Monsieur Nicolas Lebègue

Messieurs Madame Myriam Seemann

Christian Ducho

Matthias Engel

Rebecca Mesdames

Anna Déprez-Poulain

Sachez Hirsch

Bosc Damien

Camberlein Virgyl

Gealageas Ronan

Castillo-Aguilera Omar

Deprez-Poulain Deprez Benoit

Laura Medve

Ronan Gealageas

Bao Vy Lam

Valentin Guillaume

Omar Castillo-Aguilera

Virgyl Camberlein

Melissa Rossel

Charlotte Fléau

Sandrine Waremghem

Julie Charton

Julie Dumont-Ryckembusch

Damien Bosc

Florence Leroux

Benoit P Deprez. Endert Peter

Kraupner Nicolas

Karroum Bou

Lipka Nour

Deprez- Poulain Emmanuelle

Rebecca

B Deprez

R Deprez-Poulain

Poster communication

Keywords: metalloenzymes, ligand, ERAP, immune system, LasB, antimicrobial resistance, inhibitor, selectivity

Target-guided synthesis of protein ligands is an innovative strategy to discover bioactive compounds. In particular, the Kinetic Target-Guided Synthesis (KTGS) and the Dynamic Combinatorial Chemistry (DCC) have allowed, in recent years, the discovery of novel ligands for poorly explored therapeutic targets, which has enabled drug-discovery projects. This thesis project aims at using KTGS to discover and optimize ligands for two classes of metalloenzymes, namely endoplasmic reticulum aminopeptidases (ERAPs) and elastase LasB from the bacterium Pseudomonas aeruginosa.

ERAPs (1 and 2) are involved in the process of antigen maturation. These enzymes cleave peptide precursors into mature antigenic peptides so that they have an optimal size for their complexation to the major histocompatibility complex of class I and thus initiate or not the adaptive immune response. The expression levels of these proteases as well as single nucleotide polymorphisms have been associated with the development of cancers and autoimmune diseases. Thus, the modulation of these enzymes would allow to fight against pathologies associated with the immune system. P. aeruginosa is a Gram-negative bacterium with remarkable virulence and antimicrobial resistance. Today, antibiotic resistance represents a major public health issue and there is an urgent need for new therapeutics. In order to meet this need, new strategies have emerged such as targeting the virulence of bacteria to "disarm" them. LasB represents a therapeutic target of choice due to its extracellular localization and its physiopathological implications (colonization, invasion, evasion of immune response, biofilm formation, etc.).

Although there is a clear unmet medical need in these two therapeutic areas, no modulator of ERAPs or LasB has reached the market.

Thus, the use of the KTGS strategy followed by optimization phases allowed us to identify and optimize new families of ligands for these enzymes. These compounds can be considered as promising lead compounds since they present nanomolar affinities for their respective targets, selectivity and toxicity profiles as well as remarkable physicochemical properties.

Je tiens tout d'abord à remercier le Professeur Benoit Déprez, directeur de l'unité U1177.

Merci de m'avoir accueilli dans votre laboratoire, de m'avoir suivi, guidé et

fait grandir depuis ma 3 ème année de pharmacie jusqu'à cette thèse. Merci pour vos enseignements incroyables et passionnants, merci de m'avoir épaulé durant les nombreuses répétitions pour le concours de l'EDBSL, merci pour vos remarques et conseils durant toutes ces années.

A mes deux directrices de thèse, Anna et Rebecca, merci indéfiniment pour vos connaissances, vos disponibilités, vos connaissances, et votre soutien durant ces années.

Rebecca, merci de m'avoir donné l'opportunité d'avoir pu évoluer tant scientifiquement qu'humainement au sein du laboratoire à Lille, de m'avoir suivi de la 3 ème année jusqu'à aujourd'hui et de m'avoir transmis ta passion pour la chimie médicinale et cette envie de continuer en thèse. Sans toi, je n'aurais pas suivi cette voie et tout ça n'aurait pas été possible. Anna, je te suis profondément reconnaissant de m'avoir donné l'opportunité de découvrir un autre pays, un autre environnement de travail ainsi que pour ta gentillesse, ta patience, ton empathie et ton aide précieuse. Je vous remercie profondément de m'avoir accompagné durant ces années et notamment lors de la rédaction.

Un énorme merci au Dr Damien Bosc pour ton encadrement sans faille durant ces trois années. J'ai pris plaisir à travailler avec toi-même si ce fût difficile durant la méthodo. Je tiens également à te dire quand sans toi, ce travail n'aurait pas été possible. Tu as su être disponible et trouver la solution à chacun de mes problèmes rencontrés. Tu as été un parfait conseillé et tu as une part importante dans la réussite de ce projet. Je te serai indéfiniment reconnaissant pour tout ce que tu m'as apporté.

Merci au Drs Ronan Gealageas et Jelena Konstantinovic. Ronan, ça fait bizarre de t'appeler « Docteur », tout a commencé avec toi lors de mon premier stage. Je tenais également à souligner que tu as grandement participé quant à ma décision de poursuivre vers la chimie médicinale. Tu fus mon premier encadrant et tu as su me transmettre ta passion et ta motivation. Merci pour ton aide précieuse durant ces 7 années, les KTGS, les analyses RMN, les idées de chimie, tes conseils à la paillasse, tes méthodes de travail… Je n'oublie pas nos interminables débats footballistiques, nos paris ainsi que les séances du mardi midi ; Paris n'a toujours pas gagné de ligue des champions… Ce fût un réel plaisir d'avoir pu partager tous ces moments avec toi. Jelena, merci pour ton accueil lorsque je suis arrivé à Saarbrücken ainsi que pour ta disponibilité, ta patience, tes remarques et tes corrections durant la rédaction de cette thèse.

Résumé

La synthèse guidée par la cible de ligands protéiques est une stratégie innovante pour découvrir des composés bioactifs. En particulier, la Kinetic Target-Guided Synthesis (KTGS) and the Dynamic Combinatorial Chemistry (DCC) ont permis, ces dernières années, de découvrir des ligands originaux pour des cibles thérapeutiques mal explorées, ce qui a permis de lancer des projets de découverte de médicaments. Ce projet de thèse vise à utiliser la KTGS pour découvrir, puis optimiser des ligands de deux classes de métalloenzymes que sont les aminopeptidases du réticulum endoplasmiques (ERAP) et l'élastase LasB de la bactérie Pseudomonas aeruginosa.

Les ERAPs (1 et 2) participent au processus de maturation des antigènes. Ces enzymes clivent les précurseurs peptidiques en peptides antigéniques matures afin que ceux-ci disposent d'une taille optimale pour leur complexation au complexe majeur d'histocompatibilité de classe I et ainsi initient ou non la réponse immunitaire adaptative. Les niveaux d'expression de ces protéases ainsi que des polymorphismes d'un seul nucléotide ont été associé au développement de cancers et de maladies auto-immunes. Ainsi, la modulation de ces enzymes permettrait de lutter contre les pathologies associées au système immunitaire.

P. aeruginosa est une bactérie Gram negative dotée d'une virulence et d'une

résistance aux antimicrobiens remarquable. Aujourd'hui, la résistance aux antibiotiques représente un enjeu de santé publique majeur et il y a un besoin urgent en nouvelles thérapeutiques. Afin de satisfaire ce besoin, de nouvelles stratégies sont apparues comme celle consistant à cibler la virulence des bactéries afin de « désarmer » celles-ci. LasB représente une cible thérapeutique de choix de par sa localisation extracellulaire et ses implications physiopathologiques (colonisation, invasion, évasion à la réponse immunitaire, formation de biofilm, etc.).

Bien qu'il y ait un besoin médical évident non satisfait dans ces deux aires thérapeutiques, aucun modulateur des ERAPs ni de LasB n'a atteint le marché.

Ainsi, l'utilisation de la stratégie KTGS suivie de phases d'optimisation nous ont permis d'identifier et optimiser de nouvelles familles de ligands de ces enzymes. Ces composés peuvent être considérés comme des leads prometteurs puisqu'ils présentent des affinités nanomolaires pour leurs cibles respectives, des profils de sélectivité et de toxicité ainsi que des propriétés physicochimiques remarquables.

Mots-clés : KTGS, ligand, métalloenzymes, ERAP, système immunitaire, LasB, résistance antimicrobienne, inhibiteur, sélectivité. 1. Principle ............................................................................................................................................ --History and Recent Achievements ..................................................................................................

Zusammenfassung

Table of Contents

General introduction

For a drug-discovery team, the ligand identification represents a key step in the drugdiscovery process. The choice of the method to identify ligands is dependent on multiple factors such as the overall costs, the technical demands or the nature of the disease or of the target. Several hit-identification strategies have been reported (Figure 1). In the present work, we were interested in the identification of metalloenzymes ligands. As their name suggests, metalloproteins are proteins containing metal ions; the metal ion having a structural and/or functional role. If this ion is present in the active site and promotes the catalytic mechanism, we talk about metalloenzymes.

Metalloenzymes account for almost 50% of all enzymes and carry out a diverse range of biological processes like inflammation, cell proliferation, hormonal signaling or pathogen growth, depending on the nature of the reaction in which the metal ion is involved (zinc, copper, calcium, magnesium, manganese or iron). (1) High-throughput Screening (HTS)

Fragment-based drug discovery (FBDD)

Computational strategies

High-Content Screening (HCS)

Rational design

DNA Encoded Librairies (DEL)

Protein-templated strategies

Hit identification

Biophysical strategies

In particular zinc metalloenzymes are an important subclass of metalloenzymes. Some of them led to pharmacological drug classes used in major therapeutic indications:

 Angiotensin-converting enzyme (ACE) inhibitors for the treatment of high blood pressure,  Histone deacetylase (HDAC) inhibitors for the treatment of cancer and inflammatory diseases,

 Neprilysin (NEP) inhibitors for the treatment of heart failure.

In addition, many compounds targeting these enzymes are in development like TNF-α converting enzyme (TACE) inhibitors and metallobetalactamase (MBL) inhibitors to fight against inflammation and antimicrobial resistance (AMR), respectively. (2), (3) The teams of Prof. Deprez-Poulain and Prof. Hirsch target such metalloenzymes:

Insulin-degrading enzyme (IDE) and the Endoplasmic reticulum Aminopeptidases (ERAP) in Lille and Clostridial collagenases (ColH and ColQ1) and Pseudomonas aeruginosa elastase (LasB) in Saarbrücken, respectively. ( 4),( 5),( 6), (7) The objectives of this project are to use innovative protein-templated strategies, which use the target protein itself to form its own ligands (8), (9) to identify and optimize new ligands of metalloenzymes of interest in both labs (Figure 2). We will focus on the discovery of ERAP inhibitors for application in the modulation of the immune response, and LasB inhibitors to block the virulence process in P. aeruginosa infection. These ligands will serve as pharmacological tools to explore the role of these enzymes and as leads for therapeutic applications. More specifically, amongst the different target-guided synthesis approaches, dynamic combinatorial chemistry (DCC), which is thermodynamically controlled and kinetictarget guided synthesis (KTGS) which is kinetically controlled, [START_REF] Cheeseman | Receptor-assisted combinatorial chemistry: thermodynamics and kinetics in drug discovery[END_REF] we have chosen to use KTGS exclusively in this work. Even if the focus is focused on KTGS, it is important to compare the principle of the two protein-templated approaches (Figure 3).

In DCC, the target protein assembles ligands via a reversible process. The reagents and products interact continuously (dynamic combinatorial library, Figure 3A) and the thermodynamic equilibrium is displaced by the target in favor of ligands, which display the highest affinity for the protein and therefore the production of those is amplified. [START_REF] Hartman | Protein-Templated Dynamic Combinatorial Chemistry: Brief Overview and Experimental Protocol[END_REF], [START_REF] Frei | Dynamic Combinatorial Chemistry: A New Methodology Comes of Age[END_REF] In KTGS, the target catalyzes the reaction between the most affine pair of reagents for its active site and templates the formation of the ligand by an irreversible link (Figure 3B). Thus, DCC is based on the product affinity whereas KTGS is based on the reagents affinity and their proper orientation in the active site.

History and Recent Achievements

Although the first intentional example of KTGS was reported by Huc et al. in 2001 with the use of the human carbonic anhydrase II (hCAII) to catalyze thiol alkylations, (14) a previous unexpected use of this strategy was disclosed by Inglese et al. in 1991 using the glycinamide ribonucleotide transformylase. [START_REF] Inglese | Multisubstrate adduct inhibitors of glycinamide ribonucleotide transformylase: Synthetic and enzyme-assembled[END_REF] The 2000s can be considered as an important date in the development and use of this strategy thanks to the work of the Sharpless, Meldal and Fokin groups who reported the copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition.

Subsequent to this discovery, the term "in situ click chemistry" was created; the most popular example was carried out with the acetylcholinesterase (AChE). [START_REF] Lewis | Click chemistry in situ: acetylcholinesterase as a reaction vessel for the selective assembly of a femtomolar inhibitor from an array of building blocks[END_REF] Noteworthy, KTGS has greatly evolved in a short time. First, apart from the Huisgen 1,3-dipolar cycloaddition between alkynes and azides, it is possible to use other templated reaction such as the sulfo-click reaction, [START_REF] Kulkarni | Screening of protein-protein interaction modulators via sulfo-click kinetic target-guided synthesis[END_REF] hydrolysis/thio-Michael coupling, [START_REF] Oueis | First enzymatic hydrolysis/thio-Michael addition cascade route to synthesis of AChE inhibitors[END_REF] amidation, [START_REF] Jaegle | Protein-Templated Formation of an Inhibitor of the Blood Coagulation Factor Xa through a Background-Free Amidation Reaction[END_REF] Mannich reaction and more recently four-components Ugi reaction. [START_REF] Wong | The transcription factor STAT5 catalyzes Mannich ligation reactions yielding inhibitors of leukemic cell proliferation[END_REF], [START_REF] Mancini | Protein-Templated Hit Identification through an Ugi Four-Component Reaction*[END_REF] Although the majority of KTGS experiments are performed using enzymes, some experiments were published using transcriptional factors, channels and protein-protein interactions.

Finally, Deprez-Poulain et al. have demonstrated that KTGS experiments can be performed in multicomponent formats. (4) Thus, the increasing variety of targets, the chemical reactions diversity, the evolving of the KGTS format and the biological characterization of the templated ligands have contributed to the evolution of this approach, demonstrating that KTGS has undeniable potential for hit identification.

II. Design & practical aspects 1. Chemical reactions used

Among the reported reaction, the in situ click reaction between alkynes and azides is the most employed and represents around 80% of the published KTGS experiments; eight other reactions accounting for the remaining percentage (≈ 20%). (13) The high interest for the Huisgen 1,3-dipolar cycloaddition comes both from the fact that the azides and alkynes are biocompatible and that a broad range are commercially available or can be synthesized easily.

Warhead-bearing reagent

The selection and/or design of warhead-bearing reagents represent the most important step for the achievement of a KTGS experiment. Deprez et al. have analyzed the origin and nature of those in Figure 4. (13) In the majority of cases (≈ 63%), the warhead-bearing reagents come from known Structure-Activity Relationship (SAR) or previously described modulators (Figure 4A). In more than 12% of cases, the warhead-bearing reagents derive from substrates or cofactors like biotin. Interestingly, marketed drugs can also be used to design warheads (≈ 9%).

Strikingly, around 16% of cases concern warheads, which were discovered by fragment screening; demonstrating that KTGS can be complementary to other strategies for ligand identification.

Although for all ligands, the majority of warhead-bearing reagents (≈ 77%) are synthetic compounds, a non-negligible proportion derived from natural product (≈ 24%) (Figure 4B).

Finally, regarding the binding of the templated ligands, non-covalent compounds were templated in almost all cases (≈ 95%, Figure 4C). (13) Sometimes, there is a confusion between protein-templated and fragment approaches;

the protein-templated being wrongly considered as an extension of fragment approaches. In order to discriminate the two strategies, Deprez-Poulain et al. have also studied the features of

A B C
the warhead-bearing reagents and compared these characteristics with those in fragment approaches.

According to the rule of 3 (Ro3), a reagent can be considered as a fragment if it has a molecular weight (MW) lower than 300 g/mol, no more than 3 hydrogen-bond donnors (HBD), no more than 3 hydrogen-bond acceptors (HBA) and a clogP of less than 3. [START_REF] Congreve | A 'rule of three' for fragment-based lead discovery?[END_REF] If the majority of the warheads used in KTGS respect the Ro3 for the clogP (70%) and HBD (76%) criteria, the analysis is not the same for the MW and HBA criteria (Figure 5A-D).

Indeed, 62% have a MW higher than 300 g/mol (Figure 5A) and 71% display a number of HBA above 3 (Figure 5D). In conclusion, most of the reagents used in KTGS are not in accordance with the Ro3. (13) The analysis of Deprez-Poulain et al., concerning the structural features of reagents used in KTGS, showed that the number of heavy atoms differs significantly between fragments. Indeed, the majority of fragments have between 10 and 16 heavy atoms, whereas the majority of KTGS reagents have between 10 and 30 heavy atoms.

However, the reagents in KTGS and fragments differ slightly concerning the fraction of sp 3hydridized carbon atoms; both displaying a limited fraction: < 0.6 in KTGS and < 0.5 for fragments. Then, 50% of the KTGS reagents contain more rotatable bonds (between 2 and 6)

and rings (> 2) compared to fragments.

Although the main proportion of these reagents (KTGS and fragments) are neutral compounds (≈ 60% in KTGS and ≈ 70% for fragments), an important percentage of the remaining proportion of the KTGS warheads are positively charged (≈ 30%) whereas, the proportion of positively charged fragments is lower (≈ 10%); fragments with negative charge represents around 20%.

Noteworthy, theses reagents can be distinguished by their potency or affinity for the protein target as, usually, fragments display potency in a millimolar range, whereas KTGS warheads display potency in a micromolar range (≤ 500 μM). Thus, KTGS strategy cannot be considered as an extension of the fragment linking approach since many differences exist, in particular, regarding the potency of the "starting building block".

Formats, Sizes, Hit Rates and Detection Methods

Apart from the binary format, Kolb et al. and Deprez-Poulain et al. have demonstrated that KTGS experiments can be performed in multicomponent or orthogonal multicomponent formats, respectively [START_REF] Krasiński | In situ selection of lead compounds by click chemistry: target-guided optimization of acetylcholinesterase inhibitors[END_REF], (4) . Therefore the number of templated ligands increases and thus the chances of success are maximized as the pair of reagents is replaced by clusters. However, the binary format is still the most employed format in the published KTGS assays (67%, Figure 6A), partly explaining that in only one quarter of the disclosed examples, the authors investigated the formation of more than 50 putative ligands (Figure 6B).

Deprez-Poulain's group has unsurprisingly reported that the hit rate is high in KTGS.

This can be explained by the fact that the majority of assays were performed with limited pools of reagents, which were derived from known SARs or modulators. They have disclosed that 50% of KTGS experiments, in which a minimum of 30 ligands can be templated, displayed comparable hit rates to random screening (1-5%, Figure 6C).

Finally, the team has reported that mass Spectrometry (MS) methods are used in 85% of cases to detect the templated ligands (Figure 6D); X-ray, NMR or thermal shift assay can be mentioned as other methods. (13) III. Relevance of KTGS for medicinal chemistry and drug discovery

Therapeutic Areas and Protein Targets

A significant number of therapeutic areas were targeted by the published KTGS experiments, in particular infectiology and cancerology, which account for around 55%

(Figure 7A).

In the published review by Deprez-Poulain's team, KTGS experiments were performed using 26 different proteins; the most used protein family being the enzymes (73%, Figure 7B).

Among the enzymes used, we can find hydrolases, ligases, oxydoreductases, lyases and transferases. Interestingly, the team has also revealed that around 60% of the used proteins do not have marketed drugs or clinical candidates. (13)

Protein Pockets and Conformation

In Hirsch's and Deprez-Poulain's published studies, the different pockets were scored according to their size, depth, enclosure and fraction of apolar residues. They did not observe significant differences for the depth of pockets but KTGS pocket are a bit wider and contain a higher fraction of apolar amino acids (Table 1). (24) and Deprez-Poulain et al. (13) Volume ( Å3 In case of enzymes, it is quite "easy" to target the active site with the templated ligands, using warheads which can bind it. For example, Deprez-Poulain et al. used hydroxamate-bearing warheads to coordinate the zinc of IDE and therefore allow the binding of the templated ligands in the active site. (4) Thus, unsurprisingly, almost all templated ligands by enzymes target and bind the active site.

Additionally, protein-templated strategies have demonstrated that they can allow the identification of new binding pockets and/conformations, which cannot be discovered through conventional methods like X-ray crystallography or computational strategies. ( 4), (25)

Chemical Space and Ligand Efficiency of KTGS Ligands

Deprez-Poulain's team was interested in the behavior of KTGS ligands with respect to the Lipinski-Weber rules and the Doak chemical space (Figure 8). The Lipinkski-Weber rules (Rule of 5 (Ro5)) are employed to evaluate if a drug can be used as an oral drug. To comply with the Ro5, the compound must have fewer than 5 HBA and 10 HBD, a MW less than 500 g/mol and an octanol-water partition coefficient (cLogP) not greater than 5. [START_REF] Pollastri | Overview on the Rule of Five[END_REF] The Doak space corresponds to the "possible to be oral" chemical space, the compound must display fewer or equal to 15 HBA and 6 HBD, a MW less or equal to 1000, a cLogP ranging from less or equal to -2 to less or equal to 10, a Polar Surface Area (PSA) less or equal to 250 Å 2 and a number of rotatable bounds less or equal to 20. [START_REF] Doak | How Beyond Rule of 5 Drugs and Clinical Candidates Bind to Their Targets[END_REF] According to analyses, although the majority of the KTGS ligands do not comply with the with the Lipinski-Weber rules (72%), most of them display values allowing them to be in the Doak space (82%, Figure 8). Thus, thanks to the KTGS method, it is possible to explore larger chemical spaces than the one defined by Lipinski-Weber. (13) (13) Finally, Deprez-Poulain et al. have also compared the properties of the templated ligands and those of the FDA-approved drugs (Figure 9). These comparisons revealed that FDA approved drugs are lighter, less polar and less flexible than the KTGS ligands. -11 -Noteworthy, as presented on Figure 10, around 70% of templated ligands can be considered as good medchem starting points as they display a Lipophilic Ligand Efficiency (LLE) higher than 3 and, strikingly, one quarter of them can be considered as promising drug candidates as they display LLEs higher than 6 (Figure 10A). Noteworthy, in most cases, KTGS experiments led to an increase of the LLE (ΔLLE, Figure 10B). ( 13)

Insulin Degrading Enzyme (IDE)

Deprez-Poulain's team applied KTGS strategy to identify inhibitors of IDE, a zincmetalloprotease (member of the M16 group) which cleaves insulin and other bioactive peptides like amyloid-β and glucagon. (4) They performed an orthogonal multicomponent experiment from two azides (K6a and K6b) and ninety alkynes (Scheme 2). They used the information available on substrate preference and inhibition of human IDE (hIDE) to design two azides-bearing hydroxamate warheads to coordinate the zinc ion in the N-terminal part of the enzyme (K6a and K6b) while the alkynes were chosen as potential binders to the Cterminal extremity of the active site. (4) Interestingly, they observed that K6 has an influence on the open/closed equilibrium of hIDE by shifting it toward closed conformations (Figure 12).

Endothiapepsin

Hirsch et al. combined the fragment-linking approach with the protein-templated strategy to identify inhibitors of endothiapepsin (Scheme 3), a model enzyme for the studies of aspartyl proteases. [START_REF] Mondal | Fragment-Based Drug Design Facilitated by Protein-Templated Click Chemistry: Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin[END_REF]

Scheme 3 : Combination of fragment linking approach and KTGS strategy to identify endothiapepsin inhibitors

The starting fragments K7a and K7b were previously identified by Klebe et al. [START_REF] Köster | A small nonrule of 3 compatible fragment library provides high hit rate of endothiapepsin crystal structures with various fragment chemotypes[END_REF] They make H-bonds with two aspartate residues (Asp35 and Asp219) of the catalytic dyad through their hydrazide (K7a) and amidine groups (K7b) (Figure 13). As they display good physicochemical properties, promising inhibitory potency (≈ 85% inh at 1 mM) and interesting occupancy in the active site, these fragments were chosen by Hirsch et al. as good starting points for fragment linking strategy. Thanks to molecular modeling, they linked the fragments through a triazolyl moiety (K7c, Figure 13). Interestingly, the triazole ring seems to be in close proximity to the two aspartate residue and thus could interact with the catalytic dyad (Figure 13). et al. [START_REF] Mondal | Fragment-Based Drug Design Facilitated by Protein-Templated Click Chemistry: Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin[END_REF] Following the molecular modeling, they performed the KTGS through in situ click from four alkynes and nine azides which were identified by retrosynthesis of the modeled triazoles. Four hits were identified, the most potent displaying an IC 50 in the micromolaire range (K7, Scheme 3). Docking of the hit K7 showed H-bonding interactions: the triazole ring-Asp35 residue, the NH group of the piperazine-Asp119 and phenol-isoleucine 300 (Ile300), and hydrophobic interactions of the piperazine with the S3 pocket (Ileu122, Leu125 and Phe116) (Figure 14).

V. Conclusion

KTGS has proved that it can be employed as a hit identification/optimization strategy.

So far, more than 50 examples were published leading to the discovery of ligands in different

K7a

K7b

K7c therapeutic areas, especially in infectiology, cancerology and CNS disorders domains. Several target families were explored like enzymes, receptors, channels, protein-protein interactions and RNA.

Strikingly, this protein-templated strategy has the capacity to provide, in most cases, ligands which can be considered as interesting starting points for medchem programs (LLE >

3) or promising drug candidates (LLE > 6).

Additionally, KTGS allows the exploration of unknown protein conformations and therefore the discovery of new binding sites, which cannot be captured by computational strategies or NMR, showing that this strategy can be used as a valuable tool in context of drug discovery to explore the conformational space of a ligand-binding pocket.

Although this strategy presents drawbacks and limitations such as the use of biocompatible reagents or large amounts of the protein target, most of these limitations have been solved in particular through the use of multicomponents formats and development of in cellulo experiments. (13) Thus, the KTGS strategy appears as an attractive and promising approach to find ligands, and for all mentioned reasons, we decided to use it to discover new ligands of two zinc metalloenzymes, nameley the Endoplasmic Reticulum Aminopeptidases (ERAPs) and the elastase LasB of the bacterium P. aeruginosa. The next chapters of this thesis will be dedicated to the presentation of my PhD work on the ERAPs and LasB, which will be followed by a final chapter about the description of a new methodology to access 1,5disubstituted 1,2,3-triazoles, developed during my thesis.

A-Innate immunity

Innate or "non-specific" immunity is defined as the early line of defense against pathogens and is organized around constitutive and induced responses. [START_REF] Romo | Innate immunity in vertebrates: an overview[END_REF] The constitutive immune mechanisms refer to the chemical and physical barriers of the body (skin, saliva, lacrimal secretions, stomach acid, etc.), which allow an immediate response to a danger signal. However, they do not have the capacity to amplify the response and are therefore, in some cases, insufficient to eliminate the pathogen. [START_REF] Paludan | Constitutive immune mechanisms: mediators of host defence and immune regulation[END_REF] Once the pathogen is in the body, the induced immune mechanisms take over from the constitutive systems allowing the recognition and destruction of the pathogen. The Patter Recognition Receptors (PRRs), immune sensors, which discriminate self from non-self, detect

Pathogen-Associated Molecular Patterns (PAMPs) (non-self-molecules) and initiate the immune response. Cellular effectors such as Antigen-Presenting cells (APCs), macrophages, phagocytes and Natural Killer lymphocytes (NK) are thus recruited, facilitating the set-up of the defense mechanisms (inflammation, phagocytosis and complement activation here). [START_REF] Romo | Innate immunity in vertebrates: an overview[END_REF] In cases where pathogen eliminations require more powerful mechanisms, the innate immune response stimulates its collaborative system, which is the adaptive immunity.

B-Adaptive immunity

Adaptive immunity is referred as the "specific" immunity by its ability to respond selectively to numerous non-self-materials. (32) Although the APCs are innate immunity actors, they play a key role in the specific immunity in interacting and stimulating the main selfdefensive weapons of the adaptive response, which are the T (CD4 + and CD8 + ) and B lymphocytes. [START_REF] Turvey | Innate immunity[END_REF] The CD4 + T-cells, also named helper T-lymphocytes, coordinate the adaptive response in secreting key proteins like cytokines and interleukines. (34) The CD8 + T-cells, also named cytotoxic or killer T-lymphocytes, eliminate the infected cells and are thus the effectors of the adaptive cellular response. [START_REF] Zhang | CD8(+) T cells: foot soldiers of the immune system[END_REF] Once activated by the CD4 + T-lymphocytes, the B-cells differentiate into plasmocytes, which are responsible for the adaptive humoral response through the immunoglobulin secretion. [START_REF] Shapiro-Shelef | Blimp-1 is required for the formation of immunoglobulin secreting plasma cells and pre-plasma memory B cells[END_REF] Thus, when innate immunity is not sufficient to eliminate pathogens, the immune system displays a second line of defense where these pathogens are presented to more specific mechanisms; the presentation or recognition step being fundamental to initiate the response.

Immune system and antigen presentation

An antigen is a natural (protein, carbohydrate or lipid residues) or synthetic macromolecule, which, when introduced into an organism, has the capacity to stimulate the immunocompetent cells. An antigen has two main properties: its antigenicity and its immunogenicity. Antigenicity is the ability of the antigen to be recognized by the immune system and immunogenicity is its potential to induce an immune response.

The presentation or recognition step is a fundamental principle of the immunity. Numerous immune cells have the capacity to pick up these antigens and associate them with APC, thus initiating the immune response.

A-The Human Leucocyte Antigen (HLA) system

Before initiating the immune response, the immune system has to do the distinction between self-antigens and non-self-antigens. The HLA system, found on the surface of the cells, allows us to make this distinction. These HLA antigens, also named Major Histocompatibility Complex (MHC), are divided in three groups: MHC class I (MHC-I), MHC class II (MHC-II) and MHC class III (MHC-III). Only the MHC-I and MHC-II are involved in the antigenic presentation. It should be noted that these MHC systems only present protein molecules, although some polysaccharides can still be presented through this system. [START_REF] Choo | The HLA system: genetics, immunology, clinical testing, and clinical implications[END_REF] The MHC-I (Figure 16A) is composed of two polypeptide chains α and β. The polymorphic α-heavy chain, composed by three subunits (α1, α2 and α3) and encoded by the HLA genes As they display different structural characteristics, they do not have the same specificities and therefore do not bind the same antigens. Here we will focus on the HLA-I system and the implication of the ERAP enzymes in the processing and presentation of antigenic peptides.

B-Antigen-presentation process mediated by the MHC-I

The MHC-I molecules are ubiquitously expressed contrary to the MHC-II, all the nucleated cells and platelets (thrombocytes) have MHC-I complexes on their surface. The MHC-I presents the endogenous antigens. The endogenous antigens are initially present in our cells as their name suggests and are the result of protein degradation in the cytoplasm by the proteasome.

The generation of antigen-MHC-I complexes results in a multistep process (Figure 17). The proteasome, here the immunoproteasome, degrades cytoplasmic proteins further to its ubiquitination and generates peptides, which have a size from 4 to 20 amino acids. [START_REF] Kisselev | The sizes of peptides generated from protein by mammalian 26 and 20 S proteasomes. Implications for understanding the degradative mechanism and antigen presentation[END_REF] As the optimal size to be complexed with the MHC-I is between 8 and 11 residues, [START_REF] Falk | Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules[END_REF]

Immune response

However, nothing is perfect and the process of loading antigen peptides on the MHC-I can be disrupted at different steps, in particular at the level of the ERAPs.

As seen on Figure 17, the ERAPs play a key role in the process by trimming peptide precursors into mature antigenic epitopes of appropriate size for presentation on the cell surface through the MHC-I. Disruptions of their proteolytic activities can modify the presented antigenic peptide repertoire, also named immunopeptidome, altering therefore the adaptive immune response both qualitatively and quantitatively. Indeed, firstly, the nongeneration of the specific immunodominant epitopes by the ERAPs can lead to a nonpresentation to the immunocompetent cells and therefore to a suppression of existing cytotoxic responses mediated by the CD8 + T-cells resulting in an immune evasion. On the other hand, these enzymes can produce truncated peptides which, once presented, cause an abnormal stimulation of the immunocompetent cells and therefore to unusual cytotoxic responses, resulting in an over-activation of the immune system. Thus, the ERAPs appear as attractive targets to fight against the immune system associated diseases like cancer and autoimmune disorders.

II.

The ERAPs as pharmaceutical targets implicated in the antigen processing

Classification, structure and organization

The ERAPs (ERAP1 and ERAP2) are zinc metalloproteases belonging to the oxytocinase subgroup of M1 zinc metallopeptidases and are localized in the ER as their name suggests. [START_REF] Serwold | ERAAP customizes peptides for MHC class I molecules in the endoplasmic reticulum[END_REF] Both isoforms share almost 50% sequence similarity. [START_REF] Vitulano | The interplay between HLA-B27 and ERAP1/ERAP2 aminopeptidases: from anti-viral protection to spondyloarthritis[END_REF] Endoplasmic Reticulum Associated with Antigen Processing (ERAAP) is an ERAP1 homolog in mouse, while ERAP2 does not have any homolog in rodents. (42) The human ERAP genes are encoded in a short arm of the chromosome 5q15 in the opposite direction (Figure 18). The isoform 1 contains 20 exons and the other 19; both enzymes being segmented in 4 domains. The active site, containing HEXXH(X) 18 E and GAMEN motifs, which are characteristics of the M1 zinc metallopeptidases group, (43) are located in the domain II for each ERAP. The HEXXH(X) 18 E motif, containing two histidine (H), two glutamic acid and other (X) residues, is referred as a zinc-binding motif. [START_REF] Wang | Histidine residue in the zinc-binding motif of aminopeptidase A is critical for enzymatic activity[END_REF] The first glutamic acid residue is also involved in the catalytic activity by polarizing a catalytic water molecule. [START_REF] Vazeux | Identification of glutamate residues essential for catalytic activity and zinc coordination in aminopeptidase A[END_REF] The residue positions are not similar between the two ERAPs (Figure 19). (47) The GAMEN motif, containing a glycine (G), an alanine (A), a methionine (M), a glutamic acid (E) and an asparagine (N), is crucial for the enzymatic activity in interacting with the Nterminal part of substrates. [START_REF] Vazeux | A glutamate residue contributes to the exopeptidase specificity in aminopeptidase A[END_REF] As previously, the residue positions are different between the two isoforms (Figure 20).

Catalytic mechanism, substrate recognition and mechanism

The catalytic site contains a zin ion, which is coordinated by two histidine residues (H353 and 357 for ERAP1, H370 and 373 for ERAP2) and a glutamate residue (E376 for ERAP1 and E393 for ERAP2), forming the first coordination sphere (Figure 19). The second coordinate sphere is formed by a tyrosine residue (Y438 for ERAP1 and Y455 for ERAP2)

and the first glutamate residue of the HEXXH(X) 18 E motif (E354 for ERAP1 and E371 for ERAP2), the tyrosine participating in the stabilization of the tetrahedral intermediate during the catalytic mechanism and the glutamate polarizing a catalytic water molecule. [START_REF] Addlagatta | Structure of aminopeptidase N from Escherichia coli suggests a compartmentalized, gated active site[END_REF], [START_REF] Vazeux | Identification of glutamate residues essential for catalytic activity and zinc coordination in aminopeptidase A[END_REF] The Zn (II) is crucial for the catalytic activity. The ERAPs display different substrate specificities and thus complement each other in shaping the immunopeptidome.

Chang et al. have analyzed ERAP1 substrate specificity and preference. [START_REF] Chang | The ER aminopeptidase, ERAP1, trims precursors to lengths of MHC class I peptides by a 'molecular ruler' mechanism[END_REF] First, they observed that ERAP1 prefers long substrates with a length between 10 and 16 residues Then, they demonstrated that ERAP1 prefers peptides, which contain a hydrophobic or aromatic C-terminal residue (Figure 22). [START_REF] Chang | The ER aminopeptidase, ERAP1, trims precursors to lengths of MHC class I peptides by a 'molecular ruler' mechanism[END_REF] Thanks to their studies, they demonstrated that ERAP1 cleaves preferentially long and/or hydrophobic peptides thanks to a hydrophobic pocket close to the active site. They have described a "molecular ruler" mechanism for ERAP1 (Figure 23). In cases where peptides contain between 9 and 16 residues, the peptides bind optimally ERAP1 by interacting with the N-and C-terminal sites and ERAP1 trims the peptides in N-terminal direction, releasing therefore mature antigens (Figure 23A). In cases where peptides display a length below 9 residues (Figure 23A) or contain charged residues (Figure 23B), ERAP1 cannot trim them. Contrary to ERAP1, Chang and Stratikos groups have reported that ERAP2 does not follow the "molecular ruler" mechanism described on Figure 23. Indeed, through crystal structure studies, they observed that the peptides access to the substrate cavity of ERAP2 with difficulty as the interactions with this cavity appear shallow and opportunistic. This isoform showed preferences for short and/or positively charged peptides, ERAP2 activity being maximal on octameric substrates and lower on longer peptides. The ERAP2 selectivity for positively charged amino acids is due to the presence of a glutamate residue, which lines the side of the selectivity pocket of the enzyme (S1). ( 42),( 50), [START_REF] Mpakali | Structural Basis for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum (ER) Aminopeptidase 2[END_REF]

Physiopathological implications

During a physiological process (Figure 24A), the ERAPs shape the immature peptides into mature epitopes. In cases where these antigens are immunogenic, they are complexed to the MHC-I to be presented to the CD8 + T-lymphocytes and finally activate the immune response. Otherwise, they are not complexed to the MHC-I and no immune response is activated.

Disruptions of these aminopeptidases result in an abnormal antigen processing. First, the ERAP trimming can result in the formation of immunogenic epitopes, which normally are not formed and therefore cause unsuitable immune responses further to the exportation of peptide-MHC-I complexes to the surface of the cell (Figure 24B). Secondly, the ERAPs can either shape immature peptides into non-immunogenic epitopes, which normally are or destroy the "right" immunogenic epitopes leading their accumulation in the ER as they are not complexed to the MHC-I (Figure 24C). Thus, these antigens are not presented to the CD8 + Tcells with potential escape from the immune system. Consequently to their accumulation in the ER, they are finally exported to the extracellular media, provoking a local inflammatory response. [START_REF] Haroon | Endoplasmic reticulum aminopeptidases: biology and pathogenic potential[END_REF] It is important to understand that this mechanism does not only concern one antigen but the whole antigenic repertoire (immunopeptidome). The ERAPs thus exert qualitative and quantitative control on the immunopeptidome. Their expression levels and enzymatic activity are strongly linked with its composition and can therefore affect the adaptive immune response by trimming the "wrong" and/or destroying the "right" epitopes.

Two opposite consequences are linked with disruptions of these enzymes. Either the "wrong"

epitopes are shaped and presented to the CD8 + T-cells, causing an excessive stimulation of the immune system, which finally leads to autoimmune disease appearances, or, the "right" epitopes are destroyed and therefore cannot be recognized, escaping from the immune system with cancer or infectious disease developments as consequences.

Single Nucleotide Polymorphisms (SNPs) of the ERAPs, which affect enzymatic activity or protein expression, have been associated with diseases. [START_REF] Haroon | Endoplasmic reticulum aminopeptidases: biology and pathogenic potential[END_REF]

ERAPs modulation in therapy

A-Oncology

Stratikos et al. have reported that defects in the expression of the ERAP were detected in several tumors such as leukemias, lymphomas, melanomas and carcinomas of skin, breast, lung, colon, chorion, cervix, prostate, bladder and kidney. [START_REF] Stratikos | A role for naturally occurring alleles of endoplasmic reticulum aminopeptidases in tumor immunity and cancer pre-disposition[END_REF] Fruci et al. have analyzed the mRNA expression of ERAP1 and ERAP2 in around 9000 cancer samples of various origin from The Cancer Genomic Atlas and they found that the expression levels of these enzymes were altered in several tumors (Figure 25). ERAP1 is more expressed in acute myeloid leukemia (AML), stomach adenocarcinoma (STAD) and kidney renal clear cell carcinoma (KIRC), but less expressed in testicular germ cell carcinoma (TGCT), uveal melanoma (UVM) and adrenocortical carcinoma (ACC).

ERAP2 is more expressed in acute myeloid leukemia (AML), pancreatic adenocarcinoma (PAAD) and kidney renal clear cell carcinoma (KIRC), but less expressed in lung squamous cell carcinoma (LUSC), testicular germ cell carcinoma (TGCT) and uveal melanoma (UVM).

Thus, alteration of ERAP expression levels can result in a defect in the production or degradation of tumor antigens, leading to tumor escape from the immune system (Figure 26).

At physiological level, the ERAPs shape the tumor-specific antigen peptide, resulting in an activation of the cytotoxic T-cells (Figure 26A). However, when the ERAPs are altered, they do not "correctly" trim the tumor-specific mature antigens not allowing the cytotoxic T-cells activation and therefore facilitating immune evasion (Figure 26B). [START_REF] Stratikos | A role for naturally occurring alleles of endoplasmic reticulum aminopeptidases in tumor immunity and cancer pre-disposition[END_REF], [START_REF] Kuo | Endoplasmic reticulum aminopeptidase 2 involvement in metastasis of oral cavity squamous cell carcinoma discovered by proteome profiling of primary cancer cells[END_REF] Modulation of the ERAP pathway would therefore allow the "right" shaping of the immature antigens and therefore the production of the mature tumor-specific antigens which, once complexed to the MHC-I would activate the immunocompetent cells; which then would lead to the tumor suppression. (lymphoma) in mice through a mechanism involving the NK cells, [START_REF] Cifaldi | Natural killer cells efficiently reject lymphoma silenced for the endoplasmic reticulum aminopeptidase associated with antigen processing[END_REF] supporting the idea that ERAPs modulation represents a promising pathway for cancer immunotherapy.

Besides, Lim et al. have demonstrated that low levels of ERAP2 were associated with improved response to anti-PD-L1therapy, in patients with luminal bladder cancers. [START_REF] Lim | Germline genetic polymorphisms influence tumor gene expression and immune cell infiltration[END_REF] So, ERAP2 inhibitors could help to reverse resistance to checkpoint inhibitors and therefore increase the overall survival.

B-Autoimmunity

As seen previously, SNPs of the ERAPs are linked with pre-disposition to autoimmune diseases. Indeed, these SNPs modify the expression and/or the enzymatic activity of the aminopeptidases, leading in non-desired immune responses. ERAP1 is associated with Ankylosing Spondylitis, [START_REF] Evans | Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27 in disease susceptibility[END_REF] a chronic inflammatory rheumatopathy of the lower spine; the HLA-B27 being known as a high risk factor. This isoforom is also associated with Behçet disease that results in inflammation of the blood vessels; the HLA-B51 being known as a high risk factor. [START_REF] Kirino | Genome-wide association analysis identifies new susceptibility loci for Behçet's disease and epistasis between HLA-B*51 and ERAP1[END_REF] ERAP2 is associated with Ankylosing Spondylitis, Birdshot chorioretinopathy which results in a uveitis, psoriasis and Crohn disease. [START_REF] Kuiper | A genome-wide association study identifies a functional ERAP2 haplotype associated with birdshot chorioretinopathy[END_REF], (44) Thus, inhibition of mutated ERAPs would reduce the production of the pro-inflammatory antigens and therefore restore a normal immune response.

III. ERAPs modulators

In order to develop ERAP modulators, two possibilities can be envisaged: use a compound that mimics the transition state or use a compound containing a zinc Binding Group (ZBG). Several groups have the capacity to coordinate this ion like as amide, carboxylic acid, hydroxamic acid, thiol, phosphinate or phosphonate. We will focus on the ZBG strategy here.

Although some molecules display modulation of ERAPs such as leucinethiol and bestatin, these can also modulate other zinc metalloenzymes, in particular other aminopeptidases, [START_REF] Wong | The X-ray crystal structure of human aminopeptidase N reveals a novel dimer and the basis for peptide processing[END_REF] and will therefore not to be discussed here.

So far, only two families of inhibitors have been reported in the literature: aminobenzamide and phosphinic compounds; both developed by Stratikos and colleagues. [START_REF] Papakyriakou | Novel selective inhibitors of aminopeptidases that generate antigenic peptides[END_REF], [START_REF] Zervoudi | Rationally designed inhibitor targeting antigen-trimming aminopeptidases enhances antigen presentation and cytotoxic T-cell responses[END_REF] 1. Aminobenzamide

As their name suggests, these compounds contain a central aminobenzamide core.

Regarding their binding mode (Figure 27), with the isoform ERAP1 here, the 2-amide group coordinates the zinc ion monodentally. The aniline interacts with a glutamate residue (Glu354), which participates in the polarization of a water molecule involved in the catalytic activity. The free amine strongly interacts with two glutamate residues (Glu 183 and 320) through an ionic interaction. Finally, the α-substitution (Pn' groups) of the 4-amide and of the 2-amide (P1 groups) allow the interaction with other pockets and therefore maximize the binding in the active site. Stratikos's team has previously explored the selectivity profile of the S1 pocket using a library of fluorogenic substrates. This study suggested that ERAP1 exhibits selectivity for long, aromatic or hydrophobic substrates, whereas ERAP2 displays higher selectivity for positively charged substrates. [START_REF] Zervoudi | Probing the S1 specificity pocket of the aminopeptidases that generate antigenic peptides[END_REF] Based on these analyses and docking studies, they introduced an L-homo-phenylalanine moiety in S1 and they explored the flexibility of the Pn' position to obtain an acceptable selectivity for the isoform 1 (Table 2). The L-lysine-containing compounds (carboxylic acid and methyl ester) are the most potent ERAP1 inhibitors (≈ 2 μM). Interestingly, the L-tryptophan benzyl ester is fully selective for isoform 2; no ERAP1 inhibition was recovered. However, these aminobenzamide compounds exhibit limited ERAP1/ERAP2 selectivity ratios (> 10-fold for ERAP1 with respect to ERAP2) and display moderate levels of activity, in the micromolar range. These limited activities can be explained by the fact that the zinc coordination by the amide group is monodendate and therefore weak.

Phosphinic pseudopeptides

The second family of ERAPs inhibitors are phosphinic pseudopeptides (Figure 28A)

and was developed by rational design. In order to attain an acceptable selectivity, they replaced the amide by a phosphinic group as a ZBG. Indeed, the phosphinic group is known as a weak ligand of the ion zinc, and therefore the binding affinity arises mainly from specific interactions between the side chains of the peptides and the active site specificity pockets of the enzyme. As seen previously, the L-homo-phenylalanine side chain interacts with the S1 pocket through a π-π stacking interaction between the phenyl and a phenylalanine residue (Phe-450). Introduction of an isobutyl group in β-position of the phosphinic allows for interactions with the small hydrophobic S1' pocket and introduction of a tryptophan moiety in C-terminal position enables a π-π stacking interaction between the indole and a tyrosine residue (Tyr-892) of the S2' pocket (Figure 28B). The phosphinic compounds (1A, 1B, 2A and 2B) display micromolar to nanomolar activities (Table 3). Introduction of a tryptophan residue on phosphinic 1A increases significantly the potency (phosphinic 2A, ≈ 16-fold for ERAP1, ≈ 50-fold for ERAP2). For compounds containing the tryptophan residue (phosphinic 2A and 2B), the stereogenic carbon bearing isobutyl group displays a high influence on the potency; the (S)-compound (phosphinic 2A)

being more potent than the (R)-compound (phosphinic 2B) (≈ 110-fold for ERAP1 and 155fold for ERAP2). Although exhibiting a significant increase in potency, the phosphinic compounds suffer from a lack of selectivity; the compounds demonstrate same activities on both isoforms. Additionally, they display a low bioavailability, which limits their uses in vivo. Although working cooperatively and being structurally quite close, the ERAP enzymes do not present the same properties in particular regarding the substrate cleavages and therefore have different biological functions. Thus, effective and selective inhibition and in particular of one isoform with respect to the other must be developed to explore the respective roles of each ERAP and therefore to better understand the biological phenomena attributable to ERAP1 and ERAP2.

Unfortunately, there are no promising pharmacological tools yet available as the published aminobenzamide and phosphonic compounds suffer from a lack of potency and/or selectivity.

In order to develop inhibitors of the ERAPs, Deprez-Poulain's team used HTS screening and rational design approaches. The HTS screening allowed the discovery of selective ERAP2 inhibitors, which bear a carboxylic acid as ZBG (Figure 29). [START_REF] Laura | Modulators of hERAP2 discovered by high-throughput screening[END_REF] Although providing a high selectivity for the isoform 2, these compounds display a limited potency with activities in the micromolar range. On the other hand, the team has also designed hydroxamate-bearing inhibitors of the ERAPs inhibitors by rational design. The hydroxamate function, known as a strong zinc ligand through a bidendate coordination, is notably present in potent and selective inhibitors of a large number of metalloenzymes like matrix metalloproteinases, cyclooxygenases or HDACs; [START_REF] Gupta | QSAR studies on hydroxamic acids: a fascinating family of chemicals with a wide spectrum of activities[END_REF] vorinostat and panobinostat being two approved HDAC inhibitors containing this ZBG. Interestingly, the designed hydroxamates for the ERAPs inhibition by Deprez-Poulain's team display different selectivity profiles. Indeed, the team has developed equipotent inhibitors of both isoforms, inhibitors of both isoforms with preference for one with respect to the other and selective ERAP1 inhibitors; both with submicromolar activities. Unfortunately, they do not have potent selective ERAP2 inhibitors so far.

IV. Objectives and strategy

In this specific area, the goal of this work is to use KTGS to identify and optimize potent (submicromolar) and selective (in particular towards ERAP1 and IRAP) ERAP2 inhibitors that will serve to decipher ERAP2 role in cancer and autoimmune disease and as starting points for drug discovery (Figure 30).

Results

I. Design and results of KTGS experiment to identify ERAP2 inhibitors 1. Design and synthesis of the starting building blocks

To identify new ERAP2 inhibitors, we chose to apply the KTGS strategy using the Huisgen [1,3] dipolar cycloaddition. Our six azides were from the U1177 laboratory's azide library and were derived from β-amino acids (Figure 31), which contained a hydroxamate allowing us to place the ligands in the active site using zinc coordination. The alkynes were from the U1177 laboratory's alkyne library and brought the chemical diversity.

Protein-templated reaction and controls

Regarding the assay, we used ERAP2 at 1.02 μM, the azides and the alkynes at 100.0 μM. The experiment was performed in 96-wells plate, each well containing one or two azides for ten alkynes (Z1 + Z3, Z2 + Z5 and Z4, Z6).

Two control experiments were done, one without the enzyme buffer ("blank experiment") and the other with copper in place of the enzyme solution ("chemical catalysis experiment"). The blank experiment did not contain the enzyme and allowed us to identify the unspecific couplings during the protein-templated reaction and the chemical catalysis experiment allowed us to afford the 1,4-disubstutited 1,2,3-triazole controls.

In order to validate the hits, a UPLC-MS-MS analysis using a multi MRM-method was performed after three and seven days. The hits were thus identified in each cluster by mass and retention time and compared to both incubation with buffer in place of the enzyme (blank plate) and synthetically prepared triazoles (chemical catalysis plate).

Hits selection

The MS analysis led to the identification of forty six hits. An additional analysis by LC-HRMS using a time-of-flight method was performed to search for the exact mass of the expected triazoles and therefore refine the hit selection. Thus, this second HRMS analysis allowed us to confirm nineteen hits and three of these were prioritized for the chemical synthesis; these hits bearing a phenylthiophene motif (Figure 32). As the MS analysis showed perfect mass and retention time correlations between the protein-templated and copper experiments for the selected hits, we supposed that the enzyme templated the formation of the 1,4-regioisomers. Thus, we started with the chemical synthesis of the 1,4-disubstituted 1,2,3triazoles.

Figure 32: Chemical structures of the selected hits for the chemical synthesis

To synthesize these 1,4-triazoles (Hit 1-3), a copper-catalyzed click reaction was performed between the starting azides and the corresponding alkynes. Given that, I did not take part in the synthesis of these 1,4-triazoles; their chemical synthesis will not be presented and discussed here.

Biochemical evaluation of selected hits

A-Principle of the enzymatic assay

The enzymatic activity of our ERAP inhibitors was determined using fluorogenic peptide substrates. [START_REF] Harris | Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries[END_REF] L-Leucine-7-amino-4-methylcoumarine hydrochloride (L-AMC) was used for ERAP1 enzymatic activity, whereas L-Arginine-7-amino-4-methylcoumarin hydrochloride (R-AMC) was used for ERAP2 enzymatic activity. Under the action of the ERAPs, these peptides are cleaved, which releases the fluorescent AMC part (Scheme 4).

Hit E3 Hit E2 Hit E1

Enzyme inhibition leads to reduced substrate cleavage and consequently to reduced fluorescence.

B-Biochemical evaluation

Before starting the KTGS experiment, the inhibitory activity of starting azides ERAP2 activities were determined (Table 4). Only two azides (Z4 and Z6) have shown a low level of inhibition: IC 50 = 59.0 and 64.0 μM, respectively; the others having no activity.

Table 4 : Biochemical activities of the starting azides Z1-Z6

Concerning the selected, even if the aim of the KTGS was to discover new ERAP2 inhibitors, their activities on the isoform ERAP1 were also measured (Table 5).

.HCl

ERAP1

Fluorescent AMC

.HCl

ERAP2

Fluorescent AMC A. The selected hits showed IC 50 values in the micromolar range. Surprisingly, no ERAP2 inhibition was observed with hit E2, whereas activities were found for hit E1 and E3, which have the same sulfonamide moiety as hit E2 (IC 50 = 3.0 and 2.3 μM, respectively).

B.

Compound

Interestingly, no activity on ERAP1 was observed for each compound, which means the KTGS has afforded selectivity for ERAP2.

Conclusions

Although most of our warhead-bearing reagents (azide) displayed no ERAP2 activity (Table 4), the KTGS strategy allowed us to identify templated ligands with a selectivity for ERAP2 (no activity on ERAP1) and low micromolar activity, making them promising hits.

Thus, these results constitute good starting points and SARs have been carried out.

II. Preliminary Structure -Activity Relationships

After being interested in the 1,4-regioisomers, we then synthesized the 1,5-triazoles to know if they would display activity values in the same range as the other regioisomers.

1. 1,2,Compared to the 1,4-triazole, the access to the 1,5-regioisomer is difficult; that is why only the β-tryptophan and β-tyrosine analogs were synthesized. The β-napthylalanine analog was not synthesized for solubility reasons; we supposed that its solubility would be lower than the others and could be a problem for the future.

A-Chemical synthesis

The 1,5-triazoles were obtained by a succession of seven steps (Scheme 5): diazo transfer (a), amide coupling using propargylamine (b), intramolecular Huisgen cycloaddition (c) [START_REF] Balducci | Microwave-assisted intramolecular Huisgen cycloaddition of azido alkynes derived from alpha-amino acids[END_REF] , lactam hydrolysis (d) followed by esterification (e), sulfonamide formation by nucleophilic substitution (f) and aminolysis (g); the intramolecular Huisgen cycloaddition being the key step to form the 1,5-triazole by cycle constraint.

B-Biochemical evaluations

A "slight" increase of ERAP2 inhibition was observed with the 1,5-regioisomers (12) compared to the 1,4-: IC 50 = 0.8 vs 3.0 μM. Unfortunately, no ERAP2 inhibition was detected for the beta-homo-tryptophane 1,5-triazole analog ( 13) by analogy to the 1,4-regioisomer.

1: R1= -(S)-phenol (92%) 2: R1= -(R)-3-1H-indole (95%) b f 8: R1= -(S)-phenol (90%) 9: R1= -(R)-3-1H-indole (quant.) g 7: R1= -(S)-phenol (quant.) R1= -(R)-3-1H-indole (not isolated)
Interestingly, the selectivity for the isoform 2 was kept for the 1,5-triazole (Table 6).

C-Conclusion

Based on these results, the 1,5-disubstituted 1,2,3-triazole seems slightly more potent than the 1,4-analog. The β-tryptophan analog, in 1,5-configuration did not show activity as the 1,4-regioisomer.

Sulfonamide "linker"

After focusing on 1,5-triazoles, we replaced the sulfonamide to know if ERAP2 could support other linkers. N-methylsulfonamide, amide with a spacer methylene and Nmethylamide analogs were synthesized, using β-homo tyrosine. These compounds were synthesized in 1,4-configuration for chemical accessibilities reasons.

A-Chemical synthesis

The final compounds were synthesized in three steps (Scheme 6): alkyne formation

B-Biochemical evaluation

Regarding the sulfonamide linker (hit E1), its N-methylation (20) did not affect the activity significantly, whereas its replacement by amide (21) and N-methylamide (22) led to a loss of the activity. Surprisingly, an ERAP1 activity was retrieved with the amide linker (21).

The selectivity for ERAP2 was also maintained with the N-methylsufonamide linker (20) (Table 7).

C-Conclusions

Firstly, as the sulfonamide replacement by amide or N-methylamide leads to a loss of activity on ERAP2, and secondly, as its methylation decreases the solubility, the sulfonamide linker has been kept for the other modulations.

Homologation n-1: switch to α-amino acid series

The KTGS experiment was performed from β-amino acids. We therefore decided to switch to α-amino acids, in 1,4-and 1,5-configuration.

A-For 1,5-triazoles

 Chemical synthesis

The α-analog, from the L-tyrosine was synthesized using the route described in the Scheme 2 (Scheme 8). Due to lack of time, the tryptophan analog could not be synthesized.

 Biochemical evaluation

Deletion of the methylene group ( 28) was tolerated, but with a decrease in potency compared to the β-compound ( 12); ERAP2 selectivity was maintained in α-configuration (Table 8).

 Biochemical evaluation

In the D-tryptophan series, deletion of the methylene group did not restore the activity on ERAP2 (36), while it led to a four-fold gain in ERAP2 activity in the L-tyrosine (35) series: IC 50 = 0.8 vs 3.0 μM; ERAP2 selectivity being maintained in α-1,4 series (Table 9).

C-Conclusions

For the 1,5-triazoles, deletion of the methylene group is tolerated in the tyrosine series but decreases ERAP2 inhibition by a factor of 4 (12, IC 50 = 0.8 vs 3.3 μM for 28). Thus, the 1,5-disubstituted 1,2,3-triazole are more potent in β-configuration than in α-configuration (Table 10).

For the 1,4-triazoles, although deletion of the methylene group does not allow to retrieve ERAP2 inhibition in the D-tryptophan series, it is beneficial in the tyrosine series. Indeed, in the tyrosine series, this switch to α-configuration increases the ERAP2 activity by almost 4fold (35, IC 50 = 0.8 vs 3.0 μM for Hit E1). Thus, the 1,2,3-triazole 1,4-disubstituted are more potent in α-configuration than in β-configuration (Table 10).

The ERAP2 selectivity is kept in the α-series, as in β-series for the 1,4-and the 1,5-triazoles.

Thus, regarding these results, two families of ERAP2 inhibitors can be dinstinguished:

- Three parts on 22 were identified for the pharmacomodulations in this family (Figure 33): the configuration (yellow), the "south-part" corresponding to the phenythiophene group (blue), the "north-part" corresponding to the phenol group (green).

A-Configuration

To know if the configuration would be a key element for the activity and selectivity, the R-analog was synthesized from D-tyrosine.

 Biochemical evaluation

The switch to R-configuration did not affect the ERAP2 activity significantly: IC 50 for 40 = 1.3 vs 0.8 μM for 35 and it was the same for the selectivity: no hERAP1 activity was observed in R-configuration.

 Conclusions

No significant effects on the activity and selectivity were observed with the R-analog;

we decided to continue with the S-compound. The racemic mixture has to be synthesized for comparison.

B-South-part

After demonstrating that S-and R-configurations are equipotent, we continued with the modification of the phenylthiophene moiety and synthesized six corresponding analogs.

 Chemical synthesis

Before to start the synthesis of the final analogs, the alkynes needed for the triazole formation were synthesized using a nucleophilic acyl substitution by propargylamine on the corresponding sulfonyl chlorides (Scheme 9, step c), as described previously (Scheme 11).

Scheme 11 : Synthesis of alkynes 41-46

The final compounds were synthesized using a copper-catalyzed click reaction and aminolysis (Scheme 12), as also described previously.

Reagents and conditions: (a) alkyne (41-46), CuSO 4 (5H 2 O), sodium ascorbate, DMF/H

 Biochemical evaluation

First, the replacement of the phenyl by 5-(2-pyridine) did not have an influence on the activity (56, IC 50 = 0.5 μM). Next, its replacement by 5-methyl (55), 5-or 3-bromine (53 and 54) led to a decrease in the IC 50 ; 2 to 6-fold drop compared to 35. Surprisingly, the phenyl removal (57) proved to be in the same range of activity as the initial compound 35 (IC 50 = 0.6 vs 0.8 μM). Then, based on this result, the 2-thiophene was replaced by 3-thiophene (58) reducing the activity by a factor of six. In each case of active compound, the ERAP2 selectivity was kept (Table 11). -5-bromo-2-thiophene (70%) 42: R= -3-bromo-2-thiophene (89%) 43: R= -5-methyl-2-thiophene (94%) 44: R= -5-(2-pyridine)-2-thiophene (70%) 45: R= -2-thiophene (78%) 46

41: R=

: R= -3-thiophene (87%) DIPEA CH 2 Cl 2 53: R= -5-bromo-2-thiophene (38%) 54: R= -3-bromo-2-thiophene (51%) 55: R= -5-methyl-2-thiophene (59%) 56: R= -5-(2-pyridine)-2-thiophene (63%) 57: R= -2-thiophene (70%) 58: R= -3-thiophene (38%) b 47: R= -5-bromo-2-thiophene (91%) 48: R= -3-bromo-2-thiophene (72%) 49: R= -5-methyl-2-thiophene (78%) 50: R= -5-(2-pyridine)-2-thiophene (66%) 51: R= -2-thiophene (86%) 52: R= -3-thiophene (62%) a -47 -

 Conclusions

The phenyl replacement did not allow us to increase significantly the potency; 56 and 57 displaying activity values in the same range as 35.

C-Northern part

After changing the southern part, we explored the chemical space around the phenol moiety by hydroxyl substitutions: O-benzyl, O-tert-butyl and O-methyl.

 Chemical synthesis

The needed methyl ester azides for the triazole formation were synthesized according to the routes described on Scheme 9, from commercial tyrosine analogs (Scheme 13). Unfortunately, in case of the tert-butyl derivative, we observed de-tert-butylation during the esterification; that is why we decided to perform the diazo transfer directly from the azide to get the needed methyl ester azide (63, 50% yield).

The final compounds were synthesized in two steps (Scheme 14) using the routes described previously.

Reagents and conditions: a) alkyne (32 or 44), CuSO In each case, interestingly, no ERAP1 activity was observed (Table 12).

 Conclusions

The O-substitution, in particular with non-bulky groups, is beneficial for the activity and, noteworthy, did not affect the selectivity for the isoform 2. However, it drastically decreases the solubility (Table 13). Interestingly, the compounds are stable in plasma and display intermediate microsomal stability values in mouse. 2. Family 2: 1,5-disubstituted 1,2,3-triazoles in β-configuration

After studying the α-1,4-family, we continued with the exploration of the β-1,5-family.

A focused SAR from the β-tyrosine compound (12, IC 50 = 0.8 μM) was performed, in particular on the phenylthiophene moiety.

A-Chemical synthesis

The final analogs were obtained using the routes described earlier in Scheme 2 using a nucleophilic acyl substitution and aminolysis (Scheme 15).

B-Biochemical evaluation

Surprisingly, contrary to the α-1,4-family, the phenyl removal ( 74) was deleterious for the activity (IC 50 > 100 μM). As the phenyl replacement by 2-pyridyl was beneficial for the IC 50 in α-series, we reproduced the same replacement (75), which did not improve the activity here (IC 50 = 1.2 vs 0.8 μM for 12) (Table 14).

IV. Selectivity-profile studies

One compound of each family was selected for a selectivity profiling (Table 15). Each compound has been tested against eight other zinc metalloproteases included ERAP1, IRAP (Insulin-Regulated Aminopeptidase), APN (Aminopeptidase N), TACE and the MMPs -

V.

Binding mode study

To elucidate the binding mode of our compounds in the active site of ERAP2, cocrystallizations were performed with 56 (1.4-triazole) and 12 (1.5-triazole) by Prof. Stratikos in Athens; their chemical structures are presented below together with their IC 50 values (Figure 34). Concerning the binding of 56 in ERAP2, we observed a surprising folding of 56 in the active site, which can probably be explained by the flexibility provided by the methylene spacer between the triazole and the nitrogen of the sulfonamide (Figure 35). For the 1,5-triazole 12, unsurprisingly, the binding of 12 in ERAP2 is totally different as for 56 (Figure 37). -54 -

We also observed a fold of the compound in the active site allowing an interaction between the triazole and the thiophene of 12, and, between the phenol and the phenyl of 12. The phenol moiety interacts with a tyrosine residue through a π-π interaction. Additionally, the phenyl interacts with a tryptophan residue (Trp363) through a π-π interaction also (Figure 38).

Conclusion & perspectives

Thus, the use of the KTGS approach allowed us to discover two new families of selective ERAP2 inhibitors, one with nanomolar activities (α-1,4 family) and the other with submicromolar activities (β-1,5 family); these compounds are the first selective ERAP2 inhibitors with submicromolar activities.

Strikingly, the triazole formation allowed us to increase the activity by 20-fold and 70-fold in 1,4-and 1,5-substitution respectively compared to the starting azide (Z4); the 1,5-regioisomer being more potent than the 1,4. Hit E1 and 12 exhibited a LLE higher than 3 and thus represented promising starting points for the hit optimization step. SAR studies, in the α-1,4

family, led to a high increase in the potency (IC 50 = 3.0 μM for hit E1 vs 0.004 μM for 71)

especially through O-substitution of the phenol and increasing therefore the LLE (3.2 vs 7.1) (Table 16).

Concerning the β-1,5 family, due to a lack of time, only a limited number of compounds were synthesized and they displayed higher IC 50 values than the initial hit (12). Further SAR studies are required to continue the exploration and in particular to enhance the selectivity profile of our compounds in both families.

In α-1,4 family, as the O-substitution drastically decreases the solubility (11 μM), polar groups must be introduced to solve this problem.

In β-1,5 family, more analogs have to be synthesized in order to refine the selectivity profile of this family. We have to continue the exploration about the thiophene substitution and the R-compound has to be synthesized to judge its impact on the activity and the selectivity.

Moreover, the linker sulfonamide has not yet been explored and thus can be replaced through N-methylsulfonamide, amide or N-methylamide linkers as we did in 1,4-configuration.

Finally, for each family, if the hydroxamic acid becomes a problem for the selectivity in the future, it is possible to replace it by other ZBGs such as sulfamate, boronate or phosphonate. According to the European Centre for Disease Prevention and Control (ECDC), AMR is defined as "the inability or reduced ability of an antimicrobial agent to inhibit the growth of a bacterium, which, in the case of a pathogenic organism, can lead to therapy failure". [START_REF]The European Union Summary Report on Antimicrobial Resistance in zoonotic and indicator bacteria from humans, animals and food in 2018/2019[END_REF] Based on the ECDC reports, in 2019, AMR was responsible for more than 670,000 infections and 33,000 deaths in Europe (EU). [START_REF]Antimicrobial resistance in the EU/EEA (EARS-Net) -Annual Epidemiological Report 2019[END_REF] The associated cost to the health care systems was around 1.1 billion euros. [START_REF]Antimicrobial resistance in the EU/EEA (EARS-Net) -Annual Epidemiological Report for[END_REF] The World Health Organization (WHO) indicates that 10 million deaths can be expected by 2050 as a direct consequence of AMR (Figure 39). Within the scope of this PhD work, we focused on P. aeruginosa.

I. Pseudomonas aeruginosa and resistance

P. aeruginosa is an opportunistic Gram-negative bacterium, which has remarkable host persistence and virulence. P. aeruginosa infections are the leading cause of morbidity and mortality in cystic fibrosis (CF) patients and one of the leading causes of nosocomial infections. [START_REF] Moradali | Pseudomonas aeruginosa Lifestyle: A Paradigm for Adaptation, Survival, and Persistence[END_REF] Its ability to adopt a biofilm growth mode, in which the bacterium can survive the antimicrobial agents, makes its elimination even more difficult and therefore leads to the emergence of resistant strains.

Based on the ECDC study, in 2019, 30 countries in Europe have reported 20,356 isolates of P.

aeruginosa. The AMR results are reported in (Table 17) according to their antimicrobial susceptibility testing (AST). The highest EU population-weighted mean resistance percentage was reported for fluoroquinolones (18.9%), followed by piperacillin + tazobactam (16.9%), carbapenems (16.5%), ceftazidime (14.3%) and aminoglycosides (11.5%). The authors also observed that the resistance to two or more antimicrobial groups was frequent and seen in around 18.0% of all tested isolates. Important inter-country variations were observed for all antimicrobial groups; with higher resistance percentages in Southern and Eastern Europe (Figure 40). AMR is conducive to the appearance of bacterial superinfections and to the installation of chronicity. For P. aeruginosa, this phenomenon of chronic bacterial infections is commonly encountered in CF patients because this bacterium has preference for the respiratory tract. [START_REF] Ramphal | Why is Pseudomonas the colonizer and why does it persist?[END_REF] In Europe and the US, P. aeruginosa infections are among the most common in CF patients.

In Europe, 29.1% of people with CF had chronic P. aeruginosa infection in 2017. (75) In US, 27.7% of people with CF were chronically infected with this bacterium in 2019. [START_REF]Cystic Fibrosis Foundation Patient Registry Annual Report[END_REF] II.

P. aeruginosa and virulence

As mentioned previously, apart from its resistance, this bacterium has remarkable virulence.

The virulence allows bacteria to cause host damage, to adapt to hostile situations and to evade the host immune system leading to their persistence within its host. [START_REF] Balasubramanian | A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence[END_REF] Most of virulence factors are under the control of quorum sensing (QS). QS is a communication system, which is based on the diffusion of small molecules and depends on the bacterial density and various environmental signals. These small molecules are essential because, based on their quantities; they lead to the activation or repression of genes and thus allow the bacteria to coordinate their behavior. [START_REF] Lasarre | Exploiting quorum sensing to confuse bacterial pathogens[END_REF] In P. aeruginosa, four pathways form the QS: LasI-LasR, RhlI-RhlR, PqsABCDE-PqsR and AmbCDE-IqsR. Each pathway contains an enzyme with synthase activity which, depending P. aeruginosa is known for its capacity to produce numerous virulence factors, which enable the bacteria to promote their survival by playing specific roles in the pathogenesis (Figure 42). Thus, by their broad scope of harmful actions, these mechanisms appear as attractive drug targets to fight P. aeruginosa, with the idea to "disarm" bacteria rather than killing them.

In my PhD work, we focused on LasB, which is the most abundant protein in the P.

aeruginosa secretome and one of the major virulence factors due to its capacity to hydrolyze a broad spectrum of substrates. As shown previously, the regulation of LasB expression is under the control of the QS system. Located on top of P. aeruginosa QS, LasR is able to activate the three other systems and could represent a promising target. However, these systems communicate and have the capacity to activate each other and, thus, can counteract LasR inhibition. For example, LasB production can be done by a second direct pathway: the RhlR-RhlI network (Figure 41). In addition, other QS systems can indirectly influence LasB expression such as IqsR and PqsR, IqsR regulating PqsR, which, in turn, regulates RhlR (Figure 41).

III. P. aeruginosa elastase

Activity and biological role

A. Structure

Belonging to the M4 group, LasB is structurally close to thermolysin (Figure 6), with a difference in the active site; it is more open for LasB with a preference for hydrophobic amino acids in P1' position of the substrate. Its secondary structure consists of two antiparallel βsheets and seven α-helices. The active site is formed by the connection of two α-helices and a loop, which connects them. Key actor of the catalytic activity, the zinc ion is tetrahedrally coordinated by the side chain of two histidines (His 140 and His 144 ) and a glutamic acid (Glu 164 ); the remaining coordination site is being occupied by the catalytic water molecule (Figure 44). ( 1)

B. Catalytic mechanism

Regarding the catalytic mechanism (Figure 45), the carboxylic group of the glutamic acid binds to the hydrolytic water molecule by hydrogen-bonding interactions and activates it (A), facilitating its nucleophilic attack on the substrate amide group (B). This attack leads to the formation of an oxyanionic intermediate, which is stabilized by zinc coordination (C).

Subsequent to a proton rearrangement, this intermediate is degraded (D), which regenerates the ground-state enzyme and liberates the amine and carboxylic acid from the amide bond, leading to substrate inactivation €. Using this mechanism, this enzyme cleaves a large variety of host as well as bacterial substrates.

C. Substrates cleaved by LasB

 Host substrates

Concerning the host substrates cleaved by LasB, we can classify them in three categories: structural components, components of the immune system and others.

 Structural components

LasB has the ability to cleave three major components of the extracellular matrix (ECM): elastin, collagens III-IV and laminin with various consequences for the host. In fact, elastin destruction leads to the loss of tissue integrity, releasing elastin fragments able to inhibit phagocytosis. [START_REF] Yang | Mechanistic insights into elastin degradation by pseudolysin, the major virulence factor of the opportunistic pathogen Pseudomonas aeruginosa[END_REF] Moreover, collagen and laminin degradation also takes part in the loss of tissue integrity by ECM lysis. [START_REF] Heck | Degradation of soluble laminin and depletion of tissue-associated basement membrane laminin by Pseudomonas aeruginosa elastase and alkaline protease[END_REF] Apart from ECM components, this enzyme can also damage the uPar-vitronectin complex and the VE-cadherin which, respectively, participate in cell adherence and tight-junction establishment. The detachment-induced cell apoptosis (anoikis) is a result of the loss of cell adherence while the degradation of cadherins increases the epithelial permeability. [START_REF] Beaufort | The thermolysinlike metalloproteinase and virulence factor LasB from pathogenic Pseudomonas aeruginosa induces anoikis of human vascular cells[END_REF], [START_REF] Beaufort | Disruption of the endothelial barrier by proteases from the bacterial pathogen Pseudomonas aeruginosa: implication of matrilysis and receptor cleavage[END_REF] Thus, by damaging structural components, LasB promotes the invasion and the colonization. LasB is able to neutralize certain immunoglobulins such as IgA and IgGs, [START_REF] Diebel | Cleavage of SIgA by gram negative respiratory pathogens enhance neutrophil inflammatory potential[END_REF], [START_REF] Krylov | Pseudomonas bacteriophage contains an inner body in its capsid[END_REF] reducing the opsonization and, thus, the bacterial phagocytosis. It is also capable of impairing the cellmediated immunity (TNF-α, IFN-γ) and the cellular immune response (CD4). ( 87), [START_REF] Pedersen | Selective modulation of the CD4 molecular complex by Pseudomonas aeruginosa alkaline protease and elastase[END_REF] Moreover, it participates in the inflammatory terrain installation by activating the IL-1β

interleukin, an early proinflammatory molecule. [START_REF] Sun | The Pseudomonas aeruginosa protease LasB directly activates IL-1β[END_REF] Other interleukins can be inactivated by LasB such as IL-2, IL-6, and IL-8 leading, respectively, to inhibition of lymphocyte proliferation, inactivation of epithelial cell repair and disruption of neutrophil recruitment to inflammatory sites (neutrophil chemotactism). ( 90), [START_REF] Matheson | Interaction of a novel form of Pseudomonas aeruginosa alkaline protease (aeruginolysin) with interleukin-6 and interleukin-8[END_REF] Regarding the chemotactism, the elastase is able to cleave epithelial chemokines such as MPC-1, ENA-78 and RANTES. [START_REF] Leidal | Metalloproteases from Pseudomonas aeruginosa degrade human RANTES, MCP-1, and ENA-78[END_REF] Another ability of this enzyme is to target antimicrobial substrates such as the complement system (C1, C3, C5, C8 and C9), [START_REF] Schultz | Elastase of Pseudomonas aeruginosa: inactivation of complement components and complement-derived chemotactic and phagocytic factors[END_REF] the pulmonary surfactant collectins SP-A/SP-D, [START_REF] Kuang | Pseudomonas aeruginosa elastase provides an escape from phagocytosis by degrading the pulmonary surfactant protein-A[END_REF] , (95) and the LL-37 peptide. [START_REF] Schmidtchen | Proteinases of common pathogenic bacteria degrade and inactivate the antibacterial peptide LL-37[END_REF] Finally, LasB can impair the human neutrophil elastase (HNE), by targeting the α1antiprotease, which normally fights against bacterial virulence. [START_REF] Morihara | Protease and elastase of Pseudomonas aeruginosa: inactivation of human plasma alpha 1-proteinase inhibitor[END_REF] Thus, by targeting various immune components, LasB leads to immune evasion.

 Others

Among the other substrates, we find blood components including the transferrin, lactoferrin and hemoglobin, whose degradation is responsible for a release of iron and a generation of cytotoxic radicals. ( 98),( 99),( 100) Coagulation factors such as the fibrin-fibrinogenthrombin triad can also be degraded leading to hemorrhages (fibrin and fibrinogen) and inflammation (thrombin). ( 101),(102) While the inflammation and the hemorrhages are deleterious for the host, the iron acquisition is favorable for bacterial growth.

Then, some matrix metalloproteinases are targeted by LasB (MMP-1, -2, -3, and -9), causing ECM and α1-antiprotease degradation. (103)  Bacterial substrates

In addition to hydrolysis of host substrates, LasB plays an essential role in the cleavage and the activation of bacterial components. It can activate the ExoS-ExoT exotoxins and the flagellin which, respectively, increases the invasion of epithelial cells and decreases the recognition of the flagellum by the immune system. ( 104),( 105) Furthermore, it is involved in biolfilm formation by activating the NdK kinase; Ndk activation being essential for the initiation of alginate synthesis and the expression of a mucoid phenotype. (106) In conclusion, regarding the deleterious consequences of the LasB actions, this enzyme plays a crucial role in the pathogenesis of P. aeruginosa by disrupting the host response.

In order to establish a causal effect of the LasB elastase activity on the virulence, various studies on animal models of P. aeruginosa infection were performed.

Impact of LasB on virulence in animal models of P. aeruginosa infection

Gi and colleagues demonstrated that a lasB mutant was avirulent in a model of acute lung infection in mouse. (107) In this study, the mice were separated in three groups: mice infected with the lasB mutant and treated with a saline solution (ΔlasB, saline), mice infected with the P. aeruginosa PAO1 strain and treated with a saline solution (PAO1, saline) and mice infected with PAO1 and treated with a broad-spectrum metalloprotease inhibitor such as the DTPA (diethylenetriaminepentaacetic acid) (PAO1, Ca-DTPA).

The mice from the ΔlasB group treated with a saline solution remained viable during five days whereas the mice from the PAO1 group treated with a saline solution died within the five days (Figure 46A). Moreover, the mice from the PAO1 group treated with the DTPA showed a significantly higher survival rate than saline-treated mice (PAO1, saline) (Figure 46A); demonstrating that inhibition of the enzyme LasB can lead to mouse survival. Lung sections for mice infected with the lasB mutant showed normal lung architecture after hematoxylin and eosin staining (Figure 46B). In contrast, an infiltration of inflammatory cells was visible for mice infected with the PAO1 strain and not receiving DTPA. This inflammatory response was less visible in mice treated with DTPA (Figure 46B). Cigana and colleagues studied the role of LasB in a model of chronic pulmonary P.

aeruginosa infection using clinical isolates from CF patients (PAO1, RP45 and RP73). (108) Interestingly, the RP45 isolate is found in seventeen-year old and the RP73 is found at twenty-four-year old patients.

Firstly, they investigated the elastase activity in different groups: wild-type PAO1 (wt PAO1)

and ΔlasB PAO1, wt RP45 and ΔlasB RP45, wt RP73 and ΔlasB RP73, and a Leria-Bertani growth medium used as a negative control (LB). The RP45 isolate showed a strong elastolytic activity, whereas RP73 showed no activity despite the presence of the LasB enzyme (Figure 47). This absence of elastolytic activity for the RP73 isolate is consistent with the P.

aeruginosa patho-adaptation previously observed in the development of chronic infection in CF; (109) RP73 clearly does not have a functional LasB enzyme. As shown, not all lasB mutants (ΔlasB) showed elastolytic activity (Figure 47). Secondly, they wanted to test the hypothesis that LasB promotes lung colonization during chronic P. aeruginosa infections. For the RP45 strain, at day two after the infection, the lung bacterial load did not differ between wt RP45 and ΔlasB RP45 whereas, at day seven, the ΔlasB RP45 mice had significantly lower lung bacterial loads than wt RP45 (Figure 48A).

This reduced lung bacterial loads are a consequence of the less virulent ΔlasB RP45 strain, making clearance by the host easier in comparison to the wt RP45 strain (Figure 48B).

Regarding the later isolated RP73 strain, the lung bacterial load was the same as ΔlasB RP73

after seven days (Figure 48C). No difference in the incidence of colonization was observed between the two groups (Figure 48D); probably due to the absence of elastolytic activity for RP73 strains as seen previously. Finally, they investigated the role of LasB in inflammation. Levels of chemokines, cytokines, and some growth factors were measured seven days after infection for wt and ΔlasB RP45 strains (Table 18). The absence of LasB (ΔlasB RP45) led to significantly higher levels of IL-1α, IL-1β, IL-3, IL-12p70, IL-17A, eotaxin, G-CSF, macrophagic inflammatory proteins MIP-1α and 1β, RANTES. This indicates that LasB can reduce inflammation. To summarize, in this study, the authors demonstrated that:

-The production and the activity of LasB differ depending on the strain, these being important during earlier stages of the infection (RP45 strain)

-LasB promotes chronic lung colonization because the lung bacterial load was lower in the ΔlasB RP45 strain after seven days -LasB is able to decrease the inflammation, which can lead to immune evasion So, this enzyme plays an essential role in the establishment of a chronic context at the early stage of the infection.

Thus, through different studies, a causal effect between LasB and virulence has been demonstrated. Moreover, this enzyme has an extracellular localization. All these arguments make it a promising therapeutic target.

Therapeutical solutions to inhibit LasB

The main strategy to inhibit LasB is to target the zinc cation in its active site using a zinc binding group (ZBG). Various ZBGs are used and will be presented below.

A-Phosphoramidon and analogs

Phosphoramidon (IL1) is the first reported inhibitor of LasB. It is a natural dipeptide isolated from Streptomyces tanashiensis with LasB K i = 1.0 μM. [START_REF] Everett | Pseudomonas aeruginosa elastase (LasB) as a therapeutic target[END_REF] Powers and colleagues used this as a starting point for further medicinal-chemistry optimization. (110) Tryptophan replacement by a phenylalanine and removal of the glycosidic moiety led to a compound with a K i = 0.2 μM (IL2), demonstrating that the glycosidic moiety was not essential for the activity (Figure 49).

K i (LasB) = 1.0 μM IL1 IL2 K i (LasB) = 0.2 μM

B-Thiol inhibitors

Later, Kessler and colleagues replaced the phosphoramidate group by a thiol as an alternative ZBG and reversed the phenylalanine and the leucine moiety leading IL3 with K i = 0.2 μM (Figure 50). (111) Then, Cathcart and colleagues investigated the carboxylic acid in the C-terminal position and replaced it by an amide. ( 112),(113) 1,600 dipeptides were synthesized with K i values of 1.0 to 0.04 μM (IL4 and IL5) (Figure 50). These inhibitors are not built around a dipeptide skeleton; these are thiobenzamides (IL6) and N-aryl-3-mercaptosuccinimides (IL7) with IC 50 = 6.6 and 3.5 μM, respectively (Figure 51). ( 114),( 115) Unfortunately, these thiols lack potency and some of them are sensitive to oxidation. In order to solve these problems, the thiol moiety was replaced by other ZBGs such as hydroxamic acids.

C-Hydroxamic acid inhibitors

Taking inspiration from phosphoramidon and its derivatives, Dickens and colleagues designed inhibitors bearing a hydroxamic acid function as ZBG, including one with a K i value of 0.05 μM (IL8). (116) In Prof. Hartmann's team, one hydroxamic acid was synthesized. Based on the chemical structure of thiols developed previously (IL 6 and IL7), this hydroxamic acid

IL3 K i (LasB) = 0.04 μM K i (LasB) = 1.0 μM K i (LasB) = 0.2 μM

IL4 IL5

IC 50 (LasB) = 6.6 μM IC 50 (LasB) = 3.5 μM

IL7 IL6

is presented on a malonic skeleton (IL9). (117) Hydroxamate avoids the risk of oxidation observed with some thiols but it suffers from a lack of selectivity.

D-Carboxylic acid inhibitors

Carboxylic acid is another ZBG reported in the literature. A family of carboxylic acids was identified by virtual screening (IL10) and, following structure-activity relationship studies (SAR), the activity was improved by a factor of 100 (IL11) (Figure 53). ( 118)

E-Other inhibitors

A screening of a library of fragments containing different ZBGs was performed, and the results were followed by SAR studies to identify new thiopyridone and tropolone inhibitors with activities in the micromolar range (IL12 and IL13) (Figure 54). ( 119), (120) Galdino and colleagues showed that the copper complex of 1,10-phenanthroline-5,6-dione has a K i of 0.09 μM (IL14) (Figure 54). ( 121) -71 -Thus, many chemical classes have been described in the literature but no LasB inhibitor has reached the clinic due to chemical stability, selectivity or physicochemical properties issues so far. Despite these problems, these inhibitors are very good starting points for further medicinal-chemistry programs.

As seen previously, thiols and hydroxamic acids have been developed in Prof. Hirsch's and Hartmann's teams. While the thiols have been studied in detail and several subfamilies have been identified, (122) the hydroxamates have not been widely studied and their numbers are consequently limited.

IV. Strategy of the PhD project

In this specific area, as for ERAP2 project, the goal of this work is to use KTGS strategy to identify and optimize potent and selective LasB inhibitors (Figure 55).

Results

I. KTGS Las B 1. Design and synthesis of the starting building blocks

To identify new hydroxamic acid inhibitors, we chose to apply the KTGS strategy using the Huisgen [1,3] dipolar cycloaddition. The four azides (Figure 56), which bear a hydroxamate, allowed us to direct the ligands towards the active site using zinc coordination, while the alkynes introduce the chemical diversity. The alkynes come from the U1177 laboratory's alkyne library and the azides were designed specifically for this project. Regarding the design of the azides Z7 and Z8, we used the specificities of LasB for the cleavage of substrates. In fact, in a protease, there are sites and pockets for the peptide binding as described in Figure 57. The peptide cleavage takes place between S1 and S1', and at these positions, LasB has a glycine at P1 and leucine at P1' as preferences (Table 19). For these reasons, we decided to introduce an azide on the β-leucine (Z7) and on the leucine (Z8). Concerning the design of the azides Z9 and Z10, we took our inspiration from the hydroxamic acid inhibitor synthesized by Prof. Hartmann's team, which was described previously (IL9, Figure 52). For Z9, based on LasB's preferences for the substrate cleavage, we introduced an isobutyl group in the malonic position (α-position) and we put a methylazide in position 4 to explore the ligand-growing direction. For Z10, the azidobenzyl motif of Z9 was replaced by an ethyl chain for an enhanced flexibility.

The four azides were synthetized using more or less different synthetic pathways. The azide hydroxylamine (50% in water w/w), KCN (cat.), MeOH, rt., overnight, 51%.

Z7
Scheme 19 : Five-step synthesis of Z10

Protein-templated reaction and controls

In this assay, we used LasB at a concentration of 4.08 μM and the azides and the alkynes at 100 μM.

The experiment was performed in low-binding 96-wels plate, each well containing one azide and ten alkynes. As for ERAP2 KTGS, two experiment controls were done, one without the enzyme buffer ("blank experiment") and the other with copper in place of the enzyme solution ("chemical catalysis experiment"). The blank experiment did not contain the enzyme and allowed us to identify the unspecific couplings during the protein templated reaction and the chemical catalysis experiment allowed us to afford the 1,4-disubstutited 1,2,3-triazole controls.

In order to validate the hits, an UPLC-MS-MS analysis using a multi MRM-method was performed after three and six days. The hits were thus identified in each cluster by mass and retention time and compared to both incubation with buffer in place of the enzyme (blank plate) and synthetically prepared triazoles (chemical catalysis plate).

Hit selection

The MS analysis led to the identification of 264 hits. An additional analysis by LC-HRMS using a time of flight method was performed to search for the exact mass of the expected triazoles and therefore refine the hits selection. Thus, this second analysis allowed us to confirm 43 hits and ten of these were prioritized for the chemical synthesis; five from Z9 and Z10. The identified hits from Z7 and Z8 were chemically very close to the identified hits from the KTGS with ERAP2 and we did not therefore select those for intellectual property reasons.

Among the five selected hits from Z9, three were bearing a sulfonamide "linker" (Hit L1-L3), one was having a thiazolidine "linker" (Hit L4) and the last a phenol "linker" (Hit L5) (Figure 59). Concerning the chosen hits from Z10, three were with a sulfonamide function (Hit L6-L8), one with the same phenol "linker" as before (Hit L9) and the last one with an amide (Hit L10) (Figure 59). supposed that the enzyme templated the formation of the 1,4-regioisomers. Thus, we started with the chemical synthesis of the 1,4-disubstituted 1,2,3-triazoles.

A-Chemical synthesis of the selected hits from Z9

The first step consists of a monosaponification of the diethylmalonate to get the mono carboxylic acid (a). This one is activated using HBTU, in DMF with trimethylamine, to form the desired amide by reaction with the aniline (b). Then, an addition of hydrochloric acid (4N in dioxane) afforded the free amine (c) and this one reacts with the diazo transfer reagent to form the azide (d). During this step, transesterification was observed and the methyl ester was isolated. This azide engages in a copper-catalyzed [1,3] The alkynes were synthesized according to the routes described in Scheme 6. In case of the sulfonamide linkers, the alkynes (93-95) are formed by reacting propargylamine with the corresponding sulfonyl chlorides. Thiazolidine alkyne 99 was obtained in five reaction steps:

preparation of the Weinreb amide (96), the reduction to aldehyde (97), a Seyferth-Gilbert homologation (98), and finally, Boc removal followed by nucleophilic acyl substitution with B-Chemical synthesis of the selected hits from LasB Z4

Concerning the amide formation ( 84), the mono carboxylic acid is activated by a mixture of EDC/HOBt in dichloromethane with DIPEA.

Reagents and conditions : a) NaOH, EtOH/H -79 -

Unlike previously described for Hit L6-L8 and Hit L10, in case of Hit L9, the click reaction and the aminolysis were inverted (Scheme 23). In this way, we avoided the cleavage of the acetamide functional group, which took place when the aminolysis was used as the last step.

Reagents and conditions: a) aq. hydroxylamine (50% in water w/w), KCN (cat.), MeOH, rt., overnight, 86%; (b) 100, CuSO The alkynes necessary for the click reaction with the azide Z10 were synthesized according to the routes described in Scheme 9. For the sulfonamides (106-108), the reaction was performed using the same conditions as described previously. In case of the alkyne 109, we performed the coupling between the 4-pentynoic acid and tyramine (Scheme 9).

Scheme 24 : Synthesis of the alkynes for the click step (e)

Biochemical evaluations of selected hits

A-Principle of the enzymatic assay

To measure the enzymatic activity of our LasB inhibitors, we use a FRET-based proteolytic assay. In this assay, active LasB cleaves a quenched substrate, which produces an increase of fluorescence. (123) Enzyme inhibition leads to reduced substrate cleavage and consequently to reduced fluorescence (Figure 60). Phosphoramidon, a known LasB inhibitor, is used as a positive control.

B-Biochemical results from the KTGS

The biological results for the previously described LasB hits are highly promising and they are reported in Table 20.

The hits derived from Z9 are particularly interesting, showing IC 50 values in the range of 2.4-14 nM (Hit L1-Hit L5). Surprisingly, the starting azide is quite potent itself, with an IC 50 of 17 nM. We assume that the potency of this azide comes from the introduction of the isobutyl group in the malonic position. It is difficult to judge the impact of the KTGS strategy only by looking at the IC 50 , values as for the most potent compound, it only led to a slight increase of the activity (17 nM to 2.4 nM, ≈ 7-fold). However, it is important to note that this strategy does not only improve the biological activity; it can also improve other parameters such as the selectivity profile of the hits or the ADME properties.

Concerning the hits derived from the starting azide Z10, the results are less promising,

showing the activities in micromolar range, e.g., IC 50 = 1.3 μM for Hit L8. Strangely, the starting azide is more potent than the obtained KTGS hits: 0.2 μM vs 1.3 μM (≈ 6.5-fold). We

LasB

suppose that the introduction of the ethyl linker caused the flexibility of the hit structures, which turned out to be detrimental for the binding to the active site. Therefore, we decided to focus our efforts on the hits obtained from the azide Z9 and especially to further optimize the Hit L1 using conventional medicinal-chemistry strategies.

II. SAR studies

Six parts on the LasB hit 1 were identified for the pharmacomodulation (Figure 61

The amide

Concerning this part, we modified the amide position and synthesized the analog with the meta-substitution on the phenyl ring. The synthesis followed the same pathway described previously for Z9 (Scheme 25).

Reagents and conditions : a) tert-butyl N-[(3-aminophenyl)methyl]carbamate, HBTU, Et

Scheme 25 : Synthesis of the meta-analog 114

This modification did not affect the activity significantly, with compound 114 showing an IC 50 value = 9.37 ± 0.27 nM. However, this derivative proved to be chemically less stable during the last step of the synthesis, with observed degradation leading to the release of the free aniline (Figure 62). This cleavage was also observed sometimes during the synthesis of the initial KTGS hits, but less pronounced. Therefore, we decided to keep the amide in para-position to the triazole moiety.

The linker part

Concerning the linker part, we synthesized ten analogs of the Hit L1 to explore the chemical space and determine which linker is most favorable for the activity. We replaced the sulfonamide linker with a N-methylsulfonamide, a cyclic sulfonamide, an amide, a carbamate, a free amine, an aniline, a ether a thioether and we also simplified it by directly linking the triazole to a benzyl or a phenyl.

A-Chemical synthesis

The alkynes needed for the synthesis of the final compounds were synthesized as described previously (Scheme 24). For the N-methylsulfonamide alkyne (115) and the amide alkyne (116), we used the same reaction as before: nucleophilic acyl substitution of Nmethylpropargylamine and propargylamine on the desired sulfonylchloride and acyl chloride, respectively (Scheme 26, A and B). In case of, to get the cyclic sulfonamide alkyne (117), we used the reaction of the saccharin and propargylbromide (Scheme 26C). Concerning the aniline, the ether and the thioether (118-120), the corresponding aniline, ether and thioether reacted with the propargylbromide to afford the desired alkynes (Scheme 26D). Finally, to simplify the linker, we formed the alkynes 121 and 122 by a Seyferth-Gilbert homologation (Scheme 26E). For the saccharin derivative, we reversed the click reaction and the aminolysis step to prevent the cleavage between the nitrogen and the sulfur of the sulfonamide (Scheme 13).

Unfortunately, the yield of the click reaction was very low. We can explain this by the difficulties encountered during the purification; it was difficult to separate the starting azide and the final compound. Finally, the free amine 140 was obtained by Boc-deprotection from the carbamate 133 using hydrogen chloride in dioxane (Scheme 29).

B-Biochemical evaluation

First, we wanted to know if the secondary sulfonamide is essential for the activity, and therefore, we synthesized compound 131 with an N-methyl sulfonamide linker. This modification did not affect the activity (IC 50 = 2.3 ± 0.1 nM). Then, we replaced the sulfonamide by a cyclic sulfonamide and amide, which led to a decrease of the IC 50 by a factor of 10 (139 and 132). Introduction of the free amine with an aim to form stronger ionic interactions with the active site proved to be detrimental for the activity of the compound 140

(IC 50 = 55.9 ± 2.3 µM, 23-fold drop compared to Hit L1) (Table 21).

The carbamate intermediate ( 133) was also tested, showing better activity compared to the free amine analog (IC 50 = 9.8 ± 0.4 nM). Comparable activity was observed for the aniline (134) or ether (135) Surprisingly, the introduction of a thioether linker demonstrated a significant increase in activity compared to NH-and O-analogs; the compound 136 proved to be even more active than the Hit L1 (IC 50 = 1.4 ± 0.1 nM). Finally, we simplified the linker by directly connecting the triazole with a benzyl (137) or a phenyl (138) group. These modifications decreased the activities compared to the Hit L1 by factors of eight and sixteen, respectively (Table 21).

C-Conclusions

The compounds 131 and 136 looked equally promising as the initial Hit L1 and, for this reason; we decided to do the further profiling by exploring other properties such as the solubility and the metabolic stability (Table 22). Firstly, compared to Hit L1, the introduction of a tertiary sulfonamide decreases the solubility (131) and does not change significantly the metabolic stability. More importantly, the derivative with the thioether linker (136) has a poor metabolic stability in mouse microsomes (t 1/2 = 3.3 ± 0.2 min). Therefore, the sulfonamide linker was kept for the other modulations.

The nature of the sulfonamide

After demonstrating that the sulfonamide linker is the most promising, we continued with the modification of the para-iodophenyl moiety and synthesized ten corresponding analogs.

A-Chemical synthesis

The alkynes needed for the triazole formation were synthesized using a nucleophilic acyl substitution by propargylamine on the corresponding sulfonyl chlorides (Scheme 30), as described previously (Scheme 21).

Scheme 30 : Synthesis of alkynes 141-149

The final compounds were synthesized using a copper-catalyzed click reaction and aminolysis (Scheme 31), as described previously (Scheme 20 and Scheme 27). Next, we decided to modify the electronic density of the phenyl ring by introduction of an additional chlorine substituent in position 3 (165), which also did not have an influence on the activity. Finally, we continued our investigations by increasing the chain length (166), removing the substituent (167) and replacing the aromatic moiety by a cyclohexyl (168) and an isobutyl (169). In each case no improvement of the IC 50 value was observed (Table 23). As a conclusion, the nature of the substituent on the sulfonamide linker has no significant impact on the activity and a broad list of substituents is tolerated in this part of the structure. We decided to carry out further investigations, in particular regarding the ADME properties. We selected the compounds 160, 161, 165 and 169 and measured their solubility and the metabolic stabilities in mouse and human liver microsomes, as well as calculated the corresponding cLogP values (Table 24).

The replacement of 4-iodine in Hit L1 by 4-chlorine (161) increased the solubility without affecting the metabolic stability. The introduction of an additional chlorine in position 3 (165) decreased the solubility as well as the metabolic stability by a factor of three. At the same time, we managed to improve the solubility and the metabolic stability by replacing the 4iodobenzene by 4-fluorobenzene (160) and isobutyl group (169). In fact, the metabolic stability increased 3-and 4-fold for the 4-fluoro and the isobutyl compounds, respectively, being now in a satisfactory range. The metabolic stability in mouse seems to be nicely correlated with the lipophilicity, based on the calculated cLogP values (Table 24). After this small SAR study around the nature of the sulfonamide, we moved to the investigation of the malonic position.

The malonic position

For the design of the starting azides for the KTGS, we assumed that the introduction of an isobutyl chain in α-position will have a positive impact on the activity; based on the LasB preferences for the substrate cleavage and known SAR of Hirsch's team. (122) This αsubstitution could also be a key element to control the selectivity. So, the aim of this study was to verify this hypothesis. We synthesized the derivatives with no α-side chain, with an isopropyl, methylcyclopropyl and benzyl group.

A-Chemical synthesis

For the synthesis of the mentioned analogs, the synthetic pathway had to be slightly adjusted due to the known reactivity of the malonic position, particularly during the diazo transfer. The diazo transfer was performed during the first step with a quantitative yield (170).

This was followed by Boc deprotection (171) and amide formation using an acyl chloride (172) or a carboxylic acid (173-175). Finally, the last two steps were the click reaction and the aminolysis to get the final compounds without substituent (178) or with a benzyl (179) in the malonic position (Scheme 32).

Reagents and conditions : a) azide-N-diazoimidazole-1-sulfonamide hydrogen sulfate, K Unfortunately, in case of the isopropyl derivative, we observed deiodination when the aminolysis was used as the last step (Figure 63). We decided not to continue with this route due to the difficult purification and low yields.

Scheme 33 : Chemical synthesis of final compounds 182 and 183 by inverting the click reaction and the aminolysis steps

The carboxylic acids needed for the amide formation were synthesized using a monosaponification of the malonic diester (Scheme 34).

Scheme 34 : Formation of the mono carboxylic acids 173a-175a

B-Biochemical evaluation

Results of the derivatives with the variations in the malonic position are presented in compound bearing the smaller isopropyl substituent (182) proved to be in the same range of activity as the initial Hit L1 (IC 50 = 1.9 nM vs 2.4 nM).

C-Conclusions

We have demonstrated that the substitution on the malonic position is essential for the activity. However, only four compounds were synthesized. Therefore, more analogs must be explored to extend our scope of investigation and thus establish if the substitution would have an impact on the selectivity profile.

1,5-Triazole regioisomer synthesis

As presented previously, during the KTGS, the enzyme can catalyze the formation of the 1,4-disubstituted or the 1,5-disubstituted 1,2,3-triazole (or both regioisomers). For the selected hits, we supposed that LasB catalyzed the formation of the 1,4-regioisomers. In order to compare the two regioisomers, we synthesized the corresponding 1,5-triazoles.

A-Chemical synthesis

To access the 1,5-regioisomer, ruthenium-catalyzed click reaction was used. (124) For accessibility reasons, and as the carbamate 133 showed a good activity on LasB (IC 50 = 9.8 ± 0.4 nM), we first decided to perform the ruthenium-catalyzed click reaction using N-Bocpropargylamine as an alkyne. During the click reaction, only the 1,5-regioisomer formation was observed and therefore the compound was isolated in a good yield. This reaction was followed by aminolysis to give the final compound 185. 184 was engaged in a succession of

B-Biochemical evaluation

Regarding the carbamate linker (133 and 185), no significant difference in the activity was observed between the 1,4-triazole 133 and the 1,5-triazole 185 (IC 50 = 9.8 nM vs 6.5 nM, respectively). The observations were the same for the sulfonamide linkers (190-192) although the 1,4-triazoles (Hit L1, 160 and 169) display "slightly" higher activity values. As for the 1,4-triazoles, the nature of the sulfonamide did not have a significant impact on the potency; the three 1,5-triazoles displaying the same IC 50 (190-192, IC 50 ≈ 7-8 nM) (Table 26).

C-Conclusion

In order to extend our comparison study, the synthesis of 1,5-disubstituted 1,2,3triazoles with other linkers is planned.

The ZBG

In addition to the variety of previously discussed modifications, to get the full picture around the chemical space of the KTGS hits, we investigated the replacement of the hydroxamic acid with alternative ZBGs. In the literature, some cases of zinc coordination by sulfonamides were reported. (125) As our compounds contain both hydroxamic acid and a sulfonamide group, we wanted to check whether the hydroxamic acid is responsible for the coordination of the zinc cation. To verify our hypothesis, the ethyl ester 150 was tested.

Secondly, as the hydroxamic acid is known for its selectivity-related issues, it was replaced by other ZBGs such as carboxylic acid and phosphonate.

A-Chemical synthesis

The carboxylic acid was obtained by saponification of the ethyl ester using sodium hydroxide (Scheme 36A). The phosphonate was synthesized in three steps: amide formation, click reaction and ester deprotection (Scheme 36B).

B-Biochemical evaluation

As expected, the ethyl ester (150) as well as the corresponding carboxylic acid (193) did not show any activity on LasB (IC 50 > 100 μM). On the other hand, replacement of the hydroxamic acid with a phosphonate (196) resulted in the compound showing the activity in nM range, however, still being 20-fold less active than the corresponding hydroxamate derivative (IC 50 = 75.6 ± 2.6 nM).

C-Conclusions

The hydroxamate replacement by ethyl ester led to a loss of activity, which means that the zinc coordination is carried out by the hydroxamic acid and not by the sulfonamide.

Known for its poor selectivity, the hydroxamic was then replaced by carboxylic acid or phosphonate but these two ZBG did not show good activities on LasB. Therefore, we can conclude that the hydroxamate is essential for the potency. The toxicity and selectivity studies have been presented below.

III. Toxicity and selectivity profile studies

Four hydroxamic acid derivatives were selected for the selectivity and toxicity profiling: Hit L1, 160, 169, and Z9. Their chemical structures are shown below together with their biological activities on LasB (Figure 64). While identical profiles would be expected for Hit L1 and 160, 169 has been included to determine the influence of the sulfonamide nature on the selectivity, the toxicity and the antibacterial activity on P. aeruginosa. The starting Z9 has been included to rationalize the use of KTGS for the synthesis of more complex structures.

Each compound has been tested against seven other zinc metalloproteases including the MMPs -1, -2 and -3, the HDACs -3 and -8, TACE and COX-1. The cytotoxicity has been measured on three different cell-lines: HepG2, HEK293 and A549. The antibacterial activity has been measured using P. aeruginosa PA-14 strain. The results are reported in the table below. To our great pleasure, the profile of the KTGS hit (Hit L1) looks very promising: no offtarget effects, no cytotoxicity and no antibacterial activity. The analysis is the same for 160 and 169. So, it would seem that the nature of the sulfonamide does not have an impact on these parameters; but this has to be confirmed with the evaluation of more compounds.

Additionally, 160 and 169 have also been tested against the MMPs 7, 8 and 14 without demonstrating residual activities on them (IC 50 > 100 μM).

Although the selectivity profile of Z9 looks promising, the starting azide displays a micromolar activity value on TACE that is not found for Hit L1 (KTGS templated ligand).

Thus, the KTGS strategy allowed us to improve the selectivity profile.

IV. Binding mode study of hydroxamic acid inhibitor-LasB

To elucidate the binding mode of our compounds in the active site of LasB, cocrystallization was performed with 161 (resolution 2.4Å); its structure is presented below together with its IC 50 value (Figure 65). With this folding, the pocket occupancy looks extremely good and offers an explanation for the compound's excellent potency and probably explains the good selectivity profile.

Conclusion & Perspectives

For all the reasons mentioned, the use of the KTGS strategy allowed us to discover two new families of LasB inhibitors, one with nanomolar activities (family from Z9: Hit L1

to Hit L5) and the other with micromolar activities (family from Z10: Hit L6 to Hit L10).

Regarding, the compounds from Z10, we supposed that the ethyl linker probably brings a lot of flexibility which could be negative for the binding in the active site. We decided thus to stay focus on the compounds from Z9 which displayed nanomolar activity values.

Regarding the most potent hit from Hit L1, although the starting azide Z9 was surprisingly potent, a seven-fold gain in the activity was observed with the most potent KTGS hit (Hit L1) (17 nM vs 2.4 nM). This gain in the activity comes from the triazole formation (orange part) which maximizes the pocket occupancy and therefore explains the increase of the LLE. The decrease of the LE can be explained by the smaller size of the azide (Table 28). In Hit L1, we identified six parts used for the pharmacomodulation: the amide, the sulfonamide linker, the nature of this sulfonamide, the malonic position, the triazole and the ZBG.

Hit L1 has a perfect profile: nanomolar potency, no off-target effects, no cytotoxicity and no antibacterial activity. Strikingly, compared to Z9, Hit L1 does not display residual activity on TACE. The SAR studies performed on this compound did not lead to a significant increase of the IC 50 value but allowed us to improve the physicochemical properties such as the solubility and the metabolic stability. promising lead compounds. For this reason, we selected these compounds for in vivo pharmacokinetic studies and further biological evaluation. We need to investigate their activities in the model systems closer to reality, as for example, in presence of pulmonary surfactant or in the Galleria mellonella infection model.

Co-crystallization of 161 (4-chlorobenzene sulfonamide) with LasB was performed to elucidate the binding mode of the KTGS inhibitors. The complex structure demonstrated perfect pocket occupancy, presumably due to the two methylene spacers in the linker. These two CH 2 groups are responsible for the "folding" of the compound, which leads to an optimal occupancy of the pocket. This co-crystallization sets the stage for further structure-based medicinal-chemistry optimization of the initial hits.

Concerning the future modifications, first we plan to modify the two methylene spacers stated above to verify their real impact on the activity. Then, since only four analogs were synthesized with regard to the malonic position; we plan to extend the scope of possible substituents. Regarding the nature of the sulfonamide, we can introduce heterocycles (imidazole, pyridine, thiophene…). Finally, based on the compound folding, we envisage that the synthesis of macrocycles would reduce the flexibility while improving the other parameters (potency, affinity, selectivity…). All mentioned modifications are schematically presented on Figure 67. On the other side, the 1,5-regioisomer formation is more difficult. Few methods to access to the 1,5-disubstituted 1,2,3-triazoles have been reported, and for most of them with limited scope.

Step D H + Step C H + Step A

Step B

Step E -

[CuL n ] +
.

 Metal-catalyzed synthesis of 1,5-disubstituted 1,2,3-triazoles

In most cases, azide-alkyne cycloadditions either mediated by metal acetylide or metal catalysts are used. As metal catalysts, the use of ruthenium complexes is commonly encountered and was reported by Sharpless et al. (RuAAc). ( 127) Mechanistically (Scheme 38), the displacement of spectator ligands (step A) enables the formation of an activated complex which, through an oxidative coupling, is converted into a ruthenium-metallacycle (step B); the regioselectivity being controlled by this step. Then, a reductive elimination leads to the triazole formation (step C) that is finally released and the catalyst is regenerated (step D). However, most of these methodologies have restricted scopes. Moreover, most of them require the handling of isolated organic azides, which are not often commercially available and are known to be unstable, in particular those with a low molecular weight. (131)  Synthesis of 1,5-disubstituted 1,2,3-triazole through one pot reactions

To get around these drawbacks, multicomponent methodologies have been developed. ( 132),( 133),( 134),( 135),( 136),( 137) For example, a metal-and azide-free one-pot procedure using tosyl hydrazine (Scheme 42A) and, a one-pot procedure via metal, a RuAAC strategy from bromide was described (Scheme 42B) were described to produce 1,5-disubstituted 1,2,3triazoles.

Scheme 42 : Examples of one-pot strategies for the synthesis of 1,5-disubstituted 1,2,3-triazoles

Step C TsN 3

LiOtBu

Step A HOtBu

HOtBu

Step D

Step B

Step E

B. Sequential one-pot RuAAC

A. Azide-free one-pot cycloaddition using tosyl hydrazine 1.

2.

NaN 3

-104 -

These methods have the advantage to be safer by preventing the handling of organic azides.

Regrettably, they suffer from a much narrowed substrate scope, often requiring high temperatures, strict inert atmosphere conditions or an inconvenient sequential pattern, all of which have limited their use.

Thus, we investigated the feasibility to implement an efficient and safe one-pot, two-step method. This procedure would enable the in-situ conversion of bromides into azides by nucleophilic substitution with sodium azide. The azides react consecutively with alkynes by NiAAC (Scheme 43).

Results

I. Screening of reaction conditions

We started to screen different conditions for the reaction, using 4-tertbutylbenzylbromide (1a) and phenylacetylene (2A) as model substrates (Table 29). First, we performed the reaction using conditions, which are similar to those used in NiAAC reported by Kim et al.: bromide 1a (1.0 equiv), sodium azide (1.0 equiv), alkyne 2A (1.2 equiv), nickelocene (Cp 2 Ni) precatalyst (20 mol%), xantphos as ligand (20 mol%) and cesium carbonate (1.0 equiv) in water at room temperature for 2 hours. Using these conditions, conversion of the bromide 1a to azide was observed but not the cycloaddition (entry 1).

Then, in order to improve the triazole formation, we started to change the solvent. The reaction was first investigated with dimethylformamide (entry 2) because it showed efficacy in the NiAAC. Interestingly, it facilitated the triazole formation contrary to other solvents like dichloromethane, ethanol or tetrahydrofuran (entries 3-5).

After demonstrating that dimethylformamide was efficient for the reaction, we continued with the modification of the temperature. Heating the reaction mixture to 50°C under microwave irradiations for 1 hour (entry 6) resulted in a higher regioselectivity (88:12 to 93:7) but did not impact the yield (73 to 71%).

Increasing the temperature (80 or 100°C) was deleterious for the yield and the regioselectivity (entries 7 and 8), probably due to the catalyst degradation. Then, the time of the reaction was investigated. When the reaction vessel was heated under microwave irradiations for 4 h (entry 9), the yield increased (86 vs 73%) without harmful effect on the regioselectivity (ratio 92:8 vs 93:7). Finally, the bromide 1a and sodium azide were used as excess reagents (1.2 and 1.5 equiv respectively) (entry 10). Under those conditions, excellent yield (89%) and regioselectivity (ratio 92:8) were observed without the formation of by-products.

To investigate the mechanism of this one-pot reaction, 4-tert-butylbenzyl bromide 1a and sodium azide were mixed at room temperature for 2 h in DMF in presence of or without the Cp 2 Ni/Xantphos catalytic system. In both conditions, the azide was formed in quantitative yield. Thus, the Cp 2 Ni/Xantphos catalytic system has no influence on the SN2 reaction and only has beneficial effects for the azide-alkyne cycloaddition and its regioisomerism.

After optimizing the reaction conditions, we investigated the scope with various alkynes and bromides.

II. Substrate scope 1. With various alkynes

The use of aromatic (2A-2E) and aliphatic alkynes (2F-2H) in reaction with the bromide 1a was explored, and the results are reported above (Table 30). Except for alkynes 2D and 2E, the attempts to separate and isolate the two regioisomers were not productive by classical flash column chromatography, or by C-18 reversed-phase flash column chromatography with the use of classical or pentafluorophenyl columns. However, due to the high regioselectivity rates associated with this reaction (from ratio 66:34 to > 99:1), flash chromatography was still performed to afford the 1,5-triazole in mixture with the 1,4 in order to evaluate the efficiency of this one-pot multicomponent methodology.

Modest to excellent calculated yields ranging from 27% to 84% were obtained. Besides, with the aim of isolating and characterizing properly the 1,5-ring, the crude mixtures were also purified using semi-preparative supercritical fluid chromatography (SFC); with similar isolated yield (24% to 72%).

The regioselectivity of each reaction was measured by SFC analysis of the crude reaction mixtures obtained after the work-up of the reactions carried out with alkynes 2A-2E and 2H, or according to the 1 H-NMR spectra for reactions carried out with alkynes 2F and 2G, for which the separation of the two regioisomers by SFC was not optimal. Modest to excellent regioselectivities were observed. Given the regioselectivity obtained with the paranitrophenylacetylene 2B (ratio 66:34) compared to the phenylacetylene 2A (ratio 89:11), the para-fluorophenylacetylene 2C (ratio 91:9) and the para-methoxyphenylacetylene 2D (ratio > 99:1), a positive mesomeric effect on the phenyl moiety seems to have a positive contribution on the regioselectivity without impacting the yield. Interestingly, heteroaryl (2E) and alkyl (2F) chains are tolerated in this methodology. Hydroxyl (2G) and carbamates (H) groups are also compatible. Xantphos (20 mol%), Cs 2 CO 3 (1.0 equiv), DMF (2.0 mL), 50 °C (MW), 4 h. Isolated yields (%) of 3 obtained after SFC purification; 3/4 ratio determined from the crude product by SFC analysis unless noted otherwise; in brackets: calculated yields (%) of 3 obtained after flash column chromatography purification (in mixture with 4). b Isolated yield (%) of 3 obtained after flash column chromatography purification; c Determined by 1 H NMR. Boc, tert-butoxycarbonyl; Cp, cyclopentadienyl;

t-Bu, tert-butyl.

With various bromides

The use of aromatic benzyl bromides possessing either electron-donating (1a and 1b)

or electron-withdrawing (1c-1g) and non-saturated bromides (1i-1l) in reaction with the alkyne 2A was explored and the results are reported above (Table 31).

Concerning the aromatic benzyl bromide, modest to high yields both after flash chromatography purification (calculated yield: 64%-79%) and after semi-preparative SFC purification (isolated yield: 31%-84%) were obtained. The ortho-CF 3 bromide 1g afforded the triazole 3gA with the lowest yield in the series, probably due to steric effects. Besides, the position of the trifluoro group has demonstrated a minor influence on the yield without impacting the regioselectivity. Noteworthy, for these aromatic benzyl bromides, high to excellent regioselectivities were obtained (from 85:15 to 97:3 ratios). Regarding the nonsaturated bromide (1i-1l), they could be efficient in this one-pot methodology. Interestingly, functional groups such as carbamate-protected amine (1k) and ester (1l) are also welltolerated, affording the corresponding 1,5-regioisomers with high to excellent yields and regioselectivities. Moreover, no influence on enantiomerism for the triazole 3kA was observed.

Conclusion

Thus, we managed to establish a methodology to efficiently access diverse 1,5disubstituted 1,2,3-triazoles in a fast and simple manner, bypassing the encountered and known problems with the main reported procedures (Figure 68). (138) Remarkably, one of the advantages of this one-pot reaction is the in-situ generation of azides from bromides, which are commercially available with a broad diversity. Thus, this one-pot method avoids the sequential and time-consuming synthesis and isolation of azides, which could be unstable and are rarely commercially available. It makes this procedure very useful, in particular when sensitive low-molecular weight azides are needed. A diverse range of functional groups including, N-protected amines, hydroxyls and esters are tolerated in this procedure. This method will enable the synthesis of libraries of functionalizable 1,5regioisomers especially useful for drug discovery and chemical biology purposes.

Metal azide alkyne cycloaddition

Our work: one-pot NiAAC Sequential one-pot RuAAC Azide-free one-pot cycloaddition using tosyl hydrazine

General conclusion

The objectives of this work were to, first, identify new ligands of ERAP2 and LasB using the KTGS strategy and, secondly, to optimize the ligands into leads.

Regarding ERAP2, the KTGS strategy was initiated from 6 azides and 180 alkynes.

The azides derived from β-amino acids containing a hydroxamate, which allowed us to coordinate the zinc ion of ERAP2 and therefore place the ligands in the active site. The alkynes were from the U1177 alkyne library and imported the diversity on the ligands.

As for 85% of KTGS reports, mass spectrometry was used to identify the hits, allowing the identification of a micromolar compound (Hit E1, IC 50 = 3.0 μM). The SAR studies enabled us to improve the potency and led to the identification of two new selective and submicromolar ERAP2 inhibitor families: the α-1,4 and the β-1,5 families. Strikingly, these inhibitors display good ADME properties and toxicity and selectivity profiles (Figure 69). Concerning LasB, the KTGS experiment was performed from 4 azides and 190 alkynes. As for ERAP2, the azides bore a hydroxamate to coordinate the zinc ion of LasB and were designed from known SARs. The alkynes were also from the U1177 alkyne library and introduced the diversity as previously. Mass spectrometry was also used to identify the ligands. As the templated ligands from the azides Z7 and Z8 (β-leucine and leucine) were -111 -very structurally similar to those from the KTGS with ERAP2, we decided to stay focused on the hits from Z9 and Z10 (the two malonic derivatives).

Biological evaluation of the templated ligands from these two azides showed that the provided flexibility by the ethyl chain (Z4) was not very well tolerated, as the starting azide showed a higher IC 50 than the most potent hit. Even if, Z9 displayed surprisingly a high potency (17 nM), the KTGS led to a seven-fold gain in the activity for the most potent templated ligand (Ht L1, IC 50 = 2.4 nM).

Strikingly, LasB hit 1 has a promising profile: nanomolar potency, no off-target effects, no cytotoxicity and no antibacterial activity but demonstrates a limited solubility and metabolic stability. The SAR studies performed on this templated ligand did not lead to a significant increase in potency but allowed us to improve the solubility and the metabolic stability ( 142μM to > 200 μM for the solubility and 30 min to > 120 min for the metabolic stability). The most promising analogs 160 and 169 with enhanced physicochemical properties are also potent, selective, non-cytotoxic and devoid of antibacterial activity, which makes them promising compounds (Figure 70). Thus, these inhibitors have to be tested in model systems closer to reality such as in presence of pulmonary surfactant or in a Galleria mellonela infection model. Additionally, cocrystallization of 161 (4-chloro) with LasB was performed to elucidate the binding mode of the KTGS ligands and will help us to conduct further medchem optimization. Interestingly, it suggests the possibility of maximizing the pocket occupancy using a macrocycle. As explained previously, the design and the selection of the warhead-bearing reagents are a crucial step for a successful KTGS. Several methods can help the design of these reagents such as computational methods (docking, crystal structure), known SARs or approved drugs.

As we worked with zinc metalloenzymes, a ZBG (hydroxamate here) was introduced in our warheads to coordinate the zinc and therefore facilitate the binding of the templated ligands in the active sites. Noteworthy, besides providing diversity on the ligands, the alkynes can also bind protein pockets improving the pocket occupancy and therefore increasing the ligand potency. Thus, a large scope of reagents is also crucial to maximize the chances of success; more than 170 alkynes were used here thanks to the large library of the U1177's team.

Thus, we applied the KTGS strategy with success thanks to a good design of our warheadbearing reagents and the use of a large pool of reagents. Strikingly, the ERAP2 and LasB templated ligands display a LLE above 3 and comply with the Doak space (Table 32). Then, we focused on our second objective, which was to optimize the templated ligands as we improved their potency and their physicochemical properties; making them promising leads in the field of cancer and autoimmune diseases for ERAP2 and in the fight against AMR for LasB.

As perspectives, it would be interested to apply the DCC strategy with these two zinc metalloenzymes to compare the two protein-templated strategies. For example, the irreversible link through the triazole moiety could be replaced by a reversible link using an imine or an acylhydrazone (Scheme 44). Finally, additionally to the previous work, a one pot nickel-catalyzed methodology for the preparation of to 1,5-disubstituted 1,2,3-triazoles was developed (Scheme 45) and could be applied in the future for the synthesis of 1,5-regioisomers after a KTGS experiment using the Huisgen 1,3-dipolar cycloaddition.

General information

Solvents for synthesis, analysis and purification were purchased as analytical grade from commercial suppliers and used directly without further purification. Chemical reagents were purchased as reagent grade and used without further purification.

 For compounds of chapters 2 and 4

Syntheses under microwave irradiation were performed using Biotage® Initiator+.

NMR spectra were recorded on a Bruker DRX-300 spectrometer. The results were calibrated to signals from the solvent as an internal reference [e.g., 2.50 (residual DMSO-d 6 ) and 39.52 (DMSO-d 6)

ppm for 1 H and 13 C NMR spectra,respectively]. Chemical shifts (δ) are reported in parts per million (ppm). NMR coupling constants (J) are reported in Hertz (Hz), and splitting patterns are indicated as follows: (s) for singlet, (d) for doublet, (t) for triplet, (q) for quartet, (quin) for quintet, (sext) for sextuplet, (sept) for septuplet, (dd) for doublet of doublet, (ddd) for doublet of doublet of doublet, (dt)

for doublet of triplet, (qd) for quartet of doublet, (m) for multiplet, and (br) for broad, δ for chemical shift, J for coupling constant.

LC-MS Waters system was equipped with a 2747 sample manager, a 2695 separations module, a 2996 photodiode array detector (200-400 nm) and a Micromass ZQ2000 detector (scan 100-800). XBridge C 18 column (50 mm x 4.6 mm, 3.5 μm, Waters) was used. Purity (%) was determined by LC-MS, using UV detection (215 or 254 nm), and all isolated compounds showed purity greater than 95%.

HRMS analysis was performed on a LC-MS system equipped with a LCT Premier XE mass spectrometer (Waters), using a XBridge C 18 column (50 mm x 4.6 mm, 3.5 μm, Waters). Flash chromatography was performed using an Interchim Puriflash® 430 with silica columns. UV and ELSD detection were used to collect the desired product. Reverse flash chromatography was performed using a CombiFlash® C 18 Rf200 with C 18 silica gel cartridges. UV detection (215 and 254 nm) was used to collect the desired product.

 For compounds of the chapter 3

NMR spectra were recorded on a Bruker UltraShield Plus 500 MHz device. The results were calibrated and presented as before.

LC-MS analysis were measured on a LC-MS system, consisting of a Thermo Scientific Dionex UltiMate 3000 pump, autosampler, column compartment, and detector (Thermo Fisher Scientific, Dreieich, Germany) and ESI quadrupole MS (MSQ Plus or ISQ EC, Thermo Fisher Scientific, Dreieich, Germany). High-resolution mass was determined by LCMS/MS using Thermo Scientific Q Exactive Focus Orbitrap LC-MS/ MS system. Purity was determined as before. Flash chromatography was performed using either a Teledyne ISCO CombiFlash Rf+ 150 or a Teledyne ISCO CombiFlash NEXTGEN 300+ equipped with RediSepRf silica columns. Preparative HPLC was performed on a Thermo Scientific Dionex Ultimate 3000 system.

 For compounds of the chapter 4

The chromatographic system used for SFC was a SFC-PICLAB hybrid 10-20 apparatus (PIC Solution, where the used flow rate was 2 mL/min and the mobile phase was always CO2-modifier mixture with 20% of ethanol as modifier.

General procedures

General procedure A: Azide formation A suspension of amine (1 eq.), ZnCl 2 (0.06 eq.) and K 2 CO 3 (2-4 eq.) in anhydrous methanol (0.18-0.25 M) under inert atmosphere was cooled down to 0 °C with an ice-bath. Besides, anhydrous N,Ndiisopropylethylamine (1.1-2.1 eq.) was slowly added to a solution of 1H-imidazole-1-sulfonyl azide;

hydrogen sulfate or hydrogen chloride (1.2 eq.) solubilized in anhydrous methanol (0.3 M) under inert atmosphere (solution A). The azide-containing solution A was immediately added dropwise to the first mixture at 0 °C. Then, the cooling bath was removed and the white mixture was stirred at room temperature overnight. The mixture was then cooled down to 0 °C, diluted with water and carefully acidified to pH = 2 with diluted aq. HCl (1N). It was finally extracted with ethyl acetate. Combined organic layers were dried over MgSO 4 , filtered and concentrated under reduced pressure or the residue was finally purified through flash chromatography to give the desired azide.

General procedure B: Amide formation

Carboxylic acid (1 eq.) and amine (1.1-1.6 eq.) were solubilized in N,N-dimethylformamide (0.28-0.45M) or CH 2 Cl 2 (0.2 M). HBTU (1.1-1.7 eq.) or HOBt (0.1 eq.) and EDC . HCl (1.5 eq.), and trimethylamine or diisopropylethylamine (3-5 eq.) were then added and the mixture was stirred overnight. The mixture was then washed with diluted aq. HCl (1 M), sat. aq. NaHCO 3 and sat. aq.

NaCl. Combined organic layers were dried over MgSO 4 , filtered and concentrated under reduced pressure or the residue was finally purified through flash chromatography to give the desired amide.

General procedure C: Intramolecular click chemistry [START_REF] Balducci | Microwave-assisted intramolecular Huisgen cycloaddition of azido alkynes derived from alpha-amino acids[END_REF] The azido intermediate was dissolved in DMF (0.009-0.04 M) and refluxed overnight to allow cyclisation. After cooling down, the mixture was diluted in water and extracted with ethyl acetate.

Combined organic layers were dried over MgSO 4 , filtered and concentrated under reduced pressure.

The crude product was finally purified through flash chromatography to furnish the desired cyclic 1,5triazole.

General procedure D: Lactame hydrolysis [START_REF] Balducci | Microwave-assisted intramolecular Huisgen cycloaddition of azido alkynes derived from alpha-amino acids[END_REF] Pyrazinone or diazepinone 1,5-triazoles (1 eq.) was diluted in a 6 M aq. HCl solution (0.06-0.13 M) and heated under microwave irradiations at 85 °C for 1-3 hours. Then, solvents were evaporated under reduced pressure to give 1,5-triazoles as hydrochloric acid salts, which were used in the next step without further purification.

General procedure E: Esterification

Carboxylic acid (1 eq.) was dissolved in MeOH (0.1-0.5 M) and cooled down to 0 °C before thionyl chloride (2.0 eq.) was slowly added dropwise. The resulting solution was stirred overnight in the melting ice bath. Solvents were then evaporated under reduced pressure to give the desired products or residues were dissolved in a mixture of ethyl acetate and saturated aqueous NaHCO 3 and extracted twice. Combined organic layers were dried over MgSO 4 , filtered and concentrated under reduced pressure to afford desired esters.

General procedure F: Sulfonamide formation (4) The amine (1-1.5 eq.) was dissolved in N,N-dimethylformamide or dichloromethane (0.04-0.10 M) and cooled down to 0°C. R1-sulfonyl chloride (1-1.2 eq.) was then carefully added followed by N, Ndiisopropylethylamine (1.2-3 eq.). The mixture was allowed to reach room temperature and was stirred overnight. Then, the crude was diluted in aq. HCl (0.5 M) and extracted with ethyl acetate.

Combined organic layers were dried over MgSO 4 , filtered and concentrated under reduced pressure.

The residue was finally purified through flash chromatography to afford the desired sulfonamide.

General procedure G: Aminolysis

To an ester solution (1 eq.) in methanol (0.04-0.35M) was added aq. hydroxylamine (50% w/w in water, 0.04-0.35 M) and KCN (0.1-0.5 eq.). The mixture was stirred 16 to 72 h. Then, the solvents were removed under reduced pressure and the residue was purified through flash chromatography.

General procedure H: Copper-catalyzed click reaction

Azide (1 eq.) and alkyne (1.0-2.5 eq.) were dissolved in N, N-dimethylformamide (0.03-0.09 M) before an addition of copper sulfate pentahydrate (0.2-0.4 eq.) in water (0.05-0.09 M) and sodium ascorbate (0.5-1 eq.). The resulting mixture was stirred at room temperature overnight. The mixture was then diluted in water and extracted with ethyl acetate. Combined organic layers were dried over MgSO 4 , filtered and concentrated under reduced pressure. The residue was finally purified through flash chromatography affording the desired 1,4-triazole.

General procedure I: Monosaponification

Malonate diester (1 eq.) was dissolved in a mixture of methanol/water or ethanol/water (9:1-8:2, 0.32-0.58M) and sodium hydroxide (1.2 eq.) or potassium hydroxide (1.0 eq.) was added. The reaction mixture was stirred at room temperature overnight. Then, solvents were evaporated under reduced pressure and the aqueous mixture remaining was diluted with sat. aq. NaHCO 3 and washed with CH 2 Cl 2 . The aqueous layer was then carefully acidified (pH~1) with aq. HCl and extracted with CH 2 Cl 2 . Combined organic layers were dried over MgSO 4 , filtered and concentrated under reduced pressure affording the desired product.

General procedure J: Boc deprotection

The Boc-protected intermediate was dissolved in a mixture of ethanol and dichloromethane (1:1, 0.09-0.11M) and the reaction was cooled down to 0 °C before an addition of 4 N HCl in dioxane (0.18-0.22M). The mixture was stirred at room temperature overnight. Then, solvents were evaporated to give the desired compound.

General procedure K: One-pot methodology for 1,5-triazole

To a solution of bromide derivative (1.2 eq.) in N, N-dimethylformamide (0.25 M) were successively added sodium azide (1.5 eq.), alkyne (1.0 eq.), di(cyclopenta-1,3-dien-1-yl)nickel (20 mol%), xantphos (20 mol%) and cesium carbonate (1.0 eq). The brown mixture was heated under microwave irradiations (50 °C, 4 h). The dark purple mixture was filtered on a celite pad, diluted with water and extracted twice with ethyl acetate. The organic layer was dried over MgSO 4 , filtered and concentrated under reduced pressure. The residue was divided into two fractions unless noted otherwise. One fraction was purified through flash silica gel column (cHex to cHex/EtOAc: 8:2) and the other fraction was purified by SFC to afford the desired 1,5-disubstituted 1,2,3-triazole.

Compound 23 was synthesized according to the general procedure A, using L-tyrosine (226 mg, 1.25 3, 157.5, 131.4 (2C), 128.5, 116.3 (2C), 64.7, 37.8. 7.19-7.16 (m, 2H), 6.88-6.83 (m, 2H), 4.11 (dd, J = 8.4 and 5.2 Hz, 1H), 3.77 (s, 3H), 3.11 (dd, J = 14.1 and 5.2 Hz, 1H), 2.92 (dd, J = 14.1 and 8.4 Hz, 1H). 13 C NMR (75 MHz, MeOD-d 4 ) δ ppm: 173. 3, 160.2, 131.4 (2C), 129.8, 114.9 (2C), 64.7, 55.6, 37.7.

(
Compound 29 was synthesized according to the general procedure A, using D-tryptophan (255 mg, 7.12-7.07 (m, 1H), 7.04-6.99 (m, 1H) 4.19 (dd, J = 8.1 and 5.2 Hz, 1H), 3.34 (ddd, J = 14.5, 5.2 and 0.7 Hz, 1H) and 3.18 (ddd, J = 14.5, 8.1 and 0.4 Hz, 1H). 13 C NMR (75MHz, MeOD-d 4 ) δ ppm:

173.9, 138. 0, 128.6, 124.7, 122.4, 119.8, 119.1, 112.2, 110.5, 64.1, 28.7.

Methyl (3S)-3-azido-4-(4-hydroxyphenyl)butanoate (Z4-a)

Compound Z4-a was synthesized according to the general procedure E, using carboxylic acid (m, 1H). 13 C NMR (75MHz, MeOD-d 4 ) δ ppm: 173. 0, 157.4, 131.5 (2C), 129.3, 116.3 (2C), 62.4, 52.3, 40.7, 39.5. Hz, 1H), 2.90 (dd, J = 14.0 and 8.2 Hz, 1H). 13 C NMR (75 MHz, MeOD-d 4 ) δ ppm: 172. 2, 157.6, 131.3 (2C), 128.1, 116.3 (2C), 64.6, 52.9, 37.7. 1, 160.3, 131.4 (2C), 129.4, 114.9 (2C), 64.5, 55.6, 52.9, 37.7. 7.59 (m, 2H), 7.56 (d, J = 3.9 Hz, 1H), 7.46-7.38 (m, 3H), 7.29 (d, J = 3.9 Hz, 1H), 4.07 (d, J = 2.6 Hz, 2H), 2.94 (s, 3H), 2.15 (t, J = 2.5 Hz, 1H). 13 C NMR (75 MHz, CDCl 3 ) δ ppm: 151. 6, 135.5, 133.7, 132.8, 129.4 (2C), 129.3, 126.4 (2C), 123.3, 76.0, 74.4, 40.1, 34.7. 4, 152.0, 150.6, 143.6, 138.7, 134.1, 125.5, 124.7, 120.7, 79.3, 73.6, 33.4.

Methyl (2S

Methyl (2R)-

Methyl (2S)-

N-prop-

5-methyl-N-prop-2-ynyl-thiophene-2-sulfonamide (43)

Compound 43 was synthesized according to the general procedure F, using propargylamine (107 μL, (m, 4H). 13 C NMR ( 75MHz, MeOD-d4) δ ppm: 149. 0, 139.2, 133.8, 126.9, 79.4, 73.5, 33.3, 15.3.

5-bromo-N-prop-2-ynyl-thiophene-2-sulfonamide (41)

Compound 41 was synthesized according to the general procedure F, using propargylamine (81 μL, (dd, J = 14.5 and 8.3 Hz, 1H). 13 C NMR (75 MHz, MeOD-d 4 ) δ ppm: 175. 1, 157.7, 135.5, 131.9 (2C), 131.8, 127.6, 116.4 (2C), 59.8, 42.1, 36.1, 35.6. 7.51 (m, 1H), 7.36-7.32 (m, 1H), 7.12-6.99 (m, 3H), 5.13-5.05 (m, 1H) 4, 138.0, 135.5, 131.8, 128.7, 125.6, 122.6, 120.1, 119.1, 112.4, 109.6, 59.0, 36.6, 35.6, 33.1. 3.50-3.41 (m, 1H). 13 C NMR (75 MHz, DMF-d 7 ) δ ppm: 168.9, 157. 2, 150.5, 139.6, 135.3, 133.6, 133.0, 130.4 (2C), 130.5, 129.7 (2C), 129.5, 126.6, 126.4 (2C), 124.3, 115.6 (2C), 62.1, 52.8, 36.6, 36. Compound 12 was synthesized according to the general procedure D, using ester (m, 2H), 7.53 (d, J = 3.9 Hz, 1H), 7.48-7.40 (m, 4H), 7.37 (s, 1H), 6.71 (d, J = 8.6 Hz, 2H), 6.61 (d, J = 8.6 Hz, 2H), 4.97-4.91 (m, 1H), 3.97-3.82 (m, 2H), 3.14-2.97 (m, 3H), 2.84 (dd, J = 15.3 and 4.9 Hz, 1H). 13 C NMR (75 MHz, MeOD-d 4 ) δ ppm: 168. 7, 157.7, 152.4, 140.0, 136.3, 134.7, 134.0, 133.2, 131.1 (2C), 130.4 (2C), 130.2, 128.6, 127.2 (2C), 124.6, 116.5 (2C), 59.3, 41.9, 38.4, 36.5 (dd, J = 5.0 and 1.1 Hz, 1H), 7.56 (dd, J = 3.7 and 1.1 Hz, 1H), 7.32 (s, 1H), 7.16 (dd, J = 4.9 and 3.8 Hz, 1H), 6.72-6.70 (m, 2H), 6.62-6.60 (m, 2H), 4.88-4.84 (m, 1H), 3.77 (s, 2H), 3.14-2.99 (m, 3H), 2.84 (dd, J = 15.3 and 4.7 Hz, 1H). 13 C NMR (75 MHz, MeOD-d 4 ) δ ppm: 159. 2, 148.2, 132.3, 126.8, 124.2, 124.1, 123.6, 121.6 (2C), 119.3, 119.1, 107.0 (2C), 49.8, 32.4, 28.9, 27. 8, 156.1, 150.4, 150.3, 149.7, 141.0, 137.6, 133.7, 133.1, 131.9, 129.8 (2C), 126.8, 125.0, 123.9, 119.4, 115.2 (2C), 56.7, 40.2, 36.8, 35.4 7.75-7.71 (m, 2H), 7.54-7.29 (m, 8H), 7.06-7.00 (m, 1H), 6.95-6.90 (m, 1H), 6.87 (d, J = 2.3 Hz, 1H), 5.03-4.92 (m, 1H), 3.85 (d, J = 15.4 Hz, 1H), 3.71 (d, J = 15.5 Hz, 1H), 3.20-3.12 (m, 2H), 2.94-2.71 (m, 2H). 13 C NMR (75 MHz, DMSO-d 6 ) δ ppm: 166. 0, 149.5, 138.6, 136.0, 133.8, 133.1, 132.2, 131.9, 129.4 (2C), 129.2, 126.8, 126.0 (2C), 124.1, 123.8, 121.1, 118.6, 117.9, 111.5, 109.2, 56.0, 37.1, 35.5, 31. 7.4 Hz, 2H), 3.25-3.17 (m, 2H). 13 C NMR (DMSO-d 6 ) δ ppm: 163. 4, 156.1, 149.5, 139.0, 134.5, 133.0, 132.7, 132.1, 130.0 (2C), 129.4 (2C), 129.2, 126.1, 125.9 (2C), 124.1, 115.1 (2C), 60.9, 36.0, 35.6 0, 157.5, 152.5, 142.8, 136.9, 134.8, 133.9, 131.1 (2C), 130.4 (2C), 130.3, 128.3, 127.2 (2C), 125.6, 124.8, 116.3 (2C), 61.5, 52.4, 46.2, 41.6; 39.5, 35.2.

Methyl (3S)-4-(4-hydroxyphenyl)-3-[4-[[(5-phenylthiophene-2-carbonyl)amino]methyl]triazol-1yl]butanoate (18)

Compound 18 was synthesized according to the general procedure 3, 160.8, 156.1, 147.5, 144.5, 138.7, 133.1, 130.0 (2C), 129.3, 129.2 (2C), 128.5, 126.7, 125.7 (2C), 124.3, 122.3, 115.1 (2C), 58.9, 51.5, 40.0, 38.1, 34.6. 129.6, 129.3, 128.4, 128.3, 128.0, 127.0 (2C), 125.4, 124.3, 116.3 (2C), 65.2, 61.6, 52.4, 49.3, 41.7, 39.6.

Methyl (3S

Methyl (2S)-3-(4-hydroxyphenyl)-2-[4-[[(5-phenyl-2-thienyl)sulfonylamino]methyl]triazol-1yl]propanoate (33)

Compound 33 was synthesized according to the general procedure 149.7, 143.2, 142.0, 137.5, 132.6, 129.9 (2C), 125.6, 124.9, 123.8, 123.5, 119.3, 115.1 (2C), 63.3, 52.7, 38.3, 36.0.

Methyl (2S)-3-(4-hydroxyphenyl)-2-[4-[[(5-methyl-2-thienyl)sulfonylamino]methyl]triazol-1yl]propanoate (49)

Compound 49 was synthesized according to the general procedure 127.2, 127.0, 124.8, 116.4 (2C), 65.6, 53.5, 39.3, 38.1, 15.4. 5.72-5.67 (m, 1H), 4.12-4.10 (m, 2H), 3.68 (s, 3H), 3.44-3.28 (m, 2H). 13 C NMR (75 MHz, DMSOd 6 ) δ ppm: 168. 7, 156.2, 142.9, 142.3, 132.3, 131.3, 129.9 (2C), 125.6, 123.5, 118.2, 115.1 (2C), 63.3, 52.8, 38.2, 36.0.

Methyl (2S)-2-[4-[[(

Methyl (2S)-2-[4-[[(3-bromo-2-thienyl)sulfonylamino]methyl]triazol-1-yl]-3-(4hydroxyphenyl)propanoate (48)

Compound 48 was synthesized according to the general procedure 5.72-5.67 (m, 1H), 4.08 (d, J = 5.9 Hz, 2H), 3.67 (s, 3H), 3.44-3.28 (m, 2H). 13 C NMR (75 MHz, DMSO-d 6 ) δ ppm: 168. 8, 156.2, 143.2, 141.1, 132.5, 131.7, 129.9 (2C), 127.7, 125.7, 123.5, 115.1 (2C), 63.3, 52.8, 38.2, 36.0.

Methyl (2S)-3-(4-hydroxyphenyl)-2-[4-[(3-thienylsulfonylamino)methyl]triazol-1-yl]propanoate (52)

Compound 52 was synthesized according to the general procedure 1, 157.6, 145.3, 141.5, 131.6, 131.1 (2C), 129.5, 127.2, 126.4, 124.8, 116.4 (2C), 65.6, 53.5, 39.1, 38.0.

Methyl (2S)-3-(4-methoxyphenyl)-2-[4-[[[5-(2-pyridyl)-2-thienyl]sulfonylamino]methyl]triazol-1yl]propanoate (67)

Compound 67 was synthesized according to the general procedure 9, 149.0, 143.1, 139.5, 135.9, 132.7, 132.2, 129.3 (2C), 129.1, 126.7, 125.9 (2C), 123.9, 123.8, 123.4, 121.1, 118.6, 117.9, 111.5, 108.0, 62.5, 52.8, 38.3, 27.3.

(3S)-4-(4-Hydroxyphenyl)-3-[4-[[methyl-[(5-phenyl-2-thienyl)sulfonyl]amino]methyl]triazol-1yl]butanehydroxamic acid (20)

Compound 20 was synthesized according to the general procedure G, using ester 17 (69 mg, 0.13 mmol), KCN (0.9 mg, 0.01 mmol) and aq. NH 150.0, 140.4, 134.7, 133.8, 132.0, 129.8, 129.4, 129.3, 126.8, 126.0, 124.5, 124.0, 115.1, 59.5, 45.0, 40.1, 37.0, 34.4 163.8, 156.2, 149.0, 143.2, 139.7, 132.8, 132.2, 129.9 (2C), 129.3 (2C), 129.1, 125.9 (2C), 125.7, 124.0, 122.2, 115.1 (2C), 62.0, 38.3, 36.7 156.2, 143.4, 136.8, 132.6, 132.3, 130.0 (2C), 125.7, 121.9, 115.2 (2C), 112.6, 61.9, 38.1, 36.8. 7, 158.1, 150.4, 150.0, 149.7, 143.2, 142.1, 137.5, 132.6, 130.0 (2C), 127.5, 124.9, 123.7, 122.2, 119.3, 113.7 (2C), 61.9, 59.9, 38.3, 36.6 163.7, 153.9, 149.1, 143.3, 139.7, 132.8, 132.2, 130.2, 129.5 (2C), 129.4 (2C), 129.1, 125.9 (2C), 124.0, 123.4 (2C), 122.2, 77.8, 61.7, 38.3, 36.8, 28.5 (3C). HRMS-ESI+ (m/z) calcd. for C 26 H (d, J = 7.7 Hz, 1H), 7.54-7.39 (m, 5H), 7.31 (d, J = 7.7 Hz, 1H), 7.09-7.04 (m, 1H), 7.09-6.96 (m, 3H), 5.40-5.35 (m, 1H), 4,16 (s, 2H), 3.47 (dd, J = 14.4 and 7.4 Hz, 1H), 3.36 (dd, J = 14.4 and 8.4 Hz, 1H). 13 C NMR (75 MHz, DMSO-d 6 ) δ ppm: 164. 2, 149.0, 143.1, 139.6, 136.0, 132.9, 132.3, 129.4 (2C), 126.9, 125.9, 124.0 (2C), 123.9, 122.1, 121.1, 118.5, 118.3, 111.4, 108.0, 60.9, 38.3, 20.0 NMR (500 MHz, DMSO-d 6 ) δ ppm: 7.68 (t, J = 5.8 Hz, 1H), 7.46-7.45 (m, 2H), 7.42-7.40 (m, 2H), 4.40 (s, 2H), 3.79 (dd, J = 5.8 and 2.4 Hz, 2H), 3.34 (t, J = 2.4 Hz, 1H). 13 C NMR (126 MHz, DMSOd 6 ) δ ppm: 133. 0, 132.7 (2C), 129.2, 128.4 (2C), 80.5, 74.8, 57.0, 31.8.

HRMS

4-Iodo-N-prop-2-ynyl-benzenesulfonamide (93)

Compound 93 was synthesized according to the general procedure F, using propargylamine (70 μL, 7.74-7.71 (m, 2H), 7.11-7.08 (m, 2H), 3.83 (s, 3H), 3.64 (d, J = 2.5 Hz, 2H), 3.04 (t, J = 2.5 Hz, 1H). 13 C NMR (126 MHz, DMSO-d 6 ) δ ppm: 162.0, 132.1, 128.9 (2C), 114.2 (2C), 79.5, 74.6, 55.6, 31.9.

4-Methyl-N-prop-2-ynyl-benzenesulfonamide (107)

Compound 107 was synthesized according to the general procedure F, using propargylamine (111 μL, 1.73 mmol), 4-methylbenzenesulfonyl chloride (300 mg, 1.57 mmol) and N, N-diisopropylethylamine (548 μL, 3.15 mmol)

4-Propyl-N-prop-2-ynyl-benzenesulfonamide (108)

Compound 108 was synthesized according to the general procedure F, using propargylamine (97 μL, 3, 137.8, 129.0 (2C), 126.8 (2C), 79.4, 74.6, 37.0, 31.9, 23.8, 13.5.

4-Phenyl-N-prop-2-ynyl-benzenesulfonamide (95)

δ: 7.39-7.37 (m, 2H), 6.49-6.48 (m, 2H), 6.22 (t, J = 6.1 Hz, 1H), 3.83 (dd, J = 6.1 and 2.4 Hz, 2H), 3.08 (t, J = 2.4 Hz, 1H). 13 C NMR (126 MHz, DMSO-d 6 ) δ: 147. 5, 137.1 (2C), 115.5 (2C), 81.8, 77.4, 73.2, 31.9.

1-Chloro-4-prop-2-ynylsulfanyl-benzene (120)

To a solution of 4-chlorobenzenethiol (300 mg, 2. 169.8, 167.4, 139.1, 129.6 (2C), 129.0, 119.2 (2C), 60.6, 50.9, 41.8, 37.5, 25.7, 22.7, 22.1, 14.1. 7.79-7.78 (m, 1H), 7.54-7.53 (m, 1H), 7.36 (t, J = 7.9 Hz, 1H), 7.21-7.20 (m, 1H), 4.13-4.05 (m, 2H), 3.72-3.65 (m, 2H), 3.51-3.46 (m, 1H), 1.79-1.73 (m, 1H), 1.67-1.61 (m, 1H), 1.49 (sep, J = 6.6 Hz, 1H), 1.16 (t, J = 7.1 Hz, 3H), 0.90 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H). 13 C NMR (126 MHz, DMSO-d 6 ) δ ppm: 169.9, 167. 4, 139.2, 134.7, 129.2, 124.2, 119.9, 119.4, 60.7, 53.6, 50.9, 37.5, 25.7, 22.9, 22.0, 14.1.

2-[(2-Ethoxycarbonyl-4-methyl-pentanoyl)amino]ethylammonium;chloride (85)

Compound 85 was synthesized according to the general procedure J, using the Boc-protected 9, 168.8, 60.5, 50.2, 38.2, 37.3, 36.6, 25.6, 22.4, 22.2, 14.0. 8, 166.9, 137.8, 131.3, 129.0 (2C), 120.2 (2C), 61.8, 54.3, 52.4, 40.5, 26.4, 22.4, 22.1, 14.1.

Ethyl 2-[[3-(azidomethyl)phenyl]carbamoyl]-4-methyl-pentanoate (112)

Compound 112 was synthesized according to the general procedure A, using amine 111 (405 mg, 1.07 7, 167.2, 139.2, 136.4, 129.2, 123.5, 119.0, 118.9, 60.7, 53.6, 51.0, 37.4, 25.6, 22.7, 21.9, 14.0.

Ethyl 2-(2-azidoethylcarbamoyl)-4-methyl-pentanoate (86)

Compound 86 was synthesized according to the general procedure A, using amine 85 (325 mg, 1.22 (dd, J = 7.1 and 1.2 Hz, 1H), 4.13 (dd, J = 7.1 and 1.1 Hz, 1H), 3.44-3.35 (m, 4H), 3.31-3.25 (m, 1H), 1.82-1.66 (m, 2H), 1.53 (sep, J = 6.7 Hz, 1H), 1.24 (t, J = 7.1 Hz, 3H), 0.89 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H). 13 C NMR (75 MHz, CDCl 3 ) δ ppm: 172. 3, 169.2, 61.6, 51.6, 50.7, 40.1, 39.2, 26.3, 22.4, 22.1, 14.1.

Tert-butyl N-[4-(azidomethyl)phenyl]carbamate (170)

Compound 170 was synthesized according to the general procedure A, using tert-butyl N-[4-(aminomethyl) 7, 139.5, 129.1 (3C), 128.8, 118.2, 79.1, 53.3, 28.1 (3C). 7.96-7.94 (m, 2H), 7.59-7.57 (m, 2H), 7.52-7.51 (m, 2H), 7.28-7.26 (m, 2H), 5.49 (s, 2H), 4.26 (s, 2H), 4.13-4.05 (m, 2H), 3.56 (dd, J = 8.7 and 6.2 Hz, 1H), 2.64 (s, 3H), 1.77-1.72 (m, 1H), 1.67-1.62 (m, 1H), 1.49 (sep, J = 6.7

Hz, 1H), 1.15 (t, J = 7.1 Hz, 3H), 0.88 (d, J = 6.7 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H). 13 C NMR ( 126MHz, DMSO-d 6 ) δ ppm: 169. 7, 167.2, 141.8, 138.8, 138.2 (2C), 136.5, 130.9, 128.9 (2C), 128.7 (2C), 124.0, 119.5 (2C), 101.3, 60.7, 52.5, 50.9, 44.7, 37.4, 34.7, 25.7, 22.7, 21.9, 14.0. 7, 167.1, 165.4, 145.1, 138.7, 137.2 (2C), 133.5, 131.1, 129.3 (2C), 128.8 (2C), 122.9, 119.4 (2C), 98.9, 60.6, 52.4, 50.9, 37.4, 34.9, 25.6, 22.6, 21.9, 14.0.

Ethyl 2-[[4-[[4-[(tert-butoxycarbonylamino)methyl]triazol-1-yl]methyl]phenyl]carbamoyl]-4methyl-pentanoate (125)

Compound 125 was synthesized according to the general procedure H, using azide 83 (100 mg, 0.31 mmol), N-boc-propargylamine (48.7 mg, 0.31 mmol), copper (II) sulfate pentahydrate (15.7 mg, 0.06 mmol) and sodium ascorbate (31.1 mg, 0.16 mmol) in DMF (5.5 mL) and H 2 O (4.5 mL) overnight.

The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH:

(s, 1H), 7.91-7.90 (m, 1H), 7.84 (t, J = 5.7 Hz, 1H), 7.24-7.23 (m, 1H), 7.07-7.04 (m, 1H), 6.92-6.89 (m, 1H), 5.20 (s, 2H), 4.44 (t, J = 6.1 Hz, 2H), 3.52-3.50 (m, 2H) 4, 168.4, 166.6, 148.5, 142.7, 127.9, 124.8, 124.2, 122.3, 120.8, 113.2, 62.3, 49.0, 48.6, 39.0, 38.4, 25.5, 23.9, 22.4, 22.1. 1.53-1.44 (m, 1H), 1.36 (s, 9H), 1.16 (t, J = 7.1 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H). 13 C NMR (126 MHz, DMSO-d 6 ) δ ppm: 169. 7, 167.1, 155.5, 138.5, 135.7, 132.7, 130.7, 128.0 (2C), 119.4 (2C), 78.4, 60.6, 50.9, 50.2, 37.4, 32.9, 28.1 (3C), 25.6, 22.7, 21.9, 14.0. (Fremont, CA, USA). The assay was performed as described previously (145) using batimastat as a positive control according to the guidelines of the manufacturer.

 HDACs inhibition

HDAC3 and HDAC8 Inhibitor Screening Assay kits were purchased from Sigma-Aldrich. The assay was performed according to the guidelines of the manufacturer using trichostatin. Fluorescence signals were measured in a CLARIOstar plate reader (BMG Labtech).

 TACE ADAM-17 (TACE) Inhibitor Screening Assay Kit was purchased from Sigma-Aldrich. The assay was performed according to the guidelines of the manufacturer using ilomastat as a positive control.

Fluorescence signals were measured in a CLARIOstar plate reader (BMG Labtech).

 COX-1

COX-1 Inhibitor Screening Assay Kit was purchased from Abcam. The assay was performed according to the guidelines of the manufacturer. Fluorescence signals were measured in a CLARIOstar plate reader (BMG Labtech).

Cytotoxicity assay (compounds of the chapter 3):

Hep G2, HEK293 or A549 cells (2 × 10 5 cells per well) were seeded in 24-well, flat-bottomed plates.

Culturing of cells, incubations, and OD measurements were performed as described previously (Int. J.

Cancer 2007, 121, 206-210). Twenty-four hours after seeding the cells, the incubation was started by the addition of test compound in a final DMSO concentration of 1%. The living cell mass was determined after 48 h. At least two independent measurements were performed for each compound.

  Merci à l'ADME team de Lille, Alex et Catherine. Vous avez grandement participé à ce projet grâce à votre incroyable travail. Sans vous, je pense que je serais mort devant les analyses ! Merci pour ces moments de partage scientifique et humain, sans oublier les dégustations de cookies, chocolat… Aux teams ERAP et LasB pour les tests effectués sur les composés synthétisés ainsi que pour les différentes discussions. Merci au Dr Jörg Haupenthal d'avoir assuré toute la partie bio du projet LasB et de m'avoir accueilli au sein de la team LasB. Merci au Dr Andreas Kany pour ton travail remarquable concernant la partie ADME sur le projet LasB. Au Dr Emmanuelle Lipka, merci pour ces moments de partage, de m'avoir fait découvrir la SFC, de m'avoir transmis tes connaissances sur cette technique et ainsi d'avoir participé au travail de méthodologie. Au Dr Fanny Roussy et Pr Eric Deniau pour votre participation à mes comités de suivi de thèse et vos conseils. Au professeur Nathalie Azaroual et son équipe pour les analyses RMN à Lille. A l'équipe enseignante du Master Conception, Synthèse, Evaluation et Sélection des principes actifs à Lille, merci pour vos enseignements de qualité qui ont permis de renforcer mes connaissances dans le domaine des sciences du médicament. Aux secrétaires des deux laboratoires, Nathalie, Annette et Bahareh. Merci pour votre aide précieuse et d'avoir assuré tout le côté administratif de cette thèse. Je tiens également à remercier Mr François Delcroix, coordinateur de l'EDBSL pour son aide précieuse et sa réactivité. Aux post-docs, camarades thésards et autres rencontrés durant cette thèse Mélissa, Aurore, Léo, Charlotte, Chris, Andreas K, Ahmed, Mostafa, Gwen, Alex, Samira, Selina, Marine, Laetitia, Kevin, Atanaz… La liste est longue. Merci à Tony et Andreas Klein pour votre aide sur la crystallo ! Merci à Eleonora, Ioulia, Tony, Daan pour tous ces moments de partage. Un énorme merci à Lucile ! Enfin merci à Federica, Cansu et Alaa de m'avoir accueilli à Saabrücken. Merci également à mes anciens collocs Thomas, Toufik et mes anciens camarades de pharma notamment Sabrina pour ton soutien incroyable durant ces années. Enfin, merci à ma famille et en particulier mes frères Allan et Matthias et à mes parents de pour votre soutien, votre écoute et vos sacrifices pour je puisse suivre ces études.

  Die Target-Guided Synthese von Proteinliganden ist eine innovative Strategie zur Entdeckung bioaktiver Verbindungen. Insbesondere die kinetische Target-Guided Synthese (KTGS) und die dynamische kombinatorische Chemie (DCC) haben in den letzten Jahren die Entdeckung neuartiger Liganden für schlecht erforschte therapeutische Ziele ermöglicht, was Projekte zur Entdeckung von Arzneimitteln ermöglicht hat. Im Rahmen dieses Forschungsprojekts sollen mithilfe der KTGS Liganden für zwei Klassen von Metalloenzymen entdeckt und optimiert werden, nämlich die Aminopeptidasen des endoplasmatischen Retikulums (ERAPs) und die Elastase LasB aus dem Bakterium Pseudomonas aeruginosa. Die ERAPs (1 und 2) sind am Prozess der Antigenreifung beteiligt. Diese Enzyme spalten Peptidvorläufer in reife antigene Peptide, so dass diese eine optimale Größe für ihre Komplexbildung mit dem Haupt histokompatibilitätskomplex der Klasse I haben und somit die adaptive Immunantwort einführen oder nicht. Die Expressionsniveaus dieser Proteasen sowie Einzelnukleotid-Polymorphismen wurden mit der Entwicklung von Krebs und Autoimmunkrankheiten in Verbindung gebracht. Die Modulation dieser Enzyme würde es also ermöglichen, Pathologien zu bekämpfen, die mit dem Immunsystem zusammenhängen. P. aeruginosa ist ein Gramnegatives Bakterium mit bemerkenswerter Virulenz und antimikrobieller Resistenz. Die Antibiotikaresistenz stellt heute ein großes Problem für die öffentliche Gesundheit dar, und es besteht ein dringender Bedarf an neuen Therapeutika. Um diesem Bedarf gerecht zu werden, wurden neue Strategien entwickelt, die auf die Virulenz der Bakterien abzielen, um sie zu "entwaffnen". LasB ist aufgrund seiner extrazellulären Lokalisierung und seiner physiopathologischen Auswirkungen (Kolonisierung, Invasion, Umgehung der Immunantwort, Biofilmbildung usw.) ein therapeutisches Ziel der Wahl. Obwohl in diesen beiden therapeutischen Bereichen eindeutig ein ungedeckter medizinischer Bedarf besteht, ist bisher kein Modulator von ERAPs oder LasB auf dem Markt. Durch die Anwendung der KTGS-Strategie, gefolgt von Optimierungsphasen, konnten wir neue Familien von Liganden für diese Enzyme identifizieren und optimieren. Diese Verbindungen können als vielversprechende Leitverbindungen betrachtet werden, da sie. Schlüsselwörter : KTGS, ligand, metalloenzymen, ERAP, Immunsystem, LasB, antimikrobieller Resistenz, Hemmstoff, selektivität.

  GENERAL INTRODUCTION ........................................................................................................................ --CHAPTER 1: PROTEIN-TEMPLATED STRATEGIES: THE KTGS ...................................................... --I. INTRODUCTION ....................................................................................................................................... --

  ... --INTRODUCTION ............................................................................................................................................ --RESULTS ...................................................................................................................................................... --I.

  Knowledge-based programs, High-Throughput Screening or High-Content Screening have been commonly used in recent years. Progress in IT and natural sciences (biophysics, chemistry and structural biology) has led to the development of new technologies such as biophysical strategies (e.g., Nuclear Magnetic Resonance (NMR), Surface Plasmon Resonance, virtual screening, fragment screening, DNA-encoded library screening and protein-templated synthesis.

Figure 1 :

 1 Figure 1: Hit identification strategies in drug discovery

Figure 2

 2 Figure 2 : Objectives of the PhD project

Figure 3 :

 3 Figure 3 : Principles of target-guided synthesis. (A) Principle of DCC, the target protein displaces the equilibrium toward the most affine ligand. (B) Principle of KTGS, the target protein stabilizes a pair of reagents in reacting relative configurations and accelerates the irreversible formation of the ligand, which is finally detected. Adapted from Deprez-Poulain et al.(13)

Figure 4 :

 4 Figure 4 : Origin and nature of the warhead-bearing reagents. (A) Inspiration sources for the design of the warheadbearing reagents. (B) Nature of the warhead-bearing reagents. (C) Character of the templated ligand-protein binding, adapted from Deprez-Poulain et al.(13)

Figure 5 :

 5 Figure 5 : Distribution of the structural features of the warhead-bearing reagents. (A) MW, (B) cLogP, (C) HBA and (D) HBD. The proportions of warhead-bearing reagents that comply with the Ro3 are presented in red dotted lines, Adapted from Deprez-Poulain et al.(13)

Figure 6 :

 6 Figure 6 : Format (A), size (B), hit rate (C) and detection methods (D) in disclosed KTGS experiments adapted from Deprez-Poulain et al.(13)

Figure 7 :

 7 Figure 7 : (A) The therapeutic areas with at least one published KTGS example and theirs respective proportions. (B)The protein families used in KTGS experiments and theirs respective proportions adapted from Deprez-Poulain et al.(13)

Figure 8 :

 8 Figure 8 : Structural characteristics distribution of KTGS ligands and theirs behavior regarding the Ro5 and the Doak chemical space. (A) MW, (B) cLogP, (C) number of rotatable bonds, (D) number of HBA, (E) number of HBD, and (F) PSA. The proportions of templated ligands which comply with the Ro5 are in blue box and these for those in the Doak chemical space are in red dotted lines, adapted from Deprez-Poulain et al.(13)

Figure 9 :

 9 Figure 9 : Comparison of the structural and physicochemical properties of KTGS ligands and FDA approved drugs. (A) MW, (B) cLogP, HBA (C), HBD (D), (E) rotatable bonds and (F) PSA. FDA-approved drugs are in grey and KTGS ligands in blue; N=34 for KTGS and N=751 for FDA, adapted from Deprez-Poulain et al.(13)

Figure 10 : 1 .Scheme 1 :

 1011 Figure 10 : LLE (A) and ΔLLE (B) of the KTGS ligands adapted from Deprez-Poulain et al.(13)

Figure 11 :

 11 Figure 11 : X-ray structure representations of the ligand-binding pocket of EthR. a) K5a-EthR complex (PDB code: 3O8G). b) K5-EthR complex (PDB code: 3O8H). Colors legend: green (ligand) and light blue (EthR) = carbon, blue = nitrogen, red = oxygen, yellow = sulfur, dotted lines = hydrogen bonds. Key flexible residues are highlighted in orange. Adapted from Willand et al.(25)

Figure 12 :

 12 Figure 12 : Shift of the conformation equilibrium of hIDE by K6 adapted from Deprez-Poulain et al.(4)

Figure 13 :

 13 Figure 13 : X-ray crystal structure of endothiapepsin-K7a and -K7b complexes (PDB code: 3PBZ and 3PLD, respectively) and modeled potential triazole inhibitor K5c. Colors legend: purple (K7a), yellow (K7b), green (K7c) and grey (protein) = carbon, blue = nitrogen, red = oxygen and dashed lines = hydrogen bonds. Adapted from Hirschet al.[START_REF] Mondal | Fragment-Based Drug Design Facilitated by Protein-Templated Click Chemistry: Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin[END_REF]

Figure 14 :

 14 Figure 14 : Docking of K7. Colors legend: green (K7) and grey (protein) = carbon, blue = nitrogen, red = oxygen and dashed lines = hydrogen bonds and triazole is circled in red. Adapted from Hirsch et al.[START_REF] Mondal | Fragment-Based Drug Design Facilitated by Protein-Templated Click Chemistry: Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin[END_REF]

  system and defense mechanismsThe immune system displays two cooperative defense mechanisms which are the innate and the adaptive immune responses (Figure15).

Figure 15 :

 15 Figure 15 : Innate and adaptive immunity time line with their respective main actors adapted from Abbas, Lichtman and Pillai. Basic Immunology, 5th edition, 2016, Elsevier

  (A, B and C), spans the membrane. The non-polymorphic β-light chain, composed of a β2microglobulin subunit and encoded by a non-HLA gene, maintains the MHC-I conformation with the α3 subunit. The α1 and α2 subunits delimit the peptide (antigen) binding region and are thus referred to the recognition region. The β2 microglobulin and α3 bind the CD8 glycoprotein. The MHC-II (Figure 16B) is also composed of two polypeptide chains α and β, each chain being composed of two subunits (α1/α2 and β1/β2), encoded by the HLA genes (HLA-DP, HLA-DQ et HLA-DR) and spanning the membrane. The α1 and β1 subunits delimit the peptide binding region. The α2 and β2 bind the CD4 glycoprotein.

Figure 16 :

 16 Figure 16 : Structural characteristics of the MHC-I (A) and MHC-II (B) adapted from molecular biology of the cell 5eGarland science

  the extended peptides are translocated to the ER through the TAP transporter and their sizes are adjusted by the endoplasmic reticulum aminopeptidases: the ERAP enzymes. Chaperones (tapasin, calreticulin and ERp57) bind the MHC-I, maintaining its conformation and allowing an optimal binding between the peptide and MHC-I. The peptide-MHC-I complex is then transported via the Golgi to the plasma membrane where the antigenic peptide is presented to the CD8 + T-cells or NK cells, also named cytotoxic T-lymphocytes, through an interaction MHC-I-TCR.

Figure 17 :

 17 Figure 17 : The basic MHC-I antigen presentation pathway

Figure 18 :

 18 Figure 18 : Genomic organization of the human chromosome 5q15 containing the ERAPs and their respective schematic organization in four domains adapted from Babaie et al.(44)

Figure 19 :

 19 Figure 19 : Comparison of the HEXXH(X) 18 E motif between the two ERAPs. (A) Illustration of the HEXXH(X) 18 E motif in ERAP1 (PDB 3MDJ). (B) Illustration of the HEXXH(X) 18 E motif in ERAP2 (PDB 3SE6). From Paul Hermant's PharmD thesis(47)

Figure 20 :

 20 Figure 20 : Comparison of the GAMEN motif between the two ERAPs. (A) Illustration of the GAMEN motif in ERAP1 (PDB 3MDJ). (B) Illustration of the GAMEN motif in ERAP2 (PDB 3SE6). From Paul Hermant's PharmDthesis(47)

  ); ERAP1 quickly cleaving 13-residue peptides but not 9-and 5-residue peptides (Figure21B).

Figure 21 :

 21 Figure 21 : ERAP1 substrate preferences length adapted from Chang et al.[START_REF] Chang | The ER aminopeptidase, ERAP1, trims precursors to lengths of MHC class I peptides by a 'molecular ruler' mechanism[END_REF]

Figure 22 :

 22 Figure 22 : ERAP1 substrate preferences nature from Chang et al.[START_REF] Chang | The ER aminopeptidase, ERAP1, trims precursors to lengths of MHC class I peptides by a 'molecular ruler' mechanism[END_REF]

Figure 23 :

 23 Figure 23 : Schematic representation of the proposed ERAP1 cleavage model. (A) Cleavage occurs further to the binding of a substrate with an optimal length. (B) Cleavage does not occur due to the binding of a too smaller substrate. (C) Cleavage does not occur due to the presence of charged C-ter residues. Adapted from Chang et al.

Figure 24 :

 24 Figure 24 : ERAPs and antigen processing. [A] Normal processing of the antigens. [B] Abnormal processing resulting in the exportation of abnormal peptide peptide-MHC-I complexes to the surface cell when antigens are taken in charge by the MHC-I. [C] Abnormal processing resulting in their accumulation in the ER since the antigens are not taken in charge by the MHC-I. Adapted from Haroon et al.[START_REF] Haroon | Endoplasmic reticulum aminopeptidases: biology and pathogenic potential[END_REF]

Figure 25 :

 25 Figure 25 : Expression of ERAP1 and ERAP2 across The Cancer Genomic Atlas tumor tissues. Red bars are used to compare ERAP1 and ERAP2 expression levels. Red and blue asterisks respectively indicate the higher and the lower ERAP1 and ERAP2 expressing tumor types from adapted from Fruci et al.(54)

Figure 26 :

 26 Figure 26 : Schematic representation of the normal processing resulting in effective cytotoxic responses against tumor antigens (A) and of the abnormal processing resulting in an immune evasion consequently to the altered ERAPs (B).Adapted from Stratikos et al.[START_REF] Stratikos | A role for naturally occurring alleles of endoplasmic reticulum aminopeptidases in tumor immunity and cancer pre-disposition[END_REF]

Figure 27 :

 27 Figure 27 : (A) Schematic representation of an aminobenzamide compound indicating the key interacting residues in blue. (B) Molecular model of the scaffold with carbon atoms shown in orange. The zinc-coordinated residues (His-353, His-357 and Glu-376) and the catalytically important Tyr-438 are shown transparent. The three key interacting glutamic residues (Glu-183, Glu-320 and Glu-354) are shown in dark green and Zn2+ with a green sphere. All oxygen and nitrogen atoms are red and blue respectively. (C) Surface representation of the active site of ERAP1 showing the S1 and the putative S1'-S3' subsites. Adapted from Stratikos et al.(62)

Figure 28 :

 28 Figure 28 : (A) Schematic representation of phosphinic compounds indicating their key interacting residues. (B) Schematic representation of the crystal structure of phosphinic 2A bound inside the ERAP2 catalytic site. The mesh indicates the jFo-Fcj unbiased electron density at 2.5 σ calculated before ligand addition to the structure. Phosphinic 2A is shown in yellow sticks. ERAP2 residues within 4 Å of the inhibitor are shown in gray sticks. Oxygen are shown in red, nitrogen in blue, phosphorus in orange, and Zn(II) in cyan. Hydrogen bonding interactions which stabilize phosphinic 2A are indicated as dashed lines. Adapted from Stratikos et al.(63)

Figure 29 :

 29 Figure 29 : Chemical structure of one carboxylic acid selective ERAP2 inhibitor

Figure 30

 30 Figure 30 : objective and strategy for the ERAP2 PhD project

Figure 31 :

 31 Figure 31: Chemical structures of the starting azides for the KTGS with ERAP2

Scheme 4 :

 4 Scheme 4: Principle of the ERAP enzymatic assay. (A) Use of L-AMC for ERAP1. (B) Use of R-AMC for ERAP2.

  Scheme 5 : Seven-step synthesis of 1,5-triazoles

4 (Scheme 6 :Scheme 7 :

 467 Scheme 6: Chemical synthesis of the hydroxamic acids 30-31 in three steps

Table 8 :

 8 Comparison of ERAP activities between β-and αwere synthesized in four steps from the corresponding α-amino acid (Scheme 9): diazo transfer (a), esterification (b), coppercatalyzed click reaction(d) and aminolysis (e). The needed alkyne for the click reaction was also synthesized using a nucleophilic acyl substitution of the propargylamine on the sulfonyl chloride (c).

2 O 2 OH/H 2 OScheme 9 :

 2229 Scheme 9 : Chemical synthesis of the compound 35-36 in four steps

  Family 1: 1,4-disubstituted 1,2,3-triazoles in α-configuration -Family 2: 1,5-disubstituted 1,2,3-triazoles in β-configuration KTGS assay and the preliminary SARs, two families of selective ERAP2 inhibitors were identified with activity values in the submicromolar range; which makes them interesting starting points for further SARs.

Figure 33 :

 33 Figure 33 : Identified parts of compound 12 for the pharmacomodulations concerning the family 1

2 O

 2 Scheme 12 : Synthesis of the final compounds 53-58 and their respective intermediate 47-52

2 ,

 2 Scheme 13 : Synthesis of the azides 61-62

77  70 .

 7770 Scheme 14 : Synthesis of the final compounds 78-81 and their respective intermediates 74-77

Scheme 15 :

 15 Scheme 15 : Synthesis of the compounds 74-75 and their respective intermediates 72-73

Figure 34 :

 34 Figure 34 : Chemical structures of 56 and 12 and their IC 50 values on ERAP2

Figure 35 :

 35 Figure 35 : Binding mode of 56 in ERAP2. 56 is represented in yellow, the active site residues of ERAP2 in gray, the zinc as a gray sphere, oxygen and nitrogen are in red and blue, respectively.

Figure 36 :

 36 Figure 36 : 2D representation of the binding mode of 56 in ERAP2.

Figure 37 :

 37 Figure 37 : Binding mode of 12 in ERAP2. 56 is represented in green, the active site residues of ERAP2 in gray, the zinc as a gray sphere, oxygen, sulfur and nitrogen are in red, yellow and blue, respectively.

Figure 38 :

 38 Figure 38 : 2D representation of the binding mode of 12 in ERAP2.

Chapter 3 :

 3 KTGS leads to the identification of potent and selective LasB inhibitors IntroductionAlong with the vaccines, antibiotics are considered as one of the greatest advances in modern medicine. These drugs are used to fight bacterial infections, potentially fatal in some cases, and are extensively used in medical procedures which expose us to bacteria: surgery, transplants, and therapies affecting the immune system. The overuse and misuse of antibiotics in human or veterinary medicine and poor hygiene conditions and practices in healthcare settings or in the food chain, facilitate the transmission of resistant microorganisms, which boost the resistance development, therefore making the antibiotics less effective and finally useless. The spread of resistant bacteria, particularly Gram-negative bacteria, has increased to the point where, today, AMR represents a major global public health problem.

Figure 39 :

 39 Figure 39: Number of deaths per year attributable to AMR by 2050 by continent adapted from O'Neill[START_REF] O'neill | Antimicrobial Resistance:Tackling a crisis for the health and wealth of nations[END_REF]

Figure 40 :

 40 Figure 40: Percentage of invasive isolates with resistance to carbapenems (imipenem or/and meropenem) by country in Europe in 2019 from the ECDC[START_REF]Antimicrobial resistance in the EU/EEA (EARS-Net) -Annual Epidemiological Report 2019[END_REF]

  on various environmental signals (low levels of iron, oxygen, phosphate or phospholipids in the host surfactant…), leads to the production of an auto-inducer (signaling molecule) and a transcriptional receptor protein. When the auto-inducer concentration reaches a certain threshold, it activates the transcriptional receptor protein, allowing the network to control the production of many factors which are important for the bacterial growth and virulence (Figure41).

Figure 41 :

 41 Figure 41: Schematic representation of the four QS signaling network in P. aeruginosa and their respective regulation adapted from Lee et al.[START_REF] Lee | The hierarchy quorum sensing network in Pseudomonas aeruginosa[END_REF]

Figure 42 :

 42 Figure 42: Produced virulence factors by P. aeruginosa adapted from Lee et al.[START_REF] Lee | The hierarchy quorum sensing network in Pseudomonas aeruginosa[END_REF]

LasB as a therapeutic target 1 .

 1 Production and regulationLasB elastase (or pseudolysin) is a metallo-type (Zn 2+ /Ca 2+ cofactors) protease, which belongs to the thermolysin group (M4), a large group of zinc metalloproteases. It is synthesized as a preproelastase formed by three peptide domains: an N-terminal prepeptide (AA 1-23 = signal prepetide), a propeptide (AA24-197) functioning as an intramolecular chaperone for the C-terminal domain (IMC)(AA 198-498), encoding the mature elastase (33kDa) (Figure43).

Figure 43 :

 43 Figure 43: Representation of the preproelastase. The signal prepetide is in red, IMC propeptide with the chaperone (FTP) and elastase inhibitor subdomains (PepSY) in green and the mature enzyme in blue. The lysine residues subject to post-translational modification are represented in dark-red dots, the positions of the involved cysteine in the two disulfides bridges (DSB1 and DSB2) in yellow dots, the calcium-binding sites in gray, zinc-binding sites in turquoise blue and catalytic site and proton donor sites in red markers. Adapted from Everett et al.[START_REF] Everett | Pseudomonas aeruginosa elastase (LasB) as a therapeutic target[END_REF]

Figure 44 :

 44 Figure 44: (A) superposition of tertiary structures of LasB (turquoise blue, PDB entry 1EZM) and thermolysin (pink, PDB entry 1KEI), the zinc ion is represented in gray. (B) tetrahedral zinc coordination in LasB active site (PBD entry 1EZM). Adapted from Everett et al.[START_REF] Everett | Pseudomonas aeruginosa elastase (LasB) as a therapeutic target[END_REF]

Figure 45 :

 45 Figure 45: Proposed catalytic mechanism of hydrolysis of substrates by the elastase LasB adapted from Everett et al.[START_REF] Everett | Pseudomonas aeruginosa elastase (LasB) as a therapeutic target[END_REF]



  Immune system componentsBesides the structural components, this elastase can degrade numerous actors of the immune system.

Figure 46 :

 46 Figure 46: (A) Kaplan-Meier survival plot of each mice group. (B) Representative images of lung tissue of each group of mice group adapted from Gi et al. (107)

Figure 47 :

 47 Figure 47: LasB elastolytic activity in culture supernatants from each group presented previously adapted from Cigana et al. (108)

Figure 48 :

 48 Figure 48: (A) Measure of the lung CFUs after two and seven days for wt RP45 and ΔlasB RP45; LLCC (Lower Limit of Chronic Colonization) and LLQ (Lower Limit of Quantification) are indicated. (B) Incidence of clearance and capacity to establish chronic infection for wt RP45 and ΔlasB RP73. (C) Measure of the lung CFUs after two and seven days for wt RP73 and ΔlasB RP73. (D) Incidence of clearance and capacity to establish chronic infection for wt RP45 and ΔlasB RP73. Adapted from Cigana et al. (108)

Figure 49 :

 49 Figure 49 : Chemical structures of IL1 and IL2 with their K i values

Figure 50 :

 50 Figure 50 : Chemical structures of IL3, IL4 and IL5 with their K i

Figure 51 :

 51 Figure 51: Chemical structures of IL6 and IL7 and their IC 50

Figure 52 :

 52 Figure 52: Chemical structures of IL8 and IL9 and their LasB activities

Figure 53 :

 53 Figure 53: Chemical structures of IL10 and IL11 and their K i

Figure 54 : 50 IL8

 5450 Figure 54: Chemical structures of LasB inh 12, 14 with K i and LasB inh 14 with its IC 50

Figure 55 :

 55 Figure 55 : Strategy for the LasB PhD project

Figure 56 :

 56 Figure 56: Chemical structures of the starting azides for the KTGS with LasB

Figure 57 :

 57 Figure 57: Schematic representation of peptidyl side-chains and corresponding enzyme-binding sites adapted from Everett et al.[START_REF] Everett | Pseudomonas aeruginosa elastase (LasB) as a therapeutic target[END_REF]

2 , 2 O ( 4 : 1 )

 2241 Scheme 16 : Three-step synthesis of Z7

Figure 58 :

 58 Figure 58 : Chemical structure of the selected hits from Z9

Figure 59 : 4 .

 594 Figure 59: Chemical structures of the selected hits from Z10

2 OScheme 20 :

 220 Scheme 20 : Chemical synthesis of the selected hits from Z9

88 :

 88 Scheme 21 : Synthesis of the alkynes for the click step (e)

  Scheme 23 : Chemical synthesis of Hit L9

Figure 60 :

 60 Figure 60: (A) Cleavage of the fluorogenic substrate by LasB. (B) Concentration-dependent inhibition of the enzymatic reaction, phosphoramidon used as a positive control

Figure 61 :

 61 Figure 61: Identified parts of the Hit L1 for the pharmacomodulations

3 N,

 3 DMF, rt., overnight, 74%; b) HCl 4M in dioxane, EtOH, 0 °C to rt., overnight, quantit. yield; c) azide-N-diazoimidazole-1-sulfonamide hydrogen chloride, EtOH, 0 °C to rt., overnight, 31%; d) 2:1), rt., overnight, 86%; e) aq. hydroxylamine (50% in water w/w), KCN (cat.), MeOH, 48 h, overnight, 7%.

Figure 62 :

 62 Figure 62 : LC UV-spectra at 254nm of the last step crude showing the degradation of the desired compound 114 and ESI + ionization MS-spectra.

Scheme 26 :

 26 Scheme 26 : (A) Nucleophilic acyl substitution (SN) of the N-methylpropargylamine on the sulfonylchloride and reagents/conditions/yield. (B) SN of the propargylamine on the acyl chloride and reagents/conditions/yield. (C) SN of the saccharin on the propargylbromide and reagents/conditions/yield. (D) SN on the propargylbromide and reagents/conditions/yield. (E) Seyferth-Gilbert homologation and reagents/conditions/yield.

83 123:

 83 Scheme 29 : Synthesis of compound 140

Scheme 31 :

 31 Scheme 31 : Synthesis of the final compounds 160-169 and their respective intermediates 150-159

Figure 63 :

 63 Figure 63: LC-UV spectra of the purified fraction of the isopropyl derivative showing the diodination (mixture 88:12)

  Scheme 35 : Chemical synthesis of the 1,5-disubstituted 1,2,3-triazoles using ruthenium-catalyzed click reaction

Scheme 36 :

 36 Scheme 36 : (A) Synthesis of the carboxylic acid 193, (B) Synthesis of the phosphonate 196 Reagents and conditions: a) NaOH, THF/H 2 O (1:1), rt., overnight, 54%; b) 2-diethoxyphosphoryl-4-methyl-pentanoic acid, TBTU, NMM, DMF, rt., overnight, 66%; (c) 141, CuSO 4 (5H 2 O), sodium ascorbate, DMF/H 2 O (1.2:1), rt., overnight, 77%;

Figure 64 :

 64 Figure 64: Chemical structures of Hit L1, 160, 169, Z9 and their biological activities on LasB

Figure 65 :

 65 Figure 65: Chemical structure of 161 and its IC 50 value on LasB

Figure 66 :

 66 Figure 66 : Binding mode of 161 in LasB. Carbons are indicated in gray, nitrogens in blue, oxygens in red, sulfur in yellow and chlore in green. The ligand 161 is represented in cyan and the zinc as a gray sphere. (A) Surface representation of LasB bound to 161. (B) Interactions between 161 and the adjacent amino acid side chains of LasB which are represented in yellow stick

Figure 67

 67 Figure 67: Planned modifications Towards a macrocycle?

  Scheme 37 : catalytic cycle for the 1,4-disubstituted 1,2,3-triazole catalyzed by copper (I) adapted from Worrell et al. (126)

Scheme 38 :

 38 Scheme 38 : catalytic cycle for the 1,5-disubstituted 1,2,3-triazole catalyzed by ruthenium (II) adapted from Boren et al. (127)



  Scheme 40 : proposed mechanism of the 1,5-disubstituted 1,2,3-triazole using nitroolefins adapted from Maiuolo et al.(129)

Scheme 43 :

 43 Scheme 43 : Our one-pot NiAAC methodology for the synthesis of 1,5-disubstituted 1,2,3-triazoles

Figure 68 :

 68 Figure 68 : Comparison between the main strategies for the synthesis of 1,5-disubstituted 1,2,3-triazole and our onepot NiAAC strategy adapted from Camberlein et al.(138)

Figure 69 :

 69 Figure 69 : ERAP2-templated formation of Hit E1 and optimization in 12 and 71 and their respective features. The warhead is highlighted in salmon.

Figure 70 :

 70 Figure 70 : LasB-templated formation of Hit L1 and optimization in 160 and 169 and their respective features. The warhead is highlighted in salmon.

Scheme 44 :

 44 Scheme 44 : Protein-templated strategies with the enzyme ERAP2. The KTGS strategy was applied using the Huisgen 1,3-dipolar cycloaddition and led to the formation of the 1,2,3-triazole disubstituted. The DCC could also be applied, from amines and aldehydes leading to imine formations, to compare the two strategies. DCC strategy could be applied with LasB in modifying the starting amines.

Scheme 45 :

 45 Scheme 45 : Developed one pot nickel-catalyzed reaction for the synthesis of 1,5-disubstituted 1,2,3-triazoles

  Avignon, France) equipped with an autosampler comprising a 48-vial plate (model Alias, Emmen, Netherlands), three model 40 P pumps: two for CO 2 and a third for the modifier (Knauer, Berlin, Germany), a column oven with a Valco ten-position column selection valve, and a Valco six-position solvent switching valve. The system was also composed of a Smartline 2600 diode array detector (DAD) (Knauer, Berlin, Germany). Detection wavelength was set at 215 nm. The system was controlled, and the data was acquired with the SFC PicLab Analytic Online v.3.1.2 software (PIC Solution, Avignon, France). Retention times were average values of two replicate determinations. The data were processed by SFC New Data Manager V.1.8.0 software (Pic Solution, Avignon, France).The stationary phases used for SFC, and purchased from Chiral Technologies Europe (Illkirch, France), are a Chiralcel OD-H, cellulose tris (3,5dimethylphenylcarbamate) used for the purification of all compounds excepted compound 3aE, and a Chiralpak IC, cellulose tris(3,5dichlorophenylcarbamate) used for the purification of 3aE. The two columns have dimensions of 4.6x250 mm with 5 µm particle size. All SFC analyses were run in isocratic mode. The column oven temperature was 40°C and the outlet pressure was maintained at 150 bar for all experiments. For the determinations of the regioselectivity rate and the purity of the purified fraction of 1,5-disubstituted 1,2,3-triazole, the injected volume was 20 µL, the used flow rate was 4 mL/min and the mobile phase was always CO2-modifier mixture with 10% of ethanol as modifier excepted for compound 3aG

5 and 5 . 2

 552 Compound 37 was synthesized according to the general procedure A, using D-tyrosine(226 mg, 1.25 mmol), ZnCl 2 (10.2 mg, 0.08 mmol), K 2 CO 3 (691 mg, 5.0 mmol), anhydrous N,N- diisopropylethylamine (240 μL, 1.38 mmol) and diazo transfer reagent (407 mg, 1.5 mmol) in anhydrous MeOH (5mL) overnight. Compound 37 was obtained as pale brown oil (202 mg, 76%) and was used in the next step without further purification. LC tr = 1.42 min, MS (ESI-): m/z = 206 [M -H] -. 1 H NMR (300 MHz, MeOD-d 4 ) δ ppm: 7.10-7.06 (m, 2H), 6.75-6.70 (m, 2H), 4.08 (dd, J = 8.Hz, 1H), 3.07 (dd, J = 14.1 and 5.2 Hz, 1H), 2.89 (dd, J = 14.1 and 8.5 Hz, 1H). 13 C NMR (75 MHz, MeOD-d 4 ) δ ppm: 173.4, 157.5, 131.4 (2C), 128.5, 116.3 (2C), 64.7, 37.8. Compound 60 was synthesized according to the general procedure A, using O-methyl L-tyrosine analog (226 mg, 1.16 mmol), ZnCl 2 (9.5 mg, 0.07 mmol), K 2 CO 3 (641 mg, 4.64 mmol), anhydrous N,N-diisopropylethylamine (222 μL, 1.28 mmol) and diazo transfer reagent (378 mg, 1.39 mmol) in anhydrous MeOH (5mL) overnight. Compound 60 was obtained as orange oil (257 mg, 96%) and was used in the next step without further purification. LC tr = 1.83 min, MS (ESI-): m/z = 220 [M -H] -. 1 H NMR (300 MHz, MeOD-d 4 ) δ ppm:

  Compound 30 was synthesized according to the general procedure E, using carboxylic acid 23 (258 mg, 1.25 mmol) and thionyl chloride (182 μL, 2.49 mmol) in MeOH (5 mL) overnight. Compound 30 was obtained as brown oil (193 mg, 70%) and was used in the next step without further purification.LC tr = 2.40 min, MS (ESI-): m/z = 220 [M -H] -.1 H NMR (300 MHz, ) δ ppm: 7.07-7.02(m, 2H), 6.74-6.70 (m, 2H), 4.13 (dd, J = 8.1 and 5.7 Hz, 1H), 3.73 (s, 3H), 3.04 (dd, J = 14.0 and 5.7

1 .Compound 5

 15 26 mmol), 5-bromothiophene-2-sulfonyl chloride (300 mg, 1.15 mmol) and N,N-was synthesized according to the general procedure C, using 3 (1660 mg, 6.43 mmol) in DMF (200 mL) overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 9/1) affording compound 5 as a brown solid (1156 mg, 70%). LC tr = 1.48 min, MS (ESI+): m/z = 259 [M + H] + . 1 H NMR (300 MHz, MeOD-d 4 ) δ ppm: 7.57 (s, 1H), 7.03-6.99 (m, 2H), 6.74-6.70 (m, 2H), 5.05-4.97 (m, 1H), 4.41 (dd, J = 27.8 and 17.0 Hz, 2H), 3.43 (dd, J = 13.7 and 3.5 Hz, 1H), 3.15 (dd, J = 13.7 and 9.1 Hz, 1H), 3.03 (dd, J = 14.5 and 4.4 Hz, 1H), 2.95

Compound 6

 6 was synthesized according to the general procedure C, using 4 (487 mg, 1.73 mmol) in DMF (50 mL) overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 9/1) affording compound 6 as a brown oil (217 mg, 45%). LC tr = 1.90min, MS (ESI+): m/z = 282 [M + H] + .1 H NMR (300 MHz, ) δ ppm: 7.56 (s, 1H), 7.54-

13 C

 13 , 4.39(d, J = 17.0 Hz, 1H), 4.30 (d, J = 17.0 Hz, 1H), 3.69 (dd, J = 14.3 and 3.1 Hz, 1H),3.43-3.38 (m, 1H), 3.08-2.95 (m, 2H). NMR (75 MHz, MeOD-d 4 ) δ ppm: 175.

3 and 5 . 2 1 and 5 . 1

 352151 Compound 27 was synthesized according to the general procedure G, using amine 26 (100 mg, 0.26 mmol), 5-phenylthiophene-2-sulfonyl chloride(79.4 mg, 0.31 mmol) and N,N-Diisopropylethylamine(98 μL, 0.57 mmol) in DMF (5mL) overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 95/5) affording compound 27 as a yellow solid (37.7 mg, 29%). LC tr = 2.70 min, MS (ESI-): m/z = 497 [M -H] -. 1 H NMR (300 MHz, DMF-d 7 ) δ ppm: 9.51 (br s, 1H), 8.43 (br s, 1H), 7.82-7.78 (m, 2H), 7.64 (d, J = 3.9 Hz, 1H), 7.62 (d, J = 3.9Hz, 1H),7.57 (s, 1H), 7.55-7.44 (m, 3H), 7.00-6.95 (m, 2H), 6.73-6.68 (m, 2H), 5.72 (dd, J = 10.Hz, 1H), 4.18 (d, J = 16.0 Hz, 1H), 4.09 (d, J = 16.0 Hz, 1H), 3.74 (s, 3H), 3.63 (dd, J = 14.Hz, 1H),

  9 and 1.0 Hz, 1H), 7.92 (td, J = 11.5 and 1.8 Hz, 1H), 7.85 (d, J = 4.0 Hz, 1H), 7.55 (d, J = 4.0 Hz, 1H), 7.41 (ddd, J = 7.5, 4.8 and 1.0 Hz, 1H), 6.75-6.72 (m, 2H), 6.59-6.57 (m, 2H), 4.86-4.76 (m, 1H), 3.92-3.82 (m, 2H), 3.03 (dd, J = 13.6 and 6.2 Hz, 1H), 2.93 (dd, J = 13.6 and 8.7 Hz, 1H), 2.85 (dd, J = 15.5 and 10.1 Hz, 1H), 2.65 (dd, J = 15.5 and 4.5 Hz, 1H). 13 C NMR (75 MHz, DMSO-d 6 ) δ ppm: 165.

  Compound 70 was synthesized according to the general procedure G, using ester 66(60 mg, 0.11 mmol), KCN (1.4 mg, 0.02 mmol) and aq. NH 2 OH (1.5 mL, 50% w/w in water) in MeOH(1.5 mL) overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 9/1) affording compound 70 as a white solid after lyophilization (52 mg, 86%). Purity: 99%, LC tr = 2.57 min, MS (ESI+): m/z = 557 [M + H] + ; 501 [M + H -tBu] + .1 H NMR (300 MHz, ) δ ppm: 11.09 (s, 1H), 9.21 (s, 1H), 8.56 (ddd, J = 4.9, 1.7 and 0.9 Hz, 1H), 8.47 (t, J = 5.9 Hz, 1H), 8.09 (s, 1H), 8.02 (dt, J = 7.9 and 1.0 Hz, 1H), 7.88 (td, J = 7.8 and 1.7 Hz, 1H), 7.82 (d, J = 4.0 Hz, 1H), 7.58(d, J = 4.0 Hz, 1H), 7.36 (ddd, J = 7.5, 4.9 and 1.0 Hz, 1H), 7.02-6.99 (m, 2H),

  Compound 147 was synthesized according to the general procedure F, using propargylamine(94 μL, 1.47 mmol), (4-chlorophenyl)methanesulfonyl chloride (300 mg, 1.33 mmol) and N,N- diisopropylethylamine (464 μL, 2.67 mmol) in CH 2 Cl 2 (5 mL) overnight. The crude product was purified by flash chromatography on silica gel (cHex to cHex/EtOAc: 7/3) affording compound 147 as a light yellow solid (187 mg, 45%). LC tr = 3.88 min, MS (ESI-): m/z = 242 and 244[M -H] -.1 H

1 .

 1 09 mmol), 4-iodobenzenesulfonyl chloride (300 mg, 0.99 mmol) and N,N-diisopropylethylamine (345 μL, 1.98 mmol) in CH 2 Cl 2 (5 mL) overnight. The crude product was purified by flash chromatography on silica gel (cHex to cHex/EtOAc: 7/3) affording compound 93 as a white solid (207 mg, 64%). LC tr = 4.04 min, MS (ESI-): m/z = 320 [M -H] -.1 H NMR (500 MHz, DMSO-d 6 ) δ ppm: chromatography on silica gel (cHex to cHex/EtOAc: 5/5) affording compound 143 as a yellowish solid (528 mg, 76%). LC tr = 3.35 min, MS (ESI+): m/z = 226 [M + H] + . 1 H NMR (500 MHz, DMSO-d 6 ) δ ppm: 7.95 (br s, 1H),

1 .

 1 51 mmol),4-propylbenzenesulfonyl chloride (300 mg, 1.37 mmol) and N,N-diisopropylethylamine (478 μL, 2.74 mmol) in CH 2 Cl 2 (5 mL) overnight. The crude product was purified by flash chromatography on silica gel (cHex to cHex/EtOAc: 7/3) affording compound 108 as a white solid (149 mg, 45%). LC tr = 4.31 min, MS (ESI+): m/z = 238 [M + H] + . 1 H NMR (500 MHz, DMSO-d 6 ) δ ppm: 8.04 (t, J = 5.1 Hz, 1H), 7.71-7.70 (m, 2H), 7.40-7.38 (m, 2H), 3.67-3.65 (m, 2H), 3.02 (t, J = 2.5 Hz, 1H), 2.63 (t, J = 7.4 Hz, 2H), 1.60 (sep, J = 7.4 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H). 13 C NMR (126 MHz, DMSO-d 6 ) δ ppm: 147.

  07 mmol) in DMF (3 mL) were added K 2 CO 3 (573 mg, 4.15 mmol) and propargylbromide (216 µL, 2.28 mmol). The resulting solution was heated at 60°C and stirred overnight. The mixture was then diluted in water and extracted three times with ethyl acetate. Combined organic layers were dried over MgSO 4 , filtered and concentrated under reduced pressure affording the desired product as a brown solid (352 mg, 92%). LC tr = 4.81 min, MS (ESI+):m/z = no ionisation [M + H] + , (ESI-): m/z = no ionisation [M -H] -.1 H NMR (500 MHz, DMSO-d 6 ) δ ppm: 7.41 (s, 4H), 3.87 (d, J = 2.6 Hz, 2H), 3.16 (t, J = 2.6 Hz, 1H).13 C NMR (126 MHz, DMSO-To a solution of4-chlorophenol (300 mg, 2.33 mmol) in DMF (3 mL) were added K 2 CO 3 (645 mg, 4.67 mmol) and propargylbromide (243 µL, 2.57 mmol). The resulting solution was heated at 60°C and stirred overnight. The mixture was then diluted in water and extracted three times with ethyl acetate. Organic layers were combined and solvents were evaporated under reduced pressure and the residue was purified through flash chromatography on silica gel (cHex to cHex/EtOAc: 8/2) affording compound 119 as a brown solid (376 mg, 96%). LC tr = 4.55 min, MS (ESI+): m/z = no ionisation [M + H] + , (ESI-): m/z = no ionisation [M -H] -.1 H NMR (500 MHz, ) δ ppm: 7.37-7.34 (m, 2H), 7.03-6.99 (m, 2H), 4.80 (d, J = 2.4 Hz, 2H), 3.59 (t, J = 2.3 Hz, 1H).13 C NMR (126 MHz, DMSO-d 6 ) δ ppm: 156.0, 129.2 (2C), 125.0, 116.7 (2C), 78.9, 78.5, 55.7.

  Compound 82 was synthesized according to the general procedure J, using the Boc-protected intermediate 81 (440 mg, 1.12 mmol), 4 N HCl in dioxane (5 mL) in a mixture CH 2 Cl 2 /EtOH (10 mL, 5:5 v/v) overnight. Compound 82 was obtained as yellow oil (369 mg, quant. yield) and was used in the next step without further purification. LC tr = 2.15 min, MS (ESI-): m/z = 291 [M -H] -.1 H NMR (DMSO-d 6 ) δ ppm: 10.55 (s, 1H), 8.44 (br s, 3H), 7.65-7.60 (m, 2H), 7.44-7.41 (m, 2H), 4.13-4.04 (m, 2H), 3.96-3.91 (m, 2H), 3.68-3.63 (m, 1H), 1.79-1.61 (m, 2H), 1.50 (sep, J = 6.7 Hz, 1H), 1.15 (t, J = 7.1 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H), 0.87(d, J = 6.7 Hz, 3H).13 C NMR (DMSO-d 6 ) δ ppm:

[ 3 -

 3 [(2-Ethoxycarbonyl-4-methyl-pentanoyl)amino]phenyl]methylammonium;chloride (111) Compound 111 was synthesized according to the general procedure J, using the Boc-protected intermediate 110 (495 mg, 1.26 mmol), 4 N HCl in dioxane (7 mL) in a mixture CH 2 Cl 2 /EtOH (14 mL, 7:7 v/v) overnight. Compound 111 was obtained as yellow oil (415 mg, quant. yield) and was used in the next step without further purification. LC tr = 2.82 min, MS (ESI+): m/z = 293 [M + H] + , 585 [2M + H] + . 1 H NMR (500 MHz, DMSO-d 6 ) δ ppm: 10.54 (s, 1H), 8.39 (br s, 3H),

  Compound 83 was synthesized according to the general procedure A, using amine 82 (350 mg, 0.93 mmol), ZnCl 2(7.6 mg, 0.05 mmol), K 2 CO 3 (512 mg, 3.7 mmol), anhydrous N,N- diisopropylethylamine (177 μL, 1.02 mmol) and diazo transfer reagent (301 mg, 1.11 mmol) in anhydrous MeOH (5 mL) overnight. The crude product was purified by flash chromatography on silica gel (cHex to cHex/EtOAc: 8/2) affording compound 84 as a yellowish solid (176 mg, 58%). LC tr = 3.02 min, MS (ESI-): m/z = 317[M -H] -.1 H NMR (300 MHz, CDCl 3 +TMS) δ ppm: 8.75 (s, 1H),7.58-7.56 (m, 2H), 7.28-7.24 (m, 2H), 4.28 (br s, 2H), 4.26-4.19 (m, 2H), 3.46 (t, J = 7.7 Hz, 1H), 1.90-1.85 (m, 2H), 1.64 (sep, J = 6.7 Hz, 1H), 1.30 (t, J = 7.1 Hz, 3H), 0.95 (d, J = 6.7 Hz, 6H).13 C NMR (75 MHz, CDCl 3 +TMS) δ ppm: 172.

[ 4 -

 4 (Azidomethyl)phenyl]ammonium;chloride (171) Compound 171 was synthesized according to the general procedure J, using the Boc-protected intermediate 170 (550 mg, 2.22 mmol), 4 N HCl in dioxane (10 mL) in a mixture CH 2 Cl 2 /EtOH (20 mL, 10:10 v/v) overnight. Compound 171 was obtained as an orange solid (409 mg, quant. yield) and was used in the next step without further purification. LC tr = 1.40 min, MS (ESI+): m/z = 149 [M + product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 95/5) affording compound 123 as a white powder (132 mg, 59%). LC tr = 4.93 min, MS (ESI+): m/z = 654 [M + H] + . 1 H NMR (500 MHz, DMSO-d 6 ) δ ppm: 10.29 (s, 1H), 8.04 (s, 1H),

  Compound 124 was synthesized according to the general procedure H, using azide 83 (100 mg, 0.31 mmol), alkyne 116(89.5 mg, 0.31 mmol), copper (II) sulfate pentahydrate(15.7 mg, 0.06 mmol) and sodium ascorbate(31.1 mg, 0.16 mmol) in DMF(5.5 mL) and H 2O (4.5 mL) overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 98/2) affording compound 124 as a white solid (141 mg, 60%). LC tr = 4.61 min, MS (ESI+): m/z = 604 [M+H] + . 1 H NMR (500 MHz, DMSO-d 6 ) δ ppm: 10.28 (s, 1H), 9.05 (t, J = 5.6 Hz, 1H),7.98 (s, 1H), 7.85-7.83 (m, 2H), 7.65-7.63 (m, 2H), 7.57-7.55 (m, 2H), 7.30-7.28 (m, 2H), 5.49 (s, 2H), 4.47 (d, J = 5.7 Hz, 2H), 4.12-4.05 (m, 2H), 3.55 (dd, J = 8.7 and 6.2 Hz, 1H), 1.77-1.71 (m, 1H), 1.67-1.62 (m, 1H), 1.48 (sep, J = 6.7 Hz, 1H), 1.15 (t, J = 7.1 Hz, 3H), 0.88 (d, J = 6.7 Hz, 3H), 0.87(d, J = 6.7 Hz, 3H).13 C NMR (126 MHz, DMSO-d 6 ) δ ppm: 169.

  To a microwave tube filled with dioxane (6.0 mL) and degassed with N 2 during 10 min was added Cp*RuCl(COD)(16.7 mg, 0.04 mmol, 0.05 eq) and the mixture was stirred under N 2 at room temperature during 2 min. Then, 83 (280 mg, 0.88 mmol) and N-boc-propargylamine (136 mg, 0.88 mmol) were added, the tube was sealed and the reaction media was heated at 80°C and stirred overnight. After, dioxane was evaporated under reduced pressure and the crude was purified through flash chromatography on silica gel column (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 95/5) affording compound 184 as brown oil (295 mg, 70%). LC tr = 4.40 min, MS (ESI+): m/z= 947 [2M + H] + , 474 [M + H] + , 418 [M -tBu + H] + , 374 [M -Boc + H] + . 1 H NMR (500 MHz, DMSO-d 6 ) δ ppm: 10.28 (s, 1H), 7.58-7.56 (m, 2H), 7.53 (s, 1H), 7.46 (t, J = 5.5 Hz, 1H), 7.17-7.16 (m, 2H), 5.54 (s, 2H), 4.18 (d, J = 5.7 Hz, 2H), 4.13-4.06 (m, 2H), 3.56 (dd, J = 8.7 and 6.1 Hz, 1H), 1.78-1.72 (m, 1H), 1.68-1.62 (m, 1H),

  Compound 184 (290 mg, 0.61 mmol) was dissolved in ethanol and dichloromethane(5 mL, 2.5:2.5 v/v) and 4 N HCl in dioxane (2.5 mL) was added. The mixture was stirred overnight at room temperature. Then, solvents were evaporated to give compound 186 as a yellowish oil (251 mg, yield considered quantitative). LC tr = 3.04 min, MS (ESI+): m/z = 374 [M + H] + .1 H NMR (500 MHz, ) δ: 10.47 (s, 1H), 8.74 (br s, 3H),7.89 (s, 1H), 7.61-7.59 (m, 2H), 7.21-7.19 (m, 2H), 5.68

Table 1 : Characteristics of the KTGS Pockets adapted from Hirsch et al.

 1

Table 2 : Biochemical evaluation of the synthesized analogs adapted from Stratikos et al. (62)

 2

Table 3 : Biological activity of the phosphinic 1A, 1B, 2A, 2B adapted from Stratikos et al. (63) hERAP1 hERAP2

 3

	Compound	IC 50 , nM	IC 50 , nM
	Phosphinic 1A [R,S]	520 ± 75	547 ± 110
	Phosphinic 1B [R,R]	513 ± 51	571 ± 95
	Phosphinic 2A [R,S,S]	33 ± 5	11 ± 2
	Phosphinic 2B [R,R,S]	3,600 ± 500	1,700 ± 200

Table 5 : Biochemical activities of the selected hits on ERAP1 and ERAP2

 5

Table 6 : Comparison of ERAP activities between 1,4-and 1,5-triazoles

 6

Table 7 : Biochemical activities of ERAP2 hit 1 and 20-22

 7

	c

Table 9 : Comparison of ERAP activities between β-and α-1,4-triazoles

 9

Table 11 : Biochemical activities of the compounds 45 and 63-68

 11

Table 12 : Biochemical activities of the compounds 35, 56 and 68-71

 12

Table 13 : comparison between the compounds 56 and 81 according to their activity, LogD, solubility, plasma and microsomal stabilities

 13

	Compound	R1	R2	IC 50 (hERAP2) (μM)	IC 50 (hERAP1) (μM)
	35	-H	-phenyl	0.8	> 100
	56	-H	-2-pyridine	0.5	> 100
	68	-benzyl	-phenyl	> 100	> 100
	69	-tert-butyl	-phenyl	0.3	> 100
	70	-tert-butyl	-2-pyridine	0.07	> 100
	71	-methyl	-2-pyridine	0.004	> 100

Table 14 : Biochemical activities of the compound 12 and 74-75 Due

 14 Then, as in α-1,4-series, the phenol moiety needs to be investigated to know if it could have a positive impact on the activity and physicochemical properties; 12 displaying acceptable ADME profile: LogD = 2.7, solubility = 179 μM, plasma stability (mouse) t 1/2 > 6 h and microsomal stability t 1/2 = 15.8 min.

	a	b
	8
	72: R= 2-thiophene (30%)	74: R= 2-thiophene (62%)
	73: R= 5-(2-pyridyl)-2-thiophene (54%)	75: R= 5-(2-pyridyl)-2-thiophene (34%)

to a lack of time, only two analogs were synthesized; exploring the phenylthiophenyl group. Therefore, more analogs must be synthesized to extend our scope of investigation

  1, -2 and -3. Strikingly, each compound is selective for the isoform 2 of the ERAPs. 71 displays

IRAP and APN inhibition but is still selective for ERAP2 (by 50-fold compare to IRAP and -250 compared to APN); these two enzymes being involve in the hormone and hormone peptide degradations (e.g. oxytocin, vasopressin or angiotensin III). 71 did not exhibit inhibition of the MMPs -1, -2 and -3 but showed activity on TACE.

Concerning 12 (β-1,5 series), no ERAP1 inhibition was observed but it showed activities on IRAP and APN and, in particular, a lower IC 50 on IRAP than on ERAP2 (0.1 μM for IRAP vs 0.8 μM for ERAP2).

Table 15 : Selectivity profiles of 12 and 71

 15

	Compound	ERAP2	ERAP1	IRAP	APN	TACE	MMP -1, -2, -3
	71	IC 50 = 0.004 μM	IC 50 > 100 μM	IC 50 = 0.2 μM	IC 50 = 1.0 μM	88% inh @ 20μM	< 30% inh @ 20μM
	12	IC 50 = 0.8 μM	IC 50 > 100μM	IC 50 = 0.1 μM	IC 50 = 5.0 μM	To be measured	To be measured
	Thus, further SAR studies are required to refine and enhance the selectivity profile of our
	ERAP2 inhibitors, in particular on IRAP.

Table 16 : IC 50 , LE and LLE comparisons between the starting azide, the most potent hit (1,4 and 1,5-regioisomers) and a optimized compound in α-1,4 family. LE and LLE were calculated using Datawarrior software from the IC 50

 16

	Compound	Chemical structure	IC 50 (hERAP2)	Ligand Efficiency (LE)	Lipophilic Ligand Efficiency (LLE)
	Z4		59.0 μM	0.3	3.8
	Hit E1		3.0 μM	0.2	3.2
	71		0.004 μM	0.3	7.1
	12		0.8 μM	0.2	3.7

Table 17 : Total number of invasive isolates of P. aeruginosa tested and % of isolates with resistant phenotype in 2019 adapted from the ECDC (70) Antimicrobial group Total number of invasive isolates tested % with resistant phenotype

 17

	Piperacillin + tazobactam resistance	19 355	16.9
	Ceftazidime resistance	19 849	14.3
	Carbapenem (imipenem/meropenem) resistance	20 127	16.5
	Fluoroquinolone (ciprofloxacin/levofloxacin) resistance	20 273	18.9
	Aminoglycoside (gentamicin/netilmicin/tobramycin) resistance	20 109	11.5
	Combined resistance to >3 antimicrobial groups (among
	piperacillin + tazobactam, ceftazidime, carbapenems,	20 296	12.1
	fluoroquinolones and aminoglycosides)

Table 18 : Cytokine and chemokine concentrations following chronic infection by wt RP45 and ΔlasB RP45 adapted from Ciganal et al. (108)

 18

Table 19 : Table of LasB specificities according to the amino acids adapted from MeROPs base

 19

Chemical synthesis of the selected hits from Z10

	DIPEA		93: R= -4-iodobenzene (64%) 94: R= -1,7,7-trimethylnorbornan-2-one (64%)
	CH 2 Cl 2		95: R= -4-biphenyl (91%)
	N,O-dimethylhydroxylamine		Bestmann-Ohira reagent	1. HCl (4N dioxane) 2. 4-fluorobenzoylchloride,
	EDC, HOBt, Et 3 N	LiAlH 4		K 2 CO 3	Et 3 N
	DMF		THF		MeOH	CH 2 Cl 2 / MeOH
	80%		87%		74%	69%
			96	97	98	99
	propargylbromide
	K 2 CO 3
	DMF
	96%
			100
			80
			e
					Hit L6: R2= -N-ethyl-2-methyl-propane-1-
					sulfonamide (68%)
					Hit L7: R2= -N-ethyl-4-methyl-
					benzenesulfonamide (34%)
					Hit L8: R2= -N-ethyl-4-propyl-
					benzenesulfonamide (34%)
					Hit L10: R2= -N-[2-(4-hydroxyphenyl)ethyl]
					butanamide (52%)
			2 O (4:1), rt., overnight, 77%; b) tert-butyl N-(2-aminoethyl)carbamate,
	EDC.HCl, HOBt, DIPEA, CH	2 Cl	2 , rt., overnight, 80%; c) HCl 4M in dioxane, EtOH, 0 °C to rt., overnight, quantit. yield; d)
	azide-N-diazoimidazole-1-sulfonamide hydrogen chloride, K	2 CO 3 , ZnCl	2 , DIPEA, MeOH or EtOH, 0 °C to rt., overnight,

19% (if MeOH: 86), 22% (if EtOH: 101); e) alkyne (106, 107, 108 or 109), CuSO 4 (5H 2 O), sodium ascorbate, DMF/H 2 O (1.2:1), rt., overnight, 31-58%; f) aq. hydroxylamine (50% in water w/w), KCN (cat.), MeOH, rt., overnight, 34-68%. Scheme 22 :

Table 20 : Biochemical activities of the selected hits

 20

Synthesis of the final compounds 131-138 and their respective intermediates 123-130

	Eight compounds were synthesized using the same synthetic pathway described for the hits
	derived from Z9 (Scheme 20), a copper-catalyzed click reaction followed by aminolysis
	(Scheme 27).
	Reagents and conditions: a) alkyne (115, 116, 118-122, N-Boc-propargylamine), CuSO	4	(5H 2 O), sodium ascorbate,
	DMF/H	2 O (1.2:1), rt., overnight, 51-85%; b) aq. hydroxylamine (50% in water w/w), KCN (cat.), MeOH, rt., overnight-96
	h, 15-55%.
		Scheme 27 :
	A.	DIPEA			B.	Et 3 N
		CH 2 Cl 2				CH 2 Cl 2
		97%				49%
				115		116
		propargylbromide			propargylbromide
	C.	Na 2 CO 3 DMF		D.	K 2 CO 3 DMF
		89%			118: R1= -NH, R2= -I (28%)
				117		119: R1= -O, R2= -Cl (95%)
						120: R1= -S, R2= -Cl (92%)
		Bestmann-Ohira
			reagent
	E.	n	K 2 CO 3 MeOH	n	121: n=1, X=Cl (32%) 122: n=0, X=I (43%)
		n=0,1
					-84 -

Scheme 28 : Synthesis of compound 139

Reagents and conditions: a) aq. hydroxylamine (50% in water w/w),

KCN (cat.)

, MeOH, rt., overnight, 38%; (b) 117, CuSO 4 (5H 2 O), sodium ascorbate, DMF/H 2 O (1.2:1), rt., overnight, 15%.

Table 21 : Biochemical activities of the compounds 131-140

 21

Table 22 : Comparison study between Hit L1, 131 and 136 according to their activity, solubility and metabolic stability in mouse

 22

Table 23 : Biochemical activities of the compounds 160-169

 23

	a	b
	83	150: R= -4-fluorobenzene (76%)	160: R= -4-fluorobenzene (38%)
		151: R= -4-chlorobenzene (73%)	161: R= -4-chlorobenzene (47%)
		152: R= -4-methoxybenzene (76%)	162: R= -4-methoxybenzene (26%)
		153: R= -2-chlorobenzene (82%)	163: R= -2-chlorobenzene (7%)
		154: R= -3-chlorobenzene (74%)	164: R= -3-chlorobenzene (55%)
		155: R= -3,4-dichlorobenzene (77%)	165: R= -3,4-dichlorobenzene (18%)
		156: R= -4-chloro-ethylbenzene (45%)	166: R= -4-chloro-ethylbenzene (23%)
		157: R= -benzene (68%)	167: R= -benzene (17%)
		158: R= -cyclohexyl (47%)	168: R= -cyclohexyl (14%)
		159: R= -isobutyl (51%)	169: R= -isobutyl (48%)

Table 24 : Comparative study between Hit L1, 160, 161, 165 and L94 according to their activity, solubility, clogP and metabolic stability in mouse. *cLogP values were calculated using Datawarrior.

 24

	Compound	R	IC

Thus, regarding these results, the compounds 160 and 169 were chosen for in vivo pharmacokinetic studies and further biological evaluation.

50 LasB (nM) cLogP* Solubility [μM] Met Stab in mouse liver S9 t 1/2 [min]

	Hit L1	2.4 0.1	1.5	142	30.1 1.5
	161	1.7 0.1	1.6	> 200	33.9 1.0
	165	1.6 0.1	2.2	117	10.7 0.6
	160	3.8 0.1	1.1	> 200	65.5 0.7
	169	5.2 0.2	0.9	> 200	> 120

Table 25

 25

		12%		88%
	MW=486  ESI+		MW=612  ESI+ observed=613
	observed=487
	a		b
	174: R1= -isopropyl, R2= -methyl	180: R1= -isopropyl (43%)		182: R1= -isopropyl (55%)
	175: R1= -methylcyclopropyl, R2= -ethyl	181: R1= -methylcyclopropyl (45%)	183: R1= -methylcyclopropyl (47%)
		KOH
		EtOH/H 2 O
		or
		MeOH/H 2 O	173a: R1= -benzyl, R2= -ethyl (72%)
			174a: R1= -isoproyl, R1= -methyl (77%)
			175a: R1= -methylcycloproyl, R2= -ethyl (71%)

. Removal of the isobutyl group resulted in 1log loss of the activity (178). The introduction of benzyl (179) and methycyclopropyl side chains (183) led to a slight decrease in the IC 50 , value, suggesting that the corresponding binding pocket is not very deep. The

Table 25 : Biochemical activities of the compounds Hit L1, 178-179 and 182-183

 25

Table 27 : Selectivity and cytoxicity profiles of Hit L1, 160, 169 and Z9 with their antibacterial activities against the strain PA-14

 27

			Hit L1	160	169	Z9
		MMP1	> 100	> 100	> 100	> 100
		MMP2	> 100	> 100	> 100	> 100
	Selectivity	MMP3	> 100	> 100	> 100	> 100
	IC 50 (μM)	HDAC3	> 100	> 100	> 100	> 100
		HDAC8	> 100	> 100	> 100	> 100
		TACE	> 100	> 100	> 100	1.6 ± 0.2
		COX-1	> 100	> 100	> 100	> 100
		HepG2	> 100	> 100	> 100	> 100
	Cytotoxicity IC 50 (μM)	HEK293	> 100	> 100	> 100	> 100
		A549	> 100	> 100	> 100	> 100
	Antibacterial
	activity	PA-14	> 100	> 100	> 100	> 100
	IC 50 (μM)

Table 28 : IC 50 , LE and LLE comparison between Z9 and Hit L1. LE and LLE were calculated using Datawarrior software from the IC 50

 28

Table 29 : Conditions screening. a adapted from Camberlein et al. (138)

 29

			NaN 3
			Cp 2 Ni, Xantphos
			Cs 2 CO 3			1,4-regioisomer
			solvent, temp., t
		1a	2A		3aA	4aA
	Entry	Solvent	Temperature (°C)	Time (h)	cYield (%) c	Ratio 3aA/4aA d
	1	H 2 O	rt	2	-e	-e
	2	DMF	rt	2	71	88:12
	3	EtOH	rt	2	-e	-e
	4	DCM	rt	2	-e	-e
	5	THF	rt	2	-e	-e
	6	DMF	50 °C (MW)	1	73	93:7
	7	DMF	80 °C (MW)	1	49	64:36
	8	DMF	100 °C (MW)	1	-e	-e
	9	DMF	50 °C (MW)	4	86	92:8
	10 b	DMF	50°C (MW)	4	89	92:8

a Reaction conditions: Run on a 0.5 mmol scale of alkyne 2A (1.2 equiv), bromide 1a (1.0 equiv), NaN 3 (1.0 equiv), solvent (2 mL), Cp 2 Ni (20 mol%), Xantphos (20 mol%), Cs 2 CO 3 (1.0 equiv).

b Run on a 0.5 mmol scale of alkyne 2A (1.0 equiv), bromide 1a (1.2 equiv), NaN 3

(1.5 equiv)

, solvent (2 mL), Cp 2 Ni (20 mol%), xantphos (20 mol%), Cs 2 CO 3 (1.0 equiv).

c Calculated yield of 3aA determined by LCMS analysis from the crude product.

d

Determined by LCMS analysis from the crude product.

e No triazole formation. Cp, cyclopentadienyl; cYield, calculated yield; DCM, dichloromethane; DMF, dimethylformamide; MW, microwave irradiation; temp, temperature; rt, room temperature.

Table 30 : Substrate scope with various alkynes. a adapted from Camberlein et al. (138)

 30

		NaN 3
		Cp 2 Ni (20 mol%)
		Xantphos (20 mol%)
		Cs 2 CO 3 (1.0 equiv)	1,4-regioisomer
		DMF, 50 °C (MW), 4 h
	1a	2A-2H	3aA-3aH	4aA-4aH
	3aA	3aB	3aC	3aD
	72 (64)	24 (37)	34 (84)	76 b
	89:11	66:34	91:9	>99:1
	3aE	3aF	3aG	3aH
	73 b	36 (27)	56 (70)	34 (34)
	92:8	86:14 c	89:11 c	67:33
	a Reaction conditions: Run on a 0.3-0.6 mmol scale of alkyne 2 with 1a (1.2 equiv), NaN 3 (1.5 equiv), Cp 2 Ni (20 mol%),

Table 31 : Substrate scope with various bromides. a adapted from Camberlein et al. (138)

 31

		NaN 3
		Cp 2 Ni (20 mol%)
		Xantphos (20 mol%)
	R-Br	Cs 2 CO 3 (1.0 equiv)		1,4-regioisomer
		DMF, 50 °C (MW), 4 h
	1a-1l	2A	3aA-3lA	4aA-4lA
	3aA	3bA		3cA	3dA
	72 (64)	67 (69)		68 (64)	68 b
	89:11	85:15		85:15	97:3
	3eA	3fA		3gA	3hA
	84 (73)	73 (79)		31 (64)	89 (25)
	93:7	95:5		94:6	12.5:1
	3iA	3jA		3kA	3lA
	46 (65)	45 (67)		64 (90)	34 (34)
	91:9	94:6		96:4	81:19
	a Reaction conditions: Run on a 0.49 mmol scale of alkyne 2A with 1 (1.2 equiv), NaN 3 (1.5 equiv), Cp 2 Ni (20 mol%),
	xantphos (20 mol%), Cs 2 CO 3 (1.0 equiv), DMF (2.0 mL), 50 °C (MW), 4 h. Isolated yields (%) of 3 obtained after SFC

purification; 3/4 ratio determined from the crude product by SFC analysis unless noted otherwise; in brackets: calculated yields (%) of 3 obtained after flash column chromatography purification (in mixture with 4). b Isolated yield (%) of 3 obtained after flash column chromatography.

Boc, tert-butoxycarbonyl; Cp, cyclopentadienyl; Me, methyl; Ph, phenyl; t-Bu, tert- butyl.

Table 32 : Structural features of ERAP2 and LasB KTGS ligands

 32

  mmol), ZnCl 2(10.2 mg, 0.08 mmol), K 2 CO 3 (691 mg, 5.0 mmol), anhydrous N,N- diisopropylethylamine (240 μL, 1.38 mmol) and diazo transfer reagent (407 mg, 1.5 mmol) in anhydrous MeOH (5mL) overnight. Compound 23 was obtained as pale brown oil (277 mg, 93%) and was used in the next step without further purification. LC tr = 1.40 min, MS (ESI-): m/z = 206[M - H] -.1 H NMR (300 MHz, ) δ ppm: 7.10-7.06(m, 2H), 6.75-6.70 (m, 2H), 4.08 (dd, J = 8.4

	and 5.2 Hz, 1H), 3.07 (dd, J = 14.1 and 5.2 Hz, 1H), 2.89 (dd, J = 14.1 and 8.4 Hz, 1H). 13 C NMR (75
	MHz, MeOD-d 4 ) δ ppm: 173.

  1.25 mmol), ZnCl 2(10.2 mg, 0.08 mmol), K 2 CO 3 (691 mg, 5.0 mmol), anhydrous N,N- diisopropylethylamine (240 μL, 1.38 mmol) and diazo transfer reagent (407 mg, 1.5 mmol) in anhydrous MeOH (5mL) overnight. Compound 29 was obtained as brown oil (288 mg, 89%) and was used in the next step without further purification. LC tr = 1.95 min, MS (ESI-): m/z = 229[M -H] -.1 H NMR (300MHz, ) δ ppm: 7.58 (1H, m, 1H), 7.34, (dt, J = 7.9 and 1.6 Hz, 1H), 7.14 (s, 1H),

2-azido-3-(4-hydroxyphenyl)propanoate (38)

	Compound 38 was synthesized according to the general procedure E, using carboxylic acid 37 (202
	mg, 0.98 mmol) and thionyl chloride (142 μL, 1.95 mmol) in MeOH (5 mL) overnight. Compound 38
	was obtained as brown oil (130 mg, 60%) and was used in the next step without further purification.
	LC tr = 2.38 min, MS (ESI-): m/z = 220 [M -H] -. 1 H NMR (300 MHz, MeOD-d 4 ) δ ppm: 7.07-7.02
	(m, 2H), 6.74-6.70 (m, 2H), 4.14 (dd, J = 8.1 and 5.7 Hz, 1H), 3.73 (s, 3H), 3.05 (dd, J = 14.0 and 5.7
	Hz, 1H), 2.90 (dd, J = 14.0 and 8.1 Hz, 1H). 13 C NMR (75 MHz, MeOD-d 4 ) δ ppm: 172.2, 157.6,
	131.3 (2C), 128.1, 116.3 (2C), 64.6, 52.9, 37.7.
	Methyl (2S)-2-azido-3-(4-methoxyphenyl)propanoate (62)
	Compound 62 was synthesized according to the general procedure E, using carboxylic acid 60 (257
	mg, 1.16 mmol) and thionyl chloride (169 μL, 2.32 mmol) in MeOH (2.4 mL) overnight. Compound
	62 was obtained as orange oil (251 mg, 83%) and was used in the next step without further
	purification. LC tr = 2.85 min, MS (ESI+), MS (ESI-): no ionization. 1 H NMR (300 MHz, MeOD-d 4 )
	δ ppm: 7.16-7.13 (m, 2H), 6.87-6.84 (m, 2H), 4.18 (dd, J = 8.1 and 5.6 Hz, 1H), 3.77 (s, 3H), 3.74 (s,
	3H), 3.08 (dd, J = 14.0 and 5.6 Hz, 1H), 2.92 (dd, J = 14.0 and 8.1 Hz, 1H). 13 C NMR (75MHz,
	MeOD-d 4 ) δ ppm: 172.

2-azido-3-(4-benzyloxyphenyl)propanoate (61)

  LC tr = 3.08 min, MS (ESI+): m/z = 292 [M + H] + . 1 H NMR (300 MHz, CDCl 3 ) δ ppm: 7.62-

	filtered on silica gel (cHex to cHex/EtOAc: 8/2) affording compound 14 as a beige powder (95 mg,
	83%).
	Compound 61 was synthesized according to the general procedure E, using carboxylic acid 59 (357
	mg, 1.2 mmol) and thionyl chloride (175 μL, 2.4 mmol) in MeOH (5 mL) overnight. Compound 61
	was obtained as brown oil (339 mg, 77%) and was used in the next step without further purification.
	LC tr = 3.30 min, MS (ESI+): m/z = 284 [M -2N +H] + . 1 H NMR (300 MHz, MeOD-d 4 ) δ ppm: 7.44-
	7.27 (m, 5H), 7.17-7.11 (m, 2H), 6.95-6.90 (m, 2H), 5.05 (s, 2H), 4.17 (dd, J = 8.2 and 5.6 Hz, 1H),

2-ynyl-5-(2-pyridyl)thiophene-2-sulfonamide (44)

	Compound 44 was synthesized according to the general procedure F, using propargylamine (81 μL,
	1.27 mmol), 5-(2-pyridyl)thiophene-2-sulfonyl chloride (300 mg, 1.16 mmol) and N,N-
	diisopropylethylamine (241 μL, 1.39 mmol) in CH 2 Cl 2 (5 mL) overnight. The crude product was
	filtered on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 9/1) affording compound 44 as a pale yellow solid
	(226 mg, 70%). LC tr = 2.42 min, MS (ESI+): m/z = 279 [M + H] + . 1 H NMR (300 MHz, MeOD-d 4 ) δ
	ppm: 8.53 (ddd, J = 4.9, 1.7 and 1.1 Hz, 1H), 7.91-7.82 (m, 2H), 7.67 (d, J = 4.0 Hz, 1H), 7.61 (d, J =
	4.0 Hz, 1H), 7.34 (ddd, J = 7.0, 4.9 and 1.7 Hz, 1H), 3.86 (d, J = 2.5 Hz, 2H), 2.53 (t, J = 2.5 Hz, 1H).

13

C NMR (75 MHz, MeOD-d 4 ) δ ppm: 152.

  overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 95/5) affording compound 12 as a white solid after lyophilization(54 mg, 63%).Purity: 99%, LC tr = 2.37 min, MS (ESI+): m/z = 514 [M + H] + .1 H NMR (300 MHz, ) δ ppm: 7.71-7.67

	10 (100 mg, 0.2
	mmol), KCN (2.54 mg, 0.04 mmol) and aq. NH 2 OH (2.6 mL, 50% w/w in water) in MeOH (2.6 mL)
	-135 -

  . HRMS-ESI+ (m/z) calcd. for C 23 H 24 N 5 O 5 S 2 [M + H] + 514.1226, found 514.1219.

	(3S)-4-(4-Hydroxyphenyl)-3-[5-[(2-thienylsulfonylamino)methyl]triazol-1-yl]butanehydroxamic
	acid (74)
	Compound 74 was synthesized according to the general procedure D, using ester 72 (22 mg, 0.05
	mmol), KCN (0.66 mg, 0.01 mmol) and aq. NH 2 OH (1 mL, 50% w/w in water) in MeOH (1 mL)
	overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to
	CH 2 Cl 2 /MeOH: 9/1) affording compound 74 as a white solid after lyophilization (14 mg, 62%). Purity:
	99%, LC tr = 1.80 min, MS (ESI+): m/z = 437 [M + H] + . 1 H NMR (300 MHz, MeOD-d 4 ) δ ppm: 7.81

  1. HRMS-ESI+ (m/z) calcd. for C 17 H 20 N 5 O 5 S 2 [M + H] + 438.0906, found 438.0910.Compound 75 was synthesized according to the general procedure D, using ester 73 (57 mg, 0.1 mmol), KCN (1.45 mg, 0.02 mmol) and aq. NH 2 OH (1.5 mL, 50% w/w in water) in MeOH(1.5 mL) overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 9/1) affording compound 75 as a white solid after lyophilization(20 mg, 34%).

	(3S)-4-(4-Hydroxyphenyl)-3-[5-[[[5-(2-pyridyl)-2-thienyl]sulfonylamino]methyl]triazol-1-
	yl]butanehydroxamic acid (75)
	Purity: 99%, LC tr = 2.07 min, MS (ESI+): m/z = 515 [M + H] + . 1 H NMR (300 MHz, DMSO-d 6 ) δ
	ppm: 10.54 (br s, 1H), 9.26 (s, 1H), 8.79 (br s, 1H), 8.60 (ddd, J = 4.8, 1.7 and 0.9 Hz, 1H), 8.38 (br s,
	1H), 8.07 (dt, J = 7.

-4-(1H-Indol-3-yl)-3-[5-[[(5-phenyl-2-thienyl)sulfonylamino]methyl]triazol-1- yl]butanehydroxamic acid (13)

  Compound 13 was synthesized according to the general procedure D, using ester 11(116 mg, 0.22 mmol),KCN (2.82 mg, 0.04 mmol) and aq. NH 2 OH (3 mL, 50% w/w in water) in MeOH (3 mL) overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 95/5) affording compound 13 as a white solid after lyophilization (99 mg, 84%). Purity: 99%, LC tr= 2.62 min, MS (ESI+): m/z = 537 [M + H] + . 1 H NMR (300 MHz, DMSO-d 6 ) δ ppm: 10.87 (br s, 1H), 8.79 (br s, 1H),

	. HRMS-ESI+ (m/z) calcd. for
	C 22 H 23 N 6 O 5 S 2 [M + H] + 515.1171, found 515.1181.
	(3R)

  0. HRMS-ESI+ (m/z) calcd. for C 25 H 25 N 6 O 4 S 2 [M + H] + 537.1394, found 537.1379.

	(2S)-3-(4-Hydroxyphenyl)-2-[5-[[(5-phenyl-2-thienyl)sulfonylamino]methyl]triazol-1-
	yl]propanehydroxamic acid (28)
	Compound 28 was synthesized according to the general procedure D, using ester 27 (75 mg, 0.15
	mmol), KCN (1.96 mg, 0.02 mmol) and aq. NH 2 OH (5.4 mL, 50% w/w in water) in MeOH (5.4 mL)
	overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to
	CH 2 Cl 2 /MeOH: 9/1) affording compound 28 as a white solid after lyophilization (19 mg, 25%). Purity:
	100%, LC tr = 2.33 min, MS (ESI-): m/z = 498 [M -H] -. 1 H NMR (DMSO-d 6 ) δ ppm: 9.24-9.14 (m,
	2H), 7.72 (dd, J = 8.3 and 1.5 Hz, 2H), 7.55 (dd, J = 6.2 and 3.9 Hz, 2H), 7.51-7.43 (m, 4H), 6.93 (d, J
	= 8.5 Hz, 2H), 6.60 (d, J = 8.5 Hz, 2H), 5.12 (t, J =7.5 Hz, 1H), 4.21 (br s, 2H), 3.41 (dd, J = 13.9 and

-hydroxyphenyl)-3-[4-[[methyl-[(5-phenyl-2- thienyl)sulfonyl]amino]methyl]triazol-1-yl]butanoate (17)

  . HRMS-ESI+ (m/z) calcd. for C 22 H 22 N 5 O 5 S 2 [M + H]+ 500.1062, found 500.1068.

	Methyl (3S)-4-(4Compound 17 was synthesized according to the general procedure H, using azide Z4-a (84 mg, 0.36
	mmol), alkyne 14 (104 mg, 0.36 mmol), copper (II) sulfate pentahydrate (16.2 mg, 0.07 mmol) and
	sodium ascorbate (32.1 mg, 0.18 mmol) in DMF (6 mL) and H 2 O (5 mL) overnight. The crude product
	was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /EtOAc: 85/15) affording
	compound 17 as a white solid (69 mg, 37%). LC tr = 2.75 min, MS (ESI-): m/z = 525 [M -H] -. 1 H
	NMR (300 MHz, MeOD-d 4 ) δ ppm: 7.68-7.63 (m, 2H), 7.58 (s, 1H), 7.55 (d, J = 4.0 Hz, 1H), 7.45-
	7.34 (m, 4H), 6.78-6.73 (m, 2H), 6.63-6.58 (m, 2H), 5.08-4.99 (m, 1H), 4.36-4.26 (m, 2H), 3.55 (s,
	3H), 3.15-2.95 (m, 4H), 2.65 (s, 3H).

13

C NMR (75 MHz, MeOD-d 4 ) δ ppm: 172.

)-4-(4-hydroxyphenyl)-3-[4-[[methyl-(5-phenylthiophene-2- carbonyl)amino]methyl]triazol-1-yl]butanoate (19)

	Compound 19 was synthesized according to the general procedure H, using azide Z4-a (82 mg, 0.35
	mmol), alkyne 16 (89 mg, 0.35 mmol), copper (II) sulfate pentahydrate (15.8 mg, 0.06 mmol) and
	sodium ascorbate (31.4 mg, 0.16 mmol) in DMF (6 mL) and H 2 O (5 mL) overnight. The crude product
	was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /EtOAc: 7/3) affording
	compound 19 as a white solid (28 mg, 16%). LC tr = 2.58 min, MS (ESI-): m/z = 489 [M -H] -. 1 H
	NMR (300 MHz, MeOD-d 4 ) δ ppm: 7.70-7.64 (m, 3H), 7.43-7.33 (m, 5H), 6.83-6.78 (m, 2H), 6.63-
	6.59 (m, 2H), 5.14-5.05 (m, 1H), 4.72 (br s, 2H), 4.60-4.59 (m, 1H), 3.58 (s, 3H), 3.24-3.02 (m,
	6H). 13 C NMR (75 MHz, MeOD-d 4 ) δ ppm: 172.1, 157.5, 137.0, 134.6, 132.4, 131.1 (2C), 130.2 (2C),

  H, using azide 30(70 mg, 0.32 mmol), alkyne 43(68.1 mg, 0.32 mmol), copper (II) sulfate pentahydrate(15.8 mg, 0.06 mmol) and sodium ascorbate(31.3 mg, 0.16 mmol) in DMF (5.5 mL) and H 2 O (4.5 mL) overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 95/5) affording compound 49 as colorless oil (109 mg, 78%). LC tr = 2.27 min, MS (ESI+): m/z = 437 [M + H] + .

1 H NMR (300 MHz,

) δ ppm: 7.80 (s, 1H), 7.37

(d, J = 3.7 Hz, 1H), 6.90-6.86 (m, 2H), 6.77

(dq, J = 3.7 and 1.2 Hz, 1H), 6.66-6.61 (m, 2H), 5.58 (dd, J = 9.9 and 5.5 Hz, 1H), 4.19 (s, 2H), 3.72 (s, 3H), 3.46 (dd, J = 14.3 and 5.5 Hz, 1H), 3.33 (dd, J = 14.0 and 10.2 Hz, 1H), 2.48 (d, J = 1.2 Hz, 3H).

13

C NMR (75 MHz, MeOD-d 4 ) δ ppm: 170.

0, 157.6, 149.0, 145.2, 139.1, 133.6, 131.1 (2C)

,

5-bromo-2-thienyl)sulfonylamino]methyl]triazol-1-yl]-3-(4- hydroxyphenyl)propanoate (47)

	Compound 47 was synthesized according to the general procedure H, using azide 30 (63 mg, 0.29
	mmol), alkyne 41 (79.8 mg, 0.29 mmol), copper (II) sulfate pentahydrate (12.9 mg, 0.05 mmol) and
	sodium ascorbate (25.6 mg, 0.14 mmol) in DMF (5 mL) and H 2 O (4 mL) overnight. The crude product
	was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 98/2) affording

compound 47 as pale yellow oil (140 mg, 91%). LC tr = 2.52 min, MS (ESI+): m/z = 501 and 503 [M + H] + . 1 H NMR (300 MHz, DMSO-d 6 ) δ ppm: 9.25 (s, 1H), 8.52 (br t, J = 5.8 Hz, 1H), 8.03 (s, 1H), 7.40 (d, J = 4.0 Hz, 1H), 7.31 (d, J = 4.0 Hz, 1H), 6.89 (d, J = 8.5 Hz, 2H), 6.59 (d, J = 8.5 Hz, 2H), -142 -

Methyl (2S)-3-(4-hydroxyphenyl)-2-[4-[(2-thienylsulfonylamino)methyl]triazol-1-yl]propanoate (51)

  H, using azide 30 (100 mg, 0.45 mmol), alkyne 42 (127 mg, 0.45 mmol), copper (II) sulfate pentahydrate (22.6 mg, 0.09 mmol) and sodium ascorbate(44.8 mg, 0.23 mmol) in DMF (5.5 mL) and H 2 O (4.5 mL) overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl Hz, 1H),7.16 (dd, J = 3.7 and 5.0 Hz, 1H), 6.89 (d, J = 8.5 Hz, 2H), 6.59 (d, J = 8.4 Hz, 2H),

	29 (s,
	2H), 3.71 (s, 3H), 3.46 (dd, J = 14.2 and 5.3 Hz, 1H), 3.32 (dd, J = 14.0 and 10.1 Hz, 1H). 13 C NMR
	(75 MHz, MeOD-d 4 ) δ ppm: 170.0, 157.5, 145.1 (br), 137.6, 133.8, 132.6, 131.1 (2C), 127.2, 124.9
	(br), 116.4 (2C), 114.2, 65.6, 53.5, 39.0, 38.1.
	Compound 51 was synthesized according to the general procedure H, using azide 30 (63 mg, 0.29
	mmol), alkyne 45 (57.3 mg, 0.29 mmol), copper (II) sulfate pentahydrate (12.9 mg, 0.05 mmol) and

2 /MeOH: 95/5) affording compound 48 as colorless oil (165 mg, 72%). LC tr = 2.35 min, MS (ESI+): m/z = 501 and 503 [M + H] + . 1 H NMR (300 MHz, MeOD-d 4 ) δ ppm: 7.79 (br s, 1H), 7.65 (d, J = 5.2 Hz, 1H), 7.06 (d, J = 5.2 Hz, 1H), 6.89 (d, J = 8.1 Hz, 2H), 6.64 (d, J = 8.1 Hz, 2H), 5.57 (dd, J = 9.4 and 5.3 Hz, 1H), 4.sodium ascorbate (25.6 mg, 0.14 mmol) in DMF (5 mL) and H 2 O (4 mL) overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 ) affording compound 51 as brown oil (110 mg, 86%). LC tr = 2.25 min, MS (ESI+): m/z = 423 [M + H] + . 1 H NMR (300 MHz, DMSO-d 6 ) δ ppm: 9.25 (s, 1H), 8.36 (t, J = 6.0 Hz, 1H), 8.00 (s, 1H), 7.91 (dd, J = 1.3 and 5.0 Hz, 1H), 7.58 (dd, J = 1.3 and 3.7

  H, using azide 30(70 mg, 0.32 mmol), alkyne 46(63.7 mg, 0.32 mmol), copper (II) sulfate pentahydrate (15.8 mg, 0.06 mmol) and sodium ascorbate(31.3 mg, 0.16 mmol) in DMF (5.5 mL) and H 2 O (4.5 mL) overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl

	= 10.0 and 5.6 Hz, 1H), 4.18 (s, 2H), 3.72 (s, 3H), 3.47 (dd, J = 14.3 and 5.6 Hz, 1H), 3.33 (dd, J =
	14.3 and 10.0 Hz, 1H).

2 /MeOH: 95/5) affording compound 52 as brown oil (90 mg, 62%). LC tr = 2.22 min, MS (ESI+): m/z = 423 [M + H]+. 1 H NMR (300 MHz, MeOD-d 4 ) δ ppm: 8.01 (dd, J = 3.1 and 1.3 Hz, 1H), 7.77 (s, 1H), 7.51 (dd, J = 5.1 and 3.1 Hz, 1H), 7.29 (dd, J = 5.1 and 1.3 Hz, 1H), 6.91-6.84 (m, 2H), 6.66-6.62 (m, 2H), 5.57 (dd, J 13 C NMR (75 MHz, MeOD-d 4 ) δ ppm: 170.

2R)-3-(1H-indol-3-yl)-2-[4-[[(5-phenyl-2-thienyl)sulfonylamino]methyl]triazol-1- yl]propanoate (34)

	Compound 34 was synthesized according to the general procedure H, using azide 31 (144 mg, 0.3
	mmol), alkyne 32 (98 mg, 0.3 mmol), copper (II) sulfate pentahydrate (15 mg, 0.06 mmol) and sodium
	ascorbate (29.7 mg, 0.06 mmol) in DMF (6 mL) and H 2 O (5 mL) overnight. The crude product was
	purified by flash chromatography on silica gel (cHex/EtOAc: 95/5 to 4/6) affording compound 34 as
	an orange solid (127 mg, 77%). LC tr = 2.19 min, MS (ESI-): m/z = 520 [M -H] -. 1 H NMR (300MHz,
	DMSO-d 6 ) δ ppm: 10.84 (d, J = 1.4 Hz, 1H), 8.45 (br s, 1H), 8.10 (s, 1H), 7.72-7.68 (m, 2H), 7.55 (d,
	J = 3.9 Hz, 1H), 7.49-7.40 (m, 5H), 7.31-7.29 (m, 1H), 7.09-7.04 (m, 1H), 7.01-6.87 (m, 1H), 6.90
	H, using azide 62 (128 mg, 0.48
	mmol), alkyne 44 (134.2 mg, 0.48 mmol), copper (II) sulfate pentahydrate (24.1 mg, 0.1 mmol) and

sodium ascorbate

(47.8 mg, 0.24 mmol)

in DMF (9 mL) and H 2 O (8 mL) overnight. The crude product was purified by flash chromatography on silica gel (cHex/EtOAc: 95/5 to 3/7) affording compound 67 as a white solid (160 mg, 65%). LC tr = 2.70 min, MS (ESI-): m/z = 512

[M -H]

-.

1 H NMR (300MHz, Methyl ((d, J = 2.3 Hz, 1H), 5.75 (dd, J = 9.3 and 5.9 Hz, 1H), 4,14 (s, 2H), m)

.

13

C NMR (75MHz, DMSO-d 6 ) δ ppm: 168.

-Hydroxyphenyl)-2-[4-[[(5-phenyl-2-thienyl)sulfonylamino]methyl]triazol-1- yl]propanehydroxamic acid (35)

  . HRMS-ESI+ (m/z) calcd. for C 24 H 26 N 5 O 5 S 2[M + H] + 528.1375, found 528.1376. 132.9, 130.7, 129.8 (2C), 129.2 (2C), 128.5, 126.9, 125.7 (2C), 123.7, 123.2, 115.0 (2C), 59.4, 39.5, 37.2. HRMS-ESI+ (m/z) calcd. for C 25 H 26 N 5 O 4 S [M + H] + 492.1706, found 492.1702. Compound 35 was synthesized according to the general procedure G, using ester 33 (65 mg, 0.13 mmol), KCN (0.8 mg, 0.01 mmol) and aq. NH 2 OH (0.5 mL, 50% w/w in water) in MeOH (0.5 mL) overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 95/5) affording compound 35 as a white solid after lyophilization (46 mg, 71%).

	(2S)-3-(4Purity: 99%, LC t for C 22 H 22 N 5 O 5 S 2 [M + H] + 500.1062, found 500.1064.
	(2R)-3-

R = 2.47 min, MS (ESI+): m/z = 500 [M + H] + .

1 H NMR (300 MHz,

) δ ppm: 8.09 (s, 1H), 7.

73-7.71 (m, 2H), 7.57-7.53 (m, 2H), 7.49-7.38 (m, 3H), 7.91-7.89 (m, 2H), 6.62-6.59 (m, 2H)

5.20 (t, J = 7.9 Hz, 1H), 4.17 (s, 2H), 3.25-3.18 (m, 1H), 3.13-3.05 (m, 1H).

13

C NMR (75 MHz, DMSO-d 6 ) δ ppm: 163.

8, 156.2, 149.1, 143.2, 139.7, 132.8, 132.3, 130.0 (2C), 129.4 (2C), 129.1, 125.9 (2C), 125.8, 124.0, 122.2, 115.1 (2C), 62.0, 38.3, 36.8

. HRMS-ESI+ (m/z) calcd.

(4-Hydroxyphenyl)-2-[4-[[(5-phenyl-2-thienyl)sulfonylamino]methyl]triazol-1- yl]propanehydroxamic acid (40)

  J= 13.8 and 7.6 Hz, 1H), 3.09 (dd, J = 13.8 and 8.3 Hz, 1H).13 C NMR (75 MHz, DMSO-d 6 ) δ ppm:

	Compound 40 was synthesized according to the general procedure G, using ester 39 (97 mg, 0.19
	mmol), KCN (2.5 mg, 0.04 mmol) and aq. NH 2 OH (2.5 mL, 50% w/w in water) in MeOH (2.5 mL)
	overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to
	CH 2 Cl 2 /MeOH: 95/5) affording compound 40 as a white solid after lyophilization (73 mg, 74%).

Purity: 99%, LC tr = 2.37 min, MS (ESI+): m/z = 500 [M + H] + .

1 H NMR (300 MHz,

) δ ppm: 9.32 (br s, 3H), 8.09 (s, 1H), 7.72 (br d, J = 6.9 Hz, 2H), 7.

57-7.53 (m, 2H), 7.49-7.39 (m, 3H), 6.90 (br d, J = 8.3 Hz, 2H), 6.60 (br d, J = 8.3 Hz, 2H), 5.20 (t, J = 7.6 Hz, 1H), 4.17 (s, 2H), 3.22 (dd, -149 -

-Hydroxyphenyl)-2-[4-[[[5-(2-pyridyl)-2-thienyl]sulfonylamino]methyl]triazol-1- yl]propanehydroxamic acid (56)

  . HRMS-ESI+ (m/z) calcd. for C 22 H 22 N 5 O 5 S 2 [M + H] + 500.1042, found 500.1062. Compound 56 was synthesized according to the general procedure G, using ester 50 (50 mg, 0.1 mmol), KCN (1.3 mg, 0.02 mmol) and aq. NH 2 OH (2.5 mL, 50% w/w in water) in MeOH (2.5 mL) overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH

	(2S)-3-(413.8 and 7.5 Hz, 1H), 3.09 (dd, J = 13.8 and 8.2 Hz, 1H). 13 C NMR (75 MHz, DMSO-d 6 ) δ ppm:
	163.8, 156.2, 149.1, 143.2, 139.6, 132.8, 132.2, 130.0, 129.3 (2C), 129.1, 125.9 (2C), 125.7, 124.0,
	122.2, 115.1 (2C), 62.0, 38.3, 36.7. HRMS-ESI+ (m/z) calcd. for C 21 H 21 N 6 O 5 S 2 [M + H] + 501.0996,
	found 501.1015.
	(2S)

2 Cl 2 /MeOH: 9/1) affording compound 56 as a white solid after lyophilization (32 mg, 63%). Purity: 99%, LC tr = 2.08 min, MS (ESI-): m/z = 499 [M -H] -.

1

H NMR (300 MHz, DMSO-d 6 ) δ ppm: 9.27 (s, 2H), 8.09 (s, 1H), 7.74-7.71 (m, 2H), 7.57 (d, J = 3.9 Hz, 1H), 7.54 (d, J = 3.9 Hz, 1H), 7.

49-7.38 (m, 3H), 6.91-6.88 (m, 2H), 6.62-6.59 (m, 2H), 5.20 (t, J = 7.8 Hz, 1H), 4.17 (s, 2H), 3.21 (dd, J =

-3-(4-Hydroxyphenyl)-2-[4-[[(5-methyl-2-thienyl)sulfonylamino]methyl]triazol-1- yl]propanehydroxamic acid (55)

  2 Cl 2 /MeOH: 9/1) affording compound 54 as a white solid after lyophilization(50 mg, 51%). Purity: 99%, LC tr = 1.95min, MS (ESI+): m/z = 502 and 504 [M + H] + .1 H NMR (300 MHz, ) δ ppm: 11.07 (br s, 1H), 9.27-9.19 (m, 2H), 8.71 (br s, 1H), 8.00 (s, 1H), 7.91 (d, J = 5.2 Hz, 1H), 7.23(d, J = 5.2 Hz, 1H), 6.96-6.93 (m, 2H), 6.64-6.61 (m, 2H), 5.19 (t, J = 7.7 Hz, 1H), 4.21 (s, 2H), 3.23 (dd, J = 14.0 and 7.4 Hz, 1H), 3.11 (dd, J = 14.0 and 8.2 Hz, 1H).13 C NMR (DMSO-d 6 ) δ ppm: 163.7,

	Compound 55 was synthesized according to the general procedure G, using ester 49 (109 mg, 0.25

mmol), KCN (3.2 mg, 0.05 mmol) and aq. NH 2 OH (2.5 mL, 50% w/w in water) in MeOH

(2.5 mL)

overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 95/5) affording compound 55 as a white solid after lyophilization

(64 mg, 59%)

. CH

  -ESI+ (m/z) calcd. for C 16 H 17 N 5 O 5 S 2 Br [M + H] + 501.9854, found 501.9827.Compound 57 was synthesized according to the general procedure G, using ester 51(94 mg, 0.22 mmol), KCN (2.9 mg, 0.04 mmol) and aq. NH 2 OH (2.5 mL, 50% w/w in water) in MeOH(2.5 mL) overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 95/5) affording compound 57 as a white solid after lyophilization (67 mg, 70%).Compound 58 was synthesized according to the general procedure G, using ester 52(90 mg, 0.21 mmol), KCN (2.8 mg, 0.04 mmol) and aq. NH 2 OH (2.5 mL, 50% w/w in water) in MeOH(2.5 mL) overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 95/5) affording compound 58 as a white solid after lyophilization(35 mg, 38%). Purity: 99%, LC tr = 1.78 min, MS (ESI+): m/z = 424 [M + H] + . 1 H NMR (300 MHz, DMSO-d 6 ) δ ppm: 9.27 (br s, 1H), 9.14 (br s, 1H), 8.14 (dd, J = 3.0 and 1.3 Hz, 1H), 8.04 (s, 1H), 7.71 (dd, J = 5.1 J = 7.5 and 1.7 Hz, 1H), 7.82 (d, J = 4.0 Hz, 1H), 7.59 (d, J = 4.0 Hz, 1H), 7.37 (ddd, J = 7.5, 4.8 and 1.0 Hz, 1H), 7.04-7.00 (m, 2H), 6.81-6.77 (m, 2H), 5.22 (t, J = 7.9 Hz, 1H), 4.15 (s, 2H), 3.69 (m, 3H) 3.44 (dd, J = 13.8 and 7.5 Hz, 1H), 3.14 (dd, J = 13.8 and 8.2 Hz, 1H). 13 C NMR (75 MHz, DMSO-d 6 ) δ ppm: 163.

	and 3.0 Hz, 1H), 7.31 (dd, J = 5.1 and 1.3 Hz, 1H), 6.97-6.92 (m, 2H), 6.65-6.60 (m, 2H), 5.21 (t, J =
	7.8 Hz, 1H), 4.06 (s, 2H), 3.24 (dd, J = 13.9 and 7.3 Hz, 1H), 3.14 (dd, J = 13.9 and 8.4 Hz, 1H). 13 C
	(2S)-3-(4-Hydroxyphenyl)-2-[4-[(2-thienylsulfonylamino)methyl]triazol-1-yl]propanehydroxamic NMR (75 MHz, DMSO-d 6 ) δ ppm: 163.8, 156.2, 143.4, 140.3, 130.5, 130.0 (2C), 129.0, 125.8, 125.3,
	acid (57) 122.1, 115.2 (2C), 62.0, 38.1, 36.7. HRMS-ESI+ (m/z) calcd. for C 16 H 18 N 5 O 5 S 2 [M + H] + 424.0728,
	found 424.0749.
	(2S)-3-(4-Methoxyphenyl)-2-[4-[[[5-(2-pyridyl)-2-thienyl]sulfonylamino]methyl]triazol-1-
	yl]propanehydroxamic acid (71)
	Purity: 99%, LC tr = 1.82 min, MS (ESI+): m/z = 424 [M + H] + . 1 H NMR (300 MHz, DMSO-d 6 ) δ Compound 71 was synthesized according to the general procedure G, using ester 67 (150 mg, 0.29
	ppm: 11.03 (br s, 1H), 9.27 (s, 1H), 9.19 (br s, 1H), 8.33 (br s, 1H), 8.06 (s, 1H), 7.91 (dd, J = 5.0 and mmol), KCN (3.8 mg, 0.06 mmol) and aq. NH 2 OH (2.9 mL, 50% w/w in water) in MeOH (2.9 mL)
	1.3 Hz, 1H), 7.58 (dd, J = 3.7 and 1.3 Hz, 1H), 7.16 (dd, J = 5.0 and 3.7 Hz, 1H), 6.97-6.93 (m, 2H), overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to
	6.65-6.60 (m, 2H), 5.22 (t, J = 7.8 Hz, 1H), 4.10 (s, 2H), 3.24 (dd, J = 13.9 and 7.3 Hz, 1H), 3.14 (dd, CH 2 Cl 2 /MeOH: 95/5) affording compound 71 as a white solid after lyophilization (106 mg, 69%).
	J = 13.9 and 8.3 Hz, 1H). 13 C NMR (75 MHz, DMSO-d 6 ) δ ppm: 163.8, 156.2, 143.2, 141.2, 132.5, Purity: 98%, LC tr = 2.30 min, MS (ESI-): m/z = 513 [M -H] -. 1 H NMR (300 MHz, DMSO-d 6 ) δ
	131.7, 130.0 (2C), 127.7, 125.8, 122.1, 115.2 (2C), 62.0, 38.3, 36.7. HRMS-ESI+ (m/z) calcd for ppm: 11.6 (br s, 1H), 9.19 (br s, 1H), 8.56 (ddd, J = 4.8, 1.7 and 0.9 Hz, 1H), 8.46 (br s, 1H), 8.09 (s,
	C 16 H 18 N 5 O 5 S 2 [M + H] + 424.0719, found 424.0749. 1H), 8.02 (dt, J = 8.0 and 1.0 Hz, 1H), 7.88 (td,
	(2S)-3-(4-Hydroxyphenyl)-2-[4-[(3-thienylsulfonylamino)methyl]triazol-1-yl]propanehydroxamic
	acid (58)

(4-tert-Butoxyphenyl)-2-[4-[[(5-phenyl-2-thienyl)sulfonylamino]methyl]triazol-1- yl]propanehydroxamic acid (69)

  . HRMS-ESI+ (m/z) calcd. for C 22 H 23 N 6 O 5 S 2 [M + H] + 515.1178, found: 515.1178. Compound 69 was synthesized according to the general procedure G, using ester 65 (75 mg, 0.13 mmol), KCN (1.7 mg, 0.03 mmol) and aq. NH 2 OH (2.5 mL, 50% w/w in water) in MeOH (2.5 mL) overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 9/1) affording compound 69 as a white solid after lyophilization (67 mg, 90%). Purity: 99%, LC tr = 2.85 min, MS (ESI-): m/z = 554 [M -H] -. 1 H NMR (300 MHz, DMSO-d 6 ) δ ppm: 8.11 (s, 1H), 7.75-7.72 (m, 2H), 7.58(d, J = 3.9 Hz, 1H), 7.55 (d, J = 3.9 Hz, 1H), 7.50-7.38 (m, 3H), 7.03-6.99 (m, 2H), 6.85-6.81 (m, 2H), 5.26 (t, J = 7.8 Hz, 1H), 4.18 (s, 2H), 3.30 (dd, J = 13.8 and 7.4 Hz, 1H), 3.18 (dd, J = 13.8 and 8.1 Hz, 1H), 1.25 (s, 9H).13 C NMR (75 MHz, DMSO-d 6 ) δ ppm:

	(2S)-3-

  6.83-6.80 (m, 2H), 5.24(t, J = 7.8 Hz, 1H), 4.16 (d, J = 4.8 Hz, 2H), 3.28 (dd, J = 14.1 and 7.4 Hz, overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 95/5) affording compound 36 as a white solid after lyophilization (64 mg, 53%). Purity: 95%, LC tr = 2.58 min, MS (ESI-): m/z = 521 [M -H] -.1 H NMR (300 MHz, ) δ ppm: 11.17 (br s, 1H), 10.85 (br s, 1H), 9.21 (br s, 1H), 8.45 (br s, 1H),8.18 (s, 1H), 7.71-7.69 (m, 2H), 7.57

  . HRMS-ESI+ (m/z) calcd. for C 24 H 22 N 6 O 4 S 2 [M + H] + 523.1222, found 523.1223.

  in CH 2 Cl 2 (5 mL) overnight. Compound 107 was obtained as a white solid (302 mg, 89%) and used in the next step without further purifications. LC tr = 3.58 min, MS (ESI+): m/z = 210 [M + H] + . 1 H NMR (500 MHz, DMSO-d 6 ) δ ppm: 8.04 (t, J = 5.9 Hz, 1H), 7.69-7.68 (m, 2H),7.39-7.38 (m, 2H), 3.65 (dd, J = 5.9 and 2.5 Hz, 2H), 3.05 (t, J = 2.5 Hz, 1H), 2.38 (s, 3H).13 C NMR (126 MHz, DMSO-d 6 ) δ ppm: 142.8, 137.6, 129.5 (2C), 126.7 (2C), 79.4, 74.6, 31.9, 21.0.

  intermediate 84(403 mg, 1.22 mmol),4 N HCl in dioxane (6 mL) in a mixture CH 2 Cl 2 /EtOH (12 mL, 6:6 v/v) overnight. Compound 85 was obtained as colorless oil (325 mg, quant. yield) and was used in the next step without further purification. LC tr = 1.87 min, MS (ESI+): m/z= 231 [M + H] + . 1 H NMR (300 MHz, DMSO-d 6 ) δ ppm: 8.53(t, J = 5.5 Hz, 1H), 8.13 (br s, 3H), 4.09 (dd, J = 7.2 and 1.8 Hz, 1H), 4.04 (dd, J = 7.2 and 1.8 Hz, 1H),3.35-3.28 (m, 3H), 2.89-2.77 (m, 2H), 1.65-1.59 (m, 2H), 1.44 (sep, J = 6.7 Hz, 1H), 1.16 (t, J = 7.1 Hz, 3H), 0.86(d, J = 6.5 Hz, 3H), 0.85 (d, J = 6.5 Hz, 3H).

13

C NMR (75 MHz, DMSO-d 6 ) δ ppm: 169.

  mmol), ZnCl 2(8.8 mg, 0.06 mmol), K 2 CO 3 (296 mg, 2.14 mmol), anhydrous N,N- diisopropylethylamine (392 μL, 2.25 mmol) and diazo transfer reagent (270 mg, 1.29 mmol) in anhydrous MeOH (5 mL) overnight. The crude product was purified by flash chromatography on silica gel (cHex to cHex/EtOAc: 8/2) affording compound 112 as white crystals (121 mg, 31%). LC tr = 4.75 min, MS (ESI+): m/z = 319 [M + H] + . 1 H NMR (500 MHz, DMSO-d 6 ) δ ppm: 10.31 (s, 1H),

7.

65-7.64 (m, 1H), 7.55-7.53 (m, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.07-7.06 (m, 1H), 4.44 (s, 2H), 4.16- 4.06 (m, 2H)

, 3.58 (dd, J = 8.8 and 6.2 Hz, 1H), 1.78-1.74 (m, 1H), 1.69-1.63 (m, 1H), 1.50 (sep, J = 6.7 Hz, 1H), 1.16 (t, J = 7.0 Hz, 3H), 0.90

(d, J = 6.7 Hz, 3H), 0.88 (d, J = 6.7 Hz, 3H)

.

13

C NMR (

126

MHz, DMSO-d 6 ) δ ppm: 169.

  mmol), ZnCl 2 (10.0 mg, 0.07 mmol), K 2 CO 3 (674 mg, 4.87 mmol), anhydrous N,Ndiisopropylethylamine (177 μL, 1.02 mmol) and diazo transfer reagent (424 mg, 2.44 mmol) in anhydrous MeOH (5 mL) overnight. The crude product was purified by flash chromatography on silica gel (cHex/EtOAc: 9/1 to 6/4) affording compound 86 as colorless oil(72 mg, 19%). LC tr = 2.50 min, MS (ESI+): m/z = 257 [M + H] + .1 H NMR (300 MHz, CDCl 3 ) δ ppm: 6.92-6.88 (m, 1H), 4.18

  phenyl]carbamate (500 mg, 2.25 mmol), ZnCl 2(18.4 mg, 0.14 mmol), K 2 CO 3 (1240 mg, 9.0 mmol), anhydrous N,N-Diisopropylethylamine (240 μL, 2.47 mmol) and diazo transfer reagent (566 mg, 2.7 mmol) in anhydrous MeOH (5 mL) overnight. Compound 170 was obtained as orange oil (558 mg, quant. yield) and was used in the next step without further purification. LC tr = 4.72 min, MS (ESI+): m/z = no ionization [M + H] + .1 H NMR (500 MHz, DMSO-d 6 ) δ ppm: 9.42 (s, 1H),7.48-7.45 (m, 2H), 7.26-7.24 (m, 2H), 4.33 (s, 2H), 1.47 (s, 9H).13 C NMR (126 MHz, DMSOd 6 ) δ ppm: 152.

  , 2.94(t, J = 7.7 Hz, 1H), 2.07 (s, 3H), 1.57-1.46 (m, 2H), 1.35 (sep, J = 6.6 Hz, 1H), 0.80(d, J = 6.6 Hz, 3H), 0.79 (d, J = 6.6 Hz, 3H).

13

C NMR (126 MHz, DMSO-d 6 ) δ ppm: 169.

Acknowledgements

Abbreviations

AA: Amino acid ACC: Adrenocortical carcinoma Diisopropylethylamine (240 μL, 1.38 mmol) in CH 2 Cl 2 (5 mL) overnight. The crude product was purified by flash chromatography on silica gel (cHex to cHex/EtOAc: 8/2) affording compound 41 as pale yellow oil (235 mg, 70%). LC tr = 2.55 min, MS (ESI-): m/z = 278 and 280 [M -H] -. 1 H NMR (300 MHz, CDCl 3 ) δ ppm: 7.42 (d, J = 4.0 Hz, 1H), 7.08 (d, J = 3.8 Hz, 1H), 4.95-4.91 (m, 1H), 3.92 (dd, J = 6.0 and 2.5 Hz, 2H), 2.20 (t, J = 2.6 Hz, 1H). 13 C NMR (75 MHz, CDCl 3 ) δ ppm: 141. 0, 133.1, 130.5, 120.5, 77.3, 73.4, 33.1. 3-bromo-N-prop-2-ynyl-thiophene-2-sulfonamide (42) Compound 42 was synthesized according to the general procedure F, using propargylamine (81 μL, 1.26 mmol), 3-bromothiophene-2-sulfonyl chloride (300 mg, 1.15 mmol) and N, Ndiisopropylethylamine (240 μL, 1.38 mmol) in CH 2 Cl 2 (5 mL) overnight. The crude product was filtered on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 9/1) affording compound 42 as a beige solid (287 mg, 89%). LC tr = 2.32 min, MS (ESI-): m/z = 278 and 280 [M -H] -. 1 H NMR (300 MHz, ) δ ppm: 7.74 (d, J = 5.3 Hz, 1H), 7.16 (d, J = 5.3 Hz, 1H), 3.86 (d, J = 2.3 Hz, 2H), 2.45 (t, J = 2.5 Hz, 1H). 13 C NMR (75 MHz, MeOD-d 4 ) δ: 133. 8, 133.7, 132.6, 114.8, 79.0, 73.2, 33.1.

N-prop-2-ynylthiophene-2-sulfonamide (45)

Compound 45 was synthesized according to the general procedure F, using propargylamine (264 μL, 4.12 mmol), thiophene-2-sulfonyl chloride (500 mg, 2. 74 mmol) and N, N-diisopropylethylamine (572 μL, 3.29 mmol) in CH 2 Cl 2 (15 mL) overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 95/5) affording compound 45 as yellow oil (430 mg, 78%). LC tr = 2.08 min, MS (ESI-): m/z = 200 [M -H] -. 1 H NMR (300 MHz, ) δ ppm: 7.77 (dd, J = 1.4 and 5.0 Hz, 1H), 7.63 (dd, J = 1.4 and 3.8 Hz, 1H), 7.14 (dd, J = 3.0 and 5.0 Hz, 1H), 3.80 (d, J = 2.5 Hz, 2H), 2.50 (t, J = 2.5 Hz, 1H). 13 C NMR (75 MHz, MeOH-d 4 ) δ ppm: 142. 4, 133.4, 133.3, 128.4, 79.3, 73.5, 33.3.

N-prop-2-ynylthiophene-3-sulfonamide (46)

Compound 46 was synthesized according to the general procedure F, using propargylamine (116 μL, 1. 81 mmol), thiophene-3-sulfonyl chloride (300 mg, 1. 64 mmol) and N, N-diisopropylethylamine (343 μL, 1.97 mmol) in CH 2 Cl 2 (5 mL) overnight. The crude product was filtered on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 9/1) affording compound 46 as a beige solid (287 mg, 87%). LC tr = 2.02 min, MS (ESI-): m/z = 200 [M -H] -. 1 H NMR (300 MHz, ) δ ppm: 8.08 (dd, J = 3.1 and 1.3 Hz, 1H), 7.58 (dd, J = 5.2 and 3.1 Hz, 1H), 7.38 (dd, J = 5.2 and 1.3 Hz, 1H), 3.78 (dd, J = 2.5 Hz, 2H), 2.48 (t, J = 2.5 Hz, 1H). 13 C NMR (75 MHz, MeOD-d 4 ) δ ppm: 141. 6, 131.8, 129.1, 126.7, 79.5, 73.4, 33.1.

5-phenyl-N-prop-2-ynyl-thiophene-2-carboxamide (15)

Compound 15 was synthesized according to the general procedure B, using 5-phenylthiophene-2carboxylic acid (150 mg, 0.73 mmol), propargylamine (77 μL, 1.18 mmol), HBTU (456 mg, 1.18 mmol) and trimethylamine (307 μL, 2.2 mmol) in DMF (2 mL) 48h. The crude product was purified by flash chromatography on silica gel (cHex to cHex/EtOAc: 75/25) affording compound 15 as a white solid (134 mg, 76%). LC tr = 2.68 min, MS (ESI+): m/z = 242 [M + H] + . 1 H NMR (300 MHz, ) δ ppm: 8.99 (t, J = 5.2 Hz, 1H), 7.78 (d, J = 4.0 Hz, 1H), 7.73-7.70 (m, 2H), 7.55 (d, J = 4.0 Hz, 1H), 7.48-7.34 (m, 3H), 4.06 (dd, J = 2.5 and 5.6 Hz, 2H), 3.17 (t, J = 2.5 Hz, 1H). 13 C NMR (75 MHz, DMSO-d 6 ) δ ppm: 160. 7, 147.7, 138.1, 133.0, 129.5, 129.2 (2C), 128.6, 125.7 (2C), 124.4, 81.1, 73.2, 28.4.

N-methyl-5-phenyl-N-prop-2-ynyl-thiophene-2-carboxamide (16)

Compound 16 was synthesized according to the general procedure B, using 5-phenylthiophene-2carboxylic acid (150 mg, 0.73 mmol), N-methylpropargylamine (102 μL, 1.18 mmol), HBTU (456 mg, 1.18 mmol) and trimethylamine (307 μL, 2.2 mmol) in DMF (2 mL) 48h. The crude product was purified by flash chromatography on C18 column (H 2 (m, 2H), 7.48-731 (m, 4H), 7.26 (d, J = 5.7 Hz, 1H), 4.36 (d, J = 2.5 Hz, 2H), 3.29 (s, 3H), 2.36 (t, J = 2.3 Hz, 1H). 13 C NMR (75 MHz, CDCl 3 ) δ ppm: 164. 1, 148.6, 136.2, 133.5, 130.6, 129.1 (2C), 128.5, 126.2 (2C), 122.9, 78.6, 73.0, 39.6, 35.7.

(3S)-3-Azido-4-(4-hydroxyphenyl)-N-prop-2-ynyl-butanamide (3)

Compound 3 was synthesized according to the general procedure B, using azido carboxylic acid 1 (334 mg, 1.51 mmol), propargylamine (126 μL, 1.96 mmol), HBTU (630 mg, 1.66 mmol) and trimethylamine (619 μL, 4.53 mmol) in DMF (5 mL) overnight. Compound 3 was obtained as yellow oil (369 mg, 90%) and was used in the next step without further purification. LC tr = 2.07 min, MS (ESI-): m/z= 257 [M-H] -. 1 H NMR (300 MHz, ) δ ppm: 7.09-7.04 (m, 2H), 6.76-6.71 (m, 2H), 4.03-3.97 (m, 1H), 3.96-3.94 (m, 2H), 2.79 (dd, J = 13.9 and 5.7 Hz, 1H), 2.70 (dd, J = 13.9 and 8.0 Hz, 1H), 2.58 (t, J = 2.6 Hz, 1H), 2.40 (dd, J = 14.8 and 5.0 Hz, 1H), 2.31 (dd, J = 14.7 and 8.9 Hz, 1H). 13 C NMR (75 MHz, MeOD-d 4 ) δ ppm: 172. 4, 157.4, 131.5 (2C), 129.4, 116.3 (2C), 80.4, 72.2, 62.6, 41.3, 40.9, 29.5.

(3R)-3-Azido-4-(1H-indol-3-yl)-N-prop-2-ynyl-butanamide (4)

Compound 4 was synthesized according to the general procedure B, using azido carboxylic acid 2 (611 mg, 2. 50 mmol), propargylamine (208 μL, 3.3 mmol), HBTU (1040 mg, 2.8 mmol) and trimethylamine (1030 μL, 7.5 mmol) in DMF (10 mL) overnight. Compound 4 was obtained as brown oil (487 mg, 57%) and was used in the next step without further purification. LC tr = 2.50 min, MS (ESI-): m/z = 280 [M -H] -. 1 H NMR (300 MHz, ) δ ppm: 7. 59-7.56 (m, 1H), 7.36-7.33 (m, 1H), 7.13 (s, 1H), 7.10-7.00 (m, 2H), 4.19-4.11 (m, 1H), 3.94-3.93 (m, 2H), 3.07-2.95 (m, 2H), 2.57 (t, J = 2.6 Hz, 1H), 2.46 (dd, J = 14.7 and 5.0 Hz, 1H), 2.36 (dd, J = 14.7 and 8.9 Hz, 1H). 13 C NMR (75 MHz, MeOD-d 4 ) δ ppm: 172. 6, 138.1, 128.8, 124.7, 122.4, 119.9, 119.3, 112.3, 111.4, 80.4, 72.2, 61.7, 41.5, 31.4, 29.4.

(2S)-2-Azido-3-(4-hydroxyphenyl)-N-prop-2-ynyl-propanamide (24)

Compound 24 was synthesized according to the general procedure B, using azido carboxylic acid 23 (505 mg, 2.4 mmol), propargylamine (203 μL, 3.2 mmol), HBTU (1020 mg, 2.7 mmol) and trimethylamine (1000 μL, 7.3 mmol) in DMF (10 mL) overnight. Compound 24 was obtained as brown oil (345 mg, 50%) and was used in the next step without further purification. LC tr = 2.05 min, MS (ESI-): m/z = 243 [M -H] -. 1 H NMR (300 MHz, ) δ ppm: 7.09-7.03 (m, 2H), 6.74-6.69 (m, 2H), 3.98-3.92 (m, 3H), 3.08 (dd, J = 13.8 and 6.1 Hz, 1H), 2.89 (dd, J = 13.8 and 7.9 Hz, 1H), 2.57 (t, J = 2.6 Hz, 1H). 13 C NMR (75 MHz, MeOD-d 4 ) δ ppm: 171. 6, 157.6, 131.4 (2C), 128.2, 116.3 (2C), 80.1, 72.3, 65.7, 38.1, 29.4. Compound 25 was synthesized according to the general procedure C, using 24 (330 mg, 1.35 mmol) in DMF (150 mL) overnight. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 to CH 2 Cl 2 /MeOH: 85/15) affording compound 25 as an orange solid (176 mg, 48%). LC tr = 1.42 min, MS (ESI+): m/z = 245 [M + H] + . 1 H NMR (300 MHz, ) δ ppm: 9.31 (s, 1H), 8.42 (br d, J = 2.0 Hz, 1H), 7.53 (s, 1H), 6.52 (d, J = 8.5 Hz, 2H), 6.42 (d, J = 8.5 Hz, 2H), 5.38 (t, J = 3.9 Hz, 1H), 4.28 (dd, J = 16.7 and 2.7 Hz, 1H), 3.39-3.16 (m, 3H). 13 C NMR (75 MHz, DMSO-d 6 ) δ ppm: 165. 9, 156.6, 130.2 (2C), 129.2, 128.5, 124.2, 115.0 (2C), 60.0, 37.8, 35.4. (3S)-3-[5-(Aminomethyl)triazol-1-yl]-4-(4-hydroxyphenyl)butanoic acid;hydrochloride (7) Compound 7 was synthesized according to the general procedure D, using diazepinone 5 (1150 mg, 4. 45 mmol) in a 6M aq. HCl solution (30 mL) under MW irradiations (85°C, 1h). Compound 7 was obtained as brown solid (1390 mg, quant. yield) and was used in the next step without further purification. LC tr = 1.33 min, MS (ESI+): m/z = 277 [M + H] + . 1 H NMR (300 MHz, ) δ ppm: 8.64 (t, J = 6.1 Hz, 2H), 7.70 (s, 1H), 6.79 (d, J = 8.5 Hz, 2H), 6.62 (d, J = 8.5 Hz, 2H), 4.96-4.86 (m, 1H), 3.85 (dd, J = 15.2 and 5.7 Hz, 1H), 3.68 (dd, J = 15.2 and 5.7 Hz, 1H), 3.17-3.08 (m, 2H), 3.02-2.93 (m, 2H). 13 C NMR (75 MHz, DMSO-d 6 ) δ ppm: 171. 9, 156.2, 132.6, 131.7, 130.0 (2C), 126.7, 115.2 (2C), 56.8, 40.2, 38.6, 31.2 1H), 7.37 (ddd, J = 7.5, 4.9 and 1.0 Hz, 1H), 7.01-6.97 (m, 2H), 6.77-6.73 (m, 2H), 5.73 (dd, J = 9.9 and 5.7 Hz, 1H), 4.14 (s, 2H), 3.67 (m, 3H), 3.65 (m, 3H), 3.44 (dd, J = 14.3 and 5.7 Hz, 1H), 3.34 (dd, J = 14.3 and 9.9 Hz, 1H). 13 C NMR (75 MHz, DMSO-d 6 ) δ ppm: 168. 7, 158.1, 150.4, 150.0, 149.7, 143.2, 142.0, 137.5, 132.6, 129.9 (2C), 127.5, 124.9, 123.8, 123.5, 119.3, 113.7 (2C), 63.1, 54.9, 52.8, 38.3, 35.9. 5.76 (dd, J = 9.9 and 5.9 Hz, 1H), 4,13 (d, J = 5.9 Hz, 2H), 3.64 (s, 3H), 3.46 (dd, J = 14.3 and 5.9 Hz, 1H), 3.36 (dd, J = 14.3 and 9.9 Hz, 1H), 1,22 (s, 9H). 13 C NMR (75 MHz, DMSO-d 6 ) δ ppm: 168. 7, 153.9, 149.1, 143.2, 139.6, 132.8, 132.2, 130.2, 129.5 (2C), 129.4 (2C), 129.1, 125.9 (2C), 124.0, 123.6, 123.5 (2C), 77.8, 63.0, 52.8, 38.3, 36.2, 28.5 (3C).

Methyl

Methyl (2S)-3-(4-tert-butoxyphenyl)-2-[4-[[[5-(2-pyridyl)-2thienyl]sulfonylamino]methyl]triazol-1-yl]propanoate (66)

Compound 66 was synthesized according to the general procedure H, using azide 63 (99.6 mg, 0.18 mmol), alkyne 44 (50 mg, 0.18 mmol) = 7.5, 4.8 and1.0 Hz, 1H), 6.99-6.95 (m, 2H), 6.80-6.76 (m, 2H), 5.75 (dd, J = 9.8 and 6.0 Hz, 1H), 4.12 (d, J = 5.9 Hz, 2H), 3.64 (s, 3H), 3.45 (dd, J = 14.2 and 5.9 Hz, 1H), 3.35 (dd, J = 14.2 and 9.8 Hz, 1H), 1.22 (s, 9H). 13 C NMR (75 MHz, DMSO-d 6 ) δ ppm: 168. 6, 153.8, 150.4, 150.0, 149.6, 143.2, 142.0, 137.5, 132.6, 130.2, 129.5 (2C), 124.9, 123.7, 123.6, 123.5 (2C), 119.3, 77.8, 62.9, 52.7, 38.2, 36.2, 28.5.

Methyl (2S)-3-(4-benzyloxyphenyl)-2-[4-[[(5-phenyl-2-thienyl)sulfonylamino]methyl]triazol-1yl]propanoate (64)

Compound 64 was synthesized according to the general procedure H, using azide 61 (70 mg, 0.23 mmol), alkyne 32 (62.4 mg, 0.23 mmol), copper (II) sulfate pentahydrate (11.2 mg, 0.05 mmol) and sodium ascorbate (22.3 mg, 0.11 mmol) in DMF (5.5 mL) andH 168.7, 157.2, 149.1, 143.2, 139.6, 137.0, 132.8, 132.2, 130.0 (2C), 129.4 (2C), 129.1, 128.4 (2C), 127.8, 127.8, 127.7 (2C), 125.9 (2C), 124.0, 123.6, 114.6 (2C), 69.1, 63.1, 52.8, 38.3, 35.9.

Purity: 99%, LC tr = 1.97 min, MS (ESI+): m/z = 438 [M + H] + . 1 H NMR (300 MHz, ) δ ppm: 9.27 (br s, 1H), 9.13 (br s, 1H), 8.04 (s, 1H), 7.39 (d, J = 3.7 Hz, 1H), 6.96-6.93 (m, 2H), 6.88 (dd, J = 3.7 and 1.0 Hz, 1H), 6.64-6.61 (m, 2H), 5.21 (t, J = 7.8 Hz, 1H), 4.08 (s, 2H), 3.24 (dd, J = 13.9 and 7.3 Hz, 1H), 3.13 (dd, J = 13.9 and 8.3 Hz, 1H), 2.49 (s, 3H). 13 C NMR (75 MHz, DMSO- 8, 156.2, 146.7, 143.3, 138.0, 132.0, 130.0 (2C), 126.2, 125.8, 122.1, 115.2 (2C), 62.0, 38.3, 36.8, 15.1. HRMS-ESI+ (m/z) 156.2, 143.0, 142.3, 132.4, 131.3, 130.0 (2C), 125.8, 122.3, 118.3, 115.2 (2C) 5, 133.6, 132.5, 131.2, 126.4, 125.4, 79.1, 74.9, 31.9.

N-(2-Prop-2-ynoxyphenyl)acetamide (100)

To a solution of N-(2-hydroxyphenyl)

N-[2-(4-Hydroxyphenyl)ethyl]pent-4-ynamide (109)

Pentynoic acid (100 mg, 1.02 mmol), tyramine (210.0 mg, 1.53 mmol, 1.5 eq) and HBTU (464 mg, 1.22 mmol, 1.2 eq) were put in solution in DMF (5.0 mL) and triethylamine (424 µL, 3.06 mmol, 3 eq) and the mixture was stirred overnight at room temperature. The resulting mixture was then diluted with water and extracted with EtOAc (three times). Organic layers were combined and solvents were evaporated in vacuo. The residue was then purified through flash silica gel column (cH/EtOAc: 9:1 to 5:5), affording compound 109 as a colorless oil (107 mg, 47% 83.9, 71.4, 40.7, 34.4, 34.2, 14.3.

2-Azido

2-Ethoxycarbonyl-4-methyl-pentanoic acid (80)

Compound 80 was synthesized according to the general procedure I, using diethylmalonate (1000 mg, ). 13 C NMR (MeOD-d 4 ) δ ppm: 173. 0, 171.6, 62.3, 51.4, 38.8, 27.4, 22.7, 22.5, 14.4.

2-Methoxycarbonyl-3-methyl-butanoic acid (174-a)

Compound 174-a was synthesized according to the general procedure I, using dimethylmalonate (745 mg, 4. 28 mmol)

N-[4-(Azidomethyl)phenyl]-2-diethoxyphosphoryl-4-methyl-pentanamide (194)

A mixture of 2-diethoxyphosphoryl-4-methyl-pentanoic acid (328 mg, 1. H] + . 1 H NMR (500 MHz, ) δ ppm: 10.30 (s, 1H), 8.23 (s, 1H), 7.59-7.57 (m, 2H), 7.33-7.29 (m, 4H), 7.07-7.02 (m, 2H), 5.54 (s, 2H), 5.12 (s, 2H), 4.13-4.05 (m, 2H), 3.56 (dd, J = 8.7 and 6.2 Hz, 1H), 1.77-1.72 (m, 1H), 1.67-1.62 (m, 1H), 1.48 (sep, J = 6.7 Hz, 1H), 1.15 (t, J = 7.1 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H). 13 C NMR (126 MHz, DMSO-d 6 ) δ ppm: 169. 7, 167.2, 156.9, 142.7, 138.8, 130.9, 129.2 (3C), 128.8 (2C), 124.6, 119.5 (2C), 116.5 (2C), 61.4, 60.7, 52.5, 50.9, 37.4, 25.6, 22.6, 21.9, 14.0 167.1, 143.5, 138.7, 134.7, 131.0, 130.7, 130.2 (2C), 128.8 (2C), 128.5 (2C), 123.3, 119.4 (2C), 60.6, 52.4, 50.9, 37.4, 27.3, 25.6, 22.6, 21.9, 14.0.

Ethyl 2-[[4-[[4-[(4-chlorophenyl)methyl]triazol-1-yl]methyl]phenyl]carbamoyl]-4-methylpentanoate (129)

Compound 129 was synthesized according to the general procedure 8.34-8.08 (m, 1H), 7.93-7.91 (m, 2H), 7.83 (s, 1H), 7.58-7.57 (m, 2H), 7.51-7.49 (m, 2H), 7.24-7.23 (m, 2H) 9, 166.3, 143.5, 140.1, 138.8, 138.0 (2C), 130.7, 128.7 (2C), 128.3 (2C), 123.2, 119.6 (2C), 100.4, 52.4, 49.9, 38.1, 38.0, 25.7, 22.4, 22.3 6, 167.8, 168.3, 144.6, 138.7, 130.9, 128.6 (2C), 123.2, 119.5 (2C), 57.9, 52.4, 50.0, 48.6, 47.6, 42.0, 41.9, 38.0, 38.0, 26.3, 25.7, 24.5, 22.4, 22.3, 19.4, 19.2

2-(Hydroxycarbamoyl)-4-methyl-N-[4-[[4-[[(4-phenylphenyl)sulfonylamino]methyl]triazol-1yl]methyl]phenyl]pentanamide (Hit L3)

Hit L3 was synthesized according to the general procedure G, using ester ppm: 169.7, 167.1, 143.4, 139.3, 138.7, 137.2, 130.9, 129.2 (2C), 128.8 (2C), 128.5 (2C), 123.2, 119.4 (2C), 52.3, 50.9, 38.0, 37.4, 25.6, 22.6, 21.9

2-(Hydroxycarbamoyl)-N-[4-[[4-[[(4-methoxyphenyl)sulfonylamino]methyl]triazol-1yl]methyl]phenyl]-4-methyl-pentanamide (162)

Compound 162 was synthesized according to the general procedure G, using ester 152 (131 mg, 0.24 8, 166.3, 162.1, 143.7, 138.7, 131.9, 130.8, 128.7 (2C), 128.6 (2C), 123.2, 119.5 (2C), 114.3 (2C), 55.6, 52.4, 49.9, 38.1, 38.0, 25.7, 22.4, 22.3

N-[4-[[4-[[(2-Chlorophenyl)sulfonylamino]methyl]triazol-1-yl]methyl]phenyl]-2-(hydroxycarbamoyl)-4-methyl-pentanamide (163)

Compound 163 was synthesized according to the general procedure G, using ester 153 (160 mg, 0.29 mmol), KCN (5.65 mg, 0.09 mmol) and NH 2 OH (3 mL, 50% w/w in water) in MeOH (3 mL) for 72h.

The crude product was purified by flash chromatography on silica gel (CH 7.90-7.89 (m, 1H), 7.74 (s, 1H), 7.59-7.57 (m, 2H), 7.55-7.49 (m, 2H), 7.45-7.42 (m, 1H), 7.22-7.21 (m, 2H), 5.42 (s, 2H), 4.16 (s, 2H), 3.22 (t, J = 7.6 Hz, 1H), 1.69 (t, J = 7.0 Hz, 2H), 1.49 (sep, J = 6.6 Hz, 1H), 0.89 (d, J = 6.4 Hz, 3H), 0.88 (d, J = 6.4 Hz, 3H). 13 C NMR (126 MHz, DMSO-d 6 ) δ ppm: 167.9, 166. 3, 143.7, 138.7, 138.0, 133.9, 131.6, 130.8, DMSO-d 6 ) δ ppm: 169. 4, 166.6, 144.1, 123.5, 59.2, 48.9, 48.5, 39.1, 38.4, 37.7, 25.5, 24.2, 22.4, 22.2 (2C) 4, 166.6, 143.1, 142.7, 137.4, 129.6 (2C), 126.7 (2C), 123.5, 48.9, 48.4, 39.0, 38.4, 38.1, 25.5, 22.4, 22.1, 21. 7.53-7.48 (m, 3H), 7.44-7.40 (m, 2H), 4.62 (t, J = 5.4 Hz, 2H), 4.44 (t, J = 5.4 Hz, 2H), 1.89 (s, 3H). 13 C NMR (75 MHz, CDCl 3 ) δ ppm: 170. 5, 138.7, 133.4, 129.8, 129.3 (2C), 129.0 (2C), 127.0, 62.5, 47.0, 20.6

II. Biology

General protocole for Kinetic Target-guided synthesis:

Three experiments were performed, one in presence of the target enzyme, one without the target enzyme (only with the buffer solution, azides and alkynes = "blank" plate) and one using copper catalysis for the formation of the 1,4-disubstituted 1,2,3-triazoles which were used as controls ("chemical catalysis" plate).

In a 96-well plate, were mixed 0.5 μl of azide solution (10. In vitro ERAP2, ERAP1 and IRAP activity assay (compounds of the chapter 2):

ERAP2 was prepared as previously described. (142) The enzymatic activity of ERAP2 was assayed using R-AMC (L-Arginine-7-amido-4-methylcoumarin hydrochloride, Sigma). Hepes at 50 mM with 100 mM NaCl at pH 7 was used as buffer. Briefly, 60 nL of test compounds were added in 384-wells plates (dark, non-binding surface) by acoustic dispensing with Echo (Labcyte) and preincubated 30 min at ambient temperature with 10 mL of ERAP2 1 mg/mL or vehicle. The reaction was then started with the addition of 10 mL of substrate at 10 mM. The final concentration of ERAP2, substrate and DMSO was 0.5 mg/mL, 5 mM and 0.4% respectively. For the kinetic readout a Victor 3V (PerkinElmer) was used with excitation at 380 nm and emission at 450 nm. The fluorescence was measured each 3 min during 1 h.

In vitro ERAP1 and IRAP activity assays were performed as for ERAP2 using L-AMC (L-Leucine-7-Amido-4-Methylcoumarin, from Sigma) as the substrate, recombinant human ERAP1 (PILS/ARTS1 from R&D Systems, ref 2334-ZN-010) at 0.8 mg/mL final concentration or recombinant human IRAP (prepared as previously described) (143) at 0.2 mg/mL final concentration respectively.

All measurements were carried out as 8-point dose response curves and reported as the average of at least three independent measurements. Bestatin was used as a reference inhibitor (100% inhibition at 2 mM). Data analysis was performed using Xlfit® v 5.0 or GraphPad Prism® v 4.0. Nonlinear curve fitting and statistical analysis was done using built-in functions.

In vitro LasB activity assay (compounds of the chapter 3):

The LasB activity assay was performed as described previously (123) using the fluorogenic substrate 2-Aminobenzoyl-Ala-GlyLeu-Ala-4 Nitrobenzylamide, purchased from Peptides International (Louisville, KY, USA).

Fluorescence intensity was measured for 60 min at 37 °C in black 384-well microtiter plates (Greiner BioOne, Kremsmünster, Austria) using a CLARIOstar microplate reader (BMG Labtech, Ortenberg, Germany) with an excitation wavelength of 340 ± 15 nm and an emission wavelength of 415 ± 20 nm.

The assay was performed in a final volume of 50 μL of assay buffer (50 mM Tris, pH 7.2, 2.5 mM CaCl 2 , 0.075% Pluronic F-127, 5% DMSO) containing LasB at a final concentration of 10 nM (commercial batch, from Merck or Elastin Products Company) or 0.3 nM (purified batch) and the substrate at 150 μM. Before substrate addition, compounds were preincubated with the enzyme for 15 min at 37 °C. Experiments were performed in duplicates and repeated for at least two times. Blank controls without enzyme were performed. After blank subtraction, the slope of samples containing inhibitors (v) was divided by the slope of a simultaneously started uninhibited enzymatic reaction (v0).

IC50 values were determined with nonlinear regression using GraphPad Prism 5 (Graph Pad Software, San Diego, CA, USA) and are given as mean values ± standard deviation (SD). The slope factor was constrained to 1.

MIC determination assay (compounds of the chapter 3):

Assays regarding the determination of the minimum inhibitory concentration (MIC) were performed as described previously. (144) The experiments were based on the P. aeruginosa strain PA14. For the case that no MIC value could be determined due to activity reasons, percentage (%) inhibition at 100 µM (or lower, depending on the solubility of the compounds) was calculated. The OD at 600 nm was measured 16 h after inhibitor addition in a CLARIOstar Platereader (BMG Labtech, Ortenberg, Germany).

Selectivity assay:

 MMPs inhibition MMP -1, -2, -3, -7, -8 and -14 Inhibitor Screening Assay kits were purchased from AnaSpec