Synthesis and antibacterial evaluation of novel oxazolidinone derivatives containing a piperidinyl moiety

Article abstract of DOI:10.1016/j.bmcl.2019.126746

In this article, a series of novel oxazolidinone derivatives containing a piperidinyl moiety was designed and synthesized. Their antibacterial activities were measured against S. aureus, MRSA, MSSA, LREF and VRE by MIC assay. Most of them exhibited potent activity against Gram-positive pathogens comparable to linezolid. Among them, compound 9h exhibited comparable activity with linezolid against human MAO-A for safety evaluation and showed moderate metabolism in human liver microsome. The most promising compound 9h, which showed remarkable antibacterial activity against S. aureus, MRSA, MSSA, LREF and VRE pathogens with MIC value of 0.25–1 μg/mL, was an interesting candidate for further investigation.

Full text of DOI:10.1016/j.bmcl.2019.126746

Journal Pre-proofs
Synthesis and antibacterial evaluation of novel oxazolidinone derivatives con-
taining a piperidinyl moiety
Yunlei Hou, Yuhong Dong, Tianyu Ye, Jia Jiang, Liang Ding, Mingze Qin,
Xiudong Ding, Yanfang Zhao
PII:
S0960-894X(19)30709-7
DOI:
https://doi.org/10.1016/j.bmcl.2019.126746
BMCL 126746
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
6 September 2019
29 September 2019
8 October 2019
Please cite this article as: Hou, Y., Dong, Y., Ye, T., Jiang, J., Ding, L., Qin, M., Ding, X., Zhao, Y., Synthesis and
antibacterial evaluation of novel oxazolidinone derivatives containing a piperidinyl moiety, Bioorganic & Medicinal
Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126746
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Synthesis and antibacterial evaluation of novel oxazolidinone derivatives
containing a piperidinyl moiety
Yunlei Houa, Yuhong Donga, Tianyu Yea, Jia Jianga, Liang Dinga, Mingze Qina, Xiudong Dingb*, Yanfang Zhaoa**12
a
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, 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：
In this article, a series of novel oxazolidinone derivatives containing a piperidinyl moiety was designed and
synthesized. Their antibacterial activities were measured against S.aureus, MRSA, MSSA, LREF and VRE by MIC
assay. Most of them exhibited potent activity against Gram-positive pathogens comparable to linezolid. Among them,
compound 9h exhibited comparable activity with linezolid against human MAO-A for safety evaluation and showed
moderate metabolism in human liver microsome. The most promising compound 9h, which showed remarkable
antibacterial activity against S.aureus, MRSA, MSSA, LREF and VRE pathogens with MIC value of 0.25-1 μg/mL,
was an interesting candidate for further investigation.
Keywords: Oxazolidinone derivatives; Piperidinyl moiety; Antibacterial activities; Linezolid
The resistance of bacteria developed rapidly and spreads widely throughout the world due to the long-term,
inappropriate use and even abuse of antibiotics[1-3]. Among them, the resistance problem of Gram-positive bacteria
(G+) is particularly serious, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant
Enterosphere (VRE), etc. The emergence of drug-resistant bacteria has led to a significant prolongation of treatment
[4,
time and a mortality increase
drug-resistant pathogens[6].
5]. Thus, it is imperative to develop new agents with great potency against
** Corresponding author. E-mail address: yanfangzhao@126.com (Y. Zhao)
* Corresponding author. E-mail address: dingdong82111@163.com (X. Ding).

O2N
O
O2N
F
O
F
O
F
O
O
O
O
O
O
N
N
N
H
N
N
N
N
N
N
H
H
N
N
N
O
O
O
Linezolid
Ranbezolid
Compound I
Fig. 1. Chemical structures of linezolid and other oxazolidinones.
Oxazolidinones are a new class of chemically synthesized antibacterial agents against a variety of Gram-positive
bacteria, including MRSA, methicillin resistant Staphylococcus Aureus (MSSA), VRE, etc. The unique antibacterial
mechanism of oxazolidinones, which was inhibiting the synthesis of bacterial proteins by binding to the bacterial 50S
ribosomal subunit, had attracted much attention[7-10]. The first oxazolidinone antibacterial agent, linezolid (Fig. 1),
was approved for the treatment of infections caused by multi-drug resistant G+ bacteria in the United States in April
2000[11]
.
However, linezolid-resistant Staphylococcus aureus and Enterococcus spp had appeared since linezolid
was released. Linezolid was also an inhibitor of monoamine oxidases (MAOs), which may result in drug−drug
interactions with adrenergic and serotonergic agents[12-14]. Thus, there has been considerable interests in new
oxazolidinone-type drug candidates[15]. The cocrystal structure of linezolid revealed that the oxazolidinone moiety
occupied the active domain and was engaged in a hydrogen bond with G 2540[16]. And the morpholine “tail” was not
occupied the cavity, completely (Fig. 2). Accordingly, the efficient incorporation of a group into the “tail” has
attracted considerable interest among medicinal chemists.
Fig. 2. The Co-crystal structure analysis of linezolid (PDB code: 3CPW)
Ranbezolid (Fig. 1), which had a furan ring as the terminal unit, was developed by Ranbaxy Laboratories, and
its phase I clinical trial was performed in 2003[17-18]. Docking studies showed that the piperazine ring attached with a
2-methyl-5-nitrofuryl moiety had a higher total score than linezolid. Moreover, the nitrofuryl ring from the
oxazolidinone extends to C2507, G2583 and U2584, and the nitro group forms a hydrogen bond with the base of
G2583, which makes stronger interaction with the target pathogen[19]. In our previous work, a series of oxazolidinone

derivatives containing nitroheteroaromatic moieties was reported [20]. Of them, compound I (Fig. 1) inhibited S.aureu,
MRSA and VRE with MIC values of 0.5 μg/mL, 1 μg/mL and 1 μg/mL, respectively, which was more active than
Ranbezolid. However, it was contemplated that other antimicrobial compounds containing nitrofuryl group can be
catalyzed in vivo by related enzymes into the nitro group through an active nitroso radical intermediate, which in turn
produces cytotoxicity[21, 22]. At the same time, the presence of the hydrazone bond rendered the compound rigid and
insoluble, and easily hydrolyzed, which was detrimental to metabolic stability.
Delamanid was initially approved by the European Medicines Agency (EMA) in 2014 for the treatment of adult
pulmonary multi-drug resistant (MDR)-TB when an effective treatment regimen cannot otherwise be composed for
reasons of resistance or tolerability. It has been approved by several other countries/regions.[23] The
phenoxypiperidinyl “tail” of delamanid was necessary for the maintaining the antibacterial activities. In a continuing
effort to develop new drug candidates, we herein described the design, synthesis, and antibacterial activities of a
series of novel oxazolidinone derivatives. In these derivatives, the nitrohetero aromatic ring of compound I was
replaced with various substituted phenyl group or benzyl group to avoid toxic side effects, and the piperazinyl group
was replaced with a piperidinyl moiety by the bioisosteric principle caused by the hydrazone. At the same time, a NH
acted as a hydrogen bond donor was introduced as a linker between the phenyl ring and piperidinyl group to increase
the antibacterial activity (Fig. 3).
Cytotoxicity
F
O
O2N
O
F
O
H
O
N
O
X
N
N
N
N
N
N
H
O
N
n
R
Metabolic stability
O
9a-9n, X=O, NH, n=0, 1
Bioisosteric
Scaffold hopping
Compound I
N
NO2
O
N
O
F3CO
N
O
Delamanid
H bound
9a
Fig. 3. Design strategies for the novel oxazolidinone analogues containing a piperidinyl moiety.
The routes used to synthesize key intermediate 8 and compounds 9a-9n were depicted in Scheme 1 and Scheme
2, respectively. Intermediate 2 was prepared from commercially available 1-(tert-butyloxycarbonyl)piperidin-4-one 1
by deprotection with HCl in 1,4-dioxane. Subsequently, intermediate 2 was reacted with 3,4-difluoronitrobenzene in

the presence of acetonitrile to afford 3. Intermediate 3 was protected with ethylene glycol to yield the precursor 4,
which was reduced in the presence of 5% palladium on carbon in methanol and then reacted with methyl
chloroformate to yield intermediate 6. The oxazolidinone ring was constructed to the intermediate 7 by reaction of 6
with (S)-1-acetamido-3-chloropropan-2-yl acetate in a facile one-step process[24-27]. Removal of the carbonyl
protecting group of intermediate 7 with p-toluenesulfonic acid afforded key intermediate 8.
Compounds 9a−9m were prepared through reductive amination of various substituted anilines/benzylamines
with key intermediate 8 in the presence of sodium triacetoxyborohydride in DCM. Compound 9h was methylated
with iodomethane to generate compound 9n. The structures of all target compounds were confirmed by 1H NMR
and MS.
F
a
b
c
O
N Boc
O
NH
HCl
O
N
NO2
3
1
2
F
O
F
F
O
O
O
d
e
O
O
N
O
NH
N
NH2
N
4
NO2
O
O
6
5
F
O
F
O
N
O
g
f
O
O
H
H
N
N
N
N
O
N
O
O
7
8
Scheme 1. Synthetic route of key intermediate 8. Reagents and conditions: (a). hydrochloric acid, methanol,
25 °C, 5h; (b). 3,4-difluoronitrobenzene, N-ethyldiisopropylamine, acetonitrile, 80 °C, 8h; (c). Ethylene glycol,
p-toluenesulfonic acid, toluene, 110 °C,7h; (d). 5% palladium on carbon, methanol, 25 °C, 1h；(e). methyl
carbonochloridate, pyridine, dichloromethane, 25 °C, 4h; (f). (S)-1-acetamido-3-chloropropan-2-yl acetate,
t-BuOLi, methanol, dimethyl formamide, 25 °C, 23h; (g). p-toluenesulfonic acid, H2O, acetone, 70 °C, 5h.

NH2
F
O
R1
O
H
N
HN
N
N
a
O
F
O
R1
O
9a-9k
H
N
O
N
N
F
O
O
NH2
O
R2
8
H
N
HN
N
N
b
O
R2
9l-9m
F
O
F
O
N
O
O
H
H
c
N
N
HN
N
N
N
N
O
O
9h
9n
Br
Br
Scheme 2. Synthetic route of target compounds 9a-9n. Reagents and conditions: (a). Sodium
triacetoxyborohydride, acetic acid, dichloromethane, 25 °C, 24h; (b). Sodium triacetoxyborohydride, acetic
acid, dichloromethane, 25 °C, 24h; (c). Iodomethane, K2CO3, dimethyl formamide, 35 °C, 6h.
The antibacterial activities of the target compounds 9a-9n were evaluated in vitro against five bacterial strains
including S.aureus, MRSA, MSSA, LREF and VRE by MIC assay. The results were summarized in Table 1.
According to the data in Table 1, most of the tested compounds displayed potent antibacterial activity against
Gram-positive organisms and selected antibiotic-susceptible and antibiotic-resistant isolates comparable to linezolid,
and the activity ranges from 2 to 0.25 μg/mL. The preliminary SARs (structure-activity relationships) of the
oxazolidinone derivatives were studied. Compound 9h showed 64-fold more potent against LREF than that of
linezolid, which was the most active compounds having a MIC value of 0.25 μg/mL. Compound 9a with hydrogen
group and 9c with 4-OCF3 group exhibited similar levels of in vitro antibacterial activity, while compound 9d with
3-OCF3 group and 9e with 2-OCF3 group showed nearly 2-fold weaker in vitro antibacterial activity compared to
linezolid. This result indicated that the substituent on the para-position of phenyl maybe superior to ortho-position or
meta-position. This observation can be obtained on the compound 9h, 9i and 9j. By observing the antibacterial
activity of compounds 9h-9j, the potency order of antibacterial activity was para-position > ortho-position =
meta-position. In order to determine whether the extension of linker had an influence on activity, compounds 9a, 9f
and 9l, 9m were synthesized. Compounds 9l, 9m exhibited weaker activity than 9a, 9f against LREF and VRE,
respectively. These results implied that extension of linker may be not conducive to the improvement of activity.
Methylation of compound 9h on the nitrogen atom of the linker reduced the activity (9n, MIC=2 μg/mL, 2
μg/mL, 2 μg/mL, 1 μg/mL and 1 μg/mL), which deduced that the secondary amine as linker may be essential for the
activity. Thus, we speculated that the N- piperidinyl moiety may form extra hydrogen bonds and interact with the

binding site of other antibiotic. And this phenomenon could be explained by the result of molecular docking models.
Overall, most compounds exhibited moderate-to-significant antibacterial activity against Grampositive organisms and
selected antibiotic-susceptible and antibiotic-resistant isolates as compared with linezolid. Then, 9a and 9h were
selected for further clinical isolates investigation.
Table 1 The antibacterial activity of target compounds
O
O
F
F
O
F
O
O
O
N
N
N
N
HN
HN
N
N
H
N
H
H
N
N
N
R2
O
O
O
R1
Br
9l-9m
9n
9a-9k
MIC (μg/mL)
S.aureusa MRSAb MSSAc LREFd
Compound
R1/R2
VREe
1
9a
H
1
1
1
0.5
9b
4-isopropyl
2
1
2
0.5
0.5
9c
4-OCF3
1
1
1
0.5
0.5
9d
3-OCF3
2-OCF3
4-F
2
2
2
2
2
2
2
1
2
2
9e
9f
1
0.5
2
1
1
1
9g
4-Cl
4-Br
3-Br
2-Br
2-F, 4-Br
H
2
2
2
2
9h
1
0.5
2
1
0.25
2
0.25
2
9i
2
2
9j
2
2
2
2
2
9k
9l
2
2
2
1
2
2
1
1
2
2
9m
4-F
1
1
1
2
2
9n
-
2
2
2
1
1
Linezolid
Ranbezolid
-
1
1
1
＞16
0.5
2
-
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.
To ascertain their spectrum of activity against multiple strains of MDR-S. aureus, compounds 9a and 9h were
evaluated against several antibiotic-susceptible and antibiotic-resistant clinical isolates, and the results were
summarized in Table 2. Clinical strains of gram-positive bacteria 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 9a displayed moderate activity against the tested clinical isolates with MIC
values of 0.125-1 μg/mL. Significantly, compound 9h showed high activity against the tested clinical isolates with

MIC values in the range of 0.0625–1 µg/mL, which was 64-fold more potent against clinical isolates of LREF than
that of linezolid
Table 2. In vitro antibacterial activity against clinical isolates
Isolates
MRSAa (4)b
Compound
MICs (μg/mL)
0.5-1
9a
9h
0.5-1
linezolid
9a
1-4
MSSAc (4)
VREd(4)
0.5-1
9h
0.5-1
linezolid
9a
1-2
0.25-0.5
0.125-0.25
1-4
9h
linezolid
9a
LREFe(4)
0.125-0.25
0.0625-0.25
>16
9h
linezolid
a Clinical strains of methicillin-resistant Staphylococcus aureus.
b Number of bacterial strains tested are given in parentheses.
c Clinical strains of methicillin-sensitive Staphylococcus aureus.
d Clinical strains of vancomycin-resistant Enterococcus faecium.
e Clinical strains of linezolid-resistant Enterococcus faecalis.
Linezolid and other oxazolidinone antibacterial agents were reversible, nonselective inhibitors of human MAO due
to their structural similarity with toloxatone, a known MAO inhibitor. Clinical use of the oxazolidinone antibacterial
agents has shown that these agents had a side effect of hypertension because of MAOI [28]. To determine the safety
profile of compounds synthesized, we evaluated the MAO-A inhibitory activities of several representative
compounds (9a, 9h) and the results were summarized in Fig. 4. Compound 9h exhibited weaker activity than
linezolid against human MAO-A, which may be a drug candidate from a safety viewpoint.
Fig. 4. MAO-A inhibition of potent compounds
Based on the in vitro antibacterial activity and inhibition of MAO-A, 9h was selected for in vitro cytotoxicity
and metabolic stability, and the results were outlined in Table 4. Compound 9h and linezolid exhibited IC50 values >
64 μM against HepG2 cell, which indicated they were safety to hepatic cells to some extent. The human liver

microsome studies showed that 9h had a long half-life (501.99 min) and low liver microsomal clearance (3.46
mL/min/kg) in human liver microsome，which indicated that 9h was stable in human liver microsome.
Table 3. In vitro cytotoxicity and ADME results
Human liver microsome (HLM)
HepG2
cytotoxicity
IC50 (μM)
clogD
Compounds
(pH=7.4)a
T1/2
(min)
Clint
Substrate
Stability
(mL/min/kg)
remaining(%)b
9h
2.91
0.64
>64
>64
501.99
3.46
0
97
stable
stable
linezolid
∞
104.18
aCalculated using instant JChem.
bSubstrate remaining were determined in incubations with NADPH after 45 min
To understand the possible mechanism of action of compound 9h, molecular superposition of 9h and linezolid
were performed based on the crystal structure of 50S ribosome unit of E. coli with linezolid (PDB code: 3CPW [16]).
As shown in Fig. 5, 9h was found to form two hydrogen bonds via the NH of acetamide and “tail” aniline moiety
with G 2540 and U 2620, respectively. Compound 9h and linezolid have a high degree of overlap (Fig. 5D).
Furthermore, the N-piperidinyl part of compound 9h extended beyond the morpholine ring of linezolid. As shown in
Table 1, compound 9n exhibited weaker activity than 9h against the different bacteria. On the other hand, the
docking results of compound 9n suggested that the methylated N atom could not form hydrogen bond with U 2620.
Meanwhile, the introduction of methyl group resulted the forming of unfavorable bump. Therefore, the NH of played
an important role. This result was also consisted with the in vitro antibacterial activity, which the methylated product
9n displayed lower potency than compound 9h.

Fig. 5. Superposition of compound 9h and 9n. (A) and (B): Predicted binding conformation for 9h (light blue sticks)
in the binding site cavity, (C): 2D diagram of the interaction between 9h and the binding site cavity, (D) 9h
overlapping with linezolid (pink sticks), (E): 2D diagram of the interaction between 9n and the binding site cavity, (F)
predicted binding conformation for 9n (blue sticks) in the binding site cavity.
In summary, a series of novel oxazolidinone derivatives containing a piperidinyl moiety were synthesized as
1
potential antibacterial agents and their structures were confirmed by H NMR, and MS spectra. Most compounds
exhibited potent antibacterial activity against S.aureus, MRSA, MSSA, LREF and VRE pathogens as compared with
linezolid and radezolid. Compounds 9a and 9h were more potent against the tested clinical isolates of MRSA, MSSA,
VRE and LREF than linezolid. The exploration of preliminary structure-activity relationships showed that the
introduction of N-piperidinyl moiety can improve the potency against linezolid-susceptible and linezolid-resistant
Gram-positive bacteria. Compound 9h showed the most potent antibacterial activity with MIC values of 0.25-1
μg/mL against five Gram-positive bacteria, and its potency against LREF was 64-fold higher than that of linezolid.

Moreover, compound 9h was non-cytotoxic with an IC50 value of > 64 μM against HepG2 cell, and 9h exhibited
comparable inhibitory activity with linezolid against human MAO-A. 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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O
F
O
F
O
HN
N
N
O
H
O
N
N
N
H
N
O
O
9h
linezolid
Br
S.aureus MIC= 1 ug/mL
S.aureus MIC= 1 ug/mL
MRSA
MSSA
LREF
VRE
MIC= 0.5 ug/mL
MIC= 1 ug/mL
MIC= 0.25 ug/mL
MIC= 0.25 ug/mL
MRSA
MSSA
LREF
VRE
MIC= 1 ug/mL
MIC= 1 ug/mL
MIC >16 ug/mL
MIC= 2 ug/mL
Superposition of compound 9h and linezolid
[29]
Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could
have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as
potential competing interests:
[30]