Synthesis, antibacterial activities, mode of action and acute toxicity studies of new oxazolidinone-fluoroquinolone hybrids

Article abstract of DOI:10.3390/molecules24081641

To combat bacterial resistance, a series of new oxazolidinone-fluoroquinolone hybrids have been synthesized and characterized. All synthetic hybrids were preliminarily evaluated for their in vitro antibacterial activities against 6 standard strains and 3 clinical isolates. The majority of hybrids displayed excellent activities against Gram-positive bacteria, but limited activities against Gram-negative bacteria. Hybrids OBP-4 and OBP-5 were found to be the most promising compounds. Further, in vitro antibacterial activities, mode of action and acute toxicity in mice of hybrids OBP-4 and OBP-5 were investigated. Hybrids OBP-4 and OBP-5 exhibited potent activities against Gram-positive bacteria, including drug-resistant strains. Correspondingly, studies on the mode of action of hybrids OBP-4 and OBP-5 indicated a strong inhibitory activity on protein synthesis by binding the active site of 50S subunit, but a weak inhibitory action on DNA synthesis. In addition, LD₅₀ values of hybrids OBP-4 and OBP-5 in the acute oral toxicity were larger than 2000 mg/kg, suggesting a good safety profile.

Full text of DOI:10.3390/molecules24081641

molecules
Article
Synthesis, Antibacterial Activities, Mode of
Action and Acute Toxicity Studies of New
Oxazolidinone-Fluoroquinolone Hybrids
Lili Liu 1,2,3, Liping Shao 1,2,3, Jing Li 4, Haifeng Cui 4, Bing Li 1,2,3, Xuzheng Zhou 1,2,3
,
Pengyue Lv 4 and Jiyu Zhang 1,2,3,
*
1
Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture, Lanzhou 730050, China;
liull0728@163.com (L.L.); shaolp1992@163.com (L.S.); binglilb@163.com (B.L.); zhouxuzh@126.com (X.Z.)
Key Laboratory of New Animal Drug project of Gansu Province, Lanzhou 730050, China
Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural
Sciences (CAAS), Lanzhou 730050, China
2
3
4
R & D Center, Beijing Orbiepharm Co., Ltd., Beijing 100085, China; lijing1958@126.com (J.L.);
cuihf1981@163.com (H.C.); lvpenyue@163.com (P.L.)
*
Correspondence: infzjy@sina.com; Tel.: +86-931-2115278
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 26 March 2019; Accepted: 23 April 2019; Published: 25 April 2019
Abstract: To combat bacterial resistance, a series of new oxazolidinone-fluoroquinolone hybrids
have been synthesized and characterized. All synthetic hybrids were preliminarily evaluated for
their in vitro antibacterial activities against 6 standard strains and 3 clinical isolates. The majority of
hybrids displayed excellent activities against Gram-positive bacteria, but limited activities against
Gram-negative bacteria. Hybrids OBP-4 and OBP-5 were found to be the most promising compounds.
Further, in vitro antibacterial activities, mode of action and acute toxicity in mice of hybrids OBP-4 and
OBP-5 were investigated. Hybrids OBP-4 and OBP-5 exhibited potent activities against Gram-positive
bacteria, including drug-resistant strains. Correspondingly, studies on the mode of action of hybrids
OBP-4 and OBP-5 indicated a strong inhibitory activity on protein synthesis by binding the active
site of 50S subunit, but a weak inhibitory action on DNA synthesis. In addition, LD50 values of
hybrids OBP-4 and OBP-5 in the acute oral toxicity were larger than 2000 mg/kg, suggesting a good
safety profile.
Keywords: oxazolidinone-fluoroquinolone hybrids; synthesis; antibacterial activity; mode of action;
acute toxicity
1. Introduction
Although the discovery of antibiotics has had a profound positive impact on the improvements of
human health and life expectancy, the emergence of antibiotic resistance has severely limited their
clinical efficacy and applications [1,2]. Drug-resistant bacteria, especially multi-drug resistant (MDR)
bacteria, representing the most common causative agents of nosocomial infections [3,4], are difficult
to deal with using currently existing antibiotics. Worse still, the continuous evolution and spread of
antibiotic-resistant bacteria have caused enormous financial burden worldwide in terms of increased
morbidity, mortality and use of medical resources [
infections could claim 10 million deaths per year and up to 100 trillion US dollars by 2050 if nothing is
done [ ]. Nowadays, not only is antimicrobial resistance a major public concern, but more worryingly,
5–7]. It is estimated that antibiotic-resistant
8
there is an urgent need for novel antibacterials with improved potency and a low propensity for
resistance development.
Molecules 2019, 24, 1641; doi:10.3390/molecules24081641
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1641
2 of 22
In recent years, hybrid antibacterials, linking two different pharmacophores by a non-cleavable
spacer, were introduced as a promising strategy to fight drug-resistant bacteria and delay resistance
development [
antibacterials [11
9
,
10]. Such a strategy has been applied to the design and synthesis of various hybrid
14]. Fluoroquinolone, with its superior dual-target activity, chemical stability and
–
distinct structure-activity relationship (SAR), is the most popular partner in the design of hybrid
antibacterials [15]. Oxazolidinone exerted antibacterial activity by inhibiting protein synthesis [16], and
has a dissimilar mechanism of action to fluoroquinolone. The covalent combination of oxazolidinone
and fluoroquinolone may be a better choice for fighting the prevalent resistance to these two classes
of compounds [17,18]. Furthermore, their SAR investigations have shown that oxazolidinone and
fluoroquinolone have a good tolerance for structural modification and a common heterocyclic amine was
introduced at the 4- and 7- positions of the respective phenyl ring [19], which provide an advantage for
their combination. Meanwhile, previous studies have demonstrated that the oxazolidinone-quinolone
hybrids with a 4-piperidinemethanol linker exhibited improved antibacterial activities [20] and a low
propensity for spontaneous resistance development [21,22].
Inspired by these previous studies, we synthesized a series of new oxazolidinone-fluoroquinolone
hybrids with the modifications of the spacer or fluoroquinolone pharmacophore. Here, we report the
in vitro antibacterial activities of these hybrids, and the mode of action and acute toxicity of hybrids
OBP-4 and OBP-5.
2. Results and Discussion
2.1. Chemistry
The synthesis of oxazolidinone-fluoroquinolone hybrids OBP-(1–7) was performed as outlined in
Schemes 1–7. The hybrids OBP-(1–5) consisted of two parts, compound 8 and corresponding acids
(Schemes 2–5). Compound 8 was synthesized according to the following Scheme 1. The corresponding
acids of OBP-1 and OBP-2 were obtained from commercial sources, and those of OBP-(3–5) were
synthesized by another routes (Schemes 3–5). Meanwhile, we also prepared hybrids OBP-6 and OBP-7,
containing an exo-3-methyl-6-amino-3-azabicyclo [3.1.0] hexane linker between the oxazolidinone and
fluoroquinolone elements (Schemes 6 and 7). The structures of the target hybrids OBP-(1–7) were
further characterized (Supplementary Materials).
O
F
F
F
F
Fe/NH4Cl, H2O
EtOH, 90°C
CbzCl, NaHCO3
H2O, THF
BnBr, K2CO3, KI
acetone, 60°C
O
O
OBn
OBn
OH
OBn
n-BuLi, THF, -78°C-rt
H2N
CbzHN
O2N
O2N
3
1
2
2-Fluoro-4-nitrophenol
F
F
F
NH
F
NCbz
HO
O
HO
O
OBn
H2(1 atm), Pd/C, rt
OH
O
O
O
O
O
N
6
N
N
O
MeOH, Pd/C, H2
rt, 12 h
N
O
O
Na2CO3, DMF, 100°C
HO
HO
HO
HO
7
8
5
4
NCbz
Cbz
N
Trimethylsulfoxonium iodide
NaOH/DMSO, 0-55oC, 2 h
O
O
Benzyl 4-oxopiperidine-1-
carboxylate
6
Scheme 1. Synthesis of compounds 6 and 8.

Molecules 2019, 24, 1641
3 of 22
Scheme 2. Synthesis of hybrids OBP-1 and OBP-2.
Scheme 3. Synthesis of hybrid OBP-3.
Scheme 4. Synthesis of hybrid OBP-4.
Scheme 5. Synthesis of hybrid OBP-5.

Molecules 2019, 24, 1641
4 of 22
F
F
O
O
N
N
N
BrCH2NO2, K2CO3
ACN, rt, 48 h
O2N
Zn,HCl,i-PrOH
rt,overnight
NO2
NaBH4, BF3.Et2O
THF, 40°C, 4 h
N
NH2 DPIEA, ACN,
50°C, 48 h
O
O
NO2
21
20
N-benzylmaleimide
19
F
F
Boc
H
F
Boc
N
1) (Boc)2O, TEA, DMAP
DCM, 0°C, 2 h
N
N
NaHCO3, CBzCl
0°C, overnight
N
N
2) NiCl2. 6H2O
H2N
O2N
N
BzCHN
NaBH4, THF/MeOH
24
23
22
O
F
F
COOH
F
O
F
Boc
Boc
N
N
N
N
O
O
O
O
O
MeOH,H2, Pd/C
rt, 12 h
N
N
NH
N
O
n-BuLi, THF
-78°C, 2 h
O
TEA, DMSO
100℃, overnight
HO
26
25
HO
O
O
F
CO2H
O
O
F
CO2H
O
O
HO
HO
N
F
N
N
N
N
F
6N HCl/ THF
rt, 48 h
N
N
N
O
O
N
N
H
Boc
OBP-6
27
Scheme 6. Synthesis of hybrid OBP-6.
O
N
O
N
F
F
COOH
HO
O
N
F
CO2H
O
N
F
CO2H
O
O
HO
F
O
Boc
F
N
N
N
F
O
N
THF, EA/HCl
r.t, 48 h
NH
N
H
N
TEA, DMSO
100°C, overnight
O
N
Boc
OBP-7
28
HO
26
Scheme 7. Synthesis of hybrid OBP-7.
2.2. Antibacterial Activities
2.2.1. In Vitro Activities of Oxazolidinone-Fluoroquinolone Hybrids
The in vitro antibacterial activities of oxazolidinone-fluoroquinolone hybrids against a panel of
well-characterized susceptible and resistant Gram-positive and Gram-negative bacteria were evaluated
and the results were presented as MICs expressed in µg/mL (Table 1). CZD (cadazolid), LZD (linezolid)
and moxifloxacin (MXF) were used as comparator drugs. As shown in Table 1, MIC values of LZD
and MXF against standard strains were within the acceptable range, indicating that the test results
were reliable.
For Gram-positive bacteria, most oxazolidinone-fluoroquinolone hybrids with the exceptions of
OBP-6 and OBP-7 exhibited the potent antibacterial activities with MICs ranging from 0.031
g/mL, even against levofloxacin-resistant Streptococcus agalactiae (S. agalactiae), vancomycin-resistant
enterococcus (VRE) and methicillin-resistant Staphylococcus aureus (MRSA). Among these new hybrids,
OBP-4 was the most active compound against Gram-positive bacteria (MICs 0.5 g/mL), which
µg/mL to
2
µ
≤
µ
was twice as active overall than the comparator agent CZD, a oxazolidinone-fluoroquinolone hybrid
in phase III clinical trials [23]. The antibacterial efficacy of hybrid OBP-5 against Gram-positive
bacteria was comparable to that of CZD, but was approximately 4–16 times stronger than that of LZD.
The antibacterial activities of hybrids OBP-1, OBP-2 and OBP-3 against Gram-positive bacteria laid
between that of CZD and LZD. Gram-positive bacteria were less susceptible to OBP-6 and OBP-7 than
other hybrids, especially resistant strains. For Gram-negative bacteria, only Haemophilus influenzae

Molecules 2019, 24, 1641
5 of 22
(H. influenzae) was susceptible to these hybrids, while hybrids OBP-6 and OBP-7 also exhibited good
activities against Escherichia coli (E. coli) (Table 1). The above results demonstrated that the antibacterial
activities of these oxazolidinone-fluoroquinolone hybrids were mainly restricted to Gram-positive
bacteria, suggesting a more pronounced oxazolidinone-type antibacterial spectrum.
Table 1. In vitro antibacterial activities of oxazolidinone-fluoroquinolone hybrids.
MIC (µg/mL)
Organism (No.)
OBP-1
OBP-2
OBP-3
OBP-4
OBP-5
OBP-6
OBP-7
CZD
LZD
MXF
S. pneumoniae (ATCC49619)
S. agalactiaeR (13B317)
E. faecalis (ATCC29212)
VRE (13B330)
0.25
0.5
1
1
2
1
1
64
4
0.25
0.25
1
1
1
0.25
0.5
64
4
0.5
0.5
1
2
2
2
2
128
64
0.031
0.031
0.25
0.25
0.5
0.016
0.25
64
0.062
0.031
1
0.5
0.5
0.25
0.25
64
0.5
2
4
64
64
0.062
0.016
16
2
4
1
0.062
0.125
0.5
0.25
0.5
0.5
1
1
1
4
2
4
4
8
128
128
0.062
4
0.125
64
128
128
0.25
0.031
64
MRSA (13B262)
8
S. aureus (ATCC29213)
H. influenzae (ATCC49247)
P. Aeruginosa (ATCC27853)
E. coli (ATCC25922)
0.031
0.008
4
64
8
4
4
1
0.5
0.016
S. agalactiaeR (13B317), levofloxacin-resistant S. agalactiae.
Obviously, hybrids OBP-6 and OBP-7 differed greatly from other hybrids in antibacterial activities
and spacer, which indicated the spacer was a major factor influencing their antibacterial activities [20].
Meanwhile, the antibacterial activity was more or less changed due to the differences of the substituents
at N1-position of fluoroquinolone pharmacophores.
2.2.2. In Vitro Activities of Hybrids OBP-4 and OBP-5 against Clinical Isolates
As can be seen from the above data, OBP-4 and OBP-5 were the two most active
oxazolidinone-fluoroquinolone hybrids. Therefore, the susceptibility of OBP-4 and OBP-5 against
263 clinical isolates were further tested. The resulting MICs for OBP-4, OBP-5 and comparator
drugs enrofloxacin (ENR), ciprofloxacin (CIP), gatifloxacin (GAFX), nadifloxacin (NAFX), LZD and
vancomycin (VAN) were listed in Table 2. For Gram-positive isolates, hybrids OBP-4 and OBP-5 had
the lowest MICs among all tested compounds, with MIC90 values of 0.125–0.25
0.0625–0.25 g/mL for OBP-5. For Gram-negative isolates (Haemophilus parasuis), hybrids OBP-4 and
OBP-5 had relatively weak activities with MIC90 values of 4 g/mL and 2 g/mL, respectively (Table 2).
µg/mL for OBP-4 and
≤
µ
µ
µ
Table 2. In vitro activities of OBP-4 and OBP-5 against clinical isolates.
MIC (µg/mL)
Organism
(No. Tested)
CLSI a %S/%R
Agents
MIC50
MIC90
Range
S. aureus (46)
OBP-4
OBP-5
ENR
≤0.0625
≤0.0625
2
16
1
0.5
2
0.125
≤0.0625–0.25
≤0.0625–0.25
≤0.0625–8
≤0.0625–32
≤0.0625–2
≤0.0625–2
0.5–2
-/-
-/-
≤0.0625
4
32
CIP
37.9/62.1
45.5/42.4
GAFX
NDFX
LZD
VAN
OBP-4
OBP-5
ENR
2
0.5
2
100.0/-
100.0/-
-/-
0.5
0.5
0.125
≤0.0625
0.5
1
0.25
0.5
2
0.25–0.5
S. haemolyticus (32)
0.125
≤0.0625
≤0.0625
0.25
0.125
≤0.0625
0.5
≤0.0625–0.5
≤0.0625–0.125
≤0.0625–8
0.125–16
≤0.0625–2
≤0.0625–4
0.125–8
-/-
CIP
96.2/3.8
96.2/3.8
GAFX
NDFX
LZD
96.2/3.8
96.2/-
VAN
0.5
2
≤0.0625–4

Molecules 2019, 24, 1641
6 of 22
Table 2. Cont.
MIC (µg/mL)
Organism
(No. Tested)
CLSI a %S/%R
Agents
MIC50
MIC90
Range
S. epidermidis (10)
OBP-4
OBP-5
ENR
0.125
0.25
0.125
4
≤0.0625–0.25
≤0.0625–0.125
0.125–4
-/-
-/-
≤0.0625
2
CIP
8
1
1
1
16
2
2
1
2
0.25
0.125
4
8
4
0.25–16
0.125–2
20.0/60.0
30.0/50.0
GAFX
NDFX
LZD
VAN
OBP-4
OBP-5
ENR
≤0.0625–2
0.5–1
100.0/-
100.0/-
-/-
1
≤0.0625
≤0.0625
0.25
0.25
≤0.0625
≤0.0625
0.5
1–2
S. suis (9)
≤0.0625–2
≤0.0625–2
≤0.0625–8
0.125–16
≤0.0625–4
≤0.0625–4
0.5–4
-/-
CIP
GAFX
NDFX
LZD
2
4
VAN
OBP-4
OBP-5
ENR
0.25
0.25
≤0.0625
32
16
4
16
0.5
2
0.125–2
S. pneumoniae (13)
0.25
0.25
>32
>32
32
>32
0.5
32
0.25
0.125
0.5
1
0.5
1
2
0.25
0.25
32
32
8
≤0.0625–0.5
≤0.0625–0.25
2–>32
-/-
-/-
CIP
4–>32
4–32
8–>32
0.5–4
-/100.0
7.7/92.3
GAFX
NDFX
LZD
VAN
OBP-4
OBP-5
ENR
76.9/-
53.8/-
-/-
1
0.5–>32
≤0.0625–1
≤0.0625–0.5
≤0.0625–2
0.125–2
0.125–2
0.125–2
0.5–8
0.25–2
≤0.0625–1
0.5–>32
0.25–>32
0.25–8
0.5–16
0.5–16
E. faecalis (72)
E. faecium (47)
0.25
0.125
0.5
-/-
CIP
0.5
97.3/-
94.6/-
GAFX
NDFX
LZD
OBP-4
OBP-5
ENR
0.25
0.25
2
0.5
0.25
2
2
1
2
2
2
1
0.5
≤0.0625
≤0.0625
≤0.0625
≤0.0625
16
97.3/2.7
-/-
-/-
CIP
51.7/37.9
86.2/13.8
GAFX
NDFX
LZD
VAN
OBP-4
OBP-5
ENR
8
4
4
4
2
1
1
1
82.8/13.8
100.0/-
-/-
0.5–4
H. parasuis (34)
≤0.0625–4
≤0.0625–4
≤0.0625–1
≤0.0625–1
≤0.0625–1
≤0.0625–1
8–>32
-/-
CIP
GAFX
NDFX
LZD
1
>32
>32
VAN
8
2–>32
a
Criteria as published by CLSI. S, susceptible; R, resistant. Empty cells indicate that no breakpoints have
been defined.
For Staphylococcus aureus (S. aureus), Staphylococcus haemolyticus (S. haemolyticus), Staphylococcus
epidermidis (S. epidermidis) and Streptococcus suis (S. suis), most clinical isolates (>90%) can be inhibited
by hybrids OBP-4 and OBP-5 at 0.25
µg/mL, and MIC90 values of OBP-4 and OBP-5 against these
isolates were much lower than those of comparator agents (Tables 2 and 3). Against Streptococcus

Molecules 2019, 24, 1641
7 of 22
pneumoniae (S. pneumoniae) isolates, OBP-4 and OBP-5 retained the excellent activities with MIC90 of
0.25 g/mL, while comparator drugs were almost inactive with MIC90 32 g/mL except for LZD
µ
≥
µ
(Table 2). Furthermore, OBP-4 and OBP-5 showed 2- to 16-fold higher activity against Enterococcus
faecalis (E. faecalis), and 16- to 128-fold higher activity against Enterococcus faecium (E. faecium) than
comparator agents (Table 2). Although OBP-4 and OBP-5 were less active against H. parasuis than
quinolone antimicrobials, they were much better than LZD and VAN (Table 2). Therefore, hybrids
OBP-4 and OBP-5 were the most potent compounds against tested clinical isolates. Overall, OBP-5
exhibited 2-fold greater activity than OBP-4, which can be best explained by the difference of the
substituents at N1-position of fluoroquinolone pharmacophores between OBP-4 and OBP-5.
Table 3. MIC frequency distributions for tests of OBP-4 and OBP-5 against clinical isolates.
No. of Strains (Cumulative %) Inhibited at Each MIC (µg/mL)
Organism
(No. Tested)
Agents
≤0.0625
0.125
0.25
0.5
1
2
4
S. aureus (46)
S. haemolyticus (32)
S. epidermidis (10)
S. suis (9)
OBP-4
OBP-5
OBP-4
OBP-5
OBP-4
OBP-5
OBP-4
OBP-5
OBP-4
OBP-5
OBP-4
OBP-5
OBP-4
OBP-5
OBP-4
OBP-5
26(56.5)
42(91.3)
14(43.8)
30(93.4)
2(20.0)
8(80.0)
5(55.6)
5(55.6)
2(15.4)
7(53.8)
9(12.5)
17(23.6)
17(93.5)
3(97.8)
16(93.8)
2(100.0)
4(60.0)
2(20.0)
2(77.8)
3(88.9)
3(38.5)
4(84.6)
17(36.1)
49(91.7)
3(100.0)
1(100.0)
2(100.0)
4(100.0)
1(88.9)
1(100)
1(100)
S. pneumoniae (13)
E. faecalis (72)
7(92.3)
2(100.0)
43(95.8)
5(98.6)
8(17.2)
33(91.5)
3(20.6)
4(29.4)
1(100.0)
2(98.6)
1(100.0)
37(95.7)
3(97.9)
5(35.3)
6(47.1)
1(100.0)
E. faecium (47)
H. parasuis (34)
1(97.9)
1(100.0)
9(61.8)
7(67.6)
1(100.0)
3(6.4)
4(11.8)
5(14.7)
7(21.3)
1(17.6)
6(79.4)
10(97.1)
7(100.0)
1(100.0)
Meanwhile, hybrids OBP-4 and OBP-5 were also tested against a group of common resistant strains,
including MRSA, methicillin-resistant S. epidermidis (MRSE), CIP-resistant S. aureus, CIP-resistant
S. haemolyticus, VAN- and quinolone-resistant S. pneumoniae, LZD-resistant E. faecalis, and LZD or
VAN- and quinolone-resistant E. faecium. The activities of OBP-4 and OBP-5 against MRSA and MRSE
were equivalent to those of quinolone comparator agents ENR, CIP, GAFX and NDFX, but markedly
superior to those of LZD and VAN (Table 4). For other resistant organisms in the test, MICs values of
OBP-4 and OBP-5 ranged from
≤
0.0625 to 1 µg/mL, and were much lower than the corresponding
MICs values of comparator agents (Table 4). Similarly, OBP-5 displayed higher activities against tested
resistant strains than OBP-4. The superior activities of OBP-4 and OBP-5 against resistant strains
indicated that they had no cross-resistance with tested oxazolidinones or quinolones.
Table 4. In vitro activities of OBP-4 and OBP-5 against drug-resistant strains.
MIC (µg/mL)
Organism
OBP-4
OBP-5
ENR
CIP
GAFX
NDFX
LZD
VAN
MRSA
MRSE
0.25
≤0.0625
0.125
0.5
0.25
1
0.125
≤0.0625
≤0.0625
≤0.0625
≤0.0625
0.25
0.25
≤0.0625
2
8
>32
1
0.125
0.125
32
16
>32
1
0.125
≤0.0625
0.125
0.5
4
>32
1
4
0.25
2
8
0.5
8
1
1
0.25
2
>32
2
4
≤0.0625
S. aureus a
S. haemolyticus a
S. pneumoniae b
E. faecaliss c
E. faecium d
E. faecium b
a
2
2
>32
0.5
8
0.5
0.5
0.125
0.25
>32
>32
>32
>32
16
32
16
2
16
16
CIP-resistant; b VAN and quinolone-resistant; c LZD-resistant; d LZD and quinolone-resistant.

Molecules 2019, 24, 1641
8 of 22
Again, the superior antibacterial activities of hybrids OBP-4 and OBP-5 against Gram-positive
bacteria including drug-resistant strains were confirmed. The result hinted that OBP-4 and OBP-5
may have a similar mode of action with oxazolidinones. In this study, the reduced membrane-
permeability may be a leading cause of poor activities of OBP-4 and OBP-5 against Gram-negative
bacteria. Intrinsically, the weak fluoroquinolone-like characteristic of OBP-4 and OBP-5 could be
attributed to the large molecular volume and decreased liposolubility caused by the hybridization.
2.3. Mode of Action of Hybrids OBP-4 and OBP-5
2.3.1. In Vitro Enzyme Assays of Hybrids OBP-4 and OBP-5
In order to explore the mode of action of OBP-4 and OBP-5, we carried out the enzyme assays for
these two hybrids. The results were presented in Figures 1 and 2 and Table 5.
Figure 1. Inhibition assay of OBP-4 (
and DNA gyrase; Line 2(+): DNA gyrase present and compound absent; Line 3-11(9): DNA gyrase
with different concentrations of tested compound. R: relaxed DNA; S: supercoiled DNA.
A
), OBP-5 (
B
) and CIP (
C
) on DNA gyrase. Line 1( ): no compound
−
DNA gyrase and topoisomerase IV (Topo IV), two well-known targets for quinolone
antimicrobials [24], play a vital role in the replication and transcription of DNA [25]. Therefore,
the impact of OBP-4 and OBP-5 on DNA gyrase and topo IV was firstly investigated by DNA gyrase
supercoiling assay and Topo IV relaxation assay, respectively. As shown in Figure 1, OBP-4 and OBP-5
could inhibit DNA gyrase in a dose-dependent manner with IC50 values being 1–5
OBP-4 and OBP-5, respectively (Table 5). By comparison, the inhibition action of OBP-4 and OBP-5 on
DNA gyrase was evidently inferior to the reference drug CIP (IC50, <0.75 M). Similar results but with
higher IC50 values were found for hybrids OBP-4 and OBP-5 in the topo IV relaxation assay. OBP-4
(IC50, 10–15 M) was less active in inhibiting topo IV compared with CIP (IC50, 3–6 M) while OBP-5
was the least active (IC50 > 40 M) (Figure 2, Table 5). Overall, hybrid OBP-4 exhibited a much better
inhibitory activity on DNA synthesis than OBP-5, but they were significantly less active than CIP.
µM and 20 µM for
µ
µ
µ
µ

Molecules 2019, 24, 1641
9 of 22
Figure 2. Inhibition assay of OBP-4 (
and Topo IV; Line 2(+): Topo IV present and compound absent; Line 3-11(9): Topo IV with different
A
), OBP-5 (
B
) and CIP (
C
) on Topo IV. Line 1( ): no compound
−
concentrations of tested compound. R: relaxed DNA; S: supercoiled DNA.
Table 5. Activity of OBP-4 and OBP-5 against DNA gyrase, topo IV, and in an in vitro
transcription/translation assay.
IC50 (µM)
Compd.
DNA Gyrase
Topo IV
Inhib. of Protein Synthesis
OBP-4
OBP-5
CIP
1–5
20
<0.75
NT
10–15
>40
3–6
5
2
NT
5
LZD
NT
NT, not tested.
Oxazolidinones have a unique mechanism of action involving inhibition of protein synthesis
by binding the ribosomal 50S subunit [16]. Thus, the action of hybrids OBP-4, OBP-5 and the
reference drug LZD on protein synthesis was investigated with the E. coli-based cell-free protein
synthesis system. As expected, OBP-4 and OBP-5 showed potent inhibition of protein synthesis in vitro
transcription/translation assay. IC50 values of OBP-4 and OBP-5 were 5
µM for OBP-4 and 2
µM for
OBP-5, respectively (Table 5), which were equal or 2.5-fold greater than that of LZD (IC50, 5
(Table 5).
µM)
The above data demonstrated that the protein synthesis inhibition was the primary mode of action
for OBP-4 and OBP-5 whilst the activity against DNA synthesis was weak, which correlated well with
the observed antibacterial activity pattern. The results confirmed our previous speculation. For the
imbalanced dual-action mode, the possible explanation was that the introduction of oxazolidinone
substructure or spacer in hybrid changed the intrinsic binding affinity of fluoroquinolone in two targets.
2.3.2. Molecular Docking of Hybrids OBP-4 and OBP-5
As shown in Table 5, OBP-4 and OBP-5 displayed a strong inhibition action in protein synthesis.
To understand the binding mode of OBP-4 and OBP-5, the molecular docking was performed between
OBP-4 (or OBP-5) and the active site of 50S subunit. The optimal docking conformations of tested
compounds in the active site of 50S subunit were presented in Figure 3. The blue, light blue and

Molecules 2019, 24, 1641
10 of 22
red sticks represented the optimal conformations of OBP-4 (Figure 3A), OBP-5 (Figure 3B) and LZD
(Figure 3C). As expected, both OBP-4 and OBP-5 can bind to the active site of the 50S subunit. OBP-4
were docked into the active site of 50S subunit, with its oxazolidinone part forming hydrogen bonds
with U-2612 and G-2532 (Figure 3A), while OBP-5 were surrounded by G-2580, G-2610, U-2611 and
C-2479 (Figure 3B). Furthermore, OBP-4, OBP-5 and LZD have a similarity in the optimal conformations
and binding modes to the active site of 50S subunit (Figure 3C). This similarity could explain why
OBP-4 and OBP-5 have an oxazolidinone-like antibacterial spectrum and a strong inhibitory activity on
protein synthesis. Meanwhile, the better antibacterial activities of OBP-4 and OBP-5 than that of LZD
may be attributed to the interactions between fluoroquinolone pharmacophores and the 50S subunit.
The further investigations are required to clearly elucidate the mode of action of OBP-4 and OBP-5.
Figure 3.
(PDB ID: 4WFA). (
50S subunit (PDB ID: 4WFA). (
(
A
) The optimal docking conformation of OBP-4 (blue sticks) in the active site of 50S subunit
) The optimal docking conformation of OBP-5 (light blue sticks) in the active site of
) The superposition of OBP-4, OBP-5 and LZD (red sticks) in the active
B
C
site of 50S subunit (PDB ID: 4WFA). Hydrogen bonds are showed as dashed lines.
2.4. Acute Toxicity Test
The previous study has shown that CZD, structurally similar to hybrids OBP-4 and OBP-5, has
a good safety level [26]. Therefore, the oral acute toxicity of hybrids OBP-4 and OBP-5 was directly
evaluated with a limit test dose of 2000 mg/kg. No death and any toxicity signs were observed for all
animals until the experiment was finished. The oral LD50 values for hybrids OBP-4 and OBP-5 were
considered to be higher than 2000 mg/kg, suggesting that hybrids OBP-4 and OBP-5 are likely to be
safe for future application, at least with a single oral dose.

Molecules 2019, 24, 1641
11 of 22
3. Materials and Methods
3.1. General
The chemicals and reagents were acquired commercially and used without further purification.
Reactions were monitored by thin-layer chromatography (TLC) plates pre-coated with 0.2-mm-thick
silica gel GF254 (Qingdao Ocean Chemical Co., Ltd., Shandong, China). The visualized analysis of
products was achieved by UV illumination at 254 nm and staining in the solution of bismuth nitrate
and 0.05% KMnO4. The determination of the melting point was conducted with an X-4 microscopic
1
melting point apparatus (Beijing Tech Instrument Co., Ltd., Beijing, China). H-NMR and 13C-NMR
were performed on a Bruker spectrometer (Bruker BioSpin, Zürich, Switzerland) at 400 and 151 MHz,
respectively. All NMR spectra were reported in chemical shifts (δ, ppm) with residual solvents as
internal standards. Multiplicities were expressed as s (singlet), d (doublet), dd (doublet of doublets),
t (triplet) and m(multiplet), and coupling constants were expressed as J in hertz (Hz). Electrospray
ionization mass spectra (ESI-MS) were recorded with a Shimadzu mass spectrometer (Shimadzu,
Tokyo, Japan) and high resolution mass spectra (HRMS) were obtained with an Agilent Q-TOF 6530
mass spectrometer (Agilent Technologies, Palo Alto, CA, USA).
3.2. Chemistry
3.2.1. Preparation of Hybrids OBP-1, OBP-2 and OBP-3
Compound 1: a mixture of 2-fluoro-4-nitrophenol (10.00 g, 63.69 mmol, 1 eq) [27], benzyl bromide
(11.98 g, 70.00 mmol, 1.1 eq), K2CO3 (13.18 g, 95.54 mmol, 1.5 eq) and KI (1.06 g, 6.37 mmol) were
added to acetone (0.10 L) and then heated to 60 ◦C overnight. Upon cooling to room temperature
and addition of ice water, yellow solid was precipitated and dried (15.00 g, 95%), m.p., 197–198 ◦C.
1H-NMR (400 MHz, DMSO-d6)
6H), 5.35 (s, 2H). ESI-MS (m/z): 247.9 [M + H]+.
δ 8.18 (dd, J = 11.2, 2.8 Hz, 1H), 8.13 (d, J = 9.2 Hz, 1H), 7.53–7.39 (m,
Compound : compound (72.80 g, 294.74 mmol, 1 eq), reduced iron powder (99.00 g,
2
1
1768.42 mmol, 6 eq) and ammonium chloride (94.59 g, 1768.42 mmol, 6 eq) were mixed with EtOH:H2O
(2:1), and refluxed at 90 ◦C for 2 h. After cooling to room temperature, the mixture was filtered. The
filtered and concentrated to give the brown oily substance compound
(400 MHz, CDCl3) 7.42 (d, J = 7.2 Hz, 2H), 7.36 (t, J = 7.2 Hz, 2H), 7.31 (t, J = 7.0 Hz, 1H), 6.80 (t,
2
(60.00 g, 94%). 1H-NMR
δ
J = 9.0 Hz, 1H), 6.46 (dd, J = 12.4, 2.8 Hz, 1H), 6.33 (d, J = 8.8 Hz, 1H), 5.03 (s, 2H). ESI-MS (m/z): 217.9
[M + H]+.
Compound 3: a saturated sodium bicarbonate solution (0.50 L) was added to compound 2 (60.00 g,
276.50 mmol, 1 eq) in anhydrous THF (0.50 L) and cooled to 0 ◦C. The mixed solution was treated
dropwise with benzyl carbonochloridate (51.88 g, 304.15 mmol, 1.1 eq) and then stirred for 30 min at
0 ◦C. The mixture was extracted with EtOAc (3.00 L*2) and brine (2.40 L). The organic layer was dried
(Na2SO4), filtered, and concentrated in vacuo to give compound
m.p., 116–118 ◦C. 1H-NMR (400 MHz, CDCl3)
7.48–7.27 (m, 11H), 6.95–6.87 (m, 2H), 6.57 (s, 1H), 5.19
(s, 2H), 5.10 (s, 2H). ESI-MS (m/z): 351.9 [M + H]+.
3 (88.00 g, 91%) as a yellow solid,
δ
Compoun◦d
4: the suspension of compound 3 (40.00 g, 113.96 mmol, 1 eq) in THF (1.00 L) was
cooled to
−
78 C, and then n-BuLi (2.50 M in hexane 54.70 mL, 136.75 mmol, 1.2 eq) was added
◦
dropwisely and stirred at
−
78 C for 0.5 h. Subsequently, (R)-oxiran-2-ylmethyl butyrate (20.35 g,
141.31 mmol, 1.24 eq) in THF (0.50 L) was added dropwisely to the mixed solution within 30 min. The
reaction mixture was warmed up slowly and stirred at room temperature overnight. The mixture was
extracted with EtOAc (2.00 L*2) and brine (1.50 L). The organic layer was dried (MgSO4), filtered and
concentrated in vacuo to give compound
(400 MHz, DMSO-d6) 7.58 (dd, J = 13.6, 2.4 Hz, 1H), 7.44 (d, J = 7.2 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H),
7.33 (t, J = 6.8 Hz, 1H), 7.26 (t, J = 9.2 Hz, 1H), 7.22–7.16 (m, 1H), 5.22 (t, J = 5.6 Hz, 1H), 5.16 (s, 2H),
4
(31.50 g, 87%) as a yellow solid, m.p., 135–137 ◦C. 1H-NMR
δ

Molecules 2019, 24, 1641
12 of 22
4.67 (dd, J = 9.2, 5.6 Hz, 1H), 4.03 (t, J = 9.0 Hz, 1H), 3.78 (dd, J = 8.8, 6.4 Hz, 1H), 3.69–3.62 (m, 1H),
3.58–3.50 (m, 1H). ESI-MS (m/z): 317.9 [M + H]+.
Compound 5: Pd/C (1.50 g, 5%) was carefully added to a solution of compound 4 (31.00 g,
97.79 mmol, 1 eq) in MeOH (0.04 L). The reaction was proceeded with H2 at room temperature for 12 h.
The liquid was filtered, concentrated and purified by recrystallization in petroleum ether and ethyl
acetate (2:1) to give compound
DMSO-d6) 9.67 (s, 1H), 7.47 (d, J = 13.6 Hz, 1H), 7.09 (d, J = 8.8 Hz, 1H), 6.95 (t, J = 9.4 Hz, 1H), 5.20
5
(21.76 g, 98%) as a white solid, m.p., 179–181 ◦C. 1H-NMR (400 MHz,
δ
(s, 1H), 4.65 (s, 1H), 4.01 (t, J = 9.0 Hz, 1H), 3.76 (t, J = 7.4 Hz, 1H), 3.65 (d, J = 11.6 Hz, 1H), 3.54 (d,
J = 12.4 Hz, 1H). ESI-MS (m/z): 227.9 [M + H]+.
Compound 6: the sodium hydroxide (60%, 4.37 g, 109.25 mmol, 1.5 eq) was added to a solution of
trimethylsulfoxonium iodide (24.06 g, 109.32 mmol, 1.5 eq) in dry DMSO (0.10 L) at 0 ◦C, and then
stirred at 0 ◦C for 40 min. Benzyl 4-oxopiperidine-1-carboxylate (17.00 g, 72.88 mmol, 1 eq) [28] was
added to the mixture and stirred for 2 h at 55 ◦C. The reaction mixture was extracted with EtOAc
(0.18 L) and brine (0.06 L). The organic layer was dried (Na2SO4) and concentrated in vacuo. Crude
was purified by flash column chromatography (hexanes/EtOAc) on silica gel column to give compound
1
6
(14.50 g, 80%) as a yield oil. H-NMR (400 MHz, CDCl3)
δ
7.42–7.28 (m, 5H), 5.15 (s, 2H), 3.82 (s, 2H),
3.48 (t, J = 10.8 Hz, 2H), 2.70 (s, 2H), 1.83 (s, 2H), 1.45 (d, J = 10.4 Hz, 2H). ESI-MS (m/z): 247.9 [M + H]+.
Compound 7: the mixed solution of compound 5 (8.00 g, 35.24 mmol, 1 eq) and compound 6
(13.06 g, 52.86 mmol, 1.5 eq) DMF (0.05 L) was treated with Na2CO3 (7.47 g, 70.48 mmol, 2 eq). The
resulting solution was heated to 100 ◦C and stirred for 12 h. The reaction was quenched by H2O and
extracted by EtOAc (0.20 L*3). The combined organic layer was washed by brine and dried with
anhydrous Na2SO4. After evaporation and recrystallization, compound
7 (17.00 g, 90%) was obtained
as a white solid, m.p., 121–123 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ
7.61–7.54 (m, 1H), 7.40–7.30 (m,
5H), 7.23–7.16 (m, 2H), 5.21 (t, J = 5.6 Hz, 1H), 5.08 (s, 2H), 4.84 (s, 1H), 4.68 (dd, J = 9.2, 5.6 Hz, 1H),
4.04 (t, J = 9.0 Hz, 1H), 3.87–3.81 (m, 4H), 3.81–3.76 (m, 1H), 3.70–3.62 (m, 1H), 3.60–3.49 (m, 1H), 3.17
(d, J = 3.6 Hz, 2H), 1.66–1.50 (m, 4H). ESI-MS (m/z): 475.0 [M + H]+.
Compound
35.86 mmol, 1 eq) in MeOH (0.50 L). The reaction was proceeded with H2 at room temperature for 12 h.
The mixture was filtered, concentrated and purified by recrystallization to obtained compound as
a white solid (11.00 g, 92%), m.p., 179–181 ◦C. 1H-NMR (400 MHz, DMSO-d6)
7.57 (d, J = 13.2 Hz,
8: Pd/C (1.00 g, 5%) was carefully added to a solution of compound 7 (17.00 g,
8
δ
1H), 7.25–7.12 (m, 2H), 5.24 (s, 1H), 4.70–4.65 (m, 1H), 4.52 (s, 1H), 4.04 (t, J = 9.0 Hz, 1H), 3.85–3.73
(m, 3H), 3.66 (dd, J = 12.4, 3.2 Hz, 1H), 3.55 (dd, J = 12.4, 4.0 Hz, 1H), 2.81 (t, J = 10.6 Hz, 2H), 2.67 (d,
J = 12.0 Hz, 2H), 1.65–1.51 (m, 2H), 1.44 (d, J = 12.8 Hz, 2H). ESI-MS (m/z): 340.9 [M + H]+.
Compound 9: to a solution of (S)-9,10-difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de]
-1,4-benzoxazine-6-carboxylic acid (1.00 g, 3.56 mmol, 1 eq) in H2SO4 (98%, 0.02 L) [29], KNO3 (0.719 g,
7.12 mmol, 2 eq) was added and then stirred at 0 ◦C for 30 min. The reaction mixture was diluted
with ice water (0.05 L) and filtered. Compound
9 Yield (1.00 g, 86%) was obtained as a yellow solid.
1H-NMR (400 MHz, DMSO-d6)
δ
9.15 (s, 1H), 5.08 (d, J = 6.8 Hz, 1H), 4.76 (d, J = 10.8 Hz, 1H), 4.51 (d,
J = 9.6 Hz, 1H), 1.47 (d, J = 6.8 Hz, 3H).
Compound 10: Pd/C (0.05 g, 5%) was carefully added to a solution of compound
9 (1.00 g,
3.07 mmol, 1 eq) in MeOH (0.05 L). The reaction proceeded with H2 at room temperature for 12 h.
1
The liquid was filtered and concentrated to give compound 10 (0.50 g, 55%) a yellow oil. H-NMR
(400 MHz, DMSO-d6) δ 8.86 (s, 1H), 4.86 (d, J = 6.8 Hz, 1H), 4.47 (d, J = 11.2 Hz, 1H), 4.21 (d, J = 10.4 Hz,
1H), 1.40 (d, J = 6.8 Hz, 3H). ESI-MS (m/z): 300.9 [M + H]+.
General procedure for the synthesis of hybrids OBP-1, OBP-2 and OBP-3.

Molecules 2019, 24, 1641
13 of 22
A suspension of compound
8
(1.1 eq), TEA (5 eq) and corresponding acid (1 eq) in DMSO were
mixed and stirred at 120 ◦C for 20 h. The mixed solution was cooled to room temperature, adjusted pH
(<6) with 50% H2SO4, filtered and concentrated in vacuo. Finally, the target hybrids were obtained by
recrystallization in petroleum ether and ethyl acetate (2:1).
OBP-1: while solid (0.516 g, 30%), m.p., 256–257 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 15.11 (s,
1H), 8.76 (s, 1H), 7.66–7.53 (m, 2H), 7.21 (dd, J = 14.4, 5.2 Hz, 2H), 5.32 (s, 2H), 5.22 (s, 1H), 4.82 (s, 1H),
4.71–4.66 (m, 1H), 4.05 (t, J = 8.8 Hz, 1H), 3.88 (s, 2H), 3.83–3.77 (m, 1H), 3.67 (d, J = 12.8 Hz, 1H), 3.57–3.50
(m, 3H), 3.23 (s, 2H), 3.02 (s, 3H), 1.88 (t, J = 11.0 Hz, 2H), 1.65 (d, J = 12.8 Hz, 2H).13C-NMR (151 MHz,
DMSO-d6) δ 176.50 (d, J = 3.02 Hz), 165.98, 155.89 (d, J = 246.13 Hz), 154.88, 151.85 (d, J = 226.50 Hz),
145.73, 143.15 (d, J = 10.57 Hz), 138.54 (d, J = 7.55 Hz), 132.92 (d, J = 15.1 Hz), 132.74, 124.37, 119.51
(d, J = 9.06 Hz), 116.35, 114.27, 107.06 (d, J = 22.65 Hz), 106.69, 104.21 (d, J = 24.16 Hz), 83.22, 78.01,
73.57, 67.56, 62.25, 46.61, 46.51, 43.31, 34.46. ESI-MS (m/z) [M + H]+ calcd. for C28H28F2N4O9: 603.1897,
found: 603.1906.
OBP-2: while solid (0.60 g, 56%), m.p., 269–270 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 15.26 (s, 1H),
8.96 (s, 1H), 7.58 (d, J = 12.4 Hz, 2H), 7.27–7.18 (m, 2H), 5.22 (t, J = 5.6 Hz, 1H), 4.97–4.86 (m, 1H), 4.79
(s, 1H), 4.72–4.65 (m, 1H), 4.59 (d, J = 10.8 Hz, 1H), 4.39 (s, 1H), 4.05 (t, J = 9.0 Hz, 1H), 3.88 (s, 2H),
3.80 (dd, J = 8.8, 6.4 Hz, 1H), 3.72–3.62 (m, 1H), 3.58–3.46 (m, 3H), 3.24 (d, J = 11.6 Hz, 2H), 1.96–1.79
(m, 2H), 1.64 (d, J = 12.8 Hz, 2H), 1.45 (d, J = 6.8 Hz, 3H). 13C-NMR (151 MHz, DMSO-d6)
δ 176.80
(d, J = 3.02 Hz), 166.52, 156.00 (d, J = 247.64 Hz), 154.88, 151.90 (d, J = 243.11 Hz), 146.51, 143.16 (d,
J = 12.08 Hz), 140.49 (d, J = 6.04 Hz), 133.28 (d, J = 15.1Hz), 132.73, 125.27, 119.76 (d, J = 9.06 Hz), 116.34,
114.26, 107.05 (d, J = 24.16 Hz), 107.01, 103.70 (d, J = 24.16 Hz), 77.67, 73.57, 68.44, 68.15, 62.07, 55.26,
46.61, 46.55, 34.48, 18.36.ESI-MS (m/z) [M + H]+ calcd. for C29H29F2N3O9: 602.1945, found: 602.1948.
OBP-3: yellow solid (0.70 g, 83%), m.p., 255–256 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 15.09 (s,
1H), 8.68 (s, 1H), 7.59 (d, J = 14.4 Hz, 1H), 7.29–7.12 (m, 2H), 6.91 (s, 2H), 5.23 (s, 1H), 4.82–4.61 (m, 3H),
4.38 (d, J = 11.2 Hz, 1H), 4.16–3.99 (m, 2H), 3.87 (s, 2H), 3.83–3.76 (m, 1H), 3.67 (d, J = 11.2 Hz, 1H), 3.55
(d, J = 14.4 Hz, 1H), 3.50–3.42 (m, 2H), 3.24 (s, 2H), 1.93–1.75 (m, 2H), 1.63 (d, J = 12.0 Hz, 2H), 1.38
(d, J = 6.4 Hz, 3H). 13C-NMR (151 MHz, DMSO-d6)
δ 180.25 (d, J = 3.02 Hz), 166.62, 154.89, 151.90 (d,
J = 243.11 Hz), 146.78, 144.42, 143.17 (d, J = 12.08 Hz), 141.26 (d, J = 235.56 Hz), 134.31 (d, J = 15.1 Hz),
132.68 (d, J = 9.06 Hz), 127.91 (d, J = 6.04 Hz), 126.15, 124.09, 116.34 (d, J = 1.51 Hz), 114.25, 109.99,
107.06 (d, J = 24.16 Hz), 105.67, 77.66, 73.57, 68.22, 67.23, 62.06, 55.74, 46.61, 34.53, 18.31. ESI-MS (m/z)
[M + H]+ calcd. for C29H30F2N4O9: 617.2054, found: 617.2063.
3.2.2. Preparation of Hybrid OBP-4
Compound 11
:
to a stirred solution of H2SO4 (7.78 mL) in Ac2O (0.778 L, 8.21 mol),
◦
3,4-difluoroaniline (500.00 g, 3.88 mol) [30] was added at about 10 C, and then stirred at room
temperature for 30 min. The mixture was poured into ice water, filtered, the filter cake was washed
1
with water and dried to give an off-white solid (604.00 g, 91%). H-NMR (400 MHz, CDCl3)
(m, 1H), 7.53 (s, 1H), 7.12–7.03 (m, 2H), 2.17 (s, 3H). ESI-MS (m/z): 171.9 [M + H]+.
δ 7.63–7.56
Compound 12: to a stirred solution of compound 11 (588.00 g, 3.44 mol), NaOAc (338.00 g, 4.12
mol) in AcOH (1.94 L) was added dropwise t◦o a solution of Br2 (0.212 L, 4.12 mol) in AcOH (0.60 L)
◦
at 60 C, then the mixture was stirred at 80 C until SM was disappeared. After cooling to room
temperature, the mixture was poured into ice water, filtered, the filter cake was washed with water and
1
dried to give a white solid (735.00 g, 85%). H-NMR (400 MHz, CDCl3)
δ 8.40–8.30 (m, 1H), 7.60–7.48
(m, 1H), 7.41–7.35 (m, 1H), 2.24 (s, 3H). ESI-MS (m/z): 249.8 [M + H]+.
Compound 13: a mixture of compound 12 (565.00 g, 2.26 mol), sodium m-nitrobenzensulfonate
(508.00 g, 2.26 mol), iron sulfate heptahydrate (63.30 g, 158.00 mmol), boric acid (616.00 g, 9.94 mol),
water (2.16 L) and concentrated HCl (2.16 L) was refluxed for 0.5 h. Crotonaldehyde (0.278 L, 3.38 mol)
was added dropwise to the mixture at the same temperature and stirring was continued for 1 h under

Molecules 2019, 24, 1641
14 of 22
reflux. After cooling to room temperature, the insoluble matter was filtered off and the filtrate was
poured into MeOH (0.80 L). The solution was neutralized with 25% (w/v) aqueous NaOH, cooled at
ice bath. The resultant precipitates were collected by filtration, the wet solid was dissolved in DCM
(2.00 L) and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated in vacuo and
1
the crude product was recrystallized from hexane to give an off-white solid (330.00 g, 57%). H-NMR
(400 MHz, CDCl3) δ 8.31 (d, J = 8.4 Hz, 1H), 7.94–7.88 (m, 1H), 7.42 (d, J = 8.8 Hz, 1H), 2.82 (s, 3H).
Compound 14: a mixture of compound 13 (330.00 g, 1.28 mol), 10% wet Pd/C (6.60 g), and sodium
acetate (105.00 g, 1.28 mol) in 90% (v/v) aqueous AcOH (2.00 L) was hydrogenated at 50 ◦C under a
hydrogen pressure 1–5 atm. After the hydrogen absorption had ceased, the catalyst was filtered off and
the filtrate was concentrated under reduced pressure until about half of the solvent was evaporated,
the residue was poured into water, the resulting precipitates were collected by filtration and dried
to give a light red solid as the first crop (160.00 g), the filtrate was extracted with EA, the combined
organic layer was washed with saturated aqueous NaHCO3, dried over Na2SO4 and evaporated in
vacuo to give a brown solid as the second crop (65.00 g).The combined crops (225.00 g, 1.26 mol) were
dissolved in AcOH (1.10 L), to the stirred solution was added NaBH3CN (198.00 g, 3.15 mol), then the
mixture was stirred at room temperature for 6 h, to the mixture was added saturated aqueous Na2CO3
until there was no bubble produced, extracted with EA and the combined organic layer was washed
with brine, dried over anhydrous sodium sulfate and concentrated in vacuo, the residue was purified
1
by silica gel column chromatography (1% EA in PE) to give a colorless oil (180.00 g, 77%). H-NMR
(400 MHz, CDCl3) δ 6.80–6.71 (m, 1H), 6.18–6.09 (m, 1H), 3.64 (brs, 1H), 3.35–3.26 (m, 1H), 2.90–2.80
(m, 1H), 2.70–2.58 (m, 1H), 1.99–1.90 (m, 1H), 1.56–1.44 (m, 1H), 1.20 (d, J = 6.0 Hz, 3H). ESI-MS (m/z):
184.0 [M + H]+.
Compound 15: compound 14 (174.00 g, 951.00 mmol) was added to a stirred solution of L-DBTA
(187.00 g, 522.00 mmol) in EA (0.35 L) and the mixture was stirred at room temperature for 2 h. The
resulting precipitates were collected by filtration and recrystallized from 60% (v/v) aqueous MeOH.
The resulting solid was treated with aqueous NaOH to give a colorless oil (58.00 g, 33%), 100% ee as
determined by chiral HPLC. 1H-NMR (400 MHz, CDCl3)
δ 6.80–6.71 (m, 1H), 6.18–6.09 (m, 1H), 3.67 (s,
1H), 3.35- 3.26 (m, 1H), 2.90–2.80 (m, 1H), 2.70–2.58 (m, 1H), 1.99–1.90 (m, 1H), 1.56- 1.44 (m, 1H), 1.20
(d, J = 6.4 Hz, 3H).
Compound 16: A mixture of compound 15 (58.00 g, 317.00 mmol) and EMME (103.00 g,
◦
477.00 mmol) was heated at 130 C with stirring for 3 h and the reaction mixture was added
dropwise to the pre-heated PPA (161.00 g) at 130 ◦C. When the addition was complete, the mixture was
stirred at the same temperature for 1 h. Then EtOH (0.875 L), concentrated HCl (0.058 L) and water
(0.58 L) were added, the mixture was refluxed for 1 h. After cooling, water (1.13 L) was added to the
mixture and the resulting precipitates were collected by filtration, the solid was washed with water
1
then ether and dried to give an off-white solid (62.00 g, 70%). H-NMR (400 MHz, DMSO-d6))
δ 14.96
(s, 1H), 9.06 (s, 1H), 8.13–8.06 (m, 1H), 5.02–4.89 (m, 1H), 3.16–3.08 (m, 1H), 3.07–2.95 (m, 1H), 2.23–2.07
(m, 2H), 1.39 (d, J = 6.8 Hz, 3H). ESI-MS (m/z): 279.9 [M + H]+.
Compound 17: A mixture of boric anhydride (2.74 g, 39.40 mmol), Ac2O (25.58 g, 251.00 mmol),
AcOH (12.04 g, 201.00 mmol) was refluxed for 3 h. Compound 16 (20.00 g, 71.6 mmol) was added to
the resulting solution and the mixture was refluxed for 2 h, then toluene (0.12 L) was added. After
cooling to room temperature, the resulting precipitates were collected by filtration to give an off-white
1
solid (25.00 g, 86%). H-NMR (400 MHz, CDCl3)
δ
9.21 (s, 1H), 8.27–8.21 (m, 1H), 5.05–4.93 (m, 1H),
3.39–3.30 (m, 1H), 3.17–3.06 (m, 1H), 2.38–2.31 (m, 2H), 2.07 (s, 3H), 2.01 (s, 3H), 1.64 (d, J = 6.8 Hz, 3H).
ESI-MS (m/z): 405.9 [M + H]+.
OBP-4: A mixture of compound 17 (14.00 g, 34.40 mmol), compound
8 (15.20 g, 44.70 mmol)
and TEA (10.40 g, 0.103 mol) in DMF (0.25 L) was stirred at 50 ◦C for 12 h. After cooling to room
temperature, the mixture was poured into water (2.50 L), the resulting precipitates were collected by

Molecules 2019, 24, 1641
15 of 22
filtration and dried to give a yellow solid (21.60 g, 86%), the yellow solid was added to a mixture
of 1.5 N aqueous LiOH (0.08 L) and ACN (0.04 L). The mixture was stirred at room temperature for
about 2 h and then concentrated HCl was added until PH is around 2, the resulting precipitates were
collected by filtration, the obtained solid was suspended in 50% (v/v) aqueous ethanol and refluxed
for 6 h, after cooling to room temperature, the mixture was filtered to give a white solid, which was
recrystallized with ACN and EtOH to give a white solid (10.25 g, 50%), m.p., 225–226 ◦C. 1H-NMR
(400 MHz, DMSO-d6)
δ 15.32 (s, 1H), 8.96 (s, 1H), 7.87 (d, J = 12.5 Hz, 1H), 7.59 (d, J = 13.8 Hz, 1H),
7.27–7.21 (m, 2H), 5.23 (t, J = 5.5 Hz, 1H), 4.88 (br s, 1H), 4.81 (s, 1H), 4.69 (br s, 1H), 4.05 (t, J = 8.9 Hz,
1H), 3.91 (s, 2H), 3.82–3.78 (m, 1H), 3.74–3.66 (m, 2H), 3.58–3.53 (m, 1H), 3.29 (s, 1H), 3.16–2.94 (m, 4H),
2.16–1.94 (m, 3H), 1.80 (d, J = 10.3 Hz, 1H), 1.68 (t, J = 13.2 Hz, 2H), 1.42 (d, J = 6.5 Hz, 3H). 13C-NMR
(151 MHz, DMSO-d6) δ176.02 (d, J = 3.02 Hz), 166.26, 163.59, 154.87, 152.29 (d, J = 246.13 Hz), 151.91
(d, J = 243.11 Hz), 145.97 (d, J = 10.57 Hz), 143.11 (d, J = 10.57 Hz), 136.33, 132.75 (d, J = 9.06 Hz),
117.26 (d, J = 7.55 Hz), 116.32, 114.23, 112.43 (d, J = 22.65 Hz), 107.06 (d, J = 24.16 Hz), 102.72, 101.96,
77.35, 73.57, 72.72, 67.98, 62.08, 46.62, 45.91, 45.76, 33.52, 33.46, 20.77. ESI-MS (m/z) [M + H]+ calcd. for
C30H31F2N3O8: 600.2152, found: 600.2159.
3.2.3. Preparation of Hybrid OBP-5
Compound 18: to a mixture of ethyl 6,7-difluoro-1-methyl-4-oxo-4H-(1,3)-thiazeto (3,2-a)
quinoline-3-carboxylate (3.78 g, 12.10 mmol) [31], compound
8 (4.00 g, 11.80 mmol), TEA (3.57 g,
35.30 mmol) and DMSO (0.06 L) was stirred at 100 ◦C overnight. The reaction was quenched with
1
water (0.12 L), the mixture was filtered to give compound 18 (6.15 g, 83%) as a white solid. H-NMR
(400 MHz, DMSO-d6) δ 7.66–7.70 (d, J = 16 Hz,1H), 7.57–7.61 (d, J = 16 Hz,1H), 6.84–6.85 (d, J = 4 Hz,
1H), 6.17–6.21 (q, J = 12, 4Hz, 1H), 5.23–5.25 (t, J = 8 Hz, 1H), 4.86 (s, 1H), 4.68–4.70 (dd, J = 8, 4 Hz, 1H),
4.18 (q, J = 16 Hz, 2H), 4.06 (t, J = 12 Hz, 1H), 3.90 (s, 2H), 3.83 (s, 1H), 3.67 (m,1H), 3.55–3.59 (m, 1H),
3.43–3.46 (m, 2H), 3.17–3.25 (dd, J = 24, 12 Hz, 2H), 2.06 (d, J = 4 Hz, 3H), 1.88–1.93(t, J = 12 Hz,2H),
1.69–1.72 (d, J = 12 Hz, 2H), 1.24–1.26 (t, J = 8 Hz, 3H). MS (ESI): mass calcd.. for C30H31F2N3O8S
631.18, m/z found: 632.1 [M + H]+.
OBP-5: compound 18 (1.58 g, 2.50 mmol, 1eq) was dissolved in a mixture of THF/MeOH (0.02
L mL/0.02 L), and then lithium hydroxide (2.00M, 0.02 L) was slowly added and stirred at room
temperature for 2 h. The reaction mixture was filtered to give the crude of OBP-5 as a solid after
adjusting its pH (<6) with 50% H2SO4. The crude was separated on a silica gel column (DCM:
MeOH=1:1) to obtain light white solid OBP-5 (0.38 g, 25%), m.p., 250–251 ◦C. 1H-NMR (400 MHz,
DMSO-d6)
δ 14.70 (s, 1H), 7.79 (d, J = 13.6 Hz, 1H), 7.58 (d, J = 14.4 Hz, 1H), 7.27–7.17 (m, 2H), 6.96 (d,
J = 6.4 Hz, 1H), 6.40 (s, 1H), 5.23 (t, J = 5.6 Hz, 1H), 4.90 (s, 1H), 4.68 (dd, J = 8.8, 5.4 Hz, 1H), 4.04 (t,
J = 8.8 Hz, 1H), 3.90 (s, 2H), 3.82–3.75 (m, 1H), 3.71–3.62 (m, 1H), 3.57–3.50 (m, 3H), 3.29–3.23 (m, 2H),
2.11 (d, J = 5.6 Hz, 3H), 1.90 (t, J = 10.8 Hz, 2H), 1.71 (d, J = 12.8 Hz, 2H).13C-NMR (151 MHz, DMSO-d6)
δ
176.98 (d, J = 3.02 Hz), 166.55, 154.88, 151.99 (d, J = 242.11 Hz), 147.81, 143.31 (d, J = 12.08 Hz), 143.22
(d, J = 10.57 Hz), 133.45(d, J = 199.32 Hz), 132.73, 122.32, 116.56 (d, J = 6.04 Hz), 114.25 (d, J = 3.02 Hz),
109.40 (d, J = 22.65 Hz), 107.06 (d, J = 24.16 Hz), 106.94, 73.57, 68.19, 62.09, 57.54, 46.87, 46.62, 34.86,
34.33, 25.35, 20.03, 18.99. ESI-MS (m/z) [M + H]+ calcd. for C28H27F2N3O8S: 604.1560, found: 604.1565.
3.2.4. Preparation of Hybrids OBP-6 and OBP-7
Compound 19: to a mixture of N-benzylmaleimide (75.00 g, 401.07 mmol, 1 eq) [32] and BrCH2NO2
(45.00 g, 320.09 mmol, 0.8 eq), K2CO3 (44.00 g, 320.09 mmol, 0.8 eq) and ACN (0.40 L) were slowly
added and then stirred at 25 ◦C for 48 h. The reaction mixture was extracted with DCM (0.30 L*3) and
brine (0.30 L). The combined organic layers were dried with anhydrous Na2SO4 and concentrated to
1
give compound 19 (17.00 g, 17%) as a yellow oil. H-NMR (400 MHz, CDCl3)
(s, 2H), 4.50–4.46 (m, 1H), 3.35 (s, 2H). ESI-MS (m/z): 246.9 [M + H]+.
δ 7.36–7.27 (m, 5H), 4.54

Molecules 2019, 24, 1641
16 of 22
Compound 20: to a mixed solution of compound 19 (16.30 g, 66.26 mmol, 1 eq) and NaBH4 (6.29 g,
165.65 mmol, 2.5 eq) in THF (0.40 L), BF3 Et2O (8.36 mL, 66.26 mmol, 1 eq) was added dropwise at
·
0 ◦C and maintained for 1 h. The mixture was heated to 40 ◦C and kept for 4 h. After cooling, the
reaction mixture was treated with EtOH (0.40 L) under reflux for 4 h. The mixture was extracted with
DCM (0.30 L*3) and brine (0.30 L). The combined organic layers were dried with anhydrous Na2SO4.
After evaporation, compound 20 (9.00 g, 60%) was obtained by flash chromatography as a colorless oil.
1H-NMR (400 MHz, CDCl3)
δ 7.35–7.20 (m, 5H), 4.63 (s, 1H), 3.59 (s, 2H), 3.14 (d, J = 9.2 Hz, 2H), 2.51
(d, J = 8.0 Hz, 4H). ESI-MS (m/z): 218.9 [M + H]+.
Compound 21: Zn (39.20 g, 599.48 mmol, 20 eq) was slowly added to a solution of compound
20 (6.54 g, 300.00 mmol, 1 eq) and 1N HCl (0.30 L, 0.30 mol, 10 eq) in i-PrOH (0.60 L) within 1 h,
and kept overnight. Excess hydrochioric acid was neutralized using NaHCO3 until pH > 7. The
reaction was extracted with DCM (0.30 L*3) and brine (0.30 L). The combined organic layers were
dried with anhydrous Na2SO4. After evaporation, compound 21 (5.56 g crude) was obtained by flash
1
chromatography as a yellow oil. H-NMR (400 MHz, CDCl3)
δ 7.31–7.18 (m, 5H), 3.55 (s, 2H), 2.95 (d,
J = 8.8 Hz, 2H), 2.66 (s, 1H), 2.38 (d, J = 8.4 Hz, 2H), 1.34 (s, 2H).
Compound 22: DIPEA (7.63 g, 0.06 mol, 2 eq) was added dropwise to a solution of compound
21 (5.56 g, 0.03 mol, 1 eq) and 2,4-difluoronitrobenzene in ACN (0.10 L) and stirred at 50 ◦C for 48
h. After cooling to room temperature, the reaction mixture was extracted with DCM (0.30 L*3) and
brine (0.30 L). The combined organic layers were dried with anhydrous Na2SO4. After evaporation,
1
compound 22 (9.10 g, 93%) was obtained by flash chromatography as a yellow oil. H-NMR (400 MHz,
CDCl3) δ 8.00 (d, J = 8.8 Hz, 1H), 7.86 (dd, J = 11.6, 2.4 Hz, 1H), 7.38–7.28 (m, 5H), 6.93 (t, J = 8.8 Hz,
1H), 4.85 (s, 1H), 3.79–3.60 (m, 2H), 3.25 (s, 2H), 3.03 (s, 1H), 2.61 (s, 2H), 1.26 (s, 2H).
Compound 23: to a mixture of compound 22 (9.10 g, 0.03 mol, 1 eq), (Boc)2O (9.11 g, 0.04 mol,
1.5 eq) and TEA (5.60 g, 56.00 mmol, 2 eq) in DCM (0.20 L), DMAP (340.00 mg, 3.00 mmol, 0.1 eq)
was added at 0 ◦C and stirred for 2 h. The reaction mixture was extracted with DCM (0.30 L*3) and
brine (0.30 L). The combined organic layers were dried with anhydrous Na2SO4 and then concentrated
to give a yellow oil. NaBH4 (3.20 g, 80.00 mmol, 3 eq) and NiCl2 6H2O (20.00 g, 80.00 mmol, 3eq)
·
were separately added to the oil in THF/MeOH (0.05 L/0.05 L) at 0 ◦C and stirred for 2 h. The reaction
mixture was extracted with EA (0.30 L*3) and brine (0.30 L). The combined organic layers were dried
with anhydrous Na2SO4. After evaporation, compound 23 (10.00 g, 91%) was obtained by flash
1
chromatography as a yellow oil. H-NMR (400 MHz, CDCl3)
δ
7.31–7.22 (m, 5H), 6.87 (t, J = 8.2 Hz,
1H), 6.38 (d, J = 9.6 Hz, 2H), 3.72 (s, 2H), 3.55 (s, 2H), 3.25 (s, 1H), 3.03 (d, J = 8.8 Hz, 2H), 2.37 (d,
J = 7.6 Hz, 2H), 1.53 (s, 2H), 1.41 (s, 9H).
Compound 24: the saturated NaHCO3 solution was added to compound 23 (10.00 g, 30.00 mmol,
1 eq) in THF (0.50 L). CBzCl (5.16 g, 36.00 mmol, 1.2 eq) was slowly added to the mixed solution at
0 ◦C and stirred overnight. The reaction mixture was extracted with EA (0.30 L*3) and brine (0.30 L).
The combined organic layers were dried with anhydrous Na2SO4. After evaporation, compound 24
1
(10.00 g, 75%) was obtained by flash chromatography as a yellow oil. H-NMR (400 MHz, CDCl3)
δ
7.41–7.38 (m, 3H), 7.26 (s, 10H), 5.20 (s, 2H), 3.54 (s, 2H), 3.28 (s, 1H), 3.02 (d, J = 8.8 Hz, 2H), 2.35 (d,
J = 8.8 Hz, 2H), 2.02 (s, 1H), 1.56 (s, 9H), 1.34 (s, 2H).
Compound 25: the suspension of compound 24 (10.00 g, 18.83 mmol, 1 eq) in 0.30 L of THF was
cooled to
solution within 30 min. After stirring at
(0.30 L) was added dropwisely within 30 min and stirred at
room temperature, the reaction solution was continuously stirred for 12 h. The resulting mixture was
extracted with EA (0.30 L*3) and brine (0.30 L). The combined organic layers were dried with anhydrous
−
78 ◦C. n-BuLi (2.50 M, 23.00 mL, 56.50 mmol, 3 eq) was added dropwisely to the mixed
−
78 ◦C for 30 min, (R)-oxiran-2-ylmethyl butyrate in THF
78 ◦C for 30 min. After being restored to
−
MgSO4. After evaporation, compound 25 (3.50 g, 37%) was obtained by flash chromatography as a
1
yellow oil. H-NMR (400 MHz, CDCl3)
δ 7.51 (d, J = 11.2 Hz, 1H), 7.36–7.27 (m, 5H), 7.16 (s, 2H), 4.75

Molecules 2019, 24, 1641
17 of 22
(s, 1H), 4.10–3.98 (m, 3H), 3.76 (d, J = 12.4 Hz, 1H), 3.64 (s, 2H), 3.34 (s, 1H), 3.09 (s, 2H), 2.50 (s, 2H),
1.42 (s, 9H), 1.34 (s, 2H).
Compound 26: Pd/C (0.35 g, 10%) was carefully added to a solution of compound 25 (3.50 g,
7.04 mmol, 1 eq) in MeOH (0.05 L). The reaction was proceeded with H2 at room temperature for 12 h.
The liquid was filtered and concentrated to afford compound 26 (2.50 g crude, 87%) as a yellow oil.
ESI-MS (m/z): 408.0 [M + H]+.
Compound 27: to a mixture of compound 26 (1.20 g, 2.95 mmol, 1 eq) and 9,10-difluoro-3-
methyl-7-oxo-2,3-dihydro-7H-[1,3,4] oxadiazino[6,5,4-ij]quinoline-6-carboxylic acid (0.831 g, 2.95 mmol,
1 eq) [33] in DMSO (0.02 L), TEA (1.50 g, 14.74 mmol, 5 eq) was added and stirred at 100 ◦C overnight.
Excess TEA was neutralized using 50% H2SO4 until pH < 6. The filtrate was concentrated and the
1
residue was purified by silica gel column to afford compound 27 (0.35 g, 18%) as a yellow oil. H-NMR
(400 MHz, DMSO-d6) δ 15.13 (s, 1H), 8.71 (s, 1H), 7.58 (t, J = 13.6 Hz, 2H), 7.36–7.30 (m, 2H), 5.26 (s,
2H), 4.71 (s, 1H), 4.10 (t, J = 8.8 Hz, 1H), 3.91–3.81 (m, 2H), 3.71–3.60 (m, 3H), 3.57 (d, J = 3.6 Hz, 1H),
3.54 (d, J = 3.6 Hz, 1H), 3.50 (s, 2H), 2.99 (s, 3H), 1.74 (s, 2H), 1.38 (s, 9H).
Compound 28: to a mixture of compound 26 (1.30 g, 3.19 mmol, 1 eq) and 1-cyclopropyl-6,7-
difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (0.846 g, 3.19 mmol, 1 eq) [34] in DMSO (0.02 L),
◦
TEA (1.63 g, 15.97 mmol, 5 eq) was added and stirred at 100 C overnight. After cooling to room
temperature, excess TEA was neutralized using 50% H2SO4 until pH < 6. After concentration of the
solution, the residue was purified by silica gel column to afford compound 28 (0.35 g, 17%) as a white
1
solid. H-NMR (400 MHz, CDCl3)
δ 8.58 (s, 1H), 7.80 (d, J = 14.4 Hz, 1H), 7.61 (d, J = 12.4 Hz, 1H), 7.40
(t, J = 8.6 Hz, 1H), 7.34 (d, J = 10.4 Hz, 1H), 7.07 (d, J = 7.2 Hz, 1H), 5.26 (d, J = 5.2 Hz, 1H), 4.77–4.70
(m, 1H), 4.10 (t, J = 9.0 Hz, 1H), 3.8–3.80 (m, 3H), 3.75–3.64 (m, 4H), 3.58–3.50 (m, 3H), 1.93 (s, 2H), 1.39
(s, 9H), 1.23 (s, 2H), 1.12 (s, 2H).
OBP-6: to a solution of compound 27 (0.35 g, 0.52 mmol, 1 eq) in THF (0.03 L), 6N HCl (0.03 L)
was added dropwise at 0 ◦C and then stirred for 48 h at room temperature. The saturated NaHCO3
solution was added in ice bath to adjust pH (>7). The mixture was extracted with EA (0.30 L*3) and
brine (0.30 L). The combined organic layers were dried with anhydrous Na2SO4. After evaporation,
the residue was purified by silica gel column to afford hybrid OBP-6 as a yellow solid (0.28 g crude,
94%), m.p., 188–189 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 8.67 (s, 1H), 7.51 (d, J = 13.6 Hz, 1H), 7.39 (d,
J = 13.6 Hz, 1H), 7.13 (d, J = 8.8 Hz, 1H), 6.91 (t, J = 9.2 Hz, 1H), 5.92 (s, 1H), 5.21 (s, 3H), 4.64 (s, 1H),
3.98 (d, J = 8.8 Hz, 2H), 3.79–3.70 (m, 3H), 3.65 (d, J = 11.6 Hz, 2H), 3.54 (d, J = 11.2 Hz, 2H), 2.99 (s,
3H), 1.78 (s, 2H). 13C-NMR (151 MHz, DMSO-d6)
δ 176.28 (d, J = 3.02 Hz), 166.03, 154.97, 154.37 (d,
J = 206.87 Hz), 150.55 (d, J = 241.60 Hz), 145.42, 136.44 (d, J = 9.06 Hz), 133.49 (d, J = 12.08 Hz), 128.65
(d, J = 4.53 Hz), 124.37, 115.51, 113.13 (d, J = 4.53 Hz), 106.52 (d, J = 24.16 Hz), 104.35 (d, J = 24.16Hz),
82.48, 73.45, 62.11, 52.11, 46.85, 43.16, 43.01, 33.84, 31.12, 24.35, 21.58. ESI-MS (m/z) [M + H]+ calcd. for
C27H25F2N5O7: 570.1795, found: 570.1804.
OBP-7: to a solution of compound 28 (0.35 g, 0.54 mmol, 1 eq) in THF (0.01 L), EA/HCl (0.03 L) was
added dropwise and then stirred for 48 h at room temperature. The reaction mixture was filtered and
the yellow solid OBP-7 was obtained (0.25 g crude, 84%) by recrystallization in PE and EA (1:1), m.p.,
268–270 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 8.58 (s, 1H), 7.80 (d, J = 14.4 Hz, 1H), 7.41 (d, J = 14.4 Hz,
1H), 7.13 (d, J = 5.6 Hz, 2H), 6.99 (t, J = 9.2 Hz, 1H), 4.69–4.61 (m, 1H), 4.42–4.25 (m, 1H), 4.03–4.01
(m, 3H), 3.77 (d, J = 7.6 Hz, 4H), 3.69–3.61 (m, 2H), 3.61–3.48 (m, 2H), 1.96 (s, 2H), 1.33 (d, J = 6.8 Hz,
2H), 1.16 (s, 2H). 13C-NMR (151 MHz, DMSO-d6)
δ 176.21 (d, J = 3.02 Hz), 166.54, 154.97, 150.53 (d,
J = 238.58 Hz), 150.20 (d, J = 247.64 Hz), 148.18, 141.65 (d, J = 12.08 Hz), 139.96, 133.28 (d, J = 12.08 Hz),
128.79 (d, J = 9.06 Hz), 115.21, 115.18, 113.37 (d, J = 4.53 Hz), 111.12 (d, J = 24.16 Hz), 109.99, 106.62,
106.50 (d, J = 24.16 Hz), 102.02, 73.46, 62.12, 52.09, 46.83, 36.49, 36.09, 25.62, 8.21. ESI-MS (m/z) [M + H]+
calcd. for C28H26F2N4O6: 553.1893, found: 553.1901.

Molecules 2019, 24, 1641
18 of 22
3.3. Antibacterial Activities
3.3.1. Bacterial Strains
6 standard strains and 3 clinical isolates were used to the preliminary evaluation of in vitro
antibacterial activities of oxazolidinone-fluoroquinolone hybrids. For the further evaluation of hybrids
OBP-4 and OBP-5, 263 clinical isolates including S. aureus (46), S. haemolyticus(32), S. epidermidis (10),
E. faecalis (72), E. faecium (47), S. pneumoniae (13), H. parasuis (34), S. suis (9), and 8 resistant strains
including MRSA, MRSE, CIP-resistant S. aureus and S. haemolyticus, VAN and quinolone-resistant
S. pneumoniae, LZD-resistant E. faecalis, LZD/VAN and quinolone-resistant E. faecium were collected
and tested.
3.3.2. Susceptibility Testing
Susceptibility testing of oxazolidinone-fluoroquinolone hybrids against various bacteria was
performed by the broth microdilution method as outlined by the Clinical and Laboratory Standards
Institute (CLSI) [35]. Three types of media, including cation-adjusted Mueller Hinton broth,
Haemophilus Test Medium and cation-adjusted Mueller Hinton broth that contained 3%–5% lysed
horse blood, were used to culture the corresponding bacteria. All compounds including comparator
drugs were dissolved in an appropriate solvent at a concentration of 2.048 mg/mL and then serially
diluted two-fold to provide the required concentrations. A final inoculum of 5
×
105 CFU/mL was
used and plates were read after incubation for 16–24 h at 35 ◦C. The MIC was defined as the lowest
concentration of the antibacterial at which visible growth was completely inhibited. Standard quality
control strains (ATCC) were included throughout. Each determination was repeated in triplicate.
3.4. Modes of Action
3.4.1. DNA Gyrase Supercoiling Assay
The E. coli DNA Gyrase Supercoiling Assay Kit (Inspiralis Co., Ltd.) was used to investigate the
inhibition activities of hybrids OBP-4 and OBP-5 against DNA gyrase. DNA gyrase is prepared from
E. coli. One unit of DNA gyrase can convert 0.5
The final reaction system (30 L) consisted of 0.5
without compounds in assay buffer containing 35 mM Tris
2 mM DTT, 1.8 mM spermidine, 1 mM ATP, 6.5% (w/v) glycerol and 0.1 mg/mL albumin, and then was
incubated at 37 ◦C for 30 min. The reaction was terminated by adding 30
L of STEB and 30 L of
µ
g of relaxed pBR322 to the supercoiled forms in 30 min.
g relaxed pBR322 and 1 unit DNA gyrase with or
HCl (pH 7.5), 24 mM KCl, 4 mM MgCl2,
µ
µ
·
µ
µ
chloroform/isoamyl alcohol (v:v, 24:1), and then the products were resolved by gel electrophoresis on
1% (w/v) agarose gel at 80 V for 2 h in the absence of ethidium bromide. The gels were stained with
ethidium bromide (1 µg/mL in distilled water) for 15 min, and destained (5–10 min) in distilled water.
DNA bands were visualized with UV light in Bio-Rad® Chemidoc xrs and analyzed by using Quantity
One software (Version 4.6, Bio-Rad). IC50 is defined as the concentration of compound that inhibits the
enzyme activity by 50%. CIP was used as a positive control.
3.4.2. Topo IV Relaxation Assay
The E. coli Topo IV Relaxation Assay Kit (Inspiralis Co., Ltd.) was used to evaluate the effects of
hybrids OBP-4 and OBP-5 against topo IV. Topo IV was prepared by the overexpressing E. coli. One
unit of topo IV will relax 0.4
consisted of 0.4 g supercoiled pBR322 and one unit DNA Topo IV with or without compounds in
assay buffer with 40 mM Hepes-KOH (pH 7.6), 100 mM potassium glutamate, 10 mM magnesium
acetate, 10 mM DTT, 1 mM ATP and 50
g/mL albumin, and then was incubated at 37 ◦C for 30 min.
The reaction was terminated by adding 30 L of STEB and 30 L of chloroform/isoamyl alcohol (v:v,
µg of supercoiled pBR322. In the assay, the final reaction mixtures (30 µL)
µ
µ
µ
µ
24:1) after incubation for 30 min. Then, the reaction products were processed as described in a DNA
gyrase supercoiling assay.

Molecules 2019, 24, 1641
19 of 22
3.4.3. In Vitro Transcription/Translation Assay
The influence of hybrids OBP-4 and OBP-5 against protein synthesis was confirmed by the S30 T7
High-Yield Protein Expression System (Promega Co., Ltd.). The S30 T7 High-Yield Protein Expression
System is an E. coli extract-based cell-free protein expression system, which can effectively transcribe
and translate DNA fragments with a T7 promoter. The reaction system containing 1
20 L S30 Premix Plus and 18 L T7 S30 Extract with or without compounds was incubated for 1 h
at 37 ◦C with a rotational speed of 250 rpm, and terminated by cooling in ice for 5 min in a 96-well
white multi-well plate. Then, 1.25 L of the translation reaction mixture was added to 48.75 L of
Renilla Luciferase Assay Lysis Buffer, and mixed with 50 L of Renilla Luciferase Assay Reagent (cat
µg plasmid DNA,
µ
µ
µ
µ
µ
no. E2810; Promega). Renilla luciferase activity was measured on EnSpire® Multimode Plate Reader
(PerkinElmer, Waltham, MA, USA). IC50 is defined as the concentration of compound that inhibits the
protein synthesis by 50%. LZD was used as a positive control.
3.4.4. Molecular Docking Stusy
The molecular docking of hybrids OBP-4 and OBP-5 was carried out with the Discovery Studio
4.5 software. The crystal structure of the large ribosomal subunit (50S subunit) of S. aureus (PDB:
4WFA) [36] was selected as the template to perform the docking study. Prior to docking, the structures
of ligands were drawn and energy was minimized. During the process of receptor preparation, water
molecules were removed and hydrogen atoms were added [37]. Hit compounds were docked into the
active site of the receptor with an acceptable target flexibility and the docking score was calculated.
The docking complexes with the highest score were kept for further analyses. The presentation of
ligand-receptor interactions was depicted by PyMol 1.5.03 [38].
3.5. Acute Toxicity Test
Female Kunming mice (SPF grade, 18–22 g) were obtained from laboratory animal center of
Lanzhou Veterinary Research Institute (Lanzhou, China). Mice were maintained under the standard
conditions of 12 h light/dark cycle, room temperate (22
±
3 ◦C), 50–60% relative humidity. They were
fed normally with an unlimited supply of feed and water for seven days and deprived of food but not
water for 4 h prior to the experiment. The toxicity test was supported by the Animal Ethics Committee
of Lanzhou Institute of Husbandry and Pharmaceutical Sciences.
The acute toxicity of hybrids OBP-4 and OBP-5 was conducted by the limit test described in the
OECD guideline 425 [39]. The mice were divided randomly into two groups, five animals in each
group. One group of mice received a single oral dose of hybrids OBP-4 or OBP-5 at 2000 mg/kg bw,
·
and the other group of mice was given the equal volume of 0.5% CMC-Na solution (0.1 mL/10 g
of body weight). Food was restored until 4 h after administration. The observation of each animal
occurred according to the OECD guideline 425 [27], meaning continuously during the first 30 min,
periodically during the next 24 h, and then daily thereafter until 14 d after dosing. The weight changes
and mortality were calculated and recorded. The mice that survived were humanely killed at the end
of the experiment.
4. Conclusions
A series of new oxazolidinone-fluoroquinolone hybrids incorporating the structural elements of
oxazolidinone and fluoroquinolone with a linker were synthesized and evaluated for their in vitro
antibacterial activities against various Gram-positive and Gram-negative bacteria. The antibacterial
activities of most hybrids against Gram-positive bacteria were significantly higher than that of LZD,
and hybrids OBP-4 and OBP-5 were the most active two hybrids. The mode of action for hybrids OBP-4
and OBP-5 was more directed to protein synthesis inhibition instead of DNA synthesis inhibition,
showing a good consistency with the antibacterial profile. Meanwhile, a good safety profile was
also demonstrated for hybrids OBP-4 and OBP-5 with LD50 values greater than 2000 mg/kg. These

Molecules 2019, 24, 1641
20 of 22
combined data suggested that hybrids OBP-4 and OBP-5 may be promising therapeutic options for
severe nosocomial infections caused by Gram-positive bacteria.
Supplementary Materials: 1H-NMR,13C-NMR and HRMS spectra for oxazolidinone-fluoroquinolone hybrids
OBP-(1–7) are available online.
Author Contributions: Conceptualization, J.Z., L.L. and J.L.; Data curation, L.L., L.S. and H.C.; Formal analysis,
L.L., L.S., J.L., H.C., B.L., X.Z. and P.L.; Funding acquisition, J.Z.; Investigation, L.L., L.S., J.L., H.C., B.L., X.Z. and
P.L.; Project administration, J.L.; Resources, B.L. and X.Z.; Supervision, J.Z.; Validation, L.L., L.S., J.L. and H.C.;
Visualization, L.L. and L.S.; Writing—original draft, L.L., L.S. and J.Z.; Writing—review & editing, J.Z.
Funding: This study was supported by the Key Projects in the National Science & Technological Pillar Program
during the Twelfth Five-year Plan Period (2015BAD11B01) and the embarked fund for the China Agriculture
Research System (CARS-37).
Acknowledgments: We are grateful to Hongsheng Li and Zhiyong Ma for supplying strains.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
Zinner, S.H. Antibiotic use: Present and future. New Microbiol. 2007, 30, 321–325. [PubMed]
Li, B.; Webster, T.J. Bacteria antibiotic resistance: New challenges and opportunities for implant-associated
orthopedic infections. J. Orthop. Res. 2018, 36, 22–32. [CrossRef] [PubMed]
3.
4.
5.
6.
7.
8.
9.
Vivas, R.; Barbosa, A.A.T.; Dolabela, S.S.; Jain, S. Multidrug-resistant bacteria and alternative methods to
control them: An overview. Microb. Drug Resist. 2019. [CrossRef]
Watkins, R.R.; Bonomo, R.A. Overview: Global and local impact of antibiotic resistance. Infect. Dis. Clin. N.
Am. 2016, 30, 313–322. [CrossRef] [PubMed]
Ciorba, V.; Odone, A.; Veronesi, L.; Pasquarella, C.; Signorelli, C. Antibiotic resistance as a major public
health concern: Epidemiology and economic impact. Ann. Iq. 2015, 27, 562–579.
Thabit, A.K.; Crandon, J.L.; Nicolau, D.P. Antimicrobial resistance: Impact on clinical and economic outcomes
and the need for new antimicrobials. Expert Opin. Pharmacother. 2015, 16, 159–177. [CrossRef] [PubMed]
Tillotson, G.S.; Zinner, S.H. Burden of antimicrobial resistance in an era of decreasing susceptibility. Expert
Rev. Anti Infect. Ther. 2017, 15, 663–676. [CrossRef]
Sender, R.; Fuchs, S.; Milo, R. Revised Estimates for the number of human and bacteria cells in the body.
PLoS. Biol. 2016, 14, e1002533. [CrossRef]
Parkes, A.L.; Yule, I.A. Hybrid antibiotics—Clinical progress and novel designs. Expert Opin. Drug Discov.
2016, 11, 665–680. [CrossRef]
10. Barbachyn, M.R. Recent advances in the discovery of hybrid antibacterial agents. Annu. Rep. Med. Chem.
2008, 43, 281–290.
11. Robertson, G.T.; Bonventre, E.J.; Doyle, T.B.; Du, Q.; Duncan, L.; Morris, T.W.; Roche, E.D.; Yan, D.; Lynch, A.S.
In vitro evaluation of CBR-2092, a novel rifamycin-quinolone hybrid antibiotic: Studies of the mode of action
in Staphylococcus aureus. Antimicrob. Agents. Chemother. 2008, 52, 2313–2323. [CrossRef]
12. Pokrovskaya, V.; Belakhov, V.; Hainrichson, M.; Yaron, S.; Baasov, T. Design, synthesis, and evaluation of
novel fluoroquinolone-aminoglycoside hybrid antibiotics. J. Med. Chem. 2009, 52, 2243–2254. [CrossRef]
13. Blais, J.; Lewis, S.R.; Krause, K.M.; Benton, B.M. Antistaphylococcal activity of TD-1792, a multivalent
glycopeptide-cephalosporin antibiotic. Antimicrob. Agents. Chemother. 2012, 56, 1584–1587. [CrossRef]
14. Louie, T.; Nord, C.E.; Talbot, G.H.; Wilcox, M.; Gerding, D.N.; Buitrago, M.; Kracker, H.; Charef, P.;
Cornely, O.A. Multicenter, double-blind, randomized, phase 2 study evaluating the novel antibiotic cadazolid
in patients with Clostridium difficile infection. Antimicrob. Agents. Chemother. 2015, 59, 6266–6273. [CrossRef]
[PubMed]
15. Pokrovskaya, V.; Baasov, T. Dual-acting hybrid antibiotics: A promising strategy to combat bacterial resistance.
Expert Opin. Drug Discov. 2010, 5, 883–902. [CrossRef]
16. Shaw, K.J.; Barbachyn, M.R. The oxazolidinones: Past, present, and future. Ann. N. Y. Acad. Sci. 2011, 1241,
48–70. [CrossRef]

Molecules 2019, 24, 1641
21 of 22
17. Bender, J.K.; Fleige, C.; Lange, D.; Klare, I.; Werner, G. Rapid emergence of highly variable and transferable
oxazolidinone and phenicol resistance gene optrA in German Enterococcus spp. clinical isolates. Int. J.
Antimicrob. Agents 2018, 52, 819–827. [CrossRef] [PubMed]
18. De Lastours, V.; Fantin, B. Resistance to fluoroquinolones in 2013: What are the consequences in internal
medicine? Rev. Med. Interne 2014, 35, 601–608. [CrossRef]
19. Hubschwerlen, C.; Specklin, J.L.; Sigwalt, C.; Schroeder, S.; Locher, H.H. Design, synthesis and biological
evaluation of oxazolidinone-quinolone hybrids. Bioorg. Med. Chem. 2003, 11, 2313–2319. [CrossRef]
20. Hubschwerlen, C.; Specklin, J.L.; Baeschlin, D.K.; Borer, Y.; Haefeli, S.; Sigwalt, C.; Schroeder, S.; Locher, H.H.
Structure-activity relationship in the oxazolidinone-quinolone hybrid series: Influence of the central spacer
on the antibacterial activity and the mode of action. Bioorg. Med. Chem. Lett. 2003, 13, 4229–4233. [CrossRef]
[PubMed]
21. Sabine Schubert, A.D. Low propensity for development of resistance to MCB3681, the active moiety of
oxaquin (MCB3837), in Gram-positive bacteria with vancomycin-, linezolid-, methicillin- and/or ciprofloxacin
resistances. In Proceedings of the 46th ICAAC, San Francisco, CA, USA, 27–30 September 2006.
22. Locher, H.H.; Caspers, P.; Bruyere, T.; Schroeder, S.; Pfaff, P.; Knezevic, A.; Keck, W.; Ritz, D. Investigations of
the mode of action and resistance development of cadazolid, a new antibiotic for treatment of Clostridium
difficile infections. Antimicrob. Agents. Chemother. 2014, 58, 901–908. [CrossRef] [PubMed]
23. Gerding, D.N.; Hecht, D.W.; Louie, T.; Nord, C.E.; Talbot, G.H.; Cornely, O.A.; Buitrago, M.; Best, E.;
Sambol, S.; Osmolski, J.R.; et al. Susceptibility of Clostridium difficile isolates from a Phase 2 clinical trial of
cadazolid and vancomycin in C. difficile infection. J. Antimicrob. Chemother. 2016, 31, 213–219. [CrossRef]
[PubMed]
24. Correia, S.; Poeta, P.; Hebraud, M.; Capelo, J.L.; Igrejas, G. Mechanisms of quinolone action and resistance:
Where do we stand? J. Med. Microbiol. 2017, 66, 551–559. [CrossRef] [PubMed]
25. Hiasa, H.; DiGate, R.J.; Marians, K.J. Decatenating activity of Escherichia coli DNA gyrase and topoisomerases
I and III during oriC and pBR322 DNA replication in vitro. J. Biol. Chem. 1994, 269, 2093–2099. [PubMed]
26. Baldoni, D.; Gutierrez, M.; Timmer, W.; Dingemanse, J. Cadazolid, a novel antibiotic with potent activity
against Clostridium difficile: Safety, tolerability and pharmacokinetics in healthy subjects following single and
multiple oral doses. J. Antimicrob. Chemother. 2014, 69, 706–714. [CrossRef] [PubMed]
27. Clewley, R.G.; Cross, G.G.; Fischer, A.; Henderson, G.N. Formation of 4-halo-4- nitrocyclohexa-2,5-dienones
on nitration of p-halophenols and p-halophenyl acetates. Tetrahedron 1989, 45, 1299–1310. [CrossRef]
28. Wessig, P.; Möllnitz, K.; Eiserbeck, C. Oligospiroketals as novel molecular rods. Chemistry 2007, 13, 4859–4872.
[CrossRef]
29. Yang, Y.; Ji, R.; Hu, Z.; Chen, K. A new practical route to stereospecific synthesis of (S)-(-)-ofloxacin. Acta
Pharm. Sin. 1998, 33, 828–831.
30. Kose, E.; Karabacak, M.; Atac, A. The spectroscopic and quantum chemical studies of 3,4-difluoroaniline.
Spectrochim. Acta A Mol. Biomol. Spectrosc. 2015, 143, 265–280. [CrossRef] [PubMed]
31. Segawa, J.; Kitano, M.; Kazuno, K.; Matsuoka, M.; Shirahase, I.; Ozaki, M.; Matsuda, M.; Tomii, Y.;
Kise, M. Studies on pyridonecarboxylic acids.
1.
Synthesis and antibacterial evaluation of
7-substituted-6-halo-4-oxo-4H-[1,3]thiazeto[3,2-a]quinoline-3-carboxylic acids. J. Med. Chem. 1992, 35,
4727–4738. [CrossRef]
32. Matuszak, N.; Muccioli, G.G.; Labar, G.; Lambert, D.M. Synthesis and in vitro evaluation of N-substituted
maleimide derivatives as selective monoglyceride lipase inhibitors. J. Med. Chem. 2009, 52, 7410–7420.
[CrossRef] [PubMed]
33. Wan, P.H.; Kee, J.L. Baylis-Hillman route to several quinolone antibiotic intermediates. Chem. Inform. 2006,
37, 963–968.
34. Egawa, H.; Kataoka, M.; Shibamori, K.I.; Miyamato, T.; Nakano, J.; Matsumoto, J.I. A new synthetic route to
7-halo-1-cyclopropyl-6-fluoro-1,4-dihydro-4oxoquinoline-3- carboxylic acid, an intermediate for the synthesis
of quinolone antibacterial agents. J. Heterocyclic. Chem. 1987, 18, 181–185. [CrossRef]
35. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; 26th
Informational Supplement; CLSI document M100-S26; Clinical and Laboratory Standards Institute: Wayne,
PA, USA, 2016.

Molecules 2019, 24, 1641
22 of 22
36. Eyal, Z.; Matzov, D.; Krupkin, M.; Wekselman, I.; Paukner, S.; Zimmerman, E.; Rozenberg, H.; Bashan, A.;
Yonath, A. Structural insights into species-specific features of the ribosome from the pathogen Staphylococcus
aureus. Proc. Natl. Acad. Sci. USA 2015, 112, E5805–E5814. [CrossRef]
37. Taher, E.S.; Ibrahim, T.S.; Fares, M.; Al-Mahmoudy, A.M.M.; Radwan, A.F.; Orabi, K.Y.; El-Sabbagh, O.I.
Novel benzenesulfonamide and 1,2-benzisothiazol-3(2H)-one-1,1- dioxide derivatives as potential selective
COX-2 inhibitors. Eur. J. Med. Chem. 2019, 171, 372–382. [CrossRef]
38. Friesner, R.A.; Murphy, R.B.; Repasky, M.P.; Frye, L.L.; Greenwood, J.R.; Halgren, T.A.; Sanschagrin, P.C.;
Mainz, D.T. Extra precision glide: Docking and scoring incorporating a model of hydrophobic enclosure for
protein-ligand complexes. J. Med. Chem. 2006, 49, 6177–6196. [CrossRef]
39. OECD 425 Guideline for Testing of Chemical. Acute Oral Toxicity-Up-and-Down Procedure; OECD: Paris,
France, 2001. [CrossRef]
Sample Availability: Samples of the compounds are not available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).