Structure-activity relationships of antitubercular salicylanilides consistent with disruption of the proton gradient via proton shuttling

Article abstract of DOI:10.1016/j.bmc.2012.10.056

A series of salicylanilides was synthesized based on a high-throughput screening hit against Mycobacterium tuberculosis. A free phenolic hydroxyl on the salicylic acid moeity is required for activity, and the structure-activity relationship of the aniline ring is largely driven by the presence of electron withdrawing groups. We synthesized 94 analogs exploring substitutions of both rings and the linker region in this series and we have identified multiple compounds with low micromolar potency. Unfortunately, cytotoxicity in a murine macrophage cell line trends with antimicrobial activity, suggesting a similar mechanism of action. We propose that salicylanilides function as proton shuttles that kill cells by destroying the cellular proton gradient, limiting their utility as potential therapeutics.

Full text of DOI:10.1016/j.bmc.2012.10.056

Bioorganic & Medicinal Chemistry 21 (2013) 114–126
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationships of antitubercular salicylanilides consistent
with disruption of the proton gradient via proton shuttling
Ill-Young Lee a, , Todd D. Gruber b, , Amanda Samuels b, Minhan Yun a, Bora Nam a, Minseo Kang a,
Kathryn Crowley b, Benjamin Winterroth b, Helena I. Boshoff b, Clifton E. Barry III b,
⇑
a Pharmacology Research Center, Korea Research Institute of Chemical Technology, Jang-Dong100, Yuseong-Gu, Daejeon 305-600, Republic of Korea
b Tuberculosis Research Section, LCID, NIAID, NIH, 33 North Drive, Bldg. 33, Rm 2W20D, Bethesda, MD 20892, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of salicylanilides was synthesized based on a high-throughput screening hit against Mycobacte-
rium tuberculosis. A free phenolic hydroxyl on the salicylic acid moeity is required for activity, and the
structure–activity relationship of the aniline ring is largely driven by the presence of electron withdraw-
ing groups. We synthesized 94 analogs exploring substitutions of both rings and the linker region in this
series and we have identified multiple compounds with low micromolar potency. Unfortunately, cytotox-
icity in a murine macrophage cell line trends with antimicrobial activity, suggesting a similar mechanism
of action. We propose that salicylanilides function as proton shuttles that kill cells by destroying the cel-
lular proton gradient, limiting their utility as potential therapeutics.
Received 27 August 2012
Revised 26 October 2012
Accepted 31 October 2012
Available online 15 November 2012
Keywords:
Tuberculosis
Salicylanilide
Proton gradient
Antibacterial
Published by Elsevier Ltd.
1. Introduction
in vitro,8 but as yet it is unknown if this improvement carries into
animal models or humans.
Mycobacterium tuberculosis (Mtb) infects an estimated one-third
of the world’s population, causing approximately 1.7 million
deaths in 2010.1 This, coupled with the rise of multidrug resistant
and extensively drug resistant strains, necessitates the search for
novel antimycobacterial agents. We conducted a high-throughput
screen (HTS) of a subset of compounds from the National Institute
of Health’s Chemical Genomics Center (NCGC) library. In this screen
Salicylanilides have been suggested to target multiple specific
targets in both prokaryotes and eukaryotes, including two-
component systems,3 transglycosylase,9 type III secretion
systems,10 interleukin 12p40,11 and protein tyrosine kinase.12 Prior
to these studies, salicylanilides were known as potent uncouplers
of the proton gradient used to generate ATP in mitochondria;13
the precise mode of action in mycobacteria is unclear.
we
hydroxybenzamide (IMD-0354, 1) as a lead compound. IMD-0354
(1) is a potent inhibitor of I B kinase b, and is currently in Phase
identified
N-(3,5-bis(trifluoromethyl)phenyl)-5-chloro-2-
2. Results and discussion
j
I/II clinical trials for both atopic dermatitis and chronic obstructive
pulmonary disease.2 The fact that this compound is already in hu-
man trials suggested promise for repurposing the drug against
2.1. SAR of the 5-chlorosalicylate (A) ring
The A ring of the salicylanilide lead (1) was modified in an at-
tempt to improve potency and to decouple potency and cytotoxic-
ity (Table 1). An unsubstituted A ring (4) completely lacked activity
while removing only the 5-chloro substituent (2) resulted in a
small (2–4 fold) drop in potency. Removal of only the 2-hydroxy
substituent (3), however, completely abolished activity. Substitu-
tion of the 5-position with other halogens (F, 7; Br, 8; or I, 9) re-
sulted in only small (twofold) changes in potency, which we
initially interpreted to mean that the only requirement for this po-
sition was the presence of a weak electron-withdrawing group.
Unexpectedly though, the 5-methoxy ether 10 retained activity,
although less than the parent compound. The 5-nitrosalicylanilide
13 is likewise inactive suggesting that simply bearing a strong elec-
tron-withdrawing substituent is not sufficient for full activity.
Mtb, which lacks IjB kinase b. In this study, we sought to improve
on the initial potency of salicylanilide IMD-0354 against Mtb.
Salicylanilides have been shown to be active against many
pathogens, including Staphylococcus aureus,3 Escherichia coli,4 Pseu-
domonas aeruginosa,4 Aspergillus fumigatus,5 as well as being used
as anthelminthics in veterinary practice.6 Salicylanilides have
shown activity against M. tuberculosis7 although cytotoxicity in
mammalian cells is a common problem. Masking the salicylate hy-
droxyl moiety with a carbamate linkage decreases cytotoxicity
⇑
Corresponding author. Tel.: +1 301 435 7509; fax: +1 301 480 5705.
E-mail address: cbarry@niaid.nih.gov (C.E. Barry).
These authors contributed equally to this work.
0968-0896/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2012.10.056

I.-Y. Lee et al. / Bioorg. Med. Chem. 21 (2013) 114–126
115
Table 1
Table 2
SAR of salicylic acid ring (A ring)
Effect of linkers on activity
CF3
CF3
O
OH
Cl
2
Linker
3
N
H
CF3
CF3
A
5
Compd
2
3
5
MIC (
lM)
IC50 (lM)
Compd
Linker
MIC (
l
M)
IC50
7.8
(lM)
1
2
3
4
5
6
7
8
OH
OH
H
H
H
Cl
H
H
H
H
H
H
H
Cl
OH
H
H
H
H
H
H
Cl
H
H
H
3.1
6.3–13
>50
>50
3.1
5.2
12
>60
n.d.
3.1
19
50–>50
50–>50
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OAc
OMe
OBn
NH2
Cl
1-Pyrrole
20
21
22
13
CH3
F
Br
I
OMe
Cl
H
NO2
Cl
Cl
Cl
Cl
6.3–13
3.1
6.3
6.3
6.3–13
13
5.4
2.5
4.9
5.6
7.7
>60
1.7
>60
>60
>60
>60
>60
>1.7
9
>50
>50
>60
12
10
11
12
13
14
15
16
17
18
>50
50–>50
1.6–3.1
>50
>50
>50
23
24
1.6
n.d.
23
H
>50
n.d.: not determined.
MIC values determined using Mtb H37Rv. IC50 values determined using the murine
macrophage cell line J774.1.
50–>50
6.3–13
25
27
In an attempt to improve the solubility of this molecule we also
explored substitution with a 1-pyrrole moiety resulting in a more
soluble, equipotent, but still cytotoxic molecule (5).
n.d.: not determined.
Acetylation at the 2-position was tolerated (14) and cytotoxicity
was diminished, most likely because deacetylation occurs in the
mycobacterial assay but not the cytotoxicity assay. Methylation
or benzylation of the phenol (15 and 16, respectively) resulted in
loss of activity, as did substitution with either chlorine (18) or
preparation of the corresponding aniline (17). The 3,5-dichloro-
phenol (11) retained some activity while the 2,3-dihydroxy deriv-
ative (12) lost all activity (although notably increased its
cytotoxicity presumably due to facile formation of the o-quinone).
the bis(trifluoromethyl) substitution patterns that were syntheti-
cally accessible, potency was found to follow: 3,4-(30) > 3,5-
(1) = 2,5-(32) > 2,3- (31) with compound 30 being the most potent
compound identified in this series showing a 0.8–1.6 lM MIC.
Amongst mono-substitutions on the B ring, we explored only
one further substitution in the ortho position, the phenyl sulfoxide
33, which completely lacked activity. The meta position was some-
what more tolerant with simple halogens (F, 34; Br, 35) retaining
some activity (albeit 2–4 fold less than the parent 3,5-bis(trifluoro-
methyl) 1) while the corresponding nitrile (36), acid (37), ethyl es-
ter (38), unsubstituted (39) and monosubstituted (40) amides all
lost activity. The para monosubstituted anilines also showed some
interesting trends with p-CF3 > p-OCF3 > p-F (29, 42, and 41,
respectively). Para-cyano (43), phenoxy (44) and t-butyl (45) all
showed weak activity while methoxy (46), morpholino (42), car-
boxamido (48), phenylcarboxamido (49), and phenylsulfonamido
(50) all lacked activity.
Amongst the 2,3-disubstituted anilines that were synthesized
there was a slight preference against CF3 in the 2-position (com-
pare 31 with either methyl (52) or fluoro (53) substituents).
Replacing both the 2 and 3 positions with fluorine (54) resulted
in a further loss of activity. The 2,4-disubstitution pattern yielded
some highly potent compounds, including 2-chloro-4-trifluoro-
2.2. SAR of the linker region
The SAR in the linker region showed very little tolerance for
substitution (Table 2). The corresponding amine (19) was inactive
as was a derivative where the amide was replaced with a urea (20).
Simply reversing the amide linkage (22) resulted in a complete loss
of activity, as did replacing the amide with the corresponding ester
(21). Replacing the amide with a thioamide (23) resulted in slightly
improved activity. A sulfonamide linkage (24) resulted in a com-
plete loss of activity. Substituting the aniline with a benzylamine
(25) also resulted in a loss of activity although not as dramatic as
the other substitutions.
2.3. SAR of the 3,5-bis(trifluoromethyl)aniline (B) ring
methyl (57) which showed an MIC of 0.8–1.6 lM. Again there ap-
The potency on the B ring seemed to be largely driven by the
presence of electron withdrawing groups (Table 3). Removal of
one of the trifluoromethyl groups from the lead compound 1 had
only a slight effect on activity (27), whereas removing both de-
creased potency 4–8 fold (26). Changing the position of a single tri-
fluoromethyl from meta- to ortho resulted in a 4–8 fold loss in
activity (28), possibly attributable to conformational change at
the amide. Installing a single trifluoromethyl group in the para po-
sition resulted in a slight enhancement in potency (29). Amongst
peared to be a preference against CF3 in the ortho position with two
4-halo analogs carrying 2-CF3 groups showing poor activity (4-F,
55; and 4-Br, 56). Notably, the 2,4-difluoro analog (58) was the
weakest in the 2,4-disubstituted series. The 2,5-disubstitution pat-
tern also yielded some highly potent compounds, with 2-Br, 5-CF3
(61) showing submicromolar MIC. Interestingly, among the series
of three 2-halo-5-CF3 substituted salicylanilides potency was in-
versely related to their electronegativity with Br (61) > Cl (59) > F
(60). The preference at the 5-position for CF3 was not solely due

116
I.-Y. Lee et al. / Bioorg. Med. Chem. 21 (2013) 114–126
Table 3
SAR of aniline ring (B ring)
3
2
4
5
OH
Cl
O
B
N
H
6
Compd
2
3
4
5
6
MIC (
3.1
l
M)
IC50 (lM)
1
H
H
H
CF3
H
CF3
H
CF3
H
H
CF3
CF3
H
H
F
Br
CN
CO2H
CO2Et
CONH2
CONHPh
H
H
H
H
CF3
CF3
H
H
H
H
H
H
H
H
H
H
F
CF3
H
H
H
H
H
H
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
5.2
22
5.9
19
2.8
4.9
8.1
6.6
>60
15
5.7
10
>60
17
>60
>60
17
7.3
11
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
13–25
3.1–6.3
13–25
1.6–3.1
0.8–1.6
6.3–13
3.1
H
CF3
CF3
SO2Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
>50
6.3–13
6.3–13
25–50
>50
13
>50
>50
H
H
H
H
H
H
13–25
3.1–6.3
13–25
13
13–25
>50
OCF3
CN
OPh
t-Bu
OMe
43
19
>60
47
H
H
H
H
>50
n.d.
48
49
50
H
H
H
H
H
H
CONH2
CONHPh
SO2NHPh
H
H
H
H
H
H
>50
>50
>50
>60
>60
>60
51
H
H
H
H
>50
>60
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
Me
F
F
CF3
CF3
Cl
F
Cl
F
Br
CF3
Cl
F
CF3
F
H
H
H
H
H
H
H
H
CF3
CF3
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
Br
CF3
F
H
H
H
H
H
H
H
H
F
Cl
Br
CN
F
H
H
H
H
H
H
H
CF3
CF3
CF3
OMe
F
NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3.1–6.3
3.1–6.3
13
6.3–13
6.3–13
0.8–1.6
13
8.0
5.3
13
13
11
2.4
8.5
3.5
2.6
2.9
4.8
3.2
8.5
23
>60
5.3
4.5
4.5
5.8
4.4
7.7
6.3
5.8
18
3.1
6.1
4.1
21
<1.9
3.2
4.3
4.7
5.4
6.2
6.7
1.6
1.6–3.1
0.8–1.6
6.3–13
3.1–6.3
6.3
>50
>50
1.6–3.1
1.6–3.1
3.1
6.3
3.1
3.1–6.3
6.3
1.6
6.3–13
1.6
3.1–6.3
6.3–13
50–>50
0.8
3.1
1.6–3.1
1.6
CF3
CF3
CF3
CF3
Cl
F
H
H
H
CF3
OMe
Cl
F
I
Me
H
H
H
H
H
H
Me
H
Br
F
Br
CF3
Cl
F
t-Bu
Me
CF3
CF3
F
H
H
H
H
H
H
Me
H
H
F
t-Bu
Me
CF3
CF3
F
F
Cl
F
H
F
Cl
H
H
F
F
Cl
F
F
1.6
3.1–6.3
6.3
F

I.-Y. Lee et al. / Bioorg. Med. Chem. 21 (2013) 114–126
117
Table 3 (continued)
Compd
2
3
4
5
6
MIC (
l
M)
IC50 (lM)
87
88
Cl
Cl
H
H
Cl
Cl
OMe
OH
H
H
13–25
>50
8.0
32
n.d.: not determined.
Table 4
to the electron withdrawing character of this moiety since the 5-
nitro (64) derivative showed about the same weak activity as the
methoxy derivative (62). In the 2,6-disubstitution pattern only
two molecules were synthesized, 2-CF3–6-Cl (65) and 2,6-difluoro
(66) both of which were completely inactive.
5-F-Salicylic acid: aniline modifications
3
2
4
OH
O
B
5
N
H
We synthesized four 3-CF3, 4-X disubstituted compounds which
showed an activity trend of 3-CF3, 4-F (67) = 3-CF3, 4-Cl (68) >
3-CF3, 4-Br (69) > 3-CF3, 4-CN (70) although the differences in
activity were only twofold. Substitution of both groups with either
di-fluoro (72) or 3-chloro-4-fluoro (71) resulted in a slight loss of
activity as did replacement with iodine at the 3-position with sub-
stitution of a methyl group at the 4-position (73). Amongst the 3,5-
disubstituted series various combinations of electron withdrawing
groups (3-Br, 5-CF3 (74); 3,5-dichloro (76); 3,5-difluoro (77)) were
F
Compd
2
3
4
5
MIC (
l
M)
IC50 (lM)
89
90
91
92
93
94
H
Cl
H
H
F
CF3
H
F
H
H
H
H
H
CF3
H
Br
CF3
F
H
CF3
H
3.1–6.3
3.1–6.3
6.3
3.1–6.3
6.3
4.7
6.1
9.6
5.4
5.1
6.1
H
CF3
H
6.3–13
active in the range of 1.6–6.3 lM. Combining a 3-trifluoromethyl
with a 5-methoxy group (75) resulted in a loss of activity (compare
75 to 1) while replacing both trifluoromethyls with either 3,5-
dimethyl (79) or 3,5-di-t-butyl (78) either decreased (78) or
eliminated (79) activity altogether.
We also prepared a number of trisubstituted B ring analogs re-
lated to both the 3,5-disubstituted series and the 2,4-disubstituted
series. 3,4,5-Trifluoro (83), 3,4,5-trichloro (84) and 3,5-difluoro-4-
bromo (82) were all approximately the same potency as the corre-
sponding 3,5-dihalo analogs. Introducing a methyl group in the
ortho position of the 3,5-bis-CF3 parent (81) had no effect on po-
tency, while introducing a methyl group in the para position (80)
This result suggests a complex mode of action without a specific
receptor target. The inability to generate salicylanilides specific
for Mtb over J774.1 cells also suggested that there may be a com-
plex mechanism of action. A possible mechanism consistent with
the absolute requirement for a free phenolic hydroxyl is that the
salicylanilides are decoupling the proton gradient in both Mtb
and the mitochondria of J774.1 cells by acting as a proton shuttle.
Indeed, salicylanilides have previously been suggested to destroy
the proton gradient in mitochondria.13 The determinants for
strongly depolarizing salicylanilides parallel the SAR observed with
salicylanilides against Mtb: electron withdrawing groups on the B
ring and somewhat lipophilic groups on the A ring,14 although the
trend of lipophilicity has been previously shown to be less relevant
to the SAR of Mtb inhibitors.15 The salicyl phenol proton has a pKa
near physiological range. In the slightly basic interior of the Mtb
cell, the phenol could become deprotonated (Fig. 2). Stabilization
of the phenolate by a hydrogen bond to the amide NH would allow
the compound to cross the membrane to the extracellular space,
assisted by the positive electrochemical gradient. There, in the
lower pH of the media, the phenolate would become protonated
and the protonated form could transit the membrane and deposit
the acquired proton within the cell, thereby disrupting the proton
gradient.
improved potency slightly, to less than 1 lM. The 2,4,5-trifluoro
aniline derivative (85) had potency similar to the 3,4-difluoro com-
pound 72, improved compared to the 2,4-difluoro analog 58. The
2,3,4-trifluoro analog (86), in contrast, was slightly improved
compared to either the 2,3-difluoro 54 or the 2,4-difluoro 58. In
the 2,4-dichloro aniline bearing series addition of a 5-hydroxy
(88) resulted in complete loss of activity which was only slightly
rescued by methylation (87).
We synthesized six additional compounds combining a 5-F-
salicylate ring with electron withdrawing character on the aniline
ring (89–94 as shown in Table 4). While these compounds main-
tain fairly strong potency, none offers an improvement over the
parent compound in cytotoxicity.
2.4. Summary of cytotoxicity data
As mentioned above, compounds were tested for cytotoxicity in
a murine macrophage cell line (J774.1) as a prelude to treatment
studies in this model (Tables 1–4). These compounds displayed
high cytotoxicity, with IC50 values often in the low micromolar
range. Overall, the data showed that the potency in Mtb (MIC)
was modestly positively correlated to the cytotoxicity (IC50) in
J774.1 cells (Fig. 1). This suggests that there may be a common
mechanism of toxicity in both a eukaryotic cell line and Mtb. A
notable exception is compound 14, in which an acetyl group pro-
tects the salicylate 2-OH. This suggests that the bacteria cleave this
acetyl group while J774.1 cells cannot.
2.5. Possible mechanism
Despite repeated attempts to generate spontaneous mutants of
Mtb by selecting on media containing these compounds we were
never able to identify colonies with significant levels of resistance.
Figure 1. Correlation between Mtb MIC and IC50 in J774.1 macrophage cell line.
Compounds with off-scale MIC (>50 lM) or IC50 (>60 lM) values were not included.

118
I.-Y. Lee et al. / Bioorg. Med. Chem. 21 (2013) 114–126
The electron poor B ring of the salicylanilides appears to stabi-
4. Materials and methods
lize this process by modulating the pKa of the amide proton. This
explanation is further supported by the disruption of membrane
potential and pH homeostasis recently observed in Mtb using the
salicylanilide niclosamide.17 Support for the existence of these
two conformers of salicylanilides has been previously published
from NOESY NMR, IR and fluorescence spectral changes of these
compounds as a function of acid concentration.18
Disruption of the proton gradient may be a common mecha-
nism of salicylanilide action against diverse cells even if the salicy-
lanilides also targeted specific enzymes. Extensive SAR
experiments on both the enzymatic target, in vitro, and whole-cell
organism would be required to establish unique modes of action
(as was elegantly shown in Garner et al.19). In the case of Mtb,
the shared SAR between mammalian cytotoxicity values and Mtb
MIC values that parallels known SAR for strong proton gradient
decouplers argues for disruption of the proton gradient. Additional
experiments to confirm this mechanism by measuring the proton
motive force and examining transcriptional responses are
underway.
Parent salicylanilide 1 was identified from high-throughput
screening of the NIH Chemical Genomics Center (NCGC) library
using Mtb (H37Rv) expressing green fluorescent protein (GFP,
pMSP12 plasmid), using GFP fluorescence as readout.
All chemicals used were of analytical grade and commercially
available. All solvents were dried and freshly distilled prior to
use according to standard procedures. Solvents are reported as a
volume ratio. 1H NMR spectra were recorded on a Bruker spec-
trometer (300 MHz) in DMSO-d6 or CDCl3 as specified below. Melt-
ing points were performed on a Mettler Toledo MP50 melting point
apparatus. IR spectra were recorded on a Smiths Detection Identi-
fyIR FT-IR spectrometer (ATR). HRMS was obtained by APCI accu-
rate mass on
a Waters LCT Premier time-of-flight mass
spectrometer (negative mode).
4.1. General procedures
4.1.1. Procedure A
The salicylic acid (1.2 equiv) was added to a mixture of toluene
(0.3 M), aniline (1.0 equiv), phosphorus trichloride (1.1 equiv), and
pyridine (0.05 equiv) in a Radley’s Carousel reaction tube (modified
from Itai et al.20). The mixture was refluxed under nitrogen for 12 h
then cooled to room temperature. Aqueous sodium bicarbonate
was added dropwise to attain pH 6–7. The resultant mixture was
extracted with EtOAc. The organic extracts were combined, dried
(MgSO4), and concentrated under vacuum. After chromatography
(1:10 EtOAc:Hex) compounds were recrystallized (EtOAc/Hex).
3. Conclusion
We have synthesized a series of salicylanilides based on a
bis(trifluoromethyl) salicylanilide hit from
a high-throughput
screen. The electron withdrawing character of substituents on
the B ring appears to be the major driver of activity, with many
compact electron-withdrawing groups tolerated. Despite synthe-
sizing a rather large number of molecules we have been unable
to separate MIC activity from cytotoxicity in a macrophage cell
line, suggesting a common mode of action. We propose that this
mechanism involves the decoupling of the proton motive force
by proton shuttling mediated by the salicylanilides, consistent
with the observed SAR. This general mechanism likely explains
the cytotoxicity of various salicylanilides against other microbes
and suggests that further development may be challenging.
4.1.2. Procedure B
5-Chlorosalicylic acid (20 g, 116 mmol, 1.0 equiv), pyridine
(25 mL, 310 mmol, 3.0 equiv), and benzoyl chloride (25 mL,
311 mmol, 3.0 equiv) in Et2O (0.5 M) were combined and stirred
at room temperature (5 h). 1 N HCl was added dropwise until the
reaction reached pH 2–3, followed by extraction with EtOAc. The
Extracellular space
~pH 6.6
Net positive
[H+]
Intracellular space
~pH 7.7
Net negative
CF3
CF3
H+
O
H
O
O
Cl
N
H
CF3
N
H
CF3
O-
Cl
Stabilization of
negative charge
CF3
CF3
H
O
O
O
Cl
N
H
CF3
N
H
CF3
O-
H+
Cl
Figure 2. Proton shuttling mechanism for salicylanilides in killing Mtb. Through repeated cycles, the salicylanilide disrupts the proton gradient. pH values from Vandal et al.16

I.-Y. Lee et al. / Bioorg. Med. Chem. 21 (2013) 114–126
119
organic extracts were combined, dried (MgSO4), and concentrated
under vacuum. Chromatography (1:5 EtOAc:Hex) yielded 2-(ben-
zoyloxy)-5-chlorobenzoic acid (18 g, 53%). 1H NMR (300 MHz,
DMSO-d6): d 7.44 (d, J = 8.6 Hz, 1H), 7.58–7.63 (m, 2H), 7.72–7.79
(m, 2H), 7.94 (d, J = 8.6 Hz, 1H), 8.11 (m, 2H).
4.1.3.5.
N-(3,5-Bis(trifluoromethyl)phenyl)-2-hydroxy-5-(1H-
Prepared by general procedure
pyrrol-1-yl)benzamide (5)20
.
A. Brown solid. Mp >230 °C dec. IR (neat): 3231, 1637, 1552,
1520, 1379, 1274, 1128, 742 cmÀ1. Characterization (NMR, MS)
was in accordance with previously reported values.
Oxalyl chloride (3.2 mL, 36 mmol, 2.0 equiv) at 0 °C, 2-(benzoyl-
oxy)-5-chlorobenzoic acid (5 g, 18 mmol, 1 equiv), and DMF (2
drops) were combined in dichloromethane (0.3 M). The mixture
warmed to room temperature with stirring (4 h). The solvent was
removed under reduced pressure, yielding 4-chloro-2-(chlorocar-
bonyl)phenyl benzoate as a thick yellow oil (17 g), which was used
without further purification. 1H NMR (300 MHz, CDCl3): d 7.21 (d,
J = 8.6 Hz, 1H), 7.46–7.51 (m, 2H), 7.59–7.64 (m, 2H), 8.07–8.09 (m,
1H), 8.16 (d, J = 8.1 Hz, 2H).
4.1.3.6. N-(3,5-Bis(trifluoromethyl)phenyl)-2-hydroxy-5-meth-
ylbenzamide (6)20
.
Prepared by general procedure A. White
solid. Mp 145–147 °C. Yield 22%. 1H NMR (300 MHz, DMSO-d6): d
2.28 (s, 3H), 6.90 (d, J = 5.0 Hz, 1H), 7.27 (dd, J1 = 8.3 Hz,
J2 = 2.1 Hz, 1H), 7.69 (d, J = 1.7 Hz, 1H), 7.83 (s, 1H), 8.46 (s, 2H),
10.81 (s, 1H), 10.86 (s, br, 1H). IR (neat): 3290, 1655, 1571, 1383,
1274, 1114, 881 cmÀ1. HRMS (APCI), m/z calcd for C16H11F6NO2–
H: 362.0616, found 362.0621.
To a solution of 4-chloro-2-(chlorocarbonyl)phenyl benzoate
(1 mmol, 1.4 equiv) in DCM (0.2 M), amine (0.7 mmol, 1 equiv)
and triethylamine (0.6 mL, 1.2 mmol, 1.7 equiv) were added at
0 °C. The mixture was stirred at room temperature (16 h) and ex-
tracted with DCM. The organic layer was washed with water and
brine, dried (MgSO4), and the solvent was removed under vacuum.
The residue was purified by flash column chromatography.
Potassium carbonate (18 mg, 0.13 mmol, 1.2 equiv) was added
to a solution of crude product from the previous step (0.11 mmol,
1.0 equiv) in 1:1 MeOH:1,4-dioxane (0.1 M). The reaction mixture
was stirred at room temperature (2 h) and acidified with 1 N HCl,
followed by extraction with EtOAc and DCM. The organic layer
was washed with water and brine, dried (MgSO4), and the solvent
was removed under vacuum. The residue was purified by column
chromatography (1:19 MeOH:CHCl3), and recrystallized from
Et2O/Hex.
4.1.3.7. N-(3,5-Bis(trifluoromethyl)phenyl)-5-fluoro-2-hydroxy-
benz-amide (7)20
.
Prepared by general procedure A. White
solid. Mp 187–178 °C. Characterization (NMR, MS) was in accor-
dance with previously reported values.
4.1.3.8. N-(3,5-Bis(trifluoromethyl)phenyl)-5-bromo-2-hydroxy-
benz-amide (8)20
.
Prepared by general procedure A. Slightly
brown solid. Mp 194–195 °C. Characterization (NMR, MS) was in
accordance with previously reported values.
4.1.3.9. N-(3,5-Bis(trifluoromethyl)phenyl)-2-hydroxy-5-iodo-
benz-amide (9)20
.
Prepared by general procedure A. White
solid. Mp 216–219 °C. IR (neat): 3318, 3194, 1639, 1552, 1381,
1172, 1128, 881, 810 cmÀ1. Characterization (NMR, MS) was in
accordance with previously reported values.
4.1.3.10. N-(3,5-Bis(trifluoromethyl)phenyl)-2-hydroxy-5-meth-
4.1.3. Compound purity
oxy-benz-amide (10)20
.
Prepared by general procedure A.
Compound purity was assessed by LC/MS comparison of the to-
tal ion current with the peak representing the desired product on
reversed-phase chromatography as well as by the presence of
extraneous peaks in the 1H NMR. All compounds submitted for bio-
logical testing were >90% pure.
White solid. Mp 208–210 °C. Characterization (NMR, MS) was in
accordance with previously reported values.
4.1.3.11. N-(3,5-Bis(trifluoromethyl)phenyl)3,5-dichloro-2-hydroxy-
benz-amide (11)24
.
Prepared by general procedure A. White solid.
Mp 141–143 °C. Characterization (NMR, MS) was in accordance with
4.1.3.1. N-(3,5-Bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxy-
previously reported values.
benzamide (1)20
.
Prepared by general procedure A. White so-
lid (320 mg, 28%). Mp 172–173 °C. IR (neat): 3082, 1639, 1580,
1379, 1276, 1137, 888 cmÀ1. Characterization (NMR, MS) was in
accordance with previously reported values.
4.1.3.12.
N-(3,5-Bis-trifluoromethyl-phenyl)-2,3-dihydroxy-
Prepared by general procedure A. White
benzamide (12)24
.
solid. Mp 154–156 °C. IR (neat): 3507, 3343, 3137, 1657, 1272,
1141, 1073, 885 cmÀ1. Characterization (NMR, MS) was in accor-
dance with previously reported values.
4.1.3.2. N-(3,5-Bis(trifluoromethyl)phenyl)-2-hydroxybenzam-
ide (2)20
.
Prepared by general procedure A. White solid. Mp
181–182 °C [lit20: mp 179–180 °C]. IR (neat): 3322, 3103, 1623,
1568, 1381, 1272, 1155, 1125, 879 cmÀ1. Characterization (NMR,
MS) was in accordance with previously reported values.
4.1.3.13. N-(3,5-Bis-trifluoromethyl-phenyl)-2-hydroxy-5-nitro-
benz-amide (13)20
.
Prepared by general procedure A. Slightly
yellow solid. Mp 225–226 °C. IR (neat): 3388, 3338, 3096, 1568,
1470, 1340, 1276, 1164, 1118, 1075 cmÀ1
Characterization
.
4.1.3.3. N-(3,5-Bis(trifluoromethyl)phenyl)-3-chlorobenzamide
(NMR, MS) was in accordance with previously reported values.
(3)20
.
Prepared by general procedure A. White solid. Mp
189–190 °C. Yield 87%. 1H NMR (300 MHz, DMSO-d6): d 7.61 (dd,
J1 = 8.1 Hz, J2 = 8.0 Hz, 1H), 7.72 (d, J = 7.7 Hz, 1H), 7.84 (s, 1H),
7.95 (d, J = 7.8 Hz, 1H), 8.06 (s, 1H), 8.50 (s, 2H), 10.90 (s, 1H).
HRMS (APCI), m/z calcd for C15H8ClF6NO–H: 366.0120, found
366.0112.
4.1.3.14. Acetic acid 2-(3,5-bis-trifluoromethyl-phenylcarba-
moyl)-4-chloro-phenyl ester (14)20
.
Prepared by general
procedure A. White solid. Mp 117–118 °C. Characterization
(NMR, MS) was in accordance with previously reported values.
4.1.3.15. N-(3,5-Bis(trifluoromethyl)phenyl)-5-chloro-2-meth-
4.1.3.4.
(4).
N-(3,5-Bis(trifluoromethyl)phenyl)-benzamide
oxy-benzamide (15)27
White solid. Mp 148–150 °C. Characterization (NMR, MS) was in
accordance with previously reported values.
.
Prepared by general procedure A.
Prepared by general procedure A. White solid. Mp 186–
187 °C. Yield 39%. 1H NMR (300 MHz, DMSO-d6): d 7.59–7.66 (m,
3H), 7.83 (s, 1H), 8.01 (d, J = 6.9 Hz, 2H), 8.54 (s, 2H), 10.85 (s,
1H). IR (neat): 3205, 1653, 1564, 1381, 1274, 1118, 879 cmÀ1
.
4.1.3.16. 2-Benzyloxy-N-(3,5-bis-trifluoromethyl-phenyl)-5-chloro-
HRMS (APCI), m/z calcd for
C15H9F6NO–H: 332.0510, found
benzamide (16).
To a solution of compound 1 (192 mg,
332.0504.
0.5 mmol, 1.0 equiv) in DMF (1.5 mL, 0.3 M), benzyl bromide

120
I.-Y. Lee et al. / Bioorg. Med. Chem. 21 (2013) 114–126
(0.07 mL, 0.55 mmol, 1.1 equiv) and potassium carbonate (83 mg,
0.6 mmol, 1.2 equiv) were added. The solution was stirred at room
temperature (4 h). The mixture was concentrated under reduced pres-
sure and the residue was dissolved in EtOAc (20 mL) and then washed
with brine. The organic layer was dried (MgSO4) and concentrated un-
der vacuum. The crude product was purified by column chromatogra-
phy (1:15 EtOAc:Hex) to give product 16 (220 mg, 93%). White solid.
Mp 178–180 °C. 1H NMR (300 MHz, DMSO-d6): d 5.23 (s, 2H), 7.30–
7.34 (m, 4H), 7.47–7.48 (m, 2H), 7.60 (dd, J1 = 11.6 Hz, J2 = 2.7 Hz,
1H), 7.68(d, J = 2.7 Hz, 1H), 7.80 (s, 1H), 8.26 (s, 1H), 10.84 (s, 1H). IR
1134, 890 cmÀ1. Characterization (NMR, MS) was in accordance with
previously reported values.
4.1.3.21. 5-Chloro-2-hydroxy-benzoic acid 3,5-bis-trifluoro-
methyl-phenyl ester (21)24
.
Prepared by general procedure
A. White solid. Mp 98–100 °C. IR (neat): 3258, 1694, 1470, 1276,
1171, 1107, 908 cmÀ1.Characterization (NMR, MS) was in accor-
dance with previously reported values.
4.1.3.22.
methyl)benz-amide (22).
N-(5-Chloro-2-hydroxyphenyl)-3,5-bis(trifluoro-
2-Amino-4-chlorophenol (290 mg,
(neat): 3318, 2925, 1669, 1561, 1472, 1381, 1269, 1166, 1121 cmÀ1
.
HRMS (APCI), m/z calcd for C22H14ClF6NO2–H: 472.0539, found
472.0542.
2 mmol, 1.0 equiv) was dissolved in dichloromethane (8 mL,
0.3 M) under argon. A solution of 3,5-bis(trifluoromethyl)benzoyl
chloride (0.36 mL, 2 mmol, 1.0 equiv) and pyridine (0.9 mL,
10 mmol, 5 equiv) in DCM (4 mL, 0.5 M) was added dropwise on
ice, and the mixture was allowed to warm to room temperature
over 3 h. The resultant mixture was concentrated under vacuum
and resuspended in EtOAc. This solution was washed successively
with water and brine, dried (MgSO4), and concentrated under vac-
uum. The resultant residue was purified by column chromatogra-
phy (1:10 EtOAc:Hex) to give product 19 (300 mg, 39%). Red
solid. Mp 218–221 °C. 1H NMR (300 MHz, DMSO-d6) d 6.93 (d,
J = 8.7 Hz, 1H), 7.12 (d, J = 8.6 Hz, 1H), 7.66 (s, 1H), 8.37 (s, 1H),
8.58 (s, 2H), 10.15 (s, 2H). HRMS (APCI), m/z calcd for
4.1.3.17. 2-Amino-N-(3,5-bis(trifluoromethyl)phenyl)5-chloro-
benz-amide (17).
2-Amino-5-chlorobenzoic acid (1 g, 6 mmol,
1.0 equiv) and DMF (2 drops) were added to thionyl chloride
(4.4 mL, 60 mmol, 10 equiv) in 1,2-dichloroethane (20 mL, 0.3 M) at
0 °C. The mixture was stirred at room temperature (3 h). Thionyl chlo-
ride and 1,2-DCE were removed by distillation. The product was resus-
pended in DCM (20 mL) and cooled to 0 °C. To this solution was added
3,5-bis-(trifluoromethyl)aniline (7.6 mL, 48 mmol, 8.0 equiv) and tri-
ethylamine (8.4 mL, 60 mmol, 10 equiv), followed by warming to
room temperature and stirring for 16 h. The mixture was stirred at
room temperature (16 h) and diluted with DCM. The organic layer
was washed with water and brine, dried (MgSO4), and concentrated
under vacuum. The residue was purified by flash column chromatog-
raphy (1:10 EtOAc:Hex) to give product 17 (32 mg, 1.4%). White solid.
Mp 69–71 °C. 1H NMR (300 MHz, DMSO-d6): d 6.61 (s, 2H), 6.80 (d,
J = 9.0 Hz, 1H), 7.27 (dd, J1 = 9.0 Hz, J2 = 2.4 Hz, 1H), 7.77 (d,
J = 2.4 Hz, 1H), 7.79 (s, 1H), 8.45 (s, 2H), 10.76 (s, 1H). HRMS (APCI),
m/z calcd for C15H9ClF6N2O–H: 381.0229, found 381.0235.
C15H8ClF6NO2–H: 382.0070, found 382.0056.
4.1.3.23. N-(3,5-bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxy-
benzo-thioamide (23). Compound 1 (1 g, 2.6 mmol, 2.0 equiv)
was dissolved in pyridine (8 mL, 0.2 M). Phosphorus pentasulfide
(591 mg, 1.3 mmol, 1 equiv) was added, and the mixture was heated
to reflux (3 h). The mixture was concentrated under vacuum and
mixed with dilute HCl and toluene. The resulting mixture was re-
fluxed (3 h). The mixture was concentrated under vacuum. Water
was added to the residue, and it was extracted with EtOAc. The ex-
tract was purified by column chromatography (1:10 EtOAc:Hex) to
give product 25 (138 mg, 10%). yellow solid. Mp 68–70 °C. 1H NMR
(300 MHz, DMSO-d6): d 6.96 (d, J = 8.8 Hz, 1H), 7.34 (dd, J1 = 8.7 Hz,
J2 = 2.7 Hz, 1H), 7.57 (d, J = 2.7 Hz, 1H), 7.99 (s, 1H), 8.73 (s, 2H),
10.56 (s, 1H), 12.40 (s, 1H). HRMS (APCI), m/z calcd for
4.1.3.18. N-(3,5-Bis(trifluoromethyl)phenyl)2-chlorobenzamide
(18).
Prepared by general procedure A, except the reaction was
performed in acetonitrile (0.3 M) and pyridine was omitted. White
solid. Mp 142–143 °C. Yield 77%. 1H NMR (300 MHz, DMSO-d6): d
7.50–7.70 (m, 4H), 7.87 (s, 1H), 8.40 (s, 2H), 11.20 (s, 1H). IR (neat):
3251, 3087, 1660, 1379, 1276, 11,171, 1128, 883 cmÀ1. HRMS (APCI),
m/z calcd for C15H8ClF6NO–H: 366.0120, found 366.0120.
C15H8ClF6NOS–H: 397.9841, found 397.9836.
4.1.3.19. 2-[(3,5-Bis-trifluoromethyl-phenylamino)-methyl]-4-
4.1.3.24. N-(3,5-Bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxy-
benzenesulfonamide (24). Freshly distilled chlorosulfonic acid
chloro-phenol (19).
A mixture of 5-chlorosalicylaldehyde
(2.5 g, 16 mmol, 1.0 equiv) and 3,5-(trifluoromethyl)aniline
(2.5 mL, 16 mmol, 1.0 equiv) in EtOH (80 mL, 0.2 M) was refluxed
(16 h). The reaction mixture was cooled to room temperature
and the separated crystal was filtered to yield (E)-2-(((3,5-bis(tri-
fluoromethyl)phenyl)imino)-methyl)-4-chlorophenol (3.3 g, 56%).
This compound (1 g, 2.8 mmol, 1.0 equiv) was dissolved in EtOH
(3 mL, 0.9 M). Sodium borohydride (117 mg, 3.1 mmol, 1.1 equiv)
was added at 0 °C and the reaction mixture was allowed to warm
to room temperature (3 h). After complete reduction of the imine,
the pH was adjusted to 8 using 1 N HCl and the reaction mixture
was extracted with Et2O. The organic layer was washed with brine,
dried (MgSO4), and the product was purified by column chroma-
tography (1:4 EtOAc:Hex) to give product 22 (490 mg, 49%). White
solid. Mp 120–123 °C. 1H NMR (300 MHz, DMSO-d6): d 4.26 (d,
J = 5.7 Hz, 2H), 6.85 (d, J = 8.6 Hz, 1H), 7.05 (s, 1H), 7.09–7.12 (m,
4H), 7.21 (d, J = 2.6 Hz, 1H), 10.06 (s, 1H). IR (neat): 3292, 1383,
1281, 1125, 1061, 879, 819 cmÀ1. HRMS (APCI), m/z calcd for
(13.2 g, 0.1 mol, 1.0 equiv) was cooled to 15 °C in an ice-water bath
and 4-chloroanisole (27 mL, 0.2 mol, 2.0 equiv) was carefully added,
keeping the temperature of the mixture below 50 °C. The reaction
mixture was carefully poured into 250 mL ice water. The resultant
white solid (5-chloro-2-methoxybenzene-1-sulfonyl chloride) was
filtered and dried.
3,5-Bis(trifluoromethyl)aniline (2.3 g, 10 mmol, 1.0 equiv) and
4-dimethylaminopyridine (50 mg, 0.4 mmol, 0.04 equiv) were dis-
solved in anhydrous pyridine (20 mL, 0.5 M). 5-Chloro-2-methoxy-
benzene-1-sulfonyl chloride (2.41 g, 10 mmol, 1 equiv) was added.
The resulting mixture was stirred at room temperature (10 h).
Water (20 mL) was added to quench the reaction. The precipitate
formed was filtered and washed with water, cold THF, and Et2O
to afford a brown solid (N-(3,5-bis(trifluoromethyl)phenyl)-5-
chloro-2-methoxybenzene-sulfonamide). 1H NMR (300 MHz,
DMSO-d6): d 3.79 (s, 3H), 7.23 (d, J = 9.0 Hz, 1H), 7.66–7.68 (m,
3H), 7.75 (s, 1H), 7.81 (d, J = 2.6 Hz, 1H), 11.12(s, 1H).
C
15H10ClF6NO–H: 368.0277, found 368.0277. An alternate synthe-
Boron tribromide (5 mL, 1.7 mmol, 1.7 equiv) was added to a
solution of N-(3,5-bis(trifluoromethyl)phenyl)-5-chloro-2-meth-
oxybenzenesulfonamide (434 mg, 1 mmol, 1.0 equiv) in DCM
(5 mL, 0.2 M) at À70 °C and allowed to warm to room temperature
(12 h). The mixture was added dropwise to 10 mL ice water, with
stirring continued for an additional 0.5 h. The mixture was cooled
sis was additionally described previously.(2)
4.1.3.20. 1-(3,5-Bis(trifluoromethyl)phenyl)-3-(5-chloro-2-hydroxy-
phenyl)-urea (20)24
.
Prepared by general procedure A. Slightly
yellow brown. 192–193 °C. IR (neat): 3361, 1657, 1557, 1381, 1276,

I.-Y. Lee et al. / Bioorg. Med. Chem. 21 (2013) 114–126
121
to room temperature and concentrated sodium hydroxide was
added dropwise until the pH reached 6–7, followed by extraction
with DCM. The organic extracts were combined, dried (MgSO4),
concentrated under vacuum, and purified by chromatography
(1:2 EtOAc:Hex) (340 mg, 81%). Slightly yellow solid. Mp 135–
138 °C. 1H NMR (300 MHz, DMSO-d6): d 6.95 (d, J = 8.8 Hz, 1H),
7.49 (dd, J1 = 8.8 Hz, J2 = 2.3 Hz, 1H), 7.68–7.71 (m, 4H), 11.27 (s,
2H). IR (neat): 3354, 3238, 1413, 1374, 1276, 1125, 966,
solved in MeOH (160 mL, 0.1 M). 10 wt% Pd/C (220 mg) was added,
followed by stirring under hydrogen atmosphere (12 h). The resul-
tant mixture was filtered over Celite and concentrated under vac-
uum. The obtained residue was purified by column
chromatography (1:10 EtOAc:Hex) to isolate the anilines used in
the synthesis of compounds 30 (900 mg, 17%, 1H NMR (300 MHz,
CDCl3): d 4.13 (s, 2H), 6.80 (dd, J1 = 8.6 Hz, J2 = 1.9 Hz, 1H), 7.03
(d, J = 2.3 Hz, 1H), 7.58 (d, J = 8.6 Hz, 1H),) and 31 (450 mg, 8%, 1H
NMR (300 MHz, CDCl3): d 4.47 (s, 2H), 6.91 (d, J = 8.3 Hz, 1H),
7.17 (d, J = 7.7 Hz, 1H), 7.33 (br t, J = 8.0 Hz, 1H),).
826 cmÀ1
.
HRMS (APCI), m/z calcd for C14H8ClF6NO3S–H:
417.9766, found 417.9753.
4.1.3.25. N-(3,5-Bis-trifluoromethyl-benzyl)-5-chloro-2-hydroxy-
4.1.3.31. N-(3,4-Bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxy-
benz-amide (25).
1-Ethyl-3-(3-dimethylaminopropyl)carbodi-
benz-amide (30).
Prepared by general procedure A. White solid.
imide hydrochloride was added to a mixture of 5-chlorosalicylic acid
(690 mg, 4 mmol, 1.0 equiv), 3,5-bis(trifluoromethyl)benzylamine
(973 mg, 4 mmol, 1.0 equiv), 4-dimethylaminopyridine (50 mg,
0.4 mmol, 0.1 equiv) and DCM (12 mL, 0.3 M), and the mixture was
stirred at room temperature (3 h). The reaction mixture was poured
into dilute HCl (50 mL) and extracted with DCM (70 mL). After the or-
ganic layer was washed with water and brine, dried (MgSO4), and
concentrated under vacuum, the crude product was purified by col-
umn chromatography (1:4 EtOAc:Hex) to give product 23 (245 mg,
55%). White solid. Mp 125–127 °C. 1H NMR (300 MHz, DMSO-d6): d
4.68 (d, J = 5.8 Hz, 2H), 6.97 (d, J = 8.8 Hz, 1H), 7.44 (dd, J1 = 8.8,
J2 = 2.6 Hz, 1H), 7.89 (d, J = 2.7 Hz, 1H), 8.01 (s, 1H), 8.05(s, 2H),
9.41(dd, J1 = 5.9 Hz, J2 = 5.9 Hz, 1H), 12.09 (s, 1H). HRMS (APCI), m/z
calcd for C16H10ClF6NO2: 396.0226, found 396.0229.
Mp 215–217 °C. Yield 5%. 1H NMR (300 MHz, CDCl3): d 6.95 (d,
J = 8.8 Hz, 1H), 7.38 (dd, J1 = 8.8 Hz, J2 = 2.3 Hz, 1H), 7.84 (d,
J = 8.6 Hz, 1H), 7.97 (d, J = 2.3 Hz, 1H), 8.14–8.16 (m, 2H). HRMS
(APCI), m/z calcd for C15H8ClF6NO2–H: 382.0070, found 382.0082.
4.1.3.32. N-(2,3-Bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxy-
benz-amide (31).
Prepared by general procedure A. White solid.
Mp 163–164 °C. Yield 7%. 1H NMR (300 MHz, CDCl3): d 7.00–7.04 (m,
1H), 7.43–7.47 (m, 2H), 7.73–7.80 (m, 2H), 8.20 (d, J = 7.8 Hz, 1H),
8.29 (s, 1H), 11.41 (s, 1H). HRMS (APCI), m/z calcd for
C15H8ClF6NO2–H: 382.0070, found 382.0074.
4.1.3.33. N-(2,5-Bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxy-
benz-amine (32)20
Prepared by general procedure A. White so-
.
lid. Mp 202–203 °C. Characterization (NMR, MS) was in accordance
4.1.3.26. 5-Chloro-2-hydroxy-N-phenylbenzamide (26).
Pre-
with previously reported values.
pared by general procedure A.23 Slightly yellow solid. Mp 211–
212 °C. Yield 45%. 1H NMR (300 MHz, DMSO-d6): d 7.00 (d,
J = 8.8 Hz, 1H), 7.15 (dd, J1 = 7.2 Hz, J2 = 7.2 Hz, 1H), 7.34–7.39 (m,
2H), 7.46 (dd, J1 = 8.8 Hz, J2 = 2.7 Hz, 1H), 7.69 (d, J = 8.3 Hz, 2H),
7.96 (d, J = 2.6 Hz, 1H), 10.40 (s, 1H), 11.84 (s, 1H). HRMS (APCI),
m/z calcd for C13H10ClNO2–H: 246.0322, found 246.0329.
4.1.3.34. 5-Chloro-2-hydroxy-N-((2-phenylsulfonyl)phenyl)benz-
amide (33).
Prepared by general procedure A. White solid. Mp
154–156 °C. Yield 24%. 1H NMR (300 MHz, CDCl3): d 7.00 (d,
J = 8.9 Hz, 1H), 7.32–7.82 (m, 9H), 8.08(d, J = 9.0 Hz, 1H), 8.43(d,
J = 8.1 Hz, 1H), 10.62(s, 1H), 11.81 (s, 1H). IR (neat):3340, 1582,
1534, 1331, 1276, 1132, 826 cmÀ1. HRMS (APCI), m/z calcd for
4.1.3.27.
benzamide (27).
5-Chloro-N-(3-(trifluoromethyl)phenyl)-2-hydroxy-
Prepared by general procedure A.25 White
C19H14ClNO4S–H: 386.0254, found 386.0259.
solid. Mp 181–182 °C. Yield 51%. 1H NMR (300 MHz, DMSO-d6): d
7.02 (d, J = 8.8 Hz, 1H), 7.45–7.50 (m, 2H), 7.61 (dd, J1 = 8.0 Hz,
J2 = 8.0 Hz, 1H), 7.89 (d, J = 2.6 Hz, 1H), 7.94 (d, J = 8.3 Hz, 1H),
8.20 (s, 1H), 10.63 (s, 1H), 11.57 (s, br, 1H). HRMS (APCI), m/z calcd
for C14H9ClF3NO2–H: 314.0196, found 314.0202.
4.1.3.35.
(34).
5-Chloro-N-(3-fluorophenyl)-2-hydroxybenzamide
Prepared by general procedure A. White solid. Mp
>281 °C dec. Yield 38%. 1H NMR (300 MHz, DMSO-d6): d 6.92–
6.98 (m, 2H), 7.35–7.45 (m, 3H), 7.73 (d, J = 5.7 Hz, 1H), 7.85 (s,
1H), 11.06 (s, 1H). HRMS (APCI), m/z calcd for C13H9ClFNO2–H:
264.0228, found 264.0229.
4.1.3.28.
5-Chloro-2-hydroxy-N-(2-trifluoromethyl-phenyl)-
Prepared by general procedure A. White
benzamide (28)20
.
4.1.3.36.
(35).
N-(3-Bromophenyl)-5-chloro-2-hydroxybenzamide
Prepared by general procedure A. Yield 10%. White solid.
solid. Mp 155–157 °C. Characterization (NMR, MS) was in accor-
dance with previously reported values.
Mp 231–232 °C. 1H NMR (300 MHz, DMSO-d6): d 7.00 (d, J = 8.8 Hz,
1H), 7.33 (d, J = 5.2 Hz, 2H), 7.46 (dd, J1 = 8.8 Hz, J2 = 2.6 Hz, 1H),
7.63–7.66 (m, 1H), 7.88 (d, J = 2.6 Hz, 1H), 8.05 (s, 1H), 10.50 (s,
1H), 11.57 (s, 1H). HRMS (APCI), m/z calcd for C13H9BrClNO2–H:
323.9427, found 323.9433.
4.1.3.29.
5-Chloro-N-(4-(trifluoromethyl)phenyl)-2-hydroxy-
Prepared by general procedure A. White
benzamide (29)28
.
solid. Mp 222–223 °C. Characterization (NMR, MS) was in accor-
dance with previously reported values.
4.1.3.37.
(36).
5-Chloro-N-(3-cyanophenyl)-2-hydroxybenzamide
4.1.3.30. Aniline substrates for 30 and 31.
1,2-Bis(trifluoro-
Prepared by general procedure A.26 White solid. Mp
methyl)benzene (3.47 mL, 23 mmol, 1 equiv) was added slowly
to a mixture of fuming nitric acid (18.3 mL, 440 mmol, 19 equiv)
and concentrated sulfuric acid (24.5 mL, 460 mmol, 20 equiv) and
the mixture was heated to 100 °C for 4 h. The product was poured
onto ice and the aqueous phase was extracted with ether. The or-
ganic phase was washed successively with water, saturated so-
dium carbonate, brine, and then dried (MgSO4). The extract was
concentrated under vacuum, yielding a crude oil (4.5 g, 74% com-
bined yield).
240–241 °C. Yield 21%. 1H NMR (300 MHz, DMSO-d6): d 7.02 (d,
J = 8.8 Hz, 1H), 7.47 (dd, J1 = 8.8 Hz, J2 = 2.6 Hz, 1H), 7.55–7.61 (m,
2H), 7.86 (d, J = 2.6 Hz, 1H), 7.95–7.99 (m, 1H), 8.20 (s, 1H), 10.62
(s, 1H), 11.56 (s, 1H). IR (neat): 3073, 2231, 1632, 1616, 1564,
1438, 1224, 815, 787 cmÀ1
.
HRMS (APCI), m/z calcd for
C14H9ClN2O2–H: 271.0274, found 271.0283.
4.1.3.38.
(37).
3-(5-Chloro-2-hydroxybenzamido)benzoic
acid
Sodium hydroxide (130 mg, 3.3 mmol, 5 equiv) in water
The oil containing 2,3-bis(trifluoromethyl)nitrobenzene and
3,4-bis(trifluoromethyl)nitrobenzene) (4 g, 15 mmol) was dis-
(1 mL, 3 M) was added to compound 38 (208 mg, 0.65 mmol,
1 equiv) in water (1.5 mL, 0.4 M), and the mixture was stirred at

122
I.-Y. Lee et al. / Bioorg. Med. Chem. 21 (2013) 114–126
room temperature (5 h). The reaction mixture was diluted with
water (10 mL) and washed with EtOAc (20 mL). The water layer
was acidified by addition of dilute HCl, and the product was ex-
tracted with EtOAc. The EtOAc layer was washed successively with
water and brine, then dried (Na2SO4). The resultant solution was
concentrated under vacuum to give a residue which was purified
by chromatography (MeOH:CHCl3 1:5) to give product 37
(170 mg, 91%). White solid. Mp >341 °C dec. 1H NMR (300 MHz,
DMSO-d6): d 7.03 (d, J = 8.8 Hz, 1H), 7.46–7.53 (m, 2H), 7.72 (d,
J = 7.7 Hz, 1H), 7.92–7.97 (m, 2H), 8.36 (s, 1H), 10.57(s, 1H),
11.91(s, br, 1H), 12.94(s, br, 1H). IR (neat): 3069, 1694, 1591,
1568, 1454, 1294, 1221, 817,748 cmÀ1. HRMS (APCI), m/z calcd
for C14H10ClNO4-H: 290.0220, found 290.0216.
2228, 1607, 1548, 1420, 1219, 1103, 831 cmÀ1. Characterization
(NMR, MS) was in accordance with previously reported values.
4.1.3.45. 5-Chloro-2-hydroxy-N-(4-phenoxyphenyl)benzamide
(44).
Prepared by general procedure A.31 Grey solid. Mp
189–191 °C. Yield 16%. 1H NMR (300 MHz, DMSO-d6): d 6.99–
7.14 (m, 6H), 7.36–7.48(m, 3H), 7.72 (d, J = 8.9 Hz, 2H), 7.96 (d,
J = 2.6 Hz, 1H), 10.50 (s, 1H), 11.62(s, br, 1H). HRMS (APCI), m/z
calcd for C19H14ClNO3–H: 338.0584, found 338.0575.
4.1.3.46. N-(4-tert-Butylphenyl)-5-chloro-2-hydroxybenzamide
(45)7.
Prepared by general procedure A. White solid. Mp
219–220 °C [lit7: mp 221–222 °C]. Characterization (NMR, MS)
was in accordance with previously reported values.
4.1.3.39.
(38).
Ethyl
3-(5-chloro-2-hydroxybenzamido)benzoate
Prepared by general procedure A. White solid. Mp
4.1.3.47. 5-Chloro-2-hydroxy-N-(4-methoxyphenyl)benzamide
154–156 °C. Yield 52%. 1H NMR (300 MHz, DMSO-d6): d 1.31–
1.37 (m, 3H), 4.30–4.41 (m, 2H), 7.02 (d, J = 8.8 Hz, 1H), 7.46–
7.52 (m, 2H), 7.73 (d, J = 7.8 Hz, 1H), 7.94–7.97 (m, 2H), 8.37 (s,
1H), 10.61 (s, 1H), 11.70 (s, br, 1H). IR (neat): 3322, 3082, 1719,
1561, 1285, 1223, 817, 746 cmÀ1. HRMS (APCI), m/z calcd for
(46)22
.
Prepared by general procedure A. Brown solid. Mp
>207 °C dec. Yield 36%. 1H NMR (300 MHz, DMSO-d6): d 3.75 (s,
3H), 6.92–7.01 (m, 3H), 7.44–7.58 (m, 1H), 7.57–7.61 (m, 2H),
7.98 (s, 1H), 10.32 (s, 1H), 12.05 (s, 1H). HRMS (APCI), m/z calcd
for C14H12ClNO3–H: 276.0427, found 276.0434.
C16H14ClNO4-H: 318.0533, found 318.0532.
4.1.3.48.
benzamide (47).
5-Chloro-2-hydroxy-N-(4-morpholin-4-yl-phenyl)-
Prepared by general procedure A. Grey solid.
4.1.3.40. N-(3-Carbamoyl-phenyl)-5-chloro-2-hydroxy-benzam-
ide (39)28
Prepared by general procedure A. White solid. Mp.
Mp >272 °C dec. IR (neat): 3359, 3180, 1646, 1561, 1406, 1223,
1119, 828 cmÀ1. Characterization (NMR, MS) was in accordance
with previously reported values.
.
Mp >238 °C dec. Yield 9%. 1H NMR (300 MHz, DMSO-d6): d 3.05–
3.09 (m, 4H), 3.70–3.76 (m, 4H), 6.94–7.01 (m, 3H), 7.45 (dd,
J1 = 8.9 Hz, J2 = 2.7 Hz, 1H), 7.56 (d, J = 8.9 Hz, 2H), 8.00 (s, 1H),
10.40 (s, 1H). IR (neat): 3297, 2861, 1612, 1557, 1509, 1217,
1114, 922, 813 cmÀ1. HRMS (APCI), m/z calcd for C17H17ClN2O3–
H: 282.0133, found 282.0137.
4.1.3.41. 5-Chloro-2-hydroxy-N-(3-(phenylcarbamoyl)phenyl)-
benzamide (40).
Prepared by general procedure B using 3-ami-
no-N-phenylbenzamide, and the final deprotection step was modified
as follows: potassium carbonate (140 mg, 1.02 mmol, 1.2 equiv) was
added to a solution of crude product (400 mg, 0.85 mmol, 1 equiv)
from the previous step in 1:1 MeOH:1,4-dioxane (8 mL, 0.1 M). The
reaction mixture was stirred at room temperature (3 h) and acidified
with 1 N HCl, followed by extraction with EtOAc. The organic layer
was washed with water and brine, dried (MgSO4), and the solvent
was removed under vacuum and recrystallized from Et2O (200 mg,
64%). White solid. Mp 269–270 °C. 1H NMR (300 MHz, DMSO-d6): d
7.03 (d, J = 8.7 Hz, 1H), 7.11 (dd, J1 = 7.5 Hz, J2 = 7.2 Hz, 1H), 7.36 (dd,
J1 = 7.8 Hz, J2 = 7.8 Hz, 2H), 7.46–7.56 (m, 2H), 7.72–7.79 (m, 3H),
7.94–7.98 (m, 2H), 8.22 (s, 1H), 10.30 (s, 1H), 10.62 (s, 1H). IR (neat):
3281, 1646, 1607, 1525, 1424, 1324, 1221 cmÀ1. HRMS (APCI), m/z
calcd for C20H15ClN2O3–H: 365.0693, found 365.0685.
4.1.3.49. N-(4-Carbamoyl-phenyl)-5-chloro-2-hydroxy-benzam-
ide (48).
Compound 43 (50 mg, 0.18 mmol, 1 equiv) was dis-
solved in EtOH (0.8 mL,) and DMSO (0.4 mL). 1 M NaOH (1.03 mL,
5 equiv) and 30% H2O2 (1.03 mL, 50 equiv) were added and the
reaction was stirred at room temperature (16 h). After concentra-
tion under vacuum, the residue was purified by column chroma-
tography (1:19 MeOH:CHCl3) to give product 48 (19 mg, 36%).
White solid. Mp >328 °C dec. 1H NMR (300 MHz, DMSO-d6): d
7.03 (d, J = 8.7 Hz, 1H), 7.30 (s, 1H), 7.46 (dd, J1 = 8.6 Hz,
J2 = 2.4 Hz, 1H), 7.78 (d, J = 8.3 Hz, 2H), 7.88–7.91 (m, 4H), 10.55
(s, 1H), 11.69 (s, 1H). HRMS (APCI), m/z calcd for C14H11ClN2O3–
H: 289.0380, found 289.0372.
4.1.3.50.
benz-amide (49).
5-Chloro-2-hydroxy-N-(4-(phenylcarbamoyl)phenyl)
Prepared by general procedure B using 4-
4.1.3.42.
(41).
5-Chloro-N-(4-fluorophenyl)-2-hydroxybenzamide
amino-N-phenyl-benzamide. Red solid. Mp 275–277 °C. Yield 11%.
1H NMR (300 MHz, DMSO-d6): d 7.02–7.12 (m, 2H), 7.35 (dd,
J1 = 8.0 Hz, J2 = 8.0 Hz, 2H), 7.48 (dd, J1 = 8.7 Hz, J2 = 2.4 Hz, 1H), 7.78
(d, J = 8.1 Hz, 2H), 7.87 (d, J = 8.7 Hz, 2H), 7.93 (d, J = 2.1 Hz, 1H),
7.96–8.01 (m, 2H), 10.18 (s, 1H), 10.61 (s, 1H), 11.69 (s, 1H). IR (neat):
3336, 2557, 1635, 1525, 1443, 1221, 810 cmÀ1. HRMS (APCI), m/z
calcd for C20H15ClN2O3–H: 365.0693, found 365.0703.
Prepared by general procedure A.23 White solid. Mp 239–
240 °C. Yield 8%. 1H NMR (300 MHz, DMSO-d6): d 7.01 (d, J = 8.8 Hz,
1H), 7.18–7.24 (m, 2H), 7.46 (dd, J1 = 8.8 Hz, J2 = 2.7 Hz, 1H), 7.69–7.74
(m, 2H), 7.93 (d, J = 2.6 Hz, 1H), 10.46 (s, 1H), 11.77 (s, 1H). IR (neat):
3333, 2927, 1621, 1571, 1504, 1422, 1210, 1098, 819 cmÀ1. HRMS
(APCI), m/z calcd for C13H9ClFNO2–H: 264.0228, found 264.0223.
4.1.3.43. 5-Chloro-2-hydroxy-N-(4-trifluoromethoxy-phenyl)-
4.1.3.51. 5-Chloro-2-hydroxy-N-(4-(N-phenylsulfamoyl)phenyl)
benzamide (42).
Prepared by general procedure A. White so-
benz-amide (50).
Prepared by general procedure B using 4-
lid. Mp 200–202 °C. Yield 39%. 1H NMR (300 MHz, DMSO-d6): d
7.02 (d, J = 8.8 Hz, 1H), 7.39 (d, J = 8.7 Hz, 2H), 7.47 (dd,
J1 = 8.8 Hz, J2 = 2.7 Hz, 1H), 7.82 (dd, J1 = 7.0 Hz, J2 = 2.0 Hz, 2H),
7.91 (d, J = 2.7 Hz, 1H), 10.54 (s, 1H), 11.63 (s, 1H). IR (neat):
3297, 2961, 1614, 1562, 1505, 1160, 1100 cmÀ1. HRMS (APCI), m/
z calcd for C14H9ClF3NO–H: 330.0145, found 330.0156.
amino-N-phenylbenzenesulfonamide. White solid. >291 °C dec.
Yield 13%. 1H NMR (300 MHz, DMSO-d6): d 6.99–7.04 (m, 2H),
7.07–7.10 (m, 2H), 7.20–7.25 (m, 2H), 7.46 (dd, J1 = 9.0 Hz,
J2 = 3.0 Hz, 1H), 7.72 (s, 1H), 7.75 (s, 1H), 7.81–7.86 (m, 3H),
10.21 (s, 1H), 10.61 (s, 1H), 11.49 (s, 1H). HRMS (APCI), m/z calcd
for C19H15ClN2O4S–H: 401.0363, found 401.0356.
4.1.3.44.
(43)28
5-Chloro-N-(4-cyanophenyl)-2-hydroxybenzamide
4.1.3.52. 5-Chloro-2-hydroxy-N-(4-(N-(5-methylisoxazol-3-yl)-
.
Prepared by general procedure A. Slightly brown solid.
sulfamoyl)-phenyl)benzamide (51).
Prepared by general
Mp 246–247 °C [lit28: mp 242–243.5 °C]. IR (neat): 3311, 3075,
procedure B using 4-amino-N-(5-methylisoxazol-3-yl)benzenesul-

I.-Y. Lee et al. / Bioorg. Med. Chem. 21 (2013) 114–126
123
fonamide. Slightly yellow solid. Mp >288 °C dec. Yield 71%. 1H NMR
(300 MHz, DMSO-d6): d 2.26 (s, 3H), 6.13 (s, 1H), 7.00(d, J = 8.7 Hz,
1H), 7.45 (dd, J1 = 8.7 Hz, J2 = 2.7 Hz, 1H), 7.82–7.92 (m, 5H), 10.76
4.1.3.62.
N-(2-Bromo-5-(trifluoromethyl)phenyl)-5-chloro-2-
Prepared by general procedure
hydroxy-benzamide (61)24
.
A. White solid. Mp 174–176 °C. Characterization (NMR, MS) was
in accordance with previously reported values.
(s, 1H). IR (neat): 3187, 1660, 1623, 1506, 1420, 1164, 881 cmÀ1
.
HRMS (APCI), m/z calcd for C17H14ClN3O5S–H: 406.0264, found
406.0259.
4.1.3.63.
methyl)-phenyl)benzamide (62).
5-Chloro-2-hydroxy-N-(5-methoxy-2-(trifluoro-
Prepared by general proce-
4.1.3.53. 5-Chloro-N-(3-(trifluoromethyl)-2-methylphenyl)-2-
dure A. White solid. Mp 162–163 °C. Yield 35%. 1H NMR (300 MHz,
DMSO-d6): d 3.98 (s, 3H), 7.06 (d, J = 8.7 Hz, 1H), 7.29 (d, J = 8.6 Hz,
1H), 7.49 (dd, J1 = 8.7 Hz, J2 = 3.1 Hz, 2H), 7.96 (d, J = 2.8 Hz, 1H),
8.82 (d, J = 2.1 Hz, 1H), 11.05 (s, 1H), 12.18 (s, 1H). HRMS (APCI),
m/z calcd for C15H11ClF3NO3–H: 344.0301, found 344.0310.
hydroxy-benzamide (52)20
.
Prepared by general procedure
A. White solid. Mp 199–200 °C. Characterization (NMR, MS) was
in accordance with previously reported values.
4.1.3.54.
5-Chloro-N-(2-fluoro-3-(trifluoromethyl)phenyl)-2-
Prepared by general procedure
hydroxybenzamide (53)20
.
4.1.3.64.
5-Chloro-N-(2-chloro-5-fluoro-phenyl)-2-hydroxy-
A. Slightly yellow solid. Mp 250–251 °C. Characterization (NMR,
MS) was in accordance with previously reported values.
benzamide (63).
Prepared by general procedure A. White solid.
Mp 218–221 °C. Yield 26%. 1H NMR (300 MHz, DMSO-d6): d 7.04–
7.11 (m, 2H), 7.51–7.66 (m, 2H), 7.98 (d, J = 2.8 Hz, 1H), 8.38 (dd,
J1 = 11.4 Hz, J2 = 3.0 Hz, 1H), 11.09 (s, 1H), 12.40 (s, 1H). HRMS (APCI),
m/z calcd for C13H8Cl2FNO2–H: 297.9838, found 297.9834.
4.1.3.55. 5-Chloro-N-(2,3-difluorophenyl)-2-hydroxybenzamide
(54).
Prepared by general procedure A. White solid. Mp 250–
251 °C. Yield 58%. 1H NMR (300 MHz, DMSO-d6): d 7.05 (d,
J = 8.7 Hz, 1H), 7.18–7.28 (m, 2H), 7.49 (d, J1 = 8.7 Hz, 1H), 7.95–
8.04 (m, 2H), 10.77 (s, 1H), 12.17 (s, 1H). IR (neat): 3315, 3110,
1619, 1557, 1472, 1228, 1004 cmÀ1. HRMS (APCI), m/z calcd for
4.1.3.65. 5-Chloro-N-(2-fluoro-5-nitro-phenyl)-2-hydroxy-benz-
amide (64).
Prepared by general procedure A. Slightly yellow
solid. Mp 254–255 °C. Yield 19%. 1H NMR (300 MHz, DMSO-d6): d
7.07 (d, J = 8.8 Hz, 1H), 7.52 (dd, J1 = 8.8 Hz, J2 = 2.8 Hz, 1H), 7.61–
7.68 (m, 1H), 7.95 (d, J = 2.4 Hz, 1H), 8.06–8.12 (m, 1H), 9.25 (dd,
J1 = 6.7 Hz, J2 = 2.9 Hz, 1H), 11.00 (s, 1H), 12.28 (s, 1H). HRMS
(APCI), m/z calcd for C13H8ClFN2O4-H: 309.0078, found 309.0085.
C13H8ClF2NO2–H: 282.0133, found 282.0135.
4.1.3.56.
hydroxy-benzamide (55).
5-Chloro-N-(4-fluoro-2-(trifluoromethyl)phenyl)-2-
Prepared by general procedure A.
White solid. Mp 173–174 °C. Yield 6%. 1H NMR (300 MHz, DMSO-
d6): d 7.05 (d, J = 8.8 Hz, 1H), 7.52 (dd, J1 = 8.8 Hz, J2 = 2.8 Hz, 1H),
7.59–7.74 (m, 2H), 7.98 (d, J = 2.7 Hz, 1H), 8.13 (dd, J1 = 8.9 Hz,
J2 = 5.0 Hz, 1H), 10.79 (s, 1H), 12.20 (s, 1H). HRMS (APCI), m/z calcd
for C14H8ClF4NO2–H: 332.0101, found 332.0106.
4.1.3.66.
5-Chloro-N-(2-chloro-6-(trifluoromethyl)phenyl)-2-
hydroxybenzamide (65).
Prepared by general procedure A.
White solid. Mp 167–170 °C. Yield 9%. 1H NMR (300 MHz, CDCl3):
d 7.00 (d, J = 8.9 Hz, 1H), 7.48 (m, 2H), 7.57 (d, J = 2.4 Hz, 1H),
7.68–7.76 (m, 2H), 11.46 (s, 1H). HRMS (APCI), m/z calcd for
4.1.3.57.
hydroxy-benzamide (56).
N-(4-Bromo-2-(trifluoromethyl)phenyl)-5-chloro-2-
Prepared by general procedure A.
C
14H8Cl2F3NO2–H: 347.9806, found 347.9805.
Red solid. Mp 171–173 °C. Yield 31%. 1H NMR (300 MHz, DMSO-
d6): d 7.05 (d, J = 8.7 Hz, 1H), 7.50 (dd, J1 = 8.7 Hz, J2 = 2.7 Hz, 1H),
7.91–7.95 (m, 3H), 8.20 (d, J = 9.3 Hz, 1H), 10.81 (s, 1H), 12.28 (s,
1H). HRMS (APCI), m/z calcd for C14H8BrClF3NO2–H: 391.9301,
found 391.9289.
4.1.3.67. 5-Chloro-N-(2,6-difluorophenyl)-2-hydroxybenzamide
(66).
Prepared by general procedure A. Slightly yellow solid.
Mp >238 °C dec. Yield 7%. 1H NMR (300 MHz, DMSO-d6): d 7.03
(d, J = 8.8 Hz, 1H), 7.19–7.27 (m, 2H), 7.38–7.46 (m, 1H), 7.54 (m,
1H), 7.98 (d, J = 2.6 Hz, 1H), 10.23 (s, 1H), 11.87 (s, 1H). HRMS
(APCI), m/z calcd for C13H8ClF2NO2–H: 282.0133, found 282.0129.
4.1.3.58.
hydroxy-benzamide (57).
White solid. Mp 195–196 °C. Yield 43%. 1H NMR (300 MHz,
DMSO-d6): 7.14 (d, J = 8.8 Hz, 1H), 7.58 (dd, J1 = 8.8 Hz,
5-Chloro-N-(2-chloro-4-trifluoromethyl-phenyl)-2-
Prepared by general procedure A.
4.1.3.68.
5-Chloro-N-(4-fluoro-3-(trifluoromethyl)phenyl)-2-
Prepared by general procedure
hydroxybenzamide (67)20
.
d
A. White solid. Mp 203–205 °C. Characterization (NMR, MS) was
J2 = 2.9 Hz, 1H), 7.84 (dd, J1 = 8.8 Hz, J2 = 1.6 Hz, 1H), 8.02–8.05
(m, 2H), 8.78 (d, J = 8.6 Hz, 1H), 11.22 (s, 1H), 12.46 (s, 1H). HRMS
(APCI), m/z calcd for C14H8Cl2F3NO2–H: 347.9788, found 347.9797.
in accordance with previously reported values.
4.1.3.69.
5-Chloro-N-(4-chloro-3-(trifluoromethyl)phenyl)-2-
Prepared by general procedure
hydroxy-benzamide (68)30
.
4.1.3.59. 5-Chloro-N-(2,4-difluorophenyl)-2-hydroxybenzamide
A. White solid(Slightly yellow). Mp 230–232 °C. Yield 38%. 1H
NMR (300 MHz, DMSO-d6): d 7.02 (d, J = 8.7 Hz, 1H), 7.46 (d,
J = 7.5 Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H), 7.85 (s, 1H), 7.99 (d,
J = 8.4 Hz, 1H), 8.30 (s, 1H), 10.68 (s, 1H). HRMS (APCI), m/z calcd
for C14H8Cl2F3NO2–HO2–H: 347.9828, found 347.9819.
(58).
Prepared by general procedure A. Yield 12%. Grey solid.
7.03 (d,
Mp >220 °C dec. 1H NMR (300 MHz, DMSO-d6):
d
J = 8.8 Hz, 1H), 7.10–7.17 (m, 1H), 7.37–7.44 (m, 1H), 7.49 (dd,
J1 = 8.8 Hz, J2 = 2.8 Hz, 1H), 7.96 (d, J = 2.7 Hz, 1H), 8.07–8.15 (m,
1H), 10.58 (s, 1H), 12.11 (s, 1H). HRMS (APCI), m/z calcd for
C13H8ClF2NO2–H: 282.0133, found 282.0134.
4.1.3.70.
N-(4-Bromo-3-(trifluoromethyl)phenyl)-5-chloro-2-
hydroxy-benzamine (69).
Prepared by general procedure A.
4.1.3.60.
5-Chloro-N-(2-chloro-5-(trifluoromethyl)phenyl)-2-
Prepared by general procedure
White solid. Mp 238–240 °C. Yield 26%. 1H NMR (300 MHz, DMSO-
d6): d 7.02 (d, J = 8.8 Hz, 1H), 7.46 (dd, J1 = 8.8 Hz, J2 = 2.6 Hz, 1H),
7.85–7.93 (m, 3H), 8.29 (s, 1H), 10.65 (s, 1H), 11.53 (s, br, 1H). HRMS
(APCI), m/z calcd for C13H8BrClF3NO–H: 391.9301, found 391.9311.
hydroxy-benzamide (59)20
.
A. White solid. Mp 180–182 °C. Characterization (NMR, MS) was
in accordance with previously reported values.
4.1.3.61.
5-Chloro-N-(2-fluoro-5-(trifluoromethyl)phenyl)-2-
Prepared by general procedure
4.1.3.71.
hydroxy-benzamide (70).
White solid. Mp 214–216 °C. Yield 16%. 1H NMR (300 MHz,
DMSO-d6): 7.03 (d, J = 8.8 Hz, 1H), 7.48 (dd, J1 = 8.8 Hz,
5-Chloro-N-(4-cyano-3-(trifluoromethyl)phenyl)-2-
hydroxy-benzamide (60)20
.
Prepared by general procedure A.
A. White solid. Mp 199–200 °C. Characterization (NMR, MS) was
in accordance with previously reported values.
d

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I.-Y. Lee et al. / Bioorg. Med. Chem. 21 (2013) 114–126
J2 = 2.6 Hz, 1H), 7.79 (d, J = 2.6 Hz, 1H), 8.15 (s, 2H), 8.42 (s, 1H),
10.97 (s, 1H), 11.34 (s,1H). IR (neat): 3197, 2235, 1680, 1534,
1317, 1182, 1116, 1048, 890, 817 cmÀ1. HRMS (APCI), m/z calcd
for C15H8ClF3N2O2–H: 339.0148, found 339.0150.
to room temperature, water was added (100 mL) followed by extraction
with EtOAc. The crude product was purified by column chromatography
(1:10 ? 1:5 EtOAc:Hex) to give 4-bromo-3,5-bis-(trifluoromethyl)-ani-
line (340 mg) & 2-bromo-3,5-bis-(trifluoromethyl)aniline (680 mg).
4.1.3.72.
benzamide (71).
5-Chloro-N-(3-chloro-4-fluoro-phenyl)-2-hydroxy-
4.1.3.82.
5-Chloro-2-hydroxy-N-(4-methyl-3,5-bis(trifluoro-
4-Bromo-3,5-bis-(trifluo-
Prepared by general procedure A. Grey solid.
methyl)-phenyl)benzamide (80).
Mp 224–225 °C. Yield 30%. 1H NMR (300 MHz, DMSO-d6): d 7.02 (d,
J = 8.8 Hz, 1H), 7.40–7.49 (m, 2H), 7.62–7.67 (m, 1H), 7.87 (d, J = 2.6 Hz,
1H), 8.03 (dd, J1 = 6.9 Hz, J2 = 2.6 Hz, 1H), 10.51 (s, 1H), 11.61 (s, 1H).
HRMS (APCI), m/z calcd for C13H8Cl2FNO2–H: 297.9833, found 297.9835.
romethyl)aniline (616 mg, 2 mmol, 1 equiv), potassium carbonate
(829 mg, 6 mmol, 3 equiv), Pd(PPh3)4 (231 mg, 0.2 mmol,
0.1 equiv), DMF (5 mL, 0.4 M) and (MeO)3B (0.28 mL, 2 mmol,
1 equiv) were added to a flask and the contents heated to 110 °C
overnight. The reaction solution was filtered using silica gel, Celite,
and sea sand. The solvent was evaporated under reduced pressure
and the residue was purified by column chromatography (1:5
EtOAc:Hex), to give 4-methyl-3,5-bis(trifluoromethyl)aniline
(760 mg, 67%). 1H NMR (300 MHz, DMSO-d6): d 2.42 (s, 3H), 3.88
(s, 2H), 7.09 (s, 2H). This aniline was used for general procedure
A (EtOAc:Hex 1:20 ? 1:10) (630 mg, 51%). Slightly brown solid.
Mp 192–193 °C. 1H NMR (300 MHz, DMSO-d6): d 2.49 (s, 3H),
7.02 (d, J = 8.8 Hz, 1H), 7.47 (dd, J1 = 8.8 Hz, J2 = 2.7 Hz, 1H), 7.87
(d, J = 2.6, 1H), 8.41 (s, 2H), 10.75 (s, 1H), 11.48 (s, br, 1H). HRMS
(APCI), m/z calcd for C16H10ClF6NO2–H: 396.0226, found 396.0238.
4.1.3.73. 5-Chloro-N-(3,4-difluoro-phenyl)-2-hydroxy-benzam-
ide (72).
Prepared by general procedure A. Slightly yellow so-
lid. Mp 250–251 °C. Yield 25%. 1H NMR (300 MHz, DMSO-d6): d
7.01 (d, J = 8.8 Hz, 1H), 7.39–7.49 (m, 3H), 7.85–8.00 (m, 2H),
10.53 (s, 1H), 11.59 (s, 1H). HRMS (APCI), m/z calcd for
C13H8ClF2NO2–H: 282.0133, found 282.0137.
4.1.3.74.
benzamide (73).
brown solid. Mp 245–247 °C. Yield 14%. 1H NMR (300 MHz,
DMSO-d6): 2.35 (s, 3H), 7.00 (d, J = 8.7 Hz, 1H), 7.32 (d,
5-Chloro-2-hydroxy-N-(3-iodo-4-methyl-phenyl)-
Prepared by general procedure A. Yellow
d
J = 8.4 Hz, 1H), 7.46 (dd, J1 = 8.8 Hz, J2 = 2.7 Hz, 1H), 7.60 (dd,
J1 = 8.3 Hz, J2 = 2.1 Hz, 1H), 7.92 (d, J = 2.7 Hz, 1H), 8.27 (d,
J = 2.1 Hz, 1H), 10.44 (s, 1H), 11.74 (s, 1H). HRMS (APCI), m/z calcd
for C14H11ClINO2–H: 385.9445, found 385.9459.
4.1.3.83.
5-Chloro-2-hydroxy-N-(2-methyl-3,5-bis(trifluoro-
Synthesis proceeded as
methyl)-phenyl)benzamide (81).
for 80 using 2-bromo-3,5-bis-(trifluoromethyl)aniline as starting
material (330 mg, 33%). Slightly yellow solid. Mp 191–193 °C. 1H
NMR (300 MHz, DMSO-d6): d 2.46 (s, 3H), 7.07 (d, J = 8.8 Hz, 1H),
7.50 (dd, J1 = 8.7 Hz, J2 = 2.4 Hz, 1H), 7.82 (s, 1H), 7.95 (d,
J = 2.7 Hz, 1H), 8.60 (s, 1H), 10.64 (s, 1H), 12.11 (s, 1H). HRMS
(APCI), m/z calcd for C16H10ClF6NO2–H: 396.0226, found 396.0233.
4.1.3.75.
N-(3-Bromo-5-(trifluoromethyl)phenyl)-5-chloro-2-
Prepared by general procedure
hydroxy-benzamide (74)24
.
A. White solid. Mp 200–202 °C. Characterization (NMR, MS) was
in accordance with previously reported values.
4.1.3.84. N-(4-Bromo-3,5-difluorophenyl)-5-chloro-2-hydroxy-
4.1.3.76.
5-Chloro-2-hydroxy-N-(3-methoxy-5-(trifluoro-
Prepared by general pro-
benz-amide (82).
Prepared by general procedure A. White so-
methyl)-phenyl)benzamide (75)24
.
lid. Mp >271 °C dec. Yield 32%. 1H NMR (300 MHz, DMSO-d6): d
7.03 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 6.7 Hz, 1H), 7.68 (d, J = 9.5 Hz,
2H), 7.79 (s, 1H), 10.72 (s, 1H), 11.45 (s, br, 1H). HRMS (APCI), m/
z calcd for C13H7BrClF2NO2–H: 359.9238, found 359.9234.
cedure A. White solid. Mp 191–192 °C. Characterization (NMR, MS)
was in accordance with previously reported values.
4.1.3.77. 5-Chloro-N-(3,5-dichlorophenyl)-2-hydroxybenzamide
(76)20
.
Prepared by general procedure A. White solid. Mp 247–
4.1.3.85. 5-Chloro-2-hydroxy-N-(3,4,5-trifluoro-phenyl)-benz-
249 °C. Characterization (NMR, MS) was in accordance with previ-
ously reported values.
amide (83).
Prepared by general procedure A. Slightly yellow
solid. Mp 234–236 °C. Yield 52%. 1H NMR (300 MHz, DMSO-d6): d
7.03 (d, J = 8.8 Hz, 1H), 7.47 (dd, J1 = 8.8 Hz, J2 = 2.7 Hz, 1H), 7.67–
7.72 (m, 2H), 7.80 (d, J = 2.7 Hz, 1H), 10.59 (s, 1H), 11.47 (s, br,
1H). HRMS (APCI), m/z calcd for C13H7ClF3NO2–H: 300.0039, found
300.0055.
4.1.3.78. 5-Chloro-N-(3,5-difluorophenyl)-2-hydroxybenzamide
(77)21
.
Prepared by general procedure A. Slightly yellow solid.
Mp 244–246 °C. Characterization (NMR, MS) was in accordance
with previously reported values.
4.1.3.86. 5-Chloro-2-hydroxy-N-(3,4,5-trichloro-phenyl)-benz-
4.1.3.79. 5-Chloro-N-(3,5-di-tert-butylphenyl)-2-hydroxyben-
amide (84)29
.
Prepared by general procedure A. White solid.
zamide (78)19
.
Prepared by general procedure A. White solid.
Mp 287–290 °C. Yield 44%. 1H NMR (300 MHz, DMSO-d6): d 7.02
(d, J = 8.8 Hz, 1H), 7.47 (dd, J1 = 8.7 Hz, J2 = 2.5 Hz, 1H), 7.81 (d,
J = 2.5 Hz, 1H), 8.05 (s, 2H), 10.68 (s, 1H), 11.41 (s, br, 1H). HRMS
(APCI), m/z calcd for C13H7Cl4NO2–H: 347.9153, found 347.9156.
Mp 186–188 °C. Characterization (NMR, MS) was in accordance
with previously reported values.
4.1.3.80. 5-Chloro-N-(3,5-dimethylphenyl)2-hydroxybenzamide
(79).
Prepared by general procedure A. White solid. Mp 183–
4.1.3.87. 5-Chloro-2-hydroxy-N-(2,4,5-trifluorophenyl)benzam-
184 °C. Yield 22%. 1H NMR (300 MHz, DMSO-d6): d 2.49 (s, 6H),
6.79 (s, 1H), 7.00 (d, J = 8.8 Hz, 1H), 7.32 (s, 2H), 7.46 (dd,
J1 = 8.8 Hz, J2 = 2.6 Hz, 1H), 7.97 (d, J = 2.6 Hz, 1H), 10.28 (s, 1H),
ide (85).
Prepared by general procedure A. Slightly yellow so-
lid. Mp 250–251 °C. Yield 10%. 1H NMR (300 MHz, DMSO-d6): d
7.04 (d, J = 8.8 Hz, 1H), 7.50 (dd, J1 = 8.8 Hz, J2 = 2.8 Hz, 1H), 7.69–
7.79 (m, 1H), 7.93 (d, J = 2.7 Hz, 1H), 8.26–8.36 (m, 1H), 10.88 (s,
1H), 12.19 (s, br, 1H). HRMS (APCI), m/z calcd for C13H7ClF3NO2–
H: 300.0026, found 300.0032.
11.91 (s, 1H). HRMS (APCI), m/z calcd for
274.0635, found 274.0625.
C15H14ClNO2–H:
4.1.3.81. Substrates for 80 and 81: 4-bromo-3,5-bis-(trifluoro-
methyl)aniline & 2-bromo-3,5-bis-(trifluoromethyl)aniline. 3,5-
4.1.3.88. 5-Chloro-2-hydroxy-N-(2,3,4-trifluoro-phenyl)-benz-
Bis(trifluoromethyl)aniline (2 g, 8.7 mmol, 1 equiv) was dissolved in DMF
(6 mL, 1.5 M). N-bromosuccinimide (1.7 g, 9.6 mmol, 1.1 equiv) was
added, and this mixture was stirred at 50 °C for one hour. After cooling
amide (86).
Prepared by general procedure A. Slightly yellow
solid. Mp 244–246 °C. Yield 16%. 1H NMR (300 MHz, DMSO-d6): d
7.05 (d, J = 8.8 Hz, 1H), 7.32–7.42 (m, 1H), 7.51 (dd, J1 = 8.8 Hz,

I.-Y. Lee et al. / Bioorg. Med. Chem. 21 (2013) 114–126
125
J2 = 2.7 Hz, 1H), 7.85–7.94 (m, 2H), 10.67 (s, 1H), 12.08 (s, 1H).
HRMS (APCI), m/z calcd for C13H7ClF3NO2–H: 300.0026, found
300.0030.
7.02 (dd, J1 = 9.0 Hz, J2 = 4.7 Hz, 1H), 7.28–7.35 (m, 1H), 7.50 (d,
J = 8.8 Hz, 1H), 7.60 (dd, J1 = 8.1 Hz J2 = 7.9 Hz, 1H), 7.70 (dd,
J1 = 9.5 Hz, J2 = 3.2 Hz, 1H), 7.91 (d, J = 8.6 Hz, 1H), 8.22 (s, 1H),
10.63 (s, 1H), 11.38 (s, 1H). HRMS (APCI), m/z calcd for
4.1.3.89.
hydroxy-benzamide (87).
5-Chloro-N-(2,4-dichloro-5-methoxy-phenyl)-2-
Prepared by general procedure A.
C14H9F4NO2–H: 298.0491, found 298.0487.
White solid. Mp >268 °C dec. Yield 43%. 1H NMR (300 MHz,
DMSO-d6): d 3.89 (s, 3H), 7.06 (d, J = 8.7 Hz, 1H), 7.50 (dd,
J1 = 8.7 Hz, J2 = 2.7 Hz, 1H), 7.70 (s, 1H), 7.97 (d, J = 2.7 Hz, 1H),
8.38 (s, 1H), 11.20 (s, 1H), 12.49 (s, 1H). HRMS (APCI), m/z calcd
for C14H10Cl3NO3–H: 343.9648, found 343.9647.
4.2. Biological characterization
Initial minimum inhibitory concentration (MIC) screening was
performed using Mtb H37Rv(pMSP12) in the presence of 30 lg/
mL kanamycin (to maintain plasmid). Later follow-up was per-
formed using the parental wild-type strain Mtb H37Rv
(ATCC27294) since it demonstrated identical MIC values to the ori-
ginal strain. MIC values were determined in 96-well plate format at
37 °C. Mtb was grown to early log-phase (OD650 = 0.2–0.3) and di-
luted 1:1000 in Middlebrook 7H9 supplemented with 0.5% BSA,
0.2% glucose, 0.081% NaCl, 0.05% Tyloxapol (with glucose as the
sole carbon source). Two-fold dilutions were made across 96-well
plates with the final column used as negative control (no com-
pound). DMSO was used at 0.5% (v/v) final concentration and none
of the compounds appeared to be sufficiently insoluble to obscure
final MIC values although solubility may have limited the maxi-
mum value that could be tested in some cases (signified by a great-
er than sign in the data). Isoniazid was used as positive control.
Plates were read at two weeks. MIC was defined as the minimum
compound concentration causing complete cessation of visual
growth. All assays were performed at least two independent times.
Cytotoxicity was determined using the murine macrophage cell
line J774.1 (ATCC TIB-67) grown in DMEM supplemented with 10%
fetal bovine serum (FBS), 0.25 mM pyruvate, and 0.5 times MEM
nonessential amino acids (Mediatech). Cells used had fewer than
4.1.3.90.
hydroxy-benzamide (88).
5-Chloro-N-(2,4-dichloro-5-hydroxy-phenyl)-2-
To a solution of compound 88
(400 mg, 1.15 mmol, 1 equiv) in DCM (100 mL, 0.01 M) was added
a 1 M solution of BBr3 in hexane (4.5 mL, 4 equiv). The suspension
was stirred at room temperature (24 h). Water (10 mL) was added
and stirring continued for another 15 min. DCM was added and the
mixture was washed with 1 N NaOH, and dried and concentrated un-
der vacuum. The residue was purified by column chromatography
(1:5 EtOAc:Hex) to give 87 (260 mg, 67%). White solid. Mp 224–
226 °C. 1H NMR (300 MHz, DMSO-d6): d 7.08 (d, J = 8.7 Hz, 1H),7.52
(dd, J1 = 8.7 Hz, J2 = 2.7 Hz, 1H), 7.58 (s, 1H), 7.98 (d, J = 2.7 Hz, 1H),
8.31 (s, 1H), 10.68 (s, 1H), 10.88 (s, 1H), 12.31 (s, 1H). IR (neat):
3603, 3194, 1637, 1591, 1534, 1224, 826 cmÀ1. HRMS (APCI), m/z
calcd for C13H8Cl3NO3–H: 329.9492, found 329.9492
4.1.3.91.
hydroxy-benzamide (89).
N-(3-Bromo-5-trifluoromethyl-phenyl)-5-fluoro-2-
Prepared by general procedure A.
White solid. Mp 212–214 °C. Yield 40%. 1H NMR (300 MHz,
DMSO-d6): d 7.00–7.05 (m, 1H), 7.30–7.36 (m, 1H), 7.64 (dd,
J1 = 9.4 Hz, J2 = 3.2 Hz, 1H), 7.72 (s, 1H), 8.18 (s, 1H), 8.28 (s, 1H),
10.71 (s, 1H), 11.24 (s, 1H). HRMS (APCI), m/z calcd for
10 passages. Twofold dilutions of compound starting at 60 lM
were added to cells plated (5 Â 103 cells per well) onto 96-well
plates. Plates were incubated at 37 °C for 3 days (5% CO2) and were
read by Alamar Blue (Invitrogen) following the manufacturer’s
instructions.
C14H8BrF4NO2–H: 375.9596, found 375.9590.
4.1.3.92.
hydroxy-benzamide (90).
N-(2-Chloro-5-(trifluoromethyl)phenyl)-5-fluoro-2-
Prepared by general procedure A.
White solid. Mp 176–180 °C. Yield 10%. 1H NMR (300 MHz,
DMSO-d6): d 7.05–7.10 (m, 1H), 7.34–7.39 (m, 1H), 7.54 (d,
J = 8.0 Hz, 1H), 7.75 (dd, J1 = 9.4 Hz, J2 = 3.1 Hz, 1H), 7.83 (d,
J = 6.7 Hz, 1H), 8.90 (s, 1H), 11.22 (s, 1H), 12.16 (s, br, 1H). HRMS
(APCI), m/z calcd for C14H8ClF4NO2–H: 332.0090, found 332.0093.
Acknowledgments
We thank Anton Simeonov, Paul Shinn, Adam Yasgar, Carleen
Klumpp, and Ajit Jadhav for construction and help with screening
and analysis of the NCGC library. We also thank Gwendolyn Marri-
ner for helpful discussions, Noel Whittaker for HRMS data collec-
tion, and Kathryn Soucy for assistance in data collection. This
work was funded (in part) by the Intramural Research Program
of the National Institute of Allergy and Infectious Disease, National
Institutes of Health, the Bill and Melinda Gates Foundation, the
Korea Research Institute of Chemical and Technology, and the Min-
istry of Education, Science and Technology of Korea.
4.1.3.93. N-(3,5-Difluorophenyl)-5-fluoro-2-hydroxybenzamide
(91).
Prepared by general procedure A. White solid. Mp
205–206 °C. Yield 18%. 1H NMR (300 MHz, DMSO-d6): d 6.96–
7.03 (m, 2H), 7.29–7.33 (m, 1H), 7.50 (d, J = 8.7 Hz, 2H), 7.62 (d,
J = 9.3 Hz, 1H), 10.61 (s, 1H), 11.26 (s, br, 1H). HRMS (APCI), m/z
calcd for C13H8F3NO2–H: 266.0429, found 266.0439.
Supplementary data
4.1.3.94.
5-Fluoro-2-hydroxy-N-(4-trifluoromethyl)phenyl)
benzamide (92)28
.
Prepared by general procedure A. White
solid. Mp 227–230 °C [lit28: mp 222–223 °C]. Characterization
(NMR, MS) was in accordance with previously reported values.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.10.056.
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4.1.3.95.
hydroxy-benzamide (93).
5-Fluoro-N-(2-fluoro-5-(trifluoromethyl)phenyl)-2-
Prepared by general procedure A.
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Yield 29%. White solid. Mp 228–230 °C. 1H NMR (300 MHz,
DMSO-d6): d 7.05 (dd, J1 = 8.9 Hz, J2 = 4.5 Hz, 1H), 7.32–7.39 (m,
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