Aurélien Chollet

Jean-Luc Stigliani

Maria Rosalia Pasca

Giorgia Mori

Christian Lherbet

Patricia Constant

A Quemard

Jean Bernadou

Geneviève Pratviel

Vania Bernardes-Génisson
email: vania.bernardes-genisson@lcc-toulouse.fr

Annaïk Quémard

Evaluation of the inhibitory activity of (aza)isoindolinone-type compounds: toward in

Keywords: aza)isoindolinone, InhA inhibition, docking, Mycobacterium tuberculosis, structure-activity relationship, mycolic acid biosynthesis

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

Introduction

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, disease that is responsible for the death of 1.5 million people in the world annually (1). Over the years, some strains of mycobacteria have acquired simultaneous resistance to the two most used antitubercular drugs: isoniazid (INH) and rifampicin (Multi Drug Resistant strains, MDR). In ition, some strains have even developed resistance not only to the first-line drugs but also quinolones and a second-line drug (Extremely Drug Resistant strains, XDR). Importantly, the emergence of some strains resistant to all drugs used in anti-tubercular therapeutic regimen (Total Drug Resistant strains, TDR) has also been reported. In this context, erculosis represents a serious world health problem. Without effective correct treatments, the concern is that the occurrence of infections caused by MDR-strains will increase and become out of control. Thereby, the need for new anti-tubercular agents with new chemical structure and new mechanism of action appears evident and urgent.

Accepted Article

Isoniazid (INH) (Figure 1), one of the most efficient anti-tubercular medicines, is a pro-drug that requires activation by the mycobacterial catalase peroxidase enzyme (KatG) to form, after reaction with the cofactor NAD, the active metabolite (INH-NADH adduct, Figure 1). This adduct exerts its action by inhibiting InhA, an enoyl-ACP-reductase enzyme of Mtb, that is implicated in the mycolic acid biosynthesis, essential constituent of the mycobacterial cell wall. Moreover, it was shown that INH-resistant Mtb strains are frequently correlated to a default of KatG that is unable to activate INH. Direct targeting of InhA appears thus an attractive approach for the discovery of new antibiotic drugs because i) InhA is the main target of the confirmed anti-tubercular pro-drug INH, ii) enzymes belonging to the Fatty Acid Synthase II (FAS II) system, such as InhA, do not have a human ortholog and iii) InhA inhibitor, non KatG dependent, would allow to bypass resistance problems.

Lately, we have been interested in the azaisoindolinone family, designed from the core of the active metabolite of INH (Figure 1), as a promising scaffold for direct InhA inhibition. Our previous work about structure-activity relationship study on azaisoindolinone derivatives has essentially implicated modifications focusing on the hydrocarbon chain (length, position and nature of the linker of the lipophilic chain) and on the tertiary alcohol at C7 position [START_REF] Deraeve | Chemical Synthesis, Biological Evaluation and Structure-Activity Relationship Analysis of Azaisoindolinones, a Novel Class of Direct Enoyl-ACP Reductase Inhibitors as Potential Antimycobacterial Agents[END_REF][START_REF] Delaine | Development of isoniazid-NAD truncated adducts embedding a lipophilic fragment as potential bi-substrate InhA inhibitors and antimycobacterial agents[END_REF].

The thiododecyl chain placed in the meta position of the phenyl ring was optimal and the free hydroxyl at C7 revealed to be fundamental for InhA inhibition in vitro (compound 2a, Figure 1). In the continuity with this program, we describe in this paper a study about the structural lution of azaisoindolinone scaffold 2a, targeting specific regions of the molecule, as

Accepted Article

All wa To atm dio shown in Figure 2, in order to explore new structure-activity elements and get insights into important structural features to improve inhibitory enzymatic activity. The synthesis of modified azaisoindolinones, their evaluation, as direct inhibitors of the InhA activity in vitro, and the simulation of their mode of interaction with InhA by molecular docking experiments were performed. Then the compounds were tested for inhibition of whole cell Mtb growth and an investigation whether the observed inhibition might be correlated to the blockage of mycolic acid biosynthesis was also carried out.

Methods and Materials

Material chemicals were obtained from Aldrich or Acros Organics and used without further purification. The melting points were determined on an Electrothermal 9300 capillary melting point apparatus and were not corrected. TLC chromatography (silica gel F 254 nm ). Infra-red spectra were recorded on a Perkin Elmer Spectrum 100 FT-IR Spectrometer. 1 H NMR spectra were recorded on a Bruker spectrometer at 400 and 500 MHz using CDCl 3 , DMSO-d6 or MeOD-d4 as the solvent. For 1 H NMR the residual proton signal of the deuterated solvent s used as an internal reference: CDCl 3 δ= 7.29 ppm, DMSO = 2.50 ppm and MeOD = 3.31 ppm. 13 C NMR spectra were recorded on a Bruker spectrometer at 100 and 125 MHz. Mass spectra (DCI/NH 3 ) were obtained on a DSQ Thermo Fisher Scientific. For the MS-ESI a Dionex ultimate 3000 UPLC system with a ABSciex Q TRAP 4500. Flash column chromatography was performed on CombiFlash Companion with silica Redisep columns from 12 g to 80 g. All reagents were obtained from commercial suppliers and were used without further purification. Anhydrous solvents were freshly distilled before use or were obtained from the M.Braun Solvent Purification System (MB-SPS-800). The enzymatic evaluation was performed on a Cary Bio 100.

General protocol for nucleophilic addition of the dodecylthiophenyl moiety

a solution of 1-bromo-3-dodecylthiobenzene (2 eq) in dry tetrahydrofuran, under argon osphere and at -40 °C was added 2.0 M solution of n-butyllithium in cyclohexane (2 eq).

After addition, the temperature was maintained at -50 °C for 60 min. Then the temperature was decreased at -78 °C and upon this solution was added a solution of the corresponding ne (1 eq) in dry tetrahydrofuran. The reaction mixture was stirred for 30 minutes at -78 °C and was warmed to room temperature for 30 minutes more. The reaction was quenched with a

Accepted Article

(RS)-3-[3-(Dodecylsulfanyl)phenyl]-3-hydroxy-2,3-dihydro-1H-isoindol-1-one (2b)

Reagents: 1-bromo-3-dodecylthiobenzene (2.43 g, 6.8 mmol), tetrahydrofuran (5 mL), nbutyllithium 2.0 M (3.4 mL solution in cyclohexane; 6.8 mmol) and phtalimide (500 mg, 3.4 ol). The crude product was purified by flash chromatography (gradient from 100% dichloromethane to 80/20 dichloromethane/methanol) to afford a white solid (696 mg, 48%), mp: 122 °C. TLC R f : 0.59 (dichloromethane/methanol 90/10). IR (cm -1 ): 3285, 2918, 2850, 1708, 1665, 1614, 1588, 1466, 1434, 1349, 1192, 1115, 1056, 962, 790, 721, 696.

(RS)-5-Amino-3-[3-(dodecylsulfanyl)phenyl]-3-hydroxy-2-methyl-2,3-dihydro-1Hindol-1-one (2c)

Reagents: 1-bromo-3-dodecylthiobenzene (1.01 g, 2.8 mmol), dry tetrahydrofuran (5 mL), nbutyllithium solution (1.1 mL 2.5 M solution in hexanes; 2.8 mmol) and 4-amino-N-methylalimide (250 mg, 1.4 mmol). The crude product was purified by flash chromatography (gradient from 100% dichloromethane to 80/20 dichloromethane) to afford a white solid (254 mg, 40%), mp: 124 °C. TLC R f : 0.40 (dichloromethane/methanol 95/5 Hz, 3H). 13 agents: 1-bromo-3-dodecylthiobenzene (435 mg, 1.2 mmol), dry tetrahydrofuran (3 mL), nbutyllithium solution (0.48 mL 2.5 M solution in hexanes, 1.2 mmol) and of 2-hydroxy-2,5diaza-2H-indene-1,3-dione. The crude product was purified by flash chromatography (gradient from 100% dichloromethane to 80/20 dichloromethane) to afford (159 mg, 30%mixture of para and meta regioisomers 70/30). TLC R f : 0.17 (dichloromethane/methanol 95/5). IR (cm -1 ): 3104,2922,2852,1702,1616,1590,1465,1433,1376,1259,1203,1184,1135,1109,1075,973,953,902,863,841,781,745,697,653,611

Accepted Article

Separation of p-4a and m-4a by liquid chromatography

Both compounds, para and meta, were isolated using a HPLC preparative chromatography system (Waters autopurification system), a 2-ethyl-pyridine column 250 x 10.0 mm 5µm and 8% iPrOH / 92% CH 2 Cl 2 as eluant. Isocatric elution was performed at 6.0 mL/min and absorbance was followed at λ = 254.0 nm. Both regioisomers p-4a and m-4a were separated with respective retention times of t R1 = 12.4 min and t R2 = 13.2 min. c]pyridin-1-one (m-4a) 1

(RS)-1,2-Dihydroxy-1-[3-(dodecylsulfanyl)phenyl]-1,2-dihydro-3H-pyrrolo[3,4c]pyridine-3-one (p-4a)

(RS)-2,3-Dihydroxy-3-[3-(dodecylsulfanyl)phenyl]-2,3-dihydro-1H-isoindol-1-one (4b)

Reagents: 1-bromo-3-dodecylthiobenzene (879 mg, 2.45 mmol), dry tetrahydrofuran (5 mL), n-butyllithium (0.98 mL, 2.5 M solution in hexanes; 2.45 mmol) and N-hydroxyphtalimide (200 mg, 1.23 mmol). The crude product was purified by flash chromatography (gradient from 80/20 dichloromethane/methanol to 50/50) to afford a white solid (239 mg, 44%), mp: 145 °C. TLC R f : 0.17 (

(RS)-1-[3-(Dodecylsulfanyl)phenyl]-1-hydroxy-2-methoxy-1,2-dihydro-3H-pyrrolo[3,4-c] ridin-3-one (5a)

Reagents: 1-bromo-3-dodecylthiobenzene (200 mg, 0.56 mmol), dry tetrahydrofuran (1.5 mL), n-butyllithium solution (0.22 mL 2.5 M solution in hexanes; 0.56 mmol) and 2methoxy-2,5-diaza-2H-indene-1,3-dione (50 mg, 0.28 mmol). The crude product was purified by flash chromatography (gradient from 100% dichloromethane to 90/10 dichloromethane/methanol) to afford an oil (45 mg, 35% -mixture of para and meta 70/30).

A little quantity of the para compound was isolated for characterization and biological assay. agents: 1-bromo-3-dodecylthiobenzene (660 mg, 1.8 mmol), dry tetrahydrofuran (5 mL), nbutyllithium solution (0.74 mL 2.5 M solution in hexanes; 1.8 mmol) and 2-methoxy-2Hisoindole-1,3-dione (164 mg, 0.92 mmol). The crude product was purified by flash chromatography (gradient from 80/20 hexane/ethyl acetate to 60/40) to afford a white solid (101 mg, 24%), mp: 97 °C. TLC R f : 0.29 (hexane/ethyl acetate 70/30). IR (cm -1 ):

Accepted Article

(RS)-1-[3-(Dodecylsulfonyl)phenyl]-1-hydroxy-1,2-dihydro-3H-pyrrolo[3,4-c]pyridin-3e 5-oxide (3)

a solution of (RS)-1-[3-(dodecylsulfanyl)phenyl]-1-hydroxy-1,2-dihydro 3H-pyrrolo [3,4c]pyridin-3-one (63 mg, 0.15 mmol, 1 eq) in dichloromethane (5 mL) was added at room temperature 3-chloro-peroxobenzoic acid (mCPBA). The mixture was stirred for 3 hours then 10 mL of brine were added and the entitled product was extracted with dichloromethane (3 x 10 mL). The organic phases were combined, dried over sodium sulfate and concentrated under vacuum. The residue was purified by flash chromatography (gradient from 100% dichloromethane to 80/20 dichloromethane) to furnish a white solid (51 mg, 72%) mp: 175 °C. TLC R f : 0.21 (dichloromethane/methanol 95/5). IR (cm -1 ): 3117, 3036, 2957, 2922, 2852,

General protocol for the synthesis of pyridazinone containing derivatives

Corresponding N-methoxyisoindolinone type compound (1 eq), ethanol (10 mL), and hydrazine hydrate (15 eq) were added into a round-bottom flask equipped with a condenser. A nitrogen balloon was attached to the top of the condenser. After refluxing overnight, the reaction was cooled to room temperature. An off-white solid precipitated out of the solution.

After cooling to 0 °C, water was added slowly and the compound was extracted with dichloromethane (3 x 10 mL). The residue was purified by flash chromatography to furnish entitled compound. [3,4c]pyridin-3-one (5a) and its meta regioisomer (in a ratio para/meta 60/40, 64 mg, 0.14 mmol), ethanol (10 mL) and hydrazine hydrate (0.1 mL, 2.09 mmol). e crude product was purified by flash chromatography (gradient from 100% dichloromethane to 90/10 dichloromethane/methanol) to afford a yellow solid (57 mg, 96%mixture of para and meta compounds 60/40). The para compound was isolated for characterization and biological activity evaluation. TLC R f : 0.56 for the para compound and 0.66 for the meta compound (dichloromethane/methanol 95/5). IR (cm -1 ) for the mixture of a/meta 60/40: 2918,2850,1707,1666,1599,1566,1466,1410,1333,1318,1277,1217,1176,1134,1101,1082,1044,998,885,853,806,783,701,681,662,610,557

1-[3-(Dodecylsulfanyl)phenyl]-pyrido[3,4-d]pyridazin-4(3H)-one (6a)

Reagents: (RS)-1-[3-(dodecylthio)phenyl]-1-hydroxy-2-methoxy-1,2-dihydro-3H-pyrrolo

Accepted Article

4-[3-(Dodecylsulfanyl)phenyl]phthalazin-1(2H)-one (6b)

Reagents: (RS)-3-[3-(Dodecylsulfanyl)phenyl]-3-hydroxy-2-methoxy-2,3-dihydro-1Hisoindol-1-one (5b) (57 mg, 0.14 mmol), ethanol (10 mL) and hydrazine hydrate (0.11 mL,

mmol).

The crude product was purified by flash chromatography (isocratic 80/20 hexane/ethyl) to afford a white solid (15 mg, 26%). TLC R f : 0.72 (dichloromethane/methanol 95/5). IR (cm -1 ):

General protocol for addition of ethynyl-TMS group

To a solution of ethynyl-trimethylsilane (2 eq) in dry tetrahydrofuran, under argon atmosphere and at -40 °C was added n-butyllithium 2.5M solution (2 eq). After addition, the temperature was maintained at -50 °C for 60 min. Then the temperature was decreased at -78 °C and upon this solution was added a solution of the corresponding (aza)-isoindolinone starting material (1 eq) in dry tetrahydrofuran. The reaction mixture was stirred for 30 minutes at -78 °C and was warmed to room temperature for 30 minutes more. The reaction was quenched with a saturated ammonium chloride solution and the product was extracted with ethyl acetate. The organic phases were combined, dried over sodium sulfate and concentrated under vacuum.

The residue was purified by flash chromatography to furnish the entitled compound.

(RS)-1-Hydroxy-1-[2-(trimethylsilyl)ethynyl]-1,2-dihydro-3H-pyrrolo[3,4-c]pyridine-3one (7a)

Reagents: ethynyltrimethylsilane (663 mg, 0.95 mL, 6.65 mmol), dry tetrahydrofuran (10 mL), n-butyllithium solution (2.7 mL 2.5M solution in hexanes; 6.65 mmol) and 3,4pyridinedicarboximide (500 mg, 3.37 mmol). The crude product was purified by flash chromatography (gradient from 100% dichloromethane to 90/10 dichloromethane/methanol) to afford a white solid (407 mg, 49%), mp: 170 °C. TLC R f : 0.31 (dichloromethane/methanol 13

Accepted Article

General protocol for the Huisgen type cycloaddition reaction

a solution of the corresponding (trimethylsilyl)ethynylisoindolinone intermediate (1.0 eq) and alkyl azide with a defined number of carbons (1.1 eq) in methanol were added copper(II) sulfate pentahydrate (0.2 eq), sodium ascorbate (0.4 eq) and finally tetrabutylammonium fluoride (TBAF) solution (1.0 eq) to start the click chemistry reaction. The mixture was stirred for 16 hours and then was concentrated under vacuum and directly purified by flash chromatography to furnish the entitled compound.

For starting compounds with a pyridine moiety, an additional washing step with a saturated solution of ethylenediaminetetraacetic acid (EDTA) was needed to remove copper.

Accepted Article

c]pyridin-3-one (8a)

Reagents: (RS)-3-hydroxy-3-[2-(trimethylsilyl)ethynyl]-2,3-dihydro-1H-isoindol-1-one (65 mg, 0.32 mmol), 1-octyl-azide (55 mg, 0.36 mmol), methanol (10 mL), copper(II) sulfate pentahydrate (16 mg, 0.04 mmol), sodium ascorbate (26 mg, 0.13 mmol, 0.4 eq) and tetrabutylammonium fluoride solution (0.32 mL of a 1.0 M solution in tetrahydrofuran, 0.32 ol). The crude product was purified by flash chromatography (gradient from 100% dichloromethane to 90/10 dichloromethane/methanol) to afford (86 mg, 82%), mp: 175 °C. (m,2H); 1.30-1.17 (m, 14H); 0.85 (t, J = 6.8 Hz, 3H). 13 14H); 0.85 (t, J = 6.8 Hz, 3H). 13 [3,4c]pyridin-3-one (100 mg, 0.41 mmol), 1-dodecyl-azide (95 mg, 0.45 mmol), methanol (10 mL), copper(II) sulfate pentahydrate (20 mg, 0.08 mmol), sodium ascorbate (32 mg, 0.16 ol) and tetrabutylammonium fluoride solution (0.41 mL of a 1.0 M solution in tetrahydrofuran, 0.41 mmol). e crude product was purified by flash chromatography (gradient from 100% dichloromethane to 90/10 dichloromethane/methanol) to afford a yellow solid (85 mg, 54%), mp: 145 °C. TLC R f : 0.41 (dichloromethane/methanol 90/10

(RS)-3-(1-Dodecyl-1H-1,2,3-triazol-4-yl)-3-hydroxy-2,3-dihydro-1H-isoindol-1-one (10b)

Reagents: (RS)-1-hydroxy-1-[2-(trimethylsilyl)ethynyl]-1,2-dihydro-3H-pyrrolo

Biology

InhA expression and purification

The production and purification of the InhA-6xHis protein from a protease-deficient strain of E. coli BL21(DE3) transformed with the pHAT5/inhA plasmid were performed as followed. 1 mL of the bacteria was grown in 100 mL of LB medium containing ampicillin (100 mg/mL) at 37 °C. After 4 h, the solution was re-diluted in 1 L of the same medium and re-grown at 37 °C. When the proper concentration (OD 595 = 0.6-0.8) was reached, protein expression was induced by overnight incubation with 1 mM isopropyl-β-D-galactopyranoside (IPTG) at 20 °C. Cells were harvested by centrifugation at 6,000 g for 30 min at 4 °C. The dry pellet may be kept at -80°C for several months. Thawed cells (1.5 g) were sonicated in 20 mL lysis buffer (300 mM NaCl, 10 mM imidazole, 50 mM sodium phosphate buffer, pH 8.0). After centrifugation at 10,000 g for 45 min at 4 °C, the supernatant was applied onto a nickelchelated Hi-Trap HP 1 mL column (GE Healthcare) previously equilibrated with the binding buffer (50 mM NaCl, 10 mM imidazole, 50 mM sodium phosphate buffer, pH 8,0). First, the ound proteins were washed out with 10 column volume of binding buffer, and then a higher imidazole concentration (25 mM) allowed the elution of non-specifically bound proteins. The His 6 -tagged InhA protein was eluted with an imidazole gradient from 25 mM to mM over a range of 20 column volume. Fractions containing the target protein were led, concentrated to 2.0 mL and loaded on a HiLoad 16/60 Superdex 200 column (GE Healthcare) equilibrated with 150 mM NaCl, 30 mM PIPES, pH 6.8. Samples were analyzed using SDS-PAGE and Coomassie blue staining and then stored at 4 °C for short term storage co 37

InhA activity inhibition

Triclosan and NADH were obtained from Sigma-Aldrich. Stock solutions of all compounds were prepared in DMSO such that the final concentration of this co-solvent was constant at 5% v⁄v in a final volume of 1 mL for all kinetic reactions. Kinetic assays were performed using trans-2-dodecenoyl-coenzyme A (DDCoA) and wild type InhA as previously described.

Briefly, pre-incubation reactions were performed in an aqueous buffer (30 mM PIPES and mM NaCl pH 6.8) containing additionally 100 nM InhA, 250 µM cofactor (NADH) and the tested compound (50 µM or 10 µM). After 5 min of pre-incubation at 25 °C, addition of 50 µM DDCoA initiated the reaction which was followed at 340 nm (oxidation of NADH).

The inhibitory activity of each derivative was expressed as the percentage of inhibition of InhA activity (initial velocity of the reaction) with respect to the control reaction without inhibitor. Triclosan was used as a positive control. All activity assays were performed in triplicate.

Mycobacterium growth inhibition

H37Rv was used as the reference strain. The strains were grown at 37°C in Middlebrook 7H9 broth (Difco), supplemented with 0.05% Tween 80, or on solid Middlebrook 7H11 medium (Difco) supplemented with oleic acid-albumin-dextrose-catalase (OADC). MICs for the new mpounds were determined by means of the micro-broth dilution method. Dilutions of M. tuberculosis wild-type (about 10 5 -10 6 cfu/ml) were streaked onto 7H11 solid medium containing a range of drug concentrations (0.25 µg/mL to 40 µg/mL). Plates were incubated at °C for about 21 days and the growth was visually evaluated. The lowest drug dilution at which visible growth failed to occur was taken as the MIC value. Results were expressed as the average of at least three independent determinations.

Acid mycolic biosynthesis inhibition

INH, 2a and m-4a were added to broth cultures of M. tuberculosis H37Rv during the exponential growth phase at their MIC (1 µM, 25 µM and 12.5 µM respectively). The cultures were then incubated at 37 °C for 5 h before the addition of [1-14 C]acetate (1 µCi, 17 µM) to follow the biosynthesis of lipid products. Cells were incubated for 18 h and then harvested.

Fatty and mycolic acids were released from whole bacteria through saponification by incubating the latter with a mixture of 40% KOH (w/v) in methoxyethanol (1/7) for 6 h at 100 °C. After acidification by addition of 20% H 2 SO 4 , lipids including mycolic acids were extracted with diethyl ether. The crude fatty acids were methylated and methyl ester Accepted Article sea derivatives were analyzed by TLC on Silica Gel 60 (Macherey-Nagel) run in dichloromethane (4) followed by phosphorimaging (Variable Mode Imager Typhoon TRIO, Amersham Biosciences). The relative percentage of radioactivity incorporated in fatty acids (FA), in alpha-mycolic acids (α-MA) and in methoxy-and keto-mycolic acids (M+K MA, not separated during TLC) was determined for each condition using Image Quant 5.1 software. The compounds of this study were thoroughly built and fully optimized before the docking stage using the PM6 Hamiltonian (5) implemented in the MOPAC 2012 quantum chemistry package [START_REF] Stewart | Computational Chemistry[END_REF]. In most cases a chiral center was present, both configurations R and S were taken into account.

Docking

The structure 2H7M from the Brookhaven Protein Data Bank was chosen for molecular docking. The NADH cofactor was kept into the InhA structure although 2H7M ligand and water molecules were removed. The MolProbity software [START_REF] Chen | MolProbity: all-atom structure validation for macromolecular crystallography[END_REF] was used to assign the position of hydrogen atoms and the protonation state of histidines in the InhA protein.

The docking calculations were performed with the docking program Autodock Vina v1.1.2 [START_REF] Trott | Autodock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading[END_REF]. The Autodock graphical interface AutoDockTools (9) was used to keep polar hydrogens and add partial charges to the proteins using the Kollman United charges. The rch space was included in a box of 24x24x24 Å, centred on the binding site of the ligands and nicotinamide cofactor. The side chains of the six residues, Met103, Phe149, Tyr158, Met161, Met199 and Leu218, which surround the catalytic cavity, were allowed to rotate during the docking. Flexible torsions of side chains and of ligands were assigned with Autotors, an auxiliary module of AutoDockTools. For each calculation, fifteen poses ranked according to the scoring-function of Autodock Vina were obtained (VINA score). The docking procedure was followed by a post-docking optimization with the PM6-DH2 (10) method implemented into the MOPAC 2012 quantum mechanical package [START_REF] Stewart | Computational Chemistry[END_REF]. For that purpose, the docking poses were merged into the cavities of the corresponding InhA and the complexes were submitted to a minimization stage in which the ligand, a set of 12 residues (Met103, Phe149, Met155, Tyr158, Met161, Thr196, Met199, Ile202, Leu207, Ile215, Ley218 and Glu219) delimiting the binding pocket and the totality of hydrogen atoms were fully energy-minimized. The remaining residues were kept frozen. The

Accepted Article

localized molecular orbital method (keyword MOZYME) which was developed to enable calculation on large systems such as enzymes was applied [START_REF] Stewart | Application of localized molecular orbitals to the solution of semiempirical selfconsistent field equations[END_REF]. The effects of the solvation interface were simulated with the continuum model COSMO [START_REF] Klamt | Cosmo: a new approach to dielectric screening in solvents with explicit expressions for the screening energy and its gradient[END_REF] with a dielectric constant set to 78.4. The interaction energies (E opt ) were calculated with the equation: ΔE = Δ f H (ligand-receptor complex) -Δ f H (ligand alone) -Δ f H (receptor alone).

Results and discussion

Chemistry

Based on our previous results showing that the C7 hydroxyl group and the lipophilic thiododecyl chain bound to the ring C of the azaisoindolinone scaffold are essential elements for the InhA inhibitory activity, the first strategy adopted in this work was to maintain intact these two elements and to carry out modifications on the azaisoindolinone framework namely on the ring A and on the hemiamidal cycle B. For this purpose, the pyridine ring (ring A) of the azaisoindolinone scaffold was substituted by a phenyl, an aminophenyl and a N-oxide pyridyl cycles (the latter coupled with a sulfur oxidation of the chain giving the corresponding sulfone). The hemiamidal cycle B was also modified at the N8 position by subsequent introduction of a hydroxyl or a methoxy group. In a second stage, replacement of the dodecylthiophenyl motif (ring C) by an alkyl triazole ring was performed via Huisgen-type cycloaddition. Finally, a ring extension of the hemiamidal 5-membered ring to a 6-membered pyridazinone-type ring was also performed. These chemical modulations are summarized in based on a nucleophilic addition of the dodecylthiophenyl lithium intermediate over the commercial 3,4-pyridine-dicarboximide reagent (1a). The introduction of the N-oxide motif into the hit molecule 2a was carried out using mCPBA as oxidant [START_REF] Jana | Phase-vanishing methodology for efficient bromination, alkylation, epoxidation, and oxidation reactions of organic substrates[END_REF]. This reaction was accompanied by a subsequent oxidation of the sulfur atom leading to the sulfone 3. The general synthetic strategy to prepare new compounds 2b, 2c and 4b takes advantage of the ition of dodecylthiophenyl lithium intermediate upon dicarboximide starting materials embedding suitable functionalities (1b, 1c and 1d respectively). For the preparation of Nhydroxy or N-methoxy-(aza)isoindolinone derivatives 4a, 5a and 5b, the synthesis initially sisted in the reaction of hydroxylamine or methoxylamine with (aza)phthalic anhydride 1e or 1f followed by addition of the aryllithium reagent [START_REF] Nguyen | Synthesis Accepted Article of 4-Substituted chlorophtalazines, dihydrobenzoazepinediones, 2-pyrazolylbenzoic acid, and 2pyrazolylbenzohydrazide via 3-substituted 3-hydroxyisoindolin-1-ones[END_REF]. As previously observed for compound 1a [START_REF] Broussy | The first chemical synthesis of the core structure of the benzoylhydrazine-NAD adduct, a competitive inhibitor of the Mycobacterium tuberculosis enoyl reductase[END_REF], the addition of the alkylthiophenyl group occurs preferentially on the para a carbonyl group and the regioisomers formed are readily separated by flash chromatography. Except for 4a, whose the separation of isomers (ratio 70:30 para:meta) was not successful in these conditions and a HPLC preparative chromatography with a 2-ethylpyridine column was necessary. For 1c, the exocyclic amine nitrogen atom oriented the ction in a different way and promoted the addition on the meta carbonyl group with respect to the amino group. On the other hand, the replacement of the pyridine moiety by a phenyl ring suppresses the dissymmetry of the molecule discarding the formation of regioisomers during functionalization of the carbonyl group.

For the preparation of triazole compounds, addition of the lithium (trimethylsilyl)acetylide over the starting material 1a and 1b was carried out. After unsuccessful attempts on deprotection of the ethynyl group (due to the instability of the products), the intermediates 7a and 7b were directly submitted to a 1,3-cycloaddition reaction with the corresponding azides, which were prepared, in their tour, by reaction of NaN 3 with the corresponding halogen al anes [START_REF] Pardin | Reversible and competitive cinnamoyl triazole inhibitors of tissue transglutaminase[END_REF]. The trimethylsilyl cleavage step was carried out by in situ addition of tetrabutylammonium fluoride (TBAF) into the reaction mixture [START_REF] Boons | One-pot three-step synthesis of cycloaddition of azides with alkynes formed by a sonogashira cross-coupling and desilylation[END_REF]. This series of clickchemistry reactions led to a family of six compounds (8a, 8b, 9a, 9b, 10a and 10b) by varying both aromatic ring A (phenyl /pyridyl) and the length of the alkyl chain (C 8 , C 10 and C ).

Taking into account that pyridazinone compounds have potential antibacterial activities [START_REF] Husain | Synthesis and antitubercular activity of pyridazinone derivatives[END_REF][START_REF] Abubshait | An efficient synthesis and reactions of novel indolyl-pyridazinone derivatives with expected biological activity[END_REF][START_REF] Sönmez | Synthesis, antibacterial and antifungal activity of some new pyridazinone metal complexes[END_REF][START_REF] Akbas | Antibacterial and antifungal activities of new pyrazolo[3,4-D]pyridazin derivatives[END_REF], the ring extension of the hemiamidal core of compounds 5a and 5b into pyridazinone 6a and 6b, respectively, was performed in good yield by using hydrazine under reflux (96% and 76% yield, respectively) [START_REF] Wang | Structure-based design of isoindoline-1,3-diones and 2,3-dihydrophthalazine-1,4-diones as novel β-Raf inhibitors[END_REF][START_REF] Brzezinski | Concise regioselective synthesis of hydroxyazaisoindolinones and their conversion into pyridopyridazinones[END_REF]. Although this step is associated with the suppression of an Accepted Article essential substituent (C7 hydroxyl group), this new scaffold has the advantage of deleting the asymmetric carbon.

1a: R 1 = H, X = N, R 2 = NH 1b: R 1 = H, X = CH, R 2 = NH 1c: R 1 = NH 2 , X = CH, R 2 = NCH 3 1d: R 1 = H, X = CH, R 2 = NOH 1e: R 1 = H, X = N, R 2 = O 1f: R 1 = H, X = CH, R 2 = O 7a: X = N 7b: X = CH

Enzymatic tests of InhA inhibition in vitro

The InhA protein was produced and purified according to a previous reported method [START_REF] Chollet | Crystal structure of the enoyl-acp reductase of mycobacterium tuberculosis (InhA) in the apo-form and in complex with the active metabolite of isoniazid pre-formed by a biomimetic approach[END_REF]. e compounds 2a, 2b, 2c, 3, p-4a, m-4a, 4b, 5a, 5b, 6a, 6b, 8a, 8b, 9a, 9b, 10a and 10b (chiral molecules in racemic form) were evaluated for their InhA inhibitory activity by measuring the NADH consumption in the enzymatic reaction through the decrease of the absorbance at 340 nm. Target compounds were tested at 50 µM and for the more potent molecules also at 10 µM. The result is expressed as the percentage of inhibition of the InhA

Accepted Article

Th we het and ass Mo activity for the indicated concentration. Triclosan was employed as a positive control and it induced a complete loss of InhA activity at 10 µM. The data are summarized in Table 1. e hit compound 2a was re-evaluated and it exhibited 28% inhibition at 50 µM confirming the magnitude of activity according to the previous reported results [START_REF] Deraeve | Chemical Synthesis, Biological Evaluation and Structure-Activity Relationship Analysis of Azaisoindolinones, a Novel Class of Direct Enoyl-ACP Reductase Inhibitors as Potential Antimycobacterial Agents[END_REF].

All new synthesized compounds were able to inhibit InhA activity in vitro. Compounds 3, 5b, 6b, 8a, 9b and 10b displayed InhA inhibitory potencies comparable to the hit molecule 2a.

Compounds 2b, 5a, 6a, 9a and 10a were slightly more active than 2a. The best inhibitors re compounds m-4a and 4b, which displayed approximately the same percentage of InhA inhibition as 2a for 5-fold lower concentration (entries 1, 7 and 8). In contrast, compounds 2c, p-4a and 8b showed poor InhA inhibition for this series.

Structure-InhA inhibition relationship

In vitro, the fully oxidized compound 3 bearing an N-oxide pyridine and a sulfone function displayed the same InhA inhibitory activity as the hit molecule 2a. The presence of extra eroatoms in the framework of the azaisoindolinone did not contribute to improve the interaction of compound 3 within the InhA active site.

The replacement of pyridine by a phenyl ring was well tolerated. In some cases, pyridine derivatives seem to be slightly better than phenyl derivatives (entries 9 and 10, 11 and 12, 13 14, 15 and 16, 17 and 18). This suggests that pyridine nitrogen atom may be engaged in key interactions within the binding pocket of the protein. Substitution of cycle A by the amino-phenyl ring led to a deep drop in activity (entry 3). However, no conclusions could be essed on the influence of either the extra amino group or the N8-methyl bond. It is not clear whether this effect due to the presence of the extra amino group or the presence of the methyl group (or even to both of them). dification of the hemiamidal core was also investigated. Insertion of a hydroxyl group at N8 revealed a significant enhancement of the inhibitory activity of the molecule toward InhA in vitro. Indeed, azaisoindolinone-based derivatives 4a (para and meta 70/30 molar ratio) (entry 5) and 4b (entry 8) exhibited about three times more inhibitory potency toward InhA than the lead molecule 2a at 50 µM. This encouraging results observed with the mixture of two regioisomers (4a) led us to separate them by preparative HPLC to evaluate their activities separately. Surprisingly, the m-4a regioisomer was much more potent (entry 7) than the para isomer p-4a (entry 6). Both the hydroxylamino group and the position of the nitrogen atom on the pyridine ring were found to be critical for the activity of the molecule. In accordance with these observations, the replacement of the hydroxylamino group by methoxylamino group The pyridazinone-type derivatives 6a and 6b, obtained from the ring expansion of the hemiamidal moiety, showed a significant improvement of the inhibitory InhA activity when compared to 2a. In this example, it was also found that pyridine-based compound was more ive than benzene-based compound (entries 11 and 12).

Molecular Docking

Molecular docking calculations were performed in order to simulate the possible binding modes of azaisoindolinone compounds in the active site of the InhA protein, to rationalize the structure-InhA inhibition relationship observed in this work, and to define essential structural features. Although several InhA crystal structures in complex with different inhibitors are available, we chose the crystal structure PDB ID 2H7M [START_REF] He | Pyrrolidine carboxamides as a novel class of inhibitors of enoyl acyl carrier protein reductase from Mycobacterium tuberculosis[END_REF] as target for the docking study because, as previously reported by our group [START_REF] Stigliani | Cross-Docking Study on InhA Inhibitors: A Combination of Autodock Vina and PM6-DH2 Simulations to Retrieve Bio-Active Conformations[END_REF], this protein showed good ability to reproduce different modes of interaction of ligands with respect to the corresponding X-ray data. In addition, its crystal structure was solved in complex with the arylamide-type inhibitor d11 with significant inhibitory activity (d11, IC 50 InhA = 0.39 µM), and with a higholution 1.6 Å.

It s important to note that each compound with scaffold A, as defined in Table 1, is a chiral molecule (asymmetric carbon 7) and thus exists as a racemic mixture. Previous studies have wn that when R 2 = NH, a rapid racemization of C7 for both R and S enantiomers occurs in ution [START_REF] Deraeve | Chemical Synthesis, Biological Evaluation and Structure-Activity Relationship Analysis of Azaisoindolinones, a Novel Class of Direct Enoyl-ACP Reductase Inhibitors as Potential Antimycobacterial Agents[END_REF], probably through an open intermediate structure in equilibrium [START_REF] Stigliani | Binding of the tautomeric forms of isoniazid-NAD adducts to the active site of the mycobacterium tuberculosis enoyl-ACP reductase (InhA): A theoretical approach[END_REF].

Nevertheless, the cycle form (azaisoindolinone) is the unique form detected by NMR.

Consequently, the enantiomers were not separated and the compounds were used as a

Accepted Article

Ne PM im con racemate in this work. However, the docking experiments were carried out for each individual enantiomer.

w compounds 2c(R), 2c(S), m-4a(R), m-4a(S), p-4a(R), p-4a(S), 5a(R), 5a(S), 10a(R),

10a(S) were docked into the substrate binding site of the 2H7M protein in complex with NADH cofactor by using AutoDock Vina program. Due to their flexibility, these compounds show several combinations of conformation and orientation within the binding site. For each compound, the top-Vina score docking poses included into an interval of 2 kcal/mol energy were selected and were further subjected to an optimization step with the Hamiltonian method 6-DH2 as previously described. Among the final possible conformations, we retained the ones that exhibited the lower optimized energy of interaction.

For comparison, we also show the orientation of the hit molecules 2a(R) and 2a(S). The best binding mode predicted by docking calculations for different compounds is depicted in Figure 3.

The azaisoindolinone scaffold of 2a(R) (Figure 3A) is located near the NADH cofactor and forms two hydrogen bonds between the carbonyl oxygen of the hemiamidal moiety and the C-2'-hydroxyl ribose of the nicotinamide nucleotide fragment and between the tertiary alcohol and one oxygen atom of the phosphate groups of the cofactor. This conformation shows the portant role played by the tertiary alcohol in the binding of the active site and consequently, in the inhibitory activity of InhA which strengthens what has been previously revealed by the structure-activity relationship analysis (2). In the selected conformation of the 2a(S) (Figure 3B), the azaisoindolinone scaffold is also located close of the NADH. However, the carbonyl group points to the opposite side of the cofactor and does not establish hydrogen bonds with it. In contrast, the pyridine ring is close to the Tyr158 and a hydrogen bond is observed between the hydrogen of the phenol function of the tyrosine and the pyridine nitrogen. In this formation, the tertiary alcohol is also positioned in such a manner compatible with hydrogen bonding with one oxygen atom of a phosphate group. In both enantiomers of 2a, the lipophilic chain occupies the top of the substrate-binding site. 3F) adopts in the active pocket of InhA an orientation allowing the establishment of four interaction points with the cofactor the protein. These interactions take place between the tertiary alcohol at C7 and one of the phosphate oxygen, between the hydroxyl at N8 position and one of the saturated oxygen of the phosphate group, between the carbonyl group of the hemiamidal moiety and residue Thr196 and between the pyridine nitrogen atom and Tyr158 or C2'-hydroxyl ribose of the cofactor. All together, these interactions can account for a best activity observed for m-4a when compared to its regioisomer p-4a (entries 6 and 7). Docking results revealed that compound 5a(R) (Figure 3G) adopts a conformation in the InhA ive site establishing two hydrogen bond type interactions as observed for m-4a(R).

However, different of m-4a(R) the N-OMe group is not able to interact with InhA nor NADH.

On the other hand, the enantiomer 5a(S) (Figure 3H) is positioned in a completely different manner. It is located far away from the NADH cofactor and hydrogen bonds with neither NADH nor Tyr 158 could be observed. We noted an unusual interaction between the tertiary alcohol at C7 and the Met 98. For compounds 10a(R) and 10a(S) (Figure 3I and 3J), the triazole ring is positioned near the Tyr158 and the ribose moiety. It is worth to note that, even this case, the hydrogen bond between the tertiary alcohol at C7 and one of the oxygen atoms of the phosphate group is preserved. The location of the lipophilic chain is another difference between these compounds and 2a. For compounds 10a(R) and 10a(S), the chain is ated in the bottom part of the cavity. Finally, the top Vina poses selected for 2c (both R and S enantiomers), which is one of the less active compounds of the azaisoindolinone series, showed neither hydrogen bonding with NADH nor with Tyr158 (Figure 3K and3L). Only the S nantiomer showed an uncommon interaction between the tertiary alcohol and the residue t98. This lack of hydrogen bond interactions with the cofactor may explain the low inhibitory activity observed for this derivative.

In summary, binding modes allowing interactions with the nicotinamide nucleotide fragment (ribose and phosphate groups) of the cofactor NADH and with residue Tyr158 of InhA are always associated with inhibitory InhA activities in vitro.

(E.), m-4a(S) (F.), 5a(R) (G.), 5a(S) (H.), 10a(R) (I.), 10a(S) (J.), 2c(R) (K.) and 2c(S) (L.) predicted by docking calculations. Tyr158 residue and NADH cofactor molecule are respectively indicated as dark green and light green sticks.

Mycobacterium tuberculosis growth inhibition

In order to check whether the improved activity of InhA inhibiting of some compounds could be retrieved in the inhibition of Mtb growth, compounds 2a, 3, 4a, 4b, 5a, 6a and 10a were evaluated toward inhibition of Mtb H37Rv growth using the same proceeding as reported in Menendez et al [START_REF] Menendez | Chemical synthesis and biological evaluation of triazole derivatives as inhibitors of InhA and antituberculosis agents[END_REF]. The minimum inhibitory concentrations (MIC) were determined as the lowest drug concentration inhibiting bacterial growth. Triclosan was used as a positive control. The results are presented in Table 1. All tested compounds displayed an inhibitory activity toward M. tuberculosis growth globally equivalent to 2a. Although compounds m-4a and 4b were better InhA inhibitors in vitro than 2a, they did not show however, higher inhibition potency toward Mtb growth.

Accepted Article

-not determined. a. All assays were repeated three times. b. All assays were repeated three times and standards deviations ± 0.025 mg/mL.

Inhibition of the mycolic acid biosynthesis

With these results in hands, it was the question whether the inhibition of Mtb growth by the

Figure 1 .

 1 Figure 1. Structures of isoniazid, INH-NADH adduct and azaisoindolinone compound 2a.

  solution and the mixture was extracted with ethyl acetate. The organic phases were combined, dried over sodium sulfate and concentrated under vacuum.The residue was chromatographed as indicated in each case to furnish the entitled compound. bromo-3-dodecylthiobenzene (860 mg, 2.4 mmol), dry tetrahydrofuran (10 mL), n-butyllithium solution (1.7 mL 2.0 M solution in cyclohexane; 3.4 mmol) and 3,4pyridinedicarboximide (178 mg, 1.2 mmol). The crude product was purified by flash chromatography (gradient from 100% dichloromethane to 80/20 dichloromethane) to afford a ite solid (297 mg, 58%, mixture of para/meta regioisomers 80/20), mp: 86 °C. TLC R f : 0.30 (dichloromethane/methanol 95/5). IR (cm -1 ): 3159, 2917, 2849, 1698, 1614, 1587, 1464, 1347, 1205, 1067, 964, 784, 698. 1 H NMR (400 MHz, MeOD) δ (ppm): 8.94 (s, 1H); 8.75 (d, J = 5.0 Hz, 1H), 7.55 (s, 1H); 7,46 (d, J = 4.6 Hz, 1H); 7.33-7.30 (m, 3H); 2.94 (t, J = 7.0 Hz,2H); 1.63-1.59 (m, 2H); 1.50-1.25 (m, 18H); 0.91 (t, J = 6.0 Hz, 3H).13 C NMR (100 MHz, MeOD) δ (ppm): 168.3 (C); 159.0 (C); 152.7 (CH); 150.0 (C); 144.5 (CH); 140.0 (C); 138.0 ; 128.9 (CH); 128.4 (CH); 125.2 (CH); 122.5 (CH); 118.0 (CH); 87.6 (C); 70.1 (CH 2 ); 32.6 (CH 2 ); 31.7 (CH 2 ); 29.4-28.8 (6 x CH 2 ); 28.4 (CH 2 ); 22.2 (CH 2 ); 13.0 (CH 3 ). MS (ESI) m/z: 449.2 [M+Na + ]; 427.3 [M+H + ]. HRMS (ESI): for C 25 H 35 N 2 O 2 S [M+H + ]: calcd: 427.2419; found: 427.2459.

  1 H NMR (400 MHz, DMSO-d6) δ (ppm): 9.25 (s, 1H); 7.64 (d, J = 6.8 Hz, 1H); 7.56-7.46 (m, 2H); 0 (s, 1H); 7.33-7.21 (m, 4H); 6.95 (s, 1H); 2.92 (t, J = 7.2 Hz, 2H); 1.56-1.49 (m, 2H); 1.45-1.22 (m, 18H); 0.85 (t, J = 6.8 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6) δ (ppm): 168.8 (C); 151.0 (C); 143.5 (C); 137.0 (C); 132.8 (CH); 131.0 (C); 129.5 (CH); 129.4 (CH); 127.5 H); 125.2 (CH); 123.2 (CH); 123.1 (CH); 123.0 (CH); 87.6 (C); 32.4 (CH 2 ); 31.7 (CH 2 ); 29.5-29.3 (4 x CH 2 ); 29.2 (CH 2 ); 29.0 (CH 2 ); 28.9 (CH 2 ); 28.6 (CH 2 ); 22.5 (CH 2 ); 14.4 . MS (DCI/CH 4 ) m/z: 425.2 [M + ]. HRMS (DCI/CH 4 ): for C 26 H 35 NO 2 S [M + ]: calcd: 425.2393; found: 425.2389.

  C NMR (100 MHz, DMSO-d6) δ (ppm): 167.5 (C); 153.4 (C); 152.1 (C); 142.0 ; 137.3 (C); 129.7 (CH); 127.3 (CH); 125.2 (CH); 124.0 (CH); 123.2 (CH); 117.7 (C); 114.2 (CH); 107.0 (CH); 89.5 (C); 32.3 (CH 2 ); 31.7 (CH 2 ); 29.5-29.3 (4 x CH 2 ); 29.2 (CH 2 ); 29.0 (CH 2 ); 28.9 (CH 2 ); 28.6 (CH 2 ); 23.7 (CH 3 ); 22.5 (CH 2 ); 14.4 (CH 3 ). MS (DCI/CH 4 ) m/z: 455.3 [M+H + ]. HRMS (DCI/CH 4 ): for C 27 H 39 N 2 O 2 S [M+H + ]: calcd: 455.2723; found: 455.2732. (RS)-1,2-Dihydroxy-1-[3-(dodecylsulfanyl)phenyl]-1,2-dihydro-3H-pyrrolo[3,4c yridine-3-one (4a)

2 [

 2 2806, 1708, 1445, 1396, 1323, 1301, 1220, 1136, 1095, 1058, 1022, 864. 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 9.84 (s, 1H); 8.45 (s, 1H); 8.30 (dd, J = 1.6 Hz, 6.4 Hz, 1H); 8.05 (t, J = 1.6 Hz); 7.89 (d, J = 8.0 Hz, 1H); 7.83 (d, J = 8.4 Hz, 1H); 7.67 (t, J = 7.8 Hz, 1H); 7.52 (s, 1H); 7.42 (d, J = 6.8 Hz, 1H); 3.30 (t, J = 8.4 Hz, 2H); 1.57-1.47 (m, 2H); 1.32-1.15 (m, 18H); 0.85 (t, J = 6.8 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6) δ (ppm): 165.2 (C); 145.5 (C); 143.0 (CH); 142.3 (C); 140.0 (C); 134.1 (CH); 131.5 (CH); 130.9 (C); 130.3 (CH); 128.3 ; 125.1 (CH); 121.1 (CH); 87.0 (C); 54.9 (CH 2 ); 31.7 (CH 2 ); 29.4 (2 x CH 2 ); 29.3 (CH 2 ); 29.1 ( 2 x CH 2 ); 28.8 (CH 2 ); 27.8 (CH 2 ); 22.5 (CH 2 ); 14.4 (CH 3 ). MS (ESI) m/z: 475.M+H + ]. HRMS (ESI): for C 25 H 35 N 2 O 5 S: calcd: 475.2267; found: 475.2269.

. 2 (

 2 For the pure a compound 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 13.19 (s, 1H); 9.52 (s, 1H); 9.01 (d, J = 5.2 Hz, 1H); 7.54 (d, J = 5.6 Hz, 1H); 7.53-7.43 (m, 3H); 7.42-7.37 (m, 1H); 3.01 (t, J = 7.2 Hz, 2H); 1.65-1.55 (m, 2H); 1.44-1.15 (m, 18H); 0.85 (t, J = 6.8 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6) δ (ppm): 158.8 (C); 153.5 (CH); 149.9 (CH); 145.2 (C); 138.0 (C); 135.1 ; 134.3 (C); 129.7 (CH); 128.9 (CH); 128.3 (CH); 126.7 (CH); 122.3 (C); 119.1 (CH); 32.CH 2 ); 31.7 (CH 2 ); 29.5 (CH 2 ); 29.4 (CH 2 ); 29.4 (CH 2 ); 29.3 (CH 2 ); 29.1 (CH 2 ); 28.9 (CH 2 ); 28.9 (CH 2 ); 28.5 (CH 2 ); 22.5 (CH 2 ); 14.4 (CH 3 ). MS (ESI) m/z: 424.2 [M+H + ]. HRMS (ESI): for C 25 H 34 N 3 OS [M+H + ]: calcd: 424.2421; found: 424.2423.

  C NMR (100 MHz, DMSO-d5) δ (ppm): 166.6 (C); 156.2 (C); 154.1 (CH); 145.1 (CH); 125.9 (C); 118.1 (CH); 103.0 (C); 88.5 (C); 79.3 (C); 0.0 (3 x CH 3 ). MS (DCI/CH 4 ) m/z: 247.1 [M+H + ]. HRMS (DCI/CH 4 ): for C 12 H 14 N 2 O 2 Si [M+H + ]: calcd: 247.0905; found: 247.0903. (RS)-3-Hydroxy-3-[2-(trimethylsilyl)ethynyl]-2,3-dihydro-1H-isoindol-1-one (7b) Reagents: ethynyltrimethylsilane (133 mg, 0.19 mL, 1.36 mmol), dry tetrahydrofuran (2.5 mL), n-butyllithium solution (0.68 mL 2.5 M solution in hexanes, 1.36 mmol) and phtalimide (100 mg, 0.68 mmol). e crude product was purified by flash chromatography (gradient from 100% dichloromethane to 85/15 dichloromethane/methanol) to afford a white solid (97 mg, 58%), : 168 °C. TLC R f : 0.26 (dichloromethane/methanol 95/5). IR (cm -1 ): 3310, 3219, 2959, 2900, 2799, 1708, 1689, 1615, 1471, 1425, 1347, 1321, 1272, 1249, 1210, 1161, 1128, 1094, 1061, 1047, 1020, 839, 756, 694. 1 H NMR (400 MHz, CDCl 3 ) δ (ppm): 7.80 (d, J = 7.6 Hz, 1H); 7.74-7.66 (m, 2H); 7.58 (t, J = 7.2 Hz, 1H); 0.20 (s, 9H). 13 C NMR (100 MHz, CDCl 3 ) δ (ppm): 169.3 (C); 146.9 (C); 133.5 (CH); 130.4 (CH); 129.3 (C); 123.8 (CH); 122.8 (CH); .5 (C); 90.2 (C); 53.4 (C); 0.46 (3 x CH 3 ). MS (DCI/CH 4 ) m/z: 246.1 [M+H + ]. HRMS (DCI/CH 4 ): for C 13 H 15 NO 2 Si [M+H+]: calcd: 246.0947; found: 246.0950.

  -1-Hydroxy-1-(1-octyl-1H-1,2,3-triazol-4-yl)-1-hydroxy-1,2-dihydro-3H-pyrrolo[3,4-

  RS)-3-hydroxy-3-[2-(trimethylsilyl)ethynyl]-2,3-dihydro-1H-isoindol-1-one (70 mg, 0.32 mmol), 1-decyl-azide (65 mg, 0.36 mmol), methanol (10 mL), copper(II) sulfate pentahydrate (16 mg, 0.04 mmol), sodium ascorbate (26 mg, 0.13 mmol) and tetrabutylammonium fluoride solution (0.32 mL of a 1.0 M solution in tetrahydrofuran, 0.32 mmol). e crude product was purified by flash chromatography (gradient from 100% dichloromethane to 90/10 dichloromethane/methanol) to afford a yellow solid (107 mg, 94%), mp: 168 °C. TLC R f : 0.44 (dichloromethane/methanol 90/10). IR (cm -1 ): 3120, 3072, 2916, 2849, 1725, 1615, 1492, 1462, 1435, 1345, 1284, 1156, 1140, 1082, 1052, 1034, 930, 839, 3, 796, 758, 736. 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 9.53 (s, 1H); 8.87 (s, 1H); 8.78 (d, J = 5.2 Hz, 1H); 8.25 (s, 1H); 7.53 (dd, J= 1.0 Hz, 5.0 Hz, 1H); 7.21 (s, 1H); 4.34 (t, J = 7 Hz, 2H); 1.84-1.75

5 (

 5 RS)-3-hydroxy-3-[2-(trimethylsilyl)ethynyl]-2,3-dihydro-1H-isoindol-1-one (80 mg, 0.32 mmol), 1-dodecyl-azide (76 mg, 0.36 mmol), methanol (10 mL), copper(II) sulfate pentahydrate[START_REF] Pardin | Reversible and competitive cinnamoyl triazole inhibitors of tissue transglutaminase[END_REF] mg, 0.04 mmol), sodium ascorbate (26 mg, 0.13 mmol) and tetrabutylammonium fluoride solution (0.32 mL of a 1.0 M solution in tetrahydrofuran, 0.32 mmol). e crude product was purified by flash chromatography (gradient from 100% dichloromethane to 90/10 dichloromethane/methanol) to afford a yellow solid (107 mg, 87%), mp: 168 °C. TLC R f : 0.46 (dichloromethane/methanol 90/10). IR (cm -1 ): 3120, 3073, 2917, 2849, 1726, 1615, 1463, 1436, 1344, 1285, 1260, 1231, 1216, 1155, 1141, 1081, 1052, 1034, 929, 821, 796, 759, 734, 661. 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 9.52 (s, 1H); 8.87 (s, 1H); 8.78 (d, J = 5.2 Hz, 1H); 8.25 (s, 1H); 7.53 (dd, J= 1.0 Hz, 5.0 Hz, 1H); 7.20 (s, 1H); 4.33 (t, J = 7 Hz, 2H); 1.84-1.75 (m, 2H); 1.30-1.17 (m, 18H); 0.85 (t, J = 6.8 Hz, 3H). 13 C R (100 MHz, DMSO-d6) δ (ppm): 167.2 (C); 157.6 (C); 153.4 (CH); 147.9 (C); 144.9 H); 126.8 (C); 123.4 (CH); 118.6 (CH); 84.1 (C); 49.9 (CH 2 ); 31.6 (CH 2 ); 30.1 (CH 2 ); 29.CH 2 ); 29.4 (CH 2 ); 29.4 (CH 2 ); 29.3 (CH 2 ); 29.1 (CH 2 ); 28.8 (CH 2 ); 26.3 (CH 2 ); 22.5 (CH 2 ); 14.4 (CH 3 ). MS (DCI/CH 4 ) m/z: 386.2 [M+H + ]. HRMS (DCI/CH 4 ): for C 21 H 32 N 5 O 2 [M+H + ]: calcd: 386.2552; found: 386.2556.(RS)-3-Hydroxy-3-(1-octyl-1H-1,2,3-triazol-4-yl)-2,3-dihydro-1H-isoindol-1-one (8b)Reagents: (RS)-1-hydroxy-1-[2-(trimethylsilyl)ethynyl]-1,2-dihydro-3H-pyrrolo[3,4c]pyridin-3-one (52 mg, 0.21 mmol), 1-octyl-azide (36 mg, 0.23 mmol), methanol (5 mL), copper(II) sulfate pentahydrate (10 mg, 0.04 mmol), sodium ascorbate (17 mg, 0.08 mmol) and tetrabutylammonium fluoride solution (0.21 mL of a 1.0 M solution in tetrahydrofuran, 0.21 mmol). The crude product was purified by flash chromatography (gradient from 100% dichloromethane to 90/10 dichloromethane/methanol) to afford a yellow solid (28 mg, 40%), mp: 132 °C. TLC R f : 0.54 (dichloromethane/methanol 90/10

(RS)- 3 -

 3 (1-Decyl-1H-1,2,3-triazol-4-yl)-3-hydroxy-2,3-dihydro-1H-isoindol-1-one (9b)Reagents: (RS)-1-hydroxy-1-[2-(trimethylsilyl)ethynyl]-1,2-dihydro-3H-pyrrolo[3,4c]pyridin-3-one (100 mg, 0.41 mmol), 1-decyl-azide (75 mg, 0.45 mmol), methanol (10 mL), copper(II) sulfate pentahydrate (20 mg, 0.08 mmol), sodium ascorbate (32 mg, 0.16 mmol) and tetrabutylammonium fluoride solution (0.41 mL of a 1.0 M solution in tetrahydrofuran, 0.41 mmol). The crude product was purified by flash chromatography (gradient from 100% dichloromethane to 90/10 dichloromethane/methanol) to afford a yellow solid (102 mg, 70%), mp: 145 °C. TLC R f : 0.43 (dichloromethane/methanol 90/10). IR (cm -1 ): 3292, 3179, 3130, 2920, 2851, 1684, 1614, 1470, 1424, 1352, 1315, 1266, 1222, 1190, 1143, 1081, 1048, 1026, 917, 880, 854, 803, 761, 737, 720. 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 9.25 (s, 1H); 8 (s, 1H); 7.65-7.45 (m, 4H); 6.91 (s, 1H); 4.32 (t, J = 7.0 Hz, 2H); 1.80 (m, 2H); 1.31-1.18

  Molecular graphics were performed with Accelrys Discovery Studio Visualizer (Accelrys Software Inc., Discovery Studio Modeling Environment, Release 4.0, San Diego: Accelrys Software Inc., 2013).

Figure 2 .

 2 Figure 2.

Figure 2 .

 2 Figure 2. Structural evolution of azaisoindolinone scaffold 2a.

11 4a

 11 1 = H, X = N, R 2 = NH 2b: R 1 = H, X = CH, R 2 = NH 2c: R 1 = NH 2 , X = CH, R28a: X = N, n = 7 9a: X = N, n = 9 10a: X = N, n = 11 8b: X = CH, n = 7 9b: X = CH, n = 9 10b: X = CH, n =

  activity of 5a and 5b (entries 9 and 10 respectively). This result strongly supports the proposal that N8-OH may form hydrogen bond interaction into the active pocket of the InhA.The replacement of the dodecylthiophenyl (ring C) by a dodecyltriazolyl moiety in the pyridine series led to a more active compound (10a entry 17) than the reference molecule 2a.The positive effect exerted by the dodecyltriazolyl group in the place of the dodecylthiophenyl substituent on this scaffold may be explained by new hydrogen bonding possibilities afford by the heteroatoms of the aromatic cycle. Structure-InhA inhibitory activity exploration in the triazole series was also carried out by modifying the length of the hatic chain. As observed previously[START_REF] Deraeve | Chemical Synthesis, Biological Evaluation and Structure-Activity Relationship Analysis of Azaisoindolinones, a Novel Class of Direct Enoyl-ACP Reductase Inhibitors as Potential Antimycobacterial Agents[END_REF], it was shown that the length of the alkyl chain plays an important role to ensure efficient inhibitory activity toward the InhA protein (entries 13, 15 and 17).

  The p-4a(R) and p-4a(S)(Figures 3C and 3D respectively) compounds are located globally in the same position as 2a(R) and 2a(S), respectively and establish almost the same main interactions with the cofactor and the protein. It is interesting to note that, for regioisomers p-4a(R) and p-4a(S), the N-hydroxylamine group was not responsible for additionnal interactions within the active site of the protein. In contrast, the meta regioisomer, compound m-4a(R) (Figure3E), interacts with InhA by the two hydrogen bonds, which involve tertiary alcohol and carbonyl group, and a supplementary interaction between N8-OH and C2'-group. The other enantiomer m-4a(S) (Figure

  synthesized compounds comes indeed from the inhibition of the InhA target. To answer to this question, we tested compounds m-4a, 2a and INH, for comparison, toward the inhibition of mycolic acid biosynthesis in intact mycobacteria. Bacteria were treated at the MIC of each compound for 5 h and then incubated with [1-14 C]acetate for 18 h. After saponification, radiolabelled mycolic acids (MA) and fatty acids (FA) were analyzed as methyl esters by TLC and phosphorimaging. The results showed that, although these compounds inhibit InhA in vitro, they do not seem to target InhA inside Mycobacterium tuberculosis since they do not inhibit the mycolic acid biosynthesis in the used conditions. (Figure4)

Figure 4 .

 4 Figure 4. Effect of 2a and m-4b on MA biosynthesis. Bacteria in culture were treated with either INH, 2a or m-4a at their MIC and the neosynthesis of mycolic acids in vivo was followed by [1-14 C]acetate labelling (duplicates). a) TLC-phosphorimaging analysis of radiolabelled fatty acid and mycolic acid methyl esters. b) relative incorporation of radioactivity in various molecular species assessed on plate a). FA, fatty acid methyl esters; αMA, alpha-mycolic acid methyl esters; M+K MA, methoxy-and keto-mycolic acid methyl esters; INH, isoniazid.

  3223, 2917, 2849, 1694, 1614, 1588, 1467, 1435, 1357, 1205, 1115, 1093, 1061, 1023, 1005, 963, 953, 901, 884, 857, 789, 766, 738, 719, 691. 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 7.75 (d, J = 8.8 Hz, 1H); 7.66-7.50 (m, 3H); 7.36-7.20 (m , 3H); 7.07 (dt, J = 1.8 Hz, 7.2 Hz); 3.85 (s, 1H); 2.90 (t, J = 7.2 Hz, 2H); 1.55-1.46 (m, 2H); 1.35-1.18 (m, 18H); 0.85 (t, J = 6.6 Hz, 3H). CH); 90.5 (C); 65.3 (CH 3 ); 32.3 (CH 2 ); 31.7 (CH 2 ); 29.5 (CH 2 ); 29.5 (CH 2 ); 29.4 (CH 2 ); 29.3 (CH 2 ); 29.2 (CH 2 ); 29.0 (CH 2 ); 28.9 (CH 2 ); 28.5 (CH 2 ); 22.5 (CH 2 ); 14.4 (CH 3 ). MS (DCI/CH 4 ) m/z: 455.2 [M + ]. HRMS (DCI/CH 4 ): for C 27 H 37 NO 3 S [M + ]: calcd: 455.2493;

NMR (100 MHz, DMSO-d6) δ (ppm): 163.1 (C); 146.8 (C); 140.4 (C); 137.2 (C); 133.7 (CH); 130.0 (CH); 129.6 (CH); 128.2 (C); 127.8 (CH); 125.5 (CH); 123.6 (CH); 123.2 (2 x nd: 455.2494.

  2914, 2849, 1683, 1567, 1339, 1223, 1155, 995, 896, 782, 721, 697, 685. 1 H NMR (500 MHz, CH 2 ); 14.4 (CH 3 ). MS (DCI/CH 4 ) m/z: 423.6 [M+H + ]. HRMS (DCI/CH 4 ): for C H 34 N 2 OS [M+H + ]: calcd: 423.2480; found: 423.2470.

	CDCl 3 ) δ (ppm): 12.86 (s, 1H); 8.39-8.31 (m, 1H); 7.90 (t, J = 5.2 Hz, 2H); 7.68-7.63 (m,
	1H); 7.51-7.44 (m, 3H); 7.38 (d, J = 7.2 Hz, 1H); 3.01 (t, J = 7.2 Hz, 2H); 1.61 (m, 2H); 1.42-
	1.35 (m, 2H); 1.30-1.15 (m, 16H); 0.84 (t, J = 6.8 Hz, 3H). 13 C NMR (125 MHz, DMSO-d6)
	δ (ppm): 159.7 (C); 146.5 (C); 137.7 (C); 136.3 (C); 134.1 (CH); 132.2 (CH); 129.6 (CH);
	129.4 (C); 128.7 (CH); 128.6 (CH); 128.3 (C); 126.9 (CH); 126.8 (CH); 126.6 (CH); 32.2
	(CH 2 ); 31.7 (CH 2 ); 29.5 (3 x CH 2 ); 29.4 (CH 2 ); 29.3 (CH 2 ); 29.1 (CH 2 ); 28.9 (CH 2 ); 28.5
	(CH 2 ); 22.5 (
	26

  ); 28.8 (CH 2 ); 26.3 (CH 2 ); 22.5 (CH 2 ); 14.4 (CH 3 ). MS (DCI/CH 4 ) m/z: 330.2 [M+H + ]. HRMS (DCI/CH 4 ): for C 17 H 24 N 5 O 2 [M+H + ]: calcd: 330.1941; found: 330.1930.

	TLC R f : 0.42 (dichloromethane/methanol 90/10). IR (cm -1 ): 3120, 3072, 2946, 2917, 2849,
	2776, 1725, 1616, 1492, 1464, 1436, 1395, 1377, 1345, 1288, 1260, 1230, 1156, 1141, 1081,
	1052, 1034, 929, 839, 795, 734. 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 9.53 (s, 1H); 8.87
	(s, 1H); 8.78 (d, J = 5.2 Hz, 1H); 8.25 (s, 1H); 7.53 (dd, J= 1.0 Hz, 5.0 Hz, 1H); 7.21 (s, 1H);
	4.34 (t, J = 7 Hz, 2H); 1.84-1.75 (m, 2H); 1.30-1.17 (m, 10H); 0.85 (t, J = 6.8 Hz, 3H). 13 C
	R (100 MHz, DMSO-d6) δ (ppm): 167.2 (C); 157.6 (C); 153.4 (CH); 147.9 (C); 144.9
	H); 126.8 (C); 123.4 (CH); 118.6 (CH); 84.1 (C); 49.9 (CH 2 ); 31.6 (CH 2 ); 30.1 (CH 2 ); 29.0
	(CH 2

  ); 29.1 (CH 2 ); 28.8 (CH 2 ); 26.3 (CH 2 ); 22.5 (CH 2 ); 14.4 (CH 3 ). MS (DCI/CH 4 ) m/z: 358.2 [M+H + ]. HRMS (DCI/CH 4 ): for C 19 H 28 N 5 O 2 [M+H + ]: calcd: 358.2243; found:

	(CH 2
	C NMR (100 MHz, DMSO-d6) δ (ppm): 167.2 (C); 157.6 (C); 153.4 (CH); 147.9 (C); 144.9 (CH); 126.8 ]p Th NM Accepted Article (C
	(C); 123.4 (CH); 118.6 (CH); 84.1 (C); 49.9 (CH 2 ); 31.6 (CH 2 ); 30.1 (CH 2 ); 29.4 (CH 2 ); 29.3

  ). IR (cm -1 ): 3289, 3174, 3129,2923, 2853, 1683, 1614, 1468, 1424, 1378, 1353, 1316, 1266, 1222, 1199, 1179, 1143, 1081, 1061, 1048, 1025, 918, 855, 762, 737, 718. 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 9.25 (s, 1H); 7.65-7.45 (m, 4H); 6.91 (s, 1H); 4.32 (t, J = 7.0 Hz, 2H); 1.84 (m, 2H); 1.30-1.20 (m, 10H); 0.85 (t, J = 6.8 Hz, 3H).13 C NMR (100 MHz, DMSO-d6) δ (ppm): 168.5 (C); 149.9 (C); 149.1 (C); 132.7 (CH); 131.3 (C); 129.6 (CH); 123.5 (CH); 123.1 (CH); 122.8 (CH); 84.2 (C); 49.8 (CH 2 ); 31.6 (CH 2 ); 30.2 (CH 2 ); 29.0 (CH 2 ); 28.8 (CH 2 ); 26.3 (CH 2 ); 22.5 (CH 2 ); 14.4 (CH 3 ). MS (DCI/CH 4 ) m/z: 329.2 [M+H + ]. HRMS (DCI/CH 4 ): for C 18 H 25 N 4 O 2 [M+H + ]: calcd: 329.1986; found: 329.1978.

	1H); 8.18 (s,
	8.1 (m, mm Accepted Article Th

  Reagents and conditions: (a) BrC 6 H 4 SC 12 H 25 , nBuLi, THF, -78 °C, 37-58% (b) mCPBA, 72% (c) H NOMe.HCl, PTSA, toluene, reflux (d): BrC 6 H 4 SC 12 H 25 , nBuLi, THF, -78 °C, 26-36% (e) H 2 NNH 2 , EtOH, reflux, 76-99% (f): ethynyltrimethylsilane, nBuLi, -78 °C, 56-64% (g) C 8 H 17 N 3 , C 10 H 21 N 3 or C 12 H 25 N 3 , CuSO 4 .5H 2 O, NaAsc, EtOH, TBAF, 28-93% (h) for compound 4a, H 2 NOH.HCl, pyridine, reflux (i) BrC 6 H 4 SC 12 H 25 , nBuLi, THF, -78 °C, 30-44%

	Scheme 1.
	2
	Th

Table 1 .

 1 Effects of modifications upon the azaisoindolione framework on enzyme-inhibitory and antibacterial activities

				Scaffold A	Scaffold B
								InhA inhibition (%) a	MIC
	Entry Compound Scaffold	R 1	R 2	X	Y	50 µM	10 µM	(µM) a Mtb
	1	2a	A	Ph-SC 12	H	N	CH	28.1 ± 1.1	-	23.4
	2	2b	A	Ph-SC 12	H	CH	CH	42.4 ± 3.4	-	-
	3	2c	A	Ph-SC 12	CH 3	CH	CNH 2	12.3 ± 4.6	-	-
	4	3	A	Ph-S(O 2 )C 12	H	NO	CH	34.6 ± 2.8	-	21.1
	5	4a	A	Ph-SC 12	OH	N/CH	N/CH	76.1 ± 3.7	-	22.6
	6	p-4a	A	Ph-SC 12	OH	N	CH	11.4 ± 0.6	-	> 22.5
	7	m-4a	A	Ph-SC 12	OH	CH	N	80.1 ± 3.0	21	> 22.5
	8	4b	A	Ph-SC 12	OH	CH	CH	81.6 ± 4.9	36	> 22.7
	9	5a	A	Ph-SC 12	OMe	N	CH	42.8 ± 5.1	-	> 21.9
	10	5b	A	Ph-SC 12	OMe	CH	CH	25.0 ± 5.2	-	-

Acknowledgments

We thank the scientific council of the University Paul Sabatier by the doctoral fellowship of Aurélien Chollet.

Accepted Article

Conflicts of interest

The authors declare no conflict of interests.

Accepted Article

a The para carbonyl group is defined with respect to the nitrogen of the pyridine ring. The other carbonyl group is subsequently defined as meta.

Supporting information

Additional Supporting Information may be found in the online version of this article: