N-Benzyl-4-((heteroaryl)methyl)benzamides: A New Class of Direct NADH-Dependent 2-trans Enoyl-Acyl Carrier Protein Reductase (InhA) Inhibitors with Antitubercular Activity

Article abstract of DOI:10.1002/cmdc.201600020

Isoniazid (INH) remains one of the cornerstones of antitubercular chemotherapy for drug-sensitive strains of M.tuberculosis bacteria. However, the increasing prevalence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains containing mutations in the KatG enzyme, which is responsible for the activation of INH into its antitubercular form, have rendered this drug of little or no use in many cases of drug-resistant tuberculosis. Presented herein is a novel family of antitubercular direct NADH-dependent 2-trans enoyl-acyl carrier protein reductase (InhA) inhibitors based on an N-benzyl-4-((heteroaryl)methyl)benzamide template; unlike INH, these do not require prior activation by KatG. Given their direct InhA target engagement, these compounds should be able to circumvent KatG-related resistance in the clinic. The lead molecules were shown to be potent inhibitors of InhA and showed activity against M.tuberculosis bacteria. This new family of inhibitors was found to be chemically tractable, as exemplified by the facile synthesis of analogues and the establishment of structure-activity relationships. Furthermore, a co-crystal structure of the initial hit with the enzyme is disclosed, providing valuable information toward the design of new InhA inhibitors for the treatment of MDR/XDR tuberculosis.

Full text of DOI:10.1002/cmdc.201600020

DOI: 10.1002/cmdc.201600020
Full Papers
N-Benzyl-4-((heteroaryl)methyl)benzamides: A New Class
of Direct NADH-Dependent 2-trans Enoyl–Acyl Carrier
Protein Reductase (InhA) Inhibitors with Antitubercular
Activity
Ana Guardia,*[a] Gulcin Gulten,[b] Raquel Fernandez,[a] Jesus Gómez,[a] Feng Wang,[c]
Maire Convery,[d] Delia Blanco,[a] María Martínez,[a] Esther PØrez-Herrµn,[a] Marta Alonso,[a]
Fµtima Ortega,[a] Joaquín Rullµs,[a] David Calvo,[e] Lydia Mata,[e] Robert Young,[d]
James C. Sacchettini,[b] Alfonso Mendoza-Losana,[a] Modesto Remuiǵn,[a]
Lluís Ballell Pages,[a] and Julia Castro-Pichel[a]
Isoniazid (INH) remains one of the cornerstones of antitubercu-
lar chemotherapy for drug-sensitive strains of M. tuberculosis
bacteria. However, the increasing prevalence of multidrug-re-
sistant (MDR) and extensively drug-resistant (XDR) strains con-
taining mutations in the KatG enzyme, which is responsible for
the activation of INH into its antitubercular form, have ren-
dered this drug of little or no use in many cases of drug-resist-
ant tuberculosis. Presented herein is a novel family of antitu-
bercular direct NADH-dependent 2-trans enoyl–acyl carrier pro-
tein reductase (InhA) inhibitors based on an N-benzyl-4-((heter-
oaryl)methyl)benzamide template; unlike INH, these do not re-
quire prior activation by KatG. Given their direct InhA target
engagement, these compounds should be able to circumvent
KatG-related resistance in the clinic. The lead molecules were
shown to be potent inhibitors of InhA and showed activity
against M. tuberculosis bacteria. This new family of inhibitors
was found to be chemically tractable, as exemplified by the
facile synthesis of analogues and the establishment of struc-
ture–activity relationships. Furthermore, a co-crystal structure
of the initial hit with the enzyme is disclosed, providing valua-
ble information toward the design of new InhA inhibitors for
the treatment of MDR/XDR tuberculosis.
Introduction
Tuberculosis (TB) continues to be a global threat, with an addi-
tional nine million people infected—and one and a half million
still dying from the disease—each year.[1] The increasing preva-
lence of multidrug-resistant (MDR)[2–6] and extensively drug-re-
sistant (XDR)[7,8] TB strains, along with the commonly encoun-
tered co-infection in HIV patients and lack of adherence to
multidrug frontline treatment regimes are factors that contrib-
ute to this pandemic. Despite the proven efficacy of Directly
Observed Treatment Short course (DOTS),[9] which has a cure
rate of >90%, the development of new drugs with novel
modes of action for the treatment of TB remains an urgent
unmet medical need. A shortened treatment regime for both
drug-sensitive and MDR/XDR TB would have a significant
impact. The urgency for new treatments is perhaps embodied
in the profile of bedaquiline,[10] which is the first new antimyco-
bacterial drug to be approved by the US Food and Drug Ad-
ministration (FDA) in over 40 years; it was approved at the end
of 2012. Bedaquiline acts by the novel mechanism of inhibiting
mycobacterial adenosine 5’-triphosphate (ATP) synthase, with
good clinical efficacy against multiple resistant strains. Al-
though it has saved lives, it was approved with a black-box
warning, highlighting the possibility of severe cardiovascular
side effects.[10]
[a] A. Guardia, Dr. R. Fernandez, J. Gómez, D. Blanco, M. Martínez,
Dr. E. PØrez-Herrµn, M. Alonso, Dr. F. Ortega, Dr. J. Rullµs,
Dr. A. Mendoza-Losana, Dr. M. Remuiǵn, Dr. L. Ballell Pages,
Dr. J. Castro-Pichel
Diseases of the Developing World, GlaxoSmithKline
Severo Ochoa 2, 28760 Tres Cantos, Madrid (Spain)
E-mail: ana.a.guardia@gsk.com
[b] Dr. G. Gulten, Prof. J. C. Sacchettini
Department of Biochemistry and Biophysics, Texas A&M University
300 Olsen Boulevard College Station, TX 77843-2128 (USA)
[c] Dr. F. Wang
California Institute for Biomedical Research (Calibr)
11119 North Torrey Pines Road, La Jolla, CA 92037 (USA)
[d] Dr. M. Convery, Dr. R. Young
Molecular Discovery Research, GSK Medicines Research Centre
Gunnels Wood Road, Stevenage, SG1 2NY (UK)
The well-validated drug target InhA acts as an NADH-depen-
dent 2-trans enoyl–acyl carrier protein (ACP) reductase within
the type II fatty acid synthase (FASII) pathway in M. tuberculo-
sis[11] and is the primary target of the frontline antitubercular
drug isoniazid (INH).[12–14] INH is activated within the mycobac-
terium by the catalase–peroxidase enzyme KatG, forming the
[e] Dr. D. Calvo, L. Mata
Centro de Investigación Bµsica
Platform Technologies and Science, GlaxoSmithKline
Parque Tecnológico de Madrid, 28760 Tres Cantos, Madrid (Spain)
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201600020.
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isonicotinic acyl radical that has been shown to react with
NADH within the InhA active site.[15] This inhibitory adduct was
identified as the acylation product at the 4-position of the nic-
otinamide moiety of NADH. 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);[16] al-
though these findings still lack clear biochemical explanations,
60–70% of resistant isolates can be directly linked to defects in
the KatG gene (often with compensatory mutations in other
genes) and less commonly in the InhA structural gene and its
upstream promoter region.
Results and Discussion
Chemistry
The optimization of validated hit 1 was focused on generating
new analogues by modifying four identifiable structural motifs:
the right-hand side (RHS: o-chloro-p-fluorophenyl ring), the
left-hand side (LHS: pyrazole), the linker (amide bond), and the
central unit (CU: phenyl ring) (Figure 1).
There has been widespread research[16–21] into the discovery
and development of new inhibitors targeted directly at InhA
that do not require prior KatG activation, which may result in
activity against clinical isolates resistant to INH. With this goal
in mind, a high-throughput screening campaign[22] against
InhA was carried out to identify novel direct inhibitors of InhA.
Herein we report the identification of a new series of highly
selective direct InhA inhibitors based on the N-benzyl-4-((heter-
oaryl)methyl)benzamide template, including optimization at-
tempts with goals of improving whole-cell activity and the
physicochemical profile of the initial hit, as well as an in vivo
efficacy evaluation of one exemplar in an acute model of TB in-
fection. Additionally, the co-crystal structure of one representa-
tive molecule confirming its binding mode within the InhA
active site is disclosed.
Figure 1. Compound 1: medicinal chemistry optimization priorities.
RHS modifications were centered on replacement and/or
substitution of the chlorine and fluorine substituents, with var-
iation around position in the ring and also replacement of the
phenyl ring by heterocycles (see Table 4 below). Synthesis of
these compounds were carried out by amidation reactions
using pyrazolylmethylbenzoic acid 2 and the desired amine as
highlighted in Scheme 1A. Compound 16 (Scheme 1B) offered
a convenient starting point to explore extended derivatives
Scheme 1. Compounds listed in Table 4. Reagents and conditions: A) a) NatBuO (2 equiv), DMF, RT, 30 min, 74%; b) HATU (1.1 equiv), DIPEA (1.5–2 equiv), DMF,
RT, 15 min; c) RNH2 (1–2 equiv), RT, overnight, 50–100%. B) Preparation of compound 16 (Table 4): a) NaBH4 (4 equiv), BF3·Et2O (2.7 equiv), THF, 08C, overnight,
90%; b) CBr4 (1.5 equiv), PPh3 (1.5 equiv), DMF, RT, 2 h, NaN3 (3 equiv), overnight, 72%; c) PPh3 (1.5 equiv), THF/H2O, RT, overnight, quant.; d) compound 2
(2 equiv), compound 21 (1 equiv), DIPEA (2.5 equiv), EDC (1.2 equiv), DMAP (0.2 equiv), CH2Cl2/DMF (10:2), RT, overnight, 29%; e) Fe powder (9 equiv),
FeSO4·7H2O (2.2 equiv), dioxane/H2O (5:1), reflux, 6 h, 99%. C) Preparation of compounds 17 and 18 (Table 4): a) isocyanatobenzene (1.1 equiv), CH2Cl2, RT,
overnight, 44%; b) 2-phenylacetic acid (1.2 equiv), EDCI (1.2 equiv), DMAP (0.2 equiv), DMF, RT, overnight, 5%.
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with different substitution or modifications of this amino
group, enabling the preparation of a small array based on vari-
ous coupling reactions. The synthesis of the most potent com-
pounds identified using this approach (17 and 18, Table 4) is il-
lustrated in Scheme 1C.
LHS modifications were centered around two main themes
(Figure 2): 1) exploring the spacer and the connecting position
between the pyrazole and the phenyl ring (Scheme 2), and
2) replacement of the pyrazole ring with other heterocycles
(Scheme 3). Further heterocyclic variation was achieved using
Negishi couplings,[23] starting from commercially available
methyl-4-(bromomethyl)benzoate. Hydrolysis of the intermedi-
ate ester afforded the corresponding acid, which was subject-
ed to an amidation reaction with 1-(2-chloro-4-fluorophenyl)-
methanamine to yield the desired products (Scheme 3A).
Figure 2. Synthetic scheme for chemical modifications to the LHS.
An exception to this general procedure was the synthesis of
compound 32 (Scheme 3B). In this case the commercial 4-(cya-
nophenyl)boronic acid was reacted under Suzuki conditions
with 4-(chloromethyl)-2-methyl-1,3-thiazole to obtain inter-
mediate 38. Formation of the corresponding carboxylic acid by
nitrile group hydrolysis and ensuing amide formation with 1-
(2-chloro-4-fluorophenyl)methanamine afforded the desired
compound 32. Replacement of the pyrazole ring led to the
identification of compound 36 (see Table 6 below; preparation
in Scheme 3A), which provided impetus to explore heteroatom
variations of the methylene spacer (Figure 3).
Figure 3. Analogues of compound 36 (compounds 41–46).
Derivatives listed in Table 7 below (analogues of compound
36) required a customized synthetic route. Preparative details
are provided in the Supporting Information (Schemes 5–10).
Derivatives were also synthesized in order to explore modifi-
cations around the central unit (CU) following the general pro-
cedure depicted in Scheme 4. The final amide coupling reac-
tion between the corresponding modified acid and 1-(2-
chloro-4-fluorophenyl)methanamine afforded the desired com-
pounds 49–51 (Supporting Information). The exception to this
general procedure was the synthesis of 48, which was generat-
ed by mesylation of the pyridine-2-carboxamide alcohol ana-
logue to 24 followed by substitution by dimethylpyrazole
(Supporting Information).
Scheme 2. Reagents and conditions: A) Preparation of compounds 25 and 26
(Table 5): a) 1-(2-chloro-4-fluorophenyl)methanamine, HATU, DIPEA, DMF, RT,
3 h, 70%; b) NaBH4, THF, overnight, 60%; c) (MeSO2)O, DIPEA, DMF followed
by azole derivative, polymer-supported BEMP, 10%. B) Preparation of com-
pounds 27 and 28 (Table 5): a) NatBuO, 3,5-dimethyl-1H-pyrazole, DMF, RT,
2 h, 88%; b) 1-(2-chloro-4-fluorophenyl)methanamine, HATU, DIPEA, DMF, RT,
overnight, 43%; c) NaHCO3, 3,5-dimethyl-1H-pyrazole, MeOH, 1358C, 2 h, mi-
crowave, 26%; d) 1-(2-chloro-4-fluorophenyl)methanamine EDCI, DMAP, DMF,
RT, overnight, 35%.
Scheme 3. Reagents and conditions: A) Synthesis of compounds 31, 33, 34, 36, and 37 (Table 6): a) Zn/THF, reflux, 1.5 h, then Pd(PPh3)4, Ar-X, reflux, 3.5 h, 30–
40%; b) LiOH, THF/H2O, RT, overnight, 60–82%; c) 1-(2-chloro-4-fluorophenyl)methanamine, EDCI, DMAP, DMF, RT, overnight, 17–35%. B) Synthesis of com-
pound 32 (Table 6): a) Pd(PPh3)4, Na2CO3, DME, N2 atmosphere, 4-(chloromethyl)-2-methyl-1,3-thiazole, 908C, overnight, 59%; b) KOH, EtOH/H2O, overnight,
62%; c) 1-(2-chloro-4-fluorophenyl)methanamine EDCI, DMAP, DMF, RT, overnight, 40%.
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Table 3. MIC90 data for compound 1, kanamycin (KM), isoniazid (INH),
and a strain of M. bovis BCG overexpressing InhA.
Compd
MIC90 [mm]
InhA
OE/ctrl
M. bovis BCG
pATB45 (ctrl)
M. bovis BCG
pATB45–M. tub. InhA (OE)
Scheme 4. General procedure for the synthesis of compounds 49–51 (see
Table 10, Supporting Information). Reagents and conditions: a) 1-(2-chloro-4-
fluorophenyl)methanamine, EDCI, DMAP, DMF, RT, overnight, 10–48%.
1
KM
INH
31
0.62
1.0
190
0.62
12
6
1
12
To investigate the importance of the amide moiety, various
linker modifications were explored. These derivatives (com-
pound 47 in Table 8 below, and compounds 52–57 in Support-
ing Information Table 11) required specific synthetic pathways
which are described in the Supporting Information.
(Table 2) could lead to a new generation of InhA inhibitors as
powerful weapons against MDR-TB[2–6] and XDR-TB.[7,8] InhA
target engagement was supported by the increased MIC90
measured for 1 in the assay of a BCG strain overexpressing
InhA (Table 3).
Biology
SAR analysis and in vitro properties
Amongst the inhibitors identified in the high-throughput
screen against InhA,[22] a number of N-benzyl-4-((heteroaryl)me-
thyl)benzamides were highlighted. Compound 1 was identified
as a hit of particular interest for further optimization. This com-
pound exhibited good inhibitory potency against InhA (IC50 =
0.05 mm) in addition to modest antitubercular activity (MIC90 =
10 mm) without any preliminary evidence of cytotoxicity in the
HepG2 assay (Table 1).
The main goal of the medicinal chemistry program was to im-
prove the whole-cell potency of the initial hit, compound 1, by
modifications to the four different regions described above
(Figure 1). Furthermore, the optimization process also focused
on ligand efficiency metrics (LE[24] and LLE[24]) and also on phys-
ical properties (clogP and property forecast index (PFI)[25]) in
the iterative design of compounds and to help with data anal-
ysis (see Figures 7 and 8 and Table 12 in the Supporting Infor-
mation).
The development of a family of N-benzamides targeted di-
rectly at InhA which do not require prior activation by KatG
Table 1. In vitro biological profile of compound 1.
RHS SAR
RHS SAR analysis (Table 4) showed that replacement of the
phenyl ring by pyrazole or furan is tolerated (compounds 14
and 15), although they were not as potent as the parent com-
pound. The result suggests that a phenyl group is better than
pyrazole and furan for the RHS. Substituents around the
phenyl ring were essential to maintain inhibitory potency
(compound 9), and the best results were observed with ortho/
para substitution patterns in the aromatic ring, with some tol-
erance for meta substituents. Direct comparison of 1 and 4,
and 3 and 12 with compound 13, showed the para/ortho dis-
ubstitution as essential for InhA inhibitory activity.
InhA IC50 [mm][a]
0.05Æ0.01
10
6
5.16
94.63
170
<4.3
>50
M. tuberculosis H37Rv MIC90 [mm][a]
M. bovis InhA overexpressor/WT MIC90 ratio
[b]
Chrom logD7.4
HSA [%][c]
Solubility CLND [mm][d]
CYP 3A4 (VR) pIC50
HepG2 Tox50 [mm]
clogP
4
5.7
Mouse CLint [mLminÀ1 gÀ1
]
The observed tolerance of meta substitution prompted us to
consider compound 16 as a convenient starting point to ex-
plore derivatives with different modifications of the amino
group. This exercise led to the identification of the most active
molecules in terms of both enzymatic and whole-cell activity
(17 and 18, Table 4). Unfortunately, these improvements came
at the cost of high lipophilicity (clogP for compounds 17 and
18: 5.3 and 5.0; LLE: 1.68 and 2.09, respectively), and these
two compounds were considered unsuitable for further pro-
gression.
[a] Values are the mean ÆSD of four different assays. [b] pH 7.4. [c] HSA:
percent human serum albumin binding with compound. [d] CLND: chem-
iluminescent nitrogen detection, aqueous solubility.
Table 2. MIC90 values [mm] of compounds 1, 36, and 47 in H37Rv and four
clinical M. tuberculosis isolates with KatG mutations in S315T, in compari-
son with isoniazid (INH) in the same experiments.
Compd
H37Rv
KatG-1
KatG-2
KatG-3
KatG-4
1
10
31
31
0.5
31
94
31
32
12
47
31
32
12
47
23
23
16
47
31
32
LHS SAR
36
47
INH
The first set of small LHS modifications around the pyrazole
ring resulted in decreased activity (Table 5); direct comparison
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Table 4. In vitro activities of select RHS derivatives against M. tuberculosis InhA and M. tuberculosis H37Rv.
Compd
R
InhA
IC50 [mm][a]
H37Rv
MIC90 [mm][c]
Compd
R
InhA
IC50 [mm][a]
H37Rv
MIC90 [mm][c]
3
4
1.00Æ0.01[b]
1.26Æ0.03
ND
62
12
13
0.25Æ0.04
ND
ND
3.10Æ0.06[b]
5
6
1.58Æ0.08
2.51Æ0.08
125
62
14
15
0.12Æ0.02
0.09Æ0.01
31
46
7
8
1.58Æ0.01
62
62
1.58Æ0.03[b]
16
17
0.35Æ0.02
0.09Æ0.02
94
6
9
5.93Æ1.26
>125
10
11
2.51Æ0.04[b]
0.54Æ0.7
ND
23
18
0.08Æ0.01
16
[a] Values are the mean ÆSD of four different assays. [b] Values are the mean ÆSD of two different assays. [c] ND: not determined.
between compounds 1, 25, and 26 showed that a 3-methyl
group is essential for activity against the enzyme, as com-
pound 26 was found to be inactive. The additional 5-methyl
group was found to be vital to achieving enhanced potency.
In terms of the pyrazole connectivity with the CU, the para-
benzyl group was favored, whereas the two-carbon homologa-
tion was also well tolerated (27 and 28). With these data in
hand, replacement of the pyrazole motif with other heteroaro-
matic rings was investigated (Table 6). Methyl monosubstituted
1,3-thiazoles (31 and 32) were found to be very potent InhA
inhibitors, and a methyl group at the 5-position or no substitu-
tion of the ring decreased potency markedly (33 and 34).
Replacement of pyrazole motif with isomeric pyridines and
other six-membered heterocyclic systems containing two nitro-
gen atoms was then explored (Table 6). The best pyridine sub-
stitution was found to be at position 6, with a methyl group
being preferred over a trifluoromethyl substituent (36 and 37).
Several pyrazines and pyrimidines were also prepared (58–60;
see Table 9 in the Supporting Information), and were found to
be poorly active.
On the basis of the promising profile of compound 36 (IC50:
0.05 mm and M. tuberculosis MIC90: 31 mm), the impact of vari-
ous spacers linking the pyridine ring with the rest of the struc-
ture was explored (Figure 3, Table 7). The methylene linker was
first replaced by different heteroatoms. Whilst nitrogen- and
oxygen-containing structures were found to be inactive (41
and 42), the sulfur analogue 43 showed similar levels of InhA
inhibition as that of the parent compound 36. Elongation of
the methylene linker into ethyl or methylene-thioether (45 and
46) yielded inactive molecules; however, the introduction of
oxygen (compound 44) gave IC50 and MIC90 values similar to
those of 36. Unfortunately, this approach did not lead to an
improvement in the whole-cell activity of the initial hit 1.
Linker and central unit SAR
The replacements with both furan and thiophene resulted in
inactive molecules (48–51, Table 10 in the Supporting Informa-
tion), although the pyridine analogue 48 showed moderate
enzyme and whole-cell activity. To evaluate the role of the
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Table 5. In vitro activities of select pyrazole derivatives against M. tuber-
culosis InhA and M. tuberculosis H37Rv: LHS modifications.
Table 7. In vitro activities of select pyrazole derivatives against M. tuber-
culosis InhA and M. tuberculosis H37Rv: LHS modifications.
R1
R2
InhA
IC50 [mm][a]
H37Rv
MIC90 [mm]
Compd
X
InhA
IC50 [mm][a]
H37Rv
MIC90 [mm]
Compd
41
42
43
44
45
46
NH
O
S
CH2O
CH2CH2
CH2S
>10
ND
>125
125
31
>125
>125
25
26
H
H
0.5Æ0.03[b]
39
3.4Æ0.05
0.06Æ0.01
0.02Æ0.01
1.55Æ0.20
0.26Æ0.04
>10[b]
>125
27
28
H
>10
>125
[a] Values are the mean ÆSD of four different assays; ND: not deter-
mined.
H
0.32Æ0.03
62
of the amide carbonyl group (compound 57), highlighting the
importance of this interaction, which was later rationalized by
the crystal structure.
[a] Values are the mean ÆSD of four different assays. [b] Values are the
mean ÆSD of two different assays.
One of the most interesting modifications was the reversed
amide 47 (Table 8), which gave enzyme and whole-cell activity
at levels similar to those of the initial hit, as well as improved
Table 6. In vitro activities of select pyrazole derivatives against M. tuber-
culosis InhA and M. tuberculosis H37Rv: LHS modifications.
Table 8. In vitro activities of compound 47 against M. tuberculosis InhA
and M. tuberculosis H37Rv: linker modifications.
Compd
R1
InhA
IC50 [mm][a]
H37Rv
MIC90 [mm]
31
32
33
0.06Æ0.21[b]
0.09Æ0.01
3.25Æ0.66
62
125
Compd
InhA
IC50 [mm]
H37Rv
MIC90 [mm]
47
0.04Æ0.01
31
[a] Values are the mean ÆSD of four different assays.
>125
in vitro metabolic stability (CLint =2.6 mLminÀ1 gÀ1). The major
shortcomings of compound 1 were the sub-optimal physical
attributes, most notably high lipophilicity[26–28] and relatively
modest solubility, so particular efforts were made to investi-
34
36
1.40Æ0.10
0.05Æ0.01
>125
31
gate analogues with lower lipophilicity (measured as Chrom
[25]
logD7.4
;
see Figure 8, Supporting Information). This prelimi-
37
0.25Æ0.05
>125
nary SAR analysis showed how potency does not appear to be
clearly driven by lipophilicity or ligand efficiency (for example,
compounds 1, 15, 17, and 18; Supporting Information
Figure 7), and the measured data for many compounds thus
gave PFI values (Chrom logD7.4 +number of aromatic rings) of
>8, a level indicative of poor developability outcomes.
[a] Values are the mean ÆSD of four different assays. [b] Values are the
mean ÆSD of two different assays.
amide bond, various linker modifications were explored. The
urea (54, Supporting Information Table 11) gave modest inhibi-
tory activity against the enzyme. Replacement of the amide by
both an ester and a sulfonamide led to a total loss of enzyme
inhibition (52 and 53, Supporting Information Table 11) and
the same outcome was observed if the amide nitrogen was al-
kylated (55, Supporting Information Table 11) or upon removal
Despite these extensive efforts, no significant overall im-
provements from compound 1 were achieved; however, this
family of compounds did have a reasonable profile in terms of
enzyme inhibitory potency and whole-cell activity. Further-
more, exemplars showed InhA target engagement (Supporting
Information Table 8) and activity versus KatG mutant clinical
isolates (data in Table 2 for compounds 1, 36, and 47).
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In vivo properties
INH–NADH adduct, our compound did not cause flipping of
the Phe149 side chain, and so was not observed to interact
with the isonicotinic acid binding pocket.
The initial hit was progressed to pharmacokinetic (PK) studies
(Figure 4, Supporting Information), in which 1 was found to be
highly bioavailable (72%) after oral administration in mice,
reaching a Cmax value of 6.5 mgmLÀ1 with an encouraging AUC
of 39 mghmLÀ1 with sustained exposure. With this information
in hand, compound 1 was progressed to efficacy studies in
a murine model of acute TB infection, at oral doses of 200 and
500 mgkgÀ1 twice daily (Figure 5, Supporting Information). Un-
fortunately, no statistically significant decrease in colony-form-
ing units (CFU) counts in the lungs of TB-infected mice was ob-
served relative to untreated controls. The lack of in vivo effica-
cy for compound 1 could be the result of its modest antituber-
cular potency which directly impacts the efficacy drivers, AUC/
MIC90 and/or Cmax/AUC.
The crystal structure also revealed that compound 1 is
bound to the hydrophobic substrate binding pocket of InhA
which is surrounded by residues Met199, Leu207, Ile215,
Met103, Phe149, Tyr158, Ala198, and Ile202. The enzyme–inhib-
itor complex only crystallized in the presence of the NAD(H)
cofactor, which indicates the uncompetitive character of the in-
hibitor.
The direct hydrogen bonding between the carbonyl oxygen
atom of the main-chain Met98 and the amide nitrogen atom
of the inhibitor (2.9 Š, Figure 4) is the key interaction identified
with InhA that provided a rationale for the loss of activity with
N-methylation (compounds 1 and 55, Supporting Information
Table 11). The carbonyl group of compound 1 is essential for
activity (11 and 57, Supporting Information Table 11); however,
it does not itself establish any hydrogen bonding interactions
with the surrounding protein residues. It only shows van der
Waals interactions (3.6–3.8 Š) with Ala198 and Phe97.
Structure of the InhA–1 complex
Toward the end of the synthetic program, a ligand crystal
structure of the lead compound was solved; this is shown in
Figure 4 (PDB ID: 4QXM). In contrast to the previously identi-
fied InhA inhibitors such as pyrrolidine carboxamides, Gen-
zyme10850, triclosan, and NITD[21] compounds, the catalytic
residue of Tyr158 did not make any hydrogen bonds with com-
pound 1, and participated in ligand binding only through
van der Waals interactions. This way, the N-benzamide tem-
plate belongs to a new class of compounds recently described
in the literature.[16,29] In addition, in our InhA–NAD+–N-benza-
mide structure, the side chain of Phe149 adopted the identical
position as observed in the InhA–NAD(H) structure. Unlike the
The main pyrazole ring interactions are with the nicotina-
mide ring of NAD+ via p–p stacking and with the 2’-hydroxy
group of the NAD+ ribose moiety via hydrogen bonding
(2.9 Š). The 3- and 5-methyl groups contribute to compound
binding, both being important to maintain potent InhA inhibi-
tory activity. The 3-methyl group makes hydrophobic and
van der Waals interactions with the side chains of Phe149 and
Tyr158 (3.9 Š) as well as with the nicotinamide ring of NAD+,
playing an important role in the binding of the compound
with the enzyme (comparison of compounds 25 and 26,
Table 5). The presence of the 5-methyl group improves binding
with the enzyme (comparison of 1 and 25, Table 5) making ad-
ditional van der Waals interactions with Thr196 and NAD+
(3.2–3.4 Š).
In the right-hand side of the molecule, the dihalide-substi-
tuted phenyl ring interacts with the main-chain Gln100,
Met103, Met98, Gly104, Leu207, and Ile202 residues via hydro-
phobic interactions, with reasonable filling of available space.
This is consistent with the improved enzyme activity of com-
pound 1 relative to less heavily substituted analogues 3, 4, 9,
and 13. The presence of a bulkier trifluoromethyl group in 12
at the ortho position decreased activity more than one order
of magnitude relative to compound 1 (Table 4). This could be
due to steric clash with the protein, as the ortho-chlorine atom
closely packs against Met103 (3 Š).
Conclusions
We have identified a series of N-benzyl-4-((heteroaryl)methyl)-
benzamides as a novel class of direct InhA inhibitors by high-
throughput screening. These compounds demonstrated
potent activity against M. tuberculosis, maintaining activity
versus KatG mutant clinical strains and emerging as a potential
tool against MDR-TB and XDR-TB.
Figure 4. Compound 1 bound to the active site of InhA. Compound 1 is
shown in green ball-and-stick format, NAD+ in grey as thick lines, and hy-
drogen bonds as dashed lines. Helix a6, which is ordered in the structure,
along with Met98, Met103, and Tyr158 are highlighted.
Despite the thorough SAR investigation around the hit, no
compounds were obtained with significantly improved poten-
cy against M. tuberculosis H37Rv relative to compound 1. How-
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Full Papers
0.22 mmol) and HATU (90 mg, 0.25 mmol) were added and dis-
solved in DMF (15 mL). DIPEA (56 mg, 0.43 mmol) was then added.
The mixture was stirred for 10 min. 2-Chloro-4-fluorophenylme-
thanamine (69 mg, 0.43 mmol) was dissolved in DMF (0.5 mL) and
added dropwise to the initial mixture. The reaction was stirred at
room temperature for 18 h. The solvent was removed under
vacuum. The resulting residue was dissolved in CH2Cl2 (2 mL) and
washed with saturated NaHCO3 (2 mL). The organic layer was dried
over MgSO4, and volatiles were removed under vacuum. The result-
ing crude was purified by column chromatography to yield
56.5 mg of the title compound as a white solid (70%). 1H NMR
(400 MHz, [D6]DMSO): d=9.01 (t, J=5.8 Hz, 1H), 7.86 (d, J=8.1 Hz,
2H), 7.42 (dd, J=2.7, 8.7 Hz, 1H), 7.38 (dd, J=6.3, 8.6 Hz, 1H),
7.22–7.13 (m, 3H), 5.88 (s, 1H), 5.26 (s, 2H), 4.49 (d, J=5.8 Hz, 2H),
2.15 (s, 3H), 2.10 ppm (s, 3H); 13C NMR (101 MHz, [D6]DMSO): d=
166.1, 160.9 (d, J=245.9 Hz, 1C), 146.2, 141.2, 139.2, 133.1, 132.7
(d, J=3.7 Hz, 1C), 132.6 (d, J=11.0 Hz, 1C), 130.1 (d, J=9.5 Hz,
1C), 127.6 (s, 2C), 126.8 (s, 2C), 116.4 (d, J=24.9 Hz, 1C), 114.2 (d,
J=21.2 Hz, 1C), 105.2, 51.2, 40.1, 13.2, 10.6 ppm; LC–MS: tR =
1.21 min, m/z 371.12 [M+H]+; purity: >95%.
ever, several derivatives were obtained with similar InhA inhibi-
tory and antibacterial activity (e.g., compounds 11, 12, 14, 15,
18, 36, 44, and 47). Compound 1 is a potent direct InhA inhibi-
tor with moderate whole-cell activity and an encouraging
safety profile, but unfortunately it was not efficacious in an
in vivo murine model of TB infection. The SAR information pre-
sented for this new antitubercular compound series, rational-
ized by interactions observed in a co-crystal structure with
InhA, should serve as a valuable guide in the design of new
molecules toward the goals of improved levels of InhA inhibi-
tion and antitubercular whole-cell activity.
Experimental Section
Chemistry
General: All commercially available reagents and solvents were
used without further purification unless otherwise stated. High-
purity solvents were purchased from commercial vendors and
dried with molecular sieves (3–4 Š). Reactions were monitored by
thin-layer chromatography (TLC) with silica gel 60 F254 plates (thick-
ness 2 mm, Merck); analysis was carried by UV light at l 254 nm,
staining with KMnO4 and iodine. Reversed-phase liquid chromatog-
raphy/mass spectrometry (LC–MS) was performed with
a Waters 2000 chromatograph equipped with an Agilent 1100 ana-
lytical column connected to a high-resolution Micromass ZMD
spectrometer. Separation and purification of products was carried
out by column chromatography using silica gel (particle size: 30–
45 mm; Isolute). Purifications were performed automatically on
a Master-II Flash Biotage instrument. NMR spectroscopy was car-
ried out in the Structural Analysis Department of the Tres Cantos
Medicines Development Campus GlaxoSmithKline, using a Varian
Unity (300 MHz) and a Bruker DPX (400 MHz). Measurements were
made at room temperature and in the deuterated solvent indicat-
ed in each case. Chemical shifts (d) are expressed in parts per mil-
lion (ppm) using the deuterated solvent as reference (CDCl3:
7.26 ppm, [D6]DMSO: 2.49 ppm, CD3OD: 3.30 ppm); coupling con-
stants (J) are expressed in hertz (Hz). NMR signals are abbreviated
as follows: s (singlet), d (doublet), t (triplet), q (quartet), qt (quin-
tet), m (multiplet), and br (broad). Mass spectrometry data were
collected in the Structural Analysis Department of the Tres Cantos
Medicines Development Campus GlaxoSmithKline, using a Waters
ZQ2000 instrument coupled with an LC Agilent 1100 or Waters
Acquity SQD mass spectrometer. Detailed descriptions of the LC–
MS method used with the aforementioned equipment are provid-
ed in the Supporting Information.
N-(2-trifluorobenzyl)-4-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)-
benzamide (3). 2-Trifluorophenylmethanamine (47 mg, 0.43 mmol)
and carboxylic acid 2 (50 mg, 0.22 mmol) were mixed following
the procedure described for 1 to yield 72 mg of the title com-
1
pound as a white oil (86%). H NMR (400 MHz, [D6]DMSO): d=9.07
(t, J=5.8 Hz, 1H), 7.87 (d, J=8.1 Hz, 2H), 7.72 (d, J=7.6 Hz, 1H),
7.67–7.58 (m, 1H), 7.53–7.39 (m, 2H), 7.18 (d, J=8.1 Hz, 2H), 5.88
(s, 1H), 5.26 (s, 2H), 4.64 (d, J=5.6 Hz, 2H), 2.16 (s, 3H), 2.10 ppm
(s, 3H); 13C NMR (101 MHz, [D6]DMSO): d=166.2, 146.2, 141.3, 139,
137.6, 133, 132.6, 128.1, 127.6 (s, 2C), 127.3, 126.8 (s, 2C), 126.2,
125.8, 125.7 (d, J=5.9 Hz, 1C), 105.2, 51.2, 39.2, 13.3, 10.6 ppm;
LC–MS: tR =1.22 min, m/z 387.16 [M+H]+; purity: >95%.
N-(2-chlorobenzyl)-4-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)ben-
zamide (4). 2-Chlorophenylmethanamine (61.5 mg, 0.43 mmol) and
carboxylic acid 2 (50 mg, 0.22 mmol) were mixed following the
procedure described for 1 to yield 56 mg of the title compound as
1
a colorless oil (73%). H NMR (400 MHz, [D6]DMSO): d=9.00 (t, J=
5.8 Hz, 1H), 7.85 (d, J=8.3 Hz, 2H), 7.46–7.40 (m, 1H), 7.34–7.25
(m, 3H), 7.17 (d, J=8.1 Hz, 2H), 5.87 (s, 1H), 5.25 (s, 2H), 4.52 (d,
J=5.8 Hz, 2H), 2.15 (s, 3H), 2.09 ppm (s, 3H); 13C NMR (101 MHz,
[D6]DMSO): d=166.1, 146.2, 141.2, 139, 136.3, 133.1, 131.9, 129.1,
128.5, 128.5, 127.6 (s, 2C), 127.1, 126.7 (s, 2C), 105.1, 51.2, 40.5,
13.3, 10.6 ppm; LC–MS: tR =1.20 min, m/z 353.13 [M+H]+; purity:
>95%.
N-(3-propan-2-yloxybenzyl)-4-((3,5-dimethyl-1H-pyrazol-1-yl)me-
thyl)benzamide
(5).
3-Propan-2-yloxyphenylmethanamine
(71.7 mg, 0.43 mmol) and carboxylic acid 2 (50 mg, 0.22 mmol)
were mixed following the procedure described for 1 to yield
58.5 mg of the title compound as a white solid (71%). 1H NMR
(400 MHz, [D6]DMSO): d=8.95 (t, J=5.9 Hz, 1H), 7.83 (d, J=8.3 Hz,
2H), 7.23–7.16 (m, 1H), 7.15 (d, J=8.1 Hz, 2H), 6.84–6.79 (m, 2H),
6.78–6.73 (m, 1H), 5.87 (s, 1H), 5.24 (s, 2H), 4.55 (t, J=6.0 Hz, 1H),
4.41 (d, J=6.1 Hz, 2H), 2.14 (s, 3H), 2.10 (s, 3H), 1.23 (s, 3H),
1.22 ppm (s, 3H); 13C NMR (101 MHz, [D6]DMSO): d=165.9, 157.4,
146.2, 141.3, 141, 139, 133.3, 129.3, 127.5 (s, 2C), 126.7 (s, 2C), 119,
114.5, 113.5, 105.2, 68.9, 51.2, 42.5, 21.8 (s, 2C), 13.3, 10.6 ppm; LC–
MS: tR =1.22 min, m/z 377.21 [M+H]+; purity: >95%.
4-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzoic acid (2). In
a round-bottom flask, 3,5-dimethylpyrazole (500 mg, 5.2 mmol)
was dissolved in DMF (15 mL). Sodium tert-butoxide (1 g,
10.4 mmol) was added to this solution, and the mixture was stirred
at 258C for 15 min. A solution of 4-(bromomethyl)benzoic acid
(214 mg, 1 mmol) in DMF (20 mL) was then added dropwise to the
initial mixture and stirred for 30 min at 258C. The solvent was re-
moved under vacuum, and the residue dissolved in H2O (200 mL).
The solution was acidified to pH 5 with aqueous HCl (2m), and the
resulting precipitate was filtered off to afford the title compound
1
(586 mg, 74%) as a white solid. H NMR (400 MHz, [D6]DMSO): d=
12.87 (brs, 1H), 7.88 (d, J=8.0 Hz, 2H), 7.16 (d, J=8.0 Hz, 2H), 5.86
(s, 1H), 5.25 (s, 2H), 2.13 (s, 3H), 2.09 ppm (s, 3H).
N-(2-nitrobenzyl)-4-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)benza-
mide (6). 2-Nitrophenylmethanamine (66.5 mg, 0.43 mmol) and
carboxylic acid 2 (50 mg, 0.22 mmol) were mixed following the
procedure described for 1 to yield 61.6 mg of the title compound
N-(2-chloro-4-fluorobenzyl)-4-((3,5-dimethyl-1H-pyrazol-1-yl)me-
thyl)benzamide (1). In a reaction vial compound 2 (50 mg,
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694
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
1
as a brown solid (65%). H NMR (400 MHz, [D6]DMSO): d=9.07 (t,
9.01 (t, J=5.7 Hz, 1H), 7.86 (d, J=8.3 Hz, 2H), 7.64–7.58 (m, 1H),
7.37–7.32 (m, 1H), 7.31–7.27 (m, 1H), 7.25–7.18 (m, 1H), 7.17 (d,
J=8.3 Hz, 2H), 5.86 (s, 1H), 5.24 (s, 2H), 4.48 (d, J=5.8 Hz, 2H),
2.15 (s, 3H), 2.09 ppm (s, 3H); 13C NMR (101 MHz, [D6]DMSO): d=
166.1, 146.2, 141.3, 138.9, 137.8, 133.1, 132.3, 128.9, 128.5, 127.7,
127.6 (s, 2C), 126.7 (s, 2C), 122.2, 105.1, 51.2, 43, 13.3, 10.6 ppm;
LC–MS: tR =1.21 min, m/z 397.08 [M+H]+; purity: >95%.
J=5.7 Hz, 1H), 8.02 (dd, J=1.0, 8.1 Hz, 1H), 7.83 (d, J=8.3 Hz, 2H),
7.70 (dt, J=1.1, 7.6 Hz, 1H), 7.57–7.47 (m, 2H), 7.17 (d, J=8.3 Hz,
2H), 5.87 (s, 1H), 5.25 (s, 2H), 4.73 (d, J=5.8 Hz, 2H), 2.15 (s, 3H),
2.10 ppm (s, 3H); 13C NMR (101 MHz, [D6]DMSO): d=166, 148,
146.2, 141.3, 139, 134.2, 133.7, 132.9, 129.2, 128.2, 127.6 (s, 2C),
126.8 (s, 2C), 124.5, 105.2, 51.2, 39.9, 13.3, 10.6 ppm; LC–MS: tR =
1.14 min, m/z 364.15 [M+H]+; purity: >95%.
N-(4-fluoro-2-(trifluoromethyl)benzyl)-4-((3,5-dimethyl-1H-pyra-
zol-1-yl)methyl)benzamide (12). 4-Fluoro-2-(trifluoromethyl)phe-
nylmethanamine (63.5 mg, 0.434 mmol) and carboxylic acid 2
(50 mg, 0.22 mmol) were mixed following the procedure described
for 1 to yield 51.5 mg of the title compound as a white solid
(70%). 1H NMR (400 MHz, [D6]DMSO): d=9.07 (t, J=5.8 Hz, 1H),
7.85 (d, J=8.3 Hz, 2H), 7.62 (dd, J=2.5, 9.1 Hz, 1H), 7.57–7.47 (m,
2H), 7.17 (d, J=8.1 Hz, 2H), 5.87 (s, 1H), 5.25 (s, 2H), 4.60 (d, J=
5.6 Hz, 2H), 2.15 (s, 3H), 2.09 ppm (s, 3H); 13C NMR (101 MHz,
[D6]DMSO): d=166.2, 160.5 (d, J=244.4 Hz, 1C), 146.2, 141.4,
138.9, 133.8 (q, J=1.5 Hz, 1C), 132.9, 130.9 (d, J=8.1 Hz, 1C), 127.6
(s, 2C), 128 (qt, J=31.5 Hz, 1C), 126.7 (s, 2C), 125.1 (dq, J=2.9,
274.4 Hz, 1C), 119.4 (d, J=21.2 Hz, 1C), 113.4 (dd, J=5.9, 25.6 Hz,
1C), 105.1, 51.2, 38.9, 13.3, 10.6 ppm; LC–MS: tR =1.24 min, m/z
405.15 [M+H]+; purity: >95%.
N-(3-bromobenzyl)-4-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)ben-
zamide (7). 3-Bromophenylmethanamine (80 mg, 0.43 mmol) and
carboxylic acid 2 (100 mg, 0.43 mmol) were mixed following the
procedure described for 1 to yield 143 mg of the title compound
1
as a colorless oil (84%). H NMR (400 MHz, [D6]DMSO): d=9.03 (t,
J=5.9 Hz, 1H), 7.82 (d, J=8.3 Hz, 2H), 7.48 (s, 1H), 7.43 (td, J=1.8,
7.0 Hz, 1H), 7.32–7.25 (m, 2H), 7.15 (d, J=8.1 Hz, 2H), 5.86 (s, 1H),
5.24 (s, 2H), 4.45 (d, J=5.8 Hz, 2H), 2.14 (s, 3H), 2.09 ppm (s, 3H);
13C NMR (101 MHz, [D6]DMSO): d=165.9, 146.2, 142.5, 141.3, 138.9,
133.1, 130.5, 129.9, 129.6, 127.5 (s, 2C), 126.7 (s, 2C), 126.2, 121.5,
105.1, 51.2, 42, 13.3, 10.6 ppm; LC–MS: tR =1.22 min, m/z 397.08
[M+H]+; purity: >95%.
N-(3-chlorobenzyl)-4-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)ben-
zamide (8). 3-Chlorophenylmethanamine (61.5 mg, 0.434 mmol)
and carboxylic acid 2 (50 mg, 0.22 mmol) were mixed following
the procedure described for 1 to yield 47.4 mg of the title com-
N-(4-fluorobenzyl)-4-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)ben-
zamide (13). 4-Fluorophenylmethanamine (64.5 mg, 0.434 mmol)
and carboxylic acid 2 (50 mg, 0.22 mmol) were mixed following
the procedure described for 1 to yield 75.5 mg of the title com-
1
pound as a yellow solid (62%). H NMR (400 MHz, [D6]DMSO): d=
9.03 (t, J=5.9 Hz, 1H), 7.83 (d, J=8.1 Hz, 2H), 7.38–7.23 (m, 4H),
7.16 (d, J=8.3 Hz, 2H), 5.87 (s, 1H), 5.24 (s, 2H), 4.45 (d, J=5.8 Hz,
2H), 2.14 (s, 3H), 2.09 ppm (s, 3H); 13C NMR (101 MHz, [D6]DMSO):
d=166, 146.2, 142.3, 141.2, 139, 133.1, 132.9, 130.2, 127.5 (s, 2C),
127, 126.7 (s, 2C), 126.7, 125.8, 105., 51.2, 42.1, 13.3, 10.6 ppm; LC–
MS: tR =1.19 min, m/z 353.13 [M+H]+; purity: >95%.
1
pound as a yellow solid (100%). H NMR (400 MHz, [D6]DMSO): d=
9.00 (t, J=5.9 Hz, 1H), 7.82 (d, J=8.1 Hz, 2H), 7.33 (dd, J=5.8,
8.6 Hz, 2H), 7.18–7.09 (m, 4H), 5.87 (s, 1H), 5.24 (s, 2H), 4.43 (d, J=
5.8 Hz, 2H), 2.14 (s, 3H), 2.09 ppm (s, 3H); 13C NMR (101 MHz,
[D6]DMSO): d=165.9, 160.9 (d, J=241.5 Hz, 1C), 146.2, 141, 139,
135.8 (d, J=2.9 Hz, 1C), 133.3, 129.1 (d, J=8.1 Hz, 2C), 127.5 (s,
2C), 126.7 (s, 2C), 115 (d, J=21.2 Hz, 2C), 105.2, 51.2, 41.9, 13.3,
10.6 ppm; LC–MS: tR =1.15 min, m/z 337.39 [M+H]+; purity:
>95%.
N-(benzyl)-4-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzamide
(9). Phenylmethanamine (0.025 mL, 0.23 mmol) and carboxylic acid
2 (53.7 mg, 0.23 mmol) were mixed following the procedure de-
scribed for 1 to yield 110 mg of the title compound as a white
solid (100%). 1H NMR (400 MHz, [D6]DMSO): d=8.99 (t, J=5.9 Hz,
1H), 7.83 (d, J=8.3 Hz, 2H), 7.34–7.26 (m, 4H), 7.23 (td, J=2.9,
6.1 Hz, 1H), 7.15 (d, J=8.1 Hz, 2H), 5.86 (s, 1H), 5.24 (s, 2H), 4.46
(d, J=5.8 Hz, 2H), 2.14 (s, 3H), 2.09 ppm (s, 3H); 13C NMR
(101 MHz, [D6]DMSO): d=165.9, 146.2, 141.1, 139.6, 139, 133.3,
128.2 (s, 2C), 127.5 (s, 2C), 127.1, 126.7 (s, 3C), 105.1, 51.2, 42.5,
13.3, 10.6 ppm; LC–MS: tR =1.15 min, m/z 319.17 [M+H]+; purity:
>95%.
N-(4-fluorobenzyl)-4-((2-methyl-5-trifluoromethyl)furan-3-yl)me-
thyl)benzamide (14). (5-Methyl-2-(trifluoromethyl)furan-3-yl)me-
thanamine (38.9 mg, 0,21 mmol) and compound
2 (50 mg,
0.21 mmol) were mixed following the procedure described for 1 to
1
yield 50 mg of the title compound as a white solid (59%). H NMR
(400 MHz, [D6]DMSO): d=8.94 (t, J=5.8 Hz, 1H), 7.80 (d, J=8.1 Hz,
2H), 7.15 (d, J=8.3 Hz, 2H), 6.25 (s, 1H), 5.85 (s, 1H), 5.23 (s, 2H),
4.36 (d, J=4.5 Hz, 2H), 2.27 (s, 3H), 2.13 (s, 3H), 2.09 ppm (s, 3H);
13C NMR (101 MHz, [D6]DMSO): d=166, 154.8, 146.2, 141.3, 138.8,
133–132.9 (m, 1C), 133.4–132.7 (m, 1C), 127.9 (d, J=2.2 Hz, 1C),
127.5 (s, 2C), 126.7 (s, 2C), 121.3–118.5 (m, 1C), 108.9, 105.1, 51.2,
33, 13.3, 13, 10.6 ppm; LC–MS: tR =1.24 min, m/z 391.15 [M+H]+;
purity: >95%.
N-(3-methanesulfonybenzyl)-4-((3,5-dimethyl-1H-pyrazol-1-yl)-
methyl)benzamide (10). 3-Methanesulfonylphenylmethanamine
(60.4 mg, 0.434 mmol) and carboxylic acid 2 (50 mg, 0.22 mmol)
were mixed following the procedure described for 1 to yield
47.9 mg of the title compound as a white solid (55%). 1H NMR
(400 MHz, [D6]DMSO): d=9.12 (t, J=5.9 Hz, 1H), 7.87–7.77 (m, 4H),
7.65 (d, J=7.8 Hz, 1H), 7.60 (t, J=7.6 Hz, 1H), 7.17 (d, J=8.1 Hz,
2H), 5.89 (s, 1H), 5.26 (s, 2H), 4.56 (d, J=5.8 Hz, 2H), 3.18 (s, 3H),
2.15 (s, 3H), 2.10 ppm (s, 3H); 13C NMR (101 MHz, [D6]DMSO): d=
166.1, 146.2, 141.3, 141.1, 140.9, 139.2, 133.1, 132.3, 129.5, 127.6 (s,
2C), 126.8 (s, 2C), 125.4, 125.4, 105.3, 51.2, 43.5, 42.3, 13.2,
10.6 ppm; LC–MS: tR =1.04 min, m/z 397.15 [M+H]+; purity:
>95%.
N-((4-bromo-1-ethyl-1H-pyrazol-5-yl)methyl)-4-((3,5-dimethyl-1H-
pyrazol-1-yl)methyl)benzamide (15). (4-Bromo-1-ethyl-1H-pyrazol-
5-yl)methylamine (44.3 mg, 0,21 mmol) and carboxylic acid
2
(50 mg, 0.21 mmol) were mixed following the procedure described
for 1 to yield 45 mg of the title compound as a white solid (50%).
1H NMR (400 MHz, [D6]DMSO): d=8.91 (t, J=5.2 Hz, 1H), 7.83–7.74
(m, J=8.3 Hz, 2H), 7.51 (s, 1H), 7.17–7.09 (m, J=8.3 Hz, 2H), 5.84
(s, 1H), 5.22 (s, 2H), 4.51 (d, J=5.3 Hz, 2H), 4.17 (q, J=7.3 Hz, 2H),
2.13 (s, 3H), 2.08 (s, 3H), 1.26 ppm (t, J=7.2 Hz, 3H); 13C NMR
(101 MHz, [D6]DMSO): d=166, 146.2, 141.3, 138.9, 138, 136.6, 132.8,
127.6 (s, 2C), 126.7 (s, 2C), 105.1, 93.2, 51.2, 44.9, 32.5, 15.3, 13.3,
10.6 ppm; LC–MS: tR =1.13 min, m/z 415.10 [M+H]+; purity:
>95%.
N-(3-chlorobenzyl)-4-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)ben-
zamide (11). 3-Chlorophenylmethanamine (80 mg, 0.434 mmol)
and carboxylic acid 2 (100 mg, 0.43 mmol) were mixed following
the procedure described for 1 to yield 140 mg of the title com-
pound as a white solid (82%). 1H NMR (400 MHz, [D6]DMSO): d=
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(2-chloro-5-nitrophenyl)methanol (19). 2-Chloro-5-nitrobenzoic
acid (3 g, 14.9 mmol) was added to a stirred suspension of NaBH4
(2.2 g, 59.4 mmol) in dry THF (80 mL) at 08C. Then boron trifluoride
etherate (5 mL, 40 mmol) was slowly incorporated, leaving the re-
action mixture to reach room temperature overnight. The solvent
was removed under vacuum, and the resulting crude was dissolved
in CH2Cl2 and washed with HCl (1n) and brine. The organic layer
was dried over MgSO4, filtered off, and volatiles were evaporated
under vacuum. The residue was purified by column chromatogra-
phy to yield 2.5 g of the title compound as a white solid (90%).
1H NMR (300 MHz, CDCl3): d=8.46 (d, J=2.6 Hz, 1H), 8.10 (dd, J=
2.3, 8.6 Hz, 1H), 7.52 (d, J=8.8 Hz, 1H), 4.87 (d, J=4.1 Hz, 2H),
2.10 ppm (brs, 1H).
2.08 ppm (m, 3H); LC–MS: tR =2.47 min, m/z 368.14 [M+H]+;
purity: >95%.
N-(2-chloro-5-(3-phenylureido)benzyl)-4-((3,5-dimethyl-1H-pyra-
zol-1-yl)methyl)benzamide (17). A mixture of aniline 16 (50 mg,
0.13 mmol) and phenyl isocyanate (17 mg, 0.14 mmol) in CH2Cl2
(5 mL) was left under stirring at 258C overnight. The white precipi-
tate was filtered off and washed with CH2Cl2 to afford 28 mg of the
title compound as
a
white solid (44%). 1H NMR (400 MHz,
[D6]DMSO): d=9.03 (t, J=5.8 Hz, 1H), 8.78 (s, 1H), 8.53 (s, 1H),
7.89 (d, J=8.3 Hz, 2H), 7.55 (dd, J=2.5, 8.6 Hz, 1H), 7.38 (d, J=
7.6 Hz, 2H), 7.33 (d, J=8.6 Hz, 1H), 7.28–7.21 (m, 3H), 7.18 (d, J=
8.1 Hz, 2H), 6.95 (t, J=7.3 Hz, 1H), 5.86 (s, 1H), 5.25 (s, 2H), 4.48 (d,
J=5.8 Hz, 2H), 2.16 (s, 3H), 2.09 ppm (s, 3H); 13C NMR (101 MHz,
[D6]DMSO): d=166.1, 152.3, 146.2, 141.4, 139.5, 138.9, 138.9, 136.6,
133.1, 129.3, 128.7 (s, 2C), 127.6 (s, 2C), 126.8 (s, 2C), 124, 121.9,
118.2 (s, 2C), 117.8, 117.5, 105.1, 51.2, 40.4, 13.3, 10.6 ppm; LC–MS:
tR =2.72 min, m/z 487.18 [M+H]+; purity: >95%.
2-(azidomethyl)-1-chloro-4-nitrobenzene (20). Triphenylphos-
phine (5.9 g, 19.5 mmol) followed by CBr4 (6.4 g, 19.5 mmol) were
added to a stirred solution of alcohol 19 (2.4 g, 13 mmol) in DMF
(60 mL) at 08C, and the mixture was left stirring for 2 h, reaching
room temperature. NaN3 (2.53 g, 39 mmol) was then incorporated
in several portions, and the reaction was left stirring overnight. The
solvent was removed under vacuum, and the resulting crude was
dissolved in tBuOMe and washed with NaHCO3 (10%) and brine.
The organic layer was dried over MgSO4, filtered off, and the vola-
tiles were evaporated under vacuum. The residue was purified by
column chromatography to yield 2 g of the title compound as
a colorless oil (72%). 1H NMR (300 MHz, CDCl3): d=8.33 (d, J=
2.6 Hz, 1H), 8.16 (dd, J=2.4, 8.7 Hz, 1H), 7.59 (d, J=8.8 Hz, 1H),
4.62 ppm (s, 2H).
N-(2-chloro-5-(2-phenylacetamido)benzyl)-4-((3,5-dimethyl-1H-
pyrazol-1-yl)methyl)benzamide (18). A mixture of aniline 16
(50 mg, 0.13 mmol) and phenylacetic acid (22 mg, 0.16 mmol) was
treated following the procedure described for benzamide 22. The
purification in this case was performed by preparative HPLC to
1
yield 3 mg of the title compound as a white solid (4.7%). H NMR
(300 MHz, CD3OD): d=7.85 (d, J=8.3 Hz, 2H), 7.66–7.48 (m, 2H),
7.42–7.20 (m, 6H), 7.16 (d, J=8.3 Hz, 2H), 6.00 (s, 1H), 5.33 (s, 2H),
4.61 (s, 3H), 3.61 (s, 2H), 2.22 ppm (d, J=3.4 Hz, 6H); LC–MS: tR =
2.73 min, m/z 486.18 [M+H]+; purity: >95%.
N-(2-chloro-5-nitrobenzyl)-4-((3,5-dimethyl-1H-pyrazol-1-yl)me-
N-[(2-chloro-4-fluorophenyl)methyl]-4-formylbenzamide (23). 4-
Carboxybenzaldehyde (3 g, 19.9 mmol) and 2-chloro-4-fluorophe-
nylmethanamine (3.4 g, 19.9 mmol) was added following the pro-
cedure described for compound 1 to yield 5.8 g of the title com-
pound (100%). 1H NMR (400 MHz, [D6]DMSO): d=10.08 (s, 1H),
9.26 (t, J=5.5 Hz, 1H), 8.12–8.05 (m, 2H), 8.04–7.98 (m, 2H), 7.51–
7.35 (m, 2H), 7.21 (dt, J=2.5, 8.5 Hz, 1H), 4.52 ppm (d, J=5.5 Hz,
2H).
thyl)benzamide
(22).
(2-Chloro-5-nitrophenyl)methanamine
(21·H2O; 0.33 mL, 18 mmol) was added to a stirred solution of
azide derivative 20 (1.95 g, 9.2 mmol) and triphenylphosphine
(3.6 g, 13.7 mmol) in THF (50 mL) at room temperature. The mix-
ture was left under stirring overnight. The solvent was removed
under vacuum, and the resulting crude was dissolved in tBuOMe
and washed with HCl (4n). The resulting precipitate was filtered
and washed with tBuOMe to yield 0.5 g of the title compound as
a brown hydrochloride salt (quant). A solution of benzyl amine 21
(1 g, 4.3 mmol) in CH2Cl2 (2 mL) was added to a mixture of carbox-
ylic acid 2 (0.5 g, 2.17 mmol), EDCI (0.5 g, 2.6 mmol), and DIPEA
(0.72 mL, 4.5 mmol) in CH2Cl2/DMF (10 mL/2 mL). Then DMAP was
added in one portion, and the mixture was stirred at room temper-
ature overnight. The solvent was removed under vacuum. The re-
sulting residue was dissolved in EtOAc and washed with H2O. The
organic layer was dried over MgSO4, and the volatiles were re-
moved under vacuum. The resulting crude was purified by column
chromatography to yield 300 mg of the title compound (29%).
1H NMR (300 MHz, CDCl3): d=8.28 (d, J=2.6 Hz, 1H), 8.09 (dd, J=
2.5, 8.5 Hz, 1H), 7.75 (d, J=8.1 Hz, 2H), 7.55 (d, J=8.8 Hz, 1H), 7.11
(d, J=7.9 Hz, 2H), 6.83 (brs, 1H), 5.86 (s, 1H), 5.24 (s, 2H), 4.76 (d,
J=6.2 Hz, 2H), 2.24 (s, 3H), 2.14 ppm (s, 3H).
N-[(2-chloro-4-fluorophenyl)methyl]-4-(hydroxymethyl)benza-
mide (24). Aldehyde 23 (5.8 g, 19.9 mmol) was added to a stirred
suspension of NaBH4 (1.15 g, 30.4 mmol) in dry THF (30 mL) at 08C.
The mixture was left to reach room temperature for 2 h. MeOH
was then added dropwise (20 mL), and the reaction was kept
under stirring for 20 min. The solvent was removed under vacuum,
and the resulting crude was dissolved in EtOAc and washed with
H2O and brine. The organic layer was dried over MgSO4, filtered
off, and the volatiles were evaporated under vacuum to yield 3.5 g
of the title compound as a pale-yellow solid (58%). 1H NMR
(400 MHz, [D6]DMSO): d=8.99 (t, J=5.8 Hz, 1H), 7.86 (d, J=8.5 Hz,
2H), 7.44 (dd, J=2.8, 8.8 Hz, 1H), 7.40 (d, J=8.5 Hz, 2H), 7.39–7.36
(m, J=6.5 Hz, 1H), 7.20 (dt, J=3.0, 8.5 Hz, 1H), 5.30 (t, J=5.5 Hz,
1H), 4.55 (d, J=5.5 Hz, 2H), 4.49 ppm (d, J=5.5 Hz, 2H).
N-(2-chloro-5-aminobenzyl)-4-((3,5-dimethyl-1H-pyrazol-1-yl)me-
thyl)benzamide (16). A solution of nitro derivative 22 (0.25 g,
0.62 mmol) in dioxane/H2O (5 mL/1 mL) was treated with iron
powder (0.32 g, 5.6 mmol) and FeSO4·7H2O (0.38 g, 1.4 mmol) and
stirred at reflux for 6 h. It was then cooled to room temperature
and filtered through Celite. The filtrate was eluted with EtOAc and
washed with NaHCO3 (10%). The organic layer was dried over
MgSO4, and the volatiles were removed under vacuum to yield
230 mg of the title compound as a yellow solid (99%). 1H NMR
(300 MHz, CDCl3): d=7.71 (d, J=8.1 Hz, 2H), 7.17–7.05 (m, 3H),
6.76 (d, J=2.6 Hz, 1H), 6.65–6.42 (m, 2H), 5.86 (s, 1H), 5.24 (s, 2H),
4.60 (d, J=6.0 Hz, 2H), 3.65 (brs, 2H), 2.27–2.19 (m, 3H), 2.17–
N-[(2-chloro-4-fluorophenyl)methyl]-4-[(3-methyl-1H-pyrazol-1-
yl)methyl]benzamide (25). Compound 24 (60 mg, 0.2 mmol) was
dissolved in anhydrous THF at room temperature. Then methane-
sulfonic anhydride (42 mg, 0.25 mmol) and DIPEA (0.052 mL,
0.3 mmol) were added, and the mixture was stirred for 30 min. A
solution of 3-methyl-1H-pyrazole (32.8 mg, 0.4 mmol) and polymer-
supported BEMP (82.2 mg, 0.3 mmol) in CH2Cl2 was then added to
the initial mixture, and the reaction was held at reflux (608C) for
48 h. The polymer-supported BEMP was then filtered out, and the
solvent was removed under vacuum. The resulting crude was then
purified by preparative HPLC to yield 10 mg of the title compound
(10%). 1H NMR (400 MHz, CDCl3): d=7.72 (d, J=8.3 Hz, 2H), 7.43
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(dd, J=6.1, 8.6 Hz, 1H), 7.27 (d, J=2.0 Hz, 1H), 7.21 (d, J=8.3 Hz,
2H), 7.13 (dd, J=2.5, 8.3 Hz, 1H), 6.95 (dt, J=2.5, 8.3 Hz, 1H), 6.62
(t, J=5.1 Hz, 1H), 6.06 (d, J=2.3 Hz, 1H), 5.26 (s, 2H), 4.66 (d, J=
6.1 Hz, 2H), 2.28 ppm (s, 3H); 13C NMR (101 MHz, CDCl3): d=166.9,
161.9 (d, J=250.3 Hz, 1C), 149.2, 140.8, 134.3 (d, J=10.2 Hz, 1C),
133.6, 131.6–131.5 (m, 1C), 131.6 (d, J=8.0 Hz, 1C), 130.2, 127.5 (s,
2C), 127.4 (s, 2C), 117 (d, J=24.9 Hz, 1C), 114.3 (d, J=21.2 Hz, 1C),
105.8, 55.1, 41.4, 13.6 ppm; LC–MS: tR =3.67 min, m/z 358.09 [M+
H]+; purity: >95%.
160.8 (d, J=245.9 Hz, 1C), 145.7, 142.3, 138.5, 132.8 (d, J=2.9 Hz,
1C), 132.6 (d, J=11.0 Hz, 1C), 132, 130.2 (d, J=8.8 Hz, 1C), 128.8
(s, 2C), 127.3 (s, 2C), 116.3 (d, J=25.6 Hz, 1C), 114.2 (d, J=21.2 Hz,
1C), 104.2, 48.9, 40, 35.9, 13.4, 10.3 ppm; LC–MS: tR =1.23 min, m/z
385.14 [M+H]+; purity: >95%.
General procedure for the preparation of compounds 31, 33, 34,
36, and 37: methyl 4-((4-methylthiazol-2-yl)methyl)benzoate
(29). To a suspension of Zn0 (261 mg, 4 mmol) in THF (3 mL) at
1008C under Ar atmosphere, methyl 4-(bromomethyl)benzoate
(916 mg, 4 mmol) was added, and the mixture was left under stir-
ring for 1.5 h. The mixture was allowed to reach room temperature,
and a solution of 2-iodo-4-methylthiazole (300 mg, 1.5 mmol) and
tetrakis(triphenylphosphine)palladium (462 mg, 0.4 mmol) in THF
(4 mL) was added, holding the mixture at reflux for 3 h. Volatiles
were removed under vacuum, and the crude was re-dissolved in
EtOAc and washed with NaHCO3 and brine. The organic layer was
dried over MgSO4, and the volatiles were removed under vacuum.
The resulting crude was purified by column chromatography to
N-[(2-chloro-4-fluorophenyl)methyl]-4-(1H-pyrazol-1-yl)methyl)-
benzamide (26). 1H-Pyrazole (27.2 mg, 0.4 mmol) was added fol-
lowing the procedure described for compound 25 to yield the title
1
compound as a white solid. H NMR (400 MHz, [D6]DMSO): d=9.00
(t, J=5.7 Hz, 1H), 7.88–7.81 (m, 3H), 7.47 (d, J=1.8 Hz, 1H), 7.44
(dd, J=2.5, 8.8 Hz, 1H), 7.37 (dd, J=6.3, 8.6 Hz, 1H), 7.27 (d, J=
8.3 Hz, 2H), 7.18 (dt, J=2.8, 8.6 Hz, 1H), 6.28 (t, J=2.0 Hz, 1H), 5.39
(s, 2H), 4.48 ppm (d, J=5.6 Hz, 2H); 13C NMR (101 MHz, [D6]DMSO):
d=166.1, 160.9 (d, J=246.6 Hz, 1C), 141.1, 139.2, 133.2, 132.7 (d,
J=3.7 Hz, 1C), 132.6 (d, J=11.0 Hz, 1C), 130.3, 130.1 (d, J=9.5 Hz,
1C), 127.5 (s, 2C), 127.3 (s, 2C), 116.4 (d, J=24.9 Hz, 1C), 114.2 (d,
J=21.2 Hz, 1C), 105.5, 54.2, 40.1 ppm; LC–MS: tR =1.16 min, m/z
343.09 [M+H]+; purity: >95%.
1
yield 126 mg of the title compound as a yellow oil (40%). H NMR
(300 MHz, CDCl3): d =8.00 (d, J=8.3 Hz, 2H), 7.38 (d, J=8.2 Hz,
2H), 6.76 (d, J=1.0 Hz, 1H), 4.35 (s, 2H), 3.90 (s, 3H), 2.49-
2.39 ppm (m, 3H).
N-[(2-chloro-4-fluorophenyl)methyl]-3-[(3,5-dimethyl-1H-pyrazol-
1-yl)methyl]benzamide (27). To a solution of 3,5-dimethyl-1H-pyra-
zole (41.8 mg, 0.43 mmol) and NatBuO (83.6 mg, 0.87 mmol) in
DMF (1 mL) was slowly added another solution containing 3-(bro-
momethyl)benzoic acid (93,5 mg, 0.43 mmol) in DMF (1 mL); this
was left to stir at room temperature for 2 h. The solvent was re-
moved under vacuum, and the residue was dissolved in H2O and
then acidified to pH 6 with 2n HCl. It was then extracted with
EtOAc (3”100 mL). The combined extracts were dried over MgSO4,
filtered off, and the volatiles were removed under vacuum to
afford 88 mg of the intermediate 3-(3,5-dimethyl-1H-pyrazol-1-yl)-
4-((4-methylthiazol-2-yl)methyl)benzoic acid (30). To a solution of
29 (122 mg, 0.5 mmol) in THF (5 mL) was added dropwise a mixture
of LiOH (24 mg, 1 mmol) in H2O (5 mL), and the reaction was to stir
at room temperature overnight. Volatiles were removed under
vacuum, and the crude was re-dissolved in H2O and acidified to
pH 5 with HCl. The resulting precipitate was filtered off to yield
102 mg of the title compound as a brown solid (87%). 1H NMR
(300 MHz, CDCl3): d=8.06 (d, J=8.1 Hz, 2H), 7.42 (d, J=7.9 Hz,
2H), 6.77 (s, 1H), 4.38 (s, 2H), 2.44 ppm (s, 3H).
N-(2-chloro-4-fluorobenzyl)-4-((4-methylthiazol-2-yl)methyl)-
benzamide (31). 2-Chloro-4-fluorophenylmethanamine (69 mg,
0.43 mmol) and compound 30 (100 mg, 0.43 mmol) were mixed
following the procedure described for 22 to yield 40 mg of the
1
benzoic acid (88%). H NMR (400 MHz, [D6]DMSO): d=9.04 (t, J=
5.7 Hz, 1H), 7.79 (d, J=7.8 Hz, 1H), 7.68 (s, 1H), 7.49–7.40 (m, 2H),
7.38 (dd, J=6.3, 8.6 Hz, 1H), 7.23–7.16 (m, 2H), 5.85 (s, 1H), 5.23 (s,
2H), 4.48 (d, J=5.8 Hz, 2H), 2.15 (s, 3H), 2.08 ppm (s, 3H); 13C NMR
(101 MHz, [D6]DMSO): d=166.2, 160.9 (d, J=245.9 Hz, 1C), 146.1,
138.8, 138.3, 134.2, 132.7 (d, J=3.7 Hz, 1C), 132.5, 130.2 (d, J=
8.8 Hz, 1C), 129.8, 128.6, 126., 126, 116.4 (d, J=25.6 Hz, 1C), 114.2
(d, J=20.5 Hz, 1C), 105.1, 51.3, 40, 13.3, 10.7 ppm; LC–MS: tR =
1.22 min, m/z 371.12 [M+H]+; purity: >95%.
1
title compound as a yellow solid (25%). H NMR (400 MHz, CDCl3):
d=7.74 (d, J=8.1 Hz, 2H), 7.46 (dd, J=5.9, 8.5 Hz, 1H), 7.38 (d, J=
8.3 Hz, 2H), 7.14 (dd, J=2.5, 8.3 Hz, 1H), 6.96 (dt, J=2.7, 8.3 Hz,
1H), 6.75 (d, J=1.0 Hz, 1H), 6.57 (s, 1H), 4.68 (d, J=6.1 Hz, 2H),
4.33 (s, 2H), 2.42 ppm (d, J=1.0 Hz, 3H); 13C NMR (101 MHz,
CDCl3): d=168.3, 167, 162 (d, J=250.3 Hz, 1C), 152.6, 141.8, 134.3
(d, J=11.0 Hz, 1C), 132.9, 131.8, 131.6 (d, J=8.8 Hz, 1C), 129.3 (s,
2C), 127.4 (s, 2C), 116.8 (d, J=24.9 Hz, 1C), 114.3 (d, J=21.2 Hz,
1C), 113.6, 41.4, 39.4, 17.1 ppm; LC–MS: tR =1.24 min, m/z 374.07
[M+H]+; purity: >95%.
N-[(2-chloro-4-fluorophenyl)methyl]-4-[2-(3,5-dimethyl-1H-pyra-
zol-1-yl)ethyl]benzamide (28). A solution of 3,5-dimethyl-1H-pyra-
zole (146.8 mg, 1.52 mmol), 4-(2-bromoethyl)benzoic acid (350 mg,
1.52 mmol), and NaHCO3 (127 mg, 1.52 mmol) in MeOH (3 mL) was
treated under microwave conditions at 1358C for 2 h. The solvent
was removed under vacuum, and the residue was dissolved in H2O
and then acidified to pH 7 with 1n HCl. It was then extracted with
EtOAc (3”10 mL). The combined extracts were dried over MgSO4,
filtered off, and the volatiles were removed under vacuum to
afford 100 mg of the intermediate 4-(2-(3,5-dimethyl-1H-pyrazol-1-
yl)ethyl)benzoic acid as a white solid (26%). 4-(2-(3,5-Dimethyl-1H-
pyrazol-1-yl)ethyl)benzoic acid (100 mg, 0.4 mmol) and 2-chloro-4-
fluorophenylmethanamine (77,6 mg, 0.45 mmol) were dissolved in
DMF (5 mL) and reacted following the procedure described for
compound 25 to yield 50 mg of the title compound as a white
solid (35%). 1H NMR (400 MHz, [D6]DMSO): d=8.96 (t, J=5.7 Hz,
1H), 7.79 (d, J=8.1 Hz, 2H), 7.43 (dd, J=2.7, 8.7 Hz, 1H), 7.38 (dd,
J=6.2, 8.7 Hz, 1H), 7.24–7.15 (m, 3H), 5.70 (s, 1H), 4.48 (d, J=
5.8 Hz, 2H), 4.12 (t, J=7.1 Hz, 2H), 3.04 (t, J=7.2 Hz, 2H), 2.08 (s,
3H), 1.91 ppm (s, 3H); 13C NMR (101 MHz, [D6]DMSO): d =166.1,
N-[(2-chloro-4-fluorophenyl)methyl]-4-(1,3-thiazol-2-yl)methyl)-
benzamide (33). Following the procedure described for compound
31 to obtain 4-(1,3-thiazol-2-ylmethyl)benzoic acid, 2-chloro-4-fluo-
rophenylmethanamine (220 mg, 1.2 mmol) and 4-(1,3-thiazol-2-yl-
methyl)benzoic acid (190 mg, 1.2 mmol) were mixed following the
procedure described for 22 to yield 112 mg of the title compound
as a white solid (26%). 1H NMR (400 MHz, CDCl3): d=7.74 (d, J=
8.1 Hz, 2H), 7.71 (d, J=3.3 Hz, 1H), 7.45 (dd, J=6.2, 8.5 Hz, 1H),
7.38 (d, J=8.1 Hz, 2H), 7.22 (d, J=3.3 Hz, 1H), 7.13 (dd, J=2.3,
8.3 Hz, 1H), 6.96 (dt, J=2.5, 8.3 Hz, 1H), 6.58 (brs, 1H), 4.68 (d, J=
6.1 Hz, 2H), 4.38 ppm (s, 2H); 13C NMR (101 MHz, CDCl3): d=168.9,
167, 162 (d, J=250.3 Hz, 1C), 142.7, 141.7, 134.3 (d, J=11.0 Hz,
1C), 133, 131.8, 131.7 (d, J=8.8 Hz, 1C), 129.3 (s, 2C), 127.5 (s, 2C),
119.2, 117 (d, J=24.9 Hz, 1C), 114.3 (d, J=20.5 Hz, 1C), 41.4,
39.2 ppm; LC–MS: tR =1.20 min, m/z 360.05 [M+H]+; purity:
>95%.
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N-[(2-chloro-4-fluorophenyl)methyl]-4-[(dimethyl-1,3-thiazol-2-
yl)methyl]benzamide (34). Following the procedure described for
compound 31 to obtain 4-[(dimethyl-1,3-thiazol-2-yl)methyl]benzo-
ic acid, 2-chloro-4-fluorophenylmethanamine (27 mg, 0.14 mmol)
and 4-[(dimethyl-1,3-thiazol-2-yl)methyl]benzoic acid (35 mg,
0.15 mmol) were mixed following the procedure described for 22
N-[(2-chloro-4-fluorophenyl)methyl]-4-[(2-methyl-1,3-thiazol-4-
yl)methyl]benzamide (32). 2-Chloro-4-fluorophenylmethanamine
(80 mg, 0.46 mmol) and compound 39 (110 mg, 0.46 mmol) were
mixed following the procedure described for 22 to yield 70 mg of
the title compound as a white solid (40%). 1H NMR (400 MHz,
CDCl3): d=7.76 (d, J=8.3 Hz, 2H), 7.50 (dd, J=6.1, 8.6 Hz, 1H),
7.37 (d, J=8.1 Hz, 2H), 7.17 (dd, J=2.5, 8.3 Hz, 1H), 7.00 (dt, J=
2.7, 8.3 Hz, 1H), 6.68 (s, 1H), 6.61 (t, J=5.3 Hz, 1H), 4.72 (d, J=
6.1 Hz, 2H), 4.15 (s, 2H), 2.72 ppm (s, 3H); 13C NMR (101 MHz,
CDCl3): d=167.1, 166.1, 161.9 (d, J=249.6 Hz, 1C), 154.8, 143.2,
134.3, 132.2, 131.7, 131.7–131.6 (m, 1C), 129.2 (s, 2C), 127.2 (s, 2C),
116.9 (d, J=24.9 Hz, 1C), 114.3–114.1 (m, 1C), 114.2 (d, J=32.9 Hz,
1C), 41.4, 37.6, 19.2 ppm; LC–MS: tR =1.24 min, m/z 374.07 [M+
H]+; purity: >95%.
1
to yield 9 mg of the title compound as a white oil (17%). H NMR
(300 MHz, [D6]DMSO): d=9.00 (t, J=5.7 Hz, 1H), 7.85 (d, J=8.2 Hz,
2H), 7.49–7.32 (m, 4H), 7.19 (dt, J=2.6, 8.6 Hz, 1H), 4.49 (d, J=
5.7 Hz, 2H), 4.24 (s, 2H), 2.24 (s, 3H), 2.19 ppm (s, 3H); LC–MS: tR =
2.86 min, m/z 380.08 [M+H]+; purity: >95%.
N-[(2-chloro-4-fluorophenyl)methyl]-4-[(6-methylpyridin-2-yl)me-
thyl]benzamide (36). Following the procedure described for com-
pound 31 to obtain 4-[(6-methylpyridin-2-yl)methyl]benzoic acid,
2-chloro-4-fluorophenylmethanamine (52.6 mg, 0.33 mmol) and 4-
[(6-methylpyridin-2-yl)methyl]benzoic acid (64 mg, 0.28 mmol)
were mixed following the procedure described for 22 to yield
20 mg of the title compound as a yellow solid (20%). 1H NMR
(400 MHz, [D6]DMSO): d =8.95 (t, J=5.7 Hz, 1H), 7.82 (d, J=8.3 Hz,
2H), 7.58 (t, J=7.7 Hz, 1H), 7.43 (dd, J=2.5, 8.8 Hz, 1H), 7.39–7.33
(m, 3H), 7.18 (dt, J=2.7, 8.5 Hz, 1H), 7.09–7.02 (m, 2H), 4.48 (d, J=
5.6 Hz, 2H), 4.08 (s, 2H), 2.42 ppm (s, 3H); 13C NMR (101 MHz,
[D6]DMSO): d=166.2, 160.9 (d, J=259.8 Hz, 1C), 159.3, 157.4,
143.5, 136.9, 132.8 (d, J=3.7 Hz, 1C), 131.9, 130.1 (d, J=9.5 Hz,
2C), 128.8 (s, 2C), 127.4 (s, 2C), 120.7, 120, 116.3 (d, J=24.9 Hz,
1C), 114.2 (d, J=21.2 Hz, 1C), 43.5, 40, 24 ppm; LC–MS: tR =
1.23 min, m/z 368.11 [M+H]+; purity: >95%.
N-[(2-chloro-4-fluorophenyl)methyl]-4-[(6-methylpyridin-2-yl)-
amino]benzamide
(41).
6-Methyl-2-bromopyridine
(14 mL,
0.11 mmol), compound 40 (30 mg, 0.11 mmol; Supporting Informa-
tion), Pd2(dba)3 (4 mg, 0.004 mmol), dppp (3.7 mg, 0.008 mmol),
and NatBuO (15 mg, 1.5 mmol) were dissolved in toluene (3 mL).
The mixture was purged with Ar and left under stirring at 758C
overnight. H2O and tBuOMe were added. The organic layer was
dried over MgSO4, and the volatiles were removed under vacuum.
The resulting crude was purified by column chromatography to
1
yield 19 mg of the title compound as a yellow solid (53%). H NMR
(400 MHz, CDCl3): d=7.78–7.70 (m, 2H), 7.50–7.43 (m, 2H), 7.43–
7.37 (m, 2H), 7.14 (dd, J=2.5, 8.3 Hz, 1H), 6.96 (dt, J=2.7, 8.3 Hz,
1H), 6.80 (s, 1H), 6.74 (d, J=8.3 Hz, 1H), 6.69 (d, J=7.3 Hz, 1H),
6.53 (t, J=5.4 Hz, 1H), 4.68 (d, J=6.1 Hz, 2H), 2.47 ppm (s, 3H);
13C NMR (101 MHz, CDCl3): d=166.8, 161.9 (d, J=249.6 Hz, 1C),
157.2, 153.9, 144.1, 138.3, 134.3 (d, J=10.2 Hz, 1C), 131.9 (d, J=
3.7 Hz, 1C), 131.6 (d, J=8.8 Hz, 1C), 128.4 (s, 2C), 126.8, 117.8 (s,
2C), 116.9 (d, J=24.9 Hz, 1C), 115.5, 114.3 (d, J=20.5 Hz, 1C),
106.6, 41.3, 24.1 ppm; LC–MS: tR =1.27 min, m/z 369.10 [M+H]+;
purity: >95%.
N-[(2-chloro-4-fluorophenyl)methyl]-4-{[6-(trifluoromethyl)pyri-
din-2-yl]methyl}benzamide (37). Following the procedure de-
scribed for compound 31 to obtain 4-{[6-(trifluoromethyl)pyridin-2-
yl]methyl}benzoic
acid,
2-chloro-4-fluorophenylmethanamine
(33.5 mg, 0.21 mmol) and 4-{[6-(trifluoromethyl)pyridin-2-yl]me-
thyl}benzoic acid (40 mg, 0.14 mmol) were mixed following the
procedure described for 22 to yield 20 mg of the title compound
as a white solid (35%). 1H NMR (300 MHz, [D6]DMSO): d=8.98 (t,
J=5.8 Hz, 1H), 8.01 (t, J=7.8 Hz, 1H), 7.84 (d, J=8.3 Hz, 2H), 7.74
(d, J=7.6 Hz, 1H), 7.61 (d, J=7.9 Hz, 1H), 7.47–7.31 (m, 4H), 7.18
(dt, J=2.7, 8.6 Hz, 1H), 4.47 (d, J=5.7 Hz, 2H), 4.25 ppm (s, 2H);
LC–MS: tR =3.65 min, m/z 422.08 [M+H]+; purity: >95%.
N-[(2-chloro-4-fluorophenyl)methyl]-4-[(6-methylpyridin-2-yl)-
oxy]benzamide (42). Compound 61 (100 mg, 0.36 mmol; Support-
ing Information) was dissolved in DMSO (4 mL), and NaH (60% oil
suspension, 16 mg, 0.4 mmol) and finally 2-bromo-6-methylpyri-
dine (74 mg, 0.43 mmol) were added; the resulting mixture was
left to stir at 1208C overnight. EtOAc and H2O were then added.
The organic layer was dried over Na2SO4, and volatiles were re-
moved under vacuum. The resulting crude was purified by column
chromatography to yield 20 mg of the title compound as an
orange solid (20%). 1H NMR (300 MHz, CDCl3): d=7.94–7.71 (m,
2H), 7.59 (t, J=7.7 Hz, 1H), 7.46 (dd, J=6.1, 8.6 Hz, 1H), 7.23–7.09
(m, 3H), 7.07–6.87 (m, 2H), 6.68 (d, J=8.2 Hz, 2H), 4.68 (d, J=
6.0 Hz, 2H), 2.44 ppm (s, 3H); LC–MS: tR =2.95 min, m/z 370.09
[M+H]+; purity: >95%.
4-[(2-methyl-1,3-thiazol-4-yl)methyl]benzonitrile (38). Pd(Ph3)4
(76 mg, 0.07 mmol) was added to a solution of (4-cyanophenyl)bor-
onic acid (0.2 g, 1.36 mmol), 4-(chloromethyl)-2-methylthiazole
(0.25 g, 0.36 mmol) and Na2CO3 (2m, 3.4 mL, 6.8 mmol) in DME
(6 mL) previously bubbled with N2. The mixture was heated at
908C overnight. DME was evaporated, and the resulting crude was
re-dissolved in EtOAc and washed with H2O. The organic layer was
dried over MgSO4, and the volatiles were removed under vacuum.
The resulting crude was purified by column chromatography to
1
yield 0.17 g of the title compound as a colorless oil (59%). H NMR
N-[(2-chloro-4-fluorophenyl)methyl]-4-[(6-methylpyridin-2-yl)sul-
fanyl]benzamide (43). Compound 62 (70 mg, 0.24 mmol; Support-
ing Information), K2CO3 (99 mg, 0.72 mmol) and 2-bromo-6-methyl-
pyridine (0.03 mL, 0.24 mmol) were dissolved in DMF (5 mL) and
heated at 1708C 5 h. DMF was removed under high vacuum, and
the resulting crude was re-dissolved in EtOAc and washed with
H2O. The organic layer was dried over Na2SO4, and volatiles were
removed under vacuum. The resulting crude was purified by
column chromatography to yield 28 mg of the title compound as
(300 MHz, CDCl3): d=7.65–7.54 (m, J=8.2 Hz, 2H), 7.41–7.31 (m,
J=8.3 Hz, 2H), 6.71 (s, 1H), 4.13 (s, 2H), 2.69 ppm (s, 3H).
4-[(2-methyl-1,3-thiazol-4-yl)methyl]benzoic acid (39). A 5n solu-
tion of KOH was added to a solution of compound 38 (0.17 g,
0.8 mmol) in EtOH (5 mL). The mixture was left under stirring at
room temperature overnight. EtOH was evaporated, and the aque-
ous solution was extracted with tBuOMe. The aqueous layer was
then acidified to pH 5 with 2m HCl. The white precipitate was fil-
tered off and washed with H2O to obtain 0.11 g of the title com-
pound (62%). 1H NMR (300 MHz, CDCl3): d=8.02–7.90 (m, 2H),
7.34–7.27 (m, 2H), 6.65 (s, 1H), 4.16–4.03 (m, 2H), 2.71–2.60 ppm
(m, 3H).
1
a white solid (50%). H NMR (300 MHz, [D6]DMSO): d=7.76 (d, J=
8.2 Hz, 2H), 7.57 (d, J=8.2 Hz, 2H), 7.47 (dd, J=6.0, 8.5 Hz, 1H),
7.39 (t, J=7.8 Hz, 1H), 7.14 (dd, J=2.6, 8.3 Hz, 1H), 7.04–6.87 (m,
2H), 6.78 (d, J=7.9 Hz, 1H), 6.65 (t, J=5.4 Hz, 1H), 4.69 (d, J=
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6.0 Hz, 2H), 2.51 ppm (s, 3H); LC–MS: tR =2.99 min, m/z 386.07
[M+H]+; purity: >95%.
(s, 2H), 2.13 (s, 3H), 2.07 ppm (s, 3H); 13C NMR (101 MHz,
[D6]DMSO): d=167.7, 162.6–159.2 (m, 1C), 145.8, 138.5, 138.2,
134.3 (d, J=11.0 Hz, 1C), 133.4 (d, J=8.8 Hz, 1C), 132.5, 130.3 (d,
J=3.7 Hz, 1C), 127.4 (s, 2C), 119.1 (s, 2C), 116.2 (d, J=24.9 Hz, 1C),
114.1 (d, J=21.2 Hz, 1C), 104.9, 51.2, 40.2, 13.3, 10.6 ppm; LC–MS:
tR =1.22 min, m/z 371.12 [M+H]+; purity: >95%.
N-[(2-chloro-4-fluorophenyl)methyl]-4-{[(6-methylpyridin-2-yl)-
oxy]methyl}benzamide (44). Compound 63 (100 mg, 0.28 mmol;
Supporting Information), Cs2CO3 (182 mg, 0.56 mmol), and 6-meth-
ylpyridine-2-ol (30 mg, 0.28 mmol) were dissolved in CH3CN (5 mL)
and heated at 608C for 2 h. The solvent was removed under
vacuum, and the resulting crude was re-dissolved in EtOAc and
washed with H2O. The organic layer was dried over Na2SO4 and vol-
atiles were removed under vacuum. The resulting crude was puri-
fied by column chromatography to yield 45 mg of the title com-
Biology
In vitro assays
1
pound as a yellow solid (42%). H NMR (400 MHz, CDCl3): d=7.77
InhA enzyme assay. Four assays were carried out for each com-
pound, and reported data are the mean values. Enzyme activity
was measured fluorimetrically by following NADH oxidation (lex =
340 nm, lem =480 nm) using 50 mm NADH and 50 mm 2-trans-dode-
cenoyl-CoA (DDCoA) as substrates. Dose–response experiments to
determine IC50 values were performed using 5 nm InhA; percent re-
maining enzyme activity (%AR) at various compound concentra-
tions were calculated with the formula: %AR=100”{(sam-
pleÀctrl2)/(ctrl1Àctrl2)}, for which sample is the enzyme activity for
each compound concentration, ctrl1 is enzyme activity in the ab-
sence of test compound, and ctrl2 is NADH oxidation in the ab-
sence of enzyme. IC50 values were calculated by fitting %AR to
a two-parameter equation: %AR=100/{1+([compd]/IC50)”s}, in
which s is a slope factor; IC50 values were calculated using GraFit
software (ver. 5.0.12, Erithacus Software Ltd.). All reactions were
performed in 30 mm PIPES buffer (pH 6.8) at 258C.
(d, J=8.1 Hz, 2H), 7.52 (d, J=8.1 Hz, 2H), 7.50–7.41 (m, 2H), 7.14
(dd, J=2.7, 8.5 Hz, 1H), 6.97 (dt, J=2.7, 8.3 Hz, 1H), 6.73 (d, J=
7.3 Hz, 1H), 6.59 (d, J=8.3 Hz, 2H), 5.41 (s, 2H), 4.69 (d, J=6.1 Hz,
2H), 2.44 ppm (s, 3H); 13C NMR (101 MHz, CDCl3): d=167.1, 162.7,
162.2 (d, J=248.8 Hz, 1C), 156.2, 141.7, 138.9, 134.3 (d, J=10.2 Hz,
1C), 133.3, 131.7 (d, J=3.7 Hz, 1C), 131.6 (d, J=8.8 Hz, 1C), 127.9,
127, 117 (d, J=24.9 Hz, 1C), 116.1, 114.3 (d, J=21.2 Hz, 1C), 107.5,
66.5, 41.4, 24.0 ppm; LC–MS: tR =1.35 min, m/z 384.10 [M+H]+;
purity: >95%.
N-[(2-chloro-4-fluorophenyl)methyl]-4-[2-(6-methylpyridin-2-yl)-
ethyl]benzamide trifluoroacetic salt (45). Compound 61 (78 mg,
0.2 mmol; Supporting Information) was dissolved in MeOH (10 mL)
containing 2 drops of HCl 2m. Then Pd/C (7.8 mg, 10% wt) was
added, and the mixture was left under H2 (3500 kPa) with stirring
overnight. The mixture was filtered off to remove palladium under
N2 atmosphere. The volatiles were removed under vacuum, and
the corresponding crude was purified by preparative HPLC to
InhA enzyme assay (high-throughput screening methodology). Com-
pounds 3–8, 10–13, 25, 26, 31, and 55 were evaluated following
this methodology. Two assays were carried out for each com-
pound, and reported data are the mean values. Enzyme activity
was measured fluorimetrically by monitoring NADH oxidation
either directly (lex =340 nm, lem =480 nm), or by coupling with the
generation of resorufin (lex =550 nm, lem =590 nm) with diaphor-
ase.[30] Dose–response experiments to determine IC50 values were
performed using 50 mm NADH and 50 mm 2-trans-dodecenoyl-CoA
(DDCoA) as substrates, and 5 nm InhA. Percent remaining enzyme
activity (%AR) at various compound concentrations were calculat-
ed with the formula: %AR=100”{(sampleÀctrl2)/(ctrl1Àctrl2)}, for
which sample is the enzyme activity for each compound concen-
tration, ctrl1 is enzyme activity in the absence of test compound,
and ctrl2 is NADH oxidation in the absence of enzyme. IC50 values
were calculated using GraFit software (ver. 5.0.12, Erithacus Soft-
ware Ltd.) and ActivityBase software (ver. 7.4.5.2). All reactions
were run in 30 mm PIPES buffer (pH 6.8) and 0.1 mgmLÀ1 BSA at
258C.
1
afford 30 mg of the title compound as a white solid (40%). H NMR
(300 MHz, CD3OD): d=8.33 (t, J=7.9 Hz, 1H), 7.88–7.76 (m, J=
8.2 Hz, 2H), 7.76–7.62 (m, 2H), 7.41 (dd, J=6.2, 8.6 Hz, 1H), 7.36–
7.29 (m, J=8.2 Hz, 2H), 7.24 (dd, J=2.6, 8.7 Hz, 1H), 7.05 (dt, J=
2.5, 8.4 Hz, 1H), 4.61 (s, 2H), 3.21–3.13 (m, 2H), 2.74 ppm (s, 3H);
LC–MS: tR =1.96 min, m/z 382.12 [M+H]+; purity: >95%.
N-[(2-chloro-4-fluorophenyl)methyl]-4-{[(6-methylpyridin-2-yl)-
sulfanyl]methyl}benzamide (46). Compound 63 (284 mg,
0.8 mmol; Supporting Information) and 6-methylpyridine-2-thiol
(100 mg, 0.8 mmol) were mixed following the same procedure de-
scribed for 44 to yield 200 mg of the title compound as a white
solid (62%). 1H NMR (400 MHz, [D6]DMSO): d=8.97 (t, J=5.7 Hz,
1H), 7.81 (d, J=8.3 Hz, 2H), 7.55–7.48 (m, 3H), 7.43 (dd, J=2.5,
8.8 Hz, 1H), 7.37 (dd, J=6.3, 8.6 Hz, 1H), 7.18 (dt, J=2.7, 8.5 Hz,
1H), 7.07 (d, J=7.8 Hz, 1H), 6.96 (d, J=7.6 Hz, 1H), 4.47 (d, J=
5.6 Hz, 2H), 4.43 (s, 2H), 2.45 ppm (s, 3H); 13C NMR (101 MHz,
[D6]DMSO): d=166.1, 160.9 (d, J=245.9 Hz, 1C), 157.9, 156.5,
142.1, 137.0, 132.7 (d, J=3.7 Hz, 1C), 132.5 (d, J=9.5 Hz, 1C), 130.1
(d, J=8.8 Hz, 1C), 128.9 (s, 2C), 127.3 (s, 2C), 119.2, 118.6, 116.3 (d,
J=24.9 Hz, 1C), 114.2 (d, J=20.5 Hz, 1C), 40, 32.7, 24 ppm; LC–MS:
tR =1.34 min, m/z 400.08 [M+H]+; purity: >95%.
MIC90 determination on mycobacteria. For each compound the aver-
age value of duplicate determinations was calculated. We consid-
ered the duplicates to be correct if they were in the range of Æ1
dilution (1:2) in the assay. Minimum inhibitory concentration
(MIC90) values against M. tuberculosis strains for each compound
tested were determined in 96-well flat-bottom polystyrene micro-
titer plates in a final volume of 100 mL. Ten twofold drug dilutions
in neat DMSO starting at 50 mm were performed. Drug solutions
were added to Middlebrook 7H9 medium (Difco), and isoniazid
(INH; Sigma–Aldrich) was used as a positive control with twofold
dilutions of INH starting at 160 mgmLÀ1. The inoculums were stand-
ardized to ~1”107 CFUmLÀ1 and diluted 1:100 in Middlebrook 7H9
broth. The inoculums (100 mL each) were added to the entire plate,
but wells G12 and H12 were used as blank controls. All plates were
placed in a sealed box to prevent drying of the peripheral wells
and incubated at 378C without shaking for six days. A resazurin so-
2-(2-chloro-4-fluorophenyl)-N-(4-((3,5-dimethyl-1H-pyrazol-1-yl)-
methyl)phenyl)acetamide (47). Compound 65 (100 mg,
0.49 mmol; Supporting Information) was dissolved in DMF (5 mL)
and 2-chloro-4-fluorophenylacetic acid (101 mg, 0.54 mmol), HOBt
(82 mg, 0.54 mmol), and EDCI (103 mg, 0.54 mmol) were added
leaving the mixture under stirring at room temperature overnight.
DMF was removed under high vacuum, and the resulting crude
was purified by HPLC to afford 65 mg of the title compound as
a white solid (36%). 1H NMR (400 MHz, [D6]DMSO): d=10.21 (s,
1H), 7.51 (d, J=8.6 Hz, 2H), 7.47–7.39 (m, 2H), 7.19 (dt, J=2.7,
8.5 Hz, 1H), 7.03 (d, J=8.6 Hz, 2H), 5.82 (s, 1H), 5.10 (s, 2H), 3.79
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lution was prepared by dissolving one tablet of resazurin (Resazurin
Tablets for Milk Testing, Ref. 330884Y, VWR Intl. Ltd.) in 30 mL sterile
phosphate-buffered saline (PBS). Of this solution, 25 mL were
added to each well. Fluorescence was measured after 48 h (lex =
530 nm, lem =590 nm; Spectramax M5, Molecular Devices) to de-
termine MIC90 values.
In vivo assays
All animal studies were ethically reviewed and carried out in ac-
cordance with European Directive 2010/63/EU and the GSK Policy
on the Care, Welfare, and Treatment of Animals.
In vivo efficacy assay
General antimicrobial activity assay: Whole-cell antimicrobial activity
was determined by broth microdilution using the Clinical and Lab-
oratory Standards Institute (CLSI) recommended procedure, Docu-
ment M7A7, “Methods for Dilution Susceptibility Tests for Bacteria
that Grow Aerobically”. Some compounds were evaluated against
a panel of Gram-positive and Gram-negative organisms, including
Streptococcus aureus, Enterococcus faecalis, Haemophilus influenzae,
Moraxella catarrhalis, Streptococcus pneumonia, and Escherichia coli.
The MIC90 value was determined as the lowest compound concen-
tration required to produce a >80% decrease in observed fluores-
cence.
Mice. C57BL/6 specific-pathogen-free (spf) six- to eight-week-old
female mice were obtained from Harlan Iberica, (St. Feliu de Co-
dines, Spain). They were shipped under suitable travel conditions,
with the corresponding certificate of health and origin. All animals
were kept under controlled conditions in a P3 high-security facility
with sterile food and water ad libitum.
Bacteria and infection. The virulent M. tuberculosis strain H37Rv Pas-
teur was grown to mid-log phase in Proskauer–Beck medium and
stored at À708C in 2 mL aliquots. Mice were placed in the expo-
sure chamber of an airborne infection apparatus (Glas-col Inc.,
Terre Haute, IN, USA). The nebulizer compartment was filled with
7.5 mL of an M. tuberculosis suspension at a previously calculated
concentration to provide an approximate uptake of 20 viable bacil-
li within the lungs.
InhA overexpression studies. Overexpression of the target has been
shown to be a robust way to confirm the mode of action of anti-
microbial agents. To establish a link between enzymatic and
whole-cell activities, we cloned Mycobacterium smegmatis and My-
cobacterium tuberculosis inhA genes under control of the acetami-
dase promoter. M. smegmatis transformants overexpressed InhA
protein in the presence of acetamide, whereas Mycobacterium
bovis BCG constitutively overexpress the target. In both species we
observed a clear correlation between InhA expression and isoniazid
sensitivity. Therefore, we have a whole-cell mode-of-action tool to
determine if the predominant antitubercular effect of a new drug
in Mycobacterium is via inhibition of InhA.
Antibiotic formulations. Isoniazide was provided by Sigma (Madrid,
Spain) and diluted in distilled water before administration. SB-
713520 was formulated as a suspension in 1% methyl cellulose.
The products were administered for two weeks (Monday–Friday)
starting on day 11 after infection. The schedule was once daily for
isoniazid and twice daily for SB-713520.
CFU determination. The number of viable bacteria in lung and
spleen homogenates was measured at day 25 after infection by
plating serial dilutions on nutrient Middlebrook 7H11 agar (Bio-
medics s.L., Madrid, Spain) and counting bacterial colony formation
after incubation at 378C. Special care was taken to exclude hiliar
lymph nodes at the time of lung removal in order to not artificially
increase the CFU value. Lungs and spleen were immediately ex-
tracted after euthanasia.
Microsomal fraction stability experiments. Pooled mouse liver micro-
somes were purchased from Xenotech (www.xenotech.com/home).
Microsomes (final protein concentration: 0.5 mgmLÀ1), MgCl2
(5 mm final), and test compound (final substrate concentration:
0.5 mm; final DMSO concentration: 0.5%) in 0.1m phosphate buffer
pH 7.4 were pre-incubated at 378C prior to the addition of NADPH
(final concentration: 1 mm) to initiate the reaction. The final incu-
bation volume was 600 mL. All incubations were performed singu-
larly for each test compound. Each compound was incubated for
30 min, and samples (90 mL) were taken at 0, 3, 6, 9, 12, 15, 18, 21,
24, 27, and 30 min. Reactions were stopped by the addition of
sample to 200 mL CH3CN/MeOH (3:1) containing an internal stan-
dard. The terminated samples were centrifuged at 3700 rpm for
15 min at 48C to precipitate the protein. Quantitative analysis: fol-
lowing protein precipitation, the samples were analyzed using spe-
cific LC–MS/MS conditions. Data analysis: from a plot of ln[peak
area ratio], i.e., (compound peak area)/(internal standard peak
area) against time, the gradient of the line was determined. Subse-
quently, half-life (t1/2) and intrinsic clearance (CLint) were calculated
using Equations (1)–(3) below:
Animal health. Animals were supervised every day under a protocol
paying attention to weight loss, apparent good health (bristled
hair and wounded skin) and behavior (signs of aggression or isola-
tion). Animals were euthanized with halothane (Fluothane, Zeneca
Farma) overdose so as to avoid suffering. Sentinel animals were
used to determine spf conditions in the facility. Tests for 25 known
pathogens were all negative. All experimental proceedings were
approved and supervised by the Animal Care Committee of “Ger-
mans Trias i Pujol” University Hospital in agreement with the Euro-
pean Union Laws for protection of experimental animals.
Pharmacokinetics studies
C57BL/6 female mice of 18–20 g weight were used. Experimental
compounds were administered by oral gavage at 500 mgkgÀ1 per
single dose at a volume of 20 mLkgÀ1 (n=4 mice per time point).
All mice received treatment in the fed state. Compound was ad-
ministered as a 1% methyl cellulose suspension. Peripheral total
blood was the compartment chosen for the establishment of com-
pound concentrations: aliquots of 25 mL of blood were taken by
cardiac puncture for each mouse (euthanized by CO2) at 15, 30, 40,
50 min, and 1, 1.5, 3, 6, and 8 h (n=4 mice per time point). LC–MS
was used as the analytical method for establishment of compound
concentration in blood with a sensitivity of 1–5 ngmLÀ1 (LLQ) in
25 mL blood. The non-compartmental data analysis (NCA) was per-
formed with WinNonlin (ver. 5.2; Pharsight, Certara L.P.), and sup-
Elimination rate constant ðkÞ ¼ ðÀgradientÞ
t1=2 ½minŠ ¼ 0:693=k
ð1Þ
ð2Þ
ð3Þ
CLint ½mL minÀ1 fg proteingÀ1Š ¼ V  0:693=t1=2
in which V=incubation volume (mL{g microsomal protein}À1).
Human biological samples were sourced ethically, and their re-
search use was in accord with the terms of the informed consent
given by donors.
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X-ray crystallography
InhA was cloned, expressed, and purified as described previously.[31]
The protein was incubated with 2 mm NAD+ and 2 mm compound
1 prior to crystallization. The complex crystallized in 14–24%
PEG 3350 and 0.1m N-(2-acetamido)iminodiacetic acid, pH 6.8, 6%
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Acknowledgements
The authors thank Daniel lvarez for his help with the InhA bio-
chemical assay; Ana Luisa Rodriguez, Katie Hudd, Alastair Rob-
erts, Maria Cruz García Palancar at GSK-DDW Tres Cantos, and
Matthew Volfendale for their organic chemistry contributions; the
Pharmacology Group at GSK Tres Cantos, Sophie Huss, and Oscar
Atienza for in vitro DMPK studies; as well as Juan Miguel Siles at
the Core Chemistry Group. We also thank the Global Alliance for
TB Drug Development for providing funding for this investiga-
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Keywords: antimycobacterials · benzamides · drug discovery ·
InhA · medicinal chemistry · tuberculosis
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