Nusbiarylins, a new class of antimicrobial agents: Rational design of bacterial transcription inhibitors targeting the interaction between the NusB and NusE proteins

Article abstract of DOI:10.1016/j.bioorg.2019.103203

Discovery of antibiotics of a novel mode of action is highly required in the fierce battlefield with multi-drug resistant bacterial infections. Previously we have validated the protein-protein interaction between bacterial NusB and NusE proteins as an unp

Full text of DOI:10.1016/j.bioorg.2019.103203

BioorganicChemistry92(2019)103203
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Bioorganic Chemistry
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Nusbiarylins, a new class of antimicrobial agents: Rational design of
bacterial transcription inhibitors targeting the interaction between the NusB
and NusE proteins
Yangyi Qiua,b, Shu Ting Chana,b, Lin Linc, Tsun Lam Shekd, Tsz Fung Tsangc, Yufeng Zhangd,
⁎
Margaret Ipc, Paul Kay-sheung Chanc,e, Nicolas Blanchardf, Gilles Hanquetg, Zhong Zuod,
,
⁎
⁎
Xiao Yangc, , Cong Maa,b,
a The Hong Kong Polytechnic University Shenzhen Research Institute, Shenzhen, China
b State Key Laboratory of Chemical Biology and Drug Discovery, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University,
Kowloon, Hong Kong Special Administrative Region
c Department of Microbiology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong Special Administrative Region
d School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong Special Administrative Region
e Stanley Ho Centre for Emerging Infectious Diseases, The Chinese University of Hong Kong, Shatin, Hong Kong Special Administrative Region
f Université de Haute-Alsace, Université de Strasbourg, CNRS, LIMA, UMR 7042, Mulhouse 68000, France
g Laboratoire d’Innovation Moléculaire et Applications, ECPM, UMR 7042, Université de Strasbourg/Université de Haute-Alsace, CNRS, 67000 Strasbourg, France
A R T I C L E I N F O
A B S T R A C T
Keywords:
Discovery of antibiotics of a novel mode of action is highly required in the fierce battlefield with multi-drug
resistant bacterial infections. Previously we have validated the protein-protein interaction between bacterial
NusB and NusE proteins as an unprecedented antimicrobial target and reported the identification of a first-in-
class inhibitor of bacterial ribosomal RNA synthesis with antimicrobial activities. In this paper, derivatives of the
hit compound were rationally designed based on the pharmacophore model for chemical synthesis, followed by
biological evaluations. Some of the derivatives demonstrated the improved antimicrobial activity with the
minimum inhibitory concentration (MIC) at 1–2 μg/mL against clinically significant bacterial pathogens. Time-
kill kinetics, confocal microscope, ATP production, cytotoxicity, hemolytic property and cell permeability using
Caco-2 cells of a representative compound were also measured. This series of compounds were named “nus-
biarylins” based on their target protein NusB and the biaryl structure and were expected to be further developed
towards novel antimicrobial drug candidates in the near future.
Bacterial transcription
Protein-protein interaction
NusB
Inhibitor
Antimicrobial activity
1. Introduction
and mechanisms capable of eliminating MRSA is urgently required, as
they will provide alternative therapeutic choices for clinical manage-
Staphylococcus aureus is one of the most important Gram-positive
pathogens leading to a series of conditions ranging from mild skin and
soft tissue infections, respiratory infections, to fatal sepsis [1]. The re-
emergence of methicillin-resistant S. aureus (MRSA) infections which
render commonly used antibiotic treatment less effective has become a
threat to public health [2]. MRSA infections could occur in hospitals or
other health care settings, known as healthcare-associated (HA) MRSA
or community-associated among healthy populations (CA-MRSA) [2].
Discovery of structurally novel antimicrobial agents with distinct target
ment of infections caused by the resistant pathogens.
Bacterial transcription has been a proven target for antibiotics with
two drugs rifamycin and fidaxomicin in current clinical use [3]. Tran-
scription is carried out by a multi-subunit RNA polymerase (RNAP),
composed by two α subunits holding β and β’ subunits to form the
catalytic center and small subunits ω and/or ε [4,5] There are a number
of transcription factors involved in the transcription regulation, among
which is the N-utilization substances (Nus) factor family consisting of
NusA, NusB, NusE (ribosomal protein S10) and NusG [6,7]. The Nus
⁎ Corresponding authors at: The Hong Kong Polytechnic University Shenzhen Research Institute, Shenzhen, China, and State Key Laboratory of Chemical Biology
and Drug Discovery, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Kowloon, Hong Kong Special Administrative
Region (C. Ma); Department of Microbiology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong Special Administrative Region (X.
Yang); School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong Special Administrative Region (Z. Zuo).
E-mail addresses: joanzuo@cuhk.edu.hk (Z. Zuo), xiaoyang@cuhk.edu.hk (X. Yang), cong.ma@polyu.edu.hk (C. Ma).
https://doi.org/10.1016/j.bioorg.2019.103203
Received 20 May 2019; Received in revised form 15 July 2019; Accepted 14 August 2019
Availableonline16August2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

Y. Qiu, et al.
BioorganicChemistry92(2019)103203
factors were first described as part of the antitermination complex for λ
bacteriophage RNA synthesis [8–11], and they were also responsible for
the efficient transcription of bacterial ribosomal RNA (rRNA) operons
[12–14]. Other than transcription regulation, the Nus factors are re-
sponsible for the coupling of transcription and translation in bacterial
cells [15]. Recently, the Nus factors have also been shown to be in-
volved in rRNA folding and ribosome biogenesis [16].
amidation of amine 14a, and ketone 39 was obtained in 74% yield by a
palladium-catalyzed coupling reaction between iodobenzene and alde-
hyde (Scheme 2).
2.2. Antimicrobial activity and target PPI inhibition
The antimicrobial activity of the compounds was firstly tested fol-
lowing the Clinical & Laboratory Standards Institute CLSI procedures
using S. aureus ATCC 25923, which is a type strain for antimicrobial
susceptibility test [21]. Inhibition of the compounds on the in vitro
NusB-NusE interaction was assessed by an in-house designed split-lu-
ciferase assay [22]. NusB and NusE were each tagged with one of the
luciferase complementation fragments and formation of the hetero-
dimer in the presence of the inhibitors will be measured by lumines-
cence released from the reformation of native luciferase [22]. The result
was expressed as % inhibition normalized to the control groups without
compound treatment. The results of compounds 1–31 with the imine
and amine linkers are shown in Table 1. When the substituents on the
left benzene ring of compounds are an atom such as fluoride in com-
pounds 1–3 and a simple methyl group in 4–6, the MIC values ranged
4–8 μg/mL close to the hit compound 14, and the inhibition of com-
pounds on the NusB-NusE interaction was slightly inferior to 14
(Table 1). When the imine linker was reduced to amine, the resulting
compounds 17–22 demonstrated equal or decreased inhibitory and
antimicrobial activities (MICs 8–32 μg/mL; Table 1). On the contrast,
imine compounds 7–16 with bulkier substituents such as methoxy,
carboxylate, and hydroxymethyl displayed equal (Compound 8) or in-
ferior inhibitory activity and increased antimicrobial activity (MICs
2–4 μg/mL), while amine homologs always had reduced antimicrobial
activity (MICs 32–256 μg/mL; Table 1). The results suggested that the
bulky substituents were preferred for improved activity, and the flex-
ible conformation may generate the entropy penalty to affect the ac-
tivity of compounds. The only exception is compound 7 with a very
bulky t-butyl group, the reduction of imine linker to amine gave 23
which demonstrated an improved MIC of 2 μg/mL (Table 1).
The NusB and NusE proteins are relatively small in size (< 15 kDa)
and the formation of NusB-NusE heterodimer is the first step in the
formation of the bacterial rRNA operon transcription complex [17].
Since NusB and NusE are highly conserved and essential for bacterial
cell viability, we hypothesize that small molecules disrupting NusB–-
NusE heterodimer formation will result in antimicrobial activities. Re-
cently, we reported the discovery of a first-in-class inhibitor against
bacterial ribosomal RNA synthesis, targeting the NusB and NusE pro-
tein-protein interaction (PPI) [18]. The hit compound 14 was identified
by an in silico screening of a pharmacophore model designed based on
the crystal structures of NusB and NusE heterodimer [19]. 14 has been
shown to have antimicrobial activity against S. aureus strains including
MRSA [18]. In this paper, chemical derivatives of 14 were rationally
designed and synthesized and followed by the assessment of their an-
timicrobial activities.
2. Results and discussion
2.1. Design and synthesis
The chemical modifications have been designed to examine the
interaction of the left and right benzene ring of analogs of the hit
compound 14 with NusB [20], but the effect of more diverse substitu-
tion on the left benzene ring and the linker to the inhibitory and anti-
microbial activities needs to be investigated (Fig. 1A). We considered
the substitution on the left benzene ring of 14 could be altered to
achieve the improved binding with NusB, for which the linker between
the two benzene rings of 14 may also play an important role to position
the left benzene ring when the right benzene ring was anchored by two
hydrogen bonds (Fig. 1B). The nitro group and hydroxyl of phenolic
alcohol on the right benzene ring were designed to be kept intact to
maintain the important hydrogen bonds between the right benzene ring
of hit compound with Tyr16 and Glu81 of NusB, respectively (Fig. 1B).
2-hydroxy-5-nitrobenzaldehyde was subjected to condensation with
various primary amine compounds 1a-16a to provide the desired
imines 1–16 in 60–93% yields (Scheme 1). Reduction of imines furn-
ished amine analogs 17–31 in 84–94% yields.
Compounds 32–39 with various linkers have also been examined to
display diverse inhibitory and antimicrobial activities (Table 2). Com-
pared to compounds 8 and 9 with the imine linker, olefin compounds
33 and 34 expressed similar antimicrobial activity (Table 2). On the
contrast, olefins 36 and 37 exhibited lower activity than imine homo-
logs 12 and 13. The best inhibitory activity was obtained with com-
pound 38 with a 3-ethynyl group on the left benzene ring same to the
hit compound 14 (Table 2). This compound contains an amide linker to
provide an equal antimicrobial activity to 14. Finally, 39 with a car-
bonyl linker displayed a reduced inhibitory and antimicrobial activity
(Table 2). In summary, the imine linker of this class of diaryl com-
pounds can be changed to other chemical groups to increase the che-
mical diversity, but the distance of linker needs to be maintained with
at least two atoms.
To synthesize the analogs with a C]C double bond linker, the hy-
droxyl of the same starting material above was first protected as ether
32a to undergo a Horner–Wadsworth–Emmons reaction, followed by
the deprotection of 32b to give olefin 32 (Scheme 2). Ether 32a was
converted to olefin 33a by a Wittig reaction, which was subjected to
Heck reaction with substituted iodobenzenes 33b-37b to give 33c-37c
(Scheme 2). The deprotection of ether provided olefins 33–37 in
58–67% yields. Amide 38 was also synthesized in 74% yield by a simple
Based on the structural feature of diaryl group and the universal
antimicrobial activity associated to this structure, as well as the target
specificity on the surface of the NusB protein, we intended to name this
Fig. 1. (A) Binding model of 14 with its target protein NusB. (B) Design of derivatives of 14.
2

Y. Qiu, et al.
BioorganicChemistry92(2019)103203
Scheme 1. Synthesis of imines 1–16 and amines 17–31.
class of compounds as “nusbiarylins”. To better explore the potential of
nusbiarylins, we examined the antibacterial spectrum of these com-
pounds. The first bacteria set used is the top six pathogens amongst the
most recent “WHO priority pathogens list for guiding R&D of new an-
tibiotics” containing three Gram-positive and three Gram-negative
bacteria: Enterococcus faecium, S. aureus, Streptococcus pneumonia,
Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.
We used the standard strains for antibiotic susceptibility testing and the
CLSI recommended conditions to perform the experiment [20]. The
compounds demonstrated the preferred antimicrobial activity against
the Gram-positive than Gram-negative pathogens with specific activ-
ities against S. aureus, S. pneumonia and E. faecium (Table S1). Gram-
negative bacteria were less responsive to the nusbiarylin compound
treatment, which may due to reduced permeability and vigorous efflux
system. Further synthesis and biological investigation will be carried
out in the near future. Encouragingly, modest antimicrobial activity
was observed against the A. baumannii strain (MIC 32 μg/mL; Table S1).
As A. baumannii is top one bacterium of the WHO pathogen list that
requires new antibiotic development, our compounds may be devel-
oped as a specific antimicrobial agent to address this urgent need.
A series of compounds were then chosen to test against re-
presentative clinically significant Gram-positive pathogens such as
Enterococcus casseliflavus causing bacteremia [23], Staphylococcus epi-
dermidis able of growing biofilms on plastic devices, most commonly on
intravenous catheters [24], Staphylococcus saprophyticus causing urinary
tract infections [25], S. pneumoniae as the major cause of community
acquired pneumonia and meningitis [26], Streptococcus pyogenes (Group
A Streptococcus, GAS) causing pharyngitis (strep throat), localized skin
infection (impetigo), necrotizing fasciitis, and neonatal infections [27],
Streptococcus agalactiae (Group B Streptococcus, GBS) causing neonatal
infections [28]. As shown in Fig. 2A, some of the compounds displayed
antibacterial activity against all of these Gram-positive pathogens with
clinical complexity for treatment, especially compound 23 displayed a
significant antimicrobial activity against Staphylococcus saprophyticus
ATCC 15305 with an MIC of 1 μg/mL, which demonstrated the poten-
tials of our compounds.
were able to maintain stable MIC values against diverse MRSA strains,
suggesting the compounds may be developed further with improved
antimicrobial activity to treat MRSA infections.
2.3. Time-kill kinetic
The time-kill kinetic assay assesses the in vitro activity of an anti-
microbial agent against a bacterial strain over time and was performed
according to the CLSI guidelines [20]. The time kill kinetics of com-
pound 23 was measured against S. aureus ATCC 25923 in liquid culture.
23 demonstrated growth inhibition at the ¼ and 1 × MIC and started to
reduce the CFU (colony forming unit) counts at 4 × MIC (Fig. 3A).
When higher concentrations of 23 were used in this experiment, S.
aureus could be totally and efficiently eradicated below the theoretical
level of detection (200 CFU/mL) in 2 h, 16 h, and 20 h using 32, 16 and
8 × MIC, respectively (Fig. 3A). The result suggested that the com-
pounds were bacteriostatic at lower concentrations and became bac-
tericidal at higher concentration in a very short time.
2.4. ATP production
It was reported that inhibition of bacterial central metabolism is
related to antimicrobial efficacy [29]. Therefore, we have examined
compound 23 on its function to interfere with bacterial metabolism by
measuring the level of ATP in S. aureus ATCC 29523 cells over time in
the presence of 23 at various concentrations. When 23 was used at ¼ or
1 × MIC, impaired ATP production could be observed as compared to
untreated cells. As proteins already existing in cells were still capable of
functioning as normal cells even transcription is inhibited, treatment of
23 at 4 × MIC obstructed cell respiration within 2 h. Another tran-
scription-targeting antimicrobial agent, rifampicin has been shown to
suppress the oxygen consumption rate in S. aureus cells, which is con-
sistent with our observation with nusbiarylins [29].
2.5. Cellular effect of nusbiarylin compounds
MRSA represents one of the most difficult bacterial strains for
treatment due to antibiotic resistance [2]. We also tested the above
compounds against a series of globally spread HA- and CA-MRSA
strains. All of the compounds displayed consistent antibacterial activity
against the S. aureus strains with the best MIC as low as 2 μg/mL
(Fig. 2B), in which most of them developed resistance to oxacillin, the
first-line antibiotic drug used in the USA for MRSA. Our compounds
To study the mechanism of the nusbiarylin compounds at the cel-
lular level, a Bacillus subtilis strain named BS61 was employed, which
contains a green fluorescent protein (gfp) fusion to the nusB gene on the
chromosomal copy [30]. The subcellular localization of NusB fluores-
cence in the control group could be clearly identified under the confocal
microscope at the sub-nucleoid regions called transcription foci (Fig. 4A
Ctrl) [30]. When the cells were treated with compound 23 at 1/
3

Y. Qiu, et al.
BioorganicChemistry92(2019)103203
Scheme 2. Synthesis of olefins 32–37, amide 38 and ketone 39.
8 × MIC for 15 min, diffusion of the fluorescent signal into the cytosol
could already be observed although there were still localizations at the
nucleoids (Fig. 4A). When 23 was used at ¼ × MIC, the nucleoid could
barely be seen, and the cytosolic regions were filled with fluorescence
(Fig. 4A). When 23 was used at ½ or 1 × MIC, no nucleoid could be
observed, suggesting disruption of NusB localization by the nusbiarylin
compound treatment (Fig. 4A). The level of macromolecules in B subtilis
cells due to 23 treatment at a sub- MIC level (1/8 MIC and 1/4 MIC)
unaffecting cell growth was also measured (Fig. 4B). B. subtilis cells
were subject to 23 treatment at early log-phase (OD 0.2), and harvested
and lysed at mid-log phase (OD 0.5) for macromolecular quantitation.
While the DNA levels were not affected as predicted by the mode of
action, 23 showed a significant reduction in the RNA and rRNA level
compared with the untreated control cells (Fig. 4B).
2.6. In vitro cytotoxicity
Some compounds with the MIC values of 2 μg/mL against S. aureus
ATCC 25923 were chosen for in vitro cytotoxicity testing as described
previously [18]. Cell lines A549 human lung carcinoma and HaCaT
4

Y. Qiu, et al.
BioorganicChemistry92(2019)103203
Table 1
Table 2
Inhibitory
and
R
antimicrobial
activities
of
compounds
.
1–31
Inhibitory
and
R
antimicrobial
.
activities
of
compounds
32–39
Compound
MIC (μg/mL)
NusB/E PPI % Inhibition
ClogP
1
8
8
73.4
64.0
2.0
5.2
3.36
3.20
Compound
X
MIC (μg/
mL)
NusB/E PPI %
Inhibition
ClogP
17
2
4
65.4
37.5
3.2
7.1
3.36
3.20
32
33
34
35
36
37
38
39
H
(E)-CH=CH-
2
4
2
8
56.6
41.6
70.3
44.9
24.6
41.7
99.2
41.3
6.4
4.8
4.4
3.5
6.7
3.7
0.2
5.3
4.54
4.47
4.47
3.98
4.52
4.52
3.83
3.59
18
16
2-OCH3
3-OCH3
2-CN
(E)-CH=CH-
(E)-CH=CH-
(E)-CH=CH-
3
8
59.2
66.0
2.0
2.2
3.36
3.20
3-COOCH3 (E)-CH=CH- 64
4-COOCH3 (E)-CH=CH- 128
19
32
3-C^CH
-NHCO-
-CO-
8
4
4
8
53.1
46.1
1.3
6.7
3.55
3.25
H
64
20
5
4
54.9
2.8
3.55
21
16
44.4
6.2
3.25
safety of a drug. TI was defined as the ratio that compares the effective
concentration and the concentration at which the drug became toxic
and the TI was promising with 53–153 for imines, 6–9 for amine 23 and
olefin 34. Note that the TI was very preliminary by comparing the cell-
based cytotoxicity of compounds to antimicrobial activity, which needs
more validation using the in vivo animal models in further research.
Nevertheless, this class of compounds is potential for further studies.
6
4
49.2
36.1
0.9
6.4
3.55
3.25
22
16
7
4
2
51.3
39.5
1.5
2.7
4.88
4.58
23
8
2
82.6
45.2
0.8
7.9
3.07
2.85
24
32
9
2
68.0
34.9
0.8
3.07
2.85
2.7. In vitro pharmacology
25
64
10.3
With the antibacterial activity in hand, we intended to explore the in
vitro pharmacological properties of these compounds. We first in-
vestigated whether the compounds would cause hemolysis when in-
jected into the blood veins [31]. The hemolytic activities against human
blood cells of compound 23 were tested. As shown in Fig. 5, compound
23 demonstrated almost no hemolytic potential compared to the sug-
gested nonhemolytic cut-off value (< 10%) at all tested concentrations
(0.1, 1 and 10 µg/mL).
10
26
4
61.8
48.2
2.8
7.8
3.07
2.85
128
11
27
4
72.5
13.0
3.3
5.7
3.33
3.77
128
12
28
2
48.1
14.6
3.1
3.33
3.27
32
10.7
For drugs that are orally available, the intestinal absorption is cri-
tical. The human intestinal epithelial cancer cell line Caco-2 was used to
determine the permeability of compound 23 [31]. As shown in Table 4,
compound 23 possessed an excellent cell permeability, which is su-
perior to 1 × 10−5 cm/s as the standard of great intestinal epithelial
cell permeability. Furthermore, the deduced apparent permeability of
compounds had a similar magnitude to that of the predicted value by
pkCSM [32].
13
29
2
61.8
16.8
3.3
2.6
3.33
3.27
> 256
14
30
8
85.3
47.7
1.5
7.9
3.32
3.02
16
15
31
4
70.6
61.4
1.8
4.3
2.01
1.73
256
3. Conclusions
In this paper, we described the rational design and chemical
synthesis of a new class of diaryl compounds based on the previously
established hit compound and the pharmacophore model. The in-
hibitory activity at the molecular level against the in vitro NusB-NusE
heterodimer formation together with the antimicrobial activity con-
firmed the rationales of the compound design. Further characterizations
by time-kill kinetics, ATP production, and confocal microscopy pro-
vided more information on the antimicrobial activity and the me-
chanism at the cellular level. The cytotoxicity and in vitro pharmacology
study demonstrated that these compounds are suitable for further drug
development. The new series of compounds named nusbiarylin is a new
class of antimicrobial agent targeting a new PPI in bacteria, with a
distinct mechanism of action as compared with the current antibiotics
on the market. We believe further studies on these compounds will
contribute to a new era of antibiotic discovery.
16
4
72.6
2.6
2.01
human keratinocytes were used as the infections caused by S. aureus
often occur in lung and skin tissues. The 50% cytotoxic concentration
(CC50) was defined as a compound’s concentration required for the
reduction of cell viability by 50%. Compounds 8, 9, 12, 13 with the
imine linker did not show any cytotoxicity (CC50 > 200 μM, table 3),
whereas compounds 23 and 34 with an amine and olefin linkers re-
spectively demonstrated very mild cytotoxicity (CC50 18–34 μM). The
therapeutic index (TI) is a quantitative measurement of the relative
5

Y. Qiu, et al.
BioorganicChemistry92(2019)103203
Fig. 2. Antimicrobial activity evaluation of the nusbiarylin compounds with the MICs (μg/mL) shown against (A) Representative Gram-positive pathogens and (B) S.
aureus including representative globally spread HA- and CA-MRSA strains. ECAS, Enterococcus casseliflavus ATCC 25788, SEPI, Staphylococcus epidermidis ATCC
12228, SSAP, Staphylococcus saprophyticus ATCC 15305, SPNE, S. pneumoniae ATCC 49619, SPYO, Streptococcus pyogenes ATCC 19615, SAGA, Streptococcus agalactiae
ATCC 12386, CIP, ciprofloxacin, GEN, gentamicin, OXA, oxacillin, VAN, vancomycin.
4. Experimental section
4.2. Chemistry
4.1. Materials and chemicals
4.2.1. General procedure (A) for the synthesis of Schiff base type derivatives
To a solution of benzaldehydes (1 equiv) in ethanol (10 mL) was
added anilines (1 equiv), and the reaction mixture was stirred at room
temperature overnight. The solid was collected by filtration and washed
with cold ethanol and hexane.
Sodium Chloride and Phosphate Buffered Saline (tablets) were
purchased from Sigma-Aldrich (Saint Louis, Missouri). GR Grade
Dimethyl Sulfoxide (DMSO) was purchased from Duksan Pure
Chemicals (Ansan City, South Korea) and HPLC Grade Acetonitrile was
purchased from Merck KGaA (Darmstadt, Germany). For 150 mM NaCl
buffer, 4.383 g of NaCl crystals were dissolved in 500 mL of Milli-Q
water. 1x PBS was prepared by dissolving 1 PBS tablet into 200 mL of
Milli-Q water. Antibiotics were purchased from Sigma-Aldrich (Saint
Louis, Missouri).
4.2.2. General procedure (B) for the synthesis of aniline type derivatives
To a solution of Schiff base type derivatives (1 equiv) obtained ac-
cording to general procedure (A) in DCM (10 mL) was added sodium
triacetoxyborohydride (3 equiv), and the reaction mixture was stirred
at room temperature overnight. The crude reaction mixture was
Fig. 3. The effect of nusbiarylin compounds on S. aureus growth and metabolism. (A) Time kill kinetics of S. aureus treated with 23 at different concentrations. (B)
The ATP production of S. aureus treated with 23 at different concentrations.
6

Y. Qiu, et al.
BioorganicChemistry92(2019)103203
Fig. 5. In vitro hemolysis activity of compound 23 (blue line on the bottom).
Table 4
The deduced and predicted apparent permeability of compound 23.
Fig. 4. (A) Confocal microscope of B. subtilis with NusB fluorescence and (B)
the level of DNA, RNA and rRNA in B. subtilis cells treated by compound 23 at
different concentrations.
Compound
Papp Average (n = 4) (cm/s)
Papp Predicted (cm/s)
8.09 × 10−6
23
1.87
0.47 × 10−5
4.2.4. (E)-2-(((2-fluorophenyl)imino)methyl)-4-nitrophenol (1)
Table 3
Cytotoxicity of selective compounds against human A549 and HaCaT cell lines.
The title compound was prepared according to general procedure
(A) using 5-nitrosalicylaldehyde (200 mg, 1.20 mmol) and 2-fluor-
oaniline 1a (116 μL, 1.20 mmol, 1 equiv), yielding the pure product as a
yellow solid (201 mg, 65%); 1H NMR (400 MHz, Chloroform-d) δ 14.25
(s, 1H), 8.85 (s, 1H), 8.43 (d, J = 2.8 Hz, 1H), 8.31 (dd, J = 9.2, 2.8 Hz,
1H), 7.40 – 7.32 (m, 2H), 7.28 – 7.23 (m, 2H), 7.15 (d, J = 9.2 Hz, 1H);
13C NMR (100 MHz, Chloroform-d) δ 166.7, 162.4 (d, J = 3.0 Hz),
155.9 (d, J = 253.5 Hz), 140.0, 134.6 (d, J = 9.1 Hz), 129.2 (d,
J = 8.1 Hz), 128.5 (d, J = 10.1 Hz), 124.9 (d, J = 4.0 Hz), 121.3,
118.4, 118.2, 116.9 (d, J = 19.2 Hz); HRMS (ESI) calcd for C13H8FN2O3
(M - H+) 259.0524, found 259.0523; Melting Point: 166–167 °C.
Cpd.
MICa (μg/
CC50 (μM)
Therapeutic Indexb
mL)
A549
HaCaT
A549
HaCaT
8
2
2
2
2
2
2
–
454.40
232.10
446.51
510.42
27.45 438.94
24.81 124
120
53
118
114
6
9
9.46
195.82
23.08 63
25.80 134
24.41 153
12
13
23
34
DDPc
28.56 391.59
29.40 381.19
20.24
18.32
34.58
7.70
1.75
2.42
3.43
0.66
6
9
–
4.06
0.58
30.51
7.83
8
–
a
b
c
S. aureus ATCC 25923.
Calculated by CC50 (μg/mL)/½ MIC (μg/mL).
DDP: cisplatin.
4.2.5. (E)-2-(((3-fluorophenyl)imino)methyl)-4-nitrophenol (2)
The title compound was prepared according to general procedure
(A) using 5-nitrosalicylaldehyde (200 mg, 1.20 mmol) and 3-fluor-
oaniline 2a (115 μL, 1.20 mmol, 1 equiv), yielding the pure product as a
yellow solid (230 mg, 74%); 1H NMR (400 MHz, Chloroform-d) δ 14.03
(s, 1H), 8.73 (s, 1H), 8.43 (d, J = 2.8 Hz, 1H), 8.31 (dd, J = 9.1, 2.7 Hz,
1H), 7.49 – 7.43 (m, 1H), 7.15 – 7.06 (m, 4H); 13C NMR (100 MHz,
Chloroform-d) δ 166.5, 163.4 (d, J = 249.5 Hz), 161.8, 148.7 (d,
J = 8.9 Hz), 140.2, 131.0 (d, J = 9.1 Hz), 128.6 (d, J = 10.1 Hz),
118.4, 118.0, 117.1 (d, J = 3.0 Hz), 114.9 (d, J = 21.2 Hz), 108.6 (d,
J = 23.2 Hz); HRMS (ESI) calcd for C13H8FN2O3 (M - H+) 259.0524,
found 259.0828; Melting Point: 172–173 °C.
evaporated in vacuo and the pure product was obtained after flash
chromatography.
4.2.3. General procedure (C) for synthesis of (E)-stilbenes
Step 1: Hydroxyl group-protected (E)-stilbenes. To a mixture of
trimethyl(2-((4-nitro-2-vinylphenoxy)methoxy)ethyl)silane (33a)
(1.2 equiv), K3PO4 (1.4 equiv) and Pd(OAc)2 (0.03 equiv) in a sealed
tube was added iodobenzenes (1.0 equiv) and anhydrous DMF
(3 mL) in glovebox. The resulting mixture was stirred at 100 °C
overnight. After the reaction mixture was cooled to room tempera-
ture, the product was extracted with EtOAc (30 mL × 3). The or-
ganic layers were combined, washed with saturated brine, dried
over Na2SO4 and filtered. All volatiles were removed under reduced
pressure and the crude product containing 2–5% isomer was iso-
lated by flash chromatography (EtOAc/Hex) on silica gel.
4.2.6. (E)-2-(((4-fluorophenyl)imino)methyl)-4-nitrophenol (3)
The title compound was prepared according to general procedure
(A) using 5-nitrosalicylaldehyde (200 mg, 1.20 mmol) and 4-fluor-
oaniline 3a (114 μL, 1.20 mmol, 1 equiv), yielding the pure product as a
yellow solid (195 mg, 63%); 1H NMR (400 MHz, Chloroform-d) δ 14.24
(s, 1H), 8.71 (s, 1H), 8.42 (d, J = 2.8 Hz, 1H), 8.30 (dd, J = 9.2, 2.8 Hz,
1H), 7.37 – 7.34 (m, 2H), 7.22 – 7.14 (m, 2H), 7.13 (d, J = 9.2 Hz, 1H);
13C NMR (100 MHz, DMSO‑d6) δ 166.7, 161.8, 161.8 (d, J = 245.4 Hz),
143.7 (d, J = 3.0 Hz), 139.7, 128.8, 128.6, 123.9 (d, J = 9.1 Hz),
119.2, 118.6, 116.8 (d, J = 23.2 Hz); HRMS (ESI) calcd for C13H8FN2O3
(M - H+) 259.0524, found 259.0522; Melting Point: 196–197 °C.
Step 2: To a solution of crude product obtained above in MeOH
(10 mL) was added H2SO4 (100 μL). The mixture was stirred at rt
overnight. After the reaction was completed, the product was ex-
tracted with EtOAc (30 mL × 3). The organic layers were combined,
washed with saturated brine, dried over Na2SO4 and filtered. All
volatiles were removed under reduced pressure and the title product
was isolated by flash chromatography (EtOAc/Hex) on silica gel.
7

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BioorganicChemistry92(2019)103203
4.2.7. (E)-4-nitro-2-((o-tolylimino)methyl)phenol (4)
(s, 1H), 8.73 (s, 1H), 8.42 (d, J = 2.7 Hz, 1H), 8.29 (dd, J = 9.1, 2.8 Hz,
1H), 7.39 (t, J = 8.1 Hz, 1H), 7.12 (d, J = 9.2 Hz, 1H), 6.96 – 6.92 (m,
2H), 6.88 (t, J = 2.2 Hz, 1H), 3.89 (s, 3H); 13C NMR (100 MHz,
Chloroform-d) δ 166.9, 160.8, 160.7, 148.0, 140.0, 130.5, 128.4, 128.4,
118.4, 118.1, 113.8, 113.3, 107.1, 55.5; HRMS (ESI) calcd for
The title compound was prepared according to general procedure
(A) using 5-nitrosalicylaldehyde (200 mg, 1.20 mmol) and 2-methyla-
niline 4a (128 μL, 1.20 mmol, 1 equiv), yielding the pure product as a
yellow solid (215 mg, 70%); 1H NMR (400 MHz, Chloroform-d) δ 14.74
(s, 1H), 8.70 (s, 1H), 8.44 (d, J = 2.8 Hz, 1H), 8.30 (dd, J = 9.2, 2.8 Hz,
1H), 7.36 – 7.28 (m, 3H), 7.21 – 7.18 (m, 1H), 7.13 (d, J = 9.1 Hz, 1H),
2.46 (s, 3H); 13C NMR (100 MHz, Chloroform-d) δ 167.2, 160.2, 145.6,
139.9, 132.6, 131.1, 128.4, 128.3, 128.1, 127.3, 118.4, 118.2, 117.6,
18.2; HRMS (ESI) calcd for C14H11N2O3 (M - H+) 255.0775, found
255.0771; Melting Point: 169–170 °C.
C
14H11N2O4 (M - H+) 271.0724, found 271.0721; Melting Point:
167–168 °C.
4.2.13. (E)-2-(((4-methoxyphenyl)imino)methyl)-4-nitrophenol (10)
The title compound was prepared according to general procedure
(A) using 5-nitrosalicylaldehyde (200 mg, 1.20 mmol) and 4-methox-
yaniline 10a (148 mg, 1.20 mmol, 1 equiv), yielding the pure product
as a yellow solid (268 mg, 82%); 1H NMR (400 MHz, Chloroform-d) δ
14.70 (s, 1H), 8.71 (s, 1H), 8.39 (d, J = 2.8 Hz, 1H), 8.26 (dd, J = 9.1,
2.8 Hz, 1H), 7.38 – 7.34 (m, 2H), 7.10 (d, J = 9.2 Hz, 1H), 7.03 – 6.99
(m, 2H), 3.89 (s, 3H); 13C NMR (100 MHz, Chloroform-d) δ 166.9,
159.8, 158.0, 139.9, 139.4, 128.0, 127.9, 122.5, 118.3, 118.2, 114.9,
55.6; HRMS (ESI) calcd for C14H11N2O4 (M - H+) 271.0724, found
271.0727; Melting Point: 169–170 °C.
4.2.8. (E)-4-nitro-2-((m-tolylimino)methyl)phenol (5)
The title compound was prepared according to general procedure
(A) using 5-nitrosalicylaldehyde (200 mg, 1.20 mmol) and 3-methyla-
niline 5a (128 μL, 1.20 mmol, 1 equiv), yielding the pure product as a
yellow solid (230 mg, 75%); 1H NMR (400 MHz, Chloroform-d) δ 14.58
(s, 1H), 8.73 (s, 1H), 8.41 (d, J = 2.9 Hz, 1H), 8.28 (dd, J = 9.2, 2.8 Hz,
1H), 7.37 (t, J = 7.5 Hz, 1H), 7.21 – 7.15 (m, 3H), 7.11 (d, J = 9.3 Hz,
1H), 2.45 (s, 3H); 13C NMR (100 MHz, Chloroform-d) δ 167.1, 160.3,
146.6, 139.9, 139.7, 129.5, 128.9, 128.4, 128.3, 121.9, 118.4, 118.2,
118.1, 21.4; HRMS (ESI) calcd for C14H11N2O3 (M - H+) 255.0775,
found 255.0772; Melting Point: 137–138 °C.
4.2.14. Methyl (E)-2-((2-hydroxy-5-nitrobenzylidene)amino)benzoate (11)
The title compound was prepared according to general procedure
(A) using 5-nitrosalicylaldehyde (200 mg, 1.20 mmol) and methyl 2-
aminobenzoate 11a (155 μL, 1.20 mmol, 1 equiv), yielding the pure
product as a red solid (285 mg, 79%); 1H NMR (400 MHz, Chloroform-
d) δ 14.29 (s, 1H), 8.66 (s, 1H), 8.43 (d, J = 2.7 Hz, 1H), 8.30 (dd,
J = 9.2, 2.8 Hz, 1H), 8.09 (dd, J = 7.9, 1.6 Hz, 1H), 7.66 (td, J = 7.7,
1.6 Hz, 1H), 7.44 (td, J = 7.6, 1.1 Hz, 1H), 7.26 (dd, J = 8.0, 1.1 Hz,
1H), 7.14 (d, J = 9.3 Hz, 1H), 3.96 (s, 3H); 13C NMR (100 MHz,
Chloroform-d) δ 166.9, 166.3, 161.0, 147.3, 139.8, 133.7, 131.7, 128.6,
128.6, 127.4, 124.4, 119.1, 118.7, 118.3, 52.4; HRMS (ESI) calcd for
4.2.9. (E)-4-nitro-2-((p-tolylimino)methyl)phenol (6)
The title compound was prepared according to general procedure
(A) using 5-nitrosalicylaldehyde (200 mg, 1.20 mmol) and 4-methyla-
niline 6a (129 mg, 1.20 mmol, 1 equiv), yielding the pure product as a
yellow solid (248 mg, 81%); 1H NMR (400 MHz, Chloroform-d) δ 14.65
(s, 1H), 8.73 (s, 1H), 8.40 (d, J = 2.7 Hz, 1H), 8.28 (dd, J = 9.2, 2.8 Hz,
1H), 7.31 – 7.26 (m, 4H), 7.11 (d, J = 9.1 Hz, 1H), 2.43 (s, 3H); 13C
NMR (100 MHz, Chloroform-d) δ 167.0, 159.5, 143.9, 139.9, 138.4,
130.3, 128.2, 128.2, 121.1, 118.3, 118.2, 21.1; HRMS (ESI) calcd for
C
15H11N2O5 (M - H+) 299.0673, found 299.0672; Melting Point:
176–177 °C.
C
14H11N2O3 (M - H+) 255.0775, found 255.0772; Melting Point:
160–161 °C.
4.2.15. Methyl (E)-3-((2-hydroxy-5-nitrobenzylidene)amino)benzoate (12)
The title compound was prepared according to general procedure
(A) using 5-nitrosalicylaldehyde (200 mg, 1.20 mmol) and methyl 3-
aminobenzoate 12a (181 mg, 1.20 mmol, 1 equiv), yielding the pure
4.2.10. (E)-2-(((3-(tert-butyl)phenyl)imino)methyl)-4-nitrophenol (7)
The title compound was prepared according to general procedure
(A) using 5-nitrosalicylaldehyde (200 mg, 1.20 mmol) and 3-tert-bu-
tylbenzenamine 7a (179 mg, 1.20 mmol, 1 equiv), yielding the pure
product as
a
yellow solid (307 mg, 85%); 1H NMR (400 MHz,
Chloroform-d) δ 14.12 (s, 1H), 8.80 (s, 1H), 8.45 (d, J = 2.8 Hz, 1H),
8.32 (dd, J = 9.1, 2.8 Hz, 1H), 8.07 – 8.02 (m, 2H), 7.60 – 7.53 (m, 2H),
7.15 (d, J = 9.2 Hz, 1H), 3.99 (s, 3H); 13C NMR (100 MHz, DMSO‑d6) δ
166.6, 166.2, 162.8, 147.9, 139.8, 131.4, 130.5, 129.0, 128.6, 128.4,
127.0, 122.1, 119.4, 118.5, 52.8; HRMS (ESI) calcd for C15H11N2O5 (M
- H+) 299.0673, found 299.0669; Melting Point: 201–202 °C.
product as
a
yellow solid (244 mg, 68%); 1H NMR (400 MHz,
Chloroform-d) δ 14.63 (s, 1H), 8.74 (s, 1H), 8.43 (d, J = 2.7 Hz, 1H),
8.29 (dd, J = 9.2, 2.6 Hz, 1H), 7.43 – 7.41 (m, 2H), 7.36 (d, J = 2.3 Hz,
1H), 7.19 – 7.15 (m, 1H), 7.12 (d, J = 9.2 Hz, 1H), 1.40 (s, 9H); 13C
NMR (100 MHz, Chloroform-d) δ 167.1, 160.3, 153.2, 146.5, 139.9,
129.4, 128.4, 128.3, 125.2, 118.8, 118.4, 118.1, 117.7, 34.9, 31.3;
HRMS (ESI) calcd for C17H17N2O3 (M - H+) 297.1245, found 297.1242;
Melting Point: 128–129 °C.
4.2.16. Methyl (E)-4-((2-hydroxy-5-nitrobenzylidene)amino)benzoate (13)
The title compound was prepared according to general procedure
(A) using 5-nitrosalicylaldehyde (200 mg, 1.20 mmol) and methyl 4-
aminobenzoate 13a (181 mg, 1.20 mmol, 1 equiv), yielding the pure
4.2.11. (E)-2-(((2-methoxyphenyl)imino)methyl)-4-nitrophenol (8)
The title compound was prepared according to general procedure
(A) using 5-nitrosalicylaldehyde (200 mg, 1.20 mmol) and 2-methox-
yaniline 8a (135 μL, 1.20 mmol, 1 equiv), yielding the pure product as a
red solid (259 mg, 79%); 1H NMR (400 MHz, Chloroform-d) δ 15.41 (s,
1H), 8.84 (s, 1H), 8.40 (d, J = 2.8 Hz, 1H), 8.26 (dd, J = 9.3, 2.8 Hz,
1H), 7.37 – 7.32 (m, 2H), 7.09 – 7.04 (m, 3H), 3.97 (s, 3H); 13C NMR
(100 MHz, Chloroform-d) δ 168.9, 159.4, 153.0, 139.2, 133.9, 129.3,
128.5, 128.3, 121.1, 119.7, 119.0, 117.8, 112.1, 55.9; HRMS (ESI)
calcd for C14H11N2O4 (M - H+) 271.0724, found 271.0719; Melting
Point: 186–187 °C.
product as
a
yellow solid (294 mg, 82%); 1H NMR (400 MHz,
Chloroform-d) δ 13.99 (s, 1H), 8.76 (s, 1H), 8.45 (d, J = 2.8 Hz, 1H),
8.32 (dd, J = 9.2, 2.7 Hz, 1H), 8.18 – 8.16 (m, 2H), 7.40 – 7.38 (m, 2H),
7.15 (d, J = 9.1 Hz, 1H), 3.98 (s, 3H); 13C NMR (100 MHz, Chloroform-
d) δ 166.6, 166.3, 162.4, 150.8, 140.2, 131.3, 129.5, 128.9, 128.7,
121.2, 118.5, 118.0, 52.3; HRMS (ESI) calcd for C15H11N2O5 (M - H+
299.0673, found 299.0672; Melting Point: 206–207 °C.
)
4.2.17. (E)-2-(((3-ethynylphenyl)imino)methyl)-4-nitrophenol (14)
The title compound was prepared according to general procedure
(A) using 5-nitrosalicylaldehyde (200 mg, 1.20 mmol) and 3-amino-
phenylacetylene 14a (135 μL, 1.20 mmol, 1 equiv), yielding the pure
product as a yellow solid (242 mg, 76%); 1H NMR (400 MHz, DMSO‑d6)
δ 13.94 (br s, 1H), 9.16 (s, 1H), 8.66 (d, J = 2.8 Hz, 1H), 8.27 (dd,
J = 9.3, 2.9 Hz, 1H), 7.59 – 7.44 (m, 4H), 7.13 (d, J = 9.2 Hz, 1H), 4.29
(s, 1H); 13C NMR (100 MHz, DMSO‑d6) δ 166.8, 162.8, 147.8, 139.8,
4.2.12. (E)-2-(((3-methoxyphenyl)imino)methyl)-4-nitrophenol (9)
The title compound was prepared according to general procedure
(A) using 5-nitrosalicylaldehyde (200 mg, 1.20 mmol) and 3-methox-
yaniline 9a (136 μL, 1.20 mmol, 1 equiv), yielding the pure product as a
yellow solid (264 mg, 81%); 1H NMR (400 MHz, Chloroform-d) δ 14.43
8

Y. Qiu, et al.
BioorganicChemistry92(2019)103203
131.1, 130.4, 128.9, 128.7, 124.6, 123.4, 123.1, 119.3, 118.6, 83.3,
81.9; HRMS (ESI) calcd for C15H9N2O3 (M - H+) 265.0619, found
265.0620; Melting Point: 196–197 °C.
for C13H10FN2O3 (M - H+) 261.0681, found 261.0682; Melting Point:
131–132 °C.
4.2.23. 4-nitro-2-((o-tolylamino)methyl)phenol (20)
4.2.18. (E)-2-(((3-(hydroxymethyl)phenyl)imino)methyl)-4-nitrophenol
(15)
The title compound was prepared according to general procedure
(B) using 4 (100 mg, 0.39 mmol), yielding the pure product as a yellow
solid (93 mg, 92%) after flash chromatography (Hexane : EtOAc = 15 :
1); 1H NMR (400 MHz, Chloroform-d) δ 10.10 (br s, 1H), 8.17 – 8.15
(m, 2H), 7.22 – 7.18 (m, 2H), 6.98 – 6.94 (m, 2H), 6.88 (d, J = 8.1 Hz,
1H), 4.59 (s, 2H), 3.95 (br s, 1H), 2.29 (s, 3H); 13C NMR (100 MHz,
Chloroform-d) δ 163.3, 143.9, 140.8, 130.7, 127.4, 125.9, 125.4, 124.7,
123.0, 121.8, 117.1, 114.1, 48.5, 17.6; HRMS (ESI) calcd for
C14H13N2O3 (M - H+) 257.0932, found 257.0928; Melting Point:
105–106 °C.
The title compound was prepared according to general procedure
(A) using 5-nitrosalicylaldehyde (200 mg, 1.20 mmol) and 3-amino-
benzyl alcohol 15a (148 mg, 1.20 mmol, 1 equiv), yielding the pure
product as a yellow solid (277 mg, 85%); 1H NMR (400 MHz, DMSO‑d6)
δ 14.52 (br s, 1H), 9.21 (s, 1H), 8.71 (d, J = 2.9 Hz, 1H), 8.26 (dd,
J = 9.3, 2.9 Hz, 1H), 7.48 – 7.44 (m, 2H), 7.38 – 7.31 (m, 2H), 7.11 (d,
J = 9.1 Hz, 1H), 5.34 (t, J = 5.8 Hz, 1H), 4.58 (d, J = 3.9 Hz, 2H); 13C
NMR (100 MHz, DMSO‑d6) δ 167.9, 161.8, 146.5, 144.8, 139.4, 129.8,
129.0, 128.9, 126.2, 120.1, 119.7, 119.0, 63.0; HRMS (ESI) calcd for
C
14H11N2O4 (M - H+) 271.0724, found 271.0721; Melting Point:
4.2.24. 4-nitro-2-((m-tolylamino)methyl)phenol (21)
170–171 °C.
The title compound was prepared according to general procedure
(B) using 5 (100 mg, 0.39 mmol), yielding the pure product as a yellow
solid (95 mg, 94%) after flash chromatography (Hexane : EtOAc = 15 :
1); 1H NMR (400 MHz, Chloroform-d) δ 8.14 (d, J = 10.1 Hz, 2H), 7.18
(t, J = 7.6 Hz, 1H), 6.95 (d, J = 8.8 Hz, 1H), 6.84 (d, J = 7.6 Hz, 1H),
6.71 (d, J = 5.5 Hz, 2H), 4.54 (s, 2H), 2.33 (s, 3H); 13C NMR (100 MHz,
Chloroform-d) δ 163.4, 146.0, 140.8, 139.6, 129.4, 125.4, 124.5, 123.0,
123.0, 117.5, 117.1, 113.6, 49.2, 21.5; HRMS (ESI) calcd for
4.2.19. (E)-2-(((4-(hydroxymethyl)phenyl)imino)methyl)-4-nitrophenol
(16)
The title compound was prepared according to general procedure
(A) using 5-nitrosalicylaldehyde (200 mg, 1.20 mmol) and 4-amino-
benzyl alcohol 16a (148 mg, 1.20 mmol, 1 equiv), yielding the pure
product as a yellow solid (293 mg, 90%); 1H NMR (400 MHz, DMSO‑d6)
δ 14.58 (br s, 1H), 9.20 (s, 1H), 8.66 (d, J = 3.0 Hz, 1H), 8.25 (dd,
J = 9.3, 2.9 Hz, 1H), 7.48 – 7.43 (m, 4H), 7.10 (d, J = 9.3 Hz, 1H), 5.29
(br s, 1H), 4.55 (s, 2H); 13C NMR (100 MHz, DMSO‑d6) δ 167.8, 161.5,
145.1, 142.9, 139.4, 129.0, 128.8, 128.0, 121.6, 118.9, 118.9, 62.9;
HRMS (ESI) calcd for C14H11N2O4 (M - H+) 271.0724, found 271.0722;
Melting Point: 184–185 °C.
C
14H13N2O3 (M - H+) 257.0932, found 257.0928; Melting Point:
140–141 °C.
4.2.25. 4-nitro-2-((p-tolylamino)methyl)phenol (22)
The title compound was prepared according to general procedure
(B) using 6 (100 mg, 0.39 mmol), yielding the pure product as a yellow
solid (94 mg, 93%) after flash chromatography (Hexane : EtOAc = 15 :
1); 1H NMR (400 MHz, Chloroform-d) δ 10.49 (br s, 1H), 8.17 – 8.12
(m, 2H), 7.10 (d, J = 8.3 Hz, 2H), 6.94 (d, J = 8.8 Hz, 1H), 6.81 (d,
J = 8.4 Hz, 2H), 4.54 (s, 2H), 4.02 (br s, 1H), 2.31 (s, 3H); 13C NMR
(100 MHz, Chloroform-d) δ 163.5, 143.4, 140.7, 131.8, 130.1, 125.4,
124.5, 122.9, 117.1, 116.9, 49.7, 20.6; HRMS (ESI) calcd for
4.2.20. 2-(((2-fluorophenyl)amino)methyl)-4-nitrophenol (17)
The title compound was prepared according to general procedure
(B) using 1 (100 mg, 0.38 mmol), yielding the pure product as a yellow
solid (92 mg, 91%) after flash chromatography (Hexane : EtOAc = 15 :
1); 1H NMR (400 MHz, Chloroform-d) δ 9.57 (br s, 1H), 8.17 – 8.15 (m,
2H), 7.12 – 7.05 (m, 2H), 6.98 – 6.89 (m, 3H), 4.57 (s, 2H), 4.42 (s, 1H);
13C NMR (100 MHz, Chloroform-d) δ 162.8, 153.1 (d, J = 242.4 Hz),
140.9, 134.6 (d, J = 12.1 Hz), 125.5, 124.9 (d, J = 4.0 Hz), 124.7,
123.1, 121.7 (d, J = 8.1 Hz), 117.1, 116.1 (d, J = 3.0 Hz), 115.2 (d,
C
14H13N2O3 (M - H+) 257.0932, found 257.0927; Melting Point:
118–119 °C.
4.2.26. 2-(((3-(tert-butyl)phenyl)amino)methyl)-4-nitrophenol (23)
The title compound was prepared according to general procedure
(B) using 7 (100 mg, 0.34 mmol), yielding the pure product as a yellow
solid (94 mg, 93%) after flash chromatography (Hexane : EtOAc = 15 :
1); 1H NMR (400 MHz, Chloroform-d) δ 10.30 (br s, 1H), 8.17 – 8.15
(m, 2H), 7.24 (t, J = 8.0 Hz, 1H), 7.06 (d, J = 7.9 Hz, 1H), 6.97 – 6.92
(m, 2H), 6.72 (d, J = 8.1 Hz, 1H), 4.57 (s, 2H), 4.08 (s, 1H), 1.30 (s,
9H); 13C NMR (100 MHz, Chloroform-d) δ 163.4, 152.9, 145.8, 140.7,
129.3, 125.4, 124.5, 123.1, 119.4, 117.1, 114.3, 113.4, 49.4, 34.7,
31.2; HRMS (ESI) calcd for C17H19N2O3 (M - H+) 299.1401, found
299.1399; Melting Point: 112–113 °C.
J = 19.2 Hz), 48.3; HRMS (ESI) calcd for C13H10FN2O3 (M - H+
)
261.0681, found 261.0683; Melting Point: 107–108 °C.
4.2.21. 2-(((3-fluorophenyl)amino)methyl)-4-nitrophenol (18)
The title compound was prepared according to general procedure
(B) using 2 (100 mg, 0.38 mmol), yielding the pure product as a yellow
solid (90 mg, 89%) after flash chromatography (Hexane : EtOAc = 15 :
1); 1H NMR (400 MHz, Chloroform-d) δ 9.37 (br s, 1H), 8.16 – 8.14 (m,
2H), 7.22 (q, J = 7.7 Hz, 1H), 6.97 – 6.95 (m, 1H), 6.69 – 6.63 (m, 2H),
6.58 – 6.55 (m, 1H), 4.53 (s, 2H), 4.28 (br s, 1H); 13C NMR (100 MHz,
DMSO‑d6) δ 163.9 (d, J = 240.4 Hz), 162.1, 150.9 (d, J = 11.1 Hz),
140.1, 130.8 (d, J = 11.1 Hz), 127.5, 124.9, 124.3, 115.6, 108.9 (d,
J = 2.0 Hz), 102.6 (d, J = 21.2 Hz), 98.8 (d, J = 25.3 Hz), 41.1; HRMS
(ESI) calcd for C13H10FN2O3 (M - H+) 261.0681, found 261.0679;
Melting Point: 122–123 °C.
4.2.27. 2-(((2-methoxyphenyl)amino)methyl)-4-nitrophenol (24)
The title compound was prepared according to general procedure
(B) using 8 (100 mg, 0.36 mmol), yielding the pure product as a yellow
solid (88 mg, 87%) after flash chromatography (Hexane : EtOAc = 10 :
1); 1H NMR (400 MHz, Chloroform-d) δ 10.39 (br s, 1H), 8.20 – 8.11
(m, 2H), 7.02 – 6.80 (m, 5H), 4.70 (br s, 1H), 4.54 (s, 2H), 3.90 (s, 3H);
13C NMR (100 MHz, Chloroform-d) δ 163.5, 148.9, 140.7, 135.8, 125.3,
124.4, 123.3, 121.7, 121.2, 117.1, 114.8, 110.1, 55.6, 49.1; HRMS (ESI)
calcd for C14H13N2O4 (M - H+) 273.0881, found 273.0878; Melting
Point: 124–125 °C.
4.2.22. 2-(((4-fluorophenyl)amino)methyl)-4-nitrophenol (19)
The title compound was prepared according to general procedure
(B) using 3 (100 mg, 0.38 mmol), yielding the pure product as a yellow
solid (94 mg, 93%) after flash chromatography (Hexane : EtOAc = 15 :
1); 1H NMR (400 MHz, Chloroform-d) δ 10.20 (br s, 1H), 8.15 (dd,
J = 8.8, 2.8 Hz, 1H), 8.12 (d, J = 2.8 Hz, 1H), 7.02 – 6.97 (m, 2H), 6.95
(d, J = 8.8 Hz, 1H), 6.85 (dd, J = 9.0, 4.4 Hz, 2H), 4.53 (s, 2H), 4.07 (s,
4.2.28. 2-(((3-methoxyphenyl)amino)methyl)-4-nitrophenol (25)
The title compound was prepared according to general procedure
(B) using 9 (100 mg, 0.37 mmol), yielding the pure product as a yellow
solid (90 mg, 89%) after flash chromatography (Hexane : EtOAc = 15 :
1H); 13C NMR (100 MHz, Chloroform-d)
δ 163.3, 158.4 (d,
J = 242.4 Hz), 142.1 (d, J = 2.0 Hz), 140.8, 125.5, 124.5, 122.7, 118.0
(d, J = 8.1 Hz), 117.2, 116.2 (d, J = 23.2 Hz), 49.7; HRMS (ESI) calcd
9

Y. Qiu, et al.
BioorganicChemistry92(2019)103203
1); 1H NMR (400 MHz, Chloroform-d) δ 9.93 (br s, 1H), 8.17 – 8.14 (m,
2H), 7.20 (t, J = 8.1 Hz, 1H), 6.95 (d, J = 8.6 Hz, 1H), 6.56 (dd,
J = 8.2, 2.4 Hz, 1H), 6.48 (dd, J = 7.9, 2.3 Hz, 1H), 6.43 (d, J = 2.4 Hz,
1H), 4.54 (s, 2H), 3.79 (s, 3H); 13C NMR (100 MHz, Chloroform-d) δ
163.1, 160.7, 147.6, 140.8, 130.4, 125.4, 124.5, 123.1, 117.0, 109.0,
117.14, 116.90, 83.21, 77.59, 48.46; HRMS (ESI) calcd for C15H11N2O3
(M - H+) 267.0775, found 267.0773; Melting point: 134–135 °C.
4.2.34. 2-(((3-(hydroxymethyl)phenyl)amino)methyl)-4-nitrophenol (31)
The title compound was prepared according to general procedure
(B) using 15 (100 mg, 0.38 mmol), yielding the pure product as a
yellow solid (85 mg, 84%) after flash chromatography (Hexane :
EtOAc = 15 : 1); 1H NMR (400 MHz, DMSO‑d6) δ 11.29 (br s, 1H), 8.09
(d, J = 2.9 Hz, 1H), 8.03 (dd, J = 9.0, 2.9 Hz, 1H), 7.02 – 6.98 (m, 2H),
6.59 – 6.49 (m, 2H), 6.41 (dd, J = 7.9, 2.3 Hz, 1H), 5.01 (br s, 1H), 4.36
(s, 2H), 4.25 (s, 2H); 13C NMR (100 MHz, DMSO‑d6) δ 162.0, 148.7,
143.8, 140.1, 129.1, 128.3, 124.7, 124.0, 115.5, 114.9, 110.9, 110.8,
63.7, 41.3; HRMS (ESI) calcd for C14H13N2O4 (M - H+) 273.0881, found
273.0879; Melting Point: 136–137 °C.
106.8, 102.8, 55.2, 48.7; HRMS (ESI) calcd for C14H13N2O4 (M - H+
273.0881, found 273.0876; Melting Point: 98–99 °C.
)
4.2.29. 2-(((4-methoxyphenyl)amino)methyl)-4-nitrophenol (26)
The title compound was prepared according to general procedure
(B) using 10 (100 mg, 0.37 mmol), yielding the pure product as a
yellow solid (87 mg, 86%) after flash chromatography (Hexane :
EtOAc = 10 : 1); 1H NMR (400 MHz, DMSO‑d6) δ 8.09 (d, J = 2.9 Hz,
1H), 8.02 (dd, J = 9.0, 3.0 Hz, 1H), 6.99 (d, J = 8.9 Hz, 1H), 6.70 (d,
J = 8.4 Hz, 2H), 6.51 (d, J = 8.5 Hz, 2H), 4.20 (s, 2H), 3.62 (s, 3H); 13
C
NMR (100 MHz, DMSO‑d6) δ 162.1, 151.4, 142.9, 140.1, 128.4, 124.6,
124.2, 115.5, 115.1, 113.7, 55.7, 42.1; HRMS (ESI) calcd for
C14H13N2O4 (M - H+) 273.0881, found 273.0881; Melting Point:
140–141 °C.
4.2.35. 5-nitro-2-((2-(trimethylsilyl)ethoxy)methoxy)benzaldehyde (32a)
To a mixture of 5-nitrosalicylaldehyde (500 mg, 2.99 mmol) and
DIPEA (1.24 mL, 7.48 mmol, 2.5 equiv) in dry DCM (30 mL) was added
2-(trimethylsilyl)ethoxymethyl chloride (530 μL, 2.99 mmol, 1 equiv),
and the reaction mixture was stirred at room temperature until com-
plete consumption of the starting material as determined by TLC. The
product was extracted with EtOAc (20 mL × 3). The organic layers
were combined, washed with saturated brine, dried over Na2SO4 and
filtered. All volatiles were evaporated in vacuo and the product was
obtained after flash chromatography (Hexane : EtOAc = 30: 1) as a
light yellow liquid (716 mg, 80%); 1H NMR (400 MHz, Chloroform-d) δ
10.49 (s, 1H), 8.73 (d, J = 2.9 Hz, 1H), 8.42 (dd, J = 9.3, 2.9 Hz, 1H),
7.42 (d, J = 9.3 Hz, 1H), 5.48 (s, 2H), 3.85 (t, J = 8.2 Hz, 2H), 1.00 (t,
J = 8.2 Hz, 2H), 0.03 (s, 9H).
4.2.30. Methyl 2-((2-hydroxy-5-nitrobenzyl)amino)benzoate (27)
The title compound was prepared according to general procedure
(B) using 11 (100 mg, 0.33 mmol), yielding the pure product as a
yellow solid (90 mg, 89%) after flash chromatography (Hexane :
EtOAc = 15 : 1); 1H NMR (400 MHz, Chloroform-d) δ 8.65 (br s, 1H),
8.18 (d, J = 2.8 Hz, 1H), 8.14 (dd, J = 8.8, 2.8 Hz, 1H), 8.04 (br s, 1H),
8.01 (dd, J = 7.9, 1.7 Hz, 1H), 7.42 (ddd, J = 8.5, 7.3, 1.7 Hz, 1H), 6.94
(d, J = 8.8 Hz, 1H), 6.89 – 6.82 (m, 2H), 4.60 (d, J = 4.9 Hz, 2H), 3.92
(s, 3H); 13C NMR (100 MHz, DMSO‑d6) δ 168.5, 162.6, 150.7, 139.8,
135.3, 131.7, 127.0, 125.2, 124.4, 115.8, 115.4, 112.1, 110.1, 52.1,
41.2; HRMS (ESI) calcd for C15H13N2O5 (M - H+) 301.0830, found
301.0833; Melting Point: 175–176 °C.
4.2.36. (E)-4-nitro-2-styrylphenol (32)
Step 1: (E)-trimethyl(2-((4-nitro-2-styrylphenoxy)methoxy)ethyl)si-
lane (32b). To a solution of diethyl benzylphosphonate (171 μL,
0.84 mmol, 1 equiv) in dry THF (10 mL) at 0 °C was added NaH
(40 mg, 60% in mineral oil, 1.00 mmol, 1.2 equiv). The resulting
mixture was stirred at the same temperature for 30 min and 32a
(250 mg, 0.84 mmol) in dry THF (5 mL) was added dropwisely. The
mixture was stirred at room temperature overnight before extracted
with EtOAc (20 mL × 3). The organic layers were combined, wa-
shed with saturated brine, dried over Na2SO4 and filtered. All vo-
latiles were evaporated in vacuo and the crude product was used
directly in the next step without further purification.
4.2.31. Methyl 3-((2-hydroxy-5-nitrobenzyl)amino)benzoate (28)
The title compound was prepared according to general procedure
(B) using 12 (100 mg, 0.33 mmol), yielding the pure product as a
yellow solid (88 mg, 87%) after flash chromatography (Hexane :
EtOAc = 10 : 1); 1H NMR (400 MHz, DMSO‑d6) δ 11.36 (br s, 1H), 8.08
– 8.02 (m, 2H), 7.22 – 7.14 (m, 3H), 7.02 (d, J = 8.9 Hz, 1H), 6.82 (d,
J = 8.1 Hz, 1H), 6.63 (br s, 1H), 4.29 (s, 2H), 3.79 (s, 3H); 13C NMR
(100 MHz, DMSO‑d6) δ 167.1, 162.1, 149.0, 140.1, 130.8, 129.8, 127.5,
124.9, 124.1, 117.3, 117.0, 115.6, 112.9, 52.4, 41.1; HRMS (ESI) calcd
for C15H13N2O5 (M - H+) 301.0830, found 301.0831; Melting Point:
130–131 °C.
Step 2: Crude 32b obtained in last step was dissolved in 2% H2SO4-
methanol solution (10 mL). The mixture was stirred at room tem-
perature for 3 h, then quenched with water and the product was
extracted with EtOAc (20 mL × 3). The organic layers were com-
bined, washed with saturated brine, dried over Na2SO4 and filtered.
All volatiles were evaporated in vacuo and the product was obtained
after flash chromatography (Hexane : EtOAc = 10 : 1) as a yellow
solid (71 mg, 35% in two steps); 1H NMR (400 MHz, DMSO‑d6) δ
11.48 (br s, 1H), 8.48 (d, J = 2.8 Hz, 1H), 8.05 (d, J = 9.0 Hz, 1H),
7.64 – 7.62 (m, 2H), 7.47 (d, J = 16.5 Hz, 1H), 7.42 – 7.38 (m, 3H),
7.30 (t, J = 7.3 Hz, 1H), 7.06 (d, J = 8.9 Hz, 1H); 13C NMR
(100 MHz, Chloroform-d) δ 158.38, 141.90, 136.62, 132.76, 128.83,
128.46, 126.87, 125.66, 124.24, 122.96, 120.60, 116.09; HRMS
(ESI) calcd for C14H10NO3 (M - H+) 240.0666, found 240.0662;
Melting point: 136–137 °C.
4.2.32. Methyl 4-((2-hydroxy-5-nitrobenzyl)amino)benzoate (29)
The title compound was prepared according to general procedure
(B) using 13 (100 mg, 0.33 mmol), yielding the pure product as a
yellow solid (87 mg, 86%) after flash chromatography (Hexane :
EtOAc = 15 : 1); 1H NMR (400 MHz, DMSO‑d6) δ 11.38 (br s, 1H), 8.05
(d, J = 7.8 Hz, 2H), 7.69 (d, J = 8.5 Hz, 2H), 7.11 (t, J = 6.0 Hz, 1H),
7.02 (d, J = 8.1 Hz, 1H), 6.62 (d, J = 8.6 Hz, 2H), 4.33 (d, J = 5.9 Hz,
2H), 3.74 (s, 3H); 13C NMR (100 MHz, DMSO‑d6) δ 166.7, 162.1, 152.9,
140.0, 131.5, 127.1, 125.0, 124.2, 116.9, 115.7, 111.7, 51.7, 40.8;
HRMS (ESI) calcd for C15H13N2O5 (M - H+) 301.0830, found 301.0826;
Melting Point: 203–204 °C.
4.2.33. 2-(((3-ethynylphenyl)amino)methyl)-4-nitrophenol (30)
The title compound was prepared according to general procedure
(B) using 14 (100 mg, 0.38 mmol), yielding the pure product as a
yellow solid (93 mg, 92%) after flash chromatography (Hexane :
EtOAc = 15 : 1); 1H NMR (400 MHz, Chloroform-d) δ 9.58 (br s, 1H),
8.16 – 8.14 (m, 2H), 7.24 (t, J = 7.9 Hz, 1H), 7.13 (d, J = 7.8 Hz, 1H),
7.00 – 6.95 (m, 2H), 6.86 (d, J = 8.2 Hz, 1H), 4.54 (s, 2H), 4.16 (s, 1H),
3.09 (s, 1H); 13C NMR (100 MHz, Chloroform-d) δ 162.81, 146.12,
140.93, 129.59, 125.66, 125.53, 124.63, 123.34, 122.81, 119.54,
4.2.37. Trimethyl(2-((4-nitro-2-vinylphenoxy)methoxy)ethyl)silane (33a)
To
a solution of methyltriphenylphosphonium bromide (3.6 g,
10.08 mmol, 1.5 equiv) in dry THF (30 mL) at 0 °C was added NaH
(444 mg, 60% in mineral oil, 11.1 mmol, 1.65 equiv). The resulting
mixture was stirred at the same temperature for 30 min and 32a (2 g,
6.73 mmol) in dry THF (10 mL) was added dropwisely via syringe under
N2 atmosphere. The mixture was stirred at room temperature overnight
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BioorganicChemistry92(2019)103203
before extracted with EtOAc (20 mL × 3). The organic layers were
combined, washed with saturated brine, dried over Na2SO4 and filtered.
All volatiles were evaporated in vacuo and the product was obtained
after flash chromatography (Hexane : EtOAc = 100 : 1) as a white solid
1H), 7.97 (d, J = 8.1 Hz, 2H), 7.77 (d, J = 8.1 Hz, 2H), 7.55 (s, 2H),
7.07 (d, J = 9.0 Hz, 1H), 3.86 (s, 3H); 13C NMR (100 MHz, DMSO‑d6) δ
166.4, 161.8, 142.2, 140.5, 130.1, 129.0, 127.2, 125.4, 124.9, 124.6,
123.3, 116.7, 52.5; HRMS (ESI) calcd for C16H12NO5 (M - H+
298.0721, found 298.0719; Melting point: 258–259 °C.
)
(1238 mg, 62%); 1H NMR (400 MHz, Chloroform-d)
δ 8.40 (d,
J = 2.8 Hz, 1H), 8.13 (dd, J = 9.2, 2.9 Hz, 1H), 7.23 (d, J = 9.1 Hz,
1H), 7.04 (dd, J = 17.7, 11.1 Hz, 1H), 6.15 – 5.72 (m, 1H), 5.60 – 5.42
(m, 1H), 5.38 (s, 2H), 3.83 – 3.76 (m, 2H), 1.03 – 0.93 (m, 2H), 0.02 (s,
9H).
4.2.43. N-(3-ethynylphenyl)-2-hydroxy-5-nitrobenzamide (38)
To a solution a of 5-nitrosalicylic acid (50 mg, 0.27 mmol) and tri-
phenyl phosphite (86 μL, 0.33 mmol, 1.2 equiv) in toluene (5 mL) was
added 3-aminophenylacetylene (37 μL, 0.33 mmol, 1.2 equiv) in to-
luene (2 mL) via syringe under N2 atmosphere. The resulting mixture
was refluxed for 6 h. After the reaction was completed, the product was
extracted with EtOAc (20 mL × 3). The organic layers were combined,
washed with saturated brine, dried over Na2SO4 and filtered. All vo-
latiles were evaporated in vacuo and the product was obtained after
flash chromatography (Hexane : EtOAc = 20 : 1) as a yellow solid
(57 mg, 74%); 1H NMR (400 MHz, DMSO‑d6) δ 10.64 (s, 1H), 8.74 (d,
J = 3.0 Hz, 1H), 8.30 (dd, J = 9.1, 3.0 Hz, 1H), 7.91 (s, 1H), 7.74 – 7.72
(m, 1H), 7.41 (t, J = 7.9 Hz, 1H), 7.27 (d, J = 7.6 Hz, 1H), 7.17 (d,
J = 9.1 Hz, 1H), 4.23 (s, 1H); 13C NMR (100 MHz, DMSO‑d6) δ 164.7,
163.6, 139.8, 138.7, 129.7, 128.9, 128.0, 126.3, 124.0, 122.6, 121.8,
4.2.38. (E)-2-(2-methoxystyryl)-4-nitrophenol (33)
The title compound was prepared according to general procedure
(D) using 2-iodoanisole 33b (73 μL, 0.56 mmol), yielding the pure
product as a yellow solid (97 mg, 64%) after flash chromatography
(Hexane : EtOAc = 20 : 1); 1H NMR (400 MHz, Chloroform-d) δ 8.49 (d,
J = 2.7 Hz, 1H), 8.06 (dd, J = 9.0, 2.8 Hz, 1H), 7.63 (d, J = 7.8 Hz,
1H), 7.59 (d, J = 16.6 Hz, 1H), 7.34 – 7.28 (m, 2H), 7.01 (t, J = 7.5 Hz,
1H), 6.94 (dd, J = 14.9, 8.6 Hz, 2H), 6.03 (s, 1H), 3.94 (s, 3H); 13C
NMR (10 MHz, DMSO‑d6) δ 161.5, 157.2, 140.4, 129.8, 127.0, 126.0,
125.9, 125.6, 124.7, 122.7, 122.7, 121.2, 116.5, 111.9, 56.0; HRMS
(ESI) calcd for C15H12NO4 (M - H+) 270.0772, found 270.0769; Melting
point: 183–184 °C.
120.0, 118.5, 83.7, 81.2; HRMS (ESI) calcd for C15H9N2O4 (M - H+
281.0568, found 281.0571;. 181 °C (decomposed).
)
4.2.39. (E)-2-(3-methoxystyryl)-4-nitrophenol (34)
The title compound was prepared according to general procedure
(D) using 3-iodoanisole 34b (67 μL, 0.56 mmol), yielding the pure
product as a yellow solid (89 mg, 58%) after flash chromatography
(Hexane : EtOAc = 20 : 1); 1H NMR (400 MHz, Chloroform-d) δ 8.48 (d,
J = 2.8 Hz, 1H), 8.08 (dd, J = 8.9, 2.7 Hz, 1H), 7.39 – 7.29 (m, 2H),
7.23 (d, J = 16.4 Hz, 1H), 7.18 (d, J = 7.6 Hz, 1H), 7.11 (t, J = 2.2 Hz,
1H), 6.93 – 6.89 (m, 2H), 6.13 (s, 1H), 3.89 (s, 3H); 13C NMR (100 MHz,
DMSO‑d6) δ 161.6, 160.1, 140.5, 138.9, 131.3, 130.2, 125.1, 124.9,
123.0, 122.5, 119.5, 116.6, 114.3, 112.2, 55.5; HRMS (ESI) calcd for
4.2.44. (2-hydroxy-5-nitrophenyl)(phenyl)methanone (39)
To a solution of 5-nitrosalicylaldehyde (100 mg, 0.60 mmol), PdCl2
(5 mg, 0.03 mmol, 0.05 equiv), LiCl (5 mg, 0.12 mmol, 0.2 equiv) and
Na2CO3 (127 mg, 1.20 mmol, 2equiv) in DMF (5 mL) was added iodo-
benzene (135 μL, 1.2 mmol, 2equiv) via syringe under N2 atmosphere.
The resulting solution was stirred at 120 °C for 6 h. After the reaction
was completed, the product was extracted with EtOAc (20 mL × 3). The
organic layers were combined, washed with saturated brine, dried over
Na2SO4 and filtered. All volatiles were evaporated in vacuo and the
C
15H12NO4 (M - H+
)
270.0772, found 270.0771; Melting point:
product was obtained after flash chromatography (Hexane
:
158–159 °C.
EtOAc = 20 : 1) as a yellow solid (108 mg, 74%); 1H NMR (400 MHz,
Chloroform-d) δ 12.67 (s, 1H), 8.63 (d, J = 2.8 Hz, 1H), 8.42 (dd,
J = 9.2, 2.8 Hz, 1H), 7.76 – 7.69 (m, 3H), 7.61 (t, J = 7.6 Hz, 2H), 7.21
(d, J = 9.3 Hz, 1H); 13C NMR (100 MHz, Chloroform-d) δ 200.5, 168.0,
139.5, 136.4, 133.2, 130.9, 129.6, 129.3, 128.9, 119.6, 118.0; HRMS
(ESI) calcd for C13H8NO4 (M - H+) 242.0459, found 242.0454; Melting
point: 121–122 °C.
4.2.40. (E)-2-(2-hydroxy-5-nitrostyryl)benzonitrile (35)
The title compound was prepared according to general procedure
(D) using 2-iodobenzonitrile 35b (128 mg, 0.56 mmol), yielding the
pure product as a yellow solid (95 mg, 64%) after flash chromatography
(Hexane : EtOAc = 20 : 1); 1H NMR (400 MHz, DMSO‑d6) δ 11.82 (s,
1H), 8.45 (s, 1H), 8.11 – 8.02 (m, 2H), 7.87 – 7.46 (m, 3H), 7.66 – 7.49
(m, 2H), 7.08 (d, J = 9.1 Hz, 1H); 13C NMR (100 MHz, DMSO‑d6) δ
162.3, 140.6, 140.4, 133.9, 133.7, 128.9, 128.5, 127.1, 126.2, 125.8,
125.0, 124.0, 118.3, 116.9, 110.7; HRMS (ESI) calcd for C15H9N2O3 (M
- H+) 265.0619, found 265.0617; Melting point: 216–217 °C.
4.3. Determination of minimum inhibitory concentration (MIC)
The antimicrobial activity of the compounds was determined by
broth microdilution according to the CLSI guidelines [21]. The fol-
lowing bacterial strains were used in this study for the microdilution
assay: Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae
ATCC 49619, Klebsiella pneumoniae ATCC 700603, Acinetobacter bau-
mannii ATCC 19606, Pseudomonas aeruginosa PA01, Enterobacter cloacae
ATCC 13047, Escherichia coli ATCC 25922. The test medium was brain
heart infusion (BHI) for S. pneumoniae and Mueller-Hinton broth (MH)
for the rest of the organisms. Serial two-fold dilutions were performed
for the tested chemicals starting from 256 µg/mL to 0.5 µg/mL, and the
bacterial cell inoculum was adjusted to approximately 1.5 × 106 CFU
per mL. Results were taken after 16–20 hrs of incubation at 37 °C. The
MIC was defined as the lowest concentration of antibiotic with no
visible growth. Experiments were performed in duplicates.
4.2.41. Methyl (E)-3-(2-hydroxy-5-nitrostyryl)benzoate (36)
The title compound was prepared according to general procedure
(D) using methyl 3-iodobenzoate 36b (147 mg, 0.56 mmol), yielding
the pure product as a yellow solid (102 mg, 61%) after flash chroma-
tography (Hexane : EtOAc = 20 : 1); 1H NMR (400 MHz, DMSO‑d6) δ
11.53 (br s, 1H), 8.53 (d, J = 2.9 Hz, 1H), 8.19 (s, 1H), 8.06 (dd,
J = 9.0, 2.9 Hz, 1H), 7.92 (d, J = 7.9 Hz, 1H), 7.88 (d, J = 7.7 Hz, 1H),
7.61 – 7.53 (m, 2H), 7.48 (d, J = 16.6 Hz, 1H), 7.07 (d, J = 9.0 Hz, 1H),
3.89 (s, 3H); 13C NMR (100 MHz, DMSO‑d6) δ 166.6, 161.6, 140.5,
138.0, 131.5, 130.6, 130.1, 129.7, 128.8, 127.5, 125.1, 124.8, 123.3,
123.0, 116.6, 52.7; HRMS (ESI) calcd for C16H12NO5 (M - H+
298.0721, found 298.0717; Melting point: 200–201 °C.
)
4.4. Binding inhibition assay
4.2.42. Methyl (E)-4-(2-hydroxy-5-nitrostyryl)benzoate (37)
The title compound was prepared according to general procedure
(D) using methyl 4-iodobenzoate 37b (147 mg, 0.56 mmol), yielding
the pure product as a yellow solid (113 mg, 67%) after flash chroma-
tography (Hexane : EtOAc = 20 : 1); 1H NMR (400 MHz, DMSO‑d6) δ
11.60 (br s, 1H), 8.52 (d, J = 2.7 Hz, 1H), 8.07 (dd, J = 9.0, 2.8 Hz,
Previously established protocols were used for inhibitor testing
[22]. 40 mL N-LgBiT-NusB (2.5 mM in PBS) was added to 96-well plates
and then mixed with 20 mL compound. The mixture was incubated for
10 min at 37 °C. 40 mL C-SmBiT-NusE (2.5 mM in PBS) was then added
to each well, followed by incubation for 10 min at 37 °C. The final
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BioorganicChemistry92(2019)103203
concentration of the compounds was at 125 mM. After the final in-
cubation 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.
4.9. Hemolysis assay
To separate erythrocytes from human whole blood, 35 mL of human
whole blood was centrifuged at 500 × g, 4 °C for 5 mins. After cen-
trifugation, the upper layers of plasma and buffy coat were aspirated by
micropipette. 150 mM NaCl solution was added to the packed ery-
throcytes and filled up to the original volume mark, and the solution
was gently shaken to re-suspend the erythrocytes. The centrifugation
process was repeated with the same condition and after centrifugation,
the upper layers were aspirated and discarded, followed by the re-
suspension of erythrocytes using PBS (pH 7.4). A 40 × -diluted ery-
throcyte suspension was prepared by adding 1 mL of the final suspen-
sion 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. 1600 µL of the 40 × -diluted erythrocyte
suspension was added to 400 µL of the compound 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 500 × g, 4 °C, to
pellet the remaining erythrocytes and erythrocyte ghosts. Then, 100 µL
of the supernatant from 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:
4.5. Time kill kinetics
The time kill kinetics assay was performed using the CLSI guidelines
[21]. S. aureus cells were suspended to ~ 1.5 × 106 CFU/mL at log
phase in MH medium with compounds at various concentrations. As an
untreated control, bacteria were incubated in MH medium without
compounds. The cultures were grown at 37 °C with shaking at 200 rpm,
and 10 µL sample was taken at defined time points (0, 2, 4 and 6 h) for
each treatment group, followed by 10-fold serial dilutions. 5 µL sample
was taken from each dilution and spotted on a blood agar plate. After
incubation at 37 °C overnight, the number of viable bacteria in each
sample was counted and expressed as CFU/mL. The experiment was
performed in triplicate. Technical repeats were taken to ensure con-
sistent results were obtained.
4.6. Assessment of ATP production
S. aureus cells were suspended to ~1.5 × 106 CFU/mL at log phase
in MH medium with compounds at various concentrations. As an un-
treated control, bacteria were incubated in MH medium without com-
pounds. The cultures were grown at 37 °C with shaking at 200 rpm, and
10 µL sample was taken at defined time points (0, 2, 4 and 6 h) for each
treatment group. The ATP production was measured using the BacTiter-
Glo™ Microbial Cell Viability Assay Kit (Promega™) according to the
manufacturer's instructions. The experiment was performed in tripli-
cate. Technical repeats were taken to ensure consistent results were
obtained.
Asample
A
ve control
A
ve control
% of lysis =
A
+ve control
Asample: absorbance of the tested compound solution; A-ve control: absor-
bance of the vehicle solution; A+ve control: absorbance of the Triton X-
100 solution.
4.7. Confocal microscopy and macromolecular quantitation
4.10. Caco-2 permeability study
B. subtilis strain BS61 (NusB-GFP) [30] was grown on LB agar plate
containing 5 mg/mL chloramphenicol. A single colony was incubated in
LB medium supplemented with 5 mg/mL chloramphenicol at 37 °C until
OD600 ~ 0.6. Compound at 1/8, ¼, ½, 1 × MIC was then added to the
culture and allowed to incubate for further 15 min. 2.5 μL of cell culture
was placed onto 1.2% freshly made agarose plate and covered with a
coverslip prior to imaging. Leica TCS SPE confocal microscope
equipped with 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. For macromolecular quantitation,
B. subtilis stain 168 were grown on LB agar plate overnight and a single
colony was inoculated into LB medium. Compound 23 at 1/8 and ¼
MIC were added to the culture at early log-phase (OD 0.2), and cells
were harvested at mid-log phase (OD 0.5) by centrifugation. Cell lysis
and macromolecular quantitation were performed as described pre-
viously [18].
Caco-2 cells were cultured at 37 °C in Dulbecco's Modified Eagle
Medium with 10% fetal bovine serum, 1% nonessential amino acids,
and 100 U/mL penicillin, and 100 µg/mL streptomycin in an atmo-
sphere of 5% CO2 and 90% relative humidity. The cells were passaged
after 90% confluence using trypsin-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). Medium
was changed the day after seeding and every second day thereafter (AP
volume 0.5 mL and BL volume 1.5 mL). The Caco-2 cell monolayers
were used 21 to 28 days after seeding. Transepithelial electrical re-
sistance (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 apical (AP) to
basolateral (BL) direction. Cell monolayers were preincubated with
transport buffer solution (PBS+) for 20 min at 37 °C. To study ab-
sorptive transport, the donor (AP) compartment buffer was replaced
with 0.5 mL of transport buffer containing 10 µg/mL of compounds. The
pH in both AP and BL compartments was maintained at 7.4 for all
transport studies. Concentration of MC4 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 sample was drawn from the receiver compartment, and a
same volume of blank buffer was added into receiver compartment.
Concentration of compounds in sample solution was determined using
LC-MS/MS.
4.8. In vitro cytotoxicity
Human cell lines A549 lung carcinoma and HaCaT immortalized
keratinocytes were used in this study. The cells were seeded at
2.5 × 105 per well. After 24 hr incubation, the tested compounds were
added in a 2-fold serial dilution ranging from 1.562 mg/mL to 50 mg/
mL. The plates were incubated at 37 °C. At 48 hr and 72 hr after adding
the compound, the MTT assay was performed as described previously
[18]. 5-fluorouracil was used as a positive control and DMSO as a ne-
gative control.
12

Y. Qiu, et al.
BioorganicChemistry92(2019)103203
Notes
coli, Genes Cells. 7 (2002) 755–768.
[13] M. Torres, J.M. Balada, M. Zellars, C. Squires, C.L. Squires, In vivo effect of NusB and
NusG on rRNA transcription antitermination, J. Bacteriol. 186 (2004) 1304–1310.
[14] K.B. Arnvig, S. Zeng, S. Quan, A. Papageorge, N. Zhang, A.C. Villapakkam,
C.L. Squires, Evolutionary comparison of ribosomal operon antitermination func-
tion, J. Bacteriol. 190 (2008) 7251–7257.
The authors declare no competing financial interest.
Acknowledgement
[15] B.M. Burmann, K. Schweimer, X. Luo, M.C. Wahl, B.L. Stitt, M.E. Gottesman,
P. Rösch, A NusE:NusG complex links transcription and translation, Science 328
(2010) 501–504.
The research was supported by the Shenzhen Science, Technology
and Innovation Commission (SZSTI) grant JCYJ20170303155923684,
China (C.M.), Hong Kong RGC Early Career Scheme grant No.
25100017, Hong Kong (C.M.), State Key Laboratory of Chemical
Biology and Drug Discovery, and internal grant G-YBYY, Hong Kong
Polytechnic University, Hong Kong (C.M.), Hong Kong RGC General
Research Fund GRF No. 14165917, Hong Kong (X.Y.), Hong Kong Food
and Health Bureau HMRF Grant No. 17160152, Hong Kong (X.Y.) and
Chinese University of Hong Kong, Faculty of Medicine Faculty
Innovation Award FIA2018/A/03, Hong Kong (X.Y.).
[16] N. Singh, M. Bubunenko, C. Smith, D.M. Abbott, A.M. Stringer, R. Shi, D.L. Court,
J.T. Wade, SuhB associates with Nus factors to facilitate 30S ribosome biogenesis in
Escherichia coli, mBio. 7 (2016) e00114–e116.
[17] S.J. Greive, A.F. Lins, P.H. von Hippel, Assembly of an RNA-protein complex.
Binding of NusB and NusE (S10) proteins to boxA RNA nucleates the formation of
the antitermination complex involved in controlling rRNA transcription in
Escherichia coli, J. Biol. Chem. 280 (2005) 36397–36408.
[18] X. Yang, M.J. Luo, A.C.M. Yeung, P.J. Lewis, P.K.S. Chan, M. Ip, C. Ma, First-in-class
inhibitor of ribosomal RNA synthesis with antimicrobial activity against
Staphylococcus aureus, Biochemistry 56 (2017) 5049–5052.
[19] X. Luo, H.H. Hsiao, M. Bubunenko, G. Weber, D.L. Court, M.E. Gottesman,
H. Urlaub, M.C. Wahl, Structural and functional analysis of the E. coli NusB-S10
transcription antitermination complex, Mol. Cell. 32 (2008) 791–802.
[20] Y. Qiu, S.T. Chan, L. Lin, T.L. Shek, T.F. Tsang, N. Barua, Y. Zhang, M. Ip, P.K. Chan,
N. Blanchard, G. Hanquet, Z. Zuo, X. Yang, C. Ma, Design, synthesis and biological
evaluation of antimicrobial diarylimine and -amine compounds targeting the in-
teraction between the bacterial NusB and NusE proteins, Eur. J. Med. Chem. 178
(2019) 214–231.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103203.
[21] CLSI, Clinical and Laboratory Standards Institute, Performance Standards for
Antimicrobial Susceptibility Testing, Twenty-Fifth Informational Supplement
(M100S25), Clinical and Laboratory Standards Institute, Wayne, PA, 2015.
[22] T.F. Tsang, Y. Qiu, L. Lin, J. Ye, C. Ma, X. Yang, 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. 5 (2019)
521–527.
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