Synthesis and antibacterial bioactivities of cationic deacetyl linezolid amphiphiles

Article abstract of DOI:10.1016/j.ejmech.2018.06.054

Bacterial infections cause various life-threatening diseases and have become a serious public health problem due to the emergence of drug-resistant strains. Thus, novel antibiotics with excellent antibacterial activity and low cytotoxicity are urgently needed. Here, three series of novel cationic deacetyl linezolid amphiphiles bearing one lipophilic alkyl chain and one non-peptidic amide bond were synthesized and tested for antimicrobial activities. Several compounds showed excellent antibacterial activity toward drug-sensitive bacteria such as gram-negative bacteria Escherichia coli (E. coli), Salmonella enterica (S. enterica) and gram-positive Staphylococcus aureus (S. aureus), Enterococcus faecalis (E. faecalis). Moreover, these amphiphilic molecules also exhibited strong activity against drug-resistant species such as methicillin-resistant S. aureus (MRSA), KPC (Klebsiella pneumoniae carbapenemase) and NDM-1 (New Delhi metallo-β-lactamase 1) producing carbapenem-resistant Enterobacteriaceae (CRE). For example, the MICs (minimum inhibitory concentrations) of the best compound 6e, ranged from 2 to 16 μg/mL and linezolid ranged from 2 to >64 μg/mL against these strains. Therefore, 6e is a broad-spectrum antimicrobial compound that may be a suitable lead as an antibiotic. The molecule 6e were found to function primarily by permeabilization and depolarization of bacterial membranes. Importantly, bacterial resistance against compound 6e was difficult to induce, and 6e was stable under plasma conditions and showed suitable activity in mammalian plasma. Thus, these compounds can be further developed into a potential new class of broad-spectrum antibiotics.

Full text of DOI:10.1016/j.ejmech.2018.06.054

Accepted Manuscript
Synthesis and antibacterial bioactivities of cationic deacetyl linezolid amphiphiles
Peng-Yan Bai, Shang-Shang Qin, Wen-Chao Chu, Yi Yang, De-Yun Cui, Yong-Gang
Hua, Qian-Qian Yang, En Zhang
PII:
S0223-5234(18)30545-2
10.1016/j.ejmech.2018.06.054
EJMECH 10524
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 November 2017
Revised Date: 10 May 2018
Accepted Date: 22 June 2018
Please cite this article as: P.-Y. Bai, S.-S. Qin, W.-C. Chu, Y. Yang, D.-Y. Cui, Y.-G. Hua, Q.-Q. Yang,
E. Zhang, Synthesis and antibacterial bioactivities of cationic deacetyl linezolid amphiphiles, European
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.06.054.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Synthesis and antibacterial bioactivities of cationic deacetyl linezolid
amphiphiles
#
a
#a
a
a
a
a
Peng-Yan Bai , Shang-Shang Qin , Wen-Chao Chu , Yi Yang , De-Yun Cui , Yong-Gang Hua ,
a
a, b
Qian-Qian Yang , En Zhang *
a
School of Pharmaceutical Sciences; Institute of Drug Discovery and Development; Key
Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; Zhengzhou
b
University, Zhengzhou 450001, PR China. Collaborative Innovation Center of New Drug
#
Research and Safety Evaluation, Henan Province, Zhengzhou 450001, PR China. These authors
contributed equally.
E-mail: zhangen@zzu.edu.cn;
ABSTRACT
Bacterial infections cause various life-threatening diseases and have become a
serious public health problem due to the emergence of drug-resistant strains. Thus,
novel antibiotics with excellent antibacterial activity and low cytotoxicity are urgently
needed. Here, three series of novel cationic deacetyl linezolid amphiphiles bearing
one lipophilic alkyl chain and one non-peptidic amide bond were synthesized and
tested for antimicrobial activities. Several compounds showed excellent antibacterial
activity toward drug-sensitive bacteria such as gram-negative bacteria Escherichia
coli (E. coli), Salmonella enterica (S. enterica) and gram-positive Staphylococcus
aureus (S. aureus), Enterococcus faecalis (E. faecalis). Moreover, these amphiphilic
molecules also exhibited strong activity against drug-resistant species such as
methicillin-resistant S. aureus (MRSA), KPC (Klebsiella pneumoniae carbapenemase)
and NDM-1 (New Delhi metallo-β-lactamase 1) producing carbapenem-resistant
Enterobacteriaceae (CRE). For example, the MICs (minimum inhibitory
concentrations) of the best compound 6e, ranged from 2 to 16 µg/mL and linezolid
ranged from 2 to >64 µg/mL against these strains. Therefore, 6e is a broad-spectrum
antimicrobial compound that may be a suitable lead as an antibiotic. The molecule 6e
were found to function primarily by permeabilization and depolarization of bacterial
membranes. Importantly, bacterial resistance against compound 6e was difficult to
induce, and 6e was stable under plasma conditions and showed suitable activity in

ACCEPTED MANUSCRIPT
mammalian plasma. Thus, these compounds can be further developed into a potential
new class of broad-spectrum antibiotics.
Keywords: Antimicrobial; deacetyl linezolid; amphiphilic; drug-resistant
1
. Introduction
Infectious diseases caused by pathogenic bacteria is widespread and the use of
current antibiotics to treat bacterial infections is becoming ever more problematic
with the emergence of multiple antibiotic-resistant bacteria [1]. The overuse and
misuse of antimicrobial agents has given rise to multidrug-resistance bacteria, and
these strains now represent a severe threat to public health and antibiotic therapy [2].
There are many pathogenic bacteria that show resistances to antibiotics, notably the
“ESKAPE” bacteria: Enterococcus faecium (E. faecium), S. aureus, Klebsiella
pneumoniae (K. pneumonia), Acinetobacter baumanii, Pseudomonas aeruginosa and
Enterobacter species [3]. Penicillin-resistant S. aureus was first reported in the 1940s
[
4] and MRSA appeared in the 1960s [5]. Currently, MRSA is the most commonly
identified drug-resistant bacterium worldwide [6, 7]. Quaternary ammonium
compounds (QACs) were widely used as antibiotic agents because of their
biofilm-eradicating properties, but the presence of qac genes in MRSA led to the
emergence of resistance to QACs.[8-12] The carbapenem-resistant strains are also
very problematic. For example, KPC-1 and KPC-2 from a carbapenem-resistant strain
of K. pneumoniae exhibit resistance to all β-lactam compounds and other
antimicrobials [13, 14], and the NDM-producing isolates are highly resistant to most
antibiotics [15-17]. Although the number of bacteria showing antibiotic resistance
continues to grow, the number of new systemic antibacterial agents approved by the
US FDA between 1983 and 2012 has continued to decrease [18]. Thus, prevailing
bacterial resistance to antibiotics combined with stagnant antibiotic discovery has
created an urgent need for the development of new antimicrobial agents that exert
novel mechanisms of action [19, 20].
Linezolid was the first oxazolidinone antimicrobial agent [21] and this
compound represented a new synthetic class of antibacterial agents with activity
against gram-positive bacteria [22]. Linezolid inhibits initiation of protein synthesis in

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bacteria [23]. Previous studies reported that linezolid showed antibacterial activity
against several microorganisms such as S. aureus, penicillin-resistant Streptococcus
pneumoniae, MRSA and vancomycin-resistant E. faecium [24-28]. However, recent
studies reported that E. faecium, S. aureus, vancomycin-resistant E. faecium and S.
cohnii have developed resistance against linezolid [29-32]. Furthermore, linezolid is
not well suited for combating gram-negative pathogenic bacteria that are intrinsically
resistant because of efflux pumps that efficiently excrete linezolid from the cell [33,
3
4]. Resistance to linezolid by bacteria is caused mainly by modification of the
linezolid binding site on the ribosome [35, 36]. Thus, there is an extremely urgent
need to develop new antibacterial drugs that tackle drug-resistant bacteria. Besides
linezolid, Eperezolid and Tedizolid were also approved antibiotics, and some
compounds (Sutezolid, AZD5847 and Radezolid) were under clinical investigation as
potential antibacterial agents (Fig. 1) [37]. Moreover, many studies reported linezolid
derivatives with excellent antibacterial activities. For example, torezolid showed
higher in vitro antibacterial activities and better in vivo protective effects in mice [38].
In addition, 1,2,4-triazolo[4,3-a]pyrimidine oxazolidinones are potential antibacterial
agents [39].
Fig.1. Structures of approved and developmental antibacterial oxazolidinone.
Antimicrobial peptides (AMPs) have received substantial attention in recent
years owing to their membrane targeting mechanism, broad-spectrum activity against

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bacteria and low cytotoxicity [40-43]. Infections caused by antimicrobial-resistant
bacteria are currently treated with several AMPs in preclinical and clinical settings
[
44]. Encouraged by the huge achievement of AMPs, a variety of synthetic
membrane-active mimics have been developed as an alternative to antibacterial
peptides. Binaphthyl-1,2,3-triazole peptidomimetics [45], nonpeptidic xanthone
derivatives [46, 47] and other molecules with potent antibacterial and antibiofilm
activities have been developed [48-53]. Additionally, several amphiphilic derivatives
based on approved antibiotics have potent antimicrobial activity, such as vancomycin
[
54-56] and kanamycin B [57, 58]. S-galactosyl oligo(Arg) conjugates have been
shown to overcome the serious problem of passage through the E. coli cell membrane
59]. Antibacterial diamines can also target bacterial membranes[60]. Moreover,
[
guanidine and biguanide compounds had potency in preventing biofilms formation
and breaking down existing biofilms[61]. Importantly, there are currently many
antibacterial peptidomimetics, including LTX 109 [62], CSA-13 [63] and brilacidin
[
64] (Fig. 2) undergoing clinical trials. Thus, exploring the diversity of the structures
of these molecules should offer new compound leads [65].
Fig. 2. Structures of some representative AMPs.
During the course of our recent investigations for new structures with good
biological activity [66, 67], we discovered several molecules that have good
antimicrobial activity and excellent activity against drug-resistant strains[68-71].
Inspired by the antibacterial activity of linezolid and antibacterial peptide mimics, we
sought to find more effective antibacterial deacetyl linezolid derivatives. Recently,

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three series of cationic deacetyl linezolid derivatives with broad-spectrum
antibacterial activity were designed and synthesized. The final compounds were
obtained through two or three step syntheses. Moreover, these compounds possessed
positive charges, lipophilic alkyl chains and a non-peptidic amide bond. In this report,
the activity of these compounds against drug-sensitive bacteria, including S. aureus, E.
coli, E. faecalis and S. enterica, and drug-resistant bacteria such as MRSA, NDM and
KPC were studied.
(
a) K CO , chloroacetyl chloride, acetone, r. t., 0.5-1 h. (b) N, N-dimethylalkylamine, CH CN,
2
3
3
o
high-pressure reactor, 85 C, 24 h
Scheme 1. Synthesis of the deacetyl linezolid derivatives 2a–2f.
(
a) RCHO, NaBH(OAc) , 1,2-Dichloroethane, over night, r.t.. (b) 4a or 4b, DIPEA, HBTU,
3
o
DMF/CHCl (5:2), r. t., 24 h; (c) CH COCl, CH OH, 0 C-r. t., 24 h;
3
3
3
Scheme 2. Synthesis of the deacetyl linezolid derivatives 6a–6j.
2
. Results and discussion
2
.1. Synthesis and characterization
The molecules 2a–2f and 6a–6j were easily synthesized by either two or three
step syntheses using the deacetyl linezolid as the starting material (Schemes 1 and 2).
Five or six compounds in each series were synthesized to assess the importance of the
length of the lipophilic alkyl chain. To further fine-tune the structure-activity
relationship (SAR) of compounds 6a–6j, hydrophilic moieties (amino acids) were

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also varied on these lipophilic frameworks. The deacetyl linezolid was acetylated
through chloroacetyl chloride to obtain intermediate 1a. Next, compounds 2a–2f were
synthesized
N,N-dimethyl-n-alkylamines. To synthesize molecules 6a–6j, the intermediates 4a
53] and 4b [72] were prepared by known methods. The intermediates of
-alkylamines 3a–3e were accomplished by a conventional procedure. Key amide
coupling compounds 5a–5j were synthesized using N-Boc-protected amino acids 4a,
through
quaternization
by using intermediate
1a
and
[
N
4
b and 3a–3e. Finally, target compounds 6a–6j were obtained individually from 5a–
j with acetyl chloride in methanol. The products were obtained without further
5
chromatography purification in high yield. Finally, we have synthesized 16 cationic
1
deacetyl linezolid derivatives. All final compounds were characterized by H NMR,
1
3
C NMR and HRMS and were purified by reversed-phase HPLC to more than 95%
purity.
2
.2. Antibacterial activity
The antibacterial efficacy of these compounds was determined in suitable culture
medium and expressed as the MIC (minimum inhibitory concentration) (Tables 1-4).
The glycopeptide antibiotic vancomycin, the β-lactam antibiotic meropenem,
linezolid and deacetyl linezolid were used to compare the MIC results with our
compounds. These compounds displayed preferable antibacterial potency against a
wide spectrum of drug-sensitive bacteria such as S. aureus, E. coli, E. faecalis and S.
enterica (Table 1, and Figs. 3 and 4), and drug-resistant bacteria such as MRSA
(Table 2), KPC (Table 3) and NDM-1 (Table 4). Information describing MRSA,
NDM-1 and KPC strains is summarized in Table S1.
Table 1. MICs of small molecules 2a–2f, 6a–6j against drug-sensitive bacteria.
MIC (µg/mL)
E. faecalis
E. coli
S. enterica
Compound
S. aureus
2
2
a
>128
128
8
>128
64
>128
128
32
>128
128
32
b
2
c
16
2d
8
4
16
16

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2
e
4
4
16
128
64
16
8
16
16
>128
>128
128
64
2
f
4
128
32
16
4
>128
>128
128
64
6
6
a
b
6c
6d
32
32
6e
2
4
8
16
6
f
64
16
16
8
128
32
32
8
128
>128
64
>128
>128
64
6
6
g
h
6
i
16
32
6
j
4
8
8
8
a
Da-LIN
>128
2
>128
2
>128
>64
64
>128
>64
32
b
LIN
c
VAN
2
2
d
MEM
0.125
b
4
<0.125
<0.125
a
c
d
Da-LIN (deacetyl linezolid); LIN (linezolid); VAN (vancomycin); MEM (meropenem); All
experimental data was repeated twice (If the results were different, take the larger MIC).
In general, compounds 2a–2f, 6c–6e and 6i–6j (MIC: 2–16 µg/mL) showed good
activities against gram-positive S. aureus and E. faecalis (Table 1), whereas
compounds 2d–2e, 6e and 6j (MIC: 2–16 µg/mL) showed good activities against both
gram-positive and gram-negative bacteria. Moreover, compound 6e was even
comparable to vancomycin and linezolid against all bacteria tested. Compounds 2c–2e,
6
d–6e and 6i–6j (MIC: 8–32 µg/mL) were found to be more active than vancomycin
(MIC: >64 µg/mL) and linezolid (MIC: 32–64 µg/mL) against gram-negative bacteria
E. coli and S. enterica. These cationic small molecules 2a–2f, 6c–6e and 6i–6j were
found to be active against both gram-positive and gram-negative bacteria, with
activity toward gram-positive bacteria strongest. The MICs for deacetyl linezolid
were >128 µg/mL against drug-sensitive bacteria, whereas MICs were <0.125–4
µg/mL for meropenem. Clearly, these cationic deacetyl linezolid amphiphiles were
more active than deacetyl linezolid but less active than meropenem. Accordingly,
these compounds showed broad-spectrum activities, whereas vancomycin and
linezolid showed selective activities against gram-positive bacteria.
Fig. 3 presents the MICs of compounds 2a–2f and several control agents against
drug-sensitive bacteria, and clearly shows that the small molecules synthesized were

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more active against gram-positive bacteria S. aureus and E. faecalis.
Fig. 3. MICs of cationic deacetyl linezolid derivatives 2a–2f, Da-LIN, LIN, VAN
and MEM against drug-sensitive bacteria. The black, blue and yellow star represents
MICs >128, > 64 and <0.125 µg/mL. All experimental data was repeated twice.
Fig. 4. MICs of cationic deacetyl linezolid derivatives with varying alkyl chain
lengths: (a), (b) and (c) MICs of small molecules 2a–2f, 6a–6e and 6f–6j. The black
star represents MIC >128 µg/mL. All experimental data was repeated twice.
The length of the alkyl chain of compounds 2a–2f, 6a–6e and 6f–6j (Fig. 4) was

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extremely important in defining the antibacterial activity of the compounds prepared.
The MICs of compounds 2a–2f (Fig. 4a) were lower when the lipophilic chain was
C H , C H and or C H . Compounds 2c–2e were found to show much higher
9
19
12 25
14 29
activity against drug-sensitive bacteria than 2a, 2b and 2f. However, MICs of 6a–6e
Fig. 4b) and 6f–6j (Fig. 4c) were lower when the lipophilic chain was C H , C H
10 21
(
9
19
or C H . Additionally, when the lipophilic chain was C H , molecules 6e and 6j
1
1
23
11 23
displayed the strongest activities. These results were similar to those reported by other
groups [51, 53]. Furthermore, compounds 6e and 6j with the same lipophilic alkyl
chain but different hydrophilic amino acids displayed negligible differences in their
antibacterial activity. Molecules 6e and 6j had similar MICs against drug-resistant
bacteria. The SAR studies of this series of cationic deacetyl linezolid derivatives 6e
and 6j revealed that an amphiphilic structure was critical for broad-spectrum activity,
especially against gram-negative bacteria. Thus, the antibacterial activity of these
compounds was affected strongly by the length of the alkyl chain, whereas the types
of amino acids (lysine and arginine) had only a small effect on defining the
antibacterial activity of the compounds.
Table 2. MIC of small molecules against MRSA.
a
MRSA
C
ompound
M-1 M-2 M-3
M-4
16
4
M-5
4
M-6
4
M-7
4
M-8 M-9 M-10
2d
4
4
8
16
4
2
2
8
4
8
2
8
8
2
e
e
4
2
2
6
4
2
4
4
2
2
4
4
4
6
j
2
8
4
4
8
4
4
4
4
4
b
LIN
4
4
4
4
4
4
4
4
4
4
c
VAN
1
1
1
1
1
2
1
2
1
1
d
MEM
64
64
32
32
b
64
32
64
64
64
32
a
c
d
MRSA (10 different clinical isolated strains); LIN (linezolid); VAN (vancomycin); MEM (meropenem); All
experimental data was repeated twice (If the results were different, take the larger MIC).
The antibacterial activity (Table 2) of selected small molecules against MRSA
was further investigated. All selected molecules had good MICs. Compound 6e was
the most effective among the four compounds examined. In addition, this series of
small cationic amphiphiles were extremely active against MRSA (MICs: 2–16

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µg/mL). The efficiency activity of compound 6e (MICs: 2–4 µg/mL) was
comparable to linezolid (MICs: 4 µg/mL) and better than meropenem (MICs: 32–64
µg/mL), but lower than vancomycin (MICs: 1–2 µg/mL).
Table 3. MIC of small molecules against bacteria producing KPC.
a
KPC
Compound
K-1 K-2
K-3
64
16
16
8
K-4
64
32
16
8
K-5
64
K-6
32
K-7
64
K-8
32
K-9 K-10
2d
32
32
16
16
32
32
16
16
64
32
16
8
64
32
16
16
2
e
e
32
32
32
32
6
16
16
16
16
6
j
16
16
32
8
b
LIN
>64 >64
>64 >64
>64 >64
>64
>64
2
>64
64
32
>64
64
>64
>64
>64
>64
>64
>64
>64
>64
32
>64 >64
c
VAN
64
>64
64
d
MEM
32
>64
a
b
c
d
KPC (10 different clinical isolated strains); LIN (linezolid); VAN (vancomycin); MEM (meropenem); All
experimental data was repeated twice (If the results were different, take the larger MIC).
The antibacterial activity (Table 3) of small molecules against KPC was tested.
The MICs of these compounds ranged from 8 to 64 µg/mL. The MICs of the most
effective compounds 6e and 6j were 8–16 µg/mL. Most importantly, the activities of
compounds 2d, 2e, 6e and 6j (MICs: 8–64 µg/mL) were better than linezolid (MICs:
>
64 µg/mL), vancomycin (MICs: 64 or >64 µg/mL) and meropenem (MICs: 2–64 or
64 µg/mL), but worse than meropenem (MIC: 2 µg/mL) against K-3 strain.
>
Table 4. MIC of small molecules against bacteria producing NDM-1.
a
NDM-1
Compound
N-1 N-2
>64 >64
>64 >64
>64 >64
>64 >64
>64 >64
>64 >64
N-3
64
N-4
64
N-5
32
32
8
N-6
16
32
16
16
>64
64
2
N-7
32
N-8
16
N-9 N-10
2
d
16
16
32
16
2
6
e
e
32
64
16
16
16
16
16
16
16
16
6
j
16
64
16
>64
64
8
16
16
16
8
b
LIN
>64
>64
64
>64
>64
64
>64
64
>64
64
>64
64
>64
>64
>64
c
VAN
d
MEM
32
16
64
>64
d
>64
a
b
c
NDM-1 (10 different clinical isolated strains); LIN (linezolid); VAN (vancomycin); MEM (meropenem);
All experimental data was repeated twice (If the results were different, take the larger MIC).
The antibacterial activity (Table 4) of small molecules against NDM-1-producing
bacteria was investigated. The MICs ranged from 8 to 64 µg/mL and even >64 µg/mL.

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In addition, compounds 2d–2e, 6e and 6j (MICs: 8–64 µg/mL) showed higher activity
than linezolid (MICs: >64 µg/mL), vancomycin (MICs: 64 or >64 µg/mL) and
meropenem (MICs: 2–64 or >64 µg/mL) against N-3, N-4, N-7 to N-10, but displayed
lower activity than meropenem (MIC: 2 µg/mL) against N-5 and N-6 strains.
Meropenem showed better activity against N-1 and N-2 (MICs: 16–32 µg/mL) than
2
d–2e, 6e, 6j, linezolid and vancomycin with MICs >64 µg/mL.
From the MICs (Tables 1–4), compounds 2d–2e, 6e and 6j had potent activity
against both drug-sensitive bacteria and drug-resistant bacteria. Furthermore, the
antibacterial activities of compounds 2d–2e, 6e and 6j were better than the
corresponding activity of linezolid and vancomycin. Compounds 2d–2e, 6e and 6j
showed broad-spectrum antibacterial activity and 2d–2e are QACs with alkyl chains
(
-C H , –C H ) and a positive charge. Additionally, molecules 6e and 6j have the
12 25 14 29
same alkyl chain (-C H ) with an L-lysine or L-arginine residue. The positive
1
1
23
charges and the positively charged amino acids may be essential for achieving the
maximum antibacterial activity of these small cationic molecules.
Fig. 5. % of Hemolysis by cationic small molecules at different concentrations: (a)

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compound 2e; (b) compound 6e; (c) Linezolid. All experimental data were averaged
after three repetitions.
2
.3. Hemolytic activity
The hemolytic activity of the cationic small molecules 2e, 6e and linezolid are
presented in Fig. 5. The hemolysis rate of other compounds was not determined. The
ability of compounds to lyse red blood cells (RBCs) was used to evaluate the toxicity
of the compounds toward mammalian cells. The 50% hemolysis concentration (HC )
50
of 2e, 6e and linezolid were approximately 60, 100 and 550 µg/mL, respectively. In
general, the cationic small molecules 2e and 6e showed low toxicity toward RBCs. In
addition, compound 6e, one of the most potent molecules, displayed good selectivity
(
S = HC /MIC) against S. aureus (S = 50) and E. coli (S = 12.5). The hemolytic
50
toxicity study revealed that these compounds had low toxicity toward mammalian
RBCs with linezolid showing lower toxicity toward mammalian cells than compounds
2
e and 6e.
Fig. 6. Plasma stability and antibacterial activity in complex mammalian fluids of
small molecules: (a) antibacterial efficacy of compound 6e against S. aureus after
preincubating in 50% plasma for different periods of time (0, 3 and 6 h); (b)
minimum bactericidal concentrations (MBCs) of 6e in 50% plasma, serum and blood
against MRSA. The black star represents MIC >128 µg/mL. All experimental data
were averaged after two repetitions.
2
.4. Plasma stability

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Protease degradation of AMPs is a major disadvantage for their use as antibiotics
73]. To determine the stability of the cationic deacetyl linezolid derivatives under
[
plasma conditions, antibacterial efficacy of compound 6e against S. aureus after
preincubating in 50% plasma for different periods of time (0, 3 and 6 h) was evaluated.
The MBCs of compound 6e did not increase (MBC: 64 µg/mL) after 0, 3 and 6 h
treatment in 50% plasma (Fig. 6a). The above results indicate that compound 6e was
stable upon pretreatment in 50% plasma.
2
.5. Antibacterial activity in complex mammalian fluids
The loss of antibacterial activity of the compounds in the presence of complex
mammalian fluids is another serious problem when considering their suitability as
antimicrobial compounds. Antibacterial activity of compound 6e was investigated by
determining the MBCs in 50% serum, plasma and blood supplemented, respectively,
with 50% MHB against MRSA. (Fig. 6b). Compound 6e was found to be active in 50%
plasma, but was inactive in 50% serum and 50% blood. The MBCs of compound 6e
were 128 µg/mL in media and 50% plasma, whereas the MBCs were >128 µg/mL in
5
0% serum and 50% blood. The increase of the MBCs could be due to negatively
charged proteins and macromolecules in human serum or blood that bind tightly to the
cationic molecules, thereby quenching their activity toward bacterial membranes. The
above results indicate that compound 6e is active in complex mammalian fluids like
plasma, but this compound was not able to retain its antibacterial activity in serum
and blood.

ACCEPTED MANUSCRIPT
Fig. 7. Time-dependent killing. (a, c) S. aureus were grown to early exponential
phase and challenged with 6e at 4×, 6×MIC (8, 12 µg/mL) and vancomycin at
4
×MIC (8 µg/mL); (b, d) E.coli was grown to early exponential phase and
challenged with 6e at 2×, 4×MIC (16, 32 µg/mL) and moxalactam at 6×MIC (6
µg/mL). The control was treatment with sterile water. Pictures (c, d) taken after
treatment with compounds for 6 h. All experimental data were averaged after two
repetitions.
2
.6. Bactericidal time-kill kinetics
The time-kill kinetics of compound 6e was performed to determine the rate of
bactericidal action. S. aureus and E. coli, the two tested bacteria, were grown to early
exponential phase and challenged with compound 6e (2×, 4× and 6× MIC),
vancomycin (S. aureus) and moxalactam (E. coli). Compound 6e had excellent
bactericidal activity against S. aureus, showing superior activity when compared with
that of vancomycin in killing early exponential phase populations (Fig. 7a). Moreover,
6
e (6× MIC: 12 µg/mL) killed early exponential phase S. aureus at 6 h, whereas
vancomycin ( 4× MIC: 8 µg/mL) did not kill S. aureus at 6 h. Compound 6e displayed

ACCEPTED MANUSCRIPT
rapid bactericidal activity against E. coli and was superior to moxalactam in killing
early phase bacteria (Fig. 7b). Clearly, compound 6e (4× MIC: 32 µg/mL) killed E.
coli at 2 h, whereas moxalactam did not kill early exponential phase E. coli at 6 h.
Thus, compound 6e had excellent antibacterial activity against S. aureus and E. coli
(Fig. 7c,d). The vancomycin and moxalactam samples were cloudy after 6 h treatment,
but the samples treated with 6e were both clear. Thus, the results suggest that the
cationic molecule 6e kills both gram-positive and gram-negative bacteria.
Fig. 8. Antibiofilm activity of small molecule 6e. (a) cell viability in non-treated and
treated biofilms of S. aureus grown on cover slips for 24 h; (b) cell viability in
non-treated and treated biofilms of E. coli grown on cover slips for 72 h; (c) images
of the treated and non-treated biofilms of S. aureus after staining with crystal violet;
(d) images of the treated and non-treated biofilms of E. coli after staining with
crystal violet. All experimental data were averaged after three or more repetitions.
2
.7. Biofilm disruption activity
To evaluate the efficiency of this class of compounds to eradicate preformed
biofilms, compound 6e was used against established S. aureus and E. coli biofilms.

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Mature S. aureus biofilms in a 96-well plate (grown for 24 h) with an initial count of
1
6.0 log CFU/mL per well of bacteria were treated with 6e at seven different
10
concentrations. Molecule 6e was found to reduce cell viability of the biofilms (13.9,
2.9, 9.9, 8.7, 7.5, 5.9 and 4.7 log CFU per well at 2, 4, 8, 16, 32, 64 and 128 µg/mL,
1
1
0
respectively), whereas the cell viability of the non-treated biofilm increased to 18.9
log CFU per well (Fig. 8a). Compound 6e was also able to reduce the cell viability
1
0
of mature E. coli biofilms (developed for 72 h) from an initial count of 14.5 log10
CFU per well to 10.2, 8.5, 8.0, 7.1, 6.1, 6.0 and 5.3 log CFU per well at 2, 4, 8, 16,
1
0
3
2, 64 and 128 µg/mL, respectively, whereas the cell viability of the non-treated
biofilm increased to 16.1 log CFU per well (Fig. 8b). Disruption of biofilm growth
1
0
by compound 6e was visually observed by crystal violet staining (Fig. 8c,d and Fig. 9).
The MBIC (minimal bacterial inhibition concentration) and MBEC (minimal bacterial
eradicated concentration) values of compound 6e and linezolid were tested (Table 5).
The results of these tests using 6e gave MBIC (minimum concentration required to
8
0
inhibit 80% biofilm formation) values of 25.9 µg/mL against S. aureus and 61.9
µg/mL against E. coli, and the MBEC values of 6e were 64 µg/mL and 128 µg/mL
against S. aureus and E. coli, respectively. Fig. 9 shows the results of the MBEC
assay using 6e. Linezolid only inhibited S. aureus biofilm formation, and was not as
active in eradicating established biofilms when compared with the results using 6e.
The results indicate that compound 6e can inhibit bacterial biofilm formation and
eradicate established biofilms at 64 and 128 µg/mL.

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Fig. 9. Antibiofilm activity of small molecule 6e. Images of the small molecule
treated and non-treated biofilms of S. aureus and E. coli after staining with crystal
violet. All experimental data was repeated twice.
Table 5. MBIC and MBEC of compound 6e.
MBIC / MBIC (µg/mL)
MBEC / MBEC / MBEC (µg/mL)
80 90
8
0
90
Compound
S. aureus
25.9/34.1
26.8/36.1
E. coli
S. aureus
E. coli
112.8/120.3/128
>128
6
e
61.9/72.6
>128
50.2/59.8/64
>128
Linezolid
All experimental data were averaged after two repetitions.
2
.8. Mechanism of action
The molecular mechanism of action was investigated to determine whether these
cationic molecules functioned by disrupting the integrity of the bacterial cell
membrane. Gram-positive S. aureus and gram-negative E. coli were used to assess the
action mechanism of compound 6e. We also used three antibiotics (linezolid,
vancomycin and meropenem) for comparison. The mechanism of action can be

ACCEPTED MANUSCRIPT
confirmed by the following three experiments: membrane depolarization, and inner
membrane and outer membrane permeabilization (Fig. 10).
Fig. 10. Mechanism of antibacterial action of the cationic small molecule 6e. Inner
membrane permeabilization of S. aureus (a) and E. coli (b). Cytoplasmic membrane
depolarization of S. aureus (c) and E. coli (d). (e) Outer membrane permeabilization
of E. coli in the presence of amphiphilic small molecules at 10 µg/mL. All
experimental data were averaged after three repetitions.
2
.8.1. Inner membrane permeabilization
Bacterial cytoplasmic membrane permeabilization was studied using the

ACCEPTED MANUSCRIPT
fluorescent probe propidium iodide (PI). PI enters bacteria only through compromised
membranes and fluoresces upon binding to cellular DNA. After treatment with the
molecule 6e (final concentration was 20 µg/mL), an enhancement in the fluorescence
intensity was observed in S. aureus and E. coli (Fig. 10a,b). However, there was no
increase when treated with other antibiotics. Thus, the result indicate that compound
6
e can permeabilize the membrane of both gram-positive and gram-negative bacteria.
2
.8.2. Cytoplasmic membrane depolarization
The deacetyl linezolid derivatives were found to dissipate the membrane
potential of both gram-positive and gram-negative bacteria, as monitored by the
membrane-potential fluorescence sensitive dye diSC 5. Furthermore, compound 6e
3
and the three antibiotics showed different levels of dissipation of the membrane
potential. Compounds 6e (final concentration was 20 µg/mL) showed maximum
membrane depolarization, whereas the other three antibiotics did not exhibit
membrane depolarization activity against S. aureus and E. coli (Fig. 10c,d). The
results further indicated that compound 6e is able to dissipate the membrane.
2
.8.3. Outer membrane permeabilization
Outer membrane permeabilization was studied using the hydrophobic dye
N-phenyl naphthylamine (NPN). NPN is generally excluded from the outer membrane
of gram-negative bacteria. NPN can access the impaired outer membrane when this
membrane is damaged and exhibits an increase in fluorescence intensity. After
treatment with 6e and the three antibiotics (final concentration was 20 µg/mL), no
enhancement in the fluorescence intensity was observed (Fig. 10e). The above results
indicated that compound 6e was not able to permeabilize the outer membrane of E.
coli.

ACCEPTED MANUSCRIPT
Fig. 11. Inverted microscopy images of HeLa cells following treatment with
compounds for 24 h. (a), (b) and (c) cells treated with 6e (1, 2 and 4 µg/mL); (d)
non-treated cells (negative control); (e) cells treated with 0.1% Triton-X (positive
control). Scale bar is 10 µm. All experiments were repeated three times.

ACCEPTED MANUSCRIPT
Fig. 12. Fluorescence microscopy images of HeLa cells after treatment with
compounds for 24 h and staining with calcein AM and propidium iodide (PI). (a-c)
Non-treated cells (negative control); (d-f), (g-i) and (j-l) treated with 6e (1, 2 and 4
µg/mL); (m-o) treated with 0.1% Triton-X (positive control). Scale bar is 200 nm.
All experiments were repeated three times.
2
.9. Fluorescence and inverted microscopy images
HeLa cells were seeded into the wells of a 12-well plate and then treated with

ACCEPTED MANUSCRIPT
compound 6e at various concentrations (1, 2 and 4 µg/mL). The treated cell lines were
imaged by optical microscopy to visualize the morphology (Fig. 11). The treated
cells were found to have normal morphology at 1 µg/mL, 2µg/mL and 4 µg/mL (Fig.
1
1a–c), and they were similar to the untreated cell lines (Fig. 11d). In contrast, cells
treated with Triton-X were found to have completely damaged cell morphologies
Fig. 11e). Fluorescence microscopy studies using the live/dead staining method
(
showed that cells treated with compound 6e showed green fluorescence at 1, 2 and 4
µg/mL (Fig. 12d–f, g–i, j–l) and were similar to the untreated cells (Fig. 12a–c). In
contrast, cells treated with Triton-X were found to have red fluorescence (Fig. 12m–
o). The above results indicate that 6e was not toxic to mammalian cells at 2× the
MIC value (4 µg/mL).
Fig. 13. Propensity of development of bacterial resistance against compound 6e. (a)
For S. aureus where antibiotic norfloxacin and linezolid were used as the control; (b)
for E. coli where lipopeptide colistin was used as the control; (c) for MRSA where
linezolid was used as the control. All experiments was repeated twice.
2
.10. Propensity to induce bacterial resistance
It is important to evaluate the potential emergence of bacterial resistance against

ACCEPTED MANUSCRIPT
these small molecules. Compound 6e was selected as a representative compound to
evaluate the ability of these compounds to suppress the development of resistance
against both gram-positive S. aureus, MRSA and gram-negative E. coli. Three control
antibiotics norfloxacin, colistin and linezolid were chosen for S. aureus, E. coli and
MRSA, respectively. In the case of norfloxacin, colistin and linezolid, the initial MICs
were 1, 0.5 and 4 µg/mL, respectively. After the initial MIC experiment, serial
passaging was initiated by transferring the growing bacterial suspension at 1/2 MIC of
the compound or antibiotics, and was subjected to another MIC assay and the process
was repeated for 20 passages. The cationic compound 6e and linezolid showed no
change in the MIC against S. aureus even after 20 passages, whereas a 256-fold
increase in the MIC was observed for norfloxacin (Fig. 13a). Moreover, compound 6e
showed only a two-fold increase in MIC against E. coli and MRSA, whereas 32-fold
and 8-fold increases in the MIC were observed for colistin against E. coli and
linezolid against MRSA, respectively (Fig. 13b,c). The results indicate that induced
bacterial resistance by continual exposure to these small compounds is difficult and
they represent potential compounds to combat drug-resistant bacteria. In contrast,
resistance to the two positive control antibiotics norfloxacin and colistin by S. aureus
and E. coli, respectively, was easily developed (Fig. 13a,b).
3
. Conclusions
Three series of small molecules were synthesized from deacetyl linezolid. These
compounds displayed potent broad-spectrum and membrane-active antibacterial
activity against various drug-sensitive and drug-resistant bacteria. Variation of the
alkyl chain length had a great effect on their activity. The SAR studies of these
cationic small molecules revealed that the positive charges and amphiphilic structure
were critical for broad-spectrum activity, especially against gram-negative bacteria.
Additionally, the small molecules were found to be stable under plasma conditions
and showed acceptable levels of activity in mammalian plasma, but these compounds
were not able to retain antibacterial activity when incubated in serum and blood.
Moreover, these small molecules acted on bacteria rapidly and did not allow bacteria

ACCEPTED MANUSCRIPT
to develop resistance. Various spectroscopic and microscopic studies revealed that
depolarization and disruption of the bacterial cell membrane are the primary
mechanisms describing the bactericidal action of the compounds. Furthermore, an
optimized molecule showed negligible toxicity toward mammalian RBCs and HeLa
cells. Therefore, these molecules can combat drug-resistance pathogens, inhibit
bacterial biofilm formation and can eradicate established biofilms. Thus, these
compounds are potential agents that can be used to tackle infections caused by various
bacteria.
4
. Experimental section
General: Reagents and solvents were purchased from commercial sources and were
1
13
used without further purification. H NMR and C NMR spectra were recorded on a
Bruker 400 MHz and 100 MHz spectrometer respectively, and TMS as internal
standard reference. Coupling constants (J) are given in hertz (Hz). High resolution mass
spectra (HRMS) were recorded on a Waters Micromass Q-T of Micromass
spectrometer. The compounds were purified by reverse phase HPLC (Waters
Symmetry C-18 4.5×250 mm, 5 µm) using sodium dihydrogen phosphate buffer (pH
=
6-8)/acetonitrile (0−100%) as mobile phase to more than 95% purity. Analytical
thin layer chromatography (TLC) was performed on glass plates pre-coated with silica
gel (5–40 um, Qingdao Marine Chemical Factory (China)) to monitor the reactions.
Visualization was accomplished using UV light. Column chromatography was
performed on silica gel. For optical density and fluorescence measurements, Tecan
Infinite Pro series M200 Microplate Reader was used. The biological experiments were
performed with a 1300 series A Biological Safety Cabinet. TDL-5M Desktop
2
Low-speed Refrigerated Centrifuge was used in antibacterial studies. All the solvents
were of reagent grade. Drug-sensitive bacterial strains, E. coli (ATCC 25922), S.
aureus (ATCC 29213), E. faecalis (ATCC 29212) and S. enterica (ATCC 8387).
Drug-resistant bacteria, MRSA, KPC and NDM-1 were clinical isolated strains. Sheep
RBCs were used for hemolytic assay and HeLa cells were used for mammalian
cytotoxicity study.

ACCEPTED MANUSCRIPT
General procedure for the synthesis o f compound (1 a ) : To the mixture of
deacetyl linezolid (1 g, 3.39 mmol) and K CO (562 mg, 4.06 mmol) in 25 mL
2
3
acetone was added chloroacetyl chloride (306
µ
L) dropwise (about 0-3 min). The
resulting solution was stirred at room temperature for 0.5-1 h. Upon completion, the
mixture was poured into 25 mL of ice water. The precipitate formed was filtrated,
washed with water and dried under vacuum to obtain compound 1a without further
purification
.
[74]
(S)-2-chloro-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5
1
yl)methyl)acetamide (1a): White solid, yield 64.5%. H NMR (400 MHz, CDCl3)
δ
1
3
1
1
7.41 (dd,
J = 14.3, 2.6 Hz, 1H), 7.19 (s, 1H), 7.12 – 7.03 (m, 1H), 6.92 (t, J = 9.1 Hz,
H), 4.85 – 4.73 (m, 1H), 4.12 – 3.98 (m, 3H), 3.91 – 3.83 (m, 4H), 3.82 – 3.60 (m,
1
3
H), 3.13 – 2.98 (m, 4H). C NMR (100 MHz, CDCl )
δ 166.20, 155.70, 153.25,
3
53.12, 135.70, 135.61, 131.85, 131.75, 117.87, 117.83, 113.05, 113.01, 106.73,
06.47, 70.43, 65.93, 49.98, 49.95, 46.77, 41.53, 41.39.
General procedure for the synthesis o f f i n a l compounds (2 a - 2 f ) : Compound
was dissolved in CH CN, then N, N-dimethylalkylamines were added to the
1
a
3
organic solutions (1a: N, N-dimethylalkylamines = 1 : 3, molar ratio) separately in
screw top pressure tubes and the reaction mixtures were stirred at 85 °C for about 24
h. [75] After the reaction, the mixtures were allowed to cool down to room
temperature and transferred to round bottom flask. Then, the organic solvents were
removed and volume of the reaction mixtures was reduced to 1/10 to its original
volume. Finally, the products were precipitated in excess of dry diethyl ether. The
precipitates were filtered and washed repeatedly with diethyl ether. The compounds
were vacuum dried.
(S)-N-(2-(((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)amino)-2
-oxoethyl)-N,N-dimethylbutan-1-aminium (2a): Dark yellow oily liquid, yield 78.6%.
1
H NMR (400 MHz, DMSO-d6
)
δ
9.56 (t,
= 8.8, 2.1 Hz, 1H), 7.06 (t,
H), 4.24 (s, 1H), 4.11 (t, = 9.0 Hz, 1H), 3.86 (dd,
= 10.0, 5.3 Hz, 1H), 3.46 (dd, J = 11.6, 5.4 Hz, 2H), 3.39 (s, 4H),
J
= 5.6 Hz, 1H), 7.49 (dd,
J = 15.0, 2.5 Hz,
1
1
H), 7.19 (dd,
J
J
= 9.3 Hz, 1H), 4.80 (dd,
J
= 7.6, 5.5 Hz,
J
J
= 9.0, 6.6 Hz, 1H), 3.77 – 3.69
(m, 4H), 3.54 (dd,
J

ACCEPTED MANUSCRIPT
3
0
1
1
1
.20 (s, 4H), 2.98 – 2.93 (m, 4H), 1.73 – 1.62 (m, 2H), 1.26 (dd,
J
= 14.7, 7.4 Hz, 2H),
13
.90 (t,
J = 7.3 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 163.95, 155.74, 153.92,
53.31, 135.62, 135.53, 133.36, 133.26, 119.21, 119.17, 114.27, 114.24, 106.91,
06.65, 70.96, 66.13, 64.36, 61.80, 51.14, 50.68, 50.66, 47.37, 41.21, 23.82, 19.09,
+
3.37. HR-MS (ESI) Calcd for C H ClFN O [M-Cl] : 437.2559, found: 437.2563.
2
2
34
4
4
(S)-N-(2-(((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)amino)-2
1
-
oxoethyl)-N,N-dimethyloctan-1-aminium (2b): Yellow sticky solid, yield 97.0%. H
NMR (400 MHz, DMSO- 6
d
)
δ
9.24 (t,
H), 7.22 – 7.17 (m, 1H), 7.07 (t, = 9.3 Hz, 1H), 4.85 – 4.74 (m, 1H), 4.16 – 4.08 (m,
H), 3.79 (dd, = 9.1, 6.8 Hz, 1H), 3.76 – 3.71 (m, 4H), 3.59 – 3.47 (m, 2H), 3.42 (dd,
= 9.9, 7.0 Hz, 4H), 3.17 (s, 5H), 2.99 – 2.94 (m, 4H), 1.67 (s, 2H), 1.26 (s, 10H),
J = 5.7 Hz, 1H), 7.49 (dd, J = 15.0, 2.4 Hz,
1
2
J
J
J
1
3
0
1
4
.87 (t, J = 6.7 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 163.94, 153.90, 135.63,
33.36, 119.23, 114.20, 106.85, 106.60, 71.07, 66.13, 64.57, 61.73, 51.20, 50.69,
7.26, 41.23, 31.11, 28.37, 25.65, 21.99, 21.82, 13.89. HR-MS (ESI) Calcd for
+
C H ClFN O [M-Cl] : 493.3185, found: 493.3192.
26
42
4
4
(S)-N-(2-(((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)amino)-2
-oxoethyl)-N,N-dimethylnonan-1-aminium (2c): Yellow sticky solid, yield 77.4%.
1
H NMR (400 MHz, DMSO- 6
d
)
δ
9.52 (s, 1H), 7.50 (dd,
= 9.3 Hz, 1H), 4.20 (s, 1H), 4.10 (s, 1H), 3.87 – 3.81 (m, 1H),
.76 – 3.70 (m, 4H), 3.55 – 3.51 (m, 1H), 3.49 – 3.40 (m, 6H), 3.24 (dd, = 10.2, 6.7
J = 15.0, 2.5 Hz, 1H), 7.23 –
7
3
.15 (m, 1H), 7.06 (t, J
J
Hz, 1H), 3.18 (s, 4H), 3.00 (s, 2H), 2.98 – 2.93 (m, 4H), 1.69 – 1.60 (m, 2H), 1.25 (d,
1
3
J
1
5
2
5
= 5.9 Hz, 12H), 0.86 (dd, J = 6.8, 3.8 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ
63.98, 153.91, 119.20, 114.21, 106.86, 71.01, 66.12, 64.47, 62.80, 61.72, 51.13,
0.68, 49.93, 47.30, 41.16, 31.20, 28.71, 28.55, 28.44, 25.73, 25.67, 22.05, 21.82,
+
1.63, 13.91. HR-MS (ESI) Calcd for C H ClFN O [M-Cl] : 507.3341, found:
2
7
44
4
4
07.3345.
(S)-N-(2-(((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)amino)-2
-oxoethyl)-N,N-dimethyldodecan-1-aminium (2d): Yellow sticky solid, yield 69.8%.
1
H NMR (400 MHz, DMSO-d6
) δ 7.50 (dd, J = 15.0, 2.4 Hz, 1H), 7.18 (dd, J = 8.8,
1
.7 Hz, 1H), 7.05 (t, = 9.3 Hz, 1H), 4.83 – 4.76 (m, 1H), 4.24 (s, 1H), 4.09 (t, J =
J

ACCEPTED MANUSCRIPT
9
1
2
.0 Hz, 1H), 3.85 (dd,
J
= 7.6, 5.3 Hz, 1H), 3.77 – 3.71 (m, 5H), 3.69 (d, J = 4.8 Hz,
H), 3.56 – 3.49 (m, 2H), 3.17 (s, 5H), 2.98 – 2.92 (m, 5H), 2.82 (d,
.56 – 2.48 (m, 1H), 1.67 (s, 2H), 1.23 (s, 18H), 0.85 (t, = 6.7 Hz, 3H). C NMR
164.07, 153.92, 135.57, 135.48, 133.38, 133.28, 119.15,
J = 4.2 Hz, 1H),
13
J
(100 MHz, DMSO- 6
d
)
δ
1
2
14.16, 106.82, 106.56, 70.97, 66.12, 51.06, 50.67, 47.30, 31.27, 29.00, 28.92, 28.76,
8.69, 28.45, 25.69, 22.06, 21.80, 13.91. HR-MS (ESI) Calcd for C H ClFN O
4
3
0
50
4
+
[
(
-
M-Cl] : 549.3811, found: 549.3816.
S)-N-(2-(((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)amino)-2
oxoethyl)-N,N-dimethyltetradecan-1-aminium (2e): Dark yellow sticky solid, yield
1
8
7
1.8%. H NMR (400 MHz, DMSO-d6
)
δ
9.38 (s, 1H), 7.50 (dd,
= 9.4 Hz, 1H), 4.79 (s, 1H), 4.17 (s, 1H), 4.10
= 9.0, 6.8 Hz, 1H), 3.76 – 3.70 (m, 4H), 3.56 – 3.50 (m,
H), 3.45 – 3.39 (m, 2H), 3.34 (s, 3H), 3.18 (s, 4H), 2.99 – 2.94 (m, 3H), 2.93 (d,
.4 Hz, 1H), 2.81 (d, = 8.1 Hz, 1H), 1.67 (s, 2H), 1.24 (s, 22H), 0.85 (t, = 6.6 Hz,
H). C NMR (100 MHz, DMSO-d6 163.95, 153.88, 153.33, 135.52, 119.19,
J = 14.9, 2.4 Hz, 1H),
.19 (dd,
J = 8.7, 1.9 Hz, 1H), 7.06 (t, J
(t,
J
= 9.1 Hz, 1H), 3.82 (dd, J
1
3
3
1
3
2
5
J =
J
J
1
3
)
δ
14.17, 106.84, 106.58, 71.04, 66.13, 64.47, 61.71, 51.19, 50.66, 47.28, 42.38, 41.19,
1.26, 29.03, 28.99, 28.92, 28.82, 28.75, 28.67, 28.57, 28.44, 26.10, 25.67, 22.05,
+
1.83, 13.90. HR-MS (ESI) Calcd for C H ClFN O [M-Cl] : 577.4124, found:
3
2
54
4
4
77.4130.
(S)-N-(2-(((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)amino)-2
-oxoethyl)-N,N-dimethyloctadecan-1-aminium (2f): Yellow foamed solid, yield
1
8
7
2
3
0
1
6
2
1.6%. H NMR (400 MHz, DMSO-d6
)
δ
9.28 (s, 1H), 7.50 (dd,
= 12.1 Hz, 1H), 7.09 – 7.00 (m, 1H), 4.79 (s, 1H), 4.12 (dd,
H), 3.83 – 3.77 (m, 1H), 3.75 – 3.71 (m, 4H), 3.53 (dd, = 10.4, 4.8 Hz, 1H), 3.49 –
.39 (m, 4H), 3.17 (s, 4H), 2.94 (dd, = 11.0, 4.1 Hz, 5H), 1.66 (s, 2H), 1.23 (s, 30H),
.85 (t, = 6.6 Hz, 3H), 0.63 – 0.63 (m, 1H). C NMR (100 MHz, DMSO-d6
J
= 14.8, 2.2 Hz, 1H),
.17 (t,
J
J
= 17.6, 8.3 Hz,
J
J
1
3
J
)
δ
63.95, 153.88, 135.52, 133.36, 119.19, 114.14, 106.81, 106.55, 71.06, 66.12, 64.44,
1.67, 51.22, 50.66, 47.24, 41.96, 41.20, 31.25, 29.00, 28.96, 28.92, 28.75, 28.66,
8.43, 25.95, 25.66, 22.05, 21.82, 13.90. HR-MS (ESI) Calcd for C H ClFN O
3
6
62
4
4
+
[
M-Cl] : 633.4750, found: 633.4757.

ACCEPTED MANUSCRIPT
General procedure for the synthesis of intermediate molecules (3a-3e):
Alkylaldehyde (RCHO) (1.1 equiv) was added into a solution of deacetyl linezolid (1
equiv., 300 mg) and NaBH(OAc) (1.3 equiv.) in 7.5 mL 1,2-dichloroethane in a
3
round-bottom flask. The mixture solution with N protected was stirred at room
2
temperature for 12-24 h. Then saturated NaHCO was used to quench the reaction and
3
alkalified to pH = 8. The reaction solution was then extracted with dichloromethane
(
DCM). The organic layer was subsequently washed with H O (2-3 times) and brine
2
(
1-2 times). Then the organic layer was dried over anhydrous Na SO , filtrated, and
2
4
concentrated under reduced pressure and the residue was purified using column
chromatography (petroleum ether: ethyl acetate= 5:1 to 1:1 v/v and with 0.5%
triethylamine) to obtain the product.
(S)-3-(3-fluoro-4-morpholinophenyl)-5-((heptylamino)methyl)oxazolidin-2-one (3a):
1
Yellow solid, yield 28.0%. H NMR (400 MHz, CDCl )
δ
7.45 (dd,
= 9.1 Hz, 1H), 4.75 (s, 1H), 4.00 (t,
= 8.5, 6.9 Hz, 1H), 3.08 – 3.02 (m, 4H), 2.94 (dd,
= 13.6, 7.0 Hz, 2H), 1.52 (s, 1H), 1.47 (dd, = 12.8,
= 6.9 Hz, 3H). C NMR (100 MHz,
156.78, 154.56, 154.33, 136.30, 136.21, 133.55, 133.44, 118.84, 118.80,
13.82, 113.78, 107.52, 107.26, 72.35, 66.97, 52.57, 51.07, 51.04, 50.10, 48.30, 31.78,
0.08, 29.69, 29.18, 27.17, 22.60, 14.06.
J
= 14.4, 2.6 Hz,
3
1
1
J
5
H), 7.17 – 7.10 (m, 1H), 6.92 (t,
H), 3.90 – 3.85 (m, 4H), 3.82 (dd,
J
J
= 8.6 Hz,
J
= 12.8, 5.3 Hz, 2H), 2.65 (dd,
J
J
1
3
.7 Hz, 2H), 1.32 – 1.24 (m, 8H), 0.88 (t,
J
CDCl3)
δ
1
3
(S)-3-(3-fluoro-4-morpholinophenyl)-5-((octylamino)methyl)oxazolidin-2-one (3b):
1
Yellow solid, yield 26.4%. H NMR (400 MHz, CDCl )
δ
7.45 (dd,
= 9.1 Hz, 1H), 4.80 – 4.70 (m, 1H), 4.00
= 8.6 Hz, 1H), 3.89 – 3.85 (m, 4H), 3.82 (dd, = 8.4, 6.9 Hz, 1H), 3.10 – 3.01 (m,
H), 2.93 (qd, = 12.9, 5.2 Hz, 2H), 2.65 (dd, = 13.5, 6.9 Hz, 2H), 1.50 – 1.42 (m,
H), 1.28 (d, = 5.6 Hz, 10H), 0.88 (t, = 6.8 Hz, 3H). C NMR (100 MHz, CDCl )
J = 14.4, 2.6 Hz,
3
1
H), 7.14 (dd,
J = 8.8, 1.6 Hz, 1H), 6.92 (t, J
(t,
J
J
4
3
δ
1
2
J
J
1
3
J
J
3
156.79, 154.55, 154.34, 136.32, 136.23, 133.56, 133.46, 118.85, 118.81, 113.83,
13.79, 107.53, 107.27, 72.35, 66.98, 52.57, 51.08, 51.05, 50.11, 48.30, 31.81, 30.09,
9.48, 29.23, 27.22, 22.64, 14.07.