Design, synthesis, and evaluation of novel fluoroquinolone-flavonoid hybrids as potent antibiotics against drug-resistant microorganisms

Article abstract of DOI:10.1016/j.ejmech.2014.04.037

Based on a rationally conceived pharmacophore model to build a multi-target bacterial topoisomerase inhibitor, twenty-one fluoroquinolone-flavonoid hybrids were synthesized. Some obtained hybrids show excellent antibacterial activity against drug-resistant microorganisms with narigenin-ciprofloxacin being the most active, showing 8, 43, 23 and 88 times better activity than ciprofloxacin against Escherichia coli ATCC 35218, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923 and Candida albicans ATCC 90873, respectively. Drug accumulation and DNA supercoiling assays of two active analogues revealed potent inhibition of both the DNA gyrase and efflux pump, confirming the desired dual mode of action. Molecular docking study disclosed that the introduced flavonoid moiety not only provides several additional interactions but also does not disturb the binding mode of the floxacin moiety. Our data also demonstrated that development of antifungals is possible from fluoroquinolones modified at C-7 position.

Full text of DOI:10.1016/j.ejmech.2014.04.037

European Journal of Medicinal Chemistry 80 (2014) 92e100
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, and evaluation of novel fluoroquinoloneeflavonoid
hybrids as potent antibiotics against drug-resistant microorganisms
,
b
,
a
b
a
a
Zhu-Ping Xiao a , Xu-Dong Wang , Peng-Fei Wang , Yin Zhou , Jing-Wen Zhang ,
*
Lei Zhang a, Jiao Zhou a, Sha-Sha Zhou a, Hui Ouyang a, Xiao-Yi Lin a, Manzira Mustapa a,
Asaimuguli Reyinbaike 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
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on a rationally conceived pharmacophore model to build a multi-target bacterial topoisomerase
inhibitor, twenty-one fluoroquinoloneeflavonoid hybrids were synthesized. Some obtained hybrids
show excellent antibacterial activity against drug-resistant microorganisms with narigeninecipro-
floxacin being the most active, showing 8, 43, 23 and 88 times better activity than ciprofloxacin against
Escherichia coli ATCC 35218, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923 and Candida
albicans ATCC 90873, respectively. Drug accumulation and DNA supercoiling assays of two active ana-
logues revealed potent inhibition of both the DNA gyrase and efflux pump, confirming the desired dual
mode of action. Molecular docking study disclosed that the introduced flavonoid moiety not only pro-
vides several additional interactions but also does not disturb the binding mode of the floxacin moiety.
Our data also demonstrated that development of antifungals is possible from fluoroquinolones modified
at C-7 position.
Received 17 September 2013
Received in revised form
10 April 2014
Accepted 11 April 2014
Available online 13 April 2014
Keywords:
Fluoroquinolone
Flavonoid
Hybrid
DNA gyrase
Efflux pump
Inhibitor
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
the enzyme (such as b-lactamase) activity resulting in inactivation
of antibiotics [7], spontaneous chromosomal mutations [8], and
horizontal transfer of genetic elements [9]. Inhibition of the activity
of drug efflux transporters appears to be a promising approach for
restoring the activity of a drug that is the substrate of these efflux
pumps [10].
Antibiotics have a long history, beginning in the 1930s and
earlier, during which several distinct drug classes were discovered
and numerous improved analogues were made available [1]. Flu-
oroquinolones, broad-spectrum antimicrobial agents, are among
the most attractive drugs in the anti-infective chemotherapy field
[2]. These antibiotics target the bacterial type II topoisomerase
enzymes (DNA gyrase and topoisomerase IV) which are essential,
ubiquitous enzymes involved in bacterial cell growth and division
[3]. However, quinolone-resistant microbes are rapidly spreading in
hospitals and the community. In fact, antibiotic drug resistance has
become a growing problem across the globe over the past several
decades. Microbe develop drug resistance through various mech-
anisms, such as overexpression of drug efflux transporters like
multidrug and toxic compound extrusion (MATE) transporters
[4,5], changes in the target sites of antibiotics [6], optimization of
Flavonoids are a class of plant phenolic compounds that are
widely found in fruits, vegetables, seeds and herbs. A large number
of human and animal studies show that flavonoids exert beneficial
effects in the prevention of many diseases, including cancer, car-
diovascular disease and neurodegenerative disorders [11]. Because
of their considerable health related effects, flavonoids become the
subjects of much medical and biological research as investigators
try to find more biological properties and mechanisms [12].
Recently, some flavonoids received much attention due to their
unique biological properties in the modulation of multidrug resis-
tance [13]. Iris L. K. Wong et al. reported that apigenin homodimers
(I) (Scheme 1) can modulate the efflux pump P-glycoprotein ac-
tivity in human cancer [14]. Subsequently, tetramethylscutellarein
(II) and sarothrin (III) were reported as potent inhibitors of bac-
terial NorA efflux pump [15,16]. Not coincidentally, several other
flavonoids (naringenin (IV) [17], quercetin (V), kaempferol (VI) [18],
* Corresponding authors. College of Chemistry and Chemical Engineering, Jishou
University, Jishou 416000, PR China.
E-mail addresses: xiaozhuping2005@163.com (Z.-P. Xiao), zhuhl@nju.edu.cn
(H.-L. Zhu).
http://dx.doi.org/10.1016/j.ejmech.2014.04.037
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 80 (2014) 92e100
93
Scheme 1. Structures of some flavonoids and efflux pump modulators, and the scaffold of a FloxeFlav hybrid.
chrysin (VII) [13] and genistein (VIII) [19]) were also identified as
effective inhibitors of the multidrug transporters from the MRP
family.
Drug resistant bacteria constitute one of the main reasons for
chemotherapy failure, which in turn becomes a primary driver for
antibacterial research and development [1]. As widely known,
discovery of novel agents against new bacterial targets is regarded a
risky business endeavor [20]. Therefore, most development activ-
ities are focused on expanding existing antibiotic classes [21].
Recently, some fluoroquinolone hybrids were synthesized and re-
ported as good anticancer and antimicrobial agents [22e24]. Based
on this inspiration and inhibition properties of some flavonoids
against efflux pumps, hybrid analogues (IX) of flavonoidfloxacin
were designed for expecting to reduce the grave problem of fluo-
roquinolone resistance.
Scheme 2. General structures of a fluoroquinolone and a FloxeFlav hybrid.
2. Results and discussion
2.1. Chemistry
Structureeactivity relationship studies of fluoroquinolone
antibacterial agents have been widely reviewed and have demon-
strated a high tolerance for structural variations at the 7-position
(Scheme 2), including alkylations at the terminal nitrogen of the
piperazine moiety [25e27]. Specifically, it appears that increasing
bulkiness of the C-7 substituent leads to the enhancement of the
antibacterial potency against Gram-positive bacteria and the ac-
tivity against efflux-mediated resistant mutants [28]. On the basis
of this information and together with the inhibitory activity of
flavonoids against efflux pumps, a series of hybrids of floxacin and
flavonoid (FloxeFlav) were therefore synthesized for antibacterial
evaluation against resistant bacteria (Scheme 2). FloxeFlavs 4e24
were prepared by direct coupling of commercial floxacins with
Scheme 3. The synthetic route for Floxenarigenins.

94
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 80 (2014) 92e100
flavonoids at the terminal nitrogen of the 7-position through an
ethylene linker (Schemes 3 and 4).
0.14 mg/mL against E. coli, B. subtilis, S. aureus and C. albicans
respectively, which is the most active compound, showing 8- to 88-
fold more potent than the positive control ciprofloxacin. Activities
of these hybrids decreased in the sequence of ciprofloxacin, sara-
floxacin, norfloxacin, enoxacin and lomefloxacin, and this sequence
is also observed in other flavonoid series. The SAR analysis clearly
disclosed that the hybrid bearing naringenin or ciprofloxacin
showed relatively good activity. However, that bearing chrysin and
lomefloxacin (or enoxacin) was found to be a complete loss in ac-
tivity. Interestedly, some FloxeFlav hybrids acquired good inhibi-
tory activity against drug-resistance fungi, with four compounds (4,
The synthesis required only two steps from commercially
available flavonoids 1ae1e and floxacins 3ae3f. In dry DMF (N,N-
dimethylformamide), the 7-hydroxyl group of flavonoids 1ae1e
was selectively O-alkylated with 1,2-dibromoethane in the pres-
ence of K2CO3, giving the corresponding intermediates 2ae2e.
Subsequently, N-alkylation of appropriated floxacins with 2ae2e
affords FloxeFlavs 4e24 in a good yield by using DMAP (N,N-
dimethylpyridin-4-amine) as a base. The structures of 4e24 are
shown in Table 1.
5, 7 and 15) having MIC50 lower than 1
mg/mL (32.4 mg/mL for
positive control). Generally, fluoroquinolones selectively act on the
prokaryotic type II topoisomerase (DNA gyrase and topoisomerase
IV) [29] and are broad-spectrum antibacterial drugs. It is apparent
that currently available fluoroquinolones do not possess clinically
useful intrinsic antifungal activity, since they do not appear to have
significant antifungal topoisomerase activity [30]. Recently, how-
ever, S. Srinivasan et al. reported that some C-7 modified fluo-
roquinolones exhibit significant antifungal activity against
Cryptococcus neoformans [31]. In fact, several studies have
demonstrated that topoisomerases are possible targets for discov-
ering and developing fungicidal agents [32]. The results presented
herein strongly support that development of antifungals is possible
from fluoroquinolones modified at C-7 position. As for Gram-
positive bacteria, B. subtilis strain is more sensitive to FloxeFlavs
than S. aureus strain. The most active compound (7) displayed over
43-fold more potent than the clinically used antibiotics ciproflox-
acin against B. subtilis, whereas only 23-fold for S. aureus. The
excellent activity of 7 against the test organism suggested that it
may deserve further research or serve as a lead molecule for new
antibiotic drug discoveries. In the same time, this study provides a
new window for screening of new libraries of quinolone derivatives
to find antibiotics against drug-resistant microorganisms.
2.2. Antibacterial activity
The antibacterial efficacies of the obtained FloxeFlav hybrids in
comparison to ciprofloxacin were tested against drug-resistant
microorganisms, including one Gram (ꢀ), Escherichia coli, two
Gram (þ), Staphylococcus aureus and Bacillus subtilis, and one fun-
gus, Candida albicans. The results are presented in Table 2. Data for
selected pathogenic bacteria strains are reported as MIC50 (minimal
inhibitory concentration required to inhibit the growth of 50% of
organisms) values. As can be deduced from the MIC data, many of
the synthesized FloxeFlavs exhibited significant antibacterial ac-
tivity. In comparison with the positive control, improvements were
observed in 13 cases (4e8, 10e13, 15e16, 19, 24) against C. albicans
with a maximum of 88-fold increase in activity, 8 cases (4e8, 11, 13,
15) against B. subtilis with 43-fold increase in maximum, 3 cases (5,
7, 15) against S. aureus with 23-fold increase in maximum, and only
one case (7) against E. coli with about 8-fold increase. On the whole,
compound 7 was the most active derivative, showing 8, 43, 23 and
88 times better activities than ciprofloxacin against E. coli ATCC
35218, B. subtilis ATCC 6633, S. aureus ATCC 25923 and C. albicans
ATCC 90873, respectively.
In the norfloxacin series, it appears that the planarity and ri-
gidity of the flavonoid moiety has significantly influence on anti-
bacterial activity, which is illustrated by comparing the
antibacterial potency against individual strains of naringenin (4)
with that of apigenin (9). Taking apigenin (9) as a starting point,
movement of the B-ring from position 2 to 3 yields compound 12,
showing a slight to moderate increase in activity, whereas the
removal of the hydroxyl group in the 40-position resulted in a
reduction in potency (17). Substituents at 5- and 40-position were
important, which was evidenced by the 20-fold difference in MIC
between tectorigenin (12) and formononetin (23). Similar struc-
tureeactivity relationships (SAR) are observed in other floxacin
series.
2.3. Accumulation of ciprofloxacin, 5 and 7 in whole cells
Unusual high MIC50 of ciprofloxacin against methicillin-
resistant S. aureus (6.82
mg/mL) may be caused by the strain
efflux pumping ciprofloxacin with very high level (rate of
efflux ¼ 46.7%). On the contrary, accumulation of compound 5 or 7
in cells is fairly close to that in the presence of CCCP, with rate of
efflux being about 10% (Table 3). The most straightforward inter-
pretation of the present data is that introduction of a flavonoid
naringenin to the structure of a fluoroquinolone probably provides
the hybrids some inhibitory activity against efflux pump, or at least
prevents them from being the substrate of the efflux pump. These
findings suggest that covalently binding an antibiotic with an efflux
As for hybrids deriving from naringenin, compound containing
ciprofloxacin moiety (7) showed MIC value of 0.71, 0.062, 0.29 and
Scheme 4. The synthetic route for other FloxeFlavs.

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 80 (2014) 92e100
95
Table 1
Structures of the synthetic compounds.
Compound
Structure
R1
e
R2
e
R3
e
R4
H
R5
e
R6
R7
e
X
4
5
eEt
CH
CH
e
e
e
H
e
e
6
7
8
e
e
e
e
e
e
e
e
e
eMe
H
e
e
e
eEt
e
e
e
CF
CH
N
H
eEt
eEt
9
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
CH
CH
CH
CH
CH
CF
10
11
12
13
14
15
16
17
18
19
20
21
22
23
H
H
H
H
H
H
H
H
H
H
H
H
H
eEt
eEt
H
H
H
H
eMe
H
H
H
H
H
CH
N
H
H
eEt
eEt
eEt
H
H
H
H
H
H
H
H
H
H
CH
CF
eMe
H
H
H
H
H
CH
N
H
H
eEt
H
eMe
H
eMe
H
NH2
H
CF
CH
CH
H
H
H
H
eEt
H
24
H
H
H
H
CH
pump inhibitor would probably develop into a weapon against
resistant strains.
show that the hybrid compounds displayed greater inhibitory ac-
tivities than ciprofloxacin against the DNA gyrase, confirming their
strong fluoroquinolone character. These data, together with the
above mentioned inhibitory activity against efflux pump, clearly
confirm our design strategy of the FloxeFlav hybrids and the
desired dual mode of action. Notably, when compared with cipro-
floxacin, 7 with the rate of efflux of 9.8% showed an over 23-fold
increase in inhibition of DNA gyrase activity. Its potent inhibition
of both the target enzyme and efflux pump probably accounts for
2.4. DNA supercoiling activity
To investigate the possibility of a dual mode of action, the effect
of FloxeFlavs on the fluoroquinolone targets gyrase was also tested
by assaying 5, 7 and positive control ciprofloxacin against the en-
zymes isolated from E. coli (Fig. 1 and Table 4). The observed data

96
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 80 (2014) 92e100
0
ꢀ
Table 2
bonding interaction (3.440 A) was observed between 3 -H and Arg-
471. The current conformation of 7 is further fixed by the Ce
H$$$O(N) contacts between the linker (methylene group) and the
back bone residues (Glu-477, Asn-476 and DC-13). Moreover, this
linker also receives a hydrophobic interaction from Asn-476. In
comparison to gyrase-DNAeciprofloxacin complex, these newly
additional interactions may give a rational explanation for the
excellent antibacterial activity of compound 7.
Inhibitory activity (MIC50) of the synthetic compounds against microbes.
Compound
MIC50
A
(mg/mL)
B
C
D
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
28.8
17.47
>50
0.71
46.3
>50
>50
39.7
40.5
37.4
>50
26.7
>50
>50
>50
41.5
>50
>50
>50
>50
>50
5.65
0.35
0.22
1.4
7.14
0.32
28.4
0.29
21.5
26.4
14.4
8.84
19.3
9.65
>50
1.98
33.6
45.6
>50
26.6
>50
>50
28.5
34.1
19.8
6.82
0.96
0.76
4.73
0.14
2.60
35.3
24.0
9.37
8.76
4.05
48.5
0.89
17.5
>50
>50
15.9
>50
>50
43.2
42.5
30.5
32.4
0.062
0.45
22.9
14.0
2.64
3.33
1.88
35.7
1.64
16.1
41.9
>50
4.43
>50
>50
26.7
32.8
17.8
2.70
3. Conclusions
In a continuing study of our antibacterial drug development
program, we use a rationally conceived pharmacophore model to
build a multi-target bacterial topoisomerase inhibitor. This led to
the identification of some FloxeFlav hybrids with excellent anti-
bacterial activity against drug-resistant microorganisms. In general,
a fluoroquinolone hybridized with different flavonoids generalized
the hybrid with narigenin moiety being the most active. However,
in hybrids derived from a flavonoid, the compound with the cip-
rofloxacin moiety is the most potent. Among the obtained hybrids,
compound 7 is the most potent and shows 8- to 88-fold more
potent than the positive control ciprofloxacin, which was evi-
denced by the molecular docking studies. Drug accumulation and
DNA supercoiling assays of two active analogues (5 and 7) revealed
the desired dual mode of action, which could account for the
excellent antibacterial activity against resistant strains and may
suggest that a resistance development will be slow. These findings
indicate that covalently binding an antibiotic with an efflux pump
inhibitor would probably develop into a weapon against resistant
strains.
20
21
22
23
24
Ciprofloxacin
A, Multiple drug resistant Escherichia coli ATCC 35218; B, Tetracycline-resistant
Bacillus subtilis ATCC 6633; C, Methicillin-resistant Staphylococcus aureus ATCC
25923; D, amphotericin B-resistant Candida albicans ATCC 90873.
the excellent antibacterial activity against resistant strains and
suggests that resistance development may be slow to emerge
among clinical isolates.
4. Experimental section
2.5. Molecular docking
4.1. Antimicrobial activity
In order to give an explanation and understanding of the
excellent activity observed, the most potent inhibitor 7 was docked
into the ciprofloxacin binding site of gyrase from S. aureus based on
the gyrase-DNAeciprofloxacin complex structure (2xct.pdb) [33].
The binding model of compound 7 and gyrase-DNA is depicted in
Fig. 2 and the enzyme surface model is shown in Fig. 3, which
revealed that compound 7 is well filled in the ciprofloxacin binding
pocket. Ciprofloxacin moiety of 7 in our binding model nearly takes
the same pose with that observed in the X-ray structure of gyrase-
The antibacterial activities of the synthesized compounds were
tested against two Gram-positive bacterial strains (B. subtilis ATCC
6633, S. aureus ATCC 25923), one Gram-negative bacterial strain
(E. coli ATCC 35218) and one fungus (C. albicans ATCC 90873). The
MIC50s of the test compounds were determined by a colorimetric
method using the dye MTT [34e36], as described previously. The
observed MIC50s were presented in Table 2.
DNAeciprofloxacin (Fig. 3), and forms one classical hydrogen bonds
4.2. Analysis of fluoroquinolone accumulation
ꢀ
with DC-12 (2.838 A), two
p$$$p contacts between quinolinone
ꢀ
ring in ligand and pyrimidine rings in DG-9 (3.495 A) and DG-8
Fluoroquinolone accumulation in bacterial cells (S. aureus) was
determined from five independent experiments using the modified
fluorescence method described by H. Nakaminami et al. [37]. The
strains were grown in lysogeny broth (LB) to an optical density at
600 nm (OD600) of 0.6. The cells were then collected by centrifu-
gation at 6000 ꢁ g for 5 min and washed thrice with 20 mM HEPES
(pH 7.0). The cell pellets were resuspended in HEPES with final
concentration of 40 mg/mL. Subsequently, the selected fluo-
ꢀ
(4.303 A), and three nonclassical hydrogen bonds with DG-9
ꢀ
ꢀ
ꢀ
(2.383 A), Arg-458 (2.838 A) and Gly-459 (2.162 A) (Fig. 3). Addi-
tionally, 4-carbonyl and 3-carboxyl groups in ciprofloxacin moiety
bidentically anchor Mn2þ as they do in ciprofloxacin with distances
ꢀ
of 1.956 and 2.055 A. This result revealed that the introduced nar-
igenin moiety does not disturb the binding mode of the pharma-
cophore (ciprofloxacin moiety). Furthermore, the narigenin moiety
provides an additional affinity to the enzyme by several hydrogen
bonding interactions. Briefly, narigenin moiety using 5- and 40-OH
as donors forms two hydrogen bonding interactions with DC-12
roquinolone derivatives (10 mg/mL) were added to the assay mix-
tures. After incubation for 5 min at 37 ꢂC, 1 mL samples were
diluted with an equal volume of ice-cold HEPES and centrifuged at
16,000 ꢁ g for 2 min at 4 ꢂC. The resulted pellets were washed once
with 2 mL of ice-cold HEPES and resuspended in 1 mL of 100 mM
glycineeHCl (pH 3.0). The suspensions were agitated vigorously for
ꢀ
ꢀ
(3.208 A) and Arg-471 (3.291 A), respectively. It can be seen from
this that the removal (replacement) of 5- or 40-OH may be
responsible for the decrease in the activity observed in antibacterial
assay (9 vs 17, 11 vs 19, 12 vs 23 and 13 vs 24). A strong CeH$$$O
4
h at room temperature to release the intracellular fluo-
ꢀ
hydrogen bonding interaction with a distance of 2.303 A occurs
roquinolone. The samples were centrifuged at 16,000 ꢁ g for 5 min
at room temperature. The fluorescence in the supernatant was
measured with a spectrofluorometer (Jasco, Japan) using the
appropriated excitation and emission maxima. The intracellular
accumulation of the antibiotic in the samples was calculated by
between 2-H and the back bone PO of DC-13, which provides an
evidence for the significant decrease in potency of apigenineflox-
acins (9, 10 and 11) in comparison with naringenin analogues (4, 5
and 7). On the other hand, a relatively weak CeH$$$O hydrogen

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 80 (2014) 92e100
97
Table 3
clustered together and the results of the most favorable free energy
of binding were selected as the resultant complex structures.
Accumulation of selected hybrid compound by S. aureus.
Compound
Accumulatedtest
(ng/mg)
Accumulatedcontrol
(ng/mg)
Rate of efflux (%)
4.5. Statistical analysis
5
7
54.51
59.30
31.56
63.09
65.74
59.21
13.6
9.8
46.7
MIC50 and IC50 values were obtained by fitting the data in
nonlinear regression, and IC50s were reported as mean ꢃ SD of at
least 3 independent experiments. Statistical analysis was per-
formed using SPSS 16.0 software.
Ciprofloxacin
comparison with standard curves and is given in nanograms per
milligram (ng/mg) of bacteria. Carbonyl cyanide m-chlor-
ophenylhydrazone (CCCP, Sigma) with a final concentration of
4.6. Chemistry
100 mM was added for the positive control. The rates of efflux are
presented in Table 3, which were calculated based on the following
formula:
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 (200e300 mesh ASTM, E. Merck). The quantity of
silica gel used was 30e70 times the weight charged on the column.
Then, the eluates were monitored using TLC. Melting points (un-
corrected) were determined on a XT4 MP apparatus (Taike Corp.,
Beijing, China). ESI mass spectra were obtained on a Mariner Sys-
tem 5304 mass spectrometer, and 1H NMR and 13C NMR spectra
were recorded on a Bruker DPX400 or DPX300 spectrometer at
25 ꢂC with TMS and solvent signals allotted as internal standards.
accumulatedcontrolꢀaccumulatedtest
Rate of efflux ¼
ꢁ 100
accumulatedcontrol
4.3. DNA gyrase assays
DNA supercoiling activity was assayed with relaxed pBR322
Chemical shifts were reported in ppm (d). Elemental analyses were
DNA (Inspiralis Ltd) as a substrate according to the manufacturer’s
protocol. The standard reaction mixture (20 mL) contained 35 mM
performed on a CHNeO-Rapid instrument and were within ꢃ0.4%
of the theoretical values.
TriseHCl (pH 7.5), 24 mM KCl, 4 mM MgCl2, 2 mM dithiothreitol,
1.8 mM spermidine, 1 mM ATP, 6.5% glycerol, 0.1 mg/mL bovine
serum albumin (BSA), 12.5 ng/mL relaxed pBR322, and 5 U of DNA
4.6.1. General procedure for preparation of compounds 2ae2e
A flavonoid 1 (5 mmol) was dissolved in 30 mL of DMF, followed
by addition of 1,2-dibromoethane (4.3 mL, 50 mmol) and K2CO3
(20 mmol). The resulted mixture was stirred at 70 ꢂC for 10e15 h.
Three times volume of ice-cooled water was added with vigorously
gyrase (New England BioLabs). The reaction mixture was incubated
at 37 ꢂC for 40 min with the selected compound, and then was
terminated by addition of a loading dye (3.5 mL) and 20 mL chlo-
roform/isoamyl alcohol (24:1). After a brief vortex, the blue aqua
phase was analyzed by electrophoresis in 1% agarose. One unit of
supercoiling activity was defined as the amount of DNA gyrase
required to supercoil 0.5 mg of plasmid in 1 h. The IC50 was defined
as the drug concentration that reduced the enzymatic activity
observed with drug-free controls by 50% (Table 4).
4.4. Protocol of docking study
The automated docking studies were carried out using Auto-
Dock version 4.2. Arg458 and Dc12 were selected as flexible resi-
dues. 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. The three-
dimensional structure of compound 7 was constructed using
Chem3D ultra 11.0 software [Chemical Structure Drawing Standard;
Cambridge Soft corporation, USA (2009)], then it was energetically
minimized by using MOPAC with 100 iterations and minimum RMS
gradient of 0.10. The GasteigereHückel charge of the ligand was
ꢀ
assigned. A grid box of 60 ꢁ 60 ꢁ 60 A size (x, y, z) with a spacing of
ꢀ
0.375 A 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 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 ADT (AutoDockTools package, version 1.5.4) on PC which
is associated with AutoDock 4.2. Some default settings were
changed with an initial population of 50 randomly placed in-
dividuals, a maximum number of 3.0 ꢁ 106 energy evaluations, and
a maximum number of 3 ꢁ 104 generations. Results differing by less
Fig. 1. Inhibition of DNA gyrase (supercoiling) by compound 7 (A) and ciprofloxacin
(B). Lane 1, relaxed plasmid pBR322 alone; lane 2, relaxed pBR322 plus 5 U of gyrase;
lanes 3 to 8, 6 U of gyrase and 0.024, 0.098, 0.391, 1.563, 6.250, and 25.000 mg/mL
compound 7 or ciprofloxacin. nc and sc represent nicked circular and supercoiled
ꢀ
than 0.5 A in positional root-mean-square deviation (RMSD) were
forms, respectively.

98
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 80 (2014) 92e100
Table 4
4.6.2.2. Naringenin-ethylidene-sarafloxacin (5). Light yellow pow-
der, 60%, mp 170e172 ꢂC, 1H NMR (DMSO-d6): 2.59 (bs, 4H); 2.70
(bs, 2H); 3.05 (bs, 4H); 3.26e3.36 (m, 2H); 4.13 (bs, 2H); 5.48 (dd,
1H); 6.07 (d, J ¼ 2.2 Hz, 1H); 6.09 (d, J ¼ 2.2 Hz, 1H); 6.38 (d,
J ¼ 7.3 Hz, 1H); 6.79 (d, J ¼ 8.6 Hz, 2H); 7.32 (d, J ¼ 8.6 Hz, 2H); 7.53
(t, J ¼ 8.8 Hz, 2H); 7.78 (dd, J ¼ 9.0 Hz, J ¼ 4.9 Hz, 2H); 7.97 (d,
J ¼ 13.2 Hz,1H); 8.64 (s, 1H); 12.09 (s, 1H); EIMS m/z 683 (Mþ). Anal.
Calcd for C37H31F2N3O8: C, 65.00; H, 4.57; F, 5.56; N, 6.15; Found: C,
65.09; H, 4.56; F, 5.54; N, 6.16.
Activity of selected compounds as inhibitors of DNA
gyrase from E. coli.
Compound
IC50 (mM)
5
7
1.68 ꢃ 0.14
0.21 ꢃ 0.01
5.23 ꢃ 0.45
Ciprofloxacin
stirring, which was acidified with HCl and extracted with EtOAc for
three times. The combined organic layer was washed with satu-
rated brine and then dried over MgSO4. Removal of solvent under
reduced pressure yield a light yellow powder. Flash chromatog-
raphy (petroleum ether/EtOAc, from 3/1 to 1/1) furnished 2 in good
yield.
4.6.2.3. Naringenin-ethylidene-lomefloxacin (6). Light yellow pow-
der, 42%, mp 150e152 ꢂC, 1H NMR (DMSO-d6): 1.06 (d, J ¼ 4.8 Hz,
2H); 1.43 (t, 3H); 2.53e2.60 (m, 2H); 2.60e2.75 (m, 4H); 2.90e3.10
(m, 4H); 4.16 (bs, 2H); 4.57 (bs, 2H); 5.49 (dd, 1H); 6.10 (s, 1H); 6.12
(s, 1H); 6.79 (d, J ¼ 8.4 Hz, 2H); 7.32 (t, J ¼ 8.6 Hz, 2H); 7.73 (d,
J ¼ 13.1 Hz, 1H); 8.92 (s, 1H); 9.59 (bs, 1H); 12.11 (s, 1H); EIMS m/z
649 (Mþ). Anal. Calcd for C34H33F2N3O8: C, 62.86; H, 5.12; F, 5.85; N,
6.47; Found: C, 62.77; H, 5.13; F, 5.85; N, 6.48.
4.6.2. General procedure for preparation of FloxeFlavs (4e24)
A mixture of compound 2 (0.5 mmol), an appropriated fluo-
roquinolone (1 mmol) and DMAP (1.5 mmol) was dissolved in
10 mL of dry DMSO, which was stirred at 60 ꢂC for 40e50 h. The
reaction was quenched with 8e10 times volume of water in an ice-
water bath, giving a light yellow precipitate. The precipitate was
collected and was subsequently purified by column chromatog-
raphy on silica gel, eluting with CHCl3/CH3OH (V:V, 10:1 to 17:1,
containing 2% of acetic acid).
4.6.2.4. Naringenin-ethylidene-ciprofloxacin (7). Light yellow pow-
der, 61%, mp 168e170 ꢂC, 1H NMR (DMSO-d6): 1.16e1.21 (m, 2H);
1.31 (q, J ¼ 6.8 Hz, 3H); 2.71 (bs, 4H); 2.79 (t, J ¼ 5.2 Hz, 2H); 2.29e
3.37 (m, 6H); 3.80e3.89 (m, 1H); 4.20 (t, J ¼ 5.6 Hz, 2H); 4.50 (dd,
J ¼ 12.8 Hz, J ¼ 2.8 Hz,1H); 6.11 (d, J ¼ 2.0 Hz,1H); 6.14 (d, J ¼ 2.4 Hz,
1H); 6.80 (d, J ¼ 8.4 Hz, 2H); 7.33 (d, J ¼ 8.4 Hz, 2H); 7.57 (d,
J ¼ 7.6 Hz, 1H); 7.91 (d, J ¼ 13.2 Hz, 1H); 8.67 (s, 1H); 9.60 (s, 1H);
12.12 (s, 1H); 15.23 (s, 1H); 13C NMR (DMSO-d6): 8.0, 21.6, 36.3, 42.5,
49.9, 53.0, 53.4, 56.5, 66.7, 77.1, 79.1, 94.7, 95.6, 103.1, 106.9, 111.3,
111.5, 115.6, 128.9, 129.1, 139.7, 148.5, 158.3, 163.4, 163.7, 166.5, 167.1,
172.6, 197.5; EIMS m/z 629 (Mþ). Anal. Calcd for C34H32FN3O8: C,
64.86; H, 5.12; F, 3.02; N, 6.67; Found: C, 64.92; H, 5.11; F, 3.02; N,
6.66.
4.6.2.1. Naringenin-ethylidene-norfloxacin (4). Light yellow pow-
der, 72%, mp 234e236 ꢂC, 1H NMR (DMSO-d6): 1.41 (t, 3H); 2.69 (bs,
4H); 2.77 (bs, 4H); 3.20e3.40 (m, 4H); 4.19 (bs, 2H); 4.50e4.65 (m,
2H); 5.50 (dd, 1H); 6.11 (s, 1H); 6.13 (s, 1H); 6.79 (d, J ¼ 8.4 Hz, 2H);
7.12 (d, 1H); 7.33 (d, J ¼ 8.6 Hz, 2H); 7.90 (d, J ¼ 13.4 Hz, 1H); 8.94 (s,
1H); 12.11 (bs,1H); EIMS m/z 617 (Mþ). Anal. Calcd for C33H32FN3O8:
C, 64.17; H, 5.22; F, 3.08; N, 6.80; Found: C, 64.22; H, 5.21; F, 3.08; N,
6.79.
4.6.2.5. Naringenin-ethylidene-enoxacin (8). Light yellow powder,
70%, mp 242e244 ꢂC, 1H NMR (DMSO-d6): 1.39 (t, J ¼ 7.2 Hz, 3H);
2.66 (t, J ¼ 4.4 Hz, 4H); 2.76 (t, J ¼ 5.2 Hz, 2H); 3.83 (t, J ¼ 4.0 Hz,
4H); 4.19 (t, J ¼ 5.6 Hz, 2H); 4.49 (q, J ¼ 6.8 Hz, 2H); 5.49 (dd,
J ¼ 12.8 Hz, J ¼ 2.8 Hz,1H); 6.10 (d, J ¼ 2.4 Hz,1H); 6.13 (d, J ¼ 2.4 Hz,
1H); 6.80 (d, J ¼ 8.8 Hz, 2H); 7.33 (d, J ¼ 8.4 Hz, 2H); 8.09 (d,
J ¼ 13.6 Hz, 1H); 8.98 (s, 1H); 9.60 (s, 1H); 12.11 (s, 1H); 15.33 (s, 1H);
13C NMR (DMSO-d6): 15.2, 42.5, 47.1, 47.2, 47.7, 53.1, 56.4, 66.6, 79.1,
94.7, 95.6, 103.1, 108.5, 113.1, 115.6, 119.8, 120.0, 128.9, 145.3, 148.2,
150.3, 158.3, 163.3, 163.6, 166.4, 167.0, 176.8, 197.5; EIMS m/z 629
(Mþ). Anal. Calcd for C34H32FN3O8: C, 64.86; H, 5.12; F, 3.02; N, 6.67;
Found: C, 64.92; H, 5.11; F, 3.02; N, 6.66.
4.6.2.6. Apigenin-ethylidene-norfloxacin (9). White powder, 53%,
mp 250e252 ꢂC, 1H NMR (DMSO-d6): 1.41 (t, 3H); 2.73 (bs, 4H);
2.83 (bs, 2H); 3.20e3.40 (bs, 4H); 4.28 (bs, 2H); 4.50e4.60 (m, 2H);
6.44 (s, 1H); 6.71 (s, 1H); 6.83 (d, J ¼ 8.4 Hz, 2H); 7.17 (d, J ¼ 4.4 Hz,
1H); 7.39 (d, J ¼ 8.4 Hz, 2H); 7.90 (d, J ¼ 13.2 Hz, 1H); 8.42 (s, 1H);
8.93 (s, 1H); 12.97 (s, 1H); 15.35 (bs, 1H); EIMS m/z 615 (Mþ). Anal.
Calcd for C33H30FN3O8: C, 64.38; H, 4.91; F, 3.09; N, 6.83; Found: C,
64.32; H, 4.92; F, 3.09; N, 6.84.
4.6.2.7. Apigenin-ethylidene-sarafloxacin (10). White powder, 69%,
mp 177e179 ꢂC, 1H NMR (DMSO-d6): 2.63 (bs, 4H); 2.76 (t,
J ¼ 5.2 Hz, 2H); 3.07 (bs, 4H); 4.21 (t, J ¼ 5.2 Hz, 2H); 6.40 (d,
J ¼ 8.0 Hz, 1H); 6.41 (d, J ¼ 2.4 Hz, 1H); 6.67 (d, J ¼ 2.4 Hz, 1H); 6.83
(d, J ¼ 8.4 Hz, 2H); 7.40 (d, J ¼ 8.8 Hz, 2H); 7.54 (t, J ¼ 8.4 Hz, 2H);
7.79 (dd, J ¼ 8.8 Hz, J ¼ 4.8 Hz, 2H); 7.99 (d, J ¼ 13.2 Hz, 1H); 8.41 (s,
1H); 8.65 (s, 1H); 9.60 (s, 1H); 12.95 (s, 1H); 15.14 (bs, 1H); EIMS m/z
681 (Mþ). Anal. Calcd for C37H29F2N3O8: C, 65.20; H, 4.29; F, 5.57; N,
6.16; Found: C, 65.26; H, 4.29; F, 5.56; N, 6.15.
Fig. 2. Binding modes of compound 7 with gyrase from S. aureuseDNAeciprofloxacin
complex. The enzyme complex is shown as surface; while 7 (cyan) and ciprofloxacin
(orange) are shown as sticks. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 80 (2014) 92e100
99
4.6.2.12. Genistin-ethylidene-ciprofloxacin (15). White powder,
64%, decomposed at 228 ꢂC, 1H NMR (DMSO-d6): 1.19 (bs, 2H); 1.30
(bs, 2H); 2.74 (bs, 4H); 2.84 (bs, 2H); 3.33 (bs, 4H); 3.83 (bs, 1H);
4.28 (bs, 2H); 6.45 (s, 1H); 6.71 (s, 1H); 6.83 (d, J ¼ 8.4 Hz, 2H); 7.39
(d, J ¼ 8.4 Hz, 2H); 7.58 (d, J ¼ 8.0 Hz, 1H); 7.92 (d, J ¼ 13.3 Hz, 1H);
8.43 (s, 1H); 8.67 (s, 1H); 9.61 (s, 1H); 12.97 (s, 1H); 15.23 (s, 1H);
EIMS m/z 627 (Mþ). Anal. Calcd for C34H30FN3O8: C, 65.07; H, 4.82;
F, 3.03; N, 6.70; Found: C, 65.12; H, 4.81; F, 3.03; N, 6.69.
4.6.2.13. Genistin-ethylidene-enoxacin (16). White powder, 75%,
decomposed at 208 ꢂC, 1H NMR (DMSO-d6): 1.39 (t, J ¼ 7.0 Hz, 3H);
2.69 (bs, 4H); 2.80 (bs, 2H); 3.85 (bs, 4H); 4.26 (bs, 2H); 4.49 (q,
J ¼ 6.8 Hz, 2H); 6.44 (s,1H); 6.70 (s,1H); 6.82 (d, J ¼ 8.4 Hz, 2H); 7.39
(d, J ¼ 8.6 Hz, 2H); 8.09 (d, J ¼ 13.7 Hz,1H); 8.42 (s, 1H); 8.99 (s, 1H);
9.61 (s, 1H); 12.96 (s, 1H); 15.34 (s, 1H); EIMS m/z 616 (Mþ). Anal.
Calcd for C32H29FN4O8: C, 62.33; H, 4.74; F, 3.08; N, 9.09; Found: C,
62.39; H, 4.71; F, 3.09; N, 9.10.
4.6.2.14. Chrysin-ethylidene-norfloxacin (17). Light yellow powder,
37%, mp 223e225 ꢂC, 1H NMR (DMSO-d6): 1.42 (t, J ¼ 7.0 Hz, 3H);
2.74 (bs, 4H); 2.85 (bs, 2H); 3.35 (bs, 4H); 4.30 (bs, 2H); 4.59 (q,
J ¼ 6.8 Hz, 2H); 6.44 (s, 1H); 6.88 (s, 1H); 7.05 (s, 1H); 7.19 (d,
J ¼ 7.1 Hz, 1H); 7.60 (d, J ¼ 7.3 Hz, 2H); 7.62 (s, 1H); 7.93 (d,
J ¼ 13.2 Hz, 1H); 8.11 (d, J ¼ 6.8 Hz, 2H); 8.95 (s, 1H); 12.81 (s, 1H);
15.34 (s, 1H); EIMS m/z 599 (Mþ). Anal. Calcd for C33H30FN3O7: C,
66.10; H, 5.04; F, 3.17; N, 7.01; Found: C, 66.18; H, 5.03; F, 3.17; N,
7.69.
Fig. 3. Binding modes of compound 7 with gyrase. The enzyme complex is shown as
cartoon; while 7 is shown as sticks.
4.6.2.8. Apigenin-ethylidene-ciprofloxacin (11). White powder, 58%,
mp 250e252 ꢂC, 1H NMR (DMSO-d6): 1.19 (bs, 2H); 1.29e1.34 (m,
2H); 2.75 (bs, 4H); 2.84 (t, J ¼ 5.2 Hz, 2H); 3.36 (bs, 4H); 3.81e3.85
(m, 1H); 4.28 (t, J ¼ 5.6 Hz, 2H); 6.45 (d, J ¼ 2.4 Hz, 1H); 6.71 (d,
J ¼ 2.4 Hz, 1H); 6.83 (d, J ¼ 8.8 Hz, 2H); 7.40 (d, J ¼ 8.4 Hz, 2H); 7.58
(d, J ¼ 7.2 Hz, 1H); 7.91 (d, J ¼ 13.2 Hz, 1H); 8.42 (s, 1H); 8.67 (s, 1H);
9.60 (s, 1H); 12.96 (s, 1H); 15.23 (s, 1H); 13C NMR (DMSO-d6): 6.7,
8.0, 36.4, 42.1, 49.9, 53.1, 56.6, 59.0, 60.2, 66.9, 93.4, 98.9, 105.9,
106.91, 106.94, 107.2, 115.6, 121.5, 123.0, 123.8, 130.7, 141.6, 148.5,
155.0, 162.2, 164.9, 166.5, 180.9; EIMS m/z 627 (Mþ). Anal. Calcd for
4.6.2.15. Chrysin-ethylidene-lomefloxacin (18). Light yellow pow-
der, 49%, mp 251e253 ꢂC, 1H NMR (DMSO-d6): 1.10 (d, J ¼ 6.1 Hz,
3H); 1.43 (t, J ¼ 6.2 Hz, 3H); 2.55e2.85 (m, 4H); 2.96e3.18 (m, 3H);
3.34 (bs, 2H); 4.26 (bs, 2H); 4.59 (bs, 2H); 6.44 (s, 1H); 6.89 (s, 1H);
7.06 (s, 1H); 7.55e7.67 (m, 3H); 7.86 (d, J ¼ 11.9 Hz, 1H); 8.12 (d,
J ¼ 7.3 Hz, 2H); 8.93 (s, 1H); 12.81 (s, 1H); 14.93 (s, 1H); EIMS m/z
631 (Mþ). Anal. Calcd for C34H31F2N3O7: C, 64.65; H, 4.95; F, 6.02; N,
6.65; Found: C, 64.57; H, 4.96; F, 6.03; N, 6.66.
C
34H30FN3O8: C, 65.07; H, 4.82; F, 3.03; N, 6.70; Found: C, 64.98; H,
4.83; F, 3.03; N, 6.72.
4.6.2.16. Chrysin-ethylidene-ciprofloxacin (19). Light yellow pow-
der, 64%, mp 244e245 ꢂC, 1H NMR (DMSO-d6): 1.12e1.22 (m, 2H);
1.28e1.36 (m, 2H); 2.75 (bs, 4H); 2.85 (bs, 2H); 3.75e3.88 (m, 1H);
4.41 (bs, 2H); 6.45 (s, 1H); 6.89 (s, 1H); 7.06 (s, 1H); 7.53e7.68 (m,
4H); 7.91 (d, J ¼ 13.4 Hz, 1H); 8.11 (d, J ¼ 6.2 Hz, 2H); 8.67 (s, 1H);
12.82 (s, 1H); 15.23 (s, 1H); EIMS m/z 611 (Mþ). Anal. Calcd for
4.6.2.9. Genistin-ethylidene-norfloxacin (12). White powder, 54%,
mp 246e248 ꢂC, 1H NMR (DMSO-d6): 1.41 (t, J ¼ 6.9 Hz, 3H); 2.73
(bs, 4H); 2.83 (bs, 2H); 4.27 (bs, 2H); 4.59 (d, J ¼ 6.9 Hz, 2H); 6.44 (d,
J ¼ 2.0 Hz, 1H); 6.70 (d, J ¼ 1.8 Hz, 1H); 6.82 (d, J ¼ 8.6 Hz, 2H); 7.18
(d, J ¼ 7.3 Hz, 1H); 7.39 (d, J ¼ 8.6 Hz, 2H); 7.92 (d, J ¼ 13.4 Hz, 1H);
8.42 (s, 1H); 8.95 (s, 1H); 9.61 (s, 1H); 12.96 (s, 1H); 15.36 (s, 1H);
EIMS m/z 615 (Mþ). Anal. Calcd for C33H30FN3O8: C, 64.38; H, 4.91; F,
3.09; N, 6.83; Found: C, 64.32; H, 4.92; F, 3.10; N, 6.84.
C34H30FN3O7: C, 66.77; H, 4.94; F, 3.11; N, 6.87; Found: C, 66.82; H,
4.93; F, 3.11; N, 6.86.
4.6.2.17. Chrysin-ethylidene-enoxacin (20). Light yellow powder,
57%, mp 260e262 ꢂC, 1H NMR (DMSO-d6): 1.39 (t, 3H); 2.70 (bs,
4H); 2.82 (bs, 2H); 3.85 (bs, 4H); 4.28 (bs, 2H); 4.48 (q, 2H); 6.44 (s,
1H); 6.89 (s,1H); 7.06 (s,1H); 7.55e7.69 (m, 3H); 8.04e8.18 (m, 3H);
8.32 (s, 1H); 8.99 (s, 1H); 12.81 (s, 1H); 15.34 (s, 1H); EIMS m/z 600
(Mþ). Anal. Calcd for C32H29FN4O7: C, 63.99; H, 4.87; F, 3.16; N, 9.33;
Found: C, 63.91; H, 4.88; F, 3.15; N, 9.34.
4.6.2.10. Genistin-ethylidene-sarafloxacin (13). White powder, 58%,
mp 179e181 ꢂC, 1H NMR (DMSO-d6): 2.62 (bs, 4H); 2.75 (bs, 2H);
3.07 (bs, 4H); 4.20 (bs, 2H); 6.39 (d, J ¼ 6.9 Hz,1H); 6.40 (s,1H); 6.66
(s, 2H); 6.82 (d, J ¼ 8.6 Hz, 2H); 7.39 (d, J ¼ 8.6 Hz, 2H); 7.54 (t,
J ¼ 8.8 Hz, 2H); 7.76e7.82 (m, 2H); 7.99 (d, J ¼ 13.2 Hz, 1H); 8.41 (s,
1H); 8.65 (s, 1H); 9.62 (s, 1H); 12.95 (s, 1H); 15.10 (s, 1H); EIMS m/z
681 (Mþ). Anal. Calcd for C37H29F2N3O8: C, 65.20; H, 4.29; F, 5.57; N,
6.16; Found: C, 65.14; H, 4.30; F, 5.58; N, 6.18.
4.6.2.18. Chrysin-ethylidene-sparfloxacin (21). Light yellow powder,
36%, mp 223e225 ꢂC, 1H NMR (DMSO-d6): 1.00e1.19 (m, 10H);
2.72e2.97 (m, 4H); 3.05e3.20 (m, 3H); 3.95e4.05 (m, 2H); 4.18 (bs,
2H); 6.40 (s, 1H); 6.88 (s, 1H); 7.04 (s, 1H); 7.24 (bs, 2H); 7.55e7.70
(m, 3H); 8.11 (d, J ¼ 6.4 Hz, 2H); 8.49 (s, 1H); 12.80 (s, 1H); 14.68 (s,
1H); EIMS m/z 672 (Mþ). Anal. Calcd for C36H34F2N4O7: C, 64.28; H,
5.09; F, 5.65; N, 8.33; Found: C, 64.35; H, 5.09; F, 5.64; N, 8.32.
4.6.2.11. Genistin-ethylidene-lomefloxacin (14). White powder, 58%,
mp 179e181 ꢂC, 1H NMR (DMSO-d6): 1.09 (d, J ¼ 6.1 Hz, 3H); 1.43 (t,
J ¼ 6.6 Hz, 3H); 2.56e2.88 (m, 4H); 2.96e3.18 (m, 3H); 3.62 (bs, 2H);
4.26 (bs, 2H); 4.59 (bs, 2H); 6.44 (s, 1H); 6.60 (s, 1H); 6.82 (d,
J ¼ 8.4 Hz, 2H); 7.39 (d, J ¼ 8.6 Hz, 2H); 8.10 (d, J ¼ 13.9 Hz, 1H); 8.39
(s,1H); 8.93 (s,1H); 9.52 (s,1H); 12.80 (s, 1H); 15.09 (s, 1H); EIMS m/
z 647 (Mþ). Anal. Calcd for C34H31F2N3O8: C, 63.06; H, 4.82; F, 5.87;
N, 6.49; Found: C, 63.57; H, 4.82; F, 5.88; N, 6.50.
4.6.2.19. Chrysin-ethylidene-sarafloxacin (22). Light yellow powder,
40%, mp 200e202 ꢂC, 1H NMR (DMSO-d6): 2.63 (bs, 4H); 2.77 (bs,
2H); 3.07 (bs, 4H); 4.23 (bs, 2H); 4.18 (bs, 2H); 6.40 (s þ t, 2H); 6.88

100
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 80 (2014) 92e100
[9] E.B. Breidenstein, C. de la Fuente-Núñez, R.E. Hancock, Trends in Microbiology
19 (2011) 419e426.
[10] A.K. Bhardwaj, P. Mohanty, Recent Patents on Anti-infective Drug Discovery 7
(2012) 73e89.
[11] F. Perez-Vizcaino, J. Duarte, C. Santos-Buelga, Journal of the Science of Food
and Agriculture 92 (2012) 1822e1825.
[12] S.J. Hosseinimehr, Drug Discovery Today 15 (2010) 907e918.
[13] V.H. Tran, D. Marks, R.K. Duke, M. Bebawy, C.C. Duke, B.D. Roufogalis, Nutri-
tion and Cancer 63 (2011) 435e443.
[14] I.L.K. Wong, K.F. Chan, B.A. Burkett, Y. Zhao, Y. Chai, H. Sun, T.H. Chan,
L.M.C. Chow, Antimicrobial Agents and Chemotherapy 51 (2007) 930e940.
[15] G.L. Maia, V.S. Falcão-Silva, P.G. Aquino, J.X. de Araújo-Júnior, J.F. Tavares,
M.S. da Silva, L.C. Rodrigues, J.P. de Siqueira-Júnior, J.M. Barbosa-Filho, Mol-
ecules 16 (2011) 4828e4835.
[16] J.R. Bame, T.N. Graf, H.A. Junio, R.O. Bussey 3rd, S.A. Jarmusch, T. El-Elimat,
J.O. 3rd Falkinham, N.H. Oberlies, R.A. Cech, N.B. Cech, Planta Medica 79 (2013)
327e329.
[17] I.L.K. Wong, K.-F. Chan, K.H. Tsang, C.Y. Lam, Y. Zhao, T.H. Chan, L.M. Chow,
Journal of Medicinal Chemistry 52 (2009) 5311e5322.
[18] Y.Q. Han, L.J. Cao, J. Wu, H.J. Hao, Y.J. Shi, G.B. Xu, H.Y. Ren, African Journal of
Biotechnology 10 (2011) 13399e13406.
(s, 1H); 6.85 (s, 1H); 7.06 (s, 1H); 7.45e7.65 (m, 5H); 7.78 (bs, 3H);
7.98 (d, J ¼ 13.4 Hz, 1H); 8.10 (d, 2H); 8.65 (s, 1H); 12.80 (s, 1H);
15.14 (s, 1H); EIMS m/z 665 (Mþ). Anal. Calcd for C37H29F2N3O7: C,
66.76; H, 4.39; F, 5.71; N, 6.31; Found: C, 66.70; H, 4.40; F, 5.72; N,
6.30.
4.6.2.20. Formononetin-ethylidene-norfloxacin (23). White powder,
69%, mp 224e226 ꢂC, 1H NMR (DMSO-d6): 1.40 (t, J ¼ 7.2 Hz, 3H);
2.73 (bs, 4H); 2.85 (bs, 2H); 3.34 (bs, 4H); 3.77 (s, 3H); 4.30 (bs, 2H);
4.57 (q, J ¼ 7.2 Hz, 2H); 6.98 (d, J ¼ 8.7 Hz, 2H); 7.09 (d, J ¼ 8.9 Hz,
1H); 7.17 (d, J ¼ 7.0 Hz, 1H); 7.20 (s, 1H); 7.51 (d, J ¼ 8.7 Hz, 2H); 7.91
(d, J ¼ 13.5 Hz, 1H); 8.02 (d, J ¼ 9.0 Hz, 1H); 8.41 (s, 1H); 8.93 (s, 1H);
15.33 (s, 1H); EIMS m/z 613 (Mþ). Anal. Calcd for C34H32FN3O7: C,
66.55; H, 5.26; F, 3.10; N, 6.85; Found: C, 66.49; H, 5.27; F, 3.10; N,
6.86.
[19] S.K. Jaganathan, Medical Hypotheses 76 (2011) 535e537.
[20] J. Warren, British Journal of Clinical Pharmacology 71 (2011) 497e503.
[21] U. Theuretzbacher, International Journal of Antimicrobial Agents 34 (2009)
15e20.
[22] M.A. Aziz, S.E. Park, G.E.D.A.A.A. Rahma, M.A. Sayed, Y. Kwon, European
Journal of Medicinal Chemistry 69 (2013) 427e438.
[23] T. Plech, M. Wujec, U. Kosikowska, A. Malm, B. Rajtar, M.P. Dacewicz, Euro-
pean Journal of Medicinal Chemistry 60 (2013) 128e134.
[24] B. Garudachari, A.M. Isloor, M.N. Satyanarayana, H.K. Fun, N. Pavithra, A. Kulal,
European Journal of Medicinal Chemistry 68 (2013) 422e432.
[25] V. Pokrovskaya, V. Belakhov, M. Hainrichson, S. Yaron, T. Baasov, Journal of
Medicinal Chemistry 52 (2009) 2243e2254.
4.6.2.21. Formononetin-ethylidene-sarafloxacin (24). White pow-
der, 54%, mp 247e249 ꢂC, 1H NMR (DMSO-d6): 2.64 (bs, 4H); 2.79
(bs, 2H); 3.07 (bs, 4H); 3.79 (s, 3H); 4.24 (bs, 2H); 6.39 (d, J ¼ 7.2 Hz,
1H); 6.99 (d, J ¼ 8.4 Hz, 2H); 7.07 (d, J ¼ 9.2 Hz,1H); 7.18 (s, 1H); 7.52
(d, J ¼ 7.7 Hz, 4H); 7.75e7.85 (m, 2H); 8.01 (dd, J ¼ 13.8 Hz,
J ¼ 9.0 Hz, 2H); 8.42 (s, 1H); 8.65 (s, 1H); 15.13 (s, 1H); EIMS m/z 679
(Mþ). Anal. Calcd for C38H31F2N3O7: C, 67.15; H, 4.60; F, 5.59; N, 6.18;
Found: C, 67.07; H, 4.61; F, 5.60; N, 6.19.
Acknowledgment
[26] H.Y. Kim, J.A. Wiles, Q. Wang, G.C. Pais, E. Lucien, A. Hashimoto, D.M. Nelson,
J.A. Thanassi, S.D. Podos, M. Deshpande, M.J. Pucci, B.J. Bradbury, Journal of
Medicinal Chemistry 54 (2011) 3268e3282.
[27] J. Azéma, B. Guidetti, A. Korolyov, R. Kiss, C. Roques, P. Constant, M. Daffé,
M. Malet-Martino, European Journal of Medicinal Chemistry 46 (2011) 6025e
6038.
[28] B.J. Morrow, W.-P. He, K.M. Amsler, B.D. Foleno, M.J. Macielag, A.S. Lynch,
K. Bush, Antimicrobial Agents and Chemotherapy 54 (2010) 1955e1964.
[29] J.M. Fostel, D.A. Montgomery, L.L. Shen, Antimicrobial Agents and Chemo-
therapy 36 (1992) 2131e2138.
[30] A.M. Sugar, X.P. Liu, R.J. Chen, Antimicrobial Agents and Chemotherapy 41
(1997) 2518e2521.
[31] S. Srinivasan, S.R.M. Beema, P. Nithyanand, P. Manisankar, S.K. Pandian, Eu-
ropean Journal of Medicinal Chemistry 45 (2010) 6101e6105.
[32] L.L. Shen, J. Baranowski, J. Fostel, D.A. Montgomery, P.A. Lartey, Antimicrobial
Agents and Chemotherapy 36 (1992) 2778e2784.
[33] B.D. Bax, P.F. Chan, D.S. Eggleston, A. Fosberry, D.R. Gentry, F. Gorrec,
I. Giordano, M.M. Hann, A. Hennessy, M. Hibbs, J. Huang, E. Jones, J. Jones,
K.K. Brown, C.J. Lewis, E.W. May, M.R. Saunders, O. Singh, C.E. Spitzfaden,
C. Shen, A. Shillings, A.J. Theobald, A. Wohlkonig, N.D. Pearson, M.N. Gwynn,
Nature 466 (2010) 935e940.
The work was financed by grants from National Natural Science
Foundation (grant No. 21262013) of China, and by a Project sup-
ported by Hunan Provincial Natural Science Foundation of China
(grant No. 11JJ3113).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.037.
References
[1] U. Theuretzbacher, Current Opinion in Pharmacology 11 (2011) 433e438.
[2] Y. Chai, Z.-L. Wan, B. Wang, H.-Y. Guo, M.-L. Liu, European Journal of Medicinal
Chemistry 44 (2009) 4063e4069.
[3] L.B. Boyd, M.J. Maynard, S.K. Morgan-Linnell, L.B. Horton, R. Sucgang,
R.J. Hamill, J.R. Jimenez, J. Versalovic, D. Steffen, L. Zechiedrich, Antimicrobial
Agents and Chemotherapy 53 (2009) 229e234.
[4] D. Parcej, R. Tampé, Nature Chemical Biology 6 (2010) 572e580.
[5] S. Kumar, M.F. Varela, International Journal of Molecular Sciences 13 (2012)
4484e4495.
[6] L. Poirel, R.A. Bonnin, P. Nordmann, IUBMB Life 63 (2011) 1061e1067.
[7] L.I. Llarrull, S.A. Testero, J.F. Fisher, S. Mobashery, Current Opinion in Micro-
biology 13 (2010) 551e557.
[34] J. Meletiadis, J.F. Meis, J.W. Mouton, J.P. Donnelly, P.E. Verweij, Journal of
Clinical Microbiology 38 (2000) 2949e2954.
[35] Z.-P. Xiao, T.-W. Ma, M.-L. Liao, Y.-T. Feng, X.-C. Peng, J.-L. Li, Z.-P. Li, Y. Wu,
Q. Luo, Y. Deng, X. Liang, H.-L. Zhu, European Journal of Medicinal Chemistry
46 (2011) 4904e4914.
[36] Z.-P. Xiao, X.-B. He, Z.-Y. Peng, T.-J. Xiong, J. Peng, L.-H. Chen, H.-L. Zhu, Bio-
organic & Medicinal Chemistry 19 (2011) 1571e1579.
[37] H. Nakaminami, N. Noguchi, M. Sasatsu, Antimicrobial Agents and Chemo-
therapy 54 (2010) 4107e4111.
[8] D. Laurenzo, S.A. Mousa, Acta Tropica 119 (2011) 5e10.