Article

pubs.acs.org/jmc

Encoded Library Technology as a Source of Hits for the Discovery

and Lead Optimization of a Potent and Selective Class of Bactericidal
Direct Inhibitors of Mycobacterium tuberculosis InhA
Lourdes Encinas,‡ Heather O’Keefe,† Margarete Neu,§ Modesto J. Remuiñań ,‡ Amish M. Patel,†
Ana Guardia,‡ Christopher P. Davie,† Natalia Pérez-Macías,∥ Hongfang Yang,† Maire A. Convery,§
Jeff A. Messer,† Esther Pérez-Herrán,‡ Paolo A. Centrella,⊥ Daniel Á lvarez-Gómez,‡ Matthew A. Clark,⊥
Sophie Huss,‡ Gary K. O’Donovan,† Fátima Ortega-Muro,‡ William McDowell,# Pablo Castañeda,‡
Christopher C. Arico-Muendel,† Stane Pajk,∞ Joaquín Rullás,‡ Iñigo Angulo-Barturen,‡
Emilio Á lvarez-Ruíz,× Alfonso Mendoza-Losana,‡ Lluís Ballell Pages,‡ Julia Castro-Pichel,*,‡
and Ghotas Evindar*,†
†
ELT Boston, Platform Technology & Science, GlaxoSmithKline, Waltham, Massachusetts 02451, United States
‡
Diseases of the Developing World, Tres Cantos Medicines Development Campus, GlaxoSmithKline, Severo Ochoa 2, 28760 Tres
Cantos, Madrid, Spain
§
Computational and Structural Chemistry, Platform Technology & Science, GlaxoSmithKline, Stevenage SG1 2NY, Hertfordshire,
U.K.
∥
Instituto de Química Médica, Consejo Superior de Investigaciones Científicas (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
⊥
X-Chem Inc., 100 Beaver Street, Waltham, Massachusetts 02453, United States
#
Biological Reagent and Assay Development, Platform Technology & Science, GlaxoSmithKline, Stevenage SG1 2NY, Hertfordshire,
U.K.
∞
Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000 Ljubljana, Slovenia
×
Centro de Investigación Básica, GlaxoSmithKline, 28760 Tres Cantos, Madrid, Spain
*
S Supporting Information

ABSTRACT: Tuberculosis (TB) is one of the world’s oldest and

deadliest diseases, killing a person every 20 s. InhA, the enoyl-
ACP reductase from Mycobacterium tuberculosis, is the target of the
frontline antitubercular drug isoniazid (INH). Compounds that
directly target InhA and do not require activation by
mycobacterial catalase peroxidase KatG are promising candidates
for treating infections caused by INH resistant strains. The
application of the encoded library technology (ELT) to the
discovery of direct InhA inhibitors yielded compound 7 endowed
with good enzymatic potency but with low antitubercular potency.
This work reports the hit identification, the selected strategy for
potency optimization, the structure−activity relationships of a
hundred analogues synthesized, and the results of the in vivo
efficacy studies performed with the lead compound 65.

■ INTRODUCTION
Despite the existence of treatments for tuberculosis (TB), 8.7
million people fell ill with TB and 1.4 million died from TB in
2011 (latest WHO report1). The increasing prevalence of
multidrug resistant (MDR)2−7 and extremely drug resistant
(XDR)8,9 TB strains represents a threat to public health
worldwide. Resistance to TB drugs results primarily from
nonadherence by patients, incorrect drug prescription, or poor
quality drugs. The WHO sponsored implementation of DOTS10
(directly observed treatment short course) in which treatment
compliance is monitored by healthcare workers and has been
successful when appropriately implemented (cure rate of >90%).
Despite this, there is an urgent need for the development of
newer, safer, and effective antitubercular drugs with new targets
and novel modes of action (MoA).
InhA is an NADH-dependent 2-trans enoyl-acyl carrier
protein (ACP) reductase of the type II fatty acid synthase
Received: September 4, 2013
Published: January 22, 2014

© 2014 American Chemical Society 1276 dx.doi.org/10.1021/jm401326j | J. Med. Chem. 2014, 57, 1276−1288
Journal of Medicinal Chemistry
(FASII) pathway in Mycobacterium tuberculosis (Mtb).11,12 There
is a strong body of evidence indicating that it is the primary target
of the frontline antitubercular drug isoniazid (INH).13,14 Clinical
isolates and laboratory modified mycobacteria overexpressing
InhA show resistance to INH.14−16 The drug inhibits InhA
enzymatic activity, inducing an accumulation of saturated C24−
C26 fatty acids and blocking the production of longer molecules,
including mycolic acids. This inhibition correlates with
mycobacterial cell death. The essentiality of InhA has also been
demonstrated by the use of temperature sensitive mutants of
InhA in Mycobacterium smegmatis, where a shift to the
nonpermissive temperature results in rapid lysis and cell death.12
INH is a bactericidal drug and part of the first-line drug
combination regimen for antitubercular therapy and is
specifically active against M. tuberculosis. INH is activated within
the mycobacterial cell by KatG. The activated form is thought to
react with NADH within the InhA active site to form an
inhibitory adduct.
In vitro activated INH also forms adducts with NAD(P)
cofactors that bind to and inhibit InhA and other enzymes like
DHFR.17 The physiological relevance of these interactions in
vivo is clear in the case of InhA, while the potential role of other
possible targets of INH in the antitubercular activity of the drug
remains to be solidly proved.
Resistance to INH, which is one of the hallmarks of MDR
strains, has been associated with at least five different genes
(katG, inhA, ahpC, kasA, and ndh).18−20 While this finding still
remains to be clearly explained, 60−70% of resistant isolates are
directly linked to defects in the katG gene (often with
compensatory mutations in other genes) and less commonly in
the inhA structural gene and upstream promoter region.
With this background, we and others21−26 were interested in
the development of inhibitors targeted directly at InhA that do
not require previous KatG activation. Specifically, it was our goal
to develop a new agent that was able to interact with InhA
differently from the known NAD-INH complex, enabling a new
pharmacological profile when compared to INH that should
result in activity against INH resistant clinical isolates.
In search of potent and selective InhA inhibitors, we utilized
encoded library technology (ELT), a proven novel and robust
hit/lead identification platform27−29 wherein a large collection of
chemotypically diverse DNA-encoded small molecule libraries
are screened for affinity toward a desired protein target. The
broad chemical diversity and modest target protein requirements
are two of the major benefits of ELT for early drug discovery.
Successful ELT hit identification campaigns against diverse
targets have been previously reported.30−34 Herein we report the
discovery of a novel chemotype identified through ELT affinity
selection against M. tuberculosis InhA and its subsequent lead
optimization.
■
RESULTS AND DISCUSSION
As described in earlier reports, affinity selection of InhA was
performed by immobilizing biotinylated protein on a streptavidin
matrix in PhyNexus tips.27 Selections were conducted in parallel
against a panel of DNA-encoded libraries (DELs), representing a
diversity of compounds, under conditions of protein alone,
protein with NAD+, and protein with NADH. While enrichment
of specific chemotypes was observed under all selection
conditions, it was particularly evident for selections run in the
presence of NADH cofactor. A putative hit from an aminoproline
scaffold (Figure 1) was chosen for further follow-up. The DNA-
encoded library (Figure 1) utilized 22 orthogonally protected
1277
Article
Figure 1. Design of DNA-encoded library (DEL).
diamino acid scaffolds in cycle 1, followed by elaboration with
over 800 amine-capping building blocks (carboxylic acids,
aldehydes, sulfonyl chlorides, and isocyates) in cycles 2 and 3
to generate a 16.1 million compound library. The details of the
library synthesis will be the subject of a different publication in
the near future.
A cubic scatter plot in which each axis represents a cycle of
diversity in the library was used to analyze and visualize the
enriched library members for the selected chemotype (Figure
2).35 Individual points, corresponding to discrete small molecule
warheads in the library, are shown in pink and sized according to
number of unique instances recorded by DNA sequencing. The
display is set to a minimum of two unique copies per warhead,
which should indicate significantly enriched binders under these
selection conditions. In this analysis, the feature of interest is
represented by a plane defined by the cycle 1 building block
(BB1) (2S,4R)-4-aminopyrrolidine-2-carboxylic acid (1), which
was attached to DNA through its carboxylic acid functional
group. Within the selected plane, there are multiple lines due to
preferred disynthon combinations of 1 with specific cycle 3
building blocks (BB3). The most prominent line is the BB3 3-
ethyl-1-methyl-1H-pyrazole-5-carboxylic acid (2) that is high-
lighted in Figure 2. The selected disynthon combination of this
selected BB3 and the BB1 (2S,4R)-4-aminopyrrolidine-2-
carboxylic acid (1) provides the core structure of the InhA
feature selected from this library as a single stereoisomer. There
are a number of BB2s selected within the line each represented by
a single point; therefore, the BB2 is a variable component of the
selected scaffold that provides additional SAR in the form of
possible moieties tolerated by the site of interaction on the
protein. This “selection” SAR is a valuable tool in compound
prioritization for off-DNA synthesis and can provide exquisite
guidance in early chemotype lead optimization.
In preparation for the off-DNA activity confirmation, three
exemplars within the selected disynthon line were chosen to
follow up. The synthetic strategy was designed to put together
the pharmacophore earlier in the synthesis and add on the
variable components later (Scheme 1). This allowed for testing
of the intermediate in the activity assay and therefore additional
SAR exploration. Synthesis was initiated with peptide coupling of
amine 1 to acid 2 that afforded methyl ester 3. Saponification of
the ester 3 followed by another amide formation with ethylamine
gave the desired Boc-protected intermediate 4. TFA removal of
the Boc protecting group followed by acylation of the free amine
produced the desired final compounds (6, 7, and 8). In order to
submit the corresponding ester and acids for testing, the desired
compounds were prepared through Boc removal of precursor 3
followed by coupling with the appropriate carboxylic acid to give
the corresponding methyl ester 9. After saponification of methyl
ester 9 the corresponding acid 10 was accessible for testing as
well.
The prepared compounds were then assayed for their activity
against M. tuberculosis InhA as reported in Table 1. The first three
dx.doi.org/10.1021/jm401326j | J. Med. Chem. 2014, 57, 1276−1288
Journal of Medicinal Chemistry
Figure 2. (a) Spotfire cube data view of InhA selection feature in the presence of NADH. (b) Selected chemotype: BB1, cycle 1 building block; BB3,
cycle 3 building block; RCO-, variable cycle 2 building block.
compounds (6, 7, and 8) synthesized off-DNA demonstrated
inhibitory activity in the InhA assay. These data indicated some
potential for further modification of the R1 moiety with the 3-
substitued benzofuran 7 giving an IC50 potency of 34 nM.
Preliminary SAR exploration around the methylamide was
initiated with synthesis and testing of the corresponding methyl
ester and carboxylic acid. As demonstrated in Table 1, the
inhibitor potency diminished to 215 nM for the methyl ester (9)
and to 1290 nM for the corresponding carboxylic acid (10).
In order to better understand the properties of compound 7,
early activity and in vitro profiling of the hit structure were
completed (Table 2) along with obtaining a complex crystal
structure with InhA. As a key issue in any target based program,
we were able to ascertain the relationship between enzymatic
InhA inhibition and antitubercular MIC through the use of an
InhAMTB overexpressor strain.36 The compound showed a
selective antibacterial activity against M. tuberculosis and was
found to be endowed with good potency against the target and
modest, yet selective antitubercular activity. Furthermore, the
compound exhibited an encouraging balance among solubility,
lipophilicity, and permeability. The physicochemical properties
of a compound are fundamental in the drug discovery process,
mainly because of their influence on determining ADMET
(absorption, distribution, metabolism, excretion, and toxicity)
properties and the overall suitability of drug candidates. For the
oral route of administration the ‘ideal’ set of physicochemical
properties is well established.37−40 The hit was metabolically
stable when exposed to both human and mice microsomal
fractions and showed moderate intrinsic clearence in hepato-
cytes. No measurable activity was found against the common
cytochrome P450 (CYP450) isoforms (1A2, 2C9, 2D6, 3A4) or
in the hERG dofetilide binding assay (pIC50 < 4.3). Cytotoxicity
evaluation in HepG2 showed TOX50 > 50 μM.
1278
Article
Taking into account the moderate intrinsic clearance found in
the hepatocyte assay, the potential metabolites of 7 were
examined after incubation with hepatocytes and metabolite
follow-up by MS/MS. Results from N-dealkylation of the
ethylamide and pyrazole unit in addition to the formation of
oxidation products were the main detected metabolites (Figure
S1 and Table S1).
The complex crystal structure showed that compound 7 binds
to InhA in the cleft normally occupied by substrate/product14,41
(Figure 3). The pyrazole 2-nitrogen makes a hydrogen bonded
interaction to the 2′-hydroxyl group of NADH. In other InhA−
inhibitor bound structures an interaction with the NADH 2′-
hydroxyl is also present (see Figure 3B for the triclosan complex
structure14,21) typically as part of a hydrogen bond network
involving Tyr158. In the current complex a network to Tyr158 is
not formed in part because of Tyr158 maintaining the position it
occupies in the apo-enzyme.14 The substrate binding loop is
ordered in the structure with helix α6 (residues 197−206)
closing the volume of the pocket. There are direct hydrogen
bond interactions between main chain atoms of residues in the
helix and atoms flanking the central proline of the ligand.
In order to expand the explored chemical space beyond the
building blocks contained within the ELT library and also with
the idea of clarifying any possible bias introduced by the DNA
tags, we decided to introduce novel modifications in the three
positions of interest around the proline scaffold (Figure 4). The
main objectives of this initial exploration were to increase both
enzymatic activity and antitubercular potency. The 4-aminopro-
line central core was left unmodified, since we believed it
provided a desirable scaffold for the appropriate disposition of
the three different arms driving interactions within the InhA
active site. Chirality at the proline core proved to be essential for
activity (synthesis of 11’s enantiomer in Scheme S1). These
dx.doi.org/10.1021/jm401326j | J. Med. Chem. 2014, 57, 1276−1288
Journal of Medicinal Chemistry Article

Scheme 1. Off-DNA Synthesis of InhA Selected Hitsa

a
Reagents and conditions: (i) N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethyene]-N-methylmethanaminium hexafluorophosphate
N-oxide (HATU, 1.2 equiv), N,N-diisopropylethylamine (DIPEA, 3.0 equiv), N,N-dimethylformamide (DMF); (ii) lithium hydroxide (LiOH, 3.0
equiv); (iii) HATU (1.2 equiv), DIPEA (3.0 equiv); ethylamine hydrochloride (2.0 equiv); (iv) 20% trifluoroacetic acid in dichloromethane; (v)
carboxylic acid (RCOOH, 1.2 equiv), HATU (1.2 equiv), DIPEA (3.0 equiv), DMF.

conclusions were supported by the evidence generated during
the ELT selection process, where 22 orthogonally protected
diamino acid scaffolds were deselected for InhA interaction.
Preliminary SAR at P1, P2, and P3 Positions. The
molecule was divided into three different regions, and one novel
modification at a time was introduced in each position. This
strategy allowed for the quick exploration and combination of the
best chemical features in P1, P2, and P3 (Figure 4).
We first turned our attention to the introduction of new
diversity in P1. In order to confirm the importance of P1 in the
pharmacophore, this position was suppressed. Compound 12
was still able to inhibit the enzyme with moderate activity (IC50 =
0.12 μM), but no whole cell activity was found (synthesis in
Scheme S2). This evidence together with the realization that this
was the DNA-anchoring site for the ELT studies prompted us to
explore a wider chemical diversity at this site (Table 3). A
number of modifications were attempted: truncation of the side
chain to the primary amide, elongation and branching of the alkyl
chain, aliphatic cycles, introduction of different heteroatoms and
amino acids in addition to exploration of a number of aromatic
substitutions as depicted in compounds 25−48 where ring
changes, linker exploration, and variations of the substitution
pattern in the aromatic rings were explored. Synthesis of the
starting material 10 depicted in Scheme 1 allowed for a facile
access of P1 modifications.
1279
This early SAR investigation showed how the replacement of
the amide bond by an amine group (compound 13) resulted in a
complete loss of potency (synthesis in Scheme S3). N-alkylation
of the amide (compound 15) was not tolerated. A number of
replacements (Table 3) gave rise to compounds with excellent
enzymatic potency and activities in cellular assay much less
impressive or simply flat (see compounds 17, 26, 36, and 37). On
the other hand, some compounds exhibited higher IC50 values
that cannot be easily related to the potency of antitubercular
activity (see compounds 32, 34, and 35). The fact that the
context of the FAS II multienzyme complex is not truly
represented in the enzymatic assay could be involved in this
disconnection.42 Among the active compounds, 11 was 1 order
of magnitude more potent in the whole cell assay than 7 and
several times more potent than the rest and had the best
physicochemical properties in terms of molecular weight,
solubility, and lipophilicity (data not shown). This served as a
basis for further optimization at other positions.
Transformations in P2 required the synthesis of the special
precursors 54 and 55 (Scheme 2).
The effects of different heteroaromatic ring substitutions on
the P2 2,4-pyrazole ring were examined. The SAR exploration
was restricted to a small number of analogues of the pyrazole
unit. Other P2 derivatives caused a significant or complete
decrease in enzymatic activity (the SAR is summarized in Table
S2).
dx.doi.org/10.1021/jm401326j | J. Med. Chem. 2014, 57, 1276−1288
Journal of Medicinal Chemistry Article

Table 1. Activity of the First Compounds Made, Off-DNA

Figure 3. Compound 7 bound to the active site of InhA. In (A),

compound 7 is shown in green ball-and-sticks, NADH is shown as thick
lines, and hydrogen bonds are shown as dashed lines. Helix α6 and
Tyr158 are also highlighted. In (B), the complex structure of triclosan
(PDB code 1P45) is shown in gold superposed on the 7 complex
structure. The different positions of helix α6 and Tyr 158 in the two
complexes are visible.

Synthesis of the starting material 5 as depicted in Scheme 1

allowed for a facile access to P3 modifications. Among the
heteroaromatic substituents investigated as possible P3 position
replacements, only the benzofuran and benzothiophene moieties
were associated with a significant in vitro antitubercular potency
(Table S3). This tight SAR was confirmed with additional studies
as shown further below. Figure 4. Three positions of the target compound structure.

Table 2. Complete Profile of 7: In Vitro InhA Inhibition, Antitubercular Activity, Cytotoxicity, and Physicochemical Properties
and in Vitro DMPK Profile

a
Young, R. J.; Green, D. V. S.; Luscombe, C. N.; Hill, A. P. Drug Discovery Today 2011, 16, 822−830. bCLND solubility values that are within 85% of
maximum possible concentration (as determined from DMSO stock concentration).

1280 dx.doi.org/10.1021/jm401326j | J. Med. Chem. 2014, 57, 1276−1288

Journal of Medicinal Chemistry
Table 3. P1 Modifications: Structures and Potency of Compounds 11−48
Further Optimization of P1, P2, and P3 Positions. On
the basis of the preliminary established SAR, a second round of in
depth exploration of the P2 and P3 positions was initiated.
Despite the obvious ester hydrolysis liabilities,43 the glycine at P1
1281
Article
was fixed as the most suitable group in terms of potency. A
selected group of mainly nonpolar aliphatic substitutions was
evaluated around P2 (Table 4). The compounds bearing at
position 4 cyclopropyl (58) and propyl (56) groups were found
dx.doi.org/10.1021/jm401326j | J. Med. Chem. 2014, 57, 1276−1288
Journal of Medicinal Chemistry
Scheme 2. Synthesis of 54 and 55 Starting from 49a
a
Reagents and conditions: (i) ethylamine hydrochloride (1.2 equiv) or glycine methyl ester (1.1 equiv), HATU (1.2 equiv), DIPEA (3.0 equiv),
DMF; (ii) 20% trifluoroacetic acid in dichloromethane; (iii) carboxylic acid (benzofuran-3-carboxylic acid, 1.2 equiv), HATU (1.2 equiv), DIPEA
(3.0 equiv), DMF; (iv) piperidine/DMF 1:8 v/v, 0.6 M.
Table 4. SAR of the Pyrazole Derivatives: Structures and Potency of Compounds 56−62 Compared with 11
to be equipotent to ethyl (11) in MIC. In contrast, compound
57, with the larger isobutyl in the 4-pyrazole position, was found
to be inactive. With regard to possible pyrazole replacement
patterns, the 2-ethyl substitution, compound 59, showed similar
IC50 values but 1 order of magnitude improvement in terms of
MIC. The three-carbon chain propyl (60) was still active but the
polar 2-hydroxyethyl group (61) and the bulkier benzyl
substitution (62) were not tolerated. From this moment onward,
the diethylpyrazole was selected as the P2 substitution pattern of
choice.
With regard to P3, the SAR exploration is summarized in Table
S4. The synthetic route to prepare the starting material 63 that
afforded compounds 103−107 is outlined in Scheme S4. These
studies showed how nonpolar group substitutions at the 2-
benzofuran position were tolerated. The fact that none of the
replacements tested at P3 showed any significant improvement
confirmed how the ELT work around this position was able to
effectively cover a significant amount of chemical space. The
benzofuran at P3 was hence fixed ahead of further P1
optimization.
Unfortunately, the ester functionality of compound 11, which
stood out with an MIC value of 0.5 μM, was deemed unsuitable
because of the liabilities associated with enzymatic hydrolysis to
generate the likely nonpermeable carboxylic acid product (entry
1282
Article
106). With the aim of generating isosteric substitution patterns
more suitable for further development, different P1 analogues
were prepared aimed at overcoming the blood instability issues
associated with the ester group (Table S5).43 First, ester
variations were attempted with the aim of altering the rate of
hydrolysis (entries 107−113). This approach was unsuccessful
as described in Table S5. Other alternatives, such as replacements
with different amide combinations (primary, secondary or
tertiary amides; entries 114, 115, 116) or oxazole (entry 45),
were used, but all led to significant drops in whole cell potency.
Intriguingly, while low enzymatic IC50 values could be achieved,
no compounds with MIC < 1 μM could be identified.
At this stage and given the improved activity of the
diethylpyrazole P2 substitution, we chose to re-examine P1
(Table 5). The synthetic route to prepare the starting material 64
that afforded compounds 65−70 is outlined in Scheme S5. A
selection of some of the best scaffolds in terms of potency,
structural diversity, and physicochemical properties led to the
synthesis of the new compounds 65−70. However, the desirable
synergistic nature of the SAR of the diethylpyrazole and P1
substituents previously observed with the glycine derivative 59
was not transferable to the same extent to other P1 replacements.
All the compounds 65−70 showed InhA inhibition at low
concentrations combined with good activity against M. tuber-
dx.doi.org/10.1021/jm401326j | J. Med. Chem. 2014, 57, 1276−1288
Journal of Medicinal Chemistry Article

Table 5. SAR of Diethylpyrazole Derivatives: Structures and Potency of Compounds 65−70

a
Young, R. J.; Green, D. V. S.; Luscombe, C. N.; Hill, A. P. Getting physical in drug discovery II: the impact of chromatographic hydrophobicity
measurements and aromaticity. Drug Discovery Today 2011, 16, 822−830. bLigand efficiency (LE): enzyme pIC50/heavy (non-hydrogen) atom
count. cMortenson, P. N.; Murray, C. W. J. Comput.-Aided Mol. Des. 2011, 25, 663−667.

Table 6. Complete Profile of Compound 65: In Vitro InhA Inhibition, Antitubercular Activity, and Physicochemical Properties
and Early in Vitro DMPK Profile

culosis with MIC values in the range of 0.2−1 μM. We have
chosen to use the ligand lipophilicity efficiency index (LLE)
designed by Leeson and Springthorpe. This useful index helps to
rank compounds based on potency and lipophilicity along the
lead optimization.44 We have also taken into account the LLEAT,
which combines lipophilicity, size, and potency and has been
designed to have the same target value of 0.3 kcal/mol and
dynamic range as LE.45−48 As an indication of druglike
properties, compound 65 showed the best ligand efficiency
value of 0.37 kcal mol−1 atom−1, an LLEAT of 0.42. This was a
significant improvement when compared to compound 7 (LE =
0.32, LLEAT = 0.36). The good potency values, combined with a
reasonable lipophilicity and the lower molecular weight, led us to
progress this lead molecule to further in vivo evaluation.
1283
Profile of Compound 65. In order to complete its profile,
compound 65 was tested for activity against intracellular bacteria,
showing an MIC value of 0.24 μM. This significant intracellular
activity (in THP-1 cells) was accompanied by a clean P450
profile (1A2, 2C9, 2C19, 2D6, and 3A4; IC50 > 50 μM in all
cases) and good stability values in mouse and human microsomal
fractions (Table 6).
The bactericidal potential of compound 65 was assessed in
killing rate experiments with linezolid and moxifloxacin as
bacteriostatic and bactericidal controls, respectively (Figure S2).
The results show that compound 65 was able to reduce >2 log cfu
counts after 1 week of incubation very close to the moxifloxacine
behavior.
dx.doi.org/10.1021/jm401326j | J. Med. Chem. 2014, 57, 1276−1288
Journal of Medicinal Chemistry
Given its interest, the pharmacokinetic profile of 65 in
C57BL/6J mice following oral administration was evaluated at 50
mg/kg po (1% methylcellulose) (Figure 5 and Table 7).
Figure 5. Peripheral blood levels of 65 after oral administration to
C57BL/6J mice (n = 3) at a dose of 50 mg/kg as suspension in 1%
methylcellulose. Individual values and mean values for each time point
are represented in the plot.
Additionally, compound 65 was assayed by intravenous route
at 4 mg/kg and showed a high in vivo clearance (CL = 85.5 mL
min−1 kg−1) and a short half-life (t1/2 = 0.5 h) in mice (Table 7).
The oral PK profile was characterized by a Cmax value of 1.75 μg/
mL at 0.83 h postdosing and an AUC(0−∞) of 3.59 μg·h/mL
corresponding to 36% bioavailability (F).
In view of its balanced pharmacological, physicochemical, and
in vivo ADME profiles, compound 65 was progressed to both
tolerability and acute efficacy studies in a murine model of M.
tuberculosis infection.49 The aim of the preliminary safety study
was to estimate the maximal tolerable single oral dose level of 65
in female C57BL/6J mice. The MNLD (maximal nonlethal
dose) in mice treated with 65 (single oral dose formulated in 1%
methylcellulose by gavage followed by observation for 7 days)
was higher than 1000 mg/kg. No adverse clinical events were
found. The mean total exposure (AUC), obtained at 24 h,
associated with a 1000 mg/kg dose was 73.69 μg·h/mL, and the
corresponding mean Cmax was 23 μg/mL. Subsequently,
compound 65 was in vivo tested to determine the therapeutic
efficacy against a murine acute M. tuberculosis infection model
(Figure S3). No significant reduction of colony forming units
(cfu) at any dose tested was observed in the lungs of infected
mice. The positive control moxifloxacin (30 mg/kg body weight)
reduced the cfu by 3.48 log with respect to untreated mice.
Despite an acceptable in vivo pharmacokinetic profile and
good antitubercular potency (0.5 μM), compound 65 was found
to be inactive in an acute TB infection model. This result
underlines how antitubercular compounds with good in vitro
activity and aceptable PK properties can still be devoid of in vivo
activity. Further studies regarding possible explanations for this
disconnection would need to be done.
Table 7. Pharmacokinetic Parameters of 65 after Intravenous Administration of 4 mg/kg a Single Dose (20% Encapsin/%5
DMSO) and Oral Administration of 50 mg/kg b Single Dose (1% Methylcellulose) to C57BL/6J Mice (n = 3)c
route Tmax b (h) Cmax b (μg/mL) AUC(0−∞) b (μg·h/mL)
Compound 65
iv
po 0.83 (0.14) 1.75 (0.54) 3.59 (0.53)
a
t1/2, half-life; Vd, volume of distribution; CL, clearance. bTmax, time at maximum concentration; Cmax, maximum concentration; AUC(0−∞), area
under the curve for total exposure to infinite time. cValues are included as the mean and standard deviation (SD) of individual values.
■
Article
CONCLUSIONS
A class of potent and selective M. tuberculosis direct InhA
inhibitors was identified by means of the application of DNA
encoded library technology. Compound 7 was considered a
suitable starting point for further medicinal chemistry
optimization. In order to systematically investigate the chemical
space around the proline core, one modification at a time was
introduced in the molecule around the three possible positions
with the initial goal of improving potency. This approach was
chemically flexible and was able to quickly provide SAR
information. After combination of the best features in each
position, P3 was found to be the least malleable part in terms of
SAR manipulations. The nature of the group at this position was
found to be key for potent enzymatic activity. The diethylpyr-
azole at position P2 emerged as the most suitable moiety for
antitubercular potency optimizing. The SAR analysis around P1
provided the ability to improve the physicochemical and ADME
properties. Feature combination resulted in the synthesis of the
optimized lead compound 65. Despite its good balance between
InhA inhibition, antitubercular potency, and pharmacokinetic
profile, compound 65 was shown to be inactive in vivo against a
murine TB acute infection model.
Overall we presented here the optimization of a new
antitubercular hit series showing good InhA enzymatic and
antitubercular potencies and adequate physicochemical proper-
ties as reflected in attractive LE and LLE numbers. A clear
disconnection was found between the antitubercular in vitro and
in vivo profile of lead compound 65.
■
EXPERIMENTAL SECTION
General Procedures. All commercially available reagents and
solvents were used without further purification. (2S,4R)-1-tert-Butyl 2-
methyl 4-aminopyrrolidine-1,2-dicarboxylate hydrochloride 1 was
purchased from Apollo, Advanced Tech, and Tyger. 3-Ethyl-1-methyl-
1H-pyrazole-5-carboxylic acid 2 was purchased from Chembridge.
(2S,4R)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-(tert-
butoxycarbonyl)pyrrolidine-2-carboxylic acid 49 was purchased from
Aldrich and Cheminpex. Benzofuran-3-carboxylic acid 79 was purchased
from Apollo, Bionet, and Maybridge. 1,3-Diethyl-1H-pyrazole-5-
carboxylic acid 84 was purchased from Sinova SL. HATU was purchased
from Carbosynth. The rest of the reagents were obtained from Aldrich,
Maybridge, Apollo, Panreac, Chembridge, Novabiochem, Enamine,
Combiblocks, Artchem, and JW Pharmlab.
Reactions were monitored by thin layer chromatography (TLC)
using Merck 60 F254 silica gel glass backed plates. Elution was with
suitable solvent mixtures, and visualization was by UV light. Reactions
have also been followed by mass spectrometry and reversed phase
chromatography using a high resolution spectrometer Waters ZMD
2000 coupled with LC Agilent 1100 with DAD detection. The products
were purified by column chromatography or preparative HPLC.
Automated flash chromatography was performed on a Flash Biotage
Isolera SP4 system with peak detection at 254 nm, and preparative
HPLC was performed in an Agilent 1100 and 1200 with peak detection
at 254 nm. All products were obtained as amorphous solids, and melting
t1/2 a (h) Vd a (L/kg)) CL a (mL min−1 kg−1) F (%)
0.47 (0.20) 3.68 (2.08) 85.51 (15.93)
35.65 (5.07)
1284 dx.doi.org/10.1021/jm401326j | J. Med. Chem. 2014, 57, 1276−1288
Journal of Medicinal Chemistry
points were not measured. All NMR spectra were recorded on a Bruker
DPX Avance 400 MHz instrument equipped with a QNP probe.
Measurements were made at room temperature and at 80 °C and in an
appropriate deuterated solvent using residual hydrogenated solvent as
standard (CDCl3, δ = 7.26 ppm and DMSO-d6, δ = 2.50 ppm);
conditions were indicated in each case. Chemical shifts are expressed in
parts per million (ppm, δ units). Coupling constants (J) are in units of
hertz (Hz). Splitting patterns describe apparent multiplicities and are
designated as s (singlet), d (doublet), t (triplet), q (quartet), dd (double
doublet), m (multiplet), br (broad). Analytical purity was ≥95% unless
stated otherwise, as determined by 1H NMR and HPLC analyses. The
purity of final compounds was checked using a Waters ZQ2000 coupled
with LC Waters 2795 and Waters 2996 PDA detector. All mass spectra
were performed by electrospray ionization (ESI). Representative
procedures and physical properties and characterization for main
compounds are described. Characterization data for the rest of the
compounds are detailed in the Supporting Information.
Synthesis of Compounds. General Method A. General
Procedure for HATU-Mediated Coupling. To the desired carboxylic
acid (1.0 equiv) and HATU (1.2 equiv) in N,N-dimethylformamide was
added Hunig’s base (3.0 equiv). To the reaction mixture was then added
the desired amine (1.0 equiv). The reaction mixture was allowed to stir
at room temperature for 3−12 h. The reaction mixture was then diluted
with EtOAc (100 mL), washed with 10% NH4Cl (2 × 100 mL),
saturated NaHCO3 (1 × 100 mL), saturated NaCl (1 × 100 mL), dried
(MgSO4), and evaporated to dryness. The crude product was assessed
for purity and the desired mass by LCMS analysis.
General Method B. General Procedure for the Deprotection
of Methyl Esters. To the desired methyl ester starting material (1.0
equiv) in tetrahydrofuran (THF)/methanol (5:1) was added a solution
of lithium hydroxide (3.0 equiv). The reaction mixture was stirred
overnight at room temperature. The reaction was analyzed by LCMS.
The reaction mixture was diluted with EtOAc, washed with 10%
KH2SO4, saturated NaCl, dried over MgSO4, and evaporated to dryness.
General Method C. General Procedure for Boc-Deprotection
of Carbamates. To the desired Boc-protected starting material (1.0
equiv) in dichloromethane was added TFA (20% by volume).The
reaction mixture was then stirred at room temperature for 2 h.
Deprotection process was monitored by LCMS. The solvents were
evaporated in vacuo, and the product was azeotroped with 2 × CH2Cl2.
General Method D. Removal of Fmoc-Protection Group or
Fmoc Deprotection. A solution of piperidine/dimethylformamide
(1:8 v/v, 0.6 M) was added to the resin and shaken at room temperature
for 1 h. The reaction was checked by LCMS and showed that the
deprotection had been successful. The remaining piperidine and
dimethylformamide were removed by rotary evaporator heating at 80
°C. This crude was dissolved in MeOH, and a white precipitated
appeared. The solid was filtered off. The crude was added to a silica gel
column and was eluted with CH2Cl2/MeOH 8%.
(2S,4R)-1-tert-Butyl 2-Methyl 4-(3-ethyl-1-methyl-1H-pyra-
zole-5-carboxamido)pyrrolidine-1,2-dicarboxylate (3). Com-
pound 3 was synthesized from the (2S,4R)-1-tert-butyl 2-methyl-4-
aminopyrrolidine-1,2-dicarboxylate (1) and 3-ethyl-1-methyl-1H-pyr-
azole-5-carboxylic acid (2) using the same procedure as described in
general method A. The reaction produced a brown oil in 96% yield (1.57
g) with a purity of 95%. No further purification was needed. MS (ESI)
m/z [M + H]+ = 380.8.
(2S,4R)-tert-Butyl 4-(3-Ethyl-1-methyl-1H-pyrazole-5-carbox-
amido)-2-(ethylcarbamoyl)pyrrolidine-1-carboxylate (4). Com-
pound 4 was synthesized from saponification of (2S,4R)-1-tert-butyl 2-
methyl 4-(3-ethyl-1-methyl-1H-pyrazole-5-carboxamido)pyrrolidine-
1,2-dicarboxylate (3) using the same procedure as described in general
method B followed by coupling with ethylamine using the same
procedure as described in general method A. The reaction produced a
brown oil in 92% yield (1.38 g) with a purity of 95%. The crude product
was carried on to next step as is. MS (ESI) m/z [M + H]+ = 393.8; 1H
NMR (400 MHz, DMSO-d6) δ 8.42 (br s, 1H), 7.91 (br s, 1H), 6.66 (s,
1H), 4.41 (m, 1H), 4.11 (m, 1H), 3.93 (s, 3H), 3.63 (m, 2H), 3.31 (m,
1H), 3.26 (m, 2H), 3.09 (m, 2H), 2.14 (m, 1H), 2.01 (m, 1H), 1.32 (s,
9H), 1.14 (t, J = 7.2, 3H), 1.01 (t, J = 6.8, 3H).
1285
Article
3-Ethyl-N-(5-(ethylcarbamoyl)pyrrolidin-3-yl)-1-methyl-1H-
pyrazole-5-carboxamide 2,2,2-Trifluoroacetate (5). Compound 5
was synthesized from Boc-deprotection of (2S,4R)-tert-butyl 4-(3-ethyl-
1-methyl-1H-pyrazole-5-carboxamido)-2-(ethylcarbamoyl)pyrrolidine-
1-carboxylate (4) using the protocol described in general method C.
The reaction mixture was concentrated to dryness (coevaporated with
CH2Cl2 × 5, EtOAc × 1) to give the title compound (4.22 g, 128% yield)
as an orange oil. The yield was >100% because the product contained
EtOAc which could not be removed under reduced pressure. The crude
product was carried on to next step. MS (ESI) m/z [M + H]+ = 294; 1H
NMR (400 MHz, DMSO-d6) δ 9.46 (br s, 1H), 8.78 (br s, 1H), 8.56 (d, J
= 6.3 Hz, 1H), 8.50−8.47 (m, 1H), 6.65 (s, 1H), 4.51−4.43 (m, 1H),
4.36−4.26 (m, 1H), 3.95 (s, 3H), 3.62−3.52 (m, 1H), 3.25−3.12 (m,
3H), 2.57−2.49 (m, 2H), 2.39−2.27 (m, 1H), 2.18−2.08 (m, 1H),
1.19−1.13 (m, 3H), 1.05 (t, J = 7.25, 3H).
N-((3R,5S)-1-(Benzofuran-3-carbonyl)-5-(ethylcarbamoyl)-
pyrrolidin-3-yl)-3-ethyl-1-methyl-1H-pyrazole-5-carboxamide
(7). Compound 7 was synthesized from Boc-deprotection of (2S,4R)-
tert-butyl 4-(3-ethyl-1-methyl-1H-pyrazole-5-carboxamido)-2-
(ethylcarbamoyl)pyrrolidine-1-carboxylate (4) using the protocol
described in general method C followed by coupling with benzofuran-
3-carboxylic acid using the procedure described in general method A.
The reaction mixture was concentrated in vacuo, and the residue was
purified by preparative HPLC. The reaction afforded the title compound
(40 mg, 29% yield). MS (ESI) m/z [M + H]+ = 437.9; 1H NMR (400
MHz, DMSO-d6) δ 8.49 (s br, 2H), 7.98 (m, 2H), 7.64 (d, J = 7.6 Hz,
1H), 7.34 (m, 2H), 6.61 (s, 1H), 4.59 (m, 2H), 4.11 (m, 1H), 3.99 (s,
3H), 3.67 (m, 3H), 3.08 (m, 2H), 2.24 (m, 1H), 2.08 (m, 1H), 1.12 (t, J
= 7.2, 3H), 1.01 (t, J = 6.8, 3H); 13C NMR (100 MHz, DMSO-d6, 25 °C)
δ ppm 13.7, 14.7, 20.6, 33.4, 34.6, 38.4, 48.6, 53.1, 59.3, 105.3, 111.4,
116.2, 122.3, 123.6, 125.2, 126.0, 135.3, 151.3, 153.9, 159.5, 161.8, 170.8.
[α]23.2D −34.71 (c 0.68, EtOH).
N-((3R,5S)-1-(Benzofuran-3-carbonyl)-5-carbamoylpyrroli-
din-3-yl)-1,3-diethyl-1H-pyrazole-5-carboxamide (65). The title
compound was synthesized from 64 using the protocol described in
general method A. After preparative HPLC purification (XBridge-30, 35
mL/min, 10−100% acetonitrile in water, 10 nM NH4HCO3, basic, 20
min) the reaction afforded the title compound (423 mg, 77% yield). MS
(ESI) m/z [M + H]+ = 424; 1H NMR (400 MHz, DMSO-d6, 80 °C) δ
8.34 (br s, 1H), 8.28 (d, J = 6.6 Hz, 1H), 8.00 (d, J = 7.6 Hz, 1H), 7.62 (d,
J = 8.3 Hz, 1H), 7.41−7.31 (m, 2H), 7.13−6.82 (br s, 2H), 6.60 (s, 1H),
4.73 (dd, J = 8.3, 4.9 Hz, 1H), 4−62−4.57 (m, 1H), 4.40−4.35 (m, 2H),
4.11−4.07 (m, 1H), 3.71 (dd, J = 10.8, 5.6 Hz, 1H), 2.58−2.53 (m, 2H),
2.34−2.22 (m, 2H), 1.27 (t, J = 7.1 Hz, 3H), 1.19 (t, J = 7.6 Hz, 3H); 13C
NMR (100 MHz, DMSO-d6, 25 °C) δ ppm 13.7, 15.8, 20.7, 34.5, 45.4,
48.6, 53.1, 59.1, 105.4, 111.4, 116.2, 122.2, 123.6, 125.2, 126.0, 134.7,
151.4, 153.9, 159.5, 161.8, 173.2. [α]23.2D −46.53 (c 0.68, EtOH).
Materials and Methods. The human biological samples were
sourced ethically, and their research use was in accord with the terms of
the informed consents. All animal studies were ethically reviewed and
carried out in accordance with European Directive 2010/63/EU and the
GSK Policy on the Care, Welfare and Treatment of Animals.
Affinity Selection. Selections were done using streptavidin matrix
tips (PhyNexus) and biotinylated InhA protein (full length (270aa)).
Three rounds of selections were performed. Each tip in each round of
selection had 10 μg of protein loaded/attached to the matrix. The InhA
protein was chemically biotinylated using NHS-chromogenic biotin.
Biotinylated protein ran as a dimer (76 kDa) and retained activity.
Before use, tips were prewashed in selection buffer (50 mM Tris (pH
7.5), 150 mM NaCl, 0.1% Tween 20) and 1.0 mg/mL sheared salmon
sperm DNA (sssDNA, Ambion) and 10 mM β-mercaptoethanol
(BME).
For selection round 1, 10 μg of biotinylated InhA protein was
immobilized on a previously prepared streptavidin matrix tip and the tip
was washed four times with selection buffer to remove excess protein.
The selection conditions included buffer with and without NADH.
When the experimental conditions included NADH, it was added to all
relevant buffers, with the exception of elution buffer, to a final
concentration of 133μM. Then 5 nmol of pyrrolidine library was diluted
in 60 mL of selection buffer. The appropriate library samples were
dx.doi.org/10.1021/jm401326j | J. Med. Chem. 2014, 57, 1276−1288
Journal of Medicinal Chemistry
passed over the streptavidin matrix tip for 1 h at room temperature. The
tip was washed 8 times with selection buffer and 2 times with DNA free
selection buffer. Binders were eluted by passing heated (80 °C) DNA
free selection buffer over the tip for 12 min. The eluted material was
passed over a fresh streptavidin matrix tip for 10 min at room
temperature to remove any denatured protein (postclear). The postclear
step was repeated a second time to the round 1 selection output (1 μL of
the round 1 selection output was retained to be used for qPCR in order
to monitor the output from this round of selection). The remaining
round 1 selection output was brought to 60 μL by adding the necessary
volume of sssDNA and selection buffer.
For selection round 2, the above selection procedure was repeated
with fresh protein and a previously prepared fresh streptavidin matrix
tip. Then 5 μL of the round 2 selection output was retained to be used
for qPCR in order to monitor the output from this round of selection.
The remaining round 2 selection output was brought to 60 μL by adding
the necessary volume of sssDNA and selection buffer.
For selection round 3, the selection procedure was repeated with
fresh protein, a previously prepared fresh tip, and the round 2 selection
output. The postclear steps were not repeated at the end of round 3.
Quantitative PCR was run with the outputs from each round of selection
to assess selection yields for each step. The round 3 output was
sequenced using Roche/454 technology. A no-target control selection
was performed in the same fashion, using the same buffer but without
the protein input.
X-ray Crystallography. InhA was expressed in E. coli and purified in
two steps by IEC and size exclusion chromatography. The protein (4.15
mgs/mL in 20 mM Tris, pH 8.0, 0.1 M NaCl, 2 mM DTT) was
incubated with NADH and compound prior to crystallization. The
complex crystallized in 10% PEG 8000 and 0.1 M Tris, pH 8.5, at 20 °C
and was cryoprotected prior to data collection at ESRF. The structure
was solved by molecular replacement. More details, statistics, and
exemplars of the density are given in the Supporting Information.
InhA Enzymatic Assay. Enzymatic activity was measured fluori-
metrically by following NADH oxidation at λexc = 340 nm and λem= 480
nm, using 50 mM NADH and 50 mM 2-trans-dodecenoyl-CoA
(DDCoA) as substrates. Dose−response experiments to determine IC50
were performed using 5 nM InhA. The percentage of remaining
enzymatic activity (% AR) at different compound concentrations was
calculated with the formula % AR = 100 × [(sample − control 2)/
(control 1 − control 2)] where sample is the enzymatic activity for each
compound concentration, control 1 is enzyme activity in the absence of
any compound, and control 2 is NADH oxidation in absence of enzyme.
IC50 was calculated fitting % AR to a two parameter equation % AR =
100/[1 + (compound conc/IC50)s] where s is a slope factor. IC50 was
calculated using GraFit 5.0.12 software (Eritacus Software Ltd.). All
reactions were run in 30 mM PIPES buffer, pH 6.8, at 25 °C.
Strain and Growth Conditions. M. tuberculosis H37Rv
(ATC25618) wild type and H37Rv overexpressing InhA (HyrR) were
grown in Middlebrook 7H9-ADC broth (Difco) supplemented with
0.05% Tween 80 and on 7H10-OADC or 7H11-OADC agar (Difco) at
37 °C. INH and Hyg were purchased from Sigma-Aldrich. Where
required hygromycin (50 μg mL−1) was added to the culture medium.
Mycobacterium tuberculosis Inhibition Assay. The measure-
ment of the minimum inhibitory concentration (MIC) for each tested
compound was performed in 96-well flat-bottom polystyrene microtiter
plates. Ten 2-fold drug dilutions in neat DMSO were performed. An
amount of 5 μL of these drug solutions was added to 95 μL of
Middlebrook 7H9 medium (lines A−H, rows 1−10 of the plate layout).
Isoniazid was used as a positive control. Eight 2-fold dilutions of
isoniazid starting at 160 μg/mL were prepared, and 5 μL of this control
curve was added to 95 μL of Middlebrook 7H9 medium (Difco
catalogue ref 271310) (row 11, lines A−H). An amount of 5 μL of neat
DMSO was added to row 12 (growth and blank controls). The
inoculum was standardized to approximately 1 × 107 cfu/mL mL−1 and
diluted 1 in 100 in Middlebrook 7H9 broth (10% ADC (Becton
Dickinson catalog ref 211887) and 0.025% Tween 80) to produce the
final inoculum. Then 100 μL of this inoculum was added to the entire
plate except G-12 and H-12 wells (blank controls). All plates were
placed in a sealed box to prevent drying out of the peripheral wells, and
1286
Article
they were incubated at 37 °C without shaking for 6 days. A resazurin
solution was prepared by dissolving one tablet of resazurin (resazurin
tablets for milk testing; ref 330884Y′ VWR International Ltd.) in 30 mL
of sterile PBS (phosphate buffered saline). Of this solution, an amount
of 25 μL was added to each well. Fluorescence was measured
(Spectramax M5 Molecular Devices, excitation 530 nm, emission 590
nm) after 48 h to determine the MIC value.
Pharmacokinetic Studies. For pharmacokinetic studies C57BL/6
female mice of 18−20 g weight were used (n = 3 mice). Experimental
compounds were administered by intravenous (iv) bolus at 4 mg/kg at a
volume of 10 mL/kg to n = 3 mice and by oral gavage (po) at 50 mg/kg
single dose at a volume of 20 mL/kg to n = 3 mice. All mice received
treatment in the fed state. Drugs were administered as solution for iv
route in 20% encapsin and 5% DMSO and as suspension for po route in
1% methylcellulose. Peripheral total blood was the compartment chosen
for the establishment of compound concentrations. Aliquots of 25 μL of
blood were taken from the lateral tail vein for each mouse at the
following time points: for iv route, 10, 20, and 30 min and 1, 2, 4, and 8 h;
for po route, 15, 30, and 45 min and 1, 2, 4, and 8 h. LCMS was used as
the analytical method of choice for the establishment of compound
concentration in blood with a sensitivity of LLQ = 1−5 ng/mL in 25 μL
of blood. The noncompartmental data analysis (NCA) was performed
with WinNonlin Phoenix 6.3 (Pharsight, Certara L.P.), and supple-
mentary analysis was performed with GraphPad Prism 5 (GraphPad
Software, Inc.).
In Vivo Efficacy Assessment. Specific pathogen-free, 8- to 10-
week-old female C57BL/6 mice were purchased from Harlan
Laboratories and were allowed to acclimate for 1 week. The
experimental design has been previously described.49 In brief, mice
were intratracheally infected with 100 000 cfu/mouse (M. tuberculosis
H37Rv strain). Products were administered for 8 consecutive days
starting 1 day after infection. Lungs were harvested 24 h after the last
administration. All lung lobes were aseptically removed, homogenized,
and frozen. Homogenates were plated in 10% OADC-7H11 medium for
14 days at 37 °C.
■
*
ASSOCIATED CONTENT
S Supporting Information
Additional tables, schemes, figures, protocols and experimental
procedures of some assays mentioned in the main text, synthetic
procedures, and compound characterization. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Authors
*J.C.-P.: phone, +34-669-399879; fax, 34-918-070310; e-mail,
julia.p.castro@gsk.com.
*G.E.: phone, 1-781-795-4423; fax, 1-781-795-4496; e-mail,
ghotas.x.evindar@gsk.com.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors thank the Physicochemical Characterisation Group
of Analytical Chemistry, GSK Tres Cantos, Mariá Teresa Fraile-
Gabaldón, and Rubén Gómez for carrying out chromlog D
measurements for all the compounds and formulation studies,
Angel Santos for protein binding measurements, and Jesús
Gómez, Ana Á lvarez, and Raquel Gabarró for their help with
LCMS and NMR analysis. Santiago Ferrer is thanked for his
assistance with the pharmacology work. We acknowledge the
́
excellent technical expertise of Delia Blanco, Marı ́a Martinez,
́ Pedro A. Torres Gómez, and Mariá José
Rubén González del Rio,
Rebollo for biological evaluation. We gratefully acknowledge the
Laboratory Animal Science (Pre-Clinical) Department for
essential animal lab upkeep and maintenance. The research
dx.doi.org/10.1021/jm401326j | J. Med. Chem. 2014, 57, 1276−1288
Journal of Medicinal Chemistry
leading to these results has received funding from the Global
Alliance for TB Drug Development and from the European
Union’s 7th Framework Program (FP7-2007-2013) under the
Orchid Grant Agreement No. 261378. The funders had no role
in study design, data collection, analysis, decision to publish, or
preparation of the manuscript.
■
ABBREVIATIONS USED
ADMET, absorption, distribution, metabolism, excretion, and
toxicity; AUC, area under the curve; BOC, tert-butyloxycarbonyl;
CLND, chemiluminescent nitrogen detection; cfu, colony
forming unit; DEL, DNA-encoded chemical libraries; DIPEA,
N,N-diisopropylethylamine; DMF, N,N-dimethylformamide;
DMPK, drug metabolism and pharmacokinetics; DMSO,
dimethyl sulfoxide; ELT, encoded library technology; HATU,
N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-
methylene]-N-methylmethanaminium hexafluorophosphate N-
oxide; INH, isoniazid; InhA, Mycobacterium tuberculosis InhA;
LE, ligand efficiency; LLE, ligand lipophilicity efficiency; LLEAT,
ligand lipophilicity efficiency Astex; MDR-TB, multidrug-
resistant tuberculosis; MIC, minimum inhibitory concentration;
NMR, nuclear magnetic resonance; PFI, property forecast index,
therapeutics; PK, pharmacokinetics; SAR, structure−activity
relationship; TB, tuberculosis; TFA, trifluoroacetic acid; WHO,
World Health Organization; XDR, extensively drug-resistant
tuberculosis
■
REFERENCES
(1) World Health Organisation. Tuberculosis. http://www.who.int/
mediacentre/factsheets/fs104/en/index.html (accessed February 11,
2013).
(2) Becerra, M. C.; Bayona, J.; Freeman, J.; Farmer, P. E.; Kim, J. Y.
Redefining MDR-TB transmissionhot spots’ counterpoint. Int. J. Tuberc.
Lung Dis. 2000, 4, 387−394.
(3) Dye, C.; Espinal, M. A.; Watt, C. J.; Mbiaga, C.; Williams, B. G.
Worldwide incidence of multidrug-resistant tuberculosis. J. Infect. Dis.
2002, 185, 1197−1202.
(4) Espinal, M. A. The global situation of MDR-TB. Tuberculosis
(Edinburgh) 2003, 83, 44−51.
(5) Wright, A.; Zignol, M.; Van Deun, A.; Falzon, D.; Gerdes, S. R.;
Feldman, K.; Hoffner, S.; Drobniewski, F.; Barrera, L.; van Soolingen,
D.; Boulabhal, F.; Paramasivan, C. N.; Kam, K. M.; Mitarai, S.; Nunn, P.;
Raviglione, M. Epidemiology of antituberculosis drug resistance 2002-
07: an updated analysis of the global project on anti-tuberculosis drug
resistance surveillance. Lancet 2009, 373, 1861−1873.
(6) Chiang, C. Y.; Centis, R.; Migliori, G. B. Drug-resistant
tuberculosis: past, present, future. Respirology 2010, 15, 413−432.
(7) Multidrug and Extensively Drug-Resistant TB (M/XDR-TB):
2010 Global Report on Surveillance and Response (WHO/HTM/TB/
2010.3). World Health Organization: Geneva, 2010.
(8) Shah, N. S.; Wright, A.; Bai, G. H.; Barrera, L.; Boulahbal, F.;
Martin-Casabona, N.; Drobniewski, F.; Gilpin, C.; Havelkova, M.; Lepe,
R.; Lumb, R.; Metchock, B.; Portaels, F.; Rodrigues, M. F.; Rüsch-
Gerdes, S.; Van Deun, A.; Vincent, V.; Laserson, K.; Wells, C.; Cegielski,
J. P. Worldwide emergence of extensively drug-resistant tuberculosis.
Emerging Infect. Dis. 2007, 13, 380−387.
(9) Berry, M.; Kon, O. M. Multidrug- and extensively drug-resistant
tuberculosis: an emerging threat. Eur. Respir. Rev. 2009, 18, 195−197.
(10) What Is DOTS? A Guide to Understanding the WHO
Recommended TB Control Strategy Known as DOTS. Document
WHO/CDS/CPC/TB/99.270; World Health Organization: Geneva,
1999.
(11) Quemard, A.; Sacchettini, J. C.; Dessen, A.; Vilchèze, C.; Bittman,
R.; Jacobs, W. R.; Blanchard, J. S. Enzymic characterization of the target
for isoniazid in Mycobacterium tuberculosis. Biochemistry 1995, 34, 8235−
8241.
1287
Article
(12) Vilcheze, C.; Morbidoni, H. R.; Weisbrod, T. R.; Iwamoto, H.;
Kuo, M.; Sacchettini, J. C.; Jacobs, W. R., Jr. Inactivation of the inhA-
encoded fatty acid synthase II (FASII) enoyl-acyl carrier protein
reductase induces accumulation of the FASI end products and cell lysis
of Mycobacterium smegmatis. J. Bacteriol. 2000, 182, 4059−4067.
(13) Banerjee, A.; Dubnau, E.; Quemard, A.; Balasubramanian, V.; Um,
K. S.; Wilson, T.; Collins, D.; de Lisle, G.; Jacobs, W. R., Jr. InhA, a gene
encoding a target for isoniazid and ethionamide in Mycobacterium
tuberculosis. Science 1994, 263, 227−230.
(14) Dessen, A.; Quemard, A.; Blanchard, J. S.; Jacobs, W. R., Jr.;
Sacchettini, J. C. Crystal structure and function of the isoniazid target of
Mycobacterium tuberculosis. Science 1995, 267, 1638−1641.
(15) Parikh, S. L.; Xiao, G.; Tonge, P. J. Inhibition of InhA, the enoyl
reductase from Mycobacterium tuberculosis, by triclosan and isoniazid.
Biochemistry 2000, 39, 7645−7650.
(16) Scior, T.; Garces-Eisele, S. J. Isoniazid is not a lead compound for
its pyridyl ring derivatives, isonicotinoyl amides, hydrazides, and
hydrazones: a critical review. Curr. Med. Chem. 2006, 13, 2205−2219.
(17) Argyrou, A.; Vetting, M. W.; Aladegbami, B.; Blanchard, J. S.
Mycobacterium tuberculosis dihydrofolate reductase is a target for
isoniazid. Nat. Struct. Mol. Biol. 2006, 13, 408−413.
(18) Schroeder, E. K.; de Souza, O. N.; Santos, D. S.; Blanchard, J. S.;
Basso, L. A. Drugs that inhibit mycolic acid biosynthesis in
Mycobacterium tuberculosis. Curr. Pharm. Biotechnol. 2002, 3, 197−225.
(19) Basso, L. A.; Santos, D. S. Drugs that inhibit mycolic acid
biosynthesis in Mycobacterium tuberculosisan update. Med. Chem. Rev.
2005, 2, 393−413.
(20) Hazbón, M. H.; Brimacombe, M.; Bobadilla Del Valle, M.;
Cavatore, M.; Guerrero, M. I.; Varma-Basil, M.; Billman-Jacobe, H.;
Lavender, C.; Fyfe, J.; Garcia-Garcia, L.; Leon, C. I.; Bose, M.; Chaves,
F.; Murray, M.; Eisenach, K. D.; Sifuentes-Osornio, J.; Cave, M. D.;
Ponce de Leon, A.; Alland, D. Population genetics study of isoniazid
resistance mutations and evolution of multidrug-resistant Mycobacte-
rium tuberculosis. Antimicrob. Agents Chemother. 2006, 50, 2640−2649.
(21) Kuo, M. R.; Morbidoni, H. R.; Alland, D.; Sneddon, S. F.; Gourlie,
B. B.; Staveski, M. M.; Leonard, M.; Gregory, J. S.; Janjigian, A. D.; Yee,
C.; Musser, J. M.; Kreiswirth, B.; Iwamoto, H.; Perozzo, R.; Jacobs, W.
R.; Sacchettini, J. C.; Fidock, D. A. Targeting tuberculosis and malaria
through inhibition of enoyl reductase compound activity and structural
data. J. Biol. Chem. 2003, 278, 20851−20859.
(22) Lu, X. Y.; You, Q. D.; Chen, Y. D. Recent progress in the
identification and development of InhA direct inhibitors of
Mycobacterium tuberculosis. Mini-Rev. Med. Chem. 2010, 10, 182−193.
(23) Ballell Pages, L.; Castro Pichel, J.; Fernandez Menendez, R.;
Fernandez Velando, E. P.; Gonzalez del Valle S.; Leon Diaz M. L.;
Mendoza Losana, A.; Wolfendale, M. J. (Pyrazol-3-yl)-1,3,4-thiadiazole-
2-amine and (Pyrazol-3-yl)-1,3,4-thiazol-2-amine Compounds. WO/
2010/118852, 2010.
(24) Pan, P.; Tonge, P. J. Targeting InhA, the FASII enoyl-ACP
reductase: SAR studies on novel inhibitor scaffolds. Curr. Top. Med.
Chem. 2012, 12, 672−693.
(25) Castro Pichel, J.; Fernandez Menendez, R.; Fernandez Velando,
E. P.; Gonzalez del Valle S.; Mallo-Rubio, A. 3-Amino-pyrazole
Derivatives Useful against Tuberculosis. WO/2012/049161, 2012.
(26) Horlacher, O. P.; Hartkoorn, R. C.; Cole, S. T.; Altmann, K.-H.
Synthesis and antimycobacterial activity of 2,1′-dihydropyridomycins.
ACS Med. Chem. Lett. 2013, 4, 264−268.
(27) Clark, M. A.; Acharya, R. A.; Arico-Muendel, C. C.; Belyanskaya,
S. L.; Benjamin, D. R.; Carlson, N. R.; Centrella, P. A.; Chiu, C. H.;
Creaser, S. P.; Cuozzo, J. W.; Davie, C. P.; Ding, Y.; Franklin, G. J.;
Franzen, K. D.; Gefter, M. L.; Hale, S. P.; Hansen, N. J. V.; Israel, D. I.;
Jiang, J.; Kavarana, M. J.; Kelley, M. S.; Kollmann, C. S.; Li, F.; Lind, K.;
Mataruse, S.; Medeiros, P. F.; Messer, J. A.; Myers, P.; O’Keefe, H.; Oliff,
M. C.; Rise, C. E.; Satz, A. L.; Skinner, S. R.; Svendsen, J. L.; Tang, L.;
Vloten, K. V.; Wagner, R. W.; Yao, G.; Zhao, B.; Morgan, B. A. Design,
synthesis and selection of DNA-encoded small-molecule libraries. Nat.
Chem. Biol. 2009, 5, 647−654.
(28) Deng, H.; O’Keefe, H.; Davie, C. P.; Lind, K. E.; Acharya, R. A.;
Franklin, G. J.; Larkin, J.; Matico, R.; Neeb, M.; Thompson, M. M.; Lohr,
dx.doi.org/10.1021/jm401326j | J. Med. Chem. 2014, 57, 1276−1288
Journal of Medicinal Chemistry Article

T.; Gross, J. W.; Centrella, P. A.; O’Donovan, G. K.; Bedard, K. L.; methyltransferases define the specificity of the mycolic acid elongation
Vloten, K.-v.; Mataruse, S.; Skinner, S. R.; Belyanskaya, S. L.; Carpenter, complexes. PLoS One 2011, 6, e29564.
T. Y.; Shearer, T. W.; Clark, M. A.; Cuozzo, J. W.; Arico-Muendel, C. C.; (43) Koitka, M.; Hochel, J.; Gieschen, H.; Borchert, H. H. Improving
Morgan, B. A. Discovery of highly potent and selective small molecule the ex vivo stability of drug ester compounds in rat and dog serum:
ADAMTS-5 inhibitors that inhibit human cartilage degradation via inhibition of the specific esterases and implications on their identity. J.
encoded library technology (ELT). J. Med. Chem. 2012, 55, 7061−7079. Pharm. Biomed. Anal. 2010, 51, 664−678.
(29) Disch, J. S.; Evindar, G.; Chiu, C. H.; Blum, C. A.; Dai, H.; Jin, L.; (44) Leeson, P. D.; Springthorpe, B. The influence of drug-like
Schuman, E.; Lind, K. E.; Belyanskaya, S. L.; Deng, J.; Coppo, F.; concepts on decision-making in medicinal chemistry. Nat. Rev. Drug
Aquilani, L.; Graybill, T. L.; Cuozzo, J. W.; Lavu, S.; Mao, C.; Vlasuk, G. Discovery 2007, 6, 881−890.
P.; Perni, R. B. Discovery of thieno[3,2-d]pyrimidine-6-carboxamides as (45) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand efficiency: a useful
potent inhibitors of SIRT1, SIRT2, and SIRT3. J. Med. Chem. 2013, 56, metric for lead selection. Drug Discovery Today 2004, 9, 430−431.
3666−3679. (46) Kuntz, I. D.; Chen, K.; Sharp, K. A.; Kollman, P. A. The maximal
(30) Graybill, T. L.; King, B. W.; Fosbenner, D. T.; Keller, P. M.;; affinity of ligands. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 9997−10002.
Morgan, B. A.; Clark, M. A.; Cuozzo, J. DNA-Encoded Libraries: A New (47) Carr, R. A. E.; Congreve, M.; Murray, C. W.; Rees, D. C.
Resource for Innovative Hit Identification. Abstract of Papers, 237th Fragment-based lead discovery: leads by design. Drug Discovery Today
National Meeting of the American Chemical Society, Salt Lake City, UT, 2005, 10, 987−992.
U.S., March 22−26, 2009; American Chemical Society: Washington, (48) Mortenson, P. N.; Murray, C. W. Assessing the lipophilicity of
fragments and early hits. J. Comput.-Aided Mol. Des. 2011, 25, 663−667.
DC, 2009; MEDI-297.
(49) Rullas, J.; Garcia, J. I.; Beltran, M.; Cardona, P. J.; Cáceres, N.;
(31) Evindar, G.; Acharya, R. A.; Arico-Muendel, C. C.; Belyanskaya, S.
García-Bustos, J. F.; Angulo-Barturen, I. Fast standardized therapeutic-
L.; Brown, J.; Clark, A. M.; Centrella, P. A.; Cuozzo, J. W.; Davie, C. P.;
efficacy assay for drug discovery against tuberculosis. Antimicrob. Agents
Ding, Y.; Franklin, G. J.; Jeffrey, W. Gross, J. W.; Israel, D. I.; Jolivette, L.
Chemother. 2010, 54, 2262−2264.
J.; Jurewicz, A. J.; Lakdawala, A. S.; Li, F.; Li, H.; Lind, K. E.; Joseph, P.;
Marino, J. P.; Morgan, B. A. Application of Encoded Library Technology
(ELT) to the Discovery of Soluble Epoxide Hydrolase Inhibitors.
Abstracts of Papers, 238th National Meeting of the American Chemical
Society, Washington, DC, United States, August 16−20, 2009; MEDI-2.
(32) Davie, C. P.; Centrella, P. A.; O’Donovan, G. K.; Evindar, G.;
O’Keefe, H.; Patel, A. M.; Clark, M. A. Design and Synthesis of a Sixteen
Million Member DNA-Encoded Library (DEL) and Discovery of
Potent Mycobacterium tuberculosis (Mtb) InhA Inhibitors. Abstracts of
Papers, 240th National Meeting of the American Chemical Society,
Boston, MA, United States, August 22−26, 2010; American Chemical
Society: Washington, DC, 2010; MEDI-150.
(33) Ding, Y.; O’Keefe, H.; Svendsen, J. L.; Franklin, G. J.; Centrella, P.
A.; Clark, M. A.; Acharya, R. A.; Li, F.; Messer, J. A.; Chiu, C. H.; Matico,
R. E.; Murray-Thompson, M. F.; Cuozzo, J. W.; Israel, D. I.; Arico-
Muendel, C.; Morgan, B. A. Discovery of Potent and Selective Inhibitors
for ADAMTS-4 through Encoded Library Technology (ELT). Abstracts
of Papers, 240th National Meeting of the American Chemical Society,
Boston, MA, United States, August 22−26, 2010; American Chemical
Society: Washington, DC, 2010; MEDI-460.
(34) Gentile, G.; Merlo, G.; Pozzan, A.; Bernasconi, G.; Bax, B.;
Bamborough, P.; Bridges, A.; Carter, P.; Neu, M.; Yao, G.; Brough, C.;
Cutler, G.; Coffin, A.; Belyanskaya, S. 5-Aryl-4-carboxamide-1,3-
oxazoles: potent and selective GSK-3 inhibitors. Bioorg. Med. Chem.
Lett. 2012, 22, 1989−1994.
(35) For ELT data analysis and terminologies refer to refs 27−29.
(36) Mendoza-Losana, A. Gordon Research Conference: Tuber-
culosis, 2011 (personal communication).
(37) Lipinski, C. A. Drug-like properties and the causes of poor
solubility and poor permeability. J. Pharmacol. Toxicol. Methods 2000,
44, 235−249.
(38) 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.
(39) Lennernas, H.; Abrahamsson, B. The use of biopharmaceutic
classification of drugs in drug discovery and development: current status
and future extension. J. Pharm. Pharmacol. 2005, 57, 273−285.
(40) Dahan, A.; Miller, J. M.; Amidon, G. L. Prediction of solubility and
permeability class membership: provisional BCS classification of the
world’s top oral drugs. AAPS J. 2009, 11, 740−746.
(41) Rozwarski, D. A.; Vilcheze, C.; Sugantino, M.; Bittman, R.;
Sacchettini, J. C. Crystal structure of the Mycobacterium tuberculosis
enoyl-ACP reductase, InhA, in complex with NAD+ and a C16 fatty acyl
substrate. J. Biol. Chem. 1999, 274, 15582−15589.
(42) Cantaloube, S.; Veyron-Churlet, R.; Haddache, N.; Daffé, M.;
Zerbib, D. The Mycobacterium tuberculosis FAS-II dehydratases and

1288 dx.doi.org/10.1021/jm401326j | J. Med. Chem. 2014, 57, 1276−1288