G A ; Roth

C ; Johnson

A ; Abajobir

F ; Abd-Allah

S F ; Abera

G ; Abyu

M ; Ahmed

B ; Aksut

T ; Alam

K ; Alam

F ; Alla

N ; Alvis-Guzman

S ; Amrock

H ; Ansari

J ; Arnlov

H ; Asayesh

T M ; Atey

L ; Avila-Burgos

A ; Awasthi

A ; Banerjee

A ; Barac

T ; Barnighausen

L ; Barregard

N Bedi

Belay Ketema

E ; Bennett

D ; Berhe

G ; Bhutta

Z ; Bitew

S ; Carapetis

J ; Carrero

J J ; Malta

D C ; Castaneda-Orjuela

C A ; Castillo- Rivas

J ; Catala-Lopez

F ; Choi

J Y ; Christensen

H ; Cirillo

M ; Cooper

L ; Criqui

M ; Cundiff

D ; Damasceno

A ; Dandona

L ; Dandona

R ; Davletov

K ; Dharmaratne

S ; Dorairaj

P ; Dubey

M ; Ehrenkranz

R ; El

Sayed Zaki

M ; Faraon

E J A ; Esteghamati

A ; Farid

T ; Farvid

M ; Feigin

V ; Ding

E L ; Fowkes

G ; Gebrehiwot

T ; Gillum

R ; Gold

A ; Gona

P ; Gupta

R ; Habtewold

T D ; Hafezi-Nejad

N ; Hailu

T ; Hailu

G B ; Hankey

G ; Hassen

H Y ; Abate

K H ; Havmoeller

R ; Hay

S I ; Horino

M ; Hotez

P J ; Jacobsen

K ; James

S ; Javanbakht

M ; Jeemon

P ; John

D ; Jonas

J ; Kalkonde

Y ; Karimkhani

C ; Kasaeian

A ; Khader

Y ; Khan

A ; Khang

Y H ; Khera

S ; Khoja

A T ; Khubchandani

J ; Kim

D ; Kolte

D ; Kosen

S ; Krohn

K J ; Kumar

G A ; Kwan

G F ; Lal

D K ; Larsson

A ; Linn

S ; Lopez

A ; Lotufo

P A ; El

H M A ; Malekzadeh

R ; Mazidi

M ; Meier

T ; Meles

K G ; Mensah

G ; Meretoja

A ; Mezgebe

H ; Miller

T ; Mirrakhimov

E ; Mohammed

S ; Moran

A E ; Musa

K I ; Narula

J ; Neal

B ; Ngalesoni

F ; Nguyen

G ; Obermeyer

C M ; Owolabi

M ; Patton

G ; Pedro

J ; Qato

D ; Qorbani

M ; Rahimi

K ; Rai

Keywords: antiangiogenesis, cyclam, hydroxamic acids, iron complexes, nitric oxide, nitroxyl, oxidative activation mechanisms, tuberculosis, vasodilation. Résumé acides hydroxamiques, antiangiogenèse, complexes de fer, cyclam, mécanismes d'activation oxydante, nitroxyle, oxyde nitrique, tuberculose, vasodilatation

Cancer, cardiovascular diseases and tuberculosis (TB) are major therapeutic challenges, for which various drug strategies have been developed, including the use of inorganic agents such as metallo-drugs and NO • (nitric oxide)/ HNO (nitroxyl). Within this prospect, the thesis objective addresses the design, synthesis, physical-chemical studies and biological evaluation of metal-based drugs capable of releasing NO • and/or HNO with pharmacological effects. Two types of organic platforms are thus envisaged as ligands in Fe II/III complexes devised as potential sources of NO • /HNO: the spectator cyclam ligand (1,4,8,11-tetraazacyclotetradecane) and three reactive azine hydroxamic acid ligands (ArC(O)NHOH).

The first chapter deals with the chemical reactivity of a trans-[Fe(cyclam)(NO)Cl]Cl2

complex, which is shown to decompose by releasing NO • under physiological pH and temperature conditions, the process being significantly accelerated upon light irradiation at 365 nm. In contrast, in vitro experiments in the presence of glutathione revealed that the same complex produces HNO. Angiogenesis studies showed that the complex is able to significantly inhibit the hypoxia-inducible factor (HIF-1α) induced by hypoxia or addition of a NO • donor (Spermine NONOate) in breast cancer cells: this result is consistent with a release of HNO by the nitrosyl complex. On the other hand, vasodilation assays using precontracted rat aortic rings revealed that the complex exhibits a relaxation IC50 of 910 nM, vs 24 nM for the reference drug, nitroprusside. The next chapters deal with the oxidation-promoted release of HNO from aromatic hydroxamic acids. Chapter 2 focuses on the oxidation mechanism of isonicotinoic, nicotinoic and pyrazinoic hydroxamic acids, mediated by potassium ferricyanide (K3[Fe III (CN)6]) in physiological pH conditions. It is shown that oxidative activation of aryl hydroxamic acids, mediated by Fe III , involves the production of the N,Odi(di)azinoylhydroxylamine intermediate with concomitant release of HNO, instead of the putative acyl nitroso species. The free hydroxamic acids were also evaluated for different biological activities and used as controls to be compared with iron complexes described in chapter 3. These pentacyanoferrate(II) complexes, Na3Fe II (CN)5(ArCONHOH), initially devised as potential hybrid of isoniazid (INH, first-line anti-TB prodrug, for which the isonicotinoyl radical is the active metabolite) and delamanid (third-line anti-TB, for which HNO is the active metabolite), were synthesized and characterized by spectroscopic techniques, cyclic voltammetry, and DFT calculations. Using EPR and 1 H NMR spectroscopy, oxidation of these Fe II

Acknowledgment

After an intensive work, my PhD is nearing its end. In that time, my knowledge at Chemistry as well as my experimental and research skills have been significantly improved. At this moment, I take this opportunity to thank all the people who directly or indirectly helped me to finish one of the most important stages of my life.

First of all, I would like to express my most sincere and immeasurable gratitude to my supervisors, Prof. Vania Bernardes-Génisson, Prof. Luiz Gonzaga de França Lopes, Prof. Eduardo Henrique Silva de Sousa, and Prof. Remi Chauvin, for their support, encouragement, companionship, and helpfulness. Whenever I needed, they were always available to help me, both in academic and personal life.

My first steps in academic life were guided and assisted by Prof. Eduardo Sousa.

Without a doubt, he is one of my greatest references. Thank you very much for choosing me and helping me evolve. Without his help, it would not have been possible for me to complete my PhD. His contribution to this thesis is notorious.

After my graduation, I had (and still have) the honor of starting and finishing the Master's degree with Prof. Luiz Lopes, who is also my Brazilian PhD supervisor. His knowledge, enthusiasm and encouragement helped me to get firm until this moment. I am also extremely grateful for accepting me as a student and for helping me write this thesis.

Prof. Vania Bernardes-Génisson was one of the most important and significant professionals that I have had the privilege of knowing as a student. I have no words to express how grateful I am for welcoming me as a student (not to mention as a son) during my doctoral studies in France. Always concerned and attentive to whether the research was going well, but her care was not limited to academic life. On several occasions she supported me especially in the most difficult and troubled moments of my stay in France. I cannot fail to mention the dedication and professionalism in writing this thesis, it is not possible to briefly explain how much of your love and time were invested in this work. Thank you so much for everything.

Prof. Remi Chauvin, one of the greatest chemists in the world, sometimes I find myself without believing that I had the time to work and share a phase of my life with you. Your creativity and enthusiasm are contagious. Doing science by your side was an incredible experience. Thank you very much for the discussions and for helping me to evolve as a chemist and person. In addition, I could not fail to mention the good atmosphere at the lab. Thank you very much for opening the doors of your laboratory for me and for guiding me throughout this stage. From my arrival in France to writing this thesis, your contributions were and are extremely important to me. I sincerely feel honored to have been your doctoral student.

In addition, I would like to thank the members of my thesis committee: Prof. Maria Conceição F. de Oliveira ("Universidade Federal do Ceará", Brazil), Prof. Gilles Gasser ("Université Chimie Paris Tech", France), Prof. Pablo Machado ("Pontifícia Universidade Católica do Rio Grande do Sul", Brazil), and Prof. Eric Benoist ("Université de Toulouse III", France), for having accepted the invitation to participate in this defense, contributing in an insightful and significant way in improving this work.

I would like to thank all members of the "Bioinorgânica" group at the "Universidade Federal do Ceará": the professors Alda Karine, Francisco Audísio, Izaura Diógenes, Idalina Carvalho, Jackson Rodrigues, Tércio Paulo and Elisane Longhinotti, your contributions were essential in my academic life.

I have to thank all my friends from the lab. "Bioinorgânica": Aurideia, Florêncio, Gilmara, Josi, Maiara, Monilson, Ricardo, Walysson, Lucas, Patty, Alan, Giam, Iury, Leonardo, Manigat, Wellison, Karol, Dieric, Juliana, Geângela, Vitória, Carlos Daniel, Vivânia, Mikael and André Florêncio. Directly or indirectly, all of you contributed to this moment. I would like to thank Prof. Tércio Paulo for his help in several experiments both in Brazil and in France, as well as his wife, Mari. The moments we shared in France were extremely important for me, especially when it comes to a situation where you are far from your family and friends. Finally, we became good friends.

I would like to say thanks to the friends that France gave me: Cécile, Marwa, Chongwei, Dmytro, Abdelhak, Sameh, Amal, Sarah and Hend. You have helped me a lot to endure the difficulties of distance from home. In particular, I would like to extend my most sincere thanks to my "sisters" Cécile and Marwa, and my "brother" Abdelhak. I consider you as part of my family. I also have to say thanks Valerie for always being available to help me inside and outside the lab.

I also have to thank my friend Jéssica de Lima for helping, supporting and crying with me in the moments that I most needed. You are very important to me. I would like to thank the "Universidade Federal do Ceará -UFC" for all the support it has offered me during these more than 10 years. It was literally a second home for me. I also would like to thank "Laboratoire de Chimie de Coordination -LCC" and "Centre National de la Recherche Scientifique -CNRS" for providing a safe and comfortable workplace, and "Université Paul Sabatier III -UPS" for offering us various interesting and useful courses and training. I would like to say thanks to my family, especially my mother Maria Veraleide da Masceno Muniz, for always accompanying me and being present at all times. She is one of the main reasons, for which I want to become a PhD. Thank you! Finally, I have to thank the CAPES and the CAPES-COFECUB project for their financial support.

Foreword

Cancer, cardiovascular disorders, and tuberculosis are diseases that significantly affect the world population. Among the therapeutic strategies used to treat these diseases, administration of medicines is one of the most important, allowing the control of health problems, and improving the quality of patients' life. Such strategy has been increasingly adopted however, spite of the benefits brought by the use of medicines, in many cases, mainly in situations of prolonged use as for cardiovascular therapy, several side effects are observed. For cancer, the discovery for miraculous molecules allowing the cure of the disease is still anxiously waited. Moreover, the emergence of infectious diseases promoted by pathogenic strains resistant to conventional antibiotics (e.g., Mycobacterium tuberculosis towards isoniazid and rifampicin) is also a worldwide health problem urgently needing for new agents that could exhibit better efficacy and lesser side-effects.

While chemical organic drugs are largely employed to treat these affections and others, inorganic compounds have been much less developed. However, a few inorganic drugs containing metal atoms can also play a crucial role in therapeutic (e.g., LiCO3, cisplatin, sodium nitroprusside, etc.) and diagnostic (e.g., Magnevist ® and Cardiolite ® , etc.) arsenals. Among the inorganic compounds involving metals, metallo-drug is a class of molecules that exhibits a series of chemical characteristics (e.g., charge variation and metal-ligand interaction) and properties (e.g., redox activity and Lewis acid properties) that make it excellent scaffold for the development of new medicinal chemistry strategies. Additionally, another family of inorganic compounds that draw our attention is the nitric oxide (NO • ) and nitroxyl (HNO) molecules, which are both widely studied due to their relevant biological properties. As these molecules are very unstable and cannot be planned as direct drugs, pro-drugs able to release these reactive species in vivo are also of our interest. All these advances prompted us to propose new anticancer, antihypertensive and antituberculosis drug-development strategies based on the release of NO • /HNO mediated by transition-metal. In this context, this thesis focuses on the design, synthesis, physico-chemical and biological studies of new metal complexes able to release NO • /HNO in physiological conditions as a way to assist in the treatment of cancer, hypertension disorders, and tuberculosis.

The overview presented in the introduction is devoted in the first part to highlight the place of metal complexes in therapeutics and in a second part, to point out Foreword _________________________________________________________________________________ the significant biological aspects of NO • and HNO, their pharmacological importance in the context of cancer, cardiovascular disease and tuberculosis, and finally, to report their organic and inorganic donors. The results obtained under this thesis are presented in the form of scientific papers. The first part of this thesis (Chapter 1) describes the study of the reactivity of the Fe II complex, trans-[Fe(cyclam) (NO)Cl]Cl2, with regard to the release of NO • through a photochemical process and the release of HNO via the activation of the complex by biological reducing agents. The vasodilation properties of this nitrosyl complex were also examined.

The second part of this thesis is divided in two chapters (chapters 2 and 3). In the chapter 2, a mechanistic study of the oxidative activation of three (di)azine hydroxamic acids (isonicotino-, nicotino-, and pyrazino-hydroxamic acids) was described. These results led us to propose a new mechanism for the activation of these (di)azine hydroxamic acids in aqueous medium. These compounds were also evaluated for different biological activities and they were used as control molecules to be compared to the metal-drugs prepared in the chapter 3. In the third chapter, three new pentacyanoferrate(II) complexes derived from the aromatic hydroxamic acids mentioned above, originally conceived as a potential hybrid of isoniazid (front-line antitubercular, for which radical isonicotinoyl is the active metabolite) and delamanid (third-line anti-TB, for which HNO is the active metabolite), were synthesized, characterized and their ability to release dual active metabolites (NO • /HNO and aroyl radical) in the presence of H2O2, as well as their biological effects as antitubercular and cardiovascular agents were investigated.

Avant-propos

Le cancer, les troubles cardiovasculaires et la tuberculose sont des maladies qui affectent de manière significative la santé de la population mondiale. Parmi les stratégies thérapeutiques utilisées pour traiter ces maladies, l'administration de médicaments est l'une des plus importantes, permettant de pallier les problèmes de santé, et d'améliorer la qualité de vie des patients. Cette stratégie est de plus en plus adoptée, néanmoins dans de nombreux cas et principalement dans les situations d'utilisation prolongée, comme pour les maladies cardiovasculaires, plusieurs effets secondaires sont observés. Pour le cancer, la découverte de molécules « miraculeuses » Foreword _________________________________________________________________________________ 13 permettant la guérison de la maladie est encore attendue avec impatience. En outre, l'émergence de maladies infectieuses favorisées par des souches pathogènes résistantes aux antibiotiques conventionnels (par exemple Mycobacterium tuberculosis face à l'isoniazide et rifampicine) est également un problème de santé mondiale nécessitant urgemment la découverte de nouveaux agents qui pourraient présenter une meilleure efficacité et moins d'effets secondaires.

Alors que les médicaments chimiques organiques sont en grande partie utilisés pour traiter ces maux et d'autres, les composés inorganiques ont été beaucoup moins développés. Cependant, quelques médicaments inorganiques contenant des atomes métalliques peuvent également jouer un rôle crucial dans les arsenaux thérapeutiques (p. ex. : LiCO3, cisplatine, nitroprussiate de sodium, etc.) et diagnostiques (p. ex. :

Magnevist ® et Cardiolite ® , etc.). Parmi les composés inorganiques à base de métaux, ceux appelés "drogues métalliques" appartiennent à une classe de molécules qui présente une série de caractéristiques chimiques (par exemple : variation de charge et interaction métal-ligand) et de propriétés (par exemple : activité redox et propriétés de l'acide de Lewis) qui font d'elle un excellent échafaudage pour le développement de nouvelles stratégies de chimie médicinale. En outre, une autre famille de composés inorganiques qui attire notre attention est l'oxyde nitrique (NO • ) et nitroxyle (HNO), qui sont tous les deux largement étudiés en raison de leurs propriétés biologiques pertinentes. Comme ces molécules sont très instables et ne peuvent pas être administrées comme des médicaments, des pro-médicaments capables de libérer ces espèces réactives in vivo sont également de notre intérêt. Toutes ces avancées nous ont incités à proposer une nouvelle stratégie de développement de médicaments anticancéreux, antihypertenseurs et antituberculeux basée sur la libération de NO • /HNO induite par la modification de l'état d'oxydation du métal de transition. Dans ce contexte, cette thèse vise la conception, la synthèse, les études physico-chimiques et biologiques de nouveaux complexes métalliques capables de libérer NO • /HNO dans des conditions physiologiques de pH comme un moyen d'assister au traitement du cancer, des troubles de hypertension, et de la tuberculose.

L'aperçu présenté dans l'introduction consiste, dans un première temps, à faire ressortir la place des complexes métalliques en thérapeutique et dans un deuxième temps, à signaler les aspects biologiques significatifs des molécules NO • et HNO, leur importance pharmacologique dans le contexte du cancer, des maladies cardiovasculaires et de la tuberculose, et enfin, à rapporter leurs donneurs organiques et inorganiques.

Les résultats obtenus dans le cadre de cette thèse sont présentés sous forme d'articles scientifiques. La première partie de cette thèse (chapitre 1) décrit l'étude de la réactivité du complexe de Fe II , trans-[Fe(cyclam) (NO)Cl]Cl2, en ce qui concerne la libération de NO • par un processus photochimique et la libération de HNO via l'activation du complexe par des agents biologiques réducteurs. Les propriétés de vasodilatation de ce complexe de nitrosyle ont également été examinées.

La deuxième partie de cette thèse est divisée en deux chapitres (chapitres 2 et 3).

Dans le chapitre 2, une étude du mécanisme de l'activation oxydative de trois (di)azine acides hydroxamiques (acide hydroxamique isonicotinique, acide hydroxamique nicotinique et acide hydroxamique pyrazinique) a été décrite. Ces résultats nous ont amenés à proposer un nouveau mécanisme pour l'activation des acides hydroxamiques.

Ces composés ont également été évalués vis-à-vis de différentes activités biologiques et ils ont été utilisés comme des molécules témoins pour être comparés aux complexes métalliques préparés dans le chapitre 3. Dans le troisième chapitre, trois nouveaux complexes pentacyanoferrate(II) dérivés des acides hydroxamiques mentionnés cidessus, originalement conçus comme hybride potentiel de l'isoniazide (antituberculaire de première ligne, pour lequel le radical isonicotinoyle est le métabolite actif) et du délamanide (anti-TB de troisième ligne, pour lequel HNO est le métabolite actif), ont été synthétisés, caractérisés et leur capacité à libérer deux métabolites actifs (NO • /HNO et le radical aroyle) en présence de H2O2, ainsi que leurs effets biologiques (propriétés antituberculaires et cardiovasculaires) ont été étudiés. Metallo-drugs constitute a class of pharmaceutical substances that contains a metal as an active ingredient. Several substances in this class have been used in therapeutic and diagnostic medicine throughout history [1]. In terms of modern chemotherapy, the first metallo-drug used was salvarsan (Figure 1), an arsenic(III)based antimicrobial agent developed by the German physician Paul Ehrlich in 1910. It was used in the treatment of syphilis, before being replaced by penicillin after World War II [1,2]. Several other treatments based on metallo-drugs have emerged, such as gold compounds (e.g., gold cyanide (K[Au(CN)2]) and sodium aurothiomalate) for the treatment of tuberculosis during the years of 1925-1935 (described as the "Gold Decade") [3]. Despite the ineffectiveness of such complex ions in the treatment of tuberculosis in animals, the same compounds proved to be effective in the treatment of rheumatoid arthritis, culminating with the oral use of the drug auranofin (Figure 1) in 1985 [4], for the treatment of this pathology. Another milestone of modern medicine involves the metal-complex cisdiamminedichloroplatinum(II) (cisplatin or platinol) (Figure 1), first synthesized in 1845 by the Italian chemist Michele Peyrone [5]. However, it was the chemists Barnett Rosenberg and Loretta VanCamp who, in 1965, while studying the effect of electric current on Escherichia coli, reported biological actions of cisplatin. Rosenberg's work culminated with the discovery of cisplatin as one of the most well-known antitumor agent of all times, leading to development of a series of analogues, which have been used in ca. 50% of all cancer treatments [5,6].

Table of contents _________________________________________________________________________________

Table of Contents

Relevant properties of metal complexes within the therapeutic context

Metal complexes provide highly versatile platforms for drug design. In addition to the variations in the metal nature and oxidation state, metal ions adopt diverse geometries and coordination numbers, allowing the adjustment of chemical reactivity in terms of kinetics (bond exchange rates) and thermodynamics (metal-ligand bond strengths, redox potential, etc.) [7]. Not only the metal, but also the ligands are anticipated to play important roles in the biological activity, such as recognition of the outer sphere by a target, or biological action resulting from the release of the ligand [7].

According to a brief summary cited by Haas and Franz [8], the properties that make a platform of metallic complexes useful and versatile in biology are: i.

Charge Variation -"Metal ions are positively charged in aqueous solution, but that charge can be manipulated depending on the coordination environment so that a metal complexed by ligands can be cationic, anionic, or neutral."

ii.

Metal-Ligand Interaction -"Metal ions bind to ligands via interactions that are often strong and selective. The ligands impart their own functionality and can tune properties of the overall complex that are unique from those of the individual ligand or metal. The thermodynamic and kinetic properties of metal-ligand interactions influence ligand exchange reactions."

iii.

Structure and Bonding -"Metal-ligand complexes span a range of coordination geometries that give them unique shapes compared with organic molecules. The bond lengths, bond angles, and number of coordination sites can vary depending on the metal and its oxidation state." iv.

Lewis Acid Properties -"Metal ions with high electron affinity can significantly polarize groups that are coordinated to them, facilitating hydrolysis reactions."

v.

Partially Filled d-shell -"For the transition metals, the variable number of electrons in the d-shell orbitals (or f-shell for lanthanides) imparts interesting electronic and magnetic properties to transition metal complexes."

vi.

Redox Activity -"Coupled with the variability of electrons in the d-shell is the ability for many transition metals to undergo one-electron oxidation and reduction reactions."

Despite these advantages, the current diversity of metal complexes with medical therapeutic application is still limited. However, metallo-drugs are more widely represented in the category of diagnostic compounds (gadolinium, technetium, indium compounds among others) [1,[9][10][11]. As part of this thesis, the next section will focus on therapeutic metal complexes with cardiovascular, cancer and tuberculosis interests.

Anticancer metallo-drugs

Cisplatin, cis-(NH3)2PtCl2, was the first metal-based drug used clinically against cancer (Figures 1 and2) [1]. Currently, it is believed that its mode of cytotoxic action is mainly due to its ability to interact with DNA by forming stable adducts. Replacement of anionic Cl -ligands by neutral H2O ligands, mediated by the variation in chloride concentration inside of the cells, has been identified as the main route of activation. The globally cationic hydrolyzed complex [(NH3)2PtCl2-x(H3O)x] x+ , is a potent electrophile agent able to react with any nucleophile, including sulfhydryl groups on proteins and nitrogen donor atoms on nucleic acids. Cisplatin therapy including combination with other drugs has been considered as a standard protocol against many types of cancer [12]. Nevertheless, new platinum drugs were investigated due to serious side effects of cisplatin, in addition to the aim of prolonging the use inducing drug resistance. Other platinum(II)-containing anticancer drugs such as carboplatin, approved in 1989 by the Food and Drug Administration (FDA), and oxaliplatin, approved in 2002 by FDA, have thus been also used (Figure 2) [1,12]. New Pt II complexes, nedaplatin, lobaplatin, and heptaplatin (Figure 2), were prepared and clinically used in Japan, China, and South Korea, respectively [13]. Beside this, low spin 5d 6 Pt IV octahedral complexes have also been explored as potential anticancer agents, such as satraplatin (JM216) (Figure 2), the first orally administered platinum analogue, which is currently in advanced Phase III clinical trials [START_REF] Zhong | Targeting drug delivery system for platinum(Ⅳ)-Based antitumor complexes[END_REF].

Like platinum (Pt), substances containing arsenic (As) are also examples of approved drugs and in clinical studies for the treatment of cancer (Figure 3). Arsenic trioxide (trisenox) was approved by the FDA in 2000 particularly for the treatment of promyelocytic leukemia, but its toxicity, as well as the substantially reduced antitumor effects, limit its use in the treatment of other non-APL (Acute Promyelocytic Leukemia) cancers [START_REF] Xu | S-Dimethylarsino-glutathione (Darinaparsin ® ) targets histone H3.3, leading to TRAIL-induced apoptosis in leukemia cells[END_REF]. Liu and co-workers [START_REF] Kaiming | Arsenic trioxide preferentially binds to the ring finger protein PML: understanding target selection of the drug[END_REF], recently indicated that the main target of trisenox, responsible for its cytotoxic action, is the promyelocytic leukemia protein (PML). Therefore, new therapeutic agents based on As have been developed, such as Sdimethylarsino-glutathione (Darinaparsin ® , ZIO-101) (Figure 3), a new chemotherapy agent in Phases I/II of clinical trial, which has shown more potent anticancer properties against various types of solid tumors and hematologic malignancies, both in vitro and in vivo [START_REF] Xu | S-Dimethylarsino-glutathione (Darinaparsin ® ) targets histone H3.3, leading to TRAIL-induced apoptosis in leukemia cells[END_REF]. Its molecular mechanism of action remains unclear; recently, however, Sun and collaborators [START_REF] Xu | S-Dimethylarsino-glutathione (Darinaparsin ® ) targets histone H3.3, leading to TRAIL-induced apoptosis in leukemia cells[END_REF] showed that darinaparsin (Figure 3), when interacts with nucleosome, destabilizes this latter, leading to cellular apoptosis. Problems related to platinum metallo-drugs in the treatment of cancer have also stimulated the search for anticancer drugs containing ruthenium. Ruthenium complexes have emerged as a promising alternative to platinum-based chemotherapy.

Imidazolium(imidazole)(dimethylsulfoxide)tetrachlororuthenate(III) (NAMI-A) (Figure 4) was the first ruthenium complex to achieve clinical investigations in humans. It was synthesized for the first time by Mestroni and collaborators in Italy [START_REF] Barry | Exploration of the medical periodic table: towards new targets[END_REF]. In preclinical 4) was another ruthenium derivative that entered in Phase I clinical trial, however its low solubility limited its progress in the later stages. Nevertheless, its sodium salt derivative, KP1339 (Figure 4), has advanced in clinical tests [START_REF] Thota | Ru(II) Compounds: Next-Generation Anticancer Metallotherapeutics?[END_REF]. Finally, [Ru(II)(4,4′-dimethyl-2,2′-bipyridine(dmb))2(2-(2′,2′′:5′′,2′′′-terthiophene)-imidazo [4,5-f][1,10]phe-nanthroline)]Cl2 (TLD1433) (Figure 4) is the first metal-based photosensitizer, not containing a tetrapyrrolic fragment, to enter in clinical trials for non-muscle invasive bladder cancer treatment via photodynamic therapy (PDT). It is currently in Phase I/II clinical trials [START_REF] Thota | Ru(II) Compounds: Next-Generation Anticancer Metallotherapeutics?[END_REF][START_REF] Imberti | New Designs for Phototherapeutic Transition Metal Complexes[END_REF]. Gallium (Ga) and molybdenum (Mo) compounds (Figure 5) are also included in the list of chemotherapeutic agents in clinical testing phases. Some examples are:

gallium nitrate (Ga(NO3)3) (Figure 5), that displayed antineoplastic activity against non-Hodgkin's lymphoma and bladder cancers in Phase I/II clinical trials [START_REF] Chitambar | Gallium-containing anticancer compounds[END_REF], and two hexacoordinate complexes: gallium(III) tris(maltolate) (Figure 5), which is in Phases I/II of clinical tests in patients with prostatic neoplasms, multiple myeloma, bladder neoplasms and lymphoma, (ClinicalTrials.gov Identifier: NCT00050687) [21] and tris (8-quinolato)gallium(III) (KP46) (Figure 5) successfully tested in a Phase I clinical trial against kidney cancer [START_REF] Kubista | Distinct activity of the bonetargeted gallium compound KP46 against osteosarcoma cells -synergism with autophagy inhibition[END_REF]. It is believed that the antiproliferative activity of these two complexes is related to the inhibition of ribonucleotide reductase, the irondependent enzyme essential for DNA synthesis, triggering apoptosis [7]. Bis-Choline tetrathiomolybdate (ATN-224) (Figure 5), is a molybdenum(VI) metallo-drug derivative, currently in Phases I/II of clinical tests, with patients affected by various forms of cancer (melanoma, breast, colon, kidney, and relapsed prostate cancer). These studies indicate that the possible chemotherapeutic action of ATN-224 may be related to its ability to chelate and inhibit copper-transporting ATPases [START_REF] Baldari | Current Biomedical Use of Copper Chelation Therapy[END_REF].

Cardiovascular metallo-drugs

The number of metallo-drugs used for the treatment of cardiovascular disorders in clinical trials is much lower than the number of complexes developed for the treatment of cancer. Currently, the only metallic drug used for cardiovascular therapeutic purposes is sodium nitroprusside (SNP, Na2[Fe(CN)5NO]) (Figure 6), an

Fe II complex used in cardiac surgery, hypertensive crises, heart failure, vascular surgery, pediatric surgery, and other acute applications [START_REF] Hottinger | Sodium nitroprusside in 2014: A clinical concepts review[END_REF]. SNP is the first metal complex used in cardiovascular therapy, discovered by Playfair in 1849. The effectiveness of this metallo-drug, as a hypertensive agent, was only established in 1955 [START_REF] Hottinger | Sodium nitroprusside in 2014: A clinical concepts review[END_REF][START_REF] Page | Cardiovascular actions of sodium nitroprusside in animals and hypertensive patients[END_REF]. The pharmacological action of SNP is based on its ability to release NO • .

However, the rapid release of NO • , and the emergence of the cyanide toxicity, has been suggested as the main causes of the side effects observed for this prodrug [START_REF] Hottinger | Sodium nitroprusside in 2014: A clinical concepts review[END_REF]. Several other metal complexes have been developed and tested as less toxic and more efficient agents than SNP, however none of the representatives have yet participated in clinical trials yet. Some of these complexes will be described in the section 2.6. Oxacom ® , the dinitrosyl iron complex (DNIC) (Figure 6), is a promising candidate for the development of a cardiovascular metallo-drug as it has proven to be an effective hypotensive drug, with no adverse effects reported so far. It is currently in Phase I/II clinical trials [START_REF] Hsiao | Fe in biosynthesis, translocation, and signal transduction of NO: toward bioinorganic engineering of dinitrosyl iron complexes into NO-delivery scaffolds for tissue engineering[END_REF]. The endogenous effects of Oxacom ® are related to its ability to delivery NO • , with sGC (soluble guanylate cyclase) as its main target, similar to SNP. The process of sGC activation via NO • and the consequent vasorelaxation activity will be detailed in the section 2.3.2 paragraph 6. M40403, the SOD mimicking manganese(II) (pentaaza)macrocycle (Figure 6), is another example of a hypotensive metallo-drug in clinical trials (Phase I/II clinical trials currently suspended). It is indicated that its action is related to its ability to mimic the activity of superoxide dismutase, eliminating superoxide radicals and exerting beneficial effects on models of intestinal ischemia-reperfusion, and on acute inflammation [START_REF] Bonetta | Potential Therapeutic Applications of MnSODs and SOD-Mimetics[END_REF].

Antituberculosis metallo-drugs

During the years of 1925 -1935, also known as the "Gold Decade", gold cyanide, sodium aurothiomalate, and sodium aurothiosulfate Au I compounds were used for the treatment of pulmonary tuberculosis. Although auranofin is in clinical trials (Phase II) as a possible agent in host directed therapy for tuberculosis in combination with rifabutin [START_REF] Adeniji | Potential anti-TB investigational compounds and drugs with repurposing potential in TB therapy: a conspectus[END_REF], the majority of gold-based metallo-drugs available today are classified as antirheumatic drugs [1,[START_REF] Gunatilleke | Tuning the Au(I)-mediated inhibition of cathepsin B through ligand substitutions[END_REF]. At the date of this thesis, there is no metallodrug clinically approved for use in tuberculosis. Although there are a significant number of metal complexes with a potential antitubercular activity proven [START_REF] Mccarron | Unprecedented in Vitro Antitubercular Activitiy of Manganese(II) Complexes Containing 1,10-Phenanthroline and Dicarboxylate Ligands: Increased Activity, Superior Selectivity, and Lower Toxicity in Comparison to Their Copper(II) Analogs[END_REF][START_REF] Lopes | A bioinorganic chemistry perspective on the roles of metals as drugs and targets against Mycobacterium tuberculosis -a journey of opportunities[END_REF], few or none are in pre-clinical or clinical phases of study and, in most cases, their mechanisms of action have rarely been explored. Some examples of these complexes are illustrated in Figure 7, their respective minimum inhibitory concentration (MIC) values against Mtb H37Rv cells are also presented [START_REF] Mccarron | Unprecedented in Vitro Antitubercular Activitiy of Manganese(II) Complexes Containing 1,10-Phenanthroline and Dicarboxylate Ligands: Increased Activity, Superior Selectivity, and Lower Toxicity in Comparison to Their Copper(II) Analogs[END_REF][START_REF] Lopes | A bioinorganic chemistry perspective on the roles of metals as drugs and targets against Mycobacterium tuberculosis -a journey of opportunities[END_REF][START_REF] Stringer | Antimicrobial activity of organometallic isonicotinyl and pyrazinyl ferrocenyl-derived complexes[END_REF][START_REF] Plutín | Anti-Mycobacterium tuberculosis activity of platinum(II)/N,N-disubstituted-N′-acyl thiourea complexes[END_REF][START_REF] Ahmed | Synthesis and antimicrobial activity of a phenanthroline-isoniazid hybrid ligand and its Ag + and Mn 2+ complexes[END_REF][START_REF] Oliveira | Manganese(II) complexes with thiosemicarbazones as potential anti-Mycobacterium tuberculosis agents[END_REF][START_REF] Aguiar | Antitubercular activity of Ru (II) isoniazid complexes[END_REF].

Figure 7.

Promising metal-complex antitubercular compounds and their minimum inhibitory concentration (MIC) against Mtb H37Rv cells.

Nonetheless, Sousa e co-workers [START_REF] Lopes | A bioinorganic chemistry perspective on the roles of metals as drugs and targets against Mycobacterium tuberculosis -a journey of opportunities[END_REF], synthesized an isoniazid metal complex with pentacyanoferrate(II) moiety, called IQG607 (Na3[Fe(CN)5(INH)], INH = isoniazid) (Figure 7). This compound showed significantly promising inhibitory activity against Mtb H37Rv cells (MIC = 0.43 μM), in addition to the in vitro inhibition of the enoyl-ACP reductase enzyme (InhA), even without using catalase-peroxidase KatG enzyme [START_REF] Lopes | A bioinorganic chemistry perspective on the roles of metals as drugs and targets against Mycobacterium tuberculosis -a journey of opportunities[END_REF]. The mechanistic studies carried out by Sousa [START_REF] Sousa | Fe(CN)5(isoniazid)] 3-: an iron isoniazid complex with redox behavior implicated in tuberculosis therapy[END_REF] and collaborators indicated that the action of this complex may be related to its ability to be oxidized by biologically relevant agents (e.g H2O2, O2 -). Once oxidized, IQG607 is converted to its active isonicotinoyl radical form (INH active species), responsible, after reaction with NADH, for inhibition of an InhA of Mtb. Inhibition of this enzyme involves interruption of mycolic acid bio-synthesis, an essential constituent of the bacterial cell wall. Unlike INH, IQG607 could be a potential anti-TB metallo-drug targeting InhA without depending on catalase-peroxidase KatG enzyme. Although this mechanistic route may not occur in vivo [START_REF] Abbadi | Revisiting Activation of and Mechanism of Resistance to Compound IQG-607 in Mycobacterium tuberculosis[END_REF], IQG607 has exhibited exciting toxicological and pharmacological profiles, indicating also a potential to reduce the lengthy treatment once in combination with other first-line drugs [START_REF] Rodrigues-Junior | Nonclinical evaluation of IQG-607, an anti-tuberculosis candidate with potential use in combination drug therapy[END_REF]. These promising biological activities of IQG607 have stimulated the advances in pre-clinical studies [START_REF] Rodrigues-Junior | Nonclinical evaluation of IQG-607, an anti-tuberculosis candidate with potential use in combination drug therapy[END_REF][START_REF] Rodrigues-Junior | Toxicological profile of IQG-607 after single and repeated oral administration in minipigs: An essential step towards phase I clinical trial[END_REF][START_REF] Abbadi | Is IQG-607 a Potential Metallodrug or Metallopro-Drug With a Defined Molecular Target in Mycobacterium tuberculosis?[END_REF][START_REF] Rodrigues-Junior | Preclinical safety evaluation of IQG-607 in rats: Acute and repeated dose toxicity studies[END_REF][START_REF] Dadda | Preclinical pharmacokinetic profiling of IQG-607, a potential oral metallodrug to treat tuberculosis[END_REF]. • ) and nitroxyl (HNO) in medicine and their donors as therapeutic platforms

Nitric oxide (NO

Nitric oxide (NO • )

The role of nitric oxide (NO • ) in biology was elucidated in the early 1980s, when researchers understood that NO • was the endothelium-derived relaxing factor (EDRF), which is involved in the stimulation of the soluble guanylate cyclase protein (sGC) via interaction with the heme group [START_REF] Palmer; Ferrige | Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor[END_REF][START_REF] Follmann | The chemistry and biology of soluble guanylate cyclase stimulators and activators[END_REF]. These discoveries led Robert F. Furchgott, Louis J. Ignarro and Ferid Murad to be awarded the Nobel Prize in Medicine or Physiology in 1998.

Biological production of nitric oxide

This small molecule is produced endogenously via the oxidation of L-arginine to L-citrulline, catalyzed by three types of isoenzymes called nitric oxide synthases (NOS)

(Figure 8A and B) [START_REF] Knowles | Nitric oxide synthases in mammals[END_REF][START_REF] Forstermann | Nitric oxide synthases: regulation and function[END_REF]. The main isoforms are known as: neuronal (nNOS or NOS1) [START_REF] Zhou | Neuronal nitric oxide synthase: structure, subcellular localization, regulation, and clinical implications[END_REF], inducible (iNOS or NOS2) [START_REF] Taylor | Molecular regulation of the human inducible nitric oxide synthase (iNOS) gene[END_REF], and endothelial enzymes (eNOS or NOS3) [START_REF] Forstermann | Nitric oxide synthases: regulation and function[END_REF][START_REF] Raman | Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center[END_REF], but there are others [START_REF] Daff | NO synthase: Structures and mechanisms[END_REF]. Most of the known effects of NO • are directly related to the stimulation of sGC (Figure 8C) [START_REF] Follmann | The chemistry and biology of soluble guanylate cyclase stimulators and activators[END_REF][START_REF] Ritchie | The opposing roles of NO and oxidative stress in cardiovascular disease[END_REF]. This stimulus occurs through the NO • bond to the heme-iron unity of the beta subunit of sGC causing conformational changes that accelerate the conversion of guanosine-5'-triphosphate (GTP) into cyclic guanosine-3′,5′-monophosphate (cGMP) and pyrophosphate (PPi) [START_REF] Horst | Physiological activation and deactivation of soluble guanylate cyclase[END_REF][START_REF] Kang | Structural insights into the mechanism of human soluble guanylate cyclase[END_REF]. The important secondary messenger cGMP is responsible to trigger a series of downstream processes in the body [START_REF] Schermuly | Expression and function of soluble guanylate cyclase in pulmonary arterial hypertension[END_REF][START_REF] Komsuoglu-Celikyurt | Evidence for the involvement of neuronal nitric oxide synthase and soluble guanylate cyclase on cognitive functions in rats[END_REF][START_REF] Wang | Stimulation of soluble guanylate cyclase slows progression in anti-thy1-induced chronic glomerulosclerosis[END_REF][START_REF] Baltrons | Dexamethasone Up-Regulates a Constitutive Nitric Oxide Synthase in Cerebellar Astrocytes but Not in Granule Cells in Culture[END_REF][START_REF] Mülsch | Stimulation of soluble guanylate cyclase by endothelium-derived relaxing factor from cultured endothelial cells[END_REF]. Panel C shows NO • diffusion from its production site (e.g., endothelial cells) into another cell reaching soluble guanylate cyclase (sGC), whose enzymatic activity is strongly stimulated with conversion of GTP to cGMP.

A one-electron reduced form of nitric oxide, called nitroxyl (HNO) [START_REF] Miranda | The chemistry of nitroxyl (HNO) and implications in biology[END_REF], has also brought considerable attention due to some distinct biological and/or pharmacological properties. This molecule exhibits protective effects against reperfusion damage for the treatment of myocardial infarction [START_REF] Pagliaro | Nitroxyl affords thiol-sensitive myocardial protective effects akin to early preconditioning[END_REF], ability to improve myocardial contractility in the treatment of heart failure [START_REF] Katori | Peroxynitrite and myocardial contractility: in vivo versus in vitro effects[END_REF], aldehyde dehydrogenase inhibitory activity in the treatment of alcoholism [START_REF] Shoeman | Reaction of nitroxyl, an aldehyde dehydrogenase inhibitor, with N-acetyl-L-cysteine[END_REF], and tumor growth inhibitory property and antiangiogenic effect in cancer treatment [START_REF] Augustyniak | Cytotoxicity of nitroxyl (HNO/NO -) against normal and cancer human cells[END_REF][START_REF] Norris | Nitroxyl inhibits breast tumor growth and angiogenesis[END_REF].

Nitroxyl (HNO)

Nitroxyl (HNO), a small molecule whose pKa is ca. 11.4 [START_REF] Fukuto | The chemistry and biology of nitroxyl (HNO): a chemically unique species with novel and important biological activity[END_REF][START_REF] Paolocci | The pharmacology of nitroxyl (HNO) and its therapeutic potential: not just the Janus face of NO[END_REF], was first mentioned by the Italian scientist Angelo Angeli, who obtained experimental evidence for the release of HNO and nitrate from sodium trioxodinitrate salt (N2Na2O3) [START_REF] Pagliaro | Nitroglycerine and sodium trioxodinitrate: from the discovery to the preconditioning effect[END_REF][START_REF] Paolocci | The shy Angeli and his elusive creature: the HNO route to vasodilation[END_REF].

The currently known Angeli's salt (AS) was placed in evidence as the first HNO donor with unique pharmacological properties, even before the discovery of the biological roles of NO • [START_REF] Pagliaro | Nitroglycerine and sodium trioxodinitrate: from the discovery to the preconditioning effect[END_REF]. Endogenous HNO production in human is still speculative, requiring further studies, which have motivated the scientific community [START_REF] Fukuto | The chemistry and biology of nitroxyl (HNO): a chemically unique species with novel and important biological activity[END_REF][START_REF] Eberhardt | H2S and NO cooperatively regulate vascular tone by activating a neuroendocrine HNO-TRPA1-CGRP signalling pathway[END_REF][START_REF] Cortese-Krott | Key bioactive reaction products of the NO/H2S interaction are S/N-hybrid species, polysulfides, and nitroxyl[END_REF][START_REF] Li | Recognition of Exogenous and Endogenous Nitroxyl in Living Cells via a Two-Photon Fluorescent Probe[END_REF][START_REF] Donzelli | Oxidant sensor in the cGMPbinding pocket of PKGIalpha regulates nitroxyl-mediated kinase activity[END_REF][START_REF] Alvarez | The Underlying Mechanism of HNO Production by the Myoglobin-Mediated Oxidation of Hydroxylamine[END_REF]. One of the main issues contributing to this scenario is the fast reactivity of HNO, which decomposes reacting with itself to generate N2O (k = (8 ± 3) x 10 6 M -1 s -1 ) [START_REF] Shafirovich | Nitroxyl and its anion in aqueous solutions: spin states, protic equilibria, and reactivities toward oxygen and nitric oxide[END_REF], with NO • (k = (5.8 ± 0.4) x 10 6 M -1 s -1 ) [START_REF] Shafirovich | Nitroxyl and its anion in aqueous solutions: spin states, protic equilibria, and reactivities toward oxygen and nitric oxide[END_REF] or O2 ((1.8 ± 0.3) x 10 4 M -1 s -1 ) [START_REF] Smulik | Nitroxyl (HNO) reacts with molecular oxygen and forms peroxynitrite at physiological pH Biological implications[END_REF] (Scheme 1).

Scheme 1. HNO decomposition reactions followed by their respective constants [START_REF] Shafirovich | Nitroxyl and its anion in aqueous solutions: spin states, protic equilibria, and reactivities toward oxygen and nitric oxide[END_REF][START_REF] Smulik | Nitroxyl (HNO) reacts with molecular oxygen and forms peroxynitrite at physiological pH Biological implications[END_REF].

During the last decades, several pathways for HNO endogenous production have been proposed [START_REF] Alvarez | The Underlying Mechanism of HNO Production by the Myoglobin-Mediated Oxidation of Hydroxylamine[END_REF][START_REF] Paolocci | From Heaven to Heart: Nitroxyl (HNO) in the Cardiovascular System and Beyond[END_REF]. The first one is associated to the absence of the cofactor tetrahydrobiopterin (H4B) during endogenous production of NO • as a way to generate HNO [78]. This cofactor works in different ways during the synthesis of NO • , such as by stabilizing a dimeric structure of NOS (nitric oxide synthases) [79-81], accelerating the decomposition of the complex dioxygen-ferrous heme (NOS-Fe II (O2)) [78,82], and increasing the rate of consumption of NADPH (Figure 8B) [83,84]. In addition, this cofactor is essential for the biosynthesis of NO • rather than for that of HNO [START_REF] Daff | NO synthase: Structures and mechanisms[END_REF]85]. So, it is possible that under these conditions of low concentrations or in absence of H4B, the production of HNO is favored. Based on that, mechanistic studies suggested NOS-Fe II (O2 •-) would react with N-omega-hydroxy-L-arginine (NOHA), converting this hydroxamic intermediate into L-citrulline, water, ferric enzyme NOS-Fe III and HNO [START_REF] Daff | NO synthase: Structures and mechanisms[END_REF]78]. Other possible pathways for HNO biosynthesis are the decomposition of nitrosothiols [86,87], through nitrosothiol enzymatic reduction [88][START_REF] Niketić | Exposure of Mn and FeSODs, but not Cu/ZnSOD, to NO leads to nitrosonium and nitroxyl ions generation which cause enzyme modification and inactivation: an in vitro study[END_REF][START_REF] Saleem | Xanthine oxidase converts nitric oxide to nitroxyl that inactivates the enzyme[END_REF], and oxidation of hydroxylamine (NH2OH) by hemeproteins [START_REF] Alvarez | The Underlying Mechanism of HNO Production by the Myoglobin-Mediated Oxidation of Hydroxylamine[END_REF][START_REF] Paolocci | From Heaven to Heart: Nitroxyl (HNO) in the Cardiovascular System and Beyond[END_REF], which will be discussed below.

HNO endogenous synthesis by NOS

While there is a consensus on the mechanism of NO • biosynthesis, HNO endogenous production is still poorly understood. This is likely due to the high instability of HNO and the lack of effective detection methods [START_REF] Li | Recognition of Exogenous and Endogenous Nitroxyl in Living Cells via a Two-Photon Fluorescent Probe[END_REF][START_REF] Miao | Recent advances in the chemical biology of nitroxyl (HNO) detection and generation[END_REF]. Nonetheless, there are some proposed routes for HNO production in cells. As mentioned before, one of the first proposals was based on the conventional NO • biosynthesis pathways in conditions of low H4B concentration [START_REF] Daff | NO synthase: Structures and mechanisms[END_REF]78,80,[82][83][84]. In the endogenous NO • synthesis, the absence of H4B increases the probability of HNO release rather than NO Scheme 2 shows the proposed mechanism of HNO production from Larginine/NOS/NADPH system without H4B cofactor.

Scheme 2. The biological synthesis of NO • and likely production of HNO by NOS, references [START_REF] Daff | NO synthase: Structures and mechanisms[END_REF]78,80,83,84].

HNO endogenous production mediated by nitrosothiols decomposition

Another possible endogenous route for the production of HNO arises from the decomposition of nitrosothiols (RSNO), which is considered the most physiologically relevant pathway [86,87,[START_REF] Filipovic | Chemical characterization of the smallest S-nitrosothiol, HSNO; cellular cross-talk of H2S and S-nitrosothiols[END_REF]. RSNO are products of the reaction between nitric oxide and thiols (RSH). Thiols are biomolecules that play important biological roles and can be found in abundance as large (cysteine rich proteins) or small molecules [START_REF] Vos | Role of endogenous thiols in protection[END_REF][START_REF] Biswas | Redox modifications of protein-thiols: emerging roles in cell signaling[END_REF]. The homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CysGly), and glutathione (GSH) are examples of thiols performing vital functions in physiological processes. These molecules can be found in cells in different concentration ranges (from 5-15 µM for Hcy to 0.5-10 mM for GSH) [START_REF] Tian | A fluorescent probe for intracellular cysteine overcoming the interference by glutathione[END_REF][START_REF] Mansoor | Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma[END_REF][START_REF] Ueland | Total homocysteine in plasma or serum: methods and clinical applications[END_REF]. RSNO is produced by the reaction of NO • with thiols under aerobic conditions, where nitrogen oxides play a critical role (Scheme 3) [START_REF] Zhang | S-Nitrosothiols: chemistry and reactions[END_REF].

Scheme 3. Nitrosothiol (RSNO) production under aerobic conditions, reference [START_REF] Zhang | S-Nitrosothiols: chemistry and reactions[END_REF].

Some authors also reported the production of RSNO through a reaction of the nitrosonium cation (NO + ) with thiolate anion (RS -) [START_REF] Kevil | S-Nitrosothiol biology and therapeutic potential in metabolic disease[END_REF][100][101]. However, free NO + is stable only at low pH (~ 2.0) [102], while thiolate formation, dependent on the nature of the molecule and pH medium, is more abundant at pH > 7 [103-107]. Due to these properties, this route is likely less relevant in biology.

HNO production by enzymatic reduction

Enzymatic reduction of NO • mediated by superoxide dismutase (SOD) or xanthine oxidase (XO) under anaerobic conditions has been described as a potential route for HNO production. SOD can directly reduce NO • to HNO in the absence of O2 through a reversible process [88,[START_REF] Niketić | Exposure of Mn and FeSODs, but not Cu/ZnSOD, to NO leads to nitrosonium and nitroxyl ions generation which cause enzyme modification and inactivation: an in vitro study[END_REF]. In another reductive process, XO catalyzes an expected irreversible process where hypoxanthine is used as an electron donor to reduce NO • to HNO, in the absence of oxygen [START_REF] Saleem | Xanthine oxidase converts nitric oxide to nitroxyl that inactivates the enzyme[END_REF]. However, these routes of production of HNO are still under debate, particularly considering the methodology employed for HNO detection, which is susceptible to bring significant mistakes [108,109].

Another protein earlier claimed to produce HNO was the cytochrome c via a direct reduction of NO • to HNO, under anaerobic conditions [110], however, major doubts were raised due to the probe used during those studies. Later, the same team has concluded HNO was indeed not produced discarding this route as a biological option

[111].

HNO production by hydroxylamine oxidation

Another possible pathway for the biosynthesis of HNO is the oxidation of hydroxylamine (NH2OH) to HNO mediated by heme proteins using H2O2 [START_REF] Alvarez | The Underlying Mechanism of HNO Production by the Myoglobin-Mediated Oxidation of Hydroxylamine[END_REF][112][113][114].

Wink and coworkers found that heme proteins containing proximal histidine ligand (e.g., horseradish peroxidase, lactoperoxidase, myeloperoxidase, myoglobin, and hemoglobin) could catalyze the conversion of NH2OH to HNO, in the presence of hydrogen peroxide (H2O2) [112]. Recently, Álvarez and coworkers confirmed this hypothesis, reporting that myoglobin catalyzes HNO production via peroxidation of NH2OH [START_REF] Alvarez | The Underlying Mechanism of HNO Production by the Myoglobin-Mediated Oxidation of Hydroxylamine[END_REF].

Hydroxylamine is a cellular metabolic product commonly found in physiological environments [113,115,116], its oxidation into HNO is caused by the intermediate heme Fe IV• =O formed by the reaction of heme Fe III with H2O2 found in the cellular medium [117,118]. This reaction involves the heme Fe IV• =O, which oxidizes NH2OH that decomposes into HNO and H2O, restoring heme Fe III as described in Scheme 4 [112,119,120].

Scheme 4. HNO synthesis via oxidation of hydroxylamine (NH2OH) mediated by heme proteins and H2O2 [112,[117][118][119][120].

The pharmacological importance of NO • and HNO in cancer, cardiovascular and tuberculosis diseases

We have already mentioned that NO • and HNO play key roles in various physiological processes [121][122][123]. In the context of this thesis, only the implication of NO • /HNO in cancer, cardiovascular and tuberculosis affections will be considered. A summary of each pathology will be addressed below mainly in order to explain how HNO can be considered as pertinent therapeutic agent. This last approach is a known way how NO • /HNO exert their anticancer action.

Cancer treatment can occur through one or more combined treatment modalities/techniques. The combination of chemotherapy and targeted therapy, for example antiangiogenic drugs, have increased the progression-free survival (PFS, time period during and after treatment, in which a patient lives with the disease, but it does not get worse) of patients with metastatic cancer (pancreatic, colorectal, and breast), compared to treatment using chemotherapy alone [126,127,144,145]. These results helped to validate the concept of antiangiogenic agents in the treatment of cancer.

Several antiangiogenic molecules have been approved by the Food and Drug Administration (FDA) for use as anticancer agents in combination with chemotherapy [127]. Figure 9 illustrates some examples of approved antiangiogenic drugs with their respective therapeutic indications. Angiogenesis is a normal and complex process controlled by certain biomolecules produced in the body, and consists of the growth of new blood vessels from existing ones [148]. These new blood vessels will serve to take up the oxygen necessary to the growth of the cancer cell [126].

In cancer cells, the production of pro-angiogenic factors is induced in hypoxia conditions (caused by abnormal accelerated metabolism), which leads to the rapid and chaotic formation of blood vessels. Adaptation to different levels of oxygen in the cell (normal and cancer cells) is mediated by a family of transcriptional regulators, called hypoxia-inducible factors (HIFs) [127]. HIFs are heterodimers consisting of an oxygenindependent β-subunit (HIF-β) and an oxygen-dependent α-subunit (HIF-α), which has three isoforms (HIF-1α, HIF-2α and HIF-3α) [127].

Under normoxia, transcriptional activity of HIF is specifically suppressed through the degradation of the HIF-1α subunits, via a hydroxylation process mediated by an asparaginyl hydroxylase factor. Low oxygen conditions induce the stabilization of HIF-1α, promoting the upregulation of several pro-angiogenic genes including VEGF (vascular endothelial growth factor), FGF (fibroblast growth factor), and PDGF (platelet derived growth factor) [127,149]. The latters are responsible for inducing the formation of endothelial cells composing the blood vessels [148]. Thus, these different proangiogenic factors have been considered as promising targets for anticancer therapy [126,127,145].

NO • and HNO have played a key role in the regulation of HIF-1α in tumor cells.

While NO • has been indicated as a stabilizing agent for HIF-1α in normoxic conditions, HNO has been shown to act in the opposite way, blocking HIF-1α stabilization, induced by both hypoxia and NO • in normal air conditions, and hence inducing antiangiogenesis and cell death by apoptosis [START_REF] Norris | Nitroxyl inhibits breast tumor growth and angiogenesis[END_REF][150][151][152]. These properties open doors for the use of HNO donor agents as new and promising anticancer platforms. In the section 2. 10, their classification within the group of antihypertensive drugs is also indicated [165,166].

Figure 10. Some examples of cardiovascular drugs, followed in the parentheses by their pharmacological class.

Vasodilation is one of the main therapeutic strategies to obtain an antihypertensive effect. In this thesis, we are particularly interested on the antihypertensive effects of NO • /HNO. From a mechanistic point of view, after the production of NO • in the body, either endogenously or through donor drugs, NO • readily diffuses through cell membranes, until it reaches its main target, soluble guanylate cyclase (sGC) protein (Figure 11) [START_REF] Kang | Structural insights into the mechanism of human soluble guanylate cyclase[END_REF]. sGC is a heterodimeric protein made up of two subunits α and β. The β subunit has the prosthetic group (heme), which has a strong affinity for NO • . When NO • binds to Fe II -heme of sGC, it disrupts the bond between Fe II and the histidine residue (105) of the protein, a phenomenon also called trans-labilizing effect of NO • . As a result of the previous step, a significant conformational change in sGC is initiated in order to activate the catalytic site of the enzyme. This activation promotes an increase in Mg 2+ dependent catalytic activity, resulting in the conversion of GTP into PPi and cGMP [START_REF] Kang | Structural insights into the mechanism of human soluble guanylate cyclase[END_REF]. The latter regulates the effect of several biological systems such as cGMP-dependent protein kinases, ion channels, and phosphodiesterases, responsible for mediating several physiological processes including vasodilation [167]. HNO also acts as a potent vasodilator, however, despite numerous mechanisms for HNO mediated vasodilation have been proposed, including NO The treatment of TB consists of the use of vaccine and antibiotics. In the case of treatment through vaccination, Bacillus Calmette -Guérin (BCG) is the only one specifically used to prevent severe forms of tuberculosis (TB meningitis and miliary TB) in children [182]. Another alternative is the use of antibiotics that are divided into first-, second-and third-line drugs. The treatment modalities depend on the bacteria's sensitivity to the antibiotics used. Details are presented below.

iv. First-line antitubercular drugs

Treatment regimen for all new cases of all forms of pulmonary and extrapulmonary tuberculosis (except meningoencephalitis), as well as for all cases of relapse and return after default, consists of 6 months. In the first 2 months, there is a daily intake of the four first-line drugs rifampicin, isoniazid (INH), pyrazinamide (PZA) and ethambutol (Figure 12), followed by another 4 months, using only rifampicin and INH, in order to ensure the complete extermination of Mtb from the host body [183]. Within the context of this thesis, delamanid stands out because it is reported as a HNO donor prodrug with anti-TB action. Despite delamanid is known to inhibit methoxy mycolic and keto-mycolic acids biosynthetic pathways, its mode of action at molecular level remains uncertain [188]. It has been shown that to exert its anti-TB activity, an intracellular bioreductive activation via mycobacterial nitroreductase enzyme (Ddn) is required, consequently producing HNO/NO • as potential active metabolites (scheme 5) [189]. In fact, it has been proposed that HNO and NO • have anti-TB action, however the way, in which these molecules act against Mtb is still not well elucidated [190,191].

Scheme 5. Proposed mechanism for the activation reaction of delamanid by the Ddn enzyme, reference [188].

The pharmacological properties of NO • /HNO, discussed above, offer new medical perspectives for the use of NO • /HNO as a promising new anticancer, antihypertensive and anti-TB agent. As NO • /HNO are molecules highly unstable with no ideal chemical and pharmacokinetic properties to justify its use alone, the development of a new chemical family, able to act as pro-dugs and deliver NO • /HNO in vivo, is an exciting research topic, in which several researchers are working on it. In the next paragraph, an overview of NO • and HNO donors will be discussed.

Nitric oxide (NO • ) donors

Nitric oxide must reach certain biological levels to establish its effects, which can vary from signaling regulation of blood pressure to cellular apoptosis of cancer cells. This illustrates well how critical are the strategies to delivery this molecule in a controlled and selective way to guarantee proper targeting and dosing of NO • [192].

Aiming at different targets, several strategies have been used in the design NO • donors, particularly taking advantages of specific stimulus promoted by the body (e.g., pH, and enzymatic reaction) or even externally (e.g., light). Throughout the text, we will discuss the chemistry and biological properties of some NO • donors. are examples of molecules that integrate this group (Figure 15). GTN is one of the most commonly known nitric acid esters, originally used in the industry of explosives, then recognized as a drug in the treatment and prevention of angina pectoris pain [194].

Curiously, the famous Alfred Nobel who made a fortune developing explosive formulations including GTN, was indeed treated with this same compound due to his angina pectoris medical problem. Today, GTN is also used in hypertensive crises [195] and erectile dysfunction [196]. Several other organic nitrates have been used in the treatment of cardiovascular diseases [197], angina pectoris, pre-eclampsia, pulmonary hypertension, and chronic heart failure in combination with hydralazine [198][199][200]. Most of the biological effects of organic nitrates are attributed to their ability to release NO • . In general, organic nitrates are able to release NO • after reaction with enzymatic systems (e.g., cytochrome P450, xanthine oxidoreductase, glutathione-Stransferase, and aldehyde dehydrogenase 2), ferrous-heme proteins (deoxyhemoglobin and deoxymyoglobin), or thiols [193,197,211]. Glutathione is an exception because its reaction with organic nitrates must be catalyzed by glutathione-S-transferase [193].

The mechanism of NO • release through organic nitrates in physiological medium Some well-known S-nitrosothiols derivatives are S-nitroso-glutathione (GSNO), Snitroso-cysteine (SNO-Cys), S-nitroso-N-acetylpenicillamine (SNAP), and 1-nitrosothiogalactose tetraacetate (SNAG) [193], which are illustrated in Figure 19. Interestingly, despite the supposed generation of HNO in this class of molecules, the literature ignore that the observed pharmacological actions of these derivatives may also be related to HNO. A detailed analysis of the mechanism of the NO • or HNO release by this class of pro-drugs is thus necessary.

Nitroxyl (HNO) donors

Despite relatively recent reports on the biochemical, pharmacological and physiological properties of HNO, this molecule is extremely unstable, dimerizing easily into N2O (8×10 6 M -1 s -1 , pH 7.4, 22 °C). This process hinders the direct use of HNO and still raises debates about possible routes for the biosynthesis. Thus, the use of HNO donors has been essential for a better understanding of its biological role and for the appropriate use of this molecule. In addition, in analogy with NO • donors, different HNO releasing molecules have been developed and studied in order to generate new therapeutic agents, given the pharmacological potential of HNO. Differently from the NO • donor class, there are no HNO donors currently in clinical use.

Angeli's salt and primary amine diazeniumdiolate-based donors

Sodium trioxodinitrate or Angeli's salt (AS, Na2N2O3), is an inorganic salt synthesized by Angelo Angeli more than 120 years ago (Scheme 11A) [START_REF] Pagliaro | Nitroglycerine and sodium trioxodinitrate: from the discovery to the preconditioning effect[END_REF]. This inorganic compound is currently one of the most commonly used HNO donor, especially in pharmacological studies [START_REF] Paolocci | From Heaven to Heart: Nitroxyl (HNO) in the Cardiovascular System and Beyond[END_REF]. AS releases HNO through spontaneous

NO • /HNO metal-based donors

Several metal-based compounds have been developed showing potential pharmacological action [START_REF] Barry | Exploration of the medical periodic table: towards new targets[END_REF]300]. Despite that and the knowledge of the important biological roles played by metallo-proteins [START_REF] Forstermann | Nitric oxide synthases: regulation and function[END_REF][START_REF] Kang | Structural insights into the mechanism of human soluble guanylate cyclase[END_REF]301,302], supporting the therapeutic potential of metal complexes, there are still not many metallo-drugs in clinical use, particularly if compared to organic-based drugs [START_REF] Barry | Exploration of the medical periodic table: towards new targets[END_REF]300]. Related to the clinical use of NO • donor metallo-compounds, there is only one case, sodium nitroprusside (SNP, Na2[Fe(CN)5NO]). First prepared in 1849 by Playfair [START_REF] Hottinger | Sodium nitroprusside in 2014: A clinical concepts review[END_REF], SNP, a cyanoferrate complex, has been used in cardiac surgery with hypertensive crisis conditions and other acute hemodynamic emergency applications. Nonetheless, some harmful side effects are related to its administration, due to the rapid release of NO • and cyanide. This latter can be managed by the co-administration of sodium thiosulfate and hydroxocobalamin [START_REF] Hottinger | Sodium nitroprusside in 2014: A clinical concepts review[END_REF].

Recently, a material containing SNP anchored in silica-based nanoparticles was prepared, where vasorelaxation activity similar to that of isolated SNP was noticed along with a significant reduction in cytotoxicity, proving an alternative for the safer and broader use of SNP in medicine [303].

In addition to strategies based on nanomaterials, several NO • metal donors have been developed in order to provide more efficient cardiovascular activities with lower undesired side effect to the patient. Oxacom ® , a dinitrosyl iron complex (DNIC) containing glutathione as a ligand (Figures 6 and 24), is undergoing clinical trials in Phases I/II. This compound represents an exciting example of a new hypotensive drug with no adverse effects reported to date [START_REF] Hsiao | Fe in biosynthesis, translocation, and signal transduction of NO: toward bioinorganic engineering of dinitrosyl iron complexes into NO-delivery scaffolds for tissue engineering[END_REF]304].

Polypyridine ruthenium-nitrosyl complexes (Figure 24) have been considered as releasing phototherapeutic agents [305,306,308,309]. Metal complexes are versatile in terms of design and reactivity modulation, which have raised interest in their use as model photosensitizers [START_REF] Imberti | New Designs for Phototherapeutic Transition Metal Complexes[END_REF][311][312][313]. The incidence of irradiation (UV-Vis) in a metallo-nitrosyl compound induces formations of an excited state, in which π* antibonding orbitals of the nitrosyl ligand are electronically populate, leading to a decrease in the bond order between the metallic center and NO • , promoting the release of the ligand [305,306,309].

The [Ru(bpy)2(SO3)NO](PF6) complex is an interesting example on the modulation of reactivity based on the substitution of ancillary monodentate ligand.

Once sulfite, bound through S atom, is used as ligand, there was a significant change in the overall electronic density of NO • moiety if compared to that of a complex with an N-based ligand instead (e.g., isonicotinamide). This can dramatically alter the acid-base equilibrium of nitrosyl/nitrite found for these systems (Scheme 14). Depending on the equilibrium constant for this process we can have in physiological pH mainly nitrite or nitrosyl bound species, which can modulate NO • release and reactivity. NO • photorelease may occur more efficiently if the nitrosyl bound is the main species.

[M(L)x(NO)] n (aq) + 2 OH - [M(L)x(NO2)] (n-2) (aq) + H2O (aq) Scheme 14. Generic acid-base nitrosyl/nitrite equilibrium for polypyridine rutheniumnitrosyl complexes, reference [314].

By replacing isonicotinamide (isn) ligand for sulfite ion (SO3 2-) in the Another exciting case is based on the photorelease of HNO. Lee and collaborators [317] developed an nitrosyl iron complex based on the 2,2′-dimercapto-3,3′-bis(trimethylsilyl)diphenyl)phenylphosphine ( TMS PS2H2) ligand. This compound upon light irradiation (> 400 nm) showed formation of NO • that was reduced to HNO by a thiol group (-SH) already present in the structure (Figure 26). This photorelease of HNO features as a rare case via an iron complex nitrosyl, considering that in many cases NO • is found as NO + and requires two-electrons for HNO production.

One key issue for nitrosyl complexes as a photoreleaser species is the wavelength capable to promote this stimulus, which, unfortunately, in most of the cases requires highly energetic light. Despite that, any suitable phototherapy requires stable compounds with efficient activation using light from 650 to 900 nm, called therapeutic window, where light has maximum penetration in the skin with minimum issue on other biomolecules [318]. Several authors have developed strategies for the delivery of NO • to biological targets, mainly aiming at application within the therapeutic window, systems such as Roussin's Red Salt ester, Ru(salen)(Cl)NO, Cr(cyclam)(ONO)2 + , [Mn(PaPy2Q)(NO)] + , in addition to nano-structured materials, are part of these therapeutic proposals as described elsewhere [308,309,319 published and presented in chapter 1 [1].

In order to advance in the search for new NO • /HNO donor platforms for therapeutic purposes, we explored the ability of aromatic hydroxamic acid derivatives to release NO • /HNO after oxidative reaction steps. This platform could be of particular interest due to the possible mechanism of activation of this type of molecule, in addition to NO • /HNO release (metabolite active of the anti-TB drug delamanid and vasodilatator agent), an aroyl radical intermediate or an aromatic carboxylic acid might also be formed, which can be seen, respectively, as the corresponding active metabolites of isoniazid and pyrazinamide, two major antitubercular drugs. Thus, the second part of this thesis describes two chapters. The aim of the first one (chapter 2) is the investigation of the intermolecular oxidative activation mechanism of the isonicotino-, nicotino-, and pyrazino-hydroxamic acids, respective analogues of isoniazid (anti-TB), nicotinamide (vitamin B3), and pyrazinamide (anti-TB), by reaction with potassium ferricyanide (K3[Fe III (CN)6]) in pH physiological conditions. This work could assist in the design of new drugs. This study has also been published in a scientific peerreviewed journal and presented in chapter 2 [2]. Then, the third project (chapter 3) is relative to the synthesis of new Fe II complexes formulated with isonicotino-, nicotinoand pyrazino-hydroxamic acid ligands bound to the [-Fe(CN)5] n-moiety, as well as the study of the mechanism of the oxidative activation of these metal complexes by H2O2 and structural characterization of the species formed. This synthetic approach is applied aiming at development of new antituberculosis and cardiovascular agents. This latest study was equally published in a peer-reviewed journal and is presented in chapter 3 [3].

OBJECTIF

PLATFORM

The increase number of cases of cardiovascular diseases and cancer has led the scientific community to seek new treatment strategies that are more efficient and less harmful to patients. The part 1 of this manuscript presents the chemical and biological studies of a known nitrosyl Fe II complex, which has never been further investigated from a pharmacological perspective. In the context of this thesis a special emphasis on the release of NO • /HNO and on potential antiangiogenic and cardiovascular action was placed.

Introduction

Among the drugs used to treat cardiovascular diseases, the SNP is the only metal complex available [1]. As we mentioned in the introduction of this thesis, in addition to the release of CN -, another factor that limits the widespread use of this drug, is its powerful vasodilating action, due to fast release of NO • in the body [1]. In order to obtain more moderate NO • donor agents, with lower toxicity compared to SNP, several other metal complexes have been explored, for example, the DNIC 2,2'-bipyridine- [Ru(phen)2(tu)NO](PF6)3 (phen = phenanthroline, tu = thiourea) [4,5].

In addition to the development of new nitrosyl complexes with therapeutic potential related to the NO • release, the discussion about the ability of these complexes to release HNO has also emerged, since the latter shows also biological relevance.

Sousa and collaborators [6], motivated to identify the possible production of HNO by [Ru(bpy)2(SO3)NO] + , have studied the reaction of this ionic complex with glutathione (GSH). This study have shown, through the use of a selective trap to distinguish NO/HNO and cellular angiogenesis assays, the potential ability of this complex to release HNO under physiological conditions. Thus, HNO production in a physiological context has also been an important part of the reactivity studies involving nitrosyl complexes.

Holanda and his collaborators developed in 2007 a new nitrosyl iron(II) complex using 1,4,8, as ligand, called trans-[Fe(cyclam)(NO)Cl]Cl2 [7]. This complex has been studied from a chemical point of view, being indicated as a promising photoreleaser of NO • . However, such results lacked data showing NO • can be directly produced from the complex. Additionally, no results proving HNO release as well as relevant biological tests were available. Thus, the trans-[Fe(cyclam)(NO)Cl]Cl2 complex is an interesting system to be explored mainly from a biological point of view.

In view of that, the following article presents details of the reactivity study of the Dans les cellules cancéreuses, la production de facteurs pro-angiogéniques est induite dans des conditions d'hypoxie (provoquée par un métabolisme accéléré anormal). L'adaptation à différents niveaux d'oxygène dans la cellule est médiée par une famille de régulateurs transcriptionnels appelés facteurs inductibles par l'hypoxie (HIF) [5]. Sous normoxie, l'activité transcriptionnelle de HIF est spécifiquement supprimée par la dégradation de sa sous-unité α dépendante de l'oxygène (HIF-α). Des conditions de faible teneur en oxygène induisent la stabilisation de HIF-1α, favorisant l'angiogenèse cellulaire [6]. Ainsi, la protéine HIF-1α a été considérée comme une cible prometteuse pour la thérapie anticancéreuse [5].

NO The structural and electronic properties of trans-[Fe(cyclam)(NO)Cl] 2+ complex (1) were investigated using density functional theory (DFT) and time-dependent DFT (TD-DFT). Electronic transitions contributions were subsequently determined, which were used to guide interpretation of UV-Vis electronic absorption spectrum and photochemical processes. The optimized geometry of 1 obtained by DFT was compared with experimental data (Fig. S1 and Table S1) [1]. The computed geometrical parameters of such complex are in excellent agreement with crystallographic data obtained by single crystal X-ray diffraction.

Interestingly, a Fe-N-O angle of 179.3° calculated by DFT was very close to 177.4°

found by X-ray data. This value indicated nitrosyl ligand primarily exhibits major NO + character as previously reported [2]. Additionally, a plane containing O-N-Fe-Cl is not perfectly perpendicular to the one containing the nitrogen atoms of the cyclam ligand.

This distortion occurs due to interactions of the hydrogens of cyclam with axial ligands [1].

A comparison of experimental and theoretical electronic spectra of 1 is shown in supporting Fig. S2, where satisfactory agreement was observed. These calculations provided support of assigned electronic transitions based on the character of the molecular orbitals involved in this process (see Tables S2 andFig. S3-S4).

Thermal-and photo-stability of the trans-[Fe(cyclam)(NO)Cl] 2+ in the presence of metmyoglobin (Mb-Fe III )

The thermal stability of the trans-[Fe(cyclam)(NO)Cl] 2+ complex, in the presence of metmyoglobin (Mb-Fe III ), was monitored by UV-Vis spectroscopy for ca.

24 h at 37 ºC, using 134 μM of the nitrosyl complex and 6.7 μM of the Mb-Fe III dissolved in 0.1 M phosphate buffer pH 7.4 solution, under anaerobic conditions. The solutions were prepared in an argon atmosphere with the aid of an inert gas glove box.

This study was also performed with Angeli's salt (27 μM) and Mb-Fe III (6.7 μM) for comparative purposes. The experiment with Angeli's salt was monitored for ca. 1 h at 37 ºC.

The study of NO/HNO photorelease was carried out using light irradiation at 365 nm and Mb-Fe III as NO/HNO probe. The solution containing 4.6 μM of Mb-Fe III and 100.0 μM of the nitrosyl complex was prepared in anaerobic phosphate buffer (0.1 M, pH 7.4), and monitored for 1.5 h at 37 ºC by absorption spectroscopy in UV-Vis region. This experiment was also prepared in an argon atmosphere with the aid of an inert gas glove box.

Thermal stability in the presence of pentetic acid (DTPA)

The stability of the nitrosyl complex in the presence of pentetic acid (DTPA), was studied by UV-Vis spectroscopy using a solution composed of trans-

[Fe(cyclam)(NO)Cl] 2+ (2 mL at 50 μM) and of DTPA (2 mL at 75 μM), in 0.1 M phosphate buffer (pH 7.4). This study was also performed using a solution of trans-[Fe(cyclam)Cl2] + complex (2 mL at 100 μM) and DTPA (2 mL at 150 μM), under the same conditions. The solutions of these complexes were prepared and monitored for ca.

4 h at 37 ºC under aerobic conditions.

Photodecomposition in solid state

The photostability of the nitrosyl complex, in solid state, was studied by FTIR using trans-[Fe(cyclam)(NO)Cl]Cl2 salt, dispersed in KBr pellet, and irradiation at 365 nm. The study was monitored based upon the presence of the band at 1888 cm -1 , assigned to the v(NO + ), measured over a period of 6 h under aerobic conditions.

Part 1 -Nitrosyl Iron complex: cyclam as therapeutic platform _________________________________________________________________________________ 131 4.5.2. Synthetic procedures

Precursor trans-[Fe(cyclam)Cl2](PF6) complex

A solution of cis-[Fe(cyclam)Cl2]Cl (0.100 g, 0.214 mmol), in methanol/water (50%) was heated (80 ºC) and mixed for 30 min then a change of color from yellow to green was observed. A saturated solution of NH4PF6 in methanol was added dropwise.

The green solid formed was filtered, washed with ice-cold methanol, and dried by vacuum.

Yield

Nitrosyl trans-[Fe(cyclam)(NO)Cl]Cl2 complex

To a solution of cis-[Fe(cyclam)Cl2]Cl (0.100 g, 0.214 mmol), in degassed water (10 mL), it was bubbled NO gas for 60 min. The solution was concentrated by vacuum and, then, added dropwise to a mixture of ethanol/diethyl ether (70 mL, 1:1). The resulting yellow precipitate was kept overnight (16 h) at -20 ºC before it was collected by filtration, washed with ethanol/diethyl ether (1:1) ice-cold solution, and dried in a vacuum desiccator. CAUTION: since the complex is moderately light sensitive, it must be stored in a vacuum desiccator protected from light to extend their lifetime. Yield = 80% (0.080 g), yellow white solid. IR symmetric stretching (νs), symmetric bending (δs) and twisting (τ) (cm -1 ): 3092 (νs N-H), 3012 -2857 (νs C-H), 1888 (νs NO), 1452 (δs CH2), 1380 -1290 (τ CH2), 1100 -1044 (νs C-N), 835 and 885 (τ N-H), 812 (τ CH2). UV-Vis (H2O) λmax/nm (ɛ/M -1 cm -1 ) = 232 (14025), 438, [START_REF] Fukuto | The chemistry and biology of nitroxyl (HNO): a chemically unique species with novel and important biological activity[END_REF]. Cyclic voltammetry in CF3COOH/CF3COONa solution 0.1 M (pH 3.2), V = 0.1 V s -1 : E1/2 = 0.292 and 0.595 V vs NHE, Epa = 0.905 V and Epc = -0.375 V vs NHE.

Results and discussion

Thermal-and photo-stability of the trans-[Fe(cyclam)(NO)Cl]Cl2 in the presence of metmyoglobin (Mb-Fe III )

Metmyoglobin (Mb-Fe III ) can be used as a probe for nitric oxide (NO) and nitroxyl (HNO) detection. While the reaction of Mb-Fe III with HNO directly generates Part 1 -Nitrosyl Iron complex: cyclam as therapeutic platform _________________________________________________________________________________ 132 the NO-MbFe II adduct, k = 2.7 x 10 5 M -1 s -1 (Scheme 16) [1], that of NO and Mb-Fe III produces the NO-MbFe II adduct via three steps; (i) formation of NO-MbFe III adduct (unstable species), k = 5.3 x 10 4 M -1 s -1 , (ii) reaction between NO-MbFe III and OH - generating nitrite (NO2 -) and Mb-Fe II , k = 3.2 x 10 2 M -1 s -1 , (iii) and myoglobin (Mb-Fe II ) reaction with NO finally producing NO-MbFe II , k = 1.7 x 10 7 M -1 s -1 (Scheme 16) [2,3]. Thus, the thermal-and photo-stability of the ion trans-[Fe(cyclam)(NO)Cl] 2+ was studied in the presence of Mb-Fe III aiming at the discrimination of NO and HNO. Scheme 16. Production of the NO-MbFe II adduct through reacting with NO • or HNO [1][2][3].

First of all, a control experiment using Mb-Fe III and Angeli's salt, a well established direct HNO donor, was performed under physiological conditions (degassed phosphate buffer 0.1 M, pH 7.4, at 37 ºC), to obtain the UV-Vis spectral profile of the NO-MbFe II adduct (Figure 28). All anaerobic experiments were prepared in an inert gas glove box. Before the spectrophotometer analysis, the cuvettes containing the samples were efficiently sealed to prevent the entry of atmospheric air. The main differences observed in the initial and final UV-Vis spectra of the heme-protein were the displacement of the Soret band, from 408 to 420 nm, and Q bands, from 506 and 634 nm, to 548 and 580 nm, respectively. These modifications are indicative of the Mb-Fe III conversion to the NO-MbFe II form [1].

Figure 28. Spectral variation in the UV-Vis region for a mixture containing Mb-Fe III (6.7 μM) and Angeli's salt (27 μM), in anaerobic phosphate buffer (0.1 M, pH 7.4, at 37 ºC). Solution before (grey) and after 1 h (black) of reaction. A = Full spectra (Soret band) and B = Spectra zoom between 450 and 700 nm (Q bands).

The study of the trans-[Fe(cyclam)(NO)Cl] 2+ complex in the presence of Mb-Fe III , under the same conditions of the control experiment (anaerobic phosphate buffer solution, 0.1 M, pH 7.4, at 37 ºC), was monitored by UV-Vis for 24 h (Figure 29). The results did not indicate significant changes in the Mb-Fe III spectrum over the time ( 24h). Since the reaction of Mb-Fe III with NO is about fivefold slower than that with HNO, these results suggest a potential NO release, via thermal decomposition of the nitrosyl iron complex under physiological conditions, thus corroborating with the published results.

Figure 29. Spectral variation in the UV-Vis region for a mixture containing Mb-Fe III (6.7 μM) and trans-[Fe(cyclam)(NO)Cl] 2+ complex (134.0 μM), in anaerobic phosphate buffer (0.1 M, pH 7.4, at 37 ºC). Solution before (gray) and after 24 h (black) of reaction. A = Full spectra (Soret band) and B = Spectra zoom between 450 and 700 nm (Q bands). In photodecomposition study, a mixture containing the nitrosyl complex and Mb-Fe III , in anaerobic solution of phosphate buffer (0.1 M pH 7.4 at 37 °C), was monitored before and after 1.5 h of irradiation (365 nm), by absorption spectroscopy in the UV-Vis region (Figure 30). No evidences of the NO-Mb-Fe II adduct was observed over the time (1.5 h). Since the reaction of the oxidized protein with HNO is substantially faster, this result suggests that HNO was not produced in this experiment.

Photostability study of the trans-[Fe(cyclam)(NO)Cl]Cl2 in solid state

To verify the stability of the trans-[Fe(cyclam)(NO)Cl]Cl2, in solid state, when subjected to irradiation (365 nm), the nitrosyl complex, dispersed in KBr pellet, was irradiated at different times and then the respective spectra in the infrared region were recorded (Figure 32). It was observed that over the irradiation time, the stretch corresponding to the ligand NO + (1888 cm -1 ) disappears, indicating that also in a solid state, the irradiation at 365 nm can induce the decomposition of the nitrosyl complex.

Conclusion

The results shown in this section provide important information about the reactivity of the nitrosyl complex.

AS THERAPEUTIC PLATFORMS

Hydroxamic acids display several chemical and biological properties [1][2][3][4],

many of which attributed to their ability to release NO • /HNO under oxidative conditions. The pharmacological effects aligned with the HNO and NO • metabolites are diverse, the releasing moiety may also display physiological roles, which illustrates the potential use of these agents in the development of new drugs [4][5][6]. In this part of the thesis, we focused on the oxidative study of these organic molecules. From a fundamental perspective, the chapter 2 describes the oxidative mechanism involving three azine hydroxamic acid derivatives, while in the chapter 3 we explore the antituberculosis and cardiovascular properties of new Fe II hydroxamic acid complexes under oxidative conditions. HNO release is one of the key points of these two studies.

Introduction

The reaction between diethyl oxalate and hydroxylamine, producing the compound oxalohydroxamic acid (Scheme 17), carried out by H. Lossen in 1869, marks the beginning of hydroxamic acid chemistry [1,7]. From this milestone, a series of studies have begun involving new synthetic approaches and possible applications in organic and inorganic chemistry, and biochemistry as bioactive molecules [2,3].

Scheme 17. Oxalohydroxamic acid synthesis by Lossen [1,7].

It was mentioned earlier that hydroxamic acids are able to release NO • /HNO under oxidative conditions and many of their chemical and biological properties are attributed to this ability [2].

In the introduction of this thesis, we report that isoniazid (INH), pyrazinamide (PZA) and delamanid are all pro-drugs used in antituberculosis treatments [8]. The pharmacological action of these drugs is dependent on an activation step mediated by enzymes present in Mtb. INH is activated through an oxidative process mediated by the mycobacterial catalase-peroxidase enzyme, KatG, generating the isonicotinoyl radical Part 2 -Pentacyanoferrate complexes: hydroxamic acid as therapeutic platforms _________________________________________________________________________________ 140 intermediate that reacts with NAD(H) yielding an INH-NADH adduct, the ultimate active metabolite against Mtb (Scheme 18) [9]. On the other hand, the PZA activation occurs via a hydrolysis process mediated by the pyrazinamidase enzyme, PncA, yielding pyrazinoic acid as the active metabolite (Scheme 18) [8]. Delamanid is also proposed as a pro-drug that requires enzymatic activation. It has been suggested that its main active metabolite against Mtb is HNO, this latter resulting from a reductive decomposition of the nitroimidazole group by a mycobacterial nitroreductase enzyme (Ddn) (Scheme 18) [9]. Scheme 18. Enzymatic activation of INH, PZA and delamanid [8,9].

Nevertheless, the emergence of multidrug-resistant strains of Mtb has limited the use of drugs such as INH and PZA in TB therapy. In most cases, the resistance mechanism is associated with mutations in the gene that encodes for the enzymes responsible for activating drugs, as a result, mycobacteria can no longer oxidize the drugs [8]. Multidrug-resistant tuberculosis (MDR-TB) has opened up opportunities to develop new strategies to control TB. Delamanid is an example of a new pro-drug developed for use in MDR-TB cases. However, the possible strategies are not limited to organic compounds only. Motivated by a potential therapeutic approach using metal complexes, Sousa and collaborators [9], developed a strategy based on the pentacyanoferrate(II) complex of INH, called IQG607. In summary, IQG607, when oxidized to Fe III by H2O2/O2 -, produces isonicotinoyl radical, a key tool in the anti-TB action of INH (Figure 33). During this oxidation process, isonicotinic acid and isonicotinamide are also formed as final metabolites. Furthermore, this complex has shown an efficient inhibition of the InhA enzyme in vitro, even in the absence of KatG, accompanied by a very low MIC (0.2 μg/mL) against Mtb [9]. This approach could be very important, especially in cases of acquired resistance to INH, since this process would overcome the enzymatic activation of INH promoted by KatG. Thus, the use of the pentacyanoferrate(II) moiety as an alternative to enable drug activation has stimulated its use with other molecules of biological interest such as hydroxamic acids. Prior to the synthesis of Fe II complexes, we have judged necessary to study the ability of ligands alone to release NO • /HNO in oxidative conditions to better appreciate the interest to complex the hydroxamic acids (NO • /HNO donors) to a inorganic moiety . Thus, the first article presents a study of three hydroxamic acid derivatives with oxidizing agents such as hexacyanoferrate(III) complex and H2O2. In this study, when hexacyanoferrate(III) complex was used as oxidizing agent, it was possible to identify HNO as one of the final products of these reactions and N,Odi(di)azinoylhydroxylamine as intermediate. In the presence of H2O2 alone, hydroxamic acids are not oxidized. The aim of this article is to highlight the ability of hydroxamic acids to act as HNO donors, and to provide more clarity as to its mechanism.

The second article aims at the synthesis of pentacyanoferrate(II)-type complexes with the pyrazino-and isonicotino-hydroxamic acids as ligands, which are pyrazinamide's and isoniazid's analogs, respectively. The study of their oxidation mechanism and their potential to release HNO and another active metabolite (pyrazinoic acid or isonicotinoyl radical) was conducted. Finally, the antimycobacterial activity against the Mtb H37Rv and the vasodilator activity of these complexes were also investigated. Pathways, and Associated Resistance », ChemMedChem, 12, 20, 2017, pp. 1657-1676. 9.

Bibliography

______________________

Lopes, L. G. F.; Carvalho, E. M.; Sousa, E. H. S., « A bioinorganic chemistry perspective on the roles of metals as drugs and targets against Mycobacterium tuberculosis -a journey of opportunities », Dalton Transactions, 49, 2020, pp. 15988-16003. Le nitroxyle (HNO) est un acide inorganique faible (pKa = 11,4) et instable [1] qui présente plusieurs activités biologiques importantes qui sont pourtant encore très mal comprises. L'une des principales propriétés pharmacologiques de HNO est son action bénéfique sur le système cardiovasculaire [2]. HNO présente aussi une activité antiangiogénique, en opposition à son cousin NO • qui lui favorise l'angiogenèse. Cette dernière propriété citée de HNO ouvre des opportunités intéressantes pour la thérapie contre le cancer [3]. Étant donné que HNO ne peut pas être directement utilisé in vivo en raison de sa forte instabilité en milieu aqueux (dimérisation et déshydratation donnant du protoxyde d'azote, N2O, schème 19) [4] et une forte réactivité vis-à-vis des biomolécules nucléophiles (principalement avec des composés soufrés) [5], toutes les études concernant les propriétés biologiques de HNO ou son emploi comme agent thérapeutique nécessitent des composés donneurs de HNO. 8,91 (d, 1H), 8,81 (d, 1H), 7,99 (d, 1H) et 7,79 (d, 1H) ppm. Par RMN 13 C, on constate la présence de deux signaux à 167,7 et 166,0 ppm compatible avec le carbone quaternaire de deux groupes carbonylés différents, quatre signaux à 149, 7, 147,8, 123,4 et 122,7 ppm

Mechanistic insights into the in vitro metal

Schème 22. Voies de fragmentation par SM des intermédiaires proposés, N,O-di(di)

azinoylhydroxylamines, issus de l'oxydation des acides (di)azine hydroxamiques.

La RMN 1 H de la même réaction d'oxydation mais utilisant le composé 2 comme substrat a montré 8 signaux attribuables aux hydrogènes aromatiques (9,18, 9,00, 8,86, 8,68, 8,39, 8,18, 7,62, 7,49 ppm) The isonicotinohydroxamic acid (1 eq.) was added in phosphate buffer solution (0.1 M, pH 7.4) (3 mL). After solubilization of the substrate, K3[Fe(CN)6] (2.5 eq.) was added to the hydroxamic acid solution. The mixture was stirred at room temperature and analyzed by 1 H NMR spectroscopy for 322 h.

General protocol for the synthesis of N,O-diaroylhydroxylamines 4, 5 and 6

The hydroxamic acid (1 eq.) was added in phosphate buffer (0.1 M, pH 7.4) ( 3mL). After solubilization of the substrate, K3[Fe(CN)6] (2.5 eq.) was added to hydroxamic acid solution. The mixture was stirred at room temperature for 24 h. The mixture was extracted with EtOAc. The organic phase was concentrated under reduced pressure, and the solid residue was dried under vacuum before characterization.

N,O-diisonicotinoylhydroxylamine (4)

From isonicotinohydroxamic acid (0.100 g, 0.

N,O-dinicotinoylhydroxylamine (5)

From nicotinohydroxamic acid (0.100 g, 0.734 mmol) and K3[Fe(CN)6] (0.604 g, 1.84 mmol). 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 9.17 (dd, J = 2.2, 0.9 Hz, 1H), 9.00 (dd, J = 2.3, 0.9 Hz, 1H), 8.86 (dd,J = 4.8,1.7 Hz,1H),8.68 (dd,J = 4.8,1.7 Hz, 1H), 8.39 (dt, J = 8.0, 2.0 Hz, 1H), 8.18 (dt, J = 7.9, 2.0 Hz, 1H), 7.62 (ddd, J = 8.0, 4.9, 0.9

Hz, 1H), 7.50 -7.45 (m, 1H). HRMS (ESI + ): m/z calcd. for C12H9N3O3: 244.0722, found: 244.0726.

N,O-dipyrazinoylhydroxylamine (6)

From pyrazinohydroxamic acid (0.100 g, 0. General protocol for the synthesis of N-methylisonicotinamide, N-methylnicotinamide, and N-methylpyrazinamide 16, 17 and 18

The hydroxamic acid (1 eq.) and NH2CH3 40% in water (10 eq.) was added to a phosphate buffer solution (0.1 M, pH 7.4) (3 mL). After solubilization of the substrate, K3[Fe(CN)6] (2.5 eq.) was added to hydroxamic acid solution. The mixture was stirred at room temperature for 2 h. The solvent was removed by evaporation under reduced pressure. The crude material was purified by silica gel flash column chromatography using a mixture of MeOH/DCM (0-100 / 10-90) as eluent.

N-methylisonicotinamide ( 16)

From isonicotinohydroxamic acid (0. ,60.55;H,6.03;N,20.17. Found: C,60.95;H,6.13;N,19.73.

TLC Retention Factor (MeOH/DCM 10%) = 0.41. M.p. = 113 ºC.

N-methylnicotinamide [START_REF] Barry | Exploration of the medical periodic table: towards new targets[END_REF] From nicotinohydroxamic acid (0. N-methylpyrazinamide [START_REF] Thota | Ru(II) Compounds: Next-Generation Anticancer Metallotherapeutics?[END_REF] From pyrazinohydroxamic acid (0.10 g, 0.729 mmol) NH2CH3 40% in water (0.226 g, 7.29 mmol) and K3[Fe(CN)6] (0.600 g, 1.82 mmol). Yield = 69% (0.068 g), white solid.

General protocol for the synthesis of sodium isonicotinoate, nicotinoate and pyrazinoate molecules

To a mixture of the suitable carboxylic acid (1 eq.) in water (8 mL) was added a solution of NaOH (0.85 eq.) in water (2 mL). The solution was stirred at room temperature for 30 min. The solvent was removed by evaporation under reduced pressure at 50 ºC. The solid obtained was characterized.

Sodium Isonicotinoate (7b)

From isonicotinic acid (0.12 g, 0.975 mmol) and NaOH (0.033 g, 0.817 mmol). Yield = 99.0% (0.140 g), white solid. Retention Factor (MeOH/DCM 50%) = 0.6.

Sodium Nicotinoate (8b)

From nicotinic acid (0.12 g, 0.975 mmol) and NaOH (0.033 g, 0.817 mmol). Yield = 99.5 % (0.14 g), white solid. ,47.32;H,3.18;N,9.20. Found: C,47.35;H,2.96;N,9.48. TLC Retention Factor (MeOH/DCM 50%) = 0.6.

Sodium pyrazinoate (9b)

From pyrazine-2-carboxylic acid (0.12 g, 0.970 mmol) and NaOH (0.033 g, 0.817 mmol). Yield = 87.5 % (0.124 g), white solid.

Unpublished results associated with the article

The oxidative reaction of the hydroxamic acids synthesized in this work was also studied in the presence of PBN (N-tert-butyl-α-phenylnitrone) spin trap, aiming at the detection of the aroyl radical as a potential intermediate. Furthermore, the Diels-Alder reaction was also investigated as a strategy to demonstrate the possible formation of a nitroso intermediate during oxidation of the isonicotino-hydroxamic acid ligand.

An attempt to synthesize a new pentacyanoferrate(II) complex based on the hybridization of isoniazid (INH) and pyrazinamide was also made. The results obtained from these experiments are presented and discussed briefly below.

Experimental section

Attempts to detect the aroyl radical

The experiments were monitored by EPR technique. All samples used were prepared in 40 mM phosphate buffer pH 7.4, at 0 °C, by mixing appropriate amounts of studied compound and PBN dissolved in phosphate buffer solution. The reactions were initiated by the addition of the oxidant (H2O2 or [Mn(H2P2O7)3] 3-). The first spectrum was recorded ca. 2 min after initiating the reaction. In the control experiments were used solutions containing INH (1 mL at 5 mM) or IQG-607 (1 mL at 5 mM), the oxidizing agent (1 mL at 2.5 mM of [Mn(H2P2O7)3] 3-for the INH oxidation, and 1 mL at 5 mM or 15 mM of the H2O2 for the IQG-607 oxidation), and PBN (1 mL at 20 mM).

Attempt to trap the acyl nitroso intermediate via Diels-Alder reaction

The oxidative activation reaction of the isonicotino-hydroxamic acid ligand, in the presence of cyclohexa-1,3-diene, was performed using a THF:H2O (2:1, 6 mL) solution containing the isonicotino-hydroxamic acid ligand (0.05 g, 36.2 mmol) and cyclohexa-1,3-diene (48.9 μL, 54.3 mmol, 97%). K3[Fe(CN)6]•3H2O (0.333 g, 90.5 mmol) was added in the previous solution and the mixture was stirred for 96 h at room temperature [3]. The organic phase was extracted with ethyl acetate (3 x 6 mL), combined, dried over MgSO4 (anhydrous) and concentrated under vacuum. The crude was purified by flash chromatography (eluent: dichloromethane/methanol: 100/0 to 95/5).

Part 2 -Pentacyanoferrate complexes: hydroxamic acid as therapeutic platforms _________________________________________________________________________________ 169 5.5.2. Synthetic procedures

Oxidant agent [Mn(H2P2O7)3] 3-

The [Mn(H2P2O7)3] 3-was prepared following the literature procedure with some adaptations [4]. Pyrophosphoric acid (1.75 mL, 5 M) was added dropwise in a pyrophosphate sodium solution (25 mL, 200 mM). The pH was measured at 4.4. Then, Mn III (OAc)3•2H2O (0.027 g, 0.1 mmol) was added in the latter solution. The mixture was stirred overnight (16 h). After the time reaction, the concentration of the product [Mn(H2P2O7)3] 3-was determined based on literature data:  6200 M -1 cm -1 at 260 nm or 104 M -1 cm -1 at 480 nm, the valor obtained for this synthesis was 3.7 mM. The final product is stable for several months at 4 ºC.

Ligand N'-(pyridine-4-carbonyl) pyrazine-2-carbohydrazide

A suspension of pyrazinoic acid (0.104 g, 0.838 mmol) in thionyl chloride (1.2 mL, 16.6 mmol) was refluxed at 75 ºC for 2.5 h. The color of solution changed from white to strong violet color. The excess of the thionyl chloride was then removed under vacuum. The crude was resuspended in dichloromethane (2 mL) and cooled to 0 ºC. INH (0.114 g, 0.838 mmol) and triethylamine (0.28 mL, 1.68 mmol) was added in the pyrazinoic acid solution. The reaction was stirred overnight (16 h) at r.t, and the residue was concentrated under vacuum (Scheme 23). The crude was purified by flash chromatography (eluent: dichloromethane/methanol: 100/0 to 94/6). The white solid was washed several times (4x) with dichloromethane. After the control experiments, the next step was then to study the chemical activation of isonicotino-, nicotino-, and pyrazino-hydroxamic acids free ligands and complexed with Fe II (CN)5 moiety in the presence of [Mn(H2P2O7)3] 3-and PBN, in phosphate buffer (40 mM, pH 7.4). The resulting EPR spectra did not show any detectable signal (Figure 35A). In order to study the influence of the coordination to the pentacyanoferrate(II) moiety in the process of the oxidative activation, the same experiment was performed using the isonicotino-, nicotino-, and pyrazino-hydroxamic acids Fe II complexes (Figure 35B), no signal was detectable, as well. Then, similarly to the previous tests, the chemical activation of the isonicotinoand pyrazino-hydroxamic acid complexes was mediated by H2O2 and monitored by EPR in the presence of PBN, in phosphate buffer (40 mM, pH 7.4). Under these conditions, using two different proportions of complex:H2O2 (1:1 and 1:3), no signal was observed for the both complexes (Figure 36). In view of these results, experiments with the nicotino-hydroxamic acid pentacyanoferrate complex were not carried out.

Altogether these results indicate that the aroyl radical is not an intermediate of the oxidation mechanism of the hydroxamic acid derivatives studied, corroborating the mechanistic proposition of this chapter.

Attempts to trap the potential acyl nitroso intermediate by Diels-Alder reaction

As mentioned in article 2 (section 5.3), nitroso compound is one of the main intermediates proposed for hydroxamic acids oxidation. The Diels-Alder reactions, in organic solvent media, have been used as an approach for the detection of nitroso intermediate [3]. In this work, we tried to use this strategy in THF aqueous conditions (THF:H2O (2:1)). For this, the oxidation of the hydroxamic acid by [Fe(CN)6] 3- (oxidizing agent) was performed in the presence of cyclohexa-1,3-diene, aiming at production of the (2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl)(pyridin-4-yl)methanone, which could be an evidence for the nitroso intermediate formation (Scheme 24).

Scheme 24. Detection of the isonicotino nitroso intermediate by Diels-Alder reaction.

Nevertheless, the product of the reaction between the potential intermediate acyl nitroso and cyclohexa-1,3-diene was not observed. The 1 H NMR analysis of the crude, obtained at the end of the reaction, showed a higher percentage of the starting ligand and a small fraction of several other peaks non identifiable.

Complex Na6[Fe2(CN)10(N'-(pyridine-4-carbonyl) pyrazine-2-carbohydrazide)]

Bearing in mind the idea of synthesizing new hybrid-type antituberculosis prodrugs capable of exhibiting both isoniazid-(through isonicotinoyl radical potential formation) and pyrazinamide-like actions (through pyrazinoic acid potential formation), the synthesis of the binuclear complex Na6[Fe II 2(CN)10(N'-(pyridine-4-carbonyl)

pyrazine-2-carbohydrazide)] was investigated.

First, the ligand was prepared from a mixture of pyrazinoic acid and thionyl chloride (20 equiv.) at reflux (75 ºC), followed by, after solvent evaporation, addition of isoniazid and triethylamine in dichloromethane. More details are presented in the experimental part. The attempt to obtain the corresponding pentacyanoferrate complex was produced by reacting the ligand N'-(pyridine-4-carbonyl) pyrazine-2carbohydrazide with the precursor Na3[Fe II (CN)5(NH3)], following procedures already described for similar compounds [2]. Nevertheless, the desired compound was not obtained. By the 1 H-NMR technique, we observed that the product obtained corresponds to a mixture of pyridine-4-carboxylate and pyrazine-2-carboxylate compounds coordinated to Fe(CN)5 moiety ([Fe II (CN)5(pyridine-4-carboxylate)] 4-in D2O, δ (ppm): 9.03 (d, J = 5.9, 2H), 7.46 (d, J = 5.9, 2H), and [Fe II (CN)5(pyrazine-2carboxylate)] 4-in D2O, δ (ppm): 9.54 (s, 1H), 9.16 (d, J = 3.3 Hz, 1H), 8.28 (dd, J = 2.9, 1.5 Hz, 1H)), suggesting that the binuclear compound is very unstable and is rapidly oxidized and cleavage.

Conclusion

The oxidative activation of the hydroxamic acid compounds in the presence of PBN, did not show evidences for the aroyl radical formation, corroborating the activation mechanism proposed in this chapter i.e. passing through the N,O- pyrazine-2-carbohydrazide)] showed that the desired product was not obtained. On the other hand, a mixture of the pentacyanoferrate(II) complexes, coordinated with the pyridine-4-carboxylate and pyrazine-2-carboxylate ligands were produced. The formation of such products may be related to the instability of the binuclear complex formed, which after undergoing oxidative reaction could form the acid compounds as mentioned.

Chapter 3 -New pro-drugs study of Fe II hydroxamic acids complexes based: a new approach in antihypertensive and antituberculosis treatments

Article 3: Journal of Biological Inorganic Chemistry, 2020, 25, 887-901 -

Résumé de l'article en français

La tuberculose (TB), maladie due à l'infection par Mycobacterium tuberculosis (Mtb), reste aujourd'hui un problème majeur de santé publique mondial causant plus d'un million de décès par an [1]. Bien que la guérison de la tuberculose puisse être obtenue par un régime thérapeutique impliquant les médicaments de première ligne (isoniazide (INH), la rifampicine, l'éthambutol et le pyrazinamide (PYZ)) (Figure 37), ce traitement de première intention devient inefficace face à des souches de Mtb résistantes à des multiples médicaments. Le PYZ est un promédicament qui est activé par la pyrazinamidase (PncA), une enzyme du Mtb [2]. Cette activation passe par la conversion du PYZ en l'acide carboxylique correspondant (acide pyrazinoïque), ce dernier reconnu comme étant l'agent toxique pour Mtb. Cependant, l'émergence de cas de souches résistantes à ce médicament a également limité son utilisation [3]. Journal of Biological Inorganic Chemistry, 2020, 25, 887-901.

Afin de surmonter les problèmes de résistances bactériennes dans le traitement de la TB, quelques nouveaux médicaments ont été développés. L'un de ces nouveaux médicaments est le délamanide (Figure 37), utilisé en troisième intention et approuvé pour le traitement de la tuberculose résistante [4]. Fort intéressant, il y a peu de cas de résistance rapportés pour ce médicament et son action est dirigée contre des souches de

Mtb dormantes et réplicantes [5]. Le délamanide est en fait un promédicament nécessitant d'une étape d'activation enzymatique pour générer son métabolite actif, le HNO (nitroxyle). Une possible action émanant de NO • (oxyde nitrique) ne peut à ce jour être complètement exclue [3].

Outre la tuberculose, les maladies cardiovasculaires représentent également un défi dans le domaine de la santé et la situation d'urgence appelle au développement de nouveaux agents thérapeutiques, y compris ceux capables de libérer NO • et HNO. Dans la lignée des médicaments donneurs de NO • , nous pouvons mettre en évidence le nitroprussiate de sodium (SNP) comme le seul donneur de NO • , à base de métal approuvé cliniquement pour les procédures cardiovasculaires d'urgence à l'hôpital [6].

En revanche, il n'existe actuellement aucun agent donneur de HNO cliniquement disponible.

Les acides hydroxamiques ont été classiquement signalés comme des espèces libérant des NO • /HNO après une réaction d'oxydation [7]. Dans cette même ligne (νs), symmetric bending (δs), wagging (π) and twisting (τ) (cm As with complexes 3 and 6, the characterization results also indicated that compound 13 was successfully obtained. The data were very similar to that reported for 3, so that only the spectra and tables relevant to the characterization of the complex 13 are illustrated below.

NMR spectroscopy Vibrational spectroscopy H37Rv strains, with MIC values > 100 μg/mL as compounds 2, 3, 5 and 6. However, a vasodilating activity was also observed for 13 (Figure 51). The hydroxamic acid complex 13 showed a promising vasodilator profile with EC50 = 106 nM, whereas ligand 10 showed a benefic action only in high concentration with EC50 = 43 μM. This remarkable difference supports the key role of metal center, which enhanced ca. 406fold the activity. This metal complex exhibited a more moderate action compared to SNP (EC50 = 13 nM), showing an EC50 value 8-fold higher than SNP. In addition, the concentration-response curve of the vasodilation experiment shows that 13 have lower efficacy than the SNP and the other complexes 3 and 6. The cytotoxicity of the complexes (3, 6 and 13) and their respective free ligands (2, 5 and 10), was tested by Mrs Sandra Bourgeade-Delmas in Pharmadev laboratory (IRD-UPS, Toulouse), whose the director is Pr. Nicolas Fabre, using macrophage cells (J774A.1) in accord with the references [1][2][3]. The CC50 values (concentration equal to 50% of the cytotoxicity effect) obtained for the six compounds were generally higher than 60 µM, being significantly less toxic (1200 -fold) than the doxorubicin control (CC50 = 0.05 µM) (Table 3), a chemotherapeutic used in the treatment against solid tumors of several cancers [4]. These high CC50 values are indicative of a low cytotoxic activity by the hydroxamic acid derivatives studied. In an opportunistic way, the anti-leishmania and anti-malaria action of the complexes (3, 6, and 13) and the corresponding free ligands (2, 5, and 10) were also evaluated in collaboration with Pharmadev laboratory (IRD-UPS) in accord with the references [1][2][3]. The set of hydroxamic acids derivatives (ligands and complexes) tested

showed high IC50 values (> 60 µM) for leishmaniasis, being approximately 860 -fold less active than the control amphotericin B (IC50 = 0.07 µM) (Table 4), a second-line antileishmanial drug [5]. These results showed that compounds 2-13 have no promising action against this type of disease. In contrast, anti-malaria tests exhibited more promising results, particularly to the free ligands 2, 5 and 10, which presented IC50 values of 21.57, 2.53, and 3.98 µM, respectively, being relatively better than their respective Fe II complexes (Table 4) and in the case of 5, it is only 14 less active than the antimalarial drug chloroquine (IC50 = 0.18 µM) [6]. Interestingly, the presence of the [-Fe(CN)5] moiety was not responsible for beneficial effects, suggesting that biological action is not due to HNO as metabolite.

Conclusion

The complex 13 has exhibited the same chemical and biological behave as the complexes 3 and 6. Although 13 also had no action against Mtb strains, it has proven to Part 2 -Pentacyanoferrate complexes: hydroxamic acid as therapeutic platforms _________________________________________________________________________________ 219 be an interesting vasodilating agent, which stimulates future studies. In addition, none of the complexes has shown high cytotoxicity in trials with macrophages, indicating that such compounds may be safe for further in vivo studies. The anti-leishmaniasis tests do not encourage us to use such systems for this purpose. Anti-malaria studies have shown that ligands 5 and 10 have a significant action against malaria, however the introduction of the metal center has not been responsible for improving antimalarial action and therefore HNO may not be a toxic metabolite for Plasmodium falciparum.

Bibliography

______________________

General conclusion and perspectives 6. General conclusion and perspectives

Metal complexes have proven to be quite versatile tools in the development of new therapeutic approaches. As part of this thesis, the association of metal system with ligands acting as NO • or HNO donors emerged as a promising alternative in cardiovascular and anticancer drug research.

The first chapter focused on the reactivity of the trans-[Fe(cyclam)(NO)Cl]Cl2 complex. Although this complex was known to release NO • upon light stimulus, in this work we demonstrated that it also exhibits a high potential as direct NO • and HNO donor in physiological conditions. This property is associated with the antihypertensive and antiangiogenic activities observed for this complex. In this way, the results of antiangiogenic activity through the inhibition of HIF-1α suggest this nitrosyl metal complex as a strong candidate in anticancer drug design. Moreover, the vasodilation studies show that the trans-[Fe(cyclam)(NO)Cl]Cl2 complex allows a milder control of the blood pressure (IC50 = 910 nM) than the classical metal pro-drug SNP (IC50 = 24 nM), the latter being currently used in cases of hypertensive crisis. This can be considered as an advantage, the general use of the more potent vasodilator SNP being indeed limited by its abrupt capacity in inducing vasodilatation. In addition, the absence of cyanide ions in the coordination sphere of the Fe II center, which, if released, can be very toxic for patients treated by SNP, supports the potential use of the present complex in future pre-clinical studies.

The second part of the thesis has concerned the study of (di)azine hydroxamic acid derivatives, with emphasis on the ability of these molecules to release HNO after oxidative activation. A study of the oxidation of isonicotino-, nicotino-, and pyrazinohydroxamic acids by K3[Fe III (CN)6] in aqueous conditions, revealed that a N,Odi(di)azinoylhydroxylamine intermediate was formed and no nitrosocarbonyl intermediate could be detected (Figure 52). These results contrast with the frequently reported hypothesis that a nitrosocarbonyl intermediate is responsible for the release of HNO by oxidation of hydroxamic acids. By performing the same oxidative treatment in the presence of NH2CH3, we put forwards that the N,O-di(di)azinoylhydroxylamine intermediate has an electrophilic character similar to that of a putative nitrosocarbonyl intermediate. In addition, the corresponding (di)azine carboxylic acids are the final products, thus resulting from the oxidation of hydroxamic acids, not from their hydrolysis. The third chapter concerns the development of new iron-metal complexes derived from the molecules discussed above serving as ligands: the basic idea is the design of a hybrid metallic pro-drug through the association of precursors of the active metabolites of isoniazid (isonicotinoyl radical) or pyrazinamide (pyrazinoic acid) and delamanid (HNO). While in vitro treatment (at pH 7.4, 37 ºC) of the complexes 3, 6, and 13 (Figure 53) by the endogenous oxidizing agent H2O2 did not lead to any aroyl radical species, it was shown to trigger the formation of HNO and carboxylic acid derivatives, thus validating the concept of pyrazinamide-delamanid hybrid complex.

These complexes failed to inhibit the growth of non-resistant Mtb strains. However, knowing that pyrazinamide is active upon non-replicating bacilli and not upon growing strains, it should be relevant to test these compounds towards this more appropriate model. On the other hand, a promising vasodilating action was found for the three complexes studied, with emphasis on the complex 6 (EC50 = 64 nM), which is only 5fold less potent than the SNP drug (EC50 = 13 nM). As shown for the trans-[Fe(cyclam)(NO)Cl]Cl2 complex, hydroxamic acid complexes exhibit a moderate action as compared to SNP, and may thus provide a valuable advance in the design of new antihypertensive agents, since the rapid release of NO • from SNP can cause hazardous sudden drops in blood pressure. Moreover, while the complexes 3 and 6 showed efficacy similar to that of SNP, 13 was found slightly less effective. These results stimulated the consideration of complex 6 in studies of antihypertensive action in rats.

Increasing the doses of complex 6 (1, 5, and 10 mg/kg) promoted a decrease in blood pressure more efficiently on hypertensive rats (by 45, 62, and 65%, respectively) than on normotensive rats (31, 51 and 53%, respectively). Comparison of the vasodilation curves of the "free ligands" with those of their complexes clearly show that the pentacyanoferrate(II) moiety plays a crucial role in the activation of hydroxamic acids leading to HNO release. 3), isonicotino-( 6) and nicotino-( 13) hydroxamic acids pentacyanoferrate(II) complexes.

In summary, this work unveils significant systems and proposals in the area of bioinorganic chemistry. The results reported in this thesis confirm the possible use of metallo-compounds as an alternative strategy in the development of new therapeutic drugs, with special emphasis on the release of NO • and HNO. As a perspectives, further studies on the vasodilation effect of hydroxamic acid complexes may focus on mechanistic issues, e.g., using inhibitors of the soluble guanylyl cyclase (e.g., ODQ = 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one) and NOS (e.g., L-NAME = "L-N G -nitro arginine methyl ester"), which are extremely important enzymes involved in the NO •mediated vasodilation mechanism. These studies deserve to be undertaken to bring more information about the vasorelaxant activity of the Fe II complexes 3, 6 and 13.

Conclusion générale et perspectives

Les complexes métalliques se sont avérés être des outils polyvalents dans le développement de nouvelles approches thérapeutiques. Dans le cadre de cette thèse, l'association d'un système métallique à une molécule donneuse de NO ,2,4] oxadiazolo [4,3-a] quinoxalin Abstract: Cancer, cardiovascular diseases and tuberculosis (TB) are major therapeutic challenges, for which various drug strategies have been developed, including the use of inorganic agents such as metallo-drugs and NO • (nitric oxide)/ HNO (nitroxyl). Within this prospect, the thesis objective addresses the design, synthesis, physical-chemical studies and biological evaluation of metal-based drugs capable of releasing NO • and/or HNO with pharmacological effects. Two types of organic platforms are thus envisaged as ligands in Fe II/III complexes devised as potential sources of NO • /HNO: the spectator cyclam ligand (1,4,8,11-tetraazacyclotetradecane) and three reactive azine hydroxamic acid ligands (ArC(O)NHOH). The first chapter deals with the chemical reactivity of a trans-[Fe(cyclam)(NO)Cl]Cl2 complex, which is shown to decompose by releasing NO • under physiological pH and temperature conditions, the process being significantly accelerated upon light irradiation at 365 nm. In contrast, in vitro experiments in the presence of glutathione revealed that the same complex produces HNO. Angiogenesis studies showed that the complex is able to significantly inhibit the hypoxia-inducible factor (HIF-1α) induced by hypoxia or addition of a NO • donor (Spermine NONOate) in breast cancer cells: this result is consistent with a release of HNO by the nitrosyl complex. On the other hand, vasodilation assays using precontracted rat aortic rings revealed that the complex exhibits a relaxation IC50 of 910 nM, vs 24 nM for the reference drug, nitroprusside. The next chapters deal with the oxidation-promoted release of HNO from aromatic hydroxamic acids. Chapter 2 focuses on the oxidation mechanism of isonicotinoic, nicotinoic and pyrazinoic hydroxamic acids, mediated by potassium ferricyanide (K3[Fe III (CN)6]) in physiological pH conditions. It is shown that oxidative activation of aryl hydroxamic acids, mediated by Fe III , involves the production of the N,O-di(di)azinoylhydroxylamine intermediate with concomitant release of HNO, instead of the putative acyl nitroso species. The free hydroxamic acids were also evaluated for different biological activities and used as controls to be compared with iron complexes described in chapter 3. These pentacyanoferrate(II) complexes, Na3Fe II (CN)5(ArCONHOH), initially devised as potential hybrid of isoniazid (INH, first-line anti-TB prodrug, for which the isonicotinoyl radical is the active metabolite) and delamanid (third-line anti-TB, for which HNO is the active metabolite), were synthesized and characterized by spectroscopic techniques, cyclic voltammetry, and DFT calculations. Using EPR and 1 H NMR spectroscopy, oxidation of these Fe II complexes with H2O2 was shown to result in the release of HNO and corresponding azinoic acids, through a likely intramolecular electron transfer. In contrast to what was observed from the INH complex (Na3[Fe II (CN)5(INH)]), no azinoyl radical species was evidenced to be produced from the corresponding hydroxamic acid complexes. Instead, the corresponding carboxylic acids were found to be formed, in particular pyrazinoic acid which is the active metabolite of pyrazinamide, another antitubercular pro-drug: this result would be relevant for the treatment of pyrazinamide-resistant tuberculosis, as the activation could thus occur without assistance of the pyrazinamidase enzyme of Mycobacterium tuberculosis (Mtb). The free hydroxamic acids and corresponding complexes did not show any antibiotic action against an actively growing/non-resistant Mtb strain. However, the complexes showed a strong dose-dependent and reversible vasodilation activity, close to that of nitroprusside (64-250 nM vs 13 nM). The free hydroxamic acids were found ca. 19-to 76-fold less active than the corresponding complexes. On the basis of in vivo experiments, Na4[Fe II (CN)5(PyCONHO -)] also showed a promising cardiovascular activity profile for the development of a new antihypertensive agent. Key words: antiangiogenesis, cyclam, hydroxamic acids, iron complexes, nitric oxide, nitroxyl, oxidative activation mechanisms, tuberculosis, vasodilation.

I

  am also indebted to Dr. Alix Sournia Saquet and Dr. Lionel Rechignat, their involvement and contributions in the electrochemistry and EPR experiments, respectively were essential. I would like to thank Prof. David A. Wink and his team for the antiangiogenic activity experiments, Prof. Nilberto Robson Falcão do Nascimento and his team for vasodilation tests, Professors Cristiano V. Bizarro and Pr. Luiz A. Basso for anti-TB tests, and Mrs Sandra Bourgeade-Delmas and Prof. Nicolas Fabre for cytotoxicity, antileishmaniasis and anti-malaria tests.

4. PART 1 - 98 4. 2 . 4 . 3 . 4 . 4 . 4 . 5 . 2 . 5 . 3 . 5 . 4 . 5 . 5 . 5 . 7 . 5 . 8 . 1 .

 1982434445253545557581 NITROSYL IRON COMPLEX: CYCLAM AS THERAPEUTIC PLATFORM ................................................................................................................. 98 4.1. Introduction ....................................................................................................... Article 1: Journal of Inorganic Biochemistry, 2020, 210, 111133 -Résumé de l'article en français ...................................................................................................... 101 Article 1: Journal of Inorganic Biochemistry, 2020, 210, 111133 -Printed . 106 Supporting information .................................................................................. 119 Unpublished results associated with the article ............................................ 129 4.6. Conclusion ........................................................................................................ 136 5. PART 2 -PENTACYANOFERRATE COMPLEXES: HYDROXAMIC ACID AS THERAPEUTIC PLATFORMS ......................................................................... 139 5.1. Introduction ..................................................................................................... 139 5.Article 2: New Journal of Chemistry, 2020, 44, 11965-11973 -Résumé de l'article en français: .................................................................................................... 144 Article 2: New Journal of Chemistry, 2020, 44, 11965-11973 -Printed ........ 150 Support information ........................................................................................ 159 Unpublished results associated with the article ............................................ 168 5.6. Conclusion ........................................................................................................ 174 Article 3: Journal of Biological Inorganic Chemistry, 2020, 25, 887-901 -Résumé de l'article en français ................................................................................... 175 Article 3: Journal of Biological Inorganic Chemistry, 2020, 25, 887-901 -Printed .......................................................................................................................... 182 Introduction INTRODUCTION Metallo-drugs in medicine -Brief historical account

Figure 1 .

 1 Figure 1. Examples of three first prescribed metallo-drugs.

Figure 2 .

 2 Figure 2. Anticancer platinum metallo-drugs clinically used or in current clinical trials.

Figure 3 .

 3 Figure 3. Arsenic anticancer metallo-drugs: trisenox (clinically used) and darinaparsin (in clinical trials).

Figure 4 .

 4 Figure 4. Structures of ruthenium compounds in clinical trials.

Figure 5 .

 5 Figure 5. Chemotherapeutic agents based on gallium(III) and molybdenum(VI) metals assayed in clinical trials.

Figure 6 .

 6 Figure 6. Cardiovascular metallo-drugs in clinical use (SNP) and clinical trials Phases I/II (Oxacom ® and M40403 (clinical phases suspended)).

Figure 8 .

 8 Figure 8. Endogenous production of nitric oxide. Panel A shows the two-step formation of NO • from arginine to citrulline, through the intermediate N-omega-hydroxy-Larginine formation. This reaction is catalyzed by nitric oxide synthase (NOS) with participation of O2, NADPH and the cofactor tetrahydrobiopterin (H4B) (Panel B).Panel C shows NO • diffusion from its production site (e.g., endothelial cells) into another cell reaching soluble guanylate cyclase (sGC), whose enzymatic activity is strongly stimulated with conversion of GTP to cGMP.

Figure 9 .

 9 Figure 9. FDA approved antiangiogenic drugs and their targets in parentheses.

Figure 12 .

 12 Figure 12. First-line antitubercular drugs. v. Second-line antitubercular drugs The emergence of treatment-resistant bacteria using first-line antitubercular drugs has resulted in the employment of other medicines called second-line antitubercular drugs. The main second-line antitubercular drugs are: p-aminosalicylic acid, thioamides (ethionamide and prothionamide), cycloserine derivatives (cycloserine and terizidone), aminoglycosides (amikacin e streptomycin) and fluoroquinolones(levofloxacin and moxifloxacin) (Figure13). Nevertheless, these compounds are generally less potent, more toxic, and more expensive. In addition, drug-resistant TB treatment is long, ranging from 9-12 months (shorter regimen) and18-20 months (longer regimen) [184].

Figure 13 .

 13 Figure 13. Some examples of antitubercular second-line drugs. vi. Third-line antitubercular drugs Although the development of second-line drugs provides a cure for many multidrug-resistant tuberculosis (MDR-TB) patients, in some cases, the treatment is not effective beyond the high toxicity of these drugs [185]. As a way to overcome these treatment failures, new drugs with MDR-TB action, called antitubercular third-line drugs, have been developed. Three examples of third-line antitubercular drugs are bedaquiline, delamanid, and pretomanid. These drugs were the last antitubercular molecules approved for clinical use, bedaquiline in 2012 (by FDA), delamanid in 2014 (by European Medicines Agency (EMA)), and pretomanid in 2019 (by FDA) (Figure 14) [185,186]. WHO recently included bedaquiline and delamanid in the group of medicines recommended for use in longer MDR-TB regimens [187].

Figure 14 .

 14 Figure 14. Structures of bedaquiline, delamanid, and pretomanid.

2. 4 . 1 .

 41 Organic Nitrates Organic nitrite molecules are the oldest class of NO • donors [193]. Structurally they are nitric acid esters, generally represented by the formula RONO2. The glyceryl trinitrate (GTN), pentaerythrityl tetranitrate (PETN), and isosorbide dinitrate (ISDN),

Figure 15 .

 15 Figure 15. Classic organic nitrates.

Figure 16 .

 16 Figure 16. New nitrate hybrids and the goals for their development.

Scheme 6 .

 6 Scheme 6. Mechanism of nitrate organic reduction by enzymatic systems (without thiol participation) and ferrous-heme proteins (A) or enzymatic reduction involving thiols (B), references [212-214]. 2.4.2. Secondary amines-based diazeniumdiolates (NONOates) The diazeniumdiolates or NONOates compounds have a -O-N=N-O -(NONO) moiety connected to a primary or secondary amine. This type of molecule, in special the secondary amines, is considered standard NO • donors and is thus used in several experimental models. DEA NONOate, DETA NONOate, PROLI NONOate, SPER NONOate, and MAHMA NONOate are some examples of these donors (Figure 17) [215,216]. A series of studies have shown the ability of NONOates derivatives to release NO • , where these molecules were investigated for biological activities such as vasodilation [217,218], platelet aggregation inhibition [219], and modulation of angiogenesis [215].

Figure 17 .Scheme 7 .

 177 Figure 17. Structures of some NONOates derivatives molecules.

Figure 18 .

 18 Figure 18. Structure of some promising NONOates derivatives.

Figure 19 .

 19 Figure 19. Structure of some S-Nitrosothiols (RSNOs).

Figure 20 .

 20 Figure 20. Structure of some promising furoxan hybrid-based agents.

Scheme 9 .

 9 Scheme 9. Mechanism of furoxan-based molecules in releasing NO • by reaction with thiol, reference [247].

Figure 21 .Scheme 10 .

 2110 Figure 21. Structures of some sydnonimine-based compounds.

decomposition from pH 4

 4 Scheme 11. Angeli's salt (A), AcOM-IPA/NO (B), and Gal-IPA/NO (C) decomposition, references[261,265,266].

Figure 22 .Scheme 12 .Scheme 13 .

 221213 Figure 22. Examples of some hydroxylamine-based donors.

Figure 23 .

 23 Figure 23. Hydroxamic acid donors.

  another large class of NO • donors widely studied by several laboratories. Many of these compounds have also shown moderate antihypertensive activities (e.g., EC50 from 200 to 690 nM) compared to SNP[305][306][307]. These activities suggest that they may have opportunities to be used as models for antihypertensive drugs. Metal-compounds carrying NO • have also been investigated as NO • photoreleaser agents, opening further opportunities[305,306,308,309].

Figure 24 .

 24 Figure 24. Structures and in vitro vasodilation action of nitrosyl iron and ruthenium complexes (4-bzpy = 4-benzoylpyridine, ImN = imidazole, and tu = thiourea).

[

  Ru(bpy)2(L)NO] n+ system, there is a remarkable increased in the pH values for the conversion of nitrosyl into nitrite ligand, from1.45 to 10.32 (Figure 25) [314]. This major change upon binding sulfite allowed the nitrosyl species to become the main species under physiological conditions and might be a reason for many promising biological properties found for this compound. Photorelease (463 nm) of NO • from the [Ru(bpy)2(SO3)NO] + complex was validated using the selective NO • trap cPTIO (2-(4carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt)[152]. In contrast, in the presence of thiols (e.g., glutathione), the [Ru(bpy)2(SO3)NO] + complex released HNO. Other polypyridine ruthenium compounds have also showed a pH of nitrosyl/nitrite (1:1) conversion close to or higher than the physiological pH(6.4-8.0) (Figure25). Interestingly, all these metallo-nitrosyl derivatives show indeed an ancillary ligand bound to ruthenium through sulfur atom, indicating that such ligands can positively influence the acid-base nitrosyl/nitrite equilibrium. Those results showed the use of sulfur-based ligands as a relevant strategy in the development of new therapeutic platforms for NO • /HNO release[152,306,314,315].

Figure 25 .

 25 Figure 25. Structures of some metallo-nitrosyl compounds and pH of nitrosyl/nitrite conversion (isn = isonicotinamide, tu = thiourea and tbz = thiobenzamide).

Figure 26 .

 26 Figure 26. Structures of nitrosyl Fe II [Fe(NO)( TMS PS2)( TMS PS2H)] and ruthenium(II) nitrosylsulphito trans-[Ru(NH3)4(isn)(N(O)SO3)](PF6) (isn = isonicotinamide) complex.

  Compte tenu du fait que le NO • /HNO peut présenter un grand spectre d'actions thérapeutiques intéressantes et que les complexes métalliques peuvent être un outil intéressant en chimie inorganique médicinale, l'objectif de cette thèse est le développement de nouvelles stratégies de conception de médicaments impliquant la synthèse et l'étude physicochimique et biologique de complexes métalliques capables de porter et de délivrer l'agent actif biologique, NO • /HNO. Le premier chapitre vise à i) étudier la réactivité du complexe nitrosyl trans-[Fe(cyclam)(NO)Cl]Cl2, ii) étudier la stabilité de ce complexe dans des conditions physiologiques (pH 7,4 à 37 ºC) afin d'évaluer l'influence de toute décomposition spontanée possible, ainsi que l'identification des produits possibles générés, iii) déterminer l'influence de la lumière (365 nm) sur la libération possible de NO • , et iv) vérifier le potentiel biologique du trans-[Fe(cyclam)(NO)Cl]Cl2 en tant qu'agent antiangiogénique avec une propriété anticancéreuse potentielle. De plus, l'investigation du profil vasodilatateur du complexe nitrosyle est également un des objectifs de cette thèse. Les résultats de ces travaux ont déjà été publiés et sont présentés au chapitre 1[1].Afin d'avancer dans la recherche de nouveaux châssis moléculaires capables de relarguer NO • /HNO et d'avoir des intérêts thérapeutiques, nous explorons l'habilité des dérivés aromatiques azotés de l'acide hydroxamique à libérer NO • /HNO après une étape de réaction oxydative. Cette forme de châssis moléculaire pourrait avoir un intérêt tout particulier parce qu'en fonction du mécanisme d'activation de ce type de molécule, outre la libération de NO • /HNO (métabolite actif du délamanide -médicament anti-TBet agent vasodilatateur), un intermédiaire de type radical aroyle ou un acide carboxylique aromatique pourrait également être formé, ce qui pourrait être considéré, respectivement, comme le métabolite actif de l'isoniazide ou du pyrazinamide, deux pro-drogues antituberculeuses majeurs. Ainsi, la deuxième partie de cette thèse est divisée en deux projets. Le but du premier (chapitre 2) est l'étude d'un mécanisme intermoléculaire de l'activation oxydative des acides hydroxamiques dérivés des acides isonicotinoique, nicotinoique et pyrazinoique, analogues respectifs de l'isoniazide (anti-

Fe(NO) 2 [ 2 ]

 22 , SNP analogs (Kn[M(CN)5NO] (M = V, Cr, Mn and Co with n = 3, or M = Mo with n = 4)) [3], and polypyridine ruthenium derivatives such as [Ru(bpy)2(ImN)NO](PF6)3 (bpy = 2,2'-bipyridine, ImN = imidazol) and

  photochemical stimulation or reaction with GSH. By using a selective probe cPTIO(2- (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium) for UV-Vis and EPR techniques, or through other experimental (Griess reagent) and theoretical studies (density functional theory (DFT) and time-dependent DFT (TD-DFT)), we were able to confirm the NO • production from the complex via light stimulus (365 nm). On the other hand, the reaction of trans-[Fe(cyclam)(NO)Cl]Cl2 complex with GSH indicated the HNO production, as well as the formation of an intermediate adduct between the nitrosyl complex and GSH (trans-[Fe(cyclam)(Cl)N(O)SG] + )[6]. The hypothesis that this complex can release HNO in the presence of thiols has been reinforced by a cell angiogenesis assay based on the HIF-1α degradation. Additionally, the vasorelaxation capacity of the metal complex ion was assessed, in comparison to the SNP, and a more moderate vasodilation activity was noticed. These results, in addition to the evidences of its low cytotoxicity[8], made the trans-[Fe(cyclam)(NO)Cl]Cl2 complex a potential therapeutic model for cardiovascular diseases.

Figure 27 .Schème 15 .

 2715 Figure 27. Structure des complexes trans-[Fe(cyclam)(NO)Cl]Cl2 (1) et trans-[Fe(cyclam)Cl2]Cl (3).

4 . 3 .

 43 • et HNO joue un rôle clé dans la régulation de HIF-1α dans les cellules tumorales. Alors que NO • a été indiqué comme agent stabilisant pour HIF-1α dans des conditions normoxiques, HNO s'est avéré agir dans le sens inverse, bloquant la stabilisation de HIF-1α, à la fois dans des conditions induites par l'hypoxie ou par NO • , ce dernier dans des conditions normales d'air[7,8]. Basé sur ces informations, la capacité antiangiogénique du complexe trans-[Fe(cyclam)(NO)Cl] 2+ a été évaluée en utilisant le test de stabilité HIF-1α. Cette expérience, réalisée en collaboration avec l'équipe du Pr. David A. Wink où « National Cancer Institute, Cancer and Inflammation Program, Frederick -USA », a montré une diminution significative de l'accumulation de HIF-1α causée par le composé étudié. Parallèlement, le complexe 1 a également démontré une action antiangiogénique potentielle, via le test de formation du tube endothélial. Le test de formation du tube endothélial des cellules HUVEC (cellules endothéliales humaines de veine ombilicale) a démontré qu'en présence du composé 1, le nombre de vaisseaux était significativement réduit par rapport au composé précurseur sans NO + (composé 3, trans-[Fe(cyclam)Cl2] + , Figure 27). Cette partie du travail a également été réalisée avec la collaboration du Pr. David A. Wink. Les propriétés vasodilatatrices du composé 1 ont également été explorées, celui a présenté une CI50 = 910 nM (valeur correspondant à la concentration minimale nécessaire du composé pour provoquer une vasodilatation à 50%), tandis que celle de molécule de référence SNP est de 24 nM. Cette partie du travail a été réalisée avec la collaboration du Pr. Nilberto R. F. do Nascimento. Cette activité modérée, attribuée à libération de NO • ou HNO, ajoutée à la faible cytotoxicité du complexe (mesurée à travers les lignées cellulaires cancéreuses B16-F10 et HUH-7) [9], fait du composé 1 une plateforme thérapeutique prometteuse pour des molécules à propriétés antihypertensive avec des avantages par rapport au SNP. En effet, l'activité du complexe 1 peut être indicative d'une libération plus contrôlée de NO • , évitant les problèmes d'hypotension causés par la libération trop rapide de NO • associée au SNP. De plus, ce complexe n'est pas concerné par le relargage des ions cyanures toxiques. En résumé, ce composé prometteur mérite des études biologiques complémentaires afin de découvrir tout son potentiel thérapeutique, car il représente l'un des premiers composés Article 1: Journal of Inorganic Biochemistry, 2020, 210, 111133 -Printed Part 1 -Nitrosyl Iron complex: cyclam as therapeutic platform _________________________________________________________________________________ 4.4. Supporting information 4.4.1. DFT studies (Results and discussion)

Fig. S1

 S1 Fig. S1 DFT optimized structure of the complex trans-[Fe(cyclam)(Cl)(NO)] 2+ .

Fig. S2

 S2 Fig. S2 Experimental (▬), theoretical (---) electronic spectra of the trans-[Fe(cyclam)(NO)Cl] 2+ complex ion in water and vertical electronic transitions calculated by TD-DFT (▬).

Fig. S3

 S3 Fig. S3 Molecular orbitals involved in the main electronic transitions of trans-[Fe(cyclam)(Cl)(NO)] 2+ .

Fig. S7

 S7 Fig. S7 Electronic spectra of cPTIO (▬), cPTI (▬) (resulted of cPTIO in reaction with NO • , from a NO • saturated solution), cPTIO-H (▬) (resulted of cPTIO in reaction with HNO by using Angeli's salt (200 μM)), trans-[Fe(cyclam)(NO)Cl] 2+ (▬) (200 μM)after 30 min of light irradiation at 365 nm and cPTIO (▬) after 30 min of light irradiation at 365 nm. All electronic spectra were obtained in 0.1 M phosphate buffer pH 7.4 solution at 37 ºC, under aerobic conditions. The concentration of cPTIO used in all assays was 100 μM.

Fig. S8

 S8 Fig. S8 EPR monitoring of the reaction of trans-[Fe(cyclam)(NO)Cl] 2+ (400 µM) with light (365 nm) using cPTIO (200 µM) as probe, in 0.1 M phosphate buffer pH 7.4 solution at 25 ºC. A. Reaction in the presence of light (365 nm) without complex. B. Reaction with complex in absence of light. C. Reaction of trans-[Fe(cyclam)Cl2] + (400 µM) with cPTIO (200 µM) in absence of light, 0.1 M phosphate buffer pH 7.4 solution at 25 ºC. All spectra were measured under aerobic conditions.

Fig. S9 -

 S9 Fig. S9 -Electronic spectra of aqueous solution of Griess reagent (50 mM) and trans-[Fe(cyclam)(NO)Cl] 2+ (2 mM) before irradiation (gray), with 15 min of irradiation (λirrd = 365 nm) (black) and 15 min without irradiation (dotted) at room temperature (ca. 25 o C), under aerobic conditions. Inset: Solution before and after irradiation.

Fig. S11

 S11 Fig. S11 EPR monitoring of the reaction of GSH (4 mM), cPTIO (200 µM), containing DTPA (400 µM) at 25 ºC, under aerobic conditions. All spectra were recorded during 18 min at intervals of ca. 10 seconds.

  = 40% (0.040 g), green solid. IR symmetric stretching (νs), symmetric bending (δs) and twisting (τ) (cm -1 ): 3255 -3208 (νs N-H), 2980 -2925 (νs C-H), 1483 -1430 (δs CH2), 1331 -1280 (τ CH2), 1122 -1020 (νs C-N), 835 and 561 (νs P-F). UV-Vis (H2O) λmax/nm (ɛ/M -1 cm -1 ) = 238 (7635), 300, (2019), 355 (1096). Cyclic voltammetry in CF3COOH/CF3COONa solution 0.1 M (pH 3.2), V = 0.1 V s -1 : E1/2 = 0.291 V vs NHE.

Figure 30 .Figure 31 .

 3031 Figure 30. Spectral variation in the UV-Vis region for an anaerobic solution of Mb-Fe III (4.6 μM) and trans-[Fe(cyclam)(NO)Cl] 2+ (100.0 μM), in phosphate buffer 0.1 M, pH 7.4, at 37 ºC. Mixture before (gray) and after 1.5 h (black) of irradiation (365 nm). A = Full spectra (Soret band) and B = Spectra zoom between 450 and 700 nm (Q bands).

Figure 32 .

 32 Figure 32. Irradiation of the trans-[Fe(cyclam)(NO)Cl]Cl2 at 365 nm (6 h), in KBr pellet, monitoring by FTIR.

Figure 33 .

 33 Figure 33. Proposal of the redox metal-mediated activation of IQG607 in Mtb, extracted from the reference [9].

Schème 19 .Schème 20 .

 1920 Réactions de HNO en milieu aqueux ou biologique.Le HNO a été décrit comme l'un des principaux produits de l'oxydation de l'acide hydroxamique médiée par divers agents oxydants (NaIO4, metaloenzymas, Na3[Fe II (CN)5NH3]/oxydante, K3[Fe III (CN)6], ˙OH, ˙N3, etc.). Toutefois, le mécanisme exact de cette réaction d'oxydation dans des conditions physiologiques de pH ainsi que la caractérisation directe ou indirecte des éventuels intermédiaires générés, restent encore à être élucidés et font ainsi objet d'étude. Compte tenu du fort potentiel des acides hydroxamiques à générer de HNO, l'élucidation de leur mécanisme d'oxydation dans des conditions physiologiques de pH est cruciale pour assister à la conception de nouvelles molécules donneuses de HNO plus performantes. Dans le premier chapitre de cette deuxième partie (chapitre 2), nous avons examiné l'oxydation intermoléculaire directe des dérivés: acide hydroxamique isonicotinique (1), nicotinique (2) et pyrazinique (3), médiée par le ferricyanure de potassium (K3[Fe III (CN)6]) à pH = 7.4, ainsi qu'élucidé les structures des intermédiaires et des produits finaux formés (Schème 20). Réaction d'activation oxydative de l'acide hydroxamique isonicotinique (1), nicotinique (2) et pyrazinique (3), par l'ion [Fe III (CN)6] 3-à pH = 7.4. L'oxydation des acides hydroxamiques 1-3 (1 éq.) par [Fe(CN)6]

  dus à des CH aromatiques et deux pics à 146,0 et 137,5 ppm désignés comme étant les Cq des noyaux aromatiques. L'ensemble des données corrèle très bien avec la structure N,O-diisonicotinoylhydroxylamine (Schéme 21, composé 4) proposée pour l'intermédiaire. L'analyse SM (DCI-CH4) n'a pas permis l'identification du pic ion moléculaire de cet intermédiaire (composé 4). Par contre, la détection des pics à m/z = 199, attribués à l'amide 13 [M + ] (Schéme 21), m/z = 124, attribué à l'acide 7a [M + + 1] (Schème 22), et principalement à m/z = 123 qui a été attribué à l'amide 10 [M + + 1] (Schème 22) sont des forts indices d'être des fragments de l'intermédiaire di(di)azinoylhydroxylamine proposé. En effet, l'amide m/z = 123 pourrait être issu d'une étape de decarboxylation (perte de CO2) de 4. Par SMHR (mode positif ESI), un petit pic à m/z = 244,0726 a pu finalement être détecté et la masse exacte correspond bien à celle de la formule brute de 4 plus une unité [M + + 1].

du composé 5 (

 5 Schème 22). Par contre, le spectre de masse (DCI-CH4) du produit issu de la réaction d'oxydation de 2 a montré, des pics à m/z = 215 (composé 14 [M + CH4 +• ]) et à m/z = 200 (composé 14, [M + H + ]), caractéristique d'une fragmentation de 5 (Schème 22). De plus, l'analyse de masse de haute résolution (mode positif ESI) a permis de détecter d'un pic à m/z = 244,0726 correspondant au ion moléculaire [M + H + ] du composé 5. Contrairement aux études présentées pour les molécules 1 et 2, le spectre RMN 1 H de l'oxydation de 3 était plus spéculatif en raison de chevauchements de signaux, ne contribuant pas de façon significative à supporter la structure supposée pour l'intermédiaire 6. Par contre, dans le cas de l'oxydation de 3 (K3[Fe III (CN)6] + H2O2) [7] étudiée par SM haute résolution (mode positif ESI), il a été possible de détecter un pic de l'ion moléculaire à m/z = 246,0619 ([M + H + ]) cohérent avec la structure proposée pour le composé 6 (schéma 21). L'ensemble de ces caractérisations supporte N,Odi(di)azinoylhydroxylamine comme étant l'intermédiaire formé lors de l'étape d'oxydation des acides hydroxamiques in milieu aqueux et à pH physiologique. Outre les résultats de mise en évidence de l'intermédiaire N,Odi(di)azinoylhydroxylamine, nous avons aussi démontré qu'en présence de méthylamine cet intermédiaire est capable d'agir comme un agent acylant conduisant la formation de dérivés de type N-méthylamide.Enfin, les résultats des études spectroscopiques (UV-Vis et EPR), en utilisant le piège cPTIO, du suivi de la réaction d'oxydation des acides hydroxamiques par l'ion Fe III , supportent fortement la proposition de formation de HNO. En effet, les réactions des acides hydroxamiques avec l'ion [Fe III (CN)6] 3-, en présence de cPTIO, ont conduit à une diminution du quintuplet caractéristique du cPTIO présent dans le spectre de RPE et à une diminution de sa bande à 541 nm (UV-Vis). Les modifications observées lors de ces expériences, peuvent être corrélées à une réaction du cPTIO avec HNO[8]. Toutes ces approches expérimentales corroborent à une nouvelle perspective sur le mécanisme oxydatif des acides hydroxamiques aromatiques in milieu aqueux à pH physiologique. the 1 H NMR following of the K3[Fe III (CN)6] mediated oxidation of (di)azine hydroxamic acids 1, 2 and 3

  734 mmol) and K3[Fe(CN)6] (0.604 g, 1.835 mmol). 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 8.91(d, J = 4.4 Hz, 2H), 8.81 (d, J = 4.4 Hz, 2H), 7.98 (d, J = 4.4 Hz, 2H) and 7.79 (d, J = 4.4 Hz, 2H) (400 MHz, D2O) δ (ppm): 8.80 (tt, J = 4.6, 1.6 Hz, 4H), 8.22 (dt, J = 5.2, 1.6 Hz, 2H), 8.13 (dt, J = 4.6, 1.6 Hz, 2H). 13 C NMR (101 MHz, DMSO-d6) δ (ppm): 167.7 (C=O), 166.0 (C=O), 149.7 (CH x 2), 147.8 (CH x 2), 146.0 (C), 137.5 (C), 123.4 (CH x 2), 122.7 (CH x 2). IR Symmetric stretching (νs), Antisymmetric stretching (νas), Symmetric bending (s) and Twisting () (cm -1 ): 3465-3297 (νs N-H), (νs C-H), 1719 (νs C=O/ bonding to O), 1592 (νs C=O/ bonding to N) 1560 -1499 (νs C=N, nsC=C), 1420 (s N-H), 1269 (s C-H), 1059 -1015 (νas C=N), 854 ( C-H). HRMS (ESI): m/z calcd. for C12H9N3O3: 244.0722 found: 244.0726.

  719 mmol) and K3[Fe(CN)6] (0.592 g, 1.797 mmol). MS (DCI/CH4): m/z = 230 [M9 + C2H5 + ], 217 [M9 + CH3 + ], 202 [M9 + H + ] and m/z = 124 [M14 + H + ].

1 H

 1 NMR (400 MHz, DMSO-d6)  (ppm): 9.17 (d, J = 1.4 Hz, 1H), 8.89 (s, 1H), 8.85 (d, J = 2.5 Hz, 1H), 8.71 (dd, J = 2.4, 1.6 Hz, 1H), 2.83 (d, J = 4.8 Hz, 3H). 13 C NMR (101 MHz, D2O)  (ppm): 163.29 (C=O), 147.37 (CH), 144.87 (C), 143.35 (CH), 143.32 (CH), 25.98 (CH3). IR Symmetric stretching (ns), antisymmetric stretching (nas), symmetric bending (s) and twisting () (cm -1 ): 3358 (ns N-H), 2929 (ns C-H), 1669 (ns C=O), 1583 -1542 (ns C=N, ns C=C), 1404 (s N-H), 1294 (s C-H), 1054 -1024 (nas C=N), 868 ( C-H). UV-Vis (H2O) λmax/nm (ε/M -1 cm -1 ) = 310 (657), 270 (7045), 209 (8544). HRMS (DCI/CH4): m/z calcd. for [(C6H7N3O) + H + ]: 138.0667, found: 137.0662. TLC Retention Factor (MeOH/DCM 10%) = 0.58. M.p = 110 ºC.

  1 H NMR (300 MHz, D2O) δ (ppm): 8.95 (dd, J = 2.2, 0.9, 1H), 8.62 (dd, J = 5.1, 1.7, 1H), 8.34 (ddd, J = 8.0, 2.2, 1.7, 1H), 7.59 (ddd, J = 8.0, 5.1, 0.9, 1H). 13 C NMR (101 MHz, D2O) δ (ppm): 172.98 (C=O), 149.74 (CH), 148.51 (CH), 139.65 (CH), 133.54 (C), 125.04 (CH), 49.50 (reference: CH3OD). IR Symmetric stretching (ns), Antisymmetric stretching (nas), Symmetric bending (s) and Twisting () (cm -1 ): 1610 (νs C=O), 1562 -1403 (ns C=N, ns C=C), 1322 (s C-H), 1029 (nas C=N), 752 ( C-H). UV-Vis (H2O) λmax/nm (ε/M -1 cm -1 ) = 272 (2137), 265 (2819), 260 (2549), 212 (7350). Elemental Anal. calcd. for NaC6H4O2•0.4H2O: C

1 HFigure S1. 1 HFigure S2 .

 11S2 Figure S1. 1 H NMR spectra of a) sodium isonicotinoate, b) sodium nicotinoate acid, c) sodium pyrazinoate.

Figure S3 .Figure S4a .

 S3S4a Figure S3. Reactions of cPTIO with NO • or HNO, and EPR patterns of the products.

Figure S4b :

 S4b Figure S4b: EPR signal intensity of the reaction of isonicotinohydroxamic acid, nicotinohydroxamic acid and pyrazinohydroxamic acid (5 mM) with [Fe(CN)6] 3- (15 mM) in the presence of cPTIO (200 µM) at 0 and 5.5 h in phosphate buffer 40 mM (pH 7.4) and at room temperature.

Figure S5 .

 S5 Figure S5. UV-Vis absorption monitoring at 0-24 h of the evolution of a mixture of cPTIO (150 µM) and K3[Fe III (CN)6] (15 mM) in 40 mM phosphate buffer (pH 7.4) at room temperature.

Scheme 23 .

 23 Scheme 23. Synthesis of N'-(pyridine-4-carbonyl) pyrazine-2-carbohydrazide.

Figure 34 .

 34 Figure 34. EPR signals for PBN-spin trap experiments controls. A. Mixture of INH (5 mM), [Mn(H2P2O7)3] 3-(2.5 mM), and PBN (20 mM) in phosphate buffer (40 mM, pH 7.4). B. Mixture of IQG-607 (5 mM), H2O2 (5 mM) and PBN (20 mM) in phosphate buffer (40 mM, pH 7.4).

Figure 35 .

 35 Figure 35. EPR signals for PBN-spin trap experiments. A. Mixture of ligand (5 mM), [Mn(H2P2O7)3] 3-(2.5 mM) and PBN (20 mM) in phosphate buffer (40 mM, pH 7.4). B.Mixture of complex (5 mM), [Mn(H2P2O7)3] 3-(2.5 mM) and PBN (20 mM) in phosphate buffer(40 mM, pH 7.4). The vertical scale was kept identical for all spectra.

Figure 36 .

 36 Figure 36. EPR signals for PBN-spin trap experiments. A. Mixture of isonicotino hydroxamic acid complex (5 mM), H2O2 (5 (black) or 15 (green) mM) and PBN (20 mM) in phosphate buffer (40 mM, pH 7.4). B. Mixture of pyrazino hydroxamic acid complex (5 mM), H2O2 (5 (black) or 15 (green) mM) and PBN (20 mM) in phosphate buffer (40 mM, pH 7.4). The vertical scale was kept identical for all spectra.

  di(di)azinoylhydroxylamine intermediate. The reaction of isonicotino-hydroxamic acid ligand, [Fe(CN)6] 3-and cyclohexa-1,3-diene in THF aqueous conditions did not lead to the Diels Alder adduct. Two explanations are possible: i) the nitroso compound is probably not the intermediate of oxidative reaction of hydroxamic acids or ii) the Diels Alder reaction does not work well under these THF-aqueous conditions. Finally, the experimental results linked to synthesis of Na6[Fe2(CN)10(N'-(pyridine-4-carbonyl)

Figure 37 .

 37 Figure 37. Structure des promédicaments antituberculeux rifampicine, isoniazide (INH), pyrazinamide (PZY), éthambutol et délamanide.

  d'activation oxydative, il a été décrit récemment dans la littérature, comme déjà cité précédemment, un système biomimétique, alternatif capable d'activer le promédicament antituberculeux INH tout en se franchissant de l'activation enzymatique par KatG à l'intérieur du Mtb. Ce système est basé sur la réactivité redox d'un complexe de Fe II de l'INH ([Fe II (CN)5(INH)] 3-) qui en présence d'agents oxydants tels que H2O2 [8,9], génère le radical isonicotinoyle, espèce réactive de l'INH. L'activité antituberculeuse (in vitro MICMtb ~ 3,5 μM) [10], la stabilité chimique et l'absence de toxicité (indice de sélectivité SI> 4000, DL50> 2000 mg/kg) [11] du complexe [Fe II (CN)5(INH)] 3-, nous a motivé à étendre cette approche d'activation aux acides (di)azine hydroxamiques qui vont en fait servir de plateforme pour le développement de nouveaux complexes de fer capables de libérer HNO. Cet article présente la synthèse et la caractérisation de deux complexes de pentacyanoferrate(II) avec l'acide hydroxamique pyrazinique (3) (analogue du PYZ) et l'acide hydroxamique isonicotinique (6) (analogue de l'INH) (Figure 38). Ces complexes ont été capables, par l'action d'un agent oxydant (H2O2), de générer du HNO et l'acide carboxylique correspondant : l'acide pyrazinique et isonicotinique, respectivement. Les oxydations du complexe 3 et 6 par H2O2 ont été suivies par RPE (à l'aide du piège sélectif cPTIO), UV-Vis et RMN 1 H, ceci dans le but d'étudier le mécanisme de la réaction d'oxydation.

Figure 38 . 5 (

 385 Figure 38. Complexes de type pentacyanoferrate(II) synthétisés.

Figure S1 .Figure S2 -

 S1S2 Figure S1. (A) and (B) 1 H NMR spectra of 2 and 3, respectively, in D2O, 400 MHz. (C) and (D) 13 C NMR spectra of 2 and 3, respectively, in D2O, 101 MHz ( 13 C reference = MeOD). (E) and (F) 2D 1 H-15 N HMQC NMR spectrum of 2 and 3, respectively, in D2O, 50.7 MHz.

Figure S3 .Figure S4 .

 S3S4 Figure S3. UV-Visible absorption spectra of the free ligand 2 (103 µM, blue) and complex 3(45 µM, red) in water solution, at pH = 6.8.

Figure 39 .

 39 Figure 39. (A) and (B) 1 H NMR spectra of 10 and 13, respectively, in D2O, 400 MHz.

Figure 40 .

 40 Figure 40. (A) and (B) 1 H NMR spectra of 11 and 14, respectively, in D2O, 400 MHz.

Figure 41 .

 41 Figure 41. (A) and (B) 1 H NMR spectra of 12 and 15, respectively, in D2O, 400 MHz.

Figure 42 .

 42 Figure 42. (A), (B) and (C) 13 C NMR spectra of 13, 14 and 15, respectively, in D2O, 101 MHz ( 13 C reference = MeOD).

Figure 43 .Figure 44 .Figure 45 .

 434445 Figure 43. Experimental NR (A) and IR (B) spectra of the complex 13 under tetraanionic form, Na4[Fe(CN)5L], L = NicCONHO -(black line), in the solid state, and DFT-simulated spectra for [Fe(CN)5L] 4-(gray line).

Figure 46 . 3 .

 463 Figure 46. Calculated UV-Visible absorption spectra and calculated singlet-singlet transition energies of the complex 13H + taking account in a water solvent field.

Figure 47 .

 47 Figure 47. UV-Vis monitoring of the reaction of 6H + (A) and 13H + (B) (171 µM for both complexes) with H2O2 (427.5 µM) in phosphate buffer solution, 40 mM, pH 7.4, 22 ºC. Reaction at t = 0 min, before addition of H2O2, (blue line), and after 2 h (red line). Inset shows the kinetic curve based on changes at 431 nm to the 6H + , and 396 nm to the 13H + .

Figure 48 .

 48 Figure 48. 1 H-NMR spectra at 400 MHz: of the complex 6H + (20 mM) at 0 h (A) and at 52 h (B) without addition of H2O2, at 52 h after reaction of 6H + with H2O2 (50 mM) (C), and 14 complex (20 mM) (D) and isonicotinic carboxylate (11) (20 mM) (E). Solutions in 40 mM phosphate buffer, pH 7.4, at 25 ºC.

Figure 49 .

 49 Figure 49. 1 H-NMR spectra at 400 MHz: of the complex 13H + (20 mM) at 0 h (A) and at 52 h (B) without addition of H2O2, at 52 h after reaction of 13H + with H2O2 (50 mM) (C), and 15 complex (20 mM) (D) and nicotinic carboxylate (12) (20 mM) (E). Solutions in 40 mM phosphate buffer, pH 7.4, at 25 ºC.

Figure 50 .

 50 Figure 50. EPR signal of the cPTIO radical in the presence of H2O2 and the complexes (6H + (A) and 13H + (D)) (t0 = black line and t10min = red line). Control of the stability of the cPTIO EPR signal in the presence of 6H + (B) and 13H + (C) (t0 = black line and t15min = red line). Control of the stability of the cPTIO EPR signal in the presence of free ligands (5 (C) and 10 (F)) and H2O2 (t0 = black line and t15min = red line). All spectra were recorded in phosphate buffer solution, 40 mM, pH 7.4.

Figure 51 .

 51 Figure 51. Relaxation effects on aortic rings pre-contracted with 0.1 µM phenylephrine: 10 (red), 13 (blue), and SNP (black). The EC50 and respective 95% confidence interval were calculated only for 13 (106 nM, 62-181 nM).

Figure 52 .

 52 Figure 52. Mechanistic hypotheses for HNO release by oxidation of aryl hydroxamic acids.

Figure 53 .

 53 Figure 53. Structure of the pyrazino-(3), isonicotino-(6) and nicotino-(13) hydroxamic acids pentacyanoferrate(II) complexes.

Figure 1 . 18 Figure 2 .Figure 3 . 22 Figure 4 . 23 Figure 5 . 24 Figure 6 . 25 Figure 7 . 26 Figure 8 . 28 Figure 9 . 35 Figure 10 . 38 Figure 11 . 39 Figure 12 . 41 Figure 13 . 42 Figure 14 . 42 Figure 15 . 44 Figure 16 . 45 Figure 17 . 46 Figure 18 . 48 Figure 19 . 48 Figure 20 . 50 Figure 21 . 52 Figure 22 . 56 Figure 23 . 58 Figure 24 . 59 Figure 25 . 61 Figure 26 . 62 Figure 27 .Figure 28 .Figure 29 .Figure 30 .Figure 31 .Figure 32 .Figure 33 .Figure 34 .Figure 35 .Figure 36 .Figure 37 .Figure 38 .Figure 39 .Figure 40 .Figure 41 .Figure 42 .Figure 43 .Figure 44 .Figure 45 .Figure 46 .Figure 47 .Figure 48 .Figure 49 .Figure 50 .Figure 51 .Figure 52 .Figure 53 .Figure 54 .Figure 55 .Scheme 1 . 29 Scheme 2 . 30 Scheme 3 . 31 Scheme 4 . 32 Scheme 5 . 43 Scheme 6 . 46 Scheme 7 . 47 Scheme 8 . 49 Scheme 9 . 51 Scheme 10 . 52 Scheme 11 . 54 Scheme 12 . 56 Scheme 13 . 57 Scheme 14 . 60 Schème 15 .Scheme 16 .Scheme 17 .Scheme 18 .Schème 19 .Schème 20 .Schème 22 .Scheme 23 .Scheme 24 .

 118232242352462572682893510381139124113421442154416451746184819482050215222562358245925612662272829303132333435363738394041424344454647484950515253545512923033143254364674784995110521154125613571460151617181920222324 Figure 1. Examples of three first prescribed metallo-drugs. ......................................... 18 Figure 2. Anticancer platinum metallo-drugs clinically used or in current clinical trials. ........................................................................................................................................ 21 Figure 3. Arsenic anticancer metallo-drugs: trisenox (clinically used) and darinaparsin (in clinical trials). ............................................................................................................ 22Figure 4. Structures of ruthenium compounds in clinical trials..................................... 23 Figure 5. Chemotherapeutic agents based on gallium(III) and molybdenum(VI) metals assayed in clinical trials. ................................................................................................. 24 Figure 6. Cardiovascular metallo-drugs in clinical use (SNP) and clinical trials Phases I/II (Oxacom ® and M40403 (clinical phases suspended)). ............................................. 25 Figure 7. Promising metal-complex antitubercular compounds and their minimum inhibitory concentration (MIC) against Mtb H37Rv cells. .............................................. 26 Figure 8. Endogenous production of nitric oxide. Panel A shows the two-step formation of NO • from arginine to citrulline, through the intermediate N-omega-hydroxy-Larginine formation. This reaction is catalyzed by nitric oxide synthase (NOS) with participation of O2, NADPH and the cofactor tetrahydrobiopterin (H4B) (Panel B). Panel C shows NO • diffusion from its production site (e.g., endothelial cells) into another cell reaching soluble guanylate cyclase (sGC), whose enzymatic activity is strongly stimulated with conversion of GTP to cGMP. ................................................. 28 Figure 9. FDA approved antiangiogenic drugs and their targets in parentheses. .......... 35 Figure 10. Some examples of cardiovascular drugs, followed in the parentheses by their pharmacological class. .................................................................................................... 38 Figure 11. Activation mechanism of soluble guanylate cyclase by NO • , reference [170]. ........................................................................................................................................ 39 Figure 12. First-line antitubercular drugs. ..................................................................... 41 Figure 13. Some examples of antitubercular second-line drugs. ................................... 42 Figure 14. Structures of bedaquiline, delamanid, and pretomanid. ............................... 42 Figure 15. Classic organic nitrates. ............................................................................... 44 Figure 16. New nitrate hybrids and the goals for their development. ........................... 45 Figure 17. Structures of some NONOates derivatives molecules. ................................ 46 Figure 18. Structure of some promising NONOates derivatives. .................................. 48 Figure 19. Structure of some S-Nitrosothiols (RSNOs). ............................................... 48

  According to, the NOHA intermediate can be produced by NOS and L-arginine in the absence of H4B, requiring only the participation of the NADPH cofactor. This can lead to the formation of a nitroso intermediate, which can ensue with production of HNO. This nitroso intermediate can react with the heme group to produce NOS-Fe II (NO), which might release HNO without any oxidative species.

• .

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			OBJECTIVE
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Table S1 .

 S1 Continuation

		Experimental	Optimized	Deviation (±)
	Bond lengths (Å)
	N(5)-C(8)	1.484(5)	1.491	0.004
	C(1)-C(2)	1.516(6)	1.527	0.006
	C(2)-C(3)	1.522(6)	1.527	0.003
	C(4)-C(5)	1.493(6)	1.519	0.013
	C(6)-C(7)	1.519(6)	1.528	0.009
	C(7)-C(8)	1.510(6)	1.528	0.009
	C(9)-C(10)	1.498(5)	1.519	0.011
	Bond Angle (°)
	N(1)-Fe-Cl(1)	178.63(11)	178.44	0.10
	N(1)-Fe-N(2)	91.53(13)	92.45	0.46
	N(1)-Fe-N(5)	91.40(14)	93.42	1.01
	N(2)-Fe-Cl(1)	88.44(9)	88.62	0.09
	N(3)-Fe-Cl(1)	87.94(10)	88.60	0.33
	N(3)-Fe-N(2)	95.36(13)	95.08	0.14
	N(4)-Fe-Cl(1)	86.50(10)	85.61	0.45
	N(4)-Fe-N(2)	174.80(12)	174.19	0.31
	N(4)-Fe-N(3)	85.61(13)	85.45	0.08
	N(4)-Fe-N(5)	93.41(13)	93.38	0.02
	N(5)-Fe-N(3)	175.13(13)	174.08	0.53
	N(5)-Fe-N(2)	85.20(12)	85.50	0.15
	N(5)-Fe-Cl(1)	87.24(9)	85.52	0.86
	O(1)-N(1)-Fe	177.4(3)	179.3	0.95
	C(1)-N(2)-

Pentacyanoferrate complexes: hydroxamic acids as therapeutic platforms 5. PART 2 -PENTACYANOFERRATE COMPLEXES: HYDROXAMIC ACID

				Part 1 -Nitrosyl Iron complex: cyclam as therapeutic platform
	_________________________________________________________________________________
				80
	3.	Hoshino, M.; Maeda, M.; Konishi, R.; Seki, H.; Ford, P. C., « Studies on the 70 Reaction Mechanism for Reductive Nitrosylation of Ferrihemoproteins in Buffer
		60 Solutions », Journal of the American Chemical Society, 118, 24, 1996, pp. 5702-
		5707.	(%)	50
			T	40	0 h of irrad.
				30	1 h of irrad. 2 h of irrad.
				20	n NO	4 h of irrad. 6 h of irrad.
				1980 1950 1920 1890 1860 1830 1800 10
				Wavenumber (cm -1 )
		Part 2:
	Bibliography
	______________________
	1.	Zapata, A. L.; Kumar, M. R.; Pervitsky, D.; Farmer, P. J., « A singular value
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	2.
				137

The thermal stability and photoirradiation studies, involving Mb-Fe

III and trans-[Fe(cyclam)

(NO)Cl] 2+ complex, indicated that the HNO was not detected in both experiments, corroborating with the experiments reported in the publication. The presence of the DTPA chelator was not able to induce significant changes in the standard spectra of the trans-[Fe(cyclam)(NO)Cl] 2+ and trans-[Fe(cyclam)Cl2] + complexes, indicating that the presence of DTPA did not influence the experimental responses obtained. In addition, it was shown that the nitrosyl complex trans-[Fe(cyclam)(NO)Cl]Cl2, in solid state, can be photodecomposed by irradiation at 365 nm, mainly affecting the interaction between the metallic center and the ligand NO + . Sharma, V. S.; Isaacson, R. A.; John, M. E.; Waterman, M. R.; Chevion, M., « Reaction of Nitric Oxide with Heme Proteins: Studies on Metmyoglobin, Opossum Methemoglobin, and Microperoxidase », Biochemistry,

22, 16, 1983, pp. 3897-3902.

  spectres RMN1 H indiquent essentiellement des pics correspondant à la structure du carboxylate. Sur la base de ces résultats, nous postulons que l'oxydation des acides hydroxamiques dans de telles conditions se produit d'abord en produisant un radical nitroxyde instable (oxydation monoélectronique de l'acide hydroxamique), qui

	Part 2 -Pentacyanoferrate complexes: hydroxamic acid as therapeutic platforms
	_________________________________________________________________________________
	SM (basse et haute résolution (HR)). Pour le dérivé 1, l'analyse RMN 1 H a montré un
	produit présentant quatre doublets à
	L'analyse spectrale de RMN 1 H montre, en plus des signaux relatifs au produit de départ restante, l'apparition d'un nouveau composé, possédant des signaux aromatiques dupliqués. Ce nouveau composé, au fil du temps, a tendance à disparaître en faveur de la formation d'un composé final présentant des signaux à 7,61-8,70 et 7,91 ppm pour 1, 8,42 et 8,68 ppm pour 2 et 8,70-9,04 et 9,15 ppm pour 3, qui ont pu être reconnus comme étant caractéristiques des acides carboxyliques correspondants sous forme déprotonée (carboxylate). Cette proposition a été confirmée par comparaison avec les explique la duplication de signaux aromatiques observés en RMN 1 H (Schème 21). L'intermédiaire diacylhydroxylamine pourrait en effet être hydrolysé pour produire les acides carboxyliques correspondants et l'acide hydroxamique de départ qui subirait de nouveau une étape d'oxydation. Schème 21. Hypothèses mécanistiques pour la libération de HNO via oxydation des acides hydroxamiques. La possibilité d'une oxydation supplémentaire du radical nitroxyde (oxydation à 2 électrons) conduisant à la formation d'un intermédiaire nitrosocarbonylé devrait être aussi considérée puisque ce dernier pourrait réagir facilement avec H2O se décomposant en acide carboxylique correspondant et HNO (Schème 21) [6]. Cependant, les spectres RMN 1 H montrent qu'après 1 h de réaction la conversion de l'acide hydroxamique de départ conduit uniquement or très majoritairement à l'intermédiaire N,O-di(di)azinoylhydroxylamine correspondant ; le produit acide carboxylique correspondant, qui pourrait être formé à partir d'un éventuel intermédiaire nitrosocarbonylé avec de l'eau, n'étant pas détecté en début de réaction. Ensemble, ces résultats suggèrent que l'intermédiaire nitrosocarbonylé n'est pas impliqué dans la formation de HNO et d'acide carboxylique. données de RMN est converti, après dimérisation, en HNO et une N,O-di(di)azinoylhydroxylamine, ce qui Pour tenter de caractériser l'intermédiaire proposé, les réactions d'oxydation des
	147

3-(2,5 éq.)

réalisée dans une solution tampon phosphate (D2O, pH = 7,4) a été initialement suivie par RMN 1 H. Cette étude a révélé qu'après 1 h de réaction, il existe une conversion partielle du produit de départ (50%, 56% et 38% pour 1, 2 et 3, respectivement).

1

H obtenues à partir d'échantillons authentiques de sels d'acides carboxyliques. Ces résultats confirment ainsi que l'acide carboxylique est le produit final de l'oxydation -métabolite potentiel -de l'acide hydroxamique. Après 312 h de réaction, les acides hydroxamiques 1-3 ont été répétées sur une échelle préparative (acide hydroxamique (0,72 mmol) et K3[Fe III (CN)6] (2,5 éq.)) et arrêtées après 24 h. Le nouveau produit a été isolé puis caractérisé par des techniques de RMN ( 1 H et 13 C) et

. Article 3: Journal of Biological Inorganic Chemistry, 2020, 25, 887-901 -

  La réaction d'oxydation du composé 3 suivie par spectroscopie de l'UV-Vis pendant 2 h, après l'ajout de H2O2, a montré que la bande avec un maximum d'absorption à 489 nm et caractéristique de la transition TCML entre le centre métallique Fe II et le ligand 2 diminue progressivement. En effet, une fois que le centre Mtb contre les souches non résistantes et en croissance active, l'activité putative de 3 et 6 contre les souches de Mtb intracellulaires (à l'intérieur des macrophages) ou encore contre Mtb dans un état de dormance, mérite d'être examinée. La capacité de libération de NO • /HNO du composé 3 a également été évaluée par des tests de vasodilatation en collaboration avec l'équipe du Pr. Nilberto R. F. do En conclusion, les résultats obtenus dans ce chapitre indiquent le rôle clé de la partie métallique des complexes 3 et 6 dans la vasodilatation exercée par ces systèmes. Bien que les complexes 3 et 6 soient moins puissants que le SNP, cela peut être un avantage, car le SNP, libérant du NO • trop rapidement, peut provoquer d'importantes chutes de tension artérielle, nécessitant un contrôle minutieux par titrage. Sans parler encore du danger de la libération des ions cyanures par le SNP, que dans le cas des

	max/nm (ɛ/M -1 cm -1 ) = 203 (22700), 268 (6228), 478 nm (3638). Electrochemistry in 5.8Printed 0,1M Phosphate buffer pH 7.4: E1/2 0.581 V/NHE.
	5.9.2. Tables and Figures
	métallique est oxydé en Fe III , il n'y a plus d'électrons disponibles pour que cette transition TCML se produise, ce qui entraîne la disparition de cette bande. Cette expérience a indiqué que dans l'intervalle de temps étudié (2 h) l'agent H2O2 a agi efficacement dans l'oxydation du centre métallique de fer. La réaction d'oxydation par H2O2 (2,5 équivalents) a également été suivie par spectroscopie de RMN 1 H. L'analyse spectral du milieu réactionnel après 52 h de réaction a révèle que 3 est presque totalement converti en acide pyrazinoique libre et que une toute petite fraction de l'acide pyrazinoique coordonné à Fe(CN)5 en reste. La réaction entre le complexe 3 (5 mM) et H2O2 (200 mM) a également été étudiée en présence de cPTIO par RPE. Au cours de la réaction, il a été observé que les signaux caractéristiques du cPTIO (quintuplet) diminuent, indiquant ainsi qu'une réaction potentielle entre cPTIO et le HNO généré in situ a eu lieu [13]. Puisque l'acide hydroxamique non complexé au fer est inerte à H2O2 (pas de libération de HNO) nous suggérons que l'oxydation des complexes par H2O2, conduisant à la libération de HNO, passe par un transfert intramoléculaire d'électrons du ligand acide hydroxamique vers le Fe III , ce dernier issu de l'oxydation du Fe II par l'H2O2. D'autre part, vu qu'aucun radical aroyle n'a pu être détecté par RPE au cours de la réaction, nous proposons en conséquence un mécanisme réactionnel passant par l'intermédiaire nitrosocarbonylé, via lequel la formation des métabolites finaux, HNO et l'acide pyrazinoique, pourrait être expliquée. Le complexe 6 a montré le même profil d'activation chimique médiée par H2O2, et a conduit aux mêmes types de métabolites finaux : HNO et l'acide isonicotinoique. L'étude de l'activité inhibitrice de 3 et 6 ainsi que des ligands libres correspondants vis-à-vis de la croissance des souches H37Rv de Mtb, réalisée en collaboration avec Pr. Cristiano V. Bizarro et Pr. Luiz A. Basso où « Pontifícia Universidade Católica Do Rio Grande Do Sul (PUCRS), Porto Alegre, Brasil », a révélé qu'aucun des composés n'est actif (MIC > 10 μg/mL). Cependant, bien que ces testés à des fins de comparaison. Alors que les ligands libres 2 et 5 présentaient une valeur d'CE50 (concentration efficace médiane) significativement plus élevée pour l'activité de vasodilatation (19 μM) en comparaison à SNP (CE50 = 13 nM), le complexe 3 avait une valeur CE50 de 250 nM, étant ainsi 19 fois moins puissant que SNP. Le complexe 6 présente une CE50 inférieure (64 nM) à celle du composé 3 et est seulement cinq fois moins puissant que le SNP. De plus, les complexes 3 et 6 ont tous les deux montré une efficacité (effet de relaxation maximum) tout à fait comparable à celle du SNP. nouveaux complexes de type pentacyanoferrate(II) est assez réduit. Ces résultats montrent que ces complexes sont extrêmement prometteurs du point de vue cardiovasculaire et qu'ils méritent davantage d'études en vue d'une future application thérapeutique. Bibliographie ______________________ 1. Houben, R. M.; Dodd, P. J., « The Global Burden of Latent Tuberculosis Infection: A Re-estimation Using Mathematical Modelling », PLoS Medicine, 13, e1002152, 2016, pp.1-13. 2. Vandal, O. H.; Nathan, C. F.; Ehrt, S., « Acid resistance in Mycobacterium tuberculosis », Journal of Bacteriology, 191, 15, 2009, pp. 4714-4721. complexes ne présentent pas d'activité anti-Nascimento. Les ligands 2 et 5, ainsi que le SNP et le complexe 6 ont également été 3. Laborde, J.; Deraeve, C.; Bernardes-Genisson, V., « Update of Antitubercular
	Prodrugs from a Molecular Perspective: Mechanisms of Action, Bioactivation
	Pathways, and Associated Resistance », ChemMedChem, 12, 20, 2017, pp. 1657-
	1676.

Table S1 .

 S1 Selected experimental and calculated wavenumber values obtained from IR and NR spectra of solid complexes 3, and 6, and their corresponding vibrational assignments.

	Complex	λEXP /nm	λDFT /nm	Osc. Strength (f)	Key transition	Character
			496 0.0955	HOMO-1 →LUMO	MLCT
	3	360 385 0.0113 270 273 0.0147	HOMO-1 →LUMO+1 MLCT HOMO-6→LUMO IL+LLCT
		216 218 0.0504	HOMO-17→LUMO	IL+LLCT
		439 493 0.1334	HOMO-1→LUMO	MLCT
	6	266 284 0.0243	HOMO-3→LUMO	MLCT+LLCT
		233 240 0.0640	HOMO-12→LUMO	IL+LLCT

Table S2 .

 S2 Selected electronic transitions for complex 3 and 6.

		Wavenumber (cm -1 )
	3	Infrared	6	Compound	3	Raman	6	vibrational assignment
	Exp. DFT Exp. DFT Exp. DFT Exp. DFT
				508 503 507		β(FeC≡N)
	571	572 587 574 549 575 508	β(NH)OP
	654 617 697 663 653 617		Ring
	749 745		746 745		Ring
	856 828 849 828		701 722	β(CH)OP
				1033 996 1023 975	Ring
		1035 1034 1074 1023 1066		Ring+v(NO)
		1169 1135 1155 1140		β(CH)
	1029							Ring
	1176						1135	Ring
				1292 1344 1227 1191	ν(C-NH)+β(CH)
	1352 1316 1339 1358 1388 1314 1338	β(NH)+β(CH)
	1574 1415 1485				β(CH)+ν(C=O)+β(NH)
		1652 1560 1583 1542		ν(CC)ring+ν(C=O)+β(NH)
						1610 1560	ν(CC)ring
	2053 2047 2046 2071 2050 2070 2047	ν(C≡N)
	2094 2093 2065 2097 2071 2093 2065	ν(C≡N)
	Op = out-of-plane

Table 1 .

 1 Selected experimental and calculated wavenumber values obtained from IR and NR spectra of solid complex 13, and their corresponding vibrational assignments. Op = out-of-plane / ν = symmetric stretching / β = symmetric bending.

		Wavenumber (cm -1 )		Vibrational
	Infrared Exp. DFT	Raman Exp. DFT	Assignment
			497	471	β(FeC≡N)
	571	524	577	548	β(NH)op
	692	689	647	629	Ring
	912		714	746	β(CH)op
			914		Ring
			1036	1011	Ring + ν(NO)
			1204	1238	β(CH)
	1022	1042			Ring
	1176	1148			Ring
			1327	1347	ν(C-NH) + β(CH)
	1319	1348	1337	1375	β(NH) + β(CH)
	1638	1537	1599	1573	ν(CC)ring + ν(C=O) + β(NH)
			1577	1557	ν(CC)ring
	2056	2055	2094	2069	ν(C≡N)
	2100	2086	2084	2060	ν(C≡N)

Table 2 .

 2 Selected electronic transitions for complex 13H + .

	λexp (nm) λDFT (nm) Osc. Strength (f) Key transition	Character
	394	437	0.0289	HOMO-1→LUMO	MLCT
		375	0.0290	HOMO-1→LUMO+1	MLCT
	261	247	0.0227	HOMO-6→LUMO	LLCT
	215	239	0.0547	HOMO-4→LUMO+1	LLCT
		223	0.0594	HOMO-7→LUMO+1	LLCT
	LLCT = ligand-to-ligand charge transfer/ MLCT = metal-to-ligand charge transfer.

Table 3 .

 3 CC50 values to cytotoxic of complexes 3

, 6, and 13, and ligands 2, 5, and 10,

	5.10.6. Antiparasitic activity (leishmaniasis and malaria)
	measured using macrophage (J774A.1) cells.
	Compound	CC50 (µM)
	2	285.10 (± 2,22)
	3	> 106.37
	5	59.80 (± 1,26)
	6	68.26 (± 2,57)
	10	133.79 (± 0,65)
	13	> 104.60
	Doxorubicin	0.05 (± 0,01)

Table 4 .

 4 Antileishmanial and antimalarial activity from the compounds 2

, 3, 5, 6, 10, and 13.

	Compound	Anti-leishmania IC50 (µM) *	Anti-malaria IC50 (µM) *
	2	117.03	21.57
	3	82.33	38.29
	5	85.43	2.53
	6	61.57	4.81
	10	121.70	3.98
	13	66.25	5.23
	Amphotericin B	0.07	-
	Chloroquine	-	0.18

*Concentrations referring to a single measure.

  • ou HNO, de type acide hydroxamique jouant le rôle de ligand est apparue comme une alternative prometteuse dans la recherche de médicaments cardiovasculaires et anticancéreux. Par ailleurs, les résultats de l'activité antiangiogène par inhibition de HIF-1α suggèrent que ce complexe de nitrosyle est un candidat pertinent pour être étudié comme agent anticancéreux. En outre, les études de vasodilatation ont montré que le complexe trans-[Fe(cyclam)(NO)Cl]Cl2 permet un contrôle plus doux de la clairement que le centre pentacyanoferrate(II) joue un rôle crucial dans l'activation des acides hydroxamiques conduisant à la libération de HNO.

	pressions artérielle (IC50 = 910 nM) que la pro-drogue métallique classiquement utilisée
	en cas de crise d'hypertension, le SNP (IC50 = 24 nM). Ceci peut être considéré comme
	un avantage, l'utilisation du SNP, puissant vasodilatateur, étant limitée par sa capacité "abrupte" à induire la vasodilatation. De plus, l'absence d'ion cyanure dans la sphère de coordination du centre Fe II , potentiellement toxique pour le patient dans le cas du SNP, est un argument de poids pour envisager ce complexe dans de futures études précliniques. La deuxième partie de la thèse a porté sur l'étude d'acides (di)azine hydroxamiques, en mettant l'accent sur leur capacité à libérer HNO après une activation oxydante. Une étude de l'oxydation des acides hydroxamiques isonicotinique, aryl hydroxamiques. Le troisième chapitre concerne le développement de complexes métalliques à est la conception d'une pro-drogue hybride métallique issue de l'association, sur un centre métallique Fe II , de précurseurs des métabolites actif de l'isoniazide (radical isonicotinoyle) ou du pyrazinamide (acide pyrazinoique) et du délamanide (HNO). Figure 55. Structure des complexes pentacyanoferrate(II) des acides hydroxamiques nicotinique et pyrazinique par K3[Fe Figure 54. Hypothèses mécanistiques pour la libération de HNO par oxydation d'acides ligands acides hydroxamiques, présentés précédemment à l'état libre : l'objectif ultime pyrazinique (3), isonicotinique (6) et nicotinique (13).
	Toutefois, sachant que le pyrazinamide est actif sur les bacilles latents et non sur des
	souches en croissance, il serait pertinent à l'avenir de tester ces complexes sur un
	modèle plus approprié. D'autre part, des activités vasodilatatrices prometteuses ont été
	trouvées pour les trois complexes, en particulier pour le complexe 6 (EC50 = 64 nM),
	seulement 5 fois moins puissant que le SNP (EC50 = 13 nM). Comme dans le cas du
	complexe trans-[Fe(cyclam)(NO)Cl]Cl2, l'activité modérée des complexes d'acides
	hydroxamiques, en comparaison du SNP, peut être considérée comme une avancée dans
	la conception de nouveaux agents antihypertenseurs, la libération trop rapide de NO • par
	le SNP pouvant provoquer de dangereuses baisses brutales de la pression artérielle. Les
	Le premier chapitre a porté sur l'étude de la réactivité du complexe trans-complexes 3 et 6 ont montré une efficacité similaire à celle du SNP, tandis que celle du
	[Fe(cyclam)(NO)Cl]Cl2. Bien que la bibliographie indique que ce complexe libère NO • complexe 13 était moindre. Ces résultats ont encouragé l'utilisation du complexe 6 dans
	par action d'un stimulus lumineux, ce travail de thèse a permis de montrer que ce des études de l'action antihypertensive chez les rats. L'augmentation des doses du
	complexe 6 (1, 5, et 10 mg/kg) a été observée favoriser une diminution de la pression

complexe présente un fort potentiel pour le relargage direct de NO • ou HNO dans des conditions physiologiques (à pH

7,4, à 37 ºC)

. Cette propriété permet d'expliquer les activités antihypertensives et antiangiogènes ce complexe, mises en évidence au cours de cette thèse. III (CN)6] en milieu aqueux, a révélé non seulement la libération de HNO, mais aussi la formation d'intermédiaires de type N,Odi(di)azinoylhydroxylamine, aucun dérivé nitrosocarbonylé n'ayant été détecté (Figure

54

). Ces résultats sont en contradiction avec l'hypothèse fréquemment rapportée que l'intermédiaire nitrosocarbonylé est le précurseur de HNO par oxydation des acides hydroxamiques. Par le même traitement oxydant, mais en présence de NH2CH3, nous avons pu mettre en avant que l'intermédiaire N,O-di(di)azinoylhydroxylamine présente aussi un caractère électrophile, similaire à celui de l'intermédiaire nitrosocarbonylé virtuel. En outre, les acides carboxyliques correspondants ont été identifiés comme les produits finaux, résultant donc de l'oxydation des acides hydroxamiques (et non de leur hydrolyse).

Tandis que le traitement in vitro (à pH 7,4 à 37 ºC) des complexes 3, 6 et 13 (Figure

55

) par l'agent oxydant endogène H2O2, n'a pas conduit à de quelconque radicaux aroyle, il a en revanche été démontré induire la formation de HNO et de l'acide carboxylique, validant ainsi le concept de complexe hybride pyrazinamide-délamanide. Ces composés n'ont pas été observé inhiber la croissance de souches actives de Mtb non-résistantes. artérielle, et ceci plus efficacement chez les rats hypertendus (de

45, 62, et 65%, respectivement)

que chez les rats normotenseurs (

de 31, 51 and 53%, respectivement)

.

La comparaison des courbes de vasodilatation des complexes et des "ligands libres" montre En résumé, ce travail a mis au jour de nouveaux systèmes métallo-organiques capables de libérer HNO dans des conditions physiologiques, ainsi que des propositions plus générales dans le domaine de la chimie bio-inorganique. Les résultats rapportés dans cette thèse confirment le potentiel de tels complexes comme nouveaux agents thérapeutiques, en particulier comme agents cardiovasculaires. En perspectives, l'étude des propriétés vasodilatatrices des complexes d'acide hydroxamique mérite d'être complétée du point de vue mécanistique, en utilisant, par exemple, des inhibiteurs de la guanylyl cyclase soluble (par exemple le ODQ = 1H-[1

Table 1 .

 1 Selected experimental and calculated wavenumber values obtained from IR and NR spectra of solid complex 13, and their corresponding vibrational assignments. ...................................................................................................................................... 211

Table 2 .

 2 Selected electronic transitions for complex 13H + . ......................................... 213

Table 3 .

 3 CC50 values to cytotoxic of complexes 3

, 6, and 13, and ligands 2, 5, and 10, measured

  using macrophage (J774A.1) cells. .............................................................. 217

Table 4 .

 4 Antileishmanial and antimalarial activity from the compounds 2, 3, 5, 6, 10, and 13. .......................................................................................................................... 218Titre : Métallo-médicaments comme donneurs d'oxyde nitrique (NO • ) et / ou de nitroxyle (HNO) : développement de nouveaux agents et investigation des activités anticancéreuses, antihypertensives et antituberculeuses. Résumé : Le cancer, les maladies cardiovasculaires et la tuberculose (TB) sont des défis de santé majeurs pour lesquels diverses stratégies médicamenteuses ont été développées, y compris l'utilisation d'agents inorganiques tels que des complexes métalliques et l'oxyde nitrique (NO • ) ou le nitroxyle (HNO). L'objectif de la thèse porte sur la conception, la synthèse, les études physico-chimiques et l'évaluation biologique de molécules à base de métaux capables de libérer NO • et/ou HNO avec des effets pharmacologiques. Deux types de plates-formes organiques sont envisagés comme ligands des complexes de fer, conçus comme sources potentielles de NO • /HNO : le ligand spectateur cyclam(1,4,8,11tétraazacyclotétradécane) et les ligands réactifs du type azine acide hydroxamique (ArC(O)NHOH). Le premier chapitre traite de la réactivité du complexe trans-[Fe(cyclam)(NO)Cl]Cl2, qui se décompose en libérant NO • à pH physiologique, le processus est accéléré par irradiation lumineuse à 365 nm. En revanche, des expériences in vitro en présence de glutathion ont révélé que le même complexe produit HNO. Des études d'angiogenèse ont montré que le complexe est capable d'inhiber le "facteur inductible par hypoxie" (HIF-1α) induit par hypoxie ou ajout d'un donneur de NO • (Spermine NONOate) dans les cellules cancéreuses du sein, résultat cohérent avec une libération de HNO. Des tests de vasodilatation, des anneaux aortiques de rat précontractés, ont révélé que le complexe présente une CI50 de relaxation de 910 nM, contre 24 nM pour le médicament de référence nitroprussiate. Les chapitres suivants traitent de la libération de HNO par oxydation d'acides hydroxamiques aromatiques. Le chapitre 2 se concentre sur le mécanisme d'oxydation des acides hydroxamiques isonicotinoïque, nicotinoïque et pyrazinoïque par le ferricyanure de potassium à pH physiologique. Il est montré que l'activation oxydante des acides hydroxamiques par des ions Fe III implique la production de l'intermédiaire N,Odi(di)azinoylhydroxylamine avec libération concomitante de HNO, au lieu de l'espèce hypothétique acyl nitroso. Les acides hydroxamiques ont été évalués pour différentes activités biologiques et utilisés comme témoins pour être comparés aux complexes de fer décrits au chapitre 3. Les complexes de type Na3Fe II (CN)5(ArCONHOH), initialement conçus comme modèles de médicaments hybride d'isoniazide (INH, anti-TB, dont le radical isonicotinoyle est le métabolite actif) et du délamanide (anti-TB, dont HNO est le métabolite actif), ont été synthétisés et caractérisés par des techniques spectroscopiques, voltammétrie cyclique, et calculs DFT. En utilisant la spectroscopie RPE et RMN 1 H, l'oxydation de ces complexes de Fe II avec H2O2 s'est avérée entraîner la libération de HNO et des acides azinoïques correspondants, via un probable transfert d'électrons intramoléculaire. Contrairement à ce qui a été observé pour le complexe Na3[Fe II (CN)5(INH)], aucun radical azinoyle n'a été produit à partir des complexes d'acide hydroxamiques. Les acides carboxyliques correspondants ont par contre été observés, en particulier l'acide pyrazinoïque, métabolite actif du pyrazinamide, un autre promédicament anti-TB: ce résultat est a priori pertinent pour le traitement de la TB résistante au pyrazinamide, car l'activation pourrait ainsi se produire sans l'aide de l'enzyme pyrazinamidase de Mycobacterium tuberculosis (Mtb). Les acides hydroxamiques libres ou complexés n'ont montré aucune action antibiotique contre une souche de Mtb non résistante en croissance active. Cependant, les complexes ont montré une forte activité de vasodilatation dose-dépendante et réversible, proche de celle du nitroprussiate (64-250 nM vs 13 nM). Les acides hydroxamiques libres ont été 19 à 76 fois moins actifs que les complexes correspondants. Sur la base d'expériences in vivo, Na4[Fe II (CN)5(PyCONHO -)] a aussi montré un profil d'activité cardiovasculaire prometteur pour le développement d'un nouveau médicament antihypertenseur. Mots clés : acides hydroxamiques, antiangiogenèse, complexes de fer, cyclam, mécanismes d'activation oxydante, nitroxyle, oxyde nitrique, tuberculose, vasodilatation. Metallo-drugs as nitric oxide (NO • ) and/or nitroxyl (HNO) donors: development of new agents and investigation of anticancer, antihypertensive and antituberculosis activities.

Title:

Abbreviations _________________________________________________________________________________

Abbreviations

NO • : nitric oxide WHO: World Health Organization Symmetric bending (δs) and Twisting (τ) (cm -1 ): 3304-3244 (νs N-H), 1708-1660 (νs C=O), 1546-1468 (νs C=N, νs C=C), 1402 (δs N-H), 1282 (δs C-H), 1024-994 (νas C=N), 850 (τ C-H). UV-Vis (H2O) λmax/nm (ɛ/M -1 cm -1 ) = 214 (15930), 274, (12480), 324 (914), 379 (188). MS (DCI/NH3): m/z calcd. for C11H9N5O2: 243.2, found: 244.0 (M + H) + . Elemental Anal. calcd. For C11H9N5O2: C, 54.32;H,3.73;N,28.79. Found: C,53.92;H,3.34;N,28.44. TLC Retention Factor (MeOH/DCM 10%) = 0.41. M.p. = 215 °C.

Complex Na6[Fe2(CN) 10(N'-(pyridine-4-carbonyl)

pyrazine-2-carbohydrazide)]

To a solution of Na3[Fe II (CN)5(NH3)]•3H2O (0.100 g, 0.31 mmol, 2 equiv.) in degassed water (2 mL), it was slowly added a solution of the ligand N'-(pyridine-4carbonyl) pyrazine-2-carbohydrazide (0.038 g, 0.155 mmol, 1 equiv.) in degassed water (1 mL). The mixture was stirred at room temperature, under argon atmosphere and protected from light for 3 h. Then 100 mL of a cold solution of ethanol containing an excess of sodium iodide (30 equiv.) was added dropwise. The resulting suspension was kept overnight (16 h) at -20 °C before the precipitate was filtered, washed with cold ethanol, and dried in a vacuum desiccator.

Results and discussion

Attempts to detect aroyl radical

In order to check whether an aroyl radical is formed as intermediate during the hydroxamic acids oxidation, reactions using [Mn(H2P2O7)3] 3-or H2O2 as oxidizing agents and PBN (N-tert-butyl-α-phenylnitrone), a well-known trap for carbon-centered radicals [1,2], in phosphate buffer (40 mM, pH 7.4) were performed.

The INH and IQG-607 were used as positive controls. Oxidation of INH by [Mn(H2P2O7)3] 3-in the presence of PBN led to the formation of an EPR signal mainly consisting of a six-line spectrum with hyperfine splitting constants of aN = 15.5 -15.8 G and aH = 3.2 -3.6 G for a g-value of 2.006 attributed to the trapping, by PBN, of the isonicotinoyl radical (Figure 34A). Oxidation of IQG-607 by H2O2 resulted in a six-line spectrum with hyperfine splitting constants of aN = 15.5 -15.6 G and aH = 4.5 -4.6 G for a g-value of 2.006, ascribed to the acyl radical of the IQG-607 trapped by PBN, (Figure 34B), as described by Sousa and his collaborators [1,2].

5.9. Supporting information 5.9.1. Synthetic procedures Sodium pyrazine-2-carboxylate (8) To a suspension of pyrazinoic acid (0.12 g, 0.970 mmol) in water (8 mL) was added a solution of NaOH (0.033 g, 0.817 mmol) in water (2 mL). The final solution was stirred at room temperature for 30 min. The solvent was removed under vaccum at 50 °C. The solid was collected and recrystallized (solubilized in water (1mL) then addition of ethanol (50 mL)). The mixture was stirred for 30 min then the solid was collected and washed with ice-cold ethanol. Yield = 87.5% (0.124 g), white solid.

Na4[Fe II (CN)5(pyrazine-2-carboxylate)] (9)

To a solution of Na3[Fe II (CN)5(NH3)]•3H2O (0.100 g, 0.307 mmol, 1 eq.) in water (3 mL) was slowly added a solution of the ligand sodium pyrazine-2-carboxylate (0.054 g, 0.368 mmol) in water (2 mL). The mixture was stirred at room temperature, under argon atmosphere and protected from light for 3 h. Then a cold solution of ethanol (150 mL) containing an excess of sodium iodide (30 eq.) was added dropwise. The resulting precipitate was stand overnight at -20 ºC before to be collected by filtration, washed with cold ethanol and dried in a vacuum desiccator. CAUTION: since these complexes are light-and oxygen-sensitive, they must be stored in a vacuum desiccator in the dark. Yield = 64% (0.064 g), red solid.

Unpublished results associated with the article

This study allowed the development of the compound Na4[Fe II (CN)5(pyridine-3hydroxamic acid)] ( 13), as well as its characterization and the study of its oxidative activation as done for complexes 3 and 6. Despite not being included in the previous article, this complex presented chemical and biological results similar to compounds 3 and 6. The results regarding this complex are presented and discussed briefly below.

Synthetic procedures

Pyridine-3-hydroxamic acid (10) The synthesis of pyridine-3-hydroxamic acid followed the same protocol mentioned in the article for the preparation of hydroxamic acids ligands.

Yield = 82 % (0.40 g), white solid. ,52.17;H,4.38;N,20.28. Found: C,52.09;H,3.96;N,20.12. Retention Factor (methanol/dichloromethane 10%) = 0.31. M.T = 164 °C Electrochemistry (0,1 M Phosphate buffer pH 7.4) Epa = 0.641, 0.963 and 1.104 V vs NHE.

Pyridine-4-carboxylate (11) and pyridine-3-carboxylate (12)

sodium salt

The synthesis protocol of pyridine-4-carboxylate (11) and pyridine-3carboxylate ( 12) sodium salt was the same employed for the sodium pyrazine-3carboxylate (described in the supplementary material of the article).

Pyridine-4-carboxylate sodium salt (11) Yield = 99.0 % (0.140 g), white solid. Pyridine-3-carboxylate sodium salt (12) Yield = 99 % (0.14 g), white solid. 1 H NMR (300 MHz, D2O) δ (ppm): 8.95 (dd, J = 2.2, 0.9 Hz, 1H), 8.61 (dd, J = 5.0, 1.7 Hz, 1H), 8.25 (ddd, J = 7.9, 2.2, 1.7 Hz, 1H), 7.52 (ddd, J = 7.9, 5.0, 0.9 Hz, 1H). ,49.67;H,2.78;N,9.65. Found: C,49.47;H,2.07;N,9.53.

Pentacyanoferrate(II) complexes

The synthesis protocol of Na4[Fe II (CN)5(pyridine-3-hydroxamic acid)] ( 13), Na4[Fe II (CN)5(pyridine-4-carboxylate)] ( 14), and Na4[Fe II (CN)5(pyridine-3carboxylate)] ( 15) was the same employed for the complex 3 (see article).

Na4[Fe II (CN)5(pyridine-3-hydroxamic acid)] ( 13) Yield = 92 % (0.202 g), yellow solid.