precipitate formation before the medium was aspirated from the wells. 100 μL of MTT solution (0.5 mg/mL in RPMI) were then added to each well. Cells were incubated for 2 h at 37 °C and 5% CO2. After this time, the MTT solution was removed and DMSO (100 µl/well) was added to dissolve the resulting formazan crystals. Plates were shaken vigorously (300 rpm) for 5 min. The absorbance was measured at 570 nm with a microplate spectrophotometer (Eon BioTek). DMSO was used as blank. CC50 were calculated by non-linear regression analysis processed on dose-response curves, using TableCurve 2D V5 software. CC50 values represent the mean value calculated from three independent experiments.

Photodynamic activity

A colormetric assay using MTT was used to compare the cytotoxicity of the complexes in the dark and upon UV irradiation. T24, PC-3 and non-cancerous NIH3T3 cell lines were seeded in 24-well plates at a density of 10,000 cells per well in 500 μL, 24 h prior to treatment. The cells were then treated with increasing concentrations of iridium complexes (from 0.01 μM to 20 μM) for 48 h. For photocytoxicity studies, the cells were treated for 4 h with increasing concentrations of the compounds in the dark. After that, the medium was removed and replaced by