Optimization of biaryloxazolidinone as promising antibacterial agents against antibiotic-susceptible and antibiotic-resistant gram-positive bacteria

Article abstract of DOI:10.1016/j.ejmech.2019.111781

We previously discovered a series of novel biaryloxazolidinone analogues bearing a hydrazone moiety with potent antibacterial activity. However, the most potent compound OB-104 exhibited undesirable chemical and metabolic instability. Herein, novel biaryloxazolidinone analogues were designed and synthesized to improve the chemical and metabolic stability. Compounds 6a-1, 6a-3, 14a-1, 14a-3 and 14a-7 showed significant antibacterial activity against the tested Gram-positive bacteria as compared to radezolid and linezolid. Further studies indicated that most of them exhibited improved water solubility and chemical stability. Compound 14a-7 had MIC values of 0.125–0.25 μg/mL against all tested Gram-positive bacteria, and showed excellent antibacterial activity against clinical isolates of antibiotic-susceptible and antibiotic-resistant bacteria. Moreover, it was stable in human liver microsome. From a safety viewpoint, it showed non-cytotoxic activity against hepatic cell and exhibited lower inhibitory activity against human MAO-A compared to linezolid. The potent antibacterial activity and all these improved drug-likeness properties and safety profile suggested that compound 14a-7 might be a promising drug candidate for further investigation.

Full text of DOI:10.1016/j.ejmech.2019.111781

Journal Pre-proof
Optimization of biaryloxazolidinone as promising antibacterial agents against
antibiotic-susceptible and antibiotic-resistant gram-positive bacteria
Yachuang Wu, Xiudong Ding, Yifeng Yang, Yingxiu Li, Yinliang Qi, Feng Hu, Mingze
Qin, Yajing Liu, Lu Sun, Yanfang Zhao
PII:
S0223-5234(19)30933-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.111781
EJMECH 111781
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 April 2019
Revised Date: 1 June 2019
Accepted Date: 9 October 2019
Please cite this article as: Y. Wu, X. Ding, Y. Yang, Y. Li, Y. Qi, F. Hu, M. Qin, Y. Liu, L. Sun, Y. Zhao,
Optimization of biaryloxazolidinone as promising antibacterial agents against antibiotic-susceptible and
antibiotic-resistant gram-positive bacteria, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.111781.
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Optimization of
biaryloxazolidinone as
promising
antibacterial
agents
against
antibiotic-susceptible and antibiotic-resistant Gram-positive bacteria
Yachuang Wu a, 1, Xiudong Ding b, 1, Yifeng Yang a, Yingxiu Li a, Yinliang Qi a, Feng Hu c, Mingze Qin a, Yajing Liu
a, Lu Sun c**, Yanfang Zhao a*1
a Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry
of Education, 103 Wenhua Road, Shenhe District, Shenyang 110016, PR China
b Department of Clinical Laboratory, The 309th Hospital of Chinese People’s Liberation Army, Beijing 100091,
China
c School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016,
PR China
Abstract:
We previously discovered a series of novel biaryloxazolidinone analogues bearing a hydrazone moiety with
potent antibacterial activity. However, the most potent compound OB-104 exhibited undesirable chemical and
metabolic instability. Herein, novel biaryloxazolidinone analogues were designed and synthesized to improve the
chemical and metabolic stability. Compounds 6a-1, 6a-3, 14a-1, 14a-3 and 14a-7 showed significant antibacterial
activity against the tested Gram-positive bacteria as compared to radezolid and linezolid. Further studies indicated
that most of them exhibited improved water solubility and chemical stability. Compound 14a-7 had MIC values of
0.125-0.25 µg/mL against all tested Gram-positive bacteria, and showed excellent antibacterial activity against
clinical isolates of antibiotic-susceptible and antibiotic-resistant bacteria. Moreover, it was stable in human liver
microsome. From a safety viewpoint, it showed non-cytotoxic activity against hepatic cell and exhibited lower
inhibitory activity against human MAO-A compared to linezolid. The potent antibacterial activity and all these
improved drug-likeness properties and safety profile suggested that compound 14a-7 might be a promising drug
candidate for further investigation.
Keywords: structural optimization; biaryloxazolidinone; antibiotic-resistant infections; Gram-positive bacteria
1. Introduction
The emergence and outbreak of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant
Enterococci (VRE) and penicillin-resistant Streptococcus pneumoniae (PRSP) made antibiotic-resistant infections a
critical healthcare concern worldwide [1-3]. Antibiotic-resistant infections resulted in increasing patient mortality
and social economic burden. In the United States, there were approximately 19000 people dying each year from
MRSA, which is known as the first superbug [4-5]. Finding effective ways to resolve antibiotic-resistant crisis is an
urgent task. Karen B et al. summarized the approaches for resolving this global health crisis, of which, finding
antibiotics with new structural classes or mechanisms was the main concern for drug chemists [6]. Due to
developing antibiotics with new mechanisms is a long-term work, we focus our efforts on modifications of old
antibiotics with known mechanisms to overcome drug resistance.
** Corresponding author.
* Corresponding author.
E-mail address: yanfangzhao@126.com (Y. Zhao), lisunlu@126.com (L. Sun).
1 Yachuang Wu and Xiudong Ding contributed equally to this work.

The ribosomes synthesize proteins and the bacterial ribosome is one of the main targets of antibiotics [7]. Many
natural, semi-synthetic and synthetic antibiotics bind to bacterial ribosome to inhibit bacterial protein synthesis. In
recent years, important progresses on the crystal structures of antibiotics binding to ribosome have been reported,
which give drug chemists insight into the mechanism of action of ribosome-targeting antibiotics. And these
progresses are crucial for drug chemists to improve the potency and overcome bacterial resistance by further drug
optimization [8-9]. Linezolid, the first oxazolidinone drug, was approved in 2000 by FDA for the treatment of
antibiotic-resistant Gram-positive infections, including MRSA, PRSP, and VRE [10-11]. It binds to 23S RNA of the
50S ribosomal subunit to inhibit bacterial protein biosynthesis, which belongs to a ribosome-targeting antibiotic
[12]. However, bacterial resistance to linezolid has been discovered due to the overuse of linezolid. Resistance to
linezolid was mediated by a number of mechanisms, such as alteration of microbial permeability and target
mutation [7, 13].
The C-ring of linezolid and many other ribosome-targeting antibiotics, such as chloramphenicol and clindamycin,
share an overlapping region on the binding sites of ribosomal RNA, which inspired many research groups to
develop oxazolidinone derivatives with C-ring and D-ring to overcome bacterial resistance and to improve the
potency [7, 14-15]. Radezolid was developed based on the crystal structures of linezolid binding to ribosomal RNA
and it exhibited more potent activity against antibiotic-resistant Gram-positive bacteria than linezolid [16-17]. As
reported, oxazolidinone derivatives containing biaryl scaffold showed increased antibacterial potency against
Gram-positive bacteria, especially antibiotic-resistant Gram-positive bacteria compared with linezolid [18-22].
These compounds revealed outstanding antibacterial activity by forming π–π stacking or hydrogen bond
interactions with the 50S subunit.
Figure 1. Structures of linezolid and related oxazolidinone analogues
.
In our previous work, we discovered compound OB-104, a biaryloxazolidinone derivative bearing a hydrazone
moiety, showed excellent antibacterial activity against five tested Gram-positive bacteria in vitro, which were a 15-
to 30-fold increase compared to linezolid in activity against linezolid-resistant Enterococcus faecalis [23]. However,
compound OB-104 was unstable under simulated gastric acid condition (pH=1.2) and showed short half-life time
(72.85 min) in human liver microsome. Further pharmacokinetic study in rats by intragastric administration
revealed that the half-life time (T1/2) of OB-104 was 2.7 h. As shown in Figure 2, the metabolites of OB-104 in rats
were further studied by UPLC-QTOF/MS method. Four main metabolites were found in plasma of rats given
intragastric administration of OB-104. The main metabolite M-1 was confirmed by MS spectra and the reference
substance, which was formed by the hydroxylation of C-ring at C-3 position. At the same time, nitrogen
demethylation occurred on the piperazine ring to form metabolite M-2. The C=N bond of the hydrazone moiety of
OB-104 was hydrolyzed to the aldehyde metabolite M-3, which was further oxidized to the carboxyl metabolite
M-4. Metabolite M-1 with hydroxyl group at C-ring attracted our interest, and it was synthesized and its
antibacterial activity was evaluated. Remarkably, M-1 showed significant antibacterial activity against the tested
Gram-positive bacteria (data are shown in Table 1). As we know, the electronic or sterically hindered effects of

substituents in C-ring may have a large effect on the stability of hydrazone bond. Basing on the structure of M-1
and other reported biaryloxazolidinone analogues [24-25], we focused on the structural optimization of compound
OB-104 in following strategies, which are shown in Figure 3: (I) introduction of a sterically hindered substituent or
hydrogen bond donor (OH)/ hydrogen bond receptor (F) at the ortho-position of the hydrazone bond on the C-ring
(compounds 6a-6e and 16). ( ) replacement of C-ring with nitrogen-containing heterocyclic ring (compounds
14a-14b and 15a), of which, the electron-withdrawing effect of nitrogen atom was expected to improve the
chemical stability of hydrazone bond [21].
Figure 2. Predicted metabolic pathways of compound OB-104.
Figure 3. Design strategies for novel biaryloxazolidinone compounds.
2. Chemistry
The synthesis of target compounds 6 is depicted in Scheme 1. 4-Bromo-3-fluoroaniline was acylated with
methyl chloroformate to give 2 with a high yield. Intermediate 3 was obtained by cyclization reaction of
intermediate 2 with (S)-1-((acetylamino)methyl)-2-chloroethyl acetate. Intermediate 4 was prepared by

Miyaura-Ishiyama-Hartwig borylation reaction of intermediate 3 with bis(pinacolato)diboron, which was subjected
to substituted p-bromobenzaldehydes to give the corresponding biaryloxazolidinones 5 by Suzuki coupling. The
intermediates 5 were reacted with 9a-9f in the presence of acetic acid to yield the corresponding target compounds
6 with high yields. The preparation of intermediates 9a-9f was summarized in Scheme 2 and 3, as reported in our
previous work [23]. In particular, intermediate 7f was synthesized through a three-step process from
thiomorpholine. Moreover, compound 6a-5 was finally prepared by deprotection of 6a-4 in DCM, and compounds
6b-6 and 6d-4 were prepared according to the same method. Compound 6a-3 was oxidized by 3-chloroperbenzoic
acid in DCM to give compound 6a-6.
Scheme 1. Reagents and conditions: a: methyl chloroformate, pyridine, DCM, 25 °C,
2
h; b:
(S)-1-((acetylamino)methyl)-2-chloroethyl acetate, t-BuOLi, DMF, CH3OH, rt, 20 h; c: bis(pinacolato)diboron, Pd(PPh3)2Cl2,
potassium acetate, 1,4-dioxane, 90 °C, 20 h; d: substituted p-bromobenzaldehydes, Pd(PPh3)2Cl2, K2CO3, DMF/ H2O, 75 °C, 8-12 h; e:
9a-9f, acetic acid, EtOH, 78 °C, 2 h. (f) TFA, DCM, 25 °C, 5 h; (g) m-CPBA, DCM, 25 °C, 4 h.
Scheme 2. Reagents and conditions: a: NaNO2, HCl, THF/H2O, 25 °C, 4 h; b: Zn, AcOH/H2O, 0 °C, 5 h.

Scheme 3. Reagents and conditions: a: (Boc)2O, TEA, THF, 25 °C, 5 h; b: H2O2(40 %), TFA, AcOH, 50 °C, 4 h; c: TFA, DCM, 25 °C,
5 h.
Scheme 4. Reagents and conditions: a: 3-bromo-6-pyridine carboxaldehyde or 6-bromo-3-pyridine carboxaldehyde, Pd(PPh3)2Cl2,
K2CO3, DMF/ H2O, 75 °C, 12 h; b: 9a-9f, EtOH, 78 °C, 2 h; c: m-CPBA, DCM, 25 °C, 4 h; d: TFA, DCM, 25 °C, 5 h.
The synthetic route of compounds 14 is depicted in Scheme 4. Compounds 14 were synthesized from
intermediate 4 and 3-bromo-6-pyridine carboxaldehyde or 6-bromo-3-pyridine carboxaldehyde using the same
method for the synthesis of compounds 6. Moreover, Compounds 15 and 16 were prepared by acylation of 14a-5
and 6a-5 on the terminal piperazinyl ring with acid chloride using DIPEA as a base at room temperature, as shown
in Scheme 5 and 6.
O
O
O
F
F
a:
R1
Cl
O
O
N
O
N
H
H
N
N
HN
N N
N
N
N N
N
R1
O
O
14a-5
15a-1 ~ 15a-3
H C
3
15a-2: R1 =
15a-1: R =
15a-3: R1 =
H3CO
1
Scheme 5. Reagents and conditions: a: DIPEA, DMF, 25 °C, 2 h.

Scheme 6. Reagents and conditions: a: DIPEA, DMF, 25 °C, 2 h.
3. Results and discussion
3.1. Evaluation of in vitro antibacterial activity
All the target biaryloxazolidinone compounds were screened for their in vitro antibacterial activity against Gram-
positive, antibiotic-susceptible and antibiotic-resistant bacteria strains (S.aureus ATCC29213, MRSA, MSSA,
LREF and VRE), using linezolid and radezolid as positive control. The results of minimum inhibitory
concentrations (MICs, µg/mL) determined by the broth liquid microdilution method were summarized in Tables
1-2.
As shown in Table 1, we firstly synthesized the putative metabolite M-1 of OB-104, namely 6a-1. It was worth
noting that compound 6a-1 showed significantly potent antibacterial activity against five tested Gram-positive
bacteria with MIC values of 0.125-0.25 µg/mL, which was more potent than that of linezolid and radezolid. Then a
series of biaryloxazolidinone analogues with hydroxyl group (6a-2 to 6a-7) at C-ring were synthesized, and
compound 6a-3 with terminal thiomorpholine group was the most potent compound, which had the MIC values of
0.125 µg/mL against five tested Gram-positive bacteria. Based on the high activity of compound 6a-3, hydrogen
bond receptor (F) and sterically hindered substituents (OCH3, CH3 and CF3) were introduced into the C-ring at the
putative metabolic site to improve the metabolic stability. We speculated that the C-ring substituent at the
ortho-position of the hydrazone bond is related to the hydrolysis of hydrazone bond. However, most of this series
of compounds showed weaker antibacterial activities than 6a-1 and 6a-3. Compound 6b-3 with fluoro group
exhibited 2-fold to 8-fold decrease in antibacterial activity compared to compound 6a-3 with hydroxyl group.
Compounds with methoxy, trifluoromethyl or methyl groups respectively showed lower antibacterial activity
compared to compounds bearing fluorine or hydroxyl, which indicated that steric hindrance of these substituents
can reduce antibacterial activity.
Table 1. In vitro antibacterial activity of biaryloxazolidinone analogues.
MICs, µg/mL
Compounds
W
X
S.aureusa
0.125
0.25
MRSAb
0.125
0.25
MSSAc
0.125
0.25
LREFd
0.25
VREe
0.25
NCH3
O
OH
OH
OH
OH
6a-1
6a-2
6a-3
6a-5
0.25
0.25
S
0.125
0.5
0.125
0.25
0.125
0.25
0.125
0.25
0.125
0.25
NH

OH
OH
F
0.25
0.125
1
0.125
0.25
1
0.25
0.25
1
0.25
0.25
1
0.25
0.25
1
6a-6
6a-7
S O
NCH3
O
6b-1
F
0.25
0.25
0.25
0.5
2
0.25
0.25
0.25
0.5
2
0.25
0.25
0.25
0.5
2
0.25
1
0.25
1
6b-2
S
F
6b-3
CH2
NH
O
F
0.5
0.5
2
0.5
0.5
2
6b-4
F
6b-6
OCH3
OCH3
CH3
CH3
CH3
CF3
CF3
6c-1
S
2
1
1
1
1
6c-2
NCH3
S
0.5
2
0.5
1
0.5
1
0.5
2
0.5
1
6d-1
6d-2
NH
NCH3
S
0.5
2
0.5
2
0.5
2
0.5
2
0.5
2
6d-4
6e-1
2
2
2
2
2
6e-2
1
1
1
＞
16
2
linezolid
0.5
0.5
0.5
0.5
0.5
radezolid
a Standard Staphylococcus aureus (29213).
b Methicillin-resistant Staphylococcus aureus.
c Methicillin-sensitive Staphylococcus aureus.
d Linezolid-resistant Enterococcus faecalis.
e Vancomycin-resistant Enterococcus faecium.
Compound 6a-3 exhibited MIC values of 0.125 µg/mL against all tested five Gram-positive bacteria, but it
showed 2-fold decrease in antibacterial activity compared to OB-104, which indicated that the introduction of
substituent at ortho-position of the hydrazone bond is actually unfavorable for the antibacterial activity. Due to the
hydrazone moiety and biaryl scaffold were critical for antibacterial activity, then we explored the impact of C-ring
on antibacterial activity by replacing of C-ring with nitrogen-containing heterocyclic ring. We speculated that the
electron-withdrawing effect of the nitrogen atom will prevent oxidative metabolism and improve the chemical
stability. As illustrated in Table 2, when the C-ring was replaced by pydrazin-3-yl group, compound 14a showed
more potent antibacterial activity than compound 14b, of which, the C-ring was replaced by pyridine-2-yl group. It
was worth noting that compounds 14a-1, 14a-2, 14a-3 and 14a-7 exhibited MIC values of 0.125-0.25 µg/mL
against five tested Gram-positive bacteria, which was 2 to 4-fold higher than radezolid.
Compounds 6a-1 and 14a-1 showed more potent antibacterial activity compared to compounds 6a-5 and 14a-5
respectively, which indicated that substituents on the terminal piperazinyl ring could enhance the antibacterial
activity. Then we conducted further structure-activity relationship studies on the substituent of terminal piperazinyl
ring (6a-5 and 14a-5). Acyl groups were introduced to improve metabolic stability, thus avoiding demethylation.
As illustrated in Table 2, compound 15a-3 with substituent of benzoyl group exhibited reduced antibacterial
activity, while compounds with small substituents showed more potent antibacterial activity compared to
compound 15a-3. Compound 16-2 exhibited 2-fold decrease in antibacterial activity against S.aureus, MRSA and
MSSA compared to 6a-1, which indicated that introduction of large steric hindrance acyl group decreased
antibacterial activity. Compounds 6a and 14a showed significant antibacterial activity and were screened for their
antibacterial activity against clinical isolates of Gram-positive bacteria.
Table 2. In vitro antibacterial activity of biaryloxazolidinones.

Compounds
X/R1
MICs, µg/mL
S.aureusa
MRSAb
MSSAc
LREFd
VREe
NCH3
O
0.125
0.125
0.125
0.5
0.25
0.125
2
0.125
0.125
0.125
0.5
0.25
0.125
1
0.125
0.25
0.25
1
0.25
0.25
0.25
0.5
0.5
0.25
2
0.25
0.25
0.25
0.5
0.5
0.125
2
14a-1
14a-2
14a-3
14a-5
14a-6
14a-7
14b-1
14b-2
14b-4
14b-5
14b-6
15a-1
15a-2
15a-3
16-1
S
NH
0.5
0.125
2
S
O
NCH3
S
1
0.5
2
2
2
2
NH
2
2
2
2
2
1
2
2
2
2
1
1
1
1
1
1
2
1
0.5
0.5
1
1
1
1
1
1
1
2
2
0.5
0.25
1
0.5
0.25
1
0.5
0.25
1
0.5
0.25
＞16
0.5
0.5
0.25
2
16-2
linezolid
radezolid
0.5
0.5
0.5
0.5
a Standard Staphylococcus aureus (29213).
b Methicillin-resistant Staphylococcus aureus.
c Methicillin-sensitive Staphylococcus aureus.
d Linezolid-resistant Enterococcus faecalis.
e Vancomycin-resistant Enterococcus faecium.
3.2. Evaluation of inhibition of clinical isolates by potent compounds
We further evaluated antibacterial activity of our optimal compounds against four clinical isolates of MRSA (4
isolates), MSSA (4 isolates), LREF (4 isolates) and VRE (4 isolates). These four clinical isolates were
antibiotic-susceptible or antibiotic-resistant bacteria, and strain LREF was resistant to linezolid. The results were
summarized in Table 3. Compounds 6a with hydroxyl substituent at C-ring and compound 14a with the C-ring
replaced by pyridine significantly decreased the MIC values compared to linezolid. Notably, compound 6a-3
exhibited 4- to 32-fold greater in vitro inhibition of four clinical isolates than that of linezolid, with MIC values of
0.125–0.25 µg/mL. And compound 6a-3 was particularly potent against linezolid-resistant Enterococcus faecalis
(LREF).
Table 3. In vitro antibacterial activity against clinical isolates.
MICs, µg/mL
Compounds
MRSAa(4)b
MSSAc(4)
LREFd(4)
VREe(4)

0.5-1
0.125-0.25
0.5-1
1-2
0.5-1
0.125-0.25
0.5
1
0.125-0.25
0.5
1
6a-1
6a-3
0.125-0.25
0.5
1-2
0.25-0.5
1
6a-6
1-2
1-2
6a-7
2
2
0.25-0.5
0.25-0.5
0.5
14a-1
14a-2
14a-3
14a-7
16-1
0.25-0.5
0.25-0.5
0.25-0.5
2
0.25-0.5
0.25-0.5
0.25-0.5
2
1
0.25-0.5
2
0.5
2
1-2
1-2
1-2
1-2
1-4
16-2
1-4
1-4
＞16
linezolid
a Methicillin-resistant Staphylococcus aureus.
b Number of bacterial strains tested are given in parentheses.
c Methicillin-sensitive Staphylococcus aureus.
d Vancomycin-resistant Enterococcus faecium.
e Linezolid-resistant Enterococcus faecalis.
3.3. Physicochemical, ADME and drug-likeness properties prediction
Then the predictions of physicochemical, ADME parameters and drug-likeness properties of selected potent
compounds were performed using the free SwissADME web tool (http://www.swissadme.ch/), and the results are
shown in Table 4. No compounds violated Lipinski's rule of five, which indicated that all these compounds are
likely to develop into oral drugs. All compounds showed no BBB permeability and high GI values, which indicated
that these compounds were difficult to cross the blood-brain barrier to cause CNS adverse effects. High GI values
and improved chemical stability suggested that these compounds may show good oral bioavailability. Based on the
potent antibacterial activity and improved physicochemical and drug-likeness properties, further studies on
cytotoxicity and metabolic stability of these compounds were conducted.
Table 4. In silico Molecular properties prediction at SwissADME
.
Log Po/wb
Compounds MWa
Hbond
Hbond
GIe
TPSA (Å2)f Lipinskig logSh
BBB
donorc acceptord
permeanti
453.51 2.66
469.51 2.16
1
2
2
1
1
1
1
3
1
6
7
6
7
7
6
8
7
5
High
High
High
High
High
High
High
High
High
77.48
97.71
0
0
0
0
0
0
0
0
0
-4.08
-3.95
-4.36
-3.62
-3.43
-4.04
-3.42
-3.12
-2.22
No
No
No
No
No
No
No
No
No
OB-104
6a-1
472.53 2.87
454.50 1.86
441.46 1.92
457.52 2.54
489.52 1.73
438.45 2.01
337.35 1.20
119.77
90.37
6a-3
14a-1
96.36
14a-2
112.43
129.65
112.24
71.11
14a-3
14a-7
radezolid
Linezolid
a Molecular weight
b Logarithm of compound partition coefficient between n-octanol and water (< 5).

c Number of hydrogen bond donor (< 5).
d Number of hydrogen bond acceptors (< 10).
e Human gastrointestinal absorption.
f Topological polar surface area (≤ 140).
g Lipinski's rule of five.
h Predicted aqueous solubility.
i Predicted brain/blood partition coefficient.
3.4. Evaluation of acid stability and water solubility of potent compounds
Compound OB-104 showed potent antibacterial activity but poor solubility and moderate metabolic stability,
which were mainly caused by the hydrazone moiety. Therefore, optimal compounds were selected to evaluate for
chemical stability in simulated gastric acid solutions (pH= 1.2) and solubility in water, and the results are shown in
Table 5. These compounds exhibited excellent chemical stability in gastric acid solutions (pH=1.2) except for
compound 6a-1. Moreover, compounds 14a-1, 14a-3 and 14a-7 showed improved water solubility compared with
compound OB-104, which had poor solubility of 11.9 µg/mL. The solubility and chemical stability of these
compounds can be significantly improved by optimization strategy at C-ring, which may due to the introduction of
hindered and hydrophilic substituent or the change of charge distribution.
Table 5. Water solubility and acid stability of selected potent compounds.
Compounds
Solubilitya
% Remaining in simulated gastric acid (pH=1.2) at different times
(µg/mL)
0h
0.5h
1h
1.5h
2h
100
100
100
100
100
100
69.8
96.0
98.3
99.4
99.7
97.5
35.0
94.6
96.8
99.0
99.4
40.7
12.2
82.9
96.2
98.6
99.2
26.3
5.3
-
6a-1
75.9
95.8
98.0
98.7
16.1
-
6a-3
63
57
68
11.9
14a-1
14a-3
14a-7
OB-104
a Tested using UV Spectrophotometer (UV-2600).
3.5. In vitro cytotoxicity and ADME Results of selected compounds
Five of the selected potent compounds were evaluated for in vitro cytotoxicity and ADME properties, and the
results are shown in Table 6. All compounds exhibited low cytotoxicity with IC50 values more than 25 µM against
HepG2 cell, which indicated that these compounds were not toxic to hepatic cells. Furthermore, compound 14a-7
exhibited similar level of metabolic stability with a long half-life time of 409.44 min compared to linezolid, which
indicated that this compound was stable in human microsome. However, compound 14a-3 showed a short half-life
time of 19.24 min, probably because liver microsomal enzymes can oxidize the thioether moiety of compound
14a-3 to form sulfoxide or sulfone. In addition, compound 14a-1 exhibited moderate metabolic stability.
Table 6. In vitro cytotoxicity and ADME Results
Compounds
clogDa
HepG2 cytotoxicity
Human liver microsome (HLM)
Clint Substrate
IC50 (µM)
T1/2 (min)
Stabilityc

(mL/min/kg)
remaining (%)b
1.14
2.01
0.95
1.81
0.11
0.64
＞25
＞25
＞25
＞25
＞25
＞25
-
-
-
-
-
6a-1
6a-3
-
-
-
moderate metabolism
no stable
101.04
19.24
409.44
∞
17.20
90.34
4.25
0
74.88
0.62
92.99
104.18
14a-1
14a-3
14a-7
linezolid
stable
stable
aCalculated using instant JChem.bSubstrate remaining were determined in incubations with NADPH after 45 min.cT1/2 values (T1/2
>
120 min)suggested these compounds were stable in liver microsomes; T1/2 values (T1/2 = 30-120 min) suggested these compounds
showed moderate metabolism in liver microsomes; T1/2 values (T1/2 < 30 min)suggested these compounds were susceptible to
metabolism in liver microsomes.
3.6. Evaluation of inhibition of monoamine oxidases A (MAO-A) by selected compounds
We then evaluated the safety profile of selected potent compounds compared with linezolid, which was an
inhibitor of human MAO-A and may cause undesired side effect. As shown in Table 7, compounds 6a-3, 6a-7,
14a-3 and 14a-7 exhibited lower inhibition rates of human MAO-A compared to linezolid. Compounds 6a-6, 6b-2
and 14a-1 inhibited human MAO-A slightly more strongly than linezolid. Compound 6a-3 exhibited the lowest
inhibition rate of human MAO-A, and was more likely to developed into a drug candidate from a safety
perspective.
Table 7. MAO-A inhibition of potent compounds
Compounds
linezolid
6a-3
6a-6
6a-7
6b-2
14a-1
14a-3
14a-7
MAO inhibition (%) (30 µM)
76
62
88
67
80
79
75
72
3.7. Molecular superposition models of 14a-7 and linezolid
Figure 4. Superposition of compound 14a-7 and linezolid
Field-based molecular superposition of linezolid and compound 14a-7 was performed to predict the action of
14a-7 with ribosome. Obviously the conformations of 14a-7 showed the same field point pattem and perfect
overlay with linezolid, which indicated that compound 14a-7 and linezolid may bound in 50S ribosome unit with
the same pose. Furthermore, the hydrazone bond and terminal heterocycle may play a key role in the enhanced
antibacterial activity.

4. Conclusions
In summary, we described the structural optimization of biaryloxazolidinone analogue OB-104, which was
reported in our previous work. Compound OB-104 was unstable in simulated gastric acid conditions (pH= 1.2) and
human liver microsome. However, the metabolite M-1(6a-1) of OB-104 showed significant antibacterial activity,
which prompted us to conduct structural optimization strategies to improve the stability of hydrazone moiety while
maintaining or enhancing antibacterial activity. Fortunately, two series of biaryloxazolidinone analogues (6a-1,
6a-3, 14a-1, 14a-3 and 14a-7) with hydroxyl substituent at C-ring or the C-ring replaced by pyridine showed
significant antibacterial activity against Gram-positive bacteria and clinical isolates as compared to linezolid.
Further studies indicated that these compounds showed improved water solubility and chemical stability compared
with compound OB-104. Compounds 6a-1, 6a-3, 14a-1, 14a-3 and 14a-7 were not toxic to hepatic cells. The
antibacterial activity of compound 14a-7 against clinical isolates of selected antibiotic-susceptible and
antibiotic-resistant isolates was superior to that of linezolid. Moreover, compound 14a-7 was stable in human
microsome, and exhibited lower inhibition rate of human MAO-A than linezolid. The maintaining antibacterial
activity and all these improved drug-likeness properties and safety profile suggested that our strategies for
structural optimization of biaryloxazolidinone analogue OB-104 were successful. Further pharmacological studies
of these potent compounds are still in progress.
5. Experimental
5.1. Chemistry
All materials were obtained from commercial suppliers and were used without further purification unless stated
otherwise. Reactions' time of the compounds were monitored by TLC (silica gel GF254). Column chromatography
was run on silica gel (200-300 mesh) from Qingdao Ocean Chemicals (Qingdao, Shandong, China). All melting
points were measured with a Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland) and
were uncorrected. Mass spectra (MS) were taken in ESI mode on Agilent 1100 LC-MS (Agilent, Palo Alto, CA,
1
U.S.A.). H-NMR and 13C-NMR spectra were recorded on Bruker ARX-400, 400 MHz or Bruker ARX-600, 600
MHz spectrometers (Bruker Bioscience, Billerica, MA, USA) with TMS as an internal standard.
5.1.1. (4-Bromo-3-fluorophenyl)carbamic acid methyl ester (2)
4-Bromo-3-fluoroaniline (50.0 g, 0.26 mol) and pyridine (20.8 g, 0.26 mol) in DCM (250 mL) were added
dropwise to a mixture of methyl chloroformate (27.2 g, 0.29 mol) in DCM while maintaining the temperature not
more than 5 °C. Then the reaction mixture was heated to room temperature for 2 h. The reaction mixture was
washed with water (150 mL×3) for three times. The organic phase was washed with 3M HCl (200 mL), brine and
concentrated in vacuo to yield 2 as a white solid in 84.6 % yield. MS (ESI) m/z(%): 248.2 [M+Na]+.
5.1.2. N-(((5S)-3-(4-bromo-3-fluorophenyl)-2-oxo-5-oxazolidinyl)methyl)acetamide (3)
To a well-stirred solution of intermediate 2 (14.8 g, 0.06 mol) and lithium tert-butoxide (14.4 g, 0.18 mol) in dry
DMF (86 mL) was added methanol (3.84g, 0.12 mol) dropwise over 15 min while maintaining the temperature not
more than 5 °C under nitrogen and the resulted mixture was stirred at the same temperature for 1.5 h. Then a
well-stirred solution of (S)-1-((acetylamino)methyl)-2-chloroethyl acetate (22.8 g, 0.18 mol) in DMF (80 mL) was
added dropwise to this mixture and the resulted mixture was stirred at 25 ºC for another 20 h. After completion of
the reaction monitored by TLC, the mixture was cooled to 0~5 ºC, then a well-stirred solution of ammonium
chloride (6.41 g) in water (64 mL) was added while maintaining the temperature below 10 ºC. The resulted mixture
was poured into water (800 mL) to give a white precipitate, which was collected by filtration and air-dried to give 3

as a white solid in 82.5 % yield. MS (ESI) m/z(%): 353.4 [M+Na]+.
5.1.3. N-(((5S)-3-(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-oxo-5-oxazolidinyl)methyl)
acetamide (4)
To a well-stirred solution of intermediate 3 (19.8 g, 0.06 mol) and potassium acetate (17.7 g, 0.18 mol) in dry
1,4-dioxane (400 mL) was added bis(pinacolato)diboron (30.5 g, 0.12 mol) and Pd(PPh3)2Cl2 (2.1 g, 0.003 mol),
then the mixture was stirred at 90 ºC for 20 h under nitrogen . The mixture was cooled to room temperature and
filtered through celite. The filtrate was concentrated in vacuo, then 20 mL diethyl ether was added to the residue
and the mixture was stirred at room temperature for 1 h. The white precipitate was collected by filtration and
air-dried to give 4 as a white solid in 75.2 % yield. MS (ESI) m/z(%): 401.3 [M+Na]+.
5.1.4 (S)-N-(3-(2-fluoro-3'-hydroxy-4'-formylbiphenyl-4-yl)-2-oxo-1,3-oxazolidine-5-yl)acetamide (5a)
To a well-stirred solution of intermediate 4 (2.0 g, 5.29 mmol) and sodium carbonate (2.24 g, 21.16 mmol) in
DMF (20 mL) and water (2 mL) was added 4-bromo-2-hydroxybenzaldehyde (1.06 g, 5.29 mmol) and
Pd(PPh3)2Cl2 (0.18 g, 0.26 mmol), then the mixture was stirred at 75 ºC for 10 h under nitrogen. After completion
of the reaction monitored by TLC, water (50 mL) was added and the mixture was extracted with DCM (15 mL×3)
for three times. The organic phase was washed with brine and concentrated in vacuo to yield 5a as a white solid in
1
73 % yield. MS (ESI) m/z(%): 395.0 [M+Na]+. H NMR (400 MHz, DMSO) δ 9.55 (s, 1H), 8.26 (t, J = 5.6 Hz,
1H), 7.62 – 7.50 (m, 2H), 7.40 (d, J = 8.6 Hz, 1H), 7.26 (t, J = 7.8 Hz, 1H), 6.95 (d, J = 8.7 Hz, 2H), 6.79 (d, J =
8.6 Hz, 1H), 4.76 (td, J = 11.1, 5.5 Hz, 1H), 4.16 (t, J = 9.0 Hz, 1H), 3.78 (dd, J = 9.0, 6.5 Hz, 1H), 3.44 (t, J = 5.3
Hz, 2H), 1.85 (s, 3H).
5.1.4.1
(S)-N-(3-(2-fluoro-3'-hydroxy-4'-((4-methylpiperazin-1-yl)iminomethyl)biphenyl-4-yl)-2-oxo-1,3-
oxazolidie-5-ylmethyl)acetamide (6a-1)
To a well-stirred solution of intermediate 5a (2.0 g, 5.29 mmol) in ethanol (10 mL) was added
1-amino-4-methylpiperazine 9a (0.61 g, 5.29 mmol) and a drop of acetic acid, and the mixture was stirred at 78 ºC
for 2 h. The mixture was cooled to room temperature and the resulting solid was collected by filtration and purified
by column chromatography to give the target compounds 6a-1 as a yellow solid in 75% yield. M.p.: 228-230 °C;
MS (ESI), m/z (%): 470.3 [M+H]+. 1H NMR (400 MHz, DMSO) δ 11.44 (s, 1H), 8.27 (t, J = 5.7 Hz, 1H), 7.96 (s,
1H), 7.65 – 7.54 (m, 2H), 7.50 – 7.35 (m, 2H), 7.01 (s, 2H), 4.76 (td, J = 11.3, 5.4 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H),
3.79 (dd, J = 9.0, 6.5 Hz, 1H), 3.44 (t, J = 5.4 Hz, 2H), 3.13 (s, 4H), 2.51 (s, 4H), 2.24 (s, 3H), 1.84 (s, 3H).
5.1.4.2
(S)-N-(3-(2-fluoro-3'-hydroxy-4'-(morpholin-4-yliminomethyl)biphenyl-4-yl)-2-oxo-1,3-oxazolidine-5
-ylmethyl)acetamide (6a-2)
Compound 6a-2 was synthesized from intermediate 5a and 9b in the same method as described for the synthesis
of 6a-1. Yellow solid; yield: 82 %; M.p.: 253-255 °C; MS (ESI), m/z (%): 457.4 [M+H]+. 1H NMR (400 MHz,
DMSO) δ 11.33 (s, 1H), 8.27 (t, J = 5.7 Hz, 1H), 8.01 (s, 1H), 7.64 – 7.56 (m, 2H), 7.49 – 7.39 (m, 2H), 7.11 – 7.02
(m, 2H), 4.76 (td, J = 11.3, 5.4 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.81 – 3.77 (m, 5H), 3.44 (t, J = 5.4 Hz, 2H), 3.18
– 3.10 (m, 4H), 1.85 (s, 3H).
5.1.4.3
(S)-N-(3-(2-fluoro-3'-hydroxy-4'-(thiomorpholin-4-yliminomethyl)biphenyl-4-yl)-2-oxo-1,3-oxazolidine-5
-ylmethyl)acetamide (6a-3)
Compound 6a-3 was synthesized from intermediate 5a and 9c in the same method as described for the synthesis
of 6a-1. Yellow solid; yield: 79 %; M.p.: 235-237 °C; MS (ESI), m/z (%): 473.4 [M+H]+. 1H NMR (400 MHz,

DMSO) δ 11.36 (s, 1H), 8.27 (t, J = 5.7 Hz, 1H), 8.00 (s, 1H), 7.63 – 7.56 (m, 2H), 7.45 (d, J = 8.1 Hz, 1H), 7.41
(dd, J = 8.7, 1.7 Hz, 1H), 7.09 – 7.01 (m, 2H), 4.80 – 4.73 (m, 1H), 4.17 (t, J = 8.9 Hz, 1H), 3.78 (dd, J = 8.9, 6.6
Hz, 1H), 3.52 – 3.48 (m, 4H), 3.44 (t, J = 5.2 Hz, 2H), 2.81 – 2.74 (m, 4H), 1.84 (s, 3H).
5.1.4.4
(S)-N-(3-(2-fluoro-3'-hydroxy-4'-((4-tert-butoxycarbonylpiperazin-1-yl)iminomethyl)biphenyl-4-yl)-2-oxo-1,3-oxaz
olidine-5-ylmethyl)acetamide (6a-4)
Compound 6a-4 was synthesized from intermediate 5a and 9e in the same method as described for the synthesis
of 6a-1. Yellow solid; yield: 85 %.
5.1.4.5
(S)-N-(3-(2-fluoro-3'-hydroxy-4'-(piperazin-1-yliminomethyl)biphenyl-4-yl)-2-oxo-1,3-oxazolidine-5
-ylmethyl)acetamide (6a-5)
Trifluoroacetic acid (5 mL) was added to a well-stirred solution of 6a-4 (0.2 g, 0.36 mmol) in DCM(10 mL)
while maintaining the temperature below 5 °C, then the mixture was stirred at 25 ºC for 5 h. After completion of
the reaction monitored by TLC, the mixture was concentrated in vacuo and then added water (10 mL) to the residue.
Sodium carbonate solution was added dropwise to the mixture until pH was 8. The mixture was extracted with
DCM (15 mL×3) for three times. The organic phase was washed with brine and concentrated in vacuo to yield 6a-5
as a yellow solid in 68 % yield; M.p.: 214-216 °C; MS (ESI), m/z (%): 456.5 [M+H]+. 1H NMR (400 MHz, DMSO)
δ 11.49 (s, 1H), 8.28 (t, J = 5.8 Hz, 1H), 7.96 (s, 1H), 7.63 – 7.56 (m, 2H), 7.45 (d, J = 8.1 Hz, 1H), 7.40 (dd, J =
8.6, 2.0 Hz,1H), 7.06 (d, J = 8.0 Hz, 1H), 7.03 (s, 1H), 4.77 (td, J = 11.4, 5.4 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.79
(dd, J = 9.1, 6.5 Hz, 1H), 3.45 – 3.42 (m, 2H), 3.38 (s, 1H), 3.11 – 3.04 (m, 4H), 2.92 (d, J = 4.5 Hz, 4H), 1.85 (s,
3H).
5.1.4.6
(S)-N-(3-(2-fluoro-3'-hydroxy-4'-((1-oxothiomorpholin-4-yl)iminomethyl)biphenyl-4-yl)-2-oxo-1,3-
oxazolidin-5-ylmethyl)acetamide (6a-6)
3-Chloroperbenzoic acid (0.08 g, 0.46 mmol ) was added to a well-stirred solution of 6a-3 (0.2 g, 0.42 mmol) in
DCM (10 mL) while maintaining the temperature below 5 °C, then the mixture was stirred at 25 ºC for 4 h. After
completion of the reaction monitored by TLC, 10 mL saturated sodium carbonate solution was added to the mixture
and the mixture was extracted with DCM (30 mL×3) for three times. Then the combined organic extract was
washed with brine and concentrated in vacuo. The residue was purified by column chromatography to give the
target compounds 6a-6 as a yellow solid in 71 % yield. M.p.: 205-207 °C; MS (ESI) m/z(%): 511.26 [M+Na]+. 1H
NMR (400 MHz, DMSO) δ 11.22 (s, 1H), 8.27 (t, J = 5.9 Hz, 1H), 8.09 (s, 1H), 7.63 – 7.56 (m, 2H), 7.48 (d, J =
8.1 Hz, 1H), 7.41 (dd, J = 8.6, 2.0 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 7.04 (s, 1H), 4.76 (td, J = 11.3, 5.3 Hz, 1H),
4.17 (t, J = 9.0 Hz, 1H), 3.78 (dd, J = 9.1, 6.4 Hz, 1H), 3.71 – 3.66 (m, 4H), 3.43 (t, J = 5.5 Hz, 2H), 3.04 – 2.96 (m,
2H), 2.80 – 2.77 (m, 2H), 1.84 (s, 3H).
5.1.4.7
(S)-N-(3-(2-fluoro-3'-hydroxy-4'-((1,1-dioxothiomorpholin-4-yl)iminomethyl)biphenyl-4-yl)-2-oxo-1,3
-oxazolidin-5-ylmethyl)acetamide (6a-7)
Compound 6a-7 was synthesized from intermediate 5a and 9f in the same method as described for the synthesis
of 6a-1. Yellow solid; yield: 74 %; M.p.: 223-225 °C; MS (ESI), m/z (%): 505.3 [M+H]+. 1H NMR (400 MHz,
DMSO) δ 11.30 (s, 1H), 8.26 (t, J = 5.7 Hz, 1H), 8.00 (s, 1H), 7.64 – 7.55 (m, 2H), 7.47 (d, J = 8.1 Hz, 1H), 7.41
(dd, J = 8.6, 1.8 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 7.03 (s, 1H), 4.76 (td, J = 11.3, 5.4 Hz, 1H), 4.17 (t, J = 9.0 Hz,
1H), 3.78 (dd, J = 9.1, 6.5 Hz, 1H), 3.53 (s, 4H), 3.44 (t, J = 5.4 Hz, 2H), 3.12 (s, 4H), 1.84 (s, 3H).

5.1.5. (S)-N-(3-(2-fluoro-3'-fluoro-4'-formylbiphenyl-4-yl)-2-oxo-1,3-oxazolidine-5-ylmethyl)acetamide (5b)
Compound 5b was synthesized from intermediate 4 and 2-fluoro-4-bromobenzaldehyde in the same method as
described for the synthesis of 5a. Yellow solid; yield: 71%; MS (ESI) m/z(%): 373.2 [M-H]-.
5.1.5.1.
(S)-N-(3-(2-fluoro-3'-fluoro-4'-((4-methylpiperazin-1-yl)iminomethyl)biphenyl-4-yl)-2-oxo-1,3-
oxazolidin-5-ylmethyl)acetamide (6b-1)
Compound 6b-1 was synthesized from intermediate 5b and 9a in the same method as described for the synthesis
of 6a-1. Yellow solid; yield: 76 %; M.p.: 205-207 °C; MS (ESI), m/z (%): 472.5 [M+H]+. 1H NMR (400 MHz,
DMSO) δ 8.27 (t, J = 5.2 Hz, 1H), 7.85 (t, J = 8.2 Hz, 1H), 7.68 – 7.57 (m, 3H), 7.45 – 7.38 (m, 3H), 4.77 (td, J =
11.0, 5.3 Hz, 1H), 4.17 (t, J = 8.9 Hz, 1H), 3.79 (dd, J = 7.9, 7.1 Hz, 1H), 3.44 (s, 2H), 3.34 (s, 4H), 3.19 (s, 4H),
2.25 (s, 3H), 1.84 (s, 3H).
5.1.5.2
(S)-N-(3-(2-fluoro-3'-fluoro-4'-(morpholin-4-yliminomethyl)biphenyl-4-yl)-2-oxo-1,3-oxazolidine-5-
ylmethyl)acetamide (6b-2)
Compound 6b-2 was synthesized from intermediate 5b and 9b in the same manner as described for the synthesis
of 6a-1. Yellow solid; yield: 75%; M.p.: 287-289 °C; MS (ESI), m/z (%): 459.4[M+H]+. 1H NMR (400 MHz,
DMSO) δ 8.27 (t, J = 5.8 Hz, 1H), 7.86 (t, J = 8.2 Hz, 1H), 7.75 (s, 1H), 7.68 – 7.59 (m, 2H), 7.46 – 7.38 (m, 3H),
4.77 (td, J = 11.3, 5.3 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.82 – 3.74 (m, 5H), 3.44 (t, J = 5.4 Hz, 2H), 3.21 – 3.15
(m, 4H), 1.84 (s, 3H).
5.1.5.3
(S)-N-(3-(2-fluoro-3'-fluoro-4'-(thiomorpholin-4-yliminomethyl)biphenyl-4-yl)-2-oxo-1,3-oxazolidine-5
-ylmethyl)acetamide (6b-3)
Compound 6b-3 was synthesized from intermediate 5b and 9c in the same method as described for the synthesis
of 6a-1. Yellow solid; yield: 82%; M.p.: 220-222 °C; MS (ESI), m/z (%): 475.3 [M+H]+. 1H NMR (400 MHz,
DMSO) δ 8.26 (t, J = 5.8 Hz, 1H), 7.85 (t, J = 8.2 Hz, 1H), 7.72 (s, 1H), 7.67 – 7.59 (m, 2H), 7.44 – 7.39 (m, 3H),
4.77 (td, J = 11.4, 5.3 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.79 (dd, J = 9.1, 6.4 Hz, 1H), 3.59 – 3.54 (m, 4H), 3.44 (t,
J = 5.5 Hz, 2H), 2.76 – 2.73 (m, 4H), 1.84 (s, 3H).
5.1.5.4
(S)-N-(3-(2-fluoro-3'-fluoro-4'-(piperidin-1-yliminomethyl)biphenyl-4-yl)-2-oxo-1,3-oxazolidine-5-
ylmethyl)acetamide (6b-4)
Compound 6b-4 was synthesized from intermediate 5b and 9d in the same method as described for the synthesis
of 6a-1. Yellow solid; yield: 83%; M.p.: 179-182 °C; MS (ESI), m/z (%): 457.5 [M+H]+. 1H NMR (400 MHz,
DMSO) δ 8.27 (t, J = 5.7 Hz, 1H), 7.85 (t, J = 8.2 Hz, 1H), 7.67 – 7.58 (m, 3H), 7.44 – 7.37 (m, 3H), 4.77 (td, J =
11.4, 5.5 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.79 (dd, J = 9.0, 6.5 Hz, 1H), 3.44 (t, J = 5.4 Hz, 2H), 3.22 – 3.16 (m,
4H), 1.84 (s, 3H), 1.71 – 1.63 (m, 4H), 1.52 (d, J = 4.7 Hz, 2H).
5.1.5.5
(S)-N-(3-(2-fluoro-3'-fluoro-4'-((4-tert-butoxycarbonylpiperazin-1-yl)iminomethyl)biphenyl-4-yl)-2-oxo-
1,3-oxazolidine-5-ylmethyl)acetamide (6b-5)
Compound 6b-5 was synthesized from intermediate 5b and 9e in the same method as described for the synthesis
of 6a-1. Yellow solid; yield: 80%.
5.1.5.6
(S)-N-(3-(2-fluoro-3'-fluoro-4'-(piperazin-1-yliminomethyl)biphenyl-4-yl)-2-oxo-1,3-oxazolidine-5-
ylmethyl)acetamide (6b-6)
Compound 6b-6 was synthesized from intermediate 6b-5 in the same method as described for the synthesis of

6a-5. Yellow solid; yield: 75%; M.p.: 187-189 °C; MS (ESI) m/z(%):458.3 [M+H]+. 1H NMR (400 MHz, DMSO) δ
8.27 (t, J = 5.5 Hz, 1H), 8.07 (s, 1H), 7.85 (t, J = 8.1 Hz, 1H), 7.71 – 7.58 (m, 3H), 7.42 (d, J = 8.6 Hz, 3H), 4.77
(td, J = 11.8, 6.0 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.79 (dd, J = 8.2, 7.2 Hz, 1H), 3.46 – 3.42 (m, 2H), 3.14 (s, 4H),
2.91 (s, 4H), 1.84 (s, 3H).
5.1.6 (S)-N-(3-(2-fluoro-3'-methoxy-4'-formylbiphenyl-4-yl)-2-oxo-1,3-oxazolidine-5-ylmethyl)acetamide (5c)
Compound 5c was synthesized from intermediate 4 and 4-bromo-2-methoxybenzaldehyde in the same method as
described for the synthesis of 5a. Yellow solid; yield: 75 %; MS (ESI) m/z(%):387.3 [M+H]+.1H NMR (400 MHz,
DMSO) δ 10.38 (s, 1H), 8.28 (t, J = 5.7 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.72 (t, J = 8.8 Hz, 1H), 7.64 (dd, J =
13.7, 2.0 Hz, 1H), 7.46 (dd, J = 8.6, 2.0 Hz,1H), 7.36 (s, 1H), 7.27 (d, J = 8.0 Hz, 1H), 4.77 (dt, J = 11.3, 5.6 Hz,
1H), 4.19 (t, J = 9.0 Hz, 1H), 3.99 (s, 3H), 3.80 (dd, J = 9.1, 6.5 Hz, 1H), 3.45 (t, J = 5.4 Hz, 2H), 1.84 (s, 3H).
5.1.6.1
(S)-N-(3-(2-fluoro-3'-methoxy-4'-(morpholin-4-yliminomethyl)biphenyl-4-yl)-2-oxo-1,3-oxazolidine-5-
ylmethyl)acetamide (6c-1)
Compound 6c-1 was synthesized from intermediate 5c and 9b in the same method as described for the synthesis
of 6a-1. Yellow solid; yield: 71 %; M.p.: 179-181 °C; MS (ESI), m/z (%): 471.5 [M+H]+. 1H NMR (400 MHz,
DMSO) δ 8.28 (t, J = 5.6 Hz, 1H), 7.87 (s, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.66 – 7.59 (m, 2H), 7.42 (d, J = 8.4 Hz,
1H), 7.17 (s, 1H), 7.13 (d, J = 8.0 Hz, 1H), 4.77 (td, J = 11.4, 5.5 Hz, 1H), 4.18 (t, J = 9.0 Hz, 1H), 3.89 (s, 3H),
3.80 – 3.74 (m, 5H), 3.44 (t, J = 5.3 Hz, 2H), 3.16 – 3.06 (m, 4H), 1.85 (s, 3H).
5.1.6.2
(S)-N-(3-(2-fluoro-3'-methoxy-4'-(thiomorpholin-4-yliminomethyl)biphenyl-4-yl)-2-oxo-1,3-oxazolidine-
5-ylmethyl)acetamide (6c-2)
Compound 6c-2 was synthesized from intermediate 5c and 9c in the same method as described for the synthesis
of 6a-1. Yellow solid; yield: 76 %; M.p.: 207-209 °C; MS (ESI), m/z (%): 487.5 [M+H]+. 1H NMR (400 MHz,
DMSO) 8.28 (t, J = 5.8 Hz, 1H), 7.85 (s, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.67 – 7.57 (m, 2H), 7.41 (dd, J = 8.6, 2.0
Hz, 1H), 7.16 (s, 1H), 7.13 (d, J = 8.0 Hz, 1H), 4.77 (td, J = 11.4, 5.4 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.88 (s, 3H),
3.79 (dd, J = 9.1, 6.5 Hz, 1H), 3.50 – 3.46 (m, 4H), 3.44 (t, J = 5.5 Hz, 2H), 2.78 – 2.72 (m, 4H), 1.85 (s, 3H).
5.1.7 (S)-N-(3-((2-fluoro-3'-methyl-4'-formyl)biphenyl-4-yl)-2-oxo-1,3-oxazolidine-5-ylmethyl)acetamide (5d)
Compound 5d was synthesized from intermediate 4 and 4-bromo-2-methylbenzaldehyde in the same method as
described for the synthesis of 5a. Yellow solid; yield: 84 %; MS (ESI) m/z(%):393.2 [M+H]+.
5.1.7.1
(S)-N-(3-(2-fluoro-3'-methyl-4'-((4-methylpiperazin-1-yl)iminomethyl)biphenyl-4-yl)-2-oxo-1,3-
oxazolidin-5-ylmethyl)acetamide (6d-1)
Compound 6d-1 was synthesized from intermediate 5d and 9a in the same method as described for the synthesis
of 6a-1. Yellow solid; yield: 76 %; M.p.: 171-174 °C; MS (ESI), m/z (%): 468.3 [M+H]+. 1H NMR (400 MHz,
DMSO) δ 8.27 (t, J = 5.7 Hz, 1H), 7.76 (d, J = 7.9 Hz, 2H), 7.63 – 7.54 (m, 2H), 7.40 (dd, J = 8.6, 1.9 Hz, 1H),
7.35 (d, J = 6.0 Hz, 2H), 4.77 (td, J = 11.4, 5.4 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.79 (dd, J = 9.1, 6.5 Hz, 1H),
3.44 (t, J = 5.4 Hz, 2H), 3.23 – 3.12 (m, 4H), 2.57 – 2.51 (m, 4H), 2.44 (s, 3H), 2.25 (s, 3H), 1.85 (s, 3H).
5.1.7.2
(S)-N-(3-(2-fluoro-3'-methyl-4'-(thiomorpholin-4-yliminomethyl)biphenyl-4-yl)-2-oxo-1,3-oxazolidine-5
-ylmethyl)acetamide (6d-2)
Compound 6d-2 was synthesized from intermediate 5d and 9c in the same method as described for the synthesis
of 6a-1. Yellow solid; yield: 78 %; M.p.: 196-199 °C; MS (ESI), m/z (%): 471.5 [M+H]+. 1H NMR (400 MHz,

DMSO) δ 8.27 (t, J = 5.8 Hz, 1H), 7.81 (s, 1H), 7.76 (d, J = 8.7 Hz, 1H), 7.63 – 7.55 (m, 2H), 7.40 (dd, J = 8.6, 2.0
Hz, 1H), 7.38 – 7.34 (m, J = 5.8 Hz, 2H), 4.76 (td, J = 11.3, 5.4 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.79 (dd, J = 9.1,
6.5 Hz, 1H), 3.58 – 3.51 (m, 4H), 3.44 (t, J = 5.4 Hz, 2H), 2.80 – 2.72 (m, 4H), 2.45 (s, 3H), 1.85 (s, 3H).
5.1.7.3
(S)-N-(3-(2-fluoro-3'-methyl-4'-((4-tert-butoxycarbonylpiperazin-1-yl)iminomethyl)biphenyl-4-yl)-2-oxo-
1,3-oxazolidine-5-ylmethyl)acetamide (6d-3)
Compound 6d-3 was synthesized from intermediate 5d and 9e in the same method as described for the synthesis
of 6a-1. Yellow solid; yield: 82 %.
5.1.7.4
(S)-N-(3-(2-fluoro-3'-methyl-4'-(piperazin-1-yliminomethyl)biphenyl-4-yl)-2-oxo-1,3-oxazolidine-5
-ylmethyl)acetamide (6d-4)
Compound 6d-4 was synthesized from intermediate 6d-3 in the same method as described for the synthesis of
6a-5. White solid; yield: 59 %; M.p.: 202-204 °C; MS (ESI), m/z (%): 454.3 [M+H]+. 1H NMR (400 MHz, DMSO)
δ 8.92 (s, 1H), 8.29 (t, J = 5.6 Hz, 1H), 7.88 (s, 1H), 7.77 (d, J = 8.7 Hz, 1H), 7.63 – 7.55 (m, 2H), 7.44 – 7.33 (m,
3H), 4.77 (td, J = 11.3, 5.4 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.80 (dd, J = 9.0, 6.5 Hz, 1H), 3.46 – 3.42 (m, 2H),
3.38 (s, 4H), 3.23 (s, 4H), 2.47 (s, 3H), 1.85 (s, 3H).
5.1.8 (S)-N-(3-((2-fluoro-3'-trifluoromethyl-4'-formyl)biphenyl-4-yl)-2-oxo-1,3-oxazolidine-5-ylmethyl) acetamide
(5e)
Compound 5e was synthesized from intermediate 4 and 4-bromo-2-trifluoromethylbenzaldehyde in the same
method as described for the synthesis of 5a. Yellow solid; yield: 74 %; MS (ESI), m/z (%): 447.2 [M+H]+.
5.1.8.1
(S)-N-(3-(2-fluoro-3'-trifluoromethyl-4'-((4-methylpiperazin-1-yl)iminomethyl)biphenyl-4-yl)-2-oxo-
1,3-oxazolidin-5-ylmethyl)acetamide (6e-1)
Compound 6e-1 was synthesized from intermediate 5e and 9a in the same method as described for the synthesis
of 6a-1. Yellow solid; yield: 81 %; M.p.: 198-200 °C; MS (ESI), m/z (%): 522.5 [M+H]+. 1H NMR (400 MHz,
DMSO) δ 8.28 (s, 1H), 8.12 (d, J = 7.9 Hz, 1H), 7.87 – 7.78 (s, 2H), 7.73 – 7.59 (m, 3H), 7.50 – 7.41 (m, 1H), 4.78
(s, 1H), 4.18 (t, J = 8.7 Hz, 1H), 3.83 – 3.77 (m, 1H), 3.45 (s, 2H), 3.21 (s, 4H), 2.24 (s, 3H), 1.85 (s, 3H).
5.1.8.2
(S)-N-(3-(2-fluoro-3'-trifluoromethyl-4'-(thiomorpholin-4-yliminomethyl)biphenyl-4-yl)-2-oxo-1,3-
oxazolidin-5-ylmethyl)acetamide (6e-2)
Compound 6e-2 was synthesized from intermediate 5e and 9c in the same method as described for the synthesis of
6a-1. Yellow solid; yield: 76 %; M.p.: 211-213 °C; MS (ESI), m/z (%): 547.3 [M+Na]+. 1H NMR (600 MHz,
DMSO) δ 8.27 (t, J = 5.8 Hz, 1H), 8.14 (d, J = 8.8 Hz, 1H), 7.84 (s, 2H), 7.75 (s, 1H), 7.69 (t, J = 8.9 Hz, 1H), 7.64
(dd, J = 13.7, 1.7 Hz, 1H), 7.45 (dd, J = 8.6, 1.7 Hz, 1H), 4.78 (td, J = 11.4, 5.5 Hz, 1H), 4.18 (t, J = 9.0 Hz, 1H),
3.83 – 3.77 (m, 5H), 3.45 (t, J = 5.4 Hz, 2H), 3.23 – 3.18 (m, 4H), 1.85 (s, 3H).
5.1.9 (S)-N-(3-(3-fluoro-4-(6-formylpyridin-3-yl)phenyl)-2-oxo-1,3-oxazolidine-5-ylmethyl)acetamide (13a)
Compound 13a was synthesized from intermediate 4 and 3-bromo-6-pyridinecarboxaldehyde in the same method
as described for the synthesis of 5a. White solid; yield: 85%; MS (ESI), m/z (%): 358.2 [M+H]+. 1H NMR (400
MHz, DMSO) δ 10.04 (s, 1H), 9.03 (s, 1H), 8.26 (dd, J = 13.1, 7.0 Hz, 2H), 8.04 (d, J = 8.1 Hz,1H), 7.78 (t, J = 8.8
Hz, 1H), 7.69 (dd, J = 13.7, 2.0 Hz, 1H), 7.51 (dd, J = 8.6, 2.0 Hz, 1H), 4.79 (td, J = 11.3, 5.3 Hz, 1H), 4.20 (t, J =
9.0 Hz, 1H), 3.82 (dd, J = 9.1, 6.5 Hz, 1H), 3.45 (t, J = 5.4 Hz, 2H), 1.85 (s, 3H).

5.1.9.1
(S)-N-(3-(3-fluoro-4-(6-((4-methylpiperazin-1-yl)iminomethyl)pyridin-3-yl)phenyl)-2-oxo-1,3-oxazolidin
-5-ylmethyl)acetamide (14a-1)
Compound 14a-1 was synthesized from intermediate 13a and 9a in the same method as described for the
synthesis of 6a-1. Yellow solid; yield: 78%; M.p.: 211-213 °C; MS (ESI), m/z (%): 455.5 [M+H]+. 1H NMR (400
MHz, DMSO) δ 8.69 (s, 1H), 8.27 (t, J = 5.6 Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.84 (d, J = 8.3 Hz, 1H), 7.72 –
7.59 (m, 3H), 7.45 (dd, J = 8.5, 1.9 Hz, 1H), 4.78 (td, J = 11.4, 5.6 Hz, 1H), 4.19 (t, J = 9.0 Hz, 1H), 3.80 (dd, J =
9.1, 6.5 Hz, 1H), 3.46 – 3.43 (m, 2H), 3.25 (s, 4H), 2.57 (s, 4H), 2.29 (s, 3H), 1.85 (s, 3H). 13C NMR (101 MHz,
DMSO) δ 170.49, 159.65 (d, J = 245.4 Hz), 154.68, 154.45, 148.79, 140.33 (d, J = 11.4 Hz), 136.55 (d, J = 2.8 Hz),
134.45, 131.12 (d, J = 4.7 Hz), 129.15, 119.92 (d, J = 13.6 Hz), 118.55, 114.59, 106.03 (d, J = 28.3 Hz), 72.28,
54.28 (2C), 50.73 (2C), 47.66, 46.02, 41.86, 22.92.
5.1.9.2
(S)-N-(3-(3-fluoro-4-(6-(morpholin-4-yliminomethyl)pyridin-3-yl)phenyl)-2-oxo-1,3-oxazolidine
-5-ylmethyl)acetamide (14a-2)
Compound 14a-2 was synthesized from intermediate 13a and 9b in the same method as described for the
synthesis of 6a-1. Yellow solid; yield: 77%; M.p.: 207-209 °C; MS (ESI), m/z (%): 442.4 [M+H]+. 1H NMR (400
MHz, DMSO) δ 8.70 (s, 1H), 8.27 (t, J = 5.7 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.73 –
7.61 (m, 3H), 7.45 (dd, J = 8.6, 1.9 Hz, 1H), 4.78 (td, J = 11.3, 5.3 Hz, 1H), 4.18 (t, J = 9.0 Hz, 1H), 3.82 – 3.78 (m,
5H), 3.45 (t, J = 5.4 Hz, 2H), 3.26 – 3.18 (m, 4H), 1.85 (s, 3H).
5.1.9.3
(S)-N-(3-(3-fluoro-4-(6-(thiomorpholin-4-yliminomethyl)pyridin-3-yl)phenyl)-2-oxo-1,3-oxazolidine
-5-ylmethyl)acetamide (14a-3)
Compound 14a-3 was synthesized from intermediate 13a and 9c in the same method as described for the
synthesis of 6a-1. Yellow solid; yield: 74%; M.p.: 219-221 °C; MS (ESI), m/z (%): 458.4 [M+H]+. 1H NMR (400
MHz, DMSO) δ 8.69 (s, 1H), 8.27 (t, J = 5.4 Hz, 1H), 7.93 (d, J = 8.3 Hz, 1H), 7.84 (d, J = 8.3 Hz, 1H), 7.71 –
7.61 (m, 3H), 7.49 – 7.42 (m, 1H), 4.78 (dd, J = 7.8, 5.5 Hz, 1H), 4.18 (t, J = 8.9 Hz, 1H), 3.81 (dd, J = 8.8, 6.6 Hz,
1H), 3.64 – 3.58 (m, 4H), 3.45 (t, J = 5.2 Hz, 2H), 2.81 – 2.70 (m, 4H), 1.86 (s, 3H). 13C NMR (101 MHz, DMSO)
δ 170.51, 159.65 (d, J = 245.2 Hz), 154.45, 153.87, 148.65, 140.44 (d, J = 11.2 Hz), 137.16 (d, J = 2.5 Hz), 133.54,
131.10 (d, J = 4.7 Hz), 129.41, 119.69 (d, J = 13.3 Hz), 119.39, 114.63, 106.04 (d, J = 28.3 Hz), 72.30, 52.97 (2C),
47.66, 41.86, 25.74 (2C), 22.91.
5.1.9.4
(S)-N-(3-(3-fluoro-4-(6-((4-tert-butoxycarbonylpiperazin-1-yl)iminomethyl)pyridin-3-yl)phenyl)-2-oxo-
1,3-oxazolidine -5-ylmethyl)acetamide (14a-4)
Compound 14a-4 was synthesized from intermediate 13a and 9e in the same method as described for the
synthesis of 6a-1. Yellow solid; yield: 88 %.
5.1.9.5
(S)-N-(3-(3-fluoro-4-(6-(piperazin-1-yliminomethyl)pyridin-3-yl)phenyl)-2-oxo-1,3-oxazolidine
-5-ylmethyl)acetamide (14a-5)
Compound 14a-5 was synthesized from intermediate 14a-4 in the same method as described for the synthesis of
6a-5. Yellow solid; yield: 72 %; M.p.: 190-192 °C; MS (ESI), m/z (%): 441.7 [M+H]+. 1H NMR (400 MHz, DMSO)
δ 8.68 (s, 1H), 8.34 (t, J = 5.7 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.70 – 7.60 (m, 3H), 7.45
(dd, J = 8.6, 1.9 Hz, 1H), 4.78 (td, J = 11.4, 5.4 Hz, 1H), 4.18 (t, J = 9.0 Hz, 1H), 3.82 (dd, J = 9.0, 6.5 Hz, 1H),
3.44 (t, J = 5.4 Hz, 2H), 3.21 – 3.14 (m, 4H), 2.95 – 2.85 (m, 4H), 1.85 (s, 3H).

5.1.9.6
(S)-N-(3-(3-fluoro-4-(6-((1-oxothiomorpholin-4-yl)iminomethyl)pyridin-3-yl)phenyl)-2-oxo-1,3-
oxazolidin-5-ylmethyl)acetamide (14a-6)
Compound 14a-6 was synthesized from compound 14a-3 in the same method as described for the synthesis of
1
6a-6. Yellow solid; yield: 79 %; M.p.: 205-207 °C; MS (ESI), m/z (%): 474.2 [M+Na]+. H NMR (400 MHz,
DMSO) δ 8.71 (s, 1H), 8.26 (t, J = 5.7 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.75 (s, 1H),
7.71 – 7.61 (m, 2H), 7.45 (dd, J = 8.6, 1.9 Hz, 1H), 4.78 (td, J = 11.4, 5.5 Hz, 1H), 4.18 (t, J = 9.0 Hz, 1H), 3.85 –
3.74 (m, 5H), 3.44 (t, J = 5.4 Hz, 2H), 3.05 – 2.93 (m, 2H), 2.83 – 2.73 (m, 2H), 1.84 (s, 3H).
5.1.9.7
(S)-N-(3-(3-fluoro-4-(6-((1,1-dioxothiomorpholin-4-yl)iminomethyl)pyridin-3-yl)phenyl)-2-oxo-1,3
-oxazolidine-5-ylmethyl)acetamide (14a-7)
Compound 14a-7 was synthesized from intermediate 13a and 9f in the same method as described for the
synthesis of 6a-1. Yellow solid; yield: 73 %; M.p.: 229-231 °C; MS (ESI), m/z (%): 490.4 [M+H]+. 1H NMR (400
MHz, DMSO) δ 8.74 (s, 1H), 8.26 (t, J = 5.7 Hz, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.79 (s,
1H), 7.72 – 7.62 (m, 2H), 7.46 (dd, J = 8.6, 1.9 Hz, 1H), 4.78 (td, J = 11.4, 5.4 Hz, 1H), 4.19 (t, J = 9.0 Hz, 1H),
3.90 (s, 4H), 3.80 (dd, J = 9.1, 6.5 Hz, 1H), 3.44 (t, J = 5.4 Hz, 2H), 3.22 (s, 4H), 1.84 (s, 3H). 13C NMR (101 MHz,
DMSO) δ 177.19, 170.49, 159.68 (d, J = 245.7 Hz), 154.44, 153.67, 148.74, 140.52 (d, J = 11.1 Hz), 137.64,
136.95, 131.14, 129.88, 119.65, 114.67, 106.10 (d, J = 28.2 Hz), 72.29, 48.93 (2C), 48.76 (2C), 47.71, 41.88,
22.90.
5.1.10 (S)-N-(3-(3-fluoro-4-(5-formylpyridin-2-yl)phenyl)-2-oxo-1,3-oxazolidine-5-ylmethyl)acetamide (13b)
Compound 13b was synthesized from intermediate 4 and 2-bromo-5-pyridinecarboxaldehyde in the same
method as described for the synthesis of 5a. White solid; yield: 82 %; MS (ESI), m/z (%): 358.2 [M+H]+.
5.1.10.1
(S)-N-(3-(3-fluoro-4-(5-((4-methylpiperazin-1-yl)iminomethyl)pyridin-2-yl)phenyl)-2-oxo-1,3
-oxazolidin-5-ylmethyl)acetamide (14b-1)
Compound 14b-1 was synthesized from intermediate 13b and 9a in the same method as described for the
synthesis of 6a-1. Yellow solid; yield: 72%; M.p.: 226-228 °C; MS (ESI), m/z (%): 455.4 [M+H]+. 1H NMR (400
MHz, DMSO) δ 8.85 (d, J = 1.8 Hz, 1H), 8.28 (t, J = 5.8 Hz, 1H), 8.09 – 8.04 (m, 2H), 7.86 (s, 1H), 7.80 (d, J = 7.2 Hz,
1H), 7.63 (dd, J = 14.3, 2.1 Hz, 1H), 7.46 (dd, J = 8.8, 2.1 Hz, 1H), 4.78 (td, J = 11.5, 5.3 Hz, 1H), 4.19 (t, J = 9.0 Hz,
1H), 3.83 – 3.70 (m, 5H), 3.45 (t, J = 5.4 Hz, 2H), 3.04 – 2.94 (m, 2H), 2.78 (d, J = 13.4 Hz, 2H), 1.85 (s, 3H).
5.1.10.2
(S)-N-(3-(3-fluoro-4-(5-(thiomorpholin-4-yliminomethyl)pyridin-2-yl)phenyl)-2-oxo-1,3-oxazolidine-5
-ylmethyl)acetamide (14b-2)
Compound 14b-2 was synthesized from intermediate 13b and 9c in the same method as described for the
synthesis of 6a-1. Yellow solid; yield: 79%; M.p.: 232-234 °C; MS (ESI), m/z (%):458.4 [M+H]+。1H NMR (400
MHz, DMSO) δ 8.82 (d, J = 1.7 Hz, 1H), 8.28 (t, J = 5.8 Hz, 1H), 8.11 – 8.00 (m, 2H), 7.79 (d, J = 7.2 Hz, 1H),
7.74 (s, 1H), 7.62 (dd, J = 14.3, 2.1 Hz, 1H), 7.46 (dd, J = 8.8, 2.1 Hz, 1H), 4.78 (td, J = 11.5, 5.3 Hz, 1H), 4.18 (t,
J = 9.0 Hz, 1H), 3.80 (dd, J = 9.1, 6.5 Hz, 1H), 3.59 – 3.54 (m, 4H), 3.45 (t, J = 5.5 Hz, 2H), 2.77 – 2.73 (m, 4H),
1.85 (s, 3H).
5.1.10.3
(S)-N-(3-(3-fluoro-4-(5-((4-tert-butoxycarbonylpiperazin-1-yl)iminomethyl)pyridin-2-yl)phenyl)-2-oxo-
1,3-oxazolidine -5-ylmethyl)acetamide (14b-3)
Compound 14b-3 was synthesized from intermediate 13b and 9e in the same manner as described for the
synthesis of 6a-1. Yellow solid; yield: 80 %.

5.1.10.4
(S)-N-(3-(3-fluoro-4-(5-(piperazin-1-yliminomethyl)pyridin-2-yl)phenyl)-2-oxo-1,3-oxazolidine-5
-ylmethyl)acetamide (14b-4)
Compound 14b-4 was synthesized from intermediate 14b-3 in the same method as described for the synthesis of
6a-5. Yellow solid; yield: 77 %; M.p.: 250-252 °C; MS (ESI), m/z (%): 441.5 [M+H]+. 1H NMR (400 MHz, DMSO)
δ 8.85 (d, J = 1.7 Hz, 1H), 8.29 (t, J = 5.8 Hz, 1H), 8.10 – 8.04 (m, 2H), 7.86 (s, 1H), 7.81 (d, J = 7.3 Hz, 1H), 7.63
(dd, J = 14.3, 1.9 Hz, 1H), 7.47 (dd, J = 8.8, 2.0 Hz, 1H), 4.79 (td, J = 11.5, 5.4 Hz, 1H), 4.19 (t, J = 9.0 Hz, 1H),
3.86 (s, 4H), 3.81 (dd, J = 9.1, 6.6 Hz,1H), 3.46 (t, J = 5.4 Hz, 2H), 3.22 (s, 4H), 1.86 (s, 3H).
5.1.10.5
(S)-N-(3-(3-fluoro-4-(5-((1-oxothiomorpholin-4-yl)iminomethyl)pyridin-2-yl)phenyl)-2-oxo-1,3-oxazolidin-5-ylmeth
yl)acetamide (14b-5)
Compound 14b-5 was synthesized from compound 14b-2 in the same method as described for the synthesis of
6a-6. Yellow solid; yield: 70 %. M.p.: 199-201 °C; MS (ESI), m/z (%): 474.4 [M+H]+. 1H NMR (400 MHz, DMSO)
δ 8.85 (d, J = 1.8 Hz, 1H), 8.28 (t, J = 5.8 Hz, 1H), 8.09 – 8.04 (m, 2H), 7.86 (s, 1H), 7.80 (d, J = 7.2 Hz, 1H), 7.63
(dd, J = 14.3, 2.1 Hz, 1H), 7.46 (dd, J = 8.8, 2.1 Hz, 1H), 4.78 (td, J = 11.5, 5.3 Hz, 1H), 4.19 (t, J = 9.0 Hz, 1H),
3.84 – 3.69 (m, 5H), 3.45 (t, J = 5.4 Hz, 2H), 3.04 – 2.93 (m, 2H), 2.80 – 3.76 (m, 2H), 1.85 (s, 3H).
5.1.10.6
(S)-N-(3-(3-fluoro-4-(5-((1,1-dioxothiomorpholin-4-yl)iminomethyl)pyridin-2-yl)phenyl)-2-oxo-1,3
-oxazolidine-5-ylmethyl)acetamide (14b-6)
Compound 14b-6 was synthesized from intermediate 13b and 9f in the same method as described for the
synthesis of 6a-1. Yellow solid; yield: 79 %; M.p.: 242-244 °C; MS (ESI), m/z (%): 490.3 [M+H]+. 1H NMR (400
MHz, DMSO) δ 8.85 (s, 1H), 8.28 (t, J = 5.7 Hz, 1H), 8.09 – 8.04 (m, 2H), 7.86 (s, 1H), 7.81 (d, J = 7.6 Hz, 1H),
7.63 (dd, J = 14.2, 1.7 Hz, 1H), 7.47 (dd, J = 8.8, 1.7 Hz, 1H), 4.82 – 4.74 (m, 1H), 4.19 (t, J = 9.0 Hz, 1H), 3.85 (s,
4H), 3.81 (dd, J = 9.1, 6.8 Hz, 1H), 3.45 (t, J = 5.4 Hz, 2H), 3.21 (s, 4H), 1.85 (s, 3H).
5.1.11.1
(S)-N-(3-(3-fluoro-4-(6-((4-methoxyformylpiperazin-1-yl)iminomethyl)pyridin-3-yl)phenyl)-2-oxo-1,3
-oxazolidine-5-ylmethyl)acetamide (15a-1)
Methyl chloroformate (0.043 g, 0.45 mmol) was added dropwise to a well-stirred solution of DIPEA (0.07 g,
0.54 mmol) and compound 14a-5 (0.2 g, 0.45 mmol) in DMF (10 mL), then the mixture was stirred at 25 ºC for 2 h.
After completion of the reaction monitored by TLC, water (15 mL) was added and the resulting solid was collected
by filtration and purified by column chromatography to give the target intermediate 15a-1 as a white solid in 85 %
yield. M.p.: 240-242 °C; MS (ESI), m/z (%): 499.3 [M+H]+. 1H NMR (400 MHz, DMSO) δ 8.70 (s, 1H), 8.28 (s,
1H), 7.95 (d, J = 7.6 Hz, 1H), 7.85 (d, J = 8.2 Hz, 1H), 7.72 – 7.63 (m, 3H), 7.45 (d, J = 8.0 Hz, 1H), 4.82 – 4.74
(m, 1H), 4.18 (t, J = 8.8 Hz, 1H), 3.80 (dd, J = 7.7, 7.1 Hz, 1H), 3.64 (s, 3H), 3.58 (s, 4H), 3.45 (s,2H), 3.24 (s, 4H),
1.85 (s, 3H).
5.1.11.2
(S)-N-(3-(3-fluoro-4-(6-((4-propionylpiperazin-1-yl)iminomethyl)pyridin-3-yl)phenyl)-2-oxo-1,3
-oxazolidine-5-ylmethyl)acetamide (15a-2)
Compound 15a-2 was synthesized from compound 14a-6 and propionyl chloride in the same method as
described for the synthesis of 15a-1. White solid; yield: 82 %; M.p.: 225-228 °C; MS (ESI), m/z (%): 519.4
[M+Na]+. 1H NMR (400 MHz, DMSO) δ 8.70 (s, 1H), 8.28 (s, 1H), 7.95 (d, J = 7.7 Hz, 1H), 7.85 (d, J = 8.2 Hz,
1H), 7.70 – 7.63 (m, 3H), 7.45 (d, J = 8.2 Hz, 1H), 4.78 (s, 1H), 4.18 (t, J = 8.7 Hz, 1H), 3.87 – 3.75 (m, 1H), 3.65
(s, 4H), 3.45 (s, 2H), 3.27 (s, 2H), 3.21 (s, 2H), 2.38 (dd, J = 14.4, 7.1 Hz, 2H), 1.85 (s, 3H), 1.01 (t, J = 7.2 Hz,

3H).
5.1.11.3
(S)-N-(3-(3-fluoro-4-(6-((4-benzoylpiperazin-1-yl)iminomethyl)pyridin-3-yl)phenyl)-2-oxo-1,3
-oxazolidine-5-ylmethyl)acetamide (15a-3)
Compound 15a-3 was synthesized from compound 14a-6 and benzoyl chloride in the same method as described
for the synthesis of 15a-1. White solid; yield: 91 %; M.p.: 201-204 °C; MS (ESI), m/z (%): 545.4 [M+H]+. 1H
NMR (400 MHz, DMSO) δ 8.71 (s, 1H), 8.28 (t, J = 5.6 Hz, 1H), 7.95 (s, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.72 – 7.61
(m, 3H), 7.50 – 7.43 (m, 6H), 4.84 – 4.72 (m, 1H), 4.18 (t, J = 8.9 Hz, 1H), 3.93 – 3.70 (m, 3H), 3.47 (dd, J = 24.8,
19.5 Hz, 4H), 3.34 – 3.16 (m, 4H), 1.84 (s, 3H).
5.1.12.1
(S)-N-(3-(2-fluoro-3'-hydroxy-4'-((4-(methoxyformyl)piperazin-1-yl)iminomethyl)biphenyl-4-yl)-2-oxo
-1,3-oxazolidin-5-ylmethyl)acetamide (16-1)
Compound 16-1 was synthesized from compound 5b-5 and methyl chloroformate in the same method as
described for the synthesis of 15a-1. White solid; yield: 75 %; M.p.: 208-210 °C; MS (ESI), m/z (%): 514.2
[M+H]+. 1H NMR (400 MHz, DMSO) δ8.26 (t, J = 5.5 Hz, 1H), 7.78 (d, J = 8.2 Hz, 1H), 7.67 – 7.59 (m, 3H), 7.51 (d,
J = 8.2 Hz, 1H), 7.43 (d, J = 8.0 Hz, 2H), 4.77 (td, J = 11.4, 5.5 Hz, 1H), 4.17 (t, J = 8.9 Hz, 1H), 3.84 (s, 3H), 3.82 –
3.76 (m, 1H), 3.56 (s, 4H), 3.44 (t, J = 5.2 Hz, 2H), 3.15 (s, 4H), 1.84 (s, 3H).
5.1.12.2
(S)-N-(3-(2-fluoro-3'-hydroxy-4'-(((4-propionyl)piperazin-1-yl)iminomethyl)biphenyl-4-yl)-2-oxo
-1,3-oxazolidin-5-ylmethyl)acetamide (16-2)
Compound 16-2 was synthesized from compound 5b-5 and propionyl chloride in the same method as described
for the synthesis of 15a-1. White solid; yield: 77 %; M.p.: 219-221 °C; MS (ESI), m/z (%): 512.3 [M+H]+.1H NMR
(400 MHz, DMSO) δ 11.29 (s, 1H), 8.27 (t, J = 5.6 Hz,1H), 8.07 –8.01 (m, 1H), 7.66 – 7.38 (m, 5H), 7.11 – 7.02
(m, 1H), 4.80 – 4.73 (m, 1H), 4.17 (dd, J = 9.0, 6.6 Hz, 1H), 3.87 – 3.75 (m, 3H), 3.63 (s, 3H), 3.57 (d, J = 4.3 Hz,
4H), 3.44 (t, J = 5.1 Hz, 2H), 3.15 (d, J = 4.2 Hz, 4H), 1.84 (s, 3H).
5.2. Pharmacology
5.2.1. In vitro antibacterial activity
Isolates were identified on MicroScan Walk Away 96 system and antimicrobial susceptibilities were determined
by the broth liquid microdilution method according to the Clinical Laboratory Standards Institute (CLSI) guidelines
(4). Minimum inhibitory concentration (MIC) was defined as the lowest antimicrobial concentration that inhibited
bacterial growth totally. The conventional antimicrobial agents used here were penicillin, gentamycin, rifampicin,
ciprofloxacin, levofloxacin, moxifloxacin, trimethoprim-sulfamethoxazole, clindamycin, erythromycin, linezolid,
vancomycin, quinupristin-dalfopristin and tetracyclin. MRSA, VRE or LREF strains were identified according to
CLSI guidelines (5). S.aureus ATCC29213 was used as the control.
5.2.2. In vitro inhibition assay for MAO-A
The assay to test the inhibition of compounds against human recombinant MAO-A (Active Motif, 31502)
enzyme was displayed by Shanghai ChemPartner Co., LTD. using MAO-GloTM assay kit (Promega
Corporation,V1402) with modifications of the instruction manual according to preliminary optimizations. Briefly,
dose response curves or indicated concentrations of compounds were made in DMSO. A 200-nL aliquot of the
compound solution was transferred into a 384-well plate (Perkin Elmer, 6007299) by Echo® 550 (the final fraction
of DMSO was 1%). Compound was incubated with 10-uL of recombinant MAO-A solutions at room temperature

for 15 min, followed by adding 10-uL of luciferin derivative substrate to initiate the reaction. Reactions were
incubated for 60 min at room temperature and reporter luciferase detection reagent of 20 µL was added and
incubated with each reaction for 20 min to produce luminescence. Relative light units (RLU) were detected using
EnVision Multilabel Plate Reader. For 100% inhibition control (Min), 1× assay buffer was used instead of MAO-A
enzyme solution. And for no inhibition control (Max), DMSO was used instead of compound DMSO solution. The
inhibition percentage in the presence of the compound was calculated according to the equation, Percent inhibition
= (Max – ignal)/ (Mx – Min)*100%. Fit the data in GrphaPad Prism V5.0 software to obtain IC50 values using
equation, Y=Bottom + (Top – Bottom)/(1 + 10^((LogIC50 – X)*HillSlope), where Y stands for inhibition
percentage and X stands for compound concentration.
5.2.3. Cytotoxicity
The target compounds were screened in HepG2 cells by a standard MTT assay in vitro. HepG2 cells were
cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS). Approximately 4×104 cells,
suspended in MEM medium, were plated onto each well of a 96-well plate and incubated in 5% CO2 at 37 for 24
h. The tested compounds were added to the culture medium at the indicated final concentrations and the cell
cultures were continued for 48h. Fresh MTT was added to each well at a final concentration of 0.5 mg/mL and
incubated with cells at 37 for 4 h. The formazan crystals were dissolved in 150 µL DMSO per each well, and the
absorbency at 492 nm (for the absorbance of MTT formazan) and 630 nm (for the reference wavelength) was
measured with the ELISA reader. All of the compounds were tested three times in each of the cell lines. The results
expressed as IC50 (inhibitory concentration of 50%) were the mean ± SD and were calculated by using the
Ghaphapad prism 6.02.
5.2.4. Microsomal stability
30 µL of 1.5 µM compounds solution containing 0.75 mg/mL microsomes solution were dispensed to the assay
plates designated for different time points (0-, 5-, 15-, 30-, 45-min) on ice. For 0-min, 135 µL of acetonitrile
containing internal standard was added to the wells of 0-min plate and afterwards 15 µL of NADPH stock solution
(6 mM) was added. Then, pre-incubated all other plates at 37 ºC for 5 min. 15 µL of NADPH stock solution (6 mM)
was added to the plates to start the reaction and timing. At 5-min, 15-min, 30-min, and 45-min, 135 µL of
acetonitrile containing internal standard was added to the wells of corresponding plates, respectively, to stop the
reaction. After quenching, the plates at the vibrator (IKA, MTS 2/4) were shaked for 10 min (600 rpm/min) and
then centrifuged at 5594g for 15 min (Thermo Multifuge ×3R). 50 µL of the supernatant from each well was
transferred into a 96-well sample plate containing 50 lL of ultra-pure water (Millipore, ZMQS50F01) for LC/MS
analysis. All incubations were performed in duplicate. In vitro intrinsic clearance (Clint) was calculated from
half-life (T1/2) of the compounds disappearance, which was determined by the slope (k) of log-linear regression
analysis from the concentration versus time profiles, i.e., T1/2 = ln(2)/k.
5.3. Molecular superposition
The Molecular superposition studies were performed with Forge 3.0 (v10.4.2). The crystal structure of the
oxazolidinone antibiotic linezolid bound to the 50S ribosomal subunit (PDB code: 3CPW) was obtained from the
RCSB Protein Data Bank (http://www.rcsb.org/pdb/). Compound 14a-7 was drawn with Chemdraw. Before
molecular superposition, linezolid molecular was obtained from the crystal structure of 3CPW using Discovery
Studio 3.0. We selected linezolid molecules as a training set to build the model while the compound 14a-7 served

as test set. Finally, they were docked into the binding site using Discovery Studio 3.0.
Acknowledge
Supported by Basic Research Projects of Higher Education Institutions in Liaoning Province (2017LZD06), and
LiaoNing Revitalization Talents Program (XLYC1805014).
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Highlights
1, Novel biaryloxazolidinone analogues were designed to improve the metabolic stability.
2, Compound 14a-7 exhibited a MIC value of 0.125 µg/mL against S.aureus.
3, Compound 14a-7 was stable in human liver microsome.
4
, Compound 14a-7 exhibited lower inhibitory activity against human MAO-A compared to linezolid.