Design, synthesis and antibacterial evaluation of novel oxazolidinone derivatives nitrogen-containing fused heterocyclic moiety

Article abstract of DOI:10.1016/j.bmcl.2020.127660

A series of novel oxazolidinone derivatives with nitrogen-containing fused heterocyclic moiety were designed and synthesized in this article. Their antibacterial activities were measured against S. aureus, MRSA and MSSA by MIC assay. Most of them exhibite

Full text of DOI:10.1016/j.bmcl.2020.127660

Journal Pre-proofs
Designing, synthesis and antibacterial evaluation of novel oxazolidinone de‐
rivatives nitrogen-containing fused heterocyclic moiety
Jia Jiang, Yunlei Hou, Meibo Duan, Baihang Wang, Yachuang Wu, Xiudong
Ding, Yanfang Zhao
PII:
DOI:
Reference:
S0960-894X(20)30771-X
https://doi.org/10.1016/j.bmcl.2020.127660
BMCL 127660
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
19 August 2020
21 October 2020
27 October 2020
Please cite this article as: Jiang, J., Hou, Y., Duan, M., Wang, B., Wu, Y., Ding, X., Zhao, Y., Designing,
synthesis and antibacterial evaluation of novel oxazolidinone derivatives nitrogen-containing fused heterocyclic
moiety, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127660
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Designing, synthesis and antibacterial evaluation of novel
oxazolidinone derivatives nitrogen-containing fused heterocyclic
moiety
1Jia Jianga，1, Yunlei Houa，1, Meibo Duan , Baihang Wang , Yachuang Wu , Xiudong Ding ,
a
b
a
b*
a**
Yanfang Zhao
a
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, 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
1
These two authors contributed equally to this work.
Abstract：
A series of novel oxazolidinone derivatives with nitrogen-containing fused heterocyclic moiety
were designed and synthesized in this article. Their antibacterial activities were measured against
S.aureus, MRSA and MSSA by MIC assay. Most of them exhibited potent activity against Gram-
positive pathogens comparable to Linezolid and Radezolid. Compound 3b, which exhibited
significant antibacterial activity with MIC values ranging 0.5-1.0 μg/mL, might be a promising drug
candidate for further investigation.
Keywords: Oxazolidinone derivatives; Nitrogen-containing fused heterocyclic moiety; Gram-
positive bacteria; Antibacterial activities
Introduction
In recent years, with the widespread use and even abuse of antibacterial drugs, antibiotic-
resistant infections have gradually become one of the most common diseases and threatened human
health[1-5]. Among them, the resistance problem of Gram-positive bacteria (G ) is particularly
serious, such as methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus Aureus
+
(
MSSA), etc [6-7]. Thus, finding effective ways to reduce antibiotic-resistant crisis is an urgent task[8].
F
O
F
O
O
N
F
O
N
H
O
O
N
N
H
NH
N
O
N
N
N
H
N
N
N
N
O
O
N
O
H
Linezolid
Radezolid
Ⅰ
Fig. 1. Chemical structures of oxazolidinones
+
Oxazolidinones are a class of antibacterial agents against a variety of G , including MRSA,
MSSA, and vancomycin-resistant Enterosphere (VRE), which are binded to 50S ribosomal subunit
*
*
* Corresponding author. E-mail address: yanfangzhao@126.com (Y. Zhao)
Corresponding author. E-mail address: dingdong82111@163.com (X. Ding).

to inhibit bacterial protein biosynthesis[9-11]. Linezolid (Fig.1), the first clinically useful
oxazolidinone antibacterial agent, has greatly alleviated the severe situation of G infection[12].
Unfortunately, Linezolid-resistant Staphylococcus aureus and Enterococcus spp have emerged due
to the overuse of Linezolid[13-14]. Thus, it is imperative to develop new oxazolidinone-type drug
candidates against drug-resistant pathogens. The cocrystal structure of Linezolid shows that the
oxazolidinone moiety occupied the domain and was engaged in a hydrogen bond with G 2540 (Fig.
+
2
). But the morpholino moiety did not occupied the cavity completely. And we find there was no
obvious interaction between the Linezolid terminal morpholine ring and the 50S ribosomal subunit
[15].
Therefore, there has been considerable interests in the modification of the morpholine part of
Linezolid.
Fig. 2. The Co-crystal structure analysis of Linezolid
Radezolid (Fig. 1), which has a triazole ring as the “tail”, was developed by Rib-X Laboratories.
It is one of the most active and broad-spectrum antimicrobials in clinical development. The drug
+
showed excellent antibacterial activity against G such as enterococcus, staphylococcus and
streptococcus (MIC range 0.125-1 μg/mL) [16]. As reported, oxazolidinone derivatives that contains
+
+
biaryl scaffold showed increased antibacterial potency against G , especially antibiotic-resistant G
by forming π-π stacking interactions with the 50S subunit compared with Linezolid[17-18]
.
Hideyuki Suzuki and his coworkers replaced the morpholine ring of Linezolid with pyrazole
1,5-a] pyridine ring[19-21] and synthesized a series of new oxazolidone compounds, in which,
[
compound I (1, Fig. 3) ) exhibited potent antibacterial activiey with MIC values 0.25 and 0.5 μg/
mL against Staphylococcus aureus and Enterococcus faecalis, respectively. [21-22]
Hence, to overcome antibacterial resistance and discover a new oxazolidinone with the better
activity, the modification of compound I and Radezolid provide new analogues with moderate to
potent antibacterial activity. Based on our previous work in the laboratory[24-26] and the structure-
activity relationship of oxazolidone, we utilized the biaryl structure of Radezolid to keep the π-π
stacking with the 50S subunit (Fig. 4). At the same time, we introduced heterocycle moiety of
compound I into the target compounds with the aim of trying to find new possible binding mode
and occupy the cavity as far as possible. Compound 3a was synthesized firstly in our work, while
the antibacterial activity was weaker than Linezolid. Hence, there was an assumption that if
introduction of nitrogen atom could enhance the activity in the “tail” of compound or not. Then, a

series of oxazolidinone derivatives containing nitrogen-containing fused heterocycle moieties (3b-
h) were synthesized to occupy the cavity as complete as possible. Of them, compound 3b inhibited
3
S.aureu, MIRSA and MASS with MIC values of 0.5 μg/mL, 0.5 μg/mL and 0.5 μg/mL respectively,
which was more active than Linezolid.
H
N
F
O
N
O
N
N
F
O
H
NH
N
O
R
X
N
H
O
N
F
O
N
N
Radezolid
F
N
O
O
N
H
N
N
N
N
N
N
N
X
N
O
R =
N
O
X
N
O
3a
N
N
N
H
N
3b-3h
O
I
Fig. 3. Design strategies for the novel oxazolidinone analogues containing a nitrogen-
containing fused heterocycle
The synthetic route of key intermediate 4 was depicted in Scheme 1. Intermediate 2 was
prepared from commercially available 4-bromo-3-fluoroaniline 1 by acylation with chloroformate
in dichloromethane. Subsequently, intermediate 2 was reacted with (S)-1-((acetylamino)methyl)-
2
-chloroethyl acetate in the presence of t-BuOLi to afford intermediate 3. And then Miyaura
coupling occurred in intermediate 3 and bis(pinacolato)diboron to yield the key intermediate 4.
F
O
F
O
F
F
O
O
O
O
O
a
b
c
O
Br
N
H
B
N
H
N
N
Br
NH2
Br
NH
O
O
1
2
3
4
Scheme 1. Synthetic route of key intermediate 4. Reagents and conditions: (a) methyl chloroformate, pyridine,
DCM, 25 °C, 2 h; (b) (S)-1-((acetylamino)methyl)-2-chloroethyl acetate, t-BuOLi, DMF, CH OH, r.t, 20 h;
c) bis(pinacolato)diboron, Pd(PPh Cl , potassium acetate, 1,4-dioxane, 90 °C, 20 h.
3
(
3
)
2
2
The synthetic route of intermediates 7a, 7b, 9 and 10 was depicted in Scheme 2. The
intermediates 7a and 7b were prepared from 4-bromobenzonitrile by condensation and cyclization.
Intermediate 8 was prepared from intermediate 7b by reduction of pyrazine, which then underwent
N-methylation in the presence of iodomethane to form intermediate 9. Intermediate 8 reacted with
di-tert-butyl pyrocarbonate to form intermediate 10.

N
NH
N
N
X
N
d
e
X
N
Br
N
H
Br
Br
5
6
6
a: X=C
b: X=N
7a: X=C
7b: X=N
N
N
N
g
N
9
N
N
N
N
N
NH
N
Br
Br
f
N
N
N
N Boc
Br
Br
h
N
7
b
8
10
Scheme 2. Synthetic route of key intermediates 7a, 7b, 9 and 10. Reagents and conditions: (d) 2-aminopyridine
or 2-aminopyrazine, NaH, DMF, 25 °C, 10 h; (e) I , KI, K CO , DMSO, 120 °C, 8 h; (f) sodium borohydride,
Pd(PPh Cl , DCM/EtOH, 25 °C, 24 h; (g) iodomethane, K CO , DMF, 25 °C, 10 h; (h) di-tert-butyl
pyrocarbonate, DIPEA, DMAP, DMF, 25 °C, 5 h.
2
2
3
3
)
2
2
2
3
The routes to synthesize intermediates 17, 19, 21a and 21 b were depicted in Scheme 3. The
-bromino acetophenone 11 reacted with diethyl oxalate to form intermediate 12. The pyrazole ring
4
was constructed to intermediate 13 by reaction of 12 with hydrazine hydrate. Treatment of
intermediate 13 with 2-bromoethanol gave intermediate 14, which was reduced in the presence of
lithium aluminum hydride in tetrahydrofuran to give 15. Intermediate 15 reacted with PBr to yield
3
key intermediate 16. Subsequently, construction of the piperazine ring afforded the intermediate 17
and 18. Intermediate 18 was protected with di-tert-butyl pyrocarbonate to yield intermediate 19.
Intermediates 21a and 21b were constructed respectively through cyclization.
O
O
O
O
O
OEt
OEt
OEt
j
i
k
l
NH
N
O
Br
N
N
OH
Br
Br
Br
1
1
12
1
3
14
N
n
N
N
OH
Br
OH
m
Br
Br
N
N
Boc
N
5
N
17
N
NH
Br
Br
N
N
h
o
N
1
16
N
N
Br
Br
1
8
19
N
O
p
N
Br
X
Br
2
0
21a: X=C
1b: X=N
2

Scheme 3. Synthetic route of intermediates 17, 19, 21a and 21 b. Reagents and conditions: (i) Na, EtOH,diethyl
oxalate, 25 °C, 10 h; (j) hydrazine hydrate, 78 °C, 10 h; (k) 2-bromoethanol, K CO , DMF, 25 °C, 12 h; (l)
LiAlH , THF, 25 °C, 2 h; (m) PBr , DCM, 25 °C, 10 h; (n) methylamine hydrochloride, K CO , 25 °C, 6 h; (o)
NH , K CO , 25 °C, 2 h; (h) di-tert-butyl pyrocarbonate, DIPEA, DMAP, DMF, 25 °C, 5 h; (p) 2-
hydrazinylpyridine or 2-hydrazinylpyrazine, I , TBHP, K CO , 1,4-dioxane, 60 °C, 1 h.
2
3
4
3
2
3
3
2
3
2
2
3
The synthetic route of the target compounds 3a-3h was indicated in Scheme 4. Suzuki coupling
reaction of compound 4 with the above intermediates gave the target compounds 3a-3b and 3f-3h.
Compounds 3c and 3e were obtained by removing the amino protecting group of intermediate 22
and 23, respectively.
F
O
N
N
N
O
7a
N
H
N
3a
O
F
O
N
N
7b
N
O
N
H
N
N
3b
O
F
O
N
N
N
N
O
O
9
N
H
N
N
N
3
d
O
O
F
O
N
17
N
H
N
q
3f
4
F
F
O
N
N
N
O
21a
N
H
N
N
3g
O
O
N
N
N
O
21b
N
H
N
3h
O
F
O
F
O
N
N
N
N
N
N
O
O
1
0
N
O
r
N
N
H
H
N
HN
HN
N
N
Boc
Boc
2
2
3c
O
O
O
O
F
O
F
O
N
N
N
O
1
9
r
N
H
N
N
H
N
N
2
3
3e
Scheme 3. Synthetic route of target compounds 3a-3h. Reagents and conditions: (q) Pd(PPh
3 2 2 2 3
) Cl , K CO ,
DMF/ H O, 75 °C, 8-12 h; (r) TFA, DCM, 25 °C, 5h.
2
The antibacterial activities of the target compounds were evaluated in vitro against five
bacterial strains including S.aureus, MRSA, MSSA, LREF and VRE by MIC assay. The results
were shown in Table 1.
Table 1 Structures and in vitro antibacterial activities of compound 3a-3h.
F
O
O
R
N
H
N
O

R
MICs, μg/mL
Comp
ounds
S.aureusa
MRSAb
MSSAc
LREFd
VREe
N
N
N
3
a
2.0
1.0
2.0
1.0
0.5
N
N
3
3
3
3
3
b
c
d
e
f
0.5
2.0
1.0
2.0
1.0
0.5
2.0
1.0
2.0
1.0
0.5
1.0
1.0
2.0
1.0
0.5
1.0
2.0
2.0
1.0
0.5
1.0
1.0
0.5
0.5
N
N
N
N
N
HN
N
N
N
N
N
N
HN
N
N
N
N
N
N
3
3
g
N
1.0
2.0
1.0
2.0
1.0
1.0
1.0
1.0
1.0
2.0
N
N
h
N
Linez
1
0
.0
.5
1.0
0.5
1.0
0.5
＞16
2.0
0.5
olid
olid
Radez
0.5
a
b
c
d
e
Standard Staphylococcus aureus (29213).
Methicillin-resistant Staphylococcus aureus.
Methicillin-sensitive Staphylococcus aureus.
Linezolid-resistant Enterococcus faecalis.
Vancomycin-resistant Enterococcus faecium.
According to the data in Table 1, compounds 3d, 3f and 3g appeared to exhibit similar
antibacterial activity in comparison with Linezolid. Of them, compound 3b, with the MIC value 0.5
μg/mL, was comparable to Linezolid. Compound 3a and 3h with pyridinyl moiety exhibited weaker
in vitro anti-bacterial activity than other compounds. The SARs (structure-activity relationships) of
the oxazolidinone derivatives were studied. It was speculated that piperazinyl or pyrazinyl moiety
may conducive to the keep of activity. Methylation of compound 3f on the nitrogen atom of
piperazinyl moiety showed 2 fold potency advantages compared to compound 3e, which deduced
that the tertiary amine maybe conducive to the keep of activity when piperazinyl was used. In
addition, compound 3e with pyrazolyl moiety and 3c with triazolo moiety exhibited similar levels
of in vitro antibacterial activity. In conclusion, most of compounds exhibited good to moderate
+
antibacterial activity against G and selected antibiotic-susceptible and Linezolid-resistant isolates.
Compound 3b was selected for further clinical isolate investigation in the next experiment.
To elucidate the activity against multiple strains of MDR-S.aureus, compounds 3b was
evaluated against several antibiotic-susceptible and antibiotic-resistant clinical isolates, and the
+
results were summarized in Table 2. Clinical strains of G included S.aureus (4 isolates of MRSA
and 4 isolates of MSSA), E.faecium (4 isolates of VRE) and E. faecalis (4 isolates of LREF), and
all the isolates were collected in PLA 309 hospital. Compound 3b with the R-substituent group
replaced by [1,2,4]triazolo[1,5-a]pyrazine displayed increased activity against the tested clinical
isolates with MIC values of 0.5-1 μg/mL, which was 32-fold more potent against clinical iso-lates
of LREF than that of Linezolid.

Table 2. In vitro antibacterial activity against clinical isolates.
MICs, μg/mL
Compounds
a
b
c
d
e
MRSA (4)
MSSA (4)
LREF (4)
VRE (4)
3
b
0.5-1
0.5-1
0.5
0.5
Linezolid
1-4
1-4
＞16
1-4
a
b
c
d
e
.
Methicillin-resistant Staphylococcus aureus.
Number of bacterial strains tested are given in parentheses.
Methicillin-sensitive Staphylococcus aureus.
Vancomycin-resistant Enterococcus faecium.
Linezolid-resistant Enterococcus faecalis.
With the interest of the binding modes of compound 3b, the docking analysis of compound
b and Linezolid were performed depended on the crystal structure of 50S ribosome unit of E.
3
coli with linezolid (PDB code: 3CPW [15]). As shown in Fig. 4, compound 3b has the similar
binding pattern with Linezolid (Fig. 4, B). The condensed heteroaromatic moiety of 3b took
over more cavity than the morpholinyl moiety in Linezolid, and had π-π stacking interactions
with U 2619 and U 2620 (Fig. 4, A), which could result in high antibacterial activity.
A
B
Fig. 4. The Co-crystal structure analysis of 3b. (A) Predicted binding conformation for 3b (light
blue sticks) and the interaction between 3b and the binding site cavity, (B) 3b overlapping with
Linezolid (gray sticks).
Then we used the free SwissADME web tool (http://www. swissadme.ch/) to
predict physicochemical, ADME parameters and drug-likeness properties of above compounds. As
we can see from the Table 3, all the compounds fitted well with Lipinski’s rule, which means all
these compounds are hopeful to develop into oral drugs. And all the compounds showed no BBB
permeability and high GI values, which indicated that these compounds were unlikely to cause CNS
adverse effects.
Table 3. In silico Molecular properties prediction at SwissADME.
Hbond
donors
Hbond
acceptors
6
compounds MWa logPb
445.45 3.23
GIe TPSAf lipinskig logSh BBBi
-4.87 No
c
d
3
h
1
High 88.83
0

3
3
3
3
3
3
3
g
f
446.43 2.53
463.50 2.63
449.48 2.31
464.49 2.40
445.45 3.32
446.43 2.62
450.47 2.12
337.35 1.20
438.45 2.01
1
1
2
1
1
1
2
1
3
7
6
6
7
6
7
7
5
7
High 101.72
High 79.70
High 88.49
High 92.59
High 88.83
High 101.72
High 101.38
High 71.11
High 112.24
0
0
0
0
0
0
0
0
0
-4.20 No
-4.02 No
-3.65 No
-3.88 No
-4.60 No
-3.93 No
-3.51 No
-2.22 No
-3.12 No
e
d
a
b
c
Linezolid
Radezolid
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.
In summary, a series of novel oxazolidinone derivatives with nitrogen-containing fused
heterocyclic moiety was synthesized as potential antibacterial agents and their structures were
1
confirmed by H NMR and MS spectra. Their anti-bactierial activities were evaluated against S.
aureus, MRSA, MSSA, LREF and VRE pathogens and most of them exhibited potent in vitro
antibacterial activity as compared with Linezolid. Notably, compound 3b showed the most potent
+
antibacterial activity with MIC values of 0.5-1 μg/mL against five G , which was comparable to
Linezolid. This study provided the researchers more insights about the antibacterial agents.
Acknowledgements
Supported by LiaoNing Revitalization Talents Program (XLYC1805014), Basic Research
Projects of Higher Education Institutions in Liaoning Province (2017LZD06).
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H
N
F
O
N
O
N
N
F
O
H
NH
N
O
molecular
hybridization
R
X
N
H
O
F
O
N
N
N
Radezolid
F
N
O
O
N
H
N
N
N
N
N
N
N
X
N
O
R =
N
O
X
N
O
3a
N
N
N
H
N
3b-3h
O
I