Synthesis and antibacterial activity evaluation of novel biaryloxazolidinone analogues containing a hydrazone moiety as promising antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2018.09.004

A series of linezolid analogues containing a hydrazone moiety were designed, synthesized and evaluated for their antibacterial activity. Most compounds exhibited more potent antibacterial activity against S.aureus, MRSA, MSSA, LREF and VRE pathogens as compared with linezolid and radezolid. Compounds 9a, 9c, 9f, 9g, 10m and 10t were more potent against tested clinical isolates of MRSA, MSSA, VRE and LREF as compared to linezolid. Compound 9a exhibited comparable activity with linezolid against human MAO-A for safety evaluation and showed moderate metabolism in human liver microsome. The most promising compound 9a showed remarkable antibacterial activity against S.aureus, MRSA, MSSA, LREF and VRE pathogens with MIC value of 0.0675 mg/mL, respectively, which was 15- to 30-fold more potent than linezolid.

Full text of DOI:10.1016/j.ejmech.2018.09.004

Accepted Manuscript
Synthesis and antibacterial activity evaluation of novel biaryloxazolidinone analogues
containing a hydrazone moiety as promising antibacterial agents
Yachuang Wu, Xiudong Ding, Liang Ding, Yongsheng Zhang, Lei Cui, Lu Sun, Wei Li,
Di Wang, Yanfang Zhao
PII:
S0223-5234(18)30778-5
10.1016/j.ejmech.2018.09.004
EJMECH 10715
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 November 2017
Revised Date: 14 July 2018
Accepted Date: 3 September 2018
Please cite this article as: Y. Wu, X. Ding, L. Ding, Y. Zhang, L. Cui, L. Sun, W. Li, D. Wang, Y. Zhao,
Synthesis and antibacterial activity evaluation of novel biaryloxazolidinone analogues containing a
hydrazone moiety as promising antibacterial agents, European Journal of Medicinal Chemistry (2018),
doi: 10.1016/j.ejmech.2018.09.004.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and antibacterial activity evaluation of novel biaryloxazolidinone analogues
containing a hydrazone moiety as promising antibacterial agents
Yachuang Wu a,1, Xiudong Ding b,1, Liang Ding a, Yongsheng Zhang a, Lei Cui b, Lu Sun a, Wei Li a, Di Wang b*
,
Yanfang Zhao a**1
aKey Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry
of Education, 103 Wenhua Road, Shenhe District, Shenyang 110016, PR China
bDepartment of Clinical Laboratory, The 309th Hospital of Chinese People’s Liberation Army, Beijing 100091,
China
Abstract:
A series of linezolid analogues containing a hydrazone moiety were designed, synthesized and evaluated for
their antibacterial activity. Most compounds exhibited more potent antibacterial activity against S.aureus, MRSA,
MSSA, LREF and VRE pathogens as compared with linezolid and radezolid. Compounds 9a, 9c, 9f, 9g, 10m and
10t were more potent against tested clinical isolates of MRSA, MSSA, VRE and LREF as compared to linezolid.
Compound 9a exhibited comparable activity with linezolid against human MAO-A for safety evaluation and
showed moderate metabolism in human liver microsome. The most promising compound 9a showed remarkable
antibacterial activity against S.aureus, MRSA, MSSA, LREF and VRE pathogens with MIC value of 0.0675
mg/mL, respectively, which was 15- to 30-fold more potent than linezolid.
Keywords: biaryloxazolidinone; hydrazone moiety; antibacterial activity; Gram-positive organisms
1. Introduction
The worldwide spread of antibiotic-resistant infections is a critical problem which threaten global healthcare
[1-3]. The pandemic antibiotic-resistant infections caused by resistant Gram-positive bacteria such as
methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE),
vancomycin-resistant Enterococci (VRE), and penicillin-resistant Streptococcus pneumoniae (PRSP) are the main
concern [2].The rising trend of bacterial resistance stimulates significant efforts to discover new antibacterial
drugs [4].
Linezolid 1 (Zyvox; Fig.1), the first clinically useful oxazolidinone antibacterial agent, is available for the
treatment of Gram-positive infections caused by bacteria such as MRSA, PRSP, and VRE [5-6]. It binds to the
sites on the 50S ribosomal subunit to inhibit bacterial translation at the initiation phase of protein synthesis, thus
inhibits bacterial protein biosynthesis. However，linezolid-resistant bacteria such as Staphylococcus aureus,
Enterococcus faecium and other bacteria have already been observed [7-9]. Therefore, many research groups have
made significant efforts on oxazolidinones to improve the potency, reduce the toxicity and expand the antibacterial
spectrum [10].
Radezolid 3 (RX-1741, Fig.1) belongs to a new family of orally bioavailable biaryloxazolidinones that have
activity against both linezolid-susceptible and linezolid-resistant Gram-positive bacteria and Gram-negative
bacteria, such as Haemophilus influenza and Moraxella [11-12]. A p-stacking interaction may occur between the
biaryl scaffold of radezolid and the ribosome, which could enhance the binding to the bacterial ribosome.
Rifampin 4 (RMP, Fig.1) and other rifamycins [13] display activity against Mycobacteria as well as
Gram-positive bacteria, and the hydrazone moiety of these compounds plays an important role on their activity. In
** Corresponding author.
* Corresponding author.
E-mail address: yanfangzhao@126.com (Y. Zhao), wangdi_42169@163.com (D. Wang).
1 Yachuang Wu and Xiudong Ding contributed equally to this work.

addition, nicotinoyl hydrazone derivaAtivCesC5E[P14T]EanDd MquiAnoNlinUeSdCeriRvaItPivTes 6 and 7 [15-16], which contain the
hydrazone moiety, show excellent antibacterial activity. In the present work, we focused on the synthesis of a
series of biaryloxazolidinone analogues bearing a hydrazone moiety, as depicted in Figure 2.
Figure 1. Chemical structures of linezolid and other oxazolidinones.
O
F
O
N
H
N
NH
N
O
F
O
N
N
O
Radeolid (3)
N
H
H
N
X
N N
hybrid
O
OH
9a-9h, X=NCH3, O, S, CH2, N-BOC, NH, S=O,S(O2)
10a-10u, X=R1CON
OH
O
NH
O
OH OH
11a-11d, X=R2N
O
target compounds
O
N
N
O
OH
N
O
O
Rifampin (4)
Figure 2. Design strategies for the novel oxazolidinone analogues containing a hydrazone moiety
In the current study, a series of biaryloxazolidinone analogues containing a hydrazone moiety were designed
and synthesized. All biaryloxazolidinone analogues were assayed for their antibacterial activity in vitro against
five Gram-positive pathogens: S.aureus ATCC29213, MRSA, MSSA, LREF and VRE. On the basis of the
antibacterial activity results against Gram-positive pathogens, several selected compounds were evaluated for
their activities against four clinical isolates (MRSA, MSSA, VRE and LREF) in vitro and monoamine oxidase
inhibition (MAOI).
2. Chemistry
The synthesis of key intermediate 5 is depicted in Scheme 1. The methyl(3-fluorophenyl)carbamate 2 was

ACCEPTED MANUSCRIPT
prepared by acylation of commercially available material 3-fluorobenzenamine with methyl chloroformate in the
presence of pyridine with a high yield. Then, intermediate 2 was iodinated with KI-NCS-HI(45 %) complex in
water to yield 3, which was subjected to (S)-1-[(acetylamino)methyl]-2-chloroethyl acetate in the presence of
t-BuOLi to give the phenyl oxazolidinone 4 with an one-step process[18]. Intermediate 5 was prepared by suzuki
coupling of intermediate 4 with (4-formylphenyl)boronic acid.
The general synthesis of intermediates 8a-8e and 8h are depicted in Scheme 2. The N-amino product 8a-8e and
8h were prepared by nitrosation of commercial six-membered heterocycles with NaNO2, then reduction with zinc
dust in acetic acid and water. Intermediate 6h was synthesized through a three-step routes starting from
thiomorpholine, as depicted in Scheme 3. Thiomorpholine was protected with tert-butyldicarbonate (Boc2O) to
afford intermediate 10, which was oxidized by 40 % H2O2 solution to give intermediate 11. Then, 6h was prepared
from intermediate 11 by deprotection with TFA. The target compounds 9a-9e and 9h were generated via the
condensation of 5 with intermediates 8a-8e and 8h in ethanol at 78 °C with catalytic amounts of acetic acid, as
depicted in Scheme 4. Deprotection of 9e with TFA in DCM proceeded smoothly to give 9f as a white solid.
Oxidation of 9c with 3-chloroperbenzoic acid as oxidation agent yielded compound 9g. Compound 9f was reacted
with different acyl chloride in the presence of N-ethyldiisopropylamine (DIPEA) as a base, or 9f was reacted with
different acid in the presence of HATU and triethylamine at room temperature to prepare 10a-10u. Compound 11a
was prepared by acylation of 10f with methanesulfonyl chloride. Compounds 11b-11d were prepared through
nucleophilic substitution of various halogenated hydrocarbons with 9f in the presence of DIPEA in DMF.
Scheme 1. Reagents and conditions: (a) methyl chloroformate, pyridine, DCM, 25 °C, 2 h; (b) KI, NCS, HI (45%), H2O, 30 °C, 3 h;
(c) (S)-1-[(acetylamino)methyl]-2-chloroethyl acetate, t-BuOLi CH3OH, rt, 24 h; (d) (4-formylphenyl)boronic acid, Pd(PPh3)2Cl2,
K2CO3, Tol/ EtOH/ H2O, 60 °C, 12h.
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, 5h; (b) H2O2(40 %), AcOH, 50 °C, 4h; (c) TFA, DCM, 25 °C,
5h.

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Scheme 4. Reagents and conditions: (a) 8a-8e and 8h, EtOH, 78 °C, 2h; (b) TFA, DCM, 25 °C, 5h; (c) m-CPBA, DCM, 25 °C, 4h;
(d) DIPEA, DMF, 25 °C, 2h; (e) HATU, TEA, DMF, 25 °C, 8h; (f) DIPEA, DMF, 25 °C, 2h; (g) DIPEA, DMF, 25 °C, 8h.
3. Results and discussion
3.1. Evaluation of in vitro antibacterial activity
All the new synthesized oxazolidinone analogues were evaluated for in vitro antibacterial activity using
linezolid and radezolid as reference drugs. The compounds were evaluated against Gram-positive (S.aureus
ATCC29213) pathogens and selected antibiotic-susceptible and antibiotic-resistant isolates (MRSA, MSSA,
LREF and VRE). The results expressed as minimum inhibitory concentrations (MICs, ug/mL) were summarized
in Table 1 and 2.
As shown in Table 1, it is observed that 9a-9h showed significant antibacterial activity against Gram-positive
organisms and selected antibiotic-susceptible and antibiotic-resistant isolates at primary screening, and the activity
ranges from 2 to 0.0675 ug/mL. Compounds 9a, 9c and 9g showed 15- to 30-fold more potent antibacterial
activity than linezolid, which were the most active compounds having a MIC value of 0.0675 µg/mL. Compound
9e showed in vitro activities slightly weaker than linezolid. However, when deprotection of 9e with TFA,
compound 9f showed 8- to 30-fold more potent antibacteral activity than 9e. Compound 9c with thiomorpholinyl
group and 9g with 1-oxo thiomorpholinyl group exhibited similar levels of in vitro antibacterial activity, while
compound 9h with 1,1-dioxothiomorpholinyl group, showed nearly 4-fold weaker in vitro antibacterial activity
compared to 9c. In general, compounds bearing six-membered heterocyclic hydrazine moiety at the 4'-position of
the biaryl ring showed superior activity against all tested bacteria. However, the MIC values of compounds 9a-9h
indicated that atoms and substitutents on the terminal six-membered heterocycles had great effect on the
antibacterial activity. Thus, various substituents were introduced to the 4-position of the piperazinyl group of 9f
and the resulted compounds were evaluated for their antibacterial activity. The results were summarized in Table

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2.
Table 1. In vitro antibacterial activity of synthesized oxazolidinone analogues.
Compound
MICs, ug/mL
MSSAc
X
S.aureusa
0.0675
0.5
MRSAb
0.0675
0.25
LREFd
0.0675
0.25
VREe
0.0675
0.5
NCH3
O
0.0675
0.25
9a
9b
S
0.0675
0.25
0.0675
0.25
0.0675
0.25
0.0675
0.25
0.0675
0.5
9c
CH2
N-Boc
NH
9d
＞
2
＞
2
＞
2
＞
2
＞2
9e
0.0675
0.0675
0.25
1
0.0675
0.0675
0.25
1
0.0675
0.0675
0.25
1
0. 25
0.0675
0.25
0. 25
0.0675
0.25
2
9f
S=O
S(O2)
9g
9h
＞
16
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.
The oxazolidinone analogues 10a-10u and 11a bearing an acyl side chain at the terminal nitrogen-atom of
piperazine ring exhibited moderate to excellent in vitro antibacterial activity against all tested bacteria. However,
acetylated analogue 10a had 2-fold to 4-fold decrease in antibacterial activity compared to 9f, while analogues
10b-10d with an aroyl group showed similar antibacterial activity compared to 10a. Analogues 10e, 10f and 10h
with 3-pyridyl, 2-pyridyl and 2-thienoyl group respectively showed more potent antibacterial activity against
LREF and VRE than 9f, but their antibacterial activity against S.aureus, MRSA, MSSA were weaker than 9f.
Variations in substitutions of the benzoyl group of compound 10b were investigated to study the antibacterial
activity, and there seemed to have a tendency that analogue with 4-methoxy group were more active than those
with 2-methoxy or 3-methoxy group. Furthermore, when trifluoromethyl and halogen atoms were introduced to
the 4-position of benzoyl group, the resulted analogues 10l-10m and 10o showed similar activity to analogue 10i
with 4-methoxy group. Analogue 10n with 4-chlorobenzoyl group exhibited MIC value (0.0675 ug/mL) against
staphylococcal, but it had 2-fold decrease in antibacterial activity against LREF and VRE compared to 10i.
Analogues 10p-10r with propionyl, methoxycarbonyl and trifluoromethyl groups respectively showed superior
antibacterial activity to 10a. Analogues 10t and 10u were initially designed to improve aqueous solubility by
introduction of water-soluble groups, of which, 10t with hydroxyacetyl group exhibited similar level of in vitro
antibacterial activity (0.0675 ug/mL) compared to 9a. However, the analogue 10u with aminoacetyl group had
2-fold to 7.4-fold decrease in antibacterial activity compared to 10t. Introduction of methanesulfonyl group
resulted in a dramatic decrease in activity against all tested bacteria. Analogue 11b bearing a benzyl side chain at
the terminal N-atom of piperazinyl group of 9f exhibited better antibacterial activity than 10b, while analogues
11c and 11d with the allyl and propargyl group respectively showed similar antibacterial activity with 11b. These
results implied that the carbonyl group may be not essential for the activity.
Overall, most compounds exhibited

moderate-to-significant antibacterial aActiCviCtyEagPaTinEstDGrMamA-pNosUitiSveCoRrgIaPniTsms and selected antibiotic-susceptible
and antibiotic-resistant isolates as compared with linezolid and radezolid. Then, 9a, 9c, 9f, 9g, 10m and 10t were
selected for further clinical isolates investigation.
Table 2. In vitro antibacterial activity of oxazolidinones.
Compound
R1/R2
MICs, µg/mL
S.aureusa
MRSAb
MSSAc
LREFd
VREe
CH3
0.25
0.25
0.125
0.125
0.125
0.125
0.125
0.125
0.25
0.25
0.5
10a
10b
10c
Ph
0.25
0.25
0.25
0.25
0.25
0.25
0.125
0.125
0.5
2-furoyl
0.25
0.25
0.25
4-pyridyl
0.25
0.25
0.25
10d
10e
3-pyridyl
0.125
0.125
0.125
0.125
0.125
0.5
0.125
0.125
0.25
0.125
0.125
0.5
2-pyridyl
10f
2-thienyl
10g
10h
10i
2-pyrazine
4-methoxybenzoyl
2-methoxybenzoyl
3-methoxybenzoyl
4-trifluoromethyl
4-fluorobenzoyl
4-chlorobenzoyl
4-bromobenzoyl
CH3CH2
0.125
0.125
0.125
0.25
0.125
0.125
0.0675
0.25
0.125
0.125
0.125
1
0.125
0.125
0.125
1
10j
0.25
10k
10l
0.125
0.125
0.0675
0.125
0.125
0.125
0.125
0.0675
0.0675
0.125
1
0.125
0.125
0.0675
0.125
0.125
0.125
0.125
0.0675
0.0675
0.25
0.125
0.125
0.0675
0.125
0.125
0.125
0.125
0.0675
0.0675
0.25
0.125
0.125
0.25
0.125
0.125
0.25
0.125
0.125
0.125
0.125
0.25
0.0675
0.5
10m
10n
10o
10p
10q
10r
0.125
0.125
0.125
0.125
0.25
CH3O
CF3
N,N-dimethyl
OHCH2
10s
0.0675
0.5
10t
NH2CH2
10u
11a
methanesulfonyl
benzyl
0.5
1
0.5
2
0.125
0.5
0.125
0.125
0.125
1
0.125
0.125
0.125
1
0.125
0.125
0.125
0.125
0.125
0.125
2
11b
11c
allyl
propargyl
0.125
1
11d
linezolid
radezolid
＞
16
0.5
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.

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3.2. Evaluation of inhibition of clinical isolates by selected compounds
The selected compounds 9a, 9c, 9f, 9g, 10m and 10t were evaluated against several antibiotic-susceptible and
antibiotic-resistant clinical isolates, and the results were summarized in Table 3. Clinical strains of gram-positive
bacteria included S.aureus (75 isolates of MRSA and 77 isolates of MSSA), E.faecium (21 isolates of VRE) and E.
faecalis (4 isolates of LREF), and all the isolates were collected in PLA 309 hospital. All compounds were more
potent against tested Gram-positive clinical isolates of MRSA, MSSA, VRE and LREF as compared to linezolid.
Significantly, these compounds having MIC values of 0.0625-0.25 µg/mL against LREF, meanwhile the MIC
value of linezolid was higher than 16 µg/mL. 9a was the most active compound having MIC50 and MIC90 values
of 0.125 µg/mL against LREF.
Table 3. In vitro antibacterial activity against clinical isolates
MICs, µg/mL
Isolatesa
Compound
MIC range
MIC50
MIC90
0.0625-0.25
0.0625
0.125
MRSAb (75)
9a
0.0625-0.25
0.0625-0.25
0.0625-0.25
0.0625-0.25
0.0625-0.125
1-4
0.125
0.125
0.125
0.125
0.0625
2
0.25
0.25
0.25
0.25
0.125
4
9c
9f
9g
10m
10t
linezolid
MSSAc (77)
VREd(21)
LREFe(4)
9a
0.0625-0.25
0.0625-0.25
0.0625-0.25
0.0625-0.25
0.0625-0.25
0.0625-0.125
1-2
0.0625
0.125
0.125
0.125
0. 25
0.0625
2
0.125
0.25
0.25
0.25
0.25
0.125
2
9c
9f
9g
10m
10t
linezolid
0.0625-0.5
0.0625-0.5
0.0625-0.5
0.0625-0.5
0.0625-0.5
0.0625-0.5
1-4
0.25
0.15
0.25
0.125
0.25
0.125
2
0.5
0.5
0.5
0.5
0.5
0.25
4
9a
9c
9f
9g
10m
10t
linezolid
0.0625-0.25
0.0625-0.25
0.0625-0.25
0.0625-0.25
0.0625-0.25
0.0625-0.125
＞16
0.125
0.125
0. 25
0.125
0. 25
0.125
＞16
0.125
0.5
9a
9c
0.25
0.5
9f
9g
0.25
0.25
＞16
10m
10t
linezolid
a Number of bacterial strains tested are given in parentheses.

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b Methicillin-resistant Staphylococcus aureus.
c Methicillin-sensitive Staphylococcus aureus.
d Vancomycin-resistant Enterococcus faecium.
e Linezolid-resistant Enterococcus faecalis.
3.3. Evaluation of inhibition of monoamine oxidases A (MAO-A) by selected compounds
Linezolid and other oxazolidinone antibacterial agents are reversible, nonselective inhibitors of human MAO
due to their structural similarity with toloxatone, a known MAO inhibitor [19]. Clinical use of the oxazolidinone
antibacterial agents has shown that these agents had a side effect of hypertension because of MAOI. Therefore, six
compounds with potent in vitro activity were evaluated for MAO-A inhibitory activities and the results were
summarized in Table 4. The experimental data showed that these compounds exhibited comparable activity with
linezolid against human MAO-A. Of them, compound 9a may be a better drug candidate from a safety viewpoint.
Table 4. MAO-A inhibition of potent compounds
linezolid
compound
9a
9c
9f
9g
10m
10t
MAO inhibition (%) (30 µM)
74
71
81
84
88
89
80
3.4. In Vitro cytotoxicity and ADME Results of selected compounds
Based on the in vitro antibacterial activity and inhibition of MAO-A, 9a and 10t were selected for in vitro
cytotoxicity and metabolic stability, and the results were outlined in Table 5. Compound 9a and 10t exhibited
IC50 values (0.82 and 4.66 µM, respectively) against HepG2 cell, while linezolid showed IC50 value (＞25 µM),
which indicated they were toxic to hepatic cells to some extent. The human liver microsome studies revealed that
10t possessed a long half-life (663.07 min) and low liver microsomal clearance(2.62 mL/min/kg) in human liver
microsome, which indicated that 10t was stable in human liver microsome. However, 9a possessed low half-life
(72.85 min) and exhibited high Clint (23.86 mL/min/kg) and it demonstrated that 9a showed moderate metabolism
in human liver microsome.
Table 5. In Vitro cytotoxicity and ADME Results
clogDa
HepG2 cytotoxicity
Human liver microsome (HLM)
compd
IC50 (µM)
T1/2 (min)
Clint (mL/min/kg)
Substrate
remaining (%)b
64.74
Stability
9a
1.46
0.82
4.66
72.85
23.86
moderate
metabolism
stable
10t
0.25
0.64
663.07
2.62
0
93.92
＞
25
linezolid
∞
104.18
stable
aCalculated using instant JChem. bSubstrate remaining were determined in incubations with NADPH after 45 min.
3.5. Molecular superposition models of 9a and linezolid
In order to better understand the potency of the synthesized compounds, molecular superposition of 9a and
linezolid were performed based on the crystal structure of 50S ribosome unit of E. coli with linezolid (PDB:

3CPW[21]). Obviously 9a and linezoAlidCbCouEndPTinEthDe sMamAeNreUgioSnCwRitIhPaTperfect binding pose to 50S ribosome
unit (Fig. 3), and their conformation showed very good overlay. Furthermore, the N-methylpiperazinyl part of D
ring of compound 9a extended beyond the morpholine ring of linezolid. Since oxazolidinones and many other
antibiotics share an overlapping region within the binding site of RNA bases at the C-ring and D-ring portion
[11-12, 20], we speculated that the N-methylpiperazinyl part may form extra hydrogen bonds and other
interactions with the binding site of other antibiotics.
Figure 3. Superposition of compound 9a and linezolid
4. Conclusions
In summary, a series of biaryloxazolidinone analogues containing a hydrazone moiety were designed,
synthesized and evaluated for their antibacterial activity. Most compounds exhibited moderate-to-significant
antibacterial activity against Gram-positive organisms and selected antibiotic-susceptible and antibiotic-resistant
isolates as compared with linezolid and radezolid. Among them, compounds 9a, 9c, 9g and 10t were the most
active compounds having a MIC value of 0.0675 µg/mL against all tested bacteria. The selected compounds 9a,
9c, 9f, 9g, 10m and 10t were evaluated against several antibiotic-susceptible and antibiotic-resistant clinical
isolates of MRSA, MSSA, VRE and LREF, and they were more potent against tested clinical isolates as compared
to linezolid. Moreover, the potency of 9a against LREF was 15- to 30-fold higher than that of linezolid.
Compounds 9a, 9c, 9f, 9g, 10m and 10t were further examined for inhibition of MAO-A, and 9a exhibited
comparable inhibitory activity with linezolid against human MAO-A. However, compound 9a was toxic to hepatic
cells to some extent with an IC50 value of 0.82 µM against HepG2 cell and showed moderate metabolism in
human liver microsome, which led to further studies on structural optimization and pharmacological profile about
these biaryloxazolidinone analogues, and the results will be reported in future.
5. Experimental
5.1. Chemistry
Unless otherwise specified, all melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi
Labortechnik, Flawil, Switzerland) and were uncorrected. Mass spectra (MS) were taken in ESI mode on Agilent
1
1100 LC-MS (Agilent, Palo Alto, CA, 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. All materials were obtained from commercial suppliers and were used without
further purification. Reactions' time and purity of the products were monitored by TLC on FLUKA silica gel
aluminum cards (0.2 mm thickness) with fluorescent indicator 254 nm. Column chromatography was run on silica
gel (200-300 mesh) from Qingdao Ocean Chemicals (Qingdao, Shandong, China).

ACCEPTED MANUSCRIPT
5.1.1. methyl(3-fluorophenyl)carbamate (2)
Methyl chloroformate (41.4 g, 0.44 mol)) in CH2Cl2 (50 mL) was added dropwise to a well-stirred solution of
3-fluorobenzenamine (44.4 g, 0.40 mol) and pyridine (31.6 g, 0.40 mol) in CH2Cl2 (266 mL) while maintaining
the temperature between -5~0 °C. After the addition was completed, the mixture was heated to 25 ºC. The reaction
was monitored by TLC. After completion of reaction, the mixture was washed three times with water (150 mL×3).
The organic layer was washed with 1M HCl (150 mL), brine and then dried with anhydrous Na2SO4. The mixture
was filtered, and the filtrate was concentrated in vacuo to yield 2 as a white solid in 96.7% yield. MS (ESI)
m/z(%): 170.1 [M+H]+.
5.1.2. methyl N-(3-iodo-4-fluorophenyl)carbamate (3)
To a well-stirred solution of potassium iodide (54.8 g, 0.33 mol), N-chlorosuccinimide (60.0 g, 0.45 mol) and
45% hydroiodic acid (34.1 mL, 0.18 mol) in water (350 mL) at 25 ºC, intermediate 2 (50.7 g, 0.30 mol) in CH2Cl2
(200 mL) was added dropwise over 30 min. The resulted mixture was heated to 30 ºC until completion of the
reaction by TLC. Then the mixture was cooled to room temperature, quenched by adding a solution of Sodium
sulfite (58 g) in water (300 mL) over 30 mins. The layers were separated，and the aqueous layer was extracted
with CH2Cl2 (300 mL). The combined organic layer was washed with brine (100 mL)，then dried with anhydrous
Na2SO4 and concentrated. n-Heptane (150 mL) was added to the residue, and the mixture was stirred for 30 min at
0 ºC to give a white solid. The precipitate was collected by filtration, washed with n-heptane and dried to give 3 as
a white solid in 87.1% yield. MS (ESI) m/z(%): 295.9 [M+H]+.
5.1.3 N-[3-(3-fluoro-4-iodophenyl)-2-oxo-1,3-oxazolidin-5-(S)-ylmethyl]acetamide (4)
A solution of intermediate 2 (59.0 g, 0.20 mol) and potassium tert-butoxide (51.3 g, 0.64 mol) in dry DMF (240
mL) under an nitrogen atmosphere was cooled in an ice bath and treated with dry methanol (13.4 g, 0.42 mol)
dropwise over 15 mins while maintaining the temperature between 0~5 ºC, followed by stirring for 1.5 h at the
same temperature. Then (S)-1-[(acetylamino)methyl]-2-chloroethyl acetate (81.1g, 0.42mol) in DMF (170 mL)
was added dropwise, and the resulted mixture was heated to 25 ºC for another 24 h. The mixture was cooled to
0~5 ºC, then ammonium chloride (21.4 g) in water (200 mL) was added while maintaining the temperature below
10 ºC. The reaction mixture was poured into water (800 mL) to give a white precipitate, which was collected by
filtration and air-dried to give 4 as a white solid in 79.5 % yield. MS (ESI) m/z(%): 378.9 [M+H]+.
5.1.4 N-[3-(2-fluoro-4'-formylbiphenyl-4-yl)-2-oxo-1,3-oxazolidin-5-(S)-ylmethyl]acetamide (5)
Pd(PPh3)2Cl2 (2.5 g, 0.0036 mol) was added to a solution of intermediate 4 (45 g, 0.12 mol),
4-formylphenylboronic acid (28.8 g, 0.13 mmol), and sodium potassium carbonate (49.7 g, 0.36 mol) in 80 mL of
mixed solution (3:1:1= PhMe: C2H5OH: H2O) at 25 °C. The resulted mixture was refluxed under N2 until
completion determined by TLC. Cooled and the precipitate was collected by hot filtration, washed with water and
dried to give 5 as a white solid in 86.8% yield. MS (ESI) m/z(%):357.1 [M+H]+; 1H-NMR (400 MHz, DMSO-d6):
δ 10.06 (s, 1H), 8.28 (t, J = 5.8 Hz, 1H), 8.01 (d, J = 8.2 Hz, 2H), 7.80 (d, J = 8.2 Hz, 2H), 7.71-7.62 (m, 2H),
7.47 (dd, J = 8.6, 2.0 Hz, 1H), 4.78 (m, 1H), 4.19 (t, J = 9.1 Hz, 1H), 3.80 (dd, J = 9.1, 6.4 Hz, 1H), 3.45 (t, J =
5.4 Hz, 2H), 1.85 (s, 3H). 13C-NMR (151 MHz, DMSO): δ 193.10, 170.43, 160.33, 158.70, 154.36, 140.90,
140.66, 140.58, 135.48, 131.46, 131.43, 130.15, 129.65, 129.63, 121.93, 121.85, 114.42, 106.02, 105.83, 72.22,
47.56, 41.77, 22.82.
5.1.5 General procedure for the preparation of intermediates 8a-e
5.1.5.1 General procedure for the preparation of intermediates 7a-e
To a well-stirred solution of an appropriate intermediate (6a-e) (0.10 mol) in THF (100 mL) was added

ACCEPTED MANUSCRIPT
dropwise 3M HCl (60 mL) at 0 °C. Then NaNO2 (16.5 g, 0.24 mol) in water (33 mL) was added dropwise while
maintaining the same temperature. The resulted mixture was heated to 25 ºC for 3-5 h. Then the reaction mixture
was poured into water (80 mL) and extracted with CH2Cl2 (100 mL×3). The combined organic layer was washed
with brine, dried over Na2SO4, filtered, and concentra ted under reduced presusre to yield the corresponding
intermediates 7a-e.
5.1.5.2 General procedure for the preparation of intermediates 8a-e
To a well-stirred solution of an appropriate intermediate (7a-e) (0.10 mol) in a mixed solution of AcOH (100
mL) and H2O (100 mL) was added zinc powder ( 26.2 g,0.40 mol) at 0 °C, followed by stirring for 6-8 h at the
same temperature. After completion of the reaction as indicated by TLC, the resulted mixture was filtered and the
filtrate was concentrated under reduced pressure. Then ethyl acetate (100 mL) was added and the misture was
stirred for 0.5 h at the room temperature. The mixture was filtered, and the filtrate was concentrated under reduced
presusre to yield the corresponding intermediates 8a-e.
5.1.6 General procedure for the preparation of compounds 8h
5.1.6.1 tert-Butyl thiomorpholine-4-carboxylate (10)
To the mixture of thiomorpholine (5.8 g, 0.055 mol) and TEA (6.8 g, 0.067 mol) in THF (60 mL),
dibutyldicarbonate (12.8 g, 0.058 mol) was added dropwise while maintaining the temperature below 0 ºC. After
the addition was completed, the resulted mixture was stirred for 5 h at 25 °C. After completion of the reaction as
indicated by TLC, the reaction mixture was concentrated under reduced pressure. Then ethyl acetate (50 mL) was
added and the misture was washed with saturated citric acid aqueous solution and brine, dried over MgSO4,
filtered, and concentrated under reduced pressure to yield 1 as yellow oil in 88% yield.
5.1.6.2 tert-butyl thiomorpholine-4-carboxylate 1,1-dioxide (11)
To the solution of 10 (5 g, 0.024 mol) in AcOH (15 mL), H2O2 (40%, 5 mL ) was added dropwise while
maintaining the temperature below 0 ºC. After the addition was completed, the mixture was heated to 50 ºC for
another 4 h. After completion of the reaction as indicated by TLC, the reaction was quenched by adding saturated
sodium thiosulfate solution (15 mL) , Then DCM (30 mL) was added and the misture was washed with water and
brine, dried over MgSO4, filtered, and concentrated under reduced pressure to yield 1 as a light yellow solid in 88%
yield.
5.1.6.3 Thiomorpholine-1,1-oxide (6h)
To the solution of 11 (5.8 g, 0.024 mol) in DCM (50 mL) was added trifluoroacetic acid (70 mL). The resulted
mixture stirred for 5 h at 25 °C until completion of the reaction by TLC. Then the resulted mixture was
concentrated under reduced pressure. Water (30 mL) was added and the mixture basified to PH 8.0 with ammonia
solution, then the misture was extracted three times with DCM(50 mL×3). The organic extracts were combined,
washed with water and brine, dried over MgSO4, filtered, and concentrated under reduced pressure to yield 6h as
a white solid in 82% yield. MS (ESI) m/z(%): 136.0 [M+H]+.
5.1.6.4 4-Nitro sothiomorpholine-1,1-oxide (8h)
Intermediate 8h was prepared from 6h in the same manner as described for the general synthesis of
intermediates 8a-e in 82% yield.
5.1.7 N-{3-{2-fluoro-4'-[(4-methylpiperazin-1-yl)iminomethyl]-biphenyl-4-yl}-2-oxo-1,3-oxazolidine-5-(S)-ylmeth
yl}-acetamide(9a)
To a well-stirred solution of intermediate 5 (2 g, 5.6 mmol) in C2H5OH(60 mL ) was added a drop of AcOH.

The resulted mixture was refluxed for A0.5ChC. TEhPenTiEntDermMedAiaNtesU8Sa C(2R.95IPgT) was added，followed by stirring for 10
h at the same temperature. After cooled to room temperature, The resulting solid was collected by filtration and
dried to give the target compounds 9a as a light yellow solid in 82% yield. M.p.: 240-242 ºC; MS (ESI) m/z
(%):454.4 [M+H]+; 1H-NMR (400 MHz, DMSO-d6): δ 8.27 (t, J = 5.8 Hz, 1H), 7.71-7.51 (m, 7H), 7.41 (dd, J =
8.6, 2.0 Hz, 1H), 4.77 (m, 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),
3.18-3.10 (m, 4H), 2.23 (s, 3H), 1.85 (s, 3H). 13C-NMR (151 MHz, DMSO): δ 170.47, 160.20, 158.58, 154.38,
139.65, 139.57, 136.05, 134.76, 134.16, 131.05, 129.07, 129.05, 126.25, 122.92, 122.83, 114.37, 106.05, 105.85,
72.14, 54.30, 50.96, 47.56, 45.91, 41.78, 22.82.
5.1.8N-{3-[2-fluoro-4'-(morpholin-4-yliminomethyl)-biphenyl-4-yl]-2-oxo-1,3-oxazolidin-5-(S)-ylmethyl}-acetami
de (9b)
Compound 9b was prepared from intermediate 5 and 8b in the same manner as described for 9a. White solid;
yield: 78 %; M.p.: 225-227 ºC; MS (ESI) m/z (%): 441.4 [M+H]+;1H-NMR (400 MHz, DMSO-d6) : δ 8.27 (t, J =
5.7 Hz, 1H), 7.74 (s, 1H), 7.68 (s, 1H), 7.66 (s, 1H), 7.59 (ddd, J = 14.4, 10.9, 4.7 Hz, 4H), 7.42 (dd, J = 8.6, 2.0
Hz, 1H), 4.77 (m,1H), 4.17 (t, J = 9.0 Hz, 1H), 3.79 (dd, J = 10.7, 6.2 Hz, 5H), 3.44 (t, J = 5.4 Hz, 2H), 3.20-3.07
(m, 4H), 1.85 (s, 3H).
5.1.9N-{3-[2-fluoro-4'-(thiomorpholin-4-yliminomethyl)-biphenyl-4-yl]-2-oxo-1,3-oxazolidin-5-(S)-ylmethyl}-acet
amide (9c)
Compound 9c was prepared from intermediate 5 and 8c in the same manner as described for 9a. White solid;
yield: 80 %; M.p.: 212-214 ºC; MS (ESI) m/z (%):457.5 [M+H]+ ;1H-NMR (400 MHz, DMSO) δ 8.26 (t, J = 5.7
Hz, 1H), 7.72 (s, 1H), 7.66 (d, J = 8.3 Hz, 2H), 7.63-7.51 (m, 4H), 7.42 (dd, J = 8.6, 1.8 Hz,1H), 4.77 (td, J = 11.3,
5.4 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.79 (dd, J = 9.0, 6.6 Hz, 1H), 3.58-3.47 (m, 4H), 3.44 (t, J = 5.4 Hz, 2H),
2.79-2.68 (m, 4H), 1.85 (s, 3H).
5.1.10N-{3-[2-fluoro-4'-(piperidine-4-yliminomethyl)-biphenyl-4-yl]-2-oxo-1,3-oxazolidin-5-(S)-ylmethyl}-acetam
ide (9d)
Compound 9d was prepared from intermediate 5 and 8d in the same manner as described for 9a. White solid;
yield: 78 %; M.p.: 215-217 ºC; MS (ESI) m/z (%): 439.7 [M+H]+, 1H-NMR (400 MHz, DMSO-d6): δ 8.26 (t, J =
5.8 Hz, 1H), 7.61 (ddd, J = 8.9, 6.6, 3.2 Hz, 5H), 7.53 (d, J = 7.2 Hz, 2H), 7.41 (dd, J = 8.6, 2.0 Hz, 1H), 4.76 (m,
1H), 4.17 (t, J = 9.0 Hz, 1H), 3.79 (dd, J = 9.1, 6.5 Hz, 1H), 3.44 (t, J = 5.5 Hz, 2H), 3.21-3.09 (m, 4H), 1.84 (s,
3H), 1.80 (s, 4H), 1.72-1.61 (m, 4H), 1.56-1.45 (m, 2H).
5.1.11N-{3-{2-fluoro-4'-[(4-tert-butoxycarbonylpiperazin-1-yl)iminomethyl]-biphenyl-4-yl}-2-oxo-1,3-oxazolidine
-5-(S)-ylmethyl}-acetamide (9e)
Compound 9e was prepared from intermediate 5 and 8e in the same manner as described for 9a. White solid;
yield: 76 %; M.p.: 237-239 ºC; MS (ESI) m/z (%): 538.6 [M-H]-; 1H-NMR (400 MHz, DMSO) δ 8.26 (t, J = 5.8
Hz, 1H), 7.73 (s, 1H), 7.68-7.52 (m, 6H), 7.42 (dd, J = 8.6, 1.8 Hz, 1H), 4.77 (d, J = 2.5 Hz, 1H), 4.17 (t, J = 9.0
Hz, 1H), 3.79 (dd, J = 9.1, 6.5 Hz, 1H), 3.51 (d, J = 5.0 Hz, 4H), 3.44 (t, J = 5.4 Hz, 2H), 3.17-3.04 (m, 4H), 1.84
(s, 3H), 1.42 (s, 9H).
5.1.12N-{3-[2-fluoro-4'-(piperazin-1-yliminomethyl)-biphenyl-4-yl]-2-oxo-1,3-oxazolidin-5-(S)-ylmethyl}-acetami
de (9f)
To the solution of 9e (0.95 g, 0.024 mol) in DCM (8 mL) was added trifluoroacetic acid (2 mL) at 0 °C, then
the resulted mixture was stirred for 2 h at 25 °C. The reaction was monitored by TLC, after completion of reaction,
the mixture was concentrated under reduced pressure. Then water (10 mL) was added and the mixture basified to

ACCEPTED MANUSCRIPT
PH 8.0 with ammonia solution. The misture was extracted three times with DCM, and the organic extracts were
combined, washed with water and brine, dried over MgSO4, filtered, and concentrated under reduced pressure to
yield 9f as a white solid in 71 % yield. M.p.: 201-203 ºC; MS (ESI) m/z (%): 440.7 [M+H]+; 1H-NMR (600 MHz,
DMSO-d6): δ 8.30 (t, J = 5.9 Hz, 1H), 7.78 (s, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.60 (m, 4H), 7.43 (dd, J = 8.6, 2.1
Hz, 1H), 4.78 (m, 1H), 4.18 (t, J = 9.0 Hz, 1H), 3.81 (dd, J = 9.1, 6.4 Hz, 1H), 3.45 (t, J = 5.5 Hz, 2H), 3.33-3.26
(m, 4H), 3.23-3.14 (m,4H), 1.86 (s, 3H). 13C-NMR (151 MHz, DMSO): δ 170.45, 160.21, 158.59, 154.38, 139.73,
139.66, 136.48, 135.59, 134.62, 131.07, 131.04, 129.12, 129.10, 126.49, 122.84, 122.75, 114.36, 106.05, 105.86,
72.14, 49.33, 47.57, 43.27, 41.77, 22.82.
5.1.13N-{3-{2-fluoro-4'-[(4-aminoacetylpiperazin-1-yl)iminomethyl]-biphenyl-4-yl}-2-oxo-1,3-oxazolidine-5-(S)-y
lmethyl}-acetamide (9g)
To the solution of 9e (0.1 g, 0.22 mmol) in DCM (5 mL) was added 3-chloroperbenzoic acid (0.04 g, 0.23
mmol ) at 0 °C, then the resulted mixture was stirred for 2 h at 25 °C. The reaction was monitored by TLC, after
completion of reaction, saturated sodium carbonate solution (5 mL) was added. Then the misture was extracted
three times with DCM, and the organic extracts were combined, washed with water and brine, dried over MgSO4,
filtered, and concentrated under reduced pressure to yield 9g as a white solid in 82 % yield. M.p.: 222-224 ºC; MS
(ESI) m/z (%): 473.5 [M+H]+; 1H-NMR (600 MHz, DMSO) δ 8.26 (t, J = 5.8 Hz, 1H), 7.83 (s, 1H), 7.69 (d, J =
8.3 Hz, 2H), 7.63-7.54 (m, 4H), 7.42 (dd, J = 8.6, 2.0 Hz, 1H), 4.77 (td, J = 11.4, 5.3 Hz, 1H), 4.17 (t, J = 9.0 Hz,
1H), 3.79 (dd, J = 9.0, 6.5 Hz, 1H), 3.76-3.62 (m, 4H), 3.44 (t, J = 5.5 Hz, 2H), 3.04-2.89 (m, 2H), 2.77 (d, J =
13.6 Hz, 2H), 1.85 (s, 3H).
5.1.14N-{3-{2-fluoro-4'-[(1,1-dioxothiomorpholin-4-yl)iminomethyl]biphenyl-4-yl}-1,3-oxazolidine-5-(S)-ylmethyl
}-acetamide(9h)
Compound 9h was prepared from intermediate 5 and 8h in the same manner as described for 9a. White solid;
yield: 79 %; M.p.: 232-234 ºC; MS (ESI) m/z (%): 489.4 [M+H]+; 1H NMR (600 MHz, DMSO) δ 8.27 (t, J = 5.4
Hz, 1H), 7.83 (s, 1H), 7.70 (d, J = 8.0 Hz, 2H), 7.59 (dd, J = 20.2, 8.6 Hz,4H), 7.42 (d, J = 8.4 Hz, 1H), 4.78 (d, J
= 5.7 Hz, 1H), 4.17 (t, J = 8.9 Hz, 1H), 3.88-3.75 (m, 5H), 3.44 (t, J = 5.1 Hz, 2H), 3.19 (s, 4H), 1.85 (s, 3H).
5.1.15N-{3-{2-fluoro-4'-[(4-acetylpiperazin-1-yl)iminomethyl]-biphenyl-4-yl}-2-oxo-1,3-oxazolidine-5-(S)-ylmeth
yl}-acetamide (10a)
To the mixture of compound 9f (0.2 g, 0.45 mmol) and DIPEA (0.089 g, 0.68 mmol) in DMF (5 mL), methyl
chloroformate (0.056 g, 0.59 mmol) was added dropwise while maintaining the temperature below 0 ºC. After the
addition was completed, the mixture was stirred for 5 h at 25 °C. After completion of the reaction as indicated by
TLC, water (10 mL) was added. Then the resulting precipitate was filtered, washed with water and dried to get a
white solid in 58 % yield. M.p.: 242-244 ºC; MS (ESI) m/z (%):482.8 [M+H]+; 1H-NMR (400 MHz, DMSO-d6):
δ 8.27 (t, J = 5.8 Hz, 1H), 7.74 (s, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.64-7.52 (m, 4H), 7.42 (dd, J = 8.6, 2.0 Hz, 1H),
4.77 (m, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.79 (dd, J = 9.1, 6.5 Hz, 1H), 3.63 (d, J = 4.8 Hz, 4H), 3.44 (t, J = 5.4 Hz,
2H), 3.19 (d, J = 4.8 Hz, 2H), 3.14-3.06 (m, 2H), 2.05 (s, 3H), 1.85 (s, 3H).
5.1.16N-{3-{2-fluoro-4'-[(4-benzoylpiperazin-1-yl)iminomethyl]-biphenyl-4-yl}-2-oxo-1,3-oxazolidine-5-(S)-ylmet
hyl}-acetamide (10b)
Compound 10b was prepared from compound 9f and benzoyl chloride in the same manner as described for 10a.
White solid; yield: 74 %; M.p.: 213-215 ºC; MS (ESI) m/z (%): 545.0 [M+H]+; 1H-NMR (400 MHz, DMSO-d6):
δ 8.27 (t, J = 5.8 Hz, 1H), 7.75 (s, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.63-7.52 (m, 4H), 7.50-7.40 (m, 6H), 4.77 (m,
1H), 4.17 (t, J = 9.0 Hz, 1H), 3.62 (m, 7H), 3.21 (s, 4H), 1.85 (s, 3H).

ACCEPTED MANUSCRIPT
5.1.17N-{3-{2-fluoro-4'-{[4-(2-furoyl)piperazin-1-yl]iminomethyl}-biphenyl-4-yl}oxo-1,3-oxazolidin-5-(S)-ylmeth
yl}-acetamide (10c)
Compound 10c was prepared from compound 9f and 2-furoyl chloride in the same manner as described for 10a.
White solid; yield: 78 %; M.p.: 228-230 ºC; MS (ESI) m/z (%): 534.9 [M+H]+; 1H-NMR (400 MHz, DMSO-d6):
8.26 (t, J = 5.8 Hz, 1H), 7.87 (d, J = 1.0 Hz, 1H), 7.75 (s, 1H), 7.68 (d, J = 8.3 Hz, 2H), 7.64-7.52 (m, 4H), 7.42
(dd, J = 8.7, 1.9 Hz, 1H), 7.06 (d, J = 3.4 Hz, 1H), 6.65 (dd, J = 3.4, 1.7 Hz, 1H), 4.77 (m, 1H), 4.17 (t, J = 9.0 Hz,
1H), 3.86 (s, 4H), 3.79 (dd, J = 9.1, 6.5 Hz, 1H), 3.44 (t, J = 5.4 Hz, 2H), 3.28-3.20 (m, 4H), 1.85 (s, 3H).
5.1.18N-{3-{2-fluoro-4'-{[4-(4-pyridylcarboxyl)piperazin-1-yl]iminomethyl}-biphenyl-4-yl}oxo-1,3-oxazolidin-5-(
S)-ylmethyl}-acetamide (10d)
Compound 10d was prepared from compound 9f and 4-pyridyl chloride in the same manner as described for
1
10a. Yellow solid; yield: 78 %; M.p.: 207-209 ºC; MS (ESI) m/z (%):545.9 [M+H]+; H-NMR (400 MHz,
DMSO-d6): 8.70 (d, J = 5.8 Hz, 2H), 8.26 (t, J = 5.8 Hz, 1H), 7.75 (s, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.63-7.53 (m,
4H), 7.43 (ddd, J = 10.7, 6.6, 1.7 Hz, 3H), 4.77 (m, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.88-3.76 (m, 3H), 3.44 (m, 4H),
3.28 (s, 2H), 3.18 (s, 2H),1.85 (s, 3H).
5.1.19N-{3-{2-fluoro-4'-{[4-(3-pyridylcarboxyl)piperazin-1-yl]iminomethyl}-biphenyl-4-yl}oxo-1,3-oxazolidin-5-(
S)-ylmethyl}-acetamide (10e)
Compound 10d was prepared from compound 9f and 3-pyridyl chloride in the same manner as described for
1
10a. Yellow solid; yield: 73 %; M.p.: 215-217 ºC; MS (ESI) m/z (%): 546.9 [M+H]+; H-NMR (600 MHz,
DMSO-d6): 8.68 (d, J = 5.1 Hz, 2H), 8.26 (t, J = 5.7 Hz, 1H), 7.90 (d, J = 7.8 Hz, 1H), 7.75 (s, 1H), 7.67 (d, J =
8.2 Hz, 2H), 7.64-7.54 (m, 4H), 7.51 (dd, J = 7.5, 5.0 Hz, 1H), 7.42 (d, J = 8.5 Hz, 1H), 4.77 (m, 1H), 4.17 (t, J =
8.9 Hz, 1H), 3.84 (s, 2H), 3.79 (dd, J = 8.9, 6.6 Hz, 1H), 3.53 (s, 2H), 3.44 (t, J = 5.3 Hz, 2H), 3.28 (s , 2H), 3.21
(s , 2H),1.85 (s, 3H).
5.1.20N-{3-{2-fluoro-4'-{[4-(2-pyridylcarboxyl)piperazin-1-yl]iminomethyl}-biphenyl-4-yl}oxo-1,3-oxazolidin-5-(
S)-ylmethyl}-acetamide (10f)
Compound 10f was prepared from compound 9f and 2-pyridyl chloride in the same manner as described for 10e.
1
Yellow solid; yield: 76%; M.p.: 226-228 ºC; MS (ESI) m/z (%): 545.4 [M+H]+; H-NMR (600 MHz, DMSO) δ
8.91 (d, J = 1.4 Hz, 1H), 8.78 (d, J = 2.5 Hz, 1H), 8.71 (dd, J = 2.4, 1.6 Hz, 1H), 8.26 (t, J = 5.8 Hz, 1H), 7.76 (s,
1H), 7.68 (d, J = 8.4 Hz, 2H), 7.63-7.53 (m, 4H), 7.42 (dd, J = 8.6, 2.1 Hz, 1H), 4.76 (dt, J = 11.5, 5.7 Hz, 1H),
4.17 (t, J = 9.0 Hz, 1H), 3.92-3.84 (m, 2H), 3.79 (dd, J = 9.1, 6.4 Hz, 1H), 3.70-3.63 (m, 2H), 3.44 (t, J = 5.5 Hz,
2H), 3.32-3.26 (m, 2H), 3.24-3.16 (m, 2H), 1.85 (s, 3H).
5.1.21N-{3-{2-fluoro-4'-{[4-(2-thenoyl)piperazin-1-yl]iminomethyl}-biphenyl-4-yl}oxo-1,3-oxazolidin-5-(S)-ylmet
hyl}-acetamide (10g)
Compound 10g was prepared from compound 9f and 2-thiophenecarbonyl chloride in the same manner as
described for 10a. Yellow solid; yield: 78 %. M.p.: 216-218 ºC; MS (ESI) m/z (%): 550.4 [M+H]+; 1H-NMR (400
MHz, DMSO-d6): 8.29 (t, J = 5.8 Hz, 1H), 7.94-7.35 (m, 10H), 7.16 (s, 1H), 4.77 (m, 1H), 4.18 (t, J = 8.7 Hz,
1H), 3.84 (m, 5H), 3.44 (t, 2H), 3.25 (s, 4H), 1.85 (s, 3H).
5.1.22N-{3-{2-fluoro-4'-{[4-(2-pyrazinecarboxyl)piperazin-1-yl]iminomethyl}-biphenyl-4-yl}oxo-1,3-oxazolidin-5
-(S)-ylmethyl}-acetamide (10h)
Compound 10h was prepared from compound 9f and 2-pyrazinecarbonyl Chloride in the same manner as
described for 10a. Yellow solid; yield: 73 %; M.p.: 229-230 ºC; MS (ESI) m/z (%): 546.8 [M+H]+; 1H-NMR (400
MHz, DMSO) δ 8.62 (d, J = 4.6 Hz, 1H), 8.26 (t, J = 5.7 Hz, 1H), 7.95 (m, 1H), 7.75 (s, 1H), 7.72-7.53 (m, 7H),

ACCEPTED MANUSCRIPT
7.51 (dd, J = 7.1, 5.3 Hz, 1H), 7.42 (dd, J = 8.6, 1.7 Hz, 1H), 4.77 (td, J = 11.4, 5.4 Hz, 1H), 4.17 (t, J = 9.0
Hz,1H), 3.86 (s, 2H), 3.79 (dd, J = 9.0, 6.6 Hz, 1H), 3.63 (s, 2H), 3.44 (t, J = 5.4 Hz, 2H), 3.27 (s, 2H), 3.17 (s,
2H), 1.85 (s, 3H).
5.1.23N-{3-{2-fluoro-4'-{[4-(4-methoxybenzoyl)piperazin-1-yl]iminomethyl}-biphenyl-4-yl}oxo-1,3-oxazolidin-5-(
S)-ylmethyl}-acetamide (10i)
Compound 10i was prepared from compound 9f and 4-methoxybenzoyl chloride in the same manner as
1
described for 10a. Yellow solid; yield: 77 %; M.p.: 188-190 ºC; MS (ESI) m/z (%): 596.6 [M+Na]+; H-NMR
(400 MHz, DMSO-d6): δ 8.70 (d, J = 5.5 Hz, 2H), 8.27 (t, J = 5.8 Hz, 1H), 7.75 (s, 1H), 7.67 (d, J = 8.4 Hz, 2H),
7.64-7.52 (m, 4H), 7.49-7.37 (m, 3H), 4.77 (m, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.89-3.73 (m, 3H), 3.55-3.39 (m,
4H), 3.28 (s, 2H), 3.18 (s, 2H), 1.85 (s, 3H).
5.1.24N-{3-{2-fluoro-4'-{[4-(2-methoxybenzoyl)piperazin-1-yl]iminomethyl}-biphenyl-4-yl}oxo-1,3-oxazolidin-5-(
S)-ylmethyl}-acetamide (10j)
Compound 10j was prepared from compound 9f and 2-methoxybenzoyl chloride in the same manner as
described for 10a. Yellow solid; yield: 75 %; M.p.: 205-207 ºC; MS (ESI) m/z (%): 574.9 [M+H]+; 1H-NMR (600
MHz, DMSO): δ 8.26 (t, J = 5.7 Hz, 1H), 7.74 (s, 1H), 7.67 (d, J = 8.2 Hz, 2H), 7.62-7.58 (m, 2H), 7.55 (d, J =
7.6 Hz, 2H), 7.42 (dd, J = 7.1, 3.6 Hz, 2H), 7.23 (d, J = 6.3 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 7.02 (t, J = 7.4 Hz,
1H), 4.77 (td, J = 11.3, 5.5 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.89-3.73 (m, 6H), 3.44 (t, J = 5.4 Hz, 2H), 3.22 (s,
2H), 3.11 (s, 2H), 1.85 (s, 3H).
5.1.25N-{3-{2-fluoro-4'-{[4-(3-methoxybenzoyl)piperazin-1-yl]iminomethyl}-biphenyl-4-yl}oxo-1,3-oxazolidin-5-(
S)-ylmethyl}-acetamide (10k)
Compound 10k was prepared from compound 9f and 3-methoxybenzoyl chloride in the same manner as
1
described for 10a. Yellow solid; yield: 78%; M.p.: 187-188 ºC; MS (ESI) m/z (%): 596.6 [M+Na]+; H-NMR
(400 MHz, DMSO) δ 8.26 (t, J = 5.8 Hz, 1H), 7.74 (s, 1H), 7.67 (d, J = 8.3 Hz, 2H), 7.63-7.49 (m, 4H), 7.45-7.32
(m, 2H), 7.07-6.94 (m, 3H), 4.77 (dd, J = 8.4, 6.0 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.91-3.69 (m, 6H), 3.52 (s,
2H), 3.44 (t, J = 5.4 Hz, 2H), 3.21 (s, 4H), 1.85 (s, 3H).
5.1.26N-{3-{2-fluoro-4'-{[4-(4-trifluoromethylbenzoyl)piperazin-1-yl]iminomethyl}-biphenyl-4-yl}oxo-1,3-oxazoli
din-5-(S)-ylmethyl}-acetamide (10l)
Compound 10l was prepared from compound 9f and 4-(trifluoromethyl)benzoyl chloride in the same manner as
described for 10a. Yellow solid; yield: 72%; M.p.: 238-239 ºC; MS (ESI) m/z (%): 634.5 [M+Na]+; 1H-NMR (400
MHz, DMSO) δ 8.26 (t, J = 5.8 Hz, 1H), 7.84 (d, J = 8.1 Hz, 2H), 7.75 (s, 1H), 7.68 (dd, J = 8.2, 3.6 Hz, 4H),
7.63-7.53 (m, 4H), 7.42 (dd, J = 8.7, 1.9 Hz, 1H), 4.76 (dt, J = 11.4, 5.6 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.93-
3.75 (m, 4H), 3.44 (t, J = 5.4 Hz, 4H), 3.23 (d, J = 38.7 Hz, 4H), 1.85 (s, 3H).
5.1.27N-{3-{2-fluoro-4'-{[4-(4-fluorobenzoyl)piperazin-1-yl]iminomethyl}-biphenyl-4-yl}oxo-1,3-oxazolidin-5-(S)
-ylmethyl}-acetamide (10m)
Compound 10m was prepared from compound 9f and 4-fluorobenzoyl chlorid
described for 10a. Yellow solid; yield: 72 %; M.p.: 211-213 ºC; MS (ESI) m/z (%): 562.5 [M+H]+; 1H-NMR (400
MHz, DMSO d6): 8.26 (t, J = 5.8 Hz, 1H), 7.75 (s, 1H), 7.67 (d, J = 8.3 Hz, 2H), 7.64-7.49 (m, 6H), 7.42 (dd, J =
e in the same manner as
-
8.6, 1.9 Hz,1H), 7.30 (t, J = 8.8 Hz, 2H), 4.77 (m, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.88-3.39 (m, 7H), 3.22 (s, 4H),
1.85 (s, 3H).
5.1.28N-{3-{2-fluoro-4'-{[4-(4-chlorobenzoyl)piperazin-1-yl]iminomethyl}-biphenyl-4-yl}oxo-1,3-oxazolidin-5-(S)

ACCEPTED MANUSCRIPT
-ylmethyl}-acetamide (10n)
Compound 10n was prepared from compound 9f and 4-chlorobenzoyl chloride in the same manner as described
1
for 10a. Yellow solid; yield: 72 %; M.p.: 220-222 ºC; MS (ESI) m/z (%): 578.6 [M+H]+; H-NMR (400 MHz,
DMSO-d6): 8.26 (t, J = 5.8 Hz, 1H), 7.75 (s, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.64-7.50 (m, 6H), 7.42 (dd, J = 8.6,
1.9 Hz, 1H), 7.30 (t, J = 8.9 Hz, 2H), 4.77 (m, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.79 (dd, J = 9.1, 6.5 Hz, 2H), 3.79
(dd, J = 9.1, 6.5 Hz, 2H), 3.61 (s, 2H), 3.44 (t, J = 5.4 Hz, 2H), 3.22 (s, 4H), 1.85 (s, 3H).
5.1.29N-{3-{2-fluoro-4'-{[4-(4-bromobenzoyl)piperazin-1-yl]iminomethyl}-biphenyl-4-yl}oxo-1,3-oxazolidin-5-(S)
-ylmethyl}-acetamide (10o)
Compound 10n was prepared from compound 9f and 4-bromobenzoyl chloride in the same manner as described
1
for 10a. Yellow solid; yield:75 % ; M.p.: 215-217 ºC; MS (ESI) m/z (%):622.8 [M+H]+; H-NMR (400 MHz,
DMSO) δ 8.25 (t, J = 5.4 Hz, 1H), 7.74 (s, 1H), 7.67 (d, J = 8.1 Hz, 4H), 7.58 (m, 4H), 7.42 (d, J = 8.3 Hz, 3H),
4.76 (s, 1H), 4.17 (t, J = 8.9 Hz, 1H), 3.94-3.68 (m, 3H), 3.51 (s,2H), 3.44 (t, J = 5.2 Hz, 2H), 3.21 (s, 4H), 1.84 (s,
3H).
5.1.30N-{3-{2-fluoro-4'-[(4-propionylpiperazin-1-yl)iminomethyl]-biphenyl-4-yl}-2-oxo-1,3-oxazolidine-5-(S)-ylm
ethyl}-acetamide (10p)
Compound 10p was prepared from compound 9f and propionyl chloride in the same manner as described for
10a. Yellow solid; yield: 72 % ; M.p.: 209-211 ºC; MS (ESI) m/z (%):496.5 [M+H]+; 1H-NMR (600 MHz, DMSO)
δ 8.26 (t, J = 5.7 Hz, 1H), 7.68 (s, 1H), 7.66 (d, J = 8.2 Hz, 2H), 7.62-7.57 (m, 2H), 7.54 (d, J = 7.6 Hz, 2H), 7.42
(dd, J = 8.6, 1.7 Hz, 1H), 4.77 (td, J = 11.4, 5.5 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.79 (dd, J = 8.9, 6.6 Hz, 1H),
3.44 (t, J = 5.4 Hz, 2H), 3.35 (d, J = 1.9 Hz, 2H), 3.20 (s, 1H), 3.17 (s, 4H), 2.70-2.58 (m, 4H), 1.85 (s, 3H).
5.1.31N-{3-{2-fluoro-4'-[(4-methoxycarbonylpiperazin-1-yl)iminomethyl]-biphenyl-4-yl}-2-oxo-1,3-oxazolidine-5-
(S)-ylmethyl}-acetamide (10q)
Compound 10q was prepared from compound 9f and methyl chloroformate in the same manner as described for
10a. Yellow solid; yield: 58 %. M.p.: 192-194 ºC; MS (ESI) m/z (%): 498.7 [M+H]+; 1H-NMR (400 MHz, DMSO
d6): δ 8.26 (t, J = 5.8 Hz, 1H), 7.74 (s, 1H), 7.68 (s, 1H), 7.65 (s, 1H), 7.63-7.51 (m, 4H), 7.42 (dd, J = 8.6, 2.0 Hz,
1H), 4.77 (m, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.79 (dd, J = 9.1, 6.5 Hz, 1H), 3.63 (s, 3H), 3.60-3.52 (m, 4H), 3.44 (t,
J = 5.4 Hz, 2H), 3.14(m, 4H), 1.85 (s, 3H).
5.1.32N-{3-{2-fluoro-4'-[(4-trifluoromethylformylpiperazin-1-y)iminomethyl]-biphenyl-4-yl}-2-oxo-1,3-oxazolidin
e-5-(S)-ylmethyl}-acetamide (10r)
To the mixture of compound 9f (0.2 g, 0.45 mmol), TEA (0.068 g, 0.68 mmol) and trifluoroacetic acid (0.052 g
0.45 mmol) in DMF (5 mL) was added HATU (0.34 g, 0.90 mmol),Then the resulted mixture was stirred for 5 h at
25 °C. After completion of the reaction as indicated by TLC, water (15 mL) was added. The misture was extracted
three times with DCM, and the organic extracts were combined, washed with brine, dried over MgSO4, filtered,
and concentrated under reduced pressure to yield 10r as a white solid in 77 % yield. M.p.: 197-199 ºC; MS (ESI)
m/z (%): 536.55 [M+H]+; H-NMR (400 MHz, DMSO
-d6): δ 8.26 (t, J = 5.8 Hz, 1H), 7.76 (s, 1H), 7.68 (d, J =
8.4 Hz,2H), 7.63-7.54 (m, 4H), 7.42 (dd, J = 8.6, 2.0 Hz, 1H), 4.77 (m, 1H), 4.17 (t, J = 9.0 Hz, 2H), 3.78 (dd, J =
13.0, 6.9 Hz, 5H), 3.44 (t, J = 5.4 Hz, 2H), 3.27 (s, 4H), 1.85 (s, 3H).
5.1.32N-{3-{2-fluoro-4'-{[4-(N,N-dimethylformyl)piperazin-1-yl]iminomethyl}-biphenyl-4-yl}oxo-1,3-oxazolidin-5
-(S)-ylmethyl}-acetamide (10s)
Compound 10s was prepared from compound 9f and dimethylcarbamyl chloride in the same manner as
described for 10a. Yellow solid; yield: 73 %; M.p.: 212-214 ºC; MS (ESI) m/z (%): 5133.80 [M+Na]+. 1H NMR

ACCEPTED MANUSCRIPT
(400 MHz, DMSO) δ 8.26 (t, J = 5.8 Hz, 1H), 7.72 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.63-7.52 (m, 4H), 7.42 (dd,
J = 8.6, 2.0 Hz, 1H), 4.77 (dd, J = 8.6, 6.0 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), 3.32-3.24 (m,4H), 3.20-3.12 (m, 4H), 2.78 (s, 6H), 1.85 (s, 3H).
5.1.33N-{3-{2-fluoro-4'-[(4-hydroxyacetylpiperazin-1-yl)iminomethyl]-biphenyl-4-yl}-2-oxo-1,3-oxazolidine-5-(S)
-ylmethyl}-acetamide (10t)
Compound 10t was prepared from compound 9f and glycolic acide in the same manner as described for 10r.
Yellow solid; yield: 77 %; M.p.: 225-227 ºC; MS (ESI) m/z (%): 498.7[M+H]+; 1H-NMR (600 MHz, DMSO-d6):
8.27 (s, 1H), 7.75 (s, 1H), 7.68 (d, J = 7.9 Hz, 2H), 7.64-7.53 (m, 4H), 7.42 (d, J = 7.9 Hz, 1H), 4.78 (s, 1H), 4.67
(d, J = 7.9 Hz, 1H), 4.17 (dd, J = 17.5, 6.8 Hz, 3H), 3.85-3.75 (m, 1H), 3.67 (s, 2H), 3.55 (s, 2H), 3.45 (s, 2H),
3.17 (d, J = 18.3 Hz, 4H), 1.86 (s, 3H). 13C-NMR (151 MHz, DMSO): δ 170.48, 170.42, 160.21, 158.58, 154.37,
139.70, 139.63, 135.81, 134.43, 131.06, 129.96, 129.08, 126.39, 122.86, 122.78, 114.34, 106.04, 105.85, 72.15,
60.55, 51.22, 50.92, 47.56, 43.02, 41.78, 40.82, 22.83.
5.1.34N-{3-{2-fluoro-4'-[(4-aminoacetylpiperazin-1-yl)iminomethyl]-biphenyl-4-yl}-2-oxo-1,3-oxazolidine-5-(S)-y
lmethyl}-acetamide (10u)
Compound 10u was prepared from compound 9f and glycine in the same manner as described for 10r. White
solid; yield: 78%; M.p.: 222-223 ºC; MS (ESI) m/z (%): 497.5[M+H]+; 1H-NMR (600 MHz, DMSO) δ 8.29 (s,
1H), 7.76 (s, 1H), 7.67 (d, J = 7.6 Hz, 2H), 7.58 (dd, J = 26.6, 8.4 Hz, 4H), 7.42 (d, J = 8.3 Hz, 1H), 6.30 (s, 2H),
4.77 (s, 1H), 4.17 (t, J = 8.6 Hz, 1H), 3.85-3.77 (m, 1H), 3.71 (s, 4H), 3.57 (s, 2H), 3.44 (s, 2H), 3.21 (s, 2H), 3.16
(s,2H), 1.85 (s, 3H).
5.1.35N-{3-{2-fluoro-4'-[(4-methanesulfonylpiperazin-1-yl)iminomethyl]-biphenyl-4-yl}-2-oxo-1,3-oxazolidine-5-(
S)-ylmethyl}-acetamide (11a)
Compound 11a was prepared from compound 9f and methanesulfonyl chloride in the same manner as described
for 10a. Yellow solid; yield: 79 %; M.p.: 258-260 ºC; MS (ESI) m/z (%): 517.8 [M+H]+; 1H-NMR (600 MHz,
DMSO) δ 8.26 (t, J = 5.8 Hz, 1H), 7.77 (s,1H), 7.68 (d, J = 8.3 Hz, 2H), 7.62-7.54 (m, 4H), 7.42 (dd, J = 8.6, 1.9
Hz, 1H), 4.77 (td, J = 11.4, 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), 3.32-3.29 (m,4H), 3.29-3.25 (m, 4H), 2.95 (s,3H), 1.85 (s, 3H).
5.1.36N-{3-{2-fluoro-4'-[(4-benzylpiperazin-1-yl)iminomethyl]-biphenyl-4-yl}-2-oxo-1,3-oxazolidine-5-(S)-ylmeth
yl}-acetamide (11b)
To the mixture of compound 9f (0.3 g, 0.68 mmol) and CsCO3(0.45 g, 1.36 mmol) in DMF (5 mL) was added
benzyl bromide (0.17 g, 1.02 mmol), Then the resulted mixture was stirred for 4 h at 25 °C. After completion of
the reaction as indicated by TLC, water (10 mL) was added. The resulting precipitate was filtered, washed with
water and dried to get a white solid in 72 % yield. M.p.: 213-215 ºC; MS (ESI) m/z (%):530.5 [M+H]+; 1H-NMR
(400 MHz, DMSO) δ 8.26 (t, J = 5.8 Hz, 1H), 7.70-7.50 (m, 7H), 7.41 (dd, J = 8.6, 1.8 Hz, 1H), 7.34 (d, J = 4.0
Hz, 4H), 7.30-7.23 (m, 1H), 4.76 (dt, J = 11.4, 5.5 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.79 (dd, J = 9.1, 6.5 Hz, 1H),
3.54 (s, 2H), 3.44 (t, J = 5.4 Hz, 2H), 3.15 (s, 4H), 2.60-2.53 (m, 4H), 1.84 (s, 3H).
5.1.37N-{3-{2-fluoro-4'-[(4-allylpiperazin-1-yl)iminomethyl]-biphenyl-4-yl}-2-oxo-1,3-oxazolidine-5-(S)-ylmeth
yl}-acetamide (11c)
Compound 11c was prepared from compound 9f and allyl bromide in the same manner as described for 11b.
Yellow solid; yield: 79 %. M.p.: 234-235 ºC; MS (ESI) m/z (%): 480.4 [M+H]+; 1H-NMR (400 MHz, DMSO) δ
8.26 (t, J = 5.8 Hz, 1H), 7.65 (d, J = 8.1 Hz,3H), 7.62-7.56 (m, 2H), 7.54 (d, J = 7.4 Hz,2H), 7.41 (dd, J = 8.6, 2.0
Hz, 1H), 5.95-5.74 (m, 2H), 5.19 (dd, J = 22.8, 13.8 Hz, 1H), 4.76 (dt, J = 11.5, 5.6 Hz, 1H), 4.17 (t, J = 9.0 Hz,

ACCEPTED MANUSCRIPT
1H), 3.79 (dd, J = 9.1, 6.5 Hz, 1H), 3.44 (t, J = 5.4 Hz, 2H), 3.25-3.08 (m, 4H), 3.01 (d, J = 6.3 Hz, 2H), 2.58-2.52
(m, 4H), 1.85 (s, 3H).
5.1.38
N-{3-{2-fluoro-4'-[(4-propargylpiperazin-1-yl)iminomethyl]-biphenyl-4-yl}-2-oxo-1,3-oxazolidine-5-(S)-ylmethy
l}-acetamide (11d)
Compound 11d was prepared from compound 9f and allyl bromide in the same manner as described for 11b.
Yellow solid; yield: 76 %. M.p.: 225-227 ºC; MS (ESI) m/z (%): 478.5 [M+H]+; 1H-NMR (600 MHz, DMSO) δ
8.26 (t, J = 5.7 Hz, 1H), 7.68 (s, 1H), 7.66 (d, J = 8.3 Hz, 2H), 7.62-7.57 (m, 2H), 7.54 (d, J = 7.5 Hz, 2H), 7.42
(dd, J = 8.6, 1.7 Hz, 1H), 4.77 (td, J = 11.4, 5.5 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H), 3.79 (dd, J = 8.9, 6.5 Hz, 1H),
3.44 (t, J = 5.4 Hz, 2H), 3.35 (d, J = 1.9 Hz, 2H), 3.20 (s, 1H), 3.17 (s, 4H), 2.69-2.59 (m, 4H), 1.85 (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-Signal)/ (Max- 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 ℃
for24 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 absAorbCaCncEe PofTMETDT MforAmaNzaUn)SaCndR6I3P0Tnm (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
o
(6 mM) was added. Then, pre-incubated all other plates at 37 C 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) was 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 9a 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 9a 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
the Program for Ministry of Education Innovative Research Team in University and the Program for Liaoning
Innovative Research Team in University.
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Highlights
1, A series of biaryloxazolidinone analogues containing a hydrazone moiety were designed,
synthesized and evaluated for their antibacterial activity
.
2, Compound 9a exhibited a MIC value of 0.0675 µg/mL against all tested bacteria.
3, The potency of compound 9a against LREF was 64 to 265-fold higher than that of linezolid.
4, Compound 9a exhibited comparable inhibitory activity with linezolid against human MAO-A.