Discovery of meta-amido bromophenols as new antitubercular agents

Article abstract of DOI:10.1248/cpb.c18-00917

A series of meta-amido bromophenol derivatives were designed and synthesized. The compounds were found to potently inhibit the growth of Mycobacterium tuberculosis H37Ra. They also exhibited moderate inhibitory activity against Mycobacterium tuberculosis H37Rv and multidrug-resistant strains. The compounds did not show inhibitory activity against normal Gram-positive and Gram-negative bacteria. Moderate cytotoxicities and good metabolic stability were observed for the selected compounds. The results demonstrated meta-amido bromophenols as a new class of antitubercular agents with good potentials.

Full text of DOI:10.1248/cpb.c18-00917

Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/cpb.c18-00917
Advance Publication
January 26, 2019
Chem. Pharm. Bull.
Regular Article
Discovery of meta-amido bromophenols as new antitubercular agents
Jie Lianga,#, Yun-xiang Tangb,c,#, Xiang-zheng Tanga, Hua-jv Lianga, Yamin Gaob,d,
Cuiting Fangb,d, Tian-yu Zhangb,d*, Ming Yana*
a. School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China.
b. State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and
Health, Chinese Academy of Science, Guangzhou, China.
c. Institute of Physical Science and Information Technology, Anhui University, Hefei, China.
d. University of Chinese Academy of Sciences (UCAS), Beijing, China.
# These two authors contribute equally to this study.
*Corresponding authors, e-mail: zhang_tianyu@gibh.ac.cn (T. Y. Zhang);
yanming@mail.sysu.edu.cn (M. Yan)
Ⓒ 2019 The Pharmaceutical Society of Japan

Abstract
A series of m-amido bromophenol derivatives were designed and synthesized. The
compounds were found to potently inhibit the growth of Mycobacterium tuberculosis H37Ra.
They also exhibited moderate inhibitory activity against Mycobacterium tuberculosis H37Rv
and multidrug-resistant strains. The compounds did not show inhibitory activity against
normal Gram-positive and Gram-negative bacteria. Moderate cytotoxicities and good
metabolic stability were observed for the selected compounds. The results demonstrated
meta-amido bromophenols as a new class of antitubercular agents with good potentials.
Keywords
Mycobacterium tuberculosis, meta-amido bromophenol, antitubercular agent
Chemical and Pharmaceutical Bulletin Advance Publication

1. Introduction
Tuberculosis (TB), a chronic infectious disease caused by Mycobacterium tuberculosis
(Mtb), has plagued people for thousands of years and remains a huge threat to public health.
According to the report of the World Health Organization (WHO), it is estimated that over
1.3 million people died from TB in 2017.1 One third of the population in the world carries
latent TB, which can be activated upon immune system suppressed or disrupted, such as
aging and HIV co-infection.2 Moreover, Mtb has developed the resistance to many first-line
anti-TB drugs including rifampicin, isoniazid, streptomycin, ethambutol and pyrazinamide
etc.3,4 Rather limited drugs are applicable for the patients infected with multidrug-resistant
(MDR) Mtb and extensive drug-resistant (XDR) Mtb.5 The treatment period is extremely long
(typically 18-24 months) for MDR-TB and the cure rates are less than 50%.6 In recent four
decades, only two new anti-TB drugs, bedaquiline7 and delamanid8, were approved for the
treatment of MDR-TB and XDR-TB. The two drugs are still beset by the side effects. For
these reasons, anti-TB drugs with novel structures and action mechanisms are urgently
desired.9
In the recent study, we found that m-amido phenol derivatives exhibit good inhibitory
activity against Mtb in vitro.10 However, the privileged compound YZ-7 did not exert in vivo
efficacy attributed to the fast clearance in the liver. The 3-benzyl group in compound YZ-7
was supposed to be liable to the oxidation catalyzed by CYP450. The further structural
modifications were made to increase the metabolic stability (Chart 1). The replacement of
3-benzyl with a bromine led to the decreased activity. The shift of the bromine to 2-position
resulted in the complete loss of the antitubercular activity. However, to our delight, the
4-bromo derivative 3 showed good inhibitory activity, which is comparable with YZ-7. In
this paper, we report the design, synthesis and biological evaluation of a series of meta-amido
bromophenols as new antitubercular agents.
Chemical and Pharmaceutical Bulletin Advance Publication

H
N
HO
O
Br
H
H
HO
N
> 100
HO
N
MIC
μg/mL
O
2
O
Br
H
HO
Br
N
= 25
O
MIC
MIC
(Mtb H37Ra) = 5
μg/mL
μg/mL
1
YZ-7
= 5
MIC
μg/mL
3
Chart 1. Structural modifications of compound YZ-7.
2. Results and discussion
2.1 Design of new derivatives
OH
Br
O
N
H
R
Chart 2. Design of meta-amido bromophenols.
A series of meta-amido bromophenols were designed based on the structure of the lead
compound 3. The structural modifications were made at the left-side bromophenol moiety,
the amide linker, and the right-side phenyl group respectively (Chart 2). For the modification
of bromophenol moiety, the hydroxyl was removed or methylated to provide compounds 4a,
4b. The bromine was replaced with chlorine, fluorine, methyl, methoxy, ester, acetyl, and
cyano to afford derivatives 4c-4k. For the modification of the amide linker, the shift of amide
group to para- or ortho-position of the hydroxyl group led to compounds 5a and 5b.
Furthermore, the transpositional analog 5c and N-benzyl analog 5d were prepared. For the
structural modification of the right-side phenyl group, the replacement of the phenyl with
alkyl, vinyl, heteroaryl, and the introduction of various substitutents on the phenyl ring led to
the compounds 6a-6u.
Chemical and Pharmaceutical Bulletin Advance Publication

2.2 Synthesis of the target compounds
The synthesis of compounds 1-3 and 4a-4k is outlined in Chart 3. The reaction of
substituted anilines with benzoyl chloride provided the target compounds in good yields.
Compound 4i was obtained after the hydrolysis of 4h in aqueous sodium hydroxide.
R1
R1
O
3
O
Cl
3
a
R2
+
R2
1
5
1
5
N
NH2
H
1 = H, OH, OMe
2 = 4-Br, 5-Br, 6-Br,
R
R
1, 2, 3,
4a-4h, 4j, 4k
4-Cl, 4-F, 4-H, 4-Me,
4-OMe, 4-COOMe,
4-(C=O)Me, 4-CN
O
OH
O
OH
H
3C
O
O
HO
O
b
N
N
H
H
4h
4i
Chart 3. Synthesis of compounds 1, 2, 3, and 4a-4k. Reagents and conditions: (a) Et3N, THF,
0°C to rt, 67-87%; (b) NaOH, CH3OH, H2O, 50°C, 73%.
The synthesis of compounds 5a-5d is outlined in Chart 4. Compound 5a and 5b were
prepared via the reaction of 4-amino-2-bromophenol or 2-amino-6-bromophenol with
benzoic acids in the presence of HATU and DIPEA. Compound 5c was obtained via the
condensation of 4-bromo-3-methoxybenzoic acid and aniline. Compound 5d was prepared
via the benzylation of 4-bromo-3-methoxyaniline and subsequent demethylation.
Chemical and Pharmaceutical Bulletin Advance Publication

OH
OH
Br
Br
a
OH
H
R3
+
Br
N
or
COOH
HN
O
or 2-NH
3 = 4-NH2
OMe
R
2
O
5a
5b
OH
OMe
a
b
Br
Br
Br
+
H
H
N
N
NH2
COOH
O
O
5c
OMe
OMe
OH
Br
Br
Br
Br
+
c
b
N
NH2
N
H
H
5d
Chart 4. Synthesis of compounds 5a-5d. Reagents and conditions: (a) HATU, DIPEA, DMF,
rt, 71-90%; (b) BBr3, CH2Cl2, -20°C to 0°C, 38-44%; (c) K2CO3, DMF, 50°C, 72%.
The synthesis of compounds 6a-6u is outlined in Chart 5. 4-Bromo-3-methoxyaniline
was treated with BBr3 to give 4-bromo-3-hydroxyaniline. The subsequent reaction with acyl
chlorides or the direct condensation with acids in the presence of HATU and DIPEA provided
compounds 6a-6m.
OMe
OH
OH
a
Br
Br
Br
b or c
O
R4
NH2
N
NH2
H
6a-6u
Chart 5. Synthesis of 6a-6u. Reagents and conditions: (a) BBr3, CH2Cl2, 0°C to rt, 88%; (b)
R4COCl, Et3N, THF, 0°C to rt, 42%-83%; (c) R4COOH, HATU, DIPEA, DMF, rt, 76-80%.
2.3 Biological study
2.3.1 Inhibitory activity in vitro
Inhibitory activities of compounds 4a-4k were evaluated against autoluminescent Mtb H37Ra strain.11
The bacteria growth was determined by the bioluminescence intensity and the results are
summarized in Table 1. The existence of free hydroxyl is crucial for the antitubercular
Chemical and Pharmaceutical Bulletin Advance Publication

activity. Both the deletion and the methylation led to the complete loss of the inhibitory
activity (Table 1, entries 2-3). The replacement of bromine with chlorine or fluorine resulted
in the decreased activity (Table 1, entries 4-5). 4-Unsubstitutued derivative 4e is inactive
(Table 1, entry 6). The results confirmed that the 4-substitution is indispensable.
Non-halogen substituents such as methyl, methoxy, ester, carboxyl, acetyl, and cyano are
incompatible (Table 1, entries 8-13). The bromine is demonstrated to be unique for the
antitubercular activity, probably due to its suitably steric and electronic effect.
Table 1. Inhibitory activity of compounds 4a-4k against Mtb H37Ra.
R1
R2
O
N
H
MIC
(µg/mL)
5
MIC
(µg/mL)
>100
>100
>100
>100
>100
>100
2
Compound
R1
R2
Compound
R1
R2
-OH
-H
Br
Br
Br
Cl
F
-OH
-OH
-OH
-OH
-OH
-OH
Me
OMe
3
4a
4f
4g
>100
>100
12.5
50
-OMe
-OH
-OH
-OH
COOMe
COOH
COMe
CN
4b
4c
4h
4i
4d
4e
4j
H
>100
0.1
4k
INH
EMB
INH: isoniazid; EMB: ethambutol
The compounds 5a-5d with the modifications of amide linker were investigated and the
results are summarized in Table 2. The placement of amide group at the meta-position of
hydroxyl group is crucial, since the compounds 5a and 5b almost lose antitubercular
activities (Table 2, entries 1-2). The compounds 5c and 5d with the transposition of amide
group or N-benzylation showed lower activity (Table 2, entries 3-4).
Chemical and Pharmaceutical Bulletin Advance Publication

Table 2. Inhibitory activity of compounds 5a-5d against Mtb H37Ra.
Compound
Structure
Compound
Structure
MIC (µg/mL)
MIC (µg/mL)
OH
OH
Br
Br
H
N
>100
12.5
5a
5c
HN
O
O
OH
OH
Br
H
N
Br
50
12.5
2
5b
5d
N
H
O
0.1
INH
EMB
Furthermore, the antitubercular activity of compounds 6a-6u were determined and the
results are summarized in Table 3. The replacement of phenyl with methyl, vinyl and heptyl
led to the significant loss of the activity (Table 3, entries 2-4). However, the cyclohexyl
derivative 6d showed certain antitubercular activity. The morpholine derivative 6e showed
poor activity. The results suggested the strongly hydrophobic group is favorable herein. The
lower activities of heteroaryl derivatives 6f-6h (Table 3, entries 6-9) also support this
hypothesis. The effect of the substitution on the phenyl group was examined (Table 3, entries
10-23). The substitution of chlorine at ortho- and meta-position (6i, 6j) afforded the
comparable activity with compound 3, but para-chloro derivative 6k showed 4-folds increase
in the activity. The para-fluoro derivative 6l showed slightly lower activity. The para-bromo
and para-methyl analogs 6m-6n exhibited the comparable activity with 6k. Other
para-substitutents, such as MeO, CF3O, and dimethylamino also afforded good activity. The
substitution with the electron-withdrawing and hydrophobic CF3 group (6r) showed superior
activity. The MIC value is 20-folds lower than compound 3. The para-nitro derivative 6s
exhibited lower activity. The para-hydroxyl substitution (6t) led to the complete loss of
activity. The replacement phenyl with a 2-naphthyl (6u) afforded a better activity. The results
demonstrated that the hydrophobic property of the para-substitutent is favorable for
antitubercular activity. On the other hand, the activity is not sensitive to the electronic
Chemical and Pharmaceutical Bulletin Advance Publication

property of the substitutent. Both electron-donating groups and electron-withdrawing groups
provide good activity.
Table 3. Inhibitory activity of compounds 6a-6u against Mtb H37Ra.
OH
Br
O
N
R4
H
MIC
Compoun
d
MIC
Compound
R4
Ph
R4
(µg/mL)
(µg/mL)
5
1.25
2.5
3
6k
6l
Cl
-Me
100
50
6a
6b
6c
F
-CH=CH2
1.25
1.25
6m
6n
Br
100
CH3
OCH3
12.5
50
2.5
0.5
2.5
6d
6e
6f
6o
6p
6q
N
O
OCF3
N
N
25
S
12.5
25
0.25
6.25
6g
6h
6r
6s
CF3
NO2
O
Cl
5
>100
6i
6t
OH
Chemical and Pharmaceutical Bulletin Advance Publication

Cl
5
1.25
2
6j
6u
0.1
INH
EMB
2.3.2 Structure-activity relationship of
m-amido bromophenol derivatives
The structure-activity relationship (SAR) of meta-amido bromophenol derivatives is
summarized in Chart 6. The ortho-bromphenol moiety is a key pharmacophore for
antitubercyular activity. The meta-amido linker is also important. The transposition of amide
group or the replacement by N-benzyl group decreases the activity. For the right side moiety,
the hydrophobic and π-π interaction are crucial. The phenyl with 4-hydophobic substitutent
provides the best activity.
Key pharmacophore for antitubercular activity.
Phenyl with p-hydrophobic group
OH
provides better activity.
Br
O
N
H
R
The meta-amido link is important. The change of the position or
the replacement remarkably decreases the activity.
Chart 6. SAR of m-amido bromophenol derivatives.
2.3.3 Inhibitory activity against Mtb H37Rv and clinically isolated MDR-TB strains
Compounds 6p and 6r were selected for further evaluation against Mtb H37Rv and six
clinically isolated MDR-TB strains. The results are summarized in Table 4. Both compounds
exhibited moderate inhibitory activity against Mtb H37Rv and MDR-TB strains (MIC = 4 -
16 μg/mL). The activities are significantly lower than those against H37Ra. The reason is
elusive at the present stage.
Chemical and Pharmaceutical Bulletin Advance Publication

Table 4. Inhibitory activity against Mtb H37Rv and clinically isolated MDR-TB strains.
MIC (µg/mL)
Strains
6p
6r
16
RIF
INH
EMB
H37Rv
V4
8
0.03
0.41
2
8
8
8
4
8
8
16
16
16
8
<1
>10
>50
>50
>10
>10
>10
<2
--
K4
>50
>50
>10
>10
>10
K5
--
K12
K16
>4
>4
>4
16
16
K18
RIF: rifampicin
2.3.4 Evaluation of antibacterial activity
Table 5. Antibacterial activity of compounds 6p and 6r.
MIC (µg/mL)
Bacteria
6p
6r
Amoxicillin
E. coli
>50
>50
>50
>50
>50
>50
>50
>50
1.56
0.78
6.25
--a
S. aureus
E. faecalis
P. aeruginosa
a unmeasured
The current treatment of TB requires the combinations of several antitubercular agents
and a period of 6 months for drug-susceptible Mtb. Therefore, new antitubercular agents are
expected to exclusively inhibit Mtb without influencing the normal intestinal flora.12 The
antibacterial activities of compounds 6p and 6r were examined against Gram-positive
Chemical and Pharmaceutical Bulletin Advance Publication

bacteria S. aureus, E. faecalis and Gram-negative bacteria E. coli, P. aeruginosa. Amoxicillin
was used as the positive control. The results are summarized in Table 5. Compounds 6p and
6r did not show inhibitory activity (MIC > 50 μg/mL) against the tested bacteria. This result
demonstrated that the inhibitory activity of meta-amido bromophenols against Mtb is highly
exclusive.
2.3.5 Evaluation of cytotoxicity
The cytotoxicities of compounds 6p, 6r against cell lines A549 and HepG2 were evaluated
and the results are listed in Table 6. Low cytotoxicities (IC50 > 50 µg/mL) were observed
against A549, but moderate cytotoxicities were determined against HepG2. The select indexs
(SI = IC50/MIC) are in a range of 47-200 and are acceptable for the further research.
Table 6. Cytotoxicity of compounds 6p, 6r.
IC50 (µg/mL)
Compound
MIC (H37Ra)
A549 cells
HepG2
>50 (SI >100)
23.4 (SI = 47)
0.5
6p
6r
50 (SI = 200)
23.3 (SI = 93)
0.25
2.3.6 Evaluation of metabolic stability in liver microsome
The stability of compounds 6r in rat liver microsome was also examined. In comparison
with compound YZ-7 (T1/2 = 16.18 min, CLint = 85.7 mL/min/kg), 6r had a better metabolic
stability (T1/2 = 30.0 min, CLint = 69.9 mL/min/kg). The result confirmed that the
replacement of benzyl group in YZ-7 improves the metabolic stability.
3. Conclusion
We identified meta-amido bromophenols derivatives as a new class of antitubercular
agents. Several compounds exhibited potent inhibitory activity against Mtb H37Ra.
Furthermore, the selected compounds 6p and 6r showed moderate inhibitory activity against
Chemical and Pharmaceutical Bulletin Advance Publication

Mtb H37Rv and clinically isolated MDR-TB strains. The compounds did not inhibit the
representative G+ and G- bacteria. Moderate cytotoxicities were determined and the selective
indexes are acceptable. In addition, good metabolic stability in rat liver microsome was
observed. Considering the simple structure and readily modular property, meta-amido
bromophenols hold the good potentials for the further development as new antitubercular
agents.
4. Experimental
4.1 Chemistry
13
1H NMR and C NMR spectra were recorded on Bruker AVANCE 400 spectrometer.
Chemical shifts of protons are reported in parts per million downfield from tetramethylsilane.
Peaks are labeled as single (s), doublet (d), triplet (t), double doublet (dd), doublet of triplets
(dt), multiplet (m). The high-resolution mass spectra were analyzed on a SHIMADZU
LCMS-IT-TOF mass spectrometer. All chemicals were purchased from Sigma-Aldrich,
Energy and Alfa Aesar chemical companies and were used without further purification.
4.1.1 Typical procedure for the synthesis of compounds 1-3, 4a-4k
A solution of BBr3 in dichloromethane (1.0 M, 12 mL, 12 mmol) was added slowly to a
solution of 4-bromo-3-methoxyaniline (800 mg, 3.96 mmol) in methylene chloride (15 mL)
at 0°C. The resulting brown solution was warmed to room temperature and stirred for 24 h.
After saturated aqueous NaHCO3 (30 mL) was added at 0°C, the solution was extracted with
EtOAc (20 mL × 3). The combined organic layer was dried with anhydrous Na2SO4, filtered
and concentrated in vacuum. The residue was purified by flash chromatography over silica
gel (petroleum/EtOAc = 2:1) to give 5-amino-2-bromophenol (665 mg, 88%).
To a solution of 5-amino-2-bromophenol (55 mg, 0.29 mmol) and triethylamine (53 μL,
0.38 mmol) in THF (3 mL) was added slowly benzoyl chloride (0.32 mmol) at 0 °C. The
reaction mixture was then stirred at room temperature for 30 min. After the reaction was
quenched with water (10 mL), the solution was extracted with EtOAc (10 mL × 2). The
combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated in
vacuum. The residue was purified by column chromatography to afford the product 3 (74 mg,
Chemical and Pharmaceutical Bulletin Advance Publication

87%).
N-(4-bromo-3-hydroxyphenyl)benzamide (3)
1
White solid, m.p. 178.7 - 180.2 °C; H-NMR (400 MHz, DMSO-d6) δ: 10.29 (s, 1H), 10.25
(s, 1H), 7.98 – 7.87 (m, 2H), 7.66 (d, J = 2.4 Hz, 1H), 7.61 – 7.56 (m, 1H), 7.52 (t, J = 7.3 Hz,
2H), 7.41 (d, J = 8.7 Hz, 1H), 7.12 (dd, J = 8.7, 2.4 Hz, 1H); 13C-NMR (101 MHz, DMSO-d6)
δ: 166.15, 154.42, 140.04, 135.33, 132.87, 132.11, 128.86, 128.15, 113.11, 108.68, 103.73;
ESI-MS m/z: 291.9944 (Calcd for C13H10NO2Br [M+H]+: 291.9968) .
N-(3-bromo-5-hydroxyphenyl)benzamide (1)
The compound was synthesized via
a
similar procedure starting from
1
3-bromo-5-methoxyaniline. White solid, yield 80%. m.p. 184.3 - 186.2 °C; H NMR (400
MHz, DMSO-d6) δ 10.25 (s, 1H), 9.96 (s, 1H), 7.94 – 7.89 (m, 2H), 7.62 – 7.57 (m, 1H), 7.55
– 7.50 (m, 2H), 7.49 (t, J = 1.8 Hz, 1H), 7.33 (t, J = 2.0 Hz, 1H), 6.68 (t, J = 2.0 Hz, 1H); 13C
NMR (101 MHz, DMSO-d6) δ 166.24, 159.08, 141.93, 135.15, 132.23, 128.90, 128.16,
121.91, 113.96, 106.67; ESI-MS m/z: 291.9952 (Calcd for C13H10NO2Br [M+H]+:
291.9968) .
N-(2-bromo-5-hydroxyphenyl)benzamide (2)
The compound was synthesized via
a
similar procedure starting from
1
2-bromo-5-methoxyaniline. White solid, yield 52%. m.p. 195.5 - 197.2 °C; H NMR (400
MHz, DMSO-d6) δ 9.86 (d, J = 27.5 Hz, 2H), 7.97 (d, J = 6.9 Hz, 2H), 7.62 – 7.58 (m, 1H),
7.53 (dd, J = 10.8, 3.8 Hz, 2H), 7.45 (dd, J = 8.7, 2.9 Hz, 1H), 7.07 (s, 1H), 6.72 – 6.57 (m,
13
1H); C NMR (101 MHz, DMSO-d6) δ 165.32, 157.20, 136.99, 134.16, 132.91, 131.97,
128.65, 127.70, 115.26, 115.22, 108.45; ESI-MS m/z: 291.9922 (Calcd for C13H10NO2Br
[M+H]+: 291.9968) .
N-(4-bromophenyl)benzamide (4a)
The compound was synthesized via a similar procedure starting from 4-bromoaniline. White
1
solid, yield 85%. m.p. 209.9 - 212.0 °C; H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H),
7.95 (d, J = 7.2 Hz, 2H), 7.77 (d, J = 8.8 Hz, 2H), 7.66 – 7.44 (m, 5H); 13C NMR (101 MHz,
DMSO-d6) δ 165.69, 138.60, 134.72, 131.75, 131.46, 128.45, 127.70, 122.21, 115.34;
ESI-MS m/z: 297.9822 (Calcd for C13H10NOBr [M+Na]+: 297.9838) .
Chemical and Pharmaceutical Bulletin Advance Publication

N-(4-bromo-3-methoxyphenyl)benzamide (4b)
The compound was synthesized via
a
similar procedure starting from
1
4-bromo-3-methoxyaniline. White solid, yield 89%. m.p. 134.6 - 136.5 °C; H NMR (400
MHz, DMSO-d6) δ 10.35 (s, 1H), 7.96 (d, J = 7.3 Hz, 2H), 7.67 (d, J = 10.1 Hz, 1H), 7.63 –
7.48 (m, 4H), 7.40 (dd, J = 8.6, 2.1 Hz, 1H), 3.85 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ
166.13, 155.75, 140.57, 135.18, 133.03, 132.20, 128.89, 128.11, 113.98, 105.26, 104.75,
56.45; ESI-MS m/z: 306.0110 (Calcd for C14H12NO2Br [M+H]+: 306.0124) .
N-(4-chloro-3-hydroxyphenyl)benzamide (4c)
The compound was synthesized via
a
similar procedure starting from
5-amino-2-chlorophenol. White solid, yield 83%. m.p. 183.6 - 185.4 °C; 1H NMR (400 MHz,
DMSO-d6) δ 10.24 (d, J = 8.7 Hz, 2H), 7.92 (d, J = 7.2 Hz, 2H), 7.66 (d, J = 2.2 Hz, 1H),
13
7.56 (dt, J = 26.6, 7.2 Hz, 3H), 7.27 (d, J = 8.7 Hz, 1H), 7.17 (dd, J = 8.7, 2.2 Hz, 1H); C
NMR (101 MHz, DMSO-d6) δ 166.09, 153.37, 139.39, 135.36, 132.07, 129.92, 128.84,
128.15, 114.59, 112.53, 108.88; ESI-MS m/z: 248.0462 (Calcd for C13H10NO2Cl [M+H]+:
248.0473) .
N-(4-fluoro-3-hydroxyphenyl)benzamide (4d)
The compound was synthesized via a similar procedure starting from 5-amino-2-fluorophenol.
Pale yellow solid, yield 83%. m.p. 180.1 - 182.3 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.18
(s, 1H), 9.93 (s, 1H), 7.99 – 7.90 (m, 2H), 7.63 – 7.56 (m, 2H), 7.53 (t, J = 7.3 Hz, 2H), 7.13
13
(m, 2H); C NMR (101 MHz, DMSO-d6) δ 165.48, 147.45 (d, JCF = 237.4 Hz), 144.57 (d,
JCF = 13.1 Hz), 135.67(d, JCF = 2.0 Hz), 135.03, 131.57, 128.42, 127.68, 115.70 (d, JCF = 20.2
Hz), 111.19 (d, JCF = 6.0 Hz), 110.09; ESI-MS m/z: 232.0748 (Calcd for C13H10NO2F
[M+H]+: 232.0768) .
N-(3-hydroxy-4-methylphenyl)benzamide (4f)
The compound was synthesized via
a
similar procedure starting from
5-amino-2-methylphenol. White solid, yield 81%. m.p. 213.4 - 216.0 °C; 1H NMR (400 MHz,
DMSO-d6) δ 10.07 (s, 1H), 9.36 (s, 1H), 7.96 – 7.89 (m, 2H), 7.60 – 7.48 (m, 3H), 7.43 (d, J
= 1.8 Hz, 1H), 7.05 (dd, J = 8.1, 1.9 Hz, 1H), 6.99 (d, J = 8.2 Hz, 1H), 2.09 (s, 3H);13C NMR
(101 MHz, DMSO-d6) δ 165.32, 155.22, 137.78, 135.26, 131.39, 130.18, 128.35, 127.66,
Chemical and Pharmaceutical Bulletin Advance Publication

119.24, 111.08, 107.17, 15.64; ESI-MS m/z: 228.1086 (Calcd for C14H13NO2 [M+H]+:
228.1019) .
N-(3-hydroxy-4-methoxyphenyl)benzamide (4g)
The compound was synthesized via
a
similar procedure starting from
1
5-amino-2-methoxyphenol. White solid, yield 80%. m.p. 176.5 - 178.5 °C; H NMR (400
MHz, DMSO-d6) δ 10.02 (s, 1H), 9.07 (s, 1H), 7.99 – 7.87 (m, 1H), 7.60 – 7.47 (m, 1H), 7.35
(d, J = 2.4 Hz, 1H), 7.14 (dd, J = 8.7, 2.4 Hz, 1H), 6.88 (d, J = 8.7 Hz, 1H), 3.74 (s, 1H);13C
NMR (101 MHz, DMSO-d6) δ 165.09, 146.28, 144.14, 135.23, 132.73, 131.35, 128.36,
127.59, 112.26, 111.23, 108.92, 55.88; ESI-MS m/z: 244.0927 (Calcd for C14H13NO3 [M+H]+:
244.0968) .
methyl 4-benzamido-2-hydroxybenzoate (4h)
The compound was synthesized via a similar procedure starting from methyl
1
4-amino-2-hydroxybenzoate. White solid, yield 77%. m.p. 170.0 - 171.8 °C; H NMR (400
MHz, DMSO-d6) δ 10.65 (s, 1H), 10.51 (s, 1H), 7.94 (dd, J = 7.4, 5.9 Hz, 2H), 7.77 (d, J =
8.8 Hz, 1H), 7.63 – 7.57 (m, 2H), 7.57 – 7.51 (m, 2H), 7.37 (dd, J = 8.8, 2.0 Hz, 1H), 3.88 (s,
13
3H); C NMR (101 MHz, DMSO-d6) δ 169.09, 166.23, 161.11, 145.76, 134.57, 131.99,
130.63, 128.49, 127.87, 111.41, 107.77, 107.14, 52.29; ESI-MS m/z: 294.0727 (Calcd for
C15H13NO4 [M+Na]+: 294.0737) .
Synthesis of 4-benzamido-2-hydroxybenzoic acid (4i)
To a solution of compound 4h (90 mg, 0.33 mmol ) in THF ( 2 mL ) was added aqueous
NaOH (1.5 M, 8 mL). The mixture was stirred at 50 °C for 2 h. After being cooled to room
temperature, the solution was acidified with 2M HCl to pH = 3-4. The precipitation was
collected to give the compound 4h (62 mg, 73%). m.p. 263.1 - 264.7 °C; 1H NMR (400 MHz,
DMSO-d6) δ 10.48 (s, 1H), 8.05 – 7.89 (m, 2H), 7.76 (d, J = 8.7 Hz, 1H), 7.67 – 7.44 (m, 4H),
13
7.34 (dd, J = 8.7, 2.0 Hz, 1H); C NMR (101 MHz, DMSO-d6) δ 172.11, 166.64, 162.44,
146.08, 135.06, 132.40, 131.32, 128.93, 128.30, 111.59, 108.51, 107.38; ESI-MS m/z:
280.0563 (Calcd for C14H11NO4 [M+Na]+: 280.0580) .
N-(4-acetyl-3-hydroxyphenyl)benzamide (4j)
The compound was synthesized via a similar procedure starting from 1-(4-amino-2-
Chemical and Pharmaceutical Bulletin Advance Publication

1
hydroxyphenyl)ethan-1-one. Pale yellow solid, yield 82%. m.p. 204.9 - 206.6 °C; H NMR
(400 MHz, DMSO-d6) δ 12.34 (s, 1H), 10.54 (s, 1H), 7.95 (d, J = 8.0 Hz, 2H), 7.90 (d, J =
8.8 Hz, 1H), 7.65 – 7.59 (m, 1H), 7.59 – 7.50 (m, 3H), 7.37 (d, J = 8.8 Hz, 1H), 2.60 (s, 3H);
13C NMR (101 MHz, DMSO-d6) δ 203.27, 166.31, 162.34, 146.29, 134.50, 132.42, 132.07,
128.52, 127.90, 115.83, 111.06, 106.96, 26.96; ESI-MS m/z: 278.0792 (Calcd for C15H13NO3
[M+Na]+: 278.0788) .
N-(4-cyano-3-hydroxyphenyl)benzamide (4k)
The compound was synthesized via a similar procedure starting from 4-amino-2-methoxy
1
benzonitrile. Yellow brown solid, yield 67%. m.p. 151.3 - 153.6 °C; H NMR (400 MHz,
DMSO-d6) δ 8.19 – 8.10 (m, 2H), 7.78 (t, J = 7.4 Hz, 1H), 7.63 (t, J = 7.7 Hz, 2H), 7.48 (d, J
= 9.1 Hz, 1H), 6.60 – 6.54 (m, 2H), 6.45 (s, 2H); 13C NMR (101 MHz, DMSO-d6) δ 164.22,
155.14, 154.15, 134.99, 134.50, 130.38, 129.65, 128.57, 117.47, 111.90, 107.27, 90.59;
ESI-MS m/z: 261.0582 (Calcd for C14H10N2O2 [M+Na]+: 261.0634) .
4.1.2 Synthesis of compounds 5a-5c
To a solution of benzoic acid (37 mg, 0.30 mmol) and HATU (138 mg, 0.36 mmol) in
DMF (4 mL) was added DIPEA (63 μL, 0.36 mmol) and 4-amino-2-bromophenol (56 mg,
0.30 mmol) separately. The reaction mixture was stirred at room temperature for 4 h. After
water (20 mL) was added, the mixture was extracted with EtOAc (10 mL × 2). The combined
organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum. The residue
was purified by flash chromatography over silica gel (petroleum/EtOAc = 10:1 to 5:1) to give
the compound 5a.
N-(3-bromo-4-hydroxyphenyl)benzamide (5a)
1
White solid, yield 90%. m.p. 198.8 - 200.2 °C; H NMR (400 MHz, DMSO-d6) δ 10.16 (s,
1H), 10.11 (s, 1H), 7.98 (d, J = 2.5 Hz, 1H), 7.92 (dd, J = 5.2, 3.3 Hz, 2H), 7.60 – 7.47 (m,
13
4H), 6.93 (d, J = 8.8 Hz, 1H); C NMR (101 MHz, DMSO-d6) δ 165.24, 150.44, 134.83,
131.86, 131.61, 128.48, 127.61, 124.95, 121.27, 116.03, 108.58; ESI-MS m/z: 291.9941
(Calcd for C13H10NO2Br [M+H]+: 291.9968) .
N-(3-bromo-2-hydroxyphenyl)benzamide (5b)
The compound was synthesized via
a
similar procedure starting from
Chemical and Pharmaceutical Bulletin Advance Publication

2-amino-6-bromophenol. White solid, yield 71%. m.p. 127.4 - 128.6 °C; 1H NMR (400 MHz,
DMSO-d6) δ 10.08 (s, 1H), 9.76 (s, 1H), 8.01 (d, J = 7.3 Hz, 2H), 7.62 (t, J = 7.3 Hz, 1H),
13
7.55 (t, J = 7.5 Hz, 2H), 7.44 (t, J = 6.7 Hz, 2H), 6.84 (t, J = 8.0 Hz, 1H); C NMR (101
MHz, DMSO-d6) δ 166.81, 148.04, 134.23, 132.43, 130.44, 128.91, 128.39, 127.91, 125.78,
121.05, 112.13; ESI-MS m/z: 291.9932 (Calcd for C13H10NO2Br [M+H]+: 291.9968) .
4-bromo-3-hydroxy-N-phenylbenzamide (5c)
The compound was synthesized via
a
similar procedure starting from
4-bromo-3-hydroxybenzoic acid and aniline. White solid, yield 77%. m.p. 188.5 - 190.7 °C;
1H NMR (400 MHz, DMSO-d6) δ 10.64 (s, 1H), 10.25 (s, 1H), 7.74 (d, J = 7.6 Hz, 2H), 7.64
(d, J = 8.2 Hz, 1H), 7.47 (d, J = 2.0 Hz, 1H), 7.38 – 7.30 (m, 3H), 7.10 (t, J = 7.4 Hz, 1H);
13C NMR (101 MHz, DMSO-d6) δ 164.85, 154.15, 139.05, 135.66, 132.81, 128.67, 123.79,
120.39, 119.36, 115.64, 113.05; ESI-MS m/z: 291.9819 (Calcd for C13H10NO2Br [M+H]+:
291.9968) .
Synthesis of 5-(benzylamino)-2-bromophenol (5d)
To a solution of 4-bromo-3-methoxyaniline (80 mg, 0.40 mmol) and K2CO3 (110 mg,
0.80 mmol) in DMF (5 mL) was added (bromomethyl)benzene (75 mg, 0.44mmol) at room
temperature. The mixture was stirred at 55 °C for 24 h. After water (20 mL) was added, the
mixture was extracted with EtOAc (10mL × 2). The combined organic layer was dried over
anhydrous Na2SO4 and concentrated under vacuum. The crude product was purified by flash
chromatography over silica gel (petroleum/EtOAc
=
10:1 to 5:1) to give
N-benzyl-4-bromo-3-methoxyaniline (84 mg, 72%).
The compound 5d was synthesized form N-benzyl-4-bromo-3-methoxyaniline via a similar
procedure 4.1.1. White solid, yield 38%. m.p. 77.2 - 78.3 °C; 1H NMR (400 MHz, DMSO-d6)
δ 9.65 (s, 1H), 7.38 – 7.27 (m, 4H), 7.25 – 7.17 (m, 1H), 7.04 (d, J = 8.6 Hz, 1H), 6.36 (t, J =
5.9 Hz, 1H), 6.19 (d, J = 2.6 Hz, 1H), 6.02 (dd, J = 8.7, 2.6 Hz, 1H), 4.19 (d, J = 5.9 Hz, 2H);
13C NMR (101 MHz, DMSO-d6) δ 154.70, 149.67, 140.37, 132.88, 128.76, 127.54, 127.13,
106.19, 100.62, 95.37, 46.91; ESI-MS m/z: 278.0149 (Calcd for C13H12NOBr [M+H]+:
278.0175) .
4.1.3 Synthesis of compounds 6a-6u
Chemical and Pharmaceutical Bulletin Advance Publication

N-(4-bromo-3-hydroxyphenyl)acetamide (6a)
The compound was synthesized via a similar procedure 4.1.1 using acetyl chloride. White
1
solid, yield 54%. m.p. 215.7 - 216.5 °C; H NMR (400 MHz, DMSO-d6) δ 10.22 (s, 1H),
9.93 (s, 1H), 7.47 (d, J = 2.4 Hz, 1H), 7.33 (d, J = 8.6 Hz, 1H), 6.94 – 6.79 (m, 1H), 2.01 (s,
13
3H); C NMR (101 MHz, DMSO-d6) δ 168.41, 154.03, 139.74, 132.48, 111.25, 106.88,
102.39, 24.11; ESI-MS m/z: 229.9885 (Calcd for C8H8NO2Br [M+H]+: 229.9811) .
N-(4-bromo-3-hydroxyphenyl)acrylamide (6b)
The compound was synthesized via a similar procedure 4.1.1 using acryloyl chloride. Brown
1
solid, yield 63%. m.p. 183.6 - 186.0 °C; H NMR (400 MHz, DMSO-d6) δ 10.30 (s, 1H),
10.16 (s, 1H), 7.54 (d, J = 2.3 Hz, 1H), 7.38 (d, J = 8.6 Hz, 1H), 7.00 (dd, J = 8.7, 2.3 Hz,
1H), 6.43 (dd, J = 17.0, 10.1 Hz, 1H), 6.25 (dd, J = 17.0, 2.0 Hz, 1H), 5.76 (dd, J = 10.1, 2.0
Hz, 1H); 13C NMR (101 MHz, DMSO-d6) δ 163.12, 154.06, 139.37, 132.56, 131.76, 127.01,
111.64, 107.25, 103.04; ESI-MS m/z: 241.9805 (Calcd for C9H8NO2Br [M+H]+: 241.9811) .
N-(4-bromo-3-hydroxyphenyl)nonanamide (6c)
The compound was synthesized via a similar procedure 4.1.2 using octanoic acid. White solid,
1
yield 76%. m.p. 181.8 - 183.3 °C; H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 9.84 (s,
1H), 7.47 (d, J = 2.3 Hz, 1H), 7.32 (d, J = 8.6 Hz, 1H), 6.89 (dd, J = 8.7, 2.4 Hz, 1H), 2.26 (t,
J = 7.4 Hz, 2H), 1.61 – 1.49 (m, 2H), 1.26 (dd, J = 5.7, 2.5 Hz, 8H), 0.85 (d, J = 7.0 Hz, 3H);
13C NMR (101 MHz, DMSO-d6) δ 171.36, 154.00, 139.74, 132.43, 111.33, 106.97, 102.31,
36.47, 31.19, 28.65, 28.48, 25.10, 22.09, 13.97; ESI-MS m/z: 314.0734 (Calcd for
C14H20NO2Br [M+H]+: 314.0750) .
N-(4-bromo-3-hydroxyphenyl)cyclohexanecarboxamide (6d)
The compound was synthesized via a similar procedure 4.1.1 using cyclohexanecarbonyl
1
chloride. White solid, yield 80%. m.p. 194.9 - 197.2 °C; H NMR (400 MHz, DMSO-d6) δ
10.19 (s, 1H), 9.79 (s, 1H), 7.46 (d, J = 2.3 Hz, 1H), 7.31 (d, J = 8.7 Hz, 1H), 6.90 (dd, J =
8.7, 2.4 Hz, 1H), 2.33 – 2.24 (m, 1H), 1.82 – 1.69 (m, 4H), 1.43 – 1.32 (m, 2H), 1.31 – 1.09
(m, 4H); 13C NMR (101 MHz, DMSO-d6) δ 174.44, 153.98, 139.90, 132.42, 111.49, 107.12,
102.33, 44.91, 29.13, 25.42, 25.25; ESI-MS m/z: 298.0391 (Calcd for C13H16NO2Br [M+H]+:
298.0437) .
Chemical and Pharmaceutical Bulletin Advance Publication

N-(4-bromo-3-hydroxyphenyl)morpholine-4-carboxamide (6e)
To a solution of 5-amino-2-bromophenol (55 mg, 0.29 mmol) and triethylamine (53 μL,
0.38 mmol) in THF (3 mL) was added slowly morpholine-4-carbonyl chloride (0.32 mmol) at
0 °C. The mixture was stirred at 45 °C for 12 h. The reaction was quenched with water
(10 mL) and the mixture was extracted with EtOAc (10 mL × 2). The combined organic layer
was dried over anhydrous Na2SO4, filtered and concentrated under vacuum. The residue was
purified by column chromatography to afford the product 6e as a white solid (60 mg, 69%).
m.p. 193.0 - 194.8 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.08 (s, 1H), 8.54 (s, 1H), 7.31 (d,
J = 2.4 Hz, 1H), 7.27 (d, J = 8.7 Hz, 1H), 6.82 (dd, J = 8.7, 2.4 Hz, 1H), 3.62 – 3.56 (m, 4H),
13
3.42 – 3.38 (m, 4H); C NMR (101 MHz, DMSO-d6) δ 155.36, 154.22, 141.39, 132.48,
112.31, 107.88, 101.65, 66.44, 44.62; ESI-MS m/z: 301.0182 (Calcd for C11H13N2O3Br
[M+H]+: 301.0143) .
N-(4-bromo-3-hydroxyphenyl)picolinamide (6f)
The compound was synthesized via a similar procedure 4.1.2 using picolinic acid. White
1
solid, yield 80%. m.p. 202.5 - 204.4 °C; H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H),
10.33 (s, 1H), 8.79 – 8.69 (m, 1H), 8.14 (d, J = 7.8 Hz, 1H), 8.10 – 8.02 (m, 1H), 7.80 (d, J =
2.4 Hz, 1H), 7.71 – 7.63 (m, 1H), 7.42 (d, J = 8.7 Hz, 1H), 7.21 (dd, J = 8.7, 2.4 Hz, 1H); 13C
NMR (101 MHz, DMSO-d6) δ 162.57, 154.03, 149.84, 148.45, 138.74, 138.16, 132.48,
126.99, 122.46, 112.79, 108.18, 103.67; ESI-MS m/z: 292.9910 (Calcd for C12H9N2O2Br
[M+H]+: 292.9920) .
N-(4-bromo-3-hydroxyphenyl)thiophene-2-carboxamide (6g)
The compound was synthesized via a similar procedure 4.1.1 using thiophene-2-carbonyl
1
chloride. White solid, yield 66%. m.p. 222.7 - 224.5 °C; H NMR (400 MHz, DMSO-d6) δ
10.31 (s, 1H), 10.21 (s, 1H), 8.01 (d, J = 3.1 Hz, 1H), 7.84 (d, J = 4.8 Hz, 1H), 7.57 (d, J =
2.2 Hz, 1H), 7.41 (d, J = 8.7 Hz, 1H), 7.24 – 7.16 (m, 1H), 7.09 (dd, J = 8.7, 2.2 Hz, 1H); 13C
NMR (101 MHz, DMSO-d6) δ 159.88, 154.01, 139.91, 139.16, 132.50, 132.04, 129.29,
128.09, 112.60, 108.20, 103.40; ESI-MS m/z: 297.9494 (Calcd for C11H8NO2SBr [M+H]+:
297.9532) .
Chemical and Pharmaceutical Bulletin Advance Publication

N-(4-bromo-3-hydroxyphenyl)furan-2-carboxamide (6h)
The compound was synthesized via a similar procedure 4.1.1 using furan-2-carbonyl chloride.
1
White solid, yield 53%. m.p. 207.2 - 209.7 °C; H NMR (400 MHz, DMSO-d6) δ 10.32 (s,
1H), 10.19 (s, 1H), 7.93 (dd, J = 1.6, 0.7 Hz, 1H), 7.61 (d, J = 2.4 Hz, 1H), 7.43 – 7.31 (m,
13
2H), 7.09 (dd, J = 8.7, 2.4 Hz, 1H), 6.70 (dd, J = 3.5, 1.7 Hz, 1H); C NMR (101 MHz,
DMSO-d6) δ 156.22, 154.00, 147.38, 145.90, 139.00, 132.49, 114.96, 112.66, 112.22, 108.21,
103.43; ESI-MS m/z: 281.9753 (Calcd for C11H8NO3Br [M+H]+: 281.9760) .
N-(4-bromo-3-hydroxyphenyl)-2-chlorobenzamide (6i)
The compound was synthesized via a similar procedure 4.1.1 using 2-chlorobenzoyl chloride.
1
White solid, yield 79%. m.p. 204.4 - 206.7 °C; H NMR (400 MHz, DMSO-d6) δ 10.51 (s,
1H), 10.33 (s, 1H), 7.61 (d, J = 2.3 Hz, 1H), 7.56 (dd, J = 7.8, 2.5 Hz, 2H), 7.50 (m, J = 7.7,
1.7 Hz, 1H), 7.44 (m, J = 7.3, 1.1 Hz, 1H), 7.40 (d, J = 8.6 Hz, 1H), 7.01 (dd, J = 8.6, 2.3 Hz,
13
1H); C NMR (101 MHz, DMSO-d6) δ 165.02, 154.16, 139.30, 136.90, 132.66, 131.20,
129.91, 129.69, 128.95, 127.33, 111.93, 107.52, 103.43; ESI-MS m/z: 325.9586 (Calcd for
C13H9NO2ClBr [M+H]+: 325.9578) .
N-(4-bromo-3-hydroxyphenyl)-3-chlorobenzamide (6j)
The compound was synthesized via a similar procedure 4.1.1 using 3-chlorobenzoyl chloride.
White solid, yield 74%. m.p. 184.2 - 186.1 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.35 (d, J
= 3.6 Hz, 2H), 7.98 (t, J = 1.7 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.70 – 7.61 (m, 2H), 7.56 (t,
J = 7.9 Hz, 1H), 7.42 (d, J = 8.7 Hz, 1H), 7.11 (dd, J = 8.7, 2.4 Hz, 1H); 13C NMR (101 MHz,
DMSO-d6) δ 164.17, 154.03, 139.33, 136.86, 133.22, 132.50, 131.49, 130.44, 127.48, 126.58,
112.67, 108.24, 103.56; ESI-MS m/z: 325.9562 (Calcd for C13H9NO2ClBr [M+H]+:
325.9578) .
N-(4-bromo-3-hydroxyphenyl)-4-chlorobenzamide (6k)
The compound was synthesized via the procedure similar to compound 6e. White solid, yield
52%. m.p. 195.0 - 197.3 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 10.19 (s, 1H),
7.93 (dd, J = 1.6, 0.7 Hz, 1H), 7.61 (d, J = 2.4 Hz, 1H), 7.43 – 7.31 (m, 2H), 7.09 (dd, J = 8.7,
13
2.4 Hz, 1H), 6.70 (dd, J = 3.5, 1.7 Hz, 1H); C NMR (101 MHz, DMSO-d6) δ 156.22,
154.00, 147.38, 145.90, 139.00, 132.49, 114.96, 112.66, 112.22, 108.21, 103.43; ESI-MS
Chemical and Pharmaceutical Bulletin Advance Publication

m/z: 325.9565 (Calcd for C13H9NO2ClBr [M+H]+: 325.9578) .
N-(4-bromo-3-hydroxyphenyl)-4-fluorobenzamide (6l)
The compound was synthesized via the procedure similar to compound 3. White solid, yield
1
83%. m.p. 181.9 - 183.4 °C; H NMR (400 MHz, DMSO-d6) δ 10.29 (d, J = 18.2 Hz, 2H),
8.11 – 7.95 (m, 2H), 7.64 (s, 1H), 7.38 (dt, J = 16.4, 8.7 Hz, 3H), 7.11 (d, J = 8.7 Hz, 1H);
13C NMR (101 MHz, DMSO-d6) δ 164.54, 164.12 (d, JCF = 249.5 Hz), 154.01, 139.52,
132.46, 131.32, 130.50 (d, JCF = 9.1 Hz), 115.36 (d, JCF = 22.2 Hz), 112.65, 108.22, 103.35;
ESI-MS m/z: 309.9838 (Calcd for C13H9NO2FBr [M+H]+: 309.9873) .
4-bromo-N-(4-bromo-3-hydroxyphenyl)benzamide (6m)
The compound was synthesized via a similar procedure 4.1.1 using 4-bromobenzoyl chloride.
1
White solid, yield 81%. m.p. 208.5 - 210.1 °C; H NMR (400 MHz, DMSO-d6) δ 10.31 (s,
2H), 7.93 – 7.81 (m, 2H), 7.80 – 7.68 (m, 2H), 7.63 (d, J = 2.4 Hz, 1H), 7.41 (d, J = 8.7 Hz,
13
1H), 7.10 (dd, J = 8.7, 2.4 Hz, 1H); C NMR (101 MHz, DMSO-d6) δ 164.66, 154.02,
139.41, 133.94, 132.48, 131.43, 129.88, 125.44, 112.65, 108.23, 103.47; ESI-MS m/z:
369.9088 (Calcd for C13H9NO2Br2 [M+H]+: 369.9073) .
N-(4-bromo-3-hydroxyphenyl)-4-methylbenzamide (6n)
The compound was synthesized via a similar procedure 4.1.1 using 4-methylbenzoyl chloride.
1
White solid, yield 62%. m.p. 195.4 - 197.2 °C; H NMR (400 MHz, DMSO-d6) δ 10.30 (s,
1H), 10.17 (s, 1H), 7.85 (d, J = 8.2 Hz, 2H), 7.66 (d, J = 2.4 Hz, 1H), 7.40 (d, J = 8.7 Hz, 1H),
13
7.33 (d, J = 8.0 Hz, 2H), 7.11 (dd, J = 8.7, 2.4 Hz, 1H), 2.38 (s, 3H); C NMR (101 MHz,
DMSO-d6) δ 165.47, 153.98, 141.70, 139.71, 132.40, 132.01, 128.94, 127.78, 112.63, 108.20,
103.14, 21.07; ESI-MS m/z: 306.0111 (Calcd for C14H12NO2Br [M+H]+: 306.0124) .
N-(4-bromo-3-hydroxyphenyl)-4-methoxybenzamide (6o)
The compound was synthesized via a similar procedure 4.1.1 using 4-methoxybenzoyl
1
chloride. White solid, yield 81%. m.p. 207.6 - 208.9 °C; H NMR (400 MHz, DMSO-d6) δ
10.26 (s, 1H), 10.09 (s, 1H), 7.93 (d, J = 8.8 Hz, 2H), 7.65 (d, J = 2.3 Hz, 1H), 7.39 (d, J =
8.7 Hz, 1H), 7.11 (dd, J = 8.7, 2.3 Hz, 1H), 7.06 (t, J = 5.8 Hz, 2H), 3.83 (s, 3H); 13C NMR
(101 MHz, DMSO-d6) δ 164.96, 161.95, 153.93, 139.80, 132.34, 129.67, 126.88, 113.60,
112.60, 108.16, 102.96, 55.46; ESI-MS m/z: 322.0041 (Calcd for C14H12NO3Br [M+H]+:
Chemical and Pharmaceutical Bulletin Advance Publication

322.0073) .
N-(4-bromo-3-hydroxyphenyl)-4-(trifluoromethoxy)benzamide (6p)
The compound was synthesized via
a
similar procedure 4.1.1 using
1
4-(trifluoromethoxy)benzoyl chloride. White solid, yield 80%. m.p. 189.1 - 190.1 °C; H
NMR (400 MHz, DMSO-d6) δ 10.35 (d, J = 10.6 Hz, 2H), 8.13 – 8.01 (m, 2H), 7.64 (d, J =
2.4 Hz, 1H), 7.52 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 8.7 Hz, 1H), 7.11 (dd, J = 8.7, 2.4 Hz, 1H);
13C NMR (101 MHz, DMSO-d6) δ 164.49, 154.04, 139.41, 134.07, 132.51, 130.16, 120.75,
13
112.63, 108.21, 103.50, C NMR (101 MHz, DMSO-d6) δ 164.49, 154.04, 150.50, 139.41,
134.07, 132.51, 130.16, 120.75, 112.63, 108.21, 103.50; ESI-MS m/z: 375.9757 (Calcd for
C14H9NO3F3Br [M+H]+: 375.9791) .
N-(4-bromo-3-hydroxyphenyl)-4-(dimethylamino)benzamide (6q)
The compound was synthesized via
a
similar procedure 4.1.1 using
4-(dimethylamino)benzoyl chloride. White solid, yield 71%. m.p. 238.3 - 240.7 °C; 1H NMR
(400 MHz, DMSO-d6) δ 10.22 (s, 1H), 9.87 (s, 1H), 7.85 (d, J = 8.9 Hz, 2H), 7.67 (d, J = 2.3
Hz, 1H), 7.37 (d, J = 8.7 Hz, 1H), 7.11 (dd, J = 8.7, 2.3 Hz, 1H), 6.74 (d, J = 9.0 Hz, 2H),
13
2.99 (s, 6H); C NMR (101 MHz, DMSO-d6) δ 165.28, 153.89, 152.44, 140.19, 132.25,
130.96, 129.24, 120.93, 112.55, 110.76, 108.10, 102.52; ESI-MS m/z: 355.0345 (Calcd for
C15H15N2O2Br [M+H]+: 335.0390) .
N-(4-bromo-3-hydroxyphenyl)-4-(trifluoromethyl)benzamide (6r)
The compound was synthesized via
a
similar procedure 4.1.1 using
4-(trifluoromethyl)benzoyl chloride. White solid, yield 73%. m.p. 205.1 - 206.6 °C; 1H NMR
(400 MHz, DMSO-d6) δ 10.47 (s, 1H), 10.35 (s, 1H), 8.12 (d, J = 8.1 Hz, 2H), 7.90 (d, J =
8.3 Hz, 2H), 7.65 (d, J = 2.4 Hz, 1H), 7.43 (d, J = 8.7 Hz, 1H), 7.13 (dd, J = 8.7, 2.4 Hz, 1H);
13C NMR (101 MHz, DMSO) δ 164.95, 154.50, 139.70, 139.16, 132.96, 131.85 (d, J
=
CF
32.3Hz), 129.10, 125.83 (d, J CF= 3.0 Hz), 113.12, 108.72, 104.13; ESI-MS m/z: 359.9803
(Calcd for C14H9NO2F3Br [M+H]+: 359.9842) .
N-(4-bromo-3-hydroxyphenyl)-4-nitrobenzamide (6s)
The compound was synthesized via a similar procedure 4.1.1 using 4-nitrobenzoyl chloride.
Yellow solid, yield 48%. m.p. 237.0 - 239.1 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.58 (s,
Chemical and Pharmaceutical Bulletin Advance Publication

1H), 10.38 (s, 1H), 8.37 (d, J = 8.8 Hz, 2H), 8.16 (d, J = 8.8 Hz, 2H), 7.65 (d, J = 2.2 Hz, 1H),
13
7.45 (d, J = 8.7 Hz, 1H), 7.14 (dd, J = 8.7, 2.3 Hz, 1H); C NMR (101 MHz, DMSO-d6) δ
164.45, 154.52, 149.62, 141.00, 139.58, 133.02, 129.74, 124.01, 113.11, 108.69, 104.28.
ESI-MS m/z: 336.9884 (Calcd for C13H9N2O4Br [M+H]+: 336.9818) .
N-(4-bromo-3-hydroxyphenyl)-4-hydroxybenzamide (6t)
The compound was synthesized by compound 6o as the material and then the procedure
1
similar to 4.1.1. White solid, yield 32%. m.p. 215.2 - 216.7 °C; H NMR (400 MHz,
DMSO-d6) δ 10.19 (d, J = 47.7 Hz, 2H), 9.99 (s, 1H), 7.82 (d, J = 8.3 Hz, 2H), 7.64 (s, 1H),
13
7.38 (d, J = 8.7 Hz, 1H), 7.09 (d, J = 8.7 Hz, 1H), 6.85 (d, J = 8.3 Hz, 2H); C NMR
(101 MHz, DMSO-d6) δ 165.62, 161.06, 154.36, 140.37, 132.76, 130.25, 125.77, 115.36,
113.00, 108.56, 103.25; ESI-MS m/z: 307.9882 (Calcd for C13H10NO3Br [M+H]+:
307.9917) .
Synthesis of N-(4-bromo-3-hydroxyphenyl)-2-naphthamide (6u)
The compound was synthesized via a similar procedure 4.1.1 using 2-naphthoyl chloride.
1
White solid, yield 73%. m.p. 207.6 - 209.9 °C; H NMR (400 MHz, DMSO-d6) δ 10.43 (s,
1H), 10.32 (s, 1H), 8.56 (s, 1H), 8.11 – 7.97 (m, 4H), 7.72 (d, J = 2.4 Hz, 1H), 7.68 – 7.58 (m,
2H), 7.43 (d, J = 8.7 Hz, 1H), 7.17 (dd, J = 8.7, 2.4 Hz, 1H);13C NMR (101 MHz, DMSO-d6)
δ 166.17, 154.48, 140.14, 134.75, 132.92, 132.67, 132.51, 129.44, 128.49, 128.34, 128.16,
127.35, 124.96, 113.11, 108.67, 103.76; ESI-MS m/z: 342.0097 (Calcd for C17H12NO2Br
[M+H]+: 342.0124) .
4.2 Biological assays
4.2.1 Evaluation of inhibitory activity against Mtb H37Ra in vitro
Selectable marker-free autoluminescent Mtb H37Ra (UAlRa)13 broth culture in
Middlebook 7H9 medium plus 0.05% Tween80 and 10% oleic acid albumin dextrose catalase
(OADC) (7H9-Tw-OADC). Upon reaching an OD600 of 0.5-0.8, relative light unit (RLU)
value was determined by luminometer. When the RLU of 200 μL broth culture reached to
over 1 million, the test compounds were prepare with two-fold serial dilutions in a range of
final concentration from 100 μg/mL to 0.0625 μg/mL per 200 μL UAlRa of broth culture
(RLU were diluted to 2000-5000 per 200 μL). On the other hand, DMSO final concentration
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of 2% was used as negative control while the compounds (2-fold decreasing concentration
from 100 μg/mL to 0.0625 μg/mL) as well as isoniazid ( 0.1 μg/mL ) and ethambutol ( 2
μg/mL ) were used as positive controls. The RLU values were determined once daily until the
6th day. Data was analysed as MIC90 values which is basically the lowest drug concentration
that achieved a RLUdrug/RLUDMSO of less than 10% after incubation.
4.2.2 Evaluation of inhibitory activity against Mtb H37Rv and MDR-TB strains
The MICs of the test compounds were determined by the microplate alamar blue assay
(MABA)14 against Mtb H37Rv and clinically isolated MDR-TB strains isolated from the
Guangzhou Chest Hospital. Mtb H37Rv and MDR-TB strains were cultured in
7H9-Tw-OADC. The test compounds were prepared with two-fold serial dilutions in a range
of final concentration from 0.25 μg/mL to 128 μg/mL, two-fold dilutions of compounds were
prepared in 7H9-OADC medium in a 100 μL volume in 96-well microplates. INH, RIF and
EMB were used as the positive controls. Bacterial suspension of inoculum of 2×105 CFU/mL
was added 100 μL per well into the microplates, and the plates incubated 6 days at 37℃.On
the 7th day, 12.5 μL of 20% Tween 80 and 20 μL of alamar Blue (Bio-Rad) were added to the
test plate, and incubated another 24h at 37℃, and then observed the color change. If a blue
color in the well was deemed to no growth, and the blue color change to pink or violet was
interpreted as growth. The MIC was defined as the lowest drug concentration which
prevented a color change from blue to pink.
4.2.3 Determination of the antibacterial activity
To evaluated compounds exclusive of against Mtb, we selected 6p and 6r for
determination of antibacterial activity against Gram-positive and Gram-negative bacteria.
Initially, the test compounds were dissolved in DMSO to prepare the stock solutions
(10 mg/mL), and then prepared a range of concentrations from 50 to 0.4 μg/mL by means of
the standard two fold serial dilution method in 96-well microtest plates. Amoxicillin was used
as the positive controls. The bacterial suspension was adjusted with sterile saline to a
concentration of 1×105 CFU. 100 μL the bacterial suspension per well was added into the
plates and incubated at 37℃ for 24h. The MIC of compound was defined as the lowest
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concentration (the highest dilution) that completely inhibited the growth of bacteria after
incubation at 37 ℃ for 18-24 h.15
4.2.4 Determination of the cytotoxicity
A549 cell and HepG2 cell were used for determination of the cytotoxicity of compounds.
This two cell lines both were culture in RPMI 1640 Medium (with 5% fetal bovine serum
(FBS) and 1% penicillin-streptomycin), and incubated at 37℃ in 5% CO2 until they reach
log phage. The test compounds were diluted with RPMI 1640 Medium by 2-fold dilution and
the concentrations from 100 to 3.125 μg/mL. The cells suspension (1×105 cell/mL) of
100 μL were seeded in 96-well plates and then incubated at 37℃ in 5% CO overnight.16 100
2
μL different concentrations of the test compound were added to the plate by the form of
replace the cell medium and 100 μL RPMI 1640 Medium was used as the 0% inhibitor
control. Each experiment was repeated three times. After being incubated for 48 h,
10 μL/well CCK8 was added in the plates, and another 1-2 hour incubation, the plates were
read in the Envision MultilabelReader at 450 nm. Cell survival rate ( activity% ) was
calculated as follow: activity%=100*(ODcompound-ODblank)/ (ODcontrol-ODblank). The
cytotoxicities were reported as IC50 values, which were calculated by GraphPad Prism
Software version 5.23.
4.2.5 Determination of the liver microsome stability
The rat liver microsome was purchased from Xenotech company. The final incubation
volume was set at 45 μL, contained NADPH (2 mM), microsomes (0.5 mg/mL) and the
tested compound (1 μM). The incubation times were 0, 5, 15, 30, 45 and 60 min. Ice-cold
acetonitrile was added to terminate the reaction and then centrifuged at 4 ℃ for 15 min at
4000 rpm. The concentration of the tested compound in supernatant was determined by
LC-MS.17
Acknowledgements
We thank the National Natural Science Foundation of China (No. 21472248, 21772240),
the Chinese Academy of Sciences Grants (154144KYSB20150045), the National
Mega-project of China for Innovative Drugs (2018ZX09721001-003-003), and Science and
Chemical and Pharmaceutical Bulletin Advance Publication

Technology Innovation Leader of Guangdong Province (2016TX03R095) for the financial
support of this study.
Conflict of interest
The authors declare no conflict of interest.
Supplementary Materials
The online version of this article contains supplementary materials.
Chemical and Pharmaceutical Bulletin Advance Publication

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