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

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Discovery of the first Mycobacterium tuberculosis MabA (FabG1) inhibitors through a

fragment-based screening

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

PII:

S0223-5234(20)30411-6

DOI:

https://doi.org/10.1016/j.ejmech.2020.112440

EJMECH 112440

Reference:

To appear in: European Journal of Medicinal Chemistry

Received Date: 4 March 2020

Revised Date: 5 May 2020

Accepted Date: 6 May 2020

Please cite this article as: Lé. Faion, K. Djaout, R. Frita, C. Pintiala, F.-X. Cantrelle, M. Moune, A.

Vandeputte, K. Bourbiaux, C. Piveteau, A. Herledan, A. Biela, F. Leroux, L. Kremer, M. Blaise, A.

Tanina, René. Wintjens, X. Hanoulle, B. Déprez, N. Willand, A.R. Baulard, M. Flipo, Discovery of the first

Mycobacterium tuberculosis MabA (FabG1) inhibitors through a fragment-based screening, European

Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112440.

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12₁8₂0₈₀f_fr_ra_agg_mm_ene_tsnts

Stage 1

LCMSMS

Screening

on MabA

SAR

Compound 1

IC₅₀(MabA) = 430 µM

Compound 29

IC₅₀(MabA) = 38 µM

Solubility > 200 µM

logD = 3.4

Discovery of the first Mycobacterium tuberculosis MabA (FabG1) inhibitors through a Fragment-

based screening

Léo Faion^1#, Kamel Djaout^2#, Rosangela Frita², Catalin Pintiala¹, Francois-Xavier Cantrelle^3,4, Martin

Moune², Alexandre Vandeputte², Kevin Bourbiaux¹, Catherine Piveteau¹, Adrien Herledan¹, Alexandre

Biela¹, Florence Leroux¹, Laurent Kremer^5,6, Mickael Blaise⁵, Abdalkarim Tanina⁷, René Wintjens⁷,

Xavier Hanoulle^3,4, Benoit Déprez¹, Nicolas Willand¹, Alain R. Baulard², Marion Flipo*^,1

# These authors contributed equally to this work

*marion.flipo@univ-lille.fr

1. Univ. Lille, Inserm, Institut Pasteur de Lille, U1177 - Drugs and Molecules for living Systems,

F-59000 Lille, France

2. Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 9017 - CIIL - Center

for Infection and Immunity of Lille, F-59000 Lille, France

3. Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, U1167 - RID-AGE - Risk Factors and

Molecular Determinants of Aging-Related Diseases, F-59000 Lille, France

4. CNRS, ERL9002 - Integrative Structural Biology, F-59000 Lille, France

5. Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, CNRS

UMR 9004, 34293 Montpellier, France

6. INSERM, Institut de Recherche en Infectiologie de Montpellier, Montpellier, France

7. Unité Microbiologie, Chimie Bioorganique et Macromoléculaire (CP206/04), département

RD3, Faculté de Pharmacie, Université Libre de Bruxelles, B-1050 Brussels, Belgium

Abstract

Mycobacterium tuberculosis (M.tb), the etiologic agent of tuberculosis, remains the leading cause of

death from a single infectious agent worldwide. The emergence of drug-resistant M.tb strains

stresses the need for drugs acting on new targets. Mycolic acids are very long chain fatty acids

playing an essential role in the architecture and permeability of the mycobacterial cell wall. Their

biosynthesis involves two fatty acid synthase (FAS) systems. Among the four enzymes (MabA,

HadAB/BC, InhA and KasA/B) of the FAS-II cycle, MabA (FabG1) remains the only one for which

specific inhibitors have not been reported yet. The development of a new LC-MS/MS based

enzymatic assay allowed the screening of a 1280 fragment-library and led to the discovery of the first

small molecules that inhibit MabA activity. A fragment from the anthranilic acid series was optimized

into more potent inhibitors and their binding to MabA was confirmed by ¹⁹F ligand-observed NMR

experiments.

Keywords

MabA inhibitors; fragment; FabG1; tuberculosis; mycolic acid

1. 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 [7], 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.[10,11] 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-C₁₈and C₂₄-C₂₆).[12,13] 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.[11] 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).[14] 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.[8,11]

FAS-II is initiated by a Claisen condensation of malonyl-ACP and palmitoyl-CoA catalyzed by the β-

ketoacyl-ACP synthase FabH. The β-ketoacyl-ACP formed is then reduced by MabA (FabG1), a β-

ketoacyl-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-2-

enoyl-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 (C_42–62).[8,14]

InhA [15], MabA [16], HadB [17] and KasA [18] have been shown to be essential for bacterial growth,

strengthening their attractiveness as valuable chemotherapeutic targets.[19]

Several antibiotics target FAS-II. Indeed, InhA is inhibited by isoniazid as well as by two second-line

drugs, ethionamide and prothionamide.[20–22] HadAB is inhibited by isoxyl and thiacetazone [23–

25] and KasA/B are inhibited by thiolactomycin [26,27] while KasA (but not KasB) is also the target of

an indazole sulfonamide (GSK3011724A).[28] 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.[29] 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.[30] High-resolution crystal

structures of MabA from M. smegmatis in its apo, NADP⁺-bound and NADPH-bound forms were

recently reported.[31]

Structural comparison of MabA, a NADPH-dependent-ketoacyl-ACP reductase, and InhA, an NADH-

dependent enoyl-ACP reductase, revealed the presence of hydrophobic substrate binding pockets of

similar sizes.[29,32,33] 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.[34]

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.

2. 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.[35,36] 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.[37]

A

NADP⁺

NADPH

O

OH

O

CoA

S

MabA

HBCoA

AcAcCoA

end-point

B

30000

20000

10000

0

R²= 0.998

5

10

15

20

25

temps (min)

Time (min)

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.

As mentioned above, the only MabA inhibitor reported so far in the literature is the adduct of INH

with NADP⁺(Figure 2A).[34] This INH-NADP adduct can be formed in situ using Mn^III[38] 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 IC₅₀of 20 µM (Figure 2D). Triclosan [39] and pyridomycin [40,41], two known InhA

inhibitors, did not show any MabA inhibitory activity in this assay when tested up to 100 µM (data

not shown).

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).

2.2 Screening

Our 1280-fragment library is composed of commercially available molecules and sp³and spiro-

enriched compounds.[42,43] This library fulfills the ‘rule of three’ and all compounds are soluble in

DMSO at 100 mM.[44] 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 IC₅₀lower than 300 µM. Data mining of commercial

libraries identified 68 analogues of these 12 fragments. Retesting of the 80 compounds in dose-

response experiments led to the selection of 6 chemical series displaying IC₅₀s ranging from 25ꢀto

300ꢀμM (Figure 4).

threshold

negative control (without inhibitor)

positive control (INH-NADP)

A

B

100

1280 fragments

> 30% inh at 1 mM

50 fragments

IC₅₀< 300 µM

50

30

12 fragments

+ 68 analogues

0

IC₅₀< 300 µM

6 chemical series

-50

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

2.3 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 IC₅₀slightly 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 5-aminouracil 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 MIC₉₀of 300 µM.

Anthranilic acid represents a well-known privileged structure and its derivatives have been widely

used in different fields of medicinal chemistry.[47–50] 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.

2.4 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 IC₅₀was

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]

A

B

[INH-NADP adduct]

[compound 1]

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

2.5 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-tert-

butyldicarbonate (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.

a

13a

R₁

R₂

b

R₂

8-14

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.

2.5.2. 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

2-amino-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.

a

15-17

b, c

18

d

19

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.

2.5.3. 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-5-

iodobenzoic 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 2-

amino-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 [55] (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-5-iodobenzoic acid was through the synthesis of mixed anhydrides using isopropyl

chloroformate.

a

21

b,c

27, 35

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.

a

b

20, 22, 26

c, d

P

23

e

f

24, 25, 28-34, 36, 37

Scheme 4. Synthesis of compounds 20, 22-26, 28-34 and 36-37 from carboxylic acids. Reagents and conditions: (a) SOCl₂

(1.1 eq), 1H-benzotriazole (3.1 eq), carboxylic acid (1 eq), DCM, RT, overnight; (b) 2-amino-5-iodo-benzoic acid (1 eq),

DMAP (1.1 eq), CHCl₃, µW 80 °C, 15-30 min; (c) 4-phenylbutanoic acid (3 eq), PyBrop (3 eq), DIEA (6 eq), HOBt resin (1 eq),

DMF, RT, 3h; (d) 2-amino-5-iodo-benzoic acid (0.8 eq), DMF, RT, overnight; (e) isopropyl chloroformate 1 M in toluene

(1.5 eq), carboxylic acid (1eq), TEA (1.2 eq), anh. THF, RT, overnight; (f) 2-amino-5-iodo-benzoic acid (0.67 eq), Na₂CO₃

(1.33 eq), THF, H₂O, RT, 3h to overnight.

2.6 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.

2.6.1. Modifications of the substituents on the phenyl ring

1

R₁

5

4

2

R₂

3

Cpd

1

R1

R2

% inh^a

IC₅₀(µM)

I

H

100

430 ± 26

7

8

H

F

H

31

38

77

> 1000

470

9

Cl

Br

> 500

470

10

95

31

11

12

OCH₃

OPh

H

> 1000

243

100

13

14

Ph

H

194

310

5

4

Table 1. Biological activities of compounds 1, 7-14. ^aPercentage of inhibition (compounds tested at 1 mM)

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.

2.6.2. Modifications of the carbamate and the carboxylic acid

Cpd

1

R

R’

R”

% inh^aIC₅₀(µM)

100

83

tBu

H

430 ± 26

310

15

16

17

18

19

iBu

allyl

H

> 500

100

50

CH₂-Ph

tBu

H

92 ± 3.5

~ 1000

> 1000

CH₃

H

0

tBu

CH₃

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

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).

2.6.3. Replacement of the carbamate with an amide

Cpd

20

R

IC₅₀(µM)

Cpd

R

IC₅₀(µM)

Ph

145

29

30

31

32

33

3,4-Cl₂-Ph

2,4-Cl₂-Ph

4-CH₃-Ph

4-CF₃-Ph

38 ± 6

126

95

21

22

23

CH₂-Ph

(CH₂)₂-Ph

(CH₂)₃-Ph

> 500

295

69

186

4-OCF₃-Ph

84

24

25

140

109

34

3-CF₃-4-Cl-Ph

4-pyridyl

59

26

27

cyclopropyl

3-Cl-Ph

> 500

70

35

36

> 400

147

28

4-Cl-Ph

53

37

40

Table 3. Biological activities of compounds 20-36.

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

IC₅₀of 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 25), 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.

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 3,4-

dichlorophenyl ring on the amide function (IC₅₀= 38 ± 6 µM).

2.6.4. Evaluation of the interaction of the fluorinated compounds with MabA by NMR

¹⁹F ligand-observed NMR has been described as a powerful tool in detecting weak binding of small

molecules. ¹⁹F has a wide chemical shift range, therefore even small changes in binding can result in a

measurable shift in the fluorine signal.[56,57] 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 ¹⁹F cryogenic probe. The perturbations (broadening and chemical shift

changes) of ¹⁹F-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 ¹⁹F 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 ¹⁹F-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).

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

(red signal).

2.6.5. 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.[58] 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.

3. Conclusion

A new LC-MS/MS based MabA activity assay was developed and used for the screening of a 1280-

fragment 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

dose-response 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. ¹⁹F

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 IC₅₀of 38 µM and bacterial growth with a MIC₉₀of 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) ¹H

and ¹³C 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® C₁₈Rf200 with C₁₈silica 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

C₁₈Dynamax (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.

4.1.1.1. 2-(tert-butoxycarbonylamino)-5-fluoro-benzoic acid (8)

Yield: 45%. ¹H-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).

¹³C-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.

4.1.1.2. 2-(tert-butoxycarbonylamino)-5-chloro-benzoic acid (9)

Yield: 45%. ¹H-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). ¹³C-NMR (DMSO-d6): δ (ppm) 168.9,

152.3, 140.8, 134.3, 130.7, 125.5, 120.5, 117.5, 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)

1

Yield: 28%. 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). ¹³C-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

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.

4.1.2.1. 2-(tert-butoxycarbonylamino)-5-methoxy-benzoic acid (11)

Yield: 78%. ¹H-NMR (DMSO-d6): δ (ppm) 10.22 (s, NH), 8.17 (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). ¹³C-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%. ¹H-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). ¹³C-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)

1

Yield: 42%. 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). ¹³C-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.

4.1.3. 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-

1

butoxycarbonylamino)-5-phenyl-benzoic acid. Yield: 4%. 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). ¹³C-NMR (DMSO-d6): δ (ppm)

170.0, 152.5, 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

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 MgSO₄and concentrated in vacuo to give the desired

product.

4.1.4.1. 5-iodo-2-(isobutoxycarbonylamino)benzoic acid (15)

1

Yield: 62%. 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).¹³C-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.

4.1.4.2. 2-(allyloxycarbonylamino)-5-iodo-benzoic acid (16)

The residue was purified by preparative HPLC. Yield: 39%. ¹H-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). ¹³C-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.

4.1.4.3. 2-(benzyloxycarbonylamino)-5-iodo-benzoic acid (17)

1

The residue was purified by preparative HPLC. Yield: 48%. 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). ¹³C-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.

4.1.5. 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

1

reduced pressure to give 2-(tert-butoxycarbonylamino)-5-iodo-benzoic acid. Yield: 69%. 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). ¹³C-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.

4.1.6. 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 MgSO₄and concentrated in vacuo to give methyl 2-(tert-

1

butoxycarbonylamino)-5-iodo-benzoate. Yield: 28%. 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).

¹³C-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 MgSO₄and concentrated in vacuo.

4.1.7.1. 2-benzamido-5-iodo-benzoic acid (20)

The residue was precipitated in a mixture of MeCN and MeOH (1:1), then filtered and washed with

1

MeCN. Yield: 58%. 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). ¹³C-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

1

MeCN. Yield: 33%. 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). ¹³C-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%.

¹H-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). ¹³C-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)

2-phenylacetyl chloride (185 µL, 1.4 mmol, 2 eq) was added dropwise in a mixture of 2-amino-5-iodo-

benzoic 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 MgSO₄and concentrated in vacuo. The residue was purified by

1

preparative HPLC to give 5-iodo-2-[(2-phenylacetyl)amino]benzoic acid. Yield: 64%. 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). ¹³C-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)

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%. ¹H-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). ¹³C-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

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.

NaHCO₃sat. was added and the product was extracted twice with EtOAc. The organic layer was

washed once with NaHCO₃sat., once with brine, dried over MgSO₄and 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.

4.1.10.1. 2-(indane-2-carbonylamino)-5-iodo-benzoic acid (24)

Yield: 49%. ¹H-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). ¹³C-NMR (DMSO-d6): δ (ppm) 173.0, 168.8, 142.3, 141.0, 140.0, 138.6, 127.7,

126.8, 124.7, 121.1, 85.2, 47.1, 36.5. HRMS (TOF, ES-) m/z [M-H]- calculated for C17H13NO3I

405.9940, found 405.9965.

4.1.10.2. 5-iodo-2-(tetralin-2-carbonylamino)benzoic acid (25)

Yield: 42%. ¹H-NMR (DMSO-d6): δ (ppm) 14.50 (s, 1H) 8.32 (d, J = 8.7 Hz, 1H), 8.26 (d, J = 2.3 Hz, 1H),

7.58 (dd, J = 8.7 Hz, J = 2.6 Hz, 1H), 7.12 - 7.06 (m, 4H), 3.00 - 2.91 (m, 2H), 2.86 - 2.77 (m, 2H), 2.22 -

2.07 (m, 1H), 1.90 - 1.71 (m, 1H) ¹³C-NMR (DMSO-d6): δ (ppm) 173.6, 168.6, 141.0, 140.0, 138.6,

136.0, 135.7, 129.4, 129.0, 127.8, 126.1, 126.0, 121.2, 85.2, 43.2, 32.3, 28.6, 26.4. HRMS (TOF, ES-)

m/z [M-H]- calculated for C18H15NO3I 420.0097, found 420.0116.

4.1.10.3. 2-[(4-chlorobenzoyl)amino]-5-iodo-benzoic acid (28)

Yield: 49%. ¹H-NMR (DMSO-d6): δ (ppm) 12.09 (s, 1H), 8.45 (d, J = 8.9 Hz, 1H), 8.29 (d, J = 2.1 Hz, 1H),

7.98 (dd, J = 8.8 Hz, J = 2.2 Hz, 1H), 7.94 (d, J = 8.9 Hz, 2H), 7.67 (d, J = 8.8 Hz, 2H). ¹³C-NMR (DMSO-

d6): δ (ppm) 168.6, 163.7, 142.4, 140.4, 139.1, 137.2, 133.1, 129.1, 129.0, 122.3, 119.2, 86.6. HRMS

(TOF, ES-) m/z [M-H]- calculated for C14H8NO3ClI 399.9237, found 399.9267.

4.1.10.4. 2-[(3,4-dichlorobenzoyl)amino]-5-iodo-benzoic acid (29)

1

Yield: 82%. H-NMR (DMSO-d6): δ (ppm) 8.42 (d, J = 8.7 Hz, 1H), 8.31 (d, J = 2.3 Hz, 1H), 8.14 (d, J =

2.1 Hz, 1H), 7.95 (dd, J = 8.5 Hz, J = 2.1 Hz, 1H), 7.83 (d, J = 8.5 Hz, 1H), 7.65 (dd, J = 8.7 Hz, J = 2.5 Hz,

1H). ¹³C-NMR (DMSO-d6): δ (ppm) 168.3, 162.3, 140.8, 140.1, 138.9, 136.1, 134.9, 132.2, 131.6,

129.6, 128.0, 127.7, 121.2, 86.3. HRMS (TOF, ES-) m/z [M-H]- calculated for C14H7NO3Cl2I 433.8848,

found 433.8862.

4.1.10.5. 2-[(2,4-dichlorobenzoyl)amino]-5-iodo-benzoic acid (30)

1

Yield: 66%. H-NMR (DMSO-d6): δ (ppm) 8.36 (d, J = 8.6 Hz, 1H), 8.29 (d, J = 2.3 Hz, 1H), 7.74 (d, J =

1.95 Hz, 1H), 7.67-7.62 (m, 2H), 7.54 (dd, J = 8.23 Hz, J = 1.96 Hz, 1H). ¹³C-NMR (DMSO-d6): δ (ppm)

168.6, 163.8, 140.6, 140.1, 139.0, 136.4, 135.5, 131.7, 130.6, 130.1, 128.2, 127.7, 121.2, 86.5. HRMS

(TOF, ES-) m/z [M-H]- calculated for C14H7NO3Cl2I 433.8848, found 433.8866.

4.1.10.6. 5-iodo-2-[(4-methylbenzoyl)amino]benzoic acid (31)

1

Yield: 14%. H-NMR (DMSO-d6): δ (ppm) 8.49 (d, J = 8.7 Hz, 1H), 8.31 (d, J = 2.3 Hz, 1H), 7.91 (d, J =

8.2 Hz, 2H), 7.62 (dd, J = 8.7 Hz, J = 2.4 Hz, 1H), 7.34 (d, J = 7.9 Hz, 2H), 2.38 (s, 3H). ¹³C-NMR (DMSO-

d6): δ (ppm) 168.4, 164.7, 142.0, 141.2, 140.1, 138.6, 132.9, 129.7, 128.1, 127.7, 121.2, 85.5, 21.5.

HRMS (TOF, ES-) m/z [M-H]- calculated for C15H11NO3I 379.9784 found 379.9810.

4.1.10.7. 5-iodo-2-[[4-(trifluoromethyl)benzoyl]amino]benzoic acid (32)

1

Yield: 58%. H-NMR (DMSO-d6): δ (ppm) 8.46 (d, J = 8.7 Hz, 1H), 8.32 (d, J = 2.3 Hz, 1H), 8.18 (d, J =

8.2 Hz, 2H), 7.92 (d, J = 8.2 Hz, 2H), 7.66 (dd, J = 8.7 Hz, J = 2.3 Hz, 1H). ¹³C-NMR (DMSO-d6): δ (ppm)

168.6, 163.4, 140.9, 140.1, 139.4, 139.1, 131.9 (q, J = 32 Hz), 128.5, 127.4, 126.2 (q, J = 4,1 Hz), 124.3

(q, J = 272 Hz), 121.4, 86.4. HRMS (TOF, ES-) m/z [M-H]- calculated for C15H8NO3F3I 433.9501, found

433.9510.

4.1.10.8. 5-iodo-2-[[4-(trifluoromethoxy)benzoyl]amino]benzoic acid (33)

1

Yield: 37%. H-NMR (DMSO-d6): δ (ppm) 8.45 (d, J = 8.6 Hz, 1H), 8.34 (d, J = 2.4 Hz, 1H), 8.12 (d, J =

8.8 Hz, 2H), 7.66 (dd, J = 8.6 Hz, J = 2.4 Hz, 1H), 7.53 (d, J = 8.8, 2H) ¹³C-NMR (DMSO-d6): δ (ppm)

168.6, 163.4, 151.0 (q, J = 1.7 Hz), 141.0, 140.1, 138.9, 134.9, 130.0, 127.9, 121.4, 121.2, 120.2 (q, J =

256 Hz), 86.1. HRMS (TOF, ES-) m/z [M-H]- calculated for C15H8NO4F3I 449.9450, found 449.9467.

4.1.10.9. 2-[[4-chloro-3-(trifluoromethyl)benzoyl]amino]-5-iodo-benzoic acid (34)

Yield: 48%. ¹H-NMR (DMSO-d6): δ (ppm) 8.45 (d, J = 8.7 Hz, 1H), 8.39 (d, J = 1.6 Hz, 2H), 8.26 (dd, J =

8.4 Hz, J = 1.5 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.69 (dd, J = 8.6 Hz, J = 2.1 Hz, 1H). ¹³C-NMR (DMSO-

d6): δ (ppm) 168.3, 162.1, 140.9, 140.3, 139.2, 135.0, 134.4, 133.0, 132.9, 129.8 (q, J = 272 Hz), 127.7,

126.9, 126.8, 124.9, 121.2, 86.5. HRMS (TOF, ES-) m/z [M-H]- calculated for C15H7NO3F3ClI

467.9111, found 467.9113.

4.1.10.10. 2-[(6-chloropyridine-3-carbonyl)amino]-5-iodo-benzoic acid (36)

1

Yield: 69%. H-NMR (DMSO-d6): δ (ppm) 8.95 (d, J = 2.2 Hz, 1H), 8.42 (d, J = 8.7 Hz, 1H) 8.36 – 8.30

(m, 2H), 7.72 (d, J = 8.7 Hz, 1H), 7,66 (dd, J = 8.4 Hz, J = 2.3 Hz, 1H). ¹³C-NMR (DMSO-d6): δ (ppm)

168.6, 162.0, 153.3, 149.4, 140.8, 140.1, 139.1, 138.8, 130.7, 127.7, 125.0, 121.3, 86.5. HRMS (TOF,

ES-) m/z [M-H]- calculated for C13H7N2O3ClI 400.9190, found 400.9217.

4.1.10.11. 2-[[(E)-3-(4-chlorophenyl)prop-2-enoyl]amino]-5-iodo-benzoic acid (37)

Yield: 61%. ¹H-NMR (DMSO-d6): δ (ppm) 8.40 (d, 1H, J = 8.7 Hz) 8.28 (d, 1H, J = 2.3 Hz) 7.72 (d, 2H, J =

8.5 Hz), 7.60 (dd, 1H, J = 8.7 Hz, J = 2.3 Hz), 7.54 (d, 1H, J = 15.7 Hz) 7.47 (d, 2H, J = 8.5 Hz), 6.67 (d,

1H, J = 15.7 Hz) ¹³C-NMR (DMSO-d6): δ (ppm) 168.5, 163.6, 141.0, 140.1, 139.0, 138.7, 134.6, 134.1,

130.1, 129.4, 127.9, 124.8, 121.4, 85.8. HRMS (TOF, ES-) m/z [M-H]- calculated for

C16H11ClINO3 425.9394, found 425.9405.

4.1.11. General method for the synthesis of compounds 27 and 35

Step 1. To a solution of methyl 2-amino-5-iodo-benzoate (0.5 mmol, 1 eq) in anhydrous pyridine (3

mL) was added the appropriate acyl chloride (0.55 mmol, 1.1 eq) dropwise at 0 °C. The reaction

mixture was stirred at RT for 2 h. Aqueous HCl (C = 1M) was added to the solution and the product

was extracted twice with EtOAc. The organic layers were washed twice with aqueous HCl (C = 1 M),

once with brine, dried over MgSO₄and then concentrated in vacuo. The product was used in the next

without further purification.

Step 2. Lithium hydroxide (1.5 mmol, 3 eq) dissolved in water (4 mL) was added dropwise to a

solution of the methylbenzoate derivative previously formed (0.5 mmol, 1 eq) dissolved in THF (4

mL). The mixture was stirred at RT for 1 h. THF was then evaporated under reduced pressure.

4.1.11.1. 2-[(3-chlorobenzoyl)amino]-5-iodo-benzoic acid (27)

An aqueous solution of HCl (C = 1 M) was added and the product was extracted twice with EtOAc.

The organic layer was then washed once with brine, dried over MgSO₄and concentrated in vacuo.

Yield: 60 %. ¹H-NMR (DMSO-d6): δ (ppm) 14.14 (brs, OH), 12.07 (s, NH), 8.42 (d, J = 8.8 Hz, 1H), 8.28

(d, J = 2.1 Hz, 1H), 7.99 (dd, J = 8.8 Hz, J = 2.2 Hz, 1H), 7.93 (d, J = 1.7 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H),

7.73 (dd, J = 7.0, 1.8 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H). ¹³C-NMR (DMSO-d6): δ (ppm) 169.1, 163.7, 142.9,

140.6, 139.6, 136.8, 134.2, 132.6, 131.5, 127.4, 126.2, 122.8, 119.9, 87.2. HRMS (TOF, ES-) m/z [M-H]-

calculated for C14H8NO3ClI 399.9237, found 399.9260.

4.1.11.2. 5-iodo-2-(pyridine-4-carbonylamino)benzoic acid (35)

An aqueous solution of HCl (C = 1 M) was added and the product precipitated. The solid was filtered,

washed with a 1:1 mixture of diethyl ether and cyclohexane (3 x 1 mL), and then dried. Yield: 66%.

¹H-NMR (DMSO-d6): δ (ppm) 8.80 (dd, J = 4.5 Hz, J = 1.5 Hz, 2H), 8.45 (d, J = 8.6 Hz, 1H), 8.34 (d, J =

2.3 Hz, 1H), 7.89 (dd, J = 4.5, 1.6 Hz, 2H), 7.66 (dd, J = 8.6, 2.2 Hz, 1H). ¹³C-NMR (DMSO-d6): δ (ppm)

167.9, 162.9, 151.1, 142.6, 140.7, 140.1, 138.8, 128.3, 121.5, 121.3, 86.6. HRMS (TOF, ES-) m/z [M-H]-

calculated for C13H8N2O3I 366.9580, found 366.9603.

4.2 Biology

4.2.1. MabA expression and purification

E. coli BL21 (DE3) transformed with pET28a_MabA-His₆were induced with 0.5 mM IPTG at an OD of

0.6 for 4 hours at 37 °C. The bacteria were harvested by centrifugation and resuspended in lysis

buffer Tris 50 mM pH7.5, NaCl 300 mM, 10 mM imidazole, supplemented with DNase and Compete

EDTA-free protease inhibitor (Roche). The bacteria were lysed by two passages on French press.

MabA-His₆was purified on Ni²⁺-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). 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.[45] All the screening plates displayed a Z′-factor between 0.72 and 0.83.





(

x − Mean_C+×100

)





%Inh = 100 −

Mean_C−− Mean_C+





3(SD_C++ SD_C−)

Z'= 1−

Mean_C+− Mean_C−

4.2.2.2. 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.

4.2.2.3. 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.[38] 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 low-

binding 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

IC₅₀values 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.

4.2.3. ¹⁹F-NMR assay

The ¹⁹F NMR measurements were done at 298 K using 3ꢁmm tubes (160ꢁμL samples) on a Bruker

600ꢁMHz Avance III HD spectrometer equipped with a CP-QCI-F cryoprobe with optimized ¹⁹Fluorine

detection. The ¹⁹F NMR reference spectra were acquired on 160 µL samples containing a compound

(32, 33 or 34) at 100ꢁμM, in the NMR buffer [50ꢁmM Tris-Cl pH 7.5; 250ꢁmM NaCl; 1% DMSO-d6; 5%

D₂O]. Then, ¹⁹F 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 = −65.0ꢁ ppm; spectral

window sw = 20 ppm; and with ¹⁹F decoupling. The acquisition time was about 22 minutes per

sample. NMR data have been processed with Bruker Topspin 4.06.

4.2.4. 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).

4.3. 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.

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.

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, fatty-

acid 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,

structure-activity relationships; TB, tuberculosis; TEA, triethylamine.

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

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