Novel hybrids of metronidazole and quinolones: Synthesis, bioactive evaluation, cytotoxicity, preliminary antimicrobial mechanism and effect of metal ions on their transportation by human serum albumin

Article abstract of DOI:10.1016/j.ejmech.2014.08.063

A novel series of hybrids of metronidazole and quinolones as antimicrobial agents were designed and synthesized. Most prepared compounds exhibited good or even stronger antimicrobial activities in comparison with reference drugs. Furthermore, these highly active metronidazole-quinolone hybrids showed appropriate ranges of pKa, log P and aqueous solubility to pharmacokinetic behaviors and no obvious toxicity to A549 and human hepatocyte LO2 cells. Their competitive interactions with metal ions to HSA revealed that the participation of Mg²⁺ ion in compound 7d-HSA association could result in a concentration increase of free compound 7d. Molecular modeling and experimental investigation of compound 7d with DNA suggested that possible antibacterial mechanism might be in relation with multiple binding sites between bioactive molecules and topo IV-DNA complex.

Full text of DOI:10.1016/j.ejmech.2014.08.063

European Journal of Medicinal Chemistry 86 (2014) 318e334
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel hybrids of metronidazole and quinolones: Synthesis, bioactive
evaluation, cytotoxicity, preliminary antimicrobial mechanism and
effect of metal ions on their transportation by human serum albumin
Sheng-Feng Cui, Li-Ping Peng, Hui-Zhen Zhang, Syed Rasheed 1,
Kannekanti Vijaya Kumar 2, Cheng-He Zhou*
Institute of Bioorganic & Medicinal Chemistry, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715,
People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of hybrids of metronidazole and quinolones as antimicrobial agents were designed and
synthesized. Most prepared compounds exhibited good or even stronger antimicrobial activities in
comparison with reference drugs. Furthermore, these highly active metronidazoleequinolone hybrids
showed appropriate ranges of pKa, log P and aqueous solubility to pharmacokinetic behaviors and no
obvious toxicity to A549 and human hepatocyte LO2 cells. Their competitive interactions with metal ions
to HSA revealed that the participation of Mg2þ ion in compound 7deHSA association could result in a
concentration increase of free compound 7d. Molecular modeling and experimental investigation of
compound 7d with DNA suggested that possible antibacterial mechanism might be in relation with
multiple binding sites between bioactive molecules and topo IVeDNA complex.
Received 25 March 2014
Received in revised form
22 August 2014
Accepted 23 August 2014
Available online 24 August 2014
Keywords:
Quinolone
Metronidazole
Imidazole
DNA
© 2014 Elsevier Masson SAS. All rights reserved.
Antibacterial
HSA
1. Introduction
genitourinary infections, which have evolved mechanisms of
resistance to quinolones, are becoming one of the most paramount
Quinolones are an important kind of synthetic antibacterial
agents as they are generally well tolerated with excellent safety
profile, favorable pharmacokinetic characteristics, broad antibac-
terial spectrum and good treatment effectiveness [1e3]. Since the
first generation of quinolones were discovered in 1960s, four gen-
erations of quinolones have been successfully developed in suc-
cession and a large number of quinolone drugs, such as norfloxacin,
moxifloxacin, ofloxacin, temafloxacin, have been successfully and
widely used in clinic to treat genitourinary infections and common
respiratory tract pathogens. Furthermore, the antibacterial mech-
anism of quinolones has been elucidated clearly. These drugs pri-
marily target type II topoisomerases or DNA gyrase in Gram-
negative bacteria to stabilize the cleavage complex at specific
sites on DNA, then block DNA replication and finally lead to a lethal
lesion [4e5]. However, in recent years the emergence and spread of
public health threats. A growing medical concern on mechanisms
of resistance to quinolones has drawn much attention, and much
effort has been focusing on this topic [6]. Some researches [7] show
that the resistance to quinolones is principally due to mutations in
either ParC breakage-reunion or ParE TOPRIM domains of topo IV
(topoisomerase IV) and in the analogous gyrase domains [8].
Further structure activity relationship (SAR) reveals that the groups
at N-1 position of quinolones are in close proximity to the
conserved ParC helix a4 residues Ser79 and Asp83 in the topo
IVeDNA complex, whose mutation causes quinolone resistance in
pneumococci and other bacteria [9]. Therefore, the structural
modification of quinolones at N-1 position is a promising strategy
to overcome the quinolone resistance. Many kinds of functional
groups such as ethyl, cyclopropyl and various heterocyclic moieties
have been successfully introduced into N-1 position, and produced
many effective clinical quinolone drugs [10].
In recent years, azole heterocyclic compounds have been paid
special attention due to their potential applications as medicinal
agents [11e12], and an increasing effort has been directing towards
their possible medicinal potentiality [13e16]. Particularly, the
* Corresponding author.
E-mail addresses: zhouch@swu.edu.cn, zhouch6848@sina.com (C.-H. Zhou).
Postdoctoral fellow from Department of Chemistry, University of Hyderabad,
1
India.
2
Postdoctoral fellow from Indian Institute of Chemical Technology (IICT), India.
http://dx.doi.org/10.1016/j.ejmech.2014.08.063
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

S.-F. Cui et al. / European Journal of Medicinal Chemistry 86 (2014) 318e334
319
nitro-containing imidazoles are found to be a kind of important
imidazole antimicrobial derivatives, and many of them such as
benznidazole, secnidazole, metronidazole and ornidazole have
been extensively used in clinic to treat anaerobic infections [17].
Especially the structurally simple metronidazole as an effective
synthetic drug introduced in 1960 possesses strong inhibitory ef-
ficacies against Gram-negative anaerobic bacteria like Helicobacter
pylori and protozoa such as Giardia, Lamblia and Entomoeba histo-
lytic [18]. These outstanding achievements encourage numerous
researchers to focus on their research and development in nitro-
imidazoles with potential medicinal application. The mechanism
researches indicate that the nitro fragment of metronidazole plays
a significant role through the metabolic activation in exerting
biological activities [19]. Furthermore, a lot of researches disclose
that the reactive metabolic intermediates formed in microorgan-
isms by reducing nitro group in nitroimidazoles can covalently bind
with DNA and trigger the adverse effect [20]. The sterical protection
of nitro group in metronidazole has been proved to be an effective
strategy to improve the metabolism and physicochemical property
of such compounds [21]. Moreover, the incidence of resistance in
anaerobic bacteria is still very low. Therefore, the structural
modification of metronidazole has been an increasingly attractive
topic.
activities for all newly synthesized compounds were evaluated
in vitro against nine bacteria including Methicillin-Resistant Staph-
ylococcus aureus N315 (MRSA) and five fungi [29]. Cytotoxicity,
ionization constants (pKa), aqueous solubility and partition co-
efficients of some highly active target compounds were also eval-
uated to predict their pharmacokinetics behaviors. The effects of
metal ions on their transportation by HSA were also investigated in
order to preliminarily evaluate their transportation, distribution,
and metabolism by fluorescence and UVevis absorption spectros-
copy on molecular level. Furthermore, flexible ligandereceptor
docking investigation was undertaken to rationalize the antibac-
terial activity and understand the possible mechanism of the hy-
brids. The comparative study of preliminary interaction between
compound 8d and norfloxacin separately with calf thymus DNA
were also evaluated to further predict the binding ability between
these hybrids and topo IVeDNA complex.
2. Results and discussion
2.1. Chemistry
The target hybrids were synthesized according to the synthetic
route outlined in Scheme 1. Commercial ethoxymethylene malonic
ester (EMME) was reacted with a series of substituted phenyl-
amines in ethanol to afford intermediates 1aeh in almost quanti-
tative yields. The obtained compounds were then cyclized in
phenoxybenzene under reflux to produce the desired quinolones
2aeh in good yields ranging from 49.0% to 60.0%, which were
further N-alkylated by commercial racemic 2-(chloromethyl)oxir-
ane to yield ethyl 1-(oxiran-2-ylmethyl) quinoline-3-carboxylate
derivatives 3aeh in high yields (81.2e95.0%). The epoxy rings of
compounds 3aeh were opened by 4-nitroimidazole and 2-methyl-
5-nitroimidazole respectively in ethanol using sodium bicarbonate
as base to produce the corresponding racemates 4aeh and 5aeh in
satisfactory yields ranging from 80.2% to 98.5%, and then the latter
were further hydrolyzed by 3% sodium hydroxide at 100 ꢀC to afford
the target quinoloneemetronidazole hybrids 6aeh and 7aeh in
excellent yields.
In the preparation of compounds 3aeh, it was found that the
solvents and base exerted remarkable effect on their yields. In
particular, 2-(chloromethyl)oxirane as both solvent and reactant
led to relatively high yield in contrast to other common solvents
such as ethanol, methanol and acetonitrile. It was found that suit-
able base in the synthesis of intermediates 3aeh was potassium
carbonate, since other bases resulted in the low yields of target
compounds, and formation of various by-products was observed.
Our previous work has reported that the incorporation of
nitroimidazoles into berberine not only enhance the antimicrobial
activities, but also broaden antimicrobial spectrum. This type of
hybrids of berberine and nitroimidazoles exhibited anti-Gram-
negative efficacies with low MIC values ranging from 4 to 8 mg/
mL. They were also proven to effectively bind with bio-
macromolecules like DNA and Human Serum Albumin (HSA) [22].
In view of the above considerations and as an extension of our
previous work [23e28], herein we introduced the nitroimidazole
moieties into N-1 position of quinolones to generate a novel series
of hybrids of metronidazole and quinolone. Rational design and
relevant synthons were shown in Fig. 1. The nitroimidazole groups
at N-1 position of quinolones were expected to exert noncovalent
interactions with DNA base, ParC breakage-reunion or ParE TOPRIM
domains of topo IVeDNA complex to overcome the resistance and
broaden the antimicrobial spectrum. Therefore, the antimicrobial
2.2. Analysis of spectra
All the new compounds were characterized by IR, 1H NMR, 13
C
NMR, MS and HRMS spectra. Their spectral analyses were consis-
tent with the assigned structures and listed in the experimental
section. The mass spectra for new compounds gave a major frag-
ment of [MþH]þ or [MþNa]þ according to their molecular formula.
2.2.1. 1H NMR spectra
In 1H NMR spectra, it was noticed that the protons of hydroxyl
group and tertiary carbon in compounds 4e7 appeared as multiplet
peaks at d 5.20e5.95 ppm and d 4.67e4.87 ppm respectively due to
the adjacency of chiral tertiary carbon and the electron-
withdrawing character of quinolone backbones and imidazole
rings. For compounds 3aeh, because of the effect of electron-
withdrawing quinolones and epoxy rings, the protons of CH2
linked to N-1 position gave large downfield chemical shifts at
Fig. 1. Design of novel target hybrids of metronidazole and quinolones.
d 4.40e5.04 ppm. The CH proton on the epoxy ring exhibited a

320
S.-F. Cui et al. / European Journal of Medicinal Chemistry 86 (2014) 318e334
a
Scheme 1. Synthetic route of hybrids of metronidazole and quinolones. Reagents and conditions: (i) ethoxymethylene malonic ester, ethanol, reflux, 2 h; (ii) phenoxybenzene,
reflux, 1 h; (iii) 2-(chloromethyl)oxirane, K2CO3, 120 ꢀC, 3 h; (iv) 4-nitroimidazole, K2CO3, ethanol, reflux, 2 h; (v) 2-methyl-5-nitroimidazole, K2CO3, ethanol, reflux, 2 h; (vi) 3%
NaOH, 100 ꢀC, 2 h.
multiplet signal at
d
3.35e3.46 ppm in DMSO-d6 because of the
For the tested compounds 4aeh and 5aeh bearing nitro-
imidazole fragment (Supporting Information), most of them
showed better antibacterial activities than intermediates 3aeh, and
displayed more potent against Gram-negative bacteria than Gram-
positive bacteria. 4-Nitroimidazolyl quinolones 4aeb and 2-
methyl-5-nitroimidazolyl compounds 5aeb and 5d exhibited
good activities against the tested Gram-negative bacteria including
P. aeruginosa, E. coli DH52, S. dysenteriae and E. typhosa strains with
chiral carbon and the presence of strong electrophilic groups, and
the same reasons resulted in large downfield chemical shifts
(2.54e2.86 ppm) for the CH2 protons on the epoxy ring.
2.2.2. 13C NMR spectra
The 13C NMR spectral analyses were consistent with the
assigned structures. No large differences were found in 13C chem-
ical shifts for C-2, C-3, C-4 position and quaternary carbon of qui-
nolone backbones. The chemical shifts of the tertiary carbon
low MIC values in the range of 4e16 mg/mL. Notably, compound 5d
with trifluoromethyl group at 7-position of quinolone ring gave
stronger antimicrobial efficacy and broad antimicrobial spectrum in
comparison with norfloxacin and chloromycin, which was quite
sensitive to all the tested bacterial strains except for B. subtilis with
followed
(68.1e68.9
60.2e60.8 ppm)
60.6e60.8 ppm) > epoxy quinolone derivatives (
a decreased order: epoxy quinolone hydrochloride
ppm)
>
4-nitroimidazole
derivatives
(d
z
2-methyl-5-nitroimidazole derivatives
(d
d
57.2e57.7 ppm).
MIC values of 2e8
mg/mL. However, compared to 2-methyl-5-
Additionally, all the other aromatic and aliphatic carbons also
appeared at the appropriate chemical shift regions.
nitroimidazolyl quinolone
nitroimidazolyl derivative 4d gave low inhibitory concentrations
to these tested bacterial strains especially towards P. aeruginosa, E.
5d, the corresponding 4-
2.3. Biological activity
coli DH52, S. dysenteriae and E. typhosa (MIC ¼ 16e128
mg/mL).
Furthermore, except for 6,8-difluoro quinolone 4a and 6,8-dichloro
quinolone 4f and 5f, all the other target compounds could effec-
2.3.1. Antibacterial activity
The antibacterial results revealed that most of the prepared
compounds could effectively inhibit the growth of all the tested
bacteria in vitro. The synthesized intermediates 3aeh with oxiran-
2-ylmethyl group at N-1 position of quinolone showed moderate
activities against the tested bacterial strains (Supporting
Information). Moreover, the E. typhosa strain was quite sensitive
tively inhibit the growth of MRSA at the concentrations of 2e16
mL, which were more effective than clinical drug chloromycin
(MIC ¼ 16 g/mL).
mg/
m
It was also observed (Table 1) that metronidazoleequinolone
hybrids 6aeh and 7aeh displayed stronger antibacterial efficacy
than their precursor carboxylic esters 4aeh and 5aeh. The hy-
drolysis of carboxylic esters, which produced the corresponding
carboxylic acids, could enhance antimicrobial efficiency and
broaden the antimicrobial spectrum. As shown in Fig. 2, compound
7b exhibited better activities against M. luteus, MRSA, P. aeruginosa
and E. coli DH52 than its precursor 5b. Compound 7d in vitro with
to the synthetic intermediates 3aeh with MIC values of 1e8
which were more effective than standard drug chloromycin
(MIC 32 g/mL) and almost equipotent to norfloxacin
(MIC ¼ 4 g/mL). It was worthwhile to note that 6,8-dichloro-1-
mg/mL,
¼
m
m
(oxiran-2-ylmethyl) quinolone 3f and 8-fluoro-1-(oxiran-2-
ylmethyl) quinolone 3g exhibited significant activity against most
of the tested Gram-positive strains except for M. luteus and
S. dysenteriae.
MIC value of 1 mg/mL against MRSA was eight fold more active than
compound 5d. Furthermore, compound 6aeh and 7aeh showed
moderate to good anti-S. pneumoniae activity with MIC values from

S.-F. Cui et al. / European Journal of Medicinal Chemistry 86 (2014) 318e334
321
Table 1
In vitro antibacterial activities for compounds 6e7 expressed as MIC (
m
g/mL).a,b,c
Compds
Gram-positive bacteria
Gram-negative bacteria
S. pneumoniae
M. luteus
MRSA
S. aureus
B. subtilis
P. aeruginosa
E. coli DH52
S. dysenteriae
E. typhosa
6a
6b
6c
6d
6e
6f
6g
6h
7a
7b
7c
7d
7e
7f
16
32
8
16
16
8
32
64
16
4
8
4
8
16
32
64
16
1
8
8
16
8
8
8
4
256
2
1
2
16
8
8
4
8
16
4
16
2
4
2
1
4
32
4
8
8
8
16
4
16
8
16
64
16
8
8
16
16
4
8
4
4
16
4
8
16
4
8
8
16
32
8
4
4
8
8
4
8
8
128
2
2
4
4
16
8
2
512
32
1
4
4
8
2
32
8
4
64
4
1
8
8
16
8
4
16
8
8
16
4
8
1
0.5
16
16
8
32
32
4
2
4
8
4
2
16
32
32
16
8
32
8
128
32
4
8
4
1
0.5
64
16
4
512
8
32
16
8
32
16
0.5
2
32
4
64
32
16
7g
7h
A
8
32
4
16
8
B
2
1
a
Minimum inhibitory concentrations were determined by micro broth dilution method for microdilution plates.
b
c
A ¼ chloromycin, B ¼ norfloxacin.
S. pneumoniae, Streptococcus pneumoniae; M. luteus, Micrococcus luteus ATCC 4698; MRSA, Methicillin-Resistant Staphylococcus aureus N315; S. aureus, Staphylococcus
aureus ATCC 25923; B. subtilis, Bacillus subtilis ATCC 6633; P. aeruginosa, Pseudomonas aeruginosa; E. Coli DH52, Escherichia coli; S. dysenteriae, Shigella dysenteriae; E. typhosa,
Eberthella typhosa.
4 to 64
exhibited much better inhibitory potency against S. pneumoniae
strain than chloromycin (MIC 16 g/mL). Compound 7d
possessed stronger antimicrobial activities and broader antimi-
crobial spectrum than other target compounds, which was four fold
more active against M. luteus, B. subtilis, E. coli DH52 and S. dysen-
teriae in comparison with its precursor 5d.
The above results suggested that the antimicrobial efficacies
should be related to substituents on benzene ring of quinolones and
nitroimidazole ring to some extent. Among this series of hybrids,
compounds with fluoro substituent on benzene ring exerted the
most important influence on antimicrobial activities. 6,8-Difluoro
quinolone 7a, 7-trifluoromethyl 7d, 6-fluoro 7c, 7-chloro-6-fluoro
7e and 8-fluoro 7g could effectively inhibit the growth of most
m
g/mL. Especially, compound 7b and 7d (MIC ¼ 4
m
g/mL)
16
14
12
10
8
Compound 5b
Compound 7b
Compound 5d
Compound 7d
¼
m
M.luteus
B.subtilis
P.aeruginosa
S.dysenteriae
S.aureus
MRSA
E.typhosa
6
E.coli DH52
4
2
tested bacterial strains at the concentrations of 0.5e16
mg/mL,
which were more effective than 6,8-dichloro quinolone 7f and 6-
methoxyl derivative 7c. Especially 7-trifluoromethyl derivative 7d
showed significant inhibition against all the tested strains with low
inhibitory concentrations, which were comparable or even superior
to the reference drugs chloromycin and norfloxacin. To our sur-
prise, 6-methyl quinolones 7b and 6b also exhibited good activity
in comparison to standard drugs. Generally, the antibacterial ac-
tivities of these hybrids followed the order of 2-methyl-5-
nitroimidazolyl compounds > 4-nitroimidazolyl derivatives. In
contrast to 4-nitroimidazolyl compounds 6aeh, the corresponding
2-methyl-5-nitroimidazolyl quinolones 7aeh showed better anti-
bacterial activities against all the tested strains.
0
Fig. 2. The comparison of antibacterial data between compounds 5b and 7b, com-
pounds 5d and 7d respectively.
inhibit the growth of A. flavus and B. yeast strains, which gave su-
perior inhibitory potency to the first-line antifungal drug flucona-
zole (MIC ¼ 256, 16
mg/mL).
1-(Oxiran-2-ylmethyl)-quinolones 3aeh showed efficient inhi-
bition against B. yeast, C. albicans, and C. mycoderma. The fungal
strain of A. flavus was more sensitive to compounds 3aeh with
quite low MIC values (MIC ¼ 0.5e16
mg/mL) than fluconazole
(MIC ¼ 256 g/mL). Especially the activities of compounds 3d, 3f,
m
2.3.2. Antifungal activity
and 3g against A. flavus strain were 512-fold more potent than
reference drug fluconazole. Moreover, all the quinolone in-
termediates 3aeh possessed moderate to good antifungal activities
against B. yeast, C. albicans and C. mycoderma, which were com-
parable or even better in comparison with the reference drug
fluconazole.
The in vitro antifungal evaluation (Supporting Information)
revealed that the intermediates 3aeh with oxiran-2-ylmethyl
group at N-1 position of quinolones exhibited better antifungal
activities against most of the tested fungal strains than these hy-
brids of metronidazole and quinolone. Most of the prepared hy-
brids with MIC values of 64e512 mg/mL showed moderate to bad
antifungal activities against the tested fungal strains. The substi-
tution of metronidazole group in quinolones caused remarkable
decrease or total loss of antifungal properties. However, hybrids 6b
2.3.3. Cell toxicity
The highly bioactive hybrids 6b, 7b and 7d were further
examined for their cytotoxic properties on A549 cell line and
(MIC ¼ 1, 4
mg/mL) and 7b (MIC ¼ 2, 2
mg/mL) could effectively

322
S.-F. Cui et al. / European Journal of Medicinal Chemistry 86 (2014) 318e334
Table 2
normal human hepatocyte LO2 by means of CKK-8 Kit and MTT
respectively, and the absorbance of viable cells (450 nm and
490 nm) increased linearly as cells proliferate. Compounds 6b, 7b
and 7d were dissolved in DMSO to prepare the stock solutions, and
then the tested compounds were prepared in medium to obtain the
The inhibition rate of target compound 7d on human hepatocyte LO2 tested by MTT
method, each data represents an average of three parallels.
Concentration (
mg/mL)
OD values
Inhibition rate (%)
0
8
16
32
64
128
0.732 0.132
0.706 0.102
0.683 0.094
0.657 0.096
0.621 0.076
0.604 0.054
0
3.6
6.7
10.3
15.2
17.5
required concentrations of 256, 128, 64, 32, 16 and 8 mg/mL. Cells
were cultured for 24 h in these solutions. The cell viability was
examined by microplate reader. Cytotoxicity results (Fig. 3) showed
that the cell viability of all the three compounds was at least more
than 83% within concentration of 256
the tested compounds did not affect the viability of A549 cell line
within the concentration of 256 g/mL. Especially, 6-methyl hybrid
7b showed the lowest cell toxicity among the tested compounds.
Moreover, with the increase of concentrations of the tested com-
pounds, there were no obviously reducing trends to the cell
viability. As shown in Table 2, the toxicity assay suggested non-
toxicity for compound 7d on normal human hepatocyte LO2 within
mg/mL, which indicated all
m
Fig. 4 clearly showed the changes observed in the UV spectrum
of compound 7d in different buffer solutions. With the increase of
pH from 3.0 to 9.4, the molecular formula changed from I to II, and
then to III. As shown in Fig. 5, there were two parts of spectral
differences (from 245 to 272 and from 308 to 344 nm), which
resulted from the proton on the nitrogen of metronidazole and
carboxyl hydrogen of quinolone ring respectively. Herein the
spectral differences from 308 to 344 nm were chosen to analyze the
pKa values of target hybrids.
the concentration of 128 mg/mL (Inhibition rate < 17.5%).
2.3.4. Analysis of pKa values
Data analysis included normalization of the raw scans
(Abs450 nm ¼ 0), and then calculated the spectral difference be-
tween the acid spectra and the spectra obtained at every other pH
(Fig. 6). The wavelengths of maximum positive and negative de-
viations were determined graphically, and the absolute values of
the absorbance difference at the chosen wavelengths were sum-
med. The total absorbance difference was then plotted versus pH
(Fig. 7), and the data were fit to Equation (1) to obtain the pKa
values.
The ionization constant of drug is an important physicochemical
parameter to significantly influence pharmacokinetic behaviors
including absorption, distribution, metabolism, and excretion
[30e31]. The pKa values are usually used to estimate the extent of
ionization of drug molecules at different pH values, which are of
fundamental importance in the consideration of their interaction
with biological membranes. For instance, compounds with mini-
mally one charged with a pKa <4 for acid and correspondingly a
pKa >10 for base will be charged at physiological pH and display
slower diffusion rate to across biological membranes such as the
bloodebrain barrier [32].
ꢀ
ꢁ
εНА ꢁ εАꢁ ꢂ 10ðpHꢁpKaÞ
Absorbance total ¼
ꢂ ½Stꢃ
(1)
1 þ 10ðpHꢁpKaÞ
The hybrids of metronidazole and quinolone should have two
pKa values, one for the proton on the nitrogen of metronidazole and
the other one for the carboxyl hydrogen of quinolone ring. Because
the metronidazole groups and quinolone ring in these hybrid
Where εHA and εAꢁ are the extinction coefficients of the acid and
base forms of compound respectively, and [St] is the total com-
pound concentration. When using absorbance differences, the εHA
and εAꢁ are simply the minima and maxima of the curve.
As shown in Table 3, the pKa values of compounds 7aeh dis-
played an appropriate range (5.257e6.430) to pharmacokinetic
behaviors. Since the electron density on the aromatic rings could be
reduced by the electron withdrawing groups such as Cl, F and CF3
groups, the pKa values of these compounds decreased accordingly.
Meanwhile, due to their electron donating ability of methyl and
methoxyl groups, compounds 7b and 7h could destabilize anionic
form, therefore decrease the acidic character and show high pKa
values.
molecules do not possess one large
pep conjugated system, the
changes of absorbance in different pH buffer solutions should be
two relatively independent processes and take place on different
maximum absorption wavelength respectively, which followed the
reaction formulas (Fig. 4).
Compound 7d
Compound 6d
Compound 7b
120
100
80
60
40
20
0
2.3.5. Analysis of partition coefficient and solubility
Physical characteristics of drugs such as lipophilicity, hydro-
philicity and solubility have been suggested to play a decisional role
in determining where drugs are distributed and how rapidly they
are metabolized and excreted in the body. The ideal distribution
coefficient is a moderate intermediate between hydrophilic and
hydrophobic to evaluate the pharmacokinetic and pharmacody-
namic properties. The partition coefficient (log P) is frequently used
to define the lipophilic character of a drug. Therefore, the lip-
ophilicity/hydrophilicity expressed as octanol/water partition co-
efficient was determined experimentally by traditional saturation
shake flask method combining with UVevis spectrophotometric
approach (Supporting Information). As given in Table 4, the tested
compounds had suitable partition coefficient that was favorable to
permeate through biological membrane and to be delivered to the
binding sites. Moreover, for the tested compound 7aeh, most of
them were hydrophilic. Especially, compound 7d, as expected, was
1
0
2
3
4
5
64
6
7
256
8
16
32
128
Concentration of compounds (
μ
g/mL)
Fig. 3. Cytotoxic assay of target compounds 6d, 7b and 7d on A549 tested by CCK-8 Kit.
Each data bar represents an average of three parallels, and error bars indicate one
standard deviation from the mean.

S.-F. Cui et al. / European Journal of Medicinal Chemistry 86 (2014) 318e334
323
Fig. 4. Structural transposition of compound 7d in different buffer solution from 3.0 to 9.4.
values, it was suggested that the target compounds with suitable
log P values between ꢁ0.25 and 0.18, such as compounds 7b, 7d and
7e, showed better inhibition activities than others.
0.5
0.4
0.3
0.2
0.1
0.0
Normalized data
Aqueous solubility is also a governing property for how small
molecules interact with bimolecular and living systems, which
plays a critical role in every stage of drug discovery and develop-
ment. Incorrect estimation of solubility can lead to erroneous
interpretation of in vitro assay results and weaken structur-
eeactivity relationship analysis. Thereby the aqueous solubility of
target compounds was also given in this work (Table 4). In general,
all the tested compounds showed good aqueous solubility ranging
from 0.353 to 0.792 mg/mL. These obtained results were almost
consistent with the antibacterial activities. The compounds with
good aqueous solubility showed low inhibitory concentrations
against bacterial strains. For example, due to the hydrogen bonds
between water and fluoro and imidazole groups, compounds 7a
(0.57 mg/mL) and 7d (0.79 mg/mL) gave good aqueous solubility.
Meanwhile both of them exhibited significant antibacterial
activities.
7.0
7.5
8.0
8.5
8.8
9.1
9.4
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
pH
270
300
330
360
390
420
Wavelength (nm)
Fig. 5. UV spectra (
l
¼ 258e470 nm) of compound 7d in different aqueous buffer
solutions ranging from pH 3.0 to 9.4. The absorbances are normalized to zero for
2.3.6. The effect of metal ions on the transportation of compound
7d by HSA
l
¼ 460 nm.
It is well known that HSA is a kind of important and principal
extracellular protein in circulatory system, which is closely related
to the overall absorption, transportation, distribution, metabolism
of drugs and has an immense impact on the efficacy of drugs in
human body [33]. Therefore, the investigations of interactions be-
tween bioactive small molecules and HSA are not only beneficial to
provide a proper understanding of the pharmacokinetic properties
0.30
7.0
7.5
8.0
8.5
8.8
9.1
9.4
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
pH
0.15
0.00
4
pKa Determination
Spectral difference plot
3
2
1
0
-0.15
250
300
350
400
Wavelength (nm)
Fig. 6. Plot of the spectral difference between different solutions of compound 7d. The
maximum positive deviation occurred at 344 nm while the maximum negative devi-
ation occurred at 308 nm.
the most hydrophilic one because of the trifluoromethyl group on
7-position of quinolone. However, because the chloro and methoxyl
groups on benzene ring of quinolones might increase the lip-
ophilicity of compounds, compounds 7f and 7h with these groups
gave considerably larger lipophilicity in contrast to other com-
pounds. Based on the results of antibacterial activities and log P
3.0
4.5
6.0
7.5
9.0
pH
Fig. 7. The total absorbance difference (the sum of the absolute absorbance difference
values at 244 and 308 nm at each pH) was plotted against solution pH. The data was
then fit to equation.

324
S.-F. Cui et al. / European Journal of Medicinal Chemistry 86 (2014) 318e334
Table 3
compound 7d in the blood, and thus improved its antimicrobial
The pKa values of compounds 7aeh.a
efficacy [39]. The altered binding constants might be explained by
the conformational changes of HSA structure caused by binding to
metal ions or interaction between compound 7d and metal ions,
which in turn affected protein binding to drugs.
Compds 7a
7b
7c
7d
7e
7f
7g
7h
l
(nm) 308/
344
308/
342
308/
343
308/
344
308/
344
308/
344
308/
343
308/
344
pKa
5.326
0.991
6.430
0.990
5.179
0.990
5.599
0.994
5.145
0.991
5.257
0.992
5.121
0.991
5.718
0.994
Ra
3. Molecular modeling
a
R is the correlation coefficient.
To further rationalize the observed antibacterial activity and
understand the possible mechanism of the hybrids, a flexible ligand
receptor docking investigation was undertaken. The crystal struc-
ture data (Streptococcus pneumoniae topoisomerase IVeDNA com-
plexes) was obtained from the protein data bank [40], which was a
representative protein to investigate the antibacterial mechanism
of quinolones [41]. Target compounds 6aeh and 7aeh were
selected to dock with the topo IVeDNA complex. According to the
docking results, 7-trifluoromethyl hybrid of metronidazole and
quinolone 7d possessed the best score (ꢁ5.800) and lowest energy
(ꢁ6.093) among these target compounds. It was also observed that
2-methyl-5-nitroimidazolyl quinolones 7aeh got higher scores
than 4-nitroimidazolyl quinolones 6aeh. Furthermore, the sub-
stituents on benzene ring of quinolone could affect the scores of
docking results, which followed the order of 7-trifluoromethyl
of drugs, but also instructive to the design, modification and
screening of drug molecules [34]. Furthermore, some metal ions in
plasma, when drugs bind reversibly to HSA and are delivered to the
binding sites, can have an affect on the transposition ability of HSA.
The 4-oxo and 3-carboxyl groups of quinolones endow them
with excellent chelating properties with metal ions [35]. These
chelating abilities can not only influence the in vivo antimicrobial
activity, but also interfere with their pharmacokinetic properties
when co-administration of other drugs and/or some food that
contain metal ions [36]. So it is considerably reasonable for us to
further investigate the effect of metal ions including major ele-
ments like Ca2þ, Kþ, Mg2þ and Ni2þ ions on the binding constant of
these compoundeHSA complex by fluorescence and UVevis ab-
sorption spectroscopy on molecular level.
quinolones
> 6-fluoro-7-chloro derivatives >6,8-difluoro de-
Fluorescence quenching is considered as an effective approach
to investigate the transportation ability of HSA to small molecules.
Much literature discloses that the emission of Trp 214 (the single
tryptophan of HSA at residue 214) may be blue shifted if the group
is buried within a native protein, and its emission may shift to
longer wavelengths (red shift) when protein is unfolded [37,38].
The effect of compound 7d on HSA fluorescence intensity at 286 K
was shown in Fig. 8. Evidently, a progressive decrease in the fluo-
rescence intensity was caused by quenching, accompanied with the
blue shift wavelength (from 345.0 to 356.0 nm) in the albumin
spectrum. These suggested an increased hydrophobicity of the re-
gion surrounding the single Try-214 residue. The numbers of
binding sites and the equilibrium binding constants can also be
calculated according to the Scatchard Equation (2). The result
showed the compound 7d gave suitable binding constant (Fig. 9,
K ¼ 5.15 ꢂ 104 L/mol)
rivatives > 6-fluoro derivatives. These docking results of the target
hybrids were almost in accordance with the observed in vivo
antibacterial activities except for 6-methyl hybrids.
As shown in Fig. 11, the docking result of compound 7d and topo
IVeDNA complex might rationalize the possible antibacterial
mechanism. The hydroxyl group and nitrogen atom of imidazole
were in close vicinity to the phosphate of the topo IVeDNA phos-
phodiester bond, which formed two hydrogen bonds with distance
of 2.9 Å. Furthermore, the carboxyl and nitro groups of this mole-
cule were also located closest to DNA base and interacted through
hydrogen bonds with distance of 2.5 Å and 2.6 Å respectively. These
noncovalent bonds could interact with DNA base and might block
DNA replication. Moreover, the nitro and oxygen atoms at nitro
group of imidazole were in close proximity to conserve ParC helix
a4 residues Ser79 (with closest distances to the side chain atoms of
3.1 Å and 3.2 Å respectively), whose mutation caused quinolone
resistance in pneumococci and other bacteria. Therefore these
hydrogen bonds at resistance mutation region might be the
important reason that compound 7d gave strong inhibitory efficacy
against strains of quinolone-resistant bacteria such as MRSA.
.
r
Df ¼ nKb ꢁ rKb
(2)
Where Df is the molar concentration of free small molecules, r is the
moles of small molecules bound per mole of protein, n is binding
sites multiplicity per class of binding sites, and Kb is the equilibrium
binding constant. The Scatchard plots and the Kb are listed in
Table 5
4. The interaction with DNA
Based on the results of molecular modeling, the incorporating of
metronidazole might improve the interaction between target
compounds and DNA of topoisomerase IVeDNA complexes. So the
interaction between the strongest active hybrid 7d and calf thymus
deoxyribonucleic acid (DNA) was evaluated to further explore
preliminary antimicrobial mechanism. In contrast to this interac-
tion between compound 7d and DNA, the reference drug nor-
floxacin was employed to investigate the interaction with calf
thymus DNA.
The binding studies on molecular level were carried out by
UVevis spectroscopic method. In absorption spectroscopy, hypo-
chromism and hyperchromism were very important spectral fea-
tures to distinguish the change of DNA double-helical structure
[42]. With a fixed concentration of DNA, UVevis absorption
spectra were recorded as increasing amount of compound 7d. As
shown in Fig. 12, UVevis spectra displayed that the maximum ab-
sorption peak of DNA at 260 nm exhibited proportional increase
and slight blue shift along with the increasing concentration of
The effect of metal ions on the transportation of compound 7d
by HSA was evaluated by Kb/K0. As shown in Table 5, the values of
Kb/K0 were tested ranging from 0.91 to 1.51, which indicated the
metal ions including Ca2þ, Kþ, Mg2þ and Ni2þ ions had an obvious
effect on the transportation by HSA. Clearly, the value of Kb/K0 in the
presence of Ni2þ ion was approximated to 1.00, which suggested
the Ni2þ ion caused little impact on the transportation of com-
pound 7d by HSA. However the presence of metal ions such as Ca2þ
and Kþ ones considerably increased the binding constants of
compound 7deHSA complex (Kb/K0 ¼ 1.43, 1.51), thus causing
compound 7d to be slowly cleared from blood, which may lead to
more doses of compound 7d to achieve the desired therapeutic
effectiveness (Fig. 10). While the participation of Mg2þ ion reduced
the binding constants of compound 7deHSA complex, suggesting
the presence of these metal ions could decreased the concentration
of free compound 7d, shorten the storage time and half-life of

S.-F. Cui et al. / European Journal of Medicinal Chemistry 86 (2014) 318e334
325
Table 4
compound 7d. Meanwhile, the phenomenon, that the absorption
value of simply sum of free DNA and free compound 7d was less
than the measured value of DNAecompound 7d complex, was
observed in the inset of Fig. 12. The interesting observed hyper-
chromism accompanied by a slight blue shift (7 nm) suggested a
conformational change in the DNA duplex. Furthermore, it was
noticed that this hyperchromism was becoming increasingly strong
along with the increasing concentration of compound 7d. This
change was characteristic of non-covalent interactions between the
complexes and DNA resulting in some uncoiling of the DNA helix
and the exposure of some previously embedded DNA bases. The
observed spectral effects could be explained by the electrostatic
interaction of the complexes with DNA. Based on the variations in
these spectra, Equation (3) could be used to calculate the intrinsic
binding constant (K).
Log P values and solubility of compounds 7aeh.
Compds
7a
7b
7c
7d
7e
7f
7g
7h
Log P
Solubility (mg/mL)
ꢁ0.21 ꢁ0.03 ꢁ0.13 ꢁ0.25 0.18 0.22 ꢁ0.13 ꢁ0.24
0.57
0.39
0.38
0.79 0.43 0.35
0.39
0.40
2000
HSA
1500
1000
500
0
a
k
A0
A ꢁ A0
xC
xC
1
¼
þ
ꢂ
(3)
xDꢁC ꢁ xC xDꢁC ꢁ xC K½7dꢃ
A0 and A represent the absorbance of DNA in the absence and
presence of compound 7d at 260 nm, xC and xDeC were the ab-
sorption coefficients of compound 7d and DNAecompound 7d
complex respectively. The plot of A0/(A ꢁ A0) versus 1/[compound
7d] was constructed by using the absorption titration data and
linear fitting, yielding the binding constant, K ¼ 1.434 ꢂ 104 L/mol,
R ¼ 0.9997, SD ¼ 0.12 (R is the correlation coefficient. SD is standard
deviation).
As shown in Fig. 13, the similar hyperchromism of
DNAenorfloxacin complex was also observed. However, there
were some differences between norfloxacin and compound 7d.
With the gradually increasing concentration of compound 7d or
norfloxacin, the DNAecompound 7d complex showed stronger
hyperchromism than DNAenorfloxacin complex. These suggested
that the new metronidazoleequinolone hybrids have better
interaction with DNA. Therefore, this type of interaction with DNA
might improve binding ability between these hybrids and topo
IVeDNA complex.
Compound 7d /10-5 mol /L
320
340
360
380
400
Wavelength(nm)
Fig. 8. Emission spectra of HSA in the presence of various concentrations of compound
7d. c(HSA) ¼ 1.0 ꢂ 10ꢁ5 mol/L; c(compound 7d)/(10ꢁ5 mol/L), aek from 0.0 to 2.0 at
increments of 0.20; blue dash line shows the emission spectrum of compound 7d only;
T ¼ 286 K, lex ¼ 295 nm. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
HSA+Ca2+
2000
HSA
1500
a
Fig. 9. Emission spectra of HSA in the presence of various concentrations of compound
7d. c(HSA) ¼ 1.0 ꢂ 10ꢁ5 mol/L; c(compound 7d)/(10ꢁ5 mol/L), aek from 0.0 to 2.0 at
increments of 0.20; blue dash line shows the emission spectrum of compound 7d only;
T ¼ 286 K, lex ¼ 295 nm. R is the correlation coefficient. S.D. is standard deviation. (For
interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)
1000
k
500
Compound 7d only
Table 5
0
Effect of metal ions on the binding constants of compound 7deHSA complex.
320
340
360
380
400
Systems
104 Kb (L/mol)
Kb/K0
R
a
b
Wavelength (nm)
HSAecompound 7d
5.15
3.61
5.07
5.63
3.42
1.00
1.43
1.02
0.91
1.51
0.991
0.990
0.991
0.990
0.995
Fig. 10. Emission spectra of HSA þ Ca2þ in the presence of various concentrations of
compound 7d. c(HSA) 1.0 ꢂ 10ꢁ5 M; c(Ca2þ)/c(HSA) ¼ 1/1; c(compound 7d)/(10ꢁ5 M),
aek: from 0.0 to 2.0 at increments of 0.2; dash line shows the emission spectrum of
compound 7d only; T ¼ 298 K, lex ¼ 295 nm. The inset corresponds to the Scatchard
plot.
HSAecompound 7deCa2þ
HSAecompound 7deNi2þ
HSAecompound 7deMg2þ
HSAecompound 7deKþ

326
S.-F. Cui et al. / European Journal of Medicinal Chemistry 86 (2014) 318e334
preliminary interactive investigations of compound 7d with calf
thymus DNA revealed that these hybrids gave better interaction
with DNA than norfloxacin, which might improve binding ability
between these hybrids with topo IVeDNA complex.
6. Experimental
6.1. General methods
Melting points were recorded on X-6 melting point apparatus
and uncorrected. TLC analysis was done using pre-coated silica gel
plates. FT-IR spectra were carried out on Bruker RFS100/S spec-
trophotometer (Bio-Rad, Cambridge, MA, USA) using KBr pellets in
the 400e4000 cmꢁ1 range. NMR spectra were recorded on a Bruker
AV 300 spectrometer using TMS as an internal standard. The
chemical shifts were reported in parts per million (ppm), the
coupling constants (J) were expressed in hertz (Hz) and signals
were described as singlet (s), doublet (d), triplet (t), as well as
multiplet (m). The mass spectra were recorded on LCMS-2010A and
the high-resolution mass spectra (HRMS) were recorded on an
IonSpec FT-ICR mass spectrometer with ESI resource. All fluores-
cence spectra were recorded on F-7000 Spectrofluorimeter (Hita-
chi, Tokyo, Japan) equipped with 1.0 cm quartz cells, the widths of
both the excitation and emission slit were set as 2.5 nm, and the
excitation wavelength was 295 nm. The UV spectrum was recorded
at room temperature on a TU-2450 spectrophotometer (Puxi Ana-
lytic Instrument Ltd. of Beijing, China) equipped with 1.0 cm quartz
cells. HSA, CKK kit and MTT was obtained from SigmaeAldrich (St.
Louis, MO, USA). Tris, NaCl and HCl were analytical purity. HSA was
dissolved in TriseHCl buffer solution (0.05 M Tris, 0.15 M NaCl,
pH ¼ 7.4). Sample masses were weighed on a microbalance with a
resolution of 0.1 mg. All other chemicals and solvents were
commercially available, and were used without further purification.
Fig. 11. Three-dimensional conformations of compound 7d docked in topoisomerase
IVeDNA complexes.
5. Conclusion
In conclusion, starting from commercially available materials, a
novel class of hybrids of metronidazole and quinolone were
designed and synthesized in good yields by a convenient and effi-
cient procedure. All the new compounds were characterized by IR,
1H NMR, 13C NMR, MS, and HRMS spectra. The in vitro antimicrobial
evaluation against nine bacterial and five fungal strains revealed
that most of the newly synthesized compounds exhibited good
antibacterial and antifungal activities against the tested strains in
comparison with the reference drugs chloromycin, norfloxacin and
fluconazole. The prepared hybrids 7b and 7d showed significant
inhibition against all the tested bacterial strains with low inhibitory
6.1.1. General procedures for the preparation of intermediates
(1aeh) and (2aeh)
Compounds 1aeh and 2aeh were prepared according to the
literature procedures [43].
concentrations (MIC ¼ 0.5e8
mg/mL). Especially, nitroimidazole
derivatives 7aed and 7g showed comparable or superior potency
against MRSA to reference drugs. Furthermore, 1-(oxiran-2-
ylmethyl)-quinolones 3aeh exhibited better antifungal activities
against most of the tested fungal strains including A. flavus, B. yeast,
C. albicans and C. mycoderma than fluconazole. Structure activity
relationship indicated that compounds with fluoro substituent on
benzene ring exerted important effect on antimicrobial activities.
Moreover, the antibacterial activities of these hybrids were also
affected by the substituents on N-1 position of quinolone, which
followed the order of 2-methyl-5-nitroimidazolyl derivatives > 4-
nitroimidazolyl derivatives. Cell toxicity suggested most of the
target compounds did not show obvious cytotoxicity to A459 and
human hepatocyte LO2 cell lines. The pKa values of target hybrids
were also evaluated ranging from 5.145 to 6.430, which showed
appropriate range to pharmacokinetic behaviors. The target com-
pounds also gave suitable log P values ranging from ꢁ0.25 to 0.22
and good aqueous solubility (0.35e0.79 mg/mL). The metal ions’
influence on their transportation by HSA revealed that the partic-
ipation of Mg2þ ion in compound 7deHSA association could result
in the concentration increase of free compound 7d, shorten the
storage time and half-life of compound 7d in the blood, and thus
may improve its antimicrobial efficacy. Further molecular modeling
indicated the good antibacterial activity of the prepared hybrids
against quinolone-resistant bacterial strains and the possible
mechanism might be explained by the noncovalent interaction
between molecular 7d and topo IVeDNA complex, especially these
hydrogen bonds between molecule and ParC helix a4 residues
Ser79 (resistance mutation region of topo IVeDNA complex). The
6.1.2. Synthesis of ethyl 6,8-difluoro-1-(oxiran-2-ylmethyl)-4-oxo-
1,4-dihydroquinoline-3-carboxylate (3a)
A mixture of compound 2a (0.253 g, 1.0 mmol) and potassium
carbonate (0.151 g, 1.2 mmol) was stirred under reflux in 2-
(chloromethyl)oxirane (15 mL) for 3 h. When the reaction was
completed (monitored by TLC, eluent, chloroform/methanol 70/1,
V/V), the mixture was cooled to room temperature, and then the
excess 2-(chloromethyl)oxirane was evaporated under reduced
pressure. After water was added, the resulting mixture was
extracted with chloroform (3 ꢂ 20 mL). The combined organic
phase was dried over anhydrous Na2SO4, and then the solvent was
evaporated. The residue was purified via silica gel column chro-
matography (eluent, chloroform/methanol ¼ 70/1, V/V) to give
compound 3a (0.294 g) as light yellow solid. Yield: 95.1%; m.p:
>250 ꢀC. IR (KBr, cmꢁ1
(CH2, CH3), 1683, 1641 (C]O), 1609, 1559, 1493 (aromatic frame);
1H NMR (300 MHz, DMSO-d6):
8.51 (s, quinolone-2-H, 1H),
) n: 3131 (AreH), 3016 (]CeH), 2988, 2902
d
7.84e7.93 (m, quinolone-5-H, 1H), 7.79e7.81 (m, quinolone-7-H,
1H), 4.85e4.90 (m, NeCH2, 1H), 4.45e4.50 (m, NeCH2, 1H), 4.26 (q,
J ¼ 7.1 Hz, OeCH2CH3, 2H), 3.46 (s, OeCH, 1H), 2.54e2.55 (m,
OeCH2, 2H), 1.29 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR
(75 MHz, DMSO-d6):
d 171.0, 164.5, 160.2, 157.0, 154.6, 152.2, 151.3,
131.9, 130.3, 110.3, 60.4, 57.2, 50.3, 44.9, 14.7 ppm; MS (ESI): m/z 310
[MþH]þ; HRMS (ESI) calcd. for C15H13F2NO4 [MþH]þ, 310.0813;
found, 310.0811.

S.-F. Cui et al. / European Journal of Medicinal Chemistry 86 (2014) 318e334
327
120.1, 110.5, 111.1, 61.5, 57.4, 50.6, 45.1, 22.1, 14.7 ppm; MS (ESI): m/z
288 [MþH]þ; HRMS (ESI) calcd. for C16H17NO4 [MþH]þ, 288.1236;
found, 288.1234.
0.6
0.5
0.4
0.3
0.2
0.1
0.0
DNA+compound 7d
DNA-compound 7d complex
0.40
0.38
0.36
0.34
0.32
f
6.1.4. Synthesis of ethyl 6-fluoro-1-(oxiran-2-ylmethyl)-4-oxo-1,4-
dihydroquinoline-3-carboxylate (3c)
Compound 3c was prepared according to the procedure depic-
ted for compound 3a, starting from compound 2c (0.235 g,
1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-(chlor-
omethyl)oxirane (15 mL). The product 3c (0.262 g) was obtained as
white solid. Yield: 90.0%; m.p: >250 ꢀC. IR (KBr, cmꢁ1) IR (KBr,
4
8
12
16
20
a
10-6 (mol/L)
cmꢁ1
)
n
: 3131 (AreH), 3015 (]CeH), 2988, 2902 (CH2, CH3), 1725,
1641 (C]O), 1609, 1559, 1493 (aromatic frame); 1H NMR (300 MHz,
DMSO-d6):
DNA
d
8.49 (s, quinolone-2-H, 1H), 8.11 (d, J ¼ 8.0 Hz, qui-
nolone-5-H, 1H), 7.66e7.75 (m, quinolone-7-H, 1H), 7.45e7.51 (m,
quinolone-8-H, 1H), 4.51e4.89 (m, NeCH2, 2H), 4.28 (q, J ¼ 7.1 Hz,
OeCH2CH3, 2H), 3.46 (s, OeCH, 1H), 2.54e2.75 (m, OeCH2, 2H), 1.31
(t, J ¼ 7.2 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6):
250
300
350
Wavelength (nm)
d
172.0, 164.7, 151.1, 150.8, 135.5,131.1, 129.1, 125.2, 120.3,110.6, 60.4,
57.7, 50.6, 45.1, 14.7 ppm; MS (ESI): m/z 292 [MþH]þ; HRMS (ESI)
Fig. 12. UV absorption spectra of DNA with different concentrations of compound 7d
(pH ¼ 7.4, T ¼ 288 K). Inset: comparison of absorption at 260 nm between the
DNAecompound 7d complex and the sum values of free DNA and free compound 7d.
c(DNA) ¼ 7.61 ꢂ 10ꢁ5 mol/L, and c(compound 7d) ¼ 0e20 ꢂ 10ꢁ6 mol/L for curves aef
calcd. for C15H14FNO4 [MþH]þ, 292.0985; found, 292.0984.
6.1.5. Synthesis of ethyl 1-(oxiran-2-ylmethyl)-4-oxo-7-
(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylate (3d)
respectively at increment 4 ꢂ 10ꢁ6
.
Compound 3d was prepared according to the procedure depic-
ted for compound 3a, starting from compound 2d (0.285 g,
1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-(chlor-
omethyl)oxirane (15 mL). The product 3d (0.302 g) was ꢁo1btained as
0.8
DNA + norfloxacin
DNA-norfloxacin complex
white solid. Yield: 88.3%; m.p: >250 ꢀC; IR (KBr, cm
) n: 3129
(AreH), 3061 (]CeH), 2989, 2906 (CH2, CH3), 1730, 1635 (C]O),
0.7
0.6
0.5
0.4
1609, 1556, 1494 (aromatic frame); 1H NMR (300 MHz, DMSO-d6):
d
8.75 (s, quinolone-2-H, 1H), 8.45 (d, J ¼ 8.0 Hz, quinolone-5-H,
1H), 8.27 (s, quinolone-8-H, 1H), 7.82 (d, J ¼ 3.2 Hz, quinolone-6-H,
1H), 4.45e5.04 (m, NeCH2, 2H), 4.29 (q, J ¼ 7.1 Hz, OeCH2CH3, 2H),
3.41 (m, OeCH, 1H), 2.64e2.87 (m, OeCH2, 2H), 1.32 (t, J ¼ 7.2 Hz,
OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6):
d 176.8, 167.2,
151.1, 137.3, 129.7, 122.1, 124.5, 132.2, 126.5, 120.7, 112.1, 60.5, 57.4,
50.6, 45.1, 14.7 ppm; MS (ESI): m/z 342 [MþH]þ; HRMS (ESI) calcd.
for C16H14F3NO4 [MþH]þ, 342.0953; found, 342.0950.
6.1.6. Synthesis of ethyl 7-chloro-6-fluoro-1-(oxiran-2-ylmethyl)-4-
oxo-1,4-dihydroquinoline-3-carboxylate (3e)
4
8
12
16
20
Compound 3e was prepared according to the procedure depic-
ted for compound 3a, starting from compound 2e (0.270 g,1 mmol),
potassium carbonate (0.151 g, 1.2 mmol) and 2-(chloromethyl)
oxirane (15 mL). The product 3e (0.294 g) was obtained as white
10-6 (mol/L)
Fig. 13. Comparison of absorption at 260 nm between the DNAenorfloxacin complex
and the sum values of free DNA and free norfloxacin. c(DNA) ¼ 7.61 ꢂ 10ꢁ5 mol/L, and
solid. Yield: 90.5%; m.p: >250 ꢀC; IR (KBr, cmꢁ1
)
n
: 3127 (AreH),
3015 (]CeH), 2989, 2902 (CH2, CH3), 1731, 1642 (C]O), 1617, 1555,
1497 (aromatic frame); 1H NMR (300 MHz, DMSO-d6):
8.64 (s,
c(norfloxacin) ¼ 0e20 ꢂ 10ꢁ6 mol/L at increment of 4 ꢂ 10ꢁ6
.
d
quinolone-2-H, 1H), 8.28 (d, J ¼ 3.7 Hz, quinolone-5-H, 1H), 8.06 (s,
quinolone-8-H, 1H), 4.45e4.92 (m, 2H), 4.27 (q, J ¼ 7.1 Hz,
OeCH2CH3, 2H), 3.41 (m, OeCH, 1H), 2.64e2.86 (m, OeCH2, 2H),
1.31 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-
6.1.3. Synthesis of ethyl 6-methyl-1-(oxiran-2-ylmethyl)-4-oxo-1,4-
dihydroquinoline-3-carboxylate (3b)
Compound 3b was prepared according to the procedure depic-
ted for compound 3a, starting from compound 2b (0.231 g,
1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-(chlor-
omethyl)oxirane (15 mL). The product 3b (0.253 g) was ꢁo1btained as
d6): d 174.8, 167.5, 151.1, 139.7, 132.3, 129.3, 127.4, 128.7, 124.5, 114.1,
60.5, 57.7, 50.6, 45.1, 14.7 ppm; MS (ESI): m/z 326 [MþH]þ; HRMS
(ESI) calcd. for C15H13ClFNO4 [MþH]þ, 326.0595; found, 326.0593.
white solid. Yield: 88.2%; m.p: >250 ꢀC; IR (KBr, cm
) n: 3128
(AreH), 3060 (]CeH), 2989, 2905 (CH2, CH3), 1740, 1680 (C]O),
6.1.7. Synthesis of ethyl 6,8-dichloro-1-(oxiran-2-ylmethyl)-4-oxo-
1,4-dihydroquinoline-3-carboxylate (3f)
1608, 1555, 1495 (aromatic frame); 1H NMR (300 MHz, DMSO-d6):
d
8.85 (s, quinolone-2-H, 1H), 8.19 (s, quinolone-5-H, 1H), 7.98 (s,
Compound 3f was prepared according to the procedure depic-
ted for compound 3a, starting from compound 2f (0.286 g,
1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-(chlor-
omethyl)oxirane (15 mL). The product 3f (0.284 g) was ꢁo1btained as
quinolone-7-H, 1H), 7.83 (d, J ¼ 3.1 Hz, quinolone-8-H, 1H),
4.51e4.89 (m, NeCH2, 2H), 4.26 (q, J ¼ 7.1 Hz, OeCH2CH3, 2H), 3.45
(m, OeCH, 1H), 2.55e2.84 (m, OeCH2, 2H), 2.36 (s, quinolone-6-
CH3, 3H), 1.31 (t, J ¼ 7.2 Hz, OeCH2CH3, 3H) ppm; 13C NMR
white solid. Yield: 83.1%; m.p: >250 ꢀC; IR (KBr, cm
) n: 3125
(75 MHz, DMSO-d6):
d
174.0, 166.2, 134.7, 150.2, 121.4, 131.0, 125.9,
(AreH), 3015 (]CeH), 2989, 2902 (CH2, CH3), 1725, 1634 (C]O),

328
S.-F. Cui et al. / European Journal of Medicinal Chemistry 86 (2014) 318e334
1608, 1557, 1498 (aromatic frame); 1H NMR (300 MHz, DMSO-d6):
8.51 (s, quinolone-2-H, 1H), 8.08e8.19 (m, quinolone-5-H, 1H),
(300 MHz, DMSO-d6): d 8.58 (s, quinolone-2-H, H), 8.42 (s, imid-
d
azole-5-H, 1H), 7.86 (s, imidazole-2-H, 1H), 7.41e7.44 (m, quino-
lone-5-H, 1H), 7.24e7.28 (m, quinolone-7-H, 1H), 5.84 (s, eOH, 1H),
4.76 (m, CH, 1H), 4.21e4.71 (m, eCH2e, 6H), 1.28 (t, J ¼ 7.1 Hz,
7.69 (s, quinolone-8-H, 1H), 4.48e4.93 (m, 2H, NeCH2), 4.24 (q,
J ¼ 7.2 Hz, OeCH2CH3, 2H), 3.46 (s, OeCH, 1H), 2.54e2.84 (m,
OeCH2, 2H), 1.29 (t, J ¼ 7.0 Hz, OeCH2CH3, 3H) ppm; 13C NMR
OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6):
d 172.3, 164.8,
(75 MHz, DMSO-d6):
d
172.0, 164.6, 153.8, 151.2, 150.5, 131.1, 125.9,
152.9, 149.6, 147.8, 145.2, 138.6, 135.8, 131.5, 129.0, 126.9, 123.0,
109.9, 68.4, 60.8, 60.4, 51.1, 14.7 ppm; MS (ESI): m/z 423 [MþH]þ;
HRMS (ESI) calcd. for C18H16F2N4O6 [MþH]þ, 423.1116; found,
423.1114.
123.0, 120.6, 111.0, 60.5, 57.7, 50.6, 45.1, 14.7 ppm. MS (ESI): m/z 342
[MþH]þ; HRMS (ESI) calcd. for C15H13Cl2NO4 [MþH]þ, 342.0300;
found, 342.0303.
6.1.8. Synthesis of ethyl 8-fluoro-1-(oxiran-2-ylmethyl)-4-oxo-1,4-
dihydroquinoline-3-carboxylate (3g)
6.1.11. Synthesis of ethyl 1-(2-hydroxy-3-(4-nitro-1H-imidazol-1-
yl)propyl)-6-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylate
(4b)
Compound 3g was prepared according to the experimental
procedure described for compound 3a, starting from compound 2g
(0.235 g, 1 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-
(chloromethyl)oxirane (15 mL). The product 3g (0.257 g) waꢁs1ob-
Compound 4b was prepared according to the procedure depic-
ted for compound 4a, starting from compound 3b (0.287 g,
1.0 mmol), potassium carbonate (0.151 g,1.2 mmol) and 4-nitro-1H-
imidazole (0.113 g, 1.0 mmol). The product 4b (0.334 g) waꢁs1ob-
tained as yellow solid. Yield: 88.3%; m.p: >250 ꢀC; IR (KBr, cm
) n:
3127 (AreH), 3015 (]CeH), 2989, 2902 (CH2, CH3), 1735, 1638 (C]
tained as white solid. Yield: 83.5%; m.p: >250 ꢀC; IR (KBr, cm
) n:
O), 1612, 1555, 1495 (aromatic frame); 1H NMR (300 MHz, DMSO-
3350 (OeH), 3122 (AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3),
d6):
d
8.49 (s, quinolone-2-H, 1H), 8.11 (d, J ¼ 9.3 Hz, quinolone-5-H,
1708, 1682 (C]O),1609,1555, 1497,1401 (aromatic frame); 1H NMR
1H), 7.66e7.75 (m, quinolone-7-H, 1H), 7.45e7.51 (m, quinolone-6-
H, 1H), 4.51e4.89 (m, NeCH2, 2H), 4.28 (q, J ¼ 7.1 Hz, OeCH2CH3,
2H), 3.46 (m, OeCH, 1H), 2.57e2.75 (m, OeCH2, 2H), 1.31 (t,
J ¼ 7.2 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6):
(300 MHz, DMSO-d6): d 8.82 (s, quinolone-2-H, 1H), 8.41 (s, imid-
azole-5-H, 1H), 8.18 (s, quinolone-5-H, 1H), 7.98 (s, quinolone-7-H,
1H), 7.83e7.86 (m, quinolone-8-H and imidazole-2-H, 2H), 5.84 (m,
eOH, 1H), 4.73 (m, eCH, 1H), 4.17e4.43 (m, eCH2e, 6H), 2.36 (s,
d
172.0, 164.6, 153.8, 152.2, 150.5, 131.1, 129.1, 123.0, 120.3, 110.6,
quinolone-6-CH3, 3H), 1.28 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13
C
60.4, 57.5, 50.6, 45.1, 14.7 ppm; MS (ESI): m/z 292 [MþH]þ; HRMS
NMR (75 MHz, DMSO-d6): d 173.6, 161.2, 153.1, 151.2, 149.5, 140.4,
(ESI) calcd. for C15H14FNO4 [MþH]þ, 292.0985; found, 292.0982.
137.7, 135.1, 131.2, 124.5, 121.2, 115.3, 110.5, 69.0, 60.7, 60.3, 52.6,
25.6, 14.7 ppm; MS (ESI): m/z 401 [MþH]þ; HRMS (ESI) calcd. for
6.1.9. Synthesis of ethyl 6-methoxy-1-(oxiran-2-ylmethyl)-4-oxo-
1,4-dihydroquinoline-3-carboxylate (3h)
C
19H20N4O6 [MþH]þ, 401.1461; found, 401.1463.
Compound 3h was prepared according to the procedure
depicted for compound 3a, starting from compound 2h (0.247 g,
1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-(chlor-
omethyl)oxirane (15 mL). The product 3h (0.246) was obtained as
6.1.12. Synthesis of ethyl 6-fluoro-1-(2-hydroxy-3-(4-nitro-1H-
imidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate
(4c)
Compound 4c was prepared according to the procedure depic-
ted for compound 4a, starting from compound 3c (0.291 g,
1.0 mmol), potassium carbonate (0.151 g,1.2 mmol) and 4-nitro-1H-
imidazole (0.113 g, 1.0 mmol). The product 4c (0.350 g) waꢁs1ob-
white solid. Yield: 81.2%; m.p: >250 ꢀC; IR (KBr, cmꢁ1
) n: 3128
(AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1720, 1641 (C]O),
1616, 1557, 1498 (aromatic frame); 1H NMR (300 MHz, DMSO-d6):
d
8.57 (s, quinolone-2-H,1H), 7.89 (d, J ¼ 3.4 Hz, quinolone-5-H,1H),
tained as white solid. Yield: 86.6%; m.p: >250 ꢀC; IR (KBr, cm
) n:
7.68 (s, quinolone-7-H, 1H), 7.39e7.43 (m, quinolone-8-H, 1H),
4.43e4.85 (m, NeCH2, 2H), 4.26 (q, J ¼ 7.1 Hz, OeCH2CH3, 2H), 3.87
(s, quinolone-6-OCH3, 3H), 3.35 (s, OeCH, 1H), 2.57e2.85 (m,
OeCH2, 2H), 1.29 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR
3412 (OeH), 3128 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3),
1713, 1682 (C]O), 1611, 1556, 1494, 1400 (aromatic frame); 1H NMR
(300 MHz, DMSO-d6):
d 8.43 (s, quinolone-2-H, H), 8.41 (s, imid-
azole-5-H, 1H), 8.12 (d, J ¼ 3.2 Hz, quinolone-5-H, 1H), 7.85 (s,
imidazole-2-H, 1H), 7.64e7.72 (m, quinolone-7-H, 1H), 7.46e7.50
(m, quinolone-8-H, 1H), 5.78 (d, J ¼ 3.2 Hz, eOH, 1H), 4.67e4.82 (m,
eCH, 1H), 4.04e4.39 (m, eCH2e, 6H), 1.29 (t, J ¼ 7.2 Hz, OeCH2CH3,
(75 MHz, DMSO-d6):
d 172.9, 165.2, 157.0, 151.2, 131.1, 129.3, 123.5,
120.1, 108.8, 107.2, 60.2, 57.7, 55.9, 50.6, 45.1, 14.7 ppm; MS (ESI): m/
z 304 [MþH]þ; HRMS (ESI) calcd. for C16H17NO5 [MþH]þ, 304.1185;
found, 304.1183.
3H) ppm; 13C NMR (75 MHz, DMSO-d6):
d 173.3, 164.8, 151.3, 147.8,
145.2, 138.6, 131.5, 126.9, 129.0, 127.9, 122.8, 119.0, 109.9, 68.4, 60.8,
60.2, 51.1, 14.7 ppm; MS (ESI): m/z 427 [MþNa]þ; HRMS (ESI) calcd.
for C18H17FN4O6 [MþNa]þ, 427.1030; found, 427.1028.
6.1.10. Synthesis of ethyl 6,8-difluoro-1-(2-hydroxy-3-(4-nitro-1H-
imidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate
(4a)
To a stirred suspension of potassium carbonate (0.151 g,
1.2 mmol) in ethanol was added 4-nitro-1H-imidazole (0.113 g,
1.0 mmol). The mixture was stirred at 60 ꢀC for 1.5 h, and then was
cooled to room temperature. Compound 3a (0.309 g, 1.0 mmol) was
added, and the mixture was stirred on reflux for 2 h. After the re-
action came to end (monitored by TLC, eluent, chloroform/meth-
anol, 20/1, V/V), the solvent was evaporated and the residue was
treated with water and extract with chloroform (3 ꢂ 20 mL). The
combined organic extracts was dried over anhydrous sodium sul-
fate and concentrated under reduced pressure. The crude product
was purified via silica gel column chromatography (eluent, chlo-
roform/methanol ¼ 20/1, V/V) to afford compound 4a (0.408 g) as
6.1.13. Synthesis of ethyl 1-(2-hydroxy-3-(4-nitro-1H-imidazol-1-
yl)propyl)-4-oxo-7-(trifluoromethyl)-1,4-dihydroquinoline-3-
carboxylate (4d)
Compound 4d was prepared according to the procedure depic-
ted for compound 4a, starting from compound 3d (0.341 g,
1.0 mmol), potassium carbonate (0.151 g,1.2 mmol) and 4-nitro-1H-
imidazole (0.113 g, 1.0 mmol). The product 4d (0.364 g) waꢁs1ob-
tained as white solid. Yield: 80.2%; m.p: >250 ꢀC; IR (KBr, cm
) n:
3441 (OeH), 3130 (AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3),
1708, 1682 (C]O), 1631, 1555, 1497, 1400 (aromatic frame); 1H NMR
(300 MHz, DMSO-d6):
d 8.68 (s, quinolone-2-H, 1H), 8.44 (s, qui-
nolone-5-H, 1H), 8.41 (s, imidazole-5-H, 1H), 8.19 (s, quinolone-8-H,
1H), 7.86 (s, imidazole-2-H, 1H), 7.79 (d, J ¼ 8.4 Hz, quinolone-6-H,
1H), 5.83 (m, eOH, 1H), 4.75 (m, eCHe, 1H), 4.17e4.43 (m, eCH2e,
6H), 1.28 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz,
yellow solid. Yield: 96.7%; m.p: >250 ꢀC. IR (KBr, cmꢁ1
) n: 3443
(OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1771,
1676 (C]O), 1614, 1564, 1508, 1399 (aromatic frame); 1H NMR

S.-F. Cui et al. / European Journal of Medicinal Chemistry 86 (2014) 318e334
329
DMSO-d6):
d
173.6, 164.8, 121.6, 147.8, 145.2, 139.4, 138.6, 135.5,
6.1.17. Synthesis of ethyl 7-chloro-1-(2-hydroxy-3-(4-nitro-1H-
imidazol-1-yl)propyl)-6-methoxy-4-oxo-1,4-dihydroquinoline-3-
carboxylate (4h)
129.4, 129.9, 126.9, 121.2, 110.1, 109.9, 68.4, 60.8, 60.2, 51.1,
14.7 ppm; MS (ESI): m/z 455 [MþH]þ; HRMS (ESI) calcd. for
C
19H17F3N4O6 [MþH]þ, 455.1187; found, 455.1186.
Compound 4h was prepared according to the procedure
depicted for compound 4a, starting from compound 3h (0.303 g,
1.0 mmol), potassium carbonate (0.151 g,1.2 mmol) and 4-nitro-1H-
imidazole (0.113 g, 1.0 mmol). The product 4h (0.338 g) waꢁs1ob-
6.1.14. Synthesis of ethyl 7-chloro-6-fluoro-1-(2-hydroxy-3-(4-
nitro-1H-imidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3-
carboxylate (4e)
tained as white solid. Yield: 81.2%; m.p: >250 ꢀC; IR (KBr, cm
) n:
3413 (OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3),
1713, 1682 (C]O), 1611, 1556, 1494, 1400 (aromatic frame); 1H NMR
Compound 4e was prepared according to the procedure depic-
ted for compound 4a, starting from compound 3e (0.326 g,
1.0 mmol), potassium carbonate (0.151 g,1.2 mmol) and 4-nitro-1H-
imidazole (0.113 g, 1.0 mmol). The product 4e (0.348 g) waꢁs1ob-
(300 MHz, DMSO-d6):
d 8.51 (s, quinolone-2-H, H), 8.41 (s, imid-
azole-5-H, 1H), 7.85 (s, quinolone-5-H, 1H), 7.82 (s, imidazole-2-H,
1H), 7.67 (d, J ¼ 7.3 Hz, quinolone-7-H, 1H), 7.43 (m, quinolone-8-H,
1H), 5.75 (d, J ¼ 3.7 Hz, eOH, 1H), 4.82 (d, J ¼ 3.6 Hz, eCHe, 1H),
4.08e4.51(m, eCH2e, 6H), 3.87 (s, quinolone-6-OCH3, 3H), 1.28 (t,
J ¼ 7.0 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6):
tained as white solid. Yield: 79.5%; m.p: >250 ꢀC; IR (KBr, cm
) n:
3434 (OeH), 3125 (AreH), 3016 (]CeH), 2998, 2912 (CH2, CH3),
1728, 1684 (C]O), 1631, 1555, 1497, 1400 (aromatic frame); 1H NMR
(300 MHz, DMSO-d6):
d 8.57 (s, quinolone-2-H, 1H), 8.42 (s, imid-
d
172.3, 164.8, 147.7, 146.1, 145.3, 138.6, 135.6, 126.9, 122.3, 120.8,
azole-5-H, 1H), 8.23 (d, J ¼ 4.3 Hz, quinolone-5-H, 1H), 8.05 (s,
quinolone-8-H, 1H), 7.87 (s, imidazole-2-H, 1H), 5.76 (s, eOH, 1H),
4.75 (m, eCHe, 1H), 4.16e4.55 (m, eCH2e, 6H), 1.28 (t, J ¼ 7.1 Hz,
110.5, 109.0, 68.5, 60.8, 60.2, 55.9, 51.1, 25.7, 14.7 ppm; MS (ESI): m/z
417 [MþH]þ; HRMS (ESI) calcd. for C19H20N4O7 [MþH]þ, 417.1410;
found, 417.1410.
OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6):
d 176.4, 164.8,
150.3,147.8,145.2,141.8,138.6,127.9,131.5,129.0,123.9,115.0,109.9,
68.4, 60.8, 60.4, 51.0, 14.7 ppm; MS (ESI): m/z 439 [MþH]þ; HRMS
(ESI) calcd. for C18H16ClFN4O6 [MþH]þ, 439.0821; found, 439.0820.
6.1.18. Synthesis of ethyl 6,8-difluoro-1-(2-hydroxy-3-(2-methyl-5-
nitro-1H-imidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3-
carboxylate (5a)
To a stirred suspension of potassium carbonate (0.151 g,
1.2 mmol) in ethanol was added 2-methyl-5-nitro-1H-imidazole
(0.127 g, 1.0 mmol). The mixture was stirred at 60 ꢀC for 1.5 h. When
the reaction was cooled to room temperature, compound 3a
(0.309 g, 1.0 mmol) was added and stirred on reflux for 2 h. After
the reaction came to end (monitored by TLC, eluent, chloroform/
methanol, 20/1, V/V), the solvent was evaporated and the residue
was treated with water and extract with chloroform (3 ꢂ 20 mL).
The combined organic extracts was dried over anhydrous sodium
sulfate and concentrated under reduced pressure. The crude
product was purified via silica gel column chromatography (eluent,
chloroform/methanol ¼ 20/1, V/V) to give compound 5a (0.371 g) as
6.1.15. Synthesis of ethyl 6,8-dichloro-1-(2-hydroxy-3-(4-nitro-1H-
imidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate
(4f)
Compound 4f was prepared according to the procedure depic-
ted for compound 4a, starting from compound 3f (0.326 g,
1.0 mmol), potassium carbonate (0.151 g,1.2 mmol) and 4-nitro-1H-
imidazole (0.113 g, 1.0 mmol). The product 4f (0.369 g) waꢁs1ob-
tained as white solid. Yield: 81.2%; m.p: >250 ꢀC. IR (KBr, cm
) n:
3439 (OeH), 3127 (AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3),
1721, 1684 (C]O), 1621, 1555, 1497, 1400 (aromatic frame); 1H NMR
(300 MHz, DMSO-d6):
d 8.51 (s, quinolone-2-H, 1H), 8.41 (s, imid-
yellow solid. Yield: 85.2%; m.p: >250 ꢀC. IR (KBr, cmꢁ1
) n: 3443
azole-5-H, 1H), 8.23 (s, quinolone-5-H, 1H), 8.08e8.19 (m, quino-
lone-7-H, 1H), 7.69 (s, imidazole-2-H, 1H), 5.20e5.25 (m, eOH, 1H),
4.73e4.80 (m, eCHe, 1H), 4.28e4.56 (m, eCH2e, 6H), 1.29 (t,
J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6):
(OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1771,
1676 (C]O), 1614, 1564, 1508, 1399 (aromatic frame); 1H NMR
(300 MHz, DMSO-d6):
d 8.58 (s, quinolone-2-H, H), 8.32 (s, imid-
azole-4-H, 1H), 7.41e7.46 (m, quinolone-5-H, 1H), 7.24e7.28 (m,
quinolone-7-H, 1H), 5.82 (s, eOH, 1H), 4.73 (m, eCHe, 1H),
4.21e4.71 (m, eCH2e, 6H), 2.41 (s, imidazole-2-CH3, 3H), 1.28 (t,
J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6):
d
175.2, 163.7, 153.1, 146.7, 136.2, 152.1, 151.4, 148.4, 134.1, 132.1,
131.2, 124.8, 110.5, 69.0, 60.8, 60.4, 52.5, 14.8 ppm; MS (ESI): m/z
455 [MþH]þ; HRMS (ESI) calcd. for C18H16Cl2N4O6 [MþH]þ,
455.0525; found, 455.0521.
d
173.4, 164.8, 152.8, 151.3, 149.6, 145.2, 138.6, 135.7, 132.5, 131.5,
129.0, 123.0, 109.9, 68.4, 60.8, 60.2, 51.1, 21.7, 14.7 ppm; MS (ESI): m/
z 437 [MþH]þ; HRMS (ESI) calcd. for C19H18F2N4O6 [MþH]þ,
437.1273; found, 437.1270.
6.1.16. Synthesis of ethyl 8-fluoro-1-(2-hydroxy-3-(4-nitro-1H-
imidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate
(4g)
Compound 4g was prepared according to the procedure depic-
ted for compound 4a, starting from compound 3g (0.291 g,
1.0 mmol), potassium carbonate (0.151 g,1.2 mmol) and 4-nitro-1H-
imidazole (0.113 g, 1.0 mmol). The product 4g (0.398 g) waꢁs1ob-
6.1.19. Synthesis of ethyl 1-(2-hydroxy-3-(2-methyl-5-nitro-1H-
imidazol-1-yl)propyl)-6-methyl-4-oxo-1,4-dihydroquinoline-3-
carboxylate (5b)
Compound 5b was prepared according to the procedure depic-
ted for compound 5a, starting from compound 3a (0.287 g,
1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-methyl-
5-nitro-1H-imidazole (0.127 g, 1.0 mmol). The product 5b (0.367 g)
was obtained as white solid. Yield: 88.4%; m.p: >250 ꢀC; IR (KBr,
tained as white solid. Yield: 98.5%; m.p: >250 ꢀC; IR (KBr, cm
) n:
3417 (OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3),
1713, 1682 (C]O), 1614, 1557, 1494, 1400 (aromatic frame); 1H NMR
(300 MHz, DMSO-d6):
d
8.43 (d, J ¼ 1.2 Hz, quinolone-2-H, 1H), 8.41
(s, imidazole-5-H, H), 8.12 (m, quinolone-5-H, 1H), 7.86 (s, imid-
azole-2-H, 1H), 7.63e7.71 (m, quinolone-7-H, 1H), 7.43e7.50 (m,
quinolone-6-H, 1H), 5.73 (s, eOH, 1H), 4.75 (m, eCHe, 1H),
4.15e4.43 (m, eCH2e, 6H), 1.28 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm;
cmꢁ1
)
n
: 3350 (OeH), 3122 (AreH), 3016 (]CeH), 2996, 2903 (CH2,
CH3), 1708,1682 (C]O),1609,1555,1497,1401 (aromatic frame); 1H
NMR (300 MHz, DMSO-d6): 8.51 (s, quinolone-2-H, H), 8.31 (s,
d
d
imidazole-4-H, H), 7.87 (s, quinolone-5-H, 1H), 7.67 (d, J ¼ 8.3 Hz,
quinolone-7-H, 1H), 7.42 (m, quinolone-8-H, 1H), 5.85 (s, eOH, 1H),
4.83 (d, J ¼ 13.2 Hz, eCHe, 1H), 4.08e4.50 (m, eCH2e, 6H), 3.87 (s,
quinolone-6-CH3, 3H), 2.41 (s, imidazole-2-CH3, 3H), 1.28 (t,
J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6):
13C NMR (75 MHz, DMSO-d6):
d 173.3, 164.8, 152.9, 147.8, 145.2,
138.6, 126.9, 131.5, 129.0, 125.6, 122.8, 109.9, 119.0, 68.4, 60.8, 60.2,
51.1, 14.7 ppm; MS (ESI): m/z 427 [MþNa]þ; HRMS (ESI) calcd. for
C
18H17FN4O6 [MþNa]þ, 427.1030; found, 427.1028.

330
S.-F. Cui et al. / European Journal of Medicinal Chemistry 86 (2014) 318e334
d
176.3, 164.8, 149.9, 146.1, 134.6, 131.3, 133.5, 128.0, 132.8, 124.9,
6.1.23. Synthesis of ethyl 6,8-dichloro-1-(2-hydroxy-3-(2-methyl-
5-nitro-1H-imidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3-
carboxylate (5f)
120.8, 119.0, 110.5, 68.4, 60.8, 60.2, 51.1, 25.7, 21.7, 14.7 ppm; MS
(ESI): m/z 414 [MþH]þ; HRMS (ESI) calcd. for C20H22N4O6 [MþH]þ,
415.1612; found, 415.11610.
Compound 5f was prepared according to the procedure depic-
ted for compound 5a, starting from compound 3f (0.341 g,
1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-methyl-
5-nitro-1H-imidazole (0.127 g, 1.0 mmol). The product 5f (0.381 g)
was obtained as white solid. Yield: 81.2%; m.p: >250 ꢀC. IR (KBr,
6.1.20. Synthesis of ethyl 6-fluoro-1-(2-hydroxy-3-(2-methyl-5-
nitro-1H-imidazol-1-yl)propyl)-4-oxo-1,4 -dihydroquinoline-3-
carboxylate (5c)
Compound 5c was prepared according to the procedure depic-
ted for compound 5a, starting from compound 3c (0.291 g,
1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-methyl-
5-nitro-1H-imidazole (0.127 g, 1.0 mmol). The product 5c (0.345 g)
was obtained as white solid. Yield: 82.3%; m.p: >250 ꢀC; IR (KBr,
cmꢁ1
)
n
: 3439 (OeH), 3127 (AreH), 3016 (]CeH), 2996, 2903 (CH2,
CH3), 1721, 1684 (C]O), 1621, 1555, 1497, 1400 (aromatic frame); 1H
NMR (300 MHz, DMSO-d6): 8.62 (s, quinolone-2-H, 1H), 8.31 (s,
d
imidazole-4-H, 1H), 7.69 (d, J ¼ 2.1 Hz, quinolone-5-H, 1H), 7.38 (d,
J ¼ 2.1 Hz, quinolone-7-H, 1H), 5.80 (s, eOH, 1H), 4.78 (m, eCHe,
1H), 4.15e4.48 (m, eCH2e, 6H), 2.41 (s, imidazole-2-CH3, 3H), 1.28
(t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6):
cmꢁ1
)
n
: 3412 (OeH), 3128 (AreH), 3016 (]CeH), 2989, 2902 (CH2,
CH3), 1713, 1682 (C]O), 1611, 1556, 1494, 1400 (aromatic frame); 1H
NMR (300 MHz, DMSO-d6):
d
8.43 (d, J ¼ 1.2 Hz, quinolone-2-H,
d 174.4, 164.8, 149.8, 147.8, 139.8, 136.5, 134.6, 132.7, 131.5, 129.0,
1H), 8.32 (s, imidazole-4-H, H), 8.12 (d, J ¼ 3.4 Hz, quinolone-5-H,
1H), 7.64e7.72 (m, quinolone-7-H, 1H), 7.46e7.50 (m, quinolone-8-
H, 1H), 5.88 (s, eOH, 1H), 4.82 (m, eCHe, 1H), 4.04e4.39 (m,
eCH2e, 6H), 2.41 (s, imidazole-2-CH3, 3H), 1.29 (t, J ¼ 7.2 Hz,
131.1, 122.6, 109.9, 68.5, 60.8, 60.2, 51.0, 21.7, 14.7 ppm; MS (ESI): m/
z 469 [MþH]þ; HRMS (ESI) calcd. for C19H18Cl2N4O6 [MþH]þ,
469.0982; found, 469.0977.
OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6):
d 173.3, 164.8,
151.3,149.8,149.8,134.6,131.9,131.5,129.0,127.9,122.8,119.0,109.9,
68.4, 60.8, 60.2, 51.1, 21.7, 14.7 ppm; MS (ESI): m/z 441 [MþNa]þ;
HRMS (ESI) calcd. for C19H19FN4O6 [MþNa]þ, 441.1186; found,
441.1183.
6.1.24. Synthesis of ethyl 8-fluoro-1-(2-hydroxy-3-(2-methyl-5-
nitro-1H-imidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3-
carboxylate (5g)
Compound 5g was prepared according to the procedure depic-
ted for compound 5a, starting from compound 3g (0.291 g,
1 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-methyl-5-
nitro-1H-imidazole (0.127 g, 1.0 mmol). The product 5g (0.349 g)
was obtained as white solid. Yield: 83.4%; m.p: >250 ꢀC; IR (KBr,
6.1.21. Synthesis of ethyl 1-(2-hydroxy-3-(2-methyl-5-nitro-1H-
imidazol-1-yl)propyl)-4-oxo-6-(trifluoromethyl)-1,4-
dihydroquinoline-3-carboxylate (5d)
Compound 5d was prepared according to the procedure depic-
ted for compound 5a, starting from compound 3d (0.341 g,
1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-methyl-
5-nitro-1H-imidazole (0.127 g, 1.0 mmol). The product 5d (0.418 g)
was obtained as white solid. Yield: 89.3%; m.p: >250 ꢀC; IR (KBr,
cmꢁ1
)
n
: 3417 (OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2,
CH3), 1713, 1682 (C]O), 1614, 1557, 1494, 1400 (aromatic frame); 1H
NMR (300 MHz, DMSO-d6):
d
8.43 (d, J ¼ 1.2 Hz, quinolone-2-H,
1H), 8.31 (s, imidazole-4-H, 1H), 8.12 (m, quinolone-5-H, 1H),
7.63e7.72 (m, quinolone-7-H, 1H), 7.43e7.50 (m, quinolone-6-H,
1H), 5.83 (s, eOH, 1H), 4.78 (m, eCHe, 1H), 4.15e4.43 (m, eCH2e,
6H), 2.41 (s, imidazole-2-CH3, 3H),1.28 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H)
cmꢁ1
)
n
: 3441 (OeH), 3130 (AreH), 3016 (]CeH), 2996, 2903 (CH2,
CH3), 1708, 1682 (C]O),1631, 1555, 1497, 1400 (aromatic frame); 1H
NMR (300 MHz, DMSO-d6): 8.70 (s, quinolone-2-H, 1H), 8.45 (s,
d
imidazole-4-H, 1H), 8.28 (m, quinolone-5-H, 2H), 7.80 (d, J ¼ 8.0 Hz,
quinolone-6-H, 1H), 7.43 (d, J ¼ 3.0 Hz, quinolone-8-H, 1H), 5.94 (s,
eOH, 1H), 4.81 (d, J ¼ 14.4 Hz, eCHe, 1H), 4.11e4.39 (m, eCH2e,
6H), 3.42 (s, imidazole-2-CH3, 3H), 1.28 (t, J ¼ 7.1 Hz, OeCH2CH3,
ppm; 13C NMR (75 MHz, DMSO-d6):
d 174.0, 164.8, 152.9, 149.9,
146.2, 134.6, 132.7, 131.5, 129.0, 125.6, 122.8, 109.9, 119.0, 68.4, 60.6,
60.2, 51.1, 21.7, 14.7 ppm; MS (ESI): m/z 441 [MþNa]þ; HRMS (ESI)
calcd. for C19H19FN4O6 [MþNa]þ, 441.1186; found, 441.1185.
3H) ppm; 13C NMR (75 MHz, DMSO-d6):
d 173.6, 164.8, 151.7, 149.8,
145.2, 142.3, 139.4, 134.6, 135.5, 129.4, 132.6, 121.2, 110.1, 109.9, 68.4,
60.7, 60.2, 51.1, 21.7, 14.7 ppm; MS (ESI): m/z 469 [MþH]þ; HRMS
(ESI) calcd. for C20H19F3N4O6 [MþH]þ, 469.1335; found, 469.1334.
6.1.25. Synthesis of ethyl 1-(2-hydroxy-3-(2-methyl-5-nitro-1H-
imidazol-1-yl)propyl)-6-methoxy-4-oxo-1,4-dihydroquinoline-3-
carboxylate (5h)
6.1.22. Synthesis of ethyl 7-chloro-6-fluoro-1-(2-hydroxy-3-(2-
methyl-5-nitro-1H-imidazol-1-yl)propyl)-4-oxo-1,4-
dihydroquinoline-3-carboxylate (5e)
Compound 5h was prepared according to the procedure
depicted for compound 5a, starting from compound 3h (0.303 g,
1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-methyl-
5-nitro-1H-imidazole (0.127 g, 1.0 mmol). The product 5h (0.347 g)
was obtained as white solid. Yield: 80.6%; m.p: >250 ꢀC; IR (KBr,
Compound 5e was prepared according to the procedure depic-
ted for compound 5a, starting from compound 3e (0.326 g,
1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-methyl-
5-nitro-1H-imidazole (0.127 g, 1.0 mmol). The product 5e (0.381 g)
was obtained as white solid. Yield: 84.3%; m.p: >250 ꢀC; IR (KBr,
cmꢁ1
)
n
: 3413 (OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2,
CH3), 1713, 1682 (C]O), 1611, 1556, 1494, 1400 (aromatic frame); 1H
NMR (300 MHz, DMSO-d6): 8.53 (s, quinolone-2-H, H), 8.34 (s,
cmꢁ1
)
n
: 3434 (OeH), 3125 (AreH), 3016 (]CeH), 2998, 2912 (CH2,
CH3), 1728, 1684 (C]O), 1631, 1555, 1497, 1400 (aromatic frame); 1H
NMR (300 MHz, DMSO-d6): 8.57 (s, quinolone-2-H, 1H), 8.30 (s,
d
d
imidazole-4-H, H), 7.93 (d, J ¼ 9.3 Hz, quinolone-5-H, 1H), 7.67 (d,
J ¼ 2.7 Hz, quinolone-7-H, 1H), 7.40 (m, quinolone-8-H, 1H), 5.95 (s,
eOH, 1H), 4.70 (d, J ¼ 12.7 Hz, eCHe, 1H), 4.12e4.35 (m, eCH2e,
6H), 3.87 (s, quinolone-6-OCH3, 3H), 2.41 (s, imidazole-2-CH3, 3H),
1.28 (t, J ¼ 7.0 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-
imidazole-4-H, 1H), 8.23 (d, J ¼ 3.3 Hz, quinolone-5-H, 1H), 8.05 (s,
quinolone-8-H, 1H), 5.86 (s, eOH, 1H), 4.78 (m, eCHe, 1H),
4.16e4.54 (m, eCH2e, 6H), 3.42 (s, imidazole-2-CH3, 3H), 1.28 (t,
J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6):
d
176.4, 164.8, 150.3, 149.8, 145.2, 141.8, 134.6, 132.6, 131.5, 129.0,
d6): d 172.3, 164.8, 147.7, 145.3, 134.6, 135.6, 126.9, 132.7, 122.3,
123.9, 115.0, 109.9, 68.4, 60.8, 60.2, 51.0, 21.7, 14.7 ppm; MS (ESI): m/
z 453 [MþH]þ; HRMS (ESI) calcd. for C19H18ClFN4O6 [MþH]þ,
453.0977; found, 453.0976.
120.8, 110.5, 109.0, 68.4, 60.6, 60.2, 55.9, 51.1, 25.7, 21.7, 14.7 ppm;
MS (ESI): m/z 431 [MþH]þ; HRMS (ESI) calcd. for C20H22N4O7
[MþH]þ, 431.1567; found, 431.1566.

S.-F. Cui et al. / European Journal of Medicinal Chemistry 86 (2014) 318e334
331
6.1.26. Synthesis of 6,8-difluoro-1-(2-hydroxy-3-(4-nitro-1H-
imidazol-1-yl)propyl)-4-oxo-1,4 dihydroquinoline-3-carboxylic acid
(6a)
(AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3), 1708, 1682 (C]O),
1631, 1555, 1497, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-
d6):
d 15.43 (s, eCOOH, 1H), 8.68 (s, quinolone-2-H, 1H), 8.44 (s,
To a stirred solution of aqueous solution NaOH (3%, 20 mL) was
added compound 4a (0.422 g, 1.0 mmol). The mixture was stirred at
100 ꢀC for 2 h. After the reaction was completed (monitored by TLC,
eluent, chloroform/methanol ¼ 15/1, V/V), the solution was treated
with formic acid to reach pH ¼ 7. The mixture was filtered and
washed with water for three times to give compound 6a (0.337 g)
quinolone-5-H, 1H), 8.41 (s, imidazole-5-H, 1H), 8.19 (s, quinolone-
6-H, 1H), 7.86 (s, imidazole-2-H, 1H), 7.79 (d, J ¼ 8.4 Hz, quinolone-
8-H, 1H), 5.83 (s, eOH, 1H), 4.76 (m, eCHe, 1H), 4.17e4.43 (m,
eCH2e, 4H) ppm; 13C NMR (75 MHz, DMSO-d6):
d 176.5, 165.7,
151.6, 147.8,145.2,139.4,138.6,135.5,129.4,129.9,126.9,121.2, 110.1,
109.9, 68.4, 60.8, 51.1 ppm; MS (ESI): m/z 427 [MþH]þ; HRMS (ESI)
calcd. for C17H13F3N4O6 [MþH]þ, 427.0865; found, 427.0864.
as white solid. Yield: 85.2%; m.p: >250 ꢀC. IR (KBr, cmꢁ1
) n: 3443
(OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1771,
1676 (C]O), 1614, 1564, 1508, 1399 (aromatic frame); 1H NMR
6.1.30. Synthesis of 7-chloro-6-fluoro-1-(2-hydroxy-3-(4-nitro-1H-
imidazol-1-yl)propyl)-4-oxo-1,4 -dihydroquinoline-3-carboxylic
acid (6e)
(300 MHz, DMSO-d6): d 14.83 (s, eCOOH, 1H), 8.56 (s, quinolone-2-
H, 1H), 8.42 (s, imidazole-5-H, 1H), 7.86 (s, imidazole-2-H, 1H),
7.41e7.43 (m, quinolone-5-H, 1H), 7.24e7.29 (m, quinolone-7-H,
1H), 5.83 (s, eOH, 1H), 4.76 (m, eCHe, 1H), 4.21e4.71 (m, eCH2e,
Compound 6e was prepared according to the procedure depic-
ted for compound 6a, starting from compound 4e (0.438 g,
1.0 mmol). The product 6e (0.362 g) wasꢁo1btained as white solid.
4H) ppm; 13C NMR (75 MHz, DMSO-d6):
d 173.3, 165.7, 152.9, 149.6,
147.8, 145.2, 138.6, 135.8, 131.5, 129.0, 126.9, 123.0, 110.3, 68.4, 60.8,
Yield: 88.3%; m.p: >250 ꢀC; IR (KBr, cm
) n: 3434 (OeH), 3125
51.1 ppm; MS (ESI): m/z 395 [MþH]þ; HRMS (ESI) calcd. for
(AreH), 3016 (]CeH), 2998, 2912 (CH2, CH3), 1728, 1684 (C]O),
C
16H12F2N4O6 [MþH]þ, 395.0803; found, 395.0801.
1631, 1555, 1497, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-
d6):
d 14.83 (s, eCOOH, 1H), 8.57 (s, quinolone-2-H, 1H), 8.45 (s,
6.1.27. Synthesis of 1-(2-hydroxy-3-(4-nitro-1H-imidazol-1-yl)
propyl)-6-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(6b)
imidazole-5-H, 1H), 8.25 (d, J ¼ 5.3 Hz, quinolone-5-H, 1H), 8.09 (s,
quinolone-8-H, 1H), 7.87 (s, imidazole-2-H, 1H), 5.87 (s, eOH, 1H),
4.78 (m, eCHe, 1H), 4.12e4.56 (m, eCH2e, 4H) ppm; 13C NMR
Compound 6b was prepared according to the procedure depic-
ted for compound 6a, starting from compound 4b (0.400 g,
1.0 mmol). The product 6b (0.325 g) wasꢁo1btained as white solid.
(75 MHz, DMSO-d6):
d 177.2, 165.6, 150.3, 147.8, 145.2, 141.8, 138.6,
127.9, 131.5, 129.0, 123.9, 115.0, 110.0, 68.4, 60.7, 51.0 ppm; MS (ESI):
m/z 411 [MþH]þ; HRMS (ESI) calcd. for C16H12ClFN4O6 [MþH]þ,
411.0508; found, 411.0507.
Yield: 87.3%; m.p: >250 ꢀC; IR (KBr, cm
) n: 3350 (OeH), 3122
(AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3), 1708, 1682 (C]O),
1609,1555, 1497,1401 (aromatic frame); 1H NMR (300 MHz, DMSO-
6.1.31. Synthesis of 6,8-dichloro-1-(2-hydroxy-3-(4-nitro-1H-
imidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (6f)
d6):
d 14.54 (s, eCOOH, 1H), 8.51 (s, quinolone-2-H, 1H), d 8.40 (s,
imidazole-5-H, 1H), 7.87 (s, quinolone-5-H, 1H), 7.82 (s, imidazole-
2-H, 1H), 7.65 (d, J ¼ 9.3 Hz, quinolone-7-H, 1H), 7.42 (m, quinolone-
8-H, 1H), 5.75 (s, eOH, 1H), 4.76 (d, J ¼ 13.2 Hz, eCHe, 1H),
Compound 6f was prepared according to the procedure depic-
ted for compound 6a, starting from compound 4f (0.455 g,
1.0 mmol). The product 6f (0.346 g) wasꢁo1btained as white solid.
4.08e4.51 (m, eCH2e, 4H), 3.87 (s, quinolone-6-CH3, 3H) ppm; 13
C
NMR (75 MHz, DMSO-d6):
d 176.8, 165.4, 147.8, 146.1, 138.6, 131.3,
Yield: 81.1%; m.p: >250 ꢀC. IR (KBr, cm
) n: 3439 (OeH), 3127
128.0, 126.8, 124.9, 120.8, 110.6, 119.0, 68.4, 60.8, 51.1, 25.7 ppm; MS
(ESI): m/z 373 [MþH]þ; HRMS (ESI) calcd. for C16H16N4O6 [MþH]þ,
373.1148; found, 373.1148.
(AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3), 1721, 1684 (C]O),
1621, 1555, 1497, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-
d6): d15.23 (s, eCOOH, 1H), 8.44 (s, quinolone-2-H, 1H), 8.40 (s,
imidazole-5-H, 1H), 8.07e8.15 (m, quinolone-5-H, 1H), 7.87 (s,
imidazole-2-H, 1H), 7.68 (s, quinolone-7-H, 1H), 5.83 (s, eOH, 1H),
4.82e4.87 (m, eCHe,1H), 4.16e4.29 (m, eCH2e, 4H) ppm; 13C NMR
6.1.28. Synthesis of 6-fluoro-1-(2-hydroxy-3-(4-nitro-1H-imidazol-
1-yl)propyl)-4-oxo-1,4 -dihydroquinoline-3-carboxylic acid (6c)
Compound 6c was prepared according to the procedure depic-
ted for compound 6a, starting from compound 4c (0.404 g,
1.0 mmol). The product 6c (0.348 g) wasꢁo1btained as white solid.
(75 MHz, DMSO-d6):
d 177.3, 166.1, 147.8, 145.2, 139.8, 138.6, 136.5,
130.8, 131.5, 129.0, 126.9, 122.6, 109.3, 68.5, 60.8, 51.0 ppm; MS
(ESI): m/z 427 [MþH]þ; HRMS (ESI) calcd. for C16H12Cl2N4O6
[MþH]þ, 427.0212; found, 427.0210.
Yield: 92.6%; m.p: >250 ꢀC; IR (KBr, cm
) n: 3412 (OeH), 3128
(AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1713, 1682 (C]O),
1611, 1556, 1494, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-
d6):
d
14.63 (s, eCOOH, 1H), 8.53 (s, quinolone-2-H, 1H), 8.42 (s,
6.1.32. Synthesis of 8-fluoro-1-(2-hydroxy-3-(4-nitro-1H-imidazol-
1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (6g)
Compound 6g was prepared according to the procedure depic-
ted for compound 6a, starting from compound 4g (0.404 g,
1 mmol). The product 6g (0.338 g) was ꢁo1btained as white solid.
imidazole-5-H, 1H), 8.13 (d, J ¼ 3.8 Hz, quinolone-5-H, 1H), 7.85 (s,
imidazole-2-H, 1H), 7.64e7.70 (m, quinolone-7-H, 1H), 7.46e7.50
(m, quinolone-8-H, 1H), 5.84 (s, eOH, 1H), 4.67e4.80 (m, eCHe,
1H), 4.04e4.39 (m, eCH2e, 4H) ppm; 13C NMR (75 MHz, DMSO-d6):
d
176.3, 165.7, 151.3, 147.8, 145.2, 138.6, 126.9, 131.5, 129.0, 127.9,
Yield: 89.7%; m.p: >250 ꢀC; IR (KBr, cm
) n: 3417 (OeH), 3129
122.8,118.0,110.3, 68.4, 60.8, 51.1 ppm; MS (ESI): m/z 399 [MþNa]þ;
HRMS (ESI) calcd. for C16H13FN4O6 [MþNa]þ, 399.1717; found,
399.1716.
(AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1713, 1682 (C]O),
1614, 1557, 1494, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-
d6):
d
14.65 (s, eCOOH, 1H), 8.43 (d, J ¼ 1.5 Hz, quinolone-2-H, 1H),
8.40 (s, imidazole-5-H, H), 8.12 (m, quinolone-5-H, 1H), 7.86 (s,
imidazole-2-H, 1H), 7.60e7.70 (m, quinolone-7-H, 1H), 7.43e7.53
(m, quinolone-6-H, 1H), 5.78 (s, eOH, 1H), 4.75 (m, eCHe, 1H),
4.15e4.43 (m, eCH2e, 4H) ppm; 13C NMR (75 MHz, DMSO-d6):
6.1.29. Synthesis of 1-(2-hydroxy-3-(4-nitro-1H-imidazol-1-yl)
propyl)-4-oxo-6-(trifluoromethyl)-1,4-dihydroquinoline-3-
carboxylic acid (6d)
Compound 6d was prepared according to the procedure depic-
ted for compound 6a, starting from compound 4d (0.454 g,
1.0 mmol). The product 6d (0.339 g) wasꢁo1btained as white solid.
d 175.3, 165.6, 152.9, 147.8, 145.2, 138.6, 126.9, 131.5, 129.0, 125.6,
122.8, 110.3, 109.0, 68.4, 60.8, 51.1 ppm; MS (ESI): m/z 399
[MþNa]þ; HRMS (ESI) calcd. for C16H13FN4O6 [MþNa]þ, 399.1717;
found, 399.1716.
Yield: 79.5%; m.p: >250 ꢀC; IR (KBr, cm
) n: 3441 (OeH), 3130

332
S.-F. Cui et al. / European Journal of Medicinal Chemistry 86 (2014) 318e334
6.1.33. Synthesis of 1-(2-hydroxy-3-(4-nitro-1H-imidazol-1-yl)
propyl)-6-methoxy-4-oxo-1,4 -dihydroquinoline-3-carboxylic acid
(6h)
d6): d 8.52 (s, quinolone-2-H,1H), 8.32 (s, imidazole-4-H, H), 8.12 (d,
J ¼ 7.8 Hz, quinolone-5-H, 1H), 7.64e7.72 (m, quinolone-7-H, 1H),
7.46e7.50 (m, quinolone-8-H, 1H), 5.88 (s, eOH, 1H), 4.82 (m,
eCHe, 1H), 4.04e4.39 (m, eCH2e, 4H), 2.41 (s, imidazole-2-CH3,
Compound 6h was prepared according to the procedure
depicted for compound 6a, starting from compound 4h (0.416 g,
1.0 mmol). The product 6h (0.302 g) wasꢁo1btained as white solid.
3H) ppm; 13C NMR (75 MHz, DMSO-d6):
d 175.3, 165.0, 151.3, 149.8,
149.7, 134.6, 131.9, 131.5, 129.0, 127.9, 122.8, 119.0, 110.0, 68.4, 60.8,
Yield: 77.8%; m.p: >250 ꢀC; IR (KBr, cm
)
n
: 3413 (OeH), 3129
51.1, 21.7 ppm; MS (ESI): m/z 413 [MþNa]þ; HRMS (ESI) calcd. for
(AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1713, 1682 (C]O),
C
17H17FN4O4 [MþNa]þ, 413.0873; found, 413.0872.
1611, 1556, 1494, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-
d6):
d 14.43 (s, eCOOH, 1H), 8.58 (s, quinolone-2-H, 1H), 8.43 (s,
imidazole-5-H, 1H), 7.87 (s, quinolone-5-H, 1H), 7.80 (s, imidazole-
2-H, 1H), 7.67 (d, J ¼ 8.3 Hz, quinolone-7-H,1H), 7.43 (m, quinolone-
8-H, 1H), 5.85 (s, eOH, 1H), 4.82 (m, eCHe, 1H), 4.08e4.51(m,
eCH2e, 4H), 3.87 (s, quinolone-6-OCH3, 3H) ppm; 13C NMR
6.1.37. Synthesis of 1-(2-hydroxy-3-(2-methyl-5-nitro-1H-
imidazol-1-yl)propyl)-4-oxo-6-(trifluoromethyl) -1,4-
dihydroquinoline-3-carboxylic acid (7d)
Compound 7d was prepared according to the procedure depic-
ted for compound 6a, starting from compound 5d (0.468 g,
1.0 mmol). The product 7d (0.396 g) wasꢁo1btained as white solid.
(75 MHz, DMSO-d6):
d 174.8, 165.6, 147.7, 146.1, 145.3, 138.6, 135.7,
126.9, 122.3, 120.8, 110.3, 109.0, 68.4, 60.8, 55.9, 51.1, 25.7 ppm; MS
(ESI): m/z 389 [MþH]þ; HRMS (ESI) calcd. for C17H16N4O7 [MþH]þ,
389.1097; found, 389.1097.
Yield: 90.0%; m.p: >250 ꢀC; IR (KBr, cm
) n: 3441 (OeH), 3130
(AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3), 1708, 1682 (C]O),
1631, 1555, 1497, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-
6.1.34. Synthesis of 6,8-difluoro-1-(2-hydroxy-3-(2-methyl-5-
nitro-1H-imidazol-1-yl)propyl)-4-oxo-1,4 -dihydroquinoline-3-
carboxylic acid (7a)
d6): d 15.69 (s, eCOOH, 1H), 8.65 (s, quinolone-2-H, 1H), 8.45 (s,
quinolone-5-H, 1H), 8.30 (m, quinolone-6-H and imidazole-4-H,
2H), 7.80 (m, quinolone-8-H, 1H), 5.94 (s, eOH, 1H), 4.81 (m, eCHe,
1H), 4.17e4.43 (m, eCH2e, 4H), 3.42 (s, imidazole-2-CH3, 3H) ppm;
Compound 7a was prepared according to the procedure depic-
ted for compound 6a, starting from compound 5a (0.436 g,
1.0 mmol). The product 7a (0.367 g) wasꢁo1btained as white solid.
13C NMR (75 MHz, DMSO-d6):
d 175.6, 164.8, 121.6, 149.8, 145.2,
139.4, 134.6, 135.5, 129.4, 129.9, 132.6, 121.2, 110.1, 109.9, 68.4, 60.2,
Yield: 90.0%; m.p: >250 ꢀC; IR (KBr, cm
)
n
: 3443 (OeH), 3129
51.1, 21.7 ppm; MS (ESI): m/z 441 [MþH]þ; HRMS (ESI) calcd. for
(AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1771, 1676 (C]O),
C
18H15F3N4O6 [MþH]þ, 441.1202; found, 441.1203.
1614,1564,1508,1399 (aromatic frame); 1H NMR (300 MHz, DMSO-
d6):
d 14.67 (s, eCOOH, 1H), 8.59 (s, quinolone-2-H, 1H), 8.31 (s,
imidazole-4-H, 1H), 7.41e7.46 (m, quinolone-5-H, 1H), 7.24e7.28
(m, quinolone-7-H, 1H), 5.82 (s, eOH, 1H), 4.73 (m, eCHe, 1H),
6.1.38. Synthesis of 7-chloro-6-fluoro-1-(2-hydroxy-3-(2-methyl-5-
nitro-1H-imidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (7e)
4.21e4.65 (m, eCH2e, 4H), 2.41 (s, imidazole-2-CH3, 3H) ppm; 13
C
NMR (75 MHz, DMSO-d6):
d
175.4, 165.5, 152.8, 151.3, 149.6, 145.2,
Compound 7e was prepared according to the procedure depic-
ted for compound 6a, starting from compound 5e (0.452 g,
1.0 mmol). The product 7e (0.388 g) wasꢁo1btained as white solid.
138.6, 135.7, 132.5, 131.5, 129.0, 123.0, 110.4, 68.4, 60.8, 51.1,
21.7 ppm; MS (ESI): m/z 409 [MþH]þ; HRMS (ESI) calcd. for
C
17H14F2N4O6 [MþH]þ, 409.3210; found, 409.3209.
Yield: 91.5%; m.p: >250 ꢀC; IR (KBr, cm
) n: 3434 (OeH), 3125
(AreH), 3016 (]CeH), 2998, 2912 (CH2, CH3), 1728, 1684 (C]O),
6.1.35. Synthesis of 1-(2-hydroxy-3-(2-methyl-5-nitro-1H-
imidazol-1-yl)propyl)-6-methyl-4-oxo-1,4 -dihydroquinoline-3-
carboxylic acid (7b)
1631, 1555, 1497, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-
d6):
d 8.57 (s, quinolone-2-H, 1H), 8.30 (s, imidazole-4-H, 1H), 8.23
(d, J ¼ 3.3 Hz, quinolone-5-H, 1H), 8.05 (s, quinolone-8-H, 1H), 5.86
(s, eOH, 1H), 4.78 (m, eCHe, 1H), 4.26e4.57 (m, eCH2e, 4H), 3.42
(s, imidazole-2-CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6):
Compound 7b was prepared according to the procedure depic-
ted for compound 6a, starting from compound 5b (0.414 g,
1.0 mmol). The product 7b (0.341 g) wasꢁo1btained as white solid.
d
176.4, 165.9, 150.3, 149.8, 145.2, 141.8, 134.6, 132.6, 131.5, 129.0,
Yield: 88.3%; m.p: >250 ꢀC; IR (KBr, cm
)
n
: 3350 (OeH), 3122
123.9, 115.0, 109.9, 68.4, 60.7, 51.0, 21.7 ppm; MS (ESI): m/z 425
[MþH]þ; HRMS (ESI) calcd. for C17H14ClFN4O6 [MþH]þ, 425.0664;
found, 425.0662.
(AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3), 1708, 1682 (C]O),
1609, 1555, 1497, 1401 (aromatic frame); 1H NMR (300 MHz, DMSO-
d6):
d 15.38 (s, eCOOH, 1H), 8.51 (s, quinolone-2-H, 1H), d 8.34 (s,
imidazole-4-H, H), 7.86 (s, quinolone-5-H, 1H), 7.67 (d, J ¼ 5.1 Hz,
quinolone-7-H, 1H), 7.42 (m, quinolone-8-H, 1H), 5.85 (s, eOH, 1H),
4.83 (m, eCHe, 1H), 4.08e4.53 (m, eCH2e, 4H), 3.87 (s, quinolone-
6-CH3, 3H), 2.41 (s, imidazole-2-CH3, 3H) ppm; 13C NMR (75 MHz,
6.1.39. Synthesis of 6,8-dichloro-1-(2-hydroxy-3-(2-methyl-5-
nitro-1H-imidazol-1-yl)propyl)-4-oxo ꢁ1,4-dihydroquinoline-3-
carboxylic acid (7f)
DMSO-d6):
d
176.9, 165.9, 149.9, 146.1, 134.6, 131.3, 133.5, 128.0,
Compound 7f was prepared according to the procedure depic-
ted for compound 6a, starting from compound 5f (0.468 g,
1.0 mmol). The product 7f (0.391 g) wasꢁo1btained as white solid.
132.8, 124.9, 120.8, 110.3, 119.0, 68.4, 60.8, 51.1, 25.7, 21.7 ppm; MS
(ESI): m/z 387 [MþH]þ; HRMS (ESI) calcd. for C18H18N4O6 [MþH]þ,
387.1305; found, 387.1303.
Yield: 81.2%; m.p: >250 ꢀC; IR (KBr, cm
) n: 3439 (OeH), 3127
(AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3), 1721, 1684 (C]O),
6.1.36. Synthesis of 6-fluoro-1-(2-hydroxy-3-(2-methyl-5-nitro-
1H-imidazol-1-yl)propyl)-4-oxo-1,4 -dihydroquinoline-3-carboxylic
acid (7c)
1621, 1555, 1497, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-
d6):
d 15.42 (s, eCOOH, 1H), 8.48 (s, quinolone-2-H, 1H), 8.30 (s,
imidazole-4-H, 1H), 8.02e8.15 (m, quinolone-5-H, 1H), 7.68 (s,
quinolone-7-H, 1H), 5.87 (s, eOH, 1H), 4.87 (m, eCHe, 1H),
Compound 7c was prepared according to the procedure depic-
ted for compound 6a, starting from compound 5c (0.418 g,
1.0 mmol). The product 7c (0.347 g) wasꢁo1btained as white solid.
4.16e4.32 (m, eCH2e, 4H), 2.41 (s, imidazole-2-CH3, 3H) ppm; 13
C
NMR (75 MHz, DMSO-d6):
d 175.3, 164.9, 149.8, 147.8, 139.8, 136.5,
Yield: 89.0%; m.p: >250 ꢀC; IR (KBr, cm
)
n: 3412 (OeH), 3128
134.6, 132.7, 131.5, 129.0, 131.1, 122.6, 110.0, 68.5, 60.8, 51.0,
(AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1713, 1682 (C]O),
21.7 ppm; MS (ESI): m/z 441 [MþH]þ; HRMS (ESI) calcd. for
1611, 1556, 1494, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-
C
17H14Cl2N4O6 [MþH]þ, 441.0369; found, 441.0368.

S.-F. Cui et al. / European Journal of Medicinal Chemistry 86 (2014) 318e334
333
6.1.40. Synthesis of 8-fluoro-1-(2-hydroxy-3-(2-methyl-5-nitro-
1H-imidazol-1-yl)propyl)-4-oxo -1,4-dihydroquinoline-3-carboxylic
acid (7g)
prepare the stock solutions, then the tested compounds and
reference drugs were prepared in MuellereHinton broth (Guang-
dong huaikai microbial sci. & tech co., Ltd, Guangzhou, Guangdong,
China) to obtain the required concentrations of 512, 256, 128, 64,
Compound 7g was prepared according to the procedure depic-
ted for compound 6a, starting from compound 5g (0.418 g,
1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 1H-1,2,4-
triazole (0.691 g, 1.0 mmol). The product 7g (0.398 g) was ob-
32, 16, 8, 4, 2, 1, 0.5 mg/mL. These dilutions were inoculated and
incubated at 37 ꢀC for 24 h.
tained as white solid. Yield: 86.3%; m.p: >250 ꢀC; IR (KBr, cmꢁ1
) n:
6.2.2. Antifungal assays
3417 (OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3),
The newly synthesized compounds 3e7 were evaluated for their
antifungal activities against C. albicans ATCC 76615, A. fumigatus
ATCC 96918, C. utilis, S. cerevisia and A. flavus. A spore suspension in
sterile distilled water was prepared from one day old culture of the
fungi growing on Sabouraud agar (SA) media. The final spore con-
centration was 1e5 ꢂ 103 spore mLꢁ1. From the stock solutions of
the tested compounds and reference antifungal drug Fluconazole,
dilutions in sterile RPMI 1640 medium (Neuronbc Laboraton
Technology CO., Ltd, Beijing, China) were made resulting in eleven
1713, 1682 (C]O), 1614, 1557, 1494, 1400 (aromatic frame); 1H NMR
(300 MHz, DMSO-d6): d 14.58 (s, eCOOH, 1H), 8.51 (s, quinolone-2-
H, 1H), 8.31 (s, imidazole-4-H, H), 8.12 (m, quinolone-5-H, 1H),
7.65e7.72 (m, quinolone-7-H, 1H), 7.43e7.50 (m, quinolone-6-H,
1H), 5.87 (s, eOH, 1H), 4.74 (m, eCHe, 1H), 4.15e4.43 (m, eCH2e,
4H), 2.41 (s, imidazole-2-CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-
d6):
d 176.0, 165.3, 152.9, 149.9, 146.2, 134.6, 132.7, 131.5, 129.0,
125.6, 122.8, 109.5, 119.0, 68.4, 60.8, 51.1, 21.7 ppm; MS (ESI): m/z
413 [MþNa]þ; HRMS (ESI) calcd. for C17H17FN4O4 [MþNa]þ,
413.0873; found, 413.0872.
wanted concentrations (0.25e256
mg/mL) of each tested com-
pound. These dilutions were inoculated and incubated at 35 ꢀC for
24 h.
6.1.41. Synthesis of 1-(2-hydroxy-3-(2-methyl-5-nitro-1H-
imidazol-1-yl)propyl)-6-methoxy-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (7h)
6.3. Cytotoxicity assays
Compound 7h was prepared according to the procedure
depicted for compound 6a, starting from compound 5h (0.430 g,
1.0 mmol). The product 7h (0.326 g) wasꢁo1btained as white solid.
A549 cells and human hepatocyte LO2 cells were plated in 96-
well plates in 100
mL of medium and allowed to attach for 24 h, and
Yield: 81.2%; m.p: >250 ꢀC; IR (KBr, cm
)
n
: 3413 (OeH), 3129
then 100 L of medium containing an appropriate dilution of the
m
(AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1713, 1682 (C]O),
test compounds was added. Cells and compounds were incubated
at 37 ꢀC in 5% CO2 humidified incubator for 2 days. Cell viability was
determined by using CCK-8 kit and MTT respectively. Absorbance of
converted dye was measured in an ELISA plate reader at the
wavelength of 450 nm and 490 nm respectively. The cytotoxicity of
the compounds was tested for three times and calculated as the
reduction percentage of viable cells observed in the drug-free
control cell culture.
1611, 1556, 1494, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-
d6):
d 15.41 (s, eCOOH, 1H), 8.57 (s, quinolone-2-H, H), 8.34 (s,
imidazole-4-H, H), 7.93 (s, quinolone-5-H, 1H), 7.67 (d, J ¼ 4.2 Hz,
quinolone-7-H, 1H), 7.40 (m, quinolone-8-H, 1H), 5.95 (s, eOH, 1H),
4.70 (m, eCHe, 1H), 4.12e4.36 (m, eCH2e, 4H), 3.87 (s, quinolone-
6-OCH3, 3H), 2.41 (s, imidazole-2-CH3, 3H) ppm; 13C NMR (75 MHz,
DMSO-d6):
d 174.9, 165.4, 147.7, 145.3, 134.6, 135.7, 126.9, 132.7,
122.3, 120.8, 110.6, 109.0, 68.4, 60.8, 55.9, 51.1, 25.7, 21.7 ppm; MS
(ESI): m/z 403 [MþH]þ; HRMS (ESI) calcd. for C18H18N4O7 [MþH]þ,
403.1254; found, 403.1254.
6.4. Assays of pKa values
Buffer stock solutions of 500 mM were prepared and adjusted to
the final pH as follows: acetic acidesodium acetate (pH ¼ 4.0, 4.5,
and 5.0), MES (pH ¼ 5.5, 6.0, and 6.5), HEPES (pH ¼ 7.0, 7.5, and 8.0),
and CHES (pH ¼ 8.5, 8.8, 9.1, and 9.4). Analyte compound stock
solutions were prepared at 4.0 ꢂ 10ꢁ3 mol/L in DMSO. Quartz cell
6.2. Antibacterial and antifungal assays
Minimal inhibitory concentration (MIC, mg/mL) is defined as the
lowest concentration of target compounds that completely inhibit
the growth of bacteria, by means of standard two-fold serial dilu-
tion method in 96-well microtest plates according to the National
Committee for Clinical Laboratory Standards (NCCLS). The tested
microorganism strains were provided by the School of Pharma-
ceutical Sciences, Southwest University and the College of Phar-
macy, Third Military Medical University. Chloromycin, Norfloxacin
and Fluconazole were used as control drugs. DMSO with inocula-
tion bacterial not medicine was used as positive control to ensure
that the solvent had no effect on bacteria growth. All the bacteria
and fungi growth was monitored visually and spectrophotometri-
cally, and the experiments were performed in triplicate. The MIC
was loaded with 2 mL of buffer solutions. Then, 50 mL of the com-
pound stock solutions were added to the quartz cell with a
micropipette (the resulting analyte solution was premixed with the
micropipette), which was incubated at room temperature and
shaken for 10 min before the reading was performed. UV-spectra
scans were recorded between 210 nm and 400 nm (1 nm
resolution).
6.5. Assays of effect of metal ions to compoundseHSA complex
values in
Information.
m
g/mL were summarized in Table 1 and Supporting
All fluorescence spectra were recorded on F-7000 Spectrofluo-
rimeter (Hitachi, Tokyo, Japan) equipped with 1.0 cm quartz cells,
the widths of both the excitation and emission slit were set as
2.5 nm, and the excitation wavelength was 295 nm. Fluorescence
spectra were recorded at 298 K in the range of 300e450 nm. HSA
was obtained from SigmaeAldrich (St. Louis, MO, USA). Tris, NaCl,
HCl, CaCl2, KCl, MgCl2, and NiCl2 were analytical purity. HSA was
dissolved in TriseHCl buffer solution (0.05 M Tris, 0.15 M NaCl,
pH ¼ 7.4). Sample masses were weighed on a microbalance with a
resolution of 0.1 mg. All other chemicals and solvents were
commercially available, and were used without further purification.
6.2.1. Antibacterial assays
The prepared compounds 3e7 were evaluated for their anti-
bacterial activities against Gram-positive bacteria (S. aureus ATCC
6538, Methicillin-resistant Staphylococcus aureus N315 (MRSA) and
B. subtilis ATCC 21216), Gram-negative bacteria (E. coli ATCC 8099, P.
aeruginosa ATCC 27853 and B. proteus ATCC 13315). The bacterial
suspension was adjusted with sterile saline to a concentration of
1 ꢂ 105 CFU. Initially the compounds were dissolved in DMSO to

334
S.-F. Cui et al. / European Journal of Medicinal Chemistry 86 (2014) 318e334
[17] S. Khabnadideh, Z. Rezaei, N.A. Khalafi, M.H. Motazedian, Daru. J. Pharm. Sci.
Acknowledgments
15 (2007) 17e20.
[18] H.S. Sutherland, A. Blaser, I. Kmentova, S.G. Franzblau, B. Wan, Y. Wang, Z. Ma,
B.D. Palmer, W.A. Denny, A.M. Thompson, J. Med. Chem. 53 (2010) 855e866.
[19] M. Tukulula, R.K. Sharma, M. Meurillon, A. Mahajan, K. Naran, D. Warner,
J.X. Huang, B. Mekonnen, K. Chibale, ACS Med. Chem. Lett. 4 (2013) 128e131.
[20] M.N. Trivedi, S.Y. Gabhe, U.D. Vachhani, R.B. Patel, C.P. Shah, J. Chem. Pharm.
Res. 3 (2011) 313e319.
[21] L. Zhang, X.M. Peng, G.L.V. Damu, R.X. Geng, C.H. Zhou, Med. Res. Rev. 34
(2014) 340e437.
[22] L. Zhang, J.J. Chang, S.L. Zhang, G.L.V. Damu, R.X. Geng, C.H. Zhou, Bioorg. Med.
Chem. 21 (2013) 4158e4169.
[23] J.S. Lv, X.M. Peng, K.B. Bathula, C.H. Zhou, Bioorg. Med. Chem. Lett. 24 (2014)
308e313.
[24] C.H. Zhou, Y. Wang, Curr. Med. Chem. 19 (2012) 239e280.
[25] C.H. Zhou, L.L. Gan, Y.Y. Zhang, F.F. Zhang, G.Z. Wang, L. Jin, R.X. Geng, Sci.
China Ser. B Chem. 52 (2009) 415e458.
[26] H.Z. Zhang, G.L.V. Damu, G.X. Cai, C.H. Zhou, Eur. J. Med. Chem. 64 (2013)
329e344.
[27] X.M. Peng, G.L.V. Damu, C.H. Zhou, Curr. Pharm. Des. 19 (2013) 3884e3930.
[28] C.H. Zhou, Y.Y. Zhang, C.Y. Yan, K. Wan, L.L. Gan, Y. Shi, Anti-Cancer Agents
Med. Chem. 10 (2010) 371e395.
This work was partially supported by National Natural Science
Foundation of China [No. 21172181, 21372186, 81450110094,
81350110523 (the Research Fund for International Young Scientists
from International (Regional) Cooperation and Exchange Pro-
gram)], the Key Program of Natural Science Foundation of
Chongqing (CSTC2012jjB10026), the Specialized Research Fund for
the Doctoral Program of Higher Education of China (SRFDP
20110182110007) and Chongqing Special Fund for Postdoctoral
Research Proposal (Xm2014127).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.08.063.
[29] National Committee for Clinical Laboratory Standards Approved Standard
Document, M27eA2, Reference Method for Broth Dilution Antifungal Sus-
ceptibility Testing of Yeasts, National Committee for Clinical Laboratory
Standards, Wayne, PA, 2002.
[30] J.W. Tomsho, A. Pal, D.G. Hall, S.J. Benkovic, ACS Med. Chem. Lett. 3 (2012)
48e52.
[31] C.R. Martínez, C. Dardonville, ACS Med. Chem. Lett. 4 (2013) 142e145.
[32] D.T. Manallack, Perspect. Med. Chem. 1 (2007) 25e38.
[33] S.L. Zhang, J.J. Chang, G.L.V. Damu, R.X. Geng, C.H. Zhou, Med. Chem. Commun.
4 (2013) 839e846.
[34] S.L. Zhang, G.L.V. Damu, L. Zhang, R.X. Geng, C.H. Zhou, Eur. J. Med. Chem. 55
(2012) 164e175.
References
[1] S. Sabatini, F. Gosetto, G. Manfroni, O. Tabarrini, G.W. Kaatz, D. Patel,
V. Cecchetti, J. Med. Chem. 54 (2011) 5722e5736.
[2] S. Srinivasan, R.M.B. Shafreen, P. Nithyanand, P. Manisankar, S.K. Pandian, Eur.
J. Med. Chem. 45 (2010) 6101e6105.
[3] Y. Wang, G.L.V. Damu, J.S. Lv, R.X. Geng, D.C. Yang, C.H. Zhou, Bioorg. Med.
Chem. Lett. 22 (2012) 5363e5366.
[4] P.G. Higgins, A.C. Fluit, F.J. Schmitz, Curr. Drug Targets 4 (2003) 181e190.
[5] E. Leo, K.A. Gould, X.S. Pan, G. Capranico, M.R. Sanderson, M. Palumbo,
L.M. Fishe, J. Biol. Chem. 280 (2005) 14252e14263.
ꢁ
ꢀ
ꢁ ꢀ
ꢀ
[6] S. Kapic, H.C. Paljetak, S. Alihodzic, R. Antolovic, E.V. Haber, R.L. Jarvest,
D.J. Holmes, J.P. Broskey, E. Hunt, Bioorg. Med. Chem. 18 (2010) 6569e6577.
[7] D.C. Hooper, Lancet Infect. Dis. 2 (2002) 530e538.
[35] M. Zampakou, M. Akrivou, E.G. Andreadou, C.P. Raptopoulou, V. Psycharis,
A.A. Pantazaki, G. Psomas, J. Inorg. Biochem. 121 (2013) 88e99.
ꢀ
[36] A. Serafin, A. Stanczak, J. Russ. Coord. Chem. 35 (2009) 81e95.
[8] I. Laponogov, M.K. Sohi, D.A. Veselkov, X.S. Pan, R. Sawhney, A.W. Thompson,
K.E. McAuley, L.M. Fisher, Nat. Struct. Mol. Biol. 6 (2009) 667e669.
[9] K. Drlica, M. Malik, R.J. Kerns, X. Zhao, Antimicrob. Agents Chemother. 52
(2008) 385e392.
[10] C.H. David, Lancet Infect. Dis. 2 (2002) 530e538.
[11] G.L.V. Damu, Q.P. Wang, H.Z. Zhang, Y.Y. Zhang, J.S. Lv, C.H. Zhou, Sci. China
Chem. 56 (2013) 952e969.
[12] S.L. Zhang, J.J. Chang, G.L.V. Damu, B. Fang, X.D. Zhou, R.X. Geng, C.H. Zhou,
Bioorg. Med. Chem. Lett. 23 (2013) 1008e1012.
[13] X.M. Peng, G.X. Cai, C.H. Zhou, Curr. Top. Med. Chem. 13 (2013) 1963e2010.
[14] F.F. Zhang, L.L. Gan, C.H. Zhou, Bioorg. Med. Chem. Lett. 20 (2010) 1881e1884.
[15] Q.P. Wang, J.Q. Zhang, G.L.V. Damu, K. Wan, H.Z. Zhang, C.H. Zhou, Sci. China
Chem. 55 (2012) 2134e2153.
[37] T. Sen, S. Mandal, S. Haldar, K. Chattopadhyay, A. Patra, J. Phys. Chem. C. 115
(2011) 24037e24044.
[38] Y.J. Hua, Y. Wang, Y. Ou-Yang, J. Zhou, Y. Liu, J. Lumin. 130 (2010) 1394e1399.
[39] B.T. Yin, C.Y. Yan, X.M. Peng, S.L. Zhang, S. Rasheed, R.X. Geng, C.H. Zhou, Eur. J.
Med. Chem. 71 (2014) 148e159.
[40] I. Laponogov, M.K. Sohi, D.A. Veselkov, X.-S. Pan, R. Sawhney, A.W. Thompson,
K.E. McAuley, L.M. Fisher, M.R. Sanderson, Nat. Struct. Mol. Biol. 16 (2009)
667e669.
[41] E. Leo, K.A. Gould, X.-S. Pan, G. Capranico, M.R. Sanderson, M. Palumbo,
L.M. Fisher, J. Biol. Chem. 14 (2005) 14252e14263.
[42] G. Zhang, P. Fu, L. Wang, M. Hu, J. Agric. Food Chem. 59 (2011) 8944e8952.
[43] P. Vandurm, A. Guiguen, C. Cauvin, B. Georges, K.L. Van, C. Michaux,
C. Cardona, G. Mbembac, J.F. Mouscadet, L. Hevesi, C.V. Lint, J. Wouters, Eur. J.
Med. Chem. 46 (2011) 1749e1756.
[16] S.F. Cui, Y. Wang, J.S. Lv, G.L.V. Damu, C.H. Zhou, Sci. China Chem. 42 (2012)
1105e1131 (in Chinese).