Novel 3-arylfuran-2(5H)-one-fluoroquinolone hybrid: Design, synthesis and evaluation as antibacterial agent

Article abstract of DOI:10.1016/j.bmc.2014.05.018

3-Arylfuran-2(5H)-one, a novel antibacterial pharmacophore targeting tyrosyl-tRNA synthetase (TyrRS), was hybridized with the clinically used fluoroquinolones to give a series of novel multi-target antimicrobial agents. Thus, twenty seven 3-arylfuran-2(5H)-one-fluoroquinolone hybrids were synthesized and evaluated for their antimicrobial activities. Some of the hybrids exhibited merits from both parents, displaying a broad spectrum of activity against resistant strains including both Gram-negative and Gram-positive bacteria. The most potent compound (11) in antibacterial assay shows MIC₅₀ of 0.11 μg/mL against Multiple drug resistant Escherichia coli, being about 51-fold more potent than ciprofloxacin. The enzyme assays reveal that 11 is a potent multi-target inhibitor with IC₅₀ of 1.15 ± 0.07 μM against DNA gyrase and 0.12 ± 0.04 μM against TyrRS, respectively. Its excellent inhibitory activities against isolated enzymes and intact cells strongly suggest that 11 deserves to further research as a novel antibiotic.

Full text of DOI:10.1016/j.bmc.2014.05.018

Bioorganic & Medicinal Chemistry 22 (2014) 3620–3628
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel 3-arylfuran-2(5H)-one-fluoroquinolone hybrid: Design,
synthesis and evaluation as antibacterial agent
Xu-Dong Wang a, Wei Wei a, Peng-Fei Wang b, Yun-Tao Tang c, Rui-Cheng Deng a, Biao Li a, Sha-Sha Zhou a,
Jing-Wen Zhang a, Lei Zhang a, Zhu-Ping Xiao a, , Hui Ouyang a, , Hai-Liang Zhu a,b,
⇑
⇑
⇑
a College of Chemistry and Chemical Engineering, Jishou University, Jishou 416000, PR China
b State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
c College of Biology and Environmental Science, Jishou University, Jishou 416000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
3-Arylfuran-2(5H)-one, a novel antibacterial pharmacophore targeting tyrosyl-tRNA synthetase (TyrRS),
was hybridized with the clinically used fluoroquinolones to give a series of novel multi-target antimicro-
bial agents. Thus, twenty seven 3-arylfuran-2(5H)-one-fluoroquinolone hybrids were synthesized and
evaluated for their antimicrobial activities. Some of the hybrids exhibited merits from both parents, dis-
playing a broad spectrum of activity against resistant strains including both Gram-negative and Gram-
Received 12 March 2014
Revised 10 May 2014
Accepted 12 May 2014
Available online 20 May 2014
positive bacteria. The most potent compound (11) in antibacterial assay shows MIC50 of 0.11
against Multiple drug resistant Escherichia coli, being about 51-fold more potent than ciprofloxacin.
The enzyme assays reveal that 11 is a potent multi-target inhibitor with IC50 of 1.15 0.07 M against
DNA gyrase and 0.12 0.04 M against TyrRS, respectively. Its excellent inhibitory activities against iso-
lg/mL
Keywords:
3-Arylfuran-2(5H)-one
Fluoroquinolone
Hybrid
l
l
lated enzymes and intact cells strongly suggest that 11 deserves to further research as a novel antibiotic.
Ó 2014 Elsevier Ltd. All rights reserved.
Multi-target
TyrRS
DNA gyrase
1. Introduction
emergence of drug resistance, as well as the decline of the antibi-
otic discovery and development program, new antibiotics are
As microorganisms adopt diverse strategies to enhanced ability
to survive in the presence of antibiotics, antibiotic resistance is
becoming increasingly serious,1 which causes not only great
damage to the human health but also more and more economic
loss. For example, Acinetobacter baumannii which resists to amino-
glycosides, cefepime, fluoroquinolones and most of all available
antibiotics is so widespread in worldwide, and becomes a leading
cause of nosocomial infections in recent years.2,3 Methicillin-resis-
tant Staphylococcus aureus (MRSA) is a challenging pathogen capa-
ble of producing devastating disease, which leads to nearly 100,000
infections and 20,000 deaths in the United States annually.4,5 In
addition, drug resistance is also generally considered a cause of
treatment failure for HIV infection and cancer.6 Fluoroquinolones,
acting on DNA gyrase and topoisomerase IV, were introduced as
broad-spectrum antibacterial agents in the 1980s.7,8 Since their
advent, they were widely applied to treat infections. Unfortunately,
with their widely use, most of them suffered from the resistance
problem by mutations in the bacterial targets.9,10 With the
urgently needed more than ever.11,12
Recently, we have focused our research on development and
modification of 3-arylfuran-2(5H)-ones as inhibitors of tyrosyl-
tRNA synthetase (TyrRS).13,14 TyrRS is an aminoacyl-tRNA
synthetase (AaRS) which ligates specific amino acid to its cog-
nate tRNA molecule(s) and possesses an important role in bacte-
rial protein synthesis.15–17 Some of 3-arylfuran-2(5H)-ones were
found to exhibit excellent inhibitory activities against TyrRS and
intact cell. For example, IC50 of 3-(4-hydroxyphenyl)-4-(2-mor-
pholinoethoxy)furan-2(5H)-one (I, Scheme 1) against TyrRS is
0.1 lM. With respect to enzyme assay, it must be admitted that
compound I shows much less potent than the reported TyrRS
inhibitor SB-219383 (IC50 = 0.6 nM, Scheme 1). However, due to
the poor cell membrane permeability of SB-219383, compound
I (MIC50 = 0.23 lg/mL, S. aureus) in turn displays much more
active than SB-219383 (MIC50 >64 lg/mL, S. aureus) against
intact cell.18–20
Drug hybridisation is an attracting design strategy for drug
development and research, which combines the pharmacophores
of two or more existing drugs to create a single molecule with
multiple pharmacological targets.21,22 Hybrid drug has shown
some successes in chemical therapy. TD-1792 (Scheme 1), a vanco-
mycin–cephalosporin hybrid, has been completed phase II clinical
⇑
Corresponding authors. Tel./fax: +86 743 8563911 (Z.-P.X., H.O.); tel.: +86 25
83592572; fax: +86 25 83592672 (H.-L.Z.).
E-mail addresses: xiaozhuping2005@163.com (Z.-P. Xiao), oyhmail@163.com
(H. Ouyang), zhuhl@nju.edu.cn (H.-L. Zhu).
http://dx.doi.org/10.1016/j.bmc.2014.05.018
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

X.-D. Wang et al. / Bioorg. Med. Chem. 22 (2014) 3620–3628
3621
O
O
H
O
COOH
OH
O
N
N
H
O
N
OH
OH
NH2
HO
HO
O
O
OH
HOOC
HO
F
H3C
H2N
SB-219383
N
CH3
OH
3-(4-hydroxyphenyl) -4-(2-morpholino-
I
ethoxy)furan-2(5H)-one ( )
O
N
O
O
OH
O
N
OH
OH
O
O
N
O
O
N
O
Cl
O
Cl
N+
-O
O
HN
OH OH
HO
OH
N
O
O
S
O
O
H
N
H
N
H
N
O
OH
OH
O
O
NH
CH3
CH3
N
H
N
H
N
H
O
O
NH2
N
HN
O
H
N
O
H2N
N
CH3
S
O
Cl
O
O
OH
OH
HO
TD-1792
CBR-2092
Scheme 1. Structures of 3-(4-hydroxyphenyl)-4-(2-morpholinoethoxy)furan-2(5H)-one, SB-219383, TD-1792 and CBR-2092.
trial, showing potent bactericidal effects against clinically relevant
Gram-positive organisms in vitro.23,24 CBR-2092 (Scheme 1), a rif-
amycin–fluoroquinolone hybrid, has been completed the phase I
clinical trial, markedly reducing resistance frequencies by target
mutations.25
In accordance with the design proposal of hybrid drugs, similar
structures between morpholinyl group of 3-aryl-4-(2-morpholino-
ethoxy)furan-2(5H)-ones and piperazinyl group of fluoroquinolone
were merged, and twenty seven 3-arylfuran-2(5H)-one-fluoro-
quinolone hybrids were synthesized (Scheme 2). They were
subsequently evaluated for biological activities, including antimi-
crobial activities against resistant strains as well as inhibitory
activities against DNA gyrase and TyrRS. The results indicated that
some compounds show excellent antimicrobial activities and out-
standing inhibitory activities against DNA gyrase and TyrRS.
System 5304 mass spectrometer, and 1H NMR spectra were
recorded on Bruker AV-300 or AV-500 spectrometer at 25 °C with
TMS and solvent signals allotted as internal standards. Chemical
shifts were reported in ppm (d). Elemental analyses were per-
formed on a CHN-O-Rapid instrument and were within 0.4% of
the theoretical values.
2.1.1. General procedure for preparation of compounds 1a–1k
An appropriately substituted phenylacetic acid (40 mmol) was
dissolved in 0.1 M NaOH with pH in the range of 9.0 to 10.0. Then,
the solvent was removed under reduced pressure to give the corre-
sponding sodium phenylacetate. To a solution of the resulted
sodium phenylacetate (40 mmol) in DMSO (45 mL) was added
1.1 equiv of ethyl bromoacetate, which was stirred at 25 °C for
3–4 h. The mixture was then poured into ice-water (50 mL), and
extracted twice with 200 mL of EtOAc. The organic layer was dried
over MgSO4 followed by removal of the solvent under reduced
pressure. The residue was then purified by column chromatogra-
phy on silica gel to give 1a–1k in yields of 90–95%.
2. Materials and methods
2.1. Chemistry
All chemicals (reagent grade) used were purchased from Aldrich
(U.S.A.) and Sinopharm Chemical Reagent Co., Ltd (China). Separa-
tion of the compounds by column chromatography was carried out
with silica gel 60 (200–300 mesh ASTM, E. Merck). The quantity of
silica gel used was 30–70 times the weight charged on the column.
Then, the eluates were monitored using TLC. Melting points
(uncorrected) were determined on a XT4 MP apparatus (Taike
Corp., Beijing, China). ESI mass spectra were obtained on a Mariner
2.1.2. General procedure for preparation of compounds 2a–2k
A dropwise solution of compound 1 (10 mmol) in dry THF
(10 mL) was added to a suspension of NaH (24 mmol) in dry THF
(20 mL) in an ice cold bath and the stirring was maintained at room
temperature for several hours (monitored by TLC). Water (30 mL)
was added and the solution was extracted twice with ethyl ether.
The aqueous phase was cooled to 0 °C and then acidified with
concentrated hydrochloric acid to give a solid precipitate. Filtration
O
HOOC
F
N
R
X
N
O
NH
N
R
N
O
O
merge
Fluoroquinolones
N
F
X
Ar
O
COOH
N
O
O
O
O
Ar
3-Arylfuran-2(5H)-ones
Scheme 2. The rational design of compounds.

3622
X.-D. Wang et al. / Bioorg. Med. Chem. 22 (2014) 3620–3628
and washing with water furnished compound 2, which was used
without further purification.
2.1.9. 7-(4-(2-(3-(2-Fluorophenyl)furan-2(5H)-one-4-yloxy)eth-
yl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline
-3-carboxylic acid (8)
2.1.3. General procedure for preparation of compounds 3a–3k
Compound 2 (5 mmol) was dissolved in 35 mL of dry acetone,
followed by addition of 1,2-dibromoethane (4.3 mL, 50 mmol)
and triethylamine (2.2 mL, 15 mmol). The resulted mixture was
refluxed for 3–5 h. After the solvent was removed, the residue
was partitioned between EtOAc and water. The organic layer was
then dried over MgSO4 and concentrated under reduced pressure.
Flash chromatography (EtOAc/petroleum ether, from 3/2 to 2/3)
furnished Compound 3 in good yield.
Orange powder, 51.0%, mp 183–185 °C; 1H NMR (DMSO-d6,
300 Hz): 1.41 (t, 3H), 2.55–2.62 (m, 4H), 2.73 (t, 2H), 3.30–3.36
(m, 4H), 4.33 (t, 2H), 4.55–4.63 (m, 2H), 5.20 (s, 2H), 7.17 (d, 1H),
7.23–7.29 (m, 2H), 7.40–7.45 (m, 2H), 7.92 (d, 1H), 8.95 (s, 1H),
15.35 (s, 1H); MS (ESI) m/z 538.7 [MꢀH]ꢀ. Anal. Calcd for
C28H27F2N3O6: C, 62.33; H, 5.04; F, 7.04; N, 7.79; Found: C, 62.31;
H, 5.11; F, 7.01; N, 7.74.
2.1.10. 7-(4-(2-(3-(2-Fluorophenyl)furan-2(5H)-one-4-yloxy)et-
hyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydro-1,8-na-
phthyridine-3-carboxylic acid (9)
2.1.4. General procedure for preparation of 3-arylfuran-2(5H)-
one-fluoroquinolones (4–30)
Orange powder, 35.6%, mp 124–126 °C; 1H NMR (DMSO-d6,
500 Hz): 1.38 (t, 3H), 2.55–2.62 (m, 4H), 2.72 (t, 2H), 3.75–3.85
(m, 4H), 4.33 (t, 2H), 4.46–4.52 (m, 2H), 5.20 (s, 2H), 7.23–7.28
(m, 2H), 7.38–7.42 (m, 2H), 8.09 (d, 1H), 8.99 (s, 1H), 15.34 (s,
1H); MS (ESI) m/z 539.2 [MꢀH]ꢀ. Anal. Calcd for C27H26F2N4O6:
C, 60.00; H, 4.85; F, 7.03; N, 10.37; Found: C, 60.07; H, 4.81; F,
7.09; N, 10.34.
A mixture of compound 3 (1.0 mmol), floxacin (0.5 mmol), KI
(0.5 mmol) and DMAP (2.0 mmol) was added in DMSO (15 mL).
The mixture was then stirred for 48–72 h at 60 °C (monitored by
TLC). After 30 mL of water was added, the precipitate was col-
lected, which was purified by column chromatography on silica
gel, eluting with CHCl3/CH3OH (V:V, from 12/1 to 25/1, containing
0.3% of acetic acid). Yields and analytical data for the analogues
described in this report are listed below.
2.1.11. 7-(4-(2-(3-(2-Fluorophenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-(4-fluorophenyl)-6-fluoro-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid (10)
2.1.5. 7-(4-(2-(3-Phenylfuran-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (4)
White powder, 38.6%, mp 177–179 °C; 1H NMR (DMSO-d6,
300 Hz): 2.50–2.54 (m, 4H), 2.60–2.66 (m, 2H), 2.97–3.03 (m,
4H), 4.31–4.40 (m, 2H), 5.15 (s, 2H), 6.37 (d, 1H), 7.19–7.26 (m,
2H), 7.35–7.40 (m, 2H), 7.51–7.57 (m, 2H), 7.78 (dd, 2H), 7.99 (d,
1H), 8.65 (s, 1H), 15.13 (s, 1H); MS (ESI) m/z 604.4 [MꢀH]ꢀ. Anal.
Calcd for C32H26F3N3O6: C, 63.47; H, 4.33; F, 9.41; N, 6.94; Found:
C, 63.39; H, 4.22; F, 9.45; N, 6.91.
Orange powder, 54.1%, mp 234–236 °C; 1H NMR (DMSO-d6,
500 Hz): 1.42 (t, 3H), 2.46–2.54 (m, 4H), 2.70–2.80 (m, 4H),
2.85–2.88 (m, 2H), 4.43 (t, 2H), 4.59 (t, 2H), 5.16 (s, 2H), 7.16 (d,
1H), 7.23–7.27 (m, 1H), 7.36–7.40 (m, 2H), 7.89–7.93 (m, 3H),
8.95 (s, 1H), 15.33 (s, 1H); MS (ESI) m/z 520.2 [MꢀH]ꢀ. Anal. Calcd
for C28H28FN3O6: C, 64.48; H, 5.41; F, 3.64; N, 8.06; Found: C, 64.31;
H, 5.52; F, 3.67; N, 8.10.
2.1.12. 7-(4-(2-(3-(2-Fluorophenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (11)
2.1.6. 7-(4-(2-(3-Phenylfuran-2(5H)-one-4-yloxy)ethyl)piperaz-
in-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-
3-carboxylic acid (5)
White powder, 38.6%, mp 126–128 °C; 1H NMR (DMSO-d6,
500 Hz): 1.16–1.20 (m, 2H), 1.29–1.33 (m, 2H), 2.65–2.71 (m,
4H), 2.92–3.01 (m, 2H), 3.25–3.30 (m, 4H), 3.81–3.86 (m, 1H),
4.41–4.46 (m, 2H), 5.16 (s, 2H), 7.18–7.25 (m, 2H), 7.32–7.44 (m,
2H), 7.51–7.58 (m, 1H), 7.72–7.80 (m, 1H), 8.69 (s, 1H), 15.22 (s,
1H); MS (ESI) m/z 550.4 [MꢀH]ꢀ. Anal. Calcd for C29H27F2N3O6: C,
63.15; H, 4.93; F, 6.89; N, 7.62; Found: C, 63.10; H, 4.95; F, 6.86;
N, 7.73.
Orange powder, 42.0%, mp 200–203 °C; 1H NMR (DMSO-d6,
500 Hz): 1.38 (t, 3H), 2.65–2.72 (m, 4H), 2.80–2.91 (m, 2H), 3.78–
3.91 (m, 4H), 4.42–4.50 (m, 4H), 5.17 (s, 2H), 7.28 (t, 1H), 7.40 (t,
2H), 7.88 (d, 2H), 8.10 (d, 1H), 8.98 (s, 1H), 15.31 (s, 1H); MS
(ESI) m/z 521.3 [MꢀH]ꢀ. Anal. Calcd for C27H27FN4O6: C, 62.06; H,
5.21; F, 3.64; N, 10.72; Found: C, 62.12; H, 5.07; F, 3.55; N, 10.90.
2.1.7. 7-(4-(2-(3-Phenylfuran-2(5H)-one-4-yloxy)ethyl)piperaz-
in-1-yl)-1-(4-fluorophenyl)-6-fluoro-4-oxo-1,4-dihydroquinoli-
ne-3-carboxylic acid (6)
2.1.13. 7-(4-(2-(3-(4-Fluorophenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (12)
Orange powder, 39.0%, mp 192–195 °C; 1H NMR (DMSO-d6,
500 Hz): 2.50–2.57 (m, 6H), 3.15–3.23 (m, 4H), 4.19–4.21 (m,
2H), 4.92 (s, 2H), 6.28 (d, 1H), 6.67–6.85 (m, 1H), 7.26–7.35 (m,
4H), 7.43–7.48 (m, 2H), 7.75 (d, 2H), 7.94–7.99 (m, 1H), 8.56 (s,
1H), 14.89 (s, 1H); MS (ESI) m/z 586.7 [MꢀH]ꢀ. Anal. Calcd for
White powder, 50.3%, mp 137–139 °C; 1H NMR (DMSO-d6,
500 Hz): 1.19–1.22 (m, 2H), 1.27–1.35 (m, 2H), 2.66–2.73 (m,
4H), 2.94–3.05 (m, 2H), 3.26–3.31 (m, 4H), 3.80–3.87 (m, 1H),
4.40–4.47 (m, 2H), 5.19 (s, 2H), 7.35–7.46 (m, 3H), 7.57 (dd, 2H),
7.97 (d, 1H), 8.78 (s, 1H), 15.29 (s, 1H); MS (ESI) m/z 550.6 [MꢀH]ꢀ.
Anal. Calcd for C29H27F2N3O6: C, 63.15; H, 4.93; F, 6.89; N, 7.62;
Found: C, 63.27; H, 4.92; F, 6.89; N, 7.68.
C32H27F2N3O6: C, 65.41; H, 4.63; F, 6.47; N, 7.15; Found: C, 65.52;
H, 4.56; F, 6.55; N, 7.10.
2.1.8. 7-(4-(2-(3-Phenylfuran-2(5H)-one-4-yloxy)ethyl)piperaz-
in-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (7)
2.1.14. 7-(4-(2-(3-(4-Fluorophenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-(4-fluorophenyl)-6-fluoro-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid (13)
Colorless crystal, 45.1%, mp 122–124 °C; 1H NMR (DMSO-d6,
500 Hz): 1.17–1.21 (m, 2H), 1.30–1.35 (m, 2H), 3.25–3.42 (m,
8H), 3.80–3.85 (m, 1H), 4.35–4.51 (m, 4H), 5.18 (s, 2H), 7.28 (s,
1H), 7.39–7.42 (m, 2H), 7.58 (d, 1H), 7.89–7.93 (m, 3H), 8.68 (s,
1H); MS (ESI) m/z 532.9 [MꢀH]ꢀ. Anal. Calcd for C29H28FN3O6: C,
65.28; H, 5.29; F, 3.56; N, 7.88; Found: C, 65.24; H, 5.31; F, 3.52;
N, 7.91.
White powder, 35.7%, mp 228–230 °C; 1H NMR (DMSO-d6,
500 Hz): 2.60–2.65 (m, 4H), 2.80 (t, 2H), 3.05–3.11 (m, 4H), 4.37
(t, 2H), 5.15 (s, 2H), 6.39 (d, 1H), 7.23 (t, 2H), 7.54 (t, 2H), 7.80
(dd, 2H), 7.93 (dd, 2H), 8.00 (d, 1H), 8.66 (s, 1H), 15.14 (s, 1H);
MS (ESI) m/z 604.6 [MꢀH]ꢀ. Anal. Calcd for C32H26F3N3O6: C,
63.47; H, 4.33; F, 9.41; N, 6.94; Found: C, 63.42; H, 4.37; F, 9.45;
N, 6.98.

X.-D. Wang et al. / Bioorg. Med. Chem. 22 (2014) 3620–3628
3623
2.1.15. 7-(4-(2-(3-(2-Chlorophenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (14)
2.1.21. 7-(4-(2-(3-(4-Chlorophenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-(4-fluorophenyl)-6-fluoro-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid (20)
Orange powder, 42.9%, mp 188–191 °C; 1H NMR (DMSO-d6,
500 Hz): 1.43 (t, 3H), 2.49–2.53 (m, 4H), 2.97–3.03 (m, 2H),
3.72–3.76 (m, 2H), 3,89–4.03 (m, 2H), 4.44–4.63 (m, 4H), 5.16 (s,
2H), 7.26–7.52 (m, 5H), 8.00 (d, 1H), 8.98 (s, 1H), 15.27 (s, 1H);
MS (ESI) m/z 554.3 [MꢀH]ꢀ. Anal. Calcd for C28H27ClFN3O6: C,
60.49; H, 4.89; Cl, 6.38; F, 3.42; N, 7.56; Found: C, 60.38; H, 4.87;
Cl, 6.32; F, 3.33; N, 7.59.
White powder, 32.7%, mp 166–168 °C; 1H NMR (DMSO-d6,
300 Hz): 2.55–2.63 (m, 4H), 2.76–2.81 (m, 2H), 3.05–3.10 (m,
4H), 4.35–4.39 (m, 2H), 5.15 (s, 2H), 6.38 (d, 1H), 7.43–7.52 (m,
5H), 7.91–8.03 (m, 2H), 8.64 (s, 1H); MS (ESI) m/z 620.6 [MꢀH]ꢀ.
Anal. Calcd for C32H26ClF2N3O6: C, 61.79; H, 4.21; Cl, 5.70; F,
6.11; N, 6.76; Found: C, 61.62; H, 4.25; Cl, 5.68; F, 6.14; N, 6.73.
2.1.22. 7-(4-(2-(3-(4-Chlorophenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (21)
2.1.16. 7-(4-(2-(3-(2-Chlorophenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-(4-fluorophenyl)-6-fluoro-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid (15)
White powder, 44.1%, mp 235–237 °C; 1H NMR (DMSO-d6,
500 Hz): 1.17–1.23 (m, 2H), 1.25–1.35 (m, 2H), 2.70–2.78 (m,
4H), 2.82–2.90 (m, 2H), 3.31–3.40 (m, 4H), 3.80–3.85 (m, 1H),
4.43–4.49 (m, 2H), 5.20 (s, 2H), 7.45–7.51 (m, 2H), 7.57 (d, 1H),
7.85–7.98 (m, 3H), 8.67 (s, 1H), 15.23 (s, 1H); MS (ESI) m/z 566.3
[MꢀH]ꢀ. Anal. Calcd for C29H27ClFN3O6: C, 61.32; H, 4.79; Cl,
6.24; F, 3.34; N, 7.40; Found: C, 61.36; H, 4.75; Cl, 6.29; F, 3.24;
N, 7.35.
Orange powder, 44.5%, mp 172–175 °C; 1H NMR (DMSO-d6,
500 Hz): 2.52–2.57 (m, 4H), 2.63 (t, 2H), 2.97–3.03 (m, 4H), 4.83
(t, 2H), 5.17 (s, 2H), 6.37 (d, 1H), 7.33–7.43 (m, 3H), 7.51–7.58
(m, 3H), 7.80 (dd, 2H), 7.99 (d, 1H), 8.66 (s, 1H), 15.15 (s, 1H);
MS (ESI) m/z 620.0 [MꢀH]ꢀ. Anal. Calcd for C32H26ClF2N3O6: C,
61.79; H, 4.21; Cl, 5.70; F, 6.11; N, 6.76; Found: C, 61.70; H, 4.26;
Cl, 5.75; F, 6.10; N, 6.66.
2.1.17. 7-(4-(2-(3-(3-Chlorophenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-
dihydro-1,8-naphthyridine-3-carboxylic acid (16)
2.1.23. 7-(4-(2-(3-(3-Bromophenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-(4-fluorophenyl)-6-fluoro-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid (22)
White powder, 38.5%, mp 114–116 °C; 1H NMR (DMSO-d6,
500 Hz): 1.38 (t, 3H), 2.65–2.70 (m, 4H), 2.85–2.87 (m, 2H),
3.80–3.87 (m, 4H), 4.44–4.51 (m, 4H), 5.19 (s, 2H), 7.34 (d, 1H),
7.44 (dd, 1H), 7.92 (d, 1H), 7.99 (s, 1H), 8.09 (dd, 1H), 8.98 (s,
1H), 15.31 (s, 1H); MS (ESI) m/z 556.1 [MꢀH]ꢀ. Anal. Calcd for C27-
H26ClFN4O6: C, 58.22; H, 4.71; Cl, 6.37; F, 3.41; N, 10.06; Found: C,
58.25; H, 4.76; Cl, 6.17; F, 3.46; N, 10.16.
White powder, 34.6%, mp 220–222 °C; 1H NMR (DMSO-d6,
500 Hz): 2.61–2.67 (m, 4H), 2.81 (t, 2H), 3.05–3.10 (m, 4H), 4.40
(t, 2H), 5.17 (s, 2H), 6.39 (d, 1H), 7.36 (t, 1H), 7.47 (d, 1H), 7.54
(t, 2H), 7.80 (dd, 2H), 7.95 (d, 1H), 8.00 (d, 1H), 8.11 (t, 1H), 8.67
(s, 1H), 15.15 (s, 1H); MS (ESI) m/z 665.9 [MꢀH]ꢀ. Anal. Calcd for
C32H26BrF2N3O6: C, 57.67; H, 3.93; Br, 11.99; F, 5.70; N, 6.30;
Found: C, 57.56; H, 3.98; Br, 11.95; F, 5.78; N, 6.36.
2.1.18. 7-(4-(2-(3-(3-Chlorophenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-(4-fluorophenyl)-6-fluoro-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid (17)
2.1.24. 7-(4-(2-(3-(3-Bromophenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-
dihydro-1,8-naphthyridine-3-carboxylic acid (23)
White powder, 47.0%, mp 224–226 °C; 1H NMR (DMSO-d6,
300 Hz): 2.61–2.67 (m, 4H), 2.80 (t, 2H), 3.08–3.12 (m, 4H),
4.35–4.41 (m, 2H), 5.16 (s, 2H), 6.38 (d, 1H), 7.33 (d, 1H),
7.39–7.44 (m, 1H), 7.50–7.56 (m, 2H), 7.72–7.78 (m, 2H), 7.83 (d,
1H), 7.91–7.96 (m, 2H), 8.65 (s, 1H), 15.10 (s, 1H); MS (ESI) m/z
660.7 [MꢀH]ꢀ. Anal. Calcd for C32H26ClF2N3O6: C, 61.79; H, 4.21;
Cl, 5.70; F, 6.11; N, 6.76; Found: C, 61.85; H, 4.25; Cl, 5.77; F,
6.01; N, 6.72.
White powder, 39.3%, mp 135–138 °C; 1H NMR (DMSO-d6,
500 Hz): 1.37 (t, 3H), 2.68–2.71 (m, 4H), 2.86 (t, 2H), 3.85–3.90
(m, 4H), 4.44–4.49 (m,4H), 5.19 (s, 2H), 7.38 (dd, 1H), 7.47 (d,
1H), 7.96 (d, 1H), 8.08–8.13 (m, 2H), 8.97 (s,1H), 15.30 (s, 1H);
MS (ESI) m/z 598.9 [MꢀH]ꢀ. Anal. Calcd for C27H26BrFN4O6: C,
53.92; H, 4.36; Br, 13.29; F, 3.16; N, 9.32; Found: C, 53.87; H,
4.35; Br, 13.21; F, 3.23; N, 9.36.
2.1.25. 7-(4-(2-(3-(4-Bromophenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (24)
2.1.19. 7-(4-(2-(3-(4-Chlorophenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (18)
White powder, 43.1%, mp 216–218 °C; 1H NMR (DMSO-d6,
300 Hz): 1.35–1.49 (m, 4H), 2.51–2.60 (m, 4H), 2.65–2.71 (m,
4H), 2.85–2.92 (m, 2H), 4.39–4.45 (m, 2H), 4.55–4.62 (m, 2H),
5.19 (s, 2H), 7.18–7.33 (m, 1H), 7.60–7.65 (m, 2H), 7.89–7.96 (m,
3H), 8.98 (s, 1H), 15.35 (s, 1H); MS (ESI) m/z 598.2 [MꢀH]ꢀ. Anal.
Calcd for C28H27BrFN3O6: C, 56.01; H, 4.53; Br, 13.31; F, 3.16; N,
7.00; Found: C, 56.08; H, 4.59; Br, 13.36; F, 3.19; N, 7.01.
White powder, 52.5%, mp 206–208 °C; 1H NMR (DMSO-d6,
500 Hz): 1.41 (t, 3H), 2.45–2.51 (m, 4H), 2.70–2.78 (m, 4H),
2.85–2.90 (m, 2H), 4.44 (t, 2H), 4.58 (t, 2H), 5.19 (s, 2H), 7.18 (d,
1H), 7.91 (s, 1H), 7.94–7.98 (m, 2H), 8.96 (s, 1H), 15.37 (s, 1H);
MS (ESI) m/z 554.1 (MꢀH)ꢀ. Anal. Calcd for C28H27ClFN3O6: C,
60.49; H, 4.89; Cl, 6.38; F, 3.42; N, 7.56; Found: C, 60.41; H, 4.82;
Cl, 6.31; F, 3.40; N, 7.59.
2.1.26. 7-(4-(2-(3-(4-Bromophenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-(4-fluorophenyl)-6-fluoro-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid (25)
2.1.20. 7-(4-(2-(3-(4-Chlorophenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-
dihydro-1,8-naphthyridine-3-carboxylic acid (19)
Orange powder, 32.7%, mp 240–242 °C; 1H NMR (DMSO-d6,
500 Hz): 2.57–2.63 (m, 4H), 2.76–2.81 (m, 2H), 3.03–3.08 (m,
4H), 4.37 (t, 2H), 5.14 (s, 2H), 6.38 (d, 1H), 7.50–7.60 (m, 4H),
7.80 (dd, 2H), 7.86 (d, 2H), 8.00 (d, 1H), 8.66 (s, 1H), 15.15 (s,
1H); MS (ESI) m/z 664.1 [MꢀH]ꢀ. Anal. Calcd for C32H26BrF2N3O6:
C, 57.67; H, 3.93; Br, 11.99; F, 5.70; N, 6.30; Found: C, 57.58; H,
3.97; Br, 11.85; F, 5.79; N, 6.37.
White powder, 46.1%, mp 173–175 °C; 1H NMR (DMSO-d6,
300 Hz): 1.38 (t, 3H), 2.60–2.65 (m, 4H), 2.85 (t, 2H), 3.25–3.31
(m, 4H), 4.50–4.60 (m, 4H), 5.18 (s, 2H), 7.46 (d, 2H), 7.95 (d,
2H), 8.09 (d, 1H), 8.98 (s, 1H), 15.32 (s, 1H); MS (ESI) m/z 555.2
[MꢀH]ꢀ. Anal. Calcd for C27H26ClFN4O6: C, 58.22; H, 4.71; Cl,
6.37; F, 3.41; N, 10.06; Found: C, 58.29; H, 4.76; Cl, 6.30; F, 3.43;
N, 10.01.

3624
X.-D. Wang et al. / Bioorg. Med. Chem. 22 (2014) 3620–3628
2.1.27. 7-(4-(2-(3-(4-Bromophenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (26)
DMSO was prepared with different concentrations using sterilized
liquid medium (50% (v/v) of DMSO in PBS). A specified quantity of
the medium containing the test compound was added into 96-well
plates, which was replaced by the sterilized liquid medium as
blank control. Suspension of the microorganism was prepared to
contain approximately 105 cfu/mL and applied to 96-well plates
with serially diluted compounds (or blank control) to be tested
Orange powder, 39.8%, mp 228–230 °C; 1H NMR (DMSO-d6,
500 Hz): 1.15–1.20 (m, 2H), 1.25–1.32 (m, 2H), 2.74–2.81 (m,
4H), 2.88 (t, 2H), 3.32–3.40 (m, 4H), 3.80–3.85 (m, 1H), 4.45 (t,
2H), 5.19 (s, 2H), 7.55–7.63 (m, 3H), 7.89–7.94 (m, 3H), 8.67 (s,
1H), 15.24 (s, 1H); MS (ESI) m/z 610.1 [MꢀH]ꢀ. Anal. Calcd for
and incubated at 37 °C. 50
was added to each well. Incubation was continued at room temper-
ature for 4–5 h. 100 L of SDS lysis buffer with pH about 3.5 (10%
lL of PBS containing 3 mg of MTT/mL
C29H27BrFN3O6: C, 56.87; H, 4.44; Br, 13.05; F, 3.10; N, 6.86; Found:
C, 56.78; H, 4.42; Br, 13.20; F, 3.14; N, 6.81.
l
sodium dodecyl sulfate containing 5% isopropanol) was added to
extract the dye. After 8–10 h of incubation at room temperature,
the control OD value (at 570 nm) should be controlled under
1.0–2.0 to achieve reliable results. The observed MIC50s were pre-
sented in Table 1.
2.1.28. 7-(4-(2-(3-(4-Methylphenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-(4-fluorophenyl)-6-fluoro-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid (27)
White powder, 29.8%, mp 202–204 °C; 1H NMR (DMSO-d6,
300 Hz): 2.29–2.35 (m, 3H), 2.50–2.55 (m, 4H), 2.78 (d, 2H),
3.01–3.06 (m, 4H), 4.34 (t, 2H), 5.11 (s, 2H), 6.39 (d, 1H), 7.18
(d, 2H), 7.53 (dd, 2H), 7.75–7.81 (m, 4H), 7.99 (d, 1H), 8.65 (s,
1H), 15.13 (s, 1H); MS (ESI) m/z 600.5 [MꢀH]ꢀ. Anal. Calcd for
2.3. Preparation of the TyrRS and enzyme assay
S. aureus TyrRS was over-expressed in E. coli and purified to near
homogeneity (ꢁ98% as judged by SDS–PAGE) using standard puri-
fication procedures.27 TyrRS activity was measured by aminoacyla-
tion using modifications to previously described methods.28 The
assays were performed at 37 °C in a mixture containing (final con-
centrations) 100 mM Tris/Cl pH 7.9, 50 mM KCl, 16 mM MgCl2,
5 mM ATP, 3 mM DTT, 4 mg/mL E. coli MRE600 tRNA (Roche) and
C33H29F2N3O6: C, 65.88; H, 4.86; F, 6.32; N, 6.98; Found: C, 65.79;
H, 4.81; F, 6.30; N, 6.97.
2.1.29. 7-(4-(2-(3-(4-Methylphenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (28)
Orange powder, 33.9%, mp 213–215 °C; 1H NMR (DMSO-d6,
300 Hz): 1.17–1.22 (m, 2H), 1.25–1.31 (m, 2H), 2.30 (s, 3H),
2.70–2.75 (m, 4H), 2.84–2.88 (m, 2H), 3.35–3.40 (m, 4H),
3.75–3.80 (m, 1H), 4.41 (t, 2H), 5.15 (s, 2H), 7.21 (d, 2H), 7.57 (d,
1H), 7.81 (d, 2H), 7.91 (d, 1H), 8.67 (s, 1H), 15.19 (s, 1H); MS
(ESI) m/z 546.4 [MꢀH]ꢀ. Anal. Calcd for C30H30FN3O6: C, 65.80; H,
5.52; F, 3.47; N, 7.67; Found: C, 65.77; H, 5.49; F, 3.53; N, 7.69.
10
l
M
L
-tyrosine (0.3
l
M
L
-[ring-3,5-3H] tyrosine (PerkinElmer,
carrier). TyrRS
Specific activity: 1.48–2.22TBq/mmol), 10
l
M
(0.2 nM) was preincubated with a range of inhibitor concentrations
for 10 min at room temperature followed by the addition of pre-
warmed mixture at 37 °C. After specific intervals, the reaction
was terminated by adding aliquots of the reaction mix into ice-cold
7% trichloroacetic acid and harvesting onto 0.45 mm hydrophilic
Durapore filters (Millipore Multiscreen 96-well plates) and
counted by liquid scintillation. The rate of reaction in the experi-
ments was linear with respect to protein and time with less than
50% total tRNA acylation. IC50s correspond to the concentration
at which half of the enzyme activity is inhibited by the compound.
The results are presented in Table 2.
2.1.30. 7-(4-(2-(3-(4-Methoxyphenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (29)
White powder, 51.4%, mp 213–215 °C; 1H NMR (DMSO-d6,
500 Hz): 1.17–1.20 (m, 2H), 1.35–1.40 (m, 2H), 2.55–2.61 (m,
4H), 3.80 (s, 3H), 4.01–4.06 (m, 1H), 4.23–4.28 (m, 2H), 4.45–4.52
(m, 2H), 5.17 (s, 2H), 7.30–7.42 (m, 3H), 7.54 (d, 2H), 8.11(d, 1H),
9.00 (s, 1H), 15.35 (s, 1H); MS (ESI) m/z 562.3 [MꢀH]ꢀ. Anal. Calcd
for C30H30FN3O7: C, 63.94; H, 5.37; F, 3.37; N, 7.46; Found: C, 63.88;
H, 5.39; F, 3.31; N, 7.49.
2.4. DNA gyrase assay
Enzyme activity was measured by a supercoiling assay that
monitored the ATP-dependent conversion of relaxed pBR322
DNA to the supercoiled form. Specifically, 2
DNA was incubated with 0.7 L of E. coil DNA gyrase for 60 min
at 37 °C in 20
lL of relaxed pBR322
2.1.31. 7-(4-(2-(3-(2-(Benzyloxy)phenyl)furan-2(5H)-one-4-
yloxy)ethyl)piperazin-1-yl)-1-(4-fluorophenyl)-6-fluoro-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid (30)
l
l
L of the following conditions: 35 mM TrisꢂHCl (pH
7.5), 24 mM KCl, 4 mM MgCl2, 2 mM DTT, 1.8 mM Spermidine,
White powder, 49.3%, mp 187–189 °C; 1H NMR (DMSO-d6,
500 Hz): 2.40–2.47 (m, 4H), 2.50–2.55 (m, 2H), 2.87–2.95 (m,
4H), 4.10–4.16 (m, 2H), 5.05 (s, 2H), 6.33 (d, 1H), 6.93–6.97 (m,
1H), 7.08 (dd, 1H), 7.26–7.40 (m, 7H), 7.53–7.57 (m, 2H), 7.77–
7.81 (m, 2H), 8.00 (d, 1H), 15.15 (s, 1H); MS (ESI) m/z 692.2
[MꢀH]ꢀ. Anal. Calcd for C39H33F2N3O7: C, 67.53; H, 4.79; F, 5.48;
N, 6.06; Found: C, 67.47; H, 4.81; F, 5.51; N, 6.16.
1 mM ATP, 6.5% (w/v) glycerol and 0.1 mg/mL BSA. Reactions were
stopped 20 lL chloroform/iso-amyl alcohol (24/1) and 20 lL Stop
Buffer (STEB: 40% sucrose, 100 mM TrisꢂHCl (pH 7.5), 1 mM EDTA,
0.5 mg/mL bromophenol blue). Each reaction was analyzed by
electrophoresis through a 1% agarose gel (run at 50 V for 3 h).
The IC50 was defined as the drug concentration that reduced the
enzymatic activity observed with drug-free controls by 50%
(Table 2 and Fig. 1).
2.2. Antimicrobial activity
2.5. Protocol of docking study
The antibacterial activities of the synthesized compounds were
tested in LB medium against a Gram-positive bacterial strain
(Methicillin-resistant Staphylococcus aureus, ATCC 25923) and
two Gram-negative bacterial strains (Multiple drug resistant
Escherichia coli, ATCC 35218 and Tetracycline-resistant Bacillus
subtilis, ATCC 6633) with ciprofloxacin as control. The MTT
proliferation assay was used to measure the MIC50s of the test
compounds by determining the absorbance of the cells in culture.26
The automated docking studies were carried out using Auto-
Dock version 4.2. First, AutoGrid component of the program pre-
calculates a three-dimensional grid of interaction energies based
on the macromolecular target using the AMBER force field. A grid
box of 60 ꢃ 60 ꢃ 60 Å size (x, y, z) with a spacing of 0.375 Å and
grid maps were created representing the catalytic active target site
region where the native ligand was embedded. Then automated
docking studies were carried out to evaluate the binding free
A stock solution of the synthesized compound (1000 lg/mL) in

X.-D. Wang et al. / Bioorg. Med. Chem. 22 (2014) 3620–3628
3625
Table 1
Inhibitory activity (MIC50) of the synthetic compounds against microbes
O
F
COOH
N
X
N
R2
R4
R1
N
MIC50 (lg/mL)
O
R3
O
O
Entry
R1
R2
R3
R4
X
A
B
C
4
5
H
H
H
H
H
H
CH2CH3
CH2CH3
CH
N
>25
5.6
18.9
6.8
16.6
12.6
6
H
H
H
F
F
CH
1.2
0.69
0.60
7
8
9
H
F
F
H
H
H
H
H
H
CH
CH
N
1.0
2.7
2.6
0.64
3.3
2.6
0.23
2.4
1.6
CH2CH3
CH2CH3
10
F
H
H
CH
0.67
0.21
0.20
11
12
F
H
H
H
F
CH
CH
0.11
0.37
0.19
0.24
0.09
0.16
H
13
14
H
H
H
F
F
F
F
CH
CH
1.1
2.5
0.34
0.43
0.20
0.90
Cl
H
CH2CH3
CH2CH3
15
16
Cl
H
H
H
H
CH
N
0.72
2.9
0.25
8.7
0.37
15.3
Cl
17
H
Cl
H
CH
0.89
0.44
0.75
18
19
H
H
H
H
Cl
Cl
CH2CH3
CH2CH3
CH
N
19.5
9.6
15.9
24.9
18.5
13.2
20
21
22
H
H
H
H
Cl
Cl
H
F
F
CH
CH
CH
1.7
1.3
4.4
7.8
0.51
0.16
0.43
H
0.49
0.39
Br
23
24
H
H
Br
H
H
Br
CH2CH3
CH2CH3
N
CH
12.2
13.5
13.1
21.6
4.7
>25
25
26
27
H
H
H
H
H
H
Br
F
F
CH
CH
CH
4.2
3.8
2.0
11.1
8.5
2.9
Br
2.6
CH3
CH3
0.45
0.82
28
29
H
H
H
H
CH
CH
1.9
0.53
6.5
0.39
2.6
OMe
23.5
30
OBn
—
H
—
H
—
F
CH
—
2.8
2.1
0.58
2.70
Cipro
—
5.65
6.82
(A) Multiple drug resistant Escherichia coli, ATCC 35218; (B) Methicillin-resistant Staphylococcus aureus, ATCC 25923; (C) Tetracycline-resistant Bacillus subtilis, ATCC 6633.
Table 2
ADT (AutoDockTools package, version 1.5.4) on PC which is associ-
In vitro inhibitory activity data of the synthesized compounds against DNA gyrase and
ated with AutoDock 4.2. Default settings were used with an initial
population of 50 randomly placed individuals, a maximum number
of 7.5 ꢃ 106 energy evaluations, and a maximum number of
TyrRS
Entry
IC50 (lM)
DNA gyrase
TyrRS
2.7 ꢃ 104 generations. A mutation rate of 0.02 and a crossover rate
0
7
5.77 0.07
3.69 0.03
1.15 0.07
2.84 0.06
4.05 0.04
5.23 0.04
—
l
M
M
M
M
M
M
7.12 0.04
2.81 0.07
0.12 0.04
1.29 0.06
6.37 0.09
—
l
l
l
l
l
M
M
M
M
M
of 0.8 were chosen. Results differing by less than 0.5 ÅA in positional
root-mean-square deviation (RMSD) were clustered together and
the results of the most favorable free energy of binding were
selected as the resultant complex structures.
10
11
12
15
Cipro
I
l
l
l
l
l
0.10 0.03
lM
3. Results and discussion
3.1. Chemistry
I 3-(4-hydroxyphenyl)-4-(2-morpholinoethoxy)furan-2(5H)-one.
energy of the inhibitor within the macromolecules. The genetic
algorithm with local search (GA-LS) was chosen to search for the
best conformers. The parameters were set using the software
Detailed synthetic pathways to
(5H)-one-fluoroquinolone hybrids 4–30 are depicted in Scheme 3.
The intermediates 2 and 3 were synthesized according to the
a series of 3-arylfuran-2

3626
X.-D. Wang et al. / Bioorg. Med. Chem. 22 (2014) 3620–3628
Figure 1. Representative comparative data for the inhibition of DNA gyrase with Cipro and compound 11. 1% agarose gel shows the inhibitory activity of Cipro and 11 against
DNA gyrase: lane 1, relaxed DNA; lane 2, supercoiling reaction by DNA gyrase without presence of inhibitor; lanes 3–8, same as lane 1 but in the presence of 0.025, 0.10, 0.40,
0.16, 6.4, 25.6
l
g/mL inhibitor Cipro or 11.
O
O
OEt
R1
R1
R1 EtO
Br
R2
R3
R2
R3
NaOH
R2
R3
O
O
CO2H
COONa
DMSO
1a-1k
a R1=H, R2=H, R3=H
e R1=H, R2=Cl, R3=H
b R1=F, R2=H, R3=H
c R1=H, R2=H, R3=F
g R1=H, R2=Br, R3=H
d R1=Cl, R2=H, R3=H
h R1=H, R2=H, R3=Br
f
R1=H, R2=H, R3=Cl
k R1=OBn, R2=H, R3=H
i
R1=H, R2=H, R3=CH3
j
R1=H, R2=H, R3=OMe
O
F
COOH
O
O
O
O
N
X
N
Br
Br
NaH
HN
O
R4
OH
R1
R1
Br
R3
R3
R2
3a-3k
R2
2a-2k
O
O
F
O
O
R1
(See Table 1 for details)
N
N
4-30
R3
X
COOH
R2
N
R4
Scheme 3. Synthetic route of 3-arylfuran-2(5H)-one-fluoroquinolone hybrids.
previously reported method.15 The desired end products (4–30)
were obtained by amination of 3a–3k with appropriate fluoroquin-
olones in DMSO in the presence of DMAP and KI. All 3-arylfuran-
2(5H)-one-fluoroquinolone hybrids were first reported and fully
characterized by spectroscopic methods and elemental analysis.
activity (11 vs 12, 16 vs 19). In comparison with 12, replacement of
the fluoro group with a large volume group (chloro, methyl, bromo,
or methoxy group) at 4-position of benzene ring (21, 28, 26 and 29)
led to a further decrease in activity, which may suggest that these
substituents may cause a steric clash with the protein. Thus, the
compound 11, substituted by the smallest volume group (fluoro
group) at the 2-positon of benzene ring is the most active, which
shows about 30-fold more potent than ciprofloxacin, against B.
subtilis.
3.2. Antimicrobial activity
Twenty seven compounds were tested for antibacterial activi-
ties against resistant strains by using ciprofloxacin as control.
The selected resistant strains are Multiple drug resistant Esche-
richia coli (ATCC 35218, Gram-negative), tetracycline-resistant
Bacillus subtilis (ATCC 6633, Gram-negative) and Methicillin-resis-
tant Staphylococcus aureus (ATCC 25923, Gram-positive). The
MIC50s of these compounds are presented in Table 1. The most of
compounds show significant activity against all organisms and
are more potent than control drug, ciprofloxacin. On average, B.
subtilis shows slightly more sensitive response to the hybrids,
and it is followed by S. aureus. In the initial phase of our SAR stud-
ies, we focused our efforts on substitution of fluoroquinolone moi-
ety (4–7, 8–11, 18–21). The compounds with ciprofloxacin moiety
were more active than others, being illustrated by the potency
order norfloxacin, enoxacin, sarafloxacin and ciprofloxacin (4 vs 5
vs 6 vs 7, 8 vs 9 vs 10 vs 11, 18 vs 19 vs 20 vs 21 and 24 vs 25
vs 26). We then turned our attention to exploring the SAR profile
of 3-arylfuran-2(5H)-one moiety (11, 12, 21, 26, 28, 29; 10, 13,
20, 25, 27). Modification at the 2-position seems to be beneficial
to antimicrobial activity. Changing the group from the 2-position
to the 3-position or 4-position decreased the level of antimicrobial
The activities of most compounds are more potent than that of
ciprofloxacin, may be due to the introduction of the pharmaco-
phore 3-arylfuran-2(5H)-one which can effectively target the
TyrRS and possibly improve the inhibition ability of the parent
against DNA gyrase. Furthermore, the possible multi-targeting of
the hybrids may be also able to decline the emergence of
resistance.
3.3. DNA gyrase and TyrRS assay
To determine the possibility of a dual mode of action, we mea-
sured the inhibitory activities against the both possible target
enzymes DNA gyrase and TyrRS with selected hybrids (7, 10, 11,
12, and 15). The observed data show that all the selected hybrids
displayed good activities against the DNA gyrase and TyrRS (Table 2
and Fig. 1). The first information obtained in this study is that, the
potencies against DNA gyrase are similar to or better than that of
ciprofloxacin, and the hybrids with good inhibition against DNA
gyrase also exhibit strong antibacterial activities, especially for
E. coli, confirming their strong fluoroquinolone character. The

X.-D. Wang et al. / Bioorg. Med. Chem. 22 (2014) 3620–3628
3627
second is that, in comparison to ciprofloxacin, both MIC50 and IC50
values of 10, 11, 12 and 15 were decreased. It is noted that the
decreases of the MIC50 values are significantly superior to the
IC50 values of DNA gyrase. This suggests that an additional antibac-
terial mechanism may be existed, which was evidenced by TyrRS
assay. The selected hybrids also shows significant inhibition
against TyrRS, which are similar to that of 3-(4-hydroxyphenyl)-
4-(2-morpholinoethoxy)furan-2(5H)-one. These results revealed
that the hybrids of 3-arylfuran-2(5H)-one and fluoroquinolone
showed potent antibacterial activities through a dual mode of
action, effectively targeting TyrRS and DNA gyrase.
3.4. Protocol of docking study
In order to give an explanation and understanding of the mode
of action of these molecules, the most potent inhibitor 11 was
selected to dock into the binding sites of TyrRS and DNA gyrase,
which was performed on the binding model based on the TyrRS
and DNA gyrase complex structures. In both cases, compound 11
was held to the active pockets tightly by several hydrogen-bonding
interactions and hydrophobic contacts. In the binding model of
TyrRS, the fluoroquinolone moiety of 11 is oriented towards the
entrance cavity (Fig. 2) and the CH of cyclopropyl group as a donor
forms a hydrogen-bond with O atom of residue Asp40 (Fig. 3). The
piperazine moiety interacts with His50 residue by a CAHꢂ ꢂ ꢂO
hydrogen bond. Meanwhile, the CH2 group in carbon chain which
closes to piperazine moiety interacts with Gly38 and Cys37 resi-
dues through CAHꢂ ꢂ ꢂO and CAHꢂ ꢂ ꢂS hydrogen bonds. There are
also several important interactions considered as hydrogen bonds:
three of them occur between the furanone-ring moiety and
Asp195, Gln196 and Gln174 residues, while the other two occur
between the benzene-ring moiety of the molecule and residues
Gln196 and Gln190. In the binding model of DNA gyrase, the fluo-
roquinolone moiety of 11 was oriented towards the bottom of cav-
ity, which located at the same position of the active site with
ciprofloxacin (Fig. 4). Compound 11 binds the active site in an
anion form (COOꢀ), in which the oxygen ion bridges manganese
ion with metal–ligand distance of 2.637 Å (Fig. 5). The 4-carbonyl
oxygen of fluoroquinolone moiety shows hydrogen bonding inter-
action with the NH2 group of Dc12 and the F atom of fluoroquino-
lone is found to weakly interact with the CH2 group of Dc12. In
Figure 3. Binding mode of compound 11 with TyrRS from S. aureus. For clarity, only
interacting residues were labeled. Hydrogen bonding interactions are shown in
dash. This figure was made using PyMol.
Figure 4. Binding mode of Cipro and compound 11 with DNA gyrase. The structure
with green carbon atom is 11 and another with pink carbon atom is Cipro. The
enzyme complex is shown as surface; while Cipro and compound 11 are shown as
stick models. This figure was made using PyMol.
addition, the molecule is further anchored to Arg458 and Glu477
through the piperazine moiety. Furanone-ring receives two strong
hydrogen bonding interactions from the NH2 group of Asn475. The
benzene-ring using CH groups as donors receives three hydrogen
bonding interactions from Dc12 and using the F atom as an accep-
tor forms a hydrogen bond with Dc13.
Figure 2. Binding mode of compound 11 with TyrRS. The enzyme is shown as
surface; while the docking complex of 11 is shown as a stick model. This figure was
made using PyMol.
The above data strongly indicated that a high affinity of
compound 11 to the target enzymes (TyrRS and DNA gyrase) is

3628
X.-D. Wang et al. / Bioorg. Med. Chem. 22 (2014) 3620–3628
Acknowledgments
The work was financed by a grant from National Natural Sci-
ence Foundation (grant No. 21262013) of China, and by a grant
from National Natural Science Foundation (Youth Science Funds,
No. 31100421) of China.
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In this study, twenty seven novel 3-arylfuran-2(5H)-one-fluo-
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l
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51-fold more potent than ciprofloxacin, respectively. These
indicated that combining 3-arylfuran-2(5H)-one with fluoroquino-
lone could produce potent antibacterial agents by inhibiting
multi-targets (DNA gyrase and TyrRS). These newly developed
3-arylfuran-2(5H)-one-fluoroquinolone hybrids showed marked
biological activity in vitro, and deserve further research as a novel
class of antibacterial agents.