Membrane active 7-thiazoxime quinolones as novel DNA binding agents to decrease the genes expression and exert potent anti-methicillin-resistant Staphylococcus aureus activity

Article abstract of DOI:10.1016/j.ejmech.2021.113340

A novel class of 7-thiazoxime quinolones was developed as potential antimicrobial agents for the sake of bypassing resistance of quinolones. Biological assays revealed that some constructed 7-thiazoxime quinolones possessed effective antibacterial efficiency. Methyl acetate oxime derivative 6l exhibited 32-fold more active than ciprofloxacin against MRSA, which also possessed rapidly bactericidal ability and low toxicity towards mammalian cells. The combination use of 7-thiazoxime quinolone 6l and ciprofloxacin was able to improve antibacterial potency and effectively alleviate bacterial resistance. The preliminarily mechanism exploration revealed that compound 6l could destroy the cell membrane and insert into MRSA DNA to bind with DNA gyrase, then decrease the expression of gyrB and femB genes. The above results strongly suggested that methyl acetate oxime derivative 6l held a promise for combating MRSA infection.

Full text of DOI:10.1016/j.ejmech.2021.113340

European Journal of Medicinal Chemistry 217 (2021) 113340
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Membrane active 7-thiazoxime quinolones as novel DNA binding
agents to decrease the genes expression and exert potent anti-
methicillin-resistant Staphylococcus aureus activity
Jin-Ping Chen, Narsaiah Battini 2, Mohammad Fawad Ansari 1, Cheng-He Zhou*
Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Luminescence Analysis and Molecular Sensing (Southwest University), Ministry of
Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing, 400715, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of 7-thiazoxime quinolones was developed as potential antimicrobial agents for the sake of
bypassing resistance of quinolones. Biological assays revealed that some constructed 7-thiazoxime
quinolones possessed effective antibacterial efficiency. Methyl acetate oxime derivative 6l exhibited
32-fold more active than ciprofloxacin against MRSA, which also possessed rapidly bactericidal ability
and low toxicity towards mammalian cells. The combination use of 7-thiazoxime quinolone 6l and
ciprofloxacin was able to improve antibacterial potency and effectively alleviate bacterial resistance. The
preliminarily mechanism exploration revealed that compound 6l could destroy the cell membrane and
insert into MRSA DNA to bind with DNA gyrase, then decrease the expression of gyrB and femB genes.
The above results strongly suggested that methyl acetate oxime derivative 6l held a promise for
combating MRSA infection.
Received 4 December 2020
Received in revised form
30 January 2021
Accepted 25 February 2021
Available online 10 March 2021
Keywords:
Drug-resistance
Quinolone
Cell membrane
MRSA DNA
Gene
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
Quinolones are one of the most commonly used prescription
drugs to treat various infections [10,11]. In particular, the clinical
Five-membered heteroaromatic thiazole has enormously
contributed to drug applications [1,2], especially in antimicrobial
field [3,4]. As the continuous severity of antibiotic resistance [5,6],
thiazole-based clinical drugs such as cefuzonam, cefoselis, cefme-
noxime and abafungin have triggered extensive research to develop
more bioactive molecules. Introducing thiazole into the existing
drugs is one of the most widespread methods to overcome resis-
tance [7]. For instance, the cephalosporins cefdinir and tigemonam
possess excellent inhibitory efficacy to bacterial strains including
resistant ones. Our previous work revealed that the thiazole-
modified derivatives at the 3-carboxyl group of quinolone skel-
eton (Leads A, B and C, Fig.1) not only exhibited broad antimicrobial
spectrum, but also effectively retarded the development of resis-
tance [8,9]. These aforesaid stimulating properties arouse the great
enthusiasm in the further development of potential thiazolyl qui-
nolone antibacterial agents.
levofloxacin, norfloxacin and ciprofloxacin have made great dedi-
cations for human health. These drugs access the bacteria via active
transport of cell membrane and primarily target topoisomerase or
gyrase to stabilize the cleavage complex, thus blocking DNA repli-
cation [12]. However, mutations in active sites of DNA gyrase such
as gyrA and gyrB genes [13], changes in the membrane perme-
ability caused by the adjustment of the pathogen defense system
and shifts of the structure and quantity of the outer membrane
proteins all make the evolution and widespread distribution of
resistant strains [14]. The structural modification of quinolone
skeleton at the C-7 position has been proved to be a promising
strategy to circumvent antibiotic resistance mechanisms and retain
inhibitory potency against multidrug-resistant bacteria [15]. Much
work has shown that introducing azoles [16], such as imidazole
[17], triazole [18] and tetrazole [19] at C-7 site of quinolone drugs
afforded promising molecules. Nevertheless, few exemplifications
on 7-thiazolylquinolones are described as potent antibacterial
agents in the literature [20]. Therefore, we have overwhelming
interest in incorporating thiazole into the C-7 position of quino-
lones with the expectance to discovery potential new antibacterial
molecules.
* Corresponding author.
E-mail address: zhouch@swu.edu.cn (C.-H. Zhou).
Postdoctoral researchers from CSIR-Indian Institute of Integrative Medicine
2
(IIIM), India.
1
Postdoctoral fellow from Jamia Millia Islamia, Delhi, India.
https://doi.org/10.1016/j.ejmech.2021.113340
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

J.-P. Chen, N. Battini, M.F. Ansari et al.
European Journal of Medicinal Chemistry 217 (2021) 113340
Fig. 1. Previously published biologically active 3-(thiazolyl) quinolones (Leads A, B and C).
Oxime characterized with electron accepting C]N group and
donating -O- moiety is a favorable fragment in antimicrobial drugs
[21]. For instance, the fourth-generation quinolone drug gemi-
floxacin and the fifth-generation cephalosporin ceftaroline fosamil
with oxime modification exhibited excellent antibacterial potenti-
ality to resistant bacteria [22], which further pointed the way to
conduct new antibacterial agents with amendatory therapeutic
indexes. Therefore, the introduction of oxime groups into thiazolyl
quinolones is an attractive topic.
Considering all the aforementioned facts and continuing our
prior works on the thiazolyl quinolones, herein thiazoxime was
introduced into the 7-position of quinolone backbone to afford a
class of novel 7-thiazoxime quinolones with large promise as
antibacterial agents (Fig. 2). All the synthesized 7-thiazoxime
quinolones were evaluated for their antibacterial ability. Further
studies including drug resistance, time-killing kinetic, drug com-
bination and cytotoxicity assays were implemented to assess the
potential of high active molecule as new antibacterial drug. The
possible antibacterial mechanism was explored through bacterial
membrane disruption assay, interactions with MRSA DNA, molec-
ular docking and the expression of resistance-related genes.
2. Results and discussion
2.1. Chemistry
The intermediate thiazolyl oximes and target 7-thiazoxime
quinolones were prepared according to the synthetic process
illustrated in Schemes 1 and 2. Commercially available starting
material 2-acetothiazole 1 was reacted with bromine in glacial
acetic acid system to get thiazole bromide 2 with satisfactory yield.
Bromide 2 was selected to construct the 7-thiazolylquinolones
5aeb, in which the norfloxacin 4a and ciprofloxacin 4b partici-
pated in the reaction. However, the reaction of 7-
thiazolylquinolones 5aeb with hydroxylamine hydrochlorides
was difficult to produce 7-thiazoxime quinolones, in spite of using
different catalysts such as anhydrous sodium acetate, sodium car-
bonate, piperidine, pyridine, and different solvents such as meth-
anol and ethanol. This might be the consequence that the carboxyl
group had undesirable effect on the active amine. After screening
the reaction conditions, oximes 3aef were given by the conden-
sation of compound 2 with various hydroxylamine hydrochlorides
under the catalysis of anhydrous sodium acetate at room
Fig. 2. Design of novel 7-thiazoxime quinolones.
2

J.-P. Chen, N. Battini, M.F. Ansari et al.
European Journal of Medicinal Chemistry 217 (2021) 113340
Scheme 1. Synthetic scheme of thiazolyl oximes.
Scheme 2. Synthetic scheme of 7-thiazoxime quinolones.
temperature using methanol as solvent. The quinolones 4aꢀb un-
derwent nucleophilic substitution with thiazolyl oximes 3aef to
afford norfloxacin and ciprofloxacin derivatives 6aef. The modifi-
cation of ciprofloxacin analogue 4c was conducted to produce the
8-chloroquinolones 6mer in 51.4e90.7% yield, with the purpose to
investigate the effect of C-8 substitution on the growth inhibition of
microorganisms.
In general, oxime has two configurations theoretically, Z and E.
The prepared target products were cultured for single crystals to
verify the configuration of the 7-thiazoxime quinolones. Fortu-
nately, the single crystal of benzyl oxime 6p was obtained and X-ray
diffraction proved that the oxime geometry showed E configuration
(Fig. 3). This might be the reason that the Z configuration in the 7-
thiazoxime quinolones is usually unstable and can easily converted
to the E configuration.
3

J.-P. Chen, N. Battini, M.F. Ansari et al.
European Journal of Medicinal Chemistry 217 (2021) 113340
Fig. 3. Crystal structure of 7-thiazoxime ciprofloxacin 6p.
2.2. Antibacterial evaluation
6a and ethyl derivative 6b with MIC values of 0.25
suppressed the growth of MRSA, being 64-fold more potent in
comparison to norfloxacin (MIC ¼ 16 g/mL). Towards Staphylo-
mg/mL efficiently
Antibacterial evaluation of the thiazolyl quinolones were carried
out by the well-known two-fold micro-dilution standard assay
[23]. Norfloxacin and ciprofloxacin were utilized as reference
agents. Most of the newly developed 7-thiazoxime quinolones
could suppress the growth of the tested bacteria effectively. The
minimum inhibitory concentration (MIC) values were used to
represent bioactivities depicted in Table 1ꢀ4.
m
coccus aureus, Enterococcus faecalis and Acinetobacter baumannii,
the extension of alkyl chain was not conducive to improve the
antibacterial potentiality. In order to investigate the influence of
steric hindrance, tert-butyl oxime 6c was prepared. The antibacte-
rial activity of compound 6c decreased in contrast to oximes 6aꢀb.
Especially, Gram-negative bacteria was more insensitive to mole-
cule 6c obviously (Table 2). These results verified that large steric
hindrance substituents were unfavorable for antibacterial ability.
To enrich the structure-activity relationship (SAR), unsaturated
substituted oximes 6eꢀf were developed. The antimicrobial activ-
ities of allyl oxime 6e and methyl acetate oxime 6f conspicuously
strengthened in comparison with alkyl analogues 6aꢀc. The results
revealed that introducing unsaturated groups enhanced antimi-
crobial activities, which might be related to the increase in electron
cloud density. With the aim to enrich the chemical diversity, benzyl
oxime 6d was designed and synthesized. The ability of compound
6d to inhibit bacterial growth was stronger than alkyl oximes 6aꢀc,
suggesting that the electron cloud density had a more significant
effect on the antibacterial efficacies. All 7-thiazoxime norfloxacin
Thiazolyl norfloxacin molecules 5a and 6aꢀf displayed good
antibacterial activities against the tested stains in Tables 1 and 2.
The acetylthiazolyl norfloxacin 5a showed low MIC values of 1 and
4
m
g/mL respectively against standard Staphylococcus aureus ATCC
29213 and drug-resistant MRSA, being 16 and 4 folds more active in
comparison to the norfloxacin with MIC value of 16 g/mL.
m
Furthermore, the Enterococcus faecalis and Pseudomonas aeruginosa
ATCC 27853 were also sensitive to acetylthiazolyl norfloxacin 5a.
The antibacterial behavior manifested that the thiazole modified
norfloxacin derivatives gave satisfactory antibacterial potencies,
especially towards the Gram-positive bacteria. To investigate the
impact of oxime fragments on the biological activity of this class of
compounds, the alkyl oximes 6aꢀb were prepared. Methyl oxime
Table 1
Antibacterial data of 7-thiazolyl norfloxacin derivatives 5a and 6aef against Gram-positive bacteria.
Compds
MIC (mg/mL)
MRSA
Staphylococcus aureus
Staphylococcus aureus ATCC 25923
Staphylococcus aureus ATCC 29213
Enterococcus faecalis
5a
4
4
2
1
2
6a
6b
6c
6d
6e
6f
0.25
0.25
0.25
0.25
0.25
0.25
16
2
8
0.25
4
0.25
4
0.5
0.5
1
0.25
0.5
0.25
2
0.25
4
16
256
2
4
16
0.25
0.25
1
0.25
4
2
2
Norfloxacin
4
4

J.-P. Chen, N. Battini, M.F. Ansari et al.
European Journal of Medicinal Chemistry 217 (2021) 113340
Table 2
Antibacterial data of 7-thiazolyl norfloxacin derivatives 5a and 6aef against Gram-negative bacteria.
Compds
MIC (
mg/mL)
Klebsiella pneumonia Acinetobacter baumannii Escherichia coli Escherichia coli ATCC 25922 Pseudomonas aeruginosa Pseudomonas aeruginosa ATCC 27853
5a
6a
6b
6c
6d
6e
6f
32
16
0.25
2
16
1
0.25
1
2
32
64
4
2
8
8
1
1
4
0.25
8
256
256
1
128
256
256
0.25
0.25
1
0.5
0.25
32
256
256
1
0.25
0.25
2
0.5
0.25
0.25
2
64
0.5
0.25
16
Norfloxacin 4
16
molecules 6aꢀf (MIC ¼ 0.25
m
g/mL) were 64-fold more active
exert efficacy. All results demonstrated that the design of the 7-
thiazoxime quinolones was reasonable.
against MRSA in contrast with reference drug norfloxacin
(MIC ¼ 16
mg/mL), which suggested that the modified norfloxacin
derivatives were advantageous to antibacterial potencies, espe-
cially for resistant strains.
2.3. Multistep resistance study
With the aim to investigate the effectiveness of thiazoleoximes
on the antibacterial activity of other quinolones, ciprofloxacin as a
synthetic third-generation quinolone antibacterial drug was
selected to optimize. Structural modifications showed that the SAR
of thiazolyl ciprofloxacin compounds 6gꢀr were similar to nor-
floxacin derivatives, and the overall bacteriostatic efficacies of this
series of compounds were more potent than ciprofloxacin in
Tables 3 and 4. It was worth noting that the replacement of the C-8
position by a chlorine atom resulted in 8-chlorociprofloxacin de-
rivatives 6mꢀr with a significant increase in biological activity
against Gram-negative bacteria. These results showed that the 8-
The prevalence and spread of MRSA is a huge challenge in
addressing bacterial infections and drug development [24].
Therefore, it is necessary to evaluate the effect of 7-thiazoxime
quinolones on MRSA resistance. The ciprofloxacin and norfloxacin
were used as control to study the MRSA resistance of the highly
active 7-thiazoxime quinolones 6a, 6e, 6g, 6l, 6m, 6n, 6o and 6r. The
experimental results showed that the alkyl oximes 6a, 6e, 6g, 6m,
6n and 6o presented no obvious effect on the treatment of MRSA
resistance (Fig. 5A), which indicated that alkyl derivatives and
chlorine atom at C-8 were not conducive to overcoming resistance.
But the methyl acetate oxime 6l had a small chance in developing
MRSA resistance after 14 passages in contrast to ciprofloxacin.
Furthermore, when compound 6l was combined with ciprofloxa-
cin, no obvious drug resistance was generated after 16 passages in
Fig. 5B. The obtained results all supported the difficulty of MRSA
resistance development under the treatment of compound 6l
rather than norfloxacin and ciprofloxacin.
chloroquinolones
6mꢀr
exhibited
strong
efficacies
(MIC ¼ 0.25e2 g/mL) against all the evaluated microbes except for
m
Klebsiella pneumonia. In particular, all tested strains were sensitive
to methyl acetate oxime 61, especially against MRSA with a MIC
value of 0.25
mg/mL. Preliminary analysis showed that the anti-
bacterial activities of quinolone oximes tended to decrease with the
increase of Clog P values. The methyl ester compounds 6f, 6l and 6r
had the lowest ClogP values and the best antibacterial activity in
corresponding series.
2.4. Bactericidal kinetic study
Both series of 7-thiazoxime quinolones showed excellent ac-
tivities against Gram-positive bacteria. Some promising molecules
against S. aureus ATCC 25923, S. aureus ATCC 29213, S. aureus and
MRSA were more clearly emerged in Fig. 4. Notably, the anti-MRSA
activity of 7-thiazoxime quinolones remained outside the range of
the basis of our previous researches conducive to yielding potent
efficacy, which suggested that it might be an atypical mechanism to
To further evaluate the effect of quinolone methyl acetate oxime
6l on bacterial growth, the bactericidal kinetic was conducted.
MRSA was treated by compound 6l at different concentrations with
clinical antibiotic ciprofloxacin as the positive control (Fig. 6A). The
sterilization rate of ciprofloxacin was lower than the oxime deriv-
ative 6l within 6 h at the same concentration (2
mg/mL). In addition,
MRSA could be eradicated within 4 h treated by 6l (4
mg/mL). The
Table 3
Antibacterial data of 7-thiazolyl ciprofloxacin derivatives 5b and 6ger against Gram-positive bacteria.
Compds
MIC (mg/mL)
MRSA
Staphylococcus aureus
Staphylococcus aureus ATCC
Staphylococcus aureus ATCC 29213
Enterococcus faecalis
25,923
5b
16
1
4
2
8
6g
6h
6i
0.25
0.25
4
0.25
0.25
4
0.5
0.25
1
0.25
4
4
0.25
0.25
2
6j
6k
6l
6m
6n
6o
6p
6q
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
8
0.25
0.25
0.25
0.25
0.25
0.25
2
0.25
0.25
2
1
0.5
32
2
0.25
0.25
0.5
0.5
0.5
1
0.25
0.25
2
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
2
0.25
0.25
0.25
0.25
2
0.5
0.25
0.5
6r
Ciprofloxacin
5

J.-P. Chen, N. Battini, M.F. Ansari et al.
European Journal of Medicinal Chemistry 217 (2021) 113340
Table 4
Antibacterial data of 7-thiazolyl ciprofloxacin derivatives 5b and 6ger against Gram-negative bacteria.
Compds
MIC (mg/mL)
Klebsiella
pneumonia
Acinetobacter
baumannii
Escherichia coli Escherichia coli ATCC 25922 Pseudomonas
Pseudomonas aeruginosa ATCC 27853
aeruginosa
5b
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
16
2
1
1
4
4
0.25
2
128
0.25
1
4
256
32
1
1
2
0.25
0.25
1
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
1
256
0.25
64
0.25
0.25
0.25
0.25
0.25
0.25
0.5
0.25
0.25
1
1
256
0.25
0.25
0.25
256
256
0.25
256
256
8
256
0.5
0.25
0.25
256
0.5
0.25
0.25
0.5
2
0.25
0.25
0.25
0.25
0.25
0.25
0.25
4
1
0.25
0.25
4
0.25
0.25
4
0.25
0.25
0.5
Ciprofloxacin 2
1
0.5
Fig. 4. The diagram for biological activity of the most potent compounds against
MRSA, S. aureus, S. aureus ATCC 25923 and S. aureus ATCC 29213.
experiment result showed that the methyl acetate oxime 6l had a
rapid killing effect against MRSA.
2.5. Drug combination study
The adjustments of bacterial defense system lead to the failure
of single-target drugs. Combination therapy was applied as a
promising way for efficiency improvement, side-effect avoidance
and even the combat of drug resistance [25]. Therefore, the com-
bination of active compound 6l and ciprofloxacin against bacteria
was carried out. The results exhibited that the strains treated by the
combination drug were more sensitive than their individual use,
and their combined action showed synergistic and additional ef-
fects (Table 5). From the facts above, the combination of cipro-
floxacin and quinolone derivative 6l hold a significant position in
treating the microbial infective diseases.
Fig. 5. The resistance development of 7-thiazoxime quinolones 6a, 6e, 6g, 6l, 6m, 6n,
6o and 6r against MRSA.
revealed by the use of scale-up of fluorescence. MRSA cells treated
by methyl acetate oxime 6l at MIC concentration showed the in-
crease of fluorescence intensity in a time-dependent rate in com-
parison to untreated cells, which revealed that compound 6l
displayed obvious dissipation of the MRSA cell membrane potential
(Fig. 7).
2.6. Loss of membrane integrity
2.6.1. Membrane depolarization assay
The depolarization effect of the thiazolyl ciprofloxacin deriva-
tive 61 on the membrane was carried out with the fluorescent
sensitive dye diSC35 [26]. The membrane depolarization was
6

J.-P. Chen, N. Battini, M.F. Ansari et al.
European Journal of Medicinal Chemistry 217 (2021) 113340
Fig. 6. Time-dependent killing of MRSA by methyl acetate oxime 6l.
Fig. 7. Cytoplasmic membrane depolarization of MRSA treated by compound 6l.
2.6.2. Inner membrane permeabilization
hydrogen-donating and -accepting functionalities to deliver
promising likelihood to construct favorable hydrogen bond and/or
p-p interactions with the binding pocket of DNA in spatial envi-
ronment. These might display different mechanisms from con-
ventional quinolones to tackle the emergency of pathogenic
resistance. Therefore, the exploration of the interaction mode be-
tween quinolone compound 61 and DNA isolated from MRSA was
carried out by UV and fluorescence spectroscopy.
Destroying cell membranes could reduce the tendency of bac-
teria to develop resistance. In order to confirm whether these
quinolone oximes acted by disrupting the bacterial cell membrane,
the fluorescent probes calcein-AM and propidium iodide (PI) were
used to study the destruction of membrane integrity by the methyl
acetate oxime 6l through fluorescence analysis. The PI staining
experiments showed that a dose-dependent increase in fluores-
cence intensity appeared (Fig. 8A), which manifested molecule 6l
could cause extensive membrane damage. As the content of the
methyl acetate oxime 61 increased, the fluorescence intensity of
calcein-AM decreased significantly in Fig. 8B. These results indi-
cated that the active molecule 6l was capable of disrupting the
membrane integrity.
2.7.1. Influence of DNA configuration
The maximum absorption peak at 260 nm of DNA increased
proportionally with the increase of the content of molecule 6l in
Fig. 9. At the same time, the absorption of the simple addition of the
measured values of DNA and the methyl acetate oxime 6l was
slightly lesser than the 6l-DNA complex. This phenomenon might
be the result of electrostatic interaction between hybrid 6l and DNA
bases, providing strong evidence for changes in DNA duplexes and
the formation of 6l-DNA complexes. Supporting information
(Fig. S1) displayed the equation and plot of binding constant.
2.6.3. Calcein-AM/PI dual stain fluorescence microscopy
Fluorescence micrographs (Fig. 8C and D) clearly demonstrated
that the number of red fluorescent cells increased while the green
fluorescence gradually decreased, demonstrating that compound 6l
could efficiently destroy the membrane and cause MRSA death.
Generally, fluoroquinolones enter cells mainly through active
transport of carriers to exert potent activity. The dual-targeting
bactericidal effects of these two types of molecules might benefit
their synergistic or additivity bactericidal effects. These findings
provide an advisable approach to maximize antibacterial efficiency
through different modes of action.
2.7.2. Competitive interaction of compound 6l and probes with
MRSA DNA
Aiming to explore the binding mode of the compound and DNA,
commercial 40, 6-diamidino-2-phenylindole (DAPI) and marketable
acridine orange (AO), whose action modes with DNA is character-
ized by intercalation, minor groove binding or electrostatic in-
teractions, were used as spectral probes to explore the mechanism
of action, respectively [28]. The fluorescence intensity at 537 nm
was significantly weakened with the content of the methyl acetate
oxime 6l in Fig. 10, signifying that the molecule 6l could interfere
DNA-AO complex. On the contrary, the fluorescence intensity of
DAPI hardly changed with the increase of compound concentration
2.7. Interactions between the methyl acetate oxime 6l and MRSA
DNA
DNA is an important biological macromolecule considered to be
an effective drug target [27]. The methyl acetate oxime 6l contained
Table 5
Combination effects of methyl acetate oxime 6l with ciprofloxacin (MIC, mg/mL).
Bacteria
Alone
6l
In combination
6l
FIC Indexa
Effect
Ciprofloxacin
Ciprofloxacin
Klebsiella pneumonia
Escherichia coli
MRSA
0.25
0.25
0.25
0.25
2
2
8
2
0.0625
0.0625
0.125
0.25
0.125
0.125
2
0.3125
0.3125
0.5
Synergy
Synergy
Synergy
Additivity
Staphylococcus aureus ATCC 25923
1
1.5
a
The fractional inhibitory concentration (FIC) index ¼ (MIC of compound A combined/MIC of compound A alone) þ (MIC of compound B combined/MIC of compound B
alone). FIC index ꢁ0.5, synergy; 0.5 < FIC index ꢁ2, additivity; FIC index >2, antagonism.
7

J.-P. Chen, N. Battini, M.F. Ansari et al.
European Journal of Medicinal Chemistry 217 (2021) 113340
Fig. 8. Bacterial membrane permeabilization. (A, B) Estimation of membrane damage using PI and calcein-AM staining in MRSA treated with methyl acetate oxime 6l (1e4 ꢂ MIC);
(C, D) Fluorescence micrograph images of MRSA stained with calcein-AM and PI treated with compound 6l (1e4 ꢂ MIC). Scale bar for all images is 100
mm.
Fig. 9. Ultraviolet spectra of DNA with methyl acetate oxime 6l.
8

J.-P. Chen, N. Battini, M.F. Ansari et al.
European Journal of Medicinal Chemistry 217 (2021) 113340
2.9. Effects of compound 6l on MRSA genes expression
The resistance mechanism of MRSA is very complicated and
involves many genes, such as mecA and femB [30]. Recently, studies
shown that the modified molecules on the basis of existing drug
fragments could alleviate the drug resistance of the globally
apprehensive MRSA while guaranteeing the efficacy of drugs by
regulating the expression of resistance-related genes [31]. Consid-
ering that the compound could form hydrogen bonds with DNA
gyrase B, the expression levels of mecA, femB and gyrB genes in
MRSA were observed by RT-PCR. As show in Fig. 12, the gyrB gene
expression level decreased after compound 6l was cultured with
MRSA for 3 h, revealing that the quinolone oxime 6l could interact
with the gyrase to reduce the expression of related genes. Mean-
while, the relative expression of femB gene decreased rapidly,
disclosing that the most active molecule 6l might alleviate the
resistance of MRSA by inhibiting the expression of different genes.
However, compound 6l cannot inhibit mecA gene expression.
Recent studies suggested that the resistance level of MRSA has
nothing to do with the output of the PBP2a protein encoded by the
mecA gene, but is related to auxiliary genes, such as the femB gene
[32]. The results opened up a new direction for us to explore its
possiblity against MRSA mechanism in future research. All the
primers in qRT-PCR are shown in Table S1.
Fig. 10. Mode of action between methyl acetate oxime 6l MRSA DNA.
in Fig. S2. Therefore, the above experimental data showed that the
highly active compound 6l could intercalate into MRSA DNA.
2.8. Molecular modeling study
Bacterial DNA gyrase as an important target of antibacterial
quinolones plays a significant function in the process of DNA
replication [29]. Considering that the active molecule 6l could
destroy the integrity of the membrane, we deduced that the methyl
acetate oxime 6l might destroy the bacterial cell membrane, and
then interrupted the function of DNA gyrase, eventually leading to
cell death. Hence, a proper ligand-receptor docking was done
smoothly to investigate the possible antibacterial mechanism of
quinolone oximes. These obtained results of ciprofloxacin thiazole
hybrid 5b and oxime 6l binding to DNA gyrase B (PDB ID: 3U2K)
were displayed in Fig. 11. The binding energies of compounds 5b
and 6l were ꢀ8.75 and ꢀ6.72 kcal/mol, respectively. Compound 5b
could form three hydrogen bonds with the residue ARG-144 with
distance of 1.6 Å, 1.8 Å and 2.1 Å. The active molecule 6l could
interact with four amino acid residues, namely ASP-81, ARG-84,
GLY-85 and GLN-91. The supramolecular interaction by hydrogen
bonds between DNA gyrase and thiazolyl ciprofloxacin 5b or its
oxime derivative 6l might rationalize the antibacterial mechanism
of quinolone oximes.
Fig. 12. Effects of methyl acetate oxime 6l on MRSA genes expression.
Fig. 11. The comparison of ciprofloxacin thiazole hybrid 5b (A) and methyl acetate oxime 6l (B) were predicted to multisite binding with GyrB ATPase domain.
9

J.-P. Chen, N. Battini, M.F. Ansari et al.
European Journal of Medicinal Chemistry 217 (2021) 113340
2.10. Hemolysis assay
3. Conclusion
The toxicity of methyl acetate oxime 6l was further studied by
hemolysis experiment. The processed red blood cells (RBCs) were
incubated in 1% Triton X-100 (positive control) in PBS (blank) for 4 h
at 37 ꢃC and evaluated with various concentrations of compound 6l.
The results showed that the hemolysis caused by oxime 6l was less
than 3% at the effective bactericidal concentration (Fig. 13), and the
hemolysis rate was still less than 5% even at a high concentration
(64 ꢂ MIC). It was observed under the microscope that the eryth-
rocytes remained intact by treating with compound 6l (16 ꢂ MIC),
which was similar to the saline control group.
In conclusion, the present study explored a class of novel 7-
thiazoxime quinolones as potentially antimicrobial agents. Bioac-
tivity assay indicated that most of the developed compounds
exhibited good inhibitory activity against the evaluated strains and
SAR analysis was summarized in Fig. 15. Notably, methyl acetate
oxime 6l showed excellent inhibitory efficacy (MIC ¼ 0.25e1
mL) against the tested bacteria, especially against MRSA with low
MIC value of 0.25 g/mL. Furthermore, oxime 6l exerted broad
mg/
m
antimicrobial spectrum, rapidly bactericidal ability and low cyto-
toxicity to mammalian cell lines. Its combination with ciprofloxacin
could enhance the antimicrobial efficiency and abate the tendency
to induce bacterial resistance. Subsequent exploration of anti-
MRSA action mechanism revealed that 6l was able to insert into
the tested DNA after effectively damaging cell membrane to inter-
fere the DNA replication and result in femB and gryB genes to
decrease expression. All above observations provided an important
step forward for development of the 7-thiazoxime quinolones to
access significant anti-MRSA agents.
2.11. Cytotoxicity assay
Cytotoxicity is an important aspect in defining reliable drug
candidates [33]. The toxicity of oxime 6l to LO2 cells (normal he-
patocyte cell) and 293 T cells was evaluated by a colorimetric cell
proliferation MTT assay. The survival rate at more than 90% dis-
played good tolerance of LO2 and 293 T cells to compound 6l, even
if the cells were incubated for 24 h with concentration of effective
sterilization. Final result showed that molecule 6l exhibited a sig-
nificant amount of selectivity for bacterial membranes compared
with mammalian membranes (Fig. 14).
4. Experimental protocols
4.1. General methods
Sichuan Provincial People՚s Hospital (Chengdu, China) provided
the microbial strains. The PI, calcein-AM, AO and DAPI were pur-
chased from Sigma-Aldrich. The high-resolution mass spectra
(HRMS) were used the analyses of the prepared compounds on ESI-
mass spectra were recorded on IonSpec FTeCR mass spectrometer
with ESI resource and are reported in the form of (m/z). The reac-
tion was monitored at 254 and 365 nm by TLC analysis using pre-
coated silica gel plates. The synthesized compounds were analyzed
by 1H NMR and 13C NMR spectra were recorded on Bruker AV 600
spectrometer (Bruker, Germany). Melting points were taken using
basic melting point apparatus X-6 type (Focus, China) and were
uncorrected. All fluorescence spectra were recorded on F-7000
spectrofluorimeter (Hitachi, Tokyo, Japan) and UV spectra were
obtained through TU-2450 spectrophotometer (Puxi Analytic In-
strument Ltd. of Beijing, China), both of which were equipped with
1.0 cm quartz cells. Microscopic observations were made with a
positive fluorescence microscope (LW300LFT, Cewei, China). All
2.12. ADMET study
The pharmacokinetic properties of the synthesized quinolone
oximes, clinic drug cefditoren pivoxil and ciprofloxacin were pre-
dicted by using the online SwissADME and PreADMET software
[34]. The methyl ester oxime 6l and clinic drug cefditoren pivoxil
were not completely satisfied Lipinski rule in Table S3. The pre-
dictive results pointed out that the oxime 6l could not penetrate
blood-brain barrier (BBB) and had low central nervous system
toxicity. The methyl acetate oxime 6l had the highest skin perme-
ability and good human intestinal absorption. Moreover, the methyl
ester oxime 6l displayed no carcinogenicity in rat and mouse
model, indicated that compound was non-carcinogenic in nature.
These results demonstrated that methyl ester oxime 6l could act as
a promising molecule for further antibacterial development.
Fig. 13. Hemolysis assay. (A) Hemolysis rate of compound 6l to red blood cells; (B) The microscope photos of RBCs treated by 6l with 4 mg/mL (a and c) and physiological saline (b
and d).
10

J.-P. Chen, N. Battini, M.F. Ansari et al.
European Journal of Medicinal Chemistry 217 (2021) 113340
Fig. 14. Cytotoxicity of compound 6l in the 293 T and LO2 cells by MTT methodology.
Fig. 15. The antibacterial behavior of 7-thiazoxime quinolones.
chemicals are commercially available and used without prior pu-
rification unless otherwise stated.
potassium carbonate (0.45 mmol) in acetonitrile (15 mL) stirred at
50 ꢃC for 1 h, then added 2-bromo-1-(thiazol-2-yl) ethan-1-one
(0.33 mmol). After the reaction was completed (monitored by
TLC, dichloromethane/methanol (10/1, V/V). After the acetonitrile
was removed under reduced pressure, the mixture was purified by
flash silica gel column eluting with dichloromethane/methanol (1/
10e3/10, V/V) to give the pure target compound 5aeb.
4.2. Synthetic procedures and spectral data for compounds (2e6)
4.2.1. General experimental procedures for the synthesis of the
desired intermediates (2)
The desired intermediate 2 was prepared according to the pre-
viously reported procedures [35].
4.2.4. Synthesis of (E)-1-ethyl-6-fluoro-7-(4-(2-(methoxyimino)-2-
(thiazol-2-yl)ethyl)piperazin-1-yl) -4-oxo-1,4 -dihydroquinoline-3-
carboxylic acid (6a)
A mixture of norfloxacin (96 mg, 0.30 mmol) and potassium
carbonate (62 mg, 0.45 mmol) in acetonitrile (25 mL) were stirred
at 50 ꢃC for 1 h, and then cooled to room temperature. Methyl
oxime 3a (78 mg, 0.33 mmol) was added, and the resulting mixture
was stirred at 50 ꢃC for 5 h. After the completion of reaction, the
solvent was evaporated under reduced pressure. The crude product
4.2.2. General experimental procedures for the synthesis of the
desired intermediates (3aef)
The desired intermediates 3aef were prepared according to the
previously reported methods [36].
4.2.3. Synthesis of the desired intermediates (5aeb)
To a stirred solution of different quinolones (0.30 mmol) and
11

J.-P. Chen, N. Battini, M.F. Ansari et al.
European Journal of Medicinal Chemistry 217 (2021) 113340
was purified by silica gel column chromatography (eluent,
dichloromethane/methanol (V/V) ¼ 1/10e3/10) to afford target
compound 6a (81 mg) as yellow solid. Yield: 56.5%; Mp:
procedure described for compound 6a starting from intermediate
3d (93 mg, 0.33 mmol), potassium carbonate (62 mg, 0.45 mmol)
and norfloxacin (96 mg, 0.30 mmol). The target compound 6d
(66 mg) was obtained as yellow solid. Yield: 40.1%; Mp:
238.7e239.5 ꢃC; 1H NMR (600 MHz, CDCl3)
d 15.12 (bs, 1H, COOH),
8.65 (s, 1H, quinolone-2-H), 8.02 (d, J ¼ 12.8 Hz,1H, quinolone-5-H),
7.86 (d, J ¼ 3.2 Hz,1H, thiazole-5-H), 7.33 (d, J ¼ 3.2 Hz,1H, thiazole-
4-H), 6.80 (d, J ¼ 6.9 Hz, 1H, quinolone-8-H), 4.30 (dd, J ¼ 7.1, 3.4 Hz,
2H, CH2CH3), 4.19 (s, 1H, OCH3), 4.07 (s, 2H, OCH3), 3.96 (s, 2H,
piperazine-CH2), 3.34 (s, 4H, piperazine-2,2-N-(CH2)2), 2.87 (s, 4H,
piperazine-3,3-N-(CH2)2), 1.56 (t, J ¼ 8.4, 5.9 Hz, 3H, CH2CH3) ppm;
165.6e166.2 ꢃC; 1H NMR (600 MHz, CDCl3)
d 15.11 (bs, 1H, COOH),
8.64 (s,1H, quinolone-2-H), 8.01 (d, J ¼ 12.8 Hz,1H, quinolone-5-H),
7.86 (d, J ¼ 3.0 Hz, 1H, thiazole-5-H), 7.44 (dd, J ¼ 14.1, 7.3 Hz, 2H,
Ph-1, 5-2H), 7.38 (dd, J ¼ 12.7, 5.9 Hz, 2H, Ph-2, 4-2H), 7.34 (d,
J ¼ 7.0 Hz, 1H, thiazole-4-H), 7.32 (d, J ¼ 3.1 Hz, 1H, Ph-3-H), 6.78 (d,
J ¼ 6.7 Hz, 1H, quinolone-8-H), 5.44 (s, 1H, Ph-CH2), 5.30 (s, 1H, Ph-
CH2), 4.29 (dd, J ¼ 14.8, 7.4 Hz, 2H, CH2CH3), 3.97 (s, 2H, piperazine-
CH2), 3.30 (m, 4H, piperazine-2,2-N-(CH2)2), 2.80 (s, 4H, piperazine-
3,3-N-(CH2)2), 1.55 (t, J ¼ 7.2 Hz, 3H, CH2CH3) ppm; 13C NMR
13C NMR (151 MHz, CDCl3)
d 176.97 (quinolone-4-C), 167.17 (COOH),
164.25 (C]NO), (152.68, 147.04, 143.19, 142.17, 137.12, 123.16,
120.39, 112.77, 112.62, 108.35, 103.72, aromatic-C), 62.97, 57.99,
53.00, 52.68, 50.56, 14.38 ppm; HRMS (ESI, m/z) calcd for
(151 MHz, CDCl3)
d 176.98 (quinolone-4-C), 167.17 (COOH), 164.31
C
22H24FN5O4S, [M þ H]þ, 474.1611; found, 474.1594.
(C]NO), (152.68, 147.05, 143.22, 142.23, 137.11, 136.87,
128.59e128.32 (m), 128.24, 123.42, 120.45, 112.79, 112.64, 108.38,
103.73, aromatic-C), 77.52, 52.98, 52.54, 50.65, 49.90, 49.78,
14.39 ppm; HRMS (ESI, m/z) calcd. for C28H28FN5O4S [M þ H]þ,
550.1919; found, 550.1920.
4.2.5. Synthesis of (E)-7-(4-(2-(ethoxyimino) -2-(thiazol-2-yl)
ethyl)piperazin-1-yl) -1-ethyl-6-fluoro-4-oxo -1,4-
dihydroquinoline-3-carboxylic acid (6b)
Compound 6b was prepared according to the experimental
procedure described for compound 6a starting from intermediate
3b (82 mg, 0.33 mmol), potassium carbonate (62 mg, 0.45 mmol)
and norfloxacin (96 mg, 0.30 mmol). The target compound 6b
(74 mg) was obtained as yellow solid. Yield: 50.6%; Mp:
4.2.8. Synthesis of (E)-7-(4-(2-((allyloxy)imino)-2-(thiazol-2-yl)
ethyl)piperazin-1-yl) -1-ethyl-6-fluoro-4- oxo -1,4-
dihydroquinoline-3-carboxylic acid (6e)
Norfloxacin (96 mg, 0.30 mmol) was dissolved in acetonitrile
and stirred with potassium carbonate (62 mg, 0.45 mmol) for 1 h,
followed by 3e (86 mg, 0.33 mmol) for 5 h at 50 ꢃC. The reaction
mixture was concentrated under a reduced pressure, the solid was
purified by column chromatography on silica gel (300e400 mesh)
using dichloromethane/methanol (V/V ¼ 1/10e3/10) as eluent to
give 107 mg of the solid and got the yellow solid 6e. yield: 71.5%;
214.8e215.5 ꢃC; 1H NMR (600 MHz, CDCl3)
d 15.10 (bs, 1H, COOH),
8.65 (s, 1H, quinolone-2-H), 8.00 (d, J ¼ 3.1 Hz, 1H, quinolone-5-H),
7.86 (d, J ¼ 3.2 Hz, 1H, thiazole-5-H), 7.31 (d, J ¼ 3.0 Hz,1H, thiazole-
4-H), 6.80 (d, J ¼ 6.7 Hz, 1H, quinolone-8-H), 4.46 (dd, J ¼ 14.0,
7.0 Hz, 1H, OCH2CH3), 4.33 (dd, J ¼ 8.7, 5.4 Hz, 1H, OCH2CH3), 4.30
(dd, J ¼ 8.4, 6.0 Hz, 2H, NCH2CH3), 3.97 (s, 2H, piperazine-CH2), 3.35
(s, 4H, piperazine-2,2-N-(CH2)2), 2.88 (s, 4H, piperazine-3,3-N-
(CH2)2), 1.56 (t, J ¼ 7.0 Hz, 3H, CH2CH3), 1.45 (t, J ¼ 7.0 Hz, 1H,
OCH2CH3), 1.36 (t, J ¼ 7.0 Hz, 2H, OCH2CH3) ppm; 13C NMR
Mp: 185.5e186.2 ꢃC; 1H NMR (600 MHz, CDCl3)
d 15.10 (bs, 1H,
COOH), 8.65 (s, 1H, quinolone-2-H), 8.01 (d, J ¼ 12.7 Hz, 1H, qui-
nolone-5-H), 7.86 (d, J ¼ 3.0 Hz,1H, thiazole-5-H), 7.32 (d, J ¼ 3.0 Hz,
1H, thiazole-4-H), 6.80 (d, J ¼ 7.0 Hz, 1H, quinolone-8-H), 6.08 (m,
1H, CH]CH2), 5.40 (m, 1H, CH]CH2), 5.30 (dd, J ¼ 15.5, 10.8 Hz, 1H,
CH]CH2), 4.6l (d, J ¼ 5.4 Hz, 1H, OCH2), 4.77 (d, J ¼ 5.5 Hz, 1H,
OCH2), 4.33e4.27 (m, 2H, CH2CH3), 3.98 (s, 2H, piperazine-CH2),
3.34 (m, 4H, piperazine-2,2-N-(CH2)2), 2.88 (m, 4H, piperazine-3,3-
N-(CH2)2), 1.56 (t, J ¼ 6.9 Hz, 3H, CH2CH3) ppm; 13C NMR (151 MHz,
(151 MHz, CDCl3)
d 176.94 (quinolone-4-C), 167.17 (COOH), 164.62
(C]NO), (152.69, 147.05, 146.15, 143.14, 142.16, 137.12, 123.04,
120.23, 112.67, 108.33, 103.75, aromatic-C), 71.39, 71.01, 52.97,
52.63, 50.50, 49.90, 49.77, 14.68, 14.38 ppm; HRMS (ESI, m/z) calcd.
for C23H26FN5O4S [M þ H]þ, 488.1762; found, 488.1757.
4.2.6. Synthesis of (E)-7-(4-(2-(tert-butoxyimino) -2-(thiazol-2-yl)
ethyl) piperazin-1-yl) -1-ethyl-6-fluoro -4-oxo- 1,4-
dihydroquinoline-3-carboxylic acid (6c)
CDCl3) d 176.97 (quinolone-4-C), 167.16 (COOH), 164.33 (C]NO),
(152.69, 147.04, 143.20, 142.23, 137.12, 133.38, 123.25, 120.39, 118.37,
112.90e112.83 (m), 112.72, 108.38, 103.72, aromatic-C), 76.19, 57.9,
53.01, 52.62, 50.64, 14.39 ppm; HRMS (ESI, m/z) calcd. for
Compound 6c was prepared according to the experimental
procedure described for compound 6a starting from intermediate
3c (92 mg, 0.33 mmol), potassium carbonate (62 mg, 0.45 mmol)
and norfloxacin (96 mg, 0.30 mmol). The target compound 6c
(95 mg) was obtained as white solid. Yield: 61.4%; Mp:
C
24H26FN5O4S [M þ H]þ, 500.1762; found, 500.1752.
4.2.9. Synthesis of (E)-1-ethyl-6-fluoro-7-(4-(2-((2-methoxy-2-
oxoethoxy)imino) -2-(thiazol-2-yl)ethyl) piperazin -1-yl)-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid (6f)
241.5e242.3 ꢃC; 1H NMR (600 MHz, CDCl3)
d 15.14 (bs, 1H, COOH),
8.65 (s, 1H, quinolone-2-H), 8.01 (d, 1H, quinolone-5-H), 7.99 (d,
J ¼ 2.8 Hz, 1H, thiazole-5-H), 7.54 (d, J ¼ 3.1 Hz, 1H, thiazole-4-H),
6.81 (d, J ¼ 6.1 Hz, 1H, quinolone-8-H), 4.31 (q, J ¼ 7.1 Hz, 2H,
CH2CH3), 4.01 (s, 1H, piperazine-CH2), 3.97 (s, 1H, piperazine-CH2),
3.33 (m, 4H, piperazine-2,2-N-(CH2)2), 2.87 (d, J ¼ 32.0 Hz, 4H,
piperazine-3,3-N-(CH2)2), 1.56 (t, J ¼ 7.2 Hz, 3H, CH2CH3), 1.48 (s,
6H, OC(CH3)3), 1.39 (s, 3H, OC(CH3)3) ppm; 13C NMR (151 MHz,
Compound 6f was prepared according to the experimental pro-
cedure described for compound 6a starting from intermediate 3f
(97 mg, 0.33 mmol), potassium carbonate (62 mg, 0.45 mmol) and
norfloxacin (96 mg, 0.30 mmol). The target compound 6f (122 mg)
was obtained as yellow solid. Yield: 76.2%; Mp: 167.3e168.1 ꢃC; 1H
NMR (600 MHz, CDCl3)d 15.08 (bs,1H, COOH), 8.65 (s,1H, quinolone-
2-H), 8.01 (d, J ¼ 13.5 Hz, 1H, quinolone-5-H), 7.88 (d, J ¼ 3.1 Hz, 1H,
thiazole-5-H), 7.35 (d, J ¼ 3.1 Hz,1H, thiazole-4-H), 6.81 (d, J ¼ 6.2 Hz,
1H, quinolone-8-H), 4.94 (s, 1H, OCH2), 4.83 (s, 1H, OCH2), 4.30 (dd,
J ¼ 13.9, 6.8 Hz, 2H, CH2CH3), 4.01 (s, 2H, piperazine-CH2), 3.79 (s, 3H,
OCH3), 3.34 (s, 4H, piperazine-2,2-N-(CH2)2), 2.90 (s, 4H, piperazine-
3,3-N-(CH2)2), 1.56 (t, J ¼ 6.6 Hz, 3H, CH2CH3) ppm; 13C NMR
CDCl3)
d 176.98 (quinolone-4-C), 167.20 (COOH), 165.92 (C]NO),
(152.70, 147.04, 143.01, 142.04, 137.14, 122.52, 119.77, 112.74, 112.59,
108.33, 103.71, aromatic-C), 81.83, 80.95, 58.13, 52.93, 52.50, 49.69,
27.66, 14.40 ppm; HRMS (ESI, m/z) calcd. for C25H30FN5O4S [M þ
Na]þ, 538.1895; found, 538.1884.
(151 MHz, CDCl3) d 176.99 (quinolone-4-C), 169.61, 169.43 (s), 167.17
4.2.7. Synthesis of (E)-7-(4-(2-((benzyloxy) imino)-2-(thiazol-2-yl)
ethyl)piperazin-1-yl) -1-ethyl-6-fluoro-4-oxo -1,4-
dihydroquinoline-3-carboxylic acid (6d)
(COOH), 163.30 (C]NO), (152.68, 147.05, 143.34, 142.31, 137.13,
120.87, 113.37e113.19 (m), 112.71, 108.37, 103.74, aromatic-C), 71.40,
53.01, 52.52, 52.01, 50.85, 14.41 ppm; HRMS (ESI, m/z) calcd. for
Compound 6d was prepared according to the experimental
C
24H26FN5O6S [M þ H]þ, 532.1661; found, 532.1669.
12

J.-P. Chen, N. Battini, M.F. Ansari et al.
European Journal of Medicinal Chemistry 217 (2021) 113340
4.2.10. Synthesis of (E)-1-cyclopropyl-6-fluoro-7-(4-(2-
(methoxyimino)-2-(thiazol-2-yl)ethyl) piperazin-1-yl) -4- oxo-1,4-
dihydroquinoline-3-carboxylic acid (6g)
(C]NO), (152.86, (147.31, 143.00, 142.03, 139.09, 122.50, 119.75,
112.42,112.26,108.12,104.72 aromatic-C), 81.82, 58.15, 52.95, 52.52,
35.24, 27.66, 8.20 ppm; HRMS (ESI, m/z) calcd. for C26H30FN5O4S
[M þ Na]þ, 550.1990; found, 550.1886.
To a stirred solution of ciprofloxacin (100 mg, 0.30 mmol) and
potassium carbonate (62 mg, 0.45 mmol) in acetonitrile (15 mL)
stirred at 50 ꢃC for 1 h, then added compound 3a (78 mg,
0.33 mmol). After the reaction was completed (monitored by TLC,
dichloromethane/methanol (10/1, V/V). After the acetonitrile was
removed under reduced pressure, the mixture was purified by flash
silica gel column eluting with dichloromethane/methanol (1/10e3/
10, V/V) to give the pure target compound 6g as white power
(99 mg). Yield: 68.0%; Mp: > 250 ꢃC; 1H NMR (600 MHz, CDCl3)
4.2.13. Synthesis of (E)-7-(4-(2-((benzyloxy)imino)-2-(thiazol-2-yl)
ethyl) piperazin-1-yl)-1-cyclopropyl -6-fluoro -4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (6j)
Compound 6j was prepared according to the experimental
procedure described for compound 6h starting from intermediate
3d (93 mg, 0.33 mmol), potassium carbonate (62 mg, 0.45 mmol)
and ciprofloxacin (100 mg, 0.30 mmol). The target compound 6j
(79 mg) was obtained as yellow solid. Yield: 46.9%; Mp:
d
15.02 (bs, 1H, COOH), 8.73 (s, 1H, quinolone-2-H), 7.98 (d,
J ¼ 4.2 Hz, 1H, quinolone-5-H), 7.87 (d, J ¼ 3.1 Hz, 1H, thiazole-5-H),
7.34 (d, J ¼ 8.2 Hz, 1H, thiazole-4-H), 7.32 (d, J ¼ 6.9 Hz, 1H, qui-
nolone-8-H), 4.20 (s, 1H, OCH3), 4.07 (s, 2H, OCH3), 3.97 (s, 2H,
piperazine-CH2), 3.52 (d, J ¼ 3.9 Hz, 1H, cyclopropane-CH), 3.37 (m,
4H, piperazine-2,2-N-(CH2)2), 2.88 (m, 4H, piperazine-3,3-N-
(CH2)2), 1.39e1.34 (m, 2H, cyclopropane-CH2), 1.18 (s, 2H, cyclo-
145.3e146.8 ꢃC; 1H NMR (600 MHz, CDCl3)
d 14.98 (bs, 1H, COOH),
8.71 (s, 1H, quinolone-2-H), 7.97 (d, J ¼ 5.7 Hz, 1H, quinolone-5-H),
7.85 (d, J ¼ 2.9 Hz, 1H, thiazole-5-H), 7.44 (dd, J ¼ 13.0, 7.4 Hz, 2H,
Ph-1, 5-2H), 7.37 (dd, J ¼ 12.9, 6.5 Hz, 2H, Ph-2, 4-2H), 7.34 (d,
J ¼ 7.1 Hz, 1H, thiazole-4-H), 7.32 (d, J ¼ 3.1 Hz, 1H, Ph-3-H), 7.29 (d,
J ¼ 6.9 Hz, 1H, quinolone-8-H), 5.30 (s, 2H, Ph-CH2), 3.97 (s, 2H,
piperazine-CH2), 3.50 (s, 1H, cyclopropane-CH), 3.32 (m, 4H,
piperazine-2,2-N-(CH2)2), 2.80 (s, 4H, piperazine-3,3-N-(CH2)2),
1.35 (d, J ¼ 6.8 Hz, 2H, cyclopropane-CH2), 1.17 (s, 2H, cyclopropane-
propane-CH2) ppm; 13C NMR (151 MHz, CDCl3)
d 177.11 (quinolone-
4-C), 166.99 (COOH), 164.27 (C]NO), (152.85, 147.35, 143.20, 142.19,
139.08, 123.19, 120.40, 112.46, 112.31, 108.15, 104.75 aromatic-C),
62.99, 53.02, 52.69, 50.54, 35.24, 8.20 ppm; HRMS (ESI, m/z) calcd.
for C23H24FN5O4S [M þ H]þ, 486.1606; found, 486.1601.
CH2) ppm; 13C NMR (151 MHz, CDCl3)
d 177.09 (quinolone-4-C),
166.92 (COOH), 164.36 (C]NO), (152.84, 147.31, 143.20, 142.20,
139.08,136.90, 128.43 (d, J ¼ 10.3 Hz),128.21,123.34,120.40, 112.44,
112.28, 108.18, 104.72 aromatic-C), 77.51, 53.00, 52.55, 50.69, 35.22,
8.17 ppm; HRMS (ESI, m/z) calcd. for C29H28FN5O4S [M þ H]þ,
562.1619; found, 562.1615.
4.2.11. Synthesis of (E)-1-cyclopropyl-7-(4-(2-(ethoxyimino)-2-
(thiazol-2-yl)ethyl) piperazin-1-yl) -6-fluoro -4-oxo -1,4-
dihydroquinoline-3-carboxylic acid (6h)
Compound 6h was prepared according to the experimental
procedure described for compound 6h starting from intermediate
3b (82 mg, 0.33 mmol), potassium carbonate (62 mg, 0.45 mmol)
and ciprofloxacin (100 mg, 0.30 mmol). The target compound 6h
(74 mg) was obtained as white solid. Yield: 50.6%; Mp: > 250 ꢃC; 1H
4.2.14. Synthesis of (E)-7-(4-(2-((allyloxy)imino)-2-(thiazol-2-yl)
ethyl)piperazin-1-yl)-1-cyclopropyl -6-fluoro -4-oxo- 1,4-
dihydroquinoline-3-carboxylic acid (6k)
Compound 6k was prepared according to the experimental
procedure described for compound 6h starting from intermediate
3e (86 mg, 0.33 mmol), potassium carbonate (62 mg, 0.45 mmol)
and ciprofloxacin (100 mg, 0.30 mmol). The target compound 6k
(82 mg) was obtained as yellow solid. Yield: 53.4%; Mp:
NMR (600 MHz, CDCl3)
d 15.01 (bs, 1H, COOH), 8.72 (s, 1H, quino-
lone-2-H), 7.95 (d, J ¼ 13.0 Hz, 1H, quinolone-5-H), 7.86 (d,
J ¼ 2.6 Hz, 1H, thiazole-5-H), 7.33 (d, J ¼ 8.0 Hz, 1H, thiazole-4-H),
7.32 (s, 1H, quinolone-8-H), 4.46 (dd, J ¼ 13.9, 6.9 Hz, 1H, OCH2CH3),
4.33 (dd, J ¼ 13.8, 6.8 Hz, 1H, OCH2CH3), 3.98 (s, 2H, piperazine-
CH2), 3.52 (s, 1H, cyclopropane-CH), 3.36 (m, 4H, piperazine-2,2-N-
(CH2)2), 2.88 (m, 4H, piperazine-3,3-N-(CH2)2),1.45 (t, J ¼ 7.0 Hz,1H,
OCH2CH3), 1.37 (d, J ¼ 6.5 Hz, 2H, OCH2CH3), 1.36 (s, 2H, cyclopro-
pane-CH2), 1.18 (s, 2H, cyclopropane-CH2) ppm; 13C NMR (151 MHz,
203.5e204.1 ꢃC; 1H NMR (600 MHz, CDCl3)
d 14.98 (bs, 1H, COOH),
8.71 (s, 1H, quinolone-2-H), 7.95 (d, J ¼ 13.1 Hz, 1H, quinolone-5-H),
7.86 (d, J ¼ 3.1 Hz,1H, thiazole-5-H), 7.33 (d, J ¼ 9.2 Hz,1H, thiazole-
4-H), 7.31 (d, J ¼ 4.2 Hz, 1H, quinolone-8-H), 6.08 (m, 1H, CH]CH2),
5.40 (m,1H, CH]CH2), 5.29 (dd, J ¼ 14.3,11.1 Hz,1H, CH]CH2), 4.77
(d, J ¼ 5.5 Hz, 2H, OCH2), 3.98 (s, 2H, piperazine-CH2), 3.52 (d,
J ¼ 3.8 Hz, 1H, cyclopropane-CH), 3.36 (m, 4H, piperazine-2,2-N-
(CH2)2), 2.88 (m, 4H, piperazine-3,3-N-(CH2)2), 1.36 (d, J ¼ 6.3 Hz,
2H, cyclopropane-CH2), 1.18 (s, 2H, cyclopropane-CH2) ppm; 13C
CDCl3)
d 177.08 (quinolone-4-C), 166.97 (COOH), 164.67 (C]NO),
(152.85, 147.31, 143.15, 142.16, 139.08, 123.00, 120.20, 112.32 (d,
J ¼ 23.5 Hz), 112.22e112.05 (m), 108.10, 104.76 aromatic-C), 71.36,
71.00, 53.00, 52.67, 50.54, 35.25, 14.68, 8.19 ppm; HRMS (ESI, m/z)
calcd. for C24H26FN5O4S [M þ H]þ, 500.1762; found, 500.1765.
NMR (151 MHz, CDCl3)
d 177.08 (quinolone-4-C), 166.96 (COOH),
164.34 (C]NO), (154.51, 152.84, 147.32, 143.21, 142.24, 139.08,
133.43, 120.39, 118.34, 112.41, 112.26, 108.12, 104.76 aromatic-C),
76.20, 53.04, 52.64, 50.66, 35.24, 8.20 ppm; HRMS (ESI, m/z) calcd.
for C25H26FN5O4S [M þ H]þ, 512.1762; found, 512.1753.
4.2.12. Synthesis of (E)-7-(4-(2-(tert-butoxyimino)-2-(thiazol-2-yl)
ethyl)piperazin-1-yl)-1-cyclopropyl -6-fluoro -4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (6i)
Compound 6i was prepared according to the experimental
procedure described for compound 6h starting from intermediate
3c (92 mg, 0.33 mmol), potassium carbonate (62 mg, 0.45 mmol)
and ciprofloxacin (100 mg, 0.30 mmol). The target compound 6i
(97 mg) was obtained as white solid. Yield: 61.2%; Mp: > 250 ꢃC; 1H
4.2.15. Synthesis of (E)-1-cyclopropyl-6-fluoro-7-(4-(2-((2-
methoxy-2-oxoethoxy)imino) -2-(thiazol-2-yl) ethyl) piperazin-1-
yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (6l)
Compound 6l was prepared according to the experimental
procedure described for compound 6h starting from intermediate
3f (97 mg, 0.33 mmol), potassium carbonate (62 mg, 0.45 mmol)
and ciprofloxacin (100 mg, 0.30 mmol). The target hybrid 6l
(56 mg) was obtained as white solid. Yield: 34.3%; Mp:
NMR (600 MHz, CDCl3)
d 15.04 (bs, 1H, COOH), 8.75 (s, 1H, quino-
lone-2-H), 8.01 (d, J ¼ 3.1 Hz, 1H, quinolone-5-H), 7.99 (d,
J ¼ 13.0 Hz, 1H, thiazole-5-H), 7.56 (d, J ¼ 3.0 Hz, 1H, thiazole-4-H),
7.35 (d, J ¼ 6.5 Hz, 1H, quinolone-8-H), 4.00 (s, 2H, piperazine-CH2),
3.53 (d, J ¼ 3.2 Hz, 1H, cyclopropane-CH), 3.37 (m, 4H, piperazine-
2,2-N-(CH2)2), 2.89 (m, 4H, piperazine-3,3-N-(CH2)2), 1.50 (s, 6H,
OC(CH3)3), 1.41 (s, 3H, OC(CH3)3), 1.38 (d, J ¼ 6.8 Hz, 2H, cyclopro-
pane-CH2), 1.19 (d, J ¼ 2.6 Hz, 2H, cyclopropane-CH2) ppm; 13C NMR
178.5e180.1 ꢃC; 1H NMR (600 MHz, CDCl3)
d 14.99 (bs, 1H, COOH),
8.72 (s, 1H, quinolone-2-H), 7.96 (d, J ¼ 13.0 Hz, 1H, quinolone-5-H),
7.88 (d, J ¼ 3.1 Hz, 1H, thiazole-5-H), 7.35 (d, J ¼ 3.1 Hz, 1H, thiazole-
4-H), 7.33 (d, J ¼ 7.0 Hz, 1H, quinolone-8-H), 4.83 (s, 2H, OCH2), 4.02
(s, 2H, piperazine-CH2), 3.79 (s, 3H, OCH3), 3.54e3.50 (m, 1H,
(151 MHz, CDCl3)
d 177.10 (quinolone-4-C), 166.99 (COOH), 165.93
13

J.-P. Chen, N. Battini, M.F. Ansari et al.
European Journal of Medicinal Chemistry 217 (2021) 113340
cyclopropane-CH), 3.36 (s, 4H, piperazine-2,2-N-(CH2)2), 2.90 (s,
4H, piperazine-3,3-N-(CH2)2), 1.37 (q, J ¼ 6.4 Hz, 2H, cyclopropane-
CH2), 1.18 (d, J ¼ 3.1 Hz, 2H, cyclopropane-CH2) ppm; 13C NMR
(98 mg) was obtained as yellow solid. Yield: 58.1%; Mp:
143.8e144.5 ꢃC; 1H NMR (600 MHz, CDCl3)
d 14.49 (bs, 1H, COOH),
8.6l (s, 1H, quinolone-2-H), 8.02 (d, J ¼ 2.5 Hz, 1H, quinolone-5-H),
8.01 (d, J ¼ 5.7 Hz,1H, thiazole-5-H), 7.56 (d, J ¼ 3.0 Hz,1H, thiazole-
4-H), 4.35 (dt, J ¼ 10.3, 3.4 Hz, 1H, cyclopropane-CH), 3.99 (s, 2H,
piperazine-CH2), 3.43 (m, 4H, piperazine-2,2-N-(CH2)2), 2.84 (m,
4H, piperazine-3,3-N-(CH2)2), 1.50 (s, 6H, OC(CH3)3), 1.41 (s, 3H,
OC(CH3)3), 1.31 (d, J ¼ 6.9 Hz, 2H, cyclopropane-CH2), 0.96 (s, 2H,
(151 MHz, CDCl3)
d 177.10 (quinolone-4-C), 169.61, 169.42, 166.98
(COOH), 163.31 (C]NO), (152.84, 147.33, 143.34, 142.32, 139.09,
120.87, 112.43, 112.27, 108.12, 104.77 aromatic-C), 71.40, 53.03,
52.55, 52.01, 50.89, 35.25, 8.20 ppm; HRMS (ESI, m/z) calcd. for
C
25H26FN5O6S [M þ H]þ, 544.1661; found, 544.1649.
cyclopropane-CH2) ppm; 13C NMR (151 MHz, CDCl3)
d 176.81
4.2.16. Synthesis of (E)-1-cyclopropyl-6-fluoro-7-(4-(2-
(methoxyimino)-2-(thiazol-2-yl)ethyl) piperazin-1-yl) -4- oxo-1,4-
dihydroquinoline-3-carboxylic acid (6 m)
(quinolone-4-C), 166.09 (COOH), 155.24, 151.81, 143.01, 142.03,
138.12, 123.24, 122.42, 119.70, 119.12, 111.72, 111.63, 108.67, 81.71,
53.80, 53.34, 51.32, 41.17, 27.66 (d, J ¼ 8.8 Hz), 11.34 ppm; HRMS
(ESI, m/z) calcd. for C26H29ClFN5O4S [M þ H]þ, 562.1686; found,
562.1687.
A solution of compound 4c (110 mg, 0.30 mmol) and potassium
carbonate (62 mg, 0.45 mmol) in acetonitrile (20 mL) was stirred at
50 ꢃC for 1 h. Then intermediate 3a was added (78 mg, 0.33 mmol).
The mixture was stirred 50 ꢃC until TLC showed that the reaction
was completed. The solvent was removed under reduced pressure
and the residue was purified by silica gel column chromatography
(eluent, dichloromethane/methanol (V/V) ¼ 1/10e3/10) to afford
target hybrid 6m (105 mg) as yellow solid. Yield: 67.4%; Mp:
4.2.19. Synthesis of (E)-7-(4-(2-((benzyloxy)imino)-2-(thiazol-2-yl)
ethyl)piperazin-1-yl)-8-chloro-1- cyclopropyl -6 -fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (6p)
Compound 6p was prepared according to the experimental
procedure described for compound 6m starting from intermediate
3d (93 mg, 0.33 mmol), potassium carbonate (62 mg, 0.45 mmol)
and compound 4c (110 mg, 0.30 mmol). The target hybrid 6p
(115 mg) was obtained as yellow solid. Yield: 64.4%; Mp:
228.2e229.4 ꢃC; 1H NMR (600 MHz, CDCl3)
d 14.45 (bs, 1H, COOH),
8.89 (s, 1H, quinolone-2-H), 7.99 (d, J ¼ 11.6 Hz, 1H, quinolone-5-H),
7.87 (s, 1H, thiazole-5-H), 7.34 (s, 1H, thiazole-4-H), 4.34 (m, 1H,
cyclopropane-CH), 4.07 (s, 3H, OCH3), 3.93 (s, 2H, piperazine-CH2),
3.42 (m, 4H, piperazine-2,2-N-(CH2)2), 2.82 (m, 4H, piperazine-3,3-
N-(CH2)2), 1.29 (d, J ¼ 6.6 Hz, 2H, cyclopropane-CH2), 0.95 (s, 2H,
162.9e163.8 ꢃC; 1H NMR (600 MHz, CDCl3)
d 14.46 (bs, 1H, COOH),
8.88 (s, 1H, quinolone-2-H), 7.96 (d, 1H, quinolone-5-H), 7.86 (d,
J ¼ 3.0 Hz,1H, thiazole-5-H), 7.44 (d, J ¼ 7.7 Hz, 2H, Ph-1, 5-2H), 7.38
(d, J ¼ 6.9 Hz, 2H, Ph-2, 4-2H), 7.33 (d, J ¼ 3.0 Hz, 1H, thiazole-4-H),
7.27 (s, 1H, Ph-3-H), 5.31 (s, 2H, OCH2), 4.33 (dd, J ¼ 6.6, 3.2 Hz, 1H,
cyclopropane-CH), 3.95 (s, 2H, piperazine-CH2), 3.36 (s, 4H, piper-
azine-2,2-N-(CH2)2), 2.75 (s, 4H, piperazine-3,3-N-(CH2)2), 1.29 (d,
J ¼ 6.7 Hz, 2H, cyclopropane-CH2), 0.95 (s, 2H, cyclopropane-CH2)
cyclopropane-CH2) ppm; 13C NMR (151 MHz, CDCl3)
d 176.81
(quinolone-4-C), 166.05 (COOH), 164.30 (C]NO), (155.38, 151.85,
143.21, 138.09, 137.01, 128.45, 128.16, 120.44, 111.71, 111.56, 108.66
aromatic-C), 77.59, 77.44, 53.86, 53.49, 41.20, 11.36 ppm; HRMS
(ESI, m/z) calcd. for C23H23ClFN5O4S [M þ H]þ, 520.1216; found,
520.1234.
ppm; 13C NMR (151 MHz, CDCl3)
d 176.81 (quinolone-4-C), 166.05
(COOH), 164.30 (C]NO), (157.06, 155.38, 151.85, 144.63, 143.21,
142.21, 138.09, 137.01, 128.43, 128.16, 123.31, 120.44, 111.71, 111.56,
108.66 aromatic-C), 77.44, 53.86, 53.49, 51.15, 41.20, 11.36 ppm;
HRMS (ESI, m/z) calcd. for C29H27ClFN5O4S [M þ H]þ, 596.1529;
found, 596.1538.
4.2.17. Synthesis of (E)-8-chloro-1-cyclopropyl-7-(4-(2-
(ethoxyimino)-2-(thiazol-2-yl)ethyl) piperazin-1-yl) -6- fluoro-4-
oxo-1,4-dihydroquinoline-3-carboxylic acid (6n)
A solution of compound 4c (110 mg, 0.30 mmol) and potassium
carbonate (62 mg, 0.45 mmol) in acetonitrile (20 mL) was stirred at
50 ꢃC for 1 h. Ethyl oxime 3b (82 mg, 0.33 mmol) was added, and
the resulting mixture was stirred at 50 ꢃC for 5 h. The mixture was
stirred until TLC showed that the reaction was completed. The
solvent was removed under reduced pressure and the residue was
purified by silica gel column chromatography (eluent, dichloro-
methane/methanol (V/V) ¼ 1/10e3/10) to afford target hybrid 6n
(145 mg) as yellow solid. Yield: 90.7%; Mp: 176.1e177.5 ꢃC; 1H NMR
4.2.20. Synthesis of (E)-7-(4-(2-((allyloxy)imino)-2-(thiazol-2-yl)
ethyl)piperazin-1-yl)-8-chloro-1- cyclopropyl -6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (6q)
Compound 6q was prepared according to the experimental
procedure described for compound 6m starting from intermediate
3e (86 mg, 0.33 mmol), potassium carbonate (62 mg, 0.45 mmol)
and compound 4c (110 mg, 0.30 mmol). The target hybrid 6q
(84 mg) was obtained as yellow solid. Yield: 51.4%; Mp:
(600 MHz, DMSO‑d6) d 14.53 (bs, 1H, COOH), 8.82 (s, 1H, quinolone-
2-H), 7.95 (d, J ¼ 3.1 Hz, 1H, quinolone-5-H), 7.89 (d, J ¼ 11.8 Hz, 1H,
thiazole-5-H), 7.78 (d, J ¼ 3.1 Hz, 1H, thiazole-4-H), 4.38 (dd, J ¼ 9.6,
5.7 Hz, 1H, cyclopropane-CH), 4.26 (q, J ¼ 7.0 Hz, 2H, OCH2), 3.86 (s,
2H, piperazine-CH2), 3.30 (s, 4H, piperazine-2,2-N-(CH2)2), 2.68 (s,
4H, piperazine-3,3-N-(CH2)2), 1.39e1.28 (m, 3H, OCH2CH3), 1.18 (d,
J ¼ 6.2 Hz, 2H, cyclopropane-CH2), 0.97 (s, 2H, cyclopropane-CH2)
183.1e184.6 ꢃC; 1H NMR (600 MHz, CDCl3)
d 14.44 (bs, 1H, COOH),
8.88 (s, 1H, quinolone-2-H), 7.98 (d, J ¼ 11.7 Hz, 1H, quinolone-5-H),
7.87 (d, J ¼ 3.0 Hz, 1H, thiazole-5-H), 7.33 (d, J ¼ 3.1 Hz,1H, thiazole-
4-H), 6.14e6.02 (m,1H, CH]CH2), 5.41e5.27 (m,1H, CH]CH2), 4.78
(s, 2H, OCH2), 4.33 (m, 1H, cyclopropane-CH), 3.97 (s, 2H, pipera-
zine-CH2), 3.42 (m, 4H, piperazine-2,2-N-(CH2)2), 2.83 (m, 4H,
piperazine-3,3-N-(CH2)2), 1.29 (q, J ¼ 6.6 Hz, 2H, cyclopropane-
CH2), 0.95 (d, J ¼ 3.3 Hz, 2H, cyclopropane-CH2) ppm; 13C NMR
ppm; 13C NMR (151 MHz, DMSO‑d6)
d 176.59 (quinolone-4-C),
165.54 (COOH), 164.16 (C]NO), (156.83, 153.16, 144.12, 143.74,
138.49, 123.14, 122.07, 119.76, 111.05, 110.89, 108.14 aromatic-C),
70.84, 54.03, 53.52, 50.89, 41.9, 14.96, 11.25 ppm; HRMS (ESI, m/z)
calcd. for C24H25ClFN5O4S [M þ H]þ, 534.1373; found, 534.1379.
(151 MHz, CDCl3)
d 176.79 (quinolone-4-C), 166.04 (COOH), 164.27
(C]NO), 157.06, 155.38, 151.84, 151.39, 143.18, 142.21, 138.08,
133.52, 120.39, 118.21, 111.70, 111.54, 108.64, 76.14, 53.87, 53.52,
51.11, 41.20, 11.34 ppm; HRMS (ESI, m/z) calcd. for C25H25ClFN5O4S
[M þ H]þ, 546.1373; found, 546.1383.
4.2.18. Synthesis of (E)-7-(4-(2-(tert-butoxyimino)-2-(thiazol-2-yl)
ethyl)piperazin-1-yl) -8-chloro-1- cyclopropyl -6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (6o)
Compound 6o was prepared according to the experimental
procedure described for compound 6m starting from intermediate
3c (92 mg, 0.33 mmol), potassium carbonate (62 mg, 0.45 mmol)
and compound 4c (110 mg, 0.30 mmol). The target hybrid 6o
4.2.21. Synthesis of (E)-8-chloro-1-cyclopropyl-6-fluoro-7-(4-(2-
((2-methoxy-2-oxoethoxy) imino)-2- (thiazol -2 -yl)ethyl)
piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (6r)
Compound 6r was prepared according to the experimental
procedure described for compound 6m starting from intermediate
14

J.-P. Chen, N. Battini, M.F. Ansari et al.
European Journal of Medicinal Chemistry 217 (2021) 113340
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3f (97 mg, 0.33 mmol), potassium carbonate (62 mg, 0.45 mmol)
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(135 mg) was obtained as yellow solid. Yield: 78.0%; Mp:
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205.4e206.2 ꢃC; 1H NMR (600 MHz, CDCl3)
d 14.42 (bs, 1H, COOH),
8.88 (s, 1H, quinolone-2-H), 7.99 (d, J ¼ 11.6 Hz, 1H, quinolone-5-H),
7.89 (d, J ¼ 3.1 Hz, 1H, thiazole-5-H), 7.36 (d, J ¼ 3.1 Hz, 1H, thiazole-
4-H), 4.84 (s, 2H, OCH2), 4.35e4.31 (m, 1H, cyclopropane-CH), 4.01
(s, 2H, piperazine-CH2), 3.79 (s, 3H, OCH3), 3.41 (s, 4H, piperazine-
2,2-N-(CH2)2), 2.85 (s, 4H, piperazine-3,3-N-(CH2)2), 1.29 (q,
J ¼ 6.9 Hz, 2H, cyclopropane-CH2), 0.94 (q, J ¼ 6.5 Hz, 2H, cyclo-
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d 176.80 (quinolone-
4-C), 169.64 (COOH), 166.06 (C]NO), 163.20, 157.06, 155.38, 151.86,
144.70, 143.33, 138.08, 120.89, 119.31, 111.73, 111.57, 108.67, 71.40,
53.88, 51.96, 51.37, 51.19, 41.19,11.35 ppm; HRMS (ESI, m/z) calcd. for
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C
25H25ClFN5O6S [M þ H]þ, 578.1271; found, 578.1302.
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Declaration of competing interest
(b) A. Naeem, S.L. Badshah, M. Muska, N. Ahmad, K. Khan, The current case of
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The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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Acknowledgments
This research was supported in part by grants from the National
Natural Science Foundation of China (Nos. 21971212, 21672173), the
Research Funds for the Central Universities (XDJK2020C031),
Postdoctoral Science Foundation P roject of Chongqing Science and
Technology Bureau (cstc2019jcyj-bshX0124), China Postdoctoral
Science Foundation (2019M653821XB) and Chongqing Special
Foundation for Postdoctoral Research Proposal (No. XmT2018082).
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Appendix A. Supplementary data
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