Discovery of Antibacterials That Inhibit Bacterial RNA Polymerase Interactions with Sigma Factors

Article abstract of DOI:10.1021/acs.jmedchem.0c00520

Formation of a bacterial RNA polymerase (RNAP) holoenzyme by a catalytic core RNAP and a sigma (σ) initiation factor is essential for bacterial viability. As the primary binding site for the housekeeping σ factors, the RNAP clamp helix domain represents an attractive target for novel antimicrobial agent discovery. Previously, we designed a pharmacophore model based on the essential amino acids of the clamp helix, such as R278, R281, and I291 (Escherichia coli numbering), and identified hit compounds with antimicrobial activity that interfered with the core-σ interactions. In this work, we rationally designed and synthesized a class of triaryl derivatives of one hit compound and succeeded in drastically improving the antimicrobial activity against Streptococcus pneumoniae, with the minimum inhibitory concentration reduced from 256 to 1 μg/mL. Additional characterization of antimicrobial activity, inhibition of transcription, in vitro pharmacological properties, and cytotoxicity of the optimized compounds demonstrated their potential for further development.

Full text of DOI:10.1021/acs.jmedchem.0c00520

pubs.acs.org/jmc
Article
Discovery of Antibacterials That Inhibit Bacterial RNA Polymerase
Interactions with Sigma Factors
Jiqing Ye, Adrian Jun Chu, Rachel Harper, Shu Ting Chan, Tsun Lam Shek, Yufeng Zhang, Margaret Ip,
Mariya Sambir, Irina Artsimovitch, Zhong Zuo,* Xiao Yang,* and Cong Ma*
Cite This: https://dx.doi.org/10.1021/acs.jmedchem.0c00520
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Formation of a bacterial RNA polymerase (RNAP)
holoenzyme by a catalytic core RNAP and a sigma (σ) initiation
factor is essential for bacterial viability. As the primary binding site
for the housekeeping σ factors, the RNAP clamp helix domain
represents an attractive target for novel antimicrobial agent
discovery. Previously, we designed a pharmacophore model
based on the essential amino acids of the clamp helix, such as
R278, R281, and I291 (Escherichia coli numbering), and identified
hit compounds with antimicrobial activity that interfered with the
core−σ interactions. In this work, we rationally designed and
synthesized a class of triaryl derivatives of one hit compound and succeeded in drastically improving the antimicrobial activity against
Streptococcus pneumoniae, with the minimum inhibitory concentration reduced from 256 to 1 μg/mL. Additional characterization of
antimicrobial activity, inhibition of transcription, in vitro pharmacological properties, and cytotoxicity of the optimized compounds
demonstrated their potential for further development.
holoenzyme formation has been considered as a potential
INTRODUCTION
■
́
target for novel antimicrobial discovery. Andre et al. used an
Infectious diseases caused by bacteria have re-emerged to
become a crisis for human health and the global economy.1
Bacterial strains resistant to all current antibiotics have been
reported.2 Therefore, there is an urgent need for structurally
novel antimicrobial agents with targets and mechanisms
distinct from those of the existing drugs. Structure-based
drug design and synthesis have been adapted to identify novel
antimicrobial agents by exploring novel biological targets,3 and
the synthetic antimicrobial agents have several advantages over
antibiotics developed from natural products: structural
simplicity, amenability to efficient synthetic modifications,
and a lack of innate resistance mechanisms.4
Protein−protein interactions (PPIs) have been increasingly
recognized as valuable targets for drug design.5 Many bacterial
transcription factors directly bind to RNA polymerase (RNAP)
and regulate all steps in RNA synthesis.6 Among these,
initiation sigma factors are essential for transcription. They
associate with the core RNAP (Figure 1A) to form a
holoenzyme (Figure 1B),7,8 the only RNAP state that is able
to specifically recognize a promoter and initiate transcription.9
Among several domains of RNAP that form an extensive
interface with σ,8,10,11 the β′ subunit clamp helix (CH) domain
is thought to play the crucial role.12 The primary interaction
occurs between the β′CH and the region 2.2 of σ (σ2.2) (Figure
1C).13 The housekeeping σ factor (σ70 in Gram-negative
Escherichia coli; σA in Gram-positive Bacillus subtilis) is highly
conserved and essential for bacterial viability.14,15 Thus, the
affinity assay for high-throughput screening to search for hit
compounds capable of inhibiting interactions between E. coli
RNAP and σ70 and identified the SB series of compounds as a
result.16 By aligning the SB series to other compounds with
unknown or diverse binding sites on RNAP, Hinsberger et al.
designed a pharmacophore model and identified anthranilic
acid derivatives that inhibited E. coli holoenzyme formation.17
However, the exact binding sites of SB series and anthranilic
acid derivatives are still unknown.
In order to identify RNAP-σ inhibitors, we developed a
pharmacophore model based on a homology model of B.
subtilis RNAP-σA.19 We first used a house-made bis-indole
compound library and identified hit compounds by native gel
electrophoresis. The inhibitory mechanism was assessed by
isothermal calorimetry titration and in vitro and cell-based
transcription assays;20 however, the compounds’ inadequate
antimicrobial activity and solubility issues limited further
development of these indole derivatives. In pursuit of an
alternative strategy, we analyzed the major binding interface of
Received: April 3, 2020
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
© XXXX American Chemical Society
J. Med. Chem. XXXX, XXX, XXX−XXX
A

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 1. (A) E. coli RNAP core enzyme (subunits ααββ′ω) crystal structure (PDB 4LJZ with σ70 not shown);18 (B) E. coli RNAP σ70 holoenzyme
crystal structure (PDB 4LJZ); (C) amino acid sequence alignments of the β′CH region and σ2.2 of representative bacteria (red arrows indicate
amino acids important for binding);21 (D) key contacts between the E. coli β′CH and σ2.2.
Figure 2. Structures of hit compounds C3, C4, C5, and C3-005.
B. subtilis RNAP-σA holoenzyme, focusing on critical amino
acid residues identified by functional assays. The β′ subunits
with single alanine substitutions of these residues have been
tested. The results showed that R278A or R281A proteins
completely lost the affinity to σA, implicating spatially
proximate R278 and R281 as critical determinants on the σ/
β′ interface;19 other important β′CH residues have also been
identified in this analysis (Figure 1D).
According to the chemical and spatial properties of these
residues, a new pharmacophore model was developed and
employed for in silico screening of Maybridge Mini HitFinder,
a drug-like compound library composed by 2000 diverse
compounds.21 Seven compounds have been selected from top
25 hits by comparing docking models and energy-minimized
conformations. Subsequent functional studies revealed that
some of these seven compounds (C3, C4, and C5) inhibited
RNAP holoenzyme formation and RNA synthesis in vitro
(Figure 2).21 In addition, fluorescence microscopy demon-
strated that the hit compound C5 altered the RNAP
localization in the cell.
we have simply modified the aminobenzene ring of C3 to
dichlorobenzene, demonstrated a dramatic activity improve-
ment against S. pneumoniae from a minimum inhibitory
concentration (MIC) of 256 μg/mL to 8 μg/mL and
significantly attenuated S. pneumoniae toxin production.22
Encouraged by the drug-like structure, synthetic efficiency,
and modification potential of the C3 series of compounds, we
pursued systematic structural optimization. Although C3-005
demonstrated superior antimicrobial activity than C3, we still
preferred to employ the latter as the starting point for a
comprehensive derivative design, synthesis, and antimicrobial
activity assessment. The structure−activity relationship (SAR)
was discussed, and the pharmacological properties such as
hemolytic activity and cell permeability were also evaluated.
RESULTS
■
Derivative Design. The bioactive hit compound C3 2-(4-
((2-aminophenyl)thio)-3-nitrobenzoyl)benzoic acid was la-
beled as compound 1 and used as the lead in this study. It
was fitted into the pharmacophore model with the amino-
phenyl group binding to I291 (E. coli numbering) (Figure 3,
1), nitrobenzoyl group to L282, and benzoic acid to R278 or
R281 of the β′CH region.22 We planned to sequentially modify
Recently, we found that another hit compound, C3,
possessed mild antimicrobial activity against Streptococcus
pneumoniae.22 Interestingly, the derivative C3-005, in which
B
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
compounds 1 and 17 possess the same 2-amino substitution
on the left benzene ring; 17 with the amine linker lost activity
(MIC > 256 μg/mL), whereas 1 with the thioether linker
showed an MIC of 256 μg/mL. Another example is compound
23. With the amine linker, the compound had an MIC of 64
μg/mL, significantly inferior to compound 13 (MIC 8 μg/mL)
with the thioether linker when both compounds possess the
same 3,4-dichloro substituent on the left benzene ring. The
calculated log P (clog P) values of compounds varied from 0.9
(28) to 6.1 (11). Compound 13 with the greatest activity
possesses a relatively high clog P value of 5.9, suggesting a
more hydrophobic character than most of other derivatives,
which may lead to greater membrane permeability. However,
the comparison of 13 to 11 on antimicrobial activity (MIC 8
and 32 μg/mL, respectively) showed that the substitution of
the dichloro group at 3,4-position rather than 2,4-position was
more important, even the latter has a higher clog P value
(Table 1).
Next, we tested the requirement for the nitro group on the
middle benzene ring. Compound 33 was prepared from
bromobenzene. Friedel−Crafts acylation of phthalic anhydride
with bromobenzene reacted at the para-position to generate
33b,26 followed by esterification to give 33c. Palladium-
catalyzed C−S bond formation of 33c with 3,4-dichlorobenze-
nethiol provided 33d,27 which was hydrolyzed to provide
compound 33 (Scheme 2).
The synthesis of compound 34 was accomplished, as shown
in Scheme 3. Intermediate 13c obtained in the previous
experiment was reduced by Fe/NH4Cl to provide 34b, which
was then hydrolyzed to give 34.
Compounds 33−34 were evaluated for their antimicrobial
activities. As compared to nitro 13, the deletion of the nitro
group slightly decreased the antimicrobial activity (33).
Conversion of nitro to amine (34) additionally reduced the
activity, while the clog P values were maintained at 5.2−6.1.
Therefore, we decided to keep the nitro group on the middle
benzene ring unmodified for further derivative synthesis
(Table 2).
Figure 3. Docking model of 1 to β′CH (PDB 4LJZ).18
the left benzene ring of compound 1, bridging atom S, middle
nitro group, carbonyl linker between middle and right benzene
rings, and substitution on the right aromatic group, as these
groups are either involved in binding to the CH domain or
play key roles in molecular conformation. The functional
groups of compound 1 may be optimized by replacement with
isosteres or bioisosteres to better fit in the binding site, and the
prediction could be assessed by the biological activities.
Chemical Synthesis and Antimicrobial Activity
against S. pneumoniae. Scheme 1 depicts the general
procedure for the synthesis of hit compound 1 and its
derivatives 2−32 with modifications on the left benzene ring.
Commercially available 2-(4-chloro-3-nitrobenzoyl) benzoic
acid 1a reacted with methanol in the presence of thionyl
chloride formed methyl benzoate 1b.23 Subsequently, sub-
stitutions of chloride 1b by thiophenol or aniline derivatives,
piperidine, and morpholine generated benzoate 1c−32c.24 The
methyl esters were then hydrolyzed to yield compounds 1−32.
The antimicrobial activities of compounds 1−32 were
evaluated according to the Clinical & Laboratory Standards
Institute (CLSI) guidelines against S. pneumoniae ATCC
49619.25 The hit compound 1 demonstrated mild antimicro-
bial activity with a minimum inhibitory concentration (MIC)
of 256 μg/mL against S. pneumoniae.22
As shown in Table 1, most of modifications of substitution
on the left benzene ring (1−3, 5−15) maintained or improved
the activity against S. pneumoniae. The best result was obtained
in the presence of 3,4-dichlorobenzene (13), with an MIC of 8
μg/mL. The replacement of benzene ring by heteroaromatic
rings did not improve the antimicrobial activity (4, 16). When
the thioether linker was switched to amine (17−28),
methylamine (29−30), piperidine (31), or morpholine (32),
the antimicrobial activity was reduced. For example, both
Compounds 35−37, 41, and 42 were synthesized, as shown
in Scheme 4. In this series of compounds, we intended to
investigate the amide linker replacing the carbonyl group of the
hit compound 1 by maintaining the quasiplanar structure of
the middle and right benzene rings through the conjugated
system. As the linker length was extended to contain one more
N atom, we synthesized the derivatives bearing benzoic acid at
a
Scheme 1. Synthetic Route to Compounds 1−32
a
Reagents and conditions: (a) SOCl2, MeOH, reflux; (b) for 1c−16c: thiophenols, NaOAc, EtOH, reflux; for 17c−31c: amines, N,N-
diisopropylethylamine (DIPEA), DMF, 130 °C or Pd(OAc)2, xantphos, THF, 100 °C; (c) (i) THF/H2O (v/v = 2:1), NaOH, rt; (ii) 2 N HCl.
C
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
b
Table 1. Antimicrobial Activity and cLog P of Compounds 1−32
a
b
Antimicrobial activity against S. pneumoniae ATCC 29619. 1, 5, 6, 10, and 13 were previously reported.22
D
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 2. Synthetic Route to Compound 33
a
Reagents and conditions: (a) phthalic anhydride, AlCl3, toluene, reflux; (b) SOCl2, MeOH, reflux; (c) 3,4-dichlorobenzenethiol, Pd2(dba)3,
xantphos, DIPEA, dioxane, reflux; (d) (i) THF/H2O (v/v = 2:1), NaOH, rt; (ii) 2 N HCl.
a
Scheme 3. Synthetic Route to Compound 34
a
Reagents and conditions: (a) Fe, NH4Cl, EtOH/H2O (v/v = 4:1), reflux; (b) (i) THF/H2O (v:v = 2:1), NaOH, rt; (ii) 2 N HCl.
a
whether the amide linker can be accommodated by changing
the carboxylic acid position on the right benzene ring. 4-
Fluoro-3-nitrobenzoic acid 3a was converted to benzoyl
chloride 3b, followed by amide synthesis to provide 35c−
37c. Substitution of aryl fluoride and hydrolysis of methyl ester
group on the right benzene ring afforded the target compounds
35−37, 41, and 42.
Derivatives with an amine linker were also synthesized to
examine whether a flexible conformation is preferred.
Compounds 38−40, 43−44, 46−51, and 53−57 were
synthesized from 4-fluorobenzaldehyde 3a. Nitration of 3a
furnished 3b, which was subjected to reductive amination with
aniline derivatives to give 38c−40c, 46c−51c, and 53c−
57c.28,29 Using various substituted thiophenols, substitution of
Table 2. Antimicrobial Activity and cLog P of Compounds
33−34
no
R2
MIC (μg/mL)
clog P
6.09
5.23
33
34
−H
−NH2
16
32
a
Antimicrobial activity against S. pneumoniae ATCC 29619.
the ortho-(2′-), meta-(3′-), or para-(4′-) position of amide to
study the SAR by adapting the extension strategy, and assess
a
Scheme 4. Synthetic Route to Compounds 35−37, 41, and 42
a
Reagents and conditions: (a) oxalyl chloride, DMF, dichloromethane (DCM), rt; (b) anilines, tetraethylammonium, DCM, 0 °C; (c) thiophenols,
NaOAc, EtOH, reflux; (d) (i) THF/H2O (v/v = 2:1), NaOH, rt; (ii) 2 N HCl.
E
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 5. Synthetic Route to Compounds 38−40, 43−44, 46−51, and 53−57
a
Reagents and conditions: (a) HNO3, H2SO4, 0 °C; (b) anilines, NaBH4, EtOH/NaBH(OAc)3, DCM, rt; (c) thiophenols, NaOAc, EtOH, reflux;
(d) (i) THF/H2O (v/v = 2:1), NaOH, rt, or dioxane/H2O (v/v = 2:1), NaOH, 50 °C; (ii) 2 N HCl.
fluoride afforded 38d−40d, 43d−44d, 46d−50d, 51, 53, 54d,
and 55−57. After hydrolysis of methyl esters, compounds 38−
40, 43−44, 46−50, and 54 were obtained (Scheme 5).
Compounds 45 and 52 were prepared through the following
steps: 4b underwent the substitution with 3, 4-dichlorobenze-
nethiol to generate intermediate 45c, followed by reduction of
aldehyde to give alcohol 45d. The substitution of hydroxyl
using PBr3 provided bromide 45e, followed by substitution of
bromide by a substituted aniline to furnish 45f. The hydrolysis
of the methyl ester group provided the final product 45.
Starting from the same intermediate 45c, reductive amination
afforded sulfonamide 52 (Scheme 6).
As shown in Table 3, when the carbonyl linker of compound
1 was replaced by amide, the antimicrobial activity increased
fourfold (35). The benzoic acid substitution moved from
ortho- to meta- or para-position of amide reduced the activity
(36, 37). Replacement of the amide linker with flexible
methylamine did not alter the activity (38−40). Interestingly,
the 3,4-dichlorobenzene ring proved to be superior to 2-
aminobenzene for antimicrobial activity when the carbonyl
linker was present. In contrast, the derivative with both 3,4-
dichlorobenzene ring and amide linker lost activity completely
(41), and changing the substitution position of benzoic acid
barely improved the activity (42). The best antimicrobial
activity from this series of compounds was obtained when 3,4-
dichlorobenzene was combined with the methylamine linker,
resulting in an MIC of 4 μg/mL (43). Again, meta-amino
benzoic acid was not preferred (44). In summary, the
appropriate structure for binding to β′CH can be concluded
when comparing compounds 41−44. These four compounds
have close clog P values ranging from 6.4 to 7.3, but the
antimicrobial activity showed a much significant difference
(Table 3). 3,4-Dichloro group substituted on the left benzene
ring demonstrated to be a great combination with the
methylamine linker, compared to amide, while the two linkers
did not show much difference when 2-amino group was
present on the left benzene ring of the molecule.
F
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 6. Synthetic Route to Compounds 45 and 52
a
Reagents and conditions: (a) thiophenol, NaOAc, EtOH, reflux; (b) NaBH4, MeOH, 0 °C to rt; (c) PBr3, toluene, 100 °C, 1 h; (d) methyl 3-
amino-4-chlorobenzoate, DIPEA, DMF, 100 °C; (e) dioxane/H2O (v/v = 2:1), NaOH, 50 °C; (f) 3-amino benzenesulfonamide, NaBH(OAc)3,
CH2Cl2, rt, overnight.
Based on these data, we maintained both 3,4-dichloroben-
zene and methylamine linker for further structure modification
(Table 4). As benzoic acid is predicted to interact with β′
R278 or R281 (Figure 3), we reasoned that increasing the
acidity of benzoic acid by introducing electron-withdrawing
groups would improve the protein−ligand interaction. Indeed,
compared to compound 44, electron-withdrawing halides (45,
46) at para-position of benzoic acid increased the activity,
whereas electron-donating methyl (48) decreased the activity.
Compound 47 with p-fluoro substitution lost activity
completely. This anomalous phenomenon needs to be
investigated further and could be due to an impaired delivery
to the target. Changing benzoic acid to amide (51, 56),
sulfonamide (52), alcohol (53), or nitrile (57) at 2- or 3-
position of the amine linker led to a loss of the antimicrobial
activity. The best result was obtained when 5-Cl-2-benzoic acid
is present (54), with MIC of 1 μg/mL comparable to the “last
resort” antibiotic vancomycin (Table S1). Note that the
activity of 54 increased fourfold compared to 43, which may
also be partially contributed by increased bacterial membrane
permeability of 54 with a clog P value of 8.2. However, 45 and
46 with halide substitution at para-position of benzoic acid
only exhibited twofold improvement on antimicrobial activity
of 44.
In-Depth Biological Investigation. Antimicrobial Activ-
ity against Gram-Positive Pathogens. Representative com-
pounds 13, 44, 45, 54, and 55 were tested against a
representative panel of Gram-positive pathogens (Figure 4).
Compared with the reported compound 13,22 54 showed
greatly improved antimicrobial activity against S. pneumoniae,
as well as both groups A and B streptococci: Streptococcus
pyogenes (group A streptococcus, GAS) responsible for serious
throat and skin infections30 and Streptococcus agalactiae (group
B streptococcus, GBS) responsible for severe conditions in
neonates.31 Although a panel of Gram-negative pathogens was
also tested (Table S1), most of the compounds were ineffective
against Gram-negative pathogens (MIC > 256 μg/mL),
probably because of poor outer membrane permeability and/
or vigorous drug efflux.
Time-Kill Kinetics. The dose- and time-dependent relation-
ship between antimicrobial activity and bacterial growth can be
assessed by the time-kill assay. 54 was added to the liquid
culture of S. aureus and S. agalactiae at various concentrations,
and its antimicrobial profiles in both pathogens were assessed
by constructing the time-kill curves. S. aureus and S. agalactiae
(GBS) were cultured in liquid media with agitation in
accordance with the CLSI guidelines.32 54 was largely
bacteriostatic over the course of 6 h against S. aureus and
GBS even at 16 MIC with no significant decrease in CFU
counts (Figure 5A,B).
Time-kill curves were also constructed by adding 54 to S.
pneumoniae at various concentrations and measuring the in
vitro antimicrobial activity over time. S. pneumoniae cultures
were raised in liquid media with agitation in the presence of
5% CO2.33 54 was largely bacteriostatic at 1 MIC, while
increasing to 4 and 16 MICs yielded no further bactericidal
effects over time after an initial sharp drop in the cell count
(Figure 5C). Compared to trends in S. aureus and GBS, 54
appears to be able to elicit stronger bacteriostatic activity (ca.
2-fold log10 decrease) at higher concentrations in S. pneumo-
niae.
Central Metabolism. One of the characteristics of an
effective antibiotic is the arrest of bacterial respiration.34 The
ATP production of S. pneumoniae cells in the presence of 54 at
various concentrations was monitored alongside the time-kill
kinetics using the same experimental setup. A significant
decrease in S. pneumoniae ATP production rate was observed
at 1/4 MIC of 54, and increasing concentrations of the
compound further impacted cellular respiration (Figure 5D).
This mirrored previously reported trends of the transcription
inhibitor rifampicin where antimicrobial efficacy is inversely
correlated to bacterial respiration rates.33,35
Toxin Secretion. A hallmark of S. pneumoniae virulence is
the release of its exotoxin, pneumolysin, into the extracellular
milieu. Nonlytic antibiotics, such as rifampicin and macrolides,
G
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Table 3. Antimicrobial Activity and cLog P of Compounds 35−44
a
Antimicrobial activity against S. pneumoniae ATCC 29619.
have been shown to repress pneumolysin release at sub-MIC
levels.36−38 In this study, we explored the effects of 54 on
pneumolysin secretion. Overnight cultures of S. pneumoniae in
the brain−heart infusion (BHI) medium were exposed to 1/2
and 1/4 of the predetermined MICs of the test compound 54,
the nonlytic transcription inhibitor rifampicin, the bacteriolytic
drug ceftriaxone, and drug-free control. Western blot analysis
was performed using the supernatant harvested from the
overnight cultures following centrifugation.
bacteriostatic mechanism with the suppression of the exotoxin
release.
Cytotoxicity. The representative derivatives 13, 43, 45, 46,
54, and 55 with improved antimicrobial activity were subjected
to cytotoxic evaluations.40 Human HepG2 liver cancer and
A549 lung carcinoma cell lines were chosen to examine the
potential toxicity to liver cells, where drugs are normally
metabolized, and to lung cells, the primary site of S.
pneumoniae infection. The results demonstrated that all tested
compounds possess very low toxicity to these two human
cancer cell lines with 50% cytotoxic concentration (CC50)
values >70 μM (Table 5). With the improvement of
antimicrobial activity, the derivatives expressed increased the
therapeutic index value up to 57 (43 to A549), suggesting that
The control drugs performed as previously described in the
literature with ceftriaxone treatment increased, while rifampi-
cin suppressed the release of pneumolysin (Figure 6).39
A
substantial decrease in pneumolysin levels was observed in 54-
treated cultures compared to the drug-free control (Figure 6).
This suggests that 54 inhibits S. pneumoniae growth in a
H
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Study of the Mechanism of Action. β′CH−σA PPI
Inhibition. With the antimicrobial activity, cytotoxicity, and in
vitro pharmacological data in hand, we intended to assess the
molecular mechanism of this series of diaryl benzoic acid
derivatives. Several representative compounds with various
antimicrobial activities were tested for inhibition of the
interaction between β′CH−σ using our previously established
protein complementation assay (Figure S1).43 As shown in
Table 7, all of the tested compounds were able to disrupt
CH−σ interactions, in contrast to no-drug control.21,43 These
results demonstrate that structural modifications maintained
the inhibitory activity against CH−σ binding in vitro, and the
inhibitory activity approximately matched the trend of
antimicrobial activity of compounds. Compound 54 was not
able to be tested in this experiment because of insolubility
under the testing conditions, which can be reflected by its high
clog P value. Nevertheless, the inhibitory activity of 54 was
examined in the following transcription assay because of the
relatively low concentrations used.
In Vitro Transcription Initiation Assay. We next tested
compounds 13 and 54 for their ability to inhibit RNAP−σ
interactions using an in vitro transcription assay. We
preincubated the E. coli core RNAP with various concen-
trations of the compounds [or dimethyl sulfoxide (DMSO)]
and then added the σ70 subunit together with a DNA template
containing a model bacteriophage λ PR promoter and
nucleotide substrates to produce a 26-mer radiolabeled RNA
(Figure 8). We observed that although both compounds
inhibited transcription, 54 exhibited much more potent
inhibition. These results support the proposed mechanism of
action of these derivatives via the inhibition of the CH−σ
interactions. They also show that, at least for these two
representative derivatives, structural modifications leading to
stronger inhibitory effects on transcription in a purified in vitro
system also support superior antibacterial activity (Figure 4).
Confocal Microscopy. As previously reported,21 we
examined the effects of the benzoic acid compounds on
cellular RNAP localization using fluorescence microscopy. In
B. subtilis BS1048 strain, the rpoC gene is fused to gfp to
produce the green fluorescent protein (GFP)-tagged β′ subunit
of RNAP from the chromosomal locus. The fluorescently
labeled RNAP localizes to the centrally localized nucleoid, as
expected (Figure 9 Ctrl).21 The antimicrobial activity of
compound 54 against B. subtilis was measured with MIC 4 μg/
mL prior to the microscopic assay. When 54 was added at 1/2
MIC, delocalization of the fluorescence from the nucleoid
could be observed as compared to the control cells (Figure 9
1/2 MIC), suggesting that chromosomes became decon-
densed. As the concentration of 54 increased, diffusion of the
fluorescent signal into the cytosolic region became more
apparent (Figure 9 1 MIC and 2 MIC). These results suggest
that 54 inhibits transcription initiation in the cell, triggering
the diffusion of core RNAP, which cannot bind to promoters
and engage in transcription from the nucleoid.
Table 4. Antimicrobial Activity and cLog P of Compounds
45−57
no
R4
R5
MIC (μg/mL)
clog P
45
46
47
48
49
50
51
52
53
54
55
56
57
6-Cl
6-Br
6-F
6-CH3
4-Cl
6-morpholin-1-yl
H
H
H
3-COOH
3-COOH
3-COOH
3-COOH
3-COOH
3-COOH
3-CONH2
3-SO2NH2
3-CH2OH
2-COOH
2-COOH
2-CONH2
2-CN
4
4
7.56
7.71
6.99
7.21
6.96
6.23
5.48
5.16
5.58
8.19
7.59
5.88
6.76
>256
16
256
16
>256
64
>256
1
5-Cl
3-Cl
H
8
>256
>256
H
a
Antimicrobial activity against S. pneumoniae ATCC 29619.
Figure 4. Antimicrobial activity (MIC μg/mL) of selected
compounds against clinically important Gram-positive pathogens.
EFAE: Enterococcus faecalis ATCC 19433, SAURa: Staphylococcus
aureus ATCC 25923, SAURb: S. aureus ATCC 29213, SPNE: S.
pneumoniae ATCC 49619, SEPI: Streptococcus epidermidis ATCC
12228, SSAP: Streptococcus saprophyticus ATCC 15305, SPYO: S.
pyogenes ATCC 19615, SAGA: S. agalactiae ATCC 12386, VAN:
vancomycin, RIF: rifampicin.
the compounds are generally safe when assessed at the cellular
level.
In Vitro Pharmacological Evaluation. Hemolytic
Property. Three compounds 13, 43, and 45 were chosen to
assess their hemolytic property. As an important pharmaco-
logical parameter, the hemolytic property determines whether
compounds can be used in animal studies without the
destruction of blood cells.41,42 As shown in Figure 7, all
three compounds demonstrated low hemolytic potential at all
tested concentrations (0.1, 1, and 10 μg/mL). The hemolytic
values of these compounds were lower than the suggested
nonhemolytic cutoff value (<10%), suggesting very low toxic
potential to human blood cells.
Caco-2 Cell Permeability. The human intestinal epithelial
cancer cell line Caco-2 was used to determine the permeability
of compounds 13 and 43.41,42 As shown in Table 6,
compounds 13 and 43 displayed excellent human intestinal
epithelial cell permeability, which is superior to 1 × 10−5 cm/s
as the standard of great intestinal epithelial cell permeability.
The result indicated that the potential of oral availability could
be expected because of excellent gut absorption.
Cell Content Quantification of the Major Macromole-
cules. The levels of the major macromolecules in S. aureus
ATCC 29213 cells were analyzed after the treatment of 54, in
comparison with a known transcription inhibitor rifampicin. 54
and rifampicin were added to S. aureus cells in liquid culture at
1/4, and 1/8 MIC levels at the late lagging phase (OD600
=
0.2), and cells were harvested during the mid-exponential
growth (OD600 = 0.6). As shown in Figure 10A, the total levels
of DNA remained mostly unaffected by the treatments
I
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 5. Effects of 54 on the time-kill kinetics of (A) S. aureus and (B) S. agalactiae. The effects of 54 on (C) the time-kill kinetics and (D) ATP
production of S. pneumoniae when challenged at 1/4, 1, 4, and 16 MIC.
Figure 6. Effect of 54 on the expression of S. pneumoniae exotoxin. (A) Representative Western blot showing levels of pneumolysin in the culture
supernatant following overnight incubation of S. pneumoniae at 1/2 and 1/4 MICs of 54, rifampicin (Rif), and ceftriaxone (Cef) and (B) relative
intensity of the bands at 1/2 (checker bars) and 1/4 (light gray bars) MICs of the treatments normalized against the untreated control group (solid
black bar).
Table 5. Cytotoxicity of Representative Compounds
Table 6. Caco-2 Cell Permeability of Compounds 13 and 43
CC50 (μM)
therapeutic index
no.
Papp average (n = 4) (cm/s)
13
43
3.50 0.62 × 10−5
3.62 0.60 × 10−5
no.
HepG2
A549
HepG2
A549
13
43
45
46
54
55
63.8 9.3
76.7 11.4
252.2 13.3
116.3 10.8
67.7 11.3
75.7 12.5
93.3 13.0
7
38
27
24
36
12
9
57
28
18
37
11
170.1 15.4
110.3 12.4
92.6 12.6
74.4 12.5
97.7 13.1
Table 7. Inhibition of CH−σ Interactions by Representative
Compounds
no. MIC (μg/mL) IC50 (μM)
no. MIC (μg/mL) IC50 (μM)
33
37
43
16
256
4
67.0 4.7
97.0 9.6
11.6 0.6
44
45
55
8
4
8
42.9 2.5
15.0 0.7
25.1 1.5
compared to the no-drug control. Total RNA levels were
reduced by rifampicin, which was consistent with its known
mechanism and previous observations.40 Treatment with 54
also resulted in a reduction in the total level of RNA in the
staphylococcal cells (Figure 10B). Together with our
observations that 54 inhibits transcription in vitro (Figure 8)
and releases RNAP from the nucleoid (Figure 9), this result
argues that 54 inhibits transcription in target bacterial cells.
Figure 7. In vitro hemolysis study of compounds 13, 43, and 45.
J
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 8. In vitro assay of RNA synthesis inhibition by compounds 13 and 54, as compared to the DMSO control (0 sample in the middle). IC50
values were determined from three independent experiments. A representative gel is shown.
In this article, we examined a series of triaryl derivatives of a
previously identified hit compound 1, which inhibits the
formation of the RNAP holoenzyme.21 These derivatives were
obtained through pharmacophore-based rational design and
synthesis. We then carried out a systematic evaluation of the
compound series and found that a general diaryl benzoic acid
Figure 9. Confocal microscopy of B. subtilis cells monitoring RNAP-
GFP fluorescence. 54 was added to the culture at various MIC levels.
structure is required for the antimicrobial activity. This
structure also possesses desirable drug-like properties, as
shown by cytotoxicity and in vitro pharmacological assays.
Finally, we reported an improved, more potent derivative 54 of
this class of diaryl benzoic acid antimicrobials with enhanced
antimicrobial effects against S. pneumoniae with the MIC
improved to 1 μg/mL and other Gram-positive pathogens at
1−2 μg/mL, comparable to vancomycin (Table S1).
SAR Summary. Based on the antimicrobial activity against
S. pneumoniae, several important points could be drawn from
the SAR of this series of triaryl compounds (Figure 11): 3,4-
dichlorobenzene is preferred over other substituted benzene or
pyridine as the left aryl ring, probably because of increased
binding to β′CH L282 and I291 (Figure 3);18 the compounds
with left thioether linker have superior activity to amine; the
nitro group on the middle aryl ring can be deleted with slightly
reduced activity because the middle benzene ring maintains the
affinity with L282; the right methylamine linker is favored over
carbonyl or amide when the 3,4-dichloro group substituted on
the left benzene ring; 2-benzoic acid as the right aryl ring with
Reduction in total protein levels in S. aureus ATCC 29213 was
also observed following the treatment with 54 (Figure 10C), as
would be expected as a consequence of the reduced RNA
synthesis.
DISCUSSION
■
The inhibition of transcription is lethal in all cells. Bacterial
transcription is an established antimicrobial target, with
rifampicin and fidaxomicin as approved antibiotics in the
market. Other inhibitors of transcription, most of which target
RNAP, have been used extensively in mechanistic studies of
the transcription cycle.6,44 Importantly, even though human
RNAPs are structurally similar to their bacterial homologues,
sequence similarity is limited to a few key regions and
inhibitors of bacterial and human RNAP do not exhibit cross-
domain effects. Therefore, bacterial transcription represents a
valid potential target for antibiotic discovery.3
Figure 10. Effects of 54 on the levels of total (A) DNA, (B) RNA, and (C) protein of S. aureus ATCC 29213 when challenged at 1/4 × (checkered
bars) and 1/8 × (light gray bars) MICs compared to rifampicin (Rif) and the no-drug control (solid black bars).
K
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
EXPERIMENTAL SECTION
■
General. Starting materials and regents, unless otherwise stated,
were of commercial grade and were used without further purification.
All reactions were monitored by thin-layer chromatography (TLC) on
glass sheets (Silica gel F254) which can be visualized under UV light.
Flash chromatography was carried out using silica gel (200−300
mesh). 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were
measured on a Bruker AVANCE-III spectrometer with TMS as an
internal standard. Chemical shifts are expressed in δ (ppm) and
coupling constants (J) in Hz. High-resolution mass spectrometry
(MS) spectra were measured using a QTOF-2 micromass
spectrometer by electron spray ionization. The purity of all of the
compounds tested for biological activities was >95%, determined by
analytical HPLC (Agilent 1260 Infinity) on a reverse-phase column
(Agilent ZORBAX Eclipse plus C18, 4.6 × 100 mm, 5 μm particle
size) with acetonitrile/water as the eluent.45 Compounds reported
were screened for PAINS.
Figure 11. SAR of tested compounds 1−57
Synthetic Procedures for Scheme 1. General Procedure for
the Synthesis of Compounds 1c−16c. To a flask were added
compound 1b (64 mg, 0.2 mmol), benzenethiol (1.2 equiv, 0.24
mmol), NaOAc (82 mg, 1 mmol), and EtOH 5 mL. The mixture was
stirred at reflux for 4 h. After cooling to room temperature, the
precipitate was filtered, washed with EtOH and water successively,
and dried to give the titled compounds. Alternatively, water was
added, and the aqueous solution was extracted by EtOAc. The
combined organic layers were dried over anhydrous Na2SO4,
concentrated, and purified by chromatography to provide the titled
compounds.
Methyl 2-(4-chloro-3-nitrobenzoyl)benzoate (1b), methyl 2-(4-
((2-aminophenyl)thio)-3-nitrobenzoyl)benzoate (1c), methyl 2-(4-
((3-aminophenyl)thio)-3-nitrobenzoyl)benzoate (5c), methyl 2-(4-
((4-aminophenyl)thio)-3-nitrobenzoyl)benzoate (6c), methyl 2-(4-
((2-amino-4-chlorophenyl)thio)-3-nitrobenzoyl)benzoate (10c), and
methyl 2-(4-((3,4-dichlorophenyl)thio)-3-nitrobenzoyl)benzoate
(13c) were synthesized following the previously published proce-
dures.22
an electron-withdrawing group at the para-position exhibits the
greatest activity, possibly because of the improved ionic
binding interaction with R278 and R281.
The mechanism of 54 was characterized using various
methods including in vitro transcription assay, fluorescent
microscopy, and macromolecular content quantification. We
show that 54 suppresses bacterial transcription by inhibiting
the formation of bacterial RNAP holoenzyme. We also
demonstrate that 54 is largely bacteriostatic but can arrest
cellular ATP production in S. pneumoniae similar to the
transcription inhibitor rifampicin. 54 was also able to decrease
the level of secreted pneumococcal virulence factor pneumo-
lysin. The suppression is very likely to happen at the
transcription level, and the effect of 54 on the transcriptome
of S. pneumoniae cells is currently under investigation.
Although we demonstrated that the class of diaryl benzoic
acid antimicrobials could inhibit bacterial RNAP holoenzyme
formation using both in vitro and cellular assays, the possibility
of other cellular targets should not be neglected, and any off-
target effects of the compounds need to be investigated in
further studies. Detailed structural information of the diaryl
benzoic acid antimicrobials in complex with their potential
target protein will reveal critical contacts at the interface,
information necessary for rational optimization of the lead. We
are currently pursuing structural studies of the β′CH fragment
bound to representative diaryl benzoic acid compounds.
Finally, the demonstrated in vitro inhibitory properties of
these compounds need to be assessed in vivo. Analysis of the
efficacy of these compounds in treating Gram-positive
infections in small animals is currently underway in our
laboratory.
Methyl 2-(4-((2-Methoxyphenyl)thio)-3-nitrobenzoyl)benzoate
(2c). Pale yellow solid, 70 mg, 71% yield. 1H NMR (400 MHz,
CDCl3): δ 8.52 (d, J = 1.7 Hz, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.76
(dd, J = 8.5, 1.8 Hz, 1H), 7.68 (td, J = 7.5 Hz, 0.8 Hz, 1H), 7.61 (dd, J
= 12.3, 4.7 Hz, 2H), 7.56 (td, J = 7.9 Hz, 1.2 Hz, 1H), 7.38 (d, J = 7.2
Hz, 1H), 7.09 (t, J = 7.6 Hz, 1H), 7.06 (d, J = 8.3 Hz, 1H), 6.89 (d, J
= 8.6 Hz, 1H), 3.82 (s, 3H), 3.73 (s, 3H).
Methyl 2-(4-((2-Fluorophenyl)thio)-3-nitrobenzoyl)benzoate
1
(3c). Yellow solid, 70 mg, 84% yield. H NMR (400 MHz, CDCl3):
δ 8.56 (d, J = 1.6 Hz, 1H), 8.10 (d, J = 7.5 Hz, 1H), 7.79 (dd, J = 8.6,
1.7 Hz, 1H), 7.75−7.55 (m, 4H), 7.38 (d, J = 7.3 Hz, 1H), 7.33 (d, J
= 7.4 Hz, 1H), 7.27 (m, 1H), 6.92 (d, J = 8.3 Hz, 1H), 3.74 (s, 3H).
Methyl 2-(3-Nitro-4-(pyridin-2-ylthio)benzoyl)benzoate (4c). Pale
yellow solid, 70 mg, 89% yield. 1H NMR (400 MHz, CDCl3): δ 8.67
(d, J = 4.1 Hz, 1H), 8.46 (d, J = 0.8 Hz, 1H), 8.09 (d, J = 7.7 Hz, 1H),
7.79 (t, J = 7.7 Hz, 2H), 7.68 (t, J = 7.4 Hz, 1H), 7.62 (m, 2H), 7.43−
7.33 (m, 2H), 7.24 (d, J = 8.5 Hz, 1H), 3.73 (s, 3H).
Methyl 2-(4-((3-(Isopropylamino)phenyl)thio)-3-nitrobenzoyl)-
benzoate (7c). Purified by chromatography using hexane/EtOAc
(10:1). Yellow solid, 65 mg, 72% yield. 1H NMR (400 MHz, CDCl3):
δ 8.51 (d, J = 1.5 Hz, 1H), 8.10 (d, J = 7.8 Hz, 1H), 7.77 (dd, J = 8.6,
1.7 Hz, 1H), 7.68 (t, J = 7.4 Hz, 1H), 7.61 (t, J = 7.2 Hz, 1H), 7.36
(d, J = 7.3 Hz, 1H), 7.26 (t, J = 7.9 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1H),
6.86 (d, J = 7.5 Hz, 1H), 6.77 (s, 1H), 6.70 (dd, J = 8.2, 1.7 Hz, 1H),
3.74 (s, 3H), 3.69 (s, 1H), 3.64 (d, J = 6.2 Hz, 1H), 1.24 (d, J = 6.0
Hz, 6H).
CONCLUSIONS
■
In summary, these diaryl benzoic acid antimicrobials reported
in this study possess good antimicrobial activities, low
cytotoxicity, and drug-like in vitro pharmacological properties.
Therefore, they are promising candidates for further develop-
ment as novel antimicrobial agents. Our approach continues to
pave the way for a solid platform of novel antimicrobial
discovery, with a focus on bacterial transcription as a drug
target. In addition to their potential future therapeutic value,
this series of compounds could be developed as chemical
probes for studies of σ factor-mediated transcription
regulation.
Methyl 2-(4-((3-Acetamidophenyl)thio)-3-nitrobenzoyl)benzoate
1
(8c). Yellow solid, 45 mg, 50% yield. H NMR (400 MHz, DMSO-
d6): δ 10.20 (s, 1H), 8.36 (s, 1H), 8.04 (d, J = 7.7 Hz, 1H), 7.95 (s,
1H), 7.79 (m, 2H), 7.72 (m, 2H), 7.51 (t, J = 7.1 Hz, 2H), 7.32 (d, J
= 7.6 Hz, 1H), 7.01 (d, J = 8.6 Hz, 1H), 3.65 (s, 3H), 2.06 (s, 3H).
Methyl 2-(4-((3-Benzamidophenyl)thio)-3-nitrobenzoyl)-
benzoate (9c). Yellow solid, 57 mg, 56% yield. 1H NMR (400
MHz, CDCl3): δ 8.53 (s, 1H), 8.10 (d, J = 7.7 Hz, 1H), 7.95 (s, 1H),
L
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
7.89 (d, J = 7.9 Hz, 4H), 7.78 (d, J = 8.6 Hz, 1H), 7.68 (t, J = 7.3 Hz,
1H), 7.61 (m, 2H), 7.57−7.48 (m, 3H), 7.38 (m, 2H), 7.02 (d, J = 8.6
Hz, 1H), 3.74 (s, 3H).
J = 7.7 Hz, 1H), 7.95 (dd, J = 9.1, 1.7 Hz, 1H), 7.68 (t, J = 7.2, 1H),
7.61 (t, J = 8.2 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.38 (d, J = 7.4 Hz,
1H), 7.36−7.31 (m, 2H), 7.24 (m, 2H), 4.77 (d, J = 5.4 Hz, 2H), 3.77
(s, 3H).
Methyl 2-(4-((3-Chlorophenyl)amino)-3-nitrobenzoyl)benzoate
(20c). Method B, purified by chromatography using hexane/EtOAc
(10:1), yellow solid, 77 mg, 63% yield. 1H NMR (400 MHz, CDCl3):
δ 9.80 (s, 1H), 8.48 (d, J = 1.9 Hz, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.96
(dd, J = 9.0, 1.8 Hz, 1H), 7.67 (t, J = 7.4 Hz, 1H), 7.59 (t, J = 7.7 Hz,
1H), 7.42−7.35 (m, 2H), 7.33−7.27 (m, 2H), 7.21 (t, J = 9.6 Hz,
2H), 3.76 (s, 3H).
Methyl 2-(4-((3-(tert-Butyl)phenyl)amino)-3-nitrobenzoyl)-
benzoate (21c). Method A, purified by chromatography using
hexane/EtOAc (10:1), orange solid, 74 mg, 86% yield. 1H NMR
(400 MHz, CDCl3): δ 9.92 (s, 1H), 8.47 (d, J = 1.9 Hz, 1H), 8.11 (d,
J = 7.3 Hz, 1H), 7.96 (dd, J = 9.1, 1.7 Hz, 1H), 7.68 (td, J = 7.5, 1.0
Hz, 1H), 7.61 (td, J = 7.7, 1.1 Hz, 1H), 7.44−7.35 (m, 3H), 7.31 (s,
1H), 7.21 (d, J = 9.1 Hz, 1H), 7.14 (d, J = 7.4 Hz, 1H), 3.78 (s, 3H),
1.37 (s, 9H).
Methyl 2-(4-((3-Carbamoylphenyl)amino)-3-nitrobenzoyl)-
benzoate (22c). Method A, purified by chromatography using
hexane/EtOAc (8:1), orange solid, 80 mg, 95% yield. 1H NMR
(400 MHz, CDCl3): δ 9.90 (s, 1H), 8.52 (d, J = 1.9 Hz, 1H), 8.12 (d,
J = 7.7 Hz, 1H), 7.96 (dd, J = 9.0, 1.8 Hz, 1H), 7.83 (s, 1H), 7.72 (d, J
= 7.4 Hz, 1H), 7.68 (dd, J = 7.4, 1.0 Hz, 1H), 7.63 (dd, J = 7.7, 1.1
Hz, 1H), 7.61−7.55 (m, 1H), 7.50 (d, J = 8.6 Hz, 1H), 7.39 (dd, J =
7.7, 0.7 Hz, 1H), 7.23 (d, J = 9.0 Hz, 1H), 3.78 (s, 3H).
Methyl 2-(4-((3,4-Dichlorophenyl)amino)-3-nitrobenzoyl)-
benzoate (23c). Method B, purified by chromatography using
hexane/EtOAc (10:1), yellow solid, 50 mg, 37% yield. 1H NMR
(400 MHz, CDCl3): δ 9.77 (s, 1H), 8.50 (d, J = 1.9 Hz, 1H), 8.14−
8.10 (m, 1H), 8.02−7.97 (m, 1H), 7.70 (t, J = 7.2 Hz, 1H), 7.62 (t, J
= 7.3 Hz, 1H), 7.55 (d, J = 8.6 Hz, 1H), 7.46 (d, J = 2.4 Hz, 1H), 7.38
(d, J = 7.4 Hz, 1H), 7.22 (d, J = 9.0 Hz, 1H), 7.19 (dd, J = 8.6, 2.4 Hz,
1H), 3.78 (s, 3H).
Methyl 2-(4-((4-Methoxyphenyl)amino)-3-nitrobenzoyl)-
benzoate (24c). Method A, purified by chromatography using
hexane/EtOAc (8:1), orange solid, 61 mg, 75% yield. 1H NMR
(400 MHz, CDCl3): δ 9.79 (s, 1H), 8.45 (s, 1H), 8.11 (d, J = 7.7 Hz,
1H), 7.93 (d, J = 9.1 Hz, 1H), 7.68 (t, J = 7.5 Hz, 1H), 7.60 (t, J = 7.5
Hz, 1H), 7.38 (d, J = 7.3 Hz, 1H), 7.23 (d, J = 7.9 Hz, 2H), 7.03 (m,
3H), 3.88 (s, 3H), 3.77 (s, 3H).
Methyl 2-(3-Nitro-4-(phenylamino)benzoyl)benzoate (25c).
Method A, purified by chromatography using hexane/EtOAc (8:1),
orange solid, 70 mg, 93% yield. 1H NMR (400 MHz, CDCl3): δ 9.90
(s, 1H), 8.48 (d, J = 1.9 Hz, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.95 (dd, J
= 9.1, 1.8 Hz, 1H), 7.68 (td, J = 7.3, 0.7 Hz, 1H), 7.61 (td, J = 7.4, 0.8
Hz, 1H), 7.49 (t, J = 7.7 Hz, 2H), 7.41−7.29 (m, 4H), 7.21 (d, J = 9.1
Hz, 1H), 3.77 (s, 3H).
Methyl 2-(4-((2,4-Dichlorophenyl)thio)-3-nitrobenzoyl)benzoate
(11c). Yellow solid, 60 mg, 65% yield. 1H NMR (400 MHz, CDCl3):
δ 8.57 (d, J = 1.2 Hz, 1H), 8.10 (d, J = 7.7 Hz, 1H), 7.81 (dd, J = 8.4,
1.3 Hz, 1H), 7.74−7.58 (m, 4H), 7.41 (dd, J = 8.3, 2.0 Hz, 1H), 7.38
(d, J = 7.5 Hz, 1H), 6.81 (d, J = 8.6 Hz, 1H), 3.74 (s, 3H).
Methyl 2-(4-((2,4-Dimethylphenyl)thio)-3-nitrobenzoyl)benzoate
(12c). Yellow solid, 68 mg, 80% yield. 1H NMR (400 MHz, CDCl3):
δ 8.57 (d, J = 1.7 Hz, 1H), 8.08 (d, J = 7.7 Hz, 1H), 7.71 (dd, J = 8.6,
1.7 Hz, 1H), 7.66 (dd, J = 7.3, 0.8 Hz, 1H), 7.61 (d, J = 7.4 Hz, 1H),
7.45 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 7.2 Hz, 1H), 7.23 (s, 1H), 7.13
(d, J = 7.7 Hz, 1H), 6.78 (d, J = 8.6 Hz, 1H), 3.73 (s, 3H), 2.40 (s,
3H), 2.32 (s, 3H).
Methyl 2-(4-((4-(Diethylcarbamoyl)-2-nitrophenyl)thio)-3-
1
nitrobenzoyl)benzoate (14c). Yellow solid, 37 mg, 34% yield. H
NMR (400 MHz, CDCl3): δ 8.47 (d, J = 1.8 Hz, 1H), 8.13 (d, J = 8.2
Hz, 2H), 7.89 (dd, J = 8.4, 1.8 Hz, 1H), 7.72 (td, J = 7.5, 1.0 Hz, 1H),
7.68−7.62 (m, 2H), 7.58 (d, J = 8.0 Hz, 1H), 7.41 (d, J = 7.4 Hz,
1H), 7.19 (d, J = 8.4 Hz, 1H), 3.77 (s, 3H), 3.59 (s, 2H), 3.32 (s,
2H), 1.29 (d, J = 3.8 Hz, 6H).
Methyl 2-(4-((2-Methyl-6-nitrophenyl)thio)-3-nitrobenzoyl)-
benzoate (15c). Yellow solid, 72 mg, 80% yield. 1H NMR (400
MHz, CDCl3): δ 8.58 (d, J = 1.8 Hz, 1H), 8.10 (d, J = 7.7 Hz, 1H),
7.82 (dd, J = 8.5, 1.7 Hz, 1H), 7.74−7.67 (m, 2H), 7.66−7.59 (m,
3H), 7.40 (d, J = 7.3 Hz, 1H), 6.81 (d, J = 8.5 Hz, 1H), 3.74 (s, 3H),
2.47 (s, 3H).
Methyl 2-(4-((2-Methylfuran-3-yl)thio)-3-nitrobenzoyl)benzoate
(16c). Yellow solid, 45 mg, 57% yield. 1H NMR (400 MHz, CDCl3):
δ 8.54 (d, J = 1.7 Hz, 1H), 8.11 (d, J = 7.7 Hz, 1H), 7.86 (dd, J = 8.6,
1.7 Hz, 1H), 7.70 (td, J = 7.4, 0.9 Hz, 1H), 7.63 (td, J = 7.7, 1.0 Hz,
1H), 7.49 (d, J = 1.8 Hz, 1H), 7.37 (d, J = 7.2 Hz, 1H), 7.16 (d, J =
8.6 Hz, 1H), 6.39 (d, J = 1.7 Hz, 1H), 3.75 (s, 3H), 2.38 (s, 3H).
General Procedure for the Synthesis of 17c−32c. Two different
procedures were used to obtain the titled compounds:
Method A: To a flask were added compound 1b (64 mg, 0.2
mmol), amine (2 equiv, 0.4 mmol), DIPEA (99 μL, 0.6 mmol), and
dimethylformamide (DMF) (2 mL). The mixture was heated to reflux
for 6 h. After cooling to room temperature, water was added, and the
aqueous solution was extracted with EtOAc. The combined organic
layers were dried over anhydrous Na2SO4, concentrated, and purified
by chromatography to provide the titled compounds.
Method B: An oven-dried Schlenk tube was charged with
compound 1b (64 mg, 0.3 mmol), amine (1.1 equiv, 0.33 mmol),
Pd(OAc)2 (0.09 mmol, 20.2 mg), Xantphos (0.09 mmol, 52.1 mg),
Cs2CO3 (0.9 mmol, 293 mg), and tetrahydrofuran (THF) (5 mL)
and purged with nitrogen. The reaction mixture was stirred at 100 °C
for 12 h and then cooled to room temperature. Water was added, and
the aqueous phase was extracted with EtOAc. The combined organic
layers were dried over anhydrous Na2SO4, concentrated, and purified
by chromatography to provide the titled compounds.
Methyl 2-(3-Nitro-4-(quinolin-8-ylamino)benzoyl)benzoate
(26c). Method A, purified by chromatography using hexane/EtOAc
1
(8:1), dark yellow solid, 50 mg, 58% yield. H NMR (400 MHz,
CDCl3): δ 11.35 (s, 1H), 8.98 (dd, J = 4.2, 1.6 Hz, 1H), 8.53 (d, J =
2.1 Hz, 1H), 8.24 (dd, J = 8.3, 1.5 Hz, 1H), 8.13 (d, J = 7.8 Hz, 1H),
8.07 (dd, J = 9.0, 1.9 Hz, 1H), 7.85 (d, J = 8.9 Hz, 2H), 7.70 (td, J =
7.4, 1.1 Hz, 1H), 7.68−7.61 (m, 2H), 7.59 (d, J = 7.7 Hz, 1H), 7.55
(dd, J = 8.3, 4.2 Hz, 1H), 7.45−7.41 (m, 1H), 3.77 (s, 3H).
Methyl 2-(3-Nitro-4-(thiazol-2-ylamino)benzoyl)benzoate (27c).
Method B, purified by chromatography using hexane/EtOAc (10:1),
yellow solid, 72 mg, 63% yield. 1H NMR (400 MHz, CDCl3): δ 10.95
(s, 1H), 8.83 (d, J = 9.1 Hz, 1H), 8.60 (d, J = 2.0 Hz, 1H), 8.17−8.05
(m, 2H), 7.70 (td, J = 7.6, 0.9 Hz, 1H), 7.63 (td, J = 7.7, 1.0 Hz, 1H),
7.51 (d, J = 3.6 Hz, 1H), 7.40 (d, J = 7.4 Hz, 1H), 7.02 (d, J = 3.6 Hz,
1H), 3.75 (s, 3H).
Methyl 2-(4-((2-Aminocyclohexyl)amino)-3-nitrobenzoyl)-
benzoate (28c). Method A, purified by chromatography using
hexane/EtOAc (5:1), yellow solid, 55 mg, 72%. 1H NMR (400
MHz, CDCl3): δ 8.52 (d, J = 8.3 Hz, 1H), 8.41 (d, J = 1.9 Hz, 1H),
8.10 (d, J = 7.7 Hz, 1H), 8.04 (d, J = 9.1 Hz, 1H), 7.66 (d, J = 7.3 Hz,
1H), 7.60 (d, J = 7.7 Hz, 1H), 7.36 (d, J = 7.4 Hz, 1H), 7.10 (d, J =
Methyl 2-(4-((2-Aminophenyl)amino)-3-nitrobenzoyl)benzoate
(17c). Method A, purified by chromatography using hexane/EtOAc
(8:1), orange solid, 45 mg, 70% yield. 1H NMR (400 MHz, CDCl3):
δ 9.45 (s, 1H), 8.51 (d, J = 1.8 Hz, 1H), 8.10 (d, J = 7.7 Hz, 1H), 7.93
(dd, J = 9.0, 1.5 Hz, 1H), 7.67 (t, J = 7.0 Hz, 1H), 7.60 (t, J = 7.2 Hz,
1H), 7.36 (d, J = 7.3 Hz, 1H), 7.22 (t, J = 7.7 Hz, 1H), 7.14 (d, J = 7.7
Hz, 1H), 6.92−6.79 (m, 3H), 3.83 (s, 2H), 3.77 (s, 3H).
Methyl 2-(3-Nitro-4-(m-tolylamino)benzoyl)benzoate (18c).
Method A, purified by chromatography using hexane/EtOAc
(10:1), orange solid, 60 mg, 77% yield. 1H NMR (400 MHz,
CDCl3): δ 9.87 (s, 1H), 8.47 (d, J = 2.0 Hz, 1H), 8.11 (d, J = 8.1 Hz,
1H), 7.95 (dd, J = 9.0, 1.8 Hz, 1H), 7.68 (td, J = 7.5, 1.0 Hz, 1H),
7.61 (td, J = 7.5, 0.9 Hz, 1H), 7.37 (m, 2H), 7.22 (d, J = 9.1 Hz, 1H),
7.14 (m, 3H), 3.78 (s, 3H), 2.42 (s, 3H).
Methyl 2-(4-((3-(Hydroxymethyl)phenyl)amino)-3-nitrobenzoyl)-
benzoate (19c). Method A, purified by chromatography using
hexane/EtOAc (10:1), orange solid, 57 mg, 70% yield. 1H NMR
(400 MHz, CDCl3): δ 9.89 (s, 1H), 8.48 (d, J = 1.9 Hz, 1H), 8.11 (d,
M
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
9.2 Hz, 1H), 3.76 (s, 3H), 3.38 (qd, J = 9.5, 3.9 Hz, 1H), 2.83 (td, J =
9.7, 3.9 Hz, 1H), 2.13 (d, J = 12.4 Hz, 1H), 2.03 (d, J = 10.7 Hz, 1H),
1.83 (s, 2H), 1.44−1.31 (m, 4H).
2-(3-Nitro-4-(pyridin-2-ylthio)benzoyl)benzoic Acid (4). The
titled compound was prepared from the hydrolysis of 4c (70 mg,
0.18 mmol) in 1 N NaOH (0.9 mL) and THF (0.9 mL). Yellow solid,
40 mg, 58% yield, mp 205−206 °C. 1H NMR (400 MHz, DMSO-d6):
δ 13.74 (s, 1H), 8.62 (d, J = 4.0 Hz, 1H), 8.31 (d, J = 1.7 Hz, 1H),
8.02 (d, J = 7.5 Hz, 1H), 7.92 (t, J = 7.0 Hz, 1H), 7.77 (m, 2H), 7.70
(m, 2H), 7.49 (m, 2H), 7.37 (d, J = 8.4 Hz, 1H). 13C NMR (100
MHz, DMSO-d6): δ 194.5, 167.3, 153.6, 151.6, 147.1, 140.7, 139.1,
138.8, 136.0, 133.3, 132.0, 130.8, 130.5, 130.1, 128.8, 128.8, 127.9,
125.4, 124.5. HRMS (ESI): calcd for C19H11N2O5S, [M − H]−
379.0394; found, 379.0388. HPLC purity: 100.00%.
Methyl 2-(4-(Benzylamino)-3-nitrobenzoyl)benzoate (29c).
Method A, purified by chromatography using hexane/EtOAc (8:1),
orange solid, 50 mg, 64% yield. 1H NMR (400 MHz, CDCl3): δ 8.82
(t, J = 5.6 Hz, 1H), 8.48 (d, J = 1.9 Hz, 1H), 8.10 (d, J = 7.8 Hz, 1H),
7.99 (dd, J = 9.0, 1.5 Hz, 1H), 7.67 (td, J = 7.4, 0.8 Hz, 1H), 7.60 (td,
J = 7.7, 0.9 Hz, 1H), 7.45−7.38 (m, 2H), 7.36 (d, J = 6.9 Hz, 4H),
6.93 (d, J = 9.0 Hz, 1H), 4.63 (d, J = 5.6 Hz, 2H), 3.75 (s, 3H).
Methyl 2-(4-((2-Fluorobenzyl)amino)-3-nitrobenzoyl)benzoate
(30c). Method A, purified by chromatography using hexane/EtOAc
(8:1), orange solid, 53 mg, 65% yield. 1H NMR (400 MHz, CDCl3):
δ 8.79 (t, J = 4.2 Hz, 1H), 8.48 (d, J = 1.8 Hz, 1H), 8.10 (d, J = 7.8
Hz, 1H), 8.02 (dd, J = 9.0, 1.6 Hz, 1H), 7.67 (t, J = 7.4 Hz, 1H), 7.60
(t, J = 7.1 Hz, 1H), 7.40−7.31 (m, 3H), 7.16 (m, 2H), 6.95 (d, J = 9.1
Hz, 1H), 4.69 (d, J = 5.8 Hz, 2H), 3.75 (s, 3H).
2-(4-((3-(Isopropylamino)phenyl)thio)-3-nitrobenzoyl)benzoic
Acid (7). The titled compound was prepared from the hydrolysis of 7c
(65 mg, 0.14 mmol) in 1 N NaOH (0.7 mL) and THF (0.7 mL).
1
Yellow solid, 40 mg, 66% yield, mp 130−132 °C. H NMR (400
MHz, DMSO-d6): δ 13.32 (s, 1H), 8.31 (s, 1H), 8.01 (d, J = 7.5 Hz,
1H), 7.80 (d, J = 8.7 Hz, 1H), 7.75 (d, J = 7.5 Hz, 1H), 7.69 (t, J = 7.5
Hz, 1H), 7.46 (d, J = 7.3 Hz, 1H), 7.25 (t, J = 7.7 Hz, 1H), 7.08 (d, J
= 8.5 Hz, 1H), 6.78−6.69 (m, 3H), 5.86 (s, 1H), 3.53 (m, 1H), 1.12
(d, J = 6.2 Hz, 6H). 13C NMR (100 MHz, DMSO-d6): δ 194.5, 167.3,
150.3, 144.8, 144.3, 140.8, 134.4, 133.6, 133.2, 131.5, 130.7, 130.5,
130.2, 129.5, 128.8, 127.9, 126.0, 121.8, 118.6, 114.9, 43.4, 22.7.
HRMS (ESI): calcd for C23H19N2O5S, [M − H]− 435.102; found,
435.1022. HPLC purity: 97.60%.
Methyl 2-(3-Nitro-4-(piperidin-1-yl)benzoyl)benzoate (31c).
Method A, purified by chromatography using hexane/EtOAc
(15:1), yellow solid, 70 mg, 95% yield. 1H NMR (400 MHz,
CDCl3): δ 8.08 (d, J = 7.6 Hz, 1H), 8.05 (d, J = 2.0 Hz, 1H), 7.90
(dd, J = 8.8, 2.0 Hz, 1H), 7.66 (t, J = 7.2 Hz, 1H), 7.59 (t, J = 7.6 Hz,
1H), 7.36 (d, J = 7.4 Hz, 1H), 7.08 (d, J = 8.9 Hz, 1H), 3.75 (s, 3H),
3.22−3.16 (m, 4H), 1.79−1.66 (m, 6H).
Methyl 2-(4-Morpholino-3-nitrobenzoyl)benzoate (32c). Method
A, purified by chromatography using hexane/EtOAc (8:1), yellow
2-(4-((3-Acetamidophenyl)thio)-3-nitrobenzoyl)benzoic Acid (8).
The titled compound was prepared from the hydrolysis of 8c (45 mg,
0.1 mmol) in 1 N NaOH (0.5 mL) and THF (0.5 mL). Yellow solid,
32 mg, 73% yield, mp 144−146 °C. 1H NMR (400 MHz, DMSO-d6):
δ 13.33 (s, 1H), 10.21 (s, 1H), 8.34 (s, 1H), 8.01 (d, J = 7.5 Hz, 1H),
7.94 (s, 1H), 7.80−7.65 (m, 4H), 7.51 (t, J = 7.9 Hz, 1H), 7.46 (d, J =
7.3 Hz, 1H), 7.31 (d, J = 7.5 Hz, 1H), 6.99 (d, J = 8.5 Hz, 1H), 2.05
(s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 194.4, 169.2, 167.3,
144.5, 143.7, 141.6, 141.0, 134.9, 133.8, 133.3, 131.4, 130.7, 130.2,
130.0, 129.7, 128.9, 127.7, 125.8, 125.5, 121.4, 24.5. HRMS (ESI):
calcd for C22H15N2O6S, [M − H]− 435.0656; found, 435.0651. HPLC
purity: 96.56%.
1
solid, 60 mg, 81% yield. H NMR (400 MHz, CDCl3): δ 8.14−8.06
(m, 2H), 7.94 (dd, J = 8.7, 2.0 Hz, 1H), 7.68 (td, J = 7.5, 1.0 Hz, 1H),
7.61 (td, J = 7.5, 0.9 Hz, 1H), 7.41−7.33 (m, 1H), 7.10 (d, J = 8.8 Hz,
1H), 3.90−3.82 (m, 4H), 3.76 (s, 3H), 3.24−3.16 (m, 4H).
General Procedure for the Synthesis of Compounds 1−32. The
methyl esters of compounds 1c−32c were hydrolyzed with 1 M
NaOH solution in THF (1:1) at room temperature overnight. The
mixture was then diluted with a small amount of water and washed
twice with CH2Cl2. The aqueous solution was acidified by the
addition of 2 M HCl. The precipitate was collected by filtration and
washed with water to afford the titled compounds. If the compound
was not pure at this stage of the procedure, it was purified by silica gel
column chromatography.
2-(4-((3-Benzamidophenyl)thio)-3-nitrobenzoyl)benzoic Acid
(9). The titled compound was prepared from the hydrolysis of 9c
(57 mg, 0.1 mmol) in 1 N NaOH (0.5 mL) and THF (0.5 mL).
1
2-(4-((2-Aminophenyl)thio)-3-nitrobenzoyl)benzoic acid (1), 2-
(4-((3-aminophenyl)thio)-3-nitrobenzoyl)benzoic acid (5), 2-(4-((4-
aminophenyl)thio)-3-nitrobenzoyl)benzoic acid (6), 2-(4-((2-amino-
4-chlorophenyl)thio)-3-nitrobenzoyl)benzoic acid (10), and 2-(4-
((3,4-dichlorophenyl)thio)-3-nitrobenzoyl)benzoic acid (13) were
synthesized following the previously published procedures.22
2-(4-((2-Methoxyphenyl)thio)-3-nitrobenzoyl)benzoic Acid (2).
The titled compound was prepared from the hydrolysis of 2c (70
mg, 0.16 mmol) in 1 N NaOH (0.8 mL) and THF (0.8 mL). Yellow
solid, 60 mg, 92% yield, mp 209−211 °C. 1H NMR (400 MHz,
DMSO-d6): δ 13.31 (s, 1H), 8.34 (d, J = 1.7 Hz, 1H), 8.01 (d, J = 7.5
Hz, 1H), 7.75 (dd, J = 13.3, 7.2 Hz, 2H), 7.69 (m, 1H), 7.64 (m, 2H),
7.47 (d, J = 7.3 Hz, 1H), 7.26 (d, J = 8.2 Hz, 1H), 7.13 (t, J = 7.3 Hz,
1H), 6.88 (d, J = 8.5 Hz, 1H), 3.77 (s, 3H). 13C NMR (100 MHz,
DMSO-d6): δ 194.5, 167.3, 160.5, 144.4, 143.3, 140.8, 137.7, 134.6,
133.8, 133.5, 133.3, 130.7, 130.3, 128.4, 127.8, 126.0, 122.4, 116.4,
113.2, 56.5. HRMS (ESI): calcd for C21H14NO6S, [M − H]−
408.0547; found, 408.0539. HPLC purity: 97.04%.
2-(4-((2-Fluorophenyl)thio)-3-nitrobenzoyl)benzoic Acid (3). The
titled compound was prepared from the hydrolysis of 3c (60 mg, 0.15
mmol) in 1 N NaOH (0.7 mL) and THF (0.7 mL). Yellow solid, 43
mg, 72% yield, mp 175−176 °C. 1H NMR (400 MHz, DMSO-d6): δ
13.34 (s, 1H), 8.39 (s, 1H), 8.02 (d, J = 7.5 Hz, 1H), 7.76 (t, J = 7.6
Hz, 3H), 7.70 (dd, J = 13.9, 6.6 Hz, 2H), 7.49 (t, J = 8.3 Hz, 2H),
7.42 (t, J = 7.4 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H). 13C NMR (100
MHz, DMSO-d6): δ 194.4, 168.2 (d, J = 8.1 Hz), 162.8 (d, J = 248.5
Hz), 144.5, 140.7, 139.9 (d, J = 3.0 Hz), 138.4 (d, J = 10.1 Hz), 138.2,
137.2 (d, J = 3.0 Hz), 134.5 (d, J = 8.1 Hz), 133.8, 130.2, 129.8,
129.5, 128.0, 126.9 (d, J = 3.0 Hz), 126.3, 125.4, 117.5 (d, J = 22.2
Hz), 116.7 (d, J = 19.2 Hz). HRMS (ESI): calcd for C20H11FNO5S,
[M − H]− 396.0347; found, 396.0345. HPLC purity: 99.25%.
Yellow solid, 31 mg, 57% yield, mp 144−145 °C. H NMR (400
MHz, DMSO-d6): δ 13.31 (s, 1H), 10.49 (s, 1H), 8.35 (d, J = 1.6 Hz,
1H), 8.15 (s, 1H), 8.01 (d, J = 7.8 Hz, 2H), 7.95 (d, J = 7.3 Hz, 2H),
7.79 (d, J = 9.3 Hz, 1H), 7.75 (d, J = 7.2 Hz, 1H), 7.69 (t, J = 7.2 Hz,
1H), 7.62 (d, J = 7.3 Hz, 1H), 7.60−7.51 (m, 3H), 7.47 (d, J = 7.1
Hz, 1H), 7.39 (d, J = 7.5 Hz, 1H), 7.06 (d, J = 8.5 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6): δ 194.5, 167.2, 166.3, 144.6, 143.8, 141.5,
140.7, 135.0, 134.8, 133.9, 133.3, 132.3, 131.4, 130.9, 130.8, 130.5,
130.1, 129.7, 129.0, 128.9, 128.2, 127.8, 126.9, 125.9, 122.7. HRMS
(ESI): calcd for C27H17N2O6S, [M − H]− 497.0813; found, 497.0805.
HPLC purity: 95.26%.
2-(4-((2,4-Dichlorophenyl)thio)-3-nitrobenzoyl)benzoic Acid
(11). The titled compound was prepared from the hydrolysis of 11c
(60 mg, 0.13 mmol) in 1 N NaOH (0.6 mL) and THF (0.6 mL).
1
Yellow solid, 52 mg, 89% yield, mp 260−262 °C. H NMR (400
MHz, DMSO-d6): δ 13.40 (S, 1H), 8.39 (d, J = 10.3 Hz, 1H), 8.01
(dd, J = 18.5, 10.5 Hz, 2H), 7.87 (dd, J = 11.7, 8.3 Hz, 1H), 7.82−
7.60 (m, 4H), 7.49 (t, J = 8.9 Hz, 1H), 6.92 (t, J = 10.1 Hz, 1H). 13C
NMR (100 MHz, DMSO-d6): δ 194.4, 167.2, 144.7, 141.1, 140.7,
140.3, 139.8, 137.5, 135.3, 134.2, 133.4, 131.1, 130.8, 130.3, 130.0,
129.8, 128.7, 127.7, 126.0. HRMS (ESI): calcd for C20H10Cl2NO5S,
[M − H]− 445.9662; found, 445.9656. HPLC purity: 98.13%.
2-(4-((2,4-Dimethylphenyl)thio)-3-nitrobenzoyl)benzoic Acid
(12). The titled compound was prepared from the hydrolysis of 12c
(68 mg, 0.16 mmol) in 1 N NaOH (0.8 mL) and THF (0.8 mL).
1
Yellow solid, 38 mg, 58% yield, mp 240−242 °C. H NMR (400
MHz, DMSO-d6): δ 8.34 (s, 1H), 8.00 (d, J = 7.5 Hz, 1H), 7.72 (dd, J
= 6.0, 2.7 Hz, 2H), 7.66 (t, J = 7.4 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H),
7.44 (d, J = 7.3 Hz, 1H), 7.32 (s, 1H), 7.20 (d, J = 7.6 Hz, 1H), 6.77
(d, J = 8.5 Hz, 1H), 2.36 (s, 3H), 2.25 (s, 3H). 13C NMR (100 MHz,
DMSO-d6): δ 167.5, 144.5, 143.4, 143.0, 141.8, 141.1, 137.2, 134.8,
N
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
133.8, 133.0, 132.8, 130.8, 130.6, 130.2, 129.2, 127.9, 127.6, 126.1,
125.1, 21.3, 20.4. HRMS (ESI): calcd for C22H16NO5S, [M − H]−
406.0755; found, 406.0747. HPLC purity: 99.01%.
(d, J = 9.1 Hz, 1H), 5.28 (s, 1H), 4.54 (s, 2H). 13C NMR (100 MHz,
DMSO-d6): δ 193.9, 167.3, 145.9, 144.9, 141.2, 138.4, 135.5, 133.0,
132.3, 130.5, 130.4, 130.3, 129.9, 128.8, 127.9, 126.6, 124.7, 123.8,
123.3, 117.0, 62.9. HRMS (ESI): calcd for C21H15N2O6, [M − H]−
391.0936; found, 391.0932. HPLC purity: 95.83%.
2-(4-((3-Chlorophenyl)amino)-3-nitrobenzoyl)benzoic Acid (20).
The titled compound was prepared from the hydrolysis of 20c (70
mg, 0.18 mmol) in 1 N NaOH (0.9 mL) and THF (0.9 mL). Yellow
solid, 45 mg, 63% yield, mp 159−161 °C. 1H NMR (400 MHz,
DMSO-d6): δ 13.24 (s, 1H), 9.85 (s, 1H), 8.24 (d, J = 1.8 Hz, 1H),
8.01 (d, J = 7.5 Hz, 1H), 7.78 (s, 1H), 7.74 (s, 1H), 7.67 (s, 1H),
7.54−7.45 (m, 2H), 7.42 (d, J = 7.3 Hz, 1H), 7.40−7.30 (m, 2H),
7.23 (d, J = 9.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6): δ 193.9,
167.4, 144.9, 141.2, 140.5, 135.6, 134.2, 133.2, 132.9, 131.6, 130.6,
130.4, 130.3, 128.4, 127.7, 127.5, 126.2, 125.2, 123.7, 117.5. HRMS
(ESI): calcd for C20H12ClN2O5, [M − H]− 395.044; found, 395.0434.
HPLC purity: 96.87%.
2-(4-((4-(Diethylcarbamoyl)-2-nitrophenyl)thio)-3-nitrobenzoyl)-
benzoic Acid (14). The titled compound was prepared from the
hydrolysis of 14c (37 mg, 0.07 mmol) in 1 N NaOH (0.4 mL) and
THF (0.4 mL). Yellow solid, 25 mg, 69% yield, mp 129−130 °C. 1H
NMR (400 MHz, DMSO-d6): δ 13.40 (s, 1H), 8.37 (d, J = 1.7 Hz,
1H), 8.19 (s, 1H), 8.03 (d, J = 7.0 Hz, 1H), 7.86−7.62 (m, 5H), 7.51
(d, J = 7.5 Hz, 1H), 7.37 (d, J = 8.2 Hz, 1H), 3.46 (s, 2H), 3.23 (s,
2H), 1.17 (s, 3H), 1.08 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ
194.5, 167.3, 151.2, 147.2, 140.6, 139.8, 138.5, 136.8, 136.5, 134.0,
133.4, 132.6, 132.2, 130.9, 130.5, 130.2, 127.9, 127.6, 125.5, 123.8,
43.4, 14.4, 13.1. HRMS (ESI): calcd for C25H20N3O8S, [M − H]−
522.0977; found, 522.0960. HPLC purity: 97.21%.
2-(4-((2-Methyl-6-nitrophenyl)thio)-3-nitrobenzoyl)benzoic Acid
(15). The titled compound was prepared from the hydrolysis of 15c
(70 mg, 0.15 mmol) in 1 N NaOH (0.8 mL) and THF (0.8 mL).
2-(4-((3-(tert-Butyl)phenyl)amino)-3-nitrobenzoyl)benzoic Acid
(21). The titled compound was prepared from the hydrolysis of 21c
(70 mg, 0.16 mmol) in 1 N NaOH (0.8 mL) and THF (0.8 mL).
1
Yellow solid, 50 mg, 76% yield, mp 269−271 °C. H NMR (400
MHz, DMSO-d6): δ 13.40 (s, 1H), 8.41 (d, J = 1.6 Hz, 1H), 8.02 (d, J
= 7.6 Hz, 1H), 7.94 (d, J = 6.9 Hz, 1H), 7.79 (m, 4H), 7.69 (t, J = 7.4
Hz, 1H), 7.50 (d, J = 7.3 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 2.38 (s,
3H). 13C NMR (100 MHz, DMSO-d6): δ 194.4, 167.3, 155.6, 146.5,
144.9, 140.7, 135.4, 135.3, 134.3, 133.4, 133.0, 130.8, 130.3, 130.0,
128.4, 127.8, 126.0, 122.7, 120.8, 20.9. HRMS (ESI): calcd for
C21H13N2O7S, [M − H]− 437.0449; found, 437.0443. HPLC purity:
99.56%.
1
Yellow solid, 56 mg, 84% yield, mp 110−112 °C. H NMR (400
MHz, DMSO-d6): δ 13.22 (s, 1H), 9.91 (s, 1H), 8.24 (s, 1H), 8.01
(d, J = 7.6 Hz, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.77−7.70 (m, 1H),
7.67 (t, J = 7.4 Hz, 1H), 7.46−7.31 (m, 4H), 7.19 (d, J = 7.3 Hz, 1H),
7.15 (d, J = 9.1 Hz, 1H), 1.30 (s, 9H). 13C NMR (100 MHz, DMSO-
d6): δ 193.9, 167.4, 153.0, 145.9, 141.3, 138.1, 135.5, 132.8, 132.2,
130.8, 130.4, 130.3, 129.7, 128.7, 127.8, 126.6, 123.7, 122.8, 122.6,
116.9, 35.0, 31.5. HRMS (ESI): calcd for C24H21N2O5, [M − H]−
417.1456; found, 417.1455. HPLC purity: 98.35%.
2-(4-((2-Methylfuran-3-yl)thio)-3-nitrobenzoyl)benzoic Acid
(16). The titled compound was prepared from the hydrolysis of 16c
(45 mg, 0.11 mmol) in 1 N NaOH (0.5 mL) and THF (0.5 mL).
2-(4-((3-Carbamoylphenyl)amino)-3-nitrobenzoyl)benzoic Acid
(22). The titled compound was prepared from the hydrolysis of 22c
(70 mg, 0.17 mmol) in 1 N NaOH (0.8 mL) and THF (0.8 mL).
1
Yellow solid, 31 mg, 73% yield, mp 198−199 °C. H NMR (400
MHz, DMSO-d6): δ 13.45 (s, 1H), 8.35 (s, 1H), 8.02 (d, J = 7.5 Hz,
1H), 7.84 (d, J = 1.3 Hz, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.77 (t, J = 7.3
Hz, 1H), 7.70 (t, J = 7.4 Hz, 1H), 7.47 (d, J = 7.3 Hz, 1H), 7.17 (d, J
= 8.5 Hz, 1H), 6.58 (s, 1H), 2.33 (s, 3H). 13C NMR (100 MHz,
DMSO-d6): δ 194.5, 167.3, 158.8, 144.7, 143.8, 143.2, 140.8, 134.85,
133.84, 133.4, 130.8, 130.4, 130.1, 128.4, 127.8, 126.1, 115.3, 105.7,
12.0. HRMS (ESI): calcd for C19H12NO6S, [M − H]− 382.0391;
found, 382.0387. HPLC purity: 98.13%.
1
Yellow solid, 43 mg, 62% yield, mp 139−141 °C. H NMR (400
MHz, DMSO-d6): δ 13.21 (s, 1H), 9.95 (s, 1H), 8.25 (d, J = 1.6 Hz,
1H), 8.02 (s, 1H), 8.01 (d, J = 7.5 Hz, 1H), 7.86 (s, 1H), 7.80 (d, J =
6.3 Hz, 2H), 7.75 (t, J = 7.6 Hz, 1H), 7.67 (t, J = 7.4 Hz, 1H), 7.60−
7.50 (m, 2H), 7.49−7.39 (m, 2H), 7.17 (d, J = 9.1 Hz, 1H). 13C
NMR (100 MHz, DMSO-d6): δ 193.9, 167.6, 167.3, 145.6, 141.2,
138.8, 136.2, 135.6, 133.0, 132.6, 130.5, 130.4, 130.1, 128.6, 128.4,
127.8, 126.9, 125.6, 124.6, 117.2. HRMS (ESI): calcd for
C21H14N3O6, [M − H]− 404.0888; found, 404.0882. HPLC purity:
96.05%.
2-(4-((2-Aminophenyl)amino)-3-nitrobenzoyl)benzoic Acid (17).
The titled compound was prepared from the hydrolysis of 17c (40
mg, 0.1 mmol) in 1 N NaOH (0.5 mL) and THF (0.5 mL). Dark
yellow solid, 34 mg, 90% yield, mp 201−202 °C. 1H NMR (400 MHz,
DMSO-d6): δ 9.46 (s, 1H), 8.20 (d, J = 1.9 Hz, 1H), 7.98 (d, J = 7.4
Hz, 1H), 7.76 (dd, J = 9.0, 1.6 Hz, 1H), 7.65 (dt, J = 22.1, 7.1 Hz,
2H), 7.34 (d, J = 7.2 Hz, 1H), 7.14−7.04 (m, 2H), 6.82 (d, J = 7.7
Hz, 1H), 6.70−6.59 (m, 2H), 5.21 (s, 2H). 13C NMR (100 MHz,
DMSO-d6): δ 167.3, 151.4, 145.2, 143.4, 137.7, 134.6, 133.4, 133.3,
132.9, 130.6, 130.3, 130.1, 127.9, 127.6, 126.1, 117.3, 115.7, 109.0.
HRMS (ESI): calcd for C20H14N3O5, [M − H]− 376.0939; found,
376.0936. HPLC purity: 95.02%.
2-(3-Nitro-4-(m-tolylamino)benzoyl)benzoic Acid (18). The titled
compound was prepared from the hydrolysis of 18c (60 mg, 0.15
mmol) in 1 N NaOH (0.8 mL) and THF (0.8 mL). Dark yellow solid,
38 mg, 66% yield, mp 90−92 °C. 1H NMR (400 MHz, DMSO-d6): δ
13.23 (s, 1H), 9.87 (s, 1H), 8.23 (d, J = 1.9 Hz, 1H), 8.01 (d, J = 7.6
Hz, 1H), 7.78 (d, J = 10.4 Hz, 1H), 7.73 (d, J = 7.3 Hz, 1H), 7.67 (t, J
= 7.5 Hz, 1H), 7.43 (d, J = 7.4 Hz, 1H), 7.37 (t, J = 7.7 Hz, 1H),
7.22−7.10 (m, 4H), 2.34 (s, 3H). 13C NMR (100 MHz, DMSO-d6):
δ 193.8, 167.4, 145.9, 141.2, 139.7, 138.4, 135.6, 135.5, 132.9, 132.2,
130.4, 130.3, 123.0, 128.7, 127.8, 127.5, 126.6, 126.1, 122.7, 117.0,
21.4. HRMS (ESI): calcd for C21H15N2O5, [M − H]− 375.0986;
found, 375.0985. HPLC purity: 97.89%.
2-(4-((3,4-Dichlorophenyl)amino)-3-nitrobenzoyl)benzoic Acid
(23). The titled compound was prepared from the hydrolysis of 23c
(45 mg, 0.1 mmol) in 1 N NaOH (0.5 mL) and THF (0.5 mL).
1
Yellow solid, 30 mg, 70% yield, mp 115−117 °C. H NMR (400
MHz, DMSO-d6): δ 13.24 (s, 1H), 9.84 (s, 1H), 8.24 (s, 1H), 8.02
(d, J = 7.3 Hz, 1H), 7.85−7.63 (m, 5H), 7.43 (d, J = 7.3 Hz, 1H),
7.38 (d, J = 7.5 Hz, 1H), 7.27 (d, J = 9.0 Hz, 1H). 13C NMR (100
MHz, DMSO-d6): δ 193.8, 167.3, 144.5, 141.1, 139.4, 135.6, 135.5,
133.8, 132.9, 132.8, 132.3, 131.7, 130.4, 128.2, 128.1, 127.9, 127.8,
126.8, 125.1, 117.8. HRMS (ESI): calcd for C20H11Cl2N2O5, [M −
H]− 429.0051; found, 429.0041. HPLC purity: 95.09%.
2-(4-((4-Methoxyphenyl)amino)-3-nitrobenzoyl)benzoic Acid
(24). The titled compound was prepared from the hydrolysis of 24c
(61 mg, 0.15 mmol) in 1 N NaOH (0.8 mL) and THF (0.8 mL).
1
Yellow solid, 42 mg, 71% yield, mp 225−226 °C. H NMR (400
MHz, DMSO-d6): δ 13.17 (s, 1H), 9.87 (s, 1H), 8.22 (s, 1H), 8.01
(d, J = 7.6 Hz, 1H), 7.77 (d, J = 9.8 Hz, 1H), 7.73 (d, J = 7.2 Hz, 1H),
7.67 (t, J = 7.4 Hz, 1H), 7.42 (d, J = 7.3 Hz, 1H), 7.29 (d, J = 8.7 Hz,
2H), 7.05 (d, J = 8.7 Hz, 2H), 6.99 (d, J = 9.1 Hz, 1H), 3.80 (s, 3H).
13C NMR (100 MHz, DMSO-d6): δ 193.8, 167.3, 158.4, 147.0, 141.3,
135.4, 132.9, 131.5, 130.9, 130.4, 130.3, 128.9, 128.0, 127.8, 126.0,
116.7, 115.4, 55.8. HRMS (ESI): calcd for C21H15N2O6, [M − H]−
391.0936; found, 391.0929. HPLC purity: 98.67%.
2-(4-((3-(Hydroxymethyl)phenyl)amino)-3-nitrobenzoyl)benzoic
Acid (19). The titled compound was prepared from the hydrolysis of
19c (50 mg, 0.12 mmol) in 1 N NaOH (0.6 mL) and THF (0.6 mL).
Dark yellow solid, 36 mg, 75% yield, mp 132−134 °C. 1H NMR (400
MHz, DMSO-d6): δ 13.22 (s, 1H), 9.90 (s, 1H), 8.24 (d, J = 1.3 Hz,
1H), 8.01 (d, J = 7.6 Hz, 1H), 7.76 (m, 2H), 7.67 (t, J = 7.4 Hz, 1H),
7.43 (t, J = 7.0 Hz, 2H), 7.32 (s, 1H), 7.25 (t, J = 7.1 Hz, 2H), 7.17
2-(3-Nitro-4-(phenylamino)benzoyl)benzoic Acid (25). The titled
compound was prepared from the hydrolysis of 25c (70 mg, 0.18
mmol) in 1 N NaOH (0.9 mL) and THF (0.9 mL). Yellow solid, 55
mg, 82% yield, mp 184−186 °C. 1H NMR (400 MHz, DMSO-d6): δ
13.28 (s, 1H), 9.90 (s, 1H), 8.23 (d, J = 1.7 Hz, 1H), 8.01 (d, J = 7.6
O
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Hz, 1H), 7.78 (dd, J = 9.0, 1.5 Hz, 1H), 7.73 (t, J = 7.4 Hz, 1H), 7.66
(t, J = 7.3 Hz, 1H), 7.48 (t, J = 7.7 Hz, 2H), 7.41 (d, J = 7.4 Hz, 1H),
7.38 (d, J = 7.6 Hz, 2H), 7.31 (t, J = 7.3 Hz, 1H), 7.15 (d, J = 9.0 Hz,
1H). 13C NMR (100 MHz, DMSO-d6): δ 193.9, 167.5, 145.8, 141.3,
138.6, 135.5, 132.8, 132.3, 130.8, 130.4, 130.2, 130.2, 128.7, 127.7,
126.8, 126.8, 125.7, 116.9. HRMS (ESI): calcd for C20H13N2O5, [M
− H]− 361.083; found, 361.0828. HPLC purity: 100.00%.
2-(3-Nitro-4-(quinolin-8-ylamino)benzoyl)benzoic Acid (26). The
titled compound was prepared from the hydrolysis of 26c (50 mg,
0.12 mmol) in 1 N NaOH (0.6 mL) and THF (0.6 mL), deep yellow
solid, 36 mg, yield 73%, mp 151−153 °C. 1H NMR (400 MHz,
DMSO-d6): δ 11.17 (s, 1H), 8.97 (s, 1H), 8.46 (d, J = 7.4 Hz, 1H),
8.31 (s, 1H), 8.01 (d, J = 4.5 Hz, 1H), 7.91 (d, J = 6.6 Hz, 1H), 7.83
(s, 2H), 7.76 (d, J = 7.5 Hz, 1H), 7.70−7.52 (m, 4H), 7.29 (d, J = 4.2
Hz, 1H). 13C NMR (100 MHz, DMSO-d6): δ 150.2, 142.5, 141.1,
140.3, 137.2, 135.8, 135.3, 134.3, 131.0, 130.2, 129.6, 129.4, 129.1,
128.0, 127.3, 126.9, 123.5, 123.06, 117.5, 117.2. HRMS (ESI): calcd
for C23H14N3O5, [M − H]− 412.0939; found, 412.0937. HPLC
purity: 96.02%.
2-(3-Nitro-4-(thiazol-2-ylamino)benzoyl)benzoic Acid (27). The
titled compound was prepared from the hydrolysis of 27c (60 mg,
0.16 mmol) in 1 N NaOH (0.8 mL) and THF (0.8 mL). Yellow solid,
39 mg, 66% yield, mp 99−100 °C. 1H NMR (400 MHz, DMSO-d6):
δ 13.23 (s, 1H), 10.78 (s, 1H), 8.44 (d, J = 8.4 Hz, 1H), 8.18 (s, 1H),
8.03 (d, J = 7.6 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 7.77 (t, J = 7.3 Hz,
1H), 7.70 (t, J = 7.3 Hz, 1H), 7.46 (m, 2H), 7.30 (s, 1H). 13C NMR
(100 MHz, DMSO-d6): δ 194.1, 167.3, 161.9, 141.0, 140.3, 139.2,
135.9, 135.2, 133.1, 130.5, 130.5, 130.3, 129.7, 127.9, 127.1, 120.4,
114.5. HRMS (ESI): calcd for C17H10N3O5S, [M − H]− 368.0347;
found, 368.0343. HPLC purity: 98.63%.
2-(4-((2-Aminocyclohexyl)amino)-3-nitrobenzoyl)benzoic Acid
(28). The titled compound was prepared from the hydrolysis of 28c
(50 mg, 0.13 mmol) in 1 N NaOH (0.6 mL) and THF (0.6 mL). The
purification by chromatography used DCM/MeOH (10:1). Yellow
solid, 33 mg, 68% yield, decomposition temperature 265 °C. 1H NMR
(400 MHz, DMSO-d6): δ 8.15 (m, 2H), 7.94 (dd, J = 5.5, 3.4 Hz,
1H), 7.62 (d, J = 0.5 Hz, 1H), 7.47 (dd, J = 5.4, 3.3 Hz, 2H), 7.17 (d,
J = 9.2 Hz, 1H), 7.12 (dd, J = 5.3, 3.3 Hz, 1H), 3.94 (m, 1H), 3.20
(m, 1H), 1.96−1.80 (m, 2H), 1.65 (d, J = 10.3 Hz, 2H), 1.55 (m,
1H), 1.43 (m, 1H), 1.38−1.10 (m, 2H). 13C NMR (100 MHz,
DMSO-d6): δ 195.5, 169.5, 147.0, 141.3, 138.7, 135.6, 131.1, 123.0,
129.6, 128.8, 128.2, 126.7, 126.2, 115.1, 55.2, 53.7, 31.6, 31.1, 24.4,
23.9. HRMS (ESI): calcd for C20H20N3O5, [M − H]− 382.1408;
found, 382.1406. HPLC purity: 98.62%.
2-(3-Nitro-4-(piperidin-1-yl)benzoyl)benzoic Acid (31). The titled
compound was prepared from the hydrolysis of 31c (70 mg, 0.19
mmol) in 1 N NaOH (1 mL) and THF (1 mL). Yellow solid, 58 mg,
1
86% yield, mp 191−193 °C. H NMR (400 MHz, DMSO-d6): δ
13.23 (s, 1H), 8.00 (d, J = 7.6 Hz, 1H), 7.93 (d, J = 2.0 Hz, 1H), 7.72
(t, J = 7.0 Hz, 1H), 7.70−7.67 (m, 1H), 7.65 (t, J = 5.5 Hz, 1H), 7.41
(d, J = 7.3 Hz, 1H), 7.31 (d, J = 8.9 Hz, 1H), 3.14 (s, 4H), 1.60 (s,
6H). 13C NMR (100 MHz, DMSO-d6): δ 193.9, 167.4, 148.9, 141.4,
138.8, 134.0, 132.8, 130.5, 130.34, 130.26, 128.0, 127.8, 127.5, 120.4,
51.7, 25.6, 23.7. HRMS (ESI): calcd for C19H17N2O5, [M − H]−
353.1143; found, 353.1140. HPLC purity: 99.21%.
2-(4-Morpholino-3-nitrobenzoyl)benzoic Acid (32). The titled
compound was prepared from the hydrolysis of 32c (60 mg, 0.17
mmol) in 1 N NaOH (0.8 mL) and THF (0.8 mL), yellow solid, 42
mg, 69% yield, mp 192−193 °C. 1H NMR (400 MHz, DMSO-d6): δ
13.23 (s, 1H), 8.01 (d, J = 7.6 Hz, 1H), 7.98 (d, J = 1.8 Hz, 1H),
7.78−7.70 (m, 2H), 7.67 (t, J = 7.5 Hz, 1H), 7.43 (d, J = 7.3 Hz, 1H),
7.35 (d, J = 8.9 Hz, 1H), 3.70 (m, 4H), 3.17 (m, 4H). 13C NMR (100
MHz, DMSO-d6): δ 194.1, 167.3, 148.3, 141.2, 139.4, 134.2, 133.3,
130.4, 130.2, 128.5, 127.8, 120.6, 66.2, 50.9. HRMS (ESI): calcd for
C18H15N2O6, [M − H]− 355.0936; found, 355.0933. HPLC purity:
100.00%.
Synthetic Procedures for Scheme 2. 2-(4-Bromobenzoyl)-
benzoic Acid (33b). To a suspension of the phthalic anhydride (2 g,
13.5 mmol) in bromobenzene (11.38 mL, 108 mmol) was added
aluminum chloride (2.7 g, 20.3 mmol). The mixture was stirred at 90
°C for 2 h and then cooled to room temperature. The reaction
mixture was poured onto crushed ice, followed by conc. HCl (5 mL),
the mixture was extracted with DCM and basified with 10% Na2CO3
solution for product extraction into the aqueous phase. The aqueous
phase was acidified to pH 3 with conc. HCl. The resulting precipitate
was collected by filtration and dried in vacuo. 33b was obtained as a
1
white solid without further purification, 3.5 g, 85% yield. H NMR
(400 MHz, DMSO-d6): δ 13.25 (s, 1H), 8.00 (d, J = 7.5 Hz, 1H),
7.79−7.60 (m, 4H), 7.54 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 7.5 Hz,
1H).
Methyl 2-(4-Bromobenzoyl)benzoate (33c). To a solution of
compound 33b (500 mg) in MeOH (10 mL) was added SOCl2 (10
drops). The mixture was heated to reflux and stirred overnight. The
reaction mixture was cooled down to room temperature, and the
white solid precipitate was collected and dried to provide compound
33c as a white solid, 450 mg, 86% yield. 1H NMR (400 MHz,
CDCl3): δ 8.06 (d, J = 7.7 Hz, 1H), 7.69−7.63 (m, 1H), 7.63−7.55
(m, 5H), 7.39 (d, J = 7.6 Hz, 1H), 3.67 (s, 3H).
2-(4-(Benzylamino)-3-nitrobenzoyl)benzoic Acid (29). The titled
compound was prepared from the hydrolysis of 29c (50 mg, 0.13
mmol) in 1 N NaOH (0.6 mL) and THF (0.6 mL). Yellow solid, 35
Methyl 2-(4-((3,4-Dichlorophenyl)thio)benzoyl)benzoate (33d).
To a round-bottom flask were added compound 33c (64 mg, 02
mmol), i-Pr2NEt (66 μL, 0.4 mmol), and dry 1,4-dioxane. The
mixture was degassed and filled with nitrogen (3 cycles). Pd2(dba)3
(18 mg, 0.02 mmol), xantphos (23 mg, 0.04 mmol), and 3,4-
dichlorothiophenol (26 μL, 0.2 mmol) were added, and then, the
mixture was degassed twice more. The mixture was stirred at reflux
overnight. The reaction mixture was cooled down to room
temperature, filtered, and concentrated. The crude product was
purified by flash column chromatography on silica gel using hexane/
EtOAc (10:1) to afford thioether 33d as a yellow solid, 65 mg, 77.9%
yield. 1H NMR (400 MHz, CDCl3): δ 8.07 (d, J = 7.5 Hz, 1H), 7.69
(d, J = 8.4 Hz, 2H), 7.65 (dd, J = 7.5, 1.0 Hz, 1H), 7.59 (td, J = 7.6,
0.9 Hz, 1H), 7.55 (d, J = 2.1 Hz, 1H), 7.46 (d, J = 8.3 Hz, 1H), 7.43−
7.38 (m, 1H), 7.30 (d, J = 2.1 Hz, 1H), 7.26 (d, J = 8.5 Hz, 2H), 3.69
(s, 3H).
2-(4-((3,4-Dichlorophenyl)thio)benzoyl)benzoic Acid (33). To a
solution of 33d (42 mg, 0.1 mmol) in dioxane (0.5 mL) was added 1
N NaOH (0.5 mL) at room temperature. The mixture was heated to
50 °C. After stirring for 12 h, the mixture was cooled down to room
temperature and diluted with a small amount of water. The mixture
was washed with DCM, and the aqueous phase was collected and
acidified with 1 N HCl solution. The precipitate was filtered and dried
to provide compound 33 as a gray solid, 20 mg, 50% yield. mp 164−
166 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.22 (s, 1H), 7.99 (d, J
= 7.6 Hz, 1H), 7.72 (dd, J = 12.2, 7.3 Hz, 3H), 7.66 (t, J = 7.5 Hz,
1
mg, 73% yield, mp 88−90 °C. H NMR (400 MHz, DMSO-d6): δ
13.18 (s, 1H), 9.18 (t, J = 5.8 Hz, 1H), 8.22 (d, J = 1.7 Hz, 1H), 7.99
(d, J = 7.5 Hz, 1H), 7.71 (t, J = 7.1 Hz, 2H), 7.65 (t, J = 7.4 Hz, 1H),
7.42−7.32 (m, 5H), 7.27 (t, J = 6.7 Hz, 1H), 7.03 (d, J = 9.2 Hz, 1H),
4.71 (d, J = 6.1 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 193.9,
167.4, 147.7, 141.37, 138.3, 135.6, 132.8, 131.1, 130.4, 130.4, 130.2,
129.1, 128.9, 127.8, 127.7, 127.4, 125.0, 115.7, 46.3. HRMS (ESI):
calcd for C21H15N2O5, [M − H]− 375.0986; found, 375.0982. HPLC
purity: 99.55%.
2-(4-((2-Fluorobenzyl)amino)-3-nitrobenzoyl)benzoic Acid (30).
The titled compound was prepared from the hydrolysis of 30c (50
mg, 0.13 mmol) in 1 N NaOH (0.6 mL) and THF (0.6 mL). Yellow
solid, 35 mg, 73% yield, mp 100−102 °C. 1H NMR (400 MHz,
DMSO-d6): δ 13.18 (s, 1H), 9.06 (t, J = 5.9 Hz, 1H), 8.24 (d, J = 1.9
Hz, 1H), 7.99 (d, J = 7.5 Hz, 1H), 7.73 (dd, J = 18.3, 8.4 Hz, 2H),
7.65 (t, J = 7.2 Hz, 1H), 7.36 (m, 3H), 7.28−7.21 (m, 1H), 7.17 (t, J
= 7.4 Hz, 1H), 7.03 (d, J = 9.1 Hz, 1H), 4.77 (d, J = 6.1 Hz, 2H). 13C
NMR (100 MHz, DMSO-d6): δ 193.9, 167.4, 160.6 (d, J = 245.4 Hz),
147.6, 141.3, 135.8, 132.8, 131.2, 130.3 (d, J = 17.2 Hz), 129.8 (d, J =
8.1 Hz), 129.3 (d, J = 4.2 Hz), 128.9, 127.8, 125.2, 125.13, 125.10,
125.0, 124.9, 115.9 (d, J = 21.2 Hz), 115.4, 40.5 (d, J = 4.0 Hz).
HRMS (ESI): calcd for C21H14FN2O5, [M − H]− 393.0892; found,
393.0890. HPLC purity: 99.58%.
P
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
1H), 7.59 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 6.7 Hz, 2H), 7.37 (d, J =
8.3 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 196.0, 167.3, 141.6,
135.9, 134.2, 133.6, 133.0, 132.7, 132.3, 132.0, 130.3, 129.2, 127.8.
HRMS (ESI): calcd for C20H11Cl2O3S, [M − H]− 400.9811; found,
400.9806. HPLC purity 95.07%.
(d, J = 8.6 Hz, 2H), 7.77 (d, J = 8.6 Hz, 2H), 7.49 (t, J = 9.3 Hz, 1H),
3.95 (s, 3H).
Methyl 3-(4-((2-Aminophenyl)thio)-3-nitrobenzamido)benzoate
(35d). To a flask were added 35c (64 mg, 0.2 mmol), 2-
aminobenzenethiol (30 mg, 0.24 mmol), NaOAc (82 mg, 1 mmol),
and EtOH (5 mL). The mixture was stirred at room temperature
overnight. Water was added, and then, the solution was extracted with
ethyl acetate. The combined organic layers were dried over anhydrous
Na2SO4 and purified by chromatography using hexane/EtOAc (10:1)
Synthetic Procedures for Scheme 3. Methyl 2-(3-Amino-4-
((3,4-dichlorophenyl)thio)benzoyl)benzoate (34b). 13c (46 mg, 0.1
mmol), iron powder (28 mg, 0.5 mmol), and saturated ammonium
chloride solution (1 mL) and EtOH (4 mL) were added to a round-
bottom flask and heated to reflux for 1 h. The reaction mixture was
cooled down to room temperature and filtered. The precipitate was
washed with ethyl acetate, and the combined solution was then
washed twice with NaCl solution. The organic phase was dried with
anhydrous Na2SO4 and concentrated to provide 34b (35 mg) as
1
to provide 35d as a pale yellow solid, 45 mg, 53% yield. H NMR
(400 MHz, CDCl3): δ 8.27 (s, 2H), 7.94 (d, J = 8.0 Hz, 1H), 7.45 (d,
J = 7.5 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.36−7.29 (m, 2H), 6.83
(td, J = 8.0, 1.8 Hz, 3H), 6.65 (t, J = 7.5 Hz, 1H), 6.53 (d, J = 8.2 Hz,
1H), 4.49 (d, J = 5.7 Hz, 2H), 4.30 (s, 2H), 3.89 (s, 3H).
1
yellow jelly which was used without further purification. H NMR
Methyl 3-(4-((2-Aminophenyl)thio)-3-nitrobenzamido)benzoate
(36d). To a flask were added 36c (64 mg, 0.2 mmol), 2-
aminobenzenethiol (30 mg, 0.24 mmol), NaOAc (82 mg, 1 mmol),
and EtOH (5 mL). The mixture was stirred at room temperature
overnight. Water was added, and then the aqueous solution was
extracted with ethyl acetate. The combined organic layers were dried
over anhydrous Na2SO4 and purified by chromatography using
hexane/EtOAc (8:1) to provide 36d as a yellow solid, 75 mg, 89%
yield. 1H NMR (400 MHz, CDCl3): δ 9.57 (s, 1H), 8.86 (s, 1H), 8.13
(s, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.6 Hz, 1H), 7.79 (d, J =
7.6 Hz, 1H), 7.43 (t, J = 7.9 Hz, 2H), 7.34 (t, J = 7.7 Hz, 1H), 6.95
(d, J = 8.5 Hz, 1H), 6.84 (m, 2H), 4.39 (s, 2H), 3.91 (s, 3H).
Methyl 4-(4-((2-Aminophenyl)thio)-3-nitrobenzamido)benzoate
(37d). To a flask were added 37c (64 mg, 0.2 mmol), 2-
aminobenzenethiol (30 mg, 0.24 mmol), NaOAc (82 mg, 1 mmol),
and EtOH (5 mL). The mixture was stirred at room temperature
overnight. Water was added, and then, the aqueous solution was
extracted with ethyl acetate. The combined organic layers were dried
over anhydrous Na2SO4 and purified by chromatography using
hexane/EtOAc (8:1) to provide 37d as a yellow solid, 60 mg, 71%
yield. 1H NMR (400 MHz, DMSO-d6): δ 10.75 (s, 1H), 8.86 (d, J =
1.5 Hz, 1H), 8.09 (dd, J = 8.5, 1.6 Hz, 1H), 7.95 (dd, J = 22.2, 8.8 Hz,
4H), 7.39 (d, J = 7.6 Hz, 1H), 7.30 (t, J = 7.3 Hz, 1H), 6.91 (d, J = 8.6
Hz, 1H), 6.86 (d, J = 8.1 Hz, 1H), 6.68 (t, J = 7.2 Hz, 1H), 5.62 (s,
2H), 3.84 (s, 3H).
Methyl 2-(4-((3,4-Dichlorophenyl)thio)-3-nitrobenzamido)-
benzoate (41d). To a flask were added 35c (64 mg, 0.2 mmol), 3,
4-dichlorobenzenethiol (43 mg, 0.24 mmol), NaOAc (82 mg, 1
mmol), and EtOH (5 mL). The mixture was stirred at room
temperature overnight. Water was added, and then, the solution was
extracted with ethyl acetate. The combined organic layers were dried
over anhydrous Na2SO4 and purified by chromatography using
hexane/EtOAc (8:1) to provide 41d as a yellow solid, 83 mg, 87%
yield. 1H NMR (400 MHz, CDCl3): δ 12.24 (s, 1H), 8.98 (d, J = 1.6
Hz, 1H), 8.89 (d, J = 8.5 Hz, 1H), 8.13 (d, J = 7.9 Hz, 1H), 8.04 (dd,
J = 8.5, 1.8 Hz, 1H), 7.76 (d, J = 1.8 Hz, 1H), 7.65 (t, J = 8.6 Hz, 2H),
7.48 (dd, J = 8.2, 1.8 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.04 (d, J =
8.5 Hz, 1H), 4.00 (s, 3H).
Methyl 3-(4-((3,4-Dichlorophenyl)thio)-3-nitrobenzamido)-
benzoate (42d). To a flask were added 36c (64 mg, 0.2 mmol), 3,
4-dichlorobenzenethiol (43 mg, 0.24 mmol), NaOAc (82 mg, 1
mmol), and EtOH (5 mL). The mixture was stirred at room
temperature overnight. Water was added, and then, the solution was
extracted with ethyl acetate. The combined organic layers were dried
over anhydrous Na2SO4 and purified by chromatography using
hexane/EtOAc (8:1) to provide 42d as a yellow solid, 72 mg, 75%
yield. 1H NMR (400 MHz, CDCl3): δ 8.76 (d, J = 1.9 Hz, 1H), 8.19
(t, J = 2.0 Hz, 1H), 8.03 (m, 2H), 7.98 (dd, J = 8.5, 2.0 Hz, 1H), 7.88
(d, J = 7.8 Hz, 1H), 7.74 (d, J = 2.0 Hz, 1H), 7.64 (d, J = 8.3 Hz, 1H),
7.53−7.45 (m, 2H), 7.03 (d, J = 8.5 Hz, 1H), 3.95 (s, 3H).
General Procedure for the Synthesis of Compounds 35−37, 41,
and 42. The methyl esters of the titled compounds (35d−37d, 41d,
and 42d) were hydrolyzed with 1 M NaOH in THF (1:1) at room
temperature overnight. The mixture was then diluted with a small
amount of water and washed with CH2Cl2. The organic phase was
(400 MHz, CDCl3): δ 8.06 (d, J = 7.7 Hz, 1H), 7.67 (t, J = 7.3 Hz,
1H), 7.60 (t, J = 7.5 Hz, 1H), 7.45 (dd, J = 7.5, 4.0 Hz, 2H), 7.31 (d, J
= 8.4 Hz, 1H), 7.24 (d, J = 1.5 Hz, 1H), 7.17 (d, J = 2.1 Hz, 1H), 7.02
(dd, J = 8.0, 1.6 Hz, 1H), 6.95 (dd, J = 8.4, 2.1 Hz, 1H), 4.40 (s, 2H),
3.70 (s, 3H).
2-(3-Amino-4-((3,4-dichlorophenyl)thio)benzoyl)benzoic Acid
(34). To a mixture of 34b (35 mg, 0.08 mmol) in dioxane (0.5
mL) was added 1 N NaOH solution (0.5 mL). The mixture was
heated to 50 °C. After stirring for 12 h, the mixture was cooled down
to room temperature and diluted with a small amount of water. The
mixture was washed with DCM, and the aqueous phase was collected
and acidified by 1 N HCl solution. The precipitate was filtered and
dried to afford compound 34 as a yellow solid, 26 mg, 62% yield. mp
188−190 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.17 (s, 1H), 7.98
(d, J = 7.5 Hz, 1H), 7.71 (t, J = 7.2 Hz, 1H), 7.64 (t, J = 7.3 Hz, 1H),
7.55 (d, J = 8.5 Hz, 1H), 7.40 (d, J = 7.9 Hz, 2H), 7.32 (d, J = 1.9 Hz,
1H), 7.12 (d, J = 1.1 Hz, 1H), 7.02 (dd, J = 8.5, 2.0 Hz, 1H), 6.80
(dd, J = 7.9, 1.3 Hz, 1H), 5.73 (s, 2H). 13C NMR (100 MHz, DMSO-
d6): δ 167.5, 150.5, 142.3, 140.0, 137.3, 137.0, 132.7, 132.1, 131.5,
130.8, 130.1, 128.9, 128.7, 127.6, 117.0, 116.1, 115.6. HRMS (ESI):
calcd for C20H12Cl2NO3S, [M − H]− 415.992; found, 415.9923.
HPLC purity 95.38%.
Synthetic Procedures for Scheme 4. Methyl 2-(4-Fluoro-3-
nitrobenzamido)benzoate (35c). To a solution of 4-fluoro-3-
nitrobenzoic acid 3a (185 mg, 1 mmol) in dry CH2Cl2 (5 mL)
were added oxalyl chloride (2 mmol) and five drops of dry DMF
under a nitrogen atmosphere. The solution was stirred at room
temperature overnight. The solution was concentrated to remove the
excess of oxalyl chloride. The resulting acyl chloride solution was then
added dropwise to a solution of methyl 2-aminobenzoate (1 mmol)
and Et3N (0.279 mL, 2 mmol) in dry CH2Cl2 (5 mL) under a
nitrogen atmosphere, and the mixture was stirred at room
temperature overnight. The solution was then quenched with water
and extracted with CH2Cl2. The organic phase was washed with 1 M
HCl and brine successively, dried over anhydrous Na2SO4, filtered,
and concentrated under reduced pressure. The crude product was
purified via silica gel column using hexane/EtOAc (8:1) to provide
35c; yellow solid, 190 mg, 60%, yield. 1H NMR (400 MHz, CDCl3):
δ 12.30 (s, 1H), 8.88 (d, J = 8.4 Hz, 1H), 8.81 (d, J = 6.6 Hz, 1H),
8.34 (d, J = 5.6 Hz, 1H), 8.14 (d, J = 7.8 Hz, 1H), 7.67 (t, J = 7.6 Hz,
1H), 7.49 (t, J = 9.2 Hz, 1H), 7.21 (t, J = 7.5 Hz, 1H), 4.02 (s, 3H).
Methyl 3-(4-Fluoro-3-nitrobenzamido)benzoate (36c). The titled
compound was prepared by condensing methyl 3-aminobenzoate and
4-fluoro-3-nitrobenzoic acid in a similar manner as described for
compound 35c. An eluent of hexane/EtOAc (8:1) was used for
1
chromatography. Yellow solid, 200 mg, 63% yield. H NMR (400
MHz, CDCl3): δ 8.63 (dd, J = 6.8, 2.1 Hz, 1H), 8.30−8.24 (m, 1H),
8.19 (s, 1H), 8.07 (s, 1H), 8.03 (d, J = 8.2 Hz, 1H), 7.90 (d, J = 7.7
Hz, 1H), 7.55−7.44 (m, 2H), 3.95 (s, 3H).
Methyl 4-(4-Fluoro-3-nitrobenzamido)benzoate (37c). The titled
compound was prepared by condensing methyl 4-aminobenzoate and
4-fluoro-3-nitrobenzoic acid in a similar manner as described for
compound 35c. An eluent of hexane/EtOAc (8:1) was used for
1
chromatography. Yellow solid, 130 mg, 41% yield. H NMR (400
MHz, CDCl3): δ 8.61 (d, J = 4.9 Hz, 1H), 8.32−8.22 (m, 1H), 8.12
Q
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
acidified by the addition of 2 M HCl. The precipitate was filtered and
washed with water to afford the titled compounds.
white solid, 1.55 g, 92% yield. 1H NMR (400 MHz, CDCl3): δ 10.06
(s, 1H), 8.61 (dd, J = 7.0, 1.6 Hz, 1H), 8.22 (ddd, J = 8.3, 4.0, 1.9 Hz,
1H), 7.57−7.45 (m, 1H).
2-(4-((2-Aminophenyl)thio)-3-nitrobenzamido)benzoic Acid
(35). The titled compound was prepared from the hydrolysis of
35d (42 mg, 0.1 mmol) in 1 N NaOH (0.5 mL) and THF (0.5 mL).
Methyl 2-((4-Fluoro-3-nitrobenzyl)amino)benzoate (38c). In a
dried round-bottom flask, 4b (169 mg, 1 mmol) and methyl 2-
aminobenzoate (151 mg, 1 mmol) were dissolved in CH2Cl2 (10 mL)
and stirred at room temperature for 30 min. Then, the reaction was
cooled down to 0 °C, and NaBH(OAc)3 (636 mg, 3 mmol) was
added in three portions over 1 h. The reaction was allowed to stir at
room temperature overnight and quenched with a saturated aqueous
solution of sodium bicarbonate. The mixture was extracted with
CH2Cl2. The combined organic layers were dried with anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The crude
product was purified by column chromatography using hexane/
EtOAc (10:1) to give the titled compound; yellow solid, 155 mg, yield
1
Yellow solid, 30 mg, 73% yield, mp 259−261 °C. H NMR (400
MHz, DMSO-d6): δ 13.86 (s, 1H), 12.27 (s, 1H), 8.81 (s, 1H), 8.61
(d, J = 8.3 Hz, 1H), 8.06 (d, J = 7.2 Hz, 2H), 7.68 (t, J = 7.5 Hz, 1H),
7.39 (d, J = 7.4 Hz, 1H), 7.30 (t, J = 7.4 Hz, 1H), 7.24 (t, J = 7.5 Hz,
1H), 6.96 (d, J = 8.5 Hz, 1H), 6.85 (d, J = 8.1 Hz, 1H), 6.68 (t, J = 7.3
Hz, 1H), 5.65 (s, 2H). 13C NMR (100 MHz, DMSO-d6): δ 170.5,
162.7, 151.3, 145.3, 142.3, 141.0, 137.7, 134.7, 132.9, 132.2, 131.71,
131.69, 128.0, 124.9, 123.8, 120.6, 117.6, 117.3, 115.7, 109.2. HRMS
(ESI): calcd for C20H14N3O5S, [M − H]− 408.0660; found, 408.0656.
HPLC purity: 99.30%.
1
3-(4-((2-Aminophenyl)thio)-3-nitrobenzamido)benzoic Acid
(36). The titled compound was prepared from the hydrolysis of
36d (42 mg, 0.1 mmol) in 1 N NaOH (0.5 mL) and THF (0.5 mL).
51%. H NMR (400 MHz, CDCl3): δ 8.33 (s, 1H), 8.05 (d, J = 6.8
Hz, 1H), 7.97 (d, J = 7.9 Hz, 1H), 7.64 (m, 1H), 7.35−7.23 (m, 2H),
6.68 (t, J = 7.4 Hz, 1H), 6.51 (d, J = 6.9 Hz, 1H), 4.53 (d, J = 5.0 Hz,
2H), 3.91 (s, 3H).
1
Yellow solid, 35 mg, 85% yield, mp 251−252 °C. H NMR (400
MHz, DMSO-d6): δ 13.02 (s, 1H), 10.64 (s, 1H), 8.89 (d, J = 1.7 Hz,
1H), 8.39 (s, 1H), 8.10 (dd, J = 8.5, 1.7 Hz, 1H), 8.05 (d, J = 7.9 Hz,
1H), 7.70 (d, J = 7.7 Hz, 1H), 7.50 (t, J = 7.9 Hz, 1H), 7.39 (dd, J =
7.6, 1.0 Hz, 1H), 7.33−7.26 (m, 1H), 6.88 (dd, J = 19.5, 8.3 Hz, 2H),
6.68 (t, J = 7.1 Hz, 1H), 5.63 (s, 2H). 13C NMR (100 MHz, DMSO-
d6): δ 167.7, 163.6, 151.3, 145.2, 141.6, 139.4, 137.7, 133.1, 132.8,
131.8, 129.4, 127.4, 125.7, 125.2, 124.8, 121.7, 117.3, 115.7, 109.4.
HRMS (ESI): calcd for C20H14N3O5S, [M − H]− 408.0660; found,
408.0653. HPLC purity: 98.48%.
Methyl 3-((4-Fluoro-3-nitrobenzyl)amino)benzoate (39c). The
titled compound was prepared from 4b (169 mg, 1 mmol) and methyl
3-aminobenzoate (151 mg, 1 mmol) in a similar manner as described
for compound 38c. An eluent of hexane/EtOAc (10:1) was used for
1
chromatography. Yellow solid, 220 mg, 72% yield. H NMR (400
MHz, CDCl3): δ 8.06 (dd, J = 7.0, 2.0 Hz, 1H), 7.64 (m, 1H), 7.42
(d, J = 7.7 Hz, 1H), 7.28−7.25 (m, 2H), 7.22 (d, J = 7.8 Hz, 1H),
6.76 (dd, J = 8.1, 2.3 Hz, 1H), 4.45 (d, J = 5.5 Hz, 2H), 3.88 (s, 3H).
Methyl 4-((4-Fluoro-3-nitrobenzyl)amino)benzoate (40c). The
titled compound was prepared from 4b (169 mg, 1 mmol) and methyl
4-aminobenzoate (151 mg, 1 mmol) in a similar manner as described
for compound 38c. An eluent of hexane/EtOAc (10:1) was used for
4-(4-((2-Aminophenyl)thio)-3-nitrobenzamido)benzoic Acid
(37). The titled compound was prepared from the hydrolysis of
37d (42 mg, 0.1 mmol) in 1 N NaOH (0.5 mL) and THF (0.5 mL).
1
1
Yellow solid, 33 mg, 82.5% yield, mp 247−249 °C. H NMR (400
chromatography. Yellow solid, 125 mg, yield 41%. H NMR (400
MHz, DMSO-d6): δ 12.79 (s, 1H), 10.73 (s, 1H), 8.86 (s, 1H), 8.09
(d, J = 7.7 Hz, 1H), 7.95 (m, 2H), 7.90 (m, 2H), 7.39 (m, 1H), 7.30
(m, 1H), 6.90 (m, 1H), 6.85 (m, 1H), 6.67 (m, 1H), 5.63 (s, 2H).
13C NMR (100 MHz, DMSO-d6): δ 167.4, 163.9, 151.3, 145.2, 143.3,
MHz, CDCl3): δ 8.06 (d, J = 6.8 Hz, 1H), 7.88 (d, J = 8.5 Hz, 2H),
7.67−7.60 (m, 1H), 7.30−7.25 (m, 1H), 6.58 (d, J = 8.6 Hz, 2H),
4.70 (s, 1H), 4.49 (d, J = 5.7 Hz, 2H), 3.87 (s, 3H).
Methyl 4-Bromo-3-((4-fluoro-3-nitrobenzyl)amino)benzoate
(46c). In a dried round-bottom flask, 4-fluoro-3-nitrobenzaldehyde
4b, (85 mg, 0.5 mmol), methyl 3-amino-4-bromobenzoate (115 mg,
0.5 mmol), and EtOH (5 mL) were stirred at room temperature for 1
h. Then, the reaction was cooled down to 0 °C, and NaBH4 (32 mg, 1
mmol) was added in two portions over 30 min. The reaction was
allowed to stir at room temperature overnight (or until completion
monitored by TLC). The reaction was quenched with a saturated
aqueous solution of NH4Cl. After extraction with CH2Cl2, the
combined organic layers were dried with anhydrous Na2SO4, filtered,
and concentrated under reduced pressure. The crude product was
purified by column chromatography using hexane/EtOAc (10:1) to
141.8, 137.7, 133.1, 132.8, 131.8, 130.7, 127.4, 126.3, 125.8, 120.1,
117.3, 115.7, 109.4. HRMS (ESI): calcd for C20H14N3O5S, [M − H]−
408.0660; found, 408.0652. HPLC purity: 95.33%.
2-(4-((3,4-Dichlorophenyl)thio)-3-nitrobenzamido)benzoic Acid
(41). The titled compound was prepared from the hydrolysis of
41d (48 mg, 0.1 mmol) in 1 N NaOH (0.5 mL) and THF (0.5 mL).
1
Yellow solid, 27 mg, 58.3% yield, mp 279−281 °C. H NMR (400
MHz, DMSO-d6): δ 12.78 (s, 1H), 8.79 (s, 1H), 8.61 (d, J = 8.3 Hz,
1H), 8.11 (d, J = 8.5 Hz, 1H), 8.06 (d, J = 7.8 Hz, 1H), 8.03 (d, J =
1.8 Hz, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.71−7.59 (m, 2H), 7.21 (dd, J
= 14.5, 8.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 170.5,
162.3, 145.1, 141.4, 140.9, 137.2, 135.9, 134.3, 134.1, 134.0, 133.3,
132.85, 132.80, 132.7, 131.7, 131.1, 129.8, 124.8, 123.7, 120.4. HRMS
(ESI): calcd for C20H11Cl2N2O5S, [M − H]− 460.9771; found,
460.9767. HPLC purity: 95.02%.
1
give the titled compound; yellow solid, 100 mg, 52% yield. H NMR
(400 MHz, CDCl3): δ 8.08 (d, J = 6.3 Hz, 1H), 7.65 (s, 1H), 7.54 (d,
J = 7.9 Hz, 1H), 7.30 (t, J = 7.7 Hz, 2H), 7.18 (s, 1H), 4.99 (s, 1H),
4.54 (d, J = 5.0 Hz, 2H), 3.86 (s, 3H).
3-(4-((3,4-Dichlorophenyl)thio)-3-nitrobenzamido)benzoic Acid
(42). The titled compound was prepared from the hydrolysis of
42d (48 mg, 0.1 mmol) in 1 N NaOH (0.5 mL) and THF (0.5 mL).
Yellow solid, 40 mg, 86.3% yield, mp 280−282 °C. H NMR (400
Methyl 4-Fluoro-3-((4-fluoro-3-nitrobenzyl)amino)benzoate
(47c). The titled compound was prepared in an analogous fashion
as described for compound 46c using 4-fluoro-3-nitrobenzaldehyde
4b (85 mg, 0.5 mmol) and methyl 3-amino-4-fluorobenzoate (85 mg,
0.5 mmol) through the reductive amination reaction. Yellow solid, 97
1
MHz, DMSO-d6): δ 10.70 (s, 1H), 8.89 (s, 1H), 8.38 (s, 1H), 8.15
(d, J = 8.1 Hz, 1H), 8.04 (d, J = 7.6 Hz, 1H), 8.00 (s, 1H), 7.83 (d, J =
8.1 Hz, 1H), 7.70 (d, J = 7.3 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.49
(t, J = 7.6 Hz, 1H), 7.12 (d, J = 8.4 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6): δ 167.6, 163.4, 145.0, 140.9, 139.4, 137.1, 135.9, 134.2,
133.8, 133.3, 132.8, 132.7, 131.9, 131.3, 129.4, 129.3, 125.4, 125.3,
124.9, 121.7. HRMS (ESI): calcd for C20H11Cl2N2O5S, [M − H]−
460.9771; found, 460.9766. HPLC purity: 96.76%.
Synthetic Procedures for Scheme 5. 4-Fluoro-3-nitrobenzal-
dehyde (4b). 4-Fluorobenzaldehyde 4a (1.24 g, 10 mmol) was slowly
added dropwise to a solution of H2SO4 (8 mL) and HNO3 (1 mL) at
0 − 5 °C. The temperature was kept under 5 °C. After the addition
was completed, the solution was warmed to room temperature over 1
h. The reaction mixture was poured into ice; the precipitate was
filtered, washed with ice water, and dried to provide compound 4b;
1
mg, 60% yield. H NMR (400 MHz, CDCl3): δ 8.11 (d, J = 6.4 Hz,
1H), 7.67 (d, J = 7.7 Hz, 1H), 7.49−7.39 (m, 1H), 7.33 (d, J = 9.8
Hz, 1H), 7.28−7.24 (m, 1H), 7.13−7.02 (m, 1H), 4.52 (s, 3H), 3.88
(s, 3H).
Methyl 3-((4-Fluoro-3-nitrobenzyl)amino)-4-methylbenzoate
(48c). The titled compound was prepared in an analogous fashion
as described for compound 46c using 4-fluoro-3-nitrobenzaldehyde
4b (85 mg, 0.5 mmol) and methyl 3-amino-4-methylbenzoate (84 mg,
0.5 mmol) through the reductive amination reaction. An eluent of
hexane/EtOAc (10:1) was used for chromatography. Yellow solid, 73
1
mg, 46% yield. H NMR (400 MHz, CDCl3): δ 8.09 (d, J = 6.9 Hz,
1H), 7.71−7.64 (m, 1H), 7.41 (d, J = 7.6 Hz, 1H), 7.32−7.26 (m,
1H), 7.17 (s, 2H), 4.52 (d, J = 4.4 Hz, 2H), 3.86 (s, 3H), 2.27 (s,
3H).
R
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Methyl 2-Chloro-5-((4-fluoro-3-nitrobenzyl)amino)benzoate
(49c). The titled compound was prepared in an analogous fashion
as described for compound 46c using 4-fluoro-3-nitrobenzaldehyde
4b (85 mg, 0.5 mmol) and methyl 5-amino-2-chlorobenzoate (93 mg,
0.5 mmol) through the reductive amination reaction. An eluent of
hexane/EtOAc (10:1) was used for chromatography. Yellow solid,
2-((4-Fluoro-3-nitrobenzyl)amino)benzamide (56c). The titled
compound was prepared in an analogous fashion as described for
compound 46c using 4-fluoro-3-nitrobenzaldehyde 4b (85 mg, 0.5
mmol) and 2-aminobenzamide (68 mg, 0.5 mmol) through the
reductive amination reaction. An eluent of hexane/EtOAc (8:1) was
1
used for chromatography. Yellow solid, 100 mg, 69% yield. H NMR
1
150 mg, 88% yield. H NMR (400 MHz, CDCl3): δ 8.07 (d, J = 6.6
(400 MHz, CDCl3): δ 8.52 (s, 1H), 8.05 (d, J = 6.8 Hz, 1H), 7.69−
7.61 (m, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.27 (t, J = 8.9 Hz, 1H), 6.68
(t, J = 7.5 Hz, 1H), 6.51 (d, J = 8.4 Hz, 1H), 5.73 (m, 2H), 4.51 (d, J
= 5.8 Hz, 2H).
Hz, 1H), 7.68−7.59 (m, 1H), 7.31 (d, J = 10.2 Hz, 1H), 7.24 (d, J =
8.7 Hz, 1H), 7.05 (d, J = 2.6 Hz, 1H), 6.64 (dd, J = 8.6, 2.7 Hz, 1H),
4.44 (d, J = 5.3 Hz, 2H), 4.37 (s, 1H), 3.92 (s, 3H).
Methyl 3-((4-Fluoro-3-nitrobenzyl)amino)-4-morpholinoben-
zoate (50c). The titled compound was prepared in an analogous
fashion as described for compound 46c using 4-fluoro-3-nitro-
benzaldehyde 4b (85 mg, 0.5 mmol) and methyl 3-amino-4-
morpholinobenzoate (118 mg, 0.5 mmol) through the reductive
amination reaction. An eluent of hexane/EtOAc (6:1) was used for
chromatography. Yellow solid, 88 mg, 45% yield. 1H NMR (400
MHz, CDCl3): δ 8.07 (d, J = 7.1 Hz, 1H), 7.69−7.59 (m, 1H), 7.49
(dd, J = 8.1, 1.6 Hz, 1H), 7.31 (d, J = 10.4 Hz, 1H), 7.18 (d, J = 1.4
Hz, 1H), 7.08 (d, J = 8.1 Hz, 1H), 5.19 (t, J = 5.2 Hz, 1H), 4.49 (d, J
= 5.6 Hz, 2H), 3.93−3.86 (m, 4H), 3.86 (s, 3H), 3.04−2.95 (m, 4H).
3-((4-Fluoro-3-nitrobenzyl)amino)benzamide (51c). The titled
compound was prepared in an analogous fashion as described for
compound 38c using 4-fluoro-3-nitrobenzaldehyde 4b (85 mg, 0.5
mmol) and 3-aminobenzamide (68 mg, 0.5 mmol) through the
reductive amination reaction. An eluent of hexane/EtOAc (15:1) was
2-((4-Fluoro-3-nitrobenzyl)amino)benzonitrile (57c). The titled
compound was prepared in an analogous fashion as described for
compound 46c using 4-fluoro-3-nitrobenzaldehyde (4b, 85 mg, 0.5
mmol) and 2-aminobenzonitrile (59 mg, 0.5 mmol) through the
reductive amination reaction. An eluent of hexane/EtOAc (10:1) was
1
used for chromatography. Yellow solid, 92 mg, 68% yield. H NMR
(400 MHz, CDCl3): δ 8.52 (t, J = 4.7 Hz, 1H), 8.06 (dd, J = 6.9, 1.8
Hz, 1H), 7.70−7.58 (m, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.33−7.23 (m,
1H), 6.68 (t, J = 7.5 Hz, 1H), 6.51 (d, J = 8.4 Hz, 1H), 5.77 (s, 2H),
4.51 (d, J = 5.9 Hz, 2H).
Methyl 2-((4-((2-Aminophenyl)thio)-3-nitrobenzyl)amino)-
benzoate (38d). To a flask were added methyl 2-((4-fluoro-3-
nitrobenzyl)amino)benzoate (38c, 61 mg, 0.2 mmol), 2-amino-
benzenethiol (30 mg, 0.24 mmol), NaOAc (82 mg, 1 mmol), and
EtOH 2 mL. The mixture was stirred at reflux for 4 h. After cooling to
room temperature, the precipitate was filtered, washed with EtOH
and water successively, and dried to provide compound 38d as a
yellow solid, 70 mg, 85% yield. 1H NMR (400 MHz, CDCl3): δ 8.27
(s, 2H), 7.95 (d, J = 7.9 Hz, 1H), 7.45 (d, J = 7.5 Hz, 1H), 7.38 (d, J =
8.4 Hz, 1H), 7.36−7.29 (m, 2H), 6.85−6.82 (m, 3H), 6.65 (t, J = 7.5
Hz, 1H), 6.53 (d, J = 8.4 Hz, 1H), 4.48 (d, J = 5.7 Hz, 2H), 4.30 (s,
2H), 3.89 (s, 3H).
1
used for chromatography. Yellow solid, 65 mg, 45% yield. H NMR
(400 MHz, DMSO-d6): δ 8.13 (d, J = 6.7 Hz, 1H), 7.76 (s, 2H),
7.63−7.48 (m, 1H), 7.17 (s, 1H), 7.14−6.96 (m, 3H), 6.71 (d, J = 7.3
Hz, 1H), 6.57 (s, 1H), 4.40 (s, 2H).
(3-((4-Fluoro-3-nitrobenzyl)amino)phenyl)methanol (53c). The
titled compound was prepared in an analogous fashion as described
for compound 38c using 4-fluoro-3-nitrobenzaldehyde 4b (85 mg, 0.5
mmol) and (3-aminophenyl)methanol (62 mg, 0.5 mmol) through
the reductive amination reaction. An eluent of hexane/EtOAc (8:1)
Methyl 3-((4-((2-Aminophenyl)thio)-3-nitrobenzyl)amino)-
benzoate (39d). The titled compound was prepared from methyl
3-((4-fluoro-3-nitrobenzyl)amino)benzoate (39c, 64 mg, 0.2 mg) and
2-aminobenzenethiol (30 mg, 0.24 mmol) in a similar manner as
1
was used for chromatography. Yellow solid, 88 mg, 64% yield. H
NMR (400 MHz, CDCl3): δ 8.08 (d, J = 6.7 Hz, 1H), 7.71−7.61 (m,
1H), 7.28 (t, J = 9.5 Hz, 1H), 7.18 (t, J = 7.8 Hz, 1H), 6.76 (d, J = 7.4
Hz, 1H), 6.65 (s, 1H), 6.52 (d, J = 7.9 Hz, 1H), 4.62 (s, 2H), 4.44 (s,
2H), 4.29 (s, 1H), 1.62 (s, 1H).
1
described for compound 38d. Yellow solid, 66 mg, 80% yield. H
NMR (400 MHz, CDCl3): δ 8.29 (s, 1H), 7.46 (d, J = 7.6 Hz, 1H),
7.40 (t, J = 8.2 Hz, 2H), 7.34 (t, J = 7.7 Hz, 1H), 7.28 (s, 1H), 7.23 (t,
J = 7.9 Hz, 1H), 6.89−6.79 (m, 3H), 6.76 (dd, J = 8.0, 2.1 Hz, 1H),
4.42 (d, J = 5.3 Hz, 2H), 4.31 (s, 2H), 3.89 (s, 3H).
Methyl 4-Chloro-2-((4-fluoro-3-nitrobenzyl)amino)benzoate
(54c). In a dried round-bottom flask, 4-fluoro-3-nitrobenzaldehyde
4b (85 mg, 0.5 mmol), methyl 2-amino-4-chlorobenzoate (93 mg, 0.5
mmol), and EtOH (1 mL) were stirred at room temperature for 24 h.
The solvent was removed under reduced pressure. The residue was
dissolved in CH2Cl2 (3 mL). NaBH(OAc)3 (316 mg, 1.5 mmol) was
added in three portions over 1 h, and the mixture was stirred at room
temperature overnight. The reaction was quenched with a saturated
aqueous solution of NH4Cl and extracted with CH2Cl2. The
combined organic layers were dried with anhydrous Na2SO4, filtered,
and concentrated under reduced pressure. The crude product was
purified by column chromatography using hexane/EtOAc (10:1) to
give the titled compound. Yellow solid, white solid, 30 mg, yield 18%.
1H NMR (400 MHz, CDCl3): δ 8.40 (s, 1H), 8.05 (dd, J = 6.8, 1.8
Methyl 4-((4-((2-Aminophenyl)thio)-3-nitrobenzyl)amino)-
benzoate (40d). The titled compound was prepared from methyl
4-((4-fluoro-3-nitrobenzyl)amino)benzoate (40c, 61 mg, 0.2 mmol)
and 2-aminobenzenethiol (30 mg, 0.24 mmol) in a similar manner as
1
described for compound 38d. Yellow solid, 66 mg, 80% yield. H
NMR (400 MHz, CDCl3): δ 8.27 (s, 1H), 7.86 (d, J = 8.5 Hz, 2H),
7.46 (d, J = 7.6 Hz, 1H), 7.35 (m, 2H), 6.84 (m, 3H), 6.56 (d, J = 8.6
Hz, 2H), 4.60 (t, J = 5.1 Hz, 1H), 4.44 (d, J = 5.6 Hz, 2H), 4.30 (s,
2H), 3.86 (s, 3H).
Methyl 2-((4-((3,4-Dichlorophenyl)thio)-3-nitrobenzyl)amino)-
benzoate (43d). The titled compound was prepared from methyl
2-((4-fluoro-3-nitrobenzyl)amino)benzoate (38c, 64 mg, 0.2 mg) and
3,4-dichlorobenzenethiol (43 mg, 0.24 mmol) in a similar manner as
Hz, 1H), 7.90 (d, J = 8.5 Hz, 1H), 7.68−7.59 (m, 1H), 7.35−7.29 (m,
1H), 6.65 (dd, J = 8.6, 1.8 Hz, 1H), 6.51 (d, J = 1.6 Hz, 1H), 4.51 (d,
J = 5.8 Hz, 2H), 3.90 (s, 3H).
1
described for compound 38d. Yellow solid, 72 mg, 78% yield. H
NMR (400 MHz, CDCl3): δ 8.29 (t, J = 5.5 Hz, 1H), 8.23 (s, 1H),
7.96 (dd, J = 8.0, 1.4 Hz, 1H), 7.69 (d, J = 2.0 Hz, 1H), 7.56 (d, J =
8.3 Hz, 1H), 7.46−7.38 (m, 2H), 7.35−7.29 (m, 1H), 6.87 (d, J = 8.4
Hz, 1H), 6.67 (t, J = 7.5 Hz, 1H), 6.52 (d, J = 8.4 Hz, 1H), 4.51 (d, J
= 5.9 Hz, 2H), 3.90 (s, 3H).
2-Chloro-6-((4-fluoro-3-nitrobenzyl)amino)benzoic Acid (55c).
In a dried round-bottom flask, 4-fluoro-3-nitrobenzaldehyde 4b (85
mg, 0.5 mmol), 2-amino-6-chlorobenzoic acid (86 mg, 0.5 mmol),
and EtOH (5 mL) were stirred at room for 24 h. NaBH(OAc)3 (316
mg, 1.5 mmol) was added in three portions over 1 h, and the mixture
was stirred at room temperature for 2 days. The reaction was
quenched with an excess amount of saturated aqueous solution of
NaHCO3. The aqueous phase was extracted with CH2Cl2. The
organic layers were acidified, and the precipitate was filtered and dried
to provide the titled compound as a gray solid, 120 mg, yield 74%. 1H
NMR (400 MHz, CDCl3): δ 8.09−8.00 (m, 1H), 7.62 (dd, J = 4.6,
3.4 Hz, 1H), 7.31 (d, J = 10.4 Hz, 1H), 7.17 (t, J = 8.2 Hz, 1H), 6.82
(d, J = 8.0 Hz, 1H), 6.43 (d, J = 8.5 Hz, 1H), 4.51 (s, 2H).
Methyl 3-((4-((3,4-Dichlorophenyl)thio)-3-nitrobenzyl)amino)-
benzoate (44d). The titled compound was prepared from methyl
3-((4-fluoro-3-nitrobenzyl)amino)benzoate (39c, 64 mg, 0.2 mg) and
3,4-dichlorobenzenethiol (43 mg, 0.24 mmol) in a similar manner as
1
described for compound 38d. Yellow solid, 76 mg, 82% yield. H
NMR (400 MHz, CDCl3): δ 8.25 (s, 1H), 7.68 (d, J = 1.9 Hz, 1H),
7.56 (d, J = 8.3 Hz, 1H), 7.49−7.35 (m, 4H), 7.23 (t, J = 7.9 Hz, 1H),
6.87 (d, J = 8.3 Hz, 1H), 6.76 (dd, J = 8.0, 2.1 Hz, 1H), 4.44 (d, J =
5.7 Hz, 2H), 4.38 (d, J = 5.6 Hz, 1H), 3.89 (s, 3H).
S
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Methyl 4-Bromo-3-((4-((3,4-dichlorophenyl)thio)-3-nitrobenzyl)-
amino)benzoate (46d). The titled compound was prepared from
methyl 4-bromo-3-((4-fluoro-3-nitrobenzyl)amino)benzoate 46c (77
mg, 0.2 mmol) and 3,4-dichlorobenzenethiol (43 mg, 0.24 mmol) in a
similar manner as described for compound 38d. Yellow solid, 97 mg,
89% yield. 1H NMR (400 MHz, CDCl3): δ 8.27 (s, 1H), 7.71 (d, J =
1.9 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.54 (d, J = 8.2 Hz, 1H), 7.43
(d, J = 8.2 Hz, 2H), 7.31 (d, J = 1.7 Hz, 1H), 7.19 (d, J = 1.5 Hz, 1H),
6.89 (d, J = 8.4 Hz, 1H), 4.93 (t, J = 5.6 Hz, 1H), 4.53 (d, J = 5.9 Hz,
2H), 3.88 (s, 3H).
Methyl 3-((4-((3,4-Dichlorophenyl)thio)-3-nitrobenzyl)amino)-4-
fluorobenzoate (47d). The titled compound was prepared from
methyl 4-fluoro-3-((4-fluoro-3-nitrobenzyl)amino)benzoate 47c (100
mg, 0.31 mmol) and 3,4-dichlorobenzenethiol (66.7 mg, 0.37 mmol)
in a similar manner as described for compound 38d. Yellow solid, 82
mg, 55% yield. 1H NMR (400 MHz, CDCl3): δ 8.27 (s, 1H), 7.70 (s,
1H), 7.57 (dd, J = 8.2, 1.8 Hz, 1H), 7.43 (m, 3H), 7.31−7.25 (m,
1H), 7.12−6.99 (m, 1H), 6.90 (dd, J = 8.3, 1.6 Hz, 1H), 4.49 (s, 3H),
3.88 (s, 3H).
Methyl 3-((4-((3,4-Dichlorophenyl)thio)-3-nitrobenzyl)amino)-4-
methylbenzoate (48d). The titled compound was prepared from
methyl 3-((4-fluoro-3-nitrobenzyl)amino)-4-methylbenzoate 48c (63
mg, 0.2 mmol) and 3,4-dichlorobenzenethiol (43 mg, 0.24 mmol) in a
similar manner as described for compound 38d. Yellow solid, 58 mg,
61% yield. 1H NMR (400 MHz, CDCl3): δ 8.27 (s, 1H), 7.70 (d, J =
1.7 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.43 (m, 3H), 7.20−7.13 (m,
2H), 6.89 (d, J = 8.4 Hz, 1H), 4.51 (d, J = 5.7 Hz, 2H), 4.05 (t, J = 5.4
Hz, 1H), 3.87 (s, 3H), 2.25 (s, 3H).
8.22 (s, 1H), 7.80 (d, J = 7.5 Hz, 1H), 7.54 (d, J = 8.1 Hz, 1H), 7.33
(d, J = 7.4 Hz, 1H), 7.27 (dd, J = 16.9, 8.2 Hz, 2H), 6.82 (d, J = 8.0
Hz, 1H), 6.74 (d, J = 8.3 Hz, 1H), 6.63 (t, J = 7.5 Hz, 2H), 6.57 (t, J =
7.4 Hz, 1H), 5.53 (s, 2H), 4.52 (s, 2H). 13C NMR (100 MHz,
DMSO-d6): δ 170.4, 151.4, 150.7, 145.4, 138.3, 137.9, 135.9, 134.8,
133.4, 132.6, 132.2, 127.7, 124.6, 117.2, 115.6, 115.3, 112.1, 111.3,
110.1, 44.8. HRMS (ESI): calcd for C20H16N3O4S, [M − H]−
394.0867; found, 394.0862. HPLC purity: 99.32%.
3-((4-((2-Aminophenyl)thio)-3-nitrobenzyl)amino)benzoic Acid
(39). The titled compound was prepared from the hydrolysis of
methyl 3-((4-((2-aminophenyl)thio)-3-nitrobenzyl)amino)benzoate
39d (41 mg, 0.1 mmol) in 1 M NaOH (0.3 mL) and dioxane (1
mL) at 50 °C overnight. Yellow solid, 27 mg, 68% yield, mp 105−107
1
°C. H NMR (400 MHz, DMSO-d6): δ 12.66 (s, 1H), 8.25 (s, 1H),
7.55 (d, J = 8.3 Hz, 1H), 7.33 (d, J = 7.5 Hz, 1H), 7.26 (t, J = 7.6 Hz,
1H), 7.20−7.10 (m, 3H), 6.82 (d, J = 8.0 Hz, 1H), 6.75 (m, 2H), 6.63
(m, 2H), 5.53 (s, 2H), 4.34 (d, J = 5.2 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6): δ 168.3, 151.4, 148.7, 145.4, 138.7, 137.9, 135.8, 133.5,
132.6, 132.1, 129.5, 127.3, 124.6, 117.6, 117.2, 116.7, 115.6, 113.6,
110.1, 45.4. HRMS (ESI): calcd for C20H16N3O4S, [M − H]−
394.0867; found, 394.0863. HPLC purity: 97.18%.
4-((4-((2-Aminophenyl)thio)-3-nitrobenzyl)amino)benzoic Acid
(40). The titled compound was prepared from the hydrolysis of
methyl 4-((4-((2-aminophenyl)thio)-3-nitrobenzyl)amino)benzoate
40d (41 mg, 0.1 mmol) in 1 M NaOH (0.3 mL) and dioxane (1
mL) at 50 °C overnight. Yellow solid, 33 mg, 84% yield,
1
decomposition temperature 250 °C. H NMR (400 MHz, DMSO-
d6): δ 12.04 (s, 1H), 8.23 (s, 1H), 7.64 (d, J = 8.6 Hz, 2H), 7.54 (d, J
= 8.2 Hz, 1H), 7.33 (d, J = 7.3 Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H), 7.13
(t, J = 5.9 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.73 (d, J = 8.4 Hz, 1H),
6.64 (t, J = 7.3 Hz, 1H), 6.59 (d, J = 8.6 Hz, 2H), 5.52 (s, 2H), 4.38
(d, J = 5.8 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 152.2, 151.4,
145.4, 138.3, 137.9, 135.8, 133.6, 132.6, 131.6, 127.4, 124.7, 117.2,
115.6, 111.7, 110.1, 45.0. HRMS (ESI): calcd for C20H16N3O4S, [M
− H]− 394.0867; found, 394.0863. HPLC purity: 96.24%.
Methyl 2-Chloro-5-((4-((3,4-dichlorophenyl)thio)-3-nitrobenzyl)-
amino)benzoate (49d). The titled compound was prepared from
methyl 2-chloro-5-((4-fluoro-3-nitrobenzyl)amino)benzoate 49c (67
mg, 0.2 mmol) and 3,4-dichlorobenzenethiol (43 mg, 0.24 mmol) in a
similar manner as described for compound 38d. Yellow solid, 73 mg,
1
73% yield. H NMR (400 MHz, CDCl3) δ 8.24 (d, J = 0.9 Hz, 1H),
7.69 (d, J = 1.9 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.41 (td, J = 7.9,
1.7 Hz, 2H), 7.22 (d, J = 8.7 Hz, 1H), 7.04 (d, J = 2.9 Hz, 1H), 6.88
(d, J = 8.4 Hz, 1H), 6.62 (dd, J = 8.7, 2.9 Hz, 1H), 4.41 (d, J = 5.7 Hz,
2H), 4.32 (t, J = 5.6 Hz, 1H), 3.92 (s, 3H).
2-((4-((3,4-Dichlorophenyl)thio)-3-nitrobenzyl)amino)benzoic
Acid (43). The titled compound was prepared from the hydrolysis of
methyl 2-((4-((3,4-dichlorophenyl)thio)-3-nitrobenzyl)amino)-
benzoate 43d (46 mg, 0.1 mmol) in 1 M NaOH (0.3 mL) and
dioxane (1 mL) at 50 °C overnight. Yellow solid, 22 mg, 49% yield,
Methyl 3-((4-((3,4-Dichlorophenyl)thio)-3-nitrobenzyl)amino)-4-
morpholinobenzoate (50d). The titled compound was prepared
from methyl 3-((4-fluoro-3-nitrobenzyl)amino)-4-morpholinoben-
zoate 50c (78 mg, 0.2 mmol) and 3,4-dichlorobenzenethiol (43 mg,
0.24 mmol) in a similar manner as described for compound 38d.
Yellow solid, 49 mg, 45% yield. 1H NMR (400 MHz, CDCl3): δ 8.25
(s, 1H), 7.71 (s, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.48 (d, J = 8.2 Hz,
1H), 7.42 (t, J = 8.4 Hz, 2H), 7.19 (s, 1H), 7.07 (d, J = 8.1 Hz, 1H),
6.89 (d, J = 8.4 Hz, 1H), 5.16 (t, J = 5.6 Hz, 1H), 4.47 (d, J = 5.7 Hz,
2H), 3.88 (m, 7H), 3.10−2.91 (m, 4H).
1
mp 171−173 °C. H NMR (400 MHz, DMSO-d6): δ 9.56 (s, 1H),
8.17 (s, 1H), 7.91 (s, 1H), 7.84 (d, J = 7.3 Hz, 1H), 7.78 (d, J = 8.3
Hz, 1H), 7.56 (t, J = 5.7 Hz, 2H), 7.06 (t, J = 7.1 Hz, 1H), 7.00 (d, J =
8.1 Hz, 1H), 6.50−6.34 (m, 2H), 4.46 (s, 2H). 13C NMR (100 MHz,
DMSO-d6): δ 150.1, 146.0, 140.8, 136.5, 135.3, 133.8, 133.6, 133.5,
133.0, 132.6, 132.4, 132.0, 129.9, 124.2, 118.6, 114.7, 110.9, 45.2.
HRMS (ESI): calcd for C20H13Cl2N2O4S, [M − H]− 446.9979;
found, 446.9968. HPLC purity: 96.26%.
Methyl 4-Chloro-2-((4-((3,4-dichlorophenyl)thio)-3-nitrobenzyl)-
amino)benzoate (54d). The titled compound was prepared from
methyl 4-chloro-2-((4-fluoro-3-nitrobenzyl)amino)benzoate 54c (30
mg, 0.09 mmol) and 3,4-dichlorobenzenethiol (18 mg, 0.1 mmol) in a
similar manner as described for compound 38d. Yellow solid, 23 mg,
3-((4-((3,4-Dichlorophenyl)thio)-3-nitrobenzyl)amino)benzoic
Acid (44). The titled compound was prepared from the hydrolysis of
methyl 3-((4-((3,4-dichlorophenyl)thio)-3-nitrobenzyl)amino)-
benzoate 44d (46 mg, 0.1 mmol) in 1 M NaOH (0.3 mL) and
dioxane (1 mL) at 50 °C overnight. Yellow solid, 25 mg, 56% yield,
1
44% yield. H NMR (400 MHz, CDCl3): δ 8.36 (t, J = 5.4 Hz, 1H),
1
mp 224−226 °C. H NMR (400 MHz, DMSO-d6): δ 8.23 (s, 1H),
8.23 (s, 1H), 7.88 (d, J = 8.5 Hz, 1H), 7.71 (d, J = 1.9 Hz, 1H), 7.57
(d, J = 8.3 Hz, 1H), 7.43 (dd, J = 8.4, 2.0 Hz, 2H), 6.89 (d, J = 8.4 Hz,
1H), 6.64 (dd, J = 8.5, 1.8 Hz, 1H), 6.51 (d, J = 1.5 Hz, 1H), 4.48 (d,
J = 5.8 Hz, 2H), 3.89 (s, 3H).
7.92 (d, J = 1.9 Hz, 1H), 7.78 (d, J = 8.3 Hz, 1H), 7.60 (dd, J = 8.4,
1.2 Hz, 1H), 7.56 (dd, J = 8.3, 2.0 Hz, 1H), 7.16 (s, 1H), 7.13 (m,
2H), 7.01 (d, J = 8.3 Hz, 1H), 6.78−6.71 (m, 1H), 6.63 (t, J = 5.9 Hz,
1H), 4.38 (d, J = 5.8 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ
148.4, 145.9, 140.5, 136.6, 135.3, 134.0, 133.8, 133.5, 133.0, 132.6,
132.3, 129.9, 129.1, 124.3, 117.9, 115.9, 113.7, 45.5. HRMS (ESI):
calcd for C20H13Cl2N2O4S, [M − H]− 446.9979; found, 446.9975.
HPLC purity: 95.35%.
General Procedure for the Synthesis of Compounds 38−40, 43−
44, 46−51, and 54−57. The methyl esters of the intermediates
(38d−40d, 43d−44d, 46d−50d, and 54d) were hydrolyzed with 1 M
NaOH in dioxane (1:1) at 50 °C overnight. The mixture was then
diluted with a small amount of water and washed with CH2Cl2. The
solution was acidified by the addition of 2 M HCl. The precipitate was
filtered, washed with water, and dried to afford the titled compounds.
2-((4-((2-Aminophenyl)thio)-3-nitrobenzyl)amino)benzoic Acid
(38). The titled compound was prepared from the hydrolysis of
methyl 2-((4-((2-aminophenyl)thio)-3-nitrobenzyl)amino)benzoate
38d (41 mg, 0.1 mmol) in 1 M NaOH (0.3 mL) and dioxane (1
mL) at 50 °C overnight. Yellow solid, 21 mg, 53% yield, mp 201−203
4-Bromo-3-((4-((3,4-dichlorophenyl)thio)-3-nitrobenzyl)amino)-
benzoic Acid (46). The titled compound was prepared from the
hydrolysis of methyl 4-bromo-3-((4-((3,4-dichlorophenyl)thio)-3-
nitrobenzyl)amino)benzoate 46d (40 mg, 0.07 mmol) in 1 M
NaOH (0.3 mL) and dioxane (1.0 mL) at 50 °C overnight. Yellow
solid, 35 mg, 90% yield, mp 231−233 °C. 1H NMR (400 MHz,
DMSO-d6): δ 8.22 (s, 1H), 7.92 (d, J = 1.8 Hz, 1H), 7.77 (d, J = 8.3
Hz, 1H), 7.62−7.52 (m, 2H), 7.49 (d, J = 8.0 Hz, 1H), 7.07 (d, J =
1
°C. H NMR (400 MHz, DMSO-d6): δ 12.77 (s, 1H), 8.38 (s, 1H),
T
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
8.3 Hz, 1H), 7.01 (d, J = 8.1 Hz, 2H), 6.26 (s, 1H), 4.51 (d, J = 5.9
Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 168.4, 145.8, 144.8,
139.7, 136.7, 135.4, 134.4, 133.6, 133.5, 133.0, 132.8, 132.6, 132.2,
129.9, 124.1, 118.9, 113.1, 112.2, 45.4. HRMS (ESI): calcd for
C20H12BrCl2N2O4S, [M − H]− 524.9084; found, 524.9079. HPLC
purity: 99.36%.
(t, J = 6.1 Hz, 1H), 4.39 (d, J = 6.1 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6): δ 168.9, 148.5, 145.8, 140.5, 136.6, 135.7, 135.4, 134.1,
133.9, 133.6, 133.0, 132.6, 132.3, 129.8, 129.2, 124.3, 115.8, 115.6,
111.8, 45.3. HRMS (ESI): calcd for C20H14Cl2N3O3S, [M − H]−
446.0138; found, 446.0129. HPLC purity: 100.00%.
(3-((4-((3,4-Dichlorophenyl)thio)-3-nitrobenzyl)amino)phenyl)-
methanol (53). The titled compound was prepared from (3-((4-
fluoro-3-nitrobenzyl)amino)phenyl)methanol 53c (85 mg, 0.30
mmol) and 3,4-dichlorobenzenethiol (77 mg, 0.45 mmol) in a similar
manner as described for compound 38d. Yellow solid, 100 mg, 77%
3-((4-((3,4-Dichlorophenyl)thio)-3-nitrobenzyl)amino)-4-fluoro-
benzoic Acid (47). The titled compound was prepared from the
hydrolysis of methyl 3-((4-((3,4-dichlorophenyl)thio)-3-nitrobenzyl)-
amino)-4-fluorobenzoate 47d (30 mg, 0.06 mmol) in 1 M NaOH (0.2
mL) and dioxane (0.6 mL) at 50 °C overnight. Yellow solid, 21 mg,
1
yield, mp 118−120 °C. H NMR (400 MHz, CDCl3): δ 8.24 (d, J =
1
0.9 Hz, 1H), 7.67 (d, J = 2.0 Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H), 7.41
(td, J = 8.2, 1.8 Hz, 2H), 7.15 (t, J = 7.8 Hz, 1H), 6.87 (d, J = 8.4 Hz,
1H), 6.73 (d, J = 7.5 Hz, 1H), 6.63 (s, 1H), 6.50 (dd, J = 8.0, 2.0 Hz,
1H), 4.60 (d, J = 5.9 Hz, 2H), 4.41 (d, J = 5.8 Hz, 2H), 4.23 (t, J = 5.4
Hz, 1H), 1.60 (t, J = 6.0 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ
147.5, 145.5, 142.3, 138.5, 136.9, 136.1, 134.7, 134.6, 134.0, 132.5,
131.9, 131.5, 129.6, 128.9, 124.3, 116.8, 112.1, 111.4, 65.4, 46.8.
HRMS (ESI): calcd for C20H15Cl2N2O3S, [M − H]− 433.0186;
found, 433.0184. HPLC purity: 96.19%.
75% mg, mp 237−239 °C. H NMR (400 MHz, DMSO-d6): δ 8.24
(s, 1H), 7.91 (s, 1H), 7.77 (d, J = 8.3 Hz, 1H), 7.60 (d, J = 8.2 Hz,
1H), 7.56 (d, J = 8.2 Hz, 1H), 7.18 (m, 1H), 7.10 (m, 2H), 7.00 (d, J
= 8.3 Hz, 1H), 6.52 (s, 1H), 4.45 (d, J = 5.4 Hz, 2H). 13C NMR (100
MHz, DMSO-d6): δ 168.3, 153.7 (d, J = 245.4 Hz), 145.8, 134, 136.6,
136.2, 136.1, 135.4, 134.3, 133.64, 133.57, 133.0, 132.6, 132.2, 129.9,
124.2, 118.4 (d, J = 8.0 Hz), 114.8 (d, J = 18.2 Hz), 113.2 (d, J = 4.0
Hz), 45.0. HRMS (ESI): calcd for C20H12Cl2FN2O4S, [M − H]−
464.9884; found, 464.9872. HPLC purity: 100.00%.
4-Chloro-2-((4-((3,4-dichlorophenyl)thio)-3-nitrobenzyl)amino)-
benzoic Acid (54). The titled compound was prepared from the
hydrolysis of methyl 4-chloro-2-((4-((3,4-dichlorophenyl)thio)-3-
nitrobenzyl)amino)benzoate 54d (30 mg, 0.06 mmol) in 1 M
NaOH (0.2 mL) and dioxane (0.8 mL) at 50 °C overnight. Yellow
solid, 20 mg, 69% yield, mp 220−222 °C. 1H NMR (400 MHz,
DMSO-d6): δ 12.95 (s, 1H), 8.54 (s, 1H), 8.23 (s, 1H), 7.93 (d, J =
1.8 Hz, 1H), 7.79 (m, 2H), 7.58 (m, 2H), 7.02 (d, J = 8.4 Hz, 1H),
6.69 (d, J = 1.3 Hz, 1H), 6.60 (dd, J = 8.5, 1.6 Hz, 1H), 4.59 (s, 2H).
13C NMR (100 MHz, DMSO-d6): δ 169.9, 151.5, 145.8, 139.4, 139.2,
136.7, 135.5, 134.6, 134.0, 133.6, 133.0, 132.6, 132.2, 130.0, 124.4,
115.3, 111.3, 110.9, 44.6. HRMS (ESI): calcd for C20H12Cl3N2O4S,
[M − H]− 480.9589; found, 480.9579. HPLC purity: 95.83%.
2-Chloro-6-((4-((3,4-Dichlorophenyl)thio)-3-nitrobenzyl)amino)-
benzoic Acid (55). The titled compound was prepared from 2-chloro-
6-((4-fluoro-3-nitrobenzyl)amino)benzoic acid 55c (120 mg, 0.5
mmol) and 3,4-dichlorobenzenethiol (107 mg, 0.6 mmol) in a similar
manner as described for compound 38d. Yellow solid, 90 mg, 37%
yield, mp 204−206 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.47 (s,
1H), 8.21 (s, 1H), 7.91 (d, J = 1.9 Hz, 1H), 7.78 (d, J = 8.3 Hz, 1H),
7.56 (d, J = 8.3 Hz, 2H), 7.09 (t, J = 8.2 Hz, 1H), 7.00 (d, J = 8.3 Hz,
1H), 6.65 (d, J = 7.8 Hz, 1H), 6.47 (d, J = 8.4 Hz, 1H), 4.46 (s, 2H).
13C NMR (100 MHz, DMSO-d6): δ 168.0, 147.2, 145.8, 139.8, 136.6,
135.4, 134.3, 133.6, 133.0, 132.6, 132.2, 131.8, 131.6, 129.8, 124.3,
118.7, 117.4, 110.4, 45.2. HRMS (ESI): calcd for C20H12Cl3N2O4S,
[M − H]− 480.9589; found, 480.9579. HPLC purity: 95.12%.
2-((4-((3,4-Dichlorophenyl)thio)-3-nitrobenzyl)amino)-
benzamide (56). The titled compound was prepared from 2-((4-
fluoro-3-nitrobenzyl)amino)benzamide 56c (58 mg, 0.20 mmol) and
3,4-dichlorobenzenethiol (43 mg, 0.24 mmol) in a similar manner as
described for compound 38d. Yellow solid, 37 mg, 41% yield, mp
184−186 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.68 (t, J = 6.0 Hz,
1H), 8.19 (s, 1H), 7.92 (d, J = 1.9 Hz, 1H), 7.88 (s, 1H, C(
O)NH), 7.78 (d, J = 8.3 Hz, 1H), 7.62 (d, J = 7.3 Hz, 1H), 7.60−7.54
(m, 2H), 7.22 (s, C(O)NH 1H), 7.19 (t, J = 7.7 Hz, 1H), 7.01 (d, J
= 8.3 Hz, 1H), 6.62−6.50 (m, 2H), 4.49 (d, J = 6.1 Hz, 2H). 13C
NMR (100 MHz, DMSO-d6): δ 172.0, 149.5, 145.8, 140.2, 136.6,
135.4, 134.3, 133.7, 133.6, 133.04, 132.98, 132.6, 132.3, 130.0, 129.6,
124.2, 115.2, 115.1, 112.0, 45.0. HRMS (ESI): calcd for
C20H16Cl2N3O3S, [M + H]+ 448.0284; found, 448.0276. HPLC
purity: 100.00%.
3-((4-((3,4-Dichlorophenyl)thio)-3-nitrobenzyl)amino)-4-methyl-
benzoic Acid (48). The titled compound was prepared from the
hydrolysis of methyl 3-((4-((3,4-dichlorophenyl)thio)-3-nitrobenzyl)-
amino)-4-methylbenzoate 48d (42 mg, 0.09 mmol) in 1 M NaOH
(0.3 mL) and dioxane (1.0 mL) at 50 °C overnight. Yellow solid, 27
mg, 65% yield, mp 222−224 °C. 1H NMR (400 MHz, DMSO-d6): δ
8.23 (s, 1H), 7.91 (s, 1H), 7.77 (d, J = 8.3 Hz, 1H), 7.58 (m, 2H),
7.09 (m, 2H), 6.99 (d, J = 8.3 Hz, 1H), 6.91 (s, 1H), 5.95 (s, 1H),
4.45 (d, J = 5.1 Hz, 2H), 2.21 (s, 3H). 13C NMR (100 MHz, DMSO-
d6): δ 169.2, 145.94, 145.87, 140.58, 136.6, 135.4, 134.0, 133.6, 133.6,
133.0, 132.6, 132.3, 130.2, 129.9, 127.3, 124.2, 118.1, 110.2, 45.5,
18.4. HRMS (ESI): calcd for C21H15Cl2N2O4S, [M − H]− 461.0135;
found, 461.0128. HPLC purity: 98.78%.
2-Chloro-5-((4-((3,4-dichlorophenyl)thio)-3-nitrobenzyl)amino)-
benzoic Acid (49). The titled compound was prepared from the
hydrolysis of methyl 2-chloro-5-((4-((3,4-dichlorophenyl)thio)-3-
nitrobenzyl)amino)benzoate 49d (30 mg, 0.06 mmol) in 1 M
NaOH (0.2 mL) and dioxane (0.6 mL) at 50 °C overnight. Yellow
solid, 22 mg, 76% yield, mp 148−150 °C. 1H NMR (400 MHz,
DMSO-d6): δ 8.20 (d, J = 1.0 Hz, 1H), 7.92 (d, J = 2.0 Hz, 1H), 7.78
(d, J = 8.3 Hz, 1H), 7.64−7.47 (m, 2H), 7.00 (d, J = 8.3 Hz, 1H),
6.93 (d, J = 8.5 Hz, 1H), 6.62 (s, 1H), 6.50−6.34 (m, 2H), 4.31 (d, J
= 6.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 146.9, 146.8,
145.8, 140.5, 136.6, 135.4, 134.1, 133.8, 133.6, 133.0, 132.6, 132.3,
123.0, 129.8, 124.2, 117.4, 113.4, 112.9, 45.5. HRMS (ESI): calcd for
C20H12Cl3N2O4S, [M − H]− 480.9589; found, 480.9589. HPLC
purity: 98.50%.
3-((4-((3,4-Dichlorophenyl)thio)-3-nitrobenzyl)amino)-4-mor-
pholinobenzoic Acid (50). The titled compound was prepared from
the hydrolysis of methyl 3-((4-((3,4-dichlorophenyl)thio)-3-
nitrobenzyl)amino)-4-morpholinobenzoate 50d (35 mg, 0.06 mmol)
in 1 M NaOH (0.2 mL) and dioxane (0.6 mL) at 50 °C overnight.
1
Yellow solid, 22 mg, 65% yield, mp 147−149 °C. H NMR (400
MHz, DMSO-d6): δ 12.45 (s, 1H), 8.22 (s, 1H), 7.92 (s, 1H), 7.77
(d, J = 8.2 Hz, 1H), 7.56 (t, J = 5.5 Hz, 2H), 7.24 (d, J = 7.7 Hz, 1H),
7.02 (dd, J = 12.0, 8.4 Hz, 2H), 6.92 (s, 1H), 5.94 (t, J = 5.2 Hz, 1H),
4.48 (d, J = 5.1 Hz, 2H), 3.82 (s, 4H), 2.90 (s, 4H). 13C NMR (100
MHz, DMSO-d6): δ 170.3, 145.8, 141.4, 141.3, 140.9, 136.7, 135.5,
134.1, 133.6, 133.4, 133.0, 132.6, 132.2, 129.8, 123.9, 118.9, 118.7,
111.3, 67.0, 51.6, 45.9. HRMS (ESI): calcd for C24H20Cl2N3O5S, [M
− H]− 532.0506; found, 532.0499. HPLC purity: 95.73%.
3-((4-((3,4-Dichlorophenyl)thio)-3-nitrobenzyl)amino)-
benzamide (51). The titled compound was prepared from 3-((4-
fluoro-3-nitrobenzyl)amino)benzamide 51c (58 mg, 0.2 mmol) and
3,4-dichlorobenzenethiol (51 mg, 0.3 mmol) in a similar manner as
described for compound 38d. Yellow solid, 40 mg, 45% yield, mp
2-((4-((3,4-Dichlorophenyl)thio)-3-nitrobenzyl)amino)-
benzonitrile (57). The titled compound was prepared from 2-((4-
fluoro-3-nitrobenzyl)amino)benzonitrile 57c (54 mg, 0.20 mmol) and
3,4-dichlorobenzenethiol (43 mg, 0.24 mmol) in a similar manner as
described for compound 38d. Yellow solid, 50 mg, 58% yield, mp
173−175 °C. 1H NMR (400 MHz, CDCl3): δ 8.24 (s, 1H), 7.70 (d, J
= 1.8 Hz, 1H), 7.57 (d, J = 8.2 Hz, 1H), 7.50−7.39 (m, 3H), 7.36 (t, J
= 7.8 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.76 (t, J = 7.5 Hz, 1H), 6.54
(d, J = 8.5 Hz, 1H), 5.13 (t, J = 5.3 Hz, 1H), 4.51 (d, J = 5.8 Hz, 2H).
1
158−160 °C. H NMR (400 MHz, DMSO-d6): δ 8.23 (s, 1H), 7.91
(s, 1H), 7.77 (d, J = 8.3 Hz, 2H), 7.60 (dd, J = 8.4, 1.2 Hz, 1H), 7.56
(dd, J = 8.3, 1.8 Hz, 1H), 7.18 (s, 1H), 7.10 (t, J = 7.7 Hz, 1H), 7.08−
7.02 (m, 2H), 7.01 (d, J = 8.3 Hz, 1H), 6.70 (d, J = 7.4 Hz, 1H), 6.55
U
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
13C NMR (100 MHz, CDCl3): δ 149.3, 145.4, 137.0, 137.0, 136.6,
dichlorophenyl)thio)-3-nitrobenzaldehyde 45c (50 mg, 0.15 mmol),
3-aminobenzenesulfonamide (23 mg, 0.15 mmol), and CH2Cl2 (5
mL) were stirred at room temperature for 1 h. The reaction was
cooled down to 0 °C, and NaBH(OAc)3 (95 mg, 0.45 mmol) was
added in two portions over 1 min. The reaction was allowed to stir at
room temperature overnight. The reaction was quenched with a
saturated solution of NH4Cl and extracted with CH2Cl2. The
combined organic layers were dried with anhydrous Na2SO4, filtered,
and concentrated under reduced pressure. The crude product was
purified by column chromatography using hexane/EtOAc (15:1) to
give the titled compound, yellow solid, 21 mg, 29% yield, mp 191−
134.9, 134.7, 134.4, 134.1, 133.0, 132.1, 131.9, 131.2, 129.1, 124.3,
117.8, 117.6, 110.9, 96.6, 46.2. HRMS (ESI): calcd for
C20H12Cl2N3O2S, [M − H]− 429.0106; found, 428.0033. HPLC
purity: 100.00%.
Synthetic Procedures for Scheme 6. 4-((3,4-Dichlorophenyl)-
thio)-3-nitrobenzaldehyde (45c). The titled compound was prepared
from 4-fluoro-3-nitrobenzaldehyde 4b (169 mg, 1.0 mmol) and 3,4-
dichlorobenzenethiol (214 mg, 1.2 mmol) in a similar manner as
1
described for compound 38d. Yellow solid, 246 mg, 75% yield. H
NMR (400 MHz, CDCl3): δ 10.02 (s, 1H), 8.75 (d, J = 1.4 Hz, 1H),
7.90 (dd, J = 8.5, 1.5 Hz, 1H), 7.74 (d, J = 1.9 Hz, 1H), 7.64 (d, J =
8.3 Hz, 1H), 7.46 (dd, J = 8.2, 1.9 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H).
(4-((3,4-Dichlorophenyl)thio)-3-nitrophenyl)methanol (45d). In
a round-bottom flask was added 4-((3,4-dichlorophenyl)thio)-3-
nitrobenzaldehyde 45c (100 mg, 0.3 mmol) in MeOH (5 mL) and
cooled to 0 °C with an ice-water bath. NaBH4 (23 mg, 0.6 mmol) was
added, and the reaction mixture was allowed to stir at room
temperature for another 1 h. The reaction was quenched with NH4Cl
solution and extracted with EtOAc. The organic phase was combined,
washed with brine, dried with anhydrous Na2SO4, concentrated, and
purified by column chromatography using hexane/EtOAc (8:1) to
1
193 °C. H NMR (400 MHz, DMSO-d6): δ 8.24 (s, 1H), 7.91 (s,
1H), 7.79 (d, J = 8.3 Hz, 1H), 7.58 (dd, J = 15.2, 8.4 Hz, 2H), 7.21 (t,
J = 8.0 Hz, 1H), 7.17 (s, 2H), 7.05 (s, 1H), 7.00 (dd, J = 11.9, 8.4 Hz,
2H), 6.87 (t, J = 5.7 Hz, 1H), 6.71 (d, J = 7.8 Hz, 1H), 4.39 (d, J = 5.7
Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 148.8, 145.8, 145.3,
134.0, 136.6, 135.4, 134.3, 133.9, 133.6, 133.0, 132.6, 132.3, 130.0,
129.9, 124.4, 115.2, 113.6, 109.7, 45.3. HRMS (ESI): calcd for
C19H14Cl2N3O4S2, [M − H]− 481.9808; found, 481.9801. HPLC
purity: 97.55%.
Bacterial Strains and Antibiotics. The bacterial strains used in
this study for microdilution assay were as follows: E. faecalis ATCC
19433, S. aureus ATCC 25923, S. aureus ATCC 29213, S. pneumoniae
ATCC 49619, S. pyogenes ATCC 19615, S. agalactiae ATCC 12386, S.
epidermidis ATCC 12228, S. saprophyticus ATCC 15305 (The
American Type Culture Collection, Manassas, Virginia, United
States). The antibiotic controls were purchased from Sigma-Aldrich
(St. Louis, Missouri, United States).
Determination of MIC. The antimicrobial activity of the
compounds was determined by broth microdilution according to
the Clinical and Laboratory Standards Institute (CLSI) guidelines.25
The test medium was cation-adjusted Mueller−Hinton broth (MHB)
if not specified elsewhere or BHI as indicated in the main text. Serial
twofold dilutions of the tested compound were performed for the
tested chemicals starting from 256 to 0.5 μg/mL, and the bacterial cell
inoculum was adjusted to approximately 5 × 105 CFU per mL. Results
were taken after 20 h of incubation at 37 °C. The MIC was defined as
the lowest concentration of antibiotic with no visible growth.
Experiments were performed in at least duplicates.
Cytotoxicity Assay. Human HepG2 liver cancer and A549 lung
carcinoma cell lines were used in this study. The cells were seeded at
2.5 × 105 per well. After 24 h of incubation, the tested compounds
will be added in a twofold serial dilution ranging from 1.562 to 50 μg/
mL. The plates were incubated at 37 °C. At 48 h and 72 h after
adding the compound, the MTT assay was performed as described
previously.40 5-Fluorouracil was used as the positive control, and
DMSO was used as the negative control.
Hemolysis Assay. To separate erythrocytes from human whole
blood, 35 mL of human whole blood was centrifuged at 500g, 4 °C for
5 min. After centrifugation, the upper layers of plasma and buffy coat
were aspirated by a micropipette. NaCl solution (150 mM) was added
to the packed erythrocytes and filled up to the original volume mark,
and the solution was gently shaken to resuspend the erythrocytes. The
centrifugation process was repeated with the same condition, and after
centrifugation, the upper layers were aspirated and discarded,
followed by the resuspension of erythrocytes using phosphate-
buffered saline (PBS) (pH 7.4). A 40× diluted erythrocyte suspension
was prepared by adding 1 mL of the final suspension into 39 mL of
PBS (pH 7.4). Compounds with a final concentration of 0.1, 1 and 10
μg/mL were incubated at 37 °C with 2% blood solution in PBS (pH
7.4) for 45 min. The 40× diluted erythrocyte suspension (1600 μL)
was added to 400 μL of the drug solution in PBS (with 0.5% DMSO).
The mixture was vortexed gently for 10 s and inverted several times to
ensure a thorough mixing. The prepared mixture was transferred to
96-well microplates for incubation (200 μL per well, n = 6). Positive
control was Triton X-100, a known hemolytic agent, with a final
concentration of 1% v/v in PBS. Negative control is the vehicle which
is PBS (pH 7.4). After incubation, the reactions were terminated by
centrifuging the samples 10 min at 500g, 4 °C, to pellet the remaining
erythrocytes and erythrocyte ghosts. The supernatant (100 μL) from
1
provide compound 45d as a pale yellow solid, 87 mg, 88% yield. H
NMR (400 MHz, CDCl3): δ 8.26 (s, 1H), 7.69 (d, J = 2.0 Hz, 1H),
7.57 (d, J = 8.3 Hz, 1H), 7.43 (td, J = 8.2, 1.8 Hz, 2H), 6.92 (d, J =
8.4 Hz, 1H), 4.77 (d, J = 5.7 Hz, 2H).
(4-(Bromomethyl)-2-nitrophenyl)(3,4-dichlorophenyl)sulfane
(45e). Phosphorus tribromide (20 μL, 0.22 mmol) was added to a
stirred solution of (4-((3,4-dichlorophenyl)thio)-3-nitrophenyl)-
methanol 45d (60 mg, 0.18 mmol) in toluene (2 mL) at 40 °C.
The reaction was stirred at 100 °C for 30 min. The reaction was
cooled to room temperature, extracted with EtOAc, washed with
water and brine, dried with anhydrous Na2SO4, concentrated, and
purified by column chromatography using hexane/EtOAc (15:1) to
1
provide the titled compound as a yellow solid, 58 mg, 82% yield. H
NMR (400 MHz, CDCl3): δ 8.29 (d, J = 1.8 Hz, 1H), 7.71 (d, J = 2.0
Hz, 1H), 7.59 (d, J = 8.3 Hz, 1H), 7.44 (ddd, J = 8.2, 4.2, 2.0 Hz,
2H), 6.88 (d, J = 8.4 Hz, 1H), 4.49 (s, 2H).
Methyl 4-Chloro-3-((4-((3,4-dichlorophenyl)thio)-3-nitrobenzyl)-
amino)benzoate (45f). To a flask were added (4-(bromomethyl)-2-
nitrophenyl) (3,4-dichlorophenyl)sulfane (45e) (118 mg, 0.3 mmol),
methyl 3-amino-4-chlorobenzoate (56 mg, 0.3 mmol), DIPEA (149
μL, 0.9 mmol), and DMF (2 mL). The reaction was stirred at 100 °C
for 4 h. The reaction mixture was cooled to room temperature and
quenched with water and extracted with EtOAc. The organic phase
was combined, washed with brine, dried with anhydrous Na2SO4,
concentrated, and purified by column chromatography using hexane/
EtOAc (10:1) to provide the titled compound as a yellow solid, 32
1
mg, 21% yield. H NMR (400 MHz, DMSO-d6): δ 8.25 (d, J = 1.2
Hz, 1H), 7.91 (d, J = 1.9 Hz, 1H), 7.78 (d, J = 8.3 Hz, 1H), 7.59 (d, J
= 7.6 Hz, 1H), 7.57 (dd, J = 8.4, 2.0 Hz, 1H), 7.42 (d, J = 8.2 Hz,
1H), 7.16 (dd, J = 8.2, 1.6 Hz, 1H), 7.06 (d, J = 1.6 Hz, 1H), 7.02 (d,
J = 8.4 Hz, 1H), 6.61 (t, J = 6.1 Hz, 1H), 4.53 (d, J = 6.1 Hz, 2H),
3.76 (s, 3H).
4-Chloro-3-((4-((3,4-dichlorophenyl)thio)-3-nitrobenzyl)amino)-
benzoic Acid (45). The titled compound was prepared from the
hydrolysis of methyl 4-chloro-3-((4-((3,4-dichlorophenyl)thio)-3-
nitrobenzyl)amino)benzoate 45f (30 mg, 0.06 mmol) in 1 M
NaOH (0.2 mL) and dioxane (0.6 mL) at 50 °C overnight. Yellow
solid, 17 mg, 59% yield, mp 196−198 °C. 1H NMR (400 MHz,
DMSO-d6): δ 8.21 (s, 1H), 7.92 (s, 1H), 7.77 (d, J = 8.3 Hz, 1H),
7.57 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.1 Hz, 1H), 7.14 (d, J = 8.0 Hz,
1H), 7.07 (s, 1H), 6.99 (d, J = 8.3 Hz, 1H), 6.34 (s, 1H), 4.49 (d, J =
5.9 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 167.7, 145.8, 143.8,
139.7, 136.6, 135.4, 134.4, 133.6, 133.5, 133.0, 132.6, 132.2, 131.7,
123.0, 129.6, 124.2, 122.7, 118.3, 112.0, 45.1. HRMS (ESI): calcd for
C20H12Cl3N2O4S, [M − H]− 480.9589; found, 480.9577. HPLC
purity: 97.49%.
3-((4-((3,4-Dichlorophenyl)thio)-3-nitrobenzyl)amino)-
benzenesulfonamide (52). In a dried round-bottom flask, 4-((3,4-
V
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
each well was transferred to a new 96-well plate, and the absorbance
of the supernatant was measured at 540 nm, which is the hemoglobin
absorbance peak, with CLARIOstar microplate reader (BMG
Labtech, Offenburg, Germany). % of lysis was calculated with the
following equation
LB medium at 37 °C until OD600 ∼ 0.6. The compound at 1/4, 1/2,
1, 2, and 4 MIC was then added to the culture and allowed to
incubate for further 15 min. The cell culture (2.5 μL) was placed onto
1.2% freshly made agarose plate and covered with a coverslip prior to
imaging. A Leica TCS SPE confocal microscope equipped with a
63×/1.3 oil objective and mercury metal halide bulb was used to
capture the fluorescence images. The fluorescence images were
processed with LAS X software.41,42
Asample − A−ve control
% of lysis =
A+ve control − A−ve control
Quantitation of Major Macromolecules. To assess the effects
elicited by varying concentrations of treatment compounds on total
DNA, RNA, and protein levels during cell growth, a master culture of
S. aureus ATCC 29213 was first inoculated at OD600 0.1 and allowed
to grow to OD600 0.2 (early log phase) at 37 °C with agitation at 200
rpm. Upon reaching this stage, the cultures were divided into aliquots,
where compounds and drugs were added at their corresponding 1/4
and 1/8 MICs, complete with an untreated control culture. The
samples were then harvested upon reaching OD600 0.6 (the mid-log
phase of S. aureus) at 3 mL. Other samples were harvested at volumes
normalized to OD600 0.6, before being pelleted at 5000g for 5 min at 4
°C, and the supernatant discarded. Total DNA, RNA, and protein
were extracted and purified using the AllPrep Bacterial DNA/RNA/
Protein Kit (Qiagen) following the manufacturer’s guidelines. DNA
and RNA levels were measured with a Qubit DNA BR Assay Kit
(Invitrogen) and Qubit RNA BR Assay Kit (Invitrogen) coupled with
a Qubit 4 Fluorometer (Thermo Fisher), while proteins levels were
determined using an Pierce BCA Protein Assay Kit (Thermo Fisher).
The experiment was performed in triplicates.
Time-Kill Kinetic Assay. S. aureus, S. agalactiae, and S.
pneumoniae cells were suspended at ∼1.5 × 106 CFU/mL at log
phase in MHB or BHI (for Streptococcus spp.) media containing test
compounds at defined concentrations. Untreated controls were also
included. The setup was cultured at 37 °C with agitation at 200 rpm
(supplemented with 5% CO2 for Streptococcus spp.). At times 0, 2, 4,
and 6 h, 20 μL of samples was taken from each treatment group and
serially diluted in 10-folds. The number of viable bacterial cells in each
sample was quantified and expressed as CFU/mL following overnight
incubation at 37 °C with 5% CO2. The experiment was performed in
triplicates.
Caco-2 Permeability Assay. Caco-2 cells were cultured at 37 °C
in Dulbecco’s modified Eagle medium with 10% fetal bovine serum,
1% nonessential amino acids, 100 U/mL penicillin, and 100 μg/mL
streptomycin in an atmosphere of 5% CO2 and 90% relative humidity.
The cells were passaged after 90% confluence using trypsin−
ethylenediaminetetraacetic acid (EDTA) and plated at a 1:5 ratio in
75 cm2 flasks. The cells (passage number: 45) were seeded at a
density of 60,000 cells/cm2 on polycarbonate membranes of
Transwells (12 mm inner diameter, 0.4 μm pore size, 1 cm2, Corning,
NY, USA). The medium was changed the day after seeding and every
second day thereafter [apical (AP) volume 0.5 mL and BL volume 1.5
mL]. The Caco-2 cell monolayers were used 21−28 days after
seeding. Transepithelial electrical resistance (TEER) was measured to
ensure monolayer integrity. Cell monolayers with TEER values less
than 165 Ω cm2 were discarded in transport experiments. Transport
studies involved only AP to basolateral (BL) direction. Cell
monolayers were preincubated with transport buffer solution (PBS
+) for 20 min at 37 °C. To study absorptive transport, the donor
(AP) compartment buffer was replaced with 0.5 mL of transport
buffer containing 10 μg/mL compounds. The pH in both AP and BL
compartments was maintained at 7.4 for all transport studies.
Concentration of compounds in the receiver (BL) compartment
was monitored as a function of time in the linear region of transport
and under sink condition (which is the receiver concentration is <10%
of the donor concentration). At each sampling time point, 100 μL of
the sample was drawn from the receiver compartment, and a same
volume of blank buffer was added into the receiver compartment. The
concentration of compounds in sample solution was determined using
liquid chromatography−MS (LC−MS)/MS.
Assessing ATP Production. Log phase S. pneumoniae cells (∼1.5
× 106 CFU/mL) were suspended in BHI medium with test
compounds at various concentrations. An untreated control group
was also included with test compounds in medium. The setup was
grown in 37 °C with agitation at 200 rpm with 5% CO2, with 100 μL
samples being taken at time points 0, 2, 4, and 6 h, respectively, for
each treatment group. The ATP production level was quantified using
the BacTiter-Glo Microbial Cell Viability Assay Kit (Promega,
Madison, Wisconsin, United States) following the manufacturer’s
protocol. The experiment was performed in triplicates.
S. pneumoniae Toxin Release. S. pneumoniae cells were cultured
overnight without agitation on 96-well round-bottomed plates with or
without the addition of serially diluted concentrations of test
compounds. As positive controls, antibiotics were also added at serial
twofold dilutions from 2 to 0.002 μg/mL for rifampicin and
ceftriaxone. Each of the corresponding 1/2 and 1/4 MIC values
was determined following 16−20 h of incubation. The plates were
then centrifuged at 3000g for 3 min, and the supernatant transferred
to a fresh plate from the wells designated as challenged by 1/2 and 1/
4 MIC compounds, control drugs, and untreated control. The
supernatants were stored in −20 °C until use in the Western blot
assay.
Western Blot. Samples were separated in polyacrylamide gels at
80 V for 15 min and then at 150 V for 1 h. Bands were then
transferred to a PVDF membrane at 110 V for 1 h, followed by
blocking with 5% nonfat milk in TBST buffer for 1 h and overnight
incubation with 1:1000 rabbit polyclonal anti-pneumolysin primary
antibody (ab71811, Abcam, Cambridge, United Kingdom) at 4 °C
with agitation. TBST-washing cycles were performed before and after
1 h of incubation with 1:5000 goat polyclonal anti-rabbit HRP-
conjugated secondary antibody (ab97051, Abcam, Cambridge, United
Kingdom) at room temperature with agitation. Blots were developed
Binding Inhibition Assay. Previously established protocols were
used for inhibitor testing.43 Protein overproduction and purification
were performed as detailed previously.43 N-SmBiT-CH (40 μL, 2.5
μM in PBS) was added to 96-well plates and then mixed with 20 μL
of compound at desired concentrations. The mixture was incubated
for 10 min at 37 °C. C-LgBiT-σA (40 μL, 2.5 μM in PBS) was then
added to each well, followed by incubation for 10 min at 37 °C. After
the final incubation step, an equal volume of Promega Nano-Glo
Luciferase Assay Substrate was added to the reaction mixture.
Luminescence emitted was measured using a Victor X3 Multilabel
plate reader. The experiment was performed in triplicate with
technical repeats.
In Vitro Transcription Assay. Linear templates containing the λ
PR promoter followed by an initial 26 nt C-less transcribed region
were made by PCR and purified via a QIAquick PCR purification kit
(Qiagen, Valencia, CA). E. coli core RNAP (200 nM) was incubated
with 1/10 volume of each compound (or DMSO) in TGA2 (20 mM
Tris-acetate, 20 mM Na-acetate, 2 mM Mg-acetate, 5% glycerol, 1
mM dithiothreitol, 0.1 mM EDTA, pH 7.9) for 10 min at 37 °C. An
equal volume of mix containing σ70 (200 nM), DNA template (200
nM), ApU (100 μM), 5 μM UTP and ATP, and 1 μM GTP (plus 10
μCi [α32P]-GTP, 3000 Ci/mmol) in TGA2 was added, followed by
10 min incubation at 37 °C. Reactions were quenched by the addition
of an equal volume of STOP buffer (10 M urea, 60 mM EDTA, 45
mM Tris-borate; pH 8.3). Samples were heated for 2 min at 95 °C
and separated by electrophoresis in denaturing 9% acrylamide (19:1)
gels (7 M urea, 0.5× TBE). The gels were dried and RNA products
were visualized using FLA9000 phosphorimaging system and
ImageQuant Software. In vitro transcription assays were carried out
in triplicates and averaged.
Confocal Microscopy. B. subtilis strain BS1048 (RNAP β′-GFP)
was grown on an LB agar plate. A single colony was incubated in an
W
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
with Bio-Rad Clarity Western ECL substrates and imaged in a Bio-
Rad ChemiDoc Touch system in chemiluminescence mode (Bio-Rad,
Hercules, California, United States). The experiment was performed
in triplicates and representative data presented.
Irina Artsimovitch − Department of Microbiology and the
Center for RNA Biology, The Ohio State University, Columbus,
Ohio 43210, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c00520
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00520.
■
sı
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
MIC (μg/mL) of compounds 1−57 against representa-
tive Gram-positive and -negative pathogenic bacteria;
inhibitory activity of representative compounds against
the RNAP β′CH−σA PPI in the protein complementa-
tion assay; and 1H, 13C NMR, and HPLC spectra (PDF)
Molecular formula strings and biological data (CSV)
PDB coordinates for the computational model (PDB)
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the financial support from the
Research Grants Council of the Hong Kong Special
Administrative Region, China (PolyU 251000/17M, 151000/
19M, and C5008-19G to C.M., CUHK 141659/17M and
141079/19M to X.Y.), Hong Kong Polytechnic University
internal grants (G-YBYY, 1-ZVPS and large equipment fund,
the State Key Laboratory of Chemical Biology and Drug
Discovery to C.M.), Hong Kong Food and Health Bureau
HMRF (19180052 to X.Y.), and the Chinese University of
Hong Kong (Faculty of Medicine Faculty Innovation Award
FIA2018/A/03 to X.Y.). Studies in the I.A. laboratory were
supported by the National Institutes of Health grant
GM67153.
AUTHOR INFORMATION
Corresponding Authors
■
Zhong Zuo − School of Pharmacy, Faculty of Medicine, The
Chinese University of Hong Kong, New Territories, Hong Kong
SAR, People’s Republic of China; orcid.org/0000-0002-
6976-6157; Phone: +852 3943 6862; Email: joanzuo@
cuhk.edu.hk
Xiao Yang − Department of Microbiology, The Chinese
University of Hong Kong, Prince of Wales Hospital, New
Territories, Hong Kong SAR, People’s Republic of China;
Phone: +852 3505 2573; Email: xiaoyang@cuhk.edu.hk
Cong Ma − State Key Laboratory of Chemical Biology and Drug
Discovery, Department of Applied Biology and Chemical
Technology, The Hong Kong Polytechnic University, Kowloon,
Hong Kong SAR, People’s Republic of China; orcid.org/
0000-0001-9245-0356; Phone: +852 3400 8810;
Email: cong.ma@polyu.edu.hk
ABBREVIATIONS
■
RNAP, RNA polymerase; CH, clamp helix; PPI, protein−
protein interaction; MIC, minimum inhibitory concentration;
SAR, structure−activity relationship; CLSI, Clinical &
Laboratory Standards Institute; GFP, green fluorescent
protein;; CFU, colony-forming unit; BHI, brain−heart infusion
medium
Authors
Jiqing Ye − State Key Laboratory of Chemical Biology and Drug
Discovery, Department of Applied Biology and Chemical
Technology, The Hong Kong Polytechnic University, Kowloon,
Hong Kong SAR, People’s Republic of China
REFERENCES
■
(1) O’Neill, J. Antimicrobial Resistance: Tackling a Crisis for the Future
Health and Wealth of Nations; Review on Antimicrobial Resistance:
London, 2014.
Adrian Jun Chu − Department of Microbiology, The Chinese
University of Hong Kong, Prince of Wales Hospital, New
Territories, Hong Kong SAR, People’s Republic of China
Rachel Harper − Department of Microbiology, The Chinese
University of Hong Kong, Prince of Wales Hospital, New
Territories, Hong Kong SAR, People’s Republic of China
Shu Ting Chan − State Key Laboratory of Chemical Biology
and Drug Discovery, Department of Applied Biology and
Chemical Technology, The Hong Kong Polytechnic University,
Kowloon, Hong Kong SAR, People’s Republic of China
Tsun Lam Shek − School of Pharmacy, Faculty of Medicine, The
Chinese University of Hong Kong, New Territories, Hong Kong
SAR, People’s Republic of China
Yufeng Zhang − School of Pharmacy, Faculty of Medicine, The
Chinese University of Hong Kong, New Territories, Hong Kong
SAR, People’s Republic of China
Margaret Ip − Department of Microbiology, The Chinese
University of Hong Kong, Prince of Wales Hospital, New
Territories, Hong Kong SAR, People’s Republic of China
Mariya Sambir − Department of Microbiology and the Center
for RNA Biology, The Ohio State University, Columbus, Ohio
43210, United States
(2) Naylor, N. R.; Atun, R.; Zhu, N.; Kulasabanathan, K.; Silva, S.;
Chatterjee, A.; Knight, G. M.; Robotham, J. V. Estimating the burden
of antimicrobial resistance: a systematic literature review. Antimicrob.
Resist. Infect. Contr. 2018, 7, 58.
(3) Bohacek, R. S.; McMartin, C.; Guida, W. C. The art and practice
of structure-based drug design: A molecular modeling perspective.
Med. Res. Rev. 1996, 16, 3−50.
(4) Simmons, K. J.; Chopra, I.; Fishwick, C. W. G. Structure-based
discovery of antibacterial drugs. Nat. Rev. Microbiol. 2010, 8, 501−
510.
(5) Scott, D. E.; Bayly, A. R.; Abell, C.; Skidmore, J. Small molecules,
big targets: drug discovery faces the protein-protein interaction
challenge. Nat. Rev. Drug Discov. 2016, 15, 533−550.
(6) Ma, C.; Yang, X.; Lewis, P. J. Bacterial transcription as a target
for antibacterial drug development. Microbiol. Mol. Biol. Rev. 2016, 80,
139−160.
(7) Feklístov, A.; Sharon, B. D.; Darst, S. A.; Gross, C. A. Bacterial
sigma factors: a historical, structural, and genomic perspective. Annu.
Rev. Microbiol. 2014, 68, 357−376.
(8) Borukhov, S.; Nudler, E. RNA polymerase holoenzyme:
structure, function and biological implications. Curr. Opin. Microbiol.
2003, 6, 93−100.
(9) Ruff, E.; Record, M., Jr.; Artsimovitch, I. Initial events in
bacterial transcription initiation. Biomolecules 2015, 5, 1035−1062.
X
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(10) Murakami, K. S.; Masuda, S.; Campbell, E. A.; Muzzin, O.;
Darst, S. A. Structural basis of transcription initiation: an RNA
polymerase holoenzyme-DNA complex. Science 2002, 296, 1285−
1290.
(11) Bae, B.; Feklistov, A.; Lass-Napiorkowska, A.; Landick, R.;
Darst, S. A. Structure of a bacterial RNA polymerase holoenzyme
open promoter complex. eLife 2015, 4, No. e08504.
(12) Arthur, T. M.; Burgess, R. R. Localization of a σ70 binding site
on the N terminus of the Escherichia coli RNA polymerase β’ subunit.
J. Biol. Chem. 1998, 273, 31381−31387.
(13) Burgess, R. R.; Anthony, L. How sigma docks to RNA
polymerase and what sigma does. Curr. Opin. Microbiol. 2001, 4, 126−
131.
of potent nonglycosidic and nonpeptidic pan-selectin inhibitors. J.
Med. Chem. 2006, 49, 5988−5999.
(30) Watanabe, S.; Takemoto, N.; Ogura, K.; Miyoshi-Akiyama, T.
Severe invasive streptococcal infection by Streptococcus pyogenes and
Streptococcus dysgalactiae subsp. equisimilis. Microbiol. Immunol.
2016, 60, 1−9.
(31) Raabe, V. N.; Shane, A. L. Group B Streptococcus (Streptococcus
agalactiae). Microbiol. Spectr. 2019, 7, DOI: 10.1128/micro-
biolspec.GPP3-0007-2018.
(32) Clinical and Laboratory Standards Institute. Methods for
Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow
Aerobically, 11th (M07Ed11) ed.; Clinical and Laboratory Standards
Institute: Wayne, PA, 2018.
(14) Gruber, T. M.; Markov, D.; Sharp, M. M.; Young, B. A.; Lu, C.
Z.; Zhong, H. J.; Artsimovitch, I.; Geszvain, K. M.; Arthur, T. M.;
Burgess, R. R.; Landick, R.; Severinov, K.; Gross, C. A. Binding of the
initiation factor σ70 to core RNA polymerase is a multistep process.
Mol. Cell 2001, 8, 21−31.
(33) Balemans, W.; Vranckx, L.; Lounis, N.; Pop, O.; Guillemont, J.;
Vergauwen, K.; Mol, S.; Gilissen, R.; Motte, M.; Lanco̧ is, D.; De
Bolle, M.; Bonroy, K.; Lill, H.; Andries, K.; Bald, D.; Koul, A. Novel
antibiotics targeting respiratory ATP synthesis in Gram-positive
pathogenic bacteria. Antimicrob. Agents Chemother. 2012, 56, 4131−
4139.
(34) Lobritz, M. A.; Belenky, P.; Porter, C. B. M.; Gutierrez, A.;
Yang, J. H.; Schwarz, E. G.; Dwyer, D. J.; Khalil, A. S.; Collins, J. J.
Antibiotic efficacy is linked to bacterial cellular respiration. Proc. Natl.
Acad. Sci. U.S.A. 2015, 112, 8173−8180.
(15) Artsimovitch, I. Post-initiation control by the initiation factor
sigma. Mol. Microbiol. 2008, 68, 1−3.
́
(16) Andre, E.; Bastide, L.; Villain-Guillot, P.; Latouche, J.; Rouby,
J.; Leonetti, J.-P. A multiwell assay to isolate compounds inhibiting
the assembly of the prokaryotic RNA polymerase. Assay Drug Dev.
Technol. 2004, 2, 629−635.
(35) Yang, J. H.; Bening, S. C.; Collins, J. J. Antibiotic efficacy-
context matters. Curr. Opin. Microbiol. 2017, 39, 73−80.
(36) Anderson, R.; Steel, H. C.; Cockeran, R.; Smith, A. M.; von
Gottberg, A.; de Gouveia, L.; Brink, A.; Klugman, K. P.; Mitchell, T.
J.; Feldman, C. Clarithromycin alone and in combination with
ceftriaxone inhibits the production of pneumolysin by both
macrolide-susceptible and macrolide-resistant strains of Streptococcus
pneumoniae. J. Antimicrob. Chemother. 2007, 59, 224−229.
(37) Brown, L. A.; Mitchell, A. M.; Mitchell, T. J. Streptococcus
pneumoniae and lytic antibiotic therapy: are we adding insult to injury
during invasive pneumococcal disease and sepsis? J. Med. Microbiol.
2017, 66, 1253−1256.
(17) Hinsberger, S.; Husecken, K.; Groh, M.; Negri, M.;
̈
Haupenthal, J.; Hartmann, R. W. Discovery of novel bacterial RNA
polymerase inhibitors: pharmacophore-based virtual screening and hit
optimization. J. Med. Chem. 2013, 56, 8332−8338.
(18) Bae, B.; Davis, E.; Brown, D.; Campbell, E. A.; Wigneshweraraj,
S.; Darst, S. A. Phage T7 Gp2 inhibition of Escherichia coli RNA
polymerase involves misappropriation of σ70 domain 1.1. Proc. Natl.
Acad. Sci. U.S.A. 2013, 110, 19772−19777.
(19) Ma, C.; Yang, X.; Kandemir, H.; Mielczarek, M.; Johnston, E.
B.; Griffith, R.; Kumar, N.; Lewis, P. J. Inhibitors of bacterial
transcription initiation complex formation. ACS Chem. Biol. 2013, 8,
1972−1980.
(20) Yang, X.; Ma, C.; Lewis, P. J. Identification of inhibitors of
bacterial RNA polymerase. Methods 2015, 86, 45−50.
(21) Ma, C.; Yang, X.; Lewis, P. J. Bacterial transcription inhibitor of
RNA polymerase holoenzyme formation by structure-based drug
design: From in silico screening to validation. ACS Infect. Dis. 2016, 2,
39−46.
(22) Ye, J.; Chu, A. J.; Lin, L.; Yang, X.; Ma, C. First-in-class
inhibitors targeting the interaction between bacterial RNA polymerase
and sigma initiation factor affect the viability and toxin release of
Streptococcus pneumoniae. Molecules 2019, 24, 2902.
(23) Sun, Q.; Wu, R.; Cai, S.; Lin, Y.; Sellers, L.; Sakamoto, K.; He,
B.; Peterson, B. R. Synthesis and biological evaluation of analogues of
AKT (protein kinase B) inhibitor-IV. J. Med. Chem. 2011, 54, 1126−
1139.
(24) Lee, J. A.; Roh, E. J.; Oh, C.-H.; Lee, S. H.; Sim, T.; Kim, J. S.;
Yoo, K. H. Synthesis of quinolinylaminopyrimidines and quinazoli-
nylmethylaminopyrimidines with antiproliferative activity against
melanoma cell line. J. Enzyme Inhib. Med. Chem. 2015, 30, 607−614.
(25) Clinical and Laboratory Standards Institute. Performance
Standards for Antimicrobial Susceptibility Testing, 29th ed.
(M100Ed29); Clinical and Laboratory Standards Institute: Wayne,
PA, 2018.
(26) Wang, X.; Li, J.; Zhao, N.; Wan, X. A rapid and efficient access
to diaryldibenzo[b,f][1,5]diazocines. Org. Lett. 2011, 13, 709−711.
(27) Itoh, T.; Mase, T. A general palladium-catalyzed coupling of
aryl bromides/triflates and thiols. Org. Lett. 2004, 6, 4587−4590.
(28) Kuznetsov, D. M.; Kutateladze, A. G. Step-economical
photoassisted diversity-oriented synthesis: Sustaining cascade photo-
reactions in oxalyl anilides to access complex polyheterocyclic
molecular architectures. J. Am. Chem. Soc. 2017, 139, 16584−16590.
(29) Ulbrich, H. K.; Luxenburger, A.; Prech, P.; Eriksson, E. E.;
Soehnlein, O.; Rotzius, P.; Lindbom, L.; Dannhardt, G. A novel class
(38) Spreer, A.; von Ruden, C.; Mitchell, T. J.; Eiffert, H.; Nau, R.
̈
Influence of subinhibitory concentrations of protein-synthesis-
inhibiting antibiotics on production and release of the pneumococcal
virulence factor pneumolysin in vitro. Chemotherapy 2007, 53, 327−
331.
̈
(39) Spreer, A.; Kerstan, H.; Bottcher, T.; Gerber, J.; Siemer, A.;
Zysk, G.; Mitchell, T. J.; Eiffert, H.; Nau, R. Reduced release of
pneumolysin by Streptococcus pneumoniae in vitro and in vivo after
treatment with nonbacteriolytic antibiotics in comparison to
ceftriaxone. Antimicrob. Agents Chemother. 2003, 47, 2649−2654.
(40) Yang, X.; Luo, M. J.; Yeung, A. C. M.; Lewis, P. J.; Chan, P. K.
S.; Ip, M.; Ma, C. First-in-class inhibitor of ribosomal RNA synthesis
with antimicrobial activity against Staphylococcus aureus. Biochemistry
2017, 56, 5049−5052.
(41) Qiu, Y.; Chan, S. T.; Lin, L.; Shek, T. L.; Tsang, T. F.; Barua,
N.; Zhang, Y.; Ip, M.; Chan, P. K.-s.; Blanchard, N.; Hanquet, G.;
Zuo, Z.; Yang, X.; Ma, C. Design, synthesis and biological evaluation
of antimicrobial diarylimine and -amine compounds targeting the
interaction between the bacterial NusB and NusE proteins. Eur. J.
Med. Chem. 2019, 178, 214−231.
(42) Qiu, Y.; Chan, S. T.; Lin, L.; Shek, T. L.; Tsang, T. F.; Zhang,
Y.; Ip, M.; Chan, P. K.-s.; Blanchard, N.; Hanquet, G.; Zuo, Z.; Yang,
X.; Ma, C. Nusbiarylins, a new class of antimicrobial agents: Rational
design of bacterial transcription inhibitors targeting the interaction
between the NusB and NusE proteins. Bioorg. Chem. 2019, 92,
103203.
(43) Tsang, T. F.; Qiu, Y.; Lin, L.; Ye, J.; Ma, C.; Yang, X. Simple
method for studying in vitro protein-protein interactions based on
protein complementation and its application in drug screening
targeting bacterial transcription. ACS Infect. Dis. 2019, 5, 521−527.
(44) Darst, S. A. New inhibitors targeting bacterial RNA polymerase.
Trends Biochem. Sci. 2004, 29, 159−162.
Y
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(45) Qiu, Y.; Ma, C. HPLC, quantitative NMR and HRMS
spectroscopic data of nusbiarylins as a new class of antimicrobial
agents. Data Brief 2020, 29, 105313.
Z
https://dx.doi.org/10.1021/acs.jmedchem.0c00520
J. Med. Chem. XXXX, XXX, XXX−XXX