Mélina Chebaiki

Evelyne Delfourne

Rasoul Tamhaev

Saïda Danoun

Frédéric Rodriguez

Pascal Hoffmann

Emeline Grosjean

Fernanda Goncalves

Joëlle Azéma-Despeyroux

Adrián Pál

Jana Korduláková

Nadège Preuilh

Sébastien Britton

Patricia Constant

Hedia Marrakchi

Laurent Maveyraud
email: laurent.maveyraud@ipbs.fr

Lionel Mourey
email: lionel.mourey@ipbs.fr

Christian Lherbet
email: christian.lherbet@univ-tlse3.fr

Discovery of new diaryl ether inhibitors against Mycobacterium tuberculosis targeting the minor portal of InhA

Keywords: Tuberculosis, InhA, minor portal, diaryl ether, coumarin, mechanism, cytotoxicity

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

Introduction

Tuberculosis (TB) has existed for millennia and is still considered a scourge. It is the second leading causes of death due to a single pathogen worldwide, after the COVID-19 pandemic. Based on the Global Tuberculosis Report 2022, 1.6 million people died from TB and an estimated 10 million people fell ill with TB in 2021 [START_REF] Lherbet | Global Tuberculosis Report 2022[END_REF]. Although TB is considered curable, the coronavirus crisis has caused distortions in its diagnosis and treatment in many countries, which has made patients all the more vulnerable. In addition, the emergence of multidrug-resistant TB (MDR-TB) and extensively-drug resistant TB (XDR-TB) strains remains a challenge. In this context, various actions, including the discovery of new drugs, have to be taken to move towards the eradication of TB. The unique composition of the mycobacterial cell envelope has led researchers to focus on the proteins involved in the biosynthesis of its major components, mycolic acids. The biosynthesis of these essential and very specific α-alkylated β-hydroxylated fatty acids involved two fatty acid synthase (FAS) systems called FAS-I and FAS-II. In FAS-II, at least four independent enzymatic activities participate to the elongation of the FAS-I products: β-hydroxyacyl-ACP (acyl carrier protein) synthase (MabA), enoyl-ACP reductase (InhA), β-hydroxyacyl-ACP dehydratase complex (HadABC), and β-ketoacyl-ACP synthase (KasA/KasB). Isoniazid (INH) is one of the oldest drugs for tuberculosis treatment. This small molecule acts as a prodrug, requiring the prior activation by the catalase peroxidase enzyme KatG. Upon activation, the isonicotinoyl radical forms an adduct with the cofactor NAD, which inhibits InhA, leading to the arrest of mycolic acid biosynthesis and the mycobacterium death [START_REF] Banerjee | inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis[END_REF]. With the emergence of INH resistance, mainly due to mutations in KatG, new direct inhibitors of InhA have emerged. These include triclosan (TCL) [START_REF] Parikh | Inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis, by triclosan and isoniazid[END_REF] and TCL derivatives [START_REF] Boyne | Targeting fatty acid biosynthesis for the development of novel chemotherapeutics against Mycobacterium tuberculosis: evaluation of A-ring-modified diphenyl ethers as high-affinity InhA inhibitors[END_REF][START_REF] Pan | Time-dependent diaryl ether inhibitors of InhA: structure-activity relationship studies of enzyme inhibition, antibacterial activity, and in vivo efficacy[END_REF][START_REF] Rodriguez | First triclosan-based macrocyclic inhibitors of InhA enzyme[END_REF][START_REF] Stec | Biological evaluation of potent triclosan-derived inhibitors of the enoyl-acyl carrier protein reductase InhA in drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis[END_REF], GEQ and analogues [START_REF] Chollet | Design, synthesis and evaluation of new GEQ derivatives as inhibitors of InhA enzyme and Mycobacterium tuberculosis growth[END_REF][START_REF] Kuo | Targeting tuberculosis and malaria through inhibition of Enoyl reductase: compound activity and structural data[END_REF], triazoles [START_REF] Suresh | Design and synthesis of 9H-fluorenone based 1,2,3-triazole analogues as Mycobacterium tuberculosis InhA inhibitors[END_REF], thiazoles [START_REF] Shirude | Methyl-thiazoles: a novel mode of inhibition with the potential to develop novel inhibitors targeting InhA in Mycobacterium tuberculosis[END_REF], pyridomycin [START_REF] Hartkoorn | Towards a new tuberculosis drug: pyridomycin -nature's isoniazid[END_REF][START_REF] Kienle | Synthesis and Structure-Activity Relationship Studies of C2-Modified Analogs of the Antimycobacterial Natural Product Pyridomycin[END_REF], and 4-hydroxy-2-pyridones [START_REF] Manjunatha | Direct inhibitors of InhA are active against Mycobacterium tuberculosis[END_REF]. Furthermore, GlaxoSmithKline's thiadiazole GSK693 [START_REF] Martinez-Hoyos | Antitubercular drugs for an old target: GSK693 as a promising InhA direct inhibitor[END_REF] and the diazaborine AN12855 [START_REF] Xia | Discovery of a cofactor-independent inhibitor of Mycobacterium tuberculosis InhA[END_REF] were two promising leads with low toxicity, good oral bioavailability, in vivo efficacy similar to INH, and above all, activity against katG-deficient Mtb. However, none of the above compounds has been approved for clinical use. Thus, there is still a need to develop new direct inhibitors which overcome INH resistance.

In this respect, being a key target for antitubercular drug discovery, InhA has been the subject of numerous structural studies by X-ray crystallography. In 1995, its structure was the very first from the Mtb proteome to be deposited in the protein data bank (PDB) [START_REF] Dessen | Crystal structure and function of the isoniazid target of Mycobacterium tuberculosis[END_REF] and there are currently 105 deposited structures compared to 80 in the last comprehensive review published in 2018 [START_REF] Chollet | An overview on crystal structures of InhA protein: Apo-form, in complex with its natural ligands and inhibitors[END_REF]. Crystal structures corresponding to the apo-form and to complexes with its NAD cofactor, a substrate analogue and various inhibitors, including the INH-NAD adduct and the above listed direct inhibitors, have been resolved at resolution ranging from 3.4 to 1.4 Å. This allowed defining the most important structural elements with regard to the mechanisms of action and inhibition. InhA is a tetrameric enzyme with dihedral (D2) symmetry where the four subunits are related by three 2-fold axes. The tertiary fold consists of a central seven-stranded parallel β-sheet sandwiched on each side by three helices. One of the two helical faces is overhung by two additional helices, H5 and H6, which form the so-called "substrate-binding loop" (SBL). Opposite the SBL and in the extension of the β-sheet and the other helical face, there are two loops (A and B). The SBL and loops A and B line the InhA active site, which forms a deeply buried crevice. At the bottom of the crevice and spanning the C-termini of the β-strands is the cofactor binding site. Above the cofactor binding site is found the substrate binding site, one end of which is wide open to the solvent via the major portal while at the apex of the opposite side is the much smaller minor portal [START_REF] Rozwarski | Crystal structure of the Mycobacterium tuberculosis enoyl-ACP reductase, InhA, in complex with NAD+ and a C16 fatty acyl substrate[END_REF]. The catalytic triad, composed of residues Phe149-Tyr158-Lys165, is located under the entrance of the minor portal. The conformation of the SBL loop, more specifically the position of helix H6, and the orientation of the Phe149 and Tyr158 side chains are important structural descriptors related to the different states of the enzyme, i.e. apo, holo, in complex with inhibitors. All inhibitors, except the three inhibitors INH-NAD adduct, pyridomycin, and AN12855 compound, bind to the enzyme in the presence of the cofactor. A very few of the InhA inhibitors for which an X-ray structure has been deposited within the PDB reach the entrance to the minor portal: 8PP (5-octyl-2-phenoxyphenol, ligand ID 8PS in PDB 2B37) [START_REF] Sullivan | High affinity InhA inhibitors with activity against drugresistant strains of Mycobacterium tuberculosis[END_REF], PT155 (5-(4-amino-2-methylphenoxy)-2-hexyl-1-methylpyridin-4(1H)-one, ligand ID 1S5 in PDB 4OXK) [START_REF] Li | A structural and energetic model for the slow-onset inhibition of the Mycobacterium tuberculosis enoyl-ACP reductase InhA[END_REF], and AP-124 ((S)-1-(benzo[d]imidazol-1-yl)-3-(2,3-dihydro-1H-inden-5-yl)oxy)propan-2-ol, ligand ID JVZ in PDB 6R9W) [START_REF] Kamsri | Discovery of New and Potent InhA Inhibitors as Antituberculosis Agents: Structure-Based Virtual Screening Validated by Biological Assays and X-ray Crystallography[END_REF]. The alkyl diphenyl ether 5-octyl-2-phenoxyphenol is one example of the many triclosan-based InhA inhibitors, which represent one quarter of all InhA structures in the PDB [START_REF] Pan | Time-dependent diaryl ether inhibitors of InhA: structure-activity relationship studies of enzyme inhibition, antibacterial activity, and in vivo efficacy[END_REF][START_REF] Kuo | Targeting tuberculosis and malaria through inhibition of Enoyl reductase: compound activity and structural data[END_REF][START_REF] Sullivan | High affinity InhA inhibitors with activity against drugresistant strains of Mycobacterium tuberculosis[END_REF][START_REF] Li | A structural and energetic model for the slow-onset inhibition of the Mycobacterium tuberculosis enoyl-ACP reductase InhA[END_REF][START_REF] Freundlich | Triclosan Derivatives: Towards Potent Inhibitors of Drug-Sensitive and Drug-Resistant Mycobacterium tuberculosis[END_REF][START_REF] Lai | Rational Modulation of the Induced-Fit Conformational Change for Slow-Onset Inhibition in Mycobacterium tuberculosis InhA[END_REF][START_REF] Luckner | A slow, tight binding inhibitor of InhA, the enoyl-acyl carrier protein reductase from Mycobacterium tuberculosis[END_REF][START_REF] Soutter | Discovery of cofactor-specific, bactericidal Mycobacterium tuberculosis InhA inhibitors using DNA-encoded library technology[END_REF][START_REF] Spagnuolo | Evaluating the Contribution of Transition-State Destabilization to Changes in the Residence Time of Triazole-Based InhA Inhibitors[END_REF].

Here, we report the design and synthesis, based on a molecular hybridization approach, of a series of new diaryl ethers bearing various, mainly coumarinic, heterocycles (Fig. 1). This strategy, which consists in combining two molecules or molecular fragments to obtain a more affine compound for the target, was proven successful to discover new antitumor or anti-Mtb agents [START_REF] Ma | Design, Synthesis, and Structure-Activity Relationship of Novel LSD1 Inhibitors Based on Pyrimidine-Thiourea Hybrids As Potent, Orally Active Antitumor Agents[END_REF][START_REF] Bhakta | -3-yl)methyl)-4-methylpiperazine (BM212) and N-Adamantan-2-yl-N′-((E)-3,7-dimethylocta-2,6-dienyl)ethane-1,2-diamine (SQ109) Pyrrole Hybrid Derivatives: Discovery of Potent Antitubercular Agents Effective against Multidrug-Resistant Mycobacteria[END_REF]. We show that these new diaryl ethers exhibit excellent inhibition of both InhA activity and mycobacterial growth.

The mechanism of action of these compounds were also investigated through monitoring the effect on mycolic acid biosynthesis in Mtb and cytotoxic activity. Finally, the binding mode of these molecules was elucidated through the determination of high-resolution X-ray structures of complexes, which revealed that these compounds are able to occupy the minor portal, to an extent never observed before.

Results and discussion

Design of the hybrid inhibitors

First structure-activity studies on TCL have shown that this diaryl ether molecule may bind at two sites of the InhA substrate-binding pocket [START_REF] Kuo | Targeting tuberculosis and malaria through inhibition of Enoyl reductase: compound activity and structural data[END_REF]. One TCL molecule (TCL1) was found to bind through πstacking interactions between the hydroxyl-substituted ring, hereafter called " A ring", and the nicotinamide ring of the cofactor plus hydrogen bonding between the hydroxyl moiety of the A ring and the 2'-hydroxyl group of NAD + and hydroxyl group of Tyr158. The dichlorophenyl ring (or "B ring") is orthogonally oriented to the A ring. It is worth mentioning that the TCL1-binding site occupies the part of the substrate-binding site, as observed in the structure of the complex with a C16 fatty acyl substrate analogue, which is directly accessible through the major portal [START_REF] Rozwarski | Crystal structure of the Mycobacterium tuberculosis enoyl-ACP reductase, InhA, in complex with NAD+ and a C16 fatty acyl substrate[END_REF]. The second TCL molecule (TCL2) binds to the remaining part of the substrate-binding site in a rather hydrophobic region close to the minor portal. This spatial configuration of TCL1 and TCL2, which underlines the size of the InhA binding site, has been exploited to further develop TCL-based inhibitors of the enzyme [START_REF] Pan | Time-dependent diaryl ether inhibitors of InhA: structure-activity relationship studies of enzyme inhibition, antibacterial activity, and in vivo efficacy[END_REF][START_REF] Stec | Biological evaluation of potent triclosan-derived inhibitors of the enoyl-acyl carrier protein reductase InhA in drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis[END_REF][START_REF] Sullivan | High affinity InhA inhibitors with activity against drugresistant strains of Mycobacterium tuberculosis[END_REF][START_REF] Li | A structural and energetic model for the slow-onset inhibition of the Mycobacterium tuberculosis enoyl-ACP reductase InhA[END_REF][START_REF] Freundlich | Triclosan Derivatives: Towards Potent Inhibitors of Drug-Sensitive and Drug-Resistant Mycobacterium tuberculosis[END_REF][START_REF] Lai | Rational Modulation of the Induced-Fit Conformational Change for Slow-Onset Inhibition in Mycobacterium tuberculosis InhA[END_REF][START_REF] Luckner | A slow, tight binding inhibitor of InhA, the enoyl-acyl carrier protein reductase from Mycobacterium tuberculosis[END_REF][START_REF] Soutter | Discovery of cofactor-specific, bactericidal Mycobacterium tuberculosis InhA inhibitors using DNA-encoded library technology[END_REF][START_REF] Spagnuolo | Evaluating the Contribution of Transition-State Destabilization to Changes in the Residence Time of Triazole-Based InhA Inhibitors[END_REF][START_REF] Abdelaziz | Novel diaryl ether derivatives as InhA inhibitors: Design, synthesis and antimycobacterial activity[END_REF][START_REF] Ibrahim | In vitro antimycobacterial activity and physicochemical characterization of diaryl ether triclosan analogues as potential inhA reductase inhibitors[END_REF][START_REF] Rodriguez | First triclosanbased macrocyclic inhibitors of InhA enzyme[END_REF][START_REF] Tiwari | Design, synthesis, in silico and in vitro evaluation of novel diphenyl ether derivatives as potential antitubercular agents[END_REF]. In the resolved structures of InhA complexes with TCL derivatives, the diaryl ether moiety systematically binds in the same position as TCL1 whereas the various chemical substituents, at position 5 of the A ring, reach the space occupied by the TCL2 A ring (Fig. 2). The hydroxyl group of the phenol moiety revealed as essential for activity by dictating the orientation of the molecules in the substrate-binding site (for a review, see [START_REF] Vosatka | Triclosan and its derivatives as antimycobacterial active agents[END_REF]). In 2006, Sullivan et al. developed a series of alkyl diphenyl ethers bearing an alkyl chain up to 14 carbons on the A ring. The molecule showing the best inhibitory activity, both on InhA and on the growth of Mtb, bears a 8 carbon-long alkyl chain (IC50 = 5 nM, MIC(H37Rv) = 6.4 µM) [START_REF] Sullivan | High affinity InhA inhibitors with activity against drugresistant strains of Mycobacterium tuberculosis[END_REF]. Later, Freundlich et al. diversified the nature of the substituents on the A ring with branched alkyl chains or alkyl chains containing (hetero)aromatic groups, but this did not lead to improved activities against InhA [START_REF] Freundlich | Triclosan Derivatives: Towards Potent Inhibitors of Drug-Sensitive and Drug-Resistant Mycobacterium tuberculosis[END_REF]. On the other hand, Stec et al. showed that the introduction of a triazole group among various heterocycles at position 5 exhibited favorable interactions with the protein, the best compound carrying a n-butyl chain on the position 4 of the triazole [START_REF] Stec | Biological evaluation of potent triclosan-derived inhibitors of the enoyl-acyl carrier protein reductase InhA in drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis[END_REF]. Lately, Spagnuolo et al. observed that large and bulky substituents at position 5 on the A ring induce steric hindrance in the substrate-binding site resulting in the loss of key interactions [START_REF] Spagnuolo | Evaluating the Contribution of Transition-State Destabilization to Changes in the Residence Time of Triazole-Based InhA Inhibitors[END_REF][START_REF] Vosatka | Triclosan and its derivatives as antimycobacterial active agents[END_REF]. Despite this, we and others thought that bulkier substituents at the 5-position could target the space occupied by TCL2. For instance, we recently reported the synthesis of macrocyclic ditriclosan analogues which displayed good inhibitory activity against InhA (IC50 = 4.7 µM) [START_REF] Rodriguez | First triclosanbased macrocyclic inhibitors of InhA enzyme[END_REF]. Chetty et al. also developed a series of flexible di-triclosan analogues revealing inhibition in the same range [START_REF] Chetty | New InhA Inhibitors Based on Expanded Triclosan and Di-Triclosan Analogues to Develop a New Treatment for Tuberculosis[END_REF].

To go further, we decided to replace the diaryl ether corresponding to the TCL2 site with coumarin derivatives. Indeed, the coumarin skeleton is a privileged scaffold in life sciences and is present in numerous biologically relevant natural or synthetic products. Such molecules display various biological activities such as antibacterial [START_REF] Liu | Novel coumarin-thiazolyl ester derivatives as potential DNA gyrase Inhibitors: Design, synthesis, and antibacterial activity[END_REF], antifungal [START_REF] Xu | Design, Synthesis, and Antifungal Activity of 4-Amino Coumarin Based Derivatives[END_REF], anti-Alzheimer [START_REF] Yun | Synthesis and biological evaluation of 4-arylcoumarins as potential anti-Alzheimer's disease agents[END_REF], antioxidant [START_REF] Martinčič | In Silico Discovery of Novel Potent Antioxidants on the Basis of Pulvinic Acid and Coumarine Derivatives and Their Experimental Evaluation[END_REF], and antitumor [START_REF] Rubab | An Update on Synthesis of Coumarin Sulfonamides as Enzyme Inhibitors and Anticancer Agents[END_REF][START_REF] Cui | Design, synthesis and biological evaluation of coumarin derivatives as potential BRD4 inhibitors[END_REF]. Some of them have also been studied as potential InhA and FabI inhibitors (Fig. 1) [START_REF] Somagond | Click chemistry based regioselective one-pot synthesis of coumarin-3-yl-methyl-1,2,3-triazolyl-1,2,4-triazol-3(4H)-ones as newer potent antitubercular agents[END_REF][START_REF] Srivastava | Synthesis and antimycobacterial activity of 1-(β-d-Ribofuranosyl)-4-coumarinyloxymethyl-/ -coumarinyl-1,2,3-triazole[END_REF][START_REF] Hu | Synthesis and biological evaluation of coumarin derivatives containing imidazole skeleton as potential antibacterial agents[END_REF]. We speculated that coumarin moieties could mimic the second molecule of TCL as in the PDB structure 1P45 (chain A, hereafter 1P45:A) and interact with the rather hydrophobic residues defining the TCL2 site. These coumarins could be grafted to the diaryl ether scaffold with an appropriate linker.

The 1,2,3-triazole pharmacophore introduced by click-chemistry through copper(I) azide-alkyne cycloaddition (CuAAC) was chosen as a spacer, as illustrated in Fig. 1.

In silico studies of compound 7a

Preliminary molecular docking was performed to evaluate the ability of compound 7a (Fig. 3A) to bind into the substrate-binding site of the enzyme. With the aim of exploring the possible interactions and conformation of the compounds, we used two X-rays structures. The first one corresponds to the 1P45:A coordinates, where the binding of two TCL molecules has been observed with an open major portal and a closed minor portal. The second X-ray structure corresponds to the complex of InhA with PT70 (2-(o-tolyloxy)-5-hexylphenol, compound TCU in PDB 2X22, chain A, hereafter 2X22:A) in which the major portal is closed and the minor portal is opened. [START_REF] Luckner | A slow, tight binding inhibitor of InhA, the enoyl-acyl carrier protein reductase from Mycobacterium tuberculosis[END_REF] Two flexible docking protocols (MSE and OPT) were applied giving four results per ligand and two docking scores per result (see Experimental section 4.2.1. for pose selection, strength and conformity). With 1P45:A, U-shaped conformations of 7a were obtained whatever the docking protocol used (Fig. 3B). Fluctuations were limited and the hydrogen bonding network between Tyr158, the cofactor, the ligand, and the π-π interactions involving the nicotinamide group with the phenol group of 7a were similar to TCL1 (Supporting Information Fig. S1) whereas the coumarin group occupies the same place as TCL2. In contrast, Sshaped-like conformations were observed using 2X22:A, with the coumarin reaching the minor portal (Fig. 3C). The diaryl ether moiety of 7a showed the same interactions as TCL1 in 1P45:A and PT70/TCU in 2X22:A pattern (Supporting Information Fig. S2). Furthermore, by superimposing the strongest poses obtained after docking of 7a in 2X22:A, fluctuations starting from the triazole were observed in the minor portal (Fig. 3D). It has to be noted that the averaged fluctuations (positions and orientations) of coumarin were found similar using the two docking protocols. Interestingly, the docking scores regardless of protocol were better for compound 7a in 2X22:A than in 1P45:A.

Synthesis of the designed inhibitors

Based on our knowledge of the InhA binding site and docking results on compound 7a, several molecules have been conceived and are described in schemes 2 and 3. The azidomethyl derivatives 5ac were prepared in four steps from the corresponding aldehydes 1a-c, themselves obtained according to procedures previously described (Scheme 1) [START_REF] Inturi | Design, synthesis and evaluation of diphenyl ether analogues as antitubercular agents[END_REF][START_REF] Kostiuk | Unified syntheses of cavicularin and riccardin C: addressing the synthesis of an arene adopting a boat configuration[END_REF]. 5a

O R 1 N 3 O R 1 CHO O R 1 OH O R 1 Br R 2 R 3 R 2 R 3 R 2 R 3 R 2 R 3 (a) NaBH4
R 1 = OH, R 2 = R 3 = H 5b R 1 = H, R 2 = OH, R 3 = H 5c R 1 = H, R 2 = OH, R 3 = C 6 H 13 4a R 1 = OH, R 2 = R 3 = H 4b R 1 = H, R 2 = OH, R 3 = H 4c R 1 = H, R 2 = OH, R 3 = C 6 H 13 a b d 2a R 1 = OMe, R 2 = R 3 = H 2b R 1 = H, R 2 = OMe, R 3 = H 2c R 1 = H, R 2 = OMe, R 3 = C 6 H 13 1a R 1 = OMe, R 2 = R 3 = H 1b R 1 = H, R 2 = OMe, R 3 = H 1c R 1 = H, R 2 = OMe, R 3 = C 6 H 13 O R 1 Br R 2 R 3 3a R 1 = OMe, R 2 = R 3 = H 3b R 1 = H, R 2 = OMe, R 3 = H 3c R 1 = H, R 2 = OMe, R 3 = C 6 H 13 c
Scheme 1. Synthesis of the azidomethyl derivatives.

Briefly, a sequence of reduction, bromation and demethylation by BBr3 provided compounds 4a-c.

Deprotection was preferable at this stage, in terms of yield, rather than once the Huisgen's products 7a-n were obtained. Even knowing this, an effective demethylation reaction is compound-dependent.

The synthesis of compound 4a was achieved in 94%, while for compounds 4b and 4c, moderate yields were obtained with 38 and 27%, respectively. These compounds were finally transformed into their corresponding azidomethyl derivatives by action of NaN3 in DMF, synthons 5a-c being so obtained in 77, 27 and 9% overall yield respectively. Described procedures were used to dispose of the propargyl derivatives 6a-c (see Supporting Information) [START_REF] Arcau | Luminescent alkynyl-gold(i) coumarin derivatives and their biological activity[END_REF][START_REF] Ferraroni | Thioxocoumarins Show an Alternative Carbonic Anhydrase Inhibition Mechanism Compared to Coumarins[END_REF]. Compound 6d was synthesized in 38% yield, by action of propargyl bromide and potassium carbonate on 7-(diethylamino)-4-hydroxycoumarin [START_REF] Zhao | Harnessing Dual-Fluorescence Lifetime Probes to Validate Regulatory Mechanisms of Organelle Interactions[END_REF] in DMF. Compounds 6e-i were prepared involving the corresponding coumarinic acid derivatives and carbodiimidazole in dichloromethane whereas intermediates 6j-k were synthesized through wellknown protocols (see Supporting Information). The Huisgen's 1,3-dipolar azide-alkyne cycloaddition of azidomethyl derivatives 5a-c with the propargyl derivatives 6a-k proceeded in yield ranging from 44 to 87% to give the triazoles 7a-n as indicated in Scheme 2. The synthesis of 1,2,3-triazole derivatives 7an has been achieved by Cu(I)catalyzed Huisgen-Sharpless-Meldal [3+2] azide-alkyne dipolar cycloaddition (CUAAC) [START_REF] Tornøe | Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides[END_REF][START_REF] Rostovtsev | A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes[END_REF] of azidomethyl phenoxyphenols 5a-c with propargyl coumarins 6a-i and extended to propargyl benzoxadiazoles 6j-k (Scheme 2). In order to compare their inhibitory activity to that of 1,4-triazoles, two 1,5-triazole regioisomers were prepared via the ruthenium-catalyzed azide alkyne cycloaddition (RuAAC) [START_REF] Zhang | Ruthenium-Catalyzed Cycloaddition of Alkynes and Organic Azides[END_REF]. Azide 5a was reacted with alkynes 6a or 6b in the presence of Cp*RuCl(PPh3)2 as catalyst to furnish 1,5-disubstituted 1,2,3triazole derivatives 8a and 8b in low yields (Scheme 3).

O R 1 N O R 1 N 3 N N O O O O O O NH SO 2 NMe 2 N O N N H O O O NEt 2 N H O O OMe N H O O O OH N H O O O OMe 7a 7b 7c N H O O O 7d 7e 7f 7g 7h 7i 7k 6a-k R 2 R 3 5a R 1 = OH, R 2 = R 3 = H 5b R 2 = OH, R 1 = R 3 = H O O O 7l 5c R 1 = H, R 2 = OH, R 3 = C 6 H 13 N H O O O NEt 2 7m NH NO 2 N O N 7n NH NO 2 N O N 7j R 2 R 3 O O O O O O NEt 2 O 5a-c 7a-n (
O OH N N N O O O R O OH N 3 R = H R = CH 3 8a 8b (a) 6a or 6b, Cp*RuCl(PPh 3)2 , dioxane: 8a (35%), 8b (29%)
a 5a Scheme 3. Synthesis of 1,5-triazole derivatives 8a and 8b via a ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC).

InhA inhibition

In order to choose appropriate fragments for the development of inhibitors targeting the minor portal of InhA, twenty-two compounds including some alkynyl precursors, azide 5a and 1,4-and 1,5triazole derivatives were screened for their inhibitory potency against InhA. The assays were performed in the presence of 2-trans-dodecenoyl-CoA and NADH. The molecules were firstly tested at 500 µM for the alkyne precursors and 50 µM for the "click" derivatives. For the most potent inhibitors, IC50 values were determined (Table 1). The general trend shows that alkyne precursors exhibit low inhibitory activities compared to triazole products or even to the azide 5a. These results clearly show that the protein does not recognize these alkynic precursors. Diaryl ether azide 5a showed good inhibition with an IC50 of 0.843 µM. Compound 7d bearing 7-diethylaminocoumarin substituted at the position 4 showed the best inhibitory activity with 0.053 µM, a 15-fold increase in comparison to 5a.

Replacement of the 7-diethylamino substituent by hydrogen (compound 7c) resulted in a decrease of the activity. Comparison of compounds 7a and 7l, which only differ in the position of the hydroxyl group, showed the importance of this group which will dictate the positioning of the rest of the molecule by interacting with Tyr158. Coumarin-3-carboxamides 7e-7i were less active but with very important differences in inhibition depending on the substituent and its position on the coumarin. 7-Diethylamino coumarin 7f exhibited an IC50 of 3.5 µM while less bulky 7-methoxy-coumarin derivative 7g was 10 fold more active with an IC50 of 0.345 µM. Replacing coumarins by nitrobenzoxadiazole (NBD) or 4-(N,N-dimethylaminosulfonyl)benzoxadiazole (DBD) as found in 7j and 7k did not result in better inhibition. Two other compounds, 7m and 7n, bearing a C6-alkyl chain, were evaluated. These compounds are analogues to one of the most potent inhibitors of InhA, 5-hexyl-2-phenoxyphenol showing nanomolar inhibition of InhA enzyme but poor MIC (69 µM) [START_REF] Sullivan | High affinity InhA inhibitors with activity against drugresistant strains of Mycobacterium tuberculosis[END_REF]. The C6-alkyl chain plays a crucial role in interacting with the rather hydrophobic region close to the minor portal in the protein substrate-binding site. The presence of the C6 chain and the position of the hydroxyl group guide the positioning of the NBD and coumarin heterocycles toward the major portal. The two molecules showed weak inhibitory activity against the InhA enzyme (Table 1). Finally, 1,5-triazole regioisomers 8a and 8b

showed slightly weaker inhibition by comparison with 7a and 7b, with IC50 of 0.630 µM and 0.510 µM, respectively. This represents a drop in inhibition of only 2.5 to 3.8 times. These results show that 1,5triazole derivatives despite a very different conformational space compared with 1,4-triazoles have the ability to inhibit the InhA protein. NT: not tested, NI: no inhibition, NA: not applicable.

O OH N 3 O O O O O O O O O O O N H O O O N H O Et 2 N O O O O O O O OH N N N 8a-8b O OH N N N 7a-7k ED1727

Antimycobacterial activity

Table 1 lists the inhibitory activity of the growth of Mtb H37Rv for each 1.4-triazole derivatives.

Ciprofloxacine was used for comparison. MIC values obtained are very disparate, ranging from 1.25 µM to >100 µM. Two compounds, 7b and 7d, which display good inhibitory activity against InhA, were found to inhibit mycobacterial growth with MICs of 1.25 µM. Interestingly, these two active compounds are different by the positioning of the connection to the coumarin but also by the nature of the substituents that the coumarin carries. Compound 7a bearing no substituent, by comparison with compound 7b, shows a greater MIC (5 µM). 7-Des(diethylamino) compound 7c compared to 7d loses effectiveness. Coumarin amide 7e-7i presented MICs values higher than 10.0 µM. Surprisingly, compounds 7l and 7n, which displayed poor inhibitory activity against InhA, could inhibit Mtb growth with MICs of 5.0 and 2.5 µM, respectively, while 7m shows no activity. Therefore, we believe that these two compounds might have biological targets other than InhA.

O N N N O OH O 4 O Et 2 N O N N N NH OH 4 N O N O 2 N O O O O O O

Inhibition of mycolic acid biosynthesis in Mtb H37Rv

The effect of the three best anti-tubercular compounds, i.e. 7b, 7d and 7n, on the biosynthetic pathway of mycolic acids was investigated by metabolic labeling of pathogenic bacteria Mtb H37Rv with 14 C acetate. Treatment of Mtb H37Rv with three concentrations of compounds 7b and 7d corresponding to 1x, 5x, and 25x their MIC leads to a decreased production of lipids containing mycolic acids, namely trehalose monomycolates (TMM) and trehalose dimycolates (TDM) (Fig. 4A), as well as all types (alpha, methoxy, keto) of mycolic acids and accumulation of standard fatty acids (FAME) (Fig. 4B). These results are consistent with the potential of compounds 7b and 7d to inhibit the production of mycolic acids and their derivatives in mycobacterial cells. Of note, treatment with biaryl ether derivative 7n displays a distinct inhibition profile and leads to the accumulation of trehalose monomycolates and decreased production of trehalose dimycolates while it has no effect on the production of mycolic acid (Fig. 4B). This result correlates with the fact that compound 7n is a poor InhA inhibitor. This imbalance of TMM/TDM ratio in favor of TMM was reported upon the inhibition of the TMM transporter, MmpL3, using either small-molecule inhibitors [START_REF] Tahlan | SQ109 targets MmpL3, a membrane transporter of trehalose monomycolate involved in mycolic acid donation to the cell wall core of Mycobacterium tuberculosis[END_REF][START_REF] Grzegorzewicz | Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane[END_REF], or in conditional mycobacterial mmpL3 mutants [START_REF] Li | Therapeutic Potential of the Mycobacterium tuberculosis Mycolic Acid Transporter, MmpL3[END_REF].

Sensitivity of Mtb H37Ra strains overproducing KatG/InhA/HadABC

The mechanism of action of 7b, 7d, and 7n was investigated by using H37Ra strains carrying pVV16-katGsmeg, pMV261-inhA and pVV16-hadABC constructions overexpressing catalase peroxidase KatG, InhA and HadABC dehydratases, respectively. H37Ra strains carrying empty vectors pMV261 and pVV2

were used as controls. The same sensitivity of Mtb H37Ra strains overproducing KatG and HadABC comparing to the control strains means that the important HadABC component of the FASII system is not the target of the compounds and, as expected, these compounds are not activated by the KatG protein (Table 2). In contrast, the increased MIC of 7b and 7d in Mtb H37Ra overproducing InhA confirms InhA as the specific target. H37Ra pVV16-hadABC 1.25-6.25 6.25 2.5-12.5

Cytotoxic activity on human cell lines

The cytotoxic activity of compounds 7b, 7d and 7n was evaluated after 72 h of treatment on a panel of various human cell lines including the colon carcinoma HCT116 cell line, the HepG2 hepatocarcinoma cell line (which can recapitulate the metabolization of some compounds into toxic secondary metabolites), the CACO2 colorectal carcinoma cell line (used as a model of the intestinal epithelial barrier) and WI38hTERT, which are normal lung fibroblasts immortalized by telomerase expression. These assays revealed that compound 7b has only weak cytotoxic effects, especially against lung fibroblasts (>100 µM, Selectivity Index>80). Compound 7n exhibited moderate cytotoxicity against the panel tested, while 7d was clearly cytotoxic. These results show that the nature of coumarin plays an important role in the cytotoxicity of these compounds.

Table 3. Cytotoxicity of compounds 7b, 7d and 7n.

a The selectivity index corresponds for each compound to the ratio between the IC50 on the tested human cell line and the MIC on Mtb H37v. MIC and IC50 established from three independent experiments.

X-ray structures

In parallel to the characterization of the compounds for their ability to inhibit the enzymatic activity of InhA and mycobacterial growth, a massive crystallization campaign of the corresponding complexes was launched in order to rationalize the results obtained. The structures of the complexes with compounds 7b and 7e could be resolved, after incubation in the presence of NAD + , at resolutions of 1.60 and 1.96 Å, respectively (Table S1). From the more than one hundred X-ray structures of InhA available in the PDB, it appears that the protein and its complexes can be crystallized in different conditions leading to different space groups with one, two, four, six or eight molecules in the corresponding asymmetric units. The InhA/NAD + /7b complex (PDB code 8OTN) crystallized in the presence of PEG 4000 in space group (P212121 with cell parameters a=70.16 Å, b=91.18 Å, c=164.22 Å and 4 molecules in the asymmetric unit. The InhA/NAD + /7e (PDB code 8OTM) complex also crystallized in the presence of PEG 4000 and with 4 molecules in the asymmetric, but in a different space group (P21 with a=90.59 Å, b=92.78 Å, c=90.75 Å, β=119.20°). In both X-ray structures, all four molecules in the asymmetric unit are almost complete, only missing one or two residues at their N and C termini.

When superimposing based on Cα atoms, the different molecules in the asymmetric unit for each structure or the two structures between them, the calculated RMSD values do not exceed 0.3 Å,

showing that the tertiary structures of InhA in the two complexes are almost identical. Furthermore, in both structures, the SBL loop is in closed conformation and the orientation of the Phe149 and Tyr158 side chains are in the so-called out and in conformation, respectively (Fig. 5A). The NAD + is positioned in the cofactor binding site where it adopts the canonical conformation observed in all InhA structures through several hydrogen bonds and hydrophobic interactions. As expected, compounds 7b and 7e

bind similarly above the cofactor with the diaryl ether moiety in the same position as that found in the structures of InhA-TCL complexes, e.g. TCL1 in 1P45:A (see Fig. 2), the hydroxyl-substituted ring being stacked with the nicotinamide ring of the cofactor and the phenyl ring pointing towards the major portal. The orientation of the triazole with respect to the diaryl ether and its conformation is also the fluctuations are observed for the coumarin part of the ligands in the four molecules found in the respective asymmetric units (Fig. 5B). As TCL1 in 1P45:A, hydrogen bonds are found between the hydroxyl moiety of the diphenyl ether with the hydroxyl group of Tyr158 and the 2'-hydroxyl group of NAD + (Fig. 6). In addition, binding of both compounds occurs through non-bonded contacts invariably involving the cofactor and residues Gly96, Phe97, Met98, Phe149, Met155, Pro156, Ala157, Tyr158, Met161, Ala198, Met199, Ile202, Val203, Gln214, Leu217, Leu218. In some instances, the very tip of the Arg225 side chain from the same protomer and the C-terminal residues, Leu268 and Leu269, of a contacting protomer from the tetramer may complete the inhibitor-binding pocket (Fig. 6). Altogether, this allows to completely embed the diphenyl ether, the triazole and the coumarin phenyl moiety of 7b (resp. the coumarin lactone of 7e) whereas the coumarin lactone of 7b (resp. the coumarin phenyl moiety of 7e) is pointing out of the open crevice above the minor portal (Fig. 6). The overall conformations observed for 7b and 7e are reminiscent of the S-shaped conformation obtained after molecular docking of the 7a compound. It is noteworthy that the fluctuations observed for the coumarin part in the X-ray structures do not impact the positions and conformations of the InhA residues involved in non-bonded contacts, except for the side chains of residues Gln214 and Leu 217 and those of Leu268 and Leu269 of the contacting protomer, which allows fine adjustment. In the case of 7b, these fluctuations are of low amplitude (Fig. 6A,B). The flexibility of the coumarin part is more important in the case of the complex with 7e where in chain A, it is stabilized through hydrogen bonding between the carbonyl oxygen atom of the lactone and the NED2 atom in one of the two conformations of Gln214 (Fig. 6C), whereas in chain C, the C6-N1-C7-C8 amide dihedral bond adopts two alternate conformations resulting in the flipping of the C7-O3 bond without strongly affecting the orientation of the coumarin (Fig. 6D).

The X-ray structures obtained for 7b and 7e show how a diaryl ether-based hybrid inhibitor can explore the exit of the InhA minor portal in a way never seen before for triclosan derivatives. It has been previously shown that two such derivatives (i.e. compound 8PS and 1S5 in PDB 2B37 and 4OXN, respectively) can only reach the entrance of this unexplored part of the enzyme (Supporting Information Fig. S4). Future works will deal with the design of more potent diaryl ether-triazole hybrid inhibitors bearing original substituents which can provide anchors.

Conclusions

A hybridization strategy, based on a diaryl ether structural scaffold and coumarinic fragments, allows the design of new inhibitors able to explore underexploited regions in InhA. The docking results carried out on one of the targeted molecules 7a, show two binding modes in the active site of InhA, which depend on the chosen structure of InhA i.e 1P45:A or 1X22:A with major and minor portals more or less closed. Encouraged by these results, biaryl ether derivatives bearing various coumarins, or benzoxadiazoles moieties were synthesized in a few steps in high yields. Among them, two molecules 7b and 7d exhibited very good anti-Mtb efficiency and were able to block mycolic acid biosynthesis in Mtb H37Rv. Studies of their mechanism of action confirmed that InhA is their target. Two structures of the complexes InhA/NAD + with compounds 7b and 7e could be resolved. They reveal the binding of the coumarin moiety with a wide fluctuation, into the minor portal to a level never seen before. These structures represent a good opportunity for researchers interested in InhA to explore this finally accessible binding site. Further works, including structural optimization as well as pharmacological and toxicological studies, are now needed to evaluate the relevance of these molecules as anti-TB drugs.

Experimental section

Chemistry experimental 4.1.1. Material and methods

All commercially available chemical solvents and reagents were used without further purification.

Synthesis of the derivatives (4-Benzyl-3-methoxyphenyl)methanol (2a).

To a solution of aldehyde 1a (860 mg, 3.77 mmol) in methanol (43 mL) was added portion wise sodium borohydride (728 mg, 18.85 mmol). The mixture was stirred at room temperature for 3 h and water (50 mL) was added. After concentration under reduced pressure, the mixture was extracted with dichloromethane (3 x 50 mL). The combined organic layers were dried over MgSO4 and concentrated under reduced pressure. No further purification was necessary. The expected compound 2a was isolated quantitatively as a white solid, Rf = 0.34

(

4-(Bromomethyl)-2-methoxy-1-phenoxybenzene (3a

1-(4-(Bromomethyl)phenoxy)-4-hexyl-2-methoxybenzene (3c

5-(Bromomethyl)-2-phenoxyphenol (4a).

To a solution of bromide 3a (70 mg, 0.24 mmol) in dry dichloromethane (30 mL), boron tribromide (1 M solution in dichloromethane, 3 mL, 3.0 mmol) was added dropwise under argon atmosphere. The mixture was stirred at 4 °C for 4 h. Sodium bicarbonate saturated solution (40 mL) was added. The organic layer was separated and the aqueous layer was extracted with dichloromethane (2 x 40 mL). The combined organic layers were dried over MgSO4 and concentrated in vacuo. The crude product was purified by flash chromatography (petroleum ether/ethyl acetate: 90/10) affording compound 4a as a clear yellow oil (63 mg, 94%); Rf = 0.57 (dichloromethane).

2-(4-(Bromomethyl)phenoxy)-5-hexylphenol (4c).

To a solution of bromide 6c (103 mg, 0.27 mmol) in dichloromethane (5 mL) at 80 °C, was added boron tribromide (1M) (1.08 mL, 4 eq). After 5 h, methanol (5 mL) was added. The solvent was removed in vacuo and the crude product was dissolved in dichloromethane (20 mL). The organic mixture was washed with water (20 mL), dried over MgSO4, filtered and concentrated in vacuo.

5-(Azidomethyl)-2-phenoxyphenol (5a

2-(4-(Azidomethyl)phenoxy)phenol (5b).

Same procedure as for compound 5a involving bromide 4b (50 mg, 0.18 mmol), dry N,N-dimethylformamide (5 mL), sodium azide (13 mg, 0.2 mmol, 1.1 eq).

The crude product was purified by flash chromatography (petroleum ether/ethyl acetate: 80/20) affording compound 5b as a colourless oil, Rf = 0.46 (petroleum ether/ethyl acetate: 70/30).

HRMS (DCI-CH4, [M] +

) calculated for C13H11N3O2 241.0851, found 241.0869.

2-(4-(Azidomethyl)phenoxy)-5-hexylphenol (5c

7-(Diethylamino)-4-(prop-2-yn-1-yloxy)-2H-chromen-2-one (6d).

To a solution of 7-(diethylamino)-4-hydroxycoumarin (0.100 g, 0.429 mmol) in N,N-dimethylformamide, were added propargyl bromide (76 mg, 0.058 mL, 0.515 mmol) and potassium carbonate (119 mg, 0.858 mmol) at room temperature. The reaction mixture was stirred at 100 °C for overnight and then concentrated in vacuo. The crude material was dissolved in ethyl acetate and washed with water and brine. The organic layers were dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by flash chromatography (gradient dichloromethane/methanol: 100/0 to 95/5) affording compound 6d as a brown powder upon standing (0.038 g, 33%).

General procedure for the azide-alkyne cycloaddition.

A solution of azide (1.2 eq), alkyne (1 eq),

CuSO4

.5H2O (0.1 eq) and sodium ascorbate (0.4 eq) in a mixture of methanol/water was stirred at room temperature overnight. Water (100 mL) and dichloromethane (50 mL) were added. The combined organic layers were washed with water (50 mL) and brine (50 mL) and dried over MgSO4. After concentration in vacuo, the crude product was purified by flash chromatography affording the expected product.

7-((1-(3-Hydroxy-4-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (7a).

General procedure for the azide-alkyne cycloaddition involving azide 5a (

7-((1-(3-Hydroxy-4-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one (7b).

General procedure for the azide-alkyne cycloaddition involving azide 5a (100 mg, 0.416 mmol), alkyne 6b (75 mg, 0.348 mmol), CuSO4.5H2O (8.7 mg, 0.035 mmol) and sodium ascorbate (28 mg, 0.14 mmol) in a mixture methanol (4 mL), water (9 mL) and dichloromethane (20 mL). Purification by flash chromatography (dicholoromethane/methanol: 99/1) gave product 7b as a white solid (120 mg, 74%);

Rf = 0.

4-((1-(3-Hydroxy-4-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (7c).

General procedure for the azide-alkyne cycloaddition involving azide 5a (56 mg, 0.233 mmol); alkyne 6c (39 mg, 0.195 mmol), CuSO4.5H2O (4.9 mg, 0.0195 mmol) and sodium ascorbate (16 mg, 0.078 mmol) in a mixture methanol (2.5 mL), water (7.5 mL) and dichloromethane (20 mL). Purification by flash chromatography (dichloromethane/methanol: 99/1) gave compound 7c as a white solid (62 mg, 72%); Rf = 0.13 (dichloromethane/methanol: 98/2).

7-(Diethylamino)-4-((1-(3-hydroxy-4-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2Hchromen-2-one (7d).

General procedure for the azide-alkyne cycloaddition involving azide 5a (38 mg, 0.158 mmol), alkyne 6d (36 mg, 0.133 mmol), CuSO4.5H2O (4 mg, 0.00133 mmol) and sodium ascorbate (11 mg, 0.0052 mmol) in a mixture methanol (2 mL), water (5 mL) and dichloromethane (10 mL).

Purification by flash chromatography (dichloromethane/methanol: 98/2) gave product 7d as a paleyellow solid (55 mg, 81%), Rf = 0.13 (dichloromethane/methanol: 98/2).

N-((1-(3-Hydroxy-4-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2-oxo-2H-chromene-3carboxamide (7e

7-(Diethylamino)-N-((1-(3-hydroxy-4-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2-oxo-2Hchromene-3-carboxamide (7f

N-((1-(3-Hydroxy-4-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-7-methoxy-2-oxo-2Hchromene-3-carboxamide (7g).

General procedure for the azide-alkyne cycloaddition involving azide 5a (9 mg, 0.037 mmol), alkyne 6g (8 mg, 0.031 mmol), CuSO4.5H2O (0.8 mg, 0.0031 mmol) and sodium ascorbate (2.5 mg, 0.0124 mmol) in a mixture methanol (0.5 mL), water (2.5 mL) and dichloromethane (6 mL). Purification by flash chromatography (dichloromethane/methanol: 98/2) gave product 7g as a pale-yellow solid (13 mg, 87%), Rf = 0.14 (dichloromethane/methanol: 98/2).

N-((1-(3-Hydroxy-4-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-8-methoxy-2-oxo-2Hchromene-3-carboxamide (7h).

General procedure for the azide-alkyne cycloaddition involving azide 5a (21.5 mg, 0.09 mmol), alkyne 6h (19.3 mg, 0.075 mmol), CuSO4.5H2O (1.9 mg, 0.0075 mmol) and sodium ascorbate (6 mg, 0.03 mmol) in a mixture methanol (1 mL), water (4 mL) and dichloromethane (10 mL). Purification by flash chromatography (dichloromethane/methanol: 98/2) gave product 7h as a pale-yellow solid (22 mg, 59%), Rf = 0.08 (dichloromethane/methanol: 98/2).

6-Hydroxy-N-((1-(3-hydroxy-4-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2-oxo-2Hchromene-4-carboxamide (7i).

General procedure for the azide-alkyne cycloaddition involving azide 5a (27 mg, 0.11 mmol), alkyne 6i (25 mg, 0.096 mmol), CuSO4.5H2O (2.4 mg, 0.0096 mmol) and sodium ascorbate (7.7 mg, 0.038 mmol) in a mixture methanol (1.1 mL), water (6.1 mL) and dichloromethane (10 mL). The mixture was filtered affording compound 7i as a pale-yellow solid (27 mg, 48%), Rf = 0.08 (dichloromethane/methanol: 95/5).

5-((4-(((7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-2phenoxyphenol (7j

7-(((1-(3-Hydroxy-4-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-N,Ndimethylbenzo[c][1,2,5]oxadiazole-4-sulfonamide (7k).

General procedure for the azide-alkyne cycloaddition involving azide 5a (11.5 mg, 0.048 mmol), alkyne DBD 6k (11 mg, 0.04 mmol),

CuSO4.5H2O (1.02 mg, 0.0041 mmol) and sodium ascorbate (3.3 mg, 0.0167 mmol) in a mixture methanol (1 mL), water (3.5 mL) and dichloromethane (10 mL). Purification by flash chromatography (dichloromethane/methanol: 98/2) gave compound 7k as a yellow solid (15.5 mg, 75%), Rf = 0.12 (dichloromethane/methanol: 98/2).

7-((1-(4-(2-Hydroxyphenoxy)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (7l).

General procedure for the azide-alkyne cycloaddition involving azide 5b (50 mg, 0.208 mmol), alkyne 6i (35 mg, 0.174 mmol), CuSO4.5H2O (4.35 mg, 0.0174 mmol) and sodium ascorbate (14 mg, 0.696 mmol) in a mixture methanol (2 mL), water (7 mL) and dichloromethane (20 mL). Purification by flash chromatography gave compound 7l as a white solid (80 mg, 87%), Rf = 0.17 99.9% at 420 nm (tR = 6.393 min).

(

7-(Diethylamino)-N-((1-(4-(4-hexyl-2-hydroxyphenoxy)benzyl)-1H-1,2,3-triazol-4-yl)methyl)-2oxo-2H-chromene-3-carboxamide (7m

5-Hexyl-2-(4-((4-(((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)methyl)-1H-1,2,3-triazol-1yl)methyl)phenoxy)phenol (7n).

General procedure for the azide-alkyne cycloaddition involving azide 5c (13 mg, 0.04 mmol), alkyne 6k (9 mg, 0.04 mmol), CuSO4.5H2O (2 mg, 0.008 mmol, 0.2 eq) and sodium ascorbate (3 mg, 0.015 mmol, 0.4 eq) in a mixture methanol (1.5 mL), water (4.5 mL) and dichloromethane (10 mL). Purification by flash chromatography (gradient dichloromethane/methanol: 100/0 to 95/5) gave compound 7n as an orange solid (17 mg, 80%). Rf = 0.48 General procedure for the Ruthenium-catalysed azide-alkyne cycloaddition RuAAC [START_REF] Legigan | Synthesis and preliminary anticancer evaluation of new triazole bisphosphonate-based isoprenoid biosynthesis inhibitors[END_REF].

(
A solution of azide 1e (1 eq) and alkyne (1 eq) in degassed 1,4-dioxane ([alkyne] = 0.9 M) was added to a solution of Cp*RuCl(PPh3)2 (0.05 eq) in 1,4-dioxane ([Cp*RuCl(PPh3)2] = 0.009 M) under argon atmosphere. The mixture was stirred at 65 °C for 18h and the solvent was removed in-vacuo. Triazoles 5q, 5r were obtained after purification by flash-chromatography.

7-((1-(3-hydroxy-4-phenoxybenzyl)-1H-1,2,3-triazol-5-yl)methoxy)naphthalen-2(1H)-one (8a).

General procedure involving azide 2 (34.9 mg, 0.124 mmol), alkyne 6a (24.9 mg, 0.124 mmol), Cp*RuCl(PPh3)2 (5.0 mg, 6.2 µmol, 0.05 eq) in degassed 1,4-dioxane (0.8 mL). The crude product was purified by flash chromatography (Gradient petroleum ether/ethyl acetate: 70/30 to 0/100) affording the expected compound as a beige solid (19.0 mg, 35%), Rf = 0. Loop) extended region (residues 190-230) also differs, the structure of the cofactor (NAD + ) remains highly conserved. In addition to both enzyme conformations, two molecular docking protocols (MSE and OPT) and two internal scoring schemes (MolDock and ReRank) [START_REF] Thomsen | MolDock: a new technique for high-accuracy molecular docking[END_REF] were combined in a multimodal (docking) and consensus (scoring) approach. The two protocols share the same set of flexible residues:

7-((1-(3-hydroxy-4-phenoxybenzyl)-1H-1,2,3-triazol-5-yl)methoxy)-4-methylnaphthalen-2(1H)one (8b

Ala154, Ala157, Ala164, Ala198, Ala201, Ala206, Arg195, Asn231, Asp148, Asp150, Gln214, Glu219, Ile202, Ile215, Leu217, Leu218, Lys165, Met98, Met103, Met155, Met161, Met199, Met232, Phe149, Phe97, Pro193, Thr162, Thr195, Trp160, Trp22, Tyr158. Softened potentials were used with a tolerance of 1 and a strength 0.9. According to structural studies, these residues cover different cases of minor and major portal fluctuations in InhA known structures. No displaceable water molecules were taken in account inner the binding site (known as mainly hydrophobic). The corresponding NAD molecules (from 1P45:A or 2X22:A) were treated as cofactors and where set as NAD + with partial negative charges on phosphates and positive charge on nicotinamide group. The NAD is not flexible during the docking process, but treated independently of the protein: a component Ligand-Cofactor (in addition to the Ligand-Protein contribution) was used in docking scores calculations. Clustering of poses (tabu clustering) was set with an RMS threshold of 1.9 Å in order to be a little more discriminant on the best poses. Templates (pharmacophoric profile) were used with a strength of -500 and a grid resolution of 0.4 Å using conserved atom positions along InhA ligands: ether group (oxygen, hydrogen donor); alcohol group (oxygen, hydrogen donor/acceptor) and ring (carbons) of ligand TCU (PT70, taken from 2X22:A), similarity measure parameters were let at their default values. In the case of OPT (Differential evolution algorithm) protocol, the docking process used 10000 iteration steps, and a grid resolution of 0.3 Å, along 40 (1P45:A) or 30 (2X22:A) independent runs. The convergence was reached for all ligands, internal parameters (population size, crossover rate, scaling factor...) of the algorithm were let as default. A final minimization (per run) was parameterized using 4000 steps for lateral chains and 2000 steps for protein backbone followed by a minimization and optimization (hydrogen bonds) of ligands.

The same parameters were applied to the MSE (Simplex evolution algorithm) protocol, internal parameters (population size, number of iterations, energy threshold...) of the algorithm were let as default. The docking parameters used for all these simulations were benchmarked for their ability to reproduce the conformation of several co-crystalized inhibitors such TCL in 1P45:A and TCU in 2X22:A.

Typical root-mean-square deviation (RMSD) between the docking poses and X-ray structures were in the range 0.5-1.0 Å for the best poses (ReRank and MolDock combined scores). The most realistic complexes (best poses) were selected on the basis of conformity and strength. Conformity reflects the ability to reproduce the conformation of TCL-based ligands bound to InhA in known X-ray crystal structures complexes and the corresponding interaction network, mainly π-π or π-alkyl interactions with nicotinamide group of NAD, hydrogen bonding with Tyr158, hydrogen or electrostatic interactions with cofactor (OH from ribose, amido group, phosphates, charged nitrogen of pyridinium). If a given pose is found to be in the best group of values for both docking scores (ReRank and MolDock), this pose is qualified as strong.

Production and purification of InhA

Optimized InhA sequence from Mtb (UniProt P9WGR1) for Escherichia coli BL21(DE3) strain with

GenSmart TM Codon Optimization tool (Genscript) was inserted in pET15b plasmid using NdeI and BamHI restriction sites. A colony from transformed BL21(DE3) with the pET15b-InhA plasmid was grown in LB medium containing 100 µg/mL ampicillin at 37 °C 180 rpm overnight. 10 mL of this culture was added to 990 mL LB medium containing ampicillin (100 µg/mL) and incubated at 37 °C 150 rpm until an optical density (OD) at 600 nm of 0.6-0.8 was reached. Then, 1 mL of isopropyl-β-D-1thiogalactopyranoside (IPTG) 1 M was added before incubating the bacteria for 4 h at 150 rpm at 30

°C. Bacteria cells were pelleted by centrifugation at 6,000×g for 30 min at 4 °C, then resuspended with buffer A (20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 7.9) and lysed using an Emulsiflex-C5 cell disruptor (Avestin). After a centrifugation at 25,000×g for 30 min at 4 °C, the supernatant was filtered off with a 0.2 µm filter and injected into a nickel-chelated HisTrap HP column (Cytiva) equilibrated with buffer A. Unbound proteins were washed off with buffer A, then His6-InhA was eluted using buffer 20 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole, pH 7.9. Fractions containing His6-InhA

were pooled together and concentrated.

For enzymatic assays, the concentrated protein was centrifuged at 10,000×g for 10 minutes at 4

°C and injected into a preparative gel filtration SepFast 16/60 6-600 kDa pg column (BioToolomics) preequilibrated with buffer 30 mM PIPES, 150 mM NaCl, pH 6.8. His6-InhA containing fractions were flash frozen in liquid nitrogen at a final concentration of 5 µM.

For crystallographic assays, concentrated His6-InhA from affinity purification was incubated at room temperature with Thrombin (Novagen) overnight to cleave the histidine tag. Then, the solution was filtered off with a 0.2 µm filter and injected again into a nickel-chelated HisTrap HP column (Cytiva) equilibrated with buffer A and cleaved proteins were eluted with the same buffer. Fractions corresponding to the cleaved InhA were pooled together, concentrated, centrifuged at 10,000×g for 10 min at 4 °C and injected on a preparative gel filtration column SepFast 16/60 6-600 kDa pg column (BioToolomics) equilibrated with buffer 30 mM PIPES, 150 mM NaCl, pH 6.8. Fractions corresponding to InhA were collected and concentrated.

Evaluation of InhA enzyme inhibition

NADH was obtained from Sigma-Aldrich. Stock solutions of all compounds were prepared in DMSO (5% v/v). Kinetic assays were performed using trans-2-dodecenoyl-coenzyme A (DDCoA) and His6-InhA as previously described [START_REF] Doğan | Discovery of hydrazone containing thiadiazoles as Mycobacterium tuberculosis growth and enoyl acyl carrier protein reductase (InhA) inhibitors[END_REF]. Briefly, reactions were performed at 25 °C in 30 mM PIPES, 150 mM NaCl,

Crystallization and structure determination

To obtain crystals of InhA in complex with ligands, cleaved InhA (in 30 mM PIPES, 150 mM NaCl, pH 6.8) was co-incubated at either 4 or 8 mg/mL with NAD + (respectively 1.4 and 2.8 mM, corresponding to a 1:10 protein/NAD + ratio) for 1 h at 4 °C. Mixtures were then placed at room temperature 10 min before adding ligands (solubilized in DMSO), by respecting the final 5% v/v DMSO with the protein. After 2 h incubation at room temperature or 4 °C, depending on the ligand solubility, crystallization assays were performed using the vapor diffusion method at 20 °C by mixing, with the help of a Mosquito crystallization robot (SPT Labtech, Melbourn, UK), 200 nL of InhA previously incubated with NAD + and ligands with 200 nL of reservoir solution containing 14% PEG 4000, 100 mM ADA, 100 mM ammonium acetate, 5 % v/v DMSO, pH 6.8. After 1 week, crystals were cryoprotected with paraffine before being flash frozen in liquid nitrogen. Diffraction data were collected at the ESRF beamline ID30B and the ALBA beamline XALOC, processed with autoProc [START_REF] Vonrhein | Data processing and analysis with the autoPROC toolbox[END_REF], staraniso [START_REF] Tickle | STARANISO[END_REF], and XDS

[65] (Supporting Information Table S1). Structures were solved by molecular replacement with MOLREP [66] using the A chain of 4OHU in the case of the complex in the presence of 7b and 4OYR in the case of 7e. Refinement was performed using Buster/TNT [START_REF] Bricogne | BUSTER version 2.10.4[END_REF]. Dictionaries for compounds were generated using Grade2 [START_REF] Smart | Grade2 version 1[END_REF] and used in Coot (0.9.8.1) for model building [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF].

Inhibition of mycobacterial growth

Colorimetric microassay based on the reduction of tetrazolium salts (MTT, Thiazolyl Blue Tetrazolium Bromide, Sigma) to formazan by metabolically active mycobacteria was used to measure the susceptibility of Mtb H37Rv to each molecule by determining its minimum inhibitory concentration (MIC) as previously described [START_REF] Le | Drug screening approach against mycobacterial fatty acyl-AMP ligase FAAL32 renews the interest of the salicylanilide pharmacophore in the fight against tuberculosis[END_REF]. Briefly, bacterial cells growing as surface pellicle were recovered from 100 mL culture in 7H9 broth (Middlebrook 7H9 broth base BD Difco) containing 0.2% glycerol (w/v), vortexed with 4 mm glass beads, suspended in 20 mL 7H9 broth-0.2% glycerol and centrifuged at 800 rpm to eliminate residual clumps. Serial twofold dilutions of each compound to be tested (stock solution at 1 or 10 mM in DMSO) were prepared in 100 µL 7H9 broth using 96-well flat bottom microplates (Nunc). 100 µl of mycobacterial suspension (OD 600 nm 0.1) in 7H9 broth were then added to each well. After 6 days incubation at 37 °C, MTT was added (50 µl at 1 mg/ml). After one-day incubation at 37 °C, 50 µL of 20% SDS solution were added to each well to solubilize formazan crystals and the OD of each well was measured at 570 nm (microplate reader Expert plus 96, ASYS HITECH, Austria). The MIC was determined as the lowest concentration of drug that inhibited bacterial growth from at least three independent experiments. The data were normalized to the value measured in untreated wells, after subtracting the background absorbance to each value.

Inhibition of mycolic acid biosynthesis

Mtb H37Rv cells were cultivated in 7H9 broth with albumin-dextrose-catalase and 0.05% Tween 80 at 37 °C with shaking (120 rpm). INH and compounds 7b, 7d and 7n (at final concentrations of 0, 1×, 5× and 25× their MICs, stock in DMSO) were added when the culture reached O.D (600 nm) ~ 0.2.

At the same time, the 14 C-acetate (specific activity 110 mCi/mmol, ARC) was added at final concentration of 0.5 µCi/mL. After 24 h of static cultivation at 37 °C, the cultures were harvested and the fatty/mycolic acids were extracted from whole cells from 100 µL cultures and derivatized to corresponding methyl esters (FAME/MAME) [START_REF] Phetsuksiri | Antimycobacterial activities of isoxyl and new derivatives through the inhibition of mycolic acid synthesis[END_REF]. Dried extracts were dissolved in 50 µL of chloroform/methanol (2:1, v/v) and 5 µL were loaded on TLC silica gel 60 F254 plates (Merck).

FAME/MAME were separated in n-hexane/ethyl acetate (95:5, 3 runs) and visualized using an AmershamTM TyphoonTM Biomolecular Imager.

Lipids were extracted from cells from 100 μL cultures containing the same concentrations of compounds with 1.5 mL chloroform/methanol (2 :1 v/v) at 65 °C for 2 h. Then 150 µl of H2O were added, the samples were mixed, centrifuged and bottom organic phases were removed and dried under N2. Lipid extracts were subjected to biphasic Folch washing. Isolated lipids were dissolved in 30 µL of chloroform/methanol (2: 1, v/v) and 5 µL were loaded on the thin-layer chromatography (TLC) silica gel plates F254 (Merck). Lipids were separated in chloroform/methanol/water (20:4:0.5, v/v/v) and visualized using an AmershamTM TyphoonTM Biomolecular Imager.

Analysis of sensitivity of Mtb H37Ra strains overproducing KatG/InhA/HadABC

InhA protein, HadABC complex and KatGsmeg protein encoded by the gene msmeg_6384 from M. smegmatis were overproduced in Mtb H37Ra using pMV261-inhA, pVV16-hadABC or pVV16-katGsmeg constructs as already described [START_REF] Pflégr | Design and synthesis of 2-(2-isonicotinoylhydrazineylidene)propanamides as InhA inhibitors with high antitubercular activity[END_REF][START_REF] Grzegorzewicz | A common mechanism of inhibition of the Mycobacterium tuberculosis mycolic acid biosynthetic pathway by isoxyl and thiacetazone[END_REF][START_REF] Larsen | Overexpression of inhA, but not kasA, confers resistance to isoniazid and ethionamide in Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis[END_REF]. Sensitivity of Mtb H37Ra strains overproducing InhA, HadABC and KatGsmeg, as well as control strains carrying empty pMV261 and pVV16 vectors, to compounds 7b, 7d, and 7n was analyzed by drop dilution method. The culture of each strain was diluted to OD (600 nm) = 0.5. Then, 4 µl of 1x, 10x and 100x diluted samples were plated on 7H11-agar + 10 % oleic acidalbumin-dextrose-catalase containing various concentrations of tested compounds 7b and 7d (0.625, 1.25, 6.25, 12.5, 25 μM) and 7n (1.25, 2.5, 12.5, 25, 50 µM). Plates were incubated for 18 days at 37 °C.

Cell viability assays

Human cell lines were grown at 37 °C in a humidified 5 % CO2 incubator in DMEM high-glucose (Thermo Fisher Scientific) for HCT116 (Horizon Discovery), MEM containing Earle's salts, sodium pyruvate and non-essential amino acids for CACO2 (DSMZ), HepG2 (ATCC) and WI38hTERT (generous gift from Dr Olivier Sordet, CRCT, Toulouse). All media contained 10 % or 20 % (for CACO2) fetal bovine serum (Eurobio), 2.5 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin. For SRB assays, cells were plated in 96-well plates (Nunc Edge) at 1500, 2500, 9000 and 3500 cells/well respectively for HCT116, CACO2, HepG2 and WI38hTERT 24 h before treatment with the drug. Serial 2-fold dilutions were performed in medium and used to treat cells for 72 h. At the end of treatment, cells were fixed and stained with SRB as described [START_REF] Vichai | Sulforhodamine B colorimetric assay for cytotoxicity screening[END_REF]. After resuspending the dye in a 10 mM Trisbase solution, the OD at 490 nm of each well was measured (µQuant plate reader, Bio-tek) as a readout of the cell number. For calculation, background absorbance was subtracted to each value and the data were normalized to the value measured in untreated wells. Each point was measured in duplicate and the IC50 were computed from at least three independent experiments using the GraphPad Prism software to perform a non-linear regression to a four-parameter logistic curve (Inhibitor vs response; variable slope). An approximate value was provided when the curve did not fit the logistic curve.

Figure 1 .

 1 Figure 1. Design of the new diaryl ether inhibitors by a molecular hybridization approach based on TCL and coumarins.

Figure 3 .

 3 Figure 3. A. Compound 7a used for docking. B. Best poses obtained using the MSE and OPT docking protocols of 7a (black and dark gray sticks, respectively) in 1P45:A (lightblue ribbon and surface) relatively to Tyr158 (cyan). The two TCL molecules (pink) and NAD (green) of 1P45:A are also shown. C. Best poses obtained using the MSE and OPT docking protocols of 7a (black and dark gray sticks, respectively) in 2X22:A (lightblue ribbon and surface) relatively to Tyr158 (cyan). The TCU (brightorange) and NAD (green) molecules of 2X22:A are also shown. D. Fluctuations of the ten best poses (MSE and OPT docking protocols, white) of 7a in 2X22:A (lightblue ribbon and surface) relatively

Scheme 2 .

 2 Scheme 2. Synthesis of the various diaryl ether derivatives 7a-n by CuAAC click chemistry.

Figure 4 .

 4 Figure 4. TLC analysis of lipids after metabolic labeling experiment of mycobacteria treated with compounds 7b, 7d, or 7n for the analysis of lipid inhibition (A) and for the analysis of mycolic acid inhibition (B). 7b (1.25 µM 1×, 6.25 µM 5×, 31.25 µM 25×), 7d (1.25 µM 1×, 6.25 µM 5×, 31.25 µM 25×), 7n (2.5 µM 1×, 12.5 µM 5×, 62.5 µM 25×). Abbreviations: FAME: Fatty Acid Methyl Esters, MAME: Mycolic Acid Methyl Esters; TDM: Trehalose DiMycolates; TMM: Trehalose MonoMycolates; PE: PhosphatidylEthanolamine; CL: CardioLipin.

Table 2 .

 2 Sensitivity of Mtb H37Ra strains overproducing KatG/InhA/HadABC to 7b, 7d and 7n.

Figure 5 .

 5 Figure 5. X-ray structures of InhA/NAD + /7b (left) and InhA/NAD + /7e (right). A. Overall structure. Cartoon representation of InhA/NAD + /7b (8OTN, chain D) and InhA/NAD + /7e, (8OTM, chain B) with the NAD + (green), the ligand (shades of grey) and the side chains of the catalytic triad Phe149-Tyr158-Lys165 (cyan) shown as sticks. The SBL loop is shown in orange. Red and blue arrows in panel A indicate the active site entrance and minor portal, respectively. B. Two perpendicular zoom-in views showing the fluctuations of the coumarin part of the ligands in the four molecules found in the respective asymmetric unit (shown in A. and B. as different shades of grey).

Figure 6 .

 6 Figure 6. Detailed description of 7b and 7e binding to InhA/NAD + . The two most extreme conformations of the coumarin part observed for 7b (resp. 7e) are displayed in panels A. and B. (resp. C. and D.). The protein is shown as cartoon using the same color scheme as in Fig.5. The cofactor is in green. Protein residues involved in defining the inhibitor-binding pocket are shown as cyan sticks (side chain atoms involved) or spheres (only main chain atoms are involved) and labeled. Hydrogen bonds are displayed as black dotted lines.

1 H

 1 and13 C were recorded on a Bruker Avance 600 (600 MHz and 151 MHz respectively); Bruker Avance 500 (500 MHz and 125 MHz, respectively) or a Bruker Avance 300 spectrometer (300 MHz and 75 MHz, respectively). The chemical shift values are reported in ppm using the solvent residual peak as internal reference: CDCl3 δ = 7.26 ppm/77.0 ppm, DMSO-d6 δ = 2.50 ppm/39.52 ppm and the coupling constants (J) are in Hz, while multiplicities are abbreviated by s (singlet); bs (broad singlet); d (doublet); dd (doublet of doublets); ddd (doublet of doublets of doublets); t (triplet); td (triplet of doublets); tt (triplet of triplets); m (multiplet). * = the multiplicity of the signal is more complex as it is part of an AA'XX' system. High-resolution mass spectra (HRMS) were recorded on a GCT Premier spectrometer upon electron spray ionization (ESI) or chemical ionization (DCI, CH4). Follow up of the reaction outcome by thin-layer chromatography (TLC) was performed on precoated Merck silica gel 60F-254 plates using an appropriate solvent system and the different spots were detected under UV lamp (λ 254 nm or 360 nm). Flash chromatography were performed using a Puriflash®300 with silica columns.All the compounds were dissolved in acetonitrile before analysis by a Waters Acquity UHPLC-DAD-MS (Ultra Performance Liquid Chromatography with Diode Array Detector). The separations were achieved on a Waters Acquity BEH Phenyl (100 mm x 2.1 mm, particle size 1.7 µm) column. The mobile phase consisted of water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B) at a flow rate of 0.4 mL.min -1 . The temperature of the column oven was set at 30 °C. The chromatographic analysis consisted in a linear gradient program from 60% A/40% B to 0% A/100% B between 1 and 12 min. After each run, the percentage of solvents ramped to their initial composition in 2 min and then the column was re-equilibrated for 2 min. The UV detection were performed at Lambda max of each compound and UV-Vis spectra were recorded within a range of 200-800 nm. The presence of possible impurities was evaluated at different wavelengths. The data acquisition and processing were done with the Empower 2 software. All tested compounds were more than 95% pure, as established by HPLC.The synthesis of 3-methoxy-4-phenoxybenzaldehyde (1a)[START_REF] Inturi | Design, synthesis and evaluation of diphenyl ether analogues as antitubercular agents[END_REF], 4-(2-methoxyphenoxy)benzaldehyde (1b) [46], 7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (6a) [48], 4-methyl-7-(prop-2-yn-1-yloxy)-2Hchromen-2-one (6b) [47], 4-(prop-2-yn-1-yloxy)-2H-chromen-2-one (6c) [47], 7-Nitro-N-(prop-2-yn-1yl)benzo[c][1,2,5]oxadiazol-4-amine (6j) [56], and N,N-dimethyl-7-(prop-2-yn-1ylamino)benzo[c][1,2,5]oxadiazole-4-sulfonamide (6k) [57] have been previously described and the observed chemical shifts in NMR are in accordance with the published data. The synthesis and the spectroscopic data of aldehyde derivative 1c and propynyl coumarins 6e-6h are described in the Supporting Information.

pH 6 .

 6 8 containing 250 µM of the NADH cofactor, 50 µM of the DDCoA substrate and the tested compound at different concentrations. Reactions were initiated by addition of InhA (50 nM final) and NADH oxidation was followed at 340 nm. The inhibitory activity of each compound was expressed as the percentage of inhibition of the InhA activity (initial velocity of the reaction) with respect to the control reaction without inhibitor. Inhibition = ((Slope InhA alone -slope with compound) / Slope InhA alone)*100. All activity assays were performed in duplicate or triplicate. IC50 values were determined using the 4-parameter curve-fitting software XLFit (IDBS) with at least six points.

Table 1 .

 1 Activities against InhA enzyme and Mtb H37Rv strain.

	Compound	R	IC50 (

µM or % at concentration Tested) MIC H37Rv μg/mL (µM) 5a

		0.843 ± 0.094	NT
	6a	200 µM: 99% 100 µM: 30%	NT
	6b	500 µM: 90% 200 µM: 37%	NT
	6c	500 µM: NI	NT
	6e	200 µM: NI	NT
	6f	500 µM: 10%	NT
	7a	0.163 ± 0.017	2.2 (5)
	ED1726
	7b	0.205 ± 0.012	0.6 (1.25)

4-(4-Hexyl-2-methoxyphenoxy)phenyl)methanol (2c). Same

  petroleum ether/ethyl acetate: 80/20). 1 H NMR (CDCl3, 300 MHz) δ 7.33-7.27 (m, 2H); 7.07-7.02 (m,

	2H); 6.97-6.92 (m, 3H); 6.89 (dd, J = 8.2, 1.9 Hz, 1H); 4.69 (s, 2H); 3.85 (s, 3H). 13 C NMR (CDCl3, 75 MHz)
	δ 157.8; 151.3; 144.2; 137.6; 129.4 (2C); 122.4; 120.8; 119.3; 117.0 (2C); 111.5; 64.8; 55.8. HRMS (DCI-
	CH4, [M] + ) calculated for C14H14O3 230.0943, found 230.0937.
	acetate: 100/0 to 90/10) to give compound 2c as a white solid (608 mg, 90%), Rf = 0.60
	(dichloromethane/ethyl acetate: 90/10). 1 H NMR (CDCl3, 300 MHz) δ 7.26 (d*, J = 8.8 Hz, 2H); 6.93-
	6.87 (m, 3H); 6.82 (d, J = 1.9 Hz, 1H); 6.74 (dd, J = 8.0 Hz, 2.0 Hz, 1H); 4.60 (s, 2H); 3.81 (s, 3H); 2.61 (t,
	J = 8.0 Hz, 2H); 1.96 (bs, 1H); 1.63 (quint, J = 7.7 Hz, 2H); 1.42-1.30 (m, 6H); 0.91 (t, J = 7.0 Hz, 3H). 13 C
	NMR (CDCl3, 75 MHz) δ 157.8; 151.1; 142.4; 140.0; 134.6; 128.4 (2C); 121.0; 120.7; 116.8 (2C); 113.0;
	64.8; 55.9; 35.8; 31.7; 31.5; 29.0; 22.6; 14.0. HRMS (DCI-CH4, [M+H] + ) calculated for C20H27O3 315.1960,

(4-(2-Methoxyphenoxy)phenyl)methanol (2b).

Same procedure as for compound 2a, involving aldehyde 1b (340 mg, 1.49 mmol) in methanol (17 mL), sodium borohydride (290 mg, 7.45 mmol). The crude product was purified by flash chromatography (petroleum ether/ethyl acetate: 80/20) to give compound 2b as a colourless oil (320 mg, 94%); Rf = 0.08 (petroleum ether/ethyl acetate: 80/20). 1 H NMR (CDCl3, 300 MHz) δ 7.29 (d*, J = 8.7 Hz, 2H); 7.14 (ddd, J = 8.3, 7.1 Hz, 2.0 Hz, 1H); 7.03-6.89 (m, 5H); 4.65 (s, 2H); 3.84 (s, 3H). 13 C NMR (CDCl3, 75 MHz) δ 157.5; 151.4; 144.9; 134.9; 128.5 (2C); 129.9; 121.1; 121.1; 117.1 (2C); 112.8; 64.9; 55.9. HRMS (DCI-CH4, [M] + ) calculated for C14H14O3 230.0943, found 230.0954.

(procedure as for compound 2a, involving aldehyde 1c (645 mg, 2.09 mmol) in methanol (30 mL) sodium borohydride (397 mg, 10.5 mmol). The crude product was purified by flash chromatography (gradient dichloromethane/ethyl found: 315.1960.

  ). To a solution of alcohol 2a (140 mg, 0.609 mmol, 1 eq) and triphenylphosphine (416 mg, 1.58 mmol) in dry dichloromethane (7 mL) was added portion wise N-bromosuccinimide (282 mg, 1.58 mmol). The mixture was stirred at room temperature for 3 h. After concentration under reduced pressure, the crude product was purified by flash

	chromatography (petroleum ether/ethyl acetate: 70/30) affording compound 3a as a white solid (169
	mg, 95%); Rf = 0.62 (petroleum ether/ethyl acetate: 70/30). 1 H NMR (CDCl3, 300 MHz) δ 7.34-7.28 (m,
	2H); 7.07 (tt, J = 7.4 Hz, 1.4 Hz, 1H); 7.03 (d, J = 2.0 Hz, 1H); 6.98-6.92 (m, 3H); 6.87 (d, J = 8.1 Hz, 1H);
	4.51 (s, 2H); 3.87 (s, 3H). 13 C NMR (CDCl3, 75 MHz) δ 157.3; 151.2; 145.6; 134.0; 129.6 (2C); 122.9;
	121.7; 120.3; 117.7 (2C); 113.4; 56.0; 33.6. HRMS (DCI-CH4, [M+H] + ) calculated for
	C14H14BrO2 293.0177, found 293.0167.

1-(4-(Bromomethyl)phenoxy)-2-methoxybenzene (3b). Same procedure as for compound 3a

involving alcohol 2b (290 mg, 1.26 mmol), triphenylphosphine (860 mg, 3.27 mmol, 2.6 eq), dry dichloromethane (15 mL), N-bromosuccinimide (583 mg, 3.27 mmol). The crude product was purified by flash chromatography (petroleum ether/ethyl acetate: 90/10) to give compound 3b as a white solid (280 mg, 76%); Rf = 0.55 (petroleum ether/ethyl acetate: 70/30).

1

H NMR (CDCl3, 300 MHz) δ 7.31 (d*, J = 8.7 Hz, 2H); 7.16 (ddd, J = 8.4, 7.2, 2.0 Hz, 1H); 7.01 (dd, J = 8.1, 1.8 Hz, 2H); 6.94 (ddd, J = 8.1 Hz, 7.2 Hz, 1.8 Hz, 1H); 6.88 (d*, J = 8.7 Hz, 2H); 4.50 (s, 2H); 3.82 (s, 3H). 13 C NMR (CDCl3, 75 MHz) δ 158.2; 151.6; 144.3; 131.5; 130.4 (2C); 125.3; 121.6; 121.2; 116.9 (2C); 112.8; 55.9; 33.6. HRMS (DCI-CH4, [M+H] + ) calculated for C14H14BrO2 293.0177, found 293.0177.

  ). To a solution of bromide 4a (170 mg, 0.61 mmol) in dry

	N,N-dimethylformamide (2 mL), was added sodium azide (97 mg, 1.48 mmol). The mixture was stirred
	at 45 °C for 4 h. Ethyl acetate (30 mL) and water (20 mL) were added, the organic layer was washed
	with water (2 x 20 mL). The combined organic layers were dried over MgSO4 and concentrated under
	reduced pressure. The crude product was purified by flash chromatography (petroleum ether/ethyl
	acetate: 80/20) affording compound 5a as a colourless oil (125 mg, 86%); Rf = 0.60 (petroleum
	ether/ethyl acetate: 70/30). 1 H NMR (CDCl3, 300 MHz) δ 7.40-7.33 (m, 2H); 7.17-7.12 (tt, J = 7.4 Hz, 1.5
	Hz, 1H); 7.06-7.02 (m, 3H); 6.86 (d, J = 8.3 Hz, 1H); 6.79 (dd, J = 8.3 Hz, 2.3 Hz, 1H); 5.64 (s, 1H); 4.28 (s,
	2H). 13 C NMR (CDCl3, 75 MHz) δ 156.3; 147.4; 143.5; 131.7; 129.8 (2C); 123.7; 120.3; 118.6; 118.1 (2C);
	116.0; 54.2. HRMS (DCI-CH4, [M] + ) calculated for C13H11N3O2 241.0851, found 241.0845.

  ). General procedure for the azide-alkyne cycloaddition involving 5a(50 mg, 0.208

	mmol), alkyne 6e (40 mg, 0.174 mmol), CuSO4.5H2O (4.35 mg, 0.0174 mmol) and sodium ascorbate (14
	mg, 0.070 mmol) in a mixture methanol (2 mL), water (7 mL) and dichloromethane (20 mL). Purification

by flash chromatography (dichloromethane/methanol: 99/1) gave product 7e as a white solid (70 mg, 86), Rf = 0.28 (dichloromethane/methanol: 97/3). 1 H NMR (DMSO-d6, 500 MHz) δ 9.68 (s, 1H); 9.14 (t, J = 5.6 Hz, 1H); 8.89 (s, 1H); 8.06 (s, 1H); 8.00 (dd, J = 7.8 Hz, 1.5 Hz, 1H); 7.76 (ddd, J = 8.7, 7.0, 1.8 Hz, 1H); 7.51 (d, J = 8.4 Hz, 1H); 7.45 (ddd, J = 8.7 Hz, 7.1 Hz, 1.1 Hz, 1H); 7.31-7.27 (m, 2H); 7.01 (tt, J = 7.4, 1.0 Hz, 1H); 6.94 (d, J = 8.2 Hz, 1H); 6.89 (d, J = 2.1 Hz, 1H); 6.84-6.81 (m, 2H); 6.78 (dd, J = 8.2 Hz, 2.1 Hz, 1H); 5.51 (s, 2H); 4.60 (d, J = 5.6 Hz, 2H). 13 C NMR (DMSO-d6, 126 MHz) δ 161.1; 160.3; 157.7; 153.9; 149.4; 147.7; 144.4; 142.4; 134.2; 133.1; 130.3; 129.6 (2C); 125.1; 123.1; 122.0; 121.9; 119.2; 118.8; 118.4; 116.7; 116.2 (2C); 116.2; 52.3; 34.9. HRMS (ESI, [M+H] + ) calculated for C26H21N4O5 469.1512, found 469.1511. Purity (HPLC): > 99.9% (tR = 2.886 min, λmax = 300 nm).

  1 H NMR (DMSO-d6, 500 MHz ) δ 9.94 (s, 1H); 9.67 (s, 1H); 9.17 (t, J = 5.6 Hz, 1H); 8.81 (s, 1H); 8.05 (s, 1H); 7.36 (d, J = 9.0 Hz, 1H); 7.31-7.27 (m, 3H); 7.18 (dd, J = 9.0, 2.8 Hz, 1H); 7.01 (tt, J = 7.4, 1.0 Hz, 1H); 6.93 (d, J = 8.2 Hz, 1H); 6.89 (d, J = 2.0 Hz, 1H); 6.84-6.81 (m, 2H); 6.77 (dd, J = 8.2 Hz, 2.0 Hz, 1H); 5.50 (s, 2H); 4.59 (d, J = 5.6 Hz, 2H).13 C NMR (DMSO-d6, 126 MHz) δ

	161.3; 160.6; 157.7; 154.2; 149.4; 147.7; 147.4; 144.5; 142.4; 133.1; 129.6 (2C); 123.1; 122.5; 122.0;
	121.9; 119.2; 119.0; 118.6; 117.1; 116.7; 116.2 (2C); 113.8; 52.3; 34.9. HRMS (ESI, [M+H] + ) calculated
	for C26H21N4O6 485.1461, found 485.1449. Purity (HPLC): 96.5% at 290 nm (tR = 2.108 min).

  ). General procedure for the azide-alkyne cycloaddition involving azide 5a (50 mg, 0.208 mmol), alkyne 6j (38 mg, 0.174 mmol), CuSO4.5H2O (4.35 mg, 0.0174 mmol) and sodium ascorbate (14 mg, 0.070 mmol) in a mixture methanol (2 mL), water (7 mL) and dichloromethane (20 mL). The crude product was purified by flash chromatography (dichloromethane/methanol: 99/1) affording compound 7j as an orange solid (32 mg, 44%), Rf = 0.15 (dichloromethane/methanol: 98/2).

1

H NMR (DMSO-d6, 500 MHz) δ 9.85 (bs, 1H (H25)) ; 9.66 (bs, 1H); 8.52 (d, J = 8.9 Hz, 1H) ; 8.17 (s, 1H (H15)); 7.29 (tapp, J = 7.9 Hz, 2H); 7.01 (t, J = 7.3 Hz, 1H)); 6.92 (d, J = 8.1 Hz, 1H); 6.88 (d, J = 2.1 Hz, 1H); 6.81 (d, J = 8.2 Hz, 2H); 6.74 (dd, J = 8.2 Hz, 2.1 Hz, 1H) ; 6.53 (d, J = 8.9 Hz, 1H); 5.50 (s, 2H); 4.77 (s, 2H). 1 H NMR (DMSO-d6, 126 MHz) δ 157.7; 149.3; 149.0; 143.8; 142.9; 141.8; 137.7; 142.5; 133.0; 129.6 (2C); 123.6; 122.1; 121.8; 119.2; 116.7; 116.3 (2C); 110.4; 99.9; 52.4; 38.6. HRMS (ESI, [M+H] + ) calculated for C22H18N7O5 460.1369, found 460.1377. Purity (HPLC): 97.7% at 330 nm (tR = 4.718 min).

  dichloromethane/methanol: 95/5).1 H NMR (CDCl3, 600 MHz) δ 8.48 (d, J = 8.6 Hz, 1H); 7.50 (s, 1H);

	7.25-7.24 (m, 2H); 6.99 (d*, J = 8.7 Hz, 2H); 6.88 (d, J = 2.1 Hz, 1H); 6.81 (d, J = 8.2 Hz, 2H); 6.69-6.67
	(m, 2H); 6.35 (d, J = 8.6 Hz, 1H); 5.51 (s, 2H); 4.77 (d, J = 5.4 Hz, 2H); 2.56 (t, J = 7.9 Hz, 2H); 1.60-1.56
	(m, 2H); 1.36-1.29 (m, 6H); 0.88 (t, J = 7.0 Hz, 3H). 13 C NMR (CDCl3, 151 MHz) δ 158.1; 147.3; 144.3;
	143.8; 142.9; 142.5; 141.0 140.1; 136.0; 129.9 (2C); 128.5; 125.1; 121.7; 120.8; 119.6 117.7 (2C); 116.4;
	99.5; 53.9; 39.4; 35.5; 31.7; 31.3; 28.9; 22.6; 14.1. HRMS (DCI-CH4, [M+H] + ) calculated for
	C28H30N7O5 544.2308, found: 544.2316. Purity (HPLC): 95.8% at 330 nm (tR = 6.205 min).

  ). General involving azide 2 (30.8 mg, 0.124 mmol), alkyne 6b (26.5 mg, 0.124 mmol), Cp*RuCl(PPh3)2 (4.9 mg, 6.2 µmol, 0.05 eq) in degassed 1,4-dioxane (0.8 mL). The crude product was purified by flash chromatography (Gradient petroleum ether/ethyl acetate: 70/30 to 0/100) affording the expected compound as a beige solid (16.4 mg, 29%), Rf = 0.11 (petroleum ether/ethyl acetate:

	40/60). 1 H NMR (DMSO-d6, 600 MHz) δ 9.62 (s, 1H (H14)); 7.95 (s, 1H (H16)); 7.67 (d, J = 8.9 Hz, 1H (H25));
	7.30-7.27 (m, 2H (H10,12)); 7.06 (d, J = 2.5 Hz, 1H (H19)); 7.01 (tt, J = 7.4, 1.0 Hz, 1H (H11)); 6.91 (dd, J =
	8.8, 2.2 Hz, 1H (H26)); 6.89 (d, J = 8.4 Hz, 1H (H6)); 6.83-6.80 (m, 2H (H9,13)); 6.79 (d, J = 2.2 Hz, 1H (H3));
	6.67 (dd, J = 8.2, 2.2 Hz, 1H (H7)); 6.27 (d, J = 1.3 Hz, 1H (H22)); 5.62 (s, 2H (H1)); 5.36 (s, 2H (H17)); 2.39
	(d, J = 1.3 Hz, 3H (H27)); 13 C NMR (DMSO-d6, 151 MHz) δ 160.2 (C18); 160.0 (C21); 157.7 (C8); 154.5 (C20);
	153.3 (C23); 149.3 (C4); 142.2 (C5); 134.5 (C16); 133.1 (C15); 132.6 (C2); 129.6 (2C, C10,12); 126.5 (C25); 122.0
	(C11); 121.8 (C6); 118.8 (C7); 116.2 (C3); 116.2 (2C, C9,13); 113.7 (C24); 112.4 (C26); 111.5 (C22); 101.7 (C19)
	58.3 (C17); 50.8 (C1); 18.1 (C27). HRMS (DCI-CH4, [M+H + ]) for calculated for C26H22N3O5 456.1481, found
	456.1541.
	4.2. Biology experimental
	4.2.1. Molecular docking
	Molecular graphics were performed with the UCSF Chimera package [59]. The protein structures
	were downloaded from the RCSB Protein Data Bank and aligned with MatchMaker [60] on a reference
	structure 1BVR (chain A, 1BVR:A) [19]. The protein structures, were prepared (structure checks,
	rotamers, hydrogenation, splitting of chains) using Biovia (www.3dsbiovia.com) Discovery Studio
	Visualizer 2021 (DSV), UCSF Chimera and in-house Python codes. Molecular modeling studies were
	carried with Molegro Virtual Docker 6 (www.molexus.com) software using 1P45 (chain A, 1P45:A) [9]
	and 2X22 (chain A, 2X22:A) [25] PDB entries as differential targets. The 1P45:A structure is
	characterized by a open major portal and a closed minor portal, in contrast to 2X22:A in which the
	major portal is closed and the minor portal is opened, the conformations of SBL (Substrate Binding

Acknowledgments

We thank the mass spectrometry service, Institut de Chimie de Toulouse ICT-UAR 2599 (Université de Toulouse, CNRS, Toulouse, France, https://ict.cnrs.fr) for help with chemical analyses. We thank Isabelle Fabing for her help with chromatographic analyses and purification. We also thank the scientific staff of the European Synchrotron Radiation Facility (Grenoble, France), SOLEIL (Gif sur Yvette, France) and ALBA (Barcelona, Spain). We particularly thank the staff of beamlines XALOC at ALBA and ID30B at the European Synchrotron Radiation Facility, where the crystallographic experiments were conducted. We also thank Valérie Guillet for managing synchrotron radiation trips.

The UPLC, UPC2, crystallization and macromolecular crystallography equipment used in this study are part of the Integrated Screening Platform of Toulouse (PICT, IBiSA). We thank Williams R. Jacobs, Jr.

for pMV261-inhA construct and Mary Jackson for pVV16-hadABC construct. We thank Stéphanie Ballereau for giving us a sample of DBD-Cl, precursor of compound 6k. This work was supported by Slovak Research and Development Agency (grant n. APVV-19-0189), and by the OPII, ACCORD, ITMS2014+: 313021X329, co-financed by ERDF. This study received funding from the l'Université Fédérale de Toulouse Midi-Pyrénées (UFTMiP) and the Région Occitanie for a PhD grant (R. Tamhaev) and the Région Occitanie for a "Pré-maturation Prostacure" grant to S. Britton and N. Preuilh.


Maroua Bouteffeha

Cécile Dagès

Rachida Bouhlila

Jérôme Molenat

Numerical exploration of the dynamics of infiltration in hill reservoir

OMERE team is acknowledged for providing the data and maintaining the Kamech infrastructure. This wok is

Method

Data are from the Kamech catchment (Cap Bon region, Tunisia) -OMERE-(Fig. 2). Main data are the bed topography of reservoir.

Numerical exploration considers a 2D-domain representing a longitudinal cross-section of the reservoir with the underlying groundwater and uses the Hydrus 2D code (resolving Richards equation) (Fig. 3).

3 different types of water level (h) variations in the reservoir, as well as 3 values of hydraulic conductivity (Ks) of the underlying aquifer, are considered leading to 9 scenarios (Fig. 4). So every one combines one value of Ks with one type of h variations.

Introduction

Hill reservoirs are rain water-harvesting structures adopted in arid and semi-arid regions, such as North Africa, to capture and conserve runoff water and for use as alternative water resources in agricultural development. The inflows for a hill reservoir are direct rainfall and runoff (Fig. 1). Loses are evaporation and infiltration through the dam and the reservoir bed.

Evaporation was generally considered as a major reservoir loss for surface water storage structure. However, as shown by Bouteffeha et al. (2015), infiltration as a loss greatly exceeds evaporation. Knowledge of the predominant hydrological flows and their dynamics is essential in order to project for new reservoir design and also to facilitate the management of water resources, which is critical in the Mediterranean region due to the increasing scarcity of and demand for water. Knowledge of hydrological flows controlling stored volumes, especially of the surface-subsurface exchange volume remains poorly studied and insufficiently known

Results

As a validation step, the simulated monthly infiltration was compared to the estimated one (Bouteffeha et al., 2015). The fit is good for the autumn period (Fig. 5).Simulation overestimates the infiltrated volume for the period from March to May, even though the simulated infiltration reproduces the large infiltration rate in March and the slow decrease from March to May.

The relationship between infiltration and the water level was hysteretic indicating different amounts of infiltration for the rising and the recession phases (Fig. 6). Ks has an impact not only on the infiltration flow rate, but also on the width of the hysteresis. The width of hysteresis seems to increase with the decrease of Ks (Fig. 7).

Discussion

The relationship between the infiltration and the water level in the reservoir is non unequivocal: there is an hysteretic pattern. The classical linear relationship for infiltration and water level should be "revised". The calculated Hindex emphasizes the influence of Ks on the infiltration rate from the dam and particularly on the difference of the infiltration rate for the rising and the recession phases of the water level in the reservoir. This point should be investigated in order to determine the characteristic gap between the two branches of the hysteresis based on the value of Ks.

Numerical exploration of the dynamics of nfiltration in hill-resevoir

Maroua BOUTEFFEHA 1 , Cécile DAGES 2, , Jérôme MOLENAT 2 and Rachida BOUHLILA 1

1

  Tunis El Manar University, National School of Engineering of Tunis, LMHE, 1002 Tunis Belvédère, Tunisia, 2 UMR LISAH, Université de Montpellier, INRAE, IRD, Institut Agro, AgroParisTech, Montpellier, France contact : maroua.bouteffeha@enit.utm.tn INRGREF International Scientific Days 2023. 10 and 11 October, 2023 -Sciences City of Tunis, Tunisia.

Fig. 1 :Fig. 2 :FigFig. 6 :Fig. 5 :

 1265 Fig.1 : Water balance of the kamech hill resevoir for 2010-2011 (Bouteffeha et al., 2015)

Acknowledgements

partly derived from the M. Bouteffeha PhD granted by IRD.