Léo Faion

Kamel Djaout

Rosangela Frita

Catalin Pintiala

Francois-Xavier Cantrelle

Martin Moune

Alexandre Vandeputte

Kevin Bourbiaux

Catherine Piveteau

Adrien Herledan

Alexandre Biela

Florence Leroux

Laurent Kremer

Mickael Blaise

Abdalkarim Tanina

René Wintjens

Xavier Hanoulle

Benoit Déprez

Nicolas Willand

Alain R Baulard

Marion Flipo
email: marion.flipo@univ-lille.fr

Discovery of the first Mycobacterium tuberculosis MabA (FabG1) inhibitors through a fragment-based screening

Keywords: MabA inhibitors, fragment, FabG1, tuberculosis, mycolic acid

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

Introduction

Tuberculosis (TB), a disease caused by the bacillus Mycobacterium tuberculosis (M.tb), remains a major cause of death worldwide. [1] [2] In 2018, an estimated 10.0 million people became ill with TB and an estimated 1.45 million people died from the consequences of the disease (1.2 million deaths among HIV-negative people and 250 000 deaths among HIV-positive people). In addition, multidrug-resistant-TB (MDR-TB) is becoming a major threat. Drug-sensitive TB regimen has remained unchanged for 40 years [3,4] and only recently, three new chemical entities, namely bedaquiline [5], delamanid [6] and pretomanid [START_REF] Keam | Pretomanid: First Approval[END_REF], which are still in phase 3 clinical trials, have been approved for the treatment of MDR-TB. In this context, the search for new anti-TB agents acting on yet unexplored targets is thus warranted. The mycobacterial cell wall is a complex structure composed of four major biomolecules: mycolic acids, arabinogalactan, lipoarabinomannan and peptidoglycan. [8,9] Mycolic acids are very long chain fatty acids, containing between 70 and 90 carbon atoms. They play an essential role in the architecture and permeability of the cell envelope and are essential for the survival of the bacteria. [START_REF] Liu | Mycolic acid structure determines the fluidity of the mycobacterial cell wall[END_REF][START_REF] Marrakchi | Mycolic Acids: Structures, Biosynthesis, and Beyond[END_REF] The biosynthesis of mycolic acids involves two types of fatty acid synthase (FAS) systems. FAS-I cycle is a multi-functional enzyme complex, which synthesizes short-chain fatty acids (C16-C18 and C24-C26). [START_REF] Schweizer | Microbial type I fatty acid synthases (FAS): major players in a network of cellular FAS systems[END_REF]13] These fatty acids can form the α-alkyl chain (C24) found in mycolic acids or can be used as primer substrates for their subsequent elongation by the FAS-II cycle. [START_REF] Marrakchi | Mycolic Acids: Structures, Biosynthesis, and Beyond[END_REF] FAS-II system is not capable of de novo synthesis. This cycle is composed of four enzymes that act successively to elongate the acyl-CoA (C16-C18) generated by FAS-I and form the very long meromycolic acid chains (C42-C62). [START_REF] Bhatt | The Mycobacterium tuberculosis FAS-II condensing enzymes: Their role in mycolic acid biosynthesis, acid-fastness, pathogenesis and in future drug development[END_REF] Enzymatic modifications provide functional groups to the meromycolic chain like cyclopropane, keto and methoxy groups. The Claisen condensation of the modified meromycolic chain with the α-alkyl short chain (C24), followed by a reduction, leads to α-alkyl-β-hydroxy-mycolic acids. [8,[START_REF] Marrakchi | Mycolic Acids: Structures, Biosynthesis, and Beyond[END_REF] FAS-II is initiated by a Claisen condensation of malonyl-ACP and palmitoyl-CoA catalyzed by the βketoacyl-ACP synthase FabH. The β-ketoacyl-ACP formed is then reduced by MabA (FabG1), a βketoacyl-ACP reductase NADPH-dependent, to produce β-hydroxyacyl-ACP. The enzymes HadAB/BC (β-hydroxyacyl-ACP dehydratases) catalyze the dehydration of the β-hydroxyacyl-ACP into trans-2enoyl-ACP, which is then reduced by InhA (enoyl-ACP reductase NADH-dependent) to produce an acyl-ACP elongated by two carbons. The FAS-II cycle continues with a Claisen condensation of the acyl-ACP previously formed and malonyl-ACP, catalyzed by KasA/B (β-ketoacyl-ACP synthase). Several iterative cycles catalyzed by MabA, HadAB/BC, InhA and KasA/B lead to the production of elongated acyl chain (C42-62). [8,[START_REF] Bhatt | The Mycobacterium tuberculosis FAS-II condensing enzymes: Their role in mycolic acid biosynthesis, acid-fastness, pathogenesis and in future drug development[END_REF] InhA [START_REF] Vilchèze | Inactivation of the inhA-encoded fatty acid synthase II (FASII) enoyl-acyl carrier protein reductase induces accumulation of the FASI end products and cell lysis of Mycobacterium smegmatis[END_REF], MabA [START_REF] Parish | Functional complementation of the essential gene fabG1 of Mycobacterium tuberculosis by Mycobacterium smegmatis fabG but not Escherichia coli fabG[END_REF], HadB [START_REF] Brown | Identification of the dehydratase component of the mycobacterial mycolic acid-synthesizing fatty acid synthase-II complex[END_REF] and KasA [START_REF] Bhatt | Conditional depletion of KasA, a key enzyme of mycolic acid biosynthesis, leads to mycobacterial cell lysis[END_REF] have been shown to be essential for bacterial growth, strengthening their attractiveness as valuable chemotherapeutic targets. [START_REF] North | New approaches to target the mycolic acid biosynthesis pathway for the development of tuberculosis therapeutics[END_REF] Several antibiotics target FAS-II. Indeed, InhA is inhibited by isoniazid as well as by two second-line drugs, ethionamide and prothionamide. [START_REF] Banerjee | inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis[END_REF][START_REF] Kremer | Inhibition of InhA activity, but not KasA activity, induces formation of a KasA-containing complex in mycobacteria[END_REF][START_REF] Vilchèze | Transfer of a point mutation in Mycobacterium tuberculosis inhA resolves the target of isoniazid[END_REF] HadAB is inhibited by isoxyl and thiacetazone [START_REF] Grzegorzewicz | A common mechanism of inhibition of the Mycobacterium tuberculosis mycolic acid biosynthetic pathway by isoxyl and thiacetazone[END_REF][START_REF] Gannoun-Zaki | Point mutations within the fatty acid synthase type II dehydratase components HadA or HadC contribute to isoxyl resistance in Mycobacterium tuberculosis[END_REF][START_REF] Belardinelli | Mutations in the essential FAS II β-hydroxyacyl ACP dehydratase complex confer resistance to thiacetazone in Mycobacterium tuberculosis and Mycobacterium kansasii[END_REF] and KasA/B are inhibited by thiolactomycin [START_REF] Kremer | Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in Mycobacterium tuberculosis[END_REF][START_REF] Machutta | Slow onset inhibition of bacterial beta-ketoacyl-acyl carrier protein synthases by thiolactomycin[END_REF] while KasA (but not KasB) is also the target of an indazole sulfonamide (GSK3011724A). [START_REF] Abrahams | Identification of KasA as the cellular target of an antitubercular scaffold[END_REF] However, no specific inhibitors of MabA have been reported yet. The crystal structure of the apo-form MabA from M.tb was solved but several residues were not visible in the electron density. [START_REF] Cohen-Gonsaud | Crystal structure of MabA from Mycobacterium tuberculosis, a reductase involved in long-chain fatty acid biosynthesis[END_REF] A double mutant C60V/S144L of MabA was used to obtain crystal structures in the presence and absence of NADP. Nevertheless, in the holo form, the nicotinamide moiety and the ribose group were not visible in the electron density. [START_REF] Cohen-Gonsaud | Ligand-induced fit in mycobacterial MabA: The sequence-specific C-terminus locks the conformational change[END_REF] High-resolution crystal structures of MabA from M. smegmatis in its apo, NADP + -bound and NADPH-bound forms were recently reported. [START_REF] Küssau | Structural rearrangements occurring upon cofactor binding in the Mycobacterium smegmatis βketoacyl-acyl carrier protein reductase MabA[END_REF] Structural comparison of MabA, a NADPH-dependent-ketoacyl-ACP reductase, and InhA, an NADHdependent enoyl-ACP reductase, revealed the presence of hydrophobic substrate binding pockets of similar sizes. [START_REF] Cohen-Gonsaud | Crystal structure of MabA from Mycobacterium tuberculosis, a reductase involved in long-chain fatty acid biosynthesis[END_REF][START_REF] Dessen | Crystal structure and function of the isoniazid target of Mycobacterium tuberculosis[END_REF][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] However, MabA shares only 20% sequence identity with InhA or with other enoyl-ACP reductases, despite the similarity of their respective ligands. MabA has the same specificity for long-chain substrates as InhA. INH is a pro-drug which is bioactivated by the bacterial catalase peroxidase KatG to form an INH-NAD adduct that binds to and inhibits InhA. The interaction between MabA and the INH-NADP adduct has also been investigated and reported to lead to the inhibition of MabA activity in vitro. [START_REF] Ducasse-Cabanot | In Vitro Inhibition of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA by Isoniazid[END_REF] In order to discover the first drug-like MabA inhibitors, we developed a new LC-MS/MS-based enzymatic assay. To enhance the propensity to find inhibitors with physicochemical properties adequate to penetrate into mycobacteria, we used low-molecular-weight compounds, more properly called fragments, as a source of screening molecules.

Results and discussion 2.1 Development of LC-MS/MS based MabA activity assay

MabA is able to catalyze the reduction of acetoacetyl-CoA (AcAcCoA) into hydroxybutyryl-CoA (HBCoA) using NADPH as a cofactor (Figure 1A). An enzymatic assay based on the monitoring of the oxidation of NADPH by spectrophotometry was reported in the literature. [START_REF] Marrakchi | MabA (FabG1), a Mycobacterium tuberculosis protein involved in the long-chain fatty acid elongation system FAS-II[END_REF][START_REF] Silva | Mycobacterium tuberculosis β-ketoacyl-acyl carrier protein (ACP) reductase: Kinetic and chemical mechanisms[END_REF] To develop an enzymatic assay suitable for high-throughput screening in 384-well plates, we chose to detect the formation of the enzymatic reaction product HBCoA by mass spectrometry. The enzymatic reaction was stopped by adding a solution of trifluoroacetic acid in water at a final concentration of 1%, containing butyryl-CoA (BCoA) as an internal standard for MS/MS analysis. The peak areas of HBCoA and BCoA were measured by MS/MS. To determine the initial velocity phase of the enzymatic reaction, we plotted the area of HBCoA peak as a function of reaction time from 2.5 to 20 min (Figure 1B). In view of this result, we opted to quench the reaction after 15 min in all the following experiments, which insures that the assay reflects the initial velocity phase of the reaction. [START_REF] Copeland | Mechanistic considerations in high-throughput screening[END_REF] As mentioned above, the only MabA inhibitor reported so far in the literature is the adduct of INH with NADP + (Figure 2A). [START_REF] Ducasse-Cabanot | In Vitro Inhibition of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA by Isoniazid[END_REF] This INH-NADP adduct can be formed in situ using Mn III [START_REF] Nguyen | A Fast and Efficient Metal-Mediated Oxidation of Isoniazid and Identification of Isoniazid-NAD(H) Adducts[END_REF] and was used as a reference inhibitor in this assay. LC-MS/MS measurement of HBCoA peak area in the absence (Figure 2B) or in the presence (Figure 2C) of this inhibitor allowed the calculation of a percentage of inhibition and the determination of an IC50. In this assay, the INH-NADP adduct displayed an IC50 of 20 µM (Figure 2D). Triclosan [START_REF] Parikh | Inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis, by triclosan and isoniazid[END_REF] and pyridomycin [START_REF] Hartkoorn | Pyridomycin bridges the NADH-and substrate-binding pockets of the enoyl reductase InhA[END_REF][START_REF] Hartkoorn | Towards a new tuberculosis drug: Pyridomycin -nature's isoniazid[END_REF], two known InhA inhibitors, did not show any MabA inhibitory activity in this assay when tested up to 100 µM (data not shown).

Screening

Our 1280-fragment library is composed of commercially available molecules and sp 3 and spiroenriched compounds. [START_REF] Prevet | Microwave-assisted synthesis of functionalized spirohydantoins as 3-D privileged fragments for scouting the chemical space[END_REF][START_REF] Tran | Synthesis of functionalized 2isoxazolines as three-dimensional fragments for fragment-based drug discovery[END_REF] This library fulfills the 'rule of three' and all compounds are soluble in DMSO at 100 mM. [START_REF] Congreve | A "Rule of Three" for fragment-based lead discovery?[END_REF] The 1280 fragments were screened at 1 mM on MabA at a concentration of 400 nM, using the mass spectrometry enzymatic assay adapted in 384-well plates. To identify both slow and fast binding inhibitors, fragments were incubated for 10 or 30 min with MabA alone in Hepes buffer. AcAcCoA and NADPH were then added to reach the final concentration of 50 µM and the microplates were incubated 15 min at 22 °C. The reaction was stopped by the addition of a solution of trifluoroacetic acid in water containing BCoA. The peak areas of HBCoA and BCoA were measured by LC-MS/MS. To confirm the statistical validity of the screening, Z′-factors were calculated using the mean and standard deviation values of the negative (1% DMSO) and positive controls (INH-NADP adduct at 200 µM). All the screening plates displayed a Z′-factor between 0.72 and 0.83. It is frequently admitted that a Z′-factor ≥ 0.5 is acceptable for high-throughput screening. [45][46] Percentage of inhibition of each compound was calculated using the mean values of the negative and positive controls (Figure 3). Similar inhibition percentages were obtained for the two incubation conditions, suggesting that compounds are more likely fast binding inhibitors. Fifty compounds displaying an inhibition percentage greater than 30% were selected for dose response experiments, which allowed to identify 12 fragments with IC50 lower than 300 µM. Data mining of commercial libraries identified 68 analogues of these 12 fragments. Retesting of the 80 compounds in doseresponse experiments led to the selection of 6 chemical series displaying IC50s ranging from 25 to 300 μM (Figure 4).

Selection of a chemical series

An early structure-activity relationships (SAR) study was initiated on each of the six series. Briefly, whereas compound 1 of the anthranilic acid series was retested three times in duplicate and displayed an IC50 slightly higher than that obtained during the screening (430 ± 26 µM), the early SAR in this series led to compounds with improved activities that will be detailed later in this manuscript. In the 5aminouracil series (compound 2), the replacement of the pentadiene chain with other aliphatic chains led to inactive compounds (IC50 > 1 mM). For the isatine series, early SAR showed that the bromine atom of compound 3 can be removed or replaced by a fluorine atom, a chlorine atom or a methoxy group. In contrast, the 5-bromoindolin-2-one was shown inactive (IC50 > 1 mM) indicating that the ketone function is necessary for activity. Replacement of the thienopyrrole moiety of compound 4 with other fused aromatic rings was not tolerated (IC50 > 1 mM). In the thiobarbituric acid series (compound 5), the substitution of the phenyl ring is tolerated and the replacement of the sulphur atom of the thiobarbituric core with an oxygen atom decreased the activity. Finally, in the propanoic acid series, the resynthesis of compound 6 led to an inactive compound (IC50 > 1 mM) whereas the commercial batch was reconfirmed as active as during the screening (IC50 = 30 µM). This implies that the commercial product may not be the one described, although LC-MS analysis showed that it is relatively pure and that the mass is consistent with the described structure. Limited by the amount, we were unable to investigate further the structure of this compound that is no longer commercially available. The 6 hits were then tested on M.tb. Compounds 2-6 did not display antibacterial activity (MIC90 > 300 µM) whereas compound 1 showed bacterial growth inhibition with a MIC90 of 300 µM. Anthranilic acid represents a well-known privileged structure and its derivatives have been widely used in different fields of medicinal chemistry. [START_REF] Nittoli | Identification of anthranilic acid derivatives as a novel class of allosteric inhibitors of hepatitis C NS5B polymerase[END_REF][START_REF] Darakhshan | Tranilast: A review of its therapeutic applications[END_REF][START_REF] Silva | A diarylamine derived from anthranilic acid inhibits ZIKV replication[END_REF][START_REF] El-Azab | New anthranilic acid-incorporating Nbenzenesulfonamidophthalimides as potent inhibitors of carbonic anhydrases I, II, IX, and XII: Synthesis, in vitro testing, and in silico assessment[END_REF] Nevertheless, compound 1 has not yet been described in the ChEMBL database as having any biological activities. This motivated us to further optimized this scaffold.

Competition with NADPH

In order to determine whether compound 1 acts as a competitive inhibitor of NADPH, the previously described HBCoA enzymatic assay was used in the presence of two different NADPH concentrations (50 and 500 µM). This test was validated using the INH-NADP adduct in a dose-response experiment (Figure 5A). As expected, since the adduct is assumed to bind to the NADPH binding site, its IC50 was shifted towards highest concentrations in the presence of the high dose of NADPH (IC50 = 61 µM when competing with 500 µM of NADPH vs IC50 = 20.3 ± 0.6 µM (3 independent experiments) when competing with 50 µM of NADPH). In contrast, in this assay compound 1 displays a similar IC50 whatever the concentration of NADPH used, suggesting that this compound inhibits MabA in a non-competitive manner with NADPH (Figure 5B). This compound could therefore interact with the substrate binding pocket, as it has been reported for InhA inhibitors that have been co-crystallized with their target. Indeed, most of these compounds occupy the lipid-binding pocket of InhA facing the nicotinamide moiety of NAD.[51-54]

[INH-NADP adduct] [compound 1]

A B

Chemistry

In order to explore the SAR in the anthranilic acid series, the iodine atom, the Boc group and the carbamate function of compound 1 were modified.

2.5.1. Modifications of the substituents on the phenyl ring First, the importance of the iodine atom was evaluated by removing (compound 7) or replacing it by other halogen atoms (compounds 8, 9, 10). The iodine atom was also replaced by a methoxy group (compound 11) or aromatic rings such as phenoxy (compound 12) and phenyl groups (compound 13).

A naphtyl group (compound 14) was also introduced instead of the 5-iodophenyl group to evaluate the impact of the substitution by an aromatic ring. Compound 7 was commercially available and compounds 8, 9, 10 were synthesized in one step from the 5-halogenoanthranilic acid and di-tertbutyldicarbonate (Boc2O) in acetonitrile under microwave irradiations (Scheme 1). Compounds 11, 12 and 14 were synthesized according to scheme 1 in pyridine at 90 °C. The 2-amino-5-phenylbenzoic acid was not commercially available and was synthesized in one step using a Suzuki coupling (Scheme 1). These intermediate 13a was then reacted with Boc2O in acetonitrile under microwave irradiations.

Modifications of the carbamate group and the carboxylic acid

Replacement of the Boc group with other carbamate groups was evaluated and the carboxylic acid was replaced by a methyl ester. Carbamates (compounds 15-17) were synthesized in one step from 2amino-5-iodobenzoic acid and corresponding chloroformates in pyridine (Scheme 2). Compound 18 was obtained by methylation of the nitrogen atom using iodomethane. This led also to the formation of the methyl ester that was hydrolyzed in basic conditions. The methyl ester (compound 19) was obtained by reaction of the carboxylic acid with dimethylsulfate using potassium carbonate as base.

Replacement of the carbamate with an amide

The carbamate function was then replaced by an amide function variously substituted (compounds 20-37). Several methods were attempted for the coupling of the amine function of the 2-amino-5iodobenzoic acid with diverse acyl chlorides (Scheme 4) or carboxylic acids (Scheme 4). Acyl chlorides were coupled directly with 2-amino-5-iodobenzoic acid in pyridine (compound 21) or with methyl 2amino-5-iodo-benzoate and then the methyl ester was hydrolyzed in basic conditions (compounds 27 and 35). Carboxylic acids were activated either by reaction with thionyl chloride and 1H-benzotriazole (compounds 20, 22, and 26), by using an HOBt-resin [START_REF] Pop | Versatile Acylation of N-Nucleophiles Using a New Polymer-Supported 1-Hydroxybenzotriazole Derivative[END_REF] (compound 23) or by forming a mixed anhydride with isopropyl chloroformate (compounds 24, 25, 28-34 and 36-37). All these reactions led to the desired analog with moderate yields (~50%) after reverse chromatography purification. We found that the most appropriate way to activate carboxylic acids for the coupling with 2-amino-5iodobenzoic acid was through the synthesis of mixed anhydrides using isopropyl chloroformate.

Biological results

All compounds were evaluated for their inhibitory activity of MabA using our HBCoA enzymatic assay. Activities are reported in comparison to the inhibitory activity of compound 1. Replacement of the iodine atom with other halogen atoms (compounds 8-10) led to a decrease in activity related to the size of the atom. Indeed, compound 8 bearing a fluorine atom in position 5 displayed the lowest activity while compound 10 bearing a bromine atom was slightly less active than the reference compound 1. Moreover, replacement of the iodine atom with a hydrogen atom (compound 7) was detrimental for activity (IC50 > 1 mM). Introduction of a methoxy group (compound 11) led to an inactive compound whereas introduction of hydrophobic groups like phenoxy or phenyl (compounds 12 and 13) increased activity twice. Interestingly, a naphtyl ring (compound 14) instead of the 5-iodophenyl was also tolerated. These first SAR showed that replacement of the iodine atom with an aromatic ring had a favorable impact on activity. To further enhance the potency and explore the SAR, we examined the effect of changing the carbamate function with an amide function. Introduction of aliphatic chains like methyl, tert-butyl or neopentyl was not tolerated (data not shown) while compound 20 bearing a phenyl ring displayed an IC50 of 145 µM. Interestingly, the chain length between the amide function and the phenyl ring seemed important for activity. Indeed, introduction of one, two or three methylene groups (compounds 21-23) led to a decrease in activity (IC50 > 500 µM, 295 and 186 µM, respectively) compared to compound 20, whereas a more constrained linker, like a cyclopentyl (compound 24) or a cyclohexyl (compound Finally, introduction of a pyridine ring proved detrimental for activity. Indeed, replacement of the phenyl ring (compound 20) with a pyridine (compound 35) was not tolerated (IC50 > 400 µM) and introduction of a nitrogen atom in the 4-chlorophenyl ring led to compound 36, three times less active than compound 28 (IC50 = 147 µM vs IC50 = 53 µM). These SAR showed that the carbamate function could be replaced with an amide function and a phenyl ring proved optimal for activity. This led to the identification of compound 29 bearing a 3,4dichlorophenyl ring on the amide function (IC50 = 38 ± 6 µM).

Modifications of the substituents on the phenyl ring

2.6.4. Evaluation of the interaction of the fluorinated compounds with MabA by NMR 19 F ligand-observed NMR has been described as a powerful tool in detecting weak binding of small molecules. 19 F has a wide chemical shift range, therefore even small changes in binding can result in a measurable shift in the fluorine signal. [START_REF] Jordan | Fragment based drug discovery: Practical implementation based on 19F NMR spectroscopy[END_REF][START_REF] Ribeiro | Fluorinated Carbohydrates as Lectin Ligands: 19F-Based Direct STD Monitoring for Detection of Anomeric Selectivity[END_REF] Such experiments were conducted to confirm the binding of the fluorinated inhibitors to the targeted protein MabA. Three compounds (32-34) bearing a trifluoromethyl group were selected and NMR experiments were performed using a 600 MHz spectrometer equipped with a 19 F cryogenic probe. The perturbations (broadening and chemical shift changes) of 19 F-NMR signal of the three compounds in the presence of MabA confirmed the direct binding of these inhibitors to the protein (Figure 6). Moreover, the quantification of the 19 F signal perturbations (area under the red curve) in this experiment allowed to rank the compounds according to their affinity for MabA. Remarkably, the three compounds displayed a similar ranking in the NMR experiment and in the MabA enzymatic assay. The most significant 19 F-NMR-signal decrease was observed with compound 34 and among the three inhibitors this compound showed a slightly better inhibitory activity (IC50 = 59 µM vs 69 and 84 µM).

Evaluation of physico-chemical properties and assessment of antimycobacterial activity

The physico-chemical properties of the most active compound (compound 29) were measured. This compound displayed a solubility greater than 200 µM and a logD equal to 3.40. Compound 29 was then tested on M.tb grown in Middlebrook 7H9 liquid medium (pH 6.6). Despite the 10 fold-improved inhibitory activity of compound 29 compared to compound 1 in the MabA enzymatic inhibitory assay, both compounds showed the same bacterial growth inhibition (MIC90 = 300 µM, which corresponds to 130 and 110 µg/mL respectively). The antimicrobial activity of carboxylic acids, such as benzoic acid, salicylic acid, aspirin (acetyl-salicylic acid) or mefenamic acid (an anthranilic acid derivative) have been reported in the literature. [START_REF] Zhang | Susceptibility of Mycobacterium tuberculosis to weak acids[END_REF] These weak acids displayed MIC between 33 and 111 µg/mL in culture medium at pH 6.8 and have been proposed, by Zhang et al., to act by disrupting the membrane potential of the bacteria. At this stage, our data do not allow to conclude formally that the antimicrobial activity observed for compound 29 is exclusively due to MabA inhibition. Mechanism of action similar to the one hypothesized for related carboxylic acids has also to be considered. Optimization of compound 29 is in progress and further experiments will be conducted to clarify this point.

Conclusion

A new LC-MS/MS based MabA activity assay was developed and used for the screening of a 1280fragment library. This allowed identification of the first specific inhibitors of MabA, an essential enzyme involved in M.tb mycolic acid biosynthesis. From the 6 different chemical series validated by doseresponse experiments, the anthranilic acid series was chosen for structure-activity relationships studies and we were able to define the essential molecular features that governs MabA inhibition. 19 F ligand-observed NMR experiments confirmed the binding of three fluorinated analogues of this series to MabA. The most potent inhibitor, bearing a 3,4-dichlorophenyl ring on the amide function (compound 29), inhibited MabA with an IC50 of 38 µM and bacterial growth with a MIC90 of 300 µM. Optimization in this series of compounds will be pursued in order to increase their affinity to MabA as well as their ability to inhibit M.tb growth. This optimization will help to clarify the mechanism of action of these compounds in bacteria. These results will be reported in due course. 4. Experimental section 4.1. Chemistry Solvents for synthesis, analysis and purification were purchased as analytical grade from commercial suppliers and used directly without further purification. Chemical reagents were purchased from Fisher scientific, Merck, Fluorochem, Enamine or TCI as reagent grade and used without further purification. LC-MS Waters system was equipped with a 2747 sample manager, a 2695 separations module, a 2996 photodiode array detector (200-400 nm) and a Micromass ZQ2000 detector (scan 100-800). XBridge C18 column (50 mm x 4.6 mm, 3.5µm, Waters) was used. The injection volume was 20 µL. A mixture of water and acetonitrile was used as mobile phase in gradient-elution. The pH of the mobile phase was adjusted with HCOOH and NH4OH to form a buffer solution at pH 3.8. The analysis time was 5 min (at a flow rate at 2 mL/min), 10 min (at a flow rate at 1 mL/min) or 30 min (at a flow rate at 1 mL/min). Purity (%) was determined by reversed phase HPLC, using UV detection (215 nm), and all isolated compounds showed purity greater than 95%. HRMS analysis was performed on a LC-MS system equipped with a LCT Premier XE mass spectrometer (Waters), using a XBridge C18 column (, 50 mm x 4.6 mm, 3.5µm, Waters). A gradient starting from 98% H2O 5 mM Ammonium Formate pH 3.8 and reaching 100% CH3CN 5 mM Ammonium Formate pH 3.8 within 3 min at a flow rate of 1 mL/min was used. NMR spectra were recorded on a Bruker DRX-300 spectrometer. The results were calibrated to signals from the solvent as an internal reference [e.g. 2.50 (residual DMSO-d6) and 39.52 (DMSO-d6) ppm for ¹H and ¹³C NMR spectra respectively]. Chemical shifts (δ) are in parts per million (ppm) downfield from tetramethylsilane (TMS). The assignments were made using one-dimensional (1D) 1 H and 13 C spectra and two-dimensional (2D) HSQC-DEPT, COSY and HMBC spectra. NMR coupling constants (J) are reported in Hertz (Hz), and splitting patterns are indicated as follows: s for singlet, brs for broad singlet, d for doublet, t for triplet, q for quartet, quin for quintet, dd for doublet of doublet, ddd for doublet of doublet of doublet, dt for doublet of triplet, qd for quartet of doublet, m for multiplet, δ for chemical shift, J for coupling constant. Reverse flash chromatography was performed using a CombiFlash® C18 Rf200 with C18 silica gel cartridges. UV detection (215 and 254 nm) was used to collect the desired product. Preparative HPLC was performed using a Varian PRoStar system with an OmniSphere 10 µm column C18 Dynamax (250 mm × 41.4 mm) from Agilent Technologies. A gradient starting from 20% MeCN / 80% H2O / 0.1% formic acid and reaching 100% MeCN / 0.1% formic acid at a flow rate of 80 mL/min was used. UV detection (215 and 254 nm) was used to collect the desired product. Syntheses under microwave irradiations were performed using Biotage® Initiator+. 4.1.1. General method for the synthesis of compounds 8, 9 and 10 2-amino-5-halogeno-benzoic acid (1 eq), di-tert-butyl dicarbonate (1.1 eq), DMAP (0.065 eq) and TEA (1 eq) were dissolved in MeCN (C = 0.9 M). The mixture was vortexed, heated at 40 °C under microwave irradiation for 15 min. The mixture was then filtrated through cotton. The filtrate was concentrated in vacuo and the residue was purified by preparative HPLC to give the desired product.

2-(tert-butoxycarbonylamino)-5-chloro-benzoic acid (9)

Yield: 45%. 1 H-NMR (DMSO-d6): δ (ppm) 14.0 (brs, OH), 10.5 (s, NH), 8.30 (d, J = 9.0 Hz, 1H), 7.90 (d, J = 2.6 Hz, 1H), 7.63 (dd, J = 9.0 Hz, J = 2.7 Hz, 1H), 1.48 (s, 9H). 13 C-NMR (DMSO-d6): δ (ppm) 168.9, 152. 3, 140.8, 134.3, 130.7, 125.5, 120.5, 117.5, Di-tert-butyl dicarbonate (Boc2O) (1.1 eq) was added to a solution of the appropriate amine (1 eq) dissolved in anhydrous pyridine (C = 1 M). The mixture was stirred at 90 °C for 1h30, then Boc2O (0.5 eq) was added and the mixture was stirred at 90 °C for 1h. The reaction mixture was quenched with aqueous saturated NH4Cl, the product was then extracted with EtOAc (3 times) and washed twice with aqueous saturated NH4Cl, and then once with brine. The organic layer was dried over MgSO4 and concentrated in vacuo. The residue was purified by preparative HPLC to give the desired product.

2-(tert-butoxycarbonylamino)-5-methoxy-benzoic acid (11)

Yield: 78%. 1 H-NMR (DMSO-d6): δ (ppm) 10.22 (s, NH), 8.17

2-(tert-butoxycarbonylamino)-5-phenyl-benzoic acid (13)

2-amino-5-iodo-benzoic acid (263 mg, 1.0 mmol, 1 eq), phenylboronic acid (122 mg, 1.0 mmol, 1 eq), potassium carbonate (276 mg, 2.0 mmol, 2 eq), and palladium 10% on activated charcoal (0.03 eq) were dissolved in water (6 mL). The mixture was heated at reflux for 4h and then filtrated through cotton. The filtrate was acidified with an aqueous solution of HCl (1M) until pH reached 6. The aqueous layer was then extracted with EtOAc. The organic layer was dried over MgSO4, concentrated in vacuo to give 2-amino-5-phenyl-benzoic acid (Yield: 94%). The product was used in the next step without further purification. 2-amino-5-phenyl-benzoic acid (200 mg, 0.94 mmol, 1 eq), di-tert-butyl dicarbonate (409 mg, 1.8 mmol, 2 eq), DMAP (8 mg, 0.06 mmol, 0.065 eq) and TEA (131 µL, 0.94 mmol, 1 eq) were dissolved in MeCN (3 mL). The mixture was vortexed, heated at 40 °C under microwave irradiation for 15 min. The mixture was then filtrated through cotton. The filtrate was concentrated in vacuo and the residue was purified by preparative HPLC to give 2-(tert-butoxycarbonylamino)-5phenyl-benzoic acid. Yield: 4%. 1 H-NMR (DMSO-d6): δ (ppm) 10.69 (s, NH), 8.38 (d, J = 8.9 Hz, 1H), 8.21 (d, J = 2.3 Hz, 1H), 7.89 (dd, J = 8.8 Hz, J = 2.3 Hz, 1H), 7.69 -7.62 (m, 2H), 7.51 -7.43 (m, 2H), 7.36 (tt, J = 7.4 Hz, J = 1.2 Hz, 1H), 1.50 (s, 9H). 13 C-NMR (DMSO-d6): δ (ppm) 170.0, 152. The appropriate chloroformate (2 eq) was added dropwise in a mixture of 2-amino-5-iodo-benzoic acid (0.7 mmol, 1 eq) dissolved in anhydrous pyridine (3 mL). The reaction mixture was stirred at RT for 1h. The appropriate chloroformate (0-2 eq) was then added dropwise, the reaction mixture was stirred at RT for 1 h and then evaporated. An aqueous solution of HCl (1M) was added on the residue, and then the product was extracted with EtOAc. The organic layer was washed three times with an aqueous solution of HCl (1M), dried over MgSO4 and concentrated in vacuo to give the desired product.

5-iodo-2-(isobutoxycarbonylamino)benzoic acid (15)

Yield: 62%. 1 H-NMR (DMSO-d6): δ (ppm) 13.94 (brs, OH), 10.66 (s, NH), 8.20 (d, J = 2.1 Hz, 1H), 8.07 (d, J = 8.9 Hz, 1H), 7.89 (dd, J = 8.9 Hz, J = 2.2 Hz, 1H), 3.90 (d, J = 6.7 Hz, 2H), 1.92 (septuplet, J = 6.7 Hz, 1H), 0.91 (d, J = 6.8 Hz, 6H). 13 C-NMR (DMSO-d6): δ (ppm) 168.8, 153.2, 142.8, 141.3, 139.5, 120.9, 118.2, 85.1, 71.2, 27.9, 19.3. HRMS (TOF, ES-) m/z [M-H]-calculated for C12H13NO4I 361.9889, found 361.9907.

2-(allyloxycarbonylamino)-5-iodo-benzoic acid (16)

The residue was purified by preparative HPLC. Yield: 39%.

2-(benzyloxycarbonylamino)-5-iodo-benzoic acid (17)

The residue was purified by preparative HPLC. Yield: 48%. 1 H-NMR (DMSO-d6): δ (ppm) 14.04 (brs, OH), 10.73 (s, NH), 8.21 (d, J = 2.1 Hz, 1H), 8.10 (d, J = 8.8 Hz, 1H), 7.91 (dd, J = 8.9 Hz, J = 8.9 Hz, 1H), 7.46 -7.30 (m, 5H), 5.18 (s, 2H). 13 C-NMR (DMSO-d6): δ (ppm) 168.8, 153.0, 142.9, 141.1, 139.6, 136.5, 129.0, 128.7, 121.0 (2C), 118.3, 85.4, 66.9. HRMS (TOF, ES-) m/z [M-H]-calculated for C15H11NO4I 395.9733, found 395.9752.

2-(tert-butoxycarbonylamino)-5-iodo-benzoic acid (18)

To a solution of 2-(tert-butoxycarbonylamino)-5-iodo-benzoic acid (50 mg, 0.14 mmol, 1 eq) in anhydrous DMF (1 mL) was added cesium carbonate (157 mg, 0.48 mmol, 3.5 eq) followed by methyl iodide (20 µL, 0.32 mmol, 2.3 eq). The mixture was stirred at RT for 24h. LCMS analysis showed that the conversion was not complete. 150 mg of cesium carbonate (3.5 eq) and 20 µL of methyl iodide (2.3 eq) were added to the reaction mixture. The solution was stirred 24h at RT then filtered. The filtrate was poured in a tube and diluted in MeOH (4 mL). NaOH (40 mg, 1 mmol, 7.3 eq) and three drops of water were added and the mixture was stirred over the week-end at RT then evaporated. The residue was dissolved in water and the aqueous phase was washed twice with EtOAc. The aqueous phase was acidified to pH 4 with HCl then the product was extracted three times with EtOAc. The organic layers were assembled, washed once with brine, and then evaporated under reduced pressure to give 2-(tertbutoxycarbonylamino)-5-iodo-benzoic acid. Yield: 69%. 1 H-NMR (DMSO-d6): δ (ppm) 13.21 (brs, OH), 8.03 (d, J = 1.8 Hz, 1H), 7.88 (dd, J = 8.4 Hz, J = 1.8 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 3.09 (s, 3H), 1.42 (s, 2H), 1.25 (s, 7H). 13 C-NMR (DMSO-d6): δ (ppm) 166.6, 153.5, 143.0, 141.5, 138.8, 132.2, 130.7, 91.8, 80.0, 37.6, 28.5, 28.1. HRMS (TOF, ES-) m/z [M-H]-calculated for C13H15NO4I 376.0046, found 376.0070.

Methyl 2-(tert-butoxycarbonylamino)-5-iodo-benzoate (19)

2-(tert-butoxycarbonylamino)-5-iodo-benzoic acid (182 mg, 0.5 mmol, 1 eq), dimethylsulfate (95 µL, 1 mmol, 2 eq) and dipotassium carbonate (138 mg, 1 mmol, 2 eq) were dissolved in MeCN (2 mL). The mixture was stirred at RT for 2h. The solvent was evaporated in vacuo and the residue was solubilized in EtOAc. The organic layer was washed eight times with water and once with brine. The organic layer was dried over MgSO4 and concentrated in vacuo to give methyl 2-(tert-butoxycarbonylamino)-5-iodobenzoate. Yield: 28%. 1 H-NMR (DMSO-d6): δ (ppm) 10.03 (s, NH), 8.15 (d, J = 2.0 Hz, 1H), 7.97 (d, J = 8.8 Hz, 1H), 7.90 (dd, J = 8.8 Hz, J = 2.1 Hz, 1H), 3.84 (s, 3H), 1.47 (s, 9H). 13 C-NMR (DMSO-d6): δ (ppm) 167.0, 152.3, 142.8; 140.8, 138.9, 121.6, 118.4, 85.3, 81.0, 53.2, 28.3. HRMS (TOF, ES-) not determined (non ionizable compound). 4.1.7. General method for the synthesis of compounds 20, 22 and 26

Step 1. SOCl2 (1.1 mmol, 1.1 eq), 1H-benzotriazole (3.1 mmol, 3.1 eq) were mixed in DCM (2 mL) and then added dropwise to a solution of carboxylic acid (1 mmol, 1 eq) in DCM (2 mL). The reaction mixture was stirred overnight at RT and a precipitate appeared. The precipitate was filtered off and the filtrate was evaporated under reduced pressure. The product was used in the next without further purification.

Step 2. Benzotriazole derivative previously formed (0.5 mmol, 1 eq), 2-amino-5-iodo-benzoic acid (0.5 mmol, 1 eq) and DMAP (0.55 mmol, 1.1 eq) were dissolved in CHCl3 (1 mL). The mixture was vortexed, heated at 80 °C under microwave irradiations for 30 min. The reaction mixture was diluted in EtOAc then washed twice with aqueous HCl (C = 1 M) and then once with brine. The organic layer was dried over MgSO4 and concentrated in vacuo. 2-phenylacetyl chloride (185 µL, 1.4 mmol, 2 eq) was added dropwise in a mixture of 2-amino-5-iodobenzoic acid (184 mg, 0.7 mmol, 1 eq) dissolved in anhydrous pyridine (3 mL). The mixture was stirred at RT for 1h, and then 2-phenylacetyl chloride (46 µL, 0.35 mmol, 0.5 eq) was added dropwise. The reaction mixture was stirred at RT for 1h, then quenched with an aqueous solution of HCl (1M). The product was extracted with EtOAc and washed three times with an aqueous solution of HCl (1M). The organic layer was dried over MgSO4 and concentrated in vacuo. The residue was purified by preparative HPLC to give 5-iodo-2-[(2-phenylacetyl)amino]benzoic acid. Yield: 64%. 4-phenylbutanoic acid (690 mg, 4.2 mmol, 3 eq) was dissolved in EtOAc dried over molecular sieves (15 mL), with PyBrop (1.96 g, 4.2 mmol, 3 eq) and DIEA (1.46 mL, 8.4 mmol, 6 eq). The solution was added to the HOBt resin (1-Hydroxybenzotriazole-6-sulfonamidomethyl polymer-bound, 1.4 mmol, 1 eq) and the resin was shaked for 3h at RT. After the first activation step the resin was washed 3 times with DMF. The second activation step was performed under the same conditions and the resin was washed 5 times with DMF then twice with DCM and dried. The polymer-bound activated ester (146 mg, 0.25 mmol, 1.25 eq) and 2-amino-5-iodo-benzoic acid (52 mg, 0.2 mmol, 1 eq) were dissolved in DMF dried over molecular sieves (1 mL). The mixture was stirred overnight at RT. The conversion was around 70%. The supernatant was separated from the resin by filtration. The polymeric beads were washed 3 times with DMF, the washing solutions were recovered and combined with the supernatant previously recovered. The solvent was evaporated in vacuo. The residue was purified by preparative HPLC to give 5-iodo-2-(4-phenylbutanoylamino)benzoic acid. Yield: 46%. Step 1. A 1 M solution of isopropyl chloroformate in toluene (1.5 eq) was added in anhydrous THF under argon. The appropriate carboxylic acid (1 eq) was dissolved in anhydrous THF (C = 1.0 M) with TEA (1.2 eq). This solution was added dropwise to the isopropyl chloroformate solution at 0 °C. The mixture was allowed to warm to RT, stirred overnight and then evaporated under reduced pressure. NaHCO3 sat. was added and the product was extracted twice with EtOAc. The organic layer was washed once with NaHCO3 sat., once with brine, dried over MgSO4 and then evaporated under reduced pressure. The yield is supposed to be quantitative, and the product was used in the next step without further purification.

Step 2. 2-amino-5-iodo-benzoic acid (1 eq) was dissolved in water (C = 0.5 M) with sodium carbonate (2 eq). This solution was cooled to 0 °C. The previously formed anhydride (1.5 eq) dissolved in THF (C = 0.8 M) was added dropwise at 0 °C to this solution. The mixture was stirred at RT for 3 h or overnight. The solvent was then evaporated under reduced pressure. The residue was purified by reverse flash chromatography (H2O/MeOH 90/10 to 0/100 over 25 min) to give the desired product.

2-(indane-2-carbonylamino)-5-iodo-benzoic acid (24)

Yield: 49%. Plates were incubated for 10 or 30 minutes at 23 °C. The enzymatic reaction was started by adding 10 µL of a mixture of the substrate AcAcCoA and the cofactor NADPH (100 µM in Hepes buffer) using a Bravo Automated Liquid Handler. Plates were centrifuged 30 sec and then incubated for 15 minutes at 23 °C. The final concentrations were as follows: compound at 1 mM, MabA at 400 nM, AcAcCoA at 50 µM, NADPH at 50 µM, DMSO 1%. INH-NADP was used as a reference inhibitor (100% inhibition at 200 µM). The enzymatic reaction was stopped by adding 10 µL of a solution of trifluoroacetic acid in water at a final concentration of 1%, containing BCoA at a final concentration of 5 µM. Plates were sealed with a film and stored at -80 °C. Before analysis by LC-MS/MS, plates were centrifuged 4 min at 4000 rpm. Inhibition percentage of each compound was calculated using the mean values of the negative (1% DMSO) and positive controls (INH-NADP adduct at 200 µM). Z′-factors were calculated using the mean and standard deviation values (SD) of the negative and positive controls. [START_REF] Zhang | A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays[END_REF] All the screening plates displayed a Z′-factor between 0.72 and 0.83.

 

                C C C Mean Mean Mean x Inh 100 100 %         C C C C Mean Mean SD SD Z ) ( 3 1 ' 4.2.

LC-MS/MS analysis

AcAcCoA, HBCoA and BCoA were detected by multiple-reaction monitoring (MRM) using a UPLC Acquity I-class coupled with a Xevo TQD mass spectrometer (Waters). Microplates were stored at 10 °C before analysis. AcAcCoA, HBCoA and BCoA were separated using an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm, Waters) at 40 °C and a flow rate of 0.6 mL/min. Mobile phase A: ammonium acetate 10 mM in water mQ. Mobile phase B: MeOH LC-MS grade. The gradient started at 2% B, increased linearly to 14% B in 1'20 and then to 98% B in 5 sec, held isocratic at 98% B for 30 sec, and returned to initial conditions. The column was equilibrated with initial conditions for 1.0 min before each injection. Source parameters were set as follows: polarity ES+, capillary 1200 V, desolvation temperature 600 °C, cone voltage 46 V, source temperature 150 °C, cone gaz flow 50 L/h, desolvation gaz flow 1200 L/h. Transitions monitored were as follows: AcAcCoA 852.1 -342.5 (collision energy: 38 eV), HBCoA 854.1 -347.2 (collision energy: 36 eV), BCoA 838.1 -331.2 (collision energy: 34 eV). MassLynx software was used for mass spectrometry data acquisition and TargetLynx software was used for analysis (Waters). The relative amount of HBCoA in each well was determined by dividing the peak area of HBCoA by the peak area of BCoA used as an internal standard.

Preparation of INH-NADP adduct

The reference inhibitor used in this assay is the adduct of isoniazid (INH) with NADP + which can be formed in situ using a solution of [Mn III (H2P2O7)3]Na3 (Mn3PyrPh) at 5.5 mM prepared as described in the literature. [START_REF] Nguyen | A Fast and Efficient Metal-Mediated Oxidation of Isoniazid and Identification of Isoniazid-NAD(H) Adducts[END_REF] A solution of INH-NADP adduct (INH 2 mM, NADP + 2 mM, Mn3PyrPh 4 mM) was prepared by mixing 200 µL of INH (20 mM in Hepes buffer), 200 µL of NADP + (20 mM in Hepes buffer), 1450 µL of Mn3PyrPh (5.5 mM) and 150 µL of Hepes buffer. The solution was incubated at 22 °C for at least 20 min. 4.2.2.4. Dose-response experiments Inhibition percentages of compounds at different concentrations were obtained using the protocol described for screening. Compounds at 100 mM in DMSO were transferred into black 384-well lowbinding plates (Corning 3575) using an Echo 550 liquid Handler (Labcyte). Compounds were tested at 8 concentrations using 3-fold serial dilutions from 1 mM to 0.46 µM. DMSO volume was compensated so that the concentrations across all wells were equal to 1%. INH-NADP was used as a reference inhibitor (100% inhibition at 200 µM). 3.8 µL of Hepes buffer (100 mM pH 7) and 6 µL of MabA (1.33 µM in Hepes buffer) were added manually using a 16-channel VIAFLO II electronic pipette (Integra Biosciences). Compounds were incubated with MabA for 30 minutes at 22 °C before the enzymatic reaction was started by adding manually 10 µL of a mixture of the substrate AcAcCoA and the cofactor NADPH (100 µM in Hepes buffer). The final concentrations were as follows: MabA at 400 nM, AcAcCoA at 50 µM, NADPH at 50 µM. Plates were incubated for 15 minutes at 22 °C. The enzymatic reaction was stopped by adding manually 10 µL of a solution of trifluoroacetic acid in water at a final concentration of 1%, containing BCoA at a final concentration of 5 µM. Dose-response curves were analysed with GraphPad Prism using a nonlinear regression analysis. Reported IC50 values are the average of at least two experiments. 4.2.2.5. Competition experiment Inhibition percentages of compound 1 and INH-NADP adduct were obtained using the protocol described for dose-response experiments and two different NADPH concentrations (50 and 500 µM). Compound 1 was tested at 8 concentrations using 3-fold serial dilutions from 1 mM to 0.46 µM. INH-NADP adduct was tested at 8 concentrations using 3-fold serial dilutions from 200 µM to 0.09 µM.

19 F-NMR assay

The 19 F NMR measurements were done at 298 K using 3 mm tubes (160 μL samples) on a Bruker 600 MHz Avance III HD spectrometer equipped with a CP-QCI-F cryoprobe with optimized 19 Fluorine detection. The 19 F NMR reference spectra were acquired on 160 µL samples containing a compound (32, 33 or 34) at 100 μM, in the NMR buffer [50 mM Tris-Cl pH 7.5; 250 mM NaCl; 1% DMSO-d6; 5% D2O]. Then, 19 F NMR spectra of the compounds were acquired in the presence of MabA at 20 µM in the same NMR buffer. Experiments have been acquired with the following parameters: TD = 8192 points; NS = 512 scans; relaxation delay D1 = 2 seconds; carrier frequency O1P = -65.0 ppm; spectral window sw = 20 ppm; and with 19 F decoupling. The acquisition time was about 22 minutes per sample. NMR data have been processed with Bruker Topspin 4.06.

M. tuberculosis assay

Compounds dissolved in DMSO at 100 mM were transferred to a 384-well low-volume polypropylene plate (Corning, no. 3672) and used to prepare assay plates. Compounds were tested from 300 µM to 15 nM. Ten 3-fold serial dilutions of compounds were performed into black Greiner 384-well clear bottom polystyrene plates (Greiner, no. 781091) using an Echo 550 liquid Handler (Labcyte). DMSO volume was compensated so that the concentrations across all wells were equal to 0.3%. Two independent replicates were made for each setting. On the day of the experiment, 50 µL of a culture of M. tuberculosis-GFP H37Rv (grown in 7H9, with 0.2% glycerol and 0.05% Tween80 and then diluted to an OD600nm = 0.02) was transferred to each assay plate and incubated at 37 °C for 5 days. The fluorescence intensity was measured at Ex/Em = 485/535 nm using a Victor Multilabel Plate Reader (PerkinElmer). The following reference compounds were used as positive control in this assay: rifampicine (MIC = 4 ng/mL), isoniazid (MIC = 125 ng/mL) and ethionamide (MIC = 2 µg/mL).

Physico-chemical properties

These experiments were analyzed using a UPLC Acquity I-class coupled with a Xevo TQD mass spectrometer (Waters) under MRM detection. Source parameters were set as follows: polarity ES-, capillary 0.5 kV, desolvation temperature 600 °C, cone voltage 42 V, source temperature 150 °C, cone gaz flow 50 L/h, desolvation gaz flow 1200 L/h. Transitions monitored were as follows: 433.8 -389.9 (collision energy 20 eV). 4.3.1. LogD 5μL of a 10 mM solution of compound in DMSO was diluted in 245 µL of a 1/1 octanol/PBS mixture at pH 7.4. The mixture was gently shaken for 2 h at room temperature. 10 µL of each phase were diluted in 490 μL of MeOH and analyzed by LC-MS/MS. Each compound was tested in triplicate. Log D was determined as the logarithm of the ratio of concentration of product in octanol and PBS, determined by mass signals.

Figure 1 .

 1 Figure 1. A. Reduction of AcAcCoA into HBCoA and oxidation of NADPH into NADP + catalyzed by MabA. B. Progress curve of HBCoA formation as a function of enzymatic reaction time.

Figure 2 .

 2 Figure 2. A. Structure of the INH-NADP adduct. B. Mass chromatogram obtained after 15 min of enzymatic reaction without inhibitor. C. Mass chromatogram obtained after 15 min of enzymatic reaction in the presence of INH-NADP adduct at 200 µM. D. MabA inhibition with increasing doses of INH-NADP (curve was obtained using GraphPad Prism).

Figure 3 .

 3 Figure 3. A. Screening cascade. B. Screening result of fragments incubated 30 min with MabA before the initiation of the enzymatic reaction. Black crosses: percentage of inhibition of each fragment. Green: median values and standard deviations of the positive control (INH-NADP adduct at 200 µM). Red: median values and standard deviations of the negative control (1% DMSO). Blue: threshold at 30% of inhibition.

Figure 4 .

 4 Figure 4. Structures and DRCs of one hit in each chemical series identified during the screening.

Figure 5 .

 5 Figure 5. DRC of INH-NADP adduct (A) and compound 1 (B) with 50 and 500 µM of NADPH.

Scheme 1 .

 1 Scheme 1. Synthesis of Boc-2-aminobenzoic acid 8-14. Reagents and conditions: (a) PhB(OH)2 (1 eq.), K2CO3 (2 eq), Pd/C (0.03 eq), H2O, reflux, 4h. (b) Boc2O (1.1 eq), DMAP (0.065 eq), TEA (1 eq), MeCN, µW 40 °C, 15 min or Boc2O (1.6 eq), pyridine, 90 °C, 2h30.

Scheme 2 .

 2 Scheme 2. Synthesis of compounds 15-19. Reagents and conditions: (a) 2-amino-5-iodobenzoic acid (1 eq), chloroformate (2 eq), anh. pyridine, RT, 1-2h; (b) iodomethane (4.6 eq), Cs2CO3 (7 eq), anh. DMF, RT, 48 h; (c) NaOH (7 eq), MeOH, H2O, RT, 2 days; (d) dimethylsulfate (2 eq), K2CO3 (2 eq), MeCN, RT, 2h.

Scheme 3 .Scheme 4 .

 34 Scheme 3. Synthesis of compounds 21, 27 and 35 from acyl chlorides. Reagents and conditions: (a) 2-amino-5-iodo-benzoic acid (1 eq), 2-phenylacetyl chloride (2.5 eq), anh. pyridine, RT, 2h; (b) methyl 2-amino-5-iodo-benzoate (1 eq), acyl chloride (1.1 eq), anh. pyridine, RT, 2h. (c) LiOH (3 eq), H2O, THF, RT, 1h.

  ), restored activity. Replacement of the phenyl ring with an aliphatic ring like a cyclopropyl (compound 26) proved detrimental (IC50 > 500 µM). Compound 20 bearing a phenyl group directly connected to the amide function displaying the best activity, the phenyl ring was then substituted in different positions. Introduction of a chlorine atom in meta or para position (compounds 27 and 28) was favorable for activity (IC50 = 70 µM and 53, respectively) whereas introduction of electron donating or withdrawing groups like a methyl, a trifluoromethyl or a trifluoromethoxy group (compounds 31-33) slightly decreased activity. Keeping the chlorine atom in para position, we introduced an unsaturated chain between the amide function and the phenyl ring that led to a modest increase of activity (compound 37 IC50 = 40 µM). Introduction of an additional chlorine atom in position 2 of the phenyl ring (compound 30 IC50 = 126 µM) led to a compound three times less active than compound 29 bearing a second chlorine atom in position 3 (IC50 = 38 ± 6 µM), suggesting that position 2 may be sensitive to steric hindrance. Replacement of the chlorine atom in position 3 of compound 29 with a trifluoromethyl group (compound 34) led to a compound with a similar activity.

Figure 6 .

 6 Figure 6. 1D 19 F NMR spectra of compounds 32-34 at 100 µM alone (blue signal) and in the presence of MabA at 20 µM (red signal).

4 . 1 . 1 . 1 . 2 -

 41112 (tert-butoxycarbonylamino)-5-fluoro-benzoic acid (8) Yield: 45%. 1 H-NMR (DMSO-d6): δ (ppm) 13.93 (brs, OH), 10.33 (s, NH), 8.27 (dd, J = 9.3 Hz, J = 5.1 Hz, 1H), 7.66 (dd, J = 9.4 Hz, J = 3.1 Hz, 1H), 7.46 (ddd, J = 9.3 Hz, J = 7.9, Hz, J = 3.1 Hz, 1H), 1.48 (s, 9H).

13 C

 13 -NMR (DMSO-d6): δ (ppm) 168.9 (d, J = 2.4 Hz), 156.5 (d, J = 239.4 Hz), 152.6, 138.4 (d, J = 2.1 Hz), 121.6 (d, J = 21.7 Hz), 120.7 (d, J = 7.1 Hz), 117.4, 117.2 (d, J = 23.8 Hz), 28.4. HRMS (TOF, ES-) m/z [M-H]-calculated for C12H13NO4F 254.0829, found 254.0835.

  81.0, 28.3. HRMS (TOF, ES-) m/z [M-H]-calculated for C12H13NO4Cl 270.0533, found 270.0553. 4.1.1.3. 5-bromo-2-(tert-butoxycarbonylamino)benzoic acid (10) Yield: 28%. 1 H-NMR (DMSO-d6): δ (ppm) 13.99 (brs, OH), 10.45 (s, NH), 8.26 (d, J = 9.0 Hz, 1H), 8.02 (d, J = 2.5 Hz, 1H), 7.75 (dd, J = 9.0 Hz, J = 2.5 Hz, 1H), 1.48 (s, 9H). 13 C-NMR (DMSO-d6): δ (ppm) 168.8, 152.3, 141.2, 137.1, 133.6, 120.7, 117.7, 113.2, 81.0, 28.3. HRMS (TOF, ES-) m/z [M-H]-calculated for C12H13NO4Br 314.0028, found 314.0040. 4.1.2. General method for the synthesis of compounds 11, 12 and 14

  (d, J = 9.0 Hz, 1H), 7.43 (d, J = 3.0 Hz, 1H), 7.19 (dd, J = 9.1 Hz, J = 3.1 Hz, 1H), 3.75 (s, 3H), 1.47 (s, 9H). 13 C-NMR (DMSO-d6): δ (ppm) 169.7, 153.8, 152.7, 135.3, 121.0, 120.4, 117.2, 115.1, 80.2, 55.8, 28.4. HRMS (TOF, ES-) m/z [M-H]-calculated for C13H16NO5 266.1028, found 266.1038. 4.1.2.2. 2-(tert-butoxycarbonylamino)-5-phenoxy-benzoic acid (12) Yield: 36%. 1 H-NMR (DMSO-d6): δ (ppm): 13.71 (brs, OH), 10.34 (s, NH), 8.29 (d, J = 9.2 Hz, 1H), 7.50 (d, J = 3.0 Hz, 1H), 7.39 (tt, J = 8.58 Hz, J = 7.4 Hz, J = 2.1 Hz, 2H), 7.32 (dd, J = 9.1 Hz, J = 3.0 Hz, 1H), 7.14 (tt, J = 7.38, 1.1 Hz, 1H), 7.00 (dd, J = 8.58, 1.0 Hz, 2H), 1.48 (s, 9H). 13 C-NMR (DMSO-d6): δ (ppm): 168.8, 156.9, 152.1, 150.1, 137.4, 130.1, 125.3, 123.4, 120.6, 120.2, 118.2, 116.8, 80.1, 27.9. HRMS (TOF, ES-) m/z [M-H]-calculated for C18H18NO5 328.1185, found 328.1197. 4.1.2.3. 3-(tert-butoxycarbonylamino)naphthalene-2-carboxylic acid (14) Yield: 42%. 1 H-NMR (DMSO-d6): δ (ppm) 13.83 (brs, OH), 10.51 (s, NH), 8.68 (d, J = 5.7 Hz, 2H), 8.01 (d, J = 7.6 Hz, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.59 (dd, J = 7.0 Hz, J = 1.2 Hz, 1H), 7.44 (dd, J = 7.0 Hz, J = 1.2 Hz, 1H), 1.52 (s, 9H). 13 C-NMR (DMSO-d6): δ (ppm) 169.6, 152.2, 136.7, 135.8, 133.3, 129.3, 129.1, 127.5, 126.8, 125.1, 116.5, 114.3, 80.0, 28.0. HRMS (TOF, ES-) m/z [M-H]-calculated for C16 H16 N O4 286.1079, found 286.1104.

  1 H-NMR (DMSO-d6): δ (ppm) 10.71 (s, NH), 8.21 (d, J = 2.1 Hz, 1H), 8.07 (d, J = 8.8 Hz, 1H), 7.91 (dd, J = 8.9 Hz, J = 2.2 Hz, 1H), 6.07 -5.89 (m, 1H), 5.35 (dq, J = 17.3 Hz, J = 1.7 Hz, 1H), 5.25 (dq, J = 10.3 Hz, J = 1.7 Hz, 1H), 4.63 (dt, J = 5.5 Hz, J = 1.4 Hz, 2H). 13 C-NMR (DMSO-d6): δ (ppm) 168.8, 152.8, 142.9, 141.1, 139.6, 133.2, 121.0, 118.6, 118.4, 85.4, 65.9. HRMS (TOF, ES-) m/z [M-H]-calculated for C11H9NO4I 345.9576, found 345.9597.

4 . 1 . 7 . 1 . 2 -

 41712 benzamido-5-iodo-benzoic acid (20)The residue was precipitated in a mixture of MeCN and MeOH (1:1), then filtered and washed with MeCN. Yield: 58%. 1 H-NMR (DMSO-d6): δ (ppm) 14.10 (brs, OH), 12.09 (s, NH), 8.52 (d, J = 8.8 Hz, 1H), 8.30 (d, J = 2.A Hz, 1H), 8.00 -7.88 (m, 3H), 7.71 -7.51 (m, 3H). 13 C-NMR (DMSO-d6): δ (ppm) 169.1, 165.2, 142.9, 141.1, 139.6, 134.7, 132.8, 129.5, 127.5, 122.5, 112.3, 86.7. HRMS (TOF, ES-) m/z [M-H]calculated for C14H9NO3I 365.9627, found 365.9641. 4.1.7.2. 5-iodo-2-(3-phenylpropanoylamino)benzoic acid (22) The residue was precipitated in a mixture of MeCN and MeOH (1:1), then filtered and washed with MeCN. Yield: 33%. 1 H-NMR (DMSO-d6): δ (ppm) 13.94 (s, 1H), 11.04 (s, 1H), 8.28 (d, J = 8.8 Hz, 1H), 8.21 (d, J = 2.2 Hz, 1H), 7.90 (dd, J = 8.8 Hz, J = 2.2 Hz, 1H), 7.34 -7.12 (m, 5H), 2.93 (t, J = 7.5 Hz, 2H), 2.71 (t, J = 7.5 Hz, 2H). 13 C-NMR (DMSO-d6): δ (ppm) 171.0, 168.6, 142.7, 141.1, 140.7, 139.4, 128.8, 128.7, 126.5, 122.7, 119.2, 86.2, 30.9. HRMS (TOF, ES-) m/z [M-H]-calculated for C16H13NO3I 393.9940, found 393.9962.4.1.7.3. 2-(cyclopropanecarbonylamino)-5-iodo-benzoic acid (26)The product was purified by reverse flash chromatography (MeOH/H2O 20/80 to 80/20). Yield: 61%.

1 H

 1 -NMR (DMSO-d6): δ (ppm) 11.28 (s, 1H), 8.26 (d, J = 8.9 Hz, 1H), 8.21 (d, J = 2.2 Hz, 1H), 7.87 (dd, J = 8.8, 2.3 Hz, 1H), 1.71 (quin, J = 6.1 Hz, 1H), 0,85 (d, J = 6.0 Hz, 4H).13 C-NMR (DMSO-d6): δ (ppm) 171.8, 168.2, 142.1, 140.4, 138.9, 122.3, 118.6, 85.6, 16.0, 7.9. HRMS (TOF, ES-) m/z [M-H]-calculated for C11H9NO3I 329.9627, found 329.9652. 4.1.8. 5-iodo-2-[(2-phenylacetyl)amino]benzoic acid (21)

  1 H-NMR (DMSO-d6): δ (ppm) 13.90 (brs, OH), 11.08 (S, NH), 8.31 (d, J = 8.8 Hz, 1H), 8.19 (d, J = 2.2 Hz, 1H), 7.90 (dd, J = 8.8 Hz, J = 2.2 Hz, 1H), 7.41 -7.23 (m, 5H), 3.76 (s, 2H). 13 C-NMR (DMSO-d6): δ (ppm) 170.1, 168.5, 142.7, 140.8, 139.4, 135.1, 130.0, 129.1, 127.5, 122.5, 119.2, 86.4, 45.0. HRMS (TOF, ES-) m/z [M-H]calculated for C15H11NO3I 379.9784, found 379.9794. 4.1.9. 5-iodo-2-(4-phenylbutanoylamino)benzoic acid (23)

  1 H-NMR (DMSO-d6): δ (ppm)13.91 (brs, OH), 11.06 (s, NH), 8.30 (d, J = 8.8 Hz, 1H), 8.21 (d, J = 2.2 Hz, 1H), 7.90 (dd, J = 8.8 Hz, J = 2.2 Hz, 1H), 7.34 -7.14 (m, 5H), 2.63 (t, J = 7.5 Hz, 2H), 2.40 (t, J = 7.5 Hz, 2H), 1.92 (quin, J = 7.5 Hz, 2H).13 C-NMR (DMSO-d6): δ (ppm) 171.6, 168.6, 142.6, 142.0, 140.8, 139.4, 128.8, 128.8, 126.3, 122.7, 119.4, 86.2, 37.3, 34.9, 27.0. HRMS (TOF, ES-) m/z [M-H]-calculated for C17H15NO3I 408.0097, found 408.0111. 4.1.10. General method for the synthesis of compounds 24, 25, 28-34, 36 and 37

  4.3.2. Solubility 5 µL of a 10 mM of compound solution in DMSO was diluted either in 245 μL of PBS pH 7.4 or in 245 µL of MeOH in triplicate. The tubes were gently shaken 24 h at room temperature, then centrifuged for 5 min at 4000 rpm. The mixtures were filtered over 0.45 μm filters (Millex-LH Millipore). 10 µL of each solution were diluted in 490 µL of MeOH and analyzed by LC-MS/MS. The solubility was determined by the ratio of mass signal areas PBS/MeOH.

% inhibition 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279 1280

					fragments
	1280 fragments		100	positive control (INH-NADP)	negative control (without inhibitor)	threshold
	> 30% inh at 1 mM
	50 fragments IC 50 < 300 µM 12 fragments	% inhibition MabA	50 30
	+ 68 analogues		0
	IC 50 < 300 µM
	6 chemical series
			-50

Table 1 . Biological activities of compounds

 1

		R	5	1
		1
			4	2
		R
		2	3
	Cpd	R1		R2	% inh a	IC50 (µM)
	1	I		H	100	430 ± 26
	7	H		H	31	> 1000
	8	F		H	38	470
	9	Cl		H	77	> 500
	10	Br		H	95	470
	11	OCH3	H	31	> 1000
	12	OPh		H	100	243
	13	Ph		H	100	194
	14		5		100	310
			4

1, 7-14. a Percentage of inhibition (compounds tested at 1 mM)

Table 2 . Biological activities of compounds 1, 15-19. a Percentage of inhibition (compounds tested at 1 mM)

 2 2.6.2. Modifications of the carbamate and the carboxylic acid

	Cpd	R	R'	R"	% inh a IC50 (µM)
	1	tBu	H	H	100	430 ± 26
	15	iBu	H	H	100	310
	16	allyl	H	H	83	> 500
	17	CH2-Ph	H	H	100	92 ± 3.5
	18	tBu	CH3	H	50	~ 1000
	19	tBu	H	CH3	0	> 1000

In the carbamate series, replacement of the tertbutyl group with an isobutyl group (compound 15) led to a compound with a similar activity (IC50 = 310 µM). Replacement with an allyl group (compound 16) led to a less active compound (IC50 > 500 µM) whereas introduction of a benzyl group (compound 17) provided a 4-fold increase in potency (IC50 = 92 µM). Methylation of the carbamate (compound 18) or replacement of the carboxylic acid with a methyl ester (compound 19) was detrimental for activity (IC50 > 1000 µM).

Table 3 . Biological activities of compounds 20-36.

 3

  5, 141.2, 139.3, 133.5, 132.6, 129.5, 129.4, 127.8, 126.7, 119.1, 80.7, 28.4. HRMS (TOF, ES-) m/z [M-H]-calculated for C18H18NO4 312.1236, found 312.1241. 4.1.4. General method for the synthesis of compounds 15, 16 and 17

  1 H-NMR (DMSO-d6): δ (ppm) 14.50 (s, 1H), 8.30 (d, J = 8.7 Hz, 1H), 8.27 (d, J = 2.3 Hz, 1H), 7.56 (dd, J = 8.7 Hz, J = 2.40 Hz, 1H), 7.25 -7.17 (m, 2H), 7.16 -7.10 (m, 2H) 3.3 (t, J = 7.4 Hz, 1H), 3.17 (d, J = 7.5 Hz, 4H).13 C-NMR (DMSO-d6): δ (ppm) 173.0, 168.8, 142.3, 141.0, 140.0, 138.6, 127.7, 126.8, was purified on Ni 2+ -affinity column (His-trap, GE Healthcare), eluted in lysis buffer supplemented with 250 mM imidazole and dialysed against Tris 50 mM pH7.5, NaCl 300 mM. Glycerol at 10% was added to the protein for storage at -80 °C. 4.2.2. MabA enzymatic assay NADPH, AcAcCoA and BCoA were purchased from Sigma Aldrich. 4.2.2.1. Screening assay Screening was performed on EquipEx Imaginex Biomed platform at the Pasteur Institute of Lille. 200 nL of each compound at 100 mM in DMSO were transferred into black 384-well low-binding plates (Corning 3575) using an Echo 550 liquid Handler (Labcyte). 3.8 µL of Hepes buffer (100 mM pH 7) and 6 µL of MabA (1.33 µM in Hepes buffer) were added using a Bravo Automated Liquid Handler (Agilent).

Acknowledgements

We thank Eva Bannerman for technical assistance. This work was supported by Feder (12001407 (D-AL) Equipex Imaginex BioMed), l'Agence Nationale de la Recherche (ANR) France (2FightTB, ANR-13-JSV5-0010-01), Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Université de Lille, Institut Pasteur de Lille, Région Hauts-de-France. The NMR facilities were funded by the Région Hauts-de-France, CNRS, Institut Pasteur de Lille, European Community (FEDER), Ministère de l'Enseignement supérieur, de la Recherche et de l'Innovation (MESRI) and Lille University. René Wintjens is a Research Associate at the Belgian National Fund for Scientific Research (FRS-FNRS).

Abbreviations

AcAcCoA, acetoacetyl-CoA; ACP, acyl carrier protein; anh, anhydrous; BCoa, butyryl-CoA; FAS, fattyacid synthase; Boc2O, di-tert-butyldicarbonate; DCM, dichloromethane; DMAP, N,N-Dimethylpyridin-4-amine; DMF, dimethylformamide; DMSO, dimethylsulfoxide; DRC, dose-response curve; EtOAc, ethyl acetate; HBCoA, hydroxybutyryl-CoA; INH, isoniazid; MeCN, acetonitrile; MDR, multidrug-resistant; MeOH, methanol; M.tb, Mycobacterium tuberculosis; RT, room temperature; SAR, structureactivity relationships; TB, tuberculosis; TEA, triethylamine.