Discovery of novel N -phenylphenoxyacetamide derivatives as EthR inhibitors and ethionamide boosters by combining high-throughput screening and synthesis

Article abstract of DOI:10.1021/jm300377g

In this paper, we describe the screening of a 14640-compound library using a novel whole mycobacteria phenotypic assay to discover inhibitors of EthR, a transcriptional repressor implicated in the innate resistance of Mycobacterium tuberculosis to the second-line antituberculosis drug ethionamide. From this screening a new chemical family of EthR inhibitors bearing an N-phenylphenoxyacetamide motif was identified. The X-ray structure of the most potent compound crystallized with EthR inspired the synthesis of a 960-member focused library. These compounds were tested in vitro using a rapid thermal shift assay on EthR to accelerate the optimization. The best compounds were synthesized on a larger scale and confirmed as potent ethionamide boosters on M. tuberculosis-infected macrophages. Finally, the cocrystallization of the best optimized analogue with EthR revealed an unexpected reorientation of the ligand in the binding pocket.

Full text of DOI:10.1021/jm300377g

Article
pubs.acs.org/jmc
Discovery of Novel N-Phenylphenoxyacetamide Derivatives
as EthR Inhibitors and Ethionamide Boosters by Combining
High-Throughput Screening and Synthesis
Marion Flipo,†,‡,§,∥,⊥,⊞ Nicolas Willand,†,‡,§,∥,⊥,⊞ Nathalie Lecat-Guillet,†,∥,⊥,#,∇,○
Candide Hounsou,†,‡,§,∥,⊥ Matthieu Desroses,†,‡,§,∥,⊥ Florence Leroux,†,‡,§,∥,⊥ Zoe Lens,†,◆,¶
́
Vincent Villeret,†,◆ Alexandre Wohlkonig,□ Rene Wintjens,● Thierry Christophe,▲,☆
́
̈
Hee Kyoung Jeon,▲ Camille Locht,†,∥,#,∇,○ Priscille Brodin,†,∥,#,∇,○,▲ Alain R Baulard,†,∥,⊥,#,∇,○,⊞
and Benoit Deprez*,†,‡,§,∥,⊥,⊞
́
†Universite
́
Lille Nord de France, F-59000 Lille, France
‡Biostructures and Drug Discovery, Institut National de la Sante
§Universite
Droit & Sante (UDSL), F-59000 Lille, France
∥Institut Pasteur de Lille, F-59019 Lille, France
⊥Po
le de Recherche Interdisciplinaire sur le Med
#INSERM U1019, F-59000 Lille, France
́
́
et de la Recherche Medicale (INSERM) U761, F-59000 Lille, France
́
́
̂
́
icament (PRIM), F-59000 Lille, France
∇Centre National de Recherche Scientifique (CNRS) UMR8204, F-59021 Lille, France
○Center for Infection and Immunity of Lille, F-59019 Lille, France
◆Institut de Recherche Interdisciplinaire (IRI), CNRS USR3078, F-59658 Villeneuve d’Ascq, France
¶Laboratory of Molecular Virology, Institut de Biologie et de Med
6041 Gosselies, Belgium
́ ́
ecine Moleculaires (IBMM), Universite Libre de Bruxelles (ULB),
▲Biology of Intracellular Pathogens INSERM-Avenir, Institut Pasteur Korea (IPK), 463-400 Gyeonggi-do, South Korea
□Structural Biology Brussels and Molecular and Cellular Interactions, Vlaams Instituut voor Biotechnologie (VIB), 1050 Brussels, Belgium
●Laboratoire des Biopolymer
es et des Nanomateriaux Supramoleculaires, Institut de Pharmacie, ULB, 1050 Brussel, Belgium
̀ ́ ́
S
* Supporting Information
ABSTRACT: In this paper, we describe the screening of a 14640-compound library using a novel whole mycobacteria
phenotypic assay to discover inhibitors of EthR, a transcriptional repressor implicated in the innate resistance of Mycobacterium
tuberculosis to the second-line antituberculosis drug ethionamide. From this screening a new chemical family of EthR inhibitors
bearing an N-phenylphenoxyacetamide motif was identified. The X-ray structure of the most potent compound crystallized with
EthR inspired the synthesis of a 960-member focused library. These compounds were tested in vitro using a rapid thermal shift
assay on EthR to accelerate the optimization. The best compounds were synthesized on a larger scale and confirmed as potent
ethionamide boosters on M. tuberculosis-infected macrophages. Finally, the cocrystallization of the best optimized analogue with
EthR revealed an unexpected reorientation of the ligand in the binding pocket.
Received: March 19, 2012
Published: June 28, 2012
© 2012 American Chemical Society
6391
dx.doi.org/10.1021/jm300377g | J. Med. Chem. 2012, 55, 6391−6402

Journal of Medicinal Chemistry
Article
compounds twice as long as the previous 1,2,4-oxadiazoles by
reorganizing residues hiding an unexplored cavity.19 These
compounds opened new frontiers for the development of high-
affinity EthR inhibitors. Knowing that target-based approaches do
not account for permeability, we wanted to use a high-throughput
assay that would have the potential to reveal new chemotypes
with a good capacity to cross the complex mycobacterial
envelope. Here, we report the screening of a library of 14 640
compounds using a whole mycobacterial phenotypic assay that
led to the discovery of an N-phenylphenoxyacetamide family. The
optimization of this series was driven by the synthesis of a 960-
member focused library that was screened on EthR using a rapid
thermal shift assay.
INTRODUCTION
■
Tuberculosis (TB) remains a major cause of mortality, killing 1.7
million people each year.1 In 2010, TB incidence and prevalence
were estimated by the World Health Organization (WHO) at
8.8 and 12 million cases, respectively.1 Difficulties in detecting
and curing enough cases to interrupt transmission are the biggest
obstacles in controlling this infection.2 One-third of the world
population is infected with the latent form of Mycobacterium
tuberculosis, and approximately 10% will develop the disease.3
The directly observed treatment short course (DOTS), a
multidrug therapy program developed by the WHO, covers
approximately 77% of the world’s population and contributes to
controlling the epidemic.4,5 Nevertheless, the treatment success
rate remains below the target of 90%,1 particularly in Africa.6
Moreover, the global HIV infection epidemic has contributed to
the explosive increase of TB incidence and the emergence of
multi-drug-resistant (MDR) stains. In 2010, the number of
MDR-TB infections was estimated at 650 000 cases.1 MDR-TB
patients must be treated for 2−4 years with several second-line
drugs, which are less effective and poorly tolerated, weakening
observance and leading to treatment failure. In the past 40 years no
new antituberculosis drugs have succeeded in reaching the market,
and there is an urgent need for new molecules, particularly ones
that shorten the treatment time and cure patients with multi-drug-
resistant and extensively drug resistant strains.7
ASSAY DESIGN AND SCREENING
■
We previously demonstrated that the inhibition of EthR by
synthetic ligands correlates with its incapacity to bind to the DNA
operator controlling the expression of the monooxygenase coding
gene ethA.16 To exploit this as a read-out assay for the identification
of EthR inhibitors in mycobacteria, we used the nonpathogenic
fast-growing TB surrogate Mycobacterium smegmatis. The ethA
promoter/operator was inserted into a mycobacterial shuttle vector
upstream of the glucuronidase reporter gene (uidA). Introduced in
M. smegmatis, this vector led to the expression of uidA, which was
easily detected using the profluorescent substrate 4-methylumbel-
liferyl β-D-glucuronide (MUG). In contrast, when this clone was
transformed with a second plasmid overexpressing ethR of
M. tuberculosis, a total disappearance of the glucuronidase activity
was observed. A 14640-compound library purchased from Asinex
and ChemDiv was screened using this assay at 10 μM to identify
potent EthR inhibitors through their capacity to trigger the
expression of uidA (Figure 1).
BOOSTING ETHIONAMIDE APPROACH
■
Current TB therapies include a large number of prodrugs that
must be metabolically transformed by the mycobacteria to
manifest their activity. One of these, ethionamide, an antibiotic
used to treat multi-drug-resistant TB, is bioactivated by the
mycobacterial monooxygenase EthA.8,9 The active bacterial
metabolite of ethionamide10−12 inhibits InhA,13,14 the enoyl-acyl
carrier protein (ACP) reductase involved in mycolic acid
biosynthesis. The limited effectiveness of ethionamide has been
explained by the transcriptional repression of ethA exerted by
the bacterial regulator EthR.15 Thus, EthR contributes to the
innate resistance of M. tuberculosis to this antibiotic. EthR was
proposed and validated as a drug target to boost the bioactivation
of ethionamide and potentiate the drug in vivo.16 We recently
reported structure−activity relationships of a series of druglike
1,2,4-oxadiazole EthR inhibitors identified through a rational
drug design approach.17,18 This led to the discovery of highly
potent boosters both in vitro on the targeted protein EthR and
ex vivo on the human pathogen M. tuberculosis in a macrophage-
infected model. Another study performed in parallel revealed
that the ligand-binding pocket of EthR can accommodate
At the end of the screening process, 76 compounds were
identified as potent reactivators of the glucuronidase activity in
M. smegmatis through inhibition of EthR. These 76 hits and 244
chemical analogues picked out of the 14640-compound library
were selected for dose−response experiments to recover
potential false-negative compounds. Among the 320 compounds,
22 showed IC50 values below 10 μM and were tested using a
thermal shift assay on purified EthR (Figure 2).
This fluorescence-based assay was developed to confirm that
hit compounds actually bind to the target protein EthR.20 The
principle of the thermal shift assay lies in the monitoring of the
thermal unfolding of the protein using a conformation-sensitive
dye, SYPRO Orange, in which fluorescence is quenched in
aqueous solution but enhanced in a nonpolar environment, such
as the hydrophobic domain of a protein that begins to unfold.
The assay is carried out on standard Q-PCR instruments in
Figure 1. Fluorescent assay used for the screening of the library. M. smegmatis is transformed with plasmid P1 overexpressing ethR and with plasmid P2
expressing uidA under the control of the promoter/operator of ethA. In the absence of EthR inhibitors, EthR binds to the promotor/operator, which
blocks the transcription of uidA. MUG is not hydrolyzed. Compounds of the library (shown as yellow balls) that penetrate into the bacteria and inhibit
EthR release the production of UidA, which cleaves MUG in fluorescent methylumbelliferone (MU) and glucuronic acid.
6392
dx.doi.org/10.1021/jm300377g | J. Med. Chem. 2012, 55, 6391−6402

Journal of Medicinal Chemistry
Article
Figure 2. Screening cascade developed for the identification of compound 1 as an EthR inhibitor.
Figure 3. (A) X-ray structure representation of the ligand-binding pocket of EthR filled with compound 1 (PDB ID 3Q3S) surrounded by its initial omit
Fo−Fc map at the 3.0σ contour level. The Fo−Fc map was calculated prior to addition of the ligand to the model. (B) X-ray structure representation of the
homodimeric conformation of EthR filled in both monomers with compound 1. The surface of the ligand binding domain is highlighted. Hydrogen
bonds with Asn179 and Asn176 are represented with dotted lines. Color legend: blue (compound 1) or green (EthR) = carbon, dark blue = nitrogen,
red = oxygen, yellow = sulfur. The images were generated with Pymol.
96-well plates. Results are produced as graphs showing the
fluorescence intensity of SYPRO Orange as a function of the
temperature. The inflection of the sigmoid curve indicates
the melting temperature (Tm) of the protein. The variation of the
melting temperatures between holoprotein and apoprotein
(ΔTm) reflects the capacity of the ligand to stabilize the receptor
protein in a liganded conformation.21−25 From the 22
compounds identified through the glucuronidase M. smegmatis
assay, six produced a significant ΔTm (greater than 4 °C). Among
them three were members of the N-phenylphenoxyacetamide
family (Figure 2). Compound 1 (Figure 2) showed the best
activities in the two assays (IC50 = 2.9 μM and ΔTm = 6.4 °C).
Compound 1 was cocrystallized with the protein. X-ray
diffraction revealed that the N-phenyloxyacetamide motif is the
key pharmacophore for binding. Indeed, the amide function of
the ligand is hydrogen bonded to the side chains of the two
asparagines, Asn179 and Asn176(Figure3). Theo-methylphenoxy
moiety of the ligand occupies the upper hydrophobic pocket
formed by the side chains of Trp207, Thr149, Leu87, and Gly106, a
region involved in the induction of the structural reorganization of
the helix−turn−helix (HTH) motifs of EthR.20 The benzotriazole
motif faces Phe110 and Phe114, while the thirdremaining aromatic
ring in contact with Trp138 and Phe187 invades a small hydro-
phobic pocket thanks to the rotation of Phe184. The existence of
this cavity, not explored by compounds in our first series, was
previously shown by the cocrystal structure of EthR with hexadecyl
octanoate26 and by the study of EthR flexibility using in situ
click chemistry experiments.19 As expected, the crystal structure
revealed the repressor in a conformation incompatible with DNA
binding. Indeed, the distance between the two HTH motifs
(50.5 Å) is consistent with the phenotypic screening result.
To rapidly optimize the pharmacodynamic properties of this
new chemical family of EthR inhibitors, we designed a focused
library based on the N-phenylphenoxyacetamide motif. The
amide bond and the ether bond of the structure are two obvious
disconnection points amenable to high-throughput synthesis.
Diversity was thus introduced via a large set of commercially
available anilines and phenols.
6393
dx.doi.org/10.1021/jm300377g | J. Med. Chem. 2012, 55, 6391−6402

Journal of Medicinal Chemistry
Article
In the second step, phenols 3[1−24] (Table 3) were alkylated
by 2-chloro-N-phenylacetamides 2[1−32] and 2-chloro-N-
phenylpropionamides 2[33−38] to yield the final compounds.
The conditions for the alkylation were first investigated by
studying the reaction of 2-chloro-N-phenylacetamide 2[1] with
phenol 3[1]. A variety of reaction conditions were explored to
improve the conversion and avoid the alkylation of the amide
function. The chloroacetamide 2[1] was sufficiently reactive,
and the use of a catalyst such as KI, NaI, KBr, or NaBr did not
improve the reaction. K2CO3 was established as the most
efficient base for this reaction. The use of Cs2CO3 increased the
formation of the N-alkylated byproduct. On the contrary, the use
of DIEA or KOH led to complex mixtures. Polar aprotic solvents
such as CH3CN and DMF were established as the best solvents
for this reaction. DMF was preferred due to the higher solubility
of the starting materials and therefore its application in
automated parallel synthesis, especially to dispense solution. It
was found that heating was required to obtain a total conversion.
Finally, phenols 3[1−24] were deprotonated using potassium
carbonate in DMF (0.5 M) at room temperature, and the
alkylation with chloroacetamides 2[1−32] and chloropropiona-
mides 2[33−38] performed at 50 °C for 96 h led to the synthesis
of the 960-compound library (Scheme 1B). Chloroacetamides
2[19] and 2[20] were incorporated twice in the library to obtain
both protected and free carboxylic and amino derivatives thanks
to the cleavage of tert-butyl and Boc groups in acidic conditions
(50% TFA in CH2Cl2).
LIBRARY DESIGN AND SYNTHESIS
■
Anilines bearing various substituents were selected to generate
diverse pharmacophoric patterns. Secondary amines, cyclic or
acyclic, were also incorporated into the library, as well as amines
displaying two aromatic rings or anilines with acidic and basic
functions. Phenols with electron-withdrawing or electron-
donating groups on the ring were selected. Bulkier phenols
displaying naphthyl or adamantyl groups were also chosen to fill
the 3D diversity space. The 960-member virtual library was
enumerated using Pipeline Pilot. Druglike properties such as the
polar surface area (PSA), the number of rotatable bonds, clogP,
the molar mass, and the number of hydrogen bond donors and
acceptors were calculated for each member (Table 1). All the
a
Table 1. Calculated Properties of the Library Members
mean
std dev
range
molar mass (g/mol)
clogP
340.6
3.7
48.7
1.1
227.3−497.6
0.9−6.9
2−6
no. of H-bond acceptors
no. of H-bond donors
no. of rotatable bonds
PSA (Å2)
3.2
1.0
1.0
0.5
0−3
3−10
5.5
1.3
The synthesis of the library was carried out in 2D barcoded
tubes (Matrix) on a scale of 20 μmol, and the solutions of the
reactants in DMF were distributed using an automated liquid-
handling system (Tecan Genesis RSP 150). A total of 118 wells
were randomly analyzed by LC−MS to show that for 96% of
the analyzed library members the purity was higher than 80%
(see the Supporting Information).
56.2
18.1
29.5−118.4
a
All the properties were calculated using Pipeline Pilot from Accelrys.
library members were compliant with Veber’s rules and Lipinski’s
rule-of-five.27−29
The chemical synthesis of the library was developed according
to an optimized two-step synthesis protocol. The first step
consisted of an acetylation of anilines with chloroacetyl chloride
or 2-chloropropionyl chloride following a known procedure30
using dimethylacetamide as the solvent (Scheme 1A) to yield
(2-chlorophenyl)acetamide and (2-chlorophenyl)propionamide
(Table 2).
SCREENING RESULTS OF THE
N-PHENYLPHENOXYACETAMIDE LIBRARY
■
The library was screened using the thermal shift assay described
previously. Out of the 960 compounds screened at 20 μM,
a
Scheme 1. (A) Synthesis of 2-Chloroacetamide and 2-Chloropropionamide 2[1−38] and (B) Automated Parallel Synthesis of the
a
N-Phenylphenoxyacetamide Library (960 Members)
a
Reaction conditions: (a) DMA, 0 °C, then rt, 1−5 h; (b) phenol (0.5 M)/DMF (1 equiv), K2CO3 (2 equiv), 1 h, rt, then chloroacetamide (0.5 M)/
DMF (1 equiv), 96 h, 50 °C.
6394
dx.doi.org/10.1021/jm300377g | J. Med. Chem. 2012, 55, 6391−6402

Journal of Medicinal Chemistry
Article
Table 2. Structures of (2-Chlorophenyl)acetamides 2[1−32]
and (2-Chlorophenyl)propionamides 2[33−38]
Table 3. Structures of Phenols 3[1−24]
compd
R5
R6
compd
R5
R6
3[1]
3[2]
3[3]
3[4]
3[5]
3[6]
3[7]
3[8]
3[9]
3[10]
3[11]
3[12]
H
H
3[13]
3[14]
3[15]
3[16]
3[17]
3[18]
3[19]
3[20]
3[21]
3[22]
3[23]
3[24]
4-CN
H
H
H
H
H
H
H
H
H
H
H
compd
R1
R2
R3
R4
2-Me
4-Me
2-F
H
2-OMe
3-OMe
4-OMe
3-NMe2
2[1]
2[2]
2[3]
2[4]
2[5]
2[6]
2[7]
2[8]
2[9]
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-Me
2-OMe
3-OMe
4-OMe
4-Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3-F
4-F
H
H
2-Cl
3-Cl
4-Cl
3-Cl
2-CF3
4-CF3
3-CN
3-NHCOPh
3-OPh
H
H
H
H
4-Ph
3-Cl
4-Cl
4-Cl
H
4-adamantyl
4-(1,2,4-triazol-1-yl)
2-(1,3,4-oxadiazol-2-yl)
2,3-C4H4
3-CF3
4-CF3
H
H
H
2[10] 3-CN
2[11] 4-CN
2[12] 4-Ph
2[13] 4-OPh
2[14] 2-OPh
H
H
H
H
Table 4. ΔTm Distribution of EthR in Complex with the 960
H
4-OEt
Compounds of the Library
ΔTm
(°C)
no. of
compds
fraction of the
library (%)
ΔTm
(°C)
no. of
compds
fraction of the
library (%)
2[15] 3-CONHPh
2[16]
H
3,4-(−OCH2O−)
<1
421
248
166
43.9
25.8
17.3
5−7
7−9
9−11
102
21
2
10.6
2.2
2[17] 4-morpholin-4-yl
2[18] 4-(2-oxopyrrolidin-1-yl)
2[19] 4-CH2NH-Boc
H
H
H
H
H
H
H
H
H
1−3
3−5
0.2
a
2[19′] 4-CH2NH2
2[20] 4-CH2COOtBu
a
2[20′] 4-CH2COOH
or cyclohexyl was deleterious to activity (2[30], 2[31]), and sub-
stitution by a methyl group was more tolerated, but nonalkylated
analogues led to larger thermal shifts (2[1] vs 2[28] and 2[5] vs
2[29]). Interestingly, cyclization of aniline into 1,2,3,4-
tetrahydroquinoline was also tolerated (2[1] vs 2[32]). Substi-
tution on the ortho position of the phenyl was deleterious to
activity (2[3], 2[14], 2[21]). Heteroaromatic rings such as
4-benzothiazol-2-ylphenyl (2[22]) and 2-methylquinoline
(2[25]) showed good activity as well as 4-phenoxy (2[13])
and 3,4-methylenedioxy (2[16]) groups. Substitution of phenols
with 2-chloro-N-(3,4-dichlorophenyl)acetamide 2[7] led to the
maximum number of compounds that induced a positive shift in
the Tm of the protein higher than that of reference compound 1.
Smaller shifts of the melting temperature were obtained with
phenols (Figure 4B) bearing heteroaromatic substituents (3[22],
3[23]), a benzamide moiety (3[18]) or a tertiary nitrogen atom
(3[17]). The EthR ligand binding domain is a narrow hydro-
phobic tunnel, so bulky groups such as 1-naphtol (3[24]) or
trifluoro and methoxy groups in the ortho position of the phenol
(3[10] and 3[14]) were not tolerated as opposed to smaller
substituents such as a methyl group and fluorine and chlorine
atoms (3[2], 3[4], 3[6]). Para and meta substitutions were well
tolerated. 4-(Trifluoromethyl)phenol (3[11]), 3-methoxyphe-
nol (3[15]), and 3-phenoxyphenol (3[19]) derivatives showed a
maximum response.
The 23 compounds (2.4% of the library) displaying a shift of
the melting temperature higher than 7 °C (Table 4) were
selected for resynthesis on a larger scale (0.5 mmol) according to
Scheme 1 and retested to confirm the ΔTm values (Table 5).
In parallel, the ability of these 23 compounds (at 10 μM) to boost
a subactive dose of ethionamide (MIC/10) was determined by
measuring the replication of M. tuberculosis H37Rv-GFP inside
macrophages in an automated confocal microscopy assay.31−33
The results are expressed as bacterial growth inhibition (%)
(Table 5).
2[21] 2-Ph
2[22] 4-benzothiazol-2-yl
2[23] 3-pyrimidin-5-yl
2[24]
2[25]
2[26]
2[27]
2[28]
Het = 9-ethyl-9H-carbazol-3-yl
Het = 2-methylquinolin-6-yl
Het = 1-methyl-1H-indol-5-yl
Het = 2-(trifluoromethyl)-1H-benzimidazol-5-yl
H
H
CH3
2[29] 4-OMe
H
CH3
2[30]
2[31]
2[32]
2[33]
H
H
H
H
H
CH2Ph
H
cyclohexyl
2-(CH2CH2CH2−)
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
2[34] 4-Me
2[35] 4-OMe
2[36] 4-Cl
2[37] 3-Cl
2[38] 4-Ph
H
H
H
4-Cl
H
a
Chloroacetamides 2[19′] and 2[20′] were added to the table to
symbolize free carboxylic and free amino analogues of chloroaceta-
mides 2[19] and 2[20].
38 were found to induce a positive shift in the melting temp-
erature of the complex higher than the one observed with
reference compound 1. The distribution of shifts observed for
the entire library is presented in Table 4.
Figure 4 displays ranges of ΔTm according to the chloro-
acetamide and phenol building block series. Small temperature
shifts were obtained with chloropropionamides methylated
on the α-position (2[33−38]) compared to the nonmethylated
series (Figure 4A). Free carboxylic acid and amino groups
(2[19′], 2[20′]) were not well tolerated compared to their
protected analogues (2[19], 2[20]). Substitution of the amide
function on the nitrogen atom by a bulky group such as benzyl
6395
dx.doi.org/10.1021/jm300377g | J. Med. Chem. 2012, 55, 6391−6402

Journal of Medicinal Chemistry
Article
Figure 4. ΔTm distribution of EthR in complex with the 960-compound library according to (A) chloroacetamide building blocks 2[1−38] or (B)
phenol building blocks 3[1−24]. The images were generated with Miner3D.
Eight compounds (4, 5, 6, 9, 12, 14, 19, and 22) were
reconfirmed as positive as they still displayed ΔTm greater than
7 °C (Table 5). Among them, two compounds (4 and 5) were
active in vitro as potent ethionamide boosters (inh > 50%).
Compound 4 was the most active, with ΔTm = 10.6 °C and inh =
67.6%. Replacement of the lipophilic methoxy group by a more
polar cyano group on the phenol ring (compound 5) was less
tolerated (ΔTm = 8.1 °C and inh = 53%). In contrast, in the N-(2-
methylquinolin-6-yl)acetamide and N-(4-chlorophenyl)-
acetamide series, compounds 15 (inh = 83.2%) and 21 (inh =
92.6%) were active in vitro in the presence of ethionamide with
lower ΔTm values (5.1 and 6.1 °C, respectively). The most
active compounds (4, 5, 15, and 21) with inh > 50% at 10 μM
were selected for dose−response experiments performed
in the presence of ethionamide at 0.1 μg/mL (MIC/10) on
M. tuberculosis-infected macrophages (Table 6).31
All four compounds were able to boost ethionamide activity
10-fold in TB-infected macrophages with EC50 in the micro-
molar (compounds 15 and 21) or submicromolar (compounds 4
and 5) range. The N-(4-benzothiazol-2-ylphenyl)acetamide
series presented the best boosters. Compound 4 displaying
an EC50 of 0.21 μM was at least 50-fold more active than
reference compound 1 on TB-infected macrophages in
combination with a subactive dose of ethionamide. This
compound did not show any antituberculous activity when
tested alone at 10 μM (see the Supporting Information, Table 2
on p S8). The role of the phenoxy group was confirmed by the
synthesis of the sulfur analogue (compound 27) (Table 6).
Compound 27 was obtained according to Scheme 1 using
chloroacetamide 2[22] and 3-methoxythiophenol. This com-
pound displayed a ΔTm lower than that of compound 4 (6.2 °C)
and a weak activity ex vivo on TB-infected macrophages (EC50 >
10 μM).
Compound 4 was cocrystallized with EthR (Figure 5). X-ray
diffraction data revealed that, like compound 1, the amide
function of compound 4 is hydrogen bonded to the side chains of
Asn179 and Asn176. However, compound 4 has rotated by 180°
around the carbonyl function, pointing its m-methoxyphenoxy
moiety to the bottom of the ligand binding domain. While
compound 1 penetrates deeper into the binding pocket with its
p-toluyl group, filling a small hydrophobic cavity opened by the
rotation of Phe184, in the X-ray structure of EthR in complex
with compound 4, this Phe184 moves to close the gate and faces
the m-methoxyphenoxy ring, which also interacts with Trp138
and Phe114. On the other hand, at the portal of the pocket, the
phenylbenzothiazole core of compound 4 binds to the side
chains of aliphatic Met102 and Val152 and aromatic Trp103 and
Tyr148 and occupies a space that was filled in the compound
1−EthR complex with a water network. These additional
hydrophobic contacts significantly increase the ligand−protein
binding surface and thereby could explain the higher activity of
compound 4 relative to compound 1.
6396
dx.doi.org/10.1021/jm300377g | J. Med. Chem. 2012, 55, 6391−6402

Journal of Medicinal Chemistry
Article
Table 5. Biological Activities of Compounds 4−26
CONCLUSION
■
In this study, we discovered new EthR inhibitors by the screening
of a 14640-compound library on M. smegmatis. Compound 1 was
shown to be a genuine EthR ligand by X-ray diffraction of a
cocrystal. Optimization was made possible by the two-step
synthesis of a focused library of N-phenylphenoxyacetamide
derivatives. The screening of the library on the purified target
protein using a thermal shift assay followed by in vitro testing in
human macrophages infected by M. tuberculosis led to the
identification of two potent N-(4-benzothiazol-2-ylphenyl)-2-
phenoxyacetamide derivatives. Compounds 4 and 5 were at least
50-fold more active than hit compound 1 in boosting a subactive
dose of ethionamide on TB-infected macrophages. Finally, the
cocrystallization of compound 4 with EthR revealed a new
binding mode compared to that of the hit analogue 1. We believe
that the development of a screening cascade involving a whole cell
phenotypic assay and a target-based physical assay was critical to
the discovery and rapid design of new potent EthR inhibitors and
could therefore be a valuable tool for the identification of other
inhibitors of mycobacterial transcriptional regulators.
a
b
ΔTm ΔTm
(°C) (°C)
inh (%) at
c
compd
R1
R2
R5
10 μM
1
Het = 2-(4-methylphenyl)- 2-Me
2H-1,2,3-benzotriazol-5-yl
6.4
6.4
10.9 1.1
4
5
4-benzothiazol-2-yl
H
H
3-OMe
3-CN
9.9
8.2
8.9
7.0
8.1
9.6
7.7
7.7
7.0
7.5
8.8
7.2
7.2
7.3
7.6
10.6
8.1
9.1
6.8
6.1
8.1
67.6 0.4
53.4 7.6
−1.6 3.7
26.7 2.2
−0.6 0.2
−8.4 1.7
4-benzothiazol-2-yl
6
3-Cl
4-Cl 3-OPh
4-Cl 3-OMe
4-Cl 4-OMe
4-Cl 4-CF3
4-Cl 4-Cl
4-Cl 4-Me
4-Ph
7
3-Cl
8
3-Cl
9
3-Cl
10
11
12
13
14
3-Cl
6.9 −17.9 20.9
6.8 −10.4 5.7
7.0
6.2
8.2
5.1
6.6
3-Cl
3,4-(−OCH2O−)
3,4-(−OCH2O−)
3,4-(−OCH2O−)
−3.0 0.05
1.4 3.2
4-CF3
3-OPh
5.7 4.3
15 Het = 2-methylquinolin-6-yl 2-F
16 Het = 2-methylquinolin-6-yl 3-OMe
83.2 0.9
6.5 2.4
EXPERIMENTAL SECTION
■
17
18
19
20
21
22
23
24
25
26
4-OPh
4-OPh
4-CF3
4-CF3
4-Cl
H
H
H
H
H
H
H
H
H
H
3,4-diF
3-OMe
5.7 −13.5 1.0
6.7 −17.5 16.3
7.5
Biology. High-Throughput in Bacterio Assay. The shuttle plasmid
pMV261 was used to overexpress in M. smegmatis the reporter gene
uidA, which codes for the β-glucuronidase (GUS) of Escherichia coli. The
promoter/operator sequence of ethA was polymerase chain reaction
(PCR)-amplified using oligonucleotides O-315 (5′ GCC-TCT-AGA-
CAG-CGA-AGC-CTG-ACT-GG 3′) and O-316 (5′ CTC-GGC-CAT-
GGA-TCC-ACG-CTA-TCA-AC 3′), which contain restriction sites
XbaI and NcoI, respectively. The PCR product was digested and cloned
in plasmid pModTin,34 previously digested with the restriction enzymes
ScaI and NcoI. The resulting plasmid pModTin-PrethA was digested
with XbaI and EcoRV and cloned in pMV261 previously digested with
the same enzymes. These manipulations resulted in the mycobacterial
shuttle plasmid pMV261-PrethA-Tin (called P2 in Figure 1), in which
uidA is under the control of the ethA promoter/operator.
3,4-diCl 7.7
−5.4 2.8
3,4-diF
3-OMe
3-OMe
3-OPh
3-OPh
3-OPh
4-CF3
8.0
8.1
7.0
7.0
7.0
7.3
7.2
5.6 −12.5 6.9
6.1
7.4
6.1
6.7
5.7
5.0
92.6 0.3
−2.9 2.7
2.2 4.6
4-Ph
H
4-CN
3-CN
3-OMe
−8.9 9.1
−7.2 5.8
−1.5 0.4
c
a
b
ΔTm from the screening. ΔTm measured after resynthesis. Inh (%)
represents the inhibitition of M. tuberculosis growth in macrophages
with a combination of ethionamide at 0.1 μg/mL (normal MIC/10)
and each ligand at 10 μM. Inh values are the mean of two experiments.
Table 6. Biological Activities of Compounds 1, 4, 5, 15, 21, and 27
a
b
ΔTm values are the mean of two experiments. SD was equal to 0.2 °C. EC50 represents the concentration of ligand that allows ethionamide at 0.1
μg/mL (normal MIC/10) to inhibit 50% of M. tuberculosis growth in macrophages. EC50 values are the mean of two experiments. SD was <10%.
6397
dx.doi.org/10.1021/jm300377g | J. Med. Chem. 2012, 55, 6391−6402

Journal of Medicinal Chemistry
Article
Figure 5. (A) X-ray structure representation of the ligand-binding pocket of EthR filled with compound 4 (PDB ID 4DW6) surrounded by its initial
omit Fo−Fc map at the 3.0σ contour level. The Fo−Fc map was calculated prior to addition of the ligand to the model. (B) X-ray structure representation
of the homodimeric conformation of EthR filled in both monomers with compound 4. The surface of the ligand binding domain is highlighted.
Hydrogen bonds with Asn179 and Asn176 are represented with dotted lines. Color legend: blue (compound 4) or green (EthR) = carbon, dark blue =
nitrogen, red = oxygen, yellow = sulfur. The images were generated with Pymol.
In parallel, plasmid pMV261-ethR described previously8 was digested
with enzymes XbaI and SpeI, and the fragment containing ethR under the
control of the hsp60 promoter was inserted into the same restriction
sites of the single copy number plasmid pBSh-D35 to create pBSh-ethRb
(called P1 in Figure 1).
by centrifugation at 1100 rpm for 5 min, the remaining extracellular bacilli
from the infected cell suspension were killed by a 1 h amykacin (20 μM,
Sigma, A2324-5G) treatment. After a final centrifugation step, 40 μL of
M. tuberculosis H37Rv-GFP colonized macrophages were dispensed
with the Wellmate (Matrix) into 384-well Evotec plates preplated with
10 μL of compound mixture diluted in cell medium and incubated for
5 days at 37 °C, 5% CO2. Macrophages were then stained with SYTO 60
(Invitrogen, S11342) for 1 h followed by plate sealing. Confocal images
were recorded on an automated fluorescent ultra-high-throughput
microscope, Opera (Evotec). This microscope is based on an inverted
microscope architecture that allows imaging of cells cultivated in 96- or
384-well microplates (Evotec). Images were acquired with a 20× water
immersion objective (NA = 0.70). A double laser excitation (488 and
635 nm) and dedicated dichroic mirrors were used to record green
fluorescence of mycobacteria and red fluorescence of the macrophages
on two different cameras, respectively. A series of four pictures at the
center of each well were taken, and each image was then processed using
dedicated image analysis.31,32 The percentage of infected cells and the
number of cells are the two parameters extracted from image analysis as
previously reported.32 Reported data are the average of two replicates.
Potency Assay of Test Compounds on M. tuberculosis
(Ethionamide Concentration Fixed at 0.1 μg/mL, Serial Dilution of
Test Compounds). Ethionamide (Sigma, E6005-5G) was diluted in
DMSO at 10 mg/mL, and aliquots were stored frozen at −20 °C.
Test compounds were resuspended in pure DMSO at a concentration of
4 mg/mL. Ten 2-fold serial dilutions of compounds were subsequently
performed in DMSO in Greiner 384-well V-shaped polypropylene
plates (Greiner, no. 781280). Equal volumes (5 μL) of diluted com-
pounds and of ethionamide were transferred to a 384-well low-volume
polypropylene plate (Corning, no. 3672). Two independent replicates
were done for each setting. On the day of the experiment, 0.5 μL of
compound per plate was transferred by the EVOBird platform (Evotec)
to cell assay plates preplated with 10 μL of assay medium.
M. smegmatis mc215536 was transformed with plasmid P2 (pMV261-
PrethA-Tin) alone, which resulted in the high production of
glucuronidase and was used as a positive control in our screening
assay. Alternatively, M. smegmatis mc2155 was transformed conjointly
with plasmid P2 (pMV261-PrethA-Tin) and plasmid P1 (pBSh-ethRb),
in which the production of EthR represses completely the expression of
the reporter cassette PrethA-Tin, resulting in the absence of detectable
glucuronidase activity. M. smegmatis clones were cultivated in Sauton
medium supplemented with Triton WR1339.
The high-throughput screen was performed in 96-well plates
(Optiplate 96F, Perkin-Elmer) containing in each well 123 μL of the
mycobacterial culture (optical density of 0.2 at 600 nm), 10 μL of a
50 μM solution of MUG (Calbiochem), and 67 μL of a 33 μM 10%
DMSO solution of each compound of the library. The spectrophoto-
metric measure was done after 0, 24, and 48 h of incubation at 37 °C
using a VICTOR3-V (1420 multilabel counter, Perkin-Elmer) set to an
excitation wavelength of 390 nm and an emission wavelength at 405 nm.
Thermal Shift Assay. The fluorescent dye SYPRO Orange
(Invitrogen) was used to monitor protein unfolding. This dye is
environmentally sensitive and leads to an increase in fluorescence
following exposure of hydrophobic regions during protein unfolding.
The thermal shift assay was conducted in a Lightcycler 480 (Roche).
The system contained a heating/cooling device for temperature control
and a charge-coupled device (CCD) detector for real-time imaging of
the fluorescence changes in the wells of the microplate. The final sample
concentrations were 10 μM EthR, 2.5× SYPRO Orange, 1% DMSO,
and 20 μM ligand in the EthR buffer. The samples were heated from
37 to 85 °C with a heating rate of 0.04 °C/s. The fluorescence intensity
was measured at Ex/Em = 465/510 nm. The data were obtained using
the algorithmic program Wavemetrics Igor by applying the following
designed procedure: The fluorescence intensity of each well/sample is
plotted as a function of the temperature. Then the 1D numerical
derivative of these curves is calculated. At last, the maximum data values,
corresponding to the inflection points (Tm), are extracted to give Tm in a
table and in a graph.
Crystal Structure Determination of EthR−Ligand Complexes.
N-terminally hexahistidine-tagged EthR was produced in E. coli C41
(pET-15b-ethR) and purified as described. Prior to crystallization, the
protein was buffer exchanged against 10 mM Tris−HCl (pH 7.5) and
200 mM NaCl and concentrated to 9 mg/mL. A crystal was obtained by
the vapor diffusion method, 0.1 mM MES, pH 6.5, 1.4−1.65 M
ammonium sulfate (using a 0.05 M increment), and 10−15% glycerol
as the crystallization solution. Ligands were dissolved in 100% DMSO.
The EthR−ligand complexes were prepared by mixing 1 μL of ligand
(33 mM) and 9 μL of protein solution (9 mg/mL) and equilibrated for
Intracellular Assay. Raw264.7 macrophages (108 cells) were infected
with an H37Rv-GFP suspension at a multiplicity of infection (MOI) of
1:1 in 300 mL for 2 h at 37 °C with shaking (100 rpm). After two washes
6398
dx.doi.org/10.1021/jm300377g | J. Med. Chem. 2012, 55, 6391−6402

Journal of Medicinal Chemistry
Article
NH); 13C NMR (75 MHz, CDCl3) δ 55.43, 67.63, 101.60, 106.80,
108.03, 120.07, 121.62, 123.10, 125.15, 126.37, 128.49, 130.02, 130.47,
134.99, 139.28, 154.16, 158.06, 161.11, 166.34, 167.27; MS [M + H]+
m/z 391; HRMS (TOF-MS, ES+) m/z calcd for C22H18N2O3S [M + H]+
391.1116, found 391.1115.
Table 7. Data Collection Statistics
compd 1
compd 4
X-ray source
temp (K)
ESRF ID14−2
100
SLS
100
Data for N-(4-benzothiazol-2-ylphenyl)-2-(3-cyanophenoxy)-
wavelength (Å)
space group
0.93300
1.00000
1
acetamide (5): beige powder; yield 61%; purity 95%; H NMR (300
P41212
P41212
MHz, CDCl3) δ (ppm) 4.69 (s, 2H), 7.28−7.32 (m, 2H), 7.39−7.44 (m,
2H), 7.49−7.54 (m, 2H), 7.78 (d, J = 8.7 Hz, 2H), 7.93 (d, J = 7.5 Hz,
1H), 8.08 (d, J = 7.8 Hz, 1H), 8.13 (d, J = 8.7 Hz, 2H), 8,34 (br s, 1H,
NH); 13C NMR (75 MHz, CDCl3) δ 67.66, 114.03, 118.07, 118.52,
119.50, 120.17, 121.67, 123.17, 125.26, 126.46, 128.63, 130.35, 131.10,
135.07, 139.04, 154.16, 156.93, 165.17, 167.19; MS [M + H]+ m/z 386;
HRMS (TOF-MS, ES+) m/z calcd for C22H15N3O2S [M + H]+
386.0963, found 386.0966.
cell constants (Å)
a = b = 121.2, c = 33.8
2.0 (2.0−2.1)
99.8 (100.0)
6.3 (23.0)
a = b = 121.4, c = 33.8
2.0 (2.0−2.1)
99.7 (98.3)
5.9 (41.7)
a
resolution (Å)
completeness (%)
R
sym (%)
multiplicity
14.1(14.4)
8.8 (9.2)
I/σ(I)
22.6 (5.4)
27.5 (5.6)
no. of total reflns
no. of unique reflns
249020 (33903)
17607 (2348)
156698 (23020)
17743 (2498)
Data for N-(3,4-dichlorophenyl)-2-(3-phenoxyphenoxy)-
1
acetamide (6): white powder; yield 48%; purity 98%; H NMR (300
a
The number in parentheses is the statistic for the highest resolution
shell.
MHz, CDCl3) δ (ppm) 4.60 (s, 2H), 6.65 (t, J = 2.4 Hz, 1H), 6.70−6.75
(m, 2H), 7.05−7.08 (m, 2H), 7.18 (t, J = 7.5 Hz, 1H), 7.31 (t, J = 8.1 Hz,
1H), 7.37−7.44 (m, 4H), 7.85 (d, J = 1.8 Hz, 1H), 8,30 (br s, 1H, NH);
13C NMR (75 MHz, CDCl3) δ 67.50, 105.59, 108.90, 112.64, 119.31,
119.51, 121.77, 123.99, 128.21, 129.94, 130.64, 130.74, 132.95, 136.20,
156.68, 157.95, 159.09, 166.07; MS [M − H]− m/z 386.
30 min at room temperature. All crystals belong to space group P41212.
Data collection statistics are summarized in Table 7. The diffraction data
were processed with XDS. The structure has been refined using
REFMAC537 from the CCP4 suite to the resolution indicated in
Table 7. The ligands were positioned in the electron density with
Coot.38 The final Rwork (Rfree) for compounds 1 and 4 are 18.9% (23.1%)
and 18.6% (21.9%), respectively. The analysis of protein−ligand inter-
actions was performed with the aid of the LIGPLOT program.39 PDB
access codes are 3Q3S and 4DW6 for compounds 1 and 4, respectively.
Chemistry. General Information. NMR spectra were recorded on a
Bruker DRX-300 spectrometer. Chemical shifts are in parts per million
(ppm). The assignments were made using 1D 1H and 13C spectra and
2D HSQC and COSY spectra. Mass spectra were recorded with an LC−
MSMS triple-quadrupole system (Varian 1200ws) or an LC−MS
(Waters Alliance Micromass ZQ 2000). LC−MS analysis was per-
formed using a C18 TSK-GEL Super ODS 2 μm particle size column,
dimensions 50 × 4.6 mm, and a 1 mL/min flow rate. The solvent
systems used were 100% water containing 0.1% formic acid (solvent A)
and a mixture of 20% water and 80% acetonitrile containing 0.1% formic
acid (solvent B). A gradient from 100% solvent A to 100% solvent B over
7.5 min was used. The final solvent system was held constant for 1 min.
High-resolution mass spectra were recorded on an HPLC−MS-TOF
system (Waters LCT Premier). Preparative HPLC was performed using
a Varian PRoStar system using an OmniSphere 10 column (C18, 250 ×
41.4 mm, Dynamax) from Varian, Inc. and a 80 mL/min flow rate.
The solvent systems used were 100% water containing 0.1% formic acid
(solvent A) and 100% acetonitrile or methanol containing 0.1% formic
acid (solvent B). A gradient from 80% solvent A and 20% solvent B to
100% solvent B was used. Purity (%) was determined by reversed-phase
HPLC using UV detection (215 nm), and all compounds showed
purities greater than 95%. All commercial reagents and solvents were
used without further purification. Compound 1 was purchased from
ChemDiv.
Data for N-(3,4-dichlorophenyl)-2-(3-methoxyphenoxy)-
1
acetamide (7): white powder; yield 78%; purity 99%; H NMR (300
MHz, CDCl3) δ (ppm) 3.83 (s, 3H), 4.60 (s, 2H), 6.55−6.66 (m, 3H),
7.23−7.29 (m, 1H), 7.40−7.47 (m, 2H), 7.84 (d, J = 2.1 Hz, 1H), 8.28
(br s, 1H, NH); MS [M − H]+ m/z 326.
Data for N-(3,4-dichlorophenyl)-2-(4-methoxyphenoxy)-
1
acetamide (8): white powder; yield 54%; purity 98%; H NMR (300
MHz, DMSO-d6) δ (ppm) 3.69 (s, 3H), 4.63 (s, 2H), 6.86−6.89 (m,
2H), 6.93−6.96 (m, 2H), 7.56−7.63 (m, 2H), 8.03 (d, J = 2.1 Hz, 1H),
10.32 (br s, 1H, NH); MS [M + H]+ m/z 326.
Data for N-(3,4-dichlorophenyl)-2-[4-(trifluoromethyl)phenoxy]-
acetamide (9): white powder; yield 71%; purity >99%; 1H NMR (300
MHz, CDCl3) δ (ppm) 4.68 (s, 2H), 7.09 (d, J = 8.4 Hz, 2H), 7.42−7.48
(m, 2H), 7.66 (d, J = 8.4 Hz, 2H), 7.86 (d, J = 1.8 Hz, 1H), 8.24 (br s, 1H,
NH); 13C NMR (75 MHz, CDCl3) δ 67.43, 114.88, 119.31, 121.82,
124.02 (q, J = 272 Hz), 125.02 (q, J = 33 Hz), 127.50, 128.43, 130.69,
133.03, 136.06, 159.02, 165.45; MS [M − H]− m/z 362.
Data for 2-(4-chlorophenoxy)-N-(3,4-dichlorophenyl)acetamide
(10): beige powder; yield 68%; purity 98%; 1H NMR (300 MHz,
CDCl3) δ (ppm) 4.60 (s, 2H), 6.93−6.96 (m, 2H), 7.32−7.35 (m, 2H),
7.43−7.47 (m, 2H), 7.85 (d, J = 1.5 Hz, 1H), 8.25 (br s, 1H, NH); MS
[M − H]− m/z 328.
Data for N-(3,4-dichlorophenyl)-2-p-tolyloxyacetamide (11):
white powder; yield 33%; purity >99%; 1H NMR (300 MHz, DMSO-
d6) δ (ppm) 2.23 (s, 3H), 4.66 (s, 2H), 6.89 (d, J = 8.1 Hz, 2H), 7.10 (d,
J = 8.1 Hz, 2H), 7.55−7.62 (m, 2H), 8.02 (d, J = 1.5 Hz, 1H), 10.33 (br s,
1H, NH); MS [M − H]− m/z 308.
Data for N-1,3-benzodioxol-5-yl-2-(biphenyl-4-yloxy)acetamide
(12): beige powder; yield 49%; purity 95%; 1H NMR (300 MHz,
CDCl3) δ (ppm) 4.66 (s, 2H), 5.99 (s, 2H), 6.80 (d, J = 8.4 Hz, 1H),
6.91 (dd, J = 2.1 Hz, J = 8.4 Hz, 1H), 7.05−7.10 (m, 2H), 7.33 (d, J = 2.1
Hz, 1H), 7.35−7.38 (m, 1H), 7.43−4.78 (m, 2H), 7.57−7.62 (m, 4H),
8.22 (br s, 1H, NH); 13C NMR (75 MHz, CDCl3) δ 67.68, 101.41,
103.02, 108.16, 113.53, 115.14, 126.83, 127.11, 128.57, 128.85, 131.02,
135.58, 140.28, 144.78, 147.93, 156.51, 166.05; MS [M + H]+ m/z 348.
Data for N-1,3-benzodioxol-5-yl-2-[4-(trifluoromethyl)phenoxy]-
acetamide (13): beige powder; yield 66%; purity 99%; 1H NMR (300
MHz, DMSO-d6) δ (ppm) 4.77 (s, 2H), 5.98 (s, 2H), 6.86 (d, J = 8.4 Hz,
1H), 7.01 (dd, J = 2.1 Hz, J = 8.4 Hz, 1H), 7.14−7.18 (m, 2H), 7.30 (d,
J = 2.1 Hz, 1H), 7.66−7.70 (m, 2H), 10.04 (br s, 1H, NH); 13C NMR
(75 MHz, DMSO-d6) δ 67.48, 101.51, 102.32, 108.50, 113.13, 115.68,
122.14 (q, J = 33 Hz), 124.98 (q, J = 272 Hz), 127.45, 133.06, 143.75,
147.52, 161.15, 166.08; MS [M + H]+ m/z 340.
General Procedure for the Synthesis of N-Phenylphenoxyaceta-
mides 4−26. K2CO3 (2.1 equiv, 1.05 mmol, 145 mg) and appropriate
phenol (1.05 equiv, 0.53 mmol) were mixed with 2 mL of DMF. The
reaction mixture was stirred for 1 h, and then a solution of appropriate
2-chloroacetamide (1 equiv, 0.5 mmol) in 2 mL of DMF was added to
the mixture. The reaction was stirred overnight at room temperature and
then evaporated under reduced pressure. The residue was dissolved in
AcOEt, and the organic phase was washed twice with water, once with a
solution of HCl (1 N), and once with brine, then dried over MgSO4, and
evaporated under reduced pressure. The product was purified by flash
chromatography or preparative HPLC.
Data for N-(4-benzothiazol-2-ylphenyl)-2-(3-methoxyphenoxy)-
acetamide (4): beige powder; yield 61%; purity 98%; H NMR (300
1
MHz, CDCl3) δ (ppm) 3.85 (s, 3H), 4.65 (s, 2H), 6.59−6.67 (m, 3H),
7.28 (t, J = 8.1 Hz, 1H), 7.40 (dd, J = 7.65 Hz, J = 7.8 Hz, 1H), 7.51 (dd,
J = 7.65 Hz, J = 8.1 Hz, 1H), 7.78 (d, J = 8.7 Hz, 2H), 7.92 (d, J = 7.8 Hz,
1H), 8.08 (d, J = 8.1 Hz, 1H), 8.11 (d, J = 8.7 Hz, 2H), 8,44 (br s, 1H,
Data for N-1,3-benzodioxol-5-yl-2-(3-phenoxyphenoxy)-
acetamide (14): beige powder; yield 61%; purity 95%; 1H NMR (300
MHz, DMSO-d6) δ (ppm) 4.64 (s, 2H), 5.98 (s, 2H), 6.59 (dd, J =
1.8 Hz, J = 8.1 Hz, 1H), 6.65 (t, J = 2.1 Hz, 1H), 6.76 (dd, J = 2.1 Hz,
6399
dx.doi.org/10.1021/jm300377g | J. Med. Chem. 2012, 55, 6391−6402

Journal of Medicinal Chemistry
Article
J = 8.1 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 7.00−7.04 (m, 3H), 7.12−7.17
(m, 1H), 7.27−7.40 (m, 4H), 9.94 (br s, 1H, NH); MS [M + H]+ m/z 364.
Data for 2-(2-fluorophenoxy)-N-(2-methylquinolin-6-yl)-
(m, 2H), 7.11−7.16 (m, 1H), 7.30 (t, J = 8.1 Hz, 1H), 7.35−7.40 (m,
2H), 7.77−7.82 (m, 4H), 10.49 (br s, 1H, NH); 13C NMR (75 MHz,
DMSO-d6) δ 67.59, 105.80, 105.94, 110.00, 111.67, 119.36, 120.13,
124.17, 130.51, 131.07, 133.74, 143.04, 156.67, 158.35, 159.51, 167.67;
MS [M − H]− m/z 343.
1
acetamide (15): white powder; yield 13%; purity >99%; H NMR
(300 MHz, CDCl3) δ (ppm) 2.78 (s, 3H), 4.74 (s, 2H), 7.03−7.23 (m,
4H), 7.33 (d, J = 8.4 Hz, 1H), 7.68 (dd, J = 2.4 Hz, J = 9.0 Hz, 1H), 8.09
(d, J = 9.0 Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H), 8.42 (d, J = 2.4 Hz, 1H),
8.72 (br s, 1H, NH); 13C NMR (75 MHz, CDCl3) δ 24.52, 69.19,
116.30, 116.46, 116.79 (d, J = 18 Hz), 122.93, 123.45 (d, J = 7 Hz),
123.52, 124.87 (d, J = 4 Hz), 127.00, 128.65, 134.30, 136.90, 144.45,
145.35 (d, J = 10 Hz), 152.80 (d, J = 245 Hz), 158.24, 166.17; MS [M +
H]+ m/z 311; HRMS (TOF-MS, ES+) m/z calcd for C18H15FN2O2
[M + H]+ 311.1196, found 311.1191.
Data for N-(3-cyanophenyl)-2-(3-phenoxyphenoxy)acetamide
1
(25): white powder; yield 53%; purity >99%; H NMR (300 MHz,
DMSO-d6) δ (ppm) 4.72 (s, 2H), 6.60 (dd, J = 1.8 Hz, J = 8.1 Hz, 1H),
6.65 (t, J = 2.1 Hz, 1H), 6.78 (dd, J = 1.8 Hz, J = 8.1 Hz, 1H), 6.97−7.03
(m, 2H), 7.11−7.16 (m, 1H), 7.31 (t, J = 8.1 Hz, 1H), 7.35−7.41 (m,
2H), 7.49−7.57 (m, 2H), 7.85−7.91 (m, 1H), 8.09−8.11 (m, 1H), 10.39
(br s, 1H, NH); MS [M − H]− m/z 343.
Data for N-(3-methoxyphenyl)-2-[4-(trifluoromethyl)phenoxy]-
acetamide (26): white powder; yield 46%; purity 95%; 1H NMR
(300 MHz, DMSO-d6) δ (ppm) 3.72 (s, 3H), 4.81 (s, 2H), 6.64−6.68
(m, 1H), 7.15−7.35 (m, 4H), 7.31 (t, J = 2.1 Hz, 1H), 7.66−7.71 (m,
2H), 10.13 (br s, 1H, NH); MS [M − H]− m/z 324.
N-(4-Benzothiazol-2-ylphenyl)-2-[3-(methoxyphenyl)sulfanyl]-
acetamide (27). K2CO3 (1.05 equiv, 0.53 mmol, 73 mg) and
3-methoxythiophenol (1.05 equiv, 0.53 mmol) were mixed with 2 mL
of DMF. The reaction mixture was stirred for 1 h, and then a solution
of N-(4-benzothiazol-2-ylphenyl)-2-chloroacetamide (2[22]; 1 equiv,
0.5 mmol, 151 mg) in 2 mL of DMF was added to the mixture. The
reaction was stirred overnight at room temperature and then evaporated
under reduced pressure. The residue was dissolved in AcOEt, and the
organic phase was washed twice with water, once with a solution of HCl
(1 N), and once with brine, then dried over MgSO4, and evaporated
under reduced pressure. The product was purified by flash
chromatography (cyclohexane/AcOEt = 95/5 to 9/1): beige powder;
yield 72%; purity 95%; 1H NMR (300 MHz, CDCl3) δ (ppm) 3.80 (s,
3H), 3.82 (s, 2H), 6.80 (dd, J = 2.4 Hz, J = 8.1 Hz, 1H), 6.92−6.97 (m,
2H), 7.27 (t, J = 8.1 Hz, 1H), 7.39 (dd, J = 7.8 Hz, J = 8.1 Hz, 1H), 7.51
(dd, J = 7.8 Hz, J = 8.1 Hz, 1H), 7.66 (d, J = 8.7 Hz, 2H), 7.91 (d, J = 7.8
Hz, 1H), 8.07 (d, J = 7.8 Hz, 1H), 8.07 (d, J = 8.7 Hz, 2H), 8,72 (br s, 1H,
NH); 13C NMR (75 MHz, CDCl3) δ 38.46, 55.34, 112.89, 114.04,
119.85, 120.46, 121.62, 123.05, 125.15, 126.38, 128.42, 129.82, 130.46,
134.94, 135.17, 139.75, 154.08, 160.02, 166.35, 167.31; MS [M + H]+
m/z 407.
Data for 2-(3-methoxyphenoxy)-N-(2-methylquinolin-6-yl)-
1
acetamide (16): white powder; yield 13%; purity >99%; H NMR
(300 MHz, CDCl3) δ (ppm) 2.75 (s, 3H), 3.84 (s, 3H), 4.68 (s, 2H),
6.59−6.67 (m, 3H), 7.25−7.31 (m, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.65
(dd, J = 2.4 Hz, J = 9.0 Hz, 1H), 8.02 (d, J = 9.0 Hz, 1H), 8.06 (d, J = 8.4
Hz, 1H), 8.37 (d, J = 2.4 Hz, 1H), 8.48 (br s, 1H, NH); 13C NMR (75
MHz, CDCl3) δ 25.23, 55.41, 67.71, 101.61, 106.81, 108.01, 116.64,
122.73, 123.16, 126.87, 129.50, 130.51, 134.00, 136.14, 145.42, 158.12,
158.41, 161.11, 166.44; MS [M + H]+ m/z 323.
Data for 2-(3,4-difluorophenoxy)-N-(4-phenoxyphenyl)-
acetamide (17): beige powder; yield 87%; purity 96%; 1H NMR (300
MHz, CDCl3) δ (ppm) 4.59 (s, 2H), 6.71−6.76 (m, 1H), 6.84−6.91 (m,
1H), 7.00−7.05 (m, 4H), 7.10−7.21 (m, 2H), 7.35−7.38 (m, 2H),
7.54−7.58 (m, 2H), 8.16 (br s, 1H, NH); 13C NMR (75 MHz, CDCl3) δ
68.22, 105.07 (d, J = 20 Hz), 110.05, 117.78 (d, J = 20 Hz), 118.63,
119.59, 122.00, 123.31, 129.80, 131.99, 146.16 (dd, J = 13 Hz, J = 243
Hz), 150.72 (dd, J = 14 Hz, J = 249 Hz), 153.12 (d, J = 8 Hz), 154.23,
157.31, 165.39; MS [M + H]+ m/z 356.
Data for 2-(3-methoxyphenoxy)-N-(4-phenoxyphenyl)acetamide
1
(18): white powder; yield 71%; purity >99%; H NMR (300 MHz,
DMSO-d6) δ (ppm) 3.73 (s, 3H), 4.66 (s, 2H), 6.55−6.59 (m, 3H),
6.95−7.02 (m, 4H), 7.07−7.12 (m, 1H), 7.18−7.23 (m, 1H), 7.34−7.39
(m, 2H), 7.64−7.67 (m, 2H), 10.09 (br s, 1H, NH); MS [M + H]+
m/z 350.
Data for 2-(3,4-dichlorophenoxy)-N-[4-(trifluoromethyl)phenyl]-
acetamide (19): white powder; yield 76%; purity >99%; 1H NMR (300
MHz, DMSO-d6) δ (ppm) 4.82 (s, 2H), 7.01−7.09 (m, 1H), 7.30−7.36
(m, 1H), 7.52−7.60 (m, 1H), 7.66−7.73 (m, 2H), 7.82−7.88 (m, 2H),
10.46 (br s, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ 67.77, 116.05,
117.35, 120.00, 123.57, 124.21 (q, J = 32 Hz), 124.78 (q, J = 272 Hz),
126.59, 131.45, 131.99, 142.35, 157.72, 167.11; MS [M − H]− m/z 362.
Data for 2-(3,4-difluorophenoxy)-N-[4-(trifluoromethyl)phenyl]-
acetamide (20): white powder; yield 70%; purity >99%; 1H NMR (300
MHz, DMSO-d6) δ (ppm) 4.77 (s, 2H), 6.83−6.87 (m, 1H), 7.12−7.19
(m, 1H), 7.33−7.43 (m, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 8.4
Hz, 2H), 10.43 (br s, 1H, NH); MS [M − H]− m/z 330.
ASSOCIATED CONTENT
■
S
* Supporting Information
General procedure for the synthesis of 2-chloroacetamide and
2-chloropropionamide, characterization of starting materials
2[1−38], general procedure for the synthesis of the library and
purity of 118 compounds randomly selected from the library, and
effect of compound 4 on the susceptibility of M. tuberculosis
H37Rv measured by Bactec MGIT960. This material is available
free of charge via the Internet at http://pubs.acs.org.
Data for N-(4-chlorophenyl)-2-(3-methoxyphenoxy)acetamide
1
(21): white powder; yield 60%; purity >99%; H NMR (300 MHz,
DMSO-d6) δ (ppm) 3.73 (s, 3H), 4.67 (s, 2H), 6.55−6.58 (m, 3H),
7.17−7.23 (m, 1H), 7.36−7.39 (m, 2H), 7.66−7.69 (m, 2H), 10.20 (br
s, 1H, NH); 13C NMR (75 MHz, CDCl3) δ 55.60, 67.67, 101.62, 107.22,
107.39, 121.75, 127.78, 129.11, 130.50, 137.80, 159.43, 160.92, 167.17;
MS [M + H]+ m/z 292.
Data for N-biphenyl-4-yl-2-(3-methoxyphenoxy)acetamide (22):
white powder; yield 52%; purity >99%; 1H NMR (300 MHz, DMSO-d6)
δ (ppm) 3.74 (s, 3H), 4.70 (s, 2H), 6.56−6.60 (m, 3H), 7.18−7.24 (m,
1H), 7.29−7.35 (m, 1H), 7.42−7.47 (m, 2H), 7.63−7.66 (m, 4H),
7.73−7.76 (m, 2H), 10.17 (br s, 1H, NH); MS [M + H]+ m/z 334.
Data for 2-(3-phenoxyphenoxy)-N-phenylacetamide (23): white
powder; yield 45%; purity 98%; 1H NMR (300 MHz, CDCl3) δ (ppm)
4.60 (s, 2H), 6.67−6.69 (m, 1H), 6.71−6.75 (m, 2H), 7.07 (d, J = 7.8
Hz, 2H), 7.16−7.20 (m, 2H), 7.31 (t, J = 8.1 Hz, 1H), 7.35−7.42 (m,
4H), 7.60 (d, J = 7.8 Hz, 2H), 8.24 (br s, 1H, NH); MS [M + H]+ m/z
320.
Accession Codes
PDB ID codes 3Q3S and 4DW6.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: benoit.deprez@univ-lille2.fr. Phone: +33 320 964 947.
Fax: +33 320 964 709. URL: http://www.deprezlab.fr (U761);
http://www.drugdiscoverylille.org (PRIM).
Present Address
☆Galapagos N.V., General De Wittelaan L11 A3, 2800
Mechelen, Belgium.
Author Contributions
⊞These authors contributed equally to this work.
Data for N-(4-cyanophenyl)-2-(3-phenoxyphenoxy)acetamide
1
(24): white powder; yield 64%; purity >99%; H NMR (300 MHz,
Notes
DMSO-d6) δ (ppm) 4.74 (s, 2H), 6.59 (dd, J = 1.8 Hz, J = 8.1 Hz, 1H),
6.64 (t, J = 2.1 Hz, 1H), 6.77 (dd, J = 1.8 Hz, J = 8.1 Hz, 1H), 7.00−7.03
The authors declare no competing financial interest.
6400
dx.doi.org/10.1021/jm300377g | J. Med. Chem. 2012, 55, 6391−6402

Journal of Medicinal Chemistry
Article
the prodrug ethionamide in mycobacteria. J. Antimicrob. Chemother.
2006, 58, 768−772.
ACKNOWLEDGMENTS
We thank Vincent Van Geem, Dr. Bertrand Dirie,
Devassine, and Aurore Muller for technical assistance, Nicolas
Blondiaux for the Bactec experiments, Dr. Sameh H. Soror for
■
́
Step
́
hanie
(11) Hanoulle, X.; Wieruszeski, J.-M.; Rousselot-Pailley, P.; Landrieu,
I.; Baulard, A. R.; Lippens, G. Monitoring of the ethionamide pro-drug
activation in mycobacteria by (1)H high resolution magic angle spinning
NMR. Biochem. Biophys. Res. Commun. 2005, 331, 452−458.
(12) DeBarber, A. E.; Mdluli, K.; Bosman, M.; Bekker, L.-G.; Barry,
C. E. Ethionamide activation and sensitivity in multidrug-resistant
Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9677−
9682.
́
data collection, and Prof. Andre Tartar for scientific discussion.
We are grateful to the institutions that support our labora-
tory (INSERM, INSERM-Avenir fellowship to P.B., Institut
Pasteur Korea (Grants K204EA000001-08E0100-00100 and
K204EA000001-09E0100-00100), Universite
France, Institut Pasteur de Lille, CNRS, European Union, Reg
Nord-Pas de Calais, Fonds europeen de developpement regional
́
de Lille Nord de
(13) Kremer, L.; Dover, L. G.; Morbidoni, H. R.; Vilcheze, C.;
Maughan, W. N.; Baulard, A.; Tu, S.-C.; Honore, N.; Deretic, V.;
Sacchettini, J. C.; Locht, C.; Jacobs, W. R., Jr.; Besra, G. S. Inhibition of
InhA activity, but not KasA activity, induces formation of a KasA-
containing complex in mycobacteria. J. Biol. Chem. 2003, 278, 20547−
20554.
(14) Wang, F.; Langley, R.; Gulten, G.; Dover, L. G.; Besra, G. S.;
Jacobs, W. R., Jr.; Sacchettini, J. C. Mechanism of thioamide drug action
against tuberculosis and leprosy. J. Exp. Med. 2007, 204, 73−78.
(15) Engohang-Ndong, J.; Baillat, D.; Aumercier, M.; Bellefontaine, F.;
Besra, G. S.; Locht, C.; Baulard, A. R. EthR, a repressor of the TetR/
CamR family implicated in ethionamide resistance in mycobacteria,
octamerizes cooperatively on its operator. Mol. Microbiol. 2004, 51,
175−188.
́
ion
́
́
́
(FEDER) (Grants 09220019 and 09220020 PRESAGE 31510),
Agence Nationale de la Recherche (ANR; Grant ANR-06-EMPB-
033), PRIM (Projet Phare CPER TB DRUG BOOST Grant
11002859). R.W. is a Research Associate at the Belgian Fund for
Scientific Research (FNRS). We are grateful to the SLS-PX3
beamline scientists for support during data acquisition and the
European Synchrotron Radiation Facility (ESRF; Group BAG
MX-485). Data management was performed using Pipeline Pilot
from Accelrys. We thank Varian Inc. for their technical support.
RMN acquisitions were done at the Laboratoire d'Application de
Res
́
́ ́
onance Magnetique Nucleaire (LARMN), Lille, France.
(16) Willand, N.; Dirie, B.; Carette, X.; Bifani, P.; Singhal, A.; Desroses,
M.; Leroux, F.; Willery, E.; Mathys, V.; Deprez-Poulain, R.; Delcroix, G.;
Frenois, F.; Aumercier, M.; Locht, C.; Villeret, V.; Deprez, B.; Baulard,
A. R. Synthetic EthR inhibitors boost antituberculous activity of
ethionamide. Nat. Med. 2009, 15, 537−544.
ABBREVIATIONS USED
■
AcOEt, ethyl acetate; Boc, tert-butoxycarbonyl; DIEA, diisopro-
pylethylamine; DMA, dimethylacetamide; DMF, dimethylfor-
mamide; DMSO, dimethyl sulfoxide; DRC, dose−response
curve; GFP, green fluorescent protein; HTH, helix−turn−helix;
MDR, multi-drug-resistant; MeOH, methanol; MIC, minimal
inhibitory concentration; MU, 4-methylumbelliferone; MUG, 4-
methylumbelliferyl β-D-glucuronide; PBS, phosphate-buffered
saline; Ph, phenyl; rt, room temperature; SAR, structure−activity
relationship; TB, tuberculosis; TFA, trifluoroacetic acid; TSA,
thermal shift assay
(17) Flipo, M.; Desroses, M.; Lecat-Guillet, N.; Dirie,
Leroux, F.; Piveteau, C.; Demirkaya, F.; Lens, Z.; Rucktooa, P.; Villeret,
V.; Christophe, T.; Jeon, H. K.; Locht, C.; Brodin, P.; Deprez, B.;
́
B.; Carette, X.;
́
Baulard, A. R.; Willand, N. Ethionamide boosters: synthesis, biological
activity, and structure−activity relationships of a series of 1,2,4-
oxadiazole EthR inhibitors. J. Med. Chem. 2011, 54, 2994−3010.
(18) Flipo, M.; Desroses, M.; Lecat-Guillet, N.; Villemagne, B.;
Blondiaux, N.; Leroux, F.; Piveteau, C.; Mathys, V.; Flament, M.-P.;
Siepmann, J.; Villeret, V.; Wohlkonig, A.; Wintjens, R.; Soror, S. H.;
̈
Christophe, T.; Jeon, H. K.; Locht, C.; Brodin, P.; Deprez, B.; Baulard, A.
R.; Willand, N. Ethionamide boosters. 2. Combining bioisosteric
replacement and structure-based drug design to solve pharmacokinetic
issues in a series of potent 1,2,4-oxadiazole EthR inhibitors. J. Med.
Chem. 2012, 55, 68−83.
REFERENCES
■
(1) World Health Organization. Global Tuberculosis Control; World
Health Organization: Geneva, Switzerland, 2011; ISBN 978-92-4-
156438-0.
(19) Willand, N.; Desroses, M.; Toto, P.; Dirie, B.; Lens, Z.; Villeret,
V.; Rucktooa, P.; Locht, C.; Baulard, A.; Deprez, B. Exploring drug target
flexibility using in situ click chemistry: application to a mycobacterial
transcriptional regulator. ACS Chem. Biol. 2010, 5, 1007−1013.
(20) Carette, X.; Blondiaux, N.; Willery, E.; Hoos, S.; Lecat-Guillet, N.;
(2) Cox, H. S.; Ford, N.; Reeder, J. C. Are we really that good at treating
tuberculosis? Lancet Infect. Dis. 2009, 9, 138−139.
(3) Kaufmann, S. H. E.; McMichael, A. J. Annulling a dangerous
liaison: vaccination strategies against AIDS and tuberculosis. Nat. Med.
2005, 11, S33−S44.
́ ́
Lens, Z.; Wohlkonig, A.; Wintjens, R.; Soror, S. H.; Frenois, F.; Dirie, B.;
̈
(4) Elzinga, G.; Raviglione, M. C.; Maher, D. Scale up: meeting targets
in global tuberculosis control. Lancet 2004, 363, 814−819.
(5) Dye, C.; Maher, D.; Weil, D.; Espinal, M.; Raviglione, M. Targets
for global tuberculosis control [short communication]. Int. J. Tuberc.
Lung Dis. 2006, 10, 460−462.
Villeret, V.; England, P.; Lippens, G.; Deprez, B.; Locht, C.; Willand, N.;
Baulard, A. R. Structural activation of the transcriptional repressor EthR
from Mycobacterium tuberculosis by single amino acid change mimicking
natural and synthetic ligands. Nucleic Acids Res. 2012, 40, 3018−3030.
(21) Lo, M.-C.; Aulabaugh, A.; Jin, G.; Cowling, R.; Bard, J.; Malamas,
M.; Ellestad, G. Evaluation of fluorescence-based thermal shift assays for
hit identification in drug discovery. Anal. Biochem. 2004, 332, 153−159.
(22) Niesen, F. H.; Berglund, H.; Vedadi, M. The use of differential
scanning fluorimetry to detect ligand interactions that promote protein
stability. Nat. Protoc. 2007, 2, 2212−2221.
(6) Chaisson, R. E.; Martinson, N. A. Tuberculosis in Africa
combating an HIV-driven crisis. N. Engl. J. Med. 2008, 358, 1089−1092.
́
(7) Villemagne, B.; Crauste, C.; Flipo, M.; Baulard, A. R.; Deprez, B.;
Willand, N. Tuberculosis: the drug development pipeline at a glance.
Eur. J. Med. Chem. 2012, 51, 1−16.
(8) Baulard, A. R.; Betts, J. C.; Engohang-Ndong, J.; Quan, S.;
McAdam, R. A.; Brennan, P. J.; Locht, C.; Besra, G. S. Activation of the
pro-drug ethionamide is regulated in mycobacteria. J. Biol. Chem. 2000,
275, 28326−28331.
(23) Matulis, D.; Kranz, J. K.; Salemme, F. R.; Todd, M. J.
Thermodynamic stability of carbonic anhydrase: measurements of
binding affinity and stoichiometry using ThermoFluor. Biochemistry
2005, 44, 5258−5266.
(24) Senisterra, G. A.; Markin, E.; Yamazaki, K.; Hui, R.; Vedadi, M.;
Awrey, D. E. Screening for ligands using a generic and high-throughput
light-scattering-based assay. J. Biomol. Screening 2006, 11, 940−948.
(25) Vedadi, M.; Niesen, F. H.; Allali-Hassani, A.; Fedorov, O. Y.;
Finerty, P. J.; Wasney, G. A.; Yeung, R.; Arrowsmith, C.; Ball, L. J.;
Berglund, H.; Hui, R.; Marsden, B. D.; Nordlund, P.; Sundstrom, M.;
(9) Fraaije, M. W.; Kamerbeek, N. M.; Heidekamp, A. J.; Fortin, R.;
Janssen, D. B. The prodrug activator EtaA from Mycobacterium
tuberculosis is a Baeyer-Villiger monooxygenase. J. Biol. Chem. 2004,
279, 3354−3360.
(10) Hanoulle, X.; Wieruszeski, J.-M.; Rousselot-Pailley, P.; Landrieu,
I.; Locht, C.; Lippens, G.; Baulard, A. R. Selective intracellular
accumulation of the major metabolite issued from the activation of
6401
dx.doi.org/10.1021/jm300377g | J. Med. Chem. 2012, 55, 6391−6402

Journal of Medicinal Chemistry
Article
Weigelt, J.; Edwards, A. M. Chemical screening methods to identify
ligands that promote protein stability, protein crystallization, and
structure determination. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15835−
15840.
́
(26) Frenois, F.; Engohang-Ndong, J.; Locht, C.; Baulard, A. R.;
Villeret, V. Structure of EthR in a ligand bound conformation reveals
therapeutic perspectives against tuberculosis. Mol. Cell 2004, 16, 301−
307.
(27) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.
Experimental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. Adv. Drug
Delivery Rev. 2001, 46, 3−26.
(28) Lipinski, C. A. Lead- and drug-like compounds: the rule-of-five
revolution. Drug Discovery Today: Technol. 2004, 1, 337−341.
(29) Veber, D. F.; Johnson, S. R.; Cheng, H.-Y.; Smith, B. R.; Ward,
K. W.; Kopple, K. D. Molecular properties that influence the oral
bioavailability of drug candidates. J. Med. Chem. 2002, 45, 2615−2623.
(30) Cvetovich, R. J.; DiMichele, L. Formation of acrylanilides,
acrylamides, and amides directly from carboxylic acids using thionyl
chloride in dimethylacetamide in the absence of bases. Org. Process Res.
Dev. 2006, 10, 944−946.
(31) Christophe, T.; Ewann, F.; Jeon, H. K.; Cechetto, J.; Brodin, P.
High-content imaging of Mycobacterium tuberculosis-infected macro-
phages: an in vitro model for tuberculosis drug discovery. Future Med.
Chem. 2010, 2, 1283−1293.
(32) Christophe, T.; Jackson, M.; Jeon, H. K.; Fenistein, D.; Contreras-
Dominguez, M.; Kim, J.; Genovesio, A.; Carralot, J.-P.; Ewann, F.; Kim,
E. H.; Lee, S. Y.; Kang, S.; Seo, M. J.; Park, E. J.; Skovierova, H., H.;
Pham, H.; Riccardi, G.; Nam, J. Y.; Marsollier, L.; Kempf, M.; Joly-
Guillou, M.-L.; Oh, T.; Shin, W. K.; No, Z.; Nehrbass, U.; Brosch, R.;
Cole, S. T.; Brodin, P. High content screening identifies decaprenyl-
phosphoribose 2′ epimerase as a target for intracellular antimycobacte-
rial inhibitors. PLoS Pathog. 2009, 5, e1000645.
(33) Brodin, P.; Christophe, T. High-content screening in infectious
diseases. Curr. Opin. Chem. Biol. 2011, 15, 534−539.
(34) Ehrmann, M.; Bolek, P.; Mondigler, M.; Boyd, D.; Lange, R.
TnTIN and TnTAP: mini-transposons for site-specific proteolysis in
vivo. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 13111−13115.
(35) Picardeau, M.; Le Dantec, C.; Vincent, V. Analysis of the internal
replication region of a mycobacterial linear plasmid. Microbiology 2000,
146, 305−313.
(36) Snapper, S. B.; Melton, R. E.; Mustafa, S.; Kieser, T.; Jacobs, W. R.,
Jr. Isolation and characterization of efficient plasmid transformation
mutants of Mycobacterium smegmatis. Mol. Microbiol. 1990, 4, 1911−
1919.
(37) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of
macromolecular structures by the maximum-likelihood method. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 240−255.
(38) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular
graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126−
2132.
(39) Wallace, A. C.; Laskowski, R. A.; Thornton, J. M. LIGPLOT: a
program to generate schematic diagrams of protein-ligand interactions.
Protein Eng. 1995, 8, 127−134.
6402
dx.doi.org/10.1021/jm300377g | J. Med. Chem. 2012, 55, 6391−6402