Design, Synthesis, and Antimicrobial Evaluation of Novel Quinolone Imidazoles and Interactions with MRSA DNA

Article abstract of DOI:10.1111/cbdd.12532

A novel series of quinolone imidazoles as new type of antimicrobial agents were synthesized. Most compounds exhibited good bioactivities especially against MRSA even superior to reference drugs. They induced bacterial resistance more slowly than clinical drugs and gave low cytotoxicity to human cells. The pKa values of these compounds showed appropriate ranges to pharmacokinetic behaviors. The interactions between compound 8b, Cu²⁺ ion, and MRSA DNA revealed that compound 8b could intercalate into DNA through copper ion bridge to form a steady 8b-Cu²⁺-DNA ternary complex which might further block DNA replication to exert the powerful bioactivities. Study of compound 8b with human serum albumin indicated that compound 8b could be effectively stored and carried by human serum albumin.

Full text of DOI:10.1111/cbdd.12532

Received Date : 17-Oct-2014
Revised Date : 24-Dec-2014
Accepted Date : 16-Jan-2015
Article type
: Research Article
Title page
Title:
Design, synthesis and antimicrobial evaluation of novel quinolone imidazoles and interactions
with MRSA DNA
†
‡
Ling Zhang, Kannekanti Vijaya Kumar , Syed Rasheed , Rong-Xia Geng and Cheng-He Zhou*
*
Key Laboratory of Applied Chemistry of Chongqing Municipality, Institute of Bioorganic &
Medicinal Chemistry, School of Chemistry and Chemical Engineering, Southwest University,
Chongqing 400715, China.
†
Postdoctoral fellow from Indian Institute of Chemical Technology (IICT), India
‡
Postdoctoral fellow from Department of Chemistry, Hyderabad University, Hyderabad, India
Running title: Antimicrobial evaluation of quinolone imidazoles.
Keywords: Quinolone; Imidazole; anti-MRSA; DNA; Human serum albumin
*
Corresponding author: Prof. Cheng-He Zhou, School of Chemistry and Chemical Engineering,
Southwest University, Chongqing 400715, People’s Republic of China
Tel.: +86-23-68254967; Fax: +86-23-68254967
E-mail: zhouch@swu.edu.cn (Cheng-He Zhou)
Abstract:
A novel series of quinolone imidazoles as new type of antimicrobial agents were synthesized.
Most compounds exhibited good bioactivities especially against MRSA even superior to reference
drugs. They induced bacterial resistance more slowly than clinical drugs and gave low cytotoxicity
to human cells. The pKa values of these compounds showed appropriate ranges to
2
+
pharmacokinetic behaviors. The interactions of compound 8b, Cu ion and MRSA DNA revealed
that compound 8b could intercalate into DNA through copper ion bridge to form a steady
2
+
8
b–Cu –DNA ternary complex which might further block DNA replication to exert the powerful
bioactivities. Study of compound 8b with human serum albumin indicated that compound 8b
This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process
which may lead to differences between this version and the Version of Record. Please
cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12532
This article is protected by copyright. All rights reserved.

could be effectively stored and carried by human serum albumin.
Introduction
Resistance among clinical isolates has been greatest in Staphylococcus aureus and particularly
among methicillin resistant strains. It is well known that the methicillin resistant S. aureus (MRSA)
strains have become serious threatens to human health, which have resulted in weak efficacy for
most of the first-line clinical antibiotics in the treatment of infectious diseases [1]. Therefore, the
development of more effective antimicrobial agents which are possibly helpful for overcoming
drug-resistance has become an active topic for researchers [2].
Fluoroquinolones are an important kind of synthetic antibacterial agents. They exerted the
powerful biological activities by primarily inhibiting the activity of type II topoisomerases or
DNA gyrase to stabilize the cleavage complex at specific sites on DNA and preventing the
duplication of DNA [3]. However, these drugs were largely developed for treatment of infections
caused by Gram-negative bacteria, but only had limited activity against some Gram-positive
bacteria. The fluoroquinolone resistance in strains of MRSA emerged strikingly [4]. Numerous
efforts have been made in order to obtain more effective derivatives with the goal of further
extending the antibacterial spectrum and overcoming the drug-resistance. Amongst, structural
modification of the C–7 position in quinolone ring is the most important and meaningful strategy.
It revealed that the C–7 substituents could greatly influence the inhibition of DNA gyrase and cell
permeability, and ultimately impact the solubility, bioactivity, spectrum and pharmacokinetics.
The increasing interest has been directing towards the structural modification at the C–7 position
in quinolone ring by incorporating various types of substituents especially N–containing
heterocycles like pyridine [5], thiodiazole [6], piperidine [7] and triazole [8] etc.
Imidazole derivatives as antimicrobial agents have been investigated very well and shown large
potentiality in medicinal chemistry [9,10]. Especially nitro modified imidazoles, such as
metronidazole and its structurally modified derivatives including benznidazole, ornidazole,
secnidazole, nimorazole and tinidazole have been in widespread clinical use to treat diseases
caused by anaerobic bacteria. Many researches have revealed that the nitro fragment is an
important moiety to enhance lipophilicity of the target compounds which is favorable for tissue
penetration and induce bioactivities through the metabolic activation of nitro group [11]. Notably,
the product of nitroimidazole activation is mutagenic and cause DNA fragmentation in
microorganisms without forward mutation to neighboring cells. So the incidence of resistance in
anaerobic bacteria is still very low after the long term clinical use of metronidazole to treat
diseases caused by anaerobic bacteria [12]. Our previous work has reported that the incorporation
This article is protected by copyright. All rights reserved.

of nitroimidazole into berberine not only enhance the antimicrobial activities, but also broaden
antimicrobial spectrum [13]. This encourages us with special interest to continuously investigate
these nitroimidazole compounds as a novel type of potential antimicrobial agents. On the other
hand, many antimicrobial nitroimidazole agents such as nitroimidazooxazine PA-824 which is
currently under a Phase II trial were subjected to unsuitable binding propensity to proteins in
human plasma [14,15]. Therefore, the development of new types of nitroimidazoles with broad
antimicrobial spectrum and the suitable binding affinity to serum albumins has been attracting
special attention.
Many researches have revealed that combination of quinolone ring with other fragments is an
effective strategy to overcome quinolone-resistance. Previously, various heterocycle modified
quinolones as medicinal agents were widely investigated [16], but up to now no literature has
reported in the developments of metronidazole modified quinolones at C–7 position. So it will be
interesting and rational to continue our work to combine nitroimidazole moiety with C–7 position
of quinolones. Based on this, a novel type of quinolone imidazoles were designed. Benzimidazole
is a fused ring of imidazole with benzene having larger conjugated system and electron richer
properties than imidazole, this unique structure endows its derivatives to possess extensive
potentiality in medicinal chemistry especially antimicrobial aspect [17,18]. Herein we also would
like to develop this fused ring type of nitroimidazole derivatives including
2
5
-methyl-5-nitrobenzimidazole,
6-nitrobenzimidazole,
2-methylbenzimidazole,
,6-dimethylbenzimidazole, benzimidazole and 2-thiol-benzimidazole as wholly new structure
type of antimicrobial agents to investigate the effect of azole moiety on biological activities which
were expected to have large roles in the treatment of disease-caused bacteria and fungi. The
designed structures of this series of novel quinolone imidazoles are shown in Schemes 1–4.
Quinolones constructed by benzene ring fused pyridone skeleton with π–π conjugated system
possess rich endogenous fluorescence. So the study on the interaction between quinolone and
DNA plays a very important role in improving the absorption and antibacterial activity of
quinolone in body. Although previous research showed that quinolones are able to unwind the
DNA double helix in the presence of metal ion, the fluorescence spectroscopic technique [19]
failed to show any direct interaction between DNA and quinolones. It revealed that metal ion
could act as a bridge between the phosphate groups of nucleic acids and the carbonyl and carboxyl
moieties of quinolone. Quinolones only bind to DNA in the presence of an appropriate amount of
metal ion but exhibits no interaction in either an absence or excess amount of metal ion [20]. The
preliminary antimicrobial mechanism by evaluating the interaction of the prepared highly active
compound with MRSA DNA is investigated. Additionally, human serum albumins (HSA) as the
most important and abundant macromolecule proteins in the circulatory system have received
This article is protected by copyright. All rights reserved.

much attention [21]. A thorough binding analysis between drugs or bioactive small molecules and
DNA or HSA may beneficially provide useful information for the absorption, transportation,
distribution, metabolism, excretion properties and possible mechanism of agents. It might also be
significant to the design, modification and screening of drug molecules. In view of the above
observations, it is considerably reasonable for us to investigate the antimicrobial mechanism with
DNA and the further transportation behavior of the highly active prepared compound by HSA in
order to preliminarily evaluate their pharmacokinetic and transportation properties by fluorescence
and UV–vis absorption spectroscopy on molecular level.
Experimental section
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
-1
spectrophotometer (Bio-Rad, Cambridge, MA, USA) using KBr pellets in the 400–4000 cm
range. NMR spectra were recorded on a Bruker AV 300 and 600 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 other chemicals and solvents were commercially available, and were used
without further purification.
Ethyl-6-fluoro-7-(4-(oxiran-2-ylmethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (2a)
A mixture of norfloxacin (0.96 g, 0.0027 mol) and sodium hydrogen carbonate (0.69 g, 0.0048
mol) in acetonitrile (100 mL) was stirred at 50 ℃ for 1 h. After the mixture was cooled to room
temperature, 2-(chloromethyl)oxirane (1.00 g, 0.0027 mol) was added. The reaction mixture was
then heated at 45 ℃ for 10 h. After the reaction was completed (monitored by TLC,
chloroform/methanol (50/1, V/V)), the reaction was cooled to room temperature and treated with
formic acid to adjust the pH value to 5.5–6.5. After the acetonitrile was removed under reduced
pressure, the mixture was purified by flash silica gel column eluting with chloroform/methanol
(60/1, V/V) to give the pure target compound 2a as white power (0.68 g). Yield: 67.4%; mp:
-
1
1
85–186 ℃; IR (KBr, cm ) ν: 3061 (Ar–H), 1804 (aromatic frame), 1718 (C=O), 1617, 1571
1
(
aromatic frame); H NMR (300 MHz, CDCl
3
): δ 15.05 (s, 1H, COOH), 8.63 (s, 1H, quinolone
2
-H), 7.99 (d, J = 13.0 Hz, 1H, quinolone 5-H), 6.80 (d, J = 6.8 Hz, 1H, quinolone 8-H), 4.53 (t, J
8.3 Hz, 1H, CH ), 4.30 (dd, 2H, quinolone N-CH ), 3.96 (dd, J = 11.7, 6.0 Hz, 1H, O-CH), 3.59
=
2
2
This article is protected by copyright. All rights reserved.

(
t, J = 5.0 Hz, 1H, CH ), 3.32 (t, J = 14.3 Hz, 4H, piperazine N-CH ), 2.81 (dd, J = 11.5, 6.7 Hz,
2
2
4H, piperazine N-CH
2
), 2.69 (dd, J = 10.9, 5.5 Hz, 1H, CH
2
2
), 2.58 (d, J = 6.6 Hz, 1H, CH ), 1.56
1
3
(
t, J = 7.2 Hz, 3H, CH ) ppm; C NMR (151 MHz, DMSO-d ): δ 176.3 (quinolone 4-C), 165.3
3
6
(quinolone COOH), 153.7, 152.1, 142.8, 137.6, 121.2, 118.7, 112.4, 107.9, 55.8, 52.4, 49.6, 47.6,
13
4
4.9, 14.2 ppm; C NMR (151 MHz, DMSO-d ): δ 176.3 (quinolone 4-C), 165.3 (quinolone
6
COOH), 153.7, 152.1, 142.8, 137.6, 121.2, 118.7, 112.4, 107.9, 55.8, 52.4, 49.6, 47.6, 44.9, 14.2
+
+
ppm; MS (m/z): 376 [M+H] ; HRMS (TOF) calcd for C H FN O : [M+H] , 376.1673; found,
19
23
3
4
376.1674.
Compounds (2b–c) were prepared according to the procedure for the synthesis of 2a
Supplementary material).
(
Ethyl-6-fluoro-7-(4-(2-hydroxy-3-(2-methyl-5-nitro-1H-imidazol-1-yl)propyl)piperazin-1-yl)-4-ox
o-1,4-dihydroquinoline-3-carboxylic acid (3a)
A mixture of 2-methyl-5-nitroimidazole (0.35 g, 0.0027 mol) and potassium carbonate (0.69 g,
0
.0048 mol) in acetonitrile (20 mL) was stirred at 50 ℃ for 1 h. After the mixture was cooled to
room temperature, compound 2a (1.00 g, 0.0027 mol) was added. The reaction mixture was then
heated at 80 ℃ for 20 h. After the reaction was completed (monitored by TLC,
chloroform/methanol (25/1, V/V)), the reaction was cooled to room temperature and treated with
formic acid to adjust the pH value to 5.5–6.5. After the ethanol was removed under reduced
pressure, the mixture was purified by flash silica gel column eluting with chloroform/methanol
(30/1, V/V) to give the pure target compound 3a as yellow power (0.37 g). Yield: 27.4%; mp:
-
1
1
07−108 ℃; IR (KBr, cm ) ν: 3443 (OH), 3030 (Ar–H), 2940 (CH
3
), 1714 (C=O), 1628, 1540,
1
1
497, 1457 (aromatic frame); H NMR (300 MHz, CDCl
3
): δ 15.18 (s, 1H, COOH), 8.67 (s, 1H,
quinolone 2-H), 8.05 (s, J = 13.0 Hz, 1H, quinolone 5-H), 7.86 (s, 1H, imidazole 4-H), 6.86 (d, J =
.7 Hz, 1H, quinolone 8-H), 4.35 (dd, J = 13.7, 6.6 Hz, 2H, quinolone N-CH ), 4.12 (m, J = 11.9
Hz, 2H, imidazole CH ), 3.93 (m, J = 7.3 Hz, 1H, O-CH), 3.70 (m, J = 38.7, 26.7 Hz, 2H,
piperazine CH -CHOH), 3.33 (m, J = 17.4 Hz, 4H, piperazine N-CH ), 2.89–2.70 (m, J = 4.8 Hz,
H, piperazine N-CH ), 2.47 (s, 3H, imidazole 2-CH ), 1.62 (m, J = 7.1 Hz, 3H, quinolone CH
ppm; C NMR (151 MHz, DMSO-d ): δ 176.7 (quinolone 4-C), 166.5 (quinolone COOH), 150.8,
49.8, 147.4, 145.8, 138.2, 136.6, 120.4, 118.5, 110.5, 106.2, 68.0, 60.3, 53.6, 50.3, 49.8, 49.1,
6
2
2
2
2
4
2
3
3
)
1
3
6
1
1
+
+
6 6
3.4 ppm; MS (m/z): 504 [M+H] . HRMS (TOF) calcd for C23H27FN O : [M+Na] , 525.1874;
found, 525.1872.
Compounds (3b–j, 6a–j, 7a–b, 8a–e, 9a–e and 10) were prepared according to the procedure for
the synthesis of 3a (Supplementary material). Compounds (5a–b) were synthesized by the
reported procedure [22, 23].
This article is protected by copyright. All rights reserved.

Biological activity
All newly synthesized compounds 2a–c, 3a–j, 6a–j, 7a–b, 8a–e, 9a–e and 10 were evaluated
for in vitro antibacterial and antifungal activities against four Gram-positive bacteria
(
Staphylococcus aureus ATCC25923, Methicillin-resistant Staphylococcus aureus N315, Bacillus
subtilis ATCC6633 and Micrococcus luteus ATCC4698), four Gram-negative bacteria
Escherichia coli JM 109, Pseudomonas aeruginosa ATCC 27853, Bacillus proteus ATCC 13315
(
and Eberthella typhosa) and five fungi (Candida albicans ATCC 76615, Candida mycoderma,
Candida utilis, Saccharomyces cerevisia and Aspergillus flavus) using two-fold broth dilution
method in 96-well microtest plates recommended by National Committee for Clinical Laboratory
Standards (NCCLS) [24]. The biological tests were carried out in triplicate. Minimal inhibitory
concentration (MIC, nmol/L) was defined as the lowest concentration of new compounds that
completely inhibited the growth of bacteria. Currently available antimicrobial drugs such as
Chloromycin, Norfloxacin, Ciprofloxacin, Clinafloxacin and Fluconazole were used as standard
drugs.
Results and discussion
Chemistry
The target quinolone imidazoles were prepared from commercial quinolones and
2
-(chloromethyl)oxirane (Schemes 1–4). The key intermediates 2a–c were efficiently obtained in
the yields of 62.4–67.4% starting from commercial available quinolones through N–alkylation
with 2-(chloromethyl)oxirane after treatment with formic acid to adjust the pH value to 5.5–6.5.
The resulting 2a–c were reacted with corresponding azoles in acetonitrile or ethanol with
anhydrous potassium carbonate to afford target compounds 3a–j, 6a–b, 6e–g, 6j, 7a–b, 8a–e,
9
a–b, 9e and 10 in 19.3–37.6% yields after further treatment with formic acid to adjust the pH
value to 5.5–6.5. Acetic acid was reacted with o-phenylene diamine or 4-nitrobenzene-1,2-diamine
via cyclization to produce intermediates 2-methylbenzoimidazole 5a and
-methyl-5-nitrobenzoimidazole 5b in good yields of 70.4–83.7%, and then further treated by
2
intermediates 2a–c in refluxing acetonitrile or ethanol to give selectively the corresponding
compounds 6c–d, 6h–i and 9c–d with yields ranging from 13.1% to 33.4%.
Antimicrobial activities
Chloramphenicol, Norfloxacin, Ciprofloxacin and Clinafloxacin were chosen as reference
drugs which provided calibrated level of biological effects against the prepared compounds. The
obtained results, depicted in Table 1 and Table 2, revealed that most of the prepared compounds
could effectively inhibit the growth of all the tested strains in vitro. Among them, 2-nitroimidazole
functionalized quinolones 3b, 3g and 8b showed significant increase in inhibiting the growth of
This article is protected by copyright. All rights reserved.

pathogenic bacterial strains (MIC = 0.015–7.44 nmol/L), and compound 3j bearing
-methylimidazole group and compound 8d with 4-notroimidazole group also displayed fairly
4
good antibacterial activities with the MIC values in the range of 0.39–7.74 nmol/L in comparison
with reference drugs Chloramphenicol (MIC = 8.91–35.66 nmol/L) and superior to respective
Norfloxacin (MIC = 1.31–41.79 nmol/L), Ciprofloxacin (MIC = 1.26–8.79 nmol/L) and
Clinafloxacin (MIC = 0.57–1.14 nmol/L).
Excitedly, the target compounds 3b, 3e, 3g, 3j, 8b, 8d and 9c exhibited remarkable biological
activity against MRSA with quite low MIC values of 0.39–6.56 nmol/L which were more active
than Chloramphenicol (MIC = 35.66 nmol/L). Especially, quinolone 2-nitroimidazole compounds
3b (MIC = 0.85 nmol/L), 3g (MIC = 1.49 nmol/L), 8b (MIC = 0.39 nmol/L) and
4
-methylmidazole derivative 3j (MIC = 1.57 nmol/L) exerted impressive anti-MRSA activity,
superior than that of Norfloxacin (MIC = 20.89 nmol/L), Ciprofloxacin (MIC = 8.79 nmol/L) and
Clinafloxacin (MIC = 2.01 nmol/L). These implied that this new type of quinolone azoles should
have the potential to be developed as anti-MRSA agents.
It was also noteworthy that compound 8b could remarkably inhibit the growth of all the tested
bacterial strains with MIC values ranging from 0.097 to 1.38 nmol/L, which was more active than
other quinolone azoles. Particularly, compound 8b demonstrated excellent bioactivity against E.
coli with MIC value of 0.097 nmol/L, which was 128-fold more potent than the reference drug
Chloramphenicol and 8-fold more active than Clinafloxacin. Meanwhile, 2-nitroimidazoles 3b, 3g
and 4-methylimidazoles 3e, 3j also showed strong anti-E. coli activities with low MIC values of
0
.23–5.79 nmol/L, 4 times more potent than their respective control drugs. Notably, bacterium P.
aeruginosa was much more sensitive to target compounds 3b (MIC = 0.0012 nmol/L), 3g (MIC =
.0046 nmol/L) and 8b (MIC = 0.019 nmol/L) than Chloramphenicol (MIC = 35.66 nmol/L),
Norfloxacin (MIC = 41.79 nmol/L), Ciprofloxacin (MIC = 5.04 nmol/L) and Clinafloxacin (MIC
1.14 nmol/L), but only weak inhibitory activity was observed for 2-methylimidazoles 3c, 3h and
c. The corresponding 4-nitro derivatives 3d, 3i, 8d and 4-methyl ones 3e, 3j and 8e also gave
0
=
8
generally lower bioactivities. The result indicated that existence of 2-nitro group on imidazole
moiety in this series of quinolone imidazoles should be of special importance in the antibacterial
profiles, and 2-methyl group in the imidazoles seemed to be unfavorable for their antimicrobial
efficacy.
Among the fused ring type of quinolone imidazoles, only 2-methyl-5-nitrobenzimidazoles 6c,
6h and 9c and 6-nitrobenzimidazoles 6b, 6g and 9b displayed good bioactivities, others gave
overall moderate to weak bioactivities. These results demonstrated that the presence of fused ring
seemed to be unfavorable for their antimicrobial efficacy, and existence of nitro group on the
fused benzimidazole moieties in this series of quinolone imidazoles should be of special
importance in microbial inhibition.
This article is protected by copyright. All rights reserved.

The intermediates 2a–c, as shown in Table 1 and Table 2, also displayed moderate to good
activities against all the tested bacterial strains in comparison with reference drugs. Particularly,
compound 2c gave the better antimicrobial efficiencies with MIC values of 0.44 and 1.77 nmol/L
respectively against M. luteus and MRSA, than the corresponding reference drug Clinafloxacin
(MIC = 1.14 and 2.01 nmol/L, respectively). For Gram-negative P. aeruginosa, E. coli and B.
proteus strains, compound 2c (MIC = 0.89, 0.89 and 0.44 nmol/L, respectively) displayed stronger
inhibitory activity than Clinafloxacin (MIC = 1.14 nmol/L).
The in vitro antifungal evaluation revealed that the intermediates 2a–c with oxiran-2-ylmethyl
groups at C–7 position of quinolones exhibited better antifungal activities against most of the
tested fungal strains than those quinolone imidazoles (Supplementary material). Most of the
non-fused type nitroimidazoles with MIC values of 11.98–764.15 nmol/L showed moderate
antifungal activities against the tested fungal strains in comparison with reference drug
Fluconazole (MIC = 5.44–696.48 nmol/L). The substitution of non-fused nitroimidazole ring in
quinolones caused decrease of antifungal properties.
Development of resistance to compound 3b
Considering the high-level resistance of Norfloxacin to MRSA strains [25,26], we selected the
representative compound 3b to investigate the developing rate of bacterial resistance. We exposed
a standard strain of MRSA towards increasing concentrations of compound 3b from sub-MIC for
sustained passages and determined the MIC values of compound 3b for each passage of MRSA.
After 25 passages of growth in the presence of 0.85 nmol/L of compound 3b, bacterial resistance
did not increase any further, and the MIC value is only 3.41 nmol/L. By contrast, MRSA quickly
developed resistance to Norfloxacin, showing resistance to the original MIC (20.87 nmol/L) after
6
passages. This assay indicated that bacteria MRSA do not develop resistance against compound
3b as easily as they do against Norfloxacin [27].
Cytotoxicity
These highly bioactive compounds were further examined for their cytotoxic properties on
human hepatocyte LO2 cell line by means of CKK-8 Kit, [28] which could produce soluble
purple formazan in the presence of viable cells, and the absorbance increased linearly as cells
proliferate. Compounds were dissolved in DMSO to prepare the stock solutions, and then the
tested compounds were prepared in medium to obtain the required concentrations of 256, 128, 64,
3
2, 16 and 8 μg/mL. Cells were cultured for 24 h in these solutions. The cell viability was
examined by microplate reader. Cytotoxicity results showed that the cell viability of all the
compounds were at least more than 83% within concentration of 256 μg/mL, which indicated all
the tested compounds did not affect the viability of human hepatocyte LO2 cells cell line within
the concentration of 256 μg/mL.
This article is protected by copyright. All rights reserved.

Analysis of pKa values
As shown in Table 3, the pKa values of target compounds 3–10 displayed an appropriate range
(6.178–6.654) to pharmacokinetic behaviors (Supplementary material). Since the electron density
could be reduced by the electron withdrawing group like NO group, the pKa values decreased
2
accordingly, which indicated that the acidity of the carboxyl group became strong. However, due
to the electron donating ability of methyl group, compounds containing methyl groups could
destabilize anionic form, therefore decrease the acidic character and show high pKa values.
Binding discussion
Interactions with MRSA DNA
The DNA was isolated from MRSA bacteria (Supplementary material). The interactions of
compound 8b with MRSA DNA were explored in vitro by UV–vis and fluorescence spectroscopic
methods (Supplementary material).
With a fixed concentration of DNA, UV–vis absorption spectra were recorded with the
increasing amount of compound 8b. As shown in Figure 1, UV–vis spectra displayed that the
maximum absorption peak of DNA at 260 nm exhibited proportional increase and slight red shift
with the increasing concentration of compound 8b. Meanwhile the phenomenon, that the
absorption value of simply sum of free DNA and free compound 8b was a less than the measured
value of 8b–DNA complex. This suggested a conformational change in the DNA duplex. This
change is 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 can be explained by the electrostatic interaction of the complexes with
DNA [31,32].
On the basis of variations in the absorption spectra of DNA upon binding to 8b, equation 2 can
be utilized to calculate the binding constant (K).
0
A
ξC
ξC
1
equation (1)
=
+
×
0
A− A
ξD−C −ξC ξD−C − ξC K[Q]
0
A and A represent the absorbance of DNA in the absence and presence of compound 8b at 260
nm, ξ
C
and ξ
D C
- are the absorption coefficients of compound 8b and 8b–DNA complex
0
0
respectively. The plot of A /(A-A ) versus 1/[compound 8b] is constructed by using the absorption
titration data and linear fitting (Supplementary material Figure S15), yielding the binding constant,
5
K = 2.17 ×10 L/mol, R = 0.997, SD = 0.076 (R is the correlation coefficient. SD is standard
deviation).
2
+
Emission spectra of compound 8b in the presence of various concentrations of Cu ion was
shown in Figure 2. Compound 8b has very intense intrinsic fluorescence in aqueous solution at
-3
λ /λ = 280/434 nm. The results show the fluorescence intensity of 6.0×10 mol/L compound 8b
ex em
This article is protected by copyright. All rights reserved.

solution at 434 nm dropped regularly with the increasing concentration of copper(II) ion. This
may be due to a strong interaction between compound 8b and copper(II) ion.
The composition of the binary complex can be deduced from the following equations [33].
M + nL = MLn
equation (2)
log[(F /F)-1] = logK + nlog[M]
0
a
equation (3)
Where M is the quencher, L is the drug molecule with a fluorophore, ML is binary complex
n
whose resultant constant is K
a 0
. A plot of log[(F /F)-1] versus log[M] will give straight line with a
slope of n and y-axis intercept logKa.
The data in Figure 3 are well fitted to Equation 3 and the slope is 1.8, the correlation coefficient
is 8.445, respectively. The result indicates that compound 8b can form a stable 2:1 complex with
2
+
Cu ion. Research revealed that in the crystal structures of copper–quinolone complexes,
2
+
quinolones can form stable 2:1 complexes with Cu ion and metal atoms are usually bonded to
the oxygen atoms of carbonyl and carboxylic groups of the quinolones and exhibit octahedral, or
square pyramidal geometry, with counter ions or additional water molecules in remaining
coordination sites [34]. It is proposed that compound 8b in the solution form the same bonds with
copper ion.
The fluorescence spectra of compound 8b and its decreased fluorescence intensity by MRSA
2
+
DNA in the presence of Cu ion are shown in Figure 4. In the system, when MRSA DNA
solution was added, the fluorescence intensity of compound 8b was quenched obviously. And the
quenching degree was aggravated with the increasing of the concentration of DNA in aqueous
2+
solution. The result implied that there was competition between the MRSA DNA and Cu ion
when metal ion coexisted with MRSA DNA in compound 8b solution, and they also might
2
+
suggest the formation of a ternary complex among MRSA DNA, compound 8b and Cu ion.
-6
2+
The inset in Figure 4 is obtained by keeping the compound 8b (6.0 × 10 mol/L) and Cu ion
-5
(
1.0 × 10 mol/L) concentration constant and changing the concentration of DNA. The data are
well fitted to Equation 4 and the slope is 0.84, the correlation coefficient is 0.846, respectively.
2
+
The result indicates that compound 8b in the presence of Cu ion can form a stable 1:1 complex
with DNA.
From the intercalative binding mode deduced from the above fluorescence experiment results, it
2
+
indicated that a ground-state binary complex was formed between compound 8b and Cu ion, and
2
+
a ground-state ternary complex also can be formed among Cu ion, compound 8b and MRSA
DNA.
This article is protected by copyright. All rights reserved.

Steady-state emission quenching experiments using KI as quenchers may provide further
information about the binding of complexes with DNA. In the presence of DNA, the
−
Stern–Volmer plots are changed. This may be explained by repulsions of the highly negative I
−
ion from the DNA polyanion backbone which hinders access of I ion to the DNA bound
complexes. A larger slope for the Stern–Volmer curve parallels poorer protection and low binding.
KI fluorescence quenching curves of compound 8b with and without DNA are given in Figure 5
and Figure 6.
From Figure 5 and Figure 6, we can find that the addition of KI results in extensive quenching
of the fluorescence intensity. The quenching data were plotted according to the Equation 3 and the
slopes were calculated by the linear least–squares method. The observed quenching constants
were 7.02 and 7.57 L/mol with and without MRSA DNA, respectively. The quenching of the
compound 8b fluorescence was in fact enhanced when the compound 8b was bound to the DNA
helix. Ka values were used to deduce the interaction mode of the fluorescence probe with DNA.
Higher binding constants should correspond to the better protection by the DNA and a stronger
inhibition of quenching by anionic species. Thus, we can conclude that the compound 8b
intercalated into the DNA helix and it should be protected from the anionic quencher, owning to
the base pairs above and below the intercalator [35].
Interactions with HSA
In order to preliminarily evaluate the pharmacokinetic and transportation properties of the
prepared compound 8b by HSA, we further investigated the binding behaviors between HSA and
the target compound 8b. In this work, interaction information including fluorescence quenching
mechanisms, binding parameters, thermodynamic parameters and binding mode between HSA and
target compound was obtained by fluorescence and UV–vis absorption spectroscopy on molecular
level (Supplementary material) [36-38]. The interaction of compound 8b with HSA seemed to be
the presence of one high affinity binding site and the equilibrium binding constants were moderate
and the effect of temperatures was not obvious, therefore compound 8b might be stored and
carried by HSA. The fluorescence quenching mechanisms manifested that the binding was mainly
enthalpy-driven and involved an exothermic reaction, the electrostatic interactions played an
important role in the binding of compound 8b to HSA.
Conclusion
In conclusion, a series of quinolone imidazoles were successfully synthesized by a convenient and
1
13
efficient procedure. Their structures were confirmed by H NMR, C NMR, MS, IR and HRMS
spectra. The in vitro antimicrobial evaluation revealed that most of the synthesized compounds
exhibited good bioactivities against the tested Gram-positive and Gram-negative bacteria and
fungi especially against MRSA even superior to reference drugs. Moreover, these compounds
This article is protected by copyright. All rights reserved.

induced bacterial resistance more slowly compared with control. After 25 passages, the MIC
values against MRSA just changed from 0.85 nmol/L to 3.41 nmol/L. The pKa values of these
compounds showed appropriate ranges to pharmacokinetic behaviors. The interactions of
2
+
compound 8b, MRSA DNA and Cu ion were investigated and indicated a possible interaction
2
+
mechanism. Firstly, compound forms a binary complex with Cu ion, then forms a ternary
2
+
complex with DNA by intercalation binding form, and finally releases Cu ion to form interaction
between compound 8b and DNA, which might further block DNA replication to exert their
powerful antimicrobial activities. Further transportation behavior of HSA to compound 8b
manifested that compound 8b could be effectively stored and carried by HSA. The experimental
parameters indicated that the quenching mechanism of fluorescence of HSA by compound 8b was
a static quenching procedure. The association of compound 8b with HSA was spontaneous and the
electrostatic interactions played important roles in the transportation of HSA to 8b.
Acknowledgments
This work was partially supported by National Natural Science Foundation of China [(No.
21172181, 21372186), the Research Fund for International Young Scientists from International
(
Regional) Cooperation and Exchange Program NSFC (No. 81350110523, 81450110094)], 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 Foundation for Postdoctoral Research Proposal
(Xm2014127).
Conflict of Interest
The authors have declared no conflict of interest.
References
1
.
Rodvold K.A. & McConeghy K.W. (2014) Methicillin-resistant Staphylococcus aureus
therapy: past, present, and future. Clin Infect Dis 58: S20-S27.
2
.
Bielaszewska M., Mellmann A., Zhang W.L., Köck R., Fruth A., Bauwens A., Peters G. &
Karch H. (2011) Characterisation of the Escherichia coli strain associated with an outbreak of
haemolytic uraemic syndrome in Germany, 2011: a microbiological study. Lancet Infect Dis
11: 671-676.
3
4
.
.
Wu R.F. & Zhang J.P. (1991) Handbook of Import Medicine, Chinese Medical Science and
Technology Press, Beijing.
[a] David C.H. (2002) Fluoroquinolone resistance among Gram-positive cocci. Lancet Infect
Dis 2, 530-538. [b] Hidemasa N., Koko S.-N. & Norihisa N. (2014) A novel GyrB mutation in
meticillin-resistant Staphylococcus aureus (MRSA) confers a high level of resistance to
This article is protected by copyright. All rights reserved.

third-generation quinolonesInter. J Antimicrobial Agents 43: 478-479.
5
6
.
.
Han H.Y., Chen L., Xu X.R., Fan L., Yang Y. & Yang D.C. (2011) Facile synthesis and in
vitro/vivo bioactivities of amido derivatives of clinafloxacin. Sci Sin Chim 41: 461-473 (in
Chinese).
Foroumadi A., Ashraf-Askari R., Moshafi M.H., Emami S. & Zeynali A. (2003) Synthesis and
in vitro antibacterial activity of N-[5-(5-nitro-2-furyl)-1,3,4-thiadiazole-2-yl] piperazinyl
quinolone derivatives. Pharmazie 58: 432-433.
7
8
.
.
Dang Z., Yang Y.S., Ji R.Y. & Zhang S.H. (2007) Synthesis and antibacterial activity of novel
fluoroquinolones containing substituted piperidines. Bioorg Med Chem Lett 17: 4523-4526.
Wang Y., Damu G.L.V., Lv J.S., Geng R.X., Yang D.C. & Zhou C.H. (2012) Design, synthesis
and evaluation of clinafloxacin triazole hybrids as a new type of antibacterial and antifungal
agents. Bioorg Med Chem Lett 22: 5363-5366.
9
1
1
.
Zhang L., Peng X.M., Damu G.L.V., Geng R.X. & Zhou C.H. (2014) Comprehensive review
in current developments of imidazole-based medicinal chemistry. Med Res Rev 34: 340-437.
0. Peng X.M., Cai G.X. & Zhou C.H. (2013) Recent developments in azole compounds as
antibacterial and antifungal agents. Curr Top Med Chem 13: 1963-2010.
1. Sutherland H.S., Blaser A., Kmentova I., Franzblau S.G., Wan B., Wang Y., Ma Z., Palmer
B.D., Denny W.A. & Thompson A.M. (2010) Synthesis and structure-activity relationships of
antitubercular 2-nitroimidazooxazines bearing heterocyclic side chains. J Med Chem 53:
855-866.
1
2. Lau N.P., Piscitelli S.C., Wilkes L. & Danziger L.H. (1992) Clinical pharmacokinetics of
metronidazole and other nitroimidazole anti-infectives. Clin Pharmacokinet 23: 328-364.
3. Zhang L., Chang J.J., Zhang S.L., Damu G.L.V., Geng R.X. & Zhou C.H. Synthesis and
bioactive evaluation of novel hybrids of metronidazole and berberine as new type of
antimicrobial agents and their transportation behavior by human serum albumin. (2013)
Bioorg Med Chem 21: 4158-4169.
1
14. Burman W.J. (2010) Rip Van Winkle wakes up: development of tuberculosis treatment in the
21st century. Clin Infect Dis 50: S165-S172.
1
5. Tukulula M., Sharma R.K., Meurillon M., Mahajan A., Naran K., Warner D., Huang J.X.,
Mekonnen B. & Chibale K. (2013) Synthesis and antiplasmodial and antimycobacterial
evaluation of new nitroimidazole and nitroimidazooxazine derivatives. ACS Med Chem Lett 4:
128-131.
1
6. Zhou C.H. & Wang Y. (2012) Recent researches in triazole compounds as medicinal drugs.
Curr Med Chem 19: 239-280.
1
7. [a] Zhang H.Z., Damu G.LV., Cai G.X. & Zhou C.H. (2013) Design, synthesis and
antimicrobial evaluation of novel benzimidazole type of Fluconazole analogues and their
This article is protected by copyright. All rights reserved.

synergistic effects with Chloromycin, Norfloxacin and Fluconazole. Eur J Med Chem 64:
29-344; [b] Zhang H.Z., Cui S.F., Nagarajan S., Rasheed S., Cai G.X. & Zhou C.H. (2014) A
3
unique one-pot reaction via CAC cleavage from aminomethylene benzimidazoles to access
benzimidazolones with wide potentiality. Tetrahedron Lett 55: 4105-4109; [c] Zhang H.Z.,
Lin J.M., Rasheed S. & Zhou C.H. (2014) Design, synthesis, and biological evaluation of
novel benzimidazole derivatives and their interaction with calf thymus DNA and synergistic
effects with clinical drugs. Sci China Chem 57: 807-822.
1
8. [a] Zhang S.L., Damu G.L.V., Zhang L., Geng R.X. & Zhou C.H. (2012) Synthesis and
biological evaluation of novel benzimidazole derivatives and their binding behavior with
bovine serum albumin. Eur J Med Chem 55: 164-175; [b] Li S., Chen J.X., Xiang Q.X.,
Zhang L.Q., Zhou C.H., Xie J.Q., Yu L. & Li F.Z. (2014) The synthesis and activities of novel
mononuclear or dinuclear cyclen complexes bearing azole pendants as antibacterial and
antifungal agents. Eur J Med Chem 84: 677-686; [c] Zhang S.L., Chang J.J., Damu G.L.V.,
Fang B., Zhou X.D., Geng R.X. & Zhou C.H. (2013) Novel berberine triazoles: Synthesis,
antimicrobial evaluation and competitive interactions with metal ions to Human Serum
Albumin. Bioorg Med Chem Lett 23: 1008-1021; [d] Zhang S.L., Chang J.J., Damu G.L.V.,
Geng R.X. & Zhou C.H. (2013) Berberine azoles as antimicrobial agents: synthesis,
biological evaluation and their interactions with human serum albumin. Med Chem Commun
4
: 839-846.
1
2
2
9. Palu G., Valisena S., Peracchi M. & Palumbo M. (1988) Do quinolones bind to DNA?
Biochem Pharmacol 37: 1887-1888.
0. Palu G., Valisena G., Ciarrocchi G., Gatto B. & Palumbo M. (1992) Quinolone binding to
DNA is mediated by magnesium ions. Proc Natl Acad Sci USA. 89: 9671-9675.
1. Agudelo D., Bourassa P., Bruneau J., Berube G., Asselin E. & Tajmir-Riahi H.-A. (2012)
Interaction of multimicrobial synthetic inhibitor 1,2-Bis(2-benzimidazolyl)-1,2-ethanediol
with serum albumin: spectroscopic and computational studies. PLoS One 8: e53499.
2. Obermayer D., Damm M. & Kappe C.O. (2013) Simulating microwave chemistry in a
resistance-heated autoclave made of semiconducting silicon carbide ceramic. Chem-Eur J 19:
2
2
2
2
15827-15830.
3. Heravi M.M., Sadjadi S., Oskooie H.A., Shoar R.H. & Bamoharram F.F. (2007) Heteropoly
acids as green and reusable catalysts for the synthesis of 3,1,5–benzoxadiazepines. Molecules
12: 255-262.
4. National Committee for Clinical Laboratory Standards Approved standard Document,
M27-A2, Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts.
National Committee for Clinical Laboratory Standards, Wayne, PA, 2002.
5. Davidovich C., Bashan A., Auerbach-Nevo T., Yaggie R.D., Gontarek R.R. & Yonath A.
This article is protected by copyright. All rights reserved.

(2007) Induced-fit tightens pleuromutilins binding to ribosomes and remote interactions
enable their selectivity. Proc Natl Acad Sci USA. 104: 4291-4296.
2
6. Zhao Y., Tian Y., Cui Y., Liu W., Ma W. & Jiang X. (2010) Small molecule-capped gold
nanoparticles as potent antibacterial agents that target Gram-negative bacteria. J Am Chem
Soc 132: 12349-12356.
2
7. Chopra I. (2007) The increasing use of silver-based products as antimicrobial agents: a useful
development or a cause for concern? J Antimicrob Chemother 59: 587-590.
8. Frith J.E., Menzies D.J., Cameron A.R., Ghosh P., Whitehead D.L., Gronthos S., Zannettino
A.C.W. & Cooper-White J.J. Effects of bound versus soluble pentosan polysulphate in
PEG/HA-based hydrogels tailored for intervertebral disc regeneration. (2014) Biomaterials 35:
2
1150-1162.
29. [a] Tomsho J.W., Pal A., Hall D.G. & Benkovic S.J. (2012) Ring structure and aromatic
substituent effects on the pKa of the benzoxaborole pharmacophore. Med Chem Lett 3: 48-52;
[b] Martínez C.R. & Dardonville C. (2013) Rapid determination of ionization constants (pKa)
by UV spectroscopy using 96-well microtiter plates. Med Chem Lett 4: 142-145.
0. Manallack D.T. (2007) The pK(a) Distribution of drugs: Application to drug discovery.
Perspect Med Chem 1: 25-38.
3
3
1. Mote U.S., Patil S.R., Bhosale S.H., Han S.H. & Kolekar G.B. (2011) Fluorescence resonance
energy transfer from tryptophan to folic acid in micellar media and deionised water.
Photochem Photobiol B 103: 16-21.
3
3
3
3
3
2. Zhang G.W., Fu P., Wang L. & Hu M.M. (2011) Molecular spectroscopic studies of farrerol
interaction with Calf Thymus DNA. J Agric Food Chem 59: 8944-8952.
3. Song G.W., He Y. & Cai Z.X. (2004) The interaction between levofloxacine hydrochloride and
2
+
DNA mediated by Cu . J Fluoresc 14: 705-710.
4. Takacs-NovAk K., NoszAl B., Hermecz H., Keresztdri G., PodAnyi B. & SzAsz G. (1990)
Protonation equilibria of quinolone antibacterials. J Pharmacol Sci 79: 1023-1028.
5. Lerman L.S. (1961) Structural considerations in the interaction of deoxyribonucleic acid and
acridines. J Mol Biol 3: 18-30.
6. Suryawanshi V.D., Anbhule P.V., Gore A.H., Patil S.R. & Kolekar G.B. (2012) Spectroscopic
investigation
-amino-6-hydroxy-4-(3,4-dimethoxyphenyl)- pyrimidine-5-carbonitrile with Human Serum
Albumin: mechanistic and conformational study. Ind Eng Chem Res 51: 95-102.
on
the
interaction
of
pyrimidine
derivative,
2
3
7. Belatik A., Hotchandani S., Bariyanga J. & Tajmir-Riahi H.A. (2011) Binding sites of retinol
and retinoic acid with serum albumins. Eur J Med Chem 48: 114-123.
8. Lakowicz J.R. Principles of fluorescence spectroscopy, third ed., Springer, New York, 2006,
pp. 11-12.
3
This article is protected by copyright. All rights reserved.

Tables captions
Table 1 In vitro anti-Gram-positive bacterial data as MIC (nmol/L) for compounds 2–10
Table 2 In vitro anti-Gram-negtive bacterial data as MIC (nmol/L) for compounds 2–10
Table 3 The pKa values of compounds 3a–j, 6a–j, 7a–b, 8a–e, 9a–e and 10
Table 4 Binding constants and sites of 8b–HSA system at pH = 7.4
Figures captions
Figure 1 UV absorption spectra of DNA with different concentrations of compound 8b (pH = 7.4,
T = 298 K). Inset: comparison of absorption at 260 nm between the 8b–DNA complex and the
-5
sum values of free DNA and free compound 8b. c(DNA) = 3.56 × 10 mol/L, and c(compound 8b)
-5
-5
=
0–1.4 × 10 mol/L for curves a–h respectively at increment 0.2 × 10 .
2
+
Figure 2 Emission spectra of compound 8b in the presence of various concentrations of Cu ion.
-6
2+
-5
c(compound 8b) = 6.0 × 10 mol/L; c(Cu )/(1.0 × 10 mol/L), a–h: from 0.0 to 2.1 at increments
of 0.3; red line shows the emission spectrum of compound 8b only; T = 298 K, λex = 280 nm.
2
+
Figure 3 Estimation of the composition of the compound 8b–Cu complex, λex = 280 nm:
-6
c(compound 8b) = 6.0 × 10 mol/L.
2+
Figure 4 Fluorescence spectra of compound 8b and Cu system with increasing concentration of
2
+
DNA. Estimation of the composition of the compound 8b–Cu complex, λex = 280 nm:
-6
2+
-5
-6
c(compound 8b) = 6.0 × 10 mol/L, c(Cu )/(1.0 × 10 mol/L), c(DNA)/(3.3 × 10 mol/L), a–g:
from 0.0 to 0.60 at increments of 0.1; red line shows the emission spectrum of compound 8b in the
2
+
presence of Cu only; T = 298 K, λ = 280 nm. Inset: estimation of the composition of the
ex
2+
-6
compound 8b–Cu –DNA complex, c(compound 8b) = 6.0 × 10 mol/L.
Figure 5 Fluorescence spectra of compound 8b with increasing concentration of KI. c(compound
-6
-3
8
b) = 2.0 × 10 mol/L; c(KI)/(7.0 × 10 mol/L), a–h: from 0.0 to 2.1 at increments of 0.3; red line
shows the fluorescence spectrum of compound 8b only; T = 298 K, λex = 280 nm. Inset:
Stern–Volmer plot of the fluorescence titration data of compound 8b.
Figure 6 Fluorescence spectra of compound 8b and DNA system with increasing concentration of
-6
-6
-3
KI. c(compound 8b) = 2.0 × 10 mol/L; c(DNA) = 3.3 × 10 mol/L; c(KI)/(7.0 × 10 mol/L), a–h:
from 0.0 to 2.1 at increments of 0.3; red line shows the emission spectrum of compound 8b and
DNA system only; T = 298 K, λex = 280 nm. Inset: Stern–Volmer plot of the fluorescence titration
data of compound 8b and DNA system.
Schemes captions
Scheme 1 Synthetic routes of quinolone azoles.
Scheme 2 Synthetic routes of quinolone thiobenzimidazoles.
This article is protected by copyright. All rights reserved.

Scheme 3 Synthetic routes of clinafloxacin azoles.
Scheme 4 Synthetic route of clinafloxacin thiobenzimidazole.
Table 1. In vitro anti-Gram-positive bacterial data as MIC (nmol/L) for compounds 2–10
Gram-positive bacteria
Compds.
M. luteus
20.13+0.68
3.87+1.49
MRSA
9.68+0.56
S. aureus
18.94+2.05
9.38+0.54
B. subtilis
161.02+5.47
19.50+0.66
0.44+0.01
2a
2b
1.22+0.04
2
3
c
0.44+0.02
1.77+0.04
0.89+0.06
a
481.15+16.34
7.44+0.43
120.28+4.08
0.85+0.03
452.85+49.02
6.70+0.12
481.15+16.34
7.44+0.43
3b
3
c
264.24+8.97
247.48+8.41
7.95+0.46
248.70+26.92
232.92+25.21
6.56+0.14
248.70+26.92
247.48+8.41
7.95+0.46
66.06+2.24
15.46+0.52
7.16+0.14
3d
3
e
3
f
469.92+15.95
3.77+0.13
58.74+1.99
1.49+0.10
234.96+7.97
3.77+0.13
58.74+1.99
7.27+0.42
3g
3
h
64.37+2.19
241.53+8.20
4.02+0.14
128.74+4.37
15.09+0.51
1.57+0.11
60.58+0.19
60.38+2.05
3.78+0.41
32.18+1.09
241.53+8.20
7.74+0.45
3
i
3
j
6
a
489.92+16.63
448.98+15.24
218.80+7.43
119.10+4.04
231.81+7.87
119.57+4.06
0.76+0.03
122.48+4.16
897.96+30.49
109.40+3.71
119.10+4.04
115.91+3.93
29.89+1.01
1.34+0.09
244.96+8.32
224.49+7.62
102.96+11.15
119.10+4.04
102.27+3.43
105.51+3.55
0.38+0.02
244.96+8.32
224.49+7.62
437.60+14.86
59.55+2.02
245.45+7.85
26.38+1.14
0.86+0.03
6b
6
c
6d
6
e
6
f
6g
6
h
5.16+0.37
1.30+0.08
5.16+0.37
47.23+1.81
102.64+3.56
99.95+3.62
5.54+0.39
6
i
102.64+3.56
99.95+3.62
25.37+0.79
12.40+0.29
5.31+0.35
102.64+3.56
24.99+0.66
25.37+0.79
1.37+0.04
246.34+7.11
49.97+1.92
25.37+0.79
12.40+0.29
12.15+0.48
0.19+0.01
6
j
7
a
7b
24.80+0.59
12.15+0.48
0.19+0.01
8
a
12.15+0.48
0.39+0.03
8b
1.38+0.04
8
c
105.84+3.66
0.39+0.01
52.92+1.33
1.38+0.09
13.23+0.58
0.78+0.03
105.84+3.66
3.12+0.01
8d
8
9
e
52.92+1.75
3.09+0.07
52.92+1.75
1.36+0.08
13.23+0.58
12.35+0.44
2.85+0.19
254.02+7.33
5.39+0.36
a
9b
1.26+0.09
4.98+0.37
4.98+0.37
9
c
0.69+0.04
0.69+0.04
4.86+0.93
4.86+0.93
9
d
24.07+0.34
2.94+0.02
5.26+0.58
5.26+0.58
3.01+0.04
9
e
5.13+0.53
11.74+0.47
17.83+0.84
1.31+0.45
5.13+0.33
A
B
C
D
35.66+1.36
20.89+1.24
8.79+0.49
35.66+1.36
20.89+7.24
8.79+3.49
35.66+1.36
20.89+7.24
8.79+3.49
1.26+0.44
1.14+0.09
2.01+0.09
1.14+0.09
1.14+0.09
a
b
c
Minimum inhibitory concentrations were determined by micro broth dilution method for microdilution plates.
A = Chloromycin, B = Norfloxacin, C = Ciprofloxacin, D = Clinafloxacin.
M. luteus, Micrococcus luteus ATCC 4698; MRSA, Methicillin-Resistant Staphylococcus aureus N315; S. aureus,
Staphylococcus aureus ATCC25923; P. aeruginosa, Pseudomonas aeruginosa; S. dysenteriae, Shigella dysenteriae; E. coli,
Escherichia coli JM109; B. proteus, Bacillus proteus ATCC13315; B. subtilis, Bacillus subtilis ATCC 21216; E. typhosa,
Eberthella typhosa
This article is protected by copyright. All rights reserved.

Table 2. In vitro anti-Gram-negtive bacterial data as MIC (nmol/L) for compounds 2–10
Gram-negative bacteria
Compds.
P. aeruginosa
40.25+1.37
4.87+0.16
E .coli JM109
8.72+1.67
B. proteus
9.69+0.56
E. typhosa
37.89+4.10
4.59+0.49
2
2
a
b
2.35+0.13
9.38+0.54
2
3
c
0.89+0.04
0.89+0.01
0.44+0.02
1.77+0.12
a
481.15+16.34
0.0012+0.0005
247.48+8.41
123.74+4.21
16.51+0.56
234.96+7.97
0.0046+0.002
128.74+4.37
60.38+2.05
7.74+0.45
452.85+49.02
0.76+0.03
481.15+16.34
7.44+0.43
452.85+49.02
1.87+0.10
3
b
3
c
232.92+25.21
494.96+16.81
3.64+0.14
247.48+8.41
123.74+4.21
16.51+0.56
117.48+3.98
1.82+0.10
232.92+25.21
116.26+16.60
4.13+0.14
3
d
3
e
3
f
234.96+7.97
0.37+0.02
939.8+31.92
0.94+0.03
3
3
g
h
121.17+13.11
241.53+8.20
1.57+0.11
32.18+1.09
241.53+8.20
6.97+1.34
60.58+0.19
60.38+2.05
4.02+0.14
3
i
3
j
6
6
a
244.96+8.32
14.03+0.47
218.80+7.43
119.10+4.04
231.81+7.87
119.57+4.06
0.38+0.01
244.96+8.32
897.96+30.49
875.21+29.72
238.20+8.09
115.91+3.93
105.51+3.55
0.76+0.03
122.48+4.16
56.12+1.91
437.60+14.86
119.10+4.04
218.70+23.60
29.89+1.02
0.86+0.03
489.92+16.63
448.98+15.24
218.80+7.43
119.10+4.04
231.81+7.87
52.75+2.03
0.76+0.03
b
6
c
6
d
6
e
6
f
6
6
g
h
5.16+0.37
11.8+0.45
5.16+0.37
5.16+0.37
6
i
102.64+3.56
99.95+3.62
101.47+3.15
12.40+0.29
24.29+0.97
0.19+0.01
102.64+3.56
24.99+0.66
50.74+1.57
3.10+0.07
102.64+3.56
99.95+3.62
25.37+0.79
3.10+0.07
102.64+3.56
99.95+3.62
12.68+0.39
24.80+0.59
12.15+0.48
0.39+0.01
6j
7a
7b
8a
8b
24.29+0.97
0.097+0.001
52.92+1.33
3.12+0.08
3.04+0.12
0.78+0.01
8
c
105.84+3.66
3.12+0.08
105.84+3.66
3.12+0.08
52.92+1.33
3.12+0.08
8
d
8
9
e
52.92+1.33
5.39+0.14
52.92+1.33
3.09+0.07
26.46+0.87
5.39+0.14
52.92+1.33
12.35+0.48
1.26+0.09
a
9
b
4.98+0.37
4.98+0.37
1.26+0.09
9
c
4.86+0.39
1.23+0.04
4.86+0.39
4.86+0.39
9
d
24.07+0.84
5.13+0.03
24.07+0.84
2.94+0.02
12.03+0.44
5.13+0.03
96.27+3.35
11.74+0.47
17.83+0.84
9.13+0.62
9
e
A
B
C
D
35.66+1.36
41.79+1.48
5.04+0.34
8.91+0.38
35.66+1.36
20.89+0.74
8.79+0.49
41.79+1.48
8.79+0.49
5.04+0.34
1.14+0.09
1.14+0.09
1.14+0.09
0.57+0.01
a
b
c
Minimum inhibitory concentrations were determined by micro broth dilution method for microdilution plates.
A = Chloromycin, B = Norfloxacin, C = Ciprofloxacin, D = Clinafloxacin.
M. luteus, Micrococcus luteus ATCC 4698; MRSA, Methicillin-Resistant Staphylococcus aureus N315; S. aureus,
Staphylococcus aureus ATCC25923; P. aeruginosa, Pseudomonas aeruginosa; S. dysenteriae, Shigella dysenteriae; E. coli,
Escherichia coli JM109; B. proteus, Bacillus proteus ATCC13315; B. subtilis, Bacillus subtilis ATCC 21216; E. typhosa,
Eberthella typhosa
This article is protected by copyright. All rights reserved.

Table 3. The pKa values of compounds 3a–j, 6a–j, 7a–b, 8a–e, 9a–e and 10
Compds
λ(nm)
pKa
Ra
Compds
6h
6i
λ(nm)
pKa
Ra
3
3
a
259/284 6.565 0.991
259/283 6.469 0.990
259/283 6.654 0.994
259/284 6.473 0.994
259/284 6.469 0.991
259/284 6.275 0.991
259/283 6.198 0.991
259/284 6.367 0.990
259/284 6.178 0.990
259/283 6.387 0.995
259/284 6.408 0.995
259/284 6.299 0.994
259/284 6.347 0.993
259/283 6.467 0.991
259/284 6.586 0.993
259/284 6.378 0.993
259/283 6.269 0.991
259/283 6.298 0.992
259/284 6.341 0.992
259/283 6.487 0.990
259/284 6.465 0.991
259/283 6.375 0.990
259/284 6.478 0.993
259/283 6.347 0.993
259/284 6.535 0.993
259/283 6.356 0.994
259/284 6.596 0.991
259/283 6.396 0.994
259/283 6.226 0.990
259/284 6.365 0.994
259/283 6.438 0.992
259/283 6.531 0.991
259/283 6.424 0.990
b
3
c
6j
3
d
7a
7b
8a
8b
8c
3
e
3f
3
3
g
h
3
i
8d
8e
3j
6
6
a
9a
9b
9c
b
6
c
6
d
9d
9e
6
e
6f
10
6
g
a
Ra is the correlation coefficient.
This article is protected by copyright. All rights reserved.

1
2
3
4
3
a: R = CH , R = H, R = NO , R = ethyl;
3 2
1
2
3
4
O
N
3b: R = NO , R = H, R = H, R = ethyl;
2
R1
1
2
3
4
F
COOH 3c: R = CH , R = H, R = H, R = ethyl;
3
N
1
2
3
4
R2
2
3
3
3
d: R = H, R = NO , R = H, R = ethyl;
N
1
2
3
4
e: R = H, R = CH , R = H, R = ethyl;
N
3
1
2
3
4
R3
4
f: R = CH , R = H, R = NO , R = cyclopropyl;
3
2
N
R
1
2
3
4
HO
3g: R = NO , R = H, R = H, R = cyclopropyl;
2
1
2
3
4
3a-j
3h: R = CH , R = H, R = H, R = cyclopropyl;
3
1
2
3
4
3
3
i: R = H, R = NO , R = H, R = cyclopropyl;
2
ii
1
2
3
4
j: R = H, R = CH , R = H, R = cyclopropyl
3
O
R2
R3
O
N
F
COOH
F
COOH
iii
OH
N
N
N
O
4
4
N
N
N
R
N
R
2
a-b
6
a-j
R1
4
4
2
a: R = ethyl; 2b: R = cyclopropyl
1
2
3
4
6
6
a: R = H, R = H, R = H, R = ethyl;
b: R = H, R = H, R = NO , R = ethyl;
6c: R = CH , R = NO , R = H, R = ethyl;
, R = H, R = H, R = ethyl;
e: R = H, R = CH , R = CH , R = ethyl;
f: R = H, R = H, R = H, R = cyclopropyl;
, R = cyclopropyl;
h: R = CH , R = NO , R = H, R = cyclopropyl;
R = cyclopropyl;
j: R = H, R = CH , R = CH , R = cyclopropyl
1
2
3
4
i
2
1
2
3
4
O
3 2
1
2
3
4
F
COOH
6d: R = CH
6
6
6g: R = H, R = H, R = NO
6
3
1
2
2
3
3
4
4
3
3
1
N
N
1
2
3
4
4
1a-b
2
HN
R
1
2
3
4
3
2
1
2
3
4
4
4
6i: R = CH , R = H, R = H,
6
3
1
a: R = ethyl; 1b: R = cyclopropyl
1
2
3
4
3
3
R5
NH2
NH2
R5
N
iv
5
5
4
a: R = H;
5a: R = H;
CH3
5
5
N
H
4b: R = NO2; 5b: R = NO
2
4a-b
5a-b
Scheme 1. Synthetic routes of quinolone azoles. Reagents and conditions: (i) 2-(chloromethyl)oxirane,
NaHCO , CH CN, 45 ℃, 24 h; HCOOH, rt, 1 h; (ii) imidazoles, K CO , CH CN, 75 ℃, 30 h;
3
3
2
3
3
HCOOH, rt, 1 h; (iii) benzimidazoles, K CO , CH CN, 75 ℃, 30 h; HCOOH, rt, 1 h; (iv) CH COOH,
2
3
3
3
5
mol/L hydrochloric acid, 110 ℃, 20 h; NH
3
.H
2
O, rt, 1 h.
O
O
F
COOH
F
COOH
i
HN
N
O
N
N
R4
OH
N
N
R4
N
S
N
2a-b
7
a-b
4
4
4
4
2
a: R = ethyl; 2b: R = cyclopropyl
7a: R = ethyl; 7b: R = cyclopropyl
Scheme 2. Synthetic routes of quinolone thiobenzimidazoles. Reagents and conditions: (i)
-thiol-benzimidazole, K CO , CH CN, 75 ℃, 30 h; HCOOH, rt, 1 h.
2
2
3
3
This article is protected by copyright. All rights reserved.

R1
O
N
N
R2
1
2
3
F
COOH
8
8
a: R = CH , R = H, R = NO ;
3 2
N
1
2
3
b: R = NO , R = H, R = H;
2
H
N
R3
1
2
3
N
8c: R = CH , R = H, R = H;
3
HO
1
2
3
8
d: R = H,
R = NO , R = H;
Cl
2
1
2
3
8
a-e
8e: R = H,
R = CH , R = H
3
ii
R2
N
R3
O
O
F
COOH
F
COOH
O
iii
H
OH
N
H
N
N
N
N
N
N
Cl
2
c
Cl
R1
9
a-e
i
1
2
3
9
9
9
9
a: R = H, R = H,
R = H;
O
1
2
3
b: R = H, R = H,
R = NO ;
2
F
COOH
1
2
3
c: R = CH , R = NO , R = H;
3
2
1
2
3
d: R = CH , R = H, R = H;
3
H N
1
2
3
2
N
N
9e: R = H, R = CH , R = CH
3 3
Cl
1
c
Scheme 3. Synthetic routes of clinafloxacin azoles. Reagents and conditions: (i)
-(chloromethyl)oxirane, C OH, 25 ℃, 24 h; (ii) imidazoles, K CO , CH CN, 75 ℃, 24 h;
2
2
H
5
2
3
3
HCOOH, rt, 1 h; (iii) benzimidazoles, K CO , CH CN, 75 ℃, 24 h; HCOOH, rt, 1 h.
2
3
3
O
N
O
N
F
COOH
i
F
COOH
HN
N
O
H
N
OH
H
N
N
S
N
Cl
Cl
2
c
10
Scheme 4. Synthetic routes of clinafloxacin thiobenzimidazole. Reagents and conditions: (i)
-thiol-benzimidazole, K CO , CH CN, 75 ℃, 24 h; HCOOH, rt, 1 h.
2
2
3
3
This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.